RESUMO
BACKGROUND & AIMS: Disturbances in the control of ion transport lead to epithelial barrier dysfunction in patients with colitis. Enteric glia regulate intestinal barrier function and colonic ion transport. However, it is not clear whether enteric glia are involved in epithelial hyporesponsiveness. We investigated enteric glial regulation of ion transport in mice with trinitrobenzene sulfonic acid- or dextran sodium sulfate-induced colitis and in Il10(-/-) mice. METHODS: Electrically evoked ion transport was measured in full-thickness segments of colon from CD1 and Il10(-/-) mice with or without colitis in Ussing chambers. Nitric oxide (NO) production was assessed using amperometry. Bacterial translocation was investigated in the liver, spleen, and blood of mice. RESULTS: Electrical stimulation of the colon evoked a tetrodotoxin-sensitive chloride secretion. In mice with colitis, ion transport almost completely disappeared. Inhibiting inducible NO synthase (NOS2), but not neuronal NOS (NOS1), partially restored the evoked secretory response. Blocking glial function with fluoroacetate, which is not a NOS2 inhibitor, also partially restored ion transport. Combined NOS2 inhibition and fluoroacetate administration fully restored secretion. Epithelial responsiveness to vasoactive intestinal peptide was increased after enteric glial function was blocked in mice with colitis. In colons of mice without colitis, NO was produced in the myenteric plexus almost completely via NOS1. NO production was increased in mice with colitis, compared with mice without colitis; a substantial proportion of NOS2 was blocked by fluoroacetate administration. Inhibition of enteric glial function in vivo reduced the severity of trinitrobenzene sulfonic acid-induced colitis and associated bacterial translocation. CONCLUSIONS: Increased production of NOS2 in enteric glia contributes to the dysregulation of intestinal ion transport in mice with colitis. Blocking enteric glial function in these mice restores epithelial barrier function and reduces bacterial translocation.
Assuntos
Colite/metabolismo , Sistema Nervoso Entérico/citologia , Transporte de Íons , Neuroglia/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/metabolismo , Animais , Translocação Bacteriana , Colite/induzido quimicamente , Colite/genética , Modelos Animais de Doenças , Estimulação Elétrica/métodos , Fluoracetatos/administração & dosagem , Interleucina-10/deficiência , Interleucina-10/genética , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Plexo Mientérico/citologia , Neuroglia/citologiaRESUMO
Neutrophil recruitment and directional movement toward chemotactic stimuli are important processes in innate immune responses. This study examines the role of Fer kinase in neutrophil recruitment and chemotaxis to various chemoattractants in vitro and in vivo. Mice targeted with a kinase-inactivating mutation (Fer(DR/DR)) or wild type (WT) were studied using time-lapse intravital microscopy to examine leukocyte recruitment and chemotaxis in vivo. In response to keratinocyte-derived cytokine, no difference in leukocyte chemotaxis was observed between WT and Fer(DR/DR) mice. However, in response to the chemotactic peptide WKYMVm, a selective agonist of the formyl peptide receptor, a 2-fold increase in leukocyte emigration was noted in Fer(DR/DR) mice (p < 0.05). To determine whether these defects were due to Fer signaling in the endothelium or other nonhematopoietic cells, bone marrow chimeras were generated. WKYMVm-induced leukocyte recruitment in chimeric mice (WT bone marrow to Fer(DR/DR) recipients or vice versa) was similar to WT mice, suggesting that Fer kinase signaling in both leukocytes and endothelial cells serves to limit chemotaxis. Purified Fer(DR/DR) neutrophils demonstrated enhanced chemotaxis toward end target chemoattractants (WKYMVm and C5a) compared with WT using an under-agarose gel chemotaxis assay. These defects were not observed in response to intermediate chemoattractants (keratinocyte-derived cytokine, MIP-2, or LTB(4)). Increased WKYMVm-induced chemotaxis of Fer(DR/DR) neutrophils correlated with sustained PI3K activity and reduced reliance on the p38 MAPK pathway compared with WT neutrophils. Together, these data identify Fer as a novel inhibitory kinase for neutrophil chemotaxis toward end target chemoattractants through modulation of PI3K activity.
