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Cholera toxin suppresses interleukin (IL)-12 production and IL-12 receptor beta1 and beta2 chain expression.

Braun MC, He J, Wu CY, Kelsall BL - J. Exp. Med. (1999)

Bottom Line: This suppression occurred at the level of gene transcription, was maximal at low concentrations of CT, and was dependent on the A subunit of the toxin, since purified CT B subunit had minimal effect.The effects of CT were not due to autocrine production of IL-10, TGF-beta1, or prostaglandin E2.In vivo, mice given CT before systemic challenge with lipopolysaccharide had markedly reduced serum levels of IL-12 p40 and interferon gamma.

View Article: PubMed Central - PubMed

Affiliation: Immune Cell Interaction Unit, Mucosal Immunity Section, National Institutes of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
Cholera toxin (CT) is a potent mucosal vaccine adjuvant, which has been shown to induce T helper cell type 2 (Th2) responses in systemic and mucosal tissues. We report that CT inhibits the production of interleukin (IL)-12, a major Th2 counterregulatory cytokine. IL-12 p70 production by stimulated human monocytes was inhibited by CT in a dose-dependent manner. This suppression occurred at the level of gene transcription, was maximal at low concentrations of CT, and was dependent on the A subunit of the toxin, since purified CT B subunit had minimal effect. CT also inhibited the production of IL-12 p70 by monocyte-derived dendritic cells, as well as the production of tumor necrosis factor alpha, but not IL-10, IL-6, or transforming growth factor (TGF)-beta1, by stimulated monocytes. The effects of CT were not due to autocrine production of IL-10, TGF-beta1, or prostaglandin E2. CT inhibited the production of IFN-gamma by anti-CD3-stimulated human peripheral blood mononuclear cell, due in part to suppression of IL-12 production, but also to the inhibition of expression of the beta1 and beta2 chains of the IL-12 receptor on T cells. In vivo, mice given CT before systemic challenge with lipopolysaccharide had markedly reduced serum levels of IL-12 p40 and interferon gamma. These data demonstrate two novel mechanisms by which CT can inhibit Th1 immune responses, and help explain the ability of mucosally administered CT to enhance Th2-dependent immune responses.

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(A) IFN-γ production  by cultured PBMCs was significantly  reduced by CT treatment. PBMCs  (106 cells/ml) were cultured for 72 h  in the presence or absence of CT (10  ng/ml) with nothing (unstimulated), rIL-12 (1 ng/ml), anti-CD3  (1 μg/ml), anti-CD3 and anti-IL-12  (1 μg/ml), or anti-CD3 and rIL-12.  Supernatants were then harvested  and analyzed for IFN-γ production  by ELISA. Data are mean values ±  SD from a total of seven donors  from three separate experiments.  **P < 0.01; *P < 0.05. (B) CT  treatment also inhibited anti-CD3-mediated cell proliferation. PBMCs  were cultured as noted above for 72 h,  then pulsed for 6 h with [3H]thymidine, harvested, and analyzed. Data  are mean values ± SD of triplicate  samples from three different donors.  *P < 0.05.
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Figure 7: (A) IFN-γ production by cultured PBMCs was significantly reduced by CT treatment. PBMCs (106 cells/ml) were cultured for 72 h in the presence or absence of CT (10 ng/ml) with nothing (unstimulated), rIL-12 (1 ng/ml), anti-CD3 (1 μg/ml), anti-CD3 and anti-IL-12 (1 μg/ml), or anti-CD3 and rIL-12. Supernatants were then harvested and analyzed for IFN-γ production by ELISA. Data are mean values ± SD from a total of seven donors from three separate experiments. **P < 0.01; *P < 0.05. (B) CT treatment also inhibited anti-CD3-mediated cell proliferation. PBMCs were cultured as noted above for 72 h, then pulsed for 6 h with [3H]thymidine, harvested, and analyzed. Data are mean values ± SD of triplicate samples from three different donors. *P < 0.05.

Mentions: To determine the functional relevance of the effects of CT on IL-12 production, we initially examined the ability of CT to inhibit IFN-γ production by PBMCs stimulated with soluble anti-CD3. Under such stimulation conditions, T cells are activated to express CD40L, which stimulates CD40-dependent production of IL-12 by both monocytes and DCs. In turn, IL-12 drives IFN-γ production by circulating NK and T cells, and augments CD3-mediated proliferation of T cells. As shown in Fig. 7, PBMCs stimulated with anti-CD3 produced IFN-γ and proliferated in an IL-12 dependent fashion, as anti–IL-12 abrogated this response. The addition of CT inhibited IFN-γ production as well as proliferation; however, such inhibition was only partially reversed by the addition of exogenous IL-12 to the cultures. These latter findings suggested that CT may also inhibit the responsiveness of T or NK cells to IL-12. To examine this possibility, we stimulated PBMCs with anti-CD3 in the presence or absence of CT. After 72 h in culture, the level of expression of the two chains of IL-12R on T cells was determined by flow cytometry. As shown in Fig. 8, expression of both the β1 and β2 chains of IL-12R were significantly upregulated by stimulation with anti-CD3, and this enhancement was blocked by treatment with CT. Thus, CT appears to act by at least two distinct mechanisms to suppress IFN-γ production in cultured PBMCs.


