TNF-α基因多态性与慢性乙型肝炎患者干扰素治疗前后Th1/Th2细胞因子水平变化相关性研究

目的 研究TNF-α-238、308基因多态性与慢性乙型肝炎患者干扰素治疗6个月前后Th1/Th2细胞因子水平变化的相关性,探讨乙型肝炎发病机制和治疗策略.方法 以接受α干扰素正规治疗的77例慢性乙型肝炎患者作为研究对象,运用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术检测77例慢性乙型肝炎患者TNF-α-238、-308位点基因多态性,并用ELISA检测干扰素治疗前后血清细胞因子水平,最后根据患者不同应答水平及基因型分组分别进行统计分析.结果 (1)应答组治疗前血清IL-2、IFN-γ水平较无应答组高(P<0.05),IL-10水平较无应答组低(P<0.05),IL-4水平在应答组和无应答组间无显著性差异(P>0.05);(2)干扰素治疗6个月后应答组、无应答组IL-2、IFN-γ水平均升高(P<0.01),IL-4、IL-10水平均降低(P<0.01),应答组IL-2、IFN-γ水平平均升高浓度高于无应答组(P<0.05),IL-4、IL-10水平平均降低浓度也高于无应答组(P<0.05);(3)治疗前TNF-α-308G/A基因型患者血清IFN-γ水平较G/G型高(P<0.05),IL-2、IL-4、IL-10水平在两种基因型间无显著性差异(P>0.05).TNF -α-238G/G、G/A基因型患者血清IL-2、IFN-γ、IL-4和IL-10水平均无显著性差异(P>0.05);(4)TNF-α-238G/G、G/A基因型, TNF-α-308G/G、G/A基因型各组间在干扰素治疗后IL-2、IFN-γ、IL-4和IL-10平均浓度变化无显著差异(P>0.05);(5)TNF-α-238、TNF-α-308位点各基因型在α干扰素治疗应答组和无应答组的分布差异无显著性意义(P>0.05).结论 (1)干扰素治疗不同应答水平与血清Th1/Th2细胞因子水平有关;(2)干扰素治疗后不同应答水平与治疗前后Th1/Th2细胞因子变化水平有关;(3)TNF-α-308 G/A基因型可能与慢性乙型肝炎患者外周血高IFN-γ水平有关;(4) TNF-α-238、-308位点基因多态性可能与干扰素治疗后IL-2、IFN-γ、IL-4和IL-10水平变化无关;(5)TNF-α-238、-308基因多态性与干扰素治疗不同应答水平无相关性.     

细胞因子Th1/Th2失衡在AITD发病中的作用

分析Th1/Th2失衡在自身免疫性甲状腺疾病(AITD)发病中的作用。选取2013—2015年AITD患者55例,其中Graves病(GD)28例,桥本甲状腺炎(HT)27例。分析两组患者的甲状腺功能、甲状腺抗体、血清中Th1型细胞因子IL-2、TNF、IFN-γ和Th2型细胞因子IL-4、IL-5、IL-10水平。结果显示,GD组的FT3、FT4、TT3、TT4显著高于HT组,TSH显著低于HT组(P0.05)。GD组的TGAb、TPOAb显著低于HT组,TRAb显著高于HT组(P0.05)。GD组的IL-2、TNF、IFN-γ显著低于HT组,IL-4、IL-5、IL-10显著高于HT组(P0.05)。GD中,IL-4、IL-5、IL-10和TRAb呈正相关(P0.05);HT中,IL-2、TNF、IFN-γ和TGAb、TPOAb呈正相关,IL-4、IL-5和TGAb、TPOAb呈负相关(P0.05)。结果表明,HT患者Th1细胞免疫占优势,GD患者Th2细胞免疫占优势,对于鉴别诊断具有一定临床意义。     

食管癌患者外周血Th1/Th2及细胞因子表达的变化

目的 观察食管癌患者机体免疫状态的变化.方法 采用流式细胞技术检测65例哈族患者T淋巴细胞亚群(Th1、Th2、CD3+、CD4+、CD8+细胞),采用双抗体夹心酶联免疫吸附试验(ELISA)法检测血清中自细胞介素(IL)-2、干扰素(IFN)-γ、IL-4、IL10、sIL-2R和肿瘤坏死因子(TNF)-β水平.结果 食管癌患者外周血Th1/Th2细胞比值降低(P<0.05),其中Th1细胞表达的IL-2、IFN-γ等细胞因子水平低于正常对照组;Th2细胞表达的IL-4、IL10细胞因子水平高于正常对照组(P<0.05).T细胞介导的细胞免疫及血清sIL-2R和TNF-β水平存在改变和紊乱,其程度与肿瘤TNM分期相关,其特征为:CD3+、CD4+细胞减少(P<0.05),而CD8+细胞增加(P<0.01);CD4+/CD8+细胞比值下降(P<0.01),血清sIL-2R和TNF-β水平升高(P<0.05).食管癌根治术后1个月CD3+、CD4+细胞回升,CD8+细胞回降,同时血清sIL-2R和TNF-β水平降低(P<0.01).结论 食管癌患者外周血Th1/Th2细胞比值降低,细胞因子表达失衡,患者存在免疫功能抑制.     

