No abstract

This study describes the expression characteristics of a cell membrane protein recognized by a monoclonal antibody ENA1, which was obtained by immunizing mice with human umbilical vein endothelial (HUVE) cells cultured with a mixture of interleukin 1 and tumor necrosis factor-alpha. The expression of this ENA1 antigen could also be induced by lipopolysaccharide and phorbol esters. Expression was only demonstrated on HUVE cells and human umbilical arterial endothelial cells, pretreated with one or with a mixture of these reagents. No expression was detected on human fibroblasts, renal epithelial cells or on mesothelial cells derived from omental tissue, either pretreated or not with the aforementioned inducers of the antigen. Furthermore, no reactivity was observed with either polymorphonuclear cells, peripheral blood lymphocytes or the monocytic cell line U937. Time course experiments revealed that the expression of the ENA1 antigen was time dependent. Maximal expression on HUVE cells was observed after 5 h of incubation with activator, after which a decline in expression occurred. Induction of expression could be completely blocked by the mRNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide, indicating that de novo synthesis occurs. Other pharmacological reagents tested had no effect on the induction of ENA1 expression. The putative role of the newly described antigen is discussed in relation to the current knowledge of molecules involved in adhesion of immune cells in inflammatory processes.

The role of ELAM-1 in the adhesion of monocytes to HUVEC, activated for 4h with TNF, was studied using MoAb ENA2 directed against ELAM-1. In a standard adhesion assay at 37 degrees C, F(ab')2 fragments of ENA2 did not, or weakly inhibited adhesion. When metabolic activity of the monocytes was reduced by (i) fixing the monocytes, (ii) performing the adhesion assay at 4 degrees C, and (iii) combining the forementioned conditions, the adhesion of the monocytes was strongly blocked by ENA2 and less effective or not by MoAb IB4 anti-CD18. The pattern of adhesion of monocytes to HUVEC, activated with TNF assessed at 4 degrees C, paralleled ELAM-1 expression on the endothelial cells. Maximal inhibitory effect of ENA2 on adhesion was shown 5 h after activation of HUVEC, at which ELAM-1 expression was also maximal. Adhesion assessed at 37 degrees C remained enhanced for at least 24 h, whereas the inhibitory effect of ENA2 followed ELAM-1 expression. Specific involvement of ELAM-1 was also confirmed using ELAM-1 transfected COS cells. These results indicated that monocytes express a counter structure for ELAM-1 and that this counter structure is involved in adhesion.

In this study two new in vitro effects of IFN-gamma on human umbilical vein endothelial (HUVE) cells were described. First, it was shown that the expression of the adhesion molecule ELAM-1 on activated HUVE cells can be modulated by IFN-gamma. ELAM-1 is normally not expressed by HUVE cells, but its expression can rapidly be induced by TNF, IL-1, or LPS. Maximal expression is reached after 4 to 6 h of activation, and after 24 h the expression disappeared. Whereas IFN-gamma per se did not induce expression of ELAM-1, it enhanced and prolonged the expression of ELAM-1. This enhancement occurred when IFN-gamma was added before activation as well as when added simultaneously with activation. When IFN-gamma was added 6 or 9 h after the activation, the normally ongoing reduction of expression was not only retarded, but the expression increased for at least 3 h. Moreover, IFN-gamma abrogated the refractory period for restimulation. Neither IFN-beta nor IL-6 had any effect on the expression of ELAM-1. The second effect of IFN-gamma on HUVE cells is the capacity to enhance the IL-6 production by these cells. Prestimulation as well as coincubation of IFN-gamma with TNF, IL-1, or LPS resulted in a strongly augmented production of IL-6. The effects of IFN-gamma may in vivo play a role in the regulation of an inflammatory reaction, because ELAM-1 is an adhesion molecule for neutrophils, and IL-6 has an enhancing effect on the cytotoxicity of neutrophils.

Proinflammatory effects induced by the serine protease factor Xa were investigated in HUVEC. Exposure of cells to factor Xa (5–80 nM) concentration dependently stimulated the production of IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) and the expression of E-selectin, ICAM-1, and VCAM-1, which was accompanied by polymorphonuclear leukocyte adhesion. The effects of factor Xa were blocked by antithrombin III, but not by the thrombin-specific inhibitor hirudin, suggesting that factor Xa elicits these responses directly and not via thrombin. IL-1α and TNF-α were not implicated, since neither the IL-1 receptor antagonist nor a TNF-neutralizing Ab could suppress the factor Xa responses. Active site-inhibited factor Xa and factor Xa depleted from γ-carboxyglutamic acid residues were completely inactive. The effector cell protease receptor-1 (EPR-1) seems not to be involved since anti-EPR-1 Abs failed to inhibit cytokine production. Moreover, neither the factor X peptide Leu83-Leu88, representing the inter-epidermal growth factor sequence in factor Xa that mediates ligand binding to EPR-1, nor the peptide AG1, corresponding to the EPR-1 sequence Ser123-Pro137 implicated in factor Xa binding, inhibited the factor Xa-induced cytokine production. In conclusion, these findings indicate that factor Xa evokes a proinflammatory response in endothelial cells, which requires both its catalytic and γ-carboxyglutamic acid-containing domain. The receptor system involved in these responses induced by factor Xa remains to be established.