M-CSF Receptor Research Articles

IL-34 produced by human synovial fibroblast in rheumatoid arthritis supports osteoclastogenesis (117.17)

Abstract Interleukin-34 (IL-34) has a similar function with M-CSF via M-CSF receptor, c-fms, which also involves in RANKL-osteoclastogenesis. In this study, we found that IL-34 was up-regulated in the synovial fluid of RA patients with diagnostic positive rheumatoid factors. IL-34 production was much higher in the synovial fibroblast of RA patients than in that of OA patients and which was enhanced by TNFa stimulation. The expression of IL-34 mRNA and protein in synovial fibroblasts of RA patient after TNFa stimulation were determined using real-time PCR and western blot. Interestingly, TNFa induced IL-34 expression but not M-CSF dependently on NF-kB activity. The conditioned medium of synovial fibroblasts of RA patient cultured with TNFa led to chemotactic migration of human monocytes, and which was blocked by anti-IL-34 antibodies. Moreover, blocking IL-34 activity hindered the RNAKL-induced osteoclastogenesis in same conditioned medium. These results demonstrate that the increase of biologically active IL-34 in the RA synovium induces the chemotactic migration of osteoclast precursor cells and subsequently supports osteoclastogenesis in the inflammed synovium. Together, the differential regulation of IL-34 and M-CSF expression levels by TNFa further implies discrete role of IL-34 in the inflammatory condition involving RA pathogenesis.

Taming of macrophage and microglial cell activation by microRNA-124

Taming of macrophage and microglial cell activation by microRNA-124

Prostaglandin E2 Modulates Monocytes and Dendritic Cells Specific Progenitor Cell (MDP) Fate by Regulating M-CSF Receptor and Flt3 Receptor Expression.

AbstractAbstract 2636Prostaglandin E2 (PGE2), the predominant metabolite of arachidonic acid metabolism by cyclooxygenase (COX) enzymes, is an important physiological regulator of hematopoiesis. We have previously shown that PGE2 negatively regulates proliferation of macrophage colony formation (CFU-M) (Pelus et al. JEM 1979), however the mechanism through which PGE2 inhibits monocyte/macrophage generation is not known. Recently we showed that blockade of endogenous PGE2 synthesis in vivo in mice decreased overall dendritic cell (DC) number in lymphoid organs and bone marrow (Singh et al. ASH 2009). Since monocytes and dendritic cells originate in bone marrow from common hematopoietic progenitor cells termed monocyte and DC progenitor cells (MDP), having the phenotype (Lin− cKithi CD115+CX3CR1+ Flt3+), we hypothesized that PGE2 signaling may modulate MDP fate. Treatment of mice with Indomethacin, a dual COX 1 and COX2 inhibitor, for 6 days simultaneously increased bone marrow monocytes and decreased dendritic cells number compared to vehicle treated control mice. To determine whether the observed in vivo change in monocytes and dendritic cells after Indomethacin treatment was due to fate switch of MDP, we cultured FACS sorted MDP cells with Flt3L plus M-CSF, which simultaneously supports both DC and monocyte differentiation, in the absence or presence of Indomethacin for 9 days. Indomethacin reduced DC differentiation by 42±3.8% compared to vehicle control, and concomitantly increased monocyte generation in the same cultures by 25±2.8%. Moreover, as expected, addition of exogenous PGE2 to these cultures reverted the Indomethacin mediated alteration in dendritic cell and monocyte generation from MDP. To confirm whether the reduction in dendritic cells and increase in monocytes generated from MDP upon the blockade of PGE2 was due to preferential differentiation of MDP into monocyte-committed precursors rather than DC-committed precursors, we analyzed specific DC committed precursors (lin−c-kitint M-CSFR+ Flt3+) and monocyte-committed precursors (CD11−CD11b+F4/80intM-CSFR+) generated from MDP in vitro in the presence of Indomethacin. Fewer DC committed precursors were detected in Flt3L plus M-CSF cultures in the presence of Indomethacin, however monocyte precursor cell number increased compared to vehicle treated control. To further investigate whether a change in fate decision of MDP by blockade of PGE2 is due to modulation of Flt3 and M-CSF receptors on MDP, we evaluated Flt3 receptor and M-CSF receptor on bone marrow MDP from Indomethacin treated mice. Flt3 receptor expression on MDP was decreased by 30±5.2% after Indomethacin treatment compared to control, however M-CSF receptor expression on MDP was increased by 51±4.5. In conclusion, our data suggest that PGE2 regulates monocytes and dendritic cell generation by switching the differentiation fate of MDP by modulating M-CSF receptor and Flt3 receptor expression. Disclosures:No relevant conflicts of interest to declare.

