RESUMEN
Apical periodontitis (AP) is an acute or chronic inflammatory disease caused by complex interactions between infected root canal and host immune system. It results in the induction of inflammatory mediators such as chemokines and cytokines leading to periapical tissue destruction. To understand the molecular pathogenesis of AP, we have investigated inflammatory-related genes that regulate AP development. We found here that macrophage-derived CXCL9, which acts through CXCR3, is recruited by progressed AP. The inhibition of CXCL9 by a CXCR3 antagonist reduced the lesion size in a mouse AP model with decreasing IL-1ß, IL-6 and TNFα expression. The treatment of peritoneal macrophages with CXCL9 and LPS induced the transmigration and upregulation of osteoclastogenic cytokines such as IL-1ß, IL-6 and matrix metalloprotease 2, a marker of activated macrophages. This suggests that the CXCL9-CXCR3 axis plays a crucial role in the development of AP, mediated by the migration and activation of macrophages for periapical tissue destruction. Our data thus show that CXCL9 regulates the functions of macrophages which contribute to AP pathogenesis, and that blocking CXCL9 suppresses AP progression. Knowledge of the principal factors involved in the progression of AP, and the identification of related inflammatory markers, may help to establish new therapeutic strategies.
Asunto(s)
Quimiocina CXCL9/metabolismo , Activación de Macrófagos , Macrófagos/metabolismo , Periodontitis Periapical/inmunología , Receptores CXCR3/metabolismo , Animales , Línea Celular Tumoral , Ensayos de Migración de Macrófagos , Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Periodontitis Periapical/metabolismo , Periodontitis Periapical/patología , Receptores CXCR3/antagonistas & inhibidores , Raíz del Diente/patologíaRESUMEN
M2-polarized macrophages have been shown to adapt their 3D migration mode to physical properties of surrounding extracellular matrix. They migrate in the integrin-mediated adhesion and proteolytic activity-dependent "mesenchymal" mode in stiff matrices and in the integrin and protease-independent "amoeboid" mode in low density, porous environments. To find out what impact the switching between the migration modes has on expression of both protein-coding and non-coding genes we employed RNA sequencing of total RNA depleted of ribosomal RNA isolated from macrophages migrating in either mode in 3D collagens. Differentially expressed genes from both categories have been detected and the changes in expression of selected genes were further validated with RT-qPCR. The acquired data will facilitate better understanding of how mechanical properties of tissue microenvironment reflect in macrophage immune function and how the transitions between mesenchymal and amoeboid migratory modes are regulated at the gene expression level.
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Movimiento Celular , Microambiente Celular , Macrófagos , ARN Ribosómico , Ensayos de Migración de Macrófagos , Movimiento Celular/genética , Colágeno , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Análisis de Secuencia de ARNRESUMEN
Tumor migration/metastasis and immunosuppression are major obstacles in effective cancer therapy. Incidentally, these 2 hurdles usually coexist inside tumors, therefore making therapy significantly more complicated, as both oncogenic mechanisms must be addressed for successful therapeutic intervention. Our recent report highlights that the tumor expression of a TNF family member, CD70, is correlated with poor survival for primary gliomas. In this study, we investigated how CD70 expression by GBM affects the characteristics of tumor cells and the tumor microenvironment. We found that the ablation of CD70 in primary GBM decreased CD44 and SOX2 gene expression, and inhibited tumor migration, growth and the ability to attract monocyte-derived M2 macrophages in vitro. In the tumor microenvironment, CD70 was associated with immune cell infiltrates, such as T cells; myeloid-derived suppressor cells; and monocytes/macrophages based on the RNA-sequencing profile. The CD163+ macrophages were far more abundant than T cells were. This overwhelming level of macrophages was identified only in GBM and not in low-grade gliomas and normal brain specimens, implying their tumor association. CD70 was detected only on tumor cells, not on macrophages, and was highly correlated with CD163 gene expression in primary GBM. Additionally, the co-expression of the CD70 and CD163 genes was found to correlate with decreased survival for patients with primary GBM. Together, these data suggest that CD70 expression is involved in promoting tumor aggressiveness and immunosuppression via tumor-associated macrophage recruitment/activation. Our current efforts to target this molecule using chimeric antigen receptor T cells hold great potential for treating patients with GBM.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Ligando CD27/metabolismo , Glioblastoma/metabolismo , Glioblastoma/secundario , Tolerancia Inmunológica , Antígenos CD/análisis , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/análisis , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Encéfalo/citología , Neoplasias Encefálicas/inmunología , Ligando CD27/análisis , Ligando CD27/genética , Línea Celular Tumoral , Ensayos de Migración de Macrófagos/métodos , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Glioblastoma/inmunología , Glioblastoma/mortalidad , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Inmunidad Celular , Macrófagos/química , Macrófagos/citología , Macrófagos/inmunología , Metástasis de la Neoplasia , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
