Antigen-specific and nonspecific determinants of cytokine production during topical sensitization of mice to chemical allergens Artin Moussavi, PhDa,

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Presentation transcript:

Antigen-specific and nonspecific determinants of cytokine production during topical sensitization of mice to chemical allergens Artin Moussavi, PhDa, Rebecca J. Dearman, PhDb, Ian Kimber, PhDb, Kenn C. Daniel, MSca, David M. Kemeny, PhDa Journal of Allergy and Clinical Immunology Volume 106, Issue 2, Pages 357-368 (August 2000) DOI: 10.1067/mai.2000.106928 Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 1 Proliferation and IFN-γ production by allergen-primed CD4+ lymphocytes cells stimulated by allergen-activated DCs. LNCs were isolated from mice 6 days after topical exposure to 10% TMA (A, B) or 1% DNCB (C, D). CD4+ lymphocyte populations prepared by positive selection with magnetic immunobeads were cultured with increasing numbers of either DNCB-primed DCs (solid boxes) or TMA-primed DCs (open boxes). Supernatants were harvested after 72 hours of culture and analyzed for IFN-γ content by ELISA (B, D). The remaining cells were pulsed with 1 μCi of tritiated thymidine for a further 18 hours (A, C). Results are expressed as the mean ± SE of 3 independent experiments. SEs are displayed only when they exceed 0.1 ng/mL or 500 counts/min. Differences versus noncognate controls were significant (asterisk, P < .05; two asterisks, P < .01, Wilcoxon single-rank test). Journal of Allergy and Clinical Immunology 2000 106, 357-368DOI: (10.1067/mai.2000.106928) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 2 IL-10 and IL-12 p40 by hapten-primed CD4+ lymphocytes cocultured with allergen-activated DCs. CD4+ lymphocytes were isolated from the draining lymph nodes of mice 6 days after topical exposure to 10% TMA (A, B) or 1% DNCB (C, D). DNCB-primed DC (solid boxes) and TMA-primed DC (open boxes) populations were prepared from LNCs of mice sensitized 18 hours previously with chemical allergen. Supernatants were harvested after 72 hours of culture and analyzed for IL-10 (B, D) and IL-12 p40 content by ELISA (A, C). Results are expressed as the mean ± SE of 3 independent experiments. SEs are displayed only when they exceed 0.1 ng/mL. Difference versus noncognate controls was significant (asterisk, P < .05, Wilcoxon single-rank test). Journal of Allergy and Clinical Immunology 2000 106, 357-368DOI: (10.1067/mai.2000.106928) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 3 Effect of neutralizing IL-12 on DC-induced IFN-γ and IL-10 production. CD4+ lymphocytes were isolated from the draining lymph nodes of mice 6 days after topical exposure to 10% TMA (A, B) or 1% DNCB (C, D). DNCB-primed DCs (solid boxes) and TMA-primed DCs (open boxes) populations were prepared from LNCs of mice sensitized 18 hours previously with chemical allergen. TMA- and DNCB-primed CD4+ lymphocytes were cultured with 2% DCs for 72 hours in the presence of increasing concentrations of a neutralizing polyclonal antibody against IL-12. CD4+ lymphocytes were also cultured with TMA- and DNCB-primed dendritic cells, respectively, in the presence of 20 μg/mL isotype control antibody (solid triangles). Culture supernatants were analyzed for IFN-γ (A, C) and IL-10 (B, D) content by ELISA. Results are expressed as the mean ± SE of 3 independent experiments. Asterisk, Differences versus DNCB-DC were significant (P < .05, Wilcoxon single-rank test). Two asterisks, Indicate lowest concentration of anti-IL-12 at which changes in levels of assayed cytokines were significant (P < .05, Wilcoxon single-rank test). Journal of Allergy and Clinical Immunology 2000 106, 357-368DOI: (10.1067/mai.2000.106928) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 4 Effects of CD40L blockade on cytokine responses evoked by allergen-activated DCs. CD4+ lymphocytes were isolated from the LNCs of mice 6 days after topical exposure to 10% TMA or 1% DNCB. Allergen-activated DCs were prepared from LNCs of mice sensitized 18 hours previously with chemical allergen. TMA- and DNCB-primed CD4+ cells were cocultured with TMA-activated DCs (open boxes and open circles, respectively) or DNCB-activated DCs (solid boxes and solid circles, respectively). TMA- and DNCB-primed CD4+ lymphocytes were cultured alone (hatched boxes and hatched circles, respectively) or with 2% DCs for 72 hours in the presence of increasing concentrations of anti-CD40L. TMA-primed CD4+ lymphocytes were also cultured with either TMA-primed (open triangle) or DNCB-primed DCs (solid triangle) in the presence of 20 μg/mL control antibody. Culture supernatants were analyzed for IFN-γ (A), IL-10 (B), and IL-12 p40 (C) content by ELISA. The results from one of two similar experiments are displayed and represent the mean cytokine concentration of triplicate wells (SDs were less than 0.1 ng/mL). Journal of Allergy and Clinical Immunology 2000 106, 357-368DOI: (10.1067/mai.2000.106928) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 5 Effects of CD40L blockade on proliferation and cytokine responses evoked in mitogen-activated naive CD4+ T cells by allergen-activated DCs. CD4+ lymphocytes were isolated from the LNCs of naive mice and DCs were prepared from the draining lymph nodes of mice sensitized 18 hours previously with chemical allergen. CD4+ cells were cocultured with DNCB-activated DCs in the presence of soluble anti-CD3 antibody (A, B) or concanavalin A (C, D). CD4+ lymphocytes were cultured alone (hatched triangles and hatched circles ) or with 2% DCs in the presence of increasing concentrations of anti-CD40L (open symbols) or isotype-matched control antibody (closed symbols). Supernatants were harvested after 72 hours of culture and analyzed for IFN-γ (triangles) and IL-12 p40 (circles) content by ELISA (B and D ). The remaining cells were pulsed with 1 μCi tritiated thymidine for a further 18 hours to measure proliferation (A, C). The results of one of two similar experiments are displayed. Journal of Allergy and Clinical Immunology 2000 106, 357-368DOI: (10.1067/mai.2000.106928) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 6 Cognate and noncognate components of the primary DC-CD4 T-cell interaction. Topical administration of chemical allergen triggers DCs to take up (1) and present antigen to naive T cells (2), triggering expression of CD40L on the T cell surface (3). CD40-CD40L interactions induce IL-12 secretion by DCs (4), which up-regulate IFN-γ production by CD4 T cells (5). Journal of Allergy and Clinical Immunology 2000 106, 357-368DOI: (10.1067/mai.2000.106928) Copyright © 2000 Mosby, Inc. Terms and Conditions