Stress Affects Mast Cell Proteases in Murine Skin in a Model of Atopic Dermatitis-like Allergic Inflammation

Abstract

Stress exposure worsens allergic inflammatory diseases substantially. Mast cells (MCs) play a key role in peripheral immune responses to neuroendocrine stress mediators such as nerve growth factor (NGF) and substance P (SP). Mast cell proteases (MCPs) and cholinergic factors (Chrna7, SLURP1) were recently described to modulate MC stress response. We studied MCPs and Chrna7/SLURP1 and their interplay in a mouse model for noise induced stress (NiS) and atopic dermatitis-like allergic inflammation (AlD) and in cultured MC lacking Chrna7. We found that the cholinergic stress axis interacts with neuroendocrine stress mediators and stress-mediator cleaving enzymes in AlD. SP-cleaving mMCP4+ MC were upregulated in AlD and further upregulated by stress in NiS+AlD. Anti-NGF neutralizing antibody treatment blocked the stress-induced upregulation in vivo, and mMCP4+ MCs correlated with measures of AlD disease activity. Finally, high mMCP4 production in response to SP depended on Chrna7/SLURP1 in cultured MCs. In conclusion, mMCP4 and its upstream regulation by Chrna7/SLURP1 are interesting novel targets for the treatment of allergic inflammation and its aggravation by stress.

Keywords: atopic dermatitis-like allergic inflammation (AlD); mouse mast cell protease (mMCP); nicotinic acetylcholine receptor α7 (Chrna7); non-neuronal cholinergic system (NNCS); secreted Ly-6/uPAR-related protein 1 (SLURP1); stress; substance P (SP).

Figure 1

Stress (NiS) and allergic inflammation (AlD) combined dramatically enhance skin inflammation in murine back skin. Giemsa staining shows exemplary back skin full-thickness biopsies. Sections were performed along the head–tail axis as evidenced by the telogen hair follicles visible. AlD skin shows thickening of the epidermis and an increased cell density in dermis and subcutis in evidence of inflammatory infiltration. In the back skin of mice that were additionally stressed, both epidermal thickness and infiltration are dramatically increased, which is not the case in the back skin samples additionally treated with neutralizing antibodies against NGF (aNGF) or BDNF (aBDNF). Abbr.: AlD = atopic dermatitis-like allergic inflammation; BDNF = brain-derived neurotrophic factor; NGF = nerve growth factor; NiS = noise-induced stress; Pan. Carn.: = panniculus carnosus.

Figure 2

Stress and stress mediator effects on mMCP protein and mRNA expression in MCs. Data represent the mean values of the results from 5 animals (n = 5). ** = p ≤ 0.01; * = p ≤ 0.05. Immunohistomorphometric analysis was performed in 10 microscopic fields per animal on 14 µm thick skin cryosections for (a,d) mMCP4+ and mMCP6+ MCs (expressed in % of total number of FITC-Avidin+ MCs). FITC-Avidin is used as a specific MC marker in skin. (c,f) High- (black bar) and low-intensity (gray bar) staining of mMCP4+ and mMCP6+ MCs (sums up to % of mMCP+ MCs of the respective group given in (a,d)). (b,e) Representative photomicrographs of an immunohistochemical triple staining for mMCP4 and mMCP6 (red), MCs (FITC-labeled avidin, green), and cell nuclei (DAPI, blue), scale bar = 10 µm. Abbr.: AlD = atopic dermatitis-like allergic inflammation; anti-BDNF = antibody against BDNF; anti-NGF = antibody against NGF; BDNF = brain-derived neurotrophic factor; MC = mast cell; mMCP = murine mast cell protease; NGF = nerve growth factor; NiS = noise induced stress.

Figure 3

Pro-inflammatory cytokine imbalance and mMCP4+ MC percentage interacts with indicators of MC activation and allergic inflammation. Results are given in the figure. (a) IL10/TNFα mRNA expression ratio: mRNA levels were analyzed in full-thickness skin biopsies from experimental mice. Significant group differences were calculated by the Kruskal–Wallis Test. (b,c) Linear regressions were calculated, including 3–4 samples from each of the 6 experimental groups. Best-fit line and 95% confidence band are shown. * = p ≤ 0.05. (b) MC degranulation was used as a marker for stress-sensitive neurogenic inflammation. (c) Eosinophilic infiltration was used as a marker for allergic inflammation. Abbr.: AlD = atopic dermatitis-like allergic inflammation; anti-BDNF = antibody against BDNF; anti-NGF = antibody against NGF; BDNF = brain-derived neurotrophic factor; IL10 = interleukin 10; MC = mast cell; NGF = nerve growth factor; NiS = noise induced stress; TNFα = tumor necrosis factor alpha.

Figure 4

SP induces mMCP4 mRNA in the presence of Chrna7 in cultured MCs. (a,b) qRTPCR analysis of mMCP4 and mMCP6 mRNA expression in primary cultures of peritoneal MCs from Wt and α7-KO mice. Individual treatments are mentioned at the X axis of the graphs. Gene expressions were normalized to the housekeeping gene TBP. Data represent the averages of at least 3 different experiments. * = p ≤ 0.05. Abbr.: α7-KO = Chrna7 knockout; mMCP = murine mast cell protease; SLURP1 = Ly-6/uPAR-related protein 1; SP = substance P; Wt = wild type.