© 1999 American Heart Association, Inc.
Cellular Biology |
Estrogen Stimulates Neuronal Nitric Oxide Synthase Protein Expression in Human Neutrophils
From the Cardiovascular Research and Hypertension Laboratory (M.G.-D., T.d.F., A.J., M.M., J.F., L.S.d.M., F.G.-F., M.d.M.A., L.R., R.G., S.C., A.L.-F.) and Obstetrics and Gynecology Department (J.D.-R., G.G.-G.), Fundación Jiménez Díaz. Madrid, Spain.
Correspondence to Dr Antonio López-Farré, Cardiovascular Research and Hypertension Laboratory Fundación Jiménez Díaz Avda Reyes Católicos, 2. 28040 Madrid, Spain. E-mail alopez{at}fjd.es
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Abstract |
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Abstract—Recent studies have postulated the contribution of nitric oxide (NO) released by the endothelium to the beneficial effects of estrogen. Despite a neuronal-type NO synthase (nNOS) described in neutrophils, less is known about the effect of estrogen in these cells. The aim of the present study was to analyze the expression of nNOS protein in human neutrophils under different estrogenic conditions. We first analyzed nNOS expression in neutrophils obtained from premenopausal women. During the first 2 days of the follicular phase (low circulating estrogen concentrations), nNOS expression in neutrophils was reduced with respect to that found in neutrophils obtained from the same donors during the ovulatory phase (high circulating estrogen concentrations). Moreover, the expression of nNOS protein in neutrophils obtained from postmenopausal women after transdermal estrogen therapy was markedly enhanced with respect to that observed before the treatment. In vitro incubation of neutrophils derived from men for 6 hours with 17ß-estradiol (10-10 to 10-8 mol/L) upregulated the expression of nNOS protein. The 17ß-estradiol receptor antagonists, tamoxifen (10-8 mol/L) and ICI 182780 (10-8 mol/L), inhibited the upregulation of nNOS protein induced by 17ß-estradiol. The putative functional implication was denoted by a reduced expression of the CD18 antigen on the surface of 17ß-estradiol–incubated neutrophils, which was accompanied by a decreased adhesive capacity. Both effects were prevented by an NO antagonist. In conclusion, the in vivo levels of circulating estrogen concentrations seem to be associated with the level of nNOS protein expression in neutrophils from women. Moreover, low doses of 17ß-estradiol upregulate nNOS protein expression in neutrophils from men. The increased ability of 17ß-estradiol–incubated neutrophils derived from men to produce NO reduced their adhesive properties.
Key Words: estrogen • leukocyte • nitric oxide synthase • premenopause • postmenopause
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Death from cardiovascular disease is relatively rare in premenopausal women compared with age-matched men.1 However, after menopause the risk of coronary heart disease increases dramatically.2 3 Several data from ongoing clinical studies have shown that hormone replacement therapy reduces cardiovascular disease in postmenopausal women, but the final causative mechanism remains unclear.4 5 6 The benefits of estrogen have been attributed to its effects on lipids and lipoproteins. However, the change in the lipid profile reportedly accounts for only 25% to 50% of the protective effect of estrogen against cardiovascular disease,7 and new data suggest that estrogen may also exert its beneficial effects through a direct action on the vessel wall.8 9
Nitric oxide (NO) is generated by the metabolic conversion of L-arginine to L-citrulline by the activity of the enzyme NO synthase (NOS). Recent molecular cloning studies have reported that human DNA encodes 2 distinct constitutive NOS isoforms, one from neurons (nNOS) and the other from endothelial cells (eNOS), as well as an inducible NOS (iNOS) isoform that requires cytokines or endotoxin activation for its expression.10 11
Although the nNOS and eNOS isoforms were initially described as
constitutive, in recent years the inducible character of both
constitutive isoforms has been shown. In this regard, increasing
evidence indicates that nNOS expression can be regulated by various
physiological and
pathophysiological conditions, including
sympathetic activity and acute heat stress.12 13 Moreover,
tumor necrosis factor-
downregulates eNOS expression,14
which could be upregulated by several factors, including
estrogen.15
Similarly to endothelial cells, neutrophils produce NO by the activity of a constitutive NOS isoform. Recently, Wallerath et al16 have identified the constitutively expressed NOS isoform in neutrophils as neuronal type. The NO released by neutrophils has been demonstrated to prevent neutrophil adhesion to vascular endothelium, to control aggregation of neighboring platelets and, in the absence of endothelium, to produce vasodilatory effects.11 17 18
Estrogen causes short-term coronary vasodilatory effects in humans mediated by an increase in NO production.19 Moreover, long-term exposure to estrogen increases acetylcholine-mediated coronary vasodilation in nonhuman primates, male-to-female transsexuals, and postmenopausal women.20 21 22 These effects have been attributed to an increase in NOS expression by the endothelium.9 23 However, despite the growing interest in the action of estrogen on endothelial cells, less is known about the effect of estrogen on neutrophils. In this regard, several data support the involvement of estrogen in the modification of neutrophil activity during the menstrual cycles and pregnancy.24 25 Binding studies performed by Klebanoff26 demonstrated the presence of estrogen receptors (ERs) on neutrophils. However, at present there are no studies about the effect of estrogen on the NO-generating system in human neutrophils. Therefore, the aim of the present study was to evaluate the effect of estrogen on nNOS expression in human neutrophils. For this purpose, we analyzed the expression of nNOS protein in neutrophils obtained from premenopausal and postmenopausal women under different situations of circulating estrogen concentrations. Moreover, in in vitro experiments the effect of estrogen on the expression of nNOS protein in neutrophils obtained from men was also determined.
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Materials and Methods |
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Neutrophil Isolation
Neutrophils were isolated as previously described18 27 from peripheral blood from the following groups: (1) 10 healthy premenopausal women (25 to 40 years of age) during different stages of the menstrual cycle, (2) 10 postmenopausal women (45 to 65 years of age) before and after 4 months of hormone replacement therapy, and (3) from 10 healthy men (25 to 40 years of age) for in vitro estrogen incubations. The protocols were approved by the Institutional Ethics Committee.
The nNOS protein determination was performed by Western blot as reported.14 28
Neutrophils Obtained From Premenopausal Women
Neutrophils were obtained from the same donor at the following 2
different points: (1) the ovulatory phase, determined by the urinary
peak of luteinizing hormone (LH) (by disposable test sticks; Donnatest
Wyntek Diagnostic), and (2) within the first 2 days of the
follicular phase (menstruation). Serum estradiol concentrations were
determined with an ELISA kit (Oxford Biomedical Research, Inc).
In Vivo Estradiol Treatment in Postmenopausal Women
All women had not had a menstrual period for at least 4 years
and had not received any hormone replacement treatment within this
period of time. Neutrophils were obtained before and after 4 months of
hormone replacement therapy (50 µg/day estradiol transdermal patches;
Estraderm Matrix 50, Novartis Pharma SA).
Medroxyprogesterone acetate (Provera, Upjohn) was
administered orally for 10 days of each cycle (10 mg/day). Blood
samples were obtained 2 days before women received the progestin.
In Vitro Incubation of Neutrophils From Healthy Men With
Estrogen
Neutrophils (5x106 cells/tube) from men
were incubated with increasing concentrations of 17ß-estradiol
(10-10 to 10-5 mol/L) or
the solvent of 17ß-estradiol (ethanol at a final concentration
<0.01%) for 6 hours in an incubator at 37°C with 5%
CO2. ER protein expression in male neutrophils
was determined by Western blot as already described for the nNOS
protein using the polyclonal antibody MC-20 (Santa Cruz Biotechnology
Inc). The antisera may recognize a common sequence of and ß
receptor subtypes.29
Determination of NOS Activity
The NOS activity was determined as the conversion of
[3H]L-arginine into
[3H]L-citrulline as previously
described.18 27 Neutrophils from men were incubated with
17ß-estradiol (10-8 mol/L) or with the solvent
(ethanol at final concentration <0.01%), for 6 hours at 37°C.
