Macrophage Colony-stimulating Factor mRNA Research Articles

Erythropoietin Treatment in Mice Enriches Bone Marrow Osteoclast Precursors, As Reflected By Increased Macrophage Colony-Stimulating Factor (MCSF) and Elevated Expression of Osteoclast Markers in Monocytes

The impact of erythropoietin (EPO) on erythropoiesis has long been established. However, recent studies have indicated that EPO may also exert regulatory effects on other hematopoietic lineages, including the monocyte lineage. In that respect, we have previously shown that EPO treatment in mice induces bone loss. Here, we studied the effect of EPO on bone marrow (BM) monocytes and its potential implications for osteoclastogenesis. To address the dynamic changes in monocyte populations following EPO administration, we injected EPO (180U) once, or 3 times on alternating days for one week. Mice were sacrificed 16 h after EPO injection. Our results revealed a significant 20 percent increase in the monocyte population (TER119 -, LY6G -, CD11b +) 16 hours after EPO injection, and a twofold increase in the levels of these cells after one week of 3 injections of EPO. Soluble macrophage colony-stimulating factor (MCSF) protein levels in the bone marrow were increased 1.5-fold 16 h after EPO injection, while MCSF mRNA expression levels remained unchanged. After 1-week of EPO administration (3 injections), although the global BM expression of MCSF was reduced, we observed a two-fold increase in MCSF expression levels in T cells (CD3 +). To further elucidate the functional consequences of EPO-induced monocyte expansion, we isolated monocytes from mice treated with EPO for one week. We characterized expression levels of key osteoclastic markers in these cells. Indeed, we found that in BM monocytes, EPO treatment led to the upregulation of Lymphocyte function-associated antigen 1 (LFA-1) and Receptor Activator of Nuclear Factor κ B (RANK). The former is implicated in osteoclastogenesis via its interaction with intercellular adhesion molecule-1 (ICAM−1), while the latter is the surface receptor for RANK ligand which regulates osteoclast differentiation and activation. These data suggest that EPO not only influences BM monocyte expansion, but also contributes to the regulation of osteoclast differentiation, thus linking erythropoiesis to bone homeostasis. Furthermore, we investigated the early effects of EPO treatment on the expression of transcription factors implicated in monocyte differentiation. Remarkably, we found that EPO administration for 16 hours resulted in the downregulation of KLF2 and KLF4, transcription factors involved in monocytic lineage commitment. The rapid EPO-induced expansion of the monocytic population, coupled with the EPO-regulated expression of MSCF, osteoclastic markers, and transcription factors, suggests a role for EPO in modulating monocyte-to-osteoclast differentiation. The current study opens avenues for further investigating the crosstalk between erythropoiesis, myelopoiesis, and bone homeostasis.

Oncostatin M enhances osteoprotegerin synthesis but reduces macrophage colony‑stimulating factor synthesis in bFGF‑stimulated osteoblast‑like cells.

Bone remodeling is tightly controlled by various factors, including hormones, autacoids and cytokines. Among them, oncostatin M (OSM) is a multifunctional cytokine produced by osteal macrophages, which serves as an essential modulator of bone remodeling. Macrophage colony-stimulating factor (M-CSF) and osteoprotegerin are secreted by osteoblasts, and also have pivotal roles in the regulation of the bone remodeling process. The binding of basic fibroblast growth factor (bFGF), a key regulator of bone remodeling, to the corresponding receptor [fibroblast growth factor receptor (FGFR)] triggers the dimerization and activation of FGFRs, which causes the phosphorylation of FGFR substrates and subsequent activation of downstream effectors, including mitogen-activated protein kinases (MAPKs), via Grb2. bFGF can activate MAPKs, resulting in the synthesis of osteoprotegerin and vascular endothelial growth factor in osteoblast-like MC3T3-E1 cells. In the present study, the effects of OSM on bFGF-induced osteoblast activation were investigated in the synthesis of osteoprotegerin and M-CSF in osteoblasts. The release of osteoprotegerin and M-CSF were analyzed using ELISA. The mRNA expression levels of osteoprotegerin and M-CSF were analyzed using reverse transcription-quantitative PCR. Phosphorylation of p38 MAPK, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 MAPK was assessed using western blotting. OSM enhanced bFGF-induced osteoprotegerin release and bFGF-stimulated mRNA expression of osteoprotegerin. By contrast, OSM suppressed the bFGF-induced release of M-CSF and bFGF-stimulated mRNA expression of M-CSF. SB203580, a p38 MAPK inhibitor, and SP600125, a SAPK/JNK inhibitor, suppressed the bFGF-stimulated M-CSF release, whereas PD98059, an upstream kinase inhibitor of p44/p42 MAPK, failed to suppress the M-CSF release stimulated by bFGF. Furthermore, OSM enhanced the bFGF-induced phosphorylation of p38 MAPK, but attenuated the bFGF-stimulated phosphorylation of SAPK/JNK. By contrast, OSM had little effect on the bFGF-induced phosphorylation of p44/p42 MAPK. SB203580 markedly reduced the amplification of bFGF-stimulated osteoprotegerin release enhanced by OSM. These results strongly suggested that OSM may possess divergent effects on bFGF-induced osteoblast activation, upregulation of p38 MAPK and downregulation of SAPK/JNK, leading to the amplification of osteoprotegerin synthesis and the attenuation of M-CSF synthesis.

