Integrin Signalling in Neutrophils and Macrophages

Cell. Signal. Vol. 11, No. 9, pp. 621–635, 1999 Copyright  1999 Elsevier Science Inc.

ISSN 0898-6568/99 $ – see front matter PII S0898-6568(99)00003-0

TOPICAL REVIEW

Integrin Signalling in Neutrophils and Macrophages Giorgio Berton*† and Clifford A. Lowell‡ †Institute of General Pathology, University of Verona, Verona, Italy; and ‡Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA 94143, USA

ABSTRACT. Integrins have been characterized extensively as adhesion receptors capable of transducing signals inside the cell. In myelomonocytic cells, integrin-mediated adhesive interactions regulate different selective cell responses, such as transmigration into the inflammatory site, cytokine secretion, production or reactive oxygen intermediates, degranulation and phagocytosis. In the last few years, great progress has been made in elucidating mechanisms of signal transduction by integrins in neutrophils and macrophages. This review summarises the current information on the role of integrins in regulating myelomonocytic cell functions and highlights the signalling pathways activated by integrin engagement in these cells. Also, exploiting the current knowledge of mechanisms of integrin signal transduction in other cell types, we propose a model to explain how integrins transduce signals inside neutrophils and macrophages, and how signaling pathways leading to regulation of selective cell functions may be coordinated. cell signal 11;9:621–635, 1999.  1999 Elsevier Science Inc. KEY WORDS. Integrin, Neutrophil, Macrophage, Signal transduction, Tyrosine kinases

INTRODUCTION Integrins are heterodimeric transmembrane proteins which are expressed on all nucleated cells. These molecules are the primary receptors which regulate cell adhesion to extracellular matrix (ECM) proteins as well as intercellular cell–cell adhesive interactions. There are approximately 17 different integrin a chains and eight different b chains which pair together in specific patterns depending on the cell types in which they are expressed. The presence of a particular a/b pair on the cell surface confers the ability to bind the specific ECM proteins or counter receptors on other cells. Beyond their role in regulating cell adhesion, it has become clear that integrins transduce signals inside the cell which regulate the rearrangement of the actin cytoskeleton, cell movement, activation of specific cellular functions, gene transcription, cell proliferation and survival. These intracellular signals collaborate with signals transduced from growth factor receptors, cytokine receptors and other transmembrane receptors to regulate many anchorage-dependent cellular properties. Polymorphonuclear leukocytes (PMNs) and monocyte/ macrophages play a central role in innate immunity against pathogens as well as being the major cellular effectors of inflammation and tissue injury. Over the last few years, it has been appreciated that integrin-mediated adhesive interactions are essential in regulating the functions of these inflammatory cells. The purpose of this review is to summarise *Author to whom correspondence should be addressed. Tel: 139-45-8098120; fax: 139-45-809-8172. Received 27 July 1998; and accepted 14 September 1998.

the current information on the role of integrins in controlling PMN and macrophage function, with a special emphasis on the mechanisms by which integrins transduce signals inside these cells. Several recent reviews on this topic have been published which the reader can refer to for more detailed discussion of early findings [1–4]. INTEGRINS EXPRESSED BY PMNS AND MONOCYTES/MACROPHAGES Table 1 lists integrins expressed by phagocytic cells and their respective ligands. A selected number of references are included in the table and the reader can refer to other reviews for a more complete list of references (see [5–40]). The cell surface expression of many of the integrins listed in Table 1 is dramatically influenced by exposure of phagocytic cells to proinflammatory agonists. In particular the majority of PMN b2 integrins are stored in intracellular vesicles; following activation of PMNs, these vesicles fuse to the plasma membrane resulting in upregulation of b2 expression. Activation of phagocytic cells by proinflammatory mediators (bacterial products, chemokines, TNF-a, as examples) also results in conformational changes in the integrins which enhance their ability to bind ligands. This process is referred to as integrin “affinity modulation” or “inside-out” signalling. A number of cell surface receptors and integrin-associated proteins have been shown to contribute to the signalling reactions which regulate integrin affinity. These topics are not covered here but have been recently reviewed in other reports [4, 7, 41, 42]. This report

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G. Berton and C. A. Lowell TABLE 1. Integrins expressed by phagocytic cells

Integrins b1 a4/b1 (CD49d/CD29; VLA-4) a5/b1 (CD49e/CD29; VLA-5) a6/b1 (CD49f/CD29; VLA-6) b2 aL/b2 (CD11a/CD18; LFA-1) aM/b2 (CD11b/CD18; CR3; Mac-1)

ax/b2 (CD11c/CD18; gp150/95) aD/b2 (CD11d/CD18) b3 av/b3 (CD51/CD61)

Ligands Fibronectin, VCAM-1 (CD106) Fibronectin Laminin ICAM-1, ICAM-2, ICAM-3 ICAM-1, ICAM-2, C3bi, Fibrinogen, Factor X, High molecular weight kinogen, Filamentous hemagglutinin of bordetella pertussis, lipophosphoglycan and gp63 of leishmania mexicana, Histoplasma capsulatum, Lipopolysaccharide, b-glucan, Heparin, Neutrophil inhibitory factor (NIF), Oligodeoxynucleotides, Elastase Fibrinogen, Lipopolysaccharide, C3bi ICAM-3 Vitronectin, Entactin and other RGD- and KGAGDV containing ECM proteins

Ligands for b1 integrins have been reviewed in [5–8]. For expression of b1 integrins by PMNs see [9–13]. Ligands for b2 integrins are listed in [7, 14–35]. Ligands for b3 integrins are reviewed in [5, 6, 36–40].

