烧伤早期中性粒细胞对内皮单层通透性影响及CD11／CD18的作用

目的和方法：本实验应用体外灌注内皮细胞（ＥＣ）单层通透性测定方法观察了烧伤早期中性粒细胞（ＰＭＮ）对ＥＣ形态及通透性的影响。结果：烧伤早期ＰＭＮ可使ＥＣ变形，细胞收缩，细胞间间隙增大，ＥＣ单层通透性增大，应用ＣＤ１１ａ／ＣＤ１８单抗和ＣＤ１１ｂ／ＣＤ１８单抗阻断ＰＭＮ与ＥＣ粘附后，可明显减轻ＰＭＮ对ＥＣ的损伤和通透性的增高。结论：烧伤早期ＰＭＮ可引起ＥＣ损伤，ＣＤ１１／ＣＤ１８参与了此损伤     

内毒素条件下血管内皮细胞对中性粒细胞粘附性的动态改变

应用细胞微管吸吮实验系统对内毒素刺激条件下牛肺动脉血管内皮细胞（ＶＥＣ）对中性粒细胞（ＰＭＮ）的粘附性行单细胞定量测量。结果发现：在内毒素刺激下，拉离粘附于ＶＥＣ表面的ＰＭＮ所需临界分离应力Ｓｃ由正常对照的０．０８６±０．００７ｋＰａ增加到０．２８３±０．０３１ｋＰａ，随刺激过程的延长，ＶＥＣ的粘附力呈继续升高状态，１２ｈ达高峰，２４ｈ仍无明显下降趋势。结果提示ＶＥＣ在内毒素刺激诱导下对ＰＭＮ粘附性的增加既有快相反应，又有慢相反应。这种粘附性的明显增加为内毒素性损伤脏器微循环中大量ＰＭＮ滞留现象的发生提供保证     

小檗碱对中性粒细胞与血管内皮细胞粘附的影响及机制探讨

目的：探讨小檗碱对中性粒细胞及血管内皮细胞粘附作用及粘附分子的影响。方法：以人脐静脉内皮细胞（ＨＵＶＥＣ）为模型,用蛋白染料染色法观察小檗碱对中性粒细胞（ＰＭＮ）与ＨＵＶＥＣ粘附作用的影响;用细胞ＥＬＩＳＡ法、ＡＰＡＡＰ细胞免疫化学染色法观察小檗碱对ＰＭＮ－ＨＵＶＥＣ粘附过程中粘附分子ＩＣＡＭ－１及ＣＤ１８表达的影响。结果：小檗碱（０－３２～８μｇｍＬ）作用于ＨＵＶＥＣ,可抑制ＰＭＮ与ＨＵＶＥＣ间的粘附,对ＩＬ－１、ＴＮＦ（１０００μｇｍＬ）诱导的ＰＭＮ－ＨＵＶＥＣ粘附增强亦有抑制作用;而小…     

C5a对中性粒细胞和肾小球内皮细胞粘附行为的影响

目的：为揭示Ｃ５ａ和Ｃ５ａＲ对肾小球内皮细胞（ＧＶＥＣ）－中性粒细胞（ＰＭＮ）粘附的影响及其影响程度。方法：采用免疫组化胶体金银染色检测Ｃ５ａＲ在人ＧＶＥＣ上的表达，并用微管吸吮技术定量测定了经Ｃ５ａ诱导后，ＧＶＥＣ和ＰＭＮ细胞对间的粘附力，同时通过测定粘附的ＰＭＮ中髓过氧化物酶（ＭＰＯ）含量变化，观察不同剂量Ｃ５ａ对粘附的影响。结果：发现ＧＶＥＣ上分布Ｃ５ａＲ，经２０００ｎｇ／ｍｌＣ５ａ刺激，Ｇ     

