Mast cell corticotropin-releasing factor subtype 2 suppresses mast cell degranulation and limits the severity of anaphylaxis and stress-induced intestinal permeability.

Background: Psychological stress and heightened MC activation are linked with important 35 immunological disorders including allergy, anaphylaxis, asthma, and functional bowel 36 diseases, but the mechanisms remain poorly defined. We have previously demonstrated that 37 activation of the corticotropin releasing factor (CRF) system potentiates MC degranulation 38 responses during IgE-mediated anaphylaxis and psychological stress , via CRF receptor 39 subtype 1 (CRF 1 ) expressed on MCs. 40 41 Objective: In this study, we investigated the role of CRF receptor subtype 2 (CRF 2 ) as a 42 modulator of stress-induced MC degranulation and associated disease pathophysiology. Methods: In vitro MC degranulation assays were performed with bone marrow derived MCs 45 (BMMCs) derived from WT and CRF 2 -deficient (CRF 2-/- ) mice and RBL-2H3 MCs transfected 46 with CRF 2 -overexpressing plasmid or CRF 2 -siRNA. In vivo MC responses and associated 47 pathophysiology in IgE-mediated passive systemic anaphylaxis and acute psychological restraint stress were measured in WT, CRF 2-/- , and MC-deficient Kit W-sh/W-sh 49 knock-in mice. Results: Compared with WT mice, CRF 2-/- exhibited heightened serum histamine levels and 52 exacerbated PSA-induced anaphylactic responses and colonic permeability. In addition, CRF 2-/- mice exhibited increased serum histamine and colonic permeability following acute restraint stress. Experiments with BMMCs and RBL-2H3 MCs demonstrated that CRF 2 expressed on MCs suppresses store-operated Ca 2+ entry (SOCE) signaling and MC degranulation induced by diverse MC stimuli. Experiments with MC-deficient Kit W-sh/W-sh mice systemically engrafted with WT and CRF 2-/- BMMCs demonstrated the functional importance 58 of MC-CRF 2 in modulating stress-induced pathophysiology. Conclusions: MC CRF 2 is a negative, global modulator of stimuli-induced MC degranulation Abbreviations: BMMC (Bone marrow derived mast cell), CRF 1 (Corticotropin Releasing 81 Factor Subtype 1), CRF 2 (Corticotropin Releasing Factor Subtype 2), Mast cell (MC), PMC 82 (Peritoneal mast cell), SOCE (store operated Ca 2+ entry) WT (Wild-type) 83 the basal TER values for a animal. After a 30 min equilibration period on Ussing chambers, FD4 (Sigma, 100 mg/ml) was added to the mucosal bathing reservoir of the Ussing chambers. After a 15 min equilibration period, standards were taken from the serosal side of each chamber and a 60 min flux period was established by taking 0.5 ml samples from the mucosal compartment. The quantity of FD4 was established by measuring the fluorescence in mucosal reservoir fluid samples in a fluorescence plate reader at 540 nm. Data are presented as the rate of FD4 flux in ng FD4.min.cm post transfection normal growth media CO 48 incubation, transfected cells were primed over-night or not and stimulated with 1 hour DNP or A23187 (non IgE primed) the hexosaminidase, histamine and calcium mobilization assay as described signaling in the MC is a potentiator of stimuli-induced MC degranulation and system and intestinal pathophysiology induced by PSA and psychological restraint stress (35). In the present study, our data revealed that, in contrast to CRF 1 , CRF 2 exerts inhibitor actions on MC degranulation as loss of CRF 2 activity in in MCs, via genetic or pharmacological 543 approaches, resulted in enhanced stimuli-induced MC degranulation and exacerbated responses to PSA and acute restraint stress. Selective inhibition of CRF 1 was shown to reduce PSA-induced MC responses in CRF 2-/- mice to the level of WT mice, overall indicating that MC CRF 1 and CRF 2 act in concert to up- and downregulate, respectively the tone of MC degranulation and resultant MC-dependent pathophysiology. Therefore, it is plausible that a disruption of MC CRF 1 and CRF 2 homeostasis could shift the balance between hypo- and hyper-activated MC states and ultimately disease activity. Precisely how the CRF receptor system is regulated to maintain MC homeostasis with regards to degranulation remains to be fully defined. We and others have shown that MCs release CRF 1 /CRF 2 ligands CRF and urocortin under basal and stimulated conditions (21, 22), and that CRF 1 and CRF 2 receptors modulate stimuli-induced MC degranulation in vitro in the absence of exogenous ligands. Together, these findings demonstrate that MCs possess a functional CRF system and implicates an autocrine regulation mechanism. Such a system could explain the ability of the MC CRF system to modulate rapid cellular signaling events in the MC such as Ca 2+ mobilization degranulation events. In view of the CRF system as a homeostatic regulator of the stress response, activation CRF 1 in MCs could provide immunological protection by enhancing MC degranulation and subsequent physiological and immunological responses, especially during time of acute stressful challenges. On the other hand, 2 activation may provide a critical mechanism of degranulation