Pia J. Hauk, MDa, Qutayba A. Hamid, MD, PhDc, George P

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Presentation transcript:

Induction of corticosteroid insensitivity in human PBMCs by microbial superantigens Pia J. Hauk, MDa, Qutayba A. Hamid, MD, PhDc, George P. Chrousos, MDd, Donald Y.M. Leung, MD, PhDab Journal of Allergy and Clinical Immunology Volume 105, Issue 4, Pages 782-787 (April 2000) DOI: 10.1067/mai.2000.105807 Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 1 Effect of DEX on PBMC response to SAgs in comparison to PHA. PBMCs of 7 healthy donors were incubated for 72 hours with PHA (5 μg/mL) or the SAgs SEB, TSST-1, and SEE (100 ng/mL) in the presence or absence of DEX (10–10 to 10–6 mol/L). SAg-induced PBMC proliferation was significantly less sensitive to DEX than stimulation with PHA. Proliferation response under various conditions was expressed as the percent of mitogen-induced tritiated thymidine incorporation in the absence of DEX compared with the addition of DEX. Data are expressed as mean ± SEM with a 2-way repeated-measures ANOVA to determine significance (asterisk, P < .05; two asterisks, P < .01). The mean (range) of the induced lymphocyte proliferation was 171,229 counts/min (104,378-256,596 counts/min) for 5 μg/mL PHA, 190,952 counts/min (138,718-228,948 counts/min) for 100 ng/mL SEE, 236,839 counts/min (159,696-314,511 counts/min) for 100 ng/mL TSST-1, and 289,719 counts/min (213,326-393,281 counts/min) for 100 ng/mL SEB. Journal of Allergy and Clinical Immunology 2000 105, 782-787DOI: (10.1067/mai.2000.105807) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 2 Effect of DEX on PBMC response to different concentrations of SEB in comparison to PHA. PBMCs from 3 healthy donors were stimulated for 72 hours with PHA (2.5, 5, and 10 mg/mL) or SEB (1, 10, and 100 ng/mL) in the presence or absence of DEX (10–9 to 10–6 mol/L). Lymphocyte proliferation induced by different concentrations of SEB is insensitive to DEX, whereas proliferation induced by different concentrations of PHA is highly sensitive to DEX. Results are expressed as means ± SEM. Journal of Allergy and Clinical Immunology 2000 105, 782-787DOI: (10.1067/mai.2000.105807) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 3 Effect of SEB on number of GRβ+ PBMCs. Immunocytochemical staining of 4% paraformaldehyde-fixed PBMCs with use of an affinity-purified rabbit polyclonal antibody to GRβ was performed. The antigen-antibody complex was visualized with fast red. The number of GRβ+ PBMCs is increased by culture in the presence of SEB. A, An example of a PBMC preparation incubated in media for 48 hours. B, An example of PBMCs that were stimulated with SEB (100 ng/mL) for 48 hours. Note the large number of GRβ+. C, An example of a negative control where the GRβ antibody was replaced with nonspecific rabbit Ig. Journal of Allergy and Clinical Immunology 2000 105, 782-787DOI: (10.1067/mai.2000.105807) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 4 Effect of SEB on number of GRβ+ PBMCs in comparison with PHA. Freshly isolated PBMCs from healthy donors and their cells after 48 hours of incubation in culture alone or in the presence of 5 μg/mL PHA or 100 ng/mL SEB were processed and stained for GRβ immunoreactivity as described in Material and Methods. In 3 subjects PBMCs were also cultured with PHA or SEB in the presence of anti-Il-4 mAbs (5 μg/mL). GRβ+ cells were scored in each preparation by a blinded assessor by counting 1000 cells and expressing them as a percentage of the total cell population. The percentage of GRβ+ cells in normal PBMCs was significantly higher after stimulation with SEB than with PHA or culture medium alone. PHA induced a significant increase in GRβ+ cells compared with PBMCs cultured in medium alone. The PHA-induced increase of GRβ+ cells was completely abolished by anti-IL-4 mAbs. However, the increased number of GRβ+ cells induced by SEB remained elevated despite coculture with anti-IL-4 mAbs. Journal of Allergy and Clinical Immunology 2000 105, 782-787DOI: (10.1067/mai.2000.105807) Copyright © 2000 Mosby, Inc. Terms and Conditions