Expression of Interleukin-6 in Atherosclerotic Lesions of Male ApoE-Knockout Mice

Inhibition by 17ß-Estradiol

From the Departments of Cardiovascular Research (D.A.S., K.K., F.D.S., G.M.R.) and Pharmacology (V.D.V., M.H.-M.), Berlex Biosciences, Richmond, Calif.

Correspondence to Drew A. Sukovich, Department of Cardiovascular Research, Berlex Biosciences, PO Box 4099, 15049 San Pablo Ave, Richmond, CA 94804-0099. E-mail Drew_Sukovich{at}Berlex.com

	Abstract

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Abstract—Increased levels of interleukin-6 (IL-6) have been proposed to contribute to a number of pathological disorders, including osteoporosis and Alzheimer's disease. In human atherosclerotic lesions, IL-6 protein and mRNA have been detected, although the role of IL-6 in plaque formation is unknown. We have examined the expression pattern of IL-6 mRNA and secreted protein in male apolipoprotein E–knockout (apoE-KO) mice aortas. Furthermore, we have evaluated the effects of 17ß-estradiol (E2), a vasculoprotective sex steroid hormone, on the secretion of this inflammatory cytokine from isolated male apoE-KO mice aortas. The expression of IL-6 mRNA was detected by reverse transcription–polymerase chain reaction in the apoE-KO mouse aortas but not in the aortas of age-matched control mice. Similarly, the secretion of IL-6 protein from isolated apoE-KO aortic segments was significantly greater than that from aortas of age-matched control animals. The secretion of IL-6 from isolated aortic rings of apoE-KO mice ranging in age from 6 to 48 weeks showed a significant, positive correlation with percent lesion area measured in the same tissue. Immunohistochemical staining of apoE-KO mouse aortic tissue sections demonstrated colocalization of IL-6 expression with macrophages. Treatment of male apoE-KO mice with E2 for 3 weeks resulted in a statistically significant 50% reduction in IL-6 secretion from ex vivo aortic tissue segments. There was no significant change in total serum cholesterol and triglyceride levels in the E2-treated group compared with placebo-treated controls. These data demonstrate that (1) IL-6 mRNA and protein are expressed in the atherosclerotic plaques of apoE-KO mice aortas and (2) IL-6 production is suppressed by E2 treatment, which may contribute to the antiatherosclerotic effects of E2 in the apoE-KO mouse model of atherosclerosis.

Key Words: interleukin 6 • apoE-knockout mice • estrogen • atherosclerosis

	Introduction

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Atherosclerosis is a complex disease that is characterized by cholesterol deposition and monocyte infiltration into the subendothelial space, resulting in foam cell formation.¹ The presence of macrophages and T lymphocytes in the atherosclerotic lesion suggests an important role for the immune system and the inflammatory process in the pathogenesis of atherosclerosis.² A large number of proinflammatory cytokines have been shown to be expressed in human atherosclerotic lesions, particularly in association with infiltrating monocytes and macrophages.³ Although many of the functions of these cytokines are well understood in vitro, their role in the development of vascular lesions in vivo are for the most part unknown.

The mRNA transcripts of interleukin-6 (IL-6), a pleiotropic inflammatory cytokine, have been detected in human atherosclerotic lesions.⁴ This observation has been confirmed and extended by immunohistochemical studies that have demonstrated colocalized IL-6 protein expression with macrophages as well as smooth muscle cells in human atherosclerotic plaques.⁵ Moreover, IL-6 has been shown to have important effects on the cell types that are components of atherosclerotic lesions. IL-6 can "prime" THP-1 macrophage cells to produce enhanced amounts of tumor necrosis factor- in response to lipopolysaccharide (LPS),⁶ suggesting that IL-6 may play a role in stimulating macrophages to attain their full inflammatory potential. IL-6 has also been shown to stimulate the growth of vascular smooth muscle cells in a platelet-derived growth factor–dependent manner.⁷

Epidemiological studies have shown that estrogen exerts a beneficial clinical effect in postmenopausal women with Alzheimer's disease,⁸ osteoporosis,⁹ and coronary artery disease.¹⁰ The mechanism(s) for estrogen's beneficial effects on these diseases is not completely understood, although a significant link between the suppression of IL-6 gene expression by estrogen and the subsequent protective effects on bone has been established. Estradiol has been shown to inhibit IL-6 production in murine and human bone-derived cells¹¹ as well as to suppress the expression of IL-6 promoter constructs in the human osteoblast cell line U2-OS.¹² Studies on bone loss in the IL-6–knockout mouse have demonstrated that estrogen depletion does not promote the bone loss that is observed in normal littermate controls.¹³ These studies suggest an essential role for IL-6 in the bone loss associated with estrogen deficiency.

