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Endocrinology Vol. 140, No. 6 2876-2882
Copyright © 1999 by The Endocrine Society

ARTICLES

Expression of the Interleukin-6 Gene Is Constitutive and Not Regulated by Estrogen in Rat Vascular Smooth Muscle Cells in Culture1

Arlette Maret, Simone Clamens, Isabelle Delrieu, Rima Elhage, Jean-François Arnal and Francis Bayard

INSERM U-397, Institut Louis Bugnard, 31403 Toulouse Cedex 4, France

Address all correspondence and requests for reprints to: Dr. Arlette Maret, INSERM U-397, Institut Louis Bugnard, 1 avenue Jean Poulhès, 31403 Toulouse Cedex 4, France. E-mail: maret{at}rangueil.inserm.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Vascular smooth muscle cells (SMC) are major constituents of the medial layer of blood vessels and are involved in the development of atherosclerotic plaque. SMC secrete copious IL-6 under basal conditions that can be increased by cytokines such as tumor necrosis factor-{alpha} and interleukin-1ß (IL-1ß). The goal of our studies was to define the role of estrogen in IL-6 production by SMC. In a first series of experiments, the expression of specific messenger RNAs as well as the production of IL-6 bioactivity by rat SMC in culture could be demonstrated in basal and IL-1-stimulated conditions, but was unaffected by estrogen treatment. Different constructs containing deleted or mutated fragments of the human IL-6 promoter driving luciferase or chloramphenicol acetyltransferase reporter gene were then transiently transfected in these cells. A significant basal activity that was increased 2- to 4-fold after IL-1ß stimulation was observed with the total IL-6 promoter. Deletion analysis indicated that the -158/+11 region containing activator protein-1 and cAMP response element sites was apparently the minimal region of IL-6 promoter to confer both constitutive and IL-1-inducible activities. Site-directed mutagenesis experiments suggest that basal activity is dependent upon the promoter sequence -158 to -112 containing the nuclear factor (NF)-IL6(-153) and Sp1 sites, whereas IL-1ß stimulation would depend on the residual -112 nucleotides containing NF-IL6(-75) and NF-{kappa}B sites. In contrast to the down-regulation of IL-6 expression by estrogen described in osteoblasts, ethinyl estradiol as well as 17ß-estradiol did not influence stimulated IL-6 activity in our experimental conditions whatever the construct tested, even when either estrogen receptor {alpha} or ß was overexpressed. Thus, the atheroprotective properties of estrogen are probably not mediated through the regulation of IL-6 production by SMC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE INCIDENCE of cardiovascular disease, the leading cause of mortality in Western societies (1), is higher in men than in premenopausal women, but increases in postmenopausal women. An abundance of epidemiological data support a role for estrogens in this atheroprotective effect, prompting recommendations for their widespread use in postmenopausal replacement therapy (2). However, the mechanism by which this protection is mediated has remained obscure. It has traditionally been thought to be due to potentially favorable changes in blood lipids and lipoproteins (2), but a number of animal studies strongly suggest a direct effect on the vascular system (3, 4, 5, 6, 7, 8). We recently reported that cells of the vascular aortic wall are estrogen target cells that express aromatase, 17ß-estradiol hydroxysteroid dehydrogenase, and 17-ketoreductase enzyme activities as well as estrogen receptor {alpha} (9, 10). We also have been able to demonstrate that estrogens decrease the endothelial permeability barrier (11) and prevent the degradation of nitric oxide generated in endothelial cells by decreasing superoxide anion production in these cells (12), which could contribute to the atheroprotective properties of estrogen.

However, estrogens might also favorably affect the inflammatory component involved in the atherosclerotic process. Lymphokines are crucial mediators among the cellular components of the lesions, vascular smooth muscle cells, endothelial cells, macrophages, and T lymphocyte (13). Among these multipotent mediators, IL-6 is expressed in normal arteries (14) as well as in atherosclerotic lesions of genetically hyperlipidemic rabbits (15), and its secretion by smooth muscle cells (SMC) is increased by pathophysiologically relevant factors such as lipopolysaccharide, interleukin-1 (IL-1), tumor necrosis factor-{alpha} (TNF{alpha}), endothelin, hypoxia, and aging (14, 16, 17, 18, 19). IL-6 expression is under estrogen control in freshly explanted endometrial structural cells stimulated by IL-1, TNF{alpha}, and interferon-{gamma} (20) and in bone where an increased production of IL-6 in estrogen-depleted states may contribute to postmenopausal osteoporosis in women (21, 22).

