I. Introduction

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Knowledge of the mechanisms of leukocyte migration has expanded greatly in recent years. Whereas only in the last decade the first adhesion molecule was being characterized, today our understanding includes molecular descriptions for numerous adhesive receptor-ligand interactions, discovery of novel families of chemotaxins and cytokines, and the appreciation for the pivotal role adhesion pathways play in diseases from AIDS and atherosclerosis to cancer and inflammatory syndromes. Although these research efforts have clarified some issues, they also have illuminated new and perhaps difficult challenges. Indeed, the current literature on leukocyte transendothelial migration (TEM)³ portrays a complex phenomenon with many mechanisms and points of control that depend on the site of migration, the cell type, the initial inflammatory stimuli, and the presence and absence of several cellular and soluble mediators. The simple paradigm of rolling/adhering/diapedesis still holds true. However, the number of variables involved in our understanding of the process has increased dramatically.

This review will first describe the general mechanisms of TEM and the current understanding of the adhesion molecules, cytokines, and chemokines that drive leukocyte migration with an emphasis on polymorphonuclear leukocytes (PMNs; neutrophils). Also discussed are the interactions among soluble mediators that together determine the outcome of the adhesive/migratory process. Next, the special case of pulmonary neutrophil migration will be used to illustrate the dependence of the form of migration on the specific inflammatory stimuli. A summary of these observations in lung support the hypothesis that different adhesion molecule pathways are invoked by a qualitative difference in the inflammatory cytokines and chemokines present. Last is a brief discussion of current clinical and experimental strategies for pharmacologic interventions that target the adhesive process.

	II. General Mechanisms of Transendothelial Neutrophil Migration

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Circulating leukocytes can migrate from vessels into tissues under both normal and pathologic circumstances. It is well accepted that leukocyte migration from the vasculature occurs by a multistep process, dictated by the sequential activation of adhesive proteins and their ligands on both leukocytes and endothelial cells (ECs) (Lawrence and Springer, 1991; von Andrian et al., 1991; Konstantopoulos and McIntire, 1996). Monocytes, lymphocytes, and PMNs all migrate by these similar, sequence-dependent mechanisms but differ in their responses to chemotactic and inflammatory signals, particularly in their qualitative and quantitative expression of adhesion molecules (Dransfield et al., 1992; Springer, 1994; Ager, 1996; Li et al., 1996). Initiation of migration begins with the "capture" by the vessel wall of PMNs from flowing blood, and this is followed by their "rolling" along the vessel wall. This process of margination is a normal behavior of circulating PMNs. Only after appropriate stimuli are present do rolling leukocytes become firmly adhered to ECs and are thus positioned for migration from the blood vessel into tissue parenchyma.

Both the capture, or initial tethering and removal of PMNs from the flowing blood, and their rolling along the vessel wall is due to the reversible binding of transmembrane glycoprotein adhesive molecules called selectins, which are found on both PMNs and ECs (Bevilacqua and Nelson, 1993; Albelda et al., 1994; Luscinskas and Lawler, 1994; Crockett-Torabi and Fantone, 1995; Tedder et al., 1995a). Selectins have a calcium-dependent lectin domain on the extracellular NH₂ terminus that is attached to an epidermal growth factor-like domain and then to a number of short consensus sequences. A short, intracellular domain is linked to signal transduction proteins (reviewed in Crockett-Torabi and Fantone, 1995; Crockett-Torabi, 1998). Selectin-type adhesive proteins are found on most cell types of hematopoietic origin and on ECs of blood and lymph vessels.

