Low T cell production of TNF alpha and IFN gamma in ankylosing spondylitis: its relation to HLA-B27 and influence of the TNF-308 gene polymorphism

Objective-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 (IFN gamma), interleukin 4 (IL4), tumour necrosis factor alpha (TNF alpha), and IL10 at the single cell level in patients with AS in comparison with healthy HLA-B27 negative and HLA-B27 positive controls. Methods-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 IFN gamma, TNF alpha, IL4, and IL10, and analysed by flow cytometry. TNF alpha production was related to the genotype of the TNF alpha promoter at the -308 and -238 polymorphisms. Results-In peripheral blood the percentage of TNF alpha (+) 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 TNF alpha+ 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 IFN gamma+ 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 TNF alpha+ 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%). Conclusions-The lower T cell production of TNF alpha and IFN gamma 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 TNF alpha production and, therefore, might be a marker for a protective haplotype.