Neointima formation and thrombosis after vascular injury in transgenic mice overexpressing plasminogen activator inhibitor-1 (PAI-1)

Summary.  The controversial role of plasminogen activator inhibitor-1 (PAI-1) in neointima formation and restenosis was studied with the use of a vascular injury model in transgenic mice overexpressing murine PAI-1 (PAI-1 Tg) and in wild-type (WT) controls. Despite the high circulating PAI-1 levels in the PAI-1 Tg mice (52 ± 9.8 ng mL⁻¹ vs. 0.76 ± 0.17 ng mL⁻¹ in WT mice), no significant fibrin deposition was observed in non-injured femoral arteries of 8- to 12-week-old mice. Two weeks after severe electric injury, extensive and comparable fibrin deposition was observed in both genotypes, despite a significantly reduced in situ fibrinolytic activity in arterial sections of the PAI-1 Tg mice. The neointimal and medial areas were similar in WT and PAI-1 Tg mice, resulting in comparable intima/media ratios (e.g. 0.94 ± 0.25 and 1.04 ± 0.17 at the center of the injury). Nuclear cell counts in cross-sectional areas of the neointima of the injured region were also comparable in arteries from WT and PAI-1 Tg mice (224 ± 63, 233 ± 20), and the distribution pattern of α-actin-positive smooth muscle cells was similar. These findings indicate that in a vascular injury model that induces extensive and persistent fibrin deposition in femoral arteries of mice, overexpression of PAI-1 does not affect neointima formation.     

LPS对机体内源性纤溶的抑制作用及其机制研究

目的 研究细菌内毒素(LPS)对机体内源性纤溶的直接影响并探讨其作用机制。方法 以^125．纤维蛋白原进行示踪，建立大鼠肺纤维蛋白沉积模型，研究LPS对大鼠内源性纤溶的作用；同时检测LPS作用后大鼠血浆中PAI-1的水平及活性的变化。结果Batroxobin能导致纤维蛋白在大鼠肺组织沉积，表现为5min肺组织放射性记数显著升高，而30min由于内源性纤溶系统的激活，肺组织放射性记数降低；给予LPS后，抑制了30min肺同位素量的降低，与重组PAI-1的作用类似，同时测得血浆中PAI-1的水平及活性增高。结论直接证实LPS通过PAI-1途径抑制机体内源性纤溶，提示抑制PAI-1可能有助于治疗G^-菌引起的脓毒血症。     

热休克蛋白27改善小鼠内毒素血症心功能不全的机制

目的 探讨Hsp27的保护作用是否与激活P13K/Akt信号通路、减轻炎症反应有关.方法 (1)诱导小鼠内毒素血症心肌特异性高表达Hsp27转基因鼠(Hsp 27 Tg)和野生型对照鼠(WT)均腹腔注射内毒素(LPS,10ms/ks);(2)心功能测定 LPS注射后6 h,以心脏超声评价,n:6;(3)P13K/Akt信号通路活性测定 LPS注射后1 h收集心脏,以Western blot法测定Akt及Gsk-3(的磷酸化水平,n=4;(4)NF-B炎症通路活性检测 LPS注射后1 h以Western blot测定IκBα水平,n=4;同时在大鼠心肌细胞中进行相同实验,n:3;(5)心肌细胞凋亡LPS注射后24 h,以TUNEL法检测,,n=4.结果 (1)Hsp27显著改善LPS所致的心功能不全.与基础值相比,LPS处理使WT,Hsp27 Tg均出现心功能不全,但Hsp 27 Tg的心功能不全程度显著轻于WT(P<0.01或P<0.05).(2)Hsp27显著抑制LPS所致的IκBα降解.与WT比较,高表达Hsp27显著减轻了LPS诱导的小鼠心肌IκBα降解[(41.43±24.10)%vs.(72.92±9.20)%,P<0.05],培养的大鼠心肌细胞实验结果与之类似.(3)Hsp27可增强激活P13K/Akt信号通路.LPS处理后,WT和Hsp27Tg鼠心肌组织中磷酸化Akt水平分别为(3.11±0.83)和(5.13±0.73),磷酸化GSK-30水平分别为(3.19±1.04)和(5.71±1.20).与WT比较,Hsp27Tg心肌组织中的磷酸化Akt和GSK-3β水平均显著提高(P<0.05).类似结果在体外培养的细胞实验中也被证实.(4)Hsp27显著抑制LPS所致的心肌细胞凋亡.LPS处理后24 h,WT和Hsp27Tg心肌细胞凋亡率分别为(6.46±1.74)%和(2.88±0.91)%,与WT比较,心肌细胞凋亡在Hsp27Tg中被显著减轻(P<0.01).结论 高表达Hsp27对小鼠内毒素血症心功能不全有显著改善作用,其保护作用与激活P13K/Akt信号通路、抑制NFκB;依赖性炎症反应有关.     

