Endothelial cell regulation of leukocyte infiltration in inflammatory tissues

Endothelial cells play an important, active role in the onset and regulation of inflammatory and immune reactions. Through the production of chemokines they attract leukocytes and activate their adhesive receptors. This leads to the anchorage of leukocytes to the adhesive molecules expressed on the endothelial surface. Leukocyte adhesion to endothelial cells is frequently followed by their extravasation. The mechanisms which regulate the passage of leukocytes through endothelial clefts remain to be clarified. Many indirect data suggest that leukocytes might transfer signals to endothelial cells both through the release of active agents and adhesion to the endothelial cell surface. Adhesive molecules (such as PECAM) on the endothelial cell surface might also ‘direct’ leukocytes through the intercellular junction by haptotaxis. The information available on the molecular structure and functional properties of endothelial chemokines, adhesive molecules or junction organization is still fragmentary. Further work is needed to clarify how they interplay in regulating leukocyte infiltration into tissues.

rage of leukocytes to the adhesive molecules expressed on the endothelial surface. Leukocyte adhesion to endothelial cells is frequently followed by their extravasation. The mechanisms which regulate the passage of leukocytes through endothelial clefts remain to be clarified. Many indirect data suggest that leukocytes might transfer signals to endothelial cells both through the release of active agents and adhesion to the endothelial cell surface. Adhesive molecules (such as PECAM) on the endothelial cell surface might also 'direct' leukocytes through the intercellular junction by haptotaxis. The information available on the molecular structure and functional properties of endothelial chemokines, adhesive molecules or junction organization is stir fragmentary. Further work is needed to clarify how they interplay in regulating leukocyte inf'tltration into tissues.

Introduction
Circulating leukocytes migrate from the vessels and enter tissues under normal or pathological conditions. Whereas monocytes, lymphocytes and natural killer cells exhibit a significant spontaneous migration through resting endothelial cells (EC), neutrophils and eosinophils require chemotactic stimuli and/or endothelial cell activation. [1][2][3] Cell migration across endothelial monolayers involves leukocyte adherence to the endothelium, crawling on the endothelial surface and penetration between endothelial clefts.
Endothelial cells can actively regulate leukocyte infiltration in inflammatory tissues through different mechanisms such as vasodilatation, release of chemotactic cytokines, expression of adhesion molecules and opening of interendothelial junctions (Fig. 1).1 '2'4-6 All these reactions act in concert in localizing leukocytes and in facilitating their passage through the interendothelial junctions. Inflammatory stimuli are able to activate endothelial cells in,ducing their fun,ctional reprogramming toward a proinflammatory phenotype.qnterleukin-1 (ILl) or tumour necrosis factor (TNF) induce production of the vasodilatatory mediators such as prostacyclin and nitric oydde, 5''8 as well as the synthesis of a lae series of adhesive molecules and chemokines , and cause endothelial increase in permeability and alteration of junction organization. 6'9'1 In addition to 'classic' inflammatory agents, stimuli associated with the development of atherosclerotic plaques (such as minimally oxidized low-density lipoprotein [LDL]) can also modify endothelial cell reactivity and induce monocyte and lymphocyte infiltration into the vessel wall. In a general sense atherosclerotic plaque evolution presents many similarities with inflammatory reactions.
In this review we will focus concisely on the role of endothelial cells in promoting leukocyte infiltration in tissues. In particular, we will consider chemokine production, expression of adhesive molecules and regulation of junction organization. Previous reviews of this rapidly expanding area of research provide the background and framework for this contribution. ,6J,2,3

Chemokines
The proinflammatory chemokines are a family of 16 homologous low molecular weight   cyte subtypes inducing a large set of responses including change in cell shape, release of enzymes, formation of bioactive lipids, respiratory burst and most importantly activation of adhesive molecules and chemotaxis. 4'5 One of their characteristics is the presence of four cysteines, conserved in all members of the. family.
