Post-invasion events after infection with Staphylococcus aureus are strongly dependent on both the host cell type and the infecting S. aureus strain.

Host cell invasion is a major feature of Staphylococcus aureus and contributes to infection development. The intracellular metabolically active bacteria can induce host cell activation and death but they can also persist for long time periods. In this study a comparative analysis was performed of different well-characterized S. aureus strains in their interaction with a variety of host cell types. Staphylococcus aureus (strains 6850, USA300, LS1, SH1000, Cowan1) invasion was compared in different human cell types (epithelial and endothelial cells, keratinocytes, fibroblasts, osteoblasts). The number of intracellular bacteria was determined, cell inflammation was investigated, as well as cell death and phagosomal escape of bacteria. To explain strain-dependent differences in the secretome, a proteomic approach was used. Barrier cells took up high amounts of bacteria and were killed by aggressive strains. These strains expressed high levels of toxins, and possessed the ability to escape from phagolysosomes. Osteoblasts and keratinocytes ingested less bacteria, and were not killed, even though the primary osteoblasts were strongly activated by S. aureus. In all cell types S. aureus was able to persist. Strong differences in uptake, cytotoxicity, and inflammatory response were observed between primary cells and their corresponding cell lines, demonstrating that cell lines reflect only partially the functions and physiology of primary cells. This study provides a contribution for a better understanding of the pathomechanisms of S. aureus infections. The proteomic data provide important basic knowledge on strains commonly used in the analysis of S. aureus-host cell interaction.

Analysis of flow in a cone-and-plate apparatus with respect to spatial and temporal effects on endothelial cells.

Endothelial cells, covering the inner surface of vessels and the heart, are permanently exposed to fluid flow, which affects the endothelial structure and the function. The response of endothelial cells to fluid shear stress is frequently investigated in cone-plate systems. For this type of device, we performed an analytical and numerical analysis of the steady, laminar, three-dimensional flow of a Newtonian fluid at low Reynolds numbers. Unsteady oscillating and pulsating flow was studied numerically by taking the geometry of a corresponding experimental setup into account. Our investigation provides detailed information with regard to shear-stress distribution at the plate as well as secondary flow. We show that: (i) there is a region on the plate where shear stress is almost constant and an analytical approach can be applied with high accuracy; (ii) detailed information about the flow in a real cone-plate device can only be obtained by numerical simulations; (iii) the pulsating flow is quasi-stationary; and (iv) there is a time lag on the order of 10(-3) s between cone rotation and shear stress generated on the plate.

Infection and activation of monocytes by Marburg and Ebola viruses.

In this study we investigated the effects of Marburg virus and Ebola virus (species Zaire and Reston) infections on freshly isolated suspended monocytes in comparison to adherent macrophages under culture conditions. Our data showed that monocytes are permissive for both filoviruses. As is the case in macrophages, infection resulted in the activation of monocytes which was largely independent of virus replication. The activation was triggered similarly by Marburg and Ebola viruses, species Zaire and Reston, as indicated by the release of the proinflammatory cytokines interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and IL-6 as well as the chemokines IL-8 and gro-alpha. Our data suggest that infected monocytes may play an important role in the spread of filoviruses and in the pathogenesis of filoviral hemorrhagic disease.

Role of actin filaments in endothelial cell-cell adhesion and membrane stability under fluid shear stress.

