Chapter 5: Disorders of Granulocytes and Monocytes

Leukocytes, the major cells comprising inflammatory and immune responses, include neutrophils, T and B lymphocytes, natural killer (NK) cells, monocytes, eosinophils, and basophils. These cells have specific functions, such as antibody production by B lymphocytes or destruction of bacteria by neutrophils, but in no single infectious disease is the exact role of the cell types completely established. Thus, whereas neutrophils are classically thought to be critical to host defense against bacteria, they may also play important roles in defense against viral infections.

The blood delivers leukocytes to the various tissues from the bone marrow, where they are produced. Normal blood leukocyte counts are 4.3–10.8 × 10⁹/L, with neutrophils representing 45–74% of the cells, bands 0–4%, lymphocytes 16–45%, monocytes 4–10%, eosinophils 0–7%, and basophils 0–2%. Variation among individuals and among different ethnic groups can be substantial, with lower leukocyte numbers for certain African-American ethnic groups. The various leukocytes are derived from a common stem cell in the bone marrow. Three-fourths of the nucleated cells of bone marrow are committed to the production of leukocytes. Leukocyte maturation in the marrow is under the regulatory control of a number of different factors, known as colony-stimulating factors (CSFs) and interleukins (ILs). Because an alteration in the number and type of leukocytes is often associated with disease processes, total white blood cell (WBC) count (cells per μL) and differential counts are informative. This chapter focuses on neutrophils, monocytes, and eosinophils.

MATURATION

Important events in neutrophil life are summarized in Fig. 5-1. In normal humans, neutrophils are produced only in the bone marrow. The minimum number of stem cells necessary to support hematopoiesis is estimated to be 400–500 at any one time. Human blood monocytes, tissue macrophages, and stromal cells produce CSFs, hormones required for the growth of monocytes and neutrophils in the bone marrow. The hematopoietic system not only produces enough neutrophils (~1.3 × 10¹¹ cells per 80-kg person per day) to carry out physiologic functions but also has a large reserve stored in the marrow, which can be mobilized in response to inflammation or infection. An increase in the number of blood neutrophils is called neutrophilia, and the presence of immature cells is termed a shift to the left. A decrease in the number of blood neutrophils is called neutropenia.

Neutrophils and monocytes evolve from pluripotent stem cells under the influence of cytokines and CSFs (Fig. 5-2). The proliferation phase through the metamyelocyte takes about 1 week, while the maturation phase from metamyelocyte to mature neutrophil takes another week. The myeloblast is the first recognizable precursor cell and is followed by the promyelocyte. The promyelocyte evolves when the classic lysosomal granules, called the primary, or azurophil, granules are produced. The primary granules contain hydrolases, elastase, myeloperoxidase, cathepsin G, cationic proteins, and bactericidal/permeability-increasing protein, which is important for killing gram-negative bacteria. Azurophil granules also contain defensins, a family of cysteine-rich polypeptides with broad antimicrobial activity against bacteria, fungi, and certain enveloped viruses. The promyelocyte divides to produce the myelocyte, a cell responsible for the synthesis of the specific, or secondary, granules, which contain unique (specific) constituents such as lactoferrin, vitamin B₁₂–binding protein, membrane components of the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase required for hydrogen peroxide production, histaminase, and receptors for certain chemoattractants and adherence-promoting factors (CR3) as well as receptors for the basement membrane component, laminin. The secondary granules do not contain acid hydrolases and therefore are not classic lysosomes. Packaging of secondary granule contents during myelopoiesis is controlled by CCAAT/enhancer binding protein-ε. Secondary granule contents are readily released extracellularly, and their mobilization is important in modulating inflammation. During the final stages of maturation, no cell division occurs, and the cell passes through the metamyelocyte stage and then to the band neutrophil with a sausage-shaped nucleus (Fig. 5-3). As the band cell matures, the nucleus assumes a lobulated configuration. The nucleus of neutrophils normally contains up to four segments (Fig. 5-4). Excessive segmentation (more than five nuclear lobes) may be a manifestation of folate or vitamin B₁₂ deficiency or the congenital neutropenia syndrome of warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) described below. The Pelger-Hüet anomaly (Fig. 5-5), an infrequent dominant benign inherited trait, results in neutrophils with distinctive bilobed nuclei that must be distinguished from band forms. Acquired bilobed nuclei, pseudo Pelger-Hüet anomaly, can occur with acute infections or in myelodysplastic syndromes. The physiologic role of the normal multilobed nucleus of neutrophils is unknown, but it may allow great deformation of neutrophils during migration into tissues at sites of inflammation.

In severe acute bacterial infection, prominent neutrophil cytoplasmic granules, called toxic granulations, are occasionally seen. Toxic granulations are immature or abnormally staining azurophil granules. Cytoplasmic inclusions, also called Döhle bodies (Fig. 5-3), can be seen during infection and are fragments of ribosome-rich endoplasmic reticulum. Large neutrophil vacuoles are often present in acute bacterial infection and probably represent pinocytosed (internalized) membrane.

Neutrophils are heterogeneous in function. Monoclonal antibodies have been developed that recognize only a subset of mature neutrophils. The meaning of neutrophil heterogeneity is not known.

The morphology of eosinophils and basophils is shown in Fig. 5-6.

MARROW RELEASE AND CIRCULATING COMPARTMENTS

Specific signals, including IL-1, tumor necrosis factor α (TNF-α), the CSFs, complement fragments, and chemokines, mobilize leukocytes from the bone marrow and deliver them to the blood in an unstimulated state. Under normal conditions, ~90% of the neutrophil pool is in the bone marrow, 2–3% in the circulation, and the remainder in the tissues (Fig. 5-7).

