Cytokine and contact-dependent activation of natural killer cells by influenza A or Sendai virus-infected macrophages

Jukka Sirén, Timo Sareneva, Jaana Pirhonen, Mari Strengell, Ville Veckman, Ilkka Julkunen and Sampsa Matikainen

Department of Microbiology, National Public Health Institute, Helsinki, Finland

Correspondence
Jukka Sirén
jukka.siren{at}ktl.fi


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
NK cells participate in innate immune responses by secreting gamma interferon (IFN-{gamma}) 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-{gamma} 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-{gamma} gene in NK cells. IL12R{beta}2, 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-{alpha}. In contrast to IL12R{beta}2, expression of WSX-1/TCCR, a receptor for IL27, was reduced in NK cells in response to virus-induced IFN-{alpha}. 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-{alpha} in the activation of innate immunity.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
NK 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-{gamma}) drives Th1-type immunity and activates macrophages. Activated macrophages in turn produce IFN-{alpha}, 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-{gamma} production (Biron, 1998; Biron et al., 1999; Matikainen et al., 2001; Nguyen et al., 2002; Orange & Biron, 1996) resulting in a positive feedback loop between NK cells and macrophages and, ultimately, in effective innate immune responses. Among other antigen-presenting cell-derived cytokines with potential regulatory function on NK cells are the novel cytokines IL23 (Oppmann et al., 2000) and IL27 (Pflanz et al., 2002).

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 (Moretta et al., 2001). 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 virus-infected macrophages and NK cells. We demonstrate that both cell–cell contact and macrophage-derived IFN-{alpha} regulates NK cell responses during early stages of infection.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Isolation of monocytes, macrophages and NK cells.
Human primary monocytes, macrophages and NK cells were obtained from leukocyte-rich buffy coats from healthy blood donors (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland). PBMCs were isolated from buffy coats by density-gradient centrifugation using Ficoll-Paque (Amersham Pharmacia Biotech). Monocytes were allowed to adhere to plastic six-well plates (Falcon Multiwell; BD Biosciences) and were cultured for 1 day in macrophage SFM medium (Life Technologies) supplemented with 0·6 µg penicillin ml–1 and 60 µg streptomycin ml–1. To obtain macrophages, plastic-adhered monocytes were cultured for 1 week in macrophage SFM medium supplemented with antibiotics and GM-CSF (10 ng ml–1; Biosource). NK cells were purified from non-adherent PBMCs through a nylon wool column and two-step density-gradient centrifugation by Percoll (Amersham Pharmacia Biotech), followed by purification with immunomagnetic beads with anti-CD3, anti-CD14 and anti-CD19 antibodies (Abs) (Dynal). As determined by flow cytometry with anti-CD16 or anti-CD56 Abs, the procedure resulted in >90 % pure NK cells.

NK-92 cell line.
Human NK-92 cell line (Gong et al., 1994) was maintained in continuous culture in MEM{alpha} medium supplemented with 12 % horse serum (Life Technologies), 12 % FCS (Integro), 0·2 mM i-inositol, 20 mM folic acid, 40 mM 2-mercaptoethanol, 2 mM L-glutamine, antibiotics and 100 IU IL2 ml–1 (R&D Systems). Before co-culture or cytokine stimulations, NK-92 cells were cultured in IL2-free RPMI 1640 medium for 18 h.

Cytokines and virus stocks.
Affinity-purified human leukocyte IFN-{alpha} was provided by the Finnish Red Cross Blood Transfusion Service and used at 100 IU ml–1. Human recombinant (r)IL12 was purchased from R&D Systems and used at 5 ng ml–1. Neutralizing Abs against human IFN-{alpha}/{beta} have been described previously (Mogensen et al., 1975). Human pathogenic influenza virus A (strain Beijing A/353/89, H3N2) and murine Sendai virus (strain Cantell) were grown as described previously (Pirhonen et al., 1999). Monocytes/macrophages were infected with 12·8 haemagglutination units ml–1 influenza A and 20 haemagglutination units ml–1 Sendai virus.

