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Negative Regulation of Interleukin-12 Signaling by Suppressor of Cytokine Signaling-1

2002; Elsevier BV; Volume: 277; Issue: 46 Linguagem: Inglês

10.1074/jbc.m208586200

ISSN

1083-351X

Autores

Joanne L. Eyles, Donald Metcalf, Michael J. Grusby, Douglas J. Hilton, Robyn Starr,

Tópico(s)

Pharmacological Effects of Natural Compounds

Resumo

Suppressor of cytokine signaling-1 (SOCS-1) is an inhibitory protein that regulates responses to cytokines. Previously, we have shown SOCS-1 to be a key inhibitor of interferon γ (IFNγ). Recent data suggest that SOCS-1 may regulate other cytokines in vivo, in addition to IFNγ. Uncontrolled responses to interleukin-12 (IL-12), an inflammatory cytokine, could contribute to increased IFNγ production and the development of inflammatory disease in SOCS-1−/− mice. Here, we assess responses of SOCS-1-deficient cells to IL-12. Both IL-12-induced T cell proliferation and NK cytotoxic activity are enhanced in SOCS-1-deficient cells, relative to controls. To examine the contribution of continued IL-12 signaling to the SOCS-1−/− disease, we generated mice lacking both SOCS-1 and signal transducer and activator of transcription 4 (STAT4), an essential component of the IL-12 signaling pathway. SOCS-1−/− STAT4−/− mice have improved survival relative to SOCS-1−/− mice, but die between 1 and 2 months of age. We conclude that, in addition to IFNγ, SOCS-1 regulates responses to IL-12. Suppressor of cytokine signaling-1 (SOCS-1) is an inhibitory protein that regulates responses to cytokines. Previously, we have shown SOCS-1 to be a key inhibitor of interferon γ (IFNγ). Recent data suggest that SOCS-1 may regulate other cytokines in vivo, in addition to IFNγ. Uncontrolled responses to interleukin-12 (IL-12), an inflammatory cytokine, could contribute to increased IFNγ production and the development of inflammatory disease in SOCS-1−/− mice. Here, we assess responses of SOCS-1-deficient cells to IL-12. Both IL-12-induced T cell proliferation and NK cytotoxic activity are enhanced in SOCS-1-deficient cells, relative to controls. To examine the contribution of continued IL-12 signaling to the SOCS-1−/− disease, we generated mice lacking both SOCS-1 and signal transducer and activator of transcription 4 (STAT4), an essential component of the IL-12 signaling pathway. SOCS-1−/− STAT4−/− mice have improved survival relative to SOCS-1−/− mice, but die between 1 and 2 months of age. We conclude that, in addition to IFNγ, SOCS-1 regulates responses to IL-12. Cytokines are small, secreted molecules that regulate an array of cellular processes, including hematopoietic differentiation and responses to infection or injury (1Nicola N.A. Guidebook to Cytokines and Their Receptors. Oxford University Press, New York1994Google Scholar). As highly potent molecules, both cytokine secretion and responses to cytokines are tightly regulated processes. In the absence of appropriate regulation, autoimmune and autoinflammatory diseases may occur. Clearly, maintaining homeostasis of the immune system requires delicate regulation of cytokine production and action. SOCS-1 1The abbreviations used are: SOCS, suppressor of cytokine signaling; IL-12, interleukin-12; IL-12R, IL-12 receptor; IFNγ, interferon γ; STAT, signal transducer and activator of transcription; NK, natural killer; rm, recombinant murine; FACS, fluorescence-activated cell sorting; ELISA, enzyme-linked immunosorbent assay is the prototypic member of a family of cytokine inhibitors (2Hilton D.J. Richardson R.T. Alexander W.S. Viney E.M. Willson T.A. Sprigg N.S. Starr R. Nicholson S.E. Metcalf D. Nicola N.