focuses primarily on the signalling events that occur postligand binding which lead to changes in cell shape and function—the “outside-in” signal transduction pathways. IDENTIFICATION OF THE INTEGRIN aMb2 AS A MOLECULE SIGNALLING FOR PHAGOCYTOSIS The evidence that complement receptor type 3 (CR3) (i.e., the leukocyte integrin aMb2) can mediate particle engulfment by phagocytic cells represents perhaps the first demonstration of the signalling capacity of an integrin. Although the capability of receptors for complement fragments to mediate particle engulfment by macrophages was recognised over 20 years ago [43–45] the demonstration that aMb2 can mediate phagocytosis had to wait for the establishment of methods to coat model particles, such as IgM-bound sheep erythrocytes, with specific complement fragments generated by proteolytic activation of C3 to C3b and then C3bi [46–48]. Internalisation of C3bi-C3b-coated erythrocytes by macrophages is finely regulated. Resident mouse peritoneal macrophages or human PMNs and monocytes bind, but do not ingest C3b/bi-coated erythrocytes [46–48]. However, treatment of murine macrophages with T cell-derived molecules or human phagocytes with PMA, and cultivation of monocytes/macrophages on some surfaces, “activate” the C3b/bi receptors, promoting particle ingestion [46, 47, 49–51]. Importantly, “activation” of aMb2 to mediate ingestion may be induced by certain polysaccharides present on the surface of yeasts which interact with a binding site on aMb2 distinct from the C3bi binding site. Ross and colleagues characterised in detail the role of a b-glucan binding site in regulating the phagocytic ability of aMb2 [27, 52], thus elucidating why C3bi-coated yeast

particles are efficiently internalised by PMNs and macrophages, while C3bi-coated erythrocytes are not. Hence, the ability of aMb2 to mediate particle ingestion may be regulated either by “inside-out” mechanisms or via ligation of a distinct binding site present in its extracellular domain. It must be noted that the internalisation of C3bi-coated erythrocytes can be completely dissociated from triggering of other selective functions which occur in coincidence with engulfment of immunoglobulin G-coated erythrocytes, most likely due to the fact that the intracellular signal transduction pathways elicited by Fcg receptors are very different than those induced by aMb2 cross-linking. In fact, even under conditions in which aMb2 is able to promote phagocytosis of C3bi-coated erythrocytes, release of reactive oxygen intermediates or arachidonic acid does not occur [53, 54]. Again, co-ligation of the b-glucan binding site, which occurs when C3bi-coated yeast particles are used as phagocytic stimuli, renders aMb2 fully capable of triggering also phagocyte and NK cells effector functions [52]. The unique role of aMb2 in mediating phagocyte responses is also highlighted by the evidence that this receptor is physically associated with surface molecules anchored to the external leaflet of the plasma membrane by a glycosylphosphatidylinositol (GPI) linkage, such as FcgRIII (CD16), the urokinase-type plasminogen activator receptor (u-PAR, CD87) and the lipopolysaccharide receptor CD14 (reviewed in [55, 56]). Association of GPI-linked molecules with aMb2, as well as with other b2 integrins, suggests that these integrins can serve as signal transduction partners for surface molecules which are otherwise incapable of transducing signals intracellularly. Importantly, cooperation between aMb2 and FcgRIII has been demonstrated to induce FcgRII-dependent reactive oxygen intermediates (ROI) generation in PMNs [57].

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FIGURE 1. Phagocyte functions that are regulated by integrin clustering. Extracellular matrix proteins (ECM), cell counter receptors

(ICAMs-1-3, VCAM-1) or complement derived fragments (C3bi) can induce integrin clustering and the generation of intracellular signals (see text and Fig. 2) which trigger a wide array of cell responses. The cellular responses induced by integrin engagement in phagocytic cells play an essential role in innate immunity as well as inflammation-dependent tissue injury.

REGULATION OF PHAGOCYTE FUNCTIONS BY INTEGRINS After identification of aMb2 as a molecule which could signal a phagocytic response, additional findings have implicated integrins in regulation of other PMN functions (Fig. 1). Role of Integrins in Phagocyte Transmigration from Blood to Tissues There is strong evidence demonstrating an essential role for b2 integrins in PMN accumulation at sites of inflammation

in vivo. In humans, the recessive genetic disease leukocyte adhesion deficiency syndrome (LAD I) occurs because of mutations in the b2 integrin gene which cause loss of b2 expression. These patients experience recurrent infections, have a marked leukocytosis and impaired formation of inflammatory exudates [58]. In mice with knockouts of the genes encoding the b2 subunit or ICAM-1, the major b2 integrin endothelial counter-receptor, PMN recruitment into sites of inflammation is impaired [59–62]. Finally, antibodies against b2 integrins or ICAM-1 hamper PMN recruitment into tissues in animal models or inflammation

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(reviewed in [3, 8, 63, 64]). PMN movement and transmigration has also been shown to depend on b3 integrins. The integrin aVb3 dictates movement of PMNs over a vitronectin surface [65]. The 50-kDa b3 integrin associated protein (IAP, CD47), which serves as the primary signal transducer for PMN b3 integrins, is involved in PMN transendothelial migration both in vitro and in vivo as IAP-deficient mice have a defect in PMN accumulation at sites of infection [66, 67]. In monocytes, the primary adhesive interactions with endothelial cells involve b1 and b2 integrins, which recognise the counter-receptor VCAM-1 and ICAM-1, respectively. Blockade of b1 and b2 interactions by mAb treatment inhibits extravasation of monocytes into inflammatory sites [68–73]. Integrins may regulate phagocyte chemotaxis and transmigration by different mechanisms. Integrin-dependent firm adhesion on endothelial cells can be viewed as an essential first step in the process by which PMNs exit the vasculature and migrate into tissues. The process of firm cell adhesion to substratas depends on more than just the formation of high-affinity/avidity integrin-counter-receptor interactions. Integrin engagement triggers signalling that leads to the rearrangement of the actin cytoskeleton which is required for cells to spread fully over surfaces such as ECM containing basement membranes or vascular endothelial cells. The capability of PMNs and other cells to spread fully over surfaces is likely to be the major determinant of the ability of these cells to adhere firmly to the surface. Integrin signalling leading to cytoskeleton rearrangements, cell spreading and firm adhesion has just started to be elucidated and is addressed below. It is unlikely that the role of firm adhesion/spreading in PMN transmigration is simply to arrest the cells in the microcirculation thus allowing them to sense a chemotactic gradient. Integrin-mediated substratum interactions also directly contribute to the migratory capability of cells and govern the actual speed of cell migration in a fairly complex manner [74]. Integrin-transfected CHO cells migrate at low speeds when they are either fully spread or completely unspread and rounded; maximum migratory speed is attained when they are moderately spread [74]. Perturbation of integrin-induced signalling can also impair migration in vivo. For example, fibroblasts isolated from the focal adhesion kinase (pp120fak or FAK)-deficient mice have reduced migration of fibronectin-coated surfaces [75]; FAK is the major cytoplasmic tyrosine kinase recognised as a key component of integrin signal transduction in mesenchymal and epithelial cells [76, 77]. In addition, FAK overexpression increases cell migration [78] while a dominant-negative FAK form decreases it [79]. The degree of cell spreading can also regulate PMN migration. Inhibition of PMN migration through fibrin gels in response to chemoattractants correlates with enhanced spreading, again suggesting that full cell spreading and firm adhesion may lead to reduced migration [80]. Additionally, anti-b3 integrin antibodies were shown to restore the motility of neutrophils stuck on extracellular matrix proteins as

