RESUMO
Histamine is best known for its role in allergies, but it could also be involved in autoimmune diseases such as multiple sclerosis. However, studies using experimental autoimmune encephalomyelitis (EAE), the most widely used animal model for multiple sclerosis, have reported conflicting observations and suggest the implication of a nonclassical source of histamine. In this study, we demonstrate that neutrophils are the main producers of histamine in the spinal cord of EAE mice. To assess the role of histamine by taking into account its different cellular sources, we used CRISPR-Cas9 to generate conditional knockout mice for the histamine-synthesizing enzyme histidine decarboxylase. We found that ubiquitous and cell-specific deletions do not affect the course of EAE. However, neutrophil-specific deletion attenuates hypothermia caused by IgE-mediated anaphylaxis, whereas neuron-specific deletion reduces circadian activity. In summary, this study refutes the role of histamine in EAE, unveils a role for neutrophil-derived histamine in IgE-mediated anaphylaxis, and establishes a new mouse model to re-explore the inflammatory and neurologic roles of histamine.
Assuntos
Anafilaxia/imunologia , Ritmo Circadiano/imunologia , Encefalomielite Autoimune Experimental/imunologia , Histamina/imunologia , Histidina Descarboxilase/imunologia , Anafilaxia/genética , Anafilaxia/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Histamina/metabolismo , Histidina Descarboxilase/genética , Histidina Descarboxilase/metabolismo , Humanos , Estimativa de Kaplan-Meier , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Neutrófilos/citologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Medula Espinal/imunologia , Medula Espinal/metabolismoRESUMO
OBJECTIVE: Nucleotides are danger signals that activate inflammatory responses via binding P2 receptors. The nucleoside triphosphate diphosphohydrolase-8 (NTPDase8) is an ectonucleotidase that hydrolyses P2 receptor ligands. We investigated the role of NTPDase8 in intestinal inflammation. DESIGN: We generated NTPDase8-deficient (Entpd8-/-) mice to define the role of NTPDase8 in the dextran sodium sulfate (DSS) colitis model. To assess inflammation, colons were collected and analysed by histopathology, reverse transcriptase-quantitative real-time PCR (RT-qPCR) and immunohistochemistry. P2 receptor expression was analysed by RT-qPCR on primary intestinal epithelium and NTPDase8 activity by histochemistry. The role of intestinal P2Y6 receptors was assessed by bone marrow transplantation experiments and with a P2Y6 receptor antagonist. RESULTS: NTPDase8 is the dominant enzyme responsible for the hydrolysis of nucleotides in the lumen of the colon. Compared with wild-type (WT) control mice, the colon of Entpd8-/- mice treated with DSS displayed significantly more histological damage, immune cell infiltration, apoptosis and increased expression of several proinflammatory cytokines. P2Y6 was the dominant P2Y receptor expressed at the mRNA level by the colonic epithelia. Irradiated P2ry6-/- mice transplanted with WT bone marrow were fully protected from DSS-induced intestinal inflammation. In agreement, the daily intrarectal injection of a P2Y6 antagonist protected mice from DSS-induced intestinal inflammation in a dose-dependent manner. Finally, human intestinal epithelial cells express NTPDase8 and P2Y6 similarly as in mice. CONCLUSION: NTPDase8 protects the intestine from inflammation most probably by limiting the activation of P2Y6 receptors in colonic epithelial cells. This may provide a novel therapeutic strategy for the treatment of inflammatory bowel disease.
