RESUMO
Glioma is a common malignant tumor of the central nervous system (CNS) that has no effective treatment. In this study, we report that colony-stimulating factor-1 receptor (CSF-1R) is a key mediator of malignant features in glioma via modulation of the activity of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. In general, CSF-1R upregulation in glioma is associated with poor histologic grade and sursvival. Enforced expression of CSF-1R is sufficient to enhance cell growth, migration, invasion, and epithelial-mesenchymal transition, while CSF-1R silencing suppresses the above-described malignant phenotypes. Mechanistic investigations show that CSF-1R promotes activation of the ERK1/2 signaling pathway. Inhibition of the ERK1/2 pathway by SCH772984 reduces CSF-1R-induced migration, invasion, and lung metastasis of glioma cells, thus establishing a role of the ERK1/2 signaling pathway in mediating the CSF-1R effect. In summary, our results suggest that CSF-1R overexpression in gliomas contributes to the malignant behaviors of cancer cells.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Sistema de Sinalização das MAP Quinases , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Modelos Animais de Doenças , Pontos de Checagem da Fase G1 do Ciclo Celular , Glioma/genética , Glioma/patologia , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fenótipo , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Fase S , Transfecção , Regulação para CimaRESUMO
The c-fms proto-oncogene is also known as macrophage colony stimulating factor receptor (M-CSFR) or colony-stimulating factor-1 receptor (CSF-1R), and is expressed on several types of malignant tumor cells and myeloid cells. In the present study, we found that overexpression of M-CSFR was present in adult T-cell leukemia/lymphoma (ATLL) cases. M-CSFR signaling was associated with lymphoma cell proliferation, and M-CSFR inhibition induced apoptosis in lymphoma cells. The ATLL cell line ATL-T expressed M-CSF/CSF-1 and interleukin (IL)-34, which are both M-CSFR ligands. M-CSF and IL-34 expression was seen in ATLL cases, and co-expression of these ligands was detected in 11 of 13 ATLL cases. M-CSFR inhibition suppressed programmed death-1 and -2 ligand in ATL-T cells and macrophages stimulated with conditioned medium from ATL-T cells. Thus, an M-CSFR inhibitor may be useful as additional therapy against ATLL due to direct and indirect mechanisms.
Assuntos
Apoptose , Antígeno B7-H1/biossíntese , Citocinas/biossíntese , Regulação Leucêmica da Expressão Gênica , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Adulto , Linhagem Celular Tumoral , Feminino , Humanos , Leucemia-Linfoma de Células T do Adulto/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , Masculino , Proto-Oncogene Mas , Receptor de Fator Estimulador de Colônias de Macrófagos/biossínteseRESUMO
The absence of tumor necrosis factor (TNF) causes lethal infection by Leishmania major in normally resistant C57BL/6J (B6.WT) mice. The underlying pathogenic mechanism of this fatal disease has so far remained elusive. We found that B6.WT mice deficient for the tnf gene (B6.TNF-/-) displayed not only a non-healing cutaneous lesion but also a serious infection of the liver upon L. major inoculation. Infected B6.TNF-/- mice developed an enlarged liver that showed increased inflammation. Furthermore, we detected an accumulating monocyte-derived macrophage population (CD45+F4/80+CD11bhiLy6Clow) that displayed a M2 macrophage phenotype with high expression of CD206, arginase-1, and IL-6, supporting the notion that IL-6 could be involved in M2 differentiation. In in vitro experiments, we demonstrated that IL-6 upregulated M-CSF receptor expression and skewed monocyte differentiation from dendritic cells to macrophages. This was countered by the addition of TNF. Furthermore, TNF interfered with the activation of IL-6-induced gp130-signal transducer and activator of transcription (STAT) 3 and IL-4-STAT6 signaling, thereby abrogating IL-6-facilitated M2 macrophage polarization. Therefore, our results support the notion of a general role of TNF in the inflammatory activation of macrophages and define a new role of IL-6 signaling in macrophage polarization downstream of TNF.
