还原型谷胱甘肽对大鼠肝脏缺血再灌注损伤后炎性细胞因子表达的影响

目的 探讨经门静脉注射还原型谷胱甘肽(GSH)对大鼠肝脏缺血再灌注损伤后TNF-α、IL-1β和巨噬细胞炎性蛋白-2(MIP-2)表达的影响及意义.方法 72只雄性SD大鼠平均分为假手术组(SO组)、生理盐水预处理组(IR组)和GSH预处理组(GPC组).建立肝脏缺血再灌注损伤模型,检测再灌注30、60和180 min血清TNF-α、IL-1β含量,以及肝组织中TNF-α mRNA、IL-1β mRNA和MIP-2mRNA表达水平.两独立样本采用t检验,多组比较采用方差分析.结果 GPC组血清TNF-α含量于缺血再灌注180 min后显著低于IR组(t=2.512,P<0.05).而肝组织TNF-αmRNA表达水平于缺血再灌注30 min后即显著低于IR组(t=2.427,P<0.05).GPC组血清中IL-1β含量和肝组织中IL-1βmRNA表达水平于缺血再灌注后各时相点均显著低于IR组(t=2.731,3.825,4.372,3.371,3.972,4.685,P<0.05).GPC组MIP-2 mRNA表达于缺血再灌注60 min和180 min显著低于IR组(t=2.593,5.429,P<0.05).结论 TNF-α、IL-1β和MIP-2等炎性因子在肝脏缺血再灌注损伤中发挥重要作用.GSH能够抑制炎性细胞因子如TNF-α、IL-1β和MIP-2的生成,并发挥抗肝脏缺血再灌注损伤的作用.     

人参皂甙Rb1在核因子NF-E2相关因子2/血红素氧合酶-1通路减轻肠缺血再灌注致急性肺损伤中的分子机制

目的 探讨人参皂甙Rb1对肠缺血/再灌注致急性肺损伤的保护效应及核因子NF-E2相关因子2(Nrf2)/血红素氧合酶-1(HO-1)通路参与该效应的分子机制.方法 成年雄性C57BL/6J小鼠随机分为5组:假手术组(S组);肠缺血/再灌注组(I/R组);再灌注+Rb1组(I/R +Rb1组);全反式维甲酸(ATRA)+再灌注组(ATRA+ I/R组);ATRA+再灌注+Rb1组(ATRA+ I/R+Rb1组).采用肠缺血/再灌注模型,Western blot检测肺组织Nrf2、HO-1表达变化;酶联免疫吸附试验(ELISA)法检测肺组织肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6、IL-10水平;检测超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量;检测肺湿/干比及肺组织病理损伤评分.结果 与S组比较,其他4组Nrf2、HO-1蛋白表达,TNF-α、IL-6、MDA含量,肺组织湿/干重比及肺组织病理评分增高(P＜0.05);与1/R组比较,1/R+ Rb1组Nrf2,HO-1蛋白表达,TNF-α,IL-6,MDA含量,肺组织湿/干重比及肺组织病理评分降低(P＜0.05);与I/R+ Rb1组比较,ATRA+ I/R组,ATRA+ I/R+ Rb1组Nrf2、HO-1蛋白表达,NF-α、IL-6、MDA含量,肺组织湿/干重比及肺组织病理评分增高(P＜0.05).SOD活性、IL-10水平与上述变化相反.结论 肠缺血/再灌注可引起急性肺损伤,人参皂甙Rb1后处理能通过激活Nrf2/HO-1通路减轻肠缺血/再灌注所致肺损伤.     

白术多糖对缺血再灌注损伤大鼠肝脏的保护作用

目的：探讨白术多糖对大鼠肝脏缺血再灌注（I/R）损伤的保护作用。
方法：成年雄性SD大鼠随机分成3组：假手术组,I/R组,白术多糖预处理缺血再灌注组（PC组）,分别于成模后1,6,24 h 3个时段分别检测3组大鼠血清谷丙转氨酶（ALT）和谷草转氨酶（AST）水平;检测肝组织中ICAM-1 mRNA含量及IL-1的表达。
结果：PC组及I/R组ALT,AST水平以及ICAM-1 mRNA含量和IL-1表达均高于假手术组（P＜0.05）,但PC组上述4个指标均较I/R组显著降低,差异有统计学意义（P＜0.05）。
结论：白术多糖可能通过降低ICAM-1的含量而减少炎症因子IL-1的生成,从而对肝脏I/R损伤起保护作用。     

