MICROVASCULAR

RESEARCH

d-4,

127-142 (1992)

Effects of Hydrodynamics and Leukocyte-Endothelium Specificity on Leukocyte-Endothelium Interactions ERIC NAZZIOLA AND STEVEN

D. HOUSE

Department of Biology, College of Arts and Science, Seton Hall University, South Orange, New Jersey 07079 Received January 13, 1992 In vivo microscopy was used to assessthe relative contribution of hydrodynamic forces (network topography and shear rate) and the specificity for leukocytes to interact with venular endothelium as determinants of leukocyte-endothelium interactions. To ascertian this, microvascular networks in the rat and rabbit mesentery were examined under normograde and mechanically induced retrograde flows to determine the effect of reversed flow on leukocyte-endothelium interactions in arterioles and venules. The data indicate that retrograde perfusion under hemodynamic (red blood cell velocity and shear rate) states equivalent to normograde flow significantly increased leukocyte marginating flux in arterioles (from 0 to 0.5 cells/5 set) and decreased flux significantly in venules (from 1.0 to 0.2 cells/5 set). The increased flux in arterioles under retrograde conditions, however, was significantly lower than the flux in venules under nonnograde conditions and the decreased flux in venules during retrograde flow was significantly greater than the flux in arterioles during normograde flow. This apparent discrepancy appears to be the result of a heterogeneous distribution of adhesive receptors on vascular endothehum. Furthermore, marginating leukocytes in arterioles made only brief contact with the endothelium before being swept away while marginating leukocytes in venules during normal and retrograde perfusion rolled along the vascular wall, with similar velocities in both directions. In conclusion, although hydrodynamic forces are important in facilitating leukocyte margination through mechanisms of radial migration, it is leukocyte-endothelium specificity in venules that ultimately determines leukocyte-endothelium interactions. o 19~ Academic PKS, IX

INTRODUCTION Leukocyte-endothelium interactions are characterized by the tendency of leukocytes to move toward the luminal wall and contact the microvascular endothelium. In viva studies indicate that under normal physiological conditions, white blood cells (WBCs) may roll along the vessel wall, particularly in venules, at a constant velocity or may endure brief, intermittent, and multiple contacts with the endothelium in a process termed saltation (House and Lipowsky, 1987, 1988; Fiebig et al., 1991). During inflammatory reactions, however, leukocytes may adhere firmly in venules whereupon they project pseudopodia and migrate across the venular endothelium into traumatized interstitia through an event known as diapedesis (Atherton and Born, 1973; Grant, 1973; Mayrovitz et al., 1977; Fiebig et al., 1991). While leukocyte-endothelium interactions occur extensively in venules, little 127 00262862’92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.

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or no such activity is observed in arterioles (House and Lipowsky, 1987, 1991; Ley and Gaehtgens, 1991; Perry and Granger, 1991). This trend has been attributed to dramatically higher arteriolar shear rates in comparison to venular shear rates (Mayrovitz and Wiedeman, 1977; Firrell and Lipowsky, 1989). Low venular shear rates may induce the formation of red blood cell aggregates which occupy the center of the flow stream, thus promoting the radial migration of WBCs toward the vessel wall, that is, margination (Goldsmith and Spain, 1984; Nobis et al., 1985). Furthermore, at high shear rates marginating leukocytes are sloughed off the vascular endothelium, whereas at lower shear rates adhesive forces exceed shearing forces, facilitating margination and leukocyte-endothelium interactions (Lawrence et al., 1987; Mayrovitz et al., 1987; House and Lipowsky, 1988; Firrell and Lipowsky, 1989; Perry and Granger, 1991). The already lower venular shear rates may be further reduced during inflammation because of local systemic arteriolar constriction proximal to the traumatized tissue (Mayrovitz et al., 1980) and increases in venous resistance concomitant with reductions in effective diameter in the venules by leukocyte-endothelium adhesion (House and Lipowsky, 1987). Network topography may also play an important role in determining leukocyteendothelium interactions. Studies have demonstrated that the margination of leukocytes and their subsequent interaction with the endothelium occur primarily at venous confluences (Atherton and Born, 1973; Grant, 1973; Schmid-Schoenbein et al., 1975; Bagge and Karlesson, 1980). Increased radial migration at a venous confluence is the result of the faster, smaller, highly deformable erythrocytes passing the leukocytes as they enter a vessel of slightly larger diameter and forcing the leukocytes toward the vessel wall (Goldsmith, 1968; Schmid-Schoenbein et al., 1980b). Finally, preferential leukocyte-endothelium interactions in venules may be due to adhesion specificity unique to venular endothelium. In this context, leukocyteendothelium interactions are largely regulated by various membrane glycoproteins found on both leukocytes and vascular endothelium. Among the various receptors identified on leukocytes are the CDll/CDl8 complex (LFA-1, Mac-l, p150,95) (Harlan, 1985; Springer et al., 1986; Tonnesen et al., 1989; Tonnesen, 1989) and the adhesion receptor L-selectin (Kishimoto et al., 1989; Lasky, 1991, Jutila et al., 1991). The ligand on endothelial cells for CDll/CD18 is the intercellular adhesion molecule (ICAM) (Smith et al., 1989; Tonnesen et al., 1989; Tonnesen, 1989; Waltz et al., 1990) and the ligand for L-selectin is an unidentified endothelial cell surface carbohydrate. During control conditions both the CDll/CD18 complex and L-selectin are present in small amounts on leukocytes while the L-selectin ligand and ICAM are present on endothelium (Smith et al., 1989; Tonnesen, 1989; Argenbright et al., 1991; Kishimoto et al., 1989; Jutila et al., 1991). During an inflammatory response the glycoprotein complex CDll/CD18 on leukocytes is upregulated by chemotactic agents (FMLP, C5A) and lipid mediators (LTB4, PAF) (Harlan, 1985; Springer et al., 1986; Tonnesen et al., 1989; Tonnesen, 1989) while ICAM is upregulated by interleukin-1 (IL-l), tumor necrosis factor, and lipopolysaccharide (LPS) (Smith et al., 1989; Tonnesen et al., 1989; Tonnesen, 1989; Waltz et al., 1990). On the other hand, L-selectin expression is downregulated during chemotactic agent activation. Recent studies have proposed that receptor-ligands responsible for leukocyte-endothelium interactions may be ex-

