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Estrogen blocks inducible nitric oxide synthase accumulation in LPS-activated microglia cells
E. Vegeto et al. / Experimental Gerontology 35 (2000) 1309±1316
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Experimental Gerontology 35 (2000) 1309±1316 www.elsevier.nl/locate/expgero
Estrogen blocks inducible nitric oxide synthase accumulation in LPS-activated microglia cells E. Vegeto, G. Pollio, P. Ciana, A. Maggi* Center MPL, Institute of Pharmacological Sciences, Via Balzaretti 9, 20133 Milan, Italy Received 20 June 2000; received in revised form 26 June 2000; accepted 26 June 2000
Abstract Estrogens are thought to play a protective role against neurodegeneration through a variety of mechanisms including the activation of growth factors and neurotransmitter synthesis, the control of synaptic plasticity and functions, and the blockade of oxidative reactions. We here propose a novel mechanism to explain the neuroprotective effects of estradiol by showing that estrogens may antagonize nitric oxide synthase activity and reduce the accumulation of nitrites and nitrates consequent to various in¯ammatory stimuli. The potential anti-in¯ammatory activity of estradiol is analyzed in vitro in cells in culture including primary cultures of microglia and in vivo in a well-known model of in¯ammation. q 2000 Elsevier Science Inc. All rights reserved. Keywords: In¯ammation; Estrogens; Alzheimer's disease; Neurodegeneration; Nervous system
1. Introduction Estrogens modulate brain functions by binding speci®c receptors, which have been shown to be expressed in several brain areas (see Agrati et al., 2000, for a review). The ®nding of a widespread distribution of these receptors underlines the fact that these sex hormones have functions that go far beyond the control of reproduction. Recent behavioral and clinical data further support the view of an active contribution of estrogens in nonreproductive functions by demonstrating that estrogens help modulate cognitive functions and memory (Luine, 1994), play an active role in the prevention of speci®c affective disorders (Rodriguez and Grossberger, 1998) and in the delay of the onset of neurodegenerative disorders like Alzheimer's (Honjo et al., 1995; Tang et al., 1996; Birge, 1997; Doraiswamy et al., 1997; Kawas et al., 1997). * Corresponding author. Tel.: 139-2-2048-8375; fax: 139-2-2048-8249. E-mail address: [email protected] (A. Maggi). 0531-5565/00/$ - see front matter q 2000 Elsevier Science Inc. All rights reserved. PII: S 0531-556 5(00)00161-3
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E. Vegeto et al. / Experimental Gerontology 35 (2000) 1309±1316
The question to be asked relates to the mechanisms involved in such diversi®ed actions exerted by this hormone. Two main factors contribute to the cell/tissue speci®c activity of estrogens: (i) receptor multiplicity and (ii) characteristics of the target cell. With regard to the different forms of proteins binding estrogens in the brain, there are at least two intracellular receptors described so far, however, several data point to the existence of membrane receptors for ER, and other molecules binding estrogens, which may be novel forms of intracellular receptors or splicing variants of the receptors (Lu et al., 1999). The cell targets for estradiol are also varied: all the neural cells appear to express at least ERa: neurons, astrocytes, oligodendrocytes (Santagati et al., 1994), and microglia (Vegeto, submitted). Likewise, there is experimental evidence (Patrone et al., 1998), that estradiol, by binding to ERa or ERb in the same cell type may trigger the synthesis of different proteins; on the other hand, it is expected that the hormone by activating its cognate receptor in different cells may elicit quite different responses. This combination of events would lead to the observed complexity of E2 actions in neural cells. The biochemical understanding of the effects, which determine each speci®c function of estradiol is mandatory for the design of novel therapies which may mimic the positive activities of this hormone in speci®c brain regions. Several hypotheses have been put forward to explain the delay in the manifestation of Alzheimer's disease in women who had been treated with estrogen as hormonal replacement therapy after menopause. Estrogens were described to: potentiate cholinergic transmission (Gibbs et al., 1997), increase cell electrical activity (Bettini and Maggi, 1992), stimulate the growth of neurites and facilitate synapses formation (Beyer and Karolczak, 2000), act as an antioxidant agent (Behl et al., 1995; 1997; Garcia-Segura et al., 1998; Goodman et al., 1996; Green et al., 1996), regulate the metabolism of the amyloid precursor protein (Shi et al., 1998), in¯uence the cerebral blood ¯ow (Greene, 2000), and ®nally to delay neural cell apoptosis by controlling the synthesis of speci®c growth factor and of pro- and anti-apoptotic factors (Toran-Allerand et al., 1999; Meda et al., 2000). Several studies suggest a role of in¯ammatory processes in the progression of Alzheimer's and other neurodegenerative diseases (McGeer and McGeer, 1995; Aisen, 1997). We have recently shown that estradiol blocks the cytokine-induced accumulation of iNOS in smooth muscle cells in culture. In view of the relevant role played by iNOS in in¯ammatory process and in particular in the manifestation of experimental Parkinson's, we investigated on a potential anti-in¯ammatory activity of estradiol. We here show that estradiol may block iNOS accumulation in a well established model of in¯ammation and show that the same effect is exerted in primary culture of microglia. We therefore propose this anti-in¯ammatory action as a novel mechanism to explain neuroprotective effects.
