INTRODUCTION

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NEUTROPHIL RECRUITMENT is a key feature of the inflammatory response associated with ischemia-reperfusion (I/R). This is based on the observation that preventing neutrophil influx into tissues either by depleting the numbers of circulating neutrophils or by preventing neutrophil adhesion significantly reduced microvascular dysfunction (12) and tissue injury (32). More recently, it has become apparent that the recruitment of leukocytes during I/R is far more complex than initially thought and involves numerous mediators and at least two families of adhesion molecules. First, within the initial minutes of reperfusion, there is expression of P-selectin from Weibel-Palade bodies, which induces hundreds of cells to roll through postischemic vessels. Although oxidants, histamine, thrombin, and cysteinyl leukotrienes can all induce P-selectin expression and increase leukocyte rolling (9, 15, 16, 24, 35), only thrombin has been demonstrated to play a role in I/R-induced leukocyte rolling (31). The rolling cells then become activated by oxidants (10), platelet-activating factor (PAF) (22), leukotriene B₄ (34), and perhaps many other chemotactic factors, and this causes them to roll slowly and firmly adhere via ₂-integrins (2, 33). Clearly, there may be many mediators involved in the recruitment of neutrophils into reperfused tissues, and inhibiting the biological function of a single mediator or even administering a cocktail of numerous inhibitors of proinflammatory agents may not be sufficient to reduce postischemic neutrophil recruitment, vascular dysfunction, and tissue injury.

The afflicted tissue has the capacity to prevent the pathogenesis of I/R perhaps by producing endogenous anti-inflammatory molecules that can reverse the actions of the multitude of aforementioned proinflammatory mediators. This is best exemplified by the observation that it is possible to precondition tissue by briefly and temporarily reducing blood flow. This serves to render the tissue resistant to the deleterious effects of a prolonged ischemic insult (13). The vasculature itself can also be preconditioned, at least relative to neutrophil adhesion; Akimitsu et al. (1) demonstrated that preconditioning of skeletal muscle reduced reperfusion-induced neutrophil adhesion. As already stated, neutrophil rolling is a prerequisite for subsequent adhesion, which in turn is a prerequisite for vascular dysfunction in I/R. Therefore, we chose to further explore the effects of preconditioning with particular emphasis on the possibility that the regimen could reduce the first step of neutrophil recruitment, neutrophil rolling (as well as adhesion), and vascular dysfunction. Because adenosine has been implicated as the endogenous modulator of the preconditioning response (13) and adenosine has been shown to have inhibitory effects on neutrophil function (4, 7, 11), the second objective was to determine whether adenosine was also involved in the preconditioning-induced inhibition of neutrophil recruitment and vascular dysfunction associated with I/R.

Exogenous administration of adenosine has also been shown to behave as an anti-inflammatory molecule. Adenosine has been postulated to partially inhibit stimulated neutrophils from adhering to endothelium in vitro (6, 7). In vivo adenosine has been shown to reduce the second part of the recruitment cascade, neutrophil adhesion (3), in various inflammatory conditions, including I/R (11, 30); however, whether this was a direct effect was not entirely clear. Because neutrophil adhesion is dependent on the initial capture, rolling, and slowing of rolling cells, we decided to further evaluate the actions of adenosine to examine the effect of this purine on the events preceding firm adhesion. This was assessed initially using intravital microscopy in vivo and then in vitro for the first time under flow conditions to examine the direct effect of adenosine on leukocyte rolling on P-selectin and adhesion to activated endothelium under shear conditions.

