Inhaled NO reaches distal vasculatures to inhibit endothelium- but not leukocyte-dependent cell adhesion

Alison Fox-Robichaud, Derrice Payne, and Paul Kubes

Immunology Research Group, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada T2N 4N1


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Nitric oxide (NO), in addition to being a potent vasodilator, also prevents leukocyte adhesion in the microvasculature. Based on the antiadhesive properties of NO and work suggesting that NO is transported by proteins in the circulation, we tested the possibility that inhaled NO could impart antiadhesive effects in peripheral microvessels. We also determined the underlying mechanisms of actions. Three well-established models that induce local microvascular changes (either endothelium or leukocyte) were used. Hydrogen peroxide (H2O2; 100 µM) was superfused onto the cat mesentery to induce an endothelium-derived, P-selectin- and platelet-activating factor-dependent, oxidant-dependent leukocyte recruitment. In a second series of experiments, the cat mesentery was superfused with histamine (100 µM) to induce rapid endothelium-derived, P-selectin- and platelet-activating factor-dependent, oxidant-independent leukocyte recruitment. Finally, in a third series of experiments to target the leukocyte (but not the endothelium) directly in the periphery, the chemotactic molecule leukotriene B4 (20 nM) was superfused onto the cat mesentery. The above experiments were performed with and without cats breathing NO (80 parts/million). Intravital microscopy was used to visualize the mesenteric microcirculation. Inhaled NO reduced the increased leukocyte rolling and adhesion associated with H2O2 superfusion of the feline mesentery via a cGMP-dependent mechanism. In contrast, inhaled NO had no effect on the histamine-induced increase in leukocyte rolling flux but partially inhibited the subsequent adhesion. The leukocyte chemotactic mediator leukotriene B4 induced a significant increase in leukocyte adhesion, but NO inhalation did not impair this chemotactically induced leukocyte recruitment. These data suggest that inhaled NO can reach the endothelium in the distal microvasculature and alter the response to an oxidative and a nonoxidative activator of endothelium but imparts no antiadhesive effect directly on circulating leukocytes.

nitric oxide; P-selectin; platelet-activating factor; inflammation; oxidative stress; histamine; chemoattractants; leukotriene B4


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN A SERIES OF SEMINAL EXPERIMENTS, Stamler et al. (33) proposed that nitric oxide (NO) undergoes S-nitrosation with protein-bound thiol groups, forming stable S-nitrosoproteins, including S-nitrosoalbumin, that could conceivably function as a NO delivery system. Indeed, Keaney et al. (22) have reported that S-nitrosoalbumin possesses endothelium-derived relaxing factor-like properties in vitro, including vasodilatation and inhibition of platelet aggregation. Although the responses were sevenfold less potent, they lasted almost 10 min versus 19 s for nitroprusside (22). Hemoglobin was also identified as a potential carrier of NO in the form of S-nitrosohemoglobin (20, 32) and also possessed vasodilatory properties. It was demonstrated that hemoglobin can be S-nitrosated in the lung and that the NO group could dissociate in the capillaries (15). If this were the case, then continuous delivery of NO to the blood in the form of NO inhalation could conceivably produce a variety of NO adducts that could impact on the peripheral microvasculature. To support this theory, Fox-Robichaud et al. (9) have recently shown that inhaled NO at 80 parts/million (ppm) was able to prevent vasoconstriction of arterioles by local application of NO inhibitors.

Based on the aforementioned work and the fact that NO has been postulated to inhibit leukocyte recruitment in a variety of inflammatory conditions, we examined whether inhaled NO could impact on leukocyte adhesion in the periphery. Inhaled NO at 80 ppm but not at 20 ppm abolished the increased rolling, adhesion, and emigration induced by local inhibition of NO in the cat mesenteric microvasculature. Clinically relevant was the observation that inhaled NO was also able to partially inhibit leukocyte rolling, adhesion, emigration, and microvascular dysfunction in ischemia-reperfusion injury in the cat mesentery (9). This distal action of NO was seen in NO-depleted states such as ischemia-reperfusion and superfusion with N-nitro-L-arginine methyl ester but not in a NO-abundant state such as endotoxin superfusion. An important view emerged from this work: the effects of inhaled NO could no longer be purported to be limited to the pulmonary circulation. Clearly, the biological activity of NO could extend to the peripheral microvasculature either by reaching the periphery and inhibiting the activation of endothelium or by directly affecting leukocytes as they passed through the lungs and inhibiting downstream activation of these cells by postischemic endothelium.