They can be subgrouped in 0t-chemokines (Table  1) when the first two cysteines are interrupted by one amino acid (C-X-C) and ]3-chemokines when they are together (C-C). cz-Chemokines act on neutrophils, ]3-chemokines activate monocytes, eosinophils and basophils. 4'5 In vitro studies: Endothelial cells produce various chemokines in response to signals representative of inflammatory reactions, immunity and thrombosis. 5 Inflammatory cytokines (IL-1 and TNF) and bacterial endotoxins induce expression and release of IL-8 and GROcz. [16][17][18][19][20][21][22][23] Induction of IL-8 expression is associated with and depends on gene transcription, i IL-4 and IL-13 are weak inducers of IL-8 expression and amplify induc-24 25 28 tion by inflammatory cytokines. Histamine induces IL-8 production in EC. 29 Hypoxia has recently been shown to induce IL-8 and MCP-1 expression in EC, a finding potentially relevant for pathological conditions in which activation and recruitment of leukocytes may amplify tissue damage. 3'31 Platelets contain IL-1 and, when they interact with vascular EC, induce IL-8 gene expression. 32 As a result of proteolytic cleavage, IL-8 versions with a different NI-{ 2 terminus and length can be produced. 14 It has been suggested that EC release predominantly a 77-amino acid version of IL-8, which is a less active species at activating leukocytes than the most common 73-residue form. 2' The proteolytic conversion to smaller versions of the molecule can be catalysed by thrombin.
The influence of IL-8 on the interaction of polymorphonuclear cells with vascular EC has been the object of seemingly conflicting observations, which seem now to reflect different experimental protocols and, most interestingly, different functions exerted by this cytokine under different pathophysiological conditions. IL-8 increased the adhesiveness of normal polymorphonuclear leukocytes for normal EC. 4 In apparent contrast with these findings, EC-derived IL-8 was reported to inhibit binding of the polymorphonuclear leukocytes to activated EC. 2 Although it elicits polymorphonuclear leukocyte extravasation when given locally, IL-8 inhibits recruitment if administered systematically by the i.v. route. 5'6 The seemingly paradoxical antiinflammatory effects of high levels of systemic IL-8, possibly dependent upon the action of a reverse chemotactic gradient and leukocyte deactivation, may represent a feedback mechanism to control tissue damage.
The role of It-8 produced locally by vascular cells was reexamined using reconstructed vessel wall models. 7 Unequivocal evidence was obtained for the importance of IL-8 in transendothelial migration induced by inflammatory cytokines. 7'8 EC of postcapillary venules bind IL-8, possibly via heparin-like molecules. 39 EC of postcapillary venules in kidney and other tissues express the promiscuous chemokine receptor present also on erythrocytes and known as Duffy antigen. ' This receptor is present on EC of both Duffy+ and Duffy-individuals and may serve to present chemokines to circulating leukocyes. Solid phase IL-8 elicits haptotactic migration. Thus, locally t Duperray et al.
produced IL-8 may be retained on the surface of natural history of atherosclerosis. EC staining was EC and activate adhesive interactions and migraprominent in diffuse intimal thickening and in tion. 39 fatty streaks, whereas it was weak in ather-EC activated in vitro by inflammatory cytoomatous lesions. Subendothelial macrophages kines express GRO0t, which according to one were strongly positive for MCP-1 in fatty streak report, could in turn act on EC. 17 It has been lesions and in atherosclerotic plaques. In suggested that EC-bound GROa may promote plaques, a few intimal smooth muscle cells monocyte adhesion. 4 stained for MCP-1. These results suggest that EC IP10, a member of the C-X-C family but and macrophages are the major source of MCP-1 unique in that it attracts monocytes, is expressed in early atherosclerotic lesions. 53 Monocyte adhein certain endothelia of mice exposed in vivo to sion and infiltration is an early event in the IFNT or to lipopolysaccharides (tPS). 44 '45 There natural history of atherosclerosis. ' Monoare no reports on in vitro expression of this nuclear phagocyte infiltration is also a prominent chemokine in EC.
feature of vasculitis. 58 Locally produced MCP-1 EC produce substantial amounts of the C-C may play an important role in regulating extrachemokine MCP-1. 2'22 The proinflammatory vasation of leukocytes, of monocytes in partisignals IL-1, TNF and, to a lesser extent, endo-cular, in vessel wall pathology. toxin are potent stimuli for MCP-1 production. 21 The recruitment of leukocytes into sites of induce MCP-1, though we did not detect the Minflammation involves a cascade of sequential CSF receptor c-fms in EC by Northern analysis. 47 events controlled by the interaction between Given the role that lipids and monocytes play in adhesion molecules expressed by leukocytes and the natural history of atherosclerosis, it is of by the endothelium. This multi-step process is a interest that minimally modified LDL induce MCPcell to cell adhesive reaction that involves specific 1 production in EC and smooth muscle cells. 48 binding of membrane receptors on one cell to Thrombin was recently found to induce exprescounter-receptor structures on the other cell. sion of MCP-1 in monocytes and, less promi-Several adhesion receptors belonging to different nently, in EC. 49 The C-C chemokine RANTES families including integrins, selectins, and immuwas produced by EC exposed to TNF and noglobulin-like molecules have been shown to IFN7. 5 participate in this mechanism. '2 In the current The molecular basis of stimulation of chemo-model, selectins are implicated in the initial kine expression in EC has been studied to a rolling, while adhesion receptors from the integlimited extent. Induction by inflammatory signals fin family and the immunoglobulin superfamily and thrombin is protein synthesis independent in are involved in the firm attachment, flattening EC, but, interestingly, not in monocytes. 49 Direct and extravasation of leukocytes. '2'59'6 Leukodemonstration of enhanced gene transcription cytes have to adhere to the endothelium before was obtained for MCP-1 and IL-8 by nuclear run transmigrating, and it is difficult to distinguish off analysis. 2'2 between adhesion molecules involved only in adherence and proteins involved in the transmi-In vivo studies: EC at sites of delayed type hypergration process. However, several studies have sensitivity reactions and kidney allograft rejection shown that intercellular cell adhesion molecule 1 ex r the ch m kin 5 52 p ess C-C e o'e RANTES.
