Rosa Torres

E-prostanoid 2 receptors dampen mast cell degranulation via cAMP/PKA-mediated suppression of IgE-dependent signaling

The experimental administration of PGE2 for the treatment of asthma dampens clinical symptoms, and similar efficacy has been found in dust mite‐induced hypersensitivity reactions in animal models. Here, we investigate the mechanism by which PGE2 mediates suppression of MC degranulation. We find that the effect of PGE2 on FcɛRI‐dependent MC degranulation varies from activating to suppressing, depending on the relative ratio of EP2 to EP3 expression on these cells with suppression evident only in cells having increased EP2 to EP3 expression. Consistent with a role for EP2 in suppressing MC responses in vitro, we found that a selective EP2 agonist, Butaprost, inhibited MC‐mediated FcɛRI‐induced immediate hypersensitivity in a model of PCA. EP2 engagement on MCs increased cAMP production and inhibited FcɛRI‐mediated calcium influx. In addition, it also decreased the extent of FcɛRI‐induced Fyn kinase activity, leading to decreased phosphorylation of key signaling molecules such as Gab2 and Akt. Treatment with an antagonist of cAMP or shRNA down‐regulation of PKA (the principal intracellular target of cAMP) reversed the EP2‐mediated inhibitory effect on MC degranulation and restored calcium influx and phosphorylation of Akt. Collectively, the findings demonstrate that EP2 suppresses the Fyn‐mediated signals that are central to FcɛRI‐dependent MC degranulation, suggesting that engagement of the EP2 on MCs may be beneficial in dampening allergic responses.

Descubriendo el asma de origen alérgico a través del ratón. Un repaso a la patogenia de los modelos de asma alérgica en el ratón y su similitud con el asma alérgica humana

Se entiende por asma bronquial la alteración respiratoria causada por un obstrucción de los bronquios y que se acompaña de una inflamación crónica de las vías respiratorias inferiores. La obstrucción se debe en parte a la hiperreactividad bronquial, es decir, a la tendencia a una contracción excesiva de la musculatura lisa de los bronquios frente a diversos estímulos. La disminución del calibre de los bronquios dificulta el paso de aire hacia los pulmones y consecuentemente genera en el paciente una crisis de disnea. A la obstrucción bronquial contribuyen la inflamación crónica, la hipersecreción de moco y el fenómeno de reestructuración tisular o remodeling que altera la microestructura de las vías respiratorias afectadas. El cuadro descrito es en gran medida el resultado de alteraciones inmunológicas y bioquímicas en ciertos individuos con una predisposición genética. Los factores etiológicos del asma pueden ser diversos. El ejercicio, los antiinflamatorios no esteroideos, las infecciones virales y los alérgenos se encuentran entre los más frecuentes. Cuando la causa del asma es un alérgeno se habla de asma alérgica, atópica o extrínseca. Si bien el asma de origen alérgico y el asma de origen no alérgico comparten mecanismos patogénicos, es probable que los alérgenos generen un patrón de respuesta inmunológica particular especialmente en el inicio del proceso. Por eso, y porque se trata del tipo de asma más prevalente, este artículo se centra en el asma alérgica. Se estima que el asma afectará a 300 millones de personas en todo el mundo en 2010 teniendo en cuenta la incidencia actual (150 millones de personas afectadas) y la progresión anual1,2. En el 80% de los niños y adolescentes afectados, el asma es de origen alérgico y en España un 40% de los casos de asma en adultos jóvenes (20-44 años) tiene un componente alérgico según los datos preliminares del subestudio de la encuesta de Salud Respiratoria-Comisión Europea3,4. Este incremento de la prevalencia del asma alérgica especialmente en los países más desarrollados se atribuye, entre otras causas, y de acuerdo con la teoría más en boga, conocida como “hipótesis de la higiene”, a mejoras sanitarias como la menor incidencia de infecciones virales y de infestaciones parasitarias y la incorporación de nuevas vacunas a los programas5. El detonante de la hipótesis de la higiene fue la observación de que dichas mejoras se acompañan de un aumento de la incidencia de enfermedades atópicas, atribuible posiblemente a cambios en los mecanismos inmunológicos6. El asma es una enfermedad de difícil control farmacológico que supone, además de una lacra sanitaria, un problema económico de primera magnitud por absentismo laboral, gasto farmacéutico y disminución de la productividad7. La dificultad en su control estriba en que el tratamiento actual no resuelve la enfermedad y se encamina fundamentalmente a contrarrestar los episodios de broncospasmo y controlar la inflamación subyacente en el proceso crónico, una fase de la enfermedad de particular dificultad terapéutica. A las limitaciones del tratamiento se añade el riesgo de fallecimiento del paciente por ataques agudos, estimado en 18 por 1.000.000 de habitantes2. La falta de un tratamiento farmacológico más eficaz del asma alérgica se debe en gran medida al desconocimiento de las alteraciones inmunológicas o bioquímicas precisas que la originan. La investigación básica y clínica en pacientes asmáticos resulta fundamental para el avance del conocimiento de la patogenia del asma, pero hay obstáculos científicos y éticos que impiden desenmascarar determinados aspectos de la enfermedad. La aparición en la última década de modelos de asma alérgica inducida en el ratón, ha impulsado la investigación en este terreno y ofrece sin duda un valor añadido a los estudios en pacientes asmáticos, a los estudios in vivo en otros modelos animales y a los experimentos in vitro y ex vivo. En este artículo se describen las similitudes patogénicas entre el asma alérgica del humano y la de los modelos inducidos en el ratón, se destaca la particular utilidad del asma en los múridos y se recogen algunos de los datos bibliográficos que la refrendan.

