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-induced leukocyte adhesion and
microvessel permeability
1 Department of Physiology and Pharmacology, School of Medicine, West Virginia University, Morgantown, West Virginia 26506-9229; 2 Department of Human Physiology, School of Medicine, University of California, Davis 95616; and 3 Department of Laboratory Medicine, University of California, San Francisco, California 94143-0134
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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The objective of this study was to
investigate whether leukocyte adhesion and/or emigration are critical
steps in increased microvessel permeability during acute inflammation.
To conduct this study, we combined autologous blood perfusion with a
single microvessel perfusion technique, which allows microvessel
permeability to be measured precisely after the endothelium has
interacted with blood-borne stimuli. Experiments were carried out in
intact venular microvessels in rat mesenteries. Firm attachment of
leukocytes to endothelial cells was induced by intravenous injection of
TNF-
(3.5 µg/kg) and resuming autoperfusion in a precannulated
microvessel. Leukocyte emigration was facilitated by superfusion of
formyl-Met-Leu-Phe-OH. Microvessel permeability was measured as
hydraulic conductivity (Lp) or the solute
permeability coefficient to tetramethylrhodamine isothiocyanate-labeled
-lactalbumin before and after leukocyte adhesion and emigration in
individually perfused microvessels. We found that perfusion of a
microvessel with TNF- did not affect basal microvessel permeability,
but intravenous injection of TNF- caused significant leukocyte
adhesion. However, the significant leukocyte adhesion and emigration
did not cause corresponding increases in either
Lp or solute permeability. Thus our results suggest that leukocyte adhesion and emigration do not necessarily increase microvessel permeability and the mechanisms that regulate the
adhesion process act independently from mechanisms that regulate permeability. In addition, silver staining of endothelial boundaries demonstrated that leukocytes preferentially adhere at the junctions of
endothelial cells. The appearance of the silver lines indicates that
the TNF--induced firm adhesion of leukocyte to microvessel walls did
not involve apparent changes in the junctional structure of endothelial
cells, which is consistent with the results of permeability measurements.
hydraulic conductivity; permeability coefficient; leukocyte emigration; silver staining; CD11b/CD18 expression
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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AN INCREASE IN EXTRAVASATION of plasma proteins with accompanying tissue edema is one of the main characteristics of inflammatory responses that involves the initial release of proinflammatory mediators, followed by leukocyte recruitment into inflammatory sites. Certain in vivo studies have demonstrated that prevention of leukocyte adhesion with specific antibodies or induction of neutropenia provided protection against vascular dysfunction during reperfusion or acute inflammation (3, 6, 26, 27). Some in vitro studies reported that CD11/CD18-dependent firm adhesion is the trigger for the cytokine-induced respiratory burst of neutrophils (35) and that the releasing factors could injure endothelial cells directly (9, 34, 35). For decades, the interaction of leukocytes with endothelial cells has been considered the critical event leading to tissue and organ dysfunction. However, there are circumstances where albumin leakage or tissue injury has been dissociated with leukocyte adhesion and emigration (10, 12, 24, 36, 40). Moreover, several studies have shown that leukocyte adhesion did not occur at exactly the same sites as the plasma leakage (1, 2, 22). Intensive investigations have been focused on antiadhesion strategies to prevent tissue damage, but leukocyte adhesion as a critical step in the sequel leading to protein leakage and tissue damage has not been verified in experiments with consistent results, and thus the underlying mechanisms remain obscure. Therefore, the objective of our present study was to use our in vivo approach to investigate the direct contribution of the firm attachment of leukocytes to endothelia to the increased microvessel permeability during acute inflammation.
To define the role of leukocyte adhesion in microvessel
permeability, the permeability changes caused by the direct activation of endothelial cells by inflammatory mediators need to be
differentiated from those induced by the adhesion process. Our
preliminary studies found that systemic administration of TNF-
induced significant leukocyte adhesion in rat mesenteric venular
microvessels, but perfusion of TNF- alone in a single vessel did not
cause an increase in permeability (19). These differential
actions of TNF- on leukocyte adhesion and microvessel
permeability permit us to investigate the direct effects of leukocyte
adhesion and emigration on microvessel permeability independently from
the effect of inflammatory mediators.
