The alpha 4-integrin supports leukocyte rolling and adhesion in chronically inflamed postcapillary venules in vivo.

Adjuvant Immunization. Under light anesthetic (diethyl ether; BDH Inc., Toronto, ON, Canada), male Sprague-Dawley rats (175-275 g) were injected subcutaneously at the base of the tail with a solution of heat killed Mycobacteriumbutyricumin Freund's mineral oil adjuvant (0.75 mg M. butyricum in 0.1 ml adjuvant; both from Difco Laboratories Inc., Detroit, MI). This protocol has been used as a model of arthritis in other laboratories and is de- scribed in detail elsewhere (14, 16, 24). The animals were moni- tored closely and weighed at 4-d intervals. Preliminary experiments using intravital microscopy (described below) revealed a tremen- dous increase in leukocyte trafficking through postcapillary venules of the mesenteric connective tissue at 4, 8, 12, and 20 d after immunization. One group of animals was immunized with 0.1 ml of mineral oil without M. butyricum.Leukocyte traffickingin this group was indistinguishable from untreated animals, therefore immunized animals were compared directlywith untreated controls. IntravitalMicroscopy. Rats were maintained on a purified lab- oratory diet and fasted for 18-24 h before surgery. Animals were anesthetized with an intraperitoneal injection of sodium pento- barbitol (65 mg/kg body weight). The right carotid artery and jugular vein were cannulated to measure systemic arterial blood pressure (pressure transducer model P23XL; Viggo-Spectramed, Oxnard, CA; and model 7 physiologic recorder; Grass Instru- ments Co., Quincy, MA) and administer drugs and antibodies, respectively. After laparotomy, rats were placed in a supine posi- tion on an adjustable PlexiglassTM microscope stage and a segment of the mid-jejunum was exteriorized and prepared for intravital microscopy. Briefly, the mesentery was placed over an optically clear viewing pedestal and exposed tissues were covered with sa- line-soaked gauze to minimize dehydration. The temperature of the pedestal was maintained at 37~ with a constant temperature water circulator (Haake Fisons, Karlsruhe, Germany) and the rats were kept at 37~ using an infrared heat lamp. The exposed mes- entery was suffused with warmed bicarbonate-buffered saline (pH 7.4) using a peristaltic pump (Minipuls3; Villiers Le Bel, Gilson, France), while excess fluid was removed via a suction pump. This preparation has been used extensively by us and others to study leukocyte-endothelial cell interactions within the mesenteric cir- culation (1, 25-28). The mesenteric preparation was observed through an intravital microscope (Optiphot-2; Nikon Inc., Mississauga, Canada) with a * objective lens (Wetzlar L25/0.35; E. Leitz Inc., Munich, Germany) and a * 10 eyepiece. A video camera (model 5100 HS; Panasonic, Osaka, Japan) mounted on the microscope projected the image onto a color monitor (model PVM 2030; Sony, Tokyo, Ja- pan), and the images were recorded using a videocassette recorder (model AG-1790; Panasonic, Osaka, Japan) for subsequent play- back analysis. The final magnification of the image on the monitor was * t800. Single unbranched mesenteric venules (25-50 ~m in diameter) were selected for study. The same section ofvenule was observed throughout the experiment to control for variations between different regions. Venular diameter (Dv) was measured on-line using a video caliper (Microcirculation Research Institute, Texas A&M University, College Station, TX). Centerline red blood cell velocity (Vrbc) was also measured onqine using an op- tical Doppler velocimeter (Microcirculation Research Institute, Texas A&M University). Venular blood flow was calculated from the product of cross-sectional area and mean red blood cell veloc- ity (Vmean = Vrbc/1.6) assuming cylindrical geometry. Venular wall shear rate (~) was calculated based on the Newtonian defini- tion: "y = 8(Vmean/Dv), and venular wall shear stress was 7 * blood viscosity (lq), where "qwas assumed to be 0.025 poise (29). The number of rolling and adherent leukocytes was deter- mined off-hne during video playback analysis. Leukocytes were considered adherent to the venular endothelium if they remained stationary for a period of time equal to or exceeding 30 s. Rolling leukocytes were defined as those white blood cells that moved at a velocity lessthan that oferythrocytes within a given vessel. The flux of rolling leukoeytes was determined as the number of white blood cells that rolled past a fixed point in the venule during a l-rain interval using frame-by-frame analysis. Leukocyte rolling velocity was calculated from the time required for a given leukocyte to travel a fixed distance along the length of the venule. Leukocyte tethering was measured as the number of new endothelial inter- actions that were initiated within a given segment of venule dur- ing a l-rain time interval. 