Impaired Th1 cytokine production in spondyloarthropathy is restored by anti-TNFα

OBJECTIVES: To evaluate the effect of anti-TNFalpha on the Th1 and Th2 cytokines in patients with spondyloarthropathy (SpA). METHODS: Peripheral blood mononuclear cells (PBMC) were obtained from 20 patients with active SpA treated with infliximab (5 mg/kg). For comparison, PBMC were also obtained from 15 healthy controls and 19 patients with active rheumatoid arthritis (RA). After stimulation with PMA/ionomycin, the intracellular cytokines interleukin (IL)2, IL4, IL10, and interferon (IFN)gamma were determined in CD3+ T cells and in CD3+/CD56+ natural killer (NK) T cells by flow cytometry. RESULTS: At baseline the percentage of T cells positive for IFNgamma (p=0.020) and IL2 (p=0.046) was decreased in patients with SpA compared with healthy controls, while IL10 (p=0.001) was increased. This cytokine profile, confirmed by the mean fluorescence intensities (MFI), was more pronounced in CD3+/CD8- cells and contrasted with higher IL2 production in RA. NK T cells, characterised by high IL4 and IL10 numbers, were also increased in patients with SpA (p=0.017). Treatment with infliximab induced a significant and persistent increase in IFNgamma and IL2 in patients with SpA. Moreover, there was a transient decrease in IL10 and NK T cells in patients with high baseline values, resulting in values comparable with those of healthy controls. This switch in cytokine profile was seen in both the CD3+/CD8- and CD3+/CD8+ subsets. CONCLUSIONS: Before treatment patients with SpA had an impaired Th1 cytokine profile compared with healthy controls and patients with RA. TNFalpha blockade induced restoration of the Th1 cytokines, resulting in a normal cytokine balance. These data confirm the effect of anti-TNFalpha on the immune changes in SpA, and provide insights into the mechanisms involved in TNFalpha blockade.     

The immune suppressive effect of dexamethasone in rheumatoid arthritis is accompanied by upregulation of interleukin 10 and by differential changes in interferon γ and interleukin 4 production

OBJECTIVES: The influence of dexamethasone on interleukin 10 (IL10) production and the type 1 (T1)/type 2 (T2) T cell balance found in rheumatoid arthritis (RA) was studied. METHODS: Peripheral blood mononuclear cells (PB MNC) were isolated from 14 RA patients both before and 7 and 42 days after high dose dexamethasone pulse therapy. The ex vivo production of IL10, interferon gamma (IFN gamma) (T1 cell), and IL4 (T2 cell) by PB MNCs was assessed, along with parameters of disease activity (erythrocyte sedimentation rate, C reactive protein, Visual Analogue Scale, Thompson joint score). In addition, the in vitro effect of dexamethasone (0.02, 0.2, and 2 microM) on PB MNC IL10, IFN gamma, and IL4 production was studied. RESULTS: Dexamethasone pulse therapy resulted in a rapid and sustained decrease in RA disease activity. IL10 production increased after dexamethasone treatment and this was sustained for at least six weeks. A transient strong decrease in IFN gamma was seen shortly after corticosteroid treatment, while IL4 only decreased slightly. This led to an increased IL-4/IFN gamma ratio. In vitro, IL10 production was not detectable, IFN gamma and IL4 decreased, but the effect was more pronounced for IFN gamma than for IL4, which again resulted in an increased IL4/IFN gamma ratio. CONCLUSION: Dexamethasone therapy in RA patients leads to a rapid, clinically beneficial effect. The upregulation of IL10 production may be involved in the prolonged clinical benefit. The strong immunosuppressive effect is most evident in the decrease in IFN gamma, and is therefore accompanied by a relative shift towards T2 cell activity. In vitro evaluation showed that this shift in T cell balance was a direct effect of dexamethasone and thus independent of the hypothalamic-pituitary-adrenal axis.     

