Abstract
Purpose :
Autoimmune Sjögren’s syndrome is associated with chronic inflammation of the lacrimal gland and infiltration by Th1 and Th17 cells. Pathogenic effects of pro-inflammatory cytokines IFN-γ and IL-17 on glandular epithelial cells are not known. We hypothesize that inflammatory cytokines reduce the expression of immunoregulatory thrombospondin-1 (TSP-1) in glandular epithelial cells.
Methods :
Primary cultures of C57BL/6 lacrimal gland epithelial cells were generated by collagen digestion of the tissue. Cultured cells were immunostained with fluorescence-conjugated anti-pancytokeratin, anti-vimentin, anti-SMA antibodies and evaluated by fluorescence microscopy. The presence of secretory vesicles within the cells was detected by immunostaining with anti-Rab3d. Stained cells were examined by fluorescence microscopy and quantitative analysis was performed using NIH ImageJ software. Primary cultures were treated with IFN-γ or IL-17 (10 ng/ml) for 48 hr and cells were immunostained for TSP-1 and their RNA was analyzed for TSP-1 message by RT-PCR
Results :
All cells in primary cultures derived from lacrimal gland digest stained for cytokeratin supporting epithelial origin, while a small proportion (<5%) stained positively for smooth muscle actin indicating the presence of myoepithelial cells. There was no significant staining for vimentin detectable compared to isotype control staining supporting the absence of fibroblasts in primary cultures. Some cells in culture staining positively for Rab3d indicating the presence of secretory veiscles. These cells, presumably acinar cells, represented 15% of the total cells. Thus predominant cells in the culture were likely duct epithelial cells. Exposure of these epithelial cells to inflammatory cytokines IFN-γ or IL-17 for 48 hr resulted in significantly reduced immunostaining as well as reduced message for TSP-1 as compared to that detected in untreated control cells.
Conclusions :
Our results demonstrate successful primary culture of murine lacrimal gland epithelial cells and support the hypothesis that inflammatory cytokines can indeed reduce expression of immunoregulatory molecule like TSP-1 in lacrimal gland epithelial cells. Such changes in epithelial cells may contribute to immunopathogenesis of Sjögren’s syndrome.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.