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J. Zhang, A. Kumar, F.–S.X. Yu; Priming Of Human Corneal Stromal Fibroblast By Pathogen–Exposed Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2654.
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We previously showed that in response to pathogen challenge human corneal epithelial cells (HCECs) express pro–inflammatory cytokines and anti–microbial molecules in a Toll–like receptor dependent manner. The aim of this study was to test the hypothesis that the epithelium coordinates corneal innate immune response to bacterial infection by priming human corneal stromal fibroblasts (HCSFs) through induction of proinflammatory gene expression and augmenting their responsiveness to pathogen challenge.
HUCL cells, a telomerase–immortalized HCEC line, cultured in dishes or on Transwell inserts were challenged with Pseudomonas (P.) aeruginosa (PAO1 strain, cell:bacteria=1:20), purified flagellin (250ng/ml), or exo–products (1:20 dilution of M9–based P. aeruginosa conditioned medium) for 1h. The priming effect of pathogen–exposed HCECs on HCSFs was assessed by applying HUCL conditioned media collected 8 h post–pathogen exposure onto the culture of serum–starved THK cells, a telomerase–immortalized HCSF. The pathogen–exposed HUCL cells were also co–cultured in Transwell with THK cells. IΚB–α phosphorylation and degradation in the primed THK cells were detected by Western blotting. Cytokine and chemokine expression was detected with RT–PCR.
Conditioned media derived from pathogen–exposed, but not control HUCL cells induced IΚB–α phosphorylation and degradation, indicators of NF–ΚB activation, in THK cells. Co–culture of pathogen–exposed HUCL cells resulted in an increase in the expression of cytokines (IL–1ß, IL–6), chemokines (IL–8, ENA–78, MCP–1 and MCSF–2) as well as MyD88 and TLR2 in THK cells. Neutralizing antibodies of IL–1ß and TNF–α partially blocked HUCL conditioned medium induced cytokine and chemokine expression. Pre–treatment of THK cells with flagellin–challenged HUCL conditioned medium resulted in an increase in the responsiveness of THK cells to flagellin stimulation, leading to up–regulation of pro–inflammatory cytokine and chemokine expression in THK cells.
HCECs, after detecting the presence of pathogen, prime stromal fibroblasts via augmenting TLR system and/or inducing cytokine expression or both in these cells. There appears to be an amplification mechanism in the cornea, with epithelium to initiate inflammatory reaction and keratocytes to continue and/or to further enlarge the response to bacterial infection.
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