Subcutaneous prostaglandin E2 restrains airway mast cell activity in vivo and reduces lung eosinophilia and Th2 cytokine overproduction in house dust mite-sensitive mice.

Background: Prostaglandin (PG) E2 is thought to exert protective effects in the lungs. Accordingly, aerosolized PGE2 prevents the experimentally induced airway response to allergen challenge in asthmatics. In vitro evidence indicating that functional PGE2 receptors (EP) are expressed on human mast cells and that PGE2 can alter cytokine production suggests that these phenomena may be involved in its beneficial effect in asthma. However, in vivo evidence is scarce. Methods: We assessed the effects of exogenous PGE2 and of the EP1/EP3 agonist sulprostone on the murine airway response to house dust mite (HDM) allergens, a model that accurately reproduces the spontaneous exposure of allergic asthma patients to aeroallergens. We also analyzed the in vivo impact of PGE2 on production in the murine airway of mast cell protease (mMCP)-1, a specific marker of lung mast cell activity, and on local production of cytokines. Results: Exogenous PGE2, but not sulprostone, reduced eosinophilic infiltration in HDM-sensitized mice by half and led to a strong reduction in airway Th2 cytokine expression. These anti- inflammatory effects were accompanied in vivo by a substantial reduction in HDM-induced upregulation of airway mMCP-1. Neither PGE2 nor sulprostone had any effect on airway hyperresponsiveness to methacholine. Conclusions: Our results indicate that the anti-inflammatory effect of PGE2 can be reproduced in vivo in HDM-sensitized mice and suggest that this protective effect is dependent in vivo on inhibition of the allergen-triggered proinflammatory activity of bronchial mast cells. Finally, the effect of PGE2 is linked to reduced upregulation of airway Th2 cytokines.

The role of mast cells in atopy: what can we learn from canine models? A thorough review of the biology of mast cells in canine and human systems

Mast cell research has largely focused on the role of these cells in the early phase of allergic reactions. However, their involvement may well extend beyond this stage, and even reach across nonallergic conditions. Mast cells from different sources have helped advance our knowledge of their biology. Although in vitro and in vivo research in this area has mainly focused on humans, such studies are limited by the extent to which cells from certain human tissues and/or human patients can be collected or studied. While rodents also provide valuable models with which to further our understanding of the behaviour of mast cells and their contribution to allergy, reported differences between human and murine mast cells, and, in some instances, the limitations of in vivo rodent models of mast cell‐mediated allergic conditions, preclude their use. In this review, we introduce a relatively unknown mast cell population, that of the dog. Canine mast cells display many phenotypic and functional similarities with their human counterparts, and dogs develop spontaneous and induced allergic diseases that share clinical and pathophysiological features with the human condition. Therefore, the use of canine cells can shed light on the general role of mast cells, particularly in relation to allergic diseases given the potential of in vivo dog models within this field. Here we provide a detailed review of the data reported from in vitro and in vivo studies of canine mast cells, and compare them with results obtained in human systems. We also highlight direct evidence of the mast cell contribution to canine atopy. We conclude that the dog offers useful in vitro and in vivo models in which to investigate mast cell behaviour, and that its use should be considered when undertaking studies aimed either at elucidating the role of mast cells in health and disease, or at prescreening novel therapies prior to entry into man.

Mucosal mast cells mediate motor response induced by chronic oral exposure to ovalbumin in the rat gastrointestinal tract.