Macrophages and Mast Cells Infiltration Are Biomarkers of Primary Refractory Hodgkin's Lymphoma

AbstractAbstract 3881Classical Hodgkin's lymphoma is a highly curable lymphoma and about 80% of patients can be cured with modern treatment strategies. In spite of great clinical progress, a significant minority of classical Hodgkin's lymphoma experiences treatment failure after primary chemotherapy including a first line of anthracyclin-based regimen. Patients with refractory Hodgkin's lymphoma represent 5 to 10% of classical Hodgkin's Lymphoma. Many of these patients have a poor overall survival estimated at 25% at 5 years. A better biological characterization of such primary refractory patients might allow the use of early therapeutic intervention including targeted therapy. It remains, however, a challenge to identify patients whose disease will not be eradicated by standard therapy. Current prognostic models predict the outcome of treatment with imperfect accuracy, and clinically relevant biomarkers have not yet been established to improve the International Prognostic Score. For these reasons, reliable biomarkers for predicting long term survival at diagnosis are needed for such patients. Steidl et al. (Nejm,2010) recently reported the prognostic value of tumor-associated macrophages in patients with classical Hodgkin's lymphoma, showing that higher infiltration is associated with a shortened survival.The value of tumor-associated macrophages in primary refractory Hodgkin's lymphomas has not been specifically assessed. In a retrospective study (Canioni et al., Plos one 2009), we previously evaluated 59 patients (18 with primary refractory or early relapse disease and 41 responders) collected from 1997 to 2004 in two hematology centres (Necker Hospital and Gustave Roussy Institute, Paris France) for c-kit expression by immunohistochemical analysis as a marker of mast cell infiltration and correlated the results with the response to treatment. All available poor prognosis patients were first identified (18 patients with primary refractory disease or early relapse (< 1year)) and the control group (47 responders) was randomly selected. Patients received conventional chemotherapy-based treatments [(MOPP (mechlorethamine, vincristine, procarbazine, and prednisone), ABVD (doxorubicine, bleomycine, vinblastine, and dacarbazine) or the combination of both or BEACOPP (bleomycine, etoposide, doxorubicine, cyclophosphamide, vincristine, procarbazine, prednisone)] and radiotherapy in stages I and II. Most patients presented a nodular sclerosis subtype of Hodgkin's lymphoma regardless whether they were refractory (17/18cases, 94.5%) or responders (37/41cases, 90%). In this cohort, 44 (75%) patients had a localized stage, whereas 19 (25%) had a disseminated disease. The results showed that refractory Hodgkin's lymphoma were associated with an excess of mast cells infiltration in the tumor cells microenvironment. The number of mast cells stained was significantly higher in refractory Hodgkin's lymphoma (c-kit+ mast cells > 6 per field) than in responders (c-kit+ mast cells ≤ 6) (p=0.001 and 0.0194 in univariate and multivariate analysis, respectively). In this cohort of patients, using CD68 staining we also found a statistically significant higher proportion of tumor-associated macrophages in refractory Hodgkin's lymphoma as compared to responders in univariate and multivariate analyses (p=0.0048 and p=0.0041, respectively). Therefore, we confirmed a strong correlation between the number of CD68 positive macrophages in the tumor stroma and clinical outcome. The use of such markers (CD68 and c-kit) in combination with well-established clinical risk factors could improve on the predictive value of a single biomarker used alone.Therefore, in addition to mast cells, macrophages are also important players in refractory Hodgkin's lymphoma that may confer drug resistance to Hodgkin Reed Sternberg cells. Taken together, these data strongly suggest that microenvironment should be targeted in Hodgkin's lymphomas. In this regard, the use of kinase inhibitors that target both c-kit and M-CSF receptors could restore chemotherapy sensitivity in refractory Hodgkin's lymphoma and should be evaluated in clinical trials. Disclosures:No relevant conflicts of interest to declare.