Increased prevalence of regulatory T cells (Treg) is induced by pancreas adenocarcinoma

We reported earlier that patients with breast or pancreas cancer have an increased prevalence of regulatory T cells (Treg) in the blood and tumor draining lymph nodes (TDLNs) compared with healthy individuals. In the current study, we tested the hypothesis that tumor cells promote the prevalence of Treg. The transforming growth factor-beta (TGF-beta) secreting murine pancreas adenocarcinoma, Pan02 cell line was injected into syngeneic C57BL/6 mice and the prevalence of Treg in the TDLNs and tumor spleen was measured weekly. Compared with control mice, the prevalence of CD25+ CD4+ cells in TDLNs and in tumor spleen increased with tumor growth. Analysis of these CD25+ CD4+ T cells in vitro confirmed expression of the Treg marker, Foxp3. In addition, their functional activity resembled that of Treg, as evidenced by a poor proliferative capacity; suppression of proliferation of CD25- CD4 or CD8T cells and inhibition of interferon-gamma release by CD25- CD4+ T cells. Reconstitution of Pan02-bearing Rag-/- mice with naive syngeneic CD25- CD4+ T cells induced CD25+ CD4+ Foxp3+ T cells in TDLNs, but not in the spleen. In contrast, Foxp3 was not detected in unreconstituted Pan02-bearing Rag-/- mice, or reconstituted mice bearing a TGF-beta-negative esophageal tumor. Furthermore, administration of neutralizing anti-TGF-beta antibody blocked the induction of Foxp3 in reconstituted Pan02-bearing Rag-/- mice. These results mimic earlier in vitro studies showing induction of Foxp3 through CD3 plus CD28 stimulation in the presence of TGF-beta. We conclude that Pan02 tumor promotes the prevalence of Treg, in part through the secretion of TGF-beta, which may result in immune evasion.     

Regulation of murine type 1 plasminogen activator inhibitor gene expression in vivo. Tissue specificity and induction by lipopolysaccharide, tumor necrosis factor-alpha, and transforming growth factor-beta.

The regulation of type 1 plasminogen activator inhibitor (PAI-1) gene expression was studied in vivo employing a murine model system. Nuclease protection analysis revealed relatively high concentrations of PAI-1 mRNA in the aorta, adipose tissue, heart, and lungs of untreated CB6 (BalbC X C57B16) mice. Treatment of CB6 mice with LPS, TNF-alpha, or transforming growth factor-beta (TGF-beta) increased the steady-state levels of PAI-1 mRNA within 3 h in all tissues examined. However, the greatest responses to TGF-beta were observed in adipose tissue and the kidney, while LPS and TNF-alpha strongly stimulated PAI-1 gene expression in the liver, kidney, lung, and adrenals. In C3H/HeJ mice, which exhibit defective TNF-alpha release in response to LPS, the response of the PAI-1 gene to LPS was severely attenuated. However, injection of these mice with TNF-alpha increased PAI-1 mRNA in a tissue-specific pattern strikingly similar to that observed in LPS-treated CB6 mice. These results demonstrate that the PAI-1 gene is regulated in a complex and tissue-specific manner in vivo, and suggest a role for TNF-alpha in the response of the PAI-1 gene to sepsis.     

脓毒症患者辅助性T细胞17和CD4+CD25+调节性T细胞表达及血必净注射液的干预作用

目的 观察脓毒症患者外周血辅助性T细胞17(Th17)及CD4+CD25+调节性T细胞(Treg)的水平,并探讨其意义及血必净注射液的干预作用.方法 ①将64例重症监护病房(ICU)脓毒症患者按疾病严重程度分为脓毒症组(26例)、严重脓毒症组(21例)、脓毒性休克组(17例),同时选取18例健康体检者作为对照组.观察不同严重程度脓毒症患者外周血Th17和CD4+CD25+Treg的表达及其与病情严重程度的关系.②按随机原则将64例患者分为常规治疗组(25例,给予常规集束化治疗)和血必净治疗组(39例,在常规治疗基础上加用血必净注射液50 ml静脉滴注,每日2次),两组均以7 d为1个疗程.入ICU当日和治疗7 d用流式细胞术检测外周血Th17及CD4+CD25+Treg表达,观察血必净注射液的干预作用.结果 ①健康对照组Th17表达率为(0.84±0.29)%,CD4+CD25+Treg表达率为(0.43±0.20)%;脓毒症患者细胞表达均较健康对照组明显升高(均P<0.05),其中Th17表达以严重脓毒症组最高[(3.18±0.84)%],CD4+CD25+Treg 表达以脓毒性休克组最高[(3.28±0.76)%].脓毒症患者CD4+CD25+Treg与急性生理学与慢性健康状况评分系统Ⅰ(APACHE Ⅰ)评分和血乳酸均呈正相关(r1=0.519,r2=0.451,均P=0.01).②与常规治疗组比较,血必净注射液能更有效降低脓毒症患者Th17和CD4+CD25+Treg的异常表达[Th17:(1.72±0.69)%比(2.35±0.81)%,CD4+CD25+Treg:(1.78±1.00)%比(2.30±0.85)%,均P<0.05],纠正免疫平衡紊乱,缩短住ICU时间[(4.7±2.6)d比(7.5±4.3)d,P=0.0023,使脓毒症患者28 d病死率有降低趋势(20.5%比28.0%,P>0.05).结论 脓毒症患者外周血Th17和CD4+CD25+Treg表达增加,且与病情严重程度呈正相关,提示Th17和CD4+CD25+Treg在脓毒症发生发展的免疫机制中可能起着重要作用.血必净注射液能有效降低脓毒症患者Th17和CD4+CD25+Treg的异常表达,有降低脓毒症患者病死率的趋势.

