检测体外中性粒细胞自发性激活的方法学研究

从细胞形态学和细胞酶组织化学的角度出发,用伪足法和ＮＢＴ法检测体外中性粒细胞的自发性激活,研究了正常人中性粒细胞体外自发激活的时相特征,对这两处方法进行了对比分析。发现：（１）检测体外中性粒细胞自发性激活,伪足法较ＮＢＴ法更灵敏、可靠、且可实时观察;（２）体外中性粒细胞自发性激活的百分率呈现由低升高、再逐渐降低的时相过程,最高激活百分率出现在采血后第３小时,研究结果为今后深入研究中性粒细胞激活与多     

补体经典激活途径C3转化酶的体外组装及活性观察

目的：体外组装包含人C4分子的补体经典激活途径C3转化酶，并对其转化酶活性及衰变特性进行观察。方法：利用豚鼠血清功能纯C1、C2及溶血中间体EAC4^hu体外组装经典途径C3转化酶，观察不同C1、C2用量及孵育温度对C3转化酶形成和自发性衰变的影响，以及人红细胞膜抽提蛋白对C3转化酶衰变化的影响。结果：高剂量和低剂量的C1均会影响C3转化酶的形成，增加C2用量可增加C3转化酶的形成数量，C3转化酶的自发性衰变随孵育温度的升高而加速，人红细胞膜抽提蛋白可抑制C3转化酶的自发性衰变过程，结论：C1、C2用量及孵育温度是影响C3转化酶形成和自发性衰变的主要因素，体外组装的补体经典激活途径C3转化酶可应用于相关补体调控蛋白的活性检测。     

一氧化氮在中性粒细胞与激活的内皮细胞间黏附中介导的作用

目的 研究一氧化氮（NO）在大鼠中性粒细胞（PMN）与激活的大鼠肺微血管内皮细胞（PMEC）黏附中的介导作用。方法 分离和培养大鼠中性粒细胞（PMN）和大鼠肺微血管内皮细胞（PMEC），测定PMEC与PMN间黏附率，并测定一氧化氮合酶（NOS）抑制剂L-NMMA孵育的PMEC经烧伤血清或LPS激活后，与PMN间黏附率。结果 L-NMMA能增加烧伤血清或LPS激活后的PMEC与PMN间黏附率。结论 NO介导了PMN与激活的PMEC间黏附。     

槲皮素对LPS诱导的中性粒细胞产生白介素6的影响

目的 研究离体培养的人中性粒细胞在被LPS激活的情况下，槲皮素对中性粒细胞产生IL-6的影响。方法 运用ELISA法检测槲皮素对LPS诱导的中性粒细胞分泌IL-6的影响；运用流式细胞术检测槲皮素对LPS活化的中性粒细胞内IL-6的蛋白水平的影响；运用逆转录PCR（RT-PCR）技术检测槲皮素对LPS活化的中性粒细胞表达IL-6 mRNA的影响。结果 在无刺激的情况下中性粒细胞不产生IL-6，LPS（1μg／mL）以时间依赖性的方式诱导中性粒细胞表达IL-6 mRNA，合成并分泌IL-6，并在16h达到高峰。然而，预先用槲皮素（40μmol／L）处理中性粒细胞30min后，LPS诱导的中性粒细胞表达IL-6 mRNA，合成并分泌IL-6则受到了抑制。结论IL-6是炎症早期重要的促炎症因子。因此，槲皮素抑制LPS诱导中性粒细胞产生IL-6，提示槲皮素可能通过对炎症因子负性调控而发挥其抗炎作用。     

缺氧,缺血及再灌注山羊血浆对血管内皮细胞和中性粒细胞的影响

为了探讨缺氧，缺血及再灌注损伤的发生的机制，我们分别观察了缺氧（模拟海拔４０００ｍ高原）２４ｈ山羊血浆（ＨＰ），缺血１ｈ（在上述条件下于缺氧２４ｈ末放血使血压维持在６．０ｋＰａ１ｈ）山羊血浆（ＩＰ）和再灌注回输放出血液后２４ｈ）山羊血浆（ＲＰ）对中性粒细胞（ＰＭＮ）及培养的肺动脉内皮细胞（ＰＡＥＣ）的作用，ＰＭＮ在分别含ＨＰ、ＩＰ和ＲＰ的培养中温育１ｈ末，细胞活力明显升高（Ｐ〈０．００１），其升高     