Clostridium botulinum C2 toxin (C2 toxin) and purified ADP-ribosylated-alpha-actin (ADP-r-alpha-actin) cause specific actin depolymerisation in living cells. This effect was used to investigate the actin microfilament system with particular emphasis on cell-cell adhesion and plasma membrane integrity in endothelial cells. C2 toxin caused time- and dose-dependent (15-100 ng/ml) changes in endothelial surface morphology (investigated by atomic force microscopy), intercellular gap formation and cell detachment under shear stress. Low concentrations of C2 toxin (1.5 ng/ml), however, did not induce cell detachment but inhibited shear stress-dependent cell alignment. Gap formation as well as cell loss under shear stress was also observed in cells microinjected with purified ADP-r-alpha-actin. Intercellular gap formation was mediated by increased alpha-catenin solubility (40%) due to actin filament depolymerisation. Disintegration of plasma membranes (measured by LDH release) and cell fragmentation during simultaneous exposure to shear stress and C2 toxin were due to a loss of more than 50% of membrane-associated actin. These data show that small disturbances in actin dynamics inhibit shear stress-dependent cell alignment; that depolymerisation of actin filaments increases the solubility of alpha-catenin, thus resulting in cell dissociation and that actin filaments of the membrane cytoskeleton are required to protect the cells from haemodynamic injury such as shear stress. Together, the study shows a heterogeneous regulation of actin filament dynamics at subcellular locations. Junction-associated actin filaments displayed the highest sensitivity whereas stress fibres were far more stable.

Quantitative morphodynamics of endothelial cells within confluent cultures in response to fluid shear stress.

To evaluate shear stress-induced effects on cultured cells we have extended the mechanical setup of a multichannel in vitro rheological system and developed software allowing entire processing control and image data analysis. The values of cell motility, degree of orientation (alignment), and cell elongation were correlated as a function of time (morphodynamics). Collective and individual endothelial cells within confluent cultures displayed a shear stress-dependent characteristic phase behavior of the following time course: resting conditions (phase I), change of motility (phase II), onset of alignment (phase III), and finally cell elongation (phase IV). Especially cell motility was characterized by a randomized zigzag movement around mean trajectories (fluctuations) together with mean cell locomotion. Onset of shear stress caused a down-regulation of fluctuations of 30% within <10 min and simultaneously increased locomotion velocities preferring the flow direction (phase II). After a lag period of 10 to 20 min cells orientated in the direction of flow (phase III) without significant cell elongation, which finally occurs within hours (phase IV). These data provide first evidence that cells within confluent endothelial monolayers respond to shear stress with a characteristic phase behavior.

Plasma membrane plasticity of Xenopus laevis oocyte imaged with atomic force microscopy.

Proteins are known to form functional clusters in plasma membranes. In order to identify individual proteins within clusters we developed a method to visualize by atomic force microscopy (AFM) the cytoplasmic surface of native plasma membrane, excised from Xenopus laevis oocyte and spread on poly-L-lysine coated glass. After removal of the vitelline membrane intact oocytes were brought in contact with coated glass and then rolled off. Inside-out oriented plasma membrane patches left at the glass surface were first identified with the lipid fluorescent marker FM1-43 and then scanned by AFM. Membrane patches exhibiting the typical phospholipid bilayer height of 5 nm showed multiple proteins, protruding from the inner surface of the membrane, with heights of 5 to 20 nm. Modelling plasma membrane proteins as spherical structures embedded in the lipid bilayer and protruding into the cytoplasm allowed an estimation of the respective molecular masses. Proteins ranged from 35 to 2,000 kDa with a peak value of 280 kDa. The most frequently found membrane protein structure (40/microm2) had a total height of 10 nm and an estimated molecular mass of 280 kDa. Membrane proteins were found firmly attached to the poly-L-lysine coated glass surface while the lipid bilayer was found highly mobile. We detected protein structures with distinguishable subunits of still unknown identity. Since X. laevis oocyte is a generally accepted expression system for foreign proteins, this method could turn out to be useful to structurally identify specific proteins in their native environment at the molecular level.

Endothelial barrier function under laminar fluid shear stress.