Resveratrol inhibits basic fibroblast growth factor-induced macrophage colony-stimulating factor synthesis via the PI3-kinase/Akt pathway in osteoblasts.

Resveratrol is a natural polyphenol found in grapes and beneficial for human health. Resveratrol regulates basic fibroblast growth factor (bFGF)-induced osteoprotegerin synthesis through Akt pathway in osteoblast-like MC3T3-E1 cells. In this study, we investigated resveratrol effects on bFGF-induced macrophage colony-stimulating factor (M-CSF) synthesis in MC3T3-E1 cells. bFGF significantly stimulated release and mRNA expression of M-CSF, which was reduced by resveratrol and SRT1720, sirtuin 1 (SIRT1) activator. Inauhzin, SIRT1 inhibitor, reversed inhibitory effects of resveratrol on bFGF-induced mRNA expression of M-CSF. Deguelin, Akt inhibitor, and LY294002, phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, reduced bFGF-induced M-CSF synthesis. Inauhzin reversed inhibitory effects of resveratrol on bFGF-induced Akt phosphorylation. Suppressive effect of resveratrol on bFGF-induced osteoprotegerin mRNA expression was confirmed in the identical samples using in experiment of M-CSF mRNA expression. Therefore, resveratrol reduces bFGF-induced M-CSF synthesis in addition to osteoprotegerin synthesis by inhibiting PI3-kinase/Akt pathway and suppressive effects are mediated through SIRT1 activation in osteoblasts.

Oncostatin M attenuates tumor necrosis factor-α-induced synthesis of macrophage-colony stimulating factor via suppression of Akt in osteoblasts

ABSTRACT Background Oncostatin M produced by osteal macrophages, a cytokine that belongs to the interleukin-6 family, is implicated in bone fracture healing. Macrophage colony-stimulating factor (M-CSF) secreted from osteoblasts plays an important role in osteoclastogenesis. We have previously reported that tumor necrosis factor-α (TNF-α), a potent bone resorptive agent, stimulates the activation of p44/p42 mitogen-activated protein (MAP) kinase, Akt, and p70 S6 kinase in osteoblast-like MC3T3-E1 cells, and induces the synthesis of M-CSF at least in part via Akt. Objective In the present study, we investigated whether oncostatin M affects the TNF-α-induced M-CSF synthesis in MC3T3-E1 cells and the underlying mechanisms. Methods Clonal osteoblast-like MC3T3-E1 cells were treated with oncostatin M or rapamycin and then stimulated with TNF-α. M-CSF release was assessed by ELISA. M-CSF mRNA expression level was assessed by real-time RT-PCR. Phosphorylation of Akt, p44/p42 MAP kinase, and p70 S6 kinase was detected by Western blot analysis. Results Oncostatin M dose-dependently reduced the TNF-α-stimulated M-CSF release. The expression of M-CSF mRNA induced by TNF-α was significantly suppressed by oncostatin M. Rapamycin, an inhibitor of mTOR/p70 S6 kinase, had little effect on the M-CSF release by TNF-α. Oncostatin M significantly reduced the TNF-α-induced phosphorylation of Akt and p44/p42 MAP kinase. However, the p70 S6 kinase phosphorylation by TNF-α was not affected by oncostatin M. Conclusion These results strongly suggest that oncostatin M attenuates TNF-α-stimulated synthesis of M-CSF in osteoblasts, and the inhibitory effect is exerted at a point upstream of Akt and p44/p42 MAP kinase but not p70 S6 kinase.