缺血后处理对小鼠肠缺血再灌注致肾损伤时Nrf2蛋白表达的影响

目的 评价缺血后处理对小鼠肠缺血再灌注致肾损伤时核因子E2相关因子2(Nrf2)蛋白表达的影响.方法 健康雄性C57BL/6J小鼠36只,9～12周,采用随机数字表法,将其随机分为3组(n=12):假手术组(S组)、缺血再灌注组(I/R组)、缺血后处理+缺血再灌注组(IPO组).采用夹闭肠系膜上动脉根部45 min恢复灌注的方法制备小鼠肠缺血再灌注损伤模型,IPO组于缺血45 min时再灌注30s,缺血30s,重复3次后恢复灌注.于再灌注2h时采集颈动脉血样,然后处死小鼠,取肾组织,测定血清BUN、Cr和中性粒细胞明胶酶相关脂质运载蛋白(NGAL)水平,检测肾组织Nrf2和HO-1蛋白表达、MDA含量、SOD活性、TNF-α、IL-6和IL-10的含量.显微镜下观察肾组织病理学结果,并行病理学损伤评分.结果 与S组比较,I/R组血清BUN、Cr和NAGL浓度升高,肾脏组织Nrf2及HO-1蛋白表达上调,MDA含量升高,SOD活性降低,肾脏组织病理学损伤评分升高(P＜0.05);与I/R组比较,IPO组血清BUN、Cr和NAGL浓度降低,肾脏组织Nrf2及HO-1蛋白表达上调,MDA含量降低,SOD活性升高,肾脏组织病理学损伤评分降低(P＜0.05).各组肾脏组织TNF-α、IL-6和IL-10含量比较差异无统计学意义(P＞0.05).结论 缺血后处理可减轻小鼠肠缺血再灌注致肾损伤,其机制可能与促进Nrf2蛋白表达,从而上调HO-1蛋白表达有关.     

小鼠肝脏部分缺血再灌注损伤模型的建立

目的 建立小鼠肝脏部分缺血再灌注损伤模型并分析损伤评估指标的变化趋势.方法 采用96只7～8周龄的纯系C57BL/6雄性小鼠作为研究对象,建立70％肝脏缺血再灌注损伤模型.按照缺血时间将小鼠分为假手术组和缺血30、60、90 min组,每组24只.各组小鼠分别于再灌注后6、12、24和48 h处死.通过检测血清ALT、AST、TNF-α、IL-6和巨噬细胞炎性蛋白-2(MIP-2)水平以及病理组织学评分、细胞凋亡指数等方法评估各组小鼠肝组织的损伤情况.两独立样本比较采用t检验.结果 术后88只小鼠存活,8只死亡,造模成功率为91.7％ (88/96).假手术组、缺血30、60、90 min组ALT水平分别为(35±24) U/L、(1703±442) U/L、(5133±681) U/L和(8233±808) U/L,缺血30、60、90 min组ALT水平显著高于假手术组(t=6.54,12.97,17.56,P＜0.05);AST水平分别为(87±28) U/L、(2667 ±451) U/L、(6333±778)U/L和(9967±1168) U/L,缺血30、60、90 min组AST水平显著高于假手术组(t=9.89,13.89,14.65,P＜0.05);TNF-α水平分别为(14 ±5) μg/L、(83±14) μg/L、(133±17) μg/L和(202±21) μg/L,缺血30、60、90 min组TNF-α水平显著高于假手术组(t=7.78,11.82,15.34,P＜0.05);IL-6水平分别为(32 ±9) μg/L、(493±168) μg/L、(844±166) μg/L和(1345±198) μg/L,缺血30、60、90min组IL-6水平显著高于假手术组(=4.74,8.46,11.48,P＜0.05);MIP-2水平分别为(37±11) μg/L、(102±35) μg/L、(177±32)μg/L和(279±50) μg/L,缺血30、60、90 min组MIP-2水平显著高于假手术组(t=3.05,7.28,8.19,P＜0.05);细胞凋亡指数分别为1.7％±2.1％、22.7％±8.6％、54.3％±11.2％和76.3％±14.8％,缺血30、60、90 min组细胞凋亡指数显著高于假手术组(t=4.10,8.04,8.63,P＜0.05).在缺血时间相同的情况下,随着再灌注时间的延长,各监测指标呈“抛物线”样变化趋势.结论 小鼠肝脏部分缺血再灌注损伤模型能较好地反映小鼠肝组织的损伤情况.随着缺血时间的延长,小鼠肝脏的缺血再灌注损伤程度逐渐加重;随着再灌注时间的延长,ALT、AST、TNF-α、IL-6、MIP-2以及病理组织学评分和细胞凋亡指数均呈现“抛物线”样变化趋势.     