The circulating pool exists in two dynamic compartments: one freely flowing and one marginated. The freely flowing pool is about one-half the neutrophils in the basal state and is composed of those cells that are in the blood and not in contact with the endothelium. Marginated leukocytes are those that are in close physical contact with the endothelium (Fig. 5-8). In the pulmonary circulation, where an extensive capillary bed (~1000 capillaries per alveolus) exists, margination occurs because the capillaries are about the same size as a mature neutrophil. Therefore, neutrophil fluidity and deformability are necessary to make the transit through the pulmonary bed. Increased neutrophil rigidity and decreased deformability lead to augmented neutrophil trapping and margination in the lung. In contrast, in the systemic postcapillary venules, margination is mediated by the interaction of specific cell-surface molecules called selectins. Selectins are glycoproteins expressed on neutrophils and endothelial cells, among others, that cause a low-affinity interaction, resulting in “rolling” of the neutrophil along the endothelial surface. On neutrophils, the molecule L-selectin (cluster determinant [CD] 62L) binds to glycosylated proteins on endothelial cells (e.g., glycosylation-dependent cell adhesion molecule [GlyCAM1] and CD34). Glycoproteins on neutrophils, most importantly sialyl-Lewis^x (SLe^x, CD15s), are targets for binding of selectins expressed on endothelial cells (E-selectin [CD62E] and P-selectin [CD62P]) and other leukocytes. In response to chemotactic stimuli from injured tissues (e.g., complement product C5a, leukotriene B₄, IL-8) or bacterial products (e.g., N-formylmethionylleucylphenylalanine [f-met-leu-phe]), neutrophil adhesiveness increases through mobilization of intracellular adhesion proteins stored in specific granules to the cell surface, and the cells “stick” to the endothelium through integrins. The integrins are leukocyte glycoproteins that exist as complexes of a common CD18 β chain with CD11a (LFA-1), CD11b (called Mac-1, CR3, or the C3bi receptor), and CD11c (called p150,95 or CR4). CD11a/CD18 and CD11b/CD18 bind to specific endothelial receptors (intercellular adhesion molecules [ICAM] 1 and 2).

On cell stimulation, L-selectin is shed from neutrophils, and E-selectin increases in the blood, presumably because it is shed from endothelial cells; receptors for chemoattractants and opsonins are mobilized; and the phagocytes orient toward the chemoattractant source in the extravascular space, increase their motile activity (chemokinesis), and migrate directionally (chemotaxis) into tissues. The process of migration into tissues is called diapedesis and involves the crawling of neutrophils between postcapillary endothelial cells that open junctions between adjacent cells to permit leukocyte passage. Diapedesis involves platelet/endothelial cell adhesion molecule (PECAM) 1 (CD31), which is expressed on both the emigrating leukocyte and the endothelial cells. The endothelial responses (increased blood flow from increased vasodilation and permeability) are mediated by anaphylatoxins (e.g., C3a and C5a) as well as vasodilators such as histamine, bradykinin, serotonin, nitric oxide, vascular endothelial growth factor (VEGF), and prostaglandins E and I. Cytokines regulate some of these processes (e.g., TNF-α induction of VEGF, interferon [IFN] γ inhibition of prostaglandin E).

In the healthy adult, most neutrophils leave the body by migration through the mucous membrane of the gastrointestinal tract. Normally, neutrophils spend a short time in the circulation (half-life, 6–7 h). Senescent neutrophils are cleared from the circulation by macrophages in the lung and spleen. Once in the tissues, neutrophils release enzymes, such as collagenase and elastase, which may help establish abscess cavities. Neutrophils ingest pathogenic materials that have been opsonized by IgG and C3b. Fibronectin and the tetrapeptide tuftsin also facilitate phagocytosis.

With phagocytosis comes a burst of oxygen consumption and activation of the hexose-monophosphate shunt. A membrane-associated NADPH oxidase, consisting of membrane and cytosolic components, is assembled and catalyzes the univalent reduction of oxygen to superoxide anion, which is then converted by superoxide dismutase to hydrogen peroxide and other toxic oxygen products (e.g., hydroxyl radical). Hydrogen peroxide + chloride + neutrophil myeloperoxidase generate hypochlorous acid (bleach), hypochlorite, and chlorine. These products oxidize and halogenate microorganisms and tumor cells and, when uncontrolled, can damage host tissue. Strongly cationic proteins, defensins, elastase, cathepsins, and probably nitric oxide also participate in microbial killing. Lactoferrin chelates iron, an important growth factor for microorganisms, especially fungi. Other enzymes, such as lysozyme and acid proteases, help digest microbial debris. After 1–4 days in tissues, neutrophils die. The apoptosis of neutrophils is also cytokine-regulated; granulocyte colony-stimulating factor (G-CSF) and IFN-γ prolong their life span. Under certain conditions, such as in delayed-type hypersensitivity, monocyte accumulation occurs within 6–12 h of initiation of inflammation. Neutrophils, monocytes, microorganisms in various states of digestion, and altered local tissue cells make up the inflammatory exudate, pus. Myeloperoxidase confers the characteristic green color to pus and may participate in turning off the inflammatory process by inactivating chemoattractants and immobilizing phagocytic cells.

Neutrophils respond to certain cytokines (IFN-γ, granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-8) and produce cytokines and chemotactic signals (TNF-α, IL-8, macrophage inflammatory protein [MIP] 1) that modulate the inflammatory response. In the presence of fibrinogen, f-met-leu-phe or leukotriene B₄ induces IL-8 production by neutrophils, providing autocrine amplification of inflammation. Chemokines (chemoattractant cytokines) are small proteins produced by many different cell types, including endothelial cells, fibroblasts, epithelial cells, neutrophils, and monocytes, that regulate neutrophil, monocyte, eosinophil, and lymphocyte recruitment and activation. Chemokines transduce their signals through heterotrimeric G protein–linked receptors that have seven cell membrane–spanning domains, the same type of cell-surface receptor that mediates the response to the classic chemoattractants f-met-leu-phe and C5a. Four major groups of chemokines are recognized based on the cysteine structure near the N terminus: C, CC, CXC, and CXXXC. The CXC cytokines such as IL-8 mainly attract neutrophils; CC chemokines such as MIP-1 attract lymphocytes, monocytes, eosinophils, and basophils; the C chemokine lymphotactin is T cell tropic; the CXXXC chemokine fractalkine attracts neutrophils, monocytes, and T cells. These molecules and their receptors not only regulate the trafficking and activation of inflammatory cells, but specific chemokine receptors also serve as co-receptors for HIV infection (Chap. 226) and have a role in other viral infections such as West Nile infection and atherogenesis.

NEUTROPHIL ABNORMALITIES

Defects in the neutrophil life cycle can lead to dysfunction and compromised host defenses. Inflammation is often depressed, and the clinical result is often recurrent, severe bacterial and fungal infections. Aphthous ulcers of mucous membranes (gray ulcers without pus) and gingivitis and periodontal disease suggest a phagocytic cell disorder. Patients with congenital phagocyte defects can have infections within the first few days of life. Skin, ear, upper and lower respiratory tract, and bone infections are common. Sepsis and meningitis are rare. In some disorders, the frequency of infection is variable, and patients can go for months or even years without major infection. Aggressive management of these congenital diseases has extended the life span of patients well beyond 30 years.

Neutropenia

The consequences of absent neutrophils are dramatic. Susceptibility to infectious diseases increases sharply when neutrophil counts fall below 1000 cells/μL. When the absolute neutrophil count (ANC; band forms and mature neutrophils combined) falls to <500 cells/μL, control of endogenous microbial flora (e.g., mouth, gut) is impaired; when the ANC is <200/μL, the local inflammatory process is absent. Neutropenia can be due to depressed production, increased peripheral destruction, or excessive peripheral pooling. A falling neutrophil count or a significant decrease in the number of neutrophils below steady-state levels, together with a failure to increase neutrophil counts in the setting of infection or other challenge, requires investigation. Acute neutropenia, such as that caused by cancer chemotherapy, is more likely to be associated with increased risk of infection than neutropenia of long duration (months to years) that reverses in response to infection or carefully controlled administration of endotoxin (see “Laboratory Diagnosis and Management,” below).

Some causes of inherited and acquired neutropenia are listed in Table 5-1. The most common neutropenias are iatrogenic, resulting from the use of cytotoxic or immunosuppressive therapies for malignancy or control of autoimmune disorders. These drugs cause neutropenia because they result in decreased production of rapidly growing progenitor (stem) cells of the marrow. Certain antibiotics such as chloramphenicol, trimethoprim-sulfamethoxazole, flucytosine, vidarabine, and the antiretroviral drug zidovudine may cause neutropenia by inhibiting proliferation of myeloid precursors. Azathioprine and 6-mercaptopurine are metabolized by the enzyme thiopurine methyl­transferase (TPMT), hypofunctional polymorphisms in which are found in 11% of whites and can lead to accumulation of 6-thioguanine and profound marrow toxicity. The marrow suppression is generally dose-related and dependent on continued administration of the drug. Cessation of the offending agent and recombinant human G-CSF usually reverse these forms of neutropenia.

Another important mechanism for iatrogenic neutropenia is the effect of drugs that serve as immune haptens and sensitize neutrophils or neutrophil precursors to immune-mediated peripheral destruction. This form of drug-induced neutropenia can be seen within 7 days of exposure to the drug; with previous drug exposure, resulting in preexisting antibodies, neutropenia may occur a few hours after administration of the drug. Although any drug can cause this form of neutropenia, the most frequent causes are commonly used antibiotics, such as sulfa-containing compounds, penicillins, and cephalosporins. Fever and eosinophilia may also be associated with drug reactions, but often these signs are not present. Drug-induced neutropenia can be severe, but discontinuation of the sensitizing drug is sufficient for recovery, which is usually seen within 5–7 days and is complete by 10 days. Readministration of the sensitizing drug should be avoided, because abrupt neutropenia will often result. For this reason, diagnostic challenge should be avoided.

Autoimmune neutropenias caused by circulating antineutrophil antibodies are another form of acquired neutropenia that results in increased destruction of neutrophils. Acquired neutropenia may also be seen with viral infections, including infection with HIV. Acquired neutropenia may be cyclic in nature, occurring at intervals of several weeks. Acquired cyclic or stable neutropenia may be associated with an expansion of large granular lymphocytes (LGLs), which may be T cells, NK cells, or NK-like cells. Patients with large granular lymphocytosis may have moderate blood and bone marrow lymphocytosis, neutropenia, polyclonal hypergammaglobulinemia, splenomegaly, rheumatoid arthritis, and absence of lymphadenopathy. Such patients may have a chronic and relatively stable course. Recurrent bacterial infections are frequent. Benign and malignant forms of this syndrome occur. In some patients, a spontaneous regression has occurred even after 11 years, suggesting an immunoregulatory defect as the basis for at least one form of the disorder. Glucocorticoids, cyclosporine, and methotrexate are commonly used to manage these cytopenias.

