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. 2014 Jun 10;142(3):442–452. doi: 10.1111/imm.12273

Critical role of the endogenous interferon ligand–receptors in type I and type II interferons response

Ahmed Lasfar 1,2, Jeffry R Cook 3, Karine A Cohen Solal 2,4, Kenneth Reuhl 1,2, Sergei V Kotenko 5, Jerome A Langer 6, Debra L Laskin 1,2
PMCID: PMC4080960  PMID: 24597649

Abstract

Separate ligand–receptor paradigms are commonly used for each type of interferon (IFN). However, accumulating evidence suggests that type I and type II IFNs may not be restricted to independent pathways. Using different cell types deficient in IFNAR1, IFNAR2, IFNGR1, IFNGR2 and IFN-γ, we evaluated the contribution of each element of the IFN system to the activity of type I and type II IFNs. We show that deficiency in IFNAR1 or IFNAR2 is associated with impairment of type II IFN activity. This impairment, presumably resulting from the disruption of the ligand–receptor complex, is obtained in all cell types tested. However, deficiency of IFNGR1, IFNGR2 or IFN-γ was associated with an impairment of type I IFN activity in spleen cells only, correlating with the constitutive expression of type II IFN (IFN-γ) observed on those cells. Therefore, in vitro the constitutive expression of both the receptors and the ligands of type I or type II IFN is critical for the enhancement of the IFN activity. Any IFN deficiency can totally or partially impair IFN activity, suggesting the importance of type I and type II IFN interactions. Taken together, our results suggest that type I and type II IFNs may regulate biological activities through distinct as well as common IFN receptor complexes.

Keywords: cell types, constitutive interferon, interferon receptor paradigm, interferon response, T cells, type I and type II interferon receptors

Introduction

Since their discovery, the interferons (IFNs) have been considered as a distinct cytokine family. By acting directly on their targets or through the immune system, the IFNs are a first line of defence against viral infections and cancer.1–6 Currently, the IFNs include three classes of related cytokines: type I, type II and the recently identified type III IFNs.7–9 Type I IFN represents the largest cytokine subfamily with over 20 members, including 13 IFN-α subtypes in humans, IFN-β, IFN-δ, IFN-ε, IFN-κ, IFN-ω, IFN-τ and limitin. IFN-α, IFN-β, IFN-ε, IFN-κ and IFN-ω exist in humans, whereas limitin and IFN-τ have been identified in mice and in cattle (and other ruminants), respectively. However, IFN-δ homologues are also found in a variety of species of eutherian mammals10 and are reported to be closely related to limitin/IFN-ζ.9,11 The genes encoding type I IFNs are mainly clustered on chromosome 9 in humans and on chromosome 4 in mice. All type I IFNs bind a common cell-surface receptor formed by IFNAR1 and IFNAR2 known as the IFN-α/β receptor. Upon ligand and receptor interaction, janus kinase 1 (JAK1) and tyrosine kinase 2 (TYK2) kinases are activated, and the IFN-stimulated gene factor 3 (ISGF3) transcription complex is formed. ISGF3 is composed of activated signal transducer and activator of transcription 1 (STAT1) and STAT2 and the IFN regulatory factor IRF9 (ISGF3-γ or p48). In contrast, there is only one type II IFN member, IFN-γ. The functional gene encoding IFN-γ is located on chromosome 12 in humans and chromosome 10 in mice. Interaction of IFN-γ with its receptor, formed by IFNGR1 and IFNGR2, results in activation of JAK1 and JAK2 leading to the dimerization of STAT1 and gene transcription.9,12–14 In addition to the JAK/STAT pathways, other JAK-dependent signalling cascades are activated, including the mitogen-activated protein kinase p38 cascade and the phosphatidylinositol 3-kinase cascade.15,16

Although the type I and type II IFN systems have been studied extensively, the exact contribution of the ligand and receptor subunits for each of the two systems is not yet well elucidated. In addition, few studies have examined the synergisms between the two types of IFN.17 In the present studies we evaluate the effect of IFNs, IFNAR1, IFNAR2, IFNGR1 and IFNGR2 deficiency on the interaction between type I and type II IFN systems in different primary mouse cell types. The use of genomic deletions is an important step to demonstrate the synergism of IFN signalling pathways and to assess the role of constitutive IFN ligand and receptor expression. We have found that the ubiquitous expression of the type I IFN system controlled the intensity of the type II IFN response. However, the intensity of the type I IFN response might be controlled by type II IFN specifically on cells producing the ligand IFN-γ, such as activated T cells.