1. Leukocyte Selectin. Intravital microscopic analysis of the microvascular circulation in normal tissue offers visual evidence of the transient "stick and release" behavior of PMNs rolling along the vessel wall. In noninflamed tissues, the tenuous association of PMNs within postcapillary venules can be blocked by treatment with antibodies to leukocyte selectin (L-selectin) (Mel-14, LAM-1, CD62L) on circulating PMNs (Spertini et al., 1991; von Andrian et al., 1991). Constitutive expression of L-selectin is greatest on PMNs newly released from bone marrow compared with older, circulating PMNs (Matsuba et al., 1997). Loss or shedding of L-selectin from the PMN surface is due in part to metalloprotease activity, which results in rapid accumulation of bioactive L-selectin in the blood (Kishimoto et al., 1995). Autoproteolysis of L-selectin can occur after exposure to various inflammatory mediators such as lipopolysaccharide (LPS; endotoxin) and tumor necrosis factor- (TNF-), but it may also occur from normal rolling interactions with the vessel wall. For example, metalloprotease inhibitors significantly reduce rolling velocity and cleavage of L-selectin on PMNs in vitro, suggesting that L-selectin is routinely shed from circulating PMNs during normal, nonpathologic conditions (Walchek et al., 1996). Thus, the longer a PMN has been in the circulation and interacting with the vessel wall, the more L-selectin it has lost to transient binding and cleavage. Replacement of L-selectin on circulating PMNs has not been demonstrated, and a low expression level is associated with apoptosis and may be a signal for the removal of PMNs from the circulation (Matsuba et al., 1997). High plasma levels of soluble L-selectin that can occur during infection may inhibit PMN rolling at noninflamed sites. Bioactive, soluble L-selectin can bind to endothelial ligands and block their interactions with PMN-borne L-selectin (Schleiffenbaum et al., 1992; McGill et al., 1996; Ohno et al., 1997a).

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Schematic summary of adhesion molecules and respective ligands involved in TEM of neutrophils. EGF, epidermal growth factor; ESL-1, E-selectin ligand 1.

2. Platelet Selectin. At least two endothelium-bound selectins, platelet selectin (P-selectin) and endothelial cell selectin (E-selectin), can facilitate PMN-EC adhesions. These selectins are expressed only when appropriate inflammatory stimuli are present. P-selectin (granule membrane protein-140; CD62P) is stored intracellularly in Weibel-Palade bodies of ECs and in -granules of platelets (Malik and Lo, 1996). Within minutes of exposure of ECs to inflammatory mediators such as complement products, oxygen-derived free radicals, or various cytokines, P-selectin is mobilized to the cell surface where it can interact with its PMN counterpart, P-selectin glycoprotein ligand-1 (PSGL-1; CD162). Monocytes and platelets also possess PSGL-1 and can bind to P-selectin on activated ECs. Like CD34, the PSGL-1 protein is modified with O-linked sialic acid and other sugar groups. It consists of a disulfide-bonded homodimer on the PMN surface and is capable of binding two P-selectin ligands simultaneously (McEver and Cummings, 1997).

3. Endothelial Cell Selectin. A second endothelial-borne selectin, E-selectin (ELAM-1, CD62), is not stored but requires gene transcription for expression. Peak expression and activity in ECs in vitro is 4 to 6 h after exposure to inflammatory cytokines (Klein et al., 1995; Scholz et al., 1996). E-selectin can support rolling and tethering of PMNs in a fashion similar to P-selectin (Lawrence and Springer, 1994). Thus, its role in inflammation may be to maintain PMN rolling after P-selectin has been down-regulated (Malik and Lo, 1996; Yang et al., 1999a). Like L- and P-selectins, E-selectin binds in vitro to a variety of sialic acid- and fucose-containing glycoproteins, as well as to sulfated glycosaminoglycans such as heparan sulfate. However, the in vivo ligand for E-selectin may be a unique mucin modified with N-linked sugars that has been characterized in murine cells called E-selectin ligand 1 (Levinowitz et al., 1993; Steegmaier et al., 1995; Willmroth and Beaudet, 1999). Another sialylated ligand for E-selectin has been described in bovine T cells and is distinct from E-selectin ligand 1 (Walchek and Jutila, 1993). Human analogs for either murine or bovine ligands have not been reported.

The initiating signal for the next step of TEM, that of firm adhesion, is postulated to be either a receptor-mediated event in response to an inflammatory cytokine or an event propagated from signals from activated selectins. Cytoplasmic domains of bound and activated L-selectin and PSGL-1 are linked to signal transduction pathways that lead to integrin activation in PMNs (Crockett-Torabi and Fantone, 1995; Simon et al., 1995, 1999; Zimmerman et al., 1996a; McEver and Cummings, 1997; Williams and Solomkin, 1999). Thus, selectins may function to promote the orderly transition to the adhesion process by invoking integrin expression pathways in a timely, sequential manner that ensures successful migration.

1. Integrins. Integrins are a group of heterodimeric transmembrane glycoproteins found on PMNs and other hematopoietic cells that mediate cell-cell and cell-extracellular matrix adhesions (Hynes, 1992; Luscinskas and Lawler, 1994). All integrins comprise one - and one -subunit, which together form an extracellular ligand binding site. Cytoplasmic tails of integrins provide phosphorylation sites and linkages to cytoskeletal proteins involved in signal transduction (Alpin et al., 1998). There are 8 different -subunits (₁-₈) that associate with one of 16 -subunits to form at least 23 known receptors in a variety of cells, including lymphocytes, leukocytes, and platelets. Integrins are capable of mediating cell-cell binding but also are involved in cell interactions with extracellular proteins such as laminin, fibronectin, vitronectin, and fibrinogen, to name a few.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Schematic representation of the integrin heterodimer. Sites involved in ligand recognition on the -subunit include the I domain and Ca2+-binding repeat domains. An I-like domain on -subunits is also involved in ligand recognition. Intracellular phosphorylation sites modulate binding affinity.

2. Intercellular Adhesion Molecules. An important complementary endothelial ligand for Mac-1 is intercellular adhesion molecule-1 (ICAM-1) (CD54), an Ig-like molecule that exhibits low constitutive presentation on EC membranes but is markedly induced by exposure of ECs to inflammatory cytokines (Gerritsen and Bloor, 1993; Hashimoto et al., 1994; Klein et al., 1995; Scholz et al., 1996; Iigo et al., 1997). ICAM-1 is also found on tissue epithelial cells including pneumocytes (Barton et al., 1995; Burke-Gaffney and Hellewell, 1996). LFA-1 can bind to ICAM-1, but it has higher affinity to a related protein, ICAM-2, a ligand to which Mac-1 binds with less affinity. Recent in vitro studies with human umbilical vein ECs demonstrate Mac-1-mediated adhesion and TEM that is partially independent of both ICAM-1 and ICAM-2 (Issekutz et al., 1999).

Egress of PMNs through EC monolayers occurs preferentially at tricellular junctions (Burns et al., 1997a). Although the mechanism of extravasation is imperfectly understood, it is clear that selectin involvement has been down-regulated at this time and an IgG-type adhesion molecule, platelet-endothelial cell adhesion molecule-1 (PECAM-1; CD31), becomes critical for the actual passage of PMNs between ECs (Vaporciyan et al., 1993; Dejana et al., 1995; Newman, 1997).

Found on PMNs, platelets, and ECs, PECAM-1 can serve as its own ligand and form homodimers with molecules on opposing cells. PECAM-1 is evenly distributed over the surface of circulating PMNs and is concentrated at intercellular junctions of unstimulated ECs (Newman, 1997). By virtue of its junctional location, PECAM-1 is hypothesized to be a homing receptor to locate the transendothelial portal for the migrating PMN. Treatment of PMNs or endothelial monolayers with an antibody to PECAM-1 blocks transmigration in vitro (Muller et al., 1993; Muller, 1995), and similar antibodies have been effective in inhibiting PMN migration in rat models of peritonitis and alveolitis (Vaporciyan et al., 1993; Bogen et al., 1994; Muller, 1995). In both whole animal and cell systems, PECAM-1 antibody treatment does not block adhesion.

