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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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Intravenous administration of STAg causes production of IL-12  p40 that can prime an IFN-γ response. (A) Spontaneous IL-12 production by spleen cells isolated from C57BL/6 or IFN-γ KO mice injected  with STAg for the indicated times. Cells were isolated by mechanical dissociation of spleens from control mice (0 h) or mice intravenously injected with 25 μg STAg and were cultured for 24 h in medium. IL-12  p40 released into supernatants was measured by ELISA. Gray bars, B6;  hatched bars, IFN-γ KO. (B) Enhanced IFN-γ secretion by spleen cells is  dependent upon STAg-elicited IL-12 in vivo. Mice from the indicated  strains were intravenously injected with 25 μg STAg. Where indicated  (+), mice were intraperitoneally treated with 1 μg anti–IL-12 (mAb  C17.8; reference 19) immediately before injection of STAg by the intravenous route. Cells were isolated by mechanical dissociation of spleens 48 h  after injection and were restimulated with 5 μg/ml STAg for 48 h. IFN-γ  released into supernatants was measured by ELISA. Equivalent control  cultures in medium alone did not produce IFN-γ (data not shown). Data in  A and B are the average of three mice in each group except for the anti– IL-12-treated group in B, for which the data are the mean of two mice.  Error bars represent one SD from the mean.
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Figure 2: Intravenous administration of STAg causes production of IL-12 p40 that can prime an IFN-γ response. (A) Spontaneous IL-12 production by spleen cells isolated from C57BL/6 or IFN-γ KO mice injected with STAg for the indicated times. Cells were isolated by mechanical dissociation of spleens from control mice (0 h) or mice intravenously injected with 25 μg STAg and were cultured for 24 h in medium. IL-12 p40 released into supernatants was measured by ELISA. Gray bars, B6; hatched bars, IFN-γ KO. (B) Enhanced IFN-γ secretion by spleen cells is dependent upon STAg-elicited IL-12 in vivo. Mice from the indicated strains were intravenously injected with 25 μg STAg. Where indicated (+), mice were intraperitoneally treated with 1 μg anti–IL-12 (mAb C17.8; reference 19) immediately before injection of STAg by the intravenous route. Cells were isolated by mechanical dissociation of spleens 48 h after injection and were restimulated with 5 μg/ml STAg for 48 h. IFN-γ released into supernatants was measured by ELISA. Equivalent control cultures in medium alone did not produce IFN-γ (data not shown). Data in A and B are the average of three mice in each group except for the anti– IL-12-treated group in B, for which the data are the mean of two mice. Error bars represent one SD from the mean.

Mentions: To determine if IL-12 production by spleen cells in response to T. gondii also occurs in vivo, B6 mice were injected intravenously with STAg. Spleen cell suspensions from mice injected with STAg, but not from uninjected mice or from mice injected with PBS alone, spontaneously produced IL-12 p40 during overnight culture (Fig. 2 A). Maximal IL-12 p40 production could be seen as early as 3 h after systemic administration of STAg and declined progressively thereafter (Fig. 2 A). The IL-12 produced in vivo appeared to be bioactive because spleen cells from STAg-injected mice produced increased levels of IFN-γ upon in vitro restimulation of spleen cells with STAg or LPS. This enhancement of IFN-γ production was specifically dependent upon IL-12 induction in vivo because it was not seen in IL-12 p40–deficient mice or in wild type mice treated with anti–IL-12 antibodies at the time of STAg administration (Fig. 2 B). Together, these results demonstrate that spleen cells can synthesize IL-12 p40 in response to stimulation with T. gondii products in vitro or in vivo, and that IL-12 production elicited by these molecules in vivo can prime an IFN-γ response.


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)

Intravenous administration of STAg causes production of IL-12  p40 that can prime an IFN-γ response. (A) Spontaneous IL-12 production by spleen cells isolated from C57BL/6 or IFN-γ KO mice injected  with STAg for the indicated times. Cells were isolated by mechanical dissociation of spleens from control mice (0 h) or mice intravenously injected with 25 μg STAg and were cultured for 24 h in medium. IL-12  p40 released into supernatants was measured by ELISA. Gray bars, B6;  hatched bars, IFN-γ KO. (B) Enhanced IFN-γ secretion by spleen cells is  dependent upon STAg-elicited IL-12 in vivo. Mice from the indicated  strains were intravenously injected with 25 μg STAg. Where indicated  (+), mice were intraperitoneally treated with 1 μg anti–IL-12 (mAb  C17.8; reference 19) immediately before injection of STAg by the intravenous route. Cells were isolated by mechanical dissociation of spleens 48 h  after injection and were restimulated with 5 μg/ml STAg for 48 h. IFN-γ  released into supernatants was measured by ELISA. Equivalent control  cultures in medium alone did not produce IFN-γ (data not shown). Data in  A and B are the average of three mice in each group except for the anti– IL-12-treated group in B, for which the data are the mean of two mice.  Error bars represent one SD from the mean.
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Figure 2: Intravenous administration of STAg causes production of IL-12 p40 that can prime an IFN-γ response. (A) Spontaneous IL-12 production by spleen cells isolated from C57BL/6 or IFN-γ KO mice injected with STAg for the indicated times. Cells were isolated by mechanical dissociation of spleens from control mice (0 h) or mice intravenously injected with 25 μg STAg and were cultured for 24 h in medium. IL-12 p40 released into supernatants was measured by ELISA. Gray bars, B6; hatched bars, IFN-γ KO. (B) Enhanced IFN-γ secretion by spleen cells is dependent upon STAg-elicited IL-12 in vivo. Mice from the indicated strains were intravenously injected with 25 μg STAg. Where indicated (+), mice were intraperitoneally treated with 1 μg anti–IL-12 (mAb C17.8; reference 19) immediately before injection of STAg by the intravenous route. Cells were isolated by mechanical dissociation of spleens 48 h after injection and were restimulated with 5 μg/ml STAg for 48 h. IFN-γ released into supernatants was measured by ELISA. Equivalent control cultures in medium alone did not produce IFN-γ (data not shown). Data in A and B are the average of three mice in each group except for the anti– IL-12-treated group in B, for which the data are the mean of two mice. Error bars represent one SD from the mean.
Mentions: To determine if IL-12 production by spleen cells in response to T. gondii also occurs in vivo, B6 mice were injected intravenously with STAg. Spleen cell suspensions from mice injected with STAg, but not from uninjected mice or from mice injected with PBS alone, spontaneously produced IL-12 p40 during overnight culture (Fig. 2 A). Maximal IL-12 p40 production could be seen as early as 3 h after systemic administration of STAg and declined progressively thereafter (Fig. 2 A). The IL-12 produced in vivo appeared to be bioactive because spleen cells from STAg-injected mice produced increased levels of IFN-γ upon in vitro restimulation of spleen cells with STAg or LPS. This enhancement of IFN-γ production was specifically dependent upon IL-12 induction in vivo because it was not seen in IL-12 p40–deficient mice or in wild type mice treated with anti–IL-12 antibodies at the time of STAg administration (Fig. 2 B). Together, these results demonstrate that spleen cells can synthesize IL-12 p40 in response to stimulation with T. gondii products in vitro or in vivo, and that IL-12 production elicited by these molecules in vivo can prime an IFN-γ response.

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