Recombinant Macrophage Colony-stimulating Factor Research Articles

Human CD56+CD39+ dNK cells support fetal survival through controlling trophoblastic cell fate: immune mechanisms of recurrent early pregnancy loss.

Decidual natural killer (dNK) cells are the most abundant immune cells at the maternal-fetal interface during early pregnancy in both mice and humans, and emerging single-cell transcriptomic studies have uncovered various human dNK subsets that are disrupted in patients experiencing recurrent early pregnancy loss (RPL) at early gestational stage, suggesting a connection between abnormal proportions or characteristics of dNK subsets and RPL pathogenesis. However, the functional mechanisms underlying this association remain unclear. Here, we established a mouse model by adoptively transferring human dNK cells into pregnant NOG (NOD/Shi-scid/IL-2Rγnull) mice, where human dNK cells predominantly homed into the uteri of recipients. Using this model, we observed a strong correlation between the properties of human dNK cells and pregnancy outcome. The transfer of dNK cells from RPL patients (dNK-RPL) remarkably worsened early pregnancy loss and impaired placental trophoblast cell differentiation in the recipients. These adverse effects were effectively reversed by transferring CD56+CD39+ dNK cells. Mechanistic studies revealed that CD56+CD39+ dNK subset facilitates early differentiation of mouse trophoblast stem cells (mTSCs) towards both invasive and syncytial pathways through secreting macrophage colony-stimulating factor (M-CSF). Administration of recombinant M-CSF to NOG mice transferred with dNK-RPL efficiently rescued the exacerbated pregnancy outcomes and fetal/placental development. Collectively, this study established a novel humanized mouse model featuring functional human dNK cells homing into the uteri of recipients and uncovered the pivotal role of M-CSF in fetal-supporting function of CD56+CD39+ dNK cells during early pregnancy, highlighting that M-CSF may be a previously unappreciated therapeutic target for intervening RPL.

An Efficient and Rapid Two-step Purification Method for Active Human Macrophage Colony-stimulating Factor from Escherichia coli

Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that stimulates the proliferation and differentiation of mononuclear phagocytes. To be biologically active, M-CSF must form a homodimer linked by disulfide bridges. However, the refolding step of recombinant M-CSF is not only extremely laborious but also rate-limiting. Here, we describe an efficient and simplified method for refolding and purifying M-CSF from Escherichia coli. A truncated M-CSF (amino acids 36–181) was tagged with histidine, expressed, and then purified using nickel affinity columns under denaturing conditions. Our redox refolding buffer containing a mixture of reduced and oxidized glutathione and arginine correctly refolded M-CSF. Chromatography on Q-sepharose columns selectively purified the M-CSF dimer from the monomeric form. The dimeric nature of the purified M-CSF was confirmed using multi angle light scattering combined with high performance liquid chromatography. Moreover, cell proliferation and osteoclast differentiation assays using bone marrow-derived macrophages demonstrated that the recombinant M-CSF was biologically active ex vivo.

Recombinant human granulocyte macrophage colony-stimulating factor for the prevention and treatment of oral mucositis induced by concurrent chemoradiotherapy in patients with locally advanced nasopharyngeal carcinoma

