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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–producing  cells are found in the spleens of  mice injected systemically with  STAg or LPS. Mice of the indicated strains were left untreated  (A), or were intravenously injected  with 0.5 μmol OVA (C), OVA  + 40 μg LPS (D), or with 25 μg  STAg (B, E–H). Animals were  killed 6 h (B, E–H) or 4 h (C and  D) after injection. Spleens were  frozen, sectioned, and stained  with anti–IL-12 p40 as detailed  in Materials and Methods. Note  “nests” of IL-12–producing cells  after STAg or LPS injection. Arrows in C and D indicate the  central arterioles. Original magnification: A, B, and E–H,  ×100; C and D, ×200.
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Figure 3: IL-12–producing cells are found in the spleens of mice injected systemically with STAg or LPS. Mice of the indicated strains were left untreated (A), or were intravenously injected with 0.5 μmol OVA (C), OVA + 40 μg LPS (D), or with 25 μg STAg (B, E–H). Animals were killed 6 h (B, E–H) or 4 h (C and D) after injection. Spleens were frozen, sectioned, and stained with anti–IL-12 p40 as detailed in Materials and Methods. Note “nests” of IL-12–producing cells after STAg or LPS injection. Arrows in C and D indicate the central arterioles. Original magnification: A, B, and E–H, ×100; C and D, ×200.

Mentions: The ability of STAg to induce IL-12 p40 production in the spleen in vivo offered an opportunity to phenotype the IL-12–producing cells in situ. Spleen sections from STAg-injected C57BL/6 mice showed numerous intensely stained IL-12 p40+ cells (Fig. 3 B) that were not seen in sections from uninjected control mice (Fig. 3 A). Induction of IL-12 p40 was specifically dependent on exposure to the parasite extract because it was not found in sections from animals injected with PBS, hen egg lysozyme, or ovalbumin (OVA; Fig. 3 C and data not shown). Intensely stained IL-12 p40+ cells could be detected as early as 3 h after STAg injection and the staining peaked between 6 and 12 h, and then declined progressively. 24 h after injection, staining was barely visible and the sections resembled those of control animals (data not shown). IL-12 p40+ cells had dendritic profiles and formed abundant “nests” surrounding central arterioles (Fig. 4 D), suggesting that they might represent IDC. Indeed, staining of serial sections demonstrated that IL-12 p40+ cells localized exclusively in the T cell areas of the white pulp and were excluded from the red pulp or the B cell areas (Fig. 4, D–F). This was true except at 3 h after injection, when IL-12 p40+ cells showed a more diffuse location, with many cells found at the edge of the T cell area and others interspersed with B cells in the marginal zone (Fig. 4, A–C, and data not shown). This picture is consistent with the IL-12–producing cells being in the process of migrating into the T cell area (see below). LPS coinjected intravenously with OVA also induced the appearance of IL-12+ nests of dendritic profiles surrounding central arterioles in spleen sections (Fig. 3 D). These were not seen in control sections injected with OVA alone (Fig. 3 C). Nevertheless, IL-12 p40 staining of putative IDC in response to LPS injection was consistently weaker and involved fewer cells than in response to STAg (Fig. 3 D). Importantly, IL-12 staining could not be detected in MΦ in the red pulp or those in the marginal zone (including MOMA-1+ [23] metallophils and marginal zone MΦ), either at early times or up to 96 h after injection of STAg (Figs. 3 and 4, and data not shown). IL-12 p40 staining by MΦ was also not detected in LPS-injected animals (Fig. 3 D and data not shown).


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–producing  cells are found in the spleens of  mice injected systemically with  STAg or LPS. Mice of the indicated strains were left untreated  (A), or were intravenously injected  with 0.5 μmol OVA (C), OVA  + 40 μg LPS (D), or with 25 μg  STAg (B, E–H). Animals were  killed 6 h (B, E–H) or 4 h (C and  D) after injection. Spleens were  frozen, sectioned, and stained  with anti–IL-12 p40 as detailed  in Materials and Methods. Note  “nests” of IL-12–producing cells  after STAg or LPS injection. Arrows in C and D indicate the  central arterioles. Original magnification: A, B, and E–H,  ×100; C and D, ×200.
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Related In: Results  -  Collection

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Figure 3: IL-12–producing cells are found in the spleens of mice injected systemically with STAg or LPS. Mice of the indicated strains were left untreated (A), or were intravenously injected with 0.5 μmol OVA (C), OVA + 40 μg LPS (D), or with 25 μg STAg (B, E–H). Animals were killed 6 h (B, E–H) or 4 h (C and D) after injection. Spleens were frozen, sectioned, and stained with anti–IL-12 p40 as detailed in Materials and Methods. Note “nests” of IL-12–producing cells after STAg or LPS injection. Arrows in C and D indicate the central arterioles. Original magnification: A, B, and E–H, ×100; C and D, ×200.
Mentions: The ability of STAg to induce IL-12 p40 production in the spleen in vivo offered an opportunity to phenotype the IL-12–producing cells in situ. Spleen sections from STAg-injected C57BL/6 mice showed numerous intensely stained IL-12 p40+ cells (Fig. 3 B) that were not seen in sections from uninjected control mice (Fig. 3 A). Induction of IL-12 p40 was specifically dependent on exposure to the parasite extract because it was not found in sections from animals injected with PBS, hen egg lysozyme, or ovalbumin (OVA; Fig. 3 C and data not shown). Intensely stained IL-12 p40+ cells could be detected as early as 3 h after STAg injection and the staining peaked between 6 and 12 h, and then declined progressively. 24 h after injection, staining was barely visible and the sections resembled those of control animals (data not shown). IL-12 p40+ cells had dendritic profiles and formed abundant “nests” surrounding central arterioles (Fig. 4 D), suggesting that they might represent IDC. Indeed, staining of serial sections demonstrated that IL-12 p40+ cells localized exclusively in the T cell areas of the white pulp and were excluded from the red pulp or the B cell areas (Fig. 4, D–F). This was true except at 3 h after injection, when IL-12 p40+ cells showed a more diffuse location, with many cells found at the edge of the T cell area and others interspersed with B cells in the marginal zone (Fig. 4, A–C, and data not shown). This picture is consistent with the IL-12–producing cells being in the process of migrating into the T cell area (see below). LPS coinjected intravenously with OVA also induced the appearance of IL-12+ nests of dendritic profiles surrounding central arterioles in spleen sections (Fig. 3 D). These were not seen in control sections injected with OVA alone (Fig. 3 C). Nevertheless, IL-12 p40 staining of putative IDC in response to LPS injection was consistently weaker and involved fewer cells than in response to STAg (Fig. 3 D). Importantly, IL-12 staining could not be detected in MΦ in the red pulp or those in the marginal zone (including MOMA-1+ [23] metallophils and marginal zone MΦ), either at early times or up to 96 h after injection of STAg (Figs. 3 and 4, and data not shown). IL-12 p40 staining by MΦ was also not detected in LPS-injected animals (Fig. 3 D and data not shown).

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