不同剂量磷酸肌酸钠预先给药对大鼠肝缺血再灌注损伤的影响

目的 评价不同剂量磷酸肌酸钠预先给药对大鼠肝缺血再灌注损伤的影响.方法 健康雄性SD大鼠30只,体重200～250 g,采用随机数字表法分为5组(n=6):假手术组(S组)、缺血再灌注组(I/R组)、不同剂量磷酸肌酸钠预先给药组(P1～3组).采用阻断肝左中动脉及其门静脉90 min恢复灌注的方法制备大鼠肝缺血再灌注模型.P1-3组于缺血前60 min时分别尾静脉注射磷酸肌酸钠50、150和4S0mg/kg.S组和I/R组给予等容量生理盐水.于再灌注4h时采集腹主动脉血样,测定血浆ALT、AST、TNF-α和IL-1β水平.取肝组织,采用ELISA法检测MPO活性,免疫组化法检测细胞间粘附分子(ICAM-1)的表达水平,TUNEL法检测肝细胞凋亡数,电镜下观察病理学结果.结果 与S组比较,I/R组和P1～3组肝组织MPO、血浆ALT、AST活性及TNF-α、IL-1β浓度、肝细胞凋亡数升高(P＜0.05);与I/R组比较,P1～3组上述指标降低(P＜0.05);P1～3组上述指标依次降低(P＜0.05).结论 磷酸肌酸钠预先给药可呈剂量依赖性地减轻大鼠肝缺血再灌注损伤,其机制与抑制炎性反应有关.     

异丙酚对肝脏缺血再灌注大鼠血清肿瘤坏死因子-α和白细胞介素-10的影响

肝脏多发或巨大肿瘤切除术时不可避免地发生反复的肝脏缺血和再灌注,而此类肝脏缺血再灌注(I/R)可导致严重的肝脏损伤;异丙酚具有抗氧化及对脏器I/R损伤有一定的保护作用,但对反复肝脏I/R损伤的保护作用尚未定论。本研究拟采用大鼠反复肝脏I/R模型,通过观察肝脏I/R 不同时期血清肿瘤坏死因子-α(TNF-α)和白细胞介素-10 (IL-10)水平的变化,为进一步研究提供参考。     

白细胞介素12在大鼠肺缺血再灌注损伤中的作用

目的 评价白细胞介素12(IL-12)在大鼠肺缺血再灌注损伤中的作用.方法 健康成年雄性SD大鼠24只,8-10周龄,体重250-280 g,采用随机数字表法,将大鼠随机分为3组(n=8):假手术组(S组),肺缺血再灌注损伤组([/R组),IL-12单克隆抗体组(IL-12组).S组只分离肺门不夹闭；I/R组夹闭肺门60 min后,恢复灌注2 h；IL-12组于夹闭肺门前1h尾静脉注射IL-12单克隆抗体200μg/kg.于再灌注结束时采集血样和肺绀织,测定血浆TNF-α浓度、辅助性T细胞(Th)1和Th2水平、肺组织湿干重(W/D)比、髓过氧化物酶(MPO)活性、MDA含量及动脉血氧分压(PaO2)及二氧化碳分压(PaCO2).光镜下观察肺组织病理学改变.结果 与S组比较,I/R组和IL-12组肺组织W/D比、MDA含量、MPO活性、血浆TNF-α浓度升高,I/R组PaO2降低,PaCO2、Th1水平和Th1/Th2升高(P＜0.01),Th2水平差异无统计学意义(P＞0.05),IL-12组PaO2、PaCO2、Th1、Th2水平及Th1/Th2差异无统计学意义(P＞0.05)；与I/R组比较,IL-12组PaCO2、肺组织W/D比、MDA含量、MPO活性、血浆TNF-α浓度、Th1水平和Th1/Th2降低,Th2水平和PaO2升高(P＜0.01).I/R组肺组织损伤明显,IL-12组肺组织损伤程度明显减轻.结论IL-12通过使Tn1/Th2失衡而参与肺缺血再灌注损伤.     

