Kupffer cell depletion in vivo results in preferential elimination of IgG aggregates and immune complexes via specific Fc receptors on rat liver endothelial cells.

In the present study we have investigated the clearance kinetics and tissue distribution of monomeric (m) IgG and soluble aggregates of IgG (AIgG) and immune complexes (IC) in normal and Kupffer cell (KC) depleted rats. In normal rats, clearance of mIgG occurred in a biphasic manner with a first half-life (T1/2) (T1) of 36.3 +/- 6.3 min and a second T1/2 (T2) of 168.4 +/- 4.7 min. AIgG composed of 20-27 IgG molecules per aggregate were cleared significantly faster than mIgG with a T1 of 2.5 +/- 0.1 min and a T2 of 32.5 +/- 5.6 min. KC depletion did not have a significant effect on the clearance rate of mIgG (T1: 33.4 +/- 8.9 min; T2; 159.5 +/- 12.5 min), while clearance of AIgG was delayed significantly with T1 4.8 +/- 0.7 min and T2 41.2 +/- 3.2 min. Eight minutes after injection, 77% of AIgG was found in the liver in normal rats while 62% was found in the liver of KC-depleted rats. Double immunofluorescence studies indicated that AIgG in the liver was associated with KC and endothelial cells (EC) in normal rats. In KC-depleted rats, AIgG was strongly associated with EC. A similar staining pattern was observed when IgG-immune IC were administered. The clearance of AIgG in KC-depleted rats was inhibited fully by pre-administration of high concentrations of IgG but not by pretreatment with IgA. asialofetuin (ASFe) or ovalbumin (OVA). Aggregated F(ab')2IgG was cleared with a comparable rate to mIgG from the circulation, again suggesting Fc gamma receptor-mediated elimination of AIgG by EC. There was a reduced degradation of AIgG in rats depleted of KC as compared with normal rats. These data suggest binding and degradation of AIgG by EC in vivo.     

Complement enhances the clearance of large-sized soluble IgA aggregates in rats

In the present study the involvement of the complement system (C) in the clearance of soluble IgA aggregates in the rat was studied. Monoclonal monomeric IgA (mIgA) antibody (which does not activate C) or aggregated polymeric IgA (aIgA; which activates C) were administered intravenously to phosphate-buffered saline-treated and complement-depleted [Cobra venom factor (CVF)-treated] rats and assessed for clearance from the circulation. In control rats, mIgA was cleared in a biphasic fashion with a first half-life (T1/2) of 29.5 +/- 14.2 min and a second T1/2 of 230 +/- 176 min. No differences were observed in clearance of mIgA in CVF-treated rats as compared to PBS-treated rats. In PBS-treated rats, aIgA with a size between 20 S and 150 S disappeared very rapidly from the circulation with a first T1/2 of 1.1 +/- 0.4 min and a second T1/2 of 23.2 +/- 11.3 min. In CVF-treated rats the clearance of aIgA was significantly delayed as compared to that in control rats, namely with a first T1/2 of 7.3 +/- 2.6 min and a second T1/2 of 64.2 +/- 19.4 min. Immunohistochemical studies of the liver (which is the main site of clearance of aIgA) revealed that Kupffer cells (KC) are mainly responsible for the uptake of aIgA. Furthermore, in PBS-treated rats aIgA deposition was accompanied by C3 deposition in the KC. In CVF-treated rats, the percentage of KC containing aIgA was significantly lower during the first 16 min after aIgA administration as compared to PBS treated rats. In addition no detectable C3 was found in KC of CVF-treated rats. These results indicate that KC play an important role in the clearance of large molecular weight IgA in rats and that C facilitates the clearance of these complexes from the circulation.     

Kupffer cell depletion in vivo results in clearance of large-sized IgA aggregates in rats by liver endothelial cells.