基于T-bet和GATA-3表达研究特异性免疫治疗对过敏性哮喘患者治疗机制

目的 探讨屋尘螨特异性免疫治疗 (SIT)过程中,转录因子 T-bet 和 GATA-3在外周血单个核细胞 (PBMCs)中的表达变化,分析该治疗方案的作用机制.方法 纳入2015年1月至12月我院收治的过敏性哮喘患者89例,进行回顾性分析,根据治疗方案及转归划分为4组:接受SIT治疗,且显效患者纳为观察 A组,共22例;接受 SIT治疗,且未达显效标准者纳为观察 B组,共12例;未接受 SIT治疗,且显效患者纳为对照 A 组,共15例;未接受 SIT 治疗,且未达显效标准者纳为对照B组,共40例.检测对比4组治疗前、治疗12个月时 PBMCs 中 T-bet和 GATA-3 mRNA 表达水平、外周血Th1和Th2细胞比率.结果 观察 A 组、对照 A 组在治疗后 GATA-3 mRNA、Th2细胞比率明显下降,T-bet mRNA/GATA-3 mRNA、Th1/Th2明显上升,观察 A 组在治疗后 T-bet mRNA、Th1细胞比率明显升高.观察B组在治疗后T-bet mRNA、GATA-3 mRNA、Th1细胞比率、Th2细胞比率明显升高,上述差异均有统计学意义 (P <0.05).②各时点所有患者 T-bet mRNA/GATA-3 mRNA与Th1/Th2均呈显著正相关 (治疗前r=0.811,P=0.004;治疗后r=0.801,P =0.000).检测对比4组治疗前、治疗12个月时PBMCs中T-bet和GATA-3 mRNA表达水平、外周血Th1和Th2细胞比率.结论 仅采用常规疗法,有助于抑制哮喘患者 GATA-3 mRNA 过表达,协同采用特异性免疫治疗则能同时促进T-bet mRNA表达,发挥双向治疗作用,从而提升疗效.     

支气管哮喘患者CC16与气道炎症及Th1/Th2细胞因子的关系

目的探讨支气管哮喘（哮喘）患者CC16与气道炎症及Th1/Th2细胞因子的关系。方法采取病例对照研究,收集25例哮喘急性发作期患者和33例健康对照组。采外周静脉血分离血浆,提取外周血单个核细胞（PBMC）,用ELISA测定血浆中CC16、干扰素-γ（IFN-γ）、白介素-4（IL-4）的水平;用RT-PCR法检测PBMC转录因子T-bet和Gata-3mRNA表达水平。结果 1.哮喘组CC16及IFN-γ分别为（21.96±7.31）ng/ml,（118.73±22.59）pg/ml,明显低于对照组[分别为（64.88±25.27）ng/ml和（145.53±29.50）pg/ml,（均P〈0.01）],哮喘组IL-4（425.22±4.37）pg/ml高于对照组（69.72±10.15）pg/ml,（P〈0.01）。2.哮喘组T-betmRNA、T-bet/GATA-3表达水平（0.12±0.01,0.25±0.04）显著低于对照组（0.48±0.12,1.894±0.65）（均P〈0.01）,GATA-3mRNA表达（0.45±0.05）较对照组明显升高（0.30±0.08）（P〈0.01）。3.CC16与T-betmRNA表达水平、T-bet/GATA-3呈正相关（r分别为0.792,0.761,均P〈0.01）;与GATA-3mRNA无明显相关性（r=-0.146,P=0.551）。结论 CC16参与哮喘气道炎症反应,并以Th1/Th2细胞因子失衡为特点。     

肺癌Th1/Th2免疫反应状态的研究

为探讨肺癌细胞及肺癌患者外周血单个核细胞Ｔｈ１／Ｔｈ２的免疫反应状态,为肿瘤的免疫治疗提供依据,以ＩＬ－２和ＩＦＮ－ｒ代表Ｔｈ１型细胞因子,ＩＬ－４、ＩＬ－６和ＩＬ－１０代表Ｔｈ２型细胞因子,用ＲＴ－ＰＣＲ方法检测了三种人肺癌细胞株及２３例肺癌细胞患者ＰＢＭＣ中Ｔｈ１／Ｔｈ２型细胞因子ｍ－ＲＮＡ的表达。结果显示,三种肺癌细胞的Ｔｈ１型因子ＩＦＮ－ｒ和ＩＬ－２均无表达,而Ｔｈ２型因了ＩＬ－４、ＩＬ－     

