Cytokine-mediated stimulation of laminin expression and cell-growth arrestin a human submandibular gland duct-cell line (HSG)

Increased expression of laminin and various cytokines, including interferon -gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) has been de monstrated in minor salivary glands from patients with Sjogren's syndrome. Previous reports state that exposure of a human salivary-gland cell line (H SG) to IFN-gamma results in cellular changes similar to those in vivo Sjogr en's syndrome. To begin studies of the cause of increased laminin expressio n in salivary glands in Sjogren's syndrome and laminin's role in the pathol ogical process, the effects of IFN-gamma on laminin expression and growth o f HSG cells were examined here. Subconfluent cultures of HSG cells were tre ated or not with IFN-gamma (1000 units/ml) for 1, 3 or 6 days. Immunoprecip itation showed that the expression of cell-associated laminin was significa ntly greater in IFN-gamma-treated cells at 3 or 6 days than in untreated ce lls, while no significant differences in laminin counts precipitated from t he media were evident among any of the IFN-gamma-treated or untreated sampl es. Western blot analysis strongly suggested that this immunoprecipitated p roduct is a dimer of the beta- and gamma-chains of laminin. Intracellular l aminin was demonstrated immunocytochemically in a distinct, perinuclear pat tern in both cytokine-treated and untreated cells. However, only faint stai ning for type IV collagen. and no staining for fibronectin were evident in untreated and cytokine-treated cells. An RNase protection assay showed only slight upregulation of the laminin beta-chain mRNA at 3 days, but no signi ficant difference at 6 days of treatment. Taken together, these data sugges t enhanced accumulation of a dimer of laminin beta- and gamma-chains in the cytoplasm of cytokine-treated HSG cells. However, mRNA for glyceraldehyde 3-phosphate dehydrogenase was significantly reduced at 6 days of treatment, suggestive of cytokine-mediated metabolic abnormalities. IFN-gamma treatme nt also resulted in significant reductions in cell numbers over time, in ag reement with previous reports. Treatment of HSG cells for 3 days with IFN-g amma (1000 U/ml) and TNF-alpha (70 U/ml) resulted in no significant changes in cell proliferation or laminin protein and/or mRNA species compared to c ells treated with IFN-gamma alone. Karyotype analysis of HSG cells revealed human chromosomes with triploid chromosome numbers and rearrangements, cha racteristic of transformed cells. These data demonstrate that IFN-gamma inc reases the amount of intracellular laminin beta gamma dimers while decreasi ng cell growth. Further studies are required to define an interaction betwe en laminin expression and the growth and viability of HSG cells. (C) 1999 E lsevier Science Ltd. All rights reserved.