Assuntos
Fatores Quimiotáticos/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/citologia , Proteínas Tirosina Quinases/genética , Animais , Quimiocina CXCL2/farmacologia , Quimiocinas/farmacologia , Citocinas/biossíntese , Citocinas/imunologia , Expressão Gênica/efeitos dos fármacos , Contagem de Leucócitos , Leucotrieno B4/farmacologia , Camundongos , Camundongos Knockout , Microscopia de Vídeo , Neutrófilos/imunologia , Peptídeos/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Tirosina Quinases/imunologia , Receptores de Formil Peptídeo/genética , Receptores de Formil Peptídeo/imunologia , Transdução de Sinais/efeitos dos fármacos , Imagem com Lapso de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
Toll-like receptors are host sentinel receptors that signal the presence of infectious nonself and initiate protective immunity. One of the primary immune defense mechanisms is the recruitment of neutrophils from the bloodstream into the infected tissue. Although neutrophils are important in host defense, they can also be responsible for damaging pathologies associated with excessive inflammation. Here, we report that the di-acylated TLR2 ligand lipoteichoic acid can directly inhibit neutrophil recruitment in vivo. This discovery allowed us to test the concept that conventional proinflammatory TLR2 ligands can be made to act as inhibitors through specific structural modifications. Indeed, lipopeptide TLR2 ligands, when modified at their acyl chains to contain linoleate, lose their capacity to induce inflammation and yield ligands that can directly inhibit the in vivo neutrophil recruitment initiated by a wide range of proinflammatory stimuli. The inhibitory capacity of LTA and these modified ligands requires the expression of TLR2, but is independent of the TLR2 signaling adaptor, MyD88. Instead, this inhibitory effect requires functional activity of the fatty acid and nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ). Therefore, these data support a model in TLR2 biology where structural modifications of these ligands can profoundly influence host-microbial interactions. These inhibitory TLR2 ligands also have broader implications with respect to their potential use in various inflammatory disease settings.
Assuntos
Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Neutrófilos/metabolismo , PPAR gama/fisiologia , Ácidos Teicoicos/farmacologia , Receptor 2 Toll-Like/metabolismo , Acilação , Animais , Ligantes , Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Ácidos Teicoicos/metabolismoRESUMO
Sickness behaviors are host defense adaptations that arise from integrated autonomic outputs in response to activation of the innate immune system. These behaviors include fever, anorexia, and hyperalgesia intended to promote survival of the host when encountering pathogens. Cannabinoid (CB) receptor activation can induce hypothermia and attenuate LPS-evoked fever. The aim of the present study was to examine the role of CB1 receptors in the LPS-evoked febrile response. CB1 receptor-deficient (CB1(-/-)) mice did not display LPS-evoked fever; likewise, pharmacological blockade of CB1 receptors in wild-type mice blocked LPS-evoked fever. This unresponsiveness is not limited to thermogenesis, as the animals were not hyperalgesic after LPS administration. A Toll-like receptor (TLR)3 agonist and viral mimetic polyinosinic:polycytidylic acid evoked a robust fever in CB1(-/-) mice, suggesting TLR3-mediated responses are functional. LPS-evoked c-Fos activation in areas of the brain associated with the febrile response was evident in wild-type mice but not in CB1(-/-) mice. Liver and spleen TLR4 mRNA were significantly lower in CB1(-/-) mice compared with wild-type mice, and peritoneal macrophages from CB1(-/-) mice did not release proinflammatory cytokines in response to LPS. These data indicate that CB1 receptors play a critical role in LPS-induced febrile responses through inhibiting TLR4-mediated cytokine production.
Assuntos
Imunidade Inata/fisiologia , Receptor CB1 de Canabinoide/fisiologia , Receptor 4 Toll-Like/agonistas , Animais , Temperatura Corporal/fisiologia , Citocinas/biossíntese , Interpretação Estatística de Dados , Febre/induzido quimicamente , Febre/fisiopatologia , Hiperalgesia/induzido quimicamente , Hiperalgesia/genética , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Medição da Dor , Piperidinas/farmacologia , Poli I-C/farmacologia , Proteínas Proto-Oncogênicas c-fos/biossíntese , Pirazóis/farmacologia , RNA/biossíntese , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/genética , Receptor 3 Toll-Like/efeitos dos fármacosRESUMO
IL-10 is a regulatory cytokine that plays a major role in the homeostasis of the gut and this is illustrated by the fact that IL-10(-/-) mice develop spontaneous colitis. In this study, IL-10(-/-) mice were analyzed for immunological changes during colitis development. We found a reduced frequency of regulatory T cells CD4(+)CD25(+)Foxp3(+) and higher frequency of activated T cells in the colon that precedes the macroscopic signs of the disease. Production of IL-17 and IFN-γ was higher in the colon. Colitis progression culminates with the reduction of CD4(+)LAP(+) regulatory T cells in the intestine. Frequency of B1 cells and the secretory IgA production were both elevated. Despite these alterations, 16-week-old IL-10(-/-) mice could be rendered tolerant by a continuous feeding protocol. Our study provides detailed analysis of changes that precede colitis and it also suggests that oral tolerance could be used to design novel alternative therapies for the disease.
