3. Endothelial Cell Biology Adhesion Molecules Involved in the Microvascular Inflammatory Response 1 - J T. J. WILLIAMS and P. G. HELLEWELL

Introduction Adhesion of cells to each other and to the extracellular matrix is crucial to multicellular organisms in development and in the normal functioning of the immune system. One of the most studied aspects of cell adhesion is the accumulation of leukocytes in inflammation, an essential process for effective host defense against infection and injury. In order to accumulate in tissues, leukocytes must adhere to the endothelium lining the postcapillary venule, penetrate the vesselwall, migrate to the site of tissue irritation, and recognize and engulf the foreign material. At each of these stages of the inflammatory process, specificadhesiveinteractions between leukocytesand the vesselwall (and subsequently the extracellular matrix) are important. It is now clear that there are different families of cell adhesion molecules that regulate the interactions of leukocytes with the endothelium (1). These willnow be discussed. Selectlns Selectins (previously known as LEC-CAM) have a common molecular structure characterized by an N-terminallectin domain (which is homologous to a variety of Ca2+-dependent or C-type lectins), an epidermal growth factor (EGF) domain, a series of complementregulatory domains (CRP), a transmembrane domain, and a short cytoplasmic tail (1).The lectin domain is essential for cell adhesion, although the EGF domain appears to have a regulatory role. There are three members of this family that are involved in leukocyte adhesion to endothelium; endothelialleukocyteadhesion molecule-l (ELAM-l) and granule-associated membrane protein 140(GMP140), which are found on stimulated endothelium, and the peripheral lymph node homing receptor (LECAM-I), which is found on leukocytes. These have recently been renamed E-selectin, P-selectin, and L-selectin, respectively (2).

SUMMARY Accumulation of leukocytes in tissues is essential for effective host defense. To fulfill this role the cell must interact with and penetrate the vessel wall and migrate In the tissue. It is now clear that cell adhesion molecules play a crucial role In orchestrating these processes. A number of families of such adhesion molecules that mediate the Interaction of circulating leukocytes and vascular endothelial cells have been Identified. These include the leUkocyte integrins, the selectins, members of the immunoglobulin family, and certain carbohydrates. Studies in vitro haveelucidated which of these adhesion molecules are Important in the Interaction of different leUkocyte classes with the endothelium under both basal and stimulated conditions. With the aid of monoclonal antibodies, the role that these molecules play in the interaction of inflammatory cells In the microvasculature in vivo is being assessed. Studies to date have demonstrated the key role of cell adhesion molecules in inflammation. AM REV RE5PIR DI5 1992; 146:545-550

a variety of inflammatory conditions in vivo (3). Adhesion of neutrophils or HL60 promyelocytes to Cos cells transfected with a eDNA clone for E-selectin has established a role for this molecule in heterotypic cellular adhesion (4).

associated with nonlymphoid tissue in inflammation (7, 8). Both the murine peripheral lymph node receptor (gp9omeII4 , mLHRc) and the human homologue (Leu-S, lOI, LAM-I, hLHRc) have been recently renamed as L-selectin (2).

P-Selectin

Immunoglobulin Superfamily This is a large family of adhesion molecules whose structure is characterized by repeated domains similar to those found in immunoglobulins. For endothelial-leukocyte interactions, the most important members of this family are intercellular adhesion molecule-l (ICAM-l), ICAM-2, and vascular cell adhesion molecule-I (VCAM-I) (1).

P-selectin is a 140-kD 9 CRP domain glycoprotein located in the secretory granules of platelets and of endothelium. It is identical to a separately identified protein designated PADGEM (platelet-activation-dependent granule and external membrane protein), and it has also been given the cluster designation CD62. Within minutes of stimulation of endothelium by thrombin or histamine, P-selectin is expressed on the cell surface. Cytokines such as IL-I and TNF do not induce expression of P-selectin. Leukocyteadhesion induced by thrombin or histamine is blocked by anti-P-selectin mAbs, indicating that P-selectin is a functional cell adhesion molecule (5). Thrombin also stimulates endothelial cellsto synthesizeplatelet-activating factor (PAF), and there is evidence to suggest PAF as the mediator of enhanced neutrophil adhesion in response to thrombin (6). The precise link between endothelial PAF and P-selectinexpression remains to be elucidated.

E-Selectin

L-Selectin

E-selectin is a 95- to 115-kD 6 CRP domain glycoprotein expressed exclusively on endothelium activated with cytokines such as IL-I, TNF, and LPS. E-selectin expression is protein-synthesis-dependent, peaks after 4 to 6 h of cytokine stimulation, and subsequently declines to basal levels within 24 h. The pathophysiologic relevance of E-selectin has been suggested from studies showing cytokine-inducible expression in postcapillary venules of organ cultures and its presence in

Lymphocyte homing receptors, via their association with tissue-specific determinants (vascular addressins) on high endothelial venules (HEV) of lymphoid tissue, are primarily responsible for the regulation of lymphocyte recirculation. The mAb MEL-I4 recognizes an 85- to 95-kD 2 CRP domain glycoprotein on murine lymphocytes that binds to HEV in peripheral lymph nodes. However, this molecule is also found on myeloid cells, and it appears to be involved in cellular interactions

ICAM-I and ICAM-2 The intercellular adhesion molecules ICAM-I and ICAM-2 have been identified and cloned (1). ICAM-2 is truncated, possessingonly two of the five immunoglobulin (Ig) domains found in ICAM-I. Both molecules bind to the leukocyte integrin LFA-I and are involved in lymphocyte adhesion to endothelium (9). ICAM-I, which has been given the cluster designation CD54, also binds to Mac-I. These molecules are both found constitutively on a variety of different cell types in addition to endothelium. ICAM-I expression can be stimulated by y-Interferon (IFN-y) and by cytokines known to stimulate E-selectin. In

1 From the Department of Applied Pharmacology, National Heart & Lung Institute, London, United Kingdom. 2 Supported by the National Asthma Campaign, UK, and the Wellcome Trust, UK. 3 Correspondence and requests for reprints should be addressed to T. J. Williams, Department of Applied Pharmacology, National Heart & Lung Institute, Dovehouse Street, London SW36LY, UK.

