Stress and Nasal Allergy: Corticotropin-Releasing Hormone Stimulates Mast Cell Degranulation and Proliferation in Human Nasal Mucosa

Abstract

Psychological stress exacerbates mast cell (MC)-dependent inflammation, including nasal allergy, but the underlying mechanisms are not thoroughly understood. Because the key stress-mediating neurohormone, corticotropin-releasing hormone (CRH), induces human skin MC degranulation, we hypothesized that CRH may be a key player in stress-aggravated nasal allergy. In the current study, we probed this hypothesis in human nasal mucosa MCs (hM-MCs) in situ using nasal polyp organ culture and tested whether CRH is required for murine M-MC activation by perceived stress in vivo. CRH stimulation significantly increased the number of hM-MCs, stimulated both their degranulation and proliferation ex vivo, and increased stem cell factor (SCF) expression in human nasal mucosa epithelium. CRH also sensitized hM-MCs to further CRH stimulation and promoted a pro-inflammatory hM-MC phenotype. The CRH-induced increase in hM-MCs was mitigated by co-administration of CRH receptor type 1 (CRH-R1)-specific antagonist antalarmin, CRH-R1 small interfering RNA (siRNA), or SCF-neutralizing antibody. In vivo, restraint stress significantly increased the number and degranulation of murine M-MCs compared with sham-stressed mice. This effect was mitigated by intranasal antalarmin. Our data suggest that CRH is a major activator of hM-MC in nasal mucosa, in part via promoting SCF production, and that CRH-R1 antagonists such as antalarmin are promising candidate therapeutics for nasal mucosa neuroinflammation induced by perceived stress.

Keywords: CRH; mast cell; nasal mucosa; psychological stress; stem cell factor (SCF).

Figure 1

Human nasal mucosa mast cells (hM-MCs) and the mucosal epithelium in nasal polyps (NPs) express corticotropin-releasing hormone receptor type 1 (CRH-R1). (a) CRH-R1 expression on c-Kit+ or (b) tryptase+ hM-MCs in situ. An arrow denotes double+ MCs. (c) Percentage of tryptase and CRH-R1 or corticotropin-releasing hormone receptor type 2 (CRH-R2) double+ MCs. n = 5. (d) CRH expression within the mucosal epithelium in NPs (an arrow). (e) CRH-R1 and CRH-R2 expression within the mucosal epithelium in NPs. Scale bar = 20 µm. Error bars indicate the standard error of the mean (SEM). ** p < 0.01. MC, mast cell; hM-MCs, human nasal mucosa MCs; NPs, nasal polyps; CRH, corticotropin-releasing hormone; CRH-R1, CRH receptor type 1; CRH-R2, CRH receptor type 2; DAPI, diamidino-2-phenylindole.

Figure 2

CRH increased tryptase+ hM-MC numbers and stimulated their degranulation. (a) Toluidine blue histochemistry with 1 day organ-cultured human NPs treated with vehicle or CRH (10⁻⁷ M). An arrow denotes hM-MCs in the lamina propria. Top, non-degranulated MCs in vehicle-treated NPs. Bottom, degranulated MCs in CRH-treated NPs. n = 5; scale bar = 20 µm. (b) Tryptase immunohistochemistry with 1 day organ-cultured human NPs treated with vehicle or CRH (10⁻⁷ M). Scale bar = 50 µm. (c) Quantitative immunohistomorphometry of tryptase+ cells in the lamina propria of organ-cultured NPs with CRH, anti-SCF (stem cell factor neutralizing antibody; 1 μg/mL), and/or antalarmin (10⁻⁶ M); n = 6. (d) Tryptase immunohistochemistry. An arrow denotes MC degranulation induced by CRH. Scale bar = 10 µm. (e) Percentage of degranulated MCs treated with CRH, anti-SCF (1 μg/mL), and/or antalarmin (10⁻⁶ M) analyzed by tryptase immunohistochemistry; n = 6. (f) Electron microscope images of CRH-induced MC degranulation. Scale bar = 1 µm. (g) Diameter of tryptase+ hM-MC granules; n = 6; scale bar = 10 µm. Error bars indicate SEM. * p < 0.05, *** p < 0.001, **** p < 0.0001. MC, mast cell; hM-MCs, human nasal mucosa MCs; NPs, nasal polyps; CRH, corticotropin-releasing hormone; anti-SCF, stem cell factor neutralizing antibody.

