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Department of Anatomy and Neurobiology (A.J.B.-K., J.L.K.) and Sanders-Brown Center on Aging (J.N.K., F.F.H.), University of Kentucky, Lexington, Kentucky 40536; and Laboratory of Neurosciences, National Institute on Aging (S.C., M.P.M.), Baltimore, Maryland 21224
Address all correspondence and requests for reprints to: Dr. Annadora J. Bruce-Keller, Mn 210 Chandler Medical Center, University of Kentucky, Lexington, Kentucky 40536-0298. E-mail: abruce{at}pop.uky.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. Pretreatment with 17ß-estradiol, but not
17
-estradiol or progesterone, dose dependently attenuated microglial
superoxide release and phagocytic activity. Additionally,
17ß-estradiol attenuated increases in inducible nitric oxide synthase
protein expression, but did not alter nuclear factor-
B activation.
The antiinflammatory effects of 17ß-estradiol were blocked by the
antiestrogen ICI 182,780. Additionally, 17ß-estradiol induced rapid
phosphorylation of the p42/p44 mitogen-activated protein kinase (MAP
kinase), and the MAP kinase inhibitor PD 98059 blocked the
antiinflammatory effects of 17ß-estradiol. Overall, these results
suggest that estrogen receptor-dependent activation of MAP kinase is
involved in estrogen-mediated antiinflammatory pathways in microglial
cells. These results describe a novel mechanism by which estrogen may
attenuate the progression of neurodegenerative disease and suggest new
pathways for therapeutic intervention in clinical settings. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The microglial cell is a member of the monocyte/macrophage family and is the brain-resident immunocompetent cell. It is generally accepted that reactive microglia in the vicinity of neuronal injury are involved in the removal of debris from degenerating neurons (7). However, it has been hypothesized that these cells increase neuronal injury through the synthesis and secretion of agents that exacerbate the primary insult (8). For instance, microglial cells, like all phagocytic cells, are known to release short-lived cytotoxic factors, including nitric oxide, hydrogen peroxide, and superoxide radical (9). Additionally, there are numerous reports describing beneficial effects of neutralizing antibodies directed against adhesion molecules or other proinflammatory agents in experimental models of brain injury (10, 11).
Although data suggest that estrogen replacement therapy can decrease
the prevalence of and the severity of age-related neurodegenerative
disorders such as Alzheimer’s disease and stroke (12, 13), the mechanisms of estrogen’s effects are not understood.
Recent experiments using primary neuronal cultures have suggested
several potential ways in which estrogen could attenuate
neurodegeneration, including decreasing neuronal oxidative stress
(14, 15) or activating growth factor signaling pathways
(16, 17). However, even though activated glial cells are
probably a key factor in the development of brain disease, only minor
attention has been paid to the effects of estrogen on microglial cells.
To determine whether estrogen replacement therapy could protect the
brain from degeneration by modulating microglial responses, we
determined the effects of estrogen on in vitro paradigms of
microglial activation. In this report we characterize the dose- and
time-dependent effects of 17ß-estradiol on stimulus-induced
activation of primary microglial cells and N9 microglial cell lines. N9
microglial cells were used in addition to the primary microglial cells
because they represent a homogenous population of pure cells, and they
also have the advantage of a reliably high yield. Additionally, this
particular cell line has been used in the past to study many aspects of
microglial activation, including the roles of tyrosine phosphorylation
(18) and nuclear factor-B (NFB) activation
(19). Hence, in this study we characterize the effects of
17ß-estradiol on free radical release, phagocytic activity, and
inducible nitric oxide synthase (iNOS) protein expression levels; and
additionally describe estrogen-mediated signal transduction in
microglial cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Primary microglial cells were isolated from mixed glial cell cultures established from the forebrains of 1-day-old Sprague Dawley rats. In brief, forebrains were isolated under sterile conditions, the meninges were removed, and the tissue was cut into 0.5-mm pieces. The cells were dissociated by mild typsinization, followed by trituration with a fire-polished pipette. Mixed glial cells were grown to confluence in 75-cm flasks at 37 C in 5% CO2 in MEM medium supplemented with 10% heat-inactivated FCS and 2 mM glutamine. Microglial cells were removed from confluent astroglial layers flasks by differential panning (flasks rotated on an orbital shaker at 180 rpm for 20–30 min) and plated into polyethylenimine-coated 48-well plates at a density of 5 x 104 cells/cm2. After 1–2 h, the medium was changed to remove nonadherent cells, and experiments were initiated 24 h after plating. The purity of the microglial cultures (90–95%) was confirmed using antisera to CD11b (OX-42, Serotec, Oxford, UK).