	METHODS

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Intravital microscopic studies. The experimental preparation used in this study is the same as that described previously (10, 18, 21, 25). Briefly, cats (1.2-2.4 kg) were fasted for 24 h and initially anesthetized with ketamine hydrochloride (75 mg im). The jugular vein was cannulated and anesthesia was maintained by administration of pentobarbital sodium (30-40 mg/kg over the first hour and then as necessary). A tracheotomy was performed to support breathing by artificial ventilation. Systemic arterial pressure was monitored by a Statham P23A pressure transducer connected to a catheter in the left carotid artery. A midline abdominal incision was made, and a segment of small intestine was isolated from the ligament of Treitz to the ileocecal valve. The remainder of the small and large intestine was extirpated. Body temperature was maintained at 37°C using an infrared heat lamp. All exposed tissues were moistened with saline-soaked gauze to prevent evaporation. Heparin sodium (10,000 U, Elkins-Sinn, Cherry Hill, NJ) was administered, and then an arterial circuit was established between the superior mesenteric artery (SMA) and left femoral artery. SMA blood flow was continuously monitored using an electromagnetic flowmeter (Carolina Medical Electronics, Kings, NC). Blood pressure and SMA blood flow were continuously recorded with a Grass physiological recorder (Grass Instruments, Quincy, MA).

Cats were placed in a supine position on an adjustable Plexiglas microscope stage, and a segment of midjejunum was exteriorized through the abdominal incision. The mesentery was prepared for in vivo microscopic observation as previously described (10, 18, 21, 25). The mesentery was draped over an optically clear viewing pedestal that allowed for transillumination of a 3-cm segment of tissue. The temperature of the pedestal was maintained at 37°C with a constant temperature circulator (model 80, Fisher Scientific). The exposed bowel was draped with saline-soaked gauze, while the remainder of the mesentery was covered with Saran Wrap (Dow Corning). The exposed mesentery was suffused with warmed bicarbonate-buffered saline (pH 7.4) that was bubbled with a mixture of 5% CO₂-95% N₂.

The mesenteric preparation was observed through an intravital microscope (Nikon Optiphot-2, Japan) with a ×25 objective lens (Leitz Wetzlar L25/0.35) and a ×10 eyepiece. The image of the microcirculatory bed (×1,400 magnification) was recorded using a videocamera (Panasonic-Digital 5100) and a video recorder (Panasonic NV8950).

Single unbranched mesenteric venules (25-40 µm diameter, 250 µm length) were selected for each study. Venular diameter was measured online using a video caliper (Microcirculation Research Institute, Texas A & M University, College Station, TX). The number of rolling and adherent neutrophils was determined offline during playback of videotaped images. Rolling neutrophils were defined as white blood cells that moved at a velocity less than that of erythrocytes in a given vessel. The number of rolling neutrophils (flux) was counted using frame-by-frame analysis. To obtain a complete neutrophil rolling velocity profile, the rolling velocity of all neutrophils entering the vessel was measured. A neutrophil was defined as adherent to venular endothelium if it remained stationary for longer than 30 s. Adherent cells were measured at 10-min intervals as described in the experimental protocol and expressed as the number per 100-µm length of venule. Red blood cell velocity (V_RBC) was measured using an optical Doppler velocimeter (Microcirculation Research Institute, Texas A & M University), and mean red blood cell velocity (V_mean) was determined as V_RBC/1.6 (14). Wall shear rate was calculated based on the Newtonian definition: shear rate = (V_mean /D_v) × 8 (s¹), where D_v is the venular diameter.

Microvascular permeability. The degree of microvascular dysfunction was assessed as vascular albumin leakage in cat mesenteric venules. Briefly, 25 mg/kg fluorescein isothiocyanate (FITC)-labeled bovine albumin (Sigma Chemical, St. Louis, MO) were administered intravenously to animals 15 min before the start of the experimental procedure. Fluorescence intensity (excitation wavelength, 420 to 490 nm; emission wavelength, 520 nm) was detected using a silicon-intensified fluorescent camera (model C-2400-08, Hamamatsu Photonics, Hamamatsu, Japan), and images were recorded for playback analysis using a videocassette recorder. The fluorescent intensity of FITC-albumin within a defined area (10 µm × 50 µm) of the venule under study and in the adjacent perivascular interstitium (20 µm from venule) was measured under control conditions at 60 min of ischemia and at 10 and 60 min of reperfusion. This was accomplished using a video-capture board (Visionplus AT-OFG, Imaging Technology, Bedford, MA) and a computer-assisted digital imaging processor (Optimas, Bioscan, Edmonds, WA). The index of vascular albumin leakage (permeability index) was determined from the ratio: (interstitial intensity  background)/(venular intensity  background), as previously reported (8, 26).

