Modulation of conjunctival goblet cell function by inflammatory cytokines

Abstract

Ocular surface inflammation associated with Sjögren's syndrome is characterized by a loss of secretory function and alteration in numbers of mucin secreting goblet cells. Such changes are a prominent feature of ocular surface inflammatory diseases and are attributed to inflammation; however, the exact effect of the inflammatory cytokines on conjunctival goblet cell function remains largely unknown. In this study, we developed a primary culture of mouse goblet cells from conjunctival tissue and evaluated the effects on their function by inflammatory cytokines detected in the conjunctiva of mouse model of Sjögren's syndrome (Thrombospondin-1 deficient mice). We found that apoptosis of goblet cells was primarily induced by TNF-α and IFN-γ. These two cytokines also inhibited mucin secretion by goblet cells in response to cholinergic stimulation, whereas IL-6 enhanced such secretion. No changes in secretory response were detected in the presence of IL-13 or IL-17. Goblet cells proliferated to varying degrees in response to all the tested cytokines with the greatest response to IL-13 followed by IL-6. Our results therefore reveal that inflammatory cytokines expressed in the conjunctiva during an ocular surface disease directly disrupt conjunctival goblet cell functions, compromising the protective function of tears, thereby contributing to ocular surface damage.

Figure 1

Th2-type inflammatory response is increased in TSP-1 null conjunctiva. Conjunctiva tissues were collected from WT and TSP-1 null mice at 6, 8, and 12 weeks. Extracted RNA was analyzed in a real time PCR assay to determine the levels of message for the Th2 cytokine IL-13 and the Th2-associated transcription factor GATA3 *P < 0.05.

Figure 2

Primary mouse goblet cell cultures express goblet cell specific markers. (a) Cells were grown from WT conjunctival explants for 14 days, and the expression of goblet cell specific (CK-7-red and MUC5AC-green) and stratified squamous cell specific (CK-4-red) markers was analyzed by immunofluorescence. Nuclei were counterstained with DAPI (blue). Magnification = ×20. (b) Flow cytometric analysis of goblet cells for the expression of MUC5AC. Unstained cells (empty histogram), isotype controls (filled grey histogram), colonic HT-29 cells (filled green histogram), and cultured goblet cells (filled red histogram).

Figure 3

Conjunctival inflammation associated with TSP-1 deficiency prevents carbachol-mediated MUC5AC secretion. Cultured goblet cells were grown from conjunctival explants of WT and TSP-1 null mice at 4 and 22 weeks of age. Goblet cells were stimulated with the cholinergic agonist carbachol, and MUC5AC secretion evaluated by ELISA in the supernatant. *P < 0.05.

Figure 4

Expression of cytokine receptors in primary cultures of conjunctival goblet cells. The expression of the cytokine receptors IL-13Rα1, IFN-γRI, TNF-αR1, TNF-αR2, IL-6Rα, and IL-17RA was detected using flow cytometry in primary cultures of conjunctival goblet cells. Results are presented as mean fluorescence intensity (MFI) for the cytokine receptors (black bars) and the corresponding isotype controls (white bars). *P < 0.05.

Figure 5

Inflammatory cytokines alter carbachol-mediated MUC5AC secretion by goblet cells. Cultured goblet cells were treated for 24 h with IFN-γ, TNF-α, IL-6, or IL-17A (10 ng/mL), stimulated with the cholinergic agonist carbachol (10^-3M) for 1 h, and MUC5AC secretion in the supernatants was evaluated by ELISA. *P < 0.05.

Figure 6

Inflammatory cytokines alter proliferation and apoptosis rate of cultured conjunctival goblet cells. Cultured goblet cells were pulsed with BrdU and treated for 24 h with 10 ng/mL of IL-13, IFN-γ, TNF-α, IL-6, or IL-17A. Cells stained with fluorescence-conjugated anti-BrdU antibody and viability dye 7-AAD was analyzed by flow cytometry to identify stages of cell cycles. (a) Representative flow cytometry plots are shown and percentage of cells in each of the G₀-G₁, S + G₂-M phases and apoptotic cells are indicated for untreated control and IL-13 treated cells. (b) Bar graphs show changes in proliferation (S + G₂-M phases) and apoptosis relative to untreated controls.