Nitric Oxide Synthase Inhibitors Have
Opposite Effects on Acute Inflammation
Depending on Their Route of Administration
This information is current as
of June 16, 2017.
Mark J. Paul-Clark, Derek W. Gilroy, Dean Willis, Derek A.
Willoughby and Annette Tomlinson
J Immunol 2001; 166:1169-1177; ;
doi: 10.4049/jimmunol.166.2.1169
http://www.jimmunol.org/content/166/2/1169
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References
Nitric Oxide Synthase Inhibitors Have Opposite Effects on
Acute Inflammation Depending on Their Route of
Administration1
Mark J. Paul-Clark, Derek W. Gilroy,2 Dean Willis, Derek A. Willoughby, and
Annette Tomlinson
T
he signaling molecule NO is produced by the enzyme NO
synthase (NOS)3 after activation. NOS is constitutively
expressed in endothelial (ecNOS) and neuronal cells
(neuronal NOS), while a third isoform (iNOS) is induced in response to inflammatory-like stimuli and is capable of sustained
production of high levels of NO that is predominant in inflammation (for review, see Ref. 1). Although the cytostatic/cytocidal activity of NO forms part of the host defense mechanism, the excessive or inappropriate production of NO can lead to tissue damage
possibly through the formation of the potent oxidizing and nitrating agent, peroxynitrite, by a coupling of NO with superoxide
(O2⫺) (2).
NO generated from ecNOS has been reported to have both proand anti-inflammatory properties. Under physiological conditions,
NO released from the endothelium regulates vascular tone and
maintains vessel patency by helping prevent platelet aggregation
Department of Experimental Pathology, The William Harvey Research Institute, St.
Bartholomew’s and the Royal London School of Medicine and Dentistry, London,
United Kingdom
Received for publication June 28, 2000. Accepted for publication October 11, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
The work presented in this paper was supported by the Arthritis Research Campaign, United Kingdom.
2
Address correspondence and reprint requests to Dr. Derek W. Gilroy, Department of
Experimental Pathology, The William Harvey Research Institute, St. Bartholomew’s
and the Royal London School of Medicine and Dentistry, Charterhouse Square, London, EC1 M 6BQ, U.K. E-mail address: [email protected]
3
Abbreviations used in this paper: NOS, NO synthase; AE-ITU, S-(2-aminoethyl)
isothiourea; CINC, cytokine-induced neutrophil chemoattractant; CMP 48/80, compound 48/80; COX, cyclooxygenase; DPTA NoNoate, 3,3⬘-[hydroxynitrosohydrazino]bis-l-propanamine; ecNOS, endothelial cell NOS; iNOS, inducible NOS; L-NIO,
5
G
L-N (1-iminoethyl)-ornithine; L-NMMA, N -monomethyl-L-arginine; LTB4, leukotriene B4; O2⫺, superoxide; PMN, polymorphonuclear cell; SSA, superoxide-scavenging activity; TAOS, total antioxidant status; 1400W, N-(3-(aminomethyl)-benzyl)
acetamidine.
Copyright © 2001 by The American Association of Immunologists
and down-regulating adhesion molecule expression (3). However,
mediators released during the acute phase of inflammation, including histamine, 5-hydroxytryptamine, bradykinin, platelet-activating factor, and substance P, evoke the release of endothelial NO,
causing vasodilatation and vascular permeability, thus facilitating
edema formation and trafficking of inflammatory cells (4).
L-arginine analogues are the pharmacological agents most commonly used to inhibit NO production, but have poor selectivity
between NOS isoforms, thereby inhibiting iNOS and ecNOS during inflammatory insult, the latter resulting in a reduction of basal
blood flow. In the majority of experiments, L-arginine analogues
were given systemically, resulting in an amelioration of inflammation (5–7), but in some cases these anti-inflammatory actions
were reversed by vasodilators (8, 9). Therefore, we suspect that
NOS inhibitors, administered systemically, will inhibit ecNOS, resulting in vasoconstriction, followed by a reduction in blood delivery to the inflamed site. This sequence of events may ultimately
lead to reduced cellular diapedesis and exudation and thus be interpreted as anti-inflammatory.
To test the hypothesis that NOS inhibitors will have different
effects on inflammation depending on their route of administration,
we used the carrageenin-induced pleurisy. This is a well-characterized model of acute inflammation in which iNOS activity, protein expression, and nitrite production peak between 1 and 6 h after
carrageenin injection (10). The present study was designed to investigate the effects on inflammation of NOS inhibitors administered locally to the inflammatory site and to compare their effects
with NOS inhibitors given systemically. To this end, we used S-(2aminoethyl) isothiourea (AE-ITU) (11) and N-(3-(aminomethyl)benzyl) acetamidine (1400W) (12), which are structurally different
and the most selective iNOS inhibitors commercially available, as
well as the more selective ecNOS inhibitor, L-N5(1-iminoethyl)ornithine (L-NIO) (13). These drugs were administered into the
pleural or peritoneal cavity immediately before the establishment
0022-1767/01/$02.00
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The bulk of published data has shown that NO is proinflammatory. However, there also exists the conflicting notion that NO may
be protective during an inflammatory insult. In an attempt to resolve this issue, we have compared the effects on inflammation of
a range of NO synthase (NOS) inhibitors given either directly to the site of the inflammatory lesion or systemically. It was found
that in the carrageenin-induced pleurisy, a single intrapleural injection of the selective inducible NO inhibitors S-(2-aminoethyl)
isothiourea (AE-ITU; 3 and 10 mg/kg) and N-(3-(aminomethyl)-benzyl) acetamidine (1400W; 10 mg/kg) or the selective endothelial
cell NOS inhibitor L-N5(1-iminoethyl)-ornithine (10 mg/kg) not only exacerbated inflammation at the very early stages of the lesion
(1– 6 h), but also prevented inflammatory resolution. By contrast, administering NOS inhibitors systemically ameliorated the
severity of inflammation throughout the reaction. To elucidate the mechanisms by which inhibition of NO synthesis locally
worsened inflammation, we found an increase in histamine, cytokine-induced neutrophil chemoattractant, superoxide, and leukotriene B4 levels at the inflammatory site. In conclusion, this work shows that the local production of NO is protective by virtue
of its ability to regulate the release of typical proinflammatory mediators and, importantly, that NOS inhibitors have differential
anti-inflammatory effects depending on their route of administration. The Journal of Immunology, 2001, 166: 1169 –1177.
1170
NO INHIBITION IN ACUTE INFLAMMATION
of a carrageen-induced pleurisy. Edema formation, infiltrating inflammatory cell numbers, and nitrite levels in exudates were measured at various times throughout the inflammatory process.
It was found that NOS inhibitors administered locally exacerbated inflammation and prolonged resolution as a result of an increase in the proinflammatory mediators histamine, leukotriene B4
(LTB4), O2⫺, and cytokine-induced neutrophil chemoattractant
(CINC), suggesting that the local production of NO is protective.
By contrast, however, giving NOS inhibitors systemically ameliorated inflammation, thereby showing differential anti-inflammatory
properties of NOS inhibitors depending on their route of
administration.
Materials and Methods
Induction of carrageenin-induced pleurisy and measurement of
inflammatory parameters
Drug administration
Animals (n ⫽ 6 – 8 per group) were injected intrapleurally with either 3,3⬘hydroxynitrosohydrazino]bis-1-propanamine (DPTA) NoNoate, AE-ITU,
1400W, L-NIO, or saline immediately before intrapleural injection of carrageenin. For experiments examining the systemic effects of NOS inhibitors, AE-ITU or NG-monomethyl-L-arginine (L-NMMA) was injected into
the peritoneal cavity immediately before carrageenin injection into the
pleural cavity.
Mast cell depletion
Rats were depleted of mast cells using compound 48/80 CMP 48/80), as
previously described (14). Briefly, CMP 48/80 (0.1% w/v) was injected i.p.
twice daily for 4 days (0.6 mg/kg for the first six injections and 1.2 mg/kg
for the final two). Carrageenin-induced pleurisy was induced 6 h after the
last injection of CMP 48/80.
Western blot analysis
Cell pellets were sonicated on ice in protease-inhibitory buffer (0.05 M
Tris, 1 mM PMSF, 1.5 mM pepstatin A, 0.2 mM leupeptin) and centrifuged
(4000 ⫻ g, 5 min, 4°C). Protein concentrations of the supernatants were
determined by the Bradford assay. Samples were equilibrated for protein (1
mg/ml), mixed 1:1 with 2⫻ Laemmli buffer (125 mM Tris base, 2 mM
EDTA, 10% mercaptoethanol, 4% SDS, 20% glycerol, and 0.1% Coomassie brilliant blue, pH 6.8), and boiled for 5 min. Samples (30 ␮g/lane) and
m.w. color markers were resolved by gel electrophoresis on 7.5% SDSpolyacrylamide gels and transferred onto nitrocellulose membranes. iNOS
protein was detected using a specific polyclonal rabbit anti-mouse Ab (1:
5000), which does not cross-react with either ecNOS or neuronal NOS
(15). The signal was amplified with a HRP-linked goat anti-rabbit polyclonal Ab and visualized on x-ray film using ECL chemiluminescence
reagents.
iNOS activity in inflammatory cells
iNOS activity was measured as the ability of inflammatory cells to catalytically convert [3H]L-arginine to [3H]L-citrulline. The reaction mixture
consisted of NADPH (1 mM), tetrahydrobiopterin (5 ␮M), calmodulin
(300 U/ml), EGTA (1 mM), and valine (1 mM). Samples were homogenized and added to each reaction tube. The reaction was initiated by the
addition of 10 ␮l of L-arginine/[3H]L-arginine at a final concentration of 10
␮M and at a ratio of 1000:3 pmol of L-arginine:[3H]L-arginine. Samples
were incubated at 37°C for 30 min, and the reaction was terminated by the
addition of ice-cold stop buffer (2 mM EGTA, 2 mM EDTA in 20 mM
HEPES, pH 5.5). Samples were eluted through Dowex cationic exchange
resin columns with stop buffer and collected in scintillation vials. Scintil-
FIGURE 1. Time course of nitrite formation, iNOS enzyme activity,
and iNOS protein expression in the carrageenin-induced pleurisy. a, Nitrite
accumulation (f) in the cell-free inflammatory exudate was measured by
the Greiss reaction, while iNOS enzyme activity (〫) was determined by
the catalytic conversion of [3H]L-arginine to [3H]L-citrulline by inflammatory cells. b, Inflammatory cell iNOS protein expression was determined by
Western blotting, with densitometric analysis shown beneath a representative blot. Data are expressed as mean ⫾ SEM (n ⫽ 6 – 8 per time point).
lation fluid was added to each vial, and the amount of radioactive L-citrulline was determined in a liquid scintillation counter. The protein concentration of samples was measured and NOS activity expressed as pmol
L-citrulline/mg protein/30 min.
Cyclooxygenase (COX) activity in inflammatory cells
Cell pellets were sonicated on ice in protease-inhibitory buffer and incubated (37°C, 30 min) in the presence of arachidonic acid (30 ␮M), glutathione (5 mM), and adrenalin (5 mM). The reaction was terminated by
boiling, and samples were then centrifuged (10,000 ⫻ g, 30 min). PGE2 in
the supernatant was measured by RIA, and the results were expressed as ng
PGE2/mg protein/30 min.
Measurement of PGE2[infi], LTB4, 6-keto PGF1␣, histamine, and
CINC in cell-free exudates
PGE2 and LTB4 were measured by enzyme immunoassay and 6-keto
PGF1␣ (as the stable breakdown product of PGI2) by RIA. Results were
expressed as quantity (pg) of each eicosanoid in the total volume of cellfree exudate. Histamine levels were measured by RIA and results expressed as total histamine (ng) in the total volume of exudate. CINC levels
were measured using a mouse KC ELISA that had cross-reactivity with rat
rCINC-1 protein, and results expressed as total amount of CINC (pg) in
total volume of exudate.
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Male Wistar rats (180 ⫾ 20 g; T. Tuck and Sons, Batties Bridge, U.K.)
were housed with a 12-h light-dark cycle and allowed access to food and
water ad libitum. Animals were anesthetized with halothane and injected
with 0.15 ml, 1% carrageenin in saline into the pleural cavity. Pleural
exudates were collected with or without lavage (0.5 ml of 3.15% tri-sodium
citrate in saline) at various time points postcarrageenin injection. Bloodcontaminated exudates were rejected. Exudate volumes were quantified
and inflammatory cells counted (Dn coulter-counter; Coulter Electronics,
Luton, U.K.). Samples were centrifuged (800 ⫻ g, 10 min, 4°C) to separate
inflammatory cells from exudate, both of which were then stored at ⫺70°C
for subsequent procedures.
The Journal of Immunology
1171
FIGURE 3. Effects of NOS inhibitors injected locally on a rat carrageenin-induced pleurisy at 6 h. AE-ITU, 1400W, or L-NIO was injected
directly into the pleural cavity of rats immediately before carrageenin injection. Thereafter, their effects on exudate volume (a), cell number (b),
and nitrite levels (c) in the pleural exudates were determined 6 h after
carrageenin injection. Data are expressed as mean ⫾ SEM (n ⫽ 8 per
group). ⴱ, p ⱕ 0.05 in comparison with saline controls.
Nitrite levels in cell-free exudates
96-well plate. The reaction was initiated by the addition of H2O2 and conducted at 37°C. The increase in absorbance at 405 nm, a reflection of
2,2⬘-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical cation
(ABTS⫹) accumulation, was determined using an Anthos Labtech type
plate reader (Austria). Experiments were conducted in triplicate on both
cell-free inflammatory exudate as well as inflammatory cells. For the latter
determinant, values were adjusted for protein concentration.
Nitrite formation was measured by a modification of the Greiss reaction.
Briefly, 10 ␮l of NADPH (10 ␮M), glucose-6-phosphate (5 mM), glucose6-phosphate dehydrogenase (0.16 U), and PBS (10 mM) were added to
cell-free exudate in a 96-well plate. Nitrate was converted to nitrite by
addition of 10 ␮l (0.08 U) nitrate reductase and incubated for 45 min. A
total of 200 ␮l of Greiss reagent (equal volumes of 1% w/v sulfanilamide
in 5% H3PO4 and 0.1% w/v N(1-napthyl)ethylenediamine) was added and
incubated for an additional 10 min. Nitrite concentrations were determined
at 570 nm with a reference filter at 620 nm and results expressed as ␮M
nitrite in cell-free exudate.
Total antioxidant status in cells and cell-free exudate
Exudate and plasma total antioxidant status (TAOS) was measured as previously described (16). Briefly, the reaction mixture consisted of (final
concentration): 20 ␮l 2,2⬘-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)
(ABTS; 2 mM), 10 ␮l HRP (30 mU/ml), 20 ␮l H2O2 (0.1 mM), and 40 ␮l
PBS (pH 7.4), and 10 ␮l sample to make a total volume of 100 ␮l in a
Superoxide-scavenging activity in cells and cell-free exudate
Superoxide-scavenging activity (SSA) was measured as previously described (17). Briefly, the reaction mixture consisted of (final concentration): 50 ␮l ferricyctochrome c (50 ␮M), 10 ␮l xanthine oxidase (20 mU/
ml), 20 ␮l hypoxanthine (100 ␮M), and 5–10 ␮l sample with the volume
being made up to 100 ␮l with PBS (pH 7.4). The reaction was initiated by
the addition of hypoxanthine and conducted at 37°C. The increase in OD
at 550 nm was measured over a 3-min period at 30-s intervals using an
Anthos Labtech type plate reader (Salzburg, Austria). All determinants of
initial reaction rates were made with and without sample at least in
triplicate.
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FIGURE 2. -Effects of NOS inhibitors injected locally on inflammation
at 1 h. Either AE-ITU, 1400W, or L-NIO was injected directly into the
pleural cavity of rats immediately before carrageenin injection. Thereafter,
their effects on exudate volume (a), cell number (b), and nitrite (c) in
pleural exudates were determined 1 h after carrageenin injection. Data are
expressed as mean ⫾ SEM (n ⫽ 6 – 8 per group). ⴱ, p ⱕ 0.05 in comparison
with saline controls.
1172
MTT assay
Cell viability was assessed as dehydrogenase activity, measured as the
dehydrogenation of MTT to form the formazan product. A total of 50 ␮l of
MTT was added to make 0.5 mg/ml in each well and incubated until the
control lanes were developed (20 min). The cells were then washed twice
in Dulbecco’s PBS A, the formazan product was dissolved by adding 100
␮l DMSO to each well, and the plates were read at 570 nm. All samples
were measured in duplicate.
Exudate cell smears
Exudate smears (n ⫽ 4 per group, 6 h) were stained with Harris’ hematoxylin and eosin, and differential cell counts were performed in drugtreated and control animals. Mast cell numbers were assessed in drugtreated and control animals at 1 h in exudate smears stained with
toluidine blue.
Materials
Carrageenin (Viscarin) was obtained from FMC (Rockland, ME); LTB4,
PGE2 enzyme immunoassay kits, [3H]PGE2, [3H]6-keto PGF1␣, and [3H]Larginine were purchased from Amersham International (Buckinghamshire,
U.K.). Mouse KC Quantikine immunoassay kit was obtained from R&D
Systems (Oxon, U.K.). AE-ITU, 1400W, and L-NIO were purchased from
Alexis (Nottingham, U.K). iNOS Ab was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were obtained from
Sigma (Poole, U.K.).
Data analysis
Results are expressed as the mean ⫾ SEM. Statistical analysis was performed using a one-way Kruskal-Wallis nonparametric test, followed by a
Mann-Whitney U test. A p value of ⱕ0.05 was considered to be statistically significant.
Results
Profile of iNOS expression and NO production in the
carrageenin-induced pleurisy
In this model, polymorphonuclear cells (PMNs) are the principal
cell type up to 12 h after carrageenin injection and are replaced by
FIGURE 5. Effects of early NOS inhibition on inflammatory resolution
in the rat carrageenin-induced pleurisy. AE-ITU was injected directly into
the pleural cavity of rats immediately before carrageenin injection. Seventy-two hours later, at resolution, the consequence of early NOS inhibition was examined on exudate volume (a) and cell number (b). Data are
expressed as mean ⫾ SEM (n ⫽ 6 per group). ⴱ, p ⱕ 0.05 in comparison
with saline controls.
phagocytosing mononuclear cells that predominate up to resolution at 48 h (18). NO, as determined by measuring nitrite in the
cell-free inflammatory exudate, was initially high 0.5–1 h after
carrageenin injection (Fig. 1a). Thereafter, NO levels declined but
increased again at 6 h in parallel with inflammatory cell iNOS
enzyme activity. Neither NO nor iNOS activity was detectable
during resolution at 48 h. In the inflammatory cells from the pleural cavity, there was a peak in iNOS protein expression at 6 h,
mirroring iNOS activity (Fig. 1b), which gradually declined but
increased again at inflammatory resolution at 48 h. This finding is
similar to the recent report that COX 2 is also expressed in inflammatory mononuclear cells at resolution in this model (18), and
further studies are being conducted to examine the relevance of
this apparently enzymatically inactive iNOS during resolution.
Effects of administering NOS inhibitors into the pleural
cavity at 1 h
As the initial peak in exudate nitrite levels was detectable at 1 h,
this would suggest that the principal source of NO at this time was
from the postcapillary endothelial cells lining the pleural cavity
and not from an inducible source. For our first experiments, to
determine the contribution of NO to the developing inflammatory
response, we investigated the effects on inflammation of administering NOS inhibitors directly into the pleural cavity immediately
before carrageenin injection. We found that AE-ITU at 3 and 10
mg/kg significantly increased exudate volume and inflammatory
cell influx compared with untreated controls (Fig. 2, a and b).
1400W also significantly elevated exudate volume, but was without effect on cell numbers (Fig. 2, a and b). Both inhibitors reduced
nitrite levels significantly at the higher dose used (Fig. 2c). Given
their lack of selectivity, it is likely that AE-ITU and 1400W also
inhibited ecNOS at the dosing levels used. Finally, in addition to
reducing exudate nitrite levels, L-NIO (10 mg/kg) increased exudate volume compared with controls, whereas inflammatory cell
numbers were unchanged (Fig. 2a– c).
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FIGURE 4. Effects on pleural inflammation of administering an NO donor locally. DPTA NoNoate was injected directly into the pleural cavity of
rats immediately before carrageenin. Six hours after carrageenin injection,
the effects of this slow releasing NO donor were examined on exudate
volume (a) and cell number (b) in pleural exudates. Data are expressed as
mean ⫾ SEM (n ⫽ 8 per group). ⴱ, p ⱕ 0.05 in comparison with saline
controls.
NO INHIBITION IN ACUTE INFLAMMATION
The Journal of Immunology
Effects of administering NOS inhibitors into the pleural
cavity at 6 h
From these studies, it appears that the initial peak of NO generation at 1 h is anti-inflammatory. We therefore proceeded to examine whether the second peak of NO generation, apparently from
iNOS, at 6 h has a similarly protective role. To this end, the effects
of intrapleural injection of AE-ITU, 1400W, and L-NIO immediately before the establishment of a carrageenin pleurisy were determined. These experiments also showed that at doses that inhibited NO generation, NOS inhibitors significantly worsened
inflammation by increasing pleural exudates and leukocyte influx
into the pleural cavity in comparison with saline controls (Fig. 3,
a– c). By contrast, in a parallel experiment, it was found that a
slow-releasing NO donor (DPTA NoNoate), administered locally
at the time of carrageenin injection, reduced inflammatory cell
influx (Fig. 4).
We then questioned whether this exacerbated inflammation,
brought about as a consequence of NOS inhibition, represented a
transient increase at this early phase or whether in fact it resulted
in an eventual delay in inflammatory resolution. To discern this,
AE-ITU was administered into the pleural cavity immediately before carrageenin injection (as above), and its effects on inflammation were determined at 72 h, a time point at which this model is
usually resolved. It was found that pleural exudates and inflammatory cell numbers were significantly increased in AE-ITUtreated animals when compared with controls (Fig. 5). Thus, from
these experiments it appears that NO plays a critical role in determining the outcome of an inflammatory response.
FIGURE 7. Effects of NOS inhibitors injected systemically on a rat carrageenin-induced pleurisy at 36 h. Either AE-ITU or L-NMMA was injected into the peritoneal cavity just before intrapleural carrageenin injection. Thirty-six hours later, the effects of these NOS inhibitors were
determined on exudate volume (a), cell number (b), and nitrite (c) in pleural exudates. Data are expressed as mean ⫾ SEM (n ⫽ 8 per group). ⴱ, p ⱕ
0.05 in comparison with saline controls.
Effects of NOS inhibitors on carrageenin-induced pleurisy when
administered systemically
The fundamental question addressed in this study is whether there
is a differential effect on inflammation by administering NOS inhibitors locally vs systemically. To determine this, we examined
the effects on inflammation at 6 and 36 h of AE-ITU and L-NMMA
when injected into the peritoneal cavity at the time of carrageenin
injection. In keeping with the bulk of data published on the systemic effects of NOS inhibitors on inflammation, both drugs tested
reduced inflammation at 6 h (Fig. 6, a and b) at doses that inhibited
inflammatory exudate nitrite levels (Fig. 6c) and at 36 h (Fig. 7, a
and b), in which nitrite was undetectable. In contrast, AE-ITU and
L-NMMA, when injected locally into the pleural cavity immediately before carrageenin injection, increased exudate volume and
inflammatory cell number at 6 h (Fig. 2, a and b) and 36 h (Fig. 8,
a and b). These findings present highly conflicting outcomes on
inflammation depending on whether NOS inhibitors are administered either locally or systemically.
Effect of NOS inhibitors on pleural inflammation in the absence
of carrageenin
To show that AE-ITU (3 mg/kg) and 1400W (10 mg/kg) did not
exacerbate inflammation simply as a result of direct irritation, we
injected these drugs, as well as saline, into the pleural cavity in the
absence of carrageenin. Neither drug caused measurable exudate
formation or an increase in cellular influx (data not shown). Moreover, cationic compounds, including NOS inhibitors, may cause
nonspecific mast cell degranulation. Therefore, NG-nitro-D-arginine-methyl ester, the inactive enantiomer of the nonspecific NOS
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FIGURE 6. Effects of NOS inhibitors injected systemically on a rat carrageenin-induced pleurisy at 6 h. Either AE-ITU or L-NMMA was injected
into the peritoneal cavity just before intrapleural carrageenin injection. Six
hours later, the effects of these NOS inhibitors were determined on exudate
volume (a), cell number (b), and nitrite (c) in pleural exudates. Data are
expressed as mean ⫾ SEM (n ⫽ 8 per group). ⴱ, p ⱕ 0.05 in comparison
with saline controls.
1173
1174
inhibitor L-NAME, was injected intrapleurally at the same molarity
as AE-ITU (10 mg/kg) and caused no increase in inflammatory
parameters (data not shown). As a final control experiment, using
the MTT assay for the assessment of cell viability, we found that
neither AE-ITU nor L-NMMA, when injected intrapleurally,
caused toxicity to influxing inflammatory cells in the pleural cavity
(data not shown).
Mechanisms by which NOS inhibition may exacerbate
inflammation
In an attempt to elucidate the mechanisms by which inhibition of
NO synthesis worsens inflammation, we measured levels of the
most likely proinflammatory mediators after treatment with AEITU. The first candidate for investigation, histamine, was measured in cell-free exudates at 1 h, and levels were correlated with
mast cell numbers (the cellular source of histamine) in pleural
exudates. AE-ITU (10 mg/kg) significantly increased histamine
(Fig. 9a), while reducing mast cell numbers by 72%, presumably
as a consequence of increased cellular degranulation. Moreover, in
animals previously depleted of mast cells using CMP 48/80, the
increase in exudate volume observed at 1 h after treatment with
AE-ITU was partially attenuated (compare Fig. 2a with 9b) at
dosing levels that significantly reduced exudate nitrite levels (Fig.
9d). Although there was an expected reduction in exudate formation in animals treated with CMP 48/80 alone (Fig. 9b), there was
no change in cell numbers (Fig. 9c). This observation is consistent
with the fact that histamine mediates edema formation and not cell
migration.
Another candidate for investigation was CINC, a potent neutrophil chemoattractant (19). Although in control animals levels
FIGURE 9. Effects of local NOS inhibition on histamine levels in the
rat carrageenin-induced pleurisy. AE-ITU was injected into the pleural
cavity immediately before carrageenin injection. One hour later, a, histamine levels were determined in cell-free exudates. In addition, the effects
of intrapleural injection of AE-ITU on exudate volume (b), cell number (c),
and nitrite production (d) were ascertained in the pleural cavity 1 h after
depletion of mast cells using CMP 48/80. Data are expressed as mean ⫾
SEM (n ⫽ 8 per group). ⴱ, p ⱕ 0.05 in comparison with saline controls.
peaked at 1 h (Fig. 10a), the intrapleural injection of AE-ITU (3,
10 mg/kg) caused a significant increase in CINC in cell-free pleural exudates (Fig. 10b). An additional explanation for exacerbated
inflammation after NOS inhibition could be enhanced eicosanoid
synthesis, particularly as NO has been suggested to modulate COX
activity (for review, see Ref. 20). Thus, given the proinflammatory
properties of eicosanoids and their abundance in the carrageenininduced pleurisy up to 6 h (10), we examined the effects of NOS
inhibition on inflammatory cell COX activity as well as cell-free
exudate levels of PGE2, 6-keto PGF1␣ (Table I), and the neutrophil
chemoattractant, LTB4. Surprisingly, AE-ITU (3 mg/kg) had no
effect on either COX activity in inflammatory cell pellets or levels
of PGE2 and 6-keto PGF1␣. Interestingly, however, exudate levels
of LTB4 were significantly increased in comparison with controls
(Table I).
Effects of NOS inhibition on TAOS and SSA in inflammatory
cells and cell-free exudates
In a series of final experiments, TAOS and SSA activity were
measured as an indirect indication of O2⫺ and other oxidant species formation. These assays are based on the principle that oxidants formed during inflammation (21) react with antioxidants
such as glutathione, ascorbic acid, and ␣-tocopherol and reduce the
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FIGURE 8. Effects of NOS inhibitors injected locally on a rat carrageenin-induced pleurisy at 36 h. Either AE-ITU or L-NMMA was injected
into the pleural cavity of rats before carrageenin administration. Thirty-six
hours later, their effects on exudate volume (a) and cell number (b) in
pleural exudates were determined. Data are expressed as mean ⫾ SEM
(n ⫽ 8 per group). ⴱ, p ⱕ 0.05 in comparison with saline controls.
NO INHIBITION IN ACUTE INFLAMMATION
The Journal of Immunology
1175
Table I. Effects of local NOS inhibition on eicosanoid production in the
rat carrageenin-induced pleurisy at 6 ha
COX activity (ng/mg protein/30 min)
PGE2 (pg)
6-ketoPGF1a (pg)
LTB4 (pg)
Saline
AE-ITU
4.4 ⫾ 1.4
637 ⫾ 87
1631 ⫾ 364
64 ⫾ 7
5.8 ⫾ 1
852 ⫾ 77
1483 ⫾ 328
91 ⫾ 10ⴱ
a
AE-ITU (3 mg/kg) was injected into the pleural cavity of rats immediately prior
to carrageenin injection. Six hours later, levels of eicosanoids were determined in the
cell-free inflammatory exudate. Data are expressed as mean ⫾ SEM (n ⫽ 6 – 8 per
group). ⴱ, p ⱕ 0.05 in comparison to saline controls.
inflammatory exudates antioxidant capacity (22). These two parameters may provide additional support for the concept that during inflammation NO may indeed have a protective role. Administered immediately before the induction of inflammation, AE-ITU
(3, 10 mg/kg) significantly reduced both TAOS (Table II) and SSA
(Table III) in cells and cell-free exudates at 1 and 6 h in comparison with saline controls.
Discussion
In this study, we demonstrate that in acute inflammation the administration of NOS inhibitors directly into the inflamed site exacerbated the inflammatory response and prolonged resolution. Investigating the mechanisms that may account for this worsened
inflammatory response, it was found that by inhibiting NO generation there was a corresponding increase in histamine, LTB4,
CINC, and reactive oxygen species, suggesting that during inflammation the local production of NO is protective by virtue of its
ability to modulate levels of these proinflammatory mediators. By
contrast, however, we found that administrating NOS inhibitors
systemically reduced inflammation, thereby demonstrating that
NOS inhibitors have differential antiinflammatory effects depending on their route of administration.
Table II. Effect of NOS inhibition on TAOS of inflammatory cells and cell-free exudates in the carrageenin-induced pleurisy at 1 h and 6 ha
Cell (␮M L-ascorbate equivalent antioxidant
capacity/mg protein)
Control
AE-ITU (3 mg/kg)
AE-ITU (10 mg/kg)
Exudate (␮M L-ascorbate equivalent antioxidant
capacity)
1h
6h
1h
6h
1997 ⫾ 366
288 ⫾ 73ⴱ
578 ⫾ 86ⴱ
466 ⫾ 57
73 ⫾ 12ⴱ
169 ⫾ 32ⴱ
89.8 ⫾ 5.2
64.4 ⫾ 2.2ⴱ
56.2 ⫾ 2.1ⴱ
100.1 ⫾ 2.0
65.3 ⫾ 1.7ⴱ
76.1 ⫾ 2.2ⴱ
a
, AE-ITU was injected into the pleural cavity of rats immediately prior to carrageenin injection. One and 6 h later, total antioxidant status was determined in both
inflammatory cells and cell-free inflammatory exudate. Data are expressed as mean ⫾ SEM (n ⫽ 8 –10 per group). ⴱ, p ⱕ 0.05 in comparison to saline controls.
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FIGURE 10. Effects of local NOS inhibition on levels of CINC in the
rat carrageenin-induced pleurisy. The profile of CINC was determined in
the cell-free inflammatory exudate by ELISA (a). Thereafter, the effects of
AE-ITU, injected into the pleural cavity immediately before carrageenin
injection, were examined on CINC levels 1 h after carrageenin injection
(b). Data are expressed as mean ⫾ SEM (n ⫽ 6 – 8 per group). ⴱ, p ⱕ 0.05
in comparison with saline controls.
The vast majority of reports have shown that NOS inhibitors are
considered anti-inflammatory in models of both acute and chronic
inflammation. In a model of trinitrobenzene sulfonic acid-induced
colitis in rats, L-NAME reduced neutrophil and macrophage influx
(23), while in a carrageenin-induced pleurisy, L-NMMA also significantly reduced exudate formation and cellular influx (6). In
these studies, however, NOS inhibitors were administered at high
doses away from the site of inflammation, i.e., orally or i.p. Given
their lack of selectivity, it is conceivable that systemic administration may also inhibit ecNOS remote from the inflammatory locus, resulting in vasoconstriction, reduced blood delivery to the
inflamed site, and, thus, a reduction in inflammation. Indeed, it was
shown that the anti-inflammatory effects of L-arginine analogues,
given systemically, could be reversed by vasodilators (8, 9, 24). In
agreement with these studies, we also found that when NOS inhibitors were injected i.p., immediately before intrapleural injection of carrageenin, pleural exudates were significantly reduced.
Therefore, to bypass the systemic effects of NOS inhibitors, we
administered AE-ITU, 1400W, and L-NIO locally and found them
to exert a proinflammatory effect, without altering inflammatory
cell viability. In support of our findings, others have also reported
a protective role for NO. For instance, acetic acid-induced colitis
in iNOS-deficient mice resulted in increased PMN-associated tissue damage in comparison with wild-type animals (25). A similar
inflammatory cell accumulation in hepatic microvasculature was
also observed in LPS-treated iNOS knockout mice (26). Thus, the
protective effects of NO shown in these genetically modified animals may arise from the absence of only iNOS at the inflammatory
site with ecNOS being functionally active, thereby avoiding systemic perturbations.
To elucidate the mechanism by which inhibition of NO generation exacerbates inflammation, we measured levels of a number
of candidate proinflammatory mediators. As shown by others (27),
mast cell-derived histamine peaked in this model between 0.5 and
1 h after carrageenin injection. Given that histamine mediates
edema formation (27) and that NO stabilizes mast cells, thereby
preventing histamine release (28), levels of this acute inflammatory mediator were measured after NOS inhibition. We found that
1176
NO INHIBITION IN ACUTE INFLAMMATION
Table III. Effect of NOS inhibition on SSA of inflammatory cells and cell-free exudates in the carrageenin-induced pleurisy at 1 h and 6 ha
Cells (Change in rate/mg protein)
Control
AE-ITU (3 mg/kg)
AE-ITU (10 mg/kg)
Exudate (change in rate)
1h
6h
1h
6h
0.42 ⫾ 0.07
0.18 ⫾ 0.02ⴱ
0.21 ⫾ 0.02ⴱ
0.16 ⫾ 0.02
0.03 ⫾ 0.02ⴱ
0.08 ⫾ 0.01ⴱ
0.011 ⫾ 0.001
0.005 ⫾ 0.001ⴱ
0.003 ⫾ 0.001ⴱ
0.010 ⫾ 0.001
0.003 ⫾ 0.001ⴱ
0.005 ⫾ 0.001ⴱ
a
AE-ITU was injected into the pleural cavity of rats immediately prior to carrageenin injection. One and 6 h later, superoxide scavenging ability was determined in both
inflammatory cells and cell-free inflammatory exudate. Data are expressed as mean ⫾ SEM (n ⫽ 8 –10 per group). ⴱ, p ⱕ 0.05 in comparison to saline controls.
regulation of CINC has been demonstrated, an O2⫺ scavenger
N-acetylcysteine significantly reduced NF-␬B DNA binding and
CINC mRNA expression in inflamed lungs (42). Therefore, a disturbance in the balance between NO and O2⫺ production may lead
to an increase in proinflammatory mediators and provides a possible mechanism for the exacerbation of inflammation observed in
this study.
In conclusion, in the rat carrageenin-induced pleurisy, inhibition
of NO at the inflammatory site exacerbates inflammation and prolongs the pathology, suggesting a protective role for NO in this
model. Moreover, NOS inhibitors appear to have differential effects on inflammation depending on their route of administration.
Acknowledgments
We thank P. T. Gunnarsson and M. J. Carrier for help with TAOS and SSA
assays.
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observed with NOS inhibitors without altering the increase in inflammatory cells. In support of this observation, others have shown
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