The inhalation of particles and their exposure to the bronchi and alveoli constitute a major public health risk. Chemical as well as particle-related properties are important factors for the biological response but are difficult to separate from each other. Barium sulfate is a completely inert chemical compound, therefore it is ideally suited to separate these two factors. The biological response of rat alveolar macrophages (NR8383) was analyzed after exposure to barium sulfate particles with three different diameters (40 nm, 270 nm, and 1.3 µm, respectively) for 24 h in vitro (particle concentrations from 12.5 to 200 µg mL-1). The particles were colloidally stabilized as well as fluorescently-labeled by carboxymethylcellulose, conjugated with 6-aminofluorescein. All kinds of barium sulfate particles were efficiently taken up by NR8383 cells and found inside endo-lysosomes, but never in the cell nucleus. Neither an inflammatory nor a cytotoxic response was detected by the ability of dHL-60 and NR8383 cells to migrate towards a chemotactic gradient (conditioned media of NR8383 cells) and by the release of inflammatory mediators (CCL2, TNF-α, IL-6). The particles neither caused apoptosis (up to 200 µg mL-1) nor necrosis (up to 100 µg mL-1). As only adverse reaction, necrosis was found at a concentration of 200 µg mL-1 of the largest barium sulfate particles (1.3 µm). Barium sulfate particles are ideally suited as bioinert control to study size-dependent effects such as uptake mechanisms of intracellular distributions of pure particles, especially in nanotoxicology.
Asunto(s)
Sulfato de Bario/toxicidad , Quimiotaxis/efectos de los fármacos , Macrófagos Alveolares/efectos de los fármacos , Nanopartículas/toxicidad , Animales , Ensayos de Migración de Macrófagos , Células Cultivadas , Citometría de Flujo , Interleucina-6/inmunología , Pulmón/efectos de los fármacos , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Microscopía Confocal , Tamaño de la Partícula , Ratas , Estándares de Referencia , Propiedades de Superficie , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Accumulation of macrophages and neutrophil granulocytes in the lung are key events in the inflammatory response to inhaled particles. The present study aims at the time course of chemotaxis in vitro in response to the challenge of various biopersistent particles and its functional relation to the transcription of inflammatory mediators. NR8383 rat alveolar macrophages were challenged with particles of coarse quartz, barium sulfate, and nanosized silica for one, four, and 16h and with coarse and nanosized titanium dioxide particles (rutile and anatase) for 16h only. The cell supernatants were used to investigate the chemotaxis of unexposed NR8383 macrophages. The transcription of inflammatory mediators in cells exposed to quartz, silica, and barium sulfate was analyzed by quantitative real-time PCR. Challenge with quartz, silica, and rutile particles induced significant chemotaxis of unexposed NR8383 macrophages. Chemotaxis caused by quartz and silica was accompanied by an elevated transcription of CCL3, CCL4, CXCL1, CXCL3, and TNFα. Quartz exposure showed an earlier onset of both effects compared to the nanosized silica. The strength of this response roughly paralleled the cytotoxic effects. Barium sulfate and anatase did not induce chemotaxis and barium sulfate as well caused no elevated transcription. In conclusion, NR8383 macrophages respond to the challenge with inflammatory particles with the release of chemotactic compounds that act on unexposed macrophages. The kinetics of the response differs between the various particles.
Asunto(s)
Contaminantes Atmosféricos/toxicidad , Quimiocinas/metabolismo , Quimiotaxis/efectos de los fármacos , Citocinas/metabolismo , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Material Particulado/toxicidad , Animales , Sulfato de Bario/toxicidad , Línea Celular , Ensayos de Migración de Macrófagos , Perfilación de la Expresión Génica , Cinética , Nanopartículas/toxicidad , Cuarzo/toxicidad , Ratas , Dióxido de Silicio/toxicidad , Titanio/toxicidadRESUMEN
Bisphenol A (BPA), bis(2-ethylhexyl)phthalate (DEHP) and di(n-butyl)phthalate (DBP) are environmental estrogens that have been associated with a wide range of adverse health outcomes for which inflammation has also been hypothesized as a potentially involved mechanism and where macrophages play a central role. This study was carried out to evaluate if xenoestrogen (XE) treatment of classically (M1) or alternatively (M2) activated macrophages could affect their behavior. For this purpose, human peripheral blood monocyte-derived macrophages either unstimulated or activated with lipopolysaccharide (100 ng/mL, M1) or with interleukin (IL) 4 (15 ng/mL, M2) were treated with 17ß-estradiol (E2 ), BPA, DEHP and DBP alone or in combination with selective ERα or ERß antagonists. Migratory capability, cytokine release, and estrogen-associated signaling pathways were evaluated to assess macrophage function. All tested XEs had a tendency to stimulate M2 migration, an effect that followed the same direction than E2 . Moreover, all XEs significantly induced IL10 in M1 and decreased IL6 and globally decreased IL10, IL6, TNFα, and IL1ß release by M2 macrophages. However, DEHP and DBP significantly increased IL1ß release in M1 and M2 macrophages, respectively. Some of the effects described above were shown to be mediated by either ERα or ERß and were simultaneous to modulation of NF-κB, AP1, JNK, or ERK signaling pathways. We provide new evidence of the effect of XE on macrophage behavior and their mechanisms with relevance to the understanding of the action of environmental chemicals on the immune system and inflammation-associated diseases. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1496-1509, 2016.
