Chapter 66: Disorders of Neutrophil Function

CHEMOTAXIS AND MOTILITY

The similarity between neutrophil locomotion and that of amebas was noted long ago.¹ Neutrophils respond to spatial gradients of chemotaxins with differences in concentration of chemotaxin of as little as 1 percent across the cell,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, neutrophils acquire a characteristic asymmetric shape (Fig. 66–1). In the front of the cell is a pseudopodium, referred to as the lamellipodium, that advances before the body of the cell containing the nucleus and the cytoplasmic granules. At the rear of the moving cell is a knob-like tail, the uropod. The lamellipodium 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 The lamellipodium, which is very thin, forms immediately when the cell encounters a gradient of chemotactic factor. As the cell moves, the cytoplasm behind the lamellipodium streams forward, almost obliterating it. At this point, some granules appear to contact the cell periphery and release granule contents in response to chemotactic agents. The lamellipodium 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.⁷ This may also account for changes in cell viscosity. Polarity and movement is orchestrated by the cytoskeleton through signals generated from receptor associated G-proteins in an intricate network that regulates both direction and intensity of movement.^8,9,10,11

Figure 66–1.

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 (original magnification ×1200). (Reproduced with permission from Hirsch JG: Cinemicrophotographic observations on granule lysis in polymorphonuclear leucocytes during phagocytosis. J Exp Med 116:827–834,1962.)

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. 66–1. 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.⁷ 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.¹²

The formation of a lamellipodium is essential for neutrophil locomotion and is also required for ingestion. When dissolution of the lamellipodium 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 surface; thus it crawls but does not swim. Such “stickiness” is also a phase of ingestion.⁷ The neutrophil membrane adheres firmly to particles they ingest, 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

The dual neutrophil functions of immune surveillance and in situ elimination of microorganisms or cellular debris require rapid transition between a circulating nonadherent state to an adherent state, allowing the cells to migrate into tissues when necessary. Initially neutrophils appear at sites on the endothelium adjacent to the site of inflammation. Adhesion molecules on endothelium are induced by the inflammatory mediators tumor necrosis factor (TNF)-α and interleukin (IL)-1. Lipopolysaccharide (LPS), released by local activated macrophages and microorganisms, results in local extravasation of the neutrophil. In postcapillary venules or in pulmonary capillaries the slow rate of blood flow, further reduced by vessel dilatation at sites of inflammation, permits a loose and somewhat transient adhesion referred to as “tethering,” and results in the rolling of the neutrophil along the endothelium.¹³ Extensions from the rear of the neutrophil wraps around the rolling neutrophils as so called slings and provide “crawler tracks” at their front that assists in adhesion to the endothelium.¹⁴ During this tethering step, neutrophils respond to ligands, primarily chemokines dispatched on the endothelial surface by a signaling event that acts to reorganize the neutrophil surface membrane, thereby exposing adhesion molecules, which, in turn, lead to sustained adhesion and spreading (Chap. 19).

NEUTROPHIL MICROVILLI AND THEIR DYNAMICS

Circulating neutrophils contain surface microvilli of a diameter of 0.3 μm.¹⁵ Moesin, ezrin, and p205 radixin are actin-binding proteins associated with neutrophil plasma membranes and are important for organization of microvilli on the surface of the cell.^16,17 These actin binding proteins tether the primary adhesion proteins exposed on the microvilli, L-selectin and P-selectin glycoprotein ligand 1 (PSGL-1).¹⁸ L-selectin and PSGL-1 are filamentous glycosylated proteins protruding from the tips of the microvilli. E-selectin ligand 1 (ESL-1) located in the side of microvilli,¹⁹ and CD44, located on the cell body, both serve a ligands for E-selectin.²⁰ L-selection, like the other selectins, including P-selectin, which is expressed on platelets and endothelial Weibel-Palade bodies, and E-selectin, which is expressed in endothelial cells, bind with a variable affinity to sialyl fucosylated oligosaccharides including sialyl Lewis X (sLe^x), which is present on multiple specific glycolipids and glycoproteins on leukocytes and inflamed endothelial cells.²¹ When binding to their ligands, L-selectin, PSGL-1, ESL-1, and CD44 recruit Syk (spleen tyrosine kinase), a tyrosine kinase, which binds to the immunoreceptor tyrosine-based activation motif (ITAM). ITAMs are present in the cytoplasmic domains of surface membrane proteins, and Syk then orchestrates the further signaling to initiate cell activation.^22,23,24,25

ROLLING AND TETHERING

P-selectin is mobilized rapidly to the endothelial cell surface following stimulation by thrombin, histamine, or oxygen radicals and interacts with neutrophil PSGL-1 to initiate neutrophil rolling.²¹ Rolling subsequently involves newly expressed E-selectin, which appears on endothelial cells 1 to 2 hours after cell stimulation by IL-1, TNF-α, or LPS. E-selectin counterreceptors include PSGL-1, ESL-1, and CD44.^19,24 Both P- and L-selectin contribute sequentially to leukocyte rolling, but L-selectin is involved in the prolonged neutrophil sequestration on inflamed microvasculature. L-selectin is constitutively present on neutrophils and its binding capacity is rapid and transiently increased after neutrophil activation, possibly via receptor oligomerization. Activation of ADAM17, a matrix metalloproteinase expressed at the neutrophil surface, severs L-selectin from the surface of neutrophils and impairs their recruitment to endothelium.^26,27 Thus far, only one inducible L-selectin counterreceptor has been identified on inflamed endothelium.²⁸ In addition to its binding to endothelial ligands, neutrophil PSGL-1 is a counterreceptor for L-selectin, which permits previously adherent neutrophils to recruit other neutrophils to inflamed endothelium (Chap. 19).^13,21

NEUTROPHIL ADHESION AND SPREADING

Figure 66–2 shows a sequence of molecular and biophysical events leading to neutrophil activation and increased adherence during acute inflammatory response in vivo. The inflamed endothelium produces chemoattractants such as platelet-activating factor (PAF), leukotriene B₄ (LTB₄), and various chemokines, immobilized by proteoglycans on the luminal surface of endothelial cells.²⁹ Among these chemokines, IL-8 specifically attracts neutrophils. IL-8 is synthesized by endothelial cells in response to IL-1, TNF-α, or LPS, and is stored in Weibel-Palade bodies; IL-8 can be released by histamine or thrombin.³⁰ Additionally, IL-8 can be internalized by endothelial cells and transcytosed from the abluminal surface via vesicular caveolae, and presented to the tips of microvilli of the endothelial cell luminal surface.³¹ The binding of signaling molecules such as PAF and IL-8 to surface receptors on the leukocytes activates them in a juxtacrine fashion and triggers changes in affinity or avidity of β₂ integrins, leukocyte function-associated antigen (LFA)-1 (CD11a/CD18), which is constitutively expressed on neutrophil plasma membranes, and Mac-1 (CD11b/CD18), which becomes incorporated in the neutrophil plasma membrane from secretory vesicles.^13,21,32 β₂ Integrins are recognized by counterligands on endothelial cells, including members of the intercellular adhesion molecule (ICAM) family such as ICAM-1 and ICAM-2. The ICAM glycoproteins are induced by cytokines that include TNF and IL-1. The relative affinity of the β₂ integrins for ICAM is increased by exposure of neutrophils to numerous stimuli, including C5a, N-formylated bacterial peptides, IL-8, and LTB₄. The extracellular domains of unactivated integrins are in a bent position and not able to bind ligands. Intracellular signals, such as those generated via Syk as discussed above, can transform the integrins into an extended but not fully open conformation, capable of ligand binding with weak affinity permitting the integrin (LFA-1) to participate in rolling³³ and an extended and fully open form capable of ligand binding with strong affinity, mediating firm adhesion. The molecular mechanisms have been worked out in great detail. In essence, tailins and kindlins are recruited and bound to membrane near phosphotyrosine (NPxY and NxxY) motifs present on the integrin β chains and twist the cytoplasmic domains of the α and β chains. This changes the conformation of the extracellular domains from the bent to the open state, thereby permitting binding to ligands, and in so doing transmit signals from outside to inside.^21,34,35,36 Neutrophils integrate the signals of integrin engagement and those delivered simultaneously by inflammatory cytokines or chemoattractants to activate a cascade of intracellular events resulting in cell spreading (Fig. 66–2). The CD11b/CD18 integrin (MAC-1) is known to interact in cis fashion with glycosylphosphatidylinositol (GPI)-anchored membrane proteins such as FcγRIIIB (CD16), the LPS receptor CD14, and the urokinase plasminogen activator receptor (uPAR; CD87). Integrins behave as transducers mediating signals transferred by these GPI-linked receptors.³⁷ For instance, FcγRIIIB interaction with CD11b/CD18 promotes antibody-dependent phagocytosis, whereas CD14 interaction with CD11b/CD18 occurs in the presence of LPS and LPS-binding protein to generate proinflammatory mediators, and uPAR interaction with CD11b/CD18 mediates neutrophil migration by recruiting and activating the urokinase-type plasminogen activator.²⁹

Figure 66–2.

Neutrophil-mediated inflammatory response. (1) Egress of mature neutrophils from marrow to circulation. (2) Initial tethering and rolling are dominantly mediated by selectins present both on neutrophils and endothelial cells and their ligands. Invasion by bacteria stimulates tissues macrophages to secrete inflammatory cytokines, interleukin (IL)-1 and tumor necrosis factor, which, in turn, activate endothelial cells to express E- and P-selectin and IL-8. E- and P-selectin serve as counterreceptors for the neutrophil P-selectin glycoprotein ligand-1. (3) Activated endothelial cells express intercellular adhesion molecule (ICAM)-1 and ICAM-2, which serve as ligands for the neutrophil β₂ integrins. The β₂ integrins mediate tight adhesion and arrest of the leukocytes in cooperation with the selectins. Localized activation of neutrophils by juxtacrine signaling molecules or chemoattractants that bind to surface receptors is critical for inside-out signaling of β₂ integrins, making them adhesive for the ICAM ligands on the endothelium. (4) Neutrophil invasion through the vascular basement membrane with release of proteases and reactive oxidative intermediates that cause local destruction of the extracellular matrix which allows for migration of the neutrophils into tissues. (5, 6) Uptake of microorganisms into the phagocytic vacuole with concomitant degranulation both into the phagocytic vacuole (azurophil granules and specific granules) and to the exterior (specific granules and gelatinase granules). (7) A burst of transcriptional activity is initiated during diapedesis of neutrophils and during phagocytosis, which results in generation of chemokines such as IL-8, monocyte chemoattractant protein-1, macrophage inflammatory protein-1α, and IL-1β that may recruit additional cells of the immune system.⁵⁸ (8) Formation of neutrophil extracellular traps by extrusion of chromatin and cationic bactericidal granule proteins.²⁶² MΦ, macrophages; PMN, polymorphonuclear neutrophil.

TRANSENDOTHELIAL MIGRATION

Fully extended and open integrins bind ICAM-1 firmly and thus mediate attachment of neutrophils to endothelial cells.³⁸ ICAM-1 and -2 direct the motion of neutrophils to points of egress from the vascular lining. The majority are guided to points where three or more endothelial cells join. Intracellular signals from ICAMs loosen the binding between endothelial cell junctions provided by homotypic interaction of VE-cadherins.³⁹

Platelet endothelial cell adhesion molecule 1 (PECAM-1), endothelial cell-selective adhesion molecule (ESAM), junctional adhesion molecule A, B, and C (JAMs), and CD99 also form homotypic interactions between endothelial cells; however, neutrophils also express these adhesion proteins and may displace the interendothelial cell homotypic binding with neutrophil–endothelial cell binding mediated by the same proteins. In this way neutrophils can “zipper” through^40,41,42 and exit by this paracellular route. A minority of neutrophils exit by a transcellular route through so-called endothelial cups.⁴²

Pericytes are perivascular contractile cells that interact with endothelial cells and regulate vascular permeability. Neutrophils exit the vascular wall through gaps between pericytes.⁴³ Pericytes adopt different morphologies and distributions in different tissues. Such may explain differences in neutrophil recruitment to viscera.⁴⁴

Once out in tissues the forefront neutrophils generate IL-8 and LTB₄ in order to recruit an additional swarm of neutrophils to the area and recruit later incoming monocytes and macrophages.⁴⁵

NEUTROPHIL SURFACE PROTEINS

Several proteins associated with the surface of the neutrophil function in the normal housekeeping activities such as Na⁺/K⁺ adenosine triphosphatase (ATPase), but others serve specific functions such as L-selectin, PSGL-1, and integrins. The surface of neutrophils is highly dynamic as a result of the incorporation of membrane from intracellular vesicles and granules, a process that is known to add significantly to the total cell surface measured by an increase in electric capacitance.⁴⁶ A number of membrane-bound receptors are localized to secretory vesicles and incorporated into the surface membrane when secretory vesicles fuse with the plasma membrane, as occurs during diapedesis. This enhances the ability of neutrophils to respond to the signals presented by endothelial cells or present in the extravascular tissue.

Receptors for Recognition of Microbes

Neutrophils and other cells of the innate immune system recognize microbes through germline-encoded receptors, which recognize molecular patterns that are relative unique to pathogens and shared among groups of pathogens, so-called pathogen-associated molecular patterns (PAMPs). These pattern-recognition receptors (PRRs) include the membrane-bound toll-like receptors (TLRs) and C-type lectin receptors (CLRs), and the cytosolic nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and RIG-like receptors (RLRs).^47,48,49,50 Although PRRs are highly expressed in myeloid cells, they are also widely expressed in cells that are regularly exposed to microorganisms, particularly in epithelial cells.

TLRs are type 1 transmembrane signaling receptors that are activated by dimerization induced by ligand binding.⁵¹ The TLRs may dimerize both as homodimers and heterodimers. TLRs that recognize microbial membrane components are largely present on the cell surface, and include TLR2 that recognizes lipoproteins and lipopeptides in association with either TLR1 or TLR6. CD14 is known as an LPS-binding protein but is not itself able to signal and presents LPS to TLR4.⁵¹ TLR5 binds flagellin, and TLR11 binds profilin-like proteins of protozoa.⁵² TLRs that recognize viral components are largely expressed on intracellular vesicles that may fuse with phagosomes and include TLR3 (not present in neutrophils) that recognizes double-stranded RNA, TLR7/8 that binds viral single-stranded RNA,⁵³ and TLR9 that binds unmethylated GpC regions on DNA.⁵⁴

Ligand binding, that is, dimerization of TLRs leads to recruitment of one of four intracellular adaptor proteins to the TIR (toll/IL-1 receptor) domain of the TLR. These proteins include MyD88 (myeloid differentiation factor 88), Mal/TIRAP (MyD88-adaptor-like/toll-IL 1 receptor domain containing adaptor protein), TRAM (TRIF-related adaptor molecule), and TRIF (TIR domain-containing adaptor inducing IFN-β). While many TLRs (5, 7, 8, and 9) exclusively use MyD88, TLR2 requires both Mal and MyD88 and TLR4 can use either Mal (MyD88-adaptor-like) and MyD88 or TRAM and TRIF to signal to NF-κB (nuclear factor-κB) or interferon regulatory factor (IRF)-3.^55,56,47

CLRs comprise a heterogeneous group of trans-membrane receptors that bind carbohydrates such as mannose, fucose, and β-glucans present on a variety of microbes, fungi in particular. They signal largely via their cytosolic ITAMs and Syk to activate NF-κB, nuclear factor of activated T cell (NFAT), and microtubule-associated protein kinases (MAPKs) resulting in production of proinflammatory cytokines.⁴⁸

NLR proteins are cytosolic proteins that are divided into five subfamilies, NLRA, NLRB, NLRC, NLRP, and NLRX.⁵⁰ Their N terminus contains either a caspase activation and recruitment domain (CARD) or a pyrin domain (PYD). The NLRC members NOD1 and NOD2 recognize peptidoglycans of both Gram-positive and Gram-negative bacteria and signal to activate the NF-κB pathway. Other members of the NLRC and NLRP subfamily are essential in organizing the inflammasome. The NLRs multimerize through their CARDs into inflammasomes,⁵⁰ cytoplasmic structures that activate caspase-1, which, in turn, convert pro–IL-1 and pro–IL-18 to the mature proinflammatory cytokines that are secreted.⁵⁷

A variety of chemokine receptors are found on the surface of the neutrophil. These are in general G-protein–coupled receptors. Other G-protein–coupled receptors on neutrophils are the purine receptors for adenosine diphosphate (ADP) and ATP, the PAF receptor C5a, and formyl-methionyl-leucyl-phenylalanine (fMLP) receptors. Receptors not belonging to the G-protein–coupled receptor family include receptors for IL-1, IL-10, and TNF-α, and the growth factors receptors for granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Both growth factor receptors are important for myeloid development, and play an important role in enhancing neutrophil function and gene transcription in mature neutrophils. A burst of transcriptional activity is associated with the diapedesis of neutrophils into tissues, which results in downregulation of proapoptotic genes and upregulation of genes coding for antiapoptotic proteins, upregulation of genes encoding chemokines and cytokines that may recruit macrophages, T cells and additional neutrophils, and downregulation of genes encoding chemokine receptors (see Fig. 66–2).⁵⁸

Surface Components for Phagocytosis

Neutrophils express the Fc α receptor (CD89) for immunoglobulin (Ig) A and IgG receptors, FcγRIIA (CD32), and FcγRIII (CD16). Neutrophils also express receptors for the complement components, including CD1qR, CR1 (CD35), CR3 (CD11/CD18), and CR4. CR1 binds CD3b, C4b, and C3bi with decreasing affinity. CR3 recognizes C3bi (a proteolytic fragment of C3b). Of particular importance is that both Fcγ receptors and GPI-coupled receptors appear to be localized to lipid rafts. Lipid rafts are important, but elusive structures that facilitate signal transduction leading to phagocytosis by promoting several membrane protein interactions. Initially the rafts were conceptionally associated with caveolae, which are structures identified on endothelial cells and thought to be important for transendothelial cell traffic. The caveolae were identified by their high content of cholesterol lipids and the presence of the structural protein, caveolin. Rafts were subsequently identified on neutrophils, but these cells are devoid of caveolin.⁵⁹ Rafts are perhaps best viewed as patches of surface membrane that attract many hydrophobic proteins including signaling molecules such as tyrosine kinases and phosphatases. Other membrane protein receptors that are not normally associated with rafts may change their conformation and subsequently associate with rafts upon binding their ligands. This is particularly true for the Fcγ and GPI-coupled receptors.

SECRETORY VESICLES

Secretory vesicles are small intracellular vesicles that were discovered during the search for the structural basis for upregulation of a variety of surface molecules on neutrophils in response to nanomolar concentrations of fMLP and other chemotactic stimuli. They were initially identified by “latent” alkaline phosphatase.⁶⁰ Secretory vesicles of neutrophils should not be confused with the vesicles that carry cargo from endoplasmic reticulum and Golgi in the constitutive secretory pathway of other cells and that are sometimes also named secretory vesicles. Secretory vesicles of neutrophils are specialized endocytosis vesicles that are formed in the final stages of neutrophil maturation in the marrow. They contain plasma proteins, seemingly without any selectivity. Albumin thus serves as a marker for secretory vesicles and has allowed the identification of these as small intracellular vesicles that are scattered throughout the cytoplasm of neutrophils as is true for neutrophil granules. The plasma proteins inside secretory vesicles show no sign of degradation, thus no fusion takes place with lysosomal structures.⁶¹ Secretory vesicles behave like the traditional neutrophil granules. They require a specific signal for mobilization.⁶² Secretory vesicles are not important for their cargo (plasma proteins), but for their membrane which becomes fully incorporated into the plasma membrane of the neutrophil upon stimulation.^{61,63,64,65,66} Secretory vesicles host most of the neutrophil chemotactic and GPI-coupled receptors, TLRs, and one of the early acting downstream effectors, phospholipase D.⁶⁷ They enrich the plasma membrane with receptors for adhesion and signaling, and can be seen as the structural basis for transition of neutrophils from circulating quiescent cells that do not respond well to stimuli such as chemoattractants and objects to be phagocytosed, to highly responsive cells capable of establishing firm contact with endothelium. The signals generated by tethering of selectins or PSGL-1 to the endothelium are sufficient to mobilize secretory vesicles. Secretory vesicles are completely mobilized in vivo during neutrophil diapedesis.^21,66

The first identified marker of secretory vesicles, latent alkaline phosphatase, is known to be elevated in chronic myeloproliferative disorders except for chronic myelogenous leukemia (CML), but the content of secretory vesicles in neutrophils from patients with chronic myeloproliferative disorders is not different from normal neutrophils.^68,69,70 The best marker for secretory vesicles is CD35, a transmembrane protein of 160 to 250 kDa that binds complement components C3b and C4b, because CD35, in contrast to alkaline phosphatase, is absent from the plasma membrane of unstimulated neutrophils, and because it is absent from granules (in contrast to α_Mβ₂).^32,65,71 It is not known whether secretory vesicles contain lipid rafts, but most GPI-linked proteins are raft-associated⁷² and are localized to secretory vesicles in neutrophils.

