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) F-1 alpha in cultures of BAEC was measured by radioimmunoassay at 24 h after addition of either LPS (1 mu g 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-PGF(1 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 mu M for 15 min). Western blot analysis determined the expression of COX-2 protein in BAEC. 3 The accumulation of 6-oxo-PGF(1 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-PDGFF 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 mu g m 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 mu g ml(-1) for 3 h). Combination of PDGF (10 ng ml(-1)) with a low concentration of IL-1 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.