Abstract：

Objective To study the level and significance of T helper 17(Th17)and CD4+CD25+regulatory T cells(Treg)in peripheral blood of patients with sepsis and to evaluate the effects of Xuebijing of Anhui Provincial Hospital were divided into three groups:sepsis group(n=26),severe sepsis group (n=21),and septic shock group(n=17).Eighteen healthy individuals served as controls.The comparison in the expression of Th17 and CD4+CD25+Treg within groups and the correlation between their levels and group(n=25,received routine bundle treatment)and Xuebijing treatment group(n=39,received bundle treatment+Xuebijing treatment).Patients in Xuebijing treated group were given 50 ml Xuebijing injection two times per day in addition to routine bundle treatment.Seven days constituted one course of treatment.The expressions of Th17 and CD4+CD25+Treg of 64 patients on the 1 day and 7 days after treatment were detected by flow cytometry.The effects of Xuebijing injection on the patients were evaluated.Results in control group,and they were lower than that of patients with sepsis(P<0.05).The expression rate of Th17 was higher in severe sepsis group [(3.18±0.84)%]than that of other two groups(P<0.05).Moreover,The expression rate of CD4+CD25+Treg was highest [(3.28±0.76)%]in septic shock group (P<0.05),and it was positive correlated with acute physiology and chronic health evaluation Ⅰ(APACHE routine group,our study indicated that Xuebijing injection could reduce the abnormal expression of Th17[(1.72±0.69)%vs.(2.35±0.81)%,P<0.05] and CD4+CD25+Treg[(1.78±1.00)% vs.(2.30±0.85)%,P<0.05] and decrease length of stay in ICU[(4.7±2.6)days vs.(7.5±4.3)days,P=0.002].It also lowered 28-day mortality of patients with sepsis,but the difference between two groups was not significant(20.5%vs.28.0%,P>0.05).Conclusion The expression of Th17 and CD4+CD25+Treg was increased in sepsis patients and was positively correlated with severity of sepsis,suggesting that they may play an important role in pathogenesis of sepsis.Xuebijing injection could decrease the abnormal expression of Th17 and CD4+CD25+Treg and tend to decrease the fatality rate of sepsis.     