IL-6 is a pleiotropic cytokine with a variety of biological activities. On the one hand, its contribution to inflammation is strongly suggested by many studies (23); it plays a major role in the acute phase response and stimulates lymphocyte proliferation as well as differentiation of B cells and antibody production by B cells (24). Possible effects of IL-6 also include growth- and differentiation-inducing activities of nonlymphoid cells, particularly of vascular endothelial and smooth muscle cells. IL-6 modulates the proliferation of vascular smooth muscle cells in culture (25, 26), increases endothelial permeability in vitro (27), and participates in the production of the extracellular matrix constituents and of serum amyloid A (28, 29). On the other hand, recent studies have addressed another facet of IL-6 activity that is antiinflammatory (30, 31). IL-6 suppresses the generation of IL-1 and TNF{alpha} in macrophages exposed to lipopolysaccharide in culture or in mice infused with lipopolysaccharide (32, 33, 34). It stimulates the production of soluble TNF{alpha} and IL-1 receptors (35) as well as large quantities of IL-1 receptor antagonist (36), the atheroprotective activities of which have been demonstrated (37). As both pro- and antiinflammatory activities could contribute to the atherosclerotic process, we decided to investigate the regulation of IL-6 gene expression by estrogens in SMC, one of the major cellular constituents of atherosclerotic plaque, to determine whether IL-6 could be a target in the atheroprotective effect of estrogens.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant human IL-1ß was obtained from Pharma Biotechnologic (Hanover, Germany), and recombinant human IL-6 was a gift from Dr. E. Vita, Elf Biorecherche (Toulouse, France).

The DNA probe for rat IL-6 was obtained by RT-PCR using 1 µg total RNA from activated rat peritoneal macrophages and the superscript preamplification system with random hexamers for the RT step and using 20-mer oligonucleotides corresponding to the 5'- and 3'-portions of the open reading frame of the complementary DNA (cDNA; generating a PCR fragment of 1.1 kDa), 0.2 x 10-3 M deoxynucleotide, and 2.5 U AmpliTaq polymerase (Perkin Elmer, Norwalk, CT) for the amplification step.

Cell culture
Rat vascular SMC were obtained as previously described (9) from female rat aortic media (Wistar strain) and were used between passages 4 and 8. SMC were grown in DMEM supplemented with 10% calf serum, 2 mM glutamine, 0.1 mg/ml amphotericin, and 0.1 mg/ml gentamicin (Life Technologies, Grand Island, NY) at 37 C in a 10% CO2 atmosphere. Three days before the experiments, cells were switched to phenol red-free DMEM containing charcoal-treated serum (38).

Assay for IL-6 activity
Cells of the hybridoma line B9, provided by Sanofi-Elf Biorecherche (Toulouse, France), were cultured in RPMI containing L-glutamine, antibiotics, 2-mercaptoethanol (5 x 10-5 M), sodium pyruvate (2.5 x 10-3 M), recombinant IL-6 (recIL-6), and 10% FCS (B9 medium). Samples were diluted in 100 µl B9 medium. Cells were centrifuged twice in B9 medium without recIL-6 and adjusted to 50,000 cells/ml, and 100 µl of this cell suspension were added to 100 µl of the diluted samples. The cultures were incubated for 72 h and exposed for the last 4 h of culture to 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (Sigma Chemical Co., St. Louis, MO). The culture medium was then removed, and cells were dissolved in 100 µl propanol-water (2:1, vol/vol) containing 10% SDS and 0.04 N HCl and analyzed at 540 nm. The mean of triplicate cultures was determined, and biological activity evaluated by Probit analysis using the method described for IL-2 (39). The biological activity was measured using serial 4-fold dilutions of samples; recIL-6 was used as the standard in every B9 assay.

Northern blotting
SMC were lysed in RNAzol, and the RNA was extracted and precipitated twice following the protocol of Chomczynski and Sacchi (40). The RNA samples (20 µg) were fractionated on a 1.2% agarose gel containing formaldehyde, run at 100 V for 1 h, blotted on a nitrocellulose membrane by capillary transfer, and fixed by UV light on each side. After prehybridization for 2 h, the membrane was hybridized overnight using a [32P]deoxy-CDP (Amersham, Arlington Heights, IL)-labeled, random primed, IL-6 cDNA fragment. The membrane was then washed twice with 2 x SSC (standard saline citrate)-1% SDS for 30 min at 55 C. Autoradiography was performed using X-Omat AR film (Eastman Kodak Co., Rochester, NY). The nitrocellulose membrane was stripped and rehybridized with ß-actin cDNA as a control.

