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Am J Physiol Heart Circ Physiol 291: H591-H599, 2006. First published March 3, 2006; doi:10.1152/ajpheart.01228.2005
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Leukocyte-platelet aggregate adhesion and vascular permeability in intact microvessels: role of activated endothelial cells

Pingnian He, Hong Zhang, Longkun Zhu, Yanyan Jiang, and Xueping Zhou

Department of Physiology and Pharmacology, West Virginia University, Morgantown, West Virginia


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Leukocyte-platelet aggregation and aggregate adhesion have been indicated as biomarkers of the severity of tissue injury during inflammation or ischemic reperfusion. The objective of this study is to investigate the mechanisms of the aggregate adhesion and quantitatively evaluate its relationship with microvessel permeability. A combined autologous blood perfusion with single microvessel perfusion technique was employed in rat mesenteric venular microvessels. The aggregate adhesion was induced by systemic application of TNF-{alpha} plus local application of platelet-activating factor (PAF). Changes in permeability were determined by measurements of hydraulic conductivity (Lp) before and after aggregate adhesion in the same individually perfused microvessels. The compositions of the adherent aggregates were identified with fluorescent labeling and confocal imaging. In contrast to leukocyte adhesion as single cells resulting in no increase in microvessel permeability, aggregate adhesion induced prolonged increases in microvessel Lp (6.1 ± 0.9 times the control, n = 9) indicated by the initial Lp measurements after 3 h of blood perfusion, which is distinct from the transient Lp increase caused by PAF-induced endothelial activation in the absence of blood. Isoproteronol (Iso) attenuated aggregate adhesion-mediated Lp increases if applied after autologous blood perfusion and prevented the aggregate adhesion if the initial endothelial activation is inhibited by applying Iso before PAF administration but showed less effect on single leukocyte adhesion. This study demonstrated that leukocyte-platelet aggregate adhesion via a mechanism different from that of single leukocyte adhesion caused a prolonged increase in microvessel permeability. Our results also indicate that the initial activation of endothelial cells by PAF plays a crucial role in the initiation of leukocyte-platelet aggregate adhesion.

hydraulic conductivity; tumor necrosis factor-{alpha}; platelet-activating factor

ACUTE INFLAMMATION involves the initial release of proinflammatory mediators, followed by leukocyte recruitment into the inflammatory sites. Sufficient evidence has documented that inflammatory mediators can increase microvessel permeability in the absence of leukocytes via direct activation of endothelial cells (32, 33). The activation of circulating leukocytes and their interaction with microvascular endothelial cells have also been reported as central events resulting in increases in microvessel permeability (14, 38). Some in vivo studies have reported that the magnitude of albumin leakage in postischemic venules is highly correlated with the number of adherent and emigrated leukocytes, and the prevention of leukocyte adhesion with specific antibodies or induction of neutropenia provided protection against vascular dysfunction during reperfusion or acute inflammation (5, 7, 26, 27, 29). However, our previous study demonstrated that when leukocytes attached to microvessel walls as single cells following intravenous injection of TNF-{alpha}, there were no measurable increases in microvessel permeability (41). Studies in cigarette smoke-induced, leukocyte-mediated tissue damage reported that smoke-induced leukocyte adhesion did not tend to interact with endothelium as single cells but rather mostly in the form of aggregates held together by activated platelets (6, 10, 30). A significant increase in leukocyte-platelet aggregates was found in the peripheral blood of ischemic stroke and reperfusion model in rats (37). However, the mechanisms of leukocyte-platelet aggregate formation are undefined, and the corresponding changes in microvessel permeability under those conditions have not been studied. Therefore, the objectives of this study are the following: 1) establish an in vivo model to induce leukocyte-platelet aggregation and aggregate adhesion in microvessel walls; 2) identify the blood cell components of the adherent aggregates; 3) quantitatively evaluate the effect of aggregate formation and adhesion on microvessel permeability; 4) distinguish the permeability changes caused by inflammatory mediator-induced direct activation of endothelial cells from those caused by blood cell aggregate formation and adhesion; and 5) explore the mechanisms of the aggregate formation and identify strategies to prevent the increases in microvessel permeability.

Experiments were designed to employ a combined autologous blood perfusion with a single microvessel perfusion technique in rat mesenteric venular microvessels. Our preliminary study demonstrated that the combination of systemic injection of TNF-{alpha} with the application of platelet-activating factor (PAF) to local blood flow induced significant aggregated blood cell adhesion that was distinct from leukocyte adhesion as individual cells induced by the application of TNF-{alpha} alone (41). Therefore, in the present study, autologous blood perfusion with systemic and local applications of TNF-{alpha} and PAF was applied to induce aggregated blood cell adhesion to microvessel walls. Changes in microvessel permeability were determined by measuring hydraulic conductivity (Lp) before and after aggregate adhesion in the same individually perfused intact microvessel in rat mesenteries. The compositions of blood cell type in the adherent aggregates were identified by using fluorescent labeling and confocal imaging.

