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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 migrate into the inner PALS to become IDC in response to STAg. Spleen sections from C57BL/6 mice intravenously  injected 3 (A–C) or 6 h (D–G, I) previously with 25 μg STAg, or sections from uninjected mice (H), were stained for IL-12 p40 (A–G; dark brown) or  DEC-205 (H and I; dark brown) and double stained (purple) for B220 (B and E), TCR-β (C and F), or N418 (G–I). A–C and D–F are serial sections  through the same white pulp nodule. Note: IL-12–producing cells are found in the marginal zone and outer PALS 3 h after injection (A–C), but are seen  in the inner PALS (T cells area) 6 h after injection (D–F; arrows indicate the central arteriole); IL-12 p40+ cells are also positive for N418 (G, white arrow  indicating brown and purple stain) although not all N418+ cells are IL-12 p40+ (G, black arrow indicating purple only stain; see also Fig. 5); after STAg administration (H), there is an apparent increase in the number of DC in the spleen and redistribution of these cells to the inner PALS (compare H with I;  see also Table 1). Original magnification: A–F, ×200; G, ×400; H–I, ×100.
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Figure 4: IL-12–producing cells migrate into the inner PALS to become IDC in response to STAg. Spleen sections from C57BL/6 mice intravenously injected 3 (A–C) or 6 h (D–G, I) previously with 25 μg STAg, or sections from uninjected mice (H), were stained for IL-12 p40 (A–G; dark brown) or DEC-205 (H and I; dark brown) and double stained (purple) for B220 (B and E), TCR-β (C and F), or N418 (G–I). A–C and D–F are serial sections through the same white pulp nodule. Note: IL-12–producing cells are found in the marginal zone and outer PALS 3 h after injection (A–C), but are seen in the inner PALS (T cells area) 6 h after injection (D–F; arrows indicate the central arteriole); IL-12 p40+ cells are also positive for N418 (G, white arrow indicating brown and purple stain) although not all N418+ cells are IL-12 p40+ (G, black arrow indicating purple only stain; see also Fig. 5); after STAg administration (H), there is an apparent increase in the number of DC in the spleen and redistribution of these cells to the inner PALS (compare H with I; see also Table 1). Original magnification: A–F, ×200; G, ×400; H–I, ×100.

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 migrate into the inner PALS to become IDC in response to STAg. Spleen sections from C57BL/6 mice intravenously  injected 3 (A–C) or 6 h (D–G, I) previously with 25 μg STAg, or sections from uninjected mice (H), were stained for IL-12 p40 (A–G; dark brown) or  DEC-205 (H and I; dark brown) and double stained (purple) for B220 (B and E), TCR-β (C and F), or N418 (G–I). A–C and D–F are serial sections  through the same white pulp nodule. Note: IL-12–producing cells are found in the marginal zone and outer PALS 3 h after injection (A–C), but are seen  in the inner PALS (T cells area) 6 h after injection (D–F; arrows indicate the central arteriole); IL-12 p40+ cells are also positive for N418 (G, white arrow  indicating brown and purple stain) although not all N418+ cells are IL-12 p40+ (G, black arrow indicating purple only stain; see also Fig. 5); after STAg administration (H), there is an apparent increase in the number of DC in the spleen and redistribution of these cells to the inner PALS (compare H with I;  see also Table 1). Original magnification: A–F, ×200; G, ×400; H–I, ×100.
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Related In: Results  -  Collection

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Figure 4: IL-12–producing cells migrate into the inner PALS to become IDC in response to STAg. Spleen sections from C57BL/6 mice intravenously injected 3 (A–C) or 6 h (D–G, I) previously with 25 μg STAg, or sections from uninjected mice (H), were stained for IL-12 p40 (A–G; dark brown) or DEC-205 (H and I; dark brown) and double stained (purple) for B220 (B and E), TCR-β (C and F), or N418 (G–I). A–C and D–F are serial sections through the same white pulp nodule. Note: IL-12–producing cells are found in the marginal zone and outer PALS 3 h after injection (A–C), but are seen in the inner PALS (T cells area) 6 h after injection (D–F; arrows indicate the central arteriole); IL-12 p40+ cells are also positive for N418 (G, white arrow indicating brown and purple stain) although not all N418+ cells are IL-12 p40+ (G, black arrow indicating purple only stain; see also Fig. 5); after STAg administration (H), there is an apparent increase in the number of DC in the spleen and redistribution of these cells to the inner PALS (compare H with I; see also Table 1). Original magnification: A–F, ×200; G, ×400; H–I, ×100.
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