RESUMEN
BACKGROUND: Proteinase-Activated Receptor-2 (PAR2 ) is a G protein-coupled receptor activated by serine proteinases. We have shown that PAR2 activation in the airways is involved in the development of allergic inflammation and airway hyperresponsiveness (AHR) in acute murine models. We hypothesized that functional inhibition of PAR2 prevents allergic inflammation, AHR and airway remodeling in chronic allergic airway inflammation models. MATERIAL AND METHODS: We developed and used a 12 week model of cockroach extract (CE)-mediated AHR, airway inflammation and remodeling in BALB/c mice. RESULTS: Mice sensitized and challenged with CE for 12 weeks exhibit AHR, increased numbers of eosinophils in bronchoalveolar lavage (BAL) and increased collagen content in the lung tissue compared to saline controls. Administration of an anti-PAR2 antibody, SAM-11, after the initial development of airway inflammation significantly inhibited all these parameters. CONCLUSIONS: Our data demonstrate that PAR2 signaling plays a key role in CE-induced AHR and airway inflammation/remodeling in long term models of allergic airway inflammation. Targeting PAR2 activation may be a successful therapeutic strategy for allergic asthma.
Asunto(s)
Asma/inmunología , Receptor PAR-2/antagonistas & inhibidores , Receptor PAR-2/inmunología , Remodelación de las Vías Aéreas (Respiratorias)/inmunología , Animales , Hiperreactividad Bronquial/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Enfermedad Crónica , Modelos Animales de Enfermedad , Inflamación/inmunología , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: Asthma is an independent risk factor for invasive pneumococcal disease; however, the immune response of adult asthma patients to pneumococcal vaccination is unknown. We explore the serologic response of patients with moderate to severe asthma to the 23-valent pneumococcal polysaccharide vaccine (PPSV23). METHODS: Seventeen moderate to severe adult asthma patients that had not been vaccinated against pneumococcus over the 5 previous years were prospectively recruited from a tertiary care asthma clinic. Serum was analyzed for the presence of antibodies to five capsular polysaccharide (CP) antigens (6B, 9V, 19A, 19F, 23F) before and 4 weeks after PPSV23 vaccination. RESULTS: There was a wide variability in baseline anti-CP antibody concentrations. Other than for serotype 19A, our patients frequently have baseline anti-CP antibody concentrations below 1 µg/mL (35% for serotype 19F, 41% for serotypes 9V and 23F, and 59% for serotype 6B). All post-vaccination geometric mean antibody concentrations were significantly higher than baseline. In the 31 tests where the baseline antibody concentration was <1 µg/mL, 77.4% had at least a twofold increase post-vaccination. Despite this, a large proportion of post-vaccination anti-CP antibody concentrations remained <1 µg/mL (51.6% of tests). Nine patients had at least one anti-CP antibody concentration <1 µg/mL post-vaccination. There was no difference between these patients and the remaining eight patients in demographic or clinical variables. CONCLUSIONS: Patients with moderate to severe asthma have variable baseline and low post-vaccination antibody concentrations to common CP antigens included in the PPSV23 vaccine. The clinical relevance of these observations remains to be determined since the threshold concentration in adults required for clinical protection from invasive pneumococcal disease is uncertain.
RESUMEN
BACKGROUND: Allergy to the German cockroach (Blattella germanica) is a significant asthma risk factor for inner-city communities. Cockroach, like other allergens, contains trypsin-like enzyme activity that contributes to allergenicity and airway inflammation by activating proteinase-activated receptors (PARs). To date, the enzymes responsible for the proteolytic activity of German cockroach allergen have not been characterized. OBJECTIVES: We aimed to identify, isolate and characterize the trypsin-like proteinases in German cockroach allergen extracts used for clinical skin tests. For each enzyme, we sought to determine (1) its substrate and inhibitor enzyme kinetics (Km and IC50), (2) its amino acid sequence and (3) its ability to activate calcium signalling and/or ERK1/2 phosphorylation via PAR2. METHODS: Using a trypsin-specific activity-based probe, we detected three distinct enzymes that were isolated using ion-exchange chromatography. Each enzyme was sequenced by mass spectometery (deconvoluted with an expressed sequence tag library), evaluated kinetically for its substrate/inhibitor profile and assessed for its ability to activate PAR2 signalling. FINDINGS: Each of the three serine proteinase activity-based probe-labelled enzymes isolated was biochemically distinct, with different enzyme kinetic profiles and primary amino acid sequences. The three enzymes showed a 57%-71% sequence identity with a proteinase previously cloned from the American cockroach (Per a 10). Each enzyme was found to activate both Ca++ and MAPK signalling via PAR2. CONCLUSIONS AND RELEVANCE: We have identified three different serine proteinases from the German cockroach that may, via PAR2 activation, play different roles for allergen sensitization in vivo and may represent attractive therapeutic targets for asthma.
