RESUMEN
PURPOSE: The complement system is considered to play an important role in the progression of myopia, whereas the influence of complement activation on the human scleral fibroblasts (HSFs) remains unknown. Hence, the effect of complement 3a (C3a) on HSFs was investigated in this study. METHODS: HSFs were cultured with exogenous C3a at 0.1 µM for various periods following different measurement protocols, and cells without C3a treatment served as negative control (NC). Cell viability was investigated using the MTS assay after 3 days of C3a treatment. Cell proliferation was evaluated by the 5-Ethynyl-20-Deoxyuridine (EdU) assay following C3a stimulation for 24 hours. Apoptosis was assessed by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining following C3a stimulation for 48 hours and the stained cells were analysed using flow cytometry. The levels of type I collagen and matrix metalloproteinase-2 (MMP-2) were analysed using ELISA following C3a stimulation for 36 and 60 hours. The level of CD59 were analysed using western blot following C3a stimulation for 60 hours. RESULTS: The MTS assay revealed that cell viability was attenuated by 13% and 8% after C3a for 2 and 3 days, respectively (P < 0.05). The EdU assay demonstrated a 9% decrease in proliferation rate for the C3a-treated cells after 24 hours (P < 0.05). The apoptosis analysis revealed an increased percentage of cells in early apoptosis (P = 0.02) and total apoptosis (P = 0.02) in the C3a-treated group. Compared with NC group, the level of MMP-2 was increased by 17.6% (P = 0.002), whereas the levels of type I collagen and CD59 were respectively decreased by 12.5% (P = 0.024) and 21.6% (P = 0.044) with C3a treatment for 60 hours. CONCLUSIONS: These results indicated that C3a-induced complement activation is potentially involved in inducing myopic-associated scleral extracellular matrix remodelling via mediating the proliferation and function of HSFs.
Asunto(s)
Complemento C3a , Metaloproteinasa 2 de la Matriz , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/farmacología , Complemento C3a/metabolismo , Complemento C3a/farmacología , Colágeno Tipo I/metabolismo , Colágeno Tipo I/farmacología , Fibroblastos , ApoptosisRESUMEN
Pancreatic cancer is one of the deadliest cancers, against which current immunotherapy strategies are not effective. Herein, we analyzed the immune cell composition of the tumor microenvironment of pancreatic cancer samples in The Cancer Genome Atlas and found that the presence of intratumoral NK cells correlates with survival. Subsequent analysis also indicated that NK cell exclusion from the microenvironment is found in a high percentage of clinical pancreatic cancers and in preclinical models of pancreatic cancer. Mechanistically, NK cell exclusion is regulated in part by complement C3a and its receptor signaling. Inhibition of the C3a receptor enhances NK cell infiltration in syngeneic mouse models of pancreatic cancer resulting in tumor growth delay. However, tumor growth inhibition mediated by NK cells is not sufficient alone for complete tumor regression, but is potentiated when combined with radiation therapy. Our findings indicate that although C3a inhibition is a promising approach to enhance NK cell-based immunotherapy against pancreatic cancer, its combination with radiation therapy hold greater therapeutic benefit.
Asunto(s)
Complemento C3a , Neoplasias Pancreáticas , Animales , Ratones , Complemento C3a/farmacología , Neoplasias Pancreáticas/radioterapia , Células Asesinas Naturales , Inmunoterapia/métodos , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
Activation of microglia and astrocytes following germinal matrix hemorrhage and intraventricular hemorrhage (GMH-IVH) plays a detrimental role in posthemorrhagic hydrocephalus (PHH). It is still unclear whether or how an interaction occurs between microglia and astrocytes in PHH. Here, we investigated the role of the C3/C3aR pathway in microglia and astrocyte interactions and whether C3/C3aR-targeted inhibition could alleviate PHH following GMH-IVH. A total of 152 Sprague-Dawley rats at postnatal day seven (P7) were enrolled in the study, and collagenase VII was used to induce GMH-IVH. Minocycline (45 mg/kg) was administered to inhibit microglial activation. Complement C3a peptide and C3aR antagonist (SB 290157, 10 mg/kg) were used to regulate the C3/C3aR pathway. As a result, the data demonstrated that periventricular C3aR+/Iba-1+ microglia and C3+/GFAP+ astrocytes were significantly increased in GMH-IVH pups at 28 days after surgery. Intranasal C3a peptide upregulated C3aR expression in microglia. Inhibition of microglia by minocycline decreased both C3+/GFAP+ astrocytes and the colocalization volume of Iba-1 and GFAP. In addition, intraperitoneally injected C3aRA alleviated the periventricular colocalization volume of microglia and astrocytes. Compared with vehicle-treated pups, the protein level of IL-1ß, IL-6 and TNF-α in cerebral spinal fluid and brain tissue at 28 days following GMH-IVH were reduced in C3aRA-treated pups. Moreover, hydrocephalus was alleviated, and long-term cognitive ability were improved in the C3aRA-treated group. Our data presented simultaneous periventricular astrogliosis and microgliosis of pups following GMH-IVH and proved their potential interaction through the C3/C3aR pathway, indicating C3aRA as a potential pharmacological treatment of PHH in neonates.
