Chemotaxins inhibit neutrophil adherence to and transmigration across cytokine-activated endothelium: correlation to the expression of L-selectin

Non-activated neutrophils strongly adhere to cytokine-activated human umbilical vein endothelial cells (HUVE). However, activation of neutrophils by different chemotactic mediators led to potent inhibition of this endothelial-dependent interaction. For different formylated peptides, concentrations leading to maximal adherence inhibition coincided with those known for inducing maximal chemotactic migration of neutrophils. In terms of maximal adherence inhibition, a rank list was found in the order of N-formyl-Met-Leu-Phe > C5adesArg > interleukin-8 > C5a > or = leukotriene B4, whereas platelet-activating factor, and lipopolysaccharide showed no inhibition. This rank order was congruent to that of down-regulation of neutrophil L-selectin detected by the monoclonal antibody Leu-8. Moreover, the dose-dependent increase of neutrophil adherence inhibition corresponded to the loss of L-selectin expression. Concentrations higher than that required for maximal inhibition led to a dose-dependent decrease of inhibition, which was accompanied by increasing expression of neutrophil CD11/CD18. In contrast to the capacity of non-activated neutrophils to migrate across interleukin-1-activated HUVE monolayers, transmigration was significantly impaired after chemotactic activation.     

GAS6 inhibits granulocyte adhesion to endothelial cells

GAS6 is a ligand for the tyrosine kinase receptors Rse, Axl, and Mer, but its function is poorly understood. Previous studies reported that both GAS6 and Axl are expressed by vascular endothelial cells (EC), which play a key role in leukocyte extravasation into tissues during inflammation through adhesive interactions with these cells. The aim of this work was to evaluate the GAS6 effect on the adhesive function of EC. Treatment of EC with GAS6 significantly inhibited adhesion of polymorphonuclear cells (PMN) induced by phorbol 12-myristate 13-acetate (PMA), platelet-activating factor (PAF), thrombin, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), but not that induced by FMLP and IL-8. GAS6 did not affect adhesion to resting EC. Titration experiments showed that high concentrations of GAS6 were needed to inhibit PMN adhesion and that inhibition was dose-dependent at the concentration range of 0.1 to 1 microg/mL. One possibility was that high concentrations were needed to overwhelm the effect of endogenous GAS6 produced by EC. In line with this possibility, treatment of resting EC with soluble Axl significantly potentiated PMN adhesion. Analysis of localization of GAS6 by confocal microscopy and cytofluorimetric analysis showed that it is concentrated along the plasma membrane in resting EC and treatment with PAF induces depletion and/or redistribution of the molecule. These data suggest that GAS6 functions as a physiologic antiinflammatory agent produced by resting EC and depleted when proinflammatory stimuli turn on the proadhesive machinery of EC.     

Fever-range hyperthermia enhances L-selectin-dependent adhesion of lymphocytes to vascular endothelium

The L-selectin leukocyte adhesion molecule plays an important role in controlling leukocyte extravasation in peripheral lymph nodes and at sites of tissue injury or infection. Although febrile responses during infection and inflammation are associated with enhanced immune activity, the contribution of fever-range temperatures to controlling lymphocyte recruitment to tissues has not been previously examined. In this report we provide evidence that direct exposure of lymphocytes to fever-range temperatures (38-41 degrees C) in vitro for 9 to 24 h resulted in a >100% increase in L-selectin-dependent adhesion of these cells to lymph node high endothelial venules (HEV). Moreover, culture of lymphocytes under hyperthermia conditions markedly enhanced the ability of these cells to traffic in an L-selectin-dependent manner to peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches. In contrast, febrile temperatures did not increase LFA-1 function as assessed by measuring lymphocyte adhesion to ICAM-1-3T3 transfectants. Fever-range hyperthermia further did not increase L-selectin surface density on lymphocytes or L-selectin-dependent recognition of soluble carbohydrate substrates; however, a marked increase in ultrastructural immunogold-labeling of L-selectin was observed in response to thermal stimuli. These results suggest that elevated temperatures enhance L-selectin adhesion and/or avidity through the regulation of L-selectin conformation or organization in the plasma membrane. Finally, the observed thermal effects on L-selectin adhesion were attributed to soluble factors in the conditioned medium of heat-treated cells. Taken together, these data provide new insight into the potential physiologic role of the febrile response in enhancing lymphocyte recruitment to tissues through the regulation of L-selectin adhesion.     

