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

Abstract

Background: Psychological stress and heightened mast cell (MC) activation are linked with important immunologic disorders, including allergy, anaphylaxis, asthma, and functional bowel diseases, but the mechanisms remain poorly defined. We have previously demonstrated that activation of the corticotropin-releasing factor (CRF) system potentiates MC degranulation responses during IgE-mediated anaphylaxis and psychological stress through corticotropin-releasing factor receptor subtype 1 (CRF₁) expressed on MCs.

Objective: In this study we investigated the role of corticotropin-releasing factor receptor subtype 2 (CRF₂) as a modulator of stress-induced MC degranulation and associated disease pathophysiology.

Methods: In vitro MC degranulation assays were performed with bone marrow-derived mast cells (BMMCs) derived from wild-type (WT) and CRF₂-deficient (CRF₂^-/-) mice and RBL-2H3 MCs transfected with CRF₂-overexpressing plasmid or CRF₂ small interfering RNA. In vivo MC responses and associated pathophysiology in IgE-mediated passive systemic anaphylaxis and acute psychological restraint stress were measured in WT, CRF₂^-/-, and MC-deficient Kit^W-sh/W-sh knock-in mice.

Results: Compared with WT mice, CRF₂^-/- mice exhibited greater serum histamine levels and exacerbated IgE-mediated anaphylaxis and colonic permeability. In addition, CRF₂^-/- mice exhibited increased serum histamine levels and colonic permeability after acute restraint stress. Experiments with BMMCs and RBL-2H3 MCs demonstrated that CRF₂ expressed on MCs suppresses store-operated Ca²⁺ entry signaling and MC degranulation induced by diverse MC stimuli. Experiments with MC-deficient Kit^W-sh/W-sh mice systemically engrafted with WT and CRF₂^-/- BMMCs demonstrated the functional importance of MC CRF₂ in modulating stress-induced pathophysiology.

Conclusions: MC CRF₂ is a negative global modulator of stimuli-induced MC degranulation and limits the severity of IgE-mediated anaphylaxis and stress-related disease pathogenesis.

Keywords: Mast cell; allergy; anaphylaxis; corticotropin-releasing factor receptor; intestinal permeability; stress.

FIG 1.. CRF₂ deficiency exacerbates passive systemic anaphylaxis and intestinal permeability.

Wild type (CRF₂^+/+) and CRF₂^−/− female mice (6–8 weeks of age) were sensitized systemically overnight with IgE and challenged with either PBS vehicle (Control) or anti-IgE DNP to induce passive systemic anaphylaxis (PSA). A: Serum histamine levels were measured at 30 min post-vehicle or DNP challenge. B: rectal temperature was recorded at 0 and 30 min and the change in body temperature was calculated and presented as ∆ °C. C: Cardiac blood volume was measured at 0, 30 and 120 min post DNP challenge. D, E: 120 min post-DNP challenge, ex vivo colonic permeability was measured on Ussing chambers as FD4 flux rate (D) and TER (E). F-H: Tissues from WT and CRF₂^−/− mice were fixed and stained with Toluidine blue and mast cell counts were performed and presented as mast cells/hpf. G: blood basophils were counted in blood smears and expressed at the % of basophils/100 WBCs. Data are means ± SEM from a representative experiment with n=6 animals per group. Experiments were repeated in n=6 (serum histamine, body temperature) or 2 (cardiac volume, mast cell and basophil counts) independent replicates. ^#,* symbols differ by p<0.05 using a 1-Way ANOVA (A, D); Different asterisks indicate significance determine by unpaired two-tailed t-test (B, C, E),*p<0.05, **p<0.01.

FIG 2.. Localization of CRF1 and CRF2 in BMMCs and peritoneal mast cells.

CRF1 (Cy3 Red) and CRF2 (FITC Green) nuclei (DAPI blue). z-Stack overlay images show a single plane cross section through horizontal plane (XY), sagittal plane (YZ), and coronal plane (XZ). Panels A-C) murine BMMCs. Panels D-F) PMCs. White arrow indicates the nuclear staining pattern of CRF₂ (Panel C).

