F. de Mora

In vitro inhibitory effect of rupatadine on histamine and TNF-α release from dispersed canine skin mast cells and the human mast cell line HMC-1

Abstract.Objective and design: To examine the inhibitory potential of rupatadine, a new H1-antihistamine and anti-PAF agent, on histamine and TNF-α release. Comparison with an H1-antihistamine (loratadine) and a PAF-antagonist (SR-27417A).¶Material: Dispersed canine skin mast cells were used to assess the effect of the drugs tested on FcεRI-dependent and -independent histamine release; the human HMC-1 cell line was used to study TNF-α release.¶Treatment and methods: Before stimulation mast cell populations were treated with increasing concentrations of rupatadine, loratadine and SR-27417A. Histamine and TNF-α release were measured following 15-30 min and 3 h activation, respectively.¶Results: The IC50 for rupatadine in A23187, concanavalin A and anti-IgE induced histamine release was 0.7 ± 0.4 μM, 3.2 ± 0.7μM and 1.5 ± 0.4 μM, respectively whereas for loratadine the IC50 was 2.1 ± 0.9 μM, 4.0 ± 1.3 M and 1.7 ± 0.5 μM. SR-27417A exhibited no inhibitory effect. Rupatadine, loratadine and SR-27417A inhibited TNF-α release with IC50 2.0 ± 0.9 μM, 2.1 ± 1.1 M and 4.3 ± 0.6 μM, respectively.¶Conclusions: Rupatadine and loratadine showed similar inhibitory effect on histamine and TNF-α release, whereas SR-27417A only exhibited inhibitory effect against TNF-α.

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.

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.

Comparative study of histamine release from skin mast cells dispersed from atopic, ascaris-sensitive and healthy dogs

Atopic dermatitis results from the interaction between allergen and allergen-specific IgE bound to the mast cell surface receptors. This process triggers mast cell degranulation and accounts at least for early phase reaction. Furthermore, there is increasing in vitro and in vivo evidence that IgE has the ability to induce overexpression of the Fc epsilonRI receptor on the mast cell plasma membrane. In order to study the potential effect of an increase in serum IgE on mast cell activity, the histamine releasability of mature mast cells isolated from the skin of atopic, ascaris-sensitive and healthy dogs was analyzed. No histamine release was detected upon the immunological stimulation of cells that were not previously sensitized with atopic or ascaris-sensitive dog serum. However, when passively sensitized, skin mast cells were challenged with either Asc SI antigen or anti-IgE, the mast cell histamine release increased in a stimulus concentration-dependent manner. The amount of histamine released was significantly higher in response to anti-IgE than in response to Asc SI antigen. However. the difference in the percentage of mast cell histamine release between atopic (26.3+/-2.8%) and non-atopic (30.9+/-1.7%) dogs was not statistically significant, similar to what occurred when ascaris-sensitive (12.8+/-1.6%) and non-sensitive (13.2+/-1.7%) dogs were compared. Although these results could suggest that there is either little or no increase in the density of IgE receptors in atopic or ascaris-hypersensitive dogs versus controls, we strongly consider either the possibility that the digestion procedure might affect cell behaviour in vitro or that an underlying increase of receptors poorly affects the release of granule-stored mediators but influences mast cell activity in a different manner.

Stem cell factor enhances IgE-mediated histamine and TNF-α release from dispersed canine cutaneous mast cells.

Stem cell factor (SCF), the c-kit receptor ligand, plays a critical role in mast cell (MC) development and differentiation. In addition, SCF has recently been found to both modulate and induce MC activation. To investigate the effect of SCF on canine cutaneous MC function, we have characterized the ability of SCF to modulate the release by mature canine MC of preformed (histamine) and newly generated (TNF-alpha) mediators. Mature MC were isolated from skin and cultured in the absence or presence of exogenous SCF (6 ng/ml) for up to 5 days and then challenged with anti-IgE (1 microg/ml) alone for 30 min or with a combination of SCF (50 ng/ml) and anti-IgE. SCF alone failed to trigger either histamine or TNF-alpha release at any time. However, we observed that SCF used as a co-stimulus significantly potentiated histamine and TNF-alpha release in canine MC activated through Fc epsilon RI regardless of whether or not SCF was added to the medium during culturing. Thus, the mean histamine release (%) and TNF-alpha production (pg/ml) were found to be significantly higher if cells were maintained in culture in SCF-supplemented medium compared with cells cultured in the absence of exogenous SCF. We also observed that MC responsiveness to immunological stimulation increased with culture time, the percentage of histamine released being higher in cells cultured for at least 3 days when compared to freshly isolated MC. Taken together these findings suggest that canine skin MC releasability can be enhanced independently either through prolonged incubation with SCF and/or through anti-IgE and SCF co-stimulation.

Activation of the Prostaglandin E2 receptor EP2 prevents house dust mite-induced airway hyperresponsiveness and inflammation by restraining mast cells' activity.

Prostaglandin E2 (PGE2) has been proposed to exert antiasthmatic effects in patients, to prevent antigen‐induced airway pathology in murine models, and to inhibit mast cells (MC) activity in vitro.

Sensitization of Naive Beagles by Intradermal Injection of an Ascaris Antigen: Induction of a Model of Skin Allergy

Ascaris suum-hypersensitive beagles represent a unique model of skin allergy. Despite its suitability, the need to test numerous dogs prior to the selection of natural positive responders causes many complications. We hypothesized that the model could be induced in adult beagles by primary sensitization. Ten dogs were included in an intradermal sensitization program using ascaris antigen and were thereafter tested for cutaneous reaction. After 2 weeks, 60% developed a positive reaction that lasted for months. We therefore present a straightforward method to establish the Ascaris canine model of allergy that will extend its availability for research in cutaneous allergy immunomodulation.