Interleukin 12-dependent interferon gamma production by CD8alpha+ lymphoid dendritic cells - PubMed (original) (raw)

Interleukin 12-dependent interferon gamma production by CD8alpha+ lymphoid dendritic cells

T Ohteki et al. J Exp Med. 1999.

Abstract

We investigated the role of antigen-presenting cells in early interferon (IFN)-gamma production in normal and recombinase activating gene 2-deficient (Rag-2(-/-)) mice in response to Listeria monocytogenes (LM) infection and interleukin (IL)-12 administration. Levels of serum IFN-gamma in Rag-2(-/-) mice were comparable to those of normal mice upon either LM infection or IL-12 injection. Depletion of natural killer (NK) cells by administration of anti-asialoGM1 antibodies had little effect on IFN-gamma levels in the sera of Rag-2(-/-) mice after LM infection or IL-12 injection. Incubation of splenocytes from NK cell-depleted Rag-2(-/-) mice with LM resulted in the production of IFN-gamma that was completely blocked by addition of anti-IL-12 antibodies. Both dendritic cells (DCs) and monocytes purified from splenocytes were capable of producing IFN-gamma when cultured in the presence of IL-12. Intracellular immunofluorescence analysis confirmed the IFN-gamma production from DCs. It was further shown that IFN-gamma was produced predominantly by CD8alpha+ lymphoid DCs rather than CD8alpha- myeloid DCs. Collectively, our data indicated that DCs are potent in producing IFN-gamma in response to IL-12 produced by bacterial infection and play an important role in innate immunity and subsequent T helper cell type 1 development in vivo.

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Figures

Figure 1

Figure 1

IFN-γ production in the sera of NK cell–depleted Rag-2−/− mice. (A) 300 μg αASGM1 Ab were injected into B10.D2 and B10.D2–Rag-2−/− mice. Spleen cells were collected on day 3 and stained for 145-2C11–PE (anti-CD3ε) and DX5-FITC (anti-pan NK). (B) Saline, 0.5 μg IL-12, or 2 × 106 LM were intraperitoneally injected into B10.D2 or B10.D2– Rag-2−/− mice with or without treatment with αASGM1 3 d before injection (top), or into C57BL/6-γc −/−(Y)Rag-2−/− and NOD/LtSz-scid/scid mice (bottom). Sera were collected 24 h later from IL-12– injected mice and 48 h later from LM- infected and control mice, and the amounts of IFN-γ were measured by ELISA. (C) In vitro IFN-γ production from NK cell– depleted Rag-2−/− splenocytes cultured with LM. 106 collagenase-treated splenocytes were obtained from NK cell–depleted B10.D2, B10.D2–Rag-2−/−, and C57BL/ 6-γc −/−(Y)Rag-2−/− mice. The cells were infected with 4 × 105 LM and cultured for 3 d with or without either 10 μg/ml anti– IL-12 mAb (top) or 1 ng/ml IL-12 (bottom). The amounts of IFN-γ were measured by ELISA.

Figure 2

Figure 2

IFN-γ production from isolated DCs. (A) Isolation of splenic DCs. CD11c+I-Ab+ DCs were isolated from C57BL/6 splenocytes on a FACS Vantage™, and the purity of the cells was checked. (B) IFN-γ production in culture supernatants of DCs, macrophages, and NK cells. DCs (5 × 104), NK cells (5 × 104), and macrophages (2 × 104) were cultured for 3 d in the presence of 1 ng/ml IL-12. Culture supernatants were harvested and subjected to ELISA. Asterisk indicates the amount of IFN-γ produced by 5 × 104 equivalent macrophages.

Figure 2

Figure 2

IFN-γ production from isolated DCs. (A) Isolation of splenic DCs. CD11c+I-Ab+ DCs were isolated from C57BL/6 splenocytes on a FACS Vantage™, and the purity of the cells was checked. (B) IFN-γ production in culture supernatants of DCs, macrophages, and NK cells. DCs (5 × 104), NK cells (5 × 104), and macrophages (2 × 104) were cultured for 3 d in the presence of 1 ng/ml IL-12. Culture supernatants were harvested and subjected to ELISA. Asterisk indicates the amount of IFN-γ produced by 5 × 104 equivalent macrophages.

Figure 3

Figure 3

IFN-γ production by DC subpopulations. (A) Isolated splenic DCs were stained with anti-CD11c–FITC, anti-CD8α–PE, and anti-CD86– biotin, followed by streptavidin–Red 670, and subjected to cell sorting to purify CD8α−CD11c+CD86+ (myeloid) DCs and CD8α+CD11c+CD86+ (lymphoid) DCs. Purity of CD11c+CD86+ whole DCs, CD8α− DCs and CD8α+ DCs were 98, 95, and 90%, respectively. (B) Whole DCs, CD8α−CD11c+CD86+ DCs, and CD8α+ CD11c+CD86+ DCs (5 × 104) were cultured for 3 d in the presence of 1 ng/ml IL-12, and the amounts of IFN-γ in culture supernatants were measured by ELISA. No difference in viability was observed between CD8α+ and CD8α− DCs.

Figure 3

Figure 3

IFN-γ production by DC subpopulations. (A) Isolated splenic DCs were stained with anti-CD11c–FITC, anti-CD8α–PE, and anti-CD86– biotin, followed by streptavidin–Red 670, and subjected to cell sorting to purify CD8α−CD11c+CD86+ (myeloid) DCs and CD8α+CD11c+CD86+ (lymphoid) DCs. Purity of CD11c+CD86+ whole DCs, CD8α− DCs and CD8α+ DCs were 98, 95, and 90%, respectively. (B) Whole DCs, CD8α−CD11c+CD86+ DCs, and CD8α+ CD11c+CD86+ DCs (5 × 104) were cultured for 3 d in the presence of 1 ng/ml IL-12, and the amounts of IFN-γ in culture supernatants were measured by ELISA. No difference in viability was observed between CD8α+ and CD8α− DCs.

Figure 4

Figure 4

Detection of intracellular IFN-γ in IL-12–stimulated DCs. Purified DCs were cultured in the presence of 1 ng IL-12 for 3 d. After surface staining with FITC-conjugated mAb against CD11c, cells were fixed and permeabilized. Cells were then incubated with rabbit polyclonal Ab against IFN-γ (A and B) and normal rabbit serum (E and F). Freshly isolated DCs were also incubated with rabbit polyclonal Ab against IL-12R (C and D). Samples were further stained with Rhodamine-conjugated goat anti-rabbit IgG. Bars, 10 μm.

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