Reciprocal regulation of cultured human mast cell cytokine production by IL-4 and IFN-γ Hiroshi Tachimoto, MD, PhDa, Motohiro Ebisawa, MD, PhDa,b, Tomohide.

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Presentation transcript:

Reciprocal regulation of cultured human mast cell cytokine production by IL-4 and IFN-γ Hiroshi Tachimoto, MD, PhDa, Motohiro Ebisawa, MD, PhDa,b, Tomohide Hasegawa, a, Tomoko Kashiwabara, PhDc, Chisei Ra, MD, PhDd, Bruce S. Bochner, MDe, Katsushi Miura, MDe, Hirohisa Saito, MD, PhDa Journal of Allergy and Clinical Immunology Volume 106, Issue 1, Pages 141-149 (July 2000) DOI: 10.1067/mai.2000.107043 Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 1 Effect of IL-4 or IFN-γ on FcϵRI-mediated cytokine production by CHMCs. CHMCs were cultured with 0 to 10 ng/mL IL-4 (A-C) or IFN-γ (D-F) . Cells were incubated with (filled circles) or without (open circles) 1 μg/mL CRA-1 at 37°C for 6 hours. The concentrations of MIP-1α (A and D ), IL-8 (B and E ), and GM-CSF (C and F ) in the supernatants were measured by using ELISA. Values represent mean ± SEM of 4 separate experiments. *P < .05 compared with the control condition without IL-4 or IFN-γ. Journal of Allergy and Clinical Immunology 2000 106, 141-149DOI: (10.1067/mai.2000.107043) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 2 Effect of IL-4 or IFN-γ on the kinetics of FcϵRI-mediated cytokine production by CHMCs. CHMCs were cultured with 5 μg/mL IgE in the presence (IL-4, triangles ; IFN-γ, squares ) or absence (circles) of 10 ng/mL IL-4 (A-C) or IFN-γ (D-F) . Then cells were incubated with (filled symbols) or without (open symbols) 1 μg/mL CRA-1 at 37°C for 1, 3, 6, and 18 hours; supernatants were harvested; and concentrations of MIP-1α (A and D ), IL-8 (B and E ), and GM-CSF (C and F ) were determined. Values represent mean ± SEM of 4 separate experiments. *P < .05 compared with the control condition (without IL-4 or IFN-γ) at each time point. Journal of Allergy and Clinical Immunology 2000 106, 141-149DOI: (10.1067/mai.2000.107043) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 3 Kinetics of IL-4–induced enhancement of FcϵRI-mediated cytokine production by CHMCs. CHMCs were cultured with or without IL-4 (10 ng/mL, A-C ) or IFN-γ (10 ng/mL, D-F ) in the presence of SCF (100 ng/mL) and IL-6 (50 ng/mL) for 15 minutes and 1, 2, 4 or 8 days and human myeloma IgE (5 μg/mL) for 2 days. Then cells were incubated with (filled circles) or without (open circles) CRA-1 (1 μg/mL) at 37°C for 6 hours, supernatants were harvested, and concentrations of MIP-1α (A and D ), IL-8 (B and E ), and GM-CSF (C and F ) were measured. Values represent mean ± SEM of 4 separate experiments. *P < .05 compared with the control condition at day 0 with or without IL-4 or IFN-γ. Journal of Allergy and Clinical Immunology 2000 106, 141-149DOI: (10.1067/mai.2000.107043) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 4 Analysis of the effects of IL-4 and IFN-γ on expression of cytokine mRNA by using competitive RT-PCR. CHMCs were cultured with IL-4 (10 ng/mL) or IFN-γ (10 ng/mL) in the presence of SCF (100 ng/mL), IL-6 (50 ng/mL), and human myeloma IgE (5 μg/mL) for 2 days and then washed and challenged with 1 μg/mL CRA-1 at 37°C for 2 hours. cDNA levels for MIP-1α (A) , IL-8 (B) , GM-CSF (C) , and β-actin (D) are shown. M, Mimic cDNA; T, target cDNA. Lanes 1 and 8 indicate molecular weight markers. Lanes 2 to 7 show progressive 10-fold increments of mimic DNA spiked into the PCR tube with a constant amount of target cDNA. Journal of Allergy and Clinical Immunology 2000 106, 141-149DOI: (10.1067/mai.2000.107043) Copyright © 2000 Mosby, Inc. Terms and Conditions