2. Materials and methods 2.1. Cultures of smooth muscle cells and microglia Smooth muscle cells (SMC) were obtained from the intimal-medial layers of aorta of male Sprague Dawley rats (Charles River Laboratories Inc., Calco, Italy) as previously reported (Zancan et al., 1999). Cells were grown in monolayers in medium M199
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supplemented with 10% FCS, 50 U/ml penicillin, 50 mg/ml streptomycin, 2 mM glutammine and 2.2 g/l NaHCO3 and were incubated at 378C in a humidi®ed atmosphere with 5% CO2. Cells were characterized for growth and morphology and by immunostaining with monoclonal antibodies against the a form of actin, which is typically expressed in SMC. Microglial cells were isolated from cultures of newborn rat brains, as previously described (Giulian and Baker, 1986). The purity of microglia preparations was about 95±98%. For hormone treatment experiments, cells were grown for 24 h before replacing the medium with serum-free, phenol-red free DMEM. The next day, cells were treated for 4 h in the absence or presence of E2 (Sigma, Milan, Italy) as indicated in each ®gure legend in freshly added serum-free, phenol-red free DMEM, followed by a further incubation of 16 h with E. coli LPS (Sigma) without medium replacement. 2.2. Western analysis Con¯uent cells were incubated for 24 h in the presence or absence of the cytokine cocktail (10 ng/ml interlukin-1b, 10 ng interferoneg, 25 ng/ml TNFa, 10 mg/ml LPS) or 1 nM 17b-estradiol as indicated in the ®gure legends. The incubation was ended by addition of the sample buffer (0.12 M Tris±HCl pH 6.8, 40 mM EDTA, 4.6% SDS, 20% glycerol). Protein concentration was measured by Bradford's. The same amount of protein was loaded on SDS-PAGE using a discontinuous gradient (10±5%). Following electrophoretic transfer of the proteins onto nitrocellulose membranes, the blotting was obtained with the polyclonal anti-rabbit iNOS antibody at 1:1000 dilution (Transduction Laboratories, Inc.) and the secondary antibody peroxidase conjugated at 1:2000 dilution. The presence of the secondary antibody was revealed by chemiluminescence (ECL, Amersham). Scanning densitometry was then performed and care was taken to ensure that the optical density (OD) of all the bands considered were in the range of linearity previously assessed. 2.3. Nitrites/assay The nitrite concentration in the samples was measured by the Griess reaction, by adding to the 100±200 ml of sample, cell culture medium or pleural exudate, equal amount of the Griess reagent (0.1% naphthylethylenediamide dihydrochloride in H2O and 1% sulfanilamide in 5% concentrated H2PO4). The OD at 550 nm was measured using ELISA microplate reader (SLT Labinstruments, Salzburg, Austria). 2.4. Carrageenan-induced pleurosy Rats were anesthetized with iso¯urane and a skin incision was done at the level of the left sixth intercostal space. The underlying muscle was dissected and saline (0.2 ml) or saline containing 1% carrageenan were injected in the pleural cavity. The skin incision was sutured and the animals were allowed to recover. Four hours after the injection, the animals were killed by CO2 inhalation. The chest was opened, the pleural cavity rinsed with 2 ml of saline containing heparin (5 U/ml) and indomethacin (10 mg/ml). The exudate and washing solution were removed by aspiration and the total volume measured. Any exudate containing blood was discarded.
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Fig. 1. Cells were grown to con¯uence ant treated at the same time with the reagents reported in the ®gure. Estradiol was used at 1 nM concentration. The treatment was carried out for 24 h, then cells were collected as reported in Section 2 and processed for iNOS content determination by western (lower panel). The nitrite levels were measured in the growth medium. Data represent the mean ^ sem of three separate experiments run in triplicate. P , 0:01 vs. control.
3. Results We have reported that smooth muscle cells (SMC) synthesize ERa and ERb (Zancan et al., 1999). The effect of ER activation on the activity of nitric oxidase synthase (NOS) was investigated by assaying the accumulation of nitrites in the culture medium following the treatment with a cocktail of cytokines and estradiol. Fig. 1 shows that 24 h exposure to the cytokine cocktail results in a signi®cant increased accumulation of nitrites. This effect was blocked by co-treatment with 1 nM 17b-estradiol (E2). When nitrites were measured in unstimulated cells, the treatment with the hormone did not have any signi®cant consequence. Since it is well known that in SMC in¯ammatory stimuli regulate the synthesis of the inducible form of nitric oxidase synthase: iNOS, we further investigated whether E2 had any effect on iNOS levels in cytokine-stimulated cells by western analysis. As reported by several authors, the iNOS protein content is barely detectable by western blot analysis in primary cultures of SMC. Twenty-four hour treatment with the cytokines increases the signal and co-treatment with estradiol signi®cantly prevents such an increase (Fig. 1, lower panel). These observations suggest that estradiol controls iNOS synthesis in SMC at concentration compatible with ER activation, and lead to hypothesize a potential antiin¯ammatory role for this hormone. This is in contrast with the activity of E2 in the reproductive tracts, which has been traditionally associated with in¯ammation. In uterus,
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Fig. 2. Effect of E2 on nitrite and nitrate concentration in pleural exudate (left panel) and in lungs (right panel) at 4 h after carrageenan administration. Data are means ^ SE of 10 rats for each experimental group. P , 0:01 vs. control.
in fact, E2 stimulates epithelial cell proliferation, increases vascular permeability, edema and in¯ux of macrophages. To further investigate on the potential anti-in¯ammatory role of E2, we next examined the activity of E2 in a well-known model of in¯ammation (carrageenan-induced pleurosy). Carrageenan was injected into the plural activity as described (Cuzzocrea et al., 2000). At 4 h after the injection of the in¯ammatory stimuli, the animals were killed and the plural cavity was opened to obtain the exudate as described in Section 2. The production of nitrites was measured in the exudate and the iNOS activity measured in lung cells. As shown in Fig. 2, the treatment with carrageenan signi®cantly increased the content of nitrites and nitrates in the exudate as well as in lung cells. E2 pre-treatment signi®cantly prevented the carrageenan-induced elevation of nitrite and nitrates (left panel) and the iNOS activity (right panel). E2 by itself did not have any effect on both parameters (Fig. 2). These results provide further evidence of an effect of estrogens on the iNOS activity. Recent evidence suggests that toxic substances released by glial cells might be involved in the neurodegenerative processes that occur in disease like Parkinson's, Huntington's and Alzheimer's (McGeer and Rogers, 1992; McGeer and McGeer, 1995; Kalaria, 1999). Post-mortem examination reveals the presence of massive astrogliosis and activate microglia in the region where the neurodegenerative process occurs (McGeer, 1988). In addition, it has been shown that nitric oxide produced by glial cells participates in the cascade of events that leads to the degeneration of neurones in mice that are rendered parkinsonian by MPTP intoxication. We therefore investigated whether estrogens could antagonize the induction of iNOS activation caused by LPS treatment of microglial cells. Microglia was isolated from brain of 2-day-old rat brain following welldescribed procedure. The effect of E2 on iNOS activity was assessed by measuring nitrite/nitrates content in the growth medium. Fig. 3 shows that, similarly to what previously demonstrated in SMC and lung cells, 1 nM estradiol blocked LPS-induced increase of nitrite accumulation.
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Fig. 3. Microglia cells were treated as reported in the ®gure for 24 h. Data represent the mean ^ sem of three separate experiments run in triplicate. P , 0:01 vs. control.
4. Discussion The bene®cial effects of estrogens in the delay of Alzheimer's disease have been related to the hormone's neuroprotective effects preventing neuronal loss and its trophic action on neurotransmission and neuronal metabolism. The data of the present report point to a novel mechanism by which E2 might extend its neuroprotective action. E2 might, in fact, act in microglial cells and limit their pro-in¯ammatory potential. It is well known that the principal pathological feature of Alzheimer's disease is the extracellular deposition of ®brillar amyloid and its compaction into senile plaques. These plaques are the focus of a complex of cellular reactions involving the activation of both microglia and astrocytes. The plaque-associated microglia exibits an ªactivatedº phenotype. The appearance of in¯ammatory products in Alzheimer's as well as in other neurodegenerative disorders has been interpreted as evidence of chronic in¯ammatory component of the disease process. Supporting this hypothesis are reports on patients treated for extended periods with non-steroidal anti-in¯ammatory drugs, who showed a dramatic reduction in Alzheimer's risk. Who are the major players in the sustained chronic in¯ammation possibly contributing to the onset of neurodegeneration is still unknown. Liberatore et al. (1999), studying mice made parkinsonian by treatment with MPTP, provides strong evidence that nitric oxidase participates in the cascade of events leading to the degeneration of neurons. If this were the case, our study might provide relevant data for the understanding of E2 effects in neurodegeneration. We have recently shown that macrophages are a target of estrogen action (Vegeto et al., 1999). The present study shows that also microglia might be an important site of action of these powerful hormones. Studies are in progress to better de®ne the activity of this sex hormone in microglia cells both in vitro and in vivo. Acknowledgements We thank Clara Meda, Monica Rebecchi, and Paola Pizzolorusso for their excellent
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technical and secretarial support. The Italian Foundation for Cancer Research (AIRC), Telethon (E600), Istituto Superiore di SanitaÁ (Rome), Multiple Sclerosis Programme and the EC Biomed Programme (Contract No. BMH4-CT97-2286) provided ®nancial support.
