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In vivo microbial stimulation induces rapid CD40 ligand-independent production of interleukin 12 by dendritic cells and their redistribution to T cell areas.

Reis e Sousa C, Hieny S, Scharton-Kersten T, Jankovic D, Charest H, Germain RN, Sher A - J. Exp. Med. (1997)

Bottom Line: Importantly, this production of IL-12 occurs very rapidly and is independent of interferon gamma priming or of signals from T cells, such as CD40 ligand.IL-12 production by splenic DC is accompanied by an increase in number of DCs, as well as a redistribution to the T cell areas and the acquisition of markers characteristic of interdigitating dendritic cells.This model avoids the need to invoke a three-cell interaction for Th1 differentiation and points to the DC as both a sentinel for innate recognition and the dictator of class selection in the subsequent adaptive response.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-1892, USA. caetano@nih.gov

ABSTRACT
The early induction of interleukin (IL)-12 is a critical event in determining the development of both innate resistance and adaptive immunity to many intracellular pathogens. Previous in vitro studies have suggested that the macrophage (MPhi) is a major source of the initial IL-12 produced upon microbial stimulation and that this response promotes the differentiation of protective T helper cell 1 (Th1) CD4+ lymphocytes from precursors that are primed on antigen-bearing dendritic cells (DC). Here, we demonstrate by immunolocalization experiments and flow cytometric analysis that, contrary to expectation, DC and not MPhi are the initial cells to synthesize IL-12 in the spleens of mice exposed in vivo to an extract of Toxoplasma gondii or to lipopolysaccharide, two well characterized microbial stimulants of the cytokine. Importantly, this production of IL-12 occurs very rapidly and is independent of interferon gamma priming or of signals from T cells, such as CD40 ligand. IL-12 production by splenic DC is accompanied by an increase in number of DCs, as well as a redistribution to the T cell areas and the acquisition of markers characteristic of interdigitating dendritic cells. The capacity of splenic DC but not MPhi to synthesize de novo high levels of IL-12 within hours of exposure to microbial products in vivo, as well as the ability of the same stimuli to induce migration of DC to the T cell areas, argues that DC function simultaneously as both antigen-presenting cells and IL-12 producing accessory cells in the initiation of cell-mediated immunity to intracellular pathogens. This model avoids the need to invoke a three-cell interaction for Th1 differentiation and points to the DC as both a sentinel for innate recognition and the dictator of class selection in the subsequent adaptive response.

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IL-12 production by spleen cells in response to stimulation  with Toxoplasma antigens in vitro is independent of CD40L or of signals  from lymphocytes. Spleen cells from the indicated mouse strains were  cultured in the presence of STAg or live T. gondii tachyzoites as in Fig. 1,  and IL-12 p40 secreted into supernatants was measured by ELISA after 24 h.  C57BL/6 served as controls for the B6-SCID and (B6 × 129)F2 for the  CD40L KO mice. The apparently higher levels of IL-12 production from  SCID spleen probably reflect their enrichment for nonlymphoid cells relative to wild-type spleens. Spontaneous IL-12 production by spleen cells  cultured in medium alone was not detected. Results represent the mean  of triplicate cultures from two to three animals per group. Error bars represent one SD from the mean. Gray bars, STAg; hatched bars, tachyzoites.
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Figure 6: IL-12 production by spleen cells in response to stimulation with Toxoplasma antigens in vitro is independent of CD40L or of signals from lymphocytes. Spleen cells from the indicated mouse strains were cultured in the presence of STAg or live T. gondii tachyzoites as in Fig. 1, and IL-12 p40 secreted into supernatants was measured by ELISA after 24 h. C57BL/6 served as controls for the B6-SCID and (B6 × 129)F2 for the CD40L KO mice. The apparently higher levels of IL-12 production from SCID spleen probably reflect their enrichment for nonlymphoid cells relative to wild-type spleens. Spontaneous IL-12 production by spleen cells cultured in medium alone was not detected. Results represent the mean of triplicate cultures from two to three animals per group. Error bars represent one SD from the mean. Gray bars, STAg; hatched bars, tachyzoites.

