Role of endogenous IFN-gamma in macrophage programming induced by IL-12 and IL-18

Abstract

Besides the established role of interleukin-12 (IL-12) and IL-18 on interferon-gamma (IFN-gamma) production by natural killer (NK), T, and B cells, the effects of these cytokines on macrophages are largely unknown. Here, we investigated the role of IL-12/IL-18 on nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by CD11b(+) adherent peritoneal cells, focusing on the involvement of endogenously produced IFN-gamma. C57BL/6 cells released substantial amounts of NO when stimulated with IFN-gamma or lipopolysaccharide (LPS), but failed to respond to IL-12 or IL-18 or both. However, IL-12/IL-18 pretreatment was able to program these cells to release 6-8-fold more NO and TNF-alpha in response to LPS or Trypanosoma cruzi stimulation, with NO levels directly correlating with macrophage resistance to intracellular parasite growth. Analysis of IL-12/IL-18-primed cells from mice deficient in IFN-gamma, IFNGR, and IFN regulatory factor-1 (IRF-1) revealed that these molecules were essential for LPS-induced NO release, but TNF-alpha production was IFN-gamma independent. Conversely, the myeloid differentiation factor 88 (MyD88)-dependent pathway was indispensable for IL-12/IL-18-programmed LPS-induced TNF-alpha production, but not for NO release. Contaminant T and NK cells largely modulated the IL-12/IL-18 programming of LPS-induced NO response through IFN-gamma secretion. Nevertheless, a small population of IFN-gamma(+) cells with a macrophage phenotype was also identified, particularly in the peritoneum of chronically T. cruzi-infected mice, reinforcing the notion that macrophages can be an alternative source of IFN-gamma. Taken together, our data contribute to elucidate the molecular basis of the IL-12/IL-18 autocrine pathway of macrophage activation, showing that endogenous IFN-gamma plays an important role in programming the NO response, whereas the TNF-alpha response occurs through an IFN-gamma-independent pathway.

FIG. 1.

NO production by CD11b⁺ aPECs from C57BL/6 and IFN-γKO mice. Five days after starch inoculation, C57BL/6 and IFN-γKO PECs were harvested and positively selected for CD11b expression using magnetic beads. CD11b⁺ PECs (2 × 10⁵) were allowed to adhere for 4 h and then washed and cultured with medium (time 0) or stimulated with LPS, rIFN-γ, rIL-12, or rIL-12 plus rIL-18. NO was measured in 48-h supernatants as described in Materials and Methods. Experiments were repeated three times, with the same pattern of results. Data represent the mean ± SD of the different experiments. *p < 0.05, compared with values from IFN-γKO cells.

FIG. 2.

IL-12/IL-18 programming of LPS-induced NO production in CD11b⁺ aPECs from C57BL/6, 129/SV, IFN-γKO, IFNGRKO, IRF-1KO, IL-12p40KO, MyD88KO, and CD14KO mice. C57BL/6, 129/SV, IFN-γKO, IFNGRKO, IRF-1KO, IL-12p40KO, MyD88KO, and CD14KO CD11b⁺ aPECs (2 × 10⁵) were cultured for 18 h in medium without ((−)) or with rIL-12, rIL-18, rIL-12 plus rIL-18, or rIFN-γ (2.5 ng/mL of each). After washing, cells were stimulated for an additional 48 h with LPS (1 μg/mL). NO was measured in supernatants as described in Materials and Methods. Experiments were repeated three times, with the same pattern of results. Data represent the mean ± SD of the different experiments. *p < 0.05, compared with values from C57BL/6 cells; ^#p < 0.05, compared with values from cells kept only in culture medium.

FIG. 3.

IL-12/IL-18 programming of T. cruzi-induced NO response and of macrophage microbicidal activity. C57BL/6 and IFN-γKO CD11b⁺ aPECs (2 × 10⁵) were cultured for 24 h in medium without ((−)) or with rIL-12, rIL-12 plus rIL-18, or rIFN-γ (2.5 ng/mL of each). Cells were then infected with T. cruzi at a 5:1 ratio for 120 min, washed to remove extracellular parasites, and kept for an additional 48 h with cytokines or medium. After this period, cells were stained with Giemsa to count intracellular amastigotes (A and B). NO was measured in supernatants as described in Materials and Methods (A). Experiments were repeated three times, with the same pattern of results. Data represent the mean ± SD of the different experiments. *p < 0.05, compared with values from IFN-γKO cells; ^#p < 0.05, compared with values from cells kept only in culture medium.

FIG. 4.