G. Berton and C. A. Lowell

a consequence of inhibition of [Ca21]i transients [81, 82]. On the other hand, LAD I PMNs are defective in spreading, they are incapable of orienting in a chemotactic gradient and they fail to migrate [83, 84], again indicating that the balance of cell adhesion regulates cell motility. Similarly, entactin, a b3 integrin ligand, has been shown to stimulate PMN chemotaxis directly [85, 86]. Integrin-dependent activation of selective phagocyte functions may contribute to regulation of transendothelial transmigration through direct effects on the endothelial layer. Recent findings have demonstrated that PMN adhesion induces, by a yet unidentified mechanism, disorganisation of endothelial adherens junctions [87]. Integrin-Dependent Cytoskeleton Rearrangement in Phagocytic Cells Upon interaction with integrin counter-receptors present on endothelial cells and extracellular matrix proteins in the subendothelial space, phagocytes reorganise their cytoskeleton and spread over the adhesive surface. Spreading of PMNs on b2 integrin ligands requires activation of integrin affinity/avidity by chemoattractants or cytokines [41, 42]. However, human and murine mononuclear phagocytes can spread on different surfaces upon cultivation in serum-containing media even in the absence of additional stimuli, although treatment with phorbol esters enhances macrophage spreading in an integrin-dependent manner (see [4] and references contained therein). A primary requirement for b2 integrins in PMN spreading is evidenced by the fact that chemoattractant-induced cell spreading is defective in LAD I PMNs [83, 84] and anti-aMb2 antibodies hamper murine peritoneal macrophage spreading on glass in serumcontaining medium [88]. However, b3 and b1 integrins probably play the major role in phagocyte spreading over vitronectin or laminin, respectively [4, 81]. Cytoskeleton rearrangements underlying PMN spreading can also be induced in the absence of receptor agonists which induce integrin affinity modulation, thus suggesting that the signalling reactions which induce high-affinity integrin binding are separate from the signalling pathways involved in cell spreading/firm adhesion. For example, substitution of Mg21 and Ca21 of the medium with Mn21, a direct activator of integrin adhesiveness, induces PMN spreading (see [89] and references quoted therein). Moreover, plating of human and murine PMNs on surface-bound anti-b2 or -b3 integrin antibodies induces cell spreading without the need of additional stimuli [90, 91]. Cell spreading requires actin polymerisation and is blocked by cytochalasins [90, 92–94]. Besides actin, other cytoskeletal proteins are certainly involved in the formation of cytoskeletal structures underlying phagocyte spreading and the establishment of tight adhesion sites. The actinbinding protein filamin (ABP-280) binds directly to the b2 subunit [95], whereas binding of a-actinin, another actinbinding protein, requires PMN stimulation with inflammatory agonists [96]. The initiation of intracellular signalling

Integrin Signalling in Neutrophils and Macrophages

events (discussed in detail below) by tyrosine kinases is a central event in phagocyte cell spreading and actin polymerisation. This was first demonstrated using tyrosine kinase inhibitors, which block phagocyte adhesion, spreading and activation, but has been formally proved in studies using knockout mice lacking members of the Src-family kinases found in PMNs. The phagocytes from such mutant animals have an impaired ability to reorganize their actin cytoskeleton following integrin ligation, which leads to reduced adhesion-dependent respiratory burst [91] and granule secretion [91a]. The direct effect of impaired phagocyte migration in the knockout mice is demonstrated in an in vivo inflammation model in which animals are injected intraperitoneally with bacterial lipopolysaccharide and the number of PMNs invading the liver parenchyma is determined. In normal animals, large numbers of PMNs marginate along the vascular endothelium and invade the liver where they contribute significantly to liver damage during endotoxemia. This process is blocked in PMNs lacking the Src-family kinases Hck and Fgr [97] (see below for further discussion of these kinases) and as a result the mutant animals have significantly reduced tissue damage following LPS treatment. A number of cytoskeletal-associated proteins become tyrosine phosphorylated in phagocytes following integrin ligation. For example, paxillin is tyrosine phsophorylated in a b2 integrin-dependent manner in PMN [98, 99] and this correlates with spreading in cells transfected with aMb2 [98]. Other cytoskeletal proteins which have been involved in integrin-mediated phagocyte spreading include vinvulin, talin and MacMARKS [100–102]. Activation of PMN Effector Functions by Integrins A significant advance in the elucidation of the role integrins play in phagocyte activation derived from the demonstration that adherence to extracellular matrix proteins or endothelial counter-receptors enables PMNs to release ROI in response to some cytokines such as TNF, GM-CSF and G-CSF [92, 103]. Following these early studies, several reports demonstrated that ECM-protein adherent PMN stimulated with inflammatory mediators release granule constituents as well as ROI [93, 94, 104–108], (reviewed in [3]). Importantly, these responses were demonstrated to depend on b2 integrins because they were shown to be defective in LAD I PMNs and inhibited by antibodies directed against b2 integrins [104, 105, 107]. Since both integrin-mediated PMN adherence and inflammatory agonist stimulation are required to induce ROI production and granule release, the relative importance of these two signalling pathways is an open question. As discussed previously [3], the most likely role of the inflammatory agonist is to induce formation of the high-affinity state of the integrin (“inside-out” signalling) while the direct binding and clustering of integrins on the cell surface induces the signalling reactions that are required for the functional response (the “outside-in” signalling). The model that “outside-in” signalling reactions produced by integrin

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clustering are the principle mediators of PMN functional responses is supported by the observation that integrin engagement with surface-bound anti-b2 and anti-b3 mAbs is sufficient to induce PMN respiratory burst in the absence of inflammatory agonist stimulation in both human and murine cells [90, 91, 109–111]. Triggering of ROI generation by adhesion is strictly dependent on rearrangement of the actin cytoskeleton, as inhibition of actin polymerisation by cytochalasins completely inhibits ROI generation in TNF-stimulated PMNs adherent to integrin ligands or surface-bound anti-b2 mAbs [90, 92, 94]. Likewise, inhibition of proximal signalling events such as tyrosine phosphorylation, that lead to actin cytoskeletal rearrangement will also block respiratory burst in TNF-stimulated adherent PMNs [91, 99, 111]. Hence, integrin-dependent assembly of NADPH oxidase, the multicomponent enzymatic system responsible for oxygen reduction to superoxide anion [112], requires formation of a signalling complex based on the actin cytoskeleton. ROI generation in PMNs spreading over integrin ligands or anti-b2 integrin antibodies is also coupled to activation of ion fluxes. During PMN spreading a burst of intracellular acid production occurs which activates the Na1/H1 ion pump resulting in intracellular alkalinisation [113]. Interestingly, adhesion-dependent PMN responses are preceded by a striking decrease of [Cl]i, which appears to regulate both spreading and ROI generation [114]. Activation of Macrophage Gene Transcription by Integrin-Mediated Adhesion Haskill and colleagues were the first to identify adherence as a powerful inducer of gene expression in human monocytes [115, 116] (see [1, 117] for a detailed review of early studies). Several subsequent reports demonstrated selective adhesion-dependent induction of genes encoding for proinflammatory cytokines, such as TNF, IL-1, chemokines, tissue factor and transcription-associated proteins [118, 123], (reviewed in [1, 117]). Cross-linking of the b1 integrin a4b1 (VLA-4), but not b2 integrins, with soluble antibodies mimics the effect of adhesion in inducing gene expression [120, 122]. However, immobilised b2 integrin ligands or antibodies were also found to be effective either as direct stimuli or as enhancers of the monocyte transcriptional response to other agonists [118, 119, 121, 122]. Integrins and Apoptosis in Phagocytic Cells Apoptosis induced by cell detachment from the extracellular matrix, an event that is now termed “anoikis” [124, 125], has been demonstrated to occur in many different cell types. Upon cell anchorage and spreading, the intracellular signals from the integrins have been implicated in rescue from apoptosis [124–126]. Because apoptosis of PMNs has been characterised in some detail as an important determinant in resolution of inflammation [127], it is of great significance that the role of integrins in regulating PMN apoptosis has

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just started to be addressed. The first findings on this topic revealed that in mice deficient for the aMb2 integrin, PMNs elicited into the peritoneal cavity by thioglycollate injection displayed a delay in apoptosis [128]. This study also highlighted a possible mechanism responsible for enhanced survival of aMb2-deficient PMNs. The aMb2-deficient PMNs were also found to have a reduced capability to generate ROI; superoxide and other toxic oxygen radicals may directly affect the viability of PMNs. Indeed, in human PMNs in which ROI production is blocked, either by treatment with inhibitors of NADPH oxidase or in PMNs obtained from chronic granulomatous disease (CGD) patients which lack NADPH oxidase, there is a corresponding reduction in apoptosis. More recent results led, however, to a constrasting conclusion regarding the role of b2 integrins in regulation of PMN apoptosis [129]. In fact, Watson et al. [129] showed that in vivo and in vitro transendothelial transmigration results in a delay of apoptosis of LPS-treated PMNs; cross-linking of aLb2 or aMb2 also delayed apoptosis. However, neither of these effects were correlated with reduced ROI production, suggesting that the effect of integrin signalling on regulating PMN apoptosis is independent of ROI production. The possible reasons for these contrasting results are at present unclear. However, since thioglyocollate-elicited intraperitoneal accumulation of PMNs in aMb2-deficient mice is primarily dependent on aLb2 [130], and since engagement of aLb2 delays PMN apoptosis [129], the delay in apoptosis seen in aMb2 mutant PMNs may be due to a predominance of signals ensuing from aLb2. However, it must also be considered that aMb2 cross-linking has been reported to potentiate TNF-induced apoptosis [131], a finding which would be concordant with studies in aMb2-deficient mice. It is also important to note that integrin signalling involves tyrosine kinases in phagocytic cells (see below), and tyrosine phosphorylation has been shown to regulate granulocyte apoptosis in a complex manner, acting either as an inducer or an inhibitor of apoptosis [132]. The role of integrin signalling in regulating PMN apoptosis is an issue in which further investigation is clearly warranted. SIGNAL TRANSDUCTION BY INTEGRINS IN PHAGOCYTIC CELLS: THE TYROSINE KINASE CONNECTION In the last few years great progress has been made in elucidating the mechanisms of signal transduction by integrins (summarised in Fig. 2). The current knowledge points to an essential role of cytoplasmic tyrosine kinases in integrin signalling leading to cytoskeleton rearrangements, cell growth and survival, and motility [126, 133–135]. Tyrosine kinases have also been implicated in signalling by phagocyte integrins. Early studies showed that PMN spreading over integrin ligands in response to TNF or over surface-bound anti-b2 integrin antibodies is accompanied by tyrosine phosphorylation of several proteins [136, 137]. Importantly, tyrosine kinase inhibitors block integrin-dependent PMN

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spreading and ROI generation [111, 136]. Adhesion to extracellular matrix proteins or antibody engagement of b1 integrins in monocytes or monocytic cells also results in triggering of protein tyrosine phosphorylation; tyrosine kinase inhibitors block b1 integrin-mediated gene induction in these cells [138, 139]. Proteins whose tyrosine phosphorylation is increased following integrin engagement in phagocytic cells have just started to be identified. These include: the cytoskeletal associated proteins paxillin [98, 99, 140] as well as tensin and cortactin; the migogen-activated protein kinases ERK1/ ERK2 [141]; phospholipase Cg2 [142]; Vav [143, 144]; the proto-oncogene product p120c-cbl [145] and the cytoplasmic tyrosine kinase p58c-fgr [137], p72syk [139, 146] and p125FAK [140]. Binding of collagen Type I to aLb2 results in tyrosine phosphorylation of both the b and a LFA-1 subunits [14]. This finding, together with the recent evidence that the cytoplasmic tail of the b3 integrin subunit is tyrosine phosphorylated following engagement of aVb3 with ligands or antibodies [147], points to a possible role of integrin cytoplasmic tail phosphorylation in integrin signal transduction. TYROSINE KINASES IMPLICATED IN INTEGRIN SIGNALLING IN PHAGOCYTIC CELLS Three classes of tyrosine kinases have been implicated as the major signal transducers by integrins, the FAK-family kinases, p125FAK and Pyk2, the Src-family kinases, and p72syk. Although p125FAK was reported to be expressed in human PMNs, monocytes and monocytic cell lines [99, 140, 148– 150], other investigations have failed to detect either expression of this kinase in human and mouse monocyte/ macrophages [138, 151, 152] or an increase in its tyrosine phosphorylation following integrin-dependent adhesion [99] and (Suen P.W., Ilic D., Caveggion E., Berton G., Damsky C.H., Lowell C.A., unpublished observation). Hence other tyroskine kinases, possibly the FAK-homologue Pyk2, which is expressed in myeloid cells and which has been implicated in integrin signalling in hematopoietic cells [153– 156], may be playing a dominant role in integrin signalling in phagocytic cells. In addition to the FAK-family tyrosine kinases, accumulating evidence points toward a critical role for Src-family kinases in signalling by integrins in phagocytic cells. The major Src-family kinases expressed in neutrophils and monocytes/macrophages are p59/61hck (Hck), p58c-fgr (Fgr), p53/56lyn (Lyn) and p60c-src (Src). Early studies showed that the tyrosine phosphorylation and specific activity of Fgr increased during PMN spreading over fibrinogen [137]. Activation of Fgr was found to be strictly dependent on b2 integrins because it did not occur in LAD 1 PMNs and it was hampered by soluble anti-b2 integrin antibodies. Subsequent studies identified a strict correlation between PMN adhesion/spreading and Fgr or Lyn specific kinase activity [89]. Additionally, these two kinases, as well as a number of major tyrosine phosphorylated proteins, redistribute to the