References Agrati, P., Bolzoni, L., Marini, E., Meda, C., Patrone, C., Pollio, G., Rebecchi, M., Vegeto, E., Maggi, A., 2000. In vitro study of the effects of estrogen on estrogen receptor-transfected neuroblastoma cells. In: Matsumoto, A. (Ed.). Sexual Differentiation of the Brain, CRC Press, Boca Raton, FL, pp. 21±32. Aisen, P.S., 1997. In¯ammation and Alzheimer's disease: mechanisms and therapeutic strategies. Gerontology 43, 143±149. Behl, C., Widmann, M., Trapp, T., Hoslboer, F., 1995. 17-beta estradiol protects neurons from oxidative stressinduced cell death in vitro. Biochem. Biophys. Res. Commun. 216, 473±482. Behl, C., Skutella, T., Lezoualc'h, F., Post, A., Widmann, M., Newton, C.J., Holsboer, F., 1997. Neuroprotection against oxidative stress by oestrogens: structure±activity relationship. Mol. Pharmacol. 51, 535±541. Bettini, E., Maggi, A., 1992. Estrogen induction of cytochrome C oxidase subunit III in rat hippocampus. J. Neurochem. 58, 1923±1929. Beyer, C., Karolczak, M., 2000. Estrogenic stimulation of neurite growth in midbrain dopaminergic neurons depends on cAMP/protein kinase A signalling. J. Neurosci. Res. 59, 107±116. Birge, S.J., 1997. The role of estrogens in the treatment of Alzheimer's disease. Neurology 48, s36±s41. Cuzzocrea, S., Santagati, S., Sautebin, L., Mazzon, E., Calabro, G., Serraino, I., Caputi, A.P., Maggi, A., 2000. 17beta-estradiol antiin¯ammatory activity in carrageenan-induced pleurisy. Endocrinology 141, 1455±1463. Doraiswamy, P.M., Bieber, F., Kaiser, L., Krishnan, K.R., Reuning-Scherer, J., Gulanski, B., 1997. The Alzheimer's disease assessment scale: patterns and predictors of baseline cognitive performance in multicenter Alzheimer's disease trials. Neurology 48, 1511±1517. Garcia-Segura, L.M., Cardona-Gomez, P., Naftolin, F., Chowen, J.A., 1998. Estradiol upregulates Bcl-2 expression in adult brain neurons. Neuroreport 9, 593±597. Gibbs, R.B., Hashash, A., Johnson, D.A., 1997. Effects of estrogen on potassium-stimulated acetylcholine release in the hippocampus and overlying cortex of adult rats. Brain Res. 749, 143±146. Giulian, D., Baker, T.J., 1986. Characterization of ameboid microglia isolated from developing mammalian brain. J. Neurosci. 6, 22163±22178. Goodman, Y., Bruce, A.J., Cheng, B., Mattson, M.P., 1996. Oestrogens attenuate and corticosterone exacerbates excitotoxicity, oxidative injury, and amyloid b-peptide toxicity in hippocampal neurons. J. Neurochem. 66, 1836±1844. Green, P.S., Gridley, K.E., Simpkins, J.W., 1996. Estradiol protects against beta-amyloid-induced toxicity in SK-N-SH human neuroblastoma cells. Neurosci. Lett. 218, 165±168. Greene, R.A., 2000. Cerebral blood ¯ow. Fertil. Steril. 73, 143±143. Honjo, H., Tanaka, K., Kashiwagi, T., Urabe, M., Okada, H., Hayashi, M., Hayashi, K., 1995. Senile dementia Ð Alzheimer's type and estrogen. Horm. Metab. Res. 27, 204±207. Kalaria, R.N., 1999. Microglia nad Alzheimer's disease. Curr. Opin. Hematol. 6, 15±24. Kawas, C., Resnick, S., Morrison, A., Brookmeyer, R., Corrada, M., Zonderman, A., Bacal, C., Lingle, D.D., Metter, E., 1997. A prospective study of estrogen replacement therapy and the risk of developing Alzheimer's disease: the Baltimore longitudinal study of aging. Neurology 48, 1517±1521. Liberatore, G.T., Jackson-Lewis, V., Vukosavic, S., Mandir, A.S., Vila, M., McAuliffe, G., Dawson, V.L., Dawson, T.M., Prezedborski, S., 1999. Inducible nitric oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease. Nature Med. 12, 1403±1409. Lu, B., Dotzlaw, H., Leygue, E., Murphy, L.J., Watson, P.H., Murphy, L.C., 1999. Estrogen receptor-alpha mRNA variants in murine and human tissues. Mol. Cell. Endocrinol. 158, 153±161. Luine, V., 1994. Steroid hormone in¯uences on spatial memory. Ann. NY Acad. Sci. 14, 201±209. McGeer, P.L., 1988. Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains. Neurology 38, 1285±1291.
