Previous studies have shown that IL-10 can induce the expression of the suppressor of cytokine signaling 3 (SOCS-3) mRNA in human monocytes and neutrophils, suggesting that the capacity of IL-10 to inhibit the expression of LPS-inducible proinflammatory genes may depend on SOCS-3 induction. However, no direct experimental evidence has been provided to support such hypothesis. Herein, we show that stable transfection of SOCS-3 into the mouse macrophage cell line J774 resulted in an inhibition of NO, TNF-α, IL-6, and GM-CSF secretion in response to LPS at levels similar to those exerted by IL-10 in LPS-stimulated wild-type J774. Constitutive SOCS-3 expression also down-regulated the mRNA expression of inducible NO synthase and IL-6 and impaired the production of TNF-α, mainly at a post-transcriptional level. In addition, SOCS-3-transfected cells displayed a constitutive expression of the IL-1R antagonist gene, consistent with the observation that IL-10 enhances IL-1R antagonist mRNA in LPS-stimulated wild-type cells. Furthermore, in peritoneal macrophages harvested from mice carrying heterozygous disruption of the SOCS-3 gene, IL-10 was less effective in repressing LPS-stimulated TNF-α and NO production. Taken together, our data show that SOCS-3 inhibits LPS-induced macrophage activation, strongly supporting the idea that it plays a role in the molecular mechanism by which IL-10 down-modulates the effector functions of LPS-activated macrophages. Finally, we show that forced expression of SOCS-3 significantly suppresses the ability of IL-10 to trigger tyrosine phosphorylation of STAT3. Therefore, SOCS-3 functions both as an LPS signal inhibitor and as a negative feedback regulator of IL-10/STAT3 signaling.

SOCS-3 (suppressor of cytokine signaling 3) is an intracellular protein that is selectively and rapidly induced by appropriate agonists and that modulates responses of immune cells to cytokines by interfering with the Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathway. On the basis of the observations that interferon ␥ (IFN␥) up-regulates SOCS-3 gene and protein expression in primary mouse macrophages, J774 macrophage cell line and embryonal fibroblasts, we investigated which sequences of the 5 SOCS-3 gene are responsive to IFN␥. By promoter deletion analysis we identified a functional IFN␥-responsive element, located at nucleotides ؊72/؊64 upstream from the transcription initiation, whose presence and integrity is necessary to ensure responsiveness to IFN␥. This element contains a STAT consensus binding sequence (SOCS-3/STAT-binding element (SBE)) whose specific mutation totally abolished the responsiveness to IFN␥. In contrast, discrete deletion of other 5 regions of the SOCS-3 promoter did not substantially modify the inducibility by IFN␥. Electromobility shift assay analyses revealed that IFN␥ promotes specific DNA binding activities to an oligonucleotide probe containing the SOCS-3/SBE sequence. Even though IFN␥ triggered tyrosine phosphorylation of both STAT1 and STAT3 in macrophages and J774 cells, only STAT1 was appropriately activated and thus found to specifically bind to the SOCS-3/SBE oligonucleotide probe. Accordingly, IFN␥-induced SOCS-3 protein expression was not impaired in STAT3-deficient embryonal fibroblasts. Taken together, these results demonstrate that the induction of SOCS-3 by IFN␥ depends upon the presence of a STAT-binding element in the SOCS-3 promoter that is specifically activated by STAT1.

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