Hereditary neutropenias

Hereditary neutropenias are rare and may manifest in early childhood as a profound constant neutropenia or agranulocytosis. Congenital forms of neutropenia include Kostmann’s syndrome (neutrophil count <100/μL), which is often fatal and due to mutations in the antiapoptosis gene HAX-1; severe chronic neutropenia (neutrophil count of 300–1500/μL) due to mutations in neutrophil elastase (ELANE); hereditary cyclic neutropenia, or, more appropriately, cyclic hematopoiesis, also due to mutations in neutrophil elastase (ELANE); the cartilage-hair hypoplasia syndrome due to mutations in the mitochondrial RNA-processing endoribonuclease RMRP; Shwachman-Diamond syndrome associated with pancreatic insufficiency due to mutations in the Shwachman-Bodian-Diamond syndrome gene SBDS; the WHIM (warts, hypogammaglobulinemia, infections, myelokathexis [retention of WBCs in the marrow]) syndrome, characterized by neutrophil hypersegmentation and bone marrow myeloid arrest due to mutations in the chemokine receptor CXCR4; and neutropenias associated with other immune defects, such as X-linked agammaglobulinemia, Wiskott-Aldrich syndrome, and CD40 ligand deficiency. Mutations in the G-CSF receptor can develop in severe congenital neutropenia and are linked to leukemia. Absence of both myeloid and lymphoid cells is seen in reticular dysgenesis, due to mutations in the nuclear genome-encoded mitochondrial enzyme adenylate kinase-2 (AK2).

Maternal factors can be associated with neutropenia in the newborn. Transplacental transfer of IgG directed against antigens on fetal neutrophils can result in peripheral destruction. Drugs (e.g., thiazides) ingested during pregnancy can cause neutropenia in the newborn by either depressed production or peripheral destruction.

In Felty’s syndrome—the triad of rheumatoid arthritis, splenomegaly, and neutropenia—spleen-produced antibodies can shorten neutrophil life span, while large granular lymphocytes can attack marrow neutrophil precursors. Splenectomy may increase the neutrophil count in Felty’s syndrome and lower serum neutrophil-binding IgG. Some Felty’s syndrome patients also have neutropenia associated with an increased number of LGLs. Splenomegaly with peripheral trapping and destruction of neutrophils is also seen in lysosomal storage diseases and in portal hypertension.

Neutrophilia

Neutrophilia results from increased neutrophil production, increased marrow release, or defective margination (Table 5-2). The most important acute cause of neutrophilia is infection. Neutrophilia from acute infection represents both increased production and increased marrow release. Increased production is also associated with chronic inflammation and certain myeloproliferative diseases. Increased marrow release and mobilization of the marginated leukocyte pool are induced by glucocorticoids. Release of epinephrine, as with vigorous exercise, excitement, or stress, will demarginate neutrophils in the spleen and lungs and double the neutrophil count in minutes. Cigarette smoking can elevate neutrophil counts above the normal range. Leukocytosis with cell counts of 10,000–25,000/μL occurs in response to infection and other forms of acute inflammation and results from both release of the marginated pool and mobilization of marrow reserves. Persistent neutrophilia with cell counts of ≥30,000–50,000/μL is called a leukemoid reaction, a term often used to distinguish this degree of neutrophilia from leukemia. In a leukemoid reaction, the circulating neutrophils are usually mature and not clonally derived.

Abnormal neutrophil function

Inherited and acquired abnormalities of phagocyte function are listed in Table 5-3. The resulting diseases are best considered in terms of the functional defects of adherence, chemotaxis, and microbicidal activity. The distinguishing features of the important inherited disorders of phagocyte function are shown in Table 5-4.

Disorders of adhesion

Three main types of leukocyte adhesion deficiency (LAD) have been described. All are autosomal recessive and result in the inability of neutrophils to exit the circulation to sites of infection, leading to leukocytosis and increased susceptibility to infection (Fig. 5-8). Patients with LAD 1 have mutations in CD18, the common component of the integrins LFA-1, Mac-1, and p150,95, leading to a defect in tight adhesion between neutrophils and the endothelium. The heterodimer formed by CD18/CD11b (Mac-1) is also the receptor for the complement-derived opsonin C3bi (CR3). The CD18 gene is located on distal chromosome 21q. The severity of the defect determines the severity of clinical disease. Complete lack of expression of the leukocyte integrins results in a severe phenotype in which inflammatory stimuli do not increase the expression of leukocyte integrins on neutrophils or activated T and B cells. Neutrophils (and monocytes) from patients with LAD 1 adhere poorly to endothelial cells and protein-coated surfaces and exhibit defective spreading, aggregation, and chemotaxis. Patients with LAD 1 have recurrent bacterial infections involving the skin, oral and genital mucosa, and respiratory and intestinal tracts; persistent leukocytosis (resting neutrophil counts of 15,000–20,000/μL) because cells do not marginate; and, in severe cases, a history of delayed separation of the umbilical stump. Infections, especially of the skin, may become necrotic with progressively enlarging borders, slow healing, and development of dysplastic scars. The most common bacteria are Staphylococcus aureus and enteric gram-negative bacteria. LAD 2 is caused by an abnormality of fucosylation of SLe^x (CD15s), the ligand on neutrophils that interacts with selectins on endothelial cells and is responsible for neutrophil rolling along the endothelium. Infection susceptibility in LAD 2 appears to be less severe than in LAD 1. LAD 2 is also known as congenital disorder of glycosylation IIc (CDGIIc) due to mutation in a GDP-fucose transporter (SLC35C1). LAD 3 is characterized by infection susceptibility, leukocytosis, and petechial hemorrhage due to impaired integrin activation caused by mutations in the gene FERMT3.

