906
Leukotriene B4 Mediates Shear Rate-Dependent Leukocyte Adhesion in Mesenteric Venules Kristine Bienvenu, Janice Russell, and D. Neil Granger Previous studies have demonstrated that low shear rates promote leukocyte adherence to microvascular endothelium in postcapillary venules. The objective of this study was to determine whether an accumulation of inflammatory mediators such as platelet activating factor and leukotriene B4 is responsible for shear rate-dependent leukocyte-endothelial cell adhesion. Postcapillary venules (25-39 ,m in diameter) in cat mesentery were studied by intravital microscopy. Venular wall shear rate was varied over a wide range by graded occlusion of the mesenteric artery. Red blood cell velocity, vessel diameter, leukocyte rolling velocity, and the numbers of rolling and adherent leukocytes were measured at each shear rate. In one series of experiments, shear rate-dependent leukocyte adherence was monitored at different superfusion rates (1.0 and 2.5 ml/min). At the lower superfusion rate, the number of adherent leukocytes was significantly higher at any given shear rate when compared with results obtained at the higher superfusion rate. This suggests that reduced washout of inflammatory mediators contributes to shear rate-dependent leukocyte adhesion. Pretreatment with different platelet activating factor receptor antagonists (WEB 2086 or WEB 2170) had no effect on the number of adherent leukocytes normally observed at lower shear rates, suggesting that platelet activating factor does not play a major role in this process. However, shear rate-dependent leukocyte adhesion was largely prevented by pretreatment with either a leukotriene B4 receptor antagonist (SC-41930) or a leukotriene synthesis inhibitor (L663,536). The results of this study indicate that a reduced washout of leukotriene B4 is responsible for the enhanced leukocyte adherence that occurs at low venular wall shear rates. (Circulation Research 1992;71:906-911) KEY WORDS * platelet activating factor * microvascular endothelium * intestinal ischemia inflammatory mediators
A
lthough it has been known for over a century that leukocyte adhesion in postcapillary venules is an early and important component of the inflammatory process,1 only in recent years has this essential interaction between leukocytes and endothelium captured the interest of a large number of investigators. This renewed interest in leukocyte-endothelial cell interactions has led to the recognition that leukocyte adhesion in venules is a well-controlled physiological phenomenon that is influenced by both physical and chemical forces acting on cell (leukocyte and endothelium) surfaces. Physiological factors that appear to make a significant contribution to leukocyte-endothelial cell adhesion during inflammation include 1) adhesion molecules expressed on the surface of activated neutrophils and/or endothelial cells,2 2) reactive oxygen metabolites and granule-associated proteins released by activated neutrophils,3 and 3) electrostatic charge interactions between the neutrophil and endothelial cell surfaces.4 There is a growing body of evidence indicating that hydrodynamic dispersal forces (e.g., wall shear stress) generated within the microcirculation also exert
From the Department of Physiology and Biophysics, Louisiana State University Medical Center, Shreveport. Supported by grant DK-43785 from the National Institutes of Health. Address for correspondence: D. Neil Granger, PhD, Department of Physiology, LSU Medical Center, 1501 Kings Highway, Shreveport, LA 71130-3932. Received January 31, 1992; accepted June 2, 1992.
