SCID-hu mouse model
CB-17 SCID mice (Harlan, Mississauga, ON), 6–8 weeks of age were grafted with partial thickness human skin according to a previously described protocol [24, 25]. Animal protocols were approved by the local Animal Care Committee and met the guidelines of the Canadian Council for Animal Care. Briefly, partial thickness skin grafts from healthy human donors were prepared from discarded skin obtained from breast reduction surgeries using a 0.1-in. dermatome. A portion of the posterior thorax on the back (5 mm × 5 mm) was excised in halothane anesthetized recipient mice and human skin of the same size was placed and allowed to heal for 4 weeks before the mice were used for intravital microscopy. In the SCID-hu mouse model, transplanted human skin retains human dermal microvascular bed which connects with the mouse microvasculature as a result of spontaneous anastomosis .
Isolation and labelling of human leukocytes
Twenty mL of heparinised blood collected from healthy volunteers was centrifuged (1350 rpm, 7 min, room temperature) and the buffy coat was collected and washed with HBSS containing Ca2+ and Mg2+ (Gibco, Grand Isle, NY). To study the interactions between human leukocytes and human vascular endothelium of human skin graft in SCID mice using fluorescent intravital microscopy, human leukocytes were labelled with rhodamine 6-G (0.025 % final concentration, 5 min; Sigma, St. Louis, MO). Unbound rhodamine 6-G was removed by washing three times in HBSS.
Mice were administered with L-NAME (50 mg/kg b.w., i.p.; Bachem, Torrance, CA) for 4 h before viewing the human skin microvasculature using intravital microscopy. Animals were anesthetised i.p. with ketamine (160 mg/kg b.w.) and xylazine (10 mg/kg b.w.) and body temperature was regulated using a heating pad . Functional blocking antibodies and additional anesthetic was introduced through a cannulated jugular vein and infusion of rhodamine 6-G-labelled human leukocytes was through cannulated carotid artery. A flap consisting of the human skin graft was laid flat onto a pedestal, secured with suture, perfused with 37 °C-warmed saline and covered with a glass coverslip. The preparation was examined using an upright fluorescent microscope (Optiphot-2; Nikon) with a 20× water immersion objective. To identify human vessels, 100 mg of FITC-labelled Ulex europaeus (Sigma) was injected i.v. immediately before fluorescence microscopic visualization (excitation: 450–490 nm and emission: 520 nm). Rhodamine 6-G-labelled human leukocytes were visualized by excitation at 510–560 nm using a 590 nm emission filter. Images of the labelled human leukocytes and human microvessels were visualized using a silicon-intensified CCD camera (C-2400-08; Hamamatsu Photonics, Bridgewater, NJ) and recorded for playback analysis. Rolling of human leukocytes was expressed as percentage flux fraction, determined by counting the number of interacting human leukocytes in an individual vessel relative to the total number of human leukocytes passing through the vessel over the same period (determined by frame-by-frame analysis). Rhodamine 6-G-labelled human leukocytes that remained stationary on the vascular wall for at least 30 s were defined as adherent. Recording was started immediately after infusion of the labelled-leukocytes and the interactions were recorded for 30 min, a time point when the number of circulating labelled leukocytes was significantly diminished. Where indicated, the blocking mAbs (20–40 μg/mL) anti-human P-selectin G1 (BD Biosciences, Mississauga, ON) and/or anti-human E-selectin ES1 (kindly provided by Dr. KD Patel, University of Calgary, Calgary, AB), were injected i.v. as a bolus in a total volume of 100 μL of PBS after baseline interactions had been recorded. The antibodies were allowed to circulate for 2–3 min before a second bolus of human leukocytes was injected. Functional blocking is expected not to reverse leukocyte adhesion. Analysis of selectin functional blocking was thus, performed by determining the number of adherent leukocytes 5 min after the administration of blocking mAb.
Human skin samples were harvested from representative SCID mice after intravital microscopy. These samples were then frozen in OCT and cut into 5-μm thick sections. Sections were stained with polyclonal goat anti-human E-selectin antibody to examine the level of E-selectin expression and then with biotin-conjugated secondary rabbit anti-goat antibody (Jackson ImmunoResearch Laboratories, Burlington, ON). Color was developed using the ABC kit (Vector Laboratories, Burlingam, CA) and chromagen diaminobenzadine (Sigma) and the sections were then counterstained with Gill II hematoxylin. Images were captured using a CCD digital camera (Nikon).
