Browsing Electronic Theses and Dissertations by Author "Ackerman, Steven J"
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ItemThe Eosinophil Granular Leukocyte; Source and Target of High Mobility Group Protein B1Straub, Christof; Goldblum, Randall M; Ackerman, Steven J; Brasier, Allan R; Konig, Rolf; Kurosky, Alexander; Pazdrak, KonradEosinophils are associated with various inflammatory diseases in which they participate through the release of mediators. Eosinophils thereby function as effectors and immunoregulators and contribute to inflammation-associated disease symptoms. The high mobility group protein B1 (HMGB1) was discovered in the nuclear fraction of non-activated peripheral blood eosinophils through a proteomic profiling project. The protein was identified by MALDI-TOF/TOF and its presence was confirmed by Western blot analysis. Given recent reports of HMGB1’s proinflammatory capabilities and a lack of knowledge regarding its role in eosinophilic disorders, we hypothesized that eosinophil-derived HMGB1 may affect airway cells and thus be involved in the pathogenesis of eosinophilic disorders such as asthma and allergy. The subcellular expression pattern of HMGB1 was characterized. HMGB1 accumulated in the cytoplasmic fraction of eosinophils after stimulation with proinflammatory mediators, e.g. GM-CSF. Upon prolonged cytokine exposure, HMGB1 was released by eosinophils into the cell culture medium. HMGB1 release by eosinophils was thus time-dependent and cell-activation dependent. Secreted HMGB1 (sHMGB1) from activated eosinophilic cells was subsequently purified and its posttranslational modifications analyzed. We detected serine phosphorylations and lysine acetylations by Western blot analysis and ESI LTQ Orbitrap mass spectrometry was used to assign two acetylation sites to residues Lys90 and Lys114. sHMGB1 was used for cell functional studies. It induced phosphorylation of p38 MAPK and NF-κB p65 in human monocytes and led proinflammatory mediator release. In addition, sHMGB1 exerted significant chemotactic properties toward neutrophils and eosinophils. Eosinophils also responded in autocrine fashion by degranulation. Moreover, sHMGB1 caused the specific release of VEGF from bronchial epithelial cells, an important cytokine in asthma pathogenesis. My studies presented here demonstrated for the first time that activated eosinophils can be a source of HMGB1 and documented cytoplasmic accumulation and post-translational modification of in eosinophils activated by exogenous cytokines. sHMGB1 can affect immune cells and resident airway cells in proinflammatory fashion. The in vitro findings presented here support the proinflammatory role of activated eosinophils in allergy and asthma and complement recent translational studies correlating asthma severity and eosinophil lung infiltration with elevated sputum and serum HMGB1 levels in asthmatics.