Celecoxib simulates respiratory burst through pertussis toxin-sensitive G-protein, a possible signal for β₂-integrin expression on human neutrophils

The superoxide anion-generating effect of celecoxib (4-[5-(4-methylpheny)- 3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide); SC58633), a selective cyclooxygenase-2 inhibitor, on human neutrophils was evaluated in this study. Celecoxib induced superoxide anion generation in a concentration-dependent manner in human neutrophils. The EC₅₀ value of celecoxib on superoxide anion generation was 15.5±2.5 μM. A NADPH oxidase inhibitor, diphenyliodonium (20 μM), and superoxide dismutase (150 U/ml) completely inhibited the free radical generation caused by celecoxib, indicating that the respiratory burst was activated by celecoxib. 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM;10 μM) and staurosporine (200 nM) completely inhibited the superoxide anion release caused by celecoxib, respectively. These data indicated that celecoxib increased superoxide anion release by increasing intracellular calcium and protein kinase C activation. Moreover, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3- a)pyrrolo(3,4-C)-carbazole (Go-6976; 1 μM) and 3-[1-[3-(amidinothio)propyl- 1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl)maleimide, methane sulfate (Ro-31-8220; 0.5 μM), specific inhibitors of conventional protein kinase C isotypes (α, β_I and β_II), significantly inhibited superoxide anion release caused by celecoxib. Rottlerin (5 μM), a protein kinase C δ inhibitor, did not affect the free radical generation caused by celecoxib. Celecoxib caused translocation of protein kinase C α, β_I and β_II from the cytosol to the cellular membrane. 2-[2-amino-3-methoxyphenyl]-4H-1-benzopyran-4-one (PD98059; 20 μM) and wortmannin (100 nM) did not decrease the superoxide anion generation caused by celecoxib, indicating that Mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3 kinase) were not involved in the respiratory burst induced by celecoxib. Pertussis toxin (2 μg/ml), a Gi-protein sensitive inhibitor, significantly inhibited superoxide anion release. Moreover, pertussis toxin significantly inhibited intracellular calcium mobilization and protein kinase C α, β_I and β_II translocation from the cytosol to the membrane. Celecoxib increased β ₂-integrin expression on human neutrophils and this effect was inhibited by BAPTA/AM (10 μM), superoxide dismutase (150 U/ml), genistein (25 μM) and PD98059 (20 μM). This information indicated that intracellular calcium, superoxide anion, tyrosine kinase and MAP kinase are involved in β₂-integrin expression. Furthermore, BAPTA/AM, superoxide dismutase and genistein inhibited celecoxib-increased MAP kinase activity, indicating that MAP kinase is a downstream signal for β ₂-integrin expression. In conclusion, celecoxib stimulates superoxide anion release from human neutrophils by activating pertussis toxin sensitive G-protein. An increase in intracellular calcium and protein kinase C α, β_I and β_II is involved in this process. Celecoxib also regulates β₂-integrin expression through superoxide anion release, tyrosine kinase and p42/p44 MAP kinase on human neutrophils.