Phagocyte System and Disorders of Granulopoiesis and Granulocyte Function

Neutrophils

The neutrophil life span is traditionally divided into the bone marrow, circulating, and tissue phases. Approximately 14 days are spent in the bone marrow, where proliferation and the early stages of neutrophil differentiation are followed by the final stages of maturation and retention in a large, nonmitotic storage pool that is many times larger than the circulating and tissue neutrophil populations ( Table 22-1 ). Release is regulated by chemokines expressed on the cells, and their ligands are expressed by stroma cells. CXCR4 and its ligand CXCL12 retain cells, and mutations in the CXCR4 receptor account for the warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, an inherited neutropenia. Whereas CXCR2 and its ligands CXCL1 and CXCL2 promote neutrophil release. Also, the integrin α ₄ β ₁ , known as very late antigen 4 (VLA-4) may tether bone marrow neutrophils to vascular cell adhesion molecule 1 (VCAM-1) expressed on bone marrow stromal cells. Once released into the bloodstream, neutrophils have an estimated half-life of 6 to 10 hours and move between circulating and marginated pools in a reversible fashion. These estimates are based on several independent determinations using both in-vivo and in-vitro labeling techniques and transfusions, and they agree well with the estimates of neutrophils production rates of 10 cells/kg/day and circulating neutrophil counts 2.5 to 4 × 10 ⁹ /L. A recent controversial report suggests a half-life of 4 to 5 days both in humans and mice. Neutrophils exit circulation by diapedesis between or through endothelial cells into tissue sites of infection or inflammation. Once in the tissues, neutrophils are believed to live for another 1 to 2 days before undergoing apoptosis and engulfment by macrophages or they form neutrophil extracellular traps (NETs) consisting of deoxyribonucleic acid (DNA) and antibiotic proteins from nucleus, granules, and cytosol in a regulated process called netosis that traps microorganisms. This is discussed in more detail later in this chapter.

Myeloblasts are the earliest morphologically recognizable granulocyte precursors in the marrow and are identified by their relatively undifferentiated appearance with a large, oval nucleus, several prominent nucleoli, and few or no granules in a gray-blue cytoplasm in Wright-stained preparations. This stage of neutrophil differentiation is followed by the promyelocyte and myelocyte stages, which are distinguished by the appearance of distinct neutrophil granule populations ( Table 22-2 ). Azurophilic, or primary, granules are formed during the promyelocyte stage and contain myeloperoxidase (MPO), bactericidal peptides, and lysosomal enzymes. The subsequent myelocyte stage is distinguished by the formation of peroxidase-negative specific, or secondary, granules containing lactoferrin. No further cell divisions occur after the myelocyte stage. The metamyelocyte, band, and mature neutrophil exhibit progressive nuclear condensation, accumulation of glycogen, and accumulation of tertiary, gelatinase-rich granules and secretory vesicles that are endocytic vesicles marked by albumin and Complement Receptor 1 (CR1) (CD35). These neutrophil precursors can be identified and isolated by flow cytometry based on their surface antigen profile and forward and side scatter.

In Wright-stained blood smears, the mature neutrophil is 10 to 15 mm in size with a multilobed, polymorphic nucleus that has highly condensed chromatin and a yellow-pink cytoplasm containing numerous granules as well as clumps of glycogen. The mean lobe count is usually slightly less than three. Circulating neutrophils appear round with some cytoplasmic projections and surface ruffling. The morphologic changes seen with neutrophil differentiation are accompanied by temporally coordinated changes in gene expression and protein synthesis (see Fig. 22-1 ). Transcription and translation of messenger RNAs (mRNAs) for MPO and cathepsin G, which are both primary granule constituents, are restricted to myeloblasts and promyelocytes. In contrast, expression of the secondary granule proteins such as lactoferrin and transcobalamin I occurs in myelocytes and metamyelocytes. Gelatinase expression occurs even later in maturation and is first detected in bands and bone marrow neutrophils. The leukocyte β-integrin subunit CD11b is first detectable in myelocytes, and increases throughout the later stages of neutrophil differentiation. The gp91 ^phox subunit of the respiratory burst oxidase complex is expressed relatively late in neutrophil maturation, consistent with the observation that respiratory burst activity is not detected until the metamyelocyte stage.

The mature neutrophil, previously thought of as an “end-stage” cell, retains the capacity for inducible gene expression and protein synthesis even after release from the marrow cavity. Diapedesis and exposure to cytokines induce neutrophil expression of mRNA transcripts for IL-1, IL-6, TNF-α, GM-CSF, M-CSF, and IL-8, which may promote recruitment and activation of both phagocyte and lymphocyte populations in the inflammatory response.

Eosinophils

Like the neutrophil, the eosinophil is compartmentalized in the bone marrow into mitotic and storage pools; these usually constitute no more than 0.3% of the nucleated bone marrow cells. Eosinophils arise from a progenitor cell, the CFC-Eo, that is committed at a relatively early stage to differentiate into eosinophils instead of neutrophils and monocytes in the bone marrow. GATA-1, PU.1, and C/EBPs play critical roles in the transcriptional regulation of eosinophil lineage commitment and differentiation. Morphologic differentiation and maturation of the eosinophil parallel that of the neutrophil series, and its characteristic eosin-staining–specific granules are prominent by the myelocyte stage. IL-3, IL-5, and GM-CSF mediate eosinophil production in the marrow; IL-5, IL-13, chemokines (such as eotaxins [CCL11] regulated on activation, normal T cell expressed and secreted [RANTES; i.e., CCL5]), and leukotrienes [LTB4] play key roles in regulating eosinophil differentiation, chemotaxis, and functional activation.

After leaving the circulation, the majority of mature eosinophils reside in tissues, with a blood-to-tissue ratio estimated to be 1 : 300 to 1 : 500. The life span of tissue eosinophils is not known but may be several weeks, whereas the half-life in blood is around 24 hours. Eosinophils typically localize in areas exposed to the external environment, such as the tracheobronchial tree, gastrointestinal (GI) tract, mammary glands, and vagina and cervix. As discussed later, eosinophils have both immunoenhancing and immunosuppressive functions and play a role in helminthic infection, allergy, and the responses to certain tumors.

The mature eosinophil is slightly larger than the neutrophil, with a diameter of 12 to 17 µm. The nucleus is characteristically bilobed, although multiple lobes can be seen in patients with eosinophilia of diverse causes. The cytoplasm has prominent and morphologically distinctive granules that stain strongly with acid aniline dyes because of their high content of basic proteins.

Like the neutrophil, the mature eosinophil is endowed with the capacity for chemotaxis, phagocytosis, degranulation, and the synthesis of reactive oxidants and arachidonate metabolites. Eosinophils may also undergo a process similar to neutrophil netosis. Eosinophil cell surface membranes express a wide variety of molecules, including receptors for immunoglobulins and members of the immunoglobulin superfamily; cytokine receptors; adhesion molecules; chemokine, complement, and other chemotactic receptors; and major histocompatibility complex (MHC) class I and II and costimulatory molecules.

