FIGURE 3.

αvβ8-mediated SMAD4 signaling is required for IL-1β-mediated CCL20 expression. A, pSMAD2/3 phosphorylation as determined by ELISA (Cell Signaling) is increased by IL-1β and inhibited by antibody inhibition of αvβ8-mediated TGF-β activation. Human lung fibroblasts were treated with IL-1β, with isotype control or anti-αvβ8, clone B5 (7). As a control, recombinant active TGF-β (rTGF-β) was added to IL-1β-treated cells, which further increased pSMAD2/3 phosphorylation. B, conditional deletion of smad4 in mouse lung fibroblasts inhibits IL-1β-induced CCL20 expression. Primary cultures of lung fibroblasts from smad4 f/f mice were transduced with Ad-Cre or Ad-GFP control 24 h prior to treatment with or without recombinant human IL-1β, in the presence or absence of the pan-TGF-β blocking antibody (1D11) for 16 h. Harvested supernatants were assessed for CCL20 protein expression by ELISA. The results are expressed as ng/ml. *, p < 0.05; **, p < 0.01; ***, p < 0.001. C, siRNA knockdown of smad3 in mouse lung fibroblasts does not inhibit IL-1β-induced CCL20 expression. Primary cultures of lung fibroblasts from smad4 f/f mice were transduced with Ad-Cre or Ad-GFP control, as above. 16 h later, cells were transfected with siRNA to smad3 or siRNA control and after another 16 h were treated with or without recombinant human IL-1β (1 ng/ml). Harvested supernatants were assessed for CCL20 protein expression by ELISA, and the results are shown as fold induction over non-IL-1β-treated controls. The extent of inhibition of IL-1β-induced CCL20 expression by pan anti-TGF-β (1D11) is shown (filled bar). n = at least 4 per group. ***, p < 0.001.