Characterization of the unique regulatory mechanisms of phorbol ester-induced polymorphonuclear leukocyte spreading in an acidified environment

Abstract

In vitro studies have shown that acidic conditions impair spreading of polymorphonuclear leukocytes, which is prerequisite for activation of microbicidal functions. However, the mechanisms by which pH affects polymorphonuclear leukocytes functions remain obscure. Moreover, in vitro observations seem to contradict the fact that an acidic microenvironment often prevails at sites of inflammation where polymorphonuclear leukocytes must function for host defense. In the present study, we found three peculiar characteristics of porcine polymorphonuclear leukocyte that had been induced to spread over fibrinogen-coated surfaces by phorbol 12-myristate 13-acetate (PMA) in acidified medium. First, the PMA-induced spreading at acidic pH, but not at neutral/alkaline pH, was dependent on extracellular Ca 2+. Second, the spreading at acidic pH was independent of protein kinase C (PKC), whereas that at neutral/alkaline pH was strictly PKC-dependent. Finally, the spreading at acidic pH, but not at neutral/alkaline pH, was suppressed by H 2O 2 produced by activated NADPH oxidase or added exogenously. As a result, polymorphonuclear leukocyte spreading at acidic pH peaked at 30 min after PMA stimulation, and declined thereafter because of negative regulation triggered by accumulated H 2O 2, whereas that at neutral/alkaline pH was stable for at least 90 min. The NADPH oxidase inhibitor diphenyleneiodonium or the H 2O 2-degradation enzyme catalase consistently stabilized the spreading at acidic pH. We conclude that PMA-stimulated polymorphonuclear leukocytes spread in an acidic environment through a mechanism different from that under neutral/alkaline conditions. This H 2O 2-mediated negative regulation system in an acidic environment may be crucial for avoiding tissue-damaging inflammatory actions of accumulated polymorphonuclear leukocytes in vivo.