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pressed in higher concentrations on venular endothelium than on arteriolar endothelium (House and Lipowsky, 1991; Ley and Gaehtgens, 1991; Perry and Granger, 1991). The purpose of this study was to elucidate the relative contribution of hydrodynamic forces (shear rates and network topography) and the specificity for leukocytes to interact with venular endothelium as determinants of leukocyte-endothelium interaction. To ascertain this, microvascular networks in the rat and rabbit mesentery were examined under normograde and mechanically induced retrograde flows to determine the effect of a reversed network on leukocyteendothelium interactions in arterioles and venules. The data were characterized in terms of the site of leukocyte-endothelium interaction, white blood cell marginating flux, white blood cell rolling velocity, leukocyte-endothelium adhesion, red blood cell velocity, and network topography. METHODS Experiments were performed on male Wistar rats ranging from 150 to 250 g and New Zealand white rabbits of either sex ranging from 1.0 to 2.5 kg. Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (45 mg/kg) and rabbits were anesthetized with an injection into an ear vein of 30 mg/kg sodium pentobarbital. A tracheotomy was performed on the animals to facilitate respiration, and the carotid artery and jugular vein were cannulated for blood pressure monitoring and additional drug administration, respectively. Systemic arterial pressures were measured by a Statham pressure transducer. The experiments were terminated if mean arterial pressure fell below 80 mm Hg. The animal was placed on a heating pad and a midsagittal abdominal incision was made through which the jejunum and ileum could be exteriorized and then the mesentery was laid over a plexiglass stage. Preparations were continuously suffused with a bicarbonate Ringer-gelatin solution buffered to pH 7.4 and maintained at 37°C. The intestine was wrapped in cotton wool and areas outside the region of interest were covered with Saran Wrap (Dow Corning). Specimens were placed on a Nikon UM3 metallurgic microscope adapted for intravital microscopy. A Nikon extra-long-working distance 20x objective (N.A. 0.4) provided a field of view of 200 ym wide on the video monitor. All microcirculatory events were recorded using a Panasonic VWCD-52 video camera and a JVC HR-D630U videocassette recorder and viewed on a Panasonic TR-930A high-resolution television monitor. Supplemental still images were obtained using a Polaroid freeze-frame video image recorder. Microvessel length and luminal diameter were measured either on-line or offline using the video image shearing technique (IPM Model 908) (Intaglietta and Tompkins, 1973). This technique, using the current optical configuration, yields an accuracy of 25% of the measured value for lengths and diameters greater than 20 pm and ? 1 pm for measurements less than 20 pm. Red cell velocity (Vrbc) along the center line of arterioles and venules was measured either on-line or off-line using the two-slit technique (Johnson and Wayland, 1967) or the video-densitometric dual-window technique (Fagrell et al., 1974) respectively. In both applications a self-tracking correlator (Tompkins et al., 1974) was used to obtain Vrbc.

