Chapter 12: Toxic Responses of the Immune System

Immunotoxicology can be most simply defined as the study of adverse effects on the immune system resulting from occupational, inadvertent, or therapeutic exposure to drugs, environmental chemicals, and, in some instances, biological materials. Studies in animals and humans have indicated that the immune system is comprised of potential target organs, and that damage to this system can be associated with morbidity and even mortality. Indeed, in some instances, the immune system can be compromised (decreased lymphoid cellularity, alterations in lymphocyte subpopulations, decreased host resistance, and altered specific immune function responses) in the absence of observed toxicity in other organ systems. These studies coupled with tremendous advances made in immunology and molecular biology have led to a steady and exponential growth in our understanding of immunotoxicology during the past 30 years. Recognition by regulatory agencies that the immune system is an important, as well as sensitive, target organ for chemical- and drug-induced toxicity is another indication of the growth of this subdiscipline of toxicology. With the availability of sensitive, reproducible, and predictive tests, it is now apparent that the inclusion of immunotoxicity testing represents a significant adjunct to routine safety evaluations for therapeutic agents, biological agents, and chemicals now in development.

Understanding the impact of toxic responses on the immune system requires an appreciation of its role, which may be stated succinctly as the preservation of integrity. It is a series of delicately balanced, complex, multicellular, and physiological mechanisms that allow an individual to distinguish foreign material (ie, “nonself”) from “self,” and to neutralize, eliminate, and/or coexist with the foreign matter. Examples of self are all the tissues, organs, and cells of the body. Examples of nonself are a variety of opportunistic pathogens, including bacteria and viruses, and transformed cells or tissues (ie, tumors). The immune system is characterized by a virtually infinite repertoire of specificities, highly specialized effectors, complex regulatory mechanisms, and an ability to travel throughout the body. The great complexity of the mammalian immune system is an indication of the importance, as well as the difficulty, of its role. If the immune system fails to recognize as nonself an infectious entity or neoantigens expressed by a newly arisen tumor, then the host is in danger of rapidly succumbing to the unopposed invasion. This aspect of immune competence is the reason why the immune system is often synonymous with “host defense.” Alternatively, if some integral bodily tissue is not identified as self, then the immune system is capable of turning its considerable defensive capabilities against that tissue, and an autoimmune disease may be the end result. This aspect of immunocompetence emphasizes the tremendous destructive potential that is associated with the host defense mechanisms of the immune system. The cost to the host of these mistakes, made in either direction, may be quite high. The fact that mistakes can occur in either direction is an indication that immunotoxicology should be considered as a continuum (Fig. 12-1). At the center of the concept of the continuum is the recognition that immune responses in the normal human population can vary by more than two standard deviations (Luebke et al., 2004), as described in greater detail in the subsection entitled “Approaches to the Assessment of Human Immunotoxicity.” Because the cost of mistakes in immunocompetence can be so high, and because of the tremendous diversity involved in the identification of self versus nonself, a complex array of organs, cells, soluble factors, and their interactions has evolved to regulate this system and minimize the frequency of errors in either direction. Due to the potentially profound effects resulting from disruption of the delicately balanced immune system, there is a need to understand the cellular, biochemical, and molecular mechanisms of xenobiotic-induced immune modulation.

This chapter provides (1) an overview of basic concepts in immunology (structure, components, and functions), which are important to the understanding of the impact that xenobiotics may have on the exposed individual; (2) a summary of selected current methods utilized to assess immune function; and (3) a brief review of current information on the immune modulation (immune suppression, immune enhancement, hypersensitivity, and autoimmunity) induced by a variety of xenobiotics. This chapter is not meant to be an immunology textbook, nor an exhaustive review of the mechanisms of immunotoxicity of a myriad of xenobiotics. For detailed information on immunology, the reader is referred to three texts: the first edited by Paul, Fundamental Immunology (6th edition, 2008), the second written by Delves, Martin, Burton, and Roitt, Roitt’s Essential Immunology (12th edition, 2011), and the third written by Murphy, Travers, and Walport, Janeway’s Immunobiology (7th edition, 2008). For a more comprehensive review of immunotoxicology, the reader is referred to two texts: the first edited by Vohr and colleagues, Encyclopedic Reference of Immunotoxicology (2005), and the second edited by Luebke, House, and Kimber, Immunotoxicology and Immunopharmacology, Target Organ Toxicity Series (3rd edition, 2007). The reader might also find the list of abbreviations in Table 12-1 helpful.

Unlike most organ systems, the immune system has the unique quality of not being confined to a single site within the body. It comprises numerous lymphoid organs (Table 12-2) and many different cellular populations with a variety of functions. The bone marrow and thymus are referred to as primary lymphoid organs because they contain the microenvironments capable of supporting the production of mature B and T cells, respectively. Central tolerance (nonreactive to self-antigens) is also established in the primary lymphoid organs to prevent the maturation of lymphocytes that recognize self-antigens. In addition, the bone marrow is the site of origin of the pluripotent and self-renewing hematopoietic stem cell (HSC) from which all other hematopoietic cells are derived. While immunologists differ on how to define and identify the hematopoietic intermediates, Fig. 12-2 shows one model of hematopoiesis (adapted from Baltimore et al. 2008; Chaplin, 2010). During gestation, the HSC is found in the embryonic yolk sac and fetal liver; eventually, it migrates to the bone marrow. Within the bone marrow, the HSC developmentally commits to either the lymphoid or myeloid lineages by giving rise to the common lymphoid progenitor (CLP) or the common myeloid progenitor (CMP), which differentiate into various types of hematopoietic cells. CLP cells make a further commitment to become either T cells or B cells (Fig. 12-3). The maturation process for both T cells and B cells involves the establishment of central tolerance, but this process occurs at different primary lymphoid organs. For B cells, central tolerance occurs in the bone marrow and simply involves the removal of self-reactive cells. It is more complex for T cells and involves the migration of T-cell precursors from the bone marrow to the thymus where they undergo a process of both positive and negative selection resulting in mature T cells that recognize the host’s cells (ie, positive selection) but not too strongly. T cells that recognize self too strongly are deleted from the T-cell repertoire (ie, negative selection). Recognition of self is essential for T cells to initiate and regulate immune responses.

Mature, naive, or virgin lymphocytes (those T and B cells that have never undergone antigenic stimulation) are first brought into contact with exogenously derived antigens within the highly organized microenvironment of the spleen and lymph nodes, which are secondary lymphoid organs. These organs can be thought of as biological sieves. The spleen serves as a filter for the blood, removing both foreign antigens and any circulating dead cells and cellular debris. The lymph nodes are part of a network of lymphatic veins that filter antigens from the fluid surrounding the tissues of the body. In addition to the spleen and lymph nodes, other secondary lymphoid tissues associated with the skin, mucosal lamina propria, gut, bronchioles, or nasal cavity are referred to as associated lymphoid tissues (abbreviated SALT, MALT, GALT, BALT, and NALT, respectively). Included in the GALT are specialized structures in the small intestine called Peyer’s patches that collect antigens from the gastrointestinal tract. The associated lymphoid tissues tend to have more exposure to antigen and greater plasticity (ie, increase or decrease in size and/or number) than lymph nodes. The plasticity of lymphoid organs is underscored by the detection of lymphoid neogenesis in nonlymphoid organs during chronic inflammation. The new lymphoid tissue is classified as tertiary lymphoid tissue, and its development is reversible if the inflammatory response is resolved. Although studies suggest that tertiary lymphoid tissues support transient, local immune responses, there is still much to learn regarding lymphoid neogenesis and its role in the immune response particularly with clinical and experimental evidence supporting an association between lymphoid neogenesis (ie, tertiary lymphoid tissues) and pathophysiological conditions such as autoimmune disease and cancer (Drayton et al., 2006).

Antigen Recognition

Immunity

Mammalian immunity can be classified into two functional divisions: innate immunity and acquired (adaptive) immunity (Table 12-3). Innate immunity has historically been characterized as a first-line defense response with little immunological memory. Therefore, in a normal healthy adult, the magnitude of the innate immune response to a foreign organism is similar for a secondary or a tertiary challenge as it is for the primary exposure. By contrast, acquired (adaptive) immunity is characterized by specificity and immunological memory. Thus, in a normal healthy adult, the speed and magnitude of the acquired immune response to a foreign organism is greater for a secondary challenge than it is for the primary challenge.

Antigen

The primary determinant in either type of immune response is the ability of the immune system components to recognize self versus nonself. One definition of nonself, essentially, is anything other than that encoded in one’s own germline genome (Nathan, 2006). Given this broad interpretation of nonself, this includes foreign DNA, RNA, protein, and carbohydrates, and may even include aberrantly expressed or mutated self-proteins, since those are likely not contained in one’s own germline genome. A nonself substance that can be recognized by the immune system is called an antigen (also referred to as an immunogen or allergen). Antigens are usually (but not absolutely) biological molecules that can be cleaved and rearranged for presentation to other immune cells. Generally, antigens are about 10 kDa or larger in size. Smaller antigens are termed “haptens” and must be conjugated with carrier molecules (larger antigens) in order to elicit a specific response. However, once an initial response is made, the hapten can induce subsequent responses in the absence of the carrier.

Antibodies

Antibodies are produced by B cells and are defined functionally by the antigen with which they react, and by their subtype, termed “isotypes” (IgM, IgG, IgE, IgD, and IgA; Table 12-4). Thus, an IgM antibody directed against sheep red blood cells (sRBCs) is called anti-sRBC IgM. Because the immune system generates antibodies to thousands of antigens with which the host may or may not ever come into contact, antibodies of unknown specificity are referred to as immunoglobulin (eg, serum immunoglobulin or serum IgM) until they can be defined by their specific antigen (eg, anti-sRBC IgM).

The ability of the immune system to generate antibody to thousands of antigens is the result of somatic recombination, in which the germline DNA that encodes for antibodies is rearranged in B cells. All Igs are made up of heavy and light chains and of constant and variable regions. For the light chain genes, two separate gene segments (V and J) are combined to form the variable region, which is then joined to one constant region. For the heavy chain genes, three separate gene segments (V, D, and J) are combined to form the variable region, which is then joined to one constant region. There are several light chain V and J genes, and several heavy chain V, D, and J genes, which when rearranged in various combinations, contribute to the immense genetic diversity of the Ig genes (Fig. 12-4). Finally, the five types of Ig are dependent on which heavy chain constant region is transcribed and translated (heavy chain genes μ, γ, ε, δ, or α encode for the IgM, IgG, IgE, IgD, or IgA heavy chain proteins, respectively).

The variable regions, which are contained within the Fab regions of the antibody molecule, determine antibody specificity and interact with antigen (Fig. 12-5). The Fc region mediates various effector functions, such as complement activation (IgM and some IgG subclasses) and phagocyte binding (via Fc receptors). Antibodies possess several functions: (1) opsonization, which is coating of a pathogen with antibody to enhance Fc receptor-mediated endocytosis by phagocytic cells; (2) initiation of the classic pathway of complement-mediated lysis; (3) neutralization of viral infection by binding to viral particles and preventing further infection; and (4) enhancement of the specificity of effectors of cell-mediated immunity (CMI) by binding to specific antigens on target cells, which are then recognized and eliminated by effector cells such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs).

Complement

One of the consequences of antigen–antibody binding is initiation of the classical pathway of complement-mediated lysis (reviewed by Stoermer and Morrison, 2011). The complement system is a series of about 30 serum proteins whose primary functions are the destruction of membranes of infectious agents and the promotion of an inflammatory response (see the “Inflammation” section). Complement activation occurs with each component sequentially acting on others, in a manner similar to the blood-clotting cascade (Fig. 12-6). Proximal components of the cascade are often modified serine proteases, which activate the system but have limited substrate specificity. Several components are capable of binding to microbial membranes and serve as ligands for complement receptors associated with the membrane. The final components, which are related structurally, are also membrane-binding proteins that can enter into the membrane and disrupt membrane integrity, termed the membrane attack complex (MAC).

Specifically, three pathways have been identified in activation of the complement cascade. The classical complement pathway is initiated when antibody binds antigen on the microorganism. The classical pathway then proceeds by activating a C1 subunit serine protease, subsequently recruiting C4, C2, and C3. Various cleavages ultimately result in C3b surface binding to the microorganism and release of C3a, a proinflammatory mediator (see the “Inflammation” section). Microorganism-bound C3b can then be recognized by complement receptors on phagocytic cells, which engulf and destroy the microorganism. It is also at this point that the alternative pathway can be activated and amplifies the complement-mediated killing of the microorganism. Finally, C3b mediates recruitment of C5, which is cleaved, generating C5b on the surface and releasing C5a, another proinflammatory mediator (see the “Inflammation” section). Microorganism-bound C5b recruits C6 and C7, the three of which form a complex and recruit C8. C9 is ultimately recruited, polymerizes, and forms a pore in the membrane of the microorganism, causing its death. In addition to the classical and alternative pathways, complement-mediated lysis is also activated through the lectin pathway, in which binding of mannin-binding lectin (MBL) to the surface of the microorganism activates the pathway and converges with the classical pathway at C4 (Fig. 12-6).

A functional outcome of complement activation, in addition to cytolysis and generation of proinflammatory mediators, is opsonization, the process by which microorganisms are altered by opsonins (predominantly IgM and IgG and C3b), rendering them vulnerable to phagocytosis by cells (usually macrophages and neutrophils) (Dunkelberger and Song, 2010). Complement activation results in microorganism-bound C3b. Therefore, in combination with IgG antibodies recognizing extracellular antigens and C3b opsonization, phagocytic cells efficiently engulf the microbe through Fc receptors specific for IgG or complement receptors.

Antigen Processing

In order to elicit an acquired immune response to a particular antigen, that antigen must be taken up and processed by accessory cells for presentation to lymphocytes. Accessory cells that perform this function are termed antigen-presenting cells (APCs) and include macrophages, B cells, and dendritic cells (DCs). Of these, the most proficient APC is the DC. There are several subtypes of DC, including plasmacytoid (pDCs), conventional (cDCs; can be resident or migratory depending on whether they migrate to the site of infection), and specialized DCs in the skin called Langerhan’s cells. In addition, there are follicular DCs, which predominantly mediate T-cell-independent stimulation of B cells in germinal centers (Vinuesa et al., 2010) and will not be discussed further as they are beyond the scope of this chapter.

In most tissues in the absence of antigenic stimulation, DCs are in an immature state, during which they efficiently capture antigens. DCs can mature in response to antigen itself, toll-like receptor (TLR) agonists such as lipopolysaccharide (LPS), or cytokines (GM-CSF, tumor necrosis factor [TNF-α]) (Banchereau and Steinman, 1998). Upon maturation, DCs migrate to lymph nodes and present antigen to T cells. Thus, the DC–T cell interaction is critical for the development of an acquired immune response.

Immature DCs internalize the antigen either by phagocytosis, pinocytosis, or by receptor-mediated endocytosis (via antigen, Fc, or complement receptors), after which the DCs mature to express high levels of both classes of MHC (MHCI and MHCII), which can stimulate both innate and adaptive immune responses, and of costimulatory molecules to facilitate interaction with T cells (Banchereau and Steinman, 1998). Interestingly, Langerhan’s cells express high levels of major histocompatibility complex class II (MHCII) even in a resting state (ie, nonactivated) (Kaplan, 2010). Following internalization, antigen is processed (intracellular denaturation and catabolism) through several cytoplasmic compartments, and a piece of the antigen (peptide fragments about 20 amino acids in length) becomes physically associated with MHCII (Fig. 12-7). This MHCII–peptide complex is then transported to the surface of the cell and can interact in a specific manner with T lymphocytes. For most APCs, an immunogenic determinant is expressed on the surface of the APC within an hour after internalization, although this is slightly longer for B cells (three–four hours). In addition to processing and presentation, pieces of processed antigen may be expelled into the extracellular space. These pieces of processed antigen can then bind in the peptide groove of empty MHCII on the surface of other APCs for the presentation of that peptide fragment to lymphocytes.

In addition to antigen processing and presentation via MHCII, some antigens may be processed and presented via MHCI. Although the pathways share some similarities in that short peptide fragments of antigens are generated, loaded onto MHCI, and presented on the surface of cells to T lymphocytes, major differences between the MHCI and MHCII pathways are (1) antigens processed and presented via MHCI are not limited to professional APC; (2) all nucleated cells express MHCI; (3) the mechanisms by which the antigen is processed and loaded onto MHCI are slightly different than MHCII; (4) the MHCI antigenic peptides are usually smaller, often 8 to 10 amino acids in length; (5) the MHCI antigens to be processed are usually aberrantly expressed proteins, such as viral-associated proteins or mutated proteins; (6) MHCI facilitates antigen presentation to CD8⁺ T cells, whereas MHCII facilitates antigen presentation to CD4⁺ T cells. MHCI antigen processing and presentation is the major pathway by which virally infected cells are detected and killed by the acquired immune system.

Regardless of the MHC utilized to present antigens to lymphocytes, T cells are able to recognize antigen in the context of MHC with their T-cell receptor (TCR). Similar to Ig, the ability of T cells to specifically recognize thousands of antigens is due to somatic recombination. All TCRs are comprised of two different subunits, each encoded from a distinct gene (most abundant T-cell population expresses αβ, but γδ also exist). All TCR subunits are made up of constant and variable regions. For α subunits, two separate gene segments (V and J) are combined to form the variable region, which is then joined to one constant region. For β subunits, three separate gene segments (V, D, and J) are combined to form the variable region, which is then joined to one of the two constant regions. Similar to the Ig genes, there are several light chain V and J genes, and several heavy chain V, D, and J genes (Fig. 12-4).

With regard to T and B cells, key events that occur following antigen encounter are (1) specific antigen recognition either in the context of MHCI or MHCII for T cells or through the Ig receptor for B cells; (2) cellular activation and initiation of intracellular signaling cascades that contribute to production and release of cytokines and other cellular mediators; (3) clonal expansion (proliferation) of antigen-specific cells; and (4) differentiation of antigen-stimulated lymphocytes into effector and memory cells.

Innate Immunity

As stated above, innate immunity acts as a first line of defense against anything nonself. With respect to infectious agents, the innate immune system eliminates most potential pathogens before significant infection occurs. The innate immune system includes physical and biochemical barriers both inside and outside the body, as well as immune cells designed for host defense responses.

Externally, the skin provides an effective barrier, as most organisms cannot penetrate intact skin. Most infectious agents enter the body through the respiratory system, gut, or genitourinary tract. Innate defenses present to combat infection from pathogens entering through the respiratory system include mucus secreted along the nasopharynx, the presence of lysozyme in most secretions, and cilia lining the trachea and main bronchi. In addition, reflexes such as coughing, sneezing, and elevation in body temperature are also a part of innate immunity. Pathogens that enter the body via the digestive tract are met with severe changes in pH (acidic) within the stomach and a host of microorganisms living in the intestines.

Cellular Components: Neutrophils, Macrophages, Dendritic Cells, Natural Killer Cells, NKT Cells, and γδ T Cells

Several cell types are involved in innate immunity, and in fact, some also provide critical signals to initiate adaptive immune responses (Tables 12-3 and 12-5). Neutrophils (also known as polymorphonuclear cells or PMNs) are phagocytic cells that develop from the myeloid lineage of HSCs. Neutrophils enter the bloodstream where they circulate for about 10 hours and then enter the tissues where they perform effector functions for about one to two days. Neutrophils are capable of passing between (ie, extravasation) the endothelial cells of the blood vessels and thereby represent a primary line of defense against infectious agents. They are excellent phagocytic cells and can eliminate most microorganisms through the release of various reactive oxygen species (ROS), such as superoxide, singlet oxygen, ozone, hydrogen peroxide, and hydroxyl radicals. Their phagocytic activity is greatly enhanced by the presence of complement and antibody deposited on the surface of the foreign target. They are also important in the induction of an inflammatory response (see the “Inflammation” section).

Also important in the inflammatory response are macrophages. Macrophages are terminally differentiated monocytes, which develop from the myeloid lineage of HSCs. Upon exiting the bone marrow, monocytes circulate within the bloodstream for about one day. At that time, they begin to distribute to the various tissues where they can then differentiate into macrophage subsets. Tissue-specific macrophages include Kupffer cells (liver), alveolar macrophages (lung), microglial cells (CNS), and peritoneal and splenic macrophages. These tissue-specific macrophages have distinct properties and vary in the extent of surface receptors, oxidative metabolism, and expression of MHCII, due to the factors present within the microenvironment in which the monocyte differentiates. Macrophages can be classified as classically activated macrophages (M1), which are proinflammatory and participate in antigen presentation, or alternatively activated macrophages (M2), which do not present antigen well, but are efficient in apoptotic cell removal (reviewed by Martinez et al., 2008). Thus, M1 macrophages are also considered APCs.

A more proficient APC is the DC, which was introduced above in the “Antigen Processing” section. DCs are a critical part of innate immunity since they survey their environment for pathogens, then mature to become highly efficient APCs. Thus, they provide a critical bridge between initial detection of an infectious agent and elicitation of T-cell responses. DCs can be classified into several subsets and it is thought that the various subsets develop from either CLPs or CMPs, although the exact details are still not clear (reviewed by Watowich and Liu, 2010). Although not reviewed here, part of the challenge with lineage definition is due to extensive heterogeneity of cell surface protein expression of DC subsets, including varying expression of CD11b, CD11c, MHCII, CD4, CD8, and CD205 (reviewed by Watowich and Liu, 2010). All DCs possess efficient APC activity and cytokine production (Watowich and Liu, 2010), but may also possess specialized functions (pDC, for instance, secrete large amounts of Type I interferons (IFNs), such as IFN-α, which is necessary for T-cell differentiation) (Sachdeva et al., 2008).

Like other immune cells, NK cells are derived from a HSC. NK cells are part of a larger family of cells defined as innate lymphoid cells (iLCs), which play roles in innate immunity and inflammation, particularly during the initial phases of inflammation prior to T-cell differentiation (Spits and Di Santo, 2011; Vanaudenaerde et al., 2011). There are two major NK functions: cytokine production and cytolysis. NK cells are a predominant producer of IFN-γ, which helps mature DC (Vivier et al., 2011). Thus, NK cells also facilitate the innate-adaptive immunity bridge. NK cells mediate both antibody-independent and antibody-dependent cellular cytotoxicity (ADCC) using a variety of mechanisms, including perforin and granzyme, Fas L, TRAIL, and TNF-α. ADCC occurs via the FcγRIIIA, which is present on most NK cell subsets. NKT cells are one such subset of NK cells that express both NK- and T-cell markers, including Cd1d, which allows the NKT cell to present self and exogenous antigenic glycolipids (Diana and Lehuen, 2009).

Another cell type that has recently been shown to facilitate the innate-adaptive immunity bridge is the γδ T cell. These cells migrate predominantly to “exposed” tissues, including skin, lung, gut, and reproductive organs, and are also expressed highly in the liver (Bonneville et al., 2010). In part through the expression of TLRs, which are discussed in more detail below, γδ T cells can acquire effector functions, which are not unlike NK cells (cytokine production and cytotoxicity). There is also a subpopulation of B cells (ie, CD5⁺ or B-1 cells) that also bridge innate and adaptive immunity. Unlike the conventional B-2 cells, B-1 cells predominate in embryonic life and are later found mostly in the peritoneal and pleural cavities. B-1 cells are self-renewing and spontaneously produce polyspecific IgM antibodies (ie, natural antibodies) independent of T-cell help (Baumgarth, 2011).

Historically, innate immunity was defined as nonspecific. It is clear, however, that innate cells express receptors that respond to soluble components (eg, Fc or complement receptors) or to certain antigenic motifs. Pattern recognition receptors are a family of receptors that recognize pathogen-derived molecules or cell-derived molecules produced in response to cellular stress (“danger” molecules). Receptors that recognize pathogen-associated molecular patterns (PAMPs) are TLRs; receptors that recognize danger-associated molecular patterns (DAMPs) include NOD-like receptors (NLR) and RIG-like receptors (RLR) (Davis et al., 2011). Several TLRs are expressed extracellularly, and several are expressed intracellularly in endosomes. This diverse localization allows cells to respond to a variety of pathogenic components, such as bacterial cell wall lipids, single- and double-stranded nucleic acids, or fungal and parasitic products (Chang, 2010). Functional consequences of TLR engagement on cells include expression of adhesion molecules, chemokines or cytokines to stimulate T- or B-cell differentiation, enhance phagocytosis, or maturation of DC (Chang, 2010). NLRs or RLRs that recognize DAMPs are expressed on many innate cells. Several family members of NLRs participate in a complex called the inflammasome, which activates inflammatory caspases to enhance production of mature interleukin (IL)-1β and IL-18 (Martinon et al., 2009).

A common effector mechanism for many immune cells, including innate cells, is cytokine or chemokine production. Although a partial list of cytokines and a brief description of the cell types that release and are acted upon by these various mediators is provided in Table 12-6, there are also several shared characteristics of cytokines, chemokines, and IFNs that merit discussion for the purposes of understanding immunotoxicological mechanisms. First, the primary function of cytokines, chemokines, and IFNs is cell–cell communication. The resulting actions by cells receiving messages in the form of cytokines, chemokines, or IFNs are diverse and include cellular activation, initiation or termination of intracellular signaling events, proliferation, differentiation, migration, trafficking, or effector functions. Second, although some of these molecules might be constitutively expressed, most are inducible in response to antigens, cellular stressors, or other cytokines. Thus, many cytokines, chemokines, and IFNs are not stored in the cell, but rather are tightly regulated, often at the transcriptional level, so that they are quickly generated on demand. An example of a cytokine important in innate immunity is IL-1, which is rapidly transcribed in response to various stimulants using the critical transcription factor nuclear factor kappa B (NF-κB). Third, many cytokines share common receptor subunits such that should one particular subunit of a receptor be adversely affected by an immunotoxic agent, the functional outcome might be amplified. An example would be the common γ chain, which is shared by the IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, and IL-21 receptors. Although a compound might “only” affect transcription of the common γ chain, the immunological consequences of affecting this target could be quite destructive.

Other soluble components of innate immunity include the complement cascade, acute-phase proteins, granzyme and perforin, and various cytokines, chemokines and IFNs. Although the complement cascade is described elsewhere in this chapter (see “Antigen Recognition” and “Inflammation” sections), it is also important in innate immunity because of its activation through the lectin pathway. Furthermore, C3a and C5a, which are chemokines generated during the cascade, recruit phagocytic cells to the site of complement activation. Acute-phase proteins, such as serum amyloid A, serum amyloid P, and C-reactive protein, participate in an acute-phase response to infection by binding bacteria and facilitating complement activation. Granzyme and perforin work in conjunction, with perforin disrupting the target cell membrane, allowing granzyme to enter and mediate cell lysis by several mechanisms.

Acquired (Adaptive) Immunity

If the primary defenses against infection (innate immunity) are breached, the acquired arm of the immune system is activated and produces a specific immune response to each infectious agent, which usually eliminates the infection. This branch of immunity is also capable of remembering the pathogen and can protect the host from future infection by the same agent. Therefore, the two key features that define acquired immunity are specificity and memory. Acquired immunity may be further subdivided into humoral and CMI. Humoral immunity is directly dependent on the production of antigen-specific antibody by B cells and involves the coordinated interaction of APCs, T cells, and B cells. CMI is that part of the acquired immune system in which effector cells, such as phagocytic cells, helper T cells, regulatory T cells, APCs, cytotoxic T cells, or T memory cells, play the critical role(s) without antibody involvement.

Cellular Components: Antigen-Presenting Cells, T Cells, and B Cells

APCs, which were discussed previously in the “Antigen Processing” section, include professional APCs such as B cells, macrophages, and DC. Although all cells may act as APCs with internal antigen processing through the MHCI pathway, what distinguishes a professional APC from the others is the ability to internalize external antigens and process them through the MHCII pathway for presentation to T cells.

B cells are not only capable of serving as professional APCs, but they are also the effector cells of humoral immunity, producing a number of Ig isotypes with varying specificities and affinities. Like other immune cells, the B cell develops in the bone marrow from the HSC and becomes committed to the B-cell lineage when the cell begins to rearrange its Ig genes, as described in the “Antigen Recognition” section (Figs. 12-3 and 12-4). Following successful Ig rearrangement, these cells express heavy chains in their cytoplasm and are termed pre-B cells. Expression of membrane IgM and IgD indicates a mature B cell. B cells expressing CD5 (termed B-1 cells) predominate in embryonic life and are later found mostly in the intestinal mucosa (Hardy, 2006). These cells are probably associated with innate immunity and are long-lived, self-renewing cells that produce high levels of polyspecific IgM, much of which are autoantibodies suggesting a possible role of B-1 cell subtype in autoimmunity (Baumgarth, 2011). Mature B cells of the conventional B-2 subset are found in the lymph nodes, spleen, and peripheral blood. Upon antigen binding to surface immunoglobulin (part of the B-cell receptor [BCR]), the mature B cell becomes activated and, after proliferation, undergoes differentiation into either a memory B cell or an antibody-forming cell ([AFC]; also known as a plaque-forming cell [PFC]) that actively secretes antigen-specific antibody. A broad description of several B-cell characteristics can be found in Table 12-5.

At a specified time following their commitment to the T-cell lineage, T-cell precursors migrate from the bone marrow to the thymus where, in a manner analogous to B cells, they begin to rearrange their TCRs, as described in the “Antigen Recognition” section (Fig. 12-3). The TCR consists of two chains, either αβ or γδ. The majority of T cells express the αβ TCR, which is critical for the recognition of antigens in the context of MHC. A much smaller population of T cells express a γδ TCR. The γδ T cells differ from αβ T cells in that they do not require antigen processing or presentation by MHC but instead directly bind intact host and pathogen proteins. Like the B-1 lineage, the γδ T cells are expressed first in embryonic development and may be associated with innate immunity (see the “Innate Immunity” section). T cells committed to the αβ lineage will express the surface marker cluster of differentiation antigens (CD) 4 and 8. CD4 and CD8 are coreceptors expressed by αβ T cells and are involved in the interaction of the T cell with MHC. T cells expressing αβ TCR and both CD4 and CD8 are termed immature double-positive cells (CD4⁺/CD8⁺). These immature cells then undergo positive and negative selection to (1) eliminate cells that do not produce a functional TCR or produce TCRs with no affinity for self-MHC (positive selection); or (2) eliminate cells that strongly bind MHC plus self-peptide (negative selection). Double-positive cells that survive positive and negative selection will give rise to NKT cells or T cells that lose expression of either CD4 or CD8 and enter the periphery as mature single-positive cells (CD4⁺ or CD8⁺) with a high level of αβ TCR expression. This rigorous selection process produces T cells that can recognize MHC plus foreign peptides and eliminates autoreactive T cells. Additionally, expression of CD4 or CD8 will determine to which MHC class the αβ TCR will bind. CD4 will facilitate binding to MHCII expressed on APCs; T cells expressing CD4 (helper T cells, Th) help activate other cells of the adaptive immune response. CD8 will facilitate binding to MHCI, which is expressed on all nucleated cells; generally, T cells expressing CD8 mediate cell killing (CTL). A broad description of several T-cell characteristics (specific to αβ T cells) can be found in Table 12-5. In contrast to αβ T cells, the γδ T cells do not express CD4 or CD8 and therefore do not interact with MHC and do not undergo positive or negative selection. Since γδ T cells are not negatively selected for autoreactivity, these cells may be associated with the development of hypersensitivity and autoimmunity.

Mature T cells are found in the lymph nodes, spleen, and peripheral blood. Upon binding of the TCR to MHC plus antigen, the mature T cell becomes activated and, after proliferation, undergoes differentiation into either an effector cell or a memory T cell. Effector Th cells can subsequently differentiate into several phenotypes depending on the cytokine milieu (Fig. 12-8), two of which, Th1 and Th2, dictate whether CMI or humoral immunity will predominate, respectively. Th1 cells predominantly express IL-2, IFN-γ, and lymphotoxin, which promote CMI and humoral defense against intracellular invaders. Th2 cells predominantly express IL-3, IL-4, IL-5, IL-6, IL-10, and IL-13, which promote humoral defense against extracellular invaders. Although the two populations are not mutually exclusive, they do negatively regulate each other, such that a strong Th1 response suppresses a Th2 response and vice versa.

Th17, Th9, and Th22 cells have been shown to be critical for inflammatory responses (see the “Inflammation” section), and several studies including autoimmune models and clinical studies support a role of Th17 cells in inflammation and autoimmune diseases. Modulation of Th17 cellular function by xenobiotics may alter these disease states. Conversely, a better understanding of the relationship between Th17 and specific disease states may lead to new therapeutics in the treatment of autoimmune and inflammatory diseases (Hemdan et al., 2010; Kimura and Kishimoto, 2011).

The ability of APCs, B cells, and T cells to communicate with each other is dependent on a variety of receptor–ligand interactions between cell types. These interactions also help dictate the type of immune response (ie, humoral vs. CMI) and the magnitude of the immune response. For example, TCR binding by antigen presented in the context of MHC is the major interaction that must occur between APCs and T cells to initiate an acquired immune response. Subsequent to this initial interaction, costimulatory molecules must be engaged on both the T cells and APCs to sustain and direct the immune response. One of the best-characterized interactions occurs between CD28 on the T cells and CD80 or CD86 (B7.1 and B7.2) on the APCs, which provides a more robust immune response as measured by clonal expansion and cytokine (eg, IL-2) production. Later in the response (two–three days), T cells express CTLA-4, which exhibits higher affinity for CD80/CD86 than does CD28, and the CTLA-4–CD80/CD86 interaction serves to suppress the T-cell response. Other important interactions between APCs and T cells include CD40 and CD40 ligand (CD40L), which sustains clonal expansion and differentiation of the T cells and provides activation signals to the APC; inducible T-cell costimulator (ICOS [CD278], a member of the CD28-superfamily) and ligand for ICOS, which sustains clonal expansion and differentiation of the T cells and induces IL-10 production; and 4-1BB (CD137/Ly63, a member of the TNF receptor superfamily) and 4-1BB ligand, which sustains clonal expansion of the T cells and helps to activate the APC (CD40 ligand, ICOS, and 4-1BB are expressed on T cells). Therapeutic agents that bind some of these costimulatory surface molecules have been developed and utilized clinically to suppress immune function for transplantation and autoimmune therapy, which will be discussed in “Therapeutic Agents” under the “Immune Modulation by Xenobiotics” section. For simplicity, in figures throughout the chapter, only the CD28–B7 interaction is depicted.

The duration and extent of an acquired immune response is also controlled by specialized regulatory cells found in both the T-cell and B-cell lineages, and there are considerable functional similarities between the different regulatory subsets found within both lineages (reviewed by Noh and Lee, 2011) (Figs. 12-8 and 12-9). For the T-cell lineage, there is a small population of CD4⁺ cells that develop into T-regulatory cells (Tregs), which help to control various immune responses, including those directed against self. Tregs are generally identified as CD4⁺CD25⁺Foxp3⁺, which are defined as natural Tregs if they develop in the thymus (Lio and Hsieh, 2011), but can also be induced in the periphery in response to IL-2 and TGF-β (Horwitz et al., 2008). Tregs can suppress CD4⁺ and CD8⁺ IL-2 production and proliferation, and in vivo have been shown to suppress autoimmune disease (Sakaguchi et al., 2009). The mechanisms by which Tregs suppress immune responses involve direct Treg-cell contact since Tregs are unable to suppress immune function when separated from the target cell population by a semipermeable membrane or when Treg-conditioned medium is used (Takahashi et al., 1998; Thornton and Shevach, 1998). Immune suppression by Tregs is likely a multistep process, which might involve direct cell killing via granzyme or perforin, induction of intracellular cAMP, or affecting cell surface expression of critical proteins, such as CD80/CD86. For instance, Foxp3 induces high levels of CTLA-4, which would interact with target cell CD80/CD86 and prevent target cell CD80/CD86 from interacting with CD28 on other stimulatory cells (reviewed by Sakaguchi et al., 2009). It has also been suggested that the high expression level of CD25 on Tregs might act as a reservoir for IL-2 in an immune synapse microenvironment, thereby preventing IL-2 from stimulating surrounding cells (Sakaguchi et al., 2009). In addition to Tregs, other regulatory T-cell subsets have been identified such as the IL-10 producing Tr1 cells and the TGF-β producing Th3 cells; all three regulatory subsets appear to be involved in regulating immune responses (Noh and Lee, 2011). Regardless of the processes that direct Treg (or perhaps Tr1 and Th3) suppression of immune function, their induction has been identified as one mechanism by which drugs and xenobiotics might result in immune suppression (Marshall and Kerkvliet, 2010; Ohkura et al., 2011). Like the regulatory T cells, several subsets of regulatory B cells are emerging and several recent reviews have been devoted to outlining the experimental evidence for specific regulatory B-cell subpopulations and their generally suppressive role in hypersensitivity and autoimmune diseases (DiLillo et al., 2010; Mauri, 2010; Noh and Lee, 2011). Although phenotypically diverse, regulatory B cells appear to have arisen from the CD5 B-1 population (briefly described above). Noh and Lee (2011) have described three subsets of regulatory B cells based on cytokine or Foxp3 expression, which include Br1 or B10 cells that produce IL-10, Br3 that produce TGF-β, and Breg that express Foxp3. These subsets resemble the regulatory T-cell subsets, Tr1, Th3, and Treg, respectively (Fig. 12-9 adapted from Noh and Lee, 2011). The regulatory T- and B-cell subsets also appear to reciprocally activate or suppress each other and may cooperatively control immune responses. The interplay between regulatory T- and B-cell subsets and their impact on immune responses, including autoimmune and hypersensitivity diseases, are important areas of investigation that will likely lead to a better understanding of immune pathologies including those induced by drugs and other xenobiotics.

Humoral and Cell-Mediated Immunity

As described earlier, there are two branches of acquired immunity, humoral immunity, and CMI. Humoral immunity is that part of the acquired immune system in which antibody is involved. In general, B cells produce antibodies specific to an antigen, which may act to opsonize or neutralize the invader, or the antibodies act to recruit other factors, such as the complement cascade. CMI is that part of the immune system in which various effector cells perform a wide variety of functions to eliminate invaders. Often, these two branches are coordinated, such as activation of Th cells that produce specific cytokines that enhance B-cell proliferation and differentiation to secrete more antibody. A general diagram of the cellular interactions involved in a humoral immune response is given in Fig. 12-10. The production of antigen-specific IgM requires three to five days after the primary (initial) exposure to antigen (Fig. 12-11). Upon secondary antigenic challenge, the B cells undergo isotype switching, producing primarily IgG antibody, which is of higher affinity for the activating antigen. In addition, there is a higher serum antibody titer associated with a secondary antibody response. CMI functions include delayed-type hypersensitivity (DTH) and cell-mediated cytotoxicity. Cell-mediated cytotoxicity responses may occur in numerous ways: (1) MHC-dependent recognition of specific antigens (such as viral particles or tumor proteins) by CTL (Fig. 12-12); (2) indirect antigen-specific recognition by the binding of Fc receptors on NK cells to antibodies coating target cells; and (3) receptor-mediated recognition of complement-coated foreign targets by macrophages. Many of these are described in more detail in “Hypersensitivity” under the section entitled “Immune-Mediated Disease”

In cell-mediated cytotoxicity, the CTL or NK effector cell binds in a specific manner to the target cell. The majority of CTLs express CD8 and recognize either foreign MHCI on the surface of allogeneic cells, or antigen in association with self-MHCI (eg, viral particles). Fig. 12-12 shows the cell-mediated CTL response. NK cell recognition of target cells is also antigen-specific because the mechanism of recognition involves binding of Fc receptors on NK cells to the Fc portion of antigen-specific antibody that coats the target cell; a process referred to as ADCC. Once the CTL or NK cells interact with the target cell, the effector cell undergoes cytoplasmic reorientation so that cytolytic granules are oriented along the side of the effector, which is bound to the target. The effector cell then releases the contents of these granules onto the target cell. The target cell may be damaged by the perforins or enzymatic contents of the cytolytic granules. In addition, CTLs induce the target to undergo apoptosis through activation of the Fas and cytotoxic cytokine (ie, TNF and lymphotoxin) pathways. Once it has degranulated, the effector cell can release the dying target and move on to kill other target cells. Notably, many of the soluble factors critical to the innate immune system are also primary effectors in the acquired immune system, including ROS, granzyme, perforin, cytokines, and IFNs, again demonstrating the interplay among the various arms of the immune system.

Inflammation

Inflammation, simply defined, refers to a complex reaction to injury, irritation or foreign invaders characterized by pain, swelling, redness, and heat. Inflammation involves various stages, including release of chemotactic factors following the insult, increased blood flow, increased capillary permeability allowing for cellular infiltration, followed by either an acute resolution of tissue damage or persistence of the response that might contribute to fibrosis or subsequent organ failure (Serhan and Savill, 2005). It is important to emphasize that while inflammation is a natural reaction to repair tissue damage or attack foreign invaders, the process often results in destruction of adjacent cells and/or tissues. Thus, there is overwhelming evidence that inflammation plays a critical role in many diseases, including asthma, multiple sclerosis, cardiovascular disease, Alzheimer’s disease, bowel disorders, and cancer. In addition, inflammation exacerbates idiosyncratic reactions to drugs and other chemicals (reviewed by Ganey et al., 2004).

Cellular Components: Macrophages, Neutrophils, and T Cells

Many of the cellular components described in the sections above are critical to initiation and maintenance of an inflammatory response. Major cellular contributors to an inflammatory response are macrophages, neutrophils, and T cells. Neutrophils are often the first, and most numerous, responders to sites of insult. In response to either host- or pathogen-derived signals, neutrophils secrete chemotactic factors to recruit other proinflammatory cells, such as macrophages, to the area. Recently, it has been demonstrated that IL-17 produced early by various sources, including NKT cells, is a critical proinflammatory cytokine that will rapidly induce neutrophil and macrophage influx to the area of insult (Vanaudenaerde et al., 2011). Macrophages can be activated by a variety of mechanisms at the site of insult, such as activation via TLR, proinflammatory cytokines, or recognition of opsonized particles by Fc receptors or complement receptors. Macrophages and neutrophils also induce apoptosis of cells in the insult area through the release of nitric oxide and other ROS, resulting in disruption of extracellular structures that compromise tissue structure and function (reviewed by Duffield 2003). Both neutrophils and macrophages are phagocytic cells and can contribute to clearing of apoptotic cells. Later in the inflammatory response, T cells are critical for generating an adaptive immune response. T cells are attracted to the insult area by adhesion molecules and integrins, and are activated in response to antigen presented in the context of MHC, often by a DC. As described above, depending on the signals that the T cell receives from the cytokine milieu, distinct subpopulations of T cells are induced (Fig. 12-8). Chronic inflammatory states are maintained with activation and differentiation of Th2 and Th17, which drive chronic eosinophilic and neutrophilic inflammation, respectively (Jutel and Akdis, 2011). Th9 and Th22 cells also likely contribute to chronic inflammation, but their roles are not yet completely understood.

Of all Th subsets, Th17 cells have certainly been shown to play a critical role in inflammation (Kimura and Kishimoto, 2011). In contrast to cells involved in the innate response (NK or NKT cells, γδ T cells, or other iLCs) that produce IL-17, Th17 cells are part of the adaptive immune response, and produce IL-17, IL-22, and TNF-α (Vanaudenaerde et al., 2011). They are induced in the presence of IL-6 and TGF-β, conditions that are unfavorable for Tregs, thereby exacerbating the proinflammatory response through suppression of Treg differentiation and chronic neutrophilic infiltration (Awasthi and Kuchroo, 2009). Th17 cells are defined by expression of retinoid-related orphan receptors RORγ and RORα. Interestingly, Th17 cells also express high levels of aryl hydrocarbon receptor ([AHR]; Veldhoen et al., 2008), providing an additional cellular target for immune modulation by AHR ligands (see the subsection “Polychlorinated Dibenzodioxins” under “Immune Modulation by Xenobiotics”).