Direct Inhibition of Human RANK+ Osteoclast Precursors Identifies a Homeostatic Function of IL-1β

IL-1β is a key mediator of bone resorption in inflammatory settings, such as rheumatoid arthritis (RA). IL-1β promotes osteoclastogenesis by inducing RANKL expression on stromal cells and synergizing with RANKL to promote later stages of osteoclast differentiation. Because IL-1Rs share a cytosolic Toll-IL-1R domain and common intracellular signaling molecules with TLRs that can directly inhibit early steps of human osteoclast differentiation, we tested whether IL-1β also has suppressive properties on osteoclastogenesis in primary human peripheral blood monocytes and RA synovial macrophages. Early addition of IL-1β, prior to or together with RANKL, strongly inhibited human osteoclastogenesis as assessed by generation of TRAP(+) multinucleated cells. IL-1β acted directly on human osteoclast precursors (OCPs) to strongly suppress expression of RANK, of the costimulatory triggering receptor expressed on myeloid cells 2 receptor, and of the B cell linker adaptor important for transmitting RANK-induced signals. Thus, IL-1β rendered early-stage human OCPs refractory to RANK stimulation. Similar inhibitory effects of IL-1β were observed using RA synovial macrophages. One mechanism of RANK inhibition was IL-1β-induced proteolytic shedding of the M-CSF receptor c-Fms that is required for RANK expression. These results identify a homeostatic function of IL-1β in suppressing early OCPs that contrasts with its well-established role in promoting later stages of osteoclast differentiation. Thus, the rate of IL-1-driven bone destruction in inflammatory diseases, such as RA, can be restrained by its direct inhibitory effects on early OCPs to limit the extent of inflammatory osteolysis.

Growth regulation and transformation of ovarian epithelium

The discovery of peptide growth factors and cancer-causing genes (oncogenes and tumor-suppressor genes) has provided us with the exciting opportunity to begin to understand the molecular pathology of human ovarian cancer. Activation of several genes, including HER-2/neu, myc, ras, and p53 have been described in some ovarian cancers. In addition, some protooncogenes such as the epidermal growth factor receptor (erbB) and the M-CSF receptor (fms) are expressed along with the respective ligands (peptide growth factors) in some ovarian cancers. Although the studies reviewed in this paper represent a promising beginning, we remain far from a comprehensive understanding of growth regulation and transformation of human ovarian epithelium.

Pro- and anti-inflammatory control of M-CSF-mediated macrophage differentiation

Macrophages play a key role in inflammation, tissue regeneration and tolerance. Their differentiation is regulated by tissue cells derived CSF-1 (M-CSF). The ability of macrophages to use autocrine M-CSF to control their differentiation and function remained controversial. In this study we investigated the regulation of M-CSF production by Th1 and Th2 cytokines (IFN-γ and IL-4) and tolerogenic stimuli – glucocorticoid dexamethasone in primary human monocyte derived macrophages. We show that IFN-γ and IL-4 efficiently induce production of M-CSF while glucocorticoid inhibited it in a dose dependent manner. Since glucocorticoid inhibits production of inflammatory cytokines we tested whether this effect is a result of inhibited M-CSF production. We showed that exogenous M-CSF rescues the ability of glucocorticoid-treated macrophages to produce TNF and IL-6 in response to LPS. These data indicate that glucocorticoid-treated macrophages retain the ability to respond to M-CSF. Analyzing the mechanism of this responsiveness, we showed that dexamethasone up-regulates surface expression of M-CSF receptor – CSF-1R. We conclude that the ability of macrophages to produce M-CSF secures macrophage differentiation under Th1 and Th2 conditions if tissue cells are unable to supply enough M-CSF. Increased surface expression of CSF-1R in tolerogenic conditions guarantees response to minute amounts of exogenous M-CSF.

Interleukin-34 is expressed by giant cell tumours of bone and plays a key role in rankl-induced osteoclastogenesis