Expression of the cytoplasmic nucleolin for post-transcriptional regulation of macrophage colony-stimulating factor mRNA in ovarian and breast cancer cells

The formation of the mRNP complex is a critical component of translational regulation and mRNA decay. Both the 5′ and 3′UTRs of CSF-1 mRNA are involved in post-transcriptional regulation. In CSF-1 mRNA, a small hairpin loop structure is predicted to form at the extreme 5′ end (2–21nt) of the 5′UTR. Nucleolin binds the hairpin loop structure in the 5′UTR of CSF-1 mRNA and enhances translation, while removal of this hairpin loop nucleolin binding element dramatically represses translation. Thus in CSF-1 mRNA, the hairpin loop nucleolin binding element is critical for translational regulation. In addition, nucleolin interacts with the 3′UTR of CSF-1 mRNA and facilitates the miRISC formation which results in poly (A) tail shortening. The overexpression of nucleolin increases the association of CSF-1 mRNA containing short poly (A)n≤26, with polyribosomes. Nucleolin both forms an mRNP complex with the eIF4G and CSF-1 mRNA, and is co-localized with the eIF4G in the cytoplasm further supporting nucleolin's role in translational regulation. The distinct foci formation of nucleolin in the cytoplasm of ovarian and breast cancer cells implicates the translational promoting role of nucleolin in these cancers.

New Insight into Atherosclerosis in Hemodialysis Patients: Overexpression of Scavenger Receptor and Macrophage Colony-Stimulating Factor Genes

Background: Scavenger receptors (SRs) play a pivotal role in atherogenesis. The mechanism of atherosclerosis, which is specific to hemodialysis (HD) patients, was studied on the basis of SR gene expressions. Methods: The gene expressions of SR type A (SR-A) and CD36 were studied in peripheral monocytes by real-time reverse transcription polymerase chain reaction. Data were compared between HD (n = 30) and age-matched control subjects (n = 10). Serum levels of macrophage colony-stimulating factor (M-CSF) were measured with enzyme-linked immunosorbent assay to test its role in SR expression. The statistical differences and associations between two continuous variables were assessed using the Mann-Whitney U test and Pearson's correlation coefficient, respectively. Results: The relative quantities of SR mRNAs were significantly greater in HD patients than in controls [median (interquartile range): SR-A, 1.67 (0.96-2.76) vs. 0.90 (0.60-1.04), p = 0.0060; CD36, 1.09 (0.88-1.74) vs. 0.74 (0.64-0.99), p = 0.0255]. The serum concentration of M-CSF was significantly higher in HD patients than in controls [1,121 (999-1,342) vs. 176 (155-202) pg/ml, p < 0.0001]. In addition, the relative quantity of M-CSF mRNA was significantly greater in HD patients than in controls [0.79 (0.42-1.53) vs. 0.42 (0.28-0.66), p = 0.0392]. The serum M-CSF levels were positively correlated with both the relative quantity of SR-A mRNA (r² = 0.1681, p = 0.0086) and that of CD36 mRNA (r² = 0.1202, p = 0.0284) in all subjects (n = 40). Conclusion: HD patients are predisposed to atherosclerosis as a consequence of their enhanced monocyte SR expressions. SRs and M-CSF are potential therapeutic targets for atherosclerosis in this high-risk population.

Role of macrophage colony-stimulating factor in the development of neointimal thickening following arterial injury.