Our experiments were conducted in rat mesentery using a newly developed
method that combines single microvessel perfusion with autologous blood
perfusion. This method allows the mechanisms that regulate microvessel
permeability to be studied after the endothelia interact directly with
blood-borne stimuli. Leukocyte adhesion was induced by systemic
injection of TNF- and autologous blood perfusion. The emigration of
leukocytes was facilitated by topical application of
formyl-Met-Leu-Phe-OH (fMLP) in the local mesentery. Changes in
microvessel permeability were determined by paired measurements of
hydraulic conductivity (Lp) or the solute permeability coefficient to tetramethylrhodamine isothiocyanate (TRITC)-labeled -lactalbumin before and after leukocyte adhesion and
emigration in the same vessel. To correlate the morphological changes
at endothelial junctions associated with leukocyte adhesion with the
permeability measurements, we used silver staining to evaluate the
adhesion site and the endothelial junctional area to which the
leukocyte firmly attached. The changes in the expression of adhesion
molecules on leukocytes after exposure to TNF- were demonstrated
with fluorescence flow cytometry.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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Animal Preparation
Experiments were carried out in rat mesenteries. All procedures and the animal use have been approved by the Animal Care and Use Committees at West Virginia University and University of California-Davis. Female Sprague-Dawley rats (age 2-3 mo, 250-300 g, Hilltop Laboratory Animals; Scottdale, PA) were anesthetized with pentobarbital sodium given subcutaneously. The initial dosage was 65 mg/kg body wt, and an additional 3 mg/dose was given as needed. The trachea was intubated, and a midline surgical incision (1.5-2 cm) was made in the abdominal wall. The rat was then transferred to a tray and kept warm on a heating pad (17, 21). The mesentery was then gently taken out from the abdominal cavity and spread over a pillar for measurements of Lp or over a glass coverslip for measurement of solute permeability. The pillar or the glass coverslip was attached to the animal tray and placed adjacent to the rat body. The upper surface of the mesentery was superfused continuously with mammalian Ringer solution during preparation and experimentation. The temperature of the superfusate was maintained between 35 and 37°C and was monitored continuously by a thermometer probe placed at the superfusate dripper and regulated by a digitally controlled water bath. All experiments were carried out in venular microvessels, which were classified as segments where there is convergent flow, two to four branches distal to true capillaries. The mean diameter of all vessels in which the changes in permeability were measured was 42 ± 7 (SD) µm (n = 51). All of the vessels selected for experiments had brisk blood flow and were either free of or had no more than 1 adherent leukocyte/100 µm of the vessel wall.Measurement of Lp in Single Perfused Rat Mesenteric Microvessels
All measurements were based on the modified Landis technique, which measures the volume flux of water across the microvessel wall (8). The assumptions and limitations of the original method and its application in mammalian microvessels have been evaluated in detail (8, 25). In brief, a single venular microvessel was cannulated with a glass micropipette and perfused with albumin-Ringer solution containing 1% (vol/vol) of hamster red blood cells as markers. A manometer was connected with the micropipette. Depending on the downstream resistance, a pressure (range 40-60 cmH2O) was applied through the pipette to the microvessel lumen. The initial water flow per unit area of microvessel wall [(Jv/S)0] was calculated from the velocity of the marker cell after the vessel was occluded, the vessel radius, and the length between the marker cell and the occlusion site. The velocity of the marker cell was calculated after 2 s of the occlusion to avoid the effect of the compliance. Microvessel Lp was calculated from the Starling equation Lp = (Jv/S)/P, where P is the effective hydrostatic and oncotic pressure difference across the microvessel wall. Assuming the tissue hydrostatic and oncotic pressures are negligible, P represents the pressure difference between the hydrostatic pressure applied to the microvessel and the effective oncotic pressure generated from the albumin in the perfusate (BSA at 10 mg/ml has an effective oncotic pressure of 3.6 cmH2O). Lp was measured with a relatively constant pressure in each experiment. About 10-15 occlusions were conducted for baseline Lp measurement in ~30 min. The final Lp for each perfusion is the mean of the Lp calculated from each occlusion, if Lp is stable in the whole time course. Otherwise, Lp is reported as the mean peak value and the sustained level, if a transient increase in Lp is observed.Measurement of the Permeability Coefficient to
-Lactalbumin in Single Perfused Rat Mesenteric Microvessels
-lactalbumin (3 mg/ml). By
adjusting the perfusion and balance pressures, the vessel was perfused
alternately with washout or test perfusate. The perfusion status was
monitored continuously through the eyepieces of the microscope by a
20/80 optical path split. A measuring window from a fluorescence
photometer was positioned downstream from the Y branch, which covers a
segment of the vessel and the surrounding space (350 µm wide × 300-600 µm long). TRITC-labeled -lactalbumin was observed
with an excitation filter (535/50 nm), a dichroic mirror (DM 565), and
a bandpass barrier (610/75 nm, TRITC HYQ, Nikon). The fluorescence
intensity (FI) was collected by a Nikon Fluor objective [×10,
numerical aperture (NA) 0.5]. To prevent tissue damage and
photobleaching by continuous exposure to excitation, FI was measured at
2-s intervals with a 0.25-s exposure using a computer-controlled
shutter and recorded into a personal computer. A neutral density (ND)
filter (ND = 0.3) was applied to the excitation light path. The
duration for each perfusion with testing solute was 20-40 s. The
vessel diameter changes during the measuring period were negligible. The photobleaching examined in vitro with TRITC-labeled -lactalbumin was <1% in a period of 1 min after the same exposure time and interval. The apparent solute permeability coefficient (Pa)
was calculated from the relation between the initial step increase in
FI as the dye filled the microvessel lumen (
If), the
initial rate of accumulation of the fluorescent molecules in the tissue [(dIf/dt)0], and the vessel radius
(r) (23)
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Peff)
across the microvessel wall under those experimental conditions was
almost zero. Therefore, the apparent solute permeability coefficient to
-lactalbumin measured under conditions of our experiments closely
represents the true diffusive permeability coefficient. Details are
given in the APPENDIX.