Experimental protocol. After a 30-min stabilization period, baseline measurements of blood pressure, SMA blood flow, V_RBC, and vessel diameter were obtained. In the first group of animals (n = 8), the preparation was videotaped during a 10-min control period, and then the intestine was exposed to 60 min of ischemia (blood flow 20% of control) and 60 min of reperfusion. A second group of animals (n = 6) was preconditioned by occluding the SMA to mechanically reduce (Gaskell clamp) the blood flow to 20% of control for 5 min. This length of ischemic preconditioning was based on preliminary experiments (10 min caused vascular alterations) and was consistent with other reports (13). The clamp was then released to restore blood flow for 10 min. The blood flow was then reduced to 20% of control for a 1-h period. The last 10 min of ischemia were videotaped, and then the clamp was removed to restore intestinal blood flow for 1 h. The first 10 min and last 10 min of the 60-min reperfusion were also videotaped. In a third series of animals (n = 4), an identical protocol was completed; however, the mesentery was superfused with adenosine deaminase (0.25 IU/ml), an enzyme that converts extracellular adenosine to its inactive metabolite. Two additional series of experiments were performed using adenosine receptor antagonists. An A₁-receptor antagonist [8-cyclopentyl-1,3-dipropylxanthine (DPCPX); 6 mg/kg and 10 µM superfusion] and an A₂-receptor antagonist [3,7-dimethyl-1-proparbylxanthine (DMPX); 10 µM superfusion] were applied before the preconditioning protocol, and DMPX was also superfused throughout the experimental protocol. These concentrations and regimens were chosen based on previous reports that these protocols were efficacious in vivo (3, 29).

The next series of experiments were designed to examine the role of exogenous adenosine. A similar protocol was used in this third group of animals (n = 11), except no ischemic preconditioning was done. The mesentery was superfused with two different concentrations of adenosine (0.13 or 0.65 mg/l; Sigma Chemical) from 10 min before ischemia until the end of the experiment.

In vitro neutrophil rolling. Flow chamber experiments were performed in vitro to determine whether adenosine had any direct effect on neutrophil rolling, neutrophil rolling velocity, and neutrophil adhesion. Human umbilical vein endothelial cells (HUVEC) were harvested from freshly collected umbilical cords. Briefly, umbilical cord veins were rinsed of formed blood products with phosphate-buffered saline, after which the vein was filled with a collagenase solution (320 U/ml in phosphate-buffered saline). After a 20-min incubation period at 37°C, the cords were gently massaged to ensure the endothelial cells detached from the vessel wall. The digest was collected into centrifuge tubes and the collagenase inactivated with heat-inactivated fetal bovine serum (Hyclone Laboratories, Utah), after which the tube was centrifuged (400 g for 10 min at 37°C). The pellet was resuspended in medium 199 containing 20% fetal bovine serum and antibiotics but no endothelial mitogens. The cells were then seeded into fibronectin-coated T25 culture flasks (Becton Dickinson, NJ) and grown to confluence (2-5 days). When confluence was reached, HUVEC were detached from the flasks by trypsin-EDTA (GIBCO-BRL, Ontario) and seeded heavily onto fibronectin-coated glass coverslips (Fisher Scientific, Edmonton, Canada). First-passaged HUVEC were used for all in vitro endothelium experiments. The heavy seeding minimizes cell growth and thereby makes the cells most responsive to P-selectin inducers. Previous work from our laboratory has demonstrated that cultured endothelium or endothelium permitted to grow does not express P-selectin (19). Human cells were used for these experiments because harvests of cat primary cultures do not yield sufficient numbers of cells. In additional experiments, Chinese hamster ovary cells transfected with P-selectin cDNA (generously provided by Dr. R. P. McEver) were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum and grown to confluence on glass coverslips as described for HUVEC. These cells were used because they continuously express P-selectin independent of any stimulus and therefore permit direct effects of adenosine on neutrophil-P-selectin interactions.