Assuntos
Adenosina Trifosfatases/metabolismo , Colite/metabolismo , Isotiocianatos/farmacologia , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Tioureia/análogos & derivados , Adenosina Trifosfatases/genética , Animais , Apoptose , Transplante de Medula Óssea , Colo/metabolismo , Citocinas/metabolismo , Sulfato de Dextrana/farmacologia , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Humanos , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tioureia/farmacologiaRESUMO
Multiple sclerosis is a chronic inflammatory, demyelinating, and neurodegenerative disease affecting the brain, spinal cord and optic nerves. Neuronal damage is triggered by various harmful factors that engage diverse signalling cascades in neurons; thus, therapeutic approaches to protect neurons will need to focus on agents that can target multiple biological processes. We have therefore focused our attention on microRNAs: small non-coding RNAs that primarily function as post-transcriptional regulators that target messenger RNAs and repress their translation into proteins. A single microRNA can target many functionally related messenger RNAs making microRNAs powerful epigenetic regulators. Dysregulation of microRNAs has been described in many neurodegenerative diseases including multiple sclerosis. Here, we report that two microRNAs, miR-223-3p and miR-27a-3p, are upregulated in neurons in the experimental autoimmune encephalomyelitis mouse model of CNS inflammation and in grey matter-containing multiple sclerosis lesions. Prior work has shown peripheral blood mononuclear cell conditioned media causes sublethal degeneration of neurons in culture. We find overexpression of miR-27a-3p or miR-223-3p protects dissociated cortical neurons from condition media mediated degeneration. Introduction of miR-223-3p in vivo in mouse retinal ganglion cells protects their axons from degeneration in experimental autoimmune encephalomyelitis. In silico analysis revealed that messenger RNAs involved in glutamate receptor signalling are enriched as miR-27a-3p and miR-223-3p targets. We observe that antagonism of NMDA and AMPA type glutamate receptors protects neurons from condition media dependent degeneration. Our results suggest that miR-223-3p and miR-27a-3p are upregulated in response to inflammation to mediate a compensatory neuroprotective gene expression program that desensitizes neurons to glutamate by targeting messenger RNAs involved in glutamate receptor signalling.
Assuntos
Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , MicroRNAs/genética , Neurônios/patologia , Animais , Axônios/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Camundongos , MicroRNAs/metabolismo , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/genética , Medula Espinal/patologiaRESUMO
Microbial agents can aggravate inflammatory diseases, such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). An example is pertussis toxin (PTX), a bacterial virulence factor commonly used as an adjuvant to promote EAE, but whose mechanism of action is unclear. We have reported that PTX triggers an IL-6-mediated signaling cascade that increases the number of leukocytes that patrol the vasculature by crawling on its luminal surface. In the present study, we examined this response in mice lacking either TLR4 or inflammasome components and using enzymatically active and inactive forms of PTX. Our results indicate that PTX, through its ADP-ribosyltransferase activity, induces two series of events upstream of IL-6: 1) the activation of TLR4 signaling in myeloid cells, leading to pro-IL-1ß synthesis; and 2) the formation of a pyrin-dependent inflammasome that cleaves pro-IL-1ß into its active form. In turn, IL-1ß stimulates nearby stromal cells to secrete IL-6, which is known to induce vascular changes required for leukocyte adhesion. Without pyrin, PTX does not induce neutrophil adhesion to cerebral capillaries and is less effective at inducing EAE in transgenic mice with encephalitogenic T lymphocytes. This study identifies the first microbial molecule that activates pyrin, a mechanism by which infections may influence MS and a potential therapeutic target for immune disorders.
Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Inflamassomos/imunologia , Interleucina-1beta/biossíntese , Neutrófilos/efeitos dos fármacos , Toxina Pertussis/farmacologia , Animais , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Interleucina-1beta/imunologia , Interleucina-6/metabolismo , Camundongos , Esclerose Múltipla/metabolismo , Células Mieloides , Linfócitos T/imunologiaRESUMO
BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is a model of inflammatory demyelinating diseases mediated by different types of leukocytes. How these cells communicate with each other to orchestrate autoimmune attacks is not fully understood, especially in the case of neutrophils, whose importance in EAE is newly established. The present study aimed to determine the expression pattern and role of different components of the IL-36 signaling pathway (IL-36α, IL-36ß, IL-36γ, IL-36R) in EAE. METHODS: EAE was induced by either active immunization with myelin peptide, passive transfer of myelin-reactive T cells or injection of pertussis toxin to transgenic 2D2 mice. The molecules of interest were analyzed using a combination of techniques, including quantitative real-time PCR (qRT-PCR), flow cytometry, Western blotting, in situ hybridization, and immunohistochemistry. Microglial cultures were treated with recombinant IL-36γ and analyzed using DNA microarrays. Different mouse strains were subjected to clinical evaluation and flow cytometric analysis in order to compare their susceptibility to EAE. RESULTS: Our observations indicate that both IL-36γ and IL-36R are strongly upregulated in nervous and hematopoietic tissues in different forms of EAE. IL-36γ is specifically expressed by neutrophils, while IL-36R is expressed by different immune cells, including microglia and other myeloid cells. In culture, microglia respond to recombinant IL-36γ by expressing molecules involved in neutrophil recruitment, such as Csf3, IL-1ß, and Cxcl2. However, mice deficient in either IL-36γ or IL-36R develop similar clinical and histopathological signs of EAE compared to wild-type controls. CONCLUSION: This study identifies IL-36γ as a neutrophil-related cytokine that can potentially activate microglia, but that is only correlative and not contributory in EAE.