Materials and methods

Mice and interferon

Wild-type and knock-out mice were either purchased from the Jackson Laboratory (Bar Harbor, ME) or generously provided by Dr Paul Rothman (Columbia University, New York, NY). C57BL/6 and 129/sv strains were used for the study. Animals were housed in a microisolation facility in the RWJMS vivarium. Mice were fed Purina 5021 autoclavable diet and water ad libitum and were maintained at a constant 12 hr light, 12 hr dark cycle. The IFN-α and IFN-β were supplied by the manufacturer as unit/ml (PBL, Piscataway, NJ). Interferon-γ was purchased from PeproTech, Inc. (Rocky Hill, NJ) and R&D Systems (Minneapolis, MN), diluted in PBS and calibrated against IFN-α and IFN-β using an antiviral assay and IFN standard as previously described.18,19 In this antiviral assay for IFN, 1 unit/ml of IFN is the quantity necessary to produce a cytopathic effect of 50%.

Cell preparation

Kidney epithelial cells (KECs) were extracted from murine pups. Whole kidneys from each pup (2–4 days old) were removed and processed separately under sterile conditions. Each pair of whole kidneys was washed with sterile PBS, placed in 10 ml of PBS containing 2·5 mg/ml dispase II (Roche Molecular Biochemicals, Basel, Switzerland) and 2·5 μg/ml collagenase A (Roche Molecular Biochemicals, Basel, Switzerland), mechanically disrupted, and then stirred at 37°C for 30 min. Following incubation, 5 ml of Dulbecco's modified Eagle's medium plus 5% fetal bovine serum (FBS) was added, mixed by pipetting, and clumps were allowed to settle for 5 min. The KECs were collected from the supernatant after centrifugation at 400 g for 5 min.

Mouse embryonic fibroblasts (MEFs) were prepared from pregnant mice at 13 or 14 day post-coitum. The uterine horns were dissected, rinsed in 70% (volume/volume) ethanol and placed into a Falcon tube containing PBS without Ca2+ or Mg2+ (Gibco, Invitrogen, Carlsbad, CA). The uterine horns were then placed in a Petri dish (Thermo Scientific, Waltham, MA) and each embryo was separated from its placenta and embryonic sac. After enzymatic digestion in 1 ml of 0·05% trypsin/EDTA (Gibco, Invitrogen), containing 100 Kunitz units of DNase I (USB) per embryo, the tissue was transferred to 50-ml Falcon tubes and incubated for 15 min at 37° in Dulbecco's modified Eagle's medium supplemented with 10% FBS. MEFs from IFNAR1- and IFNAR2-deficient mice, generously provided by Paul Hertzog (Monash University, Clayton, Victoria, Australia) were also used in this study.

For preparation of splenocytes (SPs), spleens were extracted from mice, placed in RPMI-1640 medium with 5% FBS, and minced. After removal of large tissue clumps, the remaining cells were harvested, pelleted by centrifugation, and used for experiments.

Flow cytometric analysis

To detect changes in MHC class I antigen (H-2Kb) expression, cells were treated for 72 hr with IFNs, harvested, and incubated for 1 hr with a mouse monoclonal antibody against H-2Kb (eBiosciences, San Diego, CA), followed by incubation for 30 min with a FITC-conjugated goat anti-mouse immunoglobulin (Sigma, St Louis, MO). Stained cells (104) were analysed by Beckman Coulter Gallios Flow Cytometry (compensation was performed by Gallios software during sample acquisition).

