Suppressor of Cytokine Signaling-1 Attenuates the Duration of Interferon gamma  Signal Transduction in Vitro and in Vivo*

Marta Brysha, Jian-Guo Zhang, Patrick BertolinoDagger , Jason E. Corbin, Warren S. Alexander, Nicos A. Nicola, Douglas J. Hilton, and Robyn Starr§

From the Cooperative Research Center for Cellular Growth Factors and the Walter and Eliza Hall Institute for Medical Research, PO Royal Melbourne Hospital, VIC 3050, and the Dagger  Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag No 6, Newtown New South Wales 2042, Australia

Received for publication, March 28, 2001

    ABSTRACT
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Suppressor of cytokine signaling-1 (SOCS-1) is a cytokine-inducible intracellular protein that functions to negatively regulate cytokine signal transduction pathways. Studies in vitro have shown that constitutive overexpression of SOCS-1 inhibits signaling in response to a range of cytokines, including interferons (IFN). Mice lacking SOCS-1 die from a complex disease characterized by liver degeneration and massive inflammation. Whereas there is clear evidence of increased IFNgamma signaling in SOCS-1-/- mice, it is unclear to what extent this is due to increased IFNgamma levels or to increased IFNgamma sensitivity. Here we have used SOCS-1-/- IFNgamma -/- mice, which remain healthy and produce no endogenous IFNgamma , to demonstrate that in vitro and in vivo hepatocytes lacking SOCS-1 exhibit a prolonged response to IFNgamma and that this correlates with a dramatically increased sensitivity to the toxic effects of IFNgamma in vivo. Thus, SOCS-1 is required for the timely attenuation of IFNgamma signaling in vivo.

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SOCS-11 is an intracellular SH2 domain-containing protein that was initially isolated by screening a retroviral cDNA library for inhibitors of IL-6 signaling (1). It is also known as STAT-inducible STAT inhibitor-1 (SSI-1) (2) or JAK-binding protein (JAB) (3). SOCS-1 defines a family of eight proteins, SOCS-1 to -7 and CIS (cytokine-inducible SH2-containing protein), each of which contains a central SH2 domain and a C-terminal SOCS box (4).

In vitro studies have shown that the expression of SOCS-1, -2, -3, and CIS is inducible by a number of cytokines and that, when constitutively overexpressed, each SOCS protein can act to switch off cytokine-induced responses by inhibiting the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway (1-3). The latter studies are difficult to interpret because not only are the SOCS proteins grossly overexpressed, but they are also expressed in a temporally inappropriate manner, being present prior to the onset of signaling rather than induced as a consequence of signal transduction, which occurs physiologically.

Although both SOCS-1 and SOCS-3 act to suppress signal transduction from a similarly broad spectrum of cytokines in vitro, including IL-6, IL-4, growth hormone, IFNalpha /beta , and IFNgamma (1-3, 5-8), analyses of mice deficient in either SOCS-1 or SOCS-3 have indicated that each may play a relatively specific role in vivo. Mice lacking SOCS-3 die during embryogenesis apparently from erythrocytosis, suggesting that SOCS-3 is critical in regulating fetal liver erythropoiesis (9). SOCS-1 deletion results in mice that die, before weaning, of a complex multiorgan disease (10-12). Although the primary defect in mice lacking SOCS-3 has not been defined, the fatal neonatal disease characteristic of SOCS-1-deficient mice requires IFNgamma , because this pathology does not develop in SOCS-1-deficient mice that also lack the IFNgamma gene (13).

Interferons are fundamental components of the immune system. The predominant function of interferons is to confer cellular resistance to viral infections, although other immunomodulatory functions have also been described (14, 15). IFNgamma is produced by activated T lymphocytes and natural killer cells during viral infections and initiates an antiviral program in target cells by up-regulating the expression of genes that mediate the anti-viral response, including those encoding MHC class I antigens (14, 15). Similar to other cytokines, interferons activate the JAK/STAT pathway (16), and studies in vivo have shown that STAT1 is critical for mediating biological responses to IFNgamma (17, 18). Elevated levels of IFNgamma can be toxic, and it is clear that both IFNgamma production and responses to this cytokine must be tightly regulated in order to achieve a balance between beneficial and harmful effects (19-21). Other cytokines including IL-4, IL-10, and IL-13 contribute to some extent to this regulation by antagonizing IFNgamma functions (22-24). In addition, negative regulators such as SOCS proteins act to limit signal transduction in response to IFNgamma .