Interleukin-34 (IL-34) is a newly discovered regulator of myeloid lineage differentiation, proliferation, and survival, acting via the macrophage-colony stimulating factor receptor (M-CSF receptor, c-fms). M-CSF, the main ligand for c-fms, is required for osteoclastogenesis and has been already identified as a critical contributor of the pathogenesis of giant cell tumours of bone (GCTs), tumours rich in osteoclasts. According to the key role of M-CSF in osteoclastogenesis and GCTs, the expression of IL-34 in human GCTs was first assessed. Quantitative analysis of IL-34 mRNA expression in 14 human GCTs revealed expression of this cytokine in GCTs as well as M-CSF and c-fms. Immunohistochemistry demonstrated that osteoclast-like cells exhibited a huge immunostaining for IL-34 and that mononuclear stromal cells were slightly positive for this protein. In contrast to osteoblasts, bone-resorbing osteoclasts showed very strong staining for IL-34, suggesting its potential role in the pathogenesis of GCTs by facilitating osteoclast formation. The role of IL-34 in osteoclastogenesis was then studied in murine and human models. IL-34 was able to support RANKL-induced osteoclastogenesis in the absence of M-CSF in all models. Multinucleated cells generated in the presence of IL-34 and RANKL expressed specific osteoclastic markers and resorbed dentine. IL-34 induced phosphorylation of ERK 1/2 and Akt through the activation of c-fms, as revealed by the inhibition of signalling by a specific c-fms tyrosine kinase inhibitor. Furthermore, IL-34 stimulated RANKL-induced osteoclastogenesis by promoting the adhesion and proliferation of osteoclast progenitors, and had no effect on osteoclast survival. Overall, these data reveal that IL-34 can be entirely substituted for M-CSF in RANKL-induced osteoclastogenesis, thus identifying a new biological activity for this cytokine and a contribution to the pathogenesis of GCTs.

Abstract 5328: Expansion of CD11b+CD33lowCD15high immunosuppressive myeloid cell population in patients with bladder carcinoma

Abstract Recent studies highlighted the immunoregulatory role of CD11b myeloid cells in cancer-associated immune non-responsiveness. Increased presence of myeloid-derived suppressor cells (MDSC) is one of the major factors responsible for immune suppression in tumor hosts. Here we describe the expanded CD11b myeloid cell subset in peripheral blood obtained from patients with bladder cancer. Based on expression of CD33 and CD15 myeloid cells markers, the circulating CD11b-positive cells in patients with bladder cancer could be subdivided into two major subpopulations: CD33lowCD15high and CD33highCD15low. First subpopulation (CD11b+CD33lowCD15high cells) has granulocyte-type morphology, express G-CSF receptor (CD114) and secrete elevated amounts of pro-inflammatory cytokines and chemokines such as G-CSF, MCP-1 MIP-1alpha, MIP-1beta. Second major myeloid cell subset from cancer patients (CD11b+CD33high CD15lowcells) displays a monocyte-type cell morphology and co-express CD14, M-CSF receptor (CD115), CCR2 and HLA-DR. We show here that proportion of granulocytic CD33lowCD15high cells is highly elevated in cancer patients as compared to healthy individuals. Patients with bladder cancer but not healthy volunteers show weak T cell proliferative response after in vitro stimulation. We demonstrate that granulocyte-type CD33lowCD15high cell population is involved in T cell hyporesponsiveness, since specific depletion of this cell subset restored the T cell proliferation. Obtained results suggest that fraction of PBMC in patients with bladder cancer patients contains markedly elevated proportion of CD11b+CD33lowCD15high granulocyte-type myeloid cell population which contributes to cancer-associated inflammation and tumor-induced immune suppression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5328.

Interleukin‐34 is expressed by giant cell tumours of bone and plays a key role in RANKL‐induced osteoclastogenesis

Interleukin-34 (IL-34) is a newly discovered regulator of myeloid lineage differentiation, proliferation, and survival, acting via the macrophage-colony stimulating factor receptor (M-CSF receptor, c-fms). M-CSF, the main ligand for c-fms, is required for osteoclastogenesis and has been already identified as a critical contributor of the pathogenesis of giant cell tumours of bone (GCTs), tumours rich in osteoclasts. According to the key role of M-CSF in osteoclastogenesis and GCTs, the expression of IL-34 in human GCTs was first assessed. Quantitative analysis of IL-34 mRNA expression in 14 human GCTs revealed expression of this cytokine in GCTs as well as M-CSF and c-fms. Immunohistochemistry demonstrated that osteoclast-like cells exhibited a huge immunostaining for IL-34 and that mononuclear stromal cells were slightly positive for this protein. In contrast to osteoblasts, bone-resorbing osteoclasts showed very strong staining for IL-34, suggesting its potential role in the pathogenesis of GCTs by facilitating osteoclast formation. The role of IL-34 in osteoclastogenesis was then studied in murine and human models. IL-34 was able to support RANKL-induced osteoclastogenesis in the absence of M-CSF in all models. Multinucleated cells generated in the presence of IL-34 and RANKL expressed specific osteoclastic markers and resorbed dentine. IL-34 induced phosphorylation of ERK 1/2 and Akt through the activation of c-fms, as revealed by the inhibition of signalling by a specific c-fms tyrosine kinase inhibitor. Furthermore, IL-34 stimulated RANKL-induced osteoclastogenesis by promoting the adhesion and proliferation of osteoclast progenitors, and had no effect on osteoclast survival. Overall, these data reveal that IL-34 can be entirely substituted for M-CSF in RANKL-induced osteoclastogenesis, thus identifying a new biological activity for this cytokine and a contribution to the pathogenesis of GCTs.

