Technical Considerations in Evaluating the Adhesion of Leukocytes to Aortic Endothelium of the Rat

Comments on techniques for characterizing leukocytes adhered to the aortic endothelium of the rat are given. Alpha-naphthyl acetate esterase positive leukocytes were studied by optical microscopy of en face intima-media preparations. Results indicate 1) 1% paraformaldehyde-2% glutaraldehyde is a better fixative than formalin-calcium or 4% paraformaldehyde with or without 1.5 mM CaCl₂; the latter produces distortion of leukocytes, endothelial desquamation and enzymate inhibition, 2) washing the aorta with phosphate-buffered saline for 90 sec prior to fixation-perfusion produces a notable decrease in the number of leukocytes adhered, 3) diazotized pararosaniline is better than fast blue RR salt as coupling agent in the esterase reaction, and 4) counterstaining with 1% methyl green for 1 min, before or after the esterase reaction, is not adequate because of limited contrast and the heavy staining of smooth muscle. Counterstaining with Gill's hematoxylin No. 3 for 90 sec is adequate only when done before the esterase reaction. Inhibition of endothelial esterase activity by hematoxylin decreases background, favors contrast of adhered leukocytes and makes it possible to observe nucleus-cytoplasm relations.     

Quantitative assessment of the adherence of normal human blood mononuclear cells to vascular endothelial cell monolayers

We have developed a method to reliably quantitate the in vitro adherence of ⁵¹Cr-labeled blood mononuclear leukocytes to cultured monolayers of vascular endothelial cells from human umbilical veins. Normal mononuclear leukocytes adhered to endothelial cells more than to cover glass at all studied time periods over 4 hr with major differences seen at 2 hr (9.7 ± 1.2% vs 3.7 ± 1.1%; P < 0.01). Only a minority of cells adhering to endothelium were esterase positive. Similar patterns of binding were seen using varying concentrations of suspended mononuclear cells (1–4 × 10⁶/ml) simulating that occurring in vivo in different clinical states. This approach shows promise for in vitro approaches to lymphocyte-vascular endothelial interactions in human immune/inflammatory disorders.     

Phagocytosis of Intravenously Administered Particles by Leukocytes Adhered to the Aortic Endothelium of the Rat

Phagocytosis has been used to characterize on a functional basis leukocytes adhered to the aortic endothelium of the rat. After intravenous administration of particles, phagocytosis was observed microscopically in esterase-positive leukocytes adhered to the endothelium in whole mounts of aorta. Polybead^R blue and red, 0.5 and 1 μm particle size, were inadequate because they were insufficiently colored to be identified individually at 400. Fluoresbrite^tm YG 0.25 and 0.50 μm at doses of 0.2 and 2 ± 0.3 m1/100 g, respectively, produced endothelial lesions. The same occurred with Monastral blue B^R (MbB) at 0.3 ml/100 g, red iron at 2 ± 16 mg/100 g and India ink at different concentrations depending on the supplier. At lower particle doses, lesions were not found. Deferoxamine mesylate 1.5 mg/100 g intravenous and allopurinol 5 mg/100 g intraperitoneal administered before the particles diminished the number and intensity of lesions. In none of the cases studied was the percentage of phagocytic cells greater than 50%. Clearance curves of MbB and Fluoresbrite^tm indicated rapid disappearance of particles from the blood. Results indicate that administration of particulate suspensions is not a good method for characterizing the phagocytic leukocytes adhering to the aortic endothelium because low doses produce rapid clearance of particles, thus impeding sufficient leukocyte loading, and higher doses produce endothelial lesions that often impair reliable counting of the adhering leukocytes.     