Measurements of Lp or the Permeability Coefficient
Before and After Leukocyte Adhesion Induced by Intravenous Injection of
TNF-
-lactalbumin was measured first with
albumin-Ringer perfusate. The cannulation pipettes were then removed,
and blood flow was resumed in the same vessel. TNF- (3.5 µg/kg at
a concentration of 2 µg/ml) was then injected into the rat
bloodstream through the femoral vein using an insulin syringe through a
small skin incision and a blunt muscle separation. This amount of
TNF- was chosen to achieve a comparable plasma TNF- concentration
with a rat ischemic model (41). In addition, our
preliminary study demonstrated that this dosage was sufficient to
induce a significant leukocyte adhesion (19). Two hours
after TNF- injection, the same vessel was recannulated and perfused
with albumin-Ringer perfusate with the lowest pressure possible to
maintain the perfusion (20-30 cmH2O). The mean
velocity of the marker cells (Vmean) under that
pressure range was 200-350 µm/s. The wall shear rate calculated based on the Newtonian definition [shear rate = 8(Vmean/D), where D is the
vessel diameter] was between 28 and 40 s
1 during
perfusion. The rolling, tethering, and nonfirmly adherent leukocytes were washed away during the initial perfusion period. Changes in Lp or the permeability coefficient
were measured immediately after the recannulation. The adherent
leukocytes that could withstand that magnitude of shear rate and
remained attached to the vessel wall after the early
Lp or permeability coefficient measurements (which took ~5-10 min) were counted under the microscope and
expressed as the number per 100 µm length or per squared millimeter
of the vessel wall. Therefore, the definition of leukocyte adhesion
under our experimental conditions is different from that reported in intravital microscopy studies, such as remaining stationary for a
period >30 s. After leukocytes were counted (which took ~2 min), Lp or the permeability coefficient was measured
again for 10-15 min. Thus the total perfusion time after leukocyte
adhesion was ~30 min. An average of 90% adherent leukocytes counted
after the early measurements remained attached at the end of 30 min.
In most of the cases, the vessel seals very quickly and the blood flow was completely recovered when the cannulation pipette was pulled out. There was no continuous blood loss during experiments. If blood flow stopped, the results were discarded. One experiment was carried out per rat. The Lp measurements were conducted in a separate group of animals from the measurements of the permeability coefficient.
The plasma concentration of TNF- after the intravenous injection was
measured with ELISA (R&D kit) in another three rats. Blood samples (0.5 ml each) were taken every 30 min up to 2 h. The mean value in
samples taken at 30 min was 534 ± 8 pg/ml; at the end of 2 h, it decreased to 135 ± 7 pg/ml. This concentration range is
comparable with the plasma TNF- concentration in a rat ischemic model (41).
Effect of Leukocyte Emigration on Microvessel Lp
The effect of leukocyte transendothelial migration on permeability was studied following the same procedures used for leukocyte adhesion except fMLP (1 µM) was added to the superfusate after TNF- injection and blood flow resumption. The number of
interstitial leukocytes present in the vicinity of the vessel in which
permeability was studied was counted before and after the application
of fMLP. The area of vicinity was defined as one vessel diameter width on each side of the vessel wall. The emigrated leukocytes were expressed as the number per 100 µm of vessel length. Changes in Lp were measured first under control conditions
and then in the presence of both adherent and emigrated leukocytes.
Analysis of CD11b/CD18 Expression in Isolated Neutrophils and Whole Blood With Fluorescence Flow Cytometry
Isolation of neutrophils. Blood was collected from five donor rats (adult male Sprague-Dawley 300-350 g) by catheterization through the carotid artery and anticoagulated with heparin (10 U/ml). Neutrophils were isolated from whole blood with neutrophil isolation medium (NIM) step gradients (Cardinal Associates; Santa Fe, NM). Whole blood was first diluted with Hanks' Ca2+/Mg2+-free buffer [1:1 (vol/vol)]. Diluted blood (4 ml) was gently layered onto upper (NIM2B, 2 ml) and lower (NIM2A, 2 ml mixed with 60 µl distill water) gradient solutions and centrifuged at 1,500 g for 30 min at 20°C. The granulocyte band plus all gradients between the lower band and the red blood cell layer was collected and washed twice with buffer. The pellets (>95% neutrophils) were resuspended in buffer containing 0.5% fetal bovine serum and stored at 4°C until use.
CD11b and CD18 expression in isolated neutrophils.
Isolated neutrophils were resuspended in buffer containing
Ca2+ (1 mM) and Mg2+ (1 mM) and then warmed to
37°C before stimulation. TNF- was added to neutrophil suspensions
(total volume of 100 µl each at 2 × 106 cells/ml)
to a final concentration between 0.5 and 100 ng/ml. Cells were
incubated for 15 min at 37°C and then placed on ice. Each sample was
stained with FITC-conjugated mouse anti-rat CD18 (2.5 µg/ml) or CD11b
(12.5 µg/ml) MAb or mouse IgG (12.5 µg/ml) for 20 min in the dark
and then washed twice with buffer to eliminate excess antibody. Cells
were analyzed on a FACScan flow cytometer (Becton-Dickinson; San Diego,
CA) using CellQuest software.
Analysis of CD11b and CD18 expression in whole blood.