IL-12/IL-18 programming of LPS-induced TNF-α response in CD11b⁺ aPECs from C57BL/6, IFN-γKO, and MyD88KO mice. C57BL/6, IFN-γKO, and MyD88KO CD11b⁺ aPECs (2 × 10⁵) were cultured for 18 h in medium without ((−)) or with rIL-12, rIL-18, rIL-12 plus rIL-18, or rIFN-γ (2.5 ng/mL of each). After washing, cells were stimulated for additional 6 h with LPS (1 μg/mL). TNF-α was measured in supernatants as described in Materials and Methods. Experiments were repeated three times, with the same pattern of results. Data represent the mean ± SD of the different experiments. *p < 0.05, compared with values from C57BL/6 cells; ^#p < 0.05, compared with values from cells kept only in culture medium

FIG. 5.

IL-12/IL-18 programming of LPS-induced NO and TNF-α responses in highly purified macrophages. (A) Cells with a macrophage size were sorted by flow cytometry from C57BL/6 PECs. (B) Percentage of NK1.1⁺ and CD3⁺ cells were determined before and after sorting. (C) CD11b⁺ aPECs (2 × 10⁵) (sorted and nonsorted) of C57BL/6 mice were cultured for 12 h in medium without ((−)) or with rIL-12 (2.5 ng/mL), rIL-18 (2.5 ng/mL) or both. After washing, cells were stimulated for an additional 6 h (for TNF-α detection) or 48 h (for NO detection) with LPS (1 μg/mL). NO and TNF-α were measured in supernatants as described in Materials and Methods. Experiments were repeated three times, with the same pattern of results. Data represent the mean ± SD of the different experiments. *p < 0.05, compared with values from sorted cells; ^#p < 0.05, compared with values from cells kept only in culture medium.

FIG. 6.

Contribution of CD3⁺NK1.1⁺ and CD3⁻NK1.1⁻ (iNOSKO) cells in the IL-12/IL-18 programming of LPS-induced NO response of adherent peritoneal IFN-γKO cells. (A) iNOSKO PECs were separated according to the expression of NK1.1 and CD3 in CD3⁻NK1.1⁻ and CD3⁺NK1.1⁺ cells as described in Materials and Methods. Percentages of NK1.1⁺ and CD3⁺ cells were determined before and after cell separation. (B) aPECs (2 × 10⁵) from IFN-γKO mice were cultured for 12 h in medium without ((−)) or with rIL-12 (2.5 ng/mL) plus rIL-18 (2.5 ng/mL) in the presence of titrated numbers of CD3⁻NK1.1⁻ and CD3⁺NK1.1⁺ (iNOSKO) PECs. After washing, cells were stimulated for an additional 48 h with LPS (1 μg/mL). NO was measured in supernatants as described in Materials and Methods. Cell ratios were obtained by dividing the number of CD3⁻NK1.1⁻ or CD3⁺NK1.1⁺ (iNOSKO) cells by the numbers of IFN-γKO cells. Tendency titration curves calculated by the method of least squares were used to obtain the correspondent mathematic equations. (C) The cell ratios expected to attain a NO⁻₂ level of 10 μM in the culture supernatants were determined by using the mathematic equations obtained from tendency titration curves. Experiments were repeated three times, with the same pattern of results. Data represent the mean ± SD of the different experiments. ^#p < 0.05, compared with cells kept only in culture medium.

FIG. 7.

IFN-γ production by CD11b⁺ aPECs. (A) CD11b⁺ aPECs (2 × 10⁵) from C57BL/6 and IFN-γKO mice were cultured in medium without ((−)) or with rIL-12 and rIL-18 (2.5 ng/mL of each) for 18 h. After washing, cells were cultured for 6 h with LPS (1 μg/mL) in the presence of Golgistop containing monensin. Fixed cells were stained with fluorochrome-labeled antibodies to CD11b, MHC II, TNF-α, and IFN-γ and analyzed by confocal microscopy. (B) CD11b⁺ aPECs (2 × 10⁵) from noninfected and chronically T. cruzi-infected C57BL/6 mice were kept in culture medium without ((−)) or with rIL-12 plus rIL-18 (2.5 ng/mL of each) for 48 h. Golgistop containing monensin was added in the last 6 h. Fixed cells were stained with fluorochrome-labeled antibodies to F4/80, CD3, NK1.1, and IFN-γ and analyzed by flow citometry. Gated F4/80⁺ CD3⁻NK1.1⁻ cells are shown. Experiments were repeated three times, with the same pattern of results.