Down-regulation of the nonspecific and antigen-specific T cell cytokine response in ankylosing spondylitis during treatment with infliximab

OBJECTIVE: Treatment of active ankylosing spondylitis (AS) with the monoclonal tumor necrosis factor alpha (TNF alpha) antibody infliximab is highly clinically effective. This study was undertaken to investigate the precise mechanism of action of anti-TNF alpha treatment in AS. METHODS: Cytokine expression of CD4+ and CD8+ T cells was investigated before and 6 and 12 weeks after the start of treatment in 10 patients treated with infliximab, and before and after 6 weeks of treatment and 6 weeks after placebo was switched to infliximab in 10 patients treated initially with placebo. Peripheral blood mononuclear cells (PBMCs) were stimulated for 6 hours either nonspecifically with phorbol myristate acetate (PMA)/ionomycin or antigen specifically with a pool of 46 overlapping 18-mer peptides derived from the G1 domain of aggrecan. Cells were stained for T cell surface markers CD4 and CD8 and for the intracellular cytokines interferon-gamma (IFN gamma), TNF alpha, interleukin-4 (IL-4), and IL-10. Positive cells were quantified by flow cytometry. For monocyte-derived cytokines, PBMCs were stimulated with lipopolysaccharide (LPS) for 18 hours and TNF alpha and IL-10 in the supernatant were measured by enzyme-linked immunosorbent assay. RESULTS: Compared with baseline, infliximab treatment induced a significant decrease at 12 weeks in the number of CD4+ and CD8+ T cells that were positive for IFN gamma and TNF alpha upon PMA/ionomycin stimulation (P = 0.005). A significant reduction had already begun to occur at 6 weeks. No change in the percent IFN gamma or TNF alpha positivity among CD4+ and CD8+ subpopulations was observed after 6 weeks in patients treated with placebo. However, when these patients began infliximab treatment after 6 weeks of receiving placebo, there was a similar significant decrease in IFN gamma and TNF alpha production by CD4+ and CD8+ T cells (P < 0.05). Furthermore, infliximab treatment induced a significant reduction in the number of IFN gamma+ and TNF alpha+ CD8+ T cells (P = 0.005 at week 6 and week 12) after antigen-specific in vitro stimulation with G1-derived peptides. Between-group analysis showed that the change in the expression of IFN gamma and TNF alpha in both CD4+ and CD8+ T cells was significantly different between the infliximab and placebo groups (P = 0.001 for all variables). There was no change in the number of IL-10+ or IL-4+ T cells during treatment. No significant change in the production of TNFalpha and IL-10 upon in vitro stimulation of PBMCs with LPS was detectable during infliximab treatment. CONCLUSION: Infliximab down-regulates both IFN gamma and TNF alpha secreted by T cells but does not induce a change in cytokines produced by monocytes during 3 months of treatment. This is likely to be a relevant mechanism for the clinical efficacy of this therapy.     

Cytokine and chemokine receptor profile of peripheral blood mononuclear cells during treatment with infliximab in patients with active rheumatoid arthritis

