The Modulation of Phospho-Extracellular Signal-Regulated Kinase and Phospho-Protein Kinase B Signaling Pathways plus Activity of Macrophage-Stimulating Protein Contribute to the Protective Effect of Stachydrine on Acetaminophen-Induced Liver Injury.

Stachydrine, a prominent bioactive alkaloid derived from Leonurus heterophyllus, is a significant herb in traditional medicine. It has been noted for its anti-inflammatory and antioxidant characteristics. Consequently, we conducted a study of its hepatoprotective effect and the fundamental mechanisms involved in acetaminophen (APAP)-induced liver injury, utilizing a mouse model. Mice were intraperitoneally administered a hepatotoxic dose of APAP (300 mg/kg). Thirty minutes after APAP administration, mice were treated with different concentrations of stachydrine (0, 2.5, 5, and 10 mg/kg). Animals were sacrificed 16 h after APAP injection for serum and liver tissue assays. APAP overdose significantly elevated the serum alanine transferase levels, hepatic pro-inflammatory cytokines, malondialdehyde activity, phospho-extracellular signal-regulated kinase (ERK), phospho-protein kinase B (AKT), and macrophage-stimulating protein expression. Stachydrine treatment significantly decreased these parameters in mice with APAP-induced liver damage. Our results suggest that stachydrine may be a promising beneficial target in the prevention of APAP-induced liver damage through attenuation of the inflammatory response, inhibition of the ERK and AKT pathways, and expression of macrophage-stimulating proteins.

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.

Dimethyl fumarate suppresses granulocyte macrophage colony-stimulating factor-producing Th1 cells in CNS neuroinflammation.

OBJECTIVE: To study the immunomodulatory effect of dimethyl fumarate (DF) on granulocyte macrophage colony-stimulating factor (GM-CSF) production in CD4+ T cells in experimental autoimmune encephalomyelitis (EAE) and human peripheral blood mononuclear cells (PBMCs). METHODS: We collected splenocytes and CD4+ T cells from C57BL/6 wild-type and interferon (IFN)-γ-deficient mice. For human PBMCs, venous blood was collected from healthy donors, and PBMCs were collected using the Percoll gradient method. Cells were cultured with anti-CD3/28 in the presence/absence of DF for 3 to 5 days. Cells were stained and analyzed by flow cytometry. Cytokines were measured by ELISA in cell supernatants. For in vivo experiments, EAE was induced by myelin oligodendrocyte glycoprotein35-55 and mice were treated with oral DF or vehicle daily. RESULTS: DF acts directly on CD4+ T cells and suppresses GM-CSF-producing Th1 not Th17 or single GM-CSF+ T cells in EAE. In addition, GM-CSF suppression depends on the IFN-γ pathway. We also show that DF specifically suppresses Th1 and GM-CSF-producing Th1 cells in PBMCs from healthy donors. CONCLUSIONS: We suggest that DF exclusively suppresses GM-CSF-producing Th1 cells in both animal and human CD4+ T cells through an IFN-γ-dependent pathway. These findings indicate that DF has a better therapeutic effect on patients with Th1-dominant immunophenotype. However, future longitudinal study to validate this finding in MS is needed.

Antiosteoclastic bone resorption activity of osteoprotegerin via enhanced AKT/mTOR/ULK1-mediated autophagic pathway.

Autophagy plays a critical role in the maintenance of bone homeostasis. Osteoprotegerin (OPG) is an inhibitor of osteoclast-mediated bone resorption. However, whether autophagy is involved in the antiosteoclastogenic effects of OPG remains unclear. The present study aimed to investigate the potential mechanism of autophagy during OPG-induced bone resorption via inhibition of osteoclasts differentiated from bone marrow-derived macrophages in BALB/c mice. The results showed that after treatment with receptor activator of nuclear factor-κΒ ligand and macrophage colony-stimulating factor for 3 days, TRAP+ osteoclasts formed, representing the resting state of autophagy. These osteoclasts were treated with OPG and underwent autophagy, as demonstrated by LC3-II accumulation, acidic vesicular organelle formation, and the presence of autophagosomes. The levels of autophagy-related proteins, LC3-II increased and P62 decreased at 3 hr in OPG-treated osteoclasts. The viability, differentiation, and bone resorption activity of osteoclasts declined after OPG treatment. Treatment with OPG and chloroquine, an autophagy inhibitor, attenuated OPG-induced inhibition of osteoclastic bone resorption, whereas rapamycin (RAP), an autophagy inducer, enhanced OPG-induced inhibition of differentiation, survival, and bone resorption activity of osteoclasts. Furthermore, OPG reduced the amount of phosphorylated(p) protein kinase B (AKT) and pmTOR and increased the level of pULK, in a dose-dependant manner. LY294002, a phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT pathway inhibitor, attenuated the decline in pAKT, but enhanced the decline in pmTOR and the increase in pULK1 following OPG treatment. RAP enhanced the OPG-induced increase in pULK1. The PI3K inhibitor 3-methyladenine partly blocked OPG-induced autophagy. Thus, the results revealed that OPG inhibits osteoclast bone resorption by inducing autophagy via the AKT/mTOR/ULK1 signaling pathway.

