Selectins and mechanisms of signal transduction

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1 Selectins and mechanisms of signal transduction Elahe Crockett-Torabi Department of Surgery, Michigan State University-College of Human Medicine, East Lansing Abstract: Leukocyte emigration is essential in both lymphocyte homing, as a central part of immune surveillance, and in leukocyte invasion at sites of inflammation. The emigration of leukocytes requires the interplay of adhesion molecules of the selectin and integrin families and chemokines. Selectindependent cell-cell interaction is essential in localizing leukocytes within tissues by promoting the rolling of leukocytes along the endothelial cell surface before development of tight adhesion and subsequent transendothelial migration. Selectins also play an active role in the initiation of intracellular signaling pathways and regulation of cell-cell interactions involving monocytes, lymphocytes, platelets, and endothelial cells. This review focuses mainly on the emerging evidence of biochemical signaling mechanisms involved in the regulation of selectin-dependent leukocyte activation and adhesion, as well as the critical role played by selectins as leukocyte stimulatory molecules. This evidence has serious implications regarding the development of immune and inflammatory responses. This article will also review key structural features of the selectin receptors. A summary is provided of our current understanding of the specific molecular interaction occurring between these adhesion molecules and their counterreceptors, focusing on the critical roles they may play in the regulation of functional responses. J. Leukoc. Biol. 63: 1 14; Key Words: neutrophils leukocytes endothelial cells protein tyrosine kinase intracellular calcium superoxide anion INTRODUCTION Adhesive interactions between cells and cells with the extracellular matrix have been found to be crucial to multiple tissue functions, including embryonic development, organ morphogenesis, inflammatory responses, wound healing, immune surveillance, blood coagulation, and tumor metastasis [1 6]. Recent studies have suggested that adhesion molecules function not only to fix cells in specific locations within tissues and regulate their movement, but also to translate biochemical information from the extracellular environment through the activation of intracellular signaling pathways into specific cell functional responses [7]. Leukocyte interaction with vascular endothelial cells and extracellular matrix during immune and inflammatory reactions is essential and is dependent on a series of cellular adhesive events. Adhesion and migration of leukocytes is a dynamic process that is orchestrated by specific adhesion molecule receptors and counter-receptors expressed on leukocytes, endothelial cells, and defined regions of extracellular matrix and coagulation proteins. At least three distinct families of adhesion molecules participate in leukocyte adhesion and migration. These families include the selectins, integrins, and certain members of an immunoglobulin superfamily known as intercellular adhesion molecules (ICAM) (Table 1). Each is involved in a different phase of leukocyte emigration through the endothelium, and the synchronization of their expression and function is crucial for recruitment of leukocytes from the bloodstream to the tissue. The selectins and their counter-receptors play a major role in the initial contact of the leukocytes with the endothelium, whereas the integrins and ICAMs coordinate the subsequent adhesive interactions, including transendothelial migration and cell adhesive interactions with the extracellular matrix. Unregulated leukocyte adhesion and activation results in tissue damage associated with a number of inflammatory diseases such as arthritis, asthma, ischemia reperfusion injury, and acute lung injury [3, 5]. The physiological importance of the leukocyte adhesion molecules is clearly demonstrated by rare genetic disorders known as human leukocyte adhesion deficiency (LAD) syndromes and in experimental animal models of knockout mice that are selectin-deficient [8]. In LAD I, the 2 -integrin family is deficient, whereas in LAD II, the fucosylated ligands for selectins are absent. The leukocyte rolling and extravasation is impaired in individuals with LAD and in selectin-deficient mice. Selectins have been shown to be important in vivo in responses related to ischemia-reperfusion events [9 11], in shock [12], and in a variety of inflammatory reactions [13 15]. Interruption of the leukocyte-endothelial cell cascade has been a major focus of anti-inflammatory research and its therapeutic potential is under intense investigation [16]. A better understanding of the cellular and molecular mechanisms of leukocyte interactions and their role in both physiological and pathological cell and tissue function will assist in finding new approaches to the development of therapeutic interventions. This review is focused on the role of selectin molecules in the initiation of intracellular signal transduction and the mechanisms of selectin-dependent leukocyte activation and functional responses. Abbreviations: [Ca 2 ] i, intracellular calcium; fmlp, N-formyl-methionyl-leucylphenylalanine; HBSS, Hanks balanced salt solution; mab, monoclonal antibody; O 2, superoxide anion; PMA, phorbol myristate acetate; PTK, protein tyrosine kinase; TNF, tumor necrosis factor. Correspondence: Dr. Elahe Crockett-Torabi, Department of Surgery, B424 Clinical Center, Michigan State University, East Lansing, MI Received October 2, 1997; accepted October 2, Journal of Leukocyte Biology Volume 63, January

2 Family molecule TABLE 1. Selectins E-selectin (CD62E) (ELAM-1, LECAM-1) P-selectin (CD62P) (PADGEM, GMP140) L-selectin (CD62L) (E- and P-selection, MECA 79 antigen, MAdCAM-1, LAM-1, MEL-14, Leu-8, LECAM-1) Integrins LFA-1 (CD11a/CD18, L 2 ) Mac-1 (CD11b/CD18, MO-1, CR3, M 2 ) gp 150/95 (CD11c/CD18, X 2 ) VLA-4 ( 4 1 ) IIb 3 Immunoglobulin superfamily LFA-2 (CD2) LFA-3 (CD58) ICAM-1 (CD54) (intercellular adhesion molecule-1) ICAM-2 (CD102) ICAM-3 (CD50) VCAM-1 (CD106) (vascular cell adhesion molecule-1) PECAM-1 (CD31) (platelet endothelial cell adhesion molecule) Leukocyte Adhesion Molecules Ligand/counter-receptor Sialylated fucosylated lactosamines (i.e., sle X, sle A ), L- selectin, PSGL-1, ESL-1 Sialylated fucosylated lactosamines (i.e., sle X, sle A ), L-selectin, PSGL-1 Sgp 50 (GlyCAM-1), Sgp 90 (CD34) ICAM-1, ICAM-2, ICAM-3 ICAM-1, fibrinogen, C 3 b i, factor X Fibrinogen, C 3 b i, endothelial cells VCAM-1 Fibrinogen, fibronectin, vitronectin, thrombospondin, von Wille-brand factor LFA-3 (CD54) CD2 LFA-1, Mac-1 LFA-1 LFA-1 VLA-4 (CD49d) PECAM-1, glycosaminoglycans Studies using chimeric selectins have demonstrated that the lectin domain alone did support leukocyte binding but was insufficient for maximal binding, and that both the lectin and EGF-like domains together were required