Evidence suggests that macrophage colony-stimulating factor (M-CSF) participates critically in atherosclerosis; little is known about the role of M-CSF in the development of neointimal hyperplasia following mechanical vascular injury. We examined the expression of M-CSF and its receptor, c-fms, in rodent and rabbit models of arterial injury. Injured rat carotid arteries expressed 3- to 10-fold higher levels of M-CSF and c-fms mRNA and protein following balloon injury as compared to uninjured arteries. In the rabbit, M-CSF protein expression was greatest in neointimal smooth muscle cells (SMCs) postinjury, with some expression in medial SMCs. M-CSF-positive SMCs exhibited markers of proliferation. At 30days postinjury, neointimal SMCs in the adjacent healed area near the border between injured and uninjured zone lost both proliferative activity and overexpression of M-CSF. The presence of induced M-CSF and c-fms expression correlated with the initiation of SMCs proliferation. M-CSF stimulated incorporation of [(3)H] thymidine in human aortic smooth muscle cells in a concentration-dependent manner. Serum-free conditioned medium from aortic SMCs also promoted DNA synthesis, and this effect was blocked by M-CSF specific antibody. To test further the role of M-CSF in vivo, we induced arterial injury by placing a periadventitial collar around the carotid arteries in compound mutant mice lacking apolipoprotein apoE (apoE(-/-)) and M-CSF. Loss of M-CSF abolished the neointimal hyperplastic response to arterial injury in apoE(-/-) mice. Local delivery of M-CSF to the injured artery restored neointimal proliferation, suggesting a critical role of M-CSF for the development of neointimal thickening following arterial injury.

Evaluation of biomarkers for in vitro prediction of drug-induced nephrotoxicity: comparison of HK-2, immortalized human proximal tubule epithelial, and primary cultures of human proximal tubular cells.

There has been intensive effort to identify in vivo biomarkers that can be used to monitor drug-induced kidney damage and identify injury before significant impairment occurs. Kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and human macrophage colony stimulating factor (M-CSF) have been validated as urinary and plasma clinical biomarkers predictive of acute and chronic kidney injury and disease. Similar validation of a high throughput in vitro assay predictive of nephrotoxicity could potentially be implemented early in drug discovery lead optimization to reduce attrition at later stages of drug development. To assess these known in vivo biomarkers for their potential for in vitro screening of drug-induced nephrotoxicity, we selected a panel of nephrotoxic agents and examined their effects on the overexpression of nephrotoxicity biomarkers in immortalized (HK-2) and primary (commercially available and freshly in-house produced) human renal proximal tubule epithelial cells. Traditional cytotoxicity was contrasted with expression levels of KIM-1, NGAL, and M-CSF assessed using ELISA and real-time quantitative reverse transcription PCR. Traditional cytotoxicity assays and biomarker assays using HK-2 cells were both unsuitable for prediction of nephrotoxicity. However, increases in protein levels of KIM-1 and NGAL in primary cells were well correlated with dose levels of known nephrotoxic compounds, with limited correlation seen in M-CSF protein and mRNA levels. These results suggest that profiling compounds against primary cells with monitoring of biomarker protein levels may have potential as in vitro predictive assays of drug-induced nephrotoxicity.

Effect of lipopolysaccharides from Porphyromonas endodontalis on the expression of macrophage colony stimulating factor in mouse osteoblasts

To investigate the effects of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (Pe) on the expression of macrophage colony stimulating factor (M-CSF) mRNA and protein in MC3T3-E1 cells and the role of nucler factor-κB (NF-κB) in the process. MC3T3-E1 cells were treated with different concentrations of Pe-LPS (0-50 mg/L) and 10 mg/L Pe-LPS for different hours (0-24 h). The expression of M-CSF mRNA and protein was detected by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunoadsordent assay (ELISA). The cells untreated by Pe-LPS served as control. The expression of M- CSF mRNA and protein was also detected in 10 mg/L Pe- LPS treated MC3T3-E1 cells after pretreated with BAY 11-7082 for 1 h, a special NF-κB inhibitor. The groups were divided as follows, control group, BAY group (10 µmol/L BAY 11-7082 treated alone MC3T3-E1 cells), Pe-LPS group (10 mg/L Pe-LPS stimulated MC3T3-E1 cells for 6 h), BAY combine with Pe-LPS group (10 µmol/L BAY 11-7082 pretreated cells for 1 h and 10 mg/L of Pe-LPS stimulated MC3T3-E1 cells for 6 h). The level of M- CSF mRNA and protein increased significantly after treatment with different concentrations of Pe-LPS (0-50 mg/L), which indicated that Pe-LPS induced osteoblasts to express M-CSF mRNA and protein in dose dependent manners. The expression of M-CSF protein increased from (35 ± 2) ng/L (control group) to (170 ± 8) ng/L (50 mg/L group). Maximal induction of M-CSF mRNA expression was found in the MC3T3- E1 cells treated with 10 mg/L Pe-LPS for 6 h. After 6 h, the expression of M-CSF mRNA decreased gradually. The expression of M-CSF protein also increased with the treatment of 10 mg/L Pe-LPS for 10 h [(122 ± 4) ng/L]. After 10 h, the expression of M-CSF protein decreased gradually. The mRNA and proteins of M-CSF decreased significantly after pretreatment with 10 µmol/L BAY 11-7082 for 1 h. There was no significant difference between BAY group and the control. Pe-LPS may induce the expression of M-CSF mRNA and protein in MC3T3-E1 cells through the signaling of NF-κB.