Do antiosteoporotic drugs improve bone regeneration in vivo?

PURPOSE: Treatment of complex fractures in the elderly is a challenge for operative reconstruction due to degraded bone structure. Early peri-operative bone anabolic treatment could improve new bone formation, avoid implant loosening and accelerate fracture healing. METHODS: To compare the osteoanabolic potential of different drugs after distraction osteogenesis, 168 female Sprague-Dawley rats underwent lengthening of the right femur using a monolateral external fixator. Animals were randomly divided into six groups: vehicle-injected group, PTH(1-34), raloxifen, strontium ranelate, alendronate and simvastatin. Histomorphometry, CT-scanning, DEXA- and biomechanical analysis were performed to evaluate new bone formation, callus volume, mineralisation and biomechanical strength. Expression of bone metabolic mediators and differentiation indicators of distracted and intact bone were examined by RT-PCR and western blot. RESULTS: Histological analysis showed significant increase of the bone mass after treatment with PTH(1-34), raloxifen and strontium ranelate (p = 0.02). Raloxifen increased bone mineral content (BMC) of the whole distracted femur significantly (p = 0.007). Callus volume was significantly larger in the PTH(1-34), raloxifen and simvastatin groups (p = 0.001) compared to control. Ultimate load of distracted new formed bone was increased in PTH(1-34) and raloxifen groups. It seems that PTH(1-34) and raloxifen have a stronger effect on bone where a repair response is activated. Strontium ranelate demonstrates similar effects to PTH regarding new bone formation but shows low values for mineralisation and biomechanical strength. CONCLUSION: This study suggests that peri-operative treatment of complex and/or osteoporotic fractures with PTH(1-34) and raloxifen might be useful as a stimulator of bone formation and mineralisation to shorten the consolidation time in humans.

Elevated Gα11 expression in osteoblast lineage cells promotes osteoclastogenesis and leads to enhanced trabecular bone accrual in response to pamidronate.

Osteoblastic cells indirectly induce osteoclastogenesis in the bone microenvironment by expressing paracrine factors such as RANKL and M-CSF, leading to increased bone resorption. These cytokines can be regulated by a variety of intracellular pathways, which include G protein-coupled receptor signaling. To explore how enhanced signaling of the Gαq/11 pathway in osteoblast lineage cells may mediate osteoclast formation, we cocultured wild-type (WT) preosteoclasts with BMSCs derived from either WT or transgenic mice with osteoblast-specific overexpression of Gα11 (G11-Tg). G11-Tg cocultures had elevated osteoclast numbers with greater resorptive capacity and increased expression of Rankl, Rankl:Opg (osteoprotegerin), and M-csf compared with cocultures with WT BMSCs. As well, cocultures with G11-Tg BMSCs required a higher concentration of OPG to inhibit osteoclast formation and less angiotensin II to increase osteoclast size. These indicate that G11-Tg osteoblasts drive the increased osteoclast formation and osteopenia seen in G11-Tg mice. Pamidronate treatment of G11-Tg mice restored the trabecular bone loss phenotype, as bone mineral density, bone volume, trabecular number, separation, and expressions of osteoblastic and osteoclastic genes were comparable with WT parameters. These changes were characterized by enhanced accumulation of calcified cartilage in trabecular bone, demonstrating that resorption of the cartilaginous intermediate by osteoclasts is more affected by bisphosphonate treatment in G11-Tg mice. In conclusion, overexpression of Gα11 in osteoblastic cells promotes osteoclastogenesis by upregulation of Rankl and M-csf and bone loss by increased osteoclast resorption of the trabecular bone and cartilaginous matrix.