Electrophoretic mobility shift assay

To evaluate STAT activation, cells were treated for 15 min at 37° with various cytokines and used for electrophoretic mobility shift assay (EMSA) experiments with a GAS probe as previously described.20 EMSAs were performed with a 22-bp DNA probe (containing a Stat1α binding site) corresponding to the IFN-γ-activated sequence (GAS) element in the promoter region of the Hu-IRF-1 gene (5′-GATCGATTTCCCCGAAATCATG-3′). For STATs identification, rabbit anti-Stat1α and anti-Stat3 antibodies were used.

RT-PCR

RNA isolation and RT-PCR were performed as previously described.17,20 We used the following primers: IFN-β (F: 5′-ACT ATA AGC AGC TCC AGC TC-3′ and R: 5′-GAG TTC ATC CAG GAG ACG TA-3′). IFN-γ (F: 5′-TGT TTC TGG CTG TTA CTG CCA CCG-3′ and R: 5′-GAT TTT CAT GTC ACC ATC CTT TTG-3′). β-Actin (F: 5′-GAA TGG GTC AGA AGG ACT CCT AT-3′ and R: 5′-ATC TGG GTC ATC TTT TCA CGG TT-3′).

T-cell activation

Splenocytes were treated with murine anti-CD3 antibody (eBiosciences, San Diego CA). After 48 hr, the cells were harvested, purified on Ficoll and maintained in culture in the presence of interleukin-2 (IL-2; 250 U/ml). After 48 hr in culture, the cells were confirmed as 100% T cells by flow cytometry using an FITC-conjugated anti-CD3 antibody (eBiosciences, San Diego, CA).

Immunoblotting

To confirm STAT activation, immunoblotting was performed on cells treated for 15 min at 37° with type I and type II IFN (similarly to EMSA). Cells were harvested, washed with PBS and extracted in the presence of protease and phosphatase inhibitors (Roche Molecular Biology). Equal amounts of protein were subjected to PAGE. After transfer onto nitrocellulose membranes, immunoblots were performed using antibodies against: phosphoSTAT1 (pY701), phosphoSTAT3 (pY705), STAT1, STAT3 proteins (purchased from eBiosciences) or Actin (β-Actin, Cell Signaling Technology, Danvers, MA).

Statistics

Statistical analysis was performed using a one-way analysis of variance or Mann–Whitney U-test. Differences were considered statistically significant at P < 0·05.

Results

Cell types and differential sensitivity to IFNs

To study the contribution of each of the IFN system components to the activity of the IFN, we isolated primary cells from 129/sv mice deficient in IFNAR1, IFNAR2, IFNGR1, IFNGR2 or IFN-γ and examined their response to IFN-α, IFN-β and IFN-γ. In contrast to the IFN-γ receptor, formed by IFNGR1 and IFNGR2 and which is unique for IFN-γ, the IFN-α/β receptor, formed by IFNAR1 and IFNAR2, is shared by IFN-α and IFN-β. However, differences in the mechanism of action between IFN-α and IFN-β have been reported.21–23 Therefore, we explored potential differences between IFN-α and IFN-β in the context of the IFN-γ response. To assess the IFN responsiveness of different cell types, we used primary cultures of MEFs, KECs and SPs. After treatment with IFN-α, IFN-β or IFN-γ, we assessed the IFN response by measuring the MHC class I antigen expression and the DNA-binding activity of STAT1. We observed significant differences in the response to IFN-α, IFN-β and IFN-γ in MEFs (Fig. 1a–d) and KECs (Fig. 1e–h). We also observed a correlation between the DNA-binding activity of STAT1 and MHC class I up-regulation in MEFs (Fig. 1i) and KECs (Fig. 1j). As shown in Fig. 1(d,h), IFN-γ induced the highest expression of MHC I, with IFN-β inducing an intermediate response and IFN-α the lowest response. At least twofold differences were observed between IFN-γ and IFN-β and between IFN-β and IFN-α. However, in SPs, we only observed limited differences between IFN-α, IFN-β and IFN-γ in IFN-induced STAT1 activation and MHC I up-regulation (Fig. 1k and data not shown), implying that the response to the different types of IFNs was dependent on the cell type. Similar results were observed with cells extracted from C57BL/6 mice (data not shown).