Asunto(s)
Arginina/análogos & derivados , Astrocitos/efectos de los fármacos , Compuestos de Bencidrilo/farmacología , Hemorragia Cerebral/tratamiento farmacológico , Hemorragia Cerebral/metabolismo , Complemento C3a/farmacología , Hidrocefalia/tratamiento farmacológico , Microglía/efectos de los fármacos , Receptores Acoplados a Proteínas G/efectos de los fármacos , Animales , Animales Recién Nacidos , Arginina/administración & dosificación , Arginina/farmacología , Compuestos de Bencidrilo/administración & dosificación , Hemorragia Cerebral/complicaciones , Hemorragia Cerebral Intraventricular/complicaciones , Hemorragia Cerebral Intraventricular/tratamiento farmacológico , Hemorragia Cerebral Intraventricular/metabolismo , Complemento C3a/administración & dosificación , Modelos Animales de Enfermedad , Hidrocefalia/etiología , Hidrocefalia/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/antagonistas & inhibidoresRESUMEN
Both, aberrant mast cell responses and complement activation contribute to allergic diseases. Since mast cells are highly responsive to C3a and C5a, while Interleukin-33 (IL-33) is a potent mast cell activator, we hypothesized that IL-33 critically regulates mast cell responses to complement anaphylatoxins. We sought to understand whether C3a and C5a differentially activate primary human mast cells, and probe whether IL-33 regulates C3a/C5a-induced mast cell activities. Primary human mast cells were generated from peripheral blood precursors or isolated from healthy human lung tissue, and mast cell complement receptor expression, degranulation, mediator release, phosphorylation patterns, and calcium flux were assessed. Human mast cells of distinct origin express constitutively higher levels of C3aR1 than C5aR1, and both receptors are downregulated by anaphylatoxins. While C3a is a potent mast cell degranulation inducer, C5a is a weaker secretagogue with more delayed effects. Importantly, IL-33 potently enhances the human mast cell reactivity to C3a and C5a (degranulation, cytokine and chemokine release), independent of changes in C3a or C5a receptor expression or the level of Ca2+ influx. Instead, this reflects differential dynamics of intracellular signaling such as ERK1/2 phosphorylation. Since primary human mast cells respond differentially to anaphylatoxin stimulation, and that IL-33 is a key regulator of mast cell responses to complement anaphylatoxins, this is likely to aggravate Th2 immune responses. This newly identified cross-regulation may be important for controlling exacerbated complement- and mast cell-dependent Th2 responses and thus provides an additional rationale for targeting anti-IL33 therapeutically in allergic diseases.
Asunto(s)
Complemento C3a/farmacología , Complemento C5a/farmacología , Interleucina-33/farmacología , Mastocitos/efectos de los fármacos , Antígenos CD/biosíntesis , Antígenos CD/genética , Células Sanguíneas , Señalización del Calcio/efectos de los fármacos , Degranulación de la Célula/efectos de los fármacos , Células Cultivadas , Complemento C3a/inmunología , Complemento C5a/inmunología , Sinergismo Farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interferón gamma/farmacología , Interleucina-4/farmacología , Ligandos , Pulmón/citología , Mastocitos/inmunología , Mastocitos/metabolismo , Proteínas de la Membrana/metabolismo , Especificidad de Órganos , Fosforilación , Procesamiento Proteico-Postraduccional , Receptores de Complemento/biosíntesis , Receptores de Complemento/genéticaRESUMEN
BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) enhance islet function both in vitro and in vivo, at least in part by secreting ligands that activate islet G-protein coupled receptors (GPCRs). We assessed whether pre-treatment with a defined "cocktail" of MSC-secreted GPCR ligands enhances islet functional survival in vitro and improves the outcomes of islet transplantation in an experimental model of diabetes. METHODS: Isolated islets were cultured for 48 h with ANXA1, SDF-1 or C3a, alone or in combination. Glucose-stimulated insulin secretion (GSIS) and cytokine-induced apoptosis were measured immediately after the 48 h culture period and at 24 h or 72 h following removal of the ligands from the culture media. Islets were syngeneically transplanted underneath the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice and blood glucose levels monitored for 28 days. RESULTS: Pre-culturing islets with a cocktail of ANXA1/SDF-1/C3a potentiated GSIS and protected islet cells from cytokine-induced apoptosis in vitro. These effects were maintained for up to 72 h after the removal of the factors from the culture medium, suggesting a sustained protection of islet graft functional survival during the immediate post-transplantation period. Islets pre-treated with the cocktail of MSC secretory factors were more effective in reducing blood glucose in diabetic mice, consistent with their improved functional survival in vivo. DISCUSSION: Pre-culturing islets with a cocktail of MSC secretory products offers a well-defined, cell-free approach to improve clinical islet transplantation outcomes while avoiding many of the safety, regulatory and logistical hurdles of incorporating MSCs into transplantation protocols.