Disorders of neutrophil granules

The most common neutrophil defect is myeloperoxidase deficiency, a primary granule defect inherited as an autosomal recessive trait; the incidence is ~1 in 2000 persons. Isolated myeloperoxidase deficiency is not associated with clinically compromised defenses, presumably because other defense systems such as hydrogen peroxide generation are amplified. Microbicidal activity of neutrophils is delayed but not absent. Myeloperoxidase deficiency may make other acquired host defense defects more serious, and patients with myeloperoxidase deficiency and diabetes are more susceptible to Candida infections. An acquired form of myeloperoxidase deficiency occurs in myelomonocytic leukemia and acute myeloid leukemia.

Chédiak-Higashi syndrome (CHS) is a rare disease with autosomal recessive inheritance due to defects in the lysosomal transport protein LYST, encoded by the gene CHS1 at 1q42. This protein is required for normal packaging and disbursement of granules. Neutrophils (and all cells containing lysosomes) from patients with CHS characteristically have large granules (Fig. 5-9), making it a systemic disease. Patients with CHS have nystagmus, partial oculocutaneous albinism, and an increased number of infections resulting from many bacterial agents. Some CHS patients develop an “accelerated phase” in childhood with a hemophagocytic syndrome and an aggressive lymphoma requiring bone marrow transplantation. CHS neutrophils and monocytes have impaired chemotaxis and abnormal rates of microbial killing due to slow rates of fusion of the lysosomal granules with phagosomes. NK cell function is also impaired. CHS patients may develop a severe disabling peripheral neuropathy in adulthood that can lead to bed confinement.

Specific granule deficiency is a rare autosomal recessive disease in which the production of secondary granules and their contents, as well as the primary granule component defensins, is defective. The defect in killing leads to severe bacterial infections. One type of specific granule deficiency is due to a mutation in the CCAAT/enhancer binding protein-ε, a regulator of expression of granule components. A dominant mutation in C/EBP-ε has also been described.

Chronic granulomatous disease

Chronic granulomatous disease (CGD) is a group of disorders of granulocyte and monocyte oxidative metabolism. Although CGD is rare, with an incidence of ~1 in 200,000 individuals, it is an important model of defective neutrophil oxidative metabolism. In about two-thirds of patients, CGD is inherited as an X-linked recessive trait; 30% of patients inherit the disease in an autosomal recessive pattern. Mutations in the genes for the five proteins that assemble at the plasma membrane account for all patients with CGD. Two proteins (a 91-kDa protein, abnormal in X-linked CGD, and a 22-kDa protein, absent in one form of autosomal recessive CGD) form the heterodimer cytochrome b-558 in the plasma membrane. Three other proteins (40, 47, and 67 kDa, abnormal in the other autosomal recessive forms of CGD) are cytoplasmic in origin and interact with the cytochrome after cell activation to form NADPH oxidase, required for hydrogen peroxide production. Leukocytes from patients with CGD have severely diminished hydrogen peroxide production. The genes involved in each of the defects have been cloned and sequenced and the chromosome locations identified. Patients with CGD characteristically have increased numbers of infections due to catalase-positive microorganisms (organisms that destroy their own hydrogen peroxide) such as S. aureus, Burkholderia cepacia, and Aspergillus species. When patients with CGD become infected, they often have extensive inflammatory reactions, and lymph node suppuration is common despite the administration of appropriate antibiotics. Aphthous ulcers and chronic inflammation of the nares are often present. Granulomas are frequent and can obstruct the gastrointestinal or genitourinary tracts. The excessive inflammation is due to failure to downregulate inflammation, reflecting failure to inhibit the synthesis of, degradation of, or response to chemoattractants or residual antigens, leading to persistent neutrophil accumulation. Impaired killing of intracellular microorganisms by macrophages may lead to persistent cell-mediated immune activation and granuloma formation. Autoimmune complications such as immune thrombocytopenic purpura and juvenile rheumatoid arthritis are also increased in CGD. In addition, for unexplained reasons, discoid lupus is more common in X-linked carriers. Late complications, including nodular regenerative hyperplasia and portal hypertension, are increasingly recognized in long-term survivors of severe CGD.

Disorders of phagocyte activation

Phagocytes depend on cell-surface stimulation to induce signals that evoke multiple levels of the inflammatory response, including cytokine synthesis, chemotaxis, and antigen presentation. Mutations affecting the major pathway that signals through NF-κB have been noted in patients with a variety of infection susceptibility syndromes. If the defects are at a very late stage of signal transduction, in the protein critical for NF-κB activation known as the NF-κB essential modulator (NEMO), then affected males develop ectodermal dysplasia and severe immune deficiency with susceptibility to bacteria, fungi, mycobacteria, and viruses. If the defects in NF-κB activation are closer to the cell-surface receptors, in the proteins transducing Toll-like receptor signals, IL-1 receptor–associated kinase 4 (IRAK4), and myeloid differentiation primary response gene 88 (MyD88), then children have a marked susceptibility to pyogenic infections early in life but develop resistance to infection later.

The mononuclear phagocyte system is composed of monoblasts, promonocytes, and monocytes, in addition to the structurally diverse tissue macrophages that make up what was previously referred to as the reticuloendothelial system. Macrophages are long-lived phagocytic cells capable of many of the functions of neutrophils. They are also secretory cells that participate in many immunologic and inflammatory processes distinct from neutrophils. Monocytes leave the circulation by diapedesis more slowly than neutrophils and have a half-life in the blood of 12–24 h.