545

546

contrast, expression of ICAM-2 is not increased by cytokines. Expression of ICAM-1 in vitro is protein-synthesis-dependent, peaks after 12h, and, unlike E-selectin, is then maintained for at least a further 36 h.

VCAM-I Vascular cell adhesion molecule (VCAM-1) is a 100- to 110-kD cell surface glycoprotein expressed on the surface of endothelium and follicular dendritic cells. Basal expression of VCAM-1 on endothelium is considerably lower than that reported for ICAM-1, and it can be stimulated by some of the same mediators such as IL-1, TNF, and LPS, but not by IFN-y (10, 11). Expression is proteinsynthesis-dependent and can also be stimulated by interleukin 4; it peaks after 6 to 10 h of cytokine treatment, and it is sustained in some cases for as long as 72 h. Cloning of VCAM-1 has established that it belongs to the immunoglobulin superfamily, and there appears to be two distinct forms, one form with six Ig domains, and a predominant, alternatively spliced form with seven Ig domains. Vascular Addressins Tissue-specific determinants on high endothelial venules (HEV), which direct extravasating lymphocytes into the appropriate lymphoid or extralymphoid tissue, are known as "vascular addressins" (12).Examples of these are the mucosal and peripheral lymph node (pln) receptors, which are defined by the monoclonal antibodies MECA 367 and MECA 79, respectively(12).A number of glycoprotein speciesof distinct molecular weights bear the epitope for these mAbs. Recent evidence has shown that the MECA 79 ligand binds to L-selectin on the lymphocyte (13). The vascular addressins are also indirectly implicated in adhesion of monocytes and neutrophils to endothelium by virtue of the involvement of their cognate ligands (e.g., L-selectin, see above) in this process (8, 14). Leukocyte Integrins These are a family of multifunctional receptors for the immunoglobulin superfamily and for matrix components (15). They consist of two subunits (a and 13), which form noncovalent heterodimers in the cell membrane. There are at least 14a and eight 13 subunits, although not all of them are expressed on the same cells. Nevertheless, the opportunity for multiple specificities is apparent. With respect to endothelial-leukocyte interactions, the most important members of the family are the 132 subfamily and VLA-4. {32 Integrins The 132 integrin subfamily is found exclusively on leukocytes and is composed of three distinct but related a-chain polypeptides: CD11a (aL; 180 kD), CD11b (aM; 170 kD), and CD11c (aX; 150 kD), which are expressed on the cell surface in noncovalent association with a common 132 subunit called CD18 (95

WILLIAMS AND HELLEWELL

kD) (15). These three a/13 heterodimers are known, respectively, as LFA-1, Mac-l, and p150/95, and the whole complex is known as CDll/CD18. A defect in the 132 subunit, which results in impaired cell surface expression of this family of glycoproteins, is responsible for the congenital defect known as leukocyte adhesion deficiency (LAD) (16).Patients with this disease have severe recurrent bacterial infections, and they often have impaired wound healing. Leukocytes isolated from these patients show profound defects in adhesive, chemotactic, and phagocytic function in vitro, and they have been used extensively to define functional roles for the 132 integrins and to underscore the importance of this glycoprotein family in immune regulation. LFA-1 is expressed on the majority of leukocytes and related leukemic cell lines, and it binds to ICAM-I. Mac-I and pI50/95 glycoproteins are found mainly on monocytes, granulocytes, and certain subclasses of lymphocyte, but they are virtually absent from myelomonocytic cells such as U937 and HL60. Mac-1 binds to ICAM-I, but the endothelial receptor for p150/95 remains to be established.

VLA-4 VLA-4 is a member of the 13, integrin family. The members of this family are sometimes referred to as the very late activation antigens. They are typical a/13 heterodimeric cell surface glycoproteins, each consisting of one of six distinct a polypeptide chains in association with a common 13, subunit (CD29) (17). With the exception of VLA-4 (a4I3,), these integrins are found on many different cell types, and they function primarily as receptors for extracellular matrix proteins such as collagen, laminin, and fibronectin (IS). VLA-4 is found mainly on hematopoietic cells such as lymphocytes, monocytes and related leukemic cell lines, and it mediates cell-cell interactions in addition to functioning as an extracellular matrix receptor for fibronectin (17). VLA-4 is present on eosinophils but absent from neutrophils. An alternative binding site on VLA-4 interacts with the leukocyte adhesion molecule VCAM-1 (18). Carbohydrates It has become clear that ligands for the selectin family of adhesion molecules are carbohydrates. Two reviews summarize these findings (19, 20). Lewis x and sialylated Lewis x are a(1-3) fucosylated derivatives of polylactosamine, and expression ofthese on neutrophils and other cells correlates with their ability to bind to E-selectin. Antisialyl Lewis x mAbs inhibit neutrophil-binding to E-selectin. Moreover, cells can be converted to ELAMadhesive cells by expression of a single sugar transferase (a(I-3)-fucosyl transferase). The ligand for Pselectin, present on neutrophils and monocytes, is known to have CD15 (Lewis x) as at least part of its structure. Antibodies to CD15, or added CD15 sugar, specifically inhibit P-selectin-dependent

adhesion of platelets to monocytes or neutrophils, and they also block binding ofU937 or HL60 to Cos cells expressing P-selectin. However, this is not the only carbohydrate structure recognized by P-selectin since it has been found recently that P-selectin can also recognize the E-selectin ligand, sialyl Lewis x (21). Chemical Signals for Leukocyte Attachment to the Endothelium

