Leukocyte Flux Research Articles

Fucoidin, but not yeast polyphosphomannan PPME, inhibits leukocyte rolling in venules of the rat mesentery

Leukocyte rolling in venules is inhibited by several sulfated polysaccharides, by antibodies to the leukocyte adhesion receptor L- selectin (LECAM-1), and by recombinant soluble L-selectin. The sulfated fucose polymer fucoidin and the polyphosphomannan PPME bind to L- selectin and inhibit L-selectin-mediated lymphocyte adhesion to lymph node high endothelial venules (LN-HEV). We investigated whether fucoidin and PPME also inhibit leukocyte rolling. Rolling leukocyte flux was determined by intravital microscopy in 47 venules (diameter 21 to 50 microns) of the rat mesentery with and without micro-infusion of each reagent through 8-microns glass micropipettes. Micro-infusion (1 mg/mL) or intravenous (IV) injection (25 mg/kg) of fucoidin, but not vehicle, reduced leukocyte rolling by greater than 90%. The half- effective concentration was approximately 2.5 micrograms/mL. Stroboscopic fluorescence video microscopy showed that fucoidin decreased the fraction of rolling leukocytes from 44% of all leukocytes passing the venules in control to less than 1%. PPME micro-infusion (1 mg/mL) or IV injection (14 mg/kg) did not reduce leukocyte rolling. Hence, leukocyte rolling differs from lymphocyte homing with respect to the effect of PPME. This may be related to fucoidin binding to L- selectin with greater affinity than PPME. Alternatively, inflamed venular endothelium may express a ligand for L-selectin different from that constitutively expressed on LN-HEV.

Fucoidin, But Not Yeast Polyphosphomannan PPME, Inhibits Leukocyte Rolling in Venules of the Rat Mesentery

Leukocyte rolling in venules is inhibited by several sulfated polysaccharides, by antibodies to the leukocyte adhesion receptor L-selectin (LECAM-1), and by recombinant soluble L-selectin. The sulfated fucose polymer fucoidin and the polyphosphomannan PPME bind to L-selectin and inhibit L-selectin-mediated lymphocyte adhesion to lymph node high endothelial venules (LN-HEV). We investigated whether fucoidin and PPME also inhibit leukocyte rolling. Rolling leukocyte flux was determined by intravital microscopy in 47 venules (diameter 21 to 50 microns) of the rat mesentery with and without micro-infusion of each reagent through 8-microns glass micropipettes. Micro-infusion (1 mg/mL) or intravenous (IV) injection (25 mg/kg) of fucoidin, but not vehicle, reduced leukocyte rolling by greater than 90%. The half-effective concentration was approximately 2.5 micrograms/mL. Stroboscopic fluorescence video microscopy showed that fucoidin decreased the fraction of rolling leukocytes from 44% of all leukocytes passing the venules in control to less than 1%. PPME micro-infusion (1 mg/mL) or IV injection (14 mg/kg) did not reduce leukocyte rolling. Hence, leukocyte rolling differs from lymphocyte homing with respect to the effect of PPME. This may be related to fucoidin binding to L-selectin with greater affinity than PPME. Alternatively, inflamed venular endothelium may express a ligand for L-selectin different from that constitutively expressed on LN-HEV.

Chemoattractant-induced firm adhesion of leukocytes to vascular endothelium in vivo is critically dependent on initial leukocyte rolling.

Leukocyte rolling and firm adhesion at the venular endothelium are two discrete events in the cellular inflammatory response mediated via selectin and integrin adhesion molecules, respectively. The dependency of chemoattractant-induced firm leukocyte adhesion on the preceding rolling interaction was investigated in rat mesenteric microvessels through use of intravital microscopy. Leukocyte rolling was dose-dependently inhibited by systemic treatment with the sulphated polysaccharide fucoidin. The firm leukocyte adhesion following stimulation with the chemotactic peptide fMLP was similarly inhibited when fMLP challenge was performed subsequent to inhibition of leukocyte rolling by fucoidin. Thus, based on paired observations in single venules before and after fucoidin treatment, reduced rolling leukocyte flux prior to fMLP challenge was paralleled over a wide range by a proportional decrease in fMLP-induced leukocyte adhesion. The results demonstrate quantitatively a close relationship between the extent of leukocyte rolling and the magnitude of the subsequent firm adhesion response, and, that an initial rolling interaction is a precondition for firm adhesion to occur at physiological blood flow rates in vivo.