for an optimal recognition unit for selectin leukocyte interactions [23, 24]. However, other studies have reported data that do not support the direct involvement of the EGF-like domain in P-selectin-mediated cellular adhesion [25]. Based on the variability in the number of SCR domains and the fact that different SCR domains have been lost during recent mammalian evolution, a less critical role in ligand binding avidity or specificity for these domains has been suggested. However, the SCR domains may have a functional role in enhancing the ligand binding affinity, stabilizing receptor structure, or mediating signal transduction. It has been demonstrated that Mel-14 mab, an adhesion blocking antibody that recognizes a conformational determinant in the lectin domain of L-selectin, showed very weak binding to a construct lacking the SCR domains, suggesting that the SCR domains may be involved in induction of lectin domain conformation and enhancing its activity [26]. Other studies have shown that the mab EL-246, which recognizes both E- and L-selectin in a common region of the SCR domains effectively blocked the function of L- and E-selectin under either static or shear conditions [27]. In addition, in an in vivo homing experiment, pretreatment of lymphocytes with mab EL-246 significantly blocked their ability to home to the peripheral lymph nodes, indicating that the SCR domains are crucial for leukocyte-endothelial interactions [28]. Finally, the role of the SCR domains in L-selectin-dependent activation of signal transduction pathway and generation of second messengers has been demonstrated by cross-linking of L-selectin on neutrophil surface with mab LAM1-14, which recognizes the L-selectin SCR domains [29, 30]. SELECTIN STRUCTURE AND FUNCTION The three members of the selectin family (L-, E-, and P-selectin) are transmembrane glycoproteins that all share a common structural motif including an amino-terminal domain related to those in Ca 2 -dependent (C-type) lectins, followed by an epidermal growth factor (EGF)-like domain, a variable number (two in L-selectin, six in E-selectin, and nine in P-selectin) of short consensus repeats (SCRs) similar to those found in complementregulatory proteins, a transmembrane spanning segment, and a short cytoplasmic region [17 20]. The level of homology between the domains of selectins is 60 65% in the lectin and EGF domains, 40 45% in SCR domains, with no significant homology in transmembrane or cytoplasmic domains. The high degree of homology supports the central role of these domains in the interactions with common carbohydrate determinants on ligands. The lectin domain of selectins plays a major role in ligand recognition. This domain mediates cell-cell contact through Ca 2 -dependent interactions with cell-surface carbohydrates [21]. A small region of the E-selectin lectin domain has been identified that is critical for carbohydrate recognition [22]. The EGF-like domain also plays a role in promoting ligand recognition and cell adhesion, either in stabilizing the conformation of the lectin domain or in directly interacting with the ligand [20]. SELECTIN LIGANDS/COUNTER-RECEPTORS The importance of the carbohydrate structure of selectin ligands is underscored by the recognition of LAD II syndrome, which is characterized by an inherited inability to recruit neutrophils to sites of inflammation [31]. These patients have a defect in fucosylation and have a clinical presentation similar to patients with LAD I. Selectin molecules bind via their lectin domains to specific carbohydrate structures on ligands/counter-receptors that are composed of sialic acid, fucose, galactose, mannose, and/or an anionic sulfate or phosphate ester moiety [18, 32]. The tetrasaccharide sialyl Lewis x and its isomer sialyl-lewis a are binding determinants for the three known selectins (Table 1) [33 36]. Sialyl Lewis x and sialyl Lewis a lose binding activity on removal of sialic acid with neuraminidase or in the absence of fucose incorporation. Other molecules such as sulfated glycolipids, proteoglycans, glycoproteins, and sulfoglucuronyl glycosphingolipids lipids also exhibit ligand activity for selectins [37]. L-selectin binding to its sulfated sugar ligand is dependent on both the position of the sulfate and hydrophobic lipid in the sulfated glycolipids. Sulfoglucuronyl glycosphingolipids lipids are present on brain microvascular endothelium and on human umbilical vein endothelial cells in culture, suggesting a potential 2 Journal of Leukocyte Biology Volume 63, January 1998

3 role for these molecules in regulating tissue-specific binding of subpopulations of leukocytes. All three selectins bind weakly to sialylated, fucosylated carbohydrates. High-affinity glycoprotein ligands for all three selectins have also been identified, suggesting that in vivo each selectin preferentially interacts with specific ligands. Two sulfated glycoproteins, termed Sgp 50 and Sgp 90, have been purified from lymphoid tissues that exhibit high ligand activity for leukocyte L-selectin [38 40]. Both ligands contain many serine and threonine residues, which are potential sites for attachment of O-linked oligosaccharides. Treatment of the ligands with sialidase inhibits recognition by L-selectin. The Sgp 50, glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1), is expressed primarily in lymphoid tissues [41]. It is synthesized by the specialized endothelial cells of high endothelial venules and is secreted into the blood where it may competitively inhibit, rather than promote, L-selectin-mediated adhesion. A carbohydrate structure on GlyCAM-1 (i.e., sulfated, sialylated, and fucosylated tetrasaccharide 6 -sulfo sialyl Lewis x ) has been identified and is believed to compromise the recognition epitope for L-selectin [42]. GlyCAM-1 preferentially binds to naive lymphocytes that may play a key role in normal lymphocyte homing to lymphoid tissues [43]. The Sgp 90 ligand is identical to CD34 and is expressed by endothelial cells of many tissues as well as by hematopoietic stem cells. A third glycosylated mucin-like protein, the mucosal addressin cell adhesion molecule-1 (MadCAM-1), is a ligand for both L-selectin and specific integrin molecules [44]. A fourth sulfate-bearing high endothelial venules (HEVs) ligand for L-selectin has also been described [45]. A high-affinity ligand for P-selectin, termed P-selectin glycoprotein ligand-1 (PSGL-1), has been characterized [46, 47]. PSGL-1 is also a counter-receptor for E-selectin. PSGL-1 is expressed by all blood neutrophils, monocytes, and lymphocytes, however, specific glycosylation is required for ligand functions. Treatment of the native ligand with sialidase abolishes its interaction with its counter-receptor. Another specific glycoprotein, ESL-1 (E-selectin ligand 1), has been identified as a counter-receptor for E-selectin [48, 49]. The interaction of E-selectin with its ligand is Ca 2 -dependent. Sialidase treatment of the ligand abolishes its interaction with E-selectin. THE PHYSIOLOGICAL ROLE OF SELECTINS Leukocyte emigration is essential both in lymphocyte homing as a central part of lymphocyte immune surveillance and in leukocyte invasion at sites of inflammation. The process of leukocyte migration is a dynamic one and involves multiple steps. The adhesion receptors expressed by both leukocytes and endothelium control the multistep process of leukocyte movement from the bloodstream across vascular endothelium and into tissues. Selectins mediate the initial tethering and rolling of leukocytes along the endothelium of the vessel wall [50]. Rolling is essential for subsequent integrin-dependent adherence and transendothelial migration of leukocytes [51]. All three selectins are involved in the recruitment of leukocytes, but there are fundamental differences in their distribution, activation, and mode of expression. Rolling of leukocytes along the endothelial surface under conditions of flow is dependent on the interaction of L-selectin on leukocytes and P-and E-selectin on the endothelial cells with their respective ligands/counter-receptors. L- selectin-dependent leukocyte rolling and attachment is dependent on the activation of endothelial cells with proinflammatory mediators, which induces expression of ligand(s) on vascular endothelium [52 54]. Studies indicate that E- and P-selectin also support leukocyte rolling under conditions of flow [55 58]. Although selectin-mediated attachment accounts for the majority of leukocyte rolling activity, integrin interactions with their ligands may also contribute to leukocyte rolling initiated via the selectins. Experimental studies of knockout selectin-deficient mice suggest the presence of redundant mechanisms regulating leukocyte recruitment [41, 59 62]. In vivo studies have shown that the initial leukocyte rolling shortly after trauma (40 60 min) is P-selectin-dependent, whereas the subsequent ( min) leukocyte-endothelial cell interaction is L-selectin-dependent. The studies collectively demonstrated that there is an obligate requirement of L-selectin for leukocyte localization at sites of inflammation, and that P- and E-selectin play complementary roles in regulating leukocyte-endothelial interactions. L-selectin has a major effect on the accumulation and activation of neutrophils and monocytes at inflamed sites. Although it has been shown that preventing neutrophil migration and activation by L-selectin blockade may affect the subsequent recruitment of lymphocytes, at this time, there is no data indicating that blocking or lack of L-selectin has a major, direct effect on the infiltration of lymphocytes into different inflammatory sites [63]. Studies have shown that selectins play a role in leukocyte homing, a process by which certain subsets of lymphocytes continuously recirculate from the blood into lymphoid organs. Monocytes also leave the blood constantly, at a low rate, to replenish the tissue-macrophage populations, but their extravasation is markedly enhanced at sites of inflammation. Neutrophils, however, only leave the blood at inflammatory sites. Lymphocytes reenter the blood from the lymphoid tissues via the lymphatics and thoracic duct [64, 65]. An average lymphocyte enters the spleen, lymph nodes, or Peyer s patches at least once a day from the blood. It crosses these organs within 4 20 h, and reenters the circulation directly (spleen) or via the lymphatic and thoracic duct [63, 66]. This is the route that is mainly followed by naive, resting lymphocytes. However, to a smaller extent, naive lymphocytes recirculate through non-lymphoid tissues such as lung, liver, or gut. On activation and eventual differentiation into memory cells, the trafficking patterns of lymphocyte populations change dramatically. Their migration now occurs predominantly within non-lymphoid tissues [67]. Studies have clearly shown that L-selectin is a key receptor required for entry of lymphocytes into peripheral lymph nodes. A mab against mouse L-selectin, MEL-14, blocked lymphocyte adhesion to lymph node high endothelial venules (HEV) in vitro and completely suppressed their localization in peripheral lymph nodes in vivo [68]. Other studies have shown that L-selectin also contributes significantly to the recirculation of resting lymphocytes through Peyer s patches and the gut wall, although the blockage of immigration is not as complete as that seen for Crockett-Torabi Selectins and mechanisms of signal transduction 3

4 peripheral lymph nodes [69, 70]. A role for L-selectin has not been detected in the other tissues studied, other than lymph nodes, Peyer s patches, and intestine, at least in the absence of inflammation. It appears that, similar to neutrophil extravasation, L-selectin and its ligands mediate the initial steps in the adhesion cascade, such as tethering and rolling, which are essential in the extravasation of lymphocytes into lymph nodes. Some lines of experimental studies suggest that E-selectin participates in angiogenesis, the growth of new capillaries and postcapillary venules [71]. Angiogenesis is critical for normal growth and development and in protective responses such as wound healing and inflammation. Angiogenesis can occur in a variety of pathological conditions such as the neovascularization of solid tumors, the growth of vessels into the retina in diabetic retinopathy, and the unwanted vessel growth in chronic inflammatory diseases. In vitro studies have shown that antibodies directed against either E-selectin or sialylated fucosylated oligosaccharides inhibit the formation of capillary-like tubes [72, 73]. Moreover, synthetic analogs of sialyl Lewis x inhibit angiogenesis in vitro and in vivo [74]. Furthermore, it has been demonstrated that E-selectin is expressed in proliferating endothelial cells in infantile hemangioma tumors and in other noninflammatory angiogenic tissues such as the human placenta [75]. The levels of E-selectin in hemangiomas, but not P-selectin, correlated with angiogenesis. Other studies indicate that exogenously added soluble E-selectin induced angiogenesis in the rat cornea, and stimulated chemotaxis of human endothelial cells in vitro, which was blocked by anti-sialyl Lewis x mab [76]. Although these studies clearly indicate that E-selectin is associated with essential components of angiogenesis, endothelial cell proliferation, migration, capillary tube formation, and neovascularization, the mechanism(s) by which E-selectin contributes to these processes is unclear. Experimental models of knockout mutations in genes for selectin molecules have demonstrated that selectin-deficient mice are viable and have no developmental defects [41, 60 62]. Considering the essential role that the microvasculature plays in organ development and function, and the lack of developmental defects in selectin-deficient mice, several issues, including the possibility of overlapping functions, or compensation between genes, are highlighted [59, 