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CHAPTER 67 FUNCTIONS OF NEUTROPHILS

CHAPTER 67 FUNCTIONS OF NEUTROPHILS
Williams Hematology

CHAPTER 67 FUNCTIONS OF NEUTROPHILS

JAMES E. SMOLEN
LAURENCE A. BOXER

Chemotaxis and Motility

Ingestion

Adhesion Molecules
Granules

Types of Granules

Function of Granules
Stimulus-Response Coupling

Receptor-Ligand Interactions

G-Proteins

Phospholipid Metabolism

Calcium

Protein Kinases

Arachidonate Metabolism

Cytoskeleton

Cyclic Nucleotides

Degranulation and Membrane Fusion
Bactericidal Mechanisms

Nonoxidative Mechanisms

Oxidative Metabolism
Chapter References

Neutrophils protect the host against pyogenic infections. Their function is closely related with that of lymphocytes and macrophages, cells that are also involved in the response to infection. Chemotactic factors or chemotaxins, which are generated by the interaction of plasma proteins with antigens or pathogens, attract neutrophils from the blood to sites of infection. The diffusion of these factors creates a chemical gradient that directs the migration of neutrophils, with the cells moving toward the source of the chemotactic factor. Plasma, in addition to elaborating chemical attractants, provides antibodies and complement that coat microorganisms. This process of antibody and complement coating has been called opsonization, from the Greek word for “providing victuals.” The pathogenicity of microorganisms often results from their ability to prevent opsonization. Neutrophils ingest the opsonized microorganisms by surrounding them with moving pseudopodia, which fuse to enclose the microbe within a vesicle called the phagosome. The cytoplasmic granules of the neutrophil fuse with the phagosome and discharge their contents into it, a process called degranulation. The neutrophil reduces molecular oxygen enzymatically to generate “activated” metabolites such as superoxide and hydrogen peroxide that, together with material discharged into the phagosome from the granules, can kill ingested microbes. Granule contents and oxygen metabolites may leak from the neutrophil into extracellular fluid, where they can injure tissue as well as microbes. This leakage results from both direct secretion as well as from partially closed phagosomes (Fig. 67-1). This side effect of the attack of neutrophils against antigens or pathogens may be an important cause of tissue inflammation and in certain locations may be detrimental to the host.

FIGURE 67-1 Activities of the neutrophil.

Acronyms and abbreviations that appear in this chapter include: ADP, adenosine diphosphate; ARF, ADP-ribosylation factor; ATP, adenosine triphosphate; ATPase, adenosinetriphosphatase; BPI, bactericidal/permeability-increasing protein; BPB, bromophenacyl bromide; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; DAG, diacylglycerol; DAGK, diacylglycerol kinase; EGTA, ethylene glycol-bis(b-aminomethyl ether)-N,N-tetraacetic acid; ERK, extracellular signal-related protein kinase; FAD, flavin prosthetic group; fMet-Leu-Phe, formyl-methionyl-leucyl-phenylalanine; FMLP, formyl-methionyl-leucyl-phenylalanine; GPI, glycosylphosphatidylinositol; GTP, guanosine triphosphate; CGD, chronic granulomatous disease; HETEs, hydroxyeicosatetraenoic acids; ICAM-1, intercellular adhesion molecule-1; IL-1, interleukin-1; IL-8, interleukin-8; IP3, inositol trisphosphate; LAD I, leukocyte adhesion deficiency type I; LAD II, leukocyte adhesion deficiency type II; LTB4, leukotriene B4; LFA-1, leukocyte function-associated antigen-1; LPS, lipopolysaccharide; MAPK, microtubule-associated protein kinases; MPO, myeloperoxidase; NADPH, nicotinamide adenine dinucleotide phosphate; NEM, N-ethyl maleimide; NSF, NEM-sensitive fusion protein; PA, phosphatidic acid; PAF, platelet activating factor; PC, phosphatidylcholine; PIP1, phosphatidyl inositol-4-monophosphate; PIP2, phosphatidylinositol-4,5-bisphosphate; PKA, protein kinase A; PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospholipase C; PLD, phospholipase D; PMA, phorbol 12-myristate 13-acetate; PPH, phosphatidic acid phosphohydrolase; PT, pertussis toxin; SCAMP, secretory carrier membrane protein; SNAP, soluble NSF-attachment proteins; SNAREs, SNAP-receptors; TNF, tumor necrosis factor; VAMP-2, vesicle associated membrane protein-2

The primary function of neutrophils is to protect the host against bacterial infections. Exposure of human neutrophils to a variety of particulate and soluble stimuli evokes a series of responses, including chemotaxis, phagocytosis, degranulation, hexose monophosphate shunt stimulation, generation of reactive derivatives of oxygen, release of membrane-bound calcium, and reorganization of the cytoskeleton. All of these responses and the signal transduction processes mediating them are of considerable scientific and clinical interest. In this chapter, we will focus on the above mentioned responses, with special emphasis on stimulus-response coupling.
CHEMOTAXIS AND MOTILITY
The similarity between neutrophil locomotion and that of amebas was noted long ago.1 Neutrophils can respond to spatial gradients of chemotaxins with differences in concentration of chemotaxin of as little as 1 percent,2 although there has been contention as to whether chemotaxis also requires temporal, as well as spatial, sensing.3 Even with populations of cells as “homogenous” as neutrophils, a broad range of responsiveness is found.4 During locomotion toward a chemotactic source, these cells acquire a characteristic asymmetric shape (Fig. 67-1, Fig. 67-2). In the front of the cell is a pseudopodium that advances before the body of the cell containing the nucleus and the cytoplasmic granules. At the rear of the moving cell is a knoblike tail. The anterior pseudopodium undulates or “ruffles” as the neutrophil moves, at a rate of up to 50 µm/min. The membrane lipids also flow during locomotion,5 and enhanced cytosolic Ca2+ is observed along the membrane margin.6,7 The pseudopodium, which is very thin, forms immediately when the cell encounters a gradient of chemotactic factor. As the cell moves, the cytoplasm behind the anterior pseudopodium streams forward, almost obliterating the pseudopodium. At this point some granules appear to contact the cell periphery, and the release of granule contents, a recognized response to chemotactic agents,8 can occur. The pseudopodium extends again and the process repeats itself. A flow of cortical materials, composed particularly of actin filaments, has been proposed to account for chemotaxis as well as other cellular movements.9 This may also account for changes in cell viscosity.10

FIGURE 67-2 Cinemicrophotographic observation of granule lysis of a chicken neutrophil following phagocytosis of zymosan particles. Note the lysis of the cytoplasmic granule (G) against one of two ingested zymosan particles (Z). The dense body of the granule disappears from view in the interval of 5 s (x1200). (From JG Hirsch. J Exp Med 116:827,1962, with permission.)

INGESTION
When a neutrophil comes in contact with a particle, the pseudopodium flows around the particle, its extensions fuse, and it thereby encompasses the particle within the phagosome.1 The ingestion phase can be said to extend from recognition to the end of pseudopodium fusion. The particle thus becomes enclosed within a phagosome into which granules are rapidly discharged, as illustrated in Fig. 67-2. As with locomotion, phagocytosis results in Ca2+ being released in the vicinity of the active membranes.6 The number of ingested particles may be eventually limited by the availability of plasma membrane.11 Locomotion is not a prerequisite for ingestion: if neutrophils collide with a particle not secreting a chemotactic substance, pseudopodia form abruptly at the contact point and envelop the particle. Ingested particles gradually move toward the cell interior, where they tumble about with the nucleus and cytoplasmic granules as the cell moves off. A small number of the phagocytosed particles are actually expelled.12
The formation of a pseudopodium is essential for neutrophil locomotion. The interior cytoplasm is squeezed in the direction of the lamellopodium, possibly by the peripheral cytoplasm in the rear of the cell. The pseudopodium is also required for ingestion. When dissolution of the pseudopodium occurs, the interior contents of the cell are allowed to contact the cell membrane. Granule discharge may occur. Fusion of membranes is a common feature of (1) ingestion, where pseudopodia fuse; (2) degranulation, where granules fuse with the phagosome; and possibly (3) locomotion, where some granules may fuse with the plasma membrane. Pseudopodia form whether neutrophils are suspended in liquid medium or are attached to a surface, but the cell can only move translationally when fixed to a surface13; thus it crawls but does not swim.14 Such “stickiness” is also a phase of ingestion. The neutrophil membrane adheres firmly to particles they ingest,15 presumably to provide the frictional force needed to move pseudopodia around the particles. Thus, the formation of pseudopodia, membrane fusion, and membrane adhesiveness are all characteristics associated with the functional responses of neutrophils.
ADHESION MOLECULES
Neutrophils circulate in the blood in a nonadherent state. Upon activation, the neutrophil becomes more adhesive, enabling receptor-mediated margination to the vasculature and subsequent chemotaxis and phagocytosis.16 A number of surface proteins, most notably the b2 integrins and the L-selectins, have been identified as mediators of adherence.17,18 and 19 The bonding properties of these adherence molecules seem to govern the transient tethering of neutrophils to venules, with characteristic “rolling.”17 Adherent cells are often “primed” or sensitized, and can have substantially different functional properties as compared to cells in suspension.20 When neutrophils are in suspension, they aggregate if stimulated with chemotactic factors, a process that is distinct from adherence in many respects.21
A sequence of molecular and biophysical events leading to neutrophil activation and increased adherence during the acute inflammatory response in vivo is shown in Fig. 67-3. An activated neutrophil enters post-capillary venules adjacent to inflammatory foci and develops transient adhesive interactions with inflamed endothelium via specific classes of adhesion molecules that include the selectins.22 Endothelial cell selectins (E-selectin and P-selectin) are inducibly expressed by endothelial cells following exposure to inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1) (products of endotoxin-stimulated mononuclear phagocytes)22 (Fig. 67-3b). Specific oligosaccharide moieties expressed on neutrophil membranes serve as counter-receptors for E-selectin and P-selectin (sialyl Lewis X and Lewis X, respectively). In conjunction with neutrophil membrane L-selectin, which recognizes oligosaccharide moieties expressed by endothelial cells, they promote transient neutrophil-endothelial binding under flow conditions, termed neutrophil “rolling.”17,22