To differentiate the contributions of the direct reaction of endothelial cells to TNF-{alpha} and PAF from that of aggregate formation and adhesion-induced changes in microvessel permeability, parallel experiments were conducted to compare the magnitude and the time course of the Lp changes in the presence and absence of blood components.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal preparation. Experiments were carried out in venular microvessels in rat mesenteries. All procedures and animal use were approved by the Animal Care and Use Committee at West Virginia University. Female Sprague-Dawley rats (2–3 mo old, 220–250 g body wt, Hilltop Laboratory Animal, Scottdale, PA) were anesthetized with pentobarbital sodium given subcutaneously. Female rats were used because the mesenteric vascular bed is more developed than in male rats, making selection of a suitable venular microvessel for cannulation easier and thereby requiring the use of fewer rats. The initial dosage was 65 mg/kg body wt with an additional 3 mg per dose given as needed. The trachea was intubated and a midline surgical incision (1.5–2 cm) was made in the abdominal wall. The mesentery was gently removed from the abdominal cavity and spread over a pillar for measurements of Lp. The upper surface of the mesentery was continuously superfused with mammalian Ringer solution at 37°C. All experiments were carried out in venular microvessels with diameters ranging between 40 and 50 µm with one experiment per microvessel in each animal.

Measurement of Lp. All measurements were based on the modified Landis technique that measures the water flux across the microvessel wall (9). The assumptions and limitations of the original method and its application in mammalian microvessels have been evaluated in detail elsewhere (9, 24). Briefly, a single venular microvessel was cannulated with a glass micropipette filled with albumin-Ringer solution (control) containing hamster red blood cells as markers. The micropipette was connected to a water manometer. A hydrostatic pressure (range 30–70 cmH2O) was applied through the micropipette to the microvessel lumen to allow perfusate to continuously flow in the vessel. For each measurement, the perfused vessel was occluded with a glass rod for 5–7 s at downstream and then allowed flow freely for at least 15 s before another occlusion was made. The initial water flow per unit area of microvessel wall [(Jv/S)0, where Jv is water flux and S is unit area of the microvessel wall] was calculated from the velocity of the marker cell after the vessel was occluded, the vessel radius, and the vessel length between marker cell position and the occlusion site. Microvessel Lp was calculated from the Starling equation, Lp = (Jv/S)0/{Delta}P, where {Delta}P is the effective hydrostatic and oncotic pressure difference across the microvessel wall.

Induction of aggregated blood cell adhesion with the application of TNF-{alpha} and PAF and measurements of Lp before and after aggregate adhesion in individually perfused microvessels. Each experiment was accomplished with four steps as shown in Fig. 1. The first step is to measure baseline Lp with the albumin-Ringer perfusate. The second step includes the removal of the cannulation pipette to resume blood flow in the same vessel and the injection of TNF-{alpha} (3.5 µg/kg) into the rat blood stream through the femoral vein. This dosage of TNF-{alpha} was sufficient to induce a significant amount of leukocyte adhesion (41), and the measured plasma TNF-{alpha} concentration is comparable with that in a rat ischemic model (39). Our preliminary study indicated that the systemic application of TNF-{alpha} before PAF application is necessary to facilitate the firm adhesion of leukocyte to microvessel walls. Step 3 was performed after injection of TNF-{alpha} for 2 h. As shown in Fig. 1, a branch vessel upstream from the original cannulation site was cannulated with perfusate containing PAF (1 nM), which enabled PAF to continuously mix with the blood flow that runs through the microvessel in which the control Lp was measured. In both steps 2 and 3, blood flow remained under physiological blood flow conditions. Step 4 was conducted after 1 h of PAF application. After leukocyte-platelet aggregates adhered to the microvessel wall under normal physiological conditions, the original vessel was recannulated and perfused with albumin-Ringer solution. The rolling, tethering, and nonfirmly adherent aggregates were washed away during the initial perfusion period. Changes in Lp were measured immediately, and the adherent aggregates that could withstand the magnitude of shear rate ranging from 28 to 40 s–1 (based on the Newtonian definition) during perfusion and remained attached to the vessel wall after the early Lp measurements (which took about 5–10 min) were counted under the microscope and expressed as the number per 100-µm vessel length (41). Therefore, leukocyte-platelet aggregate adhesion under our experimental conditions is defined as adhesion under certain shear rate, which is different from the adhesion reported in some of the intravital microscopy studies, such as remaining stationary for a period >30s (8, 27). In a perfused vessel, the transparent vessel lumen allows us to fully count all of the adherent leukocytes by focusing on top and bottom of the vessel wall, which may have the advantage over vessels that filled with circulating blood cells. However, the disadvantage is that the Ringer-albumin perfusion can wash out the reagents released from adherent leukocyte-platelet aggregates with time. Therefore, the initial Lp measurements after recannulation were used to assess the Lp changes induced by adherent aggregates, which more accurately represent the Lp level if blood perfusion remains.