Mice lacking E-selectin show normal numbers of rolling leukocytes but reduced leukocyte stable arrest on cytokine-activated microvascular endothelium

OBJECTIVE: Previous work indicated that E-selectin mediates transient interactions between leukocytes and cytokine-activated endothelium in vitro. Here we examine the role of E-selectin in blood leukocyte interactions with microvascular endothelium in vivo. METHODS: E-selectin-deficient (E-/-) mice were produced by gene targeting. The effect of this null mutation on leukocyte-endothelial interactions was determined by intravital microscopy before and 4 to 5 hours after local administration of the proinflammatory cytokine tumor necrosis factor alpha (TNF alpha) in dermal microvessels with low blood flow (dorsal skin-fold chambers, intact ear skin), and after endotoxin activation in exteriorized mesenteric microvessels with higher blood flow. RESULTS: E-/- mice were viable, fertile with normal circulating leukocyte and platelet profiles. Approximately 60% of circulating leukocytes rolled in dermal microvessels of both normal (E+/+) and E-/- mice without inflammatory stimulation. After local administration of TNF alpha, rolling increased modestly and equivalently in both genotypes. The main effect of TNF alpha was a dramatic increase in leukocyte stable adhesion and, unlike rolling, this manifestation of endothelial activation was significantly reduced in E-/- animals. This reflected fewer dermal microvessels supporting higher adhesion densities in E-/- mice, and a similar trend was observed in mesenteric microvessels. CONCLUSIONS: E-selectin plays a previously unappreciated role in facilitating and/or mediating stable adhesion of leukocytes to inflamed microvascular endothelium.     

Substance P induces degranulation of mast cells and leukocyte adhesion to venular endothelium

Substance P (SP), one of the established neurotransmitters, evokes an immunoinflammatory response involving leukocyte adhesion to venular endothelium and the degranulation of mast cells. The pathogenetic relationship between these responses, however, remains unresolved. In this study, we propose to examine the changes associated with the activation of mast cells, as well as leukocyte adhesion to venular endothelium by in vivo observation of the rat mesentery. The use of an in vitro assay for intracellular Ca2+ mobilization and the degranulation of mast cells demonstrated the significant upper shift of concentration response to SP (10(-4)-10(-5) M). In vivo experiments on the mesenteric microcirculation also showed that SP induced a significant increase in the number of degranulated mast cells as well as in the number of leukocytes adherent to the venular wall. Tranilast, a mast cell stabilizer, as well as SP antagonist (CP-96,345) significantly attenuated the extent of mast cell degranulation and leukocyte adhesion elicited by SP. Although an immunoneutralization against CD18 by WT-3 significantly attenuated the leukocyte adhesion, it had no influence on the mast cell degranulation after SP superfusion. These separate in vivo observations show that SP induces leukocyte adhesion to the venular endothelium, possibly through the degranulation of mast cells.     

Transforming growth factor-beta enhances adhesion of melanoma cells to the endothelium in vitro

Melanoma invasion requires migration through the vascular barrier. An early event in this process is the adhesion of metastatic cells to the endothelium. To elucidate the role of TGF-beta in the regulation of this process, human melanoma SK-MEL24 cells were labelled with [5'-(3)H]-thymidine and co-cultured with bovine pulmonary artery endothelial-cell monolayers. Radioactivity was assumed to be proportional to the number of SK-MEL24 cells bound to the endothelium. A low number of melanoma cells adhered to endothelial cells in a time-related manner. Pretreatment for 24 hr with 0.001 to 10 ng/ml TGF-beta1 or TGF-beta2 of both cell types enhanced melanoma-endothelium adhesion in a dose-dependent manner. Both melanoma and endothelial cells expressed RI- and RII-type TGF-beta receptors. The effect of TGF-beta was abolished by co-incubation with the proteoglycan decorin. Conditioned media from melanoma-endothelium co-cultures contained latent TGF-beta and failed to affect cell-cell adhesion. However, activation of TGF-beta by heating the medium or reducing the pH, increased melanoma-endothelium adhesion to an extent similar to that of the TGF-beta administered to the cultures. Zimography demonstrated that both cell types expressed urokinase-type plasminogen activator (uPA). Addition of plasminogen to the co-cultures, which was likely to be activated to plasmin by uPA, resulted in activation of TGF-beta and parallel stimulation of melanoma-endothelium adhesion. In conclusion, TGF-beta may enhance adhesion of melanoma cells to the endothelium, playing a relevant autocrine/paracrine role in the progression of invasive melanoma.     