Cholera toxin suppresses interleukin (IL)-12 production and IL-12 receptor beta1 and beta2 chain expression.

Braun MC, He J, Wu CY, Kelsall BL - J. Exp. Med. (1999)

(A) IFN-γ production  by cultured PBMCs was significantly  reduced by CT treatment. PBMCs  (106 cells/ml) were cultured for 72 h  in the presence or absence of CT (10  ng/ml) with nothing (unstimulated), rIL-12 (1 ng/ml), anti-CD3  (1 μg/ml), anti-CD3 and anti-IL-12  (1 μg/ml), or anti-CD3 and rIL-12.  Supernatants were then harvested  and analyzed for IFN-γ production  by ELISA. Data are mean values ±  SD from a total of seven donors  from three separate experiments.  **P < 0.01; *P < 0.05. (B) CT  treatment also inhibited anti-CD3-mediated cell proliferation. PBMCs  were cultured as noted above for 72 h,  then pulsed for 6 h with [3H]thymidine, harvested, and analyzed. Data  are mean values ± SD of triplicate  samples from three different donors.  *P < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2192916&req=5

Figure 7: (A) IFN-γ production by cultured PBMCs was significantly reduced by CT treatment. PBMCs (106 cells/ml) were cultured for 72 h in the presence or absence of CT (10 ng/ml) with nothing (unstimulated), rIL-12 (1 ng/ml), anti-CD3 (1 μg/ml), anti-CD3 and anti-IL-12 (1 μg/ml), or anti-CD3 and rIL-12. Supernatants were then harvested and analyzed for IFN-γ production by ELISA. Data are mean values ± SD from a total of seven donors from three separate experiments. **P < 0.01; *P < 0.05. (B) CT treatment also inhibited anti-CD3-mediated cell proliferation. PBMCs were cultured as noted above for 72 h, then pulsed for 6 h with [3H]thymidine, harvested, and analyzed. Data are mean values ± SD of triplicate samples from three different donors. *P < 0.05.
Mentions: To determine the functional relevance of the effects of CT on IL-12 production, we initially examined the ability of CT to inhibit IFN-γ production by PBMCs stimulated with soluble anti-CD3. Under such stimulation conditions, T cells are activated to express CD40L, which stimulates CD40-dependent production of IL-12 by both monocytes and DCs. In turn, IL-12 drives IFN-γ production by circulating NK and T cells, and augments CD3-mediated proliferation of T cells. As shown in Fig. 7, PBMCs stimulated with anti-CD3 produced IFN-γ and proliferated in an IL-12 dependent fashion, as anti–IL-12 abrogated this response. The addition of CT inhibited IFN-γ production as well as proliferation; however, such inhibition was only partially reversed by the addition of exogenous IL-12 to the cultures. These latter findings suggested that CT may also inhibit the responsiveness of T or NK cells to IL-12. To examine this possibility, we stimulated PBMCs with anti-CD3 in the presence or absence of CT. After 72 h in culture, the level of expression of the two chains of IL-12R on T cells was determined by flow cytometry. As shown in Fig. 8, expression of both the β1 and β2 chains of IL-12R were significantly upregulated by stimulation with anti-CD3, and this enhancement was blocked by treatment with CT. Thus, CT appears to act by at least two distinct mechanisms to suppress IFN-γ production in cultured PBMCs.

Bottom Line: This suppression occurred at the level of gene transcription, was maximal at low concentrations of CT, and was dependent on the A subunit of the toxin, since purified CT B subunit had minimal effect.The effects of CT were not due to autocrine production of IL-10, TGF-beta1, or prostaglandin E2.In vivo, mice given CT before systemic challenge with lipopolysaccharide had markedly reduced serum levels of IL-12 p40 and interferon gamma.

View Article: PubMed Central - PubMed

Affiliation: Immune Cell Interaction Unit, Mucosal Immunity Section, National Institutes of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
Cholera toxin (CT) is a potent mucosal vaccine adjuvant, which has been shown to induce T helper cell type 2 (Th2) responses in systemic and mucosal tissues. We report that CT inhibits the production of interleukin (IL)-12, a major Th2 counterregulatory cytokine. IL-12 p70 production by stimulated human monocytes was inhibited by CT in a dose-dependent manner. This suppression occurred at the level of gene transcription, was maximal at low concentrations of CT, and was dependent on the A subunit of the toxin, since purified CT B subunit had minimal effect. CT also inhibited the production of IL-12 p70 by monocyte-derived dendritic cells, as well as the production of tumor necrosis factor alpha, but not IL-10, IL-6, or transforming growth factor (TGF)-beta1, by stimulated monocytes. The effects of CT were not due to autocrine production of IL-10, TGF-beta1, or prostaglandin E2. CT inhibited the production of IFN-gamma by anti-CD3-stimulated human peripheral blood mononuclear cell, due in part to suppression of IL-12 production, but also to the inhibition of expression of the beta1 and beta2 chains of the IL-12 receptor on T cells. In vivo, mice given CT before systemic challenge with lipopolysaccharide had markedly reduced serum levels of IL-12 p40 and interferon gamma. These data demonstrate two novel mechanisms by which CT can inhibit Th1 immune responses, and help explain the ability of mucosally administered CT to enhance Th2-dependent immune responses.

Show MeSH
Related in: MedlinePlus