OBJECTIVETo explore the expression, characteristics and roles of macrophage colony-stimulating factor receptor (M-CSF-R) in human leukemia cell lines.

METHODSPeripheral blood mononuclear cells (PBMCs) collected from 3 healthy persons, cord blood mononuclear cells (CBMCs) collected from 5 healthy persons and 4 human myelomonocytic leukemia cell lines including J6-1, J6-2, K562 and HL-60 were studied by using ABC immunoperoxidaes assay, indirect immunofluorescene staining, flow cytometry, and Western blot.

RESULTSM-CSF-R was noticed to be localized in the cytoplasm, nucleus and at the membrane in 4 human leukemia cell lines; expression of M-CSF-R was not detected in normal human PBMCs without PHA stimulation. Human PBMCs stimulated by PHA expressed a low level of M-CSF-R. Frequencies of membrane bound M-CSF-R (M-CSF-mR) expression in J6-1, J6-2, K562 and HL-60 were 78.9%, 72.6%, 54.9% and 58.0% respectively. Frequencies of cytoplasm and nucleus associated M-CSF-R (M-CSF-cnR) were 52.3%, 44.3%, 28.0% and 65.3% respectively. One form of M-CSF-R with a molecular weight of 120 000 was detected both in the cytoplasm and nucleus of HL-60 cells. The half-life of M-CSF-cnR in leukemia cells mentioned above was longer than that of corresponding M-CSF-R in stimulated CBMCs, and the half-life of M-CSF-mR in leukemia cells was extended except that of M-CSF-mR in K562 cells. Both anti-M-CSF-R monoclonal antibody and recombinant human M-CSF soluble receptor could cause the growth arrest of HL-60 cell in G(0)/G(1) phase, and could inhibit the formation of colony of HL-60 cell in soft agarose.

CONCLUSIONSExpression of M-CSF-R in leukemia cells is heterogeneous. The accumulation of cellular M-CSF-R results in the low degradation rate of cellular M-CSF-R in leukemia cells, which could be a potential mitotic signal. Signal mediated by M-CSF-R is important and necessary for the growth of HL-60 cell.

Cell Line ; HL-60 Cells ; Humans ; Leukemia ; Leukocytes, Mononuclear ; metabolism ; Macrophage Colony-Stimulating Factor ; metabolism ; Receptor, Macrophage Colony-Stimulating Factor ; Tumor Cells, Cultured

OBJECTIVETo study localization and expression of CSF-1 receptor protein, in order to discover the CSF-1 and IL-1alpha effects on CSF-1 receptor mRNA levels and to determine if the autocrine effect is inhibited through the CSF-1 receptor.

METHODSImmunolocalization of CSF-1 receptor in the cultured dental follicle cells and in mandibles of the post-natal rats from day 1 to 11 were performed. The effects of different concentrations of CSF-1, IL-1alpha on CSF-1 receptor gene expression were detected by means of RT-PCR.

RESULTSCultured dental follicle cells were immunostained for the CSF-1 receptor. In vivo, immunostaining showed that the CSF-1 receptor was present in the dental follicle of the first mandibular molar at early post-natally and was either absent or greatly reduced by day 11 post-natally. High concentrations of cvCSF-1 reduced the gene expression of the CSF-1 receptor. IL-1alpha had no effects on CSF-1 receptor mRNA levels.

CONCLUSIONSThe expression of CSF-1 receptor reaches a peak early post-natally in the dental follicle of the first mandibular molar of the rat and then subsequently declines. High concentrations of CSF-1 inhibits the expression of CSF-1 receptor, IL-1alpha has no effect on the expression of CSF-1 receptor mRNA.

Animals ; Cells, Cultured ; Dental Sac ; chemistry ; cytology ; Immunohistochemistry ; Interleukin-1 ; pharmacology ; Macrophage Colony-Stimulating Factor ; pharmacology ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptor, Macrophage Colony-Stimulating Factor ; analysis ; genetics

OBJECTIVETo explore the cellular immunology mechanism of infective endocarditis (IE), we investigated the effects of Staphylococcus aureus (S. aureus) on MCSF-1 and its receptor (c-fms) gene expression in cardiac valves.

METHODSThirty-two rabbits were divided into 4 groups: mitral or tricuspid valve artificial lesions with 5 x 10(4) CFU or 5 x 10(6) CFU S. aureus injection. Control rabbits (n = 7) received 5 x 10(6) CFU S. aureus injection. IE after operation were confirmed by naked eyes and electron microscope observations. MCSF-1, c-fms in mitral and tricuspid valves were detected by RT-PCR.

