Lacritin-induced secretion of tear proteins from cultured monkey lacrimal acinar cells.

PURPOSE: During inflammation of the ocular surface, increased proinflammatory cytokines depress tear protein secretion, suggesting that a decline in lacrimal cell function contributes to dry eye. Lacritin, a glycoprotein secreted from lacrimal acinar cells, may function as an autocrine factor to stimulate tear protein secretion. The purpose of the present experiment was to investigate lacritin-induced protein secretion in normal and cytokine-pretreated (inflammation model) monkey acinar cells. METHODS: Acinar cells from monkey lacrimal glands were cultured with or without tumor necrosis factor alpha (TNF-α) plus interferon gamma (IFN-γ). Protein secretion was induced by lacritin or carbachol (Cch, positive control). Proteins were detected and identified by immunoblotting and immunocytochemistry. Intracellular Ca(2+) was measured with the fluorophore Calcium-4, and cell damage was determined by LDH leakage into the culture medium. RESULTS: In cultured monkey acinar cells, lacritin stimulated tear protein secretion in normal cells without elevating intracellular Ca(2+). In contrast, Cch elevated intracellular Ca(2+) and release of tear proteins. This contrast suggested an alternate, calcium-independent mechanism for lacritin-induced protein secretion. TNF-α plus IFN-γ caused LDH leakage from sensitive human corneal epithelial cells, but even higher doses of TNF-α plus IFN-γ did not cause LDH leakage from monkey acinar cells, suggesting a higher tolerance against these cytokines. In cytokine-treated acinar cells, lacritin stimulated protein secretion as much as that in normal cells. In contrast, Cch-induced elevation of Ca(2+) and release of proteins were depressed by cytokines. CONCLUSIONS: Lacritin might be a useful biotechnology-based treatment agent against ocular surface diseases where endogenous lacritin is inadequate.

Mechanism for carbachol-induced secretion of lacritin in cultured monkey lacrimal acinar cells.

PURPOSE: Lacritin protein is highly expressed in the lacrimal gland, secreted into tear fluid, and detected only in primates. The mechanism for lacritin secretion has not been fully investigated, because a system for culturing primate lacrimal acinar cells had not been established. The purposes of the present study were (1) to develop a procedure to culture lacrimal acinar cells from monkey and (2) to determine the mechanism for the secretion of lacritin in the culture system. METHODS: Acinar cells from monkey lacrimal gland were cultured and characterized. Lacritin and other proteins were detected by immunohistochemistry, immunocytochemistry, and immunoblot analysis. Secreted proteins were also detected in the medium from stimulated acinar cells. mRNAs were determined by microarray and qPCR. Intracellular calcium levels were measured by calcium-4 assay. RESULTS: Acinar cells cultured for 1 day contained adequate amounts of lacritin, lactoferrin, and lipocalin for use in lacritin secretion studies. The cholinergic agonist carbachol (Cch) stimulated the secretion of lacritin and increased intracellular Ca2+. Cch-induced lacritin secretion was inhibited by the store-operated calcium (SOC) channel inhibitor YM58483 and the PKC inhibitors GF109203 and Ro-32-0432. Cch-induced lacritin secretion was not inhibited by MAPKK inhibitor U0126, although p42/p44 MAPK was phosphorylated. Cch also enhanced gene transcription, which was inhibited by U0126, GF109203, and calcium chelators. CONCLUSIONS: Successful culture of monkey lacrimal acinar cells showed that, among the prevalent tear proteins, the secretion of lacritin involved the PKC/Ca2+ pathway, not the p42/p44 MAPK pathway. Induction of transcription by Cch involved the independent p42/p44 MAPK and PKC pathways.