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FIGURE 2. Integrin signalling pathways in phagocytic cells: a working hypothesis. Integrin clustering activates cytoplasmic tyrosine

kinases by a yet unidentified mechanism (see text). Phosphorylation of different downstream targets induces activation of distinct signalling pathways. Hydrolysis of phosphatidyl inositol lipids by phosphoinositidase C-g isoforms (PLC-g) or their phosphorylation in the D3 position by phosphoinositide-3-OH kinases (PI(3) kinase) may induce activation of various protein kinase C isoforms (PKC), phospholipase D (PLD) and other protein kinases [226]. Tyrosine kinases may activate Ras and Rho GTPases though different signalling pathways (see text) as well as phosphorylate cytoskeletal proteins implicated in formation of integrin-associated multimolecular complexes. The coordinated activation of these signalling pathways underlies integrin-dependent regulation of phagocyte adhesion, cell spreading, movement and secretion of pro-inflammatory mediators.

Triton-X100-insoluble cytoskeletal fraction during PMN spreading on fibrinogen [89, 157, 158]. Importantly, the cation Mn21, an activator of integrin affinity, induced PMN spreading over fibrinogen and an increase of Fgr and Lyn kinase specific activities in the absence of additional stimuli,

thus suggesting that “outside-in” integrin signalling is sufficient to activate Src-family kinases [89]. We provided direct evidence that Src-family kinases are implicated in integrin signalling in PMNs analysing integrin-dependent spreading and respiratory burst in PMNs isolated from hck2/2 fgr2/2

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double mutant mice [91]. hck2/2fgr2/2 PMNs incubated on fibrinogen, fibronectin, collagen or ICAM-1 do not spread and fail to release significant quantities or ROIs in response to TNF or FMLP. Additionally, while wild-type PMNs spread and produce superoxide anion if plated over surfacebound anti-b2 or anti-b3 integrin antibodies, hck2/2 fgr2/2 PMNS fail to respond to integrin cross-linking. Similar integrin-mediated cell spreading defects have been observed in hck2/2fgr2/2 macrophages. Double mutant macrophages display a delay in spreading when plated over fibronectin and are defective in tyrosine phosphorylation of several proteins, including the cytoskeletal proteins paxillin, tensin and cortactin. Moreover, the actin cytoskeletal structure and the subcellular localization of paxillin, tensin and talin are altered in hck2/2fgr2/2 macrophages. A role for Src-family kinases in integrin signalling in macrophages is also suggested by studies on osteoclasts, macrophagederived polycarions specializing in bone resorption. The observation that src2/2 knockout mice develop osteopetrosis pointed to an essential role of Src in osteoclast function [159]. More recently, it has been found that adhesion of osteoclasts via integrin aVb3 to the bone matrix protein osteopontin enhances Src activity and promotes formation of complexes between aVb3, Src and PI(3)-kinase [160]. Interestingly, in murine macrophage cell lines integrin-mediated adhesion was shown to activate Src and PI3-kinase via the proto-oncogene product p120c-bcl [145], which has been identified as a Src substrate in ostoeclasts as well [161]. In primary murine peritoneal macrophages, the Src-family kinases Hck, Fgr and Lyn are principally responsible for c-Cbl tyrosine phosphorylation; in the absence of c-Cbl phosphorylation association of PI(3) kinase with c-Cbl and translocation of this complex to the cytoskeleton is blocked [162]. The impaired translocation of PI(3) kinase may impair macrophage cell spreading in a fashion similar to that which is observed with treatment of cells with the PI(3) kinase inhibitor wortmannin (see below). The tyrosine kinase p72syk, which is essential in B-cell antigen receptor signalling, was originally implicated in integrin signalling in platelets [163]. Later studies demonstrated a possible role of Syk in integrin-mediated tyrosine phosphorylation and cytokine message induction in a monocytic cell line [139]. Interestingly, this study reported that integrin-dependent tyrosine phosphorylation of Syk is independent of actin polymerisation and cell spreading because it is not affected by prior exposure of cells to cytochalasin D. In contrast, tyrosine phosphorylation of FAK and paxillin requires actin filament assembly and was strongly inhibited by cytochalasin D. Additionally, tyrosine phosphorylation of Syk, but not FAK and paxillin, was induced by anti-b1 integrin antibodies. These findings would put Syk upstream of FAK and actin polymerisation in integrin signalling in phagocytic cells, a conclusion which also derived from studies on integrin signalling in platelets [163]. Cooperation between Syk/ZAP-70 and Src-family kinases is essential in antigen receptor signalling in lymphocytes [164, 165], hence a similar interaction may occur in phagocytes. Interestingly,

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integrin signalling in PMNs activates Syk, and induces the formation of protein complexes containing Syk, Fgr, Lyn, tyrosine phosphorylated proteins and b2 integrins [146]. In addition, tyrosine phosphorylation of Syk is significantly reduced in hck2/2fgr2/2 macrophages adherent to fibronectin. Therefore, it is possible that Syk plays a direct role, in conjunction with Src-family kinases, in integrin signalling in phagocytic cells, a hypothesis which awaits validation through examination of syk2/2 PMNs and macrophages. Interestingly, a recent report has demonstrated that treatment of PMNs with the relatively Syk-specific pharmacological inhibitor piceatannol blocks spreading and ROI release [166]. MECHANISMS OF ACTIVATION OF TYROSINE KINASES BY INTEGRINS Although the studies summarised above demonstrate that activation of tyrosine kinases is essential for integrin signalling in phagocytic cells, the mechanisms by which these tyrosine kinases become enzymatically activated in adherent cells are unknown. A similar conundrum also exists in nonhematopoietic cells [133, 134]. This contrasts with the great progress which has been made in elucidating mechanisms of tyrosine kinase activation by other surface molecules such as growth factor receptors, which have an intrinsic tyrosine kinase activity, or immune receptors, which bind intracellular tyrosine kinases at their cytoplasmic tail. The difficulty in understanding mechanisms of activation of tyrosine kinases by integrins is probably due to the fact that in their “active” state (i.e., following ligand binding and aggregation) integrins become part of multimolecular complex containing cytoskeletal as well as signal transduction molecules [133, 134]. What comes first, aggregation of cytoskeletal proteins or binding and activation of tyrosine kinases? There is no evidence for a direct binding of a tyrosine kinase by an integrin in vivo. However, integrin cytoplasmic sequences can bind to FAK, as well as to cytoskeletal proteins, in vitro [167]. Moreover, both FAK and cytoskeletal proteins are rapidly redistributed to sites of integrin clustering [168–170] in adhering cells. An interesting model has been recently proposed to reconcile these differences between the in vitro and the in vivo findings [171]. According to the model, during cell adhesion/spreading, focal adhesion complexes are formed by the clustering of integrins and cytoskeletal proteins such as talin and paxillin. FAK is recruited to these complexes by interaction of its C-terminal domain with paxillin. Upon binding to paxillin (or other cytoskeletal proteins) FAK would then undergo a conformational change to allow its N-terminal domain to interact with the cytoplasmic tails of integrin b subunits. This conformational change would both activate FAK kinase activity and strengthen the interaction of FAK with the integrin-cytoskeletal protein complexes. This model is compatible with the view that integrin-dependent actin polymerisation proceeds in steps which can be distinguished based on the dependence or independence of tyrosine kinase activity. For example, in some fibroblast cell lines re-

Integrin Signalling in Neutrophils and Macrophages

distribution of FAK and a subset of cytoskeletal proteins to sites of integrin clustering is not blocked by tyrosine kinase inhibitors, although these inhibitors can effectively prevent actin polymerisation and assembly of other signalling molecules [132, 133, 168–170]. In phagocytic cells, where the Src-family kinases rather than FAK play the dominant role in integrin signalling, the mechanism of enzyme activation during adhesion is even less clear. Studies with inhibitors [111, 136] and hck2/2fgr2/2 PMNs [91] have established that Src-family kinases are essential for the formation of an actin-based cytoskeleton. Moreover, the phosphotyrosine phosphatase inhibitor vanadyl hydroperoxide, which induces an increase in tyrosine phosphorylation of several proteins, directly triggers PMN spreading [172]. In lymphocytes, the activity of Src-family kinases is believed to be primarily regulated by the tyrosine phosphatase CD45. This enzyme dephosphorylates the negative regulatory tyrosine in the kinase domain of Src-family kinases which results in activation of the kinase activity [173]. Recent results suggested the existence of a complex relationship between Src-family kinases, CD45 and regulation of cytoskeletal dynamics in macrophages. In contrast to what would otherwise be expected, Roach et al. [152] have found that Hck and Lyn are hyperactivated, not repressed, in CD452/2 macrophages. CD45 deficiency results in an increased rate of macrophage spreading and adhesion, but also in a subsequent loss of adhesion with cell detachment from the adherent surface. Thus the simple model for Src-family kinase activation that is proposed in lymphocytes is probably not occurring in adhering phagocytes. As seen with FAK in fibroblasts, redistribution of Src-family kinases and Syk to a Triton X-100-insoluble cytoskeletal fraction and formation of protein complexes containing b2 integrins and Syk has been observed upon integrin engagement in PMNs [146]. Interestingly, redistribution of some cytoskeletal proteins, Fgr, Lyn and Syk, following b2 integrin engagement with antibodies precedes de novo acting polymerisation [174], thus suggesting that in PMNs as well as in fibroblasts integrin engagement induces nucleation of cytoskeletal proteins and tyrosine kinases following which there is activation of kinase activity which signals further events leading to actin polymerisation. CA21 SIGNALLING AND PHOSPHOLIPID TURNOVER Several reports have demonstrated that engagement of b2 integrins by appropriate ligands or antibodies triggers increases in [Ca21]i in PMNs [175–179]. Additionally, the b3 integrin-associated protein (IAP) was identified as a membrane molecule mediating Ca21 influx [180]. More recently, [Ca21]i transients observed during engagement of aMb2 in PMN have been reinvestigated using confocal laser scanning microscopy [181]. This study identified two phases of integrin-mediated [Ca21]i signalling: an early increase of [Ca21]i localised at points of limited integrin engagement during the initial contact, and second global [Ca21]i in-