OBJECTIVES: To analyse immunological changes during treatment with a monoclonal anti-tumour necrosis factor alpha (TNFalpha) antibody, infliximab, in patients with rheumatoid arthritis (RA). METHODS: 25 patients with RA and 5 patients with other arthritides were studied during the first 6 weeks of treatment with infliximab. At the start of treatment and after 2 and 6 weeks, spontaneous expression of CCR3 and CCR5 on peripheral blood T cells and monocytes was studied by flow cytometry. The secretion and mRNA expression of interferon gamma (IFNgamma), interleukin (IL)4, IL5, and TNFalpha from phytohaemagglutinin (PHA) stimulated peripheral blood mononuclear cells was measured with an ELISA and RT-PCR. Plasma levels of C reactive protein, serum amyloid protein A, rheumatoid factor, and antibodies to filaggrin and citrullinated cyclic peptide were measured with an ELISA. RESULTS: The number of CD4 T cells and CD14 monocytes expressing CCR3 (p = 0.013, p = 0.009, respectively) and CD8 T cells expressing CCR5 (p = 0.040) as well as PHA stimulated secretion of IL4 and IFNgamma (p<0.05) increased during treatment in patients with RA. 15 (60%) patients with RA achieved clinical response (at least ACR20) during the first 2 weeks. The number of T cells expressing CCR3 and CCR5 was higher before treatment in non-responders than in responders (p<0.05). The number of T cells increased in responders. CONCLUSION: Increase in secretion of Th1 and Th2 cytokines together with induced expression of chemokine receptors on T cells and monocytes suggest restoration of peripheral cell mediated immunity and blockade of the accumulation of inflammatory cells in joints as response to treatment.     

Methimazole upregulates T-cell-derived cytokines without improving the existing Th1/Th2 imbalance in Graves' disease

There is probably a systemic shift of cytokine production in patients with Graves' disease (GD) toward the Th2 cytokine response. Methimazole (MMI) is the first choice for patients with GD and presumably has some direct immunomodulatory action. The aim of this study was to evaluate the balance shift in Th1/Th2 cytokines in patients with GD after 1 yr of MMI treatment, when compared to the same balance in patients with newly diagnosed GD before treatment and in healthy controls. Peripheral blood mononuclear cells (PBMC) were isolated from 17 healthy volunteers, from 18 patients with newly diagnosed GD before treatment and from 15 euthyroid patients with GD after 1 yr of MMI treatment. The PBMC were activated with ionomycin and phorbol 12-myristate 13-acetate (PMA). The concentrations of Th1/Th2 related cytokines [interferon (IFN)-gamma, interleukin (IL)-12 vs IL-4, IL-10] in the culture supernatants were measured by ELISA. PBMC from patients with GD after treatment produced significantly more IFN-gamma and IL-4 than PBMC from patients with GD before treatment, but there were no significant differences in calculated ratios of Th1 against Th2 cytokines between these two groups. When compared to PBMC from healthy controls, PBMC from patients with GD after treatment produced significantly more IL-4 and significantly less IL-12. The calculated IL-12/IL-4 ratio after treatment was significantly lower than the same ratio from healthy controls. In conclusion, our results show no significant change in the ratio between Th1 and Th2 cytokines produced by PBMC from patients with GD after 1 yr of MMI treatment, when compared to the ratio before treatment. The ongoing prevalence of the Th2 immune response after treatment speaks against the immunomodulatory action of the drug on the systemic level.     

免疫性血小板减少症患者外周血白细胞介素-18及其受体α链的表达

目的 通过检测新诊断的免疫性血小板减少症(ITP)患者用药前外周血IL-18和CD_3~+细胞表面IL-18受体α链(IL-18Rα)的表达,探讨IL-18和IL-18Rα在ITP发病中的作用机制.方法 以我院门诊及住院治疗的18例新诊断的ITP为研究对象,应用ELISA检测血浆中IL-18的含量,采用流式细胞术分析CD_3~+细胞和总淋巴细胞表面IL-18Rα的表达,应用RT-PCR检测外周血单个核细胞(PBMCs)中IL-18 mRNA、转录因子T-bet mRNA和GATA-3 mRNA的表达.选择15例与试验组匹配的健康志愿者作为正常对照组.结果 新诊断的ITP患者血浆中IL-18的含量为(468.57±141.62)pg/ml,显著高于对照组(P<0.05);CD_3~+细胞表面及淋巴细胞表面IL-18Rα的表达分别为(8.50±3.16)%和(9.16±2.98)%,两者均显著高于对照组(P<0.05);ITP患者PBMCs中IL-18 mRNA、T-bet mRNA和GATA-3 mRNA的表达分别为0.12±0.02、0.07±0.02和0.0039±0.0014,IL-18mRNA和T-bet mRNA显著高于对照组(P<0.05),而GATA-3 mRNA显著低于对照组(P<0.05),T-bet/GATA-3比例显著增高.结论 IL-18与ITP的发生发展有关,本研究从蛋白和基因水平说明IL-18和IL-18Rα可能上调ITP患者Th1类细胞的表达,通过调整T-bet/GATA-3的比例,恢复Th1/Th2的平衡,有望为ITP的治疗提供一个新的治疗策略.     