To identify whether neutrophils in whole blood have the same reaction
to TNF- as isolated neutrophils, the expression of CD11b/CD18 in
leukocytes in whole blood samples were also determined by flow
cytometry using a previously published protocol (38). Briefly, the collected blood was diluted 1:5 (vol/vol) in Hanks' buffer, stimulated with TNF- for 15 min, and stained with
FITC-labeled CD18 (2.5 µg/ml) or CD11b (12.5 µg/ml) MAb or mouse
IgG (12.5 µg/ml) for 20 min in the dark. Before analysis, the blood
samples were also stained with a red-emitting vital nucleic acid dye, LDS-751 (0.2 µg/ml). Nucleated cells were discriminated from red blood cells and platelets by an electronic threshold trigger on the red
fluorescence intensity of LDS-751. FITC green fluorescence was
determined in these cells as a function of TNF- stimulation. Background FITC signals due to nonspecific binding of FITC-conjugated mouse IgG was subtracted from each mean fluorescence value of CD11b/CD18 MAb binding in both isolated neutrophil and whole blood analysis. For each applied TNF- concentration, three analyses (using
neutrophils or whole blood from three different rats) were conducted.
Changes in the expression of CD11b/CD18 in response to TNF- were
expressed as the ratios of signals with stimulation versus the signals
under control conditions.
Measurement of Colloid Osmotic Pressure
The colloid osmotic pressures for solutions containing 50 mg/ml BSA or 3 mg/ml-lactalbumin were measured with a colloid osmometer
(4420, Wescor) at 20°C with a membrane molecular weight cut off at
10,000. Calibration was conducted physically using a water manometer
(AC-010, Wescor) with and without the presence of membrane. The
measured values were corrected for a temperature of 37°C based on
van't Hoff's law.
Silver Staining of Boundaries of Endothelial Cells Forming Microvessel Walls
To identify the locations of adherent leukocytes and illustrate the junctions of endothelial cells forming microvessel walls, silver stain was applied to individually perfused microvessels (14, 16). At the end of the experiment, microvessels with leukocyte adhesion were recannulated, perfused with AgNO3 (0.1 g/100 ml) in aqueous solution for 5-10 s, and then perfused with albumin-Ringer perfusate to delineate endothelial junctions. Details have been described (14). The numbers of adhering leukocytes overlapped with the endothelial junctions versus off junctions were counted under the microscope in 11 microvessels from 5 rats. Because the microvessels are 40-50 µm in diameter, the surfaces of the vessel wall near and distant from the lens have different focal planes. Therefore, the positions of attached leukocytes along the z-axis are readily determined by focusing the lens on upper and lower surfaces of the vessel wall. Photographs (Fig. 6) for demonstration were taken with a charge-coupled device camera (ProgRes 3012; Kontron, Japan) and a Nikon Fluor ×60, NA 1.4, oil objective.Solutions and Reagents
Mammalian Ringer solution was used for dissecting mesenteries, superfusing tissue, and preparing the perfusion solutions. The composition of the mammalian Ringer solution was (in mM) 132 NaCl, 4.6 KCl, 2 CaCl2, 1.2 MgSO4, 5.5 glucose, 5.0 NaHCO3, and 20 HEPES and Na-HEPES. The pH of the Ringer solution was maintained at 7.40-7.45 by adjusting the ratio of Na-HEPES to HEPES. All perfusates used for control and test perfusion contained BSA. Recombinant rat TNF- was purchased from Biosource
International (Camarillo, CA). The chemotactic peptide fMLP was
purchased from Calbiochem (San Diego, CA). The MAbs were from
Pharmingen (San Diego, CA), and the nucleic acid dye LDS-751 was from
Molecular Probes (Eugene, OR).
The method for labeling -lactalbumin with fluorescent molecules has
been described (23). In brief, -lactalbumin (6 mg/ml) was dissolved in borate buffer (0.05 M) containing 0.4 M NaCl. This
solution was then put into a dialysis tubing (12,000 mol weight cutoff,
Spectrapor) and dialyzed against borate buffer (0.05 M) containing 0.2 mg/ml TRITC (Research Organics) for 12 h at 15°C with stirring.
TRITC-labeled -lactalbumin was then dialyzed against glucose-free
mammalian Ringer solution, which was changed every 12 h until no
free dye was found. The final concentration of TRITC-labeled
-lactalbumin used in the experiment was 3 mg/ml in
albumin-Ringer solution. The platelet-activating factor (PAF) was
purchased from Sigma.
Data Analysis and Statistics
All values in the text are means ± SE except where noted otherwise. Changes in Lp were expressed as the ratio of testing Lp versus control Lp. The mean values of Lp or the permeability coefficient measured before and after leukocyte adhesion or treatments in the same vessel were used as paired data. The significance of the differences between groups was evaluated by paired t-test and nonparametric Wilcoxon signed-rank test. P < 0.05 was considered statistically significant.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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TNF--Induced Leukocyte Adhesion and Effect of Leukocyte Adhesion
on Microvessel Lp
7
cm · s1 · cmH2O1.