71]. TRANSMEMBRANE SIGNALING THROUGH SELECTINS Although the role of selectins in ligand binding and rolling has been extensively studied, their potential roles in intracellular signaling and leukocyte activation have only recently been described. Leukocyte rolling and adhesion to the endothelium are dynamic processes and involve multiple steps, including cell tethering and rolling, activation, and arrest (i.e., firm adhesion). As discussed earlier, the selectins mediate the initial contact and rolling of leukocytes on the vascular endothelium, which precedes firm adhesion and transendothelial migration. Firm binding to endothelium and transendothelial migration occur only when the 2 -integrin receptors become activated and bind to their ligands. However, the mechanism(s) by which the cellular environment alters the functional state of 2 -integrins is not known. In the conventional model of leukocyte recruitment, the action of inflammatory mediators (e.g., chemoattractants, cytokines) activates leukocytes, which leads to 2 -integrin-dependent firm adhesion and transendothelial migration. It was later hypothesized that binding of L-selectin molecules on leukocytes to their ligand may initiate intracellular signal(s), leading to the modulation of leukocytes 2 -integrin-dependent adhesion and responses (Fig. 1) [7, 29]. Several studies supported the hypothesis that selectins can initiate signal(s) leading to the enhanced adhesiveness of leukocytes. Initial studies demonstrated that a soluble E-selectin mutant can serve as a chemoattractant for neutrophils in a Boyden chamber assay and activate 2 -integrin (i.e., CD11b/ CD18: Mac-1) on neutrophils [77]. Interaction of unstimulated neutrophils with purified ELAM-1 on endothelial cells or with purified recombinant ELAM-1 on a culture surface enhanced the adhesive activity of Mac-1-dependent adhesion of C3bi-coated erythrocytes to neutrophils. This effect was presumed to be the result of E-selectin binding to its leukocyte-associated carbohydrate counter-receptor initiating intracellular signals, which caused conformational changes in Mac-1. Subsequent studies did support the initial observation demonstrating that crosslinking of L-selectin with anti-l-selectin mab on the neutrophil surface primed the cells and induced an up-regulation of surface Mac-1 expression on neutrophils [29]. Other studies have demonstrated that stimulus-induced 2 -integrin-mediated neutrophil aggregation was L-selectin dependent [78]. Furthermore, cross-linking of L-selectin with anti-l-selectin mab (DREG- 200) induced significant adhesion and transmigration of neutrophils across human umbilical vein endothelial cells that were 2 -integrin dependent [79]. Moreover, it has been reported that cross-linking of L-selectin on the neutrophil surface can induce a sufficient level of activation of both Mac-1 and LFA-1 to promote stationary adhesion to ICAM-1 under conditions of flow in an in vitro model [80]. Other studies have shown that cross-linking of lymphocytes L-selectin with MEL-14 mab induces homotypic lymphocyte adhesion by a mechanism independent of LFA-1 [81]. A recent study indicated that GlyCAM-1, a physiological ligand for L-selectin, induced 2 -integrin activation on naive, but not memory peripheral lymphocytes, suggesting a preferential activation of a subpopulation of lymphocytes via L-selectin [43]. As mentioned earlier, GlyCAM-1 is a ligand for L-selectin that is secreted into blood. Although premature, the results of this study may explain why under normal conditions only naive lymphocytes, and not memory T cells, bind to HEV and continuously patrol between blood and the lymphoid tissues in search of antigens. Studies have shown that L-selectin-dependent activation of neutrophils leads to the activation of respiratory burst and O 2 generation [29]. In other studies, L-selectin cross-linking potentiated the oxidative burst induced by neutrophils stimulated with fmlp and tumor necrosis factor (TNF) [82]. The generation of oxygen radicals by neutrophils on L-selectin stimulation may play an important role in neutrophil-endothelial cell interaction. Previous studies indicate that O 2 is an important mediator of leukocyte-endothelial cell interactions after reperfusion of ischemic tissues [83]. Other studies have shown that hydrogen peroxide-induced synthesis of platelet-activating factor by endo- 4 Journal of Leukocyte Biology Volume 63, January 1998

5 Fig. 1. Regulation of leukocyte adhesion and functional responses through selectins. Ligation of selectin molecules on leukocytes initiates transmembrane signals that modulate specific functional responses, including b2integrin. [Ca21]i, intracellular calcium; PTKs, protein tyrosine kinases; R, receptor. thelium is associated with enhanced endothelial-dependent neutrophil adhesion [84]. Rolling of leukocytes is diminished by superoxide dismutase and the action of reactive oxygen metabolites on endothelial cells is suggested to play a role in this process [83, 85]. The O22 generation by neutrophils after L-selectin interaction with its ligand may have a physiological role in the regulation of neutrophil transmigration rather than a pathological role. The data collectively suggest that L-selectin may regulate neutrophil-endothelial cell interactions both by direct cell-cell binding as well as indirectly secondary to reactive oxygen metabolite production by neutrophils. Ligation of neutrophil L-selectin with sulfatides has been shown to induce increased expression of TNF-a and interleukin-8 (IL-8) [86]. TNF-a plays a major role in host defense and exerts its effect on neutrophils, as well as other inflammatory cells, by binding to TNF receptors [87, 88]. Cross-linking of L-selectin on neutrophil surface with anti-l-selectin mab has been shown to down-regulate TNF receptors [89]. TNF is a proinflammatory mediator. Its role in the development of tissue injury in various experimental models of tissue injury has been clearly indicated [90]. TNF exhibits an autocrine effect on neutrophils as well as pleiotropic paracrine effects on endothelial and tissue cells. TNF-a stimulates synthesis of the endothelial adhesion protein (E-selectin). It has also been shown that TNF-a treatment induces P-selectin-dependent rolling in arterioles that requires E-selectin for rolling at normal velocities [91]. IL-8 is a cytokine with powerful chemotactic and activating effects on phagocytes, which has been implicated as a key mediator of many inflammatory disorders [92]. The major role of IL-8 is to induce, specifically, the migration of neutrophils to the site of inflammation with the subsequent activation of neutrophils leading to the generation of O22 and degranulation; processes directly associated with the inflammatory responses. Although premature, the data suggest that in vivo L-selectin ligation might activate gene transcription resulting in cytokine secretion by the inflammatory cells, leading to amplification of inflammatory responses. Several studies underscore the role of P- and E-selectin in the activation of signaling transduction pathways in leukocytes. It has been shown that P-selectin-dependent platelet adhesion to neutrophils and monocytes generated O22, which was dependent on carbohydrate ligand sialyl Lewisx interaction with P-selectin Crockett-Torabi Selectins and mechanisms of signal transduction 5

