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

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.

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.

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.

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.

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.

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.

Figure 5

IL-12 production by LOD in response to systemic administration of STAg is restricted to CD8α⁺N418⁺ DC. LOD prepared from groups of STAg- or PBS-injected C57BL/6 mice were triple stained for IL-12 p40, N418, and CD8α or for DEC-205, N418, and CD8α. (A and B). IL-12⁺ cells are only seen in STAg-injected animals and are all bright for N418 (box). (C and D) Gating on N418⁺ cells demonstrates that IL-12⁺ cells seen in response to STAg (box) are part of the CD8α⁺ DC subset. All CD8α⁺ N418⁺ cells were also positive for NLDC-145, as reported (16, 17). Data are representative of three independent experiments. Other data from the same experiment are shown in Table 1.

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.