糖皮质激素受体阻断对大鼠白细胞粘附的作用

目的明确ＧＲ阻断后对白细胞粘附的作用及可能的作用机制。方法１．在体实验。观察肠系膜微循环白细胞贴壁粘附数。 ２．高体实验。（１）ＰＭＮ与玻璃珠粘附;ＰＭＮ粘附率（％）粘附：ＰＭＮ粘附率（％）＝粘附前ＰＭＮ计数一洗脱液ＰＭＮ计数（３）ＰＭＮ粘附 粘附前ＰＭＮ计数分子ＣＤ１８：ＥＬＩＳＩＡ检测;（４）ＣＤ１８表达和ＰＭＮ与ＩＣＡＭ－１包被磁珠粘附的关系：ＰＭＮ与ＩＣＡＭ－１包被磁珠粘附,同时测定ＣＤ１８表达。结果１．在体实验。对照组仅有少量白细胞贴壁粘附（２．５０±２．１７）,阻断ＧＲ后白细胞贴壁粘附±Ｄｅｘ组的粘附率与ＴＮＦ组相比无显著差异（Ｐ＞０．０５）,Ｄｅｘ±ＴＮＦ组的粘附率与ＴＮＦ组相比有下降趋势．但差异不显著。ＴＮＦ＋Ｄｅｘ＋ＲＵ４８６组的粘附率与ＴＮＦ组和对照组相比均有差异（分别是 Ｐ＜０． ０５,Ｐ＜０． ０１）。 ２． ＰＭＮ与 ＥＣ粘附： ＴＮＦ组与ＴＮＦ＋Ｄｅｘ组结果同ＰＭＮ与玻璃珠粘附。Ｄｅｘ＋ＴＮＦ组的粘附率与ＴＮＦ组相比差异显著（Ｐ＜０．０５）。ＴＮＦ＋Ｄｅｘ＋ＲＵ４８６组的粘附率与对照组相差十分显著（Ｐ＜０．０１）,与ＴＮＦ组相比,两者无明显差异。３．ＰＭＮ与ＩＣＡＭ－１包被磁珠粘附：ＴＮＦ     

PPD诱导人内皮细胞表达粘附分子CD62E

目的探讨结核杆菌纯化蛋白衍生物（ＰＰＤ）在人脐静脉内皮细胞（ＥＣ）对外周血单个核细胞（ＰＭＮＣ ）粘附过程中的作用。方法利用培养的人脐静脉内皮细胞株,测定人ＰＭＮＣ和ＥＣ的粘附率;采用ＭｃＡｂＣＤ６２Ｅ抑制试验,检测ＰＰＤ对ＥＣ表达粘附分子的作用。结果经ＰＰＤ处理的 ＥＣ对ＰＭＮＣ的粘附率增加,并有一定的时相关系; ＭｃＡｂＣＤ６２Ｅ可以降低 ＰＰＤ的ＥＣ和ＰＭＮＣ的粘附率。结论ＰＰＤ可以直接刺激ＥＣ表达粘附分子,进一步参与介导白细胞与内皮细胞的粘附。     

TNF-α、IL-1β 、LPS对人多核白细胞、脐静脉内皮细胞血小板内皮细胞粘附分子-1表达的影响

目的：初探炎性刺激对人多核白细胞（ＰＭＮ）和脐静脉内皮细胞（ＨＵＶＥＣ）的血小板内皮细胞粘附分子－１（ＰＥＣＡＭ－１）表达的影响和ＰＥＣＡＭ－１的可能作用。方法：使用流式细胞仪和免疫组化法,观察脂多糖（ＬＰＳ）、白介素（ＩＬ）－１β和肿瘤坏死因子（ＴＮＦ）－ａ对ＰＭＮ和ＨＵＶＥＣ ＰＥＣＡＭ－１表达的变化。结果：ＬＰＳ、１Ｌ－１β和ＴＮＦ－ａ可引起ＰＭＮ表达ＰＥＣＡＭ－１蛋白减少,而对ＨＵＶＥＣ　ＰＥＣＡＭ－１的表达无显著影响,但组化显著在ＨＵ－ＶＥＣ连接部的ＰＥＣＡＭ－１染色变淡。结论：上述炎性刺激物不仅可引起ＰＭＮ表达ＰＥＣＡＭ－１减少、激活ＰＭＮ,而且可改变内皮细胞ＰＥＣＡＭ－１的分布,因此ＰＥＣＡＭ－１在炎症中可能起重要作用。     