凝血酶对狼疮肾炎末梢血单个核细胞分泌Th1/Th2细胞因子的影响

目的探讨狼疮肾炎(LN)患者凝血活性对其Th1/Th2平衡的影响。方法LN患者20例,按红斑狼疮病情活动指数(SLEDAI)分为稳定组11例(SLEDAI<9分),活动组9例(SLEDAI≥9分)。健康对照组8例。采集末梢血单个核细胞(PBMC),进行高、低浓度凝血酶刺激下体外培养。ELISA法检测培养液上清IL-10、IFN-γ水平。RT-PCR检测IL-10和IFN-γmRNA表达。结果凝血酶呈浓度依赖性上调LN患者PBMCIL-10表达,但不影响IFN-γ表达,故增加IL-10/IFN-γ比值。结论凝血酶加重狼疮肾炎患者PBMC分泌Th1/Th2细胞因子的平衡紊乱。     

Th1/Th2细胞因子在深静脉血栓形成中的变化规律

目的：研究深静脉血栓形成（DVT）中Th1/Th2细胞因子的变化规律。方法：选取DVT患者66例,按病程分为1~3 d组、4~7 d组、8~15 d组和16~30 d组,观察Th1/Th2相关因子TNF-α、IFN-γ、IL-4、IL-10水平。结果：DVT患者TNF-α、IFN-γ、IL-4、IL-10均较对照组明显升高,1~3 d组、4~7 d组的IFN-γ/IL-4比值较对照组升高,8~15 d组和16~30 d组IFN-γ/IL-4比值较对照组降低。结论：DVT中存在Th1/Th2的分化失衡,Th1/Th2细胞因子可以作为临床病情评估和疗效评价的指标。     

阳明腑实证患者外周血Th1/Th2细胞因子的变化

目的:观察阳明腑实证患者外周血Th1/Th2细胞因子变化情况,探讨其与肠源性内毒素血症的相关性.方法:25例患者随机分为两组,一组给予常规治疗作为对照组,另一组在常规治疗基础上给予复方大承气汤作为中药组,检测入院时及治疗后3d血浆内毒素(ET),血清干扰素-γ(IFN-γ)、白细胞介素-4(IL-4)的变化,用IFN-γ/IL4比值表示Th1/Th2的变化.结果:两组患者治疗后血浆ET、血清IFN-γ白细胞介素-4(IL-4)水平均降低,尤以中药治疗组降低更具有显著性(P＜0.05).Th1/Th2比值升高.结论:复方大承气汤对阳明腑实证患者血清IFN-γ、IL-4及Th1/Th2比值具有调节作用,阳明腑实证患者血清IFN-γ、IL-4及Th1/Th2比值与肠源性内毒素血症有一定的相关性.     

中药调节Th1/Th2免疫平衡在下肢深静脉血栓形成治疗中的作用

目的:探讨Th1/Th2亚群在DVT发病中的变化规律及中药复方消栓通脉颗粒调节Th1/Th2亚群平衡的作用机制。方法:60例急性DVT患者分为中西医结合组(结合组)和西药组,并设30例健康查体者为正常组,采用流式细胞术及ELISA法观察DVT患者外周血Th1、Th2细胞比例变化及血浆中TNF-α、IFN-γ、IL-4和IL-10水平。结果:消栓通脉颗粒作用后,结合组TNF-α、IFN-γ水平低于西药组(P<0.05);结合组IL-4和IL-10水平高于西药组(P<0.05);结合组治疗后肢围改善优于西药组(P<0.05)。结论:消栓通脉颗粒可以抑制TNF-α、IFN-γ转录,促进IL-4、IL-10表达,升高Th2亚群比例,优势诱导T细胞向Th2亚群偏移,减轻炎症反应,保护血管内皮细胞,促进血栓再通,改善肢体肿胀。     

氯诺昔康超前镇痛对胃癌患者围术期Th1/Th2平衡的影响

目的 观察氯诺昔康超前镇痛对胃癌患者围术期Th细胞漂移的影响.方法 30例全麻下行胃癌根治术的患者随机均分为两组.Ⅰ组患者术前给予氯诺昔康8mg.手术后PCA泵以氯诺昔康和吗啡镇痛;Ⅱ组仪在术后经PCA泵给予吗啡镇痛.分别于术前(T_0)、术毕(T_1)、术后24 h(T_2)、72 h(T_3)抽取外周血.测定血浆皮质醇(Cor)、干扰素-γ(IFN-γ)、白细胞介素-4(IL-4)浓度.结果 与T_0,时比较,Ⅱ组T_1、T_2时Cor升高.I组Cor仅在T_1时升高(P<0.05).与Ⅱ组比较,I组T_1、T_2时Cor明显降低(P<0.05).Ⅰ组T_2时IL-4低于T_0(P<0.05).与Ⅱ组比较.Ⅰ组T_3时IL-4明显降低(P<0.05).与T_0时比较,两组T_1～T_3时IFN-γ明显上升(P<0.05).Ⅰ组T_1、T_2时IFN-γ明显高于Ⅱ组(P<0.05).与T_0时比较,两组T_1～T_3.时IFN-γ/IL-4均升高(P<0.05);Ⅰ组T_2时IFN-γ/IL-4明显高于Ⅱ组(P<0.05).结论 氯诺昔康超前镇痛能减轻胃癌患者手术后Th1/Th2半衡的漂移程度.改善机体外科创伤后免疫受抑状态.     

Th1/Th2细胞因子与自身抗体在特发性少弱精子症患者血清中的表达及意义

目的:探讨特发性少弱精子症(IO)患者血清中Th1/Th2细胞因子和自身抗体表达水平及意义。方法:2017年11月至2020年4月期间,用酶联免疫吸附法(ELISA)检测48例轻、中度IO患者,48例重度IO患者和72例精液常规在正常范围的男性血清中细胞因子(IL-2、IL-4、IL-10、IL-21、TNF-α和IFN-γ)水平及抗人绒毛膜促性腺激素抗体(AhCGAb)、抗精子抗体(AsAb)、抗心磷脂抗体(AcAb)水平,并比较组间差异,构建Logistic回归模型进行回归分析。结果:与对照组比较,轻、中度IO组患者血清IL-10、IL-21和IFN-γ的含量,AhCGAb、AsAb和AcAb阳性表达率显著增高(P0.05),重度IO组患者血清中6种细胞因子含量及3种自身抗体阳性表达率均显著增高(P0.05);与轻、中度IO组比较,重度IO组患者血清中IL-2、IL-4、IL-21和TNF-α水平,AhCGAb和AsAb阳性表达率明显增加(P0.05)。Logistic多元回归分析显示,IL-21/IL-10比值增加[OR (95%CI):1.694 (0.319～4.035),P0.05]、AhCGAb阳性表达[OR (95%CI):4.357 (1.204～9.426),P0.05]和AsAb阳性[OR (95%CI):2.135 (1.902～5.429),P0.05]是IO发生的危险因素。结论:特发性少弱精子症男性血清中细胞因子IL-2、IL-4、IL-10、IL-21、TNF-α、IFN-γ和抗体AhCGAb、AsAb、AcAb的表达水平均显著不同于精液参数正常的男性对照,定量分析患者血清中的细胞因子和抗体,对于探索男性不育的致病机制具有一定价值。     

308nm准分子激光治疗对白癜风患者外周血Th1/Th2细胞因子的影响

目的：研究308nm准分子激光治疗前后白癜风患者外周血Th1型细胞因子（IFN-γ）和Th2型细胞因子（IL-10）水平,探讨308nm准分子激光治疗白癜风的免疫机制。方法：采用ELISA方法检测25例白癜风患者治疗前后和20例正常对照组外周血IFN-γ和IL-10的水平。结果：IFN-γ水平在各组之间均无统计学差异（P〉0.05）。治疗前白癜风患者外周血IL-10水平与正常对照组相比明显增加,差异有统计学意义（P〈0.05）,治疗后白癜风患者外周血IL-10水平与正常对照组相比明显增加,但是差异无统计学意义（P〉0.05）。结论：白癜风患者体内Th2型细胞因子占优势,308nm准分子激光可恢复白癜风患者Th1/Th2的平衡状态。     

趋化因子CXCL9、CXCL10、CXCL11与类风湿关节炎的研究进展

类风湿关节炎发病率、致残率高,目前确切的发病机制未明.类风湿关节炎的滑膜组织有很多趋化因子,如趋化因子CXCL9、CXCL10、CXCL11.通过查阅文献,综述趋化因子在类风湿关节炎中的作用,认为CXCL9、CXCL10、CXCL11,及其受体参与介导滑膜炎,导致骨破坏,有可能成为类风湿关节炎早期诊断的新指标及治疗的新...     

肝纤维化与肝硬化组织中趋化因子CXCL5和CXCL8的表达意义

目的 探讨肝纤维化与肝硬化组织中趋化因子CXCL5和CXCL8的表达意义.方法 收集2008年5月至2009年5月南方医科大学南方医院9例肝血管瘤患者、10例肝纤维化患者和11例肝硬化患者肝组织标本,采用ELISA法检测肝组织中的CXCL5和CXCL8的含量.采用单因素方差分析,双变量正态分布采用Pearson等级相关分析,不符合双变量正态分布的采用Spearman相关系数表示.结果 肝血管瘤、肝纤维化、肝硬化患者肝组织中CXCL5的含量分别为(0.8±0.7)、(2.0±2.0)、(17.1±4.8)ng/g;CXCL8的含量分别为(6.2±3.7)、(11.6±3.5)、(12.3±3.9)ng/g;3者比较差异有统计学意义(F=60.050,7.690,P＜0.05).CXCL5与ALT、AST、PT具有相关性(r=0.502,0.468,0.523,P＜0.05);CXCL8与ALT、AST、TBil、PT具有相关性(r=0.477,0.504,0.537,0.431,P＜0.05).结论 肝脏发生纤维化损伤时CXCL5和CXCL8的含量均显著增高,但两者的变化规律不同.CXCL5和CXCL8的变化与肝脏损伤有关,但变化的程度与肝病的损害程度不完全一致.     

