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Figure 7-1. Leukocyte recruitment. 1. Circulating leukocytes express integrins in a low-affinity conformation. 2. Exposure to

activated endothelium leads to rolling, which is mediated by L-selectin and P-selectin on the neutrophil and E-selectin on

endothelium. 3. Leukocyte exposure to cytokines released by macrophages phagocytosing pathogens induces a high-affinity

integrin conformation integrins. Tight leukocyte–endothelial adhesion involves integrin engagement with counterligand expressed

on the endothelium. 4. Subsequent exposure to chemokines leads to diapedesis, which is further mediated by the family of β1 and

β2

integrins. (Adapted from Abbas AK, Lichtman AH. Cellular and Molecular Immunology. 5th ed. Philadelphia, PA: Saunders; 2003.)

Once tightly adhered, neutrophils must diapedese between endothelial cells and across the basement

membrane to arrive at the focus of inflammation. Platelet/endothelial cell adhesion molecule 1

(PECAM-1) and integrin-associated protein are integral to transmigration (Fig. 7-1).13,14,16,17 PECAM-1

is concentrated along the intercellular junctions of endothelial cells, and both leukocyte and endothelial

PECAM-1 appear to be essential for neutrophil and monocyte diapedesis. Other candidate receptors

include the β1

integrins, or very late antigens (VLA), which possess affinity for many constituents of the

extracellular matrix, including laminin, fibronectin, and collagens, the β3

family of integrins including

the glycoprotein (gp) IIβIIIα and the vitronectin receptor.4 Further “directions” for migration to the

focus of inflammation are delivered by the concentration gradients of chemotactic factors, including

complement C5a, IL-8, LTB4

, and the bacterial product N-formylmethionyl-leucyl-phenylalanine

(fMLP).18–20

The clinical significance of even minor derangements in any aspect of this process is evident in the

disease leukocyte adhesion deficiency, characterized by complete absence of CD18, and therefore all β2

integrins. Patients usually succumb to recurrent skin and mucosal infections within the initial 10 years

of life.4

Phagocytosis

Microbial elimination commences upon first encounter with a foreign pathogen. It is facilitated by

opsonization, a process in which microbes are coated by immune globulins and/or complement, which

subsequently bind to their respective cell surface receptors, FcγRs and Mac-1.21–23 Neutrophils

constitutively express low-affinity immune globulin receptors FcγRII and FcγRIII and can be induced to

express high-affinity FcγRI by incubation with IFNγ or cross-linking β2

integrins.5,24 Complementdependent phagocytosis is mediated by interactions between the leukocyte Mac-1 receptor and the

complement opsonin iC3b.

Once engaged, FcγRs are phosphorylated on tyrosine residues within an immunoreceptor tyrosine

activation motif (ITAM) by the Src family kinases.24 These phosphorylated sites serve as docking

regions for a variety of proteins, in particular Syk. The importance of Syk is underscored by the

observation that mice deficient in Syk are incapable of ingesting IgG-opsonized particles. A series of

enzymes are subsequently activated including phosphoinositol 3-kinase, phospholipase C (PLC), and

protein kinase C. Ultimately, the actin cytoskeleton undergoes rearrangement and the local

plasmalemma is remodeled in the formation and sealing of the phagosome.4,24

This immature phagosome undergoes a series of maturation steps, whereby it acquires the machinery

necessary for the killing and disposal of internalized microorganisms. Alterations in cytosolic calcium

concentration induce the fusion of secretory vesicles and granules containing the microbicidal

armamentarium with the immature phagosome.24 Proteins effected by calcium concentration and that

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may govern phagosomal maturation include synaptotagmins, actin, calmodulin, and the Src family of

kinases.24 The soluble N-ethylmaleimide-sensitive-fusion-protein attachment protein receptor (SNARE)

proteins are thought to assist in fusion by engaging cognate receptors on the target membrane and

approximating the two membranes. Antibodies to the SNARE 5, syntaxin 6, and SNAP-23, inhibited

exocytosis of azurophilic and specific granules, respectively.24

Figure 7-2. Integrin Signaling. Integrins comprise a large family of cell surface receptors that are composed of 2 subunits, a and b,

and are activated by dimerization. The cytoplasmic tails are devoid of enzymatic activity, and hence, signal transduction is effected

by adapter proteins that connect the receptor to the cytoskeleton, cytoplasmic kinases, and transmembrane growth factor receptors.