We investigated the clearance kinetics and tissue distribution of different sized IgA in normal and macrophage-depleted rats. Rats were injected iv with liposomes containing dichloromethylene diphosphonate (DMDP). DMDP treatment resulted in complete depletion of liver macrophages 24-48 h after administration. Normal and macrophage depleted rats were injected intravenously with monomeric, dimeric, polymeric or aggregated polymeric IgA (AIgA) and assessed for blood clearance and tissue distribution. In normal rats, clearance of IgA was size dependent, i.e. a faster clearance with increasing size. No differences in clearance kinetics were observed of the different sized IgA between normal and DMDP-treated rats. TCA non-precipitable radioactivity, a measure for degradation of IgA, was found in the circulation of normal and DMDP-treated rats after AIgA administration. The liver was the main organ responsible for the clearance of IgA in normal and DMDP-treated rats. Immunofluorescence studies on liver biopsies indicated that AIgA was associated with Kupffer cells in normal rats. Electron microscopical studies revealed that the AIgA was internalized and located in vesicles in Kupffer cells. In DMDP-treated rats the AIgA was associated with endothelial cells and electron microscopy studies showed that this AIgA was taken up by endothelial cells. These data show that rat liver endothelial cells are able to bind, internalize and degrade AIgA in situations where Kupffer cells are absent, and that these cells may play an important role in the handling of AIgA and IgA-immune complexes.     

Bradykinin enhances Sindbis virus infection in human brain microvascular endothelial cells

Sindbis virus (SINV) induces inflammatory and vasoactive responses that are associated with rash and arthritis in human infections. The mechanisms underlying infection-associated microvasculopathy are still unknown. We investigated whether endothelial cells infected by SINV are differentially responsive to bradykinin (BK), a potent inducer of inflammatory edema in a broad range of infectious diseases. Human endothelial cells (HBMECs) infected with SINV presented an upregulation of bradykinin B2 receptors (BK2R) expression. Also, BK reduced SINV-induced apoptosis and enhanced virus replication in HBMECs in a way dependent on BK2R, PI3 kinase and ERK signaling. Strikingly, intracerebral infection of mice in the presence of a BK2R antagonist reduced the local viral load. Our data suggest that SINV infection renders human endothelial cells hypersensitive to BK, which increases host cell survival and viral replication. Ongoing studies may clarify if the deregulation of the kinin pathway contributes to infection-associated vasculopathies in life-threatening arbovirus infections.     

Binding and internalization of human IgG by living cultured endothelial cells

Interactions between circulating IgG and endothelial cells (EC) in humans have been described only in conditions associated with pathologic immunoglobulins and/or activated or damaged EC. In this study we provide evidence that normal human IgG includes one/some antibody species that bind to and are internalized by living EC in culture. This novel function of EC and natural autoantibodies is of potential importance for the understanding of physiologic interactions between vessels and the immune system and for the clarification of pathogenesis of vasculitis and mechanisms of action of pooled IgG used in the therapy of such conditions.     

IL‐4 induces protection of vascular endothelial cells against killing by complement and melittin through lipid biosynthesis

We have shown previously that cytokines IL‐4 and IL‐13 induce protection in porcine vascular endothelial cells (EC) against killing by the membrane attack complex (MAC) of human complement. This protection is intrinsic, not due to changes in complement regulatory proteins, and requires activation of Akt and sterol receptor element binding protein‐1 (SREBP‐1), which regulates fatty acid and phospholipid synthesis. Here we report that, compared to EC incubated in medium, IL‐4‐treated EC had a profound reduction in complement‐mediated ATP loss and in killing assessed by vital dye uptake, but only a slight reduction in permeability disruption measured by calcein release. While controls exposed to complement lost mitochondrial membrane potential and subsequently died, protected EC maintained mitochondrial morphology and membrane potential, and remained alive. SREBP‐1 and fatty acid synthase activation were required for protection and fatty acid and phospholipid synthesis, including cardiolipin, were increased after IL‐4 stimulation, without increase in cholesterol content or cell proliferation. IL‐4 also induced protection of EC from killing by the channel forming protein melittin, similar to protection observed for the MAC. We conclude that IL‐4 induced activation of Akt/SREBP‐1/lipid biosynthesis in EC, resulting in protection against MAC and melittin, in association with mitochondrial protection.     