GRANULES

Nomenclature of Neutrophil Granules

The neutrophil is known for its granules. When Paul Ehrlich introduced aniline dyes in histochemistry and discovered the different subsets of leukocytes, the neutrophil granules were divided into those that took up the azure dye, the azurophilic granules, and the others, the specific granules.^73,74 When the peroxidase reaction was introduced, the azurophil granules were found to be peroxidase-positive as a result of the presence of the major myeloid cell protein, myeloperoxidase (MPO), and the specific granules were thus named peroxidase-negative granules.^75,76 Because the azurophil granules are formed first, in the promyelocyte, and the specific granules later, in the myelocyte, these are also termed primary and secondary granules, respectively. A tertiary granule subset was identified in human neutrophils and shown to contain gelatinase,⁷⁷ but the ultrastructure was not determined until the issue of the neutrophil gelatinase (matrix metalloproteinase [MMP]-9) as a possible complex with neutrophil gelatinase-associated lipocalin (NGAL) was identified.^78,79

Granules were initially viewed of as small bags that emptied their content of bactericidal substances onto the ingested microorganisms when granules fuse with the phagocytic vacuole during phagocytosis, but it later became clear that granules are not only important for their cargo, but also for their membranes, as they contain proteins that become incorporated into the membrane of the phagocytic vacuole and into the surface membrane when the granules are mobilized.^80,81 If granules were classified by their content, both of matrix proteins and membrane proteins, the number of different granule subsets that exists in neutrophils would be meaninglessly high. Yet nature has provided a beautiful setting that allows the neutrophil to fine tune its response to a specific task. A priori, there would be two reasons for having different subsets of granules: One would be to ensure that proteins, which cannot coexist, are segregated; that is, protease-sensitive proteins are separated from proteases. The other reason would be to have proteins whose service is needed at one time separated from proteins whose service is needed at a different time.

Heterogeneity of Neutrophil Granules

Among the peroxidase-positive granules, subsets can be identified that are rich in defensins as well as some that are not.^82,83 Functionally, no difference has been identified in terms of the regulation of exocytosis of these peroxidase-positive granule subsets.⁸⁴ Other constituents include the serine proteases elastase, cathepsin G, proteinase 3, and neutrophil serine protease 4 (NSP4), and the inactive serine protease azurocidin (aka CAP 37), the antimicrobial proteins BPI (bacterial permeability-increasing protein), lysozyme, and the α defensins, which are the dominating species.⁸⁰ Defensins are also named HNPs (human neutrophil peptides). The membrane of the azurophil granules contains CD63 (granulophysin) and CD68, but their role in neutrophil function remains unclear.^85,86 Many of the proteins present in peroxidase granules are proteolytically processed both at the N-terminus and the C-terminus to the active mature forms, which are stored in the granule matrix.

Peroxidase-negative granules can be divided into three subsets based on the distribution of the two marker proteins lactoferrin and gelatinase: granules that contain lactoferrin, but no gelatinase (15 percent of peroxidase-negative granules), granules that contain both proteins (60 percent), and granules that are rich in gelatinase, but low (or absent) in lactoferrin (25 percent).⁸⁷ The latter are named gelatinase granules or tertiary granules, whereas those that contain lactoferrin are called specific or secondary granules. It is a characteristic of peroxidase-negative granules that the proteins present in their matrix are not proteolytically processed. The MMPs of peroxidase-negative granules are stored as a proform,⁸⁸ as is the major bactericidal protein hCAP-18.^89,90 No major differences have been identified in the content of membrane proteins of the peroxidase-negative granule subsets. All contain the flavocytochrome p22^phox/gp91^phox complex that is part of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and all contain the major β₂-integrin α_Mβ₂—and these are even shared with the membrane of secretory vesicles.^32,91,92 The divalent cation transporter Nramp1 is localized only to gelatinase granules,⁹³ and the membrane MMP leukolysin (MMP-25)⁹⁴ is shared between gelatinase granules and secretory vesicles. However, the subsets differ markedly in their propensity for exocytosis. Following neutrophil stimulation, gelatinase granules are exocytosed to a larger extent than granules containing both lactoferrin and gelatinase, and these are more readily mobilized than granules containing lactoferrin but lacking gelatinase. These, in turn, are mobilized more readily than peroxidase-positive granules.^{62,66,79,87,95} This organization of granule subsets with different content and different set points to trigger exocytosis allows the neutrophil to mobilize MMPs and integrins necessary for movement through the basal membrane and tissue before the bactericidal peptides and serine protease are called to play, but it puts an enormous task on the organization of the biosynthetic apparatus to secure that the right granule proteins are targeted to the granules with a given trigger for exocytosis.

The content of isolated granules has been mapped by proteome analysis.⁹⁶ High-resolution mass spectrometry has identified 1300 proteins associated with neutrophil granules, plasma membranes and secretory vesicles and confirmed that localization is largely determined by time of biosynthesis.⁹⁷

Targeting by Biosynthetic Timing

The extreme heterogeneity of neutrophil granules and their individual control of exocytosis can be explained simply by timing of their biosynthesis (Fig. 66–3). Granule proteins are synthesized during myelopoiesis from myeloblasts to band cells and segmented neutrophils in the marrow.^75,76,98 The window of biosynthesis of each granule protein is highly controlled by combinations of transcription factors that change as the cells differentiate and mature.^99,100 If all granule proteins are targeted to granules during synthesis, the content of newly formed granules would change as the cell matures because the profile of biosynthesis changes. A global view of the change in transcriptional activity of neutrophil precursors during maturation in the marrow confirmed the association between granule localization and transcriptional activity.¹⁰¹ This simple mechanism largely explains the heterogeneity of granules¹⁰² and their contents, but it does not account for the differences in exocytotic rates among individualized subsets. By timing the biosynthesis of the proteins essential for fusion^103,104 to granule membranes during maturation, it is possible to regulate the rates of exocytosis. Indeed, the v-SNARE (SNAP receptor), vesicle-associated membrane protein (VAMP)-2 is present in a higher density on gelatinase granules than on specific granules and is most highly expressed on secretory vesicles,^105,106 which correlates with the ease of releasing granule subsets from the neutrophil following activation.

Figure 66–3.

Formation of granule subsets during myelopoiesis and regulation of granule protein transcription. Difference in the appearance and disappearance of transcription factors regulate the individual window of granule protein gene transcription and translation into protein that is targeted to forming granules, explaining the heterogeneity of neutrophil granules.

Sorting between the Constitutive and Regulated Exocytotic Pathway

Although the sorting by timing can explain the granule heterogeneity of neutrophil granules, it does not provide any clues to the mechanisms responsible for diverting newly synthesized proteins to granules as opposed to immediate (constitutive) secretion. Not all granule proteins are equally efficiently directed to granules. Lysozyme is poorly retained during biosynthesis.¹⁰⁷ This explains the high concentration of lysozyme in plasma.¹⁰⁸ MPO is efficiently retained and the plasma level of MPO is consequently very low. A particularly interesting observation pertains to α defensins. These are localized exclusively to azurophil granules, but their window of biosynthesis is very similar to that of lactoferrin,^107,100 and defensins and lactoferrin are both controlled by the transcription factor C/EBPε (CCAAT/enhancer binding protein ε), which is absolutely required for biosynthesis of specific granule proteins.^109,110 The absence of defensins from specific granules, despite an active biosynthesis when other specific granule proteins are formed, is explained by a complete lack of sorting of defensins to granules in myelocytes.^99,100,107 Only defensins synthesized at the late promyelocytic stage are routed to granules, whereas defensins synthesized at the myelocyte stage are secreted from cells after biosynthesis¹⁰⁷ and is present in relatively high concentrations in plasma.¹¹¹ The defensins that are targeted to granules are processed to mature defensins, whereas the defensins that are secreted remain unprocessed. Processing of defensins removes a charge neutralizing propiece, sorting of defensins and other granule proteins to granules may depend on their ability to interact with negatively charged proteoglycans that are present in the matrix of granules.^112,113,114 Serglycin, an intracellular proteoglycan is present in Golgi and immature granules of promyelocytes and disappears as the cells mature.¹¹⁵ Serglycin is absolutely critical for confining a variety of mast cell proteins to the mast cell granules.¹¹⁶ Granulocytes from mice with a targeted disruption of the serglycin gene are morphologically normal and contain normal levels of granule proteins except elastase.¹¹⁷ CD63 was demonstrated to be involved in sorting of elastase to azurophil granules,¹¹⁸ but this may be indirectly via serglycin. An N-terminal sorting domain has been identified in serglycin and was shown to be essential for routing of serglycin to mast cell granules.¹¹⁹ No common denominator has been identified that can fully explain why neutrophil proteins are sorted to granules. Perhaps the lack of efficient sorting to granules may not solely be taken as inefficiency, but may be a way to secure a desirable level of antibiotic protein such as lysozyme¹⁰⁸ and hCAP-18¹²⁰ in plasma which renders the myeloid cells of the marrow a major secretory organ.

Control of Neutrophil Granule Protein Expression

The biosynthesis of neutrophil granule proteins is controlled at the transcriptional and not the translational level (see Fig. 66–3).^98,99,100 Not all transcription factors that are responsible for biosynthesis of granule protein have been identified, and the role of an individual transcription factor may be difficult to identify from gene knockout studies as transcription factors may work at multiple stages during myelopoiesis. The transcription factor PU.1 is essential for myelopoiesis because knockout mice do not form myeloid progenitors beyond myeloblasts^121,122; but this does not preclude PU.1 from regulating transcription of individual granule proteins at a later stage of development.^{123,124,125,126} Figure 66–3 shows the profile of important myeloid transcription factors during maturation of normal myeloid cells in the marrow in vivo. RUNX1 (AML-1), c-MYB, CASP, C/EBPα, C/EBPγ, GATA-1, and ELF-1 gene products are all strongly expressed in the myeloblast and promyelocyte, and some of these are required for azurophil granule protein expression. Then c-MYB, AML-1, GATA-1, and ELF-1 gene products are downregulated as the cells enter the myelocyte stage, heralded by a brisk and transient upregulation of C/EBPε to initiate expression of peroxidase-negative granule proteins⁹⁹ in agreement with the lack of specific granules in C/EBPε –/– mice and with the observation of a C/EBPε mutation in patients with a rare specific granule deficiency.^{109,110,127,128} PU.1, C/EBPβ, and C/EBPδ also appear at the promyelocyte myelocyte transition, but in contrast to C/EBPε, continue to increase as the cells mature to neutrophils. ELF-1 reappears at the metamyelocyte stage followed by C/EBPξ, c-Jun, and c-fos that are expressed at the band cell stage and increase in content as the cells mature.⁹⁹

MicroRNA

MicroRNAs (miRNAs) are important regulators of protein synthesis. In general, they bind to the 3′-end of mRNA and inhibit translation. Just like genes for granule proteins, mRNAs are expressed during maturation of neutrophils in the marrow depending on the stage of neutrophil maturation and can be classified into six groups, each with its characteristic expression profile.¹²⁹ So far, miRNAs have been shown to regulate proteins of importance for proliferation but not (yet) expression of individual granule proteins. Expression of the myeloid-specific miRNA-223 increases during maturation of neutrophils in the marrow, and after their release into the circulating. One of the targets of miRNA-223 is the Mef2c transcription factor. Mice that lack miRNA-223 expand granulopoiesis and the mature neutrophils mount an enhanced respiratory burst in response to phorbol myristate acetate (PMA), indicating that miRNA-223 acts as a negative regulator of granulopoiesis and neutrophil activation.¹³⁰ miRNA-130a is highly expressed in myeloblasts and promyelocytes and targets SMAD4, which, despite a high level of mRNA in promyelocytes, is not expressed at the protein level, and the cells are consequently insensitive to growth inhibition by transforming growth factor β (TGF-β)–induced growth inhibition. miRNA-130a also inhibits C/EBPε which induces exit from cell cycle and growth arrest at the myelocyte stage. Hence, miRNA-130a seems important for the expansion of myeloblasts, promyelocytes and early myelocytes.^131,132

FUNCTION OF INDIVIDUAL GRANULE PROTEINS AND THEIR ROLE IN OXIDATIVE AND NONOXIDATIVE MICROBIAL KILLING

Proteins of Azurophil Granules

Table 66–1 lists the physical-chemical and functional properties of neutrophil granules.

Table 66–1.Physical-Chemical and Functional Properties of Neutrophil Granules

Table 66–1. Physical-Chemical and Functional Properties of Neutrophil Granules

Granule Protein

Localization

Physicochemical Properties

Function

Myeloperoxidase

Azurophil granule (AG)

Heme protein, 90-kDa proform with an internal disulphide bond between the 57- and the 13.5-kDa subunits, generated by proteolytic processing that takes place during routing to granules

The MPO–halide–H₂O₂ system generates hypochlorous acid (HOCl), other chlorination products, tyrosine radicals, and reactive nitrogen intermediates, each of which can attack the surface membrane of microorganisms

Bacterial permeability-increasing (BPI) protein

55-kDa protein with high homology to the LPS-binding protein of plasma

BPI is organized into two largely symmetrical subdomains one of which is responsible for the binding of lipopolysaccharide (LPS) and for the antimicrobial activity against Gram-negative microorganisms

Defensins: three α-defensins; human neutrophil peptides (HNPs) 1–3

7-kDa proforms processed by proteolytic cleavage to mature 3-kDa defensins that share a characteristic three disulfide bond motif: 1–6, 2–4, 3–5

Defensins are small, amphipathic, pore-forming, antibacterial cationic peptides with a broad spectrum of antibacterial activity

Serine proteases of azurophil granules: elastase, cathepsin G, proteinase 3, and neutrophil serine protease 4 (NSP4); azurocidin (CAP37 or heparin-binding protein [HBP]) is enzymatically inactive

28-kDa proforms, processed to active proteases en route to azurophil granules

Serine proteases, but both elastase and cathepsin G have direct antibacterial activities that are not dependent on their enzymatic activity; proteinase 3 liberates the antibacterial peptide LL37 from hCAP-18; HBP is chemotactic for monocytes; HBP may open endothelial cell tight junctions

Lysozyme

AG ~30%; specific granules (SG) ~50%; gelatinase granules (GG) ~20%

Cationic antimicrobial peptide of 14 kDa; in contrast to many neutrophil granule proteins, lysozyme is inefficiently targeted to granules and circulates free in plasma in a substantial quantity that reflects the normal myelopoietic activity

Lysozyme cleaves peptidoglycan-polymers of bacterial cell walls and displays bactericidal activity toward the nonpathogenic Gram-positive bacteria Bacillus subtilis; a particular high serum level is characteristic for the myelomonocytic leukemias

Lactoferrin

78-kDa iron chelator; member of the transferrin protein family with a high affinity for iron and similar iron-binding characteristics as ferritin

The antibacterial activity of lactoferrin does not depend exclusively on its ability to sequester iron. Proteolytic fragments, some of which are known as lactoferricin, are directly bactericidal

Neutrophil gelatinase-associated lipocalin (NGAL) or siderochelin

25-kDa N-glycosylated member of the lipocalin protein family

NGAL is the first known siderophore-binding eukaryotic protein; NGAL binds enterochelin/enterobactin with high affinity, and blocks growth of Escherichia coli by sequestering siderophore–iron complexes

hCAP-18

18 kDa; only human member of the cathelicidin protein family

Stored and released intact; binds endotoxin; C-terminal antibactericidal peptide, LL-37 released by proteinase 3; active mainly against Gram-positive bacteria, is chemotactic for T cells, monocytes, and neutrophils, and has angiogenetic properties

Neutrophil collagenase

75-kDa matrix metalloproteinase 8 (MMP-8); like other MMPs, MMP-8 is stored inactive and must be N-terminally trimmed to remove the inhibitory peptide

Active against types I, II, and III collagen

Olfactomedin 4 (OLFM4)

65-kDa protein that forms multimers by disulfide bonding

Function is unknown, but OLFM4 is present only in a subset (25%) of neutrophils

Gelatinase

92-kDa MMP-9; stored inactive

Active against type IV collagen

Leukolysin, which is distributed among of resting neutrophils

SG ~10%; GG ~40%; secretory vesicles (SV) ~30%; plasma membranes (PM) ~20%

Leukolysin is a 56-kDa glycosylphosphatidylinositol (GPI)-anchored membrane-bound MMP (MT6-MMP/MMP-25)

Active against fibronectin, chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan

Cytochrome b₅₅₈, (gp91^phox, p22^phox)

SG ~60%; GG ~25%; SV ~15%

Heterodimeric flavoheme protein; 91-kDa glycoprotein subunit (heme-flavin binding); 22-kDa protein subunit, possibly heme binding

Together with p47^phox, p67^phox, and p40^phox, cytochrome b₅₅₈ constitutes the superoxide generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of phagocytes

CD11b/CD18 (Mac-1, Mo1, CR3, α_Mβ₂)

SG ~60%; GG ~25%; SV ~15%

Most prominent β₂-integrin in neutrophils; CD11B = α_M and is a glycoprotein of 170 kDa; CD18 = β₂ and is a glycoprotein of 95 kDa

Multifunctional integrin that functions as an adhesion receptor binding to members of the immunoglobulin family intercellular adhesion molecule (ICAM)-1, to fibronectin, collagen; is important in mediating firm adhesion to vascular endothelial cells; functions as a phagocytosis receptor for C3bi-coated particles

Pentraxin-3

Pentamer of 47-kDa subunits

Binds complement C1q, selected microorganisms

Ficolin-1

Multimer of 32-kDa subunits

Binds acetylated carbohydrates on microorganisms; may activate mannose-binding lectin-associated serine proteases

Arginase 1

37-kDa glycoprotein

Degrades arginine, the substrate for nitric oxide (NO) synthase

The protein MPO is a marker of azurophil granules. It is formed as a 90-kDa precursor with an internal disulphide bridge that forms a link between the 57- and 13.5-kDa subunits that are generated by the proteolytic processing that takes place during routing to granules. The heme group, which is necessary for the reduction-oxidation (redox) functions of MPO, associates with the 90-kDa subunit.¹³³ This seems to be a necessary prerequisite for subsequent processing.¹³⁴ MPO reacts with H₂O₂, formed by the NADPH oxidase, and increases the toxic potential of this oxidant. Through oxidation of chloride, tyrosine, and nitrite, the hydrogen peroxide (H₂O₂)-MPO system induces formation of hypochlorous acid (HOCl), other chlorination products, tyrosine radicals, and reactive nitrogen intermediates, each of which can attack the surface membrane of microorganisms.^135,136 MPO may be found on endothelial cells during inflammation and can inactivate nitric oxide (NO).¹³⁷ In addition to the activities of MPO itself, MPO is known for the anti-MPO autoantibodies that are characteristic of the pANCAs (perinuclear antineutrophil cytoplasmic antibodies) that are found in vasculitides, in particular those that primarily affect kidneys.^138,139

BPI is a 55-kDa protein with high homology to the LPS-binding protein of plasma. It is organized into two largely symmetrical subdomains, one of which is responsible for the binding of LPS and for the antimicrobial activity against Gram-negative microorganisms. In contrast to LPS-binding protein, which presents endotoxin to CD14 and elicits a proinflammatory response, BPI binds LPS independent of CD14 and neutralizes the effects of LPS.¹⁴⁰ A transgene expressing high levels of BPI has enhanced resistance against endotoxin.¹⁴¹ Important effects not related to LPS have been described, such as chemotaxis, opsonization, and dendritic cell function, as reviewed in Ref. 142.