Asunto(s)
Alérgenos/inmunología , Cucarachas/inmunología , Hipersensibilidad/inmunología , Serina Proteasas/inmunología , Secuencia de Aminoácidos , Animales , Blattellidae/inmunología , Señalización del Calcio , Línea Celular , Cromatografía por Intercambio Iónico , Humanos , Hipersensibilidad/genética , Hipersensibilidad/metabolismo , Ligandos , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , Serina Proteasas/química , Transducción de Señal , beta-Arrestinas/metabolismoRESUMEN
BACKGROUND: Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTh2) is a receptor for PGD2 and expressed by T cells, eosinophils, basophils, and ILC2 cells. CRTh2 expression by CD4(+) T cells identifies the Th2 subset, and these cells have been characterized as allergen-specific central memory Th2 cells. Recently, activation of the PGD2 -CRTh2 pathway in the lungs was associated with severe asthma. OBJECTIVE: To assess circulating levels of Th2 cells and related mediators in severe asthma and those who experience asthma exacerbations. METHODS: Peripheral blood cells expressing CRTh2 were characterized by flow cytometry and qRT-PCR. Serum IL-13 and PGD2 were measured by ELISA and compared with asthma severity and tendency to exacerbate. RESULTS: Severe asthmatics had more circulating CD4(+) CRTh2(+) T cells, CRTh2 and GATA3 mRNA, and a higher level of serum IL-13 compared to mild/moderate asthmatics. The proportion of CD4(+) CRTh2(+) T cells was associated with lower lung function and was highest in severe asthmatics that exacerbated in the last year. Circulating CD4(+) CRTh2(+) T cells, unlike eosinophils, were positively correlated with inhaled steroid dose. CONCLUSIONS AND CLINICAL RELEVANCE: Elevated levels of circulating CD4(+) CRTh2(+) T cells are a feature of severe asthma, despite high-dose corticosteroids. Tracking the systemic level of these cells may help identify type 2 severe asthmatics at risk of exacerbation.
Asunto(s)
Asma/sangre , Asma/inmunología , Subgrupos de Linfocitos T/inmunología , Células Th2/inmunología , Corticoesteroides/uso terapéutico , Adulto , Animales , Asma/diagnóstico , Asma/metabolismo , Biomarcadores , Citocinas/sangre , Citocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Inmunofenotipificación , Mediadores de Inflamación/sangre , Mediadores de Inflamación/metabolismo , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Ratones , Persona de Mediana Edad , Fenotipo , Pruebas de Función Respiratoria , Factores de Riesgo , Índice de Severidad de la Enfermedad , Transducción de Señal , Subgrupos de Linfocitos T/metabolismo , Células Th2/metabolismoRESUMEN
BACKGROUND: Proteinase-activated receptor 2 (PAR2 ) is a G protein-coupled receptor activated by trypsin-like serine proteinases. PAR2 activation has been associated with inflammation including allergic airway inflammation. We have also shown that PAR2 activation in the airways leads to allergic sensitization. The exact contribution of PAR2 in the development of eosinophilic inflammation and airway hyperresponsiveness (AHR) in sensitized individuals is not clear. OBJECTIVE: To investigate whether functional inhibition of PAR2 during allergen challenge of allergic mice would inhibit allergen-induced AHR and inflammation in mouse models of asthma. METHODS: Mice were sensitized and challenged with ovalbumin (OVA) or cockroach extract (CE). To investigate the role of PAR2 in the development of AHR and airway inflammation, we administered blocking anti-PAR2 antibodies, or a cell permeable peptide inhibitor of PAR2 signalling, pepducin, i.n. before allergen challenges and then assessed AHR and airway inflammation. RESULTS: Administration of anti-PAR2 antibodies significantly inhibited OVA- and CE-induced AHR and airway inflammation. In particular, two anti-PAR2 antibodies, the monoclonal SAM-11 and polyclonal B5, inhibited AHR, airway eosinophilia, the increase of cytokines in the lung tissue and antigen-specific T cell proliferation, but had no effect on antigen-specific IgG and IgE levels. Pepducin was also effective in inhibiting AHR and airway inflammation in an OVA model of allergic airway inflammation. CONCLUSIONS AND CLINICAL RELEVANCE: Functional blockade of PAR2 in the airways during allergen challenge improves allergen-induced AHR and inflammation in mice. Therefore, topical PAR2 blockade in the airways, through anti-PAR2 antibodies or molecules that interrupt PAR2 signalling, has the potential to be used as a therapeutic option in allergic asthma.