After blood monocytes arrive in the tissues, they differentiate into macrophages (“big eaters”) with specialized functions suited for specific anatomic locations. Macrophages are particularly abundant in capillary walls of the lung, spleen, liver, and bone marrow, where they function to remove microorganisms and other noxious elements from the blood. Alveolar macrophages, liver Kupffer cells, splenic macrophages, peritoneal macrophages, bone marrow macrophages, lymphatic macrophages, brain microglial cells, and dendritic macrophages all have specialized functions. Macrophage-secreted products include lysozyme, neutral proteases, acid hydrolases, arginase, complement components, enzyme inhibitors (plasmin, α₂-macroglobulin), binding proteins (transferrin, fibronectin, transcobalamin II), nucleosides, and cytokines (TNF-α; IL-1, -8, -12, -18). IL-1 has many functions, including initiating fever in the hypothalamus, mobilizing leukocytes from the bone marrow, and activating lymphocytes and neutrophils. TNF-α is a pyrogen that duplicates many of the actions of IL-1 and plays an important role in the pathogenesis of gram-negative shock. TNF-α stimulates production of hydrogen peroxide and related toxic oxygen species by macrophages and neutrophils. In addition, TNF-α induces catabolic changes that contribute to the profound wasting (cachexia) associated with many chronic diseases.

Other macrophage-secreted products include reactive oxygen and nitrogen metabolites, bioactive lipids (arachidonic acid metabolites and platelet-activating factors), chemokines, CSFs, and factors stimulating fibroblast and vessel proliferation. Macrophages help regulate the replication of lymphocytes and participate in the killing of tumors, viruses, and certain bacteria (Mycobacterium tuberculosis and Listeria monocytogenes). Macrophages are key effector cells in the elimination of intracellular microorganisms. Their ability to fuse to form giant cells that coalesce into granulomas in response to some inflammatory stimuli is important in the elimination of intracellular microbes and is under the control of IFN-γ. Nitric oxide induced by IFN-γ is an important effector against intracellular parasites, including tuberculosis and Leishmania.

Macrophages play an important role in the immune response. They process and present antigen to lymphocytes and secrete cytokines that modulate and direct lymphocyte development and function. Macrophages participate in autoimmune phenomena by removing immune complexes and other substances from the circulation. Polymorphisms in macrophage receptors for immunoglobulin (FcγRII) determine susceptibility to some infections and autoimmune diseases. In wound healing, they dispose of senescent cells, and they contribute to atheroma development. Macrophage elastase mediates development of emphysema from cigarette smoking.

DISORDERS OF THE MONONUCLEAR PHAGOCYTE SYSTEM

Many disorders of neutrophils extend to mononuclear phagocytes. Monocytosis is associated with tuberculosis, brucellosis, subacute bacterial endocarditis, Rocky Mountain spotted fever, malaria, and visceral leishmaniasis (kala azar). Monocytosis also occurs with malignancies, leukemias, myeloproliferative syndromes, hemolytic anemias, chronic idiopathic neutropenias, and granulomatous diseases such as sarcoidosis, regional enteritis, and some collagen vascular diseases. Patients with LAD, hyperimmunoglobulin E–recurrent infection (Job’s) syndrome, CHS, and CGD all have defects in the mononuclear phagocyte system.

Monocyte cytokine production or response is impaired in some patients with disseminated nontuberculous mycobacterial infection who are not infected with HIV. Genetic defects in the pathways regulated by IFN-γ and IL-12 lead to impaired killing of intracellular bacteria, mycobacteria, salmonellae, and certain viruses (Fig. 5-10).

Certain viral infections impair mononuclear phagocyte function. For example, influenza virus infection causes abnormal monocyte chemotaxis. Mononuclear phagocytes can be infected by HIV using CCR5, the chemokine receptor that acts as a co-receptor with CD4 for HIV. T lymphocytes produce IFN-γ, which induces FcR expression and phagocytosis and stimulates hydrogen peroxide production by mononuclear phagocytes and neutrophils. In certain diseases, such as AIDS, IFN-γ production may be deficient, whereas in other diseases, such as T cell lymphomas, excessive release of IFN-γ may be associated with erythrophagocytosis by splenic macrophages.

Autoinflammatory diseases are characterized by abnormal cytokine regulation, leading to excess inflammation in the absence of infection. These diseases can mimic infectious or immunodeficient syndromes. Gain-of-function mutations in the TNF-α receptor cause TNF-α receptor–associated periodic syndrome (TRAPS), which is characterized by recurrent fever in the absence of infection, due to persistent stimulation of the TNF-α receptor. Diseases with abnormal IL-1 regulation leading to fever include familial Mediterranean fever due to mutations in PYRIN. Mutations in cold-induced autoinflammatory syndrome 1 (CIAS1) lead to neonatal-onset multisystem autoinflammatory disease, familial cold urticaria, and Muckle-Wells syndrome. The syndrome of pyoderma gangrenosum, acne, and sterile pyogenic arthritis (PAPA syndrome) is caused by mutations in PSTPIP1. In contrast to these syndromes of overexpression of proinflammatory cytokines, blockade of TNF-α by the antagonists infliximab, adalimumab, certolizumab, golimumab, or etanercept has been associated with severe infections due to tuberculosis, nontuberculous mycobacteria, and fungi.

Monocytopenia occurs with acute infections, with stress, and after treatment with glucocorticoids. Drugs that suppress neutrophil production in the bone marrow can cause monocytopenia. Persistent severe circulating monocytopenia is seen in GATA2 deficiency, even though macrophages are found at the sites of inflammation. Monocytopenia also occurs in aplastic anemia, hairy cell leukemia, acute myeloid leukemia, and as a direct result of myelotoxic drugs.