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Estimates of wall shear rate (y) were based on the Newtonian definition: y = 8(V,,,,,,/D) (Lipowsky et al., 1978), where the value V,,,,,, was estimated from the measured V,, using the empirical relationship If,,,,,,, = V&.6 (Baker and Wayland, 1974). Measurements pertaining to marginating leukocyte flux and WBC rolling velocity were accomplished through variable speed playback of video recordings time-marked during expeiments by a video time code generator (For A, Model VT-33). Marginating leukocyte flux was determined by counting the number of leukocytes passing a line (approximately 20 pm wide) drawn perpendicular to the vessel of interest during 5-set intervals. All visible, marginating leukocytes traveling at a velocity less than that of the erythrocytes in the same stream were included in this parameter. Thus, the marginating flux included all leukocytes that made contact with the endothelium regardless of the nature of that contact, i.e., rolling along the endothelium at a constant velocity, brief, intermittent, and multiple contact with the endothelium (saltating), and leukocytes that made only a brief single contact with the endothelium (common in arterioles during retrograde flow). Because of the low magnifications used, a distinction between rolling and saltating could not be made and, thus, these categories were combined into a single group. Rolling-saltating leukocyte velocity was obtained through frameby-frame analysis of the videotape. A leukocyte was considered adhered to the endothelium if it was stationary for more than 5 set during the duration of the hemodynamic measurements. Satisfactory microvascular networks were located, and normograde flows were observed for several minutes. Reverse perfusion was induced by occluding a 30 to 50 pm venule remote from the region of observation, employing a micromanipulator and blunt micropipette (approximately 50 pm in diameter). Venular occlusion induced changes in pressure throughout the microvascular network and in some networks produced retrograde flow. Areas that demonstrated retrograde flow during occlusion and returned to normal flow on release were recorded. The term normograde flow was used to denote the normal physiological pattern of blood flow prior to any occlusion. Vessels were categorized according to their hydrodynamic topography such that diverging bifurcating vessels were identified as arterioles and converging vessels were identified as venules. Salient histological properties, such as the presence of smooth muscle in arterioles, aided in vessel classification. In most cases, retrograde perfusion changed vessels with diverging bifurcating flow patterns (arterioles) to vessels with converging flow patterns, and vessels with converging flow patterns (venules) to vessels with bifurcating flow patterns. Whereas, these vesselsremain unaltered histologically, hydrodynamically they are inverted. In a few arterioles and venules, initiation of retrograde perfusion produced reverse blood flow through the vessel length but these vessels retained their diverging or converging hydrodynamic specificity due to changes in the flow patterns through these vessels. This situation existed as the result of the highly branched microvessel topography in the rat and rabbit mesentery. These vessels were analyzed separately. The protocol involved measurement of hemodynamics during a 2-min control period (normograde) followed by a 2-min period of retrograde flow and finally a 2-min control period. Statistical analysis for differences was examined by use of

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TABLE 1 EFFECTSOF RETRCGRADEPERFUSIONON HEMODYNAMICS IN ARTERIOLES AND VENULES OF THE RAT AND RABBIT MESENTERIES

Arterioles Rat (n = 8) Normograde Diameter (pm) V,, (mm/iec) Shear rate (sec.‘) WBC marginating flux (cells/5 set) WBC rolling flux (cells/5 Set)

11.9 0.83 409.5 0.02

f f + f

0.9 0.24 133.0 0.02

Rabbit (n = 5)

Retrograde 11.9 0.69 329.7 0.67

0

2 4 2 k

Normograde

1.0 0.20 107.4 0.27*

19.7 ? 0.8 1.24 + 0.14 312.9 f 31.3 0

0

Retrograde 19.7 1.07 271.3 0.29

0

2 k 2 2

0.8 0.18 44.3 O.OS*

0

Venules Rat (n = 7) Normograde Diameter (pm) Vrk (mm/s4 Shear rate (sec.‘) WBC marginating flux (cells/5 set) WBC rolling flux (cells/5 set)

15.5 0.70 239.2 0.86

f f ” 2

1.8 0.14 50.2 0.42

0.86 2 0.42

Rabbit (n = 8)