There are several other soluble factors that contribute to inflammation that warrant discussion. In addition to ROS that are released from both macrophages and neutrophils to compromise membrane integrity, a plethora of cytokines and chemokines contribute to the inflammatory process. As mentioned above in the “Innate Immunity” section, some proinflammatory cytokines (IL-1β and IL-18) are produced in a mature form as a result of the activation of the inflammasome (Martinon et al., 2009). In fact, it has been demonstrated that inflammation as a result of disease (ie, influenza) or toxicity (ie, silicosis) depends on inflammasome activation (Cassel et al., 2008; Ichinohe et al., 2009). An additional discussion for the role of the inflammasome in immune responses to inhaled particles is provided in the subsection “Inhaled Substances” under “Immune Modulation by Xenobiotics” and in Fig. 12-22. The actions of various proinflammatory cytokines include inducing fever or activating T cells and macrophages (IL-1); stimulating B- or T-cell proliferation (IL-6); inducing neutrophilia (IL-17); and increasing vascular permeability or inducing apoptosis (TNF-α). Many proinflammatory cytokines also induce acute-phase proteins, such as C-reactive protein. C-reactive protein binds to several ligands, including phosphatidylcholine on the membranes of cells, Fc receptors, and C1q (part of the complement C1 complex), which activates the classical complement cascade (reviewed by Marnell et al., 2005). Complement participates in inflammation through inappropriate and sustained activation of the cascade, ultimately leading to destruction of cells through the MAC (Fig. 12-6). Furthermore, complement fragments, such as C3a and C5a, act as chemotactic factors to recruit other phagocytic cells to sites of insult (macrophages, neutrophils, eosinophils, or mast cells) or induce degranulation (basophils, eosinophils, or mast cells) (Dunkelberger and Song, 2010). Finally, prostaglandins and other eicosanoids possess various proinflammatory actions, including increased blood flow and pain (prostaglandin E2), increase in vascular permeability (prostacyclin I2), and stimulation of endothelial inflammatory responses (thromboxane A2) (reviewed by Ricciotti and FitzGerald, 2011). Prostaglandins contribute to hyperalgesia and it is for this reason that cyclooxygenase-2, which converts arachidonic acid to other bioactive prostaglandins, is an attractive therapeutic target to reduce inflammation and pain (see the subsection “Anti-inflammatory Agents” under “Immune modulation by Xenobiotics”).

As mentioned above, it is undisputed that chronic inflammation plays a critical role in many disease states, including asthma, multiple sclerosis, cardiovascular disease, Alzheimer’s disease, bowel disorders, and cancer. Chronic inflammation could result in high circulating levels of proinflammatory cytokines, complement fragments, prostaglandins, and thromboxanes. An inflammatory cascade can be established because many of these mediators induce expression of others (ie, TNF-α induces expression of IL-1 and IL-6 or complement fragment C3a causes chemotaxis of phagocytic cells). Moreover, the sustained presence of proinflammatory cytokines could exacerbate ROS production from neutrophils and macrophages. As seen in the next section, the underlying proinflammatory response also plays a critical role in immune-mediated disease.

Immune-Mediated Disease

As stated earlier, the purpose of the immune system is to preserve the integrity of the individual from disease states, whether infectious, parasitic, or cancerous, through both cellular and humoral mechanisms. In so doing, the ability to distinguish self from nonself plays a predominant role. However, situations arise in which the individual’s immune system responds in a manner producing tissue damage, resulting in a self-induced disease. These disease states fall into two categories: (1) hypersensitivity, or allergy, and (2) autoimmunity. Hypersensitivity reactions result from the immune system responding in an exaggerated or inappropriate manner after repeated exposure to the offending antigen. These reactions have been subdivided by Coombs and Gell (1975) into four types, which represent four different mechanisms leading to tissue damage. In the case of autoimmunity, mechanisms involve a break down in self-recognition resulting in the production of autoreactive antibodies and/or TCRs recognizing self-antigens as nonself, ultimately culminating in tissue damage and disease.

Hypersensitivity

Classification of Hypersensitivity Reactions

One characteristic common to all four types of hypersensitivity reactions is the necessity of prior exposure leading to sensitization in order to elicit a reaction upon subsequent challenge. In the case of Types I, II, and III, prior exposure to antigen leads to the production of allergen-specific antibodies (IgE, IgM, or IgG) and, in the case of Type IV, the generation of allergen-specific memory T cells. Fig. 12-13 illustrates the mechanisms of hypersensitivity reactions as classified by Coombs and Gell. Although not completely understood, regulation of Ig production is dependent in part on the characteristics of the antigen, the genetics of the individual, and environmental factors. The mechanisms of antibody production in hypersensitivity reactions are identical to those described earlier in the chapter (Fig. 12-10). A brief description of the four types of hypersensitivity reactions is presented below.

Type I (Immediate or IgE-Mediated Hypersensitivity)

Using penicillin as an example, Fig. 12-14 depicts the major events involved in a Type I hypersensitivity reaction (what most people think of as “allergy” and is clinically referred to as “atopy”). Sensitization occurs as the result of dermal exposure to antigens or by exposure to antigens through the respiratory or gastrointestinal tract. Most people would mount an IgM, IgG, or IgA immune response to these antigens and clear them without causing any allergic symptoms. It is unclear why these antigens become allergens in certain individuals who respond by mounting an IgE immune response instead, but appears to involve genetic and/or environmental determinants and likely some type of triggering event (eg, acute pathogen exposure and emotional stress). IgE production is highest in lymphatic tissues that drain sites of exposure (ie, tonsils, bronchial lymph nodes, and intestinal lymphatic tissues, including Peyer’s patches) and is low in the spleen. Compared to other Igs, IgE has a low serum concentration and a short serum half-life (Table 12-4). Once produced, soluble IgE not only binds to local tissue mast cells, but also enters the circulation, where it binds to circulating mast cells, basophils, and tissue mast cells at distant sites. Once an individual is sensitized, reexposure to the antigen results in binding to IgE on local mast cells and degranulation with the release of preformed mediators and cytokines which recruit and activate circulating eosinophils, basophils, macrophages, and neutrophils leading to the synthesis and release of more cytokines and of leukotrienes and thromboxanes. These mediators promote vasodilation, bronchial constriction, and inflammation. Clinical manifestations can vary from urticarial skin reactions (wheals and flares) to signs of hay fever, including rhinitis and conjunctivitis, to more serious diseases, such as asthma and potentially life-threatening anaphylaxis. These responses may begin within minutes of reexposure to the offending antigen; therefore, Type I hypersensitivity is often referred to as immediate hypersensitivity.

Type II (Antibody-Dependent Cytotoxic Hypersensitivity)

Type II hypersensitivity is IgG or IgM-mediated. The antibody response may be mediated by a foreign antigen attached to the surface of a cell or tissue. Conversely, an antibody response could be mediated by an autoantibody due to a breakdown in tolerance and the resulting response would be part of an autoimmune disease (eg, autoimmune hemolytic anemias and Goodpasture’s syndrome). Fig. 12-15 shows the mechanisms of action for complement-independent and complement-dependent cytotoxic reactions. Tissue damage may result from the direct action of cytotoxic cells, such as macrophages, neutrophils, or eosinophils, linked through the Fc receptor to antibody-coated target cells (complement-independent) or by antibody-induced activation of the classic complement pathway. Complement activation may result in C3b binding to the target cell surface with C3b acting as a recognition site for effector cells. Alternatively, the C5b-9 MAC may be bound to the target cell surface, resulting in direct cell lysis.

Type III (Immune Complex-Mediated Hypersensitivity)

Type III hypersensitivity reactions also involve IgG or IgM. The distinguishing feature of Type III is that, unlike Type II, in which Ig production is against specific cellular or tissue-associated antigen, Ig production is against soluble antigen in the serum (Fig. 12-16). This allows for the formation of circulating immune complexes composed of a lattice of antigen and Ig, which may result in widely distributed tissue damage in areas where immune complexes are deposited. The most common location is the vascular endothelium in the lung, joints, and kidneys. The skin and circulatory systems may also be involved. Pathology results from the inflammatory response initiated by the activation of complement. Macrophages, neutrophils, and platelets attracted to the deposition site contribute to the tissue damage. As with Type II hypersensitivity, responses similar to Type III hypersensitivity can be induced in autoimmune diseases due to autoantibodies directed against soluble antigens such as double-stranded DNA or small nuclear proteins as seen with SLE.

Type IV (Cell-Mediated Hypersensitivity)

Type IV, or DTH responses, can be divided into three classes: contact hypersensitivity, tuberculin-type hypersensitivity, and hypersensitivity pneumonitis. The following discussion is focused on contact hypersensitivity using the classical example of contact hypersensitivity seen following poison ivy exposure. Contact hypersensitivity is initiated by topical exposure, and the associated pathology is primarily epidermal. It is characterized clinically by an eczematous reaction at the site of allergen contact and, like Type I through III responses, consists of two phases: sensitization and elicitation. However, in this case sensitization is the result of the development of activated and memory T cells as opposed to antibody production (Figs. 12-17 and 12-18). Sensitization occurs when a hapten penetrates the epidermis and forms a complex with a protein carrier. The hapten–carrier complex is processed by Langerhan’s DCs that migrate out of the epidermis to the local lymph nodes. There, the APC presents the processed antigen to CD4⁺ Th cells, leading to clonal expansion and the generation of memory T cells including memory CD8⁺ CTL, (Tc) which appear to play a major role in the elicitation phase.

Upon secondary contact, Langerhan’s DCs present the processed hapten–carrier complex to memory CD8⁺ CTL in either the skin or the lymph nodes. These activated T cells then secrete cytokines that bring about further proliferation of T cells and generation of CTLs, as well as increased expression of adhesion molecules on the surface of keratinocytes and endothelial cells in the dermis. Both the expression of adhesion molecules and the secretion of proinflammatory cytokines by T cells and keratinocytes facilitate the recruitment of inflammatory cells into the skin, resulting in erythema and the formation of papules and vesicles. In addition and under certain circumstances, intracellular proteins can be modified by lipid-soluble chemicals that might readily cross the cell membrane. These cells then present modified peptides on their cell surface in conjunction with MHCI molecules. CD8⁺ cytotoxic T cells recognize these foreign peptides and cause tissue damage by either direct cytotoxic action (CTL) or the secretion of cytokines that further promote the inflammatory response.

Autoimmunity

Autoimmune disease occurs when the reactions of the immune system are directed against the body’s own tissues, and, as with hypersensitivity, is characterized by a genetic susceptibility. Autoimmune diseases may be tissue-specific, where the damage is associated with a specific type of tissue or a specific organ, or tissue-nonspecific, where the signs and symptoms are associated with several organs and tissues. The targets from the perspective of the primary sites of tissue damage in autoimmune disease are many and varied. The following organs, cells, and organelles have all been determined to be the site of autoimmune reactions: nuclei (specifically histones and/or single-stranded DNA—one of the hallmark indicators of certain types of autoimmune disease is the expression of antinuclear antibodies), red blood cells, lymphocytes, neutrophils, platelets, Igs (primarily IgG), striated muscle (cholinergic receptors), smooth muscle, mitochondria, skin (basement membranes), thyroid (thyroglobulin), kidney (glomerular and tubular basement membrane), CNS (myelin), connective tissue (synovial lining of joints), lung, and liver. Both humoral immunity and CMI can be involved as effector mechanisms in causing the damage in autoimmune conditions. Examples of autoimmune diseases include (1) myasthenia gravis, in which cholinergic receptors, especially those associated with neuromuscular junctions, are targeted; (2) multiple sclerosis, in which myelin is targeted; and (3) rheumatoid arthritis, in which connective tissue, especially the synovial lining of joints, is targeted. The terms “hypersensitivity” and “autoimmunity” are often confused and are certainly interrelated. Based on their definitions, a hypersensitivity response can be a mechanism by which an autoimmune disease is produced.

In the section on hypersensitivity presented above, two mechanisms by which host tissues are damaged by the host’s own immune system were discussed (Types II and III), creating autoimmune-like disease. In these situations, unaltered self-antigens are not the target of the immune mechanisms, but damage occurs to cells bearing hapten on membranes or to innocent bystander cells in close proximity to antigen–antibody complexes. For example, damage produced in autoimmune Goodpasture’s disease is similar to that seen in Type III hypersensitivity reactions in the lung due to trimellitic anhydride (TMA) exposure. Although the resulting pathology may be the same for autoimmune reactions and hypersensitivity, mechanisms of true autoimmune disease are distinguished from hypersensitivity. In cases of autoimmunity, self-antigens are the target, and in the case of chemical-induced autoimmunity, the disease state is induced by a modification of host tissues or immune cells by the chemical and not the chemical acting as an antigen/hapten as in hypersensitivity reactions.

Mechanisms of Autoimmunity

As discussed earlier, the rearrangement and recombination of the genes that comprise Ig and TCR result in tremendous diversity in the potential antigen recognition of B cells and T cells, respectively. Ideally, during development those lymphocytes recognizing self-antigens will largely be deleted by negative selection as central tolerance is established. Autoreactive clones that escape central tolerance and migrate to the periphery are normally controlled by peripheral tolerance mediated by various mechanisms that ultimately induce anergy or clonal deletion. For autoimmune disease to occur, an autoreactive clone must escape central tolerance, pass into the periphery, and bind with specificity to its self-antigen then mechanisms of peripheral tolerance must fail and the autoreactive clone must induce a detrimental immunological response. Clearly, induction of autoimmunity is multifaceted and likewise has been associated with several mechanisms primarily related to insufficient peripheral tolerance. Conversely, defects in central tolerance are rarely encountered. However, an example of autoimmunity due to defective central tolerance is the disease autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, which results from a defective transcriptional regulator called autoimmune regulator (AIRE). For the negative selection process in the thymus, AIRE induces the expression of a wide variety of self-antigens normally found in the periphery. Loss of AIRE results in release of autoreactive T cells to the periphery. Several mechanisms have been associated with the breakdown of peripheral tolerance and prime events for the onset of autoimmune disease. These mechanisms include inflammation; molecular mimicry by pathogen antigens; inherent defects in T or B cells including regulatory subsets, APCs, cytokines, or complement; and epitope spreading. For example, increasing evidence supports a potential breakdown of peripheral tolerance during an inflammatory response, which may be mediated by the inappropriate maturation of immature DCs that process both pathogenic antigens and self-antigens released during the inflammatory response. Self-antigens could then be presented to naive autoreactive T cells via MHC. With molecular mimicry, a pathogen presents epitopes that resemble those on self-antigens and because the pathogen will induce a strong inflammatory response, peripheral tolerance may be overridden. Defects in immune system components, such as complement, could reduce C3b-mediated clearance of self-antigen and autoantibody immune complexes (described in Types II and III hypersensitivity). Additionally, interference of normal immune regulation by Tregs or Bregs could create an environment conducive to autoimmune disease. Finally, epitope spreading may occur during an immune response due to tissue damage and the release of new epitopes that were hidden either because they were sequestered from the immune system or their expression levels were insufficient to induce negative selection or peripheral tolerance mechanisms.

Effector mechanisms involved in autoimmune disease can be the same as those described earlier for Types II and III hypersensitivity or, in the case of pathology associated with solid tissues, including organs, they may involve CD8⁺ CTL. Tissue damage associated with CTL may be the result of direct cell membrane damage and lysis, or the result of cytokines produced and released by the T cell. TNF-β has the ability to kill susceptible cells, and IFN-γ may increase the expression of MHCI on cell surfaces, making them more susceptible to CD8⁺ cells. Cytokines may also be chemotactic for macrophages, which can cause tissue damage directly or indirectly through the release of proinflammatory cytokines. As is the case with hypersensitivity reactions, autoimmune disease is often the result of more than one mechanism working simultaneously. Therefore, pathology may be the result of direct or indirect effects of CTL, antibody-dependent cytotoxicity, or complement-dependent antibody-mediated lysis.

Developmental Immunology

A sequential series of carefully timed and coordinated developmental events, beginning early in embryonic/fetal life and continuing through the early postnatal period, is required to establish a functional immune system in all mammals, including humans. The immune system develops initially from a population of pluripotent HSCs that are generated early in gestation from uncommitted mesenchymal stem cells in the intraembryonic splanchnopleure surrounding the heart. This early population of HSCs gives rise to all circulating blood cell lineages, including cells of the immune system, via migration through an orderly series of tissues, and a dynamic process that involves continual differentiation of lineage-restricted stem cells. Lymphoid-hematopoietic progenitor cells are established via the migration of these cells from intraembryonic mesenchyme to fetal liver and fetal spleen, and ultimately, the relocation of these cells in late gestation to bone marrow and thymus. The latter two organs are the primary sites of lymphopoiesis and appear to be unique in providing the microenvironment factors necessary for the development of functionally competent immune cells. These lineage-restricted stem cells expand to form a pool of highly proliferative progenitor cells that are capable of continual renewal of short-lived functional immunocompetent cells, and that ultimately provide the necessary cellular capacity for effective immune responsiveness, and the necessary breadth of the immune repertoire (Good, 1995).

It is important to recognize that immune system development does not cease at birth, and that immunocompetent cells continue to be produced from proliferating progenitor cells in the bone marrow and thymus. Mature immunocompetent cells leave these primary immune organs and migrate via the blood to the secondary immune organs: spleen, lymph nodes, and mucosal lymphoid tissues. Because birth occurs at various stages of fetal maturity, the significance of parturition as a landmark in the development of the immune system can vary from species to species (Holsapple et al., 2003). As such, direct comparison of immune functional development between humans and animals is complicated by differences in the maturity of the immune system before and after parturition. This difference has been linked to the length of gestation (Holladay and Smialowicz, 2000) in that animals with short gestation periods (eg, mice, rats, rabbits, and hamsters) have relatively immature immune systems at birth compared to humans. The onset of functional immune competence depends on the specific parameter being measured, and varies across species with striking differences noted between rodents and humans (Holsapple et al., 2003). The consensus in the scientific and regulatory arenas is that the immune system of children continues to develop postnatally until 5 to 12 years of age (Miyawaki et al., 1981). Exposure to specific antigens during the perinatal period results in a rapidly expanding accumulation of lymphocyte specificities in the pool of memory cells in secondary lymphoid tissues. As thymic function wanes and thymocytes are no longer produced in that tissue, it is this pool of memory B and T cells that maintains immune competence for the life of the individual. Senescence of immunity is associated with reductions in both innate and acquired immune responses to antigens during the last quartile of life. This failure of the immune response is due, in part, to a continual reduction in the production of newly formed cells, and to the decreased survival of long-lived memory cells in lymphoid tissues. The concept of immune senescence is discussed further below.

One feature of the developing immune system that clearly distinguishes it from the mature immune system, especially during gestation, is the role played by organogenesis. Defects in the development of the immune system due to heritable changes in the lymphoid elements have provided clinical and experimental examples of the devastating consequences of impaired immune development (Rosen et al., 1995). Therefore, the effects of chemicals on the genesis of critical immune organs in the developing fetus may be more important than effects on these tissues after having been populated by hematopoietic and lymphoid cells. However, a chemical that induces this kind of developmental structural abnormality in the fetus would legitimately be classified as a teratogen, prompting the question as to how many known teratogens would have an impact on organs essential to the normal functioning of the immune system. Interestingly, immune organs, such as the thymus, spleen, and/or bone marrow, are not typically assessed in routine developmental and reproductive toxicology studies, and it has been noted that this failure to assess developmental damage to the immune system in standard developmental and reproductive toxicology protocols should be reevaluated (Holsapple et al., 2003). Additional perspectives on the methods to assess developmental immunotoxicology (DIT) and on the emergence of regulatory guidelines that include developmental immunotoxicity testing strategies are included elsewhere in this chapter.

At the other end of the spectrum from the development of the immune system during gestation and the perinatal period is the status of the immune system in the aging population. Unlike DIT, there have been very few studies that have addressed the impact of specific xenobiotics on the aging immune system. Nonetheless, it is known that the immune system of older people is usually perceived as declining in fidelity and efficiency with age resulting in an increased susceptibility to infectious diseases and pathological conditions relating to inflammation (eg, cardiovascular disease and Alzheimer’s disease) or autoreactivity (eg, rheumatoid arthritis) (Caruso et al., 2009). Immune senescence has been defined as the impairment in immunity as a result of age-associated changes in function in a variety of cell types. It is a phenomenon of decreased function, involving changes in both innate and adaptive immunity, and a disbalance between the two arms (Pawelec et al., 2010). Virtually, all components of the immune system are affected (Ongradi and Kovesdi, 2010). The decline in the B-cell compartment is responsible for the decreased effectiveness of vaccines in older people; and the decline in immune surveillance (eg, the T-cell compartment and NK-cell function) is associated with an increased incidence in malignancy. Additionally, aging has been associated with a shift from the Th1 to the Th2 cytokine profile in response to immune stimulation, and the overproduction of Th2 cytokines could augment B-cell production of autoreactive antibodies. Studies in humans and mice have shown that TLR expression and function decline with age resulting in the decreased production of proinflammatory cytokines and chemokines. From a mechanistic standpoint, a number of factors have been proposed as contributing to the onset and severity of immune senescence including: the accumulation of damage to lymphocytes by oxidative stress, persistent microbial infections with immunosuppressive viruses that establish latency, such as cytomegalovirus and Epstein–Barr virus, and lifestyle factors such as smoking, alcohol use, and poor nutrition. An improved understanding of immune dysfunction in human aging will increase the probability of discovering means to restore appropriate function and alleviate the burden of infectious disease later in life (Ongradi and Kovesdi, 2010).

Neuroendocrine Immunology

There is overwhelming evidence that cytokines, neuropeptides, neurotransmitters, and hormones, as well as their receptors, are an integral and interregulated part of the CNS, the endocrine system, and the immune system (reviewed by Sanders and Kohm, 2002). Because receptors for neuropeptides, neurotransmitters, and hormones are present on lymphoid cells, it is reasonable to suspect that some chemicals may exert their immunomodulatory effects indirectly on the immune system by acting to modulate the activity of the nervous or endocrine systems. In addition, immune cells are capable of secreting, and do secrete, peptide hormones and neurotransmitters, which can have autocrine (immune system) and paracrine (endocrine and nervous systems) effects. Similar to the complexity of the cytokine network, the effects of various hormones and neurotransmitters are too vast to discuss here. However, it is evident, even from this brief overview of the immune system and its potential interaction with other biological systems, that immune responses are complex processes involving multiple cell types and mediators. It is not difficult to imagine then, that perturbation of leukocyte functions including the mediators they produce by drugs or chemicals could contribute to the mechanisms by which a compound is immunotoxic.

For many years, it has been widely established that xenobiotics can exert significant effects on the immune system as evidenced by changes in circulating and lymphoid organ-associated leukocyte number, proliferative capacity, and in effector functions including defense against a broad array of bacterial, parasitic, and viral pathogens. More recently, and during the establishment of the subdiscipline of immunotoxicology, a significant emphasis was placed on the development of a standardized battery of tests to evaluate immune competence. Among the unique features of the immune system is the ability of immune cells to be removed from the body and to function in vitro. This unique quality makes it possible to comprehensively evaluate the actions of xenobiotics on the immune system employing in vivo, ex vivo, and in vitro approaches to dissect the cellular, biochemical, and molecular mechanisms of action of xenobiotics. While standard toxicological endpoints such as organ weights, cellularity, and enumeration of cell populations are important components in assessing when an agent is capable of altering the immune system, by far the most sensitive indicators of immunotoxicity are the tests that challenge the various cell types within the immune system to respond functionally to exogenous stimuli (Descotes, 2004; White, 1992). Employing such a battery of functional assays whereby different cell types can be evaluated not only for their effector functions, but also in certain cases for their ability to participate as accessory cells in an immune response, can provide important mechanistic information concerning which cell type(s) within the immune system are, in fact, targeted by a xenobiotic. This section focuses on selected in vivo, ex vivo, and in vitro tests currently used for evaluating immunotoxicity, as well as models and approaches that can be utilized for elucidation of the mechanism of action.

Methods to Assess Immunocompetence

The current state-of-the-science of immunotoxicity testing was recently reviewed in a single volume of Methods in Molecular Biology. An overview of immunotoxicology testing: past and future was provided by Luster and Gerberick (2010).

General Assessment

Central to any series of studies evaluating immune competence is the inclusion of standard toxicological studies, because any immunological finding should be interpreted in conjunction with effects observed on other target organs. Standard toxicological studies that are usually evaluated include body and selected organ weights, general observations of overall animal health, selected serum chemistries, hematological parameters, and status of the bone marrow (ability to generate specific colony-forming units). In addition, histopathology of lymphoid organs, such as the spleen, thymus, and lymph nodes, may provide insight into potential immunotoxicants. General guidelines for the histopathological examination of each of these lymphoid organs was recently reviewed (Elmore, 2010).

Because of the unique nature of the immune system, there are several experimental approaches that may be taken to assess immunotoxicity and to evaluate the mechanisms of action of xenobiotics. These are depicted in Fig. 12-19 and vary with respect to in vivo or in vitro exposure, immunological challenge (ie, the stimulus employed to induce an immune response), or immunological evaluation (immune assay). As an example, Fig. 12-19 (2) is an ex vivo assay where xenobiotic exposure and antigen challenge occur in vivo and the immune response is evaluated in vitro. In contrast, as depicted in Fig. 12-19 (3), splenocytes can be isolated after in vivo treatment with a xenobiotic, sensitized with an antigen in vitro and evaluated in vitro; or as depicted in Fig. 12-19 (4), splenocytes can be removed from a naive (untreated) animal, exposed to a xenobiotic and antigen in vitro, and evaluated in vitro. If splenocytes are sensitized in vitro with sRBC to generate AFC, this is known as the Mishell–Dutton assay (Mishell and Dutton, 1967).

Functional Assessment

Innate Immunity

As described earlier, innate immunity encompasses all those immunological responses that are not induced through antigen receptors (ie, TCR or BCR) and for which there is no immunological memory. These responses include recognition of tumor cells by NK cells, phagocytosis of pathogens by macrophages, and the lytic activity of the components of the complement cascade.

To evaluate phagocytic activity, phagocytic cells, typically macrophages are harvested from the peritoneal cavity (peritoneal exudate cells) and are allowed to adhere either in a well of a tissue culture plate or in a chamber fabricated on a tissue culture slide. After adherence, macrophages are washed and incubated with fluorescently labeled latex covaspheres. At the end of incubation, cells are fixed in methanol and stained in methylene chloride. Macrophages containing five covaspheres or more are counted as positive for phagocytic activity and data are expressed as a percentage of phagocytosis (the ratio of macrophages with ≥5 covaspheres to total macrophages counted).

Phagocytosis assays are conducted in vitro after chemical exposure either in vivo or in vitro. If an in vivo assay to assess the ability of tissue macrophages to phagocytose a foreign antigen is required, the functional activity of the reticuloendothelial system can be evaluated. Intravenously injected radiolabeled sRBC (⁵¹Cr-sRBCs) are removed from the circulation by the tissue macrophage and sequestered for degradation in organs such as the liver, spleen, lymph nodes, lung, and thymus. Clearance of the ⁵¹Cr-sRBCs is monitored by sampling of the peripheral blood. When steady state has been attained, animals are euthanized and organs are removed and counted in a gamma counter to assess uptake of the ⁵¹Cr-sRBCs.

When phagocytic cells encounter antigens, especially those associated with bacteria or a virus, they undergo activation, which is induced, in part, through interactions with TLRs. Extracellular antigens are processed and presented in association with MHCII, while intracellular antigens are processed and presented in association with MHCI, as described in the “Antigen Processing” section. Concomitantly with antigen encounter by the phagocyte, there is an increase in the expression of MHCI and/or MHCII as well as the costimulatory molecules CD80/CD86. The simultaneous increase in MHC molecules in combination with CD80 and CD86 is critically important in order for the phagocytic cell to drive T-cell effectors (CD4⁺ and CD8⁺ T cells). The upregulation of MHCI and/or II and CD80 and CD86 can be readily quantified by flow cytometry (see the “Flow Cytometric Analysis” section) after the activation of phagocytic cells, primarily those of the monocytic lineage, to further assess phagocytic cell function and the influence of an immunotoxicant. These measurements are especially informative when performed in combination with cell surface markers that are capable of identifying monocytic lineage subpopulations, for example such as tissue macrophages, conventional DC, and plasmacytoid DC because they provide information on whether the immunotoxicant interferes with the activation/differentiation of the phagocytic cell and its antigen-presenting capability. An overview of the types of macrophages, their hematopoietic origin, and a general discussion of the many different assays that are used to assess their functional status was recently provided by Barnett and Brundage (2010).

Evaluation of the ability of NK cells to lyse tumor cells is achieved using the YAC-1 cell line as a tumor target for an in vitro cytotoxicity assay. YAC-1 cells are radiolabeled with ⁵¹Cr and incubated (in microtiter plates) in specific effector-to-target ratios with splenocytes from xenobiotic-exposed and nonexposed animals. During an incubation step, splenic NK cells (effectors) lyse the ⁵¹Cr-YAC-1 cells, releasing ⁵¹Cr into the supernatant. At the end of the incubation, plates are centrifuged and the supernatant is removed and counted on a gamma counter. After correcting for spontaneous release (which should be <10%), specific release of ⁵¹Cr is calculated for each effector-to-target ratio and compared to the specific release from control animals. To avoid the use of radionucleotides, a number of flow cytometry based assays have been developed utilizing fluorescent stains to identify live and dead target cells. One such assay involves the coculture of NK cells with target cells that have been stained with the fluorescent green dye 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) (Kane et al., 1996). Utilizing DiO allows for readily discriminating between target and nontarget cells while propidium iodide is used to distinguish between live and dead cells. The various methods for NK assays used in immunotoxicity testing were recently reviewed (Li, 2010).

Acquired Immunity—Humoral

The AFC assay is a sensitive indicator of immunological integrity for several reasons. It is a test of the ability of the host to mount an antibody response to a specific antigen. When a T-dependent antigen (an antigen that requires T cells to help B cells make antibody) like sRBC is used, this response requires the coordinated interaction of several different immune cells: APC (macrophages and DCs), T cells, and B cells. Therefore, an effect on any of these cell types (eg, antigen processing and presentation, cytokine production, proliferation, or differentiation) can have a profound impact on the ability of B cells to produce antigen-specific antibody. Other antigens, termed T-cell-independent antigens, such as dinitrophenyl-ficoll or trinitrophenyl-LPS, can also be used that bypass the requirement for T cells in eliciting antibody production by B cells. Although the AFC response assay was developed almost half a century ago, its sensitivity in identifying immunotoxicants has resulted in worldwide application for the purpose of hazard identification, which extends to present day (see the “Historical Perspective on Regulatory Guidance in Immunotoxicology” section).

A standard AFC assay involves immunizing control and xenobiotic-exposed mice either intravenously or intraperitoneally with sRBCs. The antigen is taken up in the spleen, and an anti-sRBC antibody response is induced. In the mouse, the peak day of antibody production is four days after immunization. Historically, the antibody response has been quantified by enumerating the number of plasma cells present in the spleen that are secreting anti-sRBC antibodies. This can be accomplished by removing the spleen, making a spleen cell suspension that is then mixed with sRBC, complement, and melted agar. This mixture is poured onto a petri dish and covered with a cover slip. After the agar hardens the petri dishes are incubated for three hours at 37°C. During this time, the B cells that have been appropriately activated by the antigen and have differentiated into plasma cells secrete anti-sRBC IgM antibody. When the complement and the secreted IgM coat the surrounding sRBCs, areas of hemolysis (plaques) appear against a lawn of sRBC that can be enumerated. At the center of each plaque is a single B cell (AFC). Data are usually presented as IgM AFC per million splenocytes. IgG AFC can also be enumerated by slight modifications of this same assay. This isotype switching (from IgM to IgG) is important in secondary responses in which memory B cells respond more quickly to an antigen. An overview of the T-cell-dependent antibody response to sRBCs was recently provided, in which this model is described as the “gold standard” for evaluating the potential adverse effects of xenobiotics on the immune system (White et al., 2010).

A second method to enumerate AFC cells is by ELISPOT. This method is similar to the plaque assay described above with the exception that the splenocytes isolated from sRBC-sensitized mice are incubated on ELISPOT mictotiter plates coated with either antimouse IgM or sRBC membranes to capture the secreted IgM. After incubation, the spleen cells are removed using a washing buffer followed by incubation with an enzyme-conjugated antimouse IgM antibody. A second washing step is used to remove unbound enzyme-conjugated antimouse IgM followed by addition of the chromogen substrate. The site at which each IgM secreting cell was present in the well of the ELISPOT microtiter plate is now visualized by the formation of a “spot” through the enzymatic conversion of the chromogen substrate. Typically, enumeration is performed using an ELISPOT reader that can rapidly quantify spot number (number of plasma cells) as well as the size and intensity (quantity of IgM being secreted) of the spots. It is noteworthy that in addition to enumeration of AFCs, the ELISPOT method can be applied to quantification of any secreted protein for which a suitable capture antibody exists. In evaluations of immunocompetent cells, the ELISPOT is especially useful for enumerating cytokine and chemokine secreting cells, and serves as an alternative to flow cytometry, where cell enumeration is critical.

A third way the anti-sRBC humoral immune response can be evaluated is ex vivo using serum from peripheral blood of immunized mice and an enzyme-linked immunosorbent assay or ELISA. Although the optimal response is delayed by one to two days (compared to the AFC assay), this approach takes into account antigen-specific antibody secreted by B cells in the spleen as well as B cells residing in the bone marrow. Like the AFC assay, mice (or other experimental animals) are immunized with sRBCs and six days postimmunization peripheral blood is collected. Serum from each sample is serially diluted and incubated in microtiter plates that have been coated with sRBC membranes. The membranes serve as the antigen to which sRBC-specific IgM or IgG will bind, as described above in the ELISPOT assay. After incubation of the test sera and a wash step, an enzyme-conjugated monoclonal antibody (the secondary antibody) against IgM (or IgG) is added. This antibody recognizes the IgM (or IgG) and binds specifically to that antibody. After incubation and a wash step, the enzyme substrate (chromogen) is added. Conversion of the substrate by the enzyme conjugated to the secondary antibody results in a color change, which can be quantified by measuring absorbance with a plate reader. Because this is a kinetic assay (color develops over time and is dependent on concentration of anti-sRBC antibody in the test sera), it is important to establish control concentration–response curves so that data can be evaluated in the linear range of the curve. Data are usually expressed in arbitrary optical density (OD) units. An advantage of the ELISA over the AFC assay and ELISPOT is the ability to attain a greater degree of flexibility, since serum samples can be stored frozen for analysis at a later date. In contrast, a disadvantage of the ELISA approach is that it provides no information on the actual number of plasma cells that were induced by sRBC sensitization. Other T-dependent antigens, including keyhole limpet hemocyanin (KLH), are routinely used to drive an antigen-specific antibody response and to assess the potential for immunotoxicity (Plitnick and Herzyk, 2010).

B cell to plasma cell differentiation can also be monitored by flow cytometry. This can be accomplished utilizing intracellular staining for IgM (or other Ig isotypes such as IgG) to identify IgM^high cells in combination with the transmembrane proteoglycan, CD138, also termed syndecan-1, which is a hallmark of a fully differentiated plasma cell (Costes et al., 1999). It is noteworthy that even in resting nonactivated B cells, background levels of intracellular IgM are readily detectable because the monomeric form of IgM serves as the B-cell antigen receptor. For this reason, the designation IgM^low typically refers to a resting B cell versus IgM^high, which is indicative of high level IgM expression as exhibited by plasma cells.

One final assay measures the ability of B cells to undergo blastogenesis and proliferation, which are critical steps in the generation of an antibody response. Lymphocyte proliferation is most often quantified either using a radioisotope-based assay or by flow cytometry. Splenocytes are stimulated in microtiter plates with a monoclonal antibody to surface Ig in the presence of IL-4, or with a B-cell mitogen such as LPS. Proliferation is evaluated two to three days after stimulation. In the radioisotope-based assay, proliferation is quantified by quantifying the incorporation of ³H-thymidine into the DNA of the cultured cells. Data are usually expressed as mean counts per minute (cpm) for each treatment group. In the flow cytometry-based assay, cells are stained with one of several membrane permeable dyes such as carboxyfluorescein succinimidyl ester (CFSE). CFSE is a colorless dye that passively diffuses into the cell. Once inside the cell, CFSE undergoes cleavage by intracellular esterases to yield carboxyfluorescein succinimidyl, which is highly fluorescent and covalently reacts with intracellular amines. As the cell divides, the intensity of staining in each of the two newly formed daughter cells decreases by approximately half, as well as in every subsequent cell division thereafter. The number of cell divisions each cell has undergone is inversely proportional to fluorescence intensity. T-cell proliferative responses can be quantified using the same methodologies as described above with the exception that T-cell-specific stimuli are used to induce T-cell proliferation. Often in broad-based immunotoxicological evaluations, B-cell proliferative responses are measured in conjunction with T-cell proliferation.

Acquired Immunity—Cell-Mediated

While there are numerous assays used to assess CMI, three primary tests are used routinely in the National Toxicology Program (NTP, 2009) test battery (see the “The National Toxicology Program Tier Approach” section later in this chapter). The test battery includes the CTL assay, DTH response, and the T-cell proliferative responses to antigens (anti-CD3 + IL-2), mitogens (phytohemaggluttinin [PHA] and concanavalin A [Con A]), and allogeneic cell antigens (mixed lymphocyte responses [MLR]).

The CTL assay measures the in vitro ability of splenic T cells to recognize allogeneic target cells (MHC mismatched) by evaluating the ability of the CTLs to proliferate and then lyse the target cells. Splenocytes are incubated with P815 mastocytoma cells, which serve as target cells. The target cells are pretreated with mitomycin C or irradiated, so that they cannot proliferate. During this sensitization phase, the CTLs recognize the targets and undergo proliferation. Five days after sensitization, the CTLs are harvested and incubated in microtiter plates with radiolabeled (⁵¹Cr) P815 mastocytoma cells. During this elicitation phase, the CTLs that have acquired memory recognize the foreign MHCI on the P815 cells and lyse the targets. At the end of the incubation, the culture plates are centrifuged, the supernatant is removed, and radioactivity released into the supernatant is quantified on a gamma counter. After correcting for spontaneous release, the percent cytotoxicity is calculated for each effector-to-target ratio and compared to that from control animals. Several flow cytometry-based methods have also been developed to assay CTL activity that avoids the use of radionucleotides. One approach is similar to the one described for the NK cell assay above and involves the labeling of target cells with a fluorescent dye (eg, DiO or CFSE) in order to discern target cells from effectors. After coculture of target cells and CTL effectors, target cell viability is assessed by propidium iodide staining or using one of several other commercially available stains to assess viability. Another alternative nonradioactive surrogate assay widely used in place of measuring direct killing of target cells is quantification of IFNγ expressing CD8⁺ T cells by flow cytometry using intracellular staining. An inherent limitation to this last approach is that direct target cell killing is not assessed and it is assumed that all IFNγ CD8⁺ cells identified are CTL effectors (see the “Flow Cytometric Analysis” section). The current state-of-the-science of using the CTL assay to evaluate the effects of xenobiotics on CMI function was recently provided by Burleson et al. (2010).

The DTH response evaluates the ability of memory T cells to recognize foreign antigen, proliferate and migrate to the site of the antigen, and secrete cytokines and chemokines, which result in the influx of other inflammatory cells. The assay itself quantifies the influx of radiolabeled mononuclear cells into the sensitization site. During xenobiotic exposure, mice are sensitized twice with KLH subcutaneously between the shoulders. On the last day of exposure, mononuclear cells are labeled in vivo with an IV injection of ¹²⁵I-5-iododeoxyuridine. One day later, mice are challenged intradermally in one ear with KLH. Twenty-four hours after challenge, animals are euthanized, the ears are biopsied, and radiolabeled cells are counted in a gamma counter. Data are expressed as a stimulation index, which represents the cpm of ¹²⁵I activity in the challenged ear divided by the cpm in the unchallenged ear. As such, the DTH functional response may be influenced by disruption of either Th1-driven, antigen-dependent T-cell development or mobilization of sensitized T cells to a local site (Dietert et al., 2010).

T cells play a central role in CMI and the ability of T cells to undergo blastogenesis and proliferation is critical to this role. Several mechanisms exist to evaluate proliferative capacity. The MLR measures the ability of T cells to recognize foreign MHCI and MHCII on splenocytes from an MHC-incompatible mouse (allogeneic cells) and undergo proliferation. For example, splenocytes from B6C3F1 mice (responders) are incubated with splenocytes from mitomycin C-treated (or irradiated) DBA/2 mice (stimulators). Proliferation is evaluated four to five days after stimulation by measuring uptake of ³H-thymidine into the DNA of the cultured responder cells. Cells are collected from each well using a cell harvester and counted in a scintillation counter. Data may be expressed as either the mean cpm for each treatment group or as a stimulation index in which the index is calculated by dividing the cpm of wells containing responders and stimulators by the cpm of wells containing responders alone. Alternatively, cell proliferation can be quantified by flow cytometry using membrane permeable dyes such as CFSE (please see the following section “Flow Cytometric Analysis”).

General T-cell proliferation can be evaluated in a manner similar to that described above for B cells (Table 12-5). Splenocytes are stimulated in microtiter plates with monoclonal antibodies directed to the CD3 complex of the TCR (anti-CD3) and the CD28 T-cell coreceptor (anti-CD28), or with the T-cell mitogens Con A or PHA. Proliferation is evaluated two to three days after stimulation by measuring uptake of ³H-thymidine into the DNA of the cultured T cells. Data are usually expressed as mean cpm for each treatment group. As discussed above, proliferation can also be quantified by flow cytometry. These studies are usually done in conjunction with B-cell proliferative responses described above.

Flow Cytometric Analysis

One of the most rapidly advancing areas and powerful tools in immunotoxicology has been the application of flow cytometry to investigations of immune modulation by xenobiotics. In the most general sense, flow cytometry is a method that employs light scatter, fluorescence, and absorbance measurements to analyze large numbers of cells (typically 5000-100,000/sample), on an individual basis. Most commonly, fluorochrome-conjugated monoclonal antibodies raised against a specific protein of interest are employed for detection. The strength of the approach is that a wide variety of measurements can be made on large numbers of cells, individually, rapidly, and with a high level of precision. In addition, methods are also available that allow for the analysis of specific proteins in cell-free preparations such as cell lysates, biological fluids, and culture supernatants. A broad selection of polyclonal and monoclonal antibodies is available to cell surface markers, intracellular proteins, and secreted proteins (Table 12-5).

The most common application of flow cytometry in immunotoxicology is to enumerate specific leukocyte populations and subpopulations. For example, antibodies are available to the T-cell surface proteins CD4, CD8, and CD3 (among others). Because flow cytometers can detect light emission of multiple wavelengths simultaneously, multiple-colored fluorochromes can be used concurrently facilitating an analysis of more than one protein simultaneously in a given sample (multicolor/parameter analysis). In this manner, the number of CD4⁺ and CD8⁺ cells can be determined simultaneously on a single sample of cells. In the thymus, dual staining can be used to assess T-cell maturation by determining the number of CD4⁺/CD8⁺ (double positive), CD4^-/CD8^- (double negative), CD4⁺/CD8^- (single positive), and CD4^-/CD8⁺ (single positive) cells residing in this organ (Fig. 12-20). This approach can be used to provide insight into which specific T-cell subsets are targeted after exposure to a xenobiotic and to identify putative effects on T-cell maturation. Similarly, antibodies are readily available to identify other leukocyte subpopulations including antibodies to surface Ig, CD19, and B220 (the CD45 phosphatase on B cells) for enumerating B cells, to Mac1 and F4/80 for macrophages, to CD16/CD56 for NK cells in humans, and to CD161 (NK-1.1) for NK cells in mice. Surface marker analysis of heterogeneous cell preparations can reveal significant alterations in lymphoid subpopulations, and in many instances, this is indicative of alterations in immunological integrity. Indeed, an indicator of disease progression in AIDS can be monitored by changes in CD4⁺ T cell numbers. Luster and coworkers (1992) have reported that in conjunction with two or three functional tests, the enumeration of lymphocyte subsets can greatly enhance the detection of immunotoxic chemicals. However, it is important to emphasize that although surface marker analysis can identify changes in leukocyte populations, functional analysis of the immune system is more definitive for the detection of immunotoxicity since the ability of immunocompetent cells to mount an effector response is assessed directly.

