树突细胞在抗原呈递和支气管哮喘发病机制中的作用

The roles of dendritic cells in antigen presentation and the pathogenesis of asthma

目的 通过对支气管哮喘 (简称哮喘 )患者和哮喘模型大鼠研究 ,探讨树突细胞 (DC)在哮喘发病机制中的作用。方法 初治未经糖皮质激素治疗的中、重度急性加重期组 (A组 )哮喘患者2 0例、缓解期组 (B组 )患者 15例、正常对照组 (C组 ) 10名 ,对哮喘患者外周血单个核细胞体外诱导培养DC ,通过流式细胞技术检测DC吞噬卵白蛋白 (OVA)的能力 ;检测细胞表面的标记物和共刺激分子 (MHC ⅡCD80 及CD86)及同种混合T淋巴细胞反应。建立哮喘气道炎症大鼠模型 ,检测大鼠支气管肺泡灌洗液 (BALF)中DC呈递功能及糖皮质激素对其影响。 2 4只SD大鼠按随机数字表法分为实验组 (D组 ,16只 )和对照组 (E组 ,8只 ) ,根据实验需要又将D组 16只大鼠分为OVA致敏激发组 (D1组 )、激发前激素预处理组 (D2 组 ) ,每组 8只。采用流式细胞技术测定D1组 (大鼠模型 )和D2组 (给予地塞米松腹腔注射 10mg/kg)和E组大鼠BALF中DC的CD80 、CD86和MHC Ⅱ的表达。结果 (1)A组患者外周血DC吞噬OVA的能力、DC细胞表达CD80 、MHC Ⅱ分别为 (41± 12 ) %、(32± 13) %、(44± 15 ) % ,B组分别为 (2 9± 10 ) %、(18± 10 ) %、(2 2± 10 ) % ,C组分别为 (2 9± 10 ) %、(14± 7) %、(18± 12 ) % ,三组间比较差异有统计学意义 (P均 <0 0 1) ;

Objective To investigate the roles of dendritic cells (DCs) in the pathogenesis of asthma. Methods (1)DCs were derived from peripheral blood monocytes of asthmatics with acute exacerbation (group A, n=20), patients at remission (group B, n=15) and healthy volunteers(group C, n=10), and cultured using media supplemented with granulocyte-macrophage colony stimulation factors (GM-CSF), interleukin-4(IL-4) and tumor necrosis factor-α (TNF-α). The antigen-uptaking function of the monocyte-derived DCs was evaluated by phagocytosis of fluorescence labeled ovalbumin (OVA), and their antigen-presenting function was determined by expression of membrane markers (MHC-Ⅱ) and co-stimulatory molecules(CD 80 and CD 86). The proliferation of T lymphocytes induced by DCs was assessed using autologous mixed T lymphocyte reaction. (2)The expression of MHC-Ⅱ and CD 80 and CD 86 of DCs were determined from the bronchoalveolar lavage (BAL) of asthmatic rats. The effects of dexamethasone on the expression of these markers and molecules were studied. Twenty-four rats were divided into the experimental group (group D) and the control group (group E). In group D, rats were subdivided into two groups with 8 rats in each. One group (group D 2) received pre-treatment with dexamethasone (10 mg/kg), and another group (group D 1) did not received dexamethasone pre-treatment, while group E was matched with the experimental group with regard to both age and weight. The rats were sensitized and challenged with OVA, and BAL was obtained. (3)All the membrane markers and co-stimulatory molecules on the surface of DCs were determined using flow cytometric method. Results (1)The phagocytosis of fluorescence labeled OVA in group A was increased [(41±12)%] compared with those in group B [(29±10)%, P<0.01] and group C [(29±10)%, P<0.01]. The expression of MHC-Ⅱ, CD 1α and CD 80 on the surface of DCs was the highest in group A [(44±15)%, (32±11)% and (32±13)%, respectively] compared with those in group B [(22±10)%, (19±9)% and (19±10)%, all P<0.01, respectively], as well as with those in group C [(18±12)%, (13±7)% and (14±7)%, all P<0.01, respectively]. There was no statistical difference in the expression of CD 86 on the cell membrane in group A, group B and group C. In autologous mixed T lymphocyte reaction study, when DC/T ratio was 1/5, DCs had the highest ability in stimulating the proliferation of T lymphocytes in group A (stimulating index=2.32±0.44) compared with those in group B(1.01±0.11,P<0.01), and those in group C (1.62±0.27,P<0.01). (2)The expression of MHC-Ⅱ in BAL cells of asthmatic rats reached peak value at 6 h after challenge, (15.2±5.0)% in group D 1, and (2.0±1.0)% in group E, P<0.01. The expression of CD 80 and CD 86 increased rapidly 2 h after challenge in group D 1 [(10.6±3.9)% and (7.5±3.8)%, respectively] compared with those in group E [(2.1±0.7)%, P<0.01 and (1.7±0.7)%, P<0.05, respectively]. The maximal inhibition effect of dexamethasone on the expression of MHC-Ⅱ was at 10 h, (7.8±2.4)% in group D 1, and (2.8±1.5)% in group D 2, P<0.01. The inhibition effect of dexamethasone on the expression of CD 80 and CD 86 was maximal at 24 h, (5.8±2.7)% and (5.5±1.5 )% respectively in group D 1, and (2.8±1.1)% and (2.9±1.6) % respectively in group D 2, P<0.05. Conclusions The antigen up-taking and presenting function of DCs were significantly enhanced in asthmatics at exacerbation and in a rat asthmatic model. Pretreatment with dexamethasone inhibited the function of DCs significantly in the animal model. The results suggest that DCs play important roles in antigen presentation in the pathogenesis of asthma, and the inhibition of glucocorticiod on DCs might be a critical mechanism in treatment of bronchial asthma.