The distinctive mature eosinophil granules are membrane-bound organelles, 0.15 to 1.5 µm in length and 0.3 to 1 µm in width that contain a variety of enzymes and cytotoxic proteins. These eosin-staining specific granules are large ovoid bodies that contain an electron-dense crystalloid core surrounded by a less dense matrix. The eosinophil major basic protein (MPB) makes up about 50% of the dense crystalloid core of the eosinophilic specific granule. MBP from eosinophils induces histamine release from basophils and mast cells and also an autocrine degranulation of eosinophils. Release of proteins from these large granules is different from the degranulation process of neutrophils, where intact granules fuse with the surface membrane and empty their entire content and the membrane of granules is incorporated into the surface membrane. Instead, because the eosinophil is active in tissues, its granules swell and a tubulovesicular system extends from the granules to the surface permitting piece meal degranulation (PMD), or the graded release of granule content. Eosinophil peroxidase plays an important role in the antihelmintic function of eosinophils and utilizes bromate to generate hypobromous acid from hydrogen peroxide.

Much less common than eosinophil-specific granules are the primary granules characterized by their content of a lysophospholipase, the Charcot-Leyden crystal (CLC) protein that can polymerize to form the bipyramidal hexagons that are CLCs. CLCs are typically found in areas of eosinophil degeneration, such as sputum from asthmatic patients, nasal mucous of patients with allergies, stools of patients with parasitic infections, and the pleural fluid of patients with pulmonary eosinophilic infiltrates. The CLC lysophospholipase catalyzes the hydrolysis and inactivation of lysophospholipids generated by phospholipase A ₂ , thus preventing the generation of proinflammatory arachidonic acid metabolites. The CLC protein composes about 5% of the total protein in eosinophils.

Slightly more numerous but distinctly smaller than primary granules are the small type granules whose content is not well characterized and secretory vesicles, which like secretory vesicles of neutrophils contain the NADPH oxidase flavocytochrome b558 , and albumin, indicating an origin as endocytic vesicles; however, in eosinophils these may also be part of the tubulovesicular system of PMD.

Eosinophil granule products, particularly MBP, eosinophil peroxidase, and eosinophil neurotoxin, are toxic to tissues, including the heart, lungs, and brain. These mediate many of the adverse clinical complications of eosinophilia and hypereosinophilic syndrome (HES), such as Löffler endocarditis and pneumonia.

Basophils and Mast Cells

Basophils and mast cells are believed to share a common progenitor cell, but mast cells leave the bone marrow to proliferate and mature in tissues, whereas full differentiation of basophils occurs in the bone marrow over 7 days before their release into the bloodstream; they are not normally found in the connective tissues. Basophils account for approximately 0.5% of the total circulating leukocytes and 0.3% of nucleated marrow cells. Mature basophils have a bilobed nucleus. Although basophils are distinctly smaller (5 to 8 µm) than mast cells (20 µm), they both contain large metachromatic granules that stain purple or bluish with Wright stain because of their high content of sulfated glycosaminoglycans. These granules are rich in heparin-type and chondroitin sulfate-type glucosaminoglycans linked to the serglycin protein backbone that is responsible for packing the cationic proteins, histamine, and kallikrein. Mast cells and basophils express high affinity receptor for the Fc portion of immunoglobulin E (IgE), which is an important trigger for release of granule contents and production of arachidonic acid metabolites in anaphylactic degranulation on their plasma membrane, and are key effector cells in certain hypersensitivity reactions. However, mast cells lack receptors for IL-2, IL-3, and CD11b/CD18 that are present on basophils. The heparin of basophils appears to have poor anticoagulant activity. Basophil granules also contain small amounts of MBP as well as serine proteases. Basophils synthesize and secrete IL-4 and IL-13 and may thus mediate a link between the innate and adaptive immune systems for generation of Th2 lymphocyte responses. Murine mast cells can secrete a wide variety of mitogenic or inflammatory cytokines (including IL-1, IL-3, IL-4, IL-5, and IL-6), chemokines, GM-CSF, and TNF-α, that are likely to play an important role in leukocyte recruitment and inflammation.

Mast cells are ordinarily distributed throughout normal connective tissue, where they are often situated adjacent to blood and lymphatic vessels, near or within nerve sheaths, and beneath epithelial surfaces that are exposed to environmental antigens such as the respiratory and GI tracts. The c-kit receptor for stem-cell factor is present on mast cells, but absent from the majority of basophils. The c-kit ligand, or stem-cell factor (SCF), is the main survival and developmental factor for mast cells, but other growth factors and cytokines such as IL-3, Il-4 and IL-9 are also supportive for mast cell development. Mature mast cells do not circulate in the blood, although circulating mast cell progenitors have been described and retain a limited proliferative capacity in the tissue compartment. In contrast to monocytes and macrophages, a transformation between the circulating and tissue forms of basophils and mast cells has not been observed.

Mast cells can be categorized into several types in mice and humans. In mice, the different types can be distinguished by their main tissue distribution and the major type of proteoglycan in their granules (heparin-containing mast cells are predominantly connective tissue and serosal mast cells, whereas chondroitin sulphate-containing mast cells are associated with mucosal surfaces). In humans two major subsets are identified based on the their content of chymase and tryptase. Both contain tryptase, but a subset that is particularly low in chymase largely secretes cytokines and chemokines and does not degranulate as opposed to the chymase rich type.

Spleen

Macrophages are distributed in all parts of the spleen, including the germinal centers where they are associated with lymphocytes. Splenic macrophages located in the red pulp and sinuses serve a clearance function, where a sluggish blood circulation maximizes the interaction between blood elements and the macrophages lining the sinus walls.

Liver

The portal circulation percolates through a labyrinthine system, the spaces of Disse, before exiting via the hepatic venous system. This hepatic circulation, although less sluggish than that of the spleen, provides considerable contact between the blood and the resident liver macrophages, known as Kupffer cells, that reside within these vascular sinuses.

Lymph Nodes

As in the spleen, macrophages exist through all regions of peripheral lymph nodes. They are most abundant in the medullary zone close to efferent lymphatic and blood capillaries. This location is likely related to the important role macrophages play in the presentation of antigens to T lymphocytes.

Lungs

Pulmonary macrophages reside both in the interstitium of alveolar sacs and free within the air spaces, where they participate in the clearance of inhaled microorganisms and particulate matter. The number of lung macrophages increases in many chronic pulmonary inflammatory disorders. Pulmonary macrophages are easily seen in lungs of smokers, where black inclusions mark the macrophage vacuoles. Hemosiderin-laden alveolar macrophages can be indicative of recurrent pulmonary hemorrhage, such as in idiopathic hemosiderosis or Goodpasture syndrome. Gastric aspiration to detect ingested iron-laden macrophages is a useful test for these disorders.

Bone Marrow

Macrophages are found throughout the bone marrow cavity. They are particularly abundant within hematopoietic islands and on the walls of the marrow sinuses. Bone marrow macrophages may have a clearance function in normal or pathologic states of ineffective hematopoiesis. The clearance function of marrow macrophages is dramatically illustrated by the lysosomal storage diseases such as Gaucher disease. Large inclusions build up within marrow macrophages (as well as hepatic and splenic macrophages) because of the inability of these cells to break down lysosomal contents. Bone marrow macrophages also support hematopoiesis by modulating mesenchymal stem cells and osteoblasts to express retention signals, in particular CXCL12, for HSCs.

