Antonio López-FarréGonzález-Fernández, María del Mar Arriero, Luis Rico, Rosa García, Santos Casado and

Jiménez, Mercedes Montón, Jerónimo Farré, Lourdes Sánchez de Miguel, Fernando Margarita García-Durán, Trinidad de Frutos, Joaquín Díaz-Recasens, Gema García-Gálvez, Ana

NeutrophilsEstrogen Stimulates Neuronal Nitric Oxide Synthase Protein Expression in Human

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 1999 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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Estrogen Stimulates Neuronal Nitric Oxide Synthase ProteinExpression in Human Neutrophils

Margarita Garcıa-Duran, Trinidad de Frutos, Joaquı´n Dıaz-Recasens, Gema Garcıa-Galvez,Ana Jimenez, Mercedes Monton, Jeronimo Farre, Lourdes Sanchez de Miguel,Fernando Gonzalez-Fernandez, Marıa del Mar Arriero, Luis Rico, Rosa Garcı´a,

Santos Casado, Antonio Lopez-Farre

Abstract—Recent studies have postulated the contribution of nitric oxide (NO) released by the endothelium to thebeneficial effects of estrogen. Despite a neuronal-type NO synthase (nNOS) described in neutrophils, less is knownabout the effect of estrogen in these cells. The aim of the present study was to analyze the expression of nNOS proteinin human neutrophils under different estrogenic conditions. We first analyzed nNOS expression in neutrophils obtainedfrom 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 donorsduring the ovulatory phase (high circulating estrogen concentrations). Moreover, the expression of nNOS protein inneutrophils obtained from postmenopausal women after transdermal estrogen therapy was markedly enhanced withrespect to that observed before the treatment. In vitro incubation of neutrophils derived from men for 6 hours with17b-estradiol (10210 to 1028 mol/L) upregulated the expression of nNOS protein. The 17b-estradiol receptor antagonists,tamoxifen (1028 mol/L) and ICI 182780 (1028 mol/L), inhibited the upregulation of nNOS protein induced by17b-estradiol. The putative functional implication was denoted by a reduced expression of the CD18 antigen on thesurface of 17b-estradiol–incubated neutrophils, which was accompanied by a decreased adhesive capacity. Both effectswere prevented by an NO antagonist. In conclusion, the in vivo levels of circulating estrogen concentrations seem to beassociated with the level of nNOS protein expression in neutrophils from women. Moreover, low doses of 17b-estradiolupregulate nNOS protein expression in neutrophils from men. The increased ability of 17b-estradiol–incubatedneutrophils derived from men to produce NO reduced their adhesive properties.(Circ Res. 1999;85:1020-1026.)

Key Words: estrogenn leukocyten nitric oxide synthasen premenopausen postmenopause

Death from cardiovascular disease is relatively rare inpremenopausal women compared with age-matched

men.1 However, after menopause the risk of coronary heartdisease increases dramatically.2,3 Several data from ongoingclinical studies have shown that hormone replacement ther-apy reduces cardiovascular disease in postmenopausalwomen, but the final causative mechanism remains un-clear.4–6 The benefits of estrogen have been attributed to itseffects on lipids and lipoproteins. However, the change in thelipid profile reportedly accounts for only 25% to 50% of theprotective effect of estrogen against cardiovascular disease,7

and new data suggest that estrogen may also exert itsbeneficial effects through a direct action on the vessel wall.8,9

Nitric oxide (NO) is generated by the metabolic conversionof L-arginine toL-citrulline by the activity of the enzyme NOsynthase (NOS). Recent molecular cloning studies havereported that human DNA encodes 2 distinct constitutiveNOS 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 itsexpression.10,11

Although the nNOS and eNOS isoforms were initiallydescribed as constitutive, in recent years the inducible char-acter of both constitutive isoforms has been shown. In thisregard, increasing evidence indicates that nNOS expressioncan be regulated by various physiological and pathophysio-logical conditions, including sympathetic activity and acuteheat stress.12,13Moreover, tumor necrosis factor-a downregu-lates eNOS expression,14 which could be upregulated byseveral factors, including estrogen.15

Similarly to endothelial cells, neutrophils produce NOby the activity of a constitutive NOS isoform. Recently,Wallerath et al16 have identified the constitutively expressedNOS isoform in neutrophils as neuronal type. The NOreleased by neutrophils has been demonstrated to preventneutrophil adhesion to vascular endothelium, to control ag-

Received March 1, 1999; accepted September 17, 1999.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.), Fundacion Jimenez Dıaz. Madrid, Spain.Correspondence to Dr Antonio Lopez-Farre, Cardiovascular Research and Hypertension Laboratory Fundacion Jimenez Dıaz Avda Reyes

Catolicos, 2. 28040 Madrid, Spain. E-mail alopez@fjd.es© 1999 American Heart Association, Inc.

