Establishment of an in vitro mast cell rapid desensitization protocol and elucidation of its mechanism for ige-dependent antigens

Resumen

Mast cells are immune effector cells that can store a wide range of inflammatory mediators in their granules, with important roles in innate and adaptive immunity. Mast cell activation can be triggered by the crosslinking of two or more high affinity receptors for IgE (Fc¿RI) by IgE and antigen/allergen binding to the alpha subunit, which induces the aggregation of the receptors and transphosphorylation of the beta and gamma subunits. This activation triggers the release of powerful mediators such as histamine, proteases, proteoglycans, prostaglandins and leukotrienes among others. These mediators act upon local and systemic tissue receptors and induce symptoms such as, flushing, puritus, hives, angioedema, bronchoconstriccion, diarrhea, vomiting, hypotension and cardiovascular collapse, which can lead to death in a few minutes (anaphylaxis) in patients sensitized to drugs, foods or environmental allergens. IgE-mediated mast cell activation through the Fc¿RI, during type I hypersensivity reactions, has been implicated in diseases such as asthma, rhinitis and drug and food allergies and anaphylaxis. The prevalence of allergic diseases has been increasing the last 30 years in developed countries with poor exposure to the sun and parasites (hygiene hypothesis). Reactions to essential drugs are also increasing because patients live longer and better due to better treatment options and targeted therapies. Thus prolonged exposure to the drugs induces sensitization in a significant proportion of this patient's population. For patients sensitized to a first line medication for the treatment of cancer or a serious infection, the allergy to such drug may increase the morbidity and reduce their life span. For patients sensitized to certain foods, allergic reactions can occur when eating cross-contaminated or cross-reactive foods even in patients who do consistent avoidance of the offending foods. To overcome avoidance for the patients in need of first line therapy, rapid drug desensitization protocols have been generated for type I hypersensitivity reactions. These protocols have become an essential tool which allows the delivery of therapeutic doses of the offending drug in a relatively short time and in an effective and safe way with minimal risk for the patients. IgE sensitized patients present positive skin test to the specific drug implicated in the reaction, which indicates that mast cells and IgE are the main cellular and molecular targets implicated in allergic reactions. After rapid drug desensitization, the specific skin test becomes negative, which demonstrates a profound inhibition of mast cell activation. Because mechanisms underlying the inhibition of mast cell responses are not completely understood and due to its clinical importance and relevance, it is critical to identify the cellular and molecular mechanisms behind the temporary tolerance induced by rapid desensitization. Initial studies of in vitro mouse mast cell desensitizations showed that incubation of mast cells with non-activating antigen doses and in the absence of calcium, induces the inhibition of the activation with optimal doses once the calcium is re-introduced. Similar results were obtained in human basophils, inducing inhibition of cellular activation with repetitive suboptimal doses of antigen or anti-IgE added at regular intervals or for a prolonged period of time. But calcium-free conditions cannot possibly be applied to human desensitizations. Few studies have undertaken physiological desensitizations of mast cells and basophils examining their releasability following hours to days of culture with low levels of antigen or anti-IgE antibody in calcium-containing medium, resulting in full desensitization. In this PhD thesis, we propose a mouse mast cell rapid desensitization protocol in physiological calcium conditions. We study and analize the desensitization process, characterizing its kinetics and proving the reproducibility, the effectiveness and the versatility of the protocol. This protocol uses two important allergens: dinitrophenyl (DNP), which has been validated in a previous study and ovalbumin (OVA), moreover, the protocol can be used for murine bone marrow mast cells (m-BMMCs) and a rat basophilic leukemia cell line (RBL-2H3) and it can be adapted to different target doses (1, 5 and 10 ng for DNP and 10 and 50 ng for OVA). Desensitized mast cells show an almost complete inhibition of degranulation, calcium flux, arachidonic acid metabolism for prostaglandin and leukotriene generation, IL-6 and TNF-¿ synthesis and STAT6, LAT and p38 MAPK phosphorylation. Thus, rapid desensitization inhibits all hallmarks of mast cell activation. The model provides insight into the specificity of the rapid desensitization process since DNP desensitized mast cells fully respond to OVA and vice versa. Even more importantly, we show that the mechanism of impaired activation is due to the luck of internalization of the antigen/IgE/Fc¿RI complex. This model for the first time offers an initial mechanistic approach to rapid desensitizations and validates the safety of human desensitizations. Furthermore, this study may constitute an important tool for the development of improved and safer protocols for drug and food desensitizations.