Defensins are small antibacterial cationic peptides with a broad spectrum of antibacterial activity.¹⁴³ They share a characteristic three-disulfide-bond motif.^82,144,145 Based on this, mammalian defensins are divided into α defensins, β defensins, and the cyclical θ defensins.¹⁴⁶ Only α defensins are found in human neutrophils, and reside exclusively in azurophil granules. They are by far the dominating proteins of azurophil granules, yet are only expressed in a subset of granules that are formed late in the promyelocyte stage.^83,100,107 Three defensins have been isolated from azurophil granules, HNP-1 to HNP-3.⁸² Large amounts of unprocessed defensing, that is, prodefensin, are secreted from late promyelocytes and myelocytes in the marrow and may serve as a marker of normal myelopoietic activity.¹¹¹ In contrast to lysozyme and MPO, prodefensins are not expressed by acute leukemia cells. A rise of prodefensin in plasma precedes detectable neutrophils by 6 days and is a measure of normal granulopoiesis which may be of clinical use in the setting of myeloablative treatment for acute leukemia.¹¹¹

The serine proteases of azurophil granules include elastase, cathepsin G, proteinase-3, and NSP4.^147,148 Azurocidin, which is also named CAP37 or heparin-binding protein (HBP), is an enzymatically inactive serine protease.^{149,150,151,152,153} Both elastase and cathepsin G have direct antibacterial activities that are not dependent on their enzymatic activity. Proteinase 3 expression leads to autoantibodies against itself in Wegener granulomatosis, which is known as cANCA (cytoplasmic antineutrophil cytoplasmic antibody).¹⁵⁴ Proteinase 3 is also bound to the surface of circulating neutrophils at levels that vary considerably amongst individuals but are highly constant throughout life in a given individual. The binding is mediated by the NB1 antigen (CD177).^155,156 A secreted precursor of proteinase 3 has been suggested to inhibit normal myelopoiesis¹⁵⁷ and to play a role in regulation of myelopoiesis. So far, the only specific substrate of proteinase 3 identified is the cathelicidin of specific granules hCAP-18. Proteinase 3 activates hCAP-18 by removing the cathelicidin part, and unleashing the antibacterial activity of the C-terminal LL-37 peptide.⁹⁰ Cathepsin C, also known as dipeptidyl peptidase 1, removes two inhibitory N-terminal amino acids from the serine proteases prior to their storage in granules.¹⁵⁸ Patients with the Papillon-Lefèvre syndrome lack cathepsin C activity and are not able to store serine proteases in their neutrophils.¹⁵⁹ The condition is characterized by severe juvenile periodontitis and keratosis in hands and feet, but not by major systemic infections,¹⁶⁰ arguing against the notion the serine proteases are important for immune defense.¹⁶¹

The membrane of azurophil granules contains CD63 (granulophysin), which is implicated in transmembrane signaling with the β₂ integrins in the activated neutrophil.^85,86,162 Also, the CD68 antigen^100,163 and presenilin appear localized exclusively to the membrane of azurophil granules,¹⁶⁴ whereas stromatin is found in the membrane of all granules¹⁶⁵ and the vacuolar-type H⁺-ATPase is shared between azurophil, gelatinase granules and secretory vesicles.¹⁶⁶ These membrane proteins will translocate to the phagocytic vacuole or to the plasma membrane when neutrophils are activated and engaged in phagocytosis.

Proteins of Peroxidase-Negative Granules

For an overview, see Table 66–1.

Lactoferrin is the dominating protein of specific granules.¹⁶⁷ It is a 78-kDa iron-chelator, member of the transferrin protein family with a high affinity for iron and similar binding characteristics as ferritin.^168,169 The antibacterial activity of lactoferrin does not depend exclusively on its ability to sequester iron because proteolytic fragments of lactoferrin, some of which are known as lactoferricin, are directly bactericidal.^170,171

NGAL, or lipocalin 2, is a 25-kDa N-glycosylated member of the lipocalin protein family.⁷⁹ Lipocalins are transport proteins that bind small and often lipophilic substances in their canonical lipocalin pocket.¹⁷² Some NGAL is associated with gelatinase (MMP-9) in a subset of specific granules,¹⁷³ but the majority is present either as a monomer or as a homodimer in specific granules. NGAL interferes with the activation and stability of MMPs,¹⁷⁴ but the major function of NGAL is to bind and sequester siderophores. NGAL binds enterochelin/enterobactin with high affinity, and blocks growth of Escherichia coli by sequestering siderophore-iron complexes,¹⁷⁵ which might not be only a neutrophil-specific antibacterial defense because NGAL can be induced in a variety of epithelial cells during inflammation by IL-1.¹⁷⁶ NGAL has been demonstrated to play a protective role in infections against E. coli,¹⁷⁷ Klebsiella pneumoniae,¹⁷⁸ Salmonella typhimurium,¹⁷⁹ and Mycobacterium tuberculosis.¹⁸⁰ NGAL has effects not explained by sequestering bacterial siderophores and was shown to worsen the outcome of pneumococcal pneumonia by deactivating macrophages.¹⁸¹ It is possible that the ability of NGAL to bind endogenous siderophore-like structures and transport iron may explain some of these effects.¹⁸² Nramp1, the cation transporter, was initially identified first in macrophages as an essential resistance factor against mycobacterial infection. It is present in membranes of both specific and gelatinase-containing neutrophil granules.^93,183

Lysozyme is a cationic antimicrobial peptide of 14 kDa.¹⁸⁴ In agreement with its biosynthetic profile, lysozyme is present in all granule subsets, with peak concentrations in specific granules.^100,108 Lysozyme cleaves peptidoglycan polymers of bacterial cell walls and displays bactericidal activity toward the nonpathogenic Gram-positive bacteria Bacillus subtilis.¹⁸⁵ Lysozyme also binds LPS¹⁸⁶ and reduces cytokine production and mortality caused by LPS in a murine model system of septic shock.¹⁸⁷ In contrast to many neutrophil granule proteins, lysozyme is inefficiently targeted to granules and circulates free in plasma in a substantial quantity that reflects the granulopoietic activity.^107,108 Lysozyme is also secreted from activated macrophages,¹⁸⁸ and a particular elevated serum level is characteristic of the leukemias with a large proportion of monocytes.¹⁸⁹

hCAP-18,⁸⁹ also known as LL-37¹⁹⁰ or CAMP, is the only human member of a family of antimicrobial peptides known as cathelicidins. Cathelicidins are typically found in peroxidase-negative granules of mammalian neutrophils.¹⁹¹ hCAP-18 is a prominent protein of neutrophil specific granules present in equimolar concentrations with lactoferrin.¹⁹² It is also present in plasma at a substantial concentration bound to lipoproteins.¹²⁰ In general, cathelicidins are proantibiotic peptides that share a common and highly conserved 14-kDa N-terminal region known as the cathelin region, whereas the C-terminal regions vary extensively among the different cathelicidins. The C-terminal peptides must be liberated from the cathelin domain by proteolysis to become antibacterial. In most species this is carried out by elastase, but in human neutrophils this is done by proteinase 3 from azurophil granules. The liberated C-terminal peptide is known as LL-37.^90,190 Like several other neutrophil proteins, hCAP-18 is formed by cells in other tissues, particularly epithelial cells.^{90,193,194,195} It is constitutively expressed in the testis and present in semen. Here, the activating protease is gastricin, a prostate protease that is active at low pH. This cleaves hCAP-18 to ALL-38, which has the same antibacterial spectrum as LL-37.¹⁹⁶ The cathelin part, which is released has some protease inhibitory activity by itself.¹⁹⁷ The LL-37 stimulates neutrophil, monocyte, and T-cell chemotaxis via the formyl peptide receptor-like-1.¹⁹⁸ In addition, hCAP-18/LL-37 has angiogenic¹⁹⁹ and endotoxin-neutralizing properties.²⁰⁰

Three MMPs have been identified in neutrophils: neutrophil collagenase (MMP-8,75 kDa), which is localized to specific granules,²⁰¹ gelatinase (MMP-9,92 kDa), which resides predominantly in gelatinase granules,^78,202 and leukolysin (MT6-MMP/MMP-25,56 kDa), which is distributed among specific granules (approximately 10 percent), gelatinase granules (approximately 40 percent), secretory vesicles (approximately 30 percent), and the plasma membrane (approximately 20 percent) of resting neutrophils.^94,203 The MMPs are stored as inactive proforms that are proteolytically activated following exocytosis. Together, the MMPs are capable of degrading major structural components of the extracellular matrix, including collagens, fibronectin, proteoglycans, and laminin, and they are believed to be of central importance for the degradation of vascular basal membranes and interstitial structures during neutrophil extravasation and migration.

Two pattern-recognition molecules, pentraxin 3 and ficolin1, are found in specific granules and gelatinase granules, respectively. Pentraxin 3, a member of the long pentraxins family, is synthesized in myelocytes and metamyelocytes and stored in specific granules of neutrophils. Pentraxin-3 binds the complement component C1q and mediates activation of the classical complement cascade. In addition, pentraxin-3 binds K. pneumoniae outer membrane protein A (KpOmpA) from Gram-negative bacteria, especially the Enterobacteriaceae species, and binds Aspergillus fumigatus conidia. Pentraxin-3 was shown to play a major role in uptake and killing of A. fumigatus conidia by neutrophils in a mouse model.^204,205

Ficolin-1 is present in gelatinase granules. Ficolin-1 binds acetylated carbohydrate structures on Gram-positive bacteria and can recruit mannose-binding lectin-associated serine proteases (MASPs) and activate the lectin complement cascade.²⁰⁶

Arginase-1 is a constituent of gelatinase granules²⁰⁷ and may participate in regulation of T-cell activities by removing arginine, the essential substrate for inducible nitrous oxide synthase. The product, proline, is essential for collagen synthesis and arginase-1 from neutrophils may thus support wound healing.

Olfactomedin 4, a 65-kDa specific granule protein, forms huge multimers, but only in approximately 25 percent of neutrophils, ranging from 5 to 40 percent between individuals and constant in each individual. The functional consequence is unknown.^208,209

Membrane proteins of peroxidase-negative granules are shared among the subsets of peroxidase-negative granules that can be distinguished based on their matrix proteins; that is, specific and azurophil granules. Two exceptions are Nramp1 and MMP-25, which are both present, predominantly in the membrane of gelatinase granules and secretory vesicles.^93,94 Cytochrome b₅₅₈, which is comprised of gp91^phox and p22^phox, forms the membrane component of the NADPH oxidase and is a prominent membrane protein of peroxidase-negative granules.^81,210 It codistributes with the major β₂-integrin of neutrophils CD11b/CD18, with the major segment in specific granules, some in gelatinase granules, and some in secretory vesicles. Secretory vesicles are rapidly mobilized, and even though only 15 percent of the total cytochrome b₅₈₈ and CD11b localizes to secretory vesicles, this is the fraction that is primarily translocated to the plasma membrane during neutrophil diapesis.^66,32 Hv1is a voltage-gated proton channel situated in the plasma membrane and membranes of peroxidase-negative granules.²¹¹ Hv1 associates with nascent phagosomes, and neutralizes the negative charge induced by transport of electrons by the NADPH oxidase.^212,213 The CD66 antigens found in the membrane of specific granules may play a role as bacterial receptors (galectin receptors) and generate signals to activate the NADPH oxidase.^214,215

STIMULUS-RESPONSE COUPLING BY NEUTROPHILS

Stimulus-response coupling 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. This chapter considers next our current understanding of the activation process, which is shown schematically in Fig. 66–4.

Figure 66–4.

Signal transduction in neutrophils. G-protein–coupled receptors are seven transmembrane receptors that couple to heterotrimeric guanosine triphosphate (GTP)-binding (G) proteins. Agonist binding to the receptor triggers exchange of guanine diphosphate (GDP) for guanine triphosphate (GTP) on the Gα subunit of the G-protein, and consequently, the disassociation of the α subunit for the βg-dimer. Both subunits can regulate the activity of multiple effectors such as phospholipase Cβ (PLCβ). PLCβ cleaves an endogenous lipid, namely phosphatidylinositol bisphosphate (PtdInsP₂), 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⁺)i. The increase in intracellular Ca²⁺ is augmented by an influx of Ca²⁺ from the extracellular space. Increased DAG, in concert with elevated Ca²⁺ can activate protein kinase isozymes α and β (PKCαβ) leading to their translocation to membranous sites. Phospholipase D (PLD) can be activated by PKC converting phosphatidylcholine to phosphatidic (PA) acid. Elevations in PA can mobilize the cytosolic proteins, p47, p67^phox, and p40^phox to bind to the membrane-bound proteins gp91^phox and p22^phox, which then reduces O₂ to O_2– in the presence of NADPH.

RECEPTOR–LIGAND INTERACTIONS

Formyl Peptide Receptor

Neutrophil responses can be evoked by a variety of particulate and soluble stimuli. Opsonized particles, immune complexes, and chemokine 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 peptide, the synthetic analogues of bacterial N-formyl peptide products, induces a variety of neutrophil responses and has been extensively employed as activating stimuli. Specific receptors for the chemotactic peptide, fMLP, have been identified on the neutrophil surface,²¹⁶ and binding of the formyl peptide to its receptor correlates with its ability to induce chemotaxis and degranulation.²¹⁷ The formyl peptide receptor,²¹⁸ like the receptors for C5a, IL-8, LTB₄, and PAF, belongs to a family of seven trans-membrane–spanning domain proteins (7TMRs) that are coupled to heterotrimeric G-proteins (containing G α and β,γ subunits).^219,213 Upon ligand binding, guanine diphosphate (GDP) bound to the Gα subunit is exchanged with guanine triphosphate (GTP) and the β,γ subunits dissociate from the receptor and mediate downstream signaling. Phosphorylation of the receptors then augments their affinity for β arrestins. The association with β arrestins block association with β,γ subunits and mediate internalization of the receptors, but may also induce additional signals via β arrestins.^220,221 The formyl peptide receptor has been studied in most detail in neutrophils. The receptor is highly glycosylated and has a relative molecular mass (Mr) of 50 to 70 kDa. It has been identified on the membranes of gelatinase granules and secretory vesicles, and shown to be mobilized to the cell surface following stimulation.²²²

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, degranulation, and superoxide generation.^222,223 Responses to C5a result from interactions with specific receptors on the cell surface.^222,224 The receptor was identified as a single polypeptide in the plasma membrane with an apparent mass of 40 to 48 kDa.^222,225 Binding studies show that there are 50,000 to 113,000 receptor sites per cell with a dissociation constant (Kd) of 2 × 10^–9 M. The C5a receptor has been isolated and cloned, and is a member of the seven transmembrane-spanning class of G-protein–coupled receptors.²²⁶

Three other important G-protein–coupled receptors are for PAF, IL-8, and LTB₄. PAF and IL-8 receptors have been cloned.^227,228 Their intracellular stores and signal transduction mechanisms are largely similar to those used by other G-protein–coupled receptors (e.g., fMLP).²²⁷ IL-8 has two related receptors, for which slightly different signal transduction pathways have been detected.²²⁹

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 increase significantly in numbers following activation with several stimuli because of incorporation from secretory vesicles (CR1 and CR3) and gelatinase and specific granules (CR3, which is the integrin Mac-1).^32,71 The C3b receptor (CR1) is a glycoprotein with a molecular weight of 205 kDa and is located in secretory vesicules.^65,71

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.²³⁰ H₂O₂ production in response to chemotaxins is influenced by monoclonal antibodies to CD11b, but not CD11a.²³¹

Fc Receptors

Neutrophils possess three different receptors for immunoglobulins. Unstimulated cells express FcγRIIA and FcγRIII, also known as CD32 and CD16, respectively. Functionally, the most important of the two is the FcγRIII for clearing immune complexes,²³² and it is attached to the membrane by a GPI linkage.²³² The linkage is relatively labile, so the amount of FcγRIII on the membrane reflects a balance between shedding and mobilization from intracellular stores. FcγRIIA is a protein that spans the plasma membrane.²³³ The signal transduction pathways initiated by FcγRIII can crosstalk with the formyl peptide receptor, with CR3, and even with each other. A direct physical linkage between CD11b and FcγRIIIB has been demonstrated by experiments in which capping of one receptor results in co-capping a substantial fraction of the other receptor. CD11b can also interact with the transmembrane FcγRII, and both of these molecules can modify each other’s signals.²³⁴

The tyrosine kinase Syk, plays a critical role in the phagocytic pathway mediated by FcγRIIA.²³⁵ A cytoplasmic amino acid motif, known as ITAM, is present on FcγRIIA and FcγRI/γ (a receptor found on IFN-γ–stimulated myeloid cells) and is essential for the phagocytic response during crosslinking of these two Fc receptors. Binding of Src family protein tyrosine kinases to the ITAM leads to activation of Src family protein tyrosine kinases and ITAM tyrosine phosphorylation. This serves to recruit phosphatidylinositol 3′-kinase (PI3K) and Syk, which when activated phosphorylates multiple substrates, including neighboring ITAMs. Syk is recruited from the cytosolic pool. The essential role for Syk-affecting signal transduction is reflected by ITAM-dependent activation of actin assembly. Other tyrosine kinases, including Src kinases, especially Lyn, facilitate the formation of the phagosome.²³⁶ Once active microfilaments are formed, they enhance the activity of phospholipase D (PLD) to generate phosphatidic acid (PA), a necessary phospholipid for phagocytosis to ensue.^237,238

Phospholipid Metabolism and Tyrosine Kinase Activation

The next step in signal transduction can be attributed to interactions of receptor-activated G proteins or through FcγRIIA and tyrosine kinases with phospholipases.^239,240 For instance, a membrane-associated phosphoinositide-specific phospholipase is activated upon stimulation with chemotactic stimuli. In particular, phospholipase C (PLC) hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP₂) and phosphatidyl inositol-4-monophosphate (PIP₁) to the putative second-messenger products inositol 1,4,5-trisphosphate (IP₃) and 1,2-diacylglycerol (DAG) (see Fig. 66–4).²⁴¹ In neutrophils, IP₃ interacts with a specific intracellular receptor and stimulates the release of Ca²⁺, as well as opens Ca²⁺ channels on the plasma membrane, resulting in rises in intracellular Ca²⁺.²⁴² Activation of the small GTP-binding proteins of the Rac, Rho, and Cdc42 families regulates actin-dependent processes such as membrane ruffling, formation of pseudopodia, and stress fibers leading to cell adhesion and motility, and appears critical in neutrophil function,^243,244 while working in concert with the phospholipases.

Even in the absence of PLC metabolism, there is a significant increase in DAG and Ca²⁺ intracellularly that accompanies phagocytosis.²⁴⁵ Ca²⁺ is necessary for granule phagosome fusion and DAG has been linked to both particle ingestion and degranulation.²⁴⁶ Both can be formed by the activation of PLD, which hydrolyzes phosphatidylcholine to produce PA and choline. Activation of PLD is mediated by Rho and/or ADP-ribosylation factor (ARF).²⁴⁷ Diacylglycerol is then generated by PA phosphohydrolase, which catalyzes the dephosphorylation of PA. The hallmark of the phosphatidylcholine-derived DAG is the presence of 1-O-alkyl linkages. During PA formation by the action of PLD on phosphatidylcholine, PA can act as a Ca²⁺ ionophore, thereby initiating fusogenic activity.²⁴⁸ Thus, the phosphatidylcholine acid generated during phagocytosis may promote fusion of neutrophil granules with newly formed phagosomes.

Another downstream target of DAG in phagocytosis is the activation of protein kinase C (PKC), particularly PKCδ, a Ca²⁺-independent isozyme of PKC found in neutrophils.²³⁷ PKCδ is one of four PKC isozymes that translocate to the plasma membrane during phagocytosis. During phagocytosis, PKCδ is translocated from the cytosol to the plasma membrane. Accompanying the translocation of PKCδ to the membrane, RAF-1 translocation is promoted. Following translocation of these two key components, mitogen-activated extracellular signal-regulated kinase (MEK) activation occurs, which is followed by activation of mitogen-activated protein (MAP) kinase/extracellular signal-related kinase (ERK)-2 and then myosin light-chain kinase.²⁴⁰ Following phosphorylation of myosin, reorganization of the actin cytoskeletal occurs leading to phagocytosis. Concomitant with the activation of PLD, ceramide is generated by a neutral sphingomyelinase activity found in the plasma membrane of neutrophils and it is most likely important in attenuating the activity of the cells through inhibition of PLD.²⁴⁰ Following engagement of the Fc receptors and Syk activation in the neutrophil, PI3K is also activated. Inhibition of PI3K activity impedes phagocytosis.²³⁷

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 neutrophils,²⁴⁹ as well as the cytosol,²⁵⁰ is activated during neutrophil stimulation, yielding arachidonic acid as one of the major end products. Arachidonic acid is not only released from stimulated neutrophils, but also serves as a regulator of phospholipase A₂ (PLA₂) activity and as a stimulus for these cells.²⁵¹ Sensitivity of the cells to other stimuli can be enhanced with arachidonic acid and other long-chain fatty acids.²⁵²

Arachidonic acid can also be metabolized by the lipoxygenase pathway to produce hydroxyeicosatetraenoic acids (HETEs), including 5-HETE, 12-HETE, and 5,12-diHETE.²⁵³ These compounds have also been shown to induce several neutrophil responses.²⁵⁴ 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.²⁵⁵ Receptors for LTB₄ have been partially purified, and their activation serves as a potent stimulus for chemotaxis and adherence.²⁵⁶

Another potent mediator of inflammation produced by stimulated neutrophils is 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphoryl choline, also known as PAF.²¹ Not only is PAF synthesized by neutrophils and activated endothelial cells, but it induces degranulation, aggregation, and superoxide generation.²⁹ Inflamed endothelium generates PAF, which serves to immobilize neutrophils on the luminal surface of the endothelial cells, thereby facilitating the interaction of the neutrophil integrin receptors with the ICAM ligands on the endothelial cells.

