【作者】 边琪；

【导师】 黎磊石； 刘志红；

【作者基本信息】 第二军医大学 ， 内科学， 2008， 博士

【摘要】 系统性红斑狼疮(systemic lupus erythematosus,SLE)是一种典型的伴有多器官损害的自身免疫性疾病,免疫调节功能的紊乱在SLE的发病中起着重要的作用。许多研究表明,SLE患者存在T、B细胞功能紊乱,而调节性T细胞(regulatory T cells,Treg)免疫抑制功能的减退进一步触发和加重了疾病的发生、发展。狼疮性肾炎(LN)是SLE最常见的脏器损害,也是影响SLE患者远期预后的主要因素。LN临床、病理表现多样,特别是表现为弥漫增殖性病变的IV型LN和表现为膜性病变的V型LN,其临床特征和病理表现相差甚大,提示其存在不同的免疫发病机制。两者之间外周血淋巴细胞亚群和Treg的分布是否存在差异目前尚未见报道。在LN的发病中肾组织局部免疫反应的发生也具有重要作用,系膜细胞、内皮细胞、足细胞及肾小管上皮细胞等肾实质细胞都参与了自身免疫反应,而肾组织淋巴细胞及巨噬细胞的浸润在LN病变形成过程发挥了不同的作用。IV型和V型LN肾组织淋巴细胞特别是调节性T细胞和巨噬细胞浸润与临床病理特点的联系以及与外周血淋巴细胞亚群的关系目前仍无相关研究。本研究分析了IV型和V型LN患者外周血淋巴细胞亚群的变化及肾组织淋巴细胞亚群和巨噬细胞的浸润情况,明确了淋巴细胞亚群、特别是调节性T细胞在IV型和V型LN中的分布特点以及与临床病理特征之间的可能联系,试图为进一步寻找不同病理类型LN免疫发病机制的差异提供一定的线索。伴有弥漫增生性病变的LN约占全部LN的60%,其病情重,治疗反应和预后较差。淋巴细胞亚群是反映SLE病情活动、监测病情变化的重要指标。既往多数研究显示,大多数活动性LN中存在CD4~+/CD8~+T细胞比例(RT)的倒置,病情缓解时RT倒置可恢复,并发现伴有弥漫增殖性病变的LN总体RT倒置更为明显。但是,我们的研究同时发现,临床中存在一部分RT比值正常甚至高于正常的活动性LN病例,其临床和病理特征是否与RT比值倒置的LN有差异,治疗及预后是否有其特殊性,目前尚不清楚。因此,我们对上述合并明显RT异常伴有弥漫增殖性病变的活动性LN患者进行研究,比较不同RT比值组临床、病理特点和预后的关系。本研究由三部分组成第一部分:IV型和V型狼疮性肾炎患者外周血淋巴细胞亚群的变化与临床意义目的:观察IV型和V型活动性狼疮性肾炎(LN)患者外周血淋巴细胞亚群及CD4~+CD25~+Foxp3~+调节性T细胞(Treg)的变化,并分析其在LN不同病理类型中的差异,以及与狼疮疾病活动性指数(SLE-DAI)的关系。方法:入选临床符合系统性红斑狼疮,根据2003年ISN/RPS病理分型标准经肾活检确诊为IV型和V型病例52例(男性11例,女性41例,年龄18～55岁),其中IV型32例,V型20例,所有IV型病例SLE-DAI≥12分,IV型病例SLE-DAI≥5分),所有病例均有完整的临床、病理及实验室检查记录。另入选20例年龄、性别匹配的志愿者作为健康对照。流式细胞仪测定外周血淋巴细胞亚群(CD3~+、CD4~+、CD8~+、CD20~+细胞)和Treg的比例与计数,计算CD4~+/CD8~+T细胞比值。比较活动性LN患者外周血CD4~+CD25~+Foxp3~+调节性T细胞和淋巴细胞亚群与正常对照的差异,并进一步分析IV型和V型活动性LN患者淋巴细胞亚群和Treg的变化特点。结果:1、活动性IV型、V型LN患者与正常人比较外周血CD4~+细胞比例、计数和CD4~+/CD8~+细胞比值及Treg细胞显著降低,CD8~+细胞及CD20~+细胞明显升高(P＜0.01)。2、IV型与V型相比:(1)外周血CD4~+细胞比例(25.9%±6.86%vs 31.0%±7.59%,P＜0.05)、计数(288±173个/ul vs 420±165个/ul,P＜0.01)和CD4+/CD8+比例降低更明显(0.74±0.31 vs 1.06±0.57,P＜0.05):(2)外周血CD20~+细胞比例无明显差异,但V型CD20~+细胞计数升高较IV型明显(185±136个/ul vs 268±179个/ul,P＜0.05):(3)Treg细胞比例(0.82%±0.4%vs 1.31%±0.7%,P=0.02)和计数(8.19±4.26个/ulvs 17.5±10.0个/ul,P＜0.01)IV型降低更明显。3、CD4~+细胞比例和计数与AI呈负相关(r分别为-0.281和-0.380,P＜0.05),CD4~+/CD8~+细胞比值与SLEDAI呈负相关(r=-0.307,P＜0.05)。Treg细胞比例和计数与SLEDAI呈显著负相关,相关系数分别为-0.411(P＜0.01)和-0.480(P＜0.01),与肾组织AI呈负相关(r为-0.325和-0.473,P＜0.01)。结论:1、活动性LN患者存在外周血淋巴细胞亚群的分布异常和Treg细胞的减少。2、IV型LN患者外周血CD4~+细胞水平、CD4~+/CD8~+细胞比例和Treg细胞水平较V型明显降低,提示淋巴细胞亚群的分布异常和Treg细胞减少的程度不同是不同病理类型LN发病机制的可能原因之一。第二部分:IV型和V型狼疮性肾炎患者肾组织淋巴细胞亚群及CD68阳性细胞的浸润与意义目的:观察IV型和V型狼疮性肾炎(LN)患者CD4~+、CD8~+、CD20~+淋巴细胞和CD68~+细胞以及Foxp3~+调节性T细胞在肾组织的浸润情况,分析其在LN不同病理类型中的差异以及与临床病理特征的联系。方法:入选临床符合系统性红斑狼疮,根据2003年ISN/RPS病理分型标准经肾活检确诊为IV型和V型病例41例(男性10例,女性31例,年龄18～55岁),其中IV型30例,V型11例,所有IV型病例SLE-DAI≥12分,V型病例SLE-DAI≥5分),所有病例均选自第一部分研究。免疫组化染色检测LN肾组织CD4、CD8、CD20和CD68阳性细胞的浸润情况,取10例移植肾供肾活检标本作为正常肾组织对照。同时入选20例LN患者(IV型和V型各10例)行调节性T细胞转录因子Foxp3和CD4或CD8双标免疫组化染色。分析IV型和V型LN肾组织包括调节性T细胞在内的淋巴细胞亚群与单核-巨噬细胞浸润的异同以及与临床病理特征的关系。