DNAM-1在系统性红斑狼疮患者T淋巴细胞的表达研究

目的：研究DNAM－1在SLE患者外周血T淋巴细胞亚群上的表达，以阐明DNAM－1抗原在SLE患者体内活化作用以及与SLE发病的关系。方法：31例SLE患者和30例健康志愿者外周血单个核细胞，在PHA刺激培养72小时后，三色荧光标记的单克隆抗体染色，利用流式细胞仪测定T淋巴细胞亚群膜表面DNAM－1抗原的表达。同时检测SLE患者抗dsDNA抗体、C3和C4补体，疾病活动性用SLEDAI记分。结果：SLE患者CD4^＋、CD8^＋淋巴细胞上DNAM－1表达率均高于正常对照组（P＜0．01）；活动期SLE组CD4^＋、CD8^＋细胞上DNAM－1表达高于正常对照组和静止期SLE组（P＜0．01），而静止期SLE组与正常对照组无明显性差异（P＞0．05）；SLE患者CD8^＋细胞DNAM－1表达与SLEDAI、抗dsDNA抗体之间呈显著正相关（P＜0．001），与C3和C4补体水平呈明显负相关（P＜0．05），CD4^＋细胞DNAM－1表达与SLEDAI、抗dsDNA抗体、C3和C4补体之间无明显相关性（P＞0．05）。结论：SLE患者内存在T细胞亚群异常活化；活动期SLE患者T淋巴细胞亚群DNA－1表达增高；SLE患者CD8^＋细胞DNAM－1表达异常与SLEDAI、抗dsDNA抗体、C3和C4补体之间有明显相关，CD8^＋细胞活化程度可能与SLE疾病严重程度有关，故DNAM－1可能参与了SLE的免疫发病机理。     

CD226在系统性红斑狼疮患者T淋巴细胞亚群上的表达

目的：研究CD226在SLE患者外周血T淋巴细胞亚群上的表达，以阐明CD226抗原在SLE患者体内T细胞活化中作用以及与SLE发病的关系。方法：31例SLE患者和30例健康志愿者外周血单个核细胞，在体外培养72h后，三色荧光标记的单克隆抗体染色，利用流式细胞仪测定T细胞亚群细胞表面CD226抗原的表达。结果；SLE患者组的CD3^ 、CD4^ 、CD8^ T淋巴细胞上CD226^ 表达均高于正常对照组（P＜0．01）；活动期SLE组、静止期SLE组的CD3^ 、CD4^ 、CD8^ T细胞上CD226^ 表达均显著高于对照组（P＜0．01），而活动期与静止期患者之间T细胞亚群上CD226^ 表达则无显著性差异（P＞0．05）。SLE患者CD3^ 、CD4^ 、CD8^ T细胞CD226^ 抗原表达水平与SLEDAI之间无明显相关性（P＞0．05）。结论：SLE患者体内存在T细胞亚群异常活化；活动期、静止期SLET淋巴细胞CD226^＋表达均增高，CD226^ 可能参与了SLE的免疫发病。     

溃疡性结肠炎黏膜的白细胞亚群和肿瘤坏死因子—α的表达

目的：揭示溃疡性结肠炎（UC）结肠黏膜肿瘤坏死因子－α（TNFα）的表达以及与溃疡性结肠炎不同时期浸润的炎细胞免疫表型之间的关系。方法：应用抗T淋巴细胞（CD45RO）、抗B淋巴细胞（CD20）、抗巨噬细胞（CD68）表面抗原的单克隆抗体、对50例溃疡性结肠炎结肠黏膜活检标本（32例活动期,18例非活动期）及5例癌旁正常组织作为对照进行了淋巴细胞亚型和分析,应用原位杂交技术,对上述病例进行了TNFα的检测分析。结果：T淋巴细胞数量在活动期高于非活动期及对照组（P＜0．01）。而固有层内B淋巴细胞数量在三组之间差异无显著性。巨噬细胞的数量在活动期高于非活动期及正常对照组（P＜0．01）。细胞因子TNFαmRNA的表达在活动期高于非活动期。结论：TNFα在溃疡性结肠炎发病机制中起到了重要作用。其可作为炎症介质,介导结肠黏膜的病理性损伤。     