Macrophage colony-stimulating factor induces prolactin expression in rat pituitary gland.

We investigated the role of macrophage colony-stimulating factor (M-CSF) in the pituitary gland to understand the effect of M-CSF on pituitary hormones and the relationship between the endocrine and immune systems. When we attempted to establish pituitary cell lines from a thyrotropic pituitary tumor (TtT), a macrophage cell line, TtT/M-87, was established. We evaluated M-CSF-like activity in conditioned media (CM) from seven pituitary cell lines using TtT/M-87 cells. TtT/M-87 proliferation significantly increased in the presence of CM from TtT/GF cells, a pituitary folliculostellate (FS) cell line. M-CSF mRNA was detected in TtT/GF and MtT/E cells by reverse transcriptase-polymerase chain reaction (RT-PCR), and its expression in TtT/GF cells was increased in a lipopolysaccharide (LPS) dose-dependent manner. M-CSF mRNA expression was also increased in rat anterior pituitary glands by LPS. M-CSF receptor (M-CSFR) mRNA was only detected in TtT/ M-87 cells and increased in the LPS-stimulated rat pituitary glands. In rat pituitary glands, M-CSF and M-CSFR were found to be localized in FS cells and prolactin (PRL)-secreting cells, respectively, by immunohistochemistry. The PRL concentration in rat sera was significantly increased at 24 h after M-CSF administration, and mRNA levels significantly increased in primary culture cells of rat anterior pituitary glands. In addition, TNF-α mRNA was increased in the primary culture cells by M-CSF. These results revealed that M-CSF was secreted from FS cells and M-CSF regulated PRL expression in rat pituitary glands.

Inflammatory Bone Loss in Experimental Periodontitis Induced by Aggregatibacter actinomycetemcomitans in Interleukin-1 Receptor Antagonist Knockout Mice

The interleukin-1 receptor antagonist (IL-1Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is not clear whether IL-1Ra plays a protective role in periodontal disease. This study was undertaken to compare experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in IL-1Ra knockout (KO) mice and wild-type (WT) mice. Computed tomography (CT) analysis and hematoxylin-and-eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining were performed. In addition, osteoblasts were isolated; the mRNA expression of relevant genes was assessed by real-time quantitative PCR (qPCR); and calcification was detected by Alizarin Red staining. Infected IL-1Ra KO mice exhibited elevated (P, <0.05) levels of antibody against A. actinomycetemcomitans, bone loss in furcation areas, and alveolar fenestrations. Moreover, protein for tumor necrosis factor alpha (TNF-α) and IL-6, mRNA for macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL) in IL-1Ra KO mouse osteoblasts stimulated with A. actinomycetemcomitans were increased (P, <0.05) compared to in WT mice. Alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN)/bone gla protein (BGP), and runt-related gene 2 (Runx2) mRNA levels were decreased (P, <0.05). IL-1α mRNA expression was increased, and calcification was not observed, in IL-1 Ra KO mouse osteoblasts. In brief, IL-1Ra deficiency promoted the expression of inflammatory cytokines beyond IL-1 and altered the expression of genes involved in bone resorption in A. actinomycetemcomitans-infected osteoblasts. Alterations consistent with rapid bone loss in infected IL-Ra KO mice were also observed for genes expressed in bone formation and calcification. In short, these data suggest that IL-1Ra may serve as a potential therapeutic drug for periodontal disease.