Asunto(s)
Alérgenos/inmunología , Asma/inmunología , Asma/metabolismo , Receptor PAR-2/antagonistas & inhibidores , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Asma/genética , Biomarcadores , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Inmunización , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Pulmón/inmunología , Pulmón/metabolismo , Masculino , Ratones , Ratones Noqueados , Ovalbúmina/inmunología , Hipersensibilidad Respiratoria/genética , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
In 2012, we received 683 submissions and published 20 editorials, 38 reviews, 11 letters and 128 original articles. This represents an acceptance rate for original papers in the range of 20%. About 30% of original papers were triaged not to go out to review, either because the editors did not feel they had sufficient priority for publication or because the topic did not feel right for the readers of the journal. We place great emphasis on obtaining sufficient high-quality reviews to make our decisions on publication fair and consistent. Inevitably, however, there is a degree of luck about what gets published and which papers miss out, and we are always happy to receive an appeal on our decisions either at the triage stage or after review. This gives us the opportunity to revisit the decision and revise it or explain in more detail to the authors the basis for the decision. Once again in 2012, we were delighted by the quality of the papers submitted and the breadth and depth of research into allergic disease that it revealed. The pattern of papers submitted was similar in previous years with considerable emphasis on all aspects of asthma and rhinitis. We were particularly pleased with our special issue on severe asthma. Elucidating mechanisms using either animal models or patients has always been a major theme of the journal, and the excellent work in these areas has been summarized by Harissios Vliagoftis with a particularly interesting section on early-life events guiding the development of allergic disease, which understandably continue to be a major theme of research. Magnus Wickman summarized the papers looking at the epidemiology of allergic disease including work from birth cohorts, which are an increasingly rich source of data on risk factors for allergic disease, and two papers on the epidemiology of anaphylaxis. Giovanni Passalacqua discussed the papers in the clinical allergy section of the journal, and Adriano Mari who runs the excellent Allergome website discussed the papers looking at allergens including characterization and the relative usefulness of allergen arrays versus single extracts in diagnosis and management.
Asunto(s)
Hipersensibilidad/diagnóstico , Hipersensibilidad/terapia , Animales , Humanos , Hipersensibilidad/epidemiología , Hipersensibilidad/etiologíaRESUMEN
BACKGROUND: Many aeroallergens contain proteinase activity and are able to induce allergic sensitization when presented to mucosal surfaces. Some of these allergens activate proteinase-activated receptor-2 (PAR2 ). OBJECTIVE: To determine the role of PAR2 activation in a murine house dust mite (HDM) allergy model. METHODS: We sensitized and challenged PAR2 -deficient mice with HDM, and examined allergic outcomes compared to wild-type animals. To focus on the role of PAR2 in allergic sensitization, we administered a PAR2 blocking antibody to wild-type animals during the sensitization phase and examined the outcomes immediately after sensitization or following subsequent allergen challenge. RESULTS: We found PAR2 -deficient mice sensitized and challenged with HDM failed to develop airway inflammation, did not produce HDM-specific IgG1 and had less IL-4 mRNA in the lungs than wild-type animals. Prevention of PAR2 activation during sensitization in wild-type mice diminished the levels of Th2 mediators, including IL-4, IL-5 and IL-13, in the lungs. Blocking PAR2 during the sensitization phase also led to decreased manifestations of allergic disease, including airway hyperresponsiveness (AHR) and airway inflammation following subsequent allergen challenge. HDM-induced proliferation of splenocytes obtained from animals sensitized in the presence of PAR2 antibody was reduced relative to those that did not receive antibody. The effect of PAR2 blockade could be transferred to naïve mice through splenic CD4(+) T cells from sensitized mice. CONCLUSIONS AND CLINICAL RELEVANCE: PAR2 activation plays a key role during the sensitization phase of our HDM allergy model, leading to increased lung cytokine production and augmented lung reactivity. PAR2 activation is a common mechanism for sensitization to a wide variety of allergens and is therefore a potential pharmacological target to prevent allergy.