结果:1、活动性LN肾组织CD4~+、CD8~+、CD20~+和CD68~+细胞浸润数量均明显高于正常对照,但外周血淋巴细胞亚群的水平与肾组织浸润情况并不平行。2、IV型LN肾小管-间质浸润的CD4~+、CD8~+、CD20~+和CD68~+细胞均高于V型病例,其中肾小管-间质浸润的CD20~+细胞IV型明显高于V型(119.3±89.7 vs36.0±36.0,P＜0.01),肾小球内浸润的CD68~+细胞也显著高于V型(12.4±8.8 vs1.7±2.31,P＜0.01)。3、肾间质CD20~+细胞数和肾小球内CD68~+细胞数与SLE-DAI呈正相关(r分别为0.507和0.393):肾间质CD20~+细胞浸润程度与AdsDNA的阳性率相关。4、肾间质CD20~+细胞数和肾小球内CD68~+细胞数与肾组织AI呈正相关(r分别为0.657和0.444):CD68~+细胞球内浸润的程度与肾小球固有细胞增殖明显相关(r=0.624,P＜0.001)。5、正常肾组织不表达Foxp3~+Treg细胞。绝大多数Foxp3表达于CD4~+细胞,偶见CD8~+Foxp3~+T细胞。LN中Foxp3~+Treg细胞主要位于肾间质,IV型LN Foxp3~+Treg细胞明显高于V型(27.6±18.0 vs 2.8±5.0,P＜0.01)。结论:1、LN肾组织淋巴细胞和巨噬细胞浸润较正常对照明显增加。2、IV型LN肾组织淋巴细胞特别是调节性T细胞和巨噬细胞浸润较V型明显升高,肾间质B细胞浸润和肾小球巨噬细胞浸润与SLE-DAI和AI呈正相关,巨噬细胞球内浸润与肾小球固有细胞增殖相关,提示不同的淋巴细胞亚群及单核巨噬细胞浸润参与了不同病理类型LN的发病。第三部分:T淋巴细胞亚群异常类型与弥漫增生型狼疮性肾炎临床病理联系和预后分析目的:探讨不同CD4~+与CD8~+T淋巴细胞比值异常与弥漫增生性狼疮性肾炎(LN)其临床免疫和病理特征的联系以及与预后的关系。方法:70例(男性5例,女性65例,年龄16～54岁)活动性狼疮性肾炎并行肾活检病理检查。肾活检病理类型为IV型,包括IVG、IVS、IV型伴V型(2003年ISN/RPS病理分型标准)。流式细胞仪测定外周血淋巴细胞亚群(CD3~+、CD4~+、CD8~+、CD20~+细胞),计算CD4~+/CD8~+细胞比值(RT比值)。根据RT比值分为低RT组(RT≤0.5,n=45)和高RT组(RT≥1.5,n=25)。采用免疫组化检测肾组织CD4、CD8、CD20和CD68阳性细胞浸润情况。比较两组临床和免疫学特征、肾活检病理改变的特点和肾组织淋巴细胞亚群浸润的异同以及和预后的关系。结果:1、低RT组CD4~+细胞显著降低、CD8~+细胞显著升高;高RT组CD4~+细胞轻度升高、CD8~+细胞降低,而CD20~+细胞明显升高。2、与高RT组比较,低RT组起病年龄明显小(26.5±10.4岁vs 32.7±9.4岁),SLEDAI高(17.6±6.0 vs 14.5±4.1);发热(51.1%vs 24.0%)、面部红斑(64.4%vs 32%)、皮肤血管炎(22.2%vs 0%)、肉眼血尿发生率较高(26.7%vs 8.0%),均有统计学意义(P＜0.05);外周血IL-10水平显著升高(15.5±11.8 vs 7.0±6.1,P＜0.01)。3、肾脏病理方面低RT组IVS型比例明显升高(11.0%vs 0%),AI升高,存在新月体、节段袢坏死和袢内血栓的比例明显高于高RT组(P＜0.05),合并狼疮性肾血管病变的发生率也升高(20%vs 4%,P＜0.05)。4、低RT组肾组织CD4、CD8、CD20、CD68阳性细胞与高RT组无显著差异。5、对部分随访时间超过6月或12月病例的研究显示,随访6月治疗有效率低RT组为70.2%、高RT组为70%,随访12月治疗有效率低RT组为86.2%、高RT组为90%,两组近期疗效无明显差异。对其中IVG型病例进一步随访分析显示,MMF治疗6个月时RT＜0.5患者有效率为66.7%、RT＞1.5患者有效率为80%,CTX治疗6个月时RT＜0.5患者有效率为55.5%、RT＞1.5患者有效率为42.9%,MMF治疗缓解率高于CTX,对于RT＞1.5的IVG型患者采用MMF诱导治疗有效率增加最明显。33例治疗有效的病例和随访初相比,低RT组RT值上升,而高RT组RT值则降低,均向正常范围靠近。结论:不同CD4、CD8 T淋巴细胞平衡失调造成IV型LN不同的肾损害特征。在CD8占绝对优势的IV型LN以炎症表现为主和血管炎改变突出,CD8升高与血管病变相关,表明CD8细胞参与病变发生。CD4占优势的IV型LN更多伴V型病变,且CD20明显升高,表明辅助性T细胞和B细胞参与了膜性病变的产生。提示RT比值异常以不同的机制参与LN的发生、发展,并影响免疫抑制治疗方案的选择。更多还原

【Abstract】 Part OneSignificance of lymphocyte subsets’ changes in peripheral blood of patients with classⅣand classⅤlupus nephritisObjective:To observe the significance of lymphocyte subsets’ changes in peripheral blood of patients with classⅣand classⅤlupus nephritis(LN).Methodology:Fifty-two patients,41 female and 11 male with age from 18 to 55 years old,diagnosed systemic lupus erythematosus(SLE) and classⅣLN(n=32) or ClassⅤLN(n=20) (ISN/RPS2003 classificatioin criteria),were enrolled in the study.Another twenty age-and sex-matched healthy volunteers served as healthy controls(HC).The