Micromolar sodium fluoride mediates anti-osteoclastogenesis in Porphyromonas gingivalis-induced alveolar bone loss.

Osteoclasts are bone-specific multinucleated cells generated by the differentiation of monocyte/macrophage lineage precursors. Regulation of osteoclast differentiation is considered an effective therapeutic approach to the treatment of bone-lytic diseases. Periodontitis is an inflammatory disease characterized by extensive bone resorption. In this study, we investigated the effects of sodium fluoride (NaF) on osteoclastogenesis induced by Porphyromonas gingivalis, an important colonizer of the oral cavity that has been implicated in periodontitis. NaF strongly inhibited the P. gingivalis-induced alveolar bone loss. That effect was accompanied by decreased levels of cathepsin K, interleukin (IL)-1ß, matrix metalloproteinase 9 (MMP9), and tartrate-resistant acid phosphatase, which were up-regulated during P. gingivalis-induced osteoclastogenesis. Consistent with the in vivo anti-osteoclastogenic effect, NaF inhibited osteoclast formation caused by the differentiation factor RANKL (receptor activator of nuclear factor κB ligand) and macrophage colony-stimulating factor (M-CSF). The RANKL-stimulated induction of the transcription factor nuclear factor of activated T cells (NFAT) c1 was also abrogated by NaF. Taken together, our data demonstrate that NaF inhibits RANKL-induced osteoclastogenesis by reducing the induction of NFATc1, ultimately leading to the suppressed expression of cathepsin K and MMP9. The in vivo effect of NaF on the inhibition of P. gingivalis-induced osteoclastogenesis strengthens the potential usefulness of NaF for treating periodontal diseases.

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

OBJECTIVE: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: Pe-LPS may induce the expression of M-CSF mRNA and protein in MC3T3-E1 cells through the signaling of NF-κB.

NOD2 Mediates Odontoblast Differentiation and RANKL Expression.

The precise regulation of odontoblast differentiation and osteoclastogenic cytokine expression in human dental pulp cells (HDPCs) is crucial for the pathology of bacteria-related pulpitis. Although the up-regulation of nucleotide-binding oligomerization domain-containing protein 2 (NOD2) has been reported in inflamed human dental pulps, the role of NOD2 in the differentiation of HDPCs remains unclear. Here, we show the involvement of NOD2 in odontoblast differentiation together with osteoclastogenic cytokine expression in HDPCs. Treatment with muramyl dipeptide (MDP), a known NOD2-agonist, significantly inhibited odontoblast differentiation of HDPCs, as revealed by reduced ALP activity, osteoblast/odontoblast marker expression, and mineralized nodule formation. Importantly, the forced down-regulation of NOD2 by small interfering RNA (siRNA) recovered MDP-down-regulated odontoblast differentiation. MDP-elicited suppression of odontoblast differentiation resulted from the increased expression of MKP-1 protein and the subsequent decline of MAPKs phosphorylation, which is a prerequisite for odontoblast differentiation. Furthermore, we found that MDP treatment elevated the expression of osteoclastogenic cytokines in HDPCs, which was also reversed by NOD2 silencing. Analysis of these data, taken together, suggests that the regulation of NOD2 expression upon MDP challenge might serve as an intrinsic mechanism that underlies the hindered dentin formation and accelerated dentin resorption in bacterial infection-mediated pulpitis.

Overexpression of developmentally regulated GTP-binding protein-2 increases bone loss.

The developmentally regulated GTP-binding protein-2 (DRG2) is a novel subclass of GTP-binding proteins. Many functional characteristics of osteoclasts (OC) are associated with small GTPases. We hypothesized that DRG2 affects bone mass via modulating OC activity. Using DRG2 transgenic mice, we investigated the role of DRG2 in bone remodeling. DRG2 overexpression caused a decrease in bone mass and an increase in the number and activity of OC in vivo. DRG2 overexpression increased fusion, spreading, survival, and resorption activity of OC in vitro. Downregulation of DRG2 by siRNA decreased fusion, spreading, and survival of OC, supporting the observations found in DRG2 transgenic OC. Transgenic mature OCs were larger, with actin rings and higher ERK, Akt, Rac1 and Rho activities than wild-type OCs. Inhibition of these proteins abolished the effects of DRG2 on formation of large OCs with actin rings, implying that DRG2 affects cytoskeleton reorganization in a Rac1/Rho/ERK/Akt-dependent manner. In summary, DRG2 is associated with survival and cytoskeleton organization of OC under influence of macrophage colony-stimulating factor, and its overexpression leads to elevated bone resorptive activity of OC, resulting in bone loss.

