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Laura Contreras-Ruiz, Arpita Gosh-Mitra, Bruce Turpie, Marie Shatos, Darlene A. Dartt, Sharmila Masli; Inflammatory Cytokines In Thrombospondin-1 Deficient Conjunctiva Block Goblet Cell Mucin Secretion. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1246.
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Ocular surface inflammation in thrombospondin-1 (TSP-1) deficient mice is associated with autoimmune Sjögren’s syndrome, which progressively develops with age. Expression of pro-inflammatory cytokines is detectable in the TSP-1null conjunctiva as the mice age. We determined if these cytokines alter mucin secretion from mouse conjunctival goblet cells.
The cholinergic agonist pilocarpine was injected intraperitoneally and tears collected from WT (C57BL/6) and TSP-1 null mice at 8 and 12 weeks of age. Tear MUC5AC content was determined by ELISA. In addition, primary cultures of mouse conjunctival goblet cells were grown from WT conjunctival explants for 14 days in complete RPMI medium. Goblet and stratified squamous cell specific marker expression was analyzed by immunofluorescence (CK-7 and MUC5AC, goblet cell; CK-4,stratified squmaous cell) and flow cytometry (MUC5AC). Cultured goblet cells were treated for 24 hrs with 10 ng/ml of each of the inflammatory cytokines previously detected in TSP-1 null inflamed conjunctiva (IFN-γ, TNF-α, IL-6, and IL-17). Cells were then stimulated with the cholinergic agonist carbachol for 1 hr and MUC5AC secretion evaluated by ELISA in the supernatants of the treated cells.
Tear MUC5AC levels were significantly lower in TSP-1 null compared to WT mice at both 8 and 12 weeks of age. Almost all cells in primary mouse goblet cell cultures expressed the goblet cell specific markers CK-7 and MUC5AC, but were negative for the stratified squamous cell marker CK-4. Treatment with IFN-γ and TNF-α, significantly inhibited and IL-6 enhanced carbachol-mediated secretion of MUC5AC. Exposure of goblet cells to IL-17 did not alter carbachol-mediated MUC5AC secretion.
The pro-inflammatory cytokines expressed in the TSP-1 null mouse conjunctiva have differential effects on the secretion of mucins produced by goblet cells in response to cholinergic stimulus resulting in altered tear quality. The loss of ocular surface protection may further exacerbate ocular surface inflammation in TSP-1 deficient mice.
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