Microvascular injury and repair in acute human bacterial pyelonephritis

Summary Acute inflammatory cell-capillary endothelial cell interactions, related to injury and repair, were investigated light and electron microscopically in acute human bacterial pyelonephritis. In inflammatory infiltrate-adjacent microvessels, the small capillaries were completely occluded by leukocyte plugs and the large capillaries were densely filled with acute inflammatory cells adhering to the endothelium. Severe damage to small and large capillaries was observed around endothelium adherent, degranulated neutrophil granulocytes containing phagocytosed bacteria. There were spaces in the endothelium, degradation of the vascular basement membrane, of the perivascular interstitial matrix and of collagen fibrils, with fibrin deposition and vessel wall fragmentation. In the small capillaries relatively distant from the interstitial infiltrates, emigration of leukocytes was frequently seen. Around the escaping cells the endothelial lining displayed occasional discontinuities, allowing leakage of vascular fluid into the interstitial space. Some small capillaries not related to the infiltrate were occluded by fibrin thrombi with apparent damage to the endothelial cells and disruption of the capillary wall. Various reparative changes were noticed in association with this change including capillary neovascularization. The findings confirm the existence of polymorphonuclear leukocyte-mediated injury of capillaries during the development of inflammatory responses in acute pyelonephritis.Some parts of this paper were presented at the XVIth Congress of the IAP, Vienna, Austria, August 31–September 5, 1986A Fellow of the Deutscher Akademischer Austauschdienst, Bonn, FRG     

Descemet's membrane detachment associated with inadvertent viscoelastic injection in viscocanalostomy

We report a case of Descemet's membrane detachment, a rare complication of viscocanalostomy. During the operation, the injection cannula was directed slightly oblique to the Schlemm's canal rather than parallel to it. Localized corneal whitening developed adjacent to the injection site during viscoelastic injection. One week postoperatively, corneal edema decreased and Descemet's membrane detachment was noted. Nine months after surgery, the cornea was clear while the Descemet's membrane detachment remained. And IOP was 19 mmHg without any medications. We think that improper cannula positioning during viscoelastic injection may cause Descemet's membrane detachment, a rare complication of viscocanalostomy.     

Functional reconstruction of corneal endothelium using nanotopography for tissue-engineering applications

Dysfunction in the corneal endothelium, which controls the hydration and transparency of the cornea, is one of the common reasons for transplantation. A tissue-engineered corneal endothelium is of interest for corneal regeneration and for in vitro testing of ocular drugs. In the native environment, corneal endothelial cells interact with the nanotopography of the underlying Descemet's membrane. This study showed that nanotopography enhanced bovine corneal endothelial cell (BCEC) responses, creating a monolayer which resembled the healthy corneal endothelium. Topographies of different geometries were first tested to identify those that would elicit the most significant responses. A BCEC monolayer was then generated on both micro- and nanoscale pillars and wells. The BCEC monolayer cultured on topographies exhibited polygonal geometries with well-developed tight junction proteins. Scanning electron microscopy revealed that cells on pillars showed a higher density of microvilli, which was similar to native corneal endothelium. BCECs on nanopillars displayed a lower coefficient of variation of area (0.31) that was within the range of healthy corneal endothelium. More importantly, a BCEC monolayer cultured on nanopillars also had an enhanced Na(+)/K(+)-ATPase immunofluorescence expression, mRNA upregulation and a higher Na(+)/K(+)-ATPase activity. These results suggest that nanopillar substrate topography may provide relevant topographical cues, which could significantly enhance the formation and function of corneal endothelium.     

Rabbit corneal damage produced by Pseudomonas aeruginosa infection.

