The functional paradox of CD43 in leukocyte recruitment: a study using CD43-deficient mice.

Animals. CD43-deficient mice (CD43 −/− ) produced by homologous recombination in embryonic stem cells were generated in a mixed background of 129/SvEv × C57BL/6 as previously described ( 11 ), and were obtained from The Jackson Laboratory (Bar Harbor, ME). Wild-type mice derived from the same background were used as controls. Animals were bred and housed in specific pathogen-free facilities and used between 6 and 12 wk of age. Mouse Cremaster Preparation. The mouse cremaster preparation was used to investigate leukocyte–endothelial cell interactions in the microcirculation ( 12 ). Mice were anesthetized by intraperitoneal injection of a mixture of xylazine hydrochloride (10 mg/kg; MTC Pharmaceuticals, Cambridge, Ontario, Canada) and ketamine hydrochloride (200 mg/kg; Rogar/STB Inc., London, Ontario, Canada). The jugular vein was cannulated and used to maintain anesthesia. The cremaster muscle was dissected free of tissues and exteriorized onto an optically clear viewing pedestal. The muscle was cut longitudinally with a cautery and held flat against the pedestal by attaching silk sutures to the corners of the tissue. The muscle was then superfused with bicarbonate-buffered saline warmed to 37°C. An intravital microscope (Axioskop, Carl Zeiss Inc. Canada, Don Mills, Ontario, Canada) has been described elsewhere. Single unbranched cremasteric postcapillary venules (25–40 μm in diameter) were selected for examination of leukocyte rolling and adhesion. Leukocytes were considered adherent to the venular endothelium if they remained stationary for a period ≤30 s. Rolling leukocytes were defined as those moving at a velocity less than that of erythrocytes within a given vessel. Leukocyte rolling velocity was calculated from the time taken for a leukocyte to roll 100 μm. Venular diameter ( D v ) was measured on-line using a video caliper (Microcirculation Research Institute, Texas A&M University, College Station, TX). Centerline red blood cell velocity ( V RBC ) was also measured on-line using an optical Doppler velocimeter (Microcirculation Research Institute) and mean red blood cell velocity ( V mean ) was determined as V RBC /1.6. Venular wall shear rate was calculated based on the Newtonian definition: γ = 8 ( V mean / D v ) ( 13 ). Experimental Protocol. To determine whether CD43 was capable of regulating leukocyte recruitment induced by an acute chemotactic stimulus, the response to the bacterial peptide N -formyl-methionyl-leucyl-phenylalanine (fMLP; Sigma Chemical Co. , St. Louis, MO) was examined. fMLP (10 μM) was superfused over the cremasteric preparations for 60 min and leukocyte rolling and adhesion in postcapillary venules determined at 15-min intervals. Leukocyte Emigration. To examine whether leukocyte emigration was impaired in CD43 −/− mice, emigration induced by platelet-activating factor (PAF), a potent promoter of leukocyte emigration in the mouse cremaster microcirculation, was studied ( 14 ). In this final group of intravital microscopy experiments, cremasteric preparations were superfused for 60 min with 100 nM PAF in a saline solution containing 0.5% BSA. PAF was used because fMLP did not induce emigration in the murine system. Peritoneal Elicitation. Mice were given a 1-ml i.p. injection of 1% oyster type II glycogen in sterile saline as previously described ( 15 ). After 4 or 24 h, cells were harvested from the peritoneal cavity by lavage via 3 ml of sterile saline, and then counted using a hemocytometer. Differential counting was performed with Wright-Giemsa staining. Finally, to ensure that incomplete harvest was not responsible for lower leukocyte yields in the peritoneum of CD43 −/− mice, peritoneums were also lavaged with heparin and EDTA. Similar results were obtained with heparin or EDTA. Flow Chamber Assay. To study murine leukocyte behavior under shear conditions in vitro, whole blood was perfused over immobilized E-selectin (5 μg/ml), using a previously described flow chamber assay ( 12 ). Coverslips were mounted into a polycarbonate chamber with parallel plate geometry ( 16 ) and observed at a magnification of 100 using an inverted microscope equipped with phase-contrast optics ( Carl Zeiss Inc. Canada). The stage area was enclosed in a warm air cabinet and maintained at 37°C. In brief, the blood was diluted 1:10 in HBSS, maintained at 37°C using a water bath, and perfused through the flow chamber at defined wall shear stresses using a syringe pump (Harvard Apparatus Inc., South Natick, MA). All of the experiments described were performed between 2 and 4 dynes/cm 2 . The blood was perfused over the substrate for 3 min and then chased with HBSS to flush out any remaining erythrocytes and noninteracting leukocytes. At this stage leukocytes interacting with the coverslip could be seen readily and were counted in at least four random fields per coverslip and expressed as the number of interacting cells per field of view. Statistical Analysis. All data are presented as mean ± SEM. The data within groups were compared using a paired Student's t test using Bonferroni corrections for multiple comparisons as required. Unpaired t tests were used to compare between groups. Statistical significance was set at P < 0.05.

Experimental Protocol. To determine whether CD43 was capable of regulating leukocyte recruitment induced by an acute chemotactic stimulus, the response to the bacterial peptide N -formyl-methionyl-leucyl-phenylalanine (fMLP; Sigma Chemical Co. , St. Louis, MO) was examined. fMLP (10 μM) was superfused over the cremasteric preparations for 60 min and leukocyte rolling and adhesion in postcapillary venules determined at 15-min intervals.