Assuntos
Interleucina-6/imunologia , Fígado/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Fator de Necrose Tumoral alfa/genética , Animais , Arginase/biossíntese , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Receptor gp130 de Citocina/metabolismo , Inflamação/imunologia , Interleucina-4/metabolismo , Interleucina-6/biossíntese , Lectinas Tipo C/biossíntese , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Fígado/parasitologia , Fígado/patologia , Ativação de Macrófagos/genética , Macrófagos/citologia , Receptor de Manose , Lectinas de Ligação a Manose/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Carga Parasitária , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Receptores de Superfície Celular/biossíntese , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT6/metabolismoRESUMO
PURPOSE: The model of oxygen-induced retinopathy (OIR) is widely used to analyze pathomechanisms in retinal neovascularization. Previous studies have shown that macrophages (MP) play a key role in vessel formation in OIR, the influence of microglia (MG) having been discussed. The aim of our study was to analyze the spatial and temporal distribution and activation of MP/MG expressing CD115 and CD11b during the process of neovascularization in OIR. METHODS: We used MacGreen mice expressing the green fluorescence protein (GFP) under the promoter for CD115. CD115+ cells were investigated in vivo by scanning laser ophthalmoscopy at postnatal days (P) 17 and 21 in MacGreen mice with OIR (75% oxygen from P7 to P12), and were compared to MacGreen room-air controls. In addition MP/MG were examined ex vivo using immunohistochemistry for CD11b+ detection on retinal flatmounts at P14, P17, and P21 of wild type mice with OIR. RESULTS: In-vivo imaging revealed the highest density of activated MP/MG in tuft areas at P17 of MacGreen mice with OIR. Tufts and regions with a high density of CD115+ cells were detected close to veins, rather to arteries. In peripheral, fully vascularized areas, the distribution of CD115+ cells in MacGreen mice with OIR was similar to MacGreen room-air controls. Correspondingly, immunohistochemical analyses of retinal flatmounts from wild type mice with OIR induction revealed that the number of CD11b+ cells significantly varies between vascular, avascular, and tuft areas as well as between the retinal layers. Activated CD11b+ cells were almost exclusively found in avascular areas and tufts of wild type mice with OIR induction; here, the proportion of activated cells related to the total number of CD11b+ cells remained stable over the course of time. CONCLUSIONS: Using two different approaches to monitor MP/MG cells, our findings demonstrated that MP/MG concentrate within pathologically vascularized areas during OIR. We were able to clarify that reactive changes of CD11b+ cell distribution to OIR primarily occur in the deep retinal layers. Furthermore, we found the highest proportion of activated CD11b+ cells in regions with pathologic neovascularization processes. Our findings support previous reports about activated MP/MG guiding revascularization in avascular areas and playing a key role in the formation and regression of neovascular tufts.
Assuntos
Macrófagos/metabolismo , Microglia/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/biossíntese , Neovascularização Retiniana/metabolismo , Animais , Animais Recém-Nascidos , Contagem de Células , Modelos Animais de Doenças , Angiofluoresceinografia , Fundo de Olho , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/patologia , Oftalmoscopia , Oxigênio/toxicidade , Neovascularização Retiniana/induzido quimicamente , Neovascularização Retiniana/patologiaRESUMO
The proliferation, differentiation, and survival of cells of the macrophage lineage depends upon signals from the macrophage colony-stimulating factor (CSF) receptor (CSF1R). CSF1R is expressed by embryonic macrophages and induced early in adult hematopoiesis, upon commitment of multipotent progenitors to the myeloid lineage. Transcriptional activation of CSF1R requires interaction between members of the E26 transformation-specific family of transcription factors (Ets) (notably PU.1), C/EBP, RUNX, AP-1/ATF, interferon regulatory factor (IRF), STAT, KLF, REL, FUS/TLS (fused in sarcoma/ranslocated in liposarcoma) families, and conserved regulatory elements within the mouse and human CSF1R locus. One element, the Fms-intronic regulatory element (FIRE), within intron 2, is conserved functionally across all the amniotes. Lineage commitment in multipotent progenitors also requires down-regulation of specific transcription factors such as MYB, FLI1, basic leucine zipper transcriptional factor ATF-like (BATF3), GATA-1, and PAX5 that contribute to differentiation of alternative lineages and repress CSF1R transcription. Many of these transcription factors regulate each other, interact at the protein level, and are themselves downstream targets of CSF1R signaling. Control of CSF1R transcription involves feed-forward and feedback signaling in which CSF1R is both a target and a participant; and dysregulation of CSF1R expression and/or function is associated with numerous pathological conditions. In this review, we describe the regulatory network behind CSF1R expression during differentiation and development of cells of the mononuclear phagocyte system.
