Different activation signals induce distinct mast cell degranulation strategies
Nicolas Gaudenzio, … , Eric Espinosa, Stephen J. Galli
Nicolas Gaudenzio, … , Eric Espinosa, Stephen J. Galli
Published September 19, 2016
Citation Information: J Clin Invest. 2016;126(10):3981-3998. https://doi.org/10.1172/JCI85538.
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Different activation signals induce distinct mast cell degranulation strategies

Abstract

Mast cells (MCs) influence intercellular communication during inflammation by secreting cytoplasmic granules that contain diverse mediators. Here, we have demonstrated that MCs decode different activation stimuli into spatially and temporally distinct patterns of granule secretion. Certain signals, including substance P, the complement anaphylatoxins C3a and C5a, and endothelin 1, induced human MCs rapidly to secrete small and relatively spherical granule structures, a pattern consistent with the secretion of individual granules. Conversely, activating MCs with anti-IgE increased the time partition between signaling and secretion, which was associated with a period of sustained elevation of intracellular calcium and formation of larger and more heterogeneously shaped granule structures that underwent prolonged exteriorization. Pharmacological inhibition of IKK-β during IgE-dependent stimulation strongly reduced the time partition between signaling and secretion, inhibited SNAP23/STX4 complex formation, and switched the degranulation pattern into one that resembled degranulation induced by substance P. IgE-dependent and substance P–dependent activation in vivo also induced different patterns of mouse MC degranulation that were associated with distinct local and systemic pathophysiological responses. These findings show that cytoplasmic granule secretion from MCs that occurs in response to different activating stimuli can exhibit distinct dynamics and features that are associated with distinct patterns of MC-dependent inflammation.

Authors

Nicolas Gaudenzio, Riccardo Sibilano, Thomas Marichal, Philipp Starkl, Laurent L. Reber, Nicolas Cenac, Benjamin D. McNeil, Xinzhong Dong, Joseph D. Hernandez, Ronit Sagi-Eisenberg, Ilan Hammel, Axel Roers, Salvatore Valitutti, Mindy Tsai, Eric Espinosa, Stephen J. Galli

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Figure 4

Pharmacological inhibition of IKK-β activity changes the signaling and degranulation pattern of anti-IgE–activated MCs.

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Pharmacological inhibition of IKK-β activity changes the signaling and d...
(A–D) IgE-sensitized PBCMCs were pretreated for 60 minutes with 100 μM BMS-345541 or DMSO and treated as described in Figure 2. (A) Upper panel: a PBCMC pretreated with DMSO; lower panel: a PBCMC pretreated with BMS-345541. White insets: higher magnification; arrows: granule structures; time scale reflects the kinetics of the responses. (B and C) Mean curves of Fluo-4 and Av.SRho MFI in PBCMCs pretreated with DMSO (B) or BMS-345541 (C). Dotted lines and arrows, ΔT. (D) Mean ΔT in PBCMCs pretreated with DMSO (blue) or BMS-345541 (red). (E and F) PBCMCs pretreated with BMS-345541 or DMSO were stimulated for 5 or 15 minutes with anti-IgE. (E) Expression of phospho–IKK-α/β in PBCMC lysate. Actin was used as loading control. (F) Immunoprecipitation of SNAP23/STX4 complexes; immunoprecipitated SNAP23 was resolved and probed for STX4. (G–J) PBCMCs were pretreated with BMS-345541 or DMSO and treated as described in Figure 3C. Time-lapse of Av.SRho (red) and dextran-FITC (pseudocolor) in PBCMCs pretreated with DMSO (G) or BMS-345541 (H); white arrowheads, increases in dextran-FITC fluorescence. (I) Mean curve of dextran-FITC MFI in PBCMCs pretreated with DMSO (blue) or BMS-345541 (red). (J) Average of dextran-FITC MFI between t = 0 and t = 5 minutes. (K) Production of TNF-α, IL-13, GM-CSF, VEGF, and MCP-1 in PBCMCs stimulated 90 minutes with vehicle (black) or anti-IgE. ns, not significant. Mean ± SEM; 2-tailed, unpaired t test; **P < 0.01; ****P < 0.0001. Data in A–D and G–J are from 3 independent experiments performed with PBCMCs from 2 donors (~30 PBCMCs analyzed per condition); data in E, F, and K are from 3 independent experiments, each of them performed with PBCMCs from a different single donor, each of which gave similar results. Scale bars: 5 μm.