Bisphosphonate- and statin-induced enhancement of OPG expression and inhibition of CD9, M-CSF, and RANKL expressions via inhibition of the Ras/MEK/ERK pathway and activation of p38MAPK in mouse bone marrow stromal cell line ST2

Osteoclast differentiation is influenced by receptor activator of the NF-κB ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and CD9, which are expressed on bone marrow stromal cells and osteoblasts. In addition, osteoprotegerin (OPG) is known as an osteoclastogenesis inhibitory factor. In this study, we investigated whether bisphosphonates and statins increase OPG expression and inhibit the expression of CD9, M-CSF, and RANKL in the bone marrow-derived stromal cell line ST2. We found that bisphosphonates and statins enhanced OPG mRNA expression and inhibited the expression of CD9, M-CSF, and RANKL mRNA. Futhermore, bisphosphonates and statins decreased the membrane localization of Ras and phosphorylated ERK1/2, and activated the p38MAPK. This indicates that bisphosphonates and statins enhanced OPG expression, and inhibited the expression of CD9, M-CSF, and RANKL through blocking the Ras/ERK pathway and activating p38MAPK. Accordingly, we believe that its clinical applications will be investigated in the future for the development of osteoporosis therapy.

Proinflammatory cytokines, IL-1β and TNF-α, induce expression of interleukin-34 mRNA via JNK- and p44/42 MAPK-NF-κB pathway but not p38 pathway in osteoblasts

The aim of this study is to investigate the induction of interleukin-34 (IL-34) and macrophage colony-stimulating factor (M-CSF) mRNA by inflammatory cytokines and the involvement of mitogen-activated protein kinases (MAPKs) in this signaling pathway in human osteoblasts as both IL-34 and M-CSF bind to the same receptor c-FMS. Among four inflammatory cytokines [(IL-1β, IL-6, IL-17, and tumor necrosis factor-α (TNF-α)], IL-34 mRNA expression level was dramatically induced by IL-1β (17-fold) and TNF-α (74-fold). IL-1β and TNF-α activated the intracellular mitogen-activated protein kinases (MAPKs): p44/42 MAPK, p38, and c-Jun N-terminal kinase (JNK) as well as nuclear factor-κB (NF-κB) in osteoblasts. IL-1β- and TNF-α-mediated induction of IL-34 mRNA expression was decreased by JNK inhibitor. Interestingly, although treatment of MEK-1/2 inhibitor showed no reduction in the increase of IL-34 mRNA expression by cytokines, combination of MEK-1/2 inhibitor and JNK inhibitor significantly inhibited IL-1β- and TNF-α-mediated IL-34 mRNA expression level compared to those by each inhibitor alone. On the other hand, M-CSF mRNA expression level was significantly induced by both IL-1β and TNF-α by up to 7- and 11-fold, respectively. IL-1β- and TNF-α-mediated induction of M-CSF mRNA was not affected by p38, JNK, and MEK-1/2 inhibitors. However, NF-κB inhibitor completely inhibited the elevation of M-CSF mRNA expression by these cytokines. These results showed that proinflammatory cytokines, IL-1β and TNF-α, induced the expression of IL-34 mRNA via JNK and p44/42 MAPK but not p38 in human osteoblasts while p38, JNK, and p44/42 MAPK were not involved in the induction of M-CSF mRNA expression by these cytokines.

Differential Effects of Trypanosoma brucei gambiense and Trypanosoma brucei brucei on Rat Macrophages