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—occurs rapidly and is highly efficient.

Fusion Proteins Over the past 20 years the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) hypothesis has become the reigning paradigm for fusion of biomembranes.¹⁰⁴ The hypothesis is centered around the protein that is sensitive to N-ethylmaleimide (designated NEM-sensitive fusion protein or NSF) and several SNAREs on the participating membranes. The SNAREs are divided into the v-SNAREs, being found on vesicles or granules, and t-SNAREs, being found on the target plasma membranes. 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. Initial docking of granules with the membrane to which they fuse is likely mediated by Rab-GTPases. Once docking is obtained, SNAREs are recruited to both membranes and interact and mediate actual fusion assisted by SNARE-interacting proteins such as sSec1/Munc18 proteins and a local rise in Ca²⁺. Disassembly of the fusion complex is mediated by NSF in an ATP-dependent process.²⁵⁷ The t-SNARE VAMP-2 is localized to the membranes of specific and gelatinase granule and secretory vesicles in resting human neutrophils,^105,106 and the t-SNARE syntaxin 4 is associated with the plasma membrane as shown by immunoelectron microscopy. Munc18–3 may interact with syntaxin 4 and regulate fusion of secondary and gelatinase granules. VAMP-7 is associated with azurophil granule fusion and Munc18–2 may interact with syntaxin 3 and regulate azurophil granule fusion.^258,259

Neutrophil Extracellular Traps

What previously was considered pus was identified as a highly bactericidal structure composed of strands of chromatin and bactericidal neutrophil granule proteins attached.^260,261 These NETs (see Fig. 66–2) are extruded from neutrophils in a process called netosis and represent one of three death programs of neutrophils: apoptosis, necrosis, and netosis. Neutrophils only undergo netosis if they have mounted a respiratory burst.²⁶² Elastase and MPO are also required for netosis.²⁶³ The NADPH oxidase activity of stimulated neutrophils thus serves two purposes, namely to generate reactive oxygen species for microbial killing and to induce formation of the bactericidal NETs after the intact neutrophil has ceased to function. This, in turn, means that patients with defective NADPH oxidase assembly (patients with chronic granulomatous disease [CGD]) lack both the ability to generate microbicidal oxygen species and the ability to form the NETs. Patients with the Papillon-Lefèvre syndrome (PLS) lack elastase and are incapable of generating NETs. PLS patients do not have a major immune defect and their symptoms are largely related to periodontal infections, in contrast to CGD patients. Negative effects of NETs have been noted as NETs may induce thrombosis.²⁶⁴ Neutrophils are able to generate NETs and maintain their structural integrity as an anucleate cell still capable of migration and phagocytosis.²⁶⁵

CLINICAL DISORDERS OF NEUTROPHIL FUNCTION

CLASSIFICATION

Neutrophil dysfunction may arise from (1) the absence of antibodies or complement components required to opsonize microorganisms, an interaction that provides a chemotactic signal; (2) the abnormalities of cytoplasmic and granule movement that alter the chemotactic response or that result in abnormalities of the plasma membrane affecting the cells in terms of capability to modulate movement; and (3) defective microbicidal capability. Comprehensive reviews of these syndromes are available to the interested reader.^266,267,268

ABNORMALITIES OF THE SIGNAL MECHANISM AS A RESULT OF ANTIBODY AND COMPLEMENT DEFECTS OR IMPAIRMENT OF PATTERN RECEPTOR RECOGNITION

Because the synergistic action of immunoglobulins and complement proteins creates the opsonins that coat microorganisms and stimulate the development of chemotactic factors, a deficiency of either one may result in impaired neutrophil function. The most profound disturbances arise from abnormalities in C3, because this protein is the focal point for generation of opsonins and chemotactic factors (Chap. 19).^269,270,271 Opsonins such as C3b, generated from cleavage of C3, serve to coat bacteria. Opsonization in general refers to the coating of pathogens by serum proteins such that they are more likely to be ingested. Activation of C3 can occur in the absence of an antibody or the classical complement components C1, C2, and C4; thus, disorders of these latter molecules result in less-severe clinical conditions. C3 deficiency is inherited as an autosomal recessive disorder. Homozygotes have undetectable serum levels of C3 and suffer from recurrent severe pyrogenic infections, whereas asymptomatic heterozygotes have half the normal values.

A functional deficiency of C3 protease resulting in severe pyrogenic infections also is seen in patients with a deficiency in C3b inactivator, a protein inhibitor of the alternative complement pathway. Unchecked activation of this pathway leads to hypercatabolism of C3 and factor B.²⁷² Properidin deficiency also results in a functional deficiency in C3.²⁷³ Properidin is a serum protein that belongs to the alternative complement pathway; it is involved in the stabilization of the enzyme complex C3bBb. The protein is a multimeric glycoprotein with a subunit Mr of 56,000, the gene of which has been cloned.²⁷⁴ Absence of properidin is associated with severe, often fatal, pyrogenic infections, often with meningococci.

Approximately 5 percent of the population have low serum levels of mannose-binding lectin (MBL),²⁷⁵ a serum lectin secreted by the liver that binds mannose sugars present and on the surface of bacteria, fungi, and some viruses. MBL is one of the soluble collectin effector proteins that contribute to the basic armamentarium of innate immunity. MBL can function as an opsonin when bound to the surfaces by activating the complement cascade. A deficiency of MBL has been reported in infants with frequent unexplained infection, chronic diarrhea, and otitis media.²⁷⁵ Other studies have identified an increased susceptibility to infection by specific pathogens in MBL-deficient individuals, including human immunodeficiency virus, Plasmodium falciparum, Cryptosporidium parvum, and Neisseria meningitidis.²⁷⁶ The deficiency in MBL largely results from three relatively common single-point mutations in exon 1 of the gene, which leads to the failure of MBL to activate complement.²⁷⁷ In addition, the protein also modulates disease severity, at least in part through complex, dose-dependent influences on cytokine production.

Phagocytes, including neutrophils, express a large number of cell surface proteins that play crucial functional roles in their biology. Microbial PRRs are an essential component of innate immunity, in which they recognize and detect PAMPs, resulting in activation of neutrophils and other phagocytes. The mammalian TLR family comprises an important class of PRRs, which recognize a wide range of microbial pathogens and pathogen-related products. At least 12 different TLRs that can be found on mononuclear phagocytes have been described.²⁷⁸ TLRs signal via MyD88, an adapter protein. MyD88 deficiency in humans can lead to recurrent infections with both Gram-positive and Gram-negative infections, thereby indicating a role for both mononuclear cells and neutrophils in host defense in the MyD88-deficient state.²⁷⁹

Because a large number of chemoattractants are generated during inflammation, it is difficult to establish the relative significance of a given individual component. Furthermore, chemotactive factors and opsonins are involved in the activity of both neutrophils and mononuclear phagocytes. Therefore, it is not clear whether the clinical consequences of disorders involving these substances are unique to one or the other of these phagocytic cells. Patients with antibody- or complement-deficient syndromes suffer mainly from infections with encapsulated pathogens such as Haemophilus influenzae, pneumococci, streptococci, and meningococci.²⁸⁰ Furthermore, splenectomized individuals deprived of an organ rich in mononuclear phagocytes have a small, but finite risk of sepsis because of the same microorganisms. Encapsulated pathogens characteristically are not associated with neutropenic states. Antibody coating of encapsulated organisms facilitates their ingestion by mononuclear phagocytes, but may be less important for their ingestion by neutrophils.

ABNORMALITIES OF THE CELLULAR RESPONSES AS THE RESULTS OF DEFECTS IN CYTOPLASMIC MOVEMENT

Degranulation Abnormalities

Chédiak-Higashi Syndrome

Definition and History This rare autosomal recessive disease was initially recognized as one in which neutrophils, monocytes, and lymphocytes contained giant cytoplasmic granules.²⁸¹ Chédiak-Higashi syndrome (CHS) is now recognized as a disorder of generalized cellular dysfunction characterized by increased fusion of cytoplasmic granules.²⁸² Pigmentary dilution affecting the hair, skin, and ocular fundi results from pathologic aggregation of melanosomes and is associated with failure of decussation of the optic and auditory nerves (Table 66–2).²⁸³ Patients with this syndrome exhibit an increased susceptibility to infection, which begins in infancy. Infections most commonly involve the skin and respiratory systems. The susceptibility to infection can be explained in part through defects in neutrophil chemotaxis, degranulation, and bactericidal activity.²⁸¹ The presence of giant granules in the neutrophil interferes with their ability to traverse narrow passages between endothelial cells. Other features of the disease include neutropenia, thrombocytopathy,²⁸⁴ natural killer cell abnormalities,^281,285 and peripheral neuropathies.²⁸⁶ Similar genetic syndromes have been described in mice, mink, cats, rats, cattle, and killer whales.²⁸⁶

Table 66–2.Clinical Disorders of Neutrophil Function

Table 66–2. Clinical Disorders of Neutrophil Function

Disorder

Etiology

Impaired Function

Clinical Consequence

DEGRANULATION ABNORMALITIES

Chédiak-Higashi syndrome

Autosomal recessive; disordered coalescence of lysosomal granules; responsible gene is CHSI/LYST which encodes a protein hypothesized to regulate granule fusion

Decreased neutrophil chemotaxis; degranulation and bactericidal activity; platelet storage pool defect; impaired NK function, failure to disperse melanosomes

Neutropenia; recurrent pyogenic infections, propensity to develop marked hepatosplenomegaly as a manifestation of the hemophagocytic syndrome

Specific granule deficiency

Autosomal recessive; functional loss of myeloid transcription factor arising from a mutation or arising from reduced expression of Gfi-1 or C/EBPε, which regulates specific granule formation

Impaired chemotaxis and bactericidal activity; bilobed nuclei in neutrophils; defensins, gelatinase, collagenase, vitamin B₁₂-binding protein, and lactoferrin

Recurrent deep-seated abscesses

ADHESION ABNORMALITIES

Leukocyte adhesion deficiency I

Autosomal recessive; absence of CD11/CD18 surface adhesive glycoproteins (β₂ integrins) on leukocyte membranes most commonly arising from failure to express CD18 mRNA

Decreased binding of C3bi to neutrophils and impaired adhesion to ICAM-1 and ICAM-2

Neutrophilia; recurrent bacterial infection associated with a lack of pus formation

Leukocyte adhesion deficiency II

Autosomal recessive; loss of fucosylation of ligands for selectins and other glycol conjugates arising from mutations of the GDP-fucose transporter

Decreased adhesion to activated endothelium expressing ELAM

Neutrophilia; recurrent bacterial infection without pus

Leukocyte adhesion deficiency III (LAD-1 variant syndrome)

Autosomal recessive; impaired integrin function arising from mutations of FERMT3 which encodes kindlin-3 in hematopoietic cells; kindlin-3 binds to β-integrin and thereby transmits integrin activation

Impaired neutrophil adhesion and platelet activation

Recurrent infections, neutropenia, bleeding tendency

DISORDERS OF CELL MOTILITY

Enhanced motile responses; FMF

Autosomal recessive gene responsible for FMF on chromosome 16, which encodes for a protein called “pyrin”; pyrin regulates caspase-1 and thereby IL-1β secretion; mutated pyrin may lead to heightened sensitivity to endotoxin, excessive IL-1β production, and impaired monocyte apoptosis

Excessive accumulation of neutrophils at inflamed sites which may be the result of excessive IL-1β production

Recurrent fever, peritonitis, pleuritis, arthritis, and amyloidosis

DEPRESSED MOTILE RESPONSES

Defects in the generation of chemotactic signals

IgG deficiencies; C3 and properdin deficiency can arise from genetic or acquired abnormalities; mannose-binding protein deficiency predominantly in neonates

Deficiency of serum chemotaxis and opsonic activities

Recurrent pyogenic infections

Intrinsic defects of the neutrophil, e.g., leukocyte adhesion deficiency, Chédiak-Higashi syndrome, specific granule deficiency, neutrophil actin dysfunction, neonatal neutrophils; direct inhibition of neutrophil mobility, e.g., drugs

In the neonatal neutrophil there is diminished ability to express β₂ integrins and there is a qualitative impairment in β₂-integrin function; ethanol, glucocorticoids, cyclic AMP

Diminished chemotaxis; impaired locomotion and ingestion; impaired adherence

Propensity to develop pyogenic infections; possible cause for frequent infections; neutrophilia seen with epinephrine arises from cyclic AMP release from endothelium

Immune complexes

Bind to Fc receptors on neutrophils in patients with rheumatoid arthritis, systemic lupus erythematosus, and other inflammatory states

Impaired chemotaxis

Recurrent pyogenic infections

Hyperimmunoglobulin-E syndrome

Autosomal dominant; responsible gene is STAT3

Impaired chemotaxis at times; impaired regulation of cytokine production

Recurrent skin and sinopulmonary infections, eczema, mucocutaneous candidiasis, eosinophilia, retained primary teeth, minimal trauma fractures, scoliosis, and characteristic facies

Hyperimmunoglobulin-E syndrome

Autosomal recessive; more than one gene likely contributes to its etiology

High IgE levels, impaired lymphocyte activation to staphylococcal antigens

Recurrent pneumonia without pneumatoceles sepsis, enzyme, boils, mucocutaneous candidiasis, neurologic symptoms, eosinophilia

MICROBICIDAL ACTIVITY

Chronic granulomatous disease

X-linked and autosomal recessive; failure to express functional gp91^phox in the phagocyte membrane in p22^phox (autosomal recessive); other autosomal recessive forms of CGD arise from failure to express protein p47^phox or p67^phox

Failure to activate neutrophil respiratory burst leading to failure to kill catalase-positive microbes

Recurrent pyogenic infections with catalase-positive microorganisms

G6PD deficiency

Less than 5% of normal activity of G6PD

Failure to activate NADPH-dependent oxidase, and hemolytic anemia

Infections with catalase-positive microorganisms

Myeloperoxidase deficiency

Autosomal recessive; failure to process modified precursor protein arising from missense mutation

H₂O₂-dependent antimicrobial activity not potentiated by myeloperoxidase

None

Rac-2 deficiency

Autosomal dominant; dominant negative inhibition by mutant protein of Rac-2–mediated functions

Failure of membrane receptor–mediated O₂ generation and chemotaxis

Neutrophilia, recurrent bacterial infections

Deficiencies of glutathione reductase and glutathione synthetase

Autosomal recessive; failure to detoxify H₂O₂

Excessive formation of H₂O₂

Minimal problems with recurrent pyogenic infections

AMP, adenosine monophosphate; C, complement; CD, cluster designation; CGD, chronic granulomatous disease; ELAM, endothelial-leukocyte adhesion molecule; FMF, familial Mediterranean fever; G6PD, glucose-6-phosphate dehydrogenase; GDP, glucose diphosphate; ICAM, intracellular adhesion molecule; Ig, immunoglobulin; IL, interleukin; LAD, leukocyte adhesion deficiency; NADPH, nicotinamide adenine dinucleotide phosphate; NK, natural killer.

Data from Remington JS Swartz MN: Current Clinical Topics in Infectious Disease, 6th ed. New York, NY: McGraw-Hill; 1985.

Although CHS carries the names of Moises Chédiak and Ototaka Higashi, the disorder was first described by Béguez César, a Cuban pediatrician in 1943. Initially characterized by neutropenia and abnormal granules in leukocytes, the syndrome was further delineated in 1948 by Steinbrinck’s description of a second case.²⁸⁷ In 1952, Chédiak reported the hematologic characteristics of the disorder,²⁸⁸ and in 1953 Higashi emphasized the giant peroxidase-containing granules within patients’ neutrophils.²⁸⁹ Besides the susceptibility to infections, patients often suffer a fatal lymphohistiocytic infiltration known as the accelerated phase occurring months from birth to several years later.²⁹⁰

Epidemiology By 2008, 300 cases worldwide had been described, with concentrations in the United States, Japan, northern Europe, and Latin America.²⁸⁶ Patients of African descent have also been described.

Etiology and Pathogenesis CHS is caused by a fundamental defect in granule morphogenesis that results in abnormally large granules in multiple tissues.^282,291 Giant granules are seen in Schwann cells, leukocytes, and macrophages of the liver and spleen, and certain cells of the pancreas, gastric mucosa, kidney, adrenal gland, and pituitary gland.²⁸⁶ Giant melanosomes form and prevent the even distribution of melanin, which results in pigmentary dilution of the hair, skin, iris, and optic fundus. Although the giant lysosomes are the primary morphologic feature of the disorder, only cells relying on the secretion by these lysosomes manifest pathologic defects. In the early stages of myelopoiesis some of the normal-size azurophil granules coalesce to form giant granules that result in large secondary lysosomes that contain reduced content of hydrolytic enzymes, including proteinases, elastase, and cathepsin G.²⁸¹ Many of the myeloid precursors die in the marrow, resulting in a moderate neutropenia, with white cell counts of about 2.5 × 10⁹/L and absolute neutrophil counts ranging from 0.5 to 2.0 × 10⁹/L.²⁹⁰ The marrow itself appears normal to hypercellular. In spite of the normal ingestion of particles and active oxygen metabolism, these neutrophils kill microorganisms relatively slowly. This delay reflects a slow and inconsistent delivery of diluted amounts of hydrolytic enzymes from the giant granules into the phagosomes, which may predispose the host to bacterial infection.^291,292 In this syndrome, monocytes have the same functional derangements as neutrophils,²⁸¹ and in an analogous fashion perforin-deficient natural killer (NK) cells show profoundly impaired cytotoxic activity and are unable to kill many targets.²⁹³

The CHS blood cell membranes are more fluid than cells of normal individuals,^281,294 and the altered membrane structure could lead to defective regulation of membrane activation, as well as promoting fusion of neutrophil azurophilic granules with each other. Conceivably, changes in membrane fluidity may affect cell function by reducing expression of Mac-1 (CD11b/CD18). The altered membrane fluidity could result in elevated levels of intracellular cyclic adenosine monophosphate, which appears in this disorder and is reflected in the reduced chemotactic responses.²⁸¹

The gene that is mutated in CHS is CHS1 (syn. LYST) found on chromosome 1q. Its size indicates a protein of more than 400 kDa is encoded.²⁹⁵ During early development, granule biogenesis is normal; with perforin in NK cells and granule enzymes in myeloid cells synthesized and routed correctly to the granules. However, once formed the granules fuse to form giant organelles.²⁹⁶ Several studies led to the suggestion that the enlarged lysosomes found in CHS cells are the results of abnormalities in membrane fusion, which could occur during the biogenesis of the lysosomes. It has been hypothesized that this CHS1 protein interacts with attachment proteins on lysosomes (v-SNAREs) and that this mutated protein leads to indiscriminate interactions with v-SNARE to yield uncontrolled fusion of lysosomes with each other.²⁹⁷

Clinical Features Characteristically patients with CHS have light skin and silvery hair. They frequently complain of solar sensitivity and photophobia. Other eye findings can include horizontal or rotatory nystagmus. Infections are common and involve the mucous membranes, skin, and respiratory tract. They are susceptible to both Gram-positive and Gram-negative bacteria, as well as fungi, with Staphylococcus aureus being the most common infecting organism.²⁶⁶ Attenuated NK function probably contributes to the increased susceptibility to infection as well. Neurologic signs and symptoms are variable in CHS and may include a peripheral and cranial neuropathy, autonomic dysfunction, weakness, and sensory deficit; and ataxia may also be a prominent feature.