Assuntos
Sistema Nervoso Central/patologia , Encefalomielite Autoimune Experimental/patologia , Interleucina-1/metabolismo , Microglia/metabolismo , Neutrófilos/metabolismo , Transferência Adotiva/efeitos adversos , Animais , Animais Recém-Nascidos , Antígenos CD/metabolismo , Encéfalo/citologia , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Glicoproteína Mielina-Oligodendrócito/toxicidade , Fragmentos de Peptídeos/toxicidade , Receptores de Interleucina-1/deficiência , Receptores de Interleucina-1/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Microglia surrounds the amyloid plaques that form in the brains of patients with Alzheimer's disease (AD), but their role is controversial. Under inflammatory conditions, these cells can express GPR84, an orphan receptor whose pathophysiological role is unknown. Here, we report that GPR84 is upregulated in microglia of APP/PS1 transgenic mice, a model of AD. Without GPR84, these mice display both accelerated cognitive decline and a reduced number of microglia, especially in areas surrounding plaques. The lack of GPR84 affects neither plaque formation nor hippocampal neurogenesis, but promotes dendritic degeneration. Furthermore, GPR84 does not influence the clinical progression of other diseases in which its expression has been reported, i.e., experimental autoimmune encephalomyelitis (EAE) and endotoxic shock. We conclude that GPR84 plays a beneficial role in amyloid pathology by acting as a sensor for a yet unknown ligand that promotes microglia recruitment, a response affecting dendritic degeneration and required to prevent further cognitive decline.
Assuntos
Doença de Alzheimer/metabolismo , Transtornos Cognitivos/metabolismo , Dendritos/metabolismo , Gliose/metabolismo , Microglia/metabolismo , Degeneração Neural/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Transtornos Cognitivos/genética , Transtornos Cognitivos/patologia , Dendritos/patologia , Modelos Animais de Doenças , Gliose/genética , Gliose/patologia , Hipocampo/metabolismo , Hipocampo/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microglia/patologia , Degeneração Neural/genética , Degeneração Neural/patologia , Receptores Acoplados a Proteínas G/genética , Regulação para CimaRESUMO
G protein-coupled receptor 84 (GPR84) is a 7-transmembrane protein expressed on myeloid cells that can bind to medium-chain free fatty acids in vitro. Here, we report the discovery of a 2-bp frameshift deletion in the second exon of the Gpr84 gene in several classical mouse inbred strains. This deletion generates a premature stop codon predicted to result in a truncated protein lacking the transmembrane domains 4-7. We sequenced Gpr84 exon 2 from 58 strains representing different groups in the mouse family tree and found that 14 strains are homozygous for the deletion. Some of these strains are DBA/1J, DBA/2J, FVB/NJ, LG/J, MRL/MpJ, NOD/LtJ, and SJL/J. However, the deletion was not found in any of the wild-derived inbred strains analyzed. Haplotype analysis suggested that the deletion originates from a unique mutation event that occurred more than 100 years ago, preceding the development of the first inbred strain (DBA), from a Mus musculus domesticus source. As GPR84 ostensibly plays a role in the biology of myeloid cells, it could be relevant 1) to consider the existence of this Gpr84 nonsense mutation in several mouse strains when choosing a mouse model to study immune processes and 2) to consider reevaluating data obtained using such strains.
Assuntos
Alelos , Receptores Acoplados a Proteínas G/genética , Animais , Sequência de Bases , Códon sem Sentido/fisiologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos NOD , Dados de Sequência Molecular , Filogenia , Receptores Acoplados a Proteínas G/fisiologia , Deleção de Sequência , Especificidade da EspécieRESUMO
Oncostatin M (OSM) is a pleiotropic cytokine of the IL-6 family and displays both pro-inflammatory and anti-inflammatory activities. We studied the impact of OSM on the gene activation profile of human synovial cells, which play a central role in the progression of inflammatory responses in joints. In synovial cells stimulated with lipopolysaccharide and recombinant human granulocyte-macrophage colony-stimulating factor, recombinant human OSM and native OSM secreted by human granulocytes both reduced the gene expression and secretion of IL-1ß and CXCL8, but increased that of IL-6 and CCL2. This impact on synovial cell activation was not obtained using IL-6 or leukaemia inhibitory factor. Signal transducer and activator of transcription-1 appeared to mediate the effects of OSM on stimulated human synovial fibroblasts. In the murine dorsal air pouch model of inflammation, OSM reduced the expression of the pro-inflammatory cytokines IL-1ß and TNF-α in lining tissues, and their presence in the cavity. These results as a whole suggest an anti-inflammatory role for OSM, guiding inflammatory processes towards resolution.
