Cytokine-mediated induction of cyclo-oxygenase-2 by activation of tyrosine kinase in bovine endothelial cells stimulated by bacterial lipopolysaccharide.

1. The induction of cyclo-oxygenase-2 (COX-2) afforded by bacterial lipopolysaccharide (LPS, endotoxin) in bovine aortic endothelial cells (BAEC) is mediated by tyrosine kinase. LPS also causes the generation of several cytokines including interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). This study investigates whether endogenous IL-1 beta, TNF-alpha, EGF or PDGF contribute to the induction of COX-2 elicited by LPS in BAEC and if their action is due to activation of tyrosine kinase. Furthermore, we have studied the induction of COX-2 by exogenous cytokines. 2. Accumulation of 6-oxo-prostaglandin (PG) F1 alpha in cultures of BAEC was measured by radioimmunoassay at 24 h after addition of either LPS (1 microgram ml-1) alone or LPS together with a polyclonal antibody to one of the various cytokines. In experiments designed to measure 'COX activity', 6-oxo-PGF1 alpha generated by BAEC activated with recombinant human IL-1 beta, TNF-alpha, EGF or PDGF for 12 h was measured after incubation of washed cells with exogenous arachidonic acid (30 microM for 15 min). Western blot analysis determined the expression of COX-2 protein in BAEC. 3. The accumulation of 6-oxo-PGF1 alpha caused by LPS in BAEC was attenuated by co-incubation with one of the polyclonal antibodies, anti-IL-1 beta, anti-TNF-alpha, anti-EGF, anti-PDGF or with the IL-1 receptor antagonist, in a dose-dependent manner. Exogenous IL-1 beta, TNF-alpha or EGF also caused an increase in COX activity, while PDGF was ineffective. The increase in COX activity elicited by IL-1,beta(10 ng ml-1), TNF-alpha (100 ng ml-1) or EGF (1000 ng ml-1) in BAEC was attenuated by erbstatin (0.005 to 5 microg ml-1), as was the expression of COX-2 protein measured by Western blot analysis.4. PDGF (10 ng ml-1) significantly augmented the rise in COX activity and COX-2 protein caused by shorter incubation of BAEC with LPS (1 microg ml-1 for 3 h). Combination of PDGF (10 ng ml-1) with a low concentration of IL-l beta (1 ng ml-1) for 12 h, also increased 'COX activity', but combination of PDGF and TNF-alpha (10 ng ml-1) did not show any increased activity.5. These results suggest that (i) the induction of COX activity and COX-2 protein elicited by LPS in BAEC is mediated by TNF-alpha with lesser contributions from PDGF, EGF or IL-1 beta; (ii) exogenous IL-1 beta,TNF-alpha or EGF alone induce COX-2 activity and protein in BAEC; (iii) PDGF synergizes with IL-1 beta,but not TNF-alpha, to cause expression of COX-2; and (iv) the induction of COX-2 protein and activity caused by these cytokines involves the activation of tyrosine kinase.