Neutrophil isolation and treatment. Human neutrophils were harvested from acetate-citrate-dextrose anticoagulated venous blood collected from healthy donors. All isolation steps were performed at room temperature. Neutrophils were purified by dextran sedimentation (Dextran 250,000, Spectrum Chemicals) followed by centrifugation through a density gradient (6.07% Ficoll type 400, Sigma Chemical) with 10% Hypaque sodium (Winthrop-Breon, NY). Isolated neutrophils were resuspended in Hanks' buffered salt solution and used at a density of 1 × 10⁶ cells/ml.

Flow chamber assay. To study neutrophil behavior under shear conditions, a flow chamber assay was established as previously described (27). Briefly, coverslips with confluent monolayers of HUVEC or P-selectin transfectants were mounted into a polycarbonate chamber with parallel plate geometry. The flow chamber was placed onto an inverted microscope stage (Zeiss, Canada) and monolayers were visualized at ×100 magnification using phase-contrast imagery. The stage area was enclosed in a warm air cabinet and maintained at 37°C. Before use, all neutrophil cell suspensions were warmed to 37°C using a water bath. A syringe pump (Harvard Apparatus, Canada) was used to draw the cell suspensions through the flow chamber at defined wall shear stresses. For playback analysis, experiments were video recorded via a CCD camera (Hatachi Denshi, Japan) that was attached to the microscope. Freshly isolated neutrophils were perfused at a shear of 2 dyn/cm² over monolayers of HUVEC and P-selectin transfectants. After an initial control period of 6 min, thrombin (1 U/ml; Sigma) was coperfused with neutrophils over HUVEC. In some experiments neutrophils and HUVEC were pretreated with adenosine (2 or 100 µM) and then thrombin was added. Thrombin was chosen as an agonist because this mediator has been shown to be an important inducer of rolling in the feline model of I/R (31).

P-selectin transfectants were also used to study the direct effect of adenosine on P-selectin-dependent neutrophil rolling. Neutrophils treated with adenosine (2 or 100 µM) were perfused over the P-selectin-expressing Chinese hamster ovary cells.

Statistical analysis. Statistical analyses were performed by conventional methods; all time points within a group were subjected to Student's paired t-test with Bonferroni corrections for multiple comparisons where necessary.

	RESULTS

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Hemodynamic factors during I/R. Figure 1 summarizes hemodynamic factors in animals before and after I/R. Blood pressure remained stable throughout the experimental protocol (Fig. 1). Intestinal blood flow was reduced by ~30% during the reperfusion period, and shear rates decreased by 50% at 60 min of reperfusion. Hematocrit and white blood cell counts were within the normal range at 60 min of reperfusion (data not shown). Preconditioning did not affect any of the aforementioned variables under control, ischemia, or reperfusion except that blood flow was less at 10 min of reperfusion in the preconditioned group.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Systemic blood pressure, intestinal blood flow, and shear rate responses to ischemia-reperfusion (I/R) in untreated animals and animals that were preconditioned with a brief 5-min ischemia/10 min reperfusion episode. * P < 0.05 relative to respective control (Con) value.  P < 0.05 relative to respective untreated value.