黄芪多糖对嗜中性粒细胞与内皮细胞粘附及粘附分子的影响

目的：通过研究黄芪多糖对嗜中性粒细胞与血管内皮细胞粘附及粘附分子的影响,探讨黄芪多糖对炎症反应的作用及其机制。方法：以黄芪多糖或黄芪多糖加ＩＬ－１ＴＮＦ处理人嗜中性粒细胞或人脐静脉内皮细胞（ＨＵＶＥＣ）后,用蛋白染料染色法研究药物对嗜中性粒细胞与内皮细胞粘附的作用,用细胞ＥＬＩＳＡ法、ＡＰＡＡＰ免疫细胞化学染色法研究药物对ＨＵＶＥＣ表面ＩＣＡＭ－１及嗜中性粒细胞表面ＣＤ１８表达的影响。结果：黄芪多糖（４０μｇｍＬ－１ｍｇｍＬ）作用于ＨＵＶＥＣ,能促进ＨＵＶＥＣ与嗜中性粒细胞粘附,并与ＩＬ－…     

恶性肿瘤患者中性粒细胞趋化性检测

恶性肿瘤患者中性粒细胞趋化性检测刘锦屏，何浩明，张亮（连云港市第一人民医院连云港２２２００２）体外中性粒细胞（ＰＭＮ）趋化试验是一种测定ＰＭＮ功能的常用方法。本文用以检测恶性肿瘤患者ＰＭＮ趋化性，现报道如下：１对象和方法１．１对象对照组为６０例健康体...     

弹性蛋白酶调节中性粒细胞炎性募集的功能研究

目的：探索中性粒细胞弹性蛋白酶（NE）对中性粒细胞炎性募集的影响。方法：采用外源性弹性蛋白酶抑制剂西维来司钠盐预处理小鼠骨髓来源的中性粒细胞,通过腹膜炎过继性迁移、细胞体外趋化、流动室黏附、免疫荧光染色、细胞伸展等实验,检测NE的抑制对中性粒细胞体内炎性募集、体外趋化、牢固黏附、细胞极化与伸展能力的影响;利用流式细胞术及鲁米诺化学发光系统检测NE的抑制对中性粒细胞吞噬及活性氧（ROS）释放能力的影响。结果：西维来司钠盐预处理显著减弱了中性粒细胞体内炎性募集（P<0.001）;减弱了中性粒细胞在体外趋化过程中对催化剂方向性的感知（P<0.05）,减慢了趋化速率（P<0.05）,降低了趋化距离（P<0.05）;减少了中性粒细胞在炎性内皮细胞表面形成牢固黏附的能力（P<0.000 1）,抑制了中性粒细胞对细菌的吞噬能力（P<0.01）;但对中性粒细胞的伸展、极化及ROS的释放均无显著影响。结论：NE的抑制显著损害了中性粒细胞在体内外的炎性募集级联反应及吞噬作用,但对中性粒细胞的伸展、极化、ROS释放等功能无显著影响。     

HNP对肺炎克雷伯杆菌粘附呼吸道上皮细胞的影响

目的：探讨呼吸道抗感染防御机制，观察人中性粒细胞α-防御素（HNP）对肺炎克雷伯杆菌粘附呼吸道上皮细胞的影响。方法：从人中性粒细胞分离纯化HNP。A549细胞与HNP（20μg·ml^-1）及两株肺炎克雷伯杆菌临床分离株共同孵育4小时，用平板培养茵落计数法测定与细胞粘附的活茵数。结果：在HNP存在的情况下，Kp03.33株细菌对肺上皮细胞的粘附提高了12倍（P〈0.01），Kp03116株细菌对上皮细胞的粘附提高了5倍（p〈0.01）结论：HNP显著增强肺炎克雷伯杆菌粘附于呼吸道上皮细胞，可能有利于呼吸道清除细菌。     