At least in vitro, SOCS-1 expression is induced by IFNgamma and overexpression of SOCS-1 inhibits IFNgamma signaling (6, 7). Clearly, IFNgamma is an essential mediator of the fatal disease seen in mice lacking SOCS-1 (13), but the precise mechanism is unclear. This report compares IFNgamma signaling in wild-type and SOCS-1-deficient mice in vivo and finds that both biochemical and biological responses to IFNgamma are attenuated by SOCS-1.

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Maintenance of Mice and Injection of IFNgamma -- SOCS-1-/- IFNgamma -/- mice were generated as described previously and were maintained on a mixed 129/Sv and C57Bl/6 genetic background (10, 13). For biological studies, neonatal mice were given daily intraperitoneal injections of 3, 0.3, or 0 µg recombinant murine IFNgamma (rmIFNgamma , PeproTech, Rocky Hill, NJ) in 0.05 ml of saline from birth and were sacrificed when moribund or at 21 days of age.

Immunoprecipitation and Western Blotting of Liver Proteins-- For biochemical studies, adult mice weighing an average of 29 g were given a single intraperitoneal injection of either 0.2 ml of saline, 2 µg of rmIFNgamma (PeproTech, Rocky Hill, NJ) or 10 µg of recombinant murine IL-6 (a gift from Dr. Richard Simpson, Ludwig Institute for Cancer Research, Melbourne, Australia). Mice were sacrificed by cervical dislocation at the indicated times after injection. Livers were dissected, immediately frozen in liquid nitrogen, and stored at -70 °C. Frozen livers were pulverized with a hammer and homogenized in RIPA buffer (1% v/v Triton X-100, 1% w/v sodium deoxycholate, 0.1% w/v SDS, 150 mM NaCl, 10 mM Tris, pH 7.5) containing 10 µg/ml leupeptin (Auspep, Parkville, Australia), 1 mM iodoacetic acid, 50 µg/ml soybean trypsin inhibitor, 20 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM EGTA (Sigma Chemical Co.) and 1 mM pervanadate. Pervanadate was prepared by adding hydrogen peroxide (20 mM final) to 1 mM sodium orthovanadate (Sigma) and incubating at room temperature for 30 min. Lysates were cleared by centrifugation and protein content was quantitated using a Coomassie Protein Assay Reagent (Pierce, Rockford IL).

For immunoprecipitations, liver lysates (12-20 mg of protein) were first precleared with protein G-Sepharose (0.060 ml, suspended 1:1 in RIPA buffer; Amersham Pharmacia Biotech, Uppsala, Sweden) for 1.5 h, then incubated with 4 µg of anti-STAT1 antibody (Transduction Laboratories, Lexington, KY) and protein G-Sepharose (0.06 ml, suspended 1:1 in RIPA buffer) for 2 h or overnight. Unbound protein was separated from the immunoprecipitate by washing 2-4 times in Nonidet P-40 lysis buffer (150 mM NaCl, 1% (v/v) Nonidet P-40, 50 mM Tris, pH 8.0) containing 10 µg/ml leupeptin, 1 mM iodoacetic acid, 50 µg/ml soybean trypsin inhibitor, 20 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM EGTA, and 1 mM pervanadate. Bound protein was eluted from the Sepharose in 0.03 ml of 2× SDS loading buffer (115 mM Tris-HCl, pH 6.8, 1.8% (w/v) SDS, 18.2% (v/v) glycerol, 2.5% (v/v) 2-mercaptoethanol, bromphenol blue) at 95 °C for 5 min, centrifuged at 13,000 rpm for 5 min, and the supernatant was loaded on SDS-PAGE gels. Eluates were split prior to SDS-PAGE to allow quantitation of protein loading. Proteins were fractionated by SDS-PAGE under reducing conditions and then electrophoretically transferred to PVDF-Plus membranes (Micron Separations Inc., Westborough, MA). Membranes were blocked with 10% skim milk for a minimum of 1 h and then incubated either for 2 h with anti-STAT1 antibody (1:250 dilution, Transduction Laboratories), or overnight with anti-phosphorylated STAT1 antibody (1:500, New England Biolabs, Beverly, MA). Antibody binding was visualized with either horseradish peroxidase-conjugated anti-rabbit or anti-mouse-Ig Fc fragment-specific (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) and the SuperSignal West Pico chemiluminescent substrate (Pierce Chemical Co.). Results shown are representative of two experiments.