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 114-119Crossref PubMed Scopus (620) Google Scholar). Originally isolated as a negative regulator of interleukin-6 (IL-6) action (3Starr R. Willson T.A. Viney E.M. Murray L.J. Rayner J.R. Jenkins B.J. Gonda T.J. Alexander W.S. Metcalf D. Nicola N.A. Hilton D.J. Nature. 1997; 387: 917-921Crossref PubMed Scopus (1816) Google Scholar, 4Endo T.A. Masuhara M. Yokouchi M. Suzuki R. Sakamoto H. Mitsui K. Matsumoto A. Tanimura S. Ohtsubo M. Misawa H. Miyazaki T. Leonor N. Taniguchi T. Fujita T. Kanakura Y. Komiya S. Yoshimura A. Nature. 1997; 387: 921-924Crossref PubMed Scopus (1234) Google Scholar, 5Naka T. Narazaki M. Hirata M. Matsumoto T. Minamoto S. Aono A. Nishimoto N. Kajita T. Taga T. Yoshizaki K. Akira S. Kishimoto T. Nature. 1997; 387: 924-929Crossref PubMed Scopus (1139) Google Scholar), subsequent studies have shown that SOCS-1 inhibits a wide range of cytokinesin vitro (6Sakamoto H. Yasukawa H. Masuhara M. Tanimura S. Sasaki A. Yuge K. Ohtsubo M. Ohtsuka A. Fujita T. Ohta T. Furukawa Y. Iwase S. Yamada H. Yoshimura A. Blood. 1998; 92: 1668-1676Crossref PubMed Google Scholar, 7Losman J.A. Chen X.P. Hilton D. Rothman P. J. Immunol. 1999; 162: 3770-3774PubMed Google Scholar, 8Sporri B. Kovanen P.E. Sasaki A. Yoshimura A. Leonard W.J. 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Starr R. J. Biol. Chem. 2001; 276: 22086-22089Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In contrast to SOCS-1−/− mice, SOCS-1−/−IFNγ−/− mice survive until adulthood and appear healthy (12Alexander W.S. Starr R. Fenner J.E. Scott C.L. Handman E. Sprigg N.S. Corbin J.E. Cornish A.L. Darwiche R. Owczarek C.M. Kay T.W. Nicola N.A. Hertzog P.J. Metcalf D. Hilton D.J. Cell. 1999; 98: 597-608Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar). These mice are not, however, completely normal and inflammatory infiltrates eventually develop, resulting in a reduced lifespan (14Metcalf D., Di Rago L. Mifsud S. Hartley L. Alexander W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9174-9179Crossref PubMed Scopus (52) Google Scholar,15Metcalf D. Mifsud S., Di Rago L. Nicola N.A. Hilton D.J. Alexander W.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 943-948Crossref PubMed Scopus (80) Google Scholar). 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Fujiwara H. J. Leukoc. Biol. 1997; 61: 80-87Crossref PubMed Scopus (85) Google Scholar). Once produced, IL-12 acts upon activated T cells and NK cells to induce their proliferation, cytotoxicity, and IFNγ production (16Kobayashi M. Fitz L. Ryan M. Hewick R.M. Clark S.C. Chan S. Loudon R. Sherman F. Perussia B. Trinchieri G. J. Exp. Med. 1989; 170: 827-845Crossref PubMed Scopus (1899) Google Scholar, 17Chan S.H. Perussia B. Gupta J.W. Kobayashi M. Pospisil M. Young H.A. Wolf S.F. Young D. Clark S.C. Trinchieri G. J. Exp. Med. 1991; 173: 869-879Crossref PubMed Scopus (962) Google Scholar, 18Chan S.H. Kobayashi M. Santoli D. Perussia B. Trinchieri G. J. Immunol. 1992; 148: 92-98PubMed Google Scholar, 19Schoenhaut D.S. Chua A.O. Wolitzky A.G. Quinn P.M. Dwyer C.M. McComas W. Familletti P.C. Gately M.K. Gubler U. J. Immunol. 1992; 148: 3433-3440PubMed Google Scholar, 30Gately M.K. Desai B.B. Wolitzky A.G. Quinn P.M. Dwyer C.M. Podlaski F.J. Familletti P.C. Sinigaglia F. Chizonnite R. 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Immunol. 