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gregation of neighboring platelets and, in the absence ofendothelium, to produce vasodilatory effects.11,17,18

Estrogen causes short-term coronary vasodilatory effects inhumans mediated by an increase in NO production.19 More-over, long-term exposure to estrogen increases acetylcholine-mediated coronary vasodilation in nonhuman primates, male-to-female transsexuals, and postmenopausal women.20–22

These effects have been attributed to an increase in NOSexpression by the endothelium.9,23 However, despite thegrowing interest in the action of estrogen on endothelial cells,less is known about the effect of estrogen on neutrophils. Inthis regard, several data support the involvement of estrogenin the modification of neutrophil activity during the menstrualcycles and pregnancy.24,25 Binding studies performed byKlebanoff26 demonstrated the presence of estrogen receptors(ERs) on neutrophils. However, at present there are no studiesabout the effect of estrogen on the NO-generating system inhuman neutrophils. Therefore, the aim of the present studywas to evaluate the effect of estrogen on nNOS expression inhuman neutrophils. For this purpose, we analyzed the expres-sion of nNOS protein in neutrophils obtained from premeno-pausal and postmenopausal women under different situationsof circulating estrogen concentrations. Moreover, in in vitroexperiments the effect of estrogen on the expression of nNOSprotein in neutrophils obtained from men was alsodetermined.

Materials and MethodsNeutrophil IsolationNeutrophils were isolated as previously described18,27 from periph-eral blood from the following groups: (1) 10 healthy premenopausalwomen (25 to 40 years of age) during different stages of themenstrual cycle, (2) 10 postmenopausal women (45 to 65 years ofage) before and after 4 months of hormone replacement therapy, and(3) from 10 healthy men (25 to 40 years of age) for in vitro estrogenincubations. The protocols were approved by the Institutional EthicsCommittee.

The nNOS protein determination was performed by Western blotas reported.14,28

Neutrophils Obtained FromPremenopausal WomenNeutrophils were obtained from the same donor at the following 2different points: (1) the ovulatory phase, determined by the urinarypeak of luteinizing hormone (LH) (by disposable test sticks; Don-natest Wyntek Diagnostic), and (2) within the first 2 days of thefollicular phase (menstruation). Serum estradiol concentrations weredetermined with an ELISA kit (Oxford Biomedical Research, Inc).

In Vivo Estradiol Treatment inPostmenopausal WomenAll women had not had a menstrual period for at least 4 years andhad not received any hormone replacement treatment within thisperiod of time. Neutrophils were obtained before and after 4 monthsof hormone replacement therapy (50mg/day estradiol transdermalpatches; Estraderm Matrix 50, Novartis Pharma SA). Medroxypro-gesterone acetate (Provera, Upjohn) was administered orally for 10days of each cycle (10 mg/day). Blood samples were obtained 2 daysbefore women received the progestin.

In Vitro Incubation of Neutrophils From HealthyMen With EstrogenNeutrophils (53106 cells/tube) from men were incubated withincreasing concentrations of 17b-estradiol (10210 to 1025 mol/L) or

the solvent of 17b-estradiol (ethanol at a final concentration,0.01%) for 6 hours in an incubator at 37°C with 5% CO2. ERprotein expression in male neutrophils was determined by Westernblot as already described for the nNOS protein using the polyclonalantibody MC-20 (Santa Cruz Biotechnology Inc). The antisera mayrecognize a common sequence ofa andb receptor subtypes.29

Determination of NOS ActivityThe NOS activity was determined as the conversion of [3H]L-arginine into [3H]L-citrulline as previously described.18,27 Neutro-phils from men were incubated with 17b-estradiol (1028 mol/L) orwith the solvent (ethanol at final concentration,0.01%), for 6 hoursat 37°C. During the last 45 minutes, neutrophils were loaded with[3H]L-arginine (1mCi/mL) followed by a 20-minute incubation at37°C.

CD18 Antigen Expression and Adhesion AssaysThe presence of the CD18 antigen in the surface of neutrophils wasdetermined by flow cytometry as described.30 Male neutrophils wereincubated with 1028 mol/L 17b-estradiol or the solvent ethanol (finalconcentration,,0.01%) for 6 hours at 37°C. For adhesion experi-ments, 2mCi/mL 51Cr (Amersham Life Science, Ltd) was added tothe incubation medium in the last 45 minutes to label neutrophils.51Cr-labeled neutrophils (0.5 mL) were added on a plastic surface for30 minutes at 37°C under static conditions as described.30

Statistical MethodsResults are expressed as mean6SEM. Unless otherwise stated, eachvalue corresponds to a minimum of 6 different experiments. Thestatistical significance was determined by ANOVA with the Bonfer-roni correction for multiple comparisons or a Studentt test, paired orunpaired. AP value,0.05 was considered statistically significant.

An expanded Materials and Methods section is available online athttp://www.circresaha.org.

ResultsnNOS Expression in Neutrophils FromPremenopausal and Postmenopausal WomenWe first determined the expression of nNOS protein byWestern blot in neutrophils obtained from premenopausalwomen at 2 different phases of the menstrual cycle, one inwhich circulating concentrations of estrogen are elevated(ovulatory phase) and the other within the first 2 days of thefollicular phase (menstruation), when circulating estrogenconcentrations fall.