Effects of Acute Exposure to O3On Rats: Sequence of Epithelial and Inflammatory Changes in the Distal Airways

AbstractThis study involves the analysis of the sequence of changes in lung epithelial permeability, free cells in the airways, prostaglandin E2 (PGE2) levels, polymorphonuclear leukocyte (PMN) flux, and alveolar lesions in rats exposed to 0.8 ppm ozone (O3) for 3 h and then studied at 4-h intervals up to 24 h postexposure. Protein content of the bronchoalveolar lavage (BAL) increased immediately after O3 exposure and returned to control levels by 16 h postexposure. Albumin concentration in the BAL also increased with time after O3 exposure, but the increase was more gradual than the protein increase. The lower albumin concentrations at 20 and 24 h postexposure were still higher than the control levels. While the total protein in the BAL could be attributed to tissue injury and increased transmucosal transport, the albumin primarily reflected elevated transport from the serum. Total cells in the BAL decreased immediately after the O3 exposure, but returned to near normal levels by 4 h. PGE, levels did not...

Imaging of leukocytes within the rat brain cortex in vivo

Confocal laser scanning microscopy was used in a rat closed cranial window preparation in order to study rhodamin 6G-labeled leukocytes within the brain cortex in vivo. Leukocytes were visualized up to 150 μm beneath the rat brain surface in noninvasive optical sections. In pial venules, leukocytes were seen flowing with the blood stream, rolling along or sticking to the endothelium, and migrating through the vessel wall. Within cerebral capillaries, leukocyte flux, velocities, and leukocyte plugging were measured. After additional intravenous administration of fluorescein, the plasma, leukocytes, and erythrocytes were visualized simultaneously. Based on stacks of optical sections of fluorescein-labeled capillaries, the individual capillaries were localized within the three-dimensional microvascular network. The usefulness of this technique was illustrated in a feasibility study in which leukocyte sticking to the vascular walls of venules, leukocyte extravasation, and intracapillary leukocyte plugging were monitored in a model of global cerebral ischemia.

Sulfated polysaccharides inhibit leukocyte rolling in rabbit mesentery venules

Before firm adhesion, leukocytes roll slowly along the walls of small venules at velocities ranging from 0.7 to 36% of mean blood flow velocity. To investigate the nature of the adhesive process underlying leukocyte rolling, synthetic (dextran sulfate) and naturally occurring sulfated polysaccharides (heparin, chondroitin sulfates, keratan sulfate, and heparan sulfate) were infused via glass micropipettes into the lumen of small venules (20-60 microns diam) of the rabbit mesentery. Leukocyte rolling was observed and quantified using both transmitted light and incident fluorescence intravital microscopy. Rolling leukocytes accounted for 27-80% of total leukocyte flux, exhibiting a wide range of individual velocities (0.01-0.84 mm/s) with a mean value of 4% of centerline velocity. Dextran sulfate (Mr 500,000) inhibited leukocyte rolling very effectively [half-effective concentration (ED50) approximately 10 micrograms/ml] and was able to almost completely abolish rolling at 500 micrograms/ml. Heparin (ED50 approximately 50 micrograms/ml), chondroitin 6-sulfate C (ED50 approximately 500 micrograms/ml), and heparan sulfate (ED50 approximately 5 mg/ml) also reduced leukocyte rolling. At 5 mg/ml, chondroitin 4-sulfate B (dermatan sulfate) was marginally effective, but chondroitin 4-sulfate A and keratan sulfate were ineffective. The present data suggest that an adhesion receptor-ligand system distinct from the leukocyte integrins may be underlying transient leukocyte adhesion (rolling). Endothelial glycoproteins or proteoglycans containing sulfated side chains may be involved in mediating this adhesive process.