Assuntos
Regulação da Expressão Gênica/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Transcrição Gênica/fisiologia , Animais , Diferenciação Celular/genética , Cromatina/genética , Desenvolvimento Embrionário/fisiologia , Humanos , Macrófagos/citologia , Camundongos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Receptores Acoplados a Proteínas G/genética , Transativadores/genéticaRESUMO
PURPOSE: Central giant cell granulomas (CGCGs) are clinically classified as nonaggressive (nA-CGCGs) and aggressive (A-CGCGs). However, histopathologically, all lesions feature spindle mononuclear cells (MCs) and multinuclear giant cells (GCs) in a hemorrhage-rich stroma. We aimed to investigate the presence of cells with a monocyte- or macrophage-related phenotype and, together with clinical variables, to examine their predictive potential for the biological behavior of CGCGs. PATIENTS AND METHODS: For our investigation, we implemented a retrospective cohort study. Sections were immunohistochemically stained for colony-stimulating factor 1 receptor (CSF-1R) (CD115), CD163, CD68, and nuclear factor κB. The clinical variables included age, gender, and location of lesions. Associations between immunostains, clinical variables, and CGCG aggressiveness were analyzed by the Wilcoxon (Mann-Whitney) exact test and t test. Significant variables were further analyzed by a logistic regression model followed by receiver operating characteristic (ROC) curve analysis for diagnostic sensitivity. Significance was set at P < .05. RESULTS: Patients with A-CGCGs (n = 36) were younger than those with nA-CGCGs (n = 31) (P = .002). Logistic regression showed that CD163-GC (ß = -0.870, P = .031) and CD115-MC (ß = -0.783, P = .027) had a significant protection effect (odds ratio, 0.419 [95% confidence interval, 0.190 to 0.925], and odds ratio, 0.457 [95% confidence interval, 0.229 to 0.913], respectively). ROC curve analysis showed that CD163-GC and CSF-1R (CD115)-MC combined were the best predictor in distinguishing nA-CGCGs from A-CGCGs (area under ROC curve, 0.814; P < .001). At the optimal cutoff value (0.408), sensitivity was 87% and specificity, 65%. CONCLUSIONS: Increasing age and high expression of CD163-GC and CSF-1R (CD115)-MC can serve as significant predictors of nA-CGCGs. A functional link between CD163-GC and the characteristic areas of extravasation of erythrocytes is discussed.
Assuntos
Antígenos CD/biossíntese , Antígenos de Diferenciação Mielomonocítica/biossíntese , Granuloma de Células Gigantes/metabolismo , Granuloma de Células Gigantes/patologia , Doenças Maxilomandibulares/metabolismo , Doenças Maxilomandibulares/patologia , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Receptores de Superfície Celular/biossíntese , Adulto , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
Patients who survive initial burn injury are susceptible to nosocomial infections. Anemia of critical illness is a compounding factor in burn patients that necessitates repeated transfusions, which further increase their susceptibility to infections and sepsis. Robust host response is dependent on an adequate number and function of monocytes/macrophages and dendritic cells. In addition to impaired RBC production, burn patients are prone to depletion of dendritic cells and an increase in deactivated monocytes. In steady-state hematopoiesis, RBCs, macrophages, and dendritic cells are all generated from a common myeloid progenitor within the bone marrow. We hypothesized in a mouse model of burn injury that an increase in myeloid-specific transcription factor V-maf musculoaponeurotic fibrosarcoma oncogene homolog B at the common myeloid progenitor stage steers their lineage potential away from the megakaryocyte erythrocyte progenitor production and drives the terminal fate of common myeloid progenitors to form macrophages vs. dendritic cells, with the consequences being anemia, monocytosis, and dendritic cell deficits. Results indicate that, even though burn injury stimulated bone marrow hematopoiesis by increasing multipotential stem cell production (LinnegSca1poscKitpos), the bone marrow commitment is shifted away from the megakaryocyte erythrocyte progenitor and toward granulocyte monocyte progenitors with corresponding alterations in peripheral blood components, such as hemoglobin, hematocrit, RBCs, monocytes, and granulocytes. Furthermore, burn-induced V-maf musculoaponeurotic fibrosarcoma oncogene homolog B in common myeloid progenitors acts as a transcriptional activator of M-CSFR and a repressor of transferrin receptors, promoting macrophages and inhibiting erythroid differentiations while dictating a plasmacytoid dendritic cell phenotype. Results from small interfering RNA and gain-of-function (gfp-globin transcription factor 1 retrovirus) studies indicate that targeted interventions to restore V-maf musculoaponeurotic fibrosarcoma oncogene homolog B/globin transcription factor 1 balance can mitigate both immune imbalance and anemia of critical illness.