脉冲高容量血液滤过对脓毒症患者细胞免疫功能的影响

目的 探讨脉冲高容量血液滤过对脓毒症患者外周血辅助性T细胞(T helper,Th)17及CD4+ CD25+ 调节性T细胞(Treg)影响及其临床价值.方法 本研究为前瞻性对照研究,将2008年1月至2010年11月在安徽省立医院ICU住院的脓毒症患者40例(男/女=24/16),年龄25～75岁,按照疾病严重程度分为3组:脓毒症组14例(男/女=8/6);严重脓毒症组15例(男/女=9/6);脓毒症休克组11例(7/4).入选和疾病严重程度分级标准:根据1992年美国胸科医师学院(ACCP)/美国危重病医学会(SCCM)共识会议制定的脓毒症诊断标准.排除标准:患有自身免疫系统疾病、急性脑卒中、心肌梗死、病毒性肝炎、HIV感染的患者以及入院前3个月内使用过激素或免疫抑制剂的患者.其中入组5 d内未行血液净化治疗患者15例(男/女=8/7)入选为A组;5 d内行脉冲高容量血液滤过的25例患者(男/女=16/9)入选为B组.两组一般资料具有可比性.连续性血液净化以24 h为1周期,两次血滤之间间隔24 h.其中高容量血液滤过(70 mL·kg-1·h-1)治疗6～8 h后续行常规CVVH治疗16～18 h剂量(35 mL·kg-1·h-1).所有入选的40例脓毒症患者在入选当天和第5天清晨空腹抽外周血送检,行流式细胞术检测血中Th17细胞及CD4+ CD25+调节性T细胞的比例.计量资料采用t检验,配对t检验和One way ANOVA分析.小样本率的比较采用确切概率法.相关分析采用Peason相关分析.另选取本院体检中心的20例健康人为健康对照组.结果 健康对照组Th17表达率为(0.91±0.38)%,CD4+ CD25+ Treg细胞表达率为(0.39±0.23)%.40例脓毒症患者在第1天这两项指标明显升高(P<0.05):其中脓毒症组分别为(2.09±0.53)%,(1.72±0.59)%;严重脓毒症组(3.90±0.80)%,(2.72±0.22)%;脓毒性休克组(1.85±0.35)%,(3.55±0.51)%.Th17表达率,严重脓毒症组最高(P<0.05).而脓毒症休克组与脓毒症组比较差异无统计学意义(P>0.05).CD4+ CD25+ 调节性T细胞表达率则呈现:脓毒症休克组>严重脓毒症组>脓毒症组(P<0.05).B组与A组比较,脉冲高容量血液滤过能显著的下调脓毒症患者Th17[(1.87±0.43)vs.(2.48±1.05),P<0.05]和CD4+ CD25+ 调节性T细胞[(1.92±0.89)vs.(2.63±0.92),P<0.05]的表达.结论 脓毒症患者外周血Th17细胞和CD4+ CD25+调节性T细胞表达增加,提示Th17细胞和CD4+ CD25+调节性T细胞在脓毒症的免疫发病机制中可能起着重要作用.脉冲高容量血液滤过能有效的调整Th17细胞和CD4+ CD25+ 调节性T细胞的表达,可作为脓毒症免疫调节治疗的重要手段之一.

Abstract：

Objective To study the effects of pulse high volume hemofiltration (PHVHF) on the changes of Th17 cells (T helper 17 cells) and CD4 + CD25 + reguratory T cells (Treg cells) in peripheral blood of patients with sepsis and to evaluate the clinical value of this intervention. Methods The patients were included in this prospective study as per the criteria of sepsis set by America Chest Physicians College/America Society for Critic Care Medicine in 1992. The patients were excluded: ① immune system disorder, ② acute stroke, ③ myocardial infarction, ④ virus hepatitis,⑤ human immunodeficiency virus infection, ⑥ under immunosuppressive therapy. Forty patients (24 males, 16 females, aged from 25 to 75years) with sepsis in ICU were enrolled from January. 2008 to November. 2010. According to the severity of disease, the patients were divided into three groups; moderate sepsis group (n = 14, 8 males, 6 females) , severe sepsis group (n = 15, 9 males, 6 females) , and septic shock group (n = 11, 7 males, 4 females). The initially clinical data of three groups were comparable. Twenty healthy individuals served as controls. According to the mode of treatment, forty patients were also divided into two groups: conventional treatment group (group A, n= 15) in which patients were treated without PHVHF within 5 days after admission and trial group (group B, n=25) in which patients were treated with pulsed high volume hemofiltration (PHVHF) within 5 days after admission. In group B, high volume hemofiltration (70 mL · kg-1 · h-1) was given to patients for 6 ～ 8 hours, and then conventional continuous vein - vein hemofiltration (35 mL · kg-1 · h-1) for 16 ~ 18 hours. The total length of period for continuum blood scavenging was 24 hours as one cycle. The interval between two cycles of blood scavenging was 24 hours. The changes of Th17 cells and CD4+ CD25 + Treg cells of 40 patients were detected with flow cytometry on the 1st day and the 5th day after admission. The data were analyzed by using SPSS version 13. 0 software. Measurement data were analyzed with Paired-samples t-test, independent-samples t-test or one way ANOVA . Ratio of small samples was compared with fisher's exact test, and the correlation was analyzed by using Pearson correlation analysis. Results The rates of Th17 cells were( 0.91 ±0.38)%, (2.09 ±0. 53)% , (3.90 ±0. 80)% , and ( 1. 85 ±0.35)% in control, moderate sepsis, severe sepsis, and septic shock groups, respectively, while the rates of CD4+ CD25+ Treg cells were (0.39 ±0.23)%, (1. 72 ±0. 59)% , (2.72 ±0. 22)% , and (3. 55 ±0. 51)% , respectively. The rate of Thl7 cells on the 1st day was higher in severe sepsis group than that in other two groups ( P < 0. 05 ) without significant difference between septic shock and moderate sepsis groups ( P > 0. 05). Moreover , the rate of CD4+ CD25 + Treg cells was up - regulated on the 1st day in the following order from high to low: septic shock group > severe sepsis group > sepsis group (P < 0.05). The rates of Th17 cells and CD4 + CD25 + Treg cells in patients of group B decreased in greater degree than that did in patients of group A (P < 0.05 ). Conclusions The changes of Th17 cells and CD4 + CD25 + Treg cells may play an important role in pathogenesis of sepsis, and the pulsed high volume hemofiltration may be one of the effective treatments for the patients with sepsis by regulating the rates of Thl7 cells and CD4 + CD25 + Treg cells.     

