Inflammatory responses of human eosinophils to cockroach are mediated through protease-dependent pathways Kota Wada, MD, Yoshinori Matsuwaki, MD, PhD,

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Presentation transcript:

Inflammatory responses of human eosinophils to cockroach are mediated through protease-dependent pathways Kota Wada, MD, Yoshinori Matsuwaki, MD, PhD, Juhan Yoon, PhD, Linda M. Benson, BS, James L. Checkel, Theresa A. Bingemann, MD, Hirohito Kita, MD Journal of Allergy and Clinical Immunology Volume 126, Issue 1, Pages 169-172.e2 (July 2010) DOI: 10.1016/j.jaci.2010.04.007 Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 The eosinophil-stimulating activities of cockroach extracts are heat-sensitive. German and Oriental cockroach extracts (50 μg/mL) were heat-treated at 37, 56, and 100°C or kept at 4°C for 30 minutes. A, Human eosinophils were incubated with these stimuli for 3 hours at 37°C; EDN concentrations in the cell-free supernatants were measured by ELISA. B, Eosinophils were incubated with stimuli for 24 hours at 37°C; IL-8 concentrations were measured by ELISA. Results show the means ± SEMs from 5 (A) or 4 (B) different donors. Significant differences compared with 4°C–treated extract (∗P < .05; ∗∗P < .01). #Significant difference compared with IL-8 production with medium alone. Journal of Allergy and Clinical Immunology 2010 126, 169-172.e2DOI: (10.1016/j.jaci.2010.04.007) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Cockroach extracts have pepstatin-sensitive aspartate protease activity that is involved in PAR-2 cleavage and eosinophil degranulation. A, German and Oriental cockroach extracts (10 or 50 μg/mL) and trypsin (1 nmol/L) were incubated with a PAR-2 fluorogenic peptide substrate, Abz-SKGRSLIGKdD (50 μmol/L), for 60 minutes at 37°C. The figure is representative of 3 experiments having similar results. B, German (5 μg/mL) and Oriental (50 μg/mL) cockroach extracts or trypsin (1 nmol/L) were incubated with pepstatin A agarose, control agarose, or medium only for 15 minutes at 4°C. After centrifugation, supernatants were incubated with Abz-SKGRSLIGKdD (50 μmol/L) as described. Results show the means ± SEMs from 3 experiments. ∗Significant differences compared with nontreated extracts (P < .05). C, German (5 μg/mL) and Oriental (50 μg/mL) cockroach extracts or trypsin (1 nmol/L) were incubated with buffer alone (control), 200 μmol/L (4-Amidinophenyl)-methanesulfonyl (APMSF) or 10 μmol/L E64 for 30 minutes at room temperature and were incubated with Abz-SKGRSLIGKdD (50 μmol/L) as described. Results show the means ± SEMs from 3 experiments. ∗Significant differences compared with control-treated extracts (P < .05). D, Cockroach extracts (50 μg/mL) and phorbol 12-myristate 13-acetate (PMA; 1 ng/mL) were preincubated with pepstatin A agarose, control agarose, or medium for 15 minutes at 4°C. Eosinophils were incubated with these treated extracts for 3 hours at 37°C. EDN concentrations were measured by ELISA. Results show the means ± SEMs from 6 donors. ∗Significant differences compared with nontreated extracts (P < .05). Journal of Allergy and Clinical Immunology 2010 126, 169-172.e2DOI: (10.1016/j.jaci.2010.04.007) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 German cockroach extract cleaves PAR-2 peptide substrate between arginine and serine. German cockroach extract samples were pretreated with pepstatin A agarose or control agarose or were untreated for 15 minutes at 4°C. Next, the fluorogenic peptide substrate, Abz-SKGRSLIGKdD, was incubated for 30 minutes at 37°C with these cockroach samples (10 μg/mL) or trypsin (1 nmol/L). Peptide products were analyzed by reverse-phase–HPLC-ESI-MS. The results are representative of 4 different experiments. Journal of Allergy and Clinical Immunology 2010 126, 169-172.e2DOI: (10.1016/j.jaci.2010.04.007) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Cockroach extracts induce eosinophil degranulation and superoxide production from eosinophils. Eosinophils were incubated with German and Oriental cockroach extracts (0-200 μg/mL) at 37°C. A, Eosinophil degranulation was detected after 3 hour of incubation by quantitating EDN concentrations in the cell-free supernatants by ELISA. B, Superoxide production was detected by the reduction of cytochrome c during the first 60 minutes of incubation. Results show the means ± SEMs from 4 different donors. Significant differences compared with medium alone (∗P < .05; ∗∗P < .01). Journal of Allergy and Clinical Immunology 2010 126, 169-172.e2DOI: (10.1016/j.jaci.2010.04.007) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions