FIGURE 1.

IL-1β increases ITGB8 mRNA and αvβ8 surface protein in lung fibroblasts and astrocytes but not dermal fibroblasts. A, multiple proinflammatory cytokines influence β8 expression on human lung fibroblasts. Shown is surface staining for αvβ8 from primary cultures of human lung fibroblasts harvested from lung samples from different patients. Cultures were treated with IL-1β (n = 33), IL-4 (n = 7), IL-5 (n = 7), and IL-13 (n = 7) for 48 h (all at 1 ng/ml). Cells were stained with anti-β8 and analyzed by flow cytometry. The percent change of anti-β8 mean fluorescence intensity induced by cytokine treatment compared with non-treated controls is shown. *, p < 0.05;**, p = 0.01, Wilcoxson signed rank test. B, immunoprecipitation of αvβ8 with anti-β8 (37E1B5) from lysates of cell surface biotin-labeled human lung fibroblasts is shown. Fibroblasts were treated with IL-1β, media only, or IL-13 for 48 h before lysis. Immunoprecipitated complexes from equal numbers of fibroblasts were resolved by 7.5% SDS-PAGE under non-reducing conditions. Shown are the relative migrations of the αv and β8 integrin subunits and the positions of the molecular mass markers (kDa). Shown is a representative experiment of three, all of which showed nearly identical results. Densitometry of the immunoprecipitated β8 bands (n = 3) is shown in C. D, shown is the time course of ITGB8 transcript and αvβ8 surface protein expression by lung fibroblasts. Cells were treated with IL-1β at time 0 and harvested at indicated time points for analysis using qPCR or flow cytometry. Values are relative to untreated controls. E, shown is a comparison of base-line ITGB8 and IL-1β-induced transcript expression by human lung fibroblasts (n = 5), astrocytes (n = 3), and dermal fibroblasts (n = 3). Gene copy numbers are normalized to GAPDH and β-actin levels and are expressed as -fold changes relative to untreated human lung fibroblasts. F and G, human astrocytes respond to IL-1β by increasing αvβ8 surface expression (F) and αvβ8-mediated activation of TGF-β (G). F, human astrocytes were treated 48 h with or without IL-1β (1 ng/ml), and αvβ8 surface expression levels were assessed by flow cytometry (n = 6). G, human astrocytes were treated for 16 h with or without IL-1β (1 ng/ml) and TGF-β activation in the presence or absence of isotype control, neutralizing anti-β8 or pan-TGF-β, determined by TGF-β bioassay (n = 8). Results are shown in arbitrary luciferase units relative to total TGF-β activation as determined with pan-anti-TGF-β. *, p < 0.05; **, p < 0.01; ***, p < 0.001.