Influence of adhesion molecule expression by human brain microvessel endothelium on cancer cell adhesion

Cultures of endothelial (En) cells derived from human brain microvessels were established in order to characterize adhesion molecule expression and to assay the adhesion properties of neoplastic cell lines to monolayers of En cells. Low constitutive expression of beta1 integrin (CD29), and ICAM-2 (CD102) was detected on human brain microvessel En cells. The beta1 chain of the VLA integrin family, ICAM-1, E-selectin (CD62E) and VCAM-1 (CD106) but not ICAM-2 and PECAM-1 (CD31) expression was upregulated by IL1-alpha, and TNF-alpha proinflammatory cytokines. High expression of PECAM-1 was found on non-activated human brain EN cells. In order to study the potential role of adhesion molecules in neoplastic cell adhesion two tumor cell lines were chosen. Adhesion of a cell line (DU145) derived from a cerebral metastasis of prostate carcinoma to human brain microvessel En cell monolayers was less pronounced compared to adhesion of a primary prostate carcinoma cell line (ND1). Adhesion of cerebral metastatic neoplastic cell line (DU145) was not significantly influenced by incubation of endothelial cells with different proinflammatory cytokines. The adhesion capability of primary prostate carcinoma line (NDI) was significantly upregulated by TNF-alpha proinflammatory cytokine. Furthermore, the adhesion of ND1 was partly inhibited using anti-E-selectin and VCAM-1 monoclonal antibodies. There was no significant effect of anti-adhesion antibodies on the adhesion characteristics of the cerebral metastatic (DU145) cell line. Our data demonstrate that different mechanisms are involved in the adhesion of neoplastic cells to cerebral En cells and turn our attention to the importance of adhesion molecule expression in the formation of metastases.     

Novel vascular molecule involved in monocyte adhesion to aortic endothelium in models of atherogenesis

Adhesion of monocytes to the endothelium in lesion-prone areas is one of the earliest events in fatty streak formation leading to atherogenesis. The molecular basis of increased monocyte adhesion is not fully characterized. We have identified a novel vascular monocyte adhesion-associated protein, VMAP-1, that plays a role in adhesion of monocytes to activated endothelium. Originally selected for its ability to block binding of a mouse monocyte-like cell line (WEHI78/24) to cytokine- or LPS-stimulated cultured mouse endothelial cells in vitro, antiVMAP-1 mAb LM151 cross-reacts with rabbit endothelium and blocks binding of human monocytes to cultured rabbit aortic endothelial cells stimulated with minimally modified low density lipoprotein, thought to be a physiologically relevant atherogenic stimulus. Most importantly, LM151 prevents adhesion of normal monocytes and monocytoid cells to intact aortic endothelium from cholesterol-fed rabbits in an ex vivo assay. VMAP-1 is a 50-kD protein. Immunohistology of vessels reveals focal constitutive expression in aorta and other large vessels. VMAP-1 is thus a novel vascular adhesion-associated protein that appears to play a critical role in monocyte adhesion to aortic endothelial cells in atherogenesis in vivo.     

Vascular endothelium in the regulation of immune response

Endothelial cells cover the entire system of arteries, veins, and lymphatics of the human body. Once viewed as a passive coat, we now know that they represent by themselves a large immunologically active system. In this work, we briefly review endothelial cell models and heterogenicity before interrelating endothelial cells with the immune system. In the latter context, we focus on the role of cytokines and that of the five families of cell adhesions molecules.     

Molecular organization and functional regulation of cell to cell junctions in the endothelium

Intercellular junctions are important structural determinants of endothelial permeability. These organelles are formed by a complex network of transmembrane proteins linked to a well developed plasmalemmal undercoat. One of the typical characteristics of endothelial junctions is their dynamic organization. Endothelial cells are able to rapidly change the architecture of the junctions to allow the passage of plasma constituents and circulating cells. This effect, in most of the cases, is quickly reversible and the endothelium is able to disorganize/reorganize the intercellular junctions within minutes. The mechanisms that regulate the opening and the closure of endothelial junctions are still obscure. It is conceivable that inflammatory agents increase permeability by binding to specific receptors generating intracellular signals which in turn cause cytoskeletal reorganization and opening of interendothelial gaps. Endothelial junctions also control leukocyte extravasation. Once leukocytes have adhered to the endothelium, a coordinated opening of interendothelial clefts occurs. The mechanism by which this takes place is unknown, but it might present characteristics similar to that triggered by soluble mediators.     