1997; 159: 1658-1665PubMed Google Scholar). Expression of the IL-12R β2 subunit is rapidly up-regulated following activation of NK and T cells, enabling these cells to respond to IL-12 (37Gately M.K. Renzetti L.M. Magram J. Stern A.S. Adorini L. Gubler U. Presky D.H. Ann. Rev. Immunol. 1998; 16: 495-521Crossref PubMed Scopus (1103) Google Scholar, 38Chakir H. Camilucci A.A. Filion L.G. Webb J.R. J. Immunol. 2000; 165: 4985-4993Crossref PubMed Scopus (28) Google Scholar). IL-12 interacts with functional IL-12R on the cell surface and activates cytoplasmic signal transduction pathways, including the Janus kinases/signal transducers and activators of transcription (JAK/STAT) signaling pathway (39Bacon C.M. McVicar D.W. Ortaldo J.R. Rees R.C. O'Shea J.J. Johnston J.A. J. Exp. Med. 1995; 181: 399-404Crossref PubMed Scopus (295) Google Scholar, 40Jacobson N.G. Szabo S.J. Weber-Nordt R.M. Zhong Z. Schreiber R.D. Darnell Jr., J.E. Murphy K.M. J. Exp. Med. 1995; 181: 1755-1762Crossref PubMed Scopus (583) Google Scholar).In vivo studies have shown that STAT4 is a critical mediator of IL-12-induced responses (41Kaplan M.H. Sun Y.L. Hoey T. Grusby M.J. Nature. 1996; 382: 174-177Crossref PubMed Scopus (1061) Google Scholar, 42Thierfelder W.E. van Deursen J.M. Yamamoto K. Tripp R.A. Sarawar S.R. Carson R.T. Sangster M.Y. Vignali D.A. Doherty P.C. Grosveld G.C. Ihle J.N. Nature. 1996; 382: 171-174Crossref PubMed Scopus (955) Google Scholar). Inflammatory diseases that appear to result from either the overproduction of IL-12 or heightened sensitivity to this cytokine demonstrate that both IL-12 production and signaling require strict regulation (25Davidson N.J. Hudak S.A. Lesley R.E. Menon S. Leach M.W. Rennick D.M. J. Immunol. 1998; 161: 3143-3149PubMed Google Scholar, 26Simpson S.J. Shah S. Comiskey M. de Jong Y.P. Wang B. Mizoguchi E. Bhan A.K. Terhorst C. J. Exp. Med. 1998; 187: 1225-1234Crossref PubMed Scopus (253) Google Scholar). Although cytokines such as IL-10 appear to regulate IL-12 production (43D'Andrea A. Aste-Amezaga M. Valiante N.M., Ma, X. Kubin M. Trinchieri G. J. Exp. Med. 1993; 178: 1041-1048Crossref PubMed Scopus (1327) Google Scholar), the mechanisms that control IL-12 signaling remain to be elucidated. One possibility is that IL-12 signaling may be regulated by inhibitory molecules such as the SOCS proteins. This study examines IL-12-induced responses in SOCS-1-deficient cells and provides evidence that SOCS-1 regulates responses to IL-12 in vivo. All mice were bred and housed in clean but not specific pathogen-free conditions at the Walter and Eliza Hall Institute of Medical Research. SOCS-1−/− mice on a 129/Sv × C57BL/6 background were generated as described previously (9Starr R. Metcalf D. Elefanty A.G. Brysha M. Willson T.A. Nicola N.A. Hilton D.J. Alexander W.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14395-14399Crossref PubMed Scopus (380) Google Scholar) and then were backcrossed at least 10 generations to C57BL/6. IFNγ−/− mice on an inbred C57BL/6 background (C57BL/6-tmlTs) were obtained from Jackson Laboratories (Bar Harbor, ME). SOCS-1+/+ IFNγ−/− and SOCS-1−/− IFNγ−/− mice were generated either on a mixed 129/Sv × C57BL/6 background as described previously (12Alexander W.S. Starr R. Fenner J.E. Scott C.L. Handman E. Sprigg N.S. Corbin J.E. Cornish A.L. Darwiche R. Owczarek C.M. Kay T.W. Nicola N.A. Hertzog P.J. Metcalf D. Hilton D.J. Cell. 1999; 98: 597-608Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar), or on a C57BL/6 background, by mating SOCS-1+/− (backcrossed 10 generations to C57BL/6) with IFNγ−/− mice. Mice were genotyped for theSOCS-1 gene by Southern blot analysis of genomic DNA obtained from tail tips, as described (9Starr R. Metcalf D. Elefanty A.G. Brysha M. Willson T.A. Nicola N.A. Hilton D.J. Alexander W.