Asunto(s)
Compuestos de Bencidrilo/toxicidad , Movimiento Celular/efectos de los fármacos , Citocinas/metabolismo , Dietilhexil Ftalato/toxicidad , Estradiol/metabolismo , Macrófagos/efectos de los fármacos , Fenoles/toxicidad , Ensayos de Migración de Macrófagos , Células Cultivadas , Quimiotaxis de Leucocito/efectos de los fármacos , Femenino , Humanos , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/fisiología , Persona de Mediana Edad , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Tenascin-C (TN-C) is an extracellular matrix protein not detected in normal adult heart, but expressed in several heart diseases closely associated with inflammation. Accumulating data suggest that TN-C may play a significant role in progression of ventricular remodeling. In this study, we aimed to elucidate the role of TN-C in hypertensive cardiac fibrosis and underlying molecular mechanisms. Angiotensin II was administered to wild-type and TN-C knockout mice for 4 weeks. In wild-type mice, the treatment induced increase of collagen fibers and accumulation of macrophages in perivascular areas associated with deposition of TN-C and upregulated the expression levels of interleukin-6 and monocyte chemoattractant protein-1 as compared with wild-type/control mice. These changes were significantly reduced in TN-C knockout/angiotensin II mice. In vitro, TN-C accelerated macrophage migration and induced accumulation of integrin αVß3 in focal adhesions, with phosphorylation of focal adhesion kinase (FAK) and Src. TN-C treatment also induced nuclear translocation of phospho-NF-κB and upregulated interleukin-6 expression of macrophages in an NF-κB-dependent manner; this being suppressed by inhibitors for integrin αVß3 and Src. Furthermore, interleukin-6 upregulated expression of collagen I by cardiac fibroblasts. TN-C may enhance inflammatory responses by accelerating macrophage migration and synthesis of proinflammatory/profibrotic cytokines via integrin αVß3/FAK-Src/NF-κB, resulting in increased fibrosis.
Asunto(s)
Regulación de la Expresión Génica , Cardiopatías/genética , Integrina alfaVbeta3/genética , Interleucina-6/genética , Activación de Macrófagos/genética , ARN Mensajero/genética , Tenascina/genética , Animales , Western Blotting , Ensayos de Migración de Macrófagos , Células Cultivadas , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis/genética , Fibrosis/metabolismo , Fibrosis/patología , Técnica del Anticuerpo Fluorescente , Cardiopatías/metabolismo , Cardiopatías/patología , Inmunohistoquímica , Integrina alfaVbeta3/biosíntesis , Interleucina-6/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteínas del Tejido Nervioso , Reacción en Cadena en Tiempo Real de la Polimerasa , Tenascina/biosíntesisRESUMEN
Vitamin D (VD) displays immunoregulatory effects and reduces adipocyte inflammation, which may participate to a reduction of adipose tissue macrophage infiltration in the context of obesity-associated low-grade inflammation. These observations have been described mainly in vitro, through the evaluation of a limited number of inflammatory markers. Here, we studied the effects of 1,25 dihydroxy-VD on chemokine network expression in adipocytes (by transcriptomic approach), and we confirm the physiological relevance of these data in vivo, by demonstrating the effect of VD on cytokine and chemokine gene expression as well as on macrophage infiltration in adipose tissue. 1,25 dihydroxy-VD down-regulated (-1.3- to -10.8-fold) the mRNA expression of 29 chemokines and limited macrophage migration in TNFα-conditioned adipocyte medium (1.5-fold; P < .05). This effect was associated with a reduction in p65 and IκB (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) phosphorylation (2-fold compared with TNFα; P < .05). The effects of VD were confirmed in mice injected ip with lipopolysaccharide (acute inflammation) and diet-induced obese mice (metabolic inflammation), where the levels of mRNA encoding proinflammatory cytokines and chemokines (â¼2-fold) were reduced in adipocytes (acute and metabolic inflammation) and adipose tissue and that macrophage infiltration was also inhibited in the adipose tissue of obese mice (metabolic inflammation). Altogether, these results showed that VD displayed a global immunoregulatory impact on adipocytes, notably via the inhibition of chemokine expression and macrophage infiltration in inflamed adipose tissue.