In vivo (ICAM-1), vascular cell adhesion molecule 1 studies on chemokines in vessel wall pathology (VCAM-1), platelet endothelial cell adhesion have largely been restricted to atherosclerosis, molecule 1 (PECAM-1) and selectins are impor-MCP-1 expression has been detected in atherotant for leukocyte diapedesis between endothelial matous lesions of rabbits, primates and man. 53-56 cells. IL-8 and MCP-1 mRNA have been detected in increased amounts of aortic aneurisms. 57  Eand P-selectins are expressed on endothelial ICAM-1 is moderately expressed on resting cells, while L-selectin expression is restricted to endothelial cells, but release of cytokines at sims leukocytes. Selectins bind to carbohydrate ligands of inflammation and immune response such as via their lectin domains. It has been shown that TNF-cz, IL-1 or IFN3, results in augmented celtetrasaccharides sialyl Lewis X and sialyl Lewis A lular expression of ICAM-1. 87 '88 The expression (sLeX, sLeA) have a ligand activity for all the of ICAM-1 has also been demonstrated on lymthree selectins. 61'64 The role of selectins in leuko-phocytes, monocytes and other non-haematocyte transmigration is still debated, poietic cells, like fibroblasts, epithelial cells and mucosal cells. 87'89 ICAM-1 is a ligand for CDlla/ E-selectin. E-selectin (CD62E) is a 115kDa glyco-CD18 (LFA-1) 9 and for CD11b/CD18 (Mac-l). 91 protein, only expressed on EC after activation by The primary binding site for CDlla/CD18 is IL-1, TNF-cz, 65 or bacterial endotoxin such as located in the NHi-terminal first Ig-like domain LPS. 66 After EC stimulation, newly synthesized Eof ICAM-1, with domain 2 also involved in this selectin is rapidly detected with a maximal interaction, 92 while the one for CD11b/CD18 is surface expression after 3-6h and a return to localized to the third Ig-like domain. 9 ICAM-1 is basal levels within 24h. 65'67 This rapid down-also a receptor for the major group of rhinoregulation, although not completely understood, viruses 94'95 and the malaria trophozoite Plasmohas been explained by the release of a soluble dium falciparum, 9 the binding site for both form of E-selectin, 68'69 and internalization of the ligands, though distinct from the LFA-1 binding molecule. 7 This regulation of E-selectin expressite, is located in the first two domains of the sion might be crucial to control leukocyte accu-ICAM-1 molecule. 92 '97 In addition, ICAM-1 is a mulation in inflammatory responses. Several receptor for CD43 98 and hyaluronan. 99 The ligands for E-selectin have been identified on leuinteraction between Mac-1/LFA-1 (CD11a,b/ kocytes, but have not yet been cloned. 71'72 CD18) and endothelial ICAM-1 is a well docu-Monoclonal antibodies specific for E-selectin mented adhesion pathway, important in the have been shown to inhibit leukocyte transmigra-adhesion and extravasation of leukotion.  It has been suggested that binding of cytes. 73'74'100-102 It has been shown recently that leukocytes to E-selectin on activated endothelium fibrinogen (Fg) is a ligand for ICAM-1, and that upregulates CDllb (Mac-l) on the leukocytes, Fg binding to ICAM-1 results in enhanced adheand induces an increased adhesion through an sion of leukocytes to EC monolayers, 1 and an ICAM-1/Mac-1 interaction. 7'77 '78 increase in their transendothelial migration. 14 These results suggest that Fg must act as a bridg-P-selectin. P-selectin (CD62P), previously termed ing molecule: for monocyte and polymorpho-PADGEM or GMP-140, is a single-chain glycopro-nuclear leukocyte adhesion, it could bind to tein of 140kDa, expressed in platelets and endo-leukocyte CD11b/CD1815 and to endothelial thelial cells. In platelets, P-selectin is stored in cz-cell ICAM-1, while for lymphocyte adhesion it granules, 79 whereas in endothelial cells it is could interact with two ICAM-1 molecules on found in Weibel-Palade bodies. '1 After activaopposing cells. This interaction between Fg and tion, P-selectin is mobilized to the external ICAM-1 was inhibited by a commercially availplasma membrane within minutes. This increase able mAb specific for ICAM-1, LB2, epitopein P-selectin expression is transient, and the mapped to the first immunoglobulin domain of protein is rapidly internalized inside the cell, 97 suggesting that this domain is involved where it is degraded or recycled. 82'83 P-selectin is in the Fg interaction with ICAM-1. However, the also upregulated transcriptionally by TNF-z. 67 inhibition obtained with LB2 on Fg-dependent Two ligands has been identified: the P-selectin adhesion was only partial, even at a high mAb glycoprotein ligand-1 (PSGL-1), expressed on concentration. This limited inhibition might a4 various leukocytes and a 120kDa ligand reflect the fact that LB2 is not reacting with the expressed only on myeloid cells. 5 P-selectin exact binding site of Fg on ICAM-1, but rather deficient mice have been shown to be deficient with a nearby site. Alternatively, an unidentified 60 in leukocyte extravasation.