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Quimiocina CXCL12/farmacología , Complemento C3a/farmacología , Trasplante de Islotes Pancreáticos/métodos , Células Madre Mesenquimatosas/metabolismo , Animales , Anexina A1/genética , Anexina A1/metabolismo , Anexina A1/farmacología , Apoptosis/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Complemento C3a/genética , Complemento C3a/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/terapia , Glucosa/metabolismo , Secreción de Insulina , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Multiple studies have identified that complement becomes activated during inflammation of the intestines yet it is unclear what roles the split complement molecules play. The epithelium, in particular, may be impacted and accordingly, we first discovered that colonic cell lines indeed possess the C5aR. Here we examined whether these cells also possess the C3aR. We determined that T84, HT-29 and Caco2 all possess C3aR mRNA and protein; T84 and HT29 were used to further explore the consequence of C3a binding the C3aR. C3a led to increased mRNA for CXCL2, CXCL8 and CXCL11. Polarized T84 monolayers responded to apically applied C3a with increased CXCL8 mRNA more rapidly than if the C3a was applied basolaterally. Polarized monolayers also increased permeability when treated with C3a. ERK1/2 was activated by C3a and the increase in CXCL8 mRNA was ERK-dependent in both T84 and HT-29. C3a resulted in activation of Gαi, determined by the ERK1/2 signal showing sensitivity to pertussis toxin. The transmembrane signal was further mapped to include Ras and c-Raf. Finally, we show that the C3aR is expressed by primary cells in mouse enteroids. We conclude that complement activation will contribute to the epithelial response during inflammation through C3a binding to the C3aR including by priming the cells to upregulate mRNA for selected chemokines.
Asunto(s)
Quimiocinas/inmunología , Complemento C3a/farmacología , Células Epiteliales/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/inmunología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Mediadores de Inflamación/inmunología , Animales , Células CACO-2 , Línea Celular Tumoral , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Colon/patología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Células HT29 , Humanos , Mediadores de Inflamación/metabolismo , Ratones Endogámicos C57BL , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/inmunología , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Candida albicans the most frequently isolated clinical fungal pathogen can cause local as well as systemic and life-threatening infections particularly in immune-compromised individuals. A better and more detailed understanding how C. albicans evades human immune attack is therefore needed for identifying fungal immune-evasive proteins and develop new therapies. Here, we identified Pra1, the pH-regulated C. albicans antigen as a hierarchical complement inhibitor that targets C3, the central human complement component. Pra1 cleaved C3 at a unique site and further inhibited effector function of the activation fragments. The newly formed C3a-like peptide lacked the C-terminal arginine residue needed for C3a-receptor binding and activation. Moreover, Pra1 also blocked C3a-like antifungal activity as shown in survival assays, and the C3b-like molecule formed by Pra1 was degraded by the host protease Factor I. Pra1 also bound to C3a and C3b generated by human convertases and blocked their effector functions, like C3a antifungal activity shown by fungal survival, blocked C3a binding to human C3a receptor-expressing HEK cells, activation of Fura2-AM loaded cells, intracellular Ca2+ signaling, IL-8 release, C3b deposition, as well as opsonophagocytosis and killing by human neutrophils. Thus, upon infection C. albicans uses Pra1 to destroy C3 and to disrupt host complement attack. In conclusion, candida Pra1 represents the first fungal C3-cleaving protease identified and functions as a fungal master regulator of innate immunity and as a central fungal immune-escape protein.