Hepatocyte Isolation-- Hepatocytes were harvested from 10-week-old mice as described previously (25). Briefly, livers were perfused retrogradely via the inferior vena cava using HBSS without calcium and magnesium and then with the same medium containing 0.5 mM EDTA. EDTA was then removed by flushing the liver with HBSS without calcium and magnesium. Hepatocytes were released by perfusing with HBSS without magnesium but containing 5 mM CaCl2 and 0.05% collagenase IV (Sigma). Viable hepatocytes were purified using a Percoll density gradient (Amersham Pharmacia Biotech, Uppsala, Sweden), and the resulting cell pellet was washed three times by 50 × g centrifugation in RPMI 1640 medium containing 10% v/v fetal calf serum and 5 × 10-5 M 2-mercaptoethanol (RF10 medium). Cells were plated in 10-cm cell culture dishes (Corning, NY) in the same medium at a density of 7.5 × 105 cells per plate and were allowed to adhere overnight at 37 °C.

Electrophoretic Mobility Shift Assays-- Adherent hepatocytes were stimulated for 10 min at 37 °C with 5 ng/ml murine IFNgamma (PeproTech) in RF10 medium, after which the cytokine was washed from the plates, and the cells were cultured in fresh RF10 at 37 °C for the remainder of the indicated time. Hepatocytes were harvested from the plates using a rubber policeman and were washed once with cold phosphate-buffered saline containing 1 mM pervanadate. Cell pellets were snap frozen on dry ice, and high salt nuclear extracts were prepared as described (26). Electrophoretic mobility gel shift assays were performed on 10 µg of nuclear protein using the m67 oligonucleotide as described (27). To identify DNA-binding complexes, protein samples were preincubated with 2 µg of antibody specific for either STAT1 (Transduction Laboratories) or STAT3 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) before binding to the oligonucleotide.

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SOCS-1 Is Required for Regulating Responses to IFNgamma in Vivo-- The neonatal mortality in SOCS-1 knockout mice has been shown to be dependent on IFNgamma , because mice lacking functional genes for both SOCS-1 and IFNgamma are viable and healthy (13). We wished to use SOCS-1-/- mice to assess the extent to which SOCS-1 regulates IFNgamma signal transduction; however, such studies are complicated by the endogenous production of IFNgamma in vitro and in vivo and compounded by the neonatal morbidity and mortality of SOCS-1-/- mice. To overcome these problems we took advantage of the observation that SOCS-1-/- IFNgamma -/- mice, which fail to produce IFNgamma , are healthy and survive to adulthood.

Previous studies have documented the toxicity of IFNgamma when administered to newborn mice (19). Further, the pathology induced by IFNgamma treatment of wild-type mice is similar to that seen in SOCS-1-/- mice (13). The phenotype of SOCS-1-/- mice may be due to increased circulating levels of IFNgamma , increased sensitivity of cells to the effects of IFNgamma because of unregulated signaling, or a combination of both. To determine whether mice are more sensitive to IFNgamma in the absence of SOCS-1, newborn SOCS-1-/- IFNgamma -/- and SOCS-1+/+ IFNgamma -/- mice received daily intraperitoneal injections of IFNgamma . These mice were monitored and were sacrificed when moribund. Mice lacking SOCS-1 were substantially more sensitive to the toxicity of IFNgamma . Injections of 3 µg of IFNgamma induced morbidity within 2 days in SOCS-1-/- IFNgamma -/- mice, whereas this dose was tolerated for ~3 weeks in SOCS-1+/+ IFNgamma -/- mice (Fig. 1). Differences in sensitivity were also seen in response to the administration of 0.3 µg of IFNgamma . In contrast to SOCS-1-/- IFNgamma -/- mice, which were all moribund by 3 weeks of age and developed the same pathological features as SOCS-1-/- mice (Fig. 1 and data not shown), SOCS-1+/+ IFNgamma -/- mice all remained healthy after receiving this dose of IFNgamma (Fig. 1).


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Fig. 1.   Mice lacking SOCS-1 are more sensitive to the effects of exogenous IFNgamma . Neonatal mice were given daily intraperitoneal injections of IFNgamma from birth. Mice were administered either 3 µg of IFNgamma (top panel), 0.3 µg of IFNgamma (middle panel) or saline (bottom panel), and their health was monitored daily. n = number of mice/group.