Urinary CXCL9 and CXCL10 Levels Correlate with the Extent of Subclinical Tubulitis

Subclinical tubulitis has been associated with the later development of interstitial fibrosis and tubular atrophy (IF/TA), leading to diminished allograft survival. The aim of this study was to investigate how concentrations of urinary CXC-receptor 3 (CXCR3) chemokines (i.e. CXCL4/9/10/11) and CCL2 relate to the extent of subclinical tubulitis. Using ELISA, urinary CXCR3 chemokines, CCL2 and tubular injury markers (i.e. urinary NGAL and α1-microglobulin [α1 m]) were measured in patients with stable estimated GFR ≥40 mL/min exhibiting normal tubular histology (n = 24), subclinical borderline tubulitis (n = 18) or subclinical tubulitis Ia/Ib (n = 22), as well as in patients with clinical tubulitis Ia/Ib (n = 17) or IF/TA (n = 10). CXCL9 and CXCL10 were significantly higher in subclinical tubulitis Ia/Ib than in subclinical borderline tubulitis (p ≤ 0.03) and normal tubular histology (p ≤ 0.0002). By contrast, NGAL, α1-m, CXCL4, CXCL11 and CCL2 were not or only marginally distinctive across these patient groups. All urinary chemokines and tubular injury markers were higher in clinical tubulitis Ia/Ib than in normal tubular histology (p ≤ 0.002), but only tubular injury markers were elevated in IF/TA. These results demonstrate a correlation of urinary CXCL9 and CXCL10 levels with the extent of subclinical tubulitis suggesting potential as noninvasive screening biomarkers.     

Urinary Chemokines CXCL9 and CXCL10 Are Noninvasive Markers of Renal Allograft Rejection and BK Viral Infection

Renal transplant recipients require periodic surveillance for immune‐based complications such as rejection and infection. Noninvasive monitoring methods are preferred, particularly for children, for whom invasive testing is problematic. We performed a cross‐sectional analysis of adult and pediatric transplant recipients to determine whether a urine‐based chemokine assay could noninvasively identify patients with rejection among other common clinical diagnoses. Urine was collected from 110 adults and 46 children with defined clinical conditions: healthy volunteers, stable renal transplant recipients, and recipients with clinical or subclinical acute rejection (AR) or BK infection (BKI), calcineurin inhibitor (CNI) toxicity or interstitial fibrosis (IFTA). Urine was analyzed using a solid‐phase bead‐array assay for the interferon gamma‐induced chemokines CXCL9 and CXCL10. We found that urine CXCL9 and CXCL10 were markedly elevated in adults and children experiencing either AR or BKI (p = 0.0002), but not in stable allograft recipients or recipients with CNI toxicity or IFTA. The sensitivity and specificity of these chemokine assays exceeded that of serum creatinine. Neither chemokine distinguished between AR and BKI. These data show that urine chemokine monitoring identifies patients with renal allograft inflammation. This assay may be useful for noninvasively distinguishing those allograft recipients requiring more intensive surveillance from those with benign clinical courses.     

Th1/Th2细胞与肿瘤复发

目的探讨T辅助淋巴细胞Ⅰ型（Th1）／T辅助淋巴细胞Ⅱ型（Th2）在肿瘤复发中的研究进展。方法复习国内、外相关文献并进行综述。结果肿瘤治疗后体内出现Th1向Th2漂移，使肿瘤细胞逃避机体的免疫监视，导致肿瘤的复发。结论Th1向Th2型漂移与肿瘤治疗后的复发有关，促使肿瘤治疗后的机体细胞因子由Th2向Th1逆转，重新达到平衡，成为肿瘤免疫治疗的新思路。     

