Differential effect of Th1- and Th2-type cytokines on rat conjunctival goblet cell function

Abstract

Purpose: To determine if Th1 and Th2 pro-inflammatory cytokines affect cultured rat conjunctival goblet cells and to measure their effects on proliferation, intracellular calcium levels ([Ca²⁺]_i) and high molecular weight glycoconjugate secretion that includes MUC5AC.

Methods: Rat conjunctival goblet cells were grown from tissue explants. Passage 1 cells were cultured in supplemented RPMI medium. The expression of goblet (CK-7) and stratified squamous (CK-4) specific cell markers was analyzed by immunofluorescence and the lectin UEA-1 was used to identify goblet cell secretory products. To evaluate proliferation cells were serum starved for 24 h, treated with the cytokines IFN-γ (3ng/ml), IL-4 (10ng/ml), IL-5 (10ng/ml) or IL-13 (10ng/ml) for 24 h and then stimulated with the cholinergic agonist carbachol (Cch) or the allergic mediator histamine for 2 h. Proliferation was measured by WST-8 assay (n=3). To measure [Ca²⁺]_i, cells were loaded with fura2 and analyzed using InCyte Im2TM Ratio Imaging System. The effect of Cch and histamine on [Ca²⁺]_i was studied in cells treated for 15 min or 24 h with cytokines or buffer (n=5). High molecular weight glycoconjugate secretion was measured in supernatants from untreated and 24 h cytokine-treated cells using an enzyme-linked lectin assay (n=3).

Results: Rat cultured cells expressed goblet cells markers (CK-7 and secretory products identified by UEA-1), but not stratified epithelial marker (CK-4). The Th1 cytokine IFN-γ decreased goblet cell proliferation by 0.79 fold, whereas Th2 cytokines IL-4, IL-5 and IL-13, and histamine increased it by 1.94, 2.65, 2.89 and 2.46 fold respectively. IFN-γ used for 15 min significantly blocked Cch-mediated increase in [Ca²⁺]_i (p = 0.009), and IL-4 and IL-13 had the same effect on histamine-mediated [Ca²⁺]_i increase after 15 min pre-incubation (p=0.006 and p=0.003, respectively). When cells were incubated with cytokines for 24 h, only IL-13 maintained the blockade (p=0.037). IFN-γ significantly blocked Cch-induced secretion (p=0.002), but Th2 cytokines did not have significant effects on histamine-induced secretion.

Conclusions: Th1- (IFN-γ) and Th2- (IL-4, IL-5 and IL-13) derived cytokines have opposite effects on proliferation and secretion from cultured rat goblet cells. These findings could explain the differences in goblet cell function found in inflammatory ocular surface diseases, such as dry eye and allergic conjunctivitis.

Keywords: 474 conjunctiva • 541 glycoconjugates/glycoproteins • 439 calcium