Figure 1
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  		Fig. 1. Experimental protocols for inducing aggregated blood cell adhesion with the application of TNF-{alpha} (step 2) and platelet-activating factor (PAF, step 3) and measuring changes in hydraulic conductivity (Lp) before (step 1) and after aggregate adhesion (step 4) in individually perfused microvessels.

 
Measurements of Lp changes before and after vessel exposure to TNF-{alpha} and PAF in the absence of blood components. To differentiate the contributions of the direct reaction of endothelial cells to TNF-{alpha} and PAF from that of blood cell adhesion-induced permeability increases, parallel experiments were conducted in the absence of blood components. In each experiment, baseline Lp was measured first as shown in step 1 of Fig. 1. Then, without autologous blood perfusion, the changes in Lp were measured while each vessel was perfused with PAF (1 nM) or perfused with TNF-{alpha} (50 ng/ml) for 2 h and followed by addition of PAF (1 nM) to the perfusate.

Platelet preparation and confocal imaging. Whole blood was collected from a carotid artery of a donor rat and anticoagulated with 3.8% sodium citrate (9:1 vol/vol). Platelet-rich plasma was obtained from multiple sequential centrifugations and each was at 120 g for 5 min. Platelets were pelleted at 550 g for 10 min and resuspended in PBS. The platelet counts and aggregation status were examined with a hemocytometer under the microscope. The single platelets in each sample were >95% in all samples. For confocal imaging experiments, the isolated platelets were resuspended to PBS containing [5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CellTracker Orange, 10 µM] for 30 min. After fluorescence labeling was completed, the suspension was centrifuged, and the pellets were resuspended in Ringer solution. A total of 8 x 108 CellTracker Orange-labeled platelets were then slowly infused through a femoral vein to the experimental rat to achieve a labeled fraction of ~13–20% of total platelet count, a calculation based on blood volume and platelet concentration (2–3 x 108 platelets/ml) of rat. TNF-{alpha} and PAF were then applied to the experimental rat following procedures described in Fig. 1, steps 2 and 3. After the aggregate adhesion, the vessel was recannulated and perfused with albumin-Ringer solution containing FITC-conjugated anti-rat CD 45 (10 µg/ml), the leukocyte common antigen, for 20 min to label the adhered leukocytes. A Leica TCS SL confocal microscope equipped with an Acosto-Optical Beam Splitter was used to acquire fluorescence confocal images. The FITC fluorescence was detected with 488 nm excitation light (argon laser), and the emission filter band was at 500–535 nm. The CellTracker Orange was detected with 543 nm excitation (HeNe laser) and 550–590 nm emission filter band. Two stacks of identical optical sections were taken at successive x-y focal planes at 0.5-µm vertical step (z-axis) with a Leica 20X (HC Plan APO, numerical aperature 0.7) objective. These two stacks of images were then merged and projected to illustrate the composition of adherent aggregates. FITC-labeled CD45 located on the surface of adherent leukocytes was observed as green fluorescent rings and CellTracker Orange-labeled platelets were observed as solid red particles. The counting of adherent leukocytes and platelets was conducted on each projected image from the half stack of volume, i.e., a projection of half of the vessel wall longitudinally. It should be noted that in a perfused vessel, there were no circulating blood cells. Therefore, all of the platelets and leukocytes showing in the image are the firmly attached ones.

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 bovine serum albumin.

Recombinant rat TNF-{alpha} was purchased from Biosource International (Camarillo, CA). 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) was purchased from Sigma-Aldrich. CellTracker Orange was from Molecular Probes. FITC-conjugated anti-rat CD 45 was from PharMingen.