Bisphosphonates modulate the expression of OPG and M-CSF in hMSC-derived osteoblasts.

Bisphosphonates have been known to suppress osteoclast activity, survival, and recruitment. In this study, we tested effects of BPs on expression of two critical genes for osteoclastogenesis, M-CSF, and OPG in the process of osteoblast differentiation from hMSC. (1) The cells were cultured in osteogenic induction medium together with 0 (control group) and 10-8 M alendronate, pamidronate for up 2 and 3 weeks (for real-time PCR) and 3 and 4 weeks (for ELISA). (2) The real-time PCR protocol for M-CSF, OPG, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) consist of 40 cycles. (3) Enzyme-linked immunosorbent assay (ELISA): the amounts of M-CSF and OPG in the culture medium were determined using commercially available ELISA kits for M-CSF and OPG. Treatment of differentiating cells with alendronate or pamidronate, nitrogen-containing BPs increase the expression of OPG, which suppresses osteoclastogenesis, whereas it decreases the expression of M-CSF, which enhances preosteoclast formation. These results suggest a new mechanism by which BPs inhibit osteoclastogenesis. Results support hypothesis that progressive accumulation of bisphosphonate in jaws causes imbalance in osteogenesis and bone absorption and collateral osteoclast-osteoblast interaction. Bisphosphonate-related osteonecrosis of jaw (BPONJ) is one of the most serious complications of bisphosphonate (BP) therapy. However, the mechanism behind the this process of BPONJ is still unclear and there are so many hypotheses. Among many hypotheses, we focused on osteoclast-osteoblast interaction in this study. The findings of this study show new light on the present BPONJ occurrence theory based on the osteoclastic activity of BPs. Also, a more advanced and developed theory for BRONJ occurrence may be obtained by combining the osteoclast inhibition mechanism and the effects on osteoblastic differentiation by BPs.

Effects of raloxifene on serum macrophage colony-stimulating factor and interleukin-18 levels in postmenopausal women younger than 60 years.

OBJECTIVE: Macrophage colony-stimulating factor (M-CSF) and interleukin-18 (IL-18) are cytokines expressed predominantly in atheromatous plaque, and overproduction of these has been found to be associated with coronary artery disease. The aim of this study was to investigate the effect of raloxifene, a selective estrogen receptor modulator, on serum M-CSF and IL-18 levels, cytokines that are presumably involved in the pathogenesis of atherosclerosis. METHODS: A total of 70 postmenopausal women (age, 56.45 ± 1.52 y) without previously confirmed cardiovascular disease were enrolled in a 6-month prospective, randomized, controlled study. Women were randomly assigned to two groups: 35 women received oral administration of 60 mg/day raloxifene for 6 months and 35 were in the control group and received no medications. Serum lipid concentrations and high-sensitivity C-reactive protein (hs-CRP), M-CSF, and IL-18 levels were measured at baseline and at the sixth month in both groups. RESULTS: Compared with the control group, the raloxifene group had a significant decrease in serum IL-18 concentrations and a 25.29% reduction in serum hs-CRP concentrations. M-CSF levels were reduced by 5.94% in the raloxifene group, but the difference was not statistically significant. At the sixth month, 60 mg/day of raloxifene significantly decreased the median serum total cholesterol and low-density lipoprotein cholesterol levels when compared with the baseline levels. CONCLUSIONS: Raloxifene reduces serum total cholesterol, low-density lipoprotein cholesterol, hs-CRP, and IL-18 levels. According to the results of our study, it is suggested that raloxifene may have a favorable effect on the prevention of cardiovascular disease in healthy postmenopausal women younger than 60 years.

The orally-active and selective c-Fms tyrosine kinase inhibitor Ki20227 inhibits disease progression in a collagen-induced arthritis mouse model.