Alterations by leukocytes of erythrocyte flow in microchannels

The effects of leukocytes on blood rheology in the microcirculation may be predicted to result from the rheological characteristics of the individual white blood cell types and their behavior at entrances of small vessels; their influence on flow resistance of blood due to their disturbance of erythrocyte flow; and the obstruction caused by their adherence to endothelial cells or geometrical hindrance of their flow at irregular or narrow regions. Deformation of leukocytes in micropipettes indicates the relative rigidity of lymphocytes and blast cells from leukemias in contrast to granulocyte viscoelastic properties, and entry times for lymphocytes in 2.6-4.6-microns channels were 11-151 s but milliseconds in 9.1-micron tubes. Lymphocytes and erythrocytes rarely (less than 1%) adhered to cultured endothelium; however, granulocytes adhered avidly at shear stresses of 10-100 dyn/cm2, typical of microcirculation. In the 9.1-microns flow tube at Hct = 17.7 +/- 2.6%, increasing [WBC] caused a linear increase in flow resistance, but above [WBC] = 1000/mm3 resistance tended to plateau. These data support the interpretation that granulocyte adherence to endothelial cells with the potential for obstruction of flow may be a more significant rheological consequence of leukocytes in blood than their resistance to flow because of their relative rigidity or their influence on flow resistance by perturbation of the erythrocyte flow.     

Iron-Hematoxylin and Safranin O As A Polychrome Stain for Epon Sections

A 3-step procedure performed at 85 C was as follows: Sections were treated with 4% aqueous ferric ammonium sulfate for 15-300 sec, washed, placed in ripened, 0.5% hematoxylin for 15-300 sec, washed, and counterstained in 1% Safranin O for 15-90 sec. This procedure produced a durable stain with clear cytoplasmic and nuclear differentiation.     

Forces Required to Initiate Membrane Tether Extrusion from Cell Surface Depend on Cell Type But Not on the Surface Molecule

When a cell adhered to another cell or substratum via surface proteins is forced to detach, lipid membrane tethers are often extruded from the cell surface before the protein bond dissociates. For example, during the inflammatory reaction leukocytes roll on the surface of activated endothelial cells. The rolling adhesion is mediated by interactions of selectins with their ligands, e.g., P-selectin glycoprotein ligand (PSGL)-1, which extrudes membrane tethers from the surfaces of both leukocytes and endothelial cells. Membrane tether extrusion has been suggested to regulate leukocyte rolling. Here we examine several factors that may affect forces required to initiate membrane tethers, or initial tether force. It was found that initial tether forces were similar regardless of the presence or absence of the cytoplasmic tail of P-selectin and regardless of whether the tethers were extruded via binding to PSGL-1 or Fcγ receptors. Initial tether forces were found to depend on the cell types tested and were greatly reduced by treatment of latrunculin A, which inhibits actin polymerization. These data provide additional insights to the control of membrane tether extrusion, which should be taken into account when cellular functions such as rolling where tether extrusion plays a regulatory role are compared using different cell types expressing the same molecule.     

Functional role of gap junctions in cytokine-induced leukocyte adhesion to endothelium in vivo

To assess the hypothesis that gap junctions (GJs) participate on leukocyte-endothelium interactions in the inflammatory response, we compared leukocyte adhesion and transmigration elicited by cytokine stimulation in the presence or absence of GJ blockers in the hamster cheek pouch and also in the cremaster muscle of wild-type (WT) and endothelium-specific connexin 43 (Cx43) null mice (Cx43e(-/-)). In the cheek pouch, topical tumor necrosis factor-alpha (TNF-alpha; 150 ng/ml, 15 min) caused a sustained increment in the number of leukocytes adhered to venular endothelium (LAV) and located at perivenular regions (LPV). Superfusion with the GJ blockers 18-alpha-glycyrrhetinic acid (AGA; 75 microM) or 18-beta-glycyrrhetinic acid (50 microM) abolished the TNF-alpha-induced increase in LAV and LPV; carbenoxolone (75 microM) or oleamide (100 microM) reduced LAV by 50 and 75%, respectively, and LPV to a lesser extent. None of these GJ blockers modified venular diameter, blood flow, or leukocyte rolling. In contrast, glycyrrhizin (75 microM), a non-GJ blocker analog of AGA, was devoid of effect. Interestingly, when AGA was removed 90 min after TNF-alpha stimulation, LAV started to rise at a similar rate as in control. Conversely, application of AGA 90 min after TNF-alpha reduced the number of previously adhered cells. In WT mice, intrascrotal injection of TNF-alpha (0.5 microg/0.3 ml) increased LAV (fourfold) and LPV (threefold) compared with saline-injected controls. In contrast to the observations in WT animals, TNF-alpha stimulation did not increase LAV or LPV in Cx43e(-/-) mice. These results demonstrate an important role for GJ communication in leukocyte adhesion and transmigration during acute inflammation in vivo and further suggest that endothelial Cx43 is key in these processes.     

Differential Effects of Cold Atmospheric Plasma in the Treatment of Malignant Glioma

Objective

Cold atmospheric plasma (CAP) has recently been shown to selectively target cancer cells with minimal effects on normal cells. We systematically assessed the effects of CAP in the treatment of glioblastoma.

Methods

Three glioma cell lines, normal astrocytes, and endothelial cell lines were treated with CAP. The effects of CAP were then characterized for viability, cytotoxicity/apoptosis, and cell cycle effects. Statistical significance was determined with student''s t-test.

Results

CAP treatment decreases viability of glioma cells in a dose dependent manner, with the ID50 between 90-120 seconds for all glioma cell lines. Treatment with CAP for more than 120 seconds resulted in viability less than 35% at 24-hours posttreatment, with a steady decline to less than 20% at 72-hours. In contrast, the effect of CAP on the viability of NHA and HUVEC was minimal, and importantly not significant at 90 to 120 seconds, with up to 85% of the cells remained viable at 72-hours post-treatment. CAP treatment produces both cytotoxic and apoptotic effects with some variability between cell lines. CAP treatment resulted in a G2/M-phase cell cycle pause in all three cell lines.

Conclusions

This preliminary study determined a multi-focal effect of CAP on glioma cells in vitro, which was not observed in the non-tumor cell lines. The decreased viability depended on the treatment duration and cell line, but overall was explained by the induction of cytotoxicity, apoptosis, and G2/M pause. Future studies will aim at further characterization with more complex pre-clinical models.     

Hematoxylin-Eosin Staining of OsO4-Fixed Epon- Embedded Tissue; Prestaining Oxidation by Acidified H2O2

Successful application of hematoxylin-eosin staining to 0.5-1 μ sections of OsO₄-fixed Epon-embedded mammalian tissue is made possible by first treating the sections for approximately 1 min at 25-30 C with 10% H₂O₂ acidified with 0.1 or 0.01 N H₂SO₄ to pH 3.2. Subsequent steps are: washing; drying; Hams hematoxylin at 50 C, 1-2 min; washing; drying; 0.2-0.3% NH₄OH in 70% ethanol, 3-5 sec, drying at 50 C; 5% aqueous eosin for 3 & 45 sec at 25-30 C, washing; drying; clearing in xylene and mounting in resin. The use of acidified H₂O₂ prevents the staining of Epon and permits the characteristic staining picture to be obtained. Sections were attached to glass slides without adhesive and processed horizontally on a rack. Slides should be well drained and blotted before each drying step, to prevent formation of precipitate on the section.     

Shear forces induce ICAM-1 nanoclustering on endothelial cells that impact on T-cell migration

The leukocyte-specific β₂-integrin LFA-1 and its ligand ICAM-1, expressed on endothelial cells (ECs), are involved in the arrest, adhesion, and transendothelial migration of leukocytes. Although the role of mechanical forces on LFA-1 activation is well established, the impact of forces on its major ligand ICAM-1 has received less attention. Using a parallel-plate flow chamber combined with confocal and super-resolution microscopy, we show that prolonged shear flow induces global translocation of ICAM-1 on ECs upstream of flow direction. Interestingly, shear forces caused actin rearrangements and promoted actin-dependent ICAM-1 nanoclustering before LFA-1 engagement. T cells adhered to mechanically prestimulated ECs or nanoclustered ICAM-1 substrates developed a promigratory phenotype, migrated faster, and exhibited shorter-lived interactions with ECs than when adhered to non mechanically stimulated ECs or to monomeric ICAM-1 substrates. Together, our results indicate that shear forces increase ICAM-1/LFA-1 bonds because of ICAM-1 nanoclustering, strengthening adhesion and allowing cells to exert higher traction forces required for faster migration. Our data also underscore the importance of mechanical forces regulating the nanoscale organization of membrane receptors and their contribution to cell adhesion regulation.     