Co-culture experiment.
The adherent monocytes or macrophages were infected with influenza A or Sendai virus. After 6 h, cells were washed with PBS and the cell culture medium was replaced with RPMI 1640 containing NK-92 or primary human NK cells. The NK cell-to-target cell ratio in co-cultures was 1 : 2. Where indicated, a porous membrane (Transwell; Corning Costar) was used to prevent a direct physical contact between cells. NK cells were harvested after 3 or 6 h of co-culture by collecting the non-adherent NK cells from the co-culture suspension, and samples for Northern blotting and ELISA were prepared. In each experiment, macrophages from three to four donors were used.

RNA isolation and Northern blot analysis.
Total cellular RNA was isolated by RNeasy kit (Qiagen) according to manufacturer's instructions. Samples containing equal amounts of RNA (10 µg) were size-separated on 1 % formaldehyde/agarose gels, transferred to a nylon membrane (Hybond; Amersham) and hybridized with IFN-{gamma} (Sareneva et al., 1994), T-bet (Strengell et al., 2002), IL12R{beta}2 (Strengell et al., 2002), IL18R{alpha} (Sareneva et al., 2000), MICB and WSX-1/TCCR cDNA probes. Probes for MICB and WSX-1/TCCR were cloned from total cellular RNA obtained from Sendai virus-infected macrophages or IL15- and IL21-treated NK-92 cells by RT-PCR using oligonucleotides 5'-CTGCTACATGGATCCCAGCGGGAA-3' and 5'-TTTGCAGGATCCAACAACAATAAA-3' (MICB), 5'-CCCCTCCAGGGATCCCCGCCATAG-3' and 5'-ACCGGCGGGGGATCCATCTCCTCC-3' (WSX-1/TCCR). Ethidium bromide staining of rRNA bands was used to ensure equal RNA loading. The probes were labelled with [{alpha}-32P]dATP [3000 Ci mmol–1 (111 TBq mmol–1); Amersham] using a random-primed DNA labelling kit (Boehringer Mannheim). The membranes were hybridized (Ultrahyb; Ambion) and washed twice at 42 °C and once at 60 °C in 1x SSC/0·1 % SDS for 30 min and exposed to Kodak AR X-omat films at –70 °C using intensifying screens.

IFN-{gamma} ELISA.
The amount of IFN-{gamma} in cell-culture supernatants was measured by ELISA (Diaclone).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Co-culture with virus-infected macrophages activates IFN-{gamma} and T-bet gene expression in NK-92 cells
To investigate the activation of NK cells by virus-infected macrophages, we cultured NK-92 cells with influenza A or Sendai virus-infected macrophages (Fig. 1). NK-92 cells were harvested after 3 or 6 h of co-culture and total cellular RNA was isolated and analysed by Northern blot. Co-culture with virus-infected macrophages resulted in a strong IFN-{gamma} gene expression in NK-92 cells, whereas no IFN-{gamma} mRNA was detected in NK-92 cells cultured with non-infected macrophages. T-bet is a Th1-specific transcription factor capable of transactivating the IFN-{gamma} gene and inducing Th1 differentiation of T cells (Szabo et al., 2000). Therefore, as another marker of NK-92 cell activation, we analysed T-bet gene expression in NK-92 cells co-cultured with virus-infected macrophages. Like IFN-{gamma}, T-bet mRNA expression was enhanced in NK-92 cells cultured with virus-infected macrophages (Fig. 1). Taken collectively, these results demonstrated that NK-92 cells are efficiently activated when they interact with virus-infected cells.



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Fig. 1. Virus-infected macrophages induce IFN-{gamma} and T-bet gene expression in NK cells. NK-92 cells were cultured together with influenza A or Sendai virus-infected macrophages. Co-culture with non-infected macrophages was used as a control. After 3 or 6 h of co-culture, NK-92 cells were harvested and total cellular RNA from NK-92 cells was isolated. Northern blot analysis with IFN-{gamma} and T-bet probes was performed. Ethidium bromide staining of rRNA bands was used to control equal RNA loading. A representative of three independent experiments is shown.

 
Cell–cell contact augments cytokine-induced IFN-{gamma} production in NK cells
Since the interaction with influenza A or Sendai virus-infected macrophages results in enhanced expression of the IFN-{gamma} gene in NK-92 cells (Fig. 1), we analysed the amount of IFN-{gamma} from co-culture supernatants by ELISA (Fig. 2). NK-92 cells were cultured with virus-infected or non-infected macrophages for 6 h (Fig. 2, +contact). Alternatively, to study the role of NK cell-activating receptors in NK-92 cell IFN-{gamma} expression, we used a porous membrane to prevent direct cellular interactions between macrophages and NK-92 cells (Fig. 2, –contact). Non-stimulated or IFN-{alpha}-stimulated NK-92 cells were used as controls. Some IFN-{gamma} production was detectable when NK-92 cells were co-cultured with non-infected macrophages. However, as shown in Fig. 2(a), NK-92 cells responded to virus-infected macrophages by secreting very high amounts of IFN-{gamma} into the cell culture supernatants. Preventing cell–cell contact between NK-92 cells and virus-infected macrophages dramatically reduced the amount of IFN-{gamma} in the co-culture supernatants (Fig. 2a). The fact that NK-92 cells produced IFN-{gamma}, albeit in reduced amounts, without direct cell–cell contact indicated that soluble factors derived from macrophages are involved in the induction of the IFN-{gamma} gene and, consequently, in IFN-{gamma} secretion from NK-92 cells. Thus, our results suggested that macrophage-derived cytokines induce IFN-{gamma} secretion from NK-92 cells and that IFN-{gamma} expression is further enhanced by cell–cell contact between NK-92 cells and virus-infected macrophages. We also studied IFN-{gamma} production by human primary NK cells in response to influenza A or Sendai virus-infected autologous monocytes (Fig. 2b). Like NK-92 cells, primary human NK cells responded efficiently to their virus-infected target cells. The amount of IFN-{gamma} produced was greater in primary NK cells co-cultured with Sendai virus-infected monocytes compared with NK cells co-cultured with influenza A virus-infected monocytes. These results demonstrated that cellular interactions between virus-infected target cells and virus-induced cytokines collectively induced maximal IFN-{gamma} production and secretion from NK cells.



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Fig. 2. Cell–cell contact enhances IFN-{gamma} production in NK cells. (a) NK-92 cells were cultured with non-infected or virus-infected macrophages (+contact) for 6 h. Alternatively, a porous membrane was used to prevent direct contact between the cells (–contact). (b) Human primary NK cells were cultured for 6 h with non-infected or virus-infected autologous monocytes (+contact). Non-stimulated or IFN-{alpha} (100 IU ml–1)-stimulated NK-92 and human primary NK cells were used as controls. The amount of IFN-{gamma} in cell culture supernatants was measured by ELISA. The mean±SD of three separate experiments (a) or four donors (b) is shown.

 
To study more closely the role of cellular interactions in NK-92 cell activation in our co-culture model, we analysed IFN-{gamma} and T-bet mRNA expression from NK-92 cells co-cultured with influenza A or Sendai virus-infected macrophages (Fig. 3). Where indicated, the cell–cell contact was allowed to happen (Fig. 3, +contact) or was prevented with a porous membrane (Fig. 3, –contact). Expression of IFN-{gamma} mRNA was high in NK-92 cells cultured with virus-infected macrophages and IFN-{gamma} gene expression was clearly reduced in NK-92 cells whose contact with macrophages was prevented. This was in accordance with the results presented in Fig. 2, highlighting the importance of cellular interactions in NK cell IFN-{gamma} production during virus infection. In contrast to IFN-{gamma} expression, T-bet mRNA expression was even higher in NK-92 cells cultured with virus-infected macrophages in the absence of cellular contacts. This suggested that T-bet expression in NK-92 cells is positively regulated, mainly by cytokines.



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Fig. 3. Cell–cell contact regulates NK cell responses. NK-92 cells were cultured with influenza A or Sendai virus-infected macrophages (+contact) for 3 h. Where indicated, a porous membrane was used to prevent cell–cell contact (–contact). Co-culture with non-infected macrophages was used as a control. After 3 h, NK-92 cells were harvested, total cellular RNA was isolated and Northern blot analysis with the indicated cDNA probes was performed. The experiment was done twice with similar results.

 
IL12, IL18, IL23 and IL27 are macrophage-derived cytokines that are known to enhance IFN-{gamma} production in NK cells (Pflanz et al., 2002; Trinchieri, 2003; Parham et al., 2002). Therefore, we analysed IL12R, IL18R, IL23R and IL27R mRNA expression in NK-92 cells co-cultured with virus-infected macrophages (Fig. 3). Expression of the IL12R{beta}2 and IL18R{alpha} genes was enhanced in NK-92 cells co-cultured with macrophages that were infected with influenza A or Sendai virus. Similar to T-bet, upregulation of these genes was enhanced by inhibition of cell–cell contact between NK-92 cells and macrophages (Fig. 3). IL23R-specific chain was not expressed in NK cells co-cultured with virus-infected macrophages (data not shown). In contrast to IL12R and IL18R, IL27R (WSX-1/TCCR) mRNA levels were downregulated in NK-92 cells co-cultured with virus-infected macrophages and inhibition of cell–cell contact did not abolish IL27R downregulation in NK-92 cells (Fig. 3). Thus, we concluded that expression of IL12R, IL18R and IL27R in NK cells is mainly regulated by macrophage-derived cytokines and that cellular interactions seem to inhibit somewhat cytokine-induced IL12R and IL18R expression in NK cells.

Macrophage-derived IFN-{alpha} regulates NK cell gene expression
Influenza A and Sendai viruses are potent inducers of IFN-{alpha} from macrophages during early times of infection (Pirhonen et al., 1999; Sareneva et al., 1998). To study the role of IFN-{alpha} in the regulation of NK cell gene expression, we added saturating amounts of neutralizing anti-IFN-{alpha}/{beta} Abs to NK cell and macrophage co-cultures. This resulted in a reduction in IFN-{gamma}, T-bet and IL12R{beta}2 mRNA expression in NK-92 cells (Fig. 4). Similarly, anti-IFN-{alpha}/{beta} Abs inhibited the downregulation of WSX-1/TCCR (IL27R) mRNA expression in NK-92 cells co-cultured with influenza A virus-infected macrophages. In contrast, anti-IFN-{alpha}/{beta} Abs had no effect on IL27R mRNA downregulation in NK-92 cells co-cultured with Sendai virus-infected macrophages (Fig. 4).



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Fig. 4. Macrophage-derived IFN-{alpha} regulates NK cell gene expression. NK-92 cells were cultured with influenza A or Sendai virus-infected macrophages with or without neutralizing anti-IFN-{alpha}/{beta} Abs. After 3 h, NK-92 cells were collected and total cellular RNA was analysed by Northern blotting with the indicated cDNA probes. Ethidium bromide staining of rRNA bands was used to control equal RNA loading.

 
In addition to IFN-{alpha}, Sendai virus-infected macrophages secrete IL12 (Pirhonen et al., 2002). Therefore, in addition to direct cellular interactions and macrophage-derived IFN-{alpha}, IL12 may influence the gene expression of NK cells co-cultured with Sendai virus-infected macrophages. To clarify the role of IFN-{alpha} and IL12 in NK cell gene expression, we stimulated NK-92 cells or human primary NK cells with IFN-{alpha} or IL12 (Fig. 5). IFN-{gamma} and T-bet gene expression was enhanced in response to IFN-{alpha} and IL12 in both cell types. Similarly, IFN-{alpha} and IL12 enhanced IL12R{beta}2 mRNA expression in NK-92 cells. Furthermore, both IFN-{alpha} and IL12 downregulated WSX-1/TCCR (IL27R) in NK-92 cells and in primary NK cells.



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Fig. 5. IFN-{alpha} and IL12 induce changes in the NK cell gene expression profile. NK-92 cells (a) or human primary NK cells from eight different blood donors (b) were treated with IFN-{alpha} (100 IU ml–1) or IL12 (5 ng ml–1) for the indicated times. Cells were collected and total cellular RNA was isolated and analysed by Northern blotting with the indicated cDNA probes. Ethidium bromide staining of rRNA bands was used to control equal RNA loading. The experiment was done twice with similar results.

 
Influenza A and Sendai viruses induce MICB gene expression in macrophages
The result showing that both cytokines and direct cellular interactions between NK-92 cells and virus-infected macrophages regulate NK-92 cell IFN-{gamma} expression led us to investigate the mechanisms of NK-92 cell activation. Influenza A and Sendai viruses elicit the production of NK cell-activating cytokines from human macrophages. However, molecules expressed on the surface of virus-infected or transformed cells also regulate NK cell activation. NK-92 cells express NK cell-activating receptor NKG2D (data not shown). Therefore, we determined the expression of NKG2D ligands MICA/B and ULBPs in influenza A or Sendai virus-infected macrophages (Fig. 6). Both influenza A and Sendai viruses induced MICB mRNA expression in macrophages and influenza A but not Sendai virus-induced MICB mRNA expression was inhibited by neutralizing antibodies against IFN-{alpha}/{beta} (Fig. 6a). In addition, stimulation of macrophages with IFN-{alpha} alone upregulated MICB mRNA expression in human macrophages (Fig. 6b). These results indicated that virus-induced IFN-{alpha}/{beta} regulates the expression of MIC genes in macrophages during virus-infections. However, type I IFNs are not the sole regulators of MIC genes as IFN-{alpha}/{beta} neutralization did not affect MICB upregulation in Sendai virus-infected macrophages. ULBP mRNA expression was not detected in influenza A or Sendai virus-infected macrophages (data not shown). Therefore, we concluded that IFN-{alpha}/{beta}-induced MICA/B on virus-infected macrophages is likely to promote NK cell IFN-{gamma} production.



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Fig. 6. (a) MICB gene expression is activated in virus-infected macrophages. Macrophages were infected with influenza A or Sendai virus. After 8 h, cells were harvested and samples for Northern blot were prepared and analysed with the MICB cDNA probe. Where indicated neutralizing antibodies against IFN-{alpha}/{beta} were used. (b) IFN-{alpha} activates MICB mRNA expression in macrophages. Macrophages were treated with IFN-{alpha} (100 IU ml–1) for the indicated times after which total cellular RNA was isolated and analysed with the MICB cDNA probe.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
NK cells respond rapidly to microbial infections by destroying virus-infected cells and by secreting IFN-{gamma}, which activates macrophages and dendritic cells (DCs) and shapes the adaptive immune responses toward Th1-type immunity. A dialogue between macrophages and NK cells, via cytokines produced by both cell types, results in effective early immune responses. In this report we have studied the interaction of NK cells and influenza A or Sendai virus-infected macrophages. The significant mortality and morbidity caused by influenza viruses makes them important pathogens and defining the exact mechanisms how cells of the immune system respond to these viruses is important. However, different viruses induce different cytokine responses; therefore, we included Sendai virus, another negative-stranded virus, in our studies. Although it is a respiratory pathogen in animals, the Sendai virus-induced cytokine pattern has been well characterized in human macrophages (Pirhonen et al., 1999, 2002). Our results showed that during influenza A or Sendai virus infection, NK cell activity was regulated by macrophage-derived cytokines and via cellular interactions between NK cells and macrophages.