Leukocyte flow dynamics in the rat liver microcirculation

The flow dynamics of leukocytes in the rat liver microcirculation was studied in the sinusoids in the three zones of the liver acinus (zone 1, periportal; zone 2, mid; and zone 3, pericentral) by means of in vivo fluorescence microscopy. Leukocytes were labeled in vivo with acridine orange. The microscopic image was videotaped and on playback of the videotapes, leukocyte velocity, leukocyte flux, erythrocyte velocity, sinusoid diameter, and sinusoid tortuosity were measured. Blood flow and flow resistance were calculated from these data. The leukocyte velocity, erythrocyte velocity, sinusoid diameter, and blood flow in the liver sinusoids in zone 3 were significantly greater than those in zone 2, which in turn were also significantly greater than those in zone 1. There were significant positive correlations between leukocyte and erythrocyte velocity, between leukocyte velocity and sinusoid diameter, and between leukocyte velocity and blood flow. Sinusoid tortuosity in zone 1 was significantly larger than that in zone 2 and zone 3. Flow resistance in zone 1 was significantly greater than that in zone 2, which was also significantly greater than that in zone 3. These results provide evidence for a “zonal gradient” of leukocyte velocity in the liver sinusoids, with increasing velocity from zone 1 to zone 3. The morphological (narrow diameter and tortuous path) and resistance factors appear to contribute to the slow velocity in zone 1.

Dimethylsulfoxide prevents chemoattractant-induced leukocyte adherence

The objective of this study was to determine whether dimethylsulfoxide (DMSO) influences chemoattractant-induced leukocyte adherence in the intestinal microcirculation. The distal colon of Sprague-Dawley rats was prepared for intravital microscopic observation of 25-35 microns diameter venules located in the muscularis externa. The number and average velocity of rolling leukocytes and the number of leukocytes adherent to the venular endothelial surface were determined from recorded video images. After a control period of observation, either N-formyl-methionyl-leucyl-phenylalanine (FMLP) or leukotriene B4 (LTB4) was added to the serosal superfusion solution. Both FMLP (1 microM) and LTB4 (1 microM) consistently caused leukocytes to adhere to venular endothelium. DMSO, at concentrations ranging between 2.5 and 140 mM, inhibited FMLP-induced leukocyte adherence in a dose-related manner. LTB4-induced leukocyte adherence was also significantly attenuated by DMSO. Leukocyte rolling velocity and leukocyte flux were not affected by DMSO. These results indicate that DMSO significantly inhibits leukocyte adherence at concentrations commonly used for its hydroxyl radical scavenging properties.

Preferential distribution of leukocytes in rat mesentery microvessel networks

Distribution of leukocytes in rat mesenteric microvessel networks was studied using intravital fluorescence video microscopy. A digital image analysis system was used to measure vessel diameters, flow velocities and leukocyte fluxes in 306 capillaries of 8 networks. Capillaries were defined as vessel segments connecting divergent to convergent branch points. Their topological position within the network was quantified by a generation number defined as the number of bifurcations between the capillary and the arteriole feeding the network. Proximal capillaries (generation numbers 4 and 5) were slightly but significantly smaller in diameter (8.9 +/- 0.4 micron, mean +/- SEM) than distal ones (generation numbers 20 and 21, 10.1 +/- 0.4 micron). Average capillary flow velocity decreased markedly from 2.0 +/- 1.0 mm.s-1 in proximal to 0.41 +/- 0.06 mm.s-1 in distal capillaries. Average leukocyte concentration was 3.4 +/- 0.5.10(9) 1(-1) and thus significantly below systemic values (6.0.10(9) 1(-1] in proximal capillaries, and above in distal ones (11.7 +/- 2.6.10(9) 1(-1). The analysis of flow and leukocyte flux partition at 138 bifurcations showed preferential distribution of leukocytes to the daughter capillary with higher flow rate. This suggests a tentative explanation for the observed leukocyte accumulation along the microvascular tree: due to their low fractional flow, proximal capillaries draw relatively leukocyte-poor blood from the arteriole feeding the network; this leads to an increased leukocyte concentration in distal capillaries. As a consequence of the concomitant increase of capillary diameter with increasing generation number, leukocytes are preferentially flowing through larger capillaries and are excluded from small ones.

Leukocyte distribution to arteriolar branches: Dependence on microvascular blood flow

Leukocyte distribution to arteriolar branches in the hamster cheek pouch microvasculature was studied by rendering the cells fluorescent via a constant intravenous infusion of acridine orange. A principal objective was to determine the major factors influencing the partitioning of the cellular flux between daughter branches at arteriolar branch points with internal diameters permitting single-file leukocyte flow. Leukocyte flux determinations (cells/sec) and individual cell velocities were made for over 8000 cells at 13 branch sites in 10 animals. The results indicate that for the branch flow ratios of 0.3 to 0.7 encountered in the present experiments, the predominant factor influencing the average leukocyte flux is the branch blood flow. Branch fractional flux dependence on fractional blood flow was analyzed using linear and nonlinear models. A linear characterization provided an excellent fit and description of the functional relationship but a slight nonlinear component was also predicted by the nonlinear sigmoidal model which also fit the data well. Because of the small degree of predicted nonlinearity and the overlap of the standard deviations of the parameter estimates, we were not able to statistically decide on which model, and hence which functional relationship, is operative. Thus, the answer to the question regarding the presence of any preferential distribution is inconclusive. However, the data and analyses suggest that if preferential distribution is operative, its magnitude over the flow range and conditions here studied is small, and that for many purposes the relationship between the in vivo average fractional cell flux and blood flow may be viewed as linear.