Neutrophils obtained from premenopausal women ex-pressed a 155-kDa nNOS-like protein (Figure 1A). As shownin Figure 1A and 1B, during the ovulatory phase (positiveurinary detection of LH), the level of nNOS expression inneutrophils was markedly enhanced with respect to that foundin the neutrophils obtained from the same donor during thefirst 2 days of the follicular phase. At ovulation, serumestradiol levels were 520630 pg/mL (1.931029 mol/L). Inthe first 2 days of the follicular phase, serum estradiolconcentration fell to 160610 pg/mL (5.9310210 mol/L).

We then determined the effect of estrogen therapy onnNOS expression in neutrophils obtained from postmeno-pausal women before and after transdermal estradiol treat-ment. In all cases, each individual was also used as her owncontrol before and after the treatment. In these experiments,neutrophils obtained after estrogen replacement therapyshowed a significant increase in nNOS protein expression(Figure 1A and 1B), which was accompanied by a higherability to produce NO ([3H]L-citrulline accumulation, pre-

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treatment, 3264%; posttreatment, 4864%;P,0.05). Serumestradiol levels after estradiol treatment were 125.466.2pg/mL, which on a molar basis corresponds approximately to5310210 mol/L of estradiol. Before treatment, estradiol serumconcentration was 15.362.1 pg/mL (5.6x10211 mol/L).

The monoclonal antibody used did not cross-react with theeNOS or iNOS isoform, because an nNOS protein band wasundetectable in homogenates of human endothelial cells andlipopolysaccharide (LPS)–treated rat macrophages (Figure 2).The monoclonal antibody used in our experiments specifi-cally recognized the nNOS isoform (155 kDa) obtained fromhomogenates of rat pituitary (Figure 2).

nNOS Protein Expression in 17b-Estradiol–Incubated Neutrophils From MenUnder basal conditions, the expression of nNOS-like protein(155 kDa) was detected in neutrophils from men (Figure 3A

and 3B). Although incubation with 17b-estradiol (1028

mol/L) did not significantly changes nNOS protein expres-sion in the first 4 hours, nNOS protein was markedlyenhanced 6 hours after the addition of 1028 mol/L 17b-estradiol and on the following times (Figure 3A and 3B). Noeffect of ethanol (final ethanol concentration,,0.01%) onnNOS expression by neutrophils from men was detected inthe different periods of incubation (data not shown).

Incubation of neutrophils derived from men with 17b-estradiol (10210 to 1028 mol/L) enhanced the expression ofnNOS protein in a dose-dependent fashion (Figure 4A and4B). The maximal increase in nNOS protein expression wasreached with 1028 mol/L 17b-estradiol (Figure 4A and 4B).Conversely, higher doses of 17b-estradiol (1027 to 1025

Figure 1. A, Representative Western blot demonstrating theexpression of nNOS in neutrophils obtained from 10 healthypremenopausal women and 15 healthy postmenopausal women.In the premenopausal women, neutrophils were obtained withinthe first 2 days of the follicular phase (foll) and at the ovulatoryphase (ovul). In the postmenopausal women, neutrophils wereobtained before (pre) and after (post) 4 months of transdermalestradiol administration (50 mg/day). B, Bar graph showing thedensitometric scanning of the Western blot. Data aremean6SEM of the women of each experimental group. *P,0.05with respect to the expression observed in postmenopausalwomen before the estrogen therapy. <P,0.05 with respect topremenopausal women in the ovulatory phase. A.U. indicatesarbitrary units.

Figure 2. Western blot demonstrating the specificity of themonoclonal antibody used in the experiments to recognizethe nNOS protein. The monoclonal antibody did not recog-nize the endothelial isoform in human umbilical endothelialcells or the inducible isoform in LPS-stimulated mouse mac-rophages, but it specifically recognized the nNOS isoform inhomogenates of rat pituitary.

Figure 3. A, Representative Western blot demonstrating theexpression of nNOS after exposure of human neutrophils to17b-estradiol (1028 mol/L) for different periods of time. B, Bargraph showing the densitometric scanning of the Western blot.Results are represented as mean6SEM of 6 different experi-ments. *P,0.05 with respect to basal.

Figure 4. A, Representative Western blot demonstrating theexpression of nNOS in human neutrophils. Neutrophils wereincubated with increasing concentrations of 17b-estradiol for 6hours. B, Bar graph showing the densitometric scanning of theWestern blot. Data are mean6SEM of 6 different experiments.*P,0.05 with respect to neutrophils incubated with the solventof 17b-estradiol, ethanol (final ethanol concentration ,0.01%).

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mol/L) tended to reduce nNOS protein expression to the basallevels (Figure 4A and 4B).

