TGF-β1-induced Chloride Channel Activity and Migration of Human Eosinophils
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Authors
Moore, Benjamin
Issue Date
2009-05
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Thesis
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en_US
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Abstract
Asthma is a chronic disease of the airways characterized by airway hyperresponsiveness, airway inflammation, and airway remodeling. Eosinophils migrate to the airways and play a significant role in the pathogenesis of asthma, particularly through their release of mediators and cytokines. Transforming growth factor (TGF)-β also induces asthmatic features in the lungs, especially airway remodeling. Eosinophils and TGF-β are closely linked to each other in the induction of airway remodeling. TGF-β activity also affects chloride channel activity, which can regulate mediator release and cell migration. Therefore,the role that TGF-β1 stimulation of eosinophils has on chloride channel activity and migration of eosinophils and the underlying mechanisms involved in this process were examined.|Human blood eosinophils were stimulated with TGF-β1, the tyrosine kinase inhibitor genistein, the protein kinase C (PKC)-δ/ε inhibitor rottlerin, the general PKC inhibitor staurosporine, and the chloride channel inhibitors 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). Protein expression of the chloride channel (CLC) family of chloride channels, PKC isozymes, and Smad transcription factors was determined using western blot, and mRNA expression of CLC was examined using RT-PCR. Chloride channel activity was measured by patch clamping, and eosinophil chemotaxis was assessed using a Boyden microchemotaxis chamber. Flow cytometry was used to determine shape change.|The results showed that TGF-β1 stimulation increased protein expression of ClC-3. There was also more CLC-3 protein expression in asthmatic eosinophils than normal eosinophils. TGF-β1 increased protein expression of PKC-β1, PKC-δ, and phospho-Smad3. TGF-β1 increased the chloride channel activity of the eosinophils, which was blocked by rottlerin. TGF-β1 induced chemotaxis of eosinophils, which was blocked by rottlerin, DIDS and NPPB. Eosinophil shape change occurred after TGF-β1 stimulation, and both DIDS and NPPB inhibited morphological changes of the cells. Rottlerin, as well as staurosporine, decreased TGF-β1-induced phosphorylation of Smad3. Genistein was more robust at reducing Smad3 phosphorylation than rottlerin or staurosporine.|These results indicate that TGF-β1 increases the chloride channel activity of eosinophils by upregulating CLC-3 via a pathway dependent on PKC (particularly PKC-δ) and possibly Smad3. TGF-β1 induced chloride channel activity also induces eosinophil migration and shape change via a PKC-δ-dependent pathway. These data offer new insights into a TGF- β1-dependent mechanism that may increase eosinophil infiltration and activation, thereby inducing airway remodeling.
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Creighton University
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Moore-thesis.pdf