Limits...
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.

Show MeSH

Related in: MedlinePlus

Comparison of IL-12 p40 production by resting PEC, thio-PEC, and spleen cells in response to T. gondii. Adherent resident or thio-PEC (2 × 105/well) or a similar number of whole spleen cells from C3H/ HeJ mice were cultured overnight in the presence or absence of T. gondii  tachyzoites (strain RH; 2 × 105/well) or STAg (5 μg/ml). Where indicated, resident PEC cultures were supplemented with 100 U/ml of IFN-γ. IL-12 p40 and TNF released into the supernatant were measured by  ELISA. Spontaneous IL-12 production by cells cultured in medium alone  was not detected. Results represent the mean of triplicate cultures. Error  bars represent one SD from the mean. Gray bars, STAg; hatched bars, tachyzoites; *, not detectable.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199158&req=5

Figure 1: Comparison of IL-12 p40 production by resting PEC, thio-PEC, and spleen cells in response to T. gondii. Adherent resident or thio-PEC (2 × 105/well) or a similar number of whole spleen cells from C3H/ HeJ mice were cultured overnight in the presence or absence of T. gondii tachyzoites (strain RH; 2 × 105/well) or STAg (5 μg/ml). Where indicated, resident PEC cultures were supplemented with 100 U/ml of IFN-γ. IL-12 p40 and TNF released into the supernatant were measured by ELISA. Spontaneous IL-12 production by cells cultured in medium alone was not detected. Results represent the mean of triplicate cultures. Error bars represent one SD from the mean. Gray bars, STAg; hatched bars, tachyzoites; *, not detectable.

Mentions: MΦ-derived IL-12 has traditionally been considered a major factor driving Th1 responses, based on the observation that MΦ produce high levels of IL-12 after exposure to microbial products or after microbial infection in vitro (1, 2). However, most experiments examining IL-12 synthesis by MΦ in response to microbial stimuli to date, including our own (10), have primarily made use of inflammatory cells such as those that can be isolated from the peritoneal cavity of mice after elicitation with thioglycollate. To study IL-12 production by resting, unactivated MΦ in response to microbial products, resident peritoneal exudate cells (PEC) from LPS hyporesponsive C3H/HeJ mice were compared to thioglycollate-elicited PEC (thio-PEC) from the same mouse strain for the ability to produce various monokines after in vitro infection with T. gondii or after exposure to a soluble T. gondii antigen extract (STAg). As shown in Fig. 1, infection of freshly isolated resident PEC with live T. gondii tachyzoites or incubation with STAg did not result in production of detectable IL-12 p40 despite the fact that the same cells produced TNF. In contrast, as previously reported (10), thio-PEC produced substantial levels of both IL-12 p40 and TNF in response to infection or exposure to STAg (Fig. 1). Addition of exogenous IFN-γ to the cultures, which dramatically augments production of IL-12 p40 by inflammatory MΦ stimulated with T. gondii (21), did not correct the selective defect in IL-12 p40 production by resident cells despite the fact that it increased their production of TNF by 10-fold (Fig. 1). Resident MΦ were able to produce IL-12 p40 upon exposure to STAg or after live infection only when incubated for 24 h before stimulation, especially if IFN-γ was included in the preculture (data not shown). Furthermore, preincubation in the presence of IFN-γ was required to obtain substantial levels of IL-12 p40 production in response to STAg with bone marrow–derived MΦ populations and monocytic cell lines (data not shown). These results suggest that resting, unprimed MΦ cannot serve as a significant source of IL-12 to drive Th1 responses to T. gondii. Thus, a model in which MΦ-derived IL-12 is a pivotal factor in driving Th1 responses to microbial infections requires that the infected MΦ be already “primed” for IL-12 production, in a manner analogous to thioglycollate elicitation or preculture in medium containing IFN-γ.


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)

Comparison of IL-12 p40 production by resting PEC, thio-PEC, and spleen cells in response to T. gondii. Adherent resident or thio-PEC (2 × 105/well) or a similar number of whole spleen cells from C3H/ HeJ mice were cultured overnight in the presence or absence of T. gondii  tachyzoites (strain RH; 2 × 105/well) or STAg (5 μg/ml). Where indicated, resident PEC cultures were supplemented with 100 U/ml of IFN-γ. IL-12 p40 and TNF released into the supernatant were measured by  ELISA. Spontaneous IL-12 production by cells cultured in medium alone  was not detected. Results represent the mean of triplicate cultures. Error  bars represent one SD from the mean. Gray bars, STAg; hatched bars, tachyzoites; *, not detectable.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199158&req=5

Figure 1: Comparison of IL-12 p40 production by resting PEC, thio-PEC, and spleen cells in response to T. gondii. Adherent resident or thio-PEC (2 × 105/well) or a similar number of whole spleen cells from C3H/ HeJ mice were cultured overnight in the presence or absence of T. gondii tachyzoites (strain RH; 2 × 105/well) or STAg (5 μg/ml). Where indicated, resident PEC cultures were supplemented with 100 U/ml of IFN-γ. IL-12 p40 and TNF released into the supernatant were measured by ELISA. Spontaneous IL-12 production by cells cultured in medium alone was not detected. Results represent the mean of triplicate cultures. Error bars represent one SD from the mean. Gray bars, STAg; hatched bars, tachyzoites; *, not detectable.
Mentions: MΦ-derived IL-12 has traditionally been considered a major factor driving Th1 responses, based on the observation that MΦ produce high levels of IL-12 after exposure to microbial products or after microbial infection in vitro (1, 2). However, most experiments examining IL-12 synthesis by MΦ in response to microbial stimuli to date, including our own (10), have primarily made use of inflammatory cells such as those that can be isolated from the peritoneal cavity of mice after elicitation with thioglycollate. To study IL-12 production by resting, unactivated MΦ in response to microbial products, resident peritoneal exudate cells (PEC) from LPS hyporesponsive C3H/HeJ mice were compared to thioglycollate-elicited PEC (thio-PEC) from the same mouse strain for the ability to produce various monokines after in vitro infection with T. gondii or after exposure to a soluble T. gondii antigen extract (STAg). As shown in Fig. 1, infection of freshly isolated resident PEC with live T. gondii tachyzoites or incubation with STAg did not result in production of detectable IL-12 p40 despite the fact that the same cells produced TNF. In contrast, as previously reported (10), thio-PEC produced substantial levels of both IL-12 p40 and TNF in response to infection or exposure to STAg (Fig. 1). Addition of exogenous IFN-γ to the cultures, which dramatically augments production of IL-12 p40 by inflammatory MΦ stimulated with T. gondii (21), did not correct the selective defect in IL-12 p40 production by resident cells despite the fact that it increased their production of TNF by 10-fold (Fig. 1). Resident MΦ were able to produce IL-12 p40 upon exposure to STAg or after live infection only when incubated for 24 h before stimulation, especially if IFN-γ was included in the preculture (data not shown). Furthermore, preincubation in the presence of IFN-γ was required to obtain substantial levels of IL-12 p40 production in response to STAg with bone marrow–derived MΦ populations and monocytic cell lines (data not shown). These results suggest that resting, unprimed MΦ cannot serve as a significant source of IL-12 to drive Th1 responses to T. gondii. Thus, a model in which MΦ-derived IL-12 is a pivotal factor in driving Th1 responses to microbial infections requires that the infected MΦ be already “primed” for IL-12 production, in a manner analogous to thioglycollate elicitation or preculture in medium containing IFN-γ.

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