Agents Actions37 (1992)
0065-4299/92/020080-05 $1.50+ 0.20/0 9 1992 Birkh~iuserVerlag, Basel
Effects of SC-41930 on leukocyte adherence and emigration in rat mesenteric venules B. J. Zimmerman, T. S. Gaginella 2 and D. N. Granger 1 1 Louisiana State University Medical Center, Department of Physiology,1501 Kings Highway,Shreveport, LA 71130,USA 2 Searle Research and Development,Skokie, IL 60077, USA
Abstract
This study demonstrates that SC-41930, 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4dihydro-8-propyl-2H-l-benzopyran-2-carboxylic acid, an orally active LTB4 receptor antagonist, reduces LTB4-induced leukocyte adhesion and emigration in rat mesenteric venules. The mesentery of Sprague Dawley rats was prepared for intravital microscopic examination and venules of 25-35 ~tm were chosen for evaluation. In control animals, LTB 4 (20 nM) was superfused over the mesentery for 30 rain. In the treatment group SC-41930 (5 ~M) was superfused for 30 rain, followed by a 30 rain superfusion with SC41930 and LTB 4. The LTB4-induced increase in leukocyte adherence and emigration in postcapillary venules was significantly attenuated by pretreatment with SC-41930. Other experiments demonstrated that platelet-activating-factor-induced leukocyte adherence was not affected by SC-41930. These results indicate that SC-41930 is a potent inhibitor of LTB4-induced leukocyte-endothelial cell adhesive interactions in postcapillary venules.
Introduction
Leukotriene B 4 (LTB4), a product of arachidonic acid, is a potent proinflammatory mediator. LTB 4 has been implicated in a variety of disease states, including psoriasis, ischemia-reperfusion, and ulcerative colitis. Elevated levels of LTB4 have been demonstrated in mucosal biopsies derived either from patients with ulcerative colitis [1] or in animal models of colonic inflammation [2]. Additionally, it has been demonstrated that (1) psoriatic skin lesions contain abnormally high amounts of LTB 4 [3], and (2) ischemia-reperfusion results in a significant increase in mucosal production of LTB4 [4]. SC-41930, an orally active LTB 4 receptor antagonist, has been shown to significantly attenuate neutrophil accumulation in animal models of ulcer-
ative colitis [5], ischemia-reperfusion [4], and psoriasis [6, 7]. Consequently, LTB 4 is frequently invoked as a mediator of the neutrophil adhesion, emigration, and activation that is associated with chronic inflammation. This has led to the proposal that LTB4-receptor antagonists may provide an effective therapeutic approach for the management of diseases associated with increased levels of LTB 4 production. In this study we assessed the possibility that SC-41930 suppresses neutrophil infiltration by interfering with leukocyte adhesion and emigration in postcapillary venules. Methods
Male Sprague-Dawley rats (150-250 g) were maintained on a purified laboratory diet and fasted 18 h
81
Agents Actions 37 (1992)
prior to the experiment. The animals were anesthetized with Na-5-ethyl-l(l'-methyl-propyl)-2-thiobarbiturate (Inactin, Byk Gulden Konstanz, Germany, 120 mg/kg body weight) and a tracheotomy was performed to facilitate breathing during the experiment. The right carotid artery was cannulated and systemic arterial pressure was measured and continuously monitored with a physiologic recorder. A midline abdominal incision was made and a segment of the jejunoileum was exteriorized through the abdominal incision. All exposed tissue was covered with saline-soaked gauze and Saran Wrap (Dow Chemical Co., Indiana) to minimize evaporation and tissue dehydration.