Abstract  We previously demonstrated that oral chronic exposure to ovalbumin (OVA) causes intestinal hypermotility in Sprague‐Dawley rats. In this study, the objective was to determine the mechanism of action of OVA and the role of mucosal mast cells in the regulation of motor activity in this model. Rats were orally exposed to OVA during 6 weeks. Intestinal mucosal mast cells (IMMCs) were counted and rat mast cell protease II (RMCPII) measured in duodenum, jejunum, ileum and colon. Anti‐OVA IgE, IgG, and IL‐4 were measured in serum. Eosinophils and IgE+ cells were counted in jejunum. In an additional study rats were treated with the mast cell stabilizer ketotifen and mast cell number, RMCPII concentration and motor activity in vitro were evaluated. OVA exposed rats showed an increase in mucosal mast cell number and in RMCPII content in small intestine and colon. However, variables of a Th2 type response were not affected by exposure to OVA: (i) neither OVA specific IgE nor IgG were found; (ii) IL‐4 did not increase and, (iii) the number of eosinophils and IgE+ cells was identical in the exposed and unexposed groups. These results brought us to hypothesize a possible non‐Ig‐mediated action of OVA on mast cells. Ketotifen significantly diminished the response to OVA: Ketotifen reduced the number of mast cells and the RMCPII content and blocked increased intestinal contractility. In addition ketotifen modified motor response in both OVA exposed and unexposed animals giving evidence of the importance of mast cells in intestine motor activity driving.

Activity of the cyclooxygenase 2-prostaglandin-E prostanoid receptor pathway in mice exposed to house dust mite aeroallergens, and impact of exogenous prostaglandin E2

BackgroundProstaglandin E2 (PGE2), experimentally administered to asthma patients or assayed in murine models, improves allergen-driven airway inflammation. The mechanisms are unknown, but fluctuations of the endogenous cyclooxygenase (COX)-2/prostaglandin/E prostanoid (EP) receptor pathway activity likely contribute to the clinical outcome. We analyzed the activity of the pathway in mice sensitized to aeroallergens, and then studied its modulation under exogenous PGE2.MethodsMice were exposed to house dust mite (HDM) aeroallergens, a model that enable us to mimic the development of allergic asthma in humans, and were then treated with either subcutaneous PGE2 or the selective EP1/3 receptor agonist sulprostone. Simultaneously with airway responsiveness and inflammation, lung COX-2 and EP receptor mRNA expression were assessed. Levels of PGE2, PGI2, PGD2 were also determined in bronchoalveolar lavage fluid.ResultsHDM-induced airway hyperreactivity and inflammation were accompanied by increased COX-2 mRNA production. In parallel, airway PGE2 and PGI2, but not PGD2, were upregulated, and the EP2 receptor showed overexpression. Subcutaneous PGE2 attenuated aeroallergen-driven airway eosinophilic inflammation and reduced endogenous PGE2 and PGI2 production. Sulprostone had neither an effect on airway responsiveness or inflammation nor diminished allergen-induced COX-2 and PGE2 overexpression. Finally, lung EP2 receptor levels remained high in mice treated with PGE2, but not in those treated with sulprostone.ConclusionThe lung COX-2/PGE2/EP2 receptor pathway is upregulated in HDM-exposed mice, possibly as an effort to attenuate allergen-induced airway inflammation. Exogenous PGE2 downregulates its endogenous counterpart but maintains EP2 overexpression, a phenomenon that might be required for administered PGE2 to exert its protective effect.

Mast cells induce upregulation of P-selectin and intercellular adhesion molecule 1 on carotid endothelial cells in a new in vitro model of mast cell to endothelial cell communication

It is suggested that mast cells contribute to cell recruitment in inflammation through the upregulation of endothelial adhesion molecules. P‐selectin and intercellular adhesion molecule (ICAM)‐1 are two key adhesion molecules that have been associated indirectly with mast cell activity. The canine C2 mastocytoma cell line and primary cultures of canine carotid endothelial cells were used to establish a new in vitro model to help study the interaction between mast cells and endothelial cells. Carotid endothelial cells were incubated with mast cell mediators to uncover their effect on endothelial ICAM‐1 and P‐selectin expression. To assess the relative contributions of tumour necrosis factor (TNF)‐α and histamine to such effect, an H1 antihistamine and a TNF‐α blocking antibody were used. Prior to activation by mast cell mediators, P‐selectin was expressed only within the cytoplasm, and ICAM‐1 was constitutively expressed on the surface of the canine carotid endothelial cells. Both adhesion molecules were enhanced significantly and strongly upon mast cell activation at various time points. Unstored TNF‐α was fully responsible for ICAM‐1 upregulation. P‐selectin was up‐regulated by both preformed and newly synthesized mast cell mediators, but neither histamine nor TNF‐α accounted for such an effect. Therefore, a new model is proposed in which the pro‐inflammatory effect of mast cells on endothelial cells can be studied in vitro. In this model, it has been demonstrated that only TNF‐α accounts for the overexpression of ICAM‐1 induced by mast cells, and that mast cells up‐regulate P‐selectin expression through a histamine‐independent mechanism.

The PGE2-EP2-mast cell axis: an antiasthma mechanism.