HIV-1感染者淋巴细胞活化与第二受体表达的研究

目的：了解HIV－1感染者体内淋巴细胞的活化情况及表达第二受体CCR5、CXCR4的淋巴细胞活化状态，分析这些指标与疾病严重程度的关系，探讨HIV感染的免疫基础。方法：用三色标记法流式细胞术检测24例HIV－1感染者及13例健康对照的抗凝血标本，分析活化标志物HLA－DR及第二受体CCR5、CXCR4的表达等指标。结果：HIV－1感染者CD8^ T淋巴细胞的HLA－DR表达高于健康对照（P＜0．001）；HIV－1感染者表达CCR5、CXCR4的CD8^ T淋巴细胞活化明显高于健康对照（P＜0．001）；表达CCR5CD4^ 、CD8^ T淋巴细胞与表达CXCR4相比HL－DR表达均明显增高（P＜0．001）；CD4^ 、CD8^ T淋巴细胞的活化状态与CD4百分率的变化明显关系。结论：HIV－1感染者CD8^ T淋巴细胞及表达不同第二受体的CD8^ T淋巴细胞活化程度明显增高，活化程度与疾病进程相关。     

青藤碱对红斑狼疮患者外周血T细胞的抑制作用及机制探讨

目的：观察青藤碱（SIN）对系统性红斑狼疮患者T细胞功能的影响，探讨其免疫抑制作用机理。方法：分离SLE患者的PBMCs，加入anti-CD3及不同浓度SIN，以ELISA和流式细胞仪方法分析青藤碱对T淋巴细胞活化和Th1／Th2细胞因子产生的影响。结果：SIN可抑制SLE患者外周血T细胞产生IFN-γ、IL-2和IL-4。SIN能够显著降低CD4^＋T和CD8^＋T细胞表面活化分子CD69和CD25的表达。结论：SIN可通过抑制T细胞的活化及细胞因子的分泌，发挥免疫抑制作用。     

肺炎支原体肺炎患儿细胞因子测定的探讨

目的：探讨肺炎支原体感染引起的小儿肺炎支原体肺炎与细胞因子的相关关系。方法：91例已确诊为肺炎支原体肺炎的患儿根据肺炎支原体IgM结果。分为重症组和轻症组；另选35例正常小儿为对照组。分别采取空腹静脉血，采用放射免疫分析法（RIA）测定血清TNF－α，IL－6的水平，并且对三组资料进行相关性分析。结果：重症组和轻症组血清TNF－α，IL－6水平明显高于正常对照组，差异有显著性（P＜0．01），重症组血清TNF－α，IL－6水平明显高于轻症组，差异有显著性（P＜0．05）。重症组和轻症组TNF－α，IL－6的资料进行直线相关分析，结果两组患儿TNF－α的相关系数为r=0.49，IL－6的相关系数为r=0.95；显示血清TNF－α，IL－6水平理肺炎支原体感染的严重程度呈正相关。结论：细胞因子TNF－α，IL－6参与肺炎支原体感染的全过程，并且血清TNF－α，IL－6水平的高低与机体感染严重程度呈正相关关系。     

再生障碍性贫血患者淋巴细胞表型变化

目的：研究再生障碍性贫血（AA）患者骨髓（BM）及外周血（PB）淋巴细胞及其活化相关分子的表达及临床意义。方法：采用单色和双色免疫荧光标记法，流式细胞仪分析AA患者的BM和PB中淋巴细胞膜分子的表达。结果：AA患者BM和BP中CD8^ 细胞增加，CD4／CD8比例下降，BM在CD25^ 细胞和HLA－DR^ 细胞增多，急性AA增加尤为显著（P＜0．01），BM中CD16^ 或CD56^ 细胞也明显增多（P＜0．05），双标记分析提示T细胞主要为CD8^ 细胞：急性AA患者CD8^ -CD25^ 细胞显著增多（P＜0．01），AA患者BM中淋巴细胞活化相关分子表达增多，尤其4－1BB^ ,CD95L^ 和CD40L＋细胞显著增多（P＜0．01），结论：AA患者BM中淋巴细胞活化相关膜分子增多，是AA免疫功能异常及最终导致造血功能衰竭的原因之一。     

应激障碍患者血清细胞因子含量水平变化分析

目的：探讨应激障碍人体血清细胞因子的影响及相互关系。方法：采用放射免疫测定69例应激障碍人及31例正常人血清白介素－2（IL－2）,肿瘤坏死因子－α(TNF-α）,粒细胞－淋巴细胞集落刺激因子（GM－CSF）水平,结果：应激障碍组IL－2,TNFα。GM－CSF水平低于对照组,各因子间呈正组关,正常组无相关性。结论：应激障碍患者的免疫功能被抑制。     