Data analysis and statistics. All values in the text are the means ± SE, except where noted otherwise. Changes in Lp were expressed as the ratio of testing Lp versus control Lp (Lptest/Lpcontrol). The mean values of Lp 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 Student t-test and nonparametric Wilcoxon Signed Rank test. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
TNF-{alpha} plus PAF-induced leukocyte-platelet aggregate adhesion in intact microvessels. After TNF-{alpha} was systemically applied for 3 h and PAF was applied to the local blood flow for 1 h (overlap for 1 h), there were significant increases in aggregated leukocyte-platelet adhesion, a pattern different from single leukocyte adhesion induced by TNF-{alpha} alone, which was illustrated in an earlier study (41). To affirm the involvement of platelet in the adherent aggregates, confocal images were acquired after aggregate adhesion in a perfused vessel (no circulating blood cells) to visualize the adherent fluorescence-labeled platelets (shown as dense red particles) and CD45-labeled leukocytes (surface labeling shown as green rings). Experiments were conducted in four microvessels in four animals. The quantitative assessments of firmly adhered leukocytes and platelets were performed off-line with projected confocal images. The mean aggregated leukocytes were 24 ± 3 per 100-µm vessel length, and the mean adherent fluorescent platelets were 16 ± 4 per 100-µm vessel length. By visualizing individual images at different focal planes, we found the fluorescent platelets either directly adhering to the vessel wall or interacting with aggregated leukocytes. With the assumption that the injected fluorescently labeled platelets and endogenous platelets have identical reactions to stimuli, the adherent fluorescent platelets (16 per 100-µm vessel length) may represent 13–20% of the total platelets. Then the estimated total number of adherent platelets that either adhered on the vessel wall or were involved in the adherent aggregates was in a range from 80 to 120 per 100-µm vessel length. One of the projected confocal images is shown in Fig. 2.


Figure 2
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  		Fig. 2. Confocal image illustrating involvement of platelet in TNF-{alpha} and PAF-induced adherent aggregates. Dense red particles are CellTracker Orange-labeled platelets, and green rings are CD45-labeled surface of leukocytes. Image acquisition was performed in albumin-Ringer-perfused vessel so that all of the platelets and leukocytes showing in the image are attached to the vessel wall. Confocal images were acquired at X-Y dimension with 0.5 µm per optical section at z dimension (vertical). This image is the projection of half stack of X-Y images.

 
TNF-{alpha} plus PAF-induced leukocyte-platelet aggregate adhesion increases microvessel permeability. Experiments were conducted in nine venular microvessels from nine animals. In each experiment, the control Lp was measured first and followed by resuming blood flow and applying TNF-{alpha} and PAF sequentially as described in MATERIALS AND METHODS. Figure 3A shows the representative video images of one of the venular microvessels under control conditions (Fig. 3A, left) and after leukocyte-platelet adhesion (Fig. 3A, right). The corresponding changes in Lp measured in that vessel are shown in Fig. 3B. The mean baseline Lp of nine vessels was 1.8 ± 0.3 x 10–7 cm·s–1·cmH2O–1. After the autologous blood perfusion for 3 h, each vessel was recannulated and perfused with albumin-Ringer perfusate. The mean adherent leukocyte-platelet aggregates was 17 ± 3 per 100-µm vessel length (Fig. 4A). Lp measured in the presence of adherent aggregates was 6.1 ± 0.9 times the control value in the first 5 min and declined to two times control in 15 min (Fig. 4B). Although the measured sixfold increase in Lp declined with time, this decline was considered as the wash-out effect by Ringer-albumin perfusion. The initial Lp more accurately represents the Lp level if blood perfusion remains. If the increased Lp was in a transient nature, there should be no such magnitude increase in Lp after 3 h of blood perfusion. Therefore, this 6.1-fold Lp increase measured in the presence of aggregate adhesion indicates a prolonged increase in microvessel permeability.


Figure 3
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  		Fig. 3. TNF-{alpha} and PAF-induced aggregated blood cell adhesion increases microvessel Lp. A: video images showing same vessel under control condition (Fig. 1, step 1) and after aggregated blood cell adhesion (Fig. 1, step 4). B: time course of Lp changes measured under vessel conditions illustrated in A. A significant increase in Lp occurred in the vessel with significant aggregated blood cell adhesion after TNF-a and PAF application and auto-blood perfusion.

 

Figure 4
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  		Fig. 4. Summary of systemic and local applications of TNF-{alpha}- and PAF-induced aggregated blood cell adhesion (A) and the associated changes in microvessel Lp (B).