Compared with the aforementioned processes of margination and adhesion, there is relatively little known of the mechanisms of PMN egress and migration through the subendothelial matrix. PMNs possess proteases capable of digesting collagen, laminin, and other extracellular components present in the vascular wall. Adhesion and migration is accompanied by release of PMN-derived proteases (Wright and Gallin, 1979; Hanlon et al., 1991), and both chemotaxis and migration through artificial substrates in vitro is inhibited by treatment with antiproteases (Lomas et al., 1995; Delclaux et al., 1996). Hence, a critical role has been proposed for PMN-derived protease activity during extravascular transit. However, protease inhibitors are ineffective in stopping PMN migration through intact EC monolayers and basement membrane matrices in vitro (Allport et al., 1997a; Mackeral et al., 1999). Furthermore, mice deficient in gelatinase B (a PMN-derived collagenase) have normal PMN emigration into the lungs, peritoneum, and skin (Betsuyaku et al., 1999). Thus, requirements for protease release and digestion of extravascular matrix components during adhesion and migration are not certain.

Leukocytes bind to matrix components such as collagen, vitronectin, and laminin via ₁-integrins. As mentioned above, activated PMNs can express the ₁-integrin VLA-4, and this allows for their binding to endothelial VCAM-1 in vitro. Interaction with ₁-integrins might also be important for transit of PMNs through the extravascular milieu. For example, PMN migration through lung or synovial fibroblast barriers has recently been shown to require not only CD18 (₂), but also VLA-4, VLA-5 (₅₁), VLA-6 (₆₁), and VLA-9 (₉₁) (Shang and Issekutz, 1997; Shang et al., 1999). VLA-5 and VLA-6 bind fibronectin and laminin, respectively. Like VLA-4, VLA-9 interacts with fibronectin and VCAM-1, but it also binds to the matrix protein tenascin. Furthermore, VLA-9 is the most highly expressed ₁-integrin on PMNs and is not expressed by either lymphocytes or monocytes (Shang et al., 1999). Fibroblasts and parenchymal tissues such as pulmonary epithelium and hepatocytes express both VCAM-1 and ICAM-1. As such, VLA-9 and Mac-1 appear to be the more critical integrins responsible for PMN migration through the subendothelium.

Lastly, there is evidence for the involvement of PECAM in extravascular transit of leukocytes. Unlike the homotypic interactions between PECAM molecules that mediate homing and diapedesis, migration through the subendothelial environment requires heterophilic binding of leukocytic PECAM-1 to an unidentified ligand (Liao et al., 1995; Wakelin et al., 1996). Antibodies to an amino-terminal domain of PECAM-1 inhibit homophilic binding, whereas antibodies to a membrane-proximal extracellular domain of PECAM blocks heterophilic interactions and movement across the basement membrane (reviewed in Muller and Randolph, 1999).

Multiple lines of evidence suggest that selectin activation and binding is required before firm adhesion can occur via Mac-1-ICAM-1 interactions (Lawrence and Springer, 1991; von Andrian et al., 1992; Ley et al., 1995). As mentioned previously, selectin-mediated signals are incorporated with other extracellular inflammatory stimuli to regulate the timely expression and engagement of PMN integrins (Crockett-Torabi and Fantone, 1995; Crockett-Torabi, 1998; Williams and Solomkin, 1999). For example, studies in vitro demonstrate that cross-linking activation of L-selectin on PMNs can lead directly to ₂-integrin-mediated adhesion in both static assays (Simon et al., 1995, 1999; Steeber et al., 1997) and in shear-flow models (Gopalan et al., 1997). Furthermore, removal of L-selectin from PMNs renders them incapable of integrin-mediated firm adhesion to endothelial monolayers (Endemann et al., 1997; Zouki et al., 1997). These results are not surprising for flow systems; L-selectin binds more rapidly and at higher shear rates than CD18 integrins (Taylor et al., 1996). However, the requirement for L-selectin in static systems suggests that selectin-binding is required for more than just physical capture of PMNs. Stimulation of leukocytes via L-selectin can activate G-proteins, tyrosine kinases, release of ceramide, and assembly of filamentous actin (Brenner et al., 1997, 1998; Simon et al., 1999), events that may be a required prelude to integrin-dependent adhesion. Firm adhesion by L-selectin activation is blocked by inhibitors of protein tyrosine kinase and protein kinase C, suggesting that these pathways link L-selectin to Mac-1 up-regulation (Steeber et al., 1997).