Other Sites

Mononuclear phagocytes associated with lymphoid cells reside throughout the alimentary tract, particularly in the submucosal tissues and small intestinal villi. They are present as microglial cells in the central nervous system (CNS), where their numbers increase after injury as monocytes emigrate across the blood-brain barrier, and they may contribute to the pathogenesis of the CNS manifestations of infection with the human immunodeficiency virus (HIV). Mammary gland macrophages released into milk during lactation have been implicated as a potential source of postnatal transmission of the HIV virus.

Dendritic Cells

Dendritic cells are specialized antigen-presenting cells with long cytoplasmic processes that are located in tissues throughout the body except for brain. They develop from a progenitor common with monocyte progenitors, supported by GM-CSF. M-CSF induces monocytic differentiation, and Flt3 ligand induces differentiation into a dendritic cell progenitor that may leave the bone marrow and home to lymphatic tissue and nonlymphoid tissue throughout life. In contrast, Langerhans cells of the epidermis, which are also antigen presenting cells, are self sustained and localize to skin during embryonic development. Some dendritic cells are also derived from the lymphoid lineage. Antigen presentation by dendritic cells, which have a high density of MHC class II molecules, is a particularly potent stimulus for T-cell mediation of the primary immune response. Plasmacytoid dendritic cells are a specialized population of dendritic cells, although derived from a shared precursor, that respond rapidly to viruses or nucleic-acid–containing complexes by secreting large amounts of type I interferon (IFN). NETs have been linked to activation of plasmacytoid dendritic cells and the pathogenesis of lupus.

Osteoclast

Osteoclasts are large, multinucleated mononuclear phagocytes that resorb mineralized cartilage and bone. Rodent transplantation studies have shown that osteoclasts can be derived from granulocyte-macrophage progenitor cells. Defects in osteoclast function result in osteopetrosis, a genetically heterogeneous group of disorders characterized by defective bone resorption. The Op/op osteopetrotic mouse mutant lacks M-CSF, which results in deficiencies of both osteoclasts and tissue macrophages. However, M-CSF levels and osteoclast numbers are normal in human infantile (“malignant”) osteopetrosis, an AR disorder with progressive obliteration of the marrow space. This severe form of osteopetrosis is caused by mutations in genes encoding a vacuolar proton pump or the chloride channel 7.

Chemokines, Small Lipids and Other Chemoattractants

A wide variety of chemoattractants for neutrophils and other circulating leukocytes are generated at sites of inflammation ( Table 22-4 ). These molecules are chemically diverse and are derived from many different sources in response to bacterial products and inflammatory mediators released as a result of tissue necrosis. This diversity provides a functional redundancy and ensures that leukocytes will be attracted to sites of injury or infection. In addition to their role as chemoattractants, the molecules listed in Table 22-4 induce the activation of many other phagocyte functions upon binding to their cognate cell-surface receptors. These include the upregulation and increased affinity of leukocyte integrin adhesion receptors to promote firm attachment to the endothelium, degranulation, and activation of the phagocyte respiratory burst. Many chemoattractants are secreted by activated phagocytes, which act as a positive feedback loop for additional recruitment and activation of inflammatory cells.

The phospholipid PAF, released by both activated phagocytes and endothelial cells, triggers platelet activation and granule release in addition to being a potent chemoattractant for neutrophils and eosinophils. Activation of phagocytes also stimulates the phospholipase A ₂ -mediated cleavage of membrane phospholipids to generate arachidonic acid, which is then converted into a variety of eicosanoid metabolites, including the chemoattractant leukotriene B ₄ (LTB ₄ ).

Chemokines (named for their combined chemo tactic and cyto kine properties) are a family of small (8 to 10 kDa) basic heparin-binding proteins that comprise an important group of phagocyte chemoattractants. Chemokines were first discovered in the late 1980s as molecules that interact relatively specifically with subsets of inflammatory leukocytes and therefore help orchestrate the sequential influx of neutrophils, monocytes, and finally lymphocytes into an inflamed tissue site. Proteoglycans on endothelial cells or in the subendothelial matrix bind chemokines to produce locally high chemokine concentrations at an inflamed site. As additional chemokines and their receptors have been identified, many other functions have emerged, including regulation of lymphoid homeostasis, hematopoiesis, and angio­genesis. Of note, SDF-1 (CXCl2) , provides a key retention signal for neutrophils in the marrow through its interaction with the CXCR4 receptor, and mutations in the CXCR4 receptor account for the WHIM syndrome, an inherited neutropenia.

Members of the chemokine family, which have a conserved structure containing two cysteine pairs, have been divided into two groups based on the disulfide sequence pattern. The CXC family, in which the first cysteine pair is separated by an intervening amino acid, include IL-8 (CXCL8), the growth-regulated oncogene (GRO) peptides ( CXCL1, 2, and 3 ), and neutrophil-activating protein 2 (NAP-2; CXCL7 ), which are all potent neutrophil activators and chemoattractants. The IL-8 and GRO chemokines are secreted by phagocytes and mesenchymal cells (including endothelial cells) in response to inflammatory mediators such as IL-1 and TNF. Fractalkine (CX ₃ CL1) is unique in having three intervening amino acids between the first two cysteine residues. In addition, rather than being soluble, fractalkine is expressed on the cell surface, because it is linked via a mucinlike stalk to a transmembrane domain. The other major family of chemokines is called the CC family, because the first two cysteines are adjacent to each other. CC chemokines include two important inducers of mononuclear phagocyte migration, monocyte chemotactic protein 1 (MCP-1; CCL2 ) and RANTES (CCL5). MCP-1 is produced by a wide variety of cells, whereas RANTES is secreted by macrophages and eosinophils. RANTES is chemotactic for eosinophils, basophils, and memory T cells as well as monocytes, and both MCP-1 and RANTES induce histamine release from basophils.

Despite the diverse chemical structures of phagocyte chemoattractants listed in Table 22-4 , the corresponding receptors all belong to the seven-transmembrane–spanning receptor (7-TMR) family, also known as heptahelical or serpentine receptors, that are coupled to heterotrimeric G proteins. For chemokines, more than six receptors for CXC chemokines and ten receptors for CC chemokines have been identified. Most chemokines bind to more than one receptor, and most chemokine receptors, particularly those for CC chemokines, recognize more than one chemokine. Neutrophils, monocyte/macrophages, eosinophils, basophils, dendritic cells, lymphocytes, and T cells each express a distinctive subset of chemokine receptors. According to one model, specific receptors are used sequentially in successive gradients of chemoattractants. Some transmit desensitizing, rather than activating, signals or even fail to signal and act instead as “decoy” receptors to downregulate inflammatory reactions. Of note, a number of chemokine receptors are coreceptors for HIV-1, including CCR5 and CCR3, whose ligands include RANTES, and CXCR4, the major receptor for stromal cell–derived factor 1 (SDF-1), which is a chemoattractant for T lymphocytes, CD34+ hematopoietic progenitor cells, and neutrophils.