Role of tissue factor in adhesion of mononuclear phagocytes to and trafficking through endothelium in vitro

An in vitro model consisting of endothelium grown on collagen was used to investigate how mononuclear phagocytes traverse endothelium in the basal-to-apical direction (reverse transmigration), a process that mimics their migration across vascular and/or lymphatic endothelium during atherosclerosis and resolution of inflammation, respectively. Monoclonal antibody (MoAb) VIC7 against tissue factor (TF) inhibited reverse transmigration by 77%. Recombinant tissue factor fragments containing at least six amino acids C-terminal to residue 202 also strongly inhibited reverse transmigration. TF was absent on resting monocytes but was induced on these cells after initial apical-to-basal transendothelial migration. Two additional observations suggest that TF is involved in adhesion between mononuclear phagocytes and endothelium: (1) when monocytes were incubated with lipopolysaccharide (LPS) to stimulate expression of TF before they were added to endothelium, VIC7 or soluble TF modestly inhibited their adhesion to the apical endothelial surface, each by about 35%; and (2) endothelial cells specifically bound to surfaces coated with TF fragments containing amino acids 202-219. This binding was blocked by anti-TF MoAb, suggesting that endothelial cells bear a receptor for TF. These data suggest that mononuclear phagocytes use TF, perhaps as an adhesive protein, to exit sites of inflammation.     

Natural human interferon-alpha inhibits the adhesion of a human carcinoma cell line to human vascular endothelium

The adhesion of cells to the microvascular endothelium is an essential step in the inflammatory response and metastasis. We have found that pretreatment of a human epidermoid carcinoma cell line, KB, with natural human interferon-alpha (IFN-alpha) inhibited the binding of the malignant cells to human umbilical vein endothelial cells (HUVEC) in a dose- and time-dependent manner. As one of several possible mechanisms for this inhibition, the expression of some revelant adhesion molecules on KB cell surfaces was examined after IFN-alpha treatment. Apart from a slight increase in the expression of integrin alpha 4 beta 1 (very late activation antigen 4, VLA-4), no changes in the expression of other adhesion molecules, such as sialyl Lewis X, CD44, and leukocyte function-associated antigen 1 (LFA-1), which is known to be a heterodimer of CD11a and CD18, were observed after treatment with IFN-alpha. In addition, the cell viability of KB was not affected by treatment of the cells with IFN-alpha, although the cell proliferation was markedly inhibited, indicating that the inhibitory effect of IFN-alpha on KB cell binding to vascular endothelium is not a result of a cytotoxic effect of IFN-alpha. Because the metastatic process requires not only the adhesion of tumor cells to vascular endothelium during their extravasation but also proliferation at distant sites, our findings from this in vitro experimental model suggest that IFN-alpha may have a potential inhibitory effect on tumor cell metastasis.     

Glycemia and regulation of endothelial adhesion molecules

Hyperglycemia is one of the most prominent factors associated with diabetic vascular disease, the development of which is initated by functional and morphological alterations of the endothelium. The review summarizes several issues of glycemia and advanced glycation end products induced changes concerning soluble as well as cell bound endothelial adhesion molecules, and further addresses the aspect of apoptosis in the light of diabetic vascular complications.     

The role of adhesion molecules in human leukocyte attachment to porcine vascular endothelium: implications for xenotransplantation

Many obstacles still prevent successful xenotransplantation of porcine donor organs. When hyperacute rejection is averted, transplanted pig organs are subject to acute vascular and cellular rejection. In autologous systems, leukocyte recruitment into inflamed tissues involves selectins, integrins, and Ig family members. To determine whether these mechanisms allow human leukocytes to effectively enter porcine grafts, the pathways by which human leukocytes adhere to TNF-alpha-stimulated porcine aortic endothelium were examined under static and physiologic flow conditions. L-selectin and E-selectin had overlapping functions in neutrophil capture and rolling, whereas Ab blockade of E-selectin and the beta2 integrins inhibited firm arrest of rolling neutrophils. Combined blockade of selectins and beta2 integrins resulted in negligible human neutrophil attachment to pig endothelium. Lymphocyte attachment to porcine endothelium was primarily L-selectin mediated, whereas beta2 integrin and VCAM-1/very late Ag-4 (VLA-4) interactions promoted static adhesion. Concurrent beta2 integrin, VLA-4, VCAM-1, and L-selectin blockade completely inhibited lymphocyte attachment. Thus, interactions between leukocyte-endothelial cell adhesion receptor pairs remained remarkably intact across the human-porcine species barrier. Moreover, disrupting the adhesion cascade may impair the ability of human leukocytes to infiltrate a transplanted porcine organ during rejection.     