Th1 and Th17 Cells Induce Proliferative Glomerulonephritis

Th1 effector CD4+ cells contribute to the pathogenesis of proliferative and crescentic glomerulonephritis, but whether effector Th17 cells also contribute is unknown. We compared the involvement of Th1 and Th17 cells in a mouse model of antigen-specific glomerulonephritis in which effector CD4+ cells are the only components of adaptive immunity that induce injury. We planted the antigen ovalbumin on the glomerular basement membrane of Rag1^−/− mice using an ovalbumin-conjugated non-nephritogenic IgG1 monoclonal antibody against α3(IV) collagen. Subsequent injection of either Th1- or Th17-polarized ovalbumin-specific CD4+ effector cells induced proliferative glomerulonephritis. Mice injected with Th1 cells developed progressive albuminuria over 21 d, histologic injury including 5.5 ± 0.9% crescent formation/segmental necrosis, elevated urinary nitrate, and increased renal NOS2, CCL2, and CCL5 mRNA. Mice injected with Th17 cells developed albuminuria by 3 d; compared with Th1-injected mice, their glomeruli contained more neutrophils and greater expression of renal CXCL1 mRNA. In conclusion, Th1 and Th17 effector cells can induce glomerular injury. Understanding how these two subsets mediate proliferative forms of glomerulonephritis may lead to targeted therapies.Although proliferative and crescentic glomerulonephritides occur in different primary renal diseases and are an important component of several systemic diseases, features of human renal biopsies suggest some common effector pathways. In most cases of rapidly progressive GN there is evidence for an important role for cellular immune effectors: T cells, macrophages, and neutrophils,^1–3 a role confirmed in animal models.^4–7 CD4+ T cells are key components of renal injury.^4,8 When activated, CD4+ cells tend to differentiate into subsets (T helper cells—Th1, Th2 and Th17) that engage immune effectors in different ways. In proliferative forms of GN, T cells direct adaptive immune responses that drive glomerular disease, but also, in rapidly progressive GN, CD4+ cells themselves accumulate in glomeruli as effectors. These effector T helper cells activate innate effector cells, predominantly neutrophils and macrophages, and activate and damage intrinsic renal cells.In GN, the variable Th1-Th2 predominance of responses influences the histologic patterns and severity of GN.⁹ Th1 cells, which secrete IFNγ and activate macrophages, are important in some forms of experimental proliferative GN. Th2 cells, characterized by IL-4 production, promote humoral immunity and are important in several forms of GN, but there is little evidence that Th2 cells play primary roles as effector cells within glomeruli in rapidly progressive GN. A binary Th1/Th2 model explains many of the differences in the patterns of immune responses in GN. However, there are discrepancies¹⁰ that might be explained by defining a role for a third major subset, Th17 cells, characterized by the production of IL-17A. Its biology has recently been comprehensively reviewed.¹¹ Th17 subset effects potentially relevant to rapidly progressive GN include direct effects on neutrophils and stimulating the production of neutrophil chemoattractants by tissue cells. Thus, in most rapidly progressive types of GN, cell-mediated injury, a key component of injury, may be directed by the Th17 subset, the Th1 subset, or both subsets.Although antigen-specific T cells are critical to adaptive immune responses, the cells themselves are relatively infrequent. T cell receptor (TcR) transgenic mice help define the contributions of different antigen-specific T helper cell subsets in organ-specific disease.^12–14 In the studies presented here we have established a new antigen-specific model of GN. Ovalbumin (OVA)-specific OT-II TcR transgenic CD4+ cells¹⁵ are polarized ex vivo under Th1- or Th17-inducing conditions. Effector cells are transferred into recombination activating gene-1 deficient (Rag1^−/−) mice with OVA planted in their glomeruli by injecting an OVA conjugate. OVA is conjugated to a mouse IgG1 mAb binding to α3(IV) collagen in murine glomerular basement membrane (GBM). The mAb-OVA conjugate dose is capable of planting significant OVA in glomeruli as an antigen to induce effector responses, but is insufficient to induce significant histologic or functional injury as an antibody. This model allows us to understand effector CD4+ T cell and Th subset-induced injury, with no effects from CD8+ cells or B cells.CD4+ cells, isolated from OVA-specific TcR transgenic (OT-II) mice and cultured under Th1 or Th17 priming conditions (see Concise Methods), were confirmed to be Th1 or Th17 by cytokine production before transfer. Th1 polarized cells expressed IFNγ, whereas no IL-17A or IL-4 production was detected (Figure 1A). Th17 polarized cells were strong IL-17A producers, showing only weak IFNγ production, with >20% of cells producing IL-17A, but few IFNγ or double-positive cells (Figure 1B). To plant OVA in glomeruli, the mAb 8D1, a non-nephritogenic, murine IgG1 binding to mouse α3(IV)NC1,¹⁶ was conjugated to OVA and purified by size-exclusion chromatography so that no free OVA or unconjugated 8D1 mAb remained, confirmed by Western blotting (Figure 1C). Antigen-specific CD4+ cells recognized OVA bound to the 8D1 anti-GBM mAb. Culture of CFSE-labeled naive OT-II cells incubated with the 8D1-OVA conjugate enhanced their survival (30% to 40% survival after 72 h versus 5% to 6% with unconjugated antibody) and induced OT-II cell proliferation (72 h: 27% of cells, up to 4 cycles, Figure 1D). OT-II cells incubated with unconjugated 8D1 did not proliferate (Figure 1E). After intravenous injection, 8D1-OVA conjugates bound to the GBM in a