The above-described effect of estradiol on nNOS proteinexpression was stereospecific, because 1028 mol/L 17b-estradiolbut not 1028 mol/L 17a-estradiol increased nNOS proteinexpression in neutrophils from men (Figure 5A and 5B).

We further analyzed the accumulation of [3H]L-citrulline in[3H]L-arginine–loaded neutrophils as a measure of NO gen-eration. After 6 hours of incubation with 17b-estradiol (1028

mol/L), neutrophils from men showed a significant increasein the conversion of [3H]L-arginine to [3H]L-citrulline ([3H]L-citrulline, basal, 3665%; 17b-estradiol treatment, 5964%;n56,P,0.05). This effect was reversed by the presence of anNO antagonist,NG-monomethyl-L-arginine (L-NMMA) (1024

mol/L) (inhibition 9364%, n56, P,0.01), demonstrating thespecificity of the reaction.

We also determined whether the increased NO productionby 17b-estradiol–stimulated neutrophils could occur throughthe expression of the iNOS isoform. Neutrophils from menfailed to express the iNOS isoform under basal conditions orafter exposure to 17b-estradiol (1028 mol/L), suggesting thatthe effects of 17b-estradiol on the ability of neutrophils frommen to produce NO occurs via the constitutive isoform

(Figure 6). The monoclonal antibody used to detect the130-kDa iNOS isoform (Transduction Laboratories, 1:1.500)reacted with a positive control obtained from homogenates ofLPS-treated mouse macrophages, whereas it failed to recog-nize a band in homogenates of human endothelial cells andrat pituitary (Figure 6).

Role of ERsTo determine the role of ER in the response to 17b-estradiolby neutrophils derived from men, nNOS protein expressionwas tested in the neutrophils under basal conditions and afterexposure to 1028 mol/L 17b-estradiol in the presence and inthe absence of 2 different ER antagonists, ICI 182780 (1028

mol/L) and tamoxifen (1028 mol/L). The ER antagonists wereadded 10 minutes before 17b-estradiol.

As mentioned above, 17b-estradiol (1028 mol/L) caused anincrease in nNOS protein expression when compared with thelevel found in basal conditions (Figure 7A and 7B). Althoughthis effect was partially inhibited by 1028 mol/L tamoxifen, itwas completely reversed by 1028 mol/L ICI 182780 (Figure7A and 7B). In the absence of 17b-estradiol, the level ofnNOS protein expression was not statistically modified byeither of the ER antagonists (Figure 7A and 7B).

Both tamoxifen and ICI 182780 reduced NO generation by1028 mol/L 17b-estradiol–incubated neutrophils, although thelevel of inhibition achieved with 1028 mol/L ICI 182780 wasgreater than with 1028 mol/L tamoxifen (inhibition, tamox-ifen, 4066%; ICI 182780, 8567%; n56,P,0.05).

We then determined whether ER protein is expressed inneutrophils from men. Western blot analysis showed the

Figure 6. Representative Western blot of the expression ofiNOS in human neutrophils before and after exposure to 1028

mol/L 17b-estradiol (17bE2) for 6 hours. The monoclonal anti-body did not recognize the endothelial isoform in human umbili-cal endothelial cells and the neuronal isoform in rat pituitary, butit specifically recognized the iNOS isoform in homogenates ofLPS-treated mouse macrophages. C indicates control.

Figure 5. A, Representative Western blot demonstrating theexpression of nNOS in human neutrophils. Neutrophils wereincubated in the presence or in the absence (control, C) of 17b-estradiol (1028 mol/L) and 17a-estradiol (1028 mol/L) for 6 hours.B, Bar graph showing the densitometric scanning of the West-ern blot. Results are represented as mean6SEM of 6 differentexperiments. *P,0.05 with respect to neutrophils incubated withthe solvent of 17b-estradiol, ethanol (final ethanol concentration,0.01).

Figure 7. A, Representative Western blot demonstrating theexpression of nNOS in human neutrophils incubated in the pres-ence and in the absence (C, control) of 17b-estradiol (17bE2,1028 mol/L) for 6 hours. Additional experiments were performedin the presence and in the absence of the ER antagoniststamoxifen (Tx, 1028 mol/L) and ICI 182780 (ICI, 1028 mol/L). B,Bar graph showing the densitometric scanning of the Westernblot. Data are mean6SEM of 6 different experiments. *P,0.05with respect to neutrophils incubated with the solvent of 17b-estradiol, ethanol (final ethanol concentration, ,0.01%).<P,0.05 with respect to 17b-estradiol in the presence of ICI182780.

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presence of a 67-kDa band for ER protein (Figure 8A and8B). Interestingly, incubation of neutrophils derived frommen for 6 hours with 17b-estradiol enhanced the expressionof the 67-kDa ER protein (Figure 8A and 8B). This effect wasinhibited by tamoxifen and ICI 182780 (Figure 8A and 8B).As it occurred for nNOS protein expression, the reduction inER expression achieved with 1028 mol/L ICI 182780 on 1028

mol/L 17b-estradiol–incubated neutrophils was of highermagnitude than that obtained with 1028 mol/L tamoxifen(Figure 8A and 8B). Under basal conditions, a weak reduc-tion in ER protein expression was observed with ICI 182780(1028 mol/L), although it did not reach statistical significance(Figure 8A and 8B). In the absence of 17b-estradiol, tamox-ifen also failed to modify ER protein expression (Figure 8Aand 8B).