With technical advancements of flow cytometers (ie, an increased number of wavelengths that can be simultaneously detected, enhanced sensitivity of detection, and more rapid analysis) coupled with a steady growth of applications, reagents, and methods, flow cytometry has become an integral tool in elucidating the cellular and molecular mechanisms of action produced by immunotoxicants by measuring their effects on a wide variety of cellular responses. Current applications go well beyond strictly evaluating cell surface markers and include measurements of cellular viability, cell cycle, cell proliferation, intracellular free calcium, induction of apoptosis, DNA strand breaks (TUNEL assay), intracellular proteins (cytosolic and nuclear), membrane potential, intracellular pH, oxidative stress, and membrane lipophilicity (reviewed by Burchiel et al., 1997, 1999; Hawley and Hawley 2004; Shapiro, 2003). Of particular utility in assessments of immune cell signaling and effector function has been the development of protocols and antibodies for intracellular staining of permeabilized cells. In the case of effector function, flow cytometry can be used to quantify the expression of multiple cytokines and/or chemokines simultaneously in individual cells by intracellular staining. Because activated leukocytes often secrete multiple regulatory factors simultaneously, termed “multifunctional,” using intracellular staining in conjunction with extracellular staining for phenotypic markers to identify specific cell populations can provide important insights into the magnitude and profile of effects exerted by an immunotoxicant on specific cell types and populations. Measurements of regulatory factors in combination with phenotypic markers can be extended to also include markers of cell activation or differentiation to identify at which stage during effector cell generation an immunotoxicant is acting. For an example of intracellular cytokine staining, refer to the study of Karmaus et al. (2011). A second application of intracellular staining in flow cytometry has been for investigating cell signaling. The complex networks and pathways that control cellular activities can be studied on an individual cell basis to provide static or kinetic information in specific cell types. For example, by using intracellular staining, the abundance of specific signaling proteins, cytosolic and nuclear, can be quantified. In addition to signaling protein abundance, post-translational modifications of proteins represent an important mechanism for transducing regulatory signals. One of the most extensively studied is protein phosphorylation and dephosphorylation, which often results in the activation or inactivation of protein function. The development of antibodies capable of distinguishing the point at which proteins possess phospho-groups at specific regulatory sites has made it possible to study modulation of distinct regulatory pathways by immunotoxicants. Because basic flow cytometers have the ability to detect six to eight colors simultaneously, with more advanced instruments possessing 18 color capacity, it is possible to measure multiple signaling events and/or network interactions in single cells. This capability coupled with the fact that the flow cytometer can evaluate thousands of cells per second, allows for accurate and reproducible detection of even rare events occurring in small subsets of leukocytes. Moreover, flow cytometry has numerous advantages over other approaches such as Western blotting and immunoprecipitation because of the rapidity of analysis and that it is not a “bulk lysis” method in which all cells in a given sample are pooled for analysis to render results that are representative of the overall mean within the entire cell population rather than in individual cells. For examples of measurement of intracellular phosphoprotein and transcription factors by flow cytometry that can be applied to study mechanisms of immune modulation, see North et al. (2010) and Lu et al. (2011).

Another major advancement in flow cytometry-based analyses has been the development of fluorescent microspheres that are individually identified by the instrument. By coating the surface of microspheres with various concentrations of two fluorescent dyes, sets of microspheres can be generated with each set possessing a unique spectral signature. Subsequently, various materials (ie, proteins, antibodies, or nucleic acids) can be covalently conjugated to the surface of these microspheres in order to create unique detection systems. This technology is being widely applied for analyzing a broad variety of soluble cellular components including proteins in cell-free preparations by flow cytometry. For example, up to 15 different cytokines can be measured simultaneously in cell supernatants or biological fluids (eg, BALF). A major advantage of this method over more traditional approaches, such as ELISA-based assays, is that only small amounts of biological samples and reagents are required because multiple proteins are assayed simultaneously. The same technology is also being applied for analyzing cell lysates for changes in protein phosphorylation in investigations of cell signaling. In addition, flow cytometry is routinely used to purify and isolate leukocyte subpopulations from heterogeneous cellular preparations. Thus, flow cytometry has become a powerful tool for characterizing the cellular and molecular mechanisms associated with immunotoxicants.

For information concerning specific flow cytometry protocols and applications beyond those provided by commercial vendors, the reader is directed to several references including the 4th edition of Practical Flow Cytometry (Shapiro, 2003), the 2nd edition of Methods in Molecular Biology: Flow Cytometry Protocols (Hawley and Hawley, 2004), and the Purdue University Cytometry Laboratories website (www.cyto.purdue.edu), which hosts the “Discussion List,” a blog to which one can submit questions to the broader community of flow cytometry users for technical assistance.

Measurements of Cytokines and Cytokine Profiling

As discussed in the earlier part of this chapter, development, maturation, differentiation, and effector responses of the immune system are highly dependent on a multitude of small, secreted proteins termed cytokines. In most cases, these immunological processes are controlled by the production of multiple cytokines, some of which are released simultaneously while others are released in a very defined temporal sequence. Many of these cytokines are produced by T cells and are the mechanism by which a wide variety of functions by T cells are mediated. Due to the importance of cytokines in regulating the immune system, xenobiotics that alter the production and release of these mediators can significantly affect immune competence. Therefore, measurement of multiple cytokines, often referred to as cytokine profiling, has become routine in immunotoxicology and can provide significant insights into the mechanisms by which a xenobiotic produces its immunotoxicity. For example, cytokine profiling has been explored as an approach for identifying chemical allergens, either contact sensitizers, which typically induce a Th1 profile of cytokines (IL-2, IFN-γ, and TNF-β), or respiratory sensitizers, which typically produce a strong Th2 cytokine profile (IL-4, IL-5, IL-6, and IL-10) (Mosmann and Coffman, 1989; Mosmann et al., 1991). Cytokines can be measured in cell culture supernatants or biological fluids (ie, serum or BALF) by ELISA or by multiplex assays. Quantification of test samples is accomplished by comparison to a standard curve employing recombinant cytokine standards. The aforementioned method, the ELISPOT assay can also be used to quantify the number of cells producing the cytokine of interest. Likewise, flow cytometry can be used to identify cytokine producing cells by intracellular staining or to quantify cytokines in media or biological fluids with the main advantage being that the source of the cytokine(s) can be identified using the former approach and that many cytokines can be assayed simultaneously from one sample using the latter approach (see the “Flow Cytometric Analysis” section). Because cytokines are regulated transcriptionally and then actively synthesized by cells at the time they are secreted, rather than existing as stored proteins, measurements of cytokine mRNA levels have become another common approach of assessing which cytokines are being expressed at a given time and the putative effects xenobiotics exert on their regulation and expression. Real time polymerase chain reaction (PCR) is most commonly used for quantifying mRNA levels of specific cytokines as well as for other genes of interest in cells or tissues. For more global assessments of mRNA levels, low-density RNA arrays or microarrays can be applied. Major advantages to quantifying cytokine expression at the mRNA rather than the protein level include significantly greater sensitivity, the ability to quantify expression in solid tissues, and the capability to rapidly design appropriate reagents (PCR primers) specific for any cytokine. A major disadvantage is that changes at the mRNA level for a given cytokine may not necessarily correlate with changes in protein. Another disadvantage over flow cytometric determinations by intracellular staining is not knowing the cell type(s) from which the cytokines are being produced. One additional limitation to cytokine measurements by ELISA or via quantification of cytokine mRNA levels is that neither of these approaches provides information concerning the biological activity of the proteins being measured. As described by Corsini and House (2010), multicytokine analysis still needs to be standardized in terms of optimum sources for analysis, protocols, and quality control issues, such as the use of reference standards and the expression of results.

Host Resistance Assays

Host resistance assays represent a way of assessing how xenobiotic exposure affects the ability of the host to combat infection by a variety of pathogens. Although host resistance studies provide significant insight into the mechanisms by which an immunotoxicant is acting, these assays are not used as a first or only choice for evaluating immune competence. The results from host resistance assays are typically more variable than other immune function assays already discussed and therefore require markedly greater numbers of animals in order to obtain statistical power. The increased number of animals required also raises ethical considerations as well as cost. In addition, as with other immune function tests, no single host resistance model can predict overall immune competence of the host, primarily because each model uses different mechanisms for elimination of various pathogens. A representative list of host resistance models is shown in Table 12-7. Typically, three challenge levels of pathogen (approximating the lethal dose 20 [LD₂₀], lethal dose 50 [LD₅₀], and lethal dose 80 [LD₈₀]) for each concentration of xenobiotic are used in order to be able to detect both increases and decreases in resistance. Endpoint analyses are lethality (for bacterial and viral pathogens), changes in tumor burden, and increased or decreased parasitemia. In host resistance studies, it is also important to consider the following: (1) strain, route of administration, and challenge size of the pathogen; (2) strain, age, and sex of the host; (3) physiological state of the host and the pathogen; and (4) time of challenge with the pathogen (prior to, during, or after xenobiotic exposure). All of these can have significant effects on the results from any individual study.

A major advantage in applying host resistance models to investigations of immunotoxicity is the ability to study the effects of an immunotoxicant within the context of the intact immune system encompassing all its diversity as it acts to protect the host against a bona fide pathogen. It is important to emphasize that the immune system possesses significant redundancy (ie, multiple mechanisms to provide defense against invading microorganisms). Therefore, even if a particular cell type or effector response has been compromised by exposure to an immunotoxicant, other components of the immune system might provide partial or complete protection to the host from pathogen challenge. In addition, certain pathogens are restricted to specific tissues or anatomical sites, for example influenza, which is typically restricted to airways. Using host resistance models permits the study of immunotoxicants and their effects within the environment and context of the tissue targeted by the pathogen (Buchweitz et al., 2007). The current state-of-the-science of a variety of host resistance models was recently reviewed including bacterial challenge models (Burleson and Burleson, 2010), viral host resistance models (Freebern, 2010), parasite challenge models (Luebke, 2010), and tumor challenge models (Ng et al., 2010).

Assessment of Developmental Immunotoxicology

Interest in DIT is predicated on the recognition that the developing immune system represents a novel target for xenobiotic-induced toxicity that presents some special considerations when it comes to assessment. The concept that any of a number of dynamic changes associated with the developing immune system might provide periods of unique susceptibility to chemical perturbation has been reviewed (Dietert et al., 2000; Holladay and Smialowicz, 2000). This unique susceptibility may be manifested as a qualitative difference, in the sense that a chemical could affect the developing immune system without affecting the adult immune system, or as a quantitative difference, in the sense that a chemical could affect the developing immune system at lower doses than the adult immune system, or as a temporal difference, in the sense that a chemical could produce either a more persistent effect in younger animals than adults, or trigger a delayed effect (ie, the consequences of early exposure are not manifested until early adulthood). As a result, while effective assessment of DIT should certainly draw upon the prior experience gained from adult-exposure immunotoxicity assessments, it is important to examine the database of known immune changes that reflect the potential for unique susceptibility and are specific for the developing immune system (Dietert and Piepenbrink, 2006b).

One of the most comprehensive reports to date attempted to compare the immunotoxicity following developmental or adult exposure to the following compounds: diethylstilbestrol, diazepam, lead, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and tributyltin oxide (Luebke et al., 2006a). The selection of these five compounds was reported to be based on the availability of some human data. The authors concluded that for all five chemicals, the developing immune system was found to be at greater risk than the adult, either because lower doses produced immunotoxicity, adverse effects were persistent, or both. However, it is noteworthy that no developmental immunotoxicants have been identified that have not also been immunomodulatory in the adult animal. Importantly, this observation may merely reflect the state-of-the-science—for example, all published DIT studies to-date have been conducted on known adult immunotoxicants, and systematic studies comparing the effects across multiple developmental windows are still uncommon.

A better understanding of the developing immune system, and in particular, an understanding of critical developmental hallmarks has prompted some to speculate about the existence of five critical windows of vulnerability (Dietert et al., 2000). The first window encompasses a period of HSC formation from undifferentiated mesenchymal cells. Exposure of the embryo to toxic chemicals during this period could result in failures of stem cell formation, abnormalities in production of all hematopoietic lineages, and altered immunocompetence. The second window is characterized by migration of hematopoietic cells to the fetal liver and thymus, differentiation of lineage-restricted stem cells, and expansion of progenitor cells for each leukocyte lineage. This developmental window is likely to be particularly sensitive to agents that interrupt cell migration, adhesion, and proliferation. The critical developmental events during the third window are the establishment of bone marrow as the primary hematopoietic site and the establishment of the bone marrow and the thymus as the primary lymphopoietic sites for B cells and T cells, respectively. The fourth window addresses the critical periods of immune system functional development, including the initial period of perinatal immunodeficiency, and the maturation of the immune system to adult levels of competence. The final window addresses the subsequent period during which mature immune responses are manifested, and functional pools of protective memory cells are established.

Most recently, considerable attention has been focused on the perinatal period (ie, prior to and just after birth) because this window of development is known to be replete with dynamic immune changes, many of which do not occur in adults (Dietert and Piepenbrink, 2006b). Indeed, one reality associated with DIT windows is that the developing immune system exists in an unbalanced state through the latter portion of gestation with certain functional CMI capacities deliberately impaired. In fact, Taylor and coworkers (2006) demonstrated that placentally induced immune skewing via the release of Fas-ligand-containing exosomes is one hallmark of a successful pregnancy brought to full term. Upon birth, restoring effective immune balance through the enhancement of Th1 capacity in the newborn is critical for protecting childhood health (Holt et al., 2005; Yun and Lee, 2005). Prenatal maturation and functional skewing of the fetal immune system followed by the rapid reversal of the imbalance at birth have features that are not effectively modeled using adult exposure-assessment (Dietert and Piepenbrink, 2006b), and DIT is best viewed as a continuum of alterations. Suppression of the developing immune system, manifested as increased susceptibility to infections and cancer, is not the only concern, and immunotoxic changes that increase the risk for allergic or autoimmune responses in later life should also be considered (Edwards and Cooper, 2006; Selgrade et al., 2006; Yeatts et al., 2006). Adding to the complexity is the demonstration that some DIT seem capable of inducing targeted immune suppression while at the same time elevating the risk of allergy and/or autoimmunity (Haggqvist et al., 2005). Because either significant immune suppression or disrupted immune regulation is a concern and needs to be detected, the most effective methodology for assessing DIT must also be capable of assessing significant changes in immune balance.

In spite of the increased interest in assessing the potential for developmental immunotoxicity, it must be emphasized that neither validated nor widely accepted methods currently exist for evaluating the effects of a chemical on the developing immune system. Several workshops have summarized consensus thinking concerning developmental immunotoxicity evaluation (Dietert et al., 2000; Holsapple, 2002b; Holsapple et al., 2005; Ladics et al., 2005; Luster et al., 2003; van Loveren and Piersma, 2004) and a number of reviews have been written (Burns-Naas et al., 2008; Dietert and Burns-Naas, 2008; Dietert and Holsapple, 2007; Dietert and Piepenbrink, 2006b; Holsapple et al., 2003, 2004, 2007; Luebke et al., 2006a; Weinstock et al., 2010; Winans et al., 2011). Additionally, other reviews have dealt with issues concerning immunotoxicity evaluation across various life stages (Germolec et al., 2004; Holsapple et al., 2005; Ladics et al., 2005; Ravel and Descotes, 2005). In light of the fact that neither validated nor established protocols presently exist to comprehensively assess whether a xenobiotic is a developmental immunotoxicant, below is a brief discussion of critical issues requiring consideration in establishing a testing framework.

In constructing a DIT testing framework, one of the first points to consider is the selection of an animal model. Consistently, the rat has been identified as the preferred species for evaluations of DIT, largely due to the fact that the rat has been utilized extensively in guideline developmental and reproductive toxicology testing. The anatomical and functional differences in the immune systems of the mouse, rat, dog, primate and human, and their use as models for DIT testing have been reviewed (Holsapple et al., 2003). The review concluded that the developing immune systems of mice and humans have been best characterized to date. In addition, the review also concluded that immune ontogeny in the mouse and rat is likely similar. Another important consideration when selecting a species is that the development of the immune system in the rodent is delayed relative to the human, and how this differential maturation will impact data extrapolation for predicting human risk. For example, some developmental landmarks observed in utero in humans occur after parturition in the rat.

A second consideration when constructing a framework for assessing DIT concerns gender-specific effects. Results from perinatal exposure to xenobiotics suggest that significant sex-based differences in immunotoxic sensitivity are common and are at least as prevalent, if not more frequent, compared with the incidence observed following adult exposure-assessment (Dietert and Piepenbrink, 2006b; Luebke et al., 2006a). As such, for the evaluation of DIT, testing of both sexes may be critical.

A third major consideration in a DIT testing framework is consideration of exposure. There is general agreement that the best exposure protocol is one where exposure occurs across all nonadult developmental windows followed either by immediate assessment or assessment after a few weeks (Holsapple et al., 2005; Ladics et al., 2005; Luster et al., 2003). Exposure to pregnant dams has been a hallmark feature of most DIT protocols, and the maternal influences on exposure to the fetus/newborn pups would be dependent on transfer of the xenobiotic either across the placenta or via lactation. The gestational (eg, transplacental) and lactational periods in the rat would result in exposure from conception to early postweaning in the pup, approximately three weeks of age. Direct exposure of pups via the diet would generally commence at about three weeks after birth. An unresolved issue is whether direct exposure of the pups, which is generally accepted as a routine procedure at around postnatal Day seven, should occur during the lactational period as well (Ladics et al., 2005); however, any decisions concerning this issue will require consideration of how humans would be exposed and the specific properties of the chemical being studied. For example, if exposure is only oral and there is no reason to believe that the lactational transfer differs significantly for a class of compounds between rats and humans, then direct dosing of pups could be initiated postweaning. In addition, information on pharmacokinetic and dosimetry could be useful in determining whether any direct dosing of pups during lactation is necessary. However, it is important to emphasize that this type of information is not routinely available for most xenobiotics.

A fourth consideration in creating a DIT testing framework concerns which specific endpoints to measure. As discussed above, immune organs, such as the thymus, spleen, and/or bone marrow, are not typically assessed in routine developmental and reproductive toxicology studies, and it has been the consensus of a number of workshops that histopathological evaluation of these immune organs could be easily integrated into these existing protocols (Holsapple et al., 2005; Luster et al., 2003). However, presently, there is uncertainty whether routine histopathology is sufficiently sensitive to detect all potential immunotoxic effects, especially when the unique characteristics of the developing immune system, as discussed above, are considered. Indeed, there are examples where morphometric histopathological findings do not predict functional impairments due to toxicity produced on the developing immune system (Hussain et al., 2005). Ultimately, while the use of specific, rather than general, histopathology has been recommended, it has been also suggested that functional tests be employed in the assessment of DIT. Unfortunately, few, if any, functional assays have been validated for detection of DIT, even for routine assays like the T-cell-dependent antibody response, which has largely been confined to adult exposure protocols. Results published on several chemicals and drugs in recent years suggest that functional tests are a front-line priority for perinatal immunotoxicity detection and that a combination of at least two functional tests, such as a multiisotype T-cell-dependent antibody response, and a CMI response assay, such as the DTH assay and/or CTL or NK cytotoxicity assays, should be paired with histopathological analysis and phenotypic analysis of lymphocyte subsets using flow cytometry (Dietert and Piepenbrink, 2006b).

One final point regarding the evaluation of developmental immunotoxicity needs to be considered. As developmental immunotoxicity protocols are inserted into existing toxicology testing regimes, such as developmental and reproductive toxicology protocols, it is likely to be necessary to incorporate immunization protocols. This approach has raised concerns among those evaluating other physiological systems (eg, reproductive and neurological) in terms of potential immunization-induced changes. However, investigations addressing this potential by determining the impact of the incorporation of immunotoxicological functional assays on standard toxicological studies in rats have been largely negative. Ladics and colleagues (1995) showed that immunization did not significantly alter the weights or morphology of routine protocol tissues with the exception of the spleen (ie, which manifested the anticipated increases in the numbers and size of germinal centers). In a subsequent study by this same group, immunization did not alter any hematological or clinical chemistry parameters, or lymphocyte subset numbers (ie, measured on peripheral blood with a flow cytometer), and did not mask the anticipated hepatotoxic effects of subchronic exposure to carbon tetrachloride (Ladics et al., 1998). An additional component of this discussion is the reality that the species being modeled, the human, has immunizations as a routine lifelong component of preventative medicine. As such, to avoid all immunizations in animal evaluation protocols does not closely simulate the childhood experience. Additionally, until the immune system is asked to respond specifically to a foreign antigen, the capacity to detect immunotoxicity may be severely limited. Therefore, immunization and a range of functional T-cell-dependent immune response evaluations seem necessary as components of an effective DIT assessment.

Assessment of Hypersensitivity Responses

As noted above, a primary role of the immune system is the discrimination of self versus nonself, and immunotoxicology can be thought of as a continuum with immunotoxic effects occurring in either direction (Fig. 12-1). The adverse consequences of exaggerated immune function would reflect an inability to recognize self, and are generally depicted as hypersensitivity and autoimmunity.

Drugs and chemicals that are capable of eliciting an immune response are generally low molecular weight substances possessing some inherent reactivity. There is a genetic susceptibility to hypersensitivity responses in that not all individuals react to the same drugs and chemicals. For the most part, the xenobiotic cannot be considered an antigen, simply because by itself it is not capable of stimulating an immune response. Instead, the xenobiotic often forms a hapten, which triggers the immune response in some tissue in the host. This property is called the sensitizing potential of the hapten and is associated with its inherent reactivity. Hapten-specific immune responses are therefore triggered only in the presence of the hapten–carrier complex and can be mediated either by humoral immunity or CMI. The damage associated with any type of hypersensitivity response can be directed against the tissue that is bound by the hapten. One of the most important and challenging problems in the field of immunotoxicology is determining the potential for chemicals to induce immune modulation and, in the current context, to demonstrate the sensitizing potential of xenobiotics. Thus, it becomes essential to have validated predictive animal models, and to understand the underlying mechanisms of action. The following is a discussion of the currently used methods of predicting the most frequently occurring hypersensitivity reactions to chemicals, Types I and IV, which are most often manifested as respiratory hypersensitivity and contact sensitization, respectively.

Assessment of Respiratory Hypersensitivity in Experimental Animals

Methods for assessing the potential to induce respiratory hypersensitivity have been reviewed (Holsapple et al., 2006). The objectives of this review were to describe the appropriate methods for identifying and characterizing respiratory hazards and risks and to identify the key data gaps and related research needs. The review addressed these objectives from the perspectives of proteins, chemicals, and drugs; and emphasized the important roles played by IgE and Th2 cell-mediated responses, while recognizing that damage to the respiratory tract can be triggered either by nonspecific irritation or by other specific (ie, non-IgE/Th2) immune-mediated mechanisms.

The state-of-the-science of animal models for respiratory hypersensitivity was also reviewed (Pauluhn, 2005). Current assays utilize two phases: induction/sensitization and challenge/elicitation. The induction phase usually includes multiple exposures to the test compound (sensitization) via the respiratory tract (ie, intranasal or intratracheal instillations, or by inhalation) or by dermal contact. There are advantages and disadvantages to each. Inhalation more closely represents environmental exposure by allowing for chemical contact with the upper as well as the lower respiratory tract. However, the equipment required is expensive and difficult to maintain. Exposure via the intranasal route is easily accomplished and allows for distribution of antigen to the upper and lower respiratory tract; however, studies have shown that a large proportion of the material can be recovered from the stomach (Robinson et al., 1996). In contrast, intratracheal instillation results in exposure to the lower respiratory tract only. The observation that a predominant respiratory sensitizer would still trigger an IgE response when applied topically has suggested an important interplay between Type I hypersensitivity reactions, manifested primarily as respiratory sensitization, and Type IV hypersensitivity reactions, manifested primarily as contact sensitization. The basis for this observation can be accounted for by a cytokine network model, which involves important cross talk between humoral immunity and CMI. Basically, a chemical with the capability of being a respiratory sensitizer will trigger an IgE response regardless of its route of exposure because it “selects” or supports the development of a Th2-dependent response, with the associated cytokine profile: IL-4, IL-5, IL-10, and IL-13. In contrast, a chemical which lacks the capability of being a respiratory sensitizer, but which can still trigger contact dermatitis, will select or support a Th1-dependent response, with the associated cytokine profile including IL-2 and IFN-γ.

The challenge (elicitation) phase involves exposure to the chemical (hapten), the homologous protein conjugate of the hapten, or the antigen. Endpoints to characterize a positive response range from the induction of Igs (ie, total IgE for chemicals or specific IgE for protein antigens), cytokines, or lymphokines in serum, to pathophysiological responses, including the influx of inflammatory cells and the onset of bronchoconstriction along with the associated changes in respiratory functional parameters. The latter may be accomplished by visual inspection of the animals’ respiratory pattern or more quantitatively by plethysmography. With plethysmography, changes in the respiratory rate, tidal volume, and plethysmographic pressure can be measured. However, plethysmography in guinea pigs, rats, and mice can be technically challenging and labor intensive. In addition, there is some controversy over the interpretation of measurements made from whole body plethysmography, and whether these measurements truly reflect changes in airway mechanics (Lundblad et al., 2002; Mitzner and Tankersley, 2003).

None of the currently applied animal models duplicates all features of human asthma, and most of the current animal models were developed for studying specific hypersensitivity responses to high molecular weight protein allergens (Pauluhn, 2005). Fewer animal models have been developed for use in the area of chemically induced respiratory allergy, which was one of the key data gaps identified in the review by Holsapple et al. (2006). One of the limitations associated with low molecular weight models is that often these compounds must conjugate with body proteins to become antigenic (ie, form hapten–carrier complexes). Often, a challenge with the conjugated chemical is necessary to induce a pulmonary response. Adding this variable can make the analysis of test results more difficult. False-negative results may occur due to variability in test article conjugation; and chemical conjugates are also necessary to measure specific immunological responses.

The majority of the current animal models are based on antibody-mediated events occurring as a reflection of the induction phase (Pauluhn, 2005). In certain cases, immunological sensitization may be confirmed by the detection of antigen-specific antibody; however, subsequent challenge does not produce clinical signs of respiratory distress. It is also possible to detect pulmonary sensitization in animal models where there is no detectable antigen-specific antibody production. In these cases, CMI or other mechanisms may be involved, or there may be difficulty in antibody detection.

Guinea pig models have been most frequently used for detection of pulmonary reactions to chemicals, because this species is known to respond vigorously to appropriate stimuli by developing an asthmatic-like bronchial spasm. In the guinea pig, as in the human, the lung is the major shock organ for anaphylactic response. Like humans, the guinea pig also demonstrates immediate- and late-onset allergic reactions as well as bronchial hyperreactivity and eosinophil influx and inflammation. The major difference in the mechanism of pulmonary responses between humans and guinea pigs is that the antibody involved in Type I reactions in humans is IgE and in guinea pigs is predominantly IgG1. The key features of this animal model involve protocols using single or repeated inhalation or cutaneous (ie, primarily topical or intradermal) exposures followed by a rest period until Day 21 (Pauluhn, 2005). After the rest period, an inhalation challenge with the hapten or antigen is performed thereby focusing on a measurement of the elicitation phase of the response (Karol et al., 1994). Respiratory patterns are often measured in whole-body plethysmographs, as discussed above. A common pathological accompaniment of increased airway hyperactivity is prolonged eosinophil-rich inflammatory leukocyte infiltration into the lungs of guinea pigs after inhalation challenge of the protein or hapten-conjugate. One of the disadvantages of using guinea pigs is the lack of reagents needed to identify cell types and mediators involved in respiratory allergy, which has hampered mechanistic studies.

Murine models, in which reagent availability is not an issue, have become more frequently utilized in the evaluation of respiratory hypersensitivity, and two approaches have been described. The first approach capitalizes on the fact that, like humans, IgE is the major anaphylactogenic antibody in mice, and focuses on the induction of total serum IgE (Dearman et al., 1998). The second approach capitalizes on the aforementioned cytokine network and has been referred to as cytokine profiling (Dearman et al., 2002, 2003; Plitnick et al., 2002, 2003). Both approaches have relied on dermal application of potential allergens/sensitizers, and on the theoretical foundation that chemical allergens induce divergent immune responses characteristic of the selective activation of discrete T-cell subpopulations (Pauluhn, 2005). Contact allergens, such as 2,4-dinitrochlorobenzene, are considered not to cause sensitization of the respiratory tract, and trigger an immune response in mice that is consistent with the preferential activation of Th1 cells (ie, little to no increase in total serum IgE and the production of IL-2 and IFN-γ in the draining lymph nodes). In contrast, topical sensitization to chemical respiratory allergens, such as TMA, triggers an immune response in mice that is consistent with the preferential activation of Th2 cells (ie, a moderate to marked increase in total serum IgE and the production of IL-4, IL-5, IL-10, and IL-13 in the draining lymph nodes). It is important to emphasize that while both the mouse total serum IgE test and cytokine profiling hold much promise, neither approach can be considered validated at this time.

Assessment of IgE-Mediated Hypersensitivity Responses in Humans

Described below are methods of human Type I hypersensitivity testing. These test results, in conjunction with a relevant history and physical exam, can be diagnostic of IgE-mediated pulmonary disease. Two skin tests are available for immediate hypersensitivity testing. In both, the measured endpoint is a wheal and flare reaction (ie, the result of edema and erythema subsequent to the release of preformed inflammatory mediators). The prick–puncture test introduces very small amounts of antigen under the skin and, owing to the reduced chance of systemic reaction, is recommended as a screening test. For test compounds not eliciting a reaction, the intradermal test using dilute concentrations of antigen may be used, but there is a higher risk of systemic reactions. The reader is referred to an additional text for a more detailed description of testing methods (Demoly et al., 1998).

In vitro serological tests, ELISAs, and radioallergosorbent tests may also be used to detect the presence of antigen-specific antibody in the patient’s serum. These tests do not pose a risk of adverse reactions and may be used in situations where standardized reagents for skin testing are not available. Serological testing is often used in population-based epidemiological studies. Additionally, bronchial provocation tests may be performed by having the patient inhale an antigen into the bronchial tree and evaluating his or her pulmonary response. In some cases, this may be the only way to demonstrate that a test article is capable of producing an asthmatic response. Care must be taken in these test situations in that it is possible to produce severe asthmatic reactions or anaphylaxis in sensitized individuals.

Assessment of Contact Hypersensitivity in Experimental Animals

Classically, the potential for a chemical to produce contact hypersensitivity has been assessed by the use of guinea pig models. These tests vary in their method of application of the test article, in the dosing schedule, and in the utilization of adjuvants. For a description of methods employed in representative tests, refer to the study of Klecak (1987). The two most commonly utilized guinea pig models, the Buehler test (Buehler, 1965) and the guinea pig maximization test (Magnusson and Kligman, 1969), are described briefly below.

In the Buehler test, the test article is applied to the shaven flank and covered with an occlusive bandage for six hours. This procedure is repeated on Days 7 and 14. On Day 28, a challenge dose of the test article is applied to a shaven area on the opposite flank and covered with an occlusive dressing for 24 hours. At 24 and 48 hours after the patch is removed, test animals are compared with vehicle-treated controls for signs of edema and erythema. The guinea pig maximization test differs in that the test article is administered by intradermal injection, an adjuvant is employed, and irritating concentrations are used. Animals are given pairs of intradermal injections at a shaven area on the shoulders. One pair of injections contains adjuvant alone, one pair contains test article alone, and one pair contains the test article mixed with adjuvant. Seven days following injection, after the area is reshaven, the test article is applied topically and an occluded patch is applied for 48 hours. In cases where the test article at the given concentration is nonirritating, the area is pretreated with 10% sodium lauryl sulfate 24 hours before the patch is applied to produce a mild inflammatory response. Two weeks following topical application, the animals are challenged on the shaven flank with a nonirritating concentration of the test article, which remains under an occluded patch for 24 hours. Then, after 24 and 48 hours, the test site is examined for signs of erythema and edema, two well-recognized indicators of cutaneous inflammation and contact dermatitis. However, the endpoints for evaluation in the guinea pig assays are visual and subjective, and it is difficult to assess irritating or colored compounds using these models.

Over the past 20 years, efforts have been made to develop and establish more quantitative and immunologically based assay methods in other species, focusing mainly on the mouse, again primarily because of the availability of reagents and techniques to conduct mechanistic studies. Gad and coworkers (1986) developed the mouse ear swelling test, which uses a quantitative measurement of ear thickness as an endpoint. Animals are sensitized by topical application of the test article for four consecutive days to abdominal skin that has been prepared by intradermal injection of adjuvant and tape stripping. On Day 10, the animals are challenged by topical application of the test article to one ear and vehicle to the contralateral ear. Measurements are made of ear thickness 24 and 48 hours later. A positive response is considered anything above a 20% increase in thickness of the treated ear over the control ear. Thorne and colleagues (1991) showed that dietary supplementation with vitamin A enhanced the mouse ear swelling assay in the absence of adjuvants, injections, or occlusive patches.

The assays described above evaluate the elicitation phase of the response in previously sensitized animals. In contrast, the mouse local lymph node assay (LLNA) identifies contact allergens as a function of the events occurring during the induction (sensitization) phase of a hypersensitivity response. The origins, development, evaluation, and eventual validation of the LLNA have been described in a comprehensive review (Kimber et al., 2002). In this assay, the induction phase of contact sensitization is measured by the incorporation of ³H-thymidine into proliferating lymphocytes in lymph nodes draining the site where the test article has been applied. Animals are dosed by topical application of the test article to the ears for three consecutive days. The animals are rested for two days and then injected intravenously with 20 μCi of ³H-thymidine. Five hours later, animals are sacrificed, the draining lymph nodes are dissected out, and single-cell suspensions are prepared and radioassayed. With consideration of dose–response and statistical significance, a threefold increase in ³H-thymidine cpm in chemically exposed animals over vehicle control animals (ie, the stimulation index) is considered to be a positive response.

The LLNA offers several advantages over the guinea pig assays in that (1) it has the potential to reduce the number of animals required and reduces animal distress; (2) it provides quantitative data that allow for statistical analysis; and (3) it provides dose–response data. The latter qualities of the LLNA have facilitated its integration into comparisons of potency across individual chemicals and/or within a chemical class. These additional advantages of the LLNA have facilitated its integration into risk assessments for contact dermatitis (Felter et al., 2003). Additionally, because the LLNA evaluates the induction phase of the immune response, it is more applicable to mechanistic studies. As an example, some compounds capable of producing contact sensitization also induce IgE production and subsequent respiratory hypersensitivity. Using three known allergenic diisocyanates—diphenylmethane-4,4’-diisocyanate, dicyclohexylmethane-4,4’-diisocyanate, and isophorone diisocyanate—Dearman and coworkers (1992) showed that all three known contact sensitizers induced lymphocyte proliferation in the draining lymph node but that only diphenylmethane-4,4’-diisocyanate, a known respiratory sensitizer, induced elevated levels of serum IgE and IgG2b. These types of results have prompted a number of investigators to propose that the LLNA could be considered as the first step in a safety evaluation process for allergenic potential (ie, as a method to identify sensitization potential of a chemical) with other methods being deployed to distinguish between skin and respiratory sensitizing activity (Holsapple et al., 2006).

Assessment of Contact Hypersensitivity in Humans

Human testing for contact hypersensitivity reactions is by skin patch testing. Patch testing allows for the diagnostic production of acute lesions of contact hypersensitivity by the application of a suspected allergen to the skin. Patches containing specified concentrations of the allergen in the appropriate vehicle are applied under an occlusive patch for 48 hours in most test protocols. Once the patch is removed and enough time elapses for the signs of mechanical irritation to resolve (approximately 30 minutes), the area is read for signs of erythema, papules, vesicles, and edema. Generally, the test is read again at 72 hours and, in some cases, signs may not appear for up to one week or more. Detailed information on patch testing has been reviewed (Mydlarski et al., 1998).

Human repeat insult patch tests are available as predictive tests in humans. Like predictive testing in animal models, there are many variations in attempts to increase the sensitivity of these procedures. These include preparation of the induction site by either tape-stripping, the application of an irritating concentration of sodium lauryl sulfate, or use of high concentrations of the test article for induction of sensitization. In general, the application of multiple occlusive patches, up to ten for 48 hours each at the same site, is followed by a rest period and then a challenge takes place under an occlusive patch at a different site. Positive reactions are scored in the same manner as for diagnostic patch tests.

Assessment of Autoimmune Responses

As described in the preceding section, exaggerated immune responses can be mediated by two entirely different types of interactions between the immune system and xenobiotics. One type of interaction was described in the preceding section in which the xenobiotic is a hapten, and the immune system plays an active role in eliciting a hypersensitivity response. The immune system can also be a passive target for the enhancing effects of drugs and chemicals, such as occurs when a xenobiotic mimics or causes the aberrant production of immunomodulatory cytokines, or when a xenobiotic disrupts the regulatory mechanisms which serve to protect self (ie, disrupt a suppressor mechanism). Additional mechanisms were presented above in the subsection “Autoimmunity” under “Immune-Mediated Disease.”

Another way that xenobiotics can enhance immune function is by acting as an adjuvant, which is defined as any substance that nonspecifically enhances the immune response to an antigen. The classic adjuvant is complete Freund’s adjuvant, which is a water-in-oil emulsion containing killed mycobacteria. The effectiveness of adjuvants in enhancing immune responses can be demonstrated by the fact that animals are often injected with complete Freund’s adjuvant to increase the production of antigen-specific antibodies, and by the desire to develop an adjuvant that is safe in humans (ie, complete Freund’s adjuvant produces severe side effects) that could be used in conjunction with vaccines or immunotherapy. The specific mechanisms for the actions of adjuvants, including complete Freund’s adjuvant, are not known. Moreover, the existence of environmental adjuvants is controversial and/or poorly studied.

In the context of testing strategies, the situation with autoimmunity is much more complex than with hypersensitivity responses. Animal models exist for a number of autoimmune diseases, and autoimmunity has been clearly demonstrated in humans. Therefore, the existence of autoimmune disease and the expected consequences cannot be denied. However, the ability of drugs and chemicals to exacerbate or trigger autoimmune disease in either animal models or humans is poorly understood. In fact, of all the possible consequences of immunotoxicity, autoimmunity is unquestionably the least understood. Primarily because of the strong genetic component in the susceptibility to autoimmunity, deciphering the exact role of xenobiotics in the induction of these conditions has proven to be very difficult. The following is a brief review of some of the currently used methods of predicting the potential of a xenobiotic to trigger or exacerbate autoimmunity leading to an inappropriate immune response to self-tissue antigens that can be associated with the generation of autoantibodies and/or autoreactive T cells.

As emphasized throughout this chapter, immunotoxicology has evolved to the point where an ever-increasing number of studies are being conducted to characterize the immunotoxicity of a variety of xenobiotics using standard immunotoxicological parameters. Depending on the specific drug/chemical, the exposure conditions, the species being tested and the immune parameter being measured, the outcomes can be manifested as decreases, increases, or no effect. While there is no question that the most frequent observation has been immune suppression, there is also no doubt that some examples of immune stimulation have been reported, a profile consistent with the concept of immunotoxicology existing as a continuum (Fig. 12-1). However, this observation can trigger some important questions. Should a treatment-related increase in an immune parameter such as the T-cell-dependent antibody response be considered an adverse response? Should an increase in the antibody response to a neoantigen be considered a harbinger of autoimmune potential? There simply is no consensus regarding the correct way to interpret an increase in immune parameters such as the T-cell-dependent antibody response, which were primarily developed to characterize the immunosuppressive potential of xenobiotics.

An assay that was specifically developed to characterize the immunostimulatory capacity of low molecular weight compounds (ie, pharmaceuticals in particular) is the popliteal lymph node assay (PLNA) (Kammuller et al., 1989). The concept for this assay was originally proposed by Gleichmann and coworkers (1984), who speculated that graft-versus-host reactions might be the basis for the pathogenetic mechanisms behind the development of drug-induced allergy and autoimmunity. The primary approach to the PLNA is to inject compounds subcutaneously into the hind footpad of either mice or rats. After six to eight days, the draining popliteal lymph nodes (PLNs) are excised and compared with PLNs from vehicle-treated animals. Differences (eg, increase) in the weight or cellularity of the treated nodes are an indication of the immunostimulatory potential of the test compound. Over the past 20 to 25 years, more than 130 chemicals have been tested in the PLNA (Pieters and Albers, 1999), yet the assay can still not be considered to be validated. Intralaboratory studies with the PLNA concluded that this method might be able to detect drugs capable of causing human drug hypersensitivity with high prevalence; but that additional development is needed to increase the reproducibility of the PLNA and to increase detection of drugs that require metabolic activation to become allergenic, or drugs for which there is dose-limiting toxicity (Weaver et al., 2005). The interpretability of the PLNA was increased by the integration of reporter antigens, trinitrophenyl-ficoll and trinitrophenyl-ovalbumin (Albers et al., 1997; Gutting et al., 1999). The reporter antigen PLNA differs from the standard PLNA in two ways. First, the reporter antigens are injected with the test substance under investigation. Second, anti-trinitrophenyl-specific antibodies are measured via ELISPOT, in addition to the comparisons of the PLNs. The profile of antibody production against the T-cell-independent antigen, trinitrophenyl-ficoll, and against trinitrophenyl-ovalbumin, an antigen recognized by T cells and B cells, enables the discrimination between immunosensitizing, and mere adjuvant or irritant potential of compounds.

The state-of-the-science of animal models of autoimmune disease has been summarized (Germolec, 2005). This review emphasized that while a wide variety of animal species have been studied, rodents have been most common, and concluded that rodent models fall into three categories: genetically predisposed animal models; animal models in which the autoimmune disease is produced by immunization with specific antigens; and animal models in which the disease is chemically induced.

Examples of genetically predisposed animal models include the nonobese diabetic (NOD) mouse, the F1 cross between the New Zealand black (NZB) and New Zealand white (NZW) mouse, and the MRL/lpr mouse. The NOD model has been used to study type 1 diabetes, specifically the T-cell autoimmune response, the role of B-cell antigen presentation, and the role of cytokines in the disease progression. The NZB × NZW F1 and the MRL/lpr mouse models have been used to study human SLE. The NZB × NZW F1 model has been used to map the specific susceptibility loci and to assess the importance of B-cell hyperactivity and T-cell involvement in autoantibody production in the development of SLE (reviewed by Germolec, 2005), while the important role of apoptosis in negative selection has been studied in the MRL/lpr mouse model, in which a genetic defect results in a mutation in the Fas gene. While both models exhibit characteristics of human SLE including high levels of serum immunoglobulins (ie, antinuclear and anti-DNA antibodies), as well as the immune-mediated nephritis, the MRL/lpr mouse model also exhibits rheumatoid factor autoantibodies and inflammatory joint disease characteristic of an arthritic response.

Arthritis can also be induced in susceptible rat strains by immunization with complete Freund’s adjuvant containing killed Mycobacterium tuberculosis in oil. Immunization of susceptible mouse strains (ie, those containing H-2^q or H-2^r alleles of the MHC) with type II collagen or cartilage glycoproteins, in the presence of adjuvant, can induce pathology similar to human rheumatoid arthritis. In collagen-induced models of arthritis, the immune response is directed against specific connective tissue antigens, while the complete Freund’s adjuvant-induced response is directed against a mycobacterium heat shock protein, with the observed pathology resulting from cross-reactive destruction of a proteoglycan found in joints (Germolec, 2005). Experimental autoimmune encephalomyelitis can be induced in a number of species by immunization with myelin basic protein and complete Freund’s adjuvant. This model has been used in rodents to characterize the role of T helper cell-mediated autoimmune disease characterized by perivascular lymphocyte infiltration of the CNS and the destruction of the myelin nerve sheath resulting in paralysis, similar to human multiple sclerosis.

One of the most commonly used models of chemically induced autoimmunity is the Brown Norway rat model, where animals are injected with mercuric chloride. While the selection of doses is such that exposure produces no overt signs of toxicity, Brown Norway rats develop an immunologically-mediated disease characterized by T-cell-mediated polyclonal B-cell activation, autoantibodies to laminin, collagen IV, and other components of the glomerular basement membrane similar to human autoimmune glomerulonephritis. Numerous mouse strains have also been used to evaluate the development of autoantibodies following exposure to mercury, gold, and cadmium (Selgrade et al., 1999). Other examples of xenobiotics that have been demonstrated to be associated with autoimmune disease will be provided below.