FIGURE 67-3 The neutrophil-mediated inflammatory response. (A) Unstimulated neutrophils (expressing L-selectin) entering a postcapillary venule. (B) Invasion of gram-negative bacteria with release of lipopolysaccharide stimulates tissue macrophages to secrete inflammatory monokines, IL-1 and TNF, which, in turn, activate endothelial cells to express E- and P-selectins. E- and P-selectins serve as counter-receptors for neutrophil L-selectin to cause low-avidity neutrophil rolling. (C) Activated endothelial cells express ICAM-1, which serves as a counter- receptor for neutrophil b2 integrin molecules leading to high-avidity leukocyte spreading and the start of transendothelial migration. Transendothelial migration of activated neutrophils is stimulated by chemotactic factors such as endothelial cell-derived IL-8 and formylated bacterial factors. Chemoattractants promote neutrophil activation with the release of L-selectin and an increase in b2 integrin affinity for ICAM-1 and for other counter-receptors promoting intravascular neutrophil aggregation. (D) Neutrophil invasion through the vascular basement membrane with the release of proteases and reactive oxidative intermediates that cause local destruction of the extracellular matrix.

Neutrophil rolling is a prerequisite for the transition to an interaction with the inflamed endothelium that is more resistant to shear stress.17,18,23 Firm adhesion is mediated by a separate class of molecules whose expression level and functional affinity are increased by high local concentrations of inflammatory stimuli.17 Specifically, on endothelial cells the ICAM-1 (CD54) glycoprotein is induced by cytokines that include TNF and IL-1. ICAM-1 serves as a recognition target for neutrophil b2 integrin counter-receptors Mac-1 (CD11b/CD18) and LFA-1 (CD11a/CD18). The relative affinity of Mac-1 and LFA-1 for ICAM-1 is increased by exposure of neutrophils to numerous stimuli, including C5a, N-formylated bacterial peptides (FMLP), IL-8 (synthesized by inflamed endothelium), and LTB418,22 (Fig. 67-3c and Fig. 67-3d). High-affinity sticking between neutrophils and endothelial cells, dependent upon b2 integrin, promotes subsequent transendothelial migration through the basement membrane. This migration continues into the extracellular matrix in response to local gradients of chemotactic factors18,24 (Fig. 67-3d). During neutrophil activation, there is a reciprocal relationship on the plasma membrane between the expression of L-selectin and the b2 integrin Mac-1. L-selectin, which initially has a high basal expression, is shed upon exposure to chemotactic stimuli. Mac-1 receptor sites are initially at a low basal expression and affinity, but increase ten- to twentyfold after stimulation.19 The changes in affinity and surface expression occur over seconds to minutes following stimulation.25
The CD11/CD18 integrins are vital to firm adherence and transmigration. Complete inhibition of CD18, the common b2 chain of the leukocyte integrins, profoundly reduces emigration of neutrophils at sites of inflammation but leads to a severe immunodeficiency syndrome termed leukocyte adhesion deficiency type I (LAD I).26 Patients with LAD I have mutations in CD18 that lead to a severe or total deficiency of the CD11/CD18 integrins from the cell surface, which include CD11a/CD18 (LFA-1, aL2), CD11b/CD18 (Mac-1, CR3, aM2), CD11c/CD18 (p150,95, aX2), and CD11d/CD18 (aD2). Another type of deficiency, LAD II, is related to defects in synthesis of the glycoprotein ligands for adhesion molecules.27
Neutrophil-mediated inflammatory injury that is dependent upon these adhesive events can occur by one of several mechanisms: (a) activation of integrin-dependent neutrophil hyperadherence promotes homotypic aggregation (mediated by CD11b/CD18) that may result in plugging of microvessels and resultant distal ischemia28; (b) high concentrations of inflammatory stimuli cause neutrophil degranulation and the release of a variety of proteases (see below) that may cause destructive proteolysis of the subadjacent extracellular matrix; and (c) the NADPH oxidase-dependent generation of oxidative metabolites from neutrophils, primed by endotoxins, inflammatory cytokines, and/or substrate adhesion and stimulated by other activating factors, may promote direct endothelial cell injury or promote the destructive effects of secreted proteases either by activating latent metalloproteinases or by deactivating oxidant-sensitive antiproteinases.29
GRANULES
TYPES OF GRANULES
PRIMARY GRANULES
Neutrophil granules serve as reservoirs for digestive and hydrolytic enzymes prior to delivery into the phagosome. Based on their biochemical and morphological properties and time of appearance during cell maturation, three major granule populations have been described (Table 67-1). Azurophil or primary granules are synthesized first during granulocytopoiesis, with secondary and tertiary granules being developed at succeeding times. It has been reported that the contents of granules are determined by the timing of their expression, rather than by signal sequences.30 Thus, granule contents become packaged into the type of granule being formed at the time they are synthesized.

