Toll-like receptors (TLRs) play a fundamental role in the recognition of bacteria and viruses. TLR3 is activated by viral dsRNA and polyinosinic-polycytidylic acid (poly(I:C)), a synthetic mimetic of viral RNA. We show that NK cells, known for their capacity to eliminate virally infected cells, express TLR3 and up-regulate TLR3 mRNA upon poly(I:C) stimulation. Treatment of highly purified NK cells with poly(I:C) significantly augments NK cell-mediated cytotoxicity. Poly(I:C) stimulation also leads to up-regulation of activation marker CD69 on NK cells. Furthermore, NK cells respond to poly(I:C) by producing proinflammatory cytokines like IL-6 and IL-8, as well as the antiviral cytokine IFN-γ. The induction of cytokine production by NK cells was preceded by activation of NF-κB. We conclude that the ability of NK cells to directly recognize and respond to viral products is important in mounting effective antiviral responses.
Natural killer cells are effector cells of the innate immune system with the ability to eliminate cancerous or virally infected cells by cytocidal action or induction of apoptosis (1). While the lack of NK cells leads to high susceptibility to viral infections in humans and rodents (2), the molecular mechanisms governing NK cells recognition of viral pathogens have just started to emerge. In contrast to B and T lymphocytes, NK cells do not express rearranged Ag-specific receptors, instead NK cell activity is thought to be regulated by a complex balance of inhibitory and stimulatory receptors (3, 4, 5). Normal autologous cells are protected from elimination by NK cells through interaction of their MHC class I molecules with inhibitory receptors on NK cells. Down-regulation of MHC class I molecules by virally infected cells is one mechanism that stimulates NK cell-mediated cytotoxicity (6). However, viruses have developed strategies to avoid that mechanism of elimination, and therefore other mechanisms of NK cell activation have evolved for effective host defense. Viral infections can indirectly activate NK cells by inducing the production of NK cell-activating cytokines from other host cells. IFN-α, a cytokine important for inhibition of viral replication, enhances NK cell-mediated cytotoxicity (2). IL-12 and IL-18 induce secretion of IFN-γ by NK cells (7, 8). IL-15 induces proliferation of NK cells, which is important for maintenance of sufficient numbers of activated NK cells (9, 10).
Recently, murine NK cells have been shown to recognize a mouse CMV (MCMV) protein via the activating receptor LY49H (11). Recognition of a virus-encoded molecule by a germline-encoded NK cell receptor is strikingly similar to other strategies of innate immune recognition, the recognition of pathogen-associated molecular patterns through TLRs, a family of evolutionary conserved receptors (12). Ten TLRs have been identified in humans and many of these have been shown to be activated by specific bacterial or viral products (13, 14, 15). TLR2 recognizes bacterial lipoproteins and several other components (12). TLR4 is activated by LPS (16), TLR5 by bacterial flagellin (17), and TLR9 by unmethylated bacterial CpG DNA (18). dsRNA, a viral product generated during the replication of many viruses, and its synthetic mimetic polyinosinic-polycytidylic acid (poly(I:C)) both activate TLR3 (19). Previously, dsRNA was thought to activate only intracellular targets, including the IFN-inducible dsRNA activated protein kinase (dsRNA-dependent protein kinase R) (20). However, the observation that cells from dsRNA-dependent protein kinase R-deficient mice still responded to dsRNA predicted the existence of another receptor (21, 22). TLR3−/− mice exhibit reduced inflammatory responses and reduced up-regulation of activation markers on splenic B-cells in response to poly(I:C) or viral dsRNA (19). In vivo inflammatory responses were also reduced in TLR3−/− mice, as demonstrated by resistance to poly(I:C)-induced shock combined with D-GalN sensitization (19).
TLR3 is known to be expressed by dendritic cells (DCs), macrophages, and epithelial cells (19, 23, 24, 25, 26). PBMC enriched for NK cells by depletion with anti-CD3, -CD14, -CD19, -CD36, and -IgE Abs have been shown to express moderate levels of TLR3 mRNA (27). Here we confirm expression of TLR3 mRNA on pure NK cells and extend that expression of TLR3 mRNA is up-regulated upon stimulation with poly(I:C). We show for the first time that in the absence of APCs NK cells directly sense dsRNA, which leads to enhanced NK cell-mediated cytotoxicity, up-regulation of activation marker CD69, and production of antiviral cytokines.
