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M. Ni, D.J. Evans, S.M. J. Fleiszig; Pseudomonas Aeruginosa Exposure Regulates Surfactant Protein D Production by Human Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):896.
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Purpose: We previously showed that SP–D was present in human tear fluid and that it protected corneal epithelial cells against Pseudomonas aeruginosa invasion in vitro. In this study, we explored the expression of SP–D in corneal epithelium and then examined the effect of P. aeruginosa exposure. Methods:Primary cultures of mouse corneal epithelial cells were prepared from female 8–12 week old wild type C57BL/6 mice and gene–targeted SP–D deficient mice. SDS–PAGE and Western blot were performed to detect the SP–D level in cultured corneal epithelial cell lysates or cell growth media. To determine whether P. aeruginosa exposure can regulate SP–D expression, SV 40–immortalized human corneal epithelial cells were stimulated with 2 x 107 or 2 x 1010 heat killed bacteria (invasive strain PAK) or were sham inoculated. After stimulation, cells were lysed and SP–D quantified using Western Blot. The effect of bacterial lipopolysaccharide (LPS) on SP–D expression was explored using two different mutants with defects in LPS core and O antigen. A fliC mutant was used to examine the role of bacterial flagellin. Results: SP–D was detected in primary cultured mouse corneal epithelial cells derived from C57BL/6 mice, but not in lysates of corneal epithelial cells derived from SP–D deficient mice. SP–D was also detected extracellularly in cultured corneal epithelial cell growth media. Cells treated with heat killed wild type P. aeruginosa showed a strong dose–dependent upregulation of SP–D production in both cell lysates and cellular secretions. LPS and flagellin mutants, however, were each defective in their ability to upregulate SP–D in cell lysates and cell secretions. Conclusions: Corneal epithelial cells were found to make and secrete SP–D and as such could contribute to tear fluid SP–D level. SP–D expression in human corneal epithelial cells was strongly upregulated when cells were exposed to P. aeruginosa, which involved bacterial LPS and flagellin. These results suggest that SP–D is an inducible factor involved in innate immunity against P. aeruginosa invasion, and that induction could involve TLR signaling.
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