linear manner; no fluorescence signal was observed after transfer of Th1 cells without 8D1-OVA (Figure 1G). Western blotting showed OVA in the kidney after injection of 8D1-OVA conjugate (Figure 1H). Lungs from 8D1-OVA-injected mice were weakly positive for mouse IgG, whereas no IgG was detected in the spleen or liver (detecting antibody titer 1:100) 3 or 21 d after injection. Mouse IgG was not detected in sera (ELISA, dilution 1:100) at day 3 or day 21. As expected (given the transfer of only CD4+ cells to Rag1^−/− mice), no anti-OVA antibodies in sera were detected in recipient mice (data not shown).Open in a separate windowFigure 1.Differentiation of OVA-specific OT-II Th1 and Th17 cells, antibody-OVA conjugation, glomerular IgG and intrarenal OVA detection, and recipient immune responses after cell transfer. (A) After stimulating naive OT-II cells with OVA in a Th1 environment, IFNγ was produced and intracellular cytokine staining of CD4+ cells demonstrated strong IFNγ staining with minimal IL-17A or IL-4. (B) Culturing cells in a Th17-stimulating environment led to strong IL-17A production, whereas cells stained positive for IL-17A but not IL-4, and only 2% of cells produced IFNγ. (C) Chromatographic profile of 8D1-OVA conjugation. The numbers 1 to 7 represent fractions collected for analyses by Western blotting, which confirmed that all OVA-conjugated fractions contained OVA and IgG (lanes 1–6), whereas unconjugated fractions (represented as “Un”) contained IgG alone. The lane labeled “M” contained molecular weight markers. (D and E): 8D1-OVA was recognized by OT-II cells because multiple cycles of proliferation of cultured naive OT-II cells (D) were seen with 8D1-OVA conjugate and (E) not seen with unconjugated antibody. Strong linear IgG staining of glomeruli was seen after (F) the administration of 8D1-OVA to Rag1^−/− mice, but not after (G) the injection of Th1 cells without antibody. Western blotting of homogenized kidney (H) 24 h after the administration of 8D1-OVA demonstrated OVA in the kidneys (labeled as OVA-Ab); this was not seen after the administration of unconjugated antibody (labeled as Un Ab). (I) Systemic immune responses of recipient Rag1^−/− mice at 21 d assessed by splenic cytokine production demonstrated enhanced IFNγ production in mice given 8D1-OVA and Th1 cells, with enhanced IL-17A production by mice receiving 8D1-OVA and Th17 cells. (J) DTH to OVA (at 21 d) was induced only in mice given 8D1-OVA and Th1 cells. *P < 0.05, ***P < 0.001.To determine whether transfer of either Th1 or Th17 antigen-specific effector cells induces glomerular injury, 8D1-OVA conjugate was administered intravenously to Rag1^−/− mice (lacking adaptive immunity). Three hours later, 5 × 10⁶ Th1 or Th17 cells were injected intravenously. Groups of mice injected with 8D1-OVA alone (without cells) or Th1 cells alone (without 8D1-OVA) served as controls. At 21 d, the injected T cells largely maintained their initial phenotype, because host splenocytes from mice given Th1 cells showed enhanced OVA-stimulated IFNγ production whereas IL-17A production was enhanced in mice given Th17 cells (Figure 1I). Dermal-delayed-type hypersensitivity (DTH) was induced by footpad injection of OVA and measured after 24 h. Only mice that received the 8D1-OVA conjugate and Th1 polarized cells developed dermal DTH (Figure 1J), a classical Th1 response.¹⁷After planting OVA in glomeruli, administration of Th1 or Th17 cells induced glomerular disease. Urinary albumin excretion was not increased in mice given 8D1-OVA conjugate alone or Th1 cells alone, but Th1 or Th17 cells with 8D1-OVA induced significant albuminuria (Figure 2A). Albuminuria was consistent throughout the time course of the study in the Th17 group, whereas in the Th1 group there was a progressive increase in albuminuria until day 21 (Figure 2B). Control mice given Th1 cells alone or the 8D1-OVA conjugate alone exhibited only mild histologic changes (no crescent formation, fibrinoid necrosis, or hyalinosis). Analysis of histologic injury demonstrated substantially more abnormal glomeruli in the mice given 8D1-OVA conjugate with Th1 or Th17 cells compared with control groups (Figure 2C). Th1 and Th17 (+8D1-OVA) recipients developed proliferative GN, [glomerular hypercellularity: 8D1-OVA and Th1 cells: 32.1 ± 1.0 cells/glomerular cross section (c/gcs), 8D1-OVA and Th17 cells: 29.8 ± 1.1 c/gcs, 8D1-OVA alone: 21.3 ± 0.2 c/gcs, Th1 cells alone: 18.9 ± 2.0 c/gcs; P < 0.001]. Representative kidney sections from each group are shown (Figure 2, D through G). Crescent formation and fibrinoid necrosis, although seen in only a few glomeruli, was observed exclusively in mice given 8D1-OVA conjugate and Th1 cells (5.5 ± 0.9% at day 21; Figure 2, H and I). No crescent formation was observed in mice receiving 8D1-OVA conjugate and Th17 cells. Mice did not develop significant renal impairment (measured by BUN; data not shown).Open in a separate windowFigure 2.Renal injury in mice injected with 8D1-OVA conjugate, then either Th1 or Th17 cells. (A) Mice given 8D1-OVA conjugate or Th1 cells alone did not develop albuminuria above values for noninjected Rag1^−/− mice (dotted line). At 21 d, albuminuria was increased in mice given 8D1-OVA and Th1 cells or 8D1-OVA and Th17 cells. (B) In mice given 8D1-OVA and Th17 cells, albuminuria had plateaued by day 7 and did not progress. In mice given 8D1-OVA and Th1 cells there was a progressive rise in albuminuria. (C) Histologic injury was significant in mice given 8D1-OVA and either Th1 or Th17 cells. Representative glomeruli from mice given (D) 8D1-OVA alone, (E) Th1 cells alone, (F) 8D1-OVA and Th1 cells, and (G) 8D1-OVA and Th17 cells are shown. (H and I) Crescentic injury and fibrinoid necrosis were only seen in mice given 8D1-OVA and Th1 cells. ***P < 0.001Recruitment and activation of leukocyte subpopulations differed in mice administered Th1 or Th17 cells (Figure 3A). Although glomerular CD4+ cell and macrophage numbers were similarly increased in mice given 8D1-OVA conjugate and either Th1 or Th17 cells at day 21, more neutrophils were found in mice given 8D1-OVA and Th17 cells compared with mice given 8D1-OVA and Th1 cells. Interstitial leukocyte infiltrates followed a similar pattern (Figure 3B). Consistent with the finding of increased neutrophils in kidneys of mice receiving Th17 cells, renal mRNA expression of the primary neutrophil attracting chemokine CXCL1 was elevated (Figure 3C). Th17 cells attract neutrophils¹⁸ and in vitro studies have shown that neutrophil recruitment is achieved via production of CXCL8, the human homologue of CXCL1, by Th17 cells.¹⁹ It is therefore likely that at least some of the Th17-induced renal injury is mediated by neutrophils. In mice receiving 8D1-OVA and Th1 cells, macrophages were likely to be more activated; only these mice developed dermal DTH and increased expression of mRNA for the macrophage chemoattractants CCL2 and CCL5 (Figure 3, D and E), which have been associated with experimental crescentic GN.²⁰ Furthermore, type 2 nitric oxide synthase (NOS2/iNOS) mRNA, a marker of macrophage activation²¹ and urinary nitrate, a marker of intrarenal macrophage NOS2 production, were increased in this group (Figure 3, F and G).Open in a separate windowFigure 3.Leukocytes in kidneys of mice with either Th1- or Th17-induced injury 21 d after cell transfer. (A) Glomerular CD4+T cells, neutrophils, and macrophages were increased in mice given 8D1-OVA and Th1/Th17 cells. Neutrophil recruitment was incrementally increased in mice given 8D1-OVA and Th17 cells compared with 8D1-OVA and Th1 cells. (B) A similar pattern of recruitment was seen in the cortical interstitium. Renal chemokine mRNA expression demonstrated (C) enhanced CXCL1 mRNA in mice given 8D1-OVA and Th17 cells, whereas (D) CCL2 and (E) CCL5 were increased in mice given 8D1-OVA and Th1 cells. (F and G) NOS2 and urinary nitrate, markers of macrophage activation, were increased in mice receiving 8D1-OVA and Th1 cells. For mRNA, values for the 8D1-OVA alone group are presented as 1. *P < 0.05, **P < 0.01, ***P < 0.001.Further studies were performed 3 d after cell transfer. At this time point, albuminuria was present in mice receiving 8D1-OVA conjugate and Th17 cells, but not 8D1-OVA conjugate and Th1 cells (Figure 4A), and a higher proportion of glomeruli were abnormal in mice that had received Th17 cells (Figure 4B). Therefore, Th17-induced glomerular injury occurred earlier than Th1-induced injury. Leukocytes were present in glomeruli (Figure 4C) with increased numbers of neutrophils in glomeruli of mice receiving Th17 cells (compared with Th1 cell recipients), whereas Th1 cell recipients exhibited more macrophages. At day 3, these findings were glomerulo-specific; differences between Th1 and Th17 cell recipients were not seen in the interstitium (Figure 4D).Open in a separate windowFigure 4.Renal disease in mice 3 d after injection with 8D1-OVA and either Th1 or Th17 cells. (A) Pathologic albuminuria (dotted line represents values for noninjected Rag1^−/− mice) and (B) increased numbers of abnormal glomeruli were evident in mice that received 8D1-OVA and Th17 cells. (C) Leukocyte recruitment to glomeruli demonstrated CD4+ cells (more in mice receiving Th1 cells), with comparatively more neutrophils in glomeruli of Th17 cell recipients and more macrophages in glomeruli of Th1 cell recipients. (D) Interstitial leukocytes were similar in Th1 and Th17 cell recipients 3 d after cell transfer. *P < 0.05, **P < 0.01, ***P < 0.001.These studies used Rag1^−/− mice as recipients of effector antigen-specific Th1 or Th17 cells. Because these mice do not possess T or B cells, OVA planted in glomeruli cannot induce CD8+ or B cell responses, and regulatory T cells are unable to influence the pattern of injury. A major advantage of this strategy is that Th1- and Th17-mediated injury can be assessed in a pure experimental system. However, T cells transferred into Rag1^−/− mice can undergo homeostatic expansion, and it is possible that the transferred Th1 cells might have expanded more rapidly than Th17 cells. Recently, studies in experimental type 1 diabetes induced by transfer of cells from a TcR transgenic mouse specific for an islet autoantigen showed conversion of Th17 cells to a Th1 phenotype after transfer.^22,23 Although our Th17 polarized OT-II cells, specific for a foreign antigen, showed some IFNγ production after 21 d, they were still capable of producing IL-17A. Furthermore, dermal DTH and renal disease were different in Th1 recipients compared with the Th17 recipients at 21 d, supporting the maintenance of separate phenotypes after transfer. Although the studies presented here are the first to demonstrate a role for Th17 and Th1 cells in the same experimental system, other studies^24–26 have used genetically deficient mice to implicate Th17 cells in experimental renal disease.These studies describe a novel model of cell-mediated proliferative GN for which adaptive components are only effector antigen-specific CD4+ T cells. They demonstrate that both Th1 and Th17 cells can induce proliferative GN. Th17 cells induce albuminuria early, with persistent accumulation of leukocytes. Administration of Th1 cells lead to a slower rise in albuminuria, but more macrophage activation and DTH-like injury, including, in some glomeruli, crescent formation and fibrinoid necrosis. It is likely that Th1 and Th17 responses play a role in proliferative forms of GN and both represent potential therapeutic targets.     

