Behavioural analysis of individual human mast cells
22 september 2017
UAntwerp - Campus Drie Eiken - Building Q - Promotiezaal - Universiteitsplein 1 - 2610 WILRIJK (route: UAntwerpen, Campus Drie Eiken
Prof D. Ebo, Prof L. De Clerck
PhD defence Nathalie Cop - Faculty of Medicine and Health Sciences
The mechanisms that govern human mast cell (MC) activation finally culminating in degranulation remain poorly understood and only partially deciphered, mainly because of shortage of eligible MC populations. Besides, most of our knowledge is based on experiments from which data only reflect an average of all stimulated cells, as degranulation is generally quantified by using traditional mediator release assays. In contrast, data on single cells remain scarce, particularly from MCs cultured out of peripheral blood.
For that reason, in this dissertation we optimize a technique to culture sufficient numbers of well-differentiated and functional MCs from CD34+ progenitor cells from small amounts of peripheral blood. Flow cytometric analysis of these cells reveals that our culture conditions generate two phenotypically distinct subpopulations: viz. immature CD117-CD203+low and mature CD117+CD203c+hi MCs. Both subpopulations express the high affinity IgE-receptor (FcεRI), contain comparable amounts of chymase, tryptase and histamine, and can successfully be uploaded with IgE and subsequently activated with anti-IgE, demonstrating release of histamine at a single cell level.
Moreover, in this thesis we provide the proof that these cells can also be activated by specific allergen that cross-links membrane-bound IgE by autologous passive sensitization experiments in birch pollen allergic patients. In addition, we demonstrate that allergen-specific degranulation can be significantly enhanced by short-term incubation with pro-inflammatory cytokines IL-33 and IL-6. Almost simultaneously, we extend functional analysis of our cells with IgE-independent activation via substance P and show that this MRGPRX2-dependent activation cannot be primed by pro-inflammatory cytokine exposure with IL-33, IL-6 or TNF-α. Next in this thesis, we demonstrate that our single cell analysis enables to further disentangle the underlying mechanisms of immediate drug hypersensitivity reactions. Hence, our mature CD117+CD203c+hi MC population can be divided into two subpopulations: viz. MRGPRX2-positive and MRGPRX2-negative MCs. Only the MRGPRX2-positive MCs degranulate in response to substance P, morphine and moxifloxacin, endorsing the hypothesis that the MRGPRX2-receptor is implicated in triggering degranulation by these compounds.
To conclude, we provide a valuable research tool to study the phenotype, intracellular content and function of individual human MCs in several auto-inflammatory, auto-immune and allergic diseases.