 
PAF-induced Lp increases in the absence of blood components. To distinguish the permeability increases caused by the direct action of TNF-{alpha} and PAF on endothelial cells from that mediated by leukocyte-platelet aggregate adhesion, we measured the changes in Lp when each vessel was perfused with TNF-{alpha} alone and TNF-{alpha} plus PAF in the absence of blood components. Perfusion of TNF-{alpha} (50 ng/ml) alone did not change the basal Lp in a 2-h perfusion period (n = 5). Each of the five microvessels was subsequently exposed to PAF (1 nM) in the presence of TNF-{alpha}. The mean peak increase in Lp was 6.7 ± 1.1 times control, which was not significantly different from the Lp changes in response to PAF alone, a study reported previously (44). The mean Lp increase declined to 2.1 ± 0.3 times the control at the end of 1 h perfusion in the presence of TNF-{alpha} and PAF. Figure 5 shows the mean time course of the Lp changes of five experiments.


Figure 5
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  		Fig. 5. Summary of time course of PAF-induced increase in Lp in the absence of blood cells. PAF was applied to microvessel lumen after TNF-{alpha} perfusion for 2 h. Pretreatment of TNF-{alpha} did not change the basal Lp and PAF response. PAF-induced Lp increase peaked at 7 min and declined to twofold in 1 h of PAF perfusion.

 
Isoproterenol prevented PAF-induced increases in microvessel permeability in the absence of blood components. Experiments were conducted in four microvessels (one vessel per animal) to examine the role of isoproterenol (Iso) in PAF-induced Lp increases in the absence of blood components. Figure 6 shows an individual representative experiment. Superfusing Iso (10 µM) before PAF application significantly attenuated PAF (1 nM)-induced increases in Lp in the absence of leukocyte adhesion. The mean peak PAF response was reduced to 1.4 ± 0.5 times the control value. After Iso was washed out for 30 min, PAF-induced increases in Lp were restored to a mean peak value of 6.2 ± 1.4 times that of the control.


Figure 6
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  		Fig. 6. Superfusion of isoproterenol attenuated PAF-induced Lp increase in the absence of blood cells. A representative experiment showing that superfusion of isoproterenol significantly attenuated PAF-induced Lp increase. When isoproterenol was washed out by normal Ringer solution, the PAF response was restored in the same vessel.

 
Iso prevented the Lp increases mediated by leukocyte-platelet aggregate adhesion. Experiments were conducted in four microvessels from four different animals to examine whether the action of Iso on endothelial cells could prevent aggregate adhesion-induced increases in microvessel permeability. After control Lp was measured, blood flow was resumed in the vessel and each rat was given TNF-{alpha} and PAF following the protocols described in Fig. 1. Iso (10 µM) was added to the superfusate 15 min before the vessel was recannulated with albumin-Ringer solution (step 4). Results showed that the pattern of platelet-leukocyte aggregation and adhesion was similar to the image shown in Fig. 3A. The mean aggregated leukocytes were 22 ± 6 per 100-µm vessel length. However, in the presence of Iso, the initial increase in Lp was significantly attenuated to 1.2 ± 0.2 times of the control value. Figure 7 shows the Lp changes in an individual experiment.


Figure 7
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  		Fig. 7. Superfusion of isoproterenol abolished aggregation adhesion-induced permeability increase. After control Lp measurements, TNF-{alpha} and PAF were applied sequentially following steps 2 and 3 with resumed blood flow. Isoproterenol was added to the superfusate after aggregate adhesion and 15 min before Lp was remeasured. Isoproterenol superfusion abolished aggregate adhesion-induced Lp increase.

 
Superfusion of Iso before application of TNF-{alpha} and PAF prevents leukocyte-platelet aggregation and aggregate adhesion. Based on the results that Iso prevented PAF-induced Lp increases in the absence of blood, experiments are designed to investigate whether leukocyte-platelet aggregate adhesion is affected if Iso is applied before TNF-{alpha} and PAF application, which prevents PAF-induced endothelial activation. In six microvessels, Iso was applied to the superfusate after control Lp was measured (before the vessel was exposed to TNF-{alpha} and PAF) and continuously presented in the superfusate throughout the experiment. After a 3-h blood perfusion with TNF-{alpha} and PAF application, there was no leukocyte-platelet aggregate adhesion. Instead, the adherent leukocytes were single leukocytes (Fig. 8), a pattern that is similar to the leukocyte adhesion induced by systemic application of TNF-{alpha} alone (41). The mean adherent leukocytes were 9 ± 1 per 100-µm vessel length. There were no significant increases in Lp under these conditions. The mean initial Lp after the recannulation was 1.2 ± 0.2 times that of the control value.


Figure 8
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  		Fig. 8. Video images of an individual microvessel under control conditions (left) and after isoproterenol superfusion applied before TNF-{alpha} and PAF application (right). When isoproterenol was applied before the application of TNF-{alpha} and PAF and the assumption of blood flow, the adhesion pattern was changed from aggregated blood cells to single attached leukocytes. Lp measured in the presence of single attached leukocytes showed no significant increase.