Assuntos
Subpopulações de Linfócitos B/metabolismo , Colite/imunologia , Inflamação/imunologia , Interleucina-10/imunologia , Mucosa Intestinal/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Subpopulações de Linfócitos B/imunologia , Colite/complicações , Colite/patologia , Colo/imunologia , Colo/patologia , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/imunologia , Tolerância Imunológica , Imunoglobulina A/biossíntese , Imunoglobulina A/imunologia , Inflamação/complicações , Inflamação/patologia , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-10/deficiência , Interleucina-10/genética , Interleucina-17/biossíntese , Interleucina-17/imunologia , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Subunidade alfa de Receptor de Interleucina-2/imunologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Linfócitos T Reguladores/imunologiaRESUMO
BACKGROUND AND AIMS: In an attempt to further investigate the role of cannabinoid (CB) system in the pathogenesis of inflammatory bowel diseases, we employed two recently developed ligands, AM841 (a covalently acting CB agonist) and CB13 (a peripherally-restricted CB agonist) to establish whether central and peripheral CB sites are involved in the anti-inflammatory action in the intestine. METHODS AND RESULTS: AM841 (0.01, 0.1 and 1 mg/kg, i.p.) significantly decreased inflammation scores in dextran sulfate sodium (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-treated mice when administered before induction of colitis or as a treatment of existing intestinal inflammation. The effect was absent in CB1, CB2 and CB(1/2)-deficient mice. A peripherally-restricted agonist CB13 did not alleviate colitis when given i.p. (0.1 mg/kg), but significantly decreased inflammation score after central administration (0.1 µg/animal). CONCLUSIONS: This is the first evidence that central and peripheral CB receptors are responsible for the protective and therapeutic action of cannabinoids in mouse models of colitis. Our observations provide new insight to CB pharmacology and validate the use of novel ligands AM841 and CB13 as potent tools in CB-related research.
Assuntos
Canabinoides/farmacologia , Doenças Inflamatórias Intestinais/prevenção & controle , Receptores de Canabinoides/efeitos dos fármacos , Animais , Dronabinol/análogos & derivados , Dronabinol/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Fagocitose/efeitos dos fármacosRESUMO
GM-CSF is well recognized as a proliferative agent for hematopoietic cells and exerts a priming function on neutrophils. The aim of this study was to determine if GM-CSF has a role as a neutrophil chemoattractant in vivo and if it can contribute to recruitment during intestinal inflammation. Initial studies in vitro, using the under-agarose gel assay, determined that GM-CSF can induce neutrophil migration at a much lower molar concentration than the fMLP-like peptide WKYMVm (33.5-134 nM vs. 1-10 µM). GM-CSF-induced neutrophil migration was ablated (<95%) using neutrophils derived from GMCSFRß(-/-) mice and significantly attenuated by 42% in PI3Kγ(-/-)neutrophils. In vivo, a significant increase in leukocyte recruitment was observed using intravital microscopy 4 h post-GM-CSF (10 µg/kg) injection, which was comparable with leukocyte recruitment induced by KC (40 µg/kg). GM-CSF-induced recruitment was abolished, and KC-induced recruitment was maintained in GMCSFRß(-/-) mice. Furthermore, in vivo migration of extravascular leukocytes was observed toward a gel containing GM-CSF in WT but not GMCSFRß(-/-) mice. Finally, in a model of intestinal inflammation (TNBS-induced colitis), colonic neutrophil recruitment, assessed using the MPO assay, was attenuated significantly in anti-GM-CSF-treated mice or GMCSFRß(-/-) mice. These data demonstrate that GM-CSF is a potent chemoattractant in vitro and can recruit neutrophils from the microvasculature and induce extravascular migration in vivo in a ß subunit-dependent manner. This property of GM-CSF may contribute significantly to recruitment during intestinal inflammation.
Assuntos
Fatores Quimiotáticos/farmacologia , Subunidade beta Comum dos Receptores de Citocinas/fisiologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Leucócitos/metabolismo , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Animais , Western Blotting , Movimento Celular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Mucosa Intestinal/metabolismo , Intestinos/patologia , Leucócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/efeitos dos fármacos , Peroxidase/genética , Peroxidase/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Cannabinoids are known to reduce intestinal inflammation. Atypical cannabinoids produce pharmacological effects via unidentified targets. We were interested in whether the atypical cannabinoid O-1602, reportedly an agonist of the putative cannabinoid receptor GPR55, reduces disease severity of dextran sulfate sodium (DSS) and trinitrobenzene sulfonic acid (TNBS)-induced colitis in C57BL/6N and CD1 mice. METHODS: DSS (2.5% and 4%) was supplied in drinking water for 1 week while TNBS (4 mg) was applied as a single intrarectal bolus. RESULTS: Both treatments caused severe colitis. Injection of O-1602 (5 mg/kg intraperitoneally) significantly reduced macroscopic and histological colitis scores, and myeloperoxidase activity. The protective effect was still present in cannabinoid receptor 1 (CB1) and 2 (CB2) double knockout mice and mice lacking the GPR55 gene. To investigate a potential mechanism underlying the protection by O-1602 we performed neutrophil chemotactic assays. O-1602 concentration-dependently inhibited migration of murine neutrophils to keratinocyte-derived chemokine (KC), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and the N-formyl-peptide receptor ligand WKYMVm. The inhibitory effect of O-1602 was preserved in neutrophils from CB1/CB2 double knockout and GPR55 knockout mice. No differences were seen in locomotor activity between O-1602-treated and control mice, indicating lack of central sedation by this compound. CONCLUSIONS: Our data demonstrate that O-1602 is protective against experimentally induced colitis and inhibits neutrophil recruitment independently of CB1, CB2, and GPR55 receptors. Thus, atypical cannabinoids represent a novel class of therapeutics that may be useful for the treatment of inflammatory bowel diseases.