Thrombotic phenotype of mice with a combined deficiency in plasminogen activator inhibitor 1 and vitronectin

OBJECTIVE: The role of vitronectin (VN) in thrombosis is not fully understood, primarily because this adhesive glycoprotein not only stabilizes plasminogen activator inhibitor 1 (PAI-1) and thus protects fibrin from premature lysis, but also because it binds to platelet integrins and may influence platelet aggregation. The absence of quantitative approaches to characterize the thrombi formed in animal models under different conditions further complicates this analysis. Methods: In this report, we describe a more comprehensive approach to assess the stability of thrombi formed in mice deficient in PAI-1 (PAI-1(-/-)), VN (VN(-/-)) or both (PAI-1(-/-)/VN(-/-)). RESULTS: We observed that all deficient mice developed unstable thrombi compared with wild type (WT) mice. Thus, only 31% of the thrombi formed in WT mice were unstable compared with 74% of PAI-1(-/-), 80% of VN(-/-), and 87% of PAI-1(-/-)/VN(-/-) mice. In this regard, the average number of emboli per WT mouse was significantly lower (0.55) compared with VN(-/-) (2.66), PAI-1(-/-) (2.1), and VN(-/-)/PAI-1(-/-) (2.35) mice. Finally, the total duration of complete vascular occlusion was higher and the rate of vascular patency was lower in the WT mice compared with the deficient mice. CONCLUSIONS: Taken together, these observations indicate that the thrombotic phenotype of mice with a combined deficiency in PAI-1 and VN does not differ significantly from the phenotype of mice with deficiencies in only PAI-1 or VN. This observation suggests that PAI-1 and VN may influence thrombus stability by regulating a common pathway.     

热休克蛋白27对内毒素血症所致心功能不全的保护作用

目的 探讨心肌特异性高表达热休克蛋白27(Hsp27)对内毒素血症所致心功能不全的影响,并初步探讨其机制.方法 所有实验均在南京医科大学第一附属医院老年医学科研究室和南京大学模式动物研究所完成.(1)心肌特异性高表达Hsp27转基因鼠(Hsp27Tg)基因型和表达鉴定:分别采用PCR法和western blot法;(2)心功能测定:HspZ7 Tg和野生型(WT)对照鼠均被腹腔注射内毒素(LPS,10 mg/kg),24 h后以超声心动图测定心功能,n=6/组;(3)死亡率:Hsp27Tg和WT鼠腹腔注射内毒素(LPS,20 mg/kg),密切监测70 h内的累积死亡率,n=37/WT,n=27/Hsp27Tg;(4)NF-kB活性测定:心肌组织样本采用凝胶迁移电泳法,细胞样本采用双报告基因法,n=4/组;(5)多组间的两两比较采用ANOVA和Seheffe检验,生存曲线分析采用log-rank检验,P＜0.05设为相差显著性界值.结果 (1)Hsp27 Tg小鼠心肌有丰富的Hsp27表达,WT则没有;(2)LPS使WT和Hsp27 Tg鼠心功能均显著下降,但与WT比较,Hsp27 Tg的心功能显著改善(P＜0.01或P＜0.05),其中EF增加27.3%.FS增加37.1%;(3)至LPS注射后70 h,WT和Hsp27Tg小鼠死亡率分别为37.84%和11.11%,与WT鼠比较,Hsp27TG生存率显著提高(P＜0.05);(4)Hsp27显著抑制LPS诱导的NF-kB激活(P＜0.01或P＜0.05).结论 Hsp27心肌特异性高表达显著抑制内毒素血症所致的心功能不全,改善生存率,其机制可能与Hsp27下调LPS诱导的NF-kB激活有关.     

Bleomycin-induced pulmonary fibrosis in fibrinogen-null mice

Mice deleted for the plasminogen activator inhibitor-1 (PAI-1) gene are relatively protected from developing pulmonary fibrosis induced by bleomycin. We hypothesized that PAI-1 deficiency reduces fibrosis by promoting plasminogen activation and accelerating the clearance of fibrin matrices that accumulate within the damaged lung. In support of this hypothesis, we found that the lungs of PAI-1(-/-) mice accumulated less fibrin after injury than wild-type mice, due in part to enhanced fibrinolytic activity. To further substantiate the importance of fibrin removal as the mechanism by which PAI-1 deficiency limited bleomycin-induced fibrosis, bleomycin was administered to mice deficient in the gene for the Aalpha-chain of fibrinogen (fib). Contrary to our expectation, fib(-/-) mice developed pulmonary fibrosis to a degree similar to fib(+/-) littermate controls, which have a plasma fibrinogen level that is 70% of that of wild-type mice. Although elimination of fibrin from the lung was not in itself protective, the beneficial effect of PAI-1 deficiency was still associated with proteolytic activity of the plasminogen activation system. In particular, inhibition of plasmin activation and/or activity by tranexamic acid reversed both the accelerated fibrin clearance and the protective effect of PAI-1 deficiency. We conclude that protection from fibrosis by PAI-1 deficiency is dependent upon increased proteolytic activity of the plasminogen activation system; however, complete removal of fibrin is not sufficient to protect the lung.     