Cell treatments
All experiments were conducted in serum-free, phenol red-free
medium. Cells (N9 microglial cells or primary rat microglial cells)
were allowed to acclimate to serum-free conditions for at least 12
h before the addition of any hormone (17ß-estradiol, 17-estradiol,
or progesterone). In pretreatment experiments, hormones were added to
the medium for 24 h before the addition of immunostimulant. In
cotreatment experiments, 17ß-estradiol was added to cells at the same
time as the immunostimulant. All hormones were solubilized as
10-mM stocks in 100% ethanol and diluted to 1
µM in sterile normal saline before application. Microglia
were activated by phorbol 12-myristate 3-acetate (PMA;
Sigma, St. Louis, MO), lipopolysaccharide (LPS; from
Escherichia coli, Sigma), or interferon-
(IFN; R&D Systems, Minneapolis, MN). PMA is an activator of protein
kinase C, and can cause microglial activation through the up-regulation
of iNOS expression and the posttranslational activation of NAPDH
oxidase (20). LPS is a major component of the cell wall of
Gram-negative bacteria, and as such is commonly used in studies of
immune cell activation and function (21). IFN is a
proinflammatory cytokine that is used experimentally to activate immune
cells, causing increased free radical production, cytokine release, and
major histocompatibility complex antigen expression
(21).
Microglial activation
Respiratory burst. Superoxide anion release in
vitro was assessed by measuring the reduction of 2 µg/ml nitro
blue tetrazolium (NBT) to a blue precipitate. Briefly, NBT was added to
culture medium 30 min after stimulation by PMA, LPS, or IFN and
allowed to incubate for another 60 min. The medium was removed, cells
were lysed in distilled water by sonication, and OD was determined
immediately at 562 nm. Excess (4 U/ml) superoxide dismutase was added
to adjacent cultures, and the amount of NBT reduced in the presence of
superoxide dismutase was subtracted as background. OD values were
converted to a percentage of the control for statistical analysis.
Control values generally did not vary by more than 10% in a given
experiment, whereas background values typically ranged from to
0.050–0.100 absorbance units.
Phagocytosis. The phagocytic activity of microglial cells was determined using blue latex beads (0.8 µm; Sigma). A suspension of beads was added directly to the culture medium 12–18 h after stimulation by LPS and allowed to incubate for 90 min. Cells were rinsed to remove nonadherent beads and lysed in distilled water using a sonicator. The OD of the cell lysate was determined immediately at 562 nm. OD values were converted to a percentage of the control for statistical analysis. Control values generally did not vary by more than 10–15% in a given experiment, whereas background values typically ranged from to 0.025–0.050 absorbance units.
iNOS expression. iNOS protein levels were measured 12–18 h after stimulation by LPS by Western blot analysis, as described below.
Western blot analysis
After treatment, cells were rinsed in ice-cold PBS, homogenized
in protein extraction buffer [0.05 M Tris-HCl, pH 7.4,
containing 1% Triton X-100, 250 mM mannitol, 100
µM sodium orthovanadate, 1 mM dithiothreitol,
5 µg/ml aprotinin, 5 µg/ml leupeptin, and 100 µM
phenylmethylsulfonylfluoride (PMSF)] and denatured in SDS, and
equivalent amounts of protein were electrophoretically separated on
12% polyacrylamide gels. The proteins were transferred to
nitrocellulose, immunoreacted with the indicated primary antisera
(iNOS, Transduction Laboratories, Inc.; phospho-ERK,
New England Biolabs, Inc., Beverley, MA), and further
processed by incubation in horseradish peroxidase-conjugated secondary
antisera. Nitrocellulose was developed using enhanced chemiluminescence
kit (Amersham Pharmacia Biotech). Images of blots were
captured with an Apple scanner, and densitometric analysis of bands was
performed using Scion software for Macintosh (Scion Corp.,
Frederick, MD). Background values were subtracted, and multiple blots
were combined for statistical analysis.