Asunto(s)
Candida albicans/enzimología , Complemento C3/antagonistas & inhibidores , Proteínas Fúngicas/fisiología , Secuencia de Aminoácidos , Unión Competitiva , Señalización del Calcio/efectos de los fármacos , Candida albicans/efectos de los fármacos , Candida albicans/inmunología , Línea Celular , Complemento C3/inmunología , Complemento C3/metabolismo , Complemento C3/farmacología , Complemento C3a/antagonistas & inhibidores , Complemento C3a/farmacología , Complemento C3b/antagonistas & inhibidores , Complemento C3b/farmacología , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/farmacología , Células HEK293 , Humanos , Interleucina-8/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Proteínas Opsoninas/inmunología , Fragmentos de Péptidos/metabolismo , Fagocitosis/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Proteolisis , Receptores de Complemento/antagonistas & inhibidores , Receptores de Complemento/metabolismo , Virulencia/inmunologíaRESUMEN
BACKGROUND: Antibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling. METHODS: Primary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003). RESULTS: Treatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement. CONCLUSIONS: Despite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Adhesión Celular/efectos de los fármacos , Inactivadores del Complemento/farmacología , Vía Clásica del Complemento/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Antígenos HLA-A/inmunología , Inmunosupresores/farmacología , Monocitos/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Complemento C3a/farmacología , Complemento C5a/farmacología , Relación Dosis-Respuesta a Droga , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Exocitosis/efectos de los fármacos , Antígenos HLA-A/metabolismo , Humanos , Antígeno de Macrófago-1/inmunología , Antígeno de Macrófago-1/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Selectina-P/inmunología , Selectina-P/metabolismoRESUMEN
Complement activation, specifically complement 3 (C3) activation and C3a generation, contributes to an imbalance between angiogenic stimulation by vascular endothelial growth factor (VEGF) and angiogenic inhibition by pigment epithelial derived factor (PEDF), leading to pathological angiogenesis. This study aimed to investigate the effects of C3a and small interfering RNA (siRNA) targeting C3 on the levels of VEGF and PEDF mRNAs in human retinal pigment epithelial (RPE) cells. ARPE-19 cells were cultured in the presence of exogenous C3a at 0.1 µM and 0.3 µM C3a for 24, 48, and 72 hours. 0.1 pmol/µL duplexes of siRNA targeting C3 were applied for C3a inhibition by transfecting ARPE-19 cells for 48 hours. RT-PCR was performed to examine the level of VEGF and PEDF mRNA. A random siRNA duplex was set for control siRNA. Results demonstrated that exogenous C3a significantly upregulated VEGF and downregulated PEDF mRNA levels in cultured ARPE-19 cells, and siRNA targeting C3 transfection reversed the above changes, significantly reducing VEGF and enhancing PEDF mRNAs level in ARPE-19 cells compared to the control. The present data provided evidence that reducing C3 activation can decreases VEGF and increase PEDF mRNA level in RPE and may serve as a potential therapy in pathological angiogenesis.
Asunto(s)
Complemento C3a/farmacología , Células Epiteliales/metabolismo , Proteínas del Ojo/genética , Factores de Crecimiento Nervioso/genética , Epitelio Pigmentado de la Retina/citología , Serpinas/genética , Factor A de Crecimiento Endotelial Vascular/genética , Línea Celular , Complemento C3a/genética , Complemento C3a/metabolismo , Células Epiteliales/efectos de los fármacos , Proteínas del Ojo/metabolismo , Humanos , Factores de Crecimiento Nervioso/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Serpinas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Astrocytes are the most numerous cells in the central nervous system with a range of homeostatic and regulatory functions. Under normal conditions as well as after ischemia, astrocytes promote neuronal survival. We have previously reported that the complement-derived peptide C3a stimulates neuronal differentiation of neural progenitor cells and protects the immature brain tissue against hypoxic-ischemic injury. Here, we studied the effects of C3a on the response of mouse cortical astrocytes to ischemia. We have found that chemical ischemia, induced by combined inhibition of oxidative phosphorylation and glycolysis, upregulates the expression of C3a receptor in cultured primary astrocytes. C3a treatment protected wild-type but not C3a receptor-deficient astrocytes from cell death induced by chemical ischemia or oxygen-glucose deprivation by reducing ERK signaling and caspase-3 activation. C3a attenuated ischemia-induced upregulation of glial fibrillary acidic protein; however, the protective effects of C3a were not dependent on the presence of the astrocyte intermediate filament system. Pre-treatment of astrocytes with C3a during recovery abrogated the ischemia-induced neuroprotective phenotype of astrocytes. Jointly, these results provide the first evidence that the complement peptide C3a modulates the response of astrocytes to ischemia and increases their ability to cope with ischemic stress.