寻常型银屑病皮肤真表皮T细胞分类比较

目的：探讨寻常型银屑病（PV）的免疫发病机制。方法：应用链霉卵白素－过氧化物酶法（SP法），比较不同病期寻常银屑病皮肤真皮T细胞表型表达及真皮微血管E－选择素表达变化等情况。结果：（1）PV进展期皮损表皮CD4^ 细胞、CD8^ 细胞及CD45RO^ 细胞，真皮CD3^ 细胞、CD4^ 细胞、CD45RO^ 细胞及CLA^ 细胞高于静止期（P均＜0．05）；（2）静止期皮损表皮CD3^ 细胞，真皮CD3^ 细胞、CD4^ 细胞、CD8^ 细胞、CD45RO^ 细胞及CLA^ 细胞高于消退期皮损，消退期皮损表皮CD8^ 细胞高于静止期（P均＜0．05）；（3）进展期皮损周边无损害皮肤真皮CD4^ 细胞高于静止期皮周（P＜0．05）；（4）PV正常皮肤与正常皮肤与正常人皮肤的T细胞各严型间差异无显著性（P＞0．05）；（5）E－选择素表达强度与PV皮损及皮损周边无损害皮肤中浸润的CLA^ 细胞数量间的关联具有显著性（P均＜0．05）。结论：PV皮损中浸润的T细胞主要表达CLA、CD45RO，CD4^ 细胞可能在PV的发生及维持中起一定作用，但不能排除CD8^ 细胞的作用，E－选择素与CLA间的相互作用在介导T细胞的皮肤归巢中发挥重要作用。     

核因子-κB对哮喘患者T淋巴细胞血红素氧合酶-1表达的调控

探讨核因子κB（NF－κB）对哮喘患者T淋巴细胞HO－1表达的转录调节机制。分离18例急性发作期哮喘患者外周血T淋巴细胞，并分成3组培养：对照组、加入NF－κB激动剂肿瘤坏死因子－α（TNF－α）组、同时加入TNF－α和NF－κB抑制剂二硫代氨基甲醇吡咯烷（PDTC）组。培养6h后留取细胞，用逆转录聚合酶链反应（RT－PCR）检测血红素氧合酶－1（HO－1）的mRNA。培养24h后留取细胞用Western印迹法检测HO－1的表达。发现TNF－α组T淋巴细胞HO－1蛋白和mRNA表达水平显著高于对照组（q=44.48、29.94,P均＜0.01)，而同时加入TNF－α和PDTC培养组T淋巴细胞HO－1蛋白和mRNA表达水平显著低于TNF－α组（q=43.23、27.99,P均＜0.01)。可见哮喘患者T淋巴细胞HO－1基因转录可能是通过激活NF－κB进行调控。T淋巴细胞NF－κB－HO氧化激活途径可能是哮喘的发病机制之一。     

CD69 and Regulatiof the Immune Function

CD69, also known as activation inducer molecule, very early activation antigen, MLR-3 and Leu-23, is a member of the natural killer (NK) cell gene complex family of signal transducing receptors. CD69 is as a type II transmembrane glycoprotein with a C-type lectin binding domain in the extracellular portion of the molecule.

CD69 expression is induced in vitro on cells of most hematopoietic lineages, including T and B lymphocytes, NK cells, murine macrophages, neutrophils and eosinophils, while it is constitutively expressed on human monocytes, platelets and epidermal Langerhans cells.

Although a specific ligand for CD69 has not been identified, its wide cellular distribution and the induction of intracellular signals upon CD69 crosslinking suggest a role for the receptor in the biology of hematopoietic cells. Moreover, certain results indicate that CD69 may be involved in the pathogenesis of such diseases as rheumatoid arthritis, chronic inflammatory liver diseases, mild asthma, and acquired immunodeficiency syndrome.     