Human umbilical vein endothelial cells (HUVECs) were harvested from fresh human umbilical cords and cultured as described previously . After confluence was reached, the cultured HUVECs were trypsinzied for detachment and then seeded onto fibronectin-coated coverslips or 48-well plates. Since senescent endothelial cells express 50 % to 75 % less eNOS mRNA compared to their primary or first-passaged counterparts , HUVECs were, thus, used at first passage for all experiments.
Isolation of human neutrophils
Human neutrophils were isolated from ACD (Acid Citrate Dextrose) anti-coagulated whole blood from healthy donors. After dextran (Spectrum Chemicals, Gardena, CA) sedimentation, isolation of neutrophils was performed at room temperature by using centrifugation through a density gradient of Ficoll Type 400 (Sigma) with 10 % Hypaque Sodium® (Sterling-Winthrop, Markham, ON). Isolated neutrophils were 97 % pure and 95 % viable. Purified human neutrophils were resuspended in HBSS with Ca2+ and Mg2+ at a concentration of 1×106 cells/mL prior to use in laminar flow chamber assay.
Laminar flow chamber assay
Coverslips with cultured HUVECs were mounted in a parallel plate flow chamber . Reagents were added to the neutrophil suspension at the indicated time. The flow chamber was placed onto the stage of an inverted microscope (Carl Zeiss, Toronto, ON) and HUVEC monolayers were visualized at 100× magnification using phase contrast imagery with a 0.45 mm2 field of view. The stage area was enclosed in a warm air cabinet and maintained at 37°C prior to perfusion using a water bath. A syringe pump (Harvard Apparatus, Laval, QC) was used to draw the cells through the flow chamber at 2 dynes/cm2 for 20 min. Experiments were recorded via a CCD camera for analysis. Interacting cells were either rolling or adherent to the surface of the cover slip. A neutrophil that remained stationary for 10 sec or more was considered as adherent.
Measurement of endothelial cell surface expression of P- and E-selectins
Surface P- and E-selectin expression on HUVECs was measured by ELISA. Cells were plated into a 48-well plate and used 24−48 h later. HUVEC monolayers were exposed to L-NAME or histamine (Sigma) for 10 min to induce P-selectin upregulation and with L-NAME or recombinant human TNFα (Collaborative Biochemical Products; Bedford, MA) for 4 h for E-selectin upregulation. After fixation (1 % formalin at 4 °C for 30 min) and rinsing with PBS, blocking was performed with 1 % BSA in PBS (30 min at 37 °C). The cells were incubated with the anti-P-selectin antibody S12 (2 μg/mL) or with the anti-E-selectin antibody EL246 (50 μg/mL) for 30 min at 37 °C, washed with 0.05 % Tween-20 followed by incubation (30 min at 37 °C) with the secondary antibody (peroxidase labelled goat-anti-mouse, 1:1000 dilution). Absorbance was measured at 450 nm after addition of TMB substrate (Dako Carpinteria, CA) and 0.01 H2SO4.
To determine the effect of L-NAME on PAF synthesis, a previously described method was used . Briefly, HUVECs were incubated with L-NAME (100 μM) for 10 min at 37 °C in the presence of 3H-acetate. Histamine (10-5 or 10-6 M) or HBSS was then added to HUVECs and further incubated for 5 min at 37°C. The cells were then incubated with acidified methanol, scraped and added to a Bligh-Dyer extraction with cold PAF. Methanol was added until a monophase which was then split. After centrifugation (800–1000 rpm), the upper phase was removed and the lower phase was dried and spotted on a TLC plate and the resultant spots counted.
Data are expressed as means ± SEM. As indicated in the figure legends, the statistical analysis in two value sets was made using paired or unpaired Student’s t test and the statistical differences among the multiple groups were analyzed by ANOVA and post hoc comparisons by the Tukey-Kramer Multiple Comparison Test. Values of p < 0.05 were considered statistically significant.