Human endothelial cell response to lipopolysaccharide, interleukin-1, and tumor necrosis factor is regulated by protein synthesis

Abstract

In this study we assessed the viability of cultured human umbilical vein endothelial cells (HUVE) treated with bacterial lipopolysaccharide (LPS), recombinant human interleukin-1 (rhIL-1), or recombinant human tumor necrosis factor-alpha (rhTNF-alpha) during inhibition of RNA or protein synthesis. Cytotoxicity was determined by 51Cr activity retained in labeled HUVE monolayers after exposure to LPS, rhIL-1 or rhTNF-alpha, and cycloheximide (Cx) or actinomycin D (Act D). Lipopolysaccharide (150 ng/ml), rhIL-1 (100 pg/ml), or rhTNF-alpha (20 ng/ml) alone was not toxic to HUVE in an 18-hr incubation. Cycloheximide alone (1 microgram/ml for 18 hr) or Act D alone (1 microgram/ml for 6 hr) was also not toxic to HUVE. However, coincubation of HUVE with Cx and LPS (150 ng/ml), rhIL-1 (10 pg/ml), or rhTNF-alpha (20 ng/ml) produced significant cytotoxicity at 18 hr (70 +/- 4% for LPS, 75 +/- 5% for rhIL-1, and 52 +/- 5% for rhTNF-alpha; mean +/- SEM of 18, 16, and 19 separate experiments, respectively). Similarly, coincubation of HUVE with Act D and LPS, rhIL-1, or rhTNF-alpha resulted in 82 +/- 5%, 85 +/- 3%, and 67 +/- 4% cytotoxicity, respectively, at 6 hr (mean +/- SEM of 5 separate experiments for LPS, and 7 separate experiments each for rhIL-1 and rhTNF-alpha). At the highest concentrations of LPS, rhIL-1, or rhTNF-alpha, cytotoxicity during coincubation with Cx or Act D was detected as early as 2 hr and was near maximal by 6 hr. In contrast to LPS, rhIL-1, or rhTNF-alpha, recombinant human interferon-gamma (up to 100 U/ml), or human alpha-thrombin (up to 10 U/ml), produced no cytotoxicity in the presence of Cx. Recombinant human lymphotoxin (up to 50 ng/ml) had a detectable cytotoxic effect in the presence of Cx although it was significantly less than that seen with rhTNF-alpha. Furthermore, coincubation of human fibroblasts and human smooth muscle cells with Cx and LPS, rhIL-1, or rhTNF-alpha produced no cytotoxicity. We conclude that under these culture conditions, LPS, rhIL-1, or rhTNF-alpha produces a lethal injury to HUVE when de novo RNA or protein synthesis is inhibited. These results suggest that LPS, rhIL-1, and rhTNF-alpha may act via a common pathway in endothelial cells and that protein synthesis is important in regulating the response to these stimuli.