Crucial role for CD69 in allergic inflammatory responses: CD69-Myl9 system in the pathogenesis of airway inflammation

CD69 has been known as an early activation marker of lymphocytes; whereas, recent studies demonstrate that CD69 also has critical functions in immune responses. Early studies using human samples revealed the involvement of CD69 in various inflammatory diseases including asthma. Moreover, murine disease models using Cd69^−/− mice and/or anti-CD69 antibody (Ab) treatment have revealed crucial roles for CD69 in inflammatory responses. However, it had not been clear how the CD69 molecule contributes to the pathogenesis of inflammatory diseases. We recently elucidated a novel mechanism, in which the interaction between CD69 and its ligands, myosin light chain 9, 12a and 12b (Myl9/12) play a critical role in the recruitment of activated T cells into the inflammatory lung. In this review, we first summarize CD69 function based on its structure and then introduce the evidence for the involvement of CD69 in human diseases and murine disease models. Then, we will describe how we discovered CD69 ligands, Myl9 and Myl12, and how the CD69-Myl9 system regulates airway inflammation. Finally, we will discuss possible therapeutic usages of the blocking Ab to the CD69-Myl9 system.     

Uncoupling activation-induced modulation of CD16 and CD69 in CD56+ cells during AIDS

The immune system of HIV+ patients is chronically activated, which has been associated with a detrimental effect on both innate and acquired immunity during AIDS. We analyzed the expression and modulation of the triggering markers CD69 and CD16 in CD56+ cells from 18 asymptomatic HIV+ individuals and 8 AIDS patients, compared with 21 seronegative subjects. We observed a diminished PMA-induced CD16 downregulation in AIDS patients (p<0.01), associated with low numbers of CD4+ cells (p<0.02). Furthermore, an enhanced unstimulated expression of CD69 in asymptomatic HIV+ patients (p<0.05) was shown. AIDS patients could not efficiently upregulate PHA-dependent CD69 expression (p<0.05), which correlated with low CD4+ counts (p< 0.05). These abnormalities in CD16 and CD69 modulation were recorded in patients under highly active antiretroviral therapy (HAART). Our results demonstrate an altered modulation of two functionally relevant receptors in CD56+ cells from AIDS patients, contributing to our understanding of the immunopathogeny of NK cell dysfunction during disease progression.     

CD69: from activation marker to metabolic gatekeeper

CD69 is a membrane‐bound, type II C‐lectin receptor. It is a classical early marker of lymphocyte activation due to its rapid appearance on the surface of the plasma membrane after stimulation. CD69 is expressed by several subsets of tissue resident immune cells, including resident memory T (TRM) cells and gamma delta (γδ) T cells, and is therefore considered a marker of tissue retention. Recent evidence has revealed that CD69 regulates some specific functions of selected T‐cell subsets, determining the migration‐retention ratio as well as the acquisition of effector or regulatory phenotypes. Specifically, CD69 regulates the differentiation of regulatory T (Treg) cells as well as the secretion of IFN‐γ, IL‐17, and IL‐22. The identification of putative CD69 ligands, such as Galectin‐1 (Gal‐1), suggests that CD69‐induced signaling can be regulated not only during cognate contacts between T cells and antigen‐presenting cells in lymphoid organs, but also in the periphery, where cytokines and other metabolites control the final outcome of the immune response. Here, we will discuss new aspects of the molecular signaling mediated by CD69 and its involvement in the metabolic reprogramming regulating TH‐effector lineages.     

The role of CD69 in acute neutrophil-mediated inflammation

The leukocyte activation marker CD69 functions as a negative regulator of the immune response, both in NK-dependent tumor rejection and in the inflammation associated with lymphocyte-dependent collagen-induced arthritis. In contrast, it has been reported that CD69-deficient mice are refractory to the neutrophil-dependent acute inflammatory response associated with anti-type II collagen antibody-induced arthritis (CAIA), suggesting a positive regulatory role for CD69 in neutrophil function during arthritis induction. To clarify this discrepancy, the CAIA response was independently analyzed in our CD69-deficient mice. In these experiments, the inflammatory response was unaffected by CD69 deficiency. Additionally, the in vivo down-regulation of CD69 expression by treatment of wild-type mice with the anti-CD69 mAb 2.2, which mimics the CD69-deficient phenotype, did not affect the course of arthritis in this model. Moreover, down-regulation of CD69 expression increased expression in arthritic joints of key inflammatory mediators, including IL-1beta, IL-6 and the chemokine MCP-1. Neutrophil accumulation in zymosan-treated air pouches and in thioglycolate-treated peritoneal cavities was also unaffected in CD69-deficient mice. In addition, CD69 expression was absent in activated neutrophils. Taken together, these results rule out a significant stimulatory role for CD69 in acute inflammatory responses mediated by neutrophils.     