6 [93, 94]. Cross-linking of the counter-receptor for P-selectin was required for the generation of a positive signal, since the soluble form of P-selectin did not mimic the effect. Other studies have shown that the leukocyte carbohydrate (CHO) Ag CD15, which is a ligand for P- and E-selectin, plays a role in leukocyte signal transduction [95]. Large amounts of complex CHO Ag are expressed on human monocyte and neutrophil surfaces, which mediate adhesion of these cells to activated platelets and endothelium [96, 97]. Cross-linking of CHO Ag on human neutrophils and monocytes induced cytoplasmic calcium fluxes, activation of the respiratory burst, and an increase in cell surface expression of granule-associated proteins (i.e., CD11b, and CD67) [95]. It has also been demonstrated that P-selectin mediates the binding of platelets and endothelial cells with monocytes and neutrophils, as well as regulating the expression of tissue factor on monocytes [98]. In addition, a juxtacrine interaction of endothelial cells with neutrophils through P- selectin molecules has been described [99, 100]. This process requires the regulated expression of P-selectin and plateletactivating factor (PAF) on the endothelial cells and the presence of neutrophils. The binding of PAF to neutrophils enhances LFA-1- and Mac-1-dependent adhesion. Other studies have demonstrated that binding of E- and P-selectins by specific antibodies induced dramatic morphological changes of IL-1- or thrombin-activated human endothelial cells, suggesting intercellular gap formation [101]. The formation of transient gaps between endothelial cells appears to be a key step in leukocyte transmigration [102, 103]. It was observed that binding of anti-e-selectin antibodies to activated endothelial cells also induced a transient increase in [Ca 2 ] i from intracellular stores, but not with the anti-p-selectin antibody. These results suggest that E- and P-selectin exhibit different signaling capacities, which may reflect differences in the structures of the transmembrane and cytoplasmic domains of these molecules. The above-noted studies collectively denote that selectins are not just passive bystander molecules that function only to tether leukocytes to endothelium but also initiate signal(s) that can activate and modulate the regulation of 2 -integrin adhesion responses, and, possibly, other molecules and functions. The biochemical signal transduction pathways utilized in response to selectin-mediated leukocyte activation and functional responses have not yet been clearly defined. One of the primary mechanisms by which leukocytes are activated may be directly initiated by the activation of receptor protein tyrosine kinases (PTKs) at the cell surface (Fig. 2) [ ]. Alternatively, signaling through receptors that lack intrinsic tyrosine activity may proceed indirectly via activation of membrane-associated cytoplasmic non-receptor PTKs. Ligandstimulated cell signaling may also be initiated through either classical heterotrimeric guanosine triphosphate (GTP)-binding proteins (G-proteins), or small Ras-like GTP-binding proteins [107]. These initial events may be linked to one of several downstream signaling elements such as phospholipase C (PLC) [ ]. Activation of PLC mediates breakdown of membrane phosphoinositides, including phosphatidyl inositol 4,5-bisphosphate (PIP 2 ), generating the second messengers 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP 3 ). IP 3 mediates the release of intracellular calcium ([Ca] i ) from intracellular stores, and DAG activates protein kinase C (PKC). Three families of PLC (i.e., PLC-, PLC-, and PLC- ) have been described. The activation of these enzymes is known to be dependent on coupling with pertussis toxin-sensitive or -insensitive G-proteins, and on the activity of tyrosine kinase(s), intrinsic to receptors or activated by receptors [110]. The mobilization of calcium and activation of PKC in stimulated leukocytes precede, and have been shown to play an important role in initiation of multiple cellular responses, including adherence, chemotaxis, respiratory burst, degranulation, and cytokine production. Other receptors may initiate signaling pathways that activate mitogen-activated protein kinases (referred to as MAP kinase or extracellular signal-regulated kinase, ERK) [114, 115]. MAP kinases are a family of 40- to 45-kDa kinases that exist as a dephosphorylated form in quiescent cells or in unstimulated cells. MAP kinases are activated in response to various mitogenic stimuli, including epidermal growth factor, platelet-derived growth factor, insulin, and phorbol esters. MAP kinase becomes activated when both tyrosine and threonine residues are phosphorylated. The direct upstream activator of MAP kinase is a serine/threonine/tyrosine kinase that is a dual-specificity kinase called MAP kinase kinase. MAP kinase is a cytoplasmic protein, which is also located within the nucleus, Thus, MAP kinase provides a physical link in the signal transduction pathway from the cytoplasm to the nucleus. Various molecules involved in signal transduction have been shown to be regulated by MAP kinase, including transcription factors, cytosolic phopholipase A 2 (cpla 2 ), and microtubule dynamics. cpla 2, is a key enzyme for the generation of arachidonic acid (AA) and its metabolism in response to extracellular stimuli plays an essential role in inflammation [110, 116]. Calcium is an intracellular messenger involved in many physiological cell functions. Changes in [Ca] i may occur secondary to the activation of multiple types of ion channels and by the release of calcium from intracellular calcium stores (e.g., specialized areas of the endoplasmic reticulum). The presence of tightly regulated local changes in intracellular calcium and the presence of [Ca] i oscillations in response to a gradient of chemotactic stimulus suggests that modulation of [Ca] i may play a critical role in regulating dynamic changes in adhesion molecule binding avidity as well as cell adhesion molecule expression during cell migration [7]. This may occur secondary to translocation of intracellular pools of adhesion molecules to the cell surface or, potentially, through modification of adhesion molecule coupling to the cytoskeleton. Recent studies have demonstrated an increase in the [Ca] i level in leukocytes mediated through selectins. Cross-linking of L-selectin on the surface of human neutrophils using Fab fragments of anti-l-selectin mabs and F(ab ) 2 fragments of a secondary cross-linking antibody resulted in a dose-dependent increase in [Ca] i and activation of the respiratory burst with O 2 generation [29, 82, 86]. Cross-linking of the mab bound to L-selectin with the secondary antibody was required for the initiation of the calcium signal. Similarly, as mentioned earlier, cross-linking of the counter-receptor for P-selectin-mediated O 2 generation was also required because the soluble form of P-selectin did not mimic the effect [93, 94]. In addition, ligation 6 Journal of Leukocyte Biology Volume 63, January 1998