Gross, light microscopic, and electron microscopic examination of the rabbit corneal destruction produced by experimental Pseudomonas aeruginosa infections revealed a combination of acute inflammation and liquefaction necrosis of the cornea. Degeneration of the epithelial cells and the start of polymorphonuclear leukocyte infiltration of the cornea occurred initially. These changes were followed by loss of the epithelium, degeneration and loss of the keratocytes and endothelium, loss of the characteristic weblike pattern of the proteoglycan ground substance, dispersal of ultrastructurally normal collagen fibrils, extensive accumulation followed by degeneration of polymorphonuclear leukocytes, and accumulation of plasma proteins and fibrin in the necrotic cornea. Histochemical examination of the cornea suggested a loss of the proteoglycan ground substance but not of collagen. Rabbit corneas injected with Clostridium histolyticum collagenase showed gross and cellular changes similar to those observed during the pseudomonal infections; however, histochemical examination suggested a loss of collagen, and electron microscopy revealed ultrastructurally abnormal collagen fibrils. The results support the idea (i) that a bacterial or host-derived collagenase is not required for extensive corneal damage during a P. aeruginosa corneal infection, and (ii) that a P. aeruginosa corneal infection may severly damage the cornea by producing extensive corneal edema and by causing the loss of the corneal proteoglycan ground substance, thus resulting in dispersal of undamaged collagen fibrils, weakening of the cornea, and subsequent descemetocele formation and corneal perforation by the anterior chamber pressure.     

房水中转化生长因子水平与角膜内皮损伤及修复的关联分析

目的 探讨房水中转化生长因子水平与角膜内皮损伤及修复的关联.方法 研究选取本院就诊患者102例,检测患者房水中总TGF-β2和活化TGF-β2的水平,监测分析角膜内皮细胞密度水平、角膜内皮六角形细胞的比例水平以及中央角膜厚度.分析讨论房水中TGF-β2水平与角膜内皮损伤及修复的关联.使用SPSS软件处理数据.结果 对治疗前总TGF-β2水平与表征角膜内皮细胞损伤状态的四项指标水平进行相关分析结果提示,治疗前研究样本总TGF-β2水平与角膜内皮细胞密度之间的相关系数为-0.123、于角膜内皮六角细胞水平之间的相关系数为-0.198,另外治疗前总TGF-β2水平与中央角膜厚度水平之间的相关系数为0.254,且均P＜0.05;治疗前活化TGF-β2水平与表征角膜内皮细胞损伤状态的指标水平相关分析结果也提示了类似结果,除了活化TGF-β2水平与内皮细胞密度的r为-0.145,未见统计学意义之外,活化TGF-β2水平与其他指标的相关系数均有统计学意义.治疗后总TGF-β2水平以及活化TGF-β2水平与四项指标的相关系数也发现了类似规律,且均P＜0.05.结论 总TGF-β2水平以及活化TGF-β2水平增高与角膜内皮损伤有关,与内皮细胞密度、角膜内皮六角细胞比例下降,中央角膜厚度则会增加密切相关.     

脱水猪角膜基质为载体体外构建猫角膜内皮组织

目的:以脱水猪角膜基质为载体体外构建猫角膜内皮组织,并观察其形态结构,以寻找体外构建角膜组织最佳载体材料及方法,最终目的是体外构建可用于移植的角膜内皮组织。方法:以脱水处理的猪角膜基质片为载体,将猫角膜内皮细胞接种于去除猪角膜内皮细胞的后弹力膜上,在添加了表皮生长因子和层粘连蛋白的培养液中培养7 d,分别观察倒置显微镜下、组织学切片及扫描电镜下内皮组织的形态结构。结果:倒置显微镜下,组织培养的猫角膜内皮细胞形成单层内皮组织,排列较规律,细胞间连接紧密;组织学观察发现,培养的猫角膜内皮细胞形成完整的内皮组织,贴附于脱水基质的后弹力膜上,与正常的角膜内皮组织结构相似;扫描电镜下,组织培养的猫角膜内皮细胞间连接紧密,细胞大小不甚一致,胞核清晰。 结论:以脱水猪角膜基质为载体,体外成功构建出猫角膜内皮组织,其形态结构近似于正常角膜内皮组织。     

Leukocyte adhesion in host defense and tissue injury.