Patients with CHS have prolonged bleeding times with normal platelet counts, resulting from impaired platelet aggregation associated with a deficiency of the storage pools of ADP and serotonin.²⁸⁴ Electron micrographs reveal normal numbers of α granules in platelets, but decreased numbers of platelet dense bodies.²⁸⁶

The accelerated phase of CHS is characterized by lymphocytic proliferation in the liver, spleen, marrow, and central nervous system. The accelerated phase may occur at any age and is now recognized as a genetic form of hemophagocytic lymphohistiocytosis (HLH).²⁹⁸ Typically, the patient develops hepatosplenomegaly and high fever in the absence of bacterial sepsis. The pancytopenia becomes worse at this stage, producing hemorrhage and an increased susceptibility to infection. The onset of the accelerated phase may be related to the inability of these patients to contain and control the Epstein-Barr virus (EBV) leading to HLH (Chap. 70). The lymphocyte expansion into the tissue is associated with excessive cytokine production and massive tissue necrosis and organ failure leading to the propensity to recurrent bacterial and viral infections, fever, and prostration usually resulting in death.²⁹⁸ At autopsy, the lymphohistiocytic infiltrates in the liver, spleen, and lymph nodes are extensive, but not neoplastic by histopathologic criteria.²⁹⁸

Laboratory Features The laboratory test diagnostic for CHS is examination of granular cell morphology. The pathognomonic feature is giant peroxidase-positive granules that can be seen in neutrophils.²⁸⁹ A microscopic examination of hair shafts reveal large, speckled pigment clumps as opposed to the normal pattern of finally divided pigment of melanin spread along the length of the shaft.²⁸⁶ Similar giant granules can occasionally be present in CML and acute myelocytic leukemia.²⁸⁶ Molecular diagnosis of CHS remains difficult and is not commercially available. Heterozygotes for CHS are considered completely normal and cannot be detected clinically or biochemically.

Differential Diagnosis The diagnosis of CHS should be considered in individuals with partial albinism, exaggerated bleeding, and recurrent infections. Patients with CHS must be distinguished from those patients with Griscelli syndrome (GS) and Hermansky-Pudlak syndrome (HPS).

GS is a rare disorder, arising from mutations in the RAB27A gene, and is defined by partial ocular and cutaneous albinism, variable cellular and humeral immunodeficiency, variable neurologic involvement, and the development of the accelerated phase. Individuals with GS lack giant granules in neutrophils and have large pigment clumps in hair shafts.²⁸⁶ HPS is a disorder of ocular and cutaneous albinism, bleeding diathesis arising from platelet dysfunction, and deposition of ceroid lipofuscin in various organs (Chap. 120). In contrast to CHS, HPS cells lack giant granules and the patients are not predisposed to recurrent infections.²⁸⁶

Therapy High-dose ascorbic acid (200 mg/day for infants, 2 g/day for adults) improves the clinical status of some patients with CHS in the stable phase.²⁸¹ Although there is controversy regarding the efficacy of ascorbic acid, given the safety of the vitamin,²⁸⁶ it is reasonable to administer it to all patients. CHS presents a therapeutic dilemma, particularly when the accelerated phase begins. Prophylactic antibiotics do not prevent infections. The only potential for curative therapy for preventing the accelerated phase is marrow transplantation.²⁹⁹ Marrow transplantation reconstitutes normal hematopoietic and immunologic function and corrects the NK deficiency in patients before entering the accelerated phase.²⁹⁹ On the other hand, if the patient is actively in the accelerated phase, stem cell transplantation from a matched unrelated donor is associated with a poor prognosis.²⁹⁹ Ocular and cutaneous albinism are not corrected after transplantation, nor does transplantation prevent progressive neuropathies from occurring.³⁰⁰

Specific Granule Deficiency Specific granule deficiency (SGD) has been described in patients of both sexes and is inherited as an autosomal recessive disorder (see Table 66–2).²⁸¹ Besides the absence of specific granules, the nuclei of the neutrophils are bilobed. Patients are afflicted with recurrent infections primarily involving the skin and lungs. S. aureus and Pseudomonas aeruginosa have been the most commonly observed pathogens, although Candida albicans also has been isolated. Specific granule-deficient neutrophils lack gelatinolytic activity in the tertiary granules; vitamin B₁₂-binding protein, lactoferrin, hCAP-18, and collagenase in the specific granules; and defensins in the primary granules.^301,302,303 This disorder also extends to eosinophils that lack the characteristic eosinophil granule proteins: major basic protein, eosinophilic cationic protein, and eosinophil-derived neurotoxin (Chap. 62).³⁰⁴ Thus, the disorder is a global defect in phagocytic granules rather than limited to specific granules, as suggested by its name. Neutrophils from these patients are defective in chemotaxis, possibly related to the absence of the intracellular pool of leukocyte adhesion molecules that normally reside in the tertiary and specific granules, and exhibit a mild defect in bactericidal activity, possibly related to the deficiency of the granule constituents, lactoferrin and defensins.^301,305 The impairment in granule protein synthesis affecting the granulocytic cells appears secondary to the functional loss of the myeloid transcription factor, C/EBPε, which was identified in two patients.^109,306 In another case of SGD, the expression of the transcription factor growth factor independence-1 (Gfi-1) was markedly reduced along with a heterozygous mutation of C/EBPε gene.³⁰⁷ It was suggested that the combined abnormalities blocked specific granule expression leading to the expression of the SGD phenotype. The defect is restricted to blood cells, as normal lactoferrin secretion has been demonstrated in the nasal secretions of an SGD patient despite the abnormality demonstrated in his neutrophils.³⁰² The diagnosis of SGD is suggested by the presence of neutrophils devoid of specific granules but containing azurophilic granules on the blood film.²⁸¹ The diagnosis can be confirmed by demonstrating a severe deficiency in either lactoferrin or hCAP-18. An acquired form of SGD can be observed in thermally injured patients or in individuals with myelodysplasia.^281,308 Treatment of SGD is symptomatic, with the administration of parenteral antibiotics for acute infections and surgical drainage of refractory infections. With aggressive medical management, patients may survive into their adult years.

Adhesion Abnormalities

Leukocyte Adhesion Deficiency

Definition and History Leukocyte adhesion deficiency type I (LAD-1) is a rare autosomal recessive disorder of leukocyte function (see Table 66–2). More than 100 cases have been reported worldwide. The disease is characterized clinically by recurrent soft-tissue infections, delayed wound healing, and severely impaired pus formation despite striking blood neutrophilia.³⁰⁹ Individuals with this disorder have decreased or absent expression of a family of structurally and functionally related leukocyte surface glycoproteins designated CD11/CD18 complex (also referred to as the β₂-integrin family of leukocyte adhesive proteins; Table 66–3). These proteins include LFA-1 (CD11a/CD18), Mo-1 or Mac-1 (CD11b/CD18), p150,95 (CD11c/CD18), and p160,95 (CD11d/CD18).³⁰⁹ The CD11 subunits are integral membrane glycoproteins, each spanning the plasma membrane only once. They are approximately 40 percent homologous, suggesting that they arise from a common primordial gene.³⁰⁹ The three distinct genes encoding the α subunits occur in a cluster on chromosome 16, whereas the gene for the β subunit is located on chromosome 21.³¹⁰

Table 66–3.Biologic and Clinical Features of Leukocyte Adherence Deficiencies 1 and 2

Table 66–3. Biologic and Clinical Features of Leukocyte Adherence Deficiencies 1 and 2

Genetic Defect

Leukocyte Functional Abnormalities

Clinical Features

Diagnosis

LAD-1

Molecular mutations affecting expression of the β₂-integrin CD18

Neutrophils; adherence spreading, homotypic aggregation, chemotaxis receptor CR3 activities: C3bi binding affecting phagocytosis, respiratory burst, and degranulation in response to C3bi-coated particles^*

Autosomal recessive; delayed umbilical cord separation; neutrophilia; defective neutrophil migration into tissue; recurrent bacterial infections; impaired wound healing

Flow cytometry for expression of CD11b/CD18 (Mac-1)

Monocytes; adherence, CR3 activities

Lymphocytes; cytotoxic

T-lymphocyte activities; NK cytotoxic activities; blastogenesis

LAD-2 (CDG-IIc)

Mutations affecting function of GDP-fucose transporter 1 resulting in defective glycosylation expression at the α_1,3-position of selectin ligands including sLe^x and other fucosylated proteins requiring fucosylation

Neutrophils; rolling mediated by sLe^x to endothelium; neutrophilia^†

Autosomal recessive; recurrent bacterial infections, periodontitis; growth retardation; developmental retardation; Bombay red cell phenotype

Flow cytometry for leukocyte sLe^x (CD15)

CDG-11c, congenital disorder of glycosylation type IIc; GDP, glucose diphosphate; NK, natural killer; sLe^x, sialyl Lewis X.

^*These functional abnormalities and clinical features are a consequence of lack of the CD11b/CD18, which includes CD11a, CD11b, CD11c, and CD11d markers of four different α chains and the common β₂-chain CD18 of Mr 95 kDa.

^†These functional abnormalities and clinical features are a consequence of lack of sLe^x expression on leukocytes.

The initial clinical description in 1979 described six children and two families with findings of delayed separation of the umbilical cord and delayed healing at the site of detachment of the cord, recurrent infections despite neutrophilia, neutrophilia persisting during infection-free periods, and impaired neutrophil chemotaxis.³¹¹ The molecular basis for LAD-1 was first suggested when neutrophils from a patient with the disorder was found to lack a high-molecular-weight membrane glycoprotein.³¹² This finding suggested that the lack of the membrane protein impaired the neutrophil’s functional responses. In 1982, another patient was evaluated and it was confirmed that the membrane glycoprotein with a Mr of 150 kDa was missing.³¹³ The normal parents and siblings of the proband exhibited intermediate quantities of the glycoprotein, which suggested the existence of a heterozygous carrier state. The disorder then became known as LAD. Subsequently, in 1984, a glycoprotein 150 was identified as one subunit of a glycoprotein that had two subunits that served as a receptor for a plasma complement component.³¹⁴ This was followed by other investigations that found that two other related leukocyte membrane glycoproteins also were deficient. Each of the three glycoproteins was then determined to be heterodimers with one common subunit and one subunit unique to each glycoprotein.³¹⁵ Synthesis of a defective subunit common to the three glycoproteins of CD11/CD18 complex resulted in loss of expression of all heterodimers.³¹⁶ This observation provided the molecular explanation for the cellular defect. In 1985, the extent of clinical severity and magnitude of the cellular abnormalities were correlated with the degree of CD11/CD18 deficiency, thereby laying the groundwork for the direct relationship between the glycoprotein deficiency and the clinical presentation.³¹⁵

Etiology and Pathogenesis Each of these molecules contains an α and a β subunit noncovalently associated in an αβ structure. They all have the same β subunit and are distinguished by their α subunits, which have different isoelectric points, molecular weights, and cell distribution (see Table 66–3).³¹⁵ The structure of CD11/CD18 has been deduced from molecular cloning of the various subunits.³¹⁵ The x-ray crystal structure and nuclear magnetic resonance analysis also reveal that activation signals lead to the separation of the α and β subunit cytoplasmic tails, thereby converting the bent conformation of each integrin with its headpiece near the plasma membrane into fully extended high-affinity structures in a switchblade-like movement.³¹⁷ These studies establish that the CD11/CD18 heterodimers are members of a large gene family involved in cell–cell and cell–matrix adhesion (integrins). Several subfamilies of integrins are described and classified according to the type of their highly homologous β subunits. The α subunits are also homologous to each other, but to a lesser degree than are the associated β subunits. Within each subfamily, a single β subunit usually is shared by several α subunits. Certain α subunits often share more than one β subunit, which alters their specificity for various ligands.³¹⁵ The molecular defect involves all four members of the CD11 integrin subfamily. In patients with LAD-1 who have been evaluated at the molecular level, absent, diminished, or structurally abnormal β subunits (CD18) were identified.³¹⁵ A heterogeneous group of mutations that are confined to the gene on chromosome 21q22.3 also was identified.³¹⁵ Many patients have point mutations that result in single amino acid substitutions in CD18, which predominantly reside between amino acids 111 and 361.³¹⁵ This peptide domain is highly conserved among all β subunits and appears to be important for interaction with the α subunit. Several affected individuals are compound heterozygotes for two different mutant alleles, whereas others are homozygotes for a single mutant allele. Messenger RNA splicing abnormalities that have been described in two kindreds can result in either deletion or insertion of amino acids in the conserved extracellular domain of CD18. Small deletions within the coding sequences of the CD18 gene disrupting the reading frame or a nucleotide substitution resulting in a premature termination signal has been described. Mutations in CD18 disrupt the association in the αβ subunits so that maturation, intracellular transport, and all cell surface assembly of functionally active αβ molecules fail to occur.³¹⁵ Approximately half of patients exhibit a low level of CD11/CD18 cell surface molecules and moderate disease, with the remainder having totally absent surface expression of these proteins, which accounts for a profound impairment of neutrophil and monocyte adherence and adhesion-dependent functions in vitro, including cell migration, phagocytosis, and complement- or antibody-dependent cytotoxicity.^315,316

The bulk of the neutrophil Mac-1 glycoprotein is stored inside the cell in the membrane of neutrophil specific and gelatinase granules and in secretory vesicles.^32,318 Exposure of neutrophils to degranulating stimuli results in a 5- to 10-fold increase in the number of Mac-1 molecules on the cell surface, which parallels the fusion of granules to the plasma membrane.³¹⁸ Neutrophils from these patients fail to augment their surface adhesive glycoproteins, as the defect in β-subunit synthesis affects both membrane and granule pools of Mac-1.³¹⁹ In contrast to Mac-1 and p150,95, LFA-1 is predominantly confined to the neutrophil plasma membrane. Consequently, the cell surface levels of LFA-1 are not enhanced by neutrophil degranulation.

Lymphocytes deficient in CD11/CD18 are able to adhere to endothelial surfaces via the expression on lymphocytes of very-late antigen-4 (VLA-4) integrin (synonym: integrin α₄β₁) receptors, which bind to the vascular cell adhesion molecule 1 (VCAM-1), found on the endothelial cells.³²⁰ This residual adhesion may account for the paucity of clinical symptoms related to lymphocyte function. The patients are not unusually susceptible to viral infection, although three patients had one or more episodes of aseptic meningitis.³¹⁵

The failure of the LAD-1 neutrophils to migrate to the sites of inflammation outside of the lung and peritoneum arises from their inability to adhere firmly to surfaces and undergo transendothelial migration from venules.^321,322,323 Failure of Mac-1–deficient neutrophils to undergo transendothelial migration occurs because β₂ integrins bind to ICAM-1 (CD54) and ICAM-2 expressed on inflamed endothelial cells.^309,324 LAD-1 neutrophils are able to accumulate in the lung, perhaps through a process of movement mediated by “chimneying,” which does not require functional integrins.³²⁵ Chemotaxis that occurs despite blockade of CD11/CD18 under special in vitro conditions has been dubbed “chimneying.” The neutrophils that do arrive at inflammatory sites in the inflamed lung by CD11/CD18-independent processes fail to recognize microorganisms coated with the opsonic complement fragment C3bi (an important stable opsonin formed by the cleavage of C3b by C3b inactivator).^313,326 Other neutrophil functions, such as degranulation and oxidative metabolism, normally triggered by C3bi binding are also diminished and markedly compromised in neutrophils from LAD-1.³⁰⁹ Similarly, the urokinase-plasminogen activator-receptor and the FcγRIII receptors, both phosphatidylinositol-linked proteins, are defective in their functions because these receptors transduce their signals through CD11/CD18.^234,327 Monocyte function is also impaired. Monocytes of affected individuals have poor fibrinogen-binding function, an activity promoted by the CD11/CD18 complex^309,328; consequently, such cells are not able to participate effectively in wound healing. Thus, impairment in neutrophil function underlies the propensity to recurrent infections, which is the clinical expression of this disease. Similar genetic syndromes have been discovered in Irish Setter dogs and Holstein cattle.³¹⁵ A CD11/CD18-deficient mouse with 2 to 6 percent of normal β₂-integrin expression has been produced by gene targeting.^321,329

Clinical Features Activated leukocytes of patients with the most-severe clinical form express less than 0.3 percent of the normal amount of the β₂ integrins, whereas those of patients with the moderate phenotype may express 2 to 7 percent of normal numbers of β₂-integrin molecules.³⁰⁹ The severely affected patients suffer from recurrent and chronic or even gangrenous soft-tissue infections (subcutaneous tissues or mucous membranes), generally by bacterial or fungal microorganisms such as S. aureus, Pseudomonas spp. and other Gram-negative enteric rods, or Candida spp. Patients with the moderate phenotype have fewer and less-severe infections. Infectious susceptibility and impaired wound healing are related to diminished or delayed infiltration of neutrophils and monocytes into extravascular inflammatory sites. In all patients surviving infancy, severe progressive generalized periodontitis is present. Individuals who are clinically well, but who are heterozygous carriers of LAD have been identified. Their stimulated neutrophils express approximately 50 percent of the normal amount of the Mac-1 α subunit and the common β subunit.³⁰⁹ The diagnosis of LAD-1 should be considered in infants with a paucity of neutrophils at sites of infection despite blood neutrophilia and have a history of delayed separation of the umbilical cord.

Laboratory Features The diagnosis is made most readily by flow cytometric measurement of surface CD11b in stimulated and unstimulated neutrophils using monoclonal antibodies directed against CD11b (Fig. 66–5). Assessment of neutrophil and monocyte adherence, aggregation, chemotaxis, C3bi-mediated phagocytosis, and cytotoxicity generally demonstrates striking abnormalities that are directly related to the molecular deficiency. Delayed-type hypersensitivity reactions are normal, and most individuals have normal specific antibody synthesis. The ability of lymphocytes to generate specific antibodies explains the self-limited course of varicella or viral respiratory infections. However, some patients have impaired T-lymphocyte–dependent antibody responses, for example, to repeat vaccination with tetanus toxoid, diphtheria toxoid, and polio virus.

Figure 66–5.

Specific diagnosis of CD11/CD18 glycoprotein deficiency by indirect immunofluorescence flow cytometric analysis. Blood neutrophils of a pediatric patient suspected of having CD11/CD18 glycoprotein deficiency and those of an abnormal individual were subjected to immunofluorescence staining for the expression of the CD11b, CD11a, CD11c, and CD18 epitope (crosshatched histograms) as compared with the background immunofluorescence staining by isotype-identical negative-control antibodies (open histograms). Neutrophils were either stained immediately after purification by Ficoll-Hypaque density centrifugation (unstimulated) or after exposure to calcium ionophore A23187 (1 mM) for 15 minutes at 37°C (A23187-stimulated). A23187 stimulation causes significant increase in CD11b and CD18 epitope staining (surface MO1 expression) by normal neutrophils as compared with unstimulated normal cells. A23187 stimulation also causes a small increase in the CD11b-epitope expression of patient cells (the CD11b crosshatched histogram becomes distinguishable from background staining after A23187 stimulation), suggesting that this patient has a “moderate” form of the disorder (capable of expressing small but detectable quantities of CD11/CD18 glycoproteins). Flow cytometric analysis was performed on a Coulter Electronics EPICS F C Flow Cytometer with a logarithmic amplifier. (Reproduced with permission from Todd R, Freyer DR: The CD11/CD18 leukocyte glycoprotein deficiency, Hematol Oncol Clin North Am 1988 Mar;2(1):13-31.)

Patients with LAD-1 usually have blood neutrophil counts of 15 to 60 × 10⁹/L. However, during infectious episodes, they commonly have neutrophil counts in excess of 100 × 10⁹/L and sometimes as high as 160 × 10⁹/L. Granulocytic hyperplasia is a feature of the marrow examination which may relate to excessive production of IL-17 and G-CSF as a result of decreased uptake of apoptotic neutrophils by tissue macrophages.^319,330 Despite elevated blood counts, there is a paucity of neutrophils in inflammatory skin windows and biopsies of infected tissues.

