E-selectin is an inducible adhesion molecule on endothelial cells. The internalization of this glycoprotein was investigated on tumor necrosis factor (TNF)-activated cultured human umbilical vein endothelial cells (HUVEC). Kinetics of intercellular adhesion molecule-1 (ICAM-1) were studied in parallel experiments. Internalization studies were performed with radioiodinated antibodies in an acid elution endocytosis assay, and by immunohistology; both approaches gave equivalent results. [125I]ENA1, a monoclonal antibody (mAb) specific for E-selectin, was internalized at a rate of approximately 1.7% of the membrane-bound [125I]mAb per minute. In contrast, less than 0.1% of membrane-bound [125I]RR1/1, an mAb specific for ICAM-1, was internalized per minute. TNF-activated HUVEC were immunostained and examined by light microscopy (LM) and electron microscopy (EM). LM revealed the presence of ENA1, but not RR1/1, after 30 minutes of incubation with these mAb in cytoplasmic vesicles, which were characterized as multivesicular bodies by EM. Without previous mAb exposure of the endothelial cells, both high amounts of E-selectin and bovine serum albumin complexed to colloidal gold, used as a marker for fluid-phase internalization, were detected in the same organelles, thus arguing against mAb interaction-induced E-selectin internalization. Furthermore, the amount of E-selectin surface expression was not influenced by ongoing mAb presence, also arguing against mAb interference with normal E-selectin kinetics. Taken together, these results indicate that TNF-activated HUVEC constitutively internalize E-selectin. Physiological significance of E-selectin internalization in the regulation of E-selectin membrane expression, and in clearing E-selectin ligands from the circulation, needs further investigation.
TNF plays a central role in septic shock induced by endotoxin or Gram-negative bacteria. Zymosan can elicit a septic shock-like syndrome in rodents in the absence of endotoxin. TNF and IL-6 release in mice treated with zymosan was investigated. One hour after intraperitoneal zymosan injection, maximal TNF levels were measured in serum, followed by IL-6 peak levels 1 h later. Treatment with a monoclonal antibody against TNF lowered zymosan-induced mortality from 63 to 11.6%, while maximal IL-6 levels were lowered by about 40%. Mechanisms triggering zymosan-induced cytokine release in murine macrophages were analysed in vitro. Cytokine release was only slightly triggered by uncoated zymosan particles. Thirty-nine per cent of TNF release by macrophages appeared to be triggered by zymosan-bound activated complement. Maximal TNF release also required the presence of natural antibodies against zymosan and zymosan-activated serum. In contrast, maximal IL-6 release was reached upon stimulation with zymosan-activated serum only, while the presence of zymosan particles lowered this response. We conclude that TNF is a crucial mediator in zymosan-induced shock. TNF release can be induced by different immunological pathways, without the need for the direct presence of endotoxins. Although IL-6 release during septic shock is partly dependent on TNF, in vitro trigger mechanisms for IL-6 and TNF differ remarkably.
We have studied culture conditions which facilitate the growth of stable, non-proliferating, human umbilical vein endothelial cell (HUVEC) monolayers. Gelatin and fibronectin coatings, with or without glutaraldehyde cross-linking, on both plastic and glass were investigated for initial attachment of HUVEC and growth characteristics. The presence during culture of intercellular (IC) junctions demonstrated by silver staining, expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and maintenance of a cobblestone appearance of HUVEC monolayers were assessed over time. Glutaraldehyde cross-linked fibronectin and gelatin coatings on glass and glutaraldehyde cross-linked gelatin or untreated fibronectin coatings on plastic served as good substrates for short term culture. Long term (20 days) cultures of HUVEC which maintained silver and PECAM-1 staining of IC junctions and a cobblestone appearance could be achieved if glutaraldehyde cross-linked gelatin coatings on glass were used as substrates.
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.
We investigated the ability of TNF-alpha to mediate damage of endothelial cells in the presence of neutrophils, by measuring detachment of cultured human umbilical vein endothelial cells (HUVEC). Endothelial cell detachment was increased from 5% to about 75% by the presence of 1-10 ng/ml TNF-alpha during incubation with neutrophils, whereas negligible endothelial cell lysis was observed as measured by 51Cr release. TNF-alpha was compared with the cytokines IL-1 alpha and IFN-gamma and with PMA and LPS. Both TNF-alpha and PMA appeared to be strong triggers for neutrophil-induced endothelial cell detachment, whilst reduced injury was seen after addition of IL-1 alpha and LPS. IFN-gamma did not induce endothelial cell detachment, but potentiated the effect of both TNF-alpha and IL-1 alpha. TNF-alpha-induced endothelial cell detachment was neutrophil dependent, since pre-incubation of neutrophils, but not pre-incubation of endothelial cells with TNF-alpha, caused endothelial cell detachment. Thus, TNF-alpha-induced increase in neutrophil-adhesiveness of HUVEC was found not to be essential for endothelial damage. Pre-incubation of neutrophils in suspension with TNF-alpha induced rapid activation, followed by nearly complete deactivation of neutrophils, as measured by their capacity to induce detachment of endothelial cells after removal of TNF-alpha. These results indicate that local presence of TNF-alpha might be critical in tissue or organ damage during early, neutrophil-mediated inflammatory processes, independent of enhanced adhesiveness of endothelium for neutrophils.
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