Influence of IL-6, IL-33, and TNF-α on human mast cell activation: Lessons from single cell analysis by flow cytometry.

BACKGROUND: Mechanisms that govern priming and degranulation of human mast cells (MCs) remain elusive. Besides, most of our knowledge is based on experiments from which data only reflect an average of all stimulated cells. This study aims at investigating the effects of proinflammatory cytokines IL-6, IL-33, and TNF-α on IgE-dependent and IgE-independent activation of individual MCs. METHODS: MCs were derived from CD34+ progenitors isolated from 50 mL whole blood from 4 healthy controls and 5 birch pollen allergic patients. Passively sensitized MCs were preincubated with IL-6, IL-33, or TNF-α and stimulated with anti-IgE/birch pollen allergen or substance P, the latter being a ligand for the G-protein-coupled MRGPRX2-receptor. Activation-i.e., upregulation of CD203c-and anaphylactic degranulation-i.e., appearance of CD63-were measured using flow cytometry. RESULTS: Preincubation with IL-33 demonstrated upregulated CD203c density without degranulation. Subsequent IgE-dependent stimulation (anti-IgE/birch pollen allergen) resulted in higher appearance of CD63 as compared to cells without preincubation, indicating IL-33 to exert a priming effect (P = 0.04). IL-6 only increased allergen-specific responses but to a lesser extent than IL-33. Combination of IL-33/IL-6 had a synergistic effect, demonstrating more degranulation in response to allergen. TNF-α had no effect on IgE-mediated activation, nor synergistic effects with IL-33. Stimulation with substance P resulted in degranulation that could not be enhanced by preincubation. CONCLUSIONS: In conclusion, IL-33, and in a lesser extent IL-6, prime individual MCs for subsequent IgE-mediated activation. Moreover, this priming effect is synergistic. In contrast, none of the cytokines had a priming effect on MRGPRX2-mediated activation of MCs. © 2017 International Clinical Cytometry Society.

Basophil activation tests: time for a reconsideration.

Challenges in in vitro allergy diagnostics lie in the development of accessible and reliable assays allowing identification of all offending allergens and cross-reactive structures. Flow-assisted analysis and quantification of in vitro activated basophils serves as a diagnostic instrument with increasing applications developed over the years. From the earliest days it was clear that the test could constitute a diagnostic asset in basophil-mediated hypersensitivity. However, utility of the basophil activation test should be reassessed regarding difficulties with preparation, characterization and validation of allergen extracts; availability and the potential of more accessible diagnostics. Today, the added value mainly lies in diagnosis of immediate drug hypersensitivity. Other potential indications are monitoring venom-immunotherapy and follow-up of natural history of food allergies. However, results in these nondiagnostic applications are preliminary. We review the most relevant clinical applications of the basophil activation test. Some personal comments and views about perspectives and challenges about flow-assisted allergy diagnosis are made.