Treatment of experimental asthma by long-term gene therapy directed against IL-4 and IL-13

The clinical manifestations of allergic asthma are believed to result from a dysregulated, T helper 2 lymphocyte (Th2)-biased response to antigen. Although asthma symptoms can be controlled acutely, there is a need for a therapy that will address the underlying immune dysfunction and provide continuous control of chronic airway inflammation. The Th2-type cytokines, IL-13 and IL-4, have been demonstrated to play a crucial role in asthma pathogenesis and their selective neutralization results in the alleviation of asthmatic symptoms in mouse models. The activity of both of these cytokines can be inhibited by a mutant IL-4 protein, IL-4 receptor antagonist (IL-4RA), and thus, continual IL-4RA therapy might be beneficial in treatment of chronic asthma. To explore the potential utility of long-term gene therapy for the treatment of asthma we used a recombinant adeno-associated virus (AAV) vector to deliver and provide sustained expression of IL-4RA in vivo. We show that AAV-mediated delivery of IL-4RA to the airways of mice reduces airway hyperresponsiveness (AHR) and airway eosinophilia triggered by either IL-13 or IL-4. Furthermore, AAV-delivered IL-4RA, expressed either systemically or in the airways of mice following allergen sensitization, significantly inhibited development of airway eosinophilia and mucus production and reduced the levels of asthma-associated Th2 cytokines and AHR in the experimental mouse model of allergic asthma. Thus, gene therapy can be a potential therapeutic option to treat and control chronic airway inflammation and asthmatic symptoms.     

Sex influences on the penetrance of IL-1beta and IL-1RN genotypes for rheumatoid arthritis in the Chinese population

Polymorphism (variable number of tandem repeats) in the second intron of the interleukin-1 receptor antagonist (IL-1Ra) gene and two single nucleotide polymorphisms at positions -511 and +3954 of the IL-1beta gene may be associated with an increased risk of rheumatoid arthritis (RA). This study used sex stratification to investigate a correlation of the three genetic polymorphisms with the risk of RA, on patients with RA and healthy controls. Polymerase chain reaction (PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were performed. The frequencies of the IL-1beta+3954 allele and genotype in female patients were significantly different compared with the controls; but in males, only the frequency of the IL-1beta+3954 allele was different. The frequency of the IL-1RN genotype in patients was not statistically different compared with the controls; however, the frequency of IL-1RN allele in female patients was different. The association of the three polymorphisms with the susceptibility to RA appears to be significantly affected by gender.     

Inhibition of cartilage destruction by double gene transfer of IL-1Ra and IL-10 involves the activin pathway

The objective of the study was to determine the effects and the molecular background of interleukin-1 receptor antagonist (IL-1Ra) and vIL-10 double gene transfer into human synovial fibroblasts from patients with rheumatoid arthritis (RA) using the SCID mouse model for cartilage erosion in RA. RA synovial fibroblasts were transduced with retro- or adenoviruses encoding IL-1Ra and/or viral IL-10 (vIL-10). SCID mice were engrafted subcutaneously with IL-1Ra and vIL-10 transduced human rheumatoid synovial fibroblasts and normal cartilage. In parallel, gene expression analysis before and after gene transfer using RNA arbitrarily primed PCR in combination with cDNA array was performed. vIL-10 and IL-1Ra double gene transfer resulted in inhibition of cartilage invasion and degradation by RA synovial fibroblasts when compared with control transduced and non-transduced implants. Expression of key genes that were altered after double gene transfer were related to the activin pathway. The results demonstrate not only that virus-based gene transfer using a combination of two joint-protective genes is a feasible approach to inhibit cartilage degradation by activated RA synovial fibroblasts, but also that the underlying molecular effects include modulation of the activin pathway.     

Release of WBC-derived IL-1 receptor antagonist into supernatants of RBCs: influence of storage time and filtration

