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Institute of Pharmacology (S.C., G.C., I.S., A.P.C.), University of Messina, Italy; Institute of Pharmacology Sciences (S.S., A.M.), University of Milano, Italy; Department of Experimental Pharmacology (L.S.), University "Federico II", Naples, Italy; Department of Biomorphology (E.M.), School of Medicine, University of Messina, Italy
Address all correspondence and requests for reprints to: Salvatore Cuzzocrea, Ph.D., Institute of Pharmacology, School of Medicine, University of Messina, Piazza XX Settembre no. 4, 98123 Messina, Italy. E-mail: salvator{at}www.unime.it
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Introduction |
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and ERß) (1). Traditionally, E2 has been
associated to a proinflammatory activity because in the reproductive
tract, which represents E2 most studied target
organ, the hormone stimulates epithelial cell proliferation, increases
vascular permeability, edema and influx of macrophages, and eosinophils
in the uterine stroma. The recent observation of an
E2-dependent blockage of cytokine-dependent
induction of inducible nitric oxide synthase (iNOS) (2) led us to
speculate that estrogens may also exert antiinflammatory activity. An
evaluation of the data present in the literature partly supports this
view. It is well known that gender affects the susceptibility to
immune-mediated inflammatory diseases such as rheumatoid arthritis and
multiple sclerosis, and women have increased risk of developing these
diseases as compared with men. Clinical remissions of both diseases
occur during pregnancy, and estrogens are of benefit in rheumatoid
arthritic disease (3, 4), leading to hypothesis that sex steroids are a
factor in its pathogenesis (4, 6). In addition, estrogens were
shown to have an antiinflammatory activity also in several animal
models (7, 8, 9, 10, 11, 12, 13, 14, 15). On the other hand, females have an impressively higher
incidence of collagen diseases like systemic lupus erythematosus (SLE),
and the severity of SLE and other inflammatory pathologies
(e.g. gingivitis) are worsened by alterations in hormonal
balance at puberty and during menses (16). This suggests that estrogens
may also play a proinflammatory role in specific diseases or districts.
The mechanisms underlying estrogen effects in these complex pathologies
are not known; their comprehension is therefore of relevance in view of
the increasing number of women chronically treated with estrogens for
oral contraception or replacement therapy. Estrogens exhibit immunomodulatory effects, in vivo and in vitro. Murine models with type II collagen-induced arthritis suggest direct effects of estrogens on T cell function (17); in other models of SLE, estrogens are shown to increase anti-DNA and antiphospholopid titers (7, 18). The administration of estradiol suppresses the delayed-type of hypersensitivity, which is an antigen-specific T cell-mediated inflammation, whereas it enhances the antigen-specific antibody response. E2 has also an antiinflammatory property that is T cell independent, not mediated by modulation of corticosteroid production, but rather, by suppression of the production of leukocytes by the bone marrow (10). These effects may be linked to the gender difference in the incidence of collagen diseases such as SLE. Finally, few studies report on an effect of estrogen on the polymorphonuclear leukocytes (PMNs), which play an important role in inflammation (20, 21, 22).
The aim of the present study was to investigate the activity of estrogens in a well-known murine model of inflammation (carrageenan-induced pleurisy). The cellular and molecular mechanism of the carrageenan-induced pleurisy is well characterized. The early phase of the carrageenan-induced inflammation is related to the production of histamine, leukotrienes, platelet-activating factor, and possibly cyclooxygenase products, while the delayed phase of the carrageenan- induced inflammatory response has been linked to neutrophil infiltration and the production of neutrophil-derived free radicals and oxidants, such as hydrogen peroxide, superoxide, and hydroxyl radical, as well as to the release of other neutrophil-derived mediators (22, 23, 24, 25, 26) that causes tissue necrosis. To characterize the effect of E2 in this model of acute inflammation, we have determined the following endpoints of the inflammatory response in ovariectomized rats: 1) exudate formation; 2) PMN infiltration; 3) peroxynitrite formation (immunohistochemistry); 4) activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP); and 5) expression of the iNOS protein (immunohistochemistry) and activity, (7, 8), lipid peroxidation, and lung injury. In addition, we have investigated the effects of the systemic administration (pretreatment) of estrogen receptor antagonists (tamoxifen and ICI) on the above parameters of inflammation.
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Materials and Methods |
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Animals
Female Sprague Dawley rats (300–350 g; Charles River Laboratories, Inc.; Milan; Italy) were housed in a controlled
environment and provided with standard rodent chow and water. Animal
care was in compliance with Italian regulations on protection of
animals used for experimental and other scientific purposes (D.M.
116192) as well as with the EEC regulations (O.J. of E.C. L 358/1
12/18/1986).
