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Influence of IL-6, IL-33, and TNF-α on human mast cell activation: Lessons from single cell analysis by flow
Nathalie Cop1, Didier G Ebo1, Chris H Bridts1
1Department of Immunology - Allergology - Rheumatology, Faculty of Medicine and Health Science, University of Antwerp, Antwerp University Hospital, Antwerp 2610, Belgium.
Insights
Interleukin-33 (IL-33) and Interleukin-6 (IL-6) prime human mast cells (MCs) for heightened IgE-mediated responses. This priming effect, particularly with IL-33, enhances degranulation, while TNF-α shows no priming effect on MRGPRX2-mediated activation.
Area of Science:
- Immunology
- Cell Biology
Background:
- Mechanisms of human mast cell (MC) priming and degranulation are not fully understood.
- Existing research often reflects average cellular responses, lacking single-cell resolution.
- This study investigates the impact of key proinflammatory cytokines on individual MC activation.
Purpose of the Study:
- To examine the effects of IL-6, IL-33, and TNF-α on IgE-dependent and IgE-independent human mast cell activation.
- To analyze single-cell responses to cytokine preincubation and subsequent stimulation.
- To elucidate the role of these cytokines in mast cell priming and degranulation.
Main Methods:
- Human MCs were derived from CD34+ progenitors.
- MCs were preincubated with IL-6, IL-33, or TNF-α.
- Cells were stimulated via IgE-dependent (anti-IgE/allergen) or IgE-independent (substance P/MRGPRX2) pathways.
- Mast cell activation (CD203c upregulation) and degranulation (CD63 expression) were quantified by flow cytometry.
Main Results:
- IL-33 preincubation upregulated CD203c without causing degranulation, priming cells for enhanced IgE-mediated degranulation (increased CD63).
- IL-6 showed a lesser priming effect on allergen-specific responses compared to IL-33.
- Combined IL-33/IL-6 demonstrated synergistic priming, significantly increasing degranulation.
- TNF-α did not affect IgE-mediated activation or show synergistic effects.
- Substance P-induced degranulation was not enhanced by cytokine preincubation.
Conclusions:
- IL-33 and, to a lesser extent, IL-6 effectively prime individual mast cells for subsequent IgE-mediated activation.
- A synergistic priming effect was observed when IL-33 and IL-6 were combined.
- Cytokines did not prime mast cells for activation via the MRGPRX2 receptor pathway.
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.