a significant influence on leukocyte-endothelial cell adhesion.5 Indeed, it has been demonstrated that reductions in shear rate result in a progressive recruitment of adherent leukocytes in venules but not in arterioles.6 This shear rate-dependent leukocyte adhesion in postcapillary venules appears to be mediated by the leukocyte adhesion glycoprotein CD11/CD18, since monoclonal antibodies directed against the glycoprotein inhibit the adhesion.6 Although the dependence of leukocyte adhesion on shear rate has been demonstrated both in vivo67 and in vitro,5 the mechanisms underlying this phenomenon remain undefined. One possible explanation for shear rate-dependent leukocyte adhesion is that as venular blood flow is reduced, there is a concomitant reduction in the washout of inflammatory mediators (e.g., leukotriene B4 [LTB4] or platelet activating factor [PAF]), which are normally produced by endothelial and/or parenchymal cells. The resultant accumulation of inflammatory mediators would then lead to activation/ upregulation of CD11/CD18 on neutrophils, thereby promoting leukocyte adherence. The principal objective of this study was to address the reduced washout hypothesis using two different approaches. One approach involved quantitation of shear rate-dependent leukocyte adhesion at different mesenteric superfusion rates, which represents an attempt to alter the washout of inflammatory mediators independent of tissue blood flow. The second approach involved the use of specific compounds that either antagonize or inhibit the biosyn-
Bienvenu et al Shear Rate-Dependent Leukocyte Adhesion thesis of certain inflammatory mediators. Our studies focused on LTB4 and PAF, because both inflammatory agents are rapid and potent initiators of neutrophil adherence in postcapillary venules2 and have been implicated as mediators of the leukocyte-endothelial cell adhesion induced by ischemia/reperfusion.8'9
Materials and Methods Surgical Procedure Thirty cats (1.5-2.5 kg) were fasted for 18-24 hours before the experiment. The animals were initially anesthetized with ketamine hydrochloride (50 mg/kg i.m.). The jugular vein was cannulated, a saline drip was established, and anesthesia was maintained with intravenous sodium pentobarbitone (30 mg/kg). A tracheotomy was performed, and the animal was ventilated by a respirator (model 665, Harvard Apparatus, South Natick, Mass.). The right carotid artery was cannulated, and systemic arterial pressure was measured with a P23A pressure transducer (Statham, Oxnard, Calif.) connected to the carotid artery cannula. Systemic blood pressure and heart rate were continuously recorded with a physiological recorder (Grass Instrument Co., Quincy, Mass.). A midline abdominal incision was made, and the large bowel (from the ileocecal valve to the distal colon) was surgically removed. The animal was heparinized (10,000 units/kg), and an arterial circuit was established between the femoral artery and the superior mesenteric artery. Superior mesenteric artery blood flow was continuously monitored with an electromagnetic flowmeter (Carolina Medical Electronics, King, N.C.) connected to a probe positioned within the arterial circuit. Superior mesenteric artery pressure was measured via a side port in the flow probe. Body temperature was maintained at 37°C by a thermistorcontrolled heat lamp. All exposed tissue was moistened with saline-soaked gauze to minimize evaporation and tissue dehydration.
Intravital Microscopy Animals were placed in a left lateral recumbent position on an adjustable Plexiglas microscope stage. A segment of midjejunum was exteriorized through the abdominal incision, with great care taken to avoid trauma to the exposed bowel and mesentery. The mesentery was prepared for microscopic observation according to the methods of House and Lipowsky.10 The mesentery was draped over an optically clear viewing pedestal that allowed for transillumination of a 3-cm2 segment of tissue. The temperature of the pedestal was maintained at 37°C with a constant temperature circulator (model 80, Fisher Scientific Co., Pittsburgh, Pa.). Rectal and mesenteric temperatures were continuously monitored with an electrothermometer. The exposed bowel wall and mesentery were covered with Saran Wrap (Dow Chemical Co., Indianapolis, Ind.). The mesentery was suffused with warmed (37°C) bicarbonate-buffered saline (pH 7.4) at a rate of 2.5 ml/min. The oxygen tension of the suffusion solution was reduced to 40 mm Hg by bubbling with a mixture of 5% C02-95% N2. An intravital microscope (Ortholux II, E. Leitz, Inc., Rockleigh, N.J.) with a x40 objective lens (Leitz Wetzlar L20/0.32, Midland, Ontario) and a x 10 eyepiece was used to observe the mesenteric microcirculation. The
907
mesentery was transilluminated with a 12-V 100-W direct current-stabilized light source. A video camera (model VK-C150, Hitachi, Japan) mounted on the microscope projected the image onto a color monitor (model PVM-2030, Sony, Japan), and the images were recorded using a videocassette recorder (model NV8950, Panasonic, Japan). A video time and date generator (model WJ810, Panasonic) projected the time, date, and stopwatch function onto the monitor. Single unbranched venules with diameters ranging between 25 and 39 ,um and a length >150 ,um were selected for study. Venular diameter was measured either on- or off-line using a video image-shearing monitor (IPM, Inc., La Mesa, Calif.). The number of adherent leukocytes was determined off-line during playback of videotaped images. A leukocyte was considered to be adherent to the venular endothelium if it remained stationary for .30 seconds." Adherent cells were expressed