Retograde 15.9 0.61 200.5 0.33

5 4.8 +- 0.36 zk 45.3 + 0.20*

0.33 r 0.20*

Normograde 23.1 1.08 237.5 1.22

-c + zi 4

0.83 0.18 43.4 0.21

1.22 2 0.21

Retrograde 23.2 0.83 177.1 0.13

2 0.92 +- 0.13 -t 27.1 f 0.07*

0.13 k 0.07*

Note. Data were obtained during a 30-set control period immediately prior to occlusion and a 30set period beginnng 30 set after retrograde perfusion. Values are mean k SEM. *Significantly different (P i 0.05) from normograde.

paired and unpaired t tests and the nonparametric Wilcoxon signed-rank and Mann-Whitney tests. In all tests, significance was assessedat the 95% confidence level (P < 0.05). RESULTS Table 1 demonstrates that similar data were obtained from the rats and rabbits in this study. Values are 30-set averages measured immediately before vessel occlusion or 30-set following occlusion. Thirty seconds of retrograde flow was sufficient in establishing new flow conditions and no additional significant changes were noted after this measurement period. There were no significant changes from control diameter in rat or rabbit arterioles or venules when retrograde flow was induced. In addition, red blood cell velocity and shear rate decreased similarly, but not significantly, in both preparations with decreases ranging from 14 to 29%. Finally, both preparations demonstrated significant increases in WBC marginating flux in arterioles (P < 0.05) and significant decreases in WBC marginating flux in venules (P < 0.05) during retrograde perfusion. Although there was a significant increase in marginating flux during retrograde perfusion in both rat and rabbit

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1.50 ‘;; s 1.25 & 1.00 6

0.75

g-1 0.50 9 0 0.25 B 0.00

AND HOUSE

500 _ 400 T; 300 ; B 200 5 cT 100 $j 0

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FIG. 1. Effect of retrograde perfusion on V,, shear rate, and WBC marginating flux in 13 arterioles. Data were obtained during a 30-set control period immediately prior to occlusion, a lo-set period immediately prior to occlusion, a lo-set period immediately following occlusion, and a 30-set period beginning 30 set after retrograde perfusion. The bars represent SEM. *Significantly different (P < 0.05) from control.

arterioles, WBCs that contacted the endothelium in these vessels did not roll or saltate along the luminal surface but were quickly swept off the endothelium. In contrast, all contact between leukocytes and venular endothelium during normal and retrograde perfusion in both rat and rabbit preparations resulted in rollingsaltating interactions. While the data for the rat and rabbit preparations yield nearly identical trends, there was one difference. In rabbits the WBC marginating flux in venules during normograde perfusion was significantly (P < 0.05) greater than the marginating flux in arterioles during retrograde perfusion, while the marginating flux in rat venules during normograde perfusion was significantly greater than the flux in arterioles during retrograde flow only at the P < 0.1 level. Although the data for the rat and rabbit have been analyzed separately, the conclusions reached are the same as those for combined data; thus, rat and rabbit data were combined for all further presentations and statistical analyses. Little or no leukocyte-endothelium adhesion was observed in these preparations during normal and retrograde perfusion. Figure 1 illustrates the effect of retrograde perfusion on Vrbc, shear rate, and WBC marginating flux in 13 arterioles that demonstrated retrograde perfusion. Data were obtained during a 30-set control period immediately prior to occlusion, a lo-set control period immediately prior to occlusion, a lo-set period immediately following retrograde perfusion, and a 30-set period beginning 30 set after retrograde perfusion. Initiation of retrograde perfusion significantly decreased (P < 0.05) red cell velocity and shear rate in arterioles (16% decrease) from control values but had no effect on WBC marginating flux. Thirty seconds of retrograde perfusion, however, significantly increased (P < 0.05) WBC marginating flux from

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200 ;

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FIG. 2. Effect of retrograde perfusion on V,h, shear rate, and WBC marginating flux in nine arterioles that demonstrated increases in marginating flux. Data were obtained during a 30-set control period immediately prior to occlusion, a lo-set period immediately prior to occlusion, a IO-set period immediately following occlusion, and a 30-set period beginning 30 set after retrograde perfusion. The bars represent SEM. *Significantly different (P < 0.05) from control.