In Vivo Leukocyte accumulation is an essential part of local host defense reactions. Detection of an inflammatory stimulus in a tissue, e.g., the presence of microbes, triggers the local extravascular generation of chemical signals that, by a complex process, initiate the interaction between the leukocyte and the lumenal surface of the microvascular endothelial cell. Signals can be derived from interstitial fluid, e.g.,the complement-derived protein fragment C5a, or host cells, e.g., LTB4, PAF, and cytokines. In addition, mechanisms have evolved to detect products of microbes themselves, e.g., formyl-methionyl peptides and endotoxin. High levels of C5a have been detected in early inflammatory exudate samples in certain in vivo models (22, 23). In these models the early phase of C5a generation was followed by the appearance of two further neutrophil chemoattractant in the exudate, IL-8 and MGSA (24-26). Once generated, it is not clear how such chemoattractants on the outside of the microvascular bed induce leukocytes in the lumen to attach to the endothelium. This attachment takes place in venules in most tissues. Uncoupling of receptors on radiolabeled neutrophils, using pertussis toxin in vitro followed by intravenous injection, prevented the local accumulation of the cells in response to intradermally injected chemoattractants (27). Administration of chemoattractants to the outside surface of venules of the hamster cheek pouch via a micropipette did not induce neutrophil attachment; however, administration to capillaries induced neutrophils to adhere downstream in venules (28). These observations, taken together, suggest that chemoattractants diffuse into the lumen of microvessels and act on chemoattractant receptors on the neutrophil. The neutrophils then increase adhesiveness and attach to venular endothelial cells, which appear to offer a preferential site of attachment. Although increased expression of integrins on neutrophils has been demonstrated in response to high concentrations of chemoattractants, experiments in vitro (29) and in vivo (30) have suggested that a conformational change is more important for increased neutrophil adhesiveness. The situation appears to differ in the pulmonary circulation. In this low pressure system neutrophils accumulate in the narrow capillaries (rather than in venules) and both adhesive forces and the me-

ADHESION MOLECULES AND THE MICROVASCULAR INFLAMMATORY RESPONSE

chanical stiffness of the leukocyte are likely to be important for retention (31). The cytokine IL-l has also been detected in inflammatory exudates (32). Experiments in rabbits haveshown that neutrophil accumulation induced by IL-l is dependent on local protein biosynthesis, whereas neutrophil accumulation induced by C5a, LTB., and FMLP is little affected by protein synthesis inhibitors (33). This may be because of a requirement for the synthesis of endothelial cell adhesion molecules in vivo or the synthesis of secondary neutrophil chemoattractants in response to IL-1. Neutrophil chemoattractants are highly potent in inducing edema formation (34). The interaction between neutrophils and the microvascular wall leads to an elevation of permeability to plasma proteins. This response can be inhibited by either depletion of circulating neutrophils (34) or using mAbs to CD18 (35, see below). It is not clear how the cell-cell interaction induces plasma protein leakage; however, the effect is rapid in onset and of long duration (34). Role of Cell Adhesion Molecules in Leukocyte Endothelial Cell Interactions A summary of the molecules involved in leukocyte adhesion to endothelium is shown in table 1.

Phagocytes Inflammatory mediators-such as C5a and LTB. induce a rapid adhesion of neutrophils to endothelium. This is mediated via LFA-l . and Mac-l binding to ICAM-l and ICAM-2 constitutively expressed on the endothelium. Treatment of endothelial cells with thrombin or histamine results in enhanced neutrophil adhesion via P-selectin. In vitro experiments suggest that this molecule may be important for the rolling of leukocytes seen in postcapillary venules under conditions of shear. Integrin II CAM-l interactions do not occur under these conditions unless the neutrophils are stimulated (35). Treatment of endothelium with cytokines (IL-I, TNF) for 6 h results in enhanced adhesion of neutrophils. At this time point, expression ofE-selectin, ICAM-l, and VCAM-I are approximately maximal. Under these con-

TABLE 1 MOLECULES INVOLVED IN LEUKOCYTE ADHESION TO ENDOTHELIUM SHOWING LIGAND-RECEPTOR PAIRS Leukocyte Adhesion Molecule

Endothelial Adhesion Molecule

CD11a CD11b CD11c VLA-4 Sialylated Lewis x Lewis x L-selectin

ICAM-1. ICAM-2 ICAM-1 (ICAM-2?)