Immunocytochemical procedures
For immunostaining, paraformaldehyde-fixed cells were exposed to
blocking solution (5% serum in PBS containing 0.1% Triton X-100) for
60 min, and then cells were incubated for 24 h in the presence of
primary antisera at 4 C. Several antibodies were used, including
antiphospho-ERK (New England Biolabs, Inc.) and OX42
(Serotec). Additionally, separate antibody solutions
directed against two distinct epitopes of estrogen receptor (ER)
were used (MC20, which recognizes an epitope at the carboxyl-terminus
of the protein, and H-184, which recognizes an epitope at the
amino-terminus of the protein; both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA). To control for nonspecific
staining, additional cells were subjected to the immunostaining
procedure without primary antisera. Cells were then incubated for
1 h in the presence of biotinylated secondary antibodies at room
temperature. After the secondary antisera, cells were exposed to
Oregon-Green- conjugated avidin D (Molecular Probes, Inc., Eugene, OR) at room temperature for 30 min and washed
three times with distilled water. Fluorescence was quantified using a
Leica Corp. (Rockleigh, NJ) TCS SP confocal laser scanning
microscope (excitation at 488 nm and a 510-nm barrier filter) with a
x63 water immersion objective. Fluorescent images were converted to
gray scale for analysis using Scion software. For each experiment,
there were four to six dishes per treatment, and three scans
(approximately six to nine cells per x63 microscope field) per dish
were taken. Relative fluorescence intensity per cell body was
quantified using Scion software.
Electrophoretic mobility shift assay
Cell extracts containing DNA-binding proteins were prepared as
previously described (22). For NFB gel shifts, equal
amounts of protein (5 µg) were incubated in a 20-µl reaction
mixture containing 20 µg BSA, 1 µg poly(dI-dC), 2 µl buffer 1
(20% glycerol, 100 mM KCl, 0.5 mM EDTA, 0.25%
Nonidet P-40, 2 mM dithiothreitol, 0.1% PMSF, and 20
mM HEPES, pH 7.9), 4 µl buffer 2 (20% Ficoll 400, 300
mM KCl, 10 mM dithiothreitol, 0.1% PMSF, and
100 mM HEPES, pH 7.9) and 20,000–50,000 cpm
32P-labeled oligonucleotide corresponding to the
B site (5'-AGT TGA GGG GAC TTT CCC AGG C-3'; Promega Corp., Madison, WI). After 20-min incubation at room
temperature, the reaction products were separated on a 7%
nondenaturing polyacrylamide gel. The specificity of binding was
examined by competition experiments, in which extracts of control and
estrogen-treated cells were incubated with the labeled probe in the
presence of 100-fold excess of unlabeled NFB or the AP-1 consensus
sequence (5'-CGC TTG ATG AGT CAG CCG GAA-3').
Statistical analyses
All data were analyzed using one-way ANOVA, followed by
Scheffés post-hoc analysis to determine statistical
significance. P < 0.05 was designated statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is a member of the nuclear receptor superfamily and can act
as a ligand-activated transcription factor to regulate the expression
of target genes. Although ERs have been demonstrated in various immune
cells, including peripheral macrophages (4), the pattern
of ER expression in microglial cells has not been characterized. To
determine whether microglia express ERs, we conducted
immunohistochemical and Western blot analyses on primary rat microglia
and the N9 microglia cell line. Primary rat microglial cultures were
prepared as described in Materials and Methods, and the
purity of the cell population was confirmed by immunoreactivity to the
microglial surface marker OX42. The presence of ER on primary
microglia was then documented by immunocytochemistry using antisera
against ER. Two different solutions of antibodies directed against
ER were used (MC20, which recognizes an epitope at the
carboxyl-terminus of the protein, and H-184, which recognizes an
epitope at the amino-terminus of the protein; both from Santa Cruz Biotechnology, Inc.) to confirm the expression of ER in
microglial cells. Examinations of culture dishes confirmed that
95–99% of cells in the microglial cell cultures exhibited specific
staining for both OX42 and ER (Fig. 1A
). Likewise, Western blot analyses of
N9 whole cell lysates revealed an approximately 62-kDa band
corresponding to ER (Fig. 1B).
