The signaling mechanism by which the anti-inflammatory cytokine IL-10 mediates suppression of proinflammatory cytokine synthesis remains largely unknown. Macrophage-specific STAT3-null mice have demonstrated that STAT3 plays a critical role in the suppression of LPS-induced TNF-α release, although the mechanism by which STAT3 mediates this inhibition is still not clear. Using an adenoviral system, we have expressed a dominant negative (DN) STAT3 in human macrophages to broaden the investigation to determine the role of STAT3 in IL-10-mediated anti-inflammatory signaling and gene expression. Overexpression of STAT3 DN completely inhibited IL-10-induced suppressor of cytokine signaling 3, tissue inhibitor of MMP-1, TNF receptor expression, and the recently identified IL-10-inducible genes, T cell protein tyrosine phosphatase and signaling lymphocyte activation molecule. STAT3 DN also blocked IL-10-mediated inhibition of MHC class II and COX2 expression. In agreement with the studies in STAT3-null mice, overexpression of the STAT3 DN completely reversed the ability of IL-10 to inhibit LPS-mediated TNF-α and IL-6 production. However, real-time PCR analysis showed that STAT3 DN expression did not affect immediate suppression of TNF-α mRNA, but did reverse the suppression observed at later time points, suggesting a biphasic regulation of TNF-α mRNA levels by IL-10. In conclusion, although STAT3 does appear to be the dominant mediator of the majority of IL-10 functions, there are elements of its anti-inflammatory activity that are STAT3 independent.

The GTPase Rho is known to mediate the assembly of integrin-containing focal adhesions and actin stress fibers. Here, we investigate the role of Rho in regulating the distribution of the monocyte-binding receptors E-selectin, ICAM-1, and VCAM-1 in human endothelial cells. Inhibition of Rho activity with C3 transferase or N19RhoA, a dominant negative RhoA mutant, reduced the adhesion of monocytes to activated endothelial cells and inhibited their spreading. Similar effects were observed after pretreatment of endothelial cells with cytochalasin D. In contrast, dominant negative Rac and Cdc42 proteins did not affect monocyte adhesion or spreading. C3 transferase and cytochalasin D did not alter the expression levels of monocyte-binding receptors on endothelial cells, but did inhibit clustering of E-selectin, ICAM-1, and VCAM-1 on the cell surface induced by monocyte adhesion or cross-linking antibodies. Similarly, N19RhoA inhibited receptor clustering. Monocyte adhesion and receptor cross-linking induced stress fiber assembly, and inhibitors of myosin light chain kinase prevented this response but did not affect receptor clustering. Finally, receptor clusters colocalized with ezrin/moesin/ radixin proteins. These results suggest that Rho is required in endothelial cells for the assembly of stable adhesions with monocytes via the clustering of monocyte-binding receptors and their association with the actin cytoskeleton, independent of stress fiber formation.

A major therapeutic challenge is how to replace bone once it is lost. Bone loss is a characteristic of chronic inflammatory and degenerative diseases such as rheumatoid arthritis and osteoporosis. Cells and cytokines of the immune system are known to regulate bone turnover by controlling the differentiation and activity of osteoclasts, the bone resorbing cells. However, less is known about the regulation of osteoblasts (OB), the bone forming cells. This study aimed to investigate whether immune cells also regulate OB differentiation. Using in vitro cell cultures of human bone marrow-derived mesenchymal stem cells (MSC), it was shown that monocytes/macrophages potently induced MSC differentiation into OBs. This was evident by increased alkaline phosphatase (ALP) after 7 days and the formation of mineralised bone nodules at 21 days. This monocyte-induced osteogenic effect was mediated by cell contact with MSCs leading to the production of soluble factor(s) by the monocytes. As a consequence of these interactions we observed a rapid activation of STAT3 in the MSCs. Gene profiling of STAT3 constitutively active (STAT3C) infected MSCs using Illumina whole human genome arrays showed that Runx2 and ALP were up-regulated whilst DKK1 was down-regulated in response to STAT3 signalling. STAT3C also led to the up-regulation of the oncostatin M (OSM) and LIF receptors. In the co-cultures, OSM that was produced by monocytes activated STAT3 in MSCs, and neutralising antibodies to OSM reduced ALP by 50%. These data indicate that OSM, in conjunction with other mediators, can drive MSC differentiation into OB. This study establishes a role for monocyte/macrophages as critical regulators of osteogenic differentiation via OSM production and the induction of STAT3 signalling in MSCs. Inducing the local activation of STAT3 in bone cells may be a valuable tool to increase bone formation in osteoporosis and arthritis, and in localised bone remodelling during fracture repair.