TABLE 67-1 NEUTROPHIL GRANULE CONSTITUENTS

Azurophil granules contain myeloperoxidase, neutral proteases (elastase, cathepsin G, cathepsin D), protease inhibitors, acid hydro-lases (b-glucuronidase, acid phosphatase, a-mannosidase, N-acetyl-glucosaminidase), and cationic proteins.31,32 and 33 Two subpopulations of azurophil granules have been identified and differ in their distribution of several lysosomal enzymes.32,34 Elastase can be preferentially released from a denser subpopulation,34 suggesting that these granule subpopulations may be under separate regulatory control. Recent studies have shown substantial heterogeneity of azurophil granule contents.35
Azurophil granule proteins possess microbicidal activity and may additionally play a significant role in the tissue destruction observed during inflammatory reactions. Defensins are microbicidal peptides that are found in these granules.31,36 These proteins are released concomitantly with other azurophil granule constituents following stimulation.37 Bactericidal/permeability-increasing protein (BPI), a cationic protein that lyses gram-negative bacteria and inhibits endotoxin, is also found in these granules.38 Myeloperoxidase (MPO) reacts with H2O2 and a halide to produce hypochlorous acid (HOCl), a potent oxidant (Clorox) believed to significantly contribute to the killing of microorganisms.39 MPO may also play a modulating role in degranulation secondary to its ability to inactivate chemotactic factors40 and inhibit the mechanisms underlying the respiratory burst and phagocytosis.41
With respect to the tissue destruction observed during inflammatory reactions, the most important azurophil constituents are the proteases. Three of these proteases have been well characterized. The serine protease, elastase, hydrolyzes typical pancreatic elastase substrates and a variety of elastin preparations from several sources.42 Cathepsin G is a chymotrypsin-like neutral protease that hydrolyzes proteoglycans and insoluble collagen.43 Finally, cathepsin D can cleave leukokinogens to generate pharmacologically active leukokinins.44 It is also active against proteoglycans. Working in concert, these proteases can exacerbate existing inflammatory reactions by generating chemotactic factors from C5.45
SPECIFIC GRANULES
Specific or secondary granules are synthesized later during granulocytopoiesis. As shown in Table 67-1, proteins packaged within these granules (in human neutrophils) include lysozyme, collagenase, vitamin B12-binding protein, heparanase, and lactoferrin.46,47,48 and 49 Lysozyme hydrolyzes cell wall proteoglycan of some bacterial species and is also found in azurophil granules. Lactoferrin is an iron-binding protein that appears to be necessary for hydroxyl radical formation50 and can influence the functions of lysozyme to kill gram-negative bacteria.51 Like certain azurophil granule proteases, several specific granule proteins may contribute to inflammatory conditions. Collagenase, in the presence of a neutral protease, hydrolyzes collagen fibrils; this enzyme may also be activated by oxidative metabolism.52 Heparanase, a heparan sulfate-degrading endoglycosidase, may be involved in extravasation of neutrophils through the subendothelial basement membrane.49 In addition, these granules also contain activators of the complement cascade.53 A modulatory role for these granules in cell locomotion is suggested by the observation that specific granule membranes contain receptors for chemoattractants, extracellular matrix proteins, and adhesion molecules.54,55,56,57,58 and 59 These granule membranes also possess FAD-containing cytochrome b60,61 and 62 that translocates to the plasma membrane upon cell stimulation, suggesting a potential for regulation of the respiratory burst. Individuals whose neutrophils are lacking in specific granule contents are susceptible to repeated staphylococcal skin and respiratory infections,63 emphasizing the importance of these proteins in host defense mechanisms (see Chap. 82). Neutrophils from these patients have impaired chemotaxis and adherence.64 The finding that these cells also do not increase expression of chemotactic receptors following activation is consistent with a role for specific granules in locomotion and may underlie their defective chemotactic response.
TERTIARY GRANULES
Tertiary granules contain gelatinase,65,66 an enzyme which bears many similarities to neutrophil collagenase,29 and heparanase.66 Tertiary granule membranes appear to contain a pool of membrane glycoproteins that participates in cell adhesion.67,68 The C3bi receptor is essential to cell adhesiveness69 and there is evidence to suggest that it is partially localized in tertiary granules.68 A tertiary granule localization has also been reported for the oxidase cytochrome b.70 Finally, the tertiary granules (like the secondary granules) constitute important intracellular pools for neutrophil adhesion molecules.66,71
PHOSPHASOMES AND SECRETORY GRANULES
As improved methods have become available for the isolation and characterization of granules and their constituents, other granule populations have been identified. For instance, “phosphasomes,” which contain alkaline phosphatase, have recently been isolated from neutrophils.72,73 These granules are lighter in density than specific granules, but do not contain gelatinase. Another granule type that has been reported is the “secretory” granule, which contains plasma proteins.74 These granules are highly labile and are most readily released in response to stimuli75; their membrane components are also rapidly recovered into vesicles.75 Secretory granules also contain complement receptor I,76 tyrosine kinases,77 and phospholipases.78
FUNCTION OF GRANULES
Granule contents play very important roles in nonoxidative mechanisms of immunity and inflammation. Secondary granules are readily mobilized to the extracellular environment in response to many stimuli, such as N-formylated peptides released by bacteria, complement proteins, and leukotriene products.29,79,80 At sites of inflammation, the neutrophils ingest microorganisms or other particles, as well as discharge granules by exocytosis.80 To a large extent, the extracellular release of specific and azurophil granules remains under separate control.79,81 Chemoattractants and other substances can be used to selectively release specific granules under conditions wherein azurophilic granule enzymes are not discharged.81,82 On the other hand, stimuli for azurophilic granule release also stimulate concomitant exocytosis of specific granules,83 with rare exceptions.84 The resistance of azurophil granules to secretion may be due to a requirement for a biochemical signal in addition to Ca2+.85 Tertiary and secretory granules are more readily discharged than specific granules during cell stimulation; thus, their contents are available to modify the response of the cell to stimuli as well as to affect function.65,68,75
Studies of the function of granule contents have depended upon the isolation and characterization of granule proteins and upon evaluation of the behavior of neutrophils with abnormal granules. For instance, the function of tertiary granules is best illustrated by patients who are deficient in a group of neutrophil membrane glycoproteins, namely the b2 integrins discussed earlier. One of these crucial integrins is CD11b/CD18, also known as Mac-1, Mo-1, or the C3bi receptor (CR3), which is normally found on neutrophil plasma membranes, specific granules, tertiary granules, and secretory granule membranes.67,71,86 Patients with a complete lack of CD18 (and hence the family of b2 integrins) present with recurrent and severe bacterial and fungal infections, impaired wound healing, diminished pus formation, and persistent neutrophilia (see Chap. 82). The severity of the clinical manifestation directly relates to the degree of deficiency of adhesive glycoproteins on the membrane.26,86 CD11b/CD18 is one of these molecules responsible for neutrophil adhesiveness.69 CD11b/CD18 is stored in gelatinase-bearing tertiary granules68 and in secretory granules.71
Release of specific granules is crucial in mobilizing mediators of inflammation, since these granules contain activators of the complement cascade, leading to the generation of the chemoattractant C5a and the opsonin C3b.53 Lactoferrin can both attenuate granulopoiesis87 and alter the functions of mature cells.88 The release of lactoferrin during degranulation diminishes the negative surface charge of cells, which may be important in sustaining cell adhesiveness.89 Lactoferrin also facilitates the generation of a highly reactive product of oxygen metabolism, namely hydroxyl radical.50 The flavoprotein cytochrome b is an important constituent of the electron transport chain for the NADPH oxidase. It is also found in specific granules and is translocated60,61 and 62,90 to the plasma membrane during cell activation, perhaps playing a key role in amplifying the respiratory burst. Recent publications have reported the presence of b cytochrome in tertiary70 and secretory91 granules as well. In addition, there is considerable evidence that the membranes of specific granules contain receptors for chemoattractants and ECM proteins.54,56,57 and 58,67 Thus, translocation of chemoattractant receptors from the granule membrane to the leading edge of the plasma membrane may be essential for cell orientation by providing fresh receptors at the leading edge of the cell during margination and diapedesis.92 In addition, membranes of specific granules contain an inhibitor of protein kinase C, which in turn may serve to dampen ongoing neutrophil activation.93,94
Azurophilic granules contain potent digestive and microbicidal enzymes. Elastase, together with the specific granule component collagenase, may facilitate the penetration of cells through extracellular matrix.95 Other lysosomal proteases serve to degrade ingested material. Lysozyme, cationic proteins, and defensins have bactericidal activity.36,96 Myeloperoxidase, a heme-containing protein, has both cytotoxic and anti-bacterial activities, through its ability to interact with halides and hydrogen peroxide to generate hydroxyl radical and hypochlorous acid, and can oxidize chemoattractant peptides and neutrophil secretory products.40,50,97 Other azurophilic granule proteins inactivate chemoattractants. Thus, the azurophilic granules mediate target cell death and modulate the inflammatory response.
STIMULUS-RESPONSE COUPLING
Secretion by neutrophils has been the subject of intense research for many years. This work has been fruitful in illuminating some of the underlying causes of defects in cell activation. Studies of neutrophil degranulation and oxidative metabolism have also revealed transduction mechanisms common to a wide variety of other important secretory cell types, thereby greatly expanding the relevance of this work. The techniques employed by researchers in this field have been extensively reviewed elsewhere.98 This review will now discuss in considerably more depth our current understanding of the activation process, which is shown schematically in Fig. 67-4.

FIGURE 67-4 Model of stimulus-response coupling in the neutrophil leading to degranulation. The signal transduction mechanisms alluded to in the text are schematically illustrated in this figure. The resting state of the cell is represented in the lower left side by an unoccupied cell surface receptor (R) coupled with an inactive Gi-like guanine nucleotide binding protein (Gi). Binding of a specific ligand to this receptor leads to conformational changes in this protein (R*) and activation of the accompanying G-protein (square figure). Activation of this G-protein is blocked by pertussis toxin and involves the binding of GTP, leading to an activated G-protein (Gi*). This G-protein then interacts with a polyphosphatidylinositol-specific phospholipase C (PLC, hexagon) leading to the activation of this enzyme (PLC*, triangle). PLC* cleaves an endogenous lipid, namely phosphatidylinositol bisphosphate (PIP2), yielding diacylglycerol (DAG) and inositol trisphosphate (IP3). IP3 is known to liberate calcium from bound intracellular stores leading to a rise in intracellular free calcium (Ca2+fr). The increase in intracellular Ca2+fr is augmented by an influx from the extracellular space (right side of figure). Increased DAG, in consort with elevated Ca2+fr, and activated protein kinase C (PKC, pentagon) leads to translocation of this enzyme to membranous sites (PKC*). In addition, phospholipase D can be activated (PLD*) by PKC*, Gi*, or Ca2+fr (details currently under investigation), converting phosphatidylcholine (PC) to phosphatidic acid (PA). PA and DAG can be interconverted through the actions of phosphatidic acid phosphohydrolase (PPH) and diacylglycerol kinase (DAGK). Elevations in intracellular Ca2+fr alone are known to induce the secretion of granule constituents to the extracellular space (lower right side) by fusion of granule membranes with the plasma membranes. This process is augmented by the presence of GTP. These transduction mechanisms are discussed in greater detail in the text.