Materials and Methods
Human blood was collected from healthy volunteers and PBMC were isolated by Hypaque Ficoll density centrifugation. Cells were preincubated with human IgG (Sigma-Aldrich, St. Louis, MO; 1 μg/106 cells) for 10 min to block FcR followed by staining with Abs to CD56 (Immunotech, Westbrook, ME), CD3, CD14, and CD11c (all Caltag Laboratories, Burlingame, CA). Cell populations were isolated by an Elite Epics cytometer (Beckman-Coulter, Seattle, WA). CD3+ T cells and (CD56+CD3−) NK cells were isolated. To ensure that there were no contaminating DCs or monocytes in the NK cell population, a negative gate was applied on CD14+ cells and CD11c+ cells for the isolation of NK cells. For some experiments CD14+ cells and CD11c+ cells were collected. The isolated cell populations yielded a purity of at least 99%. To detect any CD14- or CD11c-positive cells in the isolated cell populations, NK cells and DC/monocyte populations were stained postsort with anti-CD14-ECD (PE-Texas Red) (Immunotech; PNIM2708) or anti-CD11c-biotin (Ancell, Bayport MN; 160-030) and SA-ECD (Immunotech; PNIM3326). Different Abs to CD14 and CD11c were used for sorting and postsort staining. Postsort analysis was performed on an Epics-XL instrument (Beckman-Coulter).
Cell culture and treatments
NK cells were cultured at 37°C, 5% CO2 in RPMI 1640, 10% FCS, 2 mM l-glutamine, 1 mM sodium pyruvate, penicillin and streptomycin, either unstimulated or activated with 50 μg/ml poly(I:C) (Pharmacia, Peapack, NJ) for the times indicated. For certain experiments, NK cells were treated with human IFN-αA (Sigma-Aldrich) at 1000 U/ml for 17 h. IFN-α was blocked using a neutralizing anti-IFN-α Ab (10 μg/ml; clone 9F3, generated at Genentech, described at in Ref.28) that was added during the culture of NK cells with either poly(I:C) or IFN-α. NK92 cells were obtained from and cultured as described by American Type Culture Collection (Manassas, VA). Stimulation of NK92 cells was performed at 0.5 × 106 cells/ml with 50 μg/ml poly(I:C) for 17 h. Poly(I:C) was tested by Limulus amebocyte lysate and contained <0.01 EU/mg.
Cytokine production was measured by ELISA (Quantikine, R&D Systems, Minneapolis, MN; IFN-α and IFN-β ELISA, PBL Biomedical Laboratories, Piscataway, NJ), BD Cytometric Bead Array (BD Biosciences, Mountain View, CA) or LUMINEX Beadlyte cytokine detection system (Upstate Biotechnology, Lake Placid, NY) according to the manufacturers’ protocol.
Purified NK cells were cultured for 18 h untreated or activated with either poly(I:C) or IL-12 (R&D Systems; catalog no. 219-IL), in the absence or presence of neutralizing anti-IL-12 Ab (R&D Systems; clone no. 24910.1). After the stimulation, NK cells were cocultured with target cells (Daudi or YAC-1; American Type Culture Collection) at indicated E:T cell ratios for 4 h at 37°C, 5% CO2. Cytotoxicity was determined either by chromium release assay or colorimetrically by measuring lactate dehydrogenase (LDH) release from the cytosol, using the LDH release cytotoxicity detection kit (Roche; catalog no. 1644793). For the chromium release assay, target cells were prelabeled with chromium-51 (51Cr) (Amersham Pharmacia Biotech, Piscataway, NJ) for 2 h. Supernatants were harvested and the amount of radioactive 51Cr released into the supernatants was measured with a gamma-counter (PerkinElmer, Wellesley, MA). The LDH release cytotoxcity assay was performed according to the manufacturer’s manual. LDH activity in the cell culture supernatants was measured using an ELISA reader (at 490 nm).
Before staining, FcR on cells were blocked with human IgG (Sigma-Aldrich; 1 μg/106 cells). Sorted NK cells and T cells were stained at 5 × 105 cells per sample with anti-CD69 Abs (Caltag Laboratories) for 20 min on ice. Samples were analyzed using a FACScan cytometer (BD Biosciences) and CellQuest software.
After the indicated times total cell extracts were prepared from purified control or poly(I:C)-stimulated T cells and NK cells using a high salt/detergent buffer containing 0.1% Nonidet P-40 (29).