Early Low Urinary CXCL9 and CXCL10 Might Predict Immunological Quiescence in Clinically and Histologically Stable Kidney Recipients

We monitored the urinary C‐X‐C motif chemokine (CXCL)9 and CXCL10 levels in 1722 urine samples from 300 consecutive kidney recipients collected during the first posttransplantation year and assessed their predictive value for subsequent acute rejection (AR). The trajectories of urinary CXCL10 showed an early increase at 1 month (p = 0.0005) and 3 months (p = 0.0009) in patients who subsequently developed AR. At 1 year, the AR‐free allograft survival rates were 90% and 54% in patients with CXCL10:creatinine (CXCL10:Cr) levels <2.79 ng/mmoL and >2.79 ng/mmoL at 1 month, respectively (p < 0.0001), and 88% and 56% in patients with CXCL10:Cr levels <5.32 ng/mmoL and >5.32 ng/mmoL at 3 months (p < 0.0001), respectively. CXCL9:Cr levels also associate, albeit less robustly, with AR‐free allograft survival. Early CXCL10:Cr levels predicted clinical and subclinical rejection and both T cell– and antibody‐mediated rejection. In 222 stable patients, CXCL10:Cr at 3 months predicted AR independent of concomitant protocol biopsy results (p = 0.009). Although its positive predictive value was low, a high negative predictive value suggests that early CXCL10:Cr might predict immunological quiescence on a triple‐drug calcineurin inhibitor–based immunosuppressive regimen in the first posttransplantation year, even in clinically and histologically stable patients. The clinical utility of this test will need to be addressed by dedicated prospective clinical trials.     

Cumulative exposure to gamma interferon-dependent chemokines CXCL9 and CXCL10 correlates with worse outcome after lung transplant

Outcomes following lung transplant are suboptimal owing to chronic allograft failure termed bronchiolitis obliterans syndrome (BOS). Prior work in both mice and humans has shown that interferon gamma (IFNG)-induced chemokines, including CXCL9 and CXCL10, are elevated in patients with established BOS. We hypothesized that patients who ultimately developed BOS would have elevations in these chemokines before losing lung function. We utilized a high throughput multiplex enzyme-linked immunosorbent assay (ELISA) to measure biomarkers in bronchoalveolar lavage fluid (BALF). We modeled cumulative exposure to seven biomarkers (CXCL9, CXCL10, RANTES, IL1-RA, IL-17, MCP1 and IL-13) by calculating the 1-year area under the curve (AUC) for each biomarker in the BALF of 40 lung transplant patients who had at least four samples obtained in the first year posttransplant. Cumulative elevations in CXCL9 and CXCL10 were associated with a significant risk of subsequent graft failure after transplant (HR 9.37 and 5.52, respectively; p < 0.01 for both). Further these chemokines were also elevated in patients before the onset of BOS. CXCL9 and CXCL10 elevations were seen between 3 and 9 months before graft failure. Our data show that persistent presence of CXCL9 and CXCL10 portents worsening lung allograft function; measuring these IFNG-induced chemokines might prospectively identify patients at risk for BOS.     

The Th1 and Th2 paradigm in ANCA-associated vasculitis

In the pathogenesis of anti-neutrophil cytoplasm antibodies (ANCA)-associated vasculitis, T cell contribution is indicated by T cell-dependent ANCA production combined with the presence of T cells in inflammatory infiltrates. However, the exact pathogenic role of T cells in ANCA-associated vasculitis remains to be determined. The Th1/Th2 concept is useful for understanding T cell involvement in pathological processes. This review focuses on T cells and particularly the Th1/Th2 paradigm in ANCA-associated vasculitis. Most research has been done in Wegener's granulomatosis, where a shift in T cell response, from a Th1 pattern in localized disease towards a Th0/Th2 pattern in generalized disease, appears to occur. Although less thoroughly studied, data in Churg-Strauss syndrome and microscopic polyangiitis indicate that these diseases are predominantly associated with Th2 patterns. Further studies elucidating the true nature of the polarization towards Th1 or Th2 in ANCA-associated vasculitis are clearly needed.