Figure 1.

Figure 1

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Interferon (IFN) response in different primary cell types isolated from 129/sv mice. MHC I analysis by flow cytometry in mouse embryonic fibroblasts (MEFs) treated with 1000 U/ml of IFN-α (a), 1000 U/ml IFN-β (b) and 1000 U/ml IFN-γ (c). d. Flow cytometric histogram of three independent experiments of MHC I analysis in MEFs. MHC class I expression is presented as fold increase over baseline expression in untreated cells. Similar analysis as MEFs was performed using kidney epithelial cells (KECs) (e–h). Signal transducer and activator of transcription 1 (STAT1) signalling in MEFs (i), KECs (j) and splenocytes (SPs) (k), treated with the IFN as indicated. Cells were treated with each IFN at 1000 U/ml and cellular lysates were prepared and analysed for STAT activation by EMSA with a GAS probe. Each panel is representative of three independent experiments. Arrows indicate the positions of STAT1 (Stat1) DNA-binding complexes.

Control of IFN-γ response by IFN-α/β

We next assessed the IFN-γ response in MEFs isolated from 129/sv mice deficient in IFNAR1 and IFNAR2. As shown in Fig. 2(a–c), IFNAR1 and IFNAR2 are required for both IFN-α and IFN-β responses, as previously reported.24,25 Interestingly, although the IFN-γ induced cell signalling through the IFN-γ receptor independently of IFNAR1 and IFNAR2, deficiency of either IFNAR1 or IFNAR2 significantly impaired the IFN-γ-stimulated DNA-binding activity of STAT1 (Fig. 2a), and IFN-γ-induced MHC class I antigen stimulation (Fig. 2d). Since deficiency of either IFNAR1 or IFNAR2 decreased the responsiveness to IFN-γ, our data suggest a requirement of the functional IFN-α/β receptor (both IFNAR1 and IFNAR2) in the IFN-γ response instead of IFNAR1 alone as previously reported.17

Figure 2.

Figure 2

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Interferon (IFN) response in IFNAR1- and IFNAR2-deficient cells. (a) Mouse embryonic fibroblasts (MEFs) were treated with IFN-α (1000 U/ml), IFN-β (1000 U/ml) or two concentrations of IFN-γ (100 U/ml and 1000 U/ml). Cellular lysates were prepared and signal transducer and activator of transcription 1 (STAT1) activation was assessed by EMSA with a GAS probe. Arrows indicate the positions of the STAT1 (Stat1) DNA-binding complexes. Anti-STAT1 antibodies were used to show the identity of the STAT1 DNA-binding complexes. (b–d) MHC class I antigen expression in MEFs treated with IFN-α (b), IFN-β (c) or IFN-γ (d). MHC class I expression was presented as fold increase over baseline expression of untreated cells. (e) Expression of murine IFN-β and β-Actin mRNAs in MEFs was evaluated by RT-PCR. (f) Splenocytes (SPs) were treated with 1000 U/ml of IFN-α, IFN-β and IFN-γ. Cellular lysates were prepared and STAT1 activation was assessed by EMSA. Anti-STAT1 antibodies were used to show the identity of the STAT1 DNA-binding complexes. Arrows indicate the positions of STAT1 (Stat1) DNA-binding complexes.

The role of both IFNAR1 and IFNAR2 (IFN-α/β receptor) in IFN-γ signalling may implicate a constitutively expressed IFN-α/β, which could interact with the IFN-α/β receptor and trigger the response. In contrast to IFN-α, which includes numerous subtypes, IFN-β is unique and required for IFN-α production.26 Using RT-PCR, we found constitutive expression of IFN-β mRNA in MEFs, occurring independently of the expression of IFN-α/β receptor subunits IFNAR1 and IFNAR2 (Fig. 2e). Similarly in KECs and SPs, we observed the constitutive expression of IFN-β mRNA (data not shown) and the impairment of IFN-γ-induced STAT1 activation in IFN-α/β receptor-deficient cells (Fig. 2f). Therefore, the control of the IFN-γ response by type I IFN may depend on endogenous type I IFN ligand–receptor complexes and occurs in all cell types.