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crease due to further integrin engagement resulting from spreading of the cell. Interestingly, this study showed that while localised [Ca21]i increases depend on Ca21 mobilization from intracellular stores, the global [Ca21]i increase derives from influx from the extracellular medium. Additionally, the two [Ca21]i responses could be distinguished on the basis of their different sensitivities to cytochalasin B, the localised [Ca21]i signalling being insensitive and the global [Ca21]i signalling being inhibited by this drug. In their report, Pettit and Hallet [181] conclude that integrin-dependent [Ca21]i fluxes demonstrated in previous studies correspond to the global [Ca21]i increase they identified. This is conceivable in light of the evidence of previous studies which suggested that [Ca21]i signals deriving from integrin engagement are mainly due to influx from the extracellular medium and, on the basis of their sensitivity to cytochalsin B, are secondary to actin polymerisation (reviewed in [3]). It is likely that the two waves of [Ca21]i fluxes resulting from integrin engagement serve different functions. Because blunting of the early, localised Ca21 response by intracellular chelators blocked PMN tight adhesion and spreading [181], it seems that this type of [Ca21]i transient is required for formation of an actin-based cytoskeleton. In contrast, the second wave of [Ca21]i flux, which occurs after actin polymerisation, may be implicated in severing of actin filaments and detachment of integrin from the adherent surface at the rear of the cell (i.e., in cell movement) [65, 81]. While the role of [Ca21]i increases in regulating these latter events have been ascribed to its capability to activate the actin-filament-severing protein gelsolin and the calmodulin-dependent phosphatase calcineurin [81, 182] it is not known how a localised [Ca]i increase can induce cell spreading. Recent findings established an important link between activation of tyrosine kinases and [Ca21]i signals by integrins. Integrin engagement leads to tyrosine phosphorylation of the g2 isoform of phospholipase C (PLCg2) and the accumulation of inositol (1,4,5)P3 in PMNs [142]. Hence, PLCg2 tyrosine phosphorylation and activation may be one of the mechanisms responsible for integrin-induced phosphoinositide turnover with generation of inositol(1,4,5)P3 and release of Ca21 from intracellular stores. Ca21 release from intracellular stores may then signal Ca21 channel opening at the plasma membrane level following reorganization of the actin cytoskeleton. Integrin ligation in PMNs has been reported to induce phospholipid turnover with generation of diacylglycerol via activation of phospholipase D (PLD) [183, 184]. Additionally, integrin-dependent activation of ROI generation and lactoferrin secretion in PMNs coincides with PLD-mediated diacylglycerol formation and is inhibited by blocking PLD activity [184]. Many different signalling pathways can lead to PLD activation in PMNs and other cell types [185, 186], including increases in [Ca21]i, activation of protein kinase C (PKC), tyrosine kinases, the small GTP-binding proteins ADP-ribosylation factor (ARF) and Rho GTPases. Hence, integrin signalling may induce PLD activation by

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several mechanisms. In the light of the recent identification of a new, mammalian PLD isoform (PLD2) which regulates cytoskeleton dynamics [187], further study of integrindependent PLD activation in phagocytic cells will be needed. Accumulating evidence points toward an important role of D3 phosphorylated inositol lipids in regulation of integrin adhesiveness and integrin signalling. Early studies demonstrated aIIbb3-dependent increase in PI(3,4)P2 synthesis, and PI(3) kinase activation and redistribution to the membrane skeleton in stimulated platelets [188, 189]. Subsequent studies have also implicated PI(3) kinase in signalling leading to activation of integrin adhesiveness in other cell types [190–192]. Importantly, as discussed below in more detail, cytohesin-1, a protein associated with the b2 subunit and regulating aLb2-dependent adhesion to ICAM-1 [193], binds to PI(3,4,5)P3 via its pleckstrin homology domain. PI(3) kinase activation may also play a critical role in “outside-in” signalling ensuing from integrin ligation. Indeed, antibody cross-linking of b2 integrins induces accumulation of D3 phosphoinositides in PMN [194], and integrin-mediated adhesion induces formation of complexes between Cbl, Src-family kinase and PI3-kinase resulting in PI3-kinase activation in macrophages [145, 162]. In addition, PI(3) kinase-dependent cytoskeleton rearrangements induced by phorbol esters in platelets are in part dependent on the function of the integrin aIIbb3 [195]; likewise inhibition of PI(3) kinase blocks cytoskeletal dynamics in bone-adherent osteoclasts [196]. It is not surprising that PI(3) kinase may play a role in both “inside-out” and “outside-in” integrin signalling. In fact, PI(3) kinase has been placed upstream of Rho GTPases (see below) and Rho regulates chemoattractant induced integrin activation in leukocytes—an “inside-out” signalling pathway [197]. On the other hand, there is evidence that integrin signalling leading to cytoskeleton rearrangements—“outside-in” signalling—may require PI(3) kinase and Rho GTPase activities as well (see below). For example, tyrosine kinases may activate PI(3)-kinase, directly or indirectly, via activation of Ras (see below [198]) or phosphorylation of c-Cbl. In PMNs, integrin-mediated signalling results in activation of Lyn [89], which has been implicated in the activation PI(3) kinase [199, 200] and the Rho GTPases. In contrast, chemoattractant responses are initiated by seven-transmembrane receptors that depend on trimeric Gai-type small GTPases for PI(3) kinase activation, which in turn would activate Rho GTPases. Thus different signalling pathways may converge on PI(3) kinase activation. SMALL GTP-BINDING PROTEINS AND INTEGRIN SIGNALLING Ras-Dependent MAP Kinase Pathway Integrin ligation has been demonstrated to activate the mitogen-activated protein kinase (MAPK) cascade, primarily the extracellular regulated kinases (ERK1 and ERK2), members of the MAPK cascade, in several different cell

G. Berton and C. A. Lowell

types. For example, in monocytes, cross-linking of a4b1 increases ERK1/ERK2 tyrosine phosphorylation and activity, and treatment of these cells with inhibitors to MEK-1 (the enzyme which directly activates the ERKs) abolishes ERK activation [141]. Likewise, b2-mediated adhesion/spreading of TNF-stimulated PMNs leads to ERK2 activation [201]. The activation of this pathway is primarily dependent on Ras activation [202–207]. In PMNs, b2 cross-linking leads to an increase of GTP-bound Ras and Vav tyrosine phosphorylation, as well as a tyrosine phosphorylation-dependent association between Vav and Ras [143]. Hence, b2 integrins may signal for Ras-dependent MAP kinase pathway activation through tyrosine phosphorylation of Vav (Fig. 2). Although it is generally accepted that tyrosine phosphorylation events are involved in Ras-dependent integrin signalling to ERKs, there is still some controversy concerning which tyrosine kinases signal to Ras activation. For example, FAK, together with Src, has been recently reported to be involved in integrin signalling to ERK2 [207], but other studies excluded a role for FAK in this pathway [171, 206, 208]. Likewise, normal (or even enhanced) integrinmediated activation of ERKs is seen in hck2/2fgr2/2lyn2/2 mutant macrophages, cells which are devoid of all major Src-family kinase activity [162], questioning the role of these kinases in integrin signalling to the MAPK pathway. Rho GTPases Members of the mammalian Rho GTPase family, Rho, Rac and Cdc42, are considered to play a central role in organising the actin cytoskeleton in response to different receptor agonists such as growth factors and trimeric GTP-binding protein-coupled receptors [209, 210]. Additionally, they have been implicated in regulating integrin clustering in fibroblasts [211] and chemoattractant-stimulated integrindependent adhesion in leukocytes [197]. However, despite the established role of integrins in regulating cytoskeleton dynamics, few studies addressed whether Rho GTPases play a role within “outside-in” signalling pathways induced by integrin–ligand interactions. In fibroblasts, interaction of integrins with extracellular matrix proteins was shown to be insufficient to induce formation of actin-based focal complexes in the absence of growth factor activated Rho proteins; formation of normal focal complexes required both integrin-dependent adhesion and functionally active Rho and Rac [211]. Other studies showed that integrin crosslinking causes accumulation of Rho and Rac at sites of integrin clustering; this phenomenon depended on both the integrity of the actin cytoskeleton and tyrosine kinase activity because it was inhibited by cytochalasin D and tyrosine kinase inhibitors [169]. Additionally, it has been reported that integrin engagement induces redistribution of components of Rho GTPase signalling pathways, such as p190(RhoGAP) and p160ROCK [212–214], to focal contacts; in this system blockade of Rho activity with the C3 transferase inhibitor prevented formation of focal complexes following integrin ligation [215]. These data suggest

