Production of IL-5 and granulocyte-macrophage colony-stimulating factor by naive human mast cells activated by high-affinity IgE receptor ligation Robert.

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Presentation transcript:

Production of IL-5 and granulocyte-macrophage colony-stimulating factor by naive human mast cells activated by high-affinity IgE receptor ligation Robert B. Bressler, MDa,b, John Leskob, Margaret L. Jonesb, Matthew Wassermanb, Richard R. Dickason, PhDb, Marilyn M. Huston, PhDb, Susan W. Cook, PhDb, David P. Huston, MDa,b Journal of Allergy and Clinical Immunology Volume 99, Issue 4, Pages 508-514 (April 1997) DOI: 10.1016/S0091-6749(97)70078-2 Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 1 Human mast cells derived from long-term bone marrow MNC cultures maintained with SCF. A, Wright-Giemsa stain of a clump of cells from a 16-week-old culture. Mast cells are identified by dark staining of their numerous cytoplasmic granules. The much larger, pale-staining cells are histiocytic-appearing non-mast cells. B, Toluidine blue stain of cultured mast cells in which cytoplasmic granules exhibit meta chromasia. C, Dual immunohistochemical staining for tryptase and chymase. MCT stain blue, and MCTC stain brown-red. D, Microfluorimetric analysis of cultured human mast cells dual-stained for CD117 (y axis) and IgE binding (x axis). Journal of Allergy and Clinical Immunology 1997 99, 508-514DOI: (10.1016/S0091-6749(97)70078-2) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 1 Human mast cells derived from long-term bone marrow MNC cultures maintained with SCF. A, Wright-Giemsa stain of a clump of cells from a 16-week-old culture. Mast cells are identified by dark staining of their numerous cytoplasmic granules. The much larger, pale-staining cells are histiocytic-appearing non-mast cells. B, Toluidine blue stain of cultured mast cells in which cytoplasmic granules exhibit meta chromasia. C, Dual immunohistochemical staining for tryptase and chymase. MCT stain blue, and MCTC stain brown-red. D, Microfluorimetric analysis of cultured human mast cells dual-stained for CD117 (y axis) and IgE binding (x axis). Journal of Allergy and Clinical Immunology 1997 99, 508-514DOI: (10.1016/S0091-6749(97)70078-2) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 1 Human mast cells derived from long-term bone marrow MNC cultures maintained with SCF. A, Wright-Giemsa stain of a clump of cells from a 16-week-old culture. Mast cells are identified by dark staining of their numerous cytoplasmic granules. The much larger, pale-staining cells are histiocytic-appearing non-mast cells. B, Toluidine blue stain of cultured mast cells in which cytoplasmic granules exhibit meta chromasia. C, Dual immunohistochemical staining for tryptase and chymase. MCT stain blue, and MCTC stain brown-red. D, Microfluorimetric analysis of cultured human mast cells dual-stained for CD117 (y axis) and IgE binding (x axis). Journal of Allergy and Clinical Immunology 1997 99, 508-514DOI: (10.1016/S0091-6749(97)70078-2) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 1 Human mast cells derived from long-term bone marrow MNC cultures maintained with SCF. A, Wright-Giemsa stain of a clump of cells from a 16-week-old culture. Mast cells are identified by dark staining of their numerous cytoplasmic granules. The much larger, pale-staining cells are histiocytic-appearing non-mast cells. B, Toluidine blue stain of cultured mast cells in which cytoplasmic granules exhibit meta chromasia. C, Dual immunohistochemical staining for tryptase and chymase. MCT stain blue, and MCTC stain brown-red. D, Microfluorimetric analysis of cultured human mast cells dual-stained for CD117 (y axis) and IgE binding (x axis). Journal of Allergy and Clinical Immunology 1997 99, 508-514DOI: (10.1016/S0091-6749(97)70078-2) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 2 Kinetics for IL-5, GM-CSF, and IL-3 mRNA upregulation after FcϵRI ligation of IgE-sensitized mast cells. RNA was extracted from cultured mast cells at indicated time points. PCR was performed with IL-5–, GM-CSF–, IL-3–, and HPRT-specific primers in the presence of α32P deoxycytidine triphosphate. After polyacrylamide gel electrophoresis, PCR products were visualized by ethidium bromide staining, and radioactivity of cytokine bands was quantitated by Betascope analysis. U, Unstimulated (IgE-sensitized mast cells + anti-IgG antibody); S, stimulated (IgE-sensitized mast cells + anti-IgE antibody). Radioactivity is expressed as total counts and appears directly beneath the corresponding PCR product. Parenthetical data are ratio of cytokine counts to HPRT! (control message) counts. Journal of Allergy and Clinical Immunology 1997 99, 508-514DOI: (10.1016/S0091-6749(97)70078-2) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 2 Kinetics for IL-5, GM-CSF, and IL-3 mRNA upregulation after FcϵRI ligation of IgE-sensitized mast cells. RNA was extracted from cultured mast cells at indicated time points. PCR was performed with IL-5–, GM-CSF–, IL-3–, and HPRT-specific primers in the presence