University of Southampton Faculty of Medicine Measurement of mast cell and basophil activation in vitro as means for investigation of drug hypersensitivity by Desiree Ludwig Thesis of the degree of Master of Philosophy May 2015 University of Southampton, Faculty of Medicine, Master of Philosophy, Desiree Ludwig Measurement of mast cell and basophil activation in vitro as means for investigation of drug hypersensitivity Abstract Allergic drug reactions to drugs are becoming more frequent throughout the world and are among the most common and serious form of immuno-pathological process in modern clinical medicine. These reactions can lead to life-threatening anaphylaxis which can occur within minutes of receiving the drug. Symptoms provoked by the explosive release of mediators from mast cells and basophils release may include itchiness, angioedema, difficulty breathing and circulatory collapse. There are few reliable laboratory tests available to identify those with drug hypersensitivity who may be at risk of anaphylactic shock, or even to establish if such a reaction has occurred. Confirmation of drug hypersensitivity by skin testing or oral challenge can put the patient at risk of serious reaction, and as such are expensive and performed at few centres. The aim of the study was to seek to develop new tests for drug allergy based on assessment of mast cell or basophil activation either in vitro or in vivo. Patients were recruited who were suspected of having suffered an allergic reaction to medicine and who were undergoing skin testing or oral drug challenge. Blood was collected before and following the challenge, the nature of symptoms provoked were recorded, and clinical history and demographic data obtained. The activation of basophils in vitro in response to the drug was assessed by measurement of histamine release by immunoassay or up-regulation of CD63 by flow cytometry. In parallel studies, cells of a rat basophil leukaemia cell line (RBL-703-21) that had been transfected with the α chain of the human IgE receptor, were passively sensitised with serum and challenged with the drug implicated. The release of β-hexosaminidase in cell supernatants was measured to determine the degree of drug-induced cell activation. Mast cell tryptase was successfully purified from human lung tissue, and preliminary attempts made to optimise the detection of the basophil product basogranulin by dot blotting with specific antibodies BB1 and BB5, but time did not allow measurement of either of these markers of allergic reaction in the patients studied. Skin weals were provoked in some 40% of patients following skin testing, though other symptoms were frequently recorded in the presence or even absence of a weal. These included hives, post-nasal drip, abdominal pain, vomiting, cough, shortness of breath, wheeze and stridor, and there was considerable heterogeneity in clinical responses. In patients with a positive test outcome on skin or oral challenge, raised levels of serum IgE specific to that drug were commonly observed (where the test was available). Increased expression of CD63 was provoked by in vitro challenge of basophils in a minority of patients who responded on in vivo challenge, but no histamine release into cell supernatants was detected where this was examined. Drug challenge of RBL-703-21 cells passively sensitised with patient serum resulted in the release of only a small quantities of β-hexosaminidase, and no evidence was found for direct activation of cells by the drugs themselves. There was, however, significantly higher net release of β-hexosaminidase from patients with positive skin reactions to the neuromuscular blocking agent rocuronium than in those who did not respond on skin challenge. The reliable diagnosis of drug hypersensitivity remains a formidable challenge, and there is no ready replacement of skin or oral challenge of patients with drugs suspected of causing a reaction. The present study indicates some of the difficulties as well as ways forward in developing new laboratory tests. In vitro challenge of blood basophils or passively sensitised cells of mast cell or basophil-like cell lines hold some promise, but it will be crucial for the sensitivity of such procedures to be increased and optimised with a range of drugs. Assays for mast cell or basophil products in the circulation of patients experiencing reactions should also be valuable as diagnostic tools. 1 Contents List of figures ........................................................................................................... 4 List of Tables ............................................................................................................ 6 Declaration of Authorship ....................................................................................... 8 Acknowledgment...................................................................................................... 9 List of abbreviations .............................................................................................. 10 1. Introduction ...................................................................................................... 12 1.1 Allergic drug reactions and anaphylaxis ..................................................................... 12 1.1.1 Allergy ................................................................................................................. 12 1.1.2 Adverse drug reactions and drug hypersensitivity ............................................... 17 1.1.3 Anaphylaxis ......................................................................................................... 20 1.2 Mast cells and Basophils ........................................................................................... 23 1.2.1 Mast cells ............................................................................................................ 23 1.2.2 Basophils ............................................................................................................ 29 1.3 Mediators released after basophil and mast cell activation ........................................ 33 1.3.1 Preformed mediators ........................................................................................... 33 1.3.2 Mediators that are both preformed and newly generated ..................................... 38 1.3.3 Newly generated mediators ................................................................................. 39 1.4 Identification of drug hypersensitivity ......................................................................... 41 1.4.1 In vivo and in vitro tests for drug hypersensitivity................................................. 41 1.4.2 Limitations ........................................................................................................... 43 1.3 Tryptase and histamine as markers for anaphylaxis .................................................. 46 1.5 Aims .......................................................................................................................... 47 2. Methods ............................................................................................................ 48 2.1 Subjects and sample collection ................................................................................. 48 2.2 Cell lines and cell culture conditions ......................................................................... 49 2.3 Measuring β-hexosaminidase release from passively sensitised and non-sensitised cells ...................................................................................................................... 50 2.4 Measurement of β-hexosaminidase .......................................................................... 51 2.5 Release of histamine from whole blood basophils .................................................... 51 2 2.6 PBMC isolation ......................................................................................................... 51 2.7 Basophil purification ................................................................................................. 55 2.8 Dot blotting for basogranulin ..................................................................................... 55 2.9 Basophil activation test ............................................................................................. 56 2.10 Purification of tryptase from lung............................................................................. 57 2.11 Sodium dodecyl sulfate (SDS)-PAGE ..................................................................... 59 2.12 Western blot ........................................................................................................... 59 2.13 Statistics ................................................................................................................. 60 3. Demographic features of study population ................................................... 62 4. Identification of drug hypersensitivity ........................................................... 67 4.1 Direct actions of drugs on mast cells ......................................................................... 67 4.2 Validation of the passively sensitised mast cell model ............................................... 68 4.3 Activation of passively sensitised mast cells .............................................................. 72 4.3.1 Drug induced activation ....................................................................................... 72 4.3.2 Grass pollen-induced