Retroviral delivery of GAD-IgG fusion construct induces tolerance and modulates diabetes: a role for CD4+ regulatory T cells and TGF-beta?

Previous studies have demonstrated that antigen-specific tolerance could be induced by lipopolysaccharide (LPS)-stimulated B cells retrovirally transduced with an immunoglobulin-antigen (or epitope-containing peptide) fusion construct. To investigate the mechanism of this gene therapy system, we now adapted this approach to immunotherapy of spontaneous diabetes in nonobese diabetic (NOD) mice, a T-cell-mediated autoimmune disease triggered, in part, by a pathogenic response to glutamate decarboxylase (GAD) 65. We demonstrate that LPS-stimulated splenocytes, retrovirally transfected with GAD-IgG fusion construct, induce a significant antigen-specific hyporesponsiveness at both cellular and humoral levels and reduce the incidence of diabetes in female NOD mice. Parallel with disease protection, we observed a prolonged increase of the numbers of CD4+CD25+ T cells in the periphery of GAD-IgG-treated mice, compared to those treated with a control IgG vector, both in the prediabetic period and persisting even 8 months after gene therapy. This increase appeared to be induced by the repeated stimulation of the antigen in the periphery instead of a result of differentiation of T-cell precursor in the thymus. Moreover, CD4+CD25+ T cells induced by GAD-IgG fusion construct were capable of suppressing the proliferative response of CD4+CD25- T cells in vitro; and ablation of the activity of CD4+CD25+ T cells by blocking antibody against CD25 could reverse GAD-specific T-cell hyporesponsiveness. These results suggested that CD4+CD25+ T-cell subset induced in GAD-IgG-treated NOD mice represented the regulatory or suppressive CD4+CD25+ T cells (Treg) and might play an important role in the induction and maintenance of tolerance in NOD mice. Furthermore, the numbers of splenic CD4+CD62L+ regulatory T cells in GAD-IgG-treated mice during the prediabetic period and serum TGF-beta levels in 34-38-week-old GAD-IgG-protected mice were also increased, compared to control IgG-treated ones. Therefore, we propose that the induction of tolerance and the prevention of diabetes incidence in NOD female mice induced by the GAD-IgG fusion construct may require CD4+ regulatory T cells, and the possible mediation of TGF-beta.     

地塞米松联合IL-2对供鼠体内调节性T细胞选择性扩增作用及对急性移植物抗宿主病反应的抑制作用

目的 通过地塞米松(Dex)联合IL-2处理小鼠,建立体内扩增调节性T细胞(Treg细胞)的方法 .观察用该方法 处理的小鼠脾细胞移植后受鼠急性移植物抗宿主病(aGVHD)的发生与转归.方法 使用Dex和IL-2处理雄性C57BL/6N供鼠3 d后提取脾单个核细胞(MNC),用流式细胞术(FCM)分析CD4~+ CD25~+、CD25~+ FOXP3~+细胞的变化.,以以上方法 处理的雄性C57BL/6N小鼠为供者,对雌性BALB/c受鼠进行非清髓异基因淋巴细胞移植.移植后观测受鼠的生存率、生存时间、组织病理学变化,以及用PCR和FCM分析测定受鼠嵌合体等来评价aGVHD的发生.结果 经过Dex和IL-2联合处理,供鼠脾CD4~+ CD25~+ FOXP3~+ Treg细胞数量[(24.2±7.6)%]明显高于对照组[(4.0±0.8)%](P=0.01).Dex联合IL-2组供鼠Treg细胞与效应T细胞(Teff)的比值(0.43±0.15)显著高于对照组(0.14±0.01)(P=0.01).经过Dex联合IL-2处理供鼠后进行异基因淋巴细胞移植,受鼠aGVHD明显减轻,中位生存时间＞60 d,与对照组的中位生存时间12 d相比明显延长(P=0.0045).对照组出现典型的aGVHD表现,移植后2周Dex联合IL-2组和对照组的总体死亡率分别为29.4%和71.4%(P＜0.05).结论 经IL-2和糖皮质激素处理供鼠后进行主要组织相容性抗原复合物完全不相合脾淋巴细胞移植能明显减轻aGVHD,显著延长生存时间,可能与供者体内诱导扩增的Treg细胞增加有关.     

