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. 1998 Jun 15;187(12):2103-8.
doi: 10.1084/jem.187.12.2103.

"VSports手机版" Murine macrophages secrete interferon gamma upon combined stimulation with interleukin (IL)-12 and IL-18: A novel pathway of autocrine macrophage activation

M Munder  1 , M Mallo, K Eichmann, M Modolell
Affiliations

Murine macrophages secrete interferon gamma upon combined stimulation with interleukin (IL)-12 and IL-18: A novel pathway of autocrine macrophage activation

M Munder et al. J Exp Med. .

Abstract

Interferon (IFN)-gamma, a key immunoregulatory cytokine, has been thought to be produced solely by activated T cells and natural killer cells. In this study, we show that murine bone marrow- derived macrophages (BMMPhi) secrete large amounts of IFN-gamma upon appropriate stimulation VSports手机版. Although interleukin (IL)-12 and IL-18 alone induce low levels of IFN-gamma mRNA transcripts, the combined stimulation of BMMPhi with both cytokines leads to the efficient production of IFN-gamma protein. The macrophage-derived IFN-gamma is biologically active as shown by induction of inducible nitric oxide synthase as well as upregulation of CD40 in macrophages. Our findings uncover a novel pathway of autocrine macrophage activation by demonstrating that the macrophage is not only a key cell type responding to IFN-gamma but also a potent IFN-gamma-producing cell. .

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Figures

Figure 1
Figure 1
IL-12/IL-18–induced IFN-γ synthesis of BMMΦ. 105 AKR/N-BMMΦ were stimulated in a final volume of 200 μl in 96-well flat-bottomed plates with increasing concentrations of IL-12 alone (squares, no IL-12; circles, 0.001 ng/ml; triangles, 0.1 ng/ml; diamonds, 10 ng/ml), IL-18 alone, or both cytokines together in a final volume of 200 μl in 96-well flat-bottomed plates. At the indicated time points, culture supernatants were harvested and analyzed for IFN-γ concentration by ELISA. Data represent means of triplicate with SD indicated. Similar results were obtained in a total of three independent experiments.
Figure 3
Figure 3
Endogenously produced IFN-γ induces autocrine macrophage stimulation: iNOS induction. (A) 105 AKR/N-BMMΦ were cultivated in a final volume of 200 μl in 96-well flat-bottomed plates with medium alone (filled squares) or stimulated with 10 ng/ml IL-12 alone (circles), 50 ng/ml IL-18 alone (triangles), with both cytokines together (diamonds), or with 10 ng/ml IFN-γ (open squares) in a final volume of 200 μl in 96-well flat-bottomed plates. To allow for synergistic iNOS induction, increasing concentrations of TNF-α were titrated into each culture. After 96 h, supernatants were harvested, and IFN-γ as well as nitrites were determined as described in Materials and Methods. Data represent means of triplicate with SD indicated. Similar results were obtained in a total of four independent experiments. (B) 105 BMMΦ of IFN-γR+/+ (129Sv) or IFN-γR−/− strains of mice were stimulated with increasing concentrations of IL-12 plus IL-18 (squares, no cytokines; circles, 0.1/0.5 ng/ml; triangles, 1/ 5 ng/ml; diamonds, 10/50 ng/ml, respectively) in a final volume of 200 μl in 96-well flat-bottomed plates. Increasing concentrations of TNF-α were titrated into each culture. After 96 h, supernatants were harvested, and IFN-γ as well as nitrites were determined as described in Materials and Methods. Data represent means of triplicate with SD indicated. One of two independent experiments with similar results is shown.
Figure 2
Figure 2
IL-12/IL-18–induced IFN-γ mRNA in BMMΦ. (A) 5 × 106 AKR/N-BMMΦ were seeded in 55-mm Petriperm® hydrophob plates and either cultivated in medium alone (control, ø) or stimulated with 10 ng/ml IL-12 alone, 50 ng/ml IL-18 alone, or with both cytokines together. At the indicated time points, cells were harvested, mRNA was extracted, and cDNA was prepared as described in Materials and Methods. 1, 0.2, or 0.04 μl of input cDNA was amplified by PCR with IFN-γ–specific primers. As a control, β-actin mRNA was also amplified with 0.01 μl input cDNA. One of three independent experiments, yielding identical results, is shown. (B and C) 106 AKR/N-BMMΦ were cultivated on glass chamber slides with medium alone (B) or with 10 ng/ml IL-12 plus 50 ng/ml IL-18 (C). After 18 h, RNA in situ hybridization for IFN-γ mRNA was performed as described in Materials and Methods; ×200.
Figure 4
Figure 4
Macrophage-derived IFN-γ is biologically active: CD40 upregulation. 1.5 × 106 BMMΦ of IFN-γR+/+ (129Sv) or IFN-γR−/− strains of mice were seeded in 35-mm Petriperm® hydrophob plates in a final volume of 1.5 ml. They were either not stimulated (ø, medium only) or stimulated with 10 ng/ml IFN-γ (positive control) or with the combination of 10 ng/ml IL-12 plus 50 ng/ml IL-18. After 48 h, BMMΦ were harvested with a rubber policeman and analyzed by FACS® for CD40 upregulation as described in Materials and Methods. (*) Simultaneous determination of IFN-γ concentration (ng/ml) in the 48-h supernatant. ND, Not determined. One of two independent experiments with similar results is shown.
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