CD18 Antigen Expression in17b-Estradiol–Incubated NeutrophilsAnalysis by flow cytometry demonstrated that 17b-estradiol(1028 mol/L)–treated neutrophils from men have a reducedexpression of CD18 antigen when compared with the neutro-phils incubated with the solvent of 17b-estradiol (Figure 9).The fluorescence intensity observed in the neutrophils incu-bated with the solvent of 17b-estradiol was 16066, and itwas significantly reduced in 1028 mol/L 17b-estradiol–treated neutrophils (fluorescence intensity, 8463; n55,P,0.05). These results suggested that 17b-estradiol de-creased the expression of CD18 antigen in neutrophils de-rived from men. This effect was fully prevented by thepresence of theL-arginine–competitive analogue 1024 mol/L

L-NMMA (fluorescence intensity, 18268). On the otherhand, a decreased neutrophil adhesion to the plastic surfacewas observed 6 hours after incubation with 1028 mol/L17b-estradiol (neutrophils per well, control, 74673104; 17b-estradiol–treated neutrophils, 50653104; n55, P,0.05).The antiadhesive effect of 17b-estradiol on neutrophils wascompletely prevented by 1024 mol/L L-NMMA (17b-estradiol1L-NMMA, 80663104 neutrophils per well).

DiscussionIn the present study, we first evaluated the expression ofnNOS protein in neutrophils obtained from healthy premeno-pausal women during situations of low and high levels ofcirculating estrogen. Wallerath et al16 have previously dem-onstrated that the nNOS type is constitutively expressed inneutrophils, which was recently confirmed by other authors.31

Our results showed that in the first 2 days of the follicularphase (low serum estrogen levels), the expression of nNOSprotein in neutrophils was lower than during ovulation (highserum estrogen concentration), suggesting that there mayexist an association between the level of estrogen and nNOSexpression in neutrophils from premenopausal women. Thiswas further confirmed in the experiments with neutrophilsfrom postmenopausal women before and after transdermalestradiol application in which estrogen replacement therapy

Figure 8. A, Representative Western blot demonstrating theexpression of ERs in human neutrophils incubated in the pres-ence and in the absence (C, control) of 17b-estradiol (1028

mol/L) for 6 hours. Additional experiments were performed inthe absence and in the presence of the ER antagonists tamox-ifen (Tx, 1028 mol/L) and ICI 182780 (ICI, 1028 mol/L). B, Bargraph showing the densitometric scanning of the Western blot.Results are represented as mean6SEM of 6 different experi-ments. *P,0.05 with respect to neutrophils incubated with thesolvent of 17b-estradiol, ethanol (final ethanol concentration,,0.01%). <P,0.05 with respect to 17b-estradiol in the pres-ence of ICI 182780.

Figure 9. Flow cytometry analysis of the expression of CD18 inthe presence of 1028 mol/L 17b-estradiol (3) or the solvent of17b-estradiol, ethanol (2) (final ethanol concentration ,0.01%),for 6 hours. Additional experiments were performed adding theL-arginine antagonist L-NMMA (1024 mol/L) during the last hourof incubation (1). Figure reproduces the real trace of 1 typicalexperiment.

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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 onNO metabolism in both premenopausal and postmenopausalwomen. In this regard, Cicinelli et al32 have reported that thehighest plasma concentration of NO metabolites corre-sponded to the midcycle (the ovulatory phase). Moreover, ithas been recently suggested that transdermal administrationof estradiol in postmenopausal women induces a furthersignificant increase in NO plasma levels, postulating that thesource of NO was the endothelium.33 The present workprovides further evidence that NO released from neutrophilsstimulated by estrogen could contribute to total NO.

The in vitro experiments with neutrophils from healthymen tried to illustrate the gender specificity of the in vivofindings. Whereas low 17b-estradiol concentrations upregu-lated nNOS protein expression, concentrations above 1027

mol/L tended to reduce nNOS expression in neutrophilsobtained from men, which might suggest that the effect ofestrogen on nNOS expression could be determined by thehormonal level.

Although nNOS-encoding genes were originally consid-ered to be constitutive, it has become evident that theirexpression is regulated in different cells by a variety ofstimuli.12,13,34Moreover, estrogen has been shown to increaseeNOS expression in endothelial cells15,35; however, to ourknowledge this is the first report demonstrating the upregu-latory ability of estrogen on nNOS expression in neutrophils.