Two hours after the injection of TNF- and the resumption of blood
flow, the mean number of leukocytes firmly attached to the vessel wall
was 1,075 ± 167 leukocytes/mm2 (14.0 ± 2.0 leukocytes/100 µm). However, Lp measured in
the presence of firmly adherent leukocytes showed no changes from
control values. The mean Lp was 2.1 ± 0.4 × 107
cm · s1 · cmH2O1
(Fig. 2).
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Time-control experiments were carried out in six microvessels. The mean
control Lp was 1.6 ± 0.4 × 107
cm · s1 · cmH2O1.
Each rat was then injected with 0.5 ml of Ringer solution through the
femoral vein. After the same time course and procedure as described
above, we found that neither the number of adherent leukocytes nor the
Lp value was significantly different from
control values. The adherent leukocytes increased from 0.3 ± 0.1 to 0.8 ± 0.2 leukocytes/100 µm, and the mean
Lp value measured after 2 h was 1.4 ± 0.2 × 107
cm · s1 · cmH2O1.
Effect of TNF--Induced Leukocyte Adhesion on the Solute
Permeability Coefficient to -Lactalbumin
-lactalbumin was measured in six microvessels using the same
experimental protocol as Lp was studied with.
The mean control permeability coefficient to -lactalbumin was
5.0 ± 0.4 × 106 cm/s. Two hours after TNF-
injection and blood flow resumption, the mean number of leukocytes
adhering on the wall was 12.4 ± 1.9 leukocytes/100 µm of
vessel, or 920 ± 145 leukocytes/mm2 of vessel wall.
The permeability coefficient measured in the presence of leukocyte
adhesion was 5.2 ± 0.6 × 106 cm/s, which was
not significantly different from the control value (Fig.
3).
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Time-control experiments were conducted in seven microvessels. The mean
control permeability coefficient measured under control and 2 h
after injection of Ringer solution and resuming blood flow was 5.3 ± 0.7 × 106 and 5.4 ± 0.7 × 106 cm/s, respectively.
Effect of Emigrated Leukocytes on Microvessel Permeability
Having found no increase in either Lp or permeability to-lactalbumin associated with the adherent
leukocytes, we tested whether leukocyte emigration induced an increase
in permeability. The effect of emigrated leukocytes on microvessel
permeability was studied in five microvessels. Figure
4 shows the results. The baseline
Lp was 2.1 ± 0.5 × 107
cm · s1 · cmH2O1.
After TNF- was injected and fMLP (1 µM) was superfused on the mesentery for 2 h, the mean number of adherent leukocytes was 11.1 ± 1.1 leukocytes/100 µm of vessel length. The interstitial leukocytes presented in the vicinity of the vessel significantly increased from the baseline of 0.8 ± 0.1 to 6.7 ± 1.1 leukocytes/100 µm of vessel length. The mean
Lp measured in the presence of both adherent and
emigrated leukocytes was 1.8 ± 0.5 × 107
cm · s1 · cmH2O1.
No significant changes in Lp were observed.
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Effect of TNF- Alone on Microvessel Lp and the
Permeability Coefficient to -Lactalbumin
on endothelial cells in the
absence of blood components, the changes in Lp
or the solute permeability coefficient were measured by perfusing
TNF- alone in the microvessels. Table
1 shows the results. At concentrations between 10 ng and 10 µg/ml, TNF- had no significant effect on Lp measured immediately and at intervals up to
2 h. The mean control Lp of 18 microvessels
was 1.5 ± 0.1 × 107
cm · s1 · cmH2O1.
The solute permeability coefficient measured with 100 ng/ml TNF- in
another four microvessels also showed no significant difference from
the control. The mean control permeability coefficient was 5.8 ± 0.9 × 106 cm/s.
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To examine whether the vessel is still responsive to stimuli to
increase permeability after 2- to 3-h perfusion, we further tested the
vessel responsiveness to PAF, an agent that is known to increase
permeability (20), after the vessel was exposed to TNF-
for 2 h. Experiments were conducted in three microvessels. The mean control Lp was 1.2 ± 0.2 × 107
cm · s1 · cmH2O1.
After TNF- (50 ng/ml) was perfused for 2 h, PAF (1 nM) was applied to the perfusate. Within 5 min of exposure to PAF, the mean
peak increase in Lp was 8.5 ± 0.7 times
the control and fell to 2.2 ± 0.4 times the control within 40 min. This magnitude of Lp increase is not
significantly different from the mean peak increase in
Lp of 7.9 ± 1.4 times the control obtained
from nine microvessels that were exposed directly to PAF
(20). When each vessel was reperfused with BSA perfusate,
the mean Lp fell to 1.4 ± 0.2 times the
control in 10 min. These results demonstrated that the 2-h perfusion of
TNF- did not modify the permeability responsiveness of the
microvessel to PAF.
TNF--Induced Changes in Expressions of Adhesion Molecules
caused a significant
increase in leukocyte adhesion, it failed to induce changes in
microvessel permeability. To confirm that the TNF- was activating
leukocytes appropriately, we examined the expression of CD11b/CD18
adhesion molecules in isolated neutrophils or whole blood leukocytes
after TNF- treatment. As expected, both isolated neutrophils and the nucleated cells in whole blood showed a significant increase in the
expression of CD11b/CD18 after exposure to TNF- (Fig.