A novel beta 1 integrin-dependent mechanism of leukocyte adherence to apoptotic cells

Adherence of leukocytes to cells undergoing apoptosis has been reported to be dependent on a variety of recognition pathways. These include alpha V beta 3 (CD51/CD61, vitronectin receptor), CD36 (thrombospondin receptor), macrophage class A scavenger receptor, phosphatidylserine translocated to the outer leaflet of apoptotic cell membranes, and CD14 (LPS-binding protein). We investigated the mechanism by which leukocytes adhere to apoptotic endothelial cells (EC). Peripheral blood mononuclear leukocytes and U937 monocytic cells adhered to human or bovine aortic EC induced to undergo apoptosis by withdrawal of growth factors, treatment with the promiscuous protein kinase inhibitor staurosporine, with the protein synthesis inhibitor and protein kinase activator anisomycin, or with the combination of cycloheximide and TNF-alpha. Expression of endothelial adherence molecules such as CD62E (E-selectin), CD54 (ICAM-1), and CD106 (VCAM-1) was not induced or increased by these treatments. A mAb to alpha V beta 3, exogenous thrombospondin, or blockade of phosphatidylserine by annexin V did not inhibit leukocyte adherence. Further, leukocyte binding to apoptotic EC was completely blocked by treatment of leukocytes but not EC with mAb to beta 1 integrin. These results define a novel pathway for the recognition of apoptotic cells.     

The fluid shear stress distribution on the membrane of leukocytes in the microcirculation

Recent in-vivo and in-vitro evidence indicates that fluid shear stress on the membrane of leukocytes has a powerful control over several aspects of their cell function. This evidence raises a question about the magnitude of the fluid shear stress on leukocytes in the circulation. The flow of plasma on the surface of a leukocyte at a very low Reynolds number is governed by the Stokes equation for the motion of a Newtonian fluid. We numerically estimated the distribution of fluid shear stress on a leukocyte membrane in a microvessel for the cases when the leukocyte is freely suspended, as well as rolling along or attached to a microvessel wall. The results indicate that the fluid shear stress distribution on the leukocyte membrane is nonuniform with a sharp increase when the leukocyte makes membrane attachment to the microvessel wall. In a microvessel (10 microns diameter), the fluid shear stress on the membrane of a freely suspended leukocyte (8 microns diameter) is estimated to be several times larger than the wall shear stress exerted by the undisturbed Poiseuille flow, and increases on an adherent leukocyte up to ten times. High temporal stress gradients are present in freely suspended leukocytes in shear flow due to cell rotation, which are proportional to the local shear rate. In comparison, the temporal stress gradients are reduced on the membrane of leukocytes that are rolling or firmly adhered to the endothelium. High temporal gradients of shear stress are also present on the endothelial wall. At a plasma viscosity of 1 cPoise, the peak shear stresses for suspended and adherent leukocytes are of the order of 10 dyn/cm2 and 100 dyn/cm2, respectively.     

