‘Outside‐in’ signalling mechanisms underlying CD11b/CD18‐mediated NADPH oxidase activation in human adherent blood eosinophils

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Abstract
Incubation of human eosinophils in BSA‐coated tissue culture plates resulted in time‐dependent adhesion and attendant activation of the NADPH oxidase that were both inhibited (by >85%) by blocking antibodies raised against CD11b and CD18. SB 203580, an inhibitor of p38 mitogen‐activated protein (MAP) kinase, did not influence adhesion but inhibited superoxide anion generation (pIC50=−6.57). PP1, an inhibitor of the src‐family of protein tyrosine kinases, inhibited adhesion and CD11b/CD18‐mediated superoxide anion generation with similar potencies (pEC50s=−5.53 and −5.99 respectively) suggesting that inhibition of the NADPH oxidase was a direct consequence of blocking adhesion. The protein kinase C (PKC) inhibitors Ro‐31 8220 (broad spectrum inhibitor), GF 109203X (inhibitor of conventional and novel isoforms) and Gö 6976 (inhibitor of conventional isoforms) suppressed adhesion‐dependent NADPH oxidase activation (pIC50s=−6.61, −6.05 and −4.89 respectively) without affecting adhesion. Based upon the selectivity of these drugs PKCδ and PKCε are implicated in the suppression of oxidant production. Wortmannin, an inhibitor of phosphatidylinositol 3‐kinase (PtdIns 3‐kinase), abolished superoxide anion production in adherent eosinophils (pEC50=−9.06). Similarly, CD11b/CD18‐dependent adhesion was suppressed with the same potency (pEC50=−9.29) although the maximum effect did not exceed 50% implying that wortmannin also had an affect on those processes that govern adhesion‐driven oxidase activation. PD 098059 and piceatannol, inhibitors of MAP kinase kinase‐1 and the syk tyrosine kinase respectively, had no effect on CD11b/CD18‐mediated adhesion or NADPH oxidase activation. The results of this study demonstrate that human eosinophils adhere to BSA‐coated plastic by a CD11b/CD18‐dependent mechanism, which is responsible for activation of the NADPH oxidase. Although the signalling pathway(s) utilized by CD11b/CD18 is still to be elucidated, the data presented herein implicate p38 MAP kinase, novel PKCs and PtdIns 3‐kinase. British Journal of Pharmacology (1999) 128, 1149–1158; doi:10.1038/sj.bjp.0702892

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