RESULTSTwenty-six rabbits survived the operation and 14 rabbits developed IE (2 with 5 x 10(4) CFU and 12 with 5 x 10(6) CFU S. aureus injection) one day post operation. S. aureus injection alone did not induce IE. Compared to control rabbits, MCSF-1 mRNA was significantly upregulated and c-fms mRNA significantly downregulated after 5 x 10(4) CFU S. aureus injection with heart valve artificial lesion in mitral valves or tricuspid valves. MCSF-1 expression in mitral valves was further increased while remained unchanged in tricuspid valve after 5 x 10(6) CFU S. aureus injection compared to that in 5 x 10(4) CFU S. aureus injection group.

CONCLUSIONHigh dose bacterial invasion and heart valves lesion were the main factors for inducing infective endocarditis. Development of infective endocarditis was associated with valve MCSF-1/c-fms expression changes in this rabbit model.

Animals ; Endocarditis, Bacterial ; metabolism ; microbiology ; Macrophage Colony-Stimulating Factor ; biosynthesis ; genetics ; Mitral Valve ; metabolism ; RNA, Messenger ; biosynthesis ; Rabbits ; Receptor, Macrophage Colony-Stimulating Factor ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Staphylococcal Infections ; metabolism ; microbiology ; Staphylococcus aureus

AIMTo observe the expressional alterations of colony stimulating factor-1 receptor (CSF-1R) after ischemic injury of cerebral cortex, and study the function of colony stimulating factor-1 (CSF-1)/CSF-1R signal during the process of ischemic injury and repair of central nervous system (CNS).

METHODSWe examined the distribution and expression of CSF-1R in normal brain tissues and ischemic brain tissues by immunohistology and Western blot analysis.

RESULTSThe expression of CSF-1R in neurons could be up-regulated by ischemic injury in CNS.

CONCLUSIONCSF-1/CSF-1R might take part in the process of ischemic injury and repair.

Animals ; Brain Ischemia ; pathology ; physiopathology ; Cerebral Cortex ; blood supply ; Female ; Macrophage Colony-Stimulating Factor ; physiology ; Male ; Mice ; Mice, Inbred BALB C ; Neurons ; metabolism ; Random Allocation ; Receptor, Macrophage Colony-Stimulating Factor ; genetics ; metabolism ; physiology ; Reperfusion Injury ; metabolism ; physiopathology

OBJECTIVETo systemically investigate receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) induced differentiation of osteoclasts.

METHODSMouse protein-protein interaction(PPI) database NIA and published microarray dataset GES16749 were used to construct and analyze PPI network of RANKL and M-CSF induced mouse monocyte RAW264.7.

RESULTSIn the PPI network, transforming growth factor beta receptor 1 (TGFBR1), Rous sarcoma oncogene (SRC), myelocytomatosis oncogene(MYC) and integrin beta 3 (ITGB3) were able to interact with more proteins and they were the key nodes in the signaling transduction.

CONCLUSIONTGFBR1, SRC, MYC and ITGB3 might be the key points of RANKL and M-CSF induced differentiation of osteoclasts.

Animals ; Carrier Proteins ; Cell Differentiation ; Macrophage Colony-Stimulating Factor ; Membrane Glycoproteins ; Mice ; Osteoclasts ; Protein Interaction Maps ; RANK Ligand ; Receptor Activator of Nuclear Factor-kappa B

This study was aimed to overexpress gene hβc in NB4 cells via the method of lentivirus-mediated gene transfer, to observe the differentiation behaviour change of hβc over-expressing NB4 cells treated with IL-3 or GM-CSF, to explore the relationship between hβc gene and the differentiation behaviour of NB4 cells. The targeted hβc gene was amplified by PCR from the cloned vector carrying ORF of hβc. The PCR product containing PmeI and BstBI site introduced by primer was digested, and then cloned into lentivirus vector pRRLSIN.cPPT.PGK/IRES/GFP.WPRE to construct a lentiviral vector carrying hβc, named pLV-hβc. And the pLV-hβc plasmid was confirmed by restriction and sequencing. The recombinant lentivirus was produced by co-transfecting three plasmids into 293T packing cells. After transfection, the lentiviral supernatant was collected to transfect NB4 cells. GFP expression was examined by fluorescent microscope and the expression of hβc gene was detected by Western blot. Then, the NB4 cells over-expressing hβc were treated with IL-3 (10 ng/ml), GM-CSF (10 ng/ml), ATRA (1 µmol/L) respectively, and the CD11b expression, morphology and differentiation behaviour changes of every groups were observed by flow cytometry and microscopy, while NB4 cells transfected with blank lentivirus (NB4-blank cells) were used as controls. The results showed that the recombinant lentivirus vector carrying hβc gene could efficiently transfect NB4 cells and made NB4 cells to stably over-express hβc gene. The expression of CD11b was up-regulated in NB4-hβc cells treated with of IL-3 or GM-CSF, but it was not as obvious as the effect of ATRA, and no morphological change was observed in NB4 hβc cells treated with the IL-3 or GM-CSF. It is concluded that IL-3 or GM-CSF can induce NB4 cells over-expressing hβc to differentiate to neutrophils, but can not make them fully matured.
Cell Differentiation ; Cell Line ; Cytokine Receptor Common beta Subunit ; genetics ; Flow Cytometry ; Genetic Vectors ; Granulocyte-Macrophage Colony-Stimulating Factor ; biosynthesis ; Humans ; Interleukin-3 ; biosynthesis ; Lentivirus ; genetics ; Plasmids ; Transfection

OBJECTIVEThis study aimed to explore the effect of the up-regulation of Notch1 on osteoclastogenesis induced to osteoclasts by receptor activator for nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factors (MCSF) in vitro.

METHODSThe bone marrow stem cells (BMSCs) of Rosa(-notch1) mice were cultured and induced to osteoclasts by RANKL and MCSF. The BMSCs were transfected with the Ad-Cre-green fluorescent protein (GFP) virus or Ad-GFP virus. Total RNA from cells was extracted, and the gene expression levels of Notch1, Notch2, Notch3, Notch4, Deltal, Delta3, Delta4, Jagged1, Hes1, and tartrate resistant acid phosphatase (TRAP) were detected at the defined stage by reverse transcription-polymerase chain reaction (RT-PCR). Osteoclast formation was analyzed by TRAP assay.

RESULTSThe number of TRAP-positive multinuclear cells of the experimental group significantly decreased compared with that of the control group. The mRNA expression levels of Notch1, Notch3, Jagged1, Delta3, and Hesl of the experimental group were significantly higher than those of the control group, whereas the TRAP mRNA expression of the experimental group was significantly lower than that of the control group (P<0.05).

CONCLUSIONUp-regulation of Notch1 inhibit osteoclastogenesis of BMSCs induced by RANKL and MCSF in vitro.

Animals ; Cell Differentiation ; Cell Line ; In Vitro Techniques ; Macrophage Colony-Stimulating Factor ; Mice ; Osteoclasts ; RANK Ligand ; Receptor Activator of Nuclear Factor-kappa B ; Receptor, Notch1 ; metabolism ; Receptor, Notch2 ; Up-Regulation ; physiology

OBJECTIVEThis work aims to examine the effects of paeonol treatment on the ability of bone marrow-derived macrophage (BMM) to excrete inflammatory factors and to differentiate into osteoclasts upon induction with Porphyromonas gingivalis (P. gingivalis). This work also aims to investigate the underlying mechanisms of these abilities.

METHODSBMM culture was treated with different paeonol concentrations at for 1 h and then stimulated with P. gingivalis for 24 h before programmed death-ligand 1 (PD-L1) was quantified with flow cytometry. Tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 were detected by enzyme-linked immunosorbent assay (ELISA). The BMM culture was treated with the receptor activator for nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), and then with paeonol for 1 h prior to induction with P. gingivalis. Then, osteoclast formation was assessed using tartrate resistant acid phosphatase (TRAP) staining. The osteoclast-related proteins TRAP and receptor activator of nuclear factor-κB (RANK) were quantified by Western blotting.

RESULTSPaeonol was nontoxic to BMM within a range of 10-50 μmol·L⁻¹. Flow cytometry showed that paeonol inhibited PD-L1 expression in P. gingivalis-induced BMM in a dose-dependent manner. ELISA indicated that paeonol dose-dependently inhibited the excretion of TNF-α, IL-1β, and IL-6 by P. gingivalis-induced BMM (P<0.01). TRAP staining revealed that paenol treatment inhibited the differentiation of P. gingivalis-induced BMM into osteoclasts. Western blot results suggested that paeonol decreased the expression of TRAP and RANK in BMM.