尼古丁对炎性细胞活化及细胞间粘附分子基因表达的影响

本文观察了尼古丁对炎性细胞活化、炎性细胞与内皮细胞粘附及内皮细胞粘附分子表达的作用，同时观察７６４－３（从丹参中提取的单体）对上述部分作用的影响，将有助于阐明尼古丁在ＣＯＰＤ等慢性炎症疾病发病中的作用并为防治提供实验资料。方法：按以往报道的方法，收集大鼠腹腔ＰＭＮ，观察尼古丁作用下ＰＭＮ释放的β－ｇ及溶菌酶活性，以反映ＰＭＮ的活化；培养脐静脉内皮细胞，加入ＰＭＮ，以观察尼古丁对ＰＭＮ－内皮细胞粘附的影响；转化及扩增含ＩＣＡＭ－ＩｃＤＮＡ的质粒，酶切、ＰＥＧ法纯化及琼脂糖凝胶电泳鉴定ＩＣＡＭ－Ｉｃ…     

高糖、肿瘤坏死因子促进血管内皮细胞表面整合素（α_vβ_3）表达

本文采用ＥＬＩＳＡ方法检测培养的人脐静脉内皮细胞（ＨＵＶＥＣ）表面玻璃连接蛋白受体（ＶｉｔｒｏｎｅｃｔｉｎｒｅｃｅｐｔｏｒＶｎＲ即整合素家庭的α＿ｖβ＿３）的表达改变；用（５１）Ｃｒ－标记血小板（（５１）Ｃｒ－ｐｌａｔｅｌｅｔｅ，（５１）Ｃｒ－ｐＬ）检测ＨＵｖＥｃ的粘附功能；Ｆｕｒａ－２／Ａｍ负载ＥＣ，测定ＨＵＶＥＣ胞内游离钙离子浓度（［Ｃａ（２＋）］），观察了高糖、肿瘤坏死因子－α（Ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒ－α，ＴＮＦ－α）对ＥＣ粘附功能的影响。结果表明：①高糖（３０ｍｍｏｌ／Ｌ）可明显促进ＥＣ与ＰＬ的粘附（ＣＰＭ值：３６１±２ｌ．９３ＶＳ２ｌ９．６７±１６．２６，ｎ＝６Ｐ＜０．０１），抗β３亚单位单抗（β３ＭｃＡｂ）可部分阻断ＥＣ与ＰＬ粘附。ＴＮＦ－α（１０００μ／ｍ１）也有相同作用（ＣＰＭ值：４ｌ０．７±１７．６ＶＳ２１９．６７±１６．２６１ｎ＝６Ｐ＜０．０１），β３ＭｃＡｂ也部分阻断ＴＮＦ－α诱导的ＥＣ－ＰＬ间的粘附。②不同浓度高糖和ＴＮＦ－α不同时间可影响ＥＣ表面的α＿ｖβ＿３表达，并且在一定范围内有浓度和时间依赖性。③高糖和ＴＮＦ－α可明显增加ＥＣ的［Ｃａ（２＋）］ｉ，上述资料提示?     

单核细胞和中性粒细胞上的PECAM－1／CD31的接合可增加内细胞CR3

单核细胞和中性粒细胞上的ＰＥＣＡＭ－１／ＣＤ３１的接合可增加内细胞ＣＲ３的结合能力［英］／ＢｅｒｍａｎＭＥ…／／ＪＩｍｍｕｎｏｌ．—１９９５，１５４．—２９９～３０７中性粒细胞（ＰＭＮ）由血液中移入炎症组织需经过滚动、紧密粘附及跨内皮迁移（ＴＥＭ）３...     