Plasmid constructions and transient transfections
The reporter plasmid PrIL6Tot (Fig. 1Go) was obtained by subcloning the human genomic DNA fragment -1158 to +11, released by XbaI and XhoI digestion of the plasmid pBRgHIL-61 (supplied by Walter Fiers, Laboratory of Molecular Biology, University of Ghent, Ghent, Belgium) into pGL3-Basic vector (Promega Corp., Madison, WI), which was digested by NheI and XhoI. A deletion mutant of the 5'-flanking region was obtained by digestion of PrIL6Tot with NheI and KpnI and blunting with Klenow enzyme; the resulting plasmid contained the fragment -224 to +11 of the IL-6 gene (Fig. 1Go, PrIL6NheI). The deletion mutant containing the fragment -158 to +11 was obtained by digestion of PrIL6Tot with AatII and SacI and blunting with Klenow enzyme (Fig. 1Go, PrIL6AatII). The deletion mutant containing the fragment -112 to +11 was obtained by subcloning the HaeIII/NcoI fragment released from PrIL6Tot into pGL3-Basic vector, which was digested by SmaI and NcoI (Fig. 1Go, PrIL6HaeIII). The series of site-directed mutagenesis on the -224 to +11 fragment of the IL-6 gene upstream from the chloramphenicol acetyltransferase (CAT) reporter gene (provided by Y. Zhang, New York University, New York, NY) (41) contained no mutation (Fig. 1Go, PrIL6Z1), a single NF-IL6 site mutation (Fig. 1Go, PrIL6Z2 and PrIL6Z3) or NF{kappa}B (PrIL6Z5), double NF-IL6 site mutations (PrIL6Z4), or triple site mutations (two NF-IL6 and NF{kappa}B, PrIL6Z6).



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  		Figure 1. Schematic representation of the different constructs of the IL-6 promotor. Putative cis-acting DNA elements and their locations are indicated: one activator protein-1 (AP-1), one cAMP response element (CRE), two NF-IL6-binding sites (at positions -153 to -145 and -83 to -75), and one NF-{kappa}B-binding site (at position -72 to -63). Deletion mutants (PrIL6NheI, PrIL6AatII, and PrIL6HaeIII) and site-directed mutagenesis (41 ) (PrIL6Z1 to PrIL6Z6) are represented. +11, Cloning site for the reporter genes; LUC, luciferase.

 
For transient transfections, cells were grown to confluence, trypsinized, washed three times in DMEM, and resuspended at a density of 6 x 106 cells/ml. Cells (0.5 ml cell suspension) were transfected by electroporation with 10 µg plasmid DNA at 320 V and 960 µF using a Gene Pulser (Bio-Rad Laboratories, Inc., Richmond, CA) and then immediately diluted in complete medium (phenol red-free DMEM containing charcoal-treated serum). Cells were plated at a density of 25 x 104/well in 24-well plates (Corning Costar, Cambridge, MA). After 18 h, the medium was replaced with DMEM containing 2 µg/ml transferrin. Cells were treated with IL-1ß 16 h before the end of the experiment (48 h after electroporation). Estrogen treatment was carried out with ethinyl estradiol (EE2; 10-9 M) to avoid interference resulting from metabolism of the ligand (9), or 17ß-estradiol (E2; 10-9 M) or the vehicle ethanol (0.1%) in control cultures. Cells were also transfected with pGL3SV40LUC or psctCAT to provide a measure of transfection efficiency.

The plasmids pSG5-rat or human ER, containing the rat or human estrogen receptor cDNA, and pERE-tkLuc, containing the palindromic ERE of the vitellogenin A2 gene, have been described previously (9), and the human ERß was provided by M. Muramatsu (42).

Luciferase and CAT assays
Luciferase activity was determined following the Promega Corp. assay procedure. Cells were rinsed twice in PBS and suspended in 50 µl cell culture lysis reagent. After 15 min at room temperature, the cells were scraped and briefly centrifuged. Twenty microliters of supernatant were mixed with 100 µl luciferase assay reagent. The relative luciferase activity was determined using a luminometer.