During the last 45 minutes, neutrophils were loaded with
[3H]L-arginine (1 µCi/mL)
followed by a 20-minute incubation at 37°C.
CD18 Antigen Expression and Adhesion Assays
The presence of the CD18 antigen in the surface of neutrophils
was determined by flow cytometry as described.30 Male
neutrophils were incubated with 10-8 mol/L
17ß-estradiol or the solvent ethanol (final concentration, <0.01%)
for 6 hours at 37°C. For adhesion experiments, 2 µCi/mL
51Cr (Amersham Life Science, Ltd) was added to
the incubation medium in the last 45 minutes to label neutrophils.
51Cr-labeled neutrophils (0.5 mL) were added on a
plastic surface for 30 minutes at 37°C under static conditions as
described.30
Statistical Methods
Results are expressed as mean±SEM. Unless otherwise stated,
each value corresponds to a minimum of 6 different experiments. The
statistical significance was determined by ANOVA with the Bonferroni
correction for multiple comparisons or a Student t test,
paired or unpaired. A P value <0.05 was considered
statistically significant.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Results |
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nNOS Expression in Neutrophils From Premenopausal and Postmenopausal Women
We first determined the expression of nNOS protein by Western blot in neutrophils obtained from premenopausal women at 2 different phases of the menstrual cycle, one in which circulating concentrations of estrogen are elevated (ovulatory phase) and the other within the first 2 days of the follicular phase (menstruation), when circulating estrogen concentrations fall.
Neutrophils obtained from premenopausal women expressed a 155-kDa
nNOS-like protein (Figure 1A
). As
shown in Figure 1A and 1B, during the ovulatory phase (positive
urinary detection of LH), the level of nNOS expression in neutrophils
was markedly enhanced with respect to that found in the neutrophils
obtained from the same donor during the first 2 days of the follicular
phase. At ovulation, serum estradiol levels were 520±30 pg/mL
(1.9x10-9 mol/L). In the first 2 days of the
follicular phase, serum estradiol concentration fell to 160±10 pg/mL
(5.9x10-10 mol/L).
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P<0.05 with respect to premenopausal women in the
ovulatory phase. A.U. indicates arbitrary units.
We then determined the effect of estrogen therapy on nNOS expression in
neutrophils obtained from postmenopausal women before and after
transdermal estradiol treatment. In all cases, each individual was also
used as her own control before and after the treatment. In these
experiments, neutrophils obtained after estrogen replacement therapy
showed a significant increase in nNOS protein expression (Figure 1A
and 1B), which was accompanied by a higher ability to produce
NO ([3H]L-citrulline accumulation,
pretreatment, 32±4%; posttreatment, 48±4%; P<0.05).
Serum estradiol levels after estradiol treatment were 125.4±6.2 pg/mL,
which on a molar basis corresponds approximately to
5x10-10 mol/L of estradiol. Before treatment,
estradiol serum concentration was 15.3±2.1 pg/mL
(5.6x10-11 mol/L).
The monoclonal antibody used did not cross-react with the eNOS or iNOS
isoform, because an nNOS protein band was undetectable in
homogenates of human endothelial cells and
lipopolysaccharide (LPS)–treated rat macrophages
(Figure 2). The monoclonal antibody used
in our experiments specifically recognized the nNOS isoform (155 kDa)
obtained from homogenates of rat pituitary (Figure 2).
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nNOS Protein Expression in 17ß-Estradiol–Incubated Neutrophils
From Men
Under basal conditions, the expression of nNOS-like protein (155
kDa) was detected in neutrophils from men (Figure 3A and 3B). Although incubation with
17ß-estradiol (10-8 mol/L) did not
significantly changes nNOS protein expression in the first 4 hours,
nNOS protein was markedly enhanced 6 hours after the addition of
10-8 mol/L 17ß-estradiol and on the following
times (Figure 3A and 3B). No effect of ethanol (final ethanol
concentration, <0.01%) on nNOS expression by neutrophils from men was
detected in the different periods of incubation (data not shown).