of α32P deoxycytidine triphosphate. After polyacrylamide gel electrophoresis, PCR products were visualized by ethidium bromide staining, and radioactivity of cytokine bands was quantitated by Betascope analysis. U, Unstimulated (IgE-sensitized mast cells + anti-IgG antibody); S, stimulated (IgE-sensitized mast cells + anti-IgE antibody). Radioactivity is expressed as total counts and appears directly beneath the corresponding PCR product. Parenthetical data are ratio of cytokine counts to HPRT! (control message) counts. Journal of Allergy and Clinical Immunology 1997 99, 508-514DOI: (10.1016/S0091-6749(97)70078-2) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 2 Kinetics for IL-5, GM-CSF, and IL-3 mRNA upregulation after FcϵRI ligation of IgE-sensitized mast cells. RNA was extracted from cultured mast cells at indicated time points. PCR was performed with IL-5–, GM-CSF–, IL-3–, and HPRT-specific primers in the presence of α32P deoxycytidine triphosphate. After polyacrylamide gel electrophoresis, PCR products were visualized by ethidium bromide staining, and radioactivity of cytokine bands was quantitated by Betascope analysis. U, Unstimulated (IgE-sensitized mast cells + anti-IgG antibody); S, stimulated (IgE-sensitized mast cells + anti-IgE antibody). Radioactivity is expressed as total counts and appears directly beneath the corresponding PCR product. Parenthetical data are ratio of cytokine counts to HPRT! (control message) counts. Journal of Allergy and Clinical Immunology 1997 99, 508-514DOI: (10.1016/S0091-6749(97)70078-2) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 2 Kinetics for IL-5, GM-CSF, and IL-3 mRNA upregulation after FcϵRI ligation of IgE-sensitized mast cells. RNA was extracted from cultured mast cells at indicated time points. PCR was performed with IL-5–, GM-CSF–, IL-3–, and HPRT-specific primers in the presence of α32P deoxycytidine triphosphate. After polyacrylamide gel electrophoresis, PCR products were visualized by ethidium bromide staining, and radioactivity of cytokine bands was quantitated by Betascope analysis. U, Unstimulated (IgE-sensitized mast cells + anti-IgG antibody); S, stimulated (IgE-sensitized mast cells + anti-IgE antibody). Radioactivity is expressed as total counts and appears directly beneath the corresponding PCR product. Parenthetical data are ratio of cytokine counts to HPRT! (control message) counts. Journal of Allergy and Clinical Immunology 1997 99, 508-514DOI: (10.1016/S0091-6749(97)70078-2) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 3 IL-5 and GM-CSF protein secretion by cultured mast cells after FcϵRI ligation. Cytokine levels were measured in supernatants of IgE-sensitized mast cells at indicated time points after stimulation with anti-IgG antibody (open symbols) or anti-IgE antibody (filled symbols). Data points are expressed as means ± SEM. A, Kinetics of IL-5 production from a culture comprised of 93% mast cells, of which 60% were MCT and 40% were MCTC. Sensitized mast cells from this culture demonstrated a net histamine release of 58% after FcϵRI ligation. Similar kinetics for IL-5 protein production were obtained in three experiments with mast cells derived from independent cultures. B, Kinetics of GM-CSF protein produc! tion from two separate mast cell cultures. Culture 1 (squares) was the same culture as described above for kinetics of IL-5 protein production. In culture 2 (filled circles), stimulation was limited to anti-IgE antibody because cell numbers were insufficient for generation of control supernatants by stimulation with anti-IgG antibody. Culture 2 was comprised of 88% mast cells, of which 60% were MCT and 40% were MCTC. Journal of Allergy and Clinical Immunology 1997 99, 508-514DOI: (10.1016/S0091-6749(97)70078-2) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 3 IL-5 and GM-CSF protein secretion by cultured mast cells after FcϵRI ligation. Cytokine levels were measured in supernatants of IgE-sensitized mast cells at indicated time points after stimulation with anti-IgG antibody (open symbols) or anti-IgE antibody (filled symbols). Data points are expressed as means ± SEM. A, Kinetics of IL-5 production from a culture comprised of 93% mast cells, of which 60% were MCT and 40% were MCTC. Sensitized mast cells from this culture demonstrated a net histamine release of 58% after FcϵRI ligation. Similar kinetics for IL-5 protein production were obtained in three experiments with mast cells derived from independent cultures. B, Kinetics of GM-CSF protein produc! tion from two separate mast cell cultures. Culture 1 (squares) was the same culture as described above for kinetics of IL-5 protein production. In culture 2 (filled circles), stimulation was limited to anti-IgE antibody because cell numbers were insufficient for generation of control supernatants by stimulation with anti-IgG antibody. Culture 2 was comprised of 88% mast cells, of which 60% were MCT and 40% were MCTC. Journal of Allergy and Clinical Immunology 1997 99, 508-514DOI: (10.1016/S0091-6749(97)70078-2) Copyright © 1997 Mosby, Inc. Terms and Conditions