Ovariectomy
All surgical procedures were performed under halothane (2%)
anesthesia followed by nitrous oxygen/O2
anesthesia for about 18 min. Ovariectomy (OVX) was performed through a
single dorsal midline cutaneous incision followed by bilateral muscle
incisions.
Experimental groups
OVX animals were treated with carrageenan (carrageenan group) or
vehicle (saline) (sham group). Both of this group were subdivided into
four subgroups each treated: 1 h before carrageenan with
E2 (50 µg/kg; ip) (carrageenan +
E2 group), 1 h before
E2 with tamoxifen (50 µg/kg ip) (carrageenan +
tamoxifen group), with ICI 182 780 (500 µg/kg ip) (carrageenan + ICI
group) or with vehicle.
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) were 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 by inhalation of CO2. The
chest was carefully opened and the pleural cavity rinsed with 2 ml of
saline solution containing heparin (5 U·ml-1)
and indomethacin (10 µg·ml-1). The exudate
and washing solution were removed by aspiration and the total volume
measured. Any exudate, which was contaminated with blood, was
discarded. The amount of exudate was calculated by subtracting the
volume injected (2 ml) from the total volume recovered. The leukocytes
in the exudate were suspended in PBS and counted with an optical
microscope in a Burker’s chamber after vital Trypan blue staining.
Measurement of nitrite/nitrate
Nitrite + nitrate production, an indicator of NO synthesis, was
measured in the exudate as previously described (26). Briefly, the
nitrate in the exudate was first reduced to nitrite by incubation with
nitrate reductase (670 mU·ml-1) 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 Labinstruments, Salzburg, Austria). Nitrate
concentrations were calculated by comparison with
OD550 of standard solutions of DMEM.
Histological examination
Lung biopsies were taken at 4 h after injection of
carrageenan. The biopsies were fixed for 1 week in buffered
formaldehyde solution (10% in PBS) at room temperature, dehydrated by
graded ethanol and embedded in Paraplast (Sherwood Medical, Mahwah,
NJ). Tissue sections (thickness 7 µm) were deparaffinized with
xylene, stained with trichromic Van Gieson and studied using light
microscopy (Dialux 22 Leitz, Milan, Italy).
Immunohistochemical localization of nitrotyrosine and PARS
Tyrosine nitration, an index of the nitrosylation of proteins by
peroxynitrite and/or oxygen-derived free radicals, was determined by
immunohistochemistry as previously described (27). At the end of the
experiment, the relevant organs were fixed in 10% buffered
formaldehyde, 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. The sections were then
incubated overnight with primary antinitrotyrosine antiserum (1:1000)
or with primary antipoly (ADP-ribose) antiserum (1:500) or with control
solutions. Controls included buffer alone or nonspecific purified
rabbit IgG. Some sections were also incubated with the primary antibody
(antinitrotyrosine) in the presence of excess nitrotyrosine (10
mM) to verify the binding specificity. Specific labeling
was detected with a biotin-conjugated goat antirabbit IgG and
avidin-biotin peroxidase complex. Diaminobenzidine was used as a
cromogen.
Myeloperoxidase (MPO) activity
MPO activity, an indicator of polymorphonuclear leukocyte (PMN)
accumulation, was determined as previously described (28). Four hours
after the intrapleural injection of carrageenan, lung tissues were
obtained and weighed. Each pieces 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 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 spectrophotometrically at 650 nm. MPO 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.
Malondialdehyde (MDA) measurement
MDA levels in the lung tissue were determined as an indicator of
lipid peroxidation (29). Lung tissues, collected at the specified time,
were 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 3,000 x
g for 10 min. The absorbance of the supernatant was measured
by spectrophotometry at 650 nm.
Determination of nitric oxide synthase activity
The calcium-independent conversion of L-arginine to
L-citrulline in the homogenates of lungs (obtained 4 h
after carrageenan treatment in the presence or the absence of
E2) served as an indicator of iNOS activity (30).
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 (31). Briefly, 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 Coulter, Inc. (Milan, Italy)
scintillation counter.
Statistical analysis
All values in the figures and text are expressed as mean ±
SE (SEM) of the mean of n
observations. 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 experimental days.
The results were analyzed by one-way ANOVA followed by a Bonferroni
posthoc test for multiple comparisons. A P value
less than 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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; Fig. 1A). The volume of the exudate was
visibly smaller in E2-pretreated rats (Table 1).
