肥大细胞参与特应性皮炎表型及2型炎症因子释放的免疫活化中作用的初步研究

Preliminary studies on the role of mast cells in the phenotype and immune activation of type 2 inflammatory cytokine release of atopic dermatitis

目的研究肥大细胞在特应性皮炎(AD)表型及2型炎症因子释放的免疫活化中的作用。方法9份AD皮肤样本来自西安交通大学第二附属医院皮肤科,9份健康皮肤对照样本来自西安交通大学第二附属医院骨外科手术切除多余正常皮肤,对其进行甲苯胺蓝染色和荧光染色,明确AD皮损中肥大细胞脱颗粒活化状态。通过野生型小鼠足趾肿胀渗出实验,探究卡泊三醇(MC903)能否在体内直接激活肥大细胞;利用野生型和Kit^(W‑sh/W‑sh)肥大细胞缺失小鼠构建MC903‑AD模型,以探究肥大细胞是否影响AD小鼠的表型、组织病理及2型炎症因子水平;提取小鼠腹腔原代肥大细胞,通过钙成像、类胰蛋白酶和β‑氨基己糖苷酶释放实验、单核细胞趋化蛋白‑1(MCP‑1)和趋化因子CXC配体‑2(CXCL‑2)释放检测等,探究MC903体外激活肥大细胞释放炎症介质能力。结果与健康对照相比,AD患者皮损中处于活化状态的脱颗粒肥大细胞数目较健康对照组增多,分别为(5.40±1.14)和(2.20±0.84)个(P<0.001)。与野生型AD小鼠相比,Kit^(W‑sh/W‑sh)肥大细胞缺失AD小鼠的表型减轻,小鼠AD指数评分(ADI)分别为(10.00±0.89)和(5.50±1.05)分(P<0.001),且2型炎症因子释放减轻,白细胞介素(IL)‑4水平分别为(29.50±1.87)和(15.33±1.86)pg/mg(P<0.001),IL‑13水平分别为(6.32±0.25)和(3.93±0.22)pg/mg(P<0.001),IL‑31水平分别为(9.73±0.38)和(6.89±0.27)pg/mg(P<0.001),TSLP水平分别为(206.00±4.43)和(99.00±4.86)pg/mg(P<0.001)。MC903在体内注射可以导致小鼠足趾肿胀。MC903可以在体外激活肥大细胞。结论AD患者皮损中活化的肥大细胞数目较健康对照组增多;Kit^(W‑sh/W‑sh)肥大细胞缺失AD小鼠的表型、组织病理和2型炎症因子水平较野生型AD小鼠明显减轻;MC903可在体内和体外激活肥大细胞。肥大细胞在AD表型、免疫活化中均起到关键作用。

Objective To investigate the role of mast cells in atopic dermatitis(AD)phenotype and the immune activation of type 2 inflammatory cytokine release.Methods Nine AD skin samples were obtained from the Department of Dermatology,the Second Affiliated Hospital of Xi′an Jiaotong University,and nine healthy skin control samples were obtained from the surgical excision of excess normal skin by orthopedic surgery,the Second Affiliated Hospital of Xi′an Jiaotong University,which were subjected to toluidine blue staining and fluorescence staining to clarify the mast cell degranulation activation status of the AD skin lesions.We investigated whether MC903 could directly activate mast cells in vivo through the toe swelling and exudation assay in wild‑type mice;we constructed the MC903‑AD model using wild‑type and Kit^(W‑sh/W‑sh) mast cell‑deficient mice in order to investigate whether mast cells affected the phenotype,histopathology,and the level of type 2 inflammatory factors in AD mice;we extracted mouse peritoneal mast cells and the ability of MC903 to activate mast cells to release inflammatory mediators in vitro was explored by calcium imaging,tryptase and β‑aminohexokinase release assays,and MCP‑1 and CXCL‑2 release assays.Results The number of degranulated mast cells in an activated state was increased in skin lesions of AD patients compared to healthy controls,with(5.40±1.14)and(2.20±0.84),respectively(P<0.001).Kit^(W‑sh/W‑sh) mast cell‑deficient AD mice had an attenuated phenotype with ADI scores of(5.50±1.05),compared to wild‑type AD mice with(10.00±0.89)(P<0.001).The release of type 2 inflammatory factors in wild‑type AD mice was higher than those in Kit^(W‑sh/W‑sh) mast cell‑deficient AD mice,with IL‑4 levels of(29.50±1.87)and(15.33±1.86)pg/mg(P<0.001),IL‑13 levels were(6.32±0.25)and(3.93±0.22)pg/mg(P<0.001),IL‑31 levels were(9.73±0.38)and(6.89±0.27)pg/mg(P<0.001),and TSLP levels were(206.00±4.43)and(99.00±4.86)pg/mg(P<0.001),respectively.MC903 could cause mast cell activation in wild‑type mice,leading to increased swelling and exudation in the toes of mice,and MC903 could activate mast cells in vitro,leading to increased degranulation and release of inflammatory factors such as MCP‑1 and CXCL‑2.Conclusions The number of activated mast cells was increased in skin lesions of AD patients than in healthy controls.Kit^(W‑sh/W‑sh) mast cell‑deficient AD mice showed significantly reduced phenotype,histopathology,and type 2 inflammatory factor levels compared with wild‑type AD mice.MC903 activates mast cells in vivo and in vitro.Mast cells play a key role in AD phenotype and immune activation.