NK and T cell-derived IFN-γ is a key cytokine that stimulates innate immune responses and directs adaptive T cell response toward Th1 type. IL-15, IL-18, and IL-21 have significant roles as activators of NK and T cell functions. We have previously shown that IL-15 and IL-21 induce the expression of IFN-γ, T-bet, IL-12Rβ2, and IL-18R genes both in NK and T cells. Now we have studied the effect of IL-15, IL-18, and IL-21 on IFN-γ gene expression in more detail in human NK and T cells. IL-15 clearly activated IFN-γ mRNA expression and protein production in both cell types. IL-18 and IL-21 enhanced IL-15-induced IFN-γ gene expression. IL-18 or IL-21 alone induced a modest expression of the IFN-γ gene but a combination of IL-21 and IL-18 efficiently up-regulated IFN-γ production. We also show that IL-15 activated the binding of STAT1, STAT3, STAT4, and STAT5 to the regulatory sites of the IFN-γ gene. Similarly, IL-21 induced the binding of STAT1, STAT3, and STAT4 to these elements. IL-15- and IL-21-induced STAT1 and STAT4 activation was verified by immunoprecipitation with anti-phosphotyrosine Abs followed by Western blotting with anti-STAT1 and anti-STAT4 Abs. IL-18 was not able to induce the binding of STATs to IFN-γ gene regulatory sites. IL-18, however, activated the binding of NF-κB to the IFN-γ promoter NF-κB site. Our results suggest that both IL-15 and IL-21 have an important role in activating the NK cell-associated innate immune response.

IL-21 is a recently characterized T cell-derived cytokine that regulates NK and T cell function. IL-21R shares the common γ-chain (γc) with the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15. Despite the same γc, these cytokines have different effects on diverse cells. In this study, we have studied IL-15- and IL-21-induced gene expression in human primary NK and T cells and the NK-92 cell line. Both IL-15 and IL-21 rapidly induced mRNA synthesis for IFN-γ, T-bet, IL-2Rα, IL-12Rβ2, IL-18R, and myeloid differentiation factor 88 (MyD88), the genes that are important in activating innate immunity and Th1 response. IL-15 induced STAT5 DNA binding to the IL-2Rα IFN-γ-activated sequence (GAS), MyD88 GAS, and c-sis-inducible elements, whereas IL-21 induced STAT3 DNA binding to MyD88 GAS and c-sis-inducible elements. IL-21-induced STAT3 activation was verified by immunoprecipitation and Western blotting with anti-phosphotyrosine Ab. In addition, pretreatment of NK-92 cells with IL-15 or IL-21 strongly enhanced IL-12-induced STAT4 DNA binding to IL-2Rα GAS. The induction of IFN-γ, T-bet, IL-12Rβ2, and IL-18R gene expression in NK cells, along with STAT3 activation, suggests that IL-21 is involved in the activation of innate immune responses. Moreover, the enhanced transcription of these genes in T cells establishes a significant role for IL-21 also in the Th1 response.

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TLRs are innate immune receptors that recognize pathogen-associated structures. Binding of ligands to different TLRs can induce the production of proinflammatory cytokines in a synergistic manner. We have analyzed the molecular mechanisms of synergy in TLR ligand-stimulated human monocyte-derived macrophages and dendritic cells (moDCs). Stimulation of moDCs with the TLR8 ligand together with the TLR3 or TLR4 ligand led to synergistic IL-6, IL-10, IL-12, and TNF-alpha mRNA expression and cytokine production. DNA-binding assays showed that TLR3 and TLR8 stimulation induced binding of multiple IFN regulatory factor (IRF) and STAT transcription factors to the IL-12p35 gene promoter IFN-stimulated response element in moDCs and macrophages but with different binding profiles and kinetics. We also demonstrate that NF-kappaB, MAPKs and PI-3K pathways have an important role in TLR-induced cytokine gene expression, as pharmacological inhibitors of these signaling pathways inhibited TLR3, TLR4, and TLR8 ligand-induced cytokine mRNA expression and protein production. Especially, synergistic IL-12p70 production was abolished completely in NF-kappaB, MAPK p38, and PI-3K inhibitor-treated moDCs. Our data suggest that TLR-dependent, synergistic cytokine gene expression results from enhanced activation and cooperation among NF-kappaB, IRF, MAPK, PI-3K, and STAT signaling pathways.

NK cells participate in innate immune responses by secreting gamma interferon (IFN-c) and by destroying virus-infected cells. Here the interaction between influenza A or Sendai virus-infected macrophages and NK cells has been studied. A rapid, cell-cell contact-dependent production of IFN-c from NK cells cultured with virus-infected macrophages was observed. Expression of the MHC class I-related chain B (MICB) gene, a ligand for NK cell-activating receptor NKG2D, was upregulated in virus-infected macrophages suggesting a role for MICB in the activation of the IFN-c gene in NK cells. IL12Rb2, IL18R and T-bet mRNA synthesis was enhanced in NK cells cultured with virus-infected macrophages. Upregulation of these genes was dependent on macrophage-derived IFN-a. In contrast to IL12Rb2, expression of WSX-1/TCCR, a receptor for IL27, was reduced in NK cells in response to virus-induced IFN-a. In conclusion, these results show that virus-infected macrophages activate NK cells via cytokines and direct cellular interactions and further emphasize the role of IFN-a in the activation of innate immunity. INTRODUCTIONNK cells function as a key factor in immediate immune responses to intracellular pathogens, such as viruses (Trinchieri, 1989). The vital role of NK cells in controlling virus infections was demonstrated in a patient completely lacking NK cells and sustaining severe herpesvirus infections (Biron et al., 1989). Besides their lytic potential, activated NK cells secrete cytokines and chemokines (Cooper et al., 2001). NK cell-derived gamma interferon (IFN-c) drives Th1-type immunity and activates macrophages. Activated macrophages in turn produce IFN-a, interleukin (IL)12 and IL18 (Biron, 1999; Pirhonen et al., 1999;Sareneva et al., 1998;Trinchieri, 1997). These cytokines regulate NK cell activation and IFN-c production (Biron, 1998;Biron et al., 1999;Matikainen et al., 2001;Nguyen et al., 2002;Orange & Biron, 1996) Besides cytokines, cellular interactions regulate NK cell effector functions. NK cells express several inhibitory receptors specific for MHC class I molecules (Colonna, 1996;Long, 1999;Moretta et al., 1996;Moretta & Moretta, 1997;Ravetch & Lanier, 2000), which are frequently downregulated in virus-infected cells (Tortorella et al., 2000). In addition, activating receptors have been identified in NK cells. In humans these include the natural cytotoxicity receptors (NCRs) Nkp30, Nkp44 and Nkp46 and NKG2D . Cellular ligands for NCRs are not known, whereas NKG2D recognizes the stress-inducible MHC class I-related chain A and B (MICA/B) (Bauer et al., 1999;Wu et al., 1999) and UL-16 binding proteins (ULBPs) (Cosman et al., 2001;Sutherland et al., 2002). The balance between activating and inhibiting signals from cellular interactions and soluble mediators determines NK cell responses.Here we have investigated the interaction between virusinfected macrophages and NK cells. We demonstrate that both cell-cell contact and macrophage-derived IFN-a regulates NK cell responses during early stages of infection. METHODS...