Effects of preconditioning on postischemic vasculature. Figure 2 summarizes the data for flux of rolling neutrophils, neutrophil rolling velocity (V_WBC), and V_WBC normalized for red blood cell velocity (V_WBC/V_RBC). Ischemia followed by reperfusion induced a dramatic increase in the flux of rolling neutrophils (Fig. 2, top). Moreover, the first 10 min of reperfusion caused a very profound decrease in the velocity of neutrophil rolling (Fig. 2, middle). When neutrophil rolling velocity was normalized for V_WBC/V_RBC (Fig. 2, bottom) to establish the importance of hemodynamic forces at 10 min of reperfusion, the cells were rolling much slower than might be predicted from the small reduction in V_RBC, but at 60 min the ratio had returned to preischemic values. Preconditioning the mesenteric microvasculature caused a very large increase (more than double) in the V_WBC at 10 min reperfusion (from 14 to 35 µm/s) but failed to significantly reduce the flux of rolling neutrophils. V_WBC/V_RBC was also significantly elevated at 10 min of reperfusion above the values obtained in animals that had not been preconditioned, suggesting that the preconditioning directly prevents neutrophils from rolling slowly in reperfused microvessels (shear independent).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Neutrophil rolling flux, neutrophil rolling velocity (VWBC), and VWBC normalized for red blood cell velocity (VRBC) are presented under control conditions and following I/R in untreated animals and animals preconditioned with a brief 5-min ischemic episode followed by a 10-min reperfusion. * P < 0.05 relative to respective control value.  P < 0.05 relative to respective untreated value.

Figure 3 summarizes the data for adhesion as well as vascular permeability in untreated animals and animals that were exposed to ischemic preconditioning. Ischemia followed by reperfusion induced a significant increase in neutrophil adhesion and a large increase in FITC-albumin leakage in postcapillary venules of untreated animals. Figure 3, top, illustrates that the number of adherent neutrophils was decreased very significantly by 60 min of reperfusion in venules that were preconditioned. In fact, adhesion had returned almost to control levels. A similar pattern was observed in vascular permeability responses in preconditioned vessels (Fig. 3, bottom).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Neutrophil adhesion and microvascular permeability during control and at 60 min of reperfusion of postischemic vessels. An untreated group and a group that underwent a brief 5-min ischemia and 10-min reperfusion (preconditioning) before the prolonged (60 min) ischemic episode are presented. * P < 0.05 relative to respective control value.  P < 0.05 relative to respective untreated value.

Preconditioning and role of adenosine. Preconditioning increased V_WBC during I/R (14.0 ± 1.8 vs. 35.4 ± 8.0 µm/s). Pretreatment of animals with adenosine deaminase or the A₂-receptor antagonist did not reduce the speed with which neutrophils rolled in preconditioned vessels (Table 1). On the other hand, the A₁-receptor antagonist did indeed reverse the velocity with which neutrophils rolled. This was not the result of improved V_RBC and shear forces because the A₁-receptor antagonist also reversed the V_WBC/V_RBC preconditioning response (not shown). It is intriguing that adenosine deaminase did not reverse the preconditioning response, whereas an A₁-receptor antagonist did. This may be as a result of the adenosine deaminase not being as effective as the receptor antagonist or that blocking both the A₁ and A₂ response (by blocking adenosine via adenosine deaminase) masked the effects of just the A₁-receptor antagonist. Indeed, when the A₁- and A₂-receptor antagonists were coadministered, the A₁ response was lost (Table 1). Because neutrophil rolling flux was not affected by preconditioning, the data for the inhibitors of adenosine are not shown (they had no effect).

Table 2 illustrates that preconditioning decreased neutrophil adhesion from 21.4 ± 2.9 to 5.8 ± 2.1 cells/100 µm vessel length. Table 2 also demonstrates that neither the A₁-receptor antagonist, the A₂-receptor antagonist, nor a combination of these reagents reversed the antiadhesion response to preconditioning. The antiadhesive effect persisted under these conditions. We feel that sufficient amounts of antagonists were chosen based on 1) other studies (3, 29) and 2) the fact that this concentration of A₁-receptor antagonist at this concentration was effective in reversing the rolling velocity. Adenosine deaminase appeared to have a marginal effect on the antiadhesive property of preconditioning, but the results did not achieve significance. The microvascular permeability response to preconditioning followed a very similar pattern (Table 2). Whether the subtle differences between adenosine deaminase and the A₁ and A₂ dual receptor-antagonist blockade reflects effects of, for example, the A₃ receptor remains unknown. Clearly, vessels can be preconditioned to resist neutrophil adhesion and microvascular alterations, but endogenous adenosine has a limited role in this response. Additionally, neither preconditioning, adenosine deaminase, nor the adenosine receptor antagonists had a direct effect on baseline neutrophil-endothelial cell interactions (Table 2, Fig. 3, and data not shown), suggesting that unlike, for example, nitric oxide (5, 25), adenosine is not a significant endogenous regulator of neutrophil-endothelial cell interactions under normal conditions.