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Incubation of neutrophils derived from men with 17ß-estradiol
(10-10 to 10-8 mol/L)
enhanced the expression of nNOS protein in a dose-dependent fashion
(Figure 4A and 4B). The maximal increase
in nNOS protein expression was reached with 10-8
mol/L 17ß-estradiol (Figure 4A and 4B). Conversely, higher
doses of 17ß-estradiol (10-7 to
10-5 mol/L) tended to reduce nNOS protein
expression to the basal levels (Figure 4A and 4B).
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The above-described effect of estradiol on nNOS protein
expression was stereospecific, because 10-8
mol/L 17ß-estradiol but not 10-8 mol/L
17-estradiol increased nNOS protein expression in neutrophils from
men (Figure 5A and 5B).
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We further analyzed the accumulation of [3H]L-citrulline in [3H]L-arginine–loaded neutrophils as a measure of NO generation. After 6 hours of incubation with 17ß-estradiol (10-8 mol/L), neutrophils from men showed a significant increase in the conversion of [3H]L-arginine to [3H]L-citrulline ([3H]L-citrulline, basal, 36±5%; 17ß-estradiol treatment, 59±4%; n=6, P<0.05). This effect was reversed by the presence of an NO antagonist, NG-monomethyl-L-arginine (L-NMMA) (10-4 mol/L) (inhibition 93±4%, n=6, P<0.01), demonstrating the specificity of the reaction.
We also determined whether the increased NO production by
17ß-estradiol–stimulated neutrophils could occur through the
expression of the iNOS isoform. Neutrophils from men failed to express
the iNOS isoform under basal conditions or after exposure to
17ß-estradiol (10-8 mol/L), suggesting that
the effects of 17ß-estradiol on the ability of neutrophils from men
to produce NO occurs via the constitutive isoform (Figure 6). The monoclonal antibody used to
detect the 130-kDa iNOS isoform (Transduction Laboratories, 1:1.500)
reacted with a positive control obtained from homogenates
of LPS-treated mouse macrophages, whereas it failed to
recognize a band in homogenates of human
endothelial cells and rat pituitary (Figure 6).
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Role of ERs
To determine the role of ER in the response to 17ß-estradiol by
neutrophils derived from men, nNOS protein expression was tested in the
neutrophils under basal conditions and after exposure to
10-8 mol/L 17ß-estradiol in the presence and
in the absence of 2 different ER antagonists, ICI 182780
(10-8 mol/L) and tamoxifen
(10-8 mol/L). The ER antagonists
were added 10 minutes before 17ß-estradiol.
As mentioned above, 17ß-estradiol (10-8 mol/L)
caused an increase in nNOS protein expression when compared with the
level found in basal conditions (Figure 7A and 7B). Although this effect was
partially inhibited by 10-8 mol/L tamoxifen, it
was completely reversed by 10-8 mol/L ICI 182780
(Figure 7A and 7B). In the absence of 17ß-estradiol, the level
of nNOS protein expression was not statistically modified by either of
the ER antagonists (Figure 7A and 7B).
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Both tamoxifen and ICI 182780 reduced NO generation by 10-8 mol/L 17ß-estradiol–incubated neutrophils, although the level of inhibition achieved with 10-8 mol/L ICI 182780 was greater than with 10-8 mol/L tamoxifen (inhibition, tamoxifen, 40±6%; ICI 182780, 85±7%; n=6, P<0.05).
We then determined whether ER protein is expressed in neutrophils from
men. Western blot analysis showed the presence of a 67-kDa band
for ER protein (Figure 8A and 8B).