proportions of peripheral blood lymphocyte subsets(CD4~+,CD8~+,CD20~+ cells) and CD4~+CD25~+Foxp3~+ regulatory T cells(Treg cells) were determined in classⅣand classⅤLN patients as compared to HC by flow cytometry.Correlation analysis of the percentages and absolute cell numbers of Treg cells with clinical disease activity and pathology active index(AI) were performed.Resluts:1.Comparing with HC,the proportion and absolute cell numbers of CD4~+ cells,the ratio of CD4~+/CD8~+ T cells and the level of Treg cells in peripheral blood of active LN patients were decreased significantly(P＜0.01),meanwhile the proportion of CD8~+ cells and CD20~+ cells raised apparently in active LN patients(P＜0.01).2.Comparing with classⅤ,(1) the proportion(25.9%±6.86%vs 31.0%±7.59%, P＜0.05) and absolute cell numbers(288±173cells/ul vs 420±165cells/ul,P＜0.01) of CD4~+ cells and CD4~+/CD8~+ ratio(0.74±0.31 vs 1.06±0.57,P＜0.05) decreased more significantly in classⅣ.(2) No significant difference in the ratio of CD20~+ cells could be found between classⅣand classⅤ,but the absolute cell numbers elevated more significantly in class Ⅴ(185+136cells/ul vs 268±179cells/ul,P＜0.05).(3) Patients with classⅣLN also had statistically lower levels of Treg cells than did patients with classⅤwhen expressed as either percentages of peripheral blood lymphocyte cells(0.82±0.40%vs 1.25±0.70%, P＜0.05) or absolute cell numbers(8.19±4.26cells/ul vs 17.5±10.0 cells/ul,P＜0.05).3.Pearson correlation analysis showed the proportion and the number of CD4~+ cells were correlated with AI(r=-0.281,P＜0.05 and r=-0.380,P＜0.01 respectively).With correlation analysis the proportion and the absolute cell numbers of Treg cells were correlated with SLE-DAI(r=-0.376,P＜0.01 and r=-0.504,P＜0.01 respectively ) and AI(r=-0.278 and r=-0.489,P＜0.01 respectively).Conclusion:The level of CD4+ celIs,CD4+/CD8+ ratio and Treg cells decrease more significantly in classⅣLN patients than did in classⅤLN patients.These results suggest the difference of the immunology pathogenesis of classⅣand classⅤLN.Part twoAlterations of infiltrating lymphocyte subsets and CD68~+ cells in renal tissue of patients with ClassⅣand ClassⅤlupus nephritisObjective:To observe the status of lymphocyte subsets and CD68+ cells infiltrating in renal tissues of patients with classⅣand classⅤlupus nephritis(LN).And to analysis the difference and correlation with clinico-pathologic characteristics.Methodology:Forty-one patients(female 31 and male 10 with age from 18 to 55 years old) suffered from active systemic lupus erythematosus(SLE) and renal biopsy proved classⅣLN(n=30) or classⅤLN(n=11)(ISN/RPS2003 classificatioin