Mammalian immune responses to Trypanosoma brucei infection are important to control of the disease. In rats infected with T. brucei gambiense (Wellcome strain; WS) or T. brucei brucei (interleukin-tat 1.4 strain [ILS]), a marked increase in the number of macrophages in the spleen can be observed. However, the functional repercussions related to this expansion are not known. To help uncover the functional significance of macrophages in the context of trypanosome infection, we determined the mRNA levels of genes associated with an increase in macrophage number or macrophage function in WS- and ILS-infected rats and in cultured cells. Specifically, we assayed mRNA levels for macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), and macrophage migration inhibitory factor (MIF). Upregulation of GM-CSF and MIF mRNA levels was robust in comparison with changes in M-CSF levels in ILS-infected rats. By contrast, upregulation of M-CSF was more robust in WS-infected rats. The phagocytic activity in macrophages harvested from ILS-infected rat spleens, but not WS-infected spleens, was higher than that in macrophages from uninfected rats. These results suggest that macrophages of WS-infected rats change to an immunosuppressive type. However, when WS or ILS is cocultured with spleen macrophages or HS-P cells, a cell line of rat macrophage origin, M-CSF is upregulated relative to GM-CSF and MIF in both cell types. Anemia occurs in ILS-, but not WS-infected, rats. Treatment of spleen macrophages or HS-P cells cocultured with ILS with cobalt chloride, which mimics the effects of anemia-induced hypoxia, led to downregulation of M-CSF mRNA levels, upregulation of GM-CSF and MIF, and an increase in phagocytic activity. However, the effect of cobalt chloride on spleen macrophages and HS-P cells cocultured with WS was restricted. These results suggest that anemia-induced hypoxia in ILS-infected rats stimulates the immune system and activates macrophages.

Arterial colony stimulating factor-1 influences atherosclerotic lesions by regulating monocyte migration and apoptosis

Previous studies have shown that colony stimulating factor-1 (CSF-1) deficiency dramatically reduced atherogenesis in mice. In this report we investigate this mechanism and explore a therapeutic avenue based on inhibition of CSF-1 signaling. Lesions from macrophage colony stimulating factor-1 (Csf1)+/- mice showed increased numbers of apoptotic macrophages, decreased overall macrophage content, and inflammation. In vitro studies indicated that CSF-1 is chemotactic for monocytes. Bone marrow transplantation studies suggested that vascular cell-derived, rather than macrophage-derived, CSF-1 is responsible for the effect on atherosclerosis. Consistent with previous studies, CSF-1 affected lesion development in a dose-dependent manner, suggesting that pharmacological inhibition of CSF-1 might achieve similar results. Indeed, we observed that treatment of hyperlipidemic mice with a CSF-1 receptor kinase inhibitor inhibited plaque progression. This observation was accompanied by a reduction in the expression of adhesion factors (ICAM-1), macrophage markers (F4/80), inflammatory cytokines (Il-6, Il-1beta), and macrophage matrix degradation enzymes (MMP-9). We conclude that the M-CSF pathway contributes to monocyte recruitment and macrophage survival and that this pathway is a potential target for therapeutic intervention.

Inhibitory effect of luteolin on osteoclast differentiation and function

Osteoclasts are multinucleated cells that play a crucial role in bone resorption, and are formed by the fusion of mononuclear osteoclasts derived from osteoclast precursors of the macrophage lineage. Compounds that specifically target functional osteoclasts would be ideal candidates for anti-resorptive agents for clinical applications. In the present study, we investigated the effects of luteolin, a flavonoid, on the regulation of receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclastogenesis, functions and signaling pathway. Addition of luteolin to a coculture system of mouse bone marrow cells and ST2 cells in the presence of 10(-8) M 1alpha,25(OH)(2)D(3) caused significant inhibition of osteoclastogenesis. Luteolin had no effects on the 1alpha,25(OH)(2)D(3)-induced expressions of RANKL, osteoprotegerin and macrophage colony-stimulating factor mRNAs. Next, we examined the direct effects of luteolin on osteoclast precursors using bone marrow macrophages and RAW264.7 cells. Luteolin completely inhibited RANKL-induced osteoclast formation. Moreover, luteolin inhibited the bone resorption by mature osteoclasts accompanied by the disruption of their actin rings, and these effects were reversely induced by the disruption of the actin rings in mature osteoclasts. Finally, we found that luteolin inhibited RANKL-induced osteoclastogenesis through the suppression of ATF2, downstream of p38 MAPK and nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) expression, respectively. Taken together, the present results indicate that naturally occurring luteolin has inhibitory activities toward both osteoclast differentiation and functions through inhibition of RANKL-induced signaling pathway as well as actin ring disruption, respectively.

