Changes Of Th1/Th2 Cytokines In The Peripheral Blood Of Patients With Inflammatory Arthropathies

1. ObjectiveThl and Th2 cells have been found to play different roles in chronic inflammatory autoimmune diseases. It was demonstrated that Thl/Th2 balance is distorted in some inflammatory arthropathies such as rheumatoid arthritis (RA) and reactive arthritis (ReA) although few similar reports on ankylosing sondylitis (AS) have been published. The purpose of this study was to investigate the pattern of Thl/Th2 cytokines in the peripheral blood of patients with AS and try to understand the role of T lymphocyte subgroups in the pathogenesis of AS. We also investigated the changes of T lymphocyte subsets in the peripheral blood of RA patients during leflunomide treatment, and tried to understand the influence of leflunomide on T lymphocyte subgroups in RA patients.2. MethodsIntracellular cytokine staining and flow cytometric analysis were established and employed for intracellular cytokines (IFN-y, IL-4) and surface antigens (CD4, CDS) of T cells in the peripheral blood of 39 AS patients. Peripheral blood cells were stimulated with phorbol myristate acetate (PMA) and ionomycin in the presence of monensin. The stimulated cells were triple-stained with conjugated monoantibodies against intracellular cytokines (IFN-y and IL-4) and surface antigens (CD4 or CDS) of T cells after fixation and permeabilization. The samples were then analyzed by a flow cytometer. Sandwich ELISA was used to measure the level of IFN-y and IL-4 in the serum, and the expressions of IFN-y and IL-4 mRNA in the peripheral blood-6-mononuclear cells (PBMC) were measured by semi-quantitive RT-PCR with G3PDH as the internal standard.Intracellular cytokine staining and flow cytometric analysis was also used to investigate the T lymphocyte subsets in the peripheral blood of 25 RA patients, and the patterns of T lymphocytes in another group of 10 RA patients were analyzed before and after they had been treated with leflunomide for three months.3. ResultsThl/Th2 differentiation in the peripheral blood of AS patients The positive rates of IFN-y-producing cells among either CD4+ or CD8+ T cells were significantly higher than those of IL-4-producing cells in the peripheral blood of patients with AS (P<0.01). Although the rates of IFN-y-producing cells among CD4+ and CD8+ T cells in AS patients did not change when compared with the controls, they had a negative moderate correlation with the inflammation index (CRP, ESR and platelet counts) (P=0.01). The level of IFN-y in the serum was increased while the level of IL-4 was within the normal range, and the IFN-y/IL-4 ratio in the patients was significantly higher than that in the controls. When compared with the controls, the expressions of IFN-y and IL-4 mRNA in PBMC of AS patients was elevated as well as the IFN-y mRNA/IL-4 mRNA ratio. And the expressions of IFN-y and IL-4 mRNA had also a negative moderate correlation with CRP (PO.05).Leflunomide interferes with Thl/Th2 lymphocyte in the peripheral blood of rheumatoid arthritis patients There was no obvious changes in the percentages of Tel, Th2 and Tc2 cells in RA patients compared with the control group (P>0.05) except that of Thl, which decreased markedly (P<0.01). And the rates of these T lymphocyte subsets in RA patients had no correlation with the inflammation indices. After three months of leflunomide treatment in another group of RA patients, which led to significant improvement of clinical outcome measures such as ACR 20, the rates of Thl, Tel and Th2 decreased significantly (P<0.01), while that of Tc2 did not-7-(P>0.05). The range of decreasing Thl was larger than that of Th2, but no statistical significance was noted.4. ConclusionThere was a dominant Thl cytokine profile in the peripheral blood of our AS patients. But Thl cells seemed to be down-regulated as the inflammation deteriorated. The pathogenic factors of AS might induce a dominant Thl response and increased levels of Thl cytokines such as IFN-y, which further promoted the release of proinflammatory factors. Thl subsets decreased in the per...