It has been suggested that increasing levels of shear stress could modify endothelial permeability. This might be critical in venous grafting and in the pathogenesis of certain vascular diseases. We present a novel setup based on impedance spectroscopy that allows online investigation of the transendothelial electrical resistance (TER) under pure laminar shear stress. Shear stress-induced change in TER was associated with changes in cell motility and cell shape as a function of time (morphodynamics) and accompanied by a reorganization of catenins that regulate endothelial adherens junctions. Confluent cultures of porcine pulmonary trunk endothelial cells typically displayed a TER between 6 and 15 ohms cm2 under both resting conditions and low shear stress levels (0.5 dyn/cm2). Raising shear stress to the range of 2 to 50 dyn/cm2 caused a transient 2% to 15% increase in TER within 15 minutes that was accompanied by a reduction in cell motility. Subsequently, TER slowly decreased to a minimum of 20% below the starting value. During this period, acceleration of shape change occurred. In the ensuing period, TER values recovered, reaching control levels within hours and associated with an entire deceleration of shape change. A heterogeneous distribution of alpha-, beta-, and gamma-catenin, main components of the endothelial adherens type junctions, was also observed, indicating a differentiated regulation of shear stress-induced junction rearrangement. Additionally, catenins were partly colocalized with beta-actin at the plasma membrane, indicating migration activity of these subcellular parts. Shear stress, even at peak levels of 50 dyn/cm2, did not cause intercellular gap formation. These data show that endothelial monolayers exposed to increased levels of laminar shear stress respond with a shear stress-dependent regulation of permeability and a reorganization of junction-associated proteins, whereas monolayer integrity remains unaffected.

Characterization of human vascular endothelial cadherin glycans.

The glycosylation pattern of human vascular endothelial cadherin (VE-cadherin), purified from cultured human umbilical cord vein endothelial cells, was analyzed. VE-cadherin was metabolically radiolabeled with d-[6-(3)H]glucosamine, isolated by immunoprecipitation, purified by SDS-PAGE and in-gel digested with endoproteinase Asp N. Oligosaccharides were sequentially released from resulting glycopeptides and analyzed by chromatographic profiling. The results revealed that VE-cadherin carries predominantly sialylated diantennary and hybrid-type glycans in addition to some triantennary and high mannose-type species. Highly branched, tetraantennary oligosaccharides were found in trace amounts only. Immunohistochemical labeling of VE-cadherin and sialic acids displayed a codistribution along the intercellular junctions in endothelial cells of human umbilical arteries, veins, and cultured endothelial monolayers. Ca(2+)-depletion, performed on cultured endothelial cells, resulted in a reversible complete disappearance of VE-cadherin and of almost all sialic acid staining from the junctions. Sialidase treatment of whole cells caused a change of VE-cadherin immunofluorescence from a continuous and netlike superstructural organization to a scattered inconsistent one. Hence, cell surface sialic acids might play a role in VE-cadherin organization.

Correlation of endothelial vimentin content with hemodynamic parameters.

In mammalian species, vimentin is the sole intermediate filament protein of endothelial cells lining the chambers of the heart and the inner surface of large blood vessels. Obvious quantitative differences in the vimentin-like immunoreactivity of endothelial cells observed in different vascular segments led us to undertake a systematic survey on the endothelial content of vimentin throughout the heart chambers, the vena cava, the pulmonary trunk, and the aorta of the pig. Immunostaining and immunoblotting showed that vimentin in endothelial cells of cardiovascular segments exposed to high shear stress and blood pressure (pulmonary trunk, aorta, left ventricle) is approximately 2- to -3-fold higher than in endothelial cells exposed to lower levels of hemodynamic stress (vena cava, left and right atria, right ventricle). Throughout the aorta, an approximately 1.5-fold increase in the vimentin contents was observed in a proximal to distal direction. The total endothelial amount of vimentin was determined to be 1.2% (inferior vena cava) and 2-3.5% (aorta) of total cellular protein. These data support the notion that the endothelial vimentin cytoskeleton can adapt to different hemodynamic loads, indicating that vimentin might help endothelial cells to withstand the mechanical forces exerted by blood flow and blood pressure.

Structural and functional aspects of intercellular junctions in vascular endothelium.

Cell-to-cell-junctions of endothelial cells are specialized and differentiated areas of the plasma membrane. The main functions include the separation of the intravascular and extravascular compartments, the mechanical connection of the cells, and the maintenance of the cell polarity. Although a wide heterogeneity of endothelial cell-to-cell junctions exists in situ, they should be considered in general as adherens type junctions in which gap and tight junctions are morphologically inserted. Under certain pathological conditions, such as wound healing, angiogenesis and many types of inflammation, the interendothelial junctions have to be dissociated and reorganized in which proteins of the junctions are crucially involved. These important mechanisms predict a sophisticated regulation of junctional proteins. The present paper describes the organization and functional aspects of the occludin/ZO-1 complex typically found in tight junctions, the cadherin/catenin complex of the adherens junctions and the connection of these protein complexes to the dense peripheral band via actin filaments. In addition, special attention has been drawn on the function of junction-associated proteins with respect to their role under fluid shear stress and interendothelial gap formation during inflammation.

Role of cadherins and plakoglobin in interendothelial adhesion under resting conditions and shear stress.

The role of cadherins and the cadherin-binding cytosolic protein plakoglobin in intercellular adhesion was studied in cultured human umbilical venous endothelial cells exposed to fluid shear stress. Extracellular Ca2+ depletion (< 10(-7) M) caused the disappearance of both cadherins and plakoglobin from junctions, whereas the distribution of platelet endothelial cell adhesion molecule 1 (PECAM-1) remained unchanged. Cells stayed fully attached to each other for several hours in low Ca2+ but began to dissociate under flow conditions. At the time of recalcification, vascular endothelial (VE) cadherin and beta-catenin became first visible at junctions, followed by plakoglobin with a delay of approximately 20 min. Full fluid shear stress stability of the junctions correlated with the time course of the reappearance of plakoglobin. Inhibition of plakoglobin expression by microinjection of antisense oligonucleotides did not interfere with the junctional association of VE-cadherin, PECAM-1, and beta-catenin. The plakoglobin-deficient cells remained fully attached to each other under resting conditions but began to dissociate in response to flow. Shear stress-induced junctional dissociation was also observed in cultures of plakoglobin-depleted arterial endothelial cells of the porcine pulmonary trunk. These observations show that interendothelial adhesion under hydrodynamic but not resting conditions requires the junctional location of cadherins associated with plakoglobin. beta-Catenin cannot functionally compensate for the junctional loss of plakoglobin, and PECAM-1-mediated adhesion is not sufficient for monolayer integrity under flow.

A simple assay for quantification of protein in tissue sections, cell cultures, and cell homogenates, and of protein immobilized on solid surfaces.

The determination of total protein is often a key step for the quantitative analysis of various parameters in tissue and general biochemical research. The classical protocols are restricted to a few compatible buffers, and protocols for the determination of protein in solutions containing protein agglomerates or of protein immobilized on solid surfaces are not available. In such cases, quantification may be complicated. Here, we describe a simple sensitive method for protein quantification circumventing all these restrictions. Proteins in solution or suspension in any buffer are spotted onto cellulose acetate, dried, and stained with Amido Black. After washing off the excess dye, bound Amido Black is solubilized in an acidic solution and determined photometrically. Tissue slices (fixed or native), adherent cell cultures, or Western blots can also be stained and their protein content determined irrespective of the supporting material. A micro-version of the protocol for proteins in solution allows large numbers of samples to be evaluated at a time in microtitration plates and requires only 1-2 microl per sample. A linear concentration dependency (r2=0.950-0.999) was obtained for all samples in all cases investigated. The method presented here permits the exact determination of soluble protein in a large variety of buffers, of insoluble or immobilized protein present on a wide variety of supports, and even of whole cells or tissue slices.

Listeria monocytogenes-infected human umbilical vein endothelial cells: internalin-independent invasion, intracellular growth, movement, and host cell responses.