RECEPTOR-LIGAND INTERACTIONS
FORMYL PEPTIDE RECEPTOR
Neutrophil responses can be evoked by a variety of particulate and soluble stimuli. Opsonized particles, immune complexes and chemotactic factors produced during the inflammatory process activate neutrophils by binding to specific cell surface receptors. Of the neutrophil chemotactic receptors, the N-formyl peptide receptor is the best characterized. N-Formyl peptides, the synthetic analogs of bacterial products, induce a variety of neutrophil responses and have been extensively employed as activating stimuli. Specific receptors for the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe), have been identified on the neutrophil surface,99 and binding of the formyl peptide to its receptor correlates with its ability to induce chemotaxis and degranulation.100 The fMet-Leu-Phe receptor is stereospecific,100 has multiple affinity states,101 and has an apparent molecular mass of 50 to 70 kDa.102 An intracellular storage location has also been suggested for the fMet-Leu-Phe receptor, since neutrophil activation results in enhanced fMet-Leu-Phe binding in normal cells,103 but not in egranulated neutrophil cytoplasts.104 These receptors have been identified on the membranes of tertiary and secretory granules105 and are biochemically similar to those on the plasma membrane,106,107 suggesting that they are mobilized to the cell surface following stimulation and may thus serve a modulatory role. The formyl peptide receptor has been cloned and sequenced108,109 and belongs to a family of seven membrane-spanning domain proteins typical of G-protein coupled receptors. The receptor occurs in several forms,110 is physically associated with guanine nucleotide binding proteins (G-proteins)108,111,112 and 113 and the cytoskeleton111,114,115 (see Ref. 116 for review).
C5A RECEPTOR
Activation of the complement system generates C5a, a derivative of C5 and the most potent of the chemotactic proteins. C5a induces neutrophil chemotaxis,117 degranulation,118 and superoxide generation.119 In turn, it has been suggested that a C5a inhibitor may play a role in the regulation of inflammatory processes and that its deficiency, which is familial, may explain the attacks of sterile inflammation characteristic of Mediterranean fever.120 Responses to C5a result from interactions with specific receptors on the cell surface.121 The receptor was identified as a single polypeptide in the plasma membrane with an apparent molecular mass of 40 to 48 kDa.122 Binding studies have shown that there are 50,000 to 113,000 receptor sites per cell with a dissociation constant (Kd) of 2 × 10–9 M. Strong interactions between the C5a receptor and G-proteins has been reported.123 Like the FMLP receptor, C5a receptors have been isolated and cloned.124,125 There is some heterogeneity in both of these receptors,126 which are 20 to 35 percent identical in their amino acid sequences. The C5a receptor also belongs to the diverse class of receptors that features seven membrane-spanning domains. Expression of these receptors in other cells has been used to explore their common means of signal transduction.124,127,128
C3 RECEPTORS
Neutrophils also express receptors for the complement-derived chemotactic factors C3b and C3bi. Receptors for C3b and C3bi (also known as CR1 and CR3, respectively) are sparse on resting neutrophils but significantly increase in number following activation with several stimuli.129 Like formyl peptide receptors, the stimulus-induced increase in surface expression of C3b and C3bi receptors appears to result from the mobilization of intracellular pools,130 although the subcellular locations for these receptors appear to be distinct.54 Indeed, CR3, which is of the b2-integrin family (CD11b/CD18), may be located in tertiary granules68,131 and may be functionally linked to the cytoskeleton132 (see below). The C3b receptor (CR1) is a glycoprotein with a molecular weight of 205 kDa133 and appears to be located in secretory granules.76,134
INTEGRINS
CD11/CD18 integrins also play an important role in cell signaling. The adhesion of cells to surfaces or to other cells can either activate neutrophils directly or “prime” them for an enhanced response to other stimuli. For example, the oxidative burst of neutrophils is very different in cells that are suspended versus those that are adherent to surfaces.135 H2O2 production in response to chemotaxins has been shown to be influenced by monoclonal antibodies to CD11b, but not CD11a.136 Yet CD11a-dependent adhesion of canine neutrophils can alone trigger H2O2 production in the absence of other stimuli.137
Integrins are involved in both outside-in and inside-out signaling that induces conformational changes regulating the affinity of ligand bind-ing.138,139 and 140 In addition to traditional trans-acting receptors, there is also evidence that CD11b and CD11c can serve as cis-acting receptors that transduce signals from glycosylphosphatidylinositol (GPI)-linked receptors138 (see below).
FC RECEPTORS
Neutrophils possess three different receptors for immunoglobulins. Unstimulated cells express FcgRII and FcgRIII, also known as CD32 and CD16, respectively. Functionally, the most important of the two could be FcgRIII,141 which is attached to the membrane by a GPI linkage. This linkage is relatively labile, so the amount of FcgRIII on the membrane reflects a balance between shedding and mobilization from intracellular stores.56,58,142 FcgRII is a conventional protein that spans the plasma membrane.143 Interestingly, the signal transduction pathways initiated by FcgRIII can cross-talk with the formyl peptide receptor,144 with CR3,145 and even with each other.146 Both FcgRII and FcgRIII produce Ca2+ transients,147 although this has been disputed.148 The Fc receptors also signal through a variety of kinases, including tyrosine kinases,149 phosphatidyl inositol kinases,150 and MAP kinases.151 Finally, cytokine-stimulated neutrophils express yet another receptor FcgRI, or CD64,152 which is beginning to be understood.153 FcgRI can signal through Fcg RIIIA.154
RECEPTOR “CROSS-TALK”
Several lines of evidence suggest that CD11b participates in FcgR-mediated functions. First, neutrophils from patients with LAD I have impaired IgG-dependent phagocytosis and cytolysis.69 Also, antibodies to CD11b inhibit the phagocytosis of Ig-coated substrates.155 A direct physical linkage between CD11b and FcgRIIIB has been demonstrated by experiments in which capping of one receptor results in co-capping a substantial fraction of the other receptor.156 CD11b can also interact with the transmembrane FcgRII, and both of these molecules can modify each other’s signals.157 Finally, the uroplasminogen activator receptor (uPAR, CD187) is a GPI-anchored glycoprotein present on neutrophils with which CD11b can interact directly, but reversibly.158 In summary, although CD11b and CD11c do not directly participate in antibody-dependent recognition, these integrins provide a novel mechanism for a GPI-anchored receptor to transduce signals leading to effector responses in neutrophils.
OTHER RECEPTORS
Three other important receptors are for platelet activating factor (PAF), IL-8, and leukotriene B4 (LTB4). PAF and IL-8 receptors have been cloned124,127,159 and belong to the seven-transmembrane domain family. Their intracellular stores and signal transduction mechanisms are largely similar to those used by similar receptors (e.g., FMLP).123,127 IL-8 has two related receptors, for which slightly different signal transduction pathways have been detected.160,161 The LTB4 receptor undergoes cycling,162 as do the IL-8 receptors,163 and preliminary steps toward purification have been accomplished.164 The LTB4 receptor also signals through G-proteins.165
G-PROTEINS
Recent data strongly suggest that receptors for the chemotactic stimuli fMet-Leu-Phe, C5a, LTB4, and PAF are coupled to cellular responses through a guanine nucleotide binding protein similar to the inhibitory protein, Gi, of the adenylate cyclase system. Evidence linking a G-protein to these receptors has been provided by studies demonstrating that guanine nucleotides can regulate receptor affinity.166 A high-affinity GTPase located in neutrophil plasma membranes is stimulated by these same receptor-mediated stimuli, but not by the phorbol ester, PMA.167 This enzymatic activity is likely to be involved in terminating the activation of the guanine nucleotide binding protein. Also, direct linkages between receptors and G-proteins have been observed.108,111,112 and 113,123,162
Studies using pertussis toxin have proven instrumental in the understanding of G-protein involvement in the proposed stimulus-response coupling pathway. Pertussis toxin ADP-ribosylates the a-subunit of Gi of the adenylate cyclase system and also a 40- to 41-kDa protein in neutrophil plasma membranes.168 Initial studies demonstrated a strong correlation between the ability of the toxin to catalyze the ADP-ribosylation of the membrane protein and its ability to affect cellular responses initiated by surface receptors. However, pertussis toxin does not alter intracellular cyclic AMP levels, suggesting that the G-protein involved is distinct from Gi of the adenylate cyclase system.169 More recently, purification and characterization of a guanine nucleotide binding protein in neutrophils have shown that this protein differs both structurally and immunochemically from previously reported guanine nucleotide binding proteins.168,170,171 and 172
Not only can neutrophil responses be abolished by pertussis toxin, but stable guanine nucleotides can directly stimulate permeabilized neutrophils.173,174,175 and 176 In other cells, heterotrimeric G-proteins may also play a tonic inhibitory role in degranulation.177 While PT inhibits fMet-Leu-Phe-induced secretion from intact cells, it does not inhibit degranulation in response to guanine nucleotides, PMA, and Ca2+ in the permeabilized cell system, suggesting that a second G-protein is involved at distal sites in secretion.174 Potential candidates in this role are the family of small G-proteins (with Mw of 20-30 kDa) that has been reported in neutrophils.171,178,179 Some of these proteins are components of the bactericidal NADPH oxidase system180 that will be discussed later. Another study suggests that G-proteins sensitive to botulinum toxins C3 and D are involved in degranulation.181 Perhaps related to this, rho proteins have been shown to be involved in adhesion182 and rab5 proteins (that are probably involved in vesicular traffic) have been reported to translocate from cytosol to granules following neutrophil stimulation.183
PHOSPHOLIPID METABOLISM
The next step in signal transduction can be attributed to interactions of receptor-activated G-proteins and tyrosine kinases with phospho-lipases.172,176,184,185 and 186 Of primary importance, a membrane-associated phosphoinositide-specific phospholipase is activated upon stimulation with chemotactic stimuli. In particular, phospholipase C hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidyl inositol-4-monophosphate (PIP1)184,187 to the putative second messenger products inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG).184 In permeabilized neutrophils, IP3 has been shown to interact with a specific intracellular receptor188 and stimulate the release of Ca2+.189,190 Evidence has been presented that the Ca2+ is not derived from either the mitochondria189 or the endoplasmic reticulum,191 but rather from a distinct organelle which has been termed “calcisome.” Receptor stimulation also elicits the production of inositol 1,3,4-trisphosphate and inositol 1,3,4,5-tetrakisphosphate, but these compounds do not appear to have significant functional consequences with regard to Ca2+ release.192
Other pathways pertinent to signaling and cell activation are also involved. It has been proposed that phospholipase D, which preferentially uses phosphatidylcholine (PC) to produce phosphatidic acid (PA),172,193,194 is involved in neutrophil activation of the respiratory burst. The evidence is growing that PA generated by this enzyme is critical in cellular responses.5,193,195 Activation of PLD appears to be mediated by rho and/or ADP-ribosylation factor (ARF) along with a higher Mw factor.196,197 The evidence is growing that PA generated by this enzyme is critical in cellular responses, and particularly degranulation.176,193,195,198 Furthermore, it appears that the bulk of the DAG generated in stimulated neutrophils may be derived from the action of phosphatidate phosphohydrolase on this PA, rather than from the PLC pathway.195 The hallmark of these DAGs, which are ultimately derived from PC, is the presence of 1-O-alkyl linkages. In addition, it now appears that PA can also be synthesized de novo, from glucose.199 It has also been reported that the PA generated by the action of PLD, in combination with annexin I, promotes the Ca2+-dependent apposition of membranes.200
Fatty acids are also important in signaling and fusion. Phospholipase A2 (PLA2), found in the cytosol, membrane, and granule fractions of neutrophils,201,202 is activated during neutrophil stimulation,203 yielding arachidonic acid as a major product. The cytosolic PLA2 is important to intracellular signaling and is activated by phosphorylation204,205 and by PLD.206 Although the cytosolic enzyme per se is not Ca2+-dependent, the increases in intracellular Ca2+ occurring during cell activation result in translocation to the membrane203,204 and overall enhancement of arachidonic acid release.176,207 The role of arachidonic acid and its metabolites will be covered later in this review.
Recently, sphingolipid turnover in neutrophils has been under considerable study. Products of sphingolipid metabolism variously stimulate or inhibit cellular functions,208 including degranulation.209 The place of sphingolipid metabolism in the signal transduction scheme is now being elucidated.210,211
CALCIUM
The importance of Ca2+ as an effector of cellular function has received widespread attention in recent years. Like other cell types, the intracellular Ca2+ concentration in resting neutrophils is maintained at submicromolar levels by a plasma membrane-bound Ca2+-ATPase pump.212,213 There is substantial evidence that intracellular Ca2+ levels rise in stimulated neutrophils and that these increments may directly or indirectly bring about cell responses. Studies employing the fluorescent Ca2+ probes quin-2 and fura-2 indicate that following surface stimulation, intracellular Ca2+ rises almost immediately from a resting level of 0.1 µM to 1 µM.6,214,215 It is likely that local intracellular Ca2+ levels can reach far higher levels. Due to a variety of methodologic problems, including kinetic, spatial, and chemical limitations, conventional indicators underestimate those local subplasmalemmal Ca2+ concentrations that are the actual determinants of cellular responses.216,217 In excitable cells, it has been reported that these local concentrations could exceed 100 µM.216,217 Studies in neutrophils with new, more sensitive techniques demonstrate that local Ca2+ levels reach 1 to 5 µM,218,219 and 220 considerably higher than previously supposed.