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14395-14399Crossref PubMed Scopus (380) Google Scholar). STAT4−/− mice on a mixed 129/Sv and BALB/c genetic background were generated as described previously (41Kaplan M.H. Sun Y.L. Hoey T. Grusby M.J. Nature. 1996; 382: 174-177Crossref PubMed Scopus (1061) Google Scholar) and were mated with SOCS-1+/− mice. SOCS-1+/−STAT4+/− mice were then interbred to generate mice of the various genotypes required for the study. Mice were genotyped for STAT4 by PCR analysis of genomic DNA obtained from tail tips, using a protocol kindly provided by Dr. Mark Kaplan (Indiana University School of Medicine, Indianapolis). Briefly, wild type and knockout alleles were amplified in a single PCR reaction using a mixture of three oligonucleotide primers; a 5′ STAT4 oligo (5′-gac agc aac tgg aga aac tac agg agc-3′), a 3′ STAT4 oligo (5′-ctg agt cag ctg ctg gga gaa gag gtg-3′) and to amplify the knockout allele, a neo primer (gct acc cgt gat att gct gaa gag). PCR was performed using 40 cycles of 96 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. Wild type and knockout STAT4 alleles were amplified to produce 320 and 250 bp PCR products, respectively. Full histological analyses of mice were performed as described previously (15Metcalf D. Mifsud S., Di Rago L. Nicola N.A. Hilton D.J. Alexander W.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 943-948Crossref PubMed Scopus (80) Google Scholar). Briefly, tissues were fixed in 10% buffered formalin and sectioned and stained with hematoxylin and eosin. Unless otherwise stated, for all assays cells were cultured in mouse tonicity RPMI 1640 containing 10% (v/v) fetal calf serum and 5 × 10−5m2-mercaptoethanol, at 37 °C in a humidified cell incubator containing 10% (v/v) CO2. Recombinant murine (rm) IL-12 was purchased from R&D Systems (Minneapolis, MN). Single cell suspensions prepared from mesenteric lymph node were depleted of red blood cells and then resuspended in 50 μl of saturating amounts of 2.4G2 anti-Fcγ receptor antibody containing 10% (v/v) normal rat serum, and stained with cocktails of fluorochrome-conjugated antibodies specific for CD4, CD8, or CD44, as described previously (44Alexander W.S. Roberts A.W. Nicola N.A., Li, R. Metcalf D. Blood. 1996; 87: 2162-2170Crossref PubMed Google Scholar). Cells were analyzed using a FACScan (Becton Dickinson, Franklin Lakes, NJ) with CellQuest Software. Dead cells and erythrocytes were excluded by propidium iodide staining (1 μg/ml) and gating of forward angle and side scatter. Dispersed cell suspensions were prepared from superficial cervical, axillary, brachial, renal, mesenteric, and inguinal lymph nodes from either sick SOCS-1−/− mice (7–15 days old) and their healthy wild type littermates; or SOCS-1−/− IFNγ−/−(4–7 weeks old) and age-matched SOCS-1+/+IFNγ−/− mice. Lymph node cells from mice of the same genotype were pooled (4–6 mice/pool) and enriched for T cells using Mouse T Cell Enrichment Columns (R&D Systems) according to the manufacturer's protocol. FACS analysis showed T cell populations were typically greater than 98% pure. T cells were cultured in 96-well U-bottomed plates at a density of 5 × 105 cells/well. Appropriate wells were coated