人中性粒细胞防御素免疫细胞化学分析

采用牛血清白蛋白(BSA)——肽抗原偶联技术,制备了免抗人防御素多克隆抗血清。并以此抗血清用免疫细胞化学染色法检查了一系列正常白细胞和白血病细胞株的免疫细胞化学反应。结果显示正常人中性粒细胞、急性髓性白血病细胞株KG1和HL-60细胞呈现强阳性反应。在正常人单个核白细胞、人红白血病K562细胞株及小鼠骨髓瘤SP2/0细胞株中均为阴性反应,甚至在健康家兔及豚鼠中性粒细胞中亦呈阴性反应。本实验提示防御紊可能作为粒系白细胞分化标志。     

ADAM17 activity during human neutrophil activation and apoptosis

Substrates of the metalloprotease ADAM17 (also known as TNF-alpha converting enzyme or TACE) undergo ectodomain shedding and include various inflammatory modulators. Though polymorphonuclear leukocytes contribute significantly to inflammation, direct analyses of ADAM17 on human neutrophils are very limited. In addition, the current understanding of the processes regulating ADAM17 activity primarily relate to its rapid activation. Therefore, to extend insights into the mechanisms of ADAM17 activity, we examined its surface expression and the shedding of its substrates during extended periods of neutrophil activation and apoptosis. Contrary to studies with immortalized hematopoietic cell lines, we report that surface expression of ADAM17 is maintained by human neutrophils activated with formyl peptides or by FcR/complement receptor-mediated phagocytosis. Interestingly, bacterial phagocytosis resulted in a significant increase in ADAM17 expression several hours after pathogen engulfment. We provide novel evidence that ADAM17 surface expression is also maintained during spontaneous and anti-Fas-induced neutrophil apoptosis. The well-validated ADAM17 substrates L-selectin and proTNF-alpha were shed efficiently by neutrophils under each of the conditions tested. Our data thus indicate prolonged ADAM17 expression during neutrophil effector functions. The implications of this may be a role by ADAM17 in both the induction and down-regulation of neutrophil activity.     

Preparation of membrane fractions from human neutrophil granules: A simple method

The surface composition of the azurophilic granule of human neutrophils is largely unknown. To understand the role played by the membrane components of these organelles a simple methodology for their disruption and the isolation of suitable amounts of granule membranes is required. The method of disruption of azurophilic granules available up to now consists of seven steps of freezing-thawing and sonication, a method time-consuming when large amount of granule needs to be processed. In this paper we describe a simple, one-step method based on the granule damaging capacity of Percoll which is suitable for the disruption of large amounts of azurophilic granules. The components of granule lysates obtained with this method were resolved on sucrose gradients. Two major bands were obtained, which are shown to be different from each other judging from their content of selective markers.     

Rapid non-genomic inhibitory effects of glucocorticoids on human neutrophil degranulation

Background: Glucocorticoids acting as anti-inflammatory or immunosuppressive drugs have been shown to exert most of their effects genomically. Recent findings suggest that non-genomic activity might be relatively more important in mediating the therapeutic effects of high-dose pulsed glucocorticoid. However, few non-genomic anti-inflammatory effects were reported, much less non-genomic mechanisms.Objective: This study was performed to investigate the nongenomic effects of glucocorticoids on human neutrophil degranulation.Methods: Purified human neutrophils were pretreated with 6 -methylprednisolone or hydrocortisone for 5 min, and then primed with N-formyl-methionyl-leucyl-phenylalanine (fMLP) (10^–6 M) or phorbol myristate acetate (PMA) (50 ng/ml) in the presence of cytochalasin B. The release of two markers of neutrophil granules, lactoferrin and myeloperoxidase, was measured by ELISA and enzymology methods respectively.Results: Both 6 -methylprednisolone (10^–5–10^–4 M) and hydrocortisone (10^–4 M) showed significant inhibitory effects on neutrophil degranulation within 5 min after fMLP administration. For PMA stimulated degranulation, 6 -methylprednisolone (10^–4 M) showed significant inhibitory effects (p < 0.01), while hydrocortisone (10^–4 M) only showed an inhibitory tendency (P > 0.05). Neither RU486 (10^–5 M) nor cycloheximide (10^–4 M) could alter the inhibitory effects of glucocorticoids.Conclusion: Our results demonstrate that megadoses of glucocorticoids exert rapid inhibitory effects on human neutrophil degranulation at the cellular level via a new mechanism that is independent of corticosteroid type II receptor occupation or protein synthesis. We infer that these effects may be very important when glucocorticoids act as anti-inflammatory drugs during pulse therapy.Received 20 May 2004; returned for revision 21 July 2004; accepted by M.J. Parnham 23 September 2004L. Liu and Y. X. Wang contributed equally to this work.     

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.     