Assuntos
Fibroblastos/efeitos dos fármacos , Inflamação/tratamento farmacológico , Interleucina-1beta/metabolismo , Oncostatina M/metabolismo , Líquido Sinovial/efeitos dos fármacos , Animais , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Perfilação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Inflamação/patologia , Interleucina-1beta/genética , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Líquido Sinovial/citologia , Líquido Sinovial/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Granulocytes generally exert protective roles in the central nervous system (CNS), but recent studies suggest that they can be detrimental in experimental autoimmune encephalomyelitis (EAE), the most common model of multiple sclerosis. While the cytokines and adhesion molecules involved in granulocyte adhesion to the brain vasculature have started to be elucidated, the required chemokines remain undetermined. METHODS: CXCR2 ligand expression was examined in the CNS of mice suffering from EAE or exposed to bacterial toxins by quantitative RT-PCR and in situ hybridization. CXCL1 expression was analyzed in IL-6-treated endothelial cell cultures by quantitative RT-PCR and ELISA. Granulocytes were counted in the brain vasculature after treatment with a neutralizing anti-CXCL1 antibody using stereological techniques. RESULTS: CXCL1 was the most highly expressed ligand of the granulocyte receptor CXCR2 in the CNS of mice subjected to EAE or infused with lipopolysaccharide (LPS) or pertussis toxin (PTX), the latter being commonly used to induce EAE. IL-6 upregulated CXCL1 expression in brain endothelial cells by acting transcriptionally and mediated the stimulatory effect of PTX on CXCL1 expression. The anti-CXCL1 antibody reduced granulocyte adhesion to brain capillaries in the three conditions under study. Importantly, it attenuated EAE severity when given daily for a week during the effector phase of the disease. CONCLUSIONS: This study identifies CXCL1 not only as a key regulator of granulocyte recruitment into the CNS, but also as a new potential target for the treatment of neuroinflammatory diseases such as multiple sclerosis.
Assuntos
Encéfalo/patologia , Adesão Celular/efeitos dos fármacos , Quimiocina CXCL1/metabolismo , Encefalomielite Autoimune Experimental/patologia , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Granulócitos/fisiologia , Interleucina-6/farmacologia , Toxina Pertussis/farmacologia , Animais , Anticorpos , Anticorpos Neutralizantes/uso terapêutico , Antígenos CD/metabolismo , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Adesão Celular/fisiologia , Células Cultivadas , Quimiocina CXCL1/genética , Quimiocina CXCL1/imunologia , Quimiocina CXCL2/imunologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/etiologia , Células Endoteliais/fisiologia , Citometria de Fluxo , Proteína Glial Fibrilar Ácida/metabolismo , Granulócitos/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/deficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Receptores de Interleucina-8B/imunologiaRESUMO
Neutrophils are well-recognized phagocytes in the first line of host defense, and are also a major source of pro-inflammatory cytokines, chemokines and lipid mediators, thereby contributing to the onset and early orchestration of the inflammatory response. In contrast, recent studies indicate that neutrophils have tools to limit the magnitude and length of an inflammatory response, and may take part in engaging the resolution process. This article describes endogenous signals that may transform the phenotype of a neutrophil: from a pro-inflammatory cell to one that promotes resolution. Adenosine, an autacoid which can be found at high concentrations in inflammatory sites, inhibits several inflammatory functions of the neutrophil via engagement of the A2A receptor and reshapes the profile of lipid mediators and cytokines released, causing cells to terminate the release of pro-inflammatory signals while progressing toward resolution. These endogenous resolution pathways may represent a key target for better treatments of inflammatory diseases.