Intravital microscopy Rats were placed in a supine position on a plexiglass microscope stage and the mesentery was prepared for microscopic observation as described previously [8]. The mesentery was draped over an optically clear pedestal that allowed for observation of a 2 c m 2 segment of tissue. The temperature of the intestine was maintained at 37 ~ with a constant-temperature circulator water bath. Rectal and mesenteric temperatures were continuously monitored using an electrothermometer. The exteriorized bowel and mesentery was superfused with warmed bicarbonate-buffered saline that was bubbled with a mixture of 5% CO2 95% N 2 (pH 7.4). A biological microscope (Optiphot, Nikon Corp., Japan) with a 20 x objective lens (Leitz, Germany) and a 10 x eyepiece was used to observe the mesenteric microcirculation. The mesentery was transilluminated with a direct current light source. The image, projected by a video camera (Hitachi, Japan) onto a color monitor (Sony, Japan), was recorded using a videocassette recorder (Panasonic, Japan). A video time-date generator (Panasonic, Japan) projected the time, date and stopwatch function onto the monitor. Single unbranched venules with diameters ranging between 25 and 35 pm and a length greater than 150 ~tm were selected for study. Venular diameter (Dr) was measured on line using a video caliper (Microcirculation Research Institute, College Station, TX). The number of adherent leukocytes was determined off-line during playback of the videotaped images. A leukocyte was considered to be adherent to the venular endothelium if it remained
stationary for /> 30 s. Adherent cells are expressed as the number per 100 gm length of venule. The number of emigrated leukocytes was also determine off-line during playback of the videotaped images. Leukocyte emigration was expressed as the number per field of videotaped image. Rolling leukocytes were defined as those white cells that moved at a velocity less than that of erythrocytes. Leukocyte rolling velocity was determined from the time required for a leukocyte to traverse a 50 gm length of venule. Centerline red blood cell velocity (Vrbc) was measured using an optical Doppler velocimeter (Microcirculation Research Institute, College Station, TX) that was calibrated against a rotating glass disk coated with red blood cells. Venular blood flow was calculated from the product of mean red blood cell velocity (centerline v e l o c i t y - 1.6) and microvascular cross-sectional area, assuming cylindrical geometry. Venular wall shear rate (y) was calculated based on the Newtonian definition 7 = 8 0 0 0
( Vmean/Ov). Experimental protocols. Images from the mesenteric preparation were videotaped for 10 min after all parameters measured on-line were in a steady state. Immediately thereafter, the mesentery was superfused with bicarbonate-buffered saline containing either 20 nM LTB 4 (n=4) or 100 nM PAF (n = 5). The mesentery was superfused with either LTB4 or PAF for 30 min, with video recordings and repeat measurements of all parameters made during the final ten minutes. In additional experiments, the mesentery was superfused for 20 rain with 5 pM SC-41930 prior to the addition of either LTB 4 or PAF. 20 nM LTB 4 (n=5) or 100nM PAF (n=7) was added to the buffer containing the receptor antagonist and the mesentery was superfused for an additional 30 min, with recordings and measurements of all parameters made during the final ten minutes. Statistics. The data were analyzed using standard statistical analyses, i.e. analysis of variance with Scheffe's post hoc test. All values are reported as m e a n + S E M . Statistical significance was set at p~
0065-4299/92/020080-05 $1.50+ 0.20/0 9 1992 Birkh~iuserVerlag, Basel
Effects of SC-41930 on leukocyte adherence and emigration in rat mesenteric venules B. J. Zimmerman, T. S. Gaginella 2 and D. N. Granger 1 1 Louisiana State University Medical Center, Department of Physiology,1501 Kings Highway,Shreveport, LA 71130,USA 2 Searle Research and Development,Skokie, IL 60077, USA
Abstract
This study demonstrates that SC-41930, 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4dihydro-8-propyl-2H-l-benzopyran-2-carboxylic acid, an orally active LTB4 receptor antagonist, reduces LTB4-induced leukocyte adhesion and emigration in rat mesenteric venules. The mesentery of Sprague Dawley rats was prepared for intravital microscopic examination and venules of 25-35 ~tm were chosen for evaluation. In control animals, LTB 4 (20 nM) was superfused over the mesentery for 30 rain. In the treatment group SC-41930 (5 ~M) was superfused for 30 rain, followed by a 30 rain superfusion with SC41930 and LTB 4. The LTB4-induced increase in leukocyte adherence and emigration in postcapillary venules was significantly attenuated by pretreatment with SC-41930. Other experiments demonstrated that platelet-activating-factor-induced leukocyte adherence was not affected by SC-41930. These results indicate that SC-41930 is a potent inhibitor of LTB4-induced leukocyte-endothelial cell adhesive interactions in postcapillary venules.