7 Fig. 2. Schematic representation of signaling pathways in leukocytes. AA, arachidonic acid; [Ca]i, intracellular calcium; camp, cyclic adenosine monophosphate; DAG, 1,2-diacylglycerol; GAPs, GTPase activating proteins; GRFs, guanine nucleotide regulatory factors; IP3, inositol 1,4,5-trisphosphate; L, ligand; LT, leukotrienes; LX, lipoxins; MAP, mitogen-activated protein; NF-AT, nuclear factor of activated T cells; PA, phosphatidic acid; PC, phosphatidyl choline; PIP2, phosphatidyl inositol 4,5-bisphosphate; PKC, protein kinase C; PLC, phospholipase C; PLD, phospholipase D; PTK, protein tyrosine kinase; R, receptor. of neutrophil L-selectin with its ligand, sulfatides, has been shown to induce a dose-dependent increase in [Ca]i. The increase of [Ca]i induced by sulfatides was due to release from intracellular stores. Sulfatides are 3-sulfated galactosylceramides that bear heterogeneous fatty acyl substitution on the sphingosine moiety and are good ligands for L- and P-selectins but do not bind E-selectin [ ]. Furthermore, three other L-selectin ligands, i.e., fucoidan, mannose 6-phosphate-rich phosphomannan (PPME), and cerebrosides, did not induce any measurable [Ca]i response in neutrophils [29, 86]. In contrast, no effect on [Ca]i was observed after exposure of neutrophils to lysosulfatides (which lack the fatty acid group) or galactosylceramides (which lack the sulfate group). Similarly, no increase in the level of [Ca]i concentration was detected when L-selectin molecules on neutrophils were cross-linked with mab Leu-8, although flow cytometry analysis indicated similar levels of the antibody binding to the Crockett-Torabi Selectins and mechanisms of signal transduction 7

8 L-selectin molecules on the surface of the neutrophils compared with the other mabs that did induce calcium signals [our unpublished observation]. Moreover, the intensity of cellular responses to different clones of the mabs against L-selectin was different as reported by other studies [29, 31, 38, 45, 121]. All these data collectively suggest that distinct interactions between the ligand/mab and different regions of the L-selectin molecule are required for initiating L-selectin-dependent signal transduction. Alternatively, the differences of effects of specific mabs/ ligands may reflect variability in mab/ligand binding affinity for L-selectin molecule or the mab/ligand s accessibility to a specific region of the L-selectin molecule (e.g., structural folding of L-selectin molecule). Sulfatides, which have shown stimulatory properties, have also been shown to exhibit anti-inflammatory effects in selectindependent acute lung injury [122]. In contrast, fucoidin, which has not shown any stimulatory activity, i.e., at least for the induction of [Ca] i response in neutrophils, has shown a significant inhibitory effect on leukocyte rolling during ischemiareperfusion [123, 124]. Another study has shown that ligation of L-selectin with fucoidan induced activation of tyrosine MAP kinase in Jurkat cells [125]. These results suggest, for example in vivo, a blocking agent, which inhibits the rolling of leukocytes and migration, may indeed also alter the functional activity of the target leukocytes. In addition, a blocking agent may activate different signal transduction pathways within various cell types under different experimental conditions. For example, it has been shown that GlyCAM-1, a physiological ligand for L- selectin, preferentially binds to naive peripheral lymphocytes, but not to memory T cells, leading to 2 -integrin activation [43]. Thus, a critical interpretation of data should be considered in the evaluation of therapeutic agents that are being examined to block selectin-dependent functions. Phorbol esters such as phorbol myristate acetate (PMA) have been shown to directly activate PKC in intact cells. Neutrophils stimulated with PMA show enhanced shedding of L-selectin as well as increased Mac-1 expression and dependent binding [126]. Similarly, stimulation of lymphocytes with PMA causes increased LFA-1 and 1 -integrin-dependent adhesion [127]. Thus, PKC appears to represent one common pathway by which extracellular signals may regulate leukocyte adhesion. PKC comprises a large family of proteins with multiple isozymes that exhibit an ability to respond to multiple second messengers in vitro and therefore are capable of integrating messages transmitted through several signaling pathways (Fig. 2) [110]. Some PKC isozymes may play a role in regulating membrane functions such as down-regulation of receptors, ion channels, membrane phospholipid metabolism, gene expression, and the control of the cell cycle. For instance, it has been demonstrated that neutrophil granule exocytosis is under the control of the PKC signaling pathway and thereby may directly regulate Mac-1 expression on the cell surface [128, 129]. Furthermore, the rapid translocation of P-selectin to the surface of endothelial cells, which is controlled by the regulated secretion of Weibel-Palade bodies, is dependent on PKC activation [130]. Thus, it is likely that PKC and the subsequent phosphorylation of its substrates plays a critical role in the regulation of both selectin- and integrindependent adhesion. We have observed that pretreatment of human neutrophils with an inhibitor of PKC, staurosporine, significantly diminished O 2 generated after cross-linking of L-selectin molecules on neutrophils (Fig. 3). However, another study has demonstrated that L-selectin-induced integrin- Fig. 3. Effect of tyrosine kinase and protein kinase C inhibitors on O 2 generation by human neutrophils. Neutrophils ( ) preincubated in the absence or presence of Fab fragments of anti-l-selectin mabs (i.e., DREG-200, LAM1-14, DREG-56, or MsIgG), for 30 min on ice. After washing, the neutrophils in the absence or presence of genistein (20 µg/ml) or staurosporine (1 µm) equilibrated at 37 C for 10 min, and surface L-selectin molecules were cross-linked using F(ab ) 2 fragments of the secondary goat antimouse antibody in the presence of cytochrome C. Results are expressed as nmoles O 2 generated by neutrophils in 20 min. Solid bars, neutrophils incubated in buffer (HBSS) with no inhibitor; open bars, neutrophils pretreated with genistein; hatched bars, neutrophils pretreated with staurosporine. Final concentration of fmlp 50 nm; PMA 50 nm. Figure is representative of three separate experiments. 8 Journal of Leukocyte Biology Volume 63, January 1998