Measurement of endothelial metabolic functions in vivo

Lung metabolic functions include the interaction of microvascular endothelium with blood-borne substances such as physiologically important amine, eicosanoid, and peptide hormones and drugs. This activity is mediated by endothelial transport systems and enzymes which either synthesize or degrade these substances. Because they can alter the hormone content of aortic blood, these functions play a role in homeostasis, and their disturbance has been implicated in the pathogenesis of several forms of lung injury and disease. Both steady-state infusion and single injection, multiple indicator dilution techniques have been applied to measure endothelial metabolic functions in the intact lung. Considerable progress has been made in development of mathematical models for the processes, and their application has been tested both under normal conditions and also when the lung is perturbed experimentally. Unique experimental challenges are presented by measurement of metabolic functions in vivo, when steadystate infusion techniques cannot be used because systemic toxicity could result. In this case, the bolus injection approach has been used, with some success, both in laboratory animals and man. Although major challenges remain, their solution is essential if we are to apply knowledge of endothelial cell function in vitro to understanding lung microvascular physiology and pathophysiology in the intact animal.     

In vivo degradation of rat C1q induced by intravenous injection of soluble IgG aggregates.

Immune complexes are able to bind and activate the first component of complement, C1. Upon activation of C1, C1r and C1s are rapidly inactivated by C1-In which also forms a complex with these two subcomponents, resulting in their release from C1-immune aggregate complexes. The fate of C1q after the binding C1 to immune complexes in vivo is not clear and, therefore the clearance of radiolabelled rat C1q was investigated in normal rats and in rats receiving soluble aggregated human IgG. 125I-labelled rat C1q was cleared with a half-life (T 1/2) of 12.4 hr in normal rats. Injection of AIgG into rats that had previously received 125I-C1q accelerated the clearance of 125I-C1q, resulting, finally, in a T 1/2 of 53 min. The levels of circulating endogenous C1q were also followed using haemolytic titrations and immunochemical measurements. Directly after injection of AIgG into rats, there was a rapid decrease in C1q haemolytic activity to less than 25% of the initial value after 10 min. The rate of disappearance of C1q antigen, was, however, much slower, the lowest concentration being 30% at 2 hr. C1q haemolytic activity and the C1q antigen level returned to virtually normal values after 24 hr. Plasma samples were taken at different time intervals after the injection of AIgG and subjected to gel filtration on Sephacryl S-400 columns. It was found that, in the 10 min samples, C1q antigen and C1q haemolytic activity, each with an estimated molecular weight (MW) of 400,000, were detected together. In addition, there was C1q antigen with a MW of less than 69,000 without C1q haemolytic activity. SDS-PAGE analysis of the various serum samples indicated that the low MW C1q antigen had an apparent MW of 25,000. Measurement of uptake of 125I-C1q in various organs indicated that the main site of clearance of 125I-C1q is the liver.     

Human preformed IgG combining with membrane-bound porcine serotransferrin lyse porcine endothelial cells through antibody-dependent cellular cytotoxicity

Preformed antibodies are involved in xenograft rejection. The purpose of this work was to characterize porcine xenoantigens recognized by human preformed IgG (hpIgG), and to investigate the role of hpIgG in xenogeneic rejection. IgG eluted from porcine livers perfused with human plasma, human sera and total human IgG were immunoblotted on porcine aortic endothelial cell extracts. The amino acid sequence of a 76-kDa antigen constantly revealed was 100 % homologous with porcine serotransferrin (psTf). hpIgG from human sera, human IgG1 and IgG2 and F(ab ′ )₂ γ specifically bound to psTf. Neutralization by psTf abolished that binding. Although α1, 3-linked galactose residues (Galα1,3Gal) is the dominant epitope recognized by preformed antibodies in the swine-to-human combination, the analysis of carbohydrate composition of psTf showed that the molecule was devoid of Galα1,3Gal moieties and that preformed anti-psTf IgG bound to epitopes localized on the peptide core of the molecule. Purified human anti-psTf IgG antibodies were able to bind to psTf linked to its receptor on porcine endothelial cells, and to kill those cells through antibody-dependent cellular cytotoxicity.     