BACKGROUND: Transfusion-associated immunodepression may be related to the transfer of immunoinhibitory cytokines with blood components. STUDY DESIGN AND METHODS: After evidence of increasing concentrations of IL-1 receptor antagonist (IL-1RA) but not of IL-10 was obtained in supernatants of stored RBC units that were WBC-reduced by centrifugation (C-RBCs) in a pilot study, IL-1RA concentrations were determined weekly in supernatants of C-RBCs and in units that underwent prestorage WBC reduction by in-line filtration (F-RBCs) over a 49-day storage. For assessing total IL-1RA content, complete cell lysis by repeated freezing and thawing was done. The results were related to the changes in WBC count during storage. The dependency of IL-1RA content on preparation procedures was assessed. RESULTS: The prestorage IL-1RA concentration in C-RBCs (859 +/- 218 pg/mL) was significantly higher than in F-RBC (75 +/- 13 pg/mL). Whereas no changes were seen in F-RBCs during storage, IL-1RA levels in C-RBC supernatants drastically increased to levels about 50 times those in normal plasma (16,327 +/- 2,686 pg/mL on Day 49). Follow-up analysis revealed stringent correlation between IL-1RA release into supernatants and the current loss of WBCs (r = 0.79, n = 42; p<0.001). The total IL-1RA content did not change during storage and was directly dependent on prestorage WBC count. Preparation procedures altered the IL-1RA content only by WBC reduction. CONCLUSION: The immunosuppressive cytokine IL-1RA is transmitted by RBCs in relation to WBC content and storage time.     

白细胞介素-21及其受体在类风湿关节炎患者血清及外周血单个核细胞中的表达

目的检测类风湿关节炎(RA)患者血清、外周血单个核细胞(PBMC)中白细胞介素-21(IL-21)及其受体(IL-21R)的表达水平,探讨二者之间的相关性及其在RA病理机制中的作用。方法血清IL-21含量采用酶联免疫吸附试验检测,套式实时荧光定量聚合酶链反应法测定PBMC中IL-21R mRNA水平。结果 RA患者组血清中IL-21含量和PBMC中IL-21RmRNA表达水平均高于健康对照组,差异有统计学意义(P<0.01);RA活动期IL-21、IL-21RmRNA水平明显高于非活动期,RA患者经治疗症状改善者IL-21、IL-21R含量明显下调,分别下降21.0%、30.3%,治疗前后比较差异有统计学意义(P<0.01);IL-21和IL-21R表达水平与RA患者关节功能分级有显著相关性,Ⅲ级以上与Ⅰ级、Ⅱ级相比2项指标差异有统计学意义(P<0.01)。结论 IL-21与IL-21R水平检测对RA的诊断和治疗有重要的临床意义。     

IL-4 -588C>T polymorphism and IL-4 receptor alpha [Ex5+14A>G; Ex11+828A>G] haplotype concur in selecting H. pylori cagA subtype infections

BACKGROUND: The Th2 cytokine IL-4 might limit H. pylori associated gastric inflammation and favour H. pylori clearance. The aim of the study was to verify whether IL-4 -588C>T SNP, or two SNPs of the gene coding the alpha chain of IL-4 receptor (IL-4RA Ex5+14A>G, IL-4RA Ex11+828A>G) considered singly or as haplotypes, are correlated with H. pylori virulence genes or H. pylori associated diseases. METHODS: We studied 144 patients with non-cardia gastric cancer (NCGC)(41/50 with present or past H. pylori infection), 75 with duodenal ulcer (DU)(66 H. pylori infected) and 171 with gastritis (CG)(107 H. pylori infected). cagA gene was present in 24/28 NCGC, 45/59 DU and 56/107 CG. RESULTS: All SNPs were in Hardy-Weinberg equilibrium. IL-4RA haplotypes frequencies were estimated using Arlequin software. Neither the SNPs nor the IL-4RA haplotype correlated with disease diagnosis, H. pylori infection, degree of mucosal inflammation or intestinal metaplasia. IL-4 -588T allele (OR=3.69, 95% CI:1.34-10.16) and IL-4RA GA haplotype (p<0.05) enhanced the risk for cagA positive infections. IL-4RA GA haplotype correlated with IL-4 protein levels in H. pylori infected gastric mucosa. CONCLUSIONS: IL-4 and IL-4RA gene polymorphisms concur in selecting the H. pylori infecting strain, probably influencing the IL-4 signalling pathway.     

IL-32、MMP-13及IL-1O在类风湿关节炎中的表达及意义研究

目的探讨白细胞介素-32（IL-32）、基质金属蛋白酶-13（MMP-13）、白细胞介素-10（IL-10）在类风湿关节炎（RA）发生、病程发展中的意义。方法采用ELISA法,检测115例RA、76例对照组血清中IL-32、MMP-13及IL-10的水平,并与抗环瓜氨酸肽（CCP）抗体、类风湿因子（RF）、超敏C反应蛋白（hs-CRP）、红细胞沉降率（ESR）等指标进行相关性分析。结果 RA组与对照组比较,IL-32、MMP-13、IL-10水平升高,有统计学意义（P〈0.05）,RA重度活动期组IL-32、MMP-13显著高于稳定期组（P〈0.05）,IL-32、MMP-13水平与抗CCP抗体、ESR、hs-CRP、RF、DAS28、关节肿胀数和关节压痛数呈显著正相关（P〈0.01）,IL-10与ESR、hs-CRP呈正相关（P〈0.05或P〈0.01）。结论 IL-32、MMP-13及IL-10与RA的形成、病程发展有密切关系,可作为观察RA病情活动、疗效判断及预后的新依据。     