Focal adhesion kinase regulates metastatic adhesion of carcinoma cells within liver sinusoids

Organ-specific tumor cell adhesion to extracellular matrix (ECM) components and cell migration into host organs often involve integrin-mediated cellular processes that can be modified by environmental conditions acting on metastasizing tumor cells, such as shear forces within the blood circulation. Since the focal adhesion kinase (FAK) appears to be essential for the regulation of the integrin-mediated adhesive and migratory properties of tumor cells, its role in early steps of the metastatic cascade was investigated using in vitro and in vivo approaches. Human colon and hepatocellular carcinoma cells were used to study adhesive properties under static conditions and in a parallel plate laminar flow chamber in vitro. In addition, intravital fluorescence microscopy was used to investigate early interactions between circulating tumor cells and the microvasculature of potential target organs in vivo. Shear forces caused by hydrodynamic fluid flow induced Tyr-hyperphosphorylation of FAK in cell monolayers. Reduced expression of FAK or its endogenous inhibition by FAK-related non-kinase (FRNK) interfered with early adhesion events to extracellular matrix components under flow conditions. In contrast, tumor cell adhesion to endothelial cells under these conditions was not affected. Furthermore, down-regulation of FAK inhibited metastatic cell adhesion in vivo within the liver sinusoids. In summary, FAK appears to be involved in early events of integrin-mediated adhesion of circulating carcinoma cells under fluid flow in vitro and in vivo. This kinase may take part in the establishment of definitive adhesive interactions that enable adherent tumor cells to resist fluid shear forces, resulting in an organ-specific formation of distant metastases.     

Rapid Adhesive Responses of Endothelial Cells and of Neutrophils Induced by Leukotriene B4 are Mediated by Leucocytic Adhesion Protein CD18

Controversy has existed as to the ability of leukotriene B4 (LTB4) to enhance adhesive properties of human neutrophils (PMN) and endothelial cells. We found that LTB4 induced a rapid but transient adhesion of PMN to an albumin-coated plastic surface and to cultured human umbilical vein endothelial cells (HUVEC). Although the adhesive response of PMN to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) was longer lasting, peak hyperadherence was of similar magnitude as to LTB4 and was less susceptible to assay conditions. Adherence induced by either LTB4 or fMLP could be abrogated by the monoclonal antibody 60.3, indicating similar dependence on the leucocyte adhesion protein CD18. Lipoxin A did not induce PMN hyperadherence. Treating HUVEC with LTB4, but not with its omega-oxidized metabolites 20-OH- and 20-COOH-LTB4, lipoxin A, or with fMLP conferred a rapid, dose-related, enhanced adhesion of PMN. This effect was dependent on CD18 and on divalent cations. It disappeared with prolonged exposure to LTB4, required a metabolically active HUVEC, and was not due to passive binding of LTB4 to HUVEC. Thus, LTB4 induces a transient expression of hyperadhesiveness in HUVEC as well as in neutrophils, and both effects are dependent on expression of CD18.     

Induction of ICAM-1 expression on human airway epithelial cells by inflammatory cytokines: effects on neutrophil-epithelial cell adhesion.

Inflammation of the human airways in diseases such as chronic bronchitis, cystic fibrosis with Pseudomonas endobronchial infection, and possibly asthma during late-phase reactions involves a local influx of neutrophils (PMN) that may participate in airway epithelial injury. PMN-mediated cellular injury is most efficient under conditions of PMN-target cell adhesion. PMN express adhesive glycoproteins of the CD11/CD18 family that are counter-receptors for intercellular adhesion molecule-1 (ICAM-1), found on various cell types. We proposed that adherence by PMN to human airway epithelial cells via ICAM-1 might be an important mechanism in inflammatory airway diseases. We found that although PMN adhere poorly (less than 5%) to monolayers of human tracheal epithelial cells (TEC) in primary culture, they adhere readily (45 to 50%) to an SV40-immortalized line of human TEC, designated 9HTEo-. We also found 6-fold greater surface expression of ICAM-1 on 9HTEo- compared with primary TEC. Blocking surface ICAM-1 on 9HTEo- cells with specific monoclonal antibody inhibited PMN adherence by about 50%. Thus, ICAM-1 plays a major role in this adherence, although it is possible that other epithelial ligands contribute also. Antibodies to CD11a, CD11b, and CD18 on PMN also inhibited PMN-epithelial adherence. Treatment of primary TEC monolayers with the proinflammatory cytokines interleukin-1 (IL-1) or tumor necrosis factor-alpha (TNF-alpha) caused a 3- to 4-fold increase in both cell surface ICAM-1 expression and support of PMN adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduced intracellular oxidative metabolism promotes firm adhesion of human polymorphonuclear leukocytes to vascular endothelium under flow conditions

