Department of Microbiology, National Public Health Institute, Helsinki, Finland
Correspondence
Jukka Sirén
jukka.siren{at}ktl.fi
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ABSTRACT |
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INTRODUCTION |
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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 cellcell contact and macrophage-derived IFN- regulates NK cell responses during early stages of infection.
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METHODS |
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NK-92 cell line.
Human NK-92 cell line (Gong et al., 1994) was maintained in continuous culture in MEM
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 ml1 (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- was provided by the Finnish Red Cross Blood Transfusion Service and used at 100 IU ml1. Human recombinant (r)IL12 was purchased from R&D Systems and used at 5 ng ml1. Neutralizing Abs against human IFN-
/
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 ml1 influenza A and 20 haemagglutination units ml1 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- (Sareneva et al., 1994
), T-bet (Strengell et al., 2002
), IL12R
2 (Strengell et al., 2002
), IL18R
(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 [
-32P]dATP [3000 Ci mmol1 (111 TBq mmol1); 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- ELISA.
The amount of IFN- in cell-culture supernatants was measured by ELISA (Diaclone).
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RESULTS |
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Macrophage-derived IFN- regulates NK cell gene expression
Influenza A and Sendai viruses are potent inducers of IFN- from macrophages during early times of infection (Pirhonen et al., 1999
; Sareneva et al., 1998
). To study the role of IFN-
in the regulation of NK cell gene expression, we added saturating amounts of neutralizing anti-IFN-
/
Abs to NK cell and macrophage co-cultures. This resulted in a reduction in IFN-
, T-bet and IL12R
2 mRNA expression in NK-92 cells (Fig. 4
). Similarly, anti-IFN-
/
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-
/
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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DISCUSSION |
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Influenza A and Sendai virus-infected macrophages stimulated rapid activation of the IFN- gene and protein expression in NK-92 cells. Similarly, primary human NK cells responded to virus-infected monocytes by secreting IFN-
. IFN-
and IL12 are potent inducers of IFN-
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-
from human macrophages. However, the IFN-
-inducing activity of these viruses differs, as Sendai virus is a more potent inducer of IFN-
than influenza A virus (Pirhonen et al., 1999
). This is most likely due to the influenza A virus NS1 protein, which antagonises IFN-
/
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-
expression in NK cells. Neutralizing anti-IFN-
/
Abs reduced IFN-
mRNA synthesis in NK-92 cells cultured with virus-infected macrophages, suggesting that, in our model system, virus-induced IFN-
/
has a major role in the activation of the IFN-
gene in NK cells. However, inhibition of cellcell contact clearly reduced IFN-
production in NK-92 cells, indicating that soluble cytokines and cellular interactions synergistically enhance NK cell IFN-
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 haemagglutininneuraminidase expressed on virus-infected macrophages could contribute to NK cell IFN-
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-
(Fig. 6
). Similarly, the expression of MIC genes is upregulated by IFN-
in human DCs (Jinushi et al., 2003
). In our infection model neutralizing anti-IFN-
/
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-
/
(Matikainen et al., 2000
; Pirhonen et al., 2001
).
T-bet is a transcription factor that enhances IFN- 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 cellcell contact between virus-infected macrophages and NK-92 cells did not diminish T-bet mRNA levels in NK-92 cells. Furthermore, neutralizing anti-IFN-
/
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-
(Afkarian et al., 2002
; Lighvani et al., 2001
) and IL12 (Szabo et al., 2000
), IFN-
also upregulates T-bet gene expression in NK cells and thus enhances innate immune responses.
IL18R mRNA expression was upregulated in NK-92 cells co-cultured with virus-infected macrophages. IFN-
and IL12, in addition to activating IFN-
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-
and IL12 also indirectly enhance IFN-
production by NK cells and, consequently, promote innate and Th1 immune responses.
IL12 is the major cytokine driving Th1 differentiation and inducing IFN- 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-
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
2 mRNA expression in NK-92 cells was clearly upregulated by macrophage-derived cytokines, since inhibition of cellcell contact did not abolish IL12R
2 mRNA expression. Furthermore, IFN-
and IL12 enhanced IL12R
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
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-
/
Abs abolished WSX-1/TCCR mRNA downregulation in NK-92 cells co-cultured with influenza A virus-infected macrophages, emphasizing the role of IFN-
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-
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.
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ACKNOWLEDGEMENTS |
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Received 11 March 2004;
accepted 3 May 2004.