Influence of FK506 on experimental atherosclerosis in cholesterol-fed rabbits

Atherosclerosis 139 (1998) 95 – 106

Influence of FK506 on experimental atherosclerosis in cholesterol-fed rabbits Taro Matsumoto a,*, Eiji Saito a, Hideyuki Watanabe a, Takayuki Fujioka a, Tsutomu Yamada b, Yumiko Takahashi a, Takahiro Ueno a, Toshihiko Tochihara c, Katsuo Kanmatsuse a a

Second Department of Internal Medicine, Nihon Uni6ersity School of Medicine, 30 -1 Ohyaguchi Kami-machi, Itabashi-ku, Tokyo 173, Japan b Second Department of Pathology, Nihon Uni6ersity School of Medicine, 30 -1 Ohyaguchi Kami-machi, Itabashi-ku, Tokyo 173, Japan c Sagamihara Kyodo Hospital, 2 -8 -18 Hashimoto, Sagamihara City, Kanagawa 229, Japan Received 4 August 1997; received in revised form 16 February 1998; accepted 27 February 1998

Abstract To investigate the role of activated T lymphocytes in the formation of atherosclerotic lesions, we studied the influence of FK506, an immunosuppressant, on the development of atherosclerosis in cholesterol-fed rabbits. New Zealand White rabbits fed on a 1.5% cholesterol diet were administered FK506 at 0.05 mg/kg (n= 12), 0.1 mg/kg (n =12) or isotonic saline (as the control, n= 12) intramuscularly three times a week for 12 weeks. Although FK506 treatment did not affect plasma lipid levels, it caused an increase in the development of atherosclerotic lesions in a dose-dependent manner. Immunohistochemical analysis of the aorta after 8 weeks on the diet revealed that the ratio of T lymphocytes to the total number of cells in the plaques decreased significantly in the FK506 treated rabbits compared to the control rabbits. In culture, FK506 did not affect smooth muscle cell proliferation and cholesteryl ester formation in the macrophages. In contrast, culture medium from lymphocytes stimulated by concanavalin A decreased the accumulation of cholesteryl ester in the macrophages. This effect was inhibited by the culture medium in the presence of FK506. These findings suggest that activated T lymphocytes may inhibit intracellular cholesterol accumulation in atherosclerotic plaque. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: FK506; Atherosclerosis; T lymphocyte; Lymphokines

1. Introduction T lymphocytes as well as monocyte-derived macrophages are observed in atherosclerotic lesions. About one-third of the T lymphocytes in human atherosclerotic plaque are found to express the activation markers, HLA-DR and very late activation antigen-1 (VLA-1) [1]. In addition, a high proportion of smooth muscle cells (SMCs) and macrophages in the plaque express class II MHC, one of the cell surface proteins that is induced from activated T lymphocytes * Corresponding author. Tel: +81 3 39728111 (ext. 2413); fax: +81 3 39721098.

[2]. These observations suggest that lymphokines are released by activated T lymphocytes in atherosclerotic plaque and some of them influence plaque formation. Concerning the effect of activated T cells on atherosclerosis development, several studies have indicated that T cell activation was suppressed by cyclosporine A (CsA) in animal models [3–7], but the data have been inconsistent and frequently conflicting. This disparity in results may reflect differences in the experimental atherosclerotic models, such as cholesterol feeding or de-endothelialization, the dose of administration or the species of animal. On the other hand, the possibility exists that the experimental findings using CsA do not reflect suppression of T cell activation in the models.

0021-9150/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0021-9150(98)00066-5

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For example, it has been reported that CsA increases the plasma total and LDL cholesterol at a therapeutic dose [8–10]. Apanay et al. [11] have demonstrated that CsA increases the oxidizability of LDL in renal transplant recipients. These observations suggest that CsA can affect the development of atherosclerosis as a result of alteration of the lipid levels or lipoprotein modification. FK506 is a new immunosuppressant that is currently under clinical investigation for baseline and rescue immunosuppression in patients following organ transplantation. Although the chemical structure of FK506 is entirely different from that of CsA [12], FK506 inhibits the production of T cell-derived soluble mediators such as IL-2, IL-3, and interferon-g (IFN-g) as does CsA. It has been reported that FK506 exerts its suppression at about a 100 times lower concentration than CsA on the production of lymphokines and the expression of IL-2 receptor [13]. In addition, FK506 therapy tends to induce less hyperlipidemia when compared to CsAbased immunosuppression [14,15]. We inferred therefore that FK506 might be more useful in elucidating the effect of activated T lymphocytes on atherosclerotic plaque formation compared to CsA. In this study, the extent of the atherosclerotic lesions and the cellular composition of the intimal thickening were determined in cholesterol-fed rabbits undergoing FK506 treatment. We show that FK506 treatment can increase the development of atherosclerotic lesions independent of the plasma lipid levels. This finding suggests that activated T lymphocytes may contribute to inhibition of the development of atherosclerosis.

2. Materials and methods

2.1. Materials FK506 was obtained from Fujisawa (Osaka, Japan). Dulbecco’s modified Eagle’s medium (DMEM) and RPMI 1640 medium were purchased from Gibco (Grand Island, NY). [Methyl-3H]thymidine was purchased from Amersham (UK). [1-14C]Oleic acid and cholesteryl-1,2,6,7-[3H](N)-oleate were purchased from New England Nuclear Company (Boston, MA). Monoclonal anti-rabbit macrophage antibody (RAM-11) was purchased from Dako (Copenhagen, Denmark), and monoclonal anti-rabbit T cell antibody (CL8801) from Cedarlane Laboratories (Ontario, Canada). Concanavalin A (Con A) was purchased from EY Laboratories (San Metro, CA).

2.3. Animal model for macroscopic and microscopic studies Male New Zealand White rabbits (n=36) weighing about 2.5 kg each were purchased from the Tokyo Animal Laboratory (Tokyo, Japan). They were fed on rabbit chow supplemented with 1.5% cholesterol during the period of the experiment. Two weeks after the feeding, they were divided into three groups, adjusting the plasma cholesterol levels and body weights to be essentially similar. They were then given 0.1 mg/kg body weight (FK-high group, n= 12) or 0.05 mg/kg body weight (FK-low group, n= 12) of FK506, or isotonic saline (control group, n= 12) intramuscularly three times a week for 12 weeks, respectively. Body weight was measured every week.

2.4. Measurement of plasma lipid and blood FK506 concentrations Fasting venous blood samples were collected in heparin from the marginal ear veins every 4 weeks. The plasma was separated, and the total cholesterol (TC), triglyceride (TG) and phospholipid (PL) levels were measured by enzymatic methods. Blood samples from three rabbits each in the three groups were withdrawn 24 h after the injection, and at 2 and 8 weeks after the beginning of treatment. The whole blood and plasma concentrations of FK506 were measured by radioimmunoassay.

2.5. E6aluation of atherosclerotic lesions At the end of the 12-week experimental period, the rabbits were sacrificed by giving an overdose of intravenous pentobarbital. The thoracic aortas were removed, and opened longitudinally along the posterior side. The materials were fixed in 10% buffered formalin, and then stained with Sudan IV (Nakarai, Kyoto, Japan) for visualization of the atherosclerotic lesions. After staining, the aortas were pinned open to flatten them and photographed. The aortic surface affected by plaque was assessed quantitatively by computerized planimetry (Olympus-Avio SP-500 color image analyzer), and expressed as a percentage of the total aorta covered by the plaque involvement. Aortic arterial specimens for cross sectioning were taken from the aortas just proximal to the carotid arteries. Each specimen was embedded in paraffin and cut into 6-mm sections on a microtome. The cross sections from each specimen were then stained with hematoxylin and eosin, and examined by light microscopy.

2.2. Preparation of rabbit b-VLDL 2.6. Animal model for immunohistochemical analysis b-VLDL (dB1.006) was isolated from the plasma of 1% cholesterol-fed rabbits by ultracentrifugation [16].

Eight male New Zealand White rabbits weighing

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about 2.5 kg were fed on rabbit chow supplemented with 1.5% cholesterol for 8 weeks. Two weeks after the feeding, they were divided into two groups: an FK group (n=4) and a control group (n = 4), with matched plasma cholesterol levels and body weights. The FK group was given 0.1 mg/kg body weight of FK506 and the control group received isotonic saline intramuscularly three times a week for 6 weeks. At the end of the experiment, the rabbits were perfused with 2% paraformaldehyde (Wako, Osaka, Japan) in isotonic PBS under pentobarbital anesthesia. The thoracic aortas were removed, and tissue samples were obtained from the aortas just proximal to the carotid arteries. They were rinsed and fixed with 4% paraformaldehyde and placed in OCT embedding compound (Miles, Elkhart, IN). The tissue samples were snap-frozen in liquid nitrogen, and cut into 3-mm sections on a cryostat.

blood was withdrawn from the rabbits 1 h after the last injection. Peripheral blood lymphocytes (PBL) were isolated by gradient centrifugation over FicollPaque (Pharmacia LKB Biotechnology, Piscataway, NJ). The cells were washed three times with PBS, and then seeded at 2× 106 cells/ml into 96-well plates (Corning, NY) in 200 ml of RPMI 1640 containing 10% fetal calf serum (FCS). The cells were maintained in the culture medium in the presence of 5 mg/ml of Con A for 72 h at 37°C in 5% CO2 in air. Subsequently, 0.25 mCi of [3H]thymidine was added to each well, and the cells were incubated for an additional 16 h. The cells were harvested onto filter papers and the radioactivities were counted in a liquid scintillation counter. All results were calculated from triplicate dishes.