The signaling through 7-TMR has proven much more complicated (and powerful) than the original signaling mediated via associated heterotrimeric G proteins, which bind to specific intracellular domains of the receptor. Ligand binding to the receptor promotes the exchange of GTP for guanosine diphosphate (GDP) bound to the G protein α subunit, which in turns leads to the dissociation of the β-γ subunits and their interaction with downstream signaling effectors. In addition, 7-TMR associates with β arrestins and signals through these in a G-protein independent way. Finally, 7-TMR may transactivate tyrosine kinase growth factor receptors. Signals generated from 7-TMR activate enzymes that catalyze the production of important phospholipid second messengers at the cell membrane.

Leukocyte Adhesion and Migration into Tissues

The initial step in emigration from postcapillary venules is a low-affinity interaction between the neutrophil and the endothelium that is often referred to as rolling because of its appearance in intravital microscopy. This transient adherence, also called tethering, is mediated by the upregulation of selectin expression on endothelial cells. The selectin family of adhesion molecules are membrane-spanning glycoproteins ( Fig. 22-4 ) that bind to fucosylated structures such as Lewis X (Galβ1→4 [Fucα1→3] GlcNac→R), Sialyl-Lewis X, and other specific carbohydrates. P-selectin is important for the initial steps of neutrophil adhesion to the endothelium and is stored in the Weibel-Palade bodies and α granules of endothelial cells and platelets, respectively. Upon endothelial cell activation by histamine, thrombin, and other inflammatory molecules, these cytoplasmic storage granules fuse with the cell membrane to rapidly increase the surface expression of P-selectin. E-selectin is expressed on endothelial cells at low levels, but it is upregulated by transcriptional activation and de novo protein synthesis in response to inflammatory cytokines. E-selectin binds to three different ligands on neutrophils: P-selectin glycoprotein ligand (PSGL) 1, E-selectin ligand (ESL) 1, and CD44. These ligands allow endothelial cells to capture neutrophils by mediating tethering, rolling, and slowing of neutrophil velocity, respectively. L-selectin is expressed constitutively on the surface of neutrophils, mononuclear phagocytes, and lymphocytes and is shed within minutes of leukocyte activation by a proteolytic cleavage event near the external membrane surface insertion site. Circulating L-selectin may modulate leukocyte adhesion during inflammation. PSGL-1 is constitutively expressed on the tip of microvilli on the neutrophil surface and remains associated during activation but moves to the uropod when the neutrophil polarizes.

Rolling neutrophils can detach and return to the circulation. Others will come to a halt and within seconds adopt a flattened, adherent morphology and attach firmly to the vessel wall. This firm attachment appears in large part to be mediated by leukocyte integrin adhesion receptors binding to intracellular adhesion molecules (ICAMs) on the endothelium. In addition, complement fragments are found on the endothelial surface at inflamed sites and may also function as integrin binding sites. Leukocyte activation by chemoattractants and other inflammatory mediators is critical to the development of these strong adhesive interactions, because it leads to the upregulation of the number and avidity of cell-surface integrins (“inside-out signaling”). Exposure to locally high concentrations of chemoattractants may be enhanced by selectin-mediated tethering and by the retention of chemokines on extracellular matrix.

The integrins are a large family of adhesion proteins that are glycosylated heterodimers of a noncovalently linked α chain and β chain and are classified into subfamilies according to the type of β subunit. Many integrins mediate attachment to extracellular matrices by serving as receptors for matrix proteins. Others are involved in hemostasis, such as glycoprotein IIb/IIIa on platelets. Neutrophil β ₂ and β ₁ integrins appear to be involved in regulating neutrophil retention and release, respectively, from the bone marrow storage pool into the circulation.

The leukocyte β ₂ integrins ( Fig. 22-5 ) play a critical role in mediating adhesive interactions in inflammation, including the attachment of leukocytes to endothelial cells and are also opsonic receptors for complement fragment C3bi-coated particles. There are four different leukocyte β ₂ integrins, each having a common 95 kDa β subunit (CD18) but different α subunits, CD11a (177 kDa), CD11b (165 kDa), CD11c (150 kDa), and CD11d (160kDa) (see Fig. 22-5 ). Lymphocyte function antigen 1 (LFA-1) is expressed on the surface of all leukocytes, including lymphocytes. Mac-1 and p150,95 are expressed by granulocytes, mononuclear phagocytes, some activated T lymphocytes, and large granular lymphocytes. Mac-1 is the most prominent β ₂ integrin on neutrophils, whereas α _d β ₂ is expressed particularly in tissue macrophages. Mutations in the common β ₂ subunit result in an inherited defect in phagocyte function, leukocyte adhesion deficiency type I (LAD I) as discussed in a later section. All β ₂ integrins are absent in LAD I, indicating that the stability of each α subunit requires association with the β ₂ chain. The β ₂ subunit has a large, glycosylated extracellular domain, a single transmembrane-spanning domain, and a short cytoplasmic tail. The extracellular domain has two regions that are conserved among other β subunits. There are four cysteine-rich tandem repeats that appear to be important for the tertiary structure of the β subunit. Another conserved region, located near the N-terminus, is critical for maintenance of the α/β heterodimer and may also bind divalent cations. The α subunit is also a glycosylated integral membrane protein with a single membrane-spanning segment and a short cytoplasmic tail. The external domain contains three divalent cation-binding motifs that must be occupied for ligand binding to occur. A second important extracellular domain, the I domain (for inserted or interactive domain), can coordinate divalent cations and is also thought to be involved in ligand binding. The intracellular domain of the α subunit includes a conserved sequence that is critical for the modulation of integrin avidity (see “Leukocyte Adhesion Deficiency”).

Although β ₂ integrins are constitutively expressed on the neutrophil cell surface, a large pool of Mac-1 is stored in intracellular secretory vesicles (see Table 22-2 ). These vesicles are rapidly mobilized upon neutrophil activation by chemoattractants and fuse with the membrane to increase the cell surface expression of β ₂ integrins by about tenfold. Signaling through chemoattractant receptors also markedly increases the avidity of β ₂ integrins for their ligands, which plays an even more important role in rapidly upregulating integrin activity and promoting firm attachment to the blood vessel wall. Integrins on inactivated cells have a bent position that does not allow ligand binding. Signals generated from 7-TMRs recruit talins to the cytoplasmic part of the integrin β chains, twisting the α and β chains apart and tethering the integrins to the actin cytoskeleton. This is assisted by actin-binding protein (ABP) 1 and by the protein Kindlin 3, which is only expressed in cells of hematopoietic origin. This changes the conformation of the extracellular parts of the αβ heterodimer to an extended form, now capable of ligand binding and hence also of transducing signals from outside in. Defects in Kindlin 3 results in LAD III (see “Leukocyte Adhesion Deficiency Type III”). Ligand binding to integrins results in clustering of the integrins and the transmembrane calcium channel Orai1 that mediates store operated calcium entry and activation of tyrosine kinase and other signaling cascades, which in addition to mediating adhesion, provide important costimulatory signals to enhance migration, respiratory burst, Fcγ-mediated phagocytosis, and degranulation.