Objective To observe the effect of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on the prevention and treatment of oral mucositis induced by concurrent chemoradiotherapy in patients with locally advanced nasopharyngeal carcinoma. Methods A total of 64 patients with locally advanced nasopharyngeal carcinoma from April 2015 to April 2017 in the Second Affiliated Hospital of Xi'an Jiaotong University were enrolled. The patients were randomly divided into the observation group (32 cases) and the control group (32 cases) according to the random number table method. Both groups were treated with intensity modulated radiotherapy (IMRT) and synchronized with TP regimen (docetaxel + cisplatin) chemotherapy for 2 cycles. Both groups were treated with mouthwash at the beginning of radiotherapy to prevent oral mucositis. The observation group was given rhGM-CSF mouthwash, and the control group was given compound borax mouthwash. The oral mucositis and the oral pain during the treatment and the end of the treatment were evaluated by using American Radiation Oncology Group (RTOG)grading criteria and visual analogue scoring method (VAS) grading criteria. The time of onset of oral mucositis and the total time of radiotherapy in both groups was also recorded. Results All the patients were treated with concurrent chemoradiotherapy. The total radiotherapy time in the observation group was less than that in the control group [(46.4±1.6) vs. (48.2±3.2) d, t=-2.720, P=0.009]. The clinical total effective rate was 93.8% (30/32) in the observation group and 96.9% (31/32) in the control group respectively (χ 2=0.35, P = 0.554). The occurrence of grade 1, 2 and 3 oral mucositis in the observation group was (20.9±2.5), (29.3±2.4), and (34.5±1.8) d respectively, which was latter than that in the control group [(16.3±2.0), (24.2±2.2) and (31.0±2.2) d] respectively (t = 8.125, P = 0.000; t = 8.840, P = 0.000; t = 6.944, P = 0.001). During concurrent chemoradiotherapy, the incidence of grade 3 oral mucositis in the observation group was lower than that in the control group [31.2% (10/32) vs. 56.2% (18/32); Z = -2.197, P = 0.028]. At the end of concurrent chemoradiotherapy, the total incidence of grade 2 and grade 3 oral mucositis in the observation group was lower than that in the control group [53.1% (17/32) vs. 81.2% (26/32); Z = -2.708, P = 0.007]. The incidence of moderate and severe oral pain caused by oral mucositis in the observation group was lower than that in the control group [46.9% (15/32) and 6.2% (2/32) vs. 59.4% (19/32) and 15.6% (5/32), respectively; Z = -2.009, P = 0.045]. Conclusion rhGM-CSF mouthwash can delay the occurrence of oral mucositis, reduce the incidence of oral mucositis and oral pain to effectively prevent and treat oral mucositis induced by concurrent chemoradiotherapy in patients with locally advanced nasopharyngeal carcinoma, which is worthy of clinical application. Key words: Nasopharyngeal neoplasms; Radiotherapy; Antineoplastic combined chemotherapy protocols; Stomatitis; Granulocyte macrophage colony-stimulating factors, recombinant

The influence of macrophage growth factors on Theiler’s Murine Encephalomyelitis Virus (TMEV) infection and activation of macrophages

Macrophages are common targets for infection and innate immune activation by many pathogenic viruses including the neurotropic Theiler’s Murine Encephalomyelitis Virus (TMEV). As both infection and innate activation of macrophages are key determinants of viral pathogenesis especially in the central nervous system (CNS), an analysis of macrophage growth factors on these events was performed. C3H mouse bone-marrow cells were differentiated in culture using either recombinant macrophage colony stimulating factor (M-CSF) or granulocyte–macrophage colony-stimulating factor (GM-CSF), inoculated with TMEV (BeAn) and analyzed at various times thereafter. Cytokine RNA and protein analysis, virus titers, and flow cytometry were performed to characterize virological parameters under these culture conditions. GM-CSF-differentiated macrophages showed higher levels of TMEV viral RNA and proinflammatory molecules compared to infected M-CSF-differentiated cells. Thus, GM-CSF increases both TMEV infection and TMEV-induced activation of macrophages compared to that seen with M-CSF. Moreover, while infectious viral particles decreased from a peak at 12h to undetectable levels at 48h post infection, TMEV viral RNA remained higher in GM-CSF- compared to M-CSF-differentiated macrophages in concert with increased proinflammatory gene expression. Analysis of a possible basis for these differences determined that glycolytic rates contributed to heightened virus replication and proinflammatory cytokine secretion in GM-CSF compared to M-CSF-differentiated macrophages. In conclusion, we provide evidence implicating a role for GM-CSF in promoting virus replication and proinflammatory cytokine expression in macrophages, indicating that GM-CSF may be a key factor for TMEV infection and the induction of chronic TMEV-induced immunopathogenesis in the CNS.

Macrophage colony stimulating factor enhances non-small cell lung cancer invasion and metastasis by promoting macrophage M2 polarization

Objective: To investigate the key cytokine which polarizes M2 macrophages and promotes invasion and metastasis in non-small cell lung cancer (NSCLC). Methods: After co-culture with A549 cells in vitro, the proportion of CD14(+) CD163(+) M2 macrophages in monocytes and macrophage colony stimulating factor (M-CSF) levels in culture supernatant were detected by flow cytometry, ELISA assay and real-time qPCR, respectively. The effects of CD14(+) CD163(+) M2 macrophages on invasion of A549 cells and angiogenesis of HUVEC cells were measured by transwell assay and tubule formation assay, respectively. The clinical and prognostic significance of M-CSF expression in NSCLC was further analyzed. Results: The percentage of CD14(+) CD163(+) M2 macrophages in monocytes and the concentration of M-CSF in the supernatant followed by co-culture was (12.03±0.46)% and (299.80±73.76)pg/ml, respectively, which were significantly higher than those in control group [(2.80±1.04)% and (43.07±11.22)pg/ml, respectively, P< 0.05]. Human recombinant M-CSF promoted M2 polarization of macrophages in vitro. M2 macrophages enhanced the invasion of A549 cells (66 cells/field vs. 26 cells/field) and the angiogenesis of HUVEC cells (22 tubes/field vs. 8 tubes/field). The mRNA expression of M-CSF in stage Ⅰ-Ⅱ patients (16.23±4.83) was significantly lower than that in stage Ⅲ-Ⅳ (53.84±16.08; P<0.05). M-CSF levels were associated with poorer overall survival and disease-free survival in NSCLC patients (P<0.05). Conclusions: Tumor-derived M-CSF can induce CD14(+) CD163(+) M2 polarization of macrophages, which can further promote the metastasis and angiogenesis of NSCLC. M-CSF could be used as a potential therapeutic target of NSCLC.