Macrophage colony-stimulating factor (M-CSF) is important in the development of macrophages and osteoclasts. Previous studies have also shown that CD11b(+) myeloblasts and osteoclasts play key roles during inflammation and bone destruction in arthritic lesions. In this study, we investigated whether N-{4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl}-N'-[1-(1,3-thiazole-2-yl)ethyl] urea (Ki20227), an inhibitor of the M-CSF receptor (c-Fms), suppressed disease progression in a type II collagen (CII)-induced arthritis (CIA) mouse model. We found that Ki20227 inhibited M-CSF-dependent reactions, such as lipopolysaccharide-induced tumor necrosis factor-alpha production, which were enhanced by M-CSF in vitro. Oral administration of Ki20227 in vivo prevented inflammatory cell infiltration and bone destruction, and consequently suppressed disease progression. In addition, the number of CD11b(+), Gr-1(+), and Ly-6G(+) cells in the spleen decreased in the Ki20227-treated mice, and the CII-induced cytokine production in splenocytes isolated from the Ki20227-treated arthritic mice was also reduced. These observations indicate that Ki20227 might exert its therapeutic effects in the CIA mouse model by suppressing the M-CSF-dependent accumulation of both inflammatory and osteoclast cells, as well as by inhibiting inflammatory cytokine production. Hence, inhibitors of the c-Fms tyrosine kinase might act as anti-inflammatory or anti-osteolytic agents against arthritis.

Differential regulation of colony stimulating factor 1 and macrophage migration inhibitory factor expression by inflammatory cytokines in term human decidua: implications for macrophage trafficking at the fetal-maternal interface.

Macrophages are a major component of the leukocyte population of human pregnant endometrium. Although several crucial functions have been ascribed to these cells, the mechanisms underlying macrophage trafficking in the placental bed are poorly understood. The aim of this study was to evaluate the in vivo expression of two potentially antagonistic macrophage-targeting chemokines, colony stimulating factor 1 (CSF1, also known as M-CSF) and macrophage migration inhibitory factor (MIF), in term decidua, and to examine the effects of the inflammatory cytokines tumor necrosis factor (TNF, also known as TNF alpha) and interleukin 1beta (IL1B) on CSF1 and MIF expression in cultured decidual cells. The expression of CSF1 and MIF in term decidua was evaluated by immunohistochemistry. Cultured decidual cells were primed with estradiol (E2) or with E2+medroxyprogesterone acetate (MPA), and then incubated with corresponding steroid(s) with or without TNF or IL1B. The levels of CSF1 and MIF protein and mRNA were assessed by ELISA and quantitative RT-PCR, respectively. Immunostaining for CSF1 and MIF was observed in term decidua. The levels of secreted CSF1 and MIF were similarly unchanged whether the decidual cells were incubated with E2 or with E2+MPA. The CSF1 levels significantly increased in cultures exposed to E2 or E2+MPA plus TNF or IL1B. In contrast, the MIF levels in TNF- and IL1B-treated cells were not changed significantly from the control cultures. The ELISA data were confirmed by quantitative RT-PCR analysis. These results indicate that CSF1 and MIF are involved in regulating macrophage trafficking at the fetal-maternal interface, and suggest a mechanism by which inflammatory cytokines influence pregnancy by regulating decidual macrophage infiltration.

Different effects of simvastatin and losartan on cytokine levels in coronary artery disease.

BACKGROUND AND OBJECTIVE: Use of HMG-CoA reductase inhibitors (statins) and angiotensin II type 1 (AT(1)) receptor antagonists reduces the incidence of cardiovascular events. The cytokines macrophage colony-stimulating factor (M-CSF) and transforming growth factor (TGF)-beta may exert proatherogenic and antiatherogenic effects, respectively. In this study, we examined whether treatment with a statin or an AT(1) receptor antagonist alters M-CSF and TGF-beta levels in patients with coronary artery disease. METHODS: Twenty-seven consecutive patients with coronary artery disease were randomly assigned to the following three treatment groups for 8 weeks: simvastatin 5 mg/day (n = 10); losartan 50 mg/day (n = 9); or control (usual treatment; n = 8). Blood samples were collected before and after treatment. RESULTS: Clinical characteristics and baseline cytokine levels were comparable among the three groups. Serum levels of M-CSF were significantly decreased only in the simvastatin group (from 403 +/- 71 to 303 +/- 116 pg/mL; p = 0.009). Plasma levels of TGF-beta were significantly increased only in the losartan group (from 5.01 +/- 1.13 to 7.50 +/- 3.83 ng/mL; p = 0.021). Simvastatin decreased serum M-CSF levels independently of changes in total cholesterol or low-density lipoprotein-cholesterol. CONCLUSIONS: The results of this study indicate that simvastatin decreases serum levels of M-CSF while losartan increases plasma levels of TGF-beta, suggesting that the two drugs may have different anti-atherosclerotic properties.