IL-17/IL-17 receptor system in autoimmune disease: mechanisms and therapeutic potential

IL-17 (interleukin-17), a hallmark cytokine of Th17 (T-helper 17) cells, plays critical roles in host defence against bacterial and fungal infections, as well as in the pathogenesis of autoimmune diseases. The present review focuses on current knowledge of the regulation, functional mechanisms and targeting strategies of IL-17 in the context of inflammatory autoimmune diseases. Evidence shows that IL-17 is highly up-regulated at sites of inflammatory tissues of autoimmune diseases and amplifies the inflammation through synergy with other cytokines, such as TNF (tumour necrosis factor) α. Although IL-17 was originally thought to be produced mainly by Th17 cells, a newly defined T-cell subset with a specific differentiation programme and tight regulation, several other cell types (especially innate immune cells) are also found as important sources for IL-17 production. Although IL-17 activates common downstream signalling, including NF-κB (nuclear factor κB), MAPKs (mitogen-activated protein kinases), C/EBPs (CCAAT/enhancer-binding proteins) and mRNA stability, the immediate receptor signalling has been shown to be quite unique and tightly regulated. Mouse genetic studies have demonstrated a critical role for IL-17 in the pathogenesis of variety of inflammatory autoimmune diseases, such as RA (rheumatoid arthritis) and MS (multiple sclerosis). Importantly, promising results have been shown in initial clinical trials of monoclonal antibodies against IL-17 or its receptor (IL-17R) to block IL-17-mediated function in treating autoimmune patients with psoriasis, RA and MS. Therefore targeting IL-17/IL-17R, IL-17-producing pathways or IL-17-mediated signalling pathways can be considered for future therapy in autoimmune diseases.     

The participation of IL-8 in the synovial lesions at an early stage of rheumatoid arthritis.

Synovial tissues from Rheumatoid Arthritis (RA) were divided into three groups based on their histopathological findings and compared for their expression of IL-8 and monocyte chemotactic and activating factor (MCAF) by using immunohistochemistry and in situ hybridization. The levels of IL-8 as well as those of MCAF were markedly higher in the synovial fluid from RA joints. Synovial lining cells (SLC) and macrophages had an ability to produce IL-8 at an early phase of the disease. The presence of MCAF was restricted in macrophages at this stage. On the other hand, the production of IL-8 as well as MCAF were prominent in most components of the joints such as SLC, migrated monocytes, sublining fibroblastoid cells, endothelial cells or migrated neutrophils at an active phase. The expression of IL-8 or MCAF was low in fibrotic synovitis of RA. These data indicate that IL-8 generated from SLC and macrophages may participate to the inflammatory process in the early synovitis of RA.     

类风湿性关节炎患者血清白细胞介素的检测

为了解类风湿性关节炎（ＲＡ）患者的免疫损伤机理，应用酶联免疫吸附法（ＥＬＩＳＡ）检测３８例ＲＡ患者血清可溶性白细胞介素２受体（ｓＩＬ－２Ｒ）和白细胞介素８（ＩＬ－８）含量，并检验其相关性。结果：（１）ＲＡ患者血清ｓＩＬ－２Ｒ和ＩＬ－８水平均比正常对照组显著升高（Ｐ＜０．０１）；血清ｓＩＬ－２Ｒ浓度在类风湿因子（ＲＦ）阳性与阴性组中差异有显著性（Ｐ＜０．０１）；（２）ＲＦ阳性患者血清ｓＩＬ－２Ｒ与ＩＬ－８浓度呈正相关（ｒ＝０．７４，Ｐ＜０．０５）。提示ＲＡ患者Ｔ细胞异常活化以及免疫调节功能紊乱。     

