Absence Of Macrophage Colony-stimulating Factor Research Articles

Role of Synovial Exosomes in Osteoclast Differentiation in Inflammatory Arthritis.

This study aimed to investigate the characteristics of exosomes isolated from synovial fluid and their role in osteoclast differentiation in different types of inflammatory arthritis. Exosomes isolated from synovial fluid of rheumatoid arthritis (RA), ankylosing spondylitis (AS), gout, and osteoarthritis (OA) patients were co-incubated with CD14+ mononuclear cells from healthy donors without macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Osteoclast differentiation was evaluated via tartrate-resistant acid phosphatase (TRAP) staining and activity and F-actin ring formation. RANKL expression on synovial exosomes was assessed using flow cytometry and an enzyme-linked immunosorbent assay (ELISA). Synovial exosomes were the lowest in OA patients; these induced osteoclastogenesis in the absence of M-CSF and RANKL. Osteoclastogenesis was significantly higher with more exosomes in RA (p = 0.030) than in OA patients, but not in AS or gout patients. On treating macrophages with a specified number of synovial exosomes from RA/AS patients, exosomes induced greater osteoclastogenesis in RA than in AS patients. Synovial exosomal RANKL levels were significantly higher in RA (p = 0.035) than in AS patients. Synovial exosome numbers vary with the type of inflammatory arthritis. Synovial exosomes from RA patients may bear the disease-specific “synovial signature of osteoclastogenesis.”

PLEKHO2 is essential for M-CSF-dependent macrophage survival

Macrophage-colony stimulating factor (M-CSF) is crucial for macrophage survival; however, the mechanism associated with this signaling has not been fully elucidated. Here, we identified pleckstrin homology domain-containing family O member 2 (PLEKHO2), a protein with unknown function, as a novel regulator of macrophage survival in vitro and in vivo. We found that PLEKHO2-deficient mice exhibited severe reductions in macrophage population in the peritoneal cavity, spleen, and blood, and that PLEKHO2 expression was upregulated during macrophage differentiation and maturation. Additionally, PLEKHO2-deficient bone marrow-derived macrophages displayed increased apoptotic cell death in the absence of M-CSF, although PLEKHO2 deficiency did not affect macrophage differentiation and proliferation. Furthermore, although signaling pathways downstream of M-CSF appeared unaffected, caspase activation was elevated in PLEKHO2-deficient macrophages. Our results provided genetic evidence of roles for PLEKHO2 in promoting macrophage survival.

AG490 suppresses interleukin-34-mediated osteoclastogenesis in mice bone marrow macrophages.

Interleukin-34 (IL-34) has been recently identified as a novel cytokine, substituting for the function of macrophage colony-stimulating factor (M-CSF), a pivotal osteoclastogenic factor involved in bone-related diseases (e.g., osteomyelitis of the jaws). However, the molecular mechanisms are not fully understood. This study aimed to explore the potential mechanism of IL-34 in receptor activator of NF-kB ligand (RANKL)-induced osteoclast formation. We found that IL-34 alone significantly maintained the survival of bone marrow macrophages (BMMs) and enhanced the expression of the osteoclast-related genes TRAP, Ctsk, and NFATc1, as well as TRAP-positive multinucleated cells combined with RANKL, which can be reversed by AG490. Conversely, AG490 did not affect the M-CSF-mediated osteoclastogenesis in the presence of RANKL. The protein expression of p-STAT3 in BMMs was enhanced by IL-34 combined with RANKL compared with RANKL alone, and AG490 inhibited the expression of p-SATA3 at protein level in the IL-34 plus RANKL group, resulting in significantly increased Smad7 expression. This study demonstrated for the first time that IL-34 may play a crucial role in RANKL-induced osteoclastogenesis by promoting the proliferation and differentiation of BMMs, stimulating p-STAT3 expression, and inhibiting the expression of Smad7 in the absence of M-CSF.