Restricted control of IFN-α/β by IFN-γ

We next asked whether the deficiency in the IFN-γ system affects IFN-α and IFN-β responses. In KECs isolated from 129/sv mice deficient in IFNGR1, IFNGR2 or IFN-γ, the IFN-α response was not affected as demonstrated by MHC class I antigen expression (Fig. 3a–c). However, we did notice a slight impairment of the IFN-β response. This impairment, which occurred at high concentrations of IFN-β, was observed in IFNGR1- (Fig. 3d) and IFN-γ- (Fig. 3f) deficient cells, but not in IFNGR2-deficient cells (Fig. 3e). These results imply a possible involvement of IFN-γ and IFNGR1 in the control of IFN-β responses. We obtained similar results using KECs isolated from C57BL/6 deficient in IFNGR1, IFNGR2 or IFN-γ (data not shown). Since IFNGR1 but not IFNGR2 binds IFN-γ with high affinity, our data suggest a potential role of IFNGR1 in complex with IFN-γ. Therefore, independently of IFNGR2, the IFNGR1/IFN-γ complex can modulate IFN-β responses.

Figure 3.

Figure 3

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Biological responses of interferon-α (IFN-α) and IFN-β. Flow cytometric analysis of MHC class I antigen expression in kidney epithelial cells (KECs), isolated from wild-type mice (solid lines) and mice deficient in IFNGR1, IFNGR2 and IFN-γ (dashed lines). Cells were treated in triplicate with increasing IFN-α (a–c) or IFN-β (d–f) concentrations. MHC class I expression was presented as fold increase over baseline expression of untreated cells. *P < 0·05.

Constitutive expression of IFN-γ and IFN-α/β response

In order to investigate whether IFN-γ played a role in IFN-β activity in KECs, we analysed whether KECs constitutively express IFN-γ. RT-PCR analysis showed no IFN-γ expression in KECs isolated from 129/sv or C57BL/6 mice (data not shown). We further analysed the effects of priming cells deficient in IFN-γ and IFNGR1 with a sub-active dose of IFN-γ. IFN-γ priming did not cause any significant changes in IFN-β activity in wild-type cells (Fig. 4a) or in cells deficient in IFN-γ (Fig. 4b), IFNGR1 (Fig. 4c) or IFNGR2 (Fig. 4d). In addition to MHC class I expression, we obtained similar results on IFN-β signalling by assessing the DNA-binding activity of STAT1 (Fig. 4e). These experiments suggest that IFN-γ does not play a significant role in the response to IFN-β in KECs.

Figure 4.

Figure 4

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Modulation of the interferon-β (IFN-β) response in kidney epithelial cells (KECs). IFN-β response was evaluated by flow cytometry. The level of MHC class I antigen expression was assessed in cells treated with 1000 U/ml of IFN-β primed or not primed with a sub-active dose (0·01 U/ml, see Fig. 2d) of IFN-γ for 30 min. MHC class I antigen expression was presented as fold increase over baseline expression of untreated cells (histograms a–d): (a) wild type (Wt), (b) IFNG−/−, (c) IFNGR1−/− and (d) IFNGR2−/−. (e) signal transducer and activator of transcription 1 (STAT1) activation, assessed by EMSA, in cells treated with IFN-β (1000 U/ml) and IFN-γ (100 U/ml or 0·01 U/ml). Anti-STAT1 antibodies were used to show the identity of STAT DNA-binding complexes. The * indicates IFN-γ treatment with a sub-active dose (0·01 U/ml). Arrows indicate the positions of STAT1 (Stat1) DNA-binding complexes. Experiments are average of three independent experiments.

