Endothelial cell adhesion molecules in inflammation and postischemic reperfusion injury

Endothelial Cell Adhesion Molecules in Inflammation and Postischemic Reperfusion Injury H. Beekhuizen and J.S. van de Gevel

T

HE endothelium of the microvasculature plays an important role in the development of an inflammatory process, induced by foreign agents or injury. This process is accompanied by endothelial cell activation, loss of endothelial integrity, plasma leakage, and accumulation of leukocytes in extravascular tissue. Activation of the vascular endothelium, resulting in expression of its proinflammatory properties, has also been associated with acute leukocytemediated injury occurring in reperfused ischemic tissue.1 These proinflammatory properties include the production of inflammatory cytokines, chemokines, coagulation factors, and vasoactive agents as well as the expression of surface adhesion molecules that promote leukocyte adhesion. In the past 10 years, substantial progress has been made in the identification of mediators and adhesion events involved in the interaction of leukocytes with (activated) endothelium and their subsequent transendothelial migration into tissue. These studies revealed that the type of leukocyte that binds to the endothelial cell surface and thus accumulates in inflamed tissue depends on the nature of the inflammatory stimulus, its persistence, and the type of inflammatory reaction elicited. Almost immediately after the onset of most types of acute inflammation, neutrophils and monocytes adhere to the vascular endothelium and accumulate at the involved site, monocytes accumulating slightly later and in lower numbers than neutrophils. After 6 to 24 hours, monocytes become the predominant cell type because they continue to migrate into the inflamed tissue when neutrophil migration has virtually stopped.2–5 This shift in the type of infiltrating leukocyte correlates with an alteration of the endothelial cell phenotype, referred to as the endothelial cell activation state.6 – 8 ENDOTHELIAL CELL SURFACE MOLECULES INVOLVED IN LEUKOCYTE ADHESION

In areas of inflamed or injured tissue, leukocyte adhesion and transendothelial migration are observed in the postcapillary venules rather than in the precapillary arterioles.9,10 This local accumulation is mainly determined by the expression of specific adhesion molecules on the luminal surface of endothelial cells which serve as receptors for complementary adhesion molecules on circulating leukocytes. In addition, endothelium-derived proinflammatory agents such as platelet activating factor (PAF) as well as the

chemokines interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) have been shown to modulate the functional binding activity of the leukocyte adhesion molecules.11 Endothelial adhesion molecules implicated in leukocyte binding are E-selectin (CD62E), P-selectin (CD62P), intercellular adhesion molecule-1 (ICAM-1, CD54), ICAM-2 (CD102), vascular cell adhesion molecule-1 (VCAM-1, CD106), and platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31).8,12,13 E- and Pselectin belong to the selectin family of adhesion molecules.13,14 This family also includes L-selectin (CD62L) which is expressed on leukocytes only. All selectins contain an NH2-terminal calcium-dependent lectin domain, followed by an epidermal growth factor (EGF) domain and two to nine short consensus repeat units which are structurally related to domains found in complement binding proteins.15 The lectin- and EGF-like domains are principally involved in the binding to sialylated and fucosylated oligosaccharides, such as the sialyl-Lewis X tetrasaccharide (SLex, CD15s).16,17 SLex or related structures are linked to several glycoproteins (referred to as mucins) and glycolipids of which at present only a few have been identified on leukocytes, ie, P-selectin glycoprotein ligand-1 (PSGL-1, CD162) and L-selectin on neutrophils.15,17,18 The primary sequences of ICAM-1, ICAM-2, VCAM-1, and PECAM-1 reveal that these molecules belong to the immunoglobulin (Ig) supergene family of cell surface molecules.12,15,19 All Ig superfamily members have a variable number of repetitive extracellular Ig-like domains, followed by a transmembrane domain and a short cytoplasmic sequence. The Ig-like domains specifically recognize heterodimeric glycoproteins, designated as integrins, the third family of adhesion molecules that are expressed by leukocytes as well as many other types of cell.12,15,20

From the Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands. Supported by the Institute for Radiopathology and Radiation Protection, IRS, J.A. Cohen Institute grant 3.2.13. Address reprint requests to H. Beekhuizen, Department of Infectious Diseases, Leiden University Medical Center, C5-P, PO Box 9600, 2300 RC Leiden, The Netherlands.

© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

0041-1345/98/$19.00 PII S0041-1345(98)01405-5

Transplantation Proceedings, 30, 4251–4256 (1998)

4251

4252

BEEKHUIZEN AND VAN DE GEVEL

Fig 1. At least three consecutive events can be clearly distinguished in the interaction of leukocytes with endothelial cells at sites of inflammation or tissue injury. Leukocytes first attach to the vascular endothelium in a rolling interaction (1) and then are arrested, sticking firmly to the surface membrane of a single endothelial cell (2), before diapedesis (3). Shear forces generated by the flow of blood, endothelial selectins, and Ig-molecules as well as endothelial-derived chemokines are sequentially implicated in this process (see text).

ACTIVATED ENDOTHELIAL CELLS BIND LEUKOCYTES IN A COORDINATED FASHION

Elegant techniques such as intravital microscopy21,22 revealed that before leukocytes actually migrate through the endothelium, a process referred to as transendothelial migration or diapedesis, a coordinated sequence of adhesion events has to occur. This enables leukocytes not only to withstand the forces generated by the flow of blood, referred to as shear forces, but also to make firm contact with the vascular endothelial cells. Figure 1 illustrates the consecutive adhesion events in leukocyte emigration, ie, rolling, firm adhesion, and diapedesis, and the most likely contributions of adhesion molecules to these processes. Leukocyte Rolling

The rolling of leukocytes along the endothelial lining of small venules is observed only at sites of inflammation or vascular injury. Leukocyte rolling is the earliest visible leukocyte-endothelium interaction and involves attachment to and detachment from the endothelium.21,22 Consequently, the leukocytes are sequestered from the main vascular flow and slow down. Studies with mice lacking P-, E-, or L-selectin expression demonstrated that the initial catching or “tethering” of flowing leukocytes to the vessel wall and their subsequent rolling require the sequential interaction of P- and L-selectin with their respective glycoprotein ligands.23–25 The importance of SLex-ligand recognition in this process is evident in patients with the leukocyte adhesion deficiency type II (LAD II) syndrome. Neutrophils from these patients have a defective fucose metabolism and therefore lack SLex expression. As a consequence, the cells roll poorly and fail to adhere firmly to the endothelium.26 At present, it is assumed that to establish efficient rolling, the glycoprotein ligands for endothelial P- or E-selectin are localized and/or clustered on the tips of microvilli on leukocytes. This has now been affirmed for

L-selectin27 and PSGL-128 on neutrophils. The shear forces push the neutrophils in a forward direction. In addition to selectins, endothelial VCAM-1 has been reported to enhance rolling by binding to the a4b1-integrin VLA-4 (CD49d/CD29) which is expressed on eosinophils, monocytes, and lymphocytes.29 –31 Rolling of neutrophils, ie, cells lacking VLA-4 expression, is entirely selectin dependent. Selectin-mediated rolling is an obligatory event before leukocytes can move on to the stage of firm adhesion to the inflamed or injured endothelium. Arrest of Rolling and Firm Adhesion

The abrupt arrest and the firm adhesion of rolling leukocytes to the surface of a specific endothelial cell involve adhesive interactions that are different from those required for rolling. These high affinity interactions are mediated by integrin adhesion molecules on the leukocytes, ie, aLb2integrin LFA-1 (lymphocyte-function associated-1; CD11a/ CD18), aMb2-integrin CR3 (complement receptor 3; CD11b/CD18), aXb2-integrin p150,95 (CD11c/CD18), the a4b1-integrin VLA-4, and the a4b7-integrin LPAM-1 (lymphocyte Peyer’s patch adhesion molecule-1; CD49d/ CD).12,13 In the presence of divalent cations such as magnesium or manganese, these integrins bind either ICAM-1, ICAM-2, or VCAM-1 on the activated endothelium.20,32 Rapid upregulation of surface expression of integrins may provide a way to increase the number of leukocytes recruited to sites of inflammation. However, several studies have shown that upregulated integrin expression is neither necessary nor sufficient to promote leukocyte-endothelial cell interaction33 and that integrin molecules must be activated before they can bind with high affinity to their respective ligands.20,32 Such a conversion of the inactive to the active state occurs rapidly upon activation of the leukocyte, but the triggers of this process are still unknown. There is some evidence that