Asunto(s)
Astrocitos/enzimología , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/patología , Complemento C3a/uso terapéutico , Estrés Fisiológico , Animales , Astrocitos/efectos de los fármacos , Astrocitos/patología , Caspasa 3/metabolismo , Supervivencia Celular/efectos de los fármacos , Corteza Cerebral/patología , Técnicas de Cocultivo , Complemento C3a/farmacología , Activación Enzimática/efectos de los fármacos , Filamentos Intermedios/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones Endogámicos C57BL , Factor de Crecimiento Nervioso/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Complemento/metabolismo , Estrés Fisiológico/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
PURPOSE: Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in people 50 years of age or older in developed countries. The homozygous CC genotype in the complement factor H (CFH) Y402H single nucleotide polymorphism (SNP; rs1061170) is widely recognized as a risk factor for the development of AMD. In this study, we examined vitreal levels of granulocyte macrophage colony-stimulating factor (GM-CSF), a hematopoietic cytokine, and macrophages in the choroid of postmortem human eyes genotyped for the CFH Y402H SNP. METHODS: Twenty-two pairs of postmortem, non-diseased, human donor eyes were obtained. The vitreous and retinal tissues of the left eyes were collected for GM-CSF level measurement and CFH Y402H genotyping, respectively. The right eyes were paraffin-embedded and sectioned for immunohistochemistry using a macrophage and microglia marker, CD68. Cell cultures of RPE cells were stimulated with complement C3a, C5a, 4-hydroxynonenal (HNE), or tumor necrosis factor alpha (TNF-α), and GM-CSF expression was measured with a suspension assay or quantitative PCR. RESULTS: Eyes genotyped with the CC or the CT risk variant of the CFH Y402H SNP showed significantly increased levels of GM-CSF in the vitreous compared to eyes with the protective TT variant (mean ± standard error of mean, 607.54±85.83 pg/ml or 656.32±15.20 pg/ml versus 286.69±81.96 pg/ml, p<0.05). The choroid of eye tissues genotyped with the CC variant showed higher levels of CD68 immunoreactivity than the tissues genotyped with the TT variant (p<0.05). The GM-CSF levels detected in the supernatant of RPE cells in culture treated with HNE or TNF-α were significantly higher compared to the non-treated control (145.88±5.06 pg/ml and 149.32±3.76 pg/ml versus 123.27±4.05 pg/ml, p<0.05). Furthermore, the gene expression of GM-CSF detected in the lysate of RPE cells stimulated with complement C3a or C5a showed significantly increased fold changes compared to the non-treated control (C3a: 2.38±0.31 fold, p<0.05; C5a: 2.84±0.54 fold, p<0.01). CONCLUSIONS: Our data showed a relationship between the CFH Y402H polymorphism and GM-CSF levels in the vitreous and accumulation of choroidal macrophages in the postmortem eye. These data suggest that the at-risk variant of the CFH gene may contribute to the dysregulation of proinflammatory cytokines locally in the eye.
Asunto(s)
Coroides/metabolismo , Factor H de Complemento/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Macrófagos/citología , Polimorfismo de Nucleótido Simple , Cuerpo Vítreo/metabolismo , Aldehídos/farmacología , Sustitución de Aminoácidos , Autopsia , Células Cultivadas , Coroides/química , Coroides/citología , Complemento C3a/farmacología , Complemento C5a/farmacología , Factor H de Complemento/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Expresión Génica , Genotipo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Cuerpo Vítreo/química , Cuerpo Vítreo/citologíaRESUMEN
Multipotent mesenchymal stromal cells (MSC) exert immune-modulatory effects and support tissue regeneration in various local trauma models. In case of a polytrauma, high amounts of danger-associated molecular patterns are released, leading to a systemic increase of inflammatory mediators. The influence of such a complex inflammatory microenvironment on human MSC is mainly unknown so far. Therefore, we investigated the effects of a defined serum-free polytrauma "cocktail" containing IL beta, IL6, IL8 and the anaphylatoxins C3a and C5a, in concentrations corresponding to those measured in the blood of polytrauma patients, on human MSC in vitro. The polytrauma cocktail induced directed migration of MSC with C3a representing its major soluble chemoattractive agent. Furthermore, the polytrauma cocktail and IL1beta upregulated the expression of MMP1 indicating a potential role of IL1beta to enhance MSC migration in the tissue context. COX2, PTGES and TSG6 were also found to be upregulated upon stimulation with the polytrauma cocktail or IL1beta, but not through other single factors of the polytrauma cocktail in pathophysiologically relevant concentrations. An RNA expression array of 84 inflammation-related genes revealed that both the polytrauma cocktail and IL1beta induced C3, CSF1, TLR3 and various chemokines without major qualitative or quantitative differences. These results indicate that IL1beta is a crucial mediator of the polytrauma cocktail in terms of immune-modulation and MMP1 expression. Thus, upon encountering the primary sterile, inflammatory milieu of a polytrauma, endogenous or systemically transfused MSC might be able to migrate to sites of injury, secrete TSG6 and PGE2 and to influence macrophage biology as observed in local trauma models.
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Complemento C3a/farmacología , Mediadores de Inflamación/farmacología , Interleucina-1beta/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Células de la Médula Ósea/citología , Moléculas de Adhesión Celular/análisis , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Ciclooxigenasa 2/metabolismo , Citocinas/genética , Citocinas/metabolismo , Dinoprostona/análisis , Ensayo de Inmunoadsorción Enzimática , Humanos , Oxidorreductasas Intramoleculares/metabolismo , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Prostaglandina-E Sintasas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Inferior tendon healing can lead to scarring and tendinopathy. The role of complement in tendon healing is still unclear. The aim of this study was to understand tenocytes response to mechanical injury and whether complement is regulated by injury. Tenocytes were injured using an optimized automated scratch assay model. Using a self-assembled plotter system, 50 parallel lines of injury were created in a 6 cm diameter tenocyte cell layer. Tenocytes mitotic activity and survival post injury was assessed using FDA/ethidiumbromide assay. Furthermore, this injury model was combined with stimulation of the tenocytes with the complement split fragment C3a. Gene expression of C3aR, C5aR (CD88), CD46, CD55, tumor necrosis factor (TNF)α, interleukin (IL)-1ß, matrix metalloproteinase (MMP)-1 was analyzed. Immunolabeling for C5aR and CD55 was performed. An enhanced mitotic activity and some dead cells were detected in the vicinity of the scratches. Gene expression of the C3aR was suppressed after 4 h but induced after 24 h post injury. C5aR was down-regulated at 24 h, CD46 and CD55 were induced at 24 h in response to injury and CD55 was also elevated at 4 h. MMP-1 was upregulated by injury but both proinflammatory cytokines remained mainly unaffected. Combination of injury with C3a stimulation led to an enhanced C3aR, CD55 and TNFα gene expression. According to the gene expression data, the protein expression of C5aR was reduced and that of CD55 induced. In summary, a specific response of complement regulation was found in mechanically injured tenocytes which may be involved in healing responses.