Asunto(s)
Adipocitos/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Quimiocinas/efectos de los fármacos , Colecalciferol/farmacología , Inflamación/genética , Macrófagos/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , Vitamina D/análogos & derivados , Células 3T3-L1 , Adipocitos/metabolismo , Animales , Línea Celular , Ensayos de Migración de Macrófagos , Quimiocinas/genética , Citocinas/efectos de los fármacos , Citocinas/genética , Expresión Génica/efectos de los fármacos , Humanos , Técnicas In Vitro , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/farmacología , Vitamina D/farmacologíaRESUMEN
Inflammation is an important factor in the development of insulin resistance. SIRT1, a class 3 histone/protein deacetylase, has anti-inflammatory functions. Myeloid-specific deletion of Sirt1 promotes macrophage infiltration into insulin-sensitive organs and aggravates tissue inflammation. In this study, we investigated how SIRT1 in macrophages alters tissue inflammation in the pancreas as well as liver and adipose tissue, and further explored the role of SIRT1 in locomotion of macrophages. Myeloid-specific Sirt1-deleted mice (mS1KO) and WT littermates were fed a 60% calorie high-fat diet (HFD) for 16 weeks. Tissue inflammation and metabolic phenotypes were compared. Bone marrow macrophages (BMMs) from WT or mS1KO mice were used in in vitro chemotaxis assays and macrophage polarization studies. mS1KO mice fed a HFD exhibited glucose intolerance, reduced insulin secretion, and insulin sensitivity with a slight decrease in body weight. Consistent with these results, pancreatic islets of mS1KO mice fed a HFD displayed decreased mass with profound apoptotic cell damage and increased macrophage infiltration and inflammation. Liver and adipose tissues from mS1KO HFD mice also showed greater accumulation of macrophages and tissue inflammation. Results from in vitro experiments indicated that deletion of myeloid Sirt1 stimulated proinflammatory M1-like polarization of BMMs and augmented the adipocyte-mediated macrophage chemotaxis. The latter effect was accompanied by increased expression and acetylation of focal adhesion kinase, as well as nuclear factor kappa B. Our results indicate that myeloid SIRT1 plays a crucial role in macrophage polarization and chemotaxis, and thus regulates the development of HFD-induced pancreatic inflammation and insulin secretion, and metabolic derangements in liver and adipose tissue.
Asunto(s)
Movimiento Celular/genética , Dieta Alta en Grasa , Resistencia a la Insulina/genética , Macrófagos/inmunología , Sirtuina 1/fisiología , Tejido Adiposo/inmunología , Animales , Atrofia/inmunología , Ensayos de Migración de Macrófagos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Grasas de la Dieta/farmacología , Células HEK293 , Humanos , Inflamación/inmunología , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/patología , Hígado/inmunología , Macrófagos/efectos de los fármacos , Ratones , Ratones Noqueados , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Sirtuina 1/genéticaRESUMEN
BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT)-induced disruption of the blood-brain barrier (BBB) has been investigated as a technique for the delivery of therapeutic agents to selective regions of the brain. The purpose of this study was to determine the effects of PDT on the migration of systemically administered exogenous macrophages (Ma) loaded with iron oxide nanoparticles in non-tumor bearing rats. MATERIALS AND METHODS: A control group consisting of three Sprague-Dawley rats was injected with iron oxide-loaded rat alveolar Ma via jugular vein catheter while two animals were subjected to intracranial injection of iron oxide-loaded Ma. PDT-treated animals were injected with photosensitizer (AlPcS2a ; 1 mg/kg i.p.) followed by light irradiation (wavelength = 670 nm; light dose = 2.5 J) 48 hours later. Light irradiation was performed through the skull. Prior to light irradiation, iron oxide-loaded Ma were administered to each animal. Animals in all groups were imaged in a 7 Tesla (T) magnetic resonance (MR) imager to determine the extent of PDT-induced edema and to evaluate for the presence of iron oxide nanoparticles. Animals were sacrificed 7 days post-Ma administration and their brains analyzed for the presence of iron oxide using Perls staining. RESULTS: Significant uptake of iron oxide nanoparticles by rat alveolar Ma was observed thus providing the rationale for their use as delivery vectors. Histopathological analyses failed to find evidence of iron oxide in normal rat brain. Accumulations of iron oxide-loaded Ma were observed in both MR images and histological sections of non-tumor bearing rat brain following PDT-induced disruption of the BBB. CONCLUSIONS: MR imaging was shown to be useful for localizing iron-oxide loaded Ma in rat brains. Exogenous Ma are incapable of traversing the normal BBB and therefore, the use of Ma as delivery vehicles into the brain requires selective disruption of the BBB.