Rudwaleit M, Siegert S, Yin Z, Eick J, Thiel A, Radbruch A, Sieper J, Braun J
Rheumatology, Department of Medicine, University Hospital Benjamin Franklin, Berlin, Germany.
Ann Rheum Dis. 2001 Jan;60(1):36-42. doi: 10.1136/ard.60.1.36.
To test the hypothesis that ankylosing spondylitis (AS) is a T helper cell type 2 polarised disease by quantifying the T cell cytokines interferon gamma (IFNgamma), interleukin 4 (IL4), tumour necrosis factor alpha (TNFalpha), and IL10 at the single cell level in patients with AS in comparison with healthy HLA-B27 negative and HLA-B27 positive controls.
Peripheral blood mononuclear cells from 65 subjects (25 HLA-B27 positive patients with active AS, 18 healthy HLA-B27 positive controls, and 22 healthy HLA-B27 negative controls) were stimulated with phorbol myristate acetate/ionomycin for six hours, surface stained for CD3 and CD8, intracellularly stained for the cytokines IFNgamma, TNFalpha, IL4, and IL10, and analysed by flow cytometry. TNFalpha production was related to the genotype of the TNFalpha promoter at the -308 and -238 polymorphisms.
In peripheral blood the percentage of TNFalpha+ T cells was significantly lower in HLA-B27 positive patients with AS (median 5.1% for CD4+ T cells) than in healthy HLA-B27 negative controls (median 9.5%; p=0.008). Surprisingly, the percentage of TNFalpha+ T cells was also significantly lower in healthy HLA-B27 positive controls (median 7.48%) than in healthy HLA-B27 negative controls (p=0.034). Furthermore, the percentage of IFNgamma+ T cells was lower in patients with AS and in healthy HLA-B27 positive controls than in healthy HLA-B27 negative controls (p=0.005 and p=0.003, respectively). The percentage of IL10+/CD8+ T cells was higher in patients with AS than in both control groups. In HLA-B27 positive subjects, TNF1/2 heterozygosity at -308 (n=6) was associated with a higher percentage of TNFalpha+ T cells than TNF1/1 homozygosity (n=25; median 9.97% v 5.11% for CD4+ T cells; p=0.017). In contrast, in HLA-B27 negative controls (n=18) there was no such genotype/phenotype correlation (median 9.4% v 10.6%).
The lower T cell production of TNFalpha and IFNgamma shown at the single cell level in HLA-B27 positive patients with AS and healthy HLA-B27 positive controls may contribute to the increased susceptibility of HLA-B27 positive subjects to develop AS. Preliminary genotype-phenotype correlations suggest that in HLA-B27 positive subjects TNF2 at -308 or a linked gene results in higher TNFalpha production and, therefore, might be a marker for a protective haplotype.
通过在单细胞水平定量检测强直性脊柱炎（AS）患者与健康的HLA - B27阴性及HLA - B27阳性对照者体内的T细胞细胞因子γ干扰素（IFNγ）、白细胞介素4（IL4）、肿瘤坏死因子α（TNFα）和白细胞介素10，来验证AS是一种2型辅助性T细胞极化疾病的假说。
用佛波酯/离子霉素刺激65名受试者（25名患有活动性AS的HLA - B27阳性患者、18名健康的HLA - B27阳性对照者和22名健康的HLA - B27阴性对照者）的外周血单个核细胞6小时，进行表面CD3和CD8染色、细胞内细胞因子IFNγ、TNFα、IL4和IL10染色，并通过流式细胞术分析。TNFα的产生与TNFα启动子-308和-238多态性的基因型相关。
在外周血中，患有AS的HLA - B27阳性患者中TNFα + T细胞百分比（CD4 + T细胞中位数为5.1%）显著低于健康的HLA - B27阴性对照者（中位数为9.5%；p = 0.008）。令人惊讶的是，健康的HLA - B27阳性对照者中TNFα + T细胞百分比（中位数为7.48%）也显著低于健康的HLA - B27阴性对照者（p = 0.034）。此外，AS患者和健康的HLA - B27阳性对照者中IFNγ + T细胞百分比低于健康的HLA - B27阴性对照者（分别为p = 0.005和p = 0.003）。AS患者中IL10 + /CD8 + T细胞百分比高于两个对照组。在HLA - B27阳性受试者中，-308位点TNF1/2杂合子（n = 6）的TNFα + T细胞百分比高于TNF1/1纯合子（n = 25；CD4 + T细胞中位数为9.97%对5.11%；p = 0.017）。相反，在HLA - B27阴性对照者（n = 18）中不存在这种基因型/表型相关性（中位数为9.4%对10.6%）。
在患有AS的HLA - B27阳性患者和健康的HLA - B27阳性对照者中，单细胞水平显示的TNFα和IFNγ的T细胞产生较低，这可能导致HLA - B27阳性受试者患AS易感性增加。初步的基因型 - 表型相关性表明，在HLA - B27阳性受试者中，-308位点的TNF2或一个连锁基因导致更高水平TNFα的产生，因此可能是一种保护性单倍型的标志物。