Enhanced but hypofunctional osteoclastogenesis in an autosomal dominant osteopetrosis type II case carrying a c.1856C>T mutation in CLCN7.

Type II autosomal dominant osteopetrosis (ADO2), which is the most common form of osteopetrosis, is caused by heterozygous mutations in the chloride channel 7 (CLCN7) gene. The osteopetrosis of ADO2 has been attributed to hypofunctional osteoclasts. The mechanism underlying the abnormality in osteoclast function remains largely unknown. This study was designed to investigate gene mutations and osteoclast function in a case that was clinically diagnosed as ADO2. Genomic DNA was extracted from blood samples of this patient, and the 25 exons of CLCN7 were amplified. Peripheral blood from the ADO2 subject and a healthy age- and sex-matched control was used to evaluate osteoclastogenesis, osteoclast morphology, and bone resorption. Analysis of DNA from the patient showed a germline heterozygous missense mutation, c.1856C>T (p.P619L), in exon 20 of CLCN7. A similar homozygous mutation at this site was previously reported in a patient with autosomal recessive osteopetrosis. When cultured, the peripheral blood mononuclear cells (PBMCs) from the ADO2 patient spontaneously differentiated into mature osteoclasts in vitro. The ADO2 patient’s PBMCs formed enhanced, but heterogeneous, osteoclasts in both the presence and absence of macrophage-colony stimulating factor, and nuclear factor-ĸB ligand. Bone resorption was reduced in the ADO2 patient’s osteoclasts, which exhibited aberrant morphology and abnormal distribution of integrin avβ3. Gene analysis found increased c-fos expression and reduced RhoA and integrin beta 3 expression in ADO2 cells. In conclusion, our data suggest that enhanced, heterogeneous osteoclast induction may be an intrinsic characteristic of ADO2.

In Vitro Osteoclastogenesis and T-Cell RANKL Expression In Multiple Myeloma-Bone Disease At Diagnosis and In The Setting Of Frontline Treatment

IntroductionMultiple myeloma (MM)-bone disease occurs in 70% to 80% of patients at MM diagnosis, and up to 90% at relapse; skeletal related events cause high morbidity and mortality. MM-bone disease is mostly consisting of lytic lesions arising as a consequence of an unbalanced bone remodelling due to activation of osteoclasts (OCs), and inactivation of osteoblasts. Osteoclastogenesis may be under immunoregulation by T-cells through the production of receptor activator of NF-kB ligand (RANKL), that is the OC differentiating molecule. AimBy means of an in vitro osteoclastogenesis model derived from peripheral blood mononuclear cells (PBMCs) of patients with MM-lytic bone disease, we purposed to evaluate OC formation and T-cell RANKL expression at diagnosis, and in the setting of frontline therapy. MethodsPB was taken by venipuncture from 18 patients (7 men and 11 women), aged from 63 to 84, diagnosed as having symptomatic MM by International Myeloma Working Group (IMWG) criteria. In all of them, lytic lesions were demonstrated by skeleton standard radiography, and in some cases by nuclear magnetic resonance (NMR) too. On the basis of patients' age and fitness, initial standard therapy consisting of proteasome inhibitor (Bortezomib) associated with Melphalan and Prednisone (VMP) was administered for nine cycles. The therapy response was assessed according to IMWG criteria. The controls included PB collected from 20 healthy donors, matched for age and sex with the patients. Controls and patients gave their written informed consent to the study, that was approved by the local Ethical Committee and performed according to the Declaration of Helsinki. PB was taken from the patients at diagnosis, and following the fourth and the ninth VMP cycles, respectively. OCs were obtained from unfractionated PBMC cultures either stimulated or not with the exogenous pro-osteoclastogenic cytokines [macrophage-colony stimulating factor (M-CSF) and RANKL]. Mature OCs were identified as multinucleated tartrate-resistant acid phosphatase (TRAP) positive cells. Freshly isolated T-cells from patients' and controls' PBMCs were lysed and analyzed by Western Blotting to evaluate RANKL expression. ResultsSpontaneous osteoclastogenesis was demonstrated in the cultures derived from PBMCs isolated at diagnosis. With particular regard to the patients achieving a deep treatment response, at the end of the culture period we found that PBMCs formed few small-sized OCs, when cultured in the absence of M-CSF and RANKL. Moreover, fresh T-cells purified after four and nine VMP cycles expressed progressively lower RANKL levels than fresh T-cells purified at diagnosis. Otherwise the cultures derived from non-responsive patients' PBMCs, collected during and after treatment respectively, showed in vitro spontaneous formation of numerous and large OCs, overlapping the results at diagnosis. Fresh T-cells purified during or after treatment expressed comparable or even higher RANKL levels respect to diagnosis. ConclusionsThese findings show that in vitro spontaneous osteoclastogenesis and T-cell RANKL expression may be correlated with the response to treatment in patients with MM-lytic bone disease. However, further studies will be necessary to confirm these data, and to better translate them from a biologic to a clinical point of view. Disclosures:No relevant conflicts of interest to declare.