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Indoles/farmacología , Macrófagos Alveolares/metabolismo , Compuestos Organometálicos/farmacología , Fármacos Fotosensibilizantes/farmacología , Animales , Ensayos de Migración de Macrófagos , Medios de Contraste , Dextranos , Indoles/administración & dosificación , Imagen por Resonancia Magnética , Nanopartículas de Magnetita , Masculino , Compuestos Organometálicos/administración & dosificación , Fotoquimioterapia , Fármacos Fotosensibilizantes/administración & dosificación , Ratas , Ratas Sprague-DawleyRESUMEN
Casein kinase-2 interacting protein-1 (CKIP-1) has been identified to play an important role in cell morphology, differentiation and apoptosis. However, the role of CKIP-1 in other cellular processes is still unknown. Here we investigated transcriptome profiles of WT and CKIP-1-deficient mouse embryonic fibroblasts (MEFs), and found that innate immunity and cell migration related pathways were significantly correlated with CKIP-1 expression. As macrophage is a key cell type in innate immunity, we then used murine macrophage RAW264.7 cells to discover CKIP-1 interacting proteins by immunoprecipitation/mass spectrometry (IP/MS). Analysis of these proteins revealed migration related pathways were enriched. Further experiments indicated that knockdown of CKIP-1 in RAW264.7 cells resulted in impaired cell migration. Our study suggests that CKIP-1 is a novel regulator of macrophage migration.
Asunto(s)
Proteínas Portadoras/metabolismo , Movimiento Celular , Macrófagos/metabolismo , Animales , Proteínas Portadoras/genética , Ensayos de Migración de Macrófagos , Factores Quimiotácticos/farmacología , Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Genómica , Macrófagos/efectos de los fármacos , Ratones , N-Formilmetionina Leucil-Fenilalanina/farmacología , Mapeo de Interacción de Proteínas , Proteómica , Transducción de Señal , TranscriptomaRESUMEN
BACKGROUND: Macrophage recruitment into atherosclerotic plaques drives lesion progression, destabilization, and rupture. Chronic statin treatment reduces macrophage plaque content. Information on dynamics of macrophage recruitment would help assessing plaque vulnerability and guiding therapy. Techniques to image macrophage homing to vulnerable plaques in vivo are scarcely available. The authors tested if noninvasive fluorescence-mediated tomography (FMT) can assess plaque-stabilizing effects of short-term high-dosage atorvastatin. METHODS: Macrophages from green-fluorescent-protein-transgenic mice were labeled with a near-infrared fluorescent dye and were injected IV in apolipoprotein E-deficient mice (n=9) on Western diet 7 days after guidewire-injury of the carotid artery. FMT-scans, 2 and 7 days thereafter, quantified macrophage recruitment into carotid artery plaques. Atorvastatin was tested for macrophage adhesion, proliferation, and viability (n=5 to 6) in vitro. Fourteen mice received atorvastatin or vehicle for 4 days after 16 weeks on Western diet. FMT assessed macrophage recruitment into aortic and innominate artery lesions. Means (±SD)% are reported. RESULTS: Double-labeled macrophages were recruited into carotid artery lesions. FMT resolved fluorescence projecting on the injured carotid artery and detected a signal increase to 300% (±191) after guidewire injury. Atorvastatin reduced macrophage adhesion to activated endothelial cells by 36% (±19). In a clinically relevant proof-of-concept intervention, FMT-imaging detected that 4 days atorvastatin treatment reduced macrophage recruitment by 57% (±8) indicating plaque stabilization. Immunohistochemistry confirmed reduced macrophage infiltration. CONCLUSIONS: FMT optical imaging proved its high potential for clinical applicability for tracking recruitment of near-infrared fluorescent-labeled macrophages to vulnerable plaques in vivo. FMT-based quantification of macrophage recruitment demonstrated rapid plaque stabilization by 4-day atorvastatin treatment in apolipoprotein E-deficient mice.