SDS-PAGE and Western blotting
Extracts were analyzed by SDS-PAGE (Invitrogen) and western blot. Primary Abs used were anti-IκBα, c-21 and anti-β-actin, c-11 (Santa Cruz Biotechnology, Santa Cruz, CA). Secondary Abs were HRP-conjugated donkey anti-rabbit IgG (H+L) or donkey anti-goat IgG (H+L) (both Jackson ImmunoResearch Laboratories, West Grove, PA). Immune complexes were detected using SuperSignal West (Pierce, Rockford, IL) and Kodak XAR-5 films. Western blots were stripped in 0.1% SDS, 100 mM 2-ME in TBST for 1 h.
RNA was isolated from purified NK (CD56+CD3−), T cell (CD3+) and DC (CD11c+) populations untreated or stimulated with 50 μg/ml poly(I:C) for 17 h using the Qiagen Mini RNeasy kit (Valencia, CA). DNase treatment was included during the isolation of the RNA. TaqMan primer/probe combinations and conditions have been previously described (30). Threshold cycle numbers (Ct) were determined with Sequence Detector Software (version 1.6; Applied Biosystems) and transformed using the ΔΔCt method as described by the manufacturer using RPL19 as the calibrator gene. Expression of TLR3 in T cell, NK cell, and DC populations is displayed as a fraction of the expression measured in commercially available cDNA from human placenta (Clontech Laboratories, Palo Alto, CA).
TLR3 mRNA expression is up-regulated upon poly(I:C) stimulation
We were interested in whether NK cells expressed known innate immune receptors that would enable direct sensing of viral products. RT-PCR analysis suggested that TLR3 mRNA expression is higher in PBMC enriched for NK cells by depletion with anti-CD3, -CD14, -CD19, -CD36, and -IgE Abs than in other cell types (27). Because TLR3 is activated by dsRNA, we analyzed its expression in NK cells sorted by flow cytometer before and after poly(I:C) stimulation. TaqMan analysis of RNA isolated from purified populations of T cells, NK cells, and DC revealed presence of TLR3 message at comparable levels in NK cells and DC and up-regulation of TLR3 mRNA after stimulation with poly(I:C) in NK cells and DC, but not in T cells (Fig. 1). We confirmed the TLR3 expression data by gene expression profiling of NK cells and poly(I:C)-stimulated NK cells using Affymetrix gene chips (data not shown). Furthermore, TLR3 message was readily detectable by quantitative RT-PCR analysis of RNA isolated from the NK cell line NK92 (data not shown).
Poly(I:C) stimulation enhances NK cell cytotoxicity
NK cells are known to lyse tumor cells and nonself cells. They also lyse cells infected with certain viruses, thereby inhibiting further viral replication and spread of infection (1). Treatment of mice or humans with poly(I:C) or stimulation of PBMC cultures in vitro resulted in higher cytotoxic activity (31, 32, 33, 34). This activation was thought to be mediated by IFN-α produced by activated DCs or macrophages (31, 32, 33, 34). Similarly, the combination of IL-2 and poly(I:C) was shown to increase cytotoxic activity of PBMC (35, 36). To test whether a viral product analog could induce NK cell cytotoxicity directly, we purified CD56+CD3−CD14−CD11c− NK cells as shown in Fig. 2,A. A postsort staining and cytometric analysis of the NK cell population with anti-CD11c and anti-CD14 confirmed that there were no contaminating DCs or monocytes detectable (Fig. 2,B). As a control, sorted DCs and monocytes were detected with this stain (Fig. 2 B).
NK cells, untreated or stimulated with poly(I:C) were cocultured at various effector-to-target-cell ratios and NK cell-mediated killing of two target cell lines, Daudi and YAC-1, was analyzed. Poly(I:C) stimulation enhanced NK cell-mediated lysis of Daudi cells by >70% at effector to target cell ratios of 10:1 through 40:1 (Fig. 3,A). While YAC-1 cells were not spontaneously lysed by NK cells, poly(I:C) stimulation of NK cells induced lysis of 25% of YAC-1 cells at an effector to target cell ratio of 40:1 (Fig. 3 A).