A novel method of producing the key intermediate ASI-2 of ranirestat using a porcine liver esterase (PLE) substitute enzyme

(R)-2-amino-2-ethoxycarbonylsuccinimide (ASI-2) is a key intermediate used in the pharmaceutical industry and is valuable for the industrial synthesis of ranirestat, which is a potent aldose reductase inhibitor. ASI-2 was synthesized in a process combining chemical synthesis and bioconversion. Bioconversion in this study is a key reaction, since optically active carboxylic acid derivative ((R)-1-ethyl hydrogen 3-benzyloxycarbonylamino-3-ethoxycarbonylsuccinate, Z-MME-AE) is synthesized from a prochiral ester, diethyl 2-benzyloxycarbonylamino-2-ethoxycarbonylsuccinate, Z-MDE-AE, at a theoretical yield of 100%. Upon screening for microorganisms that asymmetrically hydrolyze Z-MDE-AE, Bacillus thuringiensis NBRC13866 was found. A novel esterase EstBT that produces Z-MME-AE was purified from Bacillus thuringiensis NBRC13866 and was stably produced in Escherichia coli JM109 cells. Using EstBT rather than porcine liver esterase (PLE), ASI-2 was synthesized with a 17% higher total yield by a novel method, suggesting that the esterase EstBT is a PLE substitute enzyme and therefore, may be of interest for future industrial applications.     

Standardization of bovine macrophage monolayers and isolation and culture of trypanosomes

We describe a method for culturing over 90% pure bovine macrophages from peripheral blood mononuclear cells separated with Nycoprep. The cells were cultured for 12 days and then stained with esterase and with anti CD14 to test for purity. The method is reproducible and ensures an adequate number of cells for immunological research. Additionally, we report the unexpected finding of Trypanosoma trypomastigotes in our macrophage cultures from bovines belonging to a geographic area from which no bovine trypanosomes had been reported before.     

快速减压对豚鼠外周微循环和大脑血流量的影响

目的：探讨动物处于减压病（ＤＣＳ）临界发病状态时微及其血流动力作用的改变。方法：采用小型化激光微综在数测量仪及ＬＤＦ－３微区血流量仪,以检测动物高压暴露前及快速减压后微循环和血流动力作用的改变。结果：快速减压后动物微血管明显收缩;毛细血管开放数量减少;微循环中可见气泡并有血栓形成;白细胞、血小板与血管内皮粘附;血流中有料多白色微小血栓;细动脉血流速度平均比正常状态减慢０．９ｍｍ／ｓ,细静脉流速减慢     

Uranyl-EDTA-hematoxylin: a new selective staining technique for nucleolar material.

This paper deals with the visualization of nuclear structures in glutaraldehyde fixed, acetic acid flattened preparations from Chironomus salivary glands, by means of an uranyl mordanting followed by hematoxylin staining. Under these conditions all the nuclear structures (bands, Balbiani rings, and nucleoli) were deeply stained. Treatment with 0.1 M EDTA for at least 30 sec after uranyl mordanting completely prevents the following hematoxylin staining in all the structures but the nucleolus. With increased EDTA extraction times (60-90 sec) the central region (composed of pars fibrosa) in spontaneously or experimentally segregated nucleoli showed the highest capacity for retaining uranyl ions. This selective staining of the nucleolar (possibly proteinic) material proved also efficient in cells from Drosophila testes and Allium roots.     

Quantitative analysis of the process and propagation of cortical granule breakdown in sea urchin eggs

Cortical granule breakdown in sea urchin eggs has been investigated with a video microscope system using Nomarski differential interference contrast optics, when induced by fertilization, microinjecting inositol 1,4,5-trisphosphate (IP3) or Ca-EGTA buffer solution into the egg, or perfusing a medium containing 1 mM Ca2+ to isolated cortices. The cortical granule increased up to 1.2 times in diameter and broke down within 40 msec. These values were almost constant among the three methods used to induce cortical granule breakdown. Upon fertilization, the cortical granule breakdown propagated over the egg surface at a speed of 3.3 microns/sec in Clypeaster japonicus eggs, which indicates that cortical granule breakdown propagated through the 3.3-microns-wide egg surface within 1 sec. In such a small area of the egg surface, however, it took much more than 1 sec for all cortical granules to break down because the maximal rate of breakdown was 7.6%/sec; that is, it took 9 sec and 18 sec for 50% and 90% respectively, of cortical granules to break down. Moreover, the rate did not simply decrease with time, and a shoulder was found during the reducing phase, which suggests that cortical granules are divided into fast and slow breakdown groups according to the responsiveness to the breakdown stimulus. The cortical granule breakdown induced by microinjecting the Ca-EGTA buffer and IP3 solutions propagated at 68 microns/sec and 35 microns/sec, respectively. The stimulus for cortical granule breakdown is discussed concerning the transient intracellular Ca2+ increase.     