VEGF, FLT3 ligand, PlGF and HGF can substitute for M-CSF to induce human osteoclast formation: implications for giant cell tumour pathobiology

Giant cell tumour of bone (GCTB) is a primary bone tumour that contains numerous very large, hyper-nucleated osteoclastic giant cells. Osteoclasts form from CD14+ monocytes and macrophages in the presence of receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage-colony stimulating factor (M-CSF). GCTB contains numerous growth factors, some of which have been reported to influence osteoclastogenesis and resorption. We investigated whether these growth factors are capable of substituting for M-CSF to support osteoclast formation from cultured human monocytes and whether they influence osteoclast cytomorphology and resorption. Vascular endothelial growth factor-A (VEGF-A), VEGF-D, FLT3 ligand (FL), placental growth factor (PlGF) and hepatocyte growth factor (HGF) supported RANKL-induced osteoclastogenesis in the absence of M-CSF, resulting in the formation of numerous TRAP+ multinucleated cells capable of lacunar resorption. Monocytes cultured in the presence of M-CSF, HGF, VEGF-A and RANKL together resulted in the formation of very large, hyper-nucleated (GCTB-like) osteoclasts that were hyper-resorptive. M-CSF and M-CSF substitute growth factors were identified immunohistochemically in GCTB tissue sections and these factors stimulated the resorption of osteoclasts derived from a subset of GCTBs. Our findings indicate that there are growth factors that are capable of substituting for M-CSF to induce human osteoclast formation and that these factors are present in GCTB where they influence osteoclast cytomorphology and have a role in osteoclast formation and resorption activity.

Treatment with at homeopathic complex medication modulates mononuclear bone marrow cell differentiation.

A homeopathic complex medication (HCM), with immunomodulatory properties, is recommended for patients with depressed immune systems. Previous studies demonstrated that the medication induces an increase in leukocyte number. The bone marrow microenvironment is composed of growth factors, stromal cells, an extracellular matrix and progenitor cells that differentiate into mature blood cells. Mice were our biological model used in this research. We now report in vivo immunophenotyping of total bone marrow cells and ex vivo effects of the medication on mononuclear cell differentiation at different times. Cells were examined by light microscopy and cytokine levels were measured in vitro. After in vivo treatment with HCM, a pool of cells from the new marrow microenvironment was analyzed by flow cytometry to detect any trend in cell alteration. The results showed decreases, mainly, in CD11b and TER-119 markers compared with controls. Mononuclear cells were used to analyze the effects of ex vivo HCM treatment and the number of cells showing ring nuclei, niche cells and activated macrophages increased in culture, even in the absence of macrophage colony-stimulating factor. Cytokines favoring stromal cell survival and differentiation in culture were induced in vitro. Thus, we observe that HCM is immunomodulatory, either alone or in association with other products.