Asunto(s)
Apolipoproteínas E/genética , Ácidos Heptanoicos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Macrófagos/metabolismo , Pirroles/farmacología , Animales , Arterias/citología , Arterias/efectos de los fármacos , Atorvastatina , Ensayos de Migración de Macrófagos , Células Cultivadas , Dieta , Fluorescencia , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Lipoproteínas LDL/farmacología , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Placa Aterosclerótica/patología , TomografíaRESUMEN
BACKGROUND: Recruitment of macrophage precursors to the adventitia plays a key role in the pathogenesis of abdominal aortic aneurysms (AAAs), but molecular mechanisms remain undefined. The innate immune signaling molecule CD14 was reported to be upregulated in adventitial macrophages in a murine model of AAA and in monocytes cocultured with aortic adventitial fibroblasts (AoAf) in vitro, concurrent with increased interleukin-6 (IL-6) expression. We hypothesized that CD14 plays a crucial role in adventitial macrophage precursor recruitment early during AAA formation. METHODS AND RESULTS: CD14(-/-) mice were resistant to AAA formation induced by 2 different AAA induction models: aortic elastase infusion and systemic angiotensin II (AngII) infusion. CD14 gene deletion led to reduced aortic macrophage infiltration and diminished elastin degradation. Adventitial monocyte binding to AngII-infused aorta in vitro was dependent on CD14, and incubation of human acute monocytic leukemia cell line-1 (THP-1) monocytes with IL-6 or conditioned medium from perivascular adipose tissue (PVAT) upregulated CD14 expression. Conditioned medium from AoAf and PVAT induced CD14-dependent monocyte chemotaxis, which was potentiated by IL-6. CD14 expression in aorta and plasma CD14 levels were increased in AAA patients compared with controls. CONCLUSIONS: These findings link CD14 innate immune signaling via a novel IL-6 amplification loop to adventitial macrophage precursor recruitment in the pathogenesis of AAA.
Asunto(s)
Aneurisma de la Aorta Abdominal/inmunología , Movimiento Celular/inmunología , Interleucina-6/inmunología , Receptores de Lipopolisacáridos/inmunología , Macrófagos/inmunología , Células Precursoras de Monocitos y Macrófagos/inmunología , Adventicia/inmunología , Animales , Línea Celular Tumoral , Ensayos de Migración de Macrófagos , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Inmunidad Innata , Macrófagos Peritoneales , Ratones , Ratones Transgénicos , Transducción de Señal/inmunologíaRESUMEN
Macrophage migration and infiltration is an important first step in many pathophysiological processes, in particular inflammatory diseases. Redox modulation of the migratory signalling processes has been reported in endothelial cells, vascular smooth muscle cells and fibroblasts. However the redox modulation of the migratory process in macrophages and in particular that from the NADPH oxidase-2 (Nox2) dependent ROS has not been established. To investigate the potential role of Nox2 in the migratory response of macrophages, bone marrow derived macrophages were obtained from WT and NOX2 knockout mice (Nox2KO) and subjected to CSF-1 stimulation. We report here that loss of Nox2 expression in BMM resulted in a significant reduction in the CSF-1 induced spreading response suggesting that Nox2 can modulate cytoskeletal events. Moreover, Nox2KO BMMs were deficient in cellular displacement in the presence of CSF-1. More significantly, when challenged with a gradient of CSF-1, Nox2KO BMMs showed a complete loss of chemotaxis accompanied by a reduction in cell migration speed and directional migration persistence. These results point to a specific role for Nox2KO downstream of CSF-1 during the BMM migratory response. Indeed, we have further found that Nox2KO BMMs display a significant reduction in the levels of ERK1/2 phosphorylation following stimulation with CSF-1.Thus Nox2 is important in BMM cellular motion to CSF-1 stimulation and necessary for their directed migration towards a CSF-1 gradient, highlighting Nox2 dependent signalling as a potential anti-inflammatory target.
Asunto(s)
Quimiotaxis/fisiología , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/fisiología , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/metabolismo , Animales , Ensayos de Migración de Macrófagos , Immunoblotting , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Microscopía Fluorescente , NADPH Oxidasa 2 , NADPH Oxidasas/genética , Imagen de Lapso de TiempoRESUMEN
BACKGROUND: Advanced glycation end products (AGEs), inflammatory-associated macrophage migration and accumulation are crucial for initiation and progression of diabetic vascular complication. Enzymatic activity of heparanase (HPA) is implicated strongly in dissemination of metastatic tumor cells and cells of the immune system. In addition, HPA enhances the phosphorylation of selected signaling molecules including AKT pathway independent of enzymatic activity. However, virtually nothing is presently known the role of HPA during macrophage migration exposed to AGEs involving signal pathway. METHODS: These studies were carried out in Ana-1 macrophages. Macrophage viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. HPA and AKT protein expression in macrophages are analysed by Western blotting and HPA mRNA expression by real time quantitative RT-PCR. Release of HPA was determined by ELISA. Macrophage migration was assessed by Transwell assays. RESULTS: HPA protein and mRNA were found to be increased significantly in AGEs-treated macrophages. Pretreatment with anti-HPA antibody which recognizes the nonenzymatic terminal of HPA prevented AGEs-induced AKT phosphorylation and macrophage migration. LY294002 (PI3k/AKT inhibitor) inhibited AGEs-induced macrophage migration. Furthermore, pretreatment with anti-receptor for advanced glycation end products (RAGE) antibody attenuated AGEs-induced HPA expression, AKT phosphorylation and macrophage migration. CONCLUSIONS: These data indicate that AGEs-induced macrophage migration is dependent on HPA involving RAGE-HPA-PI3K/AKT pathway. The nonenzymatic activity of HPA may play a key role in AGEs-induced macrophage migration associated with inflammation in diabetic vascular complication.