Assuntos
Anemia/etiologia , Queimaduras/sangue , Queimaduras/imunologia , Fator de Transcrição GATA1/fisiologia , Fator de Transcrição MafB/fisiologia , Células Progenitoras Mieloides/patologia , Mielopoese/genética , Anemia/genética , Anemia/fisiopatologia , Animais , Queimaduras/genética , Linhagem da Célula , Células Cultivadas , Estado Terminal , Células Dendríticas/patologia , Fator de Transcrição GATA1/genética , Macrófagos/patologia , Fator de Transcrição MafB/genética , Masculino , Camundongos , Monócitos/patologia , Interferência de RNA , RNA Interferente Pequeno/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Proteínas Recombinantes de Fusão/metabolismo , Transcrição GênicaRESUMO
The mononuclear phagocytic system is categorized in three major groups: monocyte-derived cells (MCs), dendritic cells and resident macrophages. During breast cancer progression the colony stimulating factor 1 (CSF-1) can reprogram MCs into tumor-promoting macrophages in the primary tumor. However, the effect of CSF-1 during colonization of the brain parenchyma is largely unknown. Thus, we analyzed the outcome of anti-CSF-1 treatment on the resident macrophage population of the brain, the microglia, in comparison to MCs, alone and in different in vitro co-culture models. Our results underline the addiction of MCs to CSF-1 while surprisingly, microglia were not affected. Furthermore, in contrast to the brain, the bone marrow did not express the alternative ligand, IL-34. Yet treatment with IL-34 and co-culture with carcinoma cells partially rescued the anti-CSF-1 effects on MCs. Further, MC-induced invasion was significantly reduced by anti-CSF-1 treatment while microglia-induced invasion was reduced to a lower extend. Moreover, analysis of lung and breast cancer brain metastasis revealed significant differences of CSF-1 and CSF-1R expression. Taken together, our findings demonstrate not only differences of anti-CSF-1 treatment on MCs and microglia but also in the CSF-1 receptor and ligand expression in brain and bone marrow as well as in brain metastasis.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias Encefálicas/patologia , Neoplasias da Mama/patologia , Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Animais , Anticorpos Monoclonais/imunologia , Encéfalo/citologia , Encéfalo/imunologia , Encéfalo/patologia , Neoplasias Encefálicas/secundário , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Humanos , Interleucina-1/farmacologia , Células MCF-7 , Fator Estimulador de Colônias de Macrófagos/biossíntese , Fator Estimulador de Colônias de Macrófagos/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Microglia/citologia , Microglia/imunologia , Microglia/patologia , Monócitos/imunologia , Invasividade Neoplásica/imunologia , Invasividade Neoplásica/patologia , Receptor de Fator Estimulador de Colônias de Macrófagos/biossínteseRESUMO
Pigmented villonodular synovitis (PVNS) is a benign, translocation-derived neoplasm. Because of its high local recurrence rate after surgery and occurrence of osteochondral destruction, a novel therapeutic target is required. The present study aimed to evaluate the significance of protein expression possibly associated with the pathogenesis during the clinical course of PVNS. In 40 cases of PVNS, positivity of colony-stimulated factor 1 (CSF1), its receptor (CSF1R), and receptor activator of nuclear factor kappa-B ligand (RANKL) were immunohistochemically determined. The relationship between the positivity and clinical outcomes was investigated. High positivity of CSF1 staining intensity was associated with an increased incidence of osteochondral lesions (bone erosion and osteoarthritis) (p = 0.009), but not with the rate of local recurrence. Positivity of CSF1R and RANKL staining was not associated with any clinical variables. The number of giant cells was not correlated with positivity of any of the three proteins, or with the clinical outcome. Focusing on knee cases, CSF1 positivity was also associated with the incidence of osteochondal change (p = 0.02). CSF1R positivity was high in cases which had local recurrence, but not significantly so (p = 0.129). Determination of CSF1 and CSF1R expression may be useful as a prognosticator of the clinical course and/or outcomes of PVNS.
Assuntos
Fator Estimulador de Colônias de Macrófagos/biossíntese , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Sinovite Pigmentada Vilonodular/genética , Adolescente , Adulto , Criança , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Fator Estimulador de Colônias de Macrófagos/genética , Masculino , Pessoa de Meia-Idade , Osteoartrite/genética , Osteoartrite/patologia , Osteoartrite/cirurgia , Prognóstico , Ligante RANK/biossíntese , Ligante RANK/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Sinovite Pigmentada Vilonodular/patologia , Sinovite Pigmentada Vilonodular/cirurgia , Resultado do TratamentoRESUMO
Acute myeloid leukemia is a clonal malignant disorder derived from a small number of leukemic stem cells (LSCs). Rearrangements of the mixed lineage leukemia (MLL) gene are found in acute myeloid leukemia associated with poor prognosis. The upregulation of Hox genes is critical for LSC induction and maintenance, but is unlikely to support malignancy and the high LSC frequency observed in MLL leukemias. The present study shows that MLL fusion proteins interact with the transcription factor PU.1 to activate the transcription of CSF-1R, which is critical for LSC activity. Acute myeloid leukemia is cured by either deletion of PU.1 or ablation of cells expressing CSF-1R. Kinase inhibitors specific for CSF-1R prolong survival time. These findings indicate that PU.1-mediated upregulation of CSF-1R is a critical effector of MLL leukemogenesis.