approximately 0 to 0.5 cells/5 set (Fig. lb). Although there was a significant increase in WBC_marginating flux 30 set after retrograde flow was induced, the WBCs made only brief contact with the endothelium before being swept away and thus WBC rolling flux was zero and a WBC rolling velocity could not be determined. Because changes in WBC marginating flux could be the result of changes in V,, and shear rate, the raw data were analyzed for arterioles that demonstrated increases in WBC marginating flux during retrograde perfusion. Nine of the thirteen vessels exhibited an increase in WBC marginating flux during reverse perfusion while four vessels remained at zero. Figure 2 illustrates the effect of retrograde perfusion on V,,,, shear rate, and WBC marginating flux in the nine arterioles that demonstrated increases in WBC marginating flux. Figure 2a demonstrates that there were significant decreases in V,, and shear rate 10 set after occlusion but velocity and shear rate returned toward control during the 30-set measurement and were not significantly different from control. The WBC marginating flux 30 set after retrograde perfusion, however, significantly increased (P < 0.05) from control (Fig. 2b). As noted above, although there was a significant increase in WBC marginating flux, the WBCs in these arterioles made only brief contact with the endothelium before being swept away, hence, the WBC rolling flux was zero. Figure 3 illustrates the effect of retrograde perfusion in venules. There were no significant changes in V,, and shear rate in these venules at any time during the retrograde perfusion (Fig. 3a). In addition, there was no significant change in WBC marginating flux during the lo-set period immediately following retro-

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1.50

500 _

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400 :;

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FIG. 3. Effect of retrograde perfusion on V,, , shear rate, and WBC marginating flux in 15 venules. Data were obtained during a 30-set control period immediately prior to occlusion, a lo-set period immediately prior to occlusion, a lo-set period immediately following occlusion, and a 30-set period beginning 30 set after retrograde perfusion. The bars represent SEM. *Significantly different (P < 0.05) from control.

grade perfusion (Fig. 3b). There was, however, a significant decrease (P < 0.05) in WBC marginating flux from approximately 1.0 to 0.2 cells/5 set during the period 30 set after occlusion. In venules, all leukocyte-endothelium contacts during both normograde and retrograde perfusion resulted in WBCs rolling-saltating along the endothelium. The effect of retrograde perfusion on WBC rolling velocity normalized to V,, is

q NORMOGRADE q RETROGRADE

CONTROL 10s 10s 30s 4. Effect of retrograde perfusion on the ratio of WBC rolling velocity to red cell velocity (Vwk/Vrbc) in 15 venules. Data were obtained during a 30-set control period immediately prior to occlusion (n = 47 observations), a IO-set period immediately prior to occlusion (n = 29), a lO-set period immediately following occlusion (n = 29), and a 30-set period beginning 30 set after retrograde perfusion (n = 17). The bars represent SEM. FIG.

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600 800 400 WALL SHEAR RATE (set-')

200

135

1000

FIG. 5. WEE marginating flux for arterioles (a) and venules (b) as a function of wall shear rate. Data are plotted for normograde (solid symbols) and retrograde perfusion (open symbols). Linear regressions with slopes significantly different from zero were obtained for arterioles during retrograde perfusion (dashed line) and venules during normograde flow (solid line).

presented in Fig. 4. Under control conditions the WBC velocity was approximately 4% of V,, . This value did not change significantly from control during retrograde perfusion, although the absolute number of marginating WBCs was significantly lower (Fig. 3b). In an attempt to make some quantitative estimates as to the relative importance of shear rate, topography, and endothelium specificity, the trends in WBC marginating flux vs shear rate in arterioles (a) and venules (b) during normograde and retrograde perfusion are illustrated in Fig. 5. Linear regressions were performed on the data yielding slopes significantly different than zero for venules during normograde perfusion (FLX = 2.0-0.004 y, r = 0.32) and arterioles during retrograde perfusion (FLX = 1.0-0.001 y, r = 0.55). These regressions suggest that there is a linear increase in WBC marginating flux as shear rate falls in arterioles and venules when vessel topography favors margination. Analysis of these relationships indicates that both the y intercept and slope are significantly (P < 0.05) greater for the venular data than for the arteriolar data. In three microcirculatory networks, initiation of retrograde perfusion produced reverse blood flow through the network. but did not change the converging hydrodynamic topography of the collecting venule in that network. Figure 6 demonstrates the branching pattern and WBC marginating flux observed in one of these networks during normal and retrograde perfusion and is consisent with the other two networks. During the 30-set normograde perfusion period, vessel segments classified as venules demonstrated average WBC marginating fluxes ranging from 0.9 to 3.6 cells/5 sec. The WBC marginating flux in the arteriole during this

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NORMOGRADE

RETROGRADE

FIG. 6. Representative microvascular network in which the collecting venule retains its converging hydrodynamic specificity during retrograde perfusion. The numbers represent WFK marginating flux (cells/5 set) during normal and retrograde conditions. Arrows denote direction of flow. See text for details.