Although IL-6 has been detected in atherosclerotic lesions in humans, a role for this cytokine in the development of vascular lesions has not been defined. A significant obstacle in this regard is the lack of an animal model that expresses IL-6 in atherosclerotic lesions similar to that observed in human plaques. Recent data have demonstrated that estrogen reduces atherosclerotic lesion development in the apolipoprotein E–knockout (apoE-KO) mouse,^{14 15} an animal model of atherosclerosis with complex lesions relevant to human pathology.^{16 17 18} This suggests that the apoE-KO mouse may be a suitable animal model to evaluate the potential role of IL-6 in lesion progression as well as its potential contribution to estrogen's antiatherosclerotic mechanism of action. We report here for the first time that IL-6 transcripts as well as the secreted protein are detected in the apoE-KO mouse aorta. We also observed a significant linear correlation between IL-6 secretion and the percent plaque area in aortic segments. Immunohistochemical analysis revealed colocalization of IL-6 protein with macrophages in atherosclerotic lesions. Furthermore, we demonstrate that estrogen treatment of male apoE-KO mice results in decreased secretion of IL-6 from isolated aortas.

	Methods

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Animals
Male homozygous apoE-KO mice on a C57Bl/6J background (N10) and C57Bl/6J mice were obtained from The Jackson Laboratory (Bar Harbor, Me) at 5 weeks of age. The animals were housed in groups of 5 mice per cage in a room with a 12-hour light/dark cycle and provided with standard mouse chow and drinking water ad libitum until they were euthanized at different ages. This study was conducted according to protocols approved by the Animal Care Committee at Berlex Biosciences in agreement with the recommendations of the American Association for the Accreditation of Laboratory Animal Care.

Tissue Preparation
Mice were killed by CO₂ inhalation. Immediately after the mice were killed, body weight was measured and blood was collected by cardiac puncture. Dissection of the thoracic aorta was performed under aseptic conditions. The vessels were carefully cleaned of adherent connective tissue under a dissecting microscope. The aortic arch was cut at the level of the semilunar valves proximally and at the level of the diaphragm distally. To measure IL-6 secretion in ex vivo organ culture, each aorta was dissected and cut into 2-mm rings unless indicated otherwise. For reverse transcription–polymerase chain reaction (RT-PCR) analysis of IL-6 gene expression, each aorta was cut into aortic arch and descending thoracic segments and immediately frozen in LN₂. For immunohistochemical analysis and determination of aortic lesion area, a 2-mm ring of the aorta from the proximal end (supravalvular region) was cut and frozen in embedding resin (Cryochrome embedding resin, Shandon Inc) and snap-frozen in LN₂-cooled isopentane. The remaining aortic tissue was then placed in cold, sterile, Dulbecco's PBS, pH 7.2 (Gibco-BRL), before the determination of plaque area by videoimage analysis or IL-6 release in organ culture (described below).

RT-PCR
Total RNA was isolated from 5 pooled aortic arch and thoracic segments from 20- to 24-week-old male apoE-KO and age-matched C57Bl/6J mice by the 1-step method of Chomczynski and Sacchi¹⁹ with the use of Ultraspec RNA reagent (Biotecx). After quantification, RNA was diluted to 250 ng/mL in DEPC-water for RT-PCR. Before RT-PCR analysis, total RNA was electrophoresed on a 1% formaldehyde agarose gel to check for intact 28S and 18S ribosomal bands. RNA samples were tested for IL-6 and ß-actin expression by using the GeneAmp thermostable rTth reverse transcriptase RNA PCR kit (Perkin-Elmer). RT-PCR primer sets for murine IL-6 and ß-actin mRNAs were obtained from Clontech. RT-PCR was performed according to the manufacturer's protocol with the following modifications: 250 ng of each total RNA sample was amplified by using 0.75 µmol/L of each primer set. The RT reaction for ß-actin was performed at 60°C for 25 minutes followed by a 2-step PCR (95°C, 10 seconds; 65°C, 20 seconds) for 25 cycles. The RT reaction for IL-6 was performed at 60°C for 30 minutes followed by a 2-step PCR (95°C, 10 seconds; 60°C, 20 seconds) for 35 cycles. Products were visualized by running 10 µL of a 100-µL total reaction volume on a 2% agarose–1% Nusieve agarose (FMC Corp) 1x TBE gel for 1.5 hours at 100 V. DNA size standards (X174 RF DNA/HaeIII fragments, Gibco-BRL) were run to determine approximate PCR product sizes.