Assuntos
Neutrófilos/fisiologia , Adenosina/fisiologia , Quimiocinas/sangue , Quimiocinas/fisiologia , Citocinas/sangue , Citocinas/fisiologia , Humanos , Inflamação/sangue , Inflamação/fisiopatologia , Inflamação/prevenção & controle , Lipídeos/sangue , Lipídeos/fisiologia , Macrófagos/fisiologia , Neutrófilos/patologia , FagocitoseRESUMO
Neutrophils contribute to demyelinating autoimmune diseases, yet their phenotype and functions have been elusive to date. Here, we demonstrate that ICAM1 surface expression distinguishes extra- from intravascular neutrophils in the mouse CNS during experimental autoimmune encephalomyelitis (EAE). Transcriptomic analysis of these 2 subpopulations indicated that neutrophils, once extravasated, acquire macrophage-like properties, including the potential for immunostimulation and MHC class II-mediated antigen presentation. In corroboration, super-resolution (3D stimulated emission-depletion [STED]) microscopy revealed neutrophils forming synapses with T and B cells in situ. Further, neutrophils specifically express the aspartic retroviral-like protease ASPRV1, which increases in the CNS during EAE and severe cases of multiple sclerosis. Without ASPRV1, mice immunized with a new B cell-dependent myelin antigen (but not with the traditional myelin oligodendrocyte glycoprotein peptide) develop a chronic phase of EAE that is less severe and even completely fades in many individuals. Therefore, ICAM1+ macrophage-like neutrophils can play both shared and nonredundant roles in autoimmune demyelination, among them perpetuating inflammation via ASPRV1.
Assuntos
Ácido Aspártico Endopeptidases/imunologia , Linfócitos B/imunologia , Encefalomielite Autoimune Experimental/imunologia , Molécula 1 de Adesão Intercelular/metabolismo , Neutrófilos/imunologia , Animais , Apresentação de Antígeno/imunologia , Doença Crônica , Sinapses Imunológicas/imunologia , Imunofenotipagem , Camundongos Endogâmicos C57BL , Medula Espinal/imunologia , Linfócitos T/imunologia , Transcriptoma/imunologiaRESUMO
Different industrial processes exist to purify allogenic bone, providing safe and cleaned blocks for bone allografting. However, they often make use of chemical reagents that can be aggressive for the bone matrix. Bone samples were processed with several soaking techniques used in industry: NaHCO3, H2O2, NaOH and H2O2+NaOH combined; the consequences on the bone matrix and cytocompoatibility were evaluated on femoral heads from osteoarthritic patients. Alterations of matrix were searched by histochemistry, atomic force microscopy (AFM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Cytocompatibility was evaluated by coculturing human osteoblast-like cells (SaOS-2) on bone slices. Collagen fibers were dramatically altered at the surface of bone treated with H2O2, NaOH (and their association), but not with NaHCO3. A marked reduction in the number of hydroxyapatite crystals was observed on the trabecular surfaces by TEM and morphological changes were evidenced in SEM and AFM. Argyrophilic proteins of the bone matrix were removed by H2O2 and NaOH (and their association), but not by NaHCO3. As a consequence, attachment, spreading, proliferation and alkaline phosphatase activity of SaOS-2 were reduced by H2O2 and NaOH treatments. Strong oxidizing reagents altered matrix integrity by modifying collagenous and non-collagenous proteins. Whether these changes have clinical consequences on the bone bonding and osseointegration in human necessitate further investigations.
Assuntos
Transplante Ósseo , Prótese de Quadril , Transplantes , Linhagem Celular Tumoral , Humanos , Técnicas de Cultura de Tecidos , Transplante HomólogoRESUMO
Proteinase-activated receptor-4 (PAR(4)) is a G-protein-coupled receptor activated by serine proteinases released during tissue repair and inflammation. We have previously shown that PAR(4) activation sensitises articular primary afferents leading to joint pain. This study examined whether mast cells contribute to this PAR(4)-induced sensitisation and consequent heightened pain behaviour. The expression of PAR(4) on synovial mast cells was confirmed with immunofluorescent staining of rat knee joint sections. Electrophysiological recordings were made from joint primary afferents in male Wistar rats during both nonnoxious and noxious rotations of the knee. Afferent firing rate was recorded for 15 minutes after close intra-arterial injection of 10(-9) to 10(-5)mol of the PAR(4) activating peptide, AYPGKF-NH(2), or the inactive peptide, YAPGKF-NH(2) (100-µl bolus). Rats were either naive or pretreated with the mast cell stabilise, cromolyn (20mg/kg). Mechanical withdrawal thresholds were determined using a dynamic planter aesthesiometer and weight bearing determined using an incapacitance tester. These behavioural measurements were taken before and after intra-articular AYPGKF-NH(2), or the inactive peptide, YAPGKF-NH(2) (100µg). Local administration of AYPGKF-NH(2) caused a significant increase in joint primary afferent firing rate and pain behaviour compared with the control peptide YAPGKF-NH(2). These effects were blocked by pretreatment with cromolyn. These data reveal that PAR(4) is expressed on synovial mast cells and the activation of PAR(4) has a pronociceptive effect that is dependent on mast cell activation. Proteinase-activated receptor-4 is expressed on synovial mast cells, and the activation of Proteinase-activated receptor-4 has a pronociceptive effect that is dependent on mast cell activation.