Molecular Biology Approaches to Immunotoxicology

As in all of the biological sciences, the continuing evolution of molecular biology-based methods and technologies have vastly expanded the tools available to immunotoxicologists. In general, molecular biology approaches have been thus far employed primarily in the investigation and elucidation of mechanisms of immunotoxicity rather than for identifying immunotoxicants. As these approaches become more refined and sophisticated with time, their application will surely expand. Presently, the primary application of molecular biology in immunotoxicology has been to identify genes whose expression has been altered by a xenobiotic, often termed gene expression profiling, and/or to quantify the magnitude to which gene expression has been changed due to some treatment. As already discussed, methods for assessing changes in gene expression have been particularly useful for studies of the immune system due to the fact that many of the immunological mediators produced by leukocytes (eg, cytokines, chemokines, and immunoglobulins) are regulated transcriptionally (ie, synthesized and secreted on demand) rather than being maintained in cells as stored products. Toward this end, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) has been the principal method currently used to assess changes in mRNA levels for specific genes in tissues and cells. With the development of real time RT-PCR, accurate quantification of gene-specific mRNA levels is readily achieved through the analysis of PCR kinetics by detecting products as they accumulate in “real time” through the application of fluorogenic probes or double-stranded DNA binding dyes. Significant advantages of RT-PCR over traditional methods for analysis of mRNA levels, principally Northern blotting, includes accuracy, sensitivity, markedly less RNA required to conduct the analysis, and less time required to complete the analysis. Recent advances in thermocycler technology and the availability of commercial reagents currently permit the quantification of up to 384 genes from one RNA sample by employing what are termed “low-density” arrays. The technology takes advantage of plates comprised of 384 wells that have been precoated with primers for up to 384 specific genes of interest.

Another approach for conducting gene expression profiling by assessing changes in mRNA levels for multiple genes simultaneously has been through the use of cDNA microarrays. The approach facilitates the analysis of hundreds to tens of thousands of genes from a single RNA sample. The principal advantage of microarrays is the ability to assay mRNA levels for a large number of genes simultaneously. The primary disadvantages are the cost associated with microarray analysis, the complexity associated with data analysis and bioinformatics, and the sensitivity of the methodology to assess moderate changes in mRNA levels. In spite of these challenges, the application of microarray analysis in immunotoxicology has been increasing (Luebke et al., 2006b).

A routinely employed methodology for characterizing effects on gene transcription has been the use of reporter assays. Reporter assays are being widely used to discern whether xenobiotic-induced changes in mRNA levels for specific genes are due to alterations at the level of transcription versus mRNA stability. Likewise, reporters can be used to characterize the effects of xenobiotics on specific transcription factors acting through defined regulatory elements. The approach involves construction of a DNA plasmid possessing the 5′ untranslated regulatory region of the gene of interest that has been ligated to the translated region of a reporter gene. Commonly used reporter genes are typically enzymes, since their expression can be easily assayed. Moreover, studies in mammalian systems commonly employ reporter genes of insect or bacterial origin, thus eliminating the need to differentiate between endogenous and ectopic expression. The most widely used reporter genes are firefly luciferase and bacterial β-galactosidase. Most often, reporter assays are performed by transient transfection into cell lines. This approach has been extensively used to study the effects of leukocyte activation stimuli and xenobiotics on the regulation of promoter and enhancer regions of cytokine and Ig genes. In spite of the important mechanistic information that can be gained from these types of studies, significant challenges often arise in utilizing reporter assays to study leukocytes. Transfection of primary leukocytes, especially lymphocytes, yielding both high transfection efficiency and good cell viability is extremely difficult. An additional complicating factor concerns the fact that lymphocytes can only be maintained viable in culture for short periods (approximately 24 hours) in the absence of activation, which limits the duration the cells can be given to recover after transfection. Likewise, it is not uncommon for T- and B-cell derived lines to be resistant to transfection in spite of the many commercial transfection reagents presently available and new refinements made to electroporation instruments. In most cases, transfection conditions must be optimized for transfection efficiency and cell viability for each cell line or preparation using a control plasmid (ie, a plasmid possessing strong constitutive expression).

RNA interference employing small interfering double-stranded RNAs (siRNA) to achieve posttranscriptional gene-specific silencing is widely applied to investigations aimed at elucidation of mechanism of immunotoxicity. RNA duplexes of 21 nucleotides possessing two nucleotide 3′ overhanging ends once transfected into the cell incorporate into a nuclease complex known as the RNA-induced silencing complex (RISC). Through an ATP-dependent mechanism, the duplexes are unwound and separated into their sense and antisense strands. RISC is directed by the unwound antisense siRNA strand homologous to the target mRNA, which then undergoes cleavage by an endonuclease termed “Slicer.” The ensuing destruction of the target gene mRNA results in the posttranslational silencing of gene expression. This approach provides a rapid mechanism by which the involvement of a specific gene product can be linked to biochemical and functional events induced by a xenobiotic in a given cell type, including leukocytes (Sandy et al., 2005). Major challenges in the application of this methodology can exist. First, identification of an effective siRNA is critical, as not all sites of a given mRNA being targeted will result in gene silencing. Numerous strategies and criteria have been developed to assist in the rationale design of siRNA (Jia et al., 2006; Khvorova et al., 2003; Schwarz et al., 2003). Second, as with the transient transfection of reporters discussed above, leukocytes, and especially lymphocytes, are often resistant to transfection resulting either in poor delivery of the siRNA into the desired cell preparation or low viability. Since siRNA are most often used in cell lines, the doubling rate of the cells can significantly affect the level of knockdown achieved based on the fact that each time a cell undergoes replication, the cellular siRNA is diluted by half. The half-life of the protein can also significantly impact on the level of knockdown achieved even if large amounts of siRNA are delivered into the cells. Also, genes that are highly expressed can be difficult to effectively knockdown due to the large amount of mRNA that must be destroyed. Likewise, genes with very low expression can be difficult to silence as the odds of the RISC complex finding rare mRNAs in the cell may be low. Lastly, antibodies must be available to the gene product being targeted for knockdown so that the magnitude of knockdown can be confirmed at the protein level. In spite of these many potential challenges, the ability to effectively achieve gene-specific silencing, in some cases of a magnitude greater than 90%, is a remarkably powerful tool when elucidating biological mechanisms for which knockout mice are not available.

A relatively new area of research that has not yet had broad application to the field of immunotoxicology is proteomics. Proteomics can be defined as research that aims to identify, quantify, and classify the function of proteins produced by given genomes. In addition to the systematic identification and characterization of proteins, a potentially important application of proteomics is in the characterization of protein–protein interactions, especially as they relate to the elucidation of signal transduction mechanisms. Strategies have been proposed for the functional analysis of signal transduction, termed phosphoproteomics (Morandell et al., 2006). Unfortunately, a major drawback in the application of proteomics includes the lack of an experimental platform currently available to systematically measure the diverse properties of proteins in a high-throughput approach. Presently, most proteomic methods are based on mass spectrometry and require expensive instrumentation, information-technology infrastructure, and highly specialized personnel. In addition, the complexity of the proteome is enormous as illustrated by Aebersold when he compared the human genome, which is comprised of approximately 30,000 genes, to human serum, which alone has been estimated to contain approximately 500,000 different protein species (Aebersold, 2003). Nevertheless, the application of proteomics to study proteins is occurring routinely in cell-based systems.

As stated above, the use of molecular biology based methods in immunotoxicology to date has been primarily for understanding mechanisms of action of known immunotoxicants. Much research is currently focused on quantifying changes in mRNA levels (eg, cytokines) and elucidating mechanisms responsible for those xenobiotic-induced changes. Similarly, changes in mRNA expression profiles may be useful as possible biomarkers of exposure. Proteomics and genomics, combined with bioinformatics, are making it possible to evaluate chemically induced alterations in entire pathways and signaling networks. The utility of molecular biology tools such as proteomics and genomics in elucidating mechanisms of action is obvious. More challenging will be the application of these tools for identifying new or suspected immunotoxicants (Luebke et al., 2006b).

Mechanistic Approaches to Immunotoxicology

Once an agent has been identified as being an immunotoxicant, it may be necessary to further characterize the mechanism by which it exerts its effects on the immune system. Toward this end, a unique aspect of the immune system that greatly facilitates investigations aimed at delineating the underlying mechanisms of action is the ability to study the immune system in the intact animal as well as remove immune cells from the intact animal and have them function in vitro. Using a combination of these two approaches, a general strategy has been successfully employed by a wide number of laboratories for characterizing mechanisms of immunotoxic action by xenobiotics and involves the following steps: (1) identifying the cell type(s) targeted by the agent; (2) determining whether the effects are mediated by the parent compound or by a metabolite of the parent; (3) determining whether the effects are mediated directly or indirectly by the xenobiotic; and (4) elucidating the molecular events responsible for altered leukocyte function. The significance of each and the experimental strategies typically employed are discussed below.

Identification of the Cell Type or Type(s) Targeted

With few exceptions, the first objective toward elucidation of the mechanism(s) responsible for immunotoxicity is the identification of the cell type(s) affected by the xenobiotic. The rationale is that the immune system is comprised of a wide variety of cell types with broad, and often, overlapping effector functions. Therefore, identification of specific cell type(s) affected allows for the selection of appropriate approaches and techniques to employ to further elucidate the mechanism of action.

The approaches available for the identification of the cell types targeted by a specific agent are numerous but typically originate with the employment of one or more of the functional assays within the immunotoxicology tier testing battery (see the “The National Toxicology Program Tier Approach” section later in this chapter). Each of the functional assays in the immunotoxicology tier testing battery provides information on accessory and/or effector cell function. For example, the AFC response to the T-cell-dependent antigen, sRBC, requires macrophages and CD4⁺ T cells (Th cells) to function as accessory cells, and B cells to serve as the effector cells. Therefore, if any one of these three cell types is adversely affected by an immunotoxicant, changes in the magnitude of the AFC response would be observed as compared to control (ie, enhancement or suppression). Similarly, the CTL response requires APC and CD4⁺ T cells (Th cells) as the accessory cells and CD8⁺ T cells, which are the effector CTL. Functional assays can be further refined to provide additional information concerning the targeted cell types by employment of various defined antigens requiring different cellular cooperativity to elicit an effector response. The AFC response has been especially useful in this respect. In addition to using a T-cell-dependent sensitizing antigen such as sRBC, T-cell-independent antigens can be employed including dinitrophenyl-ficoll or trinitrophenyl-LPS, which require macrophages, but do not require T cells, to elicit an antibody response. Likewise, B cells can be driven to differentiate into AFC in the absence of accessory cells using polyclonal B-cell activators such as LPS. By employing the three different types of antigens in order to characterize a xenobiotic, a profile of activity emerges that can distinguish between affects on the B cell, T cell, and APC.

As described in the examples above, when accessory cells are required in the elicitation of an immune response, it is often difficult to discern whether alteration of an immune response is due to the xenobiotic targeting the effector cell population or one or more of the accessory cell populations. Under such circumstances, another strategy utilized for identifying the cellular targets is a cell fractionation–reconstitution approach. Specifically, leukocytes can be isolated from treated and vehicle control animals, typically mice, and fractionated into their respective populations. The fractioned cell populations from vehicle and treated animals can then be reconstituted in various combinations to be used in functional assays to determine the population of cells that has been altered. A comprehensive discussion of the various methods that can be used to fractionate leukocyte populations and subpopulations is beyond the scope of this chapter; however, two of the most common approaches to fractionate leukocyte populations are briefly described here. The first is by cell sorting using flow cytometry. The primary advantage of this approach is that it yields an exceptionally high purity of cell populations and subpopulations. The primary disadvantages include: (1) access to a high-end flow cytometer; (2) cost of reagents and trained personnel capable of operating a flow cytometer; (3) practical limitations concerning the total number of cells that can be collected within a reasonable period of time due to the rate at which the instrument analyzes and collects the cells; and (4) positive selection (ie, the cells are bound by a cell-specific antibody) is used for identifying the desired cell population being collected. The second and more commonly used approach utilizes antibodies directed at surface antigens unique to specific leukocyte populations and subpopulations that have been covalently conjugated to magnetic beads. Using the conjugated magnetic beads and a magnet, large numbers of highly pure cell populations can be isolated rapidly, by positive or negative selection, without requirements for expensive instrumentation. As with the cell sorting approach, purified population of cells isolated from vehicle and treated animals can be isolated and reconstituted in various combinations for evaluation in functional assays.

Determination of Whether Immunotoxicity is Mediated by the Parent or by a Metabolite of the Parent Compound

A critical aspect in the elucidation of the mechanism of toxicity for any compound, regardless of the target or tissue is whether the adverse effects are mediated by the parent form of the compound or its metabolite. An understanding of the role of metabolism is especially critical when studying immunotoxicants as it will dictate the experimental approaches that can be utilized. In general, leukocytes possess very modest levels of drug-metabolizing enzymes, especially those within the cytochrome P450 family. Therefore, those agents that are metabolically bioactivated to an immunosuppressive form will in most cases not exhibit their immunotoxic profile of activity when added directly to cultured leukocytes. In fact, an important indication that metabolic activation may be a requisite mechanistic event is the observation that an agent is immunotoxic following in vivo administration while exhibiting no immunotoxic activity when added directly to cultured leukocytes. An example of such an agent is the therapeutic agent, cyclophosphamide. When administered in vivo, cyclophosphamide is a potent immunosuppressant that preferentially suppresses humoral immune responses while having no effect when directly added to cultured leukocytes.

In order to assess whether metabolic bioactivation is required for immunotoxicity, several different approaches can be employed. One approach is to determine whether pre- or co-treatment with either an inducer or an inhibitor of the enzymes known to be involved in the metabolism of the agent modify the immunotoxicity produced in vivo. Similarly, in vitro approaches have also been used to assess the role of metabolism for an immunotoxicant. Specifically, these approaches utilize various in vitro metabolic activation systems such as S9 liver homogenates or isolated liver microsomes, which can activate the xenobiotic when incorporated with leukocyte cultures. Alternatively, freshly isolated primary hepatocytes can be cocultured with leukocytes in the presence of the xenobiotic. Although primary hepatocytes most closely simulate the metabolic activity observed in vivo, this approach is also the most technically challenging since the approach is critically dependent on the isolation of viable and metabolically active hepatocytes. Cyclophosphamide is typically employed as a positive control in all three of the aforementioned in vitro activation systems to confirm metabolic activity. The metabolic activation systems discussed above can also be employed for conducting mechanistic studies in vitro that cannot be performed in the intact animal to further characterize immunotoxicants requiring metabolic bioactivation.

Determination of Whether the Effects are Mediated Directly or Indirectly by the Xenobiotic or a Metabolite of the Xenobiotic

In most instances, immunotoxicants mediate their effects by interacting directly with the immune system. These direct actions may include structural alterations in lymphoid organs or on the cellular composition of lymphoid organs, on the expression of regulatory molecules on the immune cell surface, and/or by altering intracellular biochemical or molecular events (Table 12-8). However, some xenobiotics mediate changes in immune competence through an indirect action on the immune system. Under these circumstances, changes in immune competence are mediated through the release of an immunomodulatory factor resulting from the actions of the immunotoxicant on cells or tissues other than the immune system. One tissue most often implicated in indirectly modulating the immune system is the liver, as it is the source of a broad and extremely diverse group of proteins, including acute-phase proteins. Acute-phase proteins are a family of serum proteins produced by the liver in response to inflammation or infection (see the “Inflammation” section). Acute-phase proteins are believed to have evolved as part of an immediate survival response to systemic infection. In general, acute-phase proteins are associated with downregulation of the immune system and are therefore believed to play a role in maintaining immune homeostasis. The acute-phase proteins most extensively characterized with respect to immune-modulating properties include α-fetoprotein, serum amyloid A, and C-reactive protein. Another important group of regulatory proteins involved in indirectly modulating the immune system are those that contribute to the repair and regeneration of the liver after acute injury; specifically, TGF-β1 and hepatocyte growth factor. A second example of indirect actions on the immune system is stress as well as agents that alter the regulation of the hypothalamic–pituitary–adrenal axis, thus leading to changes in hormonal homeostasis. Deregulation of hormonal homeostasis, especially increased circulating levels of glucocorticoids, can markedly decrease immune competence.

The elucidation of indirect mechanisms of action by an immunotoxicant can be challenging by virtue that the effect is indirect and mediated by one or more circulating immunomodulatory serum factors. The involvement of an immunomodulatory serum factor can be partially deduced by two distinguishing features. First, the profile of immunotoxicity observed after in vivo administration of the causative agent is different from that produced by its direct addition to leukocytes in culture. Second, the profile of immunotoxicity observed after in vivo administration of the agent can be mimicked by direct addition of serum from the treated animal to naive leukocytes in culture. Third, the involvement of a metabolite of the parent form of the chemical has been ruled out. Confirmation of the involvement of a specific serum factor is most often accomplished in vitro by abrogating the immunotoxic activity produced by serum from treated animals using neutralizing antibodies directed against the suspected serum factor.

Elucidation of the Molecular Mechanism

Numerous methodologies are available to evaluate cellular and molecular mechanisms of action and those continue to increase with the availability of new “omics” technologies, new animal models including transgenic and knockouts, and an ever-expanding list of reagents and molecular probes. Due to the broad nature of this topic area, this section is devoted to a general discussion of considerations and strategies aimed at the elucidation of molecular mechanisms. The discussion will be directed at B cells and T cells for illustrative purposes, but is certainly applicable to other cell types comprising the immune system.

When considering the molecular mechanism by which a xenobiotic alters the function of a mature lymphocyte, a practical strategy is to first identify at which stage of leukocyte function the agent is acting, antigen recognition/signaling through the antigen receptor, activation, proliferation, or differentiation. As discussed in the earlier sections of this chapter, lymphocytes have evolved a number of specialized mechanisms by which B and T cells recognize antigen. Common to both cell types is that immediately after recognition of an antigen, there are a number of biochemical events triggered, including changes in protein phosphorylation and activation of a variety of kinases, fluxes in ions, and changes in the level of cyclic nucleotides. Because lymphocytes undergo a rapid and robust rise in intracellular calcium almost immediately after antigen–receptor binding, changes in calcium flux prior to and immediately following stimulation by an antigen provide important insights into whether an agent alters the most proximal stage of leukocyte function. Similarly, changes in the phosphorylation status of the intracellular domain of membrane-associated proteins (CD3 complex, CD4 and CD8 on T cells, and CD79, CD45, and CD22 on B cells) and the kinases that mediate the phosphorylation (eg, lyk and fyn) can be investigated to study the most proximal events associated with triggering of antigen receptors. Polyclonal activators are often employed to simultaneously activate large numbers of lymphocytes facilitating the evaluation of changes in these early biochemical events. For B cells, antibodies directed against the BCR in combination with CD40L can be used. Conversely, for T cells, antibodies directed against the TCR/CD3 complex and the coreceptor CD28.

The stage termed lymphocyte activation is functionally defined and refers to that period that begins when the T cell has initiated a response to an activation stimulus and ends when there is robust transcription of the T-cell autocrine/paracrine growth factor, IL-2 and upregulation of proteins on the cell surface including CD40L, CD69, and CD25 (alpha chain of the IL-2 receptor). In B cells, it is the period that begins with the cell responding to an activation signal culminating in the upregulation of activation markers CD80 (B7.1), CD86 (B7.2), CD69, and increased MHCII. All of the aforementioned changes associated with B-cell and T-cell activation, including IL-2 synthesis, can be readily monitored and quantified in individual cells by flow cytometry. Investigations directed at elucidating the molecular events deregulated by xenobiotics during lymphocyte activation represent one of the most intensively studied areas presently in immunotoxicology as many different immunotoxicants affect this stage of lymphocyte function.

Clonal expansion is a critical phase of the immune response as it insures that a sufficient number of antigen-specific B- and T-effector cells are generated to respond effectively to combat a pathogen. In light of the importance of clonal expansion in mounting an effective immune response, the effect xenobiotics exert on lymphocyte proliferation has been widely used to determine whether a xenobiotic has immunotoxic properties. As with measurements directed at other stages of the immune response, it is often convenient to employ polyclonal B- and T-cell activators for investigating effects on proliferative responses. Due to a significant increase in the understanding of the cell cycle, coupled with the availability of reagents for measuring specific proteins involved in the regulation of cell cycle, it is now possible to functionally characterize and dissect the mechanism and specific intracellular targets affected by xenobiotics that disrupt lymphocyte proliferation. Measurements of changes in cell-cycle-associated regulatory proteins are most commonly achieved by employing flow cytometric approaches.

The final stage of the immune response involves differentiation into a mature effector or memory cell. For B and T cells, terminal differentiation into effector cells can be best assessed by measurements of effector function in conjunction with the upregulation and downregulation of proteins on the cell surface. Antibody production and upregulation of CD138 (also known as syndecan-1) are most often used as indicators of B-cell differentiation into effector cells while either cytokine production (Th cells and CTL) or lysis of target cells expressing viral or tumor proteins (CTL) are used to indicate T-cell differentiation. A number of commonly used immune function methods for assessing B- and T-cell effector functions were described earlier in this section.

Once the specific stage of lymphocyte function being altered by a specific xenobiotic has been established, experiments can be designed to identify the specific intracellular proteins affected by the xenobiotic and the molecular mechanism by which it is modulated. Cell line models can be invaluable tools for studies involving cell signaling and gene regulation.

Cell Line Models in Immunotoxicology

Cell line-based models are being widely used for identifying agents possessing the potential of producing immunotoxicity as well as for conducting in-depth investigations aimed at elucidating molecular mechanisms for immunotoxicity. Significant advantages and disadvantages exist when applying cell line-based models, some of which are unique to investigations of the immune system. A brief discussion of the strengths and limitations of cell line-based models in the context of studies of immunotoxicants is provided below.

Although there are many advantages to cell line-based models, the most important characteristic is that all of the cells are derived from the same clone thus providing a homogenous cellular preparation. The homogeneity of the model is especially useful for studies directed at characterizing signal transduction pathways as well as gene expression profiling due to the greater likelihood of obtaining reproducible results. There are a number of advantages of cell line-based models that are especially useful in immunotoxicology. Primary leukocytes whether isolated from blood or lymphoid organs are highly heterogeneous in their cellular composition. Purification of these primary cell preparations into specific cell types is expensive, can be labor intensive and with most isolation methods typically yielding 50% to 75% efficiency (ie, 25%–50% of the desired cells are lost during the purification process). Purification efficiency can become a critical issue when utilizing small rodents such as mice where the number of animals per assay can be significantly increased due to the loss of cells being recovered in the cell isolation procedure. When employing cell line models, typically there is no limitation on the number of cells available for a given study. Another important consideration in the case of primary lymphocytes is that they are only viable in culture for relatively brief periods of time (approximately 24 hours) in the absence of an activation signal. Therefore, extended preincubation periods in culture with an immunotoxicant or other response modifiers that do not activate primary lymphocytes cannot be performed. Primary leukocytes, especially lymphocytes, are also difficult to transfect, often yielding poor transfection efficiency and/or viability. The combination of poor transfection efficiency and limited culture duration in the absence of activation make reporter assays, transient ectopic gene expression, and utilization of siRNA to silence gene expression challenging in primary lymphocytes. Conversely, cell line-based models, especially of the lymphoid lineage, in most cases can be readily transfected with suitable efficiency and viability to conduct reporter assays, ectopic gene expression, and gene silencing with siRNA. It is noteworthy that primary cells of the myeloid lineage are more suited for long-term culture- and transfection-based experimental approaches as they have less stringent requirements for activation to be maintained in culture for extended periods. In such instances, it may be more advantageous to employ primary cells. Lastly, cell line-based models are also now being widely adapted for high-throughput screening due to the reproducibility of results obtained with these models and the ease in which cell lines can be maintained and manipulated.

In spite of the advantages discussed above, there are numerous disadvantages and limitations inherent in utilizing cell line models for characterizing immunotoxicants. The most important consideration when utilizing cell line models is that by definition, a cell line is an abnormal population of cells that has undergone a change rendering it capable of dividing indefinitely in culture. Since cell lines are continuously dividing, in most cases they are not good models for studying immunotoxicants that act by altering cell proliferation and/or regulators of the cell cycle. The aberrant nature of cell lines may also extend to a loss of function through one or more of its cognate receptors. Lastly, it is critical that cell lines are carefully monitored and characterized for changes in function and morphology after repeated passage in culture. Additional important considerations when selecting a cell line for mechanistic studies are the capacity of the cell line to perform a given effector function and the stimuli to which the model will respond. Toward this end, a number of cell lines have been extensively characterized and widely utilized that are capable of induced effector functions including cytokine production, antibody secretion, and release of a wide variety of mediators. Table 12-9 provides examples of some commonly used cell lines in immunotoxicology. As discussed above, with some cell lines, induction of an effector function may only be achievable by using pharmacological activators (eg, phorbol ester plus calcium ionophore) that bypass extracellular receptors or by agents that are polyclonal activators (eg, LPS) that do not activate directly through antigen receptors. Again these are characteristics of the models that need to be considered in the context of how the models will be used and for what specific purpose. In most cases, the utilization of cell line-based models can be extremely useful but results should always be confirmed, when possible, in primary leukocytes since cell lines are aberrant models.

Animal Models: Transgenics, Knockouts, and Humanized/Severe Combined Immunodeficient

The developments in molecular biology have not only permitted the evaluation of specific genes or arrays of genes but have also allowed for the manipulation of the embryonic genome, creating transgenic, and knockout mice (reviewed by IPCS, 1996). As a consequence of transgenic technology, complex immune responses can be dissected into their components. In this way, the mechanisms by which immunotoxicants act can be better understood. Mice engineered to express nonself genes (eg, human MHCI) or overexpress self genes (eg, constitutively active transcription factors) are termed “transgenic,” and can be used to address the role of a certain protein in immunotoxicology. On the other hand, mice lacking certain proteins (eg, receptors, transcription factors, or cytokines) are termed “knockouts,” and can be used for similar mechanistic studies. Numerous transgenic and knockout mice have been created and are available to investigators worldwide. Of particular interest and potential utility in the area of immunotoxicology is what has been termed “humanized” mice. Humanized mice refer to immune-deficient mice that have been reconstituted either with human HSC to support a fully human immune system, or with mature cells to evaluate immune regulation, hematopoiesis, hypersensitivity, and autoimmunity. Severe combined immunodeficient (SCID) mice, which are mouse T- and B-cell deficient due to a VDJ recombination defect, are a major strain into which human immune cells/system are established. In addition, SCID mice are backcrossed with other strains in order to eliminate various arms of the immune system (eg, β2M^null/SCID eliminate MHCI interactions) prior to reconstitution with human cells (reviewed by Thomsen et al., 2005). Although their use in mechanistic studies is obvious, SCID/hu mice have also been utilized for evaluation of efficacy of antiviral drugs for HIV/AIDS (reviewed by Taggart et al., 2004). There are still considerations, however, for broader application of these humanized mouse models for immunotoxicological assessment of compounds. For instance, one must consider how these animals compare with standard animal models with respect to time course of action, dose response, pharmacokinetics, and other factors of chemical toxicity. With the hope that these humanized mouse models can be used to identify new or suspected immunotoxicants, and mechanisms of action, one must also consider whether they are as sensitive, predictive, and/or cost-effective as traditional animal models.

Approaches to the Assessment of Human Immunotoxicity

As emphasized throughout this chapter, animal models have been extensively utilized to characterize immunotoxicity, and it is widely recognized that chemicals that produce immunotoxicity in animals have the potential to produce immune effects in the human population. The increasing emphasis on human risk assessment requires that those potential health hazards be identified whenever possible. In spite of the significant homology of the rodent and human immune systems which has facilitated our ability to make decisions on the immunotoxic potential of test materials based on the results of these experimental models (House, 1997), there is also an increasing recognition of distinct, although often subtle, differences in the composition of the immune system between animal species. These differences have primarily come to light in studies of leukocyte subpopulations, the phenotypic markers they express, and by the regulatory factors they secrete. Whether these differences in immune system composition result in significant differences across species in sensitivity to immunotoxicants is unclear. It is important to emphasize that to date there are no known cases of significant human immunotoxicity that has resulted due to false-negative results from rodent-based immunotoxicity testing. In contrast, there is extremely limited human immunotoxicology data on test materials other than those directly intended for human immunotherapy.

One of the obvious and most important goals of an experimental immunotoxicity testing strategy is to enable the best extrapolations between the results generated in the animal models and the potential risk of immunotoxicity in humans. A parallelogram approach has been used to assess relationships between animal data and human data (Selgrade, 1999; van Loveren et al., 1995) and an adaptation of which is presented in Fig. 12-21. The overall strategy is to utilize in vivo and in vitro data from animal studies and in vitro data using human leukocytes in order to predict the in vivo effects of an immunotoxicant in humans. This approach has been used to extrapolate animal to human data in an initial quantitative assessment of the risk for deleterious effects of UV radiation (van Loveren et al., 1995) and of the immunotoxicity associated with exposure to bis(tri-n-butyltin)oxide (van Loveren et al., 1998). A parallelogram approach has also been used to propose that data for ozone suggest that effects of in vivo human exposure to phosgene on alveolar macrophages phagocytosis may be predicted based on effects of in vitro exposure and in vivo animal data (reviewed by Selgrade et al., 1995).

In spite of these successful applications of the parallelogram approach, it is recognized that extrapolations of alterations in immune function observed in animals to human health is associated with various uncertainties (House, 1997). For example, experimental animals are often inbred, which certainly lessens interanimal variability thereby simplifying the statistical evaluation of observations. Additionally, laboratory studies in rodents are highly controlled for environment, diet, and health status. In contrast, humans are a highly outbred species with a high degree of interindividual variability in immune response, and any human study must take into account extreme variability in all of the controlled parameters. In fact, it has been estimated that the overall immunocompetence of the individual is affected by age, gender, genetic factors, use of certain medications, drug/alcohol use, smoking history, stress and nutritional status, and that these factors can account for variability of more than two standard deviations in the “normal” human population (Luebke et al., 2004). Luster et al. (2007) have speculated that the major gap in clarifying the shape of the dose–response curve (ie, between immune response and disease) is a lack of large-scale epidemiological studies in populations with mild-to-moderate immunodeficiency that have been monitored for immune system parameters and clinical disease taking into account potential complications by nonimmune factors which can affect infectious disease incidences.

Nonetheless, there is a clear association between suppression of immune function and an increased incidence of infectious and neoplastic disease in humans (reviewed by Biagini, 1998). However as noted above, a major limitation of immunotoxicity risk assessment has been the lack of human data (Descotes, 2006). In the context of xenobiotics, the majority of such data comes from immunotherapeutic drugs, which were intentionally designed to influence the immune system. The aforementioned association has been established primarily with a standard battery of parameters used extensively in clinical immunology studies, including routine complete blood counts and differentials, phenotypic characterization of surface markers and more detailed analysis of cellular subsets using a flow cytometer, lymphoproliferative assays (ie, using a number of stimuli including mitogens such as Con A and PHA, recall antigens such as tetanus toxoid, anti-CD3 monoclonal antibody, and allogeneic lymphocytes in an MLR), and DTH responses using a test panel of recall antigens. In contrast with the clear association demonstrated for drugs used to deliberately suppress immune function or treat cancer, the assessment of immunotoxicity in humans exposed to potentially immunotoxic chemicals is much more complicated than in experimental animals (reviewed by IPCS, 1996). One of the fallouts of this observation has been the recognition that the historic approaches that have been used in clinical immunology may not have much use in human immunotoxicology. While these endpoints are sufficient to detect immunodeficiencies associated with either congenital disorders or immunosuppressive drug therapy, they do not possess the necessary sensitivity to detect the more subtle consequences of xenobiotic-induced immunotoxicity. Interestingly, Luebke et al. (2004) noted that in contrast to individuals with severe forms of primary or secondary immunodeficiency in whom infections to opportunistic pathogens (ie, members of the herpes viruses family, certain protozoans, Candida albicans, a yeast, and Pneumocystis carinii, a fungus) are seen, humans with low to moderate suppression of immune function are more susceptible to infection with pathogens associated with infections common in the general population (ie, encapsulated bacteria like Streptococcus, and influenza viruses). As a result, several proposals have been put forth to reevaluate the way that we measure immune function in humans. Most of these testing strategies have incorporated plans to measure the primary response to a new antigen, and several of these testing strategies have recommended using newly developed vaccines as the new antigen (van Loveren et al., 2001). Vaccine response rates have been successfully used to characterize the impact of chronic stress on human immunocompetence (Glaser et al., 2000; Kiecolt-Glaser et al., 1996). Importantly, in spite of the limitations in human data, there is no question that environmental chemicals are associated with immunotoxicity in humans (Selgrade, 2007; Veraldi et al., 2006).

It is anticipated that the development, validation, and utilization of more predictive methods to assess immunotoxicology in humans will be increasingly important in the future. Notably, the application of the parallelogram approach—by definition—requires the ability to measure immunotoxic effects using in vitro approaches with human lymphocytes. As described in another section of this chapter, human lymphocytes have been previously applied to study the effects of dibenzo-p-dioxins (Lu et al., 2011, 2010; Wood and Holsapple, 1993; Wood et al., 1993). Partly because of the recognition that investigations conducted exclusively using in vitro and in vivo animals models may not always be predictive of toxicity in humans (Selgrade, 1999; Vos and van Loveren, 1998), and partly because of the tremendous increase in the development of new drugs that are known to, or suspected of targeting the human immune system as discussed elsewhere in this chapter, there has been increasing interest in establishing predictive in vitro methods using human lymphocytes. A polyclonal IgM AFC response model was developed using the combination of CD40 ligand (CD40L) plus recombinant cytokines because it effectively drives both primary human and mouse B cells in a physiologically relevant manner to mimic T-cell-dependent antibody responses in vivo (Lu et al., 2009). A second assay, the human lymphocyte activation (HuLa) assay, was developed that measures an influenza antigen-specific response using frozen-stored human peripheral blood mononuclear cells (PBMC) (Collinge et al., 2010). Although proliferation is the primary endpoint in the HuLa, flow cytometry approaches can be used to characterize the proliferating lymphocyte subsets (eg, CD4⁺ and CD8⁺ T lymphocytes and B lymphocytes), and ELISPOT can be used to measure influenza-specific AFC. A third in vitro model, termed MIMIC (Modular IMune In vitro Construct), uses the circulating immune cells of individual donors to recapitulate each individual human immune response by maintaining the autonomy of the donor and represents a physiologically accurate model of the human immune system (Higbee et al., 2009). Interestingly, a version of MIMIC was recently used to investigate the immunopotency of a number of TLR agonists on DC generation, maturation, and antigen-presenting capacity (Ma et al., 2010). Importantly, all three of these models have been used to characterize the dose–response characteristics and potency of a number of immunosuppressive drugs and nondrug chemicals.

For illustrative purposes it is useful to briefly compare and contrast the in vitro T cell-dependent IgM AFC response, which uses mouse spleen cells and is often considered one of the “gold standard” assays for characterizing immunotoxicants, to the human CD40L-induced IgM AFC assay, which utilizes purified HPB B cells. Unlike the widely used T-cell-dependent IgM AFC response assay, which uses a heterogeneous leukocyte preparation from the mouse spleen and sRBC as the sensitizing antigen, the CD40L drives IgM production using purified HPB B cells or primary mouse B cells, thus allowing for direct comparisons of sensitivity of immunotoxicants between mouse and human B cells (Lu et al., 2009). It is important to emphasize that there are a number of significant and fundamental differences between the sRBC IgM AFC response and the CD40L-induced IgM AFC response. The most significant is that the CD40L response has no accessory cell involvement (ie, T cells or APC) since CD40L stimulation is provided either through the direct addition of rCD40L or by coculturing B cells with a genetically engineered CD40L-expressing fibroblast cell line (CD40L-L cells) plus recombinant T-cell cytokines. Therefore, the CD40L assay only provides information on B-cell function. In contrast, the sRBC IgM AFC response assay, in addition to B cells, requires functional T cells and APC for IgM production. From the perspective of hazard identification, the sRBC assay has greater capacity to detect immunotoxicants since in addition to B cells, the interactions of two other cell types and their consequent accessory cell functions are required in comparison to the CD40L assay. However, mechanistically, modulation of the CD40L-induced IgM response provides definitive identification of B-cell immunotoxicants and is an assay system in which mechanisms of B-cell immunotoxicology can be investigated in the absence of potential confounders associated with the presence of other cell types. In fact, using CD40L plus cytokines, the effects of immunotoxicants on B cells can be studied temporally throughout the entire continuum from activation to differentiation using flow cytometry. Another significant difference between the sRBC and the CD40L-induced IgM response is that the former is an antigen-specific response and the latter is not. In fact, presently there is no established antibody assay that employs human primary leukocytes to drive a primary, antigen-specific, T-cell-dependent antibody response. One of the major challenges in establishing such an assay is that there are very few circulating naive B cells present in peripheral blood and even fewer that possess BCRs specific for any given antigen. Because all B cells constitutively express CD40, the CD40L-induced IgM response circumvents the requirement for antigen specificity and induces a polyclonal B-cell response. Antigen-specific secondary antibody responses are more feasible, especially in response to recall antigens, such as tetanus toxoid. Because a large portion of the human population has undergone repeated and regular vaccination throughout their life (eg, tetanus), HPB B cells possessing BCRs specific for vaccine-associated antigens in these individuals have been clonally expanded and can be readily activated and quantified in vitro. The disadvantage to this approach is that memory B cells, which are the cells that drive the secondary immune responses to recall antigens, have been found to be less sensitive to modulation by immunotoxicants. In light of this, even the CD40L-induced IgM response has been primarily conducted with naive HPB B cells after removal of memory B cells.

Recently, three extensively studied B-cell immunotoxicants in mice, arsenic, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were evaluated using the CD40L-induced IgM AFC response (Lu et al., 2009, 2010). All three immunotoxicants were found to suppress the CD40L-induced IgM AFC response in mouse splenic B cells with the magnitude of suppression being comparable to that previously reported using the sRBC IgM AFC response (Burns et al., 1991; Holsapple et al., 1986; Kawabata and White, 1989). BPDE and TCDD were also found to markedly suppress the CD40L-induced IgM AFC response in HPB B cells, and with a similar magnitude of suppression being observed between mouse and human B cells. In contrast, arsenic did not suppress the CD40L-induced IgM AFC response in HPB B cells. These limited results using only three B-cell immunotoxicants suggest that the mouse may not always serve as a reliable surrogate for predicting immune toxicity in humans.

As will be discussed in the section “The National Toxicology Program Tier Testing Approach,” a cornerstone of immunotoxicological assessments using rodents has been a tier testing battery of immune function assays capable of evaluating the effects of xenobiotics on a host of different immunological parameters. Such an approach has been necessary due to the diversity of cell types and functions that are involved in maintaining immune competence (Luster et al., 1988). In fact, there is strong historical evidence, from examination of the NTP immunotoxicology database, that no immune function assay or endpoint can reliably identify all immunotoxicants. However, if expanded to two or three key assays, a significant increase in concordance with results from the expanded testing battery can be achieved (Luster et al., 1992). Toward this end, it is conceivable that a multiassay approach, analogous to that currently used for evaluations in the mouse, could be employed for immunotoxicity evaluations in conjunction with human primary leukocytes. Until recently, a significant obstacle to such a strategy has been the absence of an in vitro assay to assess B-cell effector function (Lu et al., 2009). In fact, a number of immune function assays using primary human leukocytes have historically been widely employed such as the MLR, which provides information on CD4⁺ and CD8⁺ T cell function, in the clinical setting to determine histocompatibility between donors and recipients for tissue transplantation. Similarly, B-cell and T-cell lymphoproliferative responses, which assess lymphocyte clonal expansion, have been used for the past several decades as a preliminary approach to assess human lymphocyte function. Collectively, these assays represent a potential foundation for a human leukocyte immunotoxicology testing battery. As described in the “Biomarkers in Immunotoxicology” section, the development of more predictive biomarkers for the immune system that can provide specific information on the impact of changes in human immunocompetence, and on the susceptibility for disease associated with drug or chemical exposure is another area of intense interest. The possibility of developing better translational biomarkers could facilitate the progression from preclinical studies to clinical studies in drug development.

Regulatory Approaches to the Assessment of Immunotoxicity

The maturity and acceptance of any subdiscipline of toxicology can frequently be directly correlated to the level of interest being demonstrated by the regulatory community. This section will provide a brief review of the history of regulatory approaches to immunotoxicity. It is important to note that the regulatory approaches to immunotoxicity have evolved with the science, and the timing of the introduction of specific guidelines has generally reflected the state-of-the-science. Because of the importance of the evolution of the science, this section will begin with an overview of one of the most comprehensive databases for immunotoxicology.

The National Toxicology Program Tier Approach

Although the concept of required immunotoxicity testing is a relatively recent development, the recognition that the immune system is a potential target organ has prompted a consideration of the need to assess the potential of immunotoxicity for some time. All testing strategies to date have recognized the complexity of the immune system as a target organ, and that no single immune parameter can be used with sufficient confidence to test for the hazard of immunotoxicity. Therefore, historically immunotoxicity has been assessed using a battery of assays, which are typically structured in a multi-tiered approach. Importantly, studies conducted by the NTP have indicated that immunotoxicity can be assessed with a finite number of assays. Luster and colleagues (1988) originally described the selection of a battery of tests used by the NTP to screen for potential immunotoxic agents. The result was a tier approach to assessing immunotoxicity, which is summarized in Table 12-10.

Tier I tests provide assessment of general toxicity (eg, immunopathology, hematology, body, and organ weights) as well as some assays to assess immune functional capability (eg, proliferative responses, the T-cell-dependent antibody response assay, measured as AFC, and the NK assay). Tier I was designed to detect potential immunotoxic compounds at concentrations that do not produce overt toxicity. Tier II tests were designed to further define an immunotoxic effect, and included functional tests for CMI (eg, CTL and DTH), secondary antibody responses, enumeration of lymphocyte populations, and host resistance models.

In the original NTP effort, over 50 chemicals were studied, and included a variety of chemical classes, such as catalysts, solvents, dyes, lubricants, pesticides, disinfectants, drugs, food additives, and natural products (Luster et al., 1988). It is important to emphasize that the chemicals selected for study by the NTP were nominated for testing because of a suspicion that they would target the immune system. These studies were conducted in young adult rodents, principally the mouse, and involved comparative studies across multiple labs. The experimental design emphasized that regardless of the specific immune parameters included in a testing strategy, the interpretation for immunotoxicity could only be made in the context of a well-designed study from the perspective of the dose–response relationship. The approach by Luster and coworkers also emphasized the importance of avoiding high doses (ie, triggered less than a 10% reduction in body weight), because these types of exposures will increase the likelihood that indirect mechanisms of immunotoxicity may be involved.

Several testing configurations were ultimately defined that would minimize the number of immune tests needed, yet could still provide a high degree of sensitivity for detecting potential immunotoxicants. These configurations are depicted in Table 12-11. Specifically, the results indicated that none of the assays measured was 100% predictive alone. The top three assays in terms of detecting immunotoxicants were an assessment of lymphocyte subpopulations by flow cytometric analysis (eg, 83% concordance), the T cell-dependent antibody response (eg, 78% concordance), and an assessment of NK cell activity (eg, 69% concordance) (Luster et al., 1992). With regards to the comparison between the two most sensitive assays, it is important to note that a change in flow cytometric analysis of any of the lymphocyte subsets measured, for example, total B cells, total T cells, CD4⁺ T cells, or CD8⁺ T cells, would be sufficient to establish the concordance of flow cytometry, while the concordance of the T-cell-dependent antibody response was determined solely by the number of AFCs. Subsequent analysis indicated that an application of the full extent of the tiered approach was not necessary. The NTP studies indicated that several combinations of only two tests could give >90% concordance, and that a number of combinations of just three tests gave 100% concordance. In this regard, it is important to note that flow cytometric analysis of lymphocyte subsets, and the-T cell-dependent antibody response were consistently involved in both the 2-test concordance of >90%, and in the 3-test concordance of 100%.