内毒素介导的肾脏病变及其发生机理的实验研究

研究内毒素介导的大鼠肾功能和形态变化，并探讨其发生机理。结果发现：肾功能于注射内毒素（ＬＰＳ）后１ｈ明显减退，５ｈ达高峰，４８ｈ基本恢复。肾小球出现早期短暂的毛细血管收缩，随后扩张，肾小球内中性粒细胞和血小板聚集，内皮细胞和系膜细胞肿胀或增生，足细胞足突融合，基底膜局部增厚等形态学改变，并且有肾组织中ＴＸＢ＿２，６－ｋｅｔｏ－ＰＧＦ＿（１α）及丙二醛含量增高。作者推测，ＴＸＢ＿２、６－ｋｅｔｏ－ＰＧＦ＿（１α）含量升高和两者间比例失衡可能是ＬＰＳ介导肾功能障碍的重要起始因素，氧自由基在ＬＰＳ介导的肾功能障碍发生、发展中起重要作用。     

Characterization of the activation of the human C1r complement molecule

The proenzyme form of C1r was isolated by sequential chromatography from the euglobulin fraction of human serum on DEAE-Sepharose 6B-CL, CM-Sepharose 6B-CL and Sepharose S-300-CL. This C1r had the tendency to spontaneously activate within 60-90 min of incubation at 37 degrees C in presence of EDTA and more slowly in the presence of Ca2+. The spontaneous activation of C1r was found to be a bimolecular process and could be completely inhibited by DFP in the pH range 6-9 and in the presence of Ca2+ without affecting the hemolytic C1r activity. [14C]DFP bound to trace proteins in the 60-90 kD range, but not to C1r proenzyme. The spontaneous activation of C1r was diminished in the presence of EDTA by DFP, but could not be completely suppressed. EDTA acts by removing Ca2+ from C1r, thereby changing the conformation of the protein and causing an increased digestibility of the C1r H-chain. At temperatures above 0-4 degrees C this influence destroyed the ability of C1r proenzyme and enzyme to form macromolecular C1 and thereby abolished its hemolytic activity. We conclude from these results that the spontaneous C1r activation in the pH range 6-9 and in the presence of Ca2+ is due to contaminant proteases. C1r activated also spontaneously at higher pH values between pH 9 and 13.2, but the spontaneous activation ceased abruptly at pH 13.4. An intramolecular process of activation cannot be excluded at these high pH values. It is, however, not clear, whether this activation is a suitable model for the C1r activation in the C1 molecule, because the hemolytic activity of C1r was substantially diminished under the high pH conditions.     

Role of degranulation in activation of the respiratory burst in human neutrophils.

The neutrophil response to infection and inflammation includes membrane fusion or degranulation and activation of the membranous respiratory burst oxidase. The role of degranulation in the activation of the burst was explored in resting and activated cells. Exposed membrane proteins of intact cells were labeled with impermeant reagents. Phorbol ester-activated neutrophils and enucleated cells which are granule depleted both exhibit increased labeling with [125I]lactoperoxidase over that of resting cells. The binding of antibodies to granule membranes by cells activated with phorbol ester or treated with cytochalasin B and lithium chloride were similarly increased. These data indicate that insertion of granule membrane into the cell membrane occurs during activation and enucleation of neutrophils. Hyperosmolarity, known to inhibit degranulation, also exhibited an inhibitory effect on the respiratory burst oxidase in the presence of phorbol ester or latex. Pre-treatment of cells with phorbol ester followed by an increase in osmolarity, however, still resulted in activation. Temperatures below 17 degrees C abruptly and simultaneously abolish degranulation and activation of the respiratory burst oxidase. Pre-treatment of neutrophils with phorbol ester at 37 degrees C, followed by measurement of oxidase activity at decreased temperatures, on the other hand, revealed a linear Arrhenius plot above and below 17 degrees C. These results suggest that membrane fusion or degranulation is a step in activation of the respiratory burst.     