Simvastatin inhibits osteoclast differentiation induced by bone morphogenetic protein-2 and RANKL through regulating MAPK, AKT and Src signaling

The mevalonate pathway plays a crucial role in bone metabolism. Here we examined roles of simvastatin in osteoclast function and differentiation induced by RANKL and BMP-2 using mouse macrophage-like MLC-6 cells and human osteoclast precursor cells. MLC-6 cells expressed BMP type-I and -II receptors and Smads as well as osteoclast markers including TRAP, RANK, cathepsin-K, M-CSF receptor, MMP-9 and calcitonin receptor. Treatment with RANKL and BMP-2 acted synergistically to stimulate RANK, TRAP and cathepsin-K expression in MLC-6 cells. Simvastatin suppressed osteoclastic activity shown by increases in RANK, TRAP and cathepsin-K expression induced by RANKL and BMP-2. In contrast simvastatin alone had no effects on the osteoclastic markers in MLC-6 cells. Simvastatin activated ERK, SAPK/JNK and AKT pathways and inactivated Ras in MLC-6 cells. Simvastatin had no effect on BMP-induced Smad1/5/8 phosphorylation regardless of RANKL stimulation. Since chemical inhibition of ERK, SAPK/JNK and AKT increased TRAP and cathepsin-K expression induced by BMP-2 and RANKL, these pathways are functionally involved in inhibition of osteoclastic activity. In addition, Src phosphorylation induced by RANKL, which is involved in osteoclast differentiation, was suppressed by simvastatin. We further confirmed an inhibitory mechanism of simvastatin on osteoclast differentiation using human osteoclast precursor cells which express BMP receptor and Smad signaling machinery. Simvastatin also activated ERK pathways and inactivated Src phosphorylation in human osteoclasts differentiated by M-CSF and RANKL treatments. The inhibition of TRAP and RANK expression by simvastatin was reversed by ERK inhibition, whereas Src inhibitor enhanced simvastatin-induced suppression of osteoclast markers. Collectively, our data show that simvastatin inhibits osteoclastic differentiation through inhibiting Src as well as enhancing MAPK/AKT pathways.

Abstract C67: Hypoxia-inducible factor-2α regulates macrophage functions in both acute and tumor inflammation

Abstract Hypoxia and inflammation in the tumor microenvironment profoundly influence carcinogenesis and tumor progression. In order to connect hypoxic signaling to inflammatory responses and their roles in oncogenesis, we investigated how one of the master regulators of hypoxic hypoxia-inducible factor (HIF)-2α, adaptations, regulates macrophage functions in both acute inflammatory diseases and cancer (a disease involving various inflammatory components). Myeloid-specific deletion of HIF-2α results in defective expression of a panel of proinflammatory cytokines in macrophages responding to innate stimuli. Also, mice lacking myeloid HIF-2α are resistant to lipopolysaccharide-induced endotoxemia and display a marked inability to mount inflammation in response to several other acute irritants, revealing that this transcription factor is essential for macrophage inflammatory responses. Tumor-associated macrophages (TAMs) represent the major inflammatory cell type within tumors and HIF-2α is robustly expressed in these cells. Clinically, increased TAM HIF-2α level correlates with high tumor grade and poor prognosis in breast and uterine cervical cancer, respectively. To characterize roles of HIF-2α in TAMs, we induced hepatocellular carcinoma (HCC) and colitis-associated cancer (CAC), two established inflammation-associated tumor models, in mice lacking myeloid HIF-2α expression. Interestingly, we found that HIF-2α -deficient TAMs migrate less efficiently into tumors, leading to reduced tumor cell proliferation and decreased malignancy in mutant mice. Further studies revealed that the deficient macrophages were attributable to compromised migration capacity of HIF-2α to insufficient expression of M-CSF receptor, CXCR4 and extracellular fibronectin-1. Therefore, our study demonstrated HIF-2α as an important regulator in both acute inflammation and cancer, suggesting that the myeloid HIF-2α pathway could be exploited as therapeutic targets for treating these diseases. Citation Information: Cancer Res 2009;69(23 Suppl):C67.