Shift toward T helper 1 cytokines by type II collagen–reactive T cells in patients with rheumatoid arthritis

Objective

To investigate the impact of type II collagen (CII)–reactive T cells on the Th1/Th2 cytokine balance in patients with rheumatoid arthritis (RA).

Methods

T cell proliferative responses to bovine CII were examined in synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) by mixed lymphocyte culture. CII‐reactive T cell lines were generated from the SFMC and PBMC. Interferon‐γ (IFNγ), interleukin‐12 (IL‐12), and IL‐4 were measured by enzyme‐linked immunosorbent assay in the SF, sera, and culture supernatants of PBMC and SFMC that had been stimulated with CII.

Results

The frequency of CII‐reactive T cells was higher in the PBMC from RA patients than in that from osteoarthritis patients and healthy control subjects. In RA patients, CII‐reactive T cells were more prevalent in SFMC than in PBMC. The mean level of IFNγ and the ratio of IFNγ to IL‐4 were significantly higher in the culture supernatants of T cells stimulated with CII; these differences were more prominent in SFMC. Levels of IL‐12 in the culture supernatants of SFMC and PBMC stimulated with CII were significantly higher than those in unstimulated supernatants. T cell responsiveness correlated well with the level of type 1 cytokines in culture supernatants from RA T cells stimulated with CII. In the CII‐reactive cell lines, the increased production of IFNγ was consistent with clonal expansion.

Conclusion

CII‐reactive T cells are more abundant in SFMC than in PBMC and are strongly associated with a shift toward Th1 cytokine in the inflamed joints of RA patients. Our results suggest that a skewing toward type 1 cytokines by CII‐reactive T cells may play an important role in the chronic inflammatory process of RA.

     

Interleukin-21 enhances T-helper cell type I signaling and interferon-gamma production in Crohn's disease

BACKGROUND & AIMS: T-helper (Th)1 cells play a central role in the pathogenesis of tissue damage in Crohn's disease (CD). Interleukin (IL)-12/STAT4 signaling promotes Th1 cell commitment in CD, but other cytokines are needed to maintain activated Th1 cells in the mucosa. In this study, we examined the expression and role of IL-21, a T-cell-derived cytokine of the IL-2 family; in tissues and cells isolated from patients with inflammatory bowel disease. METHODS: IL-21 was examined by Western blotting in whole mucosa and lamina propria mononuclear cells (LPMCs) from patients with CD, ulcerative colitis (UC), and controls. We also examined the effects of exogenous IL-12 on IL-21 production, as well as the effects of blocking IL-21 with an IL-21-receptor Ig fusion protein. Interferon (IFN)-gamma was measured in the culture supernatants by enzyme-linked immunosorbent assay, and phosphorylated STAT4 and T-bet were examined by Western blotting. RESULTS: IL-21 was detected in all samples, but its expression was higher at the site of disease in CD in comparison with UC and controls. Enhanced IL-21 was seen in both ileal and colonic CD and in fibrostenosing and nonfibrostenosing disease. IL-12 enhanced IL-21 in normal lamina propria lymphocytes through an IFN-gamma-independent mechanism, and blocking IL-12 in CD LPMCs decreased anti-CD3-stimulated IL-21 expression. Neutralization of IL-21 in CD LPMC cultures decreased phosphorylated STAT4 and T-bet expression, thereby inhibiting IFN-gamma production. CONCLUSIONS: Our data suggest that IL-21 contributes to the ongoing Th1 mucosal response in CD.     