Changes in the mechanical and adhesive behaviour of human neutrophils on cooling in vitro

Changes in the rheological properties of neutrophils may influence flow in microvessels that are cooled below normal body temperature. We investigated the effects of temperature on the mechanical and adhesive properties of human neutrophils by measuring transit times for individual cells flowing through 8-microm-pores in filters, and adhesion to P-selectin for cells perfused over a monolayer of activated platelets. Pore transit time increased as temperature was decreased from 37 degrees C to 0 degrees C. Upon rapid cooling, there was an instantaneous increase attributable to changes in aqueous viscosity. Interestingly, at 10 degrees C specifically, there was an additional increase in transit time, which was abolished by the inhibitor of actin polymerization, cytochalasin B. This meant that by 15 min, transit time at 10 degrees C was greater than at 0 degrees C. Most adherent cells on P-selectin were rolling, rather than stationary, at 10, 26 or 37 degrees C. The velocity of rolling slowed with decreasing temperature. The total number of adherent cells decreased with increasing wall shear rate, but for a given shear rate there was relatively little effect of temperature on attachment. However, when adhesion at 10, 26 or 37 degrees C was compared at equal shear stress (taking into account fluid viscosity), adhesion was greatest at 10 degrees C. Measurements of immunofluorescence showed that exposure to 10 degrees C gradually increased expression of beta2-integrin CD11b/CD18, but this did not cause transformation to stationary adhesion with time in the flow assay. Thus, neutrophils show an anomalous rheological response around 10 degrees C, which may impair local microcirculation in the cold. On rewarming, "activated" cells might inhibit recovery or become released into the systemic circulation.     

In vitro activation of the hemolysin in Prevotella nigrescens ATCC 33563 and Prevotella intermedia ATCC 25611

Hemolytic activity was evaluated in the putative periodontopathogens Prevotella intermedia and Prevotella nigrescens. Whole cells of both species present weak hemolytic activity evidenced only by solid media assays after 48 h of bacterial growth or after 5 h of interaction with erythrocytes at 37 degrees C in liquid assays. In this work we show that the use of crude extract allowed the detection of a higher hemolytic activity for P. intermedia, but surprisingly not for P. nigrescens. Incubation at 37 degrees C for 9 h, or treatment with trypsin or proteinase K, increased or exposed the hemolytic activity of P. intermedia and P. nigrescens crude extract, respectively. The activation process was inhibited by TLCK and PMSF but not by EDTA, E-64 or pepstatin A, indicating the serino-protease nature of the factor involved in activation of P. intermedia and P. nigrescens hemolysins. Both the buffer and the pH employed for cell fractionation influenced the activation of hemolysin, and the best results were obtained with Universal buffer at pH 8.0. The activated hemolysins acted optimally at pH 6.5 at 37 degrees C and the maximum hemolytic activity was detected at the early log phase of growth. The results of this study show for the first time a strong hemolytic activity for P. nigrescens and evidence of proteolytic activation of hemolysins produced by periodontopathogens.     

The formylpeptide chemotactic receptor on rabbit peritoneal neutrophils: change of receptor affinity and number by L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)

Pretreatment of rabbit peritoneal neutrophils at 37 degrees with 10-35 microM L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) decreases by 20-50% the detectable number of f Met-Leu-[3H]Phe binding sites. Greater TPCK concentrations, between 50 and 100 microM, cause less of a decrease or actually increase peptide binding activity to a level greater than that of untreated cells. Furthermore, Scatchard analysis indicates that the sites detected on neutrophils after TPCK treatment have 1.2-3.2 fold lower apparent Kd (higher affinity) than those detected on untreated, control cells (1.1 +/- 1.7 X 10(-8) M vs 1.7 +/- 1.5 X 10(-8) M, P less than 0.02). Thus, TPCK treatment of rabbit peritoneal neutrophils causes both a decrease in f Met-Leu-[3H]Phe receptors and increases the affinity of the remaining sites. In addition, peritoneal neutrophils incubated at 37 degrees without TPCK were found to rapidly express additional f Met-Leu-[3H]Phe receptors. These additional sites, however, were not evident on neutrophils incubated at 37 degrees with TPCK. Concomitantly with the expression of additional sites, neutrophils placed at 37 degrees were found to spontaneously release small amounts of lysozyme. However, since equivalent amounts of lysozyme were released by cells incubated with or without TPCK, we are unable to state whether expression of the additional sites is due to neutrophil degranulation. Finally, although rabbit peripheral blood neutrophils also show an increase in binding sites at 37 degrees, treatment of these cells with TPCK does not cause a decrease in their f Met-Leu-[3H]Phe binding activity.     