1996 The cx4-Integrin Supports Leukocyte-Endothelial Interactions In Vivo ExperimentalProtocols. Upon locating a mesenteric venule 25-50 ~m in diameter, the image was recorded for 5 rain. Addi- tional 5-min recordings were made at 15-rain intervals over a 60- rain period and revealed little or no change in hemodynamic pa- rameters or leukocyte kinetics throughout the experiment. Therefore, antiadhesion molecule therapies were administered af- ter two baseline recordings (at the 20-min time point) so that each animal served as its own control. Initially, we examined leu- kocyte trafficking through mesenteric postcapillaryvenules at 4, 8, 12, or 20 d after immunization. No animals were allowed to sur- vive past 20 d due to the development of severe arthritis and other systemic complications. Leukocyte kinetics appeared to be identical at each of the days after treatment with M. butyricum,and therefore we undertook to examine the adhesive mechanisms in- volved at day 4 to avoid unnecessary discomfort related to the de- velopment of arthritis between days 8 and 20. Initially, we characterized the importance of L- and P-selectin in the increased leukocyte influx. The anti-P-selectin antibody, PB1.3 (30) (Cytel Corp., San Diego, CA) was administered at 2 mg/kg i.v. at 20 rain. We have previously demonstrated that this concentration of PB1.3 was most effective at preventing P-selectin- dependent leukocyte rolling in vivo (27). To study a role for L-selectin, animals were treated with an anti-L-selectm antibody, HRL-3 (31) (Upjohn Company, Kalamazoo, MI) at i mg/kg i.v. at 20 rain (32). An isotype-matched control antibody had no ef- fect on leukocyte rolling in the rat mesentery (27). Additionally, we examined the role of a selectin-binding polysaccharide, fucoi- din (25 mg/kg; Sigma Chemical Co., St. Louis, MO), that targets L- and P-selectin (1, 33, 34). To confirm that this concentration of fucoidin prevented selectin-dependent rolling, untreated (no M. butyricumimmunization) animals were also given the carbohy- drate moiety. This regimen has previously been shown to inhibit >85% ofroUing interactions under baseline conditions (I, 25, 26). M. butyricum-immunized animals responded only partially to anti-selectin therapy, suggesting the possibility that other rolling pathways exist in vivo. To date, in addition to selectins, only the el4-integrin has been proposed to support leukocyte rolling on endothelium (20). Therefore, in another series of experiments, immunized animals received an antibody directed against the ci4- integrin chain (TA-2, 4 mg/kg). This antibody has been shown to block lymphocyte adhesion to cytokine-stimulated rat endo- thelial cells in vitro (14, 35), and attenuate lymphocyte accumula- tion in rat dermal sites during delayed-type hypersensitivity reac- tions, without causing leukocytopenia or lymphocyte aggregation (13). Evidence that TA-2 binds the ot4-integrin has been docu- mented extensively elsewhere (35). To ensure that this antibody did not affect leukocyte trafficking nonspecificaUy, untreated ani- mals received identical doses of TA-2. To determine whether the 132-integrin was contributing to ~x4-dependent adhesion, a rat anti-CD18 antibody (WT-3, 2 mg/kg) was given to animals (36, 37). At this concentration, WT-3 inhibits leukocyte adhesion in- duced by N-formyl-met-leu-phe (fMLP) (37). Since cx4-integrins are not found on circulating neutrophils (8), it was likely that a nonneutrophil leukocyte population was inter- acting with endothelium in M. butyricum-immunized animals. To confirm this possibility, animals were given an anti-PMN serum (0.1 ml/rat; Accurate Chemical & Scientific Corp., Westbury, NY) that depletes circulating PMN populations, without affecting other leukocyte subpopulations. To confirm that the cx4-depen- dent population ofleukocytes was distinct from the PMNs, some animals were treated with the anti-PMN serum and then received the anti-oq antibody after depletion of PMNs. In a final experi- ment, animals were treated with fucoidin, and once a new base- line flux was achieved, the animals received the anti-e~4 antibody. Systemic blood samples were obtained at the beginning and end of each experiment for determination of total cell counts and population distributions. StatisticalAnalysis. All values are reported as means - SEM. The data within groups were compared using a paired Student's t test with Bonferroni corrections for multiple comparisons where appropriate. An unpaired Student's t test was used to compare be- tween groups. Statistical significance was set at P