CAT activity was determined using a previously described procedure (43). Cells were rinsed twice in PBS and suspended in 100 µl 20 mM Tris-HCl (pH 7.8), and 2 mM MgCl2. After two cycles of freezing (-80 C) and thawing, the cells were collected, incubated for 10 min at 65 C, and then centrifuged at 4 C for 10 min. Thirty-five microliters of supernatant were added to 15 µl of a solution containing 100 mM Tris-HCl (pH 8), 100 mM [14C]chloramphenicol, and 250 mM n-butyryl coenzyme A. After incubation at 37 C for 4 h, the reaction was terminated by the addition of 200 µl of a 2:1 mixture of tetramethylpentadecane-xylene and mixed vigorously by vortexing. After centrifugation for 3 min, 150 µl of the organic phase were removed and counted in 3 ml Ready Safe (Amersham).

All luciferase and CAT experiments were repeated in triplicate in three to five separate trials.

Data analysis
All values are expressed as the mean ± SD. Unpaired Student’s t tests were employed to determine the significance of changes in IL-6 production, luciferase, or CAT activities. A significant difference was attributed for P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Production of IL-6 activity by SMC
As shown in Fig. 2Go, rat cultures of SMC released significant amounts of IL-6, as reported previously by Loppnow and Libby in humans (17). Incubation with IL-1ß greatly augmented this production. EE2 treatment, in concentrations ranging from 0.01–1 x 10-8 M for 48 h, did not influence basal or stimulated activity.



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  		Figure 2. IL-6 production by SMC. SMC grown in phenol red-free DMEM supplemented with charcoal-treated serum were treated for 48 h with EE2 (or ethanol) at the indicated concentration and with IL-1ß (10 ng/ml) 16 h before the end of the experiment. Secreted IL-6 was determined using the B9 cell bioassay as described in Materials and Methods. The results of a representative experiment repeated three times are shown (mean ± SD). *, P < 0.005 vs. controls.

 
The levels of IL-6 messenger RNA (mRNA) were then measured by Northern blot analysis to examine whether the release of IL-6 protein occurred in parallel with the expression of IL-6 mRNA. As shown in Fig. 3Go, IL-6 mRNA of 1.3 and 3 kb were detected, in agreement with the report by Northemann et al. (44); both were significantly increased after stimulation with 10 ng/ml IL-1ß for 12 h. Again, no influence of EE2 was noted.



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  		Figure 3. Northern blot analysis of IL-6 mRNA in control and IL-1ß-stimulated rat SMC without or with EE2 treatment (10-9 M). Total RNA (20 µg) was separated on an agarose formaldehyde gel, transferred to nitrocellulose membrane, and probed with 32P-labeled IL-6 cDNA. Data shown are representative of three independent experiments, which gave similar results.

 
Regulation of IL-6 gene transcription in SMC
We then mapped DNA regulatory elements on the 5'-flanking region of the IL-6 gene to further investigate why the IL-6 gene was not regulated by estrogens in this cell population. Rat SMC cells were transiently transfected by the electroporation method using the different constructs described in Fig. 1Go and were subjected to luciferase or CAT assays. As shown in Fig. 4Go, a significant basal luciferase activity was displayed using the entire IL-6 promoter construct (positions -1158 to +11). Deletions of sequences down to position -158, which removed putative activator protein-1 and cAMP response element sites, did not alter the basal activity. Deletion down to position -112, which removed one of the two NF-IL6 sites (41) and a Sp1 site (45), resulted in a significant decrease in basal luciferase activity. After stimulation with IL-1ß (10 ng/ml for 16 h), luciferase activity increased 2- to 4-fold depending on the experiments with the four different constructs. A slight, but statistically significant, increase in basal activity was observed in the presence of 10-9 M EE2 or E2 for 48 h (P < 0.001). The IL-1ß-stimulated luciferase activity was unaltered by estrogen treatment. When rat SMC were cotransfected with expression plasmids for human and rat ER{alpha} (Fig. 5Go) or human ERß (not shown), EE2 was still inactive on IL-1-stimulated activity, although it was active on an estrogen response element-containing reporter gene plasmid transfected in the same population of cells (Fig. 5Go, inset, and data not shown). Similar data were obtained using the natural hormone E2 (10-9 M) for estrogen stimulation (Figs. 4Go and 5Go).



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  		Figure 4. Analysis of basal and IL-1ß-induced IL-6 promoter activity in EE2 or E2-treated or untreated rat vascular SMC. SMC were transfected by electroporation at 320 mV and 960 µF using 10 µg of the different IL-6 promoter constructs and 5 µg psctCAT (to provide a measure of transfection efficiency) and then plated at a density of 25 x 104 cells/well in 24-well plates. After 18 h, the medium was replaced with DMEM containing 2 µg/ml transferrin. Estrogen treatment was carried out for the 48 h of the experiment using EE2 (10-9 M) or E2 (10-9 M). Cells were treated with IL-1ß (10 ng/ml) 16 h before the end of the experiment. The cells were then harvested, and luciferase and CAT activities measured.

 


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  		Figure 5. IL-6 promoter activity in rat vascular SMC transfected using PrIL6Tot (10 µg) with or without pSG5-rat ER{alpha} (5 µg). Rat SMC were transfected by electroporation as described in Fig. 4Go. After 18 h, the medium was replaced with DMEM containing 2 µg/ml transferrin. Estrogen treatment was carried out for the 48 h of the experiment using EE2 (10-9 M). Cells were treated with IL-1ß (10 ng/ml) 16 h before the end of the experiment. The cells were then harvested, and luciferase activities were measured. Similar results were obtained using expression vectors for human ER{alpha} (5 µg) or ERß (5 µg) and E2 (10-9 M) as estrogen treatment. Inset, Cotransfection experiments with pSG5-rat ER{alpha} (5 µg) and pERE-tkFLuc (10 µg) containing the palindromic estrogen response element of the vitellogenin A2 gene.

 
Sequence analyses of the 5'-flanking region of the human IL-6 gene characterized two NF-IL6 at positions -153 to -145 and -83 to -75 and a NF-{kappa}B-binding site at position -72 to -63. To test whether these NF-IL6 and NF-{kappa}B binding sequences contribute to both the basal and stimulatory activities of IL-1ß in this population of cells, the series of site-directed mutagenesis (Fig. 1Go) performed by Zhang et al. (41, 46) on the -224 to +11 fragment was used in transient transfections. As shown in Fig. 6Go, compared with PrIL6Z1, mutation of the NF-IL6(-153) site with intact NF-IL6(-75) and NF-{kappa}B sites (PrIL6Z2) resulted in a significant decrease in basal luciferase activity. Nevertheless, the 4-fold stimulation of inducible activity was still observed. The basal activity was also low with the NF-IL6(-75) (PrIL6Z3) or the NF-{kappa}B site (PrIL6Z5) mutants, but the IL-6 response to IL-1ß stimulation significantly decreased. When combined mutations of the two NF-IL6 (PrIL6Z4) and NF-{kappa}B sites were created (PrIL6Z6), both basal and induced IL-6 promoter activities were barely detectable (Fig. 6Go). EE2 was constantly inactive.



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  		Figure 6. Analysis of basal and IL-1ß-induced activities of mutant constructs of the IL-6 promoter. Site-directed mutagenesis (41 ) was performed on the IL-6 DNA fragment from positions -224 to +11 as indicated in Fig. 1Go. Rat SMC were transiently transfected with these different constructs and treated with EE2 and IL-1ß as indicated in Fig. 4Go, except that in the present experiments the plasmid pGL3SV40Luc was used to measure transfection efficiency.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In agreement with the observations of Loppnow and Libby in humans (17), we found that rat SMC produced large amounts of IL-6 and expressed specific mRNA that could be increased by the administration of IL-1ß. However, in contrast to osteoblastic cell cultures, where a down-regulation has been clearly established (21), IL-6 production was not influenced by EE2 treatment in phenol red-free medium supplemented with charcoal-stripped medium for a length of time that has been shown to inhibit IL-6 secretion by osteoblastic cells (47). Similarly, transient transfections using promoter fused to reporter genes had shown that expression was inhibited by estrogens in bone marrow-derived murine stromal cell line, human osteoblastic and osteoblastoma cell lines, and HeLa as well as breast cancer MCF-7 cells (48, 49, 50). Low basal expression was observed in all of these studies, and the estrogen receptor was shown not to bind directly to the IL-6 promoter but to interfere indirectly with gene expression via protein-protein interactions with members of the CCAAT enhancer-binding protein (C/EBP) and/or NF-{kappa}B families (48, 49, 50, 51, 52). In contrast to this concordant response in these cell populations, our studies clearly show that, like the native gene, the longer IL-6 promoter constructs displayed significant basal activity that could be increased by IL-1ß treatment but was not influenced by EE2 or by E2, even when either estrogen receptor {alpha} or ß was overexpressed.

