Transforming Growth Factor-β1 Antagonizes Interleukin-5 Pro-Survival Signaling by Activating Calpain-1 in Primary Human Eosinophils.

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.