Asunto(s)
Proteínas del Sistema Complemento/inmunología , Traumatismos de los Tendones/inmunología , Tendones/inmunología , Cicatrización de Heridas/inmunología , Antígenos CD55/biosíntesis , Proliferación Celular , Supervivencia Celular/inmunología , Células Cultivadas , Complemento C3a/farmacología , Expresión Génica , Humanos , Interleucina-1beta/biosíntesis , Metaloproteinasa 1 de la Matriz/biosíntesis , Proteína Cofactora de Membrana/biosíntesis , ARN Mensajero/biosíntesis , Receptor de Anafilatoxina C5a/biosíntesis , Receptores de Complemento/biosíntesis , Tendones/citología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Acylation stimulating protein (ASP) is an adipokine derived from the immune complement system, which stimulates fat storage and is typically increased in obesity, type 2 diabetes, and cardiovascular disease. Using a diet-induced obesity (DIO) mouse model, the acute effects of ASP on energy metabolism and inflammatory processes in vivo were evaluated. We hypothesized that ASP would specifically exert proinflammatory effects. C57Bl/6 wild-type mice were put on a high-fat-high-sucrose diet for 12 weeks. Mice were then subjected to both glucose and insulin tolerance tests, each manipulation being preceded by recombinant ASP or vehicle (control) bolus injection. ASP supplementation increased whole-body glucose excursion, and this was accomplished with reduced concomitant insulin levels. However, ASP did not directly alter insulin sensitivity. ASP supplementation induced a proinflammatory phenotype, with higher levels of cytokines including IL-6 and TNF-α in plasma and in adipose tissue, liver, and skeletal muscle mRNA. Additionally, ASP increased M1 macrophage content of these tissues. ASP exerted a direct concentration-dependent role in the migration and M1 activation of cultured macrophages. Altogether, the in vivo and in vitro experiments demonstrate that ASP plays a role in both energy metabolism and inflammation, with paradoxical whole-body glucose-sensitizing yet proinflammatory effects.
Asunto(s)
Complemento C3a/farmacología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Grasas de la Dieta/efectos adversos , Humanos , Insulina/farmacología , Resistencia a la Insulina/fisiología , Interleucina-6/sangre , Macrófagos/citología , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/sangre , Obesidad/etiología , Obesidad/metabolismo , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Anaphylatoxins C3a and C5a are important modulators for dendritic cell activation and function in mice. In order to verify the significance of these observations in man, we have investigated the functional modulation of human monocytes derived DCs by C3a and C5a. Here we report that engagement of C3aR or C5aR on human monocytes derived DCs (moDCs) enhances the cell activation and their capacity for allostimulation. In addition, we show that intracellular production of cAMP is reduced and PI3K/AKT, ERK and NF-κB signalling is increased following stimulation with C3a or C5a, identifying intracellular signalling pathways that could convert cell surface C3aR and C5aR engagement into changes in moDC functions. Our data provide evidence that human DCs are equipped to react to C3a/C5a and undergo phenotypic change as well as functional modulation. Complement offers a potential route to modulate human DC function and regulate T cell mediated immunity.