Introduction
Leukotriene B 4 (LTB4), a product of arachidonic acid, is a potent proinflammatory mediator. LTB 4 has been implicated in a variety of disease states, including psoriasis, ischemia-reperfusion, and ulcerative colitis. Elevated levels of LTB4 have been demonstrated in mucosal biopsies derived either from patients with ulcerative colitis [1] or in animal models of colonic inflammation [2]. Additionally, it has been demonstrated that (1) psoriatic skin lesions contain abnormally high amounts of LTB 4 [3], and (2) ischemia-reperfusion results in a significant increase in mucosal production of LTB4 [4]. SC-41930, an orally active LTB 4 receptor antagonist, has been shown to significantly attenuate neutrophil accumulation in animal models of ulcer-
ative colitis [5], ischemia-reperfusion [4], and psoriasis [6, 7]. Consequently, LTB 4 is frequently invoked as a mediator of the neutrophil adhesion, emigration, and activation that is associated with chronic inflammation. This has led to the proposal that LTB4-receptor antagonists may provide an effective therapeutic approach for the management of diseases associated with increased levels of LTB 4 production. In this study we assessed the possibility that SC-41930 suppresses neutrophil infiltration by interfering with leukocyte adhesion and emigration in postcapillary venules. Methods
Male Sprague-Dawley rats (150-250 g) were maintained on a purified laboratory diet and fasted 18 h
81
Agents Actions 37 (1992)
prior to the experiment. The animals were anesthetized with Na-5-ethyl-l(l'-methyl-propyl)-2-thiobarbiturate (Inactin, Byk Gulden Konstanz, Germany, 120 mg/kg body weight) and a tracheotomy was performed to facilitate breathing during the experiment. The right carotid artery was cannulated and systemic arterial pressure was measured and continuously monitored with a physiologic recorder. A midline abdominal incision was made and a segment of the jejunoileum was exteriorized through the abdominal incision. All exposed tissue was covered with saline-soaked gauze and Saran Wrap (Dow Chemical Co., Indiana) to minimize evaporation and tissue dehydration.
Intravital microscopy Rats were placed in a supine position on a plexiglass microscope stage and the mesentery was prepared for microscopic observation as described previously [8]. The mesentery was draped over an optically clear pedestal that allowed for observation of a 2 c m 2 segment of tissue. The temperature of the intestine was maintained at 37 ~ with a constant-temperature circulator water bath. Rectal and mesenteric temperatures were continuously monitored using an electrothermometer. The exteriorized bowel and mesentery was superfused with warmed bicarbonate-buffered saline that was bubbled with a mixture of 5% CO2 95% N 2 (pH 7.4). A biological microscope (Optiphot, Nikon Corp., Japan) with a 20 x objective lens (Leitz, Germany) and a 10 x eyepiece was used to observe the mesenteric microcirculation. The mesentery was transilluminated with a direct current light source. The image, projected by a video camera (Hitachi, Japan) onto a color monitor (Sony, Japan), was recorded using a videocassette recorder (Panasonic, Japan). A video time-date generator (Panasonic, Japan) projected the time, date and stopwatch function onto the monitor. Single unbranched venules with diameters ranging between 25 and 35 pm and a length greater than 150 ~tm were selected for study. Venular diameter (Dr) was measured on line using a video caliper (Microcirculation Research Institute, College Station, TX). The number of adherent leukocytes was determined off-line during playback of the videotaped images. A leukocyte was considered to be adherent to the venular endothelium if it remained
stationary for /> 30 s. Adherent cells are expressed as the number per 100 gm length of venule. The number of emigrated leukocytes was also determine off-line during playback of the videotaped images. Leukocyte emigration was expressed as the number per field of videotaped image. Rolling leukocytes were defined as those white cells that moved at a velocity less than that of erythrocytes. Leukocyte rolling velocity was determined from the time required for a leukocyte to traverse a 50 gm length of venule. Centerline red blood cell velocity (Vrbc) was measured using an optical Doppler velocimeter (Microcirculation Research Institute, College Station, TX) that was calibrated against a rotating glass disk coated with red blood cells. Venular blood flow was calculated from the product of mean red blood cell velocity (centerline v e l o c i t y - 1.6) and microvascular cross-sectional area, assuming cylindrical geometry. Venular wall shear rate (y) was calculated based on the Newtonian definition 7 = 8 0 0 0
( Vmean/Ov). Experimental protocols. Images from the mesenteric preparation were videotaped for 10 min after all parameters measured on-line were in a steady state. Immediately thereafter, the mesentery was superfused with bicarbonate-buffered saline containing either 20 nM LTB 4 (n=4) or 100 nM PAF (n = 5). The mesentery was superfused with either LTB4 or PAF for 30 min, with video recordings and repeat measurements of all parameters made during the final ten minutes. In additional experiments, the mesentery was superfused for 20 rain with 5 pM SC-41930 prior to the addition of either LTB 4 or PAF. 20 nM LTB 4 (n=5) or 100nM PAF (n=7) was added to the buffer containing the receptor antagonist and the mesentery was superfused for an additional 30 min, with recordings and measurements of all parameters made during the final ten minutes. Statistics. The data were analyzed using standard statistical analyses, i.e. analysis of variance with Scheffe's post hoc test. All values are reported as m e a n + S E M . Statistical significance was set at p~