Engagement of PSGL-1 can modulate Mac-1 function. Binding of PSGL-1 to P-selectin activates tyrosine kinase and leads to the up-regulation and avidity of CD18 on PMNs (Hidari et al., 1997; Blanks et al., 1998). In addition, integrin-activating chemoattractants cause a redistribution of PSGL-1 on PMNs and concomitant loss of P-selectin binding (Lorant et al., 1995). Thus, signal transduction pathways after selectin engagement appear to promote "bond-trading" from selectins to integrins by down-regulating the former and inducing functional expression of the latter.

Activation of CD18 integrins, in turn, can lead to phosphorylation of tyrosine residues on PECAM-1 and potentially modulate the function of these adhesive proteins during PMN transmigration (Lu et al., 1996). Conversely, activation of PECAM-1 on PMNs with either cross-linking antibodies or antigen-binding fragments can increase the activity of Mac-1 (Berman and Muller, 1995), suggesting that cross-talk between the molecules occurs during TEM. Because the trailing end of the migrating PMN must detach from ECs as the leading uropod attaches, the coordination of both Mac-1-ICAM-1 and PECAM-PECAM interactions is critical for successful diapedesis. Engagement of PECAM up-regulates CD18 through phosphoinositide 3-kinase, which is a different pathway for CD18 activation from G-protein-associated activators such as fMLP, IL-8, and C5a (Jones et al., 1998a; Pellegatta et al., 1998). These studies suggest that activation of PMN adhesion molecules via integrin-ligand or selectin-ligand binding may differ from pathways initiated by cytokine-receptor binding.

Binding of Mac-1 to ICAM-1 can cause structural changes in endothelial cytoskeletal proteins associated with adherens junctions without causing EC retraction or injury to monolayers (Del Maschio et al., 1996; Allport et al., 1997b). The endothelial junctional proteins plakoglobin, cadherin, and - and -catenin dissociate within minutes of PMN binding to endothelium (Del Maschio et al., 1996). It is unclear if this reorganization is required for TEM. After prolonged exposure of endothelial monolayers to inflammatory cytokines, PECAM-1 becomes diffusely distributed throughout the cell membrane and away from intercellular junctions (Romer et al., 1995), whereas L-selectin ligands and ICAM-1 redistribute from random expression to localization at cellular junctions (Bradley and Pober, 1996). It is unknown what role this plays in PMN emigration; in each system, neither ICAM-1, PECAM-1, nor the L-selectin ligand was bound by PMN counterparts. Taken together, control of TEM is dictated in part by intracellular signaling and cross-talk between adhesive proteins and may be separate from pathways elicited by cytokines and chemoattractants that require receptor activation.

Promigratory stimuli can be classified generally as either nonchemotactic cytokines or chemoattractants. Canonical inflammatory cytokines, such as TNF- and IL-1 can engender expression of adhesive proteins on PMNs and ECs, but they are not by themselves chemotactic for PMNs. For example, TNF- and IL-1, along with other inflammatory mediators, promote the firm adhesion of PMNs to endothelium in systems in vitro (Schleimer and Rutledge, 1986; Huber et al., 1991; Burke-Gaffney and Hellewell, 1996; Komatsu et al., 1997). However, egress and migration of PMNs into extravascular spaces requires the presence of chemoattractants that cause the directed migration of PMNs through tissue. Some chemoattractants can promote expression of adhesion molecules on PMNs similar to the responses elicited by IL-1 and TNF-.