Tumor Necrosis Factor-α Alters the Modulatory Effects of Mesenchymal Stem Cells on Osteoclast Formation and Function

Mesenchymal stem cells (MSCs) are characterized by their hematopoiesis-supporting and immunosuppressive capacity, while osteoclasts are main cell components in the endosteal hematopoietic stem cell niche and pivotal players in osteoimmunology. To clarify the association of these 2 kinds of cells, mouse CD11b(+) monocytes were cultured onto MSC layers in the presence or absence of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). The results showed that MSCs independently supported osteoclast development and this effect was enhanced by M-CSF and RANKL. Interestingly, tumor necrosis factor-alpha (TNF-alpha)-stimulated MSCs turned to inhibit osteoclast formation and protect tusk slices from osteoclastic resorption. Real-time PCR and ELISA assays demonstrated that osteoprotegerin expression at both mRNA and protein levels in TNF-alpha-stimulated MSCs was up-regulated, at least partially by activating the mitogen-activated protein kinase pathway. Furthermore, TNF-alpha-stimulated MSCs maintained their immunophenotypic, multipotential differentiation and immunosuppressive characteristics. Moreover, MSCs treated with synovial fluid from rheumatoid arthritis patients modulated osteoclast generation in close relation with the TNF-alpha levels. This study suggests that MSCs exhibit dual modulatory function on osteoclasts and the result might shed light on understanding the involvement of MSCs in the inflammatory diseases.

Ceramide 1-phosphate inhibits serine palmitoyltransferase and blocks apoptosis in alveolar macrophages

We previously reported that incubation of bone-marrow derived macrophages in the absence of macrophage-colony stimulating factor (M-CSF), a cytokine that is essential for their growth and survival, resulted in stimulation of acid sphingomyelinase, accumulation of ceramides, and induction of apoptosis [A. Gomez-Munoz et al. 2004. Ceramide 1-phosphate blocks apoptosis through inhibition of acid sphingomyelinase in macrophages. J Lipid Res 45: 99–105]. Here, we show that alveolar NR8383 macrophages, which are not dependent on M-CSF for viability, undergo apoptosis when they are incubated in the absence of serum. NR8383 cells showed increased levels of ceramides under apoptotic conditions, but in contrast to bone marrow macrophage acid and neutral sphingomyelinases were only slightly activated. We found that the major mechanism for ceramide generation in NR8383 macrophages was stimulation of their synthesis de novo. This action involved activation of serine palmitoyltransferase (SPT), the key regulatory enzyme of this pathway. A relevant finding was that ceramide 1-phosphate (C1P) inhibited SPT activity and ceramide accumulation leading to inhibition of apoptosis. Furthermore, C1P enhanced the activity of antiapoptotic protein kinase B and its downstream effector nuclear factor kappa B. These observations add a new dimension to the understanding of the pro-survival actions of C1P in mammalian cells.

Macrophage colony‐stimulating factor enhances rituximab‐dependent cellular cytotoxicity by monocytes

Recent studies suggest that monocytes are the dominant effectors by which rituximab induces cell death in B-cell lymphoma. Because macrophage colony-stimulating factor (M-CSF) can enhance the cytotoxicity of monocytes, the authors examined whether this growth factor can enhance their ability to kill lymphoma cells in vitro. Monocytes derived from a healthy volunteer were cultured for 48 h in the presence or absence of M-CSF. Monocytes stimul ated with M-CSF were significantly more cytotoxic to Daudi B-cell lymphomas than unstimulated monocytes. Flow cytometry revealed that M-CSF increased monocyte expression of Fcgamma receptors III and I by 1.6- and 1.5-fold, whereas the expression of Fcgamma receptor II remained unchanged. These results suggest that pretreatment with M-CSF can improve the therapeutic efficacy of rituximab against intractable CD20(+) lymphoma.