There are two separate mechanisms involved in the stimulus-induced rise in intracellular Ca2+ levels; there is an initial IP3-mediated mobilization of Ca2+ from intracellular depots followed by an influx of extracellular Ca2+. It should also be noted that individual cells undergo periodic oscillations in intracellular Ca2+, which may be related to responsiveness.221
As discussed above, the mechanism by which Ca2+ is released following cell activation appears to involve the breakdown of membrane phosphatidylinositols222,223 that are in turn maintained by specialized enzymes.224 In permeabilized neutrophils, IP3 binds to a specific, saturable receptor188 and induces the specific release of intracellular Ca2+.189 The storage pool contributing to observed increases in Ca2+ is currently being characterized.225,226 A useful pharmacologic tool for these studies is thapsigargin, an inhibitor that blocks filling and maintenance of the storage pool.227,228 In subcellular fractions of neutrophil homogenates, IP3 stimulates Ca2+ release only from microsomes.191 Further separation of microsomal components suggests that neither the plasma membrane nor the endoplasmic reticulum is involved in sequestering IP3-sensitive Ca2+.191 Intracellular Ca2+ is sequestered in an organelle termed a “calciosome.”229 Ca2+ is stored in these sites by binding to a high-affinity calreticulin.229 However, nonmobilizable intracellular pools also exist.227
The initial mobilization of intracellular Ca2+ liberated in response to IP3 appears to directly regulate the permeability changes to Ca2+ across the plasma membrane.225,230 The repletion state of the storage pool and a soluble factor, rather than the production of inositol phosphates, are actually responsible for triggering Ca2+ influx.230,231 and 232 These findings are consistent with the previously reported observation that enhanced Ca2+ permeability during activation is dependent on mobilization of intracellular Ca2+.233 However, cell responses are not blocked by EGTA, suggesting that extracellular Ca2+ and a Ca2+ influx are not absolute requirements.234
In an effort to directly demonstrate Ca2+-dependent secretion, neutrophils have been permeabilized by a variety of means, including cholesterol-complexing agents (saponin,235 digitonin,166,189,236 and streptolysin O237), Sendai virus,174 and high-voltage electric fields,175,238 and then exposed to Ca2+ in EGTA-containing media. With some exceptions,235 these studies have shown that micromolar levels of Ca2+ alone are sufficient to induce the release of both specific and azurophil granule constituents.174,175,202,236 Specific granules require lower Ca2+ concentrations for secretion. The implication that azurophil and specific granule exocytosis differ in their Ca2+ requirements is supported by intact cell studies using quin-2 and ionomycin to establish the desired intracellular Ca2+ concentrations.85,239 While secretion can be modulated by both guanine and adenine nucleotides as well as by PKC activation, it appears that the primary intracellular trigger for exocytosis is Ca2+.240,241 Other aspects of neutrophil function require Ca2+ to various extents.242
The mechanistic significance of stimulus-induced elevations in intracellular Ca2+ is not absolutely established at present. Several investigators have reported that inducing optimal increments in intracellular Ca2+ levels is an ineffective measure for evoking some neutrophil responses.214,243 In addition, a number of responses take place independently of rises in intracellular Ca2+.244,245 These observations indicate that Ca2+ is not a sufficient or mandatory signal in some activation pathways. Elucidation of the role of this cation in signal transduction awaits further investigation.
PROTEIN KINASES
The phorbol ester, phorbol 12-myristate 13-acetate (PMA), has been used extensively as an activating stimulus to investigate the signal transduction pathways operative in neutrophils. The phosphorylation state of a wide variety of intracellular proteins changes upon stimulation with PMA.246 These changes occur in parallel with the induction of neutrophil functional responses and have also been observed with chemotactic peptide stimulation,247 suggesting the involvement of a protein kinase in the activation pathway. The ability of PMA to elicit neutrophil responses has been attributed to the activation of a Ca2+-sensitive, phospholipid-dependent protein kinase (protein kinase C or PKC). High levels of PKC have been detected in neutrophils,248 and the enzyme binds phosphatidyl serine.249 PKC is present as several isozymes,94,250 of which b and g are the most common250,251 in neutrophils. In neutrophils, PKC is located in the cytoplasm of resting cells and is redistributed to the plasma membrane following cell activation, as monitored by PMA binding, enzymatic, and immunologic activities.252
While implicated in neutrophil signal transduction, protein kinases play an undetermined role in responses. A strong case can be made for protein kinase C (PKC), since this enzyme can be activated by DAGs derived from the hydrolysis of phospholipids.184,222 This is consistent with the observation that synthetic DAGs also activate neutrophils by interacting with the same intracellular receptor as PMA.253 The close correlation between the activation of PKC and neutrophil functions, and the synergy between PKC agonists and other stimuli, have suggested a role for this enzyme in stimulus-response coupling.254
The inability of PKC inhibitors255,256 to block neutrophil activation in response to several physiologic stimuli suggests that PKC activation is not a necessary requirement for cell activation. In permeabilized neutrophils, no definitive role for PKC has been found with respect to degranulation.257,258 A direct role for PKC in degranulation can be excluded under certain circumstances.257 Indeed, neutrophils contain other kinases that may be involved in cell activation.259,260 There is growing evidence for the involvement of proteolytically modified PKC,261 cGMP-dependent kinases,262 tyrosine kinases,194,263,264 and 265 H4 histone kinases,266 and phosphatases259,264,265 in signal transduction. In addition, there is evidence that DAGs, particularly the 1-O-alkyl variety formed from PC, have other properties,267 independent of PKC activation, that can lead to cell stimulation.
Other kinases activated by low molecular weight G-proteins, particularly src and p21, are also involved in stimulus-response coupling.268,269 The JAK-STAT sequence, common to other cells of immune import, has recently been reported.270 However, the greatest concentration of research has revolved around the microtubule-associated protein kinases (MAPK) and the multiple kinases in that cascade.271,272 Such kinases have been implicated in adhesion-related signaling,273 particularly through L-selectins.272 Adhesive function in signaling through integrins has been shown to involve src kinases,274 syk,275 and tyrosine phosphorylation.276 Of particular interest to this topic is the finding that neutrophil stimulation leads to phosphorylation of PLA2.204 This enzyme appears to be regulated by the p42/p44 MAPK pathway.205,277 In macrophages, both MAPK and Ca2+ regulate PLA2 activation in response to most stimuli.278 It is likely that phosphorylation and activation of enzymes such as PLA2 are vital in regulating degranulation and membrane fusion.
ARACHIDONATE METABOLISM
In addition to their participation as putative second messenger products in the stimulus-response coupling pathway, many lipid metabolites may be released from stimulated neutrophils and in turn modulate cell function by interacting with receptors on other neutrophils. Phospholipase A2, present on both the granules and plasma membranes of neutrophils279 as well as the cytosol,280 is activated during neutrophil stimulation,185 yielding arachidonic acid as one of the major end products. Arachidonic acid is not only released from stimulated neutrophils,281 but also serves as a regulator of PLA2 activity282 and as a stimulus for these cells.283 Sensitivity of the cells to stimuli (priming) can be obtained with arachidonic acid and other long-chain fatty acids.284 Recently, arachidonic acid has been reported to be a mediator in the stimulation of a H+ pump that is associated with NADPH oxidase.285 This fatty acid can regulate other aspects of signal transduction, such as intracellular Ca2+ handling.284,286
In addition to directly affecting cell function, metabolites of arachidonic acid, generated through either the cyclooxygenase or lipoxygenase pathways, may also stimulate neutrophils. The most potent cyclooxygenase product of activated neutrophils is thromboxane B2, which, in addition to its vasoconstrictor activity, may also enhance neutrophil chemotaxis,287 aggregation,288 and adhesiveness.289
Arachidonic acid can also be metabolized by the lipoxygenase pathway to produce hydroxyeicosatetraenoic acids (HETEs), including 5-HETE, 12-HETE, and 5,12-diHETE.290,291 These compounds have also been shown to induce several neutrophil responses.292,293 Stimulated neutrophils also produce the diHETE LTB4 through the lipoxygenase pathway. LTB4 and other leukotrienes can be released in response to a variety of stimuli.294 Receptors for LTB4 have been partially purified,164,295 and their activation serves as a potent stimulus for degranulation,296 chemotaxis297 and adherence.297
Another potent mediator of inflammation produced by stimulated neutrophils is 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphoryl choline, also known as platelet-activating factor (PAF).298 Not only is PAF synthesized by neutrophils and activated endothelial cells,22 but it has been shown to induce degranulation,299,300 aggregation299 and superoxide generation.300
CYTOSKELETON
The role played by microfilaments in degranulation is suggested by the observation that in resting neutrophils, the granules are separated from the plasma membrane by filament-rich hyaline ectoplasm.301 As already discussed, surface stimulation of the cell leads to an increased mobilization of Ca2+, particularly in the periphery6,302; this increase in cytosolic Ca2+ would be expected to activate gelsolin, thereby shortening actin filaments and decreasing the viscosity in this area.303 Indeed, it has long been observed that stimulation leads to such rearrangements of microfilaments and their regulatory proteins.304,305 and 306 A decrease in viscosity of the hyaline ectoplasm could permit the granules to have access to the inner surface of the plasma membrane, thereby facilitating degranulation. The well-known ability of cytochalasins to enhance secretion307 and block locomotion308 is consonant with this model, since these alkaloids can split actin filaments.309
The enhanced turnover of microfilaments following stimulation of neutrophils is also manifested biochemically. The amount of actin associated with detergent-insoluble cytoskeleton fractions increases,305,310 as does the amount of filamentous actin (F-actin).311 This cytoskeletal network has been visualized with high-voltage electron microscopy.312 When a chemotactic peptide is used as the stimulus, these changes in cytoskeletal actin may be associated with the clearance of receptor-ligand complexes,132,310,313 probably by endocytosis. Using techniques suitable for monitoring short-term kinetics, it was shown that polymerization commences within the first few seconds following stimulation and may thus conceivably play some role in stimulus-response coupling.314,315
Recent observations indicate that this response by microfilaments is one of the earliest and most fundamental of neutrophil reactions to surface stimuli. Cell shape and F-actin content oscillate in individual neutrophils,314,316,317 and 318 as does intracellular Ca2+,221 suggesting that the two responses are closely connected. Other evidence supports this conclusion.319 Yet compelling studies have shown that F-actin and intracellular Ca2+ can be completely dissociated.244,320,321
Also, while actin assembly is generally mediated through G-proteins,322,323 fewer active G-proteins are required for F-actin assembly than for most other responses.316,324 Other recent investigations have focused on various pools of intracellular actins,303,325 regulatory interactions with actin-binding proteins,303,323,325,326 and 327 and the relationship of actin assembly to other neutrophil responses,328 particularly phosphatidylinositol metabolism.329 There is increasing evidence that polyphosphoinositides are involved in anchoring microfilaments to the plasma membrane as well as in regulating their integrity.330,331 Also, stimulation of cell surface receptors often leads to subsequent interactions of these receptors and signal transduction proteins with elements of the cytoskeleton.269,332 The possible role of other cytoskeletal proteins, such as fodrin,333 vinculin,327 and vimentin,262 are now being appreciated. While microtubules have long been studied in neutrophils, their roles are not clear, although research continues.334,335
CYCLIC NUCLEOTIDES
Exposure of neutrophils to phagocytizable particles results in a rapid doubling of cyclic adenosine 3′,5′-monophosphate (cAMP) levels.336,337 and 338 This increment is prompt (maximal within 15 s) and brief (returning to basal levels in 1–2 min). Such a rapid response, which is comparable to the earliest changes in neutrophil biochemistry and physiology, could possibly play a role in stimulus-response coupling. During the same time interval, no changes in cGMP levels are observed.336,337 The observations that changes in cAMP levels correlate closely with chemotaxis and degranulation,338 display specific desensitization after repeated exposures to the same stimulus, and that this desensitization is accompanied by a parallel decrease in O2– generation,339 suggest that changes in cyclic nucleotide levels are of mechanistic significance. However, a number of lines of evidence suggest that changes in cAMP are neither necessary340 nor sufficient82,336,338 for neutrophil responses in suspension. These findings seem to rule out cAMP in a second messenger role. However, redistribution of cAMP (without concomitant elevated levels) could still be of mechanistic significance, as immunochemical observations of cAMP show it to be localized near forming phagosomes.341