? VCAM-1 E-selectin, P-selectin P-selectin MECA 79 antigen

ditions, E-selectin-specific mAbs have been shown to inhibit adhesion of neutrophils by 40 to 60070, and the residual adhesion is blocked by anti-CDl8 mAbs (36).Preliminary results indicate that human L-selectin is also involved in CDl8-independent adhesion of neutrophils to cytokine-stirnulated endothelial cells in vitro (37). With prolonged cytokine treatment (12to 48 h), E-selectin is lost from the cell surface, and the contribution of ICAM-l to neutrophil adhesion becomes greater (36). Neutrophils do not bind VCAM because they lack the cognate ligand VLA-4. Neutrophils from patients with LAD can bind to E-selectin on activated endothelium, but they cannot migrate, adding further support for the suggestion that an integrin/ICAM interaction is important for the process of emigration rather than for capture, which occurs via a selectin. Eosinophils behave similarlyto neutrophils except that they express VLA-4 on their surface and therefore can also bind to VCAM-l (38). However, because of the relative paucity of data on eosinophils, it is difficult to assess the relative importance of each ligandreceptor pair to the overall adhesion of these cells to endothelium. Monocytes appear to express the ligands for all endothelial adhesion molecules as judged by their adhesion to immobilized P-selectin and to E-selectin, ICAM-l, and VCAM-I transfectants. However, because of this, it appears that the ligand-receptor pairs cooperate to strengthen the adhesion of monocytes to activated endothelium, and it is therefore difficult to inhibit this response. Only by blocking all the adhesive pathways is it possible to abrogate monocyte adhesion (39). Lymphocytes Lymphocytes leavethe bloodstream for either the lymphoid tissue or sites of inflammation. Lymphocyte homing via vascular address ins and homing receptors has been considered above, and it is also the subject of recent reviews (12, 40). More is known about homing to peripheral lymph nodes where L-selectin on the lymphocyte and MECA-79 antigen are involved. Lessis known about homing to mucosal lymph nodes, although CD44 or H-CAM may be involved. In the mouse, homing to the mucosal lymph nodes appears to involve the murine homologue of VLA-4, LPAM-l, on the lymphocyte. Recently, E-selectin has been proposed as a skin-specific addressin for memory T cells. Although E-selectin is expressed on inflamed endothelium in many tissues, it shows biased expressionon inflamed endothelium of skin (41). On cultured endothelial cells, ICAM-l and ICAM-2 appear to mediate the basal adhesion of lymphocytes via LFA-1. Lymphocytes accumulate at inflammatory sites in patients with LAD, and it was found that these lymphocytes were able to adhere in large numbers to cytokine-activated endothelial cells in vitro, indicating that an alternative adhesion molecule was present (42). This is now known

547

to be YCAM-1. Thus, both ICAM-l and VCAM-l are responsible for the enhanced lymphocyte adhesion. Lymphocytes do not appear to bind P-selectin. Role of Cell Adhesion Molecules in Experimental Inflammation Most focus on the mechanisms of granulocyteaccumulation and emigration in vivo has been on the role of the CDll/CDl8 on the neutrophil. This is mainly because monoclonal antibodies that recognize this adhesive molecule have been available for some time and their efficacy in vitro in blocking granulocyte adhesion to endothelium is well established, and, importantly, some of these MAbs recognizeepitopes on animal granulocytesand so they can be used in animal models of inflammatory processes. These studies are summarized in table 2 although this list is by no means comprehensive. Use of the anti-CD18 mAbs such as 60.3 and 1B4exemplifiesthis type of investigation. Intravenous administration of 60.3 dosedependently suppressed the accumulation of neutrophils into subcutaneous spongessoaked with chemoattractant (43) and into skin sites injected with similar agents (35). In other studies preincubation of radiolabeled neutrophils with 60.3 before intravenous injection blocked their accumulation in response to C5a, LTB., FMLP, and IL-l in rabbit skin (30, 33). The same procedure blocked accumulation of neutrophils into rabbit heart during reperfusion after a period of ischemia (44). Direct visual evidence (by intravital microscopy) showed that 60.3 also prevented the stimulated (but not the normal) neutrophil adherence to endothelium (35);thus, although neutrophil adherence in the venules and migration into the tissues (in response to extravascular chemoattractant) was inhibited, rolling of neutrophils along the venular endothelium was unaffected. The implication is that the mechanisms that govern the "basal" interaction of neutrophils with endothelium are CD18-independent. In support of this, recent studies have shown that inhibitors of L-selectinprevent leukocyterollingin vivo (45) and P-selectin has been implicated from in vitro studies (35). MAb 1B4 reduced tissue damage and improved the outcome of infection in a rabbit model of experimental meningitis, preventing the development of brain edema and death in animals challenged with lethal doses of Streptococcus pneumoniae (46). In other studies, pretreatment of cats with 60.3 provided protection against ischemia-induced microvascular injury of the gut (47), supporting the proposition that granulocytes play an important role in reperfusion injury (48). Subsequent studies have shown that anti-CD18 MAb-mediated inhibition of granulocyte adherence is effective at reducing injury in myocardial and whole-animal models of reperfusion injury (49, 50), although lung injury did not appear to be suppressed (50). However, in all these studies MAbs were administered

548

WILLIAMS AND HELLEWELL

TABLE 2 EXAMPLES OF THE EFFECTS OF mAbs TO CELL ADHESION MOLECULES ON EXPERIMENTAL INFLAMMATION mAb CD18 CD18 CD11b/CD18 CD18 CD18 ICAM-1 ICAM-1 ICAM-1 ICAM·1 ICAM-1 L-Selectin L-Selectin E-Selectin E-Selectin