Assuntos
Canabinoides/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Colite/tratamento farmacológico , Colo/efeitos dos fármacos , Cicloexanos/farmacologia , Infiltração de Neutrófilos/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Resorcinóis/farmacologia , Análise de Variância , Animais , Canabidiol/análogos & derivados , Canabinoides/imunologia , Canabinoides/uso terapêutico , Colite/induzido quimicamente , Colite/imunologia , Colite/patologia , Colo/metabolismo , Colo/patologia , Cicloexanos/imunologia , Cicloexanos/uso terapêutico , Sulfato de Dextrana , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Peroxidase/metabolismo , Receptor CB1 de Canabinoide/genética , Receptor CB2 de Canabinoide/genética , Receptores de Canabinoides , Receptores Acoplados a Proteínas G/genética , Resorcinóis/imunologia , Resorcinóis/uso terapêutico , Ácido TrinitrobenzenossulfônicoRESUMO
BACKGROUND: Attenuated innate immune responses to the intestinal microbiota have been linked to the pathogenesis of Crohn's disease (CD). Recent genetic studies have revealed that hypofunctional mutations of NLRP3, a member of the NOD-like receptor (NLR) superfamily, are associated with an increased risk of developing CD. NLRP3 is a key component of the inflammasome, an intracellular danger sensor of the innate immune system. When activated, the inflammasome triggers caspase-1-dependent processing of inflammatory mediators, such as IL-1ß and IL-18. METHODS: In the current study we sought to assess the role of the NLRP3 inflammasome in the maintenance of intestinal homeostasis through its regulation of innate protective processes. To investigate this role, Nlrp3(-/-) and wildtype mice were assessed in the dextran sulfate sodium and 2,4,6-trinitrobenzenesulfonic acid models of experimental colitis. RESULTS: Nlrp3(-/-) mice were found to be more susceptible to experimental colitis, an observation that was associated with reduced IL-1ß, reduced antiinflammatory cytokine IL-10, and reduced protective growth factor TGF-ß. Macrophages isolated from Nlrp3(-/-) mice failed to respond to bacterial muramyl dipeptide. Furthermore, Nlrp3-deficient neutrophils exhibited reduced chemotaxis and enhanced spontaneous apoptosis, but no change in oxidative burst. Lastly, Nlrp3(-/-) mice displayed altered colonic ß-defensin expression, reduced colonic antimicrobial secretions, and a unique intestinal microbiota. CONCLUSIONS: Our data confirm an essential role for the NLRP3 inflammasome in the regulation of intestinal homeostasis and provide biological insight into disease mechanisms associated with increased risk of CD in individuals with NLRP3 mutations.
Assuntos
Proteínas de Transporte/fisiologia , Colite/fisiopatologia , Homeostase/fisiologia , Inflamassomos/fisiologia , Intestinos/fisiologia , Animais , Apoptose/fisiologia , Quimiotaxia/fisiologia , Modelos Animais de Doenças , Furanos , Imunidade Inata/fisiologia , Interleucina-10/fisiologia , Interleucina-1beta/fisiologia , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Tiofenos , Fator de Crescimento Transformador beta/fisiologiaRESUMO
The Rap1 GTPase is a master regulator of cell adhesion, polarity, and migration. We show that both blocking Rap1 activation and expressing a constitutively active form of Rap1 reduced the ability of B16F1 melanoma cells to extravasate from the microvasculature and form metastatic lesions in the lungs. This correlated with a decreased ability of the tumor cells to undergo transendothelial migration (TEM) in vitro and form dynamic, F-actin-rich pseudopodia that penetrate capillary endothelial walls in vivo. Using multiple tumor cell lines, we show that the inability to form these membrane protrusions, which likely promote TEM and extravasation, can be explained by altered adhesion dynamics and impaired cell polarization that result when Rap1 activation or cycling is perturbed. Thus, targeting Rap1 could be a useful approach for reducing the metastatic dissemination of tumor cells that undergo active TEM.