The mechanism by which estrogen stimulated nNOS pro-tein expression in neutrophils remains to be determined,given that the 59-flanking region of nNOS does not contain acanonical estrogen response element.36 However, manyestrogen-regulated promoters do not contain perfect palin-dromic estrogen response element37,38; therefore, an investi-gation of transcriptional transactivation of nNOS by estrogenis now indicated to further elucidate the underlyingmechanisms.

In the present investigation, we also determined whetherERs are involved in the upregulatory effect of 17b-estradiolon nNOS expression and NO synthesis. Estrogen-mediatedstimulation of nNOS protein expression in neutrophils de-rived from men occurs by a receptor-dependent mechanism,given that it was inhibited by the ER antagonists tamoxifenand ICI 182780. Interestingly, at equimolar concentrations,ICI 182780 seems to be more potent to inhibit 17b-estradiol–induced nNOS expression in human neutrophils than tamox-ifen, which was confirmed in the experiments of NO produc-tion. McClelland et al39 have also recently reported a greaterinhibition of estrogen-induced events by ICI 182780 than bytamoxifen, which could be related to the known agonist/an-tagonistic effects of tamoxifen.40

The presence of ER in neutrophils has been previouslyestablished by binding assays.26 Moreover, there is recentevidence suggesting that estrogen regulates a number ofneutrophil functions by ER-dependent mechanisms that in-clude phagocytosis and chemotaxis.21,41We here demonstratefor the first time the expression of ER protein in neutrophilsfrom men by Western blot analysis. Interestingly, estrogenseems to upregulate the expression of its receptors in neutro-

phils. Similar findings have been observed in cultures of testisof a nonmammalian vertebrate and in endothelium of ovinepulmonary arteries.42,43

The ER subtypes expressed in neutrophils remain to beestablished. Both ERa and ERb are inhibited by tamoxifenand ICI 182780.44,45 Thus, the mentioned effects of estrogenon nNOS expression in neutrophils from men could bemediated by either ERa, ERb, both receptor subtypes, or ayet-unknown ER subtype.

Neutrophils play an important role in the development ofinflammation. NO released by neutrophils has been previ-ously demonstrated to prevent neutrophil adhesion by reduc-ing the expression of CD11/CD18 adhesive proteins.17,46Theflow cytometric study demonstrated that 17b-estradiol causeda reduction of CD18 antigen expression in the surface ofneutrophils from men. Moreover, 17b-estradiol–treated neu-trophils from men showed a diminished ability to interactwith a synthetic surface. The prevention of the 17b-estradioleffects on both CD18 expression and adhesion of neutrophilsby theL-arginine-competitive analogue L-NMMA supportedthe involvement of NO and suggested the functional impor-tance of the upregulation on nNOS expression by estrogen.

In summary, neutrophils obtained from premenopausalwomen, during the menstrual cycle, and from postmeno-pausal women, before and after transdermal estrogen therapy,showed different levels of nNOS, which were associated withchanges in the circulating estrogen levels. Moreover, theresults of the present study showed that the nNOS-typeprotein expressed in neutrophils from men is upregulated bylow doses of 17b-estradiol through an ER-dependent mech-anism. The increased expression of nNOS protein was asso-ciated with a decrease in the adhesion properties of theneutrophils.

The enhanced ability of neutrophils to produce NO after17b-estradiol treatment may contribute to some of the bene-ficial effects of hormone replacement therapy, particularly indiseases associated with inflammation where neutrophils playa relevant role.

AcknowledgmentsThis work was supported by grants from Fondo de InvestigacionesSanitarias de la Seguridad Social (FISS 99/0117) and Fundacio´nMapfre Medicina. M.G.-D., T.d.F., A.J., M.d.M.A., and F.G.-F. arefellows of Fundacion Conchita Rabago de Jimenez Dıaz. M.M. andL.S.d.M. are postdoctoral fellows of Bayer Laboratories and Funda-cion Jimenez Dıaz. We thank Zeneca Pharmaceuticals for providingICI 182780. We thank Marıa Begona Ibarra for secretarial assistance.

References1. Isles CG, Hole DJ, Hawthorne VM, Lever AF. Relation between coronary

risk and coronary mortality in women of the Renfew and Paisley survey:comparison with men.Lancet. 1992;339:702–706.

2. Gordon T, Kannel WB, Hjorthand MC, McNamara PM. Menopause andcoronary heart disease: the Framingham study.Ann Intern Med. 1978;89:157–161.

3. Rosenberg L, Hennekens CH, Rosner B, Belanger C, Rothman KJ,Speizer FE. Early menopause and the risk of myocardial infarction.Am JObstet Gynecol. 1981;139:47–51.

4. Stampfer MJ. A review of the epidemiology of postmenopausal estrogensand the risk of coronary heart disease. In:Sex Steroids and the Cardio-vascular System. Berlin: Springer-Verlag; 1992:145–160.

5. Grodstein F, Stampfer MJ, Manson JE, Colditz GA, Willet WC, RosnerB, Speizer FE, Hennekens CH. Postmenopausal estrogen and progestin

Garcıa-Duran et al Estrogen, NO, and Human Neutrophils 1025

by guest on July 1, 2014http://circres.ahajournals.org/Downloaded from

use and the risk of cardiovascular disease.N Engl J Med. 1996;335:453–461.