Eosinophils and neutrophils share similar morphology, many lysosomal constituents, phagocytic capacity, and oxidative metabolism. Eosinophils express a specific chemoattractant receptor and respond to a specific chemokine, eotaxin, but little is known about their required role. Eosinophils are much longer lived than neutrophils, and unlike neutrophils, tissue eosinophils can recirculate. During most infections, eosinophils appear unimportant. However, in invasive helminthic infections, such as hookworm, schistosomiasis, strongyloidiasis, toxocariasis, trichinosis, filariasis, echinococcosis, and cysticercosis, the eosinophil plays a central role in host defense. Eosinophils are associated with bronchial asthma, cutaneous allergic reactions, and other hypersensitivity states.

The distinctive feature of the red-staining (Wright’s stain) eosinophil granule is its crystalline core consisting of an arginine-rich protein (major basic protein) with histaminase activity, important in host defense against parasites. Eosinophil granules also contain a unique eosinophil peroxidase that catalyzes the oxidation of many substances by hydrogen peroxide and may facilitate killing of microorganisms.

Eosinophil peroxidase, in the presence of hydrogen peroxide and halide, initiates mast cell secretion in vitro and thereby promotes inflammation. Eosinophils contain cationic proteins, some of which bind to heparin and reduce its anticoagulant activity. Eosinophil-derived neurotoxin and eosinophil cationic protein are ribonucleases that can kill respiratory syncytial virus. Eosinophil cytoplasm contains Charcot-Leyden crystal protein, a hexagonal bipyramidal crystal first observed in a patient with leukemia and then in sputum of patients with asthma; this protein is lysophospholipase and may function to detoxify certain lysophospholipids.

Several factors enhance the eosinophil’s function in host defense. T cell–derived factors enhance the ability of eosinophils to kill parasites. Mast cell–derived eosinophil chemotactic factor of anaphylaxis (ECFa) increases the number of eosinophil complement receptors and enhances eosinophil killing of parasites. Eosinophil CSFs (e.g., IL-5) produced by macrophages increase eosinophil production in the bone marrow and activate eosinophils to kill parasites.

EOSINOPHILIA

Eosinophilia is the presence of >500 eosinophils per μL of blood and is common in many settings besides parasite infection. Significant tissue eosinophilia can occur without an elevated blood count. A common cause of eosinophilia is allergic reaction to drugs (iodides, aspirin, sulfonamides, nitrofurantoin, penicillins, and cephalosporins). Allergies such as hay fever, asthma, eczema, serum sickness, allergic vasculitis, and pemphigus are associated with eosinophilia. Eosinophilia also occurs in collagen vascular diseases (e.g., rheumatoid arthritis, eosinophilic fasciitis, allergic angiitis, and periarteritis nodosa) and malignancies (e.g., Hodgkin’s disease; mycosis fungoides; chronic myeloid leukemia; and cancer of the lung, stomach, pancreas, ovary, or uterus), as well as in Job’s syndrome, DOCK8 deficiency (see below), and CGD. Eosinophilia is commonly present in helminthic infections. IL-5 is the dominant eosinophil growth factor. Therapeutic administration of the cytokines IL-2 or GM-CSF frequently leads to transient eosinophilia. The most dramatic hypereosinophilic syndromes are Loeffler’s syndrome, tropical pulmonary eosinophilia, Loeffler’s endocarditis, eosinophilic leukemia, and idiopathic hypereosinophilic syndrome (50,000–100,000/μL). IL-5 is the dominant eosinophil growth factor and can be specifically inhibited with the monoclonal antibody mepolizumab.

The idiopathic hypereosinophilic syndrome represents a heterogeneous group of disorders with the common feature of prolonged eosinophilia of unknown cause and organ system dysfunction, including the heart, central nervous system, kidneys, lungs, gastrointestinal tract, and skin. The bone marrow is involved in all affected individuals, but the most severe complications involve the heart and central nervous system. Clinical manifestations and organ dysfunction are highly variable. Eosinophils are found in the involved tissues and likely cause tissue damage by local deposition of toxic eosinophil proteins such as eosinophil cationic protein and major basic protein. In the heart, the pathologic changes lead to thrombosis, endocardial fibrosis, and restrictive endomyocardiopathy. The damage to tissues in other organ systems is similar. Some cases are due to mutations involving the platelet-derived growth factor receptor, and these are extremely sensitive to the tyrosine kinase inhibitor imatinib. Glucocorticoids, hydroxyurea, and IFN-α each have been used successfully, as have therapeutic antibodies against IL-5. Cardiovascular complications are managed aggressively.

The eosinophilia-myalgia syndrome is a multisystem disease, with prominent cutaneous, hematologic, and visceral manifestations, that frequently evolves into a chronic course and can occasionally be fatal. The syndrome is characterized by eosinophilia (eosinophil count >1000/μL) and generalized disabling myalgias without other recognized causes. Eosinophilic fasciitis, pneumonitis, and myocarditis; neuropathy culminating in respiratory failure; and encephalopathy may occur. The disease is caused by ingesting contaminants in L-tryptophan–containing products. Eosinophils, lymphocytes, macrophages, and fibroblasts accumulate in the affected tissues, but their role in pathogenesis is unclear. Activation of eosinophils and fibroblasts and the deposition of eosinophil-derived toxic proteins in affected tissues may contribute. IL-5 and transforming growth factor β have been implicated as potential mediators. Treatment is withdrawal of products containing L-tryptophan and the administration of glucocorticoids. Most patients recover fully, remain stable, or show slow recovery, but the disease can be fatal in up to 5% of patients.

Eosinophilic neoplasms are discussed in Chapter 17.

EOSINOPENIA

Eosinopenia occurs with stress, such as acute bacterial infection, and after treatment with glucocorticoids. The mechanism of eosinopenia of acute bacterial infection is unknown but is independent of endogenous glucocorticoids, because it occurs in animals after total adrenalectomy. There is no known adverse effect of eosinopenia.