same time was 0 and 0.3 cells/5 set in the two segments studied. When retrograde perfusion was induced, the largest venule studied in this network remained a venule in terms of branching pattern, and the WBC marginating flux 30 set after retrograde perfusion was induced was measured at 2.7 cells/5 sec. The venules downstream from this venular segment, however, now demonstrated a diverging pattern. Despite the branching pattern, WBCs continued to marginate along the endothelium and in fact increased to 2.8 cells/5 set downstream. The lone arteriole in this network demonstrated increased WBC marginating flux in its initial segment (1.7 cells/5 set), although this vessel segment was downstream from a diverging bifurcation. WBC marginating flux in this arteriole fell to 0 cells/5 set further downstream, although there were no additional bifurcations or changes in Vrbf from the initial segment. There were no significant changes in V,, or diameter in these vessels during any period of normal or retrograde perfusion. DISCUSSION The inflammatory process is a complex sequence of cellular phenomena with the overall goal of protecting the body from foreign invaders, tissue damage, or both. The ability of leukocytes to interact with the endothelium and adhere at the appropriate time and place is necessary to deliver leukocytes to the inflammatory site and prevent pathogenesis in normal tissues. While inflammation is most often characterized by the number of leukocytes adhering to the endothelium, the initial step in the inflammatory process involves the rolling of WBCs along the vascular wall (Atherton and Born, 1972; Mayrovitz et al., 1987; Fiebig et al., 1991). It is obvious that the outward radial migration, or margination, of leukocytes within the lumen of the microvessel is necessary for leukocyte-endothelium interaction. The tendency of leukocytes to marginate

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in a vessel enhances the WBC flux at the wall and if adhesive bonds between the WBC and endothelium are sufficient to retard the leukocyte, rolling and adhesion may result (House and Lipowsky, 1988; Firrell and Lipowsky, 1989; Perry and Granger, 1991). In the current study, we have examined the relative contributions of the hydrodynamic forces that produce margination and the specificity of the adhesive bonds between leukocytes and arteriolar or venular endothelium as factors in the preferential leukocyte-endothelium interactions that have been observed in venular networks (Atherton and Born, 1972,1973, Firrell and Lipowsky, 1989; House and Lipowsky, 1987; Mayrovitz et al., 1977; Perry and Granger, 1991, Ley et al., 1991). To elucidate the roles of network topography, shear rate, and leukocyteendothelium specificity on leukocyte-endothelium interactions, blood flow reversal through the microcirculation of rat and rabbit mesentery was induced. Rat and Rabbit Preparations Studies of leukocyte-endothelium interactions have been performed on many different preparations including such varied tissues as the rabbit ear chamber (Schmid-Schoenbein et al., 1980b), the bat wing (Mayrovitz et al., 1977), the cat mesentery (House and Lipowsky, 1987), the rabbit omentum (House and Lipowsky, 1991), the rabbit mesentery (Perry and Granger, 1991), and the rat mesentery (Firrell and Lipowsky, 1989), to name a few. In general, the leukocyteendothelial interactions noted in each of these studies are similar. Recent work in our laboratory indicates that leukocyte-endothelium attachment-detachment processes in rabbit omentum are very similar to those in mouse lymphoid tissue, suggesting that a universal mechanism for leukocyte-endothelium adhesion may exist in all tissues (House and Lipowsky, 1991). The only difference between rat and rabbit data found in the current study was the comparison of WBC marginating flux in arterioles during retrograde perfusion with that in venules during normograde perfusion. In rabbit, the flux in arterioles was significantly less (P < 0.05) than that in venules while the difference in rat preparations was significant only at the P < 0.1 level. In light of the previous observations and the similarities observed in the current study for rabbit and rat mesenteric preparations, the data for these two preparations were combined. Hydrodynamic Factor-Topography Because leukocytes occupy two to three times the volume of erythrocytes and are considerably less deformable, WBCs traverse the microcirculation at a much slower velocity than erythrocytes (Schmid-Schoenbein et al., 1980b). Consequently, in narrow vessels, WBCs may partially obstruct the vessel lumen causing red blood cells to accumulate behind them. When these blood cells encounter a vessel of slightly increased diameter, as when postcapillary venules converge into a venule, the smaller, highly deformable erythrocytes flow around the bulkier leukocytes and migrate toward the core of the vessel (Schmid-Schoenbein, 1980b). As a result, WBCs are simultaneously displaced to the luminal periphery where they sustain contact with the endothelium (Goldsmith, 1968). This model suggests that vessel topography is responsible for the hydrodynamic forces that :initialize the interactions between leukocyte and endothelium and is in agreement with the WBC marginating flux data in the current study. The data

