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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Research Article Immunology Inflammation

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 3

Signaling events and fluorescence dequenching patterns in dextran-FITC–labeled cytoplasmic granules in MCs stimulated via FcεRI or MRGPRX2.

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Signaling events and fluorescence dequenching patterns in dextran-FITC–l...
(A–F) IgE-sensitized or nonsensitized dextran-FITC–loaded PBCMCs were stimulated with 2 μg/ml of anti-IgE (blue) or 10 μM SP (pink), respectively, or with medium alone (black), in the presence of Av.SRho. (A) Representative time-lapse sequence of Av.SRho (upper panels, red) and dextran-FITC (lower panels, pseudocolor) in a PBCMC incubated with medium alone. (B) Mean curve of pooled single-cell analyses of dextran-FITC MFI in PBCMCs incubated with medium. (C–F) Same experiment as in A and B in PBCMCs stimulated with anti-IgE (white arrowheads indicate increases in dextran-FITC fluorescence) or SP. Scale bars: 5 μm. (G) Average of dextran-FITC MFI between t = 0 and t = 5 minutes. (H–J) IgE-sensitized or nonsensitized PBCMCs were stimulated for 5 or 15 minutes with 2 μg/ml of anti-IgE or 10 μM SP or with medium alone. (H) Expression of phospho–IKK-α/β, phospho-PKC, and phospho-AKT in PBCMC lysates. Actin was used as loading control. (I) Immunoprecipitation of SNAP23/STX4 complexes in resting PBCMCs or activated with 2 μg/ml of anti-IgE or 10 μM SP. Immunoprecipitated SNAP23 was resolved and probed for STX4. (J) Immunoprecipitation of MUNC-18/STX3 complexes performed for the same times and conditions tested in I. Immunoprecipitated MUNC-18-2 was resolved and probed for STX3. Mean ± SEM; 2-tailed, unpaired t test; ****P < 0.0001. Data in A–G are from 4 independent experiments with PBCMCs from 2 donors (~35 single PBCMCs analyzed per condition); data in H and I are representative of those obtained in 6 independent experiments, each of them performed with PBCMCs from a different single donor, all of which gave similar results.