IFNgamma -induced STAT Activation Is Prolonged in Mice Lacking SOCS-1-- To assess whether SOCS-1 regulates the intensity or duration of responses to IFNgamma , the activation of downstream signaling molecules was assessed in mice lacking SOCS-1. The organ we chose to examine in these studies was the liver because it is severely affected both in SOCS-1-/- mice and in mice injected with IFNgamma . SOCS-1-/- IFNgamma -/-, SOCS-1+/+ IFNgamma -/-, and SOCS-1+/+ IFNgamma +/+ mice were given intraperitoneal injections of IFNgamma and the subsequent activation of STAT1 in the liver was monitored at various times thereafter. STAT1 phosphorylation was evident in all mice within 15 min of IFNgamma injection, with the response continuing for at least 2 h but declining to basal levels by 4 h in SOCS-1+/+ IFNgamma -/- and SOCS-1+/+ IFNgamma +/+ mice (Fig. 2A). However, phosphorylation of STAT1 was prolonged in mice lacking SOCS-1 and was maintained until 8 h after injection. This prolonged response was specific to IFNgamma , because there was no difference in the kinetics of STAT1 or STAT3 phosphorylation induced by IL-6 in mice lacking SOCS-1 (Fig. 2B and data not shown). SOCS-1 appeared to be important for regulating the duration of the response to IFNgamma , but not the intensity, because peak levels of STAT1 phosphorylation did not differ substantially between mice of different genotypes.


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Fig. 2.   IFNgamma -induced STAT1 phosphorylation is prolonged in SOCS-1-/- IFNgamma -/- mice. SOCS-1+/+ IFNgamma +/+, SOCS-1+/+ IFNgamma -/- and SOCS-1-/- IFNgamma -/- mice were injected with 2 µg of IFNgamma (A) or 10 µg of IL-6 (B) and sacrificed at the times indicated. Liver lysates were prepared and STAT1 protein immunoprecipitated with an anti-STAT1 antibody. Duplicate blots were incubated with either an antibody specific for phosphorylated STAT1 (alpha -STAT1-P, left panels) or STAT1 (right panels).

SOCS-1 Is Required for Regulating Responses to IFNgamma in Hepatocytes in Vitro-- The in vivo experiments described above indicate a role for SOCS-1 in regulating IFNgamma -induced responses in the liver but may be complicated by factors such as clearance of IFNgamma from the circulation. We therefore wished to examine IFNgamma responses in vitro using primary hepatocyte cultures. To assess the sensitivity of hepatocytes to IFNgamma , primary hepatocyte cultures were established from SOCS-1-/- IFNgamma -/- and SOCS-1+/+ IFNgamma -/- mice. Hepatocytes were stimulated for 10 min with a pulse of IFNgamma , after which time cytokine was washed from the cells, and the response was allowed to decay over varying periods. Gel shift analysis was used to monitor the DNA binding activity of STAT dimers throughout the time course. In both cell types, the response to IFNgamma peaked at 30 min after stimulation. The response decayed rapidly in SOCS-1+/+ IFNgamma -/- cells and was virtually undetectable at 2 h poststimulation (Fig. 3A). In contrast, the activity of STAT dimers was prolonged in SOCS-1-/- IFNgamma -/- cells, persisting beyond 3 h after IFNgamma treatment (Fig. 3B).


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Fig. 3.   IFNgamma -induced STAT activation is prolonged in SOCS-1-/- IFNgamma -/- hepatocytes. Electrophoretic mobility shift assays of extracts from primary hepatocytes derived from SOCS-1+/+ IFNgamma -/- (A) or SOCS-1-/- IFNgamma -/- mice, stimulated for 10 min with 5 ng/ml IFNgamma and harvested at the times indicated. In panel C, extracts from 10 min time points were pretreated with antibodies specific for either STAT1 or STAT3 before binding to the m67 oligonucleotide. Addition of an anti-STAT1 antibody abolished the formation of the two DNA/protein complexes and resulted in a supershifted complex, indicated by *. Pretreatment with an anti-STAT3 antibody did not affect the intensity of the lower DNA-protein complex, but the intensity of the upper band was reduced (bands indicated by arrows).

Supershift analyses (Fig. 3C) identified STAT1 homodimers as the predominant STAT dimers formed in response to IFNgamma (Fig. 3, A and B, lower band), with a smaller amount of STAT1/STAT3 homodimers also being activated (Fig. 3, A and B, upper band). Similar to the in vivo studies, only the duration of the response to IFNgamma was altered in SOCS-1-/- cells and not the intensity. Other experiments were performed in which hepatocytes were exposed to the stimulus for the entire time course rather than just a pulse. Active STAT dimers persisted for longer in these experiments but did not decay in cells lacking SOCS-1 throughout an 8-h culture period, whereas STAT dimer formation in SOCS-1+/+ IFNgamma -/- cells had decayed to a baseline level by 5 h of culture (data not shown).