Binding and lysis of antibody-coated human erythrocytes by activated human monocytes

Human monocytes exposed to PHA-leukocyte-conditioned medium for 24 hr acquire markedly enhanced ADCC against antibody-coated human erythrocytes. Confluent monolayers of these activated monocytes were found to bind several fold the number of 51Cr-labeled antibody-coated erythrocytes as compared to monolayers formed from control monocytes (uncoated erythrocytes were not bound). The stoichiometry of this reaction indicated that activated monolayers bind approximately 3-5 erythrocyte targets per effector monocyte, whereas control monolayers bind less than 2. Bound target cells remain intact, affixed to the monocyte surface for up to 3 hr at 25 degrees C (they were not phagocytized); incubation at 37 degrees C resulted in both target cell adherence and 51Cr release suggesting that a proportion of target cells were lysed. A substantial fraction of target cells bound at 25 degrees C were lysed (activated greater than control monocytes) if the monolayers were warmed to 37 degrees C. These results indicate that target cell target cell binding can occur at both 25 and 37 degrees C, but lysis of bound targets requires incubation at 37 degrees C. Using this temperature distinction to examine binding and lysis independently, it was found that binding was abrogated by IgG Fc fragments, but could occur over a broad pH range (5-8), and in the presence of 2-deoxy-D-glucose, colchicine, and sodium azide. Lysis of bound targets, on the other hand, was blocked by inhibitors of glycolysis, microtubule/filament organization, cellular respiration, and serine esterase activity, as well as EDTA and low pH; lysis was unaffected by scavengers of extracellular H2O2 and superoxide radicals.     

Crosstalk between inflammation and thrombosis

Inflammation shifts the hemostatic mechanisms in favor of thrombosis. Multiple mechanisms are at play including up regulation of tissue factor leading to the initiation of clotting, amplification of the clotting process by augmenting exposure of cellular coagulant phospholipids, inhibition of fibrinolysis by elevating plasminogen activator inhibitor 1 (PAI-1) and decreases in natural anticoagulant pathways, particularly targeted toward down regulation of the protein C anticoagulant pathway through multiple mechanisms. The decreased function of the natural anticoagulant pathways may be particularly problematic because these appear to play a role in dampening inflammatory responses. The protein C anticoagulant pathway provides a useful model for the impact of inflammation on coagulation. This pathway plays a major role in preventing microvascular thrombosis. The pathway is initiated when thrombin binds to thrombomodulin (TM) on the surface of the endothelium. An endothelial cell protein C receptor (EPCR) augments protein C activation by the thrombin–TM complex more than 10-fold in vivo. EPCR is shed from the endothelium by inflammatory mediators and thrombin. EPCR binds to activated neutrophils in a process that involves proteinase 3 and Mac-1 and appears to inhibit leukocyte extravisation. EPCR can undergo translocation from the plasma membrane to the nucleus where it redirects gene expression. During translocation it can carry activated protein C (APC) to the nucleus, possibly accounting for the ability of APC to modulate inflammatory mediator responses in the endothelium. TNF and other inflammatory mediators can down-regulate EPCR and TM and IL-6 can depress levels of protein S in experimental animals. Inhibition of protein C pathway function increases cytokine elaboration, endothelial cell injury and leukocyte extravisation in response to endotoxin, processes that are decreased by infusion of APC. In vitro, APC inhibits TNF elaboration from monocytes and to block leukocyte adhesion to selectins. Since thrombin can elicit many inflammatory responses in microvascular endothelium, loss of control of microvascular thrombin generation due to impaired protein C pathway function probably contributes to microvascular dysfunction in sepsis.     