Robust growth of avirulent phase II Coxiella burnetii in bone marrow-derived murine macrophages.

Published data show that murine bone marrow-derived macrophages (BMDM) restrict growth of avirulent phase II, but not virulent phase I, Coxiella burnetii. Growth restriction of phase II bacteria is thought to result from potentiated recognition of pathogen-associated molecular patterns, which leads to production of inhibitory effector molecules. Past studies have used conditioned medium from L-929 murine fibroblasts as a source of macrophage-colony stimulating factor (M-CSF) to promote differentiation of bone marrow-derived myeloid precursors into macrophages. However, uncharacterized components of conditioned medium, such as variable amounts of type I interferons, can affect macrophage activation status and their permissiveness for infection. In the current study, we show that the C. burnetii Nine Mile phase II (NMII) strain grows robustly in primary macrophages from C57BL/6J mice when bone marrow cells are differentiated with recombinant murine M-CSF (rmM-CSF). Bacteria were readily internalized by BMDM, and replicated within degradative, LAMP1-positive vacuoles to achieve roughly 3 logs of growth over 6 days. Uninfected BMDM did not appreciably express CD38 or Egr2, markers of classically (M1) and alternatively (M2) activated macrophages, respectively, nor did infection change the lack of polarization. In accordance with an M0 phenotype, infected BMDM produced moderate amounts of TNF and nitric oxide. Similar NMII growth results were obtained using C57BL/6J myeloid progenitors immortalized with an estrogen-regulated Hoxb8 (ER-Hoxb8) oncogene. To demonstrate the utility of the ER-Hoxb8 system, myeloid progenitors from natural resistance-associated macrophage protein 1 (Nramp1) C57BL/6J knock-in mice were transduced with ER-Hoxb8, and macrophages were derived from immortalized progenitors using rmM-CSF and infected with NMII. No difference in growth was observed when compared to macrophages from wild type mice, indicating depletion of metal ions by the Nramp1 transporter does not negatively impact NMII growth. Results with NMII were recapitulated in primary macrophages where C57BL/6J Nramp1+ BMDM efficiently killed Salmonella enterica serovar Typhimurium. M-CSF differentiated murine macrophages from bone marrow and conditional ER-Hoxb8 myeloid progenitors will be useful ex vivo models for studying Coxiella-macrophage interactions.

Cloning and expression of recombinant macrophage-colony stimulating factor - A progressive strategy for economical production

Macrophage-colony stimulating factor (M-CSF) has been reported to be required for the proliferation and differentiation of macrophages from hematopoietic progenitor cells. Recently, recombinant M-CSF (rM-CSF) became widely used as a biological research reagent in bone marrow stimulations, vaccine development, gene therapy approaches, and stem cell mobilization. rM-CSF is a glycoprotein that activates and enhances the differentiation and survival of macrophages, which play a key role in the osteoclastogenetic response. Here, we describe the construction of the gene encoding rM-CSF, its cloning, and expression in Escherichia coli, as well as the purification of rM-CSF protein, and its activity in a biological assay in mouse bone marrow cells. Our results show that the combination of experimental strategies employed to obtain recombinant rM-CSF can yield a biologically active protein, and may be useful when scaling-up production of other biologically similar proteins.