Subsequent results also indicated a good correlation between the results from functional tests and host resistance models (Luster et al., 1993), which indicated that the latter were not necessary to adequately identify immunosuppressive potential. The impacts of the NTP database are clear, and one only needs to consider the structure of the immunotoxicity guidelines that have been established to see the impact of the NTP results. Specifically, after several years of international debate, the importance of including functional immunotoxicity assessments in regulatory studies has been emphasized, as opposed to relying solely on histopathology as an indicator of further testing needs. Moreover, as described further below, the specific immune functional tests in most, if not all, immunotoxicity regulatory guidelines reflect the concordance results described above, and include the T-cell-dependent antibody response, immune cell phenotyping by flow cytometry, and the NK cell assay.

While the original NTP studies in the 1980s were intended to develop and validate methods to evaluate modulation of immune function, the emphasis was clearly focused on immunosuppression. The NTP contract to assess the “Potential for Environmental and Therapeutic Agents to Induce Immunotoxicity” is still assessing chemicals for potential immunomodulation at the rate of four chemicals per year for screening (eg, rangefinding) studies and two chemicals per year for definitive (eg, full protocol) studies. Importantly, the current approaches by the NTP reflect the state-of-the-science of immunotoxicology, in that two developmental immunotoxicity studies, two contact hypersensitivity studies and one study assessing the potential of a xenobiotic-associated change in an autoimmune disease model are assessed each year.

In 2009, the NTP provided another critical component to our understanding of xenobiotic-induced immunotoxicity when they released their “Explanation of Levels of Evidence for Immune System Toxicity” (NTP, 2009) as an approach to judging the quality of results in a study purporting to establish xenobiotic-induced immunotoxicity. In the document, which was prepared by a panel of experts representing the academic, government, and industry sectors of the scientific community, the NTP emphasized the importance of recognizing that the “levels of evidence” statements that are described reflected only immunological hazard, and that the actual determination of risk to humans required exposure data that was not covered in the document. Five categories of evidence of immune system toxicity were used to summarize the strength of the evidence observed in each experiment being considered: two categories for positive results (clear evidence and some evidence); one category for uncertain findings (equivocal evidence); one category for no observable effects (no evidence); and one category for experiments that cannot be evaluated because of major design or performance flaws (inadequate study). Each category included one or more bullet points for additional clarification. The category for clear evidence is worthy of specific mention.

Clear Evidence of Toxicity to the Immune System:

• Is demonstrated by data that indicate a dose-related effect (considering the magnitude of the effect and the dose–response) on more than one functional parameter and/or a disease resistance assay that is not a secondary effect of overt systemic toxicity, or

• Is demonstrated by data that indicate dose-related effects on one functional assay and additional endpoints that indicate biological plausibility. (NTP, 2009)

The significance of this category is that clear evidence of immunotoxicity can be established without the need to conduct a disease resistance assay, and that mechanistic data to establish biological plausibility is weighed very heavily. The NTP report also indicates that negative results, in which the study animals do not exhibit evidence of immunotoxicity, do not necessarily imply that a test article is not an immune system toxicant, but only that the test article is not an immune system toxicant under the specified experimental conditions. Finally, the report indicates that positive results demonstrating that a test article causes immunotoxicity in laboratory animals under the conditions of the study are assumed to be relevant in humans, unless data are available which demonstrate otherwise.

Historical Perspective on Regulatory Guidance in Immunotoxicology

The history of regulatory guidance in immunotoxicology was reviewed by House (2005). As noted above, the T-cell-dependent antibody response has been the consensus choice for a functional endpoint to identify immunotoxicity hazard in most, if not all regulatory guidelines. A reasonable question to address is what accounts for the sensitivity, predictivity, and (perceived) utility of the T-cell-dependent antibody response. The simplest answer is something that has been known within the immunology community for over 50 years. The T-cell-dependent antibody response requires the orchestrated coordination and cooperativity of multiple cell types (eg, APCs, Th cells, and B cells at the ultimate effector cell because of their ability to secrete antigen-specific antibodies) and the manifestation of most, if not all of the biological functions important to immunocompetence. As noted throughout this chapter, the discipline of immunotoxicology continues to evolve along with advances in immunology and molecular biology. However, in the context of providing an effective screen for immunotoxic hazard, nothing can compare to the time-tested and mechanistically understood specific antibody response to a well-characterized T-dependent antigen.

The Office of Prevention, Pesticides and Toxic Substances (OPPTS) of the US Environmental Protection Agency (EPA) published guidelines entitled, “Biochemicals Test Guidelines: OPPTS 880.3550 Immunotoxicity” in 1996. These guidelines described the preferred study design for an exceptionally thorough evaluation of the potential immunotoxicity in biochemical pest control agents. This guideline described a panel of tests that included standard toxicology tests as well as immune functional tests assessing both humoral immunity and CMI. OPPTS 880.3550 clearly presented a very comprehensive approach to immunotoxicity; but a second document, “Biochemicals Test Guidelines: OPPTS 880.3800 Immune Response,” was needed to provide the rationale for when these studies should be conducted. The 880 series of immunotoxicity guidelines would arguably detect any type of immunotoxic potential by pesticides. However, the comprehensive nature of these guidelines rendered them prohibitively expensive and time-consuming. The EPA released the “Health Effects Test Guidelines: OPPTS 870.7800 Immunotoxicity” in 1998 (EPA, 1998). These guidelines described the approach to immunotoxicology testing for nonbiochemical agents regulated by the EPA. The testing approach in OPPTS 870.7800 reflected the continued evolution of the science of immunotoxicology and called for a more limited, case-by-case approach than previously described by the earlier more comprehensive guidelines. The cornerstone of OPPTS 870-7800 was a functional test, the primary T-cell-dependent antibody response. If the chemical was shown to produce significant suppression of the humoral response, then surface marker assessment by flow cytometry may be performed. If the chemical was not shown to produce suppression of the humoral response, then an assessment of innate immunity (eg, specifically, the NK cell activity assay) may be performed. The specific criteria for the conduct of these “optional” tests have never been identified. The tests do not represent a comprehensive assessment of immune function but are intended to complement assessment made in routine toxicity testing (eg, hematological assessments, lymphoid organ weights, and histopathology).

Internationally, the Organization for Economic Cooperation and Development (OECD) has not as yet adopted specific guidelines for immunotoxicity assessment. As reviewed by House (2005), the OECD Guideline 407, entitled “Repeated Dose 28-day Oral Toxicity Study in Rodents” while not specific for immunotoxicology, includes a variety of toxicological endpoints that can provide early evidence of immune system alterations. House (2005) also noted at the time that over 10 years earlier, an international immunology working group recommended that functional assessments be included in standard toxicology studies when desired or when suggested by expanded histopathological results on other standard toxicology studies. However, to date, the OECD Guideline 407 has not been modified in accord with these recommendations. Moreover, while the OECD published a template for immunotoxicity studies on its website in 2011, there are still no corresponding OECD Test Guidelines for immunotoxicity.

The earliest immunotoxicity guidelines from the Food and Drug Administration (FDA) were centered on food additives as the “Draft Redbook II” in 1993. This document, although never finalized, contained an extensive description of immunotoxicology testing. In general, the Redbook guidelines reflected the tiered approach to immunotoxicology (Fig. 12-10), as described in greater detail below. Specifically, the Redbook emphasized a stepwise approach that began with expanded studies utilizing data obtained in standard toxicology testing as initial indicators of immunotoxicity. Progressively more complicated immunological tests were prescribed using an approach that was very much case-by-case, with each new level of testing predicated on positive results in the preceding level. The FDA Center for Drug Evaluation and Research (CDER) released its document entitled, “Guidance for Industry: Immunotoxicology Evaluation of Investigational New Drugs,” in 2002 (FDA, 2002). This document is arguably the most comprehensive description of approaches to immunotoxicology. Not only does the FDA CDER “Guidance for Industry” describe the full spectrum of adverse events associated with the immunotoxicology continuum including immune suppression, immunogenicity, hypersensitivity, autoimmunity and adverse immune stimulation, but also provides approaches at the level of specific methodology for evaluating each event. As with the earlier document from the FDA, the new “Guidance for Industry” advocated the use of information derived from standard repeat-dose toxicity studies to provide the earliest indicators of immunotoxicity. The review by House (2005) also addressed the current status of regulatory guidance for biologicals, vaccines, devices, and radiological agents within the FDA.

Internationally, perhaps one of the most significant advances in our approach to the assessment of immunotoxicity with human pharmaceuticals has been a guidance entitled, “S8 Immunotoxicity Studies for Human Pharmaceuticals,” which was prepared under the auspices of the International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceutical for Human Use (ICHS8, 2006). The objectives of this guideline are twofold: (1) to provide recommendations on nonclinical testing approaches to identify compounds which have the potential for immunotoxicity; and (2) to provide guidance on a weight-of-evidence decision-making approach for immunotoxicity testing. The guidance applies to unintended immune suppression and immune enhancement, excluding allergenicity or drug-specific autoimmunity. The ICH S8 guideline is based on a cause for concern approach using a review of factors such as the following: results from Standard Toxicity Studies (STS), pharmacological properties of the drug, the intended patient population, structural similarities to known immune modulators, the disposition of the drug, and clinical information.

As emphasized in the Introduction, immunotoxicity exists in a continuum (Fig. 12-1). By and large, more effort has been invested in the validation of studies to address the immunosuppressive part of the continuum, and there is no doubt that much attention is paid to immune suppression in the majority of guidance documents (House, 2005). Nonetheless, it is important to measure xenobiotic-induced changes in immune function in both directions. As noted by House (2005), it is hypersensitivity that is the most common type of immune modulation in humans resulting from exposure to xenobiotics. One of the most significant advances in our approach to assessing the potential of xenobiotics has been the LLNA. As discussed above, the LLNA was the first assay to be evaluated by the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM). The impact of this decision by ICCVAM is that the LLNA is now an accepted assay and discussed in considerable detail in the OECD 429 Guideline, “Skin Sensitization: Local Lymph Node Assay,” and the EPA document, “OPPTS 870.2600 Skin Sensitization.”

Recent Advances in Regulatory Guidance on Immunotoxicology

There have been two areas over the last few years that have impacted the status of immunotoxicology testing which are either legislative demands or because of the continued evolution in the state-of-the-science.

As noted in the sections above on “Molecular Biology Approaches to Immunotoxicology” and “Mechanistic Approaches to Immunotoxicology”, the use of in vitro and cell-based tissue culture methods have always played an important role in the science of immunotoxicology. Of course, it has always been recognized that the use of these alternative approaches are consistent with the 3Rs of reducing, replacing, and refining the use of whole animal studies. However, recent legislative actions, such as the implementation of Registration, Evaluation and Authorization of Chemicals (REACH) and of the Cosmetics Directive in Europe, have focused renewed and required attention on in vitro approaches to all types of toxicity assessment, including immunotoxicity, especially in Europe (Carfi et al., 2007; Galbiati et al., 2010; Lankveld et al., 2010). Some recent specific examples of the utility of in vitro approaches to assess immunotoxicity include an improved in vitro cytokine release using human PBMC (Kooijman et al., 2010), and some renewed interest (Fischer et al., 2011) in the Mishell–Dutton culture system (Mishell and Dutton, 1967) that was described earlier in the subsection “General Assessment” under the “Methods to Assess Immunocompetence” section. Not too surprisingly, Fischer et al. (2011) conclude that the investigation of in vitro antibody responses is a sensitive and reliable approach for the detection of a compound-specific effect on the immune system. They also conclude that the implementation of this approach in routine toxicology would enable the refinement of existing in vivo studies by reducing the number of animals. However, somewhat surprising is their conclusion that the in vitro Mishell–Dutton culture system for measuring specific antibody responses of primary cells may replace the ex vivo T-cell-dependent antibody response in the future. A significant concern pertaining to the utilization of this strictly in vitro approach is that leukocytes possess remarkably limited drug-metabolizing capability and as such, agents that are activated by metabolism to an immunotoxic form would not be detected utilizing this strategy.

As described in another section of this chapter, the interest in DIT testing has increased significantly over the last few years, with considerable attention being devoted to this area of study by the scientific and regulatory communities on a global scale. While it is important to emphasize that there are currently no regulatory guidelines for either drugs or nondrug chemicals specifically focused on assessment of DIT, but there are some general guidelines included in both developmental and reproductive toxicity (DART) and general immunotoxicology guidance documents. Evaluations of DIT began to be required as part of the US Environmental Protection Agency’s Office of Prevention, Pesticide and Toxic Substances (OPPTS) test guideline for the 2-generation reproductive toxicity study (EPA, 1998), which requires collection of spleen and thymus weights in one pup per sex per litter in F₁ and F₂ weanlings. Another example of this trend in the context of assessing the effects of chemicals on the developing immune system is the paper by Cooper et al. (2006), in which the authors described an expanded one-generation DART protocol that included the assessment of immunotoxicology—among other endpoints—following exposure during critical windows of development. More recently, Vogel et al. (2010) described a modular one-generation reproduction study—that included an immunotoxicity module—as a flexible testing system for regulatory safety assessment, and highlighted the application of such an approach to the EUs REACH program. Based on these kinds of proposed studies, the US EPA and the Dutch authorities convened a working group to revise the OECD 415 protocol to embrace some of the principles articulated by Cooper et al. (2006) and by Vogel et al. (2010). Indeed, in 2010, the OECD released a draft protocol for a revised OECD 415, extended one-generation study (OECD, 2010) that included three cohorts, with the third cohort devoted to an assessment of the potential impact of chemical exposure on developmental immunotoxicity.

In its 2002 guidance for industry, FDA noted that all drugs that are immunotoxic in adult animals and are to be given to pregnant women should be evaluated for DIT (FDA, 2002). Endpoints recommended to be assessed were hematology and lymphoid organ weights on F₁ pups in standard DART studies. More recently, a number of regulatory guidelines have also emerged to address the safety of drugs intended for pediatric indications, including the US FDA Guidance for Industry Nonclinical Safety Evaluation of Pediatric Drug Products (FDA, 2006; Tassinari et al., 2011), and the European Medicines Agency Guideline on the Need for Nonclinical Testing in Juvenile Animals on Human Pharmaceuticals for Paediatric Indications (Carleer and Karres, 2011; EMA, 2008). In Japan, the Ministry of Health, Labor, and Welfare (MHLW) started to prepare their Guideline for Nonclinical Safety Studies in Juvenile Animals in 2010 (Shimomura, 2011). In general, juvenile toxicity studies are conducted to identify potential latent signs of toxicity, to characterize the potential for chronic developmental toxicity that cannot be thoroughly assessed through standard DART protocols, and to ensure that children are not unnecessarily exposed to potentially adverse risk factors. The primary objective of juvenile animal toxicity studies of pharmaceuticals is to obtain safety data, including information on the potential for adverse effects on postnatal growth and development (Cappon et al., 2009). Indeed, the FDA Guidance from their CDER addresses “… the role and timing of animal studies in the safety evaluation of therapeutics intended for the treatment of pediatric patients,” and emphasizes the following premise, “It is thought that organ systems at highest risk for drug toxicity are those that undergo significant postnatal development” (FDA, 2006). Importantly, the immune system was identified in the FDA Guidance as an important endpoint.

While the revised OECD 415 protocol described above has been designed to assess the effects of chemical exposure in rats, more recently, evaluations of DIT for pharmaceuticals in non-human primates have been conducted as part of embryofetal development (EFD), pre-/postnatal development (PPND), or juvenile studies. With regard to critical immune system “windows,” the enhanced PPND (ePPND) study design in non-human primates (NHPs) appears optimal for DIT testing, and has been shown to have similar benefits to the testing of monoclonal antibodies (Stewart, 2009) and biopharmaceuticals (Martin et al., 2009). Additional perspective on the state-of-the-science of juvenile immunotoxicology studies for pharmaceuticals is provided in a paper by Holsapple and O’Lone (2011).

Biomarkers in Immunotoxicology

There are many potential definitions for a “biomarker.” From a toxicological perspective, the most general definition of a biomarker is a substance that can be measured in a biological system or sample that reflects a xenobiotic-induced change in cellular or biochemical components or processes, structures, or function. From a clinical perspective, a biomarker is a term often used to refer to something that can be measured whose concentration reflects the severity or presence of some disease state. As such “true” biomarkers indicate exposure to a specific chemical or drug as well as susceptibility to an adverse effect, and/or are predictive of changes in a disease state associated with chemical or drug exposure. Although traditionally biomarkers are most often associated with proteins that can be measured in the blood, biomarkers can also be associated with changes in the expression of cell-surface proteins, and, in the new world of “omics” technologies, biomarkers can not only be identified through proteomics; but through transcriptomics and metabolomics (metabonomics), as well. Translational biomarkers are those that can be effectively used in multiple species, and that can be used to bridge the effects seen in two different species so that it would be possible to directly link responses between species. In the context of drug development, translational biomarkers that can be measured in both humans and non-human species are critical so that effects seen with a drug candidate can be followed in both preclinical and clinical settings.

Based on an understanding of the immune system, especially in the context of any type of immune response, it is easy to see the potential for biomarkers in immunotoxicology. Essentially, any xenobiotic-induced change in any parameter associated with an immune response—for example, a change in an antigen-specific antibody response, or a change in the expression of a surface marker, or a change in the numbers of a particular immune cell type, or a change in the levels of a cytokine or cytokines, or a change in the expression of an immune response-related gene—could be used as a biomarker for the immunotoxicity associated with that xenobiotic. As such, a chapter entitled, “The Promise of Genomics and Proteomics in Immunotoxicology and Immunopharmacology” (Pruett et al., 2007), emphasized that further implementation of these methods could move immunotoxicology into the area of systems biology, could be beneficial with regard to mechanistic and screening applications, and could reveal new insights about immune function. Similarly, an earlier report by the National Research Council’s Committee on Biological Markers undertook to study the interrelationship of toxic exposure and immune system response at a time when the discipline of immunotoxicology was described as being “relatively young”—for example, the IL family ended with IL-8 (NRC, 1992). The NRC Subcommittee on Immunotoxicology reviewed research on currently known markers, and identified and evaluated promising technologies to find new markers.

In spite of this early effort by the NRC, and although the NTP Tier Approach for assessing whether a compound is immunotoxic has been used for many years and has served as the basis for most immunotoxicity regulatory guidelines, there is no consensus for an accepted panel of biomarkers that one can use to determine whether, and to what extent, exposure to an immunotoxic agent has occurred. Various studies have been conducted to ascertain the feasibility, predictability, and accuracy of various biomarkers of immune modulation (Karmaus et al., 2005; Luster et al., 2005; Schwab et al., 2005). Besides the potential biomarkers already mentioned, which include cytokine gene expression patterns, cell population quantification using flow cytometry, and serum antibody titers, the assessment for the presence of single nucleotide polymorphisms (SNPs) in various genes makes it possible to determine whether small populations of individuals might be more susceptible to immunotoxicants. These same approaches are being applied to the development of immunomodulators and biologics, which are either designed to target the immune system, or are likely to do so because of their mechanisms of action. As such, it is more critical than ever that translational biomarkers for the immune system be developed and tested.

Finally, while it is clear from a number of studies currently available that biomarkers for the immune system can be predictive and accurate, there are limitations of their use, especially in the context of their application in human epidemiological studies. For example, suppression of CD4⁺ T cells certainly indicates immune suppression, but it would be challenging to determine which agent(s) correlate with the magnitude of suppression, and how the exposure to multiple agents affects that magnitude. Moreover, it should be noted that immunological biomarkers might be affected by other physiological processes, such as stress (Schwab et al., 2005); and as described in the preceding subsection, “Approaches to the Assessment of Human Immunotoxicity,” stress can affect immune responses to vaccines in humans (Glaser et al., 2000; Kiecolt-Glaser et al., 1996). Certainly, the complex interplay between different physiological processes is not unique to immunotoxicology; but it will need to be considered as the concept of biomarkers in immunotoxicology is developed.

The very nature of the immune system with the different cell types, the presence of various cell types in every tissue of the body, the dependence on proliferation and differentiation for effector functions, and the necessity to maintain immune function homeostasis renders it susceptible to modulation by a wide variety of xenobiotics. Although many of the xenobiotics discussed below exhibit immunosuppressive actions, it is important to realize that many of these chemicals are actually immunomodulatory, that is, they might produce both immune suppression and immune enhancement (in the absence of true hypersensitivity or autoimmunity). Of course, one cannot ignore the chemicals that do produce true hypersensitivity and/or autoimmunity and some examples of these are discussed later in the section entitled, “Xenobiotic-Induced Hypersensitivity and Autoimmunity.” Regardless of the end effect (immune suppression, immune enhancement, hypersensitivity, or autoimmunity) of a particular xenobiotic on the immune system, several common themes exist regarding the mechanisms by which these chemicals act. First, the mechanisms by which a xenobiotic affects immune function are likely to be multifaceted, involving several proteins, signaling cascades, or receptors. In fact, for several of the xenobiotics discussed below, there is evidence to suggest that immune system effects are both xenobiotic-specific receptor-dependent and -independent. Second, whether a xenobiotic produces a particular immune effect might depend on the concentration or dose of the xenobiotic, the mode and/or magnitude of cellular stimulation, and the kinetic relationship between exposure to the xenobiotic and exposure to the immune stimulant (ie, antigen, mitogen, and pharmacological agent). Third, xenobiotic exposures rarely occur one chemical at a time; thus, the effects and/or mechanisms observed might be attributable to several chemicals or classes of chemicals. Finally, determination of immune system effects and/or mechanisms by xenobiotics in humans might be further confounded by the physiological or immunological state of the individual. Despite these variables, the application of general, functional, and mechanistic tools discussed above have driven the determination of some of the effects and mechanisms for several xenobiotics.

Halogenated Aromatic Hydrocarbons

Few classes of xenobiotics have been as extensively studied for immunotoxicity as the halogenated aromatic hydrocarbons ([HAHs]; reviewed by Holsapple, 1996; Kerkvliet, 2002; Kerkvliet and Burleson, 1994). The prototypical and most biologically potent member of this family of chemicals, which includes the polychlorinated biphenyls (PCBs), the polybrominated biphenyls (PBBs), the polychlorinated dibenzofurans (PCDFs), and the polychlorinated dibenzodioxins (PCDDs), is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin). Substantial evidence has demonstrated that the immune system is a sensitive target for toxicity by these chemicals. Derived from a variety of animal models, primarily rodents, this evidence includes thymic atrophy, pancytopenia, cachexia, immune suppression, and tumor promotion. There is also epidemiological evidence suggesting that immunotoxicity by the HAHs can also occur in humans (Weisglas-Kuperus et al., 1995, 2000, and 2004); however, significant immune suppression has not been associated conclusively with specific alterations of human immune function.

The majority of the biochemical and toxic effects produced by the HAHs are mediated via HAH binding to the cytosolic AHR. The AHR is a 95 to 110 kDa basic helix-loop-helix type of ligand-activated transcription factor (Burbach et al., 1992; Ema et al., 1992), which is associated with two heat shock proteins (hsp90) (Denis et al., 1988; Perdew, 1988; Pongratz et al., 1992), prostaglandin E synthase 3 (p23) (Cox and Miller, 2004; Kazlauskas et al., 1999) and AHR-activated 9 (ARA-9) (Carver et al., 1998; LaPres et al., 2000; Ma and Whitlock, 1997). Ligand binding induces conformational changes in the AHR, enabling the receptor–ligand complex to initially release p23 and ARA9 in the cytosol followed by nuclear translocation where it sheds its hsp90 and is transformed into a DNA-binding protein (Cuthill et al., 1987; Denis et al., 1988; Okey et al., 1980; Perdew, 1988; Pollenz et al., 1994; Pongratz et al., 1992; Probst et al., 1993). This transformation involves dimerization of the receptor–ligand complex with a structurally related 87 kDa basic helix-loop-helix protein termed the aryl hydrocarbon receptor nuclear translocator (ARNT) (Hoffman et al., 1991; Probst et al., 1993; Reyes et al., 1992). The ligand–AHR/ARNT complex acts as a transcription factor by binding to DNA at the dioxin-responsive element (DRE), also termed xenobiotic response elements (XRE), in the promoter and enhancer region of sensitive genes such as cytochrome P4501A1, aldehyde dehydrogenase-3, glutathione S-transferase, and menadione oxidoreductase (Dunn et al., 1988; Elferink et al., 1990; Fujisawa-Sehara et al., 1987; Hoffman et al., 1991; Poland and Knutson, 1982; Reyes et al., 1992; Sutter and Greenlee, 1992; Watson and Hankinson, 1992; Whitlock, 1990). Numerous lines of evidence have supported the involvement of the AHR in mediation of toxicity including immunotoxicity by HAHs. Structure–activity relationship studies have demonstrated that with few exceptions, high-affinity AHR ligands are more immunosuppressive than low-affinity ligands (Davis and Safe, 1988). In mice, allelic variation at the Ah locus has been described. These alleles code for AHR with differential binding affinities for TCDD. For example, the C57BL/6 mouse represents a strain of mice (Ah^bb), which is exquisitely sensitive to TCDD (TCDD responsive), while the DBA/2 mouse strain (Ah^dd) is much less sensitive to the toxic effects of TCDD (TCDD nonresponsive or TCDD low-responsive). More recently, AHR null mice and cell lines that do not express the AHR have collectively provided compelling evidence supporting the role of AHR in the toxicity mediated by HAHs including immunotoxicity (Sulentic et al., 2000; Vorderstrasse et al., 2001).

Polychlorinated Dibenzodioxins

By far the majority of the investigations into the immunotoxic potential and mechanisms of action of the HAHs have focused on TCDD, primarily because this chemical is the most potent of the HAHs, binding the AHR with the highest affinity. The effects of TCDD on immune function have been demonstrated to be among the earliest and most sensitive indicators of TCDD-induced toxicity (reviewed by Holsapple, 1996; Holsapple et al., 1991a, b; Kerkvliet, 2002; Kerkvliet and Burleson, 1994). TCDD is not produced commercially, except in small amounts for research purposes. Rather, it is an environmental contaminant formed primarily as a by-product of the manufacturing process that uses chlorinated phenols or during the combustion of chlorinated materials. It is usually associated with the production of herbicides such as 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), and Agent Orange (a 1:1 combination of 2,4-dichlorophenoxyacetic acid [2,4-D] and 2,4,5-T). Other sources include pulp and paper manufacturing (chlorine bleaching), automobile exhaust (leaded gasoline), combustion of municipal and industrial waste, and the production of PCBs.

Like other HAHs, exposure to TCDD results in severe lymphoid atrophy. Because thymus-derived cells play an integral role in tumor surveillance and host resistance, the earliest studies on TCDD-induced immunotoxicity focused on changes in CMI. Studies on CMI have shown that this branch of acquired immunity is sensitive to the toxic effects of TCDD after in vivo administration. CTL development and activity have been shown by numerous investigators to be significantly decreased after exposure to TCDD, an effect that appeared to be age dependent (eg, the younger the mice when exposed, the greater the sensitivity to TCDD). In an in vivo acute graft-versus-host model of T-cell immunity, the direct AHR-dependent effects of TCDD on T cells were demonstrated. In this model, T cells from C57BL/6 mice were injected into C57BL/6 × DBA/2 F1 host mice resulting in the generation of an antihost CTL response (Kerkvliet et al., 2002). By comparing the ability of TCDD to suppress the CTL response of T cells obtained from AHR^+/+ and AHR^−/− C57BL/6 mice, the role for AHR expression in T cells was assessed. These studies showed that the CTL response was suppressed by TCDD through direct effects on CD4⁺ and CD8⁺ T cells via an AHR-dependent mechanism (Kerkvliet et al., 2002). The findings are consistent with the identification of AHR expression in T cells (Lawrence et al., 1996) and structure–activity relationship studies showing a positive correlation between AHR-binding affinity of dioxin-like PCBs and suppression of in vivo CTL responses (Kerkvliet et al., 1990). More recently using the same graft-versus-host model, TCDD treatment resulted in a significant increase in the percentage of donor CD4⁺ cells that expressed high levels of CD25, low levels of CD62L as well as glucocorticoid-induced TNFR (GITR) and CTLA-4, a phenotype associated with some types of Tregs, suggesting that an important component of the immune suppression of the CTL response induced by TCDD might be the generation of Tregs (Funatake et al., 2005). In addition to suppression of CTL function, TCDD exposure also results in decreases in PHA- and Con A-induced proliferative responses, and DTH responses. Enhanced proliferative responses in juvenile mice have also been observed (Lundberg et al., 1990). Concordant with the aforementioned findings, more recently it has been reported that AHR activation by TCDD or by the tryptophan-derived endogenous AHR ligand, 2-(1′H-indole-3′carbonyl)-thiazole-4-carboxylic acid methyl ester, induced the generation of Tregs in a model of experimental autoimmune encephalomyelitis (EAE), which produced a reduction in severity of disease (Quintana et al., 2008). These findings are somewhat in contrast to the observation that 6-formylindolo[3,2-b]carbazole, also a tryptophan-derived high-affinity AHR ligand, induced IL-17 production by Th17 cells and increased the severity of disease in the same EAE autoimmune model (Quintana et al., 2008; Veldhoen et al., 2008). Importantly, Th17 cells and Tregs negatively cross regulate each other, and this observation raises what appears to be a dichotomy in that AHR activation in the same model of autoimmunity can produce very different outcomes and may be in part explained by the ligand which activates the AHR. Moreover, transcriptome analysis showed that of four types of CD4⁺ T cells (Th1, Th2, Tregs, and Th17 cells), AHR was only detected in Th17 cells suggesting that some of the effects produced by TCDD and dioxin-like compounds on T cells may occur indirectly (Veldhoen et al., 2008).

In light of the critical role DCs play in regulating T-cell-mediated responses by presenting antigen to CD4⁺ and CD8⁺ T cells as well as by providing costimulatory signals through surface expression of CD80 and CD86 to drive T-cell activation via CD28 (T-cell coreceptor), recent investigations have focused on characterizing the effects TCDD exerts on DC function. Indeed, differentiation of bone marrow-derived precursors into DCs in the presence of TCDD was found to influence the expression of several surface proteins on DCs known to play a critical role in T-cell antigen recognition and T-cell activation. Specifically, differentiation of bone marrow precursors in the presence of TCDD resulted in an increase in MHCII, CD80, and CD86 expression, when compared to vehicle controls (Bankoti et al., 2010; Simones and Shepherd, 2011), which occurred in an AHR-dependent manner. Interestingly, in spite of the dependence on the AHR these changes were not dependent on AHR-mediated changes in gene expression since DCs from mice possessing a mutation within the AHR nuclear localization signaling domain exhibited a similar profile of activity as those derived from wild-type mice. While significant TCDD-mediated increases in MHCII, CD80, and CD86 expression were observed, TCDD-treated DCs exhibited a similar capacity as vehicle-treated DCs to activate CD8⁺ T cells, which would suggest that TCDD-mediated suppression of CTL responses does not involve altered DC function. In contrast, DCs differentiated in the presence of TCDD exhibited suppressed levels of TNF-α, IL-6, and IL-12 in response to TLR-mediated (LPS or CpG) activation suggesting altered cytokine regulation (Simones and Shepherd, 2011).

Consistent with the observation that mice exposed perinatally or postnatally are more sensitive to the effects of TCDD, it has been determined that thymic involution is a result of TCDD-induced terminal differentiation of the thymic epithelium and, thus, T cells do not have a proper nutrient-filled microenvironment in which to develop (Greenlee et al., 1984, 1985). This conclusion is consistent with observations that TCDD significantly decreased the number of immature T cells (CD4⁺/CD8⁺) in the thymus (Kerkvliet and Brauner, 1990). It was also reported that in vivo administration of TCDD-induced apoptosis in mouse thymocytes (Kamath et al., 1999) and that the mechanism involved a TCDD-induced increase in Fas ligand in thymic stromal, but not thymic, T cells (Camacho et al., 2005). In these experiments, it was demonstrated that when TCDD-exposed stromal cells were mixed with untreated thymic T cells, increased apoptosis was detected in T cells that involved Fas–Fas ligand interactions. In addition, the TCDD-mediated apoptosis of thymic T cells was demonstrated to occur via an AHR-dependent mechanism, which also involved NF-κB-mediated upregulation of Fas ligand promoter activity in the thymic stromal cells (Camacho et al., 2005). It has also been shown that TCDD interferes with cell-cycle regulation during early thymocyte development, which may in part account for the profound sensitivity of thymocytes to AHR ligands (Laiosa et al., 2003). More recently, differential TCDD-induced gene expression was observed during different stages of thymocyte development providing new insights into putative targets responsible for altered thymocyte differentiation in the presence of TCDD. Interestingly, this study also revealed that the cell cycle was unaffected at the earliest stages of thymocyte development prior to commitment to the αβ T-cell lineage (Laiosa et al., 2010).

Numerous investigations have demonstrated humoral immunity, and in particular, the IgM AFC response, to be exquisitely sensitive to the toxic effects of TCDD (Sulentic and Kaminski, 2011). This effect segregates with the Ah locus (Sulentic et al., 2000; Vecchi et al., 1983) and appears to be dependent on duration and conditions of exposure. Although TCDD induces profound changes in the AFC assay, no changes have been observed in splenic cellularity (numbers of Ig⁺, CD4⁺, or Thy-1⁺ cells) either before or after antigen challenge. Cell fraction/reconstitution of splenocytes from in vivo TCDD and/or vehicle-treated mice identify the B cell as the primary cell type impaired in the AFC response, with only modest changes observed in T-cell accessory function (Dooley and Holsapple, 1988). Interestingly, unlike CMI-mediated responses, which were only affected when TCDD treatment was performed in vivo, direct addition of TCDD to leukocyte cultures was found to strongly suppress humoral immune responses to a variety of B-cell antigens and activators (ie, LPS, dinitrophenyl-ficoll, and sRBC) demonstrating the ability of TCDD to directly target B cells (Holsapple et al., 1986). Investigations using AHR null mice and cell lines that differentially express AHR have demonstrated definitively that suppression of primary humoral immune responses by TCDD is dependent on AHR activation (Sulentic et al., 2000; Vorderstrasse et al., 2001). More recently, motif searches of regulatory regions of genes involved in controlling Ig expression led to the identification of DREs in a transcriptional regulatory region (termed 3′IghRR) of the Ig heavy chain gene. The 3′IghRR plays a crucial role in mediating high-level Ig heavy chain expression and Ig class switching. Electrophoretic mobility shift assays identified TCDD-induced AHR DNA binding, and transient transfection experiments demonstrated strong suppression of 3′IghRR reporter activity by TCDD in a cell line model of B-cell differentiation (Henseler et al., 2009; Sulentic et al., 2000, 2004a, b). However, suppression of humoral immune responses by TCDD cannot be completely reconciled by altered regulation of the 3′IghRR and the Ig heavy chain as TCDD-treatment also significantly suppresses expression of the Ig light chain and Ig J chain (Yoo et al., 2004). Additional studies revealed that a bistable biological switch controlling B cell to plasma cell differentiation as formed by the transcriptional repressors, B-cell lymphoma-6 protein (BCL-6), B lymphocyte-induced maturation protein-1 (Blimp-1), and paired box protein 5 (Pax5) is significantly altered in the presence of TCDD (Bhattacharya et al., 2010; Zhang et al., 2010) resulting in elevated levels of Pax5 due to repression of Blimp-1 (North et al., 2009; Schneider et al., 2008, 2009). Importantly, Pax5 is highly expressed in resting B cells simultaneously repressing the Ig heavy chain, light chain, and J chain and is in turn repressed by Blimp-1 to allow plasma cell differentiation and Ig production. Deregulation of this bifunctional switch by TCDD is mediated in part through decreased AP-1 DNA binding within the Blimp-1 promoter (Schneider et al., 2009) in combination with direct AHR-mediated induction of Bach2 (De Abrew et al., 2011), which acts as a repressor of Blimp-1. Collectively, the aforementioned findings suggest that humoral immune responses are suppressed through the direct actions of TCDD on B cells via a AHR-dependent mechanism through multiple related, but distinct, mechanisms acting in concert which include but are likely not limited to: (1) direct action of the AHR on the 3′IghRR of the Ig heavy chain; (2) deregulation of a bifunctional switch controlling B-cell differentiation resulting in elevated Pax5 and repression of the Ig heavy chain, light chain, and J chain; and (3) impaired B-cell activation (North et al., 2010).

The effects of TCDD on innate immunity are less well studied. TCDD has been shown to suppress some functions of neutrophils, including cytolytic and cytostatic activities. This suppression has been postulated as being related to neutrophil development in the bone marrow. Results by several investigators have shown TCDD-induced alterations in serum C3, indicating that soluble mediators of innate immunity may also be targeted (White and Anderson, 1985; White et al., 1986). There have been no observed effects on macrophage-mediated cytotoxicity, NK function, or IFN production. In host resistance models, TCDD exposure has been shown to increase susceptibility to several bacterial, viral, and tumor models. The most extensively employed model of host resistance for assessing the effects of TCDD on immune competence has been influenza virus. Uniformly, all of these studies have shown impaired resistance to influenza after TCDD treatment as evidenced typically by increased mortality (Burleson et al., 1996; Nohara et al., 2002; Warren et al., 2000). However, it is noteworthy that significant differences have been reported between laboratories concerning the magnitude of impairment produced by TCDD, which may be due in part to differences in the strain of influenza virus used. In this model of host resistance, lymphocyte migration to the lung and the production of virus-specific IgG2a, IgG1, and IgG2b antibodies were markedly diminished while IgA and neutrophilia were increased in TCDD-treated mice (Vorderstrasse et al., 2003). Conversely, in this same study, no significant TCDD-associated effects were found on T-cell expansion in the lymph nodes and on the production of IFN-γ and IL-12. Collectively, the results suggested that the increase in mortality to influenza in TCDD-treated mice was due to decreased antibody production and increased pulmonary inflammation (Vorderstrasse et al., 2003).

TCDD has also been identified as a potent immunohematopoietic toxicant with the ability to alter the number of Lin-Sca-1⁺cKit⁺ (LSK) bone marrow cells, a population enriched for murine HSCs. Assessment of bone marrow cells from TCDD-treated C57BL/6J mice for hematopoietic alterations revealed increases in the number of bone marrow LSK cells, relative to control, over 24 hours through 31 days following TCDD treatment. These findings suggest that proliferation and/or differentiation processes of HSCs are affected by TCDD and that these effects contribute to a reduced capacity of bone marrow to generate pro-T lymphocytes (Murante and Gasiewicz, 2000). Activation of the AHR by TCDD was also found to elicit disruptions in the circadian rhythms of hematopoietic precursors as evidenced by an abnormal in vivo rhythm of the percentage of the total number of LSK cells in G₀ phase of the cell cycle, suggesting disruption of stem cell quiescence (Garrett and Gasiewicz, 2006). In addition, expression of AHR and ARNT mRNA within enriched hematopoietic precursors oscillated with a circadian period. Taken together, these findings demonstrate that activation of the AHR by TCDD alters the profile of hematopoietic precursors as well as the circadian rhythms associated with these precursors.

There is little doubt that TCDD and related PCDDs are immunotoxic, particularly in mice. However, extrapolation to human exposure has proven to be difficult. There are a few instances in which accidental human exposure to TCDD and related congeners has afforded the opportunity to study exposure-related human immunological responses. In children exposed to PCDDs in Seveso, Italy (1976), nearly half of the exposed study group exhibited chloracne (a hallmark of high-level human exposure to PCDDs) three years after the accident. Immune parameters measured at that time were unaffected. In a second study conducted six years later on different subjects, there was an increase in complement, which correlated with the incidence of chloracne, an increase in circulating T and B cells, and an increase in PBMC mitogenic responses. In 2002, follow-up studies in which the population in the most highly exposed zone was randomly sampled, revealed modestly decreased median serum IgG but not IgM, IgA, C3, and C4 concentrations as compared to human subjects in the surrounding noncontaminated area (Baccarelli et al., 2002). A second incident occurred in 1971 in Times Beach, Missouri, when wastes containing TCDD were sprayed on roads to prevent dust formation. Both low-risk and high-risk individuals from this area were examined for DTH responses. Slight, but statistically nonsignificant alterations were observed in high-risk compared to low-risk individuals. In addition, there was a low-level increase in mitogenic responsiveness in high-risk persons. In a second study conducted 12 years later, no alterations were observed in DTH or mitogenic responses between exposed or control individuals. In studies undertaken to evaluate the in vitro effects of TCDD on human cells, TCDD suppressed IgM secretion by human B cells in response to the superantigen toxic shock syndrome toxin-1 and the proliferation and IgG secretion of human tonsillar B cells in response to LPS and cytokines (Wood and Holsapple, 1993; Wood et al., 1993). More recently, employing an in vitro CD40L B-cell activation model, HPB B cells were found to possess similar sensitivity to TCDD, as quantified by suppression of IgM AFC response, as mouse B cells expressing the high-affinity form of the AHR (C57BL/6) (Lu et al., 2010). In addition, HPB B cells also showed a marked impairment in the upregulation of activation markers, CD80, CD86, and CD69, and rapid attenuation (15–30 minutes postactivation) of protein phosphokinases including Akt as well as the mitogen-activated protein kinase, ERK, in the presence of TCDD (Lu et al., 2011). The impairment of CD80 and CD86 by TCDD on HPB B cells is in contrast to the increased upregulation of CD80 and CD86 by TCDD on mouse bone marrow DC discussed above (Bankoti et al., 2010; Simones and Shepherd, 2011) and may reflect species and/or cell-type related differences in the actions of TCDD within the immune system. Collectively, these results suggest that human B cells are sensitive to suppression by TCDD. Moreover, the effects of TCDD on human B cells are direct and involve, at least in part, attenuation of early B-cell activation events, which culminate in impairment of plasma cell formation. These results further suggest that TCDD-mediated suppression of B-cell function, although AHR-dependent, likely involves transcriptional as well as nontranscriptional mechanisms.

Polychlorinated Dibenzofurans

Like the PCDDs, polychlorinated dibenzofurans (PCDFs) are not produced commercially but are true environmental contaminants associated with the production of chlorophenoxy acids, pentachlorophenol, and other PCB mixtures. Although higher concentrations are required to achieve observable effects, the immunotoxic profile of the PCDFs is similar in nature to that described for TCDD. In fact, most of what is known regarding the immunotoxicity of the PCDFs in animal models has been learned with structure–activity relationship studies comparing TCDD to congeners of the dibenzofurans. Tetrachlorodibenzofuran (TCDF) exposure in most species is associated with thymic atrophy and, in guinea pigs, it has been shown to suppress the DTH and lymphoproliferative responses to PHA and LPS. Suppression of the AFC response to SRBC after exposure to several PCDF congeners has also been reported. In a more recent study of simple mixtures, dose–response studies comparing TCDD to either a mixture of PCDDs and PCDFs or to a mixture of PCDFs, PCDDs, and PCBs showed that each of the two mixtures produces strikingly similar suppression of the mouse in vivo anti-sRBC IgM AFC responses as TCDD alone when normalized to the total TCDD equivalents administered (Smialowicz et al., 2008). It is noteworthy that although the immunotoxicology database is extensive for TCDD and other PCDDs, it remains limited for PCDFs.

Two important case studies of human immunotoxicology involved populations accidentally exposed to HAHs. There is evidence that the PCDFs were the primary contributors to the observed toxic effects. Greater than 1850 individuals in Japan (in 1968) and in excess of 2000 people in Taiwan (in 1979) were affected when commercial rice oil was found to be contaminated with HAHs. PCDFs were observed in the tissues of the exposed populations, and subsequent studies on immune status revealed a decrease in total circulating T cells, decreased DTH response, and enhanced lymphoproliferative responses to PHA and pokeweed mitogen. In addition, many of the exposed individuals suffered from recurring respiratory infections, suggesting that host resistance mechanisms had been compromised.