Effect in Model

References

Suppression of neutrophil accumulation in cutaneous inflammation Suppression of neutrophil accumulation in experimental meningitis Reduction in reperfusion injury Protection against hemorrhagic shock Reduction of neutrophil accumulation in E.-coli-induced lung injury Reduction of neutrophil accumulation in PMA-induced acute lung injury Reduction in reperfusion injury Prevention of renal allograft rejection Suppression of experimental asthma Prevention of hemorrhage in vasculitis Reduction of neutrophil accumulation in peritonitis Prevention of leukocyte rolling Inhibition of late-phase airway obstruction Reduction of immune-complex-induced lung injury

30, 33, 35, 43

before the onset of ischemia. The clinically relevant situation would be to provide treatment after the onset of the ischemic event but before reperfusion. Nevertheless, Vedderand coworkers (51) haveshown that treatment with MAb either before or after ischemia, but prior to reperfusion, did result in the same degree of significant protection against microvascular injury in the rabbit ear. In a similarly designed study, organ injury (particularly in the gut) after hemorrhagic shock in rhesus monkeys was dramatically protected by administration of 60.3 just prior to fluid-induced resuscitation. Perhaps the most notable observation was that in the treated group all animals survived for as long as 72 h, whereas 40070 of the control animals died (52). These 0 bservations suggest the importance ofthe COl 8 adhesion molecule on the leukocyte as a key determinant of leukocyte accumulation in blood vesselsin inflammation but not in the interaction of these cells with the endothelium during normal passage around the body (i.e.,rolling). At what stages of the process CD18 is involved is not clear, although in vitro evidence suggests that it can mediate adhesion of granulocytes to endothelium and is involved in the process of cell migration across vascular endothelium (53). This latter point is perhaps the crucial one since in vitro studies have shown that neutrophils from patients with LAD (COl8deficient) can adhere to activated endothelium via E-selectin but they cannot migrate (53). Thus, the exact relationship between COlli CD18 expression and neutrophil adhesion is not known; upregulation is not enough, and further, possible stearic modification of the molecule is also required (29). However, as alluded to above, when compared with the peripheral circulation the pulmonary circulation appears to be different. This is borne out by the observation that in certain types of pulmonary inflammation, neutrophil accumulation in the air spacesdoes not appear to be COl8-dependent (50, 54). Furthermore, postmortem examination of lungs from patients with LAD showsevidence of neutrophil migration into the air spaces.

46 44, 47, 49, 51 50, 52 54 55

56 57 59

58 8, 14, 62 45 60 61

Alternative mechanisms for accumulation of granulocytes are therefore implicated (31). With respect to other adhesion molecules, particularly those on the endothelium, fewer studies have been carried out. Nevertheless, the anti-ICAM MAb R6.5 does appear to cross-react with rabbit and primate ICAM and acts as a functional blocker, making it a valuable tool for in vivo studies. Indeed, it has been reported to reduce by approximately 70070 granulocyte infiltration into the air spaces of rabbits induced by intravenous PMA (55) and reduce the injury to ischemic hearts after reperfusion (56). The same MAb has also shown efficacy in a model of renal allograft in the monkey (57). When administered prophylactically as the sole immunosuppressive therapy for 12 days, it prevented the normal intense inflammatory response (both granulocyteand mononuclear) that leads to graft rejection and prolonged the survival from 9 to 24 days. rCAM-l also appears to be involved in mediating the hemorrhage observed in a model of experimental vasculitis (58). In a primate model of asthma, antigeninduced bronchial hyperresponsiveness and bronchial eosinophil influx were significantly attenuated by systemic administration of the anti-ICAM-l mAb R6.5 (59). Of further interest is the observation that in additional studies in the same model, CL2, an antiELAM (human) MAb, which cross-reacts with primate ELAM, had no such effect (60). However,CL2 did block neutrophil influx and the late-phase airway obstruction following a single antigen challenge with antigen (60). This suggests that the late-phase response may be the result of an influx of neutrophils dependent on E-selectin. Other studies on the role of the selectin family of adhesion molecules in regulating granulocyte accumulation and emigration in vivo are emerging slowly,but they have been hampered by the lack of tools. Another anti-Eselectin mAb that cross-reacts with the rat has been shown to reduce neutrophil accumulation in a rat model of immune-complexmediated vasculitis in the lung and skin (61).

Vascular injury was also attenuated as judged by inhibition of vascular permeability and hemorrhage (61). Based on the effects of systemic administration of mAb MEL-14 in mice, L-selectin appears to be important in regulating the extravasation of neutrophils from the bloodstream into the inflamed peritoneal cavity (8, 14).This has been confirmed in a recent study also carried out in the mouse (62). The efficacyof a solubleimmunoglobulin chimera of L-selectin was also demonstrated in this study, its intravenous administration leading to the abrogation of thioglycollateinduced peritoneal neutrophil accumulation. The same chimera (and a polyclonal anti-Lselectin antibody) also reduced rolling of leukocytes in mesenteric venules in the rat (45). This novel approach is particularly interesting because it shows that the L-selectin can be converted into a MAb-like molecule specific for the cognate ligand expressed on the endothelium. Cell Adhesion as a Target of Therapy Accumulation of leukocytes is tissues is essential for effective host defense: however, in situations where the control mechanisms fail, inflammation continues unabated, leading to destruction of healthy tissue. On the basis of the data obtained in animals, cell adhesion would appear to be an attractive target for therapy. There are a number of waysin which cell adhesion can be targeted that may lead to the development of novel drugs.

