Suppressor of cytokine signalling-3 at pathological levels does not regulate lipopolysaccharide or interleukin-10 control of tumour necrosis factor-alpha production by human monocytes

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine that suppresses the production of tumour necrosis factor-α (TNF-α) by monocytes and macrophages. Suppressor of cytokine signalling-3 (SOCS3), a negative regulator of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, is induced following IL-10 exposure but recent studies in mice suggest that SOCS3 only targets gp-130-dependent signal transduction pathways. Understanding the signalling pathways responsible for IL-10-mediated effects in primary human monocytes is relevant to human inflammatory disease and necessary for the identification of potential therapeutic targets. An adenoviral transfection system was used to express different levels of SOCS3 (quantified experimentally with its tag green fluorescent protein (GFP)) with the aim of investigating the role of SOCS3 in LPS-induced and IL-10-mediated suppression of TNF-α production by non-transformed human monocytes. SOCS3 over-expression had no effect on TNF-α mRNA levels induced by LPS or LPS plus IL-10, or on IL-10 phosphorylation of STAT3, STAT1 and ERK1/2. When data from all donors were combined, adenoviral overexpression of SOCS3 significantly reversed the suppressive effects of IL-10 on LPS-induced TNF-α production after 2 hr. However, there was a direct correlation between mean GFP intensity (extent of viral infection) and extent of reversal of IL-10's inhibitory effects. Physiological levels of SOCS3 detected in IL-10-exposed human monocytes had no effect on LPS-induced TNF-α production. Although overexpression of SOCS3 to supraphysiological levels transiently antagonized the regulatory properties of IL-10 by a post-transcriptional mechanism, these findings suggest that under pathological conditions SOCS3 does not control LPS-activation or the anti-inflammatory properties of IL-10 in primary human monocytes

Molecular mechanisms of interleukin-10-mediated inhibition of NF-κB activity: a role for p50

Nuclear factor kappa B (NF-κB) is a transcription factor pivotal for the development of inflammation. A dysregulation of NF-κB has been shown to play an important role in many chronic inflammatory diseases including rheumatoid arthritis, inflammatory bowel disease and psoriasis. Although classical NF-κB, a heterodimer composed of the p50 and p65 subunits, has been well studied, little is known about gene regulation by other hetero- and homodimeric forms of NF-κB. While p65 possesses a transactivation domain, p50 does not. Indeed, p50/p50 homodimers have been shown to inhibit transcriptional activity. We have recently shown that Interleukin-10 exerts its anti-inflammatory activity in part through the inhibition of NF-κB by blocking IκB kinase activity and by inhibiting NF-κB already found in the nucleus. Since the inhibition of nuclear NF-κB could not be explained by an increase of nuclear IκB, we sought to further investigate the mechanisms involved in the inhibition of NF-κB by IL-10. We show here that IL-10 selectively induced nuclear translocation and DNA-binding of p50/p50 homodimers in human monocytic cells. TNF-α treatment led to a strong translocation of p65 and p50, whereas pretreatment with IL-10 followed by TNF-α blocked p65 translocation but did not alter the strong translocation of p50. Furthermore, macrophages of p105/p50-deficient mice exhibited a significantly decreased constitutive production of MIP-2α and IL-6 in comparison to wild type controls. Surprisingly, IL-10 inhibited high constitutive levels of these cytokines in wt macrophages but not in p105/p50 deficient cells. Our findings suggest that the selective induction of nuclear translocation and DNA-binding of the repressive p50/p50 homodimer is an important anti-inflammatory mechanism utilized by IL-10 to repress inflammatory gene transcription