Deletion analysis indicated that the -158/+11 region contains the minimal region of IL-6 promoter to confer both constitutive and IL-1-inducible activities. It should be noted that the inhibitory effect mediated through the -224/-158 sequence in osteoblasts (52) was not observed in rat SMC. As analyzed by previous researchers, the -158/+11 region harbors two C/EBP- and one NF-{kappa}B-binding sites that have been shown to be sufficient for mediating IL-1 (53) as well as the estrogen effects through the mediation of estrogen receptor {alpha} (50, 52). Our site-directed mutagenesis experiments confirmed the synergism between NF-IL6 and NF-{kappa}B factors (50, 54). Comparison of basal and stimulated reporter gene activities in experiments using PrIL6AatII, PrIL6HaeIII, and PrIL6Z2 suggests that basal activity is dependent upon the promoter sequence -158 to -112, and IL-1ß stimulation is dependent upon the sequence -112 to +11. The -158/-112 region harbors the NF-IL6(-153) site as well as an Sp1 site (45). Sp1 activity appears to be closely related to increased cellular proliferation (55), and a role of the SMC proliferative state in IL-6 expression has been proposed (17, 56, 57). Indeed, we observed a parallel stimulation of luciferase gene expression (PrIL6Tot) and [3H]thymidine incorporation under increasing serum concentrations in the culture medium (Maret, A., unpublished observations). It has been suggested (56, 58) that expression of a constitutive NF-{kappa}B-like activity in bovine and human vascular smooth muscle cells would result from the presence of a novel and SMC-specific member of the NF-{kappa}B/Rel family. These observations were not confirmed by another group that suggested that basal NF-{kappa}B complexes in human SMC cultured in serum contain classical NF-{kappa}B, i.e. p65 and p50 (57). We propose that a cooperative C/EBP/Sp1 binding complex should be considered in interpreting the results from these experiments, similar to that already described for the CYP2D5 cytochrome P-450 gene (59). In contrast, both the NF-IL6(-75) and NF-{kappa}B sites were necessary for a significant activation by IL-1 in SMC cells. Mutation of the NF-IL6(-75)-binding site could not be rescued by the presence of a functional NF-IL6(-153) site in SMC as it can be in HeLa and MCF-7 cells (50). At the same time, estrogen insensitivity was observed. These data suggest that a second specific cooperative interaction between C/EBP and NF-{kappa}B family members is involved in IL-1ß stimulation that prevents interaction with ligand-activated ER in SMC cells.

In conclusion and in contrast to what was observed in endometrial and bone cells, estrogens do not decrease IL-6 production by rat SMC, the major source of this cytokine in the vascular wall. Estrogens, in fact, tend to increase the basal and constitutive transcriptional activities of the IL-6 promoter in this population of cells by an unknown mechanism, although this increase was not reflected by the level of cytokine expression. On the basis of recent knowledge concerning the molecular basis of the acute coronary syndromes, involving local production of inflammatory cytokines IL-1, TNF{alpha}, and interferon-{gamma} (13), it may appear that maintenance of SMC IL-6 production could, in fact, contribute to the atheroprotective effect of estrogens. Such a situation could precisely reflect the antiinflammatory, rather than the proinflammatory, activities of this cytokine. In any case, the role of IL-6 as the main mediator of the atheroprotective effect of estrogens (60) has yet to be defined. Indeed, our recent data, obtained in apolipoprotein E and IL-6 double deficient mice, do not confirm a role for IL-6 in the atheroprotective effect of estradiol (Elhage, R., S. Clamens, S. Besnard, A. Tedgui, J. F. Arnal, A. Maret, and F. Bayard, in preparation).


   Acknowledgments
 
We thank Prof. Y. Zhang for generously providing the series of site-directed mutagenesis of the IL-6 gene, Walter Fiers for providing pBRgHIL-61, Dr. N. Blaes for providing rat vascular smooth muscle cells, Prof. J. P. Besombes for helpful discussion, and M. Larribe for secretarial assistance.


   Footnotes
 
1 This work was supported by grants from INSERM and the Conseil Régional Midi-Pyrénées. Back

Received August 20, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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