Assuntos
Carcinogênese/genética , Histona-Lisina N-Metiltransferase/genética , Leucemia Mieloide Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Proto-Oncogênicas/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Transativadores/genética , Animais , Regulação Leucêmica da Expressão Gênica , Genes Homeobox , Leucemia Mieloide Aguda/mortalidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Neoplásicas , Compostos de Fenilureia/farmacologia , Prognóstico , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais , Tacrolimo/análogos & derivados , Tacrolimo/farmacologia , Tiazóis/farmacologia , Transcrição Gênica , Ativação Transcricional , Regulação para CimaRESUMO
All-lymphoid progenitors (ALPs) yield few myeloid cells in vivo, but readily generate such cells in vitro. The basis for this difference remains unknown. We hypothesized that ALPs limit responsiveness to in vivo concentrations of myeloid-promoting cytokines by reducing expression of the corresponding receptors, potentially through posttranscriptional mechanisms. Consistent with such a mechanism, ALPs express higher levels of CSF1R transcripts than their upstream precursors, yet show limited cell-surface protein expression of colony-stimulating factor 1 receptor (CSF1R). All-lymphoid progenitors and other hematopoietic progenitors deficient in A disintegrin and metalloproteinase domain 17 (ADAM17), display elevated cell surface CSF1R expression. ADAM17(-/-) ALPs, however, fail to yield myeloid cells upon transplantation into irradiated recipients. Moreover, ADAM17(-/-) ALPs yield fewer macrophages in vitro than control ALPs at high concentrations of macrophage colony stimulating factor. Mice with hematopoietic-specific deletion of ADAM17 have normal numbers of myeloid and lymphoid progenitors and mature cells in vivo. These data demonstrate that ADAM17 limits CSF1R protein expression on hematopoietic progenitors, but that compensatory mechanisms prevent elevated CSF1R levels from altering lymphoid progenitor potential.
Assuntos
Proteínas ADAM/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Linfócitos/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Proteínas ADAM/deficiência , Proteínas ADAM/genética , Proteína ADAM17 , Animais , Transplante de Medula Óssea , Linhagem da Célula , Membrana Celular/metabolismo , Regulação da Expressão Gênica , Linfopoese/efeitos dos fármacos , Linfopoese/genética , Fator Estimulador de Colônias de Macrófagos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mielopoese/efeitos dos fármacos , Mielopoese/genética , RNA Mensageiro/biossíntese , Quimera por Radiação , Receptor de Fator Estimulador de Colônias de Macrófagos/genéticaRESUMO
Pigmented or "melano-" macrophages are prominent in lymphoid and non-lymphoid tissues of poikilotherms. Though they have been extensively studied in situ only recently has a means to isolate them from other cell types been established. We provide the first in vitro characterization of isolated melanomacrophage cytochemistry and survival in culture. Unlike non-pigmented tissue macrophages melanomacrophages do not adhere to polystyrene surfaces making them easy to separate from tissue macrophages. In vitro goldfish melanomacrophages are distinguishable from tissue macrophages and neutrophils by being Sudan Black B positive (unlike tissue macrophages) and non-specific esterase positive (unlike neutrophils). Like tissue macrophages they also express acid phosphatase and CSF-1R. As sorted cells melanomacrophages only survive a few days in culture. However in coarsely disaggregated spleen and kidney tissues melanomacrophages survive for at least 3 weeks. Furthermore after 5 days culture disaggregating tissue clumps revealed encapsulated melanomacrophage clusters that remained intact for at least another week. The encapsulated clusters were resilient enough to allow for their isolation for further imaging and isolation of RNA. In some cases the clusters had either melanomacrophages or non-fluorescent cells protruding and in the latter case these could initiate outgrowths onto the plates with subsequent collapse of the cluster. These approaches for the isolation of melanomacrophages and melanomacrophage clusters should allow further study into specific cell and cluster functions.
Assuntos
Macrófagos/classificação , Macrófagos/metabolismo , Pigmentação , Fosfatase Ácida/biossíntese , Animais , Compostos Azo , Técnicas de Cultura de Células , Células Cultivadas , Citometria de Fluxo , Carpa Dourada , Rim/citologia , Naftalenos , Imagem Óptica , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Baço/citologiaRESUMO
INTRODUCTION: Although C-X-C motif chemokine 12 (CXCL12) has been shown to bind to C-X-C chemokine receptor type 7 (CXCR7), the exact molecular mechanism regulations by CXCL12/CXCR7 axis in breast tumor growth and metastasis are not well understood. CXCR7 expression has been shown to be upregulated during pathological processes such as inflammation and cancer. METHODS: Breast cancer cell lines were genetically silenced or pharmacologically inhibited for CXCR7 and/or its downstream target signal transducer and activator of transcription 3 (STAT3). 4T1 or 4T1 downregulated for CXCR7 and 4T1.2 breast cancer cell lines were injected in mammary gland of BALB/c mice to form tumors, and the molecular pathways regulating tumor growth and metastasis were assessed. RESULTS: In this study, we observed that CXCL12 enhances CXCR7-mediated breast cancer migration. Furthermore, genetic silencing or pharmacologic inhibition of CXCR7 reduced breast tumor growth and metastasis. Further elucidation of mechanisms revealed that CXCR7 mediates tumor growth and metastasis by activating proinflammatory STAT3 signaling and angiogenic markers. Furthermore, enhanced breast tumorigenicity and invasiveness were associated with macrophage infiltration. CXCR7 recruits tumor-promoting macrophages (M2) to the tumor site through regulation of the macrophage colony-stimulating factor (M-CSF)/macrophage colony-stimulating factor receptor (MCSF-R) signaling pathway. In addition, CXCR7 regulated breast cancer metastasis by enhancing expression of metalloproteinases (MMP-9, MMP-2) and vascular cell-adhesion molecule-1 (VCAM-1). We also observed that CXCR7 is highly expressed in invasive ductal carcinoma (IDC) and metastatic breast tissue in human patient samples. In addition, high CXCR7 expression in tumors correlates with worse prognosis for both overall survival and lung metastasis-free survival in IDC patients. CONCLUSION: These observations reveal that CXCR7 enhances breast cancer growth and metastasis via a novel pathway by modulating the tumor microenvironment. These findings identify CXCR7-mediated STAT3 activation and modulation of the tumor microenvironment as novel regulation of breast cancer growth and metastasis. These studies indicate that new strategies using CXCR7 inhibitors could be developed for antimetastatic therapy.