Role of IFN-γ system in the modulation of IFN-α/β response in haematopoietic cells

We extended our analysis of the role of the IFN-γ system in the IFN-α/β response in SPs. Surprisingly, we found that in contrast to the epithelial cells (KECs), SPs deficient in IFN-γ, IFNGR1 or IFNGR2 showed significant impairment of STAT1–DNA complex formation when treated with either IFN-α or IFN-β as shown by EMSA (Fig. 5a). This impairment suggests that SPs constitutively express IFN-γ. As shown in Fig. 5(b), SPs express IFN-γ in contrast to epithelial cells (Fig. 5c). Inhibition of IFN-α and IFN-β responses was neutralized after priming SPs isolated from IFN-γ-deficient mice with IFN-γ (0·01 U/ml) (Fig. 5d). In contrast, inhibition of IFN-α and IFN-β responses was not neutralized after priming SPs isolated from IFNGR1-deficient or IFNGR2-deficient mice with IFN-γ (Fig. 5d). EMSA analysis of activated T cells isolated from IFN-γ-deficient mice also revealed an impairment of the type I IFN response (Fig. 5e). This impairment was restored by IFN-γ priming (Fig. 5f). However, in contrast to STAT1, STAT3 activation was not restored by IFN-γ priming of activated T cells isolated from IFN-γ-deficient mice. In agreement with EMSA, immunobloting of protein extracts prepared from activated T cells also demonstrated an impairment of the IFN-β response (Fig. 5g, half left). Furthermore, we observed that IFN-γ priming restored STAT1 activation but not STAT3 activation (phosphorylation) in activated T cells isolated from IFN-γ-deficient mice (Fig. 5g, half right). These results suggest that the constitutive expression of IFN-γ in haematopoietic cells plays a role in the response to type I IFN.

Figure 5.

Figure 5

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Modulation of interferon-α (IFN-α) and IFN-β responses in haematopoietic cells. (a) EMSA analysis of signal transducer and activator of transcription 1 (STAT1) activation in splenocytes (SPs) isolated from wild-type, IFNG−/−, IFNGR1−/− and IFNGR2−/− mice. SPs were treated with IFN-α and IFN-β (1000 U/ml). Arrows indicate the positions of STAT1 DNA-binding complexes. Expression of IFN-β and IFN-γ mRNA was evaluated in SPs (b) and kidney epithelial cells (KECs) (c) by RT-PCR. (d) SPs were primed with a sub-active dose of IFN-γ (0·01 U/ml), treated with IFN-α and IFN-β 1000 U/ml) and analysed for STAT1 activation by EMSA. (e) Evaluation of STAT activation by EMSA in activated T cells prepared from wild type (Wt) and IFNG−/− mice. Either 10 U/ml of IFN-γ or increasing concentrations of IFN-α and IFN-β were used (10, 100 and 1000 U/ml). Arrows indicate the positions of STAT1 and STAT3 DNA-binding complexes. (f) Activated T cells were primed with a sub-active dose of IFN-γ (0·01 U/ml), treated with IFN-α and IFN-β 1000 U/ml) and analysed for STAT1 activation by EMSA. PWt and PKO indicate IFN-γ priming of activated T cells isolated from wild-type and IFN-γ knockout mice, respectively. Arrows indicate the positions of STAT1 (Stat1) and STAT3 (Stat3) DNA-binding complexes. (g) Immunoblots were performed on activated T cells primed (PWt, PKO) or not (Wt, KO) with IFN-γ (0·01 U/ml) under similar condition as EMSA. 100 U/ml of IFN-γ or IFN-β were used for cell treatment. Data are representative of three independent experiments.