Reduced intracellular oxidative metabolism promotes firm adhesion of human polymorphonuclear leukocytes to vascular endothelium under flow conditions

The interaction of polymorphonuclear leukocytes (PMN) with the vascular endothelium and their subsequent extravasation to the tissues is a key step during different physiological and pathological processes. In certain of these pathologies the oxygen tension becomes very low, leading to reduced cellular oxidative status. To evaluate the effect of lowering the intracellular redox status in the interaction of PMN with the endothelium, exposure to hypoxic conditions as well as treatment with different antioxidant agents was carried out. PMN exposure to hypoxia enhanced beta2 integrin-dependent adhesion to intercellular adhesion molecule-1-coated surfaces, concomitant with a decrease in the intracellular redox status of the cell. As occurs with hypoxia, treatment with antioxidants produced a decrease in the oxidation state of PMN. These agents enhanced adhesion of PMN to human umbilical vein endothelial cells stimulated with tumor necrosis factor-alpha (TNF-alpha), and this effect was also mediated by beta2 integrins LFA-1 and Mac-1. Adhesion studies under defined laminar flow conditions showed that the antioxidant treatment induced an enhanced adhesion mediated by beta2 integrins with a decrease in the fraction of PMN rolling on TNF-alpha-activated endothelial cells. The up-regulated PMN adhesion was correlated to an increase in the expression and activation of integrin Mac-1, without loss of L-selectin surface expression. Altogether, these results demonstrate that a reduction in the intracellular oxidative state produces an enhanced beta2 integrin-dependent adhesion of PMN to stimulated endothelial cells under conditions of flow.     

Anionic polysaccharides inhibit adhesion of sickle erythrocytes to the vascular endothelium and result in improved hemodynamic behavior

The abnormal adherence of sickle red blood cells (SS RBC) to vascular endothelium may play an important role in vasoocclusion in sickle cell anemia. Thrombospondin (TSP), unusually large molecular weight forms of von Willebrand factor, and laminin are known to enhance adhesion of SS RBC. Also, these endothelial proteins bind to sulfated glycolipids and this binding is inhibited by anionic polysaccharides. Reversible sickling may expose normally cryptic membrane sulfatides that could mediate this adhesive interaction. In this study, we have investigated the effect of anionic polysaccharides, in the presence or absence of TSP, on SS RBC adhesion to the endothelium, using cultured human umbilical vein endothelial cells (HUVEC) (for the adhesion assay) and the ex vivo mesocecum of the rat (for hemodynamic evaluation). The baseline adhesion (ie, without added TSP) of SS RBC to HUVEC was most effectively inhibited by high molecular weight dextran sulfate (HDS), whereas low molecular weight dextran sulfate (LDS) and the glycosaminoglycan chondroitin sulfate A (CSA) also had significant inhibitory effects. Heparin was mildly effective whereas other glycosaminoglycans (chondroitin sulfates B and C, heparan sulfate, and fucoidan) were ineffective. Similarly, HDS and CSA resulted in an improved hemodynamic behavior of SS RBC. Soluble TSP caused significant increases in SS RBC adhesion and in the peripheral resistance. Both HDS and CSA prevented TSP-enhanced adhesion and hemodynamic abnormalities. Thus, anionic polysaccharides can inhibit SS RBC-endothelium interaction in the presence or absence of soluble TSP. These agents may interact with RBC membrane component(s) and prevent TSP-mediated adhesion of SS RBC to the endothelium.     

Role of adhesion molecules in interactions of endothelium with leukocytes in inflammatory processes

This paper reviews recent work regarding the molecules that mediate leukocyte-endothelial cell adhesion, describes the underlying principles of leukocyte migration, and discusses a model of the sequence of events that allows a leukocyte to attach to endothelium and migrate into tissue. Pathophysiological importance of the adhesion molecules in the development of different states of inflammation has been also presented.