Neurons and astroglia govern microglial endotoxin tolerance through macrophage colony-stimulating factor receptor-mediated ERK1/2 signals

Endotoxin tolerance (ET) is a reduced responsiveness of innate immune cells like macrophages/monocytes to an endotoxin challenge following a previous encounter with the endotoxin. Although ET in peripheral systems has been well studied, little is known about ET in the brain. The present study showed that brain immune cells, microglia, being different from peripheral macrophages, displayed non-cell autonomous mechanisms in ET formation. Specifically, neurons and astroglia were indispensable for microglial ET. Macrophage colony-stimulating factor (M-CSF) secreted from these non-immune cells was essential for governing microglial ET. Neutralization of M-CSF deprived the neuron-glia conditioned medium of its ability to enable microglia to form ET when microglia encountered two lipopolysaccharide (LPS) treatments. Recombinant M-CSF protein rendered enriched microglia refractory to the second LPS challenge leading to microglial ET. Activation of microglial M-CSF receptor (M-CSFR; also known as CSF1R) and the downstream ERK1/2 signals was responsible for M-CSF-mediated microglial ET. Endotoxin-tolerant microglia in neuron-glia cultures displayed M2-like polarized phenotypes, as shown by upregulation of M2 marker Arg-1, elevated production of anti-inflammatory cytokine interleukin 10, and decreased secretion of pro-inflammatory mediators (tumor necrosis factor α, nitric oxide, prostaglandin E2 and interleukin 1β). Endotoxin-tolerant microglia protected neurons against LPS-elicited inflammatory insults, as shown by reduced neuronal damages in LPS pre-treatment group compared with the group without LPS pre-treatment. Moreover, while neurons and astroglia became injured during chronic neuroinflammation, microglia failed to form ET. Thus, this study identified a distinct non-cell autonomous mechanism of microglial ET. Interactions of M-CSF secreted by neurons and astroglia with microglial M-CSFR programed microglial ET. Loss of microglial ET could be an important pathogenetic mechanism of inflammation-associated neuronal damages.

Long-term Dose- and Time-dependent Effects of Endodontic Sealers in Human In Vitro Osteoclastogenesis

IntroductionThis study evaluates the concentration and time-dependent effects of endodontic sealers' extracts (AH Plus [Dentsply DeTrey, Konstanz, Germany], GuttaFlow [Roeko, Colténe/Whaledent, Germany], Tubliseal [Kerr/Sybron, Romulus, MI], Sealapex [Kerr/Sybron, Romulus, MI], and RealSeal [SybronEndo, Orange, CA]) in the differentiation and function of both unstimulated and stimulated osteoclast precursors, simulating, respectively, immature/undifferentiated precursors and cells undergoing osteoclastogenesis. MethodsThe sealers were mixed according to the manufacturers' instructions, freshly extracted with culture medium (1.3 cm2/mL, 24 hours, 37°C, 5% CO2/air), and diluted (1:20, 1:100, 1:500, and 1:2500). Human peripheral blood mononuclear cells were used as osteoclast precursor cells. After overnight attachment, peripheral blood mononuclear cell cultures were exposed to the sealers' extracts during 21 days in the absence (unstimulated) or presence (stimulated) of recombinant macrophage colony-stimulating factor and receptor for the activation of nuclear factor-κB ligand. Cultures performed in the absence of the extracts were used as the control. Cultures were characterized for osteoclastic differentiation and function. ResultsExtracts caused mostly inhibitory effects on osteoclastic cells, both in unstimulated and stimulated conditions, which were reflected by a decrease in tartrate-resistant acid phosphatase activity, the presence of actin rings, vitronectin and calcitonin receptors, the calcium phosphate resorbing ability, and the expression of osteoclastic genes. Also, the extracts induced alterations in the relative contribution of some intracellular signaling pathways involved in osteoclastogenic events. The sealers differed in the dose- and time-dependent profile. An adaptive cell response was noticed for the inhibitory effects after long-term exposure. ConclusionsEndodontic sealers affect the osteoclastic differentiation and activity, which is followed by an adaptive cell response. Our results suggest that the deleterious effect in the bone periapical tissues observed with the root canal sealers might involve, at least partially, a direct effect on the osteoclastic cells.

Refinement and verification of test conditions for colony-forming unit assay in rat bone marrow cells with preservable colony specimens

We established test conditions for colony-forming unit assay using rat bone marrow cells in which the colony specimens can be preserved. In our test system, all colonies can be transferred from a petri dish to a slide glass with whole agar medium because the agar medium volume is small and movable, enabling the agar medium to be dried and then fixed on the slide glass. Appropriate conditions for the colony-forming unit granulocyte–macrophage assay were as follows: 10 to 30 ng/ml recombinant murine granulocyte and macrophage colony-stimulating factor, 1 × 105 cells/dish, and culture periods of 3 days for neutrophil colonies and 8 days for macrophage colonies. Appropriate conditions for the colony-forming unit erythroid assay were as follows: 2 IU/ml recombinant human erythropoietin, 0.25 to 1 × 105 cells/dish, and culture period of 2–3 days. We also examined responses of bone marrow toxicants 5-fluorouracil and doxorubicin in these test systems, finding that both compounds decreased the numbers of colonies in a concentration-dependent manner in both assays. These findings suggest that these test systems are useful tools in evaluating bone marrow toxicity of these particular compounds.

Bone morphogenetic protein 2 enhances mouse osteoclast differentiation via increased levels of receptor activator of NF-κB ligand expression in osteoblasts

1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] induces osteoclast formation via induction of receptor activator of NF-κB ligand (RANKL, also called TNF-related activation-induced cytokine: TRANCE) in osteoblasts. In cocultures of mouse bone marrow cells and osteoblasts, 1,25(OH)(2)D(3) induced osteoclast formation in a dose-dependent manner, with maximum osteoclast formation observed at concentrations greater than 10(-9)M of 1,25(OH)(2)D(3). In the presence of bone morphogenetic protein 2 (BMP-2), the maximum formation of osteoclasts was seen with lower concentrations of 1,25(OH)(2)D(3) (greater than 10(-11)M), suggesting that BMP-2 enhances osteoclast formation induced by 1,25(OH)(2)D(3). In addition, the expressions of RANKL mRNA and proteins were induced by 1,25(OH)(2)D(3) in osteoblasts, and further upregulated by BMP-2. In mouse bone marrow cell cultures without 1,25(OH)(2)D(3), BMP-2 did not enhance osteoclast differentiation induced by recombinant RANKL and macrophage colony-stimulating factor (M-CSF), indicating that BMP-2 does not target osteoclast precursors. Furthermore, BMP-2 up-regulated the expression level of vitamin D receptor (VDR) in osteoblasts. These results suggest that BMP-2 regulates mouse osteoclast differentiation via upregulation of RANKL in osteoblasts induced by 1,25(OH)(2)D(3).

Increased Glucocorticoid Receptor Expression and Activity Mediate the LPS Resistance of SPRET/EI Mice

SPRET/Ei mice are extremely resistant to acute LPS-induced lethal inflammation when compared with C57BL/6. We found that in vivo SPRET/Ei mice exhibit strongly reduced expression levels of cytokines and chemokines. To investigate the role of the potent anti-inflammatory glucocorticoid receptor (GR) in the SPRET/Ei phenotype, mice were treated with the GR antagonist RU486 or bilateral adrenalectomy. Under such conditions, both C57BL/6 and SPRET/Ei mice were strongly sensitized to LPS, and the differences in LPS response between SPRET/Ei and C57BL/6 mice were completely gone. These results underscore the central role of GR in the LPS hyporesponsiveness of SPRET/Ei mice. Compared with C57BL/6, SPRET/Ei mice were found to express higher GR levels, which were reflected in increased GR transactivation. Using a backcross mapping strategy, we demonstrate that the high GR transcription levels are linked to the Nr3c1 (GR) locus on chromosome 18 itself. Unexpectedly, SPRET/Ei mice exhibit a basal overactivation of the hypothalamic-pituitary-adrenal axis, namely strongly increased corticosterone levels, ACTH levels, and adrenocortical size. As a consequence of the excess of circulating glucocorticoids (GCs), levels of hepatic gluconeogenic enzymes are increased, and insulin secretion from pancreatic β-cells is impaired, both of which result in hyperglycemia and glucose intolerance in SPRET/Ei mice. We conclude that SPRET/Ei mice are unique as they display an unusual combination of elevated GR expression and increased endogenous GC levels. Hence, these mice provide a new and powerful tool for the study of GR- and GC-mediated mechanisms, including immune repressive functions, neuroendocrine regulation, insulin secretion, and carbohydrate metabolism.

