Granulocyte-macrophage colony-stimulating factor and IL-5 activate mitogen-activated protein kinase through Jak2 kinase and phosphatidylinositol 3-kinase.

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Granulocyte-macrophage colony-stimulating factor and IL-5 activate mitogen-activated protein kinase through Jak2 kinase and phosphatidylinositol 3-kinase inhuman eosinophils Kazuhiro Hiraguri, MD, Satoshi Miike, MD, Hideki Sano, MD, Kazuhiro Kurasawa, MD, Yasushi Saito, MD, Itsuo Iwamoto, MD Journal of Allergy and Clinical Immunology Volume 100, Issue 6, Pages S45-S49 (December 1997) DOI: 10.1016/S0091-6749(97)70004-6 Copyright © 1997 Mosby, Inc. Terms and Conditions

FIG. 1 Time course (upper panel) and dose dependence (lower panel) of GM-CSF–induced activation of MAP kinase in human eosinophils. Upper panel, Human eosinophils were stimulated with GM-CSF (10-10 mol/L) at 37° C for 1 to 120 minutes. Cell lysates were immunoblotted with anti-MAP kinase antibody (erk1-CT). The position of MAP kinase isoforms at 40, 42, and 44 kd and their phosphorylated forms (*) are shown by the arrows. Lower panel, Eosinophils were stimulated with GM-CSF (10-12 to 10-10 mol/L) at 37° C for 3 minutes. Cell lysates were immunoblotted with anti–MAP kinase antibody. Journal of Allergy and Clinical Immunology 1997 100, S45-S49DOI: (10.1016/S0091-6749(97)70004-6) Copyright © 1997 Mosby, Inc. Terms and Conditions

FIG. 2 IL-5 induces MAP kinase activation in human eosinophils. Eosinophils were stimulated with IL-5 (10-11 to 10-9 mol/L) at 37° C for 3 minutes. Cell lysates were immunoblotted with anti–MAP kinase antibody. PMA, phorbol myristate acetate. Journal of Allergy and Clinical Immunology 1997 100, S45-S49DOI: (10.1016/S0091-6749(97)70004-6) Copyright © 1997 Mosby, Inc. Terms and Conditions

FIG. 3 In-gel kinase assay of GM-CSF– and IL-5–induced MAP kinase activation in human eosinophils. Eosinophils were stimulated with GM-CSF (10-12 to 10-10 mol/L) or IL-5 (10-11 to 10-9 mol/L) for 3 minutes, and MAP kinase activity was determined on the basis of 32P incorporation into myelin basic protein, a substrate of MAP kinase. The autoradiograms show that GM-CSF (10-10 mol/L) and IL-5 (10-9 mol/L) induce phosphorylation of major basic protein at 40 to 44 kd. PMA, phorbol myristate acetate. Journal of Allergy and Clinical Immunology 1997 100, S45-S49DOI: (10.1016/S0091-6749(97)70004-6) Copyright © 1997 Mosby, Inc. Terms and Conditions

FIG. 4 Effect of Jak2 kinase inhibitor AG-490 on GM-CSF–induced MAP kinase (MAPK) activation in human eosinophils. Eosinophils were preincubated with or without AG-490 (10-7 to 10-5 mol/L) for 30 minutes and stimulated with or without GM-CSF (10-10 mol/L) for 3 minutes. The data shown are representative of three experiments. Journal of Allergy and Clinical Immunology 1997 100, S45-S49DOI: (10.1016/S0091-6749(97)70004-6) Copyright © 1997 Mosby, Inc. Terms and Conditions

FIG. 5 Effect of tyrosine kinase inhibitor genistein on GM-CSF–induced MAP kinase (MAPK) activation in human eosinophils. Eosinophils were preincubated with or without genistein (10-7 to 10-5 mol/L) for 15 minutes and stimulated with or without GM-CSF (10-10 mol/L) for 3 minutes. The data shown are representative of four experiments. Journal of Allergy and Clinical Immunology 1997 100, S45-S49DOI: (10.1016/S0091-6749(97)70004-6) Copyright © 1997 Mosby, Inc. Terms and Conditions

FIG. 6 Effect of a PI 3-kinase inhibitor wortmannin on GM-CSF–induced MAP kinase (MAPK) activation in human eosinophils. Eosinophils were preincubated with or without wortmannin (10-8 to 10-6 mol/L) for 15 minutes and stimulated with or without GM-CSF (10-10 mol/L) for 3 minutes. The data shown are representative of four experiments. Journal of Allergy and Clinical Immunology 1997 100, S45-S49DOI: (10.1016/S0091-6749(97)70004-6) Copyright © 1997 Mosby, Inc. Terms and Conditions

FIG. 7 Effect of a protein kinase C inhibitor staurosporine on GM-CSF–induced MAP kinase (MAPK) activation in human eosinophils. Eosinophils were preincubated with or without staurosporine (10-10 to 10-8 mol/L) for 15 minutes and then stimulated with or without GM-CSF (10-10 mol/L) for 3 minutes. The data shown are representative of four experiments. Journal of Allergy and Clinical Immunology 1997 100, S45-S49DOI: (10.1016/S0091-6749(97)70004-6) Copyright © 1997 Mosby, Inc. Terms and Conditions