CONCLUSIONSPaeonol dose-dependently inhibited the excretion of the inflammatory factors TNF-α, IL-1β, and IL-6 by P. gingivalis-induced BMM in a dose-dependent manner. Moreover, paenol treatment prevented the differentiation of P. gingivalis-induced BMM differentiation into osteoclasts.
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Acetophenones ; pharmacology ; Acid Phosphatase ; Animals ; Carrier Proteins ; Cell Differentiation ; Interleukin-1beta ; Interleukin-6 ; Isoenzymes ; Macrophage Colony-Stimulating Factor ; Macrophages ; Membrane Glycoproteins ; Mice ; Osteoclasts ; Porphyromonas gingivalis ; RANK Ligand ; Receptor Activator of Nuclear Factor-kappa B ; Tumor Necrosis Factor-alpha

Osteoclasts are unique cells that degrade the bone matrix. These large multinucleated cells differentiate from the monocyte/macrophage lineage upon stimulation by two essential cytokines, macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B (NF-kappaB) ligand (RANKL). Activation of transcription factors such as microphthalmia transcription factor (MITF), c-Fos, NF-kappaB, and nuclear factor-activated T cells c1 (NFATc1) is required for sufficient osteoclast differentiation. In particular, NFATc1 plays the role of a master transcription regulator of osteoclast differentiation. To date, several mechanisms, including transcription, methylation, ubiquitination, acetylation, and non-coding RNAs, have been shown to regulate expression and activation of NFATc1. In this review, we have summarized the various mechanisms that control NFATc1 regulation during osteoclast differentiation.
Acetylation ; Bone Matrix ; Cytokines ; Gene Expression Regulation ; Macrophage Colony-Stimulating Factor ; Methylation ; Microphthalmos ; NF-kappa B ; NFATC Transcription Factors ; Osteoclasts* ; RANK Ligand ; Receptor Activator of Nuclear Factor-kappa B ; RNA, Untranslated ; T-Lymphocytes ; Transcription Factors ; Ubiquitin ; Ubiquitination

OBJECTIVETo study the application of the live cell imaging method to observe the whole process of osteoclast formation induced by monocyte macrophages in the blood system in order to clarify the origin of osteoclasts and their cytodynamics.

METHODSBlood samples (8 ml) were collected from the abdominal aorta of male SD rats weighing 280 g. Mononuclear cells were obtained by density gradient centrifugation and induced by RANKL and M-CSF. The cells were cultured and divided into four groups: inverted phase contrast microscope (IPCM) group, TRAP group, SEM group and live cell imaging (LCI) group. Images of the IPCM group were captured by a digital microscopic imaging system and recorded daily. The TRAP group was identified by enzyme activity staining after a 21-day cultivation period. The SEM group was SEM-observed after a 21-day cultivation period. The LCI group was consecutively and dynamically observed for 35 days.

RESULTSAfter 2-week cultivation, IPCM observations showed the formation of numerous apocytes. These cells displayed round, fusiform, fan-shaped, elliptic or irregular gibbous profiles. TRAP staining showed that most apocytes and monocytes had positive(+)reaction. SEM observations showed many bone absorption lacunae, hollows and channels, in which many osteoclasts with absorption activity were observed. Live cell imaging observations found that multinuclear osteoclasts originating from peripheral blood were generated by fusion of monocytes and apocytes and intercross fusion of monocytes and apocytes,which occurred at the adherent stage of the cells. Cytodynamic observations showed that the cell form of osteoclasts was complex and changeable.

CONCLUSIONRANKL and M-CSF can induce differentiation and formation from monocytes in rat peripheral blood into multinuclear osteoclasts with bone absorption activity. The osteoclasts were formed by various cell fusion processes at the adherent stage. The adherent property of osteoclasts is important for their survival and function. Osteoclasts have phagocytosis and their morphological structure is dynamically changeable, involving not only apocytes but monocytes. The osteoclast property of multinuclear giant cells formed by cell fusion may be a special biological behavior for their adaptation of functional needs and bone absorption efficiency. This experiment has further evidenced the theory of osteoclast origination in the blood system and provided new experimental clues for clarifying the cytodynamic and cytobiological properties of osteoclasts.

Acid Phosphatase ; analysis ; Animals ; Cell Survival ; Macrophage Colony-Stimulating Factor ; pharmacology ; Male ; Monocytes ; cytology ; Osteoclasts ; cytology ; RANK Ligand ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptor Activator of Nuclear Factor-kappa B ; analysis ; physiology ; Signal Transduction