Asunto(s)
Alérgenos/inmunología , Hipersensibilidad/inmunología , Hipersensibilidad/metabolismo , Pyroglyphidae/inmunología , Receptor PAR-2/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Hipersensibilidad/genética , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Noqueados , Receptor PAR-2/antagonistas & inhibidores , Receptor PAR-2/genética , Bazo/citología , Bazo/inmunología , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
BACKGROUND: CRTh2 (chemoattractant-receptor homologous molecule expressed on Th2 cells) is expressed by Th2 cells and other cells involved in allergic inflammation. Single nucleotide polymorphisms (SNPs) in CRTh2 (rs11571288, rs545659, rs634681) have been associated with various phenotypes of allergy in ethnically distinct populations. Here, we assessed the association between CRTh2 rs533116 and allergic asthma, expression of CRTh2 and Th2 cytokine production. METHODS: CRTh2 rs533116 was genotyped in an ethnically diverse population (n = 1282). The proportion of cells expressing CRTh2 was determined in peripheral blood from subjects with allergic airways disease and controls as well as with in vitro differentiated Th2 cells. Receptor function was assessed by stimulating Th2 cells with the CRTh2-specific agonist 13,14-dihydro-15-keto-PGD(2) (DK-PGD(2) ) and measuring IL-4 and IL-13 by intracellular staining and ELISA. RESULTS: CRTh2 rs533116 was associated with allergic asthma in White people (2.67 [1.09-6.55], P < 0.05), and expression of CRTh2 was higher in subjects with allergic airways disease compared to controls (P < 0.05). Among allergic individuals, the AA genotype was significantly associated with more eosinophils and higher expression of CRTh2 by both CD4(+) T cells and eosinophils (P < 0.05). In vitro, the AA genotype was associated with a higher proportion of CRTh2(+) cells during Th2 differentiation as well as more IL-4 and IL-13 expression following DK-PGD(2) stimulation (P < 0.05). CONCLUSIONS: These findings show an association between CRTh2 rs533116 and allergic asthma and suggest this may be mediated by elevated expression of CRTh2, leading to higher numbers of circulating eosinophils and Th2 cytokine production.
Asunto(s)
Asma/genética , Polimorfismo de Nucleótido Simple , Receptores Inmunológicos/genética , Receptores de Prostaglandina/genética , Adulto , Asma/inmunología , Diferenciación Celular , Citocinas/biosíntesis , Eosinófilos/fisiología , Femenino , Humanos , Masculino , Células Th2/citologíaRESUMEN
BACKGROUND: Indoleamine 2,3 dioxygenase (IDO), the rate-limiting enzyme in tryptophan catabolism, is important in generating tolerance at the foetal-maternal interface. Studies using 1-methyl-tryptophan (1-MT), the specific inhibitor of IDO, showed that this enzyme is important in interferon-gamma (IFN-gamma)-dependent inhibition of allergic inflammation in the respiratory airway during immunotherapy. AIMS OF STUDY: We investigated the role of IDO in the development of allergic sensitization, leading to allergic inflammation and airway hyper-responsiveness (AHR). METHODS: We used a mouse model to generate mucosal tolerance to lipopolysaccharide-free ovalbumin (OVA) following repeated intranasal inoculation of OVA over a 3-day period. We tested the successful induction of tolerance by subsequent intraperitoneal (i.p.) sensitization followed by intranasal challenge with OVA. A slow-release pellet of 1-MT implanted into mice was used to block IDO activity prior to repeated intranasal inoculation of OVA. We measured T-cell proliferation in response to OVA, determined airway inflammation, and measured AHR to intranasal methacholine to investigate the role of IDO in sensitization to OVA. RESULTS: Repeated intranasal administration of OVA generated tolerance and prevented a subsequent sensitization to OVA via the i.p. route. This response was inhibited in mice receiving a slow-release pellet of 1-MT. However, we successfully reconstituted tolerance in mice receiving 1-MT following intra-peritoneal injection of a mixture of kynurenine and hydroxyanthranilic acid. CONCLUSION: Our data suggest that, in addition to their role in IFN-gamma-mediated inhibition of allergic airway inflammation, products of tryptophan catabolism play an important role in the prevention of sensitization to potential allergens in the respiratory airway.