1. Cytokines: Tumor Necrosis Factor- and Interleukin-1. Two of the most important pro-adhesive cytokines that are present during most inflammatory responses are TNF- and IL-1 (Table 1). The macrophage/monocyte is the primary cellular source of both TNF- and IL-1, and LPS is perhaps their most important inducer (reviewed in Tracey and Cerami, 1993, 1994; Bemelmans et al., 1996; Di Girolamo et al., 1997). Among many pathophysiologic effects of TNF- are shock, cytotoxicity, and cachexia. Binding to two different TNF- receptors present on each cell induces the effects of TNF- on PMNs and ECs. PMNs can respond to TNF- by activating and expressing integrins, producing platelet-activating factor (PAF) and other mediators, and releasing granule contents. Likewise, ECs mobilize selectins, up-regulate ICAM-1, and activate procoagulant pathways in response to TNF- exposure. During inflammation and endotoxemia, PMNs release from their membranes a soluble TNF- receptor that can bind to and effectively inactivate circulating TNF-.

2. Chemoattractants. PMNs have at least five different receptors for chemotactic stimuli. Unique receptors exist for PAF, complement protein C5a, leukotriene B₄ (LTB₄), and bacterial peptides (e.g., fMLP) (Table 1). In addition, a growing number of chemokines and their specific receptors are being discovered and characterized as important players in inflammatory responses of PMNs (Furie and Randolph, 1995). Chemoattractants/chemokines for PMNs can be produced by a wide variety of cells, including endothelial and epithelial cells, macrophages, monocytes, lymphocytes, platelets, PMNs, and parenchymal cells. In models in vitro, chemoattractants can activate PMNs or ECs to express adhesive proteins in a manner similar to TNF- or IL-1. Thus, redundant pathways for adhesive and migratory processes probably occur in vivo (Detmers et al., 1990, 1991; Huber et al., 1991).

3. Cytokine-Chemoattractant Interaction during Migration. Although LPS, TNF-, and IL-1 are not chemotactic for PMNs, their exposure to cultured cells can induce the production of chemoattractants such as IL-8 and PAF (Huber et al., 1991; Kuijpers et al., 1992; Smart and Casale, 1994; Burns et al., 1997b). For example, IL-8 released into culture medium after TNF- stimulation of ECs can cause CD18 up-regulation on PMNs (Huber et al., 1991). In addition, EC-derived IL-8 can become localized on the cell surface where it can activate PMNs close to the vessel wall (Kuijpers et al., 1992; Rot, 1993; Imaizumi et al., 1997). Indeed, treatment of EC monolayers with antibody to IL-8 inhibits rolling PMNs from firmly adhering to the monolayer (Rainger et al., 1997), suggesting that localized IL-8 is required for adhesion. The physical nature of the IL-8-EC relationship is unclear. Rot and coworkers (Rot et al., 1996) described in situ binding of radiolabeled IL-8 to vascular endothelium in lungs, liver, and kidney. However, there is no evidence for an IL-8 receptor or binding protein on cultured or primary ECs in vitro (Petzelbauer et al., 1995; Rot et al., 1996). IL-8 likely binds to components of the vessel wall matrix such as heparan and glycosaminoglycans (Hoogewerf et al., 1997). Thus, localization might not be due to an EC component but to extracellular sites near the cell.

View larger version (49K): [in this window] [in a new window]   Fig. 3.   Schematic representation of the modulation of adhesive interactions by LPS, cytokines, chemokines, and other chemoattractants. Numerous mediators can cause PMNs to shed L-selectin and promote the mobilization of CD11/CD18 to the PMN membrane in the transition from vascular rolling to adherence. Chemokines down-regulate their receptors by phosphorylation and internalization, whereas LPS and TNF- indirectly cause proteolytic cleavage of these receptors. C5a, fMLP, and TNF- down-regulate both chemokine and certain other chemoattractant receptors as described in the text. LPS can bind to CD11/CD18 and may interfere with ligand binding and activate intracellular pathways. In ECs, LPS and cytokines cause the mobilization of Weibel-Palade bodies to the cell membrane and induce gene expression and synthesis of ICAMs and IL-8. LPS also causes the production and membrane localization of PAF. TLR-4, Toll-like receptor 4.

TEM of PMNs is a carefully orchestrated sequence of cell activation, adhesion molecule expression, and molecular cross-talk between receptors on both cell types. As such, there are several opportunities to modulate and modify the migratory response by both exogenous and endogenous factors. Several lines of evidence suggest that integrin activation needs to occur at the PMN-EC interface and not in the circulation. Premature activation of PMN integrins and chemotactic receptors by circulating mediators in vivo might be avoided by temporally and spatially controlled release and concentration of chemokines at the endothelium-blood interface as suggested by Rot and coworkers (Rot, 1993; Rot et al.,1996). Knowledge of the molecular basis of control of TEM by endogenous factors (i.e., cytokines and chemokines, etc.) may suggest how therapeutic interventions might be formulated to alter undesirable recruitment of inflammatory cells. In this regard, pharmacologic manipulation of adhesion molecules by a variety of agents has shown promise in both human and animal models of inflammation. Several therapeutic approaches are discussed in the last section of this review, but first the special case of adhesive interactions that occur during pulmonary inflammation is presented in the next section.

	III. Polymorphonuclear Leukocyte Migration in the Lungs

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Most of the known molecular and cellular mechanisms of TEM have been elucidated from studies in vitro and in systemic vessels in the mesentery and dermis. Recent work in lung, however, suggests that PMN trafficking in the pulmonary circulation is fundamentally different from events in the systemic vasculature. In animal models tested so far, PMN behavior in lungs differs with respect to 1) the marginated pool of PMNs, 2) the site of TEM, and 3) requirements of adhesion molecules for PMN extravasation (Fig. 4). Although the basic mechanisms of TEM remain incompletely understood, these three differences suggest that different paradigms are needed to explain the kinetics of PMN migration in different vascular beds.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Differences in PMN migration occurring in pulmonary circulation and in bronchial airway and systemic circulations.

Margination of PMNs is a prerequisite for their escape from the main flux of flowing blood, where they can sense and respond to inflammatory signals present in vessel wall microenvironment. The concentration of PMNs within pulmonary capillary blood is 35 to 100 times greater than in large vessels of the systemic circulation (Doerschuk et al., 1987, 1993; Gee and Albertine, 1993). In the systemic circulation, margination occurs as rolling in postcapillary venules and is mediated by L-selectin and P-selectin and their respective ligands (Butcher, 1991; Lawrence and Springer, 1991; Spertini et al., 1991; Tozeren and Ley, 1992). However, 97% of pulmonary vascular PMNs are found in the capillary network where vessels are too small to allow rolling (2-15 µm) (Doerschuk et al., 1993). Selectin-mediated rolling still occurs in pulmonary venules (>15 µm), but it makes a small contribution to the total pool of marginated PMNs in the pulmonary circulation.

Despite the lack of rolling in pulmonary capillaries, selectins may be involved in the PMN-EC interactions. For instance, treating rabbits with fucoidan, a polysaccharide made mostly of sulfated fucose that inhibits binding of all selectins, decreases the frequency of PMN stopping in capillaries by 25% and the duration of stops by 50% (Kuebler et al., 1997). Similar results are seen in fucoidan-treated rats, in which PMN localization in alveolar capillaries is decreased by 15% (Yamaguchi et al., 1997). In the same study in rats, an antibody to P-selectin was ineffective in slowing PMN transit through the lungs, whereas rolling on systemic venules was significantly inhibited. Because E-selectin is not expressed in noninflamed rat lungs, all selectin-mediated binding is therefore due to L-selectin. Furthermore, despite the expression of ICAM-1 in normal rat pulmonary capillaries, treatment with antibodies to ICAM-1 does not affect PMN transit times (Yamaguchi et al., 1997). Thus, during nonpathologic conditions, L-selectin is the only adhesion molecule likely to be involved in pulmonary PMN margination in rat.