as the number per 100-,um length of venule. Rolling leukocytes were defined as those white blood cells that moved at a velocity less than that of erythrocytes in the same stream. Leukocyte rolling velocity was determined from the time required for a leukocyte to traverse a given distance along the length of the venule. The number of rolling leukocytes present in the venule at any given moment was calculated as the flux (number of rollers passing a defined point per second) divided by the leukocyte rolling velocity. The total number of leukocytes exhibiting an adhesive interaction within the venule was determined from the sum of adherent and rolling leukocytes. In our previous studies (unpublished data), we have noted that the magnitude of the leukocyte adherence response elicited by ischemia/reperfusion or superfusion with inflammatory mediators is not influenced by the circulating leukocyte count (CLC) as long as CLC exceeds 4x 103 per cubic millimeter. Consequently, animals or experiments with CLC 500
Shear Rate(sec ) FIGURE 3. Bar graph showing effects of leukotriene B4 receptor antagonist SC-41930 and leukotriene synthesis inhibitorL663,536 on shear rate-dependent leukocyte adhesion. *p500 sec-'). tp 500
Shear Rate (sec ) FIGURE 5. Bar graph showing the influence of shear rate on the leukocyte rolling velocity/red blood cell velocity ratio (Vwbc/V,j in the presence or absence of leukotriene B4 receptor antagonist SC-41930 or leukotriene synthesis inhibitor L663,536. *p500 sec `). tp 500
Shear Rate (sec
)
FIGURE 4. Bar graph showing effects of leukotriene B4 receptor antagonist SC-41930 and leukotriene synthesis inhibitor L663,536 on shear rate-dependent recruitment of rolling leukocytes. *p500 sec-').
250-500
> 500
Shear Rate (sec ) FIGURE 6. Bar graph showing the influence of shear rate on the sum of adherent and rolling leukocytes in the presence or absence of leukotriene B4 receptor antagonist SC-41930 or leukotriene synthesis inhibitor L663,536 *p< 0. 05 compared with normal shear rate (>500 sec-'). tp
Leukotriene B4 Mediates Shear Rate-Dependent Leukocyte Adhesion in Mesenteric Venules Kristine Bienvenu, Janice Russell, and D. Neil Granger Previous studies have demonstrated that low shear rates promote leukocyte adherence to microvascular endothelium in postcapillary venules. The objective of this study was to determine whether an accumulation of inflammatory mediators such as platelet activating factor and leukotriene B4 is responsible for shear rate-dependent leukocyte-endothelial cell adhesion. Postcapillary venules (25-39 ,m in diameter) in cat mesentery were studied by intravital microscopy. Venular wall shear rate was varied over a wide range by graded occlusion of the mesenteric artery. Red blood cell velocity, vessel diameter, leukocyte rolling velocity, and the numbers of rolling and adherent leukocytes were measured at each shear rate. In one series of experiments, shear rate-dependent leukocyte adherence was monitored at different superfusion rates (1.0 and 2.5 ml/min). At the lower superfusion rate, the number of adherent leukocytes was significantly higher at any given shear rate when compared with results obtained at the higher superfusion rate. This suggests that reduced washout of inflammatory mediators contributes to shear rate-dependent leukocyte adhesion. Pretreatment with different platelet activating factor receptor antagonists (WEB 2086 or WEB 2170) had no effect on the number of adherent leukocytes normally observed at lower shear rates, suggesting that platelet activating factor does not play a major role in this process. However, shear rate-dependent leukocyte adhesion was largely prevented by pretreatment with either a leukotriene B4 receptor antagonist (SC-41930) or a leukotriene synthesis inhibitor (L663,536). The results of this study indicate that a reduced washout of leukotriene B4 is responsible for the enhanced leukocyte adherence that occurs at low venular wall shear rates. (Circulation Research 1992;71:906-911) KEY WORDS * platelet activating factor * microvascular endothelium * intestinal ischemia inflammatory mediators
A
lthough it has been known for over a century that leukocyte adhesion in postcapillary venules is an early and important component of the inflammatory process,1 only in recent years has this essential interaction between leukocytes and endothelium captured the interest of a large number of investigators. This renewed interest in leukocyte-endothelial cell interactions has led to the recognition that leukocyte adhesion in venules is a well-controlled physiological phenomenon that is influenced by both physical and chemical forces acting on cell (leukocyte and endothelium) surfaces. Physiological factors that appear to make a significant contribution to leukocyte-endothelial cell adhesion during inflammation include 1) adhesion molecules expressed on the surface of activated neutrophils and/or endothelial cells,2 2) reactive oxygen metabolites and granule-associated proteins released by activated neutrophils,3 and 3) electrostatic charge interactions between the neutrophil and endothelial cell surfaces.4 There is a growing body of evidence indicating that hydrodynamic dispersal forces (e.g., wall shear stress) generated within the microcirculation also exert
From the Department of Physiology and Biophysics, Louisiana State University Medical Center, Shreveport. Supported by grant DK-43785 from the National Institutes of Health. Address for correspondence: D. Neil Granger, PhD, Department of Physiology, LSU Medical Center, 1501 Kings Highway, Shreveport, LA 71130-3932. Received January 31, 1992; accepted June 2, 1992.