Peritoneal fluid from endometriosis patients switches differentiation of monocytes from dendritic cells to macrophages

Immunological abnormalities of cell-mediated and humoral immunity might be associated with the pathogenesis of endometriosis. This study has examined the effects of peritoneal fluid obtained from patients with endometriosis (ePF) on the phenotypic characteristics of macrophages and dendritic cells (DCs) derived from monocytes. Monocytes were obtained from healthy young volunteers and cultured with ePF ( n = 12) or a control PF (cPF) ( n = 5) in the presence or absence of macrophage-colony stimulating factor (M-CSF) or IL-4 plus granulocyte macrophage-colony stimulating factor (GM-CSF). The ePF was demonstrated to increase expression levels of CD14 and CD64 on isolated monocytes in the presence or absence of M-CSF. Compared with cPF, addition of 10% ePF to GM-CSF plus IL-4-treated monocytes significantly down-regulated CD1a expression and up-regulated CD64 expression, but did not enhance expression levels of class II MHC. ePF had no effect, however, on tumor necrosis factor-α-induced maturation of DC. Levels of IL-6, IL-10 and M-CSF production were higher in ePF-treated than cPF-treated monocytes for both cell culture conditions with GM-CSF plus IL-4 and M-CSF. A neutralizing IL-6 antibody, but not an IL-10 antibody, abrogated the ePF-induced down-regulation of CD1a, up-regulation of CD64 and secretion of M-CSF. These results suggest that ePF favorably induces monocyte differentiation toward macrophages rather than DCs, and that this effect is mediated by IL-6. A reciprocal mode of cell differentiation between macrophages and DCs in response to ePF may be related to the pathogenesis of endometriosis.

Genistein and zinc synergistically stimulate apoptotic cell death and suppress RANKL signaling‐related gene expression in osteoclastic cells

The effects of the combination of genistein and zinc, which have an anabolic effect on bone metabolism, on osteoclastic cells in mouse marrow culture system in vitro was investigated. The macrophage colony-stimulating factor (M-CSF)-dependent bone marrow macrophages were cultured in the presence of M-CSF (10 ng/ml) and receptor activator of nuclear factor kappaB (NF-kB) ligand (RANKL; 50 ng/ml) for 4 days. The osteoclastic cells formed were further cultured in medium containing either vehicle, genistein, zinc sulfate (zinc), or genistein plus zinc with or without M-CSF (10 ng/ml) and RANKL (50 ng/ml) for 24 or 72 h. The number of osteoclastic cells was significantly decreased with culture of genistein (10(-6) M) plus zinc (10(-5) M) in presence or absence of M-CSF and RANKL for 24 or 72 h as compared with the value for genistein or zinc alone. Agarose gel electrophoresis showed the presence of low-molecular weight deoxyribonucleic acid (DNA) fragments of adherent cells cultured with genistein (10(-6) M) plus zinc (10(-5) M) for 24 or 72 h, indicating that the combination of two chemicals induces apoptotic cell death. Such an effect was not seen in the case of each chemical. Genistein plus zinc-induced decrease in osteoclastic cells were significantly inhibited in the presence of caspase-3 inhibitor (10(-8) or 10(-7) M). Culture with genistein (10(-6) M) plus zinc (10(-5) M) for 72 h caused a significant increase in caspase-3 mRNA expression in the presence or absence of M-CSF and RANKL as compared with the value for each chemical alone. Genistein plus zinc-induced increase in caspase-3 mRNA expression was completely inhibited in the presence of cycloheximide (10(-7) M), an inhibitor of protein synthesis, or 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB; 10(-6) M), an inhibitor of transcription activity. The mRNA expression of tartrate-resistant acid phosphatase (TRACP) or cathepsin K was significantly decreased with culture of genistein plus zinc in the presence of M-CSF and RANKL for 72 h as compared with genistein or zinc alone. Nuclear factor of activated T cells c1 (NFATc1) mRNA expression was significantly decreased with culture of genistein plus zinc in the presence of M-CSF and RANKL for 24 or 72 h as compared with each chemical alone, while NF-kB mRNA expression was significantly changed. This study demonstrates that the combination of genistein and zinc has potent stimulatory effects on apoptotic cell death and suppressive effects on osteoclastic cell function.