ENDOTHELIAL CELL ADHESION MOLECULES

during the process of rolling crosslinking of L-selectin on the neutrophils,34 the interaction of these cells with E-selectin35 or the binding of CD14 on monocytes to its ligand on activated endothelium12 directly triggers b2integrin-mediated adherence. Such integrin-mediated firm leukocyte-endothelium interaction may also be triggered or amplified by the action of soluble factors such as LTB4, C5a, PAF, IL-8, or MCP-1 that are present at or near the endothelial surface. These factors may be derived from infiltrating leukocytes, invading microorganisms, or the endothelium itself. The binding of these factors to their seven membrane-spanning, G-proteincoupled receptors on the leukocyte triggers a signal that induces shedding of L-selectin and a conformational change in the integrin molecule, increasing its affinity for endothelial ligands.34,36 –38 For neutrophil adhesion, P-selectin and the biologically active phospholipid PAF act synergistically in inducing integrin activation. Both P-selectin and PAF are expressed on the endothelial cell surface within minutes after stimulation with histamine, thrombin, or in response to ischemia/ reperfusion. The binding of P-selectin to its ligand on neutrophils brings these cells in the proximity of endothelial cell-associated PAF, facilitating PAF binding to its receptor, which leads to the activation and integrin-mediated firm adhesion of neutrophils.38 – 40 A similar situation, referred to as juxtacrine activation,37 is postulated for endothelium-derived chemokine IL-8 and E-selectin. IL-8 is sequestered and immobilized by heparin and glycosaminoglycan moieties of proteoglycans on the luminal surface of endothelial cells. It can activate rolling of neutrophils.41,42 This notion is supported by the discovery of a novel CX3C type of human chemokine occurring as a cell surface bound glycoprotein that is induced on endothelial cells after activation by inflammatory cytokines.43 Because it is chemotactic for monocytes and lymphocytes, it is assumed that this chemokine contributes to the rapid activation of these leukocytes, thus facilitating the transition from selectinmediated rolling over to integrin-mediated firm adhesion to the activated endothelium. Firm adhesion between leukocytes and the endothelium coincides with an increase of the intercellular contact area. These leukocytes alter their shape and become flattened. Then, under the influence of chemotactic agents, they move laterally over the endothelial cell surface. Such a behavior, that is also seen in vitro in the absence of flow, is inhibited by antibodies that neutralize b2-integrins or ICAM-1.12 Diapedesis: The Actual Transendothelial Migration

Adherent leukocytes cross the vessel wall by squeezing themselves in an ameboid fashion between tightly apposed endothelial cells.44 Migration of leukocytes via the cytoplasm of the endothelial cells does not occur. During diapedesis, the barrier function of the endothelium is excellently preserved because of the close interaction between the migrating leukocyte and the endothelial cell membranes.44,45 Although reorganization of the cytoskel-

4253

etal filaments must be involved, the intracellular signals that coordinate the opening of the interendothelial junctions are still unknown. Because leukocytes must adhere to the endothelium before subsequent diapedesis, it is difficult to unravel the contribution of a particular adhesion molecule to firm adhesion of leukocytes and its role in diapedesis as such. In experimental settings, agents that inhibit firm adhesion will also reduce diapedesis. Clearly, b2-integrin molecules are involved in diapedesis. This is concluded from the profound lack in neutrophil and monocyte diapedesis observed in patients with the leukocyte adhesion deficiency type I (LAD I) syndrome, who are genetically deficient in the functional expression of leukocyte b2integrin molecules.46 Beside b2-integrins, PECAM-1 molecules may contribute to diapedesis.47 These molecules are expressed on leukocytes and in high densities on the endothelium at the intercellular junctions. A homophilic binding between leukocyte and endothelial PECAM-1 promotes diapedesis,48 whereby the leukocytes migrate in the direction of the highest density of endothelial PECAM-1, a process called “haptotaxis.” ENDOTHELIUM EXPRESSES ITS ADHESION MOLECULES UPON APPROPRIATE STIMULATION