Assuntos
Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/secundário , Melanoma Experimental/enzimologia , Melanoma Experimental/secundário , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Citoesqueleto/enzimologia , Citoesqueleto/patologia , Células Endoteliais/citologia , Células Endoteliais/enzimologia , Ativação Enzimática , Adesões Focais/enzimologia , Adesões Focais/patologia , Humanos , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/prevenção & controle , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Streptococcus pneumoniae, a gram-positive bacteria, is the most common cause of community-acquired pneumonia. It is a common cause of septic shock with multiple organ dysfunction syndrome (MODS) resulting in significant mortality. Gram-positive mouse models of sepsis with MODS are required to examine mechanisms of immune responses in severe sepsis. To assess whether lung infection due to S. pneumoniae in a nonventilated mouse model can induce multiple organ dysfunction. S. pneumoniae, SPN 15814 strain, harvested at log phase, was injected intratracheally in C57BL/6 mice at OD 600 between 0.35 and 0.63. A dose of bacteria at OD 600 = 0.63 conferred approximately 30% mortality in 36 h. Lung pneumonia was assessed by histology, lung myeloperoxidase activity, and lung bacterial load; intestinal epithelial barrier integrity was assessed by measuring blood-to-lumen clearance of Cr-EDTA; renal function was assessed by measuring plasma creatinine and urea; and myocardiac function was assessed using an isolated perfused mouse heart model. S. pneumoniae-induced pneumonia resulted in neutrophil infiltration into the lungs and increased lung bacterial load. Although relatively few bacteria gained access to the blood stream, the pneumonia was accompanied by increased intestinal epithelial barrier permeability, increased plasma creatinine, and decreased cardiac output and stroke volume. These data clearly show that intratracheal S. pneumoniae induced not only pneumonia but also MODS, despite the fact that few organisms gain access to the blood stream. This model can be used as a good gram-positive model of sepsis and MODS for further studies.
Assuntos
Insuficiência de Múltiplos Órgãos/microbiologia , Infecções Pneumocócicas/fisiopatologia , Streptococcus pneumoniae/crescimento & desenvolvimento , Animais , Modelos Animais de Doenças , Infecções por Bactérias Gram-Positivas/patologia , Infecções por Bactérias Gram-Positivas/fisiopatologia , Coração/microbiologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Pulmão/enzimologia , Pulmão/microbiologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Insuficiência de Múltiplos Órgãos/patologia , Miocárdio/patologia , Peroxidase/metabolismo , Infecções Pneumocócicas/patologia , Streptococcus pneumoniae/patogenicidade , SíndromeRESUMO
Nitric oxide (NO) derived from the inducible NO synthase (iNOS) is an important and complex mediator of inflammation in the intestine. Wnt-inducible secreted protein (WISP)-1 (CCN4), a member of the connective tissue growth factor family, is involved in tissue repair. We sought to determine the relationship between iNOS and WISP-1 in colitis. By analyzing human colonic biopsy samples, we showed that the expression of mRNA for both iNOS and WISP-1 was significantly higher in ulcerative colitis samples compared with control tissue. The upregulation of WISP-1 was positively correlated with iNOS expression in two models of colitis, induced by intrarectal trinitrobenzenesulfonic acid (TNBS) or occurring spontaneously in IL-10 deficient mice. Loss of iNOS, studied using iNOS(-/-) mice in both TNBS-induced and IL-10(-/-) colitis models, significantly attenuated the colitis-related WISP-1 increase. In human colonic epithelial cell lines, the NO donor, DETA-NONOate, elevated WISP-1 mRNA and protein expression through a beta-catenin and CREB-dependent, but Wnt-1-independent, pathway. In addition, NO-induced WISP-1 directly induced secretion of soluble collagen in colonic fibroblast cells. NO increases WISP-1 expression both in vitro and in vivo, suggesting a new role for iNOS and NO in colitis.
Assuntos
Colite Ulcerativa/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Óxido Nítrico/metabolismo , Proteínas Proto-Oncogênicas/genética , Animais , Western Blotting , Proteínas de Sinalização Intercelular CCN , Células CACO-2 , Linhagem Celular Tumoral , Colite/induzido quimicamente , Colite/genética , Colite/metabolismo , Colite Ulcerativa/genética , Colite Ulcerativa/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Células HT29 , Humanos , Interleucina-10/deficiência , Interleucina-10/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Knockout , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Compostos Nitrosos/farmacologia , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Ácido Trinitrobenzenossulfônico , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Glucagon-like peptide 2 (GLP-2) is an important intestinal growth factor with anti-inflammatory activity. We hypothesized that GLP-2 decreases mucosal inflammation and the associated increased epithelial proliferation by downregulation of Th1 cytokines attributable to reprogramming of lamina propria immune regulatory cells via an interleukin-10 (IL-10)-independent pathway. The effects of GLP-2 treatment were studied using the IL-10-deficient (IL-10(-/-)) mouse model of colitis. Wild-type and IL-10(-/-) mice received saline or GLP-2 (50 microg/kg sc) treatment for 5 days. GLP-2 treatment resulted in significant amelioration of animal weight loss and reduced intestinal inflammation as assessed by histopathology and myeloperoxidase levels compared with saline-treated animals. In colitis animals, GLP-2 treatment also reduced crypt cell proliferation and crypt cell apoptosis. Proinflammatory (IL-1beta, TNF-alpha, IFN-gamma,) cytokine protein levels were significantly reduced after GLP-2 treatment, whereas IL-4 was significantly increased and IL-6 production was unchanged. Fluorescence-activated cell sorting analysis of lamina propria cells demonstrated a decrease in the CD4(+) T cell population following GLP-2 treatment in colitic mice and an increase in CD11b(+)/F4/80(+) macrophages but no change in CD25(+)FoxP3 T cells or CD11c(+) dendritic cells. In colitis animals, intracellular cytokine analysis demonstrated that GLP-2 decreased lamina propria macrophage TNF-alpha production but increased IGF-1 production, whereas transforming growth factor-beta was unchanged. GLP-2-mediated reduction of crypt cell proliferation was associated with an increase in intestinal epithelial cell suppressor of cytokine signaling (SOCS)-3 expression and reduced STAT-3 signaling. This study shows that the anti-inflammatory effects of GLP-2 are IL-10 independent and that GLP-2 alters the mucosal response of inflamed intestinal epithelial cells and macrophages. In addition, the suggested mechanism of the reduction in inflammation-induced proliferation is attributable to GLP-2 activation of the SOCS-3 pathway, which antagonizes the IL-6-mediated increase in STAT-3 signaling.
Assuntos
Peptídeo 2 Semelhante ao Glucagon/farmacologia , Interleucina-10/fisiologia , Animais , Anti-Inflamatórios/farmacologia , Proliferação de Células/efeitos dos fármacos , Colite/tratamento farmacológico , Citocinas/metabolismo , Modelos Animais de Doenças , Interleucina-10/deficiência , Masculino , Camundongos , Camundongos Knockout , Mucosa/citologia , Mucosa/efeitos dos fármacosRESUMO
Hydrogen sulfide (H(2)S) is an endogenous gaseous mediator with the ability to modulate tissue inflammation and pain. The aim of this study was to determine the effect of an H(2)S donor (Na(2)S) on leukocyte-endothelium interactions, blood flow, and pain sensation in acutely inflamed knee joints. Acute arthritis was induced in urethane anesthetized C57bl/6 mice by intra-articular injection of kaolin/carrageenan (24-h recovery), and the effect of local administration of Na(2)S on leukocyte trafficking was measured by intravital microscopy. Synovial blood flow was measured in inflamed knees by laser Doppler perfusion imaging. Finally, the effect of an intra-articular injection of Na(2)S on joint pain in control and inflamed rats was determined by hindlimb incapacitance and von Frey hair algesiometry. Local administration of an H(2)S donor to inflamed knees caused a dose-dependent reduction in leukocyte adherence and an increase in leukocyte velocity. These effects could be inhibited by coadministration of the ATP-sensitive K(+) channel blocker glibenclamide. Local administration of Na(2)S to inflamed joints caused a pronounced vasoconstrictor response; however, there was no observable effect of Na(2)S on joint pain. These findings establish H(2)S as a novel signaling molecule in rodent knee joints. H(2)S exhibits potent anti-inflammatory properties, but with no detectable effect on joint pain.
Assuntos
Artrite/tratamento farmacológico , Artrite/imunologia , Sulfeto de Hidrogênio/metabolismo , Dor/tratamento farmacológico , Dor/imunologia , Sulfetos/farmacologia , Doença Aguda , Animais , Artrite/induzido quimicamente , Comportamento Animal/fisiologia , Carragenina/farmacologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Caulim/farmacologia , Articulação do Joelho/irrigação sanguínea , Articulação do Joelho/imunologia , Leucócitos/citologia , Leucócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dor/induzido quimicamente , Estimulação Física , Propriocepção/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologiaRESUMO
A close association between inflammatory bowel disease (IBD) and increased risk of developing adenocarcinoma exists. Moreover, chronic induction of high levels of nitric oxide (NO) produced from inducible nitric oxide synthase (iNOS) is a consistent observation in IBD. In this study we made interleukin-10/inducible nitric oxide synthase double-deficient (IL-10(-/-)/iNOS(-/-)) mice and studied the development of adenocarcinoma. Mice >6 months of age were compared with healthy wild-type (WT) controls. Inflammation was assessed using macroscopic/histological scores and myeloperoxidase activity as an indication of granulocyte infiltration. Mucosal polyps were scored macroscopically and hyperproliferation was quantified by 5-bromo-2-deoxyuridine immunohistochemistry. Dysplastic changes were assessed histologically based on cytologic and architectured atypia as well as the presence of submucosal invasion. The p53 and beta-catenin messenger RNA mRNA and protein expression were assessed using real-time polymerase chain reaction and immunohistochemistry, respectively. Inflammatory indices were significantly elevated in interleukin-10-deficient (IL-10(-/-)) over WT and not significantly different from IL-10(-/-)/iNOS(-/-) mice. The incidence of mucosal polyps was similar between IL-10(-/-) (79%) and IL-10(-/-)/iNOS(-/-) (83%) mice; however, significantly higher numbers of polyps were observed in the absence of iNOS (P < 0.05). Hyperproliferation was noted in both groups. Signs of dysplasia and submucosal invasion were significantly higher in IL-10(-/-)/iNOS(-/-) compared with IL-10(-/-) mice (P < 0.05). No significant increase in p53 and beta-catenin mRNA levels was observed in IL-10(-/-) over WT mice; however, a 2-fold (P = 0.06) and 3-fold (P < 0.05) increase, respectively, was noted in IL-10(-/-)/iNOS(-/-) mice. Our data suggest exposure to chronic NO limits abnormal p53 and beta-catenin expression and reduces incidence of adenocarcinoma in IL-10(-/-) mice.
Assuntos
Adenocarcinoma/genética , Colite/complicações , Neoplasias do Colo/genética , Óxido Nítrico Sintase Tipo II/genética , Animais , Proliferação de Células , Pólipos do Colo/genética , Interleucina-10/deficiência , Camundongos , Camundongos Mutantes , Proteína Supressora de Tumor p53/metabolismo , beta Catenina/metabolismoRESUMO
The metabotropic glutamate receptor 5 (mGluR5) is expressed by astrocytes and its expression is modulated by inflammation. Enteric glia have many similarities to astrocytes and are the most numerous cell in the enteric nervous system (ENS). We investigated whether enteric glia express a functional mGluR5 and whether expression of this receptor was altered in colitis. In both enteric plexuses of the ileum and colon of guinea pigs and mice, we observed widespread glial mGluR5 expression. Incubation of isolated segments of the guinea pig ileum with the mGluR5 specific agonist RS-2-chloro-5-hydroxyphenylglycine (CHPG) caused a dose-dependent increase in the glial expression of c-Fos and the phosphorylated form of the extracellular signal-regulated kinase 1/2. Preincubation of tissues with the group I metabotropic glutamate receptor antagonist, S-4-carboxyphenylglycine, abolished the effects of CHPG. We examined mGluR5 expression in the guinea pig trinitrobenzene sulfonic acid and the IL-10 gene-deficient (IL-10(-/-)) mouse models of colitis. In guinea pigs, mGluR5 immunoreactivity became diffusely localized over the colonic myenteric ganglia, suggesting a change in receptor distribution. In contrast, glial mGluR5 expression was significantly reduced in the colonic myenteric plexus of IL-10(-/-) mice, as assessed with both real-time quantitative RT-PCR as well as immunohistochemistry and image analysis. These changes occurred without concomitant changes to enteric ganglia or glial fibrillary acidic protein expression in the IL-10(-/-) mouse. Our data suggest that enteric glia are a functional target of the glutamatergic neurotransmitter system in the ENS and that changes in mGluR5 expression may be of physiological significance during colitis.
Assuntos
Colite/imunologia , Plexo Mientérico/imunologia , Neuroglia/fisiologia , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Plexo Submucoso/imunologia , Animais , Colite/fisiopatologia , Colo/imunologia , Colo/inervação , Expressão Gênica/imunologia , Ácido Glutâmico/fisiologia , Cobaias , Íleo/imunologia , Íleo/inervação , Imuno-Histoquímica , Masculino , Plexo Mientérico/citologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptor de Glutamato Metabotrópico 5 , Plexo Submucoso/citologiaRESUMO
BACKGROUND & AIMS: Nitric oxide (NO) is an important mediator of intestinal inflammation. Inducible NO synthase (iNOS) is the main source of NO in inflammation. Because iNOS is ubiquitously expressed, our aim was to determine which cellular source(s) of iNOS plays the central role in mediating intestinal inflammation. METHODS: Chimeric lines were produced via bone marrow (BM) transplantation following irradiation. Colitis was induced with dextran sodium sulfate (DSS) or trinitrobenzene sulfonic acid (TNBS). The severity of colitis and markers of inflammation were assessed in standard fashion. Leukocyte recruitment was assessed by intravital microscopy. RESULTS: The irradiated chimeric lines with iNOS-/- BM-derived cells were markedly more resistant to both DSS- and TNBS-induced injury. Resistance to DSS-induced colitis was lost when wild-type (wt) BM was used to reconstitute iNOS-/- mice. Neutrophils were the main source of iNOS in DSS-induced colitis. iNOS-/- chimeric lines had decreased colonic macrophage inflammatory protein 1beta and tumor necrosis factor alpha expression and increased levels of the protective growth factor, keratinocyte growth factor. LPS-mediated leukocyte recruitment was reduced in iNOS-/- mice, and there were marked changes in the inflammatory cell infiltrates between the chimeric lines with iNOS-/- vs wt BM-derived cells. Furthermore, the lamina propria CD4 +ve cells from chimeric lines with iNOS-/- BM-derived cells had reduced intracellular cytokine expression. CONCLUSIONS: iNOS produced by BM-derived cells plays a critical role in mediating the inflammatory response during colitis. Cell-specific regulation of iNOS may represent a novel form of therapy for patients with inflammatory bowel disease.
