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From the Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia.
PURPOSE. This study sought to elucidate the innate immune responses of cultured human corneal epithelial cells (HCECs) to infection by the Gram-positive bacterium Staphylococcus aureus and to determine the underlying mechanisms.
METHODS. HUCL, a telomerase-immortalized HCEC line, and primary cultures of HCECs were challenged with live or heat-killed S. aureus, its exoproducts, or cell wall components lipoteichoic acid (LTA) and peptidoglycan (PGN). I
B-
phosphorylation and degradation as well as phosphorylation of MAPKs, p38, and JNK-1/2, were assessed by Western blot analysis. The expression of interleukin (IL)-6, IL-8, TNF-, and ß-defensin-2 were determined using RT-PCR and secretion of IL-6, IL-8, TNF-, and ß-defensin were measured using enzyme-linked immunosorbent assay and immunoblot analysis of culture medium.
RESULTS. Exposure of HUCL cells to live, but not heat-killed, S. aureus resulted in NF-B activation in a time-dependent manner, as assessed by the increase in IB- phosphorylation and degradation. Live bacteria also activated the p38 and JNK pathways. The effects of live bacteria on HUCL cells may be attributable to bacterial exoproducts, since the conditioned medium of S. aureus also effectively stimulated these signaling pathways. PGN, but not LTA, activated the NF-B and MAPK pathways in a dose- and time-dependent manner. Concomitant with activation of NF-B and MAPKs, transcriptional expression of IL-6, IL-8, TNF-, and ß-defensin-2 were induced in cells challenged with bacterial exoproducts and PGN. Secretion of IL-6, IL-8, TNF-, and ß-defensin-2 were also significantly increased in HCECs in response to bacterial exoproducts and PGN challenge.
CONCLUSIONS. Corneal epithelial cells possess the ability to recognize the presence of Gram-positive bacteria and to initiate the innate immune responses by the expression and/or release of proinflammatory cytokines and ß-defensin-2 in the cornea.
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