Under normal steady state conditions, the nonthrombogenic and nonhemostatic surface properties of the endothelium, the constitutively expressed adhesion molecules ICAM-1, ICAM-2, and PECAM-1, and the narrow spaces (varying from 10 to 200 nm) between adjacent endothelial cells do not promote leukocyte adherence or transendothelial migration. The increased interaction between leukocytes and endothelial cells that is elicited during inflammation or tissue injury is mainly due to locally generated inflammatory mediators which induce or increase the expression of adhesion molecules on endothelial cells. This can be mimicked by incubation of monolayers of human endothelial cells in vitro with proinflammatory cytokines,49 such as IL-1a, tumor necrosis factor-a (TNF-a), or interferon-g (IFN-g) or even with intact bacteria.50 Table 1 summarizes changes in surface expression of adhesion molecules on cultured human umbilical cord endothelial cells upon treatment with a variety of activating or injurious agents. Our findings confirm and extend those of other investigators and reveal an often transient expression of the various adhesion molecules on the endothelial cells that depends on the duration of endothelial cell activation and the type of stimulus. For example, within minutes after exposure to IL-1, high levels of P-selectin are expressed, while there is a return to basal levels within 30 to 60 minutes. Such an effect was not found for IL-4 or IFN-g. Several hours after activation with IL-1, the expression of E-selectin is maximal. Levels return to basal within 1 day at which time ICAM-1 and VCAM-1 expression has reached a relatively stable level. These changes in surface expression of adhesion molecules correlate with a shift in extravasation from granulocytes to monocytes and lymphocytes such as is

4254

BEEKHUIZEN AND VAN DE GEVEL Table 1. Effect of Various Exogenous Stimuli on the Expression of Surface Adhesion Molecules on Cultured Vascular Endothelial Cells Endothelial Cell Surface Adhesion Molecule

Stimulus (Dose)*

None† Proinflammatory mediators Histamine (10 mmol/L)

Thrombin (1 U/mL)

IL-1a (5 ng/mL)

TNF-a (500 U/mL)

Lymphotoxin (100 U/mL)

IL-4 (30 U/mL)

IFN-g (500 U/mL)

Bacteria/bacterial products Staphylococcus aureus (live or UV killed)

Stret sanguis (live)

LPS (100 ng/mL)

Other PMA (25 ng/mL)

H2O2 (500 mmol/L)

Time of Stimulation (h)

P-Selectin (CD62P)

E-Selectin (CD62E)

ICAM-1 (CD54)

ICAM-2 (CD102)

VCAM-1 (CD106)

PECAM-1 (CD31)

Absent

Absent

Low

Moderate

Absent

High

0.5 4 24 48 0.5 4 24 48 0.5 4 24 48 0.5 4 24 48 0.5 4 24 48 0.5 4 24 48 0.5 4 24 48

1‡,§ 5 5 5 1 5 5 5 111 1 5 5 111 1 5 5 11 1 5 5 5 5 5 5 5 5 5 5

5 5 5 5 5 5 5 5 5 111 1 5 5 111 1 5 5 111 1 5 5 5 5 5 5 5 5 5

5 5 5 5 5 5 5 5 5 1 111 111 5 1 111 111 5 11 111 111 5 5 5 5 5 1 11 11

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

5 5 5 5 5 5 5 5 5 111 111 11 5 11 111 11 5 11 111 111 5 1 111 111 5 5 1 1

5 5 5 5 5 5 5 5 5 5 5 1 5 5 5 1 5 5 5 1 5 5 5 5 5 5 5 5

0.5 4 24 48 0.5 4 24 48 0.5 4 24 48

5 5 5 5 5 5 5 5 1 5 5 5

5 5 5 5 5 5 5 5 5 11 5 5

5 5 11 1 5 5 1 1 5 1 11 1

5 5 5 5 5 5 5 5 5 5 5 5

5 5 11 1 5 5 11 1 5 11 11 1

5 5 5 5 5 5 5 5 5 5 5 1

0.5 4 24 48 0.5 4 24 48

111 5 5 5 11 11 5 5

5 11 5 5 5 5 5 5

5 11 11 5 5 5 5 5

5 5 5 5 5 5 5 5

5 11 1 5 5 5 5 5

5 5 5 5 5 5 5 5

Stimuli without effect\ IL-2, IL-3, IL-5, G-CSF, GM-CSF, IL-6, IL-8, MCP-1, PAF, IL-10, TGF-b, nitric oxide, g-radiation *Monolayers of cultured human umbilical venous endothelial cells were incubated for the indicated period of time with different concentrations of human inflammatory mediators or other stimuli. Surface expression of the indicated adhesion molecules was analyzed in a flow cytometer using appropriate monoclonal antibodies. The concentration of the stimulus at which maximal induction of surface expression was seen is given between parentheses. † Basal expression of adhesion molecules on the endothelial cell surface in the absence of stimuli. ‡ Expression of adhesion molecules in the presence of stimuli is compared with that under basal unstimulated conditions (none). Data are given as: surface expression unchanged (5), slightly increased (1), modestly increased (11), or strongly increased (111) after stimulation. § Evident endothelial P-selectin expression is already observed 10 minutes after exposure of the cells to histamine. \ Endothelial cells were exposed to different concentrations of the indicated stimuli for 0.5 to 48 hours. No significant changes in surface adhesion molecule expression were observed.

ENDOTHELIAL CELL ADHESION MOLECULES

observed in vivo between 6 and 24 hours after the onset of inflammation.2– 8 In this respect, the response of cultured endothelial cells to various inflammatory mediators can be divided in at least three different ones: an immediate within minutes after stimulation; an early after several hours; and a late response after 1 to 3 days (see Table 1). The immediate response is characterized by the expression of P-selectin on the plasma membrane51 and the production of PAF, which can activate neutrophils.52 P-selectin is stored in the endothelial WeibelPalade bodies,53 that upon stimulation with histamine, thrombin, 12-myristate-13-acetate (PMA), or hydrogen peroxide fuse with the plasma membrane. This results in a rapid and transient expression of P-selectin on the luminal cell surface (Table 1). As stated earlier, P-selectin and PAF may cooperate to stimulate efficiently neutrophil transmigration.39,40 The early response to inflammatory mediators such as IL-1, TNF-a, or bacterial LPS coincides with surface expression of E-selectin and the production of the chemokines IL-8 and MCP-1.13,49,54 E-selectin expression is maximal at 4 to 6 hours after stimulation and returns to basal levels within 24 hours, even if the stimulus persists. During the early response, the expression of ICAM-1 and VCAM-1 is slightly enhanced (Table 1).49 E-selectin and IL-8 or MCP-1 may cooperate to promote efficient transmigration of neutrophils and monocytes, respectively.8,41,42 The late response to inflammation is observed 16 to 24 hours after stimulation. At this time, endothelial cells do no longer express E-selectin, but depending on the type of stimulus express ICAM-1 and/or VCAM-1 (Table 1). These changes in endothelial surface molecules together with the continuous secretion of IL-8 and MCP-1 coincide with the increased recruitment of monocytes and lymphocytes to the inflammed tissue.8,12,55 ENDOTHELIAL CELL ACTIVATION AND LEUKOCYTE ADHERENCE IN REPERFUSED ISCHEMIC TISSUE

Reperfusion with oxygen-rich blood after a period of ischemia is accompanied by activation of endothelial cells, resulting in surface expression of adhesion molecules, generation of proinflammatory mediators, and the subsequent activation and recruitment of leukocytes from the circulation, in particular neutrophils. This sequence of events closely resembles an acute inflammatory response and ultimately may lead to neutrophil-mediated microvascular dysfunction and tissue injury. A role of neutrophils and their interaction with adhesion molecules on the surface of endothelial cells after ischemia-reperfusion can be deduced from the observation that making rabbits neutropenic with polyclonal antibodies or preventing leukocyte-endothelial cell interaction with monoclonal antibodies directed against cell surface adhesion molecules affords protection against vascular postischemic injury.56 Neutrophil adhesion to the endothelium may occur already during the ischemic episode.57 This can be partially explained by a decline of shear forces as a consequence of the reduced flow of blood, a situation which allows leukocytes to settle on the endothe-