Influenza A and Sendai virus-infected macrophages stimulated rapid activation of the IFN-{gamma} gene and protein expression in NK-92 cells. Similarly, primary human NK cells responded to virus-infected monocytes by secreting IFN-{gamma}. IFN-{alpha} and IL12 are potent inducers of IFN-{gamma} production in T and NK cells (Hunter et al., 1997; Matikainen et al., 2001; Micallef et al., 1996; Okamura et al., 1995; Sareneva et al., 1998). Influenza A and Sendai viruses readily induce the secretion of IFN-{alpha} from human macrophages. However, the IFN-{alpha}-inducing activity of these viruses differs, as Sendai virus is a more potent inducer of IFN-{alpha} than influenza A virus (Pirhonen et al., 1999). This is most likely due to the influenza A virus NS1 protein, which antagonises IFN-{alpha}/{beta} production in infected cells (Garcia-Sastre et al., 1998; Wang et al., 2000). In addition, Sendai but not influenza A virus-infected macrophages secrete IL12 (Pirhonen et al., 2002), which further increases IFN-{gamma} expression in NK cells. Neutralizing anti-IFN-{alpha}/{beta} Abs reduced IFN-{gamma} mRNA synthesis in NK-92 cells cultured with virus-infected macrophages, suggesting that, in our model system, virus-induced IFN-{alpha}/{beta} has a major role in the activation of the IFN-{gamma} gene in NK cells. However, inhibition of cell–cell contact clearly reduced IFN-{gamma} production in NK-92 cells, indicating that soluble cytokines and cellular interactions synergistically enhance NK cell IFN-{gamma} production.

DCs have been found to stimulate NK cells through activating the NK cellular receptors Nkp30, Nkp46 (Ferlazzo et al., 2002; Spaggiari et al., 2001) and NKG2D (Jinushi et al., 2003). NK-92 cells do express these receptors (data not shown). Cellular ligands for Nkp30 and Nkp46 are not known, but Nkp46 has been shown to interact with the haemagglutinins on virus-infected cells (Mandelboim et al., 2001). Thus, influenza haemagglutinin or Sendai haemagglutinin–neuraminidase expressed on virus-infected macrophages could contribute to NK cell IFN-{gamma} production in our model. NK cell-activating receptor NKG2D recognizes stress-inducible molecules MICA and MICB (Bauer et al., 1999) as well as ULBPs (Cosman et al., 2001; Sutherland et al., 2002). We showed that in macrophages MICB gene expression was enhanced in response to virus-induced IFN-{alpha} (Fig. 6). Similarly, the expression of MIC genes is upregulated by IFN-{alpha} in human DCs (Jinushi et al., 2003). In our infection model neutralizing anti-IFN-{alpha}/{beta} Abs inhibited MICB mRNA expression in macrophages infected with influenza A virus, but not with Sendai virus. This is in accordance with our previous results showing that Sendai virus can directly activate a number of genes independently of IFN-{alpha}/{beta} (Matikainen et al., 2000; Pirhonen et al., 2001).

T-bet is a transcription factor that enhances IFN-{gamma} production and promotes Th1 immune response (Szabo et al., 2000, 2002). T-bet gene expression was enhanced in NK-92 cells co-cultured with virus-infected macrophages. T-bet expression was induced by macrophage-derived soluble cytokines since inhibition of cell–cell contact between virus-infected macrophages and NK-92 cells did not diminish T-bet mRNA levels in NK-92 cells. Furthermore, neutralizing anti-IFN-{alpha}/{beta} Abs clearly inhibited T-bet gene expression in NK-92 cells co-cultured with virus-infected macrophages. These results show that, in addition to IFN-{gamma} (Afkarian et al., 2002; Lighvani et al., 2001) and IL12 (Szabo et al., 2000), IFN-{alpha} also upregulates T-bet gene expression in NK cells and thus enhances innate immune responses.

