Protein kinase C activation is required for cigarette smoke-enhanced C5a-mediated release of interleukin-8 in human bronchial epithelial cells

Complement-derived anaphylatoxin C5a is a glycopolypeptide important in the regulation of inflammation. Previously, we have shown that C5a receptors ( C5aR) are constitutively expressed on human bronchial epithelial cells (HBE Cs) grown in culture. We have also shown that the expression of C5aR is inc reased upon exposure of HBECs to 5% cigarette smoke extract (CSE), and that this subtoxic dose of CSE significantly enhances C5a-stimulated interleuki n (IL)-8 release. To determine the intracellular signaling pathway of CSE C5a-mediated IL-8 release, we assayed protein kinase C (PKC) activity of H BECs after exposing the cells to CSE and/or C5a. No increase in PKC activit y was observed when HBECs were treated with 50 nM C5a for various times. Ho wever, PKC activity was increased by 2- to 3-fold in HBECs stimulated with 5% CSE for 1 h, as compared with cells incubated with medium only. No addit ional increase in PKC was observed when HBECs were treated with CSE and C5a together. When HBECs were pretreated with the PKC-specific inhibitor calph ostin C (1 mu M), no CSE-mediated PKC activation was observed. We then corr elated PKC activation with IL-8 release in the same cells. Although HBECs r equired stimulation by both CSE and C5a to release maximal levels of IL-8, preincubation of CSE-stimulated HBECs with calphostin C inhibited IL-8 rele ase by CSE + C5a. These results suggest that PKC activation by CSE alone do es not result in IL-8 release, but that CSE-stimulated PKC activation is re quired for C5a mediated IL-8 release from HBECs.