Macrophage-colony stimulating factor and interleukin-34 induce chemokines in human whole blood
Introduction
Macrophage-colony stimulating factor (M-CSF) promotes the survival, proliferation, and differentiation of mononuclear phagocyte lineages, which include monocytes, macrophages and osteoclasts [1], [2], [3]. M-CSF is synthesized by a variety of cell types, such as endothelial cells, osteoblasts, and fibroblast and binds to a single receptor, CSF-1R (c-FMS) that is encoded by the c-fms proto-oncogene and also exclusively expressed on mononuclear phagocyte, resulting in autophosphorylation by c-FMS kinase and a subsequent cascade of intracellular signals including phosphoinositol-3 (PI-3) kinase, mitogen-activated protein kinase (MAPK), and JAK/Stat pathways [4], [5]. Recently, a novel cytokine, interleukin-34 (IL-34) has been found to bind to the CSF-1R and promote monocyte viability with characteristics similar to M-CSF [6].
M-CSF plays important roles in innate immunity, cancer and inflammatory diseases, including systemic lupus erythematosus, atherosclerosis, obesity and rheumatoid arthritis (RA) [2]. M-CSF-c-FMS-pathway has reported to have a certain role in pathogenesis in RA. M-CSF is not only elevated in the synovial fluid of the patients with RA but is constitutively produced by synovial fibroblasts from RA patients [7], [8], [9]. Plasma levels of M-CSF were significantly higher in RA patients with increased number of CD16+ monocyte than in healthy controls [9]. Furthermore, M-CSF, in collaboration with nuclear factor kappa B ligand (RANKL), contributes to the differentiation of macrophages into osteoclasts, which are involved in bone destruction in RA [10]. In animal model, M-CSF-deficient mice were resistant to collagen-induced arthritis [11]. Furthermore, administration of an anti-M-CSF antibody reduced severity whereas administration of exogenous M-CSF exacerbated symptoms in mousse arthritis model [11], [12]. These indicate that M-CSF-c-FMS signaling pathway is a potential therapeutic target in RA ([2] and references therein). These extensive researches have led to the development of small molecule c-FMS kinase inhibitors showing efficacy in arthritis models in animals [13], [14], [15]. In these reports, cell proliferation assay using myeloid cell lines or primary bone marrow-derived macrophages was applied to evaluate potency of compounds as M-CSF is the predominant growth factor or survival factor for macrophage lineage cells. Also, differentiation of macrophages derived from mice with M-CSF enhances LPS-induced cytokine or chemokine production [16], [17], [18], [19]. Furthermore, M-CSF differentiated macrophage derived from human produced cytokines or chemokines in the presence or absence of LPS and also M-CSF-mediated chemokine production from macrophages was antagonized by c-FMS kinase inhibitors [19], [20], [21]. Thus, M-CSF-dependent priming for production of proinflammatory mediators has been well characterized. However, induction of proinflammatory cytokines and chemokines by M-CSF in ex vivo assay, i.e. assay using whole blood has not fully investigated to understand a role of M-CSF in the induction of proinflammatory mediators in whole blood. Furthermore, cell functional assays using downstream signals of phosphorylation of c-FMS have not yet been fully established except for cell proliferation assay using monocytic cell line to investigate translational pharmacology of c-FMS kinase inhibitors.
In current study, we investigated if M-CSF or IL-34 induces cytokines or chemokines using human whole blood (HWB) and if an M-CSF- or IL-34-induced cytokine or chemokine production from HWB is inhibited by c-FMS kinase inhibitors. Also, we investigate whether IL-34 has the function similar to M-CSF in the induction of proinflammatory mediators in HWB.
Section snippets
Human whole blood (HWB)
HWB was obtained from venous blood of donors collected anonymously with informed consent at an on-site clinic into sodium heparin tubes and used immediately.
Measurement of cytokine and chemokine level in HWB
HWB was incubated with or without GW2580, a specific c-FMS kinase inhibitor [15] for 45 min at 37 °C and then M-CSF or IL-34 (100 ng/ml) was added into the plate and the plate was further incubated for 2, 6, and 20 h at 37 °C. In the assay using recombinant human M-CSF receptor/Fc chimera, M-CSF or IL-34 was incubated with recombinant human
Cytokine or chemokine induction by M-CSF or IL-34 in HWB
Firstly, induction of cytokines or chemokines by M-CSF or IL-34 was investigated using HWB (Table 1). Among eight cytokines or growth factors tested, IL-1β, IL-6, TNFα, and GM-CSF levels in HWB were significantly elevated by M-CSF and IL-34 while the level of the elevation was small except for IL-6. Other cytokines were not elevated by M-CSF or IL-34 in HWB. Chemokine levels in HWB induced by M-CSF or IL-34 are shown in Table 2. Among seven chemokines tested, levels of IP-10/CXCL10, IL-8/CXCL8,
Discussion
In this study, we have demonstrated that proinflammatory cytokines and chemokines were induced by M-CSF and IL-34 in HWB. IL-34 is newly identified cytokine that binds to the CSF-1R and promotes monocyte viability with characteristics similar to M-CSF [6]. Also, IL-34 promotes osteoclast development in combination with RANK ligand which function is the same as that of M-CSF [22]. We elucidated that IL-34 promotes osteoclast development through M-CSF receptor (Zack M, in preparation). In this
Acknowledgements
We thank Molly K. Hall and Becky L. Hood for experimental support during HWB assay.
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