T cells and eosinophils cooperate in the induction of bronchial epithelial cell apoptosis in asthma Axel Trautmann, MDa, Peter Schmid-Grendelmeier, MDa,

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Presentation transcript:

T cells and eosinophils cooperate in the induction of bronchial epithelial cell apoptosis in asthma Axel Trautmann, MDa, Peter Schmid-Grendelmeier, MDa, Katja Krüger, a, Reto Crameri, PhDa, Mübeccel Akdis, MDa, Ahmet Akkaya, MDb, Eva-B. Bröcker, MDc, Kurt Blaser, PhDa, Cezmi A. Akdis, MDa Journal of Allergy and Clinical Immunology Volume 109, Issue 2, Pages 329-337 (February 2002) DOI: 10.1067/mai.2002.121460 Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 1 A , Death receptor expression on ECs. ECs were cultured in the absence (Control) or presence of IFN-γ, TNF-α, or both cytokines. The percentage of stained ECs after 1 day is indicated. B , Immunoprecipitation and immunoblotting with EC lysates were performed with the same mAb. β-Catenin is a control for equivalent gel loading. C , Gene expression array. cDNA probes of EC stimulated with 10 ng/mL IFN-γ and 10 ng/mL TNF-α were hybridized to gene-specific cDNA fragments spotted on GEArray membranes in duplicates. D , Sensitivity of ECs to apoptosis-inducing stimuli. ECs were treated for 3 days with apoptosis-inducing anti-Fas mAb, TNF-α, TRAIL, and TWEAK either in the absence (Alone) or presence of IFN-γ. EC viability (%) was determined by ethidium bromide exclusion and flow cytometry. Results shown in A-D represent 3 experiments. *P < .05. Journal of Allergy and Clinical Immunology 2002 109, 329-337DOI: (10.1067/mai.2002.121460) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 2 Anti-IFN-γ and anti-TNF-α mAb inhibit EC apoptosis induced by stimulated T cells. A , Phase contrast morphology (×200) of ECs 3 days after exposure to supernatants from unstimulated and stimulated CD45RO+ T cells. In parallel, ECs were stained with Hoechst (×400) to visualize all nuclei. B , Phase contrast morphology (×400) of ECs after 3 days direct coculture with unstimulated and stimulated CD45RO+ T cells. A, B, EC viability (%) was determined by ethidium bromide exclusion and flow cytometry. Mean ± SD of four separate experiments are shown in parentheses. *P < .05. Journal of Allergy and Clinical Immunology 2002 109, 329-337DOI: (10.1067/mai.2002.121460) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 3 A , Anti-TNF-α mAb inhibits EC apoptosis induced by stimulated eosinophils. Phase contrast morphology (×200) of ECs pretreated with 10 ng/mL IFN-γ 3 days after exposure to lysates from unstimulated and stimulated eosinophils. In parallel, ECs were stained with Hoechst (×400) to visualize all nuclei. B , ECP-mediated EC necrosis. Phase contrast morphology (×400) of ECs 6 hours after exposure to the indicated concentrations of ECP. C , Annexin V/propidiumiodide staining of ECs 6 hours after exposure to 10 μg/mL ECP indicate necrotic cells in quadrant 4. A, B , EC viability (%) was determined by ethidium bromide exclusion and flow cytometry. Mean ± SD of 3 separate experiments are shown in parentheses. *P < .05. Journal of Allergy and Clinical Immunology 2002 109, 329-337DOI: (10.1067/mai.2002.121460) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 4 Demonstration of apoptosic features of ECs. A , Agarose gel electrophoresis of EC DNA. ECs were cultured for 3 days with supernatants from stimulated CD45RO+ T cells or with lysates from unstimulated and stimulated eosinophils. Control, ECs alone. B , ECs were cultured with supernatants from T cells and lysates from eosinophils. The percentage of annexin V-stained ECs after 3 days is indicated. Results shown in A and B represent 3 experiments. *P < .05. Journal of Allergy and Clinical Immunology 2002 109, 329-337DOI: (10.1067/mai.2002.121460) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 5 Demonstration of apoptotic ECs in asthma. A , In addition to routine HE staining, sections were subjected to TUNEL with hematoxylin counterstaining and Hoechst staining. A , Normal bronchial mucosa as a negative control. B and C, Red nuclei indicate positive TUNEL staining; condensed and bright nuclei indicate positive Hoechst staining of apoptotic ECs in asthma. B through D , Note the detachment of apoptotic ECs with almost complete exfoliation and only a few viable residual basal cells. Results are representative of 7 asthma specimens. Journal of Allergy and Clinical Immunology 2002 109, 329-337DOI: (10.1067/mai.2002.121460) Copyright © 2002 Mosby, Inc. Terms and Conditions