© 1996 American Heart Association, Inc.
Articles |
A Balance Between Nitric Oxide and Oxidants Regulates Mast Cell–Dependent Neutrophil–Endothelial Cell Interactions
the Immunology Research Group, University of Calgary Medical Center, Calgary, Alberta, Canada.
Correspondence to Dr Paul Kubes, Immunology Research Group, Department of Medical Physiology, Faculty of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1 Canada.
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Abstract |
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Nitric oxide (NO) synthesis inhibition causes neutrophil adhesion to endothelium via a mast cell– and oxidant-dependent mechanism. The objective of this study was to delineate the cascade of events in the mast cell– and oxidant-induced neutrophil-endothelium interactions after NO synthesis inhibition. Mast cells were isolated and purified from the rat peritoneal cavity and coadministered with neutrophils to wells of endothelium. This system was treated with an NO synthesis inhibitor (NG-nitro-L-arginine methyl ester; L-NAME) for 60 minutes. L-NAME did not induce neutrophil-endothelium interactions in the absence of mast cells, but the addition of mast cells in a ratio as low as 1:50 mast cells to neutrophils was sufficient to induce a large increase in neutrophil adhesion to endothelium within 20 to 25 minutes. L-arginine, NO donors, and 8-bromo-cGMP reversed the L-NAME effect, whereas NG-nitro-D-arginine methyl ester alone had no proadhesive effect. The adhesion was inhibited by an anti-CD18 or an anti–intracellular adhesion molecule-1 antibody and a platelet-activating factor-receptor antagonist. Inhibition of NO in isolated endothelial monolayers induced oxidant release (reduction of cytochrome C) into extracellular fluid. The endothelium-derived superoxide contributed to the mast cell–induced adhesion, inasmuch as the extracellular antioxidant superoxide dismutase reduced the neutrophil adhesion response as did disruption of endothelial function. There was some direct activation of mast cells with L-NAME (independent of endothelium) inasmuch as intracellular calcium and oxidative stress increased within mast cells after L-NAME treatment, and this translated into increased neutrophil adhesion to nonendothelial substrata. These data demonstrate that depletion of NO increases oxidative stress within mast cells and endothelium and together these events promote neutrophil adhesion within the vasculature.
Key Words: endothelium • L-NAME • superoxide • integrins • platelet-activating factor
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Introduction |
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Inappropriate activation of leukocytes has been postulated to mediate the vascular dysfunction in various animal models of inflammation, including ischemia/reperfusion, hemorrhagic shock, intimal restenosis, and atherogenesis, and may underlie the pathogenesis of many human disease conditions.1 2 3 4 5 6 It is likely that the body produces various proinflammatory and anti-inflammatory mediators that constitute a very fine balance to allow for a controlled, suitable inflammatory response without the development of inappropriate inflammation. Expectedly, much emphasis over the last decade has been given to the identification and characterization of substances that promote neutrophil adhesion, but the significance of endogenous anti–adhesive molecules in normal and inflamed tissues remains unappreciated. There is a growing body of evidence that NO from both endogenous and exogenous sources limits leukocyte recruitment into normal and inflamed vessels. For example, inhibition of endogenous NO with NO inhibitors such as L-NAME promotes leukocyte adhesion in various vascular beds and species.7 8 9 10 11 12 13 These studies led investigators to conclude that endogenous NO is an important homeostatic regulator of leukocyte adhesion in postcapillary venules.
However, exposing venular endothelium and neutrophils for 60 minutes to NO inhibitors in vitro did not induce neutrophil–endothelial cell interactions.14 This observation raised the possibility that some cell type was missing in the simple in vitro system. The mast cells may be a good candidate in this regard. These cells are closely apposed to the vasculature and upon activation induce neutrophil-endothelium interactions.15 NO synthesis inhibition causes mast cell degranulation in vivo, and this event could conceivably induce leukocyte recruitment. Indeed, stabilization of mast cells prevented the mast cell degranulation and subsequent neutrophil adhesion generally observed with L-NAME.8 Moreover, inhibition of NO synthesis increased oxidative stress,13 16 raising the possibility that L-NAME perhaps via oxidants could activate mast cells.