Effects of tibolone on selectins in postmenopausal women.

OBJECTIVE: The first step in atherosclerosis is characterized by the adherence of lymphocytes and monocytes to cell adhesion molecules expressed by endothelial cells. The precise mechanism by which steroid hormones may be exerting a protective action against atherogenesis remains unclear. Therefore, we wanted to investigate the effect of tibolone on the circulating levels of various selectins in postmenopausal women. METHODS: Thirty healthy postmenopausal women were enrolled in a prospective, randomized, double blind, placebo-controlled outpatient trial. RESULTS: Patients treated with tibolone revealed a significant decrease for the variables sE-selectin, sL-selectin, and sPECAM-1 after 8 weeks of treatment. CONCLUSIONS: By reducing leukocyte adhesion molecule expression on human endothelial cells, tibolone may have the intrinsic potential to exert additional, lipid-independent, cardiovascular protective effects that may explain the clinical benefits of cardiovascular diseases in postmenopausal women.

Modulation of CSF-1-regulated post-natal development with anti-CSF-1 antibody.

Colony-stimulating factor-1 (CSF-1) regulates the survival, proliferation and differentiation of macrophages. CSF-1-deficient mice are osteopetrotic due to a lack of osteoclasts, while their tissue macrophage deficiencies and an absence of CSF-1 regulation of CSF-1 receptor-expressing cells in the female reproductive tract contribute to their pleiotropic phenotype. To further understand CSF-1 regulation of macrophages in vivo, we developed a neutralizing anti-mouse CSF-1 antibody which was expressed as a recombinant Fab' fragment and coupled to 40 kDa polyethylene glycol. As developmental regulation by CSF-1 is highest during the early post-natal period, the ability of this anti-CSF-1 reagent to inhibit development was tested by regular subcutaneous injection of mice from post-natal days 0.5-57.5. Antibody treatment decreased growth rate, decreased osteoclast number, induced osteopetrosis, decreased macrophage density in bone marrow, liver, dermis, synovium and kidney and decreased adipocyte size in adipose tissue, thereby inducing phenotypes shared by CSF-1- and CSF-1 receptor-deficient mice. While the antibody blocked macrophage development in some tissues, macrophage densities in other tissues were initially high and were reduced by treatment, proving that the antibody also blocked macrophage maintenance. Since cell surface CSF-1 is sufficient for the maintenance of normal synovial macrophage densities, these studies suggest that anti-CSF-1 Fab'-PEG efficiently neutralizes all three CSF-1 isoforms in vivo, namely the secreted proteoglycan, secreted glycoprotein and cell surface glycoprotein. Since CSF-1 has been shown to enhance chronic disease development in a number of mouse model systems, these studies demonstrate the feasibility of neutralizing CSF-1 effects in these models with an anti-CSF-1 antibody.

Cigarette smoking is associated with increased circulating proinflammatory and procoagulant markers in patients with chronic coronary artery disease: effects of aspirin treatment.