Interestingly, incubation of neutrophils derived from men for 6 hours
with 17ß-estradiol enhanced the expression of the 67-kDa ER protein
(Figure 8A and 8B). This effect was inhibited by tamoxifen and
ICI 182780 (Figure 8A and 8B). As it occurred for nNOS protein
expression, the reduction in ER expression achieved with
10-8 mol/L ICI 182780 on
10-8 mol/L 17ß-estradiol–incubated
neutrophils was of higher magnitude than that obtained with
10-8 mol/L tamoxifen (Figure 8A and 8B).
Under basal conditions, a weak reduction in ER protein expression was
observed with ICI 182780 (10-8 mol/L), although
it did not reach statistical significance (Figure 8A and 8B). In
the absence of 17ß-estradiol, tamoxifen also failed to modify ER
protein expression (Figure 8A and 8B).
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CD18 Antigen Expression in 17ß-Estradiol–Incubated
Neutrophils
Analysis by flow cytometry demonstrated that
17ß-estradiol (10-8 mol/L)–treated
neutrophils from men have a reduced expression of CD18 antigen when
compared with the neutrophils incubated with the solvent of
17ß-estradiol (Figure 9). The
fluorescence intensity observed in the neutrophils incubated
with the solvent of 17ß-estradiol was 160±6, and it was
significantly reduced in 10-8 mol/L
17ß-estradiol–treated neutrophils (fluorescence intensity,
84±3; n=5, P<0.05). These results suggested that
17ß-estradiol decreased the expression of CD18 antigen in neutrophils
derived from men. This effect was fully prevented by the presence of
the L-arginine–competitive analogue
10-4 mol/L L-NMMA (fluorescence
intensity, 182±8). On the other hand, a decreased neutrophil adhesion
to the plastic surface was observed 6 hours after incubation with
10-8 mol/L 17ß-estradiol (neutrophils per
well, control, 74±7x104;
17ß-estradiol–treated neutrophils, 50±5x104;
n=5, P<0.05). The antiadhesive effect of 17ß-estradiol on
neutrophils was completely prevented by 10-4
mol/L L-NMMA (17ß-estradiol+L-NMMA, 80±6x104
neutrophils per well).
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Discussion |
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In the present study, we first evaluated the expression of nNOS protein in neutrophils obtained from healthy premenopausal women during situations of low and high levels of circulating estrogen. Wallerath et al16 have previously demonstrated that the nNOS type is constitutively expressed in neutrophils, which was recently confirmed by other authors.31 Our results showed that in the first 2 days of the follicular phase (low serum estrogen levels), the expression of nNOS protein in neutrophils was lower than during ovulation (high serum estrogen concentration), suggesting that there may exist an association between the level of estrogen and nNOS expression in neutrophils from premenopausal women. This was further confirmed in the experiments with neutrophils from postmenopausal women before and after transdermal estradiol application in which estrogen replacement therapy increased the expression of nNOS protein by the neutrophils, providing them with a higher ability to produce NO.
Few studies have evaluated the influence of estrogen on NO metabolism in both premenopausal and postmenopausal women. In this regard, Cicinelli et al32 have reported that the highest plasma concentration of NO metabolites corresponded to the midcycle (the ovulatory phase). Moreover, it has been recently suggested that transdermal administration of estradiol in postmenopausal women induces a further significant increase in NO plasma levels, postulating that the source of NO was the endothelium.33 The present work provides further evidence that NO released from neutrophils stimulated by estrogen could contribute to total NO.
The in vitro experiments with neutrophils from healthy men tried to illustrate the gender specificity of the in vivo findings. Whereas low 17ß-estradiol concentrations upregulated nNOS protein expression, concentrations above 10-7 mol/L tended to reduce nNOS expression in neutrophils obtained from men, which might suggest that the effect of estrogen on nNOS expression could be determined by the hormonal level.
Although nNOS-encoding genes were originally considered to be constitutive, it has become evident that their expression is regulated in different cells by a variety of stimuli.12 13 34 Moreover, estrogen has been shown to increase eNOS expression in endothelial cells15 35 ; however, to our knowledge this is the first report demonstrating the upregulatory ability of estrogen on nNOS expression in neutrophils.