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McGeer, P.L., McGeer, E.G., 1995. The in¯ammatory response system of brain: implications for therapy of Alzheimer's and other neurodegenerative disorders. Brain Res. Rev. 21, 195±218. McGeer, P.L., Rogers, J., 1992. Anti-in¯ammtory agents as a therapeutic approach to Alzheimer's disease. Neurology 42, 447±449. Meda, C., Vegeto, E., Pollio, G., Ciana, P., Patrone, Pellicciari, C., Maggi, A., 2000. Estrogen prevention of neural cell death correlates with decreased expression of mRNA for the pro-apoptotic protein Nip-2. Neuroendocrinology (in press). Patrone, C., Gianazza, E., Santagati, S., Agrati, P., Maggi, A., 1998. Divergent pathways regulate ligandindependent activation of ER alpha in SK-N-BE neuroblastoma and COS-1 renal carcinoma cells. Mol. Endocrinol. 12, 835±841. Rodriguez, M.M., Grossberger, G.T., 1998. Estrogens as psychotherapeutic agents. Clinics Geriatric Med. 14, 177±189. Santagati, S., Melcangi, R., Celotti, F., Martini, L., Maggi, A., 1994. Estrogen receptor is expressed in different types og glial cells in culture. J. Neurochem. 63, 2058±2064. Shi, J., Panickar, K.S., Yang, S.H., Rabbani, O., Day, A.L., Simpkins, J.W., 1998. Estrogen attenuates overexpression of beta-amyloid precursor protein messager RNA in an animal model of focal ischemia. Brain Res. 810, 87±92. Tang, M.X., Jacobs, D., Stern, Y., Marder, K., Scho®eld, P., Gurland, B., Ancrews, H., Mayeux, R., 1996. Effect of oestrogens during menopause on risk and age at onset of Alzheimer's. Lancet 348, 429±432. Toran-Allerand, C.D., Singh, M., Setalo Jr, G., 1999. Novel mechanisms of estrogen action in the brain: new players in an old story. Frontiers Neuroendocrinol. 20 (2), 97±121. Vegeto, E., Pollio, G., Maggi, A., 1999. Estrogen and progesterone induction in survival of monoblastoid cells undergoing TNF-alpha-induced apoptosis. FASEB J. 13, 793±803. Vegeto, E., Bonincontro, E., Pollio, G., Sala, A., Ciana, P., Maggi, A. Estrogens and Microglia, J. Neurosci (submitted). Zancan, V., Santagati, S., Bolego, C., Vegeto, E., Maggi, A., Puglisi, L., 1999. 17Beta-estradiol decreases nitric oxide synthase II synthesis in vascular smooth muscle cells. Endocrinology 140, 2004±2009.