As integrins bind the extracellular matrix they become clustered and associated with the cytoskeletal proteins talin, paxilin, and

vinculin and signaling complexes. Actin stress fibers form, which increase integrin clustering. Ultimately, focal adhesion kinase

(FAK) is recruited via interactions with talin and paxillin or with the b integrin subunit. Subsequent autophosphorlyation on

tyrosine 397 provides a binding site for the Src homology 2 (SH2) domain of Src. The Src kinase phosphorylates a number of focal

adhesion components including paxillin and tensin and pl30CAS, a docking protein that recruits Crk, which can subsequently

activate proximal elements in the JNK cascade of the MAPK family. FAK may also be phosphorylated by Src on tyrosine 925,

creating a binding site for the complex of the adapter Grb2 and Ras guanosine 5'-triphosphate exchange factor mSOS. These

interactions also lead to activation of MAPK cascades, and ultimately the induction of a variety of genes. (Adapted from Giancotti F

& Ruoslahti E. Integrin Signaling. Science 1999:1028-1032)

Neutrophil Granules and Secretory Vesicles

Neutrophils exhibit an oxygen-dependent “respiratory burst” pathway that generates toxic oxygen

derivatives and an oxygen-independent pathway that utilizes toxic proteinases.25 These two microbicidal

arms are compartmentalized into four distinct granules or vesicles (Table 7-2).26 They are mobilized in a

hierarchical fashion in response to gradual elevations in the intracellular calcium level, which parallels

the current needs of the cell.27 They also contain adhesion molecules and important inflammatory

mediators that help further orchestrate the involvement of other cells.

Primary, or azurophil (affinity for the dye azure A), granules target the destruction of phagocytosed

organisms (Table 7-2). The myeloperoxidase (MPO) within these granules generates hypochlorous acid

(HOCl) from products generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and

also imparts the characteristic greenish color of pus.9,27 Other major constituents include (1) αdefensins, cytotoxic proteins that scaffold into transmembrane pores within the microbial cell wall and

perturb the maintenance of vital transmembrane gradients, and (2) bactericidal/permeability-increasing

(BPI) protein, which binds to gram-negative organisms and induces rearrangement of membrane lipids

and inhibits growth.27–30 Elastase cleaves constituents of the extracellular matrix, including

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proteoglycans, collagen (types I, III, IV), and fibronectin and, of course, elastin.31 Azurocidin is

chemotactic for monocytes and stimulates LPS-induced release of IL-6 and TNFα from monocytes.4,27

Table 7-2 Neutrophil Granules and Secretory Vesicles

Specific granules are rich in antimicrobial substances that are released extracellularly (Table 7-2).26

Lactoferrin, by sequestering iron, retards bacterial growth and can bind bacterial cell membranes and

induce irreversible membrane damage and lysis.27 Phospholipase A2

(PLA2

) participates in the

degradation of bacterial membrane phospholipids. Lysozyme, present in all granules, is a cationic

antimicrobial peptide that cleaves peptidoglycan polymers of bacterial cell walls. Gelatinase and

collagenase are other extracellular matrix degrading enzymes.27 These granules also possess receptors

for a variety of extracellular matrix proteins and cell surface ligands (i.e., β2

integrin Mac-1) that

mediates firm adhesion to the endothelium. In addition to the mechanical function of cellular

anchorage, engagement of these receptors with their respective counterligands induces phenotypic

alterations such as degranulation and enhanced ROS production.4,27

Gelatinase granules contain matrix metalloproteases, zymogens that upon proteolytic activation

degrade the interstitial matrix including collagens, fibronectin, proteoglycans, and laminin; this may

facilitate neutrophil extravasation and migration (Table 7-2). They too are a source of cell surface

adhesion molecules.4,27,30

Secretory vesicles contain many of the cell surface adhesion molecules essential for leukocyte

recruitment. Their membranes are dense with the β2

integrins LFA and Mac-1, the complement receptor

CR1, the LPS receptor CD14, and the FcγRIII. Through fusion with the plasmalemma, the cell surface is

enriched with these receptors, which facilitates firm neutrophil-endothelial engagement and the capacity

to respond to a variety of stimuli.4,27 Not surprisingly, because of the essential nature of leukocyte

recruitment, secretory vesicles possess the lowest threshold for release and thus released earliest,

followed by gelatinase, specific, and azurophil granules (Table 7-2).27

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