GM-CSF- and IRF4-Dependent Signaling Can Regulate Myeloid Cell Numbers and the Macrophage Phenotype during Inflammation.

Studies have demonstrated the importance of a GM-CSF→IFN regulatory factor 4 (IRF4)→CCL17 pathway, first identified in monocytes/macrophages, for arthritic pain and disease development. In this study, we further investigated the involvement of this new pathway in shaping the inflammatory response using the zymosan-induced peritonitis (ZIP) model. ZIP (8 mg of zymosan, i.p., day 0) was induced in C57BL/6 wild-type (WT), GM-CSF-/- , Irf4-/- , and Ccl17E/E mice. In comparison with WT mice, GM-CSF-/- and Irf4-/- mice had a reduced ZIP response, as judged by a reduced number of neutrophils and macrophages in the peritoneal cavity. Moreover, the phenotype of the ZIP macrophages was altered by a lack of GM-CSF or IRF4 (increased IL-10 secretion and Arg1 mRNA expression), with IRF4 levels being lower in GM-CSF-/- ZIP macrophages than in the WT cells. In addition, GM-CSF ̶IRF4 signaling upregulated MHC class II expression in ZIP macrophages and bone marrow-derived macrophages. Although Ccl17 mRNA expression was reduced in ZIP macrophages in the absence of either GM-CSF or IRF4, thus supporting the presence of the new pathway in inflammatory macrophages, CCL17 did not modulate the inflammatory response, both in terms of number of myeloid cells or the macrophage phenotype. Thus, during an inflammatory response, both macrophage numbers and their phenotype can depend on GM-CSF- and IRF4-dependent signaling independently of CCL17.

TNF and granulocyte macrophage-colony stimulating factor interdependence mediates inflammation via CCL17.

TNF and granulocyte macrophage-colony stimulating factor (GM-CSF) have proinflammatory activity and both contribute, for example, to rheumatoid arthritis pathogenesis. We previously identified a new GM-CSF→JMJD3 demethylase→interferon regulatory factor 4 (IRF4)→CCL17 pathway that is active in monocytes/macrophages in vitro and important for inflammatory pain, as well as for arthritic pain and disease. Here we provide evidence for a nexus between TNF and this pathway, and for TNF and GM-CSF interdependency. We report that the initiation of zymosan-induced inflammatory pain and zymosan-induced arthritic pain and disease are TNF dependent. Once arthritic pain and disease are established, blockade of GM-CSF or CCL17, but not of TNF, is still able to ameliorate them. TNF is required for GM-CSF-driven inflammatory pain and for initiation of GM-CSF-driven arthritic pain and disease, but not once they are established. TNF-driven inflammatory pain and TNF-driven arthritic pain and disease are dependent on GM-CSF and mechanistically require the same downstream pathway involving GM-CSF→CCL17 formation via JMJD3-regulated IRF4 production, indicating that GM-CSF and CCL17 can mediate some of the proinflammatory and algesic actions of TNF. Given we found that TNF appears important only early in arthritic pain and disease progression, targeting a downstream mediator, such as CCL17, which appears to act throughout the course of disease, could be effective at ameliorating chronic inflammatory conditions where TNF is implicated.

Granulocyte macrophage colony-stimulating factor induces CCL17 production via IRF4 to mediate inflammation.

Data from preclinical and clinical studies have demonstrated that granulocyte macrophage colony-stimulating factor (GM-CSF) can function as a key proinflammatory cytokine. However, therapies that directly target GM-CSF function could lead to undesirable side effects, creating a need to delineate downstream pathways and mediators. In this work, we provide evidence that GM-CSF drives CCL17 production by acting through an IFN regulatory factor 4-dependent (IRF4-dependent) pathway in human monocytes, murine macrophages, and mice in vivo. In murine models of arthritis and pain, IRF4 regulated the formation of CCL17, which mediated the proinflammatory and algesic actions of GM-CSF. Mechanistically, GM-CSF upregulated IRF4 expression by enhancing JMJD3 demethylase activity. We also determined that CCL17 has chemokine-independent functions in inflammatory arthritis and pain. These findings indicate that GM-CSF can mediate inflammation and pain by regulating IRF4-induced CCL17 production, providing insights into a pathway with potential therapeutic avenues for the treatment of inflammatory diseases and their associated pain.