Exogenous adenosine affects postischemic vasculature. In the first of this series of experiments (n = 6), adenosine (0.4 µM) was superfused onto the mesentery. Because some but not all of the neutrophil parameters were affected by this concentration of adenosine, a fivefold higher concentration of the purine (2.0 µM) was also tested (n = 5). Similar findings were noted for the higher concentration and therefore only the higher concentration is shown. Addition of adenosine to the mesenteric microvasculature did not affect systemic blood pressure, V_RBC, or shear rates under control conditions (data not shown). These baseline parameters did not differ between the adenosine group and the untreated group (Fig. 4). Systemic blood pressure was not affected throughout the experiment by the addition of exogenous adenosine, but the drop in intestinal blood flow and shear rates during reperfusion was entirely reversed by the administration of adenosine.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Systemic blood pressure, intestinal blood flow, and shear rates during control, ischemia, and 10 and 60 min of reperfusion in untreated animals and in those animals that received adenosine. * P < 0.05 relative to respective control value.  P < 0.05 relative to respective untreated value.

Figure 5 demonstrated that there was no difference between the untreated and adenosine-treated groups for flux of rolling cells, V_WBC, or V_WBC/V_RBC under control conditions, again suggesting that adenosine does not affect leukocyte behavior under baseline conditions. The flux of rolling neutrophils was also not affected by adenosine at any time during the reperfusion period. However, within the first 10 min of reperfusion, the velocity of the slow rolling neutrophils was increased and almost tripled at 60 min in the presence of adenosine. V_WBC/V_RBC was however not affected at either time point. The latter is likely due to the fact that despite the increase in V_WBC, V_RBC also increased proportionately. This is illustrated in Fig. 4, bottom; the venular shear rates returned essentially to preischemic values with adenosine.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Neutrophil rolling flux, VWBC, and VWBC normalized for VRBC are presented under control conditions and following I/R in untreated animals and animals that received adenosine throughout the experimental period.

Figure 6 summarizes the data for adhesion as well as vascular permeability in untreated animals and animals that received adenosine. Figure 6, top, illustrates that the 50% reduction in neutrophil adhesion at 10 min had not reached significance (P = 0.098); however, by 60 min the number of adherent neutrophils had decreased in venules exposed to adenosine (P = 0.0009). The microvascular permeability responses in vessels exposed to adenosine were also significantly reduced (Fig. 6, top). Adherent neutrophils are responsible for the microvascular dysfunction during reperfusion (12, 32), raising the possibility that the reduction in microvascular permeability by adenosine was likely due to its antiadhesive effects. Indeed, addition of histamine (100 µM) to adenosine-treated animals still increased microvascular permeability (data not shown), an event known to be neutrophil independent.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Neutrophil adhesion and microvascular permeability during control and at 10 and 60 min of reperfusion of postischemic vessels. In some animals adenosine was administered throughout the experimental protocol. * P < 0.05 relative to respective control value.  P < 0.05 relative to respective untreated value.