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Angiopatías Diabéticas/inmunología , Glucuronidasa/inmunología , Productos Finales de Glicación Avanzada/inmunología , Macrófagos/inmunología , Fosfatidilinositol 3-Quinasas/inmunología , Proteínas Proto-Oncogénicas c-akt/inmunología , Receptores Inmunológicos/inmunología , Animales , Western Blotting , Línea Celular , Ensayos de Migración de Macrófagos , Movimiento Celular/inmunología , Supervivencia Celular , Angiopatías Diabéticas/metabolismo , Glucuronidasa/genética , Glucuronidasa/metabolismo , Inflamación , Macrófagos/metabolismo , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunologíaRESUMEN
BACKGROUND: A previous study indicated that intrathecal administration of morphine reduces experimental inflammatory edema in rats by activating the nitric oxide/cyclic guanosine monophosphate pathway. This evidence supports the hypothesis that potassium channel opening may play an important role in mediating morphine's effect under such conditions. METHODS: Male Wistar rats received intrathecal injections of drugs (20 µL) 30 minutes before paw stimulation with carrageenan (150 µg). Edema was measured as paw volume increase (in milliliters), and plasma leakage was measured by Evans blue dye leakage. Neutrophil migration was evaluated indirectly by myeloperoxidase assay. The inflammatory infiltration and vascular congestion were observed by histologic examination. RESULTS: Morphine (37 nmol) inhibited inflammatory edema, plasma leakage, and vascular congestion but had no effect on myeloperoxidase activity or neutrophil content compared with phosphate-buffered saline. Coinjection with 4-aminopyridine (10 nmol), glibenclamide (5 nmol), and dequalinium (10 pmol) reversed, but nicorandil (0.03 nmol) enhanced the effect of morphine. CONCLUSIONS: These results support the hypothesis that the peripheral antiedematogenic effect produced by intrathecal morphine is mediated by potassium channel activation. Furthermore, this opioid effect does not involve the inhibition of acute neutrophil migration but does involve a reduction in capillary recruitment.
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Analgésicos Opioides/farmacología , Edema/tratamiento farmacológico , Morfina/farmacología , Canales de Potasio/fisiología , Analgésicos Opioides/administración & dosificación , Animales , Vasos Sanguíneos/patología , Carragenina , Ensayos de Migración de Macrófagos , Colorantes , Edema/inducido químicamente , Edema/patología , Azul de Evans , Pie/patología , Inflamación/tratamiento farmacológico , Inflamación/patología , Inyecciones Espinales , Masculino , Morfina/administración & dosificación , Nicorandil/farmacología , Nicorandil/uso terapéutico , Peroxidasa/análisis , Peroxidasa/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/agonistas , Ratas , Ratas WistarRESUMEN
Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays an important role in host defense against a variety of microorganisms including protozoan parasites. Interestingly, some microbial pathogens also express a MIF-like protein, although its role in disease pathogenesis is not well understood. The aim of this study was to compare an Eimeria-encoded MIF (E.MIF) protein with chicken MIF (C.MIF) on the basis of their structural, immunological, and biological properties. E.MIF and C.MIF proteins, each with a glutathione S-transferase epitope tag, were expressed in Escherichia coli or COS-7 cells and purified by glutathione affinity chromatography. Rabbit antisera against the purified proteins demonstrated their mutual immunological cross-reactivity on Western blots, and immunolocalized intracellular native E.MIF to the Eimeria schizont, merozoite, and oocyst life cycle stages. HD11 chicken macrophages treated in vitro with C.MIF recombinant protein expressed increased levels of transcripts encoding interleukin-6 (IL-6), IL-17, and tumor necrosis factor superfamily member 15 (TNFSF15), but decreased levels of IL-8 transcripts, compared with cells treated with the PBS control; similar treatment with E.MIF only down-regulated IL-8 transcripts. Unlike recombinant E.MIF, C.MIF exhibited in vitro chemotactic activity for HD11 cells. Conversely, E.MIF, but not C.MIF, enhanced protection against experimental Eimeria infection, compared with the PBS control. These studies provide evidence for overlapping structural and antigenic properties, but distinct immunoregulatory roles, of E.MIF and C.MIF.