6. Barret-Connor E, Bush TL. Estrogens and coronary disease.JAMA.1991;265:1851–1867.

7. Gruchow HW, Anderson AJ, Barboraik JJ, Sobocinski KA. Postmeno-pausal use of estrogen and occlusion of coronary arteries.Am Heart J.1988;115:954–963.

8. Farhat MY, Lavigne MC, Ramwell PW. The vascular protective effects ofestrogen.FASEB J. 1996;10:615–624.

9. Mendelsohn ME, Karas RH. The protective effects of estrogen on thecardiovascular system.N Engl J Med. 1999;340:1801–1811.

10. Sessa WC. The nitric oxide synthase family of proteins.J Vasc Res.1994;31:131–143.

11. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, patho-physiology and pharmacology.Pharmacol Rev. 1991;43:109–141.

12. Plochocka-Zulinska D, Krukoff TL. Increased gene expression ofneuronal nitric oxide synthase in brain of adult spontaneously hyper-tensive rats.Brain Res Mol Brain Res. 1997;48:291–297.

13. Le Greves P, Sharma HS, Westman J, Alm P, Nyberg F. Acute heat stressinduces edema and nitric oxide synthase upregulation and down-regulatesmRNA levels of the NMDAR1, NMDAR2A and NMDAR2B subunits inthe rat hippocampus.Acta Neurochir Suppl. 1997;70:275–278.

14. Alonso J, Sanchez de Miguel L, Monton M, Casado S, Lopez-Farre´ A.Endothelial cytosolic proteins bind to the 39untranslated region of en-dothelial nitric oxide synthase mRNA: regulation by tumor necrosisfactor alpha.Mol Cell Biol. 1997;17:5719–5726.

15. MacRitchie AN, Jun SS, Chen Z, German Z, Yuhanna IS, Sherman TS,Shaul PW. Estrogen upregulates endothelial nitric oxide synthase geneexpression in fetal pulmonary artery endothelium.Circ Res. 1997;81:355–362.

16. Wallerath T, Gath I, Aulitzky WE, Pollack JS, Kleinert H, ForstermannU. Identification of the NO synthase isoforms expressed in human gran-ulocytes, megakaryocytes and platelets.Thromb Haemost. 1997;77:163–167.

17. Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulatorof leukocyte adhesion.Proc Natl Acad Sci U S A. 1991;88:4651–4655.

18. Lopez-Farre A, Caramelo C, Esteban A, Alberola ML, Millas I, Monto´nM, Casado S. Effects of aspirin on platelet-neutrophil interactions: role ofnitric oxide and endothelin-1.Circulation. 1995;91:2080–2088.

19. Guetta V, Quyyumi AA, Prasad A, Panza JA, Waclawiw M, Cannon RO.The role of nitric oxide in coronary vascular effects of estrogen inpostmenopausal women.Circulation. 1997;96:2795–2801.

20. McCrohon JA, Walters WAW, Robinson JTC, McCredie RJ, Turner L,Adams MR, Handelsman DJ, Celermajer DS. Arterial reactivity isenhanced in genetic males taken high dose estrogens.J Am Coll Cardiol.1997;29:1432–1436.

21. New G, Timmins KL, Duffy SJ, Tran BT, O’Brien RC, Harper RW,Meredith IT. Long-term estrogen therapy improves vascular function inmale to female transsexuals.J Am Coll Cardiol. 1997;29:1437–1444.

22. Herrington DM, Braden GA, Williams JK, Morgan TM. Endothelial-dependent coronary vasomotor responsiveness in postmenopausal womenwith and without estrogen replacement therapy.Am J Cardiol. 1994;73:951–952.

23. Caulin-Glaser T, Garcıa-Cardena G, Sanel P, Sessa WC, Bender JR.17b-estradiol regulation of human endothelial cell basal nitric oxiderelease, independent of cytosolic Ca21 mobilization.Circ Res.1997;81:885–892.

24. Ujiokova T, Matsukawa A, Tanaka N, Matsuura K, Yoshinaga M,Okamura H. Interleukin-8 as an essential factor in the human chorionicgonadotropin-induced rabbit ovulatory process: interleukin-8 inducesneutrophil accumulation and activation in ovulation.Biol Reprod. 1998;58:526–530.

25. Rebelo I, Carvalha-Guerra F, Pereira-Leite L, Quintanilla A. Lactoferrinas a sensitive blood marker of neutrophil activation in normal preg-nancies.Eur J Obstet Gynecol Reprod Biol. 1995;62:189–194.

26. Klebanoff SJ. Estrogen binding by leukocytes during phagocytosis.J ExpMed. 1977;145:983–998.

27. Lopez-Farre A, Riesco A, Digiuni E, Mosquera JR, Caramelo C, Sanchezde Miguel L, Millas I, de Frutos T, Cernadas MR, Monton M, Alonso J,Casado S. Aspirin-stimulated nitric oxide production by neutrophils afteracute myocardial ischemia in rabbits.Circulation. 1996;94:83–87.