Interleukin 7 stimulates tumour necrosis factor alpha and Th1 cytokine production in joints of patients with rheumatoid arthritis

BACKGROUND: A large number of activated T cells are found in the joints of patients with rheumatoid arthritis (RA). Interleukin 7 (IL7), a T cell growth factor and a regulator of Th1 and Th2 cytokine production, is produced by synoviocytes from patients with RA. OBJECTIVE: To investigate the effect on proinflammatory cytokine production of synovial fluid mononuclear cells (SFMC) and the mechanism by which IL7 influences CD4+ T cell activity in patients with RA. METHODS: In a cross sectional group of patients with RA, IL7 levels were compared with those of healthy controls and related to disease activity. The effect of IL7 on cytokine production was tested by RA SFMC and on SF CD4+ T cells in the presence of mononuclear cells (MC). Production of tumour necrosis factor alpha (TNF alpha), IL1 beta, interferon gamma (IFN gamma), and IL4 was measured by enzyme linked immunosorbent assay (ELISA) and by single cell FACS analysis. Expression of the IL7 receptor alpha chain on CD4+ T cells (essential for IL7 signalling) was assessed. Direct effects of IL7 on isolated synovial fluid (SF) CD4+ T cells were studied by cytokine analysis. By neutralisation of IL12 in MC cultures, indirect effects of IL7 on T cells through accessory cells were studied. RESULTS: IL7 serum levels were higher in patients with RA than in healthy controls and correlated positively with C reactive protein levels. IL7 stimulated TNFalpha production by SFMC and very potently stimulated IFN gamma and TNF alpha production by SF CD4+ T cells. These effects were probably mediated through the IL7 receptor alpha chain, which was abundantly expressed on SF CD4+ T cells. Besides the direct stimulation of T cell cytokine production by IL7, its action was partly dependent on IL12, indicating that IL7 also stimulates accessory cell function, leading to T cell activation. CONCLUSION: IL7 stimulates proinflammatory cytokine production of intra-articular CD4+ T cells and accessory cells from patients with RA. The correlation with measures of disease activity indicates that IL7 might substantially contribute to the perpetuation of Th1 and TNF alpha mediated proinflammatory responses in patients with RA.     

Effects of Th1 and Th2 cytokines on cytokine production and ICAM-1 expression on synovial fibroblasts.

OBJECTIVES--To investigate the influence of the Th1 and Th2 lymphokines interleukins (IL)-4 and IL-13, interferon gamma (IFN gamma), and several monokines on the adhesion of mononuclear cells to synovial fibroblasts and intercellular adhesion molecule-1 (ICAM-1) expression and cytokine production of synovial fibroblasts in patients with osteoarthritis. METHODS--Synovial fibroblasts were isolated from patients with osteoarthritis and stimulated with IL-1 beta, IL-4, IL-6, IL-10, IL-12, IL-13, tumour necrosis factor alpha (TNF alpha), and IFN gamma. Subsequently, we determined the production of IL-1 alpha, IL-1 beta, IL-6, IL-10, IL-12, IFN alpha and TNF alpha, and the expression of ICAM-1 lymphocyte function associated antigen 3 (LFA-3), BB7, and major histocompatibility complex class II molecules on these cells. Furthermore, the adhesion of freshly isolated mononuclear cells from the peripheral blood was tested using a colourimetric cell-cell adhesion assay. RESULTS--Only production of IL-6 and the expression of ICAM-1 were observed. IL-1 beta and TNF alpha were the most potent stimulatory mediators of both cytokine production and ICAM-1 expression. IL-4 and IL-13 had differential effects as they upregulated cytokine production but downregulated IFN gamma induced ICAM-1 expression. In functional adhesion assays, TNF alpha, IL-1 alpha and, to a lesser extent, IFN gamma led to increased adhesion of mononuclear cells, whereas IL-4 and IL-13 had no effect. CONCLUSIONS--Our data indicate that Th1 and Th2 lymphokines can modulate the function (cytokine production and expression of adhesion molecules) of synovial fibroblasts.