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), with
concentrations of 17ß-estradiol between 0.1–10 nM
causing statistically significantly effects. Additionally, pretreatment
with 17ß-estradiol resulted in a dose-dependent attenuation of
PMA-induced superoxide production (data not shown). Concentrations of
17ß-estradiol from 10 pM to 1 µM did not
affect basal levels of superoxide production (Fig. 2).
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). Interestingly, however,
coadministration of high doses of 17ß-estradiol (1 µM)
significantly increased superoxide production in response to LPS (Table 1). Pretreatment of N9 cells with increasing concentrations of the
female sex hormone progesterone did not affect superoxide release
induced by LPS (Table 2). Likewise, the
estrogen analog 17-estradiol, which does not bind ERs, but has been
shown to have neuroprotective properties (14), did not
have any effect on superoxide release in N9 cells elicited by LPS
(Table 2).
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), and, as was observed with superoxide
release, pretreatment with 1 or 10 nM
17ß-estradiol significantly attenuated LPS-induced phagocytosis (Fig. 3). However, 100 pM 17ß-estradiol, which was
effective in preventing superoxide release, did not significantly alter
LPS-induced phagocytosis (Fig. 3).
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), although
a moderate exacerbation of LPS-induced phagocytic activity was observed
at high doses of 17ß-estradiol (1 µM). Pretreatment of
N9 cells with either progesterone or 17-estradiol did not have any
effect on LPS-induced phagocytosis (Table 2).
Effects of 17ß-estradiol on NFB activation and iNOS protein
expression
NFB is a widely expressed transcription factor that regulates
the expression of a variety of genes involved in immunity and
inflammation (see Ref. 28 for review). Increased NFB
activation occurs during microglial activation and can increase the
expression of gene products involved in the generation of neurotoxic
agents. For example, the gene encoding iNOS is responsive to NFB
activation (29). iNOS is capable of generating elevated
and sustained levels of nitric oxide, and increased iNOS expression is
characteristic of many neurodegenerative conditions (30).
To determine whether 17ß-estradiol modulates NFB activity, we
analyzed the effect of 17ß-estradiol pretreatment on LPS-induced
NFB activation. Application of 1 µg/ml LPS for 30 min caused a
robust increase in NFB activation (Fig. 4), which was not modulated by 1
nM 17ß-estradiol pretreatment (Fig. 4). In contrast,
application of 1 nM 17ß-estradiol significantly decreased
LPS-induced increases in iNOS protein levels when measured after
18 h (Fig. 5, A and B).
17ß-Estradiol did not alter basal levels of NFB activation (data
not shown) or iNOS protein expression (Fig. 5B)
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). Similarly, application of ICI
inhibited the ability of 17ß-estradiol to attenuate PMA- and IFN
-induced superoxide production (Fig. 6, C and D), but did not affect
basal levels of superoxide production (Fig. 6A). Pretreatment (24 h) of
N9 cells with ICI 182,780 by itself did not alter basal or LPS-, PMA-,
or IFN-induced superoxide production (Fig. 6, A–D).
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-induced
superoxide production (Fig. 6, B–D). Pretreatment (24 h) of N9 cells
with PD 98059 by itself did not alter basal levels or LPS-, PMA-, or
IFN--induced superoxide production (Fig. 6, A–D).
Additionally, we analyzed N9 cells for increased MAPK phosphorylation
after 17ß-estradiol treatment. As expected, within 5 min of
17ß-estradiol application, there was a significant increase in MAPK
phosphorylation (Fig. 7, A and B).
Together, these data indicate that 17ß-estradiol-mediated inhibition
of microglial activation occurs in an ER- and MAPK-dependent
manner.
