FIGURE 1.

Lacritin in normal human tears is essential for survival of stressed corneal epithelial cells. A, schematic diagram of normal tears on the eye surface as compared with skin with the protective cornified zone. B, schematic diagram of tears on the eye in dry eye. C, pool of basal human tears from 19 individuals. D, pool of dry eye human tears from 29 individuals. E, HCE-T cells were sensitized overnight with IFNG and then treated for 15 min with pooled normal tears in the presence of TNF. Cells were washed and fixed, and then FOXO3 was immunolocalized. The same approach with different tear or lacritin samples was used in F–J , M, and N. IFNG/TNF-stressed cells with lacritin-depleted tears (F), mock-depleted tears (G), pooled dry eye tears (H), dry eye tears spiked with 10 nm lacritin (I), and dry eye tears spiked with 10 nm C-25 (lacritin lacking 25 amino acids from the C terminus) (J). Bar, 10 μm. K, linear diagram of lacritin versus C-25. Yellow indicates demonstrated (15) and predicted α-helices. L, left panel, HCE-T cells were sensitized overnight with IFNG and then treated for 15 min with increasing amounts of lacritin (or FBS (middle panel)) in the presence of TNF. Viability was assessed by the MTT assay. 10 nm lacritin, but not other doses, significantly enhanced cell viability (ANOVA with Dunnett's post-test; **, p < 0.01) but is not significantly different from FBS-enhanced viability (p > 0.05). Right panel, HCE-T cells were similarly stressed with IFNG and TNF or not stressed and then left untreated or instead treated with 10 nm lacritin or C-25. Lacritin enhanced viability (ANOVA with Dunnett's post-test; **, p < 0.01). The y axis is not shown extending to zero in this and other viability assays because the assay detects subtle changes. M, lacritin (10 nm) on IFNG/TNF-stressed primary human corneal epithelial cells. Bar, 25 μm. N, C-25 (10 nm) on IFNG/TNF-stressed primary human corneal epithelial cells. Error bars represent S.E. cor, cornified; lacrt, lacritin; ep, epithelia; nml, normal.