Figure 9.
IL-1 signaling pathways contribute to augmented inflammation in macrophage-depleted wounds. A: Confluent primary keratinocytes from C57Bl/6J mice were analyzed for the release of MIP-2 in the presence or absence of cytokines (40 ng/ml IL-1β, 25 ng/ml tumor necrosis factor-α, 20 ng/ml U/ml interferon-γ) or TGF-β1 (5 ng/ml) after 20 hours as indicated. **P < 0.01 as indicated by the brackets. Bars depict means ± SD obtained from three independent cell culture experiments (n = 3). B: Confluent human HaCaT keratinocytes were stimulated with pooled wound lysates (n = 25 wounds from 5 individual animals) (f.c. 100 μg/ml) of PBS- or- DTox-treated lysM-Cre/DTR mice. Lysates were treated with an anti-TGFβ antibody (10 μg/ml), a non-specific IgG (10 μg/ml) or left untreated (w/o IgG). IL-8 was determined from cell culture supernatants after 24 by ELISA. **, ##, P < 0.01 as compared with the respective bar in PBS-control. Bars depict means ± SD obtained from three independent cell culture experiments (n = 3). C: Mink lung epithelial reporter cells were stimulated with fresh wound lysates (n = 25 wounds from 5 individual animals) (f.c. 400 μg/ml) of PBS- or- DTox-treated lysM-Cre/DTR mice in the presence or absence of an anti-TGFβ antibody (100 μg/ml). Medium containing non-specific IgG (med + IgG), or TGF-β1 (0.8 ng/ml) with non-specific IgG or an anti-TGFβ antibody (100 μg/ml) were used to control reactivity of the reporter cells. TGF-β bioactivity is expressed as stimulation of relative luminescence units (RLU) derived from luciferase enzymatic activity induced from a TGF-β-sensitive plasminogen activator inhibitor promoter in the cells. D: Confluent human HaCaT keratinocytes were stimulated with pooled wound lysates (n = 25 wounds from 5 individual animals) (f.c. 100 μg/ml) of PBS- or- DTox-treated lysM-Cre/DTR mice. Cells were pre-treated with IL-1ra (500 μg/ml) (+IL-1ra) or left untreated (w/o IL-1ra). Heated lysate (95°C) was used as a control. IL-8 was determined from cell culture supernatants after 24 by ELISA. **P < 0.01; n.s, not significant as indicated by the brackets. Bars depict means ± SD obtained from three independent cell culture experiments (n = 3).