Stimulation of osteoclast formation by inflammatory synovial fluid

Peri-articular bone resorption is a feature of arthritis due to crystal deposition and rheumatoid disease. Under these conditions, the synovial fluid contains numerous inflammatory cells that produce cytokines and growth factors which promote osteoclast formation. The aim of this study was to determine whether inflammatory synovial fluid stimulates the formation of osteoclasts. Synovial fluid from rheumatoid arthritis (RA), pyrophosphate arthropathy (PPA) and osteoarthritis (OA) patients was added to cultures (n=8) of human peripheral blood mononuclear cells (PBMCs) in the presence and absence of macrophage colony-stimulating factor (M-CSF) and the receptor activator of NF-kappaB ligand (RANKL). Osteoclast formation was assessed by the formation of cells positive for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor (VNR) and the extent of lacunar resorption. The addition of 10% OA, RA and PPA synovial fluid to PBMC cultures resulted in the formation of numerous multinucleated or mononuclear TRAP(+) and VNR(+) cells which were capable of lacunar resorption. In contrast to PBMC cultures incubated with OA synovial fluid, there was marked stimulation of osteoclast formation and resorption in cultures containing inflammatory RA and PPA synovial fluid which contained high levels of tumour necrosis factor alpha, a factor which is known to stimulate RANKL-induced osteoclast formation.

Regulation of osteoclast apoptosis by ubiquitylation of proapoptotic BH3-only Bcl-2 family member Bim

Osteoclasts (OCs) undergo rapid apoptosis without trophic factors, such as macrophage colony-stimulating factor (M-CSF). Their apoptosis was associated with a rapid and sustained increase in the pro-apoptotic BH3-only Bcl-2 family member Bim. This was caused by the reduced ubiquitylation and proteasomal degradation of Bim that is mediated by c-Cbl. Although the number of OCs was increased in the skeletal tissues of bim-/- mice, the mice exhibited mild osteosclerosis due to reduced bone resorption. OCs differentiated from bone marrow cells of bim-/- animals showed a marked prolongation of survival in the absence of M-CSF, compared with bim+/+ OCs, but the bone-resorbing activity of bim-/- OCs was significantly reduced. Overexpression of a degradation-resistant lysine-free Bim mutant in bim-/- cells abrogated the anti-apoptotic effect of M-CSF, while wild-type Bim did not. These results demonstrate that ubiquitylation-dependent regulation of Bim levels is critical for controlling apoptosis and activation of OCs.

Macrophage colony-stimulating factor promotes the survival of osteoclast precursors by up-regulating Bcl-X(L).

Macrophage colony-stimulating factor (M-CSF) is known as one of the factors essential for osteoclast development. In the present study, we examined effects of M-CSF on the apoptotic pathway of osteoclast precursors and their underlying molecular mechanisms. Osteoclast precursors underwent apoptosis in the absence of M-CSF, even in the presence of receptor activator of NF-kappakB ligand (RANKL). Active caspase-3 and -9 were detected in the osteoclast precursors and treatments of precursors with their specific inhibitors (Z-DEVD-FMK and Z-LEHD-FMK) decreased the apoptosis. M-CSF decreased apoptosis in a dose-dependent manner with decreasing in active caspases-3 and -9 levels and up-regulating Bcl-X(L). Those effects of M-CSF on inhibiting apoptosis of osteoclasts precursor by regulating anti-apoptotic signals was more effective when combined with RANKL. These results demonstrate that M-CSF acts as a survival factor for the osteoclast precursors. Furthermore, it is believed that the apoptosis of osteoclast precursors may be involved in the activation of caspase-9 and that M-CSF may promote their survival through Bcl-X(L)-induced inhibition of caspase-9 activation.

