过敏性哮喘患者树突细胞表型及分泌细胞因子的研究

A study of phenotype and function of dendritic cells and secretory cytokine in allergic asthmatic patients

目的观察过敏性哮喘患者树突细胞(DC)表达表面分子(CD1a、CD83、CD40、CD86)和分泌细胞因子(IL12和IL10)的情况,及对原始T细胞分化的影响。方法分别取过敏性哮喘患者(9例)和健康对照者(14例)外周血培养成熟DC。另取无哮喘家族史的新生儿脐血分离得到原始T细胞。将2组DC和原始T细胞共同培养。流式细胞仪测DC表面协同刺激分子CD1a、CD83、CD40、CD86的表达。ELISA测DC分泌的IL12、IL10及T细胞分泌的IFNγ、IL4的含量。结果哮喘组DC表达CD86分子比对照组显著升高(P<001)。哮喘组DC分泌IL12、IL12p40和IL10较对照组显著减少(P<001,P<005)。哮喘组T细胞释放IFNγ较对照组减少(P<005),释放IL4较对照组显著增多(P<001)。哮喘组IL12与IFNγ呈正相关(r=0758,P<005),与IL4呈负相关(r=-0756,P<005);IL10与IL4呈负相关(r=-0685,P<005);IL12与IL10呈正相关(r=0926,P<001)。结论过敏性哮喘患者DC存在缺陷,使原始T细胞向Th2优势分化,IL4等的释放增加,且不能有效地形成T细胞耐受,共同导致过敏性哮喘的发生。

Objective To investigate the deficiency of the expression of the phenotypes(CD_~1a ,CD_~83 ,CD_~40 ,CD_~86 ) and cytokines(IL-12 and IL-10) by human peripheral blood monocyte(PBMC)-derived dendritic cell (DCs) from asthmatic subjects, and their influence on nave T cell polarization.Methods Adherent cells were isolated from peripheral blood samples in asthmatic patients and in healthy volunteers, and were cultured with granulocyte-macrophage colony-stimulating factor and IL-4 as immature DC(iDC). iDCs were stimulated with lipopolysaccharide as mature DC(mDCs).Nonadherent cells were obtained from umbilical cord blood by idem methods, and nave T cells were sorted by adding anti-CD_4 and anti-CD_~45RA in nonadherent cells respectively and magnetic microbeads. Nave T cells and mDCs from two groups were co-cultured in complete RPMI1640 media respectively, and nave T cells polarized as T helper cells 1 (Th1) and Th2.The expression of the CD_~1a , CD_~83 , CD_~40 and CD_~86 on mature DCs were examined by fluorescent activated cell sorter. IL-12 and IL-10 released by mDCs and IL-4 and IFNγ produced by Th cells were measured by ELISA.Results (1) The expression of CD_~86 on dendritic cells from atopic asthmatics was higher than that from healthy control subjects(40.75±3.99 vs 29.88±1.25,P<0.01).(2)The levels of IL-12, IL-12p40 and IL-10 produced by DCs from asthmatic subjects were all significantly lower than those from healthy control group(217.79±118.65 vs 905.66±495.32, P<0.01; 2072.22±1496.37 vs 5569.43±2922.75,P<0.01; 336.89±261.52 vs 1425.00±1148.87,P<0.05, respectively).(3) IL-4 production by Th2 cells which were primed by DCs from asthmatics was significantly increased as compared to that from control group(368.56±190.72 vs 584.91±290.13,P<0.01); On the contrary, IFNγ in the patient group was reduced as compared to that in the control group(425.33±164.94 vs 49.86±18.14,P<0.05).(4) In the patient group, the level of IL-12 was positively correlated to that of IFNγ(P<0.05), negatively correlated to that of IL-4(P<0.05); IL-10 was negatively correlated to IL-4(P<0.05).(5) There was a positive correlation between IL-12 and IL-10 in the two groups(P<0.01). Conclusion Because of DC deficiency, nave T cells preferentially polarize to Th2 which synthesize more Th2-type cytokine(i. e. IL-4) and T cell tolerance cannot be induced, which may be one of the important pathogenic mechanisms for allergic asthma.