Natural killer (NK) cells are sentinel components of the innate response to pathogens, but the cell types, pathogen recognition receptors, and cytokines required for their activation in vivo are poorly defined. Here, we investigated the role of plasmacytoid dendritic cells (pDCs), myeloid DCs (mDCs), Toll-like receptors (TLRs), and of NK cell stimulatory cytokines for the induction of an NK cell response to the protozoan parasite Leishmania infantum. In vitro, pDCs did not endocytose Leishmania promastigotes but nevertheless released interferon (IFN)-α/β and interleukin (IL)-12 in a TLR9-dependent manner. mDCs rapidly internalized Leishmania and, in the presence of TLR9, produced IL-12, but not IFN-α/β. Depletion of pDCs did not impair the activation of NK cells in L. infantum–infected mice. In contrast, L. infantum–induced NK cell cytotoxicity and IFN-γ production were abolished in mDC-depleted mice. The same phenotype was observed in TLR9−/− mice, which lacked IL-12 expression by mDCs, and in IL-12−/− mice, whereas IFN-α/β receptor−/− mice showed only a minor reduction of NK cell IFN-γ expression. This study provides the first direct evidence that mDCs are essential for eliciting NK cell cytotoxicity and IFN-γ release in vivo and demonstrates that TLR9, mDCs, and IL-12 are functionally linked to the activation of NK cells in visceral leishmaniasis.
Macrophages were reported to be strong producers of interferon ␥ (IFN-␥) after stimulation by interleukin 12 (IL-12) plus IL-18, which gave rise to a novel concept of autocrine macrophage activation. Here, we show that peritoneal exudate and bone marrowderived mouse macrophages generated by conventional techniques contain small quantities of CD11b ؉ CD11c ؉ CD31 ؉ DX5 ؉ NK1. IntroductionInterferon ␥ (IFN-␥) is a key cytokine in innate and adaptive immunity. It activates macrophages for the expression of surface major histocompatibility class II (MHC II) antigens, costimulatory molecules, and inflammatory mediators and for tumoricidal and antimicrobial activity. It regulates the proliferation and death of T lymphocytes, allows the development of a type 1 T-helper cell response, and is essential for the control of many intracellular pathogens in vivo. The functions of IFN-␥ have been most convincingly demonstrated in studies that used neutralizing antibodies to IFN-␥ or IFN-␥-or IFN-␥ receptor-deficient mice. [1][2][3] IFN-␥ is typically produced by natural killer (NK) cells and CD4 ϩ T cells, but also by CD8 ϩ T cells, NKT cells, ␥␦ T cells, and B cells. [1][2][3][4][5][6] More recently, myeloid cells were reported to secrete IFN-␥. 5 Interleukin 12 (IL-12) strongly induced IFN-␥ in bone marrow (BM)-derived as well as splenic dendritic cells (DCs). 7-11 IFN-␥ mRNA or protein was also detected in mouse or human macrophages of the lung, 12,13 spleen, 7 bone marrow, 14-16 resting peritoneum, 17 or of the peritoneal exudate following a sterile inflammation. 14,18,19 Lipopolysaccharide (LPS), 14,18,20 type I interferon, 20 7,12,17 15,16,19,21 or IFN-␥ itself, 22 as well as infection with different pathogens (eg, mycobacteria, Legionella, Salmonella, and chlamydia), 12,13,23,24 stimulated the expression of IFN-␥ mRNA or protein or both in macrophages. The induction of IFN-␥ by IL-12/IL-18 received particular attention because IL-12 and IL-18 are products of macrophages, which raised the possibility of an autocrine activation loop in macrophages during the early phase of an infection. 5,15 In accordance with this hypothesis, stimulation of BM-derived macrophages (BMM⌽s) with IL-12 plus IL-18 led to the induction of inducible nitric oxide (NO) synthase and to the killing of intracellular Toxoplasma gondii, both of which were dependent on the endogenously produced IFN-␥. 15,16 The aim of the present study was to characterize the surface phenotype of macrophage subpopulations that express high amounts of IFN-␥ protein. Using multicolor flow cytometry, high-stringency cell sorting, intracellular cytokine staining (ICS), and transgenic mice devoid of any lymphoid cells, the analysis of peritonealexudate macrophages (PE-M⌽s) and BMM⌽s revealed the unexpected presence of minute populations of NK cells carrying myeloid markers or of CD8 ϩ T cells, respectively. These lymphoid cells fully accounted for the previously described strong production of IFN-␥ protein by the macrophage populations after stimulation with IL-12 and IL-1...
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