IL-9 expression by human eosinophils: Regulation by IL-1β and TNF-α

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IL-9 expression by human eosinophils: Regulation by IL-1β and TNF-α Abdelilah Soussi Gounni, PhDa, Esra Nutku, MDa, Latifa Koussih, PhDa, Fadi Aris, Bsca, Jamila Louahed, PhDb, Roy C. Levitt, MDb, Nicholas C. Nicolaides, PhDb, Qutayba Hamid, MD, PhDa Journal of Allergy and Clinical Immunology Volume 106, Issue 3, Pages 460-466 (September 2000) DOI: 10.1067/mai.2000.109172 Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 1 Detection of IL-9 transcripts in human eosinophil preparations. RNA preparation from human peripheral blood eosinophils (asthmatic subjects, lanes 2 and 3 ; normal control subjects, lanes 4 and 5 ) and eosinophil-differentiated HL-60 cells (lane 6) showed an IL-9–specific amplified fragment. Lane 1 corresponds to a PCR tube performed without cDNA. Lane 7 corresponds to a 1-kb DNA ladder molecular weight. β-Actin was used as control. Total eosinophil RNA was isolated, and first-strand cDNA synthesis was performed. Human IL-9 was amplified by using PCR and IL-9–specific primers. Journal of Allergy and Clinical Immunology 2000 106, 460-466DOI: (10.1067/mai.2000.109172) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 2 In situ localization of IL-9 mRNA expression in peripheral blood eosinophils. Representative examples of in situ hybridization of IL-9 mRNA using 35S-labeled complementary RNA probes in human peripheral blood eosinophils are shown. A, In situ hybridization of IL-9 mRNA in eosinophils from an asthmatic subject (dark field, magnification 200×). B, Positive cells show eosinophil morphology with phase-contrast microscopy (magnification 200×). No specific signal could be detected with the sense-labeled riboprobe. C, IL-9 mRNA expression in nonatopic control and atopic asthmatic subjects. Results are expressed as the percentage of positive cells. *P < .01 for comparison between asthmatic and normal control subjects. Journal of Allergy and Clinical Immunology 2000 106, 460-466DOI: (10.1067/mai.2000.109172) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 3 Detection of IL-9 protein in human blood eosinophils. Purified eosinophils from asthmatic subjects (A) and nonatopic, nonasthmatic, control subjects (C) were stained with goat polyclonal anti-IL-9 antibody. Goat IgG was used as a negative control (B and D ). E, Percentage of IL-9 immunoreactive eosinophils from asthmatic and normal control subjects. Comparison between these two groups was determined by counting 500 cells per slide (*P < .01). The staining was performed by using goat anti-IL-9 followed by biotin-labeled, rabbit anti-goat streptavidin alkaline phosphatase, and counterstaining was with hematoxylin. Journal of Allergy and Clinical Immunology 2000 106, 460-466DOI: (10.1067/mai.2000.109172) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 4 IL-9 release by human peripheral blood eosinophils. Human peripheral blood eosinophils were stimulated for 18 hours with recombinant IL-1β, TNF-α, or both at 20 ng/mL or medium alone. IL-9 release was performed by biologic assay with RA3 cell proliferation, as described in the “Methods” sections. The RA3 cell line was stimulated with serial dilution of recombinant human IL-9 or medium alone (positive and negative controls, respectively). The y axis represents the ratio between the proliferation of RA3 cell line incubated with eosinophil supernatants under different conditions or IL-9 (30 pg/mL) and their proliferation when incubated with medium alone (control). Assays were performed in triplicate from the same donor. The data corresponds to the mean ± SD and are representative of 3 independent experiments performed under the same conditions. Journal of Allergy and Clinical Immunology 2000 106, 460-466DOI: (10.1067/mai.2000.109172) Copyright © 2000 Mosby, Inc. Terms and Conditions