Signaling Crosstalk during Sequential TLR4 and TLR9 Activation Amplifies the Inflammatory Response of Mouse Macrophages

The TLR family of pattern recognition receptors is largely responsible for meditating the activation of macrophages by pathogens. Because macrophages may encounter multiple TLR ligands during an infection, signaling crosstalk between TLR pathways is likely to be important for the tailoring of inflammatory reactions to pathogens. Here, we show that rather than inducing tolerance, LPS pretreatment primed the inflammatory response (e.g., TNF production) of mouse bone marrow-derived macrophages (BMM) to the TLR9 ligand, CpG DNA. The priming effects of LPS, which correlated with enhanced Erk1/2, JNK, and p38 MAPK activation, appeared to be mediated via both c-Fms-dependent and -independent mechanisms. LPS pretreatment and inhibition of the M-CSF receptor, c-Fms, with GW2580 had comparable effects on CpG DNA-induced Erk1/2 and p38 MAPK activation. However, c-Fms inhibition did not enhance CpG DNA-induced JNK activation; also, the levels of TNF produced were significantly lower than those from LPS-primed BMM. Thus, the priming effects of LPS on TLR9 responses appear to be largely mediated via the c-Fms-independent potentiation of JNK activity. Indeed, inhibition of JNK abrogated the enhanced production of TNF by LPS-pretreated BMM. The c-Fms-dependent priming effects of LPS are unlikely to be a consequence of the inhibitory constraints of M-CSF signaling on TLR9 expression being relieved by LPS; instead, LPS may exert its priming effects via signaling molecules downstream of TLR9. In summary, our findings highlight the importance of signaling crosstalk between TLRs, as well as between TLRs and c-Fms, in regulating the inflammatory reaction to pathogens.

Inhibition of RANK Expression and Osteoclastogenesis by TLRs and IFN-γ in Human Osteoclast Precursors

TLRs have been implicated in promoting osteoclast-mediated bone resorption associated with inflammatory conditions. TLRs also activate homeostatic mechanisms that suppress osteoclastogenesis and can limit the extent of pathologic bone erosion associated with infection and inflammation. We investigated mechanisms by which TLRs suppress osteoclastogenesis. In human cell culture models, TLR ligands suppressed osteoclastogenesis by inhibiting expression of receptor activator of NF-kappaB (RANK), thereby making precursor cells refractory to the effects of RANKL. Similar but less robust inhibition of RANK expression was observed in murine cells. LPS suppressed generation of osteoclast precursors in mice in vivo, and adsorption of LPS onto bone surfaces resulted in diminished bone resorption. Mechanisms that inhibited RANK expression were down-regulation of RANK transcription, and inhibition of M-CSF signaling that is required for RANK expression. TLRs inhibited M-CSF signaling by rapidly down-regulating cell surface expression of the M-CSF receptor c-Fms by a matrix metalloprotease- and MAPK-dependent mechanism. Additionally, TLRs cooperated with IFN-gamma to inhibit expression of RANK and of the CSF1R gene that encodes c-Fms, and to synergistically inhibit osteoclastogenesis. Our findings identify a new mechanism of homeostatic regulation of osteoclastogenesis that targets RANK expression and limits bone resorption during infection and inflammation.