乳化异氟醚预处理对大鼠肾缺血再灌注损伤的影响

目的 评价乳化异氟醚预处理对大鼠肾缺血再灌注损伤的影响.方法 选择雄性SD大鼠32只,体重220～300 g,10～ 13周龄,采用随机数字表法,将其分为4组(n=8):假手术组(S组)、肾缺血再灌注组(I/R组)、乳化异氟醚预处理组(E组)和脂肪乳剂预处理组(I组).I/R组、E组和I组采用夹闭左侧肾蒂45 min后恢复再灌注的方法建立肾缺血再灌注模型.E组和I组分别静脉输注8%乳化异氟醚、30%脂肪乳剂4ml·kg-·h-30 min,洗脱15 min后制备模型.于再灌注3h时采集腹主动脉血样5ml,测定血清肌酐(Cr)、胱抑素C(Cys C)、TNF-α、IL-6、IL-10浓度;取左肾组织,行HE染色,光镜下观察病理学结果,并行肾脏近曲小管坏死程度分级.结果 与S组比较,其余3组血清Cr、Cys C、TNF-α、IL-6、IL-10浓度及肾脏近曲小管坏死程度均升高(P＜0.05);与I/R组和I组比较,E组血清Cr、Cys C、TNF-α、IL-6浓度及肾脏近曲小管坏死程度均降低(P＜0.05),血清IL-10浓度升高(P＜0.05).I/R组和I组上述各指标比较差异无统计学意义(P＞0.05).I/R组和I组肾组织病理学损伤严重,E组肾组织病理学损伤明显减轻.结论 8％乳化异氟醚预处理可减轻大鼠肾缺血再灌注损伤,其机制可能与抑制全身炎性反应有关.     

匹立尼酸对肾脏缺血再灌注大鼠血清肿瘤坏死因子-α和白细胞介素-1β的影响

目的 观察匹立尼酸(PA)对肾脏缺血再灌注大鼠血清肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)的影响.方法 健康雄性SD大鼠54只,随机均分为3组:假手术组(S组)、缺血再灌注组(I/R组)、PA组.在肾缺血60 min后再灌注建立大鼠缺血再灌注肾损伤模型,S组操作同上但不阻断血管.缺血前30 min,PA组经腹腔注射PA 5 mg/kg,I/R组和S组注射等容积生理盐水.4h后取腹主动脉血检测血清TNF-α和IL-1β的水平.结果 再灌注4h后,I/R组血清TNF-α和IL-1β水平分别为(11.65±1.15) ng/L和(230.80 ±31.82) ng/L,PA组为(7.83±1.27) ng/L和(125.74±18.03) ng/L,PA组显著低于I/R组(P＜0.05).结论 在缺血再灌注肾损伤中,预先给予PA可起到降低血清TNF-α和IL-1β水平的作用.     

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.     

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。     

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.     