Assuntos
Neoplasias da Mama/patologia , Quimiocina CXCL12/metabolismo , Neoplasias Pulmonares/secundário , Receptores CXCR/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Carcinoma Ductal de Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Ativação de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/biossíntese , Macrófagos/imunologia , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica/genética , Transplante de Neoplasias , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Receptores CXCR/antagonistas & inibidores , Receptores CXCR/biossíntese , Receptores CXCR/genética , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genética , Microambiente Tumoral , Molécula 1 de Adesão de Célula Vascular/biossínteseRESUMO
Human skeletal aging is characterized as a gradual loss of bone mass due to an excess of bone resorption not balanced by new bone formation. Using human marrow cells, we tested the hypothesis that there is an age-dependent increase in osteoclastogenesis due to intrinsic changes in regulatory factors [macrophage-colony stimulating factor (M-CSF), receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG)] and their receptors [c-fms and RANK]. In bone marrow cells (BMCs), c-fms (r = 0.61, P = 0.006) and RANK expression (r = 0.59, P = 0.008) were increased with age (27-82 years, n = 19). In vitro generation of osteoclasts was increased with age (r = 0.89, P = 0.007). In enriched marrow stromal cells (MSCs), constitutive expression of RANKL was increased with age (r = 0.41, P = 0.049) and expression of OPG was inversely correlated with age (r = -0.43, P = 0.039). Accordingly, there was an age-related increase in RANKL/OPG (r = 0.56, P = 0.005). These data indicate an age-related increase in human osteoclastogenesis that is associated with an intrinsic increase in expression of c-fms and RANK in osteoclast progenitors, and, in the supporting MSCs, an increase in pro-osteoclastogenic RANKL expression and a decrease in anti-osteoclastogenic OPG. These findings support the hypothesis that human marrow cells and their products can contribute to skeletal aging by increasing the generation of bone-resorbing osteoclasts. These findings help to explain underlying molecular mechanisms of progressive bone loss with advancing age in humans.
Assuntos
Envelhecimento/metabolismo , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Osteoclastos/metabolismo , Envelhecimento/patologia , Células da Medula Óssea/metabolismo , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Humanos , Fator Estimulador de Colônias de Macrófagos/biossíntese , Osteoclastos/patologia , Osteogênese/genética , Osteoprotegerina/biossíntese , Ligante RANK/biossíntese , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/biossíntese , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Células Estromais/metabolismoRESUMO
Macrophages contribute to innate and acquired immunity as well as many aspects of homeostasis and development. Studies of macrophage biology and function in birds have been hampered by a lack of definitive cell surface markers. As in mammals, avian macrophages proliferate and differentiate in response to CSF1 and IL34, acting through the shared receptor, CSF1R. CSF1R mRNA expression in the chicken is restricted to macrophages and their progenitors. To expedite studies of avian macrophage biology, we produced an avian CSF1R-Fc chimeric protein and generated a monoclonal antibody (designated ROS-AV170) against the chicken CSF1R using the chimeric protein as immunogen. Specific binding of ROS-AV170 to CSF1R was confirmed by FACS, ELISA and immunohistochemistry on tissue sections. CSF1 down-regulated cell surface expression of the CSF1R detected with ROS-AV170, but the antibody did not block CSF1 signalling. Expression of CSF1R was detected on the surface of bone marrow progenitors only after culture in the absence of CSF1, and was induced during macrophage differentiation. Constitutive surface expression of CSF1R distinguished monocytes from other myeloid cells, including heterophils and thrombocytes. This antibody will therefore be of considerable utility for the study of chicken macrophage biology.