A potential role for CD69 in thymocyte emigration

The early activation marker, CD69, is transiently expressed on activated mature T cells and on thymocytes that are undergoing positive or negative selection in the thymus. CD69 is a member of the NK gene complex family of C-type lectin-like signaling receptors; however, its function is unknown. In this report, we describe the characterization of mice that constitutively express high levels of surface CD69 on immature and mature T cells throughout development. Constitutive surface expression of CD69 did not affect T cell maturation, signaling through the TCR or thymocyte selection. However, phenotypically and functionally mature thymocytes accumulated in the medulla of CD69 transgenic mice and failed to be exported from the thymus. The retention of mature thymocytes correlated with transgene dose and CD69 surface levels. These results identify a potential role for CD69 in controlling thymocyte export, and suggest that the transient expression of CD69 on thymocytes and T cells may function to regulate thymocyte and T cell trafficking.     

In vivo expression of CD69 on lung eosinophils in eosinophilic pneumonia: CD69 as a possible activation marker for eosinophils.

The antigen, CD69, has been demonstrated to be expressed on activated T cells and natural killer cells. There have been no studies concerning the expression of CD69 on eosinophils. In this article, we demonstrate that lung eosinophils obtained from the bronchoalveolar lavage fluid of patients with eosinophilic pneumonia expressed significant levels of CD69, whereas peripheral blood (PB) eosinophils did not express CD69. We also activated PB eosinophils in vitro using phorbol myristate acetate and cytokines to determine whether CD69 was expressed. PB eosinophils expressed CD69 after short-term culture with phorbol myristate acetate and eosinophil hemopoietic cytokines (interleukin-3, granulocyte-macrophage--colony-stimulating factor, and interleukin-5). These findings suggest that CD69 may be a useful marker for activated eosinophils at inflammatory sites.     

Aberrant expression of CD95 and CD69 molecules among CD56 cells in women with endometriosis

PROBLEM: Activated lymphocytes can be eliminated by Fas/Fas ligand (FasL)-induced cell death. Endometrial cells express FasL. The aim of our study was to determine the expression of CD56+ cells (natural killer and natural killer T cells) Fas antigen CD95 and the early activation molecule CD69 in the peritoneal fluid of women with endometriosis. METHOD: Two-colour flow cytometry was used. RESULTS: In the early stages of endometriosis, more CD56+ cells expressed CD69 and CD95 molecules when compared with the control group. However, in case of severe endometriosis the percentage of CD95+CD56+ cells in peritoneal fluid was similar to that of the control group, but the expression of CD69 molecules remained high. CONCLUSION: Because of Fas/FasL mechanisms, in the initial stages of endometriosis the activated peritoneal fluid CD56+ cells can be intensively eliminated, thus providing conditions for the survival of ectopic endometrial cells and the development of the disease.     

CD 69 expression during selection and maturation of CD4+8+ thymocytes

We have analyzed the inducibility of protein kinase C (PKC)-dependent expression of CD 69 molecules in T cell receptor (TCR) transgenic thymocytes developing in the presence or absence of selecting, class I major histocompatibility complex (MHC) molecules. Small CD4⁺8⁺ thymocytes developing in the absence of selecting MHC molecules could not be induced to express CD 69 by TCR cross-linking even after spontaneous in vitro up-regulation of their TCR level which resulted in enhanced Ca⁺⁺ flux. In contrast, a small proportion of CD4⁺8⁺TCR^low and most TCR^high (CD4⁺8⁺ and CD4⁺8⁺) thymocytes developing in the presence of selecting MHC ligands could be induced to express CD 69 upon TCR cross-linking. Unlike the anti-TCR antibody, phorbol 12-myristate 13-acetate - a direct activator of PKC - induced the expression of CD 69 on all thymocytes. These results suggest that positive selection of CD4⁺8⁺ thymocytes results in coupling of TCR-mediated signals to the CD 69 expression pathway. In vitro analysis of thymocytes before and after positive selection suggests that (1) positive selection does not immediately result in resistance to deletion and (2) that sustained TCR ligation is needed to promote maturation of positively selected CD4⁺8⁺ thymocytes resulting in gradual loss of the sensitivity to deletion and acquisition of the ability to proliferate in response to TCR-mediated signals.