Therefore, the objectives of this study were 1) to further establish whether the actions of inhaled NO on leukocyte recruitment could occur at a localized site distal from the lung in an in vivo model, 2) to determine whether the effects were restricted to oxidant-related leukocyte recruitment, and 3) to elucidate whether the effects impacted directly on leukocytes or endothelium. To achieve these objectives, we used three separate, well-characterized models of leukocyte recruitment in vivo. We first used an oxidant-dependent [hydrogen peroxide (H2O2)-induced] model of leukocyte recruitment that stimulated P-selectin (responsible for rolling) (21, 25) and platelet-activating factor (PAF) synthesis (responsible for adhesion) in endothelium without a direct effect on leukocytes (27a). We used a second model (histamine) that also induces P-selectin-dependent rolling (1) and PAF-dependent adhesion (12, 24) by activating the endothelium but in this case is entirely independent of oxidants. The third model involved direct activation of leukocytes by leukotriene (LT) B4 as the leukocytes trafficked through the mesenteric microvasculature (36). Because leukocyte recruitment is a multistep cascade involving rolling on selectins, which is absolutely required for firm adhesion via integrins, intravital microscopy was used to visualize leukocyte recruitment in the presence of inhaled NO.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Surgery for intravital microscopy. The experimental preparation used in this study is the same as that previously described (23). Animal protocols were approved by the University of Calgary (Calgary, AB) Animal Care Committee and met the Canadian Guidelines for Animal Research. Briefly, cats (1.2-2.4 kg) were fasted for 24 h and initially anesthetized with ketamine hydrochloride (75 mg intramuscularly). The jugular vein was cannulated, and anesthesia was maintained by the administration of pentobarbital sodium. A tracheostomy was performed to support breathing by artificial ventilation. NO at 0 or 80 ppm was delivered from a certified hospital grade NO-balance N2 gas cylinder to the inhalation line of a Harvard ventilator via a high-accuracy Matheson flowmeter (Matheson Gas Products Canada, Edmonton, AB), and NO and NO2 were measured with a Pulmonox IIRC NO/NO2 electrochemical analyzer (Pulmonox Research and Development, Tofield, AB). Throughout the experiments, NO2 was <5 ppm and was not different among groups. This NO delivery setup was identical to the one used to deliver NO to newborn infants with respiratory distress in the neonatal intensive care unit of the Foothills Medical Centre (University of Calgary) except in our system, the cats were not provided with supplemental oxygen but rather were ventilated with room air. A pressure transducer (Statham P23A, Gould, Oxnard, CA) monitored systemic arterial pressure through 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 intestines was extirpated. Body temperature was maintained at 37°C with 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 the left femoral artery. SMA blood flow was continuously monitored with an electromagnetic flowmeter (Carolina Medical Electronics, King, NC). Blood pressure was continuously recorded with a physiological recorder (Grass Instruments, Quincy, MA).

The cats were placed in a supine position on an adjustable Plexiglas microscope stage, and a segment of the midjejunum was exteriorized through the abdominal incision. The mesentery was prepared for in vivo microscopic observation as previously described (23). 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, Pittsburgh, PA). The exposed bowel was draped with saline-soaked gauze while the remainder of the mesentery was covered with Saran Wrap (Dow Corning, Midland, MI). The exposed mesentery was suffused with warmed bicarbonate-buffered saline (pH 7.4) that was bubbled with a mixture of 5% CO2 and 95% N2. The mesenteric preparation was observed through an intravital microscope (Optiphot-2, Nikon, Mississauga, ON) with a ×25 objective lens (Wetzlar L25/0.35, Leitz, Munich, Germany) and a ×10 eyepiece. The image of the microcirculatory bed (×1,400 magnification) was recorded with a video camera (Digital 5100, Panasonic, Osaka, Japan) and a videorecorder (NV8950, Panasonic).