GRPR antagonist protects from drug‐induced liver injury by impairing neutrophil chemotaxis and motility

Drug‐induced liver injury (DILI) is a major cause of acute liver failure (ALF), where hepatocyte necrotic products trigger liver inflammation, release of CXC chemokine receptor 2 (CXCR2) ligands (IL‐8) and other neutrophil chemotactic molecules. Liver infiltration by neutrophils is a major cause of the life‐threatening tissue damage that ensues. A GRPR (gastrin‐releasing peptide receptor) antagonist impairs IL‐8‐induced neutrophil chemotaxis in vitro. We investigated its potential to reduce acetaminophen‐induced ALF, neutrophil migration, and mechanisms underlying this phenomenon. We found that acetaminophen‐overdosed mice treated with GRPR antagonist had reduced DILI and neutrophil infiltration in the liver. Intravital imaging and cell tracking analysis revealed reduced neutrophil mobility within the liver. Surprisingly, GRPR antagonist inhibited CXCL2‐induced migration in vivo, decreasing neutrophil activation through CD11b and CD62L modulation. Additionally, this compound decreased CXCL8‐driven neutrophil chemotaxis in vitro independently of CXCR2 internalization, induced activation of MAPKs (p38 and ERK1/2) and downregulation of neutrophil adhesion molecules CD11b and CD66b. In silico analysis revealed direct binding of GRPR antagonist and CXCL8 to the same binding spot in CXCR2. These findings indicate a new potential use for GRPR antagonist for treatment of DILI through a mechanism involving adhesion molecule modulation and possible direct binding to CXCR2.     

中性粒细胞胞外诱捕网与相关疾病的研究进展

中性粒细胞是机体防御系统的第一道防线,当机体遭受病原微生物的入侵,中性粒细胞即发挥强大的作用.研究发现,活化的中性粒细胞可以释放出非浓缩染色质形成网状支架,对病原体进行包围及限制,这一过程称为中性粒细胞胞外陷阱(neutrophil extracellular traps,NETs)形成(NETosis).NETs是由DNA骨架、组蛋白、颗粒成分以及胞浆蛋白组成的网状物,其能够网罗、杀伤病原体从而参与机体自身免疫应答,维护机体健康.然而,中性粒细胞胞外诱捕网的产生是一把双刃剑,近年来有研究表明,它可以引发代谢性疾病,如糖尿病(diabetes mellitus,DM),引起心血管疾病动脉粥样硬化(atherosclerosis,AS),参与自身免疫性疾病,如系统性红斑狼疮(system lupus erythematosus systemic lupus erythematosus,SLE)、类风湿性关节炎(rheumatoid arthritis,RA)的产生,还可以使自身凝血异常,刺激血栓形成并为其提供支架,因而研究NETs及其与疾病之间的关系具有重要意义.     

中性粒细胞胞外诱捕网在创面愈合过程中作用的研究进展

创面愈合与免疫及炎症反应密切相关。中性粒细胞作为炎症反应的主要效应细胞之一,已被证明在糖尿病足等创面愈合的病理生理过程中起着重要的作用。尤其是新近研究发现中性粒细胞在炎症、损伤等的刺激下,可以通过释放中性粒细胞胞外诱捕网(NET),抑制或阻碍创面的愈合,在创面愈合过程中起着重要的作用。然而,NET在不同类型的创面愈合中的作用还未完全阐明。本文将围绕免疫反应与炎症反应等在创面愈合中的最新研究进展,就NET在不同类型的创面愈合过程中的作用作一综述。     

CD66: role in the regulation of neutrophil effector function

Neutrophils express several heavily glycosylated carcinoembryonic antigen (CEA)-related glycoproteins (CD66 antigens) which have been implicated in adhesion to E-selectin and as receptors for the lectins galectin 3 and bacterial type-1 fimbriae. The role of the CD66 antigens in neutrophil effector function was examined using non-cross-reacting and cross-reacting domain-mapped CD66 monoclonal antibody (mAb), which recognize epitopes on biliary glycoprotein (BGP; CD66a), CEA gene family member 6 (CGM6; CD66b), nonspecific cross-reacting antigen 90 (NCA90; CD66c) or CGM1 (CD66d). We show that BGP-specific mAb which recognize an AB-domain epitope strongly augment adhesion to fibrinogen by an Fc receptor- and β2 integrin-dependent mechanism. Co-ligation of BGP with the glycophosphatidylinositol (GPI)-anchored CGM6 and NCA90 also caused increased β2 integrin-mediated adhesion, receptor clustering and priming of formyl-Met-Leu-Phe (fMLP)-induced oxidant production by neutrophils, but only a small change in expression of L-selectin and CR3 compared to the chemotactic peptide fMLP. Ligation of CGM6 or NCA90 alone did not cause activation of the neutrophil in any of the assays used and did not cause priming of fMLP-induced oxidant production even when a secondary cross-linking reagent was used. We propose that specific cross-linking of neutrophil BGP with CGM6 and NCA90 contributes significantly to the regulation of neutrophil function during neutrophil recruitment.     

