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K.F. Siemasko, A. Enriquez de Salamanca, J. Gao, Y. Diebold, M. Calonge, M.E. Stern; Effect of IFN on Muscarinic and Adrenergic Receptor Expression in a Normal Human Conjunctival (NHC) Epithelial Cell Line: Flow Cytometry Study . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3803.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the effect of inflammatory cytokines on neurotransmitter receptor expression in normal human conjunctival (NHC) epithelial cell line using Flow cytometry. Methods: Surface expression levels of muscarinic receptors (M1, M2, and M3) and adrenergic receptors (α1A-,α1B-,α1C-, α2A-, α2B-, α2C, ß1, ß2, and ß3) were analyzed in a normal human conjunctival (NHC) epithelial cell line. NHC cells were unstimulated or treated with IFNγ (500 U/ml) or TNFα (25 ng/ml) for 48 hours. Unfixed cells were incubated with specific receptor subtype antibodies followed by incubation with FITC or PE-conjugated secondary antibodies. Samples were analyzed using a Becton Dickinson FACS Calibur and Cell Quest software. Cytosolic distribution of neurotransmitter receptors was determined by intracellular staining. Fixed and Triton-X-100 permeabilized NHC cells and normal human conjunctival biopsies were analyzed for neurotransmitter receptor expression by immunofluorescence using confocal microscopy. Results: M2-muscarinic, M3-muscarinic and α1A-,α1B-,α1C-, α2A-, α2B-, α2C, ß1, and ß3 receptors were detected on the NHC cell surface by Flow cytometry. M1- muscarinic, and ß2 adrenergic receptors were not detected on the NHC cell surface. Intracellular staining for ß2 adrenergic receptors was positive as determined by Flow cytometry and confocal microscopy. Normal human conjunctival biopsies expressed all of the muscarinic and adrenergic receptors tested except α2C, as determined by confocal microscopy. Treatment of cells with IFNγ induced an increase in M2 receptor expression. No effect of IFNγ on M3-muscarinic and on any of the adrenergic receptors was detected. TNFα did not change the expression of either the muscarinic or adrenergic receptors. Conclusions: The NHC cell line tested expresses M2-muscarinic, M3-muscarinic and α1A-,α1B-,α1C-, α2A-, α2B-, α2C, ß1, and ß3 receptors on the cell surface. M1-muscarinic receptors were not detected on the surface or intracellularly. ß2- adrenergic receptor was present intracellularly only. IFNγ induced an increase in expression of the M2-muscarinic receptor. This NHC cell line will be a valuable in vitro model to investigate the neural regulation of conjunctival epithelial cell functions present in ocular surface diseases.
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