 
Effect of PAF on rat platelet aggregation. To further evaluate the initiating mechanism of leukocyte-platelet aggregate adhesion, the effect of PAF on platelet aggregation was examined in isolated rat platelets from two animals. The platelet counts and aggregation status were evaluated with a hemacytomter under the microscope. Platelets isolated from two rats were separated into 12 samples (6 samples per rat). The mean platelet count per sample from six samples (3 per rat) in PBS was 1.98 ± 0.04 x 108 platelets/ml. The other six samples were suspended to equal amount of PBS containing PAF (10 nM). The platelet counts were evaluated after 10 and 30 min exposure to PAF. The mean platelet count per sample was 1.94 ± 0.05 and 1.91 ± 0.12 x 108 platelets/ml, respectively, which are not significantly different from that of the control. The single platelets in each sample were >95% in all samples.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our present study established an in vivo experimental model that creates an acute inflammatory condition with leukocyte-platelet aggregate adhesion to microvessel walls via combined systemic and local applications of TNF-{alpha} and PAF. The uniqueness of this experimental model is that it enables us to identify the cellular components of heterotypic blood cell aggregate adhesion in the vascular wall and quantitatively measure the changes in vascular permeability that were directly associated with leukocyte-platelet aggregate adhesion in intact venular microvessels. This has not been achieved from previous studies. Our results demonstrated that, in addition to PAF-induced direct activation of endothelial cells that causes an immediate transient increase in microvessel permeability, the leukocyte-platelet aggregate adhesion caused prolonged increases in microvessel permeability. Our study also showed that enhancing endothelial cAMP levels with beta-agonist Iso not only prevented the aggregate adhesion-induced increases in Lp but also prevented the aggregate formation when Iso was applied before the PAF application while the activation of endothelial cells was prevented. These results indicated that the endothelial activation is the initiating mechanism for platelet and leukocyte activation. The aggregate adhesions are the consequences secondary to the activation of endothelial cells but are the main contributors to the prolonged increases in microvessel permeability.

Our previous study demonstrated that systemic application of TNF-{alpha} induced a significant amount of nonaggregated leukocyte adhesion without causing increases in microvessel permeability (41). The present study is the extension of our previous study to further explore the mechanisms of leukocyte and/or other blood cell involved increases in microvessel permeability during acute inflammation. It has been reported (12) previously that PAF plays a significant role in leukocyte adhesion and leukocyte-dependent increases in microvessel permeability in whole vasculature studies, but detailed mechanisms remain unclear. To evaluate the role of PAF in leukocyte-dependent increases in microvessel permeability under our experimental conditions, we first locally applied PAF (1 nM) from an upstream branch vessel to the blood stream to induce leukocyte adhesion in downstream vessels. There were significant immediate increases in leukocyte rolling and tethering, but only few leukocytes firmly attached to microvessel walls. The lack of ICAM-1 expression on endothelial cells might explain this phenomenon. TNF-{alpha} is a recognized ICAM-1 inducer. Our previous study demonstrated that systemic application of TNF-{alpha} for 2 h induced a significant number of firmly attached leukocytes to microvessel walls (41). In this study, we combined systemic application of TNF-{alpha} with local injection of PAF. This combined application induced significant firm adhesion of leukocytes and also changed the adhesion pattern from single leukocytes occurred with the application of TNF-{alpha} alone to aggregated leukocytes and platelets. Aggregate adhesion and aggregate formation in peripheral blood have been reported in both cigarette-induced inflammation (30) and ischemic stroke and reperfusion models (37), which have been considered as the biomarker to determine the extent of the tissue injury. However, no correlative permeability studies have been conducted under those conditions. This experimental model enables endothelial cells in intact microvessels to directly interact with inflamed blood cells and plasma factors, which closely mimics the clinical situation. More importantly, when the autologous blood perfusion is combined with single microvessel perfusion, it allows us to not only identify, but also for the first time, to exhibit the heterotypic aggregated blood cell interaction with endothelial cells in intact microvessel walls with fluorescent labeling and confocal imaging. It also retains the capacity of single vessel perfusion for quantitative measurements of vascular permeability under well-controlled experimental conditions. Additionally, measuring Lp changes in response to the same stimuli with and without blood cell involvement enable us to distinguish the contribution of the direct activation of endothelial cells from that of aggregate adhesion to the permeability increases.