Monoclonal Antibodies Anti-COl8 mAbs are clearly effective at blocking neutrophil accumulation in vivo, resulting in the attenuation of tissue damage in a number of animal models. One of these mAbs, 60.3, is now in clinical trial for the prevention of multiple organ failure syndrome (MOFS) in cases of traumatic injury in humans. An anti-ICAM mAb is also in clinical trial for renal allograft in humans. One potential problem with murine antibody therapy is the strong immune response against the injected mAb. This is less of a problem for acute treatments, but it precludes mAb therapy for chronic conditions such as rheumatoid arthritis. One route around this problem is to make chimeric antibodies or to humanize them. Chronic treatment with such mAbs may also result in immunosuppression. Soluble Forms of Cell Adhesion Molecules By engineering cell adhesion molecules to delete the transmembrane domain, soluble forms can be obtained. In theory, by binding to their cognate ligand on cells, these molecules can act as inhibitors by competing with their cell-bound form. In practice, such molecules may not have sufficient intrinsic affinity to compete effectively, although engineered forms coupled to immunoglobulins (immunoadhesins) do appear to work. An example of this is the L-selectin immunoglobu-

ADHESION MOLECULES AND THE MICROVASCULAR INFLAMMATORY RESPONSE

11. Wellicome SM, Thornhill MH, Thomas DS, et al. A monoclonal antibody that detects a novel antigen on endothelial cellsthat is induced by TNF, IL-l or LPS. J Immunol 1990; 144:2558-65. 12. Butcher EC. Cellular and molecular mechanisms that direct leukocyte traffic. Am J Pathol Low Molecular Weight Drugs 1990; 136:3-11. An orally administered anti-inflammatory 13. Berg EL, Robinson MK, Warnock RA, drug would be the ideal therapeutic com- Butcher EC. The human peripheral lymph node pound to emerge from the cell adhesion field. vascular addressin is a ligand for LECAM-l, the Based on structural analysis of the cell adhe- peripheral lymph node homing receptor. J Cell BioI sion molecules and their cognate ligands it 1991; 114:343-9. may be feasible to design such compounds. 14. Lewinsohn DM, Bargatze RF, Butcher EC. To date, most emphasis is being placed on Leukocyte-endothelial cell recognition: evidence of a common molecular mechanism shared by neuthe carbohydrate ligands of the selectin fam- trophils, lymphocytes, and other leukocytes. J Imily of adhesion molecules. munol 1987; 138:4313-21. 15. Ruoslahti E. Integrins. J Clin Invest 1991; Regulation of Cell Adhesion 87:1-5. 16. Anderson DC, Springer TA. Leukocyte adheMolecules One alternative approach to blocking cell sion deficiency: an inherited defect in the MAC-I, adhesion is to alter the expression of adhe- LFA-l and P150,95 glycoproteins. Annu Rev Med 1987; 38:175-94. sion molecules. Possibilitiesinclude antisense 17. Hemler ME, Elices MJ, Parker C, Takada Y. DNA and gene transcription factors. Several Structure of the integrin VLA-4and its cell-celland drugs have been demonstrated to alter leuko- cell-matrix adhesion functions. Immunol Rev 1990; cyte adhesivenessin vitro. Research into these 114:45-65. may lead to the development of useful ther- 18. ElicesMJ, Osborn L, Takada Y,et al. VCAM-l apeutic compounds that interfere with the on activated endothelium interacts with the leukoprocess of leukocyte accumulation in vivo. cyteintegrin VLA-4at a site distinct from the VLAThe platform of convincing results already 4/fibronectin binding site. Cell 1990; 60:577-84. obtained in animal models provides the ex- 19. Brandley BK, Swiedler SJ, Robbins PW. Carbohydrate ligands of the LEC Cell adhesion moleciting prospect for the development of ther- cules. Cell 1990; 63:861-3. apeutic compounds that effectively suppress 20. Springer TA, Lasky LA. Stickysugars for selecthe accumulation of selected leukocyte types tins. Nature 1991; 349:196-7. in diseases such as asthma. 21. Polley MJ, Phillips ML, WaynerE, et al. CD62 and endothelial cell-leukocyte adhesion molecule 1 (ELAM-l) recognize the same carbohydrate References ligand, sialyl-Lewisx. Proc Natl Acad SciUSA 1991; 1. Springer TA.Adhesion receptors ofthe immune 88:6224-8. 22. Jose PJ, Forrest MJ, Williams TJ. Detection system. Nature 1990; 346:425-34. 2. Bevilacqua M, Butcher E, Furie B, et al. Selec- of the complement fragment C5a in inflammatory tins: a family of adhesion receptors. Cell 1991; exudates from the rabbit peritoneal cavity using 67:223. radioimmunoassay. J Exp Med 1983;158:2177-82. 3. Ruco LP, Pomponi D, Pigott R, et al. Cytokine 23. Forrest MJ, Jose PJ, Williams TJ. Kinetics production (IL-lo., IL-lf3 and TNFa) and en- of the generation and action of chemical mediadothelial cell activation (ELAM-l and HLA-DR) tors in zymosan-induced inflammation of the rabin reactive lymphadenitis, Hodgkin's disease, and bit peritoneal cavity. Br J Pharmacol 1986; 89: in non-Hodgkin's lymphomas: an immunocyto- 719-30. chemical study. Am J Pathol 1990; 137:1163-71. 24. Collins PD, Jose PJ, WilliamsTJ. The sequen4. Bevilacqua MP, Stengelin S, Gimbrone MA, tial generation of neutrophil chemoattractant proSeed B. Endothelial leukocyte adhesion molecule teins in acute inflammation in the rabbit in vivo: 1: an inducible receptor for neutrophils related to relationship between C5a and a protein with the complement regulatory proteins and lectins. Science characteristicsof IL-8. J Immunol1991; 146:677-84. 25. Beaubien BC, Collins PD, Jose PJ, et al. A 1989; 243:1160-4. 5. Geng J-G, Bevilacqua MP, Moore KL, et al. novel neutrophil chemoattractant generated durRapid neutrophil adhesion to activated endothelium ing an inflammatory reaction in the rabbit peritomediated by GMP-140. Nature 1990; 343:757-60. neal cavity in vivo: purification, partial amino acid 6. Zimmerman GA, McIntyre TM, Mehra M, sequence and structural relationship to interleukin Prescott SM. Endothelial cell-associated platelet- 8. Biochem J 1990; 271:797-801. activating factor: a novel mechanism for signaling 26. Jose PJ, Collins PD, Perkins JA, et al. Idenintercellular adhesion. J Cell BioI 1990;110:529-40. tification of a second neutrophil chemoattractant 7. Kishimoto TK, Jutila MA, Berg EL, Butcher cytokine generated during an inflammatory reacEC. Neutrophil Mac-l and MEL-14 adhesion pro- tion in the rabbit peritoneal cavity in vivo: purifiteins inversely regulated by chemotactic factors. cation, partial amino acid sequence and structural relationship to melanoma growth stimulatory acScience 1989; 245:1238-41. 8. Jutila MA, Rott L, BergEL, Butcher EC. Func- tivity. Biochem J 1991; 278:493-7. tion and regulation of the neutrophil MEL-14 an- 27. Nourshargh S, Williams TJ. Evidence that a tigen in vivo: comparison with LFA-l and MAC-I. receptor operated event on the neutrophil mediJ Immunol 1989; 143:3318-24. ates neutrophil accumulation in vivo: pretreatment 9. Dustin ML, Springer TA. Role of lymphocyte of lllIn-neutrophils with pertussis toxin in vitro inadhesion receptors in transient interactions and cell hibits their accumulation in vivo. J ImmunoI1990; 145:2633-8. locomotion. Annu Rev Immunol 1991; 9:27-66. 10. Osborn L, Hession C, Tizard R, et al. Direct 28. Nagai K, Katori M. Possible changes in the expression cloning of vascular cell adhesion mole- leukocyte membrane as a mechanism of leukocyte cule I, a cytokine-induced endothelial protein that adhesion to the venular wallsinduced by leukotriene B4 and fMLP in the microvasculature of the hambinds to lymphocytes. Cell 1989; 59:1203-11.

