Gp120- and TNF-α–induced modulation of human B cell function: Proliferation, cyclic AMP generation, Ig production, and B-cell receptor expression Christina.

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gp120- and TNF-α–induced modulation of human B cell function: Proliferation, cyclic AMP generation, Ig production, and B-cell receptor expression Christina L. Patke, MSb, William T. Shearer, MD, PhDa Journal of Allergy and Clinical Immunology Volume 105, Issue 5, Pages 975-982 (May 2000) DOI: 10.1067/mai.2000.105315 Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 1 TNF-α–induced proliferative response and enhanced lymphoproliferation by gp120. As noted in Methods before subsequent treatments indicated in legends to Figs 1 to 5, freshly isolated HIV-1–seronegative B cells were activated with CD40 mAb plus IL-4. These activated cells were treated for 1 hour with gp120 (100 ng/mL) before addition of TNF-α (5-100 ng/mL). Activated B cells cultured in medium or gp120 alone are represented at 0 point. Tritiated thymidine incorporation was measured on day 2. Data represent mean ± SE of at least 4 experiments in quadruplicate. Journal of Allergy and Clinical Immunology 2000 105, 975-982DOI: (10.1067/mai.2000.105315) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 2 Dose-response curve of TNF-α on Ig production of activated HIV-1–seronegative human B lymphocytes with potentiation by gp120. For TNF-α dose-response, B cells were recultured with increasing doses of TNF-α alone (0.5-50 ng/mL). Activated B cells cultured in medium or gp120 (100 ng/mL) alone are represented at 0 point. Supernatants were harvested and quantitated by ELISA on (A) day 7 for IgM and (B) day 10 for IgG production. Data represent mean ± SE of 4 experiments in triplicate. Journal of Allergy and Clinical Immunology 2000 105, 975-982DOI: (10.1067/mai.2000.105315) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 3 Binding of gp120 by activated HIV-1–seronegative human B lymphocytes. These cells were cultured in the presence of (B) medium, (C) S aureus Cowan I protein plus IL-2, or (D) CD40 and IL-4 for 48 hours at 37°C. Cells were double stained with CD20-PE and gp120-FITC. Two-color immunostaining profiles of lymphocytes were gated by forward and 90-degree light scatters. Background levels are shown in A. For each quadrant the percentage of total cells is indicated. The upper right quadrant contains B cells bound by gp120. The results are a representation of 3 independent experiments. Journal of Allergy and Clinical Immunology 2000 105, 975-982DOI: (10.1067/mai.2000.105315) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 4 Modulation of TNF-α–induced B-cell cAMP generation by gp120. Activated HIV-1–seronegative human B cells were cultured for 2 hours at 37°C with medium alone, various concentrations of TNF-α (5-100 ng/mL), gp120 (100 ng/mL), or TNF-α plus gp120. The values are represented as means ± SE of 3 independent experiments performed in triplicate. Journal of Allergy and Clinical Immunology 2000 105, 975-982DOI: (10.1067/mai.2000.105315) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 5 Two-color immunofluorescence flow cytometric analysis of CD79b expression in B cells. Activated HIV-1–seronegative human B cells were treated for 24 hours with (A) medium, (B) gp120 (100 ng/mL), (C) TNF-α (100 ng/mL), or (D) TNF-α and gp120. Cells were double stained with FITC-labeled CD20 mAb and PE-labeled CD79b mAb. Two-color immunostaining profiles of lymphocytes were gated by forward-sideward scatter. For each quadrant the percentage of total cells is indicated. The results shown are a representation of 3 independent experiments. Journal of Allergy and Clinical Immunology 2000 105, 975-982DOI: (10.1067/mai.2000.105315) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 6 Hypothesis of gp120 modulation of B-cell signal transduction. The cAMP pathway activated by gp120 with augmentation by TNF-α impinges on the HIV-1 LTR CRE, resulting in a reservoir system for HIV-1 virus. Individually, cAMP and TNF-α induce NF-κB binding to HIV-1 LTR, resulting in HIV-1 viral expression with an enhanced response resulting on combination of both. Journal of Allergy and Clinical Immunology 2000 105, 975-982DOI: (10.1067/mai.2000.105315) Copyright © 2000 Mosby, Inc. Terms and Conditions