Recent work has uncovered the biochemistry leading to these rapid increases in cAMP content. Stimulated neutrophils release adenosine, which can bind to cell surface purine receptors, and which amplify adenylate cyclase activity.342 Thus, this response is outside of the mainstream of signal transduction pathways. But cAMP produced within the cells can still modulate other cellular responses and signals,343,344,345 and 346 particularly those mediated by phospholipases.344,345 cAMP-dependent protein kinase (PKA) appears to regulate a number of responses, including the respiratory burst,347 motility,348 and apoptosis.349 Signal transduction steps, such as PKC and tyrosine kinases,347,350 interact with PKA as well. Finally, there is growing evidence that cGMP-dependent protein kinases can modulate the neutrophil cytoskeleton.351
DEGRANULATION AND MEMBRANE FUSION
In stimulated cells, the signal transduction cascade activates G-proteins, followed by enhanced intracellular Ca2+, lipid remodeling, and protein kinase activation. These events culminate in secretion. This ultimate event, the fusion of granule membranes with phagosomes or the plasma membrane (with the accompanying discharge of granule contents and expression of granule membrane components on the cell surface), is coming under increased scrutiny because of its scientific and clinical importance. Degranulation is very rapid352 and apparently highly efficient, since intracellular liberation of granule contents has not been reported. This efficiency and rapidity have not yet been demonstrated with in vitro systems, hindering our comprehension of degranulation.
CALCIUM
As discussed above, the importance of elevated free Ca2+ levels in degranulation has been directly demonstrated in permeabilized neutrophils.174,175,235,352,353 How might this divalent cation mediate fusion? At the most primitive level, Ca2+ may promote membrane fusion intracellularly through its interactions with the negatively charged head groups of membrane phospholipids.354,355 Other metal cations and polylysine have been shown to induce aggregation and fusion of phospholipid vesicles in a pH-dependent fashion.354,355 The pH-dependence of fusion may be related to the ability of hydrogen ions to provide a charge-neutralizing effect.356 Thus, Ca2+ may change the physical properties of granules and membranes in such a way as to allow them to come into close apposition.357 Ca2+ may likewise interact with proteins to regulate fusion. As outlined above, Ca2+ can activate phospholipase A2278 (which can liberate membrane-fluidizing fatty acids). Alternatively, Ca2+ may regulate the interactions of annexins with phospholipids.
ANNEXINS
Annexins, which include synexin and the lipocortins, are defined functionally as Ca2+-dependent, phospholipid-binding proteins and structurally as proteins that share a conserved four-repeat, 70 amino acid sequence. Several annexins have been detected in neutrophils358,359 and 360 that also have Ca2+-dependent aggregating and fusion-promoting activity.359,360 and 361 These annexins bind neutrophil membranes and granules.200,362,363 Lipocortins I and II promote fusion of liposomes.361 By themselves, these annexins require high (millimolar) concentrations of Ca2+ to promote fusion. But they need not work alone, and it has been shown that annexin I functions synergistically with phospholipases to promote membrane apposition and fusion.200 Peptides derived from the N-terminus of annexin I also inhibit degranulation,364 suggesting a critical role for this protein. Others attempted to deplete annexins from permeabilized neutrophils.365 However, 41 percent of annexin I and 12 percent of annexin III were retained within the cells, along with most of the ability to degranulate. Restoration of these annexins also restored additional degranulation, suggesting that annexins I and III are important in secretion.
LIPIDS
Many of the lipid products that have been linked to degranulation in stimulated neutrophils are known to be fusogenic. For example, incubating Ca2+- and PLA2-treated chromaffin cell plasma membrane with chromaffin granules causes the release of granule contents,366 and cis-unsaturated fatty acids have been shown to induce the fusion of chromaffin granules aggregated by synexin and Ca2+.367 Of considerable interest are reports that DAGs, produced in stimulated neutrophils, can affect fusion.368 Furthermore, fusion may be modulated by asymmetric transmembrane distributions of phospholipids.369 The radii of curvature associated with the individual lipid components may also be important in progressing from semi-fused states (hemi-fusion) to complete fusion.370 Finally, sphingolipid metabolites are proving to have profound effects of liposome leakage and fusion.371 These data show that the physicochemical properties of lipids can greatly influence membrane fusion.
Phospholipase A2 may be particularly important in degranulation. It was reported in 1980 that the PLA2 inhibitor BPB blocks degranulation by neutrophils.372 Inhibition of this enzyme also arrests phagocytosis and disrupts vesicle trafficking in monocytes.373 While inhibitor studies must be interpreted with caution, these data suggest that PLA2 might be involved in membrane fusion. Indeed, PLA2 greatly enhances fusion of liposomes constructed to resemble biological membranes, lowering Ca2+ thresholds for fusion over 100-fold.374 PLA2 also augments fusion of these plasma membrane-like liposomes with each other and with specific granules isolated from neutrophils.374 Thus, there is good evidence for a vital role of PLA2 in neutrophil degranulation.
FUSION PROTEINS
Over the past decade, the SNARE hypothesis has become the reigning paradigm for fusion of biomembranes. Thanks to the early development of unambiguous contents-mixing assays, researchers studying a number of fusion systems (such as endosomes, Golgi, and synaptosomes) were able to readily identify and reconstitute the vital components. These systems are widespread, being found virtually complete in a wide range of species and tissues. They are centered around a protein that is sensitive to N-ethyl maleimide (designated “NEM-sensitive fusion protein” or NSF), several soluble NSF-attachment proteins (SNAP), and “SNAP-receptors” (SNAREs) on the participating membranes. The SNAREs are termed the v-SNAREs and t-SNAREs—v-SNAREs being found on vesicles or granules and the t-SNAREs being found on the target plasma membranes. This SNARE system has been the subject of many excellent reviews,375 and will not be described in detail here.
The SNARE hypothesis has proven to have great predictive value, as the constellation of fusion proteins and their interactions appears in almost all species and tissues. However, it has gradually become apparent that some cell types possess incomplete sets of proteins; indeed, it has been proposed that such an incomplete set (called SNAREpins and consisting of VAMP, syntaxin, and SNAP-25) might be functionally sufficient.376 There is increasing evidence that the fusion proteins operating in cells of the myeloid lineage are substantially different from those in neurons. For example, vesicle-associated membrane protein-2 (VAMP-2) has been detected in neutrophils and HL-60 cells using molecular techniques.377 VAMP-2 in addition to syntaxin-4 and secretory carrier membrane protein (SCAMP) were detected in neutrophils by Western blot.378 Syntaxin-4 and SCAMP are nonneuronal. These investigators also reported that they were unable to find the classical neuronal components syntaxin-1, VAMP-1, SNAP-25, synaptophysin, and cellubrevin. Another research group did find SNAP-25 protein, but it was on the “wrong” vesicle (granule membrane rather than plasma membrane).379 Human neutrophils and HL-60 cells have been reported to have two forms of SNAP-23,380 based on molecular studies. SNAP-23 is nonneuronal, and these researchers found that mRNA for this protein, unlike VAMP-2,377 increased during differentiation of HL-60 cells. Interestingly, those components that have been detected in neutrophils and HL-60 cells, namely VAMP-2, syntaxin-4, and SNAP-23,25 are homologues of the SNAREpin set.376 Thus, the proteins and lipids responsible for degranulation in neutrophils are yet to be elucidated.
BACTERICIDAL MECHANISMS
NONOXIDATIVE MECHANISMS
From the information presented in Section III, it should be apparent that neutrophil granules contain a wide variety of materials that possess bacterial activity. Such compounds include the degradative enzymes, lipases, proteases, and glycosidases as well as the more specialized antibacterial defensins and cationic proteins.381 Indeed, crude preparations of neutrophil granules themselves have been shown to possess substantial bactericidal activity in vitro.382 The bactericidal armamentarium is sufficiently broad to permit relatively efficient killing in the absence of an oxidative burst, such as is found under anaerobic conditions383 or in cells from patients with chronic granulomatous disease.384,385 The effects of cationic proteins on ingested bacteria are enhanced by increases in phagosomal acidity386; this decline in pH following phagocytosis387 may be due to both the acidity of the fusing azurophil granules387,388 and 389 and to ion pumps. Many cationic proteins and defensins involved in killing have been isolated and cloned.390,391,392,393 and 394 Proteases on the surface membrane, as well as those found in the granules, may be involved in cytolysis.395
Lysozyme, which hydrolyzes cell wall proteoglycan of some bacterial species, and lactoferrin, which sequesters the iron required for bacterial growth, must be considered as part of the nonoxidative arsenal of neutrophils.51 These agents, along with the cationic proteins found primarily in azurophil granules, would be expected to be most potent when confined to the phagolysosome. However, it is also likely that such compounds, even when released from dead neutrophils, could perform as systemic antibiotics. Furthermore, cationic proteins could function as opsonins in the extracellular space.396 The next three sections describe general classes of these microbicidal proteins.
DEFENSINS
Of the various cationic antibacterial proteins found in neutrophils, the defensins are the most common and of the lowest molecular weight. These proteins are less than 4 kDa in weight, consisting of 29-33 amino acids, and constitute from 5 to 8 percent of the total cellular protein.31,33,36 The defensins have attracted much research interest, and the major forms have been isolated and cloned391,397; the precursor molecules have also been investigated.398 Like other azurophil granule constituents, they can be released to the extracellular space following stimulation.37,399 If released into the cytoplasm of the cell, the defensins could modify signal transduction by inhibiting PKC400 and by inhibiting NADPH oxidase.401 The defensins have an unusual cyclic structure and appear to kill bacteria by disrupting their outer membranes402; however, they can also induce single-strand DNA breaks in target cells.403 There are even reports that defensins are chemotactic for phagocytes.404
CAP37 FAMILY
This group of neutrophil cationic proteins, which includes cathepsin G and azurocidin in addition to CAP37, shares considerable sequence homology to other inflammatory proteases.33,392,405,406 CAP37 is both bactericidal and chemotactic.33,405,407 An azurophil granule localization also appears likely for this protein.407 Once outside the neutrophil, it can stimulate PKC activity in endothelial cells.408 However, it has been reported that acidification of the phagolysosome can promote the antimicrobial action of CAP37.386 The antimicrobial and chemotactic activities of the CAP37 family are becoming better defined.409
BPI FAMILY
Bactericidal/permeability-increasing protein (BPI), a cationic protein that lyses gram-negative bacteria, belongs to a family that includes CAP57 and lipopolysaccharide binding protein.33 These activities have been localized to particular populations of the azurophil granules.38,393,410 The activity of BPI has been shown to reside in the amino terminus of the molecule411,412; this region is both cationic and amphipathic.390 Like the defensins, members of the BPI family have been shown to disrupt the bacterial membranes.413 Most members of this family have also been isolated and cloned.390,414 BPI itself has been shown to bind LPS, and may hence serve to buffer the serum concentration of this potent inflammatory mediator.410,411 Furthermore, the activity of BPI itself is modulated by some low molecular weight proteins (p15s) that are themselves antimicrobial.394,415
ADDITIONAL ANTIBACTERIAL PROTEINS
A number of novel microbicidal agents have recently been reported in neutrophils. Bactonectin, a dodecapeptide antibiotic, has been isolated and described.416,417 In addition to its bactericidal activity, indolicin features five tryptophan residues out of 13 amino acids,418 giving it the highest observed mole concentration among known protein sequences. A proline/arginine-rich antibacterial (and chemotactic) peptide related to PR39 has been recently described.419 Some additional low molecular weight proteins have been found in guinea pig cells.420
Most interesting of all is an extremely abundant, cytosolic, Ca2+-binding protein known as L1 or MRP8/MRP14421,422 that is a major component of pus. Initially, its very low molecular weight subunit structure hindered its detection by investigators. Its antimicrobial activity has led to the name calprotectin.421 As a cytosolic protein, MRP8/MRP14 is subject to the action of PKC423 and is translocated to the plasma membrane and cytoskeleton following stimulation.424
OXIDATIVE METABOLISM
OXIDATIVE BACTERICIDAL ACTIVITY
Activated neutrophils produce several antimicrobial oxygen metabolites, including superoxide anion (O2–), H2O2, hydroxyl radicals (OH·), hypochlorous acid (HOCl), and singlet oxygen. Even chlorine gas has been detected.425 Substantial evidence indicates that these reactive metabolites are generated by a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidase, located on the plasma membrane, which reduces molecular oxygen to O2– (Fig. 67-5). The oxidase is quiescent in resting neutrophils and is stimulated following neutrophil activation. The importance of the NADPH-oxidase to the bactericidal capacity of the cell is demonstrated in individuals with chronic granulomatous disease (CGD) whose neutrophils fail to generate O2– and related metabolites. This is a genetic disorder in which neutrophils and monocytes ingest, but do not kill, catalase-positive microorganisms. Neutrophils of CDG patients do kill pneumococci or streptococci, which do not contain catalase; these organisms generate enough H2O2 which, together with myeloperoxidase delivered into the phagosomes by degranulation, kills them. Staphylococcus aureus is the most common pathogen in this disorder, although any catalase-positive organism may be involved. Chap. 82 provides a more complete review of the disorders of neutrophil function.