Mentions: IL-12 production by both murine and human DC in vitro has been previously reported (26–32). The major mechanisms involved in IL-12 induction appear to be signaling through DC surface CD40 molecules after cross-linking by T cell–expressed CD40L, or direct signaling through MHC class II molecules on DC, cross-linked by the TCR (30, 31, 33). Spleen cells from CD40L KO mice secreted substantial levels of IL-12 p40 in response to live parasite infection or STAg stimulation in vitro, although these levels were somewhat lower than those produced by cells from (B6 × 129)F2 control mice (Fig. 6). To examine the CD40L dependence of IL-12 production by DC in response to STAg in vivo, CD40L KO mice were injected with STAg as before and spleens removed 6 h later. Staining of sections from CD40L KO mice was comparable to that of sections from B6 mice or (B6 × 129)F2 controls, suggesting that STAg-induced IL-12 production by IDC does not require cross-linking of CD40 on DC by CD40L on T cells (Fig. 3, E and F). To exclude the possibility that other cognate T cell–DC interactions or T cell–derived cytokines might be responsible for the STAg effect, SCID mice were similarly injected with STAg and analyzed in parallel to the CD40L KO mice. Again, SCID spleen sections showed IL-12 p40 staining comparable to wild-type controls (Fig. 3 H). SCID spleen cells also made high levels of IL-12 p40 in response to STAg or live infection in vitro (Fig. 6). Thus, microbe-induced IL-12 production by spleen cells in vitro or by DC in vivo does not require interactions with lymphocytes and is likely to reflect a direct effect on the cells of one or more components of the microbial extract, or an indirect effect through induction of DC-activating inflammatory cytokines produced by nonlymphoid cells.


In vivo microbial stimulation induces rapid CD40 ligand-independent production of interleukin 12 by dendritic cells and their redistribution to T cell areas.

Reis e Sousa C, Hieny S, Scharton-Kersten T, Jankovic D, Charest H, Germain RN, Sher A - J. Exp. Med. (1997)

IL-12 production by spleen cells in response to stimulation  with Toxoplasma antigens in vitro is independent of CD40L or of signals  from lymphocytes. Spleen cells from the indicated mouse strains were  cultured in the presence of STAg or live T. gondii tachyzoites as in Fig. 1,  and IL-12 p40 secreted into supernatants was measured by ELISA after 24 h.  C57BL/6 served as controls for the B6-SCID and (B6 × 129)F2 for the  CD40L KO mice. The apparently higher levels of IL-12 production from  SCID spleen probably reflect their enrichment for nonlymphoid cells relative to wild-type spleens. Spontaneous IL-12 production by spleen cells  cultured in medium alone was not detected. Results represent the mean  of triplicate cultures from two to three animals per group. Error bars represent one SD from the mean. Gray bars, STAg; hatched bars, tachyzoites.
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Related In: Results  -  Collection

