title: Inflammatory responses of corneal epithelial cells to Pseudomonas aeruginosa infection.
abstract: OBJECTIVE
  We hypothesized that corneal epithelium plays a role in the innate immune response by sensing the presence of pathogens and providing signals that activate the corneal defense system. We sought to determine the mechanisms involved in the activation of the signaling pathways and subsequent production of proinflammatory cytokines in human corneal epithelial cells (HCECs) in response to Pseudomonas aeruginosa infection.
  
  
  METHODS
  Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary cultures of HCECs were exposed to P. aeruginosa (PA01 strain) with or without the presence of the NF-kappaB inhibitor kamebakaurin, the p38 inhibitor SB203580, or the JNK inhibitor SP600125. IkappaB-alpha phosphorylation and degradation and p38 and JNK phosphorylation were assessed at different time points by Western blot analysis. Interleukin (IL)-6, IL-8, and TNF-alpha levels were determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA).
  
  
  RESULTS
  Exposure of HUCL cells and primary HCECs to P. aeruginosa resulted in rapid activation of NF-kappaB as indicated by an increase in IkappaB-alpha phosphorylation observed within 15 min and by IkappaB-alpha degradation, which peaked in 1 hr. Two stress-activated mitogen-activated protein kinases, p38 and JNK, were also activated as their phosphorylation was induced by P. aeruginosa infection. Concomitant with the activation of these Toll-like receptor-mediated signaling pathways, transcriptional expression and subsequent secretion of IL-6 and IL-8 in HUCL cells were also induced by P. aeruginosa. Presence of the NF-kappaB inhibitor kamebakaurin in culture medium blocked P. aeruginosa-induced NF-kappaB activation and inhibited IL-6, IL-8, and TNF-alpha expression and secretion. Inhibition of p38 or JNK also resulted in a decrease in bacteria-induced expression and secretion of these cytokines.
  
  
  CONCLUSIONS
  P. aeruginosa triggers an innate immune response in HCECs, and NF-kappaB and, to a lesser extent, the p38/JNK signal pathways are responsible for P. aeruginosa-induced proinflammatory cytokine production in these cells.
authors: Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia 30912, USA.
authors: Jing
authors: J
authors: Zhang
authors: Xin-Yi
authors: XY
authors: Wu
authors: Fu-Shin X
authors: FS
authors: Yu
mesh_terms: D000873:Anthracenes / Q000494:pharmacology; D015153:Blotting, Western; D018929:Cell Culture Techniques; D016207:Cytokines / Q000378:metabolism*; D004224:Diterpenes / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000378:metabolism / Q000382:microbiology*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D011725:Pyridines / Q000494:pharmacology; D012313:RNA / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000873:Anthracenes; D016207:Cytokines; D004224:Diterpenes; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D007093:Imidazoles; D015850:Interleukin-6; D016209:Interleukin-8; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D011725:Pyridines; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; C432165:pyrazolanthrone; D012313:RNA; C047382:kamebakaurin; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580