Interferon-mediated Block in the Cell Cycle and Alteration of Integrin Expression in an in Vitro Model of Sjögren's Syndrome
School of Biological Sciences. University of Missouri--Kansas City, 1999 - Salivary glands - 154 pages
The sialoadenitis seen in Sjögren's syndrome, an idiopathic, autoimmune exocrinopathy, is characterized by lymphocytic infiltration, acinar cell atrophy, and diminished salivary flow. Increased expressions of laminin, a laminin receptor, and cytokines are also noted. Several in vivo characteristics of the sialoadenitis are also evident in a cytokine-treated salivary gland ductal epithelial cell line. To elucidate mechanisms of salivary gland pathology, the effects of two cytokines, interferon gamma and tumor necrosis factor alpha, on cell proliferation and expressions of basement membrane proteins and alpha-3 integrin were evaluated in cultured salivary gland cells. In cytokine-treated cell monolayers, immunoprecipitation, immunoperoxidase, and Western Blot analysis demonstrated a moderate intracellular accumulation of an immature laminin product, but not fibronectin or collagen IV, concurrent with decreased cell proliferation. Results from RNase Protection assays suggested that the laminin accumulation was unlikely due to increased laminin beta chain gene expression. Furthermore, a significant reduction of glyceraldehyde-3-phosphate dehydrogenase expression was noted with prolonged cytokine treatment, suggesting metabolic defects. To explore the effects of cytokines on acinar cell pathology, cells were grown on Matrigel, where they formed acini with polarized nuclei. Cytokine treatment arrested cells in G1 phase of the cell cycle, as evaluated by flow cytometry, which preceded significant morphological changes and decreased viability. By immunoprecipitation, an altered form of alpha-3 integrin was evident in cultured acinar cells treated with cytokines for prolonged periods, but not in untreated cells. Cytoklines caused no significant changes in laminin expression in acinar cells. From this study, it was evident that the combination of interferon gamma and tumor necrosis factor alpha resulted in a block in G1 phase for acinar cells. This cell cycle arrest occurred prior to accumulation of the alpha-3 integrin variant or significant degenerative cellular changes. Information from the present and previous studies suggest that cytokines may alter adhesion and block cell cycle progression in acinar cells in Sjögren's syndrome sialoadenitis. Further studies may help elucidate how these cytokine-mediated cellular changes contribute to acinar cell death.
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CYTOKINEMEDIATED EFFECTS ON CELL GROWTH
CYTOKINEMEDIATED EFFECTS ON MORPHOLOGY
CYTOKINEMEDIATED EFFECTS ON EXPRESSION
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a3 integrin acinar cells acinar-like adhesion molecules basement membrane basement membrane proteins cell culture cell culture samples cell cycle cell line cell numbers cell proliferation cell types cellular chain chapter combination of IFN-y compared to untreated cultured on Matrigel cytokine cytokine effects cytokine-mediated cytokine-treated and untreated data not shown differentiation dimer ductal epithelial cells Effect of IFN-y epithelial cells fibronectin Furthermore GAPDH gene glycosylation grown on Matrigel grown on plastic HSG cell monolayers HSG cells cultured HSG cells grown IFN-y and TNF-a immunoprecipitation inhibition integrin expression intracellular laminin laminin expression laminin Py dimer lymphocytes Molecular image monoclonal antibody monolayer morphological mRNA mRNA expression nine days non-reducing conditions pathology patients with SS phases phosphorylation polyclonal antibody receptor salivary gland disease SDS-PAGE separate cell culture six days six separate cell Sjogren's syndrome SS salivary gland steady-state levels treated with IFN-y treatment of HSG units/ml untreated HSG cells up-regulation Western Blot