Despite the fact that cyclooxygenase and its products, prostaglandins, have been traditionally associated with the development of inflammation, PGE2 was implicated early on as potentially beneficial in asthma. During the 1970s and 1980s, several studies reported the bronchodilator effect of PGE2 in asthma patients. In parallel, it was being shown to exert an inhibitory effect on mast cells in vitro. In spite of this, data supporting the beneficial role for PGE2 in asthma were scarce and sometimes controversial. Many years later, in vitro and in vivo studies suggested a range of biological activities attributable to PGE2, others than the ability to relax smooth muscle, that potentially explained some of the observed positive effects in asthma. The identification and cloning of the four PGE2 receptors made available new tools with which to fine-tune investigation of the anti-inflammatory, pro-inflammatory, immunoregulatory, and bronchodilation mechanisms of PGE2. Among these, several suggested involvement of mast cells, a cell population known to play a fundamental role in acute and chronic asthma. Indeed, it has been shown that PGE2 prevents human and murine MC activity in vitro through activation of the EP2 receptor, and also that both exogenously administered and endogenous PGE2 inhibit airway MC activity in vivo in mouse models of asthma (likely through an EP2-mediated mechanism as well). In the last few years, we have furthered into the functional connection between PGE2-induced mast cells inhibition and attenuated damage, in asthma and allergy models. The validity of the findings supporting a beneficial effect of PGE2 in different asthma phases, the direct effect of PGE2 on mast cells populations, and the functional implications of the PGE2-MC interaction on airway function are some of the topics addressed in this review, under the assumption that increased understanding of the PGE2-EP2-mast cell axis will likely lead to the discovery of novel antiasthma targets.

An intranasal selective antisense oligonucleotide impairs lung cyclooxygenase-2 production and improves inflammation, but worsens airway function, in house dust mite sensitive mice

BackgroundDespite its reported pro-inflammatory activity, cyclooxygenase (COX)-2 has been proposed to play a protective role in asthma. Accordingly, COX-2 might be down-regulated in the airway cells of asthmatics. This, together with results of experiments to assess the impact of COX-2 blockade in ovalbumin (OVA)-sensitized mice in vivo, led us to propose a novel experimental approach using house dust mite (HDM)-sensitized mice in which we mimicked altered regulation of COX-2.MethodsAllergic inflammation was induced in BALBc mice by intranasal exposure to HDM for 10 consecutive days. This model reproduces spontaneous exposure to aeroallergens by asthmatic patients. In order to impair, but not fully block, COX-2 production in the airways, some of the animals received an intranasal antisense oligonucleotide. Lung COX-2 expression and activity were measured along with bronchovascular inflammation, airway reactivity, and prostaglandin production.ResultsWe observed impaired COX-2 mRNA and protein expression in the lung tissue of selective oligonucleotide-treated sensitized mice. This was accompanied by diminished production of mPGE synthase and PGE2 in the airways. In sensitized mice, the oligonucleotide induced increased airway hyperreactivity (AHR) to methacholine, but a substantially reduced bronchovascular inflammation. Finally, mRNA levels of hPGD synthase remained unchanged.ConclusionIntranasal antisense therapy against COX-2 in vivo mimicked the reported impairment of COX-2 regulation in the airway cells of asthmatic patients. This strategy revealed an unexpected novel dual effect: inflammation was improved but AHR worsened. This approach will provide insights into the differential regulation of inflammation and lung function in asthma, and will help identify pharmacological targets within the COX-2/PG system.

Locally administered prostaglandin E2 prevents aeroallergen-induced airway sensitization in mice through immunomodulatory mechanisms

Prostaglandin E2 attenuates airway pathology in asthmatic patients and exerts a protective effect in antigen-sensitized mice when administered systemically. We aimed to establish the consequences of intranasal PGE2 administration on airway reactivity to aeroallergens in mice and reveal the underlying immunoinflammatory mechanisms. PGE2 was administered either daily during a 10-day exposure to house dust mite (HDM) extracts or for limited intervals. Airway hyperreactivity was measured by whole-body and invasive plethysmography. The phenotypes of lung immune cells and cytokine production were analysed by flow cytometry and ELISA, respectively. Airway hyperreactivity was sustainably reduced only when PGE2 administration was restricted to the initial 5 days of exposure to HDM. Lung inflammation, IL-4 production, and airway mast cell activity were also prevented under this early short-term treatment with PGE2. Interestingly, a Th2 response was already committed on day 5 of exposure to HDM. This was paralleled by GM-CSF and osteopontin upregulation and a decreased number of plasmacytoid dendritic and T regulatory cells, as well as a trend towards reduced IL-10 expression. Local PGE2 administration prevented the increase of airway IL-13 and osteopontin and kept lung plasmacytoid dendritic cell counts close to baseline. GM-CSF and Tregs were unaffected by the treatment. These findings suggest that the protection provided by PGE2 is a result of the modulation of early lung immunomodulatory mechanisms, and possibly a shift in the balance of dendritic cells towards a tolerogenic profile.