5). The applied TNF- concentrations
are within the range of TNF- injected into the rat bloodstream but
differ from the plasma concentration measured with ELISA due to albumin
and soluble antibody binding in the serum and blood.
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Location of Leukocyte Adhesion on Microvessel Walls
To demonstrate the location of adherent leukocytes in relation to the endothelial junctions and evaluate the potential local structural changes associated with leukocyte adhesion and emigration, we delineated the boundaries of endothelial cells after leukocyte adhesion using the silver stain in vivo technique we developed previously (14, 16). We found that the majority of adherent leukocytes selectively overlapped with endothelial clefts (Fig. 6). There were 963 leukocytes adhering on the walls of 11 microvessels from 5 rat mesenteries, with 853 (89%) leukocytes overlapping with endothelial junctions. Most of the remaining leukocytes were located close to the side of vessel walls, where it was difficult to determine their locations from a two-dimensional view. Endothelial junctions were outlined by continuous silver lines that had no apparent interruptions, even underneath the leukocyte attachment sites. Furthermore, there was no appearance of silver dots at endothelial cell borders, as described for inflamed postcapillary venules in the rat trachea (31). Those dots have been identified by electron microscopy as endothelial gaps, which indicate the plasma extravasation sites (31).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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This study is an extension of our previous investigations of
mechanisms regulating microvessel permeability in response to inflammatory mediators in the absence of leukocytes (15, 17, 18,
21). In this study, we introduced a new approach to investigate the direct relationship between TNF--induced leukocyte adhesion, emigration, and microvessel permeability in intact microvessels by
combining autoperfusion with a single vessel perfusion technique.
This combined experimental approach closely mimics the clinical situation. Moreover, it retains the capability of single vessel perfusion to provide precise measurements of vascular functional parameters as well as its ability to differentiate the roles of individual factors contributing to a complex biological event in intact microvessels.
Previous studies using whole organs or vascular beds retained the interactions of circulating blood with endothelia, but those studies usually had limited capacity to differentiate the roles of each step or factor contributing to a multifactor involved pathological consequence. Furthermore, permeability changes in whole organ studies were mainly based on the measurement of a permeability index, which was determined as a ratio of vascular versus interstitial FI after intravenous administration of fluorescent dye-labeled albumin. In such studies, any changes in hemodynamic conditions could confound the results. Moreover, a recent whole vascular bed study reported that the protein leakage observed with FITC-labeled albumin was not found when other fluorescent dye-labeled albumin was used. The investigators observed severe hemolysis after intravenous application of FITC-labeled albumin (37), suggesting that the phototoxicity of the dye may cause increases in permeability independent of leukocyte adhesion (36). Therefore, from an in vivo study point of view, it remains unclear whether adhesion and/or migration of leukocytes are the critical events resulting in increased permeability during acute inflammation.
In contrast to previous in vivo methods, our new approach enables either Lp or solute permeability to be quantitatively measured before and after leukocyte adhesion and emigration in the same vessel in which perfusion pressure and the surface area for fluid and solute exchange are also precisely measured. This experimental approach also enables the effect of leukocyte adhesion on permeability to be investigated separately from cytokines or inflammatory mediator-induced increases in microvessel permeability.
The new findings of this study are that 1) TNF--induced
leukocyte adhesion and migration are not sufficient to cause increases in microvessel permeability; 2) increased permeability is
not a prerequisite for leukocyte adhesion in venular microvessels; and
3) the mechanisms that regulate the expression of adhesion molecules and the adhesion process act independently from the mechanisms that regulate microvessel permeability. Furthermore, applying a silver stain in vivo technique to microvessels with leukocyte adherence illustrates that leukocytes adhere preferentially at the junctions of endothelial cells in intact rat venular
microvessels. The attachment sites did not involve apparent changes in
junctional structure. This finding is consistent with the results of
permeability measurements.
Roles of TNF- in the Expression of Adhesion Molecules and in
Leukocyte Adhesion to Microvessel Walls
has been recognized as a potent proinflammatory cytokine
that increases the expression of cellular adhesion molecules and the
adhesiveness between leukocytes and endothelial cells. Its actions
include the translocation of selectins and integrins on leukocytes and
protein synthesis for the expression of E-selectin, ICAM-1, or VCAM-1
on endothelial cells (30, 32, 39). Our results showed that
TNF- at the concentration we applied to rats sufficiently elicited a
significant leukocyte adhesion. We also found that a minimum 2-h
exposure to TNF- is needed to induce a significant number of firmly
attached leukocytes on the microvessel wall. If we recannulated the
vessel sooner than 2 h, most of the rolling, tethering, or even
attached leukocytes were washed away. This time requirement also
suggests that a process of protein synthesis, rather than protein
translocation, is required for the expression of adhesion molecules on
endothelial cells that are responsible for the firm attachment of
leukocytes. Our preliminary confocal microscopy study using fluorescent
dye-labeled MAb against rat ICAM-1 in individually perfused rat venular
microvessels showed a significant increase in ICAM-1 expression in
endothelial cells forming microvessel walls after 2 h of
intravenous injection of TNF- (19). Meanwhile, our
fluorescence flow cytometry analysis demonstrated significant increases
in the expression of CD11/CD18 in both isolated neutrophils and
leukocytes in whole blood with TNF- stimulation. Therefore, from the
adhesion point of view, our results were consistent with the role of
TNF- reported in the literature.