Differential Diagnosis Eight patients (four Arab, two Turkish, one Pakistani, one Brazilian) who had neutrophilia, recurrent bacterial infections, and an inability to form pus have been described.^13,331,332 The patients also had the Bombay blood phenotype (deficiency in H blood group integrins), severe mental retardation, unusual facial appearance, microcephaly, cortical atrophy, seizures, hypotonia, and short stature (see Table 66–2). Functionally, the neutrophils were unable to adhere to E-selectin or cytokine-activated endothelial cells and exhibited impaired chemotaxis and an inability to roll on postcapillary venules in vivo. The patients are now classified as having LAD-2 or congenital disorder of glycosylation type IIc (CDG-IIc).³³³ In contrast to LAD-1, the patients’ NK cell activity is normal. The LAD-2 neutrophils express normal levels of CD18 integrins, but are deficient in the carbohydrate structure sLe^x, which renders the cells unable to roll on activated endothelial cells expressing E-selectin (see Table 66–3). Thus, the neutrophils from the patients categorized as having a LAD-2 phenotype are unable to tether to inflamed venules, which is necessary for subsequent activation (Chap. 19). The LAD-2 can be explained by a congenital disorder of fucosylation of ligands for selectins and other glycoconjugates. Each of the three selectins binds with variable affinity to sialylated and fucosylated oligosaccharides, including sLe^x, which is present on multiple specific glycolipids and glycoproteins on leukocytes and activated endothelial cells.¹³ Neutrophils from LAD-2 subjects lack sLe^x, which leads to impaired neutrophil rolling on endothelial cells. Other fucosylated determinants, including the H, Lewis, and secretor blood group antigens, are lacking as well, suggesting a global defect in fucosylation. The diminished fucosylation arises from impaired transport of GDP-fucose from the cytoplasm to the Golgi lumen.³³¹ A human GDP-fucose transporter (GFTP) that localizes to the Golgi apparatus has been demonstrated to be defective secondary to distinct mutations in the SLC35C1 gene encoding the transporter.¹³ When fibroblasts and lymphoblastoid cells derived from a LAD-2 patient were grown in the presence millimolar concentrations of fucose, cell-surface fucosylation could be restored. Following this observation oral administration of L-fucose to two Turkish patients led to normalization of neutrophil counts and functional E- and P-selectin ligands on myeloid cells accompanied by abatement of fevers and infections.¹³ Two Arab patients, in contrast to the Turkish patients who have different mutations of the gene encoding the putative GFTP, did not respond to oral fucose.³³² A Brazilian LAD-2 patient, like the Turkish patients, initially benefited from oral fucose; but, following expression of sLe^x on the myeloid cells, the patient developed autoimmune neutropenia.³³⁴ The diagnosis of LAD-2 can be made by flow cytometry analysis of CD15s (sLe^x) expression.

LAD-3, also known as LAD-1 variant syndrome, compromises two major hallmarks: a moderate LAD-1–like syndrome and severe Glanzmann-like bleeding diathesis (Chap. 120). Four patients have been described in whom the inheritance appears to be autosomal recessive and is associated with functional defects of the leukocyte and platelet integrins arising from intracellular signaling.³³⁵ The disease initially presents in early childhood and consists of the inability to form pus at sites of microbial infections, as well as a severe bleeding tendency. The neutrophils from the patients display defective adhesion and chemotaxis and are unable to undergo the respiratory burst when triggered by unopsonized zymosan. The molecular basis for LAD-3 arises from mutations in FERMT3, which encodes kindlin-3 in hematopoietic cells. Kindlin-3 binds to regions of the β-integrin tails and constitutes an essential element for transition of integrins from the bent and inactive to the extended an active conformation.³³⁶ Marrow transplantation can be curative.

Another rare cause of neutrophilia and an inability to form pus was observed in a patient with a mutation in the Rac2 GTPase, which is discussed below. The neutrophils from the patient had defects in both adhesion and chemotaxis (see Table 66–2).

Therapy, Course, and Prognosis Treatment of LAD-1 is largely supportive.^309,315 Patients with a history of recurrent infections can be maintained on prophylactic trimethoprim-sulfamethoxazole. Marrow transplantation with human leukocyte antigen (HLA)-compatible siblings or parental donors has resulted in engraftment and restoration of neutrophil function and remains the treatment of choice for patients with a severe phenotype.³³⁷

The restoration of CD11/CD18 expression in CD34 peripheral stem cells from LAD-1 following transduction with a retrovirus bearing CD18 and induced to differentiate into neutrophils with growth factors indicates that LAD-1 is caused by a defective CD18 gene and provides a basis for somatic gene therapy, which was accomplished in a dog model.^338,339 Not only did the neutrophils express the integrins, but the cells demonstrated improvement in their functional responses, such as adhesion and the respiratory burst when challenged with ligands for CD11/CD18. These results indicate that ex vivo of the transfer gene for CD18 into LAD-1 CD34+ cells followed by reinfusion of the transfused cells may represent a therapeutic approach for LAD.

The severity of infectious complications correlates with the degree of β₂ deficiency. Patients with severe deficiency may die in infancy, and those surviving infancy have a susceptibility to severe, life-threatening, systemic infections. In patients with moderate deficiency, life-threatening infections are infrequent and survival relatively long.³¹⁹ LAD-1 can be diagnosed by prenatal screening.

Neutrophil Actin Dysfunction

These patients, like patients with LAD, have recurrent pyogenic infections from birth as a result of defective chemotactic and phagocytic response (see Table 66–2). In one patient, actin isolated from blood and neutrophils could not polymerize under conditions that fully polymerized the actin of neutrophils from normal individuals.³⁴⁰ Subsequent studies on the index patient’s family confirmed that partial actin dysfunction was present in the parents and one sister.³⁴¹ One of the parents was found to be a heterozygote for LAD, and the other was not, but further studies established that LAD was not generally associated with defective actin filament assembly.^342,343 The basis of the defective polymerization of actin in the index patient remains unknown, but this disorder of phagocytes is distinct from LAD.

Defective actin polymerization has been described in a 2-month-old infant with severe recurrent bacterial infections associated with impaired chemotaxis and phagocytic response.³⁴⁴ The patient’s neutrophils showed increased expression of CD11b, distinguishing the patient’s clinical problem from LAD-1. Morphologically, the neutrophils displayed thin, filamentous projections of membrane with an underlying abnormal cytoskeletal structure. Subsequently, a 47-kDa protein was purified that inhibited actin polymerization in vitro.³⁴⁵ Further biochemical studies revealed a markedly defective actin polymerization in the patient’s neutrophils along with a severe deficiency of an 89-kDa protein and an elevated level of the 47-kDa protein. The 47-kDa protein was identified as LSP-1 (lymphocyte-specific protein-1), which is an actin-binding protein present in normal neutrophils. Overexpression of LSP-1 resulted in bundling of actin in cells, leading to an abnormal cytoskeletal structure and motility defects.³⁴⁶ Neutrophils from the patient’s parents revealed a partial defect in actin polymerization accompanied by intermediate levels of LSP-1 and the 89-kDa protein. These observations suggest that the neutrophil actin dysfunction (NAD) known as NAD47/89 is an autosomal recessive disorder. Because actin dysfunction is lethal, treatment requires restoration of normal neutrophil function by marrow replacement from a normal donor. Marrow transplantation was successful.^347,344

DISORDERS OF NEUTROPHIL MOTILITY

Familial Mediterranean Fever

Definition and History Familial Mediterranean fever (FMF) is an autosomal recessive disease that primarily affects populations surrounding the Mediterranean basin. The disease is characterized by acute limited attacks of fever often accompanied by pleuritis, peritonitis, arthritis, pericarditis, inflammation of the tunica vaginalis of the testes, and erysipelas-like skin disease (see Table 66–2). The initial description occurred in 1908, identifying a Jewish girl who had episodic abdominal pain and fever.³⁴⁸ Subsequently additional cases were identified,³⁴⁹ but it took nearly a half century to establish this disorder as familial Mediterranean fever.³⁵⁰

Epidemiology More than 10,000 patients worldwide are affected with FMF. It occurs predominantly in Sephardic Jews, Arabs, Turks, Italians, and Armenians.³⁴⁸ The disorder can occur in other populations, but it is unusual. The frequency of the susceptibility gene varies widely; it is very high among Armenians (ratio of persons with the gene to those without it is 1:7) and Sephardic Jews (1:5 to 1:16), but is lower in Ashkenazi Jews (1:135).

Etiology and Pathogenesis The pathologic findings in FMF are those of nonspecific acute inflammation affecting serosal tissues such as the pleura, peritoneum, and synovium. Neutrophilic infiltration predominates in the affected tissues. Physical and emotional stress, menstruation, and a high-fat diet may trigger the attacks.³⁵¹

The gene responsible for FMF has been identified to be located on chromosome 16. It encodes for a 781-amino-acid protein called pyrin or marenostrin.³⁵² The gene (MEFV) is predominantly expressed in neutrophils, eosinophils, monocytes, dendritic cells, and synovial and peritoneal fibroblasts, and its expression is upregulated by IFN-γ and TNF, and by the process of myeloid differentiation itself.³⁵³ Nearly all the 50 mutations in the MEFV gene are missense changes, most of which are clustered in on exon 2 and 10.³⁵⁴ Founder effects in FMF have been established, and the two most common mutations, V726A and M694V, originated in common ancestors who lived about 2500 years ago in the Middle East.³⁵²

Pyrin plays a role in controlling the activity of inflammasomes (see “Neutrophil Surface Receptors”). PYRIN, one of the four domains of pyrin, bears homology to a number of proteins involved in apoptosis and in inflammation, and is similar to a member of the six-helix-bundle death-domain superfamily that includes death domains and death effector domains known as CARDs.³⁵⁵ The PYD appears to allow for the interaction of macromolecular complexes by PYRIN–PYRIN interactions. This interaction has led to the identification of pyrin’s ability to interact specifically with another PYRIN-domain protein termed apoptosis-associated speck-like protein with a CARD (ASC).³⁵⁶ Besides the aminoterminal PYD, ASC has a C-terminal CARD domain that allows binding to the CARD of procaspase-1 (IL-1β–converting enzyme), which results in procaspase-1 autoactivation.³⁵⁵ Activated caspase-1 then converts prointerleukin-1β to IL-1β, which is, in turn, secreted and interacts with the IL-1 receptor to mediate inflammation. It has been suggested that pyrin may act as an antiinflammatory molecule by inhibiting ASC-induced IL-1 processing, which, in turn, could be defective in FMF. This hypothesis is supported by observing increased IL-1 processing and heightened sensitivity to LPS and impaired apoptosis in peritoneal macrophages from pyrin knockout mice. The puzzle, however, remains as to why serosal tissues are the main targets of inflammation in FMF.

Clinical Features Febrile episodes in FMF may begin in infancy, but by age 20 years, 90 percent of patients have had their first attack. The duration and frequency of attacks may vary considerably, even in the same patient.³⁵¹ Acute attacks frequently last 24 to 48 hours and recur once or twice a month. In some patients, attacks may recur as frequently as several times a week, or as infrequently as once a year, and symptoms may persist as long as a week during individual episodes. Some patients experience spontaneous remission that persists for years, followed by recurrence of frequent attacks. Peritonitis caused by FMF may resemble an acute abdomen, thereby leading to potential uncertainties about the clinical management of the acute abdominal episode. Attacks of pleuritic pain occur in approximately 25 to 80 percent of patients. Symptoms of pleuritis may sometimes precede abdominal pain, and some patients experience pleuritic attacks without abdominal symptoms. Recurrent pericarditis is rare. The course of peritonitis in FMF is similar to attacks at other serosal sites; however, it tends to appear at a late stage of the disease. Mild arthralgia is a common feature of febrile attacks, and monoarticular or oligoarticular arthritis may occur. Arthritis usually affects large joints, the knees in particular, and effusions are common. As many as one-third of the patients experience transient erysipelas-like skin lesions that appear typically on the lower leg, ankle, or dorsum of the foot. These lesions are circumscribed, painful, erythematous areas of swelling, which usually subsides within 24 to 48 hours.

In approximately 25 percent of affected patients, a form of renal amyloidosis develops in which the amyloid derives from a normal serum protein called serum amyloid A (amyloidosis of the AA type; Chap. 108). The amyloidosis progresses over a period of years to renal failure in almost all cases, and the cause of death in patients with FMF is usually attributed to this complication. It appears that polymorphisms in the gene for serum amyloid A increase the susceptibility to renal amyloidosis and that polymorphisms in a gene for the major histocompatibility complex class 1 α-chain influence the severity of the disease.³⁵⁰

Laboratory Features Laboratory findings in FMF are nonspecific. Nonspecific findings include increases in inflammatory mediators such as amyloid A, fibrinogen, and C-reactive protein during febrile attacks.³⁵⁰ Proteinuria greater than 0.5 g of protein per 24 hours in patients with FMF may suggest amyloidosis.

The cloning of the FMF gene now allows a reliable diagnostic test. Five founder mutations account for 74 percent of FMF carrier chromosomes from typical populations known to harbor the disease.³⁵⁷ Carrier rates for FMF mutations may be as high as 1:3 in some populations, suggesting that the disease is often underdiagnosed. Some amino acids that cause human disease are often present in wild-type in primates.³⁵⁸

Differential Diagnosis The TNF receptor–associated periodic syndrome (TRAPS) was first described in 1982 in a large Irish family.³⁵⁹ The affected family members had recurrent fever with localized myalgia and painful erythema. Differentiating this disorder from FMF was its response to corticosteroids and the autosomal dominant inheritance of the disorder. Affected patients can have attacks that last for at least 1 or 2 days, but prolonged attacks lasting longer than a week are common. Localized pain and tightness in one muscle group and a migratory pattern of the symptoms are prominent features. The disorder may be associated with colicky abdominal pain, diarrhea or constipation, nausea, and or vomiting. Painful conjunctivitis, periorbital edema, or both are common as well as chest pain secondary to sterile pleuritis.³⁵⁰ During febrile attacks, painless skin lesions may develop on the trunk or extremities and may migrate distally. Missense mutations in the gene for the type-1 TNF 55-kDa cell membrane receptor, which is required for diagnosis, have been identified. Patients with TRAPS respond dramatically to high doses of oral prednisone (>20 mg). In time, however, the responses wane, requiring higher doses of corticosteroids. Standard doses of a p75:Fc fusion protein, etanercept, administrated subcutaneously twice weekly decreases the frequency, duration, and severity of attacks; thus, etanercept may provide a safer, more effective alternative then corticosteroids in controlling the disease.

Therapy Colchicine treatment is effective in FMF and may prevent the development of amyloidosis.³⁵¹ Prophylactic colchicine, 0.6 mg orally, two to three times a day, prevents or substantially reduces the acute attacks of FMF in most patients. Some patients can abort attack with intermittent doses of colchicine beginning at the onset of attacks (0.6 mg orally every hour for 4 hours, then every 2 hours for four doses, and then every 12 hours for 2 days). In general, patients who benefit from intermittent colchicine therapy are those who experience a recognizable prodrome before developing fever and clear-cut acute symptoms.

Course and Prognosis The prognosis for normal longevity for patients has been excellent since the recognition that colchicine is an effective treatment of this disease. Most patients can be maintained almost entirely symptom-free. However, if amyloidosis develops, it may be followed by the nephrotic syndrome or uremia. Unless the patient receives a renal transplant, the likelihood of eventual death from renal failure is high.

Other Disorders of Neutrophil Motility

The directed migration of neutrophils from the circulation to an inflammatory site is a consequence of chemotaxis and leads to the accumulation of an exudate. For normal chemotaxis to occur, a complex series of events must be coordinated. Chemotactic factors must be generated in sufficient quantities to establish a chemotactic gradient. The neutrophils must have receptors for the chemotactic agents and mechanisms for discerning the direction of the chemotactic gradient. Depressed neutrophil chemotaxis has been observed in a wide variety of clinical conditions (see Table 66–2).³⁶⁰ These can be stratified as follows: (1) defects in the generation of chemotactic signals; (2) intrinsic defects of the neutrophil; and (3) direct inhibitors of neutrophil motility in response to chemotactic factors.

Older patients with chemotactic disorders may be infected by a variety of microorganisms, including fungi and Gram-positive or Gram-negative bacteria. S. aureus is the most frequent bacterial offender. Typically, the skin, gingival mucosa, and regional lymph nodes are involved. Respiratory tract infections are frequent, but sepsis is rare. Delayed or inappropriate signs and symptoms of inflammation are common. Although the cells move slowly in Boyden chambers or other chemotactic assays, they do accumulate in sufficient numbers in inflammatory sites to produce pus. However, detection of patients with neutrophils that have profound defects in chemotaxis usually is accomplished through other phagocytic assays.

Patients with the hereditary deficiency of complement factors C3, C5, or properidin exhibit an increased incidence of bacterial infections because they are unable to form the chemotactic peptide C5a.³⁶¹ The degree to which defective chemotaxis plays a role in C3 deficiency is unclear because opsonization and ingestion rates also are abnormal in these disorders. Frequently, chemotactic disorders are associated with other impaired neutrophil functions. For instance, both glycogen storage disease type 1b³⁶² and Shwachman-Diamond syndrome³⁶³ are chemotactic disorders frequently associated with an absolute neutrophil count below 0.5 × 10⁹/L. Following restoration of a normal neutrophil count with G-CSF, the patients no longer are predisposed to recurrent bacterial infections in spite of a persistent chemotactic defect. Thus, a chemotactic defect observed in vitro does not correlate invariably with decreased resistance to bacterial infections in vivo.

Among the impaired defense mechanisms of the neonate is neutrophil adherence and chemotaxis, as demonstrated by the in vitro response of neonatal neutrophils to a variety of chemotactic factors.³²² The impaired motility of the neonatal neutrophils in part arises from the diminished ability to mobilize neutrophil β₂ integrins following neutrophil activation.³⁶⁴ Additionally, the neonatal neutrophil may have a qualitative defect in β₂-integrin function, resulting in impaired neutrophil transendothelial migration for up to 1 month after birth.

At the other end of the spectrum, neutrophils from elderly loose focus during chemotaxis while their motility is unimpaired. This is caused by increased activity of PI3-K and results in less efficient bacterial killing and enhanced release of tissue destructive proteases. Inhibition of PI3K activity reverts this condition in vitro.³⁶⁵

Drugs and Extrinsic Agents That Impair Neutrophil Motility

Although many pharmacologic agents can influence neutrophil function, few drugs used in clinical medicine affect neutrophil behavior in vivo. Ethanol, an inhibitor of PLD, in concentrations that occur in human blood can inhibit neutrophil locomotion and ingestion.³⁶⁶ Glucocorticoids, especially at high and sustained doses, inhibit neutrophil locomotion, ingestion, and degranulation.³⁶⁷ Administration of glucocorticoids on alternate days does not interfere with neutrophil movement.³⁶⁸ Epinephrine does not have a direct effect on neutrophil adhesion but cyclic adenosine monophosphate (cAMP), which is released from endothelial cells following exposure to epinephrine, can depress neutrophil adherence.³⁶⁹ Similarly, elevated cAMP levels following epinephrine administration may impair neutrophil adherence, leading to diminished neutrophil margination and apparent neutrophilia. Immune complexes, as seen in patients with rheumatoid arthritis or other autoimmune diseases, also can inhibit neutrophil movement by binding to neutrophil Fc receptors.

Hyperimmunoglobulin E Syndrome

Definition and History Autosomal dominant hyperimmunoglobulin E syndrome (HIES) is a disorder characterized by markedly elevated serum IgE levels, chronic dermatitis, and serious recurrent bacterial infections.³⁷⁰ The skin infections in these patients are remarkable for their absence of surrounding erythema, leading to the formation of “cold abscesses.” The neutrophils and monocytes from patients with this syndrome exhibit a variable, but at times profound, chemotactic defect that appears extrinsic to the neutrophil (see Table 66–2).³⁷¹

The syndrome was originally described in 1966 in two red-headed, fair-skinned females who had “cold abscesses” and hyperextensible joints, which led to the appellation “Job’s syndrome.”³⁷⁰ Subsequently Buckley and coworkers documented the association of levels of immunoglobulin E with undue susceptibility to infection.³⁷²

Epidemiology Reports of more than 200 cases have been documented.^372,373 HIES occurs in persons from diverse ethnic backgrounds and does not seem to be more common in any specific population.