with 10 μg/ml anti-CD3 antibody (clone KT3-1-1) for at least 1 h at 37 °C, and then antibody was aspirated prior to adding cells. Cultures were stimulated with 0–1000 units/ml rmIL-12 in the presence of 20 μg/ml rat anti-mouse IL-2 antibody (clone JES6–1A12, R&D Systems), and cultured for 1–5 days. [H3]thymidine (1μCi/well; Amersham Biosciences) was added 17 h before harvest of the cultures. Radioactivity was measured using a scintillation counter (Packard, Meridin, CT), and the results were expressed as mean cpm ± S.D. of triplicate cultures. Lymph node T cells were purified as described above and then fixed and permeabilized using a Cytoperm/Cytofix kit (PharMingen, San Diego, CA) according to the manufacturer's protocol. In parallel, T cells were stimulated as described for the proliferation assays, with anti-CD3 antibody and either 0.5 or 50 units/ml rmIL-12. Anti-IL-2 antibody (20 μg/ml) was included in all assays. For the next 4 days, replicate samples of the cells were fixed and permeabilized. Fixed cells were incubated with 1 μg/ml of either polyclonal anti-IL-12R antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or, as a control, polyclonal anti-IL-11R antibody (Santa Cruz Biotechnology). Receptor binding was then detected using 1:200-diluted FITC-conjugated sheep anti-rabbit antibody (Amersham Biosciences) and analyzed by FACS. Spleens were harvested from SOCS-1−/− IFNγ−/− mice (5–6 weeks old) and age-matched SOCS-1+/+ IFNγ−/− mice (n = 3 mice/genotype), and cell suspensions were red blood cell-depleted. Splenocytes of the same genotype were pooled and cultured overnight at 37 °C in 6-well plates at a density of 1 × 107 cells/ml, in the presence of 0–1000 units/ml rmIL-12. The following day, splenocytes were washed twice and added to 96-well plates in graded dilutions to obtain the desired Effector:Target (E:T) ratios of 90:1, 20:1, 10:1, 5:1, and 1:1. The NK-susceptible cell line, YAC-1, was kindly provided by Dr. Mark Smyth (Peter MacCallum Cancer Institute, Melbourne). YAC-1 cells were labeled with 100 μCi of 51Cr (PerkinElmer Life Sciences) for 1 h at 37 °C, washed three times, and plated at 104 cells/well into plates containing appropriately diluted effector cells. After 4 h at 37 °C, plates were spun, and supernatants were counted in a scintillation counter (Packard). YAC-1 cells were incubated in media alone or in the presence of 10% (v/v) SDS to determine spontaneous and total release, respectively. Specific lysis was calculated by % lysis = (E − S)/(T − S) × 100, where E is the release from experimental samples, S is the spontaneous release, and T is the total release. Microsoft Excel was used for all statistical analyses. Data from in vitro assays were analyzed using a one-tailed Student's t test for independent events with the assumption of equal variance. For the analysis of SOCS-1/STAT4 mice, age-matched mice pairs were compared using a one-tailed Student's paired t test. The deleterious effects of continued IL-12 signaling are evident in inflammatory disorders such as Crohn's disease (45Parrello T. Monteleone G. Cucchiara S. Monteleone I. Sebkova L. Doldo P. Luzza F. Pallone F. J. Immunol. 