PURPOSE: To investigate the epithelial nature of primary and SV40 virus-immortalized human corneal epithelial (CEPI) cells and to study a variety of functional responses to some key inflammatory agents (bradykinin [BK], histamine, and platelet-activating factor [PAF]) and their antagonists in these cells. METHODS: Primary CEPI (P-CEPI) and clone 4 of the SV40 virus-immortalized (CEPI-17-CL4) cells were analyzed for their interaction with several monoclonal antibodies selective for various cytokeratins to define their immunocytochemical characteristics and phenotypic traits. Both cell types were tested for their ability to respond to BK, histamine, and PAF and their antagonists, using the production of [3H]inositol phosphates ([3H]IPs) as an index of receptor activation. The ability of BK, PAF, and histamine to stimulate cytokine release and the induction of mRNA for matrix metalloproteinase-1 (MMP-1) were also studied using enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction techniques, respectively. RESULTS: P-CEPI and CEPI-17-CL4 cells were both shown to possess the epithelial cell cytokeratins labeled with AE1 and AE3 antibodies. The potencies (EC50s) of BK, histamine, and PAF were similar for stimulating [3H]IPs production in P-CEPI and CEPI-17-CL4 cells: BK = 2.27 to 2.99 nM, PAF = 17.1 to 18.26 nM, and histamine = 1.65 to 5.74 microM (all n = 3 to 6). Both cell types also responded similarly to receptor-selective antagonists for BK, PAF, and histamine (Hoe-140: Ki = 10.1 to 11.9 nM; PCA-4248: Ki = 315 to 421 nM; triprolidine: Ki = 0.8 to 4.76 nM; all n = 5 to 10). Histamine (100 microM) and interleukin-1alpha (IL-1alpha, 10 ng/ml) significantly stimulated IL-6 and granulocyte macrophage colony-stimulating factor release, and histamine, BK, and PAF stimulated the mRNA for MMP-1 in these cells. CONCLUSIONS: These studies have shown that the primary and immortalized human corneal epithelial cells express functional BK (a B2 subtype), histamine (an H1 subtype), and PAF receptors and exhibit very similar immunocytochemical, signal transduction, and pharmacological properties. Therefore, the CEPI-17-CL4 cells (currently at passage 220) appear to provide a useful representative in vitro model system to study the physiological and pathologic aspects of the human corneal epithelium.
PURPOSE: To investigate the epithelial nature of primary and SV40 virus-immortalized human corneal epithelial (CEPI) cells and to study a variety of functional responses to some key inflammatory agents (bradykinin [BK], histamine, and platelet-activating factor [PAF]) and their antagonists in these cells. METHODS: Primary CEPI (P-CEPI) and clone 4 of the SV40 virus-immortalized (CEPI-17-CL4) cells were analyzed for their interaction with several monoclonal antibodies selective for various cytokeratins to define their immunocytochemical characteristics and phenotypic traits. Both cell types were tested for their ability to respond to BK, histamine, and PAF and their antagonists, using the production of [3H]inositol phosphates ([3H]IPs) as an index of receptor activation. The ability of BK, PAF, and histamine to stimulate cytokine release and the induction of mRNA for matrix metalloproteinase-1 (MMP-1) were also studied using enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction techniques, respectively. RESULTS:P-CEPI and CEPI-17-CL4 cells were both shown to possess the epithelial cell cytokeratins labeled with AE1 and AE3 antibodies. The potencies (EC50s) of BK, histamine, and PAF were similar for stimulating [3H]IPs production in P-CEPI and CEPI-17-CL4 cells: BK = 2.27 to 2.99 nM, PAF = 17.1 to 18.26 nM, and histamine = 1.65 to 5.74 microM (all n = 3 to 6). Both cell types also responded similarly to receptor-selective antagonists for BK, PAF, and histamine (Hoe-140: Ki = 10.1 to 11.9 nM; PCA-4248: Ki = 315 to 421 nM; triprolidine: Ki = 0.8 to 4.76 nM; all n = 5 to 10). Histamine (100 microM) and interleukin-1alpha (IL-1alpha, 10 ng/ml) significantly stimulated IL-6 and granulocyte macrophage colony-stimulating factor release, and histamine, BK, and PAF stimulated the mRNA for MMP-1 in these cells. CONCLUSIONS: These studies have shown that the primary and immortalized human corneal epithelial cells express functional BK (a B2 subtype), histamine (an H1 subtype), and PAF receptors and exhibit very similar immunocytochemical, signal transduction, and pharmacological properties. Therefore, the CEPI-17-CL4 cells (currently at passage 220) appear to provide a useful representative in vitro model system to study the physiological and pathologic aspects of the human corneal epithelium.
Authors: Najam A Sharif; Shouxi Xu; Peggy E Hellberg; Iok-Hou Pang; Daniel A Gamache; John M Yanni Journal: Mol Vis Date: 2009-06-06 Impact factor: 2.367