Etiology and Pathogenesis Both males and females have been affected, as well as members of succeeding generations, indicating that the disorder is autosomal dominant with an incomplete penetrance form of inheritance.³⁷⁰ STAT3 mutations cause most, if not all cases of autosomal dominant HIES. All mutations have been missense mutations or in-frame deletions, leading to the formation of full-length mutant STAT3 protein, which exerts a dominant negative effect. STAT3 is a major transduction protein affecting pathways involving wound healing angiogenesis, immunity, and cancer. The more rare autosomal recessive form is caused by mutations in dedicator of cytokinesis 8 (DOCK8), a guanine nucleotide exchange factor.³⁷⁴

The mechanism of the immune deficiencies in HIES remains clouded. Several reports with limited numbers of patients have conflicted results as to whether a chemotactic defect exists and whether there is a T-helper 1/T-helper 2 cytokine imbalance.

Clinical Features HIES may begin as early as day 1 after birth.³⁷² The syndrome is characterized by chronic eczematoid rashes, which are typically papular and pruritic. The rash generally involves the face and extensor surfaces of arms and legs; skin lesions are frequently sharply demarcated and usually lack surrounding erythema. By 5 years of age all patients have had a history of recurrent skin abscess formation with recurrent pneumonias, along with chronic otitis media and sinusitis. Patients may also develop septic arthritis, cellulitis, or osteomyelitis. The major offending pathogen is generally S. aureus. Other pathogens commonly infecting patients are C. albicans, H. influenzae, and pneumococci. Other associated features include coarse facial features, including a prominent forehead, deep set eyes, a broad nasal bridge, a wide fleshly nasal tip, mild prognathism facial asymmetry, and hemihypertrophy.³⁷⁰ There is a high incidence of scoliosis, hyperextensible joints, and delayed shedding of the primary teeth.³⁷⁰ Occasionally, unexplained osteopenia presents, which is often complicated by recurrent bone fractures. Additionally, there is an increased risk of both Hodgkin and non-Hodgkin lymphoma.

Laboratory Features Blood and sputum eosinophilia have been a consistent finding in all patients.³⁷⁰ Patient serum IgE levels range from three to 80 times the upper limit of normal. The serum IgE usually rises above 2000 IU/mL and often is elevated at birth. Upon reaching adulthood the IgE may decline over years, despite the clinical abnormalities of STAT3 deficiency. Usually patients have normal concentrations of IgG, IgA, and IgM, and may have elevated levels of IgD. Patients often have abnormally low anamnestic antibody response and poor antibody and cell-mediated responses to neoantigens. At times the neutrophils and monocytes of patients have a profound chemotactic defect.

Differential Diagnosis Autosomal recessive-HIES (AR-HIES) is a distinct clinical entity manifested by elevated IgE ligands, and recurrent skin and cutaneous viral infections and mutations in DOCK8.^370,375

Fatal sepsis occurs in AR-HIES from both Gram-positive and Gram-negative bacteria. Patients with AR-HIES have more symptomatic neurologic disease than STAT3 deficiency. Autoimmune hemolytic anemia may occur, but neutrophil chemotaxis is normal. The genetic mutation underlying AR-HIES remain unclear. Therapy remains supportive.

Therapy No known therapy is curative, and management decisions are based on the clinical findings. Prophylactic trimethoprim-sulfamethoxazole is effective in reducing infections with S. aureus.³⁷⁰ Type and route of antibiotic therapy are dictated by the results of the Gram stain and culture in patients with acute bacterial infections. Incision and drainage are essential for the management of abscesses, including superinfected pneumatoceles. Eczematoid dermatitis can be controlled with topical glucocorticoids to reduce inflammation and antihistamines to control pruritus. Intravenous immunoglobulin may decrease the number of infections for some patients. Attention needs to be paid to the scoliosis, fractures and degenerative joints by orthopedists. Retention of primary teeth requires dental expertise.

Course and Prognosis If the hyperimmunoglobulin E is recognized early in life and the patient is maintained on chronic anti-Staphylococcal antibiotic therapy, the prognosis remains good. Many such patients have reached maturity, indicating that the syndrome is compatible with prolonged survival. Conversely, if the diagnosis is delayed and the patient develops infected giant pneumatoceles, secondary fungal infections may occur, leading to a morbid state.

DEFECTS IN MICROBICIDAL ACTIVITY

Chronic Granulomatous Disease

Definition and History CGD is a genetic disorder affecting the function of neutrophils and monocytes. These phagocytic cells are able to ingest, but not kill, catalase-positive microorganisms because of an inability to generate antimicrobial oxygen metabolites (see Table 66–2). It is caused by mutations involving one of several genes encoding a component of the NADPH oxidase.³⁷⁶

In 1957, two pediatric groups caring for six male infants reported a clinical disorder of chronic suppurative lymphadenitis and recurrent fevers leading to premature deaths in the children.^377,378 In the same time period, three observations assisted in providing the framework to understand the defect in the phagocytes of patients with CGD. Scientists described first that a striking increase in oxygen consumption was found upon particle ingestion by phagocytes, which was not related to mitochondrial oxygen metabolism.³⁷⁹ Next, it was found that the process of phagocytosis was accompanied by the formation of large quantities of H₂O₂ in the cell.³⁸⁰ Subsequently, it was reported that homogenates of phagocytes consume oxygen when incubated with pyridine nucleotides.³⁸¹ These observations indicated that an oxidase enzyme or enzymes in the phagocytes were activated during phagocytosis to convert molecular oxygen into H₂O₂. It was then established that phagocytes from patients with CGD could ingest, but could not kill, the catalase-positive organisms.³⁸¹ Building on previous studies that a neutrophil oxidase mediates the increase in oxygen consumption, a pyridine-dependent oxidase was found to be deficient in neutrophils of patients with CGD, which led to their inability to reduce the dye nitroblue tetrazolium (NBT) during phagocytosis of particles.³⁸² Collectively, these studies laid the groundwork for subsequent studies to unravel the biochemical and genetic defects in CGD.

Epidemiology The incidence of CGD in the United States is 1 per 200,000 livebirths, based on data from the National Institutes of Allergy and Infectious Disease Registry.³⁸³ Data from the Registry indicates that 86 percent of patients are male and 14 percent female; 80 percent are classified as white, 11 percent as black patients, and 3 percent Asians or mixed-race patients. Of the 340 patients in the Registry with adequate information for determination genetic transmission, 70 percent had the X-linked recessive form of the disease.

Etiology and Pathogenesis Several laboratory tests are used to classify forms of CGD and aid in understanding its pathogenesis (Table 66–4). The diagnosis of CGD is based on a compatible clinical history and demonstration of a defective respiratory burst. Several methods detect the production of reactive oxidants. The NBT method relies on the intracellular reduction of NBT by superoxide anion to a blue formazan precipitate that can be seen microscopically.³⁷⁶ More sensitive methods rely on the reaction of oxidants with specific chemiluminescent and fluorescent probes. The patients with CGD may have heterogeneous array of regular symptoms and severity, depending on which subunit is defective and on the nature of the genetic mutation.

Table 66–4.Diagnostic Classification of Chronic Granulomatous Disease

Table 66–4. Diagnostic Classification of Chronic Granulomatous Disease

Affected Component

Inheritance

Subtype

Membrane-Bound Cytochrome b₅₅₈^*

Cytosol p47^{phox^*}

Cytosol p67^{phox^*}

gp91^phox

X91⁰

Not detectable

Normal

X91⁺

Normal quantity, but nonfunctional

Normal

X91^–

Defective gp91^phox, which is poorly functional or expressed in a small fraction of phagocytes

Normal

p22^phox

A22⁰

Not detectable

Normal

A22⁺

Normal quantity, but nonfunctional

Normal

p47^phox

A47⁰

Normal quantity

Not detectable

Normal

p67^phox

A67⁰

Normal

Not detectable

^*Detected by spectral analysis or immunoblotting. In this nomenclature, the first letter represents the mode of inheritance (-linked [X] or autosomal recessive [A]). The number indicates the phox component, which is genetically affected. The superscript symbols indicate whether the level of protein of the affected component is undetectable (0), diminished (–), or normal (+) as measured by immunoblot or spectral analysis.

Nicotinamide Adenine Dinucleotide Phosphate-Oxidase Function Engulfment of microbes by phagocytic cells is associated with a burst of oxygen consumption that is important for microbicidal killing and digestion. The respiratory burst is accompanied, not by mitochondrial respiration, but by a unique electron transport chain called the NADPH oxidase. Prior to stimulation, the components of the oxidase are physically separated into two major subcellular locations (Fig. 66–6). The membrane-bound portion of the NADPH oxidase contains a heterodimeric cytochrome b₅₅₈ composed of a large, heavily glycosylated subunit with a Mr of 91 kDa, known as a gp91^phox (91-kDa glycoprotein of the phagocyte oxidase), and a 22-kDa protein known as p22^phox.^376,384 Eighty to 90 percent of the cytochrome b₅₅₈ is found in specific and gelatinase granules and secretory vesicles of the neutrophil and following neutrophil activation translocates to the plasma membrane.^66,318 The heavy chain of cytochrome b contains sites for heme binding, flavin adenine dinucleotide (FAD) groups, and NADPH binding.^{385,386,387,388} The three-dimensional structure of cytochrome b₅₅₈ indicates that the carboxyl-terminal half of the peptide contains sequences for flavin and NADPH binding.³⁸⁹ The amino half of the molecule is hydrophobic and contains the histidines that coordinate heme binding.³⁹⁰ The p22^phox also contains a site for heme binding.³⁸⁵ The synthesis of the p22^phox peptide is absolutely required for stability of gp91^phox and for oxidase activity in the membrane.³⁷⁶ The p22^phox also contains proline-rich regions that display consensus protein–protein interactions that provide a binding site for p47^phox.³⁹¹ Three other proteins vital to the function of this oxidase system reside in the cytosol of the resting phagocyte. Upon stimulation, translocation of p47^phox takes place. Phosphorylated p47^phox together with two other cytoplasmic components of the oxidase, p67^phox, and a low-molecular-weight guanosine triphosphate Rac-2, translocate to the membrane, where they interact with cytoplasmic domains of the transmembrane cytochrome b₅₅₈ to form the active oxidase.^391,392 Both p47^phox and p67^phox contain SH3 (Src homology 3) domains that may participate in intramolecular and intermolecular binding with consensus proline-rich regions in p47^phox.³⁹² Phosphorylation, which occurs on serines in the cationic C-terminal region of p47^phox, serves to disrupt this intermolecular interaction, making the SH3 regions available for binding to p22^phox. Another cytoplasmic component with homology to p47^phox has been identified as p40^phox. p40^phox, like p47^phox, contains a PX domain, a motif that supports the binding to phosphoinositides on the cytosolic side of membranes.³⁹³ The p40^phox component stabilizes the cytoplasmic complexes of p67^phox and p47^phox on phagosomes. Its binding of phosphatidylinositol 3 phosphate also potentiates superoxide production upon neutrophil activation.³⁹⁴ Cytochrome b₅₅₈ spans the membrane, permitting NADPH to be oxidized at the cytoplasmic surface and oxygen to be reduced to form O₂^– on the outer surface of the plasma membrane or on the inner surface of the phagosomal membrane.³⁹⁵

Figure 66–6.

Possible mechanisms for the production of superoxide anion in neutrophils. Oxygen is reduced to superoxide (O₂^–) by an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The oxidase is 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) a low-molecular-weight cytosolic G-protein, Rac2; and (5) a membrane-bound cytochrome b₅₅₈. Cytochrome b consists of a 22-kDa protein subunit (p22) and a 91-kDa glycoprotein subunit (gp91), both of which contain heme. The gp91 subunit is a flavin adenine dinucleotide (FAD)-dependent flavoprotein that contains the NADPH binding site and ultimately shuttles electrons to molecular oxygen, forming O₂^–, and (6) the cytosol components 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 (p47) is phosphorylated upon activation of the neutrophil. The p40^phox component stabilizes the preactivation complex of p67^phox. The unstable superoxide anion (O₂^–) is converted to hydrogen peroxide (H₂O₂), either spontaneously or by the enzyme superoxide dismutase (SOD). H₂O₂ in the presence of myeloperoxidase (MPO) converts H₂O₂ to hypochlorous acid (HOCl). Both H₂O₂ and O₂^– can be transformed into hydroxyl radical (OH^–). H₂O₂ can be reduced to H₂O and O₂ by the enzyme catalase or by glutathione (GSH), a product of the hexose-monophosphate shunt. These reactive oxygen species are responsible for microbial killing. Normal oxidative function of the NADPH complex requires fully functional individual components.

Genetic Alterations Affecting Cytochrome b The most frequent form of CGD occurs in 70 percent of patients and is caused by mutations in the gp91^phox gene, termed CYBB, which is located on chromosome Xp21.1.^376,396 These mutations lead to the X-linked form of the disease. Large interstitial deletions causing other X-linked disorders such as retinitis pigmentosa, Duchenne muscular dystrophy, McLeod hemolytic anemia, and ornithine transcarbamylase deficiency, have been reported in a few patients with X-linked CGD.^{383,397,398,399} Mutation analysis of the gene encoding gp91 and a large group of X-linked CGD kindreds has documented many distinct defects, including point mutations, inversions, deletions, or insertions that disrupt the reading frame and nonsense mutations that create a premature stop codon.³⁹⁶ Some splice-site defects have also been identified. In this situation, short deletions in gp91^phox mRNA are caused by point mutations that produce partial or complete exon skipping during mRNA splicing.⁴⁰⁰ This abnormality is a common cause of X-linked CGD. In the remaining patients, point mutations have been identified that generate either premature stop codons or amino acid substitutions that apparently disrupt protein stability or function and lead to a complete lack of detectable cytochrome b₅₅₈ protein in phagocytic cells in most patients with X-linked CGD. In some situations, low levels of functional cytochrome b are present, whereas in others, normal levels of dysfunctional cytochrome b₅₅₈ occur.⁴⁰¹ In the latter situation there is some clustering of defects in regions of known function, such as the NADPH- or flavin-binding consensus regions.⁴⁰² Approximately 10 to 15 percent of X-linked CGD arises from new germline mutations.⁴⁰³

A similar array of mutations has been identified in the 5 percent of CGD patients who have abnormalities in the p22^phox gene, termed CYBA, which is located on chromosome 16q24.^376,402,404 In this autosomal disorder, mutations in the p22^phox gene result in deletions, frameshifts, and/or missense mutations. Patients with a defective p22^phox gene do not express the other cytoplasmic unit polypeptide. In one patient, p22^phox peptide was associated with normal amounts of cytochrome b with normal heme spectrum, but p47^phox translocation membrane did not occur and there was no oxidase activation because the mutation affected a proline-rich region thought to mediate binding to one of the SH3 domains of p47^phox. In gp91^phox-deficient patients, p22^phox mRNA is present, but it is not translated, which is consistent with the notion that either cytochrome subunit polypeptide is dependent upon the stable expression of the other subunit.³⁷⁶

Genetic Alterations Affecting Cytosolic Proteins Two other proteins have been identified as being vital to the function of the NADPH-oxidase system. Their absence results in the syndrome of CGD.⁴⁰⁵ These proteins have molecular masses of 47 kDa and 67 kDa, respectively, and are located in the cytosol of resting cells. Defects in the genes for p47^phox, termed NCF1, which is found on chromosome 7q11, are responsible for the majority of all cases of autosomal recessive CGD, whereas inherited defects for the gene for neutrophil p67^phox, termed NCF2, account for a small subgroup of autosomal recessive CGD.³⁷⁶ The function of p47^phox and p67^phox in regulating the respiratory burst oxidase is thought to involve activation of the electron transport function of cytochrome b₅₅₈. The mutation analysis in patients with p47^phox-deficient forms of CGD reveals an unusual pattern, in that more than 90 percent of mutant alleles have guanine-thymine dinucleotide deletion at the start of exon 2, resulting in frameshift and premature stop.^402,406 The truncated protein is unstable in that it cannot be detected immunologically. The majority of patients appear to be homozygous for this mutation without any history of consanguinity. The p47^phox gene occurs in an area of chromosome 7 that has a high degree of evolutionary duplication in normal individuals because a pseudogene highly homologous to the normal p47^phox gene exists in the normal genome in this region of duplication. The pseudogene contains the same GT deletion associated with most cases of p47^phox CGD. This implies that recombination of the normal gene and pseudogene with conversion of the normal gene to partial pseudotype sequence in that region may be responsible for the high relative rate of this specific mutation in diverse racial groups, which proved to be the case.⁴⁰⁷

A second rare form of CGD is caused by mutations in the gene for the p67^phox cytosolic component.⁴⁰¹ The p67^phox gene, which has been mapped to the long arm of chromosome 1, spans 37 kb and contains 16 exons. The mutations identified in p67^phox-deficiency CGD have included missense mutations and spliced junction mutations affecting mRNA processing, which led to nondetectable p67^phox protein by immunologic means.⁴⁰²

Mutation of NCF4, the gene encoding p40^phox, was reported in a child with granulomatous colitis. One allele had a frameshift mutation with a premature stop codon. The other had a missense mutation predicting an R105Q substitution in the PX domain which is responsible for binding to phosphatidylinositol 3 phosphate. The functional defect was inability to assemble the NADPH oxidase in the membrane of phagosomes but not on the plasma mambrane.⁴⁰⁸

Predisposition to Infection Mutations in the gene for cytochrome b₅₅₈ or the cytosolic factors involved in activating the cytochrome are associated with the CGD phenotype. Figure 66–7 shows schematically the manner in which the metabolic deficiency of the CGD neutrophil predisposes the host to infection. Normal neutrophils accumulate H₂O₂ and other oxygen metabolites in the phagosomes containing ingested microorganisms. MPO is delivered to the phagosome by degranulation and in this setting H₂O₂ acts as a substrate for MPO to oxidize halide to HOCl and chloramines, which kill the microbes. The quantity of H₂O₂ produced by the normal neutrophils is sufficient to exceed the capacity of catalase, a H₂O₂-catabolizing enzyme produced by many aerobic microorganisms, including S. aureus, most Gram-negative enteric bacteria, C. albicans, and Aspergillus spp. In contrast, H₂O₂ is not produced by CGD neutrophils, and any generated by the microbes themselves may be destroyed by their own catalase. Thus, catalase-positive microbes can multiply inside CGD neutrophils, where they are protected from most circulating antibiotics, and can be transported to distant sites and released to establish new foci of infection.⁴⁰⁵ Activation of the oxidase also has a pronounced effect on the pH within the phagocytic vacuole. It is controversial whether activation of the respiratory burst is associated with an alkaline phase, but the pH of the phagocytic vacuole becomes more acidic in CGD patients than in normal patients.^161,409 The alkaline phase may be important for the antimicrobial and digestive functions of the neutral hydrolases released from the cytoplasmic granules into the vacuole upon phagocytosis. In CGD, the phagocytic vacuoles remain acidic and the bacteria are not digested properly.⁴¹⁰ The impairment in the respiratory burst by CGD neutrophils leads to delayed neutrophil apoptosis and subsequent impaired clearance of degenerating neutrophils by CGD macrophages, which, in turn, predisposes the host to enhanced inflammation.⁴¹¹ CGD neutrophils are incapable of generating NETs and cannot trap microorganisms by this mechanism.⁴¹² The CGD macrophage is unable to clear CGD neutrophils because of a deficiency of intrinsic IL-4 production, which occurs because of defective phosphatidylserine exposure on CGD neutrophils, that is a necessary requirement to engage CGD macrophage phosphatidylserine membrane receptors and subsequent macrophage activation.⁴¹¹ In hematoxylin-and-eosin-stained sections from patients, macrophages eventually may contain a golden pigment, which reflects the abnormal accumulation of ingested material and also contributes to the diffuse granulomata that give CGD its descriptive name.⁴¹³ On the other hand, when CGD neutrophils ingest pneumococci or streptococci, these organisms generate enough H₂O₂ to result in a microbicidal effect.

Figure 66–7.

The pathogenesis of chronic granulomatous disease (CGD). The manner in which the metabolic deficiency of the CGD neutrophil predisposes the host to infection is shown schematically. Normal neutrophils accumulate hydrogen peroxide (H₂O₂) in the phagosome containing ingested Escherichia coli. Myeloperoxidase is delivered to the phagosome by degranulation, as indicated by the closed circles, and in this setting, H₂O₂ acts as a substrate for myeloperoxidase to oxidize halide to hypochlorous acid and chloramines, which kill the microbes. The quantity of H₂O₂ produced by the normal neutrophils is sufficient to exceed the capacity of catalase, a H₂O₂-catabolizing enzyme of many aerobic microorganisms, including most Gram-negative enteric bacteria, Staphylococcus aureus, Candida albicans, and Aspergillus spp. When organisms such as E. coli gain entry into the CGD neutrophils, they are not exposed to H₂O₂ because the neutrophils do not produce it, and the H₂O₂ generated by microbes themselves is destroyed by their own catalase. When CGD neutrophils ingest streptococci (Strep.) or pneumococci, these organisms generate enough H₂O₂ to result in a microbicidal effect. On the other hand, as indicated in the middle figure, catalase-positive microbes, such as E. coli, can survive within the phagosome of the CGD neutrophil.

Clinical Features Although the clinical presentation is variable, several clinical features suggest the diagnosis of CGD.³⁷⁶ Any patient with recurrent lymphadenitis should be considered to have CGD. Additionally, patients with bacterial hepatic abscesses, osteomyelitis at multiple sites or in the small bones of the hands and feet, a family history of recurrent infections, or unusual catalase-positive microbial infections all require clinical evaluation for this disorder. Table 66–5 lists the most common clinical infections that afflict CGD patients and Table 66–6 cites their prevalence.

Table 66–5.Common Infecting Organisms Isolated from Chronic Granulomatous Disease Patients

Table 66–5. Common Infecting Organisms Isolated from Chronic Granulomatous Disease Patients

Infection Type

Organism

X-Linked Recessive (%)

Autosomal Recessive (%)

Pneumonia

Aspergillus spp.

Staphylococcus spp.

Burkholderia cepacia

Nocardia spp.

Serratia spp.

Abscess

Subcutaneous

Staphylococcus spp.

Serratia spp.

Aspergillus spp.

Liver

Staphylococcus spp.

Serratia spp.

Candida spp.

Lung

Aspergillus spp.

Perirectal

Staphylococcus spp.

Brain

Aspergillus spp.

Suppurative adenitis

Staphylococcus spp.

Serratia spp.

Candida spp.

Osteomyelitis

Serratia spp.

Aspergillus spp.

Bacteremia/fungemia

Salmonella spp.

Burkholderia cepacia

Candida spp.

Staphylococcus spp.

Data from Segal BH, Leto TL, Gallin JI, et al: Genetic, biochemical, and clinical features of chronic granulomatous disease, Medicine (Baltimore) 2000 May;79(3):170–200.

Table 66–6.Prevalence of Infectious Complication of Chronic Granulomatous Disease Patients

Table 66–6. Prevalence of Infectious Complication of Chronic Granulomatous Disease Patients

Infection Type

X-Linked Recessive (%)

Autosomal Recessive (%)

Pneumonia

Abscess (all)

Subcutaneous

Liver

Lung

Brain

Perirectal

Suppurative adenitis

Osteomyelitis

Bacteremia/fungemia

Cellulitis

Data from Segal BH, Leto TL, Gallin JI, et al: Genetic, biochemical, and clinical features of chronic granulomatous disease, Medicine (Baltimore) 2000 May;79(3):170–200.

Among the various infections, only perirectal abscess, suppurative adenitis, and bacteremia/fungemia differ significantly in prevalence in the X-linked recessive and autosomal recessive CGD patients.³⁸³ Each of these conditions was twice as common in the X-linked form.

The onset of clinical signs and symptoms may occur from early infancy to young adulthood. Although the majority of patients with CGD (76 percent) are diagnosed before the age of 5 years, approximately 10 percent are not diagnosed until the second decade of life, and on rare occasions, not until the third decade or later.³⁸³ The organisms infecting CGD patients have changed considerably from those initially reported between 1957 and 1976. Staphylococcus caused most of the infections in the initial cases; Klebsiella and E. coli were then the next most common pathogens. Now Aspergillus is the prominent organism causing pneumonia and is the leading cause of death in patients.³⁷⁶ Invasive aspergillosis can occur in the first few months of life in healthy infants as well as in those with CGD. Although aspergillosis is the most common infecting fungus in CGD, Candida and several other fungal strains have been invasive in this disorder. Burkholderia cepacia is another leading cause of death in patients with CGD. Serratia marcescens is the third leading organism that commonly infects patients with CGD. Infections are characterized by microabscesses and granuloma formation. The presence of pigmented histiocytes is helpful in establishing the diagnosis. Patients may suffer from the consequences of chronic infections including the anemia of chronic disease, lymphadenopathy, hepatosplenomegaly, chronic purulent dermatitis, restrictive lung disease, gingivitis, hydronephrosis, and gastroenteric narrowing.³⁸³ Patients with CGD are also at risk for developing colitis and chorioretinitis, and discoid lupus erythematosus.³⁸³

Several mothers of patients in whom X-linked inheritance was established had an illness resembling systemic lupus erythematosus.³⁸³ Both X-linked and autosomal recessive patients with CGD also have a similar disorder.⁴¹⁴ It may be that these mothers’ and patients’ cells are unable to clear immune complexes sufficiently, which is a characteristic feature of CGD cells in vitro.⁴¹⁵ Variant alleles of MBL and FcγRIIA especially in combination are associated with rheumatologic disorders in patients with CGD.⁴¹⁶

Laboratory Findings The defect in the respiratory burst is best determined by measuring superoxide or H₂O₂ production in response to both soluble and particulate stimuli.⁴¹⁷ A test that is being employed is the use of flow cytometry using dihydrorhodamine-123 fluorescence.⁴¹⁸ Dihydrorhodamine-123 fluorescence detects oxidant production because it increases fluorescence upon oxidation.⁴¹⁸ In most cases there is no detectable superoxide or H₂O₂ generation with either type of stimulus. In the variant form of CGD, however, superoxide may be produced at rates between 0.5 and 10 percent of control.⁴¹⁹

An alternative method for measuring respiratory burst activity is the NBT test. This assay is performed by microscopically assessing the ability of individual cells to reduce NBT to purple formazan crystals following stimulation. Commonly there is no NBT reduction with most forms of CGD. In some of the variant forms, however, a high percentage of cells may contain some formazan, a finding indicative of a greatly diminished respiratory burst in most of the neutrophils. This test also permits detection of the carrier state in X-linked CGD when as few as 5 to 10 percent of the cells are NBT-negative.⁴²⁰

Most sophisticated procedures can identify the molecular defect. Cytochrome b content can be measured in extracts of detergent-disrupted neutrophils by a spectrophotometric assay.⁴²⁰ Once the diagnosis of CGD is made, the genotype can be determined. A mosaic population of oxidation that has positive and negative neutrophils in a male patient’s mother and sister strongly suggests X-linked CGD. Lack of a mosaic pattern among female relatives does not rule out the X-linked mode of inheritance because the defect can arise spontaneously. Prenatal diagnosis of CGD is established by analysis of DNA from amniocytes or chorionic villus samples.

Differential Diagnosis Leukocytes from patients with CGD have normal glucose-6-phosphate dehydrogenase (G6PD) activity. However, a few individuals with apparent CGD have been described who have neutrophils that lack or are almost lacking in G6PD activity.^421,422 The erythrocytes of these patients also lack the enzyme, and the patients have chronic hemolysis. In the cases of severe neutrophil G6PD deficiency, an attenuated respiratory burst progressively decreases as a result of the depletion of intracellular NADPH, the primary substrate for the respiratory burst oxidase. CGD and G6PD deficiency can be distinguished from each other by the hemolytic anemia seen in the latter disorder and by the fact that erythrocyte G6PD activity is normal in CGD and markedly reduced in G6PD deficiency.⁴⁰¹ A variety of studies indicate that the small GTPase Rac-2 plays an essential role in activity of the NADPH and the actin cytoskeleton in human neutrophils.³⁸³ A toddler has been described as presenting with a perirectal abscess at 5 weeks of age. This patient subsequently had necrosis of the periumbilical skin and fascia, and his surgical wounds did not heal properly. Functionally his neutrophils had multiple defective components; for example, adhesion to ligands for sLe^x, chemotaxis, release of primary azurophil granules upon stimulation with chemotactic peptide, and failure to undergo the respiratory burst using the same stimulus.^423,424 Molecular analysis identified the asparagine for aspartic acid mutation at amino acid 57 of one allele of the Rac-2 gene.^423,424 Mutant Rac-2 did not bind GTP and it inhibited and behaved as a dominant negative to impair Rac-2–mediated activation of the respiratory burst.⁴²⁴ Fortunately, the youngster was successfully transplanted with marrow from a HLA-identical older brother.⁴²⁴

Therapy, Course, and Prognosis Allogeneic hematopoietic stem cell transplantation is the only recognized curative treatment for CGD. Reduced intensity conditioning stem cell transplantation from HLA-matched donors performed in 56 patients with intractable infections and severe inflammation carried a 2-year overall survival of 96 percent.⁴²⁵ However, vigorous supportive care along with the use of recombinant IFN continues to be the foundation of treatment.³⁷⁶ Cultures must be obtained as soon as infection is suspected, as unusual organisms are commonly the source of infection and may grow promptly in vitro. Most abscesses require surgical drainage for therapeutic and diagnostic purposes, and prolonged use of antibiotics is often required. If fever occurs, it is advisable to obtain certain studies that aid in the management of septic episodes. These include roentgenograms of the chest and skeleton and a computed tomography (CT) scan of the liver because of the frequency of pneumonia, osteomyelitis, and liver abscesses.³⁸³ Arrangements should be made for prompt medical attention at the first signs of infection. With early intervention, many lesions can be managed by conservative medical means. For example, enlarging lymph nodes often regress when treated with local heat and orally administered antistaphylococcal antibiotics. It is particularly important to obtain a microbiologic diagnosis, and fine-needle aspiration may be helpful in this regard. In general, antibiotic therapy for the offending organisms is indicated and purulent masses should be drained. The cause of fever and prostration cannot always be established, and empiric treatment with broad-spectrum parenteral antibiotics is required. Often it is necessary to treat with antibiotics for a prolonged time until the initially elevated sedimentation rate approaches normal values. Aspergillus spp. infection requires treatment with amphotericin B or, in refractory cases, with granulocyte transfusions.³⁷⁶ Glucocorticoids also may be useful in the treatment of patients with antral and urethral obstruction. The risk of Aspergillus infection can be reduced by avoiding marijuana smoke and decaying plant material, such as mulch and hay, both of which contain numerous fungal spores.⁴²⁶ Long-term oral prophylaxis with trimethoprim-sulfamethoxazole (5 mg/kg per day of trimethoprim) is an accepted practice in the management of patients with CGD.³⁷⁶ Patients have prolonged infection-free periods, which result from the prevention of infections caused by S. aureus, without increasing the incidence of fungal infections. The use of itraconazole prophylactically has reduced the development of fungal infections.^427,428

IFN-γ (50 mcg/m², three times per week, subcutaneously) can reduce the number of serious bacterial and fungal infections.^427,429 IFN-γ–enhanced neutrophil function in vitro has not been correlated with improvement in the activity of the neutrophil respiratory burst in patients totally lacking the ability to generate superoxide. On the other hand, its use increases the neutrophil expression of the high-affinity Fcγ receptor 1, as well as monocyte expression of FcγRI, FcγRII, FcγRIII, CD11/CD18, and HLA-DR.⁴³⁰ The IFN-γ protective effect in patients with CGD may involve improved microbial clearance, as suggested by the enhanced phagocytic activity by neutrophils of opsonized S. aureus. In rare, X-linked CGD patients able to generate some superoxide, IFN-γ programs granulocyte cells to increase their expression of cytochrome b, which results in normal superoxide generation.⁴³¹ With the use of current prophylactic treatments, the mortality in CGD has been reduced to two patient deaths per year per 100 patients followed.³⁷⁶

CGD patients with mutations that result in 5 to 10 percent of normal-functioning amounts of NADPH have a mild phenotype and better clinical prognosis than do patients with complete absence of any NADPH-oxidase activity.⁴³² Similarly, female carriers of X-linked CGD who have only 3 to 5 percent oxidase-normal neutrophils rarely get serious infections suggestive of the CGD clinical phenotype.⁴³³ Thus, even low levels or partial correction by gene therapy of CGD is likely to provide clinical benefits. In support of that hypothesis, mouse models of X-linked and p47^phox-deficient CGD have been developed by gene targeting.^434,435 Studies in the gp91^phox- and the p47^phox-deficient mouse models of CGD show that retrovirus-mediated gene-therapy-targeting of marrow progenitor cells ex vivo can result in the correction of defects in oxidant production in vivo in blood neutrophils after radiation conditioning and transplantation of marrow stem cells.^436,437 Protection from infection challenge occurred even when the oxidase-corrected cells comprised less than 10 percent of circulating neutrophils. These promising results suggest that somatic gene therapy can be employed to correct defective phagocyte oxidase function in selected patients with CGD. In a phase I clinical trial, gene therapy for p47^phox-deficiency CGD, five adult patients received intravenous infusions of autologous blood stem cells that were ex vivo transduced using a retrovirus encoding normal p47^phox.⁴³⁸ Although conditioning therapy was not given prior to the stem cell infusion, functionally corrected neutrophils were detectable in blood for several months.³³⁹ In another study, long-term high-level clinical beneficial correction in ex vivo gene therapy of X-linked CGD occurred in two adult patients.⁴³⁹ Nonablative busulfan conditioning was used to augment gene therapy correction. There needs to be caution regarding the long-term stability and safety of gene therapy. For instance, there are concerns about gene insertion rendering patients vulnerable to developing an hematological malignancy.

Myeloperoxidase Deficiency

The functional and immunochemical absence of the enzyme MPO from granules of neutrophils and monocytes, but not eosinophils, is inherited as an autosomal recessive trait, with a prevalence of 1:2000.⁴⁴⁰ MPO, an enzyme that catalyzes the production of HOCl in the phagosome. In MPO deficiency, the microbicidal activity of the neutrophils is reduced early after ingestion of microorganisms (see Table 66–2). However, normal microbicidal activity is observed in approximately 1 hour after a variety of organisms are ingested.⁴⁴⁰ Thus, the MPO-deficient neutrophil uses an MPO-independent system for killing bacteria that is slower than the MPO–H₂O₂–halide system, but that is eventually effective in eliminating bacteria. MPO-deficient neutrophils accumulate more H₂O₂ than do normal neutrophils; the higher peroxide concentration improves the bactericidal activity of the affected neutrophils. In contrast to the retardation of bactericidal activity, candidacidal activity in MPO-deficient neutrophils is absent.⁴⁴⁰ The most significant clinical manifestation in a few patients with diabetes mellitus and MPO deficiency has been severe infection with C. albicans. Because this is such a common disorder of phagocytes, it is important to note that the vast majority of patients with this genetic disorder have not been unusually susceptible to pyogenic infections and do not require therapy.

The complementary DNA encoding human MPO has been cloned and the gene structure, including promoter and regulatory elements, delineated.⁴⁴⁰ The gene consists of 12 exons and 11 introns and is located on the long arm of chromosome 17, and its expression is finely coordinated with expression of genes encoding other lysosomal proteins. Expression of genes for human neutrophil elastase and MPO is very similar; it is low in myeloblasts, peaks during the promyelocyte stage, and eventually drops to low levels in myelocytes. MPO is a symmetric molecule composed of four peptides, where each half consists of a heavy- and a light-chain heterodimer.⁴⁴⁰ Each heavy- and light-chain heterodimer starts as a single peptide that is cleaved during the posttranslational process to yield the heavy and light chains that form half of the mature molecules. The two halves of the molecule are associated by a disulfide linkage between heavy-subunit residues at their residue C319.

The primary translation product of the gene is a single-chain peptide of 80 kDa that undergoes cotranslational glycosylation at several asparagine residues, followed by a series of modifications of these oligosaccharides. The apopromyeloperoxidase exists for a prolonged time in the endoplasmic reticulum, where it associates reversibly with several endoplasmic reticulum–resident proteins known as molecular chaperones.⁴⁴⁰ Subsequent to heme insertion, the enzymatically active promyeloperoxidase undergoes proteolytic cleavage of the pro region. Then, in a prelysosomal compartment, the single peptide is cleaved into the heavy and light subunits, which remain linked. During final sorting within the azurophil lysosome compartment, there is dimerization of half-molecules to form the mature MPO.

Most patients with MPO deficiency have a missense mutation in the gene that results in replacement of arginine 569 with tryptophan.⁴⁴⁰ The mutation results in a precursor that associates with molecular chaperones, but does not incorporate heme, resulting in a maturational arrest during processing at the stage of an inactive enzymatic apopromyeloperoxidase. Other patients are heterozygotes with one allele bearing the common mutation and the other being normal, resulting in a partial deficiency.⁴⁴¹ To date, four genotypes have been reported to cause inherited MPO deficiency, each of which results in missense mutations. In the genotype Y173C, a missense mutation results in replacement of a tyrosine at codon 173 with a cysteine residue resulting in the mutant precursor being retained in the endoplasmic reticulum by virtue of its prolonged interaction with the chaperone calnexin, and eventually undergoing degradation in a proteasome.⁴⁴⁰ In this way, the quality control system operating in the endoplasmic reticulum retrieves misfolded MPO precursors from the biosynthetic pathway and creates the biochemical phenotype of MPO deficiency. In another patient, a missense mutation resulted in an intact MPO molecule that acquired heme but failed to undergo proteolytic processing to a mature molecule.

Acquired disorders are associated with MPO deficiency. Reported states include lead intoxication, ceroid lipofuscinosis, myelodysplastic syndromes, and acute myelogenous leukemia.⁴⁴² One-half of untreated patients with acute myelogenous leukemia and 20 percent of patients with CML may have MPO deficiency.⁴⁴²

Deficiencies of Glutathione Reductase and Glutathione Synthetase

Neutrophils contain enzymes capable of inactivating potentially damaging reduced oxygen byproducts. Disposal of superoxide anion is accomplished through superoxide dismutase, a soluble enzyme that converts superoxide to a H₂O₂. H₂O₂ is detoxified by catalase and by the glutathione peroxidase–glutathione reductase system, which converts H₂O₂ to water and oxygen.⁴⁴³ In addition to the soluble enzymes, cellular vitamin E serves as an antioxidant to prevent damage to the surface of activated neutrophils when releasing H₂O₂.⁴⁴³ Single cases of profound deficiencies in glutathione reductase⁴⁴⁴ and glutathione synthetase⁴⁴³ have been associated with impaired neutrophil bactericidal activity (see Table 66–2). Both deficiencies are associated with hemolysis under conditions of oxidative stress (Chap. 48). Glutathione synthetase deficiency also has been associated with intermittent neutropenia during times of mild infection. Vitamin E has been employed to ameliorate the hemolysis and improve neutrophil function in a patient with glutathione synthetase deficiency.⁴⁴⁵ Like patients with MPO-deficient neutrophils, the patients with glutathione reductase deficiency and glutathione synthetase deficiency are not unusually susceptible to bacterial infections.

DIAGNOSTIC APPROACH TO THE PATIENT WITH SUSPECTED NEUTROPHIL DYSFUNCTION

An increased susceptibility to pyogenic infections must be viewed in light of a number of factors: (1) adequacy of host defense; (2) the microbes to which the host is exposed; and (3) the conditions of the exposure. It is not always easy to establish a diagnosis of a specific neutrophil dysfunction on clinical grounds alone. Patients with recurrent pyogenic infections often yield no clues as to why they are afflicted, and patients with established deficiency of a defense mechanism may have an unimpressive clinical history. On the other hand, patients may be suspected of having a neutrophil dysfunction if they have a history of frequent bacterial or severe infections. Recurrent pulmonary infections, hepatic abscesses, and perirectal abscesses also should alert the clinician to consider further diagnostic evaluation of neutrophil function. For example, the identification of unusual catalase-positive bacteria and fungi, such as B. cepacia, S. marcescens, Nocardia, and Aspergillus, could be indicative of CGD.

Because many of the tests of neutrophil function are bioassays with great variability, the results of the tests must be interpreted in light of the patient’s clinical condition. For instance, isolated chemotactic defects usually do not explain the propensity for a patient to have recurrent severe infections. Furthermore, variation in bioassays is often intensified by inflammation or infection. Figure 66–8 is an algorithm for evaluation of the patient with recurrent infection.

Figure 66–8.

Algorithm for the workup of patients with recurrent infections. AD, autosomal dominant; CBC, complete blood count; CVID, common variable immunodeficiency; DHR, delayed hypersensitivity reaction; G6PD, glucose-6-phosphate dehydrogenase; GSH, glutathione; Ig, immunoglobulin, LAD, leukocyte adhesion deficiency; NBT, nitroblue tetrazolium; sLe^x, sialyl Lewis X.

REFERENCES