Asunto(s)
Inmunidad Adaptativa/efectos de los fármacos , Complemento C3a/farmacología , Complemento C5a/farmacología , Células Dendríticas/efectos de los fármacos , Inflamación/inmunología , Transducción de Señal/efectos de los fármacos , Animales , Diferenciación Celular/inmunología , Complemento C3a/inmunología , Complemento C3a/metabolismo , Complemento C5a/inmunología , Complemento C5a/metabolismo , AMP Cíclico/biosíntesis , AMP Cíclico/inmunología , Citocinas/biosíntesis , Citocinas/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Masculino , Ratones , Monocitos/inmunología , Monocitos/metabolismo , FN-kappa B/inmunología , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor de Anafilatoxina C5a/inmunología , Receptor de Anafilatoxina C5a/metabolismo , Transducción de Señal/inmunologíaRESUMEN
There is a tight interaction of the bone and the immune system. However, little is known about the relevance of the complement system, an important part of innate immunity and a crucial trigger for inflammation. The aim of this study was, therefore, to investigate the presence and function of complement in bone cells including osteoblasts, mesenchymal stem cells (MSC), and osteoclasts. qRT-PCR and immunostaining revealed that the central complement receptors C3aR and C5aR, complement C3 and C5, and membrane-bound regulatory proteins CD46, CD55, and CD59 were expressed in human MSC, osteoblasts, and osteoclasts. Furthermore, osteoblasts and particularly osteoclasts were able to activate complement by cleaving C5 to its active form C5a as measured by ELISA. Both C3a and C5a alone were unable to trigger the release of inflammatory cytokines interleukin (IL)-6 and IL-8 from osteoblasts. However, co-stimulation with the pro-inflammatory cytokine IL-1ß significantly induced IL-6 and IL-8 expression as well as the expression of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) indicating that complement may modulate the inflammatory response of osteoblastic cells in a pro-inflammatory environment as well as osteoblast-osteoclast interaction. While C3a and C5a did not affect osteogenic differentiation, osteoclastogenesis was significantly induced even in the absence of RANKL and macrophage-colony stimulating factor (M-CSF) suggesting that complement could directly regulate osteoclast formation. It can therefore be proposed that complement may enhance the inflammatory response of osteoblasts and increase osteoclast formation, particularly in a pro-inflammatory environment, for example, during bone healing or in inflammatory bone disorders.
Asunto(s)
Complemento C3a/farmacología , Complemento C5a/farmacología , Interleucina-1beta/farmacología , Osteoblastos/fisiología , Osteoclastos/fisiología , Adulto , Antígenos CD/genética , Antígenos CD/metabolismo , Diferenciación Celular , Células Cultivadas , Complemento C3a/metabolismo , Complemento C3a/fisiología , Complemento C5a/metabolismo , Complemento C5a/fisiología , Expresión Génica , Humanos , Inflamación , Interleucina-1beta/fisiología , Masculino , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/fisiología , Microscopía Fluorescente , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogénesis , Osteoprotegerina/metabolismo , Proteolisis , Ligando RANK/metabolismo , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Tubulointerstitial renal fibrosis is the common end point of progressive kidney diseases, and tubular epithelial-myofibroblast transdifferentiation (TEMT) plays a key role in the progress of tubulointerstitial renal fibrosis. Anaphylatoxin C3a and C5a are identified as novel profibrotic factors in renal disease and as potential new therapeutic targets. The aim of this study was to investigate whether C3a, C5a can regulate TEMT by transforming growth factor-ß1 (TGF-ß)/connective tissue growth factor (CTGF) signaling pathway and the effects of C3a and C5a receptor antagonists (C3aRA and C5aRA) on C3a- and C5a-induced TEMT. METHODS: HK-2 cells were divided into C3a and C5a groups which were subdivided into four subgroups: control group, 10 ng/ml TGF-ß1 group, 50 nmol/L C3a group, 50 nmol/L C3a plus 1 µmol/L C3aRA group; control group, 10 ng/ml TGF-ß1 group, 50 nmol/L C5a group, 50 nmol/L C5a plus 2.5 µmol/L C5aRA group. TGF-ß1 receptor antagonist (TGF-ß1RA) 10 µg/ml was used to investigate the mechanism of C3a- and C5a-induced TEMT. Electron microscopy was used to observe the morphological changes. Immunocytochemistry staining, real-time PCR and Western blotting were used to detect the expressions of a smooth muscle actin (α-SMA), E-cadherin, Col-I, C3a receptor (C3aR), C5aR, CTGF and TGF-ß1. RESULTS: HK-2 cells cultured with C3a and C5a for 72 hours exhibited strong staining of α-SMA, lost the positive staining of E-cadherin, and showed a slightly spindle-like shape and loss of microvilli on the cell surface. The expressions of α-SMA, E-cadherin, Col-I, C3aR, C5aR, TGF-ß1 and CTGF in C3a- and C5a-treated groups were higher than normal control group (P < 0.05). C3aRA and C5aRA inhibited the expressions of α-SMA, Col-I, C3aR, C5aR, and up-regulated the expression of E-cadherin (P < 0.05). TGF-ß1 and CTGF mRNA expressions induced by C3a and C5a were partly blocked by TGF-ß1RA (P < 0.05). CONCLUSION: C3a and C5a can induce TEMT via the up-regulations of C3aR and C5aR mRNA and the activation of TGF-ß1/CTGF signaling pathway in vitro.