The major counterreceptors for the β ₂ integrins are the ICAMs ( Fig. 22-6 ), which are members of the immunoglobulin superfamily. These transmembrane proteins contain anywhere from two to six immunoglobulin domains and are present on endothelial cells, T cells, and a variety of other cell types. ICAM-1 and ICAM-2 are of particular importance in mediating binding of neutrophils and other leukocytes to the endothelium. Endothelial cell expression of ICAM-1 increases in response to inflammatory cytokines, which promotes increased cell–cell interactions with leukocytes at inflamed sites. VCAM-1 is another immunoglobulin superfamily member expressed on endothelial cells that is inducible by cytokines. VCAM-1 is the counter-receptor for the β ₁ integrin, VLA-4, and appears to be important in promoting the adherence of monocytes and eosinophils during inflammation. The β ₂ integrin Mac-1 also has an important role as an opsonic receptor for the complement fragment, C3bi (see “Recognition, Opsonization, and Phagocytosis”).

Although leukocyte β ₂ integrin-mediated adhesion is clearly important for neutrophil recruitment from the systemic microvasculature into inflammatory sites, neutrophil emigration out of the pulmonary circulation can also be mediated by alternative pathways, depending on the inflammatory stimulus. Whether the alternative pathway in pulmonary capillaries, which are in close proximity to the pulmonary epithelial cells, involves selectins or other adhesion molecules remains to be defined. TREM-1 is essential for pulmonary transepithelial migration of neutrophils.

The final steps in emigration of neutrophils from the blood vessel lumen into inflamed tissue involves squeezing between adjacent endothelial cells (diapedesis) and penetrating the basement membrane (see Fig. 22-3 ). The presence of a chemotactic gradient is required to induce the directional migration of neutrophils. Adhesive interactions between the β ₂ integrins and endothelial cell ICAM-1 are essential for neutrophil diapedesis, whereas VCAM-1 and E-selectin can mediate the transmigration of monocytes and eosinophils. Junctional adhesion molecule (JAM) 1, an immunoglobulin superfamily protein that is expressed at tight junctions of resting endothelial cells and epithelial cells, facilitates leukocyte transmigration via binding to LFA-1 (CD11a/CD18). The tight junction between endothelial cells is weakened by loss of paxillin and focal adhesion kinase in proximity to migrating neutrophils. Transendothelial migration of neutrophils is also dependent on homologous binding between neutrophil and endothelial cell platelet–endothelial cell adhesion molecule (PECAM) 1 (CD31), another immunoglobulin superfamily member expressed on the surface of leukocytes, platelets, and endothelial cells, where it is localized at the junctions between cells. Signals from ICAM-1 induce vascular permeability, activate PECAM-1 for enhanced adhesivity, and support of neutrophil transendothelial migration. The JAM-C expressed by endothelial cells seems important for directing neutrophil migration into tissues and inhibiting migration in the reverse direction. Migrating neutrophils induce increases in endothelial intracellular calcium levels and changes in actin cytoskeleton that facilitates transmigration. To find gaps between pericytes sheathing endothelial cells, neutrophils use ICAM-1 to crawl along pericyte extensions, which further guide their migration into tissues. Finally, chemoattractant-induced neutrophil degranulation results in the release of digestive enzymes, including collagenase, elastase, and gelatinase, which may facilitate basement membrane penetration.

Chemotaxis

Chemotaxis is the directional movement of a cell along a concentration gradient. Defects in neutrophil cellular motility or other steps in chemotaxis can result in decreased resistance to bacterial and fungal infections, as discussed later in this chapter. Cells respond to a chemotactic gradient by sensing constantly across their surface, and bound chemotactic receptors are continuously internalized. A migrating neutrophil has a polarized appearance, extending pseudopodia or lamellipodia, thin structures rich in actin filaments and lacking intracellular organelles, at the leading edge. The pseudopods appear to glide forward, pulling the cell body behind them. The nucleus tends to remain at the posterior half of the moving leukocyte. Migration also requires formation of a uropod at the “tail” of the leukocyte, which detaches from the underlying matrix and retracts the rear of the cell as it moves forward. Rho GTPases play an important role in establishing chemoattractant-induced polarization and migration. Ly49Q, a surface-expressed MHC I–associated receptor is associated with the SH2 domain–containing protein tyrosine phosphatase (SHP) 1 phosphatase in nonpolarized neutrophils but exchanges SHP-1 with SHP-2 in activated cells, which results in reorganization of lipid rafts and polarization.

Neutrophil movement is dependent on the dynamic assembly and disassembly of filamentous actin, which is coordinated by various ABPs whose activity is regulated by intracellular signaling molecules. Actin can exist as either a soluble monomer (globular actin) or in needlelike helical filaments (filamentous actin). Actin filaments align spontaneously in parallel bundles, but in the cell are organized into a branching network because of the presence of actin filament cross-linkers such as ABP. Agonists acting via receptors on the cell membrane trigger the generation of second messengers, which interact with ABPs to control dynamic local cycles of filamentous actin assembly. The Rho GTPases (Cdc42 and Rac) are important regulators of actin remodeling and are activated by ligand binding to chemoattractant receptors. Cdc42 activates proteins of the Wiskott-Aldrich syndrome protein (WASP) family, which then bind to a complex of seven proteins known as the Arp2/3 complex to nucleate assembly of new actin filaments at the leading edge of migrating cells.

During pseudopod extension in chemoattractant-activated neutrophils, new actin polymerization occurs at the site of membrane protrusion while the filamentous actin in the rear of the cell disassembles. How actin assembly–disassembly results in membrane extension and formation of pseudopodia is not fully understood, but it may involve localized changes in osmotic pressure caused by changes in actin polymerization. Membrane movement and retraction at the rear of the cell is mediated in part by Rho GTPase-regulated contractile proteins such as myosin 1.

Inflammasomes

Signaling through the membrane-bound PRRs in the previous paragraph generally leads to activation of the NF-κB pathway or IRF and production of proinflammatory cytokines, in particular IL-1β and IL-18 and type-1 IFNs, respectively. IL-1β and IL-18 are both without signal peptides and stored in the cytosol as proforms, but when processed by proteases, which in turn are activated by inflammasomes, these cytosolic cytokines are secreted from the cells by unconventional secretion mechanisms. IL-1β and IL-18 play a major role in activation of several steps of the inflammatory process, including endothelial cell and B-cell activation. Inflammasomes are structures that are assembled from preformed subunits in the cytosol in response to a variety of stimuli, such as signaling through cell surface (as discussed) or intracellular PRR such as the nucleotide-binding oligomerization domain–like receptors (NLRs) and retinoic acid–inducible gene 1–like receptor. Depending on the activating stimulus, different subunits become engaged in formation of the macromolecular complex known as an inflammasome and activate procaspase 1 to process the cytosolic proIL-1β and proIL-18 to the mature forms for secretion. A major cytosolic protein of neutrophils, pyrin, forms part of some inflammasomes, and mutations in the pyrin gene MEVF cause the inflammatory condition known as familial Mediterranean fever.

