Mechanisms of inhibition of IgE synthesis by nedocromil sodium: Nedocromil sodium inhibits deletional switch recombination in human B cells Richard K.S.

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Presentation transcript:

Mechanisms of inhibition of IgE synthesis by nedocromil sodium: Nedocromil sodium inhibits deletional switch recombination in human B cells Richard K.S. Loh, Haifa H. Jabara, Raif S. Geha, MD Journal of Allergy and Clinical Immunology Volume 97, Issue 5, Pages 1141-1150 (May 1996) DOI: 10.1016/S0091-6749(96)70269-5 Copyright © 1996 Mosby, Inc. Terms and Conditions

FIG. 1 Effect of NS on IgE production by PBMCs in the presence of IL-4 (50 U/ml). PBMCs at a concentration of 1.5 × 106 cells/ml were cultured for 10 days with different concentrations of NS. Supernatants were harvested after 10 days, and IgE levels were measured by radioimmunoassay. Values represent mean ± SEM net synthesis IgE (picograms per milliliter) of five experiments. Journal of Allergy and Clinical Immunology 1996 97, 1141-1150DOI: (10.1016/S0091-6749(96)70269-5) Copyright © 1996 Mosby, Inc. Terms and Conditions

FIG. 2 Cell surface expression of the CD40 ligand. T cells were left untreated (dotted line) or stimulated with PMA (100 ng/ml) plus ionomycin (5 μmol/L) (continuous line). CD40 ligand expression with or without NS was assessed at 6 hours. Journal of Allergy and Clinical Immunology 1996 97, 1141-1150DOI: (10.1016/S0091-6749(96)70269-5) Copyright © 1996 Mosby, Inc. Terms and Conditions

FIG. 3 A, Effect of NS on IgE production by T cell–depleted, B cell–enriched populations of cells in the presence of IL-4 (50 U/ml) and anti-CD40 mAb (5 μg/ml). Cells were cultured at a concentration of 1 × 106 cells/ml for 10 days with different concentrations of disodium cromoglycate. Supernatants were harvested after 10 days and IgE levels were measured by radioimmunoassay. Results represent mean ± SEM net synthesis IgE (picograms per milliliter) of three experiments. B, Effect of NS on IgE production by highly purified B cells isolated by sorting for CD19 expression. B cells were cultured as described in A. Similar results were observed in a second experiment. Journal of Allergy and Clinical Immunology 1996 97, 1141-1150DOI: (10.1016/S0091-6749(96)70269-5) Copyright © 1996 Mosby, Inc. Terms and Conditions

FIG. 3 A, Effect of NS on IgE production by T cell–depleted, B cell–enriched populations of cells in the presence of IL-4 (50 U/ml) and anti-CD40 mAb (5 μg/ml). Cells were cultured at a concentration of 1 × 106 cells/ml for 10 days with different concentrations of disodium cromoglycate. Supernatants were harvested after 10 days and IgE levels were measured by radioimmunoassay. Results represent mean ± SEM net synthesis IgE (picograms per milliliter) of three experiments. B, Effect of NS on IgE production by highly purified B cells isolated by sorting for CD19 expression. B cells were cultured as described in A. Similar results were observed in a second experiment. Journal of Allergy and Clinical Immunology 1996 97, 1141-1150DOI: (10.1016/S0091-6749(96)70269-5) Copyright © 1996 Mosby, Inc. Terms and Conditions

FIG. 4 Effect of NS on the induction of germline Cϵ transcripts in B cells stimulated with IL-4. Total RNA (10 μg) was prepared from normal peripheral blood B cells incubated with medium or IL-4 (50 U/ml) or IL-4 (50 U/ml) and NS (10-8 mol/L) for 4 days then electrophoresed on a 1% formaldehyde-agarose gel, transferred to a nitrocellulose membrane, and hybridized to a 32P-labeled 0.88 kb Hinf1 fragment. Densitometric analysis was performed on germline Cϵ transcript mRNA bands versus actin transcript mRNA. The autoradiograph was exposed for 5 days. Similar results were obtained in two additional experiments. Journal of Allergy and Clinical Immunology 1996 97, 1141-1150DOI: (10.1016/S0091-6749(96)70269-5) Copyright © 1996 Mosby, Inc. Terms and Conditions

FIG. 5 Effect of NS on the generation of Sμ→Sϵ switch fragments. Serially diluted aliquots of total cellular DNA from B cells stimulated with IL-4, IL-4 plus anti-CD40 mAb, or IL-4 plus anti-CD40 mAb and NS (10-8 mol/L) were amplified by nested PCR. The serially diluted template DNA sample amounts used in the first round of PCR are noted above the gel. The second round or nested round of PCR used 10% of the original PCR mixture as DNA template. Final PCR products were subjected to agarose gel electrophoresis. PCR-amplifiable DNA was present in all three samples, as evident by the control (C) band for which primers specific for the human IL-1 β promoter (-1323±72) yield a 1.4 kb fragment by PCR. The higher intensity of the two bands seen in the 10 ng of DNA from control B cells compared with the lower intensity of the numerous bands seen with 50 to 200 ng of DNA from control B cells is probably due to a technical artifact, which may have included lesser competition for the primers in the 10 ng DNA sample. MW, Molecular weight markers. Journal of Allergy and Clinical Immunology 1996 97, 1141-1150DOI: (10.1016/S0091-6749(96)70269-5) Copyright © 1996 Mosby, Inc. Terms and Conditions

FIG. 6 Effect of NS on IgE production by B cells in the presence of IL-4 (50 U/ml) and hydrocortisone (10-6 mol/L). B cells at a concentration of 1 × 106 cells/ml were cultured for 10 days with different concentrations of NS. Supernatants were harvested after 10 days, and IgE levels were measured by radioimmunoassay. Results represent mean ± SEM net synthesis IgE (picograms per milliliter) of five experiments. Journal of Allergy and Clinical Immunology 1996 97, 1141-1150DOI: (10.1016/S0091-6749(96)70269-5) Copyright © 1996 Mosby, Inc. Terms and Conditions

FIG. 7 Effect of NS on IgG 4 production induced in sIgG 4– B cells by IL-4 (50 U/ml) and anti-CD40 mAb (5 μg/ml). Cells (106 cells/ml) were cultured for 3 days with IL-4. IgG 4 and then anti-CD40 were added with or without NS for 10 days. IgG 4 and IgE were measured in the supernatants by ELISA and radioimmunoassay, respectively. Journal of Allergy and Clinical Immunology 1996 97, 1141-1150DOI: (10.1016/S0091-6749(96)70269-5) Copyright © 1996 Mosby, Inc. Terms and Conditions

FIG. 7 Effect of NS on IgG 4 production induced in sIgG 4– B cells by IL-4 (50 U/ml) and anti-CD40 mAb (5 μg/ml). Cells (106 cells/ml) were cultured for 3 days with IL-4. IgG 4 and then anti-CD40 were added with or without NS for 10 days. IgG 4 and IgE were measured in the supernatants by ELISA and radioimmunoassay, respectively. Journal of Allergy and Clinical Immunology 1996 97, 1141-1150DOI: (10.1016/S0091-6749(96)70269-5) Copyright © 1996 Mosby, Inc. Terms and Conditions