Role of liver endothelial and Kupffer cells in clearance of human Clq in rats

In the present study the contribution of rat liver endothelial cells (EC) and Kupffer cells (KC) in the clearance of human (hu) Clq in rats was investigated. In untreated rats and rats depleted from KC the clearance kinetics and the tissue distribution of hu Clq were measured. In untreated rats, the clearance of hu Clq occurred in a monophasic manner with a half-life of 66 ± 26.7 min. The clearance of hu Clq in KC-depleted rats was delayed significantly (p<0.001) and occurred with a half-life of 217 ± 78.8 min. Fifteen min after injection, 11 ± 3.5% of hu Clq was found in the liver of untreated rats and 8 ± 1.4% was found in the liver of KC-depleted rats. The percentage non-trichloroacetic acid precipitable activity in the circulation, as a measure for degradation of Clq, reached a level of 11.6 ± 5.6% at 240 min in untreated rats compared with 4.6 ± 5.8% in KC-depleted rats. Double immunofluorescence staining 5 min after administration of Clq in untreated rats, revealed that Clq was associated with KC and EC in the liver. Fifteen minutes after i.v. injection of hu Clq, there was an uptake of Clq in the hepatocytes. In KC-depleted rats, 5 min after administration of hu Clq, Clq was bound to the EC. Fifteen minutes after injection, Clq was also found in the hepatocytes. Electron microscopical studies revealed that Clq binds to EC, and that it is internalized in the hepatocytes and KC. The clearance of hu Clq in untreated rats was inhibited by preadministration of high concentrations of bovine C1q. These data show that rats depleted from KC are able to bind, internalize and degrade Clq, and that EC may play a role in the handling of Clq and Clq bound to immune complexes.     

Complement-dependent synergistic effects of rat monoclonal IgG antibodies in vivo

We describe studies aimed at maximizing the effector mechanisms responsible for eliminating target erythrocytes from the circulation in a fully homologous opsonization system in vivo. The effects on the subsequent fate of target erythrocytes were examined in both normal and decomplemented rats preinjected with a variety of rat IgG monoclonal antibodies (mAb) directed against different epitopes on the RTlA^a, the classical class I major histocompatibiliy complex antigen of the DA rat. In general, the clearance of both DA and (DA × PVG)F₁ erythrocytes in normal rats preinjected with various pairs of noncompetitive mAb was very rapid when compared with the overall clearance patterns seen with individual antibodies. With all mAb combinations containing IgG2b or IgG2a, an intact complement system was an essential requirement for augmenting the initial clearance and promoting hepatic sequestration of these target cells. The removal of (DA × PVG)F₁ erythrocytes, expressing half as much antigen, was considerably slower than the DA cells for each antibody pair tested although a notable degree of heterogeneity was observed in the overall behavior of both types of target cells with different mAb combinations. Our results suggest that the limiting effects of low antigen density on the target cells combined with the use of mAb of an isotype like the rat IgG2a can be overcome using pairs of mAb that recognize different epitopes on the same target antigen.     

The production of small IgG aggregates by glutaraldehyde cross-linking

Reaction conditions have been determined for the production of soluble IgG polymers in the size range 10 S to 30 S by covalent cross-linking with glutaraldehyde. This size range is comparable with that of the immune complexes which are frequently found in the circulation of patients with certain autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. The yield of IgG aggregates in this size range is far greater than has been reported for cross-linking by other bifunctional reagents or for aggregation by heating. Glutaraldehyde cross-linked IgG polymers are stable and biologically reactive. They can also be labelled with fluorescein and freeze-dried with minimal loss of integrity or reactivity.     