Complement Receptors

The activation of complement C3 to generate the cleavage fragments C3b and C3bi is the dominant source of opsonins in the absence of antibodies. Four C3 fragment receptors have been described on phagocytic leukocytes ( Table 22-5 ). The human phagocyte C3b receptor (CR1; CD35) is a high–molecular-weight, single-subunit glycoprotein that shows a substantial heterogeneity in size because of the presence of four distinct alleles in the human population that encode proteins ranging in size from 160 to 250 kDa. CR1 is responsible for the binding of C3b-opsonized particles and for initiating their ingestion. CR1 also recognizes microbes opsonized with MBL. The other major opsonic receptor, CR3, recognizes particles opsonized with C3bi. This receptor is the same as the β ₂ integrin Mac-1 (CD11b/CD18) discussed in detail in an earlier section (see Fig. 22-5 ). Binding of C3bi-opsonized particles to Mac-1 triggers both phagocytosis and, in neutrophils, the respiratory burst. The CR4 receptor is the same as the β ₂ integrin CD11c/CD18 and, on macrophages, can also initiate phagocytosis of C3bi-coated targets. Finally, a newly discovered receptor that binds the complement fragments C3b and C3bi, termed complement receptor of the immunoglobulin superfamily (CRIg) is expressed primarily in resident macrophages of the liver (Kupffer cells). CRIg appears to play a critical role in mediating phagocytosis and clearance of C3 fragment-opsonized bacteria from the circulation, at least in mice.

Immunoglobulin Receptors

Immunoglobulins are recognized by Fc receptors (FcRs), which are members of the immunoglobulin gene superfamily. FcRs bind to the “constant domain” of the antibody molecule, these being specific to each class of immunoglobulins (IgA, IgE, IgG, and IgM). The most important from the standpoint of microbial opsonization are those FcRs that recognize IgG (FCγRs) ( Table 22-6 ). The FcγRs include three distinct classes, FcγRI, FcγRII, and FcγRIII, which are encoded by at least eight genes that have evolved through gene duplication and alternative splicing, although not all give rise to detectable mRNA and/or protein. The low-affinity FcγR genes (FcγRII and III families) and two of the genes for the high-affinity IgE receptor are clustered on chromosome 1q22. The high-affinity FcγRs map to other sites on chromosome 1.

Except for FcγRIIIb, which is anchored in the membrane by a GPI moiety, the FcγR proteins have a single transmembrane domain. FcγRI and FcγRIIIa exist as oligomeric complexes with γ (in phagocytes) or ζ (in lymphocytes) chains that are important both for their stable expression and signaling functions. These accessory chains contain YXXL ITAM motifs that become tyrosine phosphorylated upon receptor cross-linking to initiate downstream signaling cascades. The cytoplasmic tail of the FcγRIIa receptor contains variant ITAM motifs, and hence this receptor does not require accessory chains for its function. There is no murine equivalent to the FcγRIIa receptor. The GPI-linked FcγRIIIb receptor must be co-ligated to either FcγRIIa or the β ₂ integrin CR3 (Mac-1) to initiate signaling functions upon ligation. FcγRIIb receptors, expressed on monocyte/macrophages, mast cells, and lymphocytes, contain ITIM sequences and instead inhibit cellular activation upon immunoglobulin cross-linking.

Each FcγR is expressed at different levels, depending on the type of phagocytic cell (see Table 22-6 ), and some FcγRII and FcγRIII family members are also expressed on lymphocytes, platelets, thymocytes, natural killer (NK) cells, and mast cells. The FcγR1 class includes the products of three highly homologous genes, denoted A, B, and C, although the protein products of the latter two have not been detected in vivo. The FcγR1a receptors are expressed by monocytes and neutrophils or eosinophils stimulated by IFN-γ or G-CSF and have a high affinity for monomeric IgG. Members of the FcγRII and FcγRIII families have a low affinity for monomeric IgG but a high affinity for clusters of IgG (e.g., several antibodies bound to the same particle) and immune complexes. Only neutrophils and IFN-γ–stimulated eosinophils appear to express FcγRIIIb, and polymorphisms in this receptor correspond to the serologically defined NA1/NA2 antigen system, which is a common antibody target in alloimmune and autoimmune neutropenia (AIN). Polymorphisms in FcγRIIa, FcγRIIIa, and FcγRIIIb are associated with an increased incidence or severity of various autoimmune or infectious diseases. For example, the 131H allele of FcγRIIa, which is the only FcγR that mediates efficient phagocytosis of IgG ₂ -opsonized bacteria, has been linked to a lower incidence of infections with encapsulated bacteria.

Cross-linking of FcγRs can trigger a wide range of functional responses, including phagocytosis of IgG-coated microbes, blood cells, or tumor cells; ingestion of immune complexes; release of inflammatory mediators; activation of the respiratory burst; and antibody-dependent cellular cytotoxicity (ADCC). The relative roles of different phagocyte FcγR classes have not been clearly delineated, although all can function as receptors for opsonized particles or immune complexes. Binding of IgG to FcγRI and FcγRIIa activates the respiratory burst, whereas secretion of granular contents is a prominent response upon binding to FcγRII and FcγRIIIb. Although the ligand-binding domains of different FcγRs all bind IgG, the specificity of the cellular response may be governed by the unique transmembrane and cytoplasmic domains of a particular FcR subtype.

Ligation and cross-linking of FcγRs initiates a cascade of biochemical signals that are initiated by activation of nonreceptor protein-tyrosine kinases of the Src family, which phosphorylate ITAM motifs in the FcγR chain and in FcγRIIa to recruit and activate the Syk tyrosine kinase and further amplify ITAM phosphorylation. Subsequent activation of several parallel pathways, including activation of PLC, PI3K, and MAP kinase (MAPK)-related pathways, and other targets leads to various cellular responses, including actin and membrane remodeling for particle ingestion, NADPH oxidase activation, and cytokine release. The DAP12 adaptor that normally potentiates these activating signaling pathways may signal for FcR and TLRs on macrophages that express TREM-2.

The opsonization of microbes with secretory IgA antibodies is likely to be important in the clearance of microbes from the mucosal surfaces of the respiratory, GI, and urogenital tracts. Neutrophils and monocytes have an FcR for IgA (FcαR) that has close structural similarities with other members of the FcR family. Ligation of the phagocyte FcαR can trigger phagocytosis, degranulation, and superoxide release. IgE antibodies can activate eosinophils and mast cells through the high-affinity FcεRI receptors.

Phagocytosis

Engagement of any of the opsonic receptors initiates phagocytosis of an opsonized particle to form a phagocytic vacuole that encloses the particle. The molecular details of this process are incompletely understood, but they involve membrane remodeling and the regulated assembly and disassembly of the actin cytoskeleton in a fashion analogous to the mechanisms that result in cell movement when chemotactic receptors are engaged. FcγR-mediated phagocytosis triggers the extension of long pseudopodia that attach in a zipperlike fashion around the particle and then fuse to form the phagosome, which is then drawn into the cell. In contrast, complement-opsonized particles attach to the cell surface only at limited points and “sink” into the cytoplasm in the absence of any pseudopod extension. These morphologic differences are associated with differences in the underlying biochemical pathways. FcR-mediated phagocytosis is sensitive to inhibitors of tyrosine kinases and requires the Rac and Cdc42 GTPases, whereas CR3-mediated phagocytosis is dependent upon protein kinase C and the Rho GTPase.

Microorganisms can also enter phagocytes by nonopsonic routes that enable them to evade the cytocidal weapons that are otherwise activated upon phagocytosis. For example, after binding to the macrophage cell surface, Salmonella typhimurium induces extensive membrane ruffling that leads to internalization of the bacterium into a macropinosome or “spacious phagosome.” Legionella pneumophila attachment to macrophages induces the formation of a pseudopod that spirals around the bacterium to form a “coiling phagosome” that does not acidify or fuse with lysosomes.