Asunto(s)
Tolerancia Inmunológica/fisiología , Inmunidad Mucosa/efectos de los fármacos , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/metabolismo , Triptófano/análogos & derivados , Alérgenos/inmunología , Animales , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferón gamma/inmunología , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Mucosa Respiratoria/inmunología , Linfocitos T/inmunología , Triptófano/metabolismoRESUMEN
Mast cells (MC) are major effector cells of IgE-mediated allergic inflammation. However, it has become increasingly clear that they also play important roles in a diversity of physiological and pathological processes. Recent advances have focused on the importance of MC in both innate and adaptive immune responses and have fostered studies of MC beyond the myopic focus on allergic reactions. MC possess a great variety of surface receptors and may be activated by inflammatory mediators, immunoglobulins, proteases, hormones, neuropeptides and bacterial products. Following activation they produce a plethora of pro-inflammatory mediators and may participate in inflammatory reactions in many organs. This review focuses on the role of MC in inflammatory reactions in mucosal surfaces with particular emphasis on their role in asthma and gastrointestinal inflammatory conditions.
Asunto(s)
Mastocitos/inmunología , Membrana Mucosa/inmunología , Apoptosis/inmunología , Asma/inmunología , Adhesión Celular/inmunología , Degranulación de la Célula , Movimiento Celular/inmunología , Cistitis Intersticial/complicaciones , Cistitis Intersticial/inmunología , Hipersensibilidad a los Alimentos/inmunología , Gastritis/inmunología , Humanos , Inmunidad Innata , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Mastocitos/metabolismo , Mastocitos/fisiología , Modelos Biológicos , Membrana Mucosa/metabolismoRESUMEN
Under physiological conditions, skin mast cells preferentially localize around nerves, blood vessels and hair follicles. This observation, which dates back to Paul Ehrlich, intuitively suggests that these enigmatic, multifacetted protagonists of natural immunity are functionally relevant to many more aspects of tissue physiology than just to the generation of inflammatory and vasodilatory responses to IgE-dependent environmental antigens. And yet, for decades, mainstream-mast cell research has been dominated by a focus on the -undisputedly prominent and important - mast cell functions in type I immune responses and in the pathogenesis and management of allergic diseases. Certainly, it is hard to believe that the very large and rather selectively distributed number of mast cells in normal, uninflamed, non-infected, non-traumatized mammalian skin or mucosal tissue simply hanging around there lazily day and night, just wait for the odd allergen or parasite-associated antigen to come by so the mast cell can finally swing into action. Indeed, the past decade has witnessed a renaissance of mast cell research 'beyond allergy', along with a more systematic exploration of the surprisingly wide range of physiological functions that mast cells may be involved in. The current debate sketches many exciting horizons that have recently come into our vision during this intriguing, ongoing search.
Asunto(s)
Mastocitos/citología , Mastocitos/fisiología , Animales , Humanos , Sistema Inmunológico , Inmunoglobulina G/química , Inflamación , Modelos Biológicos , Cicatrización de HeridasRESUMEN
BACKGROUND: Epithelium is considered an active participant in allergic inflammation. Proteinase-activated receptor (PAR) 2 is expressed in a variety of cell types, including epithelial cells, and has been implicated in inflammation. OBJECTIVE: PAR-2-mediated activation of airway epithelial cells induces the release of mediators that could promote eosinophil survival and mediate eosinophil recruitment. METHODS: PAR-2-activating peptides were used to activate the human airway epithelial cell line A549, as well as primary cultures of small airway epithelial cells (SAECs). Human peripheral blood eosinophils were cultured in the presence or absence of epithelial cell supernatants. Survival was assessed by using an Annexin V apoptosis detection kit. GM-CSF and eotaxin were measured by using ELISA. RESULTS: Eosinophils undergo apoptosis in the absence of growth factors. Supernatants from PAR-2-activated A549 epithelial cells increased eosinophil survival. Supernatants from resting SAECs also increased eosinophil survival, but supernatants from PAR-2-activated SAECs showed a greater effect. The effect of PAR-2-activated epithelial cell supernatants on eosinophil survival was completely inhibited by a neutralizing anti-GM-CSF antibody but not an anti-IL-5 antibody. Resting A549 cells did not release any detectable GM-CSF, whereas PAR-2-activated cells released 35 pg/10(6) cells. Resting SAECs released 754.3 pg/10(6) cells of GM-CSF, which was further increased to 1360.5 pg/10(6) cells after PAR-2-mediated activation. Budesonide inhibited this PAR-2 effect. PAR-2-activated epithelial cells also released eotaxin. CONCLUSION: PAR-2-mediated activation of airway epithelial cells induced release of GM-CSF, which promoted eosinophil survival and activation. It also induced release of eotaxin, which could mediate eosinophil recruitment to the airways.
Asunto(s)
Quimiocinas CC , Eosinófilos/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Pulmón/metabolismo , Receptores de Trombina/fisiología , Línea Celular , Quimiocina CCL11 , Citocinas/metabolismo , Células Epiteliales/metabolismo , Humanos , Receptor PAR-2RESUMEN
BACKGROUND: Matrix metalloproteinases (MMPs) digest extracellular matrix components and might be important mediators of tissue remodeling. Proteinase activated receptor-2 (PAR-2) is expressed in a variety of cell types including epithelial cells. PAR-2 receptors are activated by serine proteases such as trypsin and mast cell tryptase and have been implicated in inflammation. OBJECTIVE: To study the effects of PAR-2-mediated airway epithelial cell activation on the production of MMP-9. METHODS: A specific PAR-2-activating peptide and trypsin were used to activate the human airway epithelial cell line A549 as well as primary cultures of small airway epithelial cells (SAEC). MMP-2 and MMP-9 messenger RNA and enzymatic activity were evaluated by RT-PCR and gelatin zymography, respectively. RESULTS: PAR-2-activating peptides upregulated MMP-9 mRNA expression and release of MMP-9 enzymatic activity from airway epithelial cells but had no effect on MMP-2 production. Dexamethasone and budesonide (10(-6) to 10(-10) mmol) inhibited PAR-2-mediated MMP-9 release. Pretreatment with indomethacin indicated that MMP-9 release was not prostaglandin dependent. Inhibitors of the MAP kinase MEK- 1, and NFkappaB showed that both pathways are important for PAR-2-mediated MMP-9 release. Trypsin, a physiologic PAR-2 activator, upregulated MMP-9 but also MMP-2 release from airway epithelial cells. CONCLUSION: PAR-2 receptors appear to play an important role in the regulation of MMP-9 release from airway epithelial cells. As such, these receptors may be critical elements in tissue remodeling in asthma and other inflammatory conditions in the airways.
Asunto(s)
Células Epiteliales/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Receptores de Trombina/fisiología , Sistema Respiratorio/citología , Corticoesteroides/farmacología , Células Epiteliales/enzimología , Humanos , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/genética , ARN Mensajero/metabolismo , Receptor PAR-2 , Transducción de Señal/fisiología , Tripsina/farmacologíaRESUMEN
Mast cells release many biologically active molecules upon stimulation by a variety of molecules such as immunoglobulin E (IgE) and specific antigen, anaphylatoxins, as well as a number of cationic compounds which include drugs, kinins and neuropeptides. The effect of the naturally occurring polyamine spermine was studied because, even though it is polycationic, it has been implicated in the modulation of secretory processes in a variety of cells. In particular, it was previously shown that oxidation products of spermine inhibit mast cell secretion. High concentrations of spermine (5 x 10(-3) M) added at 37 degrees C induced mast cell secretion that had similar characteristics with that triggered by compound 48/80 (48/80). However, spermine inhibited mast cell secretion in a dose-dependent manner as long as it was added at 4-10 degrees C for at least 10 min in the absence of Ca++ before warming the cells to 37 degrees C and triggering them with 48/80. These findings were true both for purified rat peritoneal mast cells and for rat skin mast cells in situ. Addition of calcium after the cells had been warmed to 37 degrees C could not reverse this inhibition. The inhibition seen when spermine was added at 4 degrees C was, however, overcome if phorbol myristate acetate (PMA) or NaF, which activate PKC and G proteins respectively, were added to mast cells at 37 degrees C together with Ca++. These results indicate that polyamines could be important modulators of the activation state of mast cells and might help further define the biochemical events involved in mast cell secretion.