Fg receptor present at the surface of endothelial cells could contribute to Fg-mediated adhesion IgG superfamily: of leukocytes. Both Fg-mediated leukocyte adhesion and transendothelial migration could be ICAM-1. In general, interaction of leukocytes inhibited by a peptide from the fibrinogen 3' with ICAM-1 seems to be necessary for their chain. 16 extravasation. ICAM-1 (CD54) is a single chain. membrane glycoprotein of 80-115kDa, with five VCAM-1. VCAM-1 (CD106) is a transmembrane Ig-like repeats in its extracellular domain. 6  cytokine-activated endothelium. 7'8 A protein malemmal undercoat. -2 One of the typical containing six Ig-like domains was initially characteristics of endothelial junctions is their cloned (6D VCAM-1), m9 but this form arises dynamic organization. Endothelial cells are able from an alternative splicing of a seven Ig-like to rapidly change the architecture of the juncdomain of VCAM-1 (7D VCAM-1), which is the tions to allow the passage of circulating blood dominant form on activated endothelium. m-2 cells. This effect, in most cases, is quickly rever-VCAM-1 is a ligand for x4131 (VLA-4) and a4137 sible and the endothelium is able to disintegrins. -6 VLA-4 binds VCAM-1 through organize/reorganize its intercellular junctions the first and the fourth Ig domain. 7'8 Using within minutes. Interendothelial junctions monoclonal antibodies, several studies have present a different degree of complexity along shown that VCAM-1 is involved in the transmi-the vascular tree responding to different funcgration of monocytes and eosinophils, 74'75 but tional requirements.
For instance, they are its involvement in lymphocyte transendothelial well organized and numerous in large arteries or migration remains to be clarified. 9'2 in the blood vessels of the brain where the control of permeability must be strict, whereas PECAM-1. PECAM-1 (CD31)is a 130kDa glyco-they are very primitive in the post-capillary protein expressed on endothelial cells, platelets venules, where cell extravasation and exchange and some leukocytes. 12 CD31 is constitutively of plasma constituents need to be particularly expressed on endothelial cells, and its expression efficient. 1 is not increased by cytokines. 22 Molecular On the basis of morphological and functional cloning studies have shown that CD31 is com-characteristics at least four types of junctions posed of six extracellular Ig-like domains, a short have been described in endothelial cells. These 134 136 transmembrane region, and a relatively long cyto-are: tight junctions (TJ), adherence junc- 132 137 138 plasmic tail of 118 amino acid-containing potentions (AJ), gap junctions and syndesmos. 122 125 tial sites for posttranslational modifications.
Although there is a great deal of information Alternative splicing of the cytoplasmic tail can regarding the molecules that mediate leukocyte generate multiple CD31 isoforms that may regadhesion to the endothelium, the mechanisms by ulate phosphorylation, cytoskeletal association which leukocytes trigger the opening of endotheand ligand affinity of the potein. 126 CD31 is lial cell junctions is still obscure. In many condiheavily glycosylated and glycosylation accounts tions the passage of leukocytes through for 40% of the mass of CD31. 2 PECAM-1 endothelial junctions is a non-toxic process that appears to be able to interact both with itself in a does not increase endothelial permeability per se homophylic interaction and with other molecules or cause vascular damage. 9 in a heterophylic interaction. 25'27 In endothelial Chemoattractants and adhesive molecules sticells, CD31 is localized at intercellular junc-mulate neutrophils to secrete oxygen free raditions, 122'125 and plays an important role in adhecals, lipid metabolites and proteases, each of 125 h sion of endothelial cells.
