thapsigargin and Sjogren-s-Syndrome

The aim of this study was to examine the levels of endoplasmic reticulum (ER) stress in minor salivary glands, to investigate the interplay between ER stress-induced autophagy and apoptosis in human salivary gland (HSG) cells and to test the effect of ER stress-induced apoptosis on the cellular redistribution of the two major Sjögren's syndrome (SS) autoantigens Ro/Sjögren's syndrome-related antigen A (SSA) and La/Sjögren's syndrome-related antigen B (SSB). Minor salivary gland biopsies from SS patients and sicca controls were examined by immunohistochemistry for the expression of 78 kDa glucose-regulated protein/binding immunoglobulin protein (GRP78/BiP) as an indicator of unfolded protein response (UPR). HSG cells were treated with thapsigargin (TG) and cell viability, autophagy and apoptosis were assessed. Immunoblot was applied to detect the conversion of LC3I to LC3II and the protein levels of GRP78/BiP and X-box binding protein-1 (XBP-1). Apoptosis was evaluated by a single-stranded DNA enzyme-linked immunosorbent assay (ELISA). Ro/SSA and La/SSB localization was visualized using immunofluorescence. GRP78/BiP was expressed by acinar and ductal epithelial cells in salivary glands of patients and sicca controls. TG treatment induced autophagy, as indicated by enhanced protein expression of LC3II. The protein levels of UPR marker XBP-1 were increased after TG treatment, while GRP78/BiP levels were decreased. TG treatment resulted in induction of HSG apoptosis. Ro/SSA and La/SSB autoantigens were localized predominantly to the cytoplasm in resting cells, while they were redistributed to cell membrane and blebs in the apoptotic cells. In conclusion, ER stress is activated in minor salivary gland epithelial cells from SS patients and controls. ER stress-induced apoptosis in HSG cells leads to cell surface and apoptotic blebs relocalization of Ro/SSA and La/SSB autoantigens.

Topics: Apoptosis; Autoantigens; Autophagy; Cell Line; Cell Membrane; Cytoplasm; DNA-Binding Proteins; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Epithelial Cells; Female; Gene Expression Regulation; Heat-Shock Proteins; Humans; Microtubule-Associated Proteins; Primary Cell Culture; Protein Transport; Regulatory Factor X Transcription Factors; Ribonucleoproteins; Salivary Glands, Minor; Sjogren's Syndrome; SS-B Antigen; Thapsigargin; Transcription Factors; Unfolded Protein Response; X-Box Binding Protein 1

To determine whether chronic exposure to lymphocyte-derived cytokines could inhibit the fluid secretory mechanism in salivary gland acinar cells and so account for the loss of gland function seen in the early stages of Sjögren's syndrome.. Mouse submandibular acinar cells maintained in primary culture were exposed to a profile of cytokines produced by concanavalin A-activated splenic lymphocytes in vitro for periods up to 72 h. Agonist-evoked changes in intracellular Ca(2+) were determined microfluorimetrically in both control and cytokine-treated cells.. Acinar cells maintained in primary culture in the presence of cytokines for up to 72 h were able to mobilize intracellular calcium in response to stimulus by acetylcholine in an identical fashion to those maintained in primary culture in the absence of cytokines. Acute application of the conditioned medium produced by the activated lymphocytes had an antisecretory effect on acetylcholine-evoked Ca(2+) mobilization, which was found to be mediated by cholinesterase rather than by cytokines.. Neither chronic nor acute exposure to the profile of cytokines released by concanavalin A-activated splenic lymphocytes interfered in any way with the second messenger cascade and fluid and electrolyte secretion in acinar cells. Our data suggest an alternative hypothesis, in which elevated levels of cholinesterase can metabolize acetylcholine released within the salivary glands and thus prevent fluid secretion.

Topics: Acetylcholine; Animals; Calcium; Carbachol; Cells, Cultured; Cholinergic Agonists; Concanavalin A; Culture Media, Conditioned; Enzyme Inhibitors; Interferon-gamma; Interleukin-2; Interleukin-3; Interleukin-4; Interleukin-6; Lymphocytes; Male; Mice; Mice, Inbred Strains; Saliva; Sjogren's Syndrome; Spleen; Submandibular Gland; Thapsigargin; Vasodilator Agents