The interaction of Listeria monocytogenes with human umbilical vein endothelial cells was studied. We show that L. monocytogenes invades human umbilical vein endothelial cells independently of internalin A, internalin B, internalin C, and ActA. L. monocytogenes replicates efficiently inside the cells and moves intracellularly by the induction of actin polymerization. We further show that L. monocytogenes-infection of human umbilical vein endothelial cells induces interleukin-6 and interleukin-8 expression during the first 6 h of infection. The expression of MCP-1 and the adhesion molecules VCAM-1 and ICAM-1 was not altered under the experimental conditions used here.

Filovirus-induced endothelial leakage triggered by infected monocytes/macrophages.

The pathogenetic mechanisms underlying hemorrhagic fevers are not fully understood, but hemorrhage, activation of coagulation, and shock suggest vascular instability. Here, we demonstrate that Marburg virus (MBG), a filovirus causing a severe form of hemorrhagic fever in humans, replicates in human monocytes/macrophages, resulting in cytolytic infection and release of infectious virus particles. Replication also led to intracellular budding and accumulation of viral particles in vacuoles, thus providing a mechanism by which the virus may escape immune surveillance. Monocytes/macrophages were activated by MBG infection as indicated by tumor necrosis factor alpha (TNF-alpha) release. Supernatants of monocyte/macrophage cultures infected with MBG increased the permeability of cultured human endothelial cell monolayers. The increase in endothelial permeability correlated with the time course of TNF-alpha release and was inhibited by a TNF-alpha specific monoclonal antibody. Furthermore, recombinant TNF-alpha added at concentrations present in supernatants of virus-infected macrophage cultures increased endothelial permeability in the presence of 10 micron H2O2. These results indicate that TNF-alpha plays a critical role in mediating increased permeability, which was identified as a paraendothelial route shown by formation of interendothelial gaps. The combination of viral replication in endothelial cells (H.-J. Schnittler, F. Mahner, D. Drenckhahn, H.-D. Klenk, and H. Feldmann, J. Clin. Invest. 19:1301-1309, 1993) and monocytes/macrophages and the permeability-increasing effect of virus-induced cytokine release provide the first experimental data for a novel concept in the pathogenesis of viral hemorrhagic fever.

Angiotensin-II-induced cell hypertrophy: potential role of impaired proteolytic activity in cultured LLC-PK1 cells.

BACKGROUND: Angiotensin II-induced hypertrophy of both mesangial and tubular cells has been shown to be caused by enhanced protein synthesis. There are no data about its role on protein breakdown. Therefore, protein turnover and proteolytic activities were investigated in LLC-PK1 cells. METHODS: Protein turnover was measured by determining the incorporation and release of [14C]phenylalanine; collagenolytic and gelatinolytic activities were assayed by using fluorogenic peptidyl substrates. RESULTS: Angiotensin II (10(-8)-10(-6) M) exerted a dose-dependent inhibition of collagenolytic and gelatinolytic activities, associated with reduction of protein degradation rate. In addition angiotensin II stimulated protein synthesis in the cells. These combined effects on protein turnover resulted in an increase in both cell size and cell protein content (31.7% after 48 h). However, the rise of cell protein content was only partly (48.0%) prevented by the protein synthesis inhibitor cycloheximide (10(-5)M), which supports the role of decreased protein degradation in the angiotensin-II-induced cell hypertrophy. The angiotensin-II-induced effects on proteolytic activities as well as on cell protein content could be abolished by coincubation with the angiotensin II type I-receptor antagonist DuP 753 (10(-6)M). The calcium-channel blocker verapamil (10(-6)M) ameliorated the impairment of collagenolytic activity. On the contrary the calcium ionophore A23187 (10(-6)M) mimicked the action of angiotensin II on this enzyme activity (control 34.5 +/- 1.9; angiotensin II 24.0 +/- 2.0; A23187 23.0 +/- 2.2 and angiotensin II+verapamil, 33.8 +/- 2.6 pmol/min/micrograms DNA). The role of cytosolic [Ca2+] in the actions of angiotensin II could be finally shown by a dose-dependent rise which was partly blunted by verapamil. CONCLUSION: The angiotensin-II-induced hypertrophy in LLC-PK1 cells is caused not only by enhanced protein synthesis but also by reduced protein degradation. The concomitant decline of collagenolytic and gelatinolytic activities may contribute to the accumulation of extracellular matrix, and presumably also to cell hypertrophy. These effects are obviously mediated via angiotensin II type I receptors and seem to be [Ca2+] dependent.