Polychlorinated Biphenyls

PCBs have seen extensive commercial use for over half a century. Their unique physical and chemical properties make PCB mixtures ideal for use as plasticizers, adhesives, and as dielectric fluids in capacitors and transformers. Mixtures of PCBs (eg, Aroclors) have been commonly used to evaluate the immunotoxicity of PCBs and have been reported to suppress immune responses and decrease host resistance (reviewed by Holsapple, 1996). The first indication that PCBs produced immunotoxic effects was the observation of severe atrophy of the primary and secondary lymphoid organs in general toxicity tests and the subsequent demonstration of the reduction in numbers of circulating lymphocytes. Studies to characterize the immunotoxic action of the PCBs have primarily focused on the antibody response. This parameter is by far the one most consistently affected by PCB exposure, and effects on antibody response have been demonstrated in guinea pigs, rabbits, mice, and rhesus monkeys. PCB-exposed monkeys exhibit chloracne, alopecia, and facial edema, all classical symptoms of HAH toxicity. In an extensive characterization of the effects of PCBs on non-human primates, Tryphonas and colleagues (1991a, b) exposed rhesus monkeys to Aroclor 1254 for 23 to 55 months. The only immune parameter consistently suppressed was the AFC response to sRBC (both IgM and IgG). In addition, after 55 months of exposure, lymphoproliferative responses were dose-dependently suppressed and serum complement levels were significantly elevated. The observed elevation in serum complement has also been reported in PCDD-exposed children from Seveso, Italy (Tognoni and Boniccorsi, 1982) and in PCDD-exposed mice (White et al., 1986).

The effects of PCBs on CMI are far less clear and both suppression and enhancement have been reported. Exposure to Aroclor 1260 has been demonstrated to suppress DTH responses in guinea pigs, whereas exposure to Aroclor 1254 was reported to enhance lymphoproliferative responses in rats. In a similar study in Fischer 344 rats (Aroclor 1254), thymic weight was decreased, NK cell activity was suppressed, PHA-induced proliferative responses were enhanced, and there was no effect on the MLR proliferative response or CTL activity. Other investigators (Silkworth and Loose, 1978, 1979) have reported enhancement of graft-versus-host reactivity and the MLR proliferative response. The augmentation of selected CMI assays may reflect a PCB-induced change in T-cell subsets (as discussed above with TCDD), which contributes to immune regulation.

Studies on host resistance following exposure to PCBs indicate that the host defenses against herpes simplex virus, Plasmodium berghei, Listeria monocytogenes, and Salmonella typhimurium in mice are suppressed (reviewed by Dean et al., 1985). PCB-induced changes in tumor defenses have not been well defined, and both augmentation and suppression have been reported. This probably reflects the variability in the observed CMI responses.

The immunotoxicological effects of PCBs in humans are unclear. There are four separate reports of a longitudinal study in a cohort of Dutch children suggesting that the developing human immune system may be susceptible to immunotoxic alterations from exposure to Western European environmental levels of dioxin-like compounds, primarily PCBs (ten Tusscher et al., 2003; Weisglas-Kuperus et al., 1995, 2000, 2004). Three industrial areas were compared to a rural area with about 20% less PCBs in maternal plasma. In above studies of Dutch children, the major PCB congeners were measured in plasma in the mother and the newborn, and all dioxin-like compounds in maternal breast milk at two weeks after birth. The results showed an association between dioxin-like compound exposure and immunological changes, which included an increased number of lymphocytes, γδ T cells, CD3⁺HLA-DR⁺ (activated) T cells, CD8⁺ cells, CD4⁺CD45RO⁺ (memory T cells), and lower antibody levels to mumps and measles vaccination at preschool age (Weisglas-Kuperus et al., 1995, 2000, 2004). In addition, an association was found between dioxin/PCB prenatal exposure and decreased shortness of breath with wheeze, and the PCB burden was associated with a higher prevalence of recurrent middle-ear infections and chicken pox and a lower prevalence of allergic reactions (Weisglas-Kuperus et al., 2000). It is notable that although an association between dioxin/PCB exposure and changes in immune status was observed, all infants were found to be in the normal range. In a second study, modest but persistent changes in immune status were reported in children with perinatal exposure to dioxin-like compounds, as evidenced by a decrease in allergy and increased CD4⁺ T cells and increased CD45RA⁺ cell counts in a longitudinal subcohort of 27 healthy eight-year-old children with documented perinatal dioxin exposure (ten Tusscher et al., 2003). The original cohort at 42 months demonstrated an association among reduced vaccine titers, increased incidence of chicken pox, and increased incidence of otitis media with higher exposure to toxicity equivalents of dioxin. However, by eight years of age the more frequent recurrent ear infections were still apparent (overall), although the chicken pox frequency showed an inverse correlation with PCB/dioxin levels.

Polybrominated Biphenyls

The polybrominated biphenyls (PBBs) have been used primarily as flame retardants (Firemaster BP-6 and FF-1). While it is assumed that their profile of activity is similar to that of the PCBs, few studies have actually evaluated the action of the PBBs on immunocompetence. In Michigan (in 1973), Firemaster BP-6 was inadvertently substituted for a nutrient additive in cattle feed, resulting in widespread exposure of animals and humans to PBBs. Studies conducted on livestock following the incident indicated little if any PBB-induced alterations in immunocompetence (Kately and Bazzell, 1978; Vos and Luster, 1989). Like CMI observations involving PCBs, CMI responses in PBB-exposed individuals are not conclusive, showing both a reduction in circulating numbers of T and B cells and a suppression of selected CMI parameters or no effect on CMI at all. In a recent birth cohort study of 384 mother–infant pairs in Eastern Slovakia, PBB concentrations for selected PBB congeners were quantified in maternal and cord serum and infant blood at six months of age. Although the PBB concentrations in the Eastern Slovakia birth cohort were several-fold higher than those found in the United States, no association was observed between PBB levels, sum total or congener specific, and total serum IgG, IgA, IgM, and IgE (Jusko et al., 2011).

Polycyclic Aromatic Hydrocarbons

The polycyclic aromatic hydrocarbons (PAHs) are a ubiquitous class of environmental contaminants. They enter the environment through many routes including the burning of fossil fuels and forest fires. In addition to being carcinogenic and mutagenic, the PAHs have been found to be potent immunosuppressants. Effects have been documented on immune system development, humoral immunity, CMI, and host resistance. The most extensively studied PAHs are 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (BaP).

Early immunotoxicology studies of PAHs such as BaP, DMBA, and 3-methylcholanthrene demonstrated suppression of the antibody response to a variety of T-cell-dependent and T-cell-independent antigens. In addition, mice treated with BaP exhibited suppressed lymphoproliferative responses to mitogens, but not alloantigens (Dean et al., 1983). In Dean’s studies, host resistance to the PYB6 tumor and to L. monocytogenes were unaffected by BaP exposure, as was the DTH response and allograft rejection, suggesting that T cells and CMI were only minimally affected by BaP. In contrast to BaP, DMBA (the more potent PAH) significantly suppressed, not only AFC responses, but also NK cell activity, CTL responses, DTH responses, and alloantigen-induced lymphoproliferative responses. Therefore, DMBA exposure appears to result in long-lasting immune suppression of humoral immunity, CMI, and tumor resistance mechanisms in mice. In addition, both BaP and DMBA also produced significant effects on the bone marrow as evidenced by suppression of pre-B cell formation. A recent comprehensive immunotoxicological evaluation of 1,2:5,6-dibenzanthracene (DBA) demonstrated that a single dose via pharyngeal aspiration at doses up to 30 mg/kg had no effect on NK cell activity, anti-CD3 antibody-mediated T lymphocyte proliferation, the MLR, or B lymphocyte proliferation. In contrast, suppression of the anti-sRBC IgM AFC response was observed at 1.0 mg/kg DBA and the DTH response to C. albicans was significantly decreased at 3.0 mg/kg DBA (Smith et al., 2010). In another study, the immunosuppressive activity of various PAHs was greatly enhanced, in vitro, when combined with arsenic, a second common environmental contaminant (Li et al., 2010). Interestingly, in this same study it was observed that PAH metabolites were more potent in inducing p53 than their parent compounds.

Significant progress has been made toward elucidation of the mechanism(s) by which PAHs exert their toxicological effects including those on the immune system. Biological and toxicological activity of PAHs is generally dependent on two factors, AHR activation and the metabolism of PAHs to active metabolites by cytochrome P450 isozymes, CYP1A1, CYP1A2 and CYP1B1, which are transcriptionally induced by AHR binding to DREs in the promoter region of the genes that code for the aforementioned isozymes. Although leukocytes, in general, possess modest cytochrome P450 drug-metabolizing activity, in in vitro studies, splenocytes from naive untreated mice were found to metabolize exogenously added DMBA via P450 enzymes (Ladics et al., 1991). In addition, Ladics and colleagues (1992a, b) demonstrated that macrophages were the primary cell type in a splenic leukocyte preparation capable of metabolizing BaP to 7,8-dihydroxy-9,10-epoxy-7,8,9,10-benzo[a]pyrene (BPDE), the reactive metabolite proposed to be the ultimate carcinogenic and immunotoxic form of BaP. These data are consistent with other studies demonstrating the presence and inducibility of CYP1A1, CYP1A2, and most importantly in leukocytes, CYP1B1 (Gao et al., 2005a).

PAHs, including BaP, have also been shown to induce AHR-independent biochemical changes in lymphocytes leading to oxidative stress, activation of tyrosine kinases, and increased intracellular calcium (Mounho and Burchiel, 1998; Pessah et al., 2001). The elevation in intracellular calcium in B and T lymphocytes was shown to be mediated by BaP-7,8-dione and blocked by high concentrations of ryanodine suggesting the involvement of ryanodine receptors (Gao et al., 2005b).

Progress has also been made on the elucidation of the mechanism by which BaP and DMBA mediate bone marrow toxicity, specifically suppression of pre-B-cell formation. In vitro models demonstrated that BaP and DMBA rapidly induced apoptosis in primary pre-B cells and in the pro/pre-B cell line, BU-11 (Yamaguchi et al., 1997). The mechanism for pre-B-cell apoptosis by PAHs was shown to be dependent on bone marrow-derived stromal cells (Yamaguchi et al., 1997), CYP1B1 metabolism (Heidel et al., 1998), and the AHR (Yamaguchi et al., 1997) as well as coinciding with the induction of p53 (Yamaguchi et al., 1997). It has been demonstrated that DMBA-mediated bone marrow cytotoxicity was absent in p53 null mice and that apoptosis of primary bone marrow progenitor B cells cocultured with bone marrow stromal cells and DMBA was p53-dependent (Page et al., 2003). Collectively, these and other studies suggest that PAHs are metabolized by bone marrow stromal cells, which in turn release active PAH metabolites as part of a metabolite protein complex that induces apoptosis in pre-B cells (Allan et al., 2003).

Pesticides

Pesticides include all xenobiotics whose specific purpose is to kill another form of life, including insects (insecticides), small rodents (rodenticides), or even vegetation (herbicides). As such, these chemicals have clear biological activity and many pesticides have been studied for their effects on the immune system (Galloway and Handy, 2003). While there is much evidence that pesticides produce alterations in immune function in a variety of mammalian, amphibian, and fish animal models, the studies described here will focus on those that provide mechanistic information related to epidemiological effects in humans (reviewed by Colosio et al., 2005). Exposure to pesticides occurs most often in occupational settings, in which manufacturers, those applying the pesticides, or those harvesting treated agricultural products, are exposed. In the United States, the most widely used class of pesticides (through 2007) were the organophosphates (Grube et al., 2011), although effects of organochlorines, organotins, carbamates, and the herbicide atrazine will also be discussed.

Organophosphates

Organophosphates include malathion, parathion, methyl parathion, diazinon, and chlorpyrifos. Although the neurotoxic effects of organophosphates are well understood to occur via the inhibition of acetylcholinesterase (see Chap. 22, “Toxic Effects of Pesticides”), the mechanism by which these compounds suppress the immune system is not as well defined.

Malathion exhibits both immune suppressive and immune enhancing effects (Rodgers and Ellefson, 1990). Malathion has been shown to suppress humoral immunity as measured by the AFC response to sRBC following a subacute exposure in mice (Casale et al., 1983). In vitro exposure of either human PBMC or murine splenocytes to malathion results in decreased lymphoproliferative responses, suppressed CTL generation, and a decrease in the stimulus-induced respiratory burst in peritoneal cells (Rodgers and Ellefson, 1990). The mechanisms by which malathion is immunotoxic has been attributed to induction of oxidative stress in unstimulated mouse thymocytes (Olgun et al., 2004) or human PBMC (Ahmed et al., 2011). Parathion has attracted more attention than malathion, probably because it is more acutely toxic. This pesticide also produces mixed responses, which might depend on strain or dose (Casale et al., 1983; Crittenden et al., 1998). A comprehensive screen of various classes of pesticides that exhibit binding affinity to, and activation of, the pregnane X receptor (PXR) demonstrated that parathion activates PXR (Kojima et al., 2011), which has been shown to suppress inflammatory responses through inhibition of NF-κB (Zhou et al., 2006).

Fewer studies exist that describe the immunotoxicity of diazinon. Recently, it has been shown that diazinon produced gross changes in the spleen and thymus in response to acute doses in mice (Handy et al., 2002; Neishabouri et al., 2004). Diazinon also suppressed humoral immunity as measured by the AFC response to sRBC and CMI as measured by the DTH response following subchronic exposure to mice (Neishabouri et al., 2004).

Chlorpyrifos is one of the most widely used pesticides in the United States (Grube et al., 2011). Similar to other pesticides, its effects on immune function vary. An examination of humans exposed to chlorpyrifos demonstrated suppressed proliferative responses to Con A or PHA, but an increase in autoantibodies (Thrasher et al., 2002). Using human PBMC, it was demonstrated that chlorpyrifos modestly enhanced or suppressed IFN-γ production in response to LPS at low (1–100 μg/mL) or high (1000 μg/mL) concentrations, respectively (Duramad et al., 2006). The mechanism by which chlorpyrifos modulates immunity likely involves ERα, PXR, and AHR, as it induced reporter activity in receptor-activation assays (Kojima et al., 2004, 2011; Takeuchi et al., 2008).

Organochlorines

The organochlorines include chemicals such as chlordane, dichlorodiphenyltrichloroethane (DDT), mirex, pentachlorophenol, aldrin, dieldrin, and hexachlorobenzene. Although technically this class of compounds also includes the herbicides 2,4-D and 2,4,5-T, these compounds are considered separately under “Halogenated Aromatic Hydrocarbons.” The organochlorines are among the longer-lived pesticides and they have an increased propensity for contamination of soil and ground water, thus providing an additional route of exposure to the general population. Many organochlorine compounds also have been shown to mediate their effects via ERα (DDT, dieldrin, and chlordane), ERβ (DDT) (Kojima et al., 2004), or PXR (DDT, dieldrin, and chlordane) (Kojima et al., 2011).

Although DDT has been banned in several countries, it is still used to control malaria, typhoid, and dengue infections in some places of the world. DDT suppresses both humoral immunity and CMI. Early studies demonstrated suppression of humoral immunity following oral gavage in mice (Wiltrout et al., 1978). More recently, DDT has been shown to suppress IL-2 production from a human T-cell line and that the mechanism involved suppression of the critical transcription factor NF-κB (Ndebele et al., 2004). In another study in which occupationally exposed individuals were evaluated for cytokine levels, DDE, a primary metabolite of DDT, was associated with suppression of IL-2 and IFN-γ, and enhancement of IL-4, suggesting an imbalance of Th1/Th2 populations (Daniel et al., 2002). Finally, a correlation between DDE levels in breast milk and incidence of infant ear infections was observed, suggesting increased susceptibility to infection following DDT exposure (Dewailly et al., 2000). An increased susceptibility to infection might also involve suppression of macrophage function by DDT (Nunez et al., 2002), increased apoptosis of macrophages, reduced complement function (Dutta et al., 2008), or suppression of NK cell function (Udoji et al., 2010).

Dieldrin was used from the 1950s until 1987 when its use was banned due to environmental and human health concerns. The humoral immune response to both T-cell-dependent and T-cell-independent antigens is suppressed following exposure to dieldrin (Bernier et al., 1987), and macrophage function from dieldrin-exposed animals are depressed. The apparent effect of dieldrin on macrophages correlates with the increased susceptibility of dieldrin-exposed animals to murine hepatitis virus, which targets macrophages (Krzystyniak et al., 1985). CMI was also suppressed in mice following dieldrin exposure as measured by a suppression of the MLR and graft-versus-host disease (Hugo et al., 1988a, b). Dieldrin is also immunostimulatory under certain conditions as dieldrin enhanced the proinflammatory cytokine IL-8 in vitro and induced neutrophilic inflammation in vivo (Pelletier et al., 2001).

Chlordane refers to a group of structurally related chemicals used to control termites. The principle constituents of chlordane are heptachlor, α-chlordane, γ-chlordane, α-nonachlor, β-nonachlor, α-chlordene, β-chlordene, and γ-chlordene. Definitive immune suppression produced by chlordane was first reported by Spyker-Cranmer and colleagues (1982). In utero exposure resulted in decreased DTH responses in mice with no deficit in antibody production to sRBC. This effect correlated with an increase in resistance to influenza infection because DTH contributes to the pathology of the infection (Menna et al., 1985). In contrast to observations from mice exposed in utero, exposure of adult mice to chlordane did not result in any changes to several immune parameters, including AFC response to sRBC, MLR, DTH response, or mitogenic lymphoproliferation. In rats, immunomodulatory effects of three constituents of chlordane were noted following 28-day oral dosing (technical chlordane, cis-nonachlor, and trans-nonachlor), the most profound being increased lymphocyte numbers with decreased NK cell activity and proliferation in response to S. typhimurium (Tryphonas et al., 2003). The increase in cell numbers was consistent with a study conducted in Japan in which high concentrations of chlordane in breast milk increased CD3⁺ and CD8⁺ T-cell percentages in infants measured at approximately 10 months of age (Nagayama et al., 2007).

Many organochlorine pesticides have also been associated with increased cancer incidence (reviewed by Dich et al., 1997). The mechanism by which this occurs is unclear but is likely multifaceted, including, in part, organochlorine-induced modulation of the immune system. For instance, chlordane suppressed expression of the tumor suppressor, retinoblastoma protein, in a human B-cell/T-cell hybrid cell line (Rought et al., 1999). Furthermore, several organochlorine pesticides were demonstrated in vitro to suppress NK cell activity, which plays a role in defense against tumor formation (Reed et al., 2004). An analysis of the influence of organochlorine exposure and immune gene variants demonstrated increased risk of non-Hodgkin’s lymphoma in chlordane-exposed individuals with certain SNPs in IL-10 (Wang et al., 2007), IFN-γ, and IL-4 (Colt et al., 2009).

Organotins

Trisubstituted organotins such as tributyltin oxide are widely used as biocides and have recently been recognized as producing some immunotoxic effects. The most prominent action of tributyltin oxide is the induction of profound but reversible thymic atrophy. Studies by Vos and colleagues (1984) demonstrated a decrease in cellularity in the spleen, bone marrow, and thymus. The decrease in splenic cellularity was associated with a concomitant loss of T lymphocytes. In Jurkat T cells, tributyltin oxide increased intracellular calcium and nuclear factor of activated T cells (NFAT) translocation to the nucleus, but the increase in proliferation was not sustained, and at later times, there was decreased proliferation and increased expression of several apoptotic genes (Katika et al., 2011). In mouse thymocytes, there were increased annexin-V⁺ and Fas ligand-expressing cells following oral administration of tributyltin chloride (Chen et al., 2011), providing additional evidence that thymic atrophy is due to apoptosis.

Another sensitive target of organotin compounds is the NK cell. van Loveren and colleagues (1990) observed suppressed lung NK cytotoxicity in rats exposed orally to tributyltin oxide. The mechanism by which NK cell activity was suppressed has been investigated using human NK cells. Studies have demonstrated an increase in intracellular calcium and increased expression of various AP-1 transcription factors (Lane et al., 2009; Person and Whalen, 2010). However, overall, there was decreased AP-1 binding activity, which the authors suggested was due to increased prevalence of jun–jun homodimers, which exhibit lower affinity for the AP-1 site than other complexes, such as jun–fos heterodimers (Person and Whalen, 2010). Moreover, the mechanism by which di- and tributyltin suppressed NK cell activity might be due to its ability to interact with sulfhydryl groups since glutathione treatment reversed the suppression (Powell et al., 2009).

Carbamates

Carbamates, which include carbaryl (Sevin), aldicarb, mancozeb, and sodium methyldiothiocarbamate, are used primarily as insecticides. Similar to the organophosphates, the mechanism of action for the neurotoxic effects involves inhibition of acetylcholinesterase. In an evaluation of humoral immunity following a two-week exposure to carbaryl in rats, suppression of the IgM AFC response to sRBC was observed following inhalation exposure, but not oral or dermal exposure (Ladics et al., 1994). Following oral exposure in rats, carbaryl suppressed splenic proliferative responses, but enhanced pulmonary inflammation and IgE, in response to house dust mite, suggesting that carbaryl exhibits tissue-specific effects (Dong et al., 1998). The mechanism by which carbaryl is suppressive involves inhibition of LPS-induced signaling in macrophages (Ohnishi et al., 2008).

Pruett and coworkers (1992a) evaluated the immunotoxicity of sodium methyldithiocarbamate, and observed decreased thymus weight, depletion of the CD4⁺/CD8⁺ population of thymocytes, and profound suppression of NK cell activity following both oral and dermal exposure. They also determined that the mechanism by which sodium methyldithiocarbamate altered cytokine production from peritoneal macrophages involves inhibition of MAP kinase activity via TLR4 (Pruett et al., 2005). Pruett and coworkers (2006) further determined that the mechanism of cytokine alteration involved depletion of glutathione, alteration of copper-dependent proteins, and induction of stress.

Atrazine

Atrazine is an herbicide applied to various agricultural crops to control broad leaf weeds. It is widely used in the United States, and it has been detected in soils and ground water because of its resistance to degradation. Similar to other agents discussed, atrazine exhibits immunomodulatory effects. Using offspring of dams treated with atrazine and challenged with antigen, atrazine induced elevations in T-cell proliferation, cytolytic activity, and antigen-specific B cells in male offspring (Rowe et al., 2006). In contrast, young mice directly administered atrazine orally for 14 days exhibited suppressed thymic weight, spleen and thymic cellularity, and B cell fractions, although CD4⁺ T cell numbers increased (Filipov et al., 2005). Similarly, in adult mice, it was confirmed that atrazine suppressed thymic weight, and also suppressed splenic weight and decreased the host resistance of the mice to B16F10 melanoma tumors (Karrow et al., 2005). In vitro, atrazine suppressed cell surface expression of MHCII, CD86, CD11b, and CD11c using a mouse DC line (Pinchuk et al., 2007). Although the exact mechanism by which atrazine-induced immune suppression occurred is unclear, atrazine treatment of mice does induce corticosterone levels, indicating that activation of the hypothalamic–pituitary–adrenal axis might be involved (Pruett et al., 2003).

Metals

Generally speaking, metals target multiple organ systems and exert their toxic effects via an interaction of the free metal with targets, such as enzyme systems, membranes, or cellular organelles. In considering the immunotoxicity of most metals, it is important to remember that at high concentrations, metals usually exert immunosuppressive effects; however, at lower concentrations, immune enhancement is often observed (Koller, 1980; Vos, 1977). Furthermore, as with most immunotoxic agents, it is important to note that exposures to metals are likely not single exposures, although one metal might dominate depending on the exposure conditions (eg, high levels of mercury in fish or high levels of lead from paint).

Similar to pesticides, there is certainly a large body of literature describing the metal-induced immune alterations in mammalian, amphibian, and fish animal models, but again, the studies described here will focus on those that provide mechanistic information related to epidemiological effects in humans. Moreover, there has been a substantial increase in efforts to understand immune alterations following exposures to metal-containing nanomaterials; for these, the reader is referred to Chap. 28, “Nanotoxicology” or Chap. 23, “Toxic Effects of Metals.” Specific immunotoxic consequences of metal exposure have been reviewed (Zelikoff and Thomas, 1998), but this section focuses on the four best-studied immunotoxic metals: lead, arsenic, mercury, and cadmium.

Lead

By far the most consistent finding in studies evaluating the effects of metals on immune responses is increased susceptibility to pathogens. For lead, decreased resistance to the bacterial pathogens S. typhimurium, Escherichia coli, and L. monocytogenes has been observed (reviewed by Kasten-Jolly et al., 2010). In mechanistic studies (reviewed by Dietert and Piepenbrink, 2006a), an alteration in the ability of the macrophage to process and present antigen to antigen-primed T cells confirmed the previous observation and suggested that lead alters immune recognition. Both suppression and enhancement of the AFC response by lead has been reported.

The enhanced AFC response could be due to the observation that lead shifts the T-cell balance from Th1 to Th2 (reviewed by Dietert and Piepenbrink, 2006a). Interestingly, it has been hypothesized that lead exposure might contribute to the development of asthma (Dietert and Piepenbrink, 2006a), a predominantly Th2-mediated disease. This theory is consistent with the observation that there is increased IgE production and asthma incidence in lead-exposed children (Lutz et al., 1999) and that lead inhibits IFN-γ production, a Th1 cytokine (Heo et al., 2007). The mechanism by which lead induces a Th2 switch has been suggested to involve lead-induced alterations in bone marrow-derived DC populations that preferentially stimulate Th2 differentiation (Gao et al., 2007). Additional evidence for lead-induced alteration of APC function comes from the observation that lead-treated APCs stimulated CD4⁺ T-cell proliferation, likely via reduction of myeloid suppressor cell function (myeloid suppressor cells are macrophages that control T-cell proliferation) (Farrer et al., 2005, 2008).

Arsenic

A challenge in understanding the mechanisms by which arsenic is immunotoxic is that in addition to inconsistencies that arise from route of administration, concentrations/doses, and species/strain differences, the speciation of arsenic plays a significant role in arsenic toxicity. Moreover, early studies demonstrated the intricate interplay among the host, the pathogen, and the xenobiotic. Following exposure to the semiconductor material gallium arsenide, there was modest protection against infection of either S. pneumoniae or L. monocytogenes, but resistance to the B16F10 melanoma was reduced. It was subsequently determined that the arsenic concentrations in the blood of these animals was high enough to offer a chemotherapeutic effect against the bacterial pathogens (Burns et al., 1993).

Arsenic compounds affect human immune function. For instance, in environmentally exposed children, there was a correlation between total arsenic in urine (inorganic and arsenic metabolites) and superoxide anion production from stimulated monocytes (Luna et al., 2010) and in adult smelter workers, higher levels of urinary arsenic correlated with increased lipid peroxidation and lower vitamin E levels in blood (Escobar et al., 2010). These results suggest that arsenic exposure causes oxidative stress and in fact, using the THP-1 monocytic cell line, it was shown that arsenic trioxide decreased glutathione levels and induced heme oxygenase, which is often induced to combat oxidative stress (Wang et al., 2011).

There are also several studies in which human PBMC from unexposed individuals were treated in vitro with arsenic compounds. The percentage of IL-2-producing T cells in human PBMC was suppressed following treatment with sodium arsenite (Galicia et al., 2003). An additional study showed that sodium arsenite suppressed IL-2 production while also stimulating heme oxygenase in purified T cells, indicating that the direct T-cell impairment by arsenic occurs in parallel with oxidative stress (Martin-Chouly et al., 2011). B-cell and accessory cell (ie, macrophage) function is also compromised by arsenic compounds, as suggested by early anti-sRBC AFC responses in mice (Sikorski et al., 1991). Interestingly, in spite of a fairly extensive database showing suppression of antibody responses in murine models, in purified human B cells stimulated with CD40L, sodium arsenite did not suppress IgM production (Lu et al., 2009). Finally, treatment of primary human macrophages with arsenic trioxide resulted in decreased expression of CD14 (a PRR and coreceptor of TLRs), and altered the morphology thereby suppressing adhesion (Lemarie et al., 2006). The alterations in macrophage function were dependent on arsenic trioxide-induced activation of RhoA (small GTPase and Ras homolog) (Lemarie et al., 2006). At higher concentrations, arsenic trioxide also induced apoptosis of macrophages through inhibition of NF-κB signaling (Lemarie et al., 2006). Induction of macrophage apoptosis is the basis for the therapeutic use of arsenic trioxide to treat acute promyelocytic leukemia.

Mercury

Mercury compounds include organic (ie, methyl mercury) or inorganic forms. Mercury compounds not only suppress immunological responses, but also induce autoimmunity (see Mercury discussion under the “Autoimmunity” section). Interestingly, in one genetically susceptible mouse model of autoimmunity, subcutaneous administration of methyl mercury reduced T and B cell numbers prior to autoimmunity induction, demonstrating the immunomodulatory actions of mercury in vivo (Haggqvist et al., 2005).

Similar to arsenic, the mechanism by which mercury compounds are immunotoxic might be induction of apoptosis. Mercury also induces oxidative stress as evidenced by glutathione depletion (Mondal et al., 2005). Several studies have examined the effect of in vitro treatment of human PBMC with mercury compounds. Using mercuric chloride in PBMC stimulated with either anti-CD3/CD28/CD40 or bacterial antigen demonstrated that mercury differentially affects Th1/Th2 cytokines depending on the mode of T-cell activation (Hemdan et al., 2007). Using LPS to stimulate inflammatory cytokines, mercuric chloride enhanced an inflammatory response (Gardner et al., 2009). The induction of proinflammatory cytokines was also observed in Brazilian gold miners using mercury as an amalgam. Mercury-exposed miners demonstrated higher levels of IL-1β, TNF-α, and IFN-γ (Gardner et al., 2010). The mechanism for cytokine induction could be due to mercury-induced phosphorylation of ERK and p38 MAPKs as demonstrated using mercuric chloride in Jurkat T cells. This was due, in part, to ROS since mercury-induced phosphorylation of ERK and p38 MAPKs was abrogated by N-acetylcysteine (Haase et al., 2011).

Cadmium

Like other metals, cadmium exhibits immunomodulatory effects. Early studies demonstrated that oral administration of cadmium to mice increased susceptibility to herpes simplex type 2 virus, suppressed T and B cell proliferation, but enhanced macrophage phagocytosis (Thomas et al., 1985). As with many other immunotoxic agents, it has been suggested that the stress response, a shift to a Th2 cell population, induction of oxidative stress and induction of apoptosis all contribute to the mechanism by which cadmium suppresses humoral immunity and CMI (Hemdan et al., 2006; Lall and Dan, 1999; Pathak and Khandelwal, 2006, 2008). Using human HSCs isolated from umbilical cord blood, it was shown that in vitro treatment with cadmium chloride activated autophagy, an internal cellular degradation pathway allowing clearance of damaged cellular components (Di Gioacchino et al., 2008). Cadmium-induced oxidative stress was confirmed in PBMC isolated from a Japanese population of women living in a cadmium polluted area in which higher blood and urinary cadmium correlated with increased expression of several genes involved in oxidative stress signaling (Dakeshita et al., 2009).

It has long been known that cadmium (and other metals, such as mercury) bind to a protein called metallothionein, which is a small, cysteine-rich protein that complexes normally with divalent cations, such as copper and zinc. The role of metallothionein in metal-induced immunotoxicity has been recently reviewed (Klaassen et al., 2009), and there are several mechanisms by which the cadmium–metallothionein complex might contribute to immune modulation. The binding of cadmium to metallothionein could displace copper or zinc, altering the availability of the latter cations for biochemical processes. Alternatively, metallothionein is induced in response to several stimuli, including metals, and it has been demonstrated that metallothionein influences lymphocyte proliferation, differentiation, and various effector functions. Finally, since metallothionein is a cysteine-rich protein, it also plays a role in the oxidative homeostasis of the cell, which could be compromised under conditions of oxidative stress.

Solvents and Related Chemicals

There is limited but substantive evidence that exposure to organic solvents and their related compounds can produce immune suppression. Chemicals included in this section are aromatic hydrocarbons, such as benzene, haloalkanes and haloalkenes, glycols and glycol ethers, and nitrosamines.

Aromatic Hydrocarbons

By far the best-characterized immunotoxic effects by an organic solvent are those produced by benzene. In animal models, benzene induces anemia, lymphocytopenia, and hypoplastic bone marrow. In addition, it has been suggested that this myelotoxicity may be a result of altered differentiative capacity in bone marrow-derived lymphoid cells. Benzene (oral and inhaled) exposure has been reported to alter both humoral and CMI parameters including suppression of the anti-sRBC antibody response, decreased T- and B-cell lymphoproliferative responses (mitogens and alloantigens), and suppression of CTL activity. Benzene exposure also appears to increase the production of both IL-1 and TNF-α and to suppress the production of IL-2. With these dramatic effects on immune responses, it is not surprising that animals exposed to benzene exhibit reduced resistance to a variety of pathogens. In terms of a possible mechanism of action, Pyatt et al. (1998) demonstrated that hydroquinone, a reactive metabolite of benzene, inhibited the activity of NF-κB, a transcription factor known to regulate the expression of a number of genes critical for normal T-cell function. The authors concluded that NF-κB might be an important molecular mediator of the immunotoxicity of hydroquinone (and benzene).

A number of compounds structurally related to benzene have also been studied for their potential effects on the immune system. For example, nitrobenzene (an oxidizing agent used in the synthesis of aniline and benzene compounds) has been reported to produce immunotoxic effects (Burns et al., 1994b), with the primary targets being erythrocytes and bone marrow. Immunomodulating activity has also been observed for toluene, although most effects occur at markedly high concentrations. When compared with benzene, toluene has little to no effect on immunocompetence. However, it is noteworthy that toluene exposure effectively attenuates the immunotoxic effects of benzene (probably because of competition for metabolic enzymes). In contrast to the parent toluene, the monosubstituted nitrotoluenes (para- and meta-nitrotoluene) do significantly alter the immune system (Burns et al., 1994a, c). Exposure to p-nitrotoluene has been demonstrated to suppress the antibody response to sRBC, to decrease the number of CD4⁺ splenic T cells, and to suppress the DTH response to KLH. In addition, host resistance to L. monocytogenes was impaired, suggesting the T cell as a primary target. Similarly, m-nitrotoluene suppresses the antibody response to sRBC, the DTH response to KLH, T-cell mitogenesis, and host resistance to L. monocytogenes, again suggesting the T cell as the cellular target. The disubstituted nitrotoluene (2,4-dinitrotoluene) is also immunosuppressive (Burns et al., 1994a), with exposure resulting in suppressed humoral immunity, NK cell activity, and phagocytosis by splenic macrophages. Host resistance to bacterial challenge was also impaired. This profile of activity following exposure to 2,4-dinitrotoluene is consistent with a perturbation of the differentiation and maturation of leukocytes.

Haloalkanes and Haloalkenes

Carbon tetrachloride is widely recognized as hepatotoxic. Studies in mice revealed that carbon tetrachloride is also immunosuppressive. Mice exposed for 7 to 30 days to carbon tetrachloride (orally or intraperitoneally) exhibited a decreased T-cell-dependent antibody response (sRBC), suppressed MLR response, and lower lymphoproliferative capacity (T and B cells) (Kaminski et al., 1989a). Ex vivo activation of splenic T cells isolated from carbon tetrachloride-treated mice revealed a marked enhancement in IL-2 production. Moreover, the effect on IL-2 by carbon tetrachloride was associated with a serum factor in treated animals as direct addition of serum from carbon tetrachloride-treated mice also produced strong enhancement of IL-2 in naive activated splenic T cells. Further characterization demonstrated that exposure to carbon tetrachloride caused the induction and release of TGF-β₁ from the liver. The timing and conditions for the carbon tetrachloride-induced releases of TGF-β₁ was concomitant with the onset of immune suppression. Addition of anti-TGF-β₁ neutralizing antibodies abrogated IL-2 enhancement by serum isolated from carbon tetrachloride-treated mice, suggesting that the enhancing effects on IL-2 were mediated indirectly through TGF-β₁ released from the liver (Delaney et al., 1994; Jeon et al., 1997). Induction or inhibition of liver cytochrome P450 activity augmented and blocked, respectively, the immunotoxic actions of carbon tetrachloride, suggesting a requirement for metabolism in order for carbon tetrachloride to be immunosuppressive (Kaminski et al., 1990). Subsequently, it was demonstrated that TGF-β₁ produces profound effects on IL-2 regulation. Specifically, results in mouse spleen cells showed that TGF-β₁ exerts bifunctional effects on IL-2 and lymphoproliferation as evidenced by the fact that TGF-β₁ stimulates IL-2 production at low concentrations (0.1–1 pg/mL) and conversely suppresses IL-2 production at high concentrations (1–10 ng/mL), when activated using monoclonal antibodies directed against the CD3 complex and CD28 (McKarns and Kaminski, 2000). Additionally, concentrations of TGF-β₁ that stimulated IL-2 production concomitantly suppressed splenocyte proliferation under similar conditions. Studies have revealed that IL-2 regulation by TGF-β₁ is mediated, at least in part, via a mechanism dependent on SMAD3, a transcription factor involved in signaling through the TGF-β receptor (McKarns et al., 2004). In studies comparing acute versus subchronic carbon tetrachloride administration, acute carbon tetrachloride treatment enhanced phagocytosis and NK cell activity while subchronic treatment significantly impaired phagocytosis and NK cell activity (Jirova et al., 1996). In contrast, Fischer 344 rats exposed orally for 10 days showed no immunotoxic effects, despite signs of liver toxicity (Smialowicz et al., 1991c). The difference in sensitivity between studies in the mouse and rat may represent differences in the metabolic capabilities between these two species as well as the degree of liver injury induced, and hence the magnitude of TGF-β₁ production.

There is relatively little information on solvents and other chemicals structurally related to carbon tetrachloride. Some early studies were conducted to assess the potential immunotoxicity of a number of drinking water contaminants. Exposure to dichloroethylene (in drinking water for 90 days) has been reported to suppress the anti-sRBC antibody response in male CD-1 mice and to inhibit macrophage function in females (Shopp et al., 1985). Similarly, exposure to trichloroethylene (in the drinking water for four–six months) was reported to inhibit both humoral immunity and CMI and bone marrow colony-forming activity (Sanders et al., 1982). In those experiments, females were more sensitive than males. Exposure to 1,1,2-trichloroethane resulted in suppression of humoral immunity in both sexes. In addition, macrophage function was suppressed (males only) (Sanders et al., 1985). Inhalation studies with dichloroethane, dichloromethane, tetrachloroethane, and trichloroethene indicated that the pulmonary host resistance to Klebsiella pneumoniae was suppressed (Aranyi et al., 1986; Sherwood et al., 1987), suggesting that alveolar macrophages may be affected. More recent work with this series of solvents indicated that exposure to trichloroethylene may be an effective developmental immunotoxicant in B6C3F1 mice, suggesting that additional studies are required to determine the health risks associated with developmental exposure to this chemical (Peden-Adams et al., 2006).

Glycols and Glycol Ethers

Exposure to glycol ethers has been associated with adverse effects in laboratory animals, including thymic atrophy and mild leukopenia. Oral administration of ethylene glycol monomethyl ether for one to two weeks (House et al., 1985; Kayama et al., 1991) or its metabolite methoxyacetic acid for two weeks (House et al., 1985) produced decreased thymic weight, thymic atrophy, and a selective depletion of immature thymocytes in mice. No alterations in humoral immunity, CMI, macrophage function, or host resistance to L. monocytogenes were observed (House et al., 1985). It has also been suggested that perinatal exposure to ethylene glycol monomethyl ether may produce thymic hypocellularity and inhibition of thymocyte maturation, and that it may affect prolymphocytes in fetal liver (Holladay et al., 1994).

Oral studies (5–10 days) on the glycol ether 2-methoxyethanol have consistently shown a decrease in thymus weight in the rat (Smialowicz et al., 1991a; Williams et al., 1995). This decrease is often accompanied by alterations in lymphoproliferative responses, although suppression is seen in some cases and stimulation in others, with no clear reason for the differences in response. Alterations in spleen weight and splenic cell populations have also been observed, as well as suppression of trinitrophenyl-LPS and anti-sRBC AFC responses. Similar results have been obtained following dermal exposure to 2-methoxyethanol (Williams et al., 1995). A decrease in IL-2 production has also been reported (Smialowicz et al., 1991a). Studies using the metabolites of 2-methoxyethanol (methoxyacetaldehyde and methoxyacetic acid) or specific metabolic pathway inhibitors have shown that methoxyacetaldehyde and methoxyacetic acid are more immunotoxic than 2-methoxyethanol alone (methoxyacetaldehyde > methoxyacetic acid > 2-methoxyethanol) (Kim and Smialowicz, 1997; Smialowicz et al., 1991a, b), suggesting a role for metabolism in the observed alterations in immunocompetence. Although there was no effect following 10-day oral exposures to 2-methoxyethanol (50–200 mg/kg per day) (Smialowicz et al., 1991a), subchronic exposure for 21 days to 2000 to 6000 ppm (males) or 1600 to 4800 ppm (females) did produce an enhanced NK cell response (Exon et al., 1991) in addition to suppression of the AFC response and a decrease in IFN-γ production.

Nitrosamines

The nitrosamine family of chemicals comprises the nitrosamines, nitrosamides, and C-nitroso compounds. Exposure to nitrosamines, especially N-nitrosodimethylamine (eg, also known as dimethylnitrosamine or DMN; and the most prevalent nitrosamine) comes primarily through industrial and dietary means, and minimally through environmental exposure. DMN is used commonly as an industrial solvent in the production of dimethylhydrazine. It has been used as an antioxidant, as an additive for lubricants and gasolines, and as a softener of copolymers. The toxicity and immunotoxicity of DMN have been extensively reviewed (Myers and Schook, 1996). Single or repeated exposure to DMN inhibits T-dependent humoral immune responses (IgM and IgG), but not T-independent responses. Other symmetrical nitrosamines, such as diethylnitrosamine, dipropylnitrosamine, and dibutylnitrosamine, demonstrated similar effects on humoral immunity but were not as potent as DMN (Kaminski et al., 1989b). In fact, as the length of the aliphatic chain increased, the dose required to suppress the anti-sRBC AFC response by 50% (ED₅₀) also increased. In contrast, nonsymmetrical nitrosamines suppressed humoral immunity at comparable concentrations. Overall, the rank order of ED₅₀ values paralleled their LD₅₀ values. T-cell-mediated lymphoproliferative responses (mitogens or MLR) and DTH response were also suppressed following DMN exposure. In vivo exposure to DMN followed by challenge with several pathogens did not produce a consistent pattern of effects: decreased resistance to Streptococcus zooepidemicus and influenza, no effects on resistance to herpes simplex types 1 or 2 or Trichinella spiralis, and increased resistance to L. monocytogenes. In contrast, anti-tumor activity in DMN-exposed animals was consistently enhanced. DMN-exposed animals also have altered development of hematopoietic cells (increased macrophage precursors). Taken together, these data suggest the macrophage (or its developmental precursors) as a primary target. Mechanistic studies have demonstrated that DMN-induced alterations in CMI are associated with enhanced macrophage activity, increased myelopoietic activity, and alterations in TNF-α transcriptional activity. It has been postulated that DMN may cause the enhanced production of GM-CSF, which can have autocrine (enhanced tumoricidal and bactericidal activity) and paracrine (induced secretion of T-cell-suppressing cytokines by macrophages) activities.

Mechanistic studies have also indicated a critical role for metabolism in the immune suppression by DMN (Haggerty and Holsapple, 1990; Johnson et al., 1987; Kim et al., 1988). It is known that DMN is metabolized by the liver cytochrome P450 system to a strong alkylating agent, and studies have shown that there is a relationship between DMN-induced immune suppression and the anticipated hepatotoxicity. Interestingly, a molecular dissection of DMN-induced hepatotoxicity by mRNA differential display demonstrated an increase in transcripts for the complement protein C3 and serum amyloid A (Bhattacharjee et al., 1998). Previous work by Kaminski and Holsapple (1987) had demonstrated the potential immune suppression associated with an increase in serum amyloid A.

