Inhibition of protein kinases A and C demonstrates dual modes of response in human eosinophils stimulated with platelet-activating factor Takumi Takizawa,

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Presentation transcript:

Inhibition of protein kinases A and C demonstrates dual modes of response in human eosinophils stimulated with platelet-activating factor Takumi Takizawa, MD, PhD, Masahiko Kato, MD, PhD, Hirokazu Kimura, PhD, Masato Suzuki, MD, Atsushi Tachibana, MD, Hideru Obinata, MS, Takashi Izumi, MD, PhD, Kenichi Tokuyama, MD, PhD, Akihiro Morikawa, MD, PhD Journal of Allergy and Clinical Immunology Volume 110, Issue 2, Pages 241-248 (August 2002) DOI: 10.1067/mai.2002.126303 Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 1 Effects of protein kinase inhibitors and a cAMP analogue on PAF-induced CD11b expression in human eosinophils. Purified eosinophils (2.5 × 105 cells/sample) were stimulated with or without 1 μmol/L PAF after pretreatment with staurosporine (A), Bis I (B), H89 (C), or db-cAMP (D ; solid bar, db-cAMP alone; hatched bar, db-cAMP and H89; and shaded bar, db-cAMP and Bis I), or medium alone. CD11b expression was determined by means of flow cytometric analysis and described as mean fluorescence intensity (MFI) . Mean fluorescence intensity of control cells with or without PAF stimulation was 28.3 ± 1.63 and 10.9 ± 0.78, respectively (mean ± SEM, n = 7). *P < .05, **P < .01. Journal of Allergy and Clinical Immunology 2002 110, 241-248DOI: (10.1067/mai.2002.126303) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 2 Effects of PTX and herbimycin A on PAF-induced CD11b expression. Eosinophils were stimulated with PAF (1 μmol/L) after preincubation with PTX (A), herbimycin A (B), or medium alone. CD11b expression was determined as described in Fig 1. MFI, Mean fluorescence intensity. *P < .05, **P < .01. Journal of Allergy and Clinical Immunology 2002 110, 241-248DOI: (10.1067/mai.2002.126303) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 3 Role of PKC or PKA in PAF-induced cellular adhesion of human eosinophils. Eosinophils (5 × 104/well) were incubated with or without 1 μmol/L PAF after preincubation with Bis I (A), H89 (B), or medium alone. Percentage adhesion of control cells with or without PAF stimulation is 12.4% ± 2.40% and 6.58% ± 1.23%, respectively (mean ± SEM, n = 3). *P < .05. Journal of Allergy and Clinical Immunology 2002 110, 241-248DOI: (10.1067/mai.2002.126303) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 4 Reduction of O2− generation from PAF-stimulated human eosinophils by means of inhibition of PKC or PKA. Purified eosinophils (2.5 × 105 cells/sample) were incubated with or without 1 μmol/L PAF after preincubation with Bis I (A), myr-ψPKC (B), H89 (C), or medium alone. The O2− generation in control cells stimulated by PAF is 367.4 ± 129.8 × 104 counts (mean ± SEM, n = 6). **P < .01. Journal of Allergy and Clinical Immunology 2002 110, 241-248DOI: (10.1067/mai.2002.126303) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 5 Suppression of PAF-induced degranulation from eosinophils by inhibiting PKA. Eosinophils (5 × 104 cells/well) were incubated for 4 hours with or without PAF (1 μmol/L) after preincubation with Bis I (A), H89 (B), or medium alone. Concentrations of EPX in the supernatants were determined. The amounts of EPX release in control cells with or without PAF stimulation and total cells are 335.5 ± 251.2, 89.9 ± 59.9, and 1798.5 ± 512.7 ng/106 cells, respectively (mean ± SEM, n = 3). **P < .01. Journal of Allergy and Clinical Immunology 2002 110, 241-248DOI: (10.1067/mai.2002.126303) Copyright © 2002 Mosby, Inc. Terms and Conditions