The interaction of polymorphonuclear leukocytes (PMN) with the vascular endothelium and their subsequent extravasation to the tissues is a key step during different physiological and pathological processes. In certain of these pathologies the oxygen tension becomes very low, leading to reduced cellular oxidative status. To evaluate the effect of lowering the intracellular redox status in the interaction of PMN with the endothelium, exposure to hypoxic conditions as well as treatment with different antioxidant agents was carried out. PMN exposure to hypoxia enhanced β₂ integrin-dependent adhesion to intercellular adhesion molecule-1-coated surfaces, concomitant with a decrease in the intracellular redox status of the cell. As occurs with hypoxia, treatment with antioxidants produced a decrease in the oxidation state of PMN. These agents enhanced adhesion of PMN to human umbilical vein endothelial cells stimulated with tumor necrosis factor-α (TNF-α), and this effect was also mediated by β₂ integrins LFA-1 and Mac-1. Adhesion studies under defined laminar flow conditions showed that the antioxidant treatment induced an enhanced adhesion mediated by β₂ integrins with a decrease in the fraction of PMN rolling on TNF-α-activated endothelial cells. The up-regulated PMN adhesion was correlated to an increase in the expression and activation of integrin Mac-1, without loss of L-selectin surface expression. Altogether, these results demonstrate that a reduction in the intracellular oxidative state produces an enhanced β₂ integrin-dependent adhesion of PMN to stimulated endothelial cells under conditions of flow.     

The adhesive properties of endothelial cells on endovascular stent coated by substrates of poly-l-lysine and fibronectin

Optimizing endothelial cell growth and adhesion on the surface of metallic stents implanted in the vascular system is a fundamental issue in understanding and improving their long-term biocompatibility. The ability of the endothelial cell to attach and adhere to the luminal stent surface as well as the capacity to withstand the significant shear stress associated with blood flow are important determinants. The adhesive characteristics of human umbilical vein endothelial cellsectin (HUVEC) on stent surfaces coated with either Poly-L-Lysine (PLL) or fibron (FN) were compared with uncoated controls. Increasing concentrations of PLL and FN were measured using a micropipette aspiration system. The adhesivenamic properties of HUVECs under static flow conditions were compared to a dy environment on endovascular stents using a parallel-plate-flow chamber. A scanning electron microscope picture was used to measure the number and the adhesive cell ratio as well as the percentage of surface coverage of stent by endothelial cells. The adhesive forces of HUVECs on foreign surfaces coated with PLL and FN were higher compared to uncoated surfaces, and were dependent on incr ing concentrations. These coatings resulted in significant increase of the adhesive force of HUVECs. The influence of substrates on the adhesion of the endothelial cell monolayer under static or dynamic flow conditions was highly significant compared with controls (p<0.01). No significant differences were observed between PLL and FN substrates. Both PLL and FN coated surfaces can significantly increase the adhesion and growth of HUVECs on metallic stent surfaces.     

Mechanisms of endothelial cell-dependent leukocyte adhesion stimulated by platelet-activating factor

Platelet-activating factor (PAF) stimulates leukocyte-endothelial cell (EC) adhesion through its effects either on leukocytes or on ECs. ECs may be injured, synthesize, or express new adhesive proteins to increase leukocyte adhesion. Intermediary mediators produced by activated ECs are also likely involved in promoting leukocyte adhesion. Our experiments demonstrated that PAF induced no obvious damage to bovine pulmonary artery ECs evaluated by lactic dehydrogenase release rate, angiotensin-converiting enzyme activity, and cellular malondialdehyde content. Treatment of EC morolayers with 10^–9 M PAF increased polymorphonuclear leukocyte (PMN) adhesion. Increasing PAF concentration did not induce more PMN adherence. PAF elicited both a rapid and prolonged increment of PMN adherence to EC monolayers. The rapid adherence was greatly attenuated by pretreatment of ECs with PAF receptor antagonist SRI 63-441 but was not affected by pretreatment of PMNs with SRI 63-441, suggesting that PAF increases PMN adherence rapidly through its effects on specific receptors on ECs. Increased PMN adherence lasted if PAF treatment of ECs was sustained for 3 or 6 h. Pretreatment of ECs with actinomycin D, a protein synthesis inhibitor, significantly decreased PAF-induced sustained PMN adherence, but the inhibition is incomplete, suggesting that other mechanisms than protein synthesis also participated in the prolonged PMN adherence. We concluded from the results that PAF may induce both rapid and prolonged PMN adhesion to ECs. The effects are receptor mediated. The prolonged PMN adhesion is partly the result of protein synthesis.     

