Restraint of spreading-dependent activation of polymorphonuclear leukocyte NADPH oxidase in an acidified environment

Elucidation of the mechanisms by which environmental pH affects or regulates the functions of polymorphonuclear leukocytes (PMNs) is important because severe acidification of the microenvironment often prevails at sites of inflammation where they act in host defense. In the present study, we investigated the effect of an acidic environment on spreading-dependent activation of O2- -producing NADPH oxidase in PMNs. We found that PMNs underwent spreading spontaneously over type I collagen and plastic surfaces at both neutral and acidic pH, although spreading over fibrinogen surfaces, for which cellular stimulation with H2O2 is required, was inhibited by acidic pH. At acidic pH, however, PMNs were unable to undergo spreading-dependent production of O2-. Pharmacological experiments showed that p38 mitogen-activated protein kinase (MAPK) was involved in the signaling pathways mediating the spreading-dependent activation of NADPH oxidase, and that its spreading-dependent phosphorylation of Thr-180 and Tyr-182, a hallmark of activation, was impaired at acidic pH. Furthermore, the inhibition by acidic pH of O2- production as well as p38 MAPK phosphorylation subsequent to spreading induction was reversible; environmental neutralization and acidification after induction of spreading at acidic and neutral pH, respectively, up- and down-regulated the two phenomena. Acidic pH did not affect the O2- production activity of NADPH oxidase pre-activated by phorbol 12-myristate 13-acetate (PMA). These results suggest that, in PMNs, the p38 MAPK-mediated signaling pathway functions as a pH-sensing regulator of spreading-dependent NADPH oxidase activation.