CD36 Is a Novel Serum Amyloid A (SAA) Receptor Mediating SAA Binding and SAA-induced Signaling in Human and Rodent Cells

Serum amyloid A (SAA) is a major acute phase protein involved in multiple physiological and pathological processes. This study provides experimental evidence that CD36, a phagocyte class B scavenger receptor, functions as a novel SAA receptor mediating SAA proinflammatory activity. The uptake of Alexa Fluor 488 SAA as well as of other well established CD36 ligands was increased 5-10-fold in HeLa cells stably transfected with CD36 when compared with mock-transfected cells. Unlike other apolipoproteins that bind to CD36, only SAA induced a 10-50-fold increase of interleukin-8 secretion in CD36-overexpressing HEK293 cells when compared with control cells. SAA-mediated effects were thermolabile, inhibitable by anti-SAA antibody, and also neutralized by association with high density lipoprotein but not by association with bovine serum albumin. SAA-induced cell activation was inhibited by a CD36 peptide based on the CD36 hexarelin-binding site but not by a peptide based on the thrombospondin-1-binding site. A pronounced reduction (up to 60-75%) of SAA-induced pro-inflammatory cytokine secretion was observed in cd36(-/-) rat macrophages and Kupffer cells when compared with wild type rat cells. The results of the MAPK phosphorylation assay as well as of the studies with NF-kappaB and MAPK inhibitors revealed that two MAPKs, JNK and to a lesser extent ERK1/2, primarily contribute to elevated cytokine production in CD36-overexpressing HEK293 cells. In macrophages, four signaling pathways involving NF-kappaB and three MAPKs all appeared to contribute to SAA-induced cytokine release. These observations indicate that CD36 is a receptor mediating SAA binding and SAA-induced pro-inflammatory cytokine secretion predominantly through JNK- and ERK1/2-mediated signaling.

Liver Regeneration is Impaired in Macrophage Colony Stimulating Factor Deficient Mice After Partial Hepatectomy: The Role of M-CSF-Induced Macrophages

Macrophage colony stimulating factor (M-CSF), which induces maturation of macrophages, is notably expressed in the liver. Thus, the specific purpose of this study was to investigate the role of M-CSF in liver regeneration after partial hepatectomy (PH). Osteopetrotic (op/op) mice, genetically lacking functional M-CSF, or their littermate mice underwent 70% PH. Animals were sacrificed at the designated time points after PH, and remnant liver tissues were harvested for further investigations. Proliferation of hepatocytes was evaluated by the expression of BrdU and the liver-body weight ratio. The mRNA expression levels of TNF-α and IL-6 and protein expression levels of phosphorylated (p) STAT3 were measured. The number of Kupffer cells (KCs) was determined by immunohistochemistry. Furthermore, KCs were isolated from op/op mice or littermate mice, and mRNA expression levels of TNF- α and IL-6 were assessed after stimulation with LPS. In littermate mice, steady liver regeneration was observed. The number of KCs reduced markedly by about 60% in the op/op mice compared with littermates as reported previously. Furthermore, these cells were morphologically small and immature. In littermate mice, the peak expression levels of TNF-α and IL-6 in the liver was observed 1h after PH, which was consistent with data in previous reports. In contrast, in op/op mice, the peak expression levels were observed 3 h after PH and were significantly lower compared with littermate mice. As a result, the proliferation of hepatocytes was significantly impaired in op/op mice. The mRNA expression level of IL-6, but not TNF-α,was significantly reduced in isolated KCs from op/op mice compared with the littermates after stimulation with LPS, suggesting that the function of KCs is different between op/op mice and littermate mice. To clarify the role of M-CSF in liver regeneration, op/op mice received intraperitoneally, mouse recombinant M-CSF 2 d before PH, and liver regeneration was also assessed. As a result, the numbers of Kupffer cells and liver regeneration were recovered in the op/op mice treated with M-CSF to a similar extent to those in their littermates. Thus, M-CSF-induced hepatic macrophages play an important role in liver regeneration after PH.

Synergistic effects of basic fibroblast growth factor and insulin on Chinese hamster ovary cells under serum-free conditions

Chinese hamster ovary (CHO) cells are commonly used in the biotechnology industry to produce recombinant proteins. The development of serum-free media in order to maintain mammalian cell viability and reduce cell death in the absence of serum is highly desirable. Our aim was to find new growth factors that could maintain viability and enhance the proliferation of CHO cells under serum-free conditions. Macrophage colony-stimulating factor (M-CSF)-secreting CHO cells were used as a model cell line to evaluate the stimulatory effects of different compounds, including two growth factors, on cell proliferation. Flow cytometry and fluorescence microscopy were used to analyze the cell cycle and monitor apoptotic nuclei during serum-free culture. Among the tested compounds, insulin and basic fibroblast growth factor (bFGF), each used alone, prevented CHO cells from dying and helped maintain cell viability, but cells were unable to re-enter the cell cycle and proliferate. Simultaneous supplementation with insulin and bFGF synergistically promoted the growth of CHO cells and recombinant M-CSF synthesis in basal DMEM/F12 medium. The mitogenic effects of bFGF and insulin under serum-free conditions were confirmed in the CHO-K1 cell line. These results provide useful information for the design and development of serum-free media as well as for antiapoptotic studies of CHO cells under serum-free conditions.

Soluble TRAIL does not impair the anti‐osteoclastic activity of osteoprotegerin

Dear Editor: It has been extensively documented that osteoprotegerin (OPG), a soluble member of the TNF receptor superfamily, inhibits osteoclastogenesis by binding to receptor activator of NF-kB ligand (RANKL) and preventing interaction with its cognate transmembrane receptor RANK [reviewed in 1]. However, OPG can also interact with, and neutralize, TNF-related apoptosis inducing ligand (TRAIL), whose extracellular domain shares a 30% homology with the extracellular domain of RANKL [reviewed in 2]. Although some inconsistencies on the differential binding affinity of OPG for RANKL versus TRAIL were present in initial studies [2, 3], it has been recently demonstrated that the affinity of native OPG for native TRAIL is comparable to that for RANKL (45 nM versus 23 nM, respectively) at 37°C, as determined by plasmon surface resonance analysis [4]. Consistently with this biochemical study, OPG has been shown to act in a paracrine and autocrine manner by binding TRAIL and promoting the survival of multiple myeloma [5], prostate cancer [6], ameloblastoma cells [7] and synovial fibroblasts [8]. Interestingly, previous data from different groups have shown that recombinant TRAIL modulates the differentiation of erythroid and myeloid precursors [9–10], while inhibits both human and mouse osteoclastogenesis when added to pre-osteoclast cultures induced to differentiate with recombinant macrophage colony-stimulating factor (M-CSF) +RANKL as well as to mature osteoclasts [11–14]. On the other hand, a couple of studies suggested that recombinant soluble TRAIL might promote osteoclastogenesis [4, 15], and the proposed molecular mechanism to explain such observation was a competition between TRAIL and RANKL for OPG binding. However, it should be noticed that Vitovsky et al. used much higher concentrations of TRAIL (500 ng/ml) than RANKL (30 ng/ml) or OPG (50 ng/ml) and more importantly used mouse bone marrow pre-osteoclasts [4]. In this respect, it has been clearly shown that mouse pre-osteoclasts only express TRAIL-R2 [2], while human peripheral blood-derived pre-osteoclasts express both death receptors TRAIL-R1 and TRAIL-R2 as well as TRAIL-R4 [11–14]. Therefore, to further elucidate the important issue of the interplay between RANKL, TRAIL and OPG in human osteoclastogenesis, we have cultured adherent PBMC with M-CSF+RANKL for 14 days in the absence or presence of recombinant OPG and recombinant TRAIL, prepared as previously described [16]. TRAIL and OPG were added alone or in combination. Importantly, all cytokines were used at the same concentration (50 ng/ml). As expected [11–14], the addition of TRAIL to M-CSF+RANKL significantly (P < 0.05) inhibited osteoclast formation (Fig. 1A and B). Of note, recombinant OPG completely abrograted (P < 0.05) osteoclast formation irrespectively of the presence of recombinant TRAIL in culture (Fig. 1A and B). The anti-osteoclastic activity of OPG could not be ascribed to a low affinity of OPG for TRAIL since OPG (50 ng/ml) efficiently inhibited the apoptosis induced by TRAIL (50 ng/ml) in HL-60 leukemic cells (data not shown). Effect of combined treatment of RANKL, TRAIL and OPG on osteoclastic differentiation. Adherent PBMC were cultured with M-CSF alone for 6 days. Then cells were cultured for additional 14 days with the addition of RANKL in the absence or presence of TRAIL and/or OPG, as indicated. Cultures were analysed for osteoclastic differentiation by scoring the number of TRAP+ multinucleated cells (A), and measuring the levels of TRAP 5b (B), a specific marker of resorption activity, in culture supernatants by ELISA. Data represent the means ±SD of three different experiments performed in duplicate. Our current observations on one hand confirm that TRAIL has anti-osteoclastic activity and on the other hand indicate that it does not affect the potent anti-osteoclastic activity of OPG at least in the simplified model of osteoclastogenesis represented by human PBMC induced to differentiate by M-CSF+RANKL. Taken together with previous studies [4, 11–15], these data also suggest that the relative concentrations of TRAIL, RANKL and OPG in the local microenvironment are likely key determinant for the regulation of osteoclastogenesis.

Role of M-CSF-dependent macrophages in colitis is driven by the nature of the inflammatory stimulus

Although macrophages are considered a critical factor in determining the severity of acute inflammatory responses in the gut, recent evidence has indicated that macrophages may also play a counterinflammatory role. In this study, we examined the role of a macrophage subset in two models of colitis. Macrophage colony-stimulating factor (M-CSF)-deficient osteopetrotic mice (op/op) and M-CSF-expressing heterozygote (+/?) mice were studied following the induction of colitis by either dinitrobenzene sulfonic acid (DNBS) or dextran sulfate sodium (DSS). DNBS induced a severe colitis in M-CSF-deficient op/op mice compared with +/? mice. This was associated with increased mortality and more severe macroscopic and microscopic injury. Colonic tissue myeloperoxidase (MPO) activity as well as concentrations of TNF-alpha, IL-1beta, and IL-6 were higher and IL-10 lower in op/op mice with DNBS colitis. The severity of inflammation and mortality was attenuated in op/op mice that had received human recombinant M-CSF prior to the induction of colitis. In contrast, op/op mice appeared less vulnerable to colitis induced by DSS. Macroscopic damage, microscopic injury, MPO activity, and tissue concentrations of TNF-alpha, IL-1beta, and IL-6 were all lower in op/op mice compared with +/? mice with DSS colitis, and no changes were seen in IL-10. Macrophage inflammatory protein-1alpha concentrations were increased in op/op but not +/? mice following colitis induced by DNBS but not DSS. These results indicate that M-CSF-dependent macrophages may play either a pro- or counterinflammatory role in acute experimental colitis, depending on the stimulus used to induce colitis.

Enhanced recombinant M-CSF production in CHO cells by glycerol addition: model and validation

Addition of stimulatory chemical such as glycerol was found to increase recombinant protein production in Chinese hamster ovary (CHO) cells. However, glycerol influenced cell mitosis and reduced cell growth rate. We developed a controlled proliferation strategy to utilize the stimulation of glycerol on recombinant protein production and mitigate the problem of growth inhibition. The approach is to apply a two-stage process, where cells are cultured without glycerol for a period of time in order to obtain enough cell density and then glycerol is added to achieve high specific productivity. In addition, a model for predicting the profiles of cell proliferation and recombinant protein production was developed and validated. A two-stage process, addition of 1% glycerol after 1 day of growth, could increase the final production of macrophage-colony stimulating factor (M-CSF) by 38% compared with the value obtained without addition of glycerol.

Promotion of recombinant macrophage colony stimulating factor production by dimethyl sulfoxide addition in Chinese hamster ovary cells

A Chinese hamster ovary (CHO) cell line is the primary choice for the production of recombinant protein drugs with glycosylation modification. Recombinant protein productivity was enhanced by the addition of dimethyl sulfoxide (DMSO) in CHO cells. However, DMSO induced G0/G1 phase growth arrest and reduced cell growth rate. A two-stage process was developed to utilize the stimulatory effect of DMSO on recombinant protein production and mitigate the problem of growth inhibition in this study. In the first stage, cells are cultured without DMSO for a certain period in order to obtain a high cell density. Sequentially, DMSO is added to achieve a high specific productivity in the second stage. Using this approach, we found that by adding 1% DMSO after 24 h of growth, macrophage colony-stimulating factor (M-CSF) volumetric productivity is increased by 57% compared with the value obtained without the addition of DMSO.

Human monocytes differentiate into macrophages under the influence of human KPB-M15 conditioned medium

Culture medium conditioned by the L929 murine fibroblast cell line contains macrophage colony-stimulating factor (M-CSF), providing an alternative to recombinant M-CSF for in vitro generation of murine macrophages. No such alternative has been described for in vitro studies requiring human macrophages. We tested the differentiation of human blood monocytes into mature macrophages by culturing in media conditioned by the human KPB-M15 cell line, which produces M-CSF and interleukin 6 (IL-6). The phenotypes of macrophages cultured in KPB-M15 conditioned media and recombinant M-CSF were compared by examining viability, expression of cell surface markers, phagocytic/pinocytic activity, and cytokine/chemokine secretion in response to bacterial lipopolysaccharide (LPS). In conditioned medium, monocytes differentiated into a homogeneous population of large cells that exhibited higher expression of CD14 and the macrophage mannose receptor (CD206) than did M-CSF-cultured cells. Cells matured in KPB-M15 conditioned medium exhibited macrophage morphology, were phagocytic, and were activated in response to LPS. These data demonstrate that KPB-M15 conditioned medium can be used to differentiate human blood monocytes into macrophages in vitro.