Discussion

Interactions between the type I and type II IFN systems and the role of endogenous IFN have not been well studied. The present studies demonstrate that endogenous IFN plays a critical role in the overall signalling and activity of the IFN family. Additionally, constitutive expression of both ligands and receptors is essential for type I and type II IFN responses. When the expression of type I IFN ligand receptor is disrupted, a decrease of IFN-γ (type II IFN) signalling and activity is observed in fibroblasts, epithelial cells and splenocytes, in agreement with the previous report in MEFs deficient in IFNAR1 or IFN-β.17,27 However, using IFNAR2-deficient cells, we demonstrated that the whole type I IFN receptor rather than the IFNAR1 subunit per se played a critical role in the full activity of type II IFN.17,27–29 When the type II IFN receptor is disrupted, type I IFN is affected differentially, depending on the cell type and the IFN sub-type. Using IFN-α and non-haematopoietic primary cells, we saw no significant alterations in type I IFN signalling and activity in cells deficient in IFNGR1, IFNGR2 or IFN-γ, in agreement with previous studies using IFNGR1-deficient MEFs.17 However, using cells of haematopoietic origin, we observed an impairment of IFN activity for both IFN-α and IFN-β in cells deficient in IFNGR1, IFNGR2 or IFN-γ. This impairment may be determined by the expression of the constitutive IFN-γ, specifically expressed in cells of haematopoietic origin (Fig. 5). Therefore, depending on the cell type and the expression of IFN-γ, the amplitude of type I IFN signalling may be also controlled by the type II IFN system.

We demonstrated that priming the non-haematopoietic primary cells with a sub-active dose of IFN-γ did not increase type I IFN activity. This result suggests that the constitutive expression of IFN-γ might not be the only mechanism for the enhancement of type I IFN activity. Potential differences in the structure and organization of the IFN-γ receptor at the cell surface of haematopoietic and non-haematopoietic cells may explain this result. It has been reported that endogenous IFN-γ can profoundly affect IFN-γ receptor expression and modulate cell functions.30 After long-term culture, phytohaemagglutinin-activated T lymphoblasts (expressing IFN-γ) showed high IFNGR1 expression, whereas IFNGR2 was barely detectable.31 The IFNGR2 up-regulation was associated rather with apoptosis.32–34 We can explain the pro-apoptotic effects of type I IFN on haematopoietic cells in part by the augmentation of IFNGR2, enabling type I and type II IFN receptor interactions.17 Our results may explain why during IFN-α therapy, myelosuppression is commonly observed35 and the high efficacy of IFN-α in the treatment of haematological malignancies.36–38

In addition to the impairment of STAT1 activation by IFN-α and IFN-β, STAT3 activation was also affected in IFN-γ-deficient T cells. Although the requirement for STAT1 in IFN-α/β signalling and action is well known,39,40 the role of STAT3 in IFN signalling is not clear. In T cells, the anti-apoptotic and pro-apoptotic role of IFN-α/β is apparently controlled by the STAT1/STAT3 ratio,41 which is consistent with the role of STAT3 as a negative regulator of type I activity.42–44 Interestingly, IFN-γ restored the activation of STAT1 but not STAT3 in IFN-γ-deficient T cells, suggesting that the activation of STAT1 is directly controlled by the interaction of type I and type II IFN systems. However, the impairment of STAT3 could be more dependent on other signals inherent to T-cell activation.45

In contrast to IFN-α, we found an alteration of IFN-β activity in cells deficient in IFNGR1 and IFN-γ but not IFNGR2 (Figs 1 and 3), highlighting the difference in the activity between IFN-α and IFN-β.46,47 Although it is known that the uniqueness of IFN-β is probably a consequence of stronger affinity of IFN-β for IFNAR1,48–50 further modulation of IFN-β activity might occur in the specific context of type I and type II IFN receptor interactions.

In parallel, we investigated whether type III IFNs modulated the activity of type I and type II IFNs. In contrast to type I and type II IFNs, the recently discovered type III IFN uses the receptor chain IFN-λR1, and IL-10R2, a receptor chain shared by the non-IFN cytokines, IL-10, IL-22 and IL-26.8,20,51,52 Examination of epithelial cells (KECs) originating from IFN-λR1 knockout mice did not show any significant effect of type III IFN on the amplitude of type I and type II IFN responses (data not shown), indicating that type III IFN may not cross-talk with the type I and type II IFN systems.