The mechanism by which estrogen stimulated nNOS protein expression in neutrophils remains to be determined, given that the 5'-flanking region of nNOS does not contain a canonical estrogen response element.36 However, many estrogen-regulated promoters do not contain perfect palindromic estrogen response element37 38 ; therefore, an investigation of transcriptional transactivation of nNOS by estrogen is now indicated to further elucidate the underlying mechanisms.
In the present investigation, we also determined whether ERs are involved in the upregulatory effect of 17ß-estradiol on nNOS expression and NO synthesis. Estrogen-mediated stimulation of nNOS protein expression in neutrophils derived from men occurs by a receptor-dependent mechanism, given that it was inhibited by the ER antagonists tamoxifen and ICI 182780. Interestingly, at equimolar concentrations, ICI 182780 seems to be more potent to inhibit 17ß-estradiol–induced nNOS expression in human neutrophils than tamoxifen, which was confirmed in the experiments of NO production. McClelland et al39 have also recently reported a greater inhibition of estrogen-induced events by ICI 182780 than by tamoxifen, which could be related to the known agonist/antagonistic effects of tamoxifen.40
The presence of ER in neutrophils has been previously established by binding assays.26 Moreover, there is recent evidence suggesting that estrogen regulates a number of neutrophil functions by ER-dependent mechanisms that include phagocytosis and chemotaxis.21 41 We here demonstrate for the first time the expression of ER protein in neutrophils from men by Western blot analysis. Interestingly, estrogen seems to upregulate the expression of its receptors in neutrophils. Similar findings have been observed in cultures of testis of a nonmammalian vertebrate and in endothelium of ovine pulmonary arteries.42 43
The ER subtypes expressed in neutrophils remain to be established. Both
ER and ERß are inhibited by tamoxifen and ICI
182780.44 45 Thus, the mentioned effects of estrogen on
nNOS expression in neutrophils from men could be mediated by either
ER, ERß, both receptor subtypes, or a yet-unknown ER subtype.
Neutrophils play an important role in the development of inflammation. NO released by neutrophils has been previously demonstrated to prevent neutrophil adhesion by reducing the expression of CD11/CD18 adhesive proteins.17 46 The flow cytometric study demonstrated that 17ß-estradiol caused a reduction of CD18 antigen expression in the surface of neutrophils from men. Moreover, 17ß-estradiol–treated neutrophils from men showed a diminished ability to interact with a synthetic surface. The prevention of the 17ß-estradiol effects on both CD18 expression and adhesion of neutrophils by the L-arginine-competitive analogue L-NMMA supported the involvement of NO and suggested the functional importance of the upregulation on nNOS expression by estrogen.
In summary, neutrophils obtained from premenopausal women, during the menstrual cycle, and from postmenopausal women, before and after transdermal estrogen therapy, showed different levels of nNOS, which were associated with changes in the circulating estrogen levels. Moreover, the results of the present study showed that the nNOS-type protein expressed in neutrophils from men is upregulated by low doses of 17ß-estradiol through an ER-dependent mechanism. The increased expression of nNOS protein was associated with a decrease in the adhesion properties of the neutrophils.
The enhanced ability of neutrophils to produce NO after 17ß-estradiol treatment may contribute to some of the beneficial effects of hormone replacement therapy, particularly in diseases associated with inflammation where neutrophils play a relevant role.
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Acknowledgments |
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This work was supported by grants from Fondo de Investigaciones Sanitarias de la Seguridad Social (FISS 99/0117) and Fundación Mapfre Medicina. M.G.-D., T.d.F., A.J., M.d.M.A., and F.G.-F. are fellows of Fundación Conchita Rábago de Jiménez Díaz. M.M. and L.S.d.M. are postdoctoral fellows of Bayer Laboratories and Fundación Jiménez Díaz. We thank Zeneca Pharmaceuticals for providing ICI 182780. We thank María Begoña Ibarra for secretarial assistance.
Received March 1, 1999; accepted September 17, 1999.
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