Modeling neutrophil transport in pulmonary capillaries

Neutrophils can be retained in the pulmonary microvasculature due to their low deformability, resulting in having a higher concentration there than in the systemic circulation, even in normal lungs. It is thought that this high concentration of the cells facilitates their effective recruitment to sites of inflammation. Thus, in order to understand their role in the immune system in the lungs, where blood comes in contact with outer air via thin septa of alveoli, it is important to clarify their flow characteristics in the pulmonary capillary bed. However, in contrast to erythrocytes in systemic capillaries, little research has been performed on the flow of neutrophils in pulmonary capillaries. This may be partly because no complete rheological model of the cell has been established yet, and partly because pulmonary capillaries are very short and closely interconnected, forming a complicated three-dimensional network, in addition to difficulty in in vivo experimental observations. Moreover, the neutrophils change their mechanical properties and show active motion in response to some chemoattractants. In this article, various proposed rheological models of the neutrophil, flow models of a cell through a single capillary segment, and alveolar capillary network models are introduced, aiming at the numerical simulation of neutrophil transport in the pulmonary microvasculature.     

活性氧在中性粒细胞胞外诱捕网形成中的作用

中性粒细胞胞外诱捕网(NETs)是胞外的一种DNA网状结构,其可网罗、杀伤病原体从而参与机体固有免疫应答.然而在某些情况下NETs会引发一系列自身免疫性疾病.研究表明,在NETs形成过程中活性氧(ROS)起到至关重要的作用.阐述活性氧与NETs形成的相互作用,进一步讨论了尚未完全明确的活性氧与NETs的关系,包括还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶产生的ROS的作用,依赖NADPH氧化酶产生NETs的分子机制,以及非NADPH氧化酶产生的ROS的作用,从多方面分析ROS与NETs形成的关系.     

The actin cytoskeleton in cancer cell motility

Cancer cell metastasis is a multi-stage process involving invasion into surrounding tissue, intravasation, transit in the blood or lymph, extravasation, and growth at a new site. Many of these steps require cell motility, which is driven by cycles of actin polymerization, cell adhesion and acto-myosin contraction. These processes have been studied in cancer cells in vitro for many years, often with seemingly contradictory results. The challenge now is to understand how the multitude of in vitro observations relates to the movement of cancer cells in living tumour tissue. In this review we will concentrate on actin protrusion and acto-myosin contraction. We will begin by presenting some general principles summarizing the widely-accepted mechanisms for the co-ordinated regulation of actin polymerization and contraction. We will then discuss more recent studies that investigate how experimental manipulation of actin dynamics affects cancer cell invasion in complex environments and in vivo.     

Improving the reproducibility of Pseudomonas aeruginosa swarming motility assays

Swarming motility is a rapid and coordinated migration of a bacterial population across a semi-solid surface. This multicellular phenomenon is getting increasing attention as it is suspected to be related to biofilm development of Pseudomonas aeruginosa. Published swarm plate preparation protocols differ greatly from one study to another and no reproducible and standardized protocols have been proposed to accurately study this phenomenon. We report here a rapid and highly reproducible swarm plate protocol for P. aeruginosa and show how different key parameters affect this type of motility (i.e. agar %, drying time under laminar flow, incubation temperature and pH). Results reported here will help to standardize swarming motility assays and develop effective swarm plate protocols for other bacterial species.     

中性粒细胞凋亡的线粒体死亡机制的研究进展

中性粒细胞是血细胞中寿命最短的终末分化细胞,在炎症反应中发挥着重要的作用,是人体免疫防御系统的第一道防线。与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。中性粒细胞自发性凋亡涉及到许多复杂的生化过程,线粒体是细胞的一种重要的细胞器,也是真核生物能量代谢的中心、凋亡中心,其在中性粒细胞凋亡信号传导和病理生理过程中起着关键性的调节作用。因此,对线粒体途径的调控机制进行研究是非常重要的。     

中性粒细胞胞外诱捕网在感染性疾病中的研究进展

中性粒细胞在非特异性免疫防御系统中起重要作用.Brinkmann等首次发现中性粒细胞可以形成一种捕获并杀灭病原体的特殊网状结构,将其命名为中性粒细胞胞外诱捕网(neutrophil extracellular traps,NETs).后续研究表明,NETs的生成和降解会影响感染性疾病的病理过程.因此,揭示NETs的产生机制及其对病原体的影响为深入理解感染性疾病发生发展的免疫机制提供重要理论依据,并为疾病的干预提供新策略.现拟对NETs的构成、产生机制及其在感染性疾病中的病理生理作用等作一综述.     

A new in vitro motility assay technique to evaluate calcium sensitivity of the cardiac contractile proteins

We attempted to introduce calcium regulation into in vitro motility assay. Cardiac thin filament was reconstituted from actin and tropomyosin-troponin complex purified from rat myocardium separately. Double staining of the filaments showed tropomyosin-troponin complex was integrated along actin filaments homogeneously. The reconstituted thin filaments were made to slide on cardiac myosin fixed on a glass coverslip in the presence of MgATP while varying free Ca²⁺ concentration of the medium ([Ca²⁺]). Filaments showed only Brownian motion when [Ca²⁺] was below 10^–6.4 M. However, filaments slid at a constant velocity when [Ca²⁺] exceeded 10^–6.4 M, showing that the sliding was regulated in an on-off manner. The threshold [Ca²⁺] increased to 10^–5.0 M under acidic conditions, indicating a decrease in Ca²⁺ sensitivity of the contractile proteins. Simple actin filaments slid at a constant velocity independently of [Ca²⁺], demonstrating that the regulatory proteins were responsible for this on-off manner regulation. This new assay technique may be a powerful tool to directly evaluate the Ca²⁺ sensitivity of the contractile apparatus and to investigate how cardiac contraction is regulated by Ca²⁺.     

哮喘中性粒细胞趋化因子性质的初步研究

测定哮喘发作和缓解期患者的中性粒细胞趋化因子活性,对中性粒细胞趋化因子(NCF)的理化性质进行初步研究。发现哮喘发作期NCA显著升高,RNCF对热较稳定,对蛋白酶敏感。脂溶性NCF活性仅占总NCF活性的4.2%。Sephadex G-200层析表明有两种NCF,一种分子量大于500000,另一种分子量约10000,前者是哮喘发作时的主要NCF。     

Neutrophil-active chemokines in in vivo imaging of neutrophil trafficking

Chemokines are proinflammatory mediators that regulate leukocyte trafficking at different steps of the leukocyte recruitment cascade. Studies using new imaging approaches and new mouse models are giving new insights into the role of chemokines in neutrophil migration at sites of inflammation. Conventional rolling and adhesion paradigms as well as previously unappreciated functions of signaling pathways triggering leukocyte adhesion, intralumenal crawling and transendothelial migration are compiled and described here. In this review we will summarize recent work in this field, highlighting in vivo imaging studies that examine the behavior of neutrophils in response to chemokines.     

Computerised method for acquisition and display of gastrointestinal motility data

A computerised system is developed for the acquisition and display of gastrointestinal motility data which utilises a purpose developed software program called ‘PC-motil’, running on an IBM compatible microcomputer. ‘PC-motil’ displays data during collection, writes data to disk file and compresses all data at the end of a study on to a single monitor screen for convenient overview. Any area of interest, in single or multiple channels, may be selected and expanded for detailed examination. This system is tested by the recording of gastric and jejunal motility patterns of 11 healthy volunteers in fasting and fed states. All antral and jejunal migrating motor complexes (MMCs) in fasting studies, as well as all fed motility patterns were recognisable in both ‘compressed’ and ‘expanded’ form. The reproduction of motility patterns by the computer based system was indistinguishable from that of a conventional analogue chart recorder. This computerised system provides a convenient and cost-effective means of acquisition, storage and display of motility data in digital form.     

Effect of pulmonary surfactant on TNF-alpha-activated endothelial cells and neutrophil adhesion in vitro

Pulmonary surfactant given to infants and adults with respiratory failure is metabolized and recycled to a large extent. A small proportion also enters the circulation in cases of increased permeability of the alveolar-capillary membrane. We therefore investigated whether exogenous surfactants such as a natural bovine (natSF) or a synthetic (synSF) preparation had an impact on inflammatory conditions involving the adhesion of neutrophils to endothelial cells. Human umbilical cord vein endothelial cells (HUVEC) were plated on coverslips until confluence, activated by tumor necrosis factor-alpha and incubated with or without surfactant in the media. Human neutrophils passed the HUVEC layer in a flow chamber and interactions were visualized using a video microscope. To test if surfactant affected the expression of cell adhesion molecules, RT-PCR analyses were performed for E-selectin, vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1). Using concentrations between 50 and 300 microg/ml of surfactant in the pre-incubation media the number of adherent neutrophils increased by 10-20% at the higher concentration of the natSF (*P < 0.05) whereas the synSF had no effect. Increased neutrophil adhesion was associated with a significant up-regulation of mRNA levels for E-selectin and VCAM-1; mRNA levels for ICAM-1, however, were not affected by the presence of surfactant. These observations indicate that natSF but not synSF might have pro-inflammatory effects when higher amounts of the exogenous dose reach the circulation. This might be explained by different fatty acid profiles, e.g. the presence of arachidonic acid in the natSF or higher concentrations of surfactant-associated protein-C in the synSF.