criteria) were enrolled in the study.All cases were selected from the patients enrolled in the study of part one. CD4~+,CD8~+,CD20~+ and CD68~+ cells were detected in renal tissues by immunohistochemisty.Meanwhile,twenty patients’ renal biopsies(including LN-Ⅳand LN-V) with double immunohistochemical marker for the Treg transcription factor Foxp3,combined with a second marker for CD4 or CD8.Comparing the differences of infiltrating cells in classⅣand classⅤLN,the correlation with clinicopathology was evaluated as well.Resluts:1.Comparing with HC,the numbers of CD4~+,CD8~+,CD20~+ and CD68~+ cells in renal tissues of active LN patients were increased significantly(P＜0.01),but the level of lymphocyte subsets in peripheral blood and in diseased renal tissue was not parallel.2.Meanwhile,infiltrating CD4~+,CD8~+,CD20~+ and CD68~+ cells in renal interstitial increased more apparently in classⅣthan in classⅤ.Especially infiltrating CD68~+ cells in glomerular(12.2±8.7 vs 1.7±2.13,P＜0.01) and CD20+ cells in renal interstitial(119.3±89.7 vs 36.0±36.0,P＜0.01) increased more significantly in classⅣ.3.Infiltrating CD20~+ cells in interstitial and the degree of CD68~+ cells in glomerular were correlated with SLE-DAI(r=0.507 and 0.393,respectively).The level of infiltrating CD20~+ cells in interstitial was correlated with AdsDNA.4.Infiltrating CD20~+ cells in interstitial and the degree of CD68~+ cells in glomerular were correlated with AI(r=0.657 and 0.444,respectively).The degree of CD68~+ cells infiltrating in glomerular was correlated with proliferation of intrinsic cells in glomerular(r=0.624,P＜0.001).5.No Foxp3~+ Treg cells were found in normal renal tissue.Most Foxp3~+ cells were CD4~+.Foxp3~+ Treg cells were located in renal interstitium.Foxp3~+ Treg cells increased more significantly in classⅣthan in classⅤ(27.6±18.0 vs 2.8±5.0,P＜0.01).Conclusion:1.Infiltrating CD4~+,CD8~+,CD20~+ and CD68~+ cells in renal tissue of LN increased significantly.2.The number of infiltrating lymphocyte subsets,especially Treg cells,and macrophages in renal tissue of classⅣLN was higher than that did in classⅤLN.3.The study suggested the difference of lymphocyte subsets and macrophages infiltrating in renal tissue might participate the pathogenesis in classⅣand classⅤLN.Part three A study on clinico-pathology correlation in different types of T lymophocyte subsets’ abnormality of lupus nephritis with diffuse proliferative lesionsObjective:To analyze the clinico-pathology characteristics and prognosis in different changes of CD4~+/CD8~+ T cells ratio of lupus nephritis(LN) with diffuse proliferative lesions.Methodology:Seventy female patients(age