Mycotoxins

Mycotoxins are structurally diverse secondary metabolites of fungi that grow on feed. This class of chemicals comprises such toxins as aflatoxin, ochratoxin, and the trichothecenes, notably T-2 toxin and deoxynivalenol (vomitoxin). As a class, these toxins can produce cellular depletion in lymphoid organs, alterations in T- and B-lymphocyte function, suppression of antibody responses, suppression of NK cell activity, decreased DTH responses, and an apparent increase in susceptibility to infectious disease (reviewed by Bondy and Pestka, 2000; IPCS, 1996). T-2 toxin has also been implicated as a developmental immunotoxicant, targeting fetal lymphocyte progenitors leading the thymic atrophy often observed with these mycotoxins (Holladay et al., 1993). For ochratoxin, at least, the dose, the route of administration, and the species appear to be critical factors in results obtained in immunotoxicity studies. Past studies with aflatoxin B1 suggest that CMI and phagocytic cell functions are affected as evidenced by decreased proliferative responses to PHA and suppression of DTH responses (Raisuddin et al., 1993). In addition, in vitro experiments demonstrated that aflatoxin B1 required metabolic bioactivation in order to produce suppression of antibody responses and of mitogen-induced lymphoproliferation (Yang et al., 1986). Studies in laboratory animals have also shown increased risk to secondary infection after aflatoxin B1 treatment. The effects of aflatoxins on the human immune system have not been characterized but are of concern in light of the fact that in many parts of the world, such as in West Africa, exposure to aflatoxins is widespread as studies in Benin and Togo found that 99% of children possessed measurable aflatoxin-albumin adducts in blood (Gong et al., 2003).

For the extensively studied trichothecenes, the mechanism of immune impairment is related in part to inhibition of protein synthesis. Interestingly, trichothecenes at high doses induce leukocyte apoptosis concomitantly with immune suppression (Pestka et al., 1994). Conversely, at low doses, trichothecenes promote expression of a diverse array of cytokines including IL-1, IL-2, IL-5, and IL-6. In addition, trichothecenes activate mitogen-activated protein kinases in vivo and in vitro via a mechanism known as the ribotoxic stress response (Chung et al., 2003; Moon and Pestka, 2002; Zhou et al., 2003). Prolonged consumption of deoxynivalenol by mice was shown to induce elevation of IgA and IgA immune complex formation, and kidney mesangial IgA deposition (Pestka, 2003). It has been postulated that the enhancement in IgA production induced by deoxynivalenol may be associated with the increase in cytokine production described above. The trichothecenes are currently considered among the most potent small-molecule inhibitors of protein synthesis in eukaryotic cells, which is dichotomous to the observed increase in IgA secretion.

Adverse health effects have been associated with damp indoor environments following building envelope breech resulting from heavy rains and/or flooding, as occurred during Hurricanes Katrina and Rita in the Gulf Coast of the United States. The adverse health effects have been attributed, at least in part, to the presence of molds, most notably Stachybotrys chartarum, also known as black mold. S. chartarum produces the macrocyclic trichothecene toxin, satratoxin G, which like many of the trichothecenes is a potent inhibitor of protein synthesis. In a recent study, satratoxin G exposure of mice, 100 μg/kg for five consecutive days by intranasal instillation, induced apoptosis of olfactory sensory neurons and neutrophilic rhinitis (Islam et al., 2006). Elevated mRNA levels for proinflammatory cytokines TNF-α, IL-6, and IL-1, and the chemokine, MIP-2, were detected in nasal airways and the adjacent olfactory bulb of the brain. By Day seven, marked atrophy of the olfactory nerve and glomerular layer of the olfactory bulb was detected. These findings suggest that neurotoxicity and inflammation within the nose may be potential adverse health effects associated with Stachybotrys exposure in indoor air.

Natural and Synthetic Hormones

It is well established that a sexual dimorphism exists in the immune system. Females have higher levels of circulating Igs, a greater antibody response, and a higher incidence of autoimmune disease than males. Males appear to be more susceptible to the development of sepsis and the mortality associated with soft tissue trauma and hemorrhagic shock. Specific natural sex hormones in this dichotomy have been implicated. Immune effects of androgens and estrogens appear to be very tightly controlled within the physiological range of concentrations, and profound changes in immune activity can result from very slight changes in concentrations of hormones.

Estrogens

Diethylstilbestrol is a synthetic nonsteroidal compound possessing estrogenic activity. Diethylstilbestrol was used in men to treat prostate cancer and in women to prevent threatened abortions, as an estrogen replacement, and as a contraceptive drug. Extensive functional and host resistance studies on diethylstilbestrol (mg/kg per day range) have indicated that exposure to this chemical results in alterations in CMI and/or macrophage function and are believed to be mediated by the presence of the estrogen receptor on immune cells (Brown et al., 2006a,b; Holsapple et al., 1983; Kalland, 1980; Lai et al., 2000; Luster et al., 1980, 1984). Targeted sites of action include the thymus (thymic depletion, alteration in T-cell maturation process), T cells (decreased MLR, DTH, and lymphoproliferative responses), and macrophage (enhanced phagocytic, antitumor, and suppressor function). Pre- and neonatal exposures (mg/kg per day dose range) have also demonstrated immunotoxic effects related to T-cell dysfunction. DTH and inflammatory responses associated with diethylstilbestrol exposure in adult mice have been shown to be reversible upon cessation of exposure (Holsapple et al., 1983; Luster et al., 1980). However, effects from in utero and neonatal exposures appear to have more lasting, possibly permanent effects on immune responses (Kalland et al., 1979; Luster et al., 1979; Ways et al., 1980).

Exposure of human PBMC from men and women to 17β-estradiol (E2) increased basal IgM, IgG, and IL-10 production with no effect on IL-1α, IL-1β, IL-2, IL-4, and IL-6. Interestingly, addition of IL-10 enhanced the effect of E2 on IgM and IgG production (Kanda et al., 1999). IL-10 appears to have both positive and negative effects on immune function since in mouse models IL-10 is the mediator of a Breg subset (ie, B10 or Br1). Similar to the above results, E2 enhanced the induction of IgM and IgA AFCs from PBMC stimulated with pokeweed mitogen (testosterone had no effect) (Paavonen et al., 1981). In an autoimmune disease state, basal IgG production was more markedly induced by E2 in patients with active systemic lupus erythematosus (SLE) as compared to normal donors. In addition to an increase in total IgG, E2 also induced anti-dsDNA antibody (absent in normal donors). The profile was somewhat different for inactive SLE patients in that E2 increased total IgG production to a similar degree as SLE patients, but E2 did not induce anti-dsDNA antibody, which had a lower basal level than active SLE (Kanda et al., 1999). Furthermore, microarray analysis of T cells purified from human PBMC demonstrated E2-altered gene expression for cellular signaling proteins in activated T cells from SLE patients versus controls (Walters et al., 2009). Isoflavones (“isoflavone intervention”) administered for 16 weeks to postmenopausal women through soy milk or supplemental tablets resulted in higher frequency of B cells in peripheral blood with no effect on the frequency of CD4⁺, CD8⁺, or NK subsets. The isoflavone intervention had no effect on IL-2 or TNFα plasma levels, but IFN-γ trended toward an almost twofold nonsignificant increase (Ryan-Borchers et al., 2006). Other studies demonstrated an increase in plasma IL-6 with isoflavone diets (73 mg per day) (Jenkins et al., 2002) and increased cytotoxicity of human PBMC NK cells with isoflavone metabolites (0.1 μmol/L) (Zhang et al., 1999). In contrast to the human results with E2, in vivo administration of the phytoestrogen, genistein, suppressed anti-KLH IgG titers in mice (Yellayi et al., 2002). However, mouse cells treated with E2, diethylstilbestrol, or bisphenol A (discussed below) enhanced IgG anti-DNA antibody production and IgG immune complex deposition in the kidney, which may be a result of increased autoantibody secretion from B-1 cells (Yurino et al., 2004). Additionally, E2 has been shown to drive the expansion of the mouse Treg cell compartment and to increase Treg activity (Luo et al., 2011; Polanczyk et al., 2004). E2 was also shown in mice to inhibit recruitment and activation of inflammatory cells resulting in inhibition of TNF-α and IFN-γ production and of the inflammatory response (Salem et al., 2000). These discrepancies in the effects of E2 (activation vs. inhibition) may be due to species differences, different cellular targets, or a lack of disease state. Taken together, it is clear that E2 and phytoestrogens alter the human antibody response, an effect that is more marked in an autoimmune disease state.

A potential mechanism for the effects by estrogen and estrogenic compounds is activation of the nuclear estrogen receptor (ER, α, or β isoform), which is ubiquitously expressed in most tissues. Binding of ligand stabilizes ER dimers, which then bind to estrogen response elements (ERE) in target genes leading to transcriptional activation or inhibition. A recent and in-depth review outlines the studies demonstrating ER expression and ER-mediated effects in T cells, B cells, and APCs as well as a role of ER in murine models of autoimmunity (Cunningham and Gilkeson, 2011). In resting and activated human PBMC, CD4⁺ T cells expressed higher levels of ERα than B cells which expressed higher levels of ERβ. CD8⁺ cells express ERα and ERβ equally, but at low levels (Phiel et al., 2005). ERα and ERβ are differentially expressed in APCs (Cunningham and Gilkeson, 2011) and in human secondary lymphoid tissues (Shim et al., 2006). Additionally, estrogen and the ER appear to play a significant role in autoimmunity. Ovariectomy before sexual maturation resulted in a significant suppression in a murine model of SLE, which could be reversed by supplementary estrogen treatment (Talal, 1981). ER knockout studies have primarily corroborated this finding with knockout of ERα in three different mouse models of SLE showing attenuation of the disease and prolonged survival (Bynote et al., 2008; Svenson et al., 2008). This effect was selective for ERα since ERβ did not alter the disease state (Svenson et al., 2008). In contrast to these results, ERα (but not ERβ) knockout in a mildly autoimmune prone mouse strain led to signs of autoimmunity and spontaneous (no antigen challenge) formation of germinal centers in the spleen (Shim et al., 2004). An additional consideration in elucidating the mechanisms by which the ER mediates biological effects is the extensive cross talk that can occur between the ER and other receptor signaling pathways (eg, AHR and PPAR) and transcription factors (eg, AP-1 and NF-κB) that may be independent of the ERE.

Bisphenol A, a monomer in polycarbonate plastics and a constituent of epoxy and polystyrene resins possessing weak binding affinity for the estrogen receptor, has been recently evaluated by a number of laboratories for its potential to affect various aspects of immune function. The majority of studies to date demonstrate that leukocytes cultured in the presence of very high concentrations (>1 μM) of bisphenol A exhibit a number of alterations primarily in innate immune function responses including suppression of LPS-induced nitric oxide production and TNF-α secretion by macrophages (Kim and Jeong, 2003). The effects on nitric oxide production were shown to be correlated with a decrease in NF-κB DNA binding activity, a transcription factor critically involved in the regulation of inducible nitric oxide synthase and TNF-α. In this study, suppression by bisphenol A of LPS-induced nitric oxide production was blocked by the estrogen receptor antagonist, ICI 182,780. Bisphenol A (10–50 μM) has also been reported to enhance IL-4 production in a model of a secondary immune response (Lee et al., 2003). In vivo treatment of mice with bisphenol A (2.5 mg/kg) for seven days produced a decrease in ex vivo Con A-induced proliferation and IFN-γ secretion, but had no effect on the number of CD4⁺, CD8⁺, and CD19⁺ cells in the spleen (Sawai et al., 2003). Additional studies corroborate these findings by demonstrating an augmentation of Th1 immune responses (ie, cytokine profile and increased expression of antigen-specific IgG2a and IgA) with one study showing increased Th1 and Th2 immune responses. In these studies, bisphenol A was administered either by i.p. injection (0.1 mg/g) four times every second day (Alizadeh et al., 2006) in drinking water (10 mg/L, ad libitum) for two weeks (Goto et al., 2007) or through maternal dosing (3–3000 μg/kg) for 18 days (Yoshino et al., 2004). Presently, the putative effects of bisphenol A on immune function are poorly defined and based on the current literature, it is unclear whether the majority of the immunomodulatory effects reported are mediated through an ER-dependent mechanism. Additionally, the relatively high potential for human exposure to bisphenol A due to its wide use in plastics and other products has been of considerable concern to the public and government regulators. Despite the large number of safety-related studies published on bisphenol A, there is considerable controversy regarding its safety and the current tolerable daily intake value (0.05 mg/kg body weight per day) (Hengstler et al., 2011; Sekizawa, 2008; Vandenberg et al., 2009).

Androgens

Oxymetholone is a synthetic androgen structurally related to testosterone and was used in the past in the treatment of pituitary dwarfism and as an adjunctive therapy in osteoporosis. Its current use is limited to treatment of certain anemias. Oxymetholone was administered orally to male mice daily for 14 consecutive days (50–300 mg/kg per day) resulting in a minimal decrease in CMI (MLR and CTL response) but without altering the ability of the animals to resist infection in host resistance assays (Karrow et al., 2000). In contrast, anabolic androgenic steroids have been shown to significantly suppress the sRBC AFC response and to increase the production of proinflammatory cytokines from human PBMC.

No comprehensive studies evaluating the effects of testosterone on immune parameters have been conducted. However, it is clear that testosterone is capable of contributing to the suppression of immune function; in particular, CMI responses and macrophage activity. There are numerous reports in the clinical literature that males are more susceptible than females to infection following soft tissue trauma and hemorrhagic shock (reviewed by Catania and Chaudry, 1999). Treatment of males with agents that block testosterone (eg, flutamide) can prevent the trauma- and hemorrhage-induced depression of immunity. Similarly, treatment of females with dihydrotestosterone prior to trauma-hemorrhage results in depression of CMI similar to that of males. Furthermore, gonadectomized mice of either sex have elevated immune responses to endotoxin, which can be attenuated in either sex by the administration of testosterone. The mechanisms in these cases, including influences by the neuroendocrine system, are not clear. Other investigators have reported that, like estrogenic agents, testosterone and other androgens are capable of influencing host defense by altering lymphocyte trafficking in the body and altering the ability of the macrophage to participate in immune responses. It is uncertain if these responses are mediated by the cytosolic androgen receptor, which like the ER, is part of the nuclear receptor superfamily and regulates gene transcription by binding androgen response elements in target genes.

Glucocorticoids

The immunosuppressive actions of corticosteroids have been known for years. Following binding to a cytosolic receptor, these agents produce profound lymphoid cell depletion in rodent models. In non-human primates and humans, lymphopenia associated with decreased monocytes and eosinophils and increased neutrophils are seen. Corticosteroids induce apoptosis in rodents, and T cells are particularly sensitive. In addition, these agents suppress macrophage accessory cell function, the production of IL-1 from the macrophages, and the subsequent synthesis of IL-2 by T cells. In general, corticosteroids suppress the generation of CTL responses, MLR, NK cell activity, and lymphoproliferation. While it is clear that these drugs suppress T-cell function, their effects on B cells are not completely clear. Corticosteroids suppress humoral responses, but this appears to be due to effects on T cells, as antigen-specific antibody production by B cells to T-independent antigens does not appear to be affected by corticosteroid treatment.

In spite of the wide therapeutic use of glucocorticoids, the mechanism of action by which glucocorticoids mediate their anti-inflammatory/immunosuppressive activity is not well understood. Several mechanisms have been proposed all of which involve activation of the glucocorticoid receptor. Binding of glucocorticoids to the cytosolic glucocorticoid receptor (member of the nuclear receptor superfamily) induces the receptor to function as a ligand-activated transcription factor that undergoes homodimerization and DNA binding to glucocorticoid response elements (GREs) in the regulatory regions of glucocorticoid-responsive genes. Depending on the gene, GRE can either positively or negatively regulate transcription. For example, glucocorticoids induce annexin 1 (lipocortin 1), a calcium and phospholipid binding protein, which acts to inhibit PLA2 (Goulding and Guyre, 1992; Taylor et al., 1997). Inhibition of PLA2 results in a decrease in arachidonate formation, the precursor in the biosynthesis of inflammatory prostaglandins and leukotrienes. Similarly, glucocorticoids induce transcription of IκB, which is the endogenous inhibitor of the transcription factor, NF-κB (Auphan et al., 1995; Scheinman et al., 1995). Since transcription of many key inflammatory cytokines is regulated positively by NF-κB, induction of IκB results in retention of NF-κB in the cytosol and thus suppression of inflammatory cytokine production. Ligand-activated glucocorticoid receptors have also been found to physically interact with other transcription factors including AP-1 (Schule et al., 1990) and NF-κB (Ray and Prefontaine, 1994), to inhibit DNA binding and/or their transcriptional activity. Cross talk between the glucocorticoid receptor and other nuclear receptors may also play a role in mediating the effects of glucocorticoid ligands. Presently, it is believed that all of the above mechanisms contribute to the anti-inflammatory and immunosuppressive properties of glucocorticoids.

Therapeutic Agents

Historically speaking, very few drugs used today as immunosuppressive agents were actually developed for that purpose. In fact, if one looks closely enough, nearly all therapeutic agents possess some degree of immunomodulatory activity at some doses (Descotes, 1986). The recent explosion of knowledge regarding the function and regulation of the immune system (at the cellular, biochemical, and molecular levels) has provided investigators with a relatively new avenue for specific drug development. The following discussion focuses on those drugs used primarily for modulating the immune system: immunosuppressants (corticosteroids are described in the section “Natural and Synthetic Hormones”), AIDS therapeutics, biologics (ie, monoclonal antibodies, recombinant cytokines, and IFNs), and anti-inflammatory drugs.

Immunosuppressive Agents

Several immunosuppressive drugs are efficacious simply due to their ability to impair cellular proliferation, since proliferation is required for lymphocyte clonal expansion and, subsequently, differentiation. Other drugs inhibit specific intracellular proteins that are critical in the activation of the immune response.

Originally developed as an antineoplastic agent, cyclophosphamide (Cytoxan, CYP) is the prototypical member of a class of drugs known as alkylating agents. Upon entering the cell, the inactive drug is metabolically cleaved into phosphoramide mustard, a powerful DNA alkylating agent that leads to blockade of cell replication, and acrolein, a compound known to primarily bind to sulfhydral groups. Clinically, CYP has found use in reducing symptoms of autoimmune disease and in the pretreatment of bone marrow transplant recipients. Experimentally, this drug is often used as a positive immunosuppressive control in immunotoxicology studies because it can suppress both humoral and CMI responses. There appears to be preferential suppression of B-cell responses, possibly due to decreased production and surface expression of Igs. CMI activities that are suppressed include the DTH response, CTL, graft-versus-host disease, and the MLR. Administration of low doses of CYP prior to antigenic stimulation can produce immune enhancement of cell-mediated and humoral immune responses, which has been attributed, in part, to an inhibition of suppressor T-cell activity (Limpens et al., 1990; Limpens and Scheper, 1991). The immune-enhancing properties of CYP were demonstrated to be mediated by only one of the two major metabolites, acrolein, but not phosphoramide mustard (Kawabata and White, 1988).

Azathioprine (Imuran), one of the antimetabolite drugs, is a purine analog that is more potent than the prototype, 6-mercaptopurine, as an inhibitor of cell replication. Immune suppression likely occurs because of the ability of the drug to inhibit purine biosynthesis. It has found widespread use in the suppression of allograft rejection. It can also act as an anti-inflammatory drug and can reduce the number of neutrophils and monocytes. Clinical use of the drug is limited by bone marrow suppression and leukopenia. Azathioprine inhibits humoral immunity, but secondary responses (IgG) appear more sensitive than primary responses (IgM). Several CMI activities are also reduced by azathioprine treatment, including DTH response, MLR, and graft-versus-host disease. Although T-cell functions are the primary targets for this drug, inhibition of NK function and macrophage activities has also been reported.

Leflunomide (Arava), an isoxazole derivative, is another agent that suppresses cellular proliferation, which has been used in the treatment of rheumatic disease and transplantation (Xiao et al., 1994). Leflunomide inhibits de novo pathways of pyrimidine synthesis, thereby blocking progression from G₁ to S of the cell cycle. Thus, direct inhibition of B-cell proliferation may account for the drug’s ability to inhibit both T-cell-dependent and T-cell-independent specific antibody production. Leflunomide can also directly inhibit T-cell proliferation induced by mitogens, anti-CD3, or IL-2.

Cyclosporin A (CsA, Sandimmune) is a cyclic undecapeptide isolated from fungal organisms found in the soil. Important to its use as an immunosuppressant is the relative lack of secondary toxicity (eg, myelotoxicity) at therapeutic concentrations (Calne et al., 1981). However, hepatotoxicity and nephrotoxicity are limiting side effects. CsA acts preferentially on T cells by inhibiting the biochemical signaling pathway emanating from the TCR (reviewed by Ho et al., 1996). The result is inhibition of IL-2 gene transcription and subsequent inhibition of T-cell proliferation and clonal expansion of effector T cells. More specifically, CsA interacts with the intracellular molecule cyclophilin, an intracellular protein with peptidyl proline isomerase activity (although this enzymatic activity has nothing to do with the immunosuppressive effect of CsA). The CsA–cyclophilin complex inhibits the serine/threonine phosphatase activity of a third molecule, calcineurin. The function of calcineurin is to dephosphorylate the cytoplasmic form of the transcription factor, NFAT, therefore facilitating the transport of NFAT into the nucleus, where it can couple with nuclear components and induce the transcription of the IL-2 gene. Inhibition of calcineurin phosphatase activity by the CsA–cyclophilin complex prevents nuclear translocation of NFAT and the resulting IL-2 gene transcription.

FK506 (Tacrolimus or Prograf) is a cyclic macrolide which is structurally distinct from CsA, but which possesses a nearly identical mechanism of action (reviewed by Ho et al., 1996). Like CsA, FK506 binds intracellularly to proteins with peptidyl proline isomerase activity, the most abundant of which is FK506 binding protein-12 (FKBP12). The FK506–FKBP12 complex also binds to and inhibits calcineurin activity, thereby inhibiting IL-2 gene transcription. Clinically, FK506 inhibits T-cell proliferation, lacks myelotoxicity (although, like CsA, it does cause nephrotoxicity), and induces transplantation tolerance. In addition, the minimum effective dose appears to be approximately 10-fold lower than that of CsA.

Rapamycin (RAP, Sirolimus or Rapamune) is also a cyclic macrolide, which is structurally related to FK506. However, the mechanism by which it produces inhibition of proliferation is strikingly distinct. Unlike CsA and FK506, RAP does not inhibit TCR-dependent signaling events and IL-2 gene transcription. Rather, this compound inhibits IL-2-stimulated T-cell proliferation by blocking cell-cycle progression from late G₁ into S phase (Morice et al., 1993; Terada et al., 1993). Like FK506, RAP binds to the intracellular protein FKBP12. However, this RAP–FKBP12 complex does not bind calcineurin. Rather, the RAP–FKBP12 complex binds to the target of rapamycin, mTOR (Sabers et al., 1995), inhibiting its function. Inhibition of mTOR results in reduced cell growth and suppression of cell cycle progression and proliferation (reviewed by Fingar and Blenis, 2004). Unlike both CsA and FK506, RAP does not appear to be nephrotoxic. Due to its mechanisms of action, a significant advantage of RAP over CsA and FK506 is that it is an effective immune suppressant even after T cells have been activated, due to the fact that it blocks signaling through the IL-2 receptor. Conversely, for CsA and FK506 to be effective, T cells must encounter the drug prior to activation because once IL-2 transcription begins, neither therapeutic will provide effective suppression of the already activated T cells and IL-2 production.

AIDS Therapeutics

Traditionally, antiviral therapies have not been extremely successful in their attempt to rid the host of viral infection. This may be due to the fact that these pathogens target the DNA of the host. Thus, eradication of the infection means killing infected cells, which for HIV, are primarily CD4⁺ T cells. Numerous strategies have been developed to combat HIV, including targeting viral reverse transcriptase (nucleoside and nonnucleoside reverse transcriptase inhibitors), viral protease, viral fusion and entry, virus–T-cell interaction proteins, and stimulating immune responses (reviewed by Broder, 2010). The multidrug therapy used currently is referred to as highly active antiretroviral therapy (HAART) (reviewed by Este and Cihlar, 2010). However, eradication of this virus, and subsequently AIDS, remains a challenge because the very nature of the infection has significant immunosuppressive consequences. In addition, some of the current therapies also exhibit immunosuppressive actions. One such antiviral drug is zidovudine (Retrovir).

Zidovudine (3′-azido-3′-deoxythymidine) is a pyrimidine analog that inhibits viral reverse transcriptase. It was the first drug shown to have any clinical efficacy in the treatment of HIV-1 infection. Unfortunately, its use is limited by myelotoxicity (macrocytic anemia and granulocytopenia) (Luster et al., 1989). Early studies confirmed that the primary action of zidovudine is on innate immunity, although changes in both humoral immunity and CMI have also been observed (reviewed by Feola et al., 2006). In addition, it was shown that oral administration of high doses of zidovudine caused thymic involution and decreased responsiveness of T cells to the HIV protein, gp120 (McKallip et al., 1995). Clinically, zidovudine increases the number of circulating CD4⁺ cells and can transiently stimulate cell-mediated immune responses (lymphoproliferation, NK cell activity, and IFN-γ production). A final consideration for the immunotoxicity associated with AIDS therapeutics like zidovudine is that they are rarely administered alone and thus, drug interactions likely contribute to various immune effects.

Abacavir (ABC), a purine analog, is also an inhibitor of viral reverse transcriptase, but it has less adverse effects than zidovudine. However, in contrast to the immunosuppressive adverse effects of zidovudine, ABC induces hypersensitivity reactions in approximately 5% to 9% of patients initiating antiretroviral therapy with ABC. This adverse effect has been associated with the expression of the HLA haplotype HLA-B*5701 and elevated CD8⁺ CTL at the initiation of ABC treatment as well as an increase in CD8⁺ (not CD4⁺) T-cell proliferation in patients testing positive for ABC hypersensitivity (Easterbrook et al., 2003; Phillips et al., 2005). Based on these studies, a large study (1956 patients) was conducted to determine the effectiveness of prospective HLA-B*5701 screening in preventing hypersensitivity reactions to ABC (Mallal et al., 2008). Screening eliminated hypersensitivity reactions to ABC, and the introduction of screening prior to therapy has made a hypersensitivity reaction to ABC a rare event.

Biologics

Biologics refers to those therapies that are derived in some manner from living organisms and include monoclonal antibodies, recombinant proteins, and adoptive cell therapies. By its very nature, the immune system is often both the intended therapeutic target and unintended toxicological target of various biologics. Overall, manifestations of toxicity may include exaggerated pharmacology, effects due to biochemical cross talk, and disruptions in immune regulation by cytokine networks. Monoclonal antibodies can bind normal as well as targeted tissues, and any foreign protein may elicit the production of neutralizing antibodies against the therapeutic protein (ie, the therapeutic protein may be immunogenic). While certainly many biologics are being utilized safely, the immunotoxicological aspects of a monoclonal antibody (anti-CD3), TNF blockers, and a recombinant protein (IFN-α) will be discussed.

Monoclonal antibodies have been designed in general to suppress immune function, and include antibodies directed against certain molecules that are critical for inducing or sustaining an immune response and can be divided into the following mechanistic categories: (1) bind and neutralize specific cytokines (TNF-α; IL-6); (2) bind cell-surface molecules to trigger lysis by the adaptive immune response (CD3 on T cells; CD25, α subunit of IL-2 receptor; CD20 on mature B cells; CD52 on B cells, T cells, NK cells, monocytes, and macrophages); (3) bind cell-surface molecules and block costimulation signals from other cells (CD2 on T cells; CD80/CD86 on APCs, soluble BLyS/BAFF, CD40L on T cells); and (4) bind cell adhesion molecules and block lymphocyte trafficking (LFA-1 or α4 on leukocytes). Monoclonal antibodies directed against CD3 (Muromonab-CD3, OKT3), part of the TCR complex, have been used for acute transplant rejection. All T cells express CD3 and binding by anti-CD3 Ig opsonizes T cells for depletion by complement activation and immune clearance. Since CD3 is part of the TCR signaling complex, binding of OKT3 to CD3 can acutely induce a “cytokine release syndrome” due to a transient activation and release of cytokines, which soon after initial administration, flu-like symptoms, pulmonary edema, and hematological disorders have been reported (reviewed by Sgro, 1995). Anaphylactic reactions (Type I hypersensitivity) to the antibody can also occur. Additionally, OKT3 is a murine monoclonal antibody, therefore the therapeutic efficacy and duration of action may be decreased due to host recognition and clearance. The establishment of immunological memory will lead to more rapid clearance on subsequent administration.

Etanercept (Enbrel), adalimumab (Humira), and infliximab (Remicade) are disease-modifying antirheumatic drugs (DMARD) that block TNF to prevent (1) induction of other proinflammatory cytokines such as IL-1, IL-6, and IL-8; (2) macrophage and neutrophil activation; (3) fibroblast and endothelial activation; (4) induction of other acute-phase proteins; and (5) leukocyte migration to inflammation sites. Etanercept is a recombinant protein consisting of a dimer of the extracellular ligand-binding portion of the human 75 kD TNF receptor (TNFR2) fused to the Fc portion of human IgG1. Because TNFR2 is not selective, etanercept binds both circulating TNF-α and TNF-β preventing binding to the endogenous TNFR1 (55 kDa, TNF-α selective) and TNFR2 receptors. Adalimumab and infliximab, however, are recombinant monoclonal antibodies directed against TNF-α, which neutralize circulating TNF-α and prevent interaction with either TNFR1 or TNFR2. Hypersensitivity is a potential adverse effect of these drugs. Other immune-related adverse effects include opportunistic infections, malignancy, and autoimmune disorders which are a potential problem for all drugs that suppress the immune system. Of particular concern for TNF-α inhibitors is an association with a greater risk of tuberculosis; patients are now evaluated for tuberculosis risk factors and for latent tuberculosis infection prior to starting TNF-α inhibitor therapy (Toussirot and Wendling, 2007).

The majority of recombinant proteins have been used as immunostimulants, including IFN-α, IFN-γ, GM-CSF, erythropoietin, IL-2, and IL-12. Recombinant proteins used as immunosuppressants include IL-1 receptor antagonist (IL-1RA) and the abovementioned etanercept which is a hybrid of the extracellular ligand-binding portion of TNFR2 fused to the Fc portion of human IgG1. IFN-α, which is used as an antiviral agent, is used to treat hepatitis C and other chronic viral illnesses. The mechanism of the antiviral action of IFN-α involves, in part, direct suppression of viral replication, activation of NK cells, and enhanced expression of MHCI on virally infected cells thus increasing the likelihood of recognition by virus-specific T cells. Administration of IFN-α has been associated with autoimmune diseases, including autoimmune hypothyroidism and lupus (Vial and Descotes, 1995) and hematological disorders stemming from bone marrow suppression.

Anti-inflammatory Agents

Anti-inflammatory agents include nonselective and selective nonsteroidal anti-inflammatory drugs (NSAIDs), which suppress the production of proinflammatory soluble factors, such as prostaglandins and thromboxanes. Nonselective NSAIDs are a large class of drugs that reversibly inhibit both isoforms of cyclooxygenase (COX-1 and COX-2). The COX-2 enzyme, in particular, is induced in response to inflammatory cytokines and mediators and therefore, represents an attractive target to combat inflammatory diseases. However, due to an increased risk of cardiovascular effects in some patients (reviewed by Grosser et al., 2006), which is a side effect to varying degrees for all NSAIDs, the only COX-2 selective inhibitor currently approved for clinical use is celecoxib (celebrex). Aspirin, like nonselective NSAIDs, inhibits COX-1 and 2 enzymes, but inhibition is irreversible due to covalent binding of aspirin by acetylation to a serine residue in the COX enzyme. Aspirin is especially effective as an antiplatelet since platelets possess little biosynthesizing capacity and therefore, aspirin will inhibit COX for the life of the platelet (8–11 days).

Drugs of Abuse

Drug abuse is a social issue with extensive pathophysiological effects on the abuser. Drugs of abuse exhibit immunosuppressive actions, and in fact it has been suggested that in addition to the direct risk of HIV contraction via needle sharing or judgment lapses, abuse of some drugs has been associated with disease progression to AIDS (Rogers, 2011). Several classes of drugs will be discussed, including cannabinoids, opioids, cocaine, methamphetamine, and ethanol. Reports regarding the immune system effects of many of these drugs are often contradictory, so it should be noted that the mechanisms by which drugs of abuse suppress immune function might depend on the development of tolerance or addiction to the drugs; the immune, withdrawal, and pain status of the individual; and levels of endogenous molecules (ie, endorphins or endocannabinoids).

Cannabinoids

Much attention has been focused on the immunomodulatory effects of the cannabinoids, which can be defined as plant-derived (ie, from the marijuana plant), synthetic, or endogenous. Therapeutically, the primary psychoactive congener of marijuana, Δ⁹-tetrahydrocannabinol, is approved for use as an antiemetic in patients undergoing cancer chemotherapy and as an appetite stimulant for cachexia associated with advanced AIDS disease. Cannabinoids have also recently been approved for use in the treatment of symptoms associated with autoimmune disease, such as multiple sclerosis (Lakhan and Rowland, 2009). In addition, several states in the United States have legalized marijuana for medical use thereby increasing its use (Joffe and Yancy, 2004).

The mechanism by which Δ⁹-tetrahydrocannabinol produces psychotropic effects is through a G protein-coupled cannabinoid receptor, CB1 (Varvel et al., 2005). Peripheral tissues also express CB1, in addition to a second cannabinoid receptor, CB2. Although both receptors are expressed on immune system cells and are coupled to suppression of adenylate cyclase activity (Schatz et al., 1997), it is not entirely clear the extent to which the receptors and/or suppression of adenylate cyclase activity contributes to immune system effects by cannabinoids.

Many studies have shown that exposure to Δ⁹-tetrahydrocannabinol decreases host resistance to bacterial and viral pathogens (reviewed by Cabral and Staab, 2005). Cannabinoids alter both humoral and CMI responses as evidenced by suppression of the T-cell-dependent AFC response both in vivo and in vitro (Schatz et al., 1993) and direct suppression of T-cell function (Condie et al., 1996). With respect to the mechanism of T-cell suppression, many plant-derived compounds suppress IL-2 at the transcriptional level which is due, in part, to suppression of transcription factor activation (AP-1, NFAT, NF-κB) and ERK MAPK activity (Condie et al., 1996; Faubert and Kaminski, 2000; Herring et al., 1998). Although both cannabinoid receptors are expressed on T cells (Galiegue et al., 1995), many of the direct T-cell effects of cannabinoids have been demonstrated to occur independently of either cannabinoid receptor (Kaplan et al., 2003). On the other hand, B-cell suppression by Δ⁹-tetrahydrocannabinol was CB1 and/or CB2 receptor-dependent (Springs et al., 2008).

Cannabinoid suppression of other APC, such as macrophages and DCs, is also mediated through the CB1 and/or CB2 receptors. Δ⁹-Tetrahydrocannabinol exposure impaired lysosomal or cytochrome c processing in macrophages (Matveyeva et al., 2000; McCoy et al., 1995) likely via the CB2 receptor (Buckley et al., 2000; Chuchawankul et al., 2004). IL-12 production from DC and subsequent Th1 activation was suppressed by Δ⁹-tetrahydrocannabinol in a CB1 and/or CB2 receptor-dependent manner (Lu et al., 2006a, b). Taken together, these studies demonstrate that cannabinoid compounds alter immune function, and the mechanisms involve both cannabinoid receptor-dependent and -independent actions.

Opioids

Similar to cannabinoids, opioids refer to plant-derived, synthetic, or endogenous (endorphins) compounds that bind opioid receptors. Although technically “opioid” refers to drugs derived from the poppy plant, and “opiate” refers to agonists and antagonists with morphine-like activity (including plant-derived and synthetic compounds), they are often used interchangeably. It is well established that opioids suppress immune responses and the mechanism often involves one of the G_i-coupled opioid receptors (μ, κ, and δ receptors), but there are opioid receptor-independent mechanisms as well (Roy et al., 2011).

Early studies evaluating the immune competence of heroin addicts revealed a decrease in total T cells, which was reversed with the general opioid receptor antagonist, naloxone, suggesting a role for an opioid receptor in mediating immune suppression (McDonough et al., 1980). Later studies demonstrated that although morphine suppressed several immune parameters, there was no dose–response, suggesting that the effects were not receptor mediated, but were the result of increased circulating corticosteroids (which were significantly elevated in those animals) (LeVier et al., 1994). This conclusion is supported by the findings of other investigators as well (Pruett et al., 1992b).

Several investigators have reported decreased host resistance to viral and bacterial infections in opioid-treated animals or heroin addicts. In one study, morphine treatment of mice infected with S. pneumoniae demonstrated increased bacterial burden in the lungs and increased mortality. The mechanism by which the immune response was compromised involved, in part, suppression of NF-κB gene transcription, which likely contributes to decreased expression of inflammatory mediators, such as chemokines, reducing recruitment of neutrophils to the infection site (Wang et al., 2005a). There is also evidence that opioid use increases susceptibility to HIV infection. Although morphine and/or heroin use is associated with risk of HIV infection through shared needles, opioid use may contribute to progression of AIDS through immune suppression. Specifically, there is evidence that morphine treatment increases CCR5 expression, which is a primary receptor for HIV entry into macrophages (Guo et al., 2002). In addition, chronic morphine treatment shifts the T-cell balance toward Th2 (Azarang et al., 2007; Roy et al., 2004). Further evidence for compromised immunity toward HIV is the observation that morphine inhibited anti-HIV activity in CD8⁺ cells in an opioid receptor-dependent manner (Wang et al., 2005b).

Opioids also modulate innate immunity. Consistent with the observations that morphine and/or heroin use contributes to the progression of AIDS, Kupffer cells infected with HIV maintained in vitro in the presence of morphine resulted in a higher number of viral particles relative to untreated HIV-infected cells (Schweitzer et al., 1991). More recent studies demonstrate either suppression (Sacerdote, 2003) or enhancement (Peng et al., 2000) of cytokine production from macrophages. The differences might be due to the agonist used, in vitro versus in vivo administration, and the dosing regimen (ie, whether tolerance was induced or not). Overall, it is clear that opioids suppress immune function and that the mechanism by which this occurs is complex and likely involves the CNS, the autonomic nervous system, the hypothalamic–pituitary–adrenal axis, and one or more opioid receptors (Alonzo and Bayer, 2002).

Cocaine

Cocaine is a potent local anesthetic and central nervous system stimulant. This drug and its derivatives have been shown to alter several measures of immune competence, including humoral and cell-mediated immune responses and host resistance (Watson et al., 1983; Ou et al., 1989; Starec et al., 1991). Jeong et al. (1996) evaluated the effect of acute in vivo cocaine exposure on the generation of the anti-sRBC AFC, and determined that immune suppression was due to a cytochrome P450 metabolite of cocaine. Further studies demonstrated that sex, strain, and age differences can be detected in cocaine-induced immunodulation as assessed by the anti-SRBC AFC response (Matulka et al., 1996). Similar to other immunotoxic agents, the mechanism by which cocaine alters immune function involves a disruption of the Th1/Th2 balance and the stress response (Jankowski et al., 2010; Stanulis et al., 1997a, b). Cocaine also induces the secretion of TGF-β, which has been linked to the observation that cocaine exposure enhances replication of the HIV-1 virus in human PBMC (Chao et al., 1991; Peterson et al., 1991). A proteomic analysis was conducted in human DCs treated ex vivo with cocaine for 48 hours and demonstrated increased expression of NF-κB (Reynolds et al., 2009b), which could contribute to increased expression of various cytokines or chemokines. Cocaine was demonstrated to cause an increased HIV viral burden in a human PBL-SCID animal model, which was mediated through sigma 1 (σ1) receptors, which are informally referred to as psychoactive drug receptors (Roth et al., 2005). Although the function and role of σ1 receptors still remain to be elucidated, additional studies also suggest that cocaine effects are mediated through these receptors (Cabral, 2006; Maurice and Romieu, 2004).

Methamphetamine

Methamphetamine is a stimulant that is similar to amphetamine, although highly addictive and its use has been growing over the past several years. Relatively recently, the immunotoxicity of methamphetamine was explored (In et al., 2005). Following oral administration to mice, methamphetamine suppressed the anti-sRBC AFC response, IgG production, and mitogenic stimulation of T-cell proliferation. Even more striking was the suppression of GM-CSF-stimulated bone marrow colony growth by methamphetamine. These results indicate suppression of both CMI and humoral immunity in vivo following methamphetamine administration.

In human T cells, methamphetamine treatment ex vivo induced ROS and increased intracellular calcium (Potula et al., 2010). Consistent with this, ex vivo treatment of human DCs with methamphetamine induced MEK, which activate MAPKs (Reynolds et al., 2009a). Both of these studies demonstrate that in the absence of immune stimulation, acute methamphetamine treatment causes biochemical changes consistent with cellular activation. On the other hand, in mice treated in vivo with methamphetamine or in methamphethamine-using patients, plasma IL-6 was suppressed and IL-10 was increased, suggesting immune suppression following chronic methamphetamine use (Loftis et al., 2011).

Ethanol

Ethanol exposure has been studied both in alcoholic patients and in animal models of binge drinking. In humans, alcoholism is associated with an increased incidence of, and mortality from, pulmonary infection (reviewed by Happel and Nelson, 2005). There is also an increased incidence of bacterial infection and spontaneous bacteremia in alcoholics with cirrhosis of the liver (reviewed by Leevy and Elbeshbeshy, 2005). A consistent finding in abusers of ethanol is the significant change in PBMC. In animal models, this is observed as depletion of T and B cells in the spleen and the T cells in the thymus, particularly CD4⁺/CD8⁺ cells. The latter effect may be related in part to increased levels of corticosteroids, particularly in females (Glover et al., 2011; Han et al., 1993). In a binge-drinking animal model, ethanol suppresses innate immunity through impairment of TLR3 signaling in peritoneal macrophages. The authors also demonstrated suppression of proinflammatory cytokines (Pruett et al., 2004b). In addition to suppression of TLR3, ethanol suppresses signaling through other TLRs, contributing to pleiotropic effects of ethanol on innate immunity (Pruett et al., 2004a). Moreover, signaling alterations via TLRs depend on whether the alcohol exposure is acute or chronic (Dai and Pruett, 2006).

Inhaled Substances

Pulmonary defenses against inhaled gases and particulates are dependent on both physical and immunological mechanisms. Immune mechanisms primarily involve the complex interactions between neutrophils and alveolar macrophages and their abilities to phagocytize foreign material and produce cytokines, which not only act as local inflammatory mediators, but also serve to attract other cells into the airways.

Oxidant Gases

It is clear that exposure to oxidant gases—such as ozone (O₃), sulfur dioxide (SO₂), nitrogen dioxide (NO₂), and phosgene—alters pulmonary immunological responses and might increase the susceptibility of the host to bacterial infections (reviewed by Selgrade and Gilmour, 1994). Infiltration of both neutrophils and macrophages has been observed, resulting in the release of cellular enzyme components and free radicals, which contribute to pulmonary inflammation, edema, and vascular changes. Exposure to O₃ has been demonstrated to impair the phagocytic function of alveolar macrophages and to inhibit the clearance of bacteria from the lung. These changes were correlated with decreased resistance to S. zooepidemicus and suggest that other extracellular bacteriostatic factors may be impaired following exposure to these oxidant gases. Short-term NO₂ exposure decreases killing of several bacterial pathogens and, like O₃, this decreased resistance is probably related to changes in pulmonary macrophage function. A role for the products of arachidonic acid metabolism (specifically, the prostaglandins) has recently been implied and is supported by the fact that decreased macrophage function is associated with increased PGE₂ production and that pretreatment with indomethacin inhibits O₃-induced pulmonary hyperresponsiveness and related inflammatory responses. In humans, O₃ challenge produced a decrease in the number of macrophages in airway sputum; however, the recovered macrophages exhibited evidence of activation and enhanced antigen-presenting capability as demonstrated by increased CD80, CD86, and HLA-DR (Lay et al., 2007). In controlled studies, allergic asthmatic volunteers challenged with O₃ were found to have increased levels of inflammatory cytokines, IL-1β and IL-6, while levels of the anti-inflammatory cytokine, IL-10, were decreased in airway sputum. O₃ has also been associated with increased airway neutrophilia and eosinophilia (Peden, 2011).

