title: Protein kinase C-alpha mediates cigarette smoke extract- and complement factor 5a-stimulated interleukin-8 release in human bronchial epithelial cells.
abstract: BACKGROUND
  Cigarette smoke extract (CSE) activates protein kinase C (PKC) and augments complement factor 5a (C5a)-stimulated release of the proinflammatory cytokine IL-8 in human bronchial epithelial cells (HBEC). We hypothesized that PKC activation by alternative PKC activators will also mediate C5a-stimulated IL-8 release in HBEC.
  
  
  METHODS
  HBEC were treated with phorbol myristate acetate (100 ng/mL), calcium ionophore A23187 (2 nM), or 10 nM cholesterol-3-sulfate in the presence or absence of C5a. Interleukin-8 (IL-8) release was measured by enzyme-linked immunoadsorbent assay.
  
  
  RESULTS
  IL-8 release by PKC activators alone was significantly higher than in unstimulated cells and was further augmented in the presence of C5a. Preincubation with the PKC inhibitor calphostin C (1 microM) significantly suppressed IL-8 release in HBEC treated with CSE and C5a. Preincubation with 10 microM TMB-8 (an intracellular calcium sequester) also significantly suppressed IL-8 release in CSE- and C5a-treated HBEC, suggesting that intracellular calcium is required for CSE- and C5a-mediated IL-8 release. When HBEC were preincubated with 30 nM of the PKCbeta-specific inhibitor LY363196, CSE- and C5a-mediated IL-8 release was not inhibited. However, with higher concentrations of LY363196 (>600 nM), which exceeds the IC50 for PKCbeta greater than 100 fold, CSE- and C5a-mediated IL-8 release was significantly suppressed. Preincubation of HBEC with 100 nM of Gö 6976, a specific PKCalpha inhibitor, significantly inhibited CSE- and C5a-mediated stimulation of IL-8 release.
  
  
  CONCLUSIONS
  Collectively, these data suggest that PKC activators in addition to CSE augment C5a-stimulated IL-8 release from HBEC and that CSE and C5a stimulate IL-8 release in HBEC by activating the calcium-dependent PKCalpha isoform.
authors.affiliation: Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, Omaha 68198-5300, USA.
authors.forename: Ravindra
authors.initials: R
authors.lastname: Kashyap
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Floreani
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Heires
authors.forename: Sam D
authors.initials: SD
authors.lastname: Sanderson
authors.forename: Todd A
authors.initials: TA
authors.lastname: Wyatt
mesh_terms: D001980:Bronchi / Q000503:physiopathology*; D015936:Complement C5a / Q000378:metabolism; D004847:Epithelial Cells / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D016209:Interleukin-8 / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D012906:Smoke / Q000009:adverse effects; D012907:Smoking / Q000503:physiopathology
chemical_list: D016209:Interleukin-8; D007527:Isoenzymes; D012906:Smoke; D015936:Complement C5a; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha