RESUMEN
The constitutive heparin+ (HP) mast cells (MCs) in mice express mouse MC protease (mMCP)-5 and carboxypeptidase A (mMC-CPA). The amino acid sequence of mMCP-5 is most similar to that of human chymase-1, as are the nucleotide sequences of their genes and transcripts. Using a homologous recombination approach, a C57BL/6 mouse line was created that possessed a disrupted mMCP-5 gene. The resulting mice were fertile and had no obvious developmental abnormality. Lack of mMCP-5 protein did not alter the granulation of the IL-3/IL-9-dependent mMCP-2+ MCs in the jejunal mucosa of Trichinella spiralis-infected mice. In contrast, the constitutive HP+ MCs in the tongues of mMCP-5-null mice were poorly granulated and lacked mMC-CPA protein. Bone marrow-derived MCs were readily developed from the transgenic mice using IL-3. Although these MCs contained high levels of mMC-CPA mRNA, they also lacked the latter exopeptidase. mMCP-5 protein is therefore needed to target translated mMC-CPA to the secretory granule along with HP-containing serglycin proteoglycans. Alternately, mMCP-5 is needed to protect mMC-CPA from autolysis in the cell's granules. Fibronectin was identified as a target of mMCP-5, and the exocytosis of mMCP-5 from the MCs in the mouse's peritoneal cavity resulted in the expression of metalloproteinase protease-9, which has been implicated in arthritis. In support of the latter finding, experimental arthritis was markedly reduced in mMCP-5-null mice relative to wild-type mice in two disease models.
Asunto(s)
Artritis Experimental/patología , Quimasas/efectos adversos , Mastocitos/enzimología , Animales , Artritis Experimental/enzimología , Artritis Experimental/etiología , Carboxipeptidasas A/análisis , Carboxipeptidasas A/deficiencia , Carboxipeptidasas A/metabolismo , Quimasas/deficiencia , Quimasas/fisiología , Humanos , Mastocitos/metabolismo , Mastocitos/patología , Ratones , Ratones Endogámicos C57BL , Vesículas Secretoras/metabolismoRESUMEN
Ras guanine nucleotide-releasing protein-4 (RasGRP4) is an evolutionarily conserved calcium-regulated, guanine nucleotide exchange factor and diacylglycerol/phorbol ester receptor. While an important intracellular signaling protein for CD117+ mast cells (MCs), its roles in other immune cells is less clear. In this study, we identified a subset of in vivo-differentiated splenic CD117+ dendritic cells (DCs) in wild-type (WT) C57BL/6 mice that unexpectedly contained RasGRP4 mRNA and protein. In regard to the biologic significance of these data to innate immunity, LPS-treated splenic CD117+ DCs from WT mice induced natural killer (NK) cells to produce much more interferon-γ (IFN-γ) than comparable DCs from RasGRP4-null mice. The ability of LPS-responsive MCs to cause NK cells to increase their expression of IFN-γ was also dependent on this intracellular signaling protein. The discovery that RasGRP4 is required for CD117+ MCs and DCs to optimally induce acute NK cell-dependent immune responses to LPS helps explain why this signaling protein has been conserved in evolution.
Asunto(s)
Células Dendríticas/inmunología , Células Asesinas Naturales/inmunología , Lipopolisacáridos/farmacología , Mastocitos/inmunología , Proteínas Proto-Oncogénicas c-kit/inmunología , Factores de Intercambio de Guanina Nucleótido ras/fisiología , Animales , Técnicas de Cocultivo , Interferón gamma/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ligando OX40 , Transducción de Señal , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunología , Factores de Necrosis Tumoral/metabolismoRESUMEN
OBJECTIVE: Ras guanine nucleotide-releasing protein 4 (RasGRP-4) is a calcium-regulated guanine nucleotide exchange factor and diacylglycerol/phorbol ester receptor not normally expressed in fibroblasts. While RasGRP-4-null mice are resistant to arthritis induced by anti-glucose-6-phosphate isomerase autoantibodies, the relevance of these findings to humans is unknown. We undertook this study to evaluate the importance of RasGRP-4 in the pathogenesis of human and rat arthritis. METHODS: Synovial tissue from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were evaluated immunohistochemically for the presence of RasGRP-4 protein. Fibroblast-like synoviocytes (FLS) were isolated from synovial samples, and expression of RasGRP-4 was evaluated by real-time quantitative reverse transcription-polymerase chain reaction analyses. The proliferation potency of FLS was evaluated by exposing the cells to a RasGRP-4-specific small interfering RNA (siRNA). Finally, the ability of RasGRP-4-specific siRNAs to hinder type II collagen-induced arthritis in rats was evaluated to confirm the importance of the signaling protein in the disease. RESULTS: Unexpectedly, RasGRP-4 protein was detected in the synovial hyperplastic lining, where proliferating FLS preferentially reside. FLS isolated from tissues obtained from a subpopulation of RA patients expressed much more RasGRP-4 than did FLS from examined OA patients. Moreover, the level of RasGRP-4 transcript was correlated with the FLS proliferation rate. The ability of cultured FLS to divide was diminished when they were treated with RasGRP-4-specific siRNAs. The intraarticular injection of RasGRP-4-specific siRNAs also dampened experimental arthritis in rats. CONCLUSION: RasGRP-4 is aberrantly expressed in FLS and helps regulate their growth. This intracellular signaling protein is therefore a candidate target for dampening proliferative synovitis and joint destruction.
Asunto(s)
Artritis Reumatoide/metabolismo , Proliferación Celular/fisiología , Fibroblastos/metabolismo , Osteoartritis/metabolismo , Membrana Sinovial/metabolismo , Factores de Intercambio de Guanina Nucleótido ras/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Artritis Experimental/metabolismo , Artritis Experimental/patología , Artritis Reumatoide/patología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Fibroblastos/patología , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Osteoartritis/patología , ARN Interferente Pequeño/farmacología , Ratas , Ratas Endogámicas Lew , Membrana Sinovial/patología , Factores de TiempoRESUMEN
Mast cells (MCs) are tissue-resident immune cells that carry out protective roles against pathogens. In disease states, such as inflammatory bowel disease, these granulocytes release a diverse array of mediators that contribute to inflammatory processes. They also participate in wound repair and tissue remodeling. In this review, the composition of MCs and how their phenotypes can be altered during inflammation of the gastrointestinal tract is detailed. Animal and human clinical studies that have implicated the participation of MCs in inflammatory bowel disease are reviewed, including the contribution of the cell's mediators to clinical symptoms, stress-triggered inflammation, and fistula and strictures. Studies that have focused on negating the proinflammatory roles of MCs and their mediators in animal models suggest new targets for therapies for patients with inflammatory bowel disease.
Asunto(s)
Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Mastocitos/inmunología , Animales , HumanosRESUMEN
The sentinel roles of mammalian mast cells (MCs) in varied infections raised the question of their evolutionary origin. We discovered that the test cells in the sea squirt Ciona intestinalis morphologically and histochemically resembled cutaneous human MCs. Like the latter, C. intestinalis test cells stored histamine and varied heparin·serine protease complexes in their granules. Moreover, they exocytosed these preformed mediators when exposed to compound 48/80. In support of the histamine data, a C. intestinalis-derived cDNA was isolated that resembled that which encodes histidine decarboxylase in human MCs. Like heparin-expressing mammalian MCs, activated test cells produced prostaglandin D2 and contained cDNAs that encode a protein that resembles the synthase needed for its biosynthesis in human MCs. The accumulated morphological, histochemical, biochemical, and molecular biology data suggest that the test cells in C. intestinalis are the counterparts of mammalian MCs that reside in varied connective tissues. The accumulated data point to an ancient origin of MCs that predates the emergence of the chordates >500million years ago, well before the development of adaptive immunity. The remarkable conservation of MCs throughout evolution is consistent with their importance in innate immunity.
Asunto(s)
Evolución Biológica , Ciona intestinalis/citología , Ciona intestinalis/fisiología , Mastocitos/fisiología , Mastocitos/ultraestructura , Secuencia de Aminoácidos , Animales , Ciona intestinalis/genética , Clonación Molecular , Evolución Molecular , Femenino , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Liberación de Histamina , Histidina Descarboxilasa/genética , Histidina Descarboxilasa/metabolismo , Humanos , Inmunidad Innata , Oxidorreductasas Intramoleculares/genética , Lipocalinas/genética , Mastocitos/inmunología , Datos de Secuencia Molecular , Prostaglandina D2/biosíntesis , Vesículas Secretoras/fisiología , Homología de Secuencia de Aminoácido , Serina Proteasas/metabolismo , Especificidad de la EspecieRESUMEN
Protease serine member S31 (Prss31)/transmembrane tryptase/tryptase-γ is a mast cell (MC)-restricted protease of unknown function that is retained on the outer leaflet of the plasma membrane when MCs are activated. We determined the nucleotide sequences of the Prss31 gene in different mouse strains and then used a Cre/loxP homologous recombination approach to create a novel Prss31(-/-) C57BL/6 mouse line. The resulting animals exhibited no obvious developmental abnormality, contained normal numbers of granulated MCs in their tissues, and did not compensate for their loss of the membrane tryptase by increasing their expression of other granule proteases. When Prss31-null MCs were activated with a calcium ionophore or by their high affinity IgE receptors, they degranulated in a pattern similar to that of WT MCs. Prss31-null mice had increased baseline airway reactivity to methacholine but markedly reduced experimental chronic obstructive pulmonary disease and colitis, thereby indicating both beneficial and adverse functional roles for the tryptase. In a cigarette smoke-induced model of chronic obstructive pulmonary disease, WT mice had more pulmonary macrophages, higher histopathology scores, and more fibrosis in their small airways than similarly treated Prss31-null mice. In a dextran sodium sulfate-induced acute colitis model, WT mice lost more weight, had higher histopathology scores, and contained more Cxcl-2 and IL-6 mRNA in their colons than similarly treated Prss31-null mice. The accumulated data raise the possibility that inhibitors of this membrane tryptase may provide additional therapeutic benefit in the treatment of humans with these MC-dependent inflammatory diseases.
Asunto(s)
Colitis/enzimología , Pulmón/fisiopatología , Mastocitos/enzimología , Proteínas de la Membrana/inmunología , Enfermedad Pulmonar Obstructiva Crónica/enzimología , Triptasas/inmunología , Animales , Colitis/genética , Colitis/inmunología , Colitis/fisiopatología , Modelos Animales de Enfermedad , Humanos , Pulmón/enzimología , Pulmón/inmunología , Masculino , Mastocitos/inmunología , Proteínas de la Membrana/genética , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Triptasas/genéticaRESUMEN
Tetramer-forming tryptase (hTryptase-ß) was recently discovered to have a prominent role in preventing the internal accumulation of life-threatening fibrin deposits and fibrin-platelet clots. The anticoagulant activity of hTryptase-ß is an explanation for the presence of hemorrhagic disorders in some patients with anaphylaxis or mastocytosis. The fragments of hFibrinogen formed by the proteolysis of this prominent protein by hTryptase-ß could be used as biomarkers in the blood and/or urine for the identification and monitoring of patients with mast cell-dependent disorders. Recombinant hTryptase-ß has potential to be used in clinical settings where it is desirable to inhibit blood coagulation.
Asunto(s)
Anafilaxia/genética , Coagulación Sanguínea/genética , Regulación Neoplásica de la Expresión Génica , Mastocitos/enzimología , Mastocitosis/genética , Triptasas/genética , Anafilaxia/sangre , Fibrina/genética , Fibrina/metabolismo , Fibrinógeno/genética , Fibrinógeno/metabolismo , Histamina/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Mastocitos/patología , Mastocitosis/sangre , Multimerización de Proteína , Transducción de Señal , Piel/enzimología , Piel/patología , Trombina/genética , Trombina/metabolismo , Triptasas/química , Triptasas/metabolismoRESUMEN
We previously established a mast cell (MC)-dependent thermal injury model in mice with ulceration and scar formation that depended on nonredundant functions of mouse MC protease (mMCP)4 and mMCP5. We hypothesized that MC activation is an early event and now find by histology that exocytosis of granule contents occurred by 2 min after thermal injury in wild-type (WT) C57BL/6 mice and in the mMCP4- or mMCP5-deficient mice. The degranulation was equivalent for MCs in the dermis and hypodermis of all three strains, but only the WT mice showed an appreciable increase in epidermal thickness. There was no loss of total MCs, partially degranulated plus intact, during the 4 h of observation. By electron microscopy, MCs in all strains showed early zonal degranulation at 30 s with marked progression in magnitude by 120 s and no mitochondrial injury or cellular necrosis. Concomitantly there was an increase in intercellular spaces indicative of tight junction (TJ) disruption in WT mice but not in the mMCP4- or mMCP5-deficient strains. The desmosomes were intact in all strains. Immunodetection of the TJ protein claudin 4 in WT and mMCP5-deficient mice indicated a significant reduction after scald injury whereas mMCP4(-/-) mice showed no significant changes. Taken together, these findings reveal that a second-degree burn injury can initiate an immediate novel zonal degranulation of MCs throughout all skin layers and a disruption of the epidermal TJs dependent on the nonredundant presence of mMCP4 and mMCP5.
Asunto(s)
Quimasas/deficiencia , Epidermis/metabolismo , Serina Endopeptidasas/deficiencia , Uniones Estrechas/metabolismo , Animales , Quemaduras/genética , Quemaduras/metabolismo , Degranulación de la Célula , Quimasas/genética , Claudina-4/metabolismo , Epidermis/lesiones , Epidermis/ultraestructura , Exocitosis , Técnica del Anticuerpo Fluorescente , Mastocitos/metabolismo , Mastocitos/fisiología , Mastocitos/ultraestructura , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Serina Endopeptidasas/genética , Temperatura , Uniones Estrechas/patología , Uniones Estrechas/ultraestructura , Factores de TiempoRESUMEN
Mast cells (MCs) are active participants in blood coagulation and innate and acquired immunity. This review focuses on the development of mouse and human MCs, as well as the involvement of their granule serine proteases in inflammation and the connective tissue remodeling that occurs during the different phases of the healing process of wounded skin and other organs. The accumulated data suggest that MCs, their tryptases, and their chymases play important roles in tissue repair. While MCs initially promote healing, they can be detrimental if they are chronically stimulated or if too many MCs become activated at the same time. The possibility that MCs and their granule serine proteases contribute to the formation of keloid and hypertrophic scars makes them potential targets for therapeutic intervention in the repair of damaged skin.
Asunto(s)
Diferenciación Celular/inmunología , Inflamación/enzimología , Inflamación/inmunología , Mastocitos/enzimología , Mastocitos/inmunología , Triptasas/fisiología , Cicatrización de Heridas/inmunología , Animales , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/inmunología , Feto , Humanos , Inflamación/patología , Hígado/citología , Hígado/enzimología , Hígado/inmunología , Mastocitos/patología , RatonesRESUMEN
Mouse mast cell protease (mMCP)-6-null C57BL/6 mice lost less aggrecan proteoglycan from the extracellular matrix of their articular cartilage during inflammatory arthritis than wild-type (WT) C57BL/6 mice, suggesting that this mast cell (MC)-specific mouse tryptase plays prominent roles in articular cartilage catabolism. We used ex vivo mouse femoral head explants to determine how mMCP-6 and its human ortholog hTryptase-ß mediate aggrecanolysis. Exposure of the explants to recombinant hTryptase-ß, recombinant mMCP-6, or lysates harvested from WT mouse peritoneal MCs (PMCs) significantly increased the levels of enzymatically active matrix metalloproteinases (MMP) in cartilage and significantly induced aggrecan loss into the conditioned media, relative to replicate explants exposed to medium alone or lysates collected from mMCP-6-null PMCs. Treatment of cartilage explants with tetramer-forming tryptases generated aggrecan fragments that contained C-terminal DIPEN and N-terminal FFGVG neoepitopes, consistent with MMP-dependent aggrecanolysis. In support of these data, hTryptase-ß was unable to induce aggrecan release from the femoral head explants obtained from Chloe mice that resist MMP cleavage at the DIPEN↓FFGVG site in the interglobular domain of aggrecan. In addition, the abilities of mMCP-6-containing lysates from WT PMCs to induce aggrecanolysis were prevented by inhibitors of MMP-3 and MMP-13. Finally, recombinant hTryptase-ß was able to activate latent pro-MMP-3 and pro-MMP-13 in vitro. The accumulated data suggest that human and mouse tetramer-forming tryptases are MMP convertases that mediate cartilage damage and the proteolytic loss of aggrecan proteoglycans in arthritis, in part, by activating the zymogen forms of MMP-3 and MMP-13, which are constitutively present in articular cartilage.
Asunto(s)
Agrecanos/metabolismo , Cartílago Articular/metabolismo , Mastocitos/inmunología , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Animales , Artritis/metabolismo , Células Cultivadas , Precursores Enzimáticos/metabolismo , Matriz Extracelular/metabolismo , Inflamación , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Triptasas/deficiencia , Triptasas/genética , Triptasas/metabolismoRESUMEN
BACKGROUND: Cigarette smoke-induced chronic obstructive pulmonary disease (COPD) is a life-threatening inflammatory disorder of the lung. The development of effective therapies for COPD has been hampered by the lack of an animal model that mimics the human disease in a short timeframe. OBJECTIVES: We sought to create an early-onset mouse model of cigarette smoke-induced COPD that develops the hallmark features of the human condition in a short time-frame. We also sought to use this model to better understand pathogenesis and the roles of macrophages and mast cells (MCs) in patients with COPD. METHODS: Tightly controlled amounts of cigarette smoke were delivered to the airways of mice, and the development of the pathologic features of COPD was assessed. The roles of macrophages and MC tryptase in pathogenesis were evaluated by using depletion and in vitro studies and MC protease 6-deficient mice. RESULTS: After just 8 weeks of smoke exposure, wild-type mice had chronic inflammation, mucus hypersecretion, airway remodeling, emphysema, and reduced lung function. These characteristic features of COPD were glucocorticoid resistant and did not spontaneously resolve. Systemic effects on skeletal muscle and the heart and increased susceptibility to respiratory tract infections also were observed. Macrophages and tryptase-expressing MCs were required for the development of COPD. Recombinant MC tryptase induced proinflammatory responses from cultured macrophages. CONCLUSION: A short-term mouse model of cigarette smoke-induced COPD was developed in which the characteristic features of the disease were induced more rapidly than in existing models. The model can be used to better understand COPD pathogenesis, and we show a requirement for macrophages and tryptase-expressing MCs.
Asunto(s)
Modelos Animales de Enfermedad , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Humo/efectos adversos , Triptasas/inmunología , Remodelación de las Vías Aéreas (Respiratorias) , Animales , Macrófagos/inmunología , Mastocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad Pulmonar Obstructiva Crónica/patología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Pruebas de Función Respiratoria , Nicotiana , Triptasas/deficiencia , Triptasas/genéticaRESUMEN
Mast cells (MCs) are heterogeneous cells whose phenotype is modulated by signals received from the local microenvironment. Recent studies have identified the mesenchymal-derived cytokine IL-33 as a potent direct activator of MCs, as well as regulator of their effector phenotype, and have implicated this activity in the ability of mast cells to contribute to murine experimental arthritis. We explored the hypothesis that IL-33 enables participation of synovial MCs in murine K/BxN arthritis by promoting their activation by IgG immune complexes. Compared to wild-type (WT) control mice, transgenic animals lacking the IL-33 receptor ST2 exhibited impaired MC-dependent immune complex-induced vascular permeability (flare) and attenuated K/BxN arthritis. Whereas participation of MCs in this model is mediated by the activating IgG receptor FcγRIII, we pre-incubated bone marrow-derived MCs with IL-33 and found not only direct induction of cytokine release but also a marked increase in FcγRIII-driven production of critical arthritogenic mediators including IL-1ß and CXCL2. This "priming" effect was associated with mRNA accumulation rather than altered expression of Fcγ receptors, could be mimicked by co-culture of WT but not ST2(-/-) MCs with synovial fibroblasts, and was blocked by antibodies against IL-33. In turn, WT but not ST2(-/-) MCs augmented fibroblast expression of IL-33, forming a positive feedback circuit. Together, these findings confirm a novel role for IL-33 as an amplifier of IgG immune complex-mediated inflammation and identify a potential MC-fibroblast amplification loop dependent on IL-33 and ST2.
Asunto(s)
Complejo Antígeno-Anticuerpo/fisiología , Interleucinas/fisiología , Mastocitos/metabolismo , Animales , Retroalimentación Fisiológica , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/genética , Interleucinas/metabolismo , Mastocitos/inmunología , Ratones , Ratones Transgénicos , ARN Mensajero/metabolismo , Receptores de IgG/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina/fisiologíaRESUMEN
RasGRP4 (Ras guanine nucleotide-releasing protein-4) is an intracellular, calcium-regulated guanine nucleotide exchange factor and diacylglycerol/phorbol ester receptor expressed in mast cells (MCs) and their progenitors. To study the function of this signaling protein in inflammatory disorders, a homologous recombination approach was used to create a RasGRP4-null C57BL/6 mouse line. The resulting transgenic animals had normal numbers of MCs in their tissues that histochemically and morphologically resembled those in WT C57BL/6 mice. MCs could also be generated from RasGRP4-null mice by culturing their bone marrow cells in IL-3-enriched conditioned medium. Despite these data, the levels of the transcripts that encode the proinflammatory cytokines IL-1ß and TNF-α were reduced in phorbol 12-myristate 13-acetate-treated MCs developed from RasGRP4-null mice. Although inflammation was not diminished in a Dermatophagoides farinae-dependent model of allergic airway disease, dextran sodium sulfate-induced colitis was significantly reduced in RasGRP4-null mice relative to similarly treated WT mice. Furthermore, experimental arthritis could not be induced in RasGRP4-null mice that had received K/BxN mouse serum. The latter findings raise the possibility that the pharmacologic inactivation of this intracellular signaling protein might be an effective treatment for arthritis or inflammatory bowel disease.
Asunto(s)
Artritis Experimental/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Transducción de Señal , Factores de Intercambio de Guanina Nucleótido ras/metabolismo , Animales , Antígenos Dermatofagoides/toxicidad , Artritis Experimental/genética , Artritis Experimental/patología , Artritis Experimental/terapia , Asma/inducido químicamente , Asma/genética , Asma/metabolismo , Asma/patología , Carcinógenos/farmacología , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Citocinas/farmacología , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/patología , Enfermedades Inflamatorias del Intestino/terapia , Ratones , Ratones Noqueados , Acetato de Tetradecanoilforbol/farmacología , Factores de Intercambio de Guanina Nucleótido ras/genéticaRESUMEN
T-cell intracellular antigen-1 (TIA-1) is a translational repressor that dampens the production of proinflammatory cytokines and enzymes. In this study we investigated the role of TIA-1 in a mouse model of pulmonary inflammation induced by exposure to the allergenic extract (Df) of the house dust mite Dermatophagoides farinae. When intranasally challenged with a low dose of Df, mice lacking TIA-1 protein (Tia-1(-/-)) showed more severe airway and tissue eosinophilia, infiltration of lung bronchovascular bundles, and goblet cell metaplasia than wild-type littermates. Tia-1(-/-) mice also had higher levels of Df-specific IgE and IgG(1) in serum and ex vivo restimulated Tia-1(-/-) lymph node cells and splenocytes transcribed and released more Th2/Th17 cytokines. To evaluate the site of action of TIA-1, we studied the response to Df in bone marrow chimeras. These experiments revealed that TIA-1 acts on both hematopoietic and non-hematopoietic cells to dampen pulmonary inflammation. Our results identify TIA-1 as a negative regulator of allergen-mediated pulmonary inflammation in vivo. Thus, TIA-1 might be an important player in the pathogenesis of bronchial asthma.
Asunto(s)
Dermatophagoides farinae/inmunología , Eosinofilia/inmunología , Pulmón/inmunología , Neumonía/inmunología , Proteínas de Unión al ARN/inmunología , Células Th17/inmunología , Células Th2/inmunología , Administración Intranasal , Alérgenos/administración & dosificación , Alérgenos/inmunología , Animales , Antígenos Dermatofagoides/administración & dosificación , Antígenos Dermatofagoides/inmunología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/patología , Movimiento Celular , Quimera/inmunología , Citocinas/biosíntesis , Citocinas/inmunología , Dermatophagoides farinae/química , Modelos Animales de Enfermedad , Eosinofilia/inducido químicamente , Eosinofilia/complicaciones , Eosinofilia/patología , Células Caliciformes , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Noqueados , Neumonía/inducido químicamente , Neumonía/complicaciones , Neumonía/patología , Proteínas de Unión al ARN/genética , Antígeno Intracelular 1 de las Células T , Células Th17/metabolismo , Células Th2/metabolismoRESUMEN
The mouse and human TPSB2 and TPSAB1 genes encode tetramer-forming tryptases stored in the secretory granules of mast cells (MCs) ionically bound to heparin-containing serglycin proteoglycans. In mice these genes encode mouse MC protease-6 (mMCP-6) and mMCP-7. The corresponding human genes encode a family of serine proteases that collectively are called hTryptase-ß. We previously showed that the α chain of fibrinogen is a preferred substrate of mMCP-7. We now show that this plasma protein also is highly susceptible to degradation by hTryptase-ß· and mMCP-6·heparin complexes and that Lys(575) is a preferred cleavage site in the protein α chain. Because cutaneous mouse MCs store substantial amounts of mMCP-6·heparin complexes in their secretory granules, the passive cutaneous anaphylaxis reaction was induced in the skin of mMCP-6(+)/mMCP-7(-) and mMCP-6(-)/mMCP-7(-) C57BL/6 mice. In support of the in vitro data, fibrin deposits were markedly increased in the skin of the double-deficient mice 6 h after IgE-sensitized animals were given the relevant antigen. Fibrinogen is a major constituent of the edema fluid that accumulates in tissues when MCs degranulate. Our discovery that mouse and human tetramer-forming tryptases destroy fibrinogen before this circulating protein can be converted to fibrin changes the paradigm of how MCs hinder fibrin deposition and blood coagulation internally. Because of the adverse consequences of fibrin deposits in tissues, our data explain why mice and humans lack a circulating protease inhibitor that rapidly inactivates MC tryptases and why mammals have two genes that encode tetramer-forming serine proteases that preferentially degrade fibrinogen.
Asunto(s)
Coagulación Sanguínea , Fibrina/metabolismo , Fibrinógeno/metabolismo , Heparina/metabolismo , Mastocitos/enzimología , Proteolisis , Vesículas Secretoras/enzimología , Trombina/metabolismo , Triptasas/metabolismo , Anafilaxia/inducido químicamente , Anafilaxia/enzimología , Anafilaxia/genética , Anafilaxia/patología , Animales , Edema/enzimología , Edema/genética , Edema/patología , Fibrina/genética , Fibrinógeno/genética , Heparina/genética , Humanos , Inmunoglobulina E/metabolismo , Mastocitos/patología , Ratones , Ratones Noqueados , Vesículas Secretoras/genética , Piel/enzimología , Piel/patología , Trombina/genética , Triptasas/genéticaRESUMEN
RATIONALE: Mast cells (MCs) contribute to the formation of abdominal aortic aneurysms (AAAs) by producing biologically active mediators. Tryptase is the most abundant MC granule protein and participates in MC activation, protease maturation, leukocyte recruitment, and angiogenesis-all processes critical to AAA pathogenesis. OBJECTIVE: To test the hypothesis that tryptase participates directly in AAA formation. METHODS AND RESULTS: Immunohistochemistry demonstrated enhanced tryptase staining in media and adventitia of human and mouse AAA lesions. Serum tryptase levels correlated significantly with the annual expansion rate of AAA before (r = 0.30, P = 0.003) and after (r = 0.29, P = 0.005) adjustment for common AAA risk factors in a patient follow-up study, and associated with risks for later surgical repair or overall mortality before (P = 0.009, P = 0.065) and after (P = 0.004, P = 0.001) the adjustment. Using MC protease-6-deficient mice (Mcpt6(-/-)) and aortic elastase perfusion-induced experimental AAAs, we proved a direct role of this tryptase in AAA pathogenesis. Whereas all wild-type (WT) mice developed AAA at 14 or 56 days postperfusion, Mcpt6(-/-) mice were fully protected. AAA lesions from Mcpt6(-/-) mice had fewer inflammatory and apoptotic cells, and lower chemokine levels, than did those from WT mice. MC from WT mice restored reduced AAA lesions and lesion inflammatory cell content in MC-deficient Kit(W-sh/W-sh) mice, but those prepared from Mcpt6(-/-) mice did not. Mechanistic studies demonstrated that tryptase deficiency affected endothelial cell (EC) chemokine and cytokine expression, monocyte transmigration, smooth-muscle cell apoptosis, and MC and AAA lesion cysteinyl cathepsin expression and activities. CONCLUSIONS: This study establishes the direct participation of MC tryptase in the pathogenesis of experimental AAAs, and suggests that levels of this protease can serve as a novel biomarker for abdominal aortic expansion.
Asunto(s)
Aneurisma de la Aorta Abdominal , Mastocitos/enzimología , Triptasas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Aorta Abdominal/inmunología , Aorta Abdominal/metabolismo , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/inmunología , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Apoptosis/fisiología , Biomarcadores/sangre , Catepsinas/genética , Catepsinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Granulocitos/inmunología , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Mutantes , Triptasas/genéticaRESUMEN
Patients with inflammatory bowel disease (IBD) have increased numbers of human tryptase-ß (hTryptase-ß)-positive mast cells (MCs) in the gastrointestinal tract. The amino acid sequence of mouse mast cell protease (mMCP)-6 is most similar to that of hTryptase-ß. We therefore hypothesized that this mMCP, or the related tryptase mMCP-7, might have a prominent proinflammatory role in experimental colitis. The dextran sodium sulfate (DSS) and trinitrobenzene sulfonic acid (TNBS) colitis models were used to evaluate the differences between C57BL/6 (B6) mouse lines that differ in their expression of mMCP-6 and mMCP-7 with regard to weight loss, colon histopathology, and endoscopy scores. Microarray analyses were performed, and confirmatory real-time PCR, ELISA, and/or immunohistochemical analyses were carried out on a number of differentially expressed cytokines, chemokines, and matrix metalloproteinases (MMPs). The mMCP-6-null mice that had been exposed to DSS had significantly less weight loss as well as significantly lower pathology and endoscopy scores than similarly treated mMCP-6-expressing mice. This difference in colitis severity was confirmed endoscopically in the TNBS-treated mice. Evaluation of the distal colon segments revealed that numerous proinflammatory cytokines, chemokines that preferentially attract neutrophils, and MMPs that participate in the remodeling of the ECM were all markedly increased in the colons of DSS-treated WT mice relative to untreated WT mice and DSS-treated mMCP-6-null mice. Collectively, our data show that mMCP-6 (but not mMCP-7) is an essential MC-restricted mediator in chemically induced colitis and that this tryptase acts upstream of many of the factors implicated in IBD.
Asunto(s)
Enfermedades Inflamatorias del Intestino/inmunología , Mastocitos/inmunología , Triptasas/inmunología , Animales , Quimiocinas/inmunología , Colon/metabolismo , Colon/patología , Citocinas/inmunología , Sulfato de Dextran/toxicidad , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/enzimología , Enfermedades Inflamatorias del Intestino/patología , Mastocitos/metabolismo , Metaloproteinasas de la Matriz/inmunología , Ratones , Ratones Endogámicos C57BL , Análisis por Micromatrices , Reacción en Cadena de la Polimerasa , Ácido Trinitrobencenosulfónico/toxicidad , Triptasas/metabolismoRESUMEN
A second-degree epidermal scald burn in mice elicits an inflammatory response mediated by natural IgM directed to nonmuscle myosin with complement activation that results in ulceration and scarring. We find that such burn injury is associated with early mast cell (MC) degranulation and is absent in WBB6F1-Kit(W)/Kit(Wv) mice, which lack MCs in a context of other defects due to a mutation of the Kit receptor. To address further an MC role, we used transgenic strains with normal lineage development and a deficiency in a specific secretory granule component. Mouse strains lacking the MC-restricted chymase, mouse MC protease (mMCP)-4, or elastase, mMCP-5, show decreased injury after a second-degree scald burn, whereas mice lacking the MC-restricted tryptases, mMCP-6 and mMCP-7, or MC-specific carboxypeptidase A3 activity are not protected. Histologic sections showed some disruption of the epidermis at the scald site in the protected strains suggesting the possibility of topical reconstitution of full injury. Topical application of recombinant mMCP-5 or human neutrophil elastase to the scalded area increases epidermal injury with subsequent ulceration and scarring, both clinically and morphologically, in mMCP-5-deficient mice. Restoration of injury requires that topical administration of recombinant mMCP-5 occurs within the first hour postburn. Importantly, topical application of human MC chymase restores burn injury to scalded mMCP-4-deficient mice but not to mMCP-5-deficient mice revealing nonredundant actions for these two MC proteases in a model of innate inflammatory injury with remodeling.
Asunto(s)
Quemaduras/inmunología , Quimasas/inmunología , Cicatriz/inmunología , Epidermis/inmunología , Mastocitos/inmunología , Modelos Inmunológicos , Serina Endopeptidasas/inmunología , Animales , Quemaduras/enzimología , Quemaduras/genética , Quemaduras/patología , Carboxipeptidasas A/genética , Carboxipeptidasas A/inmunología , Carboxipeptidasas A/metabolismo , Degranulación de la Célula/genética , Degranulación de la Célula/inmunología , Quimasas/genética , Quimasas/metabolismo , Quimasas/farmacología , Cicatriz/enzimología , Cicatriz/genética , Cicatriz/patología , Epidermis/enzimología , Epidermis/patología , Humanos , Inmunoglobulina M/genética , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Inflamación , Elastasa de Leucocito/genética , Elastasa de Leucocito/inmunología , Elastasa de Leucocito/metabolismo , Elastasa de Leucocito/farmacología , Mastocitos/enzimología , Mastocitos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Mutantes , Miosinas/genética , Miosinas/inmunología , Miosinas/metabolismo , Proteínas Proto-Oncogénicas c-kit , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/farmacología , Triptasas/genética , Triptasas/inmunología , Triptasas/metabolismo , Triptasas/farmacologíaRESUMEN
A characteristic feature of tissue resident human mast cells (MCs) is their hTryptase-beta-rich cytoplasmic granules. Mouse MC protease-6 (mMCP-6) is the ortholog of hTryptase-beta, and we have shown that this tetramer-forming tryptase has beneficial roles in innate immunity but adverse roles in inflammatory disorders like experimental arthritis. Because the key tissue factors that control tryptase expression in MCs have not been identified, we investigated the mechanisms by which fibroblasts mediate the expression and granule accumulation of mMCP-6. Immature mouse bone marrow-derived MCs (mBMMCs) co-cultured with fibroblast-like synoviocytes (FLS) or mouse 3T3 fibroblasts markedly increased their levels of mMCP-6. This effect was caused by an undefined soluble factor whose levels could be increased by exposing FLS to tumor necrosis factor-alpha or interleukin (IL)-1beta. Gene expression profiling of mBMMCs and FLS for receptor.ligand pairs of potential relevance raised the possibility that IL-33 was a sought after fibroblast-derived factor that promotes tryptase expression and granule maturation via its receptor IL1RL1/ST2. MCs lacking IL1RL1 exhibited defective fibroblast-driven tryptase accumulation, whereas recombinant IL-33 induced mMCP-6 mRNA and protein accumulation in wild-type mBMMCs. In agreement with these data, synovial MCs from IL1RL1-null mice exhibited a marked reduction in mMCP-6 expression. IL-33 is the first factor shown to modulate tryptase expression in MCs at the mRNA and protein levels. We therefore have identified a novel pathway by which mesenchymal cells exposed to inflammatory cytokines modulate the phenotype of local MCs to shape their immune responses.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Interleucinas/metabolismo , Receptores de Interleucina/metabolismo , Triptasas/metabolismo , Animales , Técnicas de Cocultivo , Citocinas/metabolismo , Citoplasma/metabolismo , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Sistema Inmunológico , Inmunohistoquímica/métodos , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , Ligandos , Ratones , Ratones TransgénicosRESUMEN
BACKGROUND: Mast cell chymase may participate in the pathogenesis of human abdominal aortic aneurysm (AAA), yet a direct contribution of this serine protease to AAA formation remains unknown. METHODS AND RESULTS: Human AAA lesions had high numbers of chymase-immunoreactive mast cells. Serum chymase level correlated with AAA growth rate (P=0.009) in a prospective clinical study. In experimental AAA produced by aortic elastase perfusion in wild-type (WT) mice or those deficient in the chymase ortholog mouse mast cell protease-4 (mMCP-4) or deficient in mMCP-5 (Mcpt4(-/-), Mcpt5(-/-)), Mcpt4(-/-) but not Mcpt5(-/-) had reduced AAA formation 14 days after elastase perfusion. Even 8 weeks after perfusion, aortic expansion in Mcpt4(-/-) mice fell by 50% compared with that of the WT mice (P=0.0003). AAA lesions in Mcpt4(-/-) mice had fewer inflammatory cells and less apoptosis, angiogenesis, and elastin fragmentation than those of WT mice. Although Kit(W-sh/W-sh) mice had protection from AAA formation, reconstitution with mast cells from WT mice, but not those from Mcpt4(-/-) mice, partially restored the AAA phenotype. Mechanistic studies suggested that mMCP-4 regulates expression and activation of cysteine protease cathepsins, elastin degradation, angiogenesis, and vascular cell apoptosis. CONCLUSIONS: High chymase-positive mast cell content in human AAA lesions, greatly reduced AAA formation in Mcpt4(-/-) mice, and significant correlation of serum chymase levels with human AAA expansion rate suggests participation of mast cell chymase in the progression of human and mouse AAA.