秦息痛片联合甲氨蝶呤治疗类风湿关节炎的临床效果及对血清IL-17、IL-6水平的影响

目的探讨秦息痛片联合甲氨蝶呤治疗类风湿关节炎(RA)的临床效果及对血清IL-17、IL-6水平的影响。方法按照随机数表法将80例RA患者分为对照组和观察组,各40例。对照组给予甲氨蝶呤治疗,观察组给予甲氨蝶呤+秦息痛片治疗。比较两组的治疗效果,并对血清促炎因子与RA疾病活动度进行相关性分析。结果观察组的治疗总有效率为92.5%,明显高于对照组的72.5%(P<0.05)。治疗后,两组关节压痛数、关节肿胀数、DAS28评分、ESR、CRP、血清IL-17、IL-6水平均显著降低,且观察组低于对照组(P<0.05)。Pearson相关性分析结果显示,RA患者血清IL-17、IL-6表达水平与DAS28评分呈显著正相关(P<0.001)。结论秦息痛片联合甲氨蝶呤治疗RA的临床效果显著,可明显改善患者临床症状,可能的内在机制为降低血清IL-17、IL-6的表达。     

A proinflammatory role for IL-18 in rheumatoid arthritis

IL-18 is a novel cytokine with pleiotropic activities critical to the development of T-helper 1 (Th1) responses. We detected IL-18 mRNA and protein within rheumatoid arthritis (RA) synovial tissues in significantly higher levels than in osteoarthritis controls. Similarly, IL-18 receptor expression was detected on synovial lymphocytes and macrophages. Together with IL-12 or IL-15, IL-18 induced significant IFN-gamma production by synovial tissues in vitro. IL-18 independently promoted GM-CSF and nitric oxide production, and it induced significant TNF-alpha synthesis by CD14(+) macrophages in synovial cultures; the latter effect was potentiated by IL-12 or IL-15. TNF-alpha and IFN-gamma synthesis was suppressed by IL-10 and TGF-beta. IL-18 production in primary synovial cultures and purified synovial fibroblasts was, in turn, upregulated by TNF-alpha and IL-1beta, suggesting that monokine expression can feed back to promote Th1 cell development in synovial membrane. Finally, IL-18 administration to collagen/incomplete Freund's adjuvant-immunized DBA/1 mice facilitated the development of an erosive, inflammatory arthritis, suggesting that IL-18 can be proinflammatory in vivo. Together, these data indicate that synergistic combinations of IL-18, IL-12, and IL-15 may be of importance in sustaining both Th1 responses and monokine production in RA.     

IL-17 mediates articular hypernociception in antigen-induced arthritis in mice

IL-17 is an important cytokine in the physiopathology of rheumatoid arthritis (RA). However, its participation in the genesis of nociception during RA remains undetermined. In this study, we evaluated the role of IL-17 in the genesis of articular nociception in a model of antigen (mBSA)-induced arthritis. We found that mBSA challenge in the femur–tibial joint of immunized mice induced a dose- and time-dependent mechanical hypernociception. The local IL-17 concentration within the mBSA-injected joints increased significantly over time. Moreover, co-treatment of mBSA challenged mice with an antibody against IL-17 inhibited hypernociception and neutrophil recruitment. In agreement, intraarticular injection of IL-17 induced hypernociception and neutrophil migration, which were reduced by the pre-treatment with fucoidin, a leukocyte adhesion inhibitor. The hypernociceptive effect of IL-17 was also reduced in TNFR1^−/− mice and by pre-treatment with infliximab (anti-TNF antibody), a CXCR1/2 antagonist or by an IL-1 receptor antagonist. Consistent with these findings, we found that IL-17 injection into joints increased the production of TNF-α, IL-1β and CXCL1/KC. Treatment with doxycycline (non-specific MMPs inhibitor), bosentan (ET_A/ET_B antagonist), indomethacin (COX inhibitor) or guanethidine (sympathetic blocker) inhibited IL-17-induced hypernociception. IL-17 injection also increased PGE₂ production, MMP-9 activity and COX-2, MMP-9 and PPET-1 mRNA expression in synovial membrane. These results suggest that IL-17 is a novel pro-nociceptive cytokine in mBSA-induced arthritis, whose effect depends on both neutrophil migration and various pro-inflammatory mediators, as TNF-α, IL-1β, CXCR1/2 chemokines ligands, MMPs, endothelins, prostaglandins and sympathetic amines. Therefore, it is reasonable to propose IL-17 targeting therapies to control this important RA symptom.     