Bone marrow adipocytes support dexamethasone-induced osteoclast differentiation

The purpose of this study was to examine the ability of bone marrow adipocytes to support osteoclast differentiation in vitro. The primary bone marrow adipocytes were obtained from bone marrow fluid during prosthesis insertion. NFκ-B ligand (RANKL), Osteoprotegerin (OPG), and macrophage colony stimulating factor (M-CSF) expressions in bone marrow adipocytes with or without dexamethasone were examined. In a co-culture system with bone marrow adipocytes and osteoclast precursor cells, osteoclast differentiation was assessed by the expression of titrate-resistant acid phosphatase (TRAP) staining. RANKL, OPG, and M-CSF mRNA expressions were confirmed in all individuals. Dexamethasone significantly induced RANKL and OPG expression. The RANKL/OPG ratio was increased by dexamethasone and was significant at 10 −7 M dexamethasone. With 10 −7 M dexamethasone, osteoclast precursor cells differentiated into multinucleated TRAP-positive cells when co-cultured with bone marrow adipocytes. The present study demonstrates for the first time that bone marrow adipocytes can support osteoclast differentiation in vitro.

Expression of Macrophage Colony-Stimulating Factor (M-CSF) and Its Receptor in Streptozotocin-Induced Diabetic Rats

Purpose: To demonstrate the expression and location of macrophage colony-stimulating factor (M-CSF) and its receptor (CSF-1R) in the retinas of diabetic rats, as well as in vitreous human proliferative diabetic retinopathy (PDR). Methods: The retinas of streptozotocin-induced diabetic rat were studied. Real-time PCR was applied to evaluate M-CSF and its receptor CSF-1R mRNA expression in the retinas. The protein levels of M-CSF and CSF-1R were evaluated by Western blot analysis. Cellular sources of M-CSF and CSF-1R were determined by double-immunofluorescence staining. M-CSF levels in vitreous samples from patients with PDR were measured by ELISA. Results: M-CSF and CSF-1R mRNA were upregulated in the retinas as early as two weeks after the onset of diabetes and increased over time. A similar pattern was observed for M-CSF/CSF-1R protein expression levels. Double-immunofluorescence staining revealed that increased M-CSF immunoreactivity occurred mainly in the nerve fiber layer in diabetic retinas, co-localizing with glial fibrillary acidic protein. Increased CSF-1R immunoreactivity was observed in OX-42-labeled microglia and ganglion cells in the ganglion cell layer. The vitreous level of M-CSF was elevated in patients with PDR compared to control subjects. Conclusions: The early upregulation of MCSF/CSF-1R signaling may be an important regulatory pathway among neurons, microglia, and glia in diabetic retinopathy.

Increase in the level of macrophage colony-stimulating factor in patients with systemic lupus erythematosus

Macrophage colony-stimulating factor (M-CSF) primarily regulates the survival, proliferation and function of macrophages.1 Studies on MRL- Faslpr mice demonstrate that M-CSF plays a pivotal role in kidney injury.2–4 Increased glomerular...

Inhibition of Interleukin-12 Production by Trypanosoma brucei in Rat Macrophages

The immune response of a host infected with Trypanosoma brucei is modulated by trypomastigotes. We examined the changes in cytokine production in T. brucei gambiense (Wellcome strain; WS) infected rats and the influence on production of interleukin (IL)-12 by macrophages. The blood concentration of interferon-gamma, tumor necrosis factor-alpha, and IL-10 increased beginning the second day after infection. However, an increase in IL-12p40 was not observed until 4 days after infection. When spleen macrophages and Kupffer cells harvested from uninfected rats and HS-P cells (a rat macrophagelike cell line) were cocultured with WS, IL-12p40 production did not change. When HS-P cells were cultured with WS, transport of nuclear factor-kappaB into the nucleus increased. Levels of macrophage colony-stimulating factor (M-CSF) and granulocyte macrophage colony-stimulating factor mRNA in the spleens and livers of WS-infected rats were high in comparison with uninfected rats, suggesting that the WS promotes macrophage proliferation. The level of IL-12p40 mRNA in HS-P cells cocultured with WS increased in response to transfection with a small interfering RNA against M-CSF or addition of anti-M-CSF antibody. These results suggest that the WS inhibits IL-12p40 mRNA production by promoting production of macrophage colony-stimulating factor by macrophages.