a significant influence on leukocyte-endothelial cell adhesion.5 Indeed, it has been demonstrated that reductions in shear rate result in a progressive recruitment of adherent leukocytes in venules but not in arterioles.6 This shear rate-dependent leukocyte adhesion in postcapillary venules appears to be mediated by the leukocyte adhesion glycoprotein CD11/CD18, since monoclonal antibodies directed against the glycoprotein inhibit the adhesion.6 Although the dependence of leukocyte adhesion on shear rate has been demonstrated both in vivo67 and in vitro,5 the mechanisms underlying this phenomenon remain undefined. One possible explanation for shear rate-dependent leukocyte adhesion is that as venular blood flow is reduced, there is a concomitant reduction in the washout of inflammatory mediators (e.g., leukotriene B4 [LTB4] or platelet activating factor [PAF]), which are normally produced by endothelial and/or parenchymal cells. The resultant accumulation of inflammatory mediators would then lead to activation/ upregulation of CD11/CD18 on neutrophils, thereby promoting leukocyte adherence. The principal objective of this study was to address the reduced washout hypothesis using two different approaches. One approach involved quantitation of shear rate-dependent leukocyte adhesion at different mesenteric superfusion rates, which represents an attempt to alter the washout of inflammatory mediators independent of tissue blood flow. The second approach involved the use of specific compounds that either antagonize or inhibit the biosyn-
Bienvenu et al Shear Rate-Dependent Leukocyte Adhesion thesis of certain inflammatory mediators. Our studies focused on LTB4 and PAF, because both inflammatory agents are rapid and potent initiators of neutrophil adherence in postcapillary venules2 and have been implicated as mediators of the leukocyte-endothelial cell adhesion induced by ischemia/reperfusion.8'9
Materials and Methods Surgical Procedure Thirty cats (1.5-2.5 kg) were fasted for 18-24 hours before the experiment. The animals were initially anesthetized with ketamine hydrochloride (50 mg/kg i.m.). The jugular vein was cannulated, a saline drip was established, and anesthesia was maintained with intravenous sodium pentobarbitone (30 mg/kg). A tracheotomy was performed, and the animal was ventilated by a respirator (model 665, Harvard Apparatus, South Natick, Mass.). The right carotid artery was cannulated, and systemic arterial pressure was measured with a P23A pressure transducer (Statham, Oxnard, Calif.) connected to the carotid artery cannula. Systemic blood pressure and heart rate were continuously recorded with a physiological recorder (Grass Instrument Co., Quincy, Mass.). A midline abdominal incision was made, and the large bowel (from the ileocecal valve to the distal colon) was surgically removed. The animal was heparinized (10,000 units/kg), and an arterial circuit was established between the femoral artery and the superior mesenteric artery. Superior mesenteric artery blood flow was continuously monitored with an electromagnetic flowmeter (Carolina Medical Electronics, King, N.C.) connected to a probe positioned within the arterial circuit. Superior mesenteric artery pressure was measured via a side port in the flow probe. Body temperature was maintained at 37°C by a thermistorcontrolled heat lamp. All exposed tissue was moistened with saline-soaked gauze to minimize evaporation and tissue dehydration.