类风湿性关节炎IL-17检测的临床应用价值研究

目的研究类风湿性关节炎患者关节液和血清中IL-17的含量与其它非免疫性关节损伤IL-17含量差异性,为探讨RA患者的关节损伤机制提供科学依据。方法应用ELISA方法定量检测40例RA组关节液和血清IL-17的含量;检测40例半月板损伤组关节液和血清IL-17的含量;检测40例正常对照组血清IL-17的含量。应用t检验比较各组间的差异性。结果 RA组中,关节液的IL-17含量明显高于血清中的IL-17含量（P〈0.01）;RA组关节液中的IL-17含量高于半月板损伤组（病例对照组）关节液中的IL-17含量（P〈0.01）;血清IL-17定量检测结果显示,RA组明显高于病例对照组和正常对照组（P〈0.01）;而病例对照组与正常对照组比较,IL-17含量无显著性差异（P〉0.05）。结论 RA组关节液和血清中均具有较高的IL-17检测值,证实RA患者体内存在细胞免疫应答异常,IL-17是RA患者关节滑膜损伤的重要因素之一。     

类风湿关节炎患者血清白介素-20水平及影响因素研究

目的：探讨类风湿关节炎患者疾病活动与血清中白介素-20（IL-20）水平的关系。方法：用酶标记免疫吸附测定（enzyme-linked immunosorbent assay,ELISA）方法检测60例类风湿关节炎患者及30例正常对照者外周血血清中IL-20水平。分析IL-20水平与RA发病和类风湿因子（rheumatoid factor,RF）、C反应蛋白（C reactive protein,CRP）、血沉（erythrocyte sedimentation rate,ESR）及同期手X光片分期的相关性。结果：活动期类风湿关节炎患者外周血中IL-20水平明显高于正常对照组（P〈0.01）。类风湿关节炎患者IL-20水平与其RF、CRP、血沉及同期手X光片分期无明显相关性（P〉0.2）。结论：活动期类风湿关节炎患者外周血中IL-20水平明显高于正常对照组,但与RF、CRP、血沉及同期手X光片分期无明显相关性。IL-20可能参与类风湿关节炎的发病过程,提示需进一步研究。     

血清IL-22水平在类风湿性关节炎患者不同时期的临床意义

目的：探讨IL-22在类风湿关节炎（rheumatoid arthritis,RA）不同时期患者中的临床意义。方法：用酶联免疫吸附法检测35例活动组RA患者、35例缓解组 RA 患者及30例健康人血清 IL-22的水平,并分析它与活动度和实验室指标的关系。结果：RA活动组和 RA 缓解组IL-22水平均明显高于对照组,差异均有统计学意义（p〈0.05）,而 RA 活动组与 RA 缓解组相比,差异亦有统计学意义（p〈0.05）。RA 患者血清 IL-22与 DAS28评分、血沉、C 反应蛋白、类风湿因子均呈正相关关系（p〈0.05）。结论：IL-22可能参与了 RA 的发病过程,并可将其血清水平作为RA活动参考指标。     

IL-2 enhancing factor(s) in B cell supernatants from patients with rheumatoid arthritis or systemic lupus erythematosus

Culture supernatants of B cells from patients with rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) in the active stage enhanced interleukin 2 (IL-2) dependent proliferation of CTLL A/J cells. This activity, designated B cell-derived growth-enhancing factor-2 (BGEF-2), was recovered by gel filtration of a molecular weight between 15,000 and 20,000. BGEF-2 itself did not show IL-2 activity nor IL-1 activity, and BGEF-2 activity was not detected in the following cytokines: Interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma), tumor necrosis factor (TNF), interleukin 4 (IL-4), interleukin 5 (IL-5) and interleukin 6 (IL-6). Furthermore, BGEF-2 was distinguishable from B cell-derived growth-enhancing factor described in a previous paper [Kang et al. (1987) J. Immunol., 139, 1154-1160]. BGEF-2 was produced by B cells from patients with RA or SLE only when the patients were in the active stage. BGEF-2 enhanced IL-2-dependent growth of peripheral blood T cells from patients with active RA, but did not enhance the growth of T cells from healthy volunteers. These results suggest that BGEF-2 is a B cell-derived lymphokine which plays an important role in the pathogenesis of RA and SLE.     

IL-25与自身免疫炎症性疾病

IL-25是新近发现的IL-17家族成员,具有免疫调节功能。IL-25可参与2型免疫反应,促进IL-4、IL-5、IL-9、IL-13等细胞因子分泌,也可与IL-17A及其受体IL-17RA结合,亦能抑制关节软骨的形成,刺激一氧化氮的释放以及前炎症因子IL-6、IL-8的产生,进而参与自身免疫炎症疾病。