Granulocyte-macrophage colony-stimulating factor is a key mediator in inflammatory and arthritic pain.

OBJECTIVES: Better therapies are needed for inflammatory pain. In arthritis the relationship between joint pain, inflammation and damage is unclear. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is important for the progression of a number of inflammatory/autoimmune conditions including arthritis; clinical trials targeting its action in rheumatoid arthritis are underway. However, its contribution to inflammatory and arthritic pain is unknown. The aims of this study were to determine whether GM-CSF controls inflammatory and/or arthritic pain. METHODS: A model of inflammatory pain (complete Freund's adjuvant footpad), as well as two inflammatory arthritis models, were induced in GM-CSF(-/-) mice and development of pain (assessment of weight distribution) and arthritic disease (histology) was assessed. Pain was further assessed in a GM-CSF-driven arthritis (methylated bovine serum albumin/GM-CSF) model and the cyclooxygenase-dependence determined using indomethacin. RESULTS: GM-CSF was absolutely required for pain development in both the inflammatory pain and arthritis models, including for IL-1-dependent arthritic pain. Pain in a GM-CSF-driven arthritis model, but not the disease itself, was abolished by the cyclooxygenase inhibitor, indomethacin, indicating separate pathways downstream of GM-CSF for pain and arthritis control. CONCLUSIONS: GM-CSF is key to the development of inflammatory and arthritic pain, suggesting that pain alleviation could result from trials evaluating its role in inflammatory/autoimmune conditions.

Hypoxia enhances the proliferative response of macrophages to CSF-1 and their pro-survival response to TNF.

In chronic inflammatory lesions there are increased numbers of macrophages with a possible contribution of enhanced survival/proliferation due, for example, to cytokine action; such lesions are often hypoxic. Prior studies have found that culture in low oxygen can promote monocyte/macrophage survival. We show here, using pharmacologic inhibitors, that the hypoxia-induced pro-survival response of macrophages exhibits a dependence on PI3-kinase and mTOR activities but surprisingly is suppressed by Akt and p38 MAPK activities. It was also found that in hypoxia at CSF-1 concentrations, which under normoxic conditions are suboptimal for macrophage proliferation, macrophages can proliferate more strongly with no evidence for alteration in CSF-1 receptor degradation kinetics. TNF promoted macrophage survival in normoxic conditions with an additive effect in hypoxia. The enhanced hypoxia-dependent survival and/or proliferation of macrophages in the presence of CSF-1 or TNF may contribute to their elevated numbers at a site of chronic inflammation.

Granulocyte-macrophage colony-stimulating factor is a key mediator in experimental osteoarthritis pain and disease development.

INTRODUCTION: Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to be important in the development of inflammatory models of rheumatoid arthritis and there is encouraging data that its blockade may have clinical relevance in patients with rheumatoid arthritis. The aims of the current study were to determine whether GM-CSF may also be important for disease and pain development in a model of osteoarthritis. METHODS: The role of GM-CSF was investigated using the collagenase-induced instability model of osteoarthritis. We studied both GM-CSF-/- mice and wild-type (C57BL/6) mice treated prophylactically or therapeutically with a monoclonal antibody to GM-CSF. Disease development (both early and late) was evaluated by histology and knee pain development was measured by assessment of weight distribution. RESULTS: In the absence of GM-CSF, there was less synovitis and matrix metalloproteinase-mediated neoepitope expression at week 2 post disease induction, and less cartilage damage at week 6. GM-CSF was absolutely required for pain development. Therapeutic neutralization of GM-CSF not only abolished the pain within 3 days but also led to significantly reduced cartilage damage. CONCLUSIONS: GM-CSF is key to the development of experimental osteoarthritis and its associated pain. Importantly, GM-CSF neutralization by a therapeutic monoclonal antibody-based protocol rapidly and completely abolished existing arthritic pain and suppressed the degree of arthritis development. Our results suggest that it would be worth exploring the importance of GM-CSF for pain and disease in other osteoarthritis models and perhaps clinically for this form of arthritis.