Histone Deacetylase Inhibitor M344 Inhibits Cell Proliferation and Induces Apoptosis in Human THP-1 Leukemia Cells

Histone acetylation plays an important role in the silencing and activation of genes involved in tumoregenesis. Trichostatin A, originally identified as an anti-fungal drug, is a potent inhibitor of histone deacetylase (HDAC) with potential anti-tumor activity. In this study, we investigated the effect of M344, an amide analogues of trichostatin A, on the growth and differentiation of THP-1 human leukemia cells. We showed that at low doses, (< 0.2 muM), M344 could inhibit the growth of THP-1 cells at G1 phase in vitro with low cytotoxic effect. Low dose of M344 exerted some differentiating effect on THP-1 cells as judged by the expression of c-fms proto-oncogene (M-CSF receptor) and appearance of adherent cells. Growth arrest induced by M344 is associated with increased levels of cyclin-dependent protein kinase inhibitor p21 and cyclin E, in agreement with G1 phase arrest. At higher doses (2 muM), M344 could induce THP-1 cells to undergo apoptosis, which was associated with the cleavage of PARP, cytochrome c release and activation of both caspases-8, -9, followed by the activation of caspase-3. In addition, M344 could increase the levels of pro-apoptotic protein Bax but decreased the levels of anti-apoptotic protein XIAP. M344 is a potent activator of NF-kappaB transcription factor. RT-PCR assay showed that the M344 could transiently increase IL-1 expression yet markedly decreased TNF-alpha expression. Our results show that M344 is a potent growth inhibitor and inducer of apoptosis in human leukemia cells and suggest potential therapeutic strategies of HDAC inhibitors for patients with leukemias.

An M-CSF Receptor c-Fms Antibody Inhibits Mechanical Stress–Induced Root Resorption during Orthodontic Tooth Movement in Mice

To examine the effect of anti-c-Fms antibody on odontoclastogenesis and root resorption in an orthodontic tooth movement mouse model. We used orthodontic tooth movement in which an Ni-Ti coil spring was inserted between the upper incisors and the upper first molar. Root resorption occurred in this model. Anti-c-Fms antibody was injected daily into a local site for 12 days during mechanical loading. Odontoclastogenesis and root resorption were assessed by histology and scanning electron microscopy. The anti-c-Fms antibody significantly inhibited odontoclastogenesis and root resorption during orthodontic tooth movement. M-CSF and/or its receptor is a potential therapeutic target in mechanical stress- induced odontoclastogenesis, and injection of an anti-c-Fms antibody might be useful for inhibition of mechanical stress-induced root resorption during orthodontic tooth movement.

CSF-1R, DAP12 and β-catenin: a ménage à trois

DAP12-coupled receptors influence signals emanating from Toll-like receptors, integrins and receptors for cytokines and growth factors. New findings indicate that DAP12 also facilitates the ability of CSF-1R, the receptor for M-CSF, to induce the stabilization and nuclear translocation of beta-catenin.

Preventive and therapeutic effects of imatinib in Wistar-Kyoto rats with anti-glomerular basement membrane glomerulonephritis

Imatinib is a selective tyrosine kinase inhibitor that can block activity of the platelet-derived growth factor receptor (PDGFR) and that has immunomodulatory effects on various cell types. Here we measured the protective effects of imatinib in Wistar-Kyoto rats with nephrotoxic serum nephritis, a kidney disease model where CD8+ T cells and macrophages play pathogenetic roles. Groups of animals were given imatinib from one day before up to 13 days following induction of nephritis and from day 7 to 20 following disease induction. Compared to control rats, at each time point imatinib treatment caused significantly less proteinuria, lowered serum blood urea nitrogen and creatinine, and decreased the number of glomeruli with necrosis, crescents, and fibrin deposits. Imatinib-treated rats had a significant reduction in glomerular macrophage accumulation and reduced renal cortical PDGFR-beta and M-CSF receptor mRNA expression. Using colocalization we found that glomerular macrophages had reduced IL-1beta and MCP-1 protein expression. Late imatinib treatment significantly reduced proteinuria, serum blood urea nitrogen, and creatinine, and reversed renal histopathological changes. We show that imatinib has renoprotective and therapeutic properties and provide pre-clinical work that will need to be confirmed in patients with crescentic glomerulonephritis.

Identification of a Myeloid Pathway of NK Cell Development and a Myeloid- NK Cell Precursor