Trypan blue exclusion counting in the exudate revealed 87.3 ±
4.97 x 106/rat polymorphonuclear leukocyte
in comparison to sham rat (2 ± 0.8 x
106/rat) (Fig. 1A). Sham animals demonstrated no
abnormalities in the pleural cavity or fluid. The polymorphonuclear
migration was significantly reduced in rats pretreated with
E2 (Fig. 1A). E2
pretreatment did not cause significant changes in these parameters in
sham rats (Table 1; Fig. 1A). Coadministration of
E2 and tamoxifen or ICI significantly blocked the
effect of E2 (Table 1; Fig. 1A).
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, myeloperoxidase activity
(99.26 ± 11.7 mU/100 mg wet tissue) was significantly
(P < 0.01) increased at 4 h after carrageenan
injection compared with sham rats (15.6 ± 3.5 mU/100 mg wet
tissue). E2 pretreatment prevented the
carrageenan effect only in the absence of the two antagonists (Fig. 2B).
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) as well as infiltration of
the bronchial (Fig. 2B) and perivascular space (Fig. 2C) with PMNs.
E2 pretreatment reduced both tissue injury (Fig. 2D) and infiltration of the bronchial (Fig. 2E) and perivascular space
(Fig. 2F). Tamoxifen and ICI treatments significantly blocked the
effect of E2 (data not shown).
NOx levels were also significantly (P < 0.01)
increased in the exudate after carrageenan challenge (76.3 ± 4.3
nmol/rat vs. 9.98 ± 0.77 nmol/sham rat) (Fig. 3A). In the lungs obtained from animals
subjected to carrageenan-induced pleurisy, a significant increase of
inducible NO synthase activity was detected at 4 h (204 ±
2.97 fmol/mg/min) (Fig. 3B). NOx levels and iNOS activity were
significantly reduced in rats pretreated with E2
(Fig. 3, A and B). Tamoxifen and ICI treatments significantly blocked
the effect of E2 (Fig. 3, A and B).
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). E2 pretreatment
prevented the carrageenan effect only if administered in the absence of
the two antagonists (Fig. 4).
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) and in the apical portion of the
epithelial cell (Fig. 5B) as well as in inflammatory cells (Fig. 5C).
No positive staining was found in perivascular (Fig. 5D) and in the
apical portion of the epithelial cell (Fig. 5E) as well as in
inflammatory cells (Fig. 5F) in the lungs of the carrageenan-treated
rats when they were pretreated with E2.
Immunohistochemical analysis for PARS from lung sections obtained from
rats treated with carrageenan also revealed a positive brown staining
(see arrows) in perivascular (Fig. 6A) and in the apical portion of the
epithelial cell (Fig. 6B) as well as in inflammatory cells (Fig. 6C)
(Fig. 6A). In contrast, no positive staining for PARS was found in
perivascular (Fig. 6D) and in the apical portion of the epithelial cell
(Fig. 6E) as well as in inflammatory cells (Fig. 6F) in the lungs of
carrageenan-treated rats, which had been pretreated with
E2. Tamoxifen and ICI treatments significantly
blocked the effect of E2 (data not shown). Please
note that there was no staining for either nitrotyrosine or PARS in
lungs obtained from sham-operated rats (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We here show that E2 decreases iNOS activity and the consequent formation of free radical species. Previous studies have shown that NOS inhibitors reduce the development of carrageenan-induced inflammation and support a role for NO in the pathophysiology associated with this model of inflammation (27, 35, 36, 37, 38).
In addition, we demonstrate that E2 attenuates the nitrosilation of proteins in the lung of rats treated with carrageenan. 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 (39). There is, however, recent evidence 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 (40). Increased nitrotyrosine staining is considered, therefore, as an indication of "increased nitrosative stress" rather than a specific marker of the generation of peroxynitrite.
Reactive oxygen species (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 activates 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 called the PARS Suicide Hypothesis. There is recent evidence that the activation of PARS may also play an important role in inflammation (27, 42, 43). We demonstrate here that E2 attenuates the increase in PARS activity caused by carrageenan in the lung. Thus, we propose that the antiinflammatory effects of E2 reported here are, at least in part, due to the prevention of the activation of PARS.
Estrogens have been reported to have antioxidative activities that might in part explain some of the findings reported in the present study. However, the antioxidant activity of E2 is observed at pharmacological concentrations of the hormone and is not blocked by antagonists of the estrogen receptors. We thus believe that the lack of lung tissue injury in E2 -pretreated rats here reported is not due to estrogens antioxidant action because observed in rats treated with low doses of the hormone and prevented by the coadministration of two antagonists of estrogen receptor: tamoxifen and ICI 182,780. The effects here reported therefore are most likely receptor mediated.
In conclusion, our study shows that E2 may have an antiinflammatory activity in a well known model for the study of antiinflammatory drugs. This model will hopefully be useful for the dissection of the mechanisms by which estrogen might influence a number of human pathologies.
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Acknowledgments |
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Received August 24, 1999.
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