Background Alarmins are molecular ‘danger signals’ released by injured cells that can contribute to the innate immune response by activating immune cells via multiple receptors including Toll-like receptors (TLR), Nod-like receptors (NLR) and the receptor for advanced glycationend products (RAGE). Pulmonary fibrosis is associated with the upregulation of alarmins such as Interleukin 1 α (IL-1α) and High mobility group box 1 (HMGB1) in bronchoalveolar lavage fluid (BAL), however it remains unclear whether alarmins can contribute directly to the fibrogenic process by interacting with fibroblasts. We hypothesised that alarmins released from damaged epithelial cells act as damage associated molecular patterns (DAMPs) which are recognised by fibroblasts and lead to their activation.
Methods The 16HBE14o- human bronchial epithelial cell line was damaged by hydrogen peroxide (H2O2) induced oxidative stress and alarmin release (Heat Shock Protein 60 (HSP-60), HMGB1, IL-1α) measured via ELISA. MRC5 human lung fibroblasts were treated with media from damaged lung epithelia and cell proliferation (XTT proliferation assay), phosphorylation of downstream TLR signalling molecules (interleukin 1 receptor associated kinase 1 (IRAK1), TGFβ associated kinase 1 (TAK1)—western blotting) and gene expression of proinflammatory cytokines (interleukin 6 (IL-6) and Interferon β (IFNβ)—qRT-PCR) assessed.
Results Conditioned media from 16HBE14o- cells damaged with 200 μM H2O2 contained elevated concentrations of HSP-60 (16.7 vs 0.64 ng/ml; p<0.05, n=3), HMGB1 (71 vs 12 ng/ml; p<0.001, n=3) and IL-1α (434 vs 130 pg/ml; p<0.001, n=3) compared to untreated controls. Treatment of MRC5 cells with media from damaged lung epithelia enhanced cell proliferation by 29% (p<0.01, n=3), increased TAK1 and IRAK1 phosphorylation and increased IL-6 and IFNβ gene expression 2.7-fold (p<0.001, n=3) and threefold (p<0.001, n=3) respectively. Blocking IL-1R (500 ng/ml of IL-1R antagonist (IL-1Ra)) diminished IL-6 (85%; p<0.01, n=3) and IFNβ (34%; p<0.05, n=3) gene expression compared to treatment with conditioned media from damaged cells alone.
Conclusions The results suggest that alarmins such as the intrerleukin-1 family, released by damaged human lung epithelia may be implicated in activation of fibroblasts and could contribute to fibrogenic responses in lung disease. However, further studies are required to confirm relative importance within the family and reveal mechanisms of action.
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