Radial distribution of white cells during blood flow in small tubes

The radial distribution of white blood cells (WBC) in blood flowing through glass tubes (i.d. 69 μm) was studied as a function of wall shear stress (range 0.1–2.5 Pa) and suspending medium (plasma, buffered saline, high-molecular-weight dextran solution). It was found that, irrespective of the choice of suspending medium, the highest leukocyte flux at high shear stresses was found in the tube center. WBC redistribution was seen upon lowering the shear stresses: A significant shift of WBC flux toward the marginal fluid layers occurred at the expense of the axial region. After replacement of plasma by other media the flow-dependent redistribution of WBCs was qualitatively unaffected. However, suspension of cells in dextran solution (inducing strong red cell aggregation) resulted in enhanced WBC margination, while in saline (no red cell aggregation) axial accumulation was accentuated. The results support the concept of size-dependent radial distribution of particles in flow of mixed suspensions. If applied to the living microcirculation, the data serve to explain WBC margination in microvessels (the first step in the series of events leading to emigration) in terms of a hydrodynamic phenomenon resulting from red cell/white cell interaction. The pronounced flow dependence of WBC margination results primarily from the effect of shear on red cell aggregation which leads to an alteration of the effective particle size distribution in the flowing blood.

Factors Influencing Leukocyte Adherence in Microvessels

In vivo studies of the microcirculation of an untraumatized and unanesthetized animal preparation has shown that leukocyte adherence to vascular endothelium is an extremely rare occurrence. Induction of leukocyte adherence can be produced in a variety of ways including direct trauma to the vessels, remote tissue injury via laser irradiation, and denuding the epithelium overlying the observed vessels. The role of blood flow and local hemodynamics on the leukocyte adherence process is quite complex and still not fully understood. From the results reported it may be concluded that blood flow stasis will not produce leukocyte adherence but will augment pre-existing adherence. Studies using 2 quantitative measures of adherence, leukocyte flux and leukocyte velocity have shown these parameters to be affected differently by local hemodynamics. Initial adherence appears to be critically dependent on the magnitude of the blood shear stress at the vessel wall as evidenced by the lack of observable leukocyte flux above some threshold value. Subsequent behavior of the leukocytes as characterized by their average rolling velocity shows no apparent relationship to shear stress but, for low velocities, may be related to the linear blood velocity.

31 The 'Impotent Neutrophil' Syndrome

Leukocyte function was studied in a 2–2,5-year-old Negro male with the history of repeated abscesses, cervical and inguinal adenitis, recurrent pneumonia, seborrheic eczema of the scalp, hepatosplenomegaly and anemia. An appropriate leukocytosis with neutrophilia occurred with each infection, absolute neutrophil counts ranging from 3,900 to 24,500/mm3. IgG, IgA and IgM globulins were present in increased amounts. Lymph node biopsy contained caseating granuloma. Skin tests and cultures for all types of acid fast bacilli and fungi were repeatedly negative. These findings are characteristic of the syndrome of chronic granulomatous disease of childhood. There was a normal flux of leukocytes into Rebuck skin windows and exudate fluid. Leukocytes appeared normal with alkaline phosphatase, peroxidase and Wright's stain and were normally vacuolated after bacterial phagocytosis. Normal leukocyte motility and phagocytosis followed by degranulation were seen with phase microscopy. Decreased bactericidal activity of the patient's leukocytes during in vitro incubation with Staphylococcus aureus and Aerobacter aerogenes was repeatedly demonstrated in the presence of the patient's serum or normal serum. To determine the nature of this defect a citric acid extract of the patient's leukocyte granules was compared with control specimens and decreased bactericidal activity was found. The deficient or abnormal phagocytin activity of the patient's leukocytes, demonstrated by this study, could account for his clinical syndrome. (SPR)