FIGURE 67-5 Possible mechanisms for the production of superoxide anion in polymorphonuclear leukocytes. Oxygen is reduced to superoxide (O2–) by an NADPH oxidase. The oxidase appears to be a composite of (1) a 47-kDa cytosolic protein (p47); (2) a 67-kDa cytosolic protein (p67); (3) a 40-kDa cytosolic protein (p40); (4) one or more low molecular weight cytosolic G-proteins, such as Rac1 and Rap1A; and (5) a membrane-bound cytochrome b-558. Cytochrome b consists of a 22 kDa protein subunit and a 91-kDa glycoprotein subunit, both of which contain heme. The gp91 subunit is an FAD-dependent flavoprotein which contains the NADPH binding site and ultimately shuttles electrons to molecular oxygen, forming O2–. The cytosol components have also been reported to translocate to the membrane and may serve to alter the tertiary structure of cytochrome b, to permit the flow of electrons from NADPH to O2. The p47 subunit can be phosphorylated to various extents, but the significance of this phosphorylation is unclear. The low molecular weight G-protein is also important in stabilizing the oxidase complex. (This figure is reproduced with permission from DeLeo, F.R. and Quinn, M.T. Assembly of the phagocyte NADPH oxidase: Molecular interaction of oxidase proteins. J Leukocyte Biol 60(6):677–691, 1996.)