Our results indicated that the microvessel walls experienced two types of Lp increases in this experimental model. One is the immediate transient Lp increase resulting from the direct activation of endothelial cells by PAF that peaked at 7 min and declined to two times that of the control value at the end of 1 h of PAF perfusion (based on the Lp measurements in the absence of blood). This transient pattern, though the mechanism remains unidentified, occurs in a variety of inflammatory mediator-induced permeability increases in the absence of blood (15–17, 19). The 6.1-fold Lp increase measured after TNF-{alpha} and PAF application and autologous blood perfusion for 3 h indicates a prolonged increase in microvessel permeability that is distinct from the transient Lp increase caused by the direct action of PAF on endothelium. Although the measured 6.1-fold increase in Lp also declined with time, it was due to the wash-out effect by albumin-Ringer perfusate. The initial Lp measurements immediately after recannulation more accurately represent the Lp level if blood perfusion remains. This prolonged increase in microvessel permeability must attribute to agents released from adherent leukocyte-platelet aggregates. These results also indicate that the increased permeability was not an irreversible damage in the vessel wall, which could be recovered with Ringer-albumin perfusion.

The main differences with regard to the applied stimuli and the involved blood cell components between TNF-{alpha}-induced single leukocyte adhesion with no changes in microvessel permeability (41) and leukocyte-platelet aggregate adhesion with prolonged increases in microvessel permeability are the additional application of PAF and the involvement of platelets. Identifying the key target of PAF that induces leukocyte-platelet aggregation may lead us to define the critical mechanism responsible for leukocyte-platelet-dependent increases in microvessel permeability.

The name of PAF obviously indicates its main function. However, rat platelets have been reported as exhibiting low affinity or no receptor for PAF binding (21, 34). Those studies indicated that PAF-induced platelet aggregation and serotonin release is highly species selective. Platelets from humans, dogs, and rabbits are highly sensitive to PAF, but no effects were elicited on rat platelets. Our observation that isolated rat platelets in suspension did not aggregate with PAF addition was consistent with those reports. Therefore, the possibility that the aggregate formation and adhesion is the result of the direct action of PAF on platelets is excluded.

Sufficient evidence implicated that platelets normally do not adhere to the vascular endothelium unless the normal properties of either endothelial cells or platelets have been altered. Because platelets do not react to PAF directly, the other potential target of PAF is endothelial cells lining the microvessel walls. Our present and previous studies demonstrated that exposure of microvessels to PAF induced transient increases in endothelial intracellular Ca2+ concentration and microvessel permeability in the absence of blood cells (2, 44). Studies in the rat cremaster microvascular bed identified that venular endothelium has a higher level of PAF receptor than any other segment of microvasculature and topical application of PAF induced prompt openings of the venular and capillary junctions (36). At the electron microscopy level, the investigators revealed intramural deposition at the openings of intercellular junctions. The deposits were mainly tracers, erythrocytes, and platelets that escaped through the gaps but were retained by the basement membranes (36). Another electron microscopy study (2) in the rat mesenteric venular microvessels confirmed PAF-induced gap formation at the peak of Lp increase with serial sections in individually perfused venular microvessels. Among a variety of agonists that are able to activate platelets, collagen serves as primary activator at sites of vascular injury. An in vivo study in injured arterial wall demonstrated that subendothelial collagens are the major trigger of arterial thrombus formation, and collagen-induced activation of platelets plays a crucial role for platelet adhesion and aggregation via a platelet collagen receptor (31). With the information taken together, we reasoned that the subendothelial collagen exposure as the result of PAF-induced gap formation might serve as the primary activator for platelets and leukocyte-platelet aggregate adhesion. Additionally, endothelial P-selectin expression and cytokine production upon endothelial activation by PAF may also play important roles in leukocyte and platelet activations and aggregate adhesion. We, therefore, hypothesize that PAF-induced endothelial activation serves as the primary trigger for the activation of platelets and leukocytes resulting in leukocyte-platelet aggregate adhesion and prolonged increases in microvessel permeability.

This hypothesis is fully supported by our results with the application of Iso. The activation of adenylate cyclase-induced cAMP production by beta-adrenergic receptor agonist has been shown to enhance endothelial barrier functions and prevent a variety of inflammatory mediator-induced increases in microvessel permeability (4, 16, 18). One of the structural endpoints of stimulated cAMP levels is an increase in the mean number of tight-junction strands between endothelial cells (1). Our results showed that the application of Iso not only attenuated or abolished platelet-leukocyte aggregate adhesion-induced prolonged increases in permeability if it was applied after aggregate adhesion but also changed the adhesion pattern from leukocyte-platelet aggregate adhesion to single leukocyte adhesion if applied before the PAF application while endothelial activation is inhibited. Although beta-adrenergic receptor agonist may also prevent platelet aggregation, superfusion of Iso mainly affects endothelial cells with little, if any, effect on platelet or leukocytes due to rapid blood flow. These results implicated that the endothelial activation is a prerequisite for leukocyte-platelet aggregate adhesion under our experimental conditions. The fact that TNF-{alpha}-induced leukocyte adhesion is only single leukocytes without the involvement of platelet and leukocyte-platelet aggregation also supports this notion, because TNF-{alpha} does not cause an initial increase in microvessel permeability and gap formation (41). These results together suggest that single leukocyte adhesion that occurs in the absence of endothelial activation is regulated by a mechanism different from that which initiates leukocyte-platelet aggregation.