Assuntos
Artralgia/metabolismo , Articulação do Joelho/metabolismo , Mastócitos/metabolismo , Nociceptores/fisiologia , Oligopeptídeos/metabolismo , Receptores de Trombina/metabolismo , Animais , Artralgia/patologia , Articulação do Joelho/inervação , Articulação do Joelho/patologia , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/patologia , Nociceptores/efeitos dos fármacos , Oligopeptídeos/fisiologia , Ratos , Ratos Wistar , Receptores de Trombina/fisiologia , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/metabolismo , Membrana Sinovial/patologiaRESUMO
Tissue engineering of autologous bone combined with osteoprogenitor cells is a suitable strategy for filling large bone defects. The aim of this study was to evaluate the osteogenicity of a xenogenic bone graft cultured with allogenic bone marrow stromal cells (BMSC) in a mouse critical size craniotomy. Bovine trabecular bone grafts were made free of bone marrow cells or debris and were delipidated. BMSC were harvested from C57BL/6-Tg(ACTbEGFP)1Osb/J mice (GFP+ cells) and were cultured 14 days on bone grafts in control or osteogenic medium. Engineered grafts were implanted in calvarial defect in C57BL/6 mice. Four groups were studied: graft with BMSC differentiated in osteoblasts (G-Ob), graft with BMSC (G-BMSC), graft without cells (G) and no graft. Calvariae were studied 2 and 8 weeks after implantation by radiographic and histomorphometric analyses. G group: the bone ingrowth was limited to the edges of the defect. The center of the graft was filled by a fibrovascular connective tissue. G-BMSC or G-Ob groups: bone formation occurred early in the center of the defect and did not increase between 2 and 8 weeks; the newly formed woven bone was partially replaced by lamellar bone. The preoperative osteoblastic differentiation of BMSC did not allow faster and better bone regeneration. After 2 weeks, GFP+ cells were observed around the grafted bone but no GFP+ osteocyte was present in the newly formed bone. No GFP+ cell was noted after 8 weeks. However, pre-implantation culture of the biomaterial with allogenic BMSC greatly enhanced the bone regeneration.
Assuntos
Regeneração Óssea , Transplante Ósseo/métodos , Células Estromais/citologia , Engenharia Tecidual/métodos , Animais , Células da Medula Óssea , Bovinos , Diferenciação Celular , Técnicas de Cocultura , Implantes Experimentais , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/citologia , Osteogênese , Transplante HeterólogoRESUMO
Adenosine, prostaglandin E(2), or increased intracellular cyclic AMP concentration each elicit potent anti-inflammatory events in human neutrophils by inhibiting functions such as phagocytosis, superoxide production, adhesion and cytokine release. However, the endogenous molecular pathways mediating these actions are poorly understood. In the present study, we examined their impact on the gene expression profile of stimulated neutrophils. Purified blood neutrophils from healthy donors were stimulated with a cocktail of inflammatory agonists in the presence of at least one of the following anti-inflammatory agents: adenosine A(2A) receptor agonist CGS 21680, prostaglandin E(2), cyclic-AMP-elevating compounds forskolin and RO 20-1724. Total RNA was analyzed using gene chips and real-time PCR. Genes encoding transcription factors, enzymes and regulatory proteins, as well as secreted cytokines/chemokines showed differential expression. We identified 15 genes for which the anti-inflammatory agents altered mRNA levels. The agents affected the expression profile in remarkably similar fashion, suggesting a central mechanism limiting cell activation. We have identified a set of genes that may be part of important resolution pathways that interfere with cell activation. Identification of these pathways will improve understanding of the capacity of tissues to terminate inflammatory responses and contribute to the development of therapeutic strategies based on endogenous resolution.