Adenosine does not directly reduce neutrophil-endothelium interactions in vitro. Because I/R-induced neutrophil rolling is dependent on thrombin (31) and P-selectin (23), we examined the direct effect of adenosine on thrombin-induced P-selectin-dependent neutrophil recruitment under defined shear conditions (2 dyn/cm²) in vitro. In some experiments endothelium was treated with thrombin (1 U/ml), and neutrophil rolling, V_WBC, and adhesion were determined (Figs. 7-9). Pretreatment of the endothelium and neutrophils with adenosine at either a concentration equivalent to that used in vivo (2 µM) or a higher concentration (100 µM; not shown) did not reduce neutrophil rolling (Fig. 7), rolling velocity (Fig. 8), or adhesion (Fig. 9) in response to thrombin, suggesting that the effects of adenosine in vivo were not a result of direct effects on neutrophil rolling or on P-selectin expression per se. Pretreatment of neutrophils or endothelium alone with adenosine also did not impact on neutrophil-endothelium interactions (data not shown). Although we present data with thrombin, histamine-induced rolling was also not reduced by adenosine (data not shown). Moreover, adenosine did not disrupt neutrophil rolling on P-selectin constitutively expressed on transfected Chinese hamster ovary cells (data not shown), suggesting that adenosine has no direct effect on neutrophil-P-selectin interactions.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Adenosine (100 µM) pretreatment of human umbilical vein endothelium and neutrophils before addition of thrombin (1 U/ml). Neutrophils were coperfused with adenosine over human umbilical vein endothelial cell (HUVEC) monolayer. At t = 6 min thrombin (1 U/ml) was added to neutrophil suspension and coperfused over HUVEC, and neutrophil rolling flux was shown in untreated and adenosine-treated cells.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Neutrophil rolling velocity on HUVEC in presence and absence of adenosine. Details are in Fig. 7.

View larger version (39K): [in this window] [in a new window]   Fig. 9.   Neutrophil adhesion to HUVEC in presence and absence of adenosine. Details are in Fig. 7.

	DISCUSSION

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A recent study (1) has demonstrated that in addition to the fact that an organ can be preconditioned to resist subsequent reperfusion injury, the microvasculature can also be preconditioned to resist neutrophil adhesion. Our study confirms this observation but also extends this work to demonstrate for the first time that single vessels can be preconditioned to reduce subsequent endothelial barrier dysfunction in response to a prolonged ischemic episode. Moreover, we demonstrate that the V_WBC was dramatically increased with preconditioning, whereas the flux of cells was not affected. This latter point is worth noting because it provides insight into the complexity of both the preconditioning response and the reperfusion-induced dysfunction within the microvasculature; mediators such as PAF responsible for V_WBC, neutrophil adhesion, and microvascular dysfunction are susceptible to preconditioning, whereas thrombin- and selectin-dependent neutrophil rolling are resistant to the protective mediators released during preconditioning.

Although others have shown a clear role for adenosine as a protective mediator of the preconditioning response in the heart (13) and at the level of at least skeletal muscle microvasculature (1), our data at the level of single vessels are far less convincing in this regard. Although there may have been some reversal of the adhesion and microvascular dysfunction in response to preconditioning with adenosine deaminase (at a concentration shown to be efficacious), the beneficial effect was only partial. Akimitsu et al. (1) were able to abolish the preconditioning response (to neutrophil adhesion) with adenosine deaminase. Clearly, in the mouse skeletal muscle, adenosine played a far more important role as an antiadhesive agent than it did in the cat mesentery during preconditioning. These dichotomous results raise some issues about the potential importance of adenosine in preconditioning of the microvasculature of different tissues and different species, including perhaps humans. Although Akimitsu et al. (1) did not examine the importance of different adenosine receptors, in this study the adenosine-receptor antagonists were even less effective at reversing the benefit of the preconditioning response than adenosine deaminase. This may reflect complete inhibition of adenosine with adenosine deaminase versus inhibition of just the A₁- and A₂-receptor antagonists, but not A₃-receptor antagonists with the receptor inhibition strategy.

Interestingly, A₁-receptor blockade consistently and completely prevented the rise in V_WBC with preconditioning, suggesting that adenosine was being released into the preconditioned vessel and that it had a dramatic yet exclusive effect on this particular parameter. Moreover, an identical response was observed if instead of preconditioning, adenosine was superfused during I/R. Although most studies have focussed on the number of rolling leukocytes as an indicator of whether adhesion would ensue, the novel observation that adenosine had a dramatic effect on V_WBC in postischemic vessels may be very significant in regulating adhesion. This contention is based on the fact that recent work from our laboratory demonstrated that neutrophils rolling at a slow rate adhered in response to an order of magnitude lower concentration of proinflammatory agent (PAF) than did fast rolling neutrophils (16). However, as our in vitro work clearly demonstrates that adenosine could not affect V_WBC or rolling flux, it suggests a lack of direct effect of this purine on neutrophil-endothelial cell interactions. This is based on the fact that pretreatment of neutrophils with adenosine at 2 µM or even at pharmacological doses (100 µM) displayed absolutely no effect on neutrophil flux or rolling velocity on P-selectin-transfected Chinese hamster ovary cells (data not shown), wherein P-selectin is constitutively expressed. Moreover, no effect was seen on thrombin-treated endothelium where P-selectin expression through the thrombin receptor is a necessary event. Moreover, pretreatment of the neutrophils with adenosine also revealed no reduction in neutrophil rolling or V_WBC, suggesting that adenosine did not cause alterations of adhesion molecules on the neutrophil surface.

Unlike the less than equivocal results obtained for endogenous adenosine in the preconditioning response, the protective effect of exogenous adenosine as it related to neutrophil adhesion and the associated microvascular dysfunction were far more convincing, suggesting that adenosine at sufficient concentrations could indeed impact on the reperfusion response. The likely beneficial mechanism of action of exogenous adenosine on microvascular dysfunction has been reported to be in part dependent on its antiadhesive property for neutrophils, similar to that previously reported for monoclonal antibodies directed against CD18 (12, 20). One possible explanation may be the direct effect of adenosine on neutrophils; acadesine and adenosine have been reported to prevent the upregulation of CD11b on the surface of neutrophils (28), and investigators have observed approximately a 25-50% inhibition in neutrophil adhesion in vitro under static shear-independent conditions, suggesting a direct effect on neutrophil adhesion (4). In our in vitro assay, which is done under flow conditions to more closely mimic the in vivo condition, there was absolutely no effect of adenosine on neutrophil adhesion to endothelium (a CD18-dependent adhesion). These conditions mimic our in vivo model, wherein thrombin induced the leukocyte recruitment in I/R (31). Therefore, the lack of direct effect of adenosine on thrombin-induced adhesion in our study and the 25-50% reduction in neutrophil adhesion in static assays cannot explain the far more effective inhibition (90%) noted in postischemic vessels in vivo. Clearly, other potential mechanisms, including perhaps inhibitory effects of adenosine on other cells such as mast cells (previously evoked in I/R; see Ref. 17), need to be considered in the future.

Direct effects of adenosine on the flux of rolling cells have not been described to date, but in vitro work might predict a role for adenosine in reducing neutrophil rolling in vivo. For example, Firestein et al. (7) reported that adenosine decreased by 30% L-selectin-dependent binding in a static assay and predicted a greater effect under flow conditions. Because in our feline model of I/R, a component of the leukocyte rolling was L-selectin-dependent (23) yet no effect of adenosine was seen in the same model, the possibility that adenosine affects L-selectin-dependent rolling seems highly unlikely. Additionally, a similar yet even more convincing argument can be made for a lack of effect of adenosine on P-selectin-dependent rolling; adenosine had no effect on the flux of rolling cells in vivo, which is 50% or more P-selectin dependent, and absolutely no effect on neutrophil rolling on P-selectin constitutively expressed on transfected cells or on endothelium induced to express P-selectin.

In conclusion, we report that single postcapillary microvessels can be preconditioned to resist subsequent neutrophil and vascular responses to I/R. This includes a clear inhibitory effect on V_WBC as well as neutrophil adhesion and microvascular permeability alterations. Although exogenous adenosine was able to mimic the preconditioning effect, the preconditioning response per se did not appear to be dependent on endogenous adenosine. Finally, our observation that leukocyte adhesion in postischemic vessels can be attenuated with adenosine is consistent with the original observation of Grisham et al. (11) in the gut and subsequent work in skeletal muscle (30); however, our results question whether the adenosine-induced decrease in adhesion is due to direct effects of this purine on neutrophils and/or endothelium.