Interleukin-6 production by endothelial cells via stimulation of protease-activated receptors is amplified by endotoxin and tumor necrosis factor-alpha

Human endothelial cells respond to extracellular proteases, endotoxin (lipo polysaccharide, LPS), and inflammatory cytokines, Endothelial cells express several protease-activated receptors (PAR), including the thrombin-activat ed receptors PAR-1 and PAR-3 and a thrombin-independent, protease-activated receptor, PAR-2, To examine the potential cooperation between PAR and infl ammatory stimuli, we investigated the effects of the PAR-1 agonist peptide Ser-Phe-Leu-Leu-Arg-Asn (SFLLRN) and PAR-2 agonist peptide Ser-Leu-Ile-Gly- Lys-Val (SLIGKV) on endothelial cells. Human umbilical vein endothelial cel ls (HUVEC) were cultured in vitro with SFLLRN or SLIGKV in the presence and absence of LPS or tumor necrosis factor-alpha (TNF-alpha), and interleukin -6 (IL-6) levels in the culture supernatants were assayed. Both SFLLRN and SLIGKV induced detectable levels of IL-6 production in a dose-dependent fas hion, with the PAR-1 receptor agonist being more potent. In the presence of all stimulatory concentrations of LPS or TNF-alpha tested, both peptides w ere found to further enhance IL-6 production. The effects of SFLLRN and SLI GKV were specific, as related peptides with identical amino acid compositio ns, but lacking in consensus sequences, were biologically inactive either a lone or in the presence of LPS. Both the direct and the amplifying effects of PAR agonist peptides on IL-6 production were pertussis toxin sensitive a nd caused an increase in the intracellular levels of calcium, implicating G -proteins and calcium mobilization in these pathways. Furthermore, the ampl ifying effect of LPS or TNF-alpha on PAR-mediated cytokine production was a ssociated with corresponding increases in nuclear NF-kappaB proteins. The r esults demonstrate significant potentiation of PAR-induced signaling by LPS and TNF-alpha and indicate the potential cooperation of proteases and infl ammatory stimuli in amplifying vascular inflammation.