9 dependent T cell adhesion to recombinant immobilized cell adhesion molecules (i.e., ICAM-1, ICAM-3, VCAM-1, and fibronectin) was relatively independent of PKC activity, but dependent on PTK activity [131]. Although premature, these results may reflect differential signal transduction pathway activation via L-selectin in the regulation of various functional responses. The importance of kinase activation and protein phosphorylation in induction of E-selectin has been suggested by the observation of the inhibitory effect of the kinase inhibitor on the expression of the gene encoding E-selectin in response to TNF-, IL-1, and LPS [132]. It has been reported that activation of protein kinase A (PKA) inhibits E-selectin induction by both TNF- and IL-1 [133, 134]. The physiological relevance of this observation is emphasized by the finding that the binding of neutrophils to endothelial cells, induced with LPS, is inhibited by drugs that elevate cellular camp levels [135]. Other studies indicate that adenosine and adenylate cylase agonists (e.g., prostaglandin E 1 and prostacyclin) will also inhibit neutrophil O 2 production, degranulation, adhesion to endothelial cell monolayers in vitro, as well as decrease neutrophil recruitment and tissue injury in a variety of neutrophildependent inflammatory conditions [136, 137] (Fig. 2). Thus, it is clear that the local modulation of adenosine and camp within leukocytes and endothelial cells represents a potential target for therapeutic intervention for modulating leukocyte recruitment at sites of tissue injury. Recent studies indicate that the protein tyrosine kinasedependent pathway plays a key role in selectin-dependent leukocyte activation [30, 125, 131, 138, 139]. Ligation of L-selectin with sulfatides or cross-linking of L-selectin with anti-l-selectin mabs (i.e., LAM1-14, DREG-200, and GREG- 56) on the surface of neutrophils induced tyrosine phosphorylation of a variety of cellular proteins including MAP kinase, and PLC [30, 139, and unpublished results]. The responses were significantly inhibited by the protein tyrosine kinase inhibitor, genistein. Similarly, genistein blocked the transient increase in [Ca] i and the O 2 induced by the ligation of L-selectin with sulfatides or cross-linking of L-selectin with anti-l-selectin mab (Fig. 3). The kinetics of tyrosine phosphorylation, which preceded changes in the [Ca] i level and the inhibition of [Ca] i with the tyrosine kinase inhibitor, genistein, suggest that the [Ca] i signal is dependent on, and is located downstream from, PTK activation. In addition, U73122, an inhibitor of PLC, blocked the transient increase in [Ca] i, indicating that the IP3 generated via PLC activation mediated the calcium response [140] (Fig. 2). Furthermore, this finding supports the previous report, which demonstrated that L-selectin mediated the transient increase in [Ca] i due to release of calcium from intracellular stores [86]. One possible intracellular substrate candidate for PTK activation is PLC, which is present in neutrophils and is regulated by tyrosine kinases [141]. Indeed, our recent studies have demonstrated the association of tyrosine phosphorylation of PLC with L-selectin-mediated signal transduction, leading to phosphoinositide hydrolysis, [Ca] i changes, and O 2 generation by human neutrophils [unpublished results]. Similarly, it has been demonstrated that in human T cells, L-selectin, in association with T cell receptor (TCR)/CD3 complex, enhanced phosphatidylinositol hydrolysis and protein tyrosine phosphorylation [142]. Stimulation of PLC activity by the ligand receptor complex is dependent on the activation of G-proteins in leukocytes. Studies have shown that a pertussis toxin-sensitive G-protein is required for lymphocyte emigration in mouse peripheral lymph node and Peyer s patch HEVs [143]. Although pertussis toxin treatment had no effect on the initial rolling interaction of lymphocytes with HEVs, it completely inhibited an activation-dependent sticking event required for lymphocyte arrest and emigration [144]. Similarly, the rise in [Ca] i after exposure of neutrophils to sulfatides or L-selectin mab (DREG-200) immobilized to Staphylococcus aureus was insensitive to pertussis toxin treatment [86]. These results indicate the presence of multiple signaling pathways in leukocytes coupled to L-selectin-dependent binding and subsequent cell rolling and adherence. Moreover, as noted above, several studies noted the importance of the biochemical compositions of the ligands in the induction of L-selectin-dependent adhesion. It has also been demonstrated that soluble and insoluble stimuli activate neutrophils through two different pathways; pertussis toxin-sensitive and pertussis toxin-insensitive G-protein-dependent pathways, respectively [145]. Therefore, depending on the nature of stimuli and the experimental conditions, a cautious analysis of the results in the evaluation of the role of G-proteins in L-selectin-dependent leukocyte activation should be considered. Although the role of P-selectin in the initiation of signal transduction has been suggested, the mechanism of signaling has not been identified. It has been shown that degranulation of platelets and endothelial cells is accompanied by the phosphorylation of P-selectin [98]. Phosphorylation of P-selectin occurs on serine, threonine, tyrosine, and histidine residues upon platelet activation. However, the physiological role of this event is not currently known. L-selectin-mediated signal transduction has also been demonstrated in canine neutrophils. Cross-linking of canine neutrophil L-selectin using anti-l-selectin antibody induced a rapid and transient increase in [Ca] i levels and O 2 generation, which were dependent on the extent of L-selectin cross-linking. The responses were significantly inhibited by the protein PTK inhibitor, genistein, suggesting that ligation of canine neutrophil L-selectin is coupled to intracellular signal transduction pathways and the generation of second messengers, which are associated with tyrosine kinase activation [138]. It has also been shown that L-selectin is involved in the initial adhesion of canine neutrophils to cytokine-stimulated endothelial cells under flow conditions [146]. Canine models have been extensively used to study the role of neutrophils in mediating myocardial ischemia-reflow injury and the contribution of the adhesion molecules to neutrophil adhesion and activation in the myocardial inflammatory responses [11, 147, 148]. Studies have reported success in protecting the tissue damage after ischemia-reperfusion by administrating neutralizing antibodies to selectin or 2 -integrin molecules [11, ]. These studies indicate that canine neutrophils behave similarly to human neutrophils with regard to L-selectin-dependent activation, which provide a suitable animal model for the study of the role of neutrophils and their adhesion interactions in mediating inflammatory tissue injury. Crockett-Torabi Selectins and mechanisms of signal transduction 9