Improved in vitro rheological system for studying the effect of fluid shear stress on cultured cells.

A rheological in vitro system has been developed to study and quantify cellular adhesion under precisely defined external shear forces. The system is similar to a cone-and-plate viscosimeter. A rotating transparent cone produces both steady and pulsatile flow profiles on cultured cells. Direct visualization of cells by phase-contrast or fluorescence optics and connection of the optical system to a computer-controlled x/y-linear stage allows automatic recording of any point of the cell cultures. With the use of up to 12 individual rheological units, this setup allows the quantitative analysis of cell substrate adhesion by determination of cell detachment kinetics. Two examples of application of this rheological system have been studied. First, we show that the extracellular matrix protein laminin strongly increases endothelial cell adhesion under fluid shear stress. In a second approach, we obtained further support for the concept that shear stress-induced formation of actin filament stress fibers is important for endothelial cells to resist the fluid shear stress; inhibition of stress fiber formation by doxorubicin resulted in significant detachment of endothelial cells exposed to medium levels of fluid shear stress (5 dyn/cm2). No detachment was seen under resting conditions.

Replication of Marburg virus in human endothelial cells. A possible mechanism for the development of viral hemorrhagic disease.

Marburg and Ebola virus, members of the family Filoviridae, cause a severe hemorrhagic disease in humans and primates. The disease is characterized as a pantropic virus infection often resulting in a fulminating shock associated with hemorrhage, and death. All known histological and pathophysiological parameters of the disease are not sufficient to explain the devastating symptoms. Previous studies suggested a nonspecific destruction of the endothelium as a possible mechanism. Concerning the important regulatory functions of the endothelium (blood pressure, anti-thrombogenicity, homeostasis), we examined Marburg virus replication in primary cultures of human endothelial cells and organ cultures of human umbilical cord veins. We show here that Marburg virus replicates in endothelial cells almost as well as in monkey kidney cells commonly used for virus propagation. Our data support the concept that the destruction of endothelial cells resulting from Marburg virus replication is a possible mechanism responsible for the hemorrhagic disease and the shock syndrome typical of this infection.

Role of actin and myosin in the control of paracellular permeability in pig, rat and human vascular endothelium.

1. We have investigated the endothelial actomyosin system with particular emphasis on its possible role in actively opening a paracellular route for permeability. 2. Actin and myosin comprised 16% of total endothelial protein with a molar actin/myosin ratio of 16.2 which is close to the actin/myosin ratio of muscle (studies on freshly isolated pig pulmonary arterial endothelial cells, PAEC). 3. By immunocytochemistry at the light and electron microscope levels the bulk of actin and myosin was colocalized in close vicinity to the intercellular clefts of both micro- and macrovascular endothelial cells in situ and in vitro. 4. Calcium-ionophore-induced rise in permeability of human umbilical venous endothelial cells (HUVEC) and PAEC monolayers grown on filters in a two-chamber permeability system was caused by opening of intercellular gaps. Gap formation depended on the rise in intracellular Ca2+ and could be blocked by the calmodulin-binding drugs trifluperazine (TFP) and W7. 5. In skinned monolayers of cultured PAEC and in isolated sheets of HUVEC gap formation was shown to require ATP and occurred only when free myosin binding sites were available on endothelial actin filaments (experiments with myosin subfragment 1 modified by N-ethylmaleimide, S1-NEM). 6. These experiments suggest that actin and myosin in endothelial cells play a central role in regulating the width of the intercellular clefts, thereby controlling the paracellular pathway of vascular permeability.