It is clear that exposure to oxidant gases can also augment pulmonary allergic reactions. This may be a result of increased lung permeability (leading to greater dispersion of the antigen) and to the enhanced influx of antigen-specific IgE-producing cells in the lungs. In studies involving O₃ exposure and challenge with L. monocytogenes, decreased resistance to the pathogen correlated not only with changes in macrophage activity, but also with alterations in T-cell-derived cytokine production (which enhances phagocytosis). In support of an effect on T cells, other cell-mediated changes were observed including changes in the T- to B-cell ratio in the lung, decreased DTH response, enhanced allergic responses, and changes in T-cell proliferative responses.

Particles: Asbestos, Silica, and nanoparticles

It is believed that alterations in both humoral immunity and CMI occur in individuals exposed to asbestos and exhibiting asbestosis. Decreased DTH response and fewer T cells circulating in the periphery as well as decreased T-cell proliferative responses have been reported to be associated with asbestosis (reviewed by Miller and Brown, 1985; Warheit and Hesterberg, 1994). Autoantibodies and increased serum Ig levels have also been observed. Within the lung, alveolar macrophage activity has been implicated as playing a significant role in asbestos-induced changes in immune competence. Fibers of asbestos that are deposited in the lung are phagocytized by macrophages, resulting in macrophage lysis and release of lysosomal enzymes and subsequent activation of other macrophages. It has been hypothesized that the development of asbestosis in animal models occurs by the following mechanism: fibers of asbestos deposited in the alveolar space recruit macrophages to the site of deposition. Some fibers may migrate to the interstitial space where the complement cascade becomes activated, releasing C5a, a potent macrophage activator and chemoattractant for other inflammatory cells. Recruited interstitial and resident alveolar macrophages phagocytize the fibers and release cytokines, which cause the proliferation of cells within the lung and the release of collagen. A sustained inflammatory response could then contribute to the progressive pattern of fibrosis, which is associated with asbestos exposure.

The primary adverse consequence of silica exposure, like that to asbestos, is the induction of lung fibrosis (silicosis). However, several immune alterations have been associated with silica exposure in experimental animals, including decreased antibody-mediated and CMI parameters (reviewed by IPCS, 1996). Alterations in both T- and B-cell parameters have been reported, although T-cell-dependent responses appear to be more affected than B-cell-dependent responses. Dose and route of antigen exposure appear to be important factors in determining silica-induced immunomodulation. Silica is toxic to macrophages and neutrophils, and exposure is correlated with increased susceptibility to infectious pathogens. The significance of these immunological alterations for the pathogenesis of silicosis remains to be determined. The association of this disease with the induction of autoantibodies is discussed in the subsection “Silica” under “Xenobiotic-Induced Hypersensitivity and Autoimmunity.”

More recently, there has been growing interest in the effects of nanomaterials, especially airborne nanoparticles on lung-related diseases and the role of the immune system in the etiology of these diseases. The term “nanomaterial” is extremely broad and only signifies that the material is less than 100 nm in size. Among these nanomaterials, nanosized silicas are members. Although much is known concerning the toxicity of crystalline silica, little is known about the toxicity of nanosized silicas, as is the case for the majority of other nanosized materials. Significant effort is currently being directed toward understanding the influence of shape, charge, composition, specific functional groups, catalytic activity, and other properties on the biological and toxicological potential of these nanomaterials. In more recent studies, it has been reported that airway exposure to nanoparticles can induce a number of proinflammatory cytokines including IL-1β, MCP-1, MIP-2, GM-CSF, and MIP-1α. The mechanism by which exposure to nanoparticles results in induction of proinflammatory cytokines is presently poorly understood, but is believed, in part, to involve induction of oxidative stress (Di Gioacchino et al., 2011) and, in part, through the activation of the NALP3-inflammasome (Martinon et al., 2009). The NALP3-inflammasome is a cytosolic multiprotein complex, which when activated promotes the production of inflammatory cytokines (Fig. 12-22). In fact, pulmonary inflammation is a common response induced by nanoparticles in airways that cannot be explained by their surface chemistry and composition (Sager et al., 2008). A concern with exposure to nanoparticles is the possibility they can enhance immune responses to airborne antigens due to their ability to induce pulmonary inflammation. Indeed, several recent studies have demonstrated with a number of different nanoparticles, including multiwalled carbon nanotubes and titanium oxide, that when administered to experimental animals in combination with ovalbumin, the inflammatory and immunological responses, as measured by cytokine production, cellular infiltrate, and ovalbumin-specific antibodies, were significantly increased when compared to ovalbumin alone (Larsen et al., 2010; Ryman-Rasmussen et al., 2009). Exposure to nanoparticles can occur in the occupational setting, or through environmental exposure to ultrafine particles, which are, for example, by-products of combustion engines and common constituents of urban air pollutants. Further investigation is needed to understand the risks associated with exposure to nanoparticles, especially on respiratory disease.

Pulmonary Irritants

Chemicals such as formaldehyde, silica, and ethylenediamine have been classified as pulmonary irritants and may produce hypersensitivity-like reactions. Macrophages from mice exposed to formaldehyde vapor exhibit increased synthesis of hydroperoxide (Dean et al., 1984). This may contribute to enhanced bactericidal activity and potential damage to local tissues. Although silica is usually thought of for its potential to induce silicosis in the lung (a condition similar to asbestosis), its immunomodulatory effects have also been documented (Levy and Wheelock, 1975). Silica decreased reticuloendothelial system clearance and suppressed both humoral immunity (AFC response) and the CMI response (CTL) against allogeneic fibroblasts. Both local and serum factors were found to play a role in silica-induced alterations in T-cell proliferation. Silica exposure may also inhibit phagocytosis of bacterial antigens (related to reticuloendothelial system clearance) and inhibit tumoricidal activity (Thurmond and Dean, 1988).

Ultraviolet Radiation

Ultraviolet radiation (UVR), especially midrange UVB (290–340 nm), is an important environmental factor affecting human health with both beneficial effects including vitamin D production, tanning, and adaptation to UV, and adverse effects including sunburn, skin cancer, and ocular damage. Most, if not all of the chemicals, drugs and other materials discussed in this chapter were selected because they have been associated with immunotoxic effects, and because there is the chance for human exposure. And indeed, UVR has also been demonstrated to modulate immune responses in animals and humans. In fact, UVR is cited elsewhere in this chapter as one of the examples of where the parallelogram approach has been effectively used for a human risk assessment (van Loveren et al, 1995). It is important to emphasize that all humans encounter lifetime exposure to this ubiquitous environmental immunotoxicant (Ullrich, 2007a). The effects of UV exposure on the immune system have been reviewed (Garssen and van Loveren, 2001). While UV-induced immunomodulation has been shown to have some beneficial effects on some skin diseases, such as psoriasis, and has been demonstrated to impair some allergic and autoimmune diseases in both animals and humans, UV-induced immunomodulation can also lead to several adverse health consequences, including a pivotal role during the process of skin carcinogenesis. Kripke (1981) provided the first evidence that UVR was immunosuppressive. UV-induced skin tumors were removed and transplanted into normal age- and sex-matched syngeneic recipient mice. Interestingly, the tumors did not grow in normal mice, and progressive tumor growth was only seen when the tumors were transplanted into recipient mice that were immunosuppressed. These results were explained by proposing that exposure to UVR had two effects: induction of skin tumors and induction of immune suppression. Studies have shown that these two events are related (Ullrich, 2007b) based on a series of observations. First, UVR-induced keratinocyte-derived platelet activating factor was shown to play a role in the induction of immunosuppression. Second, cis-urocanic acid, a skin-derived immunosuppressive compound, mediates immune suppression by binding to serotonin receptors on target cells. Finally, studies showed that blocking the binding of these compounds to their receptors not only suppresses UVR-induced immune suppression, but that this approach also interferes with skin cancer induction.

There have been a number of studies to further characterize the mechanism of action for UV-induced immunomodulation. The first step is the absorption of UV photons by chromophores, so-called “photoreceptors,” such as DNA and urocanic acid (Garssen et al., 1997). As a consequence of UV absorption by chromophores, epidermal and dermal cells, including keratinocytes, melanocytes, Langerhan’s cells, mast cells, dermal fibroblasts, endothelial cells, as well as skin-infiltrating cells (ie, granulocytes and macrophages), produce and/or release many immunoregulatory mediators, including cytokine, chemokines, and neurohormones (Sleijffers et al., 2004). The mediators include both pro- and anti-inflammatory cytokines, such as TNF-α, IL-1, IL-6, and IL-10, which can modify directly or indirectly the function of APCs. Langerhan’s cells, the major APC in the skin, change phenotypically and functionally, which ultimately impacts the activity of T cells at the time of antigen presentation, both locally and systemically. One early explanation for UV-induced immunomodulation is that UVR induced a switch from a predominantly Th1 response (favoring DTH responses) to a Th2 response (favoring antibody responses). This hypothesis was supported by findings of altered cytokine secretion patterns indicative of a Th1 to Th2 switch (Araneo et al., 1989; Simon et al., 1990). Indeed, the majority of studies dealing with the effects of UVR indicated that Th1-mediated immune responses are especially sensitive to UV exposure. However, as noted above, UVR has been demonstrated to be associated with a suppression of certain allergic and autoimmune reactions. Indeed, more recent studies have demonstrated that Ig isotypes that are linked to either Th1 or Th2 cells can be suppressed by UVR and that UV exposure not only impairs Th1 responses; but also some Th2 responses (Sleijffers et al., 2004).

Schwartz (2008) summarized 25 years of studies by noting that in contrast to the general immunosuppression associated with the use of conventional immunosuppressive drugs, UVR suppresses the immune system in an antigen-specific fashion via the induction of immunotolerance. Several investigators have noted that this effect is mostly mediated via Tregs induced by UVR, and that induction of Tregs, expressing CD4 and CD25, is an active process, which requires antigen presentation by UV damage. Once activated in an antigen-specific manner, these Tregs can suppress immune responses in a general fashion via the release of IL-10. Ullrich (2007a) noted that this model is consistent with the fact that UVR is absorbed in the upper layers of the skin, does not penetrate into the underlying tissues and internal organs, and that T cells are not directly targeted by UVR in vivo because few T cells are found in normal skin.

When an individual’s immune system responds in a manner producing tissue damage, it could result in hypersensitivity or autoimmunity, which could be exacerbated, or even induced by, a xenobiotic. Fig. 12-23 is a schematic delineating the possible cascade of effects that can occur when a chemical produces an immune-mediated disease.

Hypersensitivity

Polyisocyanates

Polyisocyanates have a widespread use in industry and are responsible for occupationally related lung disease. These chemicals are used in the production of adhesives, paint hardeners, elastomers, and coatings. Occupational exposure is by inhalation and skin contact. Polyisocyanates are known to induce all four types of hypersensitivity responses.

Compounds in this group include toluene diisocyanate, methylene diphenyl diisocyanate, and hexamethylene diisocyanate. They are highly reactive compounds that readily conjugate with endogenous proteins, such as albumin, forming neoantigens responsible for hypersensitivity (Wisnewski et al., 2011). Pulmonary sensitization to these compounds can occur through either topical or inhalation exposure. In murine models employing intranasal or intratracheal sensitization and challenge with toluene diisocyanate, significant induction of Th2 cytokines, IgE, and eosinophilia has been demonstrated (Ban et al., 2006; Matheson et al., 2005; Plitnick et al., 2005). It has also been demonstrated that albumin-conjugated methylene diphenyl diisocyanate challenge via intranasal instillation following initial dermal exposure caused increased airway inflammation as characterized by eosinophil and lymphocyte infiltration into the BALF (Wisnewski et al., 2011).

In humans diagnosed with occupational asthma associated with diisocyanate exposure, there was increased production of IL-8, MCP-1, TNF-α, and RANTES (chemokine ligand CCL5) in PBMC stimulated ex vivo with albumin-conjugated diisocyanate (Lummus et al., 1998). Moreover, an examination of humans exposed to various diisocyanates demonstrated an association between incidence of occupational asthma from hexamethylene diisocyanate and specific SNPs in genes encoding IL-4 receptor α, IL-13, and CD14 (Bernstein et al., 2006, 2011). It has also been shown that diisocyanate-induced IgE is not as robust as that observed in allergic asthma, suggesting that distinct pathophysiological mechanisms control occupational versus allergic asthma (Bernstein et al., 2002). In a mouse model in which toluene diisocyanate plus ovalbumin was used to induce asthma, PI3 kinase was critical for the upregulation of IL-17 and pulmonary inflammation (Kim et al., 2007). The mechanism by which cytokines and chemokines are upregulated also likely involves activation of p38 MAPK (Mitjans et al., 2008).

Acid Anhydrides

The acid anhydrides are reactive organic compounds used in the manufacturing of paints, varnishes, coating materials, adhesives, and casting and sealing materials. Trimellitic acid anhydride (TMA) is one of the most widely used compounds in this group, and it causes all four hypersensitivity reactions. Similar to the diisocyanates, acid anhydrides bind to serum proteins, such as albumin (Valstar et al., 2006).

In an attempt to understand mechanisms by which TMA induced asthma following occupational exposure, a comparison between topical versus intranasal sensitization with TMA was conducted (Farraj et al., 2006). Intranasal challenge with TMA in mice that were either topically or intranasally sensitized with TMA produced a marked allergic rhinitis of similar severity, characterized by an influx of eosinophils and lymphocytes. Both the topical and intranasal routes of sensitization also produced significant increases in total serum IgE after intranasal challenge with TMA. In addition, both the topical and intranasal routes of sensitization induced significant increases in the mRNA expression of the Th2 cytokines IL-4, IL-5, and IL-13 (Farraj et al., 2006). These findings are significant as they suggest that dermal exposure represents a potential route of sensitization of the respiratory tract to chemical allergens.

Toxicogenomic analyses have been conducted in an attempt to identify gene expression profiles for TMA-induced occupational asthma and/or contact dermatitis (Regal et al., 2007). In one study, although overlap between genes induced in response to ovalbumin (allergic asthma model) or TMA (occupational asthma model) in mice occurred, over 100 unique TMA-induced genes were identified (Regal et al., 2007). Another study examined the gene expression profile in auricular lymph nodes following dermal exposure to TMA to determine changes in gene expression that correlated with the LLNA (Boverhof et al., 2009). Over 1000 genes were expressed in response to the highest dose of TMA following dermal exposure, including IL-4, IL-21, Mki67 (involved in cell division), and Aicda (involved in B cell class switch recombination) (Boverhof et al., 2009).

Cellular assays are also being developed to identify respiratory sensitizers, particularly to determine if low dose exposures contribute to occupational asthma. For instance, in response to a low dose of intratracheal administration of TMA, IgE⁺ and MHCII⁺ B cells were identified in the lung-associated lymph nodes (Fukuyama et al., 2010). The increase in IgE⁺MHCII⁺ B cells correlated with increased serum and BALF IgE and IgG1, and increased inflammatory cells in the BALF (eosinophils, neutrophils) (Fukuyama et al., 2010).

Metals

Metals and metallic substances, including metallic salts and metal-containing nanomaterials, are responsible for producing contact and pulmonary hypersensitivity reactions. For additional information on the mechanisms by which metal-containing nanomaterials induce hypersensitivity, the reader is referred to “Chap. 28, Nanotoxicology.” Exposure to these compounds may occur via inhalation or due to their solubility in aqueous media. Although many metals induce contact dermatitis (Forte et al., 2008), platinum, cobalt, chromium, nickel, and beryllium will be discussed here.

Platinum

Exposure to platinum-group elements occurs occupationally in the mining, dentistry, and jewelry industries. There may also be acute hypersensitivity to platinum-containing chemotherapeutics in up to 20% of patients (Syrigou et al., 2010). Platinum salts are allergenic and induce Type 1 and IV hypersensitivity reactions such as contact dermatitis and occupational asthma.

Exposure to the platinum salt sodium hexachloroplatinate by intradermal sensitization followed by intranasal challenge as a model of platinum-induced occupational asthma demonstrated that IL-4, IL-5, and IL-13 were modestly induced following ex vivo stimulation of lung-associated lymph nodes with Con A (Ban et al., 2010). Serum IgE levels and BALF neutrophils and eosinophils were also increased. These results are consistent with increased IgE in peripheral blood of platinum-sensitized patients (Raulf-Heimsoth et al., 2000).

Cobalt

Cobalt exposure comes from metal-on-metal replacement prostheses, or occupationally in superalloy production and pigment manufacturing. Cobalt also induces Type 1 and IV hypersensitivity reactions such as contact dermatitis, DTH in peri-implant areas in hip replacement patients, and occupational asthma (Thyssen and Menne, 2010).

There are several studies in which hip replacement patients who received metal-on-metal arthroplasty had increased metal-reactive T-cell responses. In one study, 7 out of 16 hip replacement patients demonstrated skin reactivity to cobalt, and of those 7 patients, 3 had elevated serum IgE (Thomas et al., 2009). Moreover, PBMC from 7 out of 16 patients proliferated ex vivo in response to cobalt chloride stimulation (Thomas et al., 2009). Part of this mechanism might involve creation of metal–protein complexes, which act as haptens (Mabilleau et al., 2008).

In workers exposed to hard metal dust (a cobalt-containing alloy), there was increased incidence of asthma and higher serum IgE levels (Shirakawa et al., 1992), indicating cobalt-induced occupational asthma. In a mouse model in which control or hypoxia inducible factor-1α (HIF-1α) was postnatally deleted in the lung, oropharyngeal aspiration of cobalt chloride induced a robust Th2-mediated asthma-like response characterized by eosinophilia, mucus cell metaplasia, and altered cytokine profile (Saini et al., 2010a, b). These results suggest that hypoxia signaling via HIF-1α might contribute to cobalt-induced occupational asthma.

Chromium

Chromium is another metal in which exposure occurs either from metal-on-metal prosthesis, or occupationally in the electroplating, leather tanning, and paint, cement, and paper pulp production industries. Chromium predominantly elicits contact dermatitis (Shelnutt et al., 2007), a Type IV hypersensitivity reaction, but can also induce hypersensitivity in patients with metal-on-metal hip arthroplasty, similar to those observed in response to cobalt (Thomas et al., 2009). Chromium exists in several oxidation states, including chromium (III) or chromium (VI), but chromium (VI) more readily permeates the skin barrier (Forte et al., 2008). However, once absorbed, chromium (VI) is reduced to chromium (III), which is most likely responsible for chromium toxicity (Shelnutt et al., 2007).

Upon the reduction of chromium (VI) to chromium (III), ROS are produced. Production of ROS is one mechanism by which hypersensitivity reactions are exacerbated (Roychowdhury and Svensson, 2005). Indeed, chromium has been shown to increase ROS production in several cell types. In human dermal fibroblasts treated with potassium dichromate, heme oxygenase-1 expression (HMO-1) was induced in a MAPK-dependent manner (Joseph et al., 2008). Similarly, in a HaCT keratinocyte cell line, potassium dichromate increased ROS formation, and activated Akt, NF-κB, and MAPK, ultimately contributing to the induction of IL-1α and TNF-α (Wang et al., 2010).

Nickel

Exposure to nickel occupationally occurs in the mining, milling, smelting, and refinishing industries. Consumers are exposed through clothing fasteners or body piercings, with adult women having the highest prevalence of nickel allergy (up to 17%) (Thyssen and Menne, 2010). Nickel causes both Type I and IV hypersensitivity. Similar to chromium, higher oxidation states of nickel have greater potential to sensitize individuals (Kasper-Sonnenberg et al., 2011), which could also be due to higher potential to generate ROS when reduced in vivo.

Most people are sensitized to nickel dermally; however, there have been reports that exposure of children to relatively high ambient air nickel concentrations was associated with a positive skin test for nickel (Kasper-Sonnenberg et al., 2011). Nickel sulfate treatment of human DC isolated from PBMC demonstrated robust induction of IL12, which was due to nickel sulfate activation of NF-κB, p38 MAPK, and IFN regulatory factor-1 (IRF-1) (Antonios et al., 2010). Induction of IL-12 helps to stimulate T cells and in fact, nickel-reactive T cells can be isolated from PBMC of nickel-sensitized individuals (Moed et al., 2004). However, a recent study demonstrated that nickel chloride treatment of purified CD3⁺ T cells activated with anti-CD3/CD28 impaired T-cell activation likely through inhibition of intracellular calcium concentration and NFAT activation (Saito et al., 2011). The authors suggested that the discrepancy in T-cell effects by nickel is either due to differences in nickel concentrations and/or differences in the ability of nickel to target different transcription factors (Saito et al., 2011).

Beryllium

Beryllium exposure occurs most frequently in high-technology ceramics and dental alloy manufacturing, and in the electronics, nuclear, and aerospace industries. Although its use in the manufacturing of fluorescent bulbs has been discontinued, chronic beryllium disease (CBD) was originally identified in 1946 in a group of fluorescent lamp manufacturing workers (Forte et al., 2008). Beryllium produces Type IV hypersensitivity reactions. Skin contact has been found to produce lesions of contact hypersensitivity, whereas lesions produced by penetration of splinters of beryllium under the skin are granulomatous in nature. Inhalation of beryllium can result in acute beryllium disease (ABD such as pneumonitis, tracheobronchitis), CBD, and increased risk of lung cancer (Cummings et al., 2009; McCleskey et al., 2009).

CBD is a pulmonary disease characterized by granulomas. Most patients with CBD also possess lymphocytes that proliferate in response to ex vivo beryllium treatment (Rosenman et al., 2011). One possible mechanism for the sensitization and subsequent development of disease is that beryllium can alter peptide binding to MHC molecules. In fact, it has been reported that there is a glutamic acid substitution in one of the MHCII molecules in humans both sensitized to beryllium and those diagnosed with CBD, indicating a genetic susceptibility to beryllium-induced hypersensitivity (Rosenman et al., 2011).

Therapeutic Agents

Hypersensitivity responses to therapeutic drugs are among the major types of unpredictable drug reactions, accounting for up to 10% of all adverse effects. Drugs that commonly induce hypersensitivity include sulfa drugs, barbiturates, anticonvulsants, insulin, iodine (used in many X-ray contrast dyes), and platinum-containing chemotherapeutics. Penicillin is the most common agent involved in drug allergy and is discussed here as an example. The high incidence of allergic reaction to penicillin is in part due to widespread exposure to the compound. Not only has there been indiscriminant use of the drug, but exposure occurs through food products including milk from treated animals and the use of penicillin as an antimicrobial in the production of vaccines. Efforts are still being made to reduce unnecessary exposure.

The mechanism by which hypersensitivity to penicillin occurs is through the formation of a neoantigen. The formation of the primary penicillin neoantigen occurs during the breakdown of penicillin, in which the β-lactam ring opens, forming a reactive intermediate that reacts with other proteins. The resultant penicilloylated protein now acts as a hapten to which the immune system mounts a response. As is the case with other haptens, subsequent exposures to penicillin may not absolutely require the formation of penicilloylated proteins to elicit secondary responses.

Reactions to penicillin are varied and may include any of the four types of hypersensitivity reactions (reviewed by Chang et al., 2011). The most commonly seen clinical manifestation of Type I reactions is urticaria; however, anaphylactic reactions occur in about 10 to 40 of every 100,000 patients receiving injections. Clinical signs of rhinitis and asthma are much less frequently observed. Blood dyscrasias can occur due to the production of IgG against penicillin metabolites bound to the surface of red blood cells (Type II reaction). Penicillin has also been implicated in Type III reactions leading to serum-sickness-like symptoms. Owing to the high frequency of Type IV reactions when penicillin is applied topically, especially to inflamed or abraded skin, products are no longer available for topical application. Type IV reactions generally result in an eczematous skin reaction, but a rare, life-threatening form of dermal necrosis may result. In these cases, there is severe erythema and a separation of the epidermis at the basal layer. This reaction, which gives the clinical appearance of severe scalding, is thought to be a severe delayed reaction.

Latex

Natural rubber latex is derived from the rubber tree Hevea brasiliensis and is used in the manufacture of over 40,000 products including examination and surgical gloves, among other medical products. Allergic reactions to natural rubber latex products have become an important occupational health concern with increased use of universal precautions, particularly latex gloves, to combat the spread of bloodborne pathogens. Hypersensitivity to latex usually occurs via a Type I or Type IV reaction. Dermatological reactions to latex include irritant dermatitis due to chemical additives or mechanical abrasion and the occlusive conditions caused by wearing gloves; contact dermatitis due to the chemical additives used in the glove manufacturing (eg, thiurams, carbamates, mercapto compounds, and phenylenediamines), and potentially more serious IgE-mediated responses due to residual latex proteins that remained in the finished products (reviewed by Shah and Chowdhury, 2011). The IgE responses may manifest as urticaria, asthma, or life-threatening anaphylaxis. Several latex proteins have been identified, and antibodies to most can be detected in latex-allergic individuals (Ahmed et al., 2004; Lehto et al., 2007).

Food and Genetically Modified Organisms

Awareness of hypersensitivity reactions to foods and genetically modified organisms (or crops; GMOs) has increased in the last several years. The most common food allergens are milk, egg, peanuts and other tree nuts, fish, shellfish, soy, and wheat. Peanut allergies are relatively common, can be severe, thus, current information regarding the mechanism of peanut hypersensitivity is provided as an example. Hypersensitivity to peanuts occurs primarily via a Type I reaction, and the IgE responses may include shortness of breath, asthma, and anaphylaxis. At least 11 peanut proteins have been identified and antibodies to most can be detected in peanut-allergic patients (reviewed by Finkelman, 2010). More recent studies have shown that peanut extract treatment of mouse or human plasma induced complement C3a, which could contribute to anaphylaxis (Khodoun et al., 2009). In addition, peanut-reactive T cells have been isolated from the blood of peanut-allergic individuals, suggesting that the hypersensitivity to peanuts also involves a Type IV reaction (de Jong et al., 1996; DeLong et al., 2011).

Exposure to GMOs is becoming more widespread as biotechnological advances in food production are used, for example, to confer insect resistance or provide desired nutrients. Allergenic determinants in GMOs result from the expression of novel proteins that might be recognized as nonself by the immune system. There are several considerations in determining potential hypersensitivity to a GMO. Bioinformatic tools are being developed in order to establish whether the introduced protein is allergenic and/or whether its amino acid sequence is similar enough to known allergens to be considered potentially allergenic (Goodman and Tetteh, 2011; Ladics et al., 2011). In addition, the appropriate test must be selected (eg, radioallergosorbent tests and Ig levels) in order to avoid false positives or false negatives. Finally, ideally, hypersensitivity to GMOs will be tested on subjects prior to release, but it is also important to survey reactions in the general public following widespread availability (reviewed by Germolec et al., 2003).

Formaldehyde

Formaldehyde is used as a preservative, sterilant, and fumigant. Additional exposures come from the textile industry, where it is used to improve wrinkle resistance, and in the furniture, auto upholstery, and resins industries. This low molecular weight compound is extremely soluble in water and forms haptens with human proteins easily (Maibach, 1983). Human predictive testing with 1% to 10% formalin (formalin is 37% formaldehyde) for induction and 1% formalin for challenge showed sensitization rates of 4.5% to 7.8% (Marzulli and Maibach, 1987), and Basketter et al. examined 10 aldehydes of varying degrees of allergenicity in humans using the LLNA. The results confirmed that formaldehyde was the strongest allergen and is a contact sensitizer (Basketter et al., 2001).

Occupational exposure to formaldehyde has been associated with the occurrence of asthma (Thompson and Grafstrom, 2008) and increased formaldehyde exposure has now also been associated with increased incidence of childhood asthma (McGwin et al., 2010). Formaldehyde can enhance the respiratory allergic response to other stimuli. For example, Sadakane and coworkers (2002) showed that formaldehyde exposure (0.5% mist once a week for four weeks) in ICR mice enhanced the eosinophilic airway inflammation following the intratracheal instillation with a dust mite allergen. Fujimaki and colleagues (2004) exposed C3H/He mice to formaldehyde at 0, 80, 400, or 2000 ppb formaldehyde for 12 weeks. When mice were immunized with ovalbumin and then exposed to formaldehyde, the total number of BALF cells, macrophages, and eosinophils were significantly increased at the highest concentration. Exposure to 400 ppb formaldehyde induced significant decreases in antiovalbumin IgG1 and IgG3 antibodies, but there was no effect on antiovalbumin IgE antibody. In contrast, in response to a two-week formaldehyde exposure in the absence of another stimulation, total IgE was increased in the serum of C57BL/6 mice (Jung et al., 2007). Lung and liver IL-1β, IL-4, and IL-5 were also increased, and histopathology showed eosinophil infiltration, and inflammatory cell influx (Jung et al., 2007). It has been postulated that part of the mechanism for formaldehyde-increased pulmonary inflammation involves disruption of airway thiol levels, which could affect levels of reactive oxygen and nitrogen species (Staab et al., 2008; Thompson and Grafstrom, 2008).

Autoimmunity

There are numerous reports of xenobiotics that have been associated with autoimmunity; however, firm evidence for their involvement is difficult to obtain. Presently, there are very few instances of human autoimmune diseases for which an environmental trigger has been definitely identified (Rose, 2005). These relationships may be causative through direct mechanisms, or they may be indirect, acting as an adjuvant. In the area of xenobiotic-induced autoimmunity, exact mechanisms of action are not always known. Chemical exposure may also serve to exacerbate a preexisting autoimmune state (Coleman and Sim, 1994; Kilburn and Warshaw, 1994). Table 12-12 lists chemicals known to be associated with autoimmunity and identifies the proposed self-antigenic determinant for each chemical except for silica, which most likely acts as an adjuvant. Table 12-13 shows chemicals that have been implicated in autoimmune reactions, but in these cases the mechanism of autoimmunity has not been as clearly defined or confirmed. The list includes both drug and nondrug chemicals. The heterogeneity of these structures and biological activities illustrate the breadth of potential for the induction of chemically mediated autoimmune disease. A number of recent papers have reviewed some specific examples of xenobiotics associated with autoimmune disease (Pollard et al., 2010), including drugs/immunotherapeutics (D’Cruz, 2000; Pichler, 2003; Vial et al., 2002), vaccines (Descotes et al., 2002; Vial and Descotes, 2004), environmental chemicals (D’Cruz, 2000; Hess, 2002), and pesticides (Holsapple, 2002a). A brief discussion of selected drug and nondrug chemicals is provided.

Therapeutic Agents

Methyldopa

Methyldopa is a centrally acting sympatholytic drug that has been widely used for the treatment of essential hypertension; but with the advent of newer antihypertensive drugs, the use of methyldopa has declined. Platelets and erythrocytes are targeted by the immune system in individuals treated with this drug (Garratty, 2010). In the case of thrombocytopenia, antibodies are detected against platelets, which are indicative of immune recognition of a self- or altered self-antigen. Hemolytic anemia occurs in at least 1% of individuals treated with methyldopa, and up to 30% of these individuals develop antibodies to erythrocytes as manifest in a positive Coombs test. Interestingly, the antibodies are not directed against the chemical or a chemical membrane conjugate.

Hydralazine, Isoniazid, and Procainamide

Hydralazine is a direct-acting vasodilator drug used in the treatment of hypertension. Isoniazid is an antimicrobial drug used in the treatment of tuberculosis. Procainamide is a drug that selectively blocks sodium channels in myocardial membranes, making it useful in the treatment of cardiac arrhythmias. All three drugs produce autoimmunity, which is manifested as a SLE-like syndrome. Indeed, procainamide represents one of the best examples for a clear association between exposure to a xenobiotic and the onset or progression of an autoimmune disease. The association between procainamide and the SLE-like condition is based on the finding that the disease remits when the drug is discontinued and recurs when the drug is re-administered. Antibodies to DNA have been detected in individuals showing this syndrome. Studies with hydralazine and isoniazid indicate that the antigenic determinant is myeloperoxidase. Although the actual mechanism by which these drugs induce autoimmunity requires further elucidation, studies suggest a breakdown of central tolerance to low affinity self-antigens in the thymus and altered peripheral tolerance due to inhibition of DNA methylation (reviewed by Chang and Gershwin, 2010). There is no evidence indicating that the immune system is recognizing the chemical or a chemical conjugate. In addition, these drugs have also been shown to produce hypersensitivity responses not associated with the SLE syndrome.

Halothane

Halothane, one of the most widely studied of the drugs inducing autoimmunity, is an inhalation anesthetic that can induce autoimmune hepatitis (Reichle and Conzen, 2003). The incidence of this iatrogenic disease in humans is about 1 in 20,000. The pathogenesis of the hepatitis results from the chemical altering a specific liver protein to such a degree that the immune system recognizes the altered protein and antibodies are produced. Studies using rat microsomes show that halothane has to be oxidized by cytochrome P450 enzymes to trifluoroacetylhalide before it binds to the protein. Investigations indicate that in affected individuals antibodies to specific microsomal proteins are produced.

Vinyl Chloride

Vinyl chloride, which is used in the plastics industry as a refrigerant and in the synthesis of organic chemicals, is a known carcinogen and is also associated with a scleroderma-like syndrome. The disease affects multisystemic collagenous tissues, manifesting itself as pulmonary fibrosis, skin sclerosis, and/or fibrosis of the liver and spleen. Ward et al. reported on 320 exposed workers, showing that 58 (18%) had a scleroderma-like syndrome (Ward et al., 1976). The individuals who showed the disease were in a group genetically similar (ie, HLA-DR5) to patients with classic idiopathic scleroderma. Although the exact mechanism whereby this chemical produces autoimmunity is unclear, it is presumed that vinyl chloride acts as an amino acid and is incorporated into protein. Because this would produce a structurally abnormal protein, which would be antigenic, an immune response would be directed against tissues with the modified protein present.

Other occupational exposures suspected to induce scleroderma-like reactions include solvents, particularly organic solvents; but the evidence is still limited. For example, several epidemiological studies found an increased relative risk for scleroderma-like reactions following exposure to solvents when compared to the general population. However, the association was weak and not reproduced in other studies, and these studies frequently assessed exposure to solvents in general without providing details on specific solvents (Garabrant and Dumas, 2000; Garabrant et al., 2003).

Mercury

This widely used metal is known to have several target systems, including the CNS and renal system. Mercury also has two different actions with respect to the immune system. The first action is direct injury, described previously in the subsection “Metals” under “Immunomodulation by Xenobiotics.” The second action is mercury-induced autoimmune disease that is manifested as glomerular nephropathy. Antibodies produced to laminin are believed to be responsible for damage to the basement membrane of the kidney. Mice and rats exposed to mercury also show antinuclear antibodies. The role of these antibodies in the autoimmune disease is not clear; however, they represent a known biomarker of autoimmunity. Studies in the brown Norway rat point to a mercury-induced autoreactive CD4⁺ cell as being responsible for the polyclonal antibody response. Mercury chloride induces an increase in the expression of MHCII molecules on B lymphocytes as well as shifting the T helper cell population along the Th2 line. It is the Th2 cell that promotes antibody production. The imbalance between Th1 and Th2 cells is believed to be caused by the depletion of cysteine and the reduced form of glutathione in Th1 cells. Reduced glutathione is known to be important in the synthesis of and responsiveness to IL-2 in T cells. Thus, Th1 cells that synthesize and respond to IL-2 would be at a greater risk than Th2 cells.

Mercury-induced autoimmunity has a strong genetic component. This has been extensively studied in the rat and mouse. For example, some strains of rats, such as the Lewis rat, are completely resistant, while others, such as the brown Norway, are exquisitely sensitive. Susceptibility appears to be linked to 3 or 4 genes, one of which is the MHC. A number of reviews addressing the role of mercury in autoimmunity have been prepared (Bigazzi, 1999; Pelletier et al., 1994; Schiraldi and Monestier, 2009).

Silica

Crystalline silica (silicon dioxide) is a primary source of elemental silicon and is used commercially in large quantities as a constituent of building materials, ceramics, concretes, and glasses. Experimental animals as well as humans exposed to silica may have perturbations in the immune system. Depending on the length of exposure, dose, and route of administration of silica, it may kill macrophages or may act as an immunostimulant. Silica has been shown to be associated with an increase in scleroderma in silica-exposed workers (Kilburn and Warshaw, 1994). This effect is believed to be mediated via an adjuvant mechanism. Adjuvancy as a mechanism of causing autoimmunity has been implicated with a number of other chemicals, including paraffin and silicones. Inherent in adjuvancy as a mechanism of producing autoimmunity is that the population affected by these chemicals must already be at risk for the autoimmune disease. This is supported by the data indicating a genetic component to many autoimmune diseases.

Brown and colleagues (2004) developed a model in which apoptosis plays a critical role in silica-induced autoimmune diseases. As described by the authors, inhalation of crystalline silica results in concurrent activation and apoptosis of the alveolar macrophage resulting in an environment of inflammation and apoptosis. This environment may provide excess self-antigen that is further ingested by activated macrophages or DCs that are able to migrate to local lymph nodes. Within these local lymph nodes, these APCs, laden with apoptotic material, activate T cells and B cells thereby inducing an autoimmune response.

Hexachlorobenzene

As noted above, autoimmunity induced by pesticides has been previously reviewed (Holsapple, 2002a). There is little doubt that the pesticide most extensively studied in the context of autoimmunity is hexachlorobenzene. Hexachlorobenzene is a low molecular weight compound that was used in the past as a fungicide for seed grains. Even though its use as a pesticide was prohibited in most countries in the 1970s, it is still generated as a by-product of several industrial processes and trace amounts of hexachlorobenzene are present as contaminants in some chlorine-containing pesticides. Although emissions of hexachlorobenzene have decreased dramatically compared to the 1970s, residues can still be found throughout the environment due to its stability and persistence.

One of the drivers for including hexachlorobenzene in this brief presentation of xenobiotics associated with autoimmune disease is based on an accidental poisoning incident that occurred in Turkey in 1955 to 1959. Approximately, 3000 to 5000 people ingested seed grain contaminated with hexachlorobenzene. Patients developed a disease characterized by hepatic porphyria, called porphyria turcica, which was manifested as bullous skin lesions, mainly in sun-exposed skin that ultimately healed with severe scars. The skin lesions have been attributed to the phototoxicity associated with the elevated levels of porphyrins. In addition to the dermatological changes, other clinical manifestations included neurological symptoms, hepatomegaly, enlarged thyroid, splenomegaly, hyperpigmentation, hirsutism, enlarged lymph nodes, and painful arthritis of the hands. For many of the clinical symptoms exhibited by the victims, an immune etiology was considered.

Indeed, the autoimmunogenic potential of hexachlorobenzene has been characterized in a number of laboratory studies, which have been reviewed (Ezendam et al., 2004; Michielsen et al., 1999). The former review emphasized a striking difference in the profile of activity in rats, where the predominant effect was immune enhancement, and in mice, where the predominant effect was immune suppression (Michielsen et al., 1999). Only the former results will be discussed further in this section. Exposure to a variety of strains of rats produced increases in the following types of parameters: peripheral blood counts, serum IgM and IgG levels, autoantibodies, spleen and lymph node weights, marginal zones and follicles of spleens, and primary and secondary antibody responses to tetanus toxoid. Interestingly, exposure to hexachlorobenzene caused opposite effects on two induced autoimmune models in Lewis rats, causing an increase in the severity of experimental allergic encephalomyelitis, and a decrease in the severity of adjuvant arthritis (Michielsen et al., 1999). These findings suggest that comparative studies using different genetically autoimmune-prone models may be needed to investigate the role of xenobiotics in the onset and progression of autoimmunity.

Concerning a possible mechanism of action, Ezendam et al. (2005), proposed that after exposure to hexachlorobenzene, its deposition can directly induce cell damage or elicit damage by interfering with the integrity of cell membranes due to its lipophilic nature. Ultimately, hexachlorobenzene exposure triggers proinflammatory mediators, such as TNF-α, IL-1, IL-6, ROS, and chemokines. These proinflammatory mediators serve as adjuvant signals that induce a systemic inflammatory response with influxes of neutrophils and macrophages into various nonimmune and immune organs. Subsequently, this leads to polyclonal activation of T and B cells, eosinophilia, and eventually to visible clinical effects.

As noted throughout this chapter, the immune system has unquestionably been identified as a potential target organ for drugs and chemicals. With the demonstration that (1) chemicals can perturb the immune system of animals; (2) perturbation of immune function is correlated with an increased risk of infectious disease; and (3) perturbations in immune function can occur in the absence of any clinically observable effect, attention has focused, and will continue to focus, on the risk to the human population following exposure to chemicals that can alter immune function in animals. In fact, the characterization of the risk associated with xenobiotic-induced immunotoxicity arguably represents one of the key challenges for this discipline in the immediate future.

Risk can be defined as the probability that an adverse event/effect will manifest itself. Risk must also incorporate the hazard, including dose–response relationships, and exposure. Exposure is a function of the amount of chemical involved and the time of its interaction with people and/or the environment. As such, assessment of risk is often an assessment of the probability for exposure. However, most papers in the immunotoxicology literature that are identified as “risk assessment” papers have focused on just one of the above components, most often, hazard identification. Thus, risk assessment in immunotoxicology remains a “New Frontier.”

The science of immunotoxicology continues to evolve, and any overview, including this chapter, must consider the discipline as a “snapshot” in time. Just during the period of time since this chapter was last published, immunotoxicology has experienced significant advancement. This, in part, has been driven by the tremendous growth in knowledge within immunology and cell biology coupled with an explosion in methodological and technological capabilities. New tests reflecting a variety of potential impacts of immunotoxicity have emerged, and traditional tests have been improved. In light of the aforementioned, there are several specific areas within the subdiscipline of immunotoxicology that are currently on the forefront, but will likely see significant advancements and changes. The first, as emphasized earlier in this chapter, will be the continued evolution and application of human primary leukocytes in mechanistic studies of immunotoxicology. In spite of the similarities between the human immune system and that of other animal species, there is an increasing appreciation that often subtle but potentially important differences exist. Advancements in technology, especially flow cytometry have already and will continue to greatly facilitate the application of human primary leukocytes in studies of immunotoxicology. A second area which will see significant changes will be the application of human-derived cell lines, and validation of assays using these cell lines for the purpose of evaluating and screening potential immunotoxicants. A significant driver for broader employment of cell lines in immunotoxicity testing are cost, ethical considerations to reduce the use of animals in toxicity testing, and a fundamental belief that toxicants which alter one or more of the major signaling pathways regulating cell function can be identified in this manner. A recent report by a National Academy of Sciences committee on “Toxicology in the 21st Century” as well as recent initiatives with US Federal agencies such as ToxCast have brought the strategy of using cell line for hazard identification to the forefront. It is highly likely that a significant shift toward the employment of cell lines in immunotoxicology from current strategies utilizing in vivo animal models will require the establishment of a panel of well-characterized cell lines and probably the development of new cell lines in order to make up such a cell line panel. The third area of emphasis in immunotoxicology, as well in other areas of toxicology, will be the application of computational biology to better understand and describe the underlying molecular mechanisms by which an immunotoxicant alters immune function. The application of computational biology has tremendous potential in estimating the potential risk from exposure to immunotoxicants as well as predicting the risk associated with exposure to multiple immunotoxicants simultaneously. The last area on the forefront of immunotoxicology, and in all biomedical disciplines, is increased use of transcriptome analysis. This change will be primarily driven by major advancements and applications of next generation sequencing which will likely make microarrays obsolete due to the significantly greater sensitivity of this technology, decreased cost, and open platform (ie, capable of quantifying the entire transcriptome and not restricted to the DNA tiled on a chip). Moreover, the applications of next generation sequencing beyond studies of the transcriptome are considerable and span uses such as identification of SNPs associated with sensitive subpopulations, and applications in personalized medicine to identification and analysis of DNA methylation for studies of epigenetics.

In spite of these advances, significant challenges remain to be addressed within the discipline of immunotoxicology and include: (1) how to interpret the significance of minor or moderate immunotoxic effects in animal models in relation to human risk assessment; (2) how to better integrate a consideration of exposure, especially to multiple agents simultaneously, into immunotoxicological risk assessment; (3) how to design better human studies to assess the impact on the immune system in the context of risk assessment; (4) how to identify and establish sensitive translational biomarkers of immunotoxicity that can be used to bridge studies conducted in preclinical species to studies in humans; and (5) how to gain a better understanding of the role of genetics in identifying sensitive subpopulations to immune-altering agents. Many of the challenges identified above are not unique to immunotoxicology, but nevertheless are critical, and will need to be addressed through concerted and systematic efforts to improve human immune testing strategies.