SUMMARYEfforts to identify the signal transduction pathways used by interleukin-10 (IL-10) have resulted in limited success. The anti-inflammatory effects elicited by IL-10, and the mechanisms by which these are mediated, are still relatively unknown. Understanding the signalling mechanisms behind the suppression of cytokine expression by IL-10 could be of potential therapeutic interest. Although the consensus is that the Janus kinase, Jak1, as well as the signal transducer and activator of transcription STAT3 are central, much controversy exists about the participation and roles of many other signalling pathways targeted by IL-10. The mechanisms of cytokine suppression proposed by various groups have included transcriptional, post-transcriptional and post-translational regulation of IL-10 target genes; nevertheless no unifying model has emerged thus far. Here we would like to highlight novel findings and discuss their implications in the context of current understanding of IL-10 signalling.

The bacterial endotoxin LPS is a potent stimulator of monocyte and macrophage activation and induces adhesion of monocytes. Morphological changes in response to LPS have not been characterized in detail, however, nor have the signaling pathways mediating LPS-induced adhesion been elucidated. We have found that LPS rapidly induced adhesion and spreading of peripheral blood monocytes, and that this was inhibited by the Src family kinase inhibitor PP1 and the phosphatidylinositide 3-kinase inhibitor LY294002. LPS also stimulated actin reorganization, leading to the formation of filopodia, lamellipodia, and membrane ruffles in Bac1 mouse macrophages. Proline-rich tyrosine kinase 2 (Pyk2), a tyrosine kinase related to focal adhesion kinase, and paxillin, a cytoskeletal protein that interacts with Pyk2, were both tyrosine phosphorylated in response to LPS in monocytes and macrophages. Both tyrosine phosphorylation events were inhibited by PP1 and LY294002. Adhesion also stimulated tyrosine phosphorylation of Pyk2 and paxillin in monocytes, and this was further enhanced by LPS. Finally, Pyk2 and paxillin colocalized within membrane ruffles in LPS-stimulated cells. These results indicate that LPS stimulation of monocytes and macrophages results in rapid morphological changes and suggest that Pyk2 and/or paxillin play a role in this response.

