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Daniela Marcano, Ghanashyam Acharya, Stephen C Pflugfelder; IFN-γ inhibits cell proliferation and outgrowth from cultured lacrimal gland tissues.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2491.
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Increased expression of interleukin-1, interferon-γ (IFN-γ), and tumor necrosis factor-α has been measured in autoimmune dacryoadenitis. These pro-inflammatory cytokines stimulate the production of ROS and RNS species that can induce cell death. This study tested the hypothesis that IFN-γ inhibits cell outgrowth from murine lacrimal gland (LG) explants and stimulates production of pro-apoptotic factors.
LGs of healthy wild type (WT) and IFN-γ KO C57BL/6 male mice (9-10 weeks old) were removed and used for RT-PCR and tissue and cell cultures. Total RNA was extracted, cDNA synthesized and amplified for inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), and caspase 3. LG cells were obtained by digesting minced glands in a media supplemented with collagenase type I. These cells were incubated with WST-1 reagent for 1h at days 3 and day 14 and absorbance at 440 nm was measured. LG explants were obtained after digesting small pieces of gland in collagenase for 20 min and cultured on plastic dishes. At day 25, cultures were fixed and immunofluorescence staining performed for cytokeratins 8 and 14 (K8 and K14), Vimentin, and iNOS.
Compared to IFN-γ KO, higher expression of iNOS, IL-1β, and caspase 3 was found in WT LGs. Remarkably, iNOS expression in the IFN-γ KO was 5 times higher than WT. Significantly greater proliferation was noted in cultured glandular cells from IFN-γ KO compared to WT, suggesting that IFN-γ suppresses it. This finding is also supported by observed cell outgrowth in 49% of cultured IFN-γ-KO explants compared to only 34% of WT. Conspicuously, outgrowth from the IFN-γKO explants is usually greater than the WT explants. Because fibroblasts are a typical source of contamination in primary cultures, we used immunofluorescence to characterize the cellular outgrowth, identifying epithelial cells by K8 and K14 positivity, while spindle-like cells were confirmed by Vimentin staining. In all cases, cells growing from explants were positive for either K8 or K14 and negative for vimentin. iNOS was strongly detected at the leading front of the cell-outgrowth from IFN-γ KO and WT explants, but immunoreactivity of cells adjacent to the explants was less in the IFN-γ KO.
IFN-γ suppresses growth and increases production of pro-apoptotic factors in the LG. Increased iNOS expression in WT may lead to production of free radicals that reduce cellular viability.
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