Th1/Th2 balance in childhood idiopathic nephrotic syndrome

AIMS: In view of the conflicting evidence of helper T cell type 1 (Th1) or type 2 (Th2) pattern of cytokine synthesis in childhood idiopathic nephrotic syndrome (INS) this study examined the balance of Th1 and Th2 which are characterized by intracellular cytokine production of interferon-gamma (IFNgamma) and interleukin-4 (IL-4), respectively. SUBJECTS AND METHODS: Sixteen children with steroid-sensitive INS (mean age 9.0 years) were included in this study, together with 15 healthy normal children (mean age 7.9 years) for the control group. Intracellular production of both IFNgamma and IL-4 in helper T cell (CD4+ cell) was investigated by a 3-color flow cytometry. RESULTS: The cross-sectional data showed no significant differences of percentages of Th0 (IFNgamma+ IL-4+ CD4+ cell), Th1 (IFNgamma+ lL-4- CD4+ cell) and Th2 (IFNgamma- IL-4+ CD4+ cell) in CD4+ cells (p > 0.05). The Th1/Th2 ratio during nephrotic relapse did not differ from those during nephrotic remission and in normal healthy children (p > 0.05). CONCLUSION: We conclude that there is no significant skew of Th1/Th2 balance in childhood INS and that the cardinal immunological abnormality does not lie in helper T cells but in other cells, such as suppressor/cytotoxic T cells, natural killer cells or monocytes/macrophage. To clarify the pathogenesis of INS, comprehensive studies for these cells would be worthwhile.     

Th1/Th2细胞因子对器官移植排斥反应的影响

目的:研究Th1/Th2细胞因子对同种异系小鼠心脏移植存活时间的影响.方法:使用野生型BALB/c小鼠作为供体,野生型B6小鼠、IL-4基因去除B6小鼠及IFN-γ基因去除B6小鼠作为受体,行腹部异位小鼠心脏移植.部分IL-4基因去除小鼠、IFN-γ基因去除小鼠联合应用α-半乳糖神经酰胺(α-galactosylceramide,α-GalCer),以获得更强的Th1/Th2偏移.比较移植物存活时间.向野生型、IL-4基因去除及IFN-γ基因去除B6小鼠腹腔内注射供体小鼠脾细胞,提取受体小鼠脾脏CD8<'8>T细胞行淋巴细胞毒试验.结果:IFN-γ基因去除组小鼠的移植物存活时间为(6.40±0.55)d,联合应用α-GalCer组移植物存活时间为(8.00±1.15)d.IL-4基因去除组小鼠的移植物存活时间为(8.00±1.00)d,联合应用α-GalCer组移植物存活时间为(8.60±1.34)d.淋巴细胞毒试验显示IFN-γ基因去除小鼠的CD8+T细胞毒性明显增强.结论:Th1/Th2细胞因子与排斥反应之间并不存在简单的对应关系.     

Influence of Th1, Th2, and Th3 cytokines during the early phase after liver transplantation

To investigate the change of Th1, Th2, Th3 cytokines during early liver transplantation. IFN-r, IL-4, TGF-beta production by CD4+ T cells were determined by fluorescence activated cell sorter analysis. Comparing the acute rejection with the non-acute rejection groups on the 7th, 14th, and 28th day showed that high interferon-gamma production associated with acute rejection in the early posttransplant period. There was no evidence of significant changes in interleukin-4 and transforming growth factor beta (TGF-beta) levels between non-acute rejection groups with acute rejection groups. Th1 cytokine high production is significantly associated with acute rejection in liver transplant recipients.     

儿童原发性肾病综合征与Th1/Th2细胞失衡

目的 探讨Th1/Th2细胞失衡在原发性肾病综合征（PNS）患儿发病中的作用。方法运用三色荧光标记法流式细胞术检测21例初发和16例缓解期NS患儿外周血Th1和Th2细胞百分率（％）,并以10例正常儿童作对照。结果 正常儿童外周血Th1、Th2和Th0细胞百分率分别为（13.42±4.36）％,（2.53±1.97）％和（1.25±0.92）％。初发的PNS患儿均明显减低,分别为（2.34±2.09）％,（1.02±0.96）％和（0.40±0.38）％（P<0.05）。缓解期PNS患儿,分别为（11.96±4.75）％,（2.87±2.46）％和（1.31±0.87）％,与正常儿童比,差异均无显著性意义。由于初发的PNS患儿Th1细胞减低相对于Th2细胞而言更显著,因而导致Th1/Th2比值也较缓解期PNS患儿和正常儿童明显下降（2.43±2.65比4.17±2.32和5.41±2.77,P均<0.05）。结论 儿童PNS是一种以Th2细胞占优势的免疫介导的肾小球疾病,源于Th1细胞减低所致的Th1/Th2细胞失衡在原发性肾病综合征发病过程中可能起着重要作用。