Assuntos
Anticorpos Monoclonais/imunologia , Galinhas/imunologia , Macrófagos/imunologia , Receptor de Fator Estimulador de Colônias de Macrófagos/imunologia , Animais , Células da Medula Óssea/imunologia , Células CHO , Diferenciação Celular/imunologia , Linhagem Celular , Linhagem da Célula/imunologia , Cricetulus , Feminino , Fator Estimulador de Colônias de Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/imunologia , RNA Mensageiro/biossíntese , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Receptor de Fator Estimulador de Colônias de Macrófagos/genéticaRESUMO
Macrophage-lineage cells are indispensable to vertebrate homeostasis and immunity. In turn, macrophage development is largely regulated through colony-stimulating factor-1 (CSF1) binding to its cognate receptor (CSF1R). To study amphibian monopoiesis, we identified and characterized the X. laevis CSF1R cDNA transcript. Quantitative analysis revealed that CSF1R tissue gene expression increased with X. laevis development, with greatest transcript levels detected in the adult lung, spleen and liver tissues. Notably, considerable levels of CSF1R mRNA were also detected in the regressing tails of metamorphosing animals, suggesting macrophage involvement in this process, and in the adult bone marrow; corroborating the roles for this organ in Xenopus monopoiesis. Following animal infections with the ranavirus Frog Virus 3 (FV3), both tadpole and adult X. laevis exhibited increased kidney CSF1R gene expression. Conversely, while FV3-infected tadpoles increased their spleen and liver CSF1R mRNA levels, the FV3-challenged adults did not. Notably, FV3 induced elevated bone marrow CSF1R expression, and while stimulation of tadpoles with heat-killed E. coli had no transcriptional effects, bacterial stimulation of adult frogs resulted in significantly increased spleen, liver and bone marrow CSF1R expression. We produced the X. laevis CSF1R in recombinant form (rXlCSF1R) and determined, via in vitro cross-linking studies, that two molecules of rXlCSF1R bound the dimeric rXlCSF1. Finally, administration of rXlCSF1R abrogated the rXlCSF1-induced tadpole macrophage recruitment and differentiation as well as bacterial and FV3-elicited peritoneal leukocyte accumulation. This work marks a step towards garnering greater understanding of the unique mechanisms governing amphibian macrophage biology.
Assuntos
Diferenciação Celular/genética , Larva/crescimento & desenvolvimento , Macrófagos/citologia , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Sequência de Aminoácidos , Animais , Medula Óssea/metabolismo , Infecções por Vírus de DNA , Técnicas de Cultura Embrionária , Rim/metabolismo , Larva/virologia , Fígado/metabolismo , Macrófagos/imunologia , Dados de Sequência Molecular , RNA Mensageiro/genética , Ranavirus , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência , Baço/metabolismo , Xenopus laevis/embriologiaRESUMO
BACKGROUND: Microglia are the primary immune cells of the brain whose phenotype largely depends on their surrounding micro-environment. Microglia respond to a multitude of soluble molecules produced by a variety of brain cells. Macrophage colony-stimulating factor (M-CSF) is a cytokine found in the brain whose receptor is expressed by microglia. Previous studies suggest a critical role for M-CSF in brain development and normal functioning as well as in several disease processes involving neuroinflammation. METHODS: Using biopsy tissue from patients with intractable temporal epilepsy and autopsy tissue, we cultured primary adult human microglia to investigate their response to M-CSF. Mixed glial cultures were treated with 25 ng/ml M-CSF for 96 hours. Proliferation and phagocytosis assays, and high through-put immunocytochemistry, microscopy and image analysis were performed to investigate microglial phenotype and function. RESULTS: We found that the phenotype of primary adult human microglia was markedly changed following exposure to M-CSF. A greater number of microglia were present in the M-CSF- treated cultures as the percentage of proliferating (BrdU and Ki67-positive) microglia was greatly increased. A number of changes in protein expression occurred following M-CSF treatment, including increased transcription factors PU.1 and C/EBPß, increased DAP12 adaptor protein, increased M-CSF receptor (CSF-1R) and IGF-1 receptor, and reduced HLA-DP, DQ, DR antigen presentation protein. Furthermore, a distinct morphological change was observed with elongation of microglial processes. These changes in phenotype were accompanied by a functional increase in phagocytosis of Aß1-42 peptide. CONCLUSIONS: We show here that the cytokine M-CSF dramatically influences the phenotype of adult human microglia. These results pave the way for future investigation of M-CSF-related targets for human therapeutic benefit.
Assuntos
Proliferação de Células/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Microglia/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Fatores de Transcrição/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Antimetabólitos , Autopsia , Biópsia , Bromodesoxiuridina , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Células Cultivadas , Antígenos HLA/biossíntese , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Microglia/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Transativadores/biossíntese , Transativadores/genéticaRESUMO
BACKGROUND: Tumor-associated macrophages (TAMs) have high impact on the cancer development because they can facilitate matrix invasion, angiogenesis, and tumor cell motility. It gives cancer cells the capacity to invade normal tissues and metastasize. The signaling of colony-stimulating factor-1 receptor (CSF-1R) which is an important regulator of proliferation and differentiation of monocytes and macrophages regulates most of the tissue macrophages. However, CSF-1R is expressed also in breast epithelial tissue during some physiological stages i.g.: pregnancy and lactation. Its expression has been also detected in various cancers. Our previous study has showed the expression of CSF-1R in all examined canine mammary tumors. Moreover, it strongly correlated with grade of malignancy and ability to metastasis. This study was therefore designed to characterize the role of CSF-1R in canine mammary cancer cells proliferation, apoptosis, migration, and invasion. As far as we know, the study presented hereby is a pioneering experiment in this field of veterinary medicine. RESULTS: We showed that csf-1r silencing significantly increased apoptosis (Annexin V test), decreased proliferation (measured as Ki67 expression) and decreased migration ("wound healing" assay) of canine mammary cancer cells. Treatment of these cells with CSF-1 caused opposite effect. Moreover, csf-1r knock-down changed growth characteristics of highly invasive cell lines on Matrigel matrix, and significantly decreased the ability of these cells to invade matrix. CSF-1 treatment increased invasion of cancer cells. CONCLUSION: The evidence of the expression and functional role of the CSF-1R in canine mammary cancer cells indicate that CSF-1R targeting may be a good therapeutic approach.
Assuntos
Doenças do Cão/fisiopatologia , Neoplasias Mamárias Animais/fisiopatologia , Receptor de Fator Estimulador de Colônias de Macrófagos/fisiologia , Animais , Apoptose/fisiologia , Western Blotting/veterinária , Linhagem Celular Tumoral , Proliferação de Células , Cães , Feminino , Citometria de Fluxo/veterinária , Invasividade Neoplásica/fisiopatologia , Metástase Neoplásica/fisiopatologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Receptor de Fator Estimulador de Colônias de Macrófagos/biossínteseRESUMO
Macrophage and dendritic cell (DC) progenitors (MDPs) and common DC progenitors (CDPs) are bone marrow (BM) progenitors with DC differentiation potential. However, both MDPs and CDPs give rise to large numbers of conventional DCs (cDCs) and few plasmacytoid DCs (pDCs), implying that more dedicated pDC progenitors remain to be identified. Here we have described DC progenitors with a prominent pDC differentiation potential. Although both MDPs and CDPs express the macrophage colony stimulating factor (M-CSF) receptor (M-CSFR), the progenitors were confined to a M-CSFR(-) fraction, identified as Lin(-)c-Kit(int/lo)Flt3(+)M-CSFR(-), and expressed high amounts of E2-2 (also known as Tcf4) an essential transcription factor for pDC development. Importantly, they appeared to be directly derived from either CDPs or lymphoid-primed multipotent progenitors (LMPPs). Collectively, our findings provide insight into DC differentiation pathways and may lead to progenitor-based therapeutic applications for infection and autoimmune disease.
Assuntos
Células Dendríticas/metabolismo , Células Progenitoras Linfoides/metabolismo , Linfopoese/imunologia , Macrófagos/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/biossíntese , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Células da Medula Óssea/metabolismo , Diferenciação Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Células Progenitoras Linfoides/imunologia , Linfopoese/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Receptor de Fator Estimulador de Colônias de Macrófagos/deficiência , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator de Transcrição 4 , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Transcription intermediary factor 1γ (TIF1γ) was suggested to play a role in erythropoiesis. However, how TIF1γ regulates the development of different blood cell lineages and whether TIF1γ is involved in human hematological malignancies remain to be determined. Here we have shown that TIF1γ was a tumor suppressor in mouse and human chronic myelomonocytic leukemia (CMML). Loss of Tif1g in mouse HSCs favored the expansion of the granulo-monocytic progenitor compartment. Furthermore, Tif1g deletion induced the age-dependent appearance of a cell-autonomous myeloproliferative disorder in mice that recapitulated essential characteristics of human CMML. TIF1γ was almost undetectable in leukemic cells of 35% of CMML patients. This downregulation was related to the hypermethylation of CpG sequences and specific histone modifications in the gene promoter. A demethylating agent restored the normal epigenetic status of the TIF1G promoter in human cells, which correlated with a reestablishment of TIF1γ expression. Together, these results demonstrate that TIF1G is an epigenetically regulated tumor suppressor gene in hematopoietic cells and suggest that changes in TIF1γ expression may be a biomarker of response to demethylating agents in CMML.