4255

lial surface. However, such adherence of leukocytes will only hold when it is accompanied by the expression of appropriate adhesion molecules on the surface of activated endothelial cells.57,58 Upon reperfusion, however, endothelial cell activation is even more pronounced and neutrophil recruitment is enhanced. The magnitude of neutrophil adhesion and migration elicited by reperfusion positively correlates with the duration and the degree of ischemia.59 The rapid adherence of neutrophils to endothelial cells during postischemic reperfusion is explained by the rapid translocation of P-selectin from the intracellular WeibelPalade bodies to the endothelial cell surface together with an enhanced expression of endothelial cell-associated PAF. Wehrich et al60 have shown that the burst of oxygen radicals generated by endothelial xanthine oxidase or other enzymes61 at the onset of reperfusion precedes the induction of P-selectin and PAF. Exposure in vitro of monolayers of endothelial cells to oxidants like H2O2 induces P-selectin expression during several hours (Table 1).62 This is in sharp contrast to the transient P-selectin expression in response to histamine or thrombin and is most likely due to aberrant reinternalization of P-selectin. Protection against postischemic reperfusion injury has been observed in animals treated with anti P-selectin antibodies,60 synthetic drugs that downregulate P-selectin surface expression,63 and after administration of PAF-receptor antagonists.64 Furthermore, intravascular infusion of PAF to mimic the elevated PAF levels in plasma that are normally associated with ischemia and reperfusion induces subsequent neutrophil infiltration.64 Similar to the events as described earlier for the immediate endothelial inflammatory response, the PAF-induced activation of neutrophils results in a higher binding activity of their b2-(CD11/CD18) integrin molecules, which in turn will bind to endothelial ICAM-1 to establish firm adhesion and transendothelial migration of the neutrophil.52 In several in vivo and in vitro models, antibodies against either CD18, CD11a, or CD11b on the neutrophils or against ICAM-1 on the endothelial cells have been demonstrated to attenuate neutrophil adhesion and microvascular injury.64 Taken together, these findings are evidence that early P-selectin-mediated rolling along the endothelium positions the neutrophil for appropriate activation by endothelialassociated PAF and subsequent induction of b2-integrin/ ICAM-1-mediated adhesion. This then may initiate the sequence of neutrophil-mediated harmful events accompanied by the release of reactive oxygen metabolites (eg, H2O2, O2 2 ) and proteases which ultimately result in microvascular dysfunction and tissue injury.65

REFERENCES 1. Land W, Meßmer K: Transplant Rev 10:108, 1996 2. Issekutz AC, Movat HZ: Lab Invest 42:310, 1980 3. Issekutz TB, Issekutz AC, Movat HZ: Am J Pathol 103:47, 1981 4. Paz RA, Spector WG: J Pathol Bacteriol 84:85, 1962

4256 5. van Furth R, Diesselhoff-den Dulk MMC, Mattie H: J Exp Med 138:1314, 1997 6. Munro JM, Pober JS, Cotran RS: Lab Invest 64:295, 1991 7. Henseleit U, Steinbrink K, Sunderko ¨tter C, et al: Exp Dermatol 3:249, 1994 8. Malik AB, Lo SK: Pharmacol Rev 48:213, 1996 9. Atherton A, Born GVR: J Physiol 233:157, 1973 10. Ley K, Gaehtgens P: Circ Res 69:1034, 1991 11. Ebnett K, Kaldjian EP, Anderson AO, et al: Annu Rev Immunol 14:155, 1996 12. Beekhuizen H, van Furth R: J Leukoc Biol 54:363, 1993 13. Carlos TM, Harlan JM: Blood 84:2068, 1994 14. Tedder TF, Steeber DA, Chen A, et al: FASEB J 9:866, 1995 15. Springer TA: Nature 346:425, 1990 16. Murphy JF, McGregor L: Biochem J 303:619, 1994 17. Varki A: Proc Natl Acad Sci USA 91:7390, 1994 18. McEver RP, Moore KL, Cummings RD: J Biol Chem 270:11025, 1995 19. Williams AF, Barclay AN: Annu Rev Immunol 6:381, 1988 20. Hynes RO: Cell 69:11, 1992 21. von Andrian UH, Chambers JD, McEnvoy L, et al: Proc Natl Acad Sci USA 88:7538, 1991 22. von Andrian UH, Hansell P, Chambers JD, et al: Am J Physiol 263:H1034, 1992 23. Mayadas TN, Johnson RC, Rayburn H, et al: Cell 74:541, 1993 24. Tedder TF, Piscueta P: J Exp Med 181:2259, 1995 25. Ley KE, Bullard D, Arbones ML, et al: J Exp Med 181:669, 1995 26. Etzioni A, Frydman MD, Pollack S, et al: N Engl J Med 327:1789, 1992 27. Picker LJ, Warnock RA, Burns AR: Cell 66:921, 1991 28. Moore KL, Patel KD, Bruehl RE: J Cell Biol 128:661, 1995 29. Sriramarao P, von Andrian UH, Butcher EC, et al: J Immunol 153:4238, 1994 30. Berlin C, Bargatze RF, Campbell JJ, et al: Cell 80:413, 1995 31. Alon R, Kassner PD, Woldemar Carr M, et al: J Cell Biol 128:1243, 1995 32. Humphries MJ: Curr Opin Cell Biol 8:632, 1996 33. Vedder NB, Harlan JM: J Clin Invest 81:676, 1988 34. Simon SI, Burns AR, Taylor AD, et al: J Immunol 155:1502, 1995 35. Lo SK, Lee S, Ramos SA, et al: J Exp Med 173:1493, 1991 36. Murphy PM: Annu Rev Immunol 12:593, 1994 37. Bokoch GM: Blood 86:1649, 1995 38. del Pozo MA, Sa´nchez-Mateos P, Sa ´nchez-Madrid F: Immunol Today 17:127, 1996