Assuntos
Células da Medula Óssea/metabolismo , Colite/metabolismo , Colite/fisiopatologia , Óxido Nítrico Sintase Tipo II/fisiologia , Animais , Células da Medula Óssea/patologia , Transplante de Medula Óssea/métodos , Quimiocinas/metabolismo , Colite/induzido quimicamente , Citocinas/metabolismo , Sulfato de Dextrana , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica , Leucócitos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ácido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Ácido TrinitrobenzenossulfônicoRESUMO
Lymphocyte localization to inflammatory sites is paramount for developing and maintaining an immune response. Rolling is the first step in recruitment, but our knowledge of its mechanisms in Th1 and Th2 CD4(+) lymphocytes is incomplete. Whereas initial studies suggested that Th1 but not Th2 lymphocytes used P-selectin for recruitment, more recent studies have proposed that both subtypes bind selectins. We used intravital microscopy to demonstrate in vivo that polarized Th1 and Th2 lymphocytes both use P-selectin to roll and adhere to cytokine [tumor necrosis factor (TNF)-alpha or interleukin (IL)-4]-activated intestinal microvasculature. The majority of Th1 lymphocyte flux in TNF-alpha- and IL-4-treated animals was P-selectin-dependent. Th1 lymphocytes also interacted with E-selectin to control rolling velocity after TNF-alpha stimulation. Th2 lymphocytes, which make IL-4 but not interferon-gamma, bound P-selectin ex vivo, with more than 95% rolling on P-selectin in vivo. Both Th1 and Th2 lymphocytes regulated rolling velocity by interacting with alpha(4)-integrin. Furthermore, in a model of spontaneous intestinal inflammation (ie, IL-10-deficient mice), both Th1 and Th2 lymphocytes rolled, adhered, and ultimately emigrated into the local microenvironment. These results suggest that both Th1 and Th2 lymphocytes use P-selectin in the initial rolling step in vivo in response to a global activator of the vasculature (TNF), a subtle inducer of P-selectin (IL-4), and pathological inflammation (IL-10-deficient mice).
Assuntos
Endotélio Vascular/fisiologia , Intestinos/irrigação sanguínea , Migração e Rolagem de Leucócitos/fisiologia , Microcirculação/fisiologia , Selectina-P/genética , Células Th1/fisiologia , Células Th2/fisiologia , Animais , Biomarcadores , Primers do DNA , Replicação do DNA , Endotélio Vascular/citologia , Regulação da Expressão Gênica , Interleucina-10/deficiência , Interleucina-10/fisiologia , Interleucina-4/farmacologia , Camundongos , Microscopia , Infiltração de Neutrófilos/fisiologia , Reação em Cadeia da Polimerase , RNA/isolamento & purificação , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Mast cells have been implicated as the central effectors in allergic responses, yet a fatal anaphylactic response can be induced in mast cell-deficient mice. In this study, we examined the immediate hypersensitivity response in wild-type (WT) and mast cell-deficient mice (W/W(v)) in two different tissues (skin and skeletal muscle). Vascular permeability and leukocyte recruitment were studied after immediate challenge or 4 h postchallenge in OVA-sensitized mice. In skin, immediate challenge induced a significant increase in vascular permeability (75%) within 30 min and was accompanied by increased leukocyte adhesion 4 h postchallenge. In the absence of mast cells, no changes in vascular permeability or leukocyte recruitment were observed in skin. In WT skeletal muscle, immediate challenge induced a rapid increase (80%) in vascular permeability within 5 min and significant leukocyte recruitment after 4 h. Surprisingly, in W/W(v), a gradual increase in vascular permeability was observed, reaching a maximum (50%) within 30 min. Despite the absence of mast cells, subsequent leukocyte emigration was similar to that observed in WT mice. Pretreatment with anti-platelet serum in W/W(v) returned Ag-induced vascular permeability and leukocyte recruitment to baseline. Platelets were shown to interact with endothelium in skeletal muscle, but not dermal microvasculature. These data illustrate that mast cells play a prominent role in vascular permeability and leukocyte recruitment in skin in response to Ag, however, in skeletal muscle; these changes can occur in the absence of mast cells, and are mediated, in part, by the presence of platelets.