28. Gonzalez-Fernandez F, Lopez-Farre A, Rodriguez-Feo JA, Farre´ J,Guerra J, Fortes J, Millas I, Garcıa-Duran M, Rico L, Mata P, Sanchez deMiguel L, Casado S. Expression of inducible nitric oxide synthase afterendothelial denudation of the rat carotid artery: role of platelets.Circ Res.1998;83:1080–1087.

29. Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilson S, Gustarsson JA.Cloning of a novel estrogen receptor expressed in rat prostate and ovary.Proc Natl Acad Sci U S A. 1996;93:5925–5930.

30. Lopez-Farre A, Riesco A, Espinosa G, Digiuni E, Cernadas MR, A´ lvarezV, Monton M, Rivas F, Gallego MJ, Egido J, Casado S, Caramelo C.Effect of endothelin-1 on neutrophil adhesion to endothelial cells andperfused heart.Circulation. 1993;88:1166–1171.

31. Greenberg SS, Ouyang J, Zhao X, Giles TD. Human and rat neutrophilsconstitutively express neuronal nitric oxide synthase mRNA.NitricOxide. 1998;2:203–212.

32. Cicinelli E, Ignarro LJ, Lograno M, Galantino P, Balzano G, SchomauerLM. Circulating levels of nitric oxide in fertile women in relation to themenstrual cycle.Fertil Steril. 1996;66:1036–1038.

33. Cicinelli E, Ignarro LJ, Schomauer LM, Matteo MG, Galantino P,Balzano G. Effects of short-term transdermal estradiol administration onplasma levels of nitric oxide in postmenopausal women.Fertil Steril.1998;69:58–61.

34. Caggiano AO, Kraig RP. Neuronal nitric oxide synthase expression isinduced in neocortical astrocytes after spreading depression.J CerebBlood Flow Metab. 1998;18:75–87.

35. Hayashi T, Yamada K, Esaki T, Kuzuya M, Satake S, Ishikawa T, HidakaH, Iguchi A. Estrogen increases endothelial nitric oxide by a receptor-mediated system.Biochem Biophys Res Commun. 1995;214:847–855.

36. Fostermann U, Boissel JP, Kleinert H. Expressional control of the “con-stitutive” isoforms of nitric oxide synthase (NOSI and NOSIII).FASEB J.1998;12:773–790.

37. Kato S, Tora L, Yamanchi J, Masushige S, Bellard M, Chambon P. A farupstream estrogen response element of the ovoalbumin gene containsseveral half palindromic 59-TGACC-39 motifs acting synergistically.Cell. 1992;68:731–742.

38. Murdoch FE, Byrne LM, Amazi EA, Furlow DJ, Meier DA, Gorski J.Estrogen receptor binding to DNA: affinity for nonpalindromic elementsfrom the rat prolactin gene.Biochemistry. 1995;34:9144–9150.

39. McClelland RA, Manning DL, Gee JM, Anderson E, Clarke R, Howell A,Dowsett M, Robertson JF. Effects of short-term antiestrogen treatment ofprimary breast cancer on estrogen receptor mRNA and protein expressionand on estrogen-regulated genes.Breast Cancer Res Treat. 1996;41:31–41.

40. Barkhem T, Calsson B, Nilsson Y, Enmark E, Gustafsson JA, Nilsson S.Differential response of estrogen receptora and estrogen receptorb topartial estrogen agonists/antagonists.Mol Pharmacol. 1998;54:105–112.

41. Genskens M, Burglen MJ, Uriel J. In vitro binding of3H-estradiol toeosinophil and neutrophil granulocytes in various tissues (normal andneoplastic) of newborn and adult rats.Virchows Arch B Cell Pathol.1977;24:67–78.

42. Cardone A, Angelini F, Varriale B. Autoregulation of estrogen andandrogen receptor mRNAs and down regulation of androgen receptormRNA by estrogen in primary cultures of lizard testis cells.Gen CompEndocrinol. 1998;110:227–236.

43. Jun SS, Chen Z, Pace MC, Shaul PW. Estrogen upregulatescyclooxigenase-1 gene expression in ovine fetal pulmonary artery endo-thelium.J Clin Invest. 1998;102:176–183.

44. Mendelson ME, Karas RH. Estrogen and the blood vessel wall.Curr OpinCardiol. 1994;9:619–626.

45. Mosselman S, Polman J, Dijkema R. Erb: identification and character-ization of a novel human estrogen receptor.FEBS Lett. 1999;392:49–53.

46. Chello M, Mastroroberto P, Marchese AR, Maltese G, Santangelo E,Amantea B. Nitric oxide inhibits neutrophil adhesion during experimentalextracorporeal circulation.Anesthesiology. 1998;89:443–448.

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