Defining GM-CSF- and macrophage-CSF-dependent macrophage responses by in vitro models.

GM-CSF and M-CSF (CSF-1) induce different phenotypic changes in macrophage lineage populations. The nature, extent, and generality of these differences were assessed by comparing the responses to these CSFs, either alone or in combination, in various human and murine macrophage lineage populations. The differences between the respective global gene expression profiles of macrophages, derived from human monocytes by GM-CSF or M-CSF, were compared with the differences between the respective profiles for macrophages, derived from murine bone marrow cells by each CSF. Only 17% of genes regulated differently by these CSFs were common across the species. Whether a particular change in relative gene expression is by direct action of a CSF can be confounded by endogenous mediators, such as type I IFN, IL-10, and activin A. Time-dependent differences in cytokine gene expression were noted in human monocytes treated with the CSFs; in this system, GM-CSF induced a more dramatic expression of IFN-regulated factor 4 (IRF4) than of IRF5, whereas M-CSF induced IRF5 but not IRF4. In the presence of both CSFs, some evidence of "competition" at the level of gene expression was observed. Care needs to be exercised when drawing definitive conclusions from a particular in vitro system about the roles of GM-CSF and M-CSF in macrophage lineage biology.

Extracellular proteomes of M-CSF (CSF-1) and GM-CSF-dependent macrophages.

Macrophage colony-stimulating factor (M-CSF) (also known as CSF-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have distinct effects on macrophage lineage populations, which are likely to be contributing to their functional heterogeneity. A comparative proteomic analysis of proteins released into culture media from such populations after M-CSF and GM-CSF exposure was carried out. Adherent macrophage populations, termed bone marrow-derived macrophage (BMM) and GM-BMM, were generated after treatment of murine bone marrow precursors with M-CSF and GM-CSF, respectively. Proteins in 16-h serum-free conditioned media (CM) were identified by two-dimensional gel electrophoresis and mass spectrometry. Respective protein profiles from BMM and GM-BMM CM were distinct and there was the suggestion of a switch from primarily signal peptide-driven secretion to non-classical secretion pathways from BMM to GM-BMM. Extracellular expression of cathepsins (lysosomal proteases) and their inhibitors seems to be a characteristic difference between these macrophage cell types with higher levels usually observed in BMM-CM. Furthermore, we have identified a number of proteins in BMM-CM and GM-BMM-CM that could be involved in various tissue regeneration and inflammatory (immune) processes, respectively. The uncharacterized protein C19orf10, a protein found at high levels in the synovial fluid of arthritis patients, was also differentially regulated; its extracellular levels were upregulated in the presence of GM-CSF.

Low dose metal particles can induce monocyte/macrophage survival.

Aseptic loosening results in pain, loss of function, and ultimately prosthetic joint failure and revision surgery. The generation of wear particles from the prosthesis is a major factor in local osteolysis. We investigated the effects of such wear particles on the survival of monocytes and macrophages, populations implicated in wear particle-driven pathology. Particles from titanium aluminum vanadium (TiAlV) and cobalt chromium (CoCr) alloys were generated in-house and were equivalent in size (0.5-3 microm) to those seen in patients. Human CD14(+) monocytes and murine bone marrow-derived macrophages (BMM) were treated with TiAlV and CoCr particles in vitro, and cell survival was assayed. Both particles increased monocyte and macrophage survival in a dose-dependent manner, with an optimal concentration of around 10(7) particles/mL. Conditioned media from particle-treated BMM also increased macrophage survival. Studies with antibody blockade and gene-deficient mice suggest that particle-induced BMM survival is independent of endogenous CSF-1 (M-CSF), GM-CSF, and TNFalpha. These data indicate that wear particles can promote monocyte/macrophage survival in vitro possibly via an endogenous mediator. If this phenomenon occurs in vivo, it could mean that increased numbers of macrophages (and osteoclasts) would be found at a site of joint implant failure, which could contribute to the local inflammatory reaction and osteolysis.