NK cells are CD56+CD3− innate immune effector cells that play important roles in tumor surveillance. Since the identification of a common lymphoid progenitor (CLP) and the demonstration that NK cells can be derived from CLPs, the ontogeny of NK cells has been assumed to be exclusively lymphoid. Our laboratory has been studying NK cell development using CD34+ hematopoietic progenitors (HPCs) cultured on a murine fetal liver stromal cell line (EL08.1D2) in the presence of cytokines (IL-3 for the first week, IL-7, IL-15, SCF and FLT-3L) and hydrocortisone (HDC). After 3–4 weeks under these conditions, NK cells develop. We set out to determine the impact of stroma and HDC on NK cell development. Previous studies had identified antigens on CD34 HPCs that were associated with a greater propensity to become NK cells when cultured in cytokines (including CD7, CD161, CD45RA and intergrin b7). FACS purifying CD34 cells that lacked these receptors (CD34+NKlin−) identified a subset of CD34 cells that was absolutely dependent on stroma or HDC for functional NK cell development (using limiting dilution assays, p<0.05). In the absence of stroma and HDC, however, CD34+NKlin− cells showed myeloid lineage (CD13 and CD33) outgrowth. Likewise, under the influence of cytokines, HDC and stroma, CD34+NKlin− cells appeared to first acquire CD13 and/or CD33, then CD56. These results lead us to hypothesize that the mechanism of HDC and stroma was to “skew” myeloid cells to the NK lineage. To test this, CD34+NKlin− cells were cultured for 2 wks and CD13low/−, CD13int and CD13high cells were FASC sorted. Culture of sorted populations with cytokines alone did not give rise to NK cells. The addition of either HDC or stroma resulted in the development of NK cells and their combination was additive (p<0.05). Importantly, there was a quantitative difference in NK cell generation from the three fractions, with decreasing NK cell growth with advancing myeloid commitment. In further experiments CD34+NKlin− HPCs were cultured for two weeks and then CD14+ cells were isolated. NK cells again developed with the combination of cytokines, HDC and stroma, but not with cytokines alone. To further query whether myeloid cells could become NK cells, we isolated CFU-GMs from 3 wk methylcellulose colonies and cultured them with cytokines or cytokines, HDC and stroma. NK cell developed only in the presence of cytokines, HDC and stroma (0/36 vs. 9/51, p<0.05). Collectively, these results suggested a myeloid-NK progenitor. The M-CSF receptor (M-CSFR, CD115) is expressed by granulo-monocytic precursors, promoting monocytic differentiation in response to M-CSF. Therefore, M-CSFR is not merely a phenotypic marker, but is functionally linked with the monocytic differentiation. CD117highCD56−CD115+ and CD115− cells were isolated and tested for their ability to give rise to NK cells. Strikingly, both could give rise to NK cells, but CD115+ progenitors required HDC and stroma for full NK cell maturation. Addition of high doses of M-CSF to CD115+ progenitors abolished NK cell development. Lower doses of M-CSF lead to NK cells that co-expressed CD14, supporting the existence of a common NK-myeloid progenitor. Lastly, the NK cells derived from the myeloid progenitors (CD117highCD56−CD115+) were compared to the remaining “lymphoid” fraction. The myeloid derived NK cells expressed significantly more KIR receptors and had remarkably higher killing against B-LCL cells (721-221) (p<0.05 for both, n=4). Collectively, these studies show that cells at seemingly late stages of myeloid development cells can become NK cells under the influence of stroma and HDC. Thus, like dendritic cells, NK cells can be derived from both myeloid and lymphoid developmental pathways. Such NK cells differ both functionally and phenotypically depending upon their developmental origin. Lastly, we demonstrate the existence of a previously unknown common myeloid-NK progenitor (CD117highCD56−CD115+).

THOC5 couples M-CSF receptor signaling to transcription factor expression

THOC5 is a nuclear/cytoplasmic protein member of the spliceosome complex which potentiates C/EBP expression in adipocyte differentiation. As C/EBP family members are important regulators of myelopoiesis and THOC5 is highly expressed in neutrophil/macrophage progenitor cells we assessed the role of THOC5 in cytokine-stimulated monocytic development. M-CSF stimulated maturation of the NFS60 cell line was associated with enhanced THOC5 expression and phosphorylation. THOC5 was also shown to form a complex with C/EBPβ. Ectopic expression of THOC5 mimicked M-CSF mediated cell maturation and enhanced protein expression of the myeloid transcription factors C/EBPβ, C/EBPα, Pu-1 and also GAB2 (a PI-3 Kinase and macrophage development regulator). Increased THOC5 expression also mimicked M-CSF stimulated increases in the lipid second messenger PtdInsP 3. Inhibition of THOC5-induced increases in PtdInsP 3 levels abrogated the elevated levels of C/EBPβ. Thus THOC5 expression can potentiate receptor signalling to transcription factor expression and monocyte differentiation.