大鼠胰岛微血管内皮细胞分离、纯化与培养的改进

目的：建立稳定可靠的大鼠胰岛微血管内皮细胞分离培养方法。方法：分离纯化大鼠胰岛后进行内皮细胞选择性培养，使用UEA-1包被的免疫磁珠对胰岛微血管内皮细胞进行纯化。免疫荧光法检测经典的内皮细胞标志物第Ⅷ因子相关抗原（vWF）和CD34的表达和吞噬Dil标记的乙酰化低密度脂蛋白（Dil-Ac-LDL）能力。结果：胰岛培养4-5 d后，可见内皮细胞从贴壁的胰岛内爬出，通过UEA-1包被的免疫磁珠筛选出胰岛微血管内皮细胞接种后24 h细胞开始贴壁。该细胞具有单层生长、接触抑制的特性。大鼠胰岛微血管内皮细胞表达vWF和CD34，可摄取Dil-Ac-LDL。结论：本研究方法是一种较为高效的分离、纯化和培养大鼠胰岛微血管内皮细胞的方法。     

内毒素休克小鼠肝脏血管内皮细胞特异性结合肽的体内筛选及初步鉴定

目的：利用体内噬菌体展示技术筛选能与内毒素休克小鼠肝脏血管特异性结合的短肽。方法:尾静脉注射噬菌体环七肽库至内毒素休克小鼠体内，循环10 min后，回收肝脏中的噬菌体。将回收的噬菌体扩增、纯化，并以此为起始物进行下一轮筛选。经过4轮筛选后，将所得文库与正常小鼠做一次差减筛选。随机挑取噬菌体单克隆进行测序，序列分析后，进行噬菌体的体内回输实验及免疫组化分析，验证所筛得噬菌体克隆的导向情况。结果:经过4轮体内筛选，肝脏中噬菌体回收率逐步提高，证明噬菌体文库在肝脏血管中得到有效富集。DNA测序后发现，10条序列中有5条为LTTWAPA，体内回输实验和免疫组化实验均证实表达该序列的噬菌体能有效地靶向于休克小鼠肝脏血管内皮细胞。结论:筛选到的短肽LTTWAPA能特异性结合于内毒素休克小鼠肝脏血管内皮细胞，有可能对内毒素休克的治疗具有重要意义。     

Paracrine mechanisms of endothelial progenitor cells in vascular repair

Endothelial progenitor cells (EPCs) play an important role in repairing damaged blood vessels and promoting neovascularization. However, the specific mechanism of EPCs promoting vascular repair is still unclear. Currently, there are two different views on the repair of damaged vessels by EPCs, one is that EPCs can directly differentiate into endothelial cells (ECs) and integrate into injured vessels, the other is that EPCs act on cells and blood vessels by releasing paracrine substances. But more evidence now supports the latter. Therefore, the paracrine mechanisms of EPCs are worth further study. This review describes the substances secreted by EPCs, some applications based on paracrine effects of EPCs, and the studies of paracrine mechanisms in cardiovascular diseases--all of these are to support the view that EPCs repair blood vessels through paracrine effects rather than integrating directly into damaged vessels.     

黄酒对同型半胱氨酸诱导的内皮祖细胞功能障碍的影响

目的:探索同型半胱氨酸(Hcy)诱导的内皮祖细胞(EPCs)的功能障碍能否被黄酒所逆转。方法:密度梯度离心法从大鼠骨髓细胞悬液中获取单个核细胞,将其接种在人纤维连接蛋白包被的培养板,诱导单个核细胞向EPCs分化,孵箱中培养7 d后,收集贴壁细胞用于实验。激光共聚焦显微镜鉴定Di I-ac-LDL和FITC-UEA-I双染色阳性细胞被认定是正在分化的EPCs。上述细胞培养24 h后收集样品,采用MTT比色法、Transwell小室、凋亡试剂盒和体外血管生成试剂盒分别观察EPCs的活力、迁移能力、凋亡和体外血管生成能力。结果:与对照组相比,Hcy干预下EPCs的活力、迁移和体外血管生成能力均显著减弱(P0.01);黄酒或红酒的干预则能明显改善Hcy对EPCs上述功能的影响(P0.01);进一步与对照组比较后,发现黄酒或红酒的干预还能使EPCs的活力、迁移和体外血管生成能力较对照组也有明显改善(P0.05);乙醇组EPCs的上述功能较Hcy组没有明显的变化。各组对EPCs的凋亡没有影响。结论:Hcy能显著减弱EPCs的活力、迁移能力和体外血管生成能力,小剂量的黄酒能改善EPCs的上述功能。     

