(Circulation. 1996;93:318-326.)
© 1996 American Heart Association, Inc.
Articles |
Nitric Oxide Inhibits Numerous Features of Mast Cell–Induced Inflammation
From the Immunological Sciences Research Group, University of Calgary Medical Centre, Calgary, Alberta, Canada.
Correspondence to Dr Paul Kubes, Immunology Research Group, Department of Medical Physiology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1. E-mail pkubes@acs.ucalgary.ca.
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Abstract |
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Background We previously reported that mast cell degranulation causes histamine and P-selectin–dependent leukocyte rolling and platelet-activating factor (PAF)- and CD18-associated leukocyte adhesion, whereas others have reported serotonin-induced edema formation. The purpose of the present study was to determine whether nitric oxide (NO) could inhibit the mast cell–induced multistep recruitment of leukocytes and the associated microvascular dysfunction in single inflamed venules.
Methods and Results Intravital fluorescence microscopy was used to demonstrate increased leukocyte rolling and adhesion and increased albumin extravasation in single 25- to 40-µm venules that were treated with the mast cell–degranulating agent compound 48/80 (CMP 48/80). The mast cell–induced histamine-dependent rolling and PAF-dependent adhesion were completely inhibited by the addition of the NO donor spermine NO. However, spermine NO did not directly inhibit histamine-induced leukocyte rolling and only partly affected PAF-induced leukocyte adhesion. Compound 48/80–activated mast cells evoked a significant increase in PAF-dependent neutrophil adhesion in vitro. Spermine-NO prevented the mast cell–dependent neutrophil adhesion but failed to affect direct adhesion with PAF. The mast cell–induced albumin leakage was also inhibited by the NO donor.
Conclusions Taken together, these results suggest that exogenous NO can modulate leukocyte recruitment and microvascular permeability alterations elicited by mast cell activation and raises the possibility that the use of NO donors may be a reasonable therapeutic approach to reducing mast cell–dependent inflammation.
Key Words: cardiovascular diseases • leukocytes • edema • microcirculation
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Inflammation is characterized by hyperemia, increased microvascular permeability to plasma proteins and fluid, and enhanced recruitment of leukocytes to the site of injury. There is a growing body of evidence to implicate the mast cell as a key player in this cascade of events. Mast cells have been demonstrated to lie in close proximity to the microvasculature and, on stimulation, are a rich source of numerous inflammatory mediators.1 These mediators include preformed molecules such as vasoactive amines and proteases and agents that are synthesized, including leukotrienes, prostaglandins, PAF, and a variety of cytokines.2 Thus, on activation, mast cells have the capacity to evoke all of the key microvascular features associated with acute inflammation. This may be of critical importance clinically because mast cells have been implicated in many disease states, including IgE-mediated anaphylaxis,3 4 5 ischemia/reperfusion of various organs,6 7 8 rheumatoid arthritis,9 10 and atherosclerosis.11 Therefore, prevention of mast cell reactivity could be a potential therapeutic strategy in controlling inflammation.
Evidence suggests that endogenously produced NO may regulate mast cell reactivity and thereby influence microvascular alterations. For example, inhibition of endogenous NO synthesis with the L-arginine analogue L-NAME causes mast cell degranulation, an increase in microvascular permeability, and an increase in leukocyte infiltration.12 13 Moreover, prevention of the L-NAME–associated mast cell degranulation prevented the L-NAME–induced leukocyte recruitment. These data raise the possibility that endogenous NO is an homeostatic regulator or depressor of mast cell reactivity that otherwise leads to leukocyte recruitment and inflammation. In vitro data further confirm this notion; inhibition of NO synthesis with L-NMMA significantly augmented histamine release from activated mast cells, suggesting that removal of NO from mast cells may enhance their reactivity.14 Also, delivery of exogenous NO may suppress mast cell reactivity; PAF and histamine release from mast cells stimulated with CMP 48/80 or the calcium ionophore A23187 could be prevented with the use of exogenous NO in vitro.14 15 Therefore, it is conceivable that exogenous NO can directly affect the mast cell–induced sequelae of inflammation in microvessels.
To test this hypothesis, we used intravital microscopy, which allows for visualization of leukocyte behavior and vascular dysfunction in single postcapillary venules, and examined the direct effect of NO donors in a well-characterized model16 of mast cell–induced leukocyte recruitment and microvascular permeability. In this model, perivenular mast cells are directly activated with CMP 48/80, and leukocyte rolling, leukocyte adhesion, and FITC-albumin leakage are observed. Concomitantly, in vitro experiments were performed to assess the role of NO directly on mast cell–induced leukocyte/endothelial cell interactions.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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All animal experiments were approved by and conducted in accordance with the guidelines established by the Canadian Council on Animal Care.
Intravital Microscopy
Male Sprague-Dawley rats (200 to 300 g)
were
maintained on a purified laboratory diet and fasted for 24 hours before
each experiment. The animals were initially anesthetized
intraperitoneally with sodium pentobarbital (65
mg/kg body wt), and the right jugular vein was cannulated for drug and
additional anesthetic administration. The right carotid artery was
cannulated, and systemic arterial pressure was monitored
with the use of a Statham P23 XL pressure transducer and Grass
physiological recorder connected to the right
carotid artery cannula. A midline abdominal incision was made, the rats
were placed in a supine position on an adjustable Plexiglas microscope
stage, a segment of the midjejunum was exteriorized through the
abdominal incision, and the mesentery was prepared for in vivo
microscopic observation as previously described.16 17
Briefly, the mesentery was draped over an optically clear viewing pedestal that allows for transillumination of a 2-cm2 segment of tissue. The temperature of the pedestal was maintained at 37°C with a constant temperature circulator (model 80, Fisher Scientific). Rectal and mesenteric temperatures were monitored with an electrothermometer. The exposed bowel wall was covered with saline-soaked gauze to minimize tissue dehydration, and the exposed mesentery was then superfused with the use of a Minipuls 2 suffusion pump (Gilson) with warmed bicarbonate-buffered saline (37°C, pH 7.4). We observed the mesenteric microcirculation by using an intravital microscope (Nikon Optiphot-2) with a 25x objective lens (Leitz Wetzlar L25/0.35) and a 10x eyepiece. A video camera mounted on the microscope projected the image onto a color monitor, and the images were recorded for playback analysis using a videocassette recorder. A video time/date generator projected the time, date, and stopwatch function onto the monitor.
Single
unbranched mesenteric venules (25 to 40 µm in diameter) were
selected for study. Venular diameter was measured on-line using a
video calliper (Microcirculation Research Institute, Texas A&M
University). Centerline red blood cell velocity (Vrbc) was
also measured on-line using an optical Doppler
velocimeter (Microcirculation Research Institute).
Venular blood flow was calculated from the product of mean red
blood cell velocity (Vmean=centerline velocity/1.6) and
venular cross-sectional area, assuming cylindrical geometry.
Venular wall shear rate (
) was calculated based on the newtonian
definition: =8(Vmean/Dv), where
Dv is venular diameter.18
The number of
adherent leukocytes were determined off-line during
playback of videotaped images. A leukocyte was defined as adherent to
venular endothelium if it remained stationary for a
period of 
30 seconds. Adherent cells were expressed as the number per
100-µm length of venule. Rolling leukocytes were defined as white
blood cells that move at a velocity less than that of erythrocytes in
the same vessel. Flux of rolling leukocytes was measured as cells per
minute that could be seen moving past a defined reference point in the
vessel. The same reference point was used throughout the experiment
because leukocytes may roll for only a section of the vessel before
rejoining the flow of blood or firmly adhering.
The degree of vascular albumin leakage in rat mesenteric venules was quantified according to a previously published protocol.13 Briefly, 25 mg/kg FITC-labeled bovine albumin (Sigma Chemical Co) was administered intravenously to the animals 15 minutes before the start of the experimental procedure. Fluorescence intensity (excitation wavelength, 420 to 490 nm; emission wavelength, 520 nm) was detected with the use of a silicon-intensified fluorescent camera (model C-2400-08, Hamamatsu Photonics), and images were recorded for playback analysis with a videocassette recorder. The fluorescent intensity of FITC-albumin within a defined area (10x50 µm) of the venule under study and in the adjacent perivascular interstitium (20 µm from venule) was measured at 15-minute intervals after administration of FITC-albumin. This was accomplished with a video capture board (Visionplus AT-OFG, Imaging Technology Inc) and a computer-assisted digital imaging processor (Optimas, Bioscan Inc). The index of vascular albumin leakage (permeability index) was determined from the following ratio: (interstitial intensity minus background)/(venular intensity minus background), as previously reported.13
Experimental Protocol
In untreated control animals, the
mesentery was superfused with
warmed bicarbonate-buffered saline for 90 minutes, and mesenteric
recordings were performed at 30-minute intervals. Leukocyte
kinetics reached a steady state over the first 30 minutes, and so in
all experimental groups, preparations were allowed to stabilize for 30
minutes before the protocol was begun. To examine the role of
connective tissue mast cells and establish the effect of mast cell
degranulation on leukocyte kinetics, rat mesenteric venules were
superfused with CMP 48/80 (mast cell–degranulating agent, 1
µg/mL; Sigma) for 60 minutes. This concentration of CMP 48/80 has
been shown to elicit optimal increases in the flux and adhesion of
leukocytes in rat mesenteric venules.16
To determine the effect of exogenous NO on mast cell–mediated leukocyte recruitment and microvascular dysfunction, two groups of animals were treated with either of the NO donors SP-NO and SIN-1. SP-NO (100 µmol/L; supplied by Dr Larry Keefer, National Cancer Institute) was added to the superfusion buffer 15 minutes before the start of CMP 48/80 superfusion. SIN-1 (1 mg/kg; Casella AG) was given as a bolus 15 minutes before the start of the experiment. These doses and routes of administration have been shown to reduce leukocyte adhesion and microvascular permeability alterations.19 20 To control for potential effects of spermine, in another group of animals, spermine (100 µmol/L; Sigma) was added to the superfusion buffer 15 minutes before the beginning of the experimental protocol.
We previously reported that the mast cell–induced leukocyte rolling was histamine dependent and that the adhesion was PAF related. Therefore, in another series of experiments, we examined the direct effect of SP-NO on histamine-induced leukocyte rolling or PAF-induced leukocyte adhesion. In one series of experiments, histamine was superfused onto the mesentery (100 µmol/L) for 60 minutes in the presence or absence of SP-NO (100 µmol/L). The preparation used was identical to one previously described by our group21 and a slight modification of the aforementioned setup. Briefly, before any surgical intervention, animals are administered sodium cromoglycate (5 mg/kg; Sigma) IV before treatment to stabilize mast cells. This modification results in rapid, consistent increases in histamine-induced P-selectin–dependent leukocyte rolling and allowed us to directly examine the role of NO on mast cell–independent, histamine-induced leukocyte rolling. In a second series of experiments, PAF (10 nmol/L) was superfused onto the rat mesentery in the presence or absence of SP-NO. Because recent unpublished work from our laboratory suggests that serotonin is responsible for the mast cell–dependent microvascular dysfunction, serotonin (10 µmol/L; Sigma) was superfused onto the rat mesentery in the presence and absence of SP-NO (100 µmol/L) for 60 minutes, and the leakage of FITC-albumin was determined as described above.
In Vitro Adhesion Studies
Experiments were conducted on HUVEC
that were grown to
confluence or on protein-coated plastic, and neutrophil adhesion to
this biological substratum was tested in the presence or absence of
mast cells and/or CMP 48/80. 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 was incubated for 20 minutes at 25°C. The cords
were gently massaged to ensure detachment of
endothelial cells from the vessel wall. The digest was
collected, the collagenase was inactivated with
fetal calf serum, and the cell suspension was centrifuged
(400g for 10 minutes at 37°C). The pellet was resuspended
in M199 containing 10% fetal calf serum and antibiotics plated in
25-cm2 flasks. Cultures were incubated in 5%
CO2 at 37°C with 96% humidity, expanded by
trypsinization, and grown to confluence in 48-well plates. The identity
of some of the cultures was checked by indirect staining with
FITC-labeled factor VIII antibody22 and by uptake of
acetylated LDL23 according to established
techniques.
Mast cells were isolated from male Sprague-Dawley rats according to a previously published method.24 Briefly, rats were anesthetized with ether, killed by cervical dislocation, and bled. After lavage of the peritoneal cavity with 15 mL of 4°C HEPES-buffered Tyrode's solution, peritoneal mast cells were purified from the peritoneal cell suspension by centrifugation through a two-step discontinuous gradient of percoll. RPMI supplemented with 10% fetal calf serum and HEPES (10 mmol/L) was used to prepare 30% and 80% concentrations of percoll. A gradient was formed by layering 20 mL 30% percoll over 15 mL 80% percoll. The cells were placed on this gradient and centrifuged at 1600 rpm for 20 minutes, and the pellet was washed and resuspended in HEPES-buffered Tyrode's solution. This procedure yielded mast cells that were more than 90% pure and 97% viable.
Neutrophils from healthy donors were purified by dextran sedimentation, followed by hypotonic lysis and histopaque centrifugation as previously described.25 26 The cells were radiolabeled by incubating purified neutrophils (2x107 cells/mL) with 30 µCi/mL of Na51CrO4 at 37°C for 60 minutes. The cells were washed three times and resuspended in PBS. The adhesion assay was conducted in an identical manner as previously described.25 Endothelial cell monolayers were exposed to neutrophils and either Hanks' buffered saline solution (control), CMP 48/80 (1 µg/mL), mast cells (2x105 cells/well), or a combination of CMP 48/80 plus mast cells in the presence and absence of NO donors. Previous work has suggested that PAF was responsible for the mast cell–induced neutrophil adhesion.16 These data were confirmed in this study with a PAF receptor antagonist, WEB 2086, and in some experiments neutrophils were directly exposed to PAF in the presence of the NO donors. The supernatant of each well was then aspirated, and the well was gently washed with 200 µL PBS. The cells that remained adherent were then lysed with an overnight incubation with 0.5 mL NaOH (2 mol/L). The cell lysate was collected, and the lysate and supernatant were assayed for 51Cr activity. Neutrophil adherence was calculated as the ratio of radioactivity in the cell lysate to the radioactivity in the cell lysate plus supernatant.
The data were analyzed using standard statistical analyses, ie, ANOVA and Student's t test with Bonferroni's correction for multiple comparisons where appropriate. All values are reported as mean±SEM. Statistical significance was set at P<.05.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Fig 1
illustrates shear rates, flux of rolling
leukocytes, leukocyte adhesion, and vascular permeability in untreated
control animals and in animals treated with CMP 48/80. In untreated
control animals, shear rates, flux of rolling leukocytes, and leukocyte
adhesion did not change over the 60-minute experimental protocol.
Superfusion of the rat mesentery with CMP 48/80 did not affect venular
wall shear rate (Fig 1A) but significantly increased the flux
of
rolling leukocytes compared with untreated controls (Fig 1B).
Leukocyte
adhesion was also significantly increased at 30 (twofold) and 60
minutes (fourfold) of CMP 48/80 superfusion (Fig 1C). CMP
48/80–induced mast cell activation also resulted in a rapid (at 15
minutes) and prolonged increase in vascular albumin leakage
(Fig 1D), whereas in the untreated control group, there was no
rise in
vascular protein leakage until 60 minutes. It should be noted that a
significant phase of the early rise in FITC-albumin leakage in
the CMP 48/80–treated animals preceded leukocyte infiltration.
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P<.05, relative
to respective untreated value.
To evaluate the effects of exogenous NO on mast cell–induced
leukocyte recruitment and microvascular permeability alterations, we
treated two additional series of animals with the NO donors SP-NO or
SIN-1. Fig 2 illustrates the time course of flux of
rolling leukocytes in CMP 48/80–treated animals and in animals treated
with either CMP 48/80 plus SP-NO (Fig 2A) or CMP 48/80 plus
SIN-1 (Fig 2B). Both SP-NO and SIN-1 were able to significantly
reduce the
increased flux of rolling leukocytes associated with CMP 48/80
superfusion. Fig 3 demonstrates that the NO donor SP-NO
was able to completely prevent the CMP 48/80–induced leukocyte
adhesion at both 30 and 60 minutes (Fig 3A). Although the other
NO
donor, SIN-1, had no effect on the degree of CMP 48/80–induced
leukocyte adhesion observed at 30 minutes, it did reduce the leukocyte
adhesion observed at 60 minutes by 50% (Fig 3B).
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The Table demonstrates that when SP-NO was added to the
CMP 48/80 superfusate, shear rates did not change at 30
minutes, but by 60 minutes, shear rates had increased by 40%
(P<.05). SIN-1 did not significantly change shear rates
from those observed in CMP 48/80–treated animals. Because of the slow
and regulated release of NO from SP-NO (t1/2=39
minutes)27 and the potential complication that SIN-1
releases superoxide in addition to NO,28 SP-NO was used
for the remainder of experiments.
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To determine whether the addition of NO could prevent the mast
cell–mediated increases in microvascular permeability, the degree
of FITC-albumin leakage was evaluated in the presence of SP-NO.
Fig 4 demonstrates that CMP 48/80 induced a rapid (15
minutes) and prolonged increase in FITC-albumin leakage from
the postcapillary venules under study. The addition of SP-NO to the CMP
48/80 superfusate completely prevented the rise in
microvascular permeability associated with mast cell activation. In the
animals treated with CMP 48/80 plus SP-NO, the degree of
albumin leakage remained at levels observed under control
conditions. This was not due to a reduction in local blood perfusion as
SP-NO did not affect venular blood flow at 30 minutes and increased
this parameter at 60 minutes. The addition of spermine
to the CMP 48/80 superfusate had no effect on microvascular
parameters associated with mast cell activation (data not
shown).
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To establish whether NO was affecting leukocyte rolling and adhesion
directly or as a result of its effects on mast cells, we performed in
vivo and in vitro experiments. We previously identified that the mast
cell–induced leukocyte rolling and adhesion in this model were
modulated by histamine and PAF, respectively.16 Moreover,
we identified (unpublished observations) that the mast cell mediator
that modulates a significant portion of the increased microvascular
permeability is serotonin. Therefore, we examined the
direct effect of SP-NO on (1) histamine-induced leukocyte rolling,
(2) PAF-induced leukocyte adhesion, and (3)
serotonin-induced microvascular dysfunction. Fig 5 illustrates
that SP-NO did not affect
histamine-induced leukocyte rolling (Fig 5A) or
serotonin-induced microvascular dysfunction (Fig 5C).
Histamine did not affect leukocyte adhesion, whereas
serotonin did not affect leukocyte rolling or adhesion
(data not shown). SP-NO did attenuate PAF-induced leukocyte adhesion
(Fig 5B) (by 
50%). The latter observation is consistent
with a report that other NO donors inhibit PAF-induced leukocyte
adhesion by 50%,19 an event that has been proposed to be
related to NO inactivation of superoxide. Superoxide mediates 50%
of the PAF-associated adhesive response (based on superoxide dismutase
inhibition).
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An in vitro adhesion assay system was used to determine the direct
effect of SP-NO on mast cell–induced leukocyte adhesion. Fig
6 illustrates that CMP 48/80–treated mast cells caused
an increase in neutrophil adhesion to HUVEC monolayers, whereas CMP
48/80 or mast cells alone did not increase
neutrophil/endothelial cell interactions (inset). The
mast cell–dependent increase in leukocyte adhesion was inhibited
by 70% via PAF/receptor antagonist WEB 2086 at a
concentration that completely prevented PAF-induced leukocyte adhesion.
SP-NO reduced the mast cell–dependent leukocyte adhesion to the
same degree as WEB 2086, but the NO donor was not able to directly
affect PAF-induced leukocyte adhesion. SP-NO was also able to
significantly attenuate the degree of neutrophil adhesion to
protein-coated plastic, further supporting the concept that this NO
donor was eliciting its effects on the mast cell and not the
endothelium (Fig 7). Again, SP-NO did
not directly affect PAF-induced leukocyte adhesion to
protein-coated plastic.
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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NO, an endogenous autacoid produced by a variety of cells, including the endothelium, has been shown by various laboratories to have profound anti-inflammatory properties in various acute inflammatory conditions, including ischemia/reperfusion and cardiac anaphylaxis.20 29 30 Interestingly, in both of these conditions, mast cells have also been implicated as playing an important role in the associated inflammation.29 30 31 32 In one study, NO administration greatly reduced histamine release from isolated, anaphylactic hearts, suggesting a potential link between NO and mast cell reactivity.30 Furthermore, mast cell stabilization, much like NO administration, reduces ischemia/reperfusion–induced leukocyte rolling,32 33 leukocyte adhesion and emigration,20 32 33 and vascular dysfunction.20 34 Although a causal relation between NO and mast cell reactivity is a tempting supposition to explain the protective effect of NO in these acute inflammatory conditions, direct evidence in vivo is lacking to implicate NO as a modulator of mast cell reactivity in inflamed microvessels. In the present study, we demonstrated that an exogenous NO donor can (1) reduce neutrophil recruitment and (2) attenuate the profound increase in microvascular permeability associated with direct activation of perivenular mast cells.
The pattern of leukocyte recruitment to sites of injury follows a distinct series of events. After their displacement from the mainstream of blood, leukocytes make initial contact with the endothelium described as leukocyte rolling. This rolling response is mediated by a family of adhesion molecules called selectins found on both leukocytes and endothelial cells.35 36 37 A likely selectin candidate in the early recruitment of rolling leukocytes is P-selectin, which is mobilized from Weibel-Palade bodies by various mast cell–derived mediators, including histamine, leukotriene C4, and oxidants.38 39 40 We recently reported that mast cell–derived histamine mediates P-selectin–dependent leukocyte rolling in vivo.16 In the present study, we demonstrated that NO donors completely inhibit the enhanced flux of rolling leukocytes associated with mast cell activation. However, this was not the case with exogenously administered histamine. Because histamine, much like mast cells, also elicits a significant P-selectin–dependent increase in leukocyte rolling,21 41 these data suggest that the mode of action of NO is not direct inhibition of P-selectin expression or effects on leukocyte/endothelial cell, P-selectin/ligand interactions. A more likely interpretation of these data is that NO functions directly to prevent histamine release from the mast cells and thereby inhibits the recruitment of leukocyte rolling.
Superfusion of the mesentery with CMP 48/80 allows us to examine the role of mast cells surrounding a single venule and avoids problems that would be encountered with systemic administration of CMP 48/80. However, due to the very small amount of mesentery that is exposed to CMP 48/80, we are unable to directly measure levels of histamine in this tissue. Nevertheless, reports by others lend credence to our hypothesis that NO directly reduces histamine release from mast cells. Salvemini et al14 previously demonstrated that NO can reduce the release of histamine from mast cells activated with CMP 48/80 or the calcium ionophore A23187. Moreover, NO synthesis inhibitors augment the release of histamine from activated mast cells.42 Taken together, these data suggest that NO dampens the release of histamine from mast cells and thereby reduces the leukocyte rolling in single postcapillary venules observed in the present study. Because mast cell stabilization reduces ischemia/reperfusion–induced leukocyte rolling,32 the inhibitory effect of NO on mast cells may also explain the antirolling effect of NO donors in ischemia/reperfusion reported by Gauthier et al.33
Once leukocyte rolling is established, firm adhesion of the leukocytes to the venular endothelium is the next step in leukocyte recruitment during an inflammatory response.43 Previous work has demonstrated a role for PAF and CD18 in causing the mast cell–induced leukocyte adhesion in this model of inflammation.16 The NO donor SP-NO was able to completely prevent the increased leukocyte adhesion elicited by mast cell activation. Because leukocyte rolling is an essential prerequisite for leukocyte adhesion, the possibility exists that in inhibiting leukocyte rolling, SP-NO subsequently prevented leukocyte adhesion. Diphenhydramine, the histamine (H1)-receptor antagonist that prevented CMP 48/80–induced leukocyte rolling was almost as effective at inhibiting leukocyte adhesion as either the PAF receptor antagonist or an anti-CD18 monoclonal antibody.16 To test the possibility that SP-NO specifically inhibits histamine-induced rolling and thereby prevents subsequent adhesion, we examined the role of SP-NO on leukocyte adhesion in a static in vitro assay system in which rolling is not a prerequisite for adhesion. In our in vitro adhesion assay, mast cells activated with CMP 48/80 promote leukocyte adhesion to endothelium primarily via PAF as WEB 2086 (a PAF-receptor antagonist) reduced neutrophil/endothelium interactions by 70%. SP-NO also prevented mast cell–derived, PAF-induced leukocyte adhesion in the same system, suggesting that exogenous NO inhibits the PAF-induced leukocyte/endothelium interaction. However, SP-NO was not capable of reducing leukocyte adhesion induced with exogenously administered PAF, suggesting that SP-NO does not directly interfere in the PAF-induced upregulation and activation of CD18 on the surface of leukocytes. Moreover, these data also discount the possibility that the effects of SP-NO were simply a result of toxicity to the leukocytes or endothelium.
An alternative explanation for the antiadhesive property of SP-NO may
be related to the fact that SP-NO increased shear forces in
postcapillary venules at 60 minutes of CMP 48/80 superfusion. It is
well known that decreased shear enhances the propensity of a leukocyte
to adhere in postcapillary venules44 ; however, whether
this relation holds true as shear forces increase above normal levels,
ie, increased shear reduces leukocyte adhesion, has not been examined.
Although it is likely that the SP-NO–induced increase in shear forces
contributed to the antiadhesive effect, there is ample evidence in this
study to suggest that other, shear-independent mechanisms also
exist. For example, SP-NO reduced
neutrophil/endothelial cell interactions in vitro under
static conditions. Moreover, when we compared adhesion results from the
compound 48/80 group with those from the CMP 48/80 plus SP-NO group at
a given shear (400 to 600 s-1, and removed data
outside this shear range), the adhesion was significantly higher in the
CMP 48/80 group than in the SP-NO–treated group. Finally, a second NO
donor, SIN-1, did not significantly increase shear, yet leukocyte
adhesion was significantly reduced (Fig 3
). Therefore, these
data
as a whole suggest that NO may exert antiadhesive effects that extend
beyond the simple effect of increased shear forces.
Although it is tempting to conclude that SP-NO also prevents the release of PAF from mast cells, an alternative explanation exists. It is conceivable that mast cells release various proinflammatory molecules that in turn activate the endothelium to synthesize PAF. It is well known that the mast cell can produce cysteinyl leukotrienes, histamine, and even oxidants,1 2 and these molecules have been demonstrated to activate PAF synthesis in endothelium.45 46 Therefore, it is possible that SP-NO prevents the synthesis of endothelium-derived PAF by modulating endothelial cell function. Heller et al47 demonstrated that various nitrovasodilators inhibit thrombin-induced PAF production in endothelial cells via increased intracellular cGMP levels. However, our data suggest that at least a portion of the antiadhesive property of NO was related to its ability to regulate PAF synthesis and release from mast cells. This contention is based on the observation that mast cell activation caused neutrophil adhesion to protein-coated plastic (no endothelium) and that NO prevented this response. Nevertheless, a direct inhibitory role for NO on endothelium cannot be discounted.
We demonstrated that in addition to preventing leukocyte adhesion, SP-NO profoundly inhibits FITC-albumin leakage from microvessels exposed to CMP 48/80. It is well known that leukocyte adhesion can cause microvascular dysfunction.48 Therefore, the reduction in microvascular dysfunction with SP-NO might be explained by the reduction in leukocyte adhesion. However, because the mast cell–induced, increased FITC-albumin leakage preceded the leukocyte influx, it is unlikely that the protective effect of SP-NO could simply be explained by its inhibition of leukocyte adhesion. Preliminary data from our laboratory suggest that a monoclonal antibody (CL26) that prevents leukocyte adhesion had no effect on the early (first 30 minutes) rise in albumin leakage but did attenuate albumin leakage induced by CMP 48/80 in the final 30 minutes (P. Kubes and J. Gaboury, unpublished observations). Therefore, it appears that SP-NO inhibited the early leukocyte-independent increase in microvascular permeability (when no leukocytes were present) as well as the delayed vascular dysfunction that appears to be associated with leukocyte adhesion. Again, the commonality between these results and those in ischemia/reperfusion are striking. Kurose et al20 demonstrated that NO donors prevented both the early neutrophil-independent and the later neutrophil-dependent vascular dysfunction and the associated reperfusion-induced mast cell degranulation. The possibility that degranulating mast cells were responsible for the reperfusion-induced increase in microvascular permeability was not addressed. However, based on our study, the possibility exists that SP-NO affects mast cells to prevent reperfusion-induced FITC-albumin leakage out of the vasculature.
If NO were an important regulator of mast cell reactivity, then removal of endogenous NO might cause mast cell activation and subsequent leukocyte recruitment and increased microvascular permeability. Superfusion of NO synthesis inhibitors onto the rat mesentery caused all of the sequelae of inflammation observed in the present study and in ischemia/reperfusion studies: mast cell degranulation,12 49 increased leukocyte rolling,50 increased leukocyte adhesion,12 13 51 and increased microvascular permeability.13 It is therefore tempting to speculate that in disease conditions where NO production is impaired, such as atherosclerosis, reperfusion injury, and other vascular diseases,52 53 microvascular dysfunction may be at least in part related to increased mast cell reactivity. Therefore, NO supplementation with either NO donors or perhaps the precursor of NO L-arginine may prevent mast cell degranulation and thereby explain the benefit of this form of therapy in models of atherogenesis and/or reperfusion injury.53 54 55
In the present study, we demonstrated directly that NO has a profound inhibitory effect on mast cell–induced sequelae of inflammation. Because numerous studies have documented anti-inflammatory effects of NO donors in various inflammatory conditions, including models of stroke,56 myocardial infarction,57 and reperfusion of various other organs,20 58 59 the possibility exists that exogenous NO may reduce mast cell reactivity and thereby inhibit the pathogenesis of these conditions. Also, this concept may extend to various other models of mast cell–associated inflammation, including hypersensitivity reactions,3 4 5 60 rheumatoid arthritis,9 10 psoriasis,61 arthus reaction,62 and anaphylaxis.29 30
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by a grant from the Alberta Heritage Foundation for Medical Research (AHFMR) and the Heart and Stroke Foundation of Canada. Dr Kubes is an AHFMR and Medical Research Council Scholar. Dr Gaboury is supported by a Heart and Stroke Foundation of Canada Research Traineeship.
Received June 9, 1995; revision received August 10, 1995; accepted September 4, 1995.
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References |
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TopAbstractIntroductionMethods Results Discussion References |
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1. Galli SJ. New concepts about the mast cell. N Engl J Med. 1993;328:257-265.
2. Crowe SE, Perdue MH. Gastrointestinal food hypersensitivity: basic mechanisms of pathophysiology. Gastroenterology. 1992;103:1075-1095. [Medline] [Order article via Infotrieve]
3. Tannenbaum S, Oertel H, Henderson W, Kaliner M. The biologic activity of mast cell granules, I: elicitation of inflammatory responses in rat skin. J Immunol. 1980;125:325-335. [Medline] [Order article via Infotrieve]
4. Gleich GJ. The late phase of the immunoglobulin E-mediated reaction: a link between anaphylaxis and common allergic disease? J Allergy Clin Immunol. 1982;70:160-169. [Medline] [Order article via Infotrieve]
5. Wershil BK, Wang ZS, Gordon JR, Galli SJ. Recruitment of neutrophils during IgE-dependent cutaneous late phase reactions in the mouse is mast cell-dependent. J Clin Invest. 1991;87:446-453.
6.
Keller AM, Clancy RM, Barr ML. Acute
reoxygenation injury in the isolated rat heart:
role of resident cardiac mast cells. Circ
Res. 1988;63:1044-1052.
7. Parenteau GL, Clark RE. Prevention of ischemia-reperfusion injury by the allergy drug lodoxamide tromethamine. Ann Thorac Surg. 1991;52:832-838. [Abstract]
8. Su M, Chi EY, Bishop MJ, Henderson WR Jr. Lung mast cells increase in number and degranulate during pulmonary artery occlusion/reperfusion injury in dogs. Am Rev Respir Dis. 1993;147:448-456. [Medline] [Order article via Infotrieve]
9. Wasserman SI. The mast cell and synovial inflammation: or, what's a nice cell like you doing in a joint like this? Arthritis Rheum. 1984;27:841-844. [Medline] [Order article via Infotrieve]
10. Bridges AJ, Malone DG, Jicinsky J, Chen M, Ory P, Engber W, Graziano FM. Human synovial mast cell involvement in rheumatoid arthritis and osteoarthritis: relationship to disease type, clinical activity and antirheumatic therapy. Arthritis Rheum. 1991;34:1116-1124. [Medline] [Order article via Infotrieve]
11. Atkinson JB, Harlan CW, Harlan GC, Virmani R. The association of mast cells and atherosclerosis: a morphologic study of early atherosclerotic lesions in young people. Hum Pathol. 1994;25:154-159. [Medline] [Order article via Infotrieve]
12. Kubes P, Kanwar S, Niu X-F, Gaboury J. Nitric oxide synthesis inhibition induces leukocyte adhesion via superoxide and mast cells. FASEB J. 1993;7:1293-1299. [Abstract]
13.
Kurose I, Wolf R, Grisham MB, Aw TY, Specian RD,
Granger DN. Microvascular responses to inhibition of nitric
oxide production: role of active oxidants.
Circ Res. 1995;76:30-39.
14. Salvemini D, Masini E, Pistelli A, Mannaioni PF, Vane J. Nitric oxide: a regulatory mediator of mast cell reactivity. J Cardiovasc Pharmacol. 1991;17(suppl 3):S258-S264.
15. Hogaboam CM, Befus AD, Wallace JL. Modulation of rat mast cell reactivity by IL-1 beta: divergent effects on nitric oxide and platelet-activating factor release. J Immunol. 1993;151:3767-3774. [Abstract]
16. Gaboury JP, Johnston B, Niu X-F, Kubes P. Mechanisms underlying acute mast cell-induced leukocyte rolling and adhesion in vivo. J Immunol. 1995;154:804-813. [Abstract]
17.
Gaboury JP, Kubes P. Reductions in physiologic
shear rates lead to CD11/CD18-dependent, selectin-independent
leukocyte rolling in vivo. Blood. 1994;83:345-350.
18. House SD, Lipowsky HH. Leukocyte-endothelium adhesion: microhemodynamics in mesentery of the cat. Microvasc Res. 1987;34:363-379. [Medline] [Order article via Infotrieve]
19.
Gaboury J, Woodman RC, Granger DN, Reinhardt P, Kubes
P. Nitric oxide prevents leukocyte adherence: role of
superoxide. Am J Physiol. 1993;265:H862-H867.
20.
Kurose I, Wolf R, Grisham MB, Granger DN.
Modulation of ischemia/reperfusion-induced
microvascular dysfunction by nitric oxide. Circ
Res. 1994;74:376-382.
21. Kubes P, Kanwar S. Histamine induces leukocyte rolling in post-capillary venules: a P-selectin-mediated event. J Immunol. 1994;152:3570-3577. [Abstract]
22.
Inauen W, Granger DN, Meininger CJ, Schelling ME,
Granger HJ, Kvietys PR.
Anoxia-reoxygenation-induced,
neutrophil-mediated endothelial cell injury: role
of elastase. Am J Physiol. 1990;259:H925-H931.
23.
Voyta JC, Via DP, Butlerfield CE, Zetter BR.
Identification and isolation of endothelial
cells based on their increased uptake of acetylated low density
lipoprotein. J Cell Biol. 1984;99:2034-2040.
24. Shanahan F, Denburg JA, Fox J, Bienenstock J, Befus D. Mast cell heterogeneity: effects of neuroenteric peptides on histamine release. J Immunol. 1985;135:1331-1337. [Abstract]
25.
Niu X-F, Smith CW, Kubes P. Intracellular
oxidative stress induced by nitric oxide synthesis inhibition increases
endothelial cell adhesion to neutrophils.
Circ Res. 1994;74:1133-1140.
26.
Woodman RC, Reinhardt PH, Kanwar S, Johnston FL, Kubes
P. The effects of human neutrophil elastase (HNE) on
neutrophil function in vitro and in inflamed microvessels.
Blood. 1993;82:2188-2195.
27. Maragos CM, Morley D, Wink DA, Dunams TM, Saavedra JE, Hoffman A, Bove AA, Issac L, Hrabie JA, Keefer LK. Complexes of NO with nucleophiles as agents for the controlled biological release of nitric oxide: vasorelaxant effects. J Med Chem. 1991;34:3242-3247. [Medline] [Order article via Infotrieve]
28. Hogg N, Darley-Usmar VM, Wilson MT, Moncada S. Production of hydroxyl radicals from the simultaneous generation of superoxide and nitric oxide. Biochem J. 1992;281:419-424.
29. Thelen KI, Dembinska-Kiec A, Pallapies D, Simmet T, Peskar BA. Effect of 3-morpholinosydnonimine (SIN-1) and NG-nitro-L-arginine (NNA) on isolated perfused anaphylactic guinea-pig hearts. Naunyn-Schmiedebergs Arch Pharmacol. 1992;345:93-99. [Medline] [Order article via Infotrieve]
30. Masini E, Gambassi F, Di Bello MG, Mugnai L, Raspanti S, Mannaioni PF. Nitric oxide modulates cardiac and mast cell anaphylaxis. Agents Actions [Suppl]. 1994;41:C89-C90.
31.
Kanwar S, Kubes P. Mast cells contribute
to ischemia/reperfusion-induced granulocyte infiltration
and intestinal dysfunction. Am J Physiol. 1994;267:G316-G321.
32. Kanwar S, Kubes P. Ischemia/reperfusion-induced granulocyte influx is a multistep process mediated by mast cells. Microcirculation. 1994;1:175-182. [Medline] [Order article via Infotrieve]
33.
Gauthier TW, Davenpeck KL, Lefer AM. Nitric
oxide attenuates leukocyte-endothelial interaction
via P-selectin in splanchnic ischemia-reperfusion.
Am J Physiol. 1994;267:G562-G568.
34. Lynch MJ, Grum CM, Gallagher KP, Bolling SF, Deeb GM, Morganroth ML. Xanthine oxidase inhibition attenuates ischemic-reperfusion lung injury. J Surg Res. 1988;44:538-544. [Medline] [Order article via Infotrieve]
35.
Ley K, Gaehtgens P, Fennie C, Singer MS, Lasky LA,
Rosen SD. Lectin-like cell adhesion molecule 1 mediates
leukocyte rolling in mesenteric venules in vivo.
Blood. 1991;77:2553-2555.
36. Lawrence MB, Springer TA. Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell. 1991;65:859-873. [Medline] [Order article via Infotrieve]
37. Abbassi O, Kishimoto TK, McIntire LV, Anderson DC, Smith CW. E-selectin supports neutrophil rolling in vitro under conditions of flow. J Clin Invest. 1993;92:2719-2730.
38.
Patel KD, Zimmerman GA, Prescott SM, McEver RP,
McIntyre TM. Oxygen radicals induce human
endothelial cells to express GMP-140 and bind
neutrophils. J Cell Biol. 1991;112:749-759.
39.
Hattori R, Hamilton KK, Fugate RD, McEver RP, Sims PJ.
Stimulated secretion of endothelial von
Willebrand factor is accompanied by rapid redistribution to the
cell surface of the intracellular granule membrane protein
GMP-140. J Biol Chem. 1989;264:7768-7771.
40. Zimmerman GA, Prescott SM, McIntyre TM. Leukotrienes C4 and D4: inflammatory agonists that induce PAF synthesis and endothelial cell-dependent neutrophil adhesion. In: Fritsch J, Müller-Peddinghaus R, eds. Advances in Rheumatology and Inflammation. Basel, Switzerland: Eular Publishers; 1992:49-58.
41. Asako H, Kurose I, Wolf R, DeFrees S, Zheng Z-L, Phillips ML, Paulson JC, Granger DN. Role of H1 receptors and P-selectin in histamine-induced leukocyte rolling and adhesion in postcapillary venules. J Clin Invest. 1994;93:1508-1515.
42. Masini E, Salvemini D, Pistelli A, Mannaioni PF, Vane JR. Rat mast cells synthesize a nitric oxide like-factor which modulates the release of histamine. Agents Actions. 1991;33:61-63. [Medline] [Order article via Infotrieve]
43.
Von Andrian UH, Chambers JD, McEvoy LM, Bargatze RF,
Arfors KE, Butcher EC. Two-step model of
leukocyte-endothelial cell interaction in
inflammation: distinct roles for LECAM-1 and the leukocyte
ß2 integrins in vivo. Proc Natl Acad Sci
U S A. 1991;88:7538-7542.
44. Perry MA, Granger DN. Role of CD11/CD18 in shear-rate dependent leukocyte-endothelial cell interactions in cat mesenteric venules. J Clin Invest. 1991;87:1798-1804.
45. Lewis MS, Whatley RE, Cain P, McIntyre TM, Prescott SM, Zimmerman GA. Hydrogen peroxide stimulates the synthesis of platelet activating factor by endothelium and induces endothelial cell-dependent neutrophil adhesion. J Clin Invest. 1988;82:2045-2055.
46. Zimmerman GA, Whatley RE, McIntyre TM, Prescott SM. Production of platelet-activating factor, a biologically active lipid by vascular endothelial cells. Am Rev Resp Dis. 1987;136:204-207. [Medline] [Order article via Infotrieve]
47. Heller R, Bussolino F, Ghigo D, Garbarino G, Pescarmona G, Till U, Bosia A. Nitrovasodilators inhibit thrombin-induced platelet-activating factor synthesis in human endothelial cells. Biochem Pharmacol. 1992;44:223-229. [Medline] [Order article via Infotrieve]
48.
Arfors KE, Lundberg C, Lindbom L, Lundberg K, Beatty
PG, Harlan JM. A monoclonal antibody to the membrane
glycoprotein CD18 inhibits polymorphonuclear leukocytes
accumulation and plasma leakage in vivo. Blood. 1987;69:338-340.
49.
Kanwar S, Wallace JL, Befus D, Kubes P. Nitric
oxide synthesis inhibition increases epithelial permeability via mast
cells. Am J Physiol. 1994;266:G222-G229.
50. Davenpeck KL, Gauthier TW, Lefer AM. Inhibition of endothelial-derived nitric oxide promotes P-selectin expression and actions in the rat microcirculation. Gastroenterology. 1994;107:1050-1058. [Medline] [Order article via Infotrieve]
51.
Suematsu M, Tamatani T, Delano FA, Miyasaka M, Forrest
M, Suzuki H, Schmid-Schönbein GW. Microvascular oxidative
stress preceding leukocyte activation elicited by in vivo nitric oxide
suppression. Am J Physiol. 1994;266:H2410-H2415.
52.
Tsao PS, Aoki N, Lefer DJ, Johnson G, Lefer AM.
Time course of endothelial dysfunction and
myocardial injury during myocardial ischemia and reperfusion in
the cat. Circulation. 1990;82:1402-1412.
53.
Lefer AM, Ma X-L. Decreased basal nitric oxide
release in hypercholesterolemia increases
neutrophil adherence to rabbit coronary artery
endothelium.
Arteriosclerosis Thromb. 1993;13:771-776.
54.
Weyrich AS, Xin-Liang M, Lefer AM. The role of
L-arginine in ameliorating reperfusion injury after
myocardial ischemia in the cat.
Circulation. 1992;86:279-288.
55.
Morikawa E, Huang Z, Moskowitz MA.
L-Arginine decreases infarct size caused by middle
cerebral arterial occlusion in SHR. Am J
Physiol. 1992;263:H1632-H1635.
56. Zhang F, White JG, Iadecola C. Nitric oxide donors increase blood flow and reduce brain damage in focal ischemia: evidence that nitric oxide is beneficial in the early stages of cerebral ischemia. J Cereb Blood Flow Metab. 1994;14:217-226. [Medline] [Order article via Infotrieve]
57. Johnson G, Tsao PS, Lefer AM. Cardioprotective effects of authentic nitric oxide in myocardial ischemia with reperfusion. Crit Care Med. 1990;19:244-252.
58.
Kubes P. Ischemia/reperfusion in the
feline small intestine: a role for nitric oxide. Am J
Physiol. 1993;264:G143-G149.
59.
Payne D, Kubes P. Nitric oxide donors reduce the
rise in reperfusion-induced intestinal mucosal
permeability. Am J Physiol. 1993;265:G189-G195.
60. Busse W. Mechanisms of inflammation and their therapeutic implications in the asthmatic patient. Ann Allergy. 1992;69:261-266. [Medline] [Order article via Infotrieve]
61. Brody I. Dermal and epidermal involvement in the evolution of acute eruptive guttate psoriasis vulgaris. J Invest Dermatol. 1984;82:465-470. [Medline] [Order article via Infotrieve]
62. Zhang Y, Ramos BF, Jakschik BA. Augmentation of reverse arthus reaction by mast cells in mice. J Clin Invest. 1991;88:841-846.
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