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N.M. Said, J. Zhang, F.X. Yu; Signaling Pathways and Innate Responses to Pseudomonas Aeruginosa Infection in Human Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3239.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Pseudomonas aeruginosa (PA) is an organism commonly involved in bacterial keratitis associated with contact lens wear. The aim of this study was to elucidate the signaling pathways activated in PA-infected corneal epithelial cells. Methods: HUCL cells (a telomerase-immortalized human corneal epithelial cell line) cultured in keratinocyte basic medium (lack of growth factors) were infected with PA01 strain. IΚB-α phosphorylation and degradation, ERK2 and ERK1/2 phosphorylation were assessed by Western blot. Activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies and Western blotting with phosphotyrosine antibody. Interleukin-8 and defensin expression was assessed using semiquantitative RT-PCR. Results: PA infection of HUCL cells resulted in rapid activation of NF-kB, as indicated by the increase in IΚB-α phosphorylation and degradation within 30 min of PA-epithelial contact. A transient activation of ERK1/2 phosphorylation was also observed within the period. EGFR activation, on the other hand, was detectable 1 h and reached the peak 4 h post PA-epithelial contact. PA infection-induced EGFR phosphorylation was sensitive to MMP inhibition, suggesting that the release of endogenous ligand(s) of EGFR was MMP-dependent. PA infection of corneal epithelial cells also caused up-regulation of IL-8, ß-defense-2, α-defensin 5 and -6. While ß-defense-2expression was up-regulated in 1 h of PA-epithelial contact, elevation of IL-8, α-defensin 5 and -6 mRNA level was observed in 4 h of PA-epithelial contact. Conclusions: Multiple signaling pathways including NF-ΚB and EGFR transactivation are activated in corneal epithelial cells in response to PA infection, leading release of antimicrobial peptide defensins and proinflammatory cytokine IL-8.
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