Single unbranched mesenteric venules (25-40 µm in diameter, 250 µm in length) were selected for each study. Venular diameter was measured on- or off-line with a video caliper (Microcirculation Research Institute, Texas A & M University, College Station, TX). The number of rolling and adherent leukocytes was determined off-line during playback analysis. Rolling leukocytes were defined as white blood cells that moved at a velocity less than that of erythrocytes in a given vessel. The number of rolling leukocytes (flux) was counted by frame-by-frame analysis. To obtain a complete leukocyte rolling velocity profile, the rolling velocity of all leukocytes entering the vessel was measured. A leukocyte was defined as adherent to venular endothelium if it remained stationary for >30 s. Adherent cells are expressed as the number of cells per 100-µm length of venule. Red blood cell velocity (VRBC) was measured with an optical Doppler velocimeter (Microcirculation Research Institute), and mean VRBC (Vmean) was determined as VRBC/1.6 (18). Wall shear rate was calculated based on the Newtonian definition: shear rate = (Vmean/Dv) × (8 s-1), where Dv 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 of fluorescein isothiocyanate (FITC)-labeled bovine albumin (Sigma, St. Louis, MO) were administered intravenously to animals 15 min before the start of the experimental procedure. Fluorescence intensity (excitation wavelength, 420-490 nm; emission wavelength, 520 nm) was detected with a silicon-intensified fluorescent camera (model C-2400-08, Hamamatsu Photonics, Hamamatsu, Japan), and images were recorded for playback analysis on a videocassette recorder. The fluorescence intensity of FITC-albumin within a defined area (10 × 50 µm) of the venule under study and in the adjacent perivascular interstitium (20 µm from venule) was measured during the control period and at various times after application of mediator. This was accomplished with 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 of (interstitial intensity - background) to (venular intensity - background) as previously reported (13, 26).

Experimental protocol. The animals were ventilated with room air with either 0 or 80 ppm NO. Baseline measurements of blood pressure, SMA blood flow, VRBC, and vessel diameter were obtained. Leukocyte parameters including leukocyte rolling velocity, flux of rolling leukocytes, leukocyte adhesion, and leukocyte emigration were measured. FITC-albumin leakage from the venule under study was also determined with the computer-assisted imaging program. In six separate series of experiments, the mesenteric microvasculature was locally superfused with hydrogen peroxide (H2O2; 100 µM), histamine (100 µM), or LTB4 (20 nM) in the presence and absence of inhaled NO.

Finally, to further elucidate the remote effects of inhaled NO, we repeated the H2O2 experiments in presence of the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). As before, NO inhalation began with a tracheostomy and ventilation. After a control period, the mesentery was superfused for 30 min with 10 µM ODQ (8), and then H2O2 (100 µM) was superfused for 2 h.

Statistics. The data were analyzed with standard statistical analysis, i.e., ANOVA and Student's t-test, with Bonferroni correction for multiple comparisons where appropriate. All values are means ± SE. Significance was set at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

H2O2 superfusion resulted in a gradual sixfold increase in the number of rolling cells, which was maximal by 60 min (Fig. 1A). By 120 min, the rolling flux had begun to decline with H2O2 superfusion. When the cats were ventilated with NO, there was no rise in the number of rolling cells (Fig. 1A) at any of the times examined and was significantly reduced from the H2O2 group at 60 min. H2O2-induced adhesion increased more than fourfold over the 120-min value (Fig. 1B). With NO inhalation, leukocyte adhesion was dramatically reduced over the 2 h of H2O2 superfusion. There is some good evidence that NO was not simply inactivating H2O2 because all responses to H2O2 should be inhibited in the inhaled NO group. Figure 1C demonstrates that direct application of H2O2 to the mesentery caused microvascular dysfunction (FITC-albumin leakage) that was not affected by inhaled NO.


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Fig. 1.   Effect of inhaled nitric oxide (NO) on hydrogen peroxide (H2O2)-induced flux of rolling leukocytes (A), leukocyte adhesion (B), and vascular leakage (C) before (con) and at 60 and 120 min of H2O2 superfusion in animals ventilated with room air alone (n = 5; ) or room air plus 80 ppm inhaled NO (n = 5; ). Significant difference (P < 0.05) compared with: * control; dagger  H2O2 alone.

Figure 2 shows representative captured images demonstrating the antiadhesive ability of inhaled NO. Figure 2A shows leukocyte rolling and adhesion in the mesenteric venule after 60 min of H2O2 superfusion. In contrast, in the animal ventilated with 80 ppm NO, there are no leukocytes visible within the venule 60 min into H2O2 superfusion (Fig. 2B). Finally, Table 1 demonstrates that shear rates within the microvasculature declined to ~50% of control levels with H2O2, whereas with inhaled NO, the value only dropped ~25% (P < 0.05). Total intestinal blood flow dropped ~20-25% in both groups of animals. Because the mesenteric tissue (not the entire intestine) was exposed to H2O2 and this tissue comprises only a small portion of total intestinal blood flow, it is expected that total intestinal blood flow would not differ between the two groups.


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Fig. 2.   Captured images of mesenteric vessels during H2O2 superfusion. A: mesenteric venule (V) after 60 min of H2O2 superfusion showing several rolling (arrowhead) and adherent (arrow) leukocytes within the venule. B: mesenteric arteriole (A) and venule from an animal ventilated with 80 ppm NO after 60 min of H2O2 superfusion. There are no leukocytes visible in venule.


                              
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Table 1.   Hemodynamic effects of H2O2 in presence and absence of inhaled NO

To confirm that inhaled NO was indeed acting distally through a cGMP-dependent mechanism, we repeated the H2O2 experiments in the presence of the locally applied guanylyl cyclase inhibitor ODQ (8). As shown in Fig. 3, superfusion with ODQ completely reversed the distal action of inhaled NO. Oxidant-induced leukocyte rolling flux in the presence of ODQ and inhaled NO gradually increased over the first 60 min (Fig. 3A), similar to the response seen with H2O2 alone. As shown in Fig. 3B, ODQ treatment resulted in a marked increase in H2O2-induced adhesion. At 60 min, adhesion had increased 10-fold with guanylyl cyclase inhibition. However, as before, ODQ had no effect on vascular leakage, confirming that the oxidant-induced microvasculature permeability changes are not altered by inhaled NO (Fig. 3C).


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Fig. 3.   Effect of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and inhaled NO on H2O2-induced flux of rolling leukocytes (A), leukocyte adhesion (B), and vascular leakage (C) before and at 60 and 120 min of H2O2 superfusion in animals ventilated with 80 ppm NO in presence (n = 4; ) and absence (n = 5; ) of 10 µM ODQ. Significant difference (P < 0.05) compared with: * control; dagger  H2O2 alone.

Figure 4 summarizes the data for histamine-induced leukocyte rolling, adhesion, and vascular permeability. The flux of rolling leukocytes increased approximately five- to sevenfold above baseline over the 2 h of superfusion (Fig. 4A). In the group of cats breathing NO, an identical increase in leukocyte rolling flux was noted in response to histamine, suggesting differences in the ability of inhaled NO to impact on oxidant- and nonoxidant-dependent P-selectin-associated leukocyte rolling. With histamine superfusion, leukocyte adhesion rose from a mean of <1 cell to 8-12 cells over the subsequent 2 h (Fig. 4B). Interestingly, the histamine-induced adhesion was inhibited by 50% with inhaled NO. A very dramatic increase in vascular leakage was induced by histamine superfusion (~75%) within the first 30 min and persisted throughout the experiment (Fig. 4C). Inhaled NO at 80 ppm did not prevent this rapid and sustained leakage. Histamine superfusion of the feline mesentery had no effect on intestinal blood flow and shear rate over the 2-h period in either group of animals (Table 2).


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Fig. 4.   Effect of inhaled NO on histamine-induced flux of rolling leukocytes (A), leukocyte adhesion (B), and vascular leakage (C) before and at 60 and 120 min of histamine superfusion in animals ventilated with room air alone (n = 6; ) or room air plus 80 ppm inhaled NO (n = 5; ). Significant difference (P < 0.05) compared with: * control; dagger  H2O2 alone.


                              
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Table 2.   Hemodynamic effects of histamine in presence and absence of inhaled NO

The above experiments focused on the ability of inhaled NO to reduce endothelium-dependent leukocyte recruitment. LTB4, which activates leukocytes directly to adhere (36), did not significantly increase the leukocyte rolling flux above the control level (Table 3), consistent with the view that this is a chemoattractant with minimal effects on endothelium. LTB4 induced leukocyte adhesion to rise from 1 to 12 cells/100 µm within 60 min. LTB4-induced leukocyte adhesion was not reduced in the presence of inhaled NO. Although LTB4 superfusion was terminated after 1 h, adhesion persisted for an additional 1 h, and again, inhaled NO had no effect on this parameter. LTB4 (like histamine or H2O2) also induced microvascular dysfunction, which was not altered by NO inhalation (Table 3). The hemodynamic effects of LTB4 on intestinal blood flow and venular shear rate are shown in Table 4. Superfusion of LTB4 alone or in the presence of inhaled NO had no effect on either intestinal blood flow or shear rate (Table 4). To confirm these results, a second chemotactic agent, N-formyl-methionyl-leucyl-phenylalanine, was superfused onto the mesentery, and although the responses to the bacterial peptide were quite variable, leukocyte adhesion occurred in the presence and absence of NO inhalation (data not shown).

                              
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Table 3.   Effect of inhaled NO on LTB4-induced rolling, adhesion, and vascular permeability


                              
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Table 4.   Hemodynamic effects of LTB4 in presence and absence of inhaled NO


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Therapeutically, inhalation of NO has been used to target the pulmonary vasculature and, specifically, to reduce pulmonary hypertension. More recently, the work has been extended to suggest that inhaled NO may affect neutrophil recruitment in the lung. Inhaled NO has been shown to prevent hemodialysis-induced neutrophil accumulation in the lungs (28), LPS-induced neutrophil accumulation in the lungs (4), ischemia-reperfusion-induced neutrophil infiltration and capillary leak in the lungs (3), IL-1-induced neutrophil infiltration in the pulmonary vasculature (16), and neutrophil infiltration in lung allografts (10). However, because the prevailing view has been that inhaled NO is rapidly inactivated in the lung capillaries by reaction with oxyhemoglobin (6), little attention has been given to the potential effects in peripheral microvessels. In 1998, Fox-Robichaud et al. (9) reported that inhaled NO could indeed reverse the vasoconstriction and leukocyte recruitment in the mesentery associated with NO synthesis inhibition, suggesting that a mechanism must be in place to transport NO to the periphery and/or impart effects in the lung that impact on events in peripheral microvessels. It was also demonstrated that inhaled NO could reduce ischemia-reperfusion-induced leukocyte recruitment in the mesentery, although the mechanism remained entirely unclear. In the present study, we used three extremely well-defined models of leukocyte recruitment. First, we demonstrated that inhalation of NO could reduce leukocyte recruitment associated with a localized oxidative stress (H2O2) within a peripheral microvasculature. Unexpectedly, some of the biological effects of inhaled NO extended beyond oxidative stress on the endothelium: inhaled NO decreased leukocyte adhesion to histamine-stimulated endothelium. Finally, the data for the first time reveal that direct stimulation of leukocytes with LTB4 or N-formyl-methionyl-leucyl-phenylalanine (data not shown) in no way impaired adhesion in peripheral microvessels in the presence of NO inhalation.

It is known that the oxidative stress imparted by H2O2 can be reduced or countered by NO both in the form of exogenous NO donors and from endogenous sources (21). Two critical observations were made in the present study. First, H2O2 exposure of a tissue localized at a significant distance from the lungs can be targeted with inhaled NO. Second, this distal action of inhaled NO can be blocked by the local inhibition of guanylyl cyclase. Additionally, inhaled NO appeared to inhibit some but not all of the biological effects of H2O2. For example, leukocyte rolling and adhesion were essentially abolished by inhaled NO, but the endothelial dysfunction measured as an increase in FITC-albumin leakage occurred regardless of the presence of inhaled NO. The mechanism by which NO may inhibit H2O2 remains unclear. NO most likely functions in a selective downstream manner either by inhibiting or terminating lipid peroxidation after it has been initiated (30) or by inhibiting H2O2-dependent signaling of P-selectin expression (responsible for leukocyte rolling) and/or PAF synthesis (responsible for firm adhesion), perhaps by increasing levels of cGMP.

Histamine-induced leukocyte rolling is also P-selectin dependent as a result of activation of histamine H1 receptors on the endothelium (24), yet it is intriguing that inhaled NO only inhibited H2O2-induced, but not histamine-induced, P-selectin-dependent rolling. It has been postulated that H2O2 and histamine mobilize P-selectin via very different intracellular pathways, although few details are available regarding these mechanisms of action (30a). It is interesting, however, that H2O2 decreases endogenous NO production from endothelial cells (19), whereas histamine causes the release of NO from the endothelium (31). Because NO synthesis inhibition is known to mobilize P-selectin to the endothelial cell surface (1a), it is possible that inhaled NO simply replaces NO in the H2O2 model and prevents P-selectin mobilization. With histamine, P-selectin is expressed independent of NO levels so that additional sources of NO may not affect P-selectin expression (13). It is noteworthy that inhaled NO can reduce leukocyte recruitment in ischemia-reperfusion (9), which is dependent on oxidants (27) but not on histamine (Kubes, unpublished observations).

The leukocyte adhesion that occurs in both the histamine- and H2O2-dependent leukocyte recruitment model has been demonstrated to occur via PAF. This phospholipid is rapidly synthesized at the endothelial surface so that it can activate rolling leukocytes to firmly adhere. PAF-receptor antagonists do not affect leukocyte rolling (12) but dramatically inhibit the increase in leukocyte adhesion associated with histamine or H2O2 (12, 30a). Inhaled NO consistently inhibited both histamine- and H2O2-induced leukocyte adhesion, suggesting that NO may impact on the ability of the endothelium to synthesize PAF. Indeed, Heller et al. (17) demonstrated that nitrovasodilators were able to inhibit thrombin-induced PAF synthesis by endothelium in vitro. The mechanism of action appeared to include a reduction in the enzymatic activity of both phospholipase A2 and acetyltransferase, two enzymes critical for the synthesis of PAF. In this study, we propose for the first time in vivo that this mechanism may also be operative for histamine- and H2O2-induced PAF synthesis. As a final point, however, sufficient amounts of inhaled NO did reach the mesentery to inhibit histamine-induced leukocyte adhesion but not microvascular permeability. Because the latter can be inhibited if sufficiently high levels of NO are applied directly to the mesentery (13), either inhaled NO is simply not able to deliver sufficient amounts of NO or NO donors function differently from NO adducts formed by NO inhalation.

Our data also demonstrate that in an animal breathing NO, the leukocytes that pass through the NO-enriched lung maintain their ability to adhere avidly even when the leukocytes are stimulated directly with chemotactic agents such as LTB4. The concentration of NO used in this study has been reported to be sufficient to prevent platelet aggregation (29), suggesting that this level of inhaled NO can have effects on circulating platelets but not on circulating leukocytes. The lack of a direct effect of NO on leukocytes is consistent with data by Fox-Robichaud et al. (9) that NO in vitro is unable to inhibit leukocyte adhesion but is not consistent with the work of Banick et al. (2), who reported that NO could inhibit neutrophil CD18-dependent adhesion in vitro but in a very low narrow range between ~10 and 50 nmol NO generated/min. Higher or lower concentrations of NO had little effect, and if our levels of inhaled NO happened to fall outside this narrow range, it may explain the lack of effect in our study. It is intriguing that there is a report that 4 days of inhaled NO in patients with adult respiratory distress syndrome reduced CD18 upregulation on the surface of neutrophils (5). CD18 is the molecule primarily responsible for firm adhesion. However, in that study, the cytokine levels were also reduced in patients breathing NO relative to those in untreated patients, perhaps explaining the lower CD18 levels. To avoid the many complications of interpretation associated with isolating leukocytes and exposing them to various artificial substratum in vitro, we for the first time directly examined leukocyte adhesivity in the microcirculation of animals while they were in the process of inhaling NO and observed absolutely no impairment in the ability of these leukocytes to respond to chemotactic stimuli. These data strongly support the view that there appears not to be a direct effect of inhaled NO of leukocyte function in vivo.

A final point that should be discussed is the concentration of inhaled NO (80 ppm) in this study versus the many adult clinical studies that generally use <40 ppm inhaled NO. We have no evidence that 80 ppm inhaled NO is toxic; NO2 levels were continuously read on-line and never exceeded the permissible 5 ppm. In fact, the levels never rose under these conditions. Additionally, 80 ppm NO has been used in neonate studies without any detrimental effects. For example, Wessel et al. (34) used 80 ppm NO on 99 adult and pediatric patients for up to 10 h without any detrimental effects. When we ventilated the animals with 300 ppm inhaled NO, then NO2 levels (and methemoglobin levels) did rise and cardiovascular complications were noted (unpublished observations). We did not use lower concentrations because the previous study by Fox-Robichaud et al. (9) revealed that 80 ppm was optimal in our model of ischemia-reperfusion and lower concentrations were not sufficient to impact on the distal microvasculature. It should be noted that all previous studies have focused on the effects of inhaled NO at the level of the lung, and so 80 ppm inhaled NO was not necessary. We are proposing that 80 ppm NO will need to be used to impact on the peripheral vasculature, and this may be a very useful means for targeting increased oxidative stress in, for example, stroke, myocardial infarct, and other pathological conditions wherein oxidants and/or leukocytes could conceivably play an inappropriate role.


    ACKNOWLEDGEMENTS

This study was supported by a grant from the Medical Research Council of Canada.


    FOOTNOTES

A. Fox-Robichaud is a Medical Research Council/Canadian Association of Gastroenterology/Schering Canada Fellow. P. Kubes is a Medical Research Council Scientist and Senior Scholar of the Alberta Heritage Foundation for Medical Research.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. Kubes, Immunology Research Group, Univ. of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta, Canada T2N 4N1 (E-mail: pkubes{at}ucalgary.ca).

Received 5 February 1999; accepted in final form 6 August 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 277(6):L1224-L1231
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