2000; 165: 7234-7239Crossref PubMed Scopus (115) Google Scholar). Therefore uncontrolled responses to IL-12 may contribute to the severe inflammatory disease in SOCS-1−/− mice. To investigate this, in vitro assays were developed to measure IL-12 responses by the two major IL-12-responsive cell types, NK and T cells. Firstly, we assessed IL-12-induced proliferation of SOCS-1−/− T cells. In order to measure proliferation in response to IL-12 alone, a neutralizing antibody to IL-2 was included in all assays. Initially, we assayed purified lymph node T cells from SOCS-1−/− and control mice in the absence of activation with anti-CD3. Under these conditions, T cells are expected to be in a resting state and unresponsive to IL-12. As shown in Fig.1 A, purified T cells did not proliferate in the absence of exogenous cytokine, and IL-12 induced negligible proliferation of T cells from control mice. In contrast, the proliferative response of SOCS-1−/− T cells to IL-12 was significantly greater, suggesting that these cells were activated and expressed functional IL-12R in vivo (Fig.1 A). Due to the poor health and complex phenotype of SOCS-1−/−mice and the activated phenotype of SOCS-1−/− T cells, we decided to use T cells from healthy, adult SOCS-1−/−IFNγ−/− mice to assess IL-12 responses. T cells from age-matched SOCS-1+/+ IFNγ−/− mice were used as a control. T cells from both SOCS-1−/−IFNγ−/− and SOCS-1+/+IFNγ−/− mice responded poorly to IL-12 treatment alone, most likely due to low IL-12R expression by resting T cells (data not shown). Therefore IL-12-induced proliferation was assayed in the presence of a comitogen, anti-CD3, in order to activate T cells and induce IL-12R expression. T cells of both genotypes failed to respond to anti-CD3 alone (Fig. 1 B). The combination of anti-CD3 and IL-12, however, induced a dose-dependent proliferation by both SOCS-1−/− IFNγ−/− and SOCS-1+/+ IFNγ−/− T cells (Fig.1 B). SOCS-1−/− IFNγ−/− T cells showed significantly greater proliferation in response to IL-12 than SOCS-1+/+ IFNγ−/− T cells at all IL-12 concentrations tested, and proliferated in response to as little as 0.5 units/ml IL-12 (Fig. 1 B). To determine which cells were expanding in response to IL-12, the T cell composition of the proliferating cells was analyzed by FACS and was expressed as a ratio of CD8/CD4 cells. The CD8/CD4 ratios in initial populations were ∼1:1 for T cells of either genotype (Fig.1 C). These ratios changed slightly after 5 days in culture in the absence of exogenous cytokine (1.3:1 for SOCS-1−/−IFNγ−/−, and 0.5:1 for SOCS-1+/+IFNγ−/− T cells). The T cell composition of SOCS-1+/+ IFNγ−/− samples did not change substantially after culture with IL-12 for 5 days (Fig. 1 C). In contrast, IL-12 induced a dose-dependent increase in the ratio of CD8/CD4 cells (to ∼4.5:1) from SOCS-1−/−IFNγ−/− mice, suggesting that CD8 T cells were more responsive to IL-12 than CD4 T cells (Fig. 1 C). NK and NKT cells were present in negligible numbers in the purified lymph node preparations and did not expand substantially in response to IL-12 (data not shown). Increased IL-12-induced proliferation of SOCS-1−/− IFNγ−/− lymph node T cells may be due to uncontrolled IL-12 signaling in the absence of SOCS-1 and/or as a result of increased IL-12R expression (46Chang J.T. Shevach E.M. Segal B.M. J. Exp. Med. 1999; 189: 969-978Crossref PubMed Scopus (86) Google Scholar). To distinguish these possibilities, IL-12R expression was analyzed by FACS in both freshly isolated lymph node T cells and cells stimulated with IL-12 in combination with anti-CD3 (Fig. 2). IL-12R expression was similar in freshly isolated SOCS-1−/− IFNγ−/− and SOCS-1+/+ IFNγ−/− lymph node T cells (Fig.2). The combination of IL-12 and anti-CD3 was sufficient to upregulate IL-12R expression on T cells of both genotypes, however, SOCS-1−/− IFNγ−/− T cells up-regulated IL-12R