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Figure 6: IL-12 production by spleen cells in response to stimulation with Toxoplasma antigens in vitro is independent of CD40L or of signals from lymphocytes. Spleen cells from the indicated mouse strains were cultured in the presence of STAg or live T. gondii tachyzoites as in Fig. 1, and IL-12 p40 secreted into supernatants was measured by ELISA after 24 h. C57BL/6 served as controls for the B6-SCID and (B6 × 129)F2 for the CD40L KO mice. The apparently higher levels of IL-12 production from SCID spleen probably reflect their enrichment for nonlymphoid cells relative to wild-type spleens. Spontaneous IL-12 production by spleen cells cultured in medium alone was not detected. Results represent the mean of triplicate cultures from two to three animals per group. Error bars represent one SD from the mean. Gray bars, STAg; hatched bars, tachyzoites.
Mentions: IL-12 production by both murine and human DC in vitro has been previously reported (26–32). The major mechanisms involved in IL-12 induction appear to be signaling through DC surface CD40 molecules after cross-linking by T cell–expressed CD40L, or direct signaling through MHC class II molecules on DC, cross-linked by the TCR (30, 31, 33). Spleen cells from CD40L KO mice secreted substantial levels of IL-12 p40 in response to live parasite infection or STAg stimulation in vitro, although these levels were somewhat lower than those produced by cells from (B6 × 129)F2 control mice (Fig. 6). To examine the CD40L dependence of IL-12 production by DC in response to STAg in vivo, CD40L KO mice were injected with STAg as before and spleens removed 6 h later. Staining of sections from CD40L KO mice was comparable to that of sections from B6 mice or (B6 × 129)F2 controls, suggesting that STAg-induced IL-12 production by IDC does not require cross-linking of CD40 on DC by CD40L on T cells (Fig. 3, E and F). To exclude the possibility that other cognate T cell–DC interactions or T cell–derived cytokines might be responsible for the STAg effect, SCID mice were similarly injected with STAg and analyzed in parallel to the CD40L KO mice. Again, SCID spleen sections showed IL-12 p40 staining comparable to wild-type controls (Fig. 3 H). SCID spleen cells also made high levels of IL-12 p40 in response to STAg or live infection in vitro (Fig. 6). Thus, microbe-induced IL-12 production by spleen cells in vitro or by DC in vivo does not require interactions with lymphocytes and is likely to reflect a direct effect on the cells of one or more components of the microbial extract, or an indirect effect through induction of DC-activating inflammatory cytokines produced by nonlymphoid cells.

Bottom Line: Importantly, this production of IL-12 occurs very rapidly and is independent of interferon gamma priming or of signals from T cells, such as CD40 ligand.IL-12 production by splenic DC is accompanied by an increase in number of DCs, as well as a redistribution to the T cell areas and the acquisition of markers characteristic of interdigitating dendritic cells.This model avoids the need to invoke a three-cell interaction for Th1 differentiation and points to the DC as both a sentinel for innate recognition and the dictator of class selection in the subsequent adaptive response.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-1892, USA. caetano@nih.gov

ABSTRACT
The early induction of interleukin (IL)-12 is a critical event in determining the development of both innate resistance and adaptive immunity to many intracellular pathogens. Previous in vitro studies have suggested that the macrophage (MPhi) is a major source of the initial IL-12 produced upon microbial stimulation and that this response promotes the differentiation of protective T helper cell 1 (Th1) CD4+ lymphocytes from precursors that are primed on antigen-bearing dendritic cells (DC). Here, we demonstrate by immunolocalization experiments and flow cytometric analysis that, contrary to expectation, DC and not MPhi are the initial cells to synthesize IL-12 in the spleens of mice exposed in vivo to an extract of Toxoplasma gondii or to lipopolysaccharide, two well characterized microbial stimulants of the cytokine. Importantly, this production of IL-12 occurs very rapidly and is independent of interferon gamma priming or of signals from T cells, such as CD40 ligand. IL-12 production by splenic DC is accompanied by an increase in number of DCs, as well as a redistribution to the T cell areas and the acquisition of markers characteristic of interdigitating dendritic cells. The capacity of splenic DC but not MPhi to synthesize de novo high levels of IL-12 within hours of exposure to microbial products in vivo, as well as the ability of the same stimuli to induce migration of DC to the T cell areas, argues that DC function simultaneously as both antigen-presenting cells and IL-12 producing accessory cells in the initiation of cell-mediated immunity to intracellular pathogens. This model avoids the need to invoke a three-cell interaction for Th1 differentiation and points to the DC as both a sentinel for innate recognition and the dictator of class selection in the subsequent adaptive response.

Show MeSH
Related in: MedlinePlus