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

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

doi:10.1074/jbc.m208586200

Abstract

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 gamma (IFNgamma). Recent data suggest that SOCS-1 may regulate other cytokines in vivo, in addition to IFNgamma. Uncontrolled responses to interleukin-12 (IL-12), an inflammatory cytokine, could contribute to increased IFNgamma 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 IFNgamma, SOCS-1 regulates responses to IL-12.

Highlights

Cytokines are small, secreted molecules that regulate an array of cellular processes, including hematopoietic differentiation and responses to infection or injury (1)
These mice are not, completely normal and inflammatory infiltrates eventually develop, resulting in a reduced lifespan (14, 15). These findings suggest that Suppressor of cytokine signaling-1 (SOCS-1) may be an essential physiological regulator of other cytokines in addition to IFN␥ and that dysregulated responses to these cytokines may contribute to disease development in SOCS-1Ϫ/Ϫ mice
Uncontrolled responses to IL-12 may contribute to the severe inflammatory disease in SOCS-1Ϫ/Ϫ mice

Summary

EXPERIMENTAL PROCEDURES

Mice were genotyped for the SOCS-1 gene by Southern blot analysis of genomic DNA obtained from tail tips, as described (9). 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). Flow Cytometry—Single cell suspensions prepared from mesenteric lymph node were depleted of red blood cells and resuspended in 50 ␮l of saturating amounts of 2.4G2 anti-Fc␥ 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 (44). Splenocytes of the same genotype were pooled and cultured overnight at 37 °C in 6-well plates at a density of 1 ϫ 107 cells/ml, in the presence of 0 –1000 units/ml rmIL-12. For the analysis of SOCS-1/STAT4 mice, age-matched mice pairs were compared using a one-tailed Student’s paired t test

RESULTS

Thymus cortex atrophy

DISCUSSION