THE CELL-FREE NADPH-DEPENDENT OXIDASE SYSTEM
Considerable attention has been given to the molecular factors underlying activation of the NADPH-oxidase, and multiple pathways appear to exist. Stimulation of the oxidase enzyme results in the generation of O2– after a lag period of several seconds,426 thus allowing sufficient time for the intervention of many of the aforementioned biochemical events in the activation pathway. Studies conducted in intact cells have been extended to broken cells. Cell-free oxidase activity was first reported in homogenates from resting guinea pig peritoneal macrophages.427 Activation of the NADPH-oxidase from macrophages requires both particulate and soluble fractions and certain unsaturated fatty acids or anionic detergents.428 Cell-free oxidase activity has also been obtained from neutrophil homogenates and appears to have similar requirements.429 The oxidase has also recently been fully solubilized from neutrophil membranes and displays kinetic properties similar to the oxidase of intact cells.430 In some forms of autosomal recessive CGD, one or another of the cytosolic factors has been found to be defective.431 Studies employing this system proved valuable in characterizing the mechanism(s) underlying activation of the NADPH-dependent oxidase.432 The next section will detail the advances made in understanding the oxidase, advances that are directly attributable to the development of the in vitro assay system.
COMPONENTS OF THE NADPH-DEPENDENT OXIDASE
The biochemistry of the NADPH oxidase has come under intense scrutiny in recent years. The oxidase appears to be a composite of (1) a 47-kDa cytosolic protein, termed “p47-phox,” where “phox” refers to “phagocyte oxidase”433,434 and 435; (2) a 67-kDa cytosolic protein, termed “p67-phox”433,435; (3) two low molecular weight cytosolic G-proteins, identified variously as rac1/2 (rac2 in human neutrophils) and rap1A436,437; (4) a 40-kDa protein, termed “p40-phox”438; and (5) a membrane-bound cytochrome b-558,439,440 and 441 which is an FAD-dependent flavoprotein.440,442 (The designation “b-558” is based on spectral properties. The cytochrome is also termed “b-245” on the basis of electrochemical potential.) The cytosolic constituents are believed to activate an electron transport chain in cytochrome b, which serves to ferry electrons from NADPH at the binding site, through a flavin prosthetic group (FAD), and finally to molecular oxygen, forming superoxide.443,444 NADPH is subsequently regenerated through the hexose monophosphate shunt (Fig. 67-5).445 That these constituents may indeed be intrinsic to the oxidase has been recognized in studies utilizing neutrophils from individuals with the genetic disorder CGD. Patients with the X-linked form of the disease lack cytochrome b442,445,446 and 447 and contain reduced levels of flavoprotein.442,445,446 Levels of both oxidase components are normal in patients with the autosomally transmitted form,442,446 but the enzyme cannot be activated448,449 due to dysfunctions of either p47-phox or p67-phox.
The subcellular localization of the NADPH-oxidase as well as its mechanism of activation have been disputed. Cytochrome b consists of a 22-kDa protein subunit and a 91-kDa glycoprotein subunit,447,450,451 both of which contain heme.452 The 91-kDa subunit is missing in X-linked CGD, and the 22-kDa subunit is missing in a very rare form of autosomal recessive CGD.447,453 The well-known flavoprotein component454,455 and NADPH binding site have recently been attributed to the b cytochrome.60,61,455 This cytochrome has been reported to be distributed between the plasma membrane and the membranes of cytoplasmic granules in resting cells.62,70,439,456,457 and 458 Stimulation results in the translocation of the cytochrome from granule fractions to the plasma membrane.62,457,459 Based on these observations, several investigators have advanced the hypothesis that the granules are involved in activation of the oxidase.445,458,459 However, the relevance of the translocation phenomenon to oxidase activation is obscure.457,458 The cytosol components have also been reported to translocate to the membrane460,461 and may serve to alter the tertiary structure of cytochrome b, to permit the flow of electrons from NADPH to O2. Following assembly, the entire oxidase complex is associated with the cytoskeleton.462
The oxidase components assemble in a sequential fashion. Using the b cytochrome on the membrane as a nucleus, the oxidase complex assembles first with p47-phox.460,461 The p67-phox then binds to the p47-phox moiety,460 which then alters the cytochrome b to allow binding of NADPH.61 The low molecular weight G-protein rac2 serves to stabilize this complex.461,463,464 and 465 Another low molecular weight G-protein, rap1A, appears to be associated with the b cytochrome.437,464,466 Other soluble oxidase components are also suspected467; however, these other components can only play modulatory roles since the oxidase has been reconstituted using recombinant p47-phox, p67-phox, b cytochrome, and rac1436,468 or rac2 (in humans).465 Absence of either p47-phox or p67-phox leads to CGD (see Chap. 82). An accessory role is likely for p40-phox, which associates with both p47-phox and p67-phox via SH3 and PC motifs.438,469,470 The role of protein kinases in oxidase activation is still unclear. On one hand, in whole neutrophils471 and in the reconstituted system,472 several PKC inhibitors are ineffective in abolishing stimulus-induced activation of the oxidase. Furthermore, the “requirement” for ATP noted by some investigators actually reflects the GTP requirement for the low molecular weight G-protein.473 On the other hand, p47-phox can be phosphorylated to various extents,474 and the amount of phosphorylation is correlated with translocation of the oxidase components475,476 and 477 and with oxidase activity476,478,479; however, in some cases, hyperphosphorylation results in inhibition.480 A variety of protein kinases481 may be involved in phosphorylating p47-phox, including PKC,482,483 PKA,483 MAP kinase,483 p38,484 ERK,484 proline-directed kinases,485 and other novel kinases.486,487 In fact, the in vitro oxidase system can be activated by PKC488 or by a phosphatidic acid–regulated protein kinase.489 The various phosphorylation sites on p47-phox have been extensively mapped and the essential serine residues identified.479,490 Even p67-phox is phosphorylated during neutrophil activation, by both PKC and other enzymes.491 Phosphorylation of the rap1A oxidase protein can actually inhibit activity.466 The b cytochrome may also be phosphorylated,492 but the significance of this is unclear. Finally, there also appears to be a role for phosphatidic acid and diacylglycerols in oxidase assembly.493,494
A final aspect of the oxidase is its ubiquity. Components of the phagocyte oxidase, or their homologues, can be found in a wide variety of tissues. For example, the full oxidase complex can be found in B lymphocytes495 and the only reason for low enzymatic activity in those cells is a transcriptional block in the synthesis of B cytochrome.496 A fully active oxidase complex has also been found in fibroblasts497 and keratinocytes.498 Even plants can generate H2O2 and have proteins immunologically related to p47-phox and p67-phox.499
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Books@Ovid
Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology

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5 comments on “CHAPTER 67 FUNCTIONS OF NEUTROPHILS

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