CA2-DEPENDENT PRODUCTION AND RELEASE OF IL-8 IN HUMAN NEUTROPHILS()

IL-8, a potent neutrophil chemoattractant that is elevated about 200-f ind in exudative neutrophils isolated from localized inflammatory site s in vivo, is thought to play a major role in recruitment of neutrophi ls to inflammatory sites. Incubation of peripheral blood neutrophils w ith thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-seque stering-ATPase, causes a dose-dependent induction of IL-8 synthesis th at continues for up to 8 h, Cycloheximide inhibits the thapsigargin-in duced IL-8 production, suggesting the induction of protein synthesis d e novo. In addition, Northern blot analysis of mRNA isolated from neut rophils indicates that thapsigargin treatment increases IL-8 mRNA in a time- and dose-dependent manner. Thapsigargin also induces a biphasic rise in the intracellular Ca2+ concentration, [Ca2+](i), which is com posed of an initial (within 15 s) EGTA-insensitive elevation in [Ca2+] (i), followed by a delayed (2-min) EGTA-sensitive component. Addition of EGTA before thapsigargin inhibited the induction of IL-8 production . Experiments in which EGTA was added at various times after thapsigar gin treatment indicated that a sustained Ca2+ influx was required for maximum IL-8 production. Ascomycin and cyclosporin A, inhibitors of th e Ca2+-dependent phosphatase, calcineurin, also inhibited thapsigargin -induced IL-8 production, Thus, in neutrophils, a prolonged Increase i n [Ca2+](i) stimulates IL-8 transcription and synthesis, possibly thro ugh a calcineurin-dependent pathway.