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* Institute of Pharmacology,
Department of Biomorphology, School of Medicine, and
Institute of General Surgery, University of Messina, Italy
1Correspondence: Institute of Pharmacology, School of Medicine, University of Messina, Torre Biologica-Policlinico Universitario Via C. Valeria-Gazzi, 98100 Messina, Italy. E-mail: salvator{at}www.unime.it
ABSTRACT
Oxidative stress has been suggested as a potential mechanism in the
pathogenesis of lung inflammation. The pharmacological profile of
n-acetylcysteine (NAC), a free radical scavenger, was
evaluated in an experimental model of lung injury (carrageenan-induced
pleurisy). Injection of carrageenan into the pleural cavity of rats
elicited an acute inflammatory response characterized by fluid
accumulation in the pleural cavity that contained many neutrophils
(PMNs), an infiltration of PMNs in lung tissues and subsequent lipid
peroxidation, and increased production of nitrite/nitrate, tumor
necrosis factor 
, and interleukin 1ß. All parameters of
inflammation were attenuated by NAC treatment. Furthermore, carrageenan
induced an up-regulation of the adhesion molecules ICAM-1 and
P-selectin, as well as nitrotyrosine and poly (ADP-ribose) synthetase
(PARS), as determined by immunohistochemical analysis of lung tissues.
The degree of staining for the ICAM-1, P-selectin, nitrotyrosine, and
PARS was reduced by NAC. In vivo NAC treatment significantly reduced
peroxynitrite formation as measured by the oxidation of the fluorescent
dihydrorhodamine-123, prevented the appearance of DNA damage, an
decrease in mitochondrial respiration, and partially restored the
cellular level of NAD+ in ex vivo macrophages harvested from the
pleural cavity of rats subjected to carrageenan-induced pleurisy. A
significant alteration in the morphology of red blood cells was
observed 24 h after carrageenan administration. NAC treatment has
the ability to significantly diminish the red blood cell alteration.
Our results clearly demonstrate that NAC treatment exerts a protective
effect and clearly indicate that NAC offers a novel therapeutic
approach for the management of lung injury where radicals have been
postulated to play a role.—Cuzzocrea, S., Mazzon, E., Dugo, L.,
Serraino, I., Ciccolo, A., Centorrino, T., De Sarro, A., Caputi,
A. P. Protective effects of n-acetylcysteine on
lung injury and red blood cell modification induced by carrageenan in
the rat.
Key Words: inflammation • nitric oxide • peroxynitrite • n-acetylcysteine • superoxide
OXIDATIVE STRESS RESULTS from an oxidant/antioxidant
imbalance, an excess of oxidants, and/or a depletion of antioxidants.
Oxidative stress is thought to play an important role in the
pathogenesis of lung disease not only through direct injurious effects,
but also by involvement in the molecular mechanisms that control lung
inflammation. Studies have shown an increased oxidant burden and
increased markers of oxidative stress in the air spaces, breath, blood,
and urine in smokers and in patients with pulmonary inflammation. The
presence of oxidative stress has important consequences for the
pathogenesis of lung inflammation. These include oxidative inactivation
of antiproteinases, airspace epithelial injury, increased sequestration
of neutrophils in the pulmonary microvasculature, and gene expression
of proinflammatory mediators. With regard to the latter, oxidative
stress has a role in enhancing the inflammation that occurs in smokers
and patients with lung inflammation through the activation of
redox-sensitive transcriptions factors such as nuclear factor 
B
(NF-B) and activator protein 1, which regulate the genes for
proinflammatory mediators and protective antioxidant gene expression.
Sources of the increased oxidative stress in patients with lung
inflammation are derived from the increased burden of oxidants present
in cigarette smoke or from the increased amounts of reactive oxygen
species released from leukocytes both in the airspace and in the blood.
Antioxidant depletion or deficiency in antioxidants may contribute to
oxidative stress.
The development of airflow limitation is related to dietary deficiency
of antioxidants, hence dietary supplementation may be a beneficial
therapeutic intervention. Antioxidants that have good bioavailability
or molecules with antioxidant enzyme activity may not only protect
against the direct injurious effects of oxidants, but also may
fundamentally alter the inflammatory events that play an important part
in the pathogenesis of lung inflammation. Neutrophil infiltration into
inflamed tissue plays a crucial role in the destruction of foreign
antigens and in the breakdown and remodeling of injured tissue
(1)
.
The first step in the recruitment of neutrophils to the airspace is the
sequestration of these cells in lung microcirculation (2)
.
The sequestration of neutrophils in the pulmonary capillaries allows
time for the neutrophils to interact with the pulmonary capillary
endothelium, resulting in their adherence to the endothelium and then
their transmigration across the alveolar capillary membrane to the
interstitium and airspace of the lungs in response to inflammation or
infection. Neutrophils can be activated while in transit in the
pulmonary microcirculation by mediators, including cytokines released
from resident lung cells, alveolar macrophages, and epithelial and
endothelial cells. Recently it has been demonstrated that several
mechanisms involving oxidants cause neutrophil sequestration in the
pulmonary microcirculation (3
4
5)
.
There has been considerable interest recently in the systemic effects
of lung inflammation. One manifestation of a systemic effect is the
presence of markers of oxidative stress in the blood in patients with
lung inflammation. This is reflected in increased sequestration of
neutrophils in the pulmonary microcirculation during lung inflammation
as an oxidant-mediated event (6
7
8
9)
. Rahman and colleagues
(9)
demonstrated increased production of superoxide anion
from peripheral blood neutrophils obtained from patients with acute
exacerbations of lung inflammation, which returned to normal when the
patients were restudied when clinically stable. Other studies have
shown that circulating neutrophils from patients with lung inflammation
have up-regulated surface adhesion molecules, which may also be an
oxidant-mediated effect (9
10
11)
. Neutrophil activation may
be even more pronounced in neutrophils that are sequestered in the
pulmonary microcirculation in patients with lung inflammation, since
animal models of lung inflammation have shown that neutrophils
sequestered in the pulmonary microcirculation release more reactive
oxygen species (ROS) than circulating neutrophils in the same animal
(11)
. Thus, neutrophils that are sequestered in the
pulmonary microcirculation may be a source of oxidative stress, which
may have a role of inducing airway injury in lung inflammation.
It has been demonstrated that
.O2- has some
proinflammatory properties: recruitment of neutrophils at sites of
inflammation, formation of chemotactic factors (12
, 13)
,
DNA damage, depolymerization of hyaluronic acid and collagen
(14
15
16)
, lipid peroxidation, release of cytokines such
tumor necrosis factor (TNF-) and interleukin 1ß (IL-1ß), and
formation of peroxinitrite (ONOO-). Peroxynitrite itself is a highly
reactive oxidant produced by the combination of
.O2- and nitric oxide (NO)
at rates approaching the diffusion limit (15
16
17)
.
Moreover, peroxynitrite can also cause DNA damage (18
, 19)
, resulting in the activation of the nuclear enzyme poly
(ADP-ribose) synthetase (PARS), depletion of NAD and ATP, and
ultimately cell death (20)
. ONOO- inhibits the activity of
the endogenous SOD enzymes, a factor that contributes to increased
formation of .O2-
(21
, 22)
.
Recent in vitro experiments have shown that thiol antioxidants such as
n-acetylcysteine (NAC) and n-acystelin block the
release of these inflammatory mediators from epithelial cells and
macrophages by a mechanism involving increasing intracellular GSH and
decreasing NF-B activation (23
, 24)
.
An association between dietary intake of antioxidant vitamins and lung
function has been demonstrated in the general population. Britton and
co-workers (25)
showed in a population of 2633 subjects an
association between dietary intake of the antioxidant vitamin E and
lung function, supporting the hypothesis that this antioxidant may have
a role in protecting against the development of lung inflammation;
hence, vitamin supplementation may be a possible preventive therapy
against the development of lung inflammation. Even though intervention
studies have been difficult to perform (26)
, some evidence
suggests that antioxidant vitamin supplementation reduces oxidant
stress, measured as a decrease in pentane levels in breath as an
assessment of lipid peroxides (27)
.
Here we investigated the effects of NAC on the inflammatory response (pleurisy) caused by injection of carrageenan in the rat. We have investigated the effects of NAC on lung injury (histology), red blood cell (RBC) damage, as well as increases in nitrotyrosine (immunohistochemistry) and PARS activity caused by carrageenan in the lung.
MATERIALS AND METHODS
Animals
Male Sprague-Dawley rats (300–350 g; Charles River, Milan,
Italy) were housed in a controlled environment and provided with
standard rodent chow and water. Animal care complied with Italian
regulations on protection of animals used for experimental and other
scientific purposes (D.M. 116192) as well as with EEC regulations
(O.J. of E.C. L 358/1 12/18/1986)
Carrageenan-induced pleurisy
Rats were anesthetized with isoflurane and submitted to a skin
incision at the level of the left sixth intercostal space. The
underlying muscle was dissected and saline (0.2 ml) or saline
containing 1% 
-carrageenan (0.2 ml) was injected into the pleural
cavity. The skin incision was closed with a suture and the animals were
allowed to recover. NAC (20 mg/kg) or an equivalent volume (0.3 ml) of
vehicle (saline) was injected intraperitoneally (i.p.) 3, 6, and
12 h after carrageenan. At 24 h after the injection of
carrageenan, the animals were killed by inhalation of
CO2. The chest was opened carefully and the
pleural cavity rinsed with 2 ml of saline solution containing heparin
(5 U/ml) and indomethacin (10 µg/ml). The exudate and washing
solution were removed by aspiration and the total volume was measured.
Any exudate contaminated with blood was discarded. The amount of
exudate was calculated by subtracting the volume injected (2 ml) from
the total volume recovered. Leukocytes in the exudate were suspended in
phosphate-buffered saline (PBS) and counted with an optical microscope
in a Burker’s chamber after vital trypan blue staining. Cytokines
(TNF- and IL-1ß) were measured in the exudates by ELISAs using
commercially available kits (Calbiochem-Novabiochem Corporation, San
Diego, Calif.).
Measurement of lung tissue myeloperoxidase activity and
malondialdehyde
Myeloperoxidase (MPO) activity, a hemoprotein located in
azurophil granules of neutrophils, has been used as a biochemical
marker for neutrophil infiltration into tissues (28)
. In
the present study, MPO was measured photometrically by a method similar
to that described previously (29)
. At 4 h after the
intrapleural injection of carrageenan, lung tissues were obtained and
weighed. Each piece of tissue was homogenized in a solution containing
0.5% hexa-decyl-trimethyl-ammonium bromide dissolved in 10 mM
potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000
g at 4°C. An aliquot of the supernatant was then allowed
to react with a solution of tetramethylbenzidine (1.6 mM) and 0.1 mM
H2O2. The rate of change in
absorbance was measured spectrophotometrically at 650 nm. MPO activity
was defined as the quantity of enzyme degrading 1 µmol of
peroxide/min at 37°C and was expressed in milliunits per 100 mg
weight of wet tissue. Malondialdehyde (MDA) levels in the lung tissue
were determined as an indicator of lipid peroxidation
(30)
. Lung tissue collected at the specified time was
homogenized in 1.15% KCl solution. An aliquot (100 µl) of the
homogenate was added to a reaction mixture containing 200 µl of 8.1%
SDS, 1500 µl of 20% acetic acid (pH 3.5), 1500 µl of 0.8%
thiobarbituric acid, and 700 µl distilled water. Samples were then
boiled for 1 h at 95°C and centrifuged at 3000 g for
10 min. The absorbance of the supernatant was measured by
spectrophotometry at 650 nm.
Immunofluorescence localization of ICAM-1, P-selectin,
nitrotyrosine, and PARS
Indirect immunofluorescence staining was performed on 7
µm-thick sections of unfixed rat lung. Sections were cut in with a
Slee and London cryostat at -30°C, transferred onto clean glass
slides, and dried overnight at room temperature. Sections were
permeabilized with acetone at -20°C for 10 min and rehydrated in PBS
(150 mM NaCI, 20 MM sodium phosphate, pH 7.2) at room temperature for
45 min. Sections were incubated overnight with 1) rabbit
anti-human polyclonal antibody directed at P-selectin (CD62P), which
react with rat and with mouse anti-rat antibody directed at ICAM-1
(CD54) (1:500 in PBS, v/v) (DBA, Milan, Italy) or 2)
anti-nitrotyrosine rabbit polyclonal antibody (1:500 in PBS, v/v) or
anti-poly (ADP-ribose) goat polyclonal antibody rat (1:500 in PBS,
v/v). Sections were washed with PBS and incubated with secondary
antibody (TRITC-conjugated anti-rabbit and with FITC-conjugated
anti-mouse (Jackson, West Grove, Pa.) or with TRITC-conjugated
anti-goat antibody (1:80 in PBS, v/v) for 2 h at room temperature.
Sections were washed as before, mounted with 90% glycerol in PBS, and
observed with a Nikon RCM8000 confocal microscope equipped with a 40 x
oil objective.
Histological examination
Lung biopsies were taken at 24 h after injection of
carrageenan. The biopsies were fixed for 1 wk in buffered formaldehyde
solution (10% in PBS) at room temperature, dehydrated by graded
ethanol and embedded in Paraplast (Sherwood Medical, Mahwah, N.J.).
Tissue sections (thickness 7 µm) were deparaffinized with xylene,
stained with trichromic Van Gieson, and studied using light microscopy
(Dialux 22 Leitz). Blood was passed on the slide, fixed at 37°C,
stained with May Grunward-Giensa, and studied using light microscopy.
Isolation of pleural macrophages
Resident pleural cell macrophages were collected 24 h after
the carrageenan injection from rats treated or not with NAC
(31)
. After exsanguination, the pleural cavity, was opened
and the cells in it were collected by repeated washing with 2 ml of
medium (11.8 mM Tris-HCL buffer saline, containing 2.6 mM KCl, 1.0 mM
MgCl2, 0.4 mM
NaH2P04 5.4 mM Glucose, 1.5
mM EDTA, pH 7.4). Total leukocyte counts in the exudate were measured
with a Neubauer cell count plate after fixation with Turk’s solution.
Differential counts of the exuded leukocytes were performed after smear
preparation and staining with Wright-Giemsa methods as described
previously by Ogino et al. (32)
. Cells
(106/ml), consisting mainly of macrophages
(
70%) were cultured in Dulbecco’s modified Eagle medium
supplemented with L-glutamine (3.5 mM), penicillin (50/ml),
streptomycin (50 µg/ml), and heparin sodium (10 U/ml) in 12-well
plates. Cells were allowed to adhere for 2 h at 37°C in a
humidified 5% CO2 incubator. Nonadherent cells
were removed by rinsing the plates three times with 5% dextrose water.
After removing nonadherent cells (10%), adherent macrophages were
scraped for the measurement of inducible nitric oxide synthase (iNOS)
activity, DNA single-strand breaks and cellular levels of
NAD+. In another series of experiments, cells
were made to adhere (for 2 h) as described above and the levels of
nitrite/nitrate (NOx) and ONOO- formation from
the cells was measured in the supernatant.
Measurement of NOx and peroxynitrite from
pleural macrophages
NOx production, an indicator of NO
synthesis, was measured in cell supernatants as described previously
(31)
. The nitrate in the supernatant was first reduced to
nitrite by incubation with nitrate reductase (670 mU/ml) and NADPH (160
µM) at room temperature for 3 h. The nitrite concentration in
the samples was then measured by the Griess reaction by adding 100 µl
of Griess reagent (0.1% naphthylethylenediamide dihydrochloride in
H2O and 1% sulfanilamide in 5% concentrated
H2PO4; vol. 1:1) to 100
µl samples. The optical density at 550 nm
(OD550) was measured using ELISA microplate
reader (SLT; Lab Instruments, Salzburg, Austria). The formation of
peroxynitrite was measured by the peroxynitrite-dependent oxidation of
dihydrorhodamine-123 to rhodamine-123 (33)
as described
previously (31)
. Cells were rinsed with PBS and the medium
was then replaced with PBS containing 5 µM dihydrorhodamine-123.
After a 60 min incubation at 37°C, the fluorescence of rhodamine-123
was measured using a fluorometer at an excitation wavelength of 500 nm
and emission wavelength of 536 nm (slit widths, 2.5 and 3.0 nm,
respectively). This method is an indirect measurement of peroxynitrite
production since other oxidant species can oxidize dihydrorhodamine-123
(34
, 35)
.
Measurement of mitochondrial respiration in pleural macrophages
Cell respiration was assessed by measuring the
mitochondrial-dependent reduction of MTT [3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to
formazan as described previously (31)
. Cells in 96-well
plates were incubated at 37°C with MTT (0.2 mg/ml) for 1 h.
Culture medium was removed by aspiration and the cells were solubilized
in DMSO (100 µl). The extent of reduction of MTT to formazan within
cells was quantified by the measurement of OD550.
As discussed (36
, 38)
, measurement of the reduction of MTT
appears to be mainly by mitochondrial complexes I and II, but may also
involve NADH- and NADPH-dependent energetic processes that occur
outside the mitochondrial inner membrane. Thus, this method cannot be
used to separate the effect of free radicals, oxidants, or other
factors on the individual enzymes in the mitochondrial respiratory
chain. It is, however, a useful technique to monitor changes in the
general energetic status of the cells (36
, 37)
.
Determination of DNA single-strand breaks in pleural macrophages
The formation of DNA strand breaks in double-stranded DNA was
determined by the alkaline unwinding method (31
, 36
, 37)
.
Cells in 12-well plates were scraped into 0.2 ml of solution A buffer
(myoinositol 250 mM,
NaH2PO3 10 mM,
MgCl2 1 mM, pH 7.2). The cell lysate was then
transferred into plastic tubes designated T (maximum fluorescence), P
(fluorescence in sample used to estimate extent of DNA unwinding), or B
(background fluorescence). To each tube, 0.2 ml of solution B (alkaline
lysis solution: NaOH 10 mM, urea 9 M, ethylenediaminetetraacetic acid
2.5 mM, sodium dodecyl sulfate 0.1%) was added and incubated at 4°C
for 10 min to allow cell lysis and chromatin disruption; 0.1 ml each of
solutions C (0.45 volume solution B in 0.2 N NaOH) and D (0.4 volume
solution B in 0.2 N NaOH) were then added to the P and B tubes.
Solution E (0.1 ml neutralizing solution: glucose 1 M, mercaptoethanol
14 mM) was added to the T tubes before solutions C and D were added.
From this point on, all incubations were carried out in the dark. A 30
min incubation period at 0°C was then allowed during which the alkali
diffused into the viscous lysate. As the neutralizing solution,
solution E was added to the T tubes before addition of the alkaline
solutions C and D; the DNA in the T tubes was never exposed to a
denaturing pH. At the end of the 30 min incubation, the contents of the
B tubes were sonicated for 30 s to ensure rapid denaturation of
DNA in the alkaline solution. All tubes were then incubated at 15°C
for 10 min. Denaturation was stopped by chilling to 0°C and adding
0.4 ml of solution E to the P and B tubes; 1.5 ml of solution F
(ethidium bromide 6.7 µg/ml in 13.3 mM NaOH) was added to all the
tubes and fluorescence (excitation: 520 nm, emission: 590 nm) was
measured by a fluorometer. Under the conditions used, in which ethidium
bromide binds preferentially to double-stranded DNA, the percentage of
double-stranded DNA (D) may be determined using the equation: % D = 100 x [F(P) - F(B)]/[F(T) - F (B)], where F(P) is
the fluorescence of the sample, F(B) the background fluorescence, i.e.,
fluorescence due to all cell components other than double-stranded DNA,
and F(T) the maximum fluorescence.
Measurement of cellular NAD+ levels in pleural
macrophages
Activation of PARS results in the depletion of its substrate
NAD+ and a reduction in the rate of glycolysis
(38)
. As NAD+ functions as a
cofactor in glycolysis and the tricarboxylic acid cycle,
NAD+ depletion leads to a rapid fall in
intracellular ATP levels (39
, 40)
. We measured
NAD+ levels as a mean to indirectly evaluate PARS
activation as previously done by others (39
, 40)
. Cells in
12-well plates were extracted in 0.25 ml of 0.5 N
HClO4, scraped, neutralized with 3 M KOH, and
centrifuged for 2 min at 10.000 g. The supernatant was
assayed for NAD+ using a modification of the
colorimetric method (20
, 31)
in which NADH produced by
enzymatic cycling with alcohol dehydrogenase reduces MTT to formazan
through the intermediation of phenazine methosulfate. The rate of MTT
reduction is proportional to the concentration of the coenzyme. The
reaction mixture contained 10 µl of a solution of 2.5 mg/ml MTT, 20
µl of a solution of 4 mg/ml phenazine methosulfate, 10 µl of a
solution of 0.6 mg/ml alcohol dehydrogenase (300 U/mg), and 190 µl of
0.065 M glycyl-glycine buffer, pH 7.4, which contained 0.1 M
nicotinamide and 0.5 M ethanol. The mixture was warmed to 37°C for 10
min, then the reaction was started by the addiction of 20 µl of the
sample. The rate of increase in absorbance was read immediately after
the addition of NAD+ samples and after 10 and 20
min incubation at 37°C against blank at 560 nm in the ELISA
microplate reader (Lab Instruments).
Determination of nitric oxide synthase activity (pleural
macrophages and lung tissue)
The calcium-independent conversion of L-arginine to
L-citrulline in the homogenates of either pleural
macrophages or lungs (obtained 4 h after carrageenan treatment in
the presence or the absence of NAC) served as an indicator of iNOS
activity (42)
. Cells or lung tissue were scraped into a
homogenization buffer composed of 50 mM Tris.HCl, 0.1 mM EDTA, and 1 mM
phenylmethylsulfonyl fluoride (pH 7.4) and homogenized in the buffer on
ice using a tissue homogenizer. Conversion of
[3H]-L-arginine to
[3H]-L-citrulline was measured in
the cell/lung homogenates as described previously (31)
.
Homogenates (30 µl) were incubated in the presence of
[3H]-L-arginine (10 µM, 5 kBq per
tube), NADPH (1 mM), calmodulin (30 nM), tetrahydrobiopterin (5 µM),
and EGTA (2 mM) for 20 min at 22°C. Reactions were stopped by
dilution with 0.5 ml of ice-cold HEPES buffer (pH 5.5) containing EGTA
(2 mM) and EDTA (2 mM). Reaction mixtures were applied to Dowex 50W
(Na+ form) columns and the eluted
[3H]-L-citrulline activity was
measured by a Beckman scintillation counter.
Preparation of cytosolic fractions and Western blot analysis for
IB-
Extracts of pleural macrophages collected 24 h after the
carrageenan injection from rats treated with or without NAC were
prepared as described (43)
. Harvested cells
(2x107) were washed twice with ice-cold PBS and
centrifuged at 180 g for 10 min at 4°C. The cell pellet
was resuspended in 100 µl of ice-cold hypotonic lysis buffer (10 mM
HEPES, 1.5 mM MgC12, 10 mM KCI, 0.5 mM phenylmethylsulfonyl fluoride,
1.5 µg/ml soybean trypsin inhibitor, pepstatin A 7 µg/ml, leupeptin
5 µg/ml, 0.1 mM benzamidine, 0.5 mM DTT) and incubated in ice for 15
min. The cells were lysed by rapid passage through a syringe needle
five or six times and the cytoplasmic fraction was then obtained by
centrifugation for 1 min at 13,000 g for 1 min. Protein
concentration was determined with the Bio-Rad protein assay kit.
Immunoblotting analysis of IB- proteins was performed on a
cytosolic fraction. Cytosolic fraction proteins were mixed with gel
loading buffer (50 mM Tris/10% SDS/10% glycerol/10%
2-mercaptoethanol/2 mg bromphenol per ml) in a ratio of 1:1, boiled for
3 min, and centrifuged at 10,000 g for 10 min. Protein
concentration was determined and equivalent amounts (75 µg) of each
sample were electrophoresed in a 12% discontinuous polyacrylamide mini
gel. The proteins were transferred. onto nitrocellulose membranes,
according to the manufacturer’s instructions (Bio-Rad, Hercules, CA).
The membranes were saturated by incubation at 4°C overnight with 10%
nonfat dry milk in PBS and incubated with anti-IB- (1:1000) for
1 h at room temperature. The membranes were washed three times
with 1% Triton X-100 in PBS and incubated with anti-rabbit
immunoglobulins coupled to peroxidase (1: 1000). The immune complexes
were visualized by the ECL chemiluminescence method (Amersham, Little
Chalfont, UK).
Scanning electron microscopy
Blood samples (taken from femoral vein) for RBC evaluation were
taken at 24 h after the carrageenan administration. The
morphological alteration of RBCs were followed by scanning electron
microscopy. Blood samples were fixed (at + 4'C) in modified Karnovsky
fixative (1.5% glutaraldehyde and 1.5% paraformaldehyde in 0.1 M
cacodylate buffer). The blood was then transferred to ice-cold 0.1 M
phosphate buffer and postfixed in 1% OS04 in 0.1 M cacodylate buffer
for I h. After thorough rinsing in 0.1 M phosphate buffer, blood
samples were dehydrated in a graded series of ethanols and transferred
into liquid C02 in a critical point dryer. The
dried specimens were mounted, sputter-coated with gold and examined in
a scanning electron microscope at 20 W.
Materials
Cell culture medium, heparin, and fetal calf serum were obtained
from Sigma (Milan, Italy). Perchloric acid was obtained from Aldrich
(Milan, Italy). Primary anti-nitrotyrosine antibody was from Upstate
Biotech (DBA, Milan, Italy). All other reagents and compounds used were
obtained from Sigma Chemical Company (Sigma, Milan, Italy).
Data analysis
All values in the figures and text are expressed as mean ±
standard error of the mean (SE) for n
observations. For the in vitro studies, data represent the number of
wells studied (6–9 wells from 2–3 independent experiments). For the
in vivo studies, n represents the number of animals studied.
The results were analyzed by one-way ANOVA, followed by a Bonferroni
post hoc test for multiple comparisons. A P value of less
than 0.05 was considered significant. In the experiments involving
histology or immunohistochemistry, the figures shown are representative
of at least three experiments performed on different experimental days.
RESULTS
Effects of NAC in carrageenan-induced pleurisy
Histological examination of lung sections revealed significant
tissue damage (Fig. 1B
). Thus, when compared with lung sections taken from
saline-treated animals (Fig. 1A
), histological examination
of lung sections of rats treated with carrageenan showed edema, tissue
injury, and infiltration of the tissue with neutrophils (PMNs) (Fig. 1B
). NAC significantly reduced the degree of injury as well
as the infiltration of PMNs (Fig. 1C
). Furthermore,
injection of carrageenan into the pleural cavity of rats elicited an
acute inflammatory response characterized by the accumulation of fluid
(edema) that contained large amounts of PMNs (Fig. 2A
, B
). Neutrophils also infiltrated in the lung
tissues (Fig. 3A
); this was associated with lipid peroxidation of lung
tissues, as evidenced by an increase in the levels of malonyldialdehyde
(Fig. 3B
). Edema, neutrophil infiltration in lung
tissue, and lipid peroxidation were attenuated by the i.p. injection of
NAC (n=10) (Figs. 2, 3).
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Effects of NAC on the expression of adhesion molecules (ICAM-1,
P-selectin)
Staining of lung tissue sections obtained from
saline-treated rats with anti-ICAM-1 antibody showed specific staining
along bronchial epithelium, demonstrating that ICAM-1 is constitutively
expressed (Fig. 4A
). At 4 h after carrageenan injection, the staining
intensity substantially increased along the bronchial epithelium (Fig. 4D
). Lung tissue section obtained from carrageenan-treated
rats showed positive staining for P-selectin localized in the bronchial
epithelium (Fig. 4F
). No positive staining for ICAM-1
or P-selectin was found in the lungs of carrageenan-treated rats that
received an i.p. injection of NAC (Fig. 4G
, H
).
To verify the binding specificity for ICAM-1 or P-selectin, some
sections were also incubated with only the primary antibody (no
secondary) or with only the secondary antibody (no primary). In these
situations, no positive staining was found in the sections, indicating
that the immunoreaction was positive in all the experiments carried
out.
|
Effects of NAC on nitrotyrosine and PARS
At 4 h after carrageenan injection, lung sections were
taken in order to determine the immunohistological staining for
nitrotyrosine or PARS. Sections of lung from saline-treated rats did
not reveal any immunoreactivity for nitrotyrosine (Fig. 5A
) or PARS (Fig. 5B
) within the normal
architecture. A positive staining for nitrotyrosine (Fig. 5D
) and PARS (Fig. 5E
) was localized primarily in
the vessels and in the bronchial epithelium. NAC reduced the staining
for both nitrotyrosine and PARS (Fig. 5G
, H
). To
confirm that the immunoreaction for the nitrotyrosine was specific,
some sections were also incubated with the primary antibody
(anti-nitrotyrosine) in the presence of excess nitrotyrosine (10 mM) to
verify the binding specificity. To verify the binding specificity for
PARS, sections were also incubated with only the primary antibody (no
secondary) or with only the secondary antibody (no primary). In
these situations, no positive staining was found in the sections,
indicating that the immunoreaction was positive in all the experiments
carried out.
|
Effects of NAC on the release of cytokine
When compared with controls at 24 h after the
injection of carrageenan, an increase in the levels of TNF- and
IL-1ß was observed in pleural exudates (Fig. 6A
, B
). NAC treatment attenuated the release of
TNF- and IL-1ß (Fig. 6A
, B
),
|
Effects of NAC on nitric oxide production
The levels of NOx were
significantly (P<0.01) increased in the exudate from
carrageenan-treated rats (Fig. 7A
). In contrast, levels of NOx were
significantly lower in the exudate of carrageenan-treated rats treated
with NAC (Fig. 7A
). In the lungs obtained from animals
subjected to carrageenan-induced pleurisy, a significant increase in
iNOS activity was detected at 24 h (Fig. 7B
). The iNOS
activity was significantly (P<0.01) lower in rats treated
with NAC (Fig. 7B
).
|
Effects of NAC on RBC alteration
At 24 h after the injection of carrageenan a
significant alteration in the RBC morphology was observed (Figs. 8B
and Fig. 9B
). RBC alteration was correlated with a significant
reduction in the amount of HGB (8.2±0.4 g/dl) 24 h after the
injection of carrageenan. NACs significantly prevent RBC alteration and
prevent the loss of Hb contest (15±0.8 g/dl) (Fig. 8C
and
Fig. 9C
)
|
|
Effects of NAC on the increase in peroxynitrite formation, NO
production, DNA damage, PARS activation, and injury of macrophages
obtained from the pleural cavity of carrageenan-treated rats
In pleural macrophages obtained from rats at 24 h after
carrageenan injection, a significant nitrate/nitrite production was
detectable in comparison to sham (Fig. 10A
). A rapid and sustained production of peroxynitrite
compared with sham was also observed after carrageenan-induced pleurisy
(n=4; Fig. 10B
). There was a marked increase in
DNA single-strand breakage in peritoneal macrophages cells from
carrageenan-treated rats compared with sham (Fig. 10C
). Carrageenan-mediated disruption of cellular
energetic pool was indicated by a significant decreased in cellular
respiration (Fig. 10D
) and intracellular concentration of
NAD+ (Fig. 10E
) vs. sham.
|
In vivo treatment of the animals with NAC significantly inhibited NO
production (Fig. 10A
). NAC treatment also caused a reduction
of dihydrorhodamine-123 oxidation in cells from carrageenan-treated
rats (Fig. 10B
) and prevented the carrageenan-induced DNA
single-strand breakage (Fig. 10C
). In vivo treatment
of the animals with NAC significantly inhibited the decrease in
cellular respiration and restored the depletion of intracellular levels
of NAD+ (Figs. 10D
, E
). Pleural
macrophages were also analyzed for PARS immunoreactivity. Cells
obtained from carrageenan-treated rats showed intense positive staining
for PARS (Fig. 11A
), which was markedly reduced in carrageenan-treated
rats treated with NAC (Fig. 11B
). Staining was absent in
pleural macrophages obtained from sham-operated rats (data not shown).
|
Effect of NAC on IB- degradation
The appearance of IB- in the cytosolic fractions was
investigated by immunoblotting analysis. A basal level of IB- was
detectable in the cytosolic fraction of unstimulated cells, whereas
24 h after carrageenan administration, IB- disappeared. NAC
in vivo treatment prevented IB- degradation; in fact, the
IB- band remained unchanged at 24 h after carrageenan
administration (Fig. 12
).
|
DISCUSSION
ROS have been implicated in a wide variety of diseases. Evidence
for increased oxidant stress in lung inflammation is emerging (5
, 9
, 44)
. For example, Postma and colleagues (45)
have shown a correlation between 0 and 2 release by peripheral blood
neutrophils and bronchial hyperreactivity in patients with lung
inflammation (45)
. Several earlier studies suggested that
oxidant stress may reflect the inflammatory component of lung
inflammation (44)
. For example, lung inflammation is
usually characterized by extensive infiltration of pulmonary tissue by
PMNs, which is more marked in bronchoalveolar lavage fluid during
acute, infectious exacerbations. Neutrophil activation represents an
important source of ROS (46)
.
Furthermore, there is much evidence that the production of ROS such as
hydrogen peroxide, superoxide, and hydroxyl radicals at the site of
inflammation contributes to tissue damage (12
, 31
, 47
48
49
50
51)
. Inhibitors of NOS activity reduce the development of
carrageenan-induced inflammation and support a role for NO in the
pathophysiology associated with this model of inflammation (31
, 50
51
52
53)
. In addition to NO, peroxynitrite is also generated in
carrageenan-induced inflammation (30
, 37
, 49
, 51)
. The
biological activity and decomposition of peroxynitrite depend a great
deal on the cellular or chemical environment (presence of proteins,
thiols, glucose, the ratio of NO and superoxide, carbon dioxide levels,
and other factors), and these factors influence its toxic potential
(15
, 54
55
56)
. We have now demonstrated that NAC reduces
1) the development of carrageenan-induced pleurisy,
2) the infiltration of the lung with PMNs (histology and MPO
activity), 3) the degree of lipid peroxidation in the lung,
and 4) the degree of lung injury (histology) in rats treated
with carrageenan. All these findings support the view that NAC
attenuates the degree of inflammation and lung injury caused by
carrageenan in the rat. What, then, is the mechanism by which NAC
protects the lung against this inflammatory injury?
NAC exerts its effect both as a source of sulfhydryl groups (repletion
of intracellular reduced glutathione) and through a direct reaction
with hydroxyl radical (57)
. Recently, studies of animals
suggest benefits from acetylcysteine in the context of systemic
inflammatory response syndrome caused by severe sepsis model. In a pig
gram-negative sepsis model, an infusion of acetylcysteine reduced
pulmonary capillary leak without reducing mortality (58)
.
Acetylcysteine also beneficially modulates inflammatory cell function
in animals. Endotoxin-induced neutrophil activation in sheep lung is
reduced (58)
.
NAC may have additional protective ability to reduce oxyradical-related
oxidant processes by directly interfering with the oxidants,
up-regulating antioxidant systems such as superoxide dismutase
(58)
, or enhancing the catalytic activity of glutathione
peroxidase (59)
. Therefore, oxygen radical scavengers
administered before or at the onset of sepsis were shown to improve the
survival in animal models of sepsis (60)
. NAC has an
antioxidant property (61)
and, as a sulfhydryl donor, may
contribute to the regeneration of endothelium-derived relaxing
factor and glutathione (62)
. Increasing evidence
indicates that the action of NAC is pertinent to microcirculatory blood
flow and tissue oxygenation. NAC was shown to enhance oxygen
consumption via increased oxygen extraction in patients 18 h after
the onset of fulminant liver failure (62)
. It has been
speculated that NAC could also exert beneficial effects on impaired
nutritive blood flow in patients with severe sepsis (62)
.
In addition, in this study we show that the antioxidant NAC can block
RBC modification in vivo. RBC modification is by the lung injury, which
in turn is correlated with less oxygen exchange. Recently it has been
demonstrated in vitro that NAC protection of oxidative damage leads to
decreased RBC dense cell formation. This effect was correlated with the
presence in the dense cells of the highest percent ISCs, which provides
a partial explanation of NAC’s effect on ISC formation. But it does
not directly address the molecular mechanism of NAC’s effect on ISC
formation. Dense cells have decreased fetal hemoglobin and increased
hemoglobin S concentration, leading to greater polymerization of HbS
(6
, 7)
. This would cause many of the high-density SS
erythrocytes to become sickled in shape. NAC, as the N-acetyl
derivative of L-cysteine, is an antioxidant
(63)
. It is highly permeable to cell membranes and is
converted within the cytoplasm to L-cysteine, which is a
precursor to GSH. It could therefore protect thiols from oxidative
damage by its antioxidant capacity and by raising the levels of GSH
(64)
.
There are a number of sites where NAC can interfere with the
inflammatory process (see Fig. 13
). The results of the current study show that neither the inhibition of
plasma NOx production nor the inhibition of
NOx levels in the exudate were completely
inhibited by NAC. This reduction in iNOS is related to the inhibitory
effect of NAC on activation of the NF-B (57)
, since
this transcription factor is involved in the process of iNOS expression
(65
, 66)
. Recent evidence suggested that the activation of
NF-B may also be under the control of oxidant/antioxidant balance
(67)
. This hypothesis is based on the observation that low
doses of peroxides, including
H2O2 and
tert-butyl-hydroperoxide, induce NF-B activation whereas some
antioxidants prevent it (68
, 69)
. Our results agree with
this hypothesis, since it is conceivable that NAC inhibited NF-B
activation through an antioxidant mechanism. Moreover, the transient
loss of IB- that occurs in pleural macrophages from
carrageenan-treated rats was prevented by NAC treatment, suggesting
that these compounds inhibit NF-B activation by stabilizing
IB-
|
At the dose used (20 mg/kg), NAC therefore fully inhibited the appearance of nitrotyrosine staining in the inflamed lung.
Nitrotyrosine formation along with its detection by immunostaining was
initially proposed as a relatively specific marker for the detection of
the endogenous formation ‘footprint’ of peroxynitrite
(60)
. Recent evidence, however, indicates that certain
other reactions can also induce tyrosine nitration; e.g., the reaction
of nitrite with hypochlorous acid and the reaction of myeloperoxidase
with hydrogen peroxide can lead to the formation of nitrotyrosine
(61)
. Increased nitrotyrosine staining is therefore
considered an indication of ‘increased nitrosative stress’ rather
than a specific marker of the generation of peroxynitrite.
ROS and peroxynitrite produce cellular injury and necrosis via several
mechanisms including peroxidation of membrane lipids, protein
denaturation, and DNA damage. ROS produce strand breaks in DNA that
trigger energy-consuming DNA repair mechanisms and activate the nuclear
enzyme PARS, resulting in the depletion of its substrate NAD in vitro
and a reduction in the rate of glycolysis. As NAD functions as a
cofactor in glycolysis and the tricarboxylic acid cycle, NAD depletion
leads to a rapid fall in intracellular ATP. This process has been
termed the ‘PARS suicide hypothesis’. There is recent evidence that
the activation of PARS may also play an important role in inflammation
(31
, 37
, 41
, 62)
. We demonstrate here that NAC attenuates
the increase in PARS activity caused by carrageenan in the lung.
Similarly, NAC attenuates the formation of peroxynitrite by macrophages
(ex vivo) obtained from rats that had been injected with NAC. In these
macrophages, NAC also attenuated the fall in NAD associated with the
enhanced formation of peroxynitrite. Thus, we propose that the
anti-inflammatory effects of NAC reported here are due at least in part
to the prevention of the activation of PARS.
In conclusion, this study demonstrated that the stable nitroxide
radical NAC attenuates the pleurisy caused by carrageenan
administration in the rat. We speculate that the observed
anti-inflammatory effects of NAC may depend on a combination of the
following pharmacological properties of this agent: 1) NAC
inhibit NF-B activation; 2) NAC scavenges and inactivates
superoxide anions that would prevent the formation of peroxynitrite.
This in turn prevents the activation of PARS and the associated tissue
injury. 3) In addition to superoxide anions, NAC also
scavenges other ROS, including hydroxyl radicals. It is not clear
whether NAC is able to scavenge peroxynitrite. 4) In
addition, NAC reduces the recruitment of PMNs into the inflammatory
site. This effect of NAC is very likely secondary to the prevention by
NAC of endothelial oxidant injury and, hence, preservation of
endothelial barrier function. These results support the view that the
overproduction of reactive oxygen or nitrogen free radicals contributes
to acute inflammation. Finally, we propose that small molecules such as
NAC may be useful in the therapy of conditions associated with local or
systemic inflammation.
ACKNOWLEDGMENTS
This study was supported by a grant from Consiglio Nazionale delle Ricerche. The authors thank Giovanni Pergolizzi and Carmelo La Spada for their excellent technical assistance during this study, Mrs. Caterina Cutrona for secretarial assistance, and Miss Valentina Malvagni for editorial assistance with the manuscript.
Received for publication August 10, 2000. Revision received November 8, 2000. REFERENCES
B activation. Am. J. Resp. Crit. Care Med. 157,A889(abstr.)