The contribution of CD4+ CD25+ T-regulatory-cells to immune suppression in sepsis

Studies have indicated that there is a development of generalized immune dysfunction after septic insult. However, the mechanisms responsible for these changes remain unclear. Recently, accumulating evidence shows that several lymphocyte subpopulations such as NKT-, CD4(+)-Th2-T-, CD8(+)-T-, gammadelta-T-, and CD4+ CD25+ T regulatory cells are capable of actively contributing to the induction of septic immune suppression. Thus, our aim was to investigate the contribution of CD4+ CD25+ cells to the immune dysfunction seen in sepsis. To study this, C57BL/6J, C57BL/6-Il6(tm1Kopf) (interleukin [IL] 6 -/-), and C57BL/6-Il10(tm1Cgn) (IL-10 -/-) mice were subjected to cecal ligation and puncture (CLP) or sham operations. Twenty-four hours later, blood was collected, and splenocytes were isolated. Phenotypic expression of CD4/CD25 (by fluorescence-activated cell sorter), cell proliferation (presented as proliferation index = [with anti-CD3]/[without anti-CD3]), and immune suppressive capacity (by in vitro add-back experiments) were assessed. The results indicate a marked elevation in CD4+ CD25+ cell levels and their proliferation index after sepsis in background mice. CD4+ CD25- cells from sham and CLP mice proliferated equally. However, coculture of CD4+ CD25- with CD4+ CD25+ cells suppressed their proliferation in both sham and CLP mice. Depletion of CD25+ cells in vivo before CLP markedly restored CD4+ CD25- proliferative capacity and Th1 cytokine release while not altering plasma proinflammatory cytokine levels. Subsequently, IL-6 -/- and IL-10 -/- mice were used to elucidate the possible mediator(s) regulating the changes seen after sepsis. Although CD4+ CD25+ cells increased after septic insult in both C57BL/6J and IL-6 -/- mice, this was not observed in IL-10 -/- mice. Similarly, in vitro proliferation studies showed that proliferation index increased in CD4+ CD25+ cells from septic C57BL/6J and IL6 -/- mice, but it remained the same in IL-10 -/- mice. Surprisingly, depletion of CD25+ cells before inducing sepsis did not alter septic mortality. Together, these findings suggest that although CD4+ CD25+ T regulatory cells induced by IL-10 seem to contribute to aspects of sepsis-induced lymphoid immune suppression, the oblation of CD25+ cells does not provide a survival advantage or disadvantage.     

Inhibition of Nur77/Nurr1 leads to inefficient clonal deletion of self- reactive T cells

The Nur77/Nurr1 family of DNA binding proteins has been reported to be required for the signal transduction of CD3/T cell receptor (TCR)- mediated apoptosis in T cell hybridomas. To determine the role of this family of DNA-binding proteins in thymic clonal deletion, transgenic (Tg) mice bearing a dominant negative mutation were produced. The transgene consisted of a truncated Nur77 (deltaNur77) gene encoding the DNA-binding domain of Nur77 ligated to a TCR-beta enhancer resulting in early expression in thymocytes. Apoptosis of CD4+CD8+ thymocytes mediated by CD3/TCR signaling was greatly inhibited in the deltaNur77 Tg mice, compared with non-Tg littermates, after treatment with anti- CD3 or anti-TCR antibody in vivo and in vitro. Clonal deletion of self- reactive T cells was investigated in deltaNur77-Db/HY TCR-alpha/beta double Tg mice. There was a five-fold increase in the total number of thymocytes expressing self-reactive Db/HY TCR-alpha/beta in the deltaNur77-TCR-alpha/beta double Tg male mice. Deficient clonal deletion of self-reactive thymocytes was demonstrated by a 10-fold increase in the CD4+CD8+ thymocytes that expressed Tg TCR-alpha/beta. There was an eightfold increase in the CD8+, Db/HY TCR-alpha/beta T cells in the lymph nodes (LN) of delta Nur77-Db/HY TCR-alpha/beta double Tg compared with Db/HY TCR-alpha/beta Tg male mice. In spite of defective clonal deletion, the T cells expressing the Tg TCR were functionally anergic. In vivo analysis revealed increased activation and apoptosis of T cells associated with increased expression of Fas and Fas ligand in LN of deltaNur77-Db/HY TCR-alpha/beta double male mice. These results indicate that inhibition of Nur77/Nurr1 DNA binding in T cells leads to inefficient thymic clonal deletion, but T cell tolerance is maintained by Fas-dependent clonal deletion in LN and spleen.     