Complement activation by neutrophil granulocytes

Complement plays a vital role in the body's defence systems. Cardiopulmonary bypass induces a detrimental inflammatory reaction in which the complement system is known to participate through direct effects as well as through activation of neutrophils, platelets and endothelial cells. On the other hand, it has been suggested that in the setting of cardiopulmonary bypass, complement may be activated by neutrophils, perhaps due to fragmentation caused by the heart–lung machine. We therefore investigated whether intact or fragmented neutrophils were able to activate the complement system, and whether neutrophil–platelet interaction could influence such complement activation. Lepirudin-anticoagulated plasma was incubated at 37 °C with resting or activated intact neutrophils or neutrophils combined with platelets, or increasing amounts of fragmented neutrophils. Complement activation was evaluated by measurement of C1rs-C1 inhibitor complexes, C4bc, C3bBbP, C3bc, C5a and sC5b-9. We found significant activation of complement only by unphysiological doses of fragmented neutrophils or supernatant from fragmented neutrophils, consistent with a limited clinical significance related to neutrophil destruction during cardiopulmonary bypass. Unstimulated neutrophils induced C3bPBb formation but little formation of other activation products, indicating an increased C3 hydrolysis which was kept under control by regulatory mechanisms. Neutrophils and platelets combined increased classical activation and decreased alternative activation, similar to the findings with platelets alone. Our data confirm that in the setting of acute neutrophil fragmentation or activation, complement activation is much more important in the inflammatory network as an event upstream to neutrophil activation than vice versa.     

Effect of hirudin versus heparin on hemocompatibility of blood contacting biomaterials: an in vitro study

Hirudin serves as an alternative anticoagulant for extracorporeal blood circulation. Comparing anticoagulation with hirudin (2.5 or 5.0 microg/mL) and heparin (2.0 or 4.0 IU/mL) human blood was circulated in a modified 'Chandler System' using PVC-tubes for 2 hours at 37 degrees C. Activation of coagulation (thrombin-antithrombin III-complex, prothrombin fragment 1+2 and D-Dimer), platelet (platelet factor 4 - PF4) and complement systems was analyzed. Both heparin concentrations and 5.0 microg/dL hirudin led to as significantly less activated plasmatic coagulation as 2.5 microg/dL hirudin. Decreased levels of PF4 and anaphylatoxin C5a (p<0.05) as well as terminal complement complex demonstrated improved hemocompatibility after anticoagulation with heparin in contrast to hirudin. Because initial coagulation cascade, platelet activation and complement activation is less influenced by hirudin than by heparin, hemocompatibility is more dependent on the characteristics of the biomaterials used. This predestines hirudin as anticoagulant for in vitro studies analyzing hemocompatibility of biomaterials or surface modifications.     

Influence of collection and separation of blood samples on plasma IL-1, IL-6 and TNF-alpha concentrations.

The anticoagulant used for the collection of blood was found to influence in vitro cytokine production in whole blood. Lithium heparin in certain collection tubes was found to contain endotoxin and induced cytokine synthesis in a time-dependent manner whereas endotoxin-free lithium heparin did not. No induction of cytokine occurred in the presence of EDTA which was also able to inhibit endotoxin-induced cytokine synthesis. Synthesis or absence of cytokine correlated with the induction of messenger RNA. Investigation of the kinetics of cytokine induction in whole blood revealed that tumour necrosis factor alpha (TNF) was detectable after 2 h of incubation at 37 degrees C and interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) after 3 h. In certain samples IL-1 and IL-6 were detectable in plasma separated immediately from blood collected into endotoxin-free lithium heparin, presumably reflecting in vivo synthesis, and similar concentrations were detected after 3 h of incubation of whole blood at 37 degrees C. These data indicate that as long as blood is collected into endotoxin-free anticoagulant then cytokine measurements will reflect the in vivo status.