IL18R{alpha} mRNA expression was upregulated in NK-92 cells co-cultured with virus-infected macrophages. IFN-{alpha} and IL12, in addition to activating IFN-{gamma} gene expression, also enhance IL18R and Myd88 adapter mRNA expression in human NK and T cells (Sareneva et al., 2000). The results presented in this report further suggest that IL18R gene expression is activated during virus infection via cytokines. In this way, IFN-{alpha} and IL12 also indirectly enhance IFN-{gamma} production by NK cells and, consequently, promote innate and Th1 immune responses.

IL12 is the major cytokine driving Th1 differentiation and inducing IFN-{gamma} production. Recently two cytokines, IL23 and IL27, which bear functional and structural resemblance to IL12, have been characterized (Oppmann et al., 2000; Pflanz et al., 2002). Like IL12, IL23 and IL27 promote a Th1 response by inducing IFN-{gamma} production from NK and T cells (Pflanz et al., 2002; Trinchieri, 2003; Parham et al., 2002). We have shown that Sendai but not influenza A virus-infected macrophages produce IL12 and IL23 (Pirhonen et al., 2002). In addition, both IL27 subunits, EBI3 and p28, are expressed by activated antigen-presenting cells (Pflanz et al., 2002). Therefore, we studied IL12R, IL23R and IL27R gene expression in NK-92 cells co-cultured with virus-infected macrophages. IL12R{beta}2 mRNA expression in NK-92 cells was clearly upregulated by macrophage-derived cytokines, since inhibition of cell–cell contact did not abolish IL12R{beta}2 mRNA expression. Furthermore, IFN-{alpha} and IL12 enhanced IL12R{beta}2 mRNA synthesis in NK-92 cells, as has been previously reported in T cells (Rogge et al., 1997). Parham et al. (2002) have shown, using RT-PCR-based methods, that certain NK cell lines express IL23R. However, we were not able to detect IL23R mRNA expression by Northern blot analysis from NK-92 or primary human NK cells (data not shown). This suggests that IL23R is expressed at very low levels in NK cells. A recent report shows that IL23 is a critical and broad regulator of late-stage inflammatory processes (Cua et al., 2003). Therefore, it is conceivable that IL23R is expressed at a low level in NK cells.

WSX-1/TCCR is a receptor for IL27 (Pflanz et al., 2002). In contrast to IL12R{beta}2, IL27R mRNA levels were downregulated in NK-92 cells co-cultured with influenza A or Sendai virus-infected macrophages. This inhibition was dependent on macrophage-derived cytokines. Neutralizing anti-IFN-{alpha}/{beta} Abs abolished WSX-1/TCCR mRNA downregulation in NK-92 cells co-cultured with influenza A virus-infected macrophages, emphasizing the role of IFN-{alpha} in modulation of cytokine responsiveness during virus infection. IL27 drives clonal expansion of naïve CD4+ T cells (Pflanz et al., 2002), suggesting that IL27 acts at early times of anti-microbial immune responses. From this perspective, it is conceivable that a rapid downregulation of WSX-1/TCCR occurs in response to IFN-{alpha} and IL12. Previously, it has been shown that WSX-1/TCCR expression is downregulated during IL12-induced Th1 cell differentiation (Chen et al., 2000). In conclusion, our results suggest that virus-infected macrophages enhance the IL12 response and diminish the IL27 response in NK cells during virus infection.

Taken together, our results demonstrate the diverse and rapid response of NK cells to virus-infected macrophages. Both macrophage-derived cytokines and cellular interactions stimulate NK cell activation and effector functions, leading to NK cell activation and, ultimately, to an effective immune response to eradicate an invading pathogen.


   ACKNOWLEDGEMENTS
 
This work was supported by the Medical Research Council of the Academy of Finland, the Sigrid Juselius Foundation and the Finnish Cancer Foundation. The authors wish to thank Hanna Valtonen, Mari Aaltonen and Teija Westerlund for expert technical assistance.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 11 March 2004; accepted 3 May 2004.