Sepsis is characterized by the increases in percentages of circulating CD4+CD25+ regulatory T cells and plasma levels of soluble CD25

The function of immune system is to protect hosts from invading microorganisms by destroying infected cells while minimizing damage to tissues. Among immune cells, CD4(+)CD25(+) regulatory T cells (Treg cells) control immune responses by limiting infectious processes. However, it remains unclear whether Treg cells are induced in systemic inflammatory response syndrome (SIRS) or infectious SIRS (i.e. sepsis). SIRS and sepsis are associated with stressful inflammatory conditions. We therefore measured CD25(+) T cells and circulating CD4(+) T cells, along with plasma levels of CD25, interleukin (IL)-6, and IL-10, in 20 septic patients (64 +/- 11 years), 16 SIRS patients (59 +/- 16 years), and control subjects: 13 elderly (60 +/- 16 years) and 14 young volunteers (28 +/- 3 years). Septic patients (23.3 +/- 11.8%, p < 0.01) showed significantly higher percentages of CD25(+) cells among CD4(+) T cells (i.e. Treg cells) than did either young (10.6 +/- 3.7%) or elderly volunteers (11.1 +/- 3.8%). The percentages of Treg cells in septic patients were higher than those in SIRS patients (12.4 +/- 6.9%, p < 0.01). Moreover, plasma levels of soluble CD25 were significantly higher in septic patients, compared to the levels in SIRS patients or volunteers (p < 0.01). No significant difference in plasma levels of IL-6 or IL-10 was found between septic patients and SIRS patients. Thus, sepsis is associated with the increased percentages of Treg cells and elevated plasma level of soluble CD25. The elevation of these parameters might be a useful marker of infections in SIRS.     

Regulatory T cells selectively express toll-like receptors and are activated by lipopolysaccharide

Regulatory CD4 T cells (Treg) control inflammatory reactions to commensal bacteria and opportunist pathogens. Activation of Treg functions during these processes might be mediated by host-derived proinflammatory molecules or directly by bacterial products. We tested the hypothesis that engagement of germline-encoded receptors expressed by Treg participate in the triggering of their function. We report that the subset of CD4 cells known to exert regulatory functions in vivo (CD45RB(low) CD25(+)) selectively express Toll-like receptors (TLR)-4, -5, -7, and -8. Exposure of CD4(+) CD25(+) cells to the TLR-4 ligand lipopolysaccharide (LPS) induces up-regulation of several activation markers and enhances their survival/proliferation. This proliferative response does not require antigen-presenting cells and is augmented by T cell receptor triggering and interleukin 2 stimulation. Most importantly, LPS treatment increases CD4(+) CD25(+) cell suppressor efficiency by 10-fold and reveals suppressive activity in the CD4(+) CD45RB(low) CD25(-) subset that when tested ex-vivo, scores negative. Moreover, LPS-activated Treg efficiently control naive CD4 T cell-dependent wasting disease. These findings provide the first evidence that Treg respond directly to proinflammatory bacterial products, a mechanism that likely contributes to the control of inflammatory responses.     

Receptor-interacting protein kinase 3 deficiency inhibits immune cell infiltration and attenuates organ injury in sepsis

Introduction

Sepsis is defined as a systemic hyper-inflammatory immune response, with a subsequent immune-suppressive phase, which leads to multiple organ dysfunction and late lethality. Receptor-interacting protein kinase 3 (RIPK3)-dependent necrosis is implicated in driving tumor necrosis factor alpha (TNF-α)- and sepsis-induced mortality in mice. However, it is unknown if RIPK3 deficiency has any impact on immune cell trafficking, which contributes to organ damage in sepsis.

Methods

To study this, male wild-type (WT) and RIPK3-deficient (Ripk3^-/-) mice on C57BL/6 background were subjected to sham operation or cecal ligation and puncture (CLP)-induced sepsis. Blood and tissue samples were collected 20 hours post-CLP for various measurements.

Results

In our severe sepsis model, the mean survival time of Ripk3^-/- mice was significantly extended to 68 hours compared to 41 hours for WT mice. Ripk3^-/- mice had significantly decreased plasma levels of TNF-α and IL-6 and organ injury markers compared to WT mice post-CLP. In the lungs, Ripk3^-/- mice preserved better integrity of microscopic structure with reduced apoptosis, and decreased levels of IL-6, macrophage inflammatory protein (MIP)-2 and keratinocyte-derived chemokine (KC), compared to WT. In the liver, the levels of MIP-1, MIP-2 and KC were also decreased in septic Ripk3^-/- mice. Particularly, the total number of neutrophils in the lungs and liver of Ripk3^-/- mice decreased by 59.9% and 66.7%, respectively, compared to WT mice post-CLP. In addition, the number of natural killer (NK) and CD8T cells in the liver decreased by 64.8% and 53.4%, respectively, in Ripk3^-/- mice compared to WT mice post-sepsis.

Conclusions

Our data suggest that RIPK3 deficiency modestly protected from CLP-induced severe sepsis and altered the immune cell trafficking in an organ-specific manner attenuating organ injury. Thus, RIPK3 acts as a detrimental factor in contributing to the organ deterioration in sepsis.