The effect of Rho drugs on mast cell activation and degranulation

Abstract

Mast cells are tissue-resident immune cells that produce potent proinflammatory mediators, which are stored in cytoplasmic granules. Stimulation triggers degranulation, a process that mobilizes granules to dock and fuse to the plasma membrane, releasing mediators. Mast cell degranulation has an important role in immunity but can also intensify inflammation and contribute to allergic disorders. Hence, it is important to understand signaling pathways that regulate mast cell degranulation. Here, we examined the role of Rho proteins in regulating mast cell activation leading to degranulation. RBL-2H3 cells and bone marrow-derived mast cells (BMMCs) were stimulated through aggregation of FcεRI receptors. Stimulated cells showed a large increase in the levels of activated Rac and, to a lesser extent, RhoA. Drugs were used to acutely inhibit the function of specific Rho proteins. The Rac inhibitor EHT-1864 and the RhoA inhibitor rhosin inhibited degranulation. Microscopic characterization showed that, upon stimulation, RBL-2H3 cells formed surface ridges that grew into large protrusions reminiscent of circular dorsal ruffles, which flattened into large lamellipodia. LysoTracker-labeled cells showed granules stream into peripheral protrusions. EHT-1864 reduced granule motility, whereas rhosin increased motility; both drugs affected the formation of peripheral protrusions. These results showed that, in response to stimuli, Rho proteins control discrete cytoskeletal remodeling processes that are needed for granule exocytosis. Rac is required to stimulate the remodeling of mast cells, triggering actin-mediated flattening of the cell periphery to create an active degranulation zone, whereas RhoA controls the streaming of highly motile granules into the active zone.

RBL cells as models for in vitro studies of mast cells and basophils

“…To gain insight into the factors that govern these cytoskeletal changes, a pharmacological screen of Rho family inhibitors was recently conducted testing the impact of inhibitors of members of the Rho family of GTPases on MC secretion . The results of this study conducted on both FcεRI‐activated BMMCs and RBL cells revealed similar drug sensitivities, therefore implicating similar actin signaling linked with BMMC‐ or RBL‐stimulated exocytosis . These results are consistent with previous studies of direct measurements of actin rearrangements that occur during exocytosis of BMMC or RBL that documented similar actin remodeling …”

“…79 The results of this study conducted on both FcεRI-activated BMMCs and RBL cells revealed similar drug sensitivities, therefore implicating similar actin signaling linked with BMMC-or RBL-stimulated exocytosis. 79 These results are consistent with previous studies of direct measurements of actin rearrangements that occur during exocytosis of BMMC or RBL that documented similar actin remodeling. 80,81…”

“…MC exocytosis is associated with remarkable cytoskeletal changes. To gain insight into the factors that govern these cytoskeletal changes, a pharmacological screen of Rho family inhibitors was recently conducted testing the impact of inhibitors of members of the Rho family of GTPases on MC secretion . The results of this study conducted on both FcεRI‐activated BMMCs and RBL cells revealed similar drug sensitivities, therefore implicating similar actin signaling linked with BMMC‐ or RBL‐stimulated exocytosis .…”

RhoA/Rho‐kinases in asthma: from pathogenesis to therapeutic targets

“…Studies in our laboratory demonstrated a significant infiltration of mast cells in the lung tissues of asthma mouse model 92 and in nasal eosinophilic polyps of patients with chronic rhinosinusitis with nasal polyps (CRSwNP) 93 . Interestingly, recent studies provided evidence that RhoA signalling is associated with mast cell activation 94 . In particular, Sheshachalam et al 94 showed that RhoA signalling controls mast cell morphology that transitions to an activated form and granule movement for degranulation.…”

“…Interestingly, recent studies provided evidence that RhoA signalling is associated with mast cell activation 94 . In particular, Sheshachalam et al 94 showed that RhoA signalling controls mast cell morphology that transitions to an activated form and granule movement for degranulation. Sibilano et al reported a significant impairment in mast cell degranulation in bone marrow‐derived mast cells (BMMCs) with RhoA knockdown 95 .…”