Asunto(s)
Calcio/metabolismo , Mastocitos/efectos de los fármacos , Espermina/farmacología , Animales , Toxina del Cólera/farmacología , Depresión Química , Técnicas In Vitro , Masculino , Mastocitos/metabolismo , Cavidad Peritoneal/citología , Ratas , Ratas Sprague-Dawley , Serotonina/metabolismo , Estaurosporina/farmacología , Temperatura , Acetato de Tetradecanoilforbol/farmacología , Factores de Virulencia de Bordetella/farmacología , p-Metoxi-N-metilfenetilamina/farmacologíaRESUMEN
The proopiomelanocortin (POMC)-derived neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) is known to modulate some aspects of inflammation through direct effects on T cells, B cells, and monocytes. To determine whether alpha-MSH might similarly influence mast cell responsiveness, mast cells were examined to see if they expressed the receptor for alpha-MSH, melanocortin-1 (MC-1), and whether alpha-MSH altered mast cell function. We thus first identified MC-1 on bone marrow cultured murine mast cells (BMCMC) and a murine mast cell line (MCP-5) employing flow cytometry and through detection of specific binding. Subsequent treatment of mast cells with alpha-MSH increased the cAMP concentration in a characteristic biphasic pattern, demonstrating that alpha-MSH could affect intracellular processes. We next examined the effect of alpha-MSH on mediator release and cytokine expression. IgE/DNP-human serum albumin-stimulated histamine release from mast cells was inhibited by approximately 60% in the presence of alpha-MSH. Although activation of BMCMC induced the expression of mRNAs for the inflammatory cytokines IL-1beta, IL-4, IL-6, TNF-alpha, and the chemokine lymphotactin, mRNAs for IL-1beta, TNF-alpha, and lymphotactin were down-modulated in the presence of alpha-MSH. Finally, IL-3-dependent proliferative activity of BMCMC was slightly but significantly augmented by alpha-MSH. Taken together, these observations suggest that alpha-MSH may exert an inhibitory effect on the mast cell-dependent component of a specific inflammatory response.
Asunto(s)
Mastocitos/inmunología , Mastocitos/metabolismo , Receptores de Corticotropina/fisiología , alfa-MSH/fisiología , Animales , División Celular/inmunología , Células Cultivadas , Quimiocinas/antagonistas & inhibidores , Quimiocinas/biosíntesis , Quimiocinas/genética , AMP Cíclico/metabolismo , Citocinas/antagonistas & inhibidores , Citocinas/biosíntesis , Citocinas/genética , Regulación hacia Abajo/inmunología , Liberación de Histamina , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , Receptores de Corticotropina/biosíntesis , Receptores de Melanocortina , alfa-MSH/metabolismoRESUMEN
BACKGROUND: Because of the close association between mast cells and fibroblasts in the microenvironment and the importance of connexins (Cxs) in fibroblast communication with other cells, we hypothesized that mast cells also express Cxs, allowing them to similarly communicate with other cells through gap junctions. OBJECTIVES: We sought to identify the expression of Cxs (particularly Cx43, Cx32, and Cx26) by murine mast cells. METHODS: The expression of Cxs was studied by RT-PCR, Northern blot analysis, Western blot analysis, flow cytometry, and confocal laser scanning microscopy. RESULTS: In this report we demonstrate that murine bone marrow cultured mast cells and the growth factor-independent murine mast cell line C57, express Cx43 and Cx32 as assessed by RT-PCR, Northern blot analysis, Western blot analysis, and flow cytometry, but do not express Cx26. We also show, by confocal laser scanning microscopy, that Cx43 localizes to the cytoplasmic membrane of mast cells in a pattern similar to that seen in fibroblasts. CONCLUSIONS: Mast cells express Cx43 and Cx32, and Cx43 is associated with the cytoplasmic membrane, suggesting that mast cells have the potential to communicate with other cells in their microenvironment in part through gap junctions.