Roles of TNF--Induced Leukocyte Adhesion and Emigration in
Microvessel Permeability
Another possibility is that the local damage associated with adhesion,
if any, was not sufficient to increase permeability. Silver nitrate
staining of endothelial boundaries in inflamed rat tracheas
demonstrated that plasma extravasation was associated with the
appearance of silver dots, which have been identified by electron
microscopy as silver deposits at endothelial gaps (31).
Our silver stain of endothelial boundaries with adherent leukocytes
showed continuous silver lines at endothelial junctions without
apparent interruption with silver dot deposition. If silver dots are a
reliable index for endothelial gap formation, our results suggest that
there is no local gap formation associated with leukocyte adhesion
under our experimental conditions, which is in accordance with the
permeability measurements. Therefore, our results suggested that
TNF--induced leukocyte adhesion did not trigger the activation of
endothelial cells leading to increases in microvessel permeability.
Systemic injection of TNF- induced a significant leukocyte adhesion,
but the leukocyte migration was minimal under our experimental conditions. When the chemotactic peptide fMLP was applied to the superfusate after TNF- injection, the number of emigrated leukocytes significantly increased from 0.8 to 6.7 leukocytes/100 µm of vessel length during a 2-h period. However, Lp measured
in the presence of that magnitude of emigrated leukocytes still did not
show a significant increase. If the disruption of the integrity of
endothelial junctions by leukocyte emigration is reversible, it is not
surprising that there is no measurable increase in
Lp because not all of the leukocytes emigrated
simultaneously. This observation is consistent with an in vitro study
using cultured endothelial monolayers (5). The
investigators reported that tight junctions from endothelial borders
appeared intact during and immediately after neutrophil transendothelial migration and that no widespread proteolytic loss of
the tight junctions was found.
Roles of TNF- in Microvessel Permeability in the Absence of
Blood Components
alone (10 ng-10 µg/ml)
showed no increases in Lp and permeability to
-lactalbumin. These results indicate that the stimuli required to
activate endothelial cells and increase permeability are different from
those required to elicit the adhesion of leukocytes to endothelial
cells. Although the inflammatory mediator-induced increases in
permeability may facilitate the adhesion process, leukocyte adhesion
can occur in microvessels that have no increases in permeability. These results suggest that increased permeability is not a prerequisite for
leukocyte adhesion.
Dissociation Between Leukocyte Adhesion and Changes in Permeability Reported by Other Investigators
Much evidence in the literature documented that leukocyte adhesion during inflammation and ischemia-reperfusion is the critical step leading to protein leakage and tissue edema (3, 6, 26-28). However, the dissociation between leukocyte adhesion and changes in permeability in the presence of inflammatory stimuli has also been reported in several studies (1, 2, 22, 43). For example, administration of P-selectin antibody (13) or leukotriene receptor antagonist (29) abolished the PAF-mediated plasma leakage without the reduction of adherent leukocytes in venules. An inhibition of histamine-associated leukocyte adhesion was found without affecting the leakage formation (43). Studies in the rat tracheal mucosa found that 94% of the gap formations were distinct from sites of leukocyte adhesion or migration in the leaky venules (1, 2). These studies provided indirect evidence that leukocyte adhesion might not be the critical event leading to increased permeability. Our present study demonstrates the direct evidence that leukocyte adhesion does not necessarily result in permeability increase. Our results suggest that the critical event or process that contributes to the increased microvessel permeability during inflammation or ischemia-reperfusion may involve more than leukocyte endothelial cell interaction.Preferential Location of Leukocyte Adhesion in Intact Microvessel Walls
Our previous study of leukocyte adhesion in combination with staining endothelial boundaries using silver nitrate in frog mesenteric venular microvessels quantitatively demonstrated that the adherent leukocytes were preferentially attached to the junctional area of endothelial cells (16). Our present study in rat mesenteries further demonstrates that >89% of adherent leukocytes overlapped with endothelial junctions. Because the junctions of endothelial cells occupy a relatively large portion of the vessel wall, we conducted a calculation to determine whether 89% junctional adherence of leukocyte is significantly different from a random distribution on the vessel wall. On the basis of measurements reported by McDonald (31), the junctional length per luminal surface of rat postcapillary venules is 105 ± 3 mm/mm2. If we expand this cleft length to a band with a 3 µm width that equals one-half of the contact length of the leukocytes, the calculated band area is 31.5% of the total area of the vessel wall without the exclusion of the overlap at the tricellular corner. On the basis of this calculation, leukocytes have <31.5% probability of attaching to the junctions by a random process. Thus the over 89% junctional adherence is significantly distinguishable from a random distribution.An in vitro study using cultured endothelial monolayers demonstrated that the preferential adhesion of neutrophils was P-selectin dependent (4). However, a direct correlation between this preferential leukocyte adhesion and a corresponding distribution of adhesion molecules in intact microvessels has not been identified. Further studies are needed to demonstrate the temporal and spatial distribution of adhesion molecules on endothelial cells under the same experimental conditions whereby leukocyte adhesion and permeability were studied.