Functional maturation of the capillary wall in the fetal and neonatal rat heart: permeability characteristics of developing myocardial capillaries

The development of the functional components of the myocardial capillary wall was characterized by time-course studies of transendothelial transport of intravascularly injected probes of graded size from 16 days of gestation in the fetal rat to seven days postpartum. Despite the morphological changes occurring in the developing endothelial cells, the interaction of the probes was similar throughout the developmental period studied. The carbon particles were retained within the capillary lumina without any association with interendothelial junctions or with plasmalemmal vesicles. Carbon also was associated with coated vesicles. In contrast to carbon, ferritin was localized sequentially, over 60 sec of circulation, in plasmalemmal vesicles on the lumenal surface, in the cytoplasm, and on the ablumenal surface of the endothelial cells as well as in the interstitial space. Ferritin was located also in coated pits and vesicles and, after 90 sec of circulation, in multivesicular bodies. Within 30 sec of circulation, reaction product of myoglobin was located in plasmalemmal vesicles, coated vesicles, and transendothelial cell channels. Also within 30 sec, myoglobin partially filled the interendothelial space from the capillary lumina to the level of the tight junction. At all developmental ages studied, the interendothelial cell junctions appeared structurally tight and were impermeable to all of the probes. Once ferritin or myoglobin had reached the ablumenal space, the basal lamina did not appear to restrain the passage of the probes. Plasmalemmal vesicles are the capillary structures which transendothelially transport ferritin and myoglobin in developing myocardial capillaries.     

A new method for the purification of human eosinophils and neutrophils, and a comparison of the ability of these cells to damage schistosomula of Schistosoma mansoni.

Centrifugation of human white blood cells over either Ficoll-Hypaque or slightly hypertonic Metrizamide discontinuous gradients reliably produces separate fractions that are enriched for either neutrophilic or eosinophilic granulocytes. This single step purification routinely yields 90 to 100% pure neutrophils and 85 to 100% pure eosinophils. Metrizamide gradients, in particular, reproducibly provide high yields of 90 to 100% pure eosinophils from normal subjects with 2 to 3% eosinophils in their peripheral blood. The method does not damage cells as judged by morphologic or functional criteria. The purified cell populations were tested for their ability to damage antibody-coated schistosomula either by the measurement of 51Cr release from labeled organisms, or by direct morphologic assessment. Neutrophils were superior in their ability to release 51Cr from labeled organisms, but eosinophils adhered to the organisms to a greater extent and induced microscopically detectable damage.     

Tannic acid, Iron hematoxylin, Alcian blue, and Basic fuchsin for staining islets and reticular fibers of the pancreas

In this technique alpha cells are stained by basic fuchsin, beta cells by iron-hematoxylin, reticular fibers by ferric tannate, and much by alcian blue. Among 6 commonly used fixatives tested, Bouin's fluid fixation (8-12 hr) gave the best staining results. Procedure: paraffin sections to water; 0.5% Li₂CO₃ to remove picric acid; 20% tannic acid, 15 min; wash well; 2-4 sec in 0.5% basic fuchsin containing 10% alcohol; rinse, then differentiate in 1% aniline in 90% alcohol until alpha cells are red and beta cells pink; 1% phosphomolybdic acid, 1 min; 5% hematoxylin in 2% iron alum, 0.5 min; wash well; 1% filtered alcian blue SGX, 15 sec; rinse, dehydrate, clear, and mount in synthtic resin. Results: reticular fibers, black; acinar cells, orange to gray; alpha cells, red; collagenous fibers, red; beta cells, gray granules; ducts, bluish-green. The method was tested on rat, rabbit, dog, hamster, cow and man.