Intravital Microscopy Animals were placed in a left lateral recumbent position on an adjustable Plexiglas microscope stage. A segment of midjejunum was exteriorized through the abdominal incision, with great care taken to avoid trauma to the exposed bowel and mesentery. The mesentery was prepared for microscopic observation according to the methods of House and Lipowsky.10 The mesentery was draped over an optically clear viewing pedestal that allowed for transillumination of a 3-cm2 segment of tissue. The temperature of the pedestal was maintained at 37°C with a constant temperature circulator (model 80, Fisher Scientific Co., Pittsburgh, Pa.). Rectal and mesenteric temperatures were continuously monitored with an electrothermometer. The exposed bowel wall and mesentery were covered with Saran Wrap (Dow Chemical Co., Indianapolis, Ind.). The mesentery was suffused with warmed (37°C) bicarbonate-buffered saline (pH 7.4) at a rate of 2.5 ml/min. The oxygen tension of the suffusion solution was reduced to 40 mm Hg by bubbling with a mixture of 5% C02-95% N2. An intravital microscope (Ortholux II, E. Leitz, Inc., Rockleigh, N.J.) with a x40 objective lens (Leitz Wetzlar L20/0.32, Midland, Ontario) and a x 10 eyepiece was used to observe the mesenteric microcirculation. The
907
mesentery was transilluminated with a 12-V 100-W direct current-stabilized light source. A video camera (model VK-C150, Hitachi, Japan) mounted on the microscope projected the image onto a color monitor (model PVM-2030, Sony, Japan), and the images were recorded using a videocassette recorder (model NV8950, Panasonic, Japan). A video time and date generator (model WJ810, Panasonic) projected the time, date, and stopwatch function onto the monitor. Single unbranched venules with diameters ranging between 25 and 39 ,um and a length >150 ,um were selected for study. Venular diameter was measured either on- or off-line using a video image-shearing monitor (IPM, Inc., La Mesa, Calif.). The number of adherent leukocytes was determined off-line during playback of videotaped images. A leukocyte was considered to be adherent to the venular endothelium if it remained stationary for .30 seconds." Adherent cells were expressed as the number per 100-,um length of venule. Rolling leukocytes were defined as those white blood cells that moved at a velocity less than that of erythrocytes in the same stream. Leukocyte rolling velocity was determined from the time required for a leukocyte to traverse a given distance along the length of the venule. The number of rolling leukocytes present in the venule at any given moment was calculated as the flux (number of rollers passing a defined point per second) divided by the leukocyte rolling velocity. The total number of leukocytes exhibiting an adhesive interaction within the venule was determined from the sum of adherent and rolling leukocytes. In our previous studies (unpublished data), we have noted that the magnitude of the leukocyte adherence response elicited by ischemia/reperfusion or superfusion with inflammatory mediators is not influenced by the circulating leukocyte count (CLC) as long as CLC exceeds 4x 103 per cubic millimeter. Consequently, animals or experiments with CLC 500
Shear Rate(sec ) FIGURE 3. Bar graph showing effects of leukotriene B4 receptor antagonist SC-41930 and leukotriene synthesis inhibitorL663,536 on shear rate-dependent leukocyte adhesion. *p500 sec-'). tp 500
Shear Rate (sec ) FIGURE 5. Bar graph showing the influence of shear rate on the leukocyte rolling velocity/red blood cell velocity ratio (Vwbc/V,j in the presence or absence of leukotriene B4 receptor antagonist SC-41930 or leukotriene synthesis inhibitor L663,536. *p500 sec `). tp 500
Shear Rate (sec
)
FIGURE 4. Bar graph showing effects of leukotriene B4 receptor antagonist SC-41930 and leukotriene synthesis inhibitor L663,536 on shear rate-dependent recruitment of rolling leukocytes. *p500 sec-').
250-500
> 500
Shear Rate (sec ) FIGURE 6. Bar graph showing the influence of shear rate on the sum of adherent and rolling leukocytes in the presence or absence of leukotriene B4 receptor antagonist SC-41930 or leukotriene synthesis inhibitor L663,536 *p< 0. 05 compared with normal shear rate (>500 sec-'). tp