The hyperimmunoglobulin E–recurrent infection syndrome, or Job’s syndrome, is a rare multisystem disease in which the immune and somatic systems are affected, including neutrophils, monocytes, T cells, B cells, and osteoclasts. Autosomal dominant mutations in signal transducer and activator of transcription 3 (STAT3) lead to inhibition of normal STAT signaling with broad and profound effects. Patients have characteristic facies with broad nose, kyphoscoliosis, and eczema. The primary teeth erupt normally but do not deciduate, often requiring extraction. Patients develop recurrent sinopulmonary and cutaneous infections that tend to be much less inflamed than appropriate for the degree of infection and have been referred to as “cold abscesses.” Characteristically, pneumonias cavitate, leading to pneumatoceles. Coronary artery aneurysms are common, as are cerebral demyelinated plaques that accumulate with age. Importantly, IL-17–producing T cells, which are thought responsible for protection against extracellular and mucosal infections, are profoundly reduced in Job’s syndrome. Despite very high IgE levels, these patients do not have elevated levels of allergy. An important syndrome with clinical overlap with STAT3 deficiency is due to autosomal recessive defects in dedicator of cytokinesis 8 (DOCK8). In DOCK8 deficiency, IgE elevation is joined to severe allergy, viral susceptibility, and increased rates of cancer.

Initial studies of WBC and differential and often a bone marrow examination may be followed by assessment of bone marrow reserves (steroid challenge test), marginated circulating pool of cells (epinephrine challenge test), and marginating ability (endotoxin challenge test) (Fig. 5-7). In vivo assessment of inflammation is possible with a Rebuck skin window test or an in vivo skin blister assay, which measures the ability of leukocytes and inflammatory mediators to accumulate locally in the skin. In vitro tests of phagocyte aggregation, adherence, chemotaxis, phagocytosis, degranulation, and microbicidal activity (for S. aureus) may help pinpoint cellular or humoral lesions. Deficiencies of oxidative metabolism are detected with either the nitroblue tetrazolium (NBT) dye test or the dihydrorhodamine (DHR) oxidation test. These tests are based on the ability of products of oxidative metabolism to alter the oxidation states of reporter molecules so that they can be detected microscopically (NBT) or by flow cytometry (DHR). Qualitative studies of superoxide and hydrogen peroxide production may further define neutrophil oxidative function.

Patients with leukopenias or leukocyte dysfunction often have delayed inflammatory responses. Therefore, clinical manifestations may be minimal despite overwhelming infection, and unusual infections must always be suspected. Early signs of infection demand prompt, aggressive culturing for microorganisms, use of antibiotics, and surgical drainage of abscesses. Prolonged courses of antibiotics are often required. In patients with CGD, prophylactic antibiotics (tri­methoprim-sulfamethoxazole) and antifungals (itraconazole) markedly diminish the frequency of life-threatening infections. Glucocorticoids may relieve gastrointestinal or genitourinary tract obstruction by granulomas in patients with CGD. Although TNF-α-blocking agents may markedly relieve inflammatory bowel symptoms, extreme caution must be exercised in their use in CGD inflammatory bowel disease, because it profoundly increases these patients’ already heightened susceptibility to infection. Recombinant human IFN-γ, which nonspecifically stimulates phagocytic cell function, reduces the frequency of infections in patients with CGD by 70% and reduces the severity of infection. This effect of IFN-γ in CGD is additive to the effect of prophylactic antibiotics. The recommended dose is 50 μg/m² subcutaneously three times weekly. IFN-γ has also been used successfully in the treatment of leprosy, nontuberculous mycobacteria, and visceral leishmaniasis.

Rigorous oral hygiene reduces but does not eliminate the discomfort of gingivitis, periodontal disease, and aphthous ulcers; chlorhexidine mouthwash and tooth brushing with a hydrogen peroxide–sodium bicarbonate paste help many patients. Oral antifungal agents (fluconazole, itraconazole, voriconazole, posaconazole) have reduced mucocutaneous candidiasis in patients with Job’s syndrome. Androgens, glucocorticoids, lithium, and immunosuppressive therapy have been used to restore myelopoiesis in patients with neutropenia due to impaired production. Recombinant G-CSF is useful in the management of certain forms of neutropenia due to depressed neutrophil production, including those related to cancer chemotherapy. Patients with chronic neutropenia with evidence of a good bone marrow reserve need not receive prophylactic antibiotics. Patients with chronic or cyclic neutrophil counts <500/μL may benefit from prophylactic antibiotics and G-CSF during periods of neutropenia. Oral trimethoprim-sulfamethoxazole (160/800 mg) twice daily can prevent infection. Increased numbers of fungal infections are not seen in patients with CGD on this regimen. Oral quinolones such as levofloxacin and ciprofloxacin are alternatives.

In the setting of cytotoxic chemotherapy with severe, persistent lymphocyte dysfunction, trimethoprim-sulfamethoxazole prevents Pneumocystis jiroveci pneumonia. These patients, and patients with phagocytic cell dysfunction, should avoid heavy exposure to airborne soil, dust, or decaying matter (mulch, manure), which are often rich in Nocardia and the spores of Aspergillus and other fungi. Restriction of activities or social contact has no proven role in reducing risk of infection for phagocyte defects.

Although aggressive medical care for many patients with phagocytic disorders can allow them to go for years without a life-threatening infection, there may still be delayed effects of prolonged antimicrobials and other inflammatory complications. Cure of most congenital phagocyte defects is possible by bone marrow transplantation, and rates of success are improving (Chap. 31). The identification of specific gene defects in patients with LAD 1, CGD, and other immunodeficiencies has led to gene therapy trials in a number of genetic white cell disorders.