Integrin Signalling in Neutrophils and Macrophages

that Rho family GTPases act within signalling pathways induced by integrin ligation. The possible implication of Rho GTPases in “outside-in” integrin signalling in phagocytic cells has not been as extensively studied as in non-hematopoietic cells. Spreading and actin organization in macrophages has been shown to be regulated by Cdc42, Rac and Rho [216, 217]. In leukocytes, regulation of Rho GTPase activity may be accomplished by the proto-oncogene Vav, which acts to catalyse GDP/GTP exchange on Rac following tyrosine phosphorylation [218]. As stated previously, several reports have described tyrosine phosphorylation of Vav following integrin engagement [143, 144, 219]. Additionally, T-cells from Vav-deficient mice show defects in actin polymerisation following cross-linking on the T-cell antigen receptor [220]. However, it must be noted that several signalling pathways triggered by integrins in phagocytic cells may converge in activation of Rho GTPases (Fig. 2). For example, integrins can activate Ras in different cell types, including neutrophils (see above) and Rac may be a downstream target of Ras [209, 210]. Importantly, in different cell types, including PMNs, Ras activation by receptors coupled to trimeric GTP-binding proteins may be accomplished through cytoplasmic tyrosine kinases such as Src, Lyn, Syk, and Pyk2 [199, 221–223] (i.e., the same kinases implicated in integrin signalling, see above). In this context, it is of great significance that Rac1 appears to be placed downstream of the tyrosine kinases Fgr and Hck in a signalling pathway triggered by engagement of the PMN surface molecule CD66 [224]. In analogy with Ras, tyrosine kinases could activate Rho GTPases directly, for example via Vav tyrosine phosphorylation and activation of nucleotide exchange factor activity [218]. As stated previously, Rho GTPases may also become activated in integrin- and tyrosine kinase-dependent pathways by the upstream activation of PI(3)-kinase. ADP-Ribosylation Factor (ARF) Using a yeast two-hybrid interaction screen, Kolanus et al. [193] recently identified a 47-kDa protein, named cytohesin-1, which associates with the cytoplasmic tail of the b2 integrin. This molecule has two domains with sequence motifs identified in other proteins, a C-terminal pleckstrin homology (PH) and an N-terminal domain related to the yeast Sec7 gene. Interestingly, it was found that expression of the Sec7 domain in Jurkat T-cells enhanced constitutive cell adhesion to the aLb2 counter-receptor ICAM-1, while expression of the PH domain blocked Jurkat-ICAM-1 interaction stimulated by anti-T-cell receptor antibodies [193]. The PH domain has been found to be present in different proteins involved in signal transduction and it has been shown to mediate binding of proteins with inositol lipids phosphorylated either at the D4/D5 or the D3 position [225, 226]. Recent findings highlighted the role of the PH domain in signalling through D3 phosphoinositides [225]. The Sec7 domain has been identified in three mammalian PH-domain containing proteins, namely Grp1, ARNO and

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cytohesin-1. The Sec7 region can catalise guanine nucleotide exchange in members of ADP-ribosylation factor (ARF) family of small GTPases [227–232]. At present, the mechanism by which cytohesin-1 regulates b2 integrinmediated adhesion of terminally differentiated cells such as PMN or macrophages to ICAM-1 is unknown. It has been speculated that, due to the capability of the PH domain to bind D3 phosphorylated inositol lipids, cytohesin-1 may be a downstream effector of PI(3) kinase-dependent activation of cell adhesion [233]. In this model, activation of PI(3) kinase leads to the accumulation of D3 phosphoinositides at the membrane which recruit cytoadhesin-1 from the cytoplasm to the membrane. In this new subcellular location, cytoadhesin-1 could catalyse GTP/GDP exchange on ARFs. It is also important to note that ARFs, which have been recognised as regulating membrane trafficking [234], have also been implicated in PMN degranulation [235]. Hence, integrin-dependent PMN degranulation (see above) may be a functional consequence of the signalling involving b2 integrins, cytohesin-1 and ARFs. CONCLUSION It is now well recognized that integrin-mediated cell adhesion in both leukocytes as well as non-hematopoietic cells leads to signalling events which affect cell activation, cell motility, cell proliferation and apoptosis. Disruption of these “outside-in” signalling pathways leads to impaired leukocyte function much in the same fashion as does loss of integrins (or integrin counter-receptors) expression. The signalling pathways elicited by integrins are extremely complicated; undoubtedly many of these pathways contribute independently to aspects of the biologic response to cell adhesion (for example regulating gene transcription without affecting cytoskeletal rearrangements) while other physiologic responses will require the coordinated action of several different integrin signalling pathways. The relative contribution of each of the signalling responses to the physiological functions elicited in leukocytes following integrinmediated adhesion will be an avenue of much future work. Nevertheless, a better understanding of these signalling pathways is critical because of the central role that monocytes, macrophages and PMNs play in tissue injury during inflammatory disease. The potential exists that specific inhibition of integrin-mediated signalling responses will offer a completely new approach to the treatment of inflammation. The authors’ work has been supported by grants from Associazione Italiana Ricerca sul Cancro (AIRC) and Fondazione Cariverona (Progetto Sanita`) to G. B., and NIH grants DK502267 and HL54476 to C. A. L.

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