However, many issues remain unresolved with respect to the view that oxidants activate mast cells to recruit neutrophils after NO synthesis inhibition. First, the evidence of a role for mast cells is entirely dependent upon the use of mast cell–stabilizing drugs. The specificity of these compounds has been challenged. Second, the sequence of activating events involving mast cells and oxidants that leads to leukocyte recruitment in an NO-deficient microenvironment is presently unknown. Third, the source of the oxidants after NO synthesis inhibition remains unknown. Fourth, the factor or factors released from mast cells that cause neutrophil adhesion to endothelium have not been identified. Clearly, resolving these issues may provide insight into the pathogenesis of disease states in which NO is suppressed and significant circulatory pathologies, including leukocyte–endothelial cell interactions, are noted. These may include hypercholesterolemia,17 ischemia/reperfusion,10 intimal restenosis,18 19 and atherogenesis.3
Therefore, the first objective of this study was to determine whether mast cells were critical to neutrophil–endothelial cell interactions in an environment depleted of NO and if so to elucidate the mediators involved. The second objective was to determine whether oxidative stress either in endothelial cells or mast cells was contributing to the increased neutrophil adhesion. Since oxidants were implicated in the neutrophil–endothelial cell interaction, the last objective was to determine whether L-NAME was affecting mast cells directly or in a more circuitous manner by first inducing endothelium to produce oxidants that would subsequently activate the mast cells to induce neutrophil–endothelial cell interactions.
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Materials
MAb R6.5 (anti–ICAM-1) was kindly provided by Dr C.W. Smith. MAb IB4 (anti-CD18) was a kind gift of Dr K.E. Arfors (Experimental Medicine Inc, Princeton, NJ). The PAF-receptor antagonist (WEB 2086) was generously provided by Boehringer Ingelheim (Burlington, Ontario, Canada), and SIN-1 was donated by Dr Henning (Cassella AG, Frankfurt). Spermine-NO was from Cayman Chemical Company. L-NAME and D-NAME were purchased from Bachem. FCS, HBSS, and M199 were obtained from GIBCO Laboratories, and all other reagents were purchased from Sigma Chemical Co.
Endothelial Cell Monolayers
HUVECs were grown to confluence in 48-well plates, and neutrophil adhesion to this biological substratum was tested as previously described.8 14 20 Briefly, umbilical cord veins were rinsed of formed blood elements with PBS-containing antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin, and 1 µg/mL amphotericin B). Collagenase (2.5 mg/mL; 149 U/mg) was instilled into the vein and the cord incubated for 20 minutes at 37°C. The cords were gently massaged to ensure detachment of endothelial cells from the vessel wall. The digest was collected into centrifuge tubes, the collagenase inactivated with FCS, and centrifuged (400g for 10 minutes at 25°C). The pellet was resuspended in M199 containing 10% FCS and antibiotics plated in 25-cm2 flasks. Cultures were incubated in 5% CO2 at 37°C and 96% humidity, expanded by trypsinization, and grown to confluence in 48-well plates. Confluent monolayers of endothelium limited to the first three passages were used for these experiments. The identity of some cultures was checked by indirect staining with FITC-labeled factor VIII antibody21 and by the uptake of acetylated low-density lipoprotein22 according to established techniques.
Mast Cell Purification
Mast cells were isolated according to a method described previously.15 23 24 Briefly, Sprague-Dawley rats were anesthetized with ether, killed by cervical dislocation, and bled. After lavage and massage of the peritoneal cavity with 20 mL of 4°C HTB, rat peritoneal mast cells were purified from the peritoneal suspension by centrifugation through a two-step discontinuous gradient of Percoll. M199 supplemented with 10% FCS and HEPES (10 mmol/L) was used to prepare a 30% and 80% concentration of Percoll. A gradient was formed by layering 20 mL of 30% Percoll over 15 mL of 80% Percoll. The cells were placed on this gradient and centrifuged at 1600 rpm for 20 minutes, then the pellet was washed and resuspended in HTB. This procedure yielded mast cells that were 98% pure and 97% viable, as previously reported.15
Neutrophil Isolation and Adherence Assay
Neutrophils from healthy volunteers were purified by dextran sedimentation followed by hypotonic lysis and centrifuged on Histopaque, as previously described.14 20 This procedure yields a population of neutrophils that is more than 95% viable (trypan blue exclusion) and more than 98% pure. The neutrophil adhesion assay was a modification of the method of Fehr and Dahinden.25 Briefly, neutrophils (2x107/mL) were radiolabed with Na51CrO4 (30 µCi/mL) at 30°C for 30 minutes. The cells were washed three times with cold PBS and then resuspended at 2x107 cells per mL in PBS. Neutrophils were incubated with activated mast cells on endothelial monolayers or protein-coated plastic. The fluid and loose cells from each well were carefully aspirated and the wells were gently washed once with HBSS. The cells that remained adherent were then lysed by an overnight incubation with 2 mol/L NaOH. The lysate was assayed for 51Cr activity, and neutrophil adherence was calculated as the ratio of counts in the lysate to counts in the lysate plus supernatant and washed solution.
Experimental Protocol
Initially, to ensure that no direct interactions existed between the mast cells, endothelium, and neutrophils, endothelium and neutrophils were incubated in the presence and absence of unactivated mast cells. The mast cell number (103 to 106 cells/well) that caused optimal activation of neutrophil-endothelium interactions was next established. Additionally, mast cells were pretreated with various concentrations of CMP 48/80 to establish optimal mast cell–induced neutrophil-endothelium interactions. Establishing optimal conditions for adhesion allowed direct comparison of the ability of L-NAME to induce mast cell–dependent neutrophil-endothelium interactions. In the next series of experiments, mast cells, neutrophils, and endothelium were treated with different concentrations of L-NAME (1 µmol/L to 1 mmol/L) or L-NAME plus L-arginine (1 mmol/L). The experiments were also repeated with D-NAME at the same concentrations as L-NAME. Additionally, NO donors SIN-1 (100 µmol/L), spermine-NO (100 µmol/L), and CAS 754 (100 µmol/L) and the cGMP analogue 8-bromo-cGMP were coincubated with L-NAME. A temporal series of experiments was also performed wherein mast cells, endothelium, and neutrophils were exposed to L-NAME, and adhesion was determined at 5-minute intervals (up to 30 minutes).
The molecular mechanisms underlying the neutrophil adhesion induced by activated mast cells were assessed in the presence of MAbs directed against adhesion molecules on endothelial cells and neutrophils. MAb IB4 (anti-CD18; 20 µg/mL), R6.5 (anti–ICAM-1; 20 µg/mL), or the PAF-receptor antagonist WEB 2086 (20 µg/mL) was incubated in our culture system in the presence of L-NAME.
Although we have previously determined that L-NAME induces oxidative stress within endothelium,14 whether these oxidants enter the extracellular environment or whether they stay intracellular remains unclear. Therefore, in some experiments, endothelium was grown to confluence in six-well plates and washed, and ferricytochrome C was placed in each well. Then endothelium was exposed to either L-NAME or D-NAME for 30 minutes, and we measured SOD-inhibitable reduction of ferricytochrome C in an end-point assay as previously described.20
To determine whether endothelial cell–derived oxidants were contributing to the mast cell–induced adhesion, in a complementary series of experiments, mast cells, endothelium, and neutrophils were incubated with L-NAME, L-NAME plus SOD, or L-NAME plus sodium azide (0.1 mmol/L). In addition, in some experiments the endothelium was removed from the system entirely (protein-coated plastic was used) or endothelium was fixed and L-NAME was added to mast cells and neutrophils. Sodium azide (0.1 mmol/L) was used because it has previously been shown to inhibit the oxidative stress generated by endothelium in response to L-NAME.14 We further investigated the importance of endothelium-derived oxidants in the L-NAME–induced mast cell–dependent neutrophil-endothelium interaction; in some wells only endothelium was exposed to L-NAME for 30 minutes, the wells were washed, and naive mast cells and neutrophils were subsequently added.
Does L-NAME Directly Activate Mast Cells?