FLT3 ligand can substitute for macrophage colony-stimulating factor in support of osteoclast differentiation and function

AbstractAlthough bone resorption and osteoclast numbers are reduced in osteopetrotic (op/op) mice, osteoclasts are nevertheless present and functional, despite the absence of macrophage colony-stimulating factor (M-CSF). This suggests that alternative factors can partly compensate for the crucial actions of M-CSF in osteoclast induction. It was found that when nonadherent bone marrow cells were incubated in RANKL with Flt3 ligand (FL) without exogenous M-CSF, tartrate-resistance acid phosphatase (TRAP)–positive cells were formed, and bone resorption occurred. Without FL, only macrophagelike TRAP-negative cells were present. Granulocyte-macrophage CSF, stem cell factor, interleukin-3, and vascular endothelial growth factor could not similarly replace the need for M-CSF. TRAP-positive cell induction in FL was not due to synergy with M-CSF produced by the bone marrow cells themselves because FL also enabled their formation from the hemopoietic cells of op/op mice, which lack any M-CSF. FL appeared to substitute for M-CSF by supporting the differentiation of adherent cells that express mRNA for RANK and responsiveness to RANKL. To determine whether FL can account for the compensation for M-CSF deficiency that occurs in vivo, FL signaling was blockaded in op/op mice by the injection of soluble recombinant Flt3. It was found that the soluble receptor induced a substantial decrease in osteoclast number, strongly suggesting that FL is responsible for the partial compensation for M-CSF deficiency that occurs in these mice.

Effects of macrophage colony-stimulating factor (M-CSF) on protease production from monocyte, macrophage and foam cell in vitro: a possible mechanism for anti-atherosclerotic effect of M-CSF

M-CSF is a growth factor that stimulates proliferation and differentiation of monocyte/macrophage-lineage cells. In our previous studies, M-CSF regresses atherosclerotic lesions preformed in aorta of high cholesterol-fed rabbit. Immunohistochemical analysis indicated that extracellular matrix (ECM), such as collagen, was especially eliminated in the intima of atherosclerotic lesion. To define the collagen-lowering potential of M-CSF, we have studied the effects of M-CSF on production of collagen-degrading proteases, such as MMP-1, -9 and urokinase in vitro. Monocytes freshly isolated from human peripheral blood produced MMP-9, but not urokinase, and M-CSF enhanced MMP-9 production. Macrophages were prepared by culturing monocytes for 10 days in the presence or absence of M-CSF, and protease production was assayed. M-CSF augmented production of MMP-9 and urokinase in a dose-dependent manner. M-CSF also enhanced MMP-1 production of macrophages, but not significantly. Foam cells were prepared by culturing macrophages in the presence of acetyl LDL, and protease production from these cells were also elevated by M-CSF. These results suggest that M-CSF exogenously administered in atherosclerotic rabbits might regress the thickened intima by activating macrophages to degrade collagen accumulated in the lesion.

Combination of β-cryptoxanthin and zinc has potent effects on apoptotic cell death and suppression of bone resorption-related gene expression in osteoclastic cells

