Divergent Mechanisms Utilized by SOCS3 to Mediate Interleukin-10 Inhibition of Tumor Necrosis Factor α and Nitric Oxide Production by Macrophages

Pooran Qasimi,Andrew Ming-Lum,Ali Ghanipour,Christopher J Ong,Michael E Cox,James Ihle,Nicolas Cacalano,Akihiko Yoshimura,Alice L-F Mui

doi:10.1074/jbc.m508608200

Abstract

The cytokine interleukin-10 (IL-10) potently inhibits macrophage function through activation of the transcription factor STAT3. The expression of SOCS3 (suppressor of cytokine signaling-3) has been shown to be induced by IL-10 in a STAT3-dependent manner. However, the relevance of SOCS3 expression to the anti-inflammatory effect of IL-10 on macrophages has been controversial. Through kinetic analysis of the requirement for SOCS3 in IL-10 inhibition of lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNFalpha) transcription and translation, SOCS3 was found to be necessary for TNFalpha expression during the early phase, but not the late phase of IL-10 action. SOCS3 was essential for IL-10 inhibition of LPS-stimulated production of iNOS (inducible nitric-oxide synthase) protein and nitric oxide (NO). To determine the domains of SOCS3 protein important in mediating these effects, SOCS3-/- macrophages were reconstituted with SOCS3 mutated for the SH2, KIR, SOCS box domains, and tyrosines 204 (Tyr204) and 221 (Tyr221). The SH2 domain, SOCS box, and both Tyr204 and Tyr221 were required for IL-10 inhibition of TNFalpha mRNA and protein expression, but interestingly the KIR domain was necessary only for IL-10 inhibition of TNFalpha protein expression. In contrast, Tyr204 and Tyr221 were the only structural features of SOCS3 that were necessary in mediating IL-10 inhibition of iNOS protein expression and NO production. These data define SOCS3 as an important mediator of IL-10 inhibition of macrophage activation and that SOCS3 interferes with distinct LPS-stimulated signal transduction events through differing mechanisms.

Highlights

Macrophages form the first line of defense against foreign pathogens by producing pro-inflammatory mediators that either directly attack the invading organism, or recruit and activate other cells of the innate and acquired immune systems [1]
A number of IL-10-regulated genes have been identified as being regulated by STAT3, including the NF-␬B family member BCL3, which is important in inhibition of TNF␣ transcription [20], and the cell cycle inhibitors p19INK4d and p21WIF1, which are required for IL-10 inhibition of macrophage proliferation [19]
Induction of SOCS3 Message by IL-10 in a STAT3-dependent Manner— From a survey of SOCS family members, we found that IL-10 treatment of J774.1 macrophage cells induces SOCS3 mRNA expression

Summary

Introduction

Macrophages form the first line of defense against foreign pathogens by producing pro-inflammatory mediators that either directly attack the invading organism, or recruit and activate other cells of the innate and acquired immune systems [1]. A number of IL-10-regulated genes have been identified as being regulated by STAT3, including the NF-␬B family member BCL3, which is important in inhibition of TNF␣ transcription [20], and the cell cycle inhibitors p19INK4d and p21WIF1, which are required for IL-10 inhibition of macrophage proliferation [19]. The SOCS family of proteins is transcriptionally regulated by a broad range of extracellular ligands and function in a classic feedback loop to negatively regulate signal transduction through multiple cytokine and growth factor receptors (24 –26). SOCS proteins have been reported to target signaling molecules for ubiquitin-mediated degradation through their SOCS box-mediated interaction with Elongins B and C [27] and this complex recruits the E2 ubiquitin-conjugating enzyme, forming the E3 ubiquitin-ligase [33]. The proximity of this ubiquitin ligase to SOCS-recruited signaling molecules can result in the proteasomal degradation of the latter [34, 35], association with Elongins B/C has been shown in some systems to protect SOCS protein from degradation (33, 36 –38)

Methods

Results

Conclusion