10 In T lymphocytes antigen-dependent activation occurs secondary to binding to the TCR, and stimulation of receptor-coupled PTKs (Fig. 2). Several tyrosine kinases of the src (i.e., lck and fyn) and syk (i.e. Syk and ZAP-70) families play key roles in T cell signaling [112, 151]. Activation of PTKs in T lymphocytes has induced the phosphorylation of PLC- 1 and stimulation of phosphoinositide metabolism leading to increases in [Ca] i and PKC activity [ ]. Both PKC activation and increases in [Ca] i are associated with increases in LFA-1 and 1 -integrin binding avidity in T lymphocytes [156]. Furthermore, increases in [Ca] i result in enhanced activity of the calmodulin-dependent protein serine/threonine phosphatase calcineurin (Fig. 2). Calcineurin is the target of the immunosuppression drug cyclosporin and acts on the transcription factor, nuclear factor of activated T cells (NF-AT), facilitating its transport to the nucleus where it regulates transcription of several genes, including interleukin-2 (IL-2). A second T cell intracellular signaling pathway that is activated by the TCR is the p21 ras /Raf-1/MAP kinase system (Fig. 2) [114, 115]. The RAS family of proteins are guanine nucleotide-binding proteins whose activity is determined by the rate of exchange of bound GDP for GTP and the rate of hydrolysis of bound GTP. The rate of exchange of bound GDP for GTP is regulated by a family of guanine nucleotide regulatory factors (GRFs), whereas the hydrolysis of GTP bound to RAS is controlled by GTPase-activating proteins (GAPs). In T lymphocytes, TCR-associated PTKs activate p21 ras independent of the calcium and PKC pathways. This activation potentially occurs secondary to phosphorylation-dependent stimulation of GRFs (e.g., Vav) or inhibition of p21 ras -GAPs. Activation of GTP-bound p21 ras leads to the subsequent activation of a cascade of mitogen-activated protein kinases, including a MAP kinase kinase kinase (MAPKKK; e.g., RAF-1), MAP kinase kinase (MAPKK), and MAP kinase (MAPK, e.g., ERK1 and ERK2). The MAP kinases phosphorylate and modulate the activity of specific transcription factors and thereby regulate gene expression. Furthermore, there are mitogen kinase phosphatases (e.g., MKP-1) that may function in vivo as regulators of RAS/MAPK gene activation. The TCR/RAS/MAPK pathway has been suggested as playing a key role in regulating antigen-dependent lymphokine production in T lymphocytes. Although it has been clear that TCR-dependent activation of T lymphocytes results in increased LFA-1-dependent adhesion, it was not known until recently whether biochemical mechanisms similar to TCR stimulation of calcineurin or the RAS/MAPK pathway might regulate adhesion molecule expression and turnover in T lymphocytes or other leukocyte populations. A recent study has demonstrated that L-selectin activates the Ras/MAPK pathway via the tyrosine kinase p56 lck in human peripheral blood lymphocytes as well as in Jurkat cells [125]. Cross-linking of L-selectin on lymphocytes or Jurkat cells with anti-l-selectin mab, i.e., Dreg 56, or stimulation with fucoidan or sialyl-lewis x, initiated a signaling cascade from L-selectin via the tyrosine kinase p56 lck to Sos, Ras, MAPK, and Rac2, resulting in O 2 generation. The data suggested that first, p56 lck functions upstream of Ras and Rac2, second, that Ras is upstream of Rac2 and O 2 generation, and third, that O 2 generation is regulated by Rac2 on L-selectin triggering. It appears that p56 lck plays a role in rolling of leukocytes because rolling of p56 lck -deficient JCaM1.6 cells exhibited an almost complete absence of rolling in a fucoidan-coated flow channel, whereas Jurkat cells and p56 lck -reconstituted JCaM1.6 cells showed rolling on fucoidan in vitro. Rac-2 is one of the critical cytosolic components of the NADPH oxidase that interacts with a membrane-bound cytochrome b to generate O 2 [157]. It has been shown that tyrosine kinase activity is required for activation of Rac-2 in human neutrophils for the generation of O 2 [158]. These results demonstrate a potentially important role of tyrosine phosphorylation in the signal transduction mechanisms that underlie selectin-dependent leukocyte-endothelial interactions. The importance of MAP kinases in a wide variety of intracellular signaling pathways has been demonstrated [115]. As noted above, two separate studies have demonstrated that cross-linking of L-selectin molecules with anti-l-selectin mab or ligation of L-selectin with sulfatides induced tyrosine phosphorylation of multiple proteins including MAP kinase in human neutrophils. The responses were inhibited by the pretreatment of neutrophils with the tyrosine kinase inhibitor, genistein. Another study has shown tyrosine phosphorylation of MAP kinase in Jurkat cells on L-selectin stimulation [125]. The significance of the activation of MAP kinase in L-selectin-dependent leukocyte activation is as yet unknown. As mentioned earlier, MAP kinases are involved in signal transduction regulating various molecules, including transcription factors, microtubule dynamics, and cytosolic phospholipase A 2 (cpla 2 ; Fig. 2). MAP kinases regulate the activity of cpla 2, a key enzyme for the generation of AA and its metabolites that include prostaglandins, thromboxanes, leukotrienes (LT), lipoxins (LX), as well as other oxygenated fatty acids. The central role of AA metabolites in hypersensitivity reaction and inflammation has been extensively studied. Leukotriene B 4, is a chemotactic factor for neutrophils and causes downregulation of surface L-selectin and activation of Mac-1 on neutrophils, thereby potentially regulating L-selectin function(s) [159]. It is interesting to note that an inverse relationship between the amount of leukotrienes and lipoxins biosynthesis has been established in human leukocytes in that, during lipoxin formation, leukotriene biosynthesis is blocked [160]. Lipoxins are potent anti-inflammatory molecules. Lipoxin A4 blocks the vasoconstrictive action of leukotriene-d 4 and inhibits leukocytedependent inflammation [160, 161]. In addition, 15-HpETE, an AA metabolite (i.e., lipoxygenase pathway), inhibits formation of TNF, thus serving as the down-regulator of a key proinflammatory cytokine [162, 163]. Interest in AA biosynthesis pathways is heightened by the finding that aspirin actually triggers biosynthesis of lipoxins, i.e., 15-epi-LX. It appears that these novel aspirin-triggered compounds mediate, at least in part, some of the beneficial aspects of aspirin by diminishing leukocyte adhesion [164]. The role played by these intracellular signaling molecules in selectin-dependent adhesion and activation of leukocytes, and the regulation of functional responses and leukocyte-endothelial interactions are key topics for the future. CONCLUSIONS Over the past 10 years there has been a rapid expansion in our knowledge regarding the molecular interactions involved in the 10 Journal of Leukocyte Biology Volume 63, January 1998