BEEKHUIZEN AND VAN DE GEVEL 39. Lorant DE, Patel KD, McIntyre TM, et al: J Cell Biol 115:223, 1991 40. Zimmerman GA, Prescott SM, McIntyre TM: Immunol Today 13:93, 1992 41. Rot A: Immunol Today 13:291, 1992 42. Tanaka Y, Adams DH, Shaw S: Immunol Today 14:111, 1993 43. Bazan JF, Bacon KB, Hardiman G, et al: Nature 385:640, 1997 44. MacLeod AG: In Thomas BE (ed): Aspects of Acute Inflammation. Kalamazoo, MI: Upjohn Company, 1975 45. Dejana E, Corada M, Lampugnani MG: FASEB J 9:910, 1995 46. Arnaout MA: Immunol Rev 114:147, 1990 47. Muller WA, Weigl SA, Deng X, et al: J Exp Med 178:449, 1993 48. DeLisser HM, Newman PJ, Albelda SM: Immunol Today 15:490, 1994 49. Pober JS, Gimbrone MA, Lapierre LA, et al: J Immunol 137:1893, 1986 50. Beekhuizen H, van de Gevel JS, Olsson B, et al: J Immunol 158:774, 1997 51. Celi A, Furie B, Furie BC: Proc Soc Exp Biol Med 198:703, 1991 52. Zimmerman GA, McIntyre TM, Mehra M, et al: J Cell Biol 110:529, 1990 53. Hattori R, Hamilton KK, Fugate RD, et al: J Biol Chem 264:7768, 1989 54. Bevilacqua MP, Stengelin S, Gimbrone MA, et al: Science 243:1160, 1989 55. Carlos TM, Schwartz BR, Kovach NL, et al: Blood 76:965, 1990 56. Hernandez LA, Grisham MB, Twohig B, et al: Am J Physiol 253:H699, 1987 57. Milhoan KA, Lane TA, Bloor CM: Am J Physiol 263:H956, 1992 58. Ginis I, Mentzer SJ, Li X, et al: J Immunol 155:802, 1995 59. Rainger GE, Fisher A, Shearman C, et al: Am J Physiol 269:H1398, 1995 60. Wehrich AS, Ma X, Lefer DJ, et al: J Clin Invest 91:2620, 1993 61. Bulkley GB: Lancet 344:934, 1994 62. Patel KD, Zimmerman GA, Prescott SM, et al: J Clin Invest 82:2045, 1988 63. Scalia R, Murohara T, Delyani JA, et al: J Leukoc Biol 59:317, 1996 64. Granger DN, Kvietys PR: Can J Physiol Pharmacol 71:67, 1993 65. Ward PA, Varani J: J Leuk Biol 48:97, 1990