from 16 to 54 years old) suffered from active LN and renal biopsy proved classⅣ,including IVG,IVS and classⅤ+Ⅳ(ISN/RPS2003 classificatioin criteria),were enrolled in the study.Proportions of peripheral blood lymphocyte subsets(CD4~+,CD8~+,CD20~+ cells) were determined by flow cytometry. According to CD4~+/CD8~+ ratio(RT) these patients were divided to low RT group(RT≤0.5,n=45)and high RT group(RT≥1.5,n=25).CD4~+,CD8~+,CD20~+ and CD68~+ cells were detected in renal tissues by immunohistochemisty.The clinico-pathology characteristics and prognosis were compared between the two groups.Resluts:1.In low RT group,the level of CD4~+ T cells decreased significantly,CD8~+ T cells increased apparently.However,in high RT group,the level of CD4~+ T cells raised slightly, CD8~+ T cells decreased apparently and CD20~+ B cells increased significantly.2.Comparing to high RT group,in low RT group onset age of SLE was younger(26.5±10.4y vs 32.7±9.4y,P＜0.05),SLEDAI(17.2±6.4 vs 14.8±4.4,P=0.09) and the prevalence of fever(51.1%vs 24.0%),malar rash(64.4%vs 32%),skin vasculitis(22.2% vs 0%) and gorss hematuria(26.7%vs 8.0%) were higher;and the level of serum IL-10 was increased significantly(15.5±11.8 vs 7.0±6.1,P＜0.01).3.Renal biopsy showed in low RT group the percentage of class IVS was higher,meanwhile,the proportion of patiens with renal vascular lesion(20%vs 4%,P＜0.05) and AI(9.16±4.34 vs 7.17±3.10,P＜0.05) were increased more signifcantly than in high RT group.4.There were no significance differences of infiltrating CD4~+,CD8~+,CD20~+ and CD68~+ cells in renal tissues of the two groups. 5.Follow-up study showed there were no difference in complete or partial remission rate at the 6th and 12th months in different RT groups.To patients with class IVG LN,in both low RT group and high RT group MMF induction therapy was more effective than CTX induction therapy,especially for the patients in high RT group.Accompany with disease remission,the abnormal RT was ameliorated.Conclusion:The imbalance of CD4~+,CD8~+ T lymphocytes induce the different characteristics of renal lesions in LN-Ⅳ.ClassⅣwith CD8 predominance manifests inflammation and vasculitis,and the increase of CD8~+ T cells is associated with vascular lesion.ClassⅣwith CD4 predominance frequently accompany with membranous lesion,and the level of CD20~+ cells increase significantly at same time,that is suggested Th cells and B cells participate the development of membranous lesions.Our study suggests distinct abnormal RT probably participate the onset and development of LN through different pathophysiology,and these might be associated with the selection of immunotherapy protocols.更多还原