Another factor to account for the dramatic extent of pulmonary PMN margination may be the discrepancy between neutrophil and capillary diameters (Downey et al., 1990). PMNs that are 7 to 8 µm in diameter must deform to an elongated shape to pass through capillaries, which average 5 to 6 µm and can be as small as 2 µm (Hogg, 1987; Doerschuk et al., 1993; Wiggs et al., 1994; Gebb et al., 1995). Shape change in PMNs takes much longer than in erythrocytes, and this likely accounts for their longer pulmonary transit time compared with erythrocytes (2.7 versus 1.3 s) (Hogg et al., 1988; Lien et al., 1991).

A dramatic increase in pulmonary margination is accompanied by neutropenia when animals are given i.v. LPS or zymosan-activated serum (ZAS; a source of complement activation products; C5a) (Haslett et al., 1987; Doerschuk, 1992). Capillary sequestration in these models may be due to lack of PMN deformability, inasmuch as these same agents cause actin polymerization and PMN stiffness in vitro (Erzurum et al., 1992).

Given their slow transit and intimate contact with capillary endothelium, PMNs are well positioned to respond to inflammatory signals generated within airspaces. Thus, in contrast to migration predominately from postcapillary venules in the systemic circulation, PMNs primarily extravasate from the alveolar capillary network in rabbits, rats, and mice (Doerschuk et al., 1989; Walker et al., 1991; Doerschuk, 1992; Downey et al., 1993). It is curious, therefore, that P-selectin is constitutively expressed on pulmonary arterioles and venules in rabbits and not on pulmonary capillary ECs where margination and extravasation occurs (Mulligan et al., 1992b). In rat, by contrast, P-selectin is absent in the normal lung, but ICAM-1 is constitutively expressed on capillary and venular endothelium (Yamaguchi et al., 1997). As discussed previously, ICAM-1 associates with CD18 to mediate the firm adhesion of PMNs and usually requires induction for full responses. Although ICAM-1 has a low constitutive level of expression in most tissues, it is 30-fold higher in lung tissue (Panes et al., 1995). Thus, pulmonary leukostasis after infusion of LPS or ZAS may be due to the high level of ICAM-1 in lung (Haslett et al., 1987; Doerschuk et al., 1989; Doerschuk, 1992). Both LPS and ZAS are capable of mobilizing CD18 on isolated PMNs, and similar up-regulation of CD18 in vivo would provide ligands for lung ICAM-1 (Erzurum et al., 1992; Klut et al., 1997). Therefore, by virtue of the density in adhesion molecules and slow PMN transit times, PMN adhesion and extravasation is favored in the pulmonary capillaries over downstream venules.

C. Adhesion Molecule Requirements

1. Integrins. Another important difference between the pulmonary and systemic circulations is in the mechanics of PMN diapedesis, or egress of the PMN through the vessel wall. In all models tested so far, systemic PMN migration from postcapillary venules requires CD18 integrins. However, pulmonary PMN migration can occur independently of CD18 adhesion molecules. Whether or not migration depends on CD18 varies with the intrapulmonary stimulus. IL-1, phorbol myristate acetate, and Gram-negative bacterial stimuli, including LPS, elicit migration via pathways predominately mediated by CD18 (Doerschuk et al., 1990; Hellewell et al., 1994; Qin et al., 1996; Ramamoorthy et al., 1997). By contrast, Gram-positive bacteria, hydrochloric acid, and C5a elicit pulmonary PMN recruitment that is mostly independent of CD18. The adhesion molecules needed for CD18-independent migration in lung are unknown.

2. Selectins. It is unclear if selectins are responsible for CD18-independent migration in vivo. Studies using knockout mice and blocking antibodies that target selectins have not provided definitive answers. Mice deficient in L-, P-, or E-selectins exhibit PMN rolling behavior that is compromised in both normal and inflamed venules in the systemic circulation (Mayadas et al., 1993; Johnson et al., 1995; Kunkel and Ley, 1996). In a model of pneumonia using Streptococcus pneumo