During their life span, leukocytes adhere transiently to one another, to other cell types, such as vascular endothelial cells, and to extracellular matrix proteins. This adhesiveness is mediated by families of specific cell surface adhesion molecules, namely, integrins, immunoglobulin superfamily molecules, and selectins. Adhesion is required for leukocyte-mediated cytotoxicity, phagocytosis, chemotaxis, and induction of lymphocyte proliferation and maturation. It also participates in recirculation and homing of lymphocytes into lymphoid organs and in leukocyte migration from the vascular compartment to extravascular tissues. Adhesion underlies the beneficial or detrimental role of leukocytes in immune and inflammatory responses. In animals, blocking monoclonal antibodies to adhesion molecules dramatically reduce vascular and tissue injury in several organs following ischemia-reperfusion, and delay renal allograft rejection. Moreover, expression of particular adhesion molecules is induced or increased in cells which are targets for allergic or autoimmune reactions and in inflamed tissues. On the other hand, a congenital deficiency of the CD11/CD18 integrins (Leu-CAMs) leads to recurrent, and sometimes fatal, bacterial infections, and lack of particular cell-adhesion molecules on Burkitt's lymphoma cells may enable these cells to escape immunosurveillance.     

兔角膜内皮细胞载体的体外培养及移植的研究

以特定曲率的壳聚糖-硫酸软骨素共混膜为载体，构建兔角膜内皮。研究了共混膜的透光性、体外酶降解性以及兔角膜内皮细胞在载体上的细胞贴附性、细胞形态及膜强度等性质。结果表明，共混膜的透光率达90％以上，生物降解性良好。该共混膜有良好的细胞贴附性及机械强度；兔角膜内皮细胞可在共混膜上长成良好的单层，细胞形态较好，并贴附牢固，振荡后细胞无脱落，膜片保持完整。将培养好的载体植入到去除内皮层的兔角膜中，术眼在56天内基本保持透明，说明体外构建的内皮可执行角膜内皮层的部分功能。     

Will Descemet’s stripping with automated endothelial keratoplasty (DSAEK) lower the rates of allograft rejection in corneal transplants for endothelial failure?

BACKGROUND: Allograft corneal rejection occurs in a substantial number of full thickness transplants in spite of the relative immune privilege enjoyed by the cornea. Compared to other layers of the cornea, endothelial rejection has most disastrous consequences on graft survival. In the last few years, a new technique, Descemet's stripping with automated endothelial keratoplasty (DSAEK) is being used of selective transplantation of the endothelium. It involves stripping diseased endothelium (and Descemet's) and replacing it by a small lamella fashioned from a cadaveric donor cornea, which consists of endothelium, Descemet's membrane and a part of posterior stoma. HYPOTHESIS: We hypothesize that DSAEK might substantially reduce the incidence of allograft immune rejection in corneal transplant done for cases with endothelial failure. EVALUATION OF THE HYPOTHESIS: In published reports of consisting of more than 300 surgeries and three years experience with DSAEK, no case of graft rejection has been reported. In our opinion, this advantage of DSAEK compared to conventional full thickness keratoplasty could be due to four factors: (a) The transplanted tissue is placed in the anterior chamber and has no exposure to the surface, where the antigen presenting cells (APC) and antibodies are present. (b) Significant reduction in the number of sutures connecting the host and donor tissue may lead to lesser suture related rejection episodes. (c) Absence of direct contact between the host stroma vessels and the transplanted tissue disrupts the immune affecter and effecter arcs. (d) Reduced immunogenicity of the donated tissue due to absence of epithelium. CONSEQUENCES OF THE HYPOTHESIS: If this hypothesis stands true in subsequent studies, it could lead to substantial reduction in the socioeconomic resources involved in management of graft rejection. Even if this hypotheses fails the test of well controlled studies, this would broaden the current understanding of the ocular immunology and the immune privilege with which the anterior chamber of the eye is normally associated with.     

Morphologic correlates of glomerular oxidant injury induced by the myeloperoxidase-hydrogen peroxide-halide system of the neutrophil

Neutrophilic polymorphonuclear leukocytes can mediate glomerulonephritis by releasing reactive oxygen species such as H2O2. We have previously demonstrated that H2O2-mediated glomerular injury can be potentiated by reaction with polymorphonuclear leukocyte myeloperoxidase (MPO). When MPO was perfused into renal arteries of rats, it bound to the glomerular capillary wall due to its cationic charge. Subsequent perfusion with nontoxic concentrations of H2O2 and halides resulted in acute glomerular injury, halogenation of the glomerular basement membrane, and proteinuria. The studies reported here document the morphologic changes that accompany MPO-mediated glomerular injury. Acutely, there is severe injury to the endothelium with cell swelling and lysis. Within 10 minutes, a marked platelet influx occurs. Platelets frequently occlude capillary lumens and bind to areas of denuded glomerular basement membrane where platelet degranulation results. By 4 days, the platelet infiltration has ceased, and a reparative phase develops characterized by marked proliferation of resident endothelial cells and possibly mesangial cells. By 21 days postperfusion, the glomerular lesion had largely resolved. In contrast, control rats perfused with MPO alone, H2O2 alone, or buffered saline alone demonstrate minimal glomerular injury at all times studied. MPO-mediated glomerular disease results in endothelial and mesangial cell injury, activation of platelets, and a subsequent proliferative response. These morphologic changes resemble those seen in several forms of inflammatory and proliferative glomerulonephritis in man.     

Autoradiographic study of corneal neovascularization induced by chemical cautery

The corneas of adult rats were cauterized chemically, and the responses of the pericorneal blood vessels and the cellular constituents of the cornea were followed by light microscopic autoradiography after labeling with 3H-thymidine. As in previous experiments, this injury elicited a neovascularization as capillaries sprouted and extended centripetally from the corneoscleral limbus to the cautery site. Chemical cautery induced a response in the epithelium, endothelium, and fibroblasts of the cornea as well as in the vascular cells. Elevated labeling indices for the corneal epithelium and endothelium began at 18 and 21 hours after injury, respectively. In all of these corneal cell types, the labeling index returned to control values by 75 hours. The onset and decline of DNA synthesis in corneal fibroblasts paralleled that of the corneal epithelium and endothelium. Labeling indices of vascular cells (endothelial cells and pericytes) increased 21 hours after injury, reached a maximal level at 45 hours, and returned to control values by 1 month after cautery. The first mitoses in vascular endothelial cells and pericytes were noted 36 hours after injury, and the initial capillary sprouts appeared at 39 hours. This study demonstrates that the thymidine incorporation by cells in the pericorneal blood vessels occurs early within the postcauterization period, at least 15 hours before the first mitotic figures are detected in these same vascular cells. The significance of the temporally related elevations in labeling indices of the vascular cells and the cellular constituents of the corneal cells is uncertain, but there are many potential interrelationships between the controls of cell division and migration for cells of the vessels, epithelium, endothelium, and corneal stroma.     

A case of Peters' anomaly in a springer spaniel.

An 8-week-old springer spaniel presented with a large central corneal opacity of the left globe, which was accompanied by cords of tissue spanning from the iris collarette to the posterior cornea. A posterior cortical cataract was noted in the right eye. At the owner's request the puppy was humanely destroyed, and a necropsy was performed. Upon sectioning the left globe in the vertical plane, a circle of pigmented strands of tissue was observed spanning the anterior chamber from the iris to the posterior aspect of the cornea. The right globe appeared normal when inspected grossly. Histologically, a membrane of pigmented tissue covered the posterior aspect of the broad central corneal leukoma of the left globe. This membrane and the cords traversing the anterior chamber were composed of vascular uveal tissue. Descemet's membrane and the corneal endothelium were reduced or absent in the zone of corneal opacity. Other than the changes associated with cataract, the right globe was histologically normal. The clinical and histological findings in the left globe were identical with those described for Peters> anomaly in human beings.     

Hydrodynamic injury of the endothelium in acute aortic stenosis.

The acute effects of increased shear stress on the endothelium were studied by reducing the lumen of the rat aorta to 20-25% of normal by means of metal clips. Intimal damage in the stenotic area was assessed by light microscopy after perfusion with AgNo3 and study of the endothelium en face. Most of the endothelium was lost within 3 minutes; the extent of the damage was not increased after 1 hour. Electron-microscopic examination showed that some endothelial cells became permeable to tracers (thorium dioxide and horseradish peroxidase); platelets adhered to the exposed internal elastic membrane. Focal endothelial changes were represented by myelin figures of various kinds arising from the luminal surface and by "cellular ulcers," superficial erosions of the endothelial cells accompanied by localized cytoplasmic changes. These "ulcers" occurred more frequently over the nucleus and near junctions; they have not been described in other forms of arterial injury.     

Electron microscopy of the canine corneal basement membranes

The purpose of this study was to characterize the surface topographical features of the epithelial and endothelial (Descemet's) basement membranes of the canine cornea. Corneas were obtained from young, healthy dogs (<2 years old) with no history or evidence of previous ocular disease. The epithelium and endothelium was carefully removed preserving the anterior and posterior basement membranes. The specimens were examined by transmission electron microscopy and scanning electron microscopy. The epithelial and endothelial basement membrane surface topography is an intricate meshwork of pores and fibers measuring in the nanometer size range. The features of the endothelial basement membrane overall are smaller in size than the epithelial basement membrane. These surface topographical features may incite changes in epithelial and endothelial cell behavior.     

丹参单体IH764-3通过抑制白细胞激活产物减轻人脑微血管内皮细胞的缺氧-复氧损伤

目的探讨丹参单体IH764-3在白细胞介导的脑内皮细胞缺氧-复氧损伤中的作用及机制。方法通过MTT法检测人脑微血管内皮细胞(HBMVEC)的存活;明胶酶谱法检测白细胞MMP-9的释放;荧光定量法检测白细胞内活性氧水平;ELISA法检测白细胞释放细胞因子的水平。结果缺氧-复氧使HBMVEC存活下降,当复氧时加入白细胞激活产物使损伤加重,同时加入IH764-3则具有保护作用。缺氧-复氧使白细胞释放MMP-9增加;细胞内活性氧水平升高;释放的TNF-α、IL-1α、IL-2、IFN-γ增加。IH764-3可呈剂量依赖性减少由缺氧-复氧所致的白细胞分泌MMP-9、ROS和细胞因子增加。结论IH764-3能够减轻白细胞介导的HBMVEC的缺氧-复氧损伤,在脑缺血前及缺血后干预中具有潜在应用价值。     

Exposure of endothelial cells to free heme potentiates damage mediated by granulocytes and toxic oxygen species

Endothelial damage may follow exposure to toxic oxygen species generated by closely apposed ("marginated") granulocytes. Because iron markedly catalyzes oxidant damage in diverse systems, we wondered whether intercalculated heme, and/or its constituent iron, might potentiate oxidant damage of endothelium. Cultured monolayers of porcine aortic endothelial cells were exposed for brief periods to purified hemin. Uptake of heme was rapid, dose dependent, and not reversible by buffer or serum washes. Despite high levels of cell-associated heme, no direct heme-mediated cytotoxicity occurred, but heme-loaded endothelium became highly sensitive to oxidant challenge by (a) reagent H2O2, (b) enzymatically generated oxidants (xanthine/xanthine oxidase), or (c) phorbol-activated polymorphonuclear leukocytes. An increase in endothelial cell lipid peroxidation accompanied heme-augmented oxidant cytolysis, and both parameters were reduced in parallel by micromolar amounts of the hydrophobic oxygen radical scavenger/iron chelator U74500A. Endothelial uptake of heme was inhibited by a specific heme-binding protein, hemopexin. Concomitantly, hemopexin completely blocked augmented H2O2- and polymorphonuclear leukocyte-mediated cytotoxicity but only if added simultaneously and stoichiometrically with hemin. Significant loss of protection occurred if hemopexin addition was delayed 15 minutes, and protection was completely lost after a 60-minute interval. The iron moiety of heme was critical to oxidant sensitization because neither iron-free protoporphyrin IX nor tin-protoporphyrin was able to sensitize endothelial cells to H2O2 or activated polymorphonuclear leukocytes. These results may provide mechanistic insights into atherogenesis, reperfusion injury, and the organ injury accompanying hemoglobinemia or myoglobinemia.     

Demonstration of inflammatory mediator-induced inflammation and endothelial cell damage in the anterior segment of the eye.

Although some investigations have demonstrated the ability of inflammatory mediators, including vasopermeability and chemotactic factors, to induce acute inflammatory reactions in vivo, little is known about the response of various elements of the anterior segment to the direct effects of inflammatory mediators. These studies were initiated to develop models for the investigation of inflammatory responses in this region of the eye. Acute inflammatory reactions were induced within the rabbit anterior chamber by intracameral injection of 50 microliters of various inflammatory mediators and were evaluated by clinical grade, leukocyte influx into the aqueous humor, and morphologic changes in the corneal endothelium. Peak responses were recorded following injection of 10(-4) M formyl-methionyl-leucyl-phenylalanine (fMLP); 5 ED50 C5fr; 0.5 mg/ml C5; undiluted anti-red blood cell (RBC) serum; and 10(-5) M histamine. The number of leukocytes per milliliter of aqueous humor induced by each mediator was quantitated by comparison with the number of leukocytes induced by buffer instillation into a separate group of rabbits (mediator-induced influx/buffer-induced influx). Comparisons were made 24 hours after instillation of mediators. The results of these studies were as follows: buffer alone, 1.0; fMLP, 3.1 C5fr, 61.0; C5, 8.7; anti-RBC, 91.0; and histamine, 24.0. Clinical grades correlated well with these ratios. In addition, differences were noted when the time kinetics of acute responses induced by two different mediators (10(-4) M fMLP, a synthetic preformed chemotactic factor; and a 1:5 dilution of anti-RBC, which binds to vascular and corneal endothelial cells) were directly compared over 48 hours. Responses induced with fMLP peaked between 5 and 8 hours and resolved rapidly, whereas anti-RBC-induced responses peaked between 8 and 12 hours and resolved very slowly. Histopathologic analysis indicated that both fMLP and anti-RBC induced a similar sequence of changes in the corneal endothelium. Within 2-3 hours after instillation of either mediator, the endothelial cells exhibited prominent vacuolization/retraction phenomena. At the peak of leukocyte influx PMNs filled these vacuoles, then migrated back into the aqueous humor within several hours. Normal morphologic features were recovered following clearance of leukocytes from the anterior chamber. We believe that these models will be useful in identifying the roles of individual mediators in acute and chronic endocular inflammation and in the injury of corneal endothelium.     

An evaluation of the role of leukocytes in the pathogenesis of experimentally induced corneal vascularization. II. Studies on the effect of leukocytic elimination on corneal vascularization.

Investigations on several experimental models in the past have supported the hypotheses that corneal vascularization is a manifestation of the inflammatory response and that leukocytes perform an essential role in stimulating corneal vascular ingrowth. To evaluate the possible role of leukocytes further in this phenomenon, the effect of leukocyte elimination on corneal vascularization induced by silver nitrate cauterization was investigated. Weanling Fischer albino rats received doses of total body x-irradiation ranging from 1100 to 2100 rads to deplete circulating leukocytes, and corneal silver nitrate cauterization was performed 4 days later. In this model, animals that received 1500 rads or more total body x-irradiation became severely leukopenic within 4 days. As a rule, neither leukocytes nor blood vessels invaded the cauterized corneas, whereas both a leukocytic and vascular invasion occurred at lower doses of irradiation that did not totally eliminate circulating leukocytes. Corneal vascularization ensued if the corneal cauterization was performed immediately after total body x-irradiation with 1500 rads before the leukopenic effect of x-irradiation occurred. Control studies in which the cornea was cauterized 4 days after only the head received 1500 rads x-irradiation ruled out the possibility of irradiation-induced limbal endothelial damage as the explanation for the vascular suppression observed by x-ray treatment. In nonirradiated rats, silver nitrate cauterization of the cornea consistently induced corneal vascularization by 2 to 3 days. In further experiments, methylprednisolone acetate was administered subconjunctivally after corneal cauterization. This corticosteroid inhibited the infiltration of leukocytes and the subsequent vascular invasion into the corneal stroma, if administered immediately after silver nitrate cauterization. However, when the same glucocorticoid was administered 1 day after cauterization, both a leukocytic infiltration and vascular ingrowth occurred but to a less severe degree than in non-glucocorticoid-treated cauterized corneas. These investigations together demonstrated that a vascular ingrowth of the cornea did not follow corneal cauterization with silver nitrate in the absence of leukocytes, and gives further support to the hypothesis that leukocytes serve a crucial function in corneal vascularization.     

Kinetics of thrombomodulin release and endothelial cell injury by neutrophil-derived proteases and oxygen radicals

Thrombomodulin is a transmembranous glycoprotein of endothelial cells. In vitro it is a marker of endothelial cell injury. In vivo the levels of serum thrombomodulin are regarded as a parameter of activity in vasculitides. The latter are pathophysiologically determined by neutrophil-derived inflammation and endothelial cell injury caused by secretion of proteases and hydrogen peroxide. It was the objective of this study to determine whether thrombomodulin is only a late marker of advanced endothelial cell injury or whether it indicates also earlier stages of cell alterations. Over 24 hr endothelial cell cultures were incubated with hydrogen peroxide or the neutrophil proteases proteinase-3, elastase and cathepsin G. The time-dependent increase of thrombomodulin in the supernatant was determined by enzyme-linked immunosorbent assay and immunoblot. In addition the viability (eosin, tetrazolium dye assay), detachment (crystal-violet assay), and apoptosis (4',6-diamine-2'-phenylindole-dihydrochloride assay) of the respective endothelial cells were determined for adherent and non-adherent cells. A rapid thrombomodulin increase was found under all experimental conditions. The additional immunoblotting analysis showed the pattern of proteolytic cleavage caused by the protease reactivity. In case of hydrogen peroxide the thrombomodulin increase was closely correlated with the loss of cell viability and lysis. The incubation of endothelial cells with the different proteases resulted in a time-dependent detachment of primarily viable cells. In addition to cell necrosis apoptotic cell death was found in the subgroup of detached endothelial cells after prolonged incubation over 24 hr with proteinase-3 (23%), elastase (31%), and cathepsin G (19%). In contrast, still adhering cells did not show any signs of necrosis or apoptosis. In summary these studies confirm in vitro that soluble thrombomodulin is not only a parameter of advanced endothelial cell destruction itself but also in addition an early marker of initial endothelial cell membrane changes induced by neutrophil derived proteases and oxygen radicals.     

Injury to endothelial cells by phagocytosing polymorphonuclear leukocytes and modulatory role of lipoxygenase products.

Phagocytosis of microorganisms by polymorphonuclear leukocytes (PMN) is accompanied by inadvertent extracellular release of microbicidal products; this could result in tissue damage. We investigated whether PMN damages endothelial cells when phagocytosis of Staphylococcus aureus occurs on the endothelial surface and how this damage might be modulated. Damage was assayed by the measurement of cell detachment or cell lysis of cultured endothelial cells that were radiolabeled with 51Cr. Uptake of bacteria was accompanied by nonlytic detachment of endothelial cells from the monolayer. This effect was inhibited by alpha-1-antitrypsin but remained unaffected by scavengers of toxic oxygen species. During phagocytosis, PMN adhered to the endothelial cells. Adherence could be prevented by inhibition of the lipoxygenase pathway of arachidonic acid metabolism of the PMN with nordihydroguaiaretic acid. This inhibition also resulted in a marked decrease of the detaching activity of the PMN. The addition of exogenous leukotriene B4 during phagocytosis greatly enhanced the damage to the endothelial monolayer. These results indicate that phagocytosis of staphylococci by PMN is accompanied by injury to endothelial cell monolayers due to released lysosomal proteases and that products of the lipoxygenase pathway of PMN play a modulatory role in this injury.