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 (Ahbb), which is exquisitely sensitive to TCDD (TCDD responsive), while the DBA/2 mouse strain (Ahdd) 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).
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.
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.
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.
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 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 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).
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).
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, 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 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).
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.
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).
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 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).
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.
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.
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 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.
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).
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.
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.
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.
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.
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 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 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).
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).
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.
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 Gi-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 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 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 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).
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.
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.
Activation of the inflammasome by nanomaterials. Nanomaterials can be adjuvants through effects in APCs. For example, in a macrophage, nanomaterials can induce reactive oxygen species (ROS) to enhance expression of MHCII or B7 proteins or activate the inflammasome. Upon activation, the inflammasome produces active IL-1β and IL-18, which can recruit other monocytes or DCs, or enhance T-cell proliferation.
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 (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.