RESUMO
The purpose of this research was to investigate the role of neurogenic involvement in the etiopatho-genesis of ocular surface inflammation, with the final goal of identifying new potential anti-inflammatory agents. We describe the presence of two "classic" markers of inflammation (HLA-DR and ICAM-1) and some neuroreceptors in cultured human conjunctival epithelial cells under basal and pro-inflammatory conditions. Two markers of inflammation (HLA-DR, ICAM-1) and several neuroreceptor subtypes (M1-, M2-, and M3-muscarinic; alpha1A-, alpha1B-, alpha1D-, alpha2A-, alpha2B-, alpha2C-, beta1-, beta2-, and beta3-adrenergic) were analyzed in a normal human conjunctival epithelial cell line (IOBA-NHC). These markers were studied in basal conditions and under the influence of two pro-inflammatory cytokines: tumor necrosis factor alpha (TNF-alpha) and/or interferon gamma (IFN-gamma). Immunofluorescence (confocal microscopy), western blotting, or flow cytometry techniques were used. In basal conditions, epithelial cells expressed all inflammatory markers except HLA-DR. The addition of IFN-gamma enhanced expression of HLA-DR, ICAM-1, and M2-muscarinic receptor. TNF-alpha up-regulated the expression of ICAM-1. When epithelial cells were incubated in the presence of both cytokines together, the cell surface expression of HLA-DR, ICAM-1, alpha1B-, and alpha2B-adrenergic receptors was increased. Cultured human conjunctival epithelial cells have been shown to be susceptible to up-regulation of the expression of inflammatory markers and cell membrane expression of some neuroreceptors under pro-inflammatory conditions. Consequently, pharmacologic neuro-modulation could have a role in the comprehensive management of ocular surface inflammation.
RESUMO
PURPOSE: To characterize a new nontransfected, spontaneously immortalized epithelial cell line from normal human conjunctiva (IOBA-NHC), both morphologically and functionally, to determine whether the differentiated phenotype of conjunctival epithelial cells is preserved. METHODS: Outgrowing cells from explanted conjunctival tissue were successively passaged and preliminarily characterized at passage 3 to assess epithelial origin. The cells were further characterized at passages 15 to 20, 40, 60, and 100 by analyzing (1) proliferation and in vitro behavior (viability, plating efficiency, colony forming efficiency and colony size, and Ki-67 protein expression), (2) karyotype and G-banding, (3) epithelial marker expression (cytokeratins, desmoplakins, EGF receptor), (4) absence of contaminating cell types, (5) expression of conjunctival differentiation markers (mucin gene expression), and (6) functional capability in response to proinflammatory stimuli. IOBA-NHC cells were analyzed by light and electron (transmission and scanning) microscopy, immunohistochemistry, electrophoresis and Western blot analysis, flow cytometry, and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: IOBA-NHC cells showed high proliferative ability in vitro and typical epithelial morphology. Cytokeratins and GalNAc, GluNAc, mannose, and sialic acid residues were immunodetected in these cells. No contaminating cell types were found. MUC1, -2, and -4, but not -5AC or -7 mucin genes were expressed in every cell passage tested. Exposure of cells to inflammatory mediators (IFNgamma and/or TNFalpha) resulted in increased expression of intercellular adhesion molecule (ICAM)-1 and HLA-DR. CONCLUSIONS: Morphologic and functional characterization of the nontransfected, spontaneously immortalized IOBA-NHC cell line shows that this new cell line may be a useful experimental tool in the field of ocular surface cell biology.
