Chemical constituents of diesel exhaust particles induce IL-4 production and histamine release by human basophils Gilles Devouassoux, MDa, Andrew Saxon,

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Presentation transcript:

Chemical constituents of diesel exhaust particles induce IL-4 production and histamine release by human basophils Gilles Devouassoux, MDa, Andrew Saxon, MDb, Dean D. Metcalfe, MDc, Calman Prussin, MDc, Maurice G. Colomb, MDd, Christian Brambilla, MDa, David Diaz-Sanchez, PhDb Journal of Allergy and Clinical Immunology Volume 109, Issue 5, Pages 847-853 (May 2002) DOI: 10.1067/mai.2002.122843 Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 1 DEPex induces basophil IL-4 expression. PBMCs were incubated in the absence (A) or presence (B) of DEPex (1 μg/mL) or ionomycin (1 μmol/L) (C) for 4 hours; fixed; stained with antibodies to IgE, lineage markers, and IL-4; and analyzed by means of flow cytometry. After gating on IgEhigh, lineagelow basophils, a cytokine versus IgE dot plot was generated. Statistical markers were placed on the basis of isotype-matched control staining. The percentages given in each panel (A , B , and C ) represent the percentages of basophils producing IL-4. D , Means ± SD of 5 allergic donors (filled column) and 4 nonallergic donors (open column) are shown. Results are expressed as the percentage of maximal IL-4 production. The mean maximal IL-4 expression (100%) was 82%. Statistically significant differences are noted: *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology 2002 109, 847-853DOI: (10.1067/mai.2002.122843) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 2 Concentration dependence of DEPex activity. PBMCs and enriched basophils were incubated with DEPex (0-2 μg/mL) to evaluate IL-4 expression (open circles) by means of flow cytometry and degranulation by means of histamine assay (filled circles) . Means ± SD of 6 and 5 independent experiments are shown for IL-4 expression and histamine release, respectively. Results are expressed as the percentages of maximal basophil IL-4 expression and histamine release. The mean maximal IL-4 expression and histamine release were 75.1% and 27 mmol/L, respectively. Statistically significant differences are noted: *P2 < .05 and **P < .01. Journal of Allergy and Clinical Immunology 2002 109, 847-853DOI: (10.1067/mai.2002.122843) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 3 Basophils are a target of DEPex. Enriched basophils (A) were incubated with or without DEPex (1 μg/mL; B and C ) for 4 hours. Purity and frequency of IL-4-producing basophils were determined by means of flow cytometry. The percentage of basophil purity is given in A (circle) , and percentages in B and C represent the percentages of basophils producing IL-4. Similar data were obtained in 4 independent experiments. Basophil IL-4 (filled column) and histamine (open column) releases into the supernatant were analyzed by means of ELISA and histamine assay, respectively (mean ± SD, n = 4, D ). Each experiment was performed in duplicate. Statistically significant differences are noted: **P < .01. Journal of Allergy and Clinical Immunology 2002 109, 847-853DOI: (10.1067/mai.2002.122843) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 4 DEPex does not synergize with antigen to increase basophil IL-4 production and histamine release. PBMCs from allergic subjects were incubated with media, antigen, DEPex (1 μg/mL), or antigen plus DEPex. IL-4 production was analyzed by means of flow cytometry (filled column) and histamine release by means of histamine assay (open column) . The mean ± SD of 4 independent experiments is shown, and results are expressed as the percentage of maximal IL-4 expression. The mean maximal IL-4 expression and histamine release were 77.5% and 24.8 mmol/L, respectively. Statistically significant differences are noted: **P < .01. NS , Not significant; Ag , antigen. Journal of Allergy and Clinical Immunology 2002 109, 847-853DOI: (10.1067/mai.2002.122843) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 5 Kinetics of cytokine expression by means of DEPex-stimulated basophils. PBMCs from allergic subjects were stimulated in vitro with media (open squares) , antigen (closed circles) , or DEPex (open circles) for 0 to 20 hours at 37°C; fixed; stained for IgE, lineage, and IL-4; and analyzed by means of flow cytometry. Means ± SD of 3 independent experiments with 3 donors are shown. Results are expressed as the percentage of maximal IL-4 expression. The mean maximal IL-4 expression was 77%. Statistically significant differences are noted: **P < .01. NS , Not significant; Ag , antigen. Journal of Allergy and Clinical Immunology 2002 109, 847-853DOI: (10.1067/mai.2002.122843) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 6 NAC abrogates IL-4 expression on DEPex-activated basophils. PBMCs were preincubated with media or NAC (10 mmol/L) for 1 hour at 37°C and then activated with DEPex (1000 ng/mL, filled column ), antigen (open column) , or ionomycin (shaded column) for 4 hours. IL-4 production was analyzed by means of flow cytometry, and the frequency of IL-4+ basophils was determined. Means ± SD of 5 independent experiments are shown, and results are expressed as the percentage of basophil IL-4 expression. Statistically significant differences are noted: **P < .01. Journal of Allergy and Clinical Immunology 2002 109, 847-853DOI: (10.1067/mai.2002.122843) Copyright © 2002 Mosby, Inc. Terms and Conditions