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Cultured human endothelial cells (EC) resist tumor necrosis factor (TNF)-mediated apoptosis. However, the combination of TNF and the protein synthesis inhibitor cycloheximide (CHX) induces apoptosis in up to 50% of EC within 24 hours. TNF + CHX killing is effectively blocked by transfected CrmA protein or treatment with Z-VAD.fmk peptide-both inhibitors of interleukin-1-converting enzyme-like proteases-but not by transfected antiapoptotic proteins Bcl-2, Bcl-XL, or A1. C6-ceramide (cer) can also sensitize EC to TNF-induced apoptosis. TNF + cer killing, which can affect more than 50% of EC, is not effectively inhibited by CrmA or Z-VAD frank, but can be readily blocked by Bcl-2, Bcl-XL, or A1. Both TNF + CHX and TNF+ cer killing are induced by a TNF mutein that only interacts with the type 1 TNF receptor, and both responses can be inhibited by the antiapoptotic protein A20. These data suggest that TNF activates two biochemically separable pathways of EC injury.

Tumor necrosis factor (TNF) activates transcription of endothelial leukocyte adhesion molecule-1 (CD62E) in endothelial cells (ECs) through the binding to the gene promoter of the p50/p65 heterodimeric form of nuclear factor-kappa B (NF-kappa B) and of the N-terminal phosphorylated form of the ATF2/c-Jun transcription factor, which is phosphorylated by Jun N-terminal kinase (JNK). However, the intracellular signaling pathways that activate endothelial NF-kappa B and JNK in TNF-induced responses are unknown. In this study we have examined the role of a recently described TNF signaling pathway involving sphingomyelin activation to generate ceramide, a potential intracellular mediator. We find that concentrations of TNF that strongly activate NF-kappa B and JNK within 15 minutes do not produce either a measurable decline in sphingomyelin or a measurable generation of ceramide in cultured human umbilical vein ECs at any time examined. Stimulation of ECs with purified sphingomyelinase (SMase) enzyme causes a rapid 60% to 80% decrease in cellular sphingomyelin content and a large increase in ceramide. However, SMase treatment only minimally activates NF-kappa B, achieving levels that are insufficient to initiate gene transcription. Extracellular SMase does not have access to intracellular sphingomyelin, but treatment of ECs with membrane-permeant ceramide analogues still completely fails to activate NF-kappa B and only activates JNK at late times. Neither SMase nor ceramide analogues induce gene transcription or surface expression of endothelial leukocyte adhesion molecules that are readily induced by TNF. Strikingly, low concentrations of membrane-permeant ceramide cause programmed cell death in ECs, a finding not observed at any concentrations of TNF tested. We conclude that ceramide is not an important second messenger for TNF signaling of gene transcription in ECs but may be a second messenger for cell death in response to as-yet-unidentified signals.

Tumor necrosis factor-alpha (TNF-alpha) interacts with two distinct membrane receptor proteins, p55 and p75, which are variably expressed on different cell types. We have examined the function of p55 and p75 on human endothelial cells (EC). Both receptor types are detected on cultured EC by FACS analysis. A mutagenized recombinant human TNF (R32W-TNF), which binds selectively to p55, is equipotent with human recombinant wild-type TNF (wt-TNF) in upregulating several different leukocyte adhesion molecules as well as class I major histocompatibility complex molecules. R32W-TNF also fully desensitizes EC to wt-TNF, as assessed by inhibition of re-induction of endothelial leukocyte adhesion molecule-1 (ELAM-1). At low wt-TNF concentrations, induction of ELAM-1 is partly inhibited by blocking monoclonal antibodies to either p55 or p75 and to a greater extent by a combination of both monoclonal antibodies. In contrast, ELAM-1 induction by R32W-TNF is only inhibited by anti-p55. We conclude that both TNF receptors (p55 and p75) can contribute to TNF-induced activation of EC, but that signaling through p55 is sufficient.

We have investigated the role of cAMP as a signal transducer for TNF-induction of leukocyte adhesion molecule expression in cultured human umbilical vein endothelial cells (EC). Forskolin, a stimulator of adenylate cyclase, either alone or in combination with isobutyl methylxanthine (IBMX), an inhibitor of phosphodiesterase, fails to induce expression of endothelial leukocyte adhesion molecule 1 (ELAM-1 or E-selectin), of vascular cell adhesion molecule 1 (VCAM-1) or of intercellular adhesion molecule 1 (ICAM-1 or CD54). Unexpectedly, this combination of cAMP-elevating drugs inhibits TNF induction of ELAM-1 and VCAM-1 but not ICAM-1 expression. Similar results were observed with the membrane-permeant cAMP mimetics 8 bromoadenosine 3':5' cyclic monophosphate (8Br-cAMP) and N(6)2'-O-dibutyryladenosine 3':5'-cyclic monophosphate. Inhibition was greater at lower TNF concentrations (< 10 U/ml), at higher 8 Br-cAMP concentrations (> 100 microM), and at early times (2 h). Forskolin plus IBMX selectively inhibits TNF-induced increases in ELAM-1 and VCAM-1 mRNA, indicating that the action of cAMP is to block synthesis of these molecules. TNF, through stimulation of prostaglandin synthesis, produces slight elevations in the levels of endothelial cAMP. However, these increases in cAMP appear too small compared to those induced by forskolin plus IBMX to inhibit adhesion molecule expression. Indeed, complete inhibition of the TNF-mediated rise in cAMP, achieved by blocking cyclooxygenase with indomethacin, does not alter ELAM-1 expression. We conclude that cAMP is neither an intracellular mediator nor a physiological regulator of TNF-induced adhesion molecule expression in EC. However, our findings suggest that pharmacological elevations of cAMP in EC, by inhibiting TNF-induced synthesis of ELAM-1 and VCAM-1, could serve to limit inflammation.