FIG 3.. Genetic deficiency and overexpression of CRF₂ induces divergent effects on stimuli induced MC degranulation. A:

β-hex % release from IgE-sensitized WT and CRF₂^−/− BMMCs stimulated with DNP (32 ng/mL). B, C: β-hex release from WT and CRF₂^−/− BMMCs stimulated with A23187 (B) and c48/80 (C). D: Transmission electron microscopy analysis of WT and CRF₂^−/− BMMCs stimulated with IgE/DNP. E,F: β-hex release from RBL-2H3 MCs transfected with CRF₂-overexpressing plasmid or eGFP control or CRF₂-siRNA. Data are means ± SEM from a representative experiment with n=3 (bone marrow donors) per group. Experiments were repeated in n=6 (A-C) or 2 (E-F) independent replicates. *Significance between groups was determined by an unpaired two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

FIG 4.. CRF₂ selective antagonism enhances mast cell degranulation in rodent and human mast cells.

β-hex % release from RBL-2H3 mast cells (A) and human LAD2 cells (B) following pre-treatment with the CRF₂ antagonist drug Astressin 2B (A2B) at indicated concentrations. **p<0.01 1-Way ANOVA (n=6 replicates/treatment). Experiments repeated in n=3 independent experiments.

FIG 5.. CRF2−/− BMMCs exhibit exacerbated Ca²⁺ mobilization responses to diverse mast cell stimuli.

Murine BMMCs from female mice were loaded with Fluo 4 Ca²⁺ indicator and stimulated with indicated MC degranulation stimuli as described in the Material and Methods Section. Intracellular Ca²⁺ mobilization (presented as ∆F480/F540 or as representative traces) in response to indicated concentrations of IgE/DNP (A,E), A23187 (B,F) and c48/80 (C,G) and Carbachol (D,H). Data shown are the means ± SE and are representative of 3 independent experiments performed in triplicate. Data were analyzed using a 1-way ANOVA *P < 0.05, **P < 0.001, ***P < 0.0001.

FIG 6.. CRF2−/− BMMCs exhibit heightened intracellular Ca²⁺ store release and expression of SOCE channels.

BMMCs derived from WT and CRF₂^−/− mice were loaded with Fluo 4, and intracellular Ca²⁺ levels were measured following stimulation with IgE/DNP. A,B: Representative intracellular Ca²⁺ traces for experiments conducted under Ca²⁺-replete (A) or Ca²⁺-free (1 mM EDTA; B) conditions. C: Mean peak change in fluorescence following IgE/DNP stimulus presented as ∆ peak fluorescence. D-I: Representative Western blots and densitometry analysis for STIM1 (D, G), TRPC1 (E, H), and Orai (F, I) in WT and CRF₂^−/− BMMCs. *Significance between groups was determined by an unpaired two-tailed t-test (C, G-I),*p<0.05, **p<0.01.

FIG 7.. CRF2−/− mice exhibit heightened serum histamine levels and intestinal permeability following acute restraint stress.

Wild type and CRF₂^−/− mice female mice (8–10 weeks of age) underwent 1 h of restraint stress or housed under normal cage conditions (Control) as described in Materials and Methods. Serum histamine (A) and colonic permeability (B) was measured by ELISA following 1 h of restraint stress. Data are means ± SEM (n=8 mice/group). Data were analyzed using a 2-way ANOVA, *p<0.05, **p<0.01, ^#P=0.07.

FIG 8.. CRF₂ expressed on mast cells is a critical modulator of stress-induced intestinal permeability.

Mast cell deficient Kit^W-sh/W-sh mice were engrafted with BMMCs derived from WT or CRF₂^−/− mice. Twelve weeks post-engraftment, mice were exposed to restraint stress (RS) for 3 hours. FD4 flux rate (A) and TER (B) measured from colon mounted on Ussing chambers from WT and CRF₂^−/− BMMC-engrafted Kit^W-sh/W-sh mice. C: MC numbers, determined with Toluidine blue staining in intestinal mesenteric windows from WT and CRF2−/− engrafted Kit^W-sh/W-sh mice. Data are means ± SEM (n=8 mice/group). Experiments were repeated in two independent studies. Data were analyzed using a 2-way ANOVA,*p<0.05.