With regard to what factor or agents are responsible for the prolonged increases in microvessel permeability, we consider the amplified reactive oxygen species (ROS) release from the aggregates plays a major role. For a decade, the physical contact between leukocyte and endothelium is considered the critical step for endothelial barrier damage, resulting in protein leakage and tissue edema, and that the adhesion process was the trigger for the neutrophil respiratory burst (7, 11, 13, 28). However, we and others found dissociation between leukocyte adhesion and the increases in microvessel permeability (3, 20, 41–43). Our previous study demonstrated that neither exposure of isolated neutrophils to TNF-{alpha} alone nor TNF-{alpha}-induced leukocyte adhesion in intact microvessels is sufficient to induce leukocyte respiratory burst resulting in permeability increase (41). Instead, preexposure leukocytes to TNF-{alpha} in vivo or in vitro potentiated oxidative reaction of leukocytes to additional stimulus such as formyl-Met-Leu-Phe-OH, indicating a priming role of TNF-{alpha} in neutrophil oxidative activity (42, 43). Studies by others indicated a similar situation for PAF (22, 25, 40). Even though both TNF-{alpha} and PAF, acting as priming agents for neutrophils, could not directly trigger neutrophil respiratory burst, the releasing agents from the activated endothelial cells can serve as the secondary stimuli resulting in enhanced ROS production from either circulating or adherent leukocytes. Additionally, it was reported that collagen-induced platelet aggregation is associated with a burst of H2O2 that serves as a second messenger by activating arachidonic acid metabolism and phospholipase C pathway (35). The released H2O2 and the activation of other signaling pathways may also serve as secondary stimuli that trigger enhanced ROS production from TNF-{alpha}- and PAF-primed neutrophils resulting in an amplified chain reaction for platelet and leukocyte activation and recruitment. Our previous study demonstrated quantitative correlation between the magnitude of ROS release from either suspended or adherent leukocytes, and the magnitude of permeability increases in intact microvessels (42, 43). We consider the amplified production of ROS from leukocyte-platelet aggregation is the main cause for prolonged increases in microvessel permeability.

Currently extensive studies were focused on the roles of either platelet or leukocytes in heterotypic blood cell interactions with endothelium in sepsis and ischemic-reperfusion models. Immunoneutralization of platelet glycoprotein Ib{alpha}. showed significant attenuation of endotoxin-induced platelet and leukocyte interactions with rat venular endothelium in vivo, indicating the important role of platelets in the heterotypic blood cell interaction with endothelium (23). Other studies reported that the application of antibody for leukocyte adhesion prevented high dosage of PAF-induced increased filtration, indicating leukocyte-dependent platelet activation in an ischemic-reperfusion model (28). Although further investigations are needed to clarify whether the activations of platelets and leukocytes occur simultaneously or sequentially, we predict that the amplified ROS release from both activated platelets and leukocytes contributes to the prolonged increases in microvessel permeability. Instead of focusing on individual roles of platelet or leukocyte in these prolonged permeability increases, our present study brought the attention to the activated endothelial cells and presented a novel mechanism of activated endothelium-dependent leukocyte-platelet aggregation. Our results demonstrated that even both platelets and leukocytes were present, inhibiting the initial activation of endothelium by PAF prevented the aggregate formation and the prolonged permeability increases. This is the first study demonstrating the critical roles of the activated endothelial cells and the initial increases in vascular permeability in the formation and adhesion of leukocyte-platelets aggregates, a mechanism that has not been elucidated previously. Most importantly, our results indicated that preventing the initial inflammatory mediator-induced activation of endothelial cells by enhancing endothelial barrier functions might be a more efficient strategy to prevent the activated blood cell-mediated prolonged increases in microvessel permeability and prevent tissue edema and organ dysfunction during acute inflammation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the National Heart, Lung, and Blood Institute Grant HL-56237 and American Heart Association Ohio Valley Affiliate Grant-In-Aid.


   ACKNOWLEDGMENTS
 
H. Zhang's current address is Department of Laboratory Medicine, University of California, San Francisco, CA 94143-0134.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Dr. 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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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