大鼠骨髓内皮祖细胞的分离、培养及鉴定

目的 研究大鼠骨髓来源的内皮祖细胞(EPC)的分离、鉴定、培养方法以及向内皮细胞分化的诱导条件.方法 密度梯度离心法分离SD大鼠股骨及胫骨单个核细胞,经差速贴壁后取2次贴壁细胞置于纤连蛋白铺被的培养板中.在血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)及表皮生长因子(EGF)诱导下培养两周,观察其形态学改变,以免疫细胞化学染色及Dil-acLDL、FITC-UEA-1双荧光染色法对其进行鉴定.结果 贴壁细胞呈现铺路石、团簇样生长,呈梭形、线样排列特殊形态.免疫细胞化学结果显示,贴壁细胞CD133、CD34、Flk-1、血管性假血友病因子(vWF)在不同时段呈阳性表达.诱导培养第2天,CD133阳性表达率为(79.4±4.5)%.自诱导培养的第6天开始,Flk-1与vWF呈高表达,阳性率为(74.2±3.5)%和(72.2±4.3)%.结论 用Percoll密度梯度离心法在VEGF、bFGF及EGF培养体系下可以获得较高纯度的EPC,该细胞具有内皮细胞的特性,经过体外诱导可以分化为内皮细胞.     

血管内皮生长因子对外周血内皮祖细胞功能的影响

目的: 观察不同浓度血管内皮生长因子(VEGF）对体外培养人外周血内皮祖细胞（EPCs）生物学功能的影响，探讨VEGF促进EPCs分裂生长的合适浓度。方法: 密度梯度离心法获取人外周血单个核细胞（MNCs），接种至人纤维连接蛋白（HFN）包被的培养板上，培养4 d后收集贴壁细胞，换用配有不同浓度（对照组、10 μg/L、20 μg/L、50 μg/L）VEGF的培养基继续培养3 d后进行细胞免疫组化鉴定EPCs，采用MTT比色法、改良的Boyden小室和黏附能力测定实验，观察EPCs的增殖、迁移和黏附能力。结果: 从人外周血能成功分离EPCs细胞，并能分化为血管内皮细胞；从冠心病患者分离的EPCs增殖能力较非冠心病患者弱；VEGF在较低浓度（10 μg/L和20 μg/L）时即能显著促进EPCs生长的各项生物学指标，但高浓度（50 μg/L）时并不能进一步增强这一效应；低浓度（10 μg/L）下VEGF对冠心病患者作用较非冠心病患者弱，高浓度时对两者促进作用相近。结论: 冠心病患者EPCs功能显著减弱，较低浓度的VEGF即可显著增强EPCs的各项生物学功能，可能对损伤血管的再内皮化有益。     

大鼠心肌微血管内皮细胞的培养及基因芯片分析的研究

目的：用血管内皮细胞生物学功能基因芯片研究培养的大鼠心肌微血管内皮细胞特征。 方法： 用植块法培养大鼠心肌微血管内皮细胞，利用倒置显微镜、扫描电子显微镜和透射电子显微镜观察培养细胞的形态学特征；利用免疫细胞化学方法显示血管内皮细胞特异性表面标志；用细胞计数方法绘制细胞生长曲线；用血管内皮细胞生物学功能基因芯片研究细胞基因表达谱，并比较原代细胞和传代细胞基因表达的变化。 结果： 利用植块法培养的大鼠心肌微血管内皮细胞具备多种典型微血管内皮细胞（MVEC）特征：细胞呈多边形或梭形并呈铺路石样生长；存在管腔样结构（TLS）和毛细血管网络，细胞表面有丰富的微绒毛； CD34、CD31、CD105 、vW因子和Tie-2阳性；多种与正常血管功能密切相关的基因不同程度表达，前2代细胞特征稳定。 结论： 用植块法培养的大鼠心肌微血管内皮细胞，具备微血管内皮细胞的特征，原代和第2代培养细胞特征稳定，可用于心血管疾病的基础与临床研究。