中性粒细胞自发性凋亡的研究概况

中性粒细胞是血细胞中寿命最短的终末分化细胞，也是机体最主要的炎症细胞。同其它的血细胞和组织细胞在凋亡的发生机制上有所不同，中性粒细胞从分化成熟开始，就启动它的自发性凋亡程序(spontaneous apoptosis program)。机体的炎症反应是一把双刃剑，在清除异物的同时也带来损伤。中性粒细胞这一最主要的炎症细胞则可以通过自发性凋亡的机制调控和减轻炎症损伤，构成炎症反应的主要收敛机制之一。中性粒细胞的自发性凋亡是一个受自身多基因调控和众多细胞因子及其它多种因素调节的复杂过程。     

ONO-AE-248诱发的中性粒细胞非凋亡性程序化死亡相关蛋白的初步研究

中性粒细胞(Polymorphonuclear neutrophil,PMN)是机体重要的炎症细胞,它对炎症的发生、发展及转归起着关键的作用.其中,中性粒细胞自发性凋亡是维持机体自身稳定的一个重要因素,炎症部位中性粒细胞的结局将关系到许多疾病的发生、发展和转归.     

ASC在ONO-AE-248诱导的中性粒细胞非凋亡、非坏死性死亡中的表达

目的:探讨凋亡相关斑点样蛋白(Apoptosis-associated speck-like protein,ASC)在ONO-AE-248所诱发的中性粒细胞非凋亡、非坏死性死亡中的作用及意义.方法:TUNEL法标记凋亡细胞,结合激光共聚焦扫描显微镜观察细胞核形态以及DNA片段化发生的情况;Western blot法检测不同药物刺激组(LPS延迟凋亡组、TNF-α促进凋亡组、ONO-AE-248刺激组以及自发性凋亡组)的ASC蛋白的表达差异性.结果:TUNEL法检测ONO-AE-248培养12小时后的中性粒细胞,未见TUNEL阳性细胞.Western blot结果显示ONO-AE-248刺激后中性粒细胞ASC蛋白的表达一直处于下调状态.结论:ONO-AE-248诱导人中性粒细胞死亡过程中细胞核DNA断裂方式明显不同于自发性凋亡组,核内染色体可能只发生DNA较大片段的断裂,而没有发生小片段化.ONO-AE-248引起的ASC表达的下调可能是这种新型细胞死亡方式区别于自发性凋亡的重要差异点.     

ONO-AE-248诱导中性粒细胞非凋亡性程序化死亡线粒体形态结构的变化

目的：探讨线粒体形态结构的变化在ONO-AE-248诱导的中性粒细胞非凋亡性程序化死亡中的作用及意义。方法：Ficoll法分离健康志愿者外周静脉血中性粒细胞，与ONO-AE-248共同培养，透射电子显微镜观察线粒体形态结构的改变；流式细胞术检测线粒体膜势能的变化。结果：ONO-AE-248刺激6小时，中性粒细胞线粒体显著肿胀，嵴断裂，数目增多；ONO-AE-248早期迅速导致中性粒细胞线粒体膜势能的溃散。结论：线粒体变化是ONO-AE-248诱导中性粒细胞非凋亡性程序化死亡区别于凋亡的早期形态学特征事件之一。线粒体可能在ONO-AE-248诱导的中性粒细胞非凋亡性程序化细胞死亡中扮演更为关键性的角色。     

蛋白激酶C在中性粒细胞凋亡信号转导中的作用

蛋白激酶C(PKC)是一类ca2+、磷脂依赖性的丝氨酸/苏氨酸蛋白激酶,由12种具有不同生物学特性的同工酶组成.PKC通过催化多种蛋白质上Ser/Thr的磷酸化,在细胞下游信号转导和调节细胞功能中发挥重要的作用.中性粒细胞(PMN)是参与机体固有免疫的主要细胞,一旦分化成熟便启动它的自发性凋亡程序,从而维持机体的内环境稳定和促成炎症反应的无损伤性收敛.PMN凋亡的调控是一个受多基因、多因子、多条信号转导通路独立或交联作用的复杂网络.作为细胞内信号转导重要递质的PKC,在调控PMN的自发性凋亡进程中起着重要作用.     

抗类风湿病药金诺芬对中性粒细胞自发性凋亡的影响

目的 研究抗类风湿病药金诺芬(AF)对中性粒细胞自发性凋亡的影响。方法 运用流式细胞技术和MTS细胞活性测定技术对不同浓度的金诺芬影响下的中性粒细胞活性及其凋亡进行观察，并用低分子质量DNA“梯形条带”(DNA ladder)检测技术对其凋亡进行鉴定。结果 低浓度金诺芬(1μmol／L)可通过延迟中性粒细胞的自发性凋亡而增加其生命期限；但当金诺芬浓度大于5μmol／L时，中性粒细胞的坏死则明显增加，因而缩短了其生命期限。结论 金诺芬临床治疗量时的血清浓度一般在0．7μmol／L左右，在此浓度下，中性粒细胞通过延迟自发性凋亡来延长其功能性生命期限，所以金诺芬对类风湿关节炎患者的抗炎、抗风湿作用不可能通过直接清除炎症局部的中性粒细胞来完成；金诺芬的抗炎、抗风湿作用可能存在更为复杂的作用机制。     

全身炎性反应综合征中性粒细胞线粒体细胞色素c的研究进展

全身炎性反应综合征(systemic inflammation response syndrome,SIRS)是多种疾病发展至多器官功能障碍综合征(multiple organs dysfunction syndrome,MODS)的前期表现.中性粒细胞,又名多形核白细胞(polymorphonuclear neutrophil,PMN)是重要的炎症效应细胞,其凋亡延迟或障碍进而导致炎症反应持续发展和加剧是SIRS的主要病理生理过程.线粒体细胞色素c(cytochrome c,Cyt-c)是重要的凋亡蛋白,通过内源性和外源性等多种凋亡途径参与PMN凋亡.深入探讨SIRS时PMN凋亡异常的分子机制,研究线粒体Cyt-c在PMN凋亡中的作用,寻找诱导PMN适时、适度凋亡的有效方法,对于限制组织损伤、控制炎症反应、减轻SIRS有重要意义.     

ONO-AE-248诱发线粒体膜势能降低导致中性粒细胞非凋亡性程序化细胞死亡

目的研究线粒体在ONO—AE-248诱导中性粒细胞非凋亡性程序化细胞死亡中形态结构及功能的变化情况，为进一步确立这一新的死亡形式和找寻其独特的死亡信号转导途径提供实验依据。方法体外新鲜分离人外周血中性粒细胞与ONO—AE-248共同培养，流式细胞术检测其线粒体膜势能的变化；激光共聚焦扫描显微镜观察线粒体形态结构和分布情况。结果ONO—AE-248迅速导致中性粒细胞线粒体膜势能的溃散；ONO—AE-248刺激后中性粒细胞在线粒体形态、结构、分布和胞浆内染色特性等方面均与阴性对照有明显差异。结论线粒体变化是ONO—AE-248诱导中性粒细胞非凋亡性程序化细胞死亡早期形态学上区别于凋亡的显著变化之一。线粒体膜通透性转变以及线粒体膜势能下降可能是ONO—AE-248诱导的非凋亡性程序化细胞死亡的主要原因。     

中性粒细胞非凋亡性程序化细胞死亡的研究概况

中性粒细胞是机体固有免疫应答的主要效应细胞，也是机体内重要的炎症细胞。中性粒细胞除了凋亡和坏死外，还存在一种非凋亡性程序化细胞死亡形式。该形式具有空泡化，线粒体通透性增大等形态学特征，具有caspase-3、caspase-8非依赖性以及无DNA片段化的生物化学特征。目前尚不知其确切的生物学意义，但它与发育和许多生理／病理现象有关。这些研究成果对于重新认识和定位中性粒细胞程丰化细胞死亡的多样性和复杂性提出了新的思路。     

线粒体、内质网与细胞凋亡

细胞凋亡是近年来研究的热点,对其发生机制的研究对于肿瘤等多种疾病的治疗和免疫调节都具有重要价值。线粒体与内质网是细胞凋亡信号传导途径中起重要作用的细胞器。本文综述了凋亡过程中有关线粒体及内质网途径信号传导、调控机制的研究进展。     

Redox regulation of neutrophil apoptosis

The persistence of a neutrophil-mediated inflammatory response is due in part to a delay in their spontaneous rates of apoptosis or cell death. Regulating apoptosis has important implications for the resolution of inflammatory disorders, such as the systemic inflammatory response syndrome or acute respiratory distress syndrome. Neutrophils through their primary function of killing bacteria generate large concentrations of reactive oxygen intermediates and have alterations in the levels of antioxidants. Reactive oxygen intermediates and antioxidants are important regulators of the apoptotic caspases, but the mechanisms involved are still under debate and investigation. This review addresses the role of the cellular redox status of neutrophils on the apoptotic cascade leading to cell death.     

Clearance of apoptotic neutrophils and resolution of inflammation

The engulfment of apoptotic cells by phagocytes, a process referred to as efferocytosis, is essential for maintenance of normal tissue homeostasis and a prerequisite for the resolution of inflammation. Neutrophils are the predominant circulating white blood cell in humans, and contain an arsenal of toxic substances that kill and degrade microbes. Neutrophils are short-lived and spontaneously die by apoptosis. This review will highlight how the engulfment of apoptotic neutrophils by human phagocytes occurs, how heterogeneity of phagocyte populations influences efferocytosis signaling, and downstream consequences of efferocytosis. The efferocytosis of apoptotic neutrophils by macrophages promotes anti-inflammatory signaling, prevents neutrophil lysis, and dampens immune responses. Given the immunomodulatory properties of efferocytosis, understanding pathways that regulate and enhance efferocytosis could be harnessed to combat infection and chronic inflammatory conditions.     

Association of gastric epithelial apoptosis with the ability of Helicobacter pylori to induce a neutrophil oxidative burst

Both polymorphonuclear cell infiltration and increased epithelial apoptosis are seen in gastric mucosa in the presence of Helicobacter pylori infection. This study examined the association between bacterial ability to stimulate an oxidative burst in neutrophils and epithelial apoptosis. Biopsy specimens were obtained from 15 patients to detect apoptotic cells by the TUNEL method. H. pylori strains isolated from corresponding stomach biopsy samples were tested for the ability to stimulate an oxidative burst in human neutrophils. Neutrophils were isolated from healthy subjects without H. pylori infection and the oxidative burst was measured by flow cytometry with dichlorofluorescein diacetate. Stimulation with H. pylori increased both the percentage of activated cells and fluorescence intensity. There was a significant positive correlation between the number of epithelial apoptotic cells and fluorescence intensity. Increased neutrophil oxidative burst stimulated by H. pylori may play a role in enhanced gastric mucosal DNA damage and consequent atrophic gastritis and gastric cancer.     

Target antigens for anti-neutrophil cytoplasmic autoantibodies (ANCA) are on the surface of primed and apoptotic but not unstimulated neutrophils

The reaction of ANCA with ANCA antigens on the surface of neutrophils may play a critical role in the pathogenesis of ANCA vasculitis. Therefore, an understanding of the circumstances that result in surface expression of these antigens is important for an understanding of pathogenic mechanisms. In this study we investigated the surface expression of ANCA antigens on quiescent, primed, and apoptotic neutrophils. ANCA antigens and other granule constituents were not detected on the surface of neutrophils in freshly heparinized blood. ANCA antigens were on the surface of neutrophils primed by in vitro incubation for 4 h and 8 h. These cells did not show evidence of apoptosis. After 24 h incubation, about 30% of the neutrophils were apoptotic, and ANCA antigens and other granule constituents were present on the surface of both apoptotic and non-apoptotic cells. Our data indicate that there are no ANCA antigens on the surface of quiescent neutrophils, but that they are on the surface of primed neutrophils before the cells become apoptotic, and remain on the surface of cells after they become apoptotic. Based on these observations, we hypothesize that ANCA can react in vivo with primed but not quiescent neutrophils. Previously published observations indicate that the interaction of ANCA with primed neutrophils results in neutrophil activation, which may be involved in the pathogenesis of ANCA vasculitis.     

细胞凋亡机制研究进展

细胞凋亡是一种重要的生物学过程,在细胞生长发育以及对外界刺激的反应中有关键的作用。就近几年细胞凋亡信号传递机制、酶学机制、线粒体在细胞凋亡中的作用以及基因调控机制的研究作一简要论述。     

Life of neutrophil: From stem cell to neutrophil extracellular trap

Neutrophils are one of the main types of effector cells in the innate immune system. Neutrophils play a major role in fighting diseases and are recruited almost immediately to sites of infection. The neutrophils have a variety of defensive mechanisms and their high affinity to chemotactic agents makes them ideal in the defense against pathogens. New functions of neutrophils have been discovered over the years. The latest role of neutrophils is neutrophil traps, which are a new component of innate anti-microbial immunity. Before neutrophils can effectively kill microorganisms they undergo a series of complex developmental processes.     

Neutrophil secondary necrosis is induced by LL-37 derived from cathelicidin

Neutrophils represent the most common granulocyte subtype present in blood. The short half-life of circulating neutrophils is regulated by spontaneous apoptosis, and tissue infiltrating neutrophils die by apoptosis and secondary necrosis. The mechanism of neutrophil apoptosis has been the subject of many studies; however, the mechanism of neutrophil secondary necrosis is less clear. Human cathelicidin cationic peptide 18, proteolytically processed to its active form, LL-37, is secreted by neutrophils and epithelial cells and shown to have effects in addition to bacterial lysis. We demonstrate here that LL-37 affects neutrophil lifespan by the pathway of secondary necrosis, rapidly converting annexin V-positive (AV(+)), propidium iodide-negative (PI(-); apoptotic) cells into PI(+) (necrotic) cells with the release of IL-8, IL-1R antagonist, ATP, and intact granules. The effects of LL-37 on apoptotic neutrophils are neither energy-dependent nor affected by pretreatment with G-CSF, GM-CSF, TNF-alpha, and LPS and are partially inhibited by human serum. Moreover, LL-37 decreases CXCR2 expression of AV(-)PI(-) (live) neutrophils, suggesting an effect on the neutrophil response to its chemotactic factors, including IL-8. Thus, the lifespan and inflammatory functions of neutrophils are directly affected by LL-37.     

Effect of exogenous opioid peptides on TNF-alpha-induced human neutrophil apoptosis in vitro

Polymorphonuclear leukocyte (neutrophil) apoptosis is an important mechanism regulating the life span and some functions of neutrophils at inflamed sites. Opioid peptides are present in the peripheral circulation and their concentrations rapidly increase as a result of stress and inflammation. The effect of opioid peptides such as met-enkephalin (M-ENK) and beta-endorphin (beta-END) on tumor necrosis factor (TNF)-alpha-induced apoptosis in human neutrophils in vitro was investigated. Neutrophils isolated from peripheral blood were cultured in the absence or presence of 10(-6)-10(-10) M of opioid peptides for 8, 12 and 18 h. Features of apoptotic neutrophils were measured by a flow cytometric method based on analysis of the apoptotic nuclei (DNA content). We found that M-ENK and beta-END enhanced both uninduced and TNF-alpha-induced neutrophil apoptosis in vitro in a dose-dependent manner. The effect of opioid peptides on the modulation of neutrophil apoptosis was not reversed by the opioid-receptor antagonist naloxone. The results suggest that M-ENK and beta-END can regulate neutrophil life span via apoptosis and in this way may participate in the resolution of inflammation.     

Role of human immunodeficiency virus in leukocytes apoptosis from infected patients

The hallmark of the immunodeficiency virus infection is a progressive detriment of the immune response which has been associated to a gradual loss of its responsible components, in particularly, CD4 positive T cells. Although this cell population is considered the main target of the virus, there is a recent deal of interest in studying other components that may not be targets of the virus, but are important elements to control infectious microorganisms and that have been demonstrated to be altered during HIV infection. Neutrophils (PMN) are innate immune components that play a fundamental role against HIV infection and these cells have been described as functionally altered during AIDS. It has been suggested that such a dysfunction could be attributed to an increased susceptibility of these cells to accelerated spontaneous apoptosis. However, the underlying mechanisms that induce programmed cell death of neutrophils remain unknown. In previous works we have explored some events involved during cell death of neutrophils from HIV infected patients. It is the purpose of this work to review the current knowledge of apoptosis signals in neutrophils and to discuss our own data about some mechanisms involved in spontaneous and Fas mediated apoptosis, which may contribute to understand neutrophils dysfunction during HIV infection.