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Estrogen blocks inducible nitric oxide synthase accumulation in LPS-activated microglia cells E. Vegeto, G. Pollio, P. Ciana, A. Maggi* Center MPL, Institute of Pharmacological Sciences, Via Balzaretti 9, 20133 Milan, Italy Received 20 June 2000; received in revised form 26 June 2000; accepted 26 June 2000
Abstract Estrogens are thought to play a protective role against neurodegeneration through a variety of mechanisms including the activation of growth factors and neurotransmitter synthesis, the control of synaptic plasticity and functions, and the blockade of oxidative reactions. We here propose a novel mechanism to explain the neuroprotective effects of estradiol by showing that estrogens may antagonize nitric oxide synthase activity and reduce the accumulation of nitrites and nitrates consequent to various in¯ammatory stimuli. The potential anti-in¯ammatory activity of estradiol is analyzed in vitro in cells in culture including primary cultures of microglia and in vivo in a well-known model of in¯ammation. q 2000 Elsevier Science Inc. All rights reserved. Keywords: In¯ammation; Estrogens; Alzheimer's disease; Neurodegeneration; Nervous system
1. Introduction Estrogens modulate brain functions by binding speci®c receptors, which have been shown to be expressed in several brain areas (see Agrati et al., 2000, for a review). The ®nding of a widespread distribution of these receptors underlines the fact that these sex hormones have functions that go far beyond the control of reproduction. Recent behavioral and clinical data further support the view of an active contribution of estrogens in nonreproductive functions by demonstrating that estrogens help modulate cognitive functions and memory (Luine, 1994), play an active role in the prevention of speci®c affective disorders (Rodriguez and Grossberger, 1998) and in the delay of the onset of neurodegenerative disorders like Alzheimer's (Honjo et al., 1995; Tang et al., 1996; Birge, 1997; Doraiswamy et al., 1997; Kawas et al., 1997). * Corresponding author. Tel.: 139-2-2048-8375; fax: 139-2-2048-8249. E-mail address: [email protected] (A. Maggi). 0531-5565/00/$ - see front matter q 2000 Elsevier Science Inc. All rights reserved. PII: S 0531-556 5(00)00161-3
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The question to be asked relates to the mechanisms involved in such diversi®ed actions exerted by this hormone. Two main factors contribute to the cell/tissue speci®c activity of estrogens: (i) receptor multiplicity and (ii) characteristics of the target cell. With regard to the different forms of proteins binding estrogens in the brain, there are at least two intracellular receptors described so far, however, several data point to the existence of membrane receptors for ER, and other molecules binding estrogens, which may be novel forms of intracellular receptors or splicing variants of the receptors (Lu et al., 1999). The cell targets for estradiol are also varied: all the neural cells appear to express at least ERa: neurons, astrocytes, oligodendrocytes (Santagati et al., 1994), and microglia (Vegeto, submitted). Likewise, there is experimental evidence (Patrone et al., 1998), that estradiol, by binding to ERa or ERb in the same cell type may trigger the synthesis of different proteins; on the other hand, it is expected that the hormone by activating its cognate receptor in different cells may elicit quite different responses. This combination of events would lead to the observed complexity of E2 actions in neural cells. The biochemical understanding of the effects, which determine each speci®c function of estradiol is mandatory for the design of novel therapies which may mimic the positive activities of this hormone in speci®c brain regions. Several hypotheses have been put forward to explain the delay in the manifestation of Alzheimer's disease in women who had been treated with estrogen as hormonal replacement therapy after menopause. Estrogens were described to: potentiate cholinergic transmission (Gibbs et al., 1997), increase cell electrical activity (Bettini and Maggi, 1992), stimulate the growth of neurites and facilitate synapses formation (Beyer and Karolczak, 2000), act as an antioxidant agent (Behl et al., 1995; 1997; Garcia-Segura et al., 1998; Goodman et al., 1996; Green et al., 1996), regulate the metabolism of the amyloid precursor protein (Shi et al., 1998), in¯uence the cerebral blood ¯ow (Greene, 2000), and ®nally to delay neural cell apoptosis by controlling the synthesis of speci®c growth factor and of pro- and anti-apoptotic factors (Toran-Allerand et al., 1999; Meda et al., 2000). Several studies suggest a role of in¯ammatory processes in the progression of Alzheimer's and other neurodegenerative diseases (McGeer and McGeer, 1995; Aisen, 1997). We have recently shown that estradiol blocks the cytokine-induced accumulation of iNOS in smooth muscle cells in culture. In view of the relevant role played by iNOS in in¯ammatory process and in particular in the manifestation of experimental Parkinson's, we investigated on a potential anti-in¯ammatory activity of estradiol. We here show that estradiol may block iNOS accumulation in a well established model of in¯ammation and show that the same effect is exerted in primary culture of microglia. We therefore propose this anti-in¯ammatory action as a novel mechanism to explain neuroprotective effects.
2. Materials and methods 2.1. Cultures of smooth muscle cells and microglia Smooth muscle cells (SMC) were obtained from the intimal-medial layers of aorta of male Sprague Dawley rats (Charles River Laboratories Inc., Calco, Italy) as previously reported (Zancan et al., 1999). Cells were grown in monolayers in medium M199
E. Vegeto et al. / Experimental Gerontology 35 (2000) 1309±1316
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supplemented with 10% FCS, 50 U/ml penicillin, 50 mg/ml streptomycin, 2 mM glutammine and 2.2 g/l NaHCO3 and were incubated at 378C in a humidi®ed atmosphere with 5% CO2. Cells were characterized for growth and morphology and by immunostaining with monoclonal antibodies against the a form of actin, which is typically expressed in SMC. Microglial cells were isolated from cultures of newborn rat brains, as previously described (Giulian and Baker, 1986). The purity of microglia preparations was about 95±98%. For hormone treatment experiments, cells were grown for 24 h before replacing the medium with serum-free, phenol-red free DMEM. The next day, cells were treated for 4 h in the absence or presence of E2 (Sigma, Milan, Italy) as indicated in each ®gure legend in freshly added serum-free, phenol-red free DMEM, followed by a further incubation of 16 h with E. coli LPS (Sigma) without medium replacement. 2.2. Western analysis Con¯uent cells were incubated for 24 h in the presence or absence of the cytokine cocktail (10 ng/ml interlukin-1b, 10 ng interferoneg, 25 ng/ml TNFa, 10 mg/ml LPS) or 1 nM 17b-estradiol as indicated in the ®gure legends. The incubation was ended by addition of the sample buffer (0.12 M Tris±HCl pH 6.8, 40 mM EDTA, 4.6% SDS, 20% glycerol). Protein concentration was measured by Bradford's. The same amount of protein was loaded on SDS-PAGE using a discontinuous gradient (10±5%). Following electrophoretic transfer of the proteins onto nitrocellulose membranes, the blotting was obtained with the polyclonal anti-rabbit iNOS antibody at 1:1000 dilution (Transduction Laboratories, Inc.) and the secondary antibody peroxidase conjugated at 1:2000 dilution. The presence of the secondary antibody was revealed by chemiluminescence (ECL, Amersham). Scanning densitometry was then performed and care was taken to ensure that the optical density (OD) of all the bands considered were in the range of linearity previously assessed. 2.3. Nitrites/assay The nitrite concentration in the samples was measured by the Griess reaction, by adding to the 100±200 ml of sample, cell culture medium or pleural exudate, equal amount of the Griess reagent (0.1% naphthylethylenediamide dihydrochloride in H2O and 1% sulfanilamide in 5% concentrated H2PO4). The OD at 550 nm was measured using ELISA microplate reader (SLT Labinstruments, Salzburg, Austria). 2.4. Carrageenan-induced pleurosy Rats were anesthetized with iso¯urane and a skin incision was done at the level of the left sixth intercostal space. The underlying muscle was dissected and saline (0.2 ml) or saline containing 1% carrageenan were injected in the pleural cavity. The skin incision was sutured and the animals were allowed to recover. Four hours after the injection, the animals were killed by CO2 inhalation. The chest was opened, the pleural cavity rinsed with 2 ml of saline containing heparin (5 U/ml) and indomethacin (10 mg/ml). The exudate and washing solution were removed by aspiration and the total volume measured. Any exudate containing blood was discarded.
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Fig. 1. Cells were grown to con¯uence ant treated at the same time with the reagents reported in the ®gure. Estradiol was used at 1 nM concentration. The treatment was carried out for 24 h, then cells were collected as reported in Section 2 and processed for iNOS content determination by western (lower panel). The nitrite levels were measured in the growth medium. Data represent the mean ^ sem of three separate experiments run in triplicate. P , 0:01 vs. control.
3. Results We have reported that smooth muscle cells (SMC) synthesize ERa and ERb (Zancan et al., 1999). The effect of ER activation on the activity of nitric oxidase synthase (NOS) was investigated by assaying the accumulation of nitrites in the culture medium following the treatment with a cocktail of cytokines and estradiol. Fig. 1 shows that 24 h exposure to the cytokine cocktail results in a signi®cant increased accumulation of nitrites. This effect was blocked by co-treatment with 1 nM 17b-estradiol (E2). When nitrites were measured in unstimulated cells, the treatment with the hormone did not have any signi®cant consequence. Since it is well known that in SMC in¯ammatory stimuli regulate the synthesis of the inducible form of nitric oxidase synthase: iNOS, we further investigated whether E2 had any effect on iNOS levels in cytokine-stimulated cells by western analysis. As reported by several authors, the iNOS protein content is barely detectable by western blot analysis in primary cultures of SMC. Twenty-four hour treatment with the cytokines increases the signal and co-treatment with estradiol signi®cantly prevents such an increase (Fig. 1, lower panel). These observations suggest that estradiol controls iNOS synthesis in SMC at concentration compatible with ER activation, and lead to hypothesize a potential antiin¯ammatory role for this hormone. This is in contrast with the activity of E2 in the reproductive tracts, which has been traditionally associated with in¯ammation. In uterus,
E. Vegeto et al. / Experimental Gerontology 35 (2000) 1309±1316
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Fig. 2. Effect of E2 on nitrite and nitrate concentration in pleural exudate (left panel) and in lungs (right panel) at 4 h after carrageenan administration. Data are means ^ SE of 10 rats for each experimental group. P , 0:01 vs. control.
in fact, E2 stimulates epithelial cell proliferation, increases vascular permeability, edema and in¯ux of macrophages. To further investigate on the potential anti-in¯ammatory role of E2, we next examined the activity of E2 in a well-known model of in¯ammation (carrageenan-induced pleurosy). Carrageenan was injected into the plural activity as described (Cuzzocrea et al., 2000). At 4 h after the injection of the in¯ammatory stimuli, the animals were killed and the plural cavity was opened to obtain the exudate as described in Section 2. The production of nitrites was measured in the exudate and the iNOS activity measured in lung cells. As shown in Fig. 2, the treatment with carrageenan signi®cantly increased the content of nitrites and nitrates in the exudate as well as in lung cells. E2 pre-treatment signi®cantly prevented the carrageenan-induced elevation of nitrite and nitrates (left panel) and the iNOS activity (right panel). E2 by itself did not have any effect on both parameters (Fig. 2). These results provide further evidence of an effect of estrogens on the iNOS activity. Recent evidence suggests that toxic substances released by glial cells might be involved in the neurodegenerative processes that occur in disease like Parkinson's, Huntington's and Alzheimer's (McGeer and Rogers, 1992; McGeer and McGeer, 1995; Kalaria, 1999). Post-mortem examination reveals the presence of massive astrogliosis and activate microglia in the region where the neurodegenerative process occurs (McGeer, 1988). In addition, it has been shown that nitric oxide produced by glial cells participates in the cascade of events that leads to the degeneration of neurones in mice that are rendered parkinsonian by MPTP intoxication. We therefore investigated whether estrogens could antagonize the induction of iNOS activation caused by LPS treatment of microglial cells. Microglia was isolated from brain of 2-day-old rat brain following welldescribed procedure. The effect of E2 on iNOS activity was assessed by measuring nitrite/nitrates content in the growth medium. Fig. 3 shows that, similarly to what previously demonstrated in SMC and lung cells, 1 nM estradiol blocked LPS-induced increase of nitrite accumulation.
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Fig. 3. Microglia cells were treated as reported in the ®gure for 24 h. Data represent the mean ^ sem of three separate experiments run in triplicate. P , 0:01 vs. control.
4. Discussion The bene®cial effects of estrogens in the delay of Alzheimer's disease have been related to the hormone's neuroprotective effects preventing neuronal loss and its trophic action on neurotransmission and neuronal metabolism. The data of the present report point to a novel mechanism by which E2 might extend its neuroprotective action. E2 might, in fact, act in microglial cells and limit their pro-in¯ammatory potential. It is well known that the principal pathological feature of Alzheimer's disease is the extracellular deposition of ®brillar amyloid and its compaction into senile plaques. These plaques are the focus of a complex of cellular reactions involving the activation of both microglia and astrocytes. The plaque-associated microglia exibits an ªactivatedº phenotype. The appearance of in¯ammatory products in Alzheimer's as well as in other neurodegenerative disorders has been interpreted as evidence of chronic in¯ammatory component of the disease process. Supporting this hypothesis are reports on patients treated for extended periods with non-steroidal anti-in¯ammatory drugs, who showed a dramatic reduction in Alzheimer's risk. Who are the major players in the sustained chronic in¯ammation possibly contributing to the onset of neurodegeneration is still unknown. Liberatore et al. (1999), studying mice made parkinsonian by treatment with MPTP, provides strong evidence that nitric oxidase participates in the cascade of events leading to the degeneration of neurons. If this were the case, our study might provide relevant data for the understanding of E2 effects in neurodegeneration. We have recently shown that macrophages are a target of estrogen action (Vegeto et al., 1999). The present study shows that also microglia might be an important site of action of these powerful hormones. Studies are in progress to better de®ne the activity of this sex hormone in microglia cells both in vitro and in vivo. Acknowledgements We thank Clara Meda, Monica Rebecchi, and Paola Pizzolorusso for their excellent
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technical and secretarial support. The Italian Foundation for Cancer Research (AIRC), Telethon (E600), Istituto Superiore di SanitaÁ (Rome), Multiple Sclerosis Programme and the EC Biomed Programme (Contract No. BMH4-CT97-2286) provided ®nancial support.
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