Allergic sensitivity to the aeroallergen, Alternaria, has been linked to the development and persistence of asthma. Sensitivity to an allergen, like Alternaria, is classically thought to be regulated via an IgE mediated pathway and IgE itself has been shown to be associated with asthma. This link between asthma and IgE is not always evident; hence, it is possible that the activation of inflammatory responses due to allergen exposure in susceptible individuals occurs via a non-IgE mediated pathway. The capacity of Alternaria to activate innate immune response directly from airway epithelial cells is not well characterized. In addition, proteases associated with allergens have been suggested as contributors to asthma pathogenesis. Hence, this study hypothesizes that Alternaria alternata directly activates inflammatory mediators from human airway epithelial cells via a mechanism that is independent of IgE and involves Alternaria-derived proteases. Alternaria was shown to contain measurable protease activity and capable of dose-dependently inducing an early inflammatory mediator response from airway epithelial cells (16HBE14o-). Mediators that were induced included IL-6, IL-8, TNF-α, IL-1α, FGF-basic, GMCSF, GCSF, IL-1ra and VEGF. Overall, these responses were shown to be driven by heat-labile protein constituents of Alternaria with some of the mediators being inhibited in the presence of a serine specific protease inhibitor, AEBSF. Protease constituents of Alternaria were also shown to have direct inhibitory effects on (cell-free) ENA-78 and RANTES. Furthermore, Alternaria was shown to be capable of cleaving the PAR-2 receptor which is abundantly expressed on 16HBE14o-cells. This cleavage was partially inhibited using AEBSF and abolished by heat-inactivating Alternaria. Supporting the involvement of PAR-2 in these Alternaria-induced responses, PAR-2 agonist, SLIGRL-NH2, also elicited a response which was impressively similar to that of Alternaria stimulation. PAR-2 antagonist, FSLLRY-NH2 was able to antagonize some Alt-induced responses but the efficacy of this antagonist is questionable. The use of low PAR-2 expressing HeLa cells, also demonstrated that minimal PAR-2 expression is necessary to induce a PAR-2 linked inflammatory response. Overall, Alternaria-induced inflammatory responses from human airway epithelial cells is due to heat-labile constituents at least some of which have protease activity that acts via a PAR-2 mechanism.
|Commitee:||Bloom, John, Boitano, Scott, French, Edward, Lai, Josephine|
|School:||The University of Arizona|
|School Location:||United States -- Arizona|
|Source:||DAI-B 71/11, Dissertation Abstracts International|
|Subjects:||Molecular biology, Microbiology|
|Keywords:||Alternaria, Cytokine expression, Epithelial cells, Inflammatory responses, Proteases|
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