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indicate that WBC marginating flux in venules was approximately 1.0 cell/5 set during control conditions when vessel topography favored WBC migration. When retrograde perfusion was induced, however, there was a significant decrease in WBC marginating flux 30 set after reverse flow. There were no changes in hemodynamics during the reverse perfusion, suggesting that flow reversal was responsible for the decrease in WBC marginating flux. Similarly, the data indicate that WBC marginating flux in arterioles significantly increased from approximately zero during normal flow conditions to greater than 0.5 cells/5 set during retrograde perfusion. While this increase could be attributed to decreases in shear rate (see below), the increase was more likely the result of increased WBC migration resulting from the favorable hydrodynamic interactions which occur when blood flows from the smaller capillaries to the larger arterioles. In fact, when only arterioles that demonstrated increases in WBC marginating flux were studied (Fig. 2), the data indicated that there was a significant increase in marginating flux in arterioles, although there was no significant change in shear rate. These findings clearly indicate that the mechanically reversed converging vessel system promotes leukocyte-endothelium interactions in arterioles. If hydrodynamic forces produced by the vessel topography were the only forces responsible for WBC margination and leukocyte-endothelium interactions, then blood flow reversal should produce similar values for WBC marginating flux in normograde venules and retrograde arterioles and similar values in retrograde venules and normograde arterioles. The combined data indicate, however, that the increased WBC flux in arterioles 30 set after retrograde perfusion was significantly lower (P < 0.05) than the original WBC flux in venules under normograde conditions (0.5 + 0.2 vs 1.0 2 0.2 cell/5 set, respectively). Furthermore, the WBC marginating flux in venules during retrograde perfusion was significantly greater (P < 0.05) than the WBC marginating flux in arterioles under normograde conditions (0.2 + 0.1 vs 0.01 + 0.05 cells/5 set, respectively). This apparent discrepancy may be due to lower shear rates in venules than in arterioles or the result of a heterogeneous distribution of adhesive receptors on vascular endothelium between arterioles and venules. Hydrodynamic

Factor-Shear

Force

Preferential margination of WBCs and leukocyte-endothelium interactions in venules may be the result of hydrodynamic forces related to blood flow-induced shear forces. Atherton and Born in 1973 suggested that the higher shear rates in arterioles prevented leukocyte adhesion and rolling in these vessels. Additionally, in vitro studies indicate that the low shear rates in venules may produce red blood cell aggregation, forcing the leukocytes toward the endothelium (Goldsmith and Spain, 1984; Nobis et al., 1985). The current data indicate that reductions in shear force enhance margination and leukocyte-endothelium interactions in venules during normograde flow and arterioles during retrograde flow. These findings are in agreement with several in vivo studies (Mayrovitz and Wiedeman, 1977; Firrell and Lipowsky, 1989, Perry and Granger, 1991). The observation that decreases in shear rate did not increase WBC marginating flux in arterioles during normograde perfusion is consistent with recent work by Ley and Gaehtgens (1991) which indicates that prolonged reduced shear rates did not increase leukocyteendothelium interactions in arterioles.