lin chimera. This may not be necessary for all adhesion moleculesbecause soluble Mac-l (Cfrl lb) does appear to inhibit neutrophil adhesion to activated endothelium.

549,

ster cheek pouch. Int J Microcirc 1988;7:305-14. 29. VedderNB, Harlan JM. Increased surface expression of CDllb/CDl8 (Mac-I) is not required for stimulated neutrophil adherence to cultured endothelium. J Clin Invest 1988; 81:676-82. 30. Nourshargh S, Rampart M, Hellewell PG, et al. Accumulation of 111 In-neutrophils in rabbit skin in allergicand non-allergic inflammatory reactions in vivo: inhibition by neutrophil pretreatment in vitro with a monoclonal antibody recognising the CDl8 antigen. J Immunol 1989; 142:3193-8. 31. Worthen GS, Schwab B, Elson EL, Downey GP. Mechanics of stimulated neutrophils: cell stiffening induces retention in capillaries. Science 1989; 245:183-6. 32. Bochner BS, Charlesworth EN, Lichtenstein LM, et al. Interleukin-l is released at sites of human cutaneous allergic reactions. J Allergy Clin Immunol 1990; 86:830-9. 33. Rampart M, Williams TJ. Evidence that neutrophil accumulation induced by interleukin-l requires both local protein biosynthesis and neutrophil CDl8 antigen expression in vivo. Br J Pharmacol 1988; 94:1143-8. 34. Wedmore CV, Williams TJ. Control of vascular permeability by polymorphonuclear leukocytes in inflammation. Nature 1981; 289:646-50. 35. Lawrence MB, Springer TA. Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell 1991; 65:859-73. 36. Luscinskas FW, Brock AF, Arnaout MA, Gimbrone MA. Endothelial-leukocyte adhesion molecule-l-dependent and leukocyte(CDll/CDl8)dependent mechanisms contribute to polymorphonuclear leukocyte adhesion to cytokineactivated human vascular endothelium. J Immunol 1989; 142:2257-63. 37. Hallmann R, Jutila MA, Smith CW, Anderson DC, Kishimoto TK, Butcher EC. The peripherallymph node homing receptor, LECAM-l, is involved in CDl8-independent adhesion of human neutrophils to endothelium. Biochem Biophys Res Commun 1991; 174:236-43. 38. Weller PF, Rand TH, Goelz SE, Chi-Rosso G, Lobb RR. Human eosinophil adherence to vascular endothelium mediated by binding to vascular cell adhesion molecule 1 and endothelial leukocyte adhesion molecule 1. Proc Natl Acad Sci USA 1991; 88:7430-3. 39. Carlos TM, Schwartz BR, Kovach NL, et al. Vascular cell adhesion molecule-l mediates lymphocyte adherence to cytokine-activated cultured human endothelial cells. Blood 1990; 76:965-70. 40. Michl J, Qiu Q-Y, Kuerer HM. Homing receptors and addressins. CUff Opin Immunol 1991; 3:373-82. 41. Picker LJ, Kishimoto TK, Smith CW, Warnock RA, Butcher EC. ELAM-l is an adhesion molecule for skin-homing T cells. Nature 1991; 349:796-38. 42. Schwartz BR, Wayner EA, Carlos TM, Ochs HD, Harlan JM. Identification of surface proteins mediating adherence of CDll/CDl8-deficient Iymphoblastoid cells to cultured human endothelium. J Clin Invest 1990; 85:2019-22. 43. Price TH, Beatty PG, Corpuz SR. In vivo inhibition of neutrophil function in the rabbit using monoclonal antibody to CDl8. J Immunol 1987; 139:4174-7. 44. Williams FM, Collins PD, Tanniere-Zeller M, Williams TJ. The relationship between neutrophils and increasedmicrovascularpermeabilityin a model of myocardialischaemia and reperfusion in the rabbit. Br J Pharmacol 1990; 100:729-34. 45. Ley K, Gaehtgens P, Fennie C, Singer MS, Lasky LA, Rosen SD. Lectin-likecelladhesion molecule 1mediates leukocyte rolling in mesentericven-