Role of the cytoskeleton in adhesion stabilization of human colorectal carcinoma cells to extracellular matrix components under dynamic conditions of laminar flow

Adhesion stabilization of malignant cells in the microcirculation is necessary for successful metastasis formation. The adhesion of colon carcinoma cells to microcirculation extracellular matrix (ECM) components is mediated, in part, by integrins that can be intracellularly linked to cytoskeletal proteins. Thus the functional status of at least certain integrins can be regulated by complex interactions with cytosolic, cytoskeletal and membrane-bound proteins. Wall shear stress caused by fluid flow also influences cellular functions, such as cell morphology, cytoskeletal arrangements and cell signaling. Using a parallel plate laminar flow chamber dynamic adhesion of human HT-29 colon carcinoma cells to collagen was investigated and compared with cell adhesion under static conditions. Cells were pretreated with cytochalasin D, nocodazole, colchicine or acrylamide to disrupt actin filaments, microtubules or intermediate filaments. Disruption of actin filaments completely inhibited all types of adhesive interactions. In contrast, impairment of tubulin polymerization or disruption of intermediate filaments resulted in different effects on static and dynamic adhesion. Treatment with acrylamide did not interfere with dynamic cell adhesion, whereas under static conditions it partially reduced adhesion rates. Under dynamic conditions increased initial adhesive interactions between HT-29 cells and collagen were found after disruption of microtubules, and the adherent cells demonstrated extensive crawling on collagen surfaces. In contrast, under static adhesion disrupting microtubules did not affect cell adhesion rates. Cytochalasin D and acrylamide were found to inhibit Tyr-phosphorylation of FAK and paxillin, whereas microtubule disrupting agents at low but not high concentrations increased phosphorylation of these focal adhesion proteins. Our results revealed that cytoskeletal components appear to be involved in adhesion stabilization of HT-29 cells to ECM components, and hydrodynamic shear forces modulate this involvement. Tyr-phosphorylation of focal adhesion proteins, such as paxillin and FAK, appears to be a part of this cytoskeleton-mediated process. This revised version was published online in July 2006 with corrections to the Cover Date.     

Antibodies to CD18 influence neutrophil migration through extracellular matrix

Mac-1 (CD11b/CD18) is known to be involved in neutrophil (PMN) adhesion to endothelial cells and extracellular matrix. Although antibodies to CD 18 are being tested for therapy in humans, their role in PMN migration through the extracellular matrix is unknown. We used direct visualization to quantify PMN motility through reconstituted, three-dimensional gels of collagen type I. Gels were prepared with different concentrations of collagen (ranging from 0.1 to 1.0 mg/mL) and PMN migration was examined in the presence and absence of antibodies to CD18 (anti-CD18), with and without stimulation by N-formyl peptides. In low-concentration gels (<0.6 mg/mL), anti-CD18 had a significant influence on PMN migration, increasing motility in unstimulated PMN by 90% at 0.3 mg/mL collagen, and decreasing motility in N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN by 70% at 0.4 mg/mL collagen. But antiCD18 had no effect on the rate of cell migration through high-concentration collagen gels (>0.6 mg/mL). PMN migration through collagen gels is CD18-dependent but only under conditions of high hydration, suggesting that CD18-mediated effects (e.g., adhesion to gel fibers) are only important when the fiber density is relatively low. Anti-CD18 inhibited, but did not eliminate, the adhesion of fMLP-stimulated PMN to the surface of collagen gels, suggesting that cells use multiple mechanisms for gaining traction within the gel. Because of the multiple modes of interaction between motile cells and the deformable fiber matrix, blockade of one component, such as CD18, can enhance the rate of cell migration under one set of conditions, and inhibit under another.