Figure 3

CRH increased tryptase+ hM-MC proliferation but not the apoptosis of hM-MCs. CRH-R1 gene silencing showed that CRH-R1 small interfering RNA (siRNA) inhibited the increase in tryptase+ hM-MC number and degranulation by CRH in situ. (a) Tryptase/Ki-67 double immunofluorescence. The arrow denotes tryptase and Ki-67 double+ cells within human nasal polyps treated with CRH; n = 6; scale bar = 10 µm. (b) Quantitative immunohistomorphometry of tryptase/proliferating cell nuclear antigen (PCNA) double+ cells; n = 4. (c) Quantitative immunohistomorphometry of tryptase/terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) double+ cells; n = 4. (d) Quantitative immunohistomorphometry of tryptase+ cells in the lamina propria of organ-cultured NPs with CRH-R1 siRNA-treated human NPs; n = 4. (e) Percentage of degranulated hM-MCs in the lamina propria with CRH-R1 siRNA-treated human NPs; n = 4. Error bars indicate SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, N.S. = not significant. MC, mast cell; hM-MCs, human nasal mucosa MCs; NPs, nasal polyps; CRH, corticotropin-releasing hormone; CRH-R1, CRH receptor type 1; CRH-R1 siRNA, CRH-R1 siRNA-treated NPs; SCR, scrambled siRNA-treated NPs; PCNA, proliferating cell nuclear antigen; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling.

Figure 4

CRH increased stem cell factor (SCF) expression in the mucosal epithelium in NPs. (a) SCF immunofluorescence of organ-cultured human NPs treated with vehicle or CRH (10⁻⁷ M). CRH significantly increased SCF expression within the epithelium of 1 day organ-cultured human NPs. The arrow denotes SCF-positive immunoreactivity within the epithelium. n = 6; scale bar = 50 µm. (b) Double immunofluorescence for AE1/3/SCF and (c) tryptase/SCF of CRH-treated NPs. Most of the SCF+ cells were epithelial cells. Some tryptase+ hM-MCs within the epithelium expressed SCF. The arrow denotes SCF+ hM-MCs; n = 4 in (b) and 6 in (c). Scale bar = 10 µm in (b), 20 µm in (c). (d) SCF immunoreactivity in NP epithelium with CRH-R1 siRNA-treated human NPs. The arrow denotes SCF-positive immunoreactivity within the epithelium. n = 4; scale bar = 20 µm. Error bars indicate SEM. *p < 0.05, ** p < 0.01. MC, mast cell; SCF, stem cell factor; hM-MCs, human nasal mucosa MCs; NPs, nasal polyps; CRH, corticotropin-releasing hormone; CRH-R1, CRH receptor type 1; CRH-R1 siRNA, CRH-R1 siRNA-treated NPs; SCR, scrambled siRNA-treated NPs; DAPI, diamidino-2-phenylindole.

Figure 5

CRH increased the expression of CRH-R1 on tryptase+ hM-MCs and upregulated tryptase immunoreactivity within the hM-MCs. Percentage of (a) tryptase+ and (b) c-Kit+ MCs with CRH-R1 in the lamina propria of organ-cultured human NPs, analyzed by double immunofluorescence of tryptase or c-Kit/CRH-R1. n = 7 in (a) and 4 in (b). (c) Tryptase immunoreactivity within the hM-MCs in NPs treated with vehicle or CRH. CRH treatment significantly increased tryptase immunoreactivity within hM-MCs. n = 5; scale bar = 10 µm. Error bars indicate SEM. * p < 0.05, N.S. = not significant. MC, mast cell; hM-MCs, human nasal mucosa MCs; NPs, nasal polyps; CRH, corticotropin-releasing hormone; CRH-R1, CRH receptor type 1.

Figure 6

Restraint stress increased mice nasal mucosa MC (mM-MC) numbers and degranulation. (a) Toluidine blue histochemistry of murine nasal mucosa. The arrow denotes mM-MCs in the lamina propria, and the arrowhead denotes a degranulated mM-MC. Scale bar = 20 µm. (b) Quantitative immunohistomorphometry of mM-MC. (c) The percentage of degranulated mM-MCs in the nasal mucosa after restraint stress and nasal application of antalarmin. Both acute and chronic restraint stress significantly increased mM-MC numbers in the nasal mucosa and stimulated their degranulation. The increased number of mM-MCs and their degranulation in stressed mouse nasal mucosa was significantly inhibited by nasal application of antalarmin. Control group, n = 4 (without stress); stress group, n = 4; stress + antalarmin (5 μg/g/day) group, n = 5; and antalarmin group, n = 4 (without stress). Error bars indicate SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. MC, mast cell; mM-MCs, mice nasal mucosa MCs.