Asunto(s)
Transdiferenciación Celular/efectos de los fármacos , Complemento C3a/farmacología , Complemento C5a/farmacología , Miofibroblastos/citología , Western Blotting , Cadherinas/genética , Línea Celular , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/ultraestructura , Humanos , Inmunohistoquímica , Microscopía Electrónica de Rastreo , Miofibroblastos/efectos de los fármacos , Miofibroblastos/ultraestructura , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Activation of the complement system by injury increases inflammation by producing complement fragments C5a and C3a which are able to recruit and activate immune cells. Complement activation may contribute to pain after inflammation and injury. In this study, we examined whether C5a and C3a elicit nociception when injected into mouse hind paws in vivo, and whether C5a and C3a activate and/or sensitize mechanosensitive nociceptors when applied on peripheral terminals in vitro. We also examined the dorsal root ganglia (DRG) for C5a receptor (C5aR) mRNA and effects of C5a and C3a on intracellular Ca(2+) concentration ([Ca(2+)](i)) using Ca(2+) imaging. Heat hyperalgesia was elicited by intraplantar injection of C5a, and mechanical hyperalgesia by C5a and C3a. After exposure to either C5a or C3a, C-nociceptors were sensitized to heat as evidenced by an increased proportion of heat responsive fibers, lowered response threshold to heat and increased action potentials during and after heat stimulation. A-nociceptors were activated by complement. However, no change was observed in mechanical responses of A- and C-nociceptors after C5a and C3a application. The presence of C5aR mRNA was detected in DRG. C5a and C3a application elevated [Ca(2+)](i) and facilitated capsaicin-induced [Ca(2+)](i) responses in DRG neurons. The results suggest a potential role for complement fragments C5a and C3a in nociception by activating and sensitizing cutaneous nociceptors.
Asunto(s)
Complemento C3a/farmacología , Complemento C5a/farmacología , Factores Inmunológicos/farmacología , Nociceptores/efectos de los fármacos , Umbral del Dolor/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Análisis de Varianza , Animales , Calcineurina/metabolismo , Calcio/metabolismo , Capsaicina/farmacología , Complemento C5a/genética , Complemento C5a/metabolismo , Relación Dosis-Respuesta a Droga , Conducta Exploratoria/efectos de los fármacos , Ganglios Espinales/citología , Humanos , Hiperalgesia/clasificación , Hiperalgesia/fisiopatología , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Fibras Nerviosas/efectos de los fármacos , Fibras Nerviosas/fisiología , Nociceptores/fisiología , Dimensión del Dolor/métodos , Estimulación Física/métodos , ARN Mensajero/metabolismo , Tiempo de Reacción/efectos de los fármacos , Células Receptoras Sensoriales/efectos de los fármacos , Piel/inervaciónRESUMEN
Anaphylatoxin C3a is a third complement component (C3)-derived peptide, the multiple functions of which range from stimulation of inflammation to neuroprotection. In a previous study, we have shown that signaling through C3a receptor positively regulates in vivo neurogenesis in adult mouse brain. Here, we studied the direct effects of C3a on adult mouse whole brain-derived neural progenitor cells (NPCs) in vitro. Our results demonstrate that NPCs bind C3a in a specific and reversible manner and that C3a stimulates neuronal differentiation of NPCs. Furthermore, C3a stimulated the migration of NPCs induced by low concentrations of stromal cell-derived factor (SDF)-1alpha, whereas it inhibited NPC migration at high concentration of SDF-1alpha. In the same manner, C3a modulated SDF-1alpha-induced extracellular-signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation in these cells. In addition, C3a had inhibitory effect on SDF-1alpha-induced neuronal differentiation of NPCs. These data show that C3a modulates SDF-1alpha-induced differentiation and migration of these cells, conceivably through the regulation of ERK1/2 phosphorylation. Our results provide the first evidence that C3a regulates neurogenesis by directly affecting the fate and properties of NPCs.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Complemento C3a/farmacología , Factores Inmunológicos/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Células Madre/citología , Animales , Western Blotting , Células Cultivadas , Quimiocina CXCL12/farmacología , Humanos , Inmunoprecipitación , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , ARN Mensajero , Células Madre/metabolismoRESUMEN
C5L2 is a recently identified receptor for C5a/C5adesArg, C3a and C3adesArg (ASP). C5a/C5adesArg bind with high affinity, with no identified activation. By contrast, some studies demonstrate C3a/ASP binding/activation to C5L2; others do not. Our aim is to critically evaluate ASP/C3adesArg-C5L2 binding and bioactivity. Cell-associated fluorescent-ASP (Fl-ASP) binding to C5L2 increased from transiently transfectedAsunto(s)
Adipocitos/metabolismo
, Complemento C3a/metabolismo
, Ácidos Grasos/metabolismo
, Receptores de Quimiocina/metabolismo
, Células 3T3-L1
, Animales
, Células CHO
, Complemento C3a/química
, Complemento C3a/genética
, Complemento C3a/farmacología
, Cricetinae
, Cricetulus
, Ácidos Grasos/química
, Humanos
, Ratones
, Ratones Noqueados
, Unión Proteica/fisiología
, Receptor de Anafilatoxina C5a
, Receptores de Quimiocina/química
, Receptores de Quimiocina/genética
, Proteínas Recombinantes/química
, Proteínas Recombinantes/genética
, Proteínas Recombinantes/metabolismo
, Proteínas Recombinantes/farmacología
, Viperidae