expression faster (by day 2 of culture) and in response to lower doses of IL-12 (0.5 units/ml) than SOCS-1+/+IFNγ−/− T cells (Fig. 2). Again, this demonstrates that cells from SOCS-1-deficient mice are hypersensitive to IL-12 and suggests that the enhanced IL-12-induced proliferation of SOCS-1-deficient cells may be due both to increased levels of IL-12R expression and to an intrinsic hyper-responsiveness to IL-12. A key function of IL-12 is to induce cytotoxic activity in NK cells (32Robertson M.J. Soiffer R.J. Wolf S.F. Manley T.J. Donahue C. Young D. Herrmann S.H. Ritz J. J. Exp. Med. 1992; 175: 779-788Crossref PubMed Scopus (364) Google Scholar). We analyzed IL-12-induced NK cytotoxic activity in unsorted splenocytes by measuring lysis of the NK susceptible cell line, YAC-1. Previous analyses have shown that the proportion of splenic NK cells is similar in SOCS-1−/−IFNγ−/− and SOCS-1+/+IFNγ−/−mice. 2R. Starr, A. Cornish, W. Alexander, M. Chong, T. Kay, and D. Hilton, manuscript in preparation. Negligible cytotoxicity activity of the splenocytes was observed when cultured in media alone (Fig. 3). IL-12 induced a dose-dependent increase in NK activity in both SOCS-1−/− IFNγ−/− and SOCS-1+/+ IFNγ−/− splenocytes. SOCS-1−/− IFNγ−/− splenocytes induced maximal specific lysis of target cells when stimulated with 100 units/ml IL-12. In contrast, SOCS-1+/+IFNγ−/− splenocytes required stimulation with a 10-fold higher dose of IL-12 to achieve this effect. Since IL-12 promotes IFNγ production (16Kobayashi M. Fitz L. Ryan M. Hewick R.M. Clark S.C. Chan S. Loudon R. Sherman F. Perussia B. Trinchieri G. J. Exp. Med. 1989; 170: 827-845Crossref PubMed Scopus (1899) Google Scholar, 17Chan S.H. Perussia B. Gupta J.W. Kobayashi M. Pospisil M. Young H.A. Wolf S.F. Young D. Clark S.C. Trinchieri G. J. Exp. Med. 1991; 173: 869-879Crossref PubMed Scopus (962) Google Scholar, 18Chan S.H. Kobayashi M. Santoli D. Perussia B. Trinchieri G. J. Immunol. 1992; 148: 92-98PubMed Google Scholar, 19Schoenhaut D.S. Chua A.O. Wolitzky A.G. Quinn P.M. Dwyer C.M. McComas W. Familletti P.C. Gately M.K. Gubler U. J. Immunol. 1992; 148: 3433-3440PubMed Google Scholar), an increase in IL-12 production could drive the elevated IFNγ levels seen in SOCS-1−/− mice. We compared the levels of IL-12 in the serum of sick SOCS-1−/− mice and their wild type littermates and in healthy SOCS-1−/−IFNγ−/− and SOCS-1+/+IFNγ−/− mice to determine whether IL-12 levels are elevated in the absence of SOCS-1 and if so, whether it occurs independently of IFNγ. Mouse serum samples were analyzed using an ELISA with a detection sensitivity of ∼12 pg/ml IL-12 (data not shown). Detectable levels of IL-12 were found in the serum of 15% SOCS-1−/− mice (n = 20; data not shown). In contrast, IL-12 was not detected in the serum of SOCS-1−/− IFNγ−/− mice (n= 10), SOCS-1+/+ IFNγ−/− mice (n = 10), or wild type mice (n = 10; data not shown), suggesting that the elevated IL-12 levels measured in some SOCS-1−/− mice may be secondary to disease development. The in vitro functional assays suggest IL-12 responses are elevated in the absence of SOCS-1. We wished to examinein vivo whether IL-12 contributes to disease in SOCS-1−/− mice. STAT4 is believed to the primary mediator of the biological functions of IL-12 (41Kaplan M.H. Sun Y.L. Hoey T. Grusby M.J. Nature. 1996; 382: 174-177Crossref PubMed Scopus (1061) Google Scholar, 42Thierfelder W.E. van Deursen J.M. Yamamoto K. Tripp R.A. Sarawar S.

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