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Background Eosinophils rapidly undergo apoptosis unless exposed to prosurvival cytokines such as interleukin 5 (IL-5) or granulocyte-macrophage colony stimulating factor (GM-CSF). In vivo, eosinophils are exposed to TGF-β 1 which can induce apoptosis suggesting it may function to counteract the effects of IL-5 or GM-CSF and limit, in vivo tissue eosinophilia. Objective The objective of this study was to investigate the proapoptotic effects of TGF-β alone and in combination with IL-5 on eosinophils. Methods Peripheral blood eosinophil (PBEos) viability was assessed using flow cytometry after exposure to TGF-β1 and IL-5. Calpain-1 activation was determined in cell extracts by western blot analysis of endogenous substrates and with a fluorogenic α-spectrin substrate. Molecular interactions between calpain1 and calpastatin were assessed by immunoprecipitation and western blotting. Results Physiologic concentrations of TGF-β1 significantly antagonized the prosurvival effects of IL-5. TGF-β1-induced apoptosis was suppressed by inhibitors of calpain, or its downstream target, caspase 3. TGF-β1 signaling through Smad3 was unaffected by IL-5 and was required for the pro-apoptotic effects of TGF-β1. However, IL-5 induced Akt phosphorylation was inhibited by TGF-β1 and was associated with accelerated calpain cleavage and eosinophil death. Conclusion TGF-β1 induces calpain-1 activation through antagonism of Akt which induces caspase activation and eosinophil apoptosis.

Normal pulmonary function requires precise control over the content and composition of the extracellular matrix (ECM). During the pathogenesis of lung fibrosis, a net increase in ECM content displaces epithelial cells and causes severe reductions of pulmonary function. This process is often associated with elevated levels of TGF-β1 which enhances ECM production by fibroblasts. However, the signaling mechanisms that coordinate ECM production remain unclear. Here, we show that Pin1, a peptidyl-prolyl isomerase which isomerizes pSer/pThr-Pro peptide bonds, reciprocally controls the production of pro- and anti-fibrogenic ECM. Pin1 knockout mice treated with bleomycin, which induces pulmonary fibrosis by elevating TGF-β1, showed significantly attenuated expression of collagens (I, III and V), TIMPs (TIMP1 and 4) and fibrogenic cytokines (TGF-β1, IL-1β and CTGF) but increased MMPs (MMP2 and 3). In primary lung fibroblasts, Pin1 was required for TGF-β1-induced phosphorylation and subsequent nuclear transactivation of Smad3. These functions were mediated by TGF-β1 dependent interactions of Pin1 with inhibitory Smad6. In Pin1 knockout cells, Smad6 remained predominantly cytoplasmic, likely attenuating Smad3 phosphorylation and transcriptional activity. These data support and extend our previous observations that Pin1 blockade in vivo significantly reduced total collagen deposition in the lungs of animal models of asthma and lung transplantation.

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