The hig level of which is a potential agonist of endothelial perconstitutive expression of PECAM-1 in endothelial meability.
However, experimental evidence cells suggests that its function might be regulated, suggests that these reactive agents are not necesand phosphorylation of the cytoplasmic domain sary for neutrophil extravasation. has been demonstrated. 28 PECAM-1 is directly In patients with chronic granulomatous disease involved in the process of leukocyte diapedesis leukocytes are unable to make oxygen metabobetween endothelial cells, as demonstrated by lites but can extravasate and infiltrate in areas of inhibition studies using anti-PECAM-1 monoclonal inflammation and form pus. 39 antibodies and soluble recombinant PECAM-1. 29 Inhibitors of proteases do not affect neutrophil Leukocytes blocked in transmigration by anti-extravasation in different experimental condi-PECAM-1 antibodies remained attached to the tions. 139 In addition, the possibility that leukocyte endothelium, clearly implicating PECAM-1 in dia-passage through the endothelium requires proh 129 pedesis rather than in ad es'on, tease digestion of membrane proteins seems unlikely in view of the very rapid, within seconds, closure and reorganization of the junctions after Regulation of endothelial cell-to-cell leukocyte diapedesis. junctions These observations, however, do not exclude the possibility that oxygen free radicals and pro-Circulating cells infiltrate into tissues migrating teases might act as contributing factors in leukothrough intercellular junctions. These organelles cyte extravasation, inducing endothelial cell are formed by a complex network of transmem-damage and mediating oedema during sustained brane proteins linked to a well developed plasinflammatory reactions.
The question of how leukocytes pass through endothelial clefts remains open. An interesting possibility is that leukocyte adhesion to endothelial cells could cause a cascade of events that resembles that induced by soluble agonists of endothelial permeability. In particular, leukocyte ligation to endothelial adhesive molecules (such as selectins, VCAM or ICAM-1) could generate intracellular signals similar to those induced by permeability increasing agents. It has been found 14 that endothelial cells respond to neutrophil contact and migration by increasing intracellular calcium. Similarly, inhibitors of intracellular Ca 2 + block neutrophil transmigration. In addition, ICAM-1 activation by specific antibodies leads to cortactin phosphorylation. 14 This or other signals could induce changes in cleft molecular organization (see above), leading to the opening of gaps between endothelial cells. According to this hypothesis endothelial cells would not only play an important role in regulating leukocyte attachment to their surface but also actively modulate their extravasation.
Leukocytes might find preferential pathways for their passage through the interendothelial clefts. As discussed above, TJ and AJ comprise a system of discrete ion selective pores rather than an absolute seal around the cells. 134 In endothelial cells, the presence of areas of junctionless clefts that regulate the transendothelial transport of high molecular weight proteins has been described. 142 Leukocytes might be directed to these pores through the concentration gradient of specific adhesive molecules such as PECAM. Their passage would require them to squeeze through the pores accompanied by a rearrangement of the endothelial cell cytoskeleton organization around these structures.
There might be differences comparing the modalities of leukocyte extravasation for different types of vessels, for example lymphatic versus large vessels, where the clefts present different levels of complexity. There might also be distinct mechanisms regulating the passage of the different types of leukocytes or of other types of cells.

Concluding remarks
We begin now to understand that leukocytes and endothelial cells are able to communicate and reciprocally modulate their responses. Following inflammatory stimulation endothelial cells attract and localize leukocytes through the release of chemokines and expression of adhesive molecules. Leukocytes in turn might transfer signals to the endothelium releasing soluble mediators, such as cytokines, oxidation products and lytic enzymes. Adhesion of leukocytes to endothelial adhesive molecules might also cause endothelial cell activation facilitating leukocyte passage through interendothelial clefts. Leukocyte extravasation is not always accompanied by endothelial cell damage and an increase in permeability. In contrast, it seems that the opening of endothelial junctions is a well regulated process that is frequently reversible. Future work is required to fully understand how we might modulate the cross-talk between endothelial cells and leukocytes. This appears to be a difficult task considering the complexity and the number of soluble and membrane bound molecules which involved interplay.