By contrast, i.d. 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- during IgE-dependent stimulation TLR2-IN-C29 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 PCdependent 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. Introduction Secretory granule exocytosis is a tightly regulated process, shared by mast cells (MCs) and other eukaryotic cells, that influences the outcome of diverse physiological and pathological processes (1). MC degranulation can contribute to resistance to venoms (2C4), bacteria (5), and parasites (6, 7) but also to the morbidity and mortality associated with allergic diseases (8, 9). Aggregation of the high-affinity IgE receptor (Fc?RI) on the MC plasma membrane, induced when specific antigens cross-link Fc?RI-bound IgE, activates a complex intracellular signaling pathway resulting in secretion of cytoplasmic granule content into the extracellular environment (10), which can orchestrate local or systemic inflammation (11C16). However, stimuli that can activate MCs via various receptors that are distinct from those binding antibodies also can contribute to inflammatory processes (17C19). Examples of such stimuli include complement anaphylatoxins (e.g., C3a and C5a) (19), the vasoconstrictor peptide endothelin 1 (ET1) (17), and a panel of cationic substances such as the neuropeptide substance P (SP) (20) and drugs associated with pseudoallergic reactions (e.g., icatibant and cetrorelix) (21, 22). Although important progress has been made in the analysis of MC degranulation in situ (23C28), technical constraints have limited the spatiotemporal resolution of this process, which has hampered analysis of the dynamics and quantitative characteristics of granule exteriorization in real time at the single-cell level. We developed a dynamic imaging system that, in contrast to static structural imaging (such as conventional transmission electron microscopy [TEM]), can follow in real time the spatially complex, rapidly evolving features of MCs undergoing activation. By combining newly designed granule detection and modeling techniques, we demonstrate that both human primary MCs in TLR2-IN-C29 vitro and mouse dermal MCs in vivo can respond to distinct stimuli of activation by finely regulating the dynamics and features of MC granule secretion. Results MCs differentially exteriorize secretory granules in response to different stimuli. We compared MC responses to (a) SP, an endogenous cationic 11Camino acid neuropeptide implicated in various inflammatory conditions (18, 29, 30) and a strong activator of the receptor MRGPRX2 (the ortholog of MRGPRB2: the receptor for cationic secretagogues in the mouse) (30C32), and (b) an antibody-dependent stimulus, anti-IgE, which activates IgE-bearing MCs by cross-linking Fc?RI-bound IgE. Similar levels of degranulation of primary human peripheral bloodCderived cultured MCs (PBCMCs) (33, 34), measured by release of the granule-stored mediator -hexosaminidase, were induced when PBCMCs were stimulated with 2 g/ml of anti-IgE or with 10 M SP (Figure 1A). Except as otherwise TLR2-IN-C29 noted, we used these conditions of stimulation for Rabbit Polyclonal to NOX1 all subsequent studies analyzing PBCMC activation via FcRI or MRGPRX2. While anti-IgE stimulation dose-dependently induced strong de novo secretion of lipid mediators (e.g., prostaglandins D2 and E2) and several inflammatory cytokines and chemokines, SP triggered secretion of only low amounts of lipid mediators and VEGF (Figure 1, BCE). Thus, when we used SP or anti-IgE under conditions that resulted in the same extent of PBCMC degranulation, activation via MRGPRX2 versus FcRI triggered distinct patterns of secretion of MC mediators not stored in the granules. Open in a separate window Figure 1 Human MC activation by.