Human corneal epithelial cells express functional PAR-1 and PAR-2.

PURPOSE: The objective of this study was to examine whether HCECs express functional proteinase-activated receptor (PAR)-1 and -2 and evaluate the effects of receptor activation on corneal epithelial cell proinflammatory cytokine production. METHODS: Expression of PAR-1 and -2 mRNAs was determined by RT-PCR in cultured primary human corneal epithelial cells (HCECs) and the human corneal epithelial cell line HCE-T. Localization of PAR-1 and -2 in whole normal human corneas was determined by immunofluorescence with PAR-1 and -2 antibodies. The functional competence of PAR-1 and -2 in corneal epithelial cells was assessed by measuring the rapid induction of intracellular [Ca(2+)] in response to thrombin, trypsin, and specific receptor-activating peptides derived from the tethered ligands of the PAR receptors. HCE-T expression of cytokines (IL-6, IL-8, and TNFalpha) in response to activation of PAR-1 and -2 was measured by quantitative RT-PCR and ELISA. RESULTS: Functional PAR-1 and -2 were expressed in both HCECs and HCE-T cells. Immunoreactivity for PAR-1 and -2 was detected in the outer epithelial layer of the cornea in whole human corneal sections. Activation of PAR-1 and -2 led to upregulation in HCE-T cells of both expression of mRNA and secretion of the proinflammatory cytokines IL-6, IL-8, and TNFalpha. CONCLUSIONS: The results show for the first time that functional PAR-1 and -2 are present in human cornea. Activation of these receptors results in the production of various corneal epithelial cell proinflammatory cytokines. These observations indicate that PAR-1 and -2 may play an important role in modulating corneal inflammatory and wound-healing responses. These receptors may be useful therapeutic targets in several corneal disease processes.

Human keratinocytes express functional CD14 and toll-like receptor 4.

CD14 and the toll-like receptor 4 have been known to play an important role in lipopolysaccharide-induced cellular responses in bacterial infections. Although CD14 and toll-like receptor 4 expression has been demonstrated in a number of myeloid cells, much less is known about the expression and function of these lipopolysaccharide receptors on nonleukocytes. In this study, we demonstrate that human keratinocytes are capable of expressing functional CD14 and toll-like receptor 4. Keratinocytes were found to constitutively express CD14 and toll-like receptor 4 mRNA that was augmented by exposure to lipopolysaccharide. Cell surface expression of keratinocyte CD14 and toll-like receptor 4 was detected by flow cytometry. Lipopolysaccharide binding to keratinocyte CD14 and toll-like receptor 4 resulted in a rapid intracellular Ca2+ response, nuclear factor-kappaB nuclear translocation, and the secretion of proinflammatory cytokines and chemokines. These results have important implications for our understanding of cutaneous innate immunity to bacterial infections of the skin.