To test whether the enhanced cytotoxicity observed in poly(I:C)-stimulated NK cells could have been mediated indirectly by IL-12 produced by potential undetectable contaminating APCs, we blocked IL-12 with neutralizing Abs. No difference in cytotoxicity mediated by poly(I:C)-stimulated NK cells was observed in the presence of neutralizing Abs to IL-12 (Fig. 3 B). This indicated that NK cell activation by poly(I:C) does not occur via IL-12 producing APCs.
NK cells up-regulate activation markers in response to poly(I:C) stimulation
CD69 is a cell surface marker that has been shown to be up-regulated on NK cells after activation by cytokines like IL-2, IL-12, and IFN-α (37). Staining of sorted NK cells and T cells that were cultured in the presence or absence of poly(I:C) for 17 h with anti-CD69 Abs demonstrates CD69 significant up-regulation of CD69 on poly(I:C)-stimulated NK cells, but not on T cells (Fig. 4).
NK cell activation by poly(I:C) is independent of type I IFNs
Type I IFNs are produced by DCs and macrophages in response to poly(I:C) and they are known to activate NK cells (38). To investigate whether the mechanism of activation of NK cells in response to poly(I:C) is dependent on type I IFNs, we analyzed the production of IFN-α and IFN-β in supernatants of NK cells cultured with or without poly(I:C). As shown in Fig. 5,A, poly(I:C) stimulation does not induce IFN-α or IFN-β secretion by NK cells. Fig. 5,B shows that CD69 up-regulation in poly(I:C)-stimulated NK cells is independent of IFN-α, because addition of a neutralizing anti-IFN-α Ab did not influence the expression level of CD69. In contrast, the anti-IFN-α Ab did block the CD69 up-regulation in the control, IFN-α-stimulated NK cells (Fig. 5 B).
Treatment with poly(I:C) induces production of cytokines
To investigate other effects of poly(I:C) on NK cell function, we examined the production of cytokines by purified NK cells after stimulation with poly(I:C). Untreated NK cells did not produce any IL-6 or IL-8, whereas NK cells treated with poly(I:C) for 17 h produced IL-6 and IL-8 (Fig. 6,A). Shown are five of six donors that were analyzed at the same time. The sixth donor showed induction of the same cytokines in response to poly(I:C) stimulation, only at higher amounts (740 pg/ml IL-6; 940 pg/ml IL-8). In a fraction of the donors TNF-α (one of six) and IL-1β (four of six) were induced after poly(I:C) stimulation and in none of the six donors was IL-10 or IL-12 detected. IFN-γ, a cytokine known for its antiviral activities, was induced after 17 h of stimulation with poly(I:C) and up-regulated maximally by 48 h (Fig. 6 A).
To categorically exclude the possibility of contamination of primary NK cells with undetectable numbers of APCs known to respond to dsRNA, we also tested IL-6 production in a NK cell line, NK92. Untreated NK92 did not produce any IL-6, however stimulation with poly(I:C) induced IL-6 secretion dramatically (Fig. 6 B). Thus, NK cells are innately responsive to dsRNA.
Stimulation of NK cells with poly(I:C) leads to activation of NF-κB
To test whether poly(I:C) stimulation induces signaling in NK cells, we studied the activation of the NF-κB pathway. NK cells and T cells were cultured without treatment or stimulated with poly(I:C) for 30, 60, or 90 min. Activation of NF-κB signaling was appraised by analysis of degradation of its inhibitor IκBα by western blot. IκΒα degradation was detected after 60 min of stimulation with poly(I:C) and by 90 min most of the IκΒα protein was degraded (Fig. 7). As a control for protein loading, the blots were reprobed for β-actin. The kinetics of NF-κB activation in NK cells is similar to that seen in other cell types (19, 21). IκBα degradation was not detected in T lymphocytes after poly(I:C) treatment (Fig. 7). This observation is consistent with the fact that stimulation of TLRs results in NF-κB activation.
In this study we demonstrate that NK cells express TLR3 permitting direct detection of dsRNA. Purified NK cells are activated by poly(I:C) as evidenced by strongly enhanced cytotoxicity toward a variety of target cell lines, up-regulation of CD69, and secretion of the proinflammatory cytokines IL-6 and IL-8, and the antiviral cytokine IFN-γ. Production of these cytokines has been shown in various cell types to involve NF-κB, a major stress and immune response regulator (39). Our data show that poly(I:C)-induced IL-6, IL-8, and IFN-γ secretion by NK cells is preceded by NF-κB activation. Taken together, these findings suggest that viral recognition via TLR3 equips NK cells to play a fundamental role in the first line of defense against viral infections.