BACKGROUND: Smoking is associated with endothelial dysfunction. Cytokines released by injured endothelium promote vascular interactions with leukocytes and platelets. We investigated whether (a) cigarette smoking is linked to increased cytokine production, which may mediate platelet activation and thrombin generation in chronic coronary artery disease (CAD), and (b) aspirin treatment inhibits smoking-related changes on cytokines, platelets, and thrombin. METHODS AND RESULTS: Plasma macrophage-colony-stimulating factor (M-CSF) and C-reactive protein (CRP) were measured in 100 patients with chronic CAD, 60 of whom were chronic smokers. Prothrombin fragments 1+2 and urinary 11-dehydro-thromboxane B2 (TXB2) were additionally measured in 60 of 100 patients (30 of whom were smokers) and in 24 healthy controls. Smokers (n = 20) matched for age, myocardial ischemia, and other risk factors with 20 nonsmokers entered a double-blind crossover trial of aspirin (300 mg/d for 3 weeks) versus placebo. Blood and urine measurements were repeated after each treatment. Compared with nonsmokers, smokers had 3-fold median M-CSF (1499 vs 476 pg/mL), 2-fold CRP (1.5 vs 0.8 mg/L), and higher 11-dehydro-TXB 2 (3.6 vs 2.1 ng/mg creatinine, P < .01 for all comparisons). After aspirin treatment, M-CSF, CRP, 11-dehydro-TXB 2 , and prothrombin fragments 1+2 remained higher in smokers compared with nonsmokers despite a significant reduction of these markers by aspirin (P < .05). M-CSF remained related to 11-dehydro-TXB 2 excretion during both treatment phases (P < .01) suggesting that cytokine-mediated thromboxane A 2 production was not altered by aspirin. CONCLUSIONS: Smoking is associated with increased M-CSF, CRP, and platelet activity. Although aspirin treatment reduces the proinflammatory and procoagulant markers in smokers, it does not abolish the proinflammatory effects of smoking in patients with chronic CAD.

Interferon-gamma switches monocyte differentiation from dendritic cells to macrophages.

Human monocytes differentiate into dendritic cells (DCs) or macrophages according to the nature of environmental signals. Monocytes stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin 4 (IL-4) yield DCs. We tested here whether interferon-gamma (IFN-gamma), a potent activator of macrophages, may modulate monocyte differentiation. Addition of IFN-gamma to IL-4 plus GM-CSF-stimulated monocytes switches their differentiation from DCs to CD14(-)CD64(+) macrophages. IFN-gamma increases macrophage colony-stimulating factor (M-CSF) and IL-6 production by IL-4 plus GM-CSF-stimulated monocytes by acting at the transcriptional level and acts together with IL-4 to up-regulate M-CSF but not IL-6 production. IFN-gamma also increases M-CSF receptor internalization. Results from neutralizing experiments show that both M-CSF and IL-6 are involved in the ability of IFN-gamma to skew monocyte differentiation from DCs to macrophages. Finally, this effect of IFN-gamma is limited to early stages of differentiation. When added to immature DCs, IFN-gamma up-regulates IL-6 but not M-CSF production and does not convert them to macrophages, even in the presence of exogenous M-CSF. In conclusion, IFN-gamma shifts monocyte differentiation to macrophages rather than DCs through autocrine M-CSF and IL-6 production. These data show that IFN-gamma controls the differentiation of antigen-presenting cells and thereby reveals a new mechanism by which IFN-gamma orchestrates the outcome of specific immune responses.

Expression of macrophage colony-stimulating factor and its receptor in microglia activation is linked to teratogen-induced neuronal damage.

Prenatal exposure to teratogen agents is linked to the pathogenesis of neurodevelopment disorders, but the mechanisms leading to the neurodevelopmental disturbance are poorly understood. To elucidate this, an in vitro model of microglial activation induced by neuronal injury has been characterized. In this connection, exposure of primary microglial cells to the conditioned medium from the neuronal damage induced by teratogen, cyclophosphamide, is accompanied by a reactive microgliosis as assessed by reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, lectin histochemistry, double labeling immunohistochemistry and in situ hybridization. Our results showed that reactive microglia were capable of releasing various cytokines such as tumor necrosis factor-alpha, interleukin-1, interleukin-6, transforming growth factor-beta and nitric oxide. Also, we have shown that macrophage colony-stimulating factor (M-CSF) was in fact produced by the reactive microglia. Concomitant to this was the increased expression of M-CSF receptor in these cells following the teratogen-induced neuronal injury. The up-regulation of M-CSF receptor suggests that the cells are capable of responding to self-derived M-CSF in an autocrine fashion. Results with antibody neutralization further suggest that microglial proinflammatory response, as manifested by cytokine expression in culture, is mediated by M-CSF, which acts as a molecular signal that initiates a microglial reaction. We therefore suggest that microglial activation following cyclophosphamide treatment is not only a response to the neuronal damage, but is also a cause of the damage during pathogenesis of neurodevelopment disorders. To this end, the increased expression of M-CSF and its receptor on microglia would be directly linked to the active cell proliferation and proinflammatory response in the teratogen-induced injury.