To explain the cell response to treatment by type I and type II IFNs, we propose an alternative IFN model (Fig. 6c). In contrast to the classical type I and type II IFN models53–55 (Fig. 6a and b), our model takes into consideration the constitutive expression of type I and type II IFN (Figs 2 and 5). Physical interaction between IFNAR1 and IFNGR217 and the heterodimerization of IFNGR1 and IFNGR256 are also represented. By interacting with its cognate receptor, the constitutive IFN of one IFN system amplifies the response to the other IFN system. As indicated (Fig. 6c), amplification of the IFN response may result from a super receptor organization of the type I and type II IFN systems. This organization enables high increases of local concentrations of IFN receptor complexes, which are crucial for downstream IFN receptor signalling. The presence of this receptor organization between the type I and type II IFN systems may be considered as a unique mechanism for amplifying and regulating cellular responsiveness to IFN.

Figure 6.

Figure 6

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Interferon (IFN) receptor paradigms (view from outside the cell surface). (a) Established model of the type I IFN system. The receptor is formed by two subunits, IFNAR1 and IFNAR2. Type I IFN ligands such as IFN-α/β bind to the receptor and induce cell signalling. (b) Established model of the type II IFN system. The receptor is formed by two subunits, IFNGR1 and IFNGR2. In contrast to type I IFN, there is only one type II IFN member (IFN-γ). IFN-γ binds as a dimer to the heterodimer receptor (IFNGR1/IFNGR2-IFNGR1/IFNGR2). After the binding of IFN-γ to the receptor, cell signalling is induced. (c) A new paradigm for type I and type II IFN systems. This paradigm incorporates the constitutive expression of type I (IFN-α/β) and type II IFN (IFN-γ) and the interaction between types I and II IFN receptors (IFNAR1-IFNGR2). In contrast to type II IFN (IFN-γ), the constitutive expression of type I IFN (IFN-α/β) is ubiquitous. IFN-γ is expressed by haematopoietic cells such as T cells. By interacting with their cognate receptors, the constitutively expressed IFN-α/β or IFN-γ facilitate the recruitment of the IFN receptors, the Janus kinases (JAKs) and the signal transducers and activators of transcription (STATs), and enable the amplification of IFN signalling by the exogenous IFN-α/β or IFN-γ.

In the classical model, IFN-γ, IFNGR1 and IFNGR2 are mutually dependent for their functions (Fig. 6b). However, in T lymphocytes, distinct functions for IFNGR1 and IFNGR2 have been reported. In contrast to IFNGR1-deficient mice,57 T lymphocytes isolated from IFN-γ-58 or IFNGR2-59 deficient mice are not able to generate type 1 T helper (Th1) cells. Since the genetic background of mice may not be the sole cause for the functional disparity in the type II IFN system (IFN-γ, IFNGR1 and IFNGR2),60 we hypothesize that specific interactions between the members of the type I and type II IFN systems may in part explain those differences in T cells. A specific interaction between IFNGR2 and IFNAR1 has been reported.17 As represented in the new IFN paradigm, this interaction enables the clustering of the type I and type II IFN systems and the modulation of IFN functions (Fig. 6c). This model implies that in the absence of IFNGR1, IFN-γ may interact with IFNGR2 associated with IFNAR1 and facilitate the generation of T helper type 1 cells. However, the lack of either IFN-γ or IFNGR2 may prevent the functional interaction between the type I and type II IFN systems, resulting in the induction of T helper type 1 differentiation. This new IFN paradigm could serve as a model for understanding the intricate role of other cell surface ligand–receptor complexes, and will be of interest for designing new drugs such as antibodies to target the constitutive expression of ligands and receptors so as to modulate the functions of the type I and type II IFN systems.

Acknowledgments

This work was supported in part by a CINJ Award and NIH grants RO1 051139, ES004738, CA132624, AR055073 and ES005022. We are thankful to Dr. S. Pestka for his support and advice.

Disclosures

The authors disclose no potential conflicts of interest.

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