Validity of Measurement of Lp With Adherent Leukocytes on the Microvessel Wall
In mesenteric microvessels with continuous endothelium, the principal pathway for water and solutes lies between the endothelial cells through the interendothelial cell cleft (33). The adherence of leukocyte on the microvessel wall has the potential to affect Lp measurements by two factors: the volume in the vessel lumen and the surface area of the vessel wall. We estimated that the volume exclusion for the average number of adherent leukocytes (1,100 leukocytes/mm2) was ~3% of the vessel lumen volume, assuming cylindrical vascular geometry. This calculation was based on the estimated volume of leukocyte (299 µm3) measured by Ting Beall et al. (42) and the mean radius (21 µm) of the microvessels we used for the study. This 3% volume exclusion may results in ~1.5% overestimation of the Lp value.On the other hand, because the primary water pathway is at endothelial clefts and the majority of adherent leukocytes are at the junctional area, the adherent leukocytes may affect the water transport by reducing the surface area. The contact length of adherent leukocytes under the shear rate we applied to vessel is ~6 µm based on the measurements in rat mesenteric venules by Firell and Lipowsky (11). If the contact area between leukocyte and endothelium completely overlap with the junctions, the maximum junctional length occupied by 1,100 leukocytes/mm2 is equivalent to 6% of the total junction length based on 105 mm junction length/mm2 luminal surface reported in rat venular microvessels (31). Even if we assume that the adherent leukocytes completely block the water pathway at the attachment site, which is unlikely, the uncorrected Lp value is underestimated by 6% due to the reduction of the surface area. If both assumptions exist, the final outcome is about 4.5% underestimation of the measured Lp value. A similar estimation also applies to the measurements of the permeability coefficient. Even if we correct all of the Lp and permeability coefficient values measured in the presence of leukocyte adhesion with this potential error, the significance of the data comparison and the conclusions presented in this paper are not affected. On the basis of these calculations, we consider that the effect of leukocyte adhesion on the measurement of Lp and the permeability coefficient under our experimental conditions is negligible.
In summary, this study introduces a new experimental approach that enables the direct effect of leukocyte adhesion on permeability to be investigated independently from cytokine- or inflammatory mediator-induced increases in microvessel permeability. The results demonstrated that leukocyte adhesion and emigration do not necessarily cause increased permeability and that the mechanisms that regulate the adhesion process act independently from mechanisms that regulate microvessel permeability. The critical event or process that contributes to the increased microvessel permeability during inflammation or ischemia-reperfusion remains to be identified.
| |
APPENDIX |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
|
|---|
The apparent permeability coefficient (Pa) measured
under our experimental conditions is determined by the true diffusive permeability coefficient of the microvessel wall (Pd) and
the convective component due to solvent drag. The magnitude of solvent drag contributing to the total flux is determined by the hydraulic conductivity (Lp), the solute reflection
coefficient (
), and the effective filtration pressure
(Peff) across the microvessel wall (7)
|
|
|
Peff can be expressed as
|
is
the osmotic reflection coefficient, and 
p is the colloid
osmotic pressure of the perfusate. Under our experimental conditions, the mean P value was 15 cmH2O (varied between 10 and 20 cmH2O, n = 17). The reflection coefficient
to BSA (BSA) was estimated as 0.94 in rat mesenteric
microvessels by Kendall and Michel (25). The colloid
osmotic pressure for perfusate containing 50 mg/ml BSA
(pBSA) measured with a colloid osmometer was
21 cmH2O at 37°C (the actual BSA concentration measured
with a refractometer was 45 mg/ml). The reflection coefficient to
-lactalbumin (
-lactalbumin) was estimated as 0.35, assuming a similar value to that in frog mesenteric microvessels; the
colloid osmotic pressure for -lactalbumin (p
-lactalbumin; 3 mg/ml) measured with an
osmometer was 3.5 cmH2O. When hydrostatic pressure was
between 10 and 20 cmH2O, with perfusate containing 50 mg/ml
BSA and 3 mg/ml -lactalbumin, Peff was less than or
close to zero; Pe was negligible and Pe/[exp(Pe) 
1] was close to
1. Therefore, the apparent permeability coefficient measured under our
experimental conditions was close to the true diffusive permeability coefficient.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. F. E. Curry for suggestions regarding permeability coefficient measurement in rat microvessels to overcome solvent drag problems. We also thank Dr. Charles Michel for valuable comments on this manuscript and Dr. Scott Simon for technical support for the preliminary flow cytometer analysis.
| |
FOOTNOTES |
|---|
This study was supported by American Heart Association National Center Grant-In-Aid 96011510, by a West Virginia University School of Medicine Research grant, and by National Heart, Lung, and Blood Institute Grant HL-56237.
Address for reprint requests and other correspondence: P. He, Dept. of Physiology and Pharmacology, School of Medicine, Health Sciences Center North, West Virginia Univ., Morgantown, WV 26506-9229 (E-mail: phe{at}hsc.wvu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
August 8, 2002;10.1152/ajpheart.00787.2001
Received 30 June 2001; accepted in final form 8 July 2002.
| |
REFERENCES |
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DISCUSSION
APPENDIX
REFERENCES
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