TLR3 recognizes double-stranded RNA, a product associated with viral infections. Many details of TLR3-induced mechanisms have emerged from gene-targeted mice or inhibition studies in transformed cell lines. However, the pathways activated in human immune cells or cells from disease tissue are less well understood. We have investigated TLR3-induced mechanisms of human primary cells of the innate immune system, including dendritic cells (DCs), macrophages (MØs), endothelial cells (ECs), and synovial fibroblasts isolated from rheumatoid arthritis joint tissue (RA-SFs). Here, we report that while these cells all express TLR3, they differ substantially in their response to TLR3 stimulation. The key antiviral response chemokine IP-10 was produced by all cell types, while DCs and MØs failed to produce the proinflammatory cytokines TNF␣ and IL-6. Unexpectedly, TNF␣ was found secreted by TLR3-stimulated RA-SF. Furthermore, TLR3 stimulation did not activate NFB, MAPKs, or IRF-3 in DCs and MØs, but was able to do so in ECs and RA-SF. These findings were specific for human cells, thereby revealing a complexity not previously expected. This is the first report of such cell type-and species-specific response for any TLR stimulation and helps to explain important difficulties in correlating murine models of inflammatory diseases and human inflammation. IntroductionThe discovery of Toll-like receptors (TLRs) uncovered a key mechanism used by the immune system to detect infections and tissue damage. 1 There are 10 known human TLRs that recognize "molecular patterns" produced as a result of pathogenic infections. TLR3 is a member of this receptor family that is activated by double-stranded RNA (dsRNA), 2 an intermediate formed by most viruses during their replication. The viral dsRNA functions as a danger signal released in the extracellular environment from dying virally infected cells, alerting inflammatory cells and contributing to systemic disease. 3,4 In addition to microbial products, there is growing evidence that TLRs also recognize endogenous ligands found at sites of tissue destruction and cell death, 5 and TLR3 has been shown to recognize mRNA released from dying cells. 6 Although there are concerns that some of the data on endogenous ligands could be a result of contamination with microbial products, the detection of endogenous ligands would be useful in alerting the host of the presence of tissue injury induced by infection or other means. However, there is a potential drawback in that the same endogenous ligands are also found at sites of chronic inflammation and could further drive the inflammatory response in a TLRdependent manner. There is increasing evidence that TLRs could be associated with chronic inflammatory diseases such as rheumatoid arthritis (RA). 5,7 In this context, TLR3 expression is increased in the RA synovium, 8,9 and the presence of TLR3 ligands might affect several aspects of the disease by acting on cells within the joint. In support of this, dsRNA can induce joint inflammation and cause a...

IL-10 is an important immunosuppressive cytokine that can down-regulate expression of other cytokines and has been shown to down-regulate itself. We show, in this study, that treatment of human monocyte-derived macrophages with IL-10 induces IL-10 mRNA in a dose- and time-dependent manner with an optimum induction at 100 ng/ml and at 6 h, whereas IL-10-induced IL-10 protein can be detected at 18 h. In the same cells, IL-10 can partially suppress IL-10 mRNA induced by LPS, but only down to the level of IL-10-induced IL-10. An adenoviral luciferase reporter construct driven by the −195 IL-10 promoter, which contains a Stat motif, was readily induced by both IL-10 and LPS. Mutation of this Stat motif ablated IL-10 activation of this promoter, but not the LPS activation. Finally, we show that overexpression of a dominant-negative Stat3 protein will prevent IL-10 induction, but not LPS induction, of IL-10 mRNA. These data show that IL-10 induces IL-10 in monocyte-derived macrophages in an autocrine manner via activation of the transcription factor Stat3.

IL-10 is a potent anti-inflammatory cytokine and inhibitor of TNF-α production. The molecular pathways by which IL-10 inhibits TNF-α production are obscure, with diverse mechanisms having been published. In this study, a new approach has been taken for the study of human cells. Adenovirus was used to deliver TNF-α promoter-based luciferase reporter genes to primary human monocytic cells. The reporter genes were highly responsive to macrophage activation and appeared to mirror the behavior of the endogenous TNF-α gene. When added, either with or after the stimulus, IL-10 required the 3′ untranslated region of the TNF-α gene to inhibit luciferase mRNA and protein expression, indicating a posttranscriptional mechanism. However, if macrophages were incubated with IL-10 before activation, inhibition of gene expression was also mediated by the 5′ promoter, suggesting a transcriptional mechanism. To our knowledge, this is the first time that a dual mechanism for IL-10 function has been demonstrated. Studies to elucidate the mechanisms underlying the inhibition of TNF-α production addressed the effect of IL-10 on the activation of p38 mitogen-activated protein kinase and NF-κB. However, these studies could demonstrate no requirement for the inhibition of p38 mitogen-activated protein kinase or NF-κB activation as potential mechanisms. Overall, these results may explain the diversity previously ascribed to the complex mechanisms of IL-10 anti-inflammatory activity.