Determination of Aortic Lesion Area
Atherosclerotic plaque areas (fatty lesions) were visualized by transilluminating the intact, freshly isolated aortas without fixation and staining. Images were saved as TIF files and analyzed for plaque area in C-image (Compix Inc). In brief, the total projected vascular area was outlined by using a computer mouse cursor, and lesion area was determined by using a computerized thresholding function to count only the highest-density pixels. A single observer who was blinded to the age of the animal from which the aorta had been dissected performed this procedure. The ratio of pixels representing the projected area of plaques to those representing the projected area of the vessels was calculated for each aorta and expressed in percent. Immediately after the images of the aortas were obtained, the vessels were cut into 2-mm rings and placed in a 24-well plate containing organ culture medium for IL-6 protein secretion determination.

Ex Vivo Aorta Organ Culture and IL-6 Secretion Assay
The aortic segments from 20- to 24-week-old male apoE-KO (n=5) and age-matched C57Bl/6J (n=5) mice were dissected, cut into 2-mm rings, and placed into 1 mL of DMEM (Gibco-BRL) containing 1x ITS (insulin, transferrin, and selenium; Gibco-BRL) and 0.1% BSA (Sigma Chemical Co). Serum samples were also taken when the animals were euthanized and stored at -70°C. At time 0, aorta samples were washed with PBS several times, and fresh medium was added. Tissue was incubated for 4 hours at 37°C in a 5% CO₂ atmosphere, and the medium was then removed and frozen at -70°C before analysis. Aorta samples were weighed and genomic DNA was isolated (QiaAMP tissue kit No. 29304, Qiagen). Tissue culture supernatants were assayed for IL-6 by using a commercially available murine IL-6 ELISA (Biotrak IL-6 ELISA kit, Amersham Life Sciences). IL-6 values were normalized to genomic DNA content. In addition, serum IL-6 levels were also determined by ELISA.

Correlation of Aortic Lesion Area and IL-6 Secretion
The aortic segments from 6- (n=5), 16- (n=3), 32- (n=3), and 48- (n=5) week-old male apoE-KO mice were dissected (total n=16) and photographed intact by using a computerized imaging system for plaque lesion area determination as described above. Each aorta was then cut into 2-mm rings and placed into organ culture medium for IL-6 protein secretion determination by the organ culture method as described above.

Immunohistochemistry
To determine the cellular localization of IL-6 in the vessel wall of the apoE-KO mice, aortic rings from the supravalvular region of the aortas of 20- to 24-week-old male apoE-KO mice (n=5) and age-matched control mice (n=5) were cryosectioned at 5-µm thickness at -24°C. Sections were thaw-mounted onto glass slides (ProbeOn Plus, Fisher Biotech), postfixed in cold (-20°C) acetone for 1 minute, and then washed in PBS, pH 7.2. Double immunofluorescence staining was performed by using a CD68 primary antibody at a concentration of 4.5 µg/mL (KP1 antibody, DAKO) and a rat anti-mouse IL-6 monoclonal antibody at a concentration of 4 µg/mL (Genzyme Diagnostics). In brief, the sections were blocked in 1% goat serum in calcium- and magnesium-free PBS plus 0.005% Triton X-100 for 30 minutes. Tissue sections were then incubated with the primary antibody for 1 hour at 37°C. After they were washed, immunofluorescent secondary antibodies were incubated with the tissue slides (Texas red–conjugated goat anti-rat and FITC-conjugated goat anti-mouse; Molecular Probes) as described by the manufacturer. Digitized fluorescent images were obtained on an inverted confocal microscope (model No. 2010, Molecular Dynamics). Selected images were saved as TIF files, which were then imported into an Adobe Photoshop file (Adobe Systems) for image processing and printing. Hematoxylin- and eosin-stained sections were digitally photographed with a Fuji HC-2000 camera attached to an Aioskop bright-field microscope (Zeiss). All digital images were printed on a Fuji Pictography 3000 color printer after image processing to enhance contrast and adjust background.

Estrogen Effects on Ex Vivo IL-6 Secretion
Twenty- to 24-week-old male apoE-KO mice were treated with a 0.25-mg, 21-day, slow-release 17ß-estradiol (E2) or placebo pellet (Innovative Research of America) for 3 weeks. Animals were euthanized and the aortas dissected as described above. Serum was collected for lipid profile determination and estradiol and testosterone measurements. IL-6 expression was determined by using the ex vivo organ culture secretion assay described above.

Serum Lipid and Sex Hormone Levels
Blood samples were collected in serum collection tubes (Microtainer serum separator tubes, Becton Dickinson). Serum estradiol and testosterone levels were determined from pooled plasma samples by standardized radioimmunoassay methods through a contract service at the Veterinary Diagnostic Laboratory, New York State College of Veterinary Medicine (Cornell University, Ithaca, NY). Serum total cholesterol and triglycerides were measured at Consolidated Veterinary Diagnostics, Inc (West Sacramento, Calif).

Statistical Analysis
For the comparison of ex vivo IL-6 secretion from control versus apoE-KO mice and between estrogen-treated and placebo-treated apoE-KO mice, Student's t test comparison was used. For determination of correlation between IL-6 secretion and percent lesion area, the Spearman rank order and F tests were used.

	Results

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IL-6 mRNA Is Expressed in the ApoE-KO Mouse Aorta
IL-6 mRNA expression was analyzed by RT-PCR with the use of total RNA from 5 pooled aortic arch and thoracic aorta segments of 20- to 24-week-old male apoE-KO mice and age-matched C57Bl/6J (genetic control) mice. To assess for variations in RNA quality and quantity, RT-PCR was performed by using ß-actin primers as a control. In all RNA samples examined, ß-actin expression levels were similar (Figure 1A). In contrast to ß-actin expression, IL-6 mRNA expression was detected in the apoE-KO mouse aortic arch only (Figure 1B). No IL-6 mRNA was detected in the control aorta RNA samples or the apoE-KO mouse thoracic aorta segments. The ß-actin and IL-6 RT-PCRs produced the expected product sizes of 540 and 638 bp, respectively, compared with RT-PCR product from control reactions (data not shown).

View larger version (42K): [in this window] [in a new window]   Figure 1. A, RT-PCR analysis of ß-actin expression in pooled aortic tissue of five 20- to 24-week-old male mice. Lane 1, DNA size standards; 2, C57Bl/6J control mouse aortic arch RNA; 3, C57Bl/6J control mouse thoracic aorta RNA; 4, apoE-KO aortic arch RNA; and 5, apoE-KO thoracic aorta RNA. B, RT-PCR analysis of IL-6 expression in pooled aortic tissue of five 20- to 24-week-old male mice. Lane 1, DNA size standards; 2, C57Bl/6J control aortic arch RNA; 3, C57Bl/6J control thoracic aorta RNA; 4, apoE-KO aortic arch RNA; and 5, apoE-KO thoracic aorta RNA. RT-PCR was performed as described in Methods. RT-PCR products were run on a 2% agarose–1% Nusieve agarose 1x TBE gel and then visualized by UV light. Note uniform expression of ß-actin mRNA but selective expression of IL-6 mRNA in aortic arch of apoE-KO mice only.

Secretion of IL-6 Protein From Isolated Aortas of ApoE-KO Mice
To confirm the RT-PCR expression analysis and develop a more quantitative method to measure changes in the level of IL-6 secretion, we developed an ex vivo organ culture assay by using isolated aortas from 20- to 24-week-old male apoE-KO and age-matched control mice. The amount of IL-6 protein released from the aortic tissue was measured in the organ culture medium by ELISA. To control for differences in tissue size and total cellular composition, each aorta was weighed, and total genomic DNA was extracted after the 4-hour incubation. The isolated apoE-KO aorta released a statistically significant (P<0.01) 10-fold-greater amount of IL-6 than that released from age-matched control mice aortas, after normalization to genomic DNA content (Figure 2). In a separate study, when aortic arch and thoracic aortic segments from the same aorta were cultured separately, the secretion of IL-6 was detected predominantly in the supernatants of aortic arch but not the thoracic aorta organ cultures (data not shown). This result corroborates our original observation that IL-6 mRNA is found predominantly in the aortic arch. No IL-6 was detected in the serum samples from any of the apoE-KO mice examined in this study (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 2. Ex vivo IL-6 secretion in aortic segments isolated from apoE-KO and control mice. Aortas from 20- to 24-week-old male apoE-KO (n=5) and age-matched control (n=5) mice were dissected and cut into 2-mm rings. Each set of rings was placed into 1 mL of DMEM supplemented with 1x ITS (insulin, transferrin, and selenium) and 0.1% BSA and then incubated at 37°C in a 5% CO2 atmosphere for 4 hours. Secreted IL-6 was measured in medium by ELISA and is expressed relative to genomic DNA content of incubated aortic tissue. Results are mean±SEM of 5 experiments and show a statistically significant (*P<0.01) 10-fold increase in IL-6 secretion from apoE-KO aortas (stippled bar) versus age-matched controls (black bar).

Ex Vivo IL-6 Secretion Correlates With Atherosclerotic Lesion Area
The relationship between IL-6 secretion and the extent of plaque area was evaluated in aortas isolated from male apoE-KO mice at distinct ages ranging from 6 to 48 weeks. There was significant visible progression of lesion area in the apoE-KO mouse aorta within the time frame examined. By the age of 48 weeks, almost all of the aortic arch and some of the thoracic aorta contained visible lesions. A graph of the amount of IL-6 released from the apoE-KO mouse aorta against the percent lesion area of the same tissue revealed a statistically significant linear correlation (R=0.94, P<0.0001) (Figure 3). A statistically significant linear correlation was also observed between IL-6 secretion from isolated aortas and total lesion area (R=0.95, P<000.1) (data not shown). No significant amount of IL-6 was detected in the organ culture supernatants of isolated aortas from similar age-matched C57Bl/6J mice (data not shown).

View larger version (13K): [in this window] [in a new window]   Figure 3. Amount of ex vivo IL-6 secretion correlates with lesion area of the same apoE-KO aorta. Aortas from 6- (n=5), 16- (n=3), 32- (n=3), and 48- (n=5) week-old male apoE-KO mice (total n=16) were dissected and photographed intact on a computerized imaging system for percent lesion area determination. Each aorta was then cut into 2-mm rings and placed into medium for ex vivo IL-6 secretion determination as described in Figure 2. Correlation of IL-6 secretion (pg/µg DNA) versus percent lesion area (lesion area divided by total aorta area) is shown. Data presented are segregated on the basis of age at 6 (), 16 (), 32 (), and 48 () weeks. Spearman rank order correlation test reveals an R value of 0.94 that is statistically significant at P<0.001.

IL-6 Expression Colocalizes With Macrophages
Immunofluorescent labeling of frozen tissue sections from the supravalvular region of the apoE-KO mouse aorta revealed faint IL-6 staining in discrete areas within the atherosclerotic plaque (Figure 4D). In contrast, the macrophage marker CD68 strongly stained the aortic plaque tissue, indicating that a large number of cells within the plaque were monocytes or macrophages (Figure 4C). Most if not all of the cells positive for IL-6 staining colocalized with the staining for CD68 (Figure 4B). In contrast, not all cells that were CD68-positive stained with the IL-6 antibody. No staining for IL-6 or CD68 was detected in the method control slides (no primary antibodies) or in the aortas of age-matched control mice without atherosclerotic lesions (data not shown).

View larger version (137K): [in this window] [in a new window]   Figure 4. Double immunofluorescent staining of IL-6 and CD68 in vascular lesions of the apoE-KO mouse. A, Hematoxylin and eosin stain of frozen section from proximal aorta (supravalvular region) of a male 20- to 24-week-apoE-KO mouse. Medial wall is thinned and there is a moderately sized atheroma composed of macrophages and collagen-producing cells. Confocal microscopy in B shows colocalization (yellow) of antibody label for macrophages (CD68, red) and IL-6 (rat anti-mouse monoclonal, green). C and D show intensity of staining for CD68 and IL-6 antibodies, respectively, in pseudocolor (red and white are highest intensity areas). Bar=20 µm.

E2 Treatment of ApoE-KO Mice Inhibits Ex Vivo IL-6 Secretion
To determine whether E2 could exert an effect on IL-6 secretion, randomly selected 20- to 24-week-old male apoE-KO mice were treated with an E2 pellet (0.25 mg, 21-day, slow-release form) or a placebo pellet for 3 weeks. After the animals were killed, the ex vivo secretion of IL-6 from isolated aortic tissue as well as total cholesterol, triglycerides, estrogen, and testosterone levels in the serum were determined. The estrogen levels were 10-fold greater and testosterone levels 4-fold lower in the E2-treated versus placebo-treated animals (the Table). Treatment with E2 resulted in a statistically significant 50% reduction in IL-6 secretion from isolated apoE-KO mice aortas compared with aortas from age-matched control mice (P<0.01, Figure 5). There was no statistically significant difference in the total cholesterol and triglyceride levels in the sera of the apoE-KO mice treated with E2 compared with the placebo control group (the Table).

View larger version (18K): [in this window] [in a new window]   Figure 5. Effect of E2 on ex vivo IL-6 secretion in aortic segments isolated from apoE-KO mice. Twenty- to 24-week-old male apoE-KO mice were treated with 0.25 mg, 21-day, slow-release E2 (n=5) or placebo (n=5) pellets for 3 weeks. Aortas were dissected, and ex vivo IL-6 secretion was determined by assaying organ culture medium by ELISA. Amount of IL-6 protein secreted into medium is shown as mean±SEM of 5 experiments. Treatment with E2 (stippled bar) resulted in statistically significant (*P<0.01) 50% reduction in IL-6 secretion compared with placebo-treated controls (black bar).

	Discussion

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IL-6 is a multifunctional cytokine that plays a critical role in the acute-phase inflammatory response and is an inducer of B-cell proliferation and maturation (for reviews see References 20 through 22^{20 21 22} ). IL-6 is a 26-kDa protein that is produced by a number of cells, such as monocytes, lymphocytes, and hepatocytes, and is a member of an extended family of cytokines that utilize the shared signal transducer gp130.²² The dysregulation of IL-6 expression has been linked to a number of diseases, including multiple myeloma,²³ lymphoma,²⁴ rheumatoid arthritis,^{25 26} Castleman's disease,^{27 28} osteoporosis,²⁹ and Alzheimer's disease.³⁰ Recently, it has been found that unlike most cytokines, IL-6 levels in the serum increase with age in humans, mice, and monkeys, suggesting that the dysregulated control of IL-6 protein expression may contribute to a number of age-related diseases.²¹

In an effort to study the potential role of IL-6 in atherosclerosis, we have initiated studies to characterize the expression patterns of IL-6 mRNA and secreted protein in the aortas of apoE-KO mice. We detected IL-6 mRNA transcripts in the aortas of 20- to 24-week-old male apoE-KO mice but not in the aortas of age-matched control mice. Most of the apoE-KO mice at this age have extensive vascular lesions in the aortic arch and very little (if any) in the thoracic segment. Similar to the distribution of lesions within the aortic segment, IL-6 mRNA was found predominantly in regions containing a large amount of visible lesion (arch) but not in segments from the same aorta with little visible lesion (thoracic).

To confirm our initial observation that IL-6 mRNA is expressed in the apoE-KO mouse aorta, we developed an ex vivo aorta organ culture assay that measured the amount of IL-6 secreted from the isolated aorta into the medium. Using this method, we demonstrated that IL-6 protein secretion was substantially greater in the apoE-KO versus age-matched control mouse aorta. We also observed that IL-6 was secreted mainly from the aortic arch, confirming the RT-PCR expression data. These 2 experiments established that the amount of IL-6 mRNA and protein is substantially increased in the vascular lesions of the apoE-KO mouse, similar to that observed in humans.^{4 5}

We also evaluated the relationship between the extent of plaque in an isolated aorta segment and the amount of IL-6 secreted from the same tissue. We observed a significant, positive, linear relationship between these 2 variables in male apoE-KO mice between the ages of 6 and 48 weeks. To determine the potential cellular source of IL-6 in the apoE-KO mouse aortic lesions, we performed double immunofluorescence staining of tissue sections taken from the supravalvular region of the aorta. Faint but discrete staining was observed in cells that also stained for the monocyte/macrophage marker CD68. This is similar to the staining pattern observed in human atherosclerotic tissue.⁵ Except for the Watanabe heritable hyperlipidemic rabbit model,³¹ no other animal model has been characterized for the presence of IL-6 in atherosclerotic lesions. In the Watanabe heritable hyperlipidemic rabbit model, IL-6 transcripts were detected by in situ hybridization,³² which did not allow the identification of the cell types expressing IL-6 or the pattern of expression during lesion progression. Our data indicate that the apoE-KO mouse is a suitable animal model for evaluating the role of IL-6 in atherosclerotic lesion development.

Several lines of evidence have demonstrated that estrogen is an important determinant of cardiovascular risk in women. In epidemiological studies, estrogen replacement therapy is associated with reduced risk of cardiovascular morbidity and mortality in postmenopausal women.¹⁰ Multiple regression analysis of several large-scale studies indicate that only 25% to 50% of the observed risk reduction due to estrogen replacement therapy can be associated with beneficial changes in the lipoprotein profile.³³ The results from these studies suggest that other mechanisms or factors are also involved, including the direct effects of estrogen on various cellular components present in vascular lesions. Similar to the observation in humans, estrogen has been shown to reduce atherosclerotic lesion development in the apoE-KO mouse model of atherosclerosis,^{14 15} and this effect can only partially be explained through the effects on plasma lipoprotein levels.¹⁴ It is possible that similar to the antiosteoporotic action of estrogen,¹³ the suppression of IL-6 secretion from cells within the vessel wall (eg, macrophages) is a component of estrogen's antiatherosclerotic mechanism of action. In support of this hypothesis, estrogen treatment has been shown to suppress IL-6 secretion in vitro¹¹ and in peritoneal macrophages isolated from LPS-stimulated mice.³⁴ Indeed, our results demonstrate that 3 weeks of treatment with E2 results in the significant reduction of IL-6 secretion from isolated aortas of male apoE-KO mice. This observation can be explained as the result of the reduction of the plaque itself, a decrease in the number of macrophages within the plaque, or less secretion of IL-6 by the same number of macrophages by E2. Although our studies do not address these specific mechanisms, several reports have detected the classic estrogen receptor in macrophage cells and cell lines.^{35 36} In addition, estrogen inhibits the expression of JE/monocyte chemotactic protein 1 mRNA in LPS-stimulated murine macrophages that also express cytosolic estrogen receptors.³⁷ Recently, it has been observed that IL-6 can induce monocyte chemotactic protein-1 in peripheral blood mononuclear cells and in the U937 cell line.³⁸ Taken together, these observations suggest the possibility that the suppression of IL-6 secretion by macrophages within the vessel wall by E2 may be protective by inhibiting the autocrine stimulation of potent chemokines involved in monocyte recruitment into the subendothelial space, resulting in less foam cell formation.

Alternatively, the suppression of IL-6 secretion can be due to the decrease in testosterone observed in the estrogen-treated animals. However, this possibility may be unlikely, since several lines of evidence suggest that testosterone can suppresses IL-6 gene expression and secretion in vivo and in vitro. Analysis of trabecular bone loss in orchidectomized wild-type and IL-6–KO mice has demonstrated that testosterone depletion does not promote the bone loss observed in normal littermate controls, suggesting that IL-6 mediates the bone loss caused by androgen deficiency.³⁹ This is similar to that observed with estrogen depletion in IL-6–KO mice.¹³ These studies suggest that estrogen and testosterone exert positive effects on bone through the suppression of IL-6. Testosterone has also been shown to suppresses endogenous IL-6 gene expression and IL-6 promoter constructs in prostate tumor cell lines.⁴⁰

In summary, the expression of IL-6 mRNA and protein in the apoE-KO mouse aorta reflects the pathophysiological expression of this cytokine in human atherosclerotic lesions. Therefore, the apoE-KO mouse may serve as an appropriate animal model to study the role of IL-6 in atherosclerotic lesion development and progression. Furthermore, this animal model provides an in vivo system to determine whether the suppression of IL-6 directly contributes to the vasculoprotective mechanism of this ovarian steroid hormone.

	Acknowledgments

 
The authors would like to thank Neil Miyamoto (Berlex Biosciences) for helpful comments on the manuscript. We would also like to thank Jeff Davis and Tim Kenrick (Berlex Biosciences) for their expert help with animal husbandry.

Received November 5, 1997; accepted April 2, 1998.

	References

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