While expression of TLR3 was first reported to be restricted to DC among human PBMC (24), the expression of TLR3 that we detected in unstimulated NK cells is in accordance with Hornung et al. (27) who reported moderate levels of TLR3 RNAin NK cells. Furthermore, we found that poly(I:C) stimulation increased TLR3 RNA levels in NK cells, as well as in DC. This increase in TLR3 message seems to be specific for the TLR3 activator poly(I:C) since other inflammatory signals like LPS, IL-1β, and TNF-α were reported to decrease TLR3 levels (24). While LPS and proinflammatory cytokines were found to augment the levels of TLR4 mRNA in monocytes, the levels of TLR2 RNA were not augmented in response to LPS, suggesting that TLRs are differentially regulated (24).
Our data demonstrate that the activation of human NK cells in response to poly(I:C) is independent of type I IFNs because poly(I:C) stimulation does not induce the secretion of IFN-α or IFN-β in NK cells. Accordingly, IFN-α neutralizing Abs did not block the up-regulation of the activation marker CD69 on poly(I:C)-stimulated NK cells.
The injection of mice with poly(I:C) has been shown to enhance NK cell-mediated cytotoxicity (40). In contrast to human NK cells, the cytolytic activity of mouse NK cells was dependent on type I IFNs, because the effect of poly(I:C) was completely abrogated in mice lacking the type I IFN receptor or STAT-1 (40). This finding suggests that the mechanism of activation of NK cells by poly(I:C) is different in humans and mice. In accordance with that, we also found that in vitro stimulation of NK cells isolated from mice with poly(I:C) failed to enhance their cytolytic activity (data not shown).
Recently, Toll/IL-1R (TIR) domain-containing adapter inducing IFN-β/TIR-containing adapter molecule 1, a novel adaptor molecule that preferentially mediates TLR-3 signaling has been described (41, 42). TIR-containing adapter molecule 1 RNA is expressed in immature DCs, macrophages and NK cells (42). The fact that NK cells, like DCs and macrophages express the signaling machinery important for TLR3 downstream signaling, supports our findings.
Other TLRs are also likely to play an important role in the defense against viruses. TLR7 and TLR8 mediate the effects of imidazoquinoline compounds, which have been therapeutically used to treat human Papillomavirus infections (36, 43). In addition to LPS, TLR4 is activated by the respiratory syncytial virus F protein (44). TLR4-deficient mice challenged with respiratory syncytial virus showed higher viral titers in their lungs, deficient NK trafficking and function, as well as delayed virus clearance compared with wild-type mice (45). TLR4 also interacts with mouse mammary tumor virus and Moloney-murine leukemia virus envelope protein (46). LPS hyporesponsive C3H/HeJ mice express a missense mutation in TLR4 and have a reduced incidence and increased latency of mouse mammary tumor virus-induced tumors (46).
Recently, a MCMV encoded MHC-like protein (m157) was shown to engage Ly49H, an activating NK cell receptor, in MCMV-resistant mice (11). Interestingly, m157 engaged an inhibitory NK cell receptor in certain MCMV-susceptible mice (11). In humans, functional Ly49 genes are not present; however, the killer cell Ig-like receptor family of human NK cell receptors presumably mediates similar functions. It will be interesting to determine whether activating killer cell Ig-like receptors can also recognize other pathogen-encoded ligands.
This study shows that human NK cells can directly sense TLR3 activator dsRNA. It is likely that dsRNA-induced activation of NK cells via TLR3 plays a crucial role in the prevention of pathogenesis of many viral infections in humans. Furthermore, a detailed understanding of this pathway in NK cells and its effect on other cell types has potentially important implications for the control of inflammatory responses to infections. Autoimmune diseases are known to be exacerbated by infections, often by influenza virus. In this respect altering the course of viral infections and tumors by manipulation of the expression or signaling of TLR3 may be therapeutically useful.
We thank Peter Schow, Katharine Grimmer, and Wendy Tombo for cell sorting; Mark Nagel and Phil Hass for protein purification; and Drs. Dan Eaton, Sherman Fong, and Andy Chan for critical reading of the manuscript.
Abbreviations used in this paper: DC, dendritic cell; MCMV, mouse CMV; poly(I:C), polyinosinic-polycytidylic acid; LDH, lactate dehydrogenase; TIR, Toll/IL1R.