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in the current study, little or no leukocyte-endothelium adhesion was observed in arterioles or venules during normograde and retrograde perfusion. This suggests that the adhesive forces generated under these conditions were not sufficient to overcome the hydrodynamic dispersal forces (shear rates of approximately 300 set-’ in both arterioles and venules). The observation that WBCs marginated and rolled along the venular endothelium during both normograde and retrograde perfusion, although the numbers were significantly reduced during retrograde perfusion, suggeststhat the adhesive forces generated under these conditions were sufficient to permit WBC rolling. In contrast, WBCs marginated in arterioles during retrograde perfusion but made only brief contact with the endothelium and did not roll along the endothelium before being swept away. The lack of rolling WBCs in arterioles under conditions that favor margination and leukocyteendothelium interactions suggests that hydrodynamic dispersal forces were greater than the adhesive forces in these vessels. This could be attributed to shear rates that prevented leukocyte-endothelium interactions, a lack of adhesive receptors necessary to overcome dispersal forces, or a combination of these two factors. Because shear rates in these arterioles were not significantly greater than venular shear rates and at comparable reduced shear rates there was significantly more leukocyte-endothelium interaction in venules than in arterioles 0, intercepts of 2.0 + 0.42 (SD) and 0.97 + 25, respectively), a reduced number of adhesion receptors in the arterioles is highly likely. Endothelial Specificity Adhesive glycoprotein molecules have been found on the surface of both leukocytes and endothelial cells (Harlan, 1985; Springer et al., 1986; Tonnesen et al., 1989; Tonnesen, 1989; McEver, 1991). Recent studies indicate that rolling of unactivated WBCs is mediated by the leukocyte receptor L-selectin (Kishimoto et al., 1989; Jutila et al., 1991). Adhesion during inflammation, however, is due to rapid expression of the endothelial membrane glycoprotein P-selectin following exposure to the mediators thrombin or histamine (McEver, 1991). P-selectin is believed to tether the WBC to the endothelium and facilitate leukocyte activation and increased expression of leukocyte adhesion molecules from the glycoprotein family CDll/CD18 (LFA-1, Mac-l, p150,95) (Harlan, 1985; Springer et al., 1986; Tonnesen et al., 1989; Tonnesen, 1989). Both the CDll/CD18 complex and Lselectin are present in small amounts on unactivated WBCs while their ligands, ICAM and an unidentified endothelial surface carbohydrate, respectively, are present on unactivated endothelium (Smith et al., 1989; Tonnesen, 1989; Argenbright et al., 1991; Kishimoto et al., 1989; Waltz et al., 1990; Jutila et al., 1991). While it is beyond the scope of the present study to identify specific receptors, the present observations suggest that receptor-ligands responsible for leukocyteendothelium interactions may be expressed in higher concentrations on venular endothelium than on arteriolar endothelium. The idea that adhesion receptors may be unique to venous endothelium is supported indirectly by several studies. Work on histamine binding sites indicates that these receptors are present to a much greater degree in venules than in arterioles (Heltianu et al., 1982; Bundgaard, 1988). Furthermore, expression of an activation antigen induced by IL-l or LPS is localized primarily in postcapillary venules (Messadi et al., 1987) and the endothelial cell protein P-selectin is also found primarily in postcapillary venules

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(McEver, 1991). These studies indicate that there is tissue specificity for certain receptors and support the hypothesis that ligands for receptors responsible for leukocyte-endothelium interactions are found on venular endothelium but not, or at least’ many fewer ligands, on arteriolar endothelium. In vessels that demonstrated retrograde flow, the lo-set normograde WBC marginating flux and lo-set retrograde flux were not significantly different. In the case of arterioles, little or no leukocyte-endothelium interaction occurred immediately following occlusion because reverse flow had not existed long enough for the hydrodynamic conditions favoring these interactions to be sufficiently established. In contrast, leukocyte-endothelium interactions in venules were maintained during the lo-set period after retrograde perfusion because leukocytes that were already in contact with the endothelium and adhesion receptor-ligands continued to marginate and roll along the wall in the opposite direction. In the latter case, hydrodynamic conditions which favored the margination of leukocytes had been removed, and thus the number of marginating WBCs in venules soon decreased. The data obtained from venular networks which partially maintained their converging hydrodynamic topography during retrograde perfusion (Fig. 6) further illustrate the importance of endothelial specificity. The data indicate that during retrograde perfusion, WBCs that make contact with the venular endothelium roll along the endothelium with increases in leukocyte marginating flux down the length of the venule despite the WBCs passing through a hydrodynamically unfavorable diverging bifurcation. As the rolling leukocytes penetrate downstream into the arterioles, however, they are released from the endothelium. The release occurs in the arterioles, although there are no additional bifurcations and no changes in diameter or shear forces. This suggests that the release of the WBCs from the endothelium in arterioles is a consequence of sparse endothelium adhesion receptor-ligand concentration in the arterioles. Hence, while favorable vessel hydrodynamics are essential for the initial contact of leukocytes with vascular endothelium, the presence of leukocyte-endothelium receptor-ligands is the ultimate determinant of leukocyte-endothelium interactions. Finally, the current study indicates that the ratio of WBC rolling velocity to RBC velocity in venules is not different during normograde and retrograde perfusion. In light of prior theoretical analyses by Schmid-Schoenbein et al., (1987), which suggests that the ratio of WBC to RBC velocity may be used as a measure of the energy of adhesion between the WBC and endothelium, the current observations suggest that the balance of force (adhesive and dispersal) acting on the WBC in contact with venular endothelium is identical during normal and retrograde perfusion. Such a conclusion suggests that the ability of adhesion receptors on the endothelium to interact with leukocytes is not dependent on the direction of leukocyte movement.

ACKNOWLEDGMENTS This work was supported in part by FIRST Award R29 HL-44914 to S. D. House and a Seton Hall Research Council Award. The authors express their appreciation to Dr. H. H. Lipowsky of Penn State University for his helpful comments and suggestions.

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