We investigated whether the effect of beta-cryptoxanthin (CRP) on osteoclastic cells formed in the mouse marrow culture system in vitro is enhanced by culture with zinc. Bone marrow cells were isolated from mice. The macrophage colony-stimulating factor (M-CSF)-dependent bone marrow macrophages were cultured in the presence of M-CSF (10 ng/ml) and receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL; 50 ng/ml) for 96 h. The osteoclastic cells formed were further cultured for 24 or 72 h in a medium containing either vehicle, CRP, zinc sulfate (zinc), or CRP plus zinc with or without M-CSF (10 ng/ml) and RANKL (50 ng/ml). The number of osteoclastic cells was significantly decreased after culture with the combination of CRP (10(-7) M) and zinc (10(-5) M) in the presence or absence of M-CSF and RANKL for 24 or 72 h as compared with the value for CRP or zinc alone. Agarose gel electrophoresis showed the presence of low-molecular weight deoxyribonucleic acid (DNA) fragments of adherent cells cultured with CRP (10(-7) M) plus zinc (10(-5) M) for 24 or 72 h in the presence of M-CSF and RANKL, indicating that the combination of the two chemicals induces apoptotic cell death. CRP plus zinc-induced decrease in osteoclastic cells was significantly inhibited in the presence of caspase-3 inhibitor (10(-8) or 10(-7) M). Culture with CRP (10(-7) M) plus zinc ((10(-5) M) for 24 or 72 h caused a significant increase in caspase-3 mRNA expression in the presence or absence of M-CSF and RANKL as compared with the value for each chemical alone. CRP plus zinc-induced increase in caspase-3 mRNA expression was completely inhibited in the presence of cycloheximide (10(-7) M), an inhibitor of protein synthesis, or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DBR; 10(-6) M), an inhibitor of transcription activity. The mRNA expression of tartrate-resistant acid phosphatase (TRACP) and cathepsin K was significantly decreased after culture with CRP plus zinc in the presence or absence of M-CSF and RANKL for 72 h as compared with CRP or zinc alone. Nuclear factor of activated T cells c1 (NFATc1) mRNA expression was significantly decreased after culture with CRP plus zinc in the presence or absence of M-CSF and RANKL for 72 h as compared with each chemical alone, while NF-kappaB mRNA expression was not significantly changed. This study demonstrated that the combination of CRP and zinc has potent suppressive effects on osteoclastic cells in vitro.

Development, differentiation, and phenotypic heterogeneity of murine tissue macrophages

In murine ontogeny, macrophage precursor cells develop in the yolk sac and fetal liver. Primitive macrophages also appear in the yolk sac, migrate to various tissues, and differentiate into several fetal macrophage populations. Because the development of the monocytic cell lineage is incomplete in the early stage of fetal hematopoiesis, primitive/fetal macrophages are considered to originate from granulocyte-macrophage colony forming cells or earlier macrophage precursors, bypassing the early monocytic cell series. In adult mice rendered severely monocytopenic by administration of strontium-89, resident macrophages are maintained by self-renewal. In contrast, administration of liposome-encapsulated dichloromethylene diphosphonate (clodronate) results in the elimination of various tissue macrophage populations. The repopulation of affected macrophages is dependent on the increase of precursors in the liver and spleen during the period of macrophage depletion. Such precursors reconstitute heterogeneous macrophage subpopulations. In mice homozygous for the osteopetrosis (op) mutation, the absence of macrophage colony-stimulating factor (M-CSF) activity results in a deficiency of monocytes and monocyte-derived macrophages. However, immature macrophages are present in various tissues. Administration of M-CSF to op/op mice induces the increased proliferative capacity and the morphological maturation of macrophages. However, the responses of individual tissue macrophage subpopulations to M-CSF are different. These results indicate that macrophage development, differentiation, and proliferation are regulated by the tissue microenvironment including the in situ production of macrophage growth factors in both fetal and adult life.

Cytokine production by calvariae of osteopetrotic mice.

In the osteopetrotic op/op mouse, the absence of macrophage colony-stimulating factor (M-CSF) prevents the growth of macrophages and osteoclasts and, consequently, bone resorption. In the present study, we investigated whether this deficiency in M-CSF production alters the production of cytokines in op/op bones. Calvariae of phenotypically normal (+/?) and op/op mice were stimulated in vitro with lipopolysaccharide or Pasteurella multocida toxin to produce cytokines. Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) synthesis was the same both in calvaria from osteopetrotic and phenotypically normal animals. However, the production of granulocyte colony-stimulating factor (G-CSF), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF alpha) was lower in calvaria from op/op animals than was the case in +/? calvaria. Thus, the lack of biologically active M-CSF causes defects which are not compensated by cells independent of M-CSF.