Asunto(s)
Membrana Celular/metabolismo , Conexinas/biosíntesis , Mastocitos/metabolismo , Animales , Northern Blotting , Western Blotting , Línea Celular , Células Cultivadas , Conexina 26 , Conexina 43/biosíntesis , Fibroblastos/fisiología , Citometría de Flujo , Uniones Comunicantes/fisiología , Mastocitos/fisiología , Ratones , Microscopía Confocal , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína beta1 de Unión ComunicanteRESUMEN
Activation of mast cells results in the generation and release of bioactive mediators which in turn initiate allergic inflammation. Mast cell function is enhanced following stimulation in part because of the induction of specific genes and their products. To identify additional genes induced in mast cells that support this process, we thus constructed an activation-specific mast cell subtraction library. To date, we have isolated 26 novel inducible murine mast cell (imc) cDNA clones. Among them, a full-coding region of the murine gene imc-415 was found to have a greater than 90% nucleotide sequence homology and a 97.5% amino acid sequence homology to both a human beta4 integrin-binding protein (p27(BBP)) and a human translation initiation factor 6 (eIF6), which in turn are identical. In vitro translation of the imc-415 gene yielded a band of an approximately 26 kDa. This is the same as the calculated molecular weight of murine IMC-415 protein based on the predicted amino acid sequence and is the molecular weight of p27(BBP)/eIF6. Murine imc-415 message was also induced in inflamed lung tissues in a mouse model of asthma. These results suggest a role for murine imc-415 in allergic inflammation where it may enhance protein synthesis. Human eIF6/p27(BBP) may also play a role in allergic diseases based on the similarities in sequence and in gene expression patterns.
Asunto(s)
Mastocitos/metabolismo , Factores de Iniciación de Péptidos/genética , Secuencia de Aminoácidos , Animales , Antígenos CD/metabolismo , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular , ADN Complementario , Drosophila/genética , Factores Eucarióticos de Iniciación , Femenino , Biblioteca de Genes , Humanos , Integrina beta4 , Proteínas de Filamentos Intermediarios/química , Proteínas de Filamentos Intermediarios/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Peso Molecular , Factores de Iniciación de Péptidos/biosíntesis , Factores de Iniciación de Péptidos/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Fas ligand (FasL, CD95L, Apo-1L), a type II membrane protein belonging to the tumour necrosis factor family, induces apoptosis in Fas-bearing cells. As murine mast cells have been shown to express Fas antigen, we hypothesized that mast cells might also express FasL. To explore this possibility, we first demonstrated FasL mRNA in mast cells by reverse transcription-polymerase chain reaction and FasL protein by immunoblot analysis. FasL protein was shown to be exclusively located within the cell by flow cytometry. In agreement with this observation, bone marrow cultured mast cells were unable to kill Jurkat T cells. Our results demonstrate that FasL is expressed in murine mast cells and suggest that this murine mast cell FasL is not lytic, owing to the intracellular localization.
Asunto(s)
Mastocitos/inmunología , Receptor fas/análisis , Animales , Células Cultivadas , Técnicas de Cocultivo , Citometría de Flujo , Humanos , Immunoblotting , Células Jurkat , Ratones , Ratones Endogámicos BALB C , Ratones Mutantes , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Receptor fas/genéticaRESUMEN
OBJECTIVE: This article reviews the current understanding of the pathophysiologic role of intestinal mast cells. DATA SOURCE: Up to date English language publications on mast cell characteristics, heterogeneity and functions were used. Recent articles were used to develop and extend novel concepts about the role of intestinal mast cells. STUDY SELECTION: Reference sources were selected because of their pertinence to the pathophysiological effects of mast cells in intestinal hypersensitivity. Recent publications on the following topics were emphasized: mast cell proteases in intestinal anaphylaxis; effects of nitric oxide in gastrointestinal pathophysiology; involvement of cytokines derived from mast cells in tissue damage and repair. RESULTS: Mast cells are clearly implicated in the pathology of intestinal disease. Growing evidence suggests physiological roles for mast cells in the protection of tissues from inflammatory damage, and in intestinal maturation. Mast cells can release cytokines, such as tumour necrosis factor-alpha and interleukin-10, which were originally thought to contribute to inflammatory damage, but which may also have anti-inflammatory properties. Interestingly, mast cell function can be regulated by nitric oxide, and mast cells themselves are sources of this important mediator. Nitric oxide has protective as well as detrimental effects in the intestine. CONCLUSIONS: Intestinal mast cells have physiologic regulatory effects in addition to their pathologic effects. However, relatively little is known about the mechanisms of these regulatory effects. Mast cells are likely in an ongoing fluctuating balance between physiological functions and pathological effects in normal individuals. Poorly known factors can create an imbalance and lead to pathologic reactions.