Divergent mechanisms utilized by SOCS3 to mediate IL-10 inhibition of TNF-a and nitric oxide production by macrophages

The cytokine, interleukin-10 (IL-10), inhibits activation o f macrophages by activators such as lipopolysacchride (LPS). However the mechanism by which IL-10 interferes with LPS signalling is still unclear. One well-characterized signalling pathway activated b y IL-10 is that of Stat3 (Signal transducer and activator o f transcription), which is essential for the antiproliferative and anti-inflammatory actions of IL-10 on macrophages. In our search for IL-10- induced, Stat3-regulated genes, we found a candidate belonging to SOCS (suppressor of cytokine signalling) family of negative regulators of cytokine signalling. Using mutant IL-10 receptor and dominant negative Stat3, we show that IL-10 induces SOCS3 message and protein in a Stat3-dependent manner. However mere expression o f SOCS3 protein in macrophages was not sufficient to inhibit TNF-α protein production in response to LPS, suggesting that additional IL-10-induced signals are required. And indeed we find IL-10 stimulation induces phosphorylation of tyrosine 204 o f SOCS3 protein. In order to determine the role o f SOCS3 in IL-10 inhibition of macrophage activation, we derived cell lines from SOCS3⁻[sup /]⁻ and SOCS3⁺[sup /]⁻ mice. SOCS3⁻[sup /]⁻ macrophages respond to LPS in a manner similar to wild-type cells, but IL-10 is less effective in inhibiting LPS-induced TNF-α and NO production in the SOCS3⁻[sup /]⁻ cells as compared to SOCS3⁺[sup /]⁻ macrophages. Reconstitution of SOCS3⁻[sup /]⁻ cells with a wild-type SOCS3 cDNA restored IL-10 responsiveness. In order to determine which SOCS3 domain is important in IL-10 signalling, SOCS3⁻[sup /]⁻ macrophages were reconstituted with various SOCS3 domain mutants. IL-10 required all domains of SOCS3 protein for the inhibition of TNF-α protein expression. However, for inhibition of TNF-α mRNA expression, IL-10 does not seem to require the KIR domain of SOCS3 protein. In contrast, only the two tyrosine residues, 204 and 221, located in the SOCS-box domain are required for IL-10 inhibition of LPS-induced iNOS protein expression and subsequent NO production. These studies demonstrate the importance of SOCS3 protein in the anti-inflammatory action of IL-10 and that inhibition of NO and TNF-α by IL-10 depends on different domains of SOCS3. Characterization of SOCS3 signalling domains and its immediate downstream targets will allow development of therapeutic strategies, which replicate the beneficial anti- inflammatory action of IL-10.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat