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* Institute of Pharmacology, University of Messina, Italy;
Department of Cellular and Structural Biology, The University of Texas Health Center at San Antonio, San Antonio, Texas, USA; and
Biomorphology School of Medicine, 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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: inflammation • nitric oxide • peroxynitrite • superoxide • hydroxyl radical
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. The systemic inflammatory response is also
associated with the production of oxygen-derived free radicals
(4
, 5)
, and there is now substantial evidence that much of
the cytotoxicity of these radicals is due to a concerted action of
oxygen- and nitrogen-derived free radicals and oxidants. Peroxynitrite,
a cytotoxic oxidant species formed from the reaction of NO and
superoxide (6)
, may mediate part of the oxidative injury
associated with simultaneous production of NO and oxyradicals.
Peroxynitrite formation has been demonstrated in various inflammatory
disorders (3
, 7
8
9)
and in circulatory shock
(10)
. Peroxynitrite is a potent oxidant, and therefore it
is conceivable that endogenous antioxidant mechanisms counteract its
toxicity. In in vitro studies, it has been established that
antioxidants such as glutathione, ascorbic acid, and alpha-tocopherol
are scavengers of peroxynitrite and inhibitors of its oxidant capacity
(11
, 12)
.
Melatonin is an indole that is synthesized in and secreted from the
pineal gland during the night (13)
. Its lipophilicity
ensures that melatonin rapidly enters cells, where it may accumulate in
the nucleus (14)
. Recently, it was demonstrated that
melatonin is a free radical scavenger (15
16
17
18
19
20)
, an
antioxidant that protects cells against the damage induced by several
oxidative agents including paraquat (21)
and carbon
tetrachloride (22)
. Melatonin is also a scavenger of
peroxynitrite (23)
and inhibits the production of NO
(9
, 24
, 25)
.
In this study we investigated the role of endogenous melatonin against the peroxynitrite-induced injury in a model of local inflammation caused by carrageenan. Specifically, we have investigated whether depletion of endogenous melatonin in rats exposed on 24 h light cycle for 1 wk affects the inflammatory response (pleural exudate formation, cellular infiltration) and cellular injury in ex vivo macrophages harvested from the pleural cavity of the rats subjected to carrageenan-induced pleurisy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-carrageenan was injected into the pleural cavity. The skin
incision was closed with a suture and the animals were allowed to
recover. At 4 h after the injection of carrageenan, the animals
were killed under CO2 vapor. The chest was
carefully opened and the pleural cavity washed with 2 ml of saline
solution with heparin (5 U/ml) and indomethacin 10 µg/ml). The
exudate and washing were removed by aspiration and the total volume was
measured. Exudates contaminated with blood were discarded. The results
were 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 optical microscope by
Burker's chamber after trypan blue stain. Carrageenan (or vehicle) was
given to groups of animals (MEL -/-) maintained on a 24 h light
cycle for 1 wk to deplete melatonin and to animals (MEL +/+) exposed to
a 14:10 light/dark cycle. The following groups of animals were used:
control + MEL +/+, control + MEL -/-, carrageenan + MEL +/+, and
carrageenan + MEL -/- (n=10 rats in each group). In a
second group of experiments, melatonin (MEL, 15 mg/kg i.p.) was
administered to MEL -/- rat as a single bolus 15 min before
carrageenan.
Cell culture
Pleural macrophages from rats were harvested by pleural lavage
with DMEM medium supplemented with L-glutamine (3.5 mM), penicillin (50
U/ml), streptomycin (50 µg/ml), and heparin sodium (10 U/ml). The
cells were collected 4 h after the carrageenan injection from MEL
+/+ or MEL -/- rats. The cells were plated on 12-well plates at 1
million cells/ml and incubated for 2 h at 37°C in a humidified
5% CO2 incubator. After incubation, supernatant
was collected for the measurement of nitrite and nitrate. Nonadherent
cells were removed by rinsing the plates three times with 5% dextrose
water. After removing nonadherent cells, adherent macrophages were
scraped in order to measure DNA strand breaks and cellular
NAD+. Mitochondrial respiration and peroxynitrite
formation were measured in the adherent cells in the subsequent 1-h
period.
Measurement of nitrite/nitrate
Nitrite/nitrate production, an indicator of NO synthesis, was
measured in supernatant samples as described previously
(26)
. After 3 h incubation with nitrate reductase
(670 mU/ml) and NADPH (160 µM) at room temperature, the total nitrite
concentration in the samples was measured by the Griess reaction. The
optical density at 550 nm (OD550) was measured
using enzyme-linked immunoassay (ELISA) microplate reader
(SLT-Labinstruments, Salzburg, Austria).
Measurement of peroxynitrite-induced oxidation of dihydrorhodamine
123.
The formation of peroxynitrite was measured by the
peroxynitrite-dependent oxidation of dihydrorhodamine 123 to rhodamine
123, as described previously (26)
. After a 60 min
incubation with 5 µM dihydrorhodamine 123 at 37°C, the
fluorescence of rhodamine 123 was measured using a fluorometer at an
excitation wavelength of 500 nm, emission wavelength of 536 nm (slit
widths 2.5 and 3.0 nm, respectively).
Nitrotyrosine Western blotting
Immunoblotting analysis of nitrotyrosine was performed using
rabbit antinitrotyrosine antibody (DBA, Milan, Italy) in 1 µg/ml in
PBS-T (PBS with 0.05% Tween 20) overnight at 4°C, as described
previously (26)
.
Measurement of mitochondrial respiration.
Cell respiration was assessed by the mitochondrial-dependent
reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] to formazan (26)
. Cells in 96-well plates were
incubated at 37°C with MTT (0.2 mg/ml) for 1 h. The extent of
reduction of MTT to formazan within cells was quantitated by measuring
OD550. 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, but is useful to
monitor changes in the general energetic status of the cell
(26)
.
Determination of DNA single-strand breaks.
The formation of DNA strand breaks in double-stranded DNA was
determined by the alkaline unwinding methods as described previously
(26)
. 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.
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 x g. The supernatant
was assayed for NAD+ using a modification of the
colorimetric method (26)
, in which NADH produced by
enzymatic cycling with alcohol dehydrogenase reduces MTT to formazan
through the intermediation of phenazine methosulfate. 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
(SLT-Labinstruments).
Immunohistochemical localization of nitrotyrosine
Tyrosine nitration was detected as described previously
(3)
in lung sections by immunohistochemistry. At the
specified time after injection of carrageenan, tissues were fixed in
10% buffered formalin and 8 µM sections were prepared from paraffin
embedded tissues. After deparaffinization, endogenous peroxidase was
quenched with 0.3% H2O2 in
60% methanol for 30 min. The sections were permeabilized with 0.1%
Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by
incubating the section in 2% normal goat serum in PBS for 20 min.
Endogenous biotin or avidin binding sites were blocked by sequential
incubation for 15 min with avidin and biotin (biotin blocking kit;
DBA). The sections were then incubated overnight with 1:1000 dilution
of primary antinitrotyrosine antibody (DBA) or with control solutions.
Controls included buffer alone or nonspecific purified rabbit
immunoglobulin G. Specific labeling was detected with a
biotin-conjugated goat anti-rabbit immunoglobulin G and avidin-biotin
peroxidase complex (Vectastain Elite ABC kit; DBA).
Myeloperoxidase activity
Myeloperoxidase (MPO) activity, an index of polymorphonuclear
leukocyte (PMN) accumulation, was determined as described previously
(27)
. Lungs tissues, collected at the specified time, were
homogenized in a solution containing 0.5% hexadecyl-trimethyl-ammonium
bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and
centrifuged for 30 min at 20,000 x g at 4°C. An
aliquot of the supernatant was then allowed to react with a solution of
tetra-methyl-benzidine (1.6 mM) and 0.1 mM
H2O2. The rate of change in
absorbance was measured by spectrophotometer at 650 nm. Myeloperoxidase
activity was defined as the quantity of enzyme degrading 1 µmol of
peroxide min-1 at 37°C and was expressed in
milliunits per gram weight of wet tissue.
Lipid peroxidation measurement
Malondialdehyde (MDA) levels in the lung tissue were determined
as an index of lipid peroxidation, as described by Okhawa et al.
(28)
. Lung tissue, collected at 4 h after the
injection of carrageenan, 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% sodium dodecyl sulfate, 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 x g for 10 min. The absorbance
of the supernatant was measured by spectrophotometry at 650 nm.
Nitric oxide synthase assay
Calcium-independent conversion of L-arginine to L-citrulline in
homogenates of the pleural macrophage cells and of the lungs (obtained
4 h after carrageenan treatment in the presence or the absence of
melatonin) served as an indicator of iNOS activity (29)
.
Cells were scraped into a homogenization buffer composed of 50 mM
Tris·HCl, 0.1 mM EDTA and 1 mM phenylmethylsulphonyl 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
homogenates as described (30)
. Briefly, homogenates (30
µl) were incubated in the presence of
[3H]-L-arginine (10 µM, 5 kBq/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.
Melatonin measurement
Melatonin levels in the pineal gland and the plasma were
determined as described previously (31)
. Pineals were
removed from all the animals, frozen on solid
CO2, and stored at -70°C until assayed for
melatonin content, using the procedure of Webb et al.
(31)
. The concentration of melatonin in the plasma samples
was determined using a highly specific antibody (Guildhay Stockgrand
Antisera, Guilford, U.K.) in a direct radioimmunoassay, as described
previously (32)
.
Cell culture medium, heparin, and fetal calf serum were obtained from Sigma (Milan, Italy). Perchloric acid was obtained from Aldrich (Milan, Italy). Primary antinitrotyrosine antibody was from Upstate Biotech (DBA). All other reagents and compounds used were obtained from Sigma.
Data analysis
All values in the figures and text are expressed as mean ±
standard error (SEM) of the mean of triplicate observations
from a single experiment. 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. In the experiments involving
histology or immunohistochemistry, the figures shown are representative
of at least three experiments performed on different days. The results
were analyzed by one-way analysis of variance, followed by a Bonferroni
post-hoc test for multiple comparisons. A P value
of less than 0.05 was considered significant
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RESULTS |
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). The effect of light exposure on melatonin levels observed in the
present paper agrees with previous observations (31)
.
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Endogenous melatonin protects against carrageenan-induced pleurisy
All carrageenan-injected rats developed an acute pleurisy,
producing 1.34 ± 0.13 ml of turbid exudate (Fig. 1
A). Trypan blue stain revealed 85 ± 2 x
106 PMNs/rat in comparison to sham rat
(2.4±0.2x 106/rat) (Fig. 1B
).
NOx levels were also significantly
(P<0.01) increased in the exudate after carrageenan
challenge (57±2.8 nmol/rat vs. 5.1±0.6 nmol/sham rat) (Fig. 2
A). Sham animals demonstrated no abnormalities in the pleural
cavity or fluid. The degree of peritoneal exudation and
polymorphonuclear migration was significantly enhanced in MEL-/- rats
(Fig. 1)
. The absence of melatonin did not cause significant changes in
these parameters in sham rats (Fig. 1)
. Melatonin treatment
significantly reversed the effect of melatonin depletion (Fig. 1)
. In
the MEL-/- rats, the carrageenan-induced exudate nitrate/nitrite was
unaffected (Fig. 2A
).
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In the lungs obtained from animals subjected to carrageenan-induced
pleurisy, a significant increase of iNOS activity was detected at
4 h (195±13 fmol/mg/min) (Fig. 2B
). iNOS activity was
unaffected in the MEL-/- rats (Fig. 2B
).
At 4 h after carrageenan administration, lungs were examined for
MPO activity (the latter being indicative of neutrophil infiltration)
and MDA in order to estimate lipid peroxidation. As shown in Fig. 3
, MPO activity and MDA levels (81±4 mU/100 mg wet tissue, 290 ± 7
µg/g wet tissue, respectively) were significantly
(P<0.01) increased in the lung at 4 h after
carrageenan injection when compared to sham rats (19 ±3.5 mU/100
mg wet tissue, 180 ± 4 µg/g wet tissue, respectively). MPO
activity and MDA levels were significantly (P<0.01)
enhanced to 126 ± 2.1 mU/100 mg wet tissue and 439 ± 6.2
µg/g wet tissue, respectively, in the MEL-/- rats (Fig. 3)
.
Melatonin treatment significantly reversed the effect of melatonin
depletion (Fig. 3)
.
|
At 4 h after the intrapleural injection of carrageenan, lung
sections were analyzed for the presence of nitrotyrosine.
Immunohistochemical analysis, using a specific antinitrotyrosine
antibody, revealed a positive staining in lungs from
carrageenan-treated rats (Fig. 4
A). Nitrotyrosine staining was substantially more pronounced
in the lungs of the carrageenan-treated MEL-/- rats (Fig. 4B
). Staining was absent in control tissue (data not shown).
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Endogenous melatonin protects against the cellular energetic
failure
In pleural macrophages obtained from rats at 4 h after
carrageenan injection, a significant nitrate/nitrite production was
detectable (41±4.2 µM) and was correlated with a significant
increase in iNOS activity (33±1.4 fmol mg/min, Fig. 5
). Using Western blotting, immunoreactivity for nitrotyrosine, an index
of the nitrosylation of proteins, was also detected. In pleural cells
from control animals (lane A) and the MEL-/- rat (lane B), no
positive immunoreactivity was found (Fig. 6
). However, there was a marked increase in the nitrotyrosine
immunoreactivity in pleural cells from carrageenan-treated rats (lane
C, Fig. 6
). Nitrotyrosine immunoreactivity was substantially more
pronounced in the pleural cell of the carrageenan-treated MEL-/- rats
(lane D, Fig. 6
). The carrageenan injection induced the nitration of
several proteins, most notably proteins of ~60 kDa and 135 kDa and in
several others in the range of 110 kDa (lane A, Fig. 3
). A rapid and
sustained production of peroxynitrite (51±1.9
pmol/min/106 cells) was also observed after
carrageenan-induced pleurisy (Fig. 7
A). This was associated with a significant increase in the
occurrence of single-strand breaks in the DNA (Fig. 7B
), a
reduction in mitochondrial respiration (Fig. 8
A) and a significant fall in the intracellular levels of NAD
(Fig. 8B
) in these cells. The absence of endogenous
melatonin significantly increased dihydrorhodamine 123 oxidation and
enhanced the carrageenan-induced DNA single-strand breakage (Fig. 7)
,
the decrease in cellular respiration, and in part the depletion of
intracellular levels of NAD+ (Fig. 8)
. Melatonin treatment
significantly reversed the effect of melatonin depletion (Figs. 6
and 7)
. The depletion of endogenous melatonin levels did not affect NO
production (Fig. 5)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 34)
and can exacerbate
ischemia/reperfusion injury in some (35)
but not other
(36)
experimental models, to our knowledge this is the
first investigation to study the importance of endogenous melatonin in
the development of acute inflammation (carrageenan-induced pleurisy).
Effect of in vivo depletion of endogenous melatonin
synthesis on nitrotyrosine formation in carrageenan-induced acute
inflammation
Reactive oxidants such as hydrogen peroxide, superoxide, and
hydroxyl radical contribute to tissue damage in inflammation (3
, 8
, 9
, 37)
. Pharmacological inhibitors of NOS have been shown to
reduce the development of carrageenan-induced inflammation and support
a role of NO in this model of inflammation (3
, 8
, 9
, 38)
.
More recent studies have shown the formation of peroxynitrite in
carrageenan-induced inflammation (3
, 8
, 9
, 39)
. Using
nitrotyrosine immunohistochemistry, this study has confirmed the
production of peroxynitrite in the lung of rats subjected to
carrageenan-induced pleurisy.
Moreover, we have observed that in the animals depleted of melatonin, a much more pronounced nitrotyrosine staining was seen, suggesting the presence of more biologically active peroxynitrite in the alveolar macrophage and in the airway epithelial cells. The more pronounced nitrotyrosine staining was not due to increased production of NO, as demonstrated by the measurement of lung iNOS activity.
Endogenous melatonin protects against pleural macrophage
dysfunction
It is well known that acute inflammatory processes, in which
vascular permeability increases and leukocyte migration occurs, several
mediators are involved, including neutrophil-derived free radicals such
as hydrogen peroxide, superoxide, and hydroxyl radical (3
, 8)
. It is proposed that reactive oxygen species, including
oxygen radicals, and nonradicals that are either oxidizing agents
and/or are easily converted into radicals, such as HOCl, ozone,
peroxynitrite, single oxygen, and
H2O2, can cause structural
alteration in DNA (40)
with consequent cellular
dysfunction (30)
. In ex vivo macrophages
harvested from the pleural cavity of rats subjected to
carrageenan-induced pleurisy, we recently reported the production of
NO, superoxide and peroxynitrite, concomitant with inhibition of
suppression of mitochondrial respiration, DNA single-strand breakage,
NAD depletion, and ATP depletion (26)
. Using
pharmacological inhibitors and scavengers, it appears that the most
important cytotoxic species under these conditions is peroxynitrite and
not NO or superoxide per se. This conclusion is based on the
simultaneous protective effects of NOS inhibitors (3
, 8
, 9)
and a cell-permeable superoxide dismutase scavenger compound
(39)
against the suppression of mitochondrial respiration
and by the protective effects of various peroxynitrite scavengers
(3
, 9)
Although a variety of endogenous antioxidant
systems in the cell are actively involved during the inflammatory
process, it is remarkable that depletion of melatonin alone exerted a
marked potentiating effect of peroxynitrite-induced cytotoxicity. These
findings agree with recent suggestions that endogenous antioxidant
systems play an important role against the oxidant-induced injury and,
specifically, against the peroxynitrite-induced injury (12
, 39)
. Several data support this hypothesis: 1)
enhancement of the appearance of DNA strand breaks; 2)
demonstration of a further decrease in the conversion of MTT to
formazan; and 3) the partially enhanced reduction of the
intracellular levels of NAD+. A variety of additive or synergistic
cytotoxic processes triggered by peroxynitrite may contribute to acute
and delayed cytotoxicity, and depletion of melatonin may also interfere
with these pathways.
Role of melatonin on NO, oxyradicals, and peroxynitrite formation
in carrageenan-induced acute inflammation
Melatonin is secreted principally by the pineal gland, and levels
are highest at night (13)
. Melatonin is involved in
various physiological functions, including the regulation of seasonal
reproduction, circadian rhythms, sleep, mood, performance, and the
immune response, which makes it likely that the pineal hormone may be a
factor in aging (13
, 41)
. Recently, numerous reports have
demonstrated the protective effects of melatonin in various models of
ischemia-reperfusion injury (42
, 43)
, inflammatory bowel
disease (44)
, and neurotoxicity (45
, 46)
.
Moreover, melatonin protects against shock induced by bacterial
lipopolysaccharide (20
, 43)
to inhibit thirst and fever
induced by endotoxin (47)
and to protect against
inflammation (9
, 24
, 25)
.
Melatonin is an effective scavenger of the hydroxyl radical and the
peroxyl radical (16
, 48)
, and may stimulate some important
antioxidative enzymes such as superoxide dismutase, glutathione
peroxidase, and glutathione reductase (16)
. Melatonin also
acts as a peroxynitrite scavenger and protects cultured cells against
peroxynitrite-induced injury (23)
. Thus, the mechanism of
the observed inflammatory alterations in the melatonin depleted animals
theoretically may be related to peroxynitrite, oxyradicals, NO, or a
combination of these.
In vitro studies in macrophages and other cell types have
established that endogenous antioxidants (such as glutathione) protect
only against a very large amount of NO, but not against lower levels of
NO production (49
, 50)
such as those relevant to the
ex vivo or in vivo conditions in our experiments.
It is conceivable that a more pronounced inhibition of mitochondrial
respiration by oxygen-derived free radicals and oxidants can lead to a
dysfunctional electron transfer, with more superoxide production from
the mitochondria. This effect would also lead to an enhancement of
peroxynitrite production, with subsequent increased cytotoxicity. It is
noteworthy that the production of superoxide, not the production of NO,
is the rate-limiting factor in peroxynitrite formation during
endotoxemia (49)
.
Furthermore, hydrogen peroxide prolongs the half-life of peroxynitrite
(51)
. In addition, recent reports have shown that
nitrotyrosine formation may also result from the reaction between
nitrite and myeloperoxidase (52)
. Thus, it is possible
that the cytotoxic effects observed in response to carrageenan
represent the sum of a complex interaction between various oxygen- and
nitrogen-derived radicals and oxidants.
In conclusion, this study demonstrates that endogenous melatonin plays an important role against the carrageenan-induced inflammation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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