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IFNgamma is critical for mediating the multiorgan pathology of SOCS-1-/- mice, because mice lacking both SOCS-1 and IFNgamma are viable and healthy (13). However, the specific relationship between SOCS-1 and IFNgamma has yet to be defined. Is the phenotype of SOCS-1-/- mice due to increased sensitivity of cells to IFNgamma in the absence of SOCS-1 or to increased production of IFNgamma ? Both appear to contribute to the phenotype. A previous study has shown that circulating levels of IFNgamma are increased in mice lacking SOCS-1 (12). The data presented here clearly establish that in the absence of SOCS-1, mice and cells isolated from them are more sensitive to the effects of IFNgamma . Neonatal mice lacking SOCS-1 were substantially more sensitive to the toxic effects of administered IFNgamma . Further, we have directly established a biochemical basis for this hypersensitivity and have shown that SOCS-1 attenuates signal transduction pathways in vivo, consistent with in vitro data. STAT1 activation was prolonged in the liver of SOCS-1-/- IFNgamma -/- mice after injection of IFNgamma . This increased sensitivity in vivo was paralleled in vitro by prolonged STAT1 activation in primary hepatocytes in response to IFNgamma .

Although forced expression of SOCS-1 has been shown to limit the intensity of STAT activation in response to cytokine (1-3), SOCS-1 expression is temporally inappropriate in these studies. In the present study, which relies on more physiological expression, SOCS-1 appears to be important for limiting the duration of responses to cytokine and not the magnitude of the response. This result is consistent with the current model of SOCS action in which SOCS expression is induced by cytokine and then functions to switch off the cytokine-induced signal (1). The relative contributions to the SOCS-1-/- phenotype of increased IFNgamma production and prolonged IFNgamma responses are difficult to delineate. In situations in which IFNgamma is limiting, for instance in SOCS-1-/- IFNgamma +/- mice, pathological changes including uncontrolled macrophage activation are still evident and may result predominantly from inappropriate and prolonged signaling in response to more normal levels of circulating IFNgamma (28). Further, serum IFNgamma is undetectable in some SOCS-1-/- mice (data not shown). Our data suggest that hypersensitivity to IFNgamma may be driving the typical SOCS-1-/- phenotype seen in these mice.

Although the JAK/STAT pathway has been well characterized using both primary and continuous cell lines, the kinetics of JAK/STAT activation in vivo following cytokine stimulation has not been comprehensively studied. In the course of this study we have developed assays to monitor the phosphorylation of STATs from mouse liver, resulting in a detailed study of the kinetics of IFNgamma signaling in vivo.

The phenotype of SOCS-1-/- mice demonstrates a specific non-redundant role for SOCS-1 in determining the rate at which interferon signaling is attenuated. The lack of effect on IL-6 signaling in vivo may indicate that other SOCS proteins can compensate for the absence of SOCS-1 in other cytokine pathways. Whereas IFNgamma signaling is clearly prolonged by several hours in SOCS1-/- mice, STAT1 activation is still switched off, albeit later than normal. This residual negative regulation of signal transduction may be mediated by other members of the SOCS family or by other inhibitory mechanisms such as phosphatases (29) and the protein inhibitor of activated STAT (PIAS) (30). The results presented in this study using wild-type and SOCS-1-deficient cells provide a benchmark against which cells from other compound-knockout mice can be compared.

    ACKNOWLEDGEMENTS

We thank Dr. Richard Simpson and Robert Moritz for their generous gift of recombinant murine IL-6 and Katya Gray, Kathy Hanzinikolas, and Andrew Naughton for expert animal husbandry.

    FOOTNOTES

* This work was supported by the Anti-Cancer Council of Victoria, Melbourne, Australia; AMRAD Operations Pty. Ltd., Melbourne, Australia; the National Health and Medical Research Council, Canberra, Australia; the J. D. and L. Harris Trust; National Institutes of Health Grant CA-22556; and the Australian Federal Government Cooperative Research Centers Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a Queen Elizabeth II Fellowship from the Australian Research Council. To whom correspondence should be addressed. Tel.: 61 3 9345 2525; Fax: 61 3 9347 0852; E-mail: starr@wehi.edu.au.

Published, JBC Papers in Press, April 16, 2001, DOI 10.1074/jbc.M102737200

    ABBREVIATIONS

The abbreviations used are: SOCS, suppressor of cytokine signaling; JAK, Janus kinase; STAT, signal transducer and activator of transcription; IL, interleukin; IFN, interferon; RIPA, radioimmune precipitation buffer; PAGE, polyacrylamide gel electrophoresis; HBSS, Hank's buffered saline solution.

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