Oxygen-Independent Toxicity

Phagocyte granules supply preformed cytotoxic and digestive compounds that play a key role in oxygen-independent killing and digestion of microbes, senescent cells, and particulate debris. In neutrophils, azurophilic (primary) and specific (secondary) granules serve as the main storage reservoir of these compounds. Oxygen-independent pathways complement those dependent on the respiratory burst (see “Oxygen Dependent Toxicity”), and are also important for phagocyte antimicrobial activity under the adverse conditions of hypoxia and acidosis often encountered locally at the site of infection.

Numerous cationic antimicrobial proteins are contained within neutrophil azurophilic granules. Defensins are small (29-25 amino acids) basic peptides that constitute more than 5% of the total cellular protein of human neutrophils, although they are absent in murine neutrophils. These peptides exhibit antimicrobial effects against a broad range of gram-positive and gram-negative organisms, fungi, mycobacteria, and some enveloped viruses. Defensins are also cytotoxic to mammalian cells. Defensins kill target cells by insertion into the cellular membrane and formation of voltage-regulated channels. Defensinlike peptides have also been found in small intestinal Paneth cells and in tracheal epithelium. Bactericidal/permeability-increasing pro­tein (BPI) is a 55-kD cationic protein that has potent cytotoxic effects toward gram-negative bacteria. BPI binds avidly to LPS, leading to both bacterial killing by damaging the cell membrane and to the neutralization of endotoxin associated with the bacterial cell wall and in serum. Serprocidins are a family of 29 kD glycoproteins that are homologous to members of the serine protease superfamily and include azurocidin (CAP37) and four serine proteases (cathepsin G, elastase, proteinase 3, and the recently identified NSP4). In human neutrophils, serprocidins are even more potent than the defensins in antimicrobial activity and have a broad spectrum of cytotoxicity that is, with few exceptions, unrelated to proteolytic activity. Cathepsin G, elastase, and proteinase 3 are often referred to as neutral proteases because the optimal pH for their proteolytic activity is approximately 7. Azurocidin can bind to endotoxin, which appears to account for its activity for gram-negative bacteria. Azurocidin also is a potent chemoattractant for monocytes as well as fibroblasts and T cells. Exogenous administration of antimicrobial peptides is being studied as an alternative or adjunctive therapy to conventional antibiotics in a number of settings.

Both azurophilic and specific granules contain lysozyme, which hydrolyzes the cell wall of saprophytic gram-positive organisms and may also assist in the nonlytic killing of other organisms. hCAP-18 is a member of the cathelcidin family of antimicrobial peptides, which is cleaved by proteinase-3 after exocytosis. The N-terminal region is homologous to other cathelicidins, whereas the 37-amino acid C-terminal fragment (LL-37) has additional activities, including acting as chemoattractant as well as effects on apoptosis. Specific granules also contain the iron-binding glycoprotein lactoferrin, which has direct bacteriocidal activity both related and unrelated to the chelation of iron compounds required for bacterial metabolism. Lactoferrin may also catalyze the nonenzymatic formation of hydroxyl radicals (OH•) during the respiratory burst (see “Oxygen-Dependent Toxicity”). Vitamin B ₁₂ (cobalamin)–binding protein has been proposed to bind the analogous family of compounds found in bacteria to exert an antimicrobial effect. Lipocalin 2, also known as NGAL, interferes with bacterial iron utilization by binding to bacterial ferric-siderophore complexes. NGAL-deficient mice demonstrate a major reduction in defense against both Klebsiella pneumoniae and Mycobacterium tuberculosis, but NGAL, like most other neutrophil granule proteins, is highly expressed in epithelial cells during inflammation, and the role of neutrophil-delivered proteins versus epithelial-cell–fabricated proteins has not been determined. Human neutrophil NGAL has, however, been associated with defense against M. tuberculosis.

Azurophilic granules contain a variety of hydrolases (see Table 22-2 ) that have a lower pH optimum (<6), consistent with the lysosomal character of these granules. Studies using indicator dyes and biochemical techniques suggest that after a transient rise, the pH of the phagocytic vacuole falls below 6, which would enhance the activity of these enzymes upon their discharge into the vacuole. The acid hydrolases serve primarily a digestive rather than a microbicidal function. Azurophilic granules also contain MPO, which is an important enzyme in microbicidal oxygen-dependent reactions that are described in “Oxygen-Dependent Toxicity.”

Inherited partial or complete deficiency of MPO, which occurs in 0.05% of the population, can occasionally result in increased susceptibility to infection (see “Myelooxidase Deficiency”). Deficiencies in other individual neutrophil granule proteins have not yet been described in humans, but gene-targeted mice lacking the neutrophil granule serine proteases elastase or cathepsin G have impaired host defense against gram-negative sepsis and fungal infections. A few rare disorders involving defects in granule formation (SGD) or degranulation (CHS) are associated with recurrent bacterial infections. Inherited mutations in neutrophil elastase are present in patients with cyclic neutropenia (CyN) and severe congenital neutropenia (SCN). The mutant forms of elastase may have abnormal properties that exert a toxic effect on granulopoiesis, likely by eliciting an unfolded protein response that induces apoptosis.

Oxygen-Dependent Toxicity

The resting neutrophil relies primarily on glycolysis for energy and hence consumes relatively little oxygen. However, within seconds after contacting opsonized microbes or high concentrations of chemoattractants, oxygen consumption increases dramatically, often by more than 100-fold. This “extra respiration of phagocytosis” was first observed in 1933, but it was almost 30 years before it was appreciated that this process was insensitive to mitochondrial poisons and thus not related to increased energy demands. The enzyme complex responsible for this phenomenon, referred to as the NADPH or respiratory burst oxidase, is associated with the plasma and phagolysosomal membranes and catalyzes the transfer of an electron from NADPH to molecular oxygen, thereby forming the superoxide radical (O ₂ −) ( Fig. 22-8 ). Superoxide, although itself a relatively weak microbicidal agent, is the precursor to a family of potent oxidants that are essential for the killing of many microorganisms (see Fig. 22-8 ). The importance of the respiratory burst to normal host defense is underscored by the recurrent and often life-threatening infections seen in patients with chronic granulomatous disease (CGD), who are genetically deficient in respiratory burst oxidase activity.

The respiratory burst oxidase is a multi-subunit enzyme complex assembled from membrane-bound and soluble proteins upon phagocyte activation ( Fig. 22-9 ). Five polypeptides, gp91 ^phox , p22 ^phox , p47 ^phox , p67 ^phox , and p40 ^phox , that are essential for normal respiratory burst function have been identified ( Table 22-7 ), and mutations in the corresponding genes are responsible for five different genetic subgroups of CGD. The oxidase subunits have been given the designation phox , for ph agocyte ox idase. Neutrophils have the highest expression level of the NADPH oxidase, followed by monocytes, macrophages, eosinophils, and dendritic cells. B lymphocytes also express the NAPDH oxidase, but at very low levels, and even smaller amounts are reported in T lymphocytes.