S50 ules in vivo. Blood 1991; 77:2553-5. 46. Tuomanen EI, Saukkonen K, Sande S, Cioffe C, Wright SD. Reduction of inflammation, tissue damage and mortality in bacterial meningitis in rabbits treated with monoclonal antibodies against adhesion-promoting receptors of leucocytes. J Exp Med 1989; 170:959-68. 47. Hernandez LA, Grisham MB, Twohig B, Arfors KE, Harlan JM, Granger DN. Role of neutrophils in ischemia-reperfusion-induced microvascular injury. Am J Physiol 1987; 253:699-703. 48. Mullane KM, Smith CWo The role of leukocytes in ischemic damage, reperfusion injury and repair of the myocardium. In: Piper HM, ed. Pathophysiology of severe ischemic myocardial injury. The Netherlands: Kluwer Academic Publishers, 1990; 239-67. 49. Simpson PJ, Todd RF III, Fantone JC, Mickelson JK, Griffin JD, Lucchesi BR. Reduction of experimental canine myocardial reperfusion injury by a monoclonal antibody (Anti-Mol,Anti-CDllb) that inhibits leukocyte adhesion. J Clin Invest 1988; 81:624-9. 50. VedderNB,Winn RK, Rice CL, Chi EY,Arfors KE, Harber JM. A monoclonal antibody to the adherence-promoting leukocyte glycoprotein, CDI8, reduces organ injury and improves survival from hemorrhagic shock and resuscitation in rab-

WILLIAMS AND HELLEWELL

bits. J Clin Invest 1988; 81:939-44. 51. VedderNB, Winn RK, RiceCL, Chi EY,Arfors KE, Harlan JM. Inhibition of leukocyte adherence by anti-CDl8 monoclonal antibody attenuates reperfusion injury in the rabbit ear. Proc Nat! Acad Sci USA 1990; 87:2643-6. 52. Mileski WJ, Winn RK, Vedder NB, Pohlman TH, Harlan JM, Rice CL. Inhibition of CDl8dependent neutrophil adherence reduces organ injury after hemorrhagic shock in primates. Surgery 1990; 108:206-12. 53. Smith CW, Rothlein R, Hughes BJ, et aI. Recognition of an endothelial determinant for CDI8dependent human neutrophil adherence and transendothelial migration. J Clin Invest 1988; 82: 1746-56. 54. Doerschuk CM, Winn RK, Coxson HO, Harlan JM. CDl8-dependent and -independent mechanisms of neutrophil emigration in the pulmonary and systemic microcirculation of rabbits. J Immunol 1990; 144:2327-33. 55. Barton RW, Rothlein R, Ksiazer J, Kennedy C. The effect of anti-intercellular adhesion molecule-Ion phorbol-ester-induced rabbit lung inflammation. J Immunol 1989; 143:1278-82. 56. Seewaldt-BeckerE, Rothlein R, Dammgen rw CDwl8 dependent adhesion of leukocytes to endothelium and its relevancefor cardiac reperfusion.

In: Springer TA, Anderson DC, Rosenthal AS, Rothlein R, eds. Leukocyte adhesion molecules. New York: Springer-Verlag, 1990; 138-48. 57. Cosimi AB, Conti D, Delmonico FL, et at. In vivo effects of monoclonal antibody to lCAM-l (CD54)in nonhuman primates with renal allografts. J Immunol 1990; 144:4604-12. 58. Argenbright LW, Barton RW. The Shwartzman response: a model of ICAM-I dependent vasculitis. Agents Actions 1991; 34:208-10. 59. Wegner CD, Gundel RH, ReillyP, Haynes N, Letts LG, Rothlein R. Intercellular adhesion molecule-I(ICAM-l) in the pathogenesisof asthma. Science 1990; 247:456-9. 60. Gundel RH, Wegner CD, Torcellini CA, et al. Endothelial leukocyte adhesion molecule-I mediates antigen-induced acute airway inflammation and late phase airway obstruction in monkeys. J Clin Invest 1991; 88:1407-11. 61. Mulligan MS, Varani J, Dame MK, et al. Role of endothelial-leukocyte adhesion molecule I (ELAM-I) in neutrophil-mediated lung injury in rats. J Clin Invest 1991; 88:1396-406. 62. Watson SR, Fennie C, Lasky LA. Neutrophil influx in to an inflammatory site inhibited by a soluble homing receptor-IgG chimaera. Nature 1991; 349:164-6.