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, A and B). Likewise,
the ability of 17ß-estradiol to inhibit superoxide release was
significantly attenuated by both the ER blocker ICI 182,780 and the
MAPK inhibitor PD 98059 (Fig. 8, A and B), and was not observed if
17ß-estradiol and PMA were coadministered (data not shown). Lastly,
application of 1 nM 17ß-estradiol for 30 min increased
MAPK phosphorylation in primary microglia (Fig. 9, A and B), and 17ß-estradiol-induced
increases in MAPK phosphorylation were blocked by 30-min pretreatments
with ICI 182,780 or PD 98059 (Fig. 9B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-estradiol or progesterone and is dependent on
ER activation and subsequent activation of the p42/44 MAPK family of
transcription factors. Overall, these results describe a novel
mechanism by which estrogen could prevent neurodegeneration in the
brain and suggest that 17ß-estradiol’s antiinflammatory properties
may be partially responsible for the reported increases in brain
function elicited by estrogen replacement therapy in clinical
settings Although estrogen has been shown to have neuroprotective effects in numerous experimental models, the majority of clinical data supports the protective effects of estrogen replacement therapy in women who may at risk for Alzheimer’s disease (AD) (12, 13). It is well documented that women have a greater risk than men to develop AD, and considering that the mean age for menopause in women is 54 yr, it is possible that women spend 25–33% of their lives in as estrogen-deficient state. Conversely, men have a continuing endogenous source of an endogenous estrogen substrate until very late in life through the intracerebral aromatization of testosterone to estrogen. This suggests that postmenopausal estrogen deprivation has a role in the development of AD, and accordingly, retrospective studies have demonstrated an inverse correlation between estrogen replacement therapy and incidence of AD (12, 13). Although the mechanisms of estrogen-related neuroprotection are not yet known, several possibilities exist. Estrogen is directly neuroprotective in vitro (14, 15, 16, 17) and also has vascular effects (31) that could be important. Additionally, it has been shown that estrogen treatment increases ß-amyloid precursor protein expression while decreasing toxic ß peptide generation in neurons (32, 33). Although all of these possibilities are important and may represent aspects of estrogen-mediated neuroprotection, a central confounding issue in AD research is the presence of diffuse amyloid plaques in normal aged brain, which are not associated with any pathology or dementia. Therefore, it seems likely that AD is either caused or accelerated by a process that transforms diffuse amyloid deposits into senile plaques, and a role for activated microglia has been proposed for this exact process (9, 34, 35, 36). Hence, the ability of estrogen to modulate microglial activation could be a key mechanism in estrogen-mediated attenuation of AD progression. Results from this study along with reports describing the antiinflammatory properties of endogenous estrogen outside of the brain (4), support the hypothesis that nanomolar levels of estrogen could delay the progression of AD by attenuating microglial activation.
Interestingly, in this study high (micromolar) concentrations of
17ß-estradiol exacerbated LPS-induced microglial activation when the
drugs were coadministered, highlighting both the dose and time
sensitivity of estrogen treatment. Such divergent effects of different
doses of 17ß-estradiol have also been reported for macrophage and T
cell activation (4). The sensitive dose and time
dependence of 17ß-estradiol on immune cells may be a reflection of
multiple signal transduction pathways that can be harnessed by estrogen
under varying circumstances. Likewise, different cell types in the
immune system do not respond in the same way to 17ß-estradiol
treatment (4). The divergent effects of 17ß-estradiol on
different cells is also highlighted by the observation that although
nanomolar concentrations of 17ß-estradiol did not alter NFB
activation in this study, micromolar doses (1–10 µM)
have been shown to block LPS-induced NFB activation in astroglial
cells (37). Additionally, although our data concerning
iNOS activation in microglial cells are in agreement with previous
reports using showing 17ß-estradiol-induced decreases in iNOS
(38, 39), other studies have shown that 17ß-estradiol
actually increases the expression of endothelial and neuronal subtypes
of nitric oxide synthase (39, 40). This divergence in the
effects of 17ß-estradiol on different subtypes of NOS could be a
reflection of the different mechanisms necessary to activate the
various enzymes, but also suggests that modulation of endogenous
estrogen could have clinically divergent results across different cell
types. All of these experimental observations underlie the necessity to
examine and fully characterize the signal transduction mechanisms of
estrogen signaling in different cell types, so that appropriate
therapeutic agents can be devised.
There are potentially multiple mechanisms, both genomic and nongenomic,
by which estrogen could modulate microglial activation. In
characterizing the signal transduction of 17ß-estradiol’s effects in
microglial cells, this study highlights the importance of several key
pathways, beginning with the role of ER. 17-Estradiol, which does
not activate ER, but has been shown to be neuroprotective
(14), did not exhibit any antiinflammatory effects in our
studies. Additionally, the effects of 17ß-estradiol were nearly
completely blocked by ICI 182,780, suggesting that activation of ER is
a necessary step in mediating microglial activation. Accordingly, our
studies demonstrate the presence of ER on microglial cells. Although
other reports have documented ER expression in cultured astrocytes
and oligodendrocytes (41), this is the first report of
ER on microglial cells. The expression of ERß was not examined in
this study. However, ERß is also expressed on primary cultured
microglial cells (42), begging the question as to whether
the reported effects of 17ß-estradiol on microglial cells are
mediated through ER or ERß. Genetically engineered mice deficient
in one or both receptors would be an excellent source of primary
microglia to use to determine whether the antiinflammatory effects of
17ß-estradiol are mediated through either ER or ERß, or an
entirely novel estradiol-binding receptor.
Although estrogen is generally thought to act via nuclear translocation of its receptors (1), increasing evidence now demonstrates that estrogen has rapid effects on intracellular signal transduction pathways. For example, estradiol has been shown to increase cAMP levels in breast cancer cells (43), modulate neuronal calcium (44) and kainate (45) currents, and increase phosphorylation of the cAMP response element binding protein in rat brain (46). Additionally, 17ß-estradiol has been shown to activate several kinase pathways, including stress-activated kinases (JNK/SAPK) (47) and p42/44 MAPKs (16, 47). Indeed, 17ß-estradiol-induced activation of the MAPK pathway in neurons has been shown to be especially important for estrogen-mediated neuroprotection in cortical cells (16, 17). Likewise, our results demonstrate that in microglial cells, 17ß-estradiol causes rapid, ER-dependent activation of MAPK and further indicate that such activation is necessary for the subsequent antiinflammatory effects of 17ß-estradiol. Interestingly, in immunocompetent cells, MAPK pathways are activated in response to activation of high affinity IgG receptors (48), colony-stimulating factors (49), and amyloid ß-peptides (24). Hence, estrogen-mediated intracellular signaling and inflammatory pathways in microglia could intersect at the MAPK pathway, leading to an attenuation of inflammatory responses dependent on MAPK activation. In this regard, neurotropins, which signal through MAPK activation, have been shown to decrease parameters of microglial activation, including CD40 (50) and major histocompatibility complex expression (51), and nitric oxide release (52). However, the exact mechanisms by which increased MAPK activation elicits a decrease in subsequent microglial activation is not known, but may involve negative feedback of MAPK activation via up-regulation of MAPK phosphatase (53) or increased expression of genes of unidentified antiinflammatory proteins. This last possibility seems likely, in that our results suggest that the antiinflammatory effects of 17ß-estradiol rely at least in part on changes in gene expression. For instance, no attenuation in microglial activation was observed when 17ß-estradiol was coadministered with LPS, even with relatively long-term changes such as increased phagocytic activity or iNOS expression.
The mechanism by which estrogen causes the activation of MAPK is also unclear. In our studies the antiestrogen ICI 182,780 blocked both antiinflammatory effects and activation of MAPK, indicating that the estrogen/ER complex mediates MAPK activation. This is in agreement with published reports on the effects of estrogen on MAPK activation in neurons (16, 17) and breast cancer cells (54). These data clearly indicate a cytosolic action of ER, and recent reports further suggest that ER can exist in cytosolic complexes with heat shock protein 90, Raf, or MEK (17). On the other hand, sequence analyses have also indicated that ER are phosphorylated by Src kinases in vitro (55, 56), and furthermore, that activation of the MAPK pathway may regulate ER DNA binding and transcriptional activity (56, 57, 58). Taken as a whole, these studies suggest multiple mechanisms of cross-talk between estrogen and MAPK pathways, which are seemingly important for the manifestations of estrogen’s effects on microglial cells. However, future studies are needed to delineate the exact mechanisms of these interactions and their implication for human disease.
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Acknowledgments |
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Footnotes |
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Received February 10, 2000.
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)
or 1 µg/ml LPS (
). Superoxide production was quantified by
measuring NBT oxidation 90 min after the administration of LPS. Values
are expressed as a percentage of the control and represent the mean and
SEM of five to seven experiments, with treatments measured
in quadruplicate, followed by ANOVA with Scheffés
post-hoc tests. **, Significant (P
< 0.01) decreases in PMA-evoked superoxide production by
17ß-estradiol.