An unusual b-type flavocytochrome, located in the membrane of secretory vesicles, gelatinase granules, and specific granules, mediates electron transfer in the oxidase complex. It is often referred to as flavocytochrome b ₅₅₈ , for its spectral peak of light absorbance at 558 nm, or as flavocytochrome b ₂₄₅ in reference to its midpoint potential of −245 mV, which is extremely low. This flavocytochrome is a heterodimer that contains a 91-kD glycosylated protein, gp91 ^phox , and a nonglycosylated subunit, p22 ^phox . The gene for gp91 ^phox , which is the site of mutations in the X-linked form of CGD, was one of the first human disease–associated genes to be identified by positional cloning. gp91 ^phox is the redox center of the oxidase and contains both flavoprotein and heme-binding domains in its cytosolic and membrane-spanning portions, respectively. This subunit is also sometimes called NADPH oxidase (NOX) 2, referring to its number in a series of homologous flavocytochromes. The p22 ^phox subunit is also an integral membrane protein and provides an important docking site for p47 ^phox during NADPH oxidase assembly. Heterodimer formation with gp91 ^phox is essential for heme incorporation and intracellular stability of both flavocytochrome subunits.

The p47 ^phox , p67 ^phox , and p40 ^phox subunits are found in the cytosol as a complex that is stabilized by intermolecular interactions that include those mediated by SH3 domains and proline-rich SH3 binding motifs within these proteins. Phagocyte activation induces translocation of this complex to the flavocytochrome, which is triggered by the phosphorylation of p47 ^phox to expose additional SH3 domains that bind to a proline-rich target SH3-binding sequence in p22 ^phox . The p47 ^phox subunit is necessary for translocation of p67 ^phox to the membrane of activated neutrophils based on studies of p47 ^phox -deficient CGD neutrophils. However, substantial amounts of superoxide can be generated from neutrophil membranes in vitro in the absence of p47 ^phox , provided that high concentrations of p67 ^phox and Rac-GTP are supplied. Hence, p47 ^phox acts as an “adaptor” protein to mediate translocation of p67 ^phox and to position it correctly in the active NADPH oxidase complex. The p40 ^phox plays a specialized role in stimulating high levels of superoxide production on phagosome membranes via a domain that binds to the membrane lipid phosphatidylinositol 3-phosphate. p40 ^phox may also help with stabilization of p67 ^phox on flavocytochrome b–containing granule membranes as they are recruited to the phagosomes.

The active NADPH oxidase also requires the GTP-bound form of the Rho GTPase, Rac. The main target of Rac-GTP is the p67 ^phox subunit, which contains a Rac-binding domain created by four α-helical tetratricopeptide repeat motifs. The GTP-bound form of Rac also appears to interact with flavocytochrome b .

Oxidase assembly is triggered by receptor-mediated binding of many soluble chemoattractants (see Table 22-4 ), which requires higher concentrations of these molecules compared with the initiation of chemotaxis. The binding of opsonized microbes to Fcγ and com­plement receptors or of fungi to β-glucan receptors are other major physiologic triggers of the respiratory burst, which is activated at sites of contact. Two critical events downstream of receptor binding are the phosphorylation of p47 ^phox and the activation of Rac to its GTP-bound state. The functional oxidase complex is assembled at the plasma membrane in response to soluble agonists. During phagocytosis, additional flavocytochrome b for NADPH oxidase assembly is delivered to the phagosome by the membrane fusion of specific granules, which contain the majority of the neutrophil's supply of flavocytochrome b . Since release of O ₂ ⁻ occurs largely at the extracellular side of the membrane, oxidants are released at sites of microbial contact or within the phagocytic vacuole, where they can interact with granule contents to potentiate their microbicidal effects.

Based on the redox properties of the flavocytochrome b ₅₅₈ , the following pathway has been proposed for transfer of electrons from NADPH to oxygen (O ₂ ) in the respiratory burst (see reaction 1 in Fig. 22-8 ):

The flavocytochrome spans membrane, so that NADPH is oxidized at the cytoplasmic surface and oxygen is reduced to form O ₂ ⁻ on the outer surface of the plasma membrane (or inner surface of the phagosomal membrane).

Once formed, the O ₂ ⁻ radical is first converted, either spontaneously or by means of superoxide dismutase, into hydrogen peroxide (H ₂ O ₂ ; see reaction 2 in Fig. 22-8 ). Azurophilic granule (derived MPO) in the presence of halides catalyzes the conversion of H ₂ O ₂ to hypochlorous acid (HOCl), the active agent in household bleach (see reaction 4 in Fig. 22-8 ). H ₂ O ₂ may also be converted into OH• in a nonenzymatic reaction with O ₂ ⁻ catalyzed by either iron or copper ions (see reaction 3 in Fig. 22-8 ). H ₂ O ₂ , HOCl, and OH• are all strong oxidants that participate in microbial killing within the phagocytic vacuole. Reactive oxidants also regulate phagocyte proteolytic activity by activating latent phagocyte metalloproteinases (such as collagenase and gelatinase) and inactivating plasma antiproteinases. Enhanced phagocyte proteolysis at localized sites may be important for facilitating cellular migration into inflamed tissues, destruction of microbes, and removal of cellular debris.

Other enzymatic pathways related to oxidant generation include the detoxification of H ₂ O ₂ by glutathione peroxidase and reductase (see reactions 6 and 7, Fig. 22-8 ). Glutathione is produced from γ-glutamyl cysteine by the enzyme glutathione synthetase (see reaction 9, Fig. 22-8 ). Other important antioxidant systems in phagocytes and other tissues include catalase, which catalyzes the conversion of H ₂ O ₂ into O ₂ and water; ascorbic acid; and α tocopherol (Vitamin E). The generation of NADPH is important in providing a source of reducing equivalents for the glutathione detoxification pathway as well as the respiratory burst itself. NADPH is replenished from NADP ⁺ by leukocyte glucose-6-phosphate dehydrogenase (G6PD; see reaction 8, Fig. 22-8 ) in the hexose monophosphate shunt.

A second oxygen-dependent pathway with antimicrobial effects involves the generation of NO from the oxidation of L-arginine to L-citrulline. This reaction is catalyzed by NO synthase (NOS), with molecular oxygen supplying the oxygen in NO. There are three different NOSs, two of which are constitutively expressed in a variety of tissues, including endothelium, brain, and neutrophils. Expression of a third, high-output isoform of NOS (inducible NOS [iNOS]) is inducible by inflammatory stimuli in a variety of cells, including macrophages and neutrophils, where it has a wide spectrum of antitumor and antimicrobial activity against bacteria, parasites, helminths, viruses, and tumor cells. NO can also interact with superoxide to form peroxynitrite, which mediates tyrosine nitration of cellular and bacterial proteins. High levels of iNOS-catalyzed NO production are readily elicited in normal mouse macrophages by exposure, for example, to IFN-γ and endotoxin. In human monocytes and macrophages, IFN-α/β, IL-4 plus anti-CD23, or chemokines can induce iNOS expression. The expression of iNOS has been detected in a variety of inflammatory and infectious diseases in humans, including malaria, hepatitis C, tuberculosis, tuberculoid leprosy, and acquired immunodeficiency syndrome dementia. Cytokine-activated human neutrophils also exhibit inducible production of NO that leads to nitration of ingested bacteria.