Since L-NAME–treated mast cells were able to induce neutrophil adhesion even if endothelium was removed from the system, we initially examined whether L-NAME was directly affecting the mast cells. First, oxidative stress was measured in mast cells. A stock solution of DHR 123 was diluted in PBS just prior to study. Mast cells were resuspended in HTB at a concentration of 4x105 cells/mL and preloaded with DHR 123 (5 µmol/L) at 37°C for 20 minutes, with gentle mixing every 5 minutes. After incubation, the mast cells were washed with cold HTB (800 rpm for 4 minutes) and resuspended in HTB. Aliquots of cells were then removed and incubated with L-NAME, L-NAME plus L-arginine, or L-NAME plus various antioxidants, including SOD (60 µg/mL), butylated hydroxytoluene (0.1 mmol/L), 
, '-dipyridyl (2 mmol/L), and the iron chelator desferrioxamine (0.2 mmol/L), for 30 minutes at 37°C. Since mitochondria are known to convert 1% to 4% of all oxygen to superoxide and H2O2,26 sodium azide (0.1 mmol/L) was used to inhibit mitochondrial respiration as previously described.27 The concentrations of these antioxidants have been established elsewhere.14 Florescence was measured at peak excitation (500 nm) and emission (536 nm).
To further confirm that L-NAME was affecting mast cells, in another series of experiments we determined whether L-NAME could directly activate mast cells. Mast cells were treated with L-NAME or D-NAME, and intracellular Ca2+ levels were measured. CMP 48/80 was used as a positive control. Briefly, mast cells were incubated with fluo-3 acetoxymethyl ester (AM; Molecular Probes, 2 µmol/L) for 30 minutes at 37°C, washed, and resuspended in buffer containing 1 mmol/L CaCl2. Changes in cytosolic Ca2+ were measured using a FACScan (Becton Dickson Systems Inc) as previously described.28
In a final series of experiments the membrane-permeable antioxidants butylated hydroxytoluene, ,'-dipyridyl, the iron chelator desferrioxamine, or sodium azide was added to the mast cell–endothelium-neutrophil coculture to determine whether these antioxidants (which inhibit both extracellular and intracellular oxidants) were more effective than the extracellular antioxidant SOD.
Statistical Analysis
All values were expressed as mean±SEM, and means were compared by Student's t test with Bonferroni correction for multiple comparison. Statistical significance was set at P<.05.
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Fig 1
summarizes mast cell–activation data for optimal adhesion of neutrophils to endothelium. One million neutrophils were added to a monolayer of endothelium, and varying numbers of mast cells stimulated with 1 µmol/L CMP 48/80 were then added to the well. Approximately 20 000 mast cells were required to induce peak levels of adhesion of neutrophils to endothelial cell monolayers. However, 5000 cells/well was sufficient to induce significant neutrophil-endothelium interactions. The lower panel of Fig 1 demonstrates that mast cells respond to CMP 48/80 in a dose-dependent fashion and that peak levels are reached between 0.1 and 1.0 µg/mL. The CMP 48/80 response was not observed if mast cells were not added to the neutrophil-endothelium coculture. Moreover, unactivated mast cells added simultaneously to neutrophils and endothelium also did not cause an increase in neutrophil–endothelial cell interactions, suggesting that the three cell types were in a quiescent state and that no direct activation between cells occurred in the absence of a stimulus. Therefore, this is a mast cell–dependent neutrophil adhesion assay to endothelium, and maximal adhesion can be achieved with 20 000 mast cells stimulated with 1.0 µg of CMP 48/80. These values were directly compared with L-NAME–induced responses.
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Exposure of endothelium and neutrophils to L-NAME (0.001 to 1 mmol/L) for 60 minutes did not promote neutrophil adhesion to endothelium (Fig 2) as previously reported.8 However, in our study, addition of mast cells to the neutrophil-endothelium culture in the presence of the NO synthesis inhibitor L-NAME caused neutrophils to adhere avidly to endothelium (Fig 2). This occurred in a dose-dependent fashion, reaching peak levels at 10 to 100 µmol/L of L-NAME. It is noteworthy that CMP 48/80 at optimal concentrations caused only 
15% more neutrophils to adhere to endothelium, suggesting that L-NAME is a potent activator of mast cell–induced neutrophil adhesion to endothelium. To ensure that this was the result of inhibition of NO, some wells received L-arginine (1 mmol/L); L-NAME–induced adhesion was reversed at all concentrations tested (Table). This was unrelated to a nonspecific effect of L-arginine since this amino acid did not affect CMP 48/80–induced neutrophil adhesion (data not shown). D-NAME, the enantiomer of L-NAME, was unremarkable for its effect on neutrophil–endothelial cell interactions in the presence of mast cells (Table). Fig 3 illustrates that L-NAME caused neutrophil adhesion between 20 and 30 minutes in the presence of mast cells. Finally, the addition of spermine-NO or SIN-1, two NO donors, was able to completely prevent the L-NAME–induced neutrophil–endothelial cell interaction (Fig 4), as did the cGMP analogue 8-bromo-cGMP. CAS 754, a third NO donor, was not effective at reversing the L-NAME response (Fig 3). We speculate that this may be related to the rapidity with which NO is released from this donor.29
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P<.05 relative to mast cell (MC) + L-NAME only value.
Fig 5 demonstrates that the adhesion was mediated by PAF because the PAF-receptor antagonist WEB 2086 completely prevented the response. Since PAF is a potent activator of the ß2-integrin (CD11/CD18), we also tested the role of CD18 and its ligand ICAM-1. Essentially all of the adhesion was prevented with anti-CD18 MAb and to a lesser degree with the anti–ICAM-1 antibody (Fig 5).
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We have previously reported increased oxidant production within endothelium in response to L-NAME,14 and therefore we tested this cell type as a potential source of extracellular oxidant production. Indeed, L-NAME–treated endothelium but not D-NAME–treated endothelium produced 5 nmol/L superoxide over 60 minutes as assessed by cytochrome C reduction (Fig 6). This is one order of magnitude less superoxide than would be expected from similar amounts of neutrophils.20 To test whether endothelial cell–derived superoxide could contribute to the mast cell–induced neutrophil adhesion, the mast cell–endothelium-neutrophil coculture was incubated with L-NAME, L-NAME plus SOD, or L-NAME plus sodium azide–treated endothelium. Fig 7 demonstrates that both interventions significantly reduced the mast cell–induced neutrophil adhesion in response to L-NAME; adhesion on intact HUVECs increased more than 3.5-fold, whereas neutrophil adhesion in the presence of SOD or sodium azide–treated endothelium was significantly reduced. Also shown in Fig 7 is the fact that L-NAME induced adhesion to protein-coated plastic (no endothelium) also increased only 2-fold as did adhesion to fixed endothelium (data not shown). Additionally, L-NAME was added to just the endothelium and washed before the neutrophils and the mast cells were exposed to the endothelium. Treating endothelium alone caused a significant increase in neutrophil adhesion that was inhibitable by SOD, suggesting that inhibition of NO synthesis in endothelium is sufficient to activate mast cells and induce adhesion (data not shown).
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It should be noted, however, that L-NAME–treated mast cells still induced some neutrophil adhesion to protein-coated plastic, suggesting a potential direct effect of L-NAME on mast cells. Indeed, loading mast cells with DHR 123 revealed increased oxidant levels within L-NAME–treated cells (Fig 8). The increased oxidative stress induced by L-NAME could be entirely inhibited by three different lipophilic antioxidants, by the iron chelator desferrioxamine, but not by the extracellular antioxidant SOD. Further confirmation that NO synthesis inhibition directly activates mast cells is the observation that L-NAME but not D-NAME increased intracellular Ca2+ influx in mast cells (Fig 9) In these experiments, the mast cells had to be exposed to L-NAME for a minimum of 15 to 20 minutes to observe biological responses. Unlike L-NAME, CMP 48/80 increased intracellular Ca2+ within the first few minutes of exposure (data not shown).
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The membrane-permeable antioxidants butylated hydroxytoluene and , '-dipyridyl, the iron chelator desferrioxamine, and sodium azide when added to the mast cell–endothelium-neutrophil coculture revealed essentially complete inhibition of neutrophil adhesion, whereas the extracellular antioxidant SOD inhibited the adhesion by 60% (Fig 10).
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To determine whether the mast cell–induced adhesion in the absence of endothelium was also dependent upon PAF, WEB 2086 was added to some protein-coated wells with mast cells and neutrophils. Fig 11 demonstrates that all of the L-NAME–stimulated, mast cell–dependent neutrophil adhesion was inhibitable by WEB 2086, suggesting a PAF-dependent adhesive interaction. This proadhesive response was also inhibitable by 8-bromo-cGMP, suggesting that the cGMP analogue was directly modulating the activity of the mast cells (Fig 11).
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Discussion |
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Previous work in vivo has revealed that NO synthesis inhibition leads to adhesion of neutrophils to postcapillary venules in a very rapid (20 to 30 minutes) manner,7 and this effect was almost entirely inhibitable with the extracellular antioxidant SOD.8 However, in a simple neutrophil–endothelial cell culture system, the responses associated with NO synthesis inhibition in vitro were very different14 from the adhesive response observed in vivo. For example, the increase in neutrophil adhesion to endothelium that was apparent within the first hour of L-NAME exposure in the intact vasculature8 required 4 hours to fully develop in the in vitro system.14 Incubating neutrophils and endothelium with L-NAME for only 1 hour did not increase adhesive interactions in vitro,8 14 raising the possibility that this in vitro system was lacking a cell that contributed critically to the neutrophil-endothelium interaction. In the present study, we demonstrate that addition of as few as 20 000 mast cells to this coculture (a ratio of 50:1 neutrophils to mast cells) significantly increased neutrophil adhesion to endothelium within 20 minutes of L-NAME administration. Although L-NAME is cationic and may directly activate mast cells independent of NO to induce neutrophil adhesion,30 this appears unlikely inasmuch as L-arginine (also a cationic molecule) reversed the effects of L-NAME and the stereospecific enantiomer D-NAME did not cause an increase in neutrophil adhesion. Finally, providing a continuous, exogenous source of NO reversed the effects of L-NAME. These data are consistent with the view that removal of NO from mast cells and/or endothelium causes an increase in neutrophil adhesion, with mast cells playing an essential role in this biological response.
The observation that mast cells are important for L-NAME–induced neutrophil adhesion raises the possibility that NO produced from mast cells functions in an autocrine fashion to regulate mast cell reactivity and that removal of this autacoid causes mast cell activation. We provide three pieces of evidence to support this view. The first piece of evidence to suggest that L-NAME caused mast cell activation was the increased intracellular Ca2+ levels after exposure of mast cells to L-NAME, an event that appears essential for mediator release from mast cells. Second, removal of NO from mast cells caused detectable levels of intracellular oxidative stress that could be inhibited by various intracellular antioxidants but not by the extracellular antioxidant SOD. Therefore, it is unlikely that the few non–mast cell types contaminating our mast cell population or the mast cells themselves were releasing oxidants into the surrounding milieu to cause oxidative stress within neighboring cells. It is more likely that the increased oxidative stress originated from within the mast cells per se after NO synthesis inhibition. It is noteworthy that it has previously been reported that activation of mast cells with non–oxidant activating agents such as CMP 48/80 or A23187 also increases intracellular oxidative stress,28 suggesting perhaps that intracellular oxidant production may be a component of mast cell activation. Even more interesting is the observation that 8-bromo-cGMP, which is not an antioxidant, also inhibited the increased intracellular oxidative stress (data not shown) and neutrophil adhesion, suggesting that the increased oxidant production may be an intracellular signal to activate mast cells.
The third piece of evidence to suggest that L-NAME directly activated mast cells is the observation that inhibition of NO synthesis in mast cells caused subsequent increase in neutrophil adhesion to protein-coated plastic. This would suggest a negative feedback mechanism of NO on the mast cell. Other work is consistent with the autocrine hypothesis; Salvemini et al31 have demonstrated that removal of L-NAME from mast cells augmented histamine release from these cells, suggesting an autocrine-suppressive role for NO in these immunocytes. These data together suggest that continuous production of NO by mast cells depresses their level of reactivity under normal, nonpathological conditions.
The endothelium also contributed significantly to L-NAME–induced neutrophil adhesion. In the absence of endothelium, mast cells treated with L-NAME increased neutrophil adhesion to a lesser degree than when endothelium was present. One possible explanation is that endothelium treated with L-NAME further activated mast cells. Indeed, in our mast cell, endothelium, and neutrophil cell culture system treated with L-NAME, the increased adhesion could be attenuated by selectively treating the endothelium with sodium azide or by fixing the endothelium with paraformaldehyde. These data support the view that in the absence of NO, the endothelium releases some factor that contributes significantly to the mast cell–induced neutrophil adhesion.
Our data would support the contention that the intercellular signal between endothelium and mast cells was superoxide. First, when the endothelium was treated with L-NAME, significant cytochrome C reduction could be detected in the fluid bathing the endothelium (Fig 6
). Hence, not only was the endothelium producing superoxide but the oxidant was reaching the extracellular space to a significant degree. This source of superoxide may then stimulate mast cells to release proadhesive agents, including PAF, which results in neutrophil binding to endothelium. Indeed, support for this view lies in the fact that the extracellular antioxidant SOD was able to significantly reduce neutrophil adhesion. It is noteworthy that in many tissues there is a significant proportion of mast cells that makes intimate contact with vessels and would almost certainly be able to detect changes around the endothelium. This form of communication between endothelium and mast cells may serve as a very important detector mechanism to recruit neutrophils to sites of endothelial dysfunction and injury but may also account for some of the inappropriate pathology associated with excessive leukocyte–endothelial cell interactions in ischemia/reperfusion, atherogenesis, and intimal restenosis. Interestingly, increased oxidant production and depressed NO levels are key features of these disease states.18 32 33
It is noteworthy that even 1 to 10 µmol/L L-NAME caused some neutrophil adhesion response to endothelium in the presence of mast cells. Since it is unlikely that this concentration of L-NAME completely inhibited cellular NO synthase activity, it raises the possibility that even subtle alterations in NO production may be sufficient to alter the balance between NO and superoxide to promote neutrophil adhesion. In other words, complete inhibition of NO synthesis may not be necessary to induce the adhesion responses. Alternatively, L-NAME may inhibit the enzymatic activity of NO synthase more effectively in cells in culture than in vivo. A contributing factor may be the fact that in vitro endothelium loses its NO synthase activity with passage,34 which may allow for lower levels of L-NAME to inhibit NO synthase activity in culture. In our laboratory, the increased oxidative stress from endothelium was evident only in passages 1 through 3, suggesting that NO synthase activity was indeed lost by passage 4.
In conclusion, our working hypothesis is that in a system in which NO is depleted there is increased oxidative stress within mast cells, in part from the mast cells themselves as well as from the endothelium. The mast cells, particularly those closely apposed to the vasculature, will be activated by the oxidants, releasing various proadhesive molecules to recruit neutrophils and perhaps other leukocytes. Although our aim was to examine the very early responses to L-NAME, it is intriguing that mast cells are a rich source of cytokines including tumor necrosis factor (TNF). Moreover, mast cell–derived TNF has been shown to increase E-selectin, ICAM-1, and vascular cell adhesion molecule-1 (VCAM-1) expression on endothelium.35 36 Clearly, NO deprivation could conceivably lead to both prolonged neutrophil adhesion and (with the expression of VCAM-1 and other cytokines) the recruitment of other leukocytes including monocytes and lymphocytes. These cells would further contribute to the vasculopathies associated with numerous diseases. It is intriguing that mast cell activation and degranulation have been documented in early atherosclerotic lesions in humans,37 a disease hallmarked by increased leukocyte-endothelium interactions.
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Acknowledgments |
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This study was supported by a grant from the Heart and Stroke Foundation of Canada. Dr Kubes is an AHFMR and Medical Research Council Scholar.
Received February 13, 1996; accepted July 29, 1996.
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