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Pollos/inmunología , Coccidiosis/veterinaria , Eimeria/inmunología , Factores Inhibidores de la Migración de Macrófagos/inmunología , Secuencia de Aminoácidos , Animales , Ensayos de Migración de Macrófagos , Quimiotaxis , Pollos/parasitología , Coccidiosis/inmunología , Reacciones Cruzadas , Citocinas/inmunología , Macrófagos/inmunología , Datos de Secuencia Molecular , Recuento de Huevos de Parásitos , Proteínas Recombinantes/inmunología , Aumento de PesoRESUMEN
Ewing sarcoma-primitive neuroectodermal tumor (EWS) is associated with the most unfavorable prognosis of all primary musculoskeletal tumors. The objective of the present study was to investigate whether tumor-associated macrophages (TAMs) affect the development of EWS. TAMs were isolated from mouse xenografts using CD11b magnetic beads and examined for their cytokine expression and osteoclastic differentiation. To evaluate the role of TAMs in xenograft formation, liposome-encapsulated clodronate was used to deplete TAMs in mice. Macrophage infiltration and tumor microvascular density were histologically evaluated in 41 patients with EWS, and association with prognosis was examined using Kaplan-Meier survival analysis. In mouse EWS xenografts, TAMs expressed higher concentrations of cytokines including interleukin-6, keratinocyte-derived chemokine, and monocyte chemotactic protein-1. TAMs were more capable than normal monocytes of differentiating into tartrate-resistant acid phosphatase-positive giant cells. Depleting macrophages using liposome-encapsulated clodronate significantly inhibited development of EWS xenografts. In human EWS samples, higher levels of CD68-positive macrophages were associated with poorer overall survival. In addition, enhanced vascularity, increase in the amount of C-reactive protein, and higher white blood cell counts were also associated with poor prognosis and macrophage infiltration. TAMs seem to enhance the progression of EWS by stimulating both angiogenesis and osteoclastogenesis. Further investigation of the behavior of TAMs may lead to development of biologically targeted therapies for EWS.
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Neoplasias Óseas/patología , Macrófagos/patología , Sarcoma de Ewing/patología , Adolescente , Adulto , Anciano , Animales , Neoplasias Óseas/mortalidad , Línea Celular Tumoral , Ensayos de Migración de Macrófagos , Transformación Celular Neoplásica/patología , Niño , Citocinas/metabolismo , Femenino , Humanos , Estimación de Kaplan-Meier , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Trasplante de Neoplasias , Pronóstico , Sarcoma de Ewing/mortalidad , Trasplante Heterólogo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adulto JovenRESUMEN
We previously reported an increase in matrix metalloproteinase-9 (MMP-9) levels in the olfactory bulb immediately after nerve transection; however, its role remains unknown. In this study, we determined the source of MMP-9 by monitoring the infiltration of inflammatory leukocytes in the olfactory bulb after nerve transection. We used myeloperoxidase to identify neutrophils and CD68 to identify macrophages at days 1, 7, and 10. MMP-9 colocalized with neutrophils at all three time points but was not contained in macrophages. This is the first study to demonstrate that MMP-9 is associated with early inflammatory response after olfactory injury, and provides insight into mechanisms underlying olfactory injury and recovery processes.
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Inflamación/enzimología , Inflamación/patología , Metaloproteinasa 9 de la Matriz/metabolismo , Bulbo Olfatorio/enzimología , Bulbo Olfatorio/lesiones , Traumatismos del Nervio Olfatorio , Nervio Olfatorio/enzimología , Animales , Biomarcadores , Ensayos de Migración de Macrófagos , Inmunohistoquímica , Macrófagos/metabolismo , Macrófagos/fisiología , Ratones , Microscopía Confocal , Neutrófilos/enzimología , Neutrófilos/fisiología , Bulbo Olfatorio/patología , Nervio Olfatorio/patología , Peroxidasa/metabolismoRESUMEN
OBJECTIVE: We examined whether the modulatory effect of pregnancy on multiple sclerosis (MS) is associated with changes in the apoptotic molecules in sera. SUBJECTS AND METHODS: The serum levels of tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL), sFas, Fas ligand (sFasL) and macrophage migration inhibitory factor were analyzed from 19 MS patients and 14 controls during late pregnancy and post-partum. The obtained results were related to disease activity and the progression of MS. RESULTS: Disease activity decreased during pregnancy. The levels of sTRAIL and sFasL increased from late pregnancy to post-partum situation in both MS patients and controls, but in MS patients the changes in the levels of sTRAIL from late pregnancy to post-partum were smaller than in controls. CONCLUSIONS: Post-partum upregulation of TRAIL and FasL seems to be caused by physiologic reactivation of the mother's immune system after pregnancy. An increased risk of relapses in MS post-partum may be associated with changes in the immunomodulatory potential of these apoptotic molecules.