2.7. Immunohistochemistry

Rabbit aortic medial SMCs were isolated by the explant method. The cells (passages 4 to 7) were suspended at 6×104 cells/ml in DMEM containing 10% FCS, 100 mg/ml of streptomycin, and 100 U/ml of penicillin. The cells were dispensed as 200-ml portions into 96-well plates, and incubated for 24 h at 37°C in 5% CO2 in air. The medium was changed to DMEM containing 0.4% FCS, and the cells were incubated for 48 h to synchronize with the G0 stage. The cells were maintained in culture medium (DMEM+10% FCS) alone or in the presence of various concentrations of FK506 ranging from 1 pg/ml to 100 mg/ml for 24 h at 37°C. Then, 0.4 mCi of [3H]thymidine was added to each well, and the cells were incubated for an additional 24 h. After the cells had been washed with PBS, 100 ml of 0.02% EDTA and 100 ml of trypsin (Gibco) were added to each well. The suspended cells were harvested onto filter papers and the radioactivities were counted in a liquid scintillation counter. The results were calculated from triplicate dishes. The data are presented as percentage changes based on the response in the control wells cultured in the absence of FK506.

Sections were incubated with 3% hydrogen peroxide in PBS to block endogenous peroxidase activity. After repeated rinsing with PBS, the sections were preincubated with 20% normal goat serum for 30 min at room temperature. They were then incubated with either the monoclonal anti-rabbit macrophage antibody (RAM-11) or monoclonal anti-rabbit T lymphocyte antibody (CL8801) overnight at 4°C. The sections were next incubated with biotinylated anti-mouse IgG (DAKO) for 30 min and subsequently with peroxidase-conjugated streptavidin (DAKO) at room temperature. The peroxidase was visualized by incubation with 3% 3-amino-9-ethylcarbazole (AEC, Sigma, St. Louis, MO) in N,N-dimethylformamide (Sigma) and 0.01% hydrogen peroxide in 0.1 M acetate buffer (pH 5.2). The materials were then counterstained with hematoxylin and mounted. The cells existing in the atherosclerotic plaque and the T lymphocyte positive cells in the plaque or adhering to the surface were counted under high-power magnification by light microscopy. The total number of cells in the plaque was determined by counting all the hematoxylin positive nuclei. The percentage of T lymphocyte positive cells to total cells in the plaque and the percentage of T lymphocyte positive cells to total cells adhering to the surface of the intima were calculated and compared between the FK group and control group.

2.8. Lymphocyte proliferation assay Male New Zealand White rabbits were given FK506 at 0.1 mg/kg body weight (FK-high, n = 1) or 0.05 mg/kg body weight (FK-low, n = 1) or isotonic saline (control, n= 1) intramuscularly three times a week for 1 week, respectively. Fresh heparinized

2.9. SMC proliferation assay

2.10. Esterified cholesterol synthesis Rabbit PBL were isolated from heparinized blood by gradient centrifugation over Ficoll-Paque. Cells were washed three times with PBS, and incubated in a 60-mm diameter dish in RPMI 1640 containing 10% FCS for 2 h. The non-adherent mononuclear cells, at a final concentration of 2×106 cells/ml, were then cultured in RPMI 1640 medium containing 10% FCS with 20 mg/ml of Con A. Next, with or without addition of 100 ng/ml of FK506, they were incubated at 37°C for 72 h. After centrifugation, the media were collected as lymphocyte conditioned media (LCM).

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Fig. 1. Time course of body weights in FK506 treated and untreated control rabbits on a cholesterol diet. NZW rabbits fed on a 1.5% cholesterol diet were administered FK506 at 0.05 mg/kg (FK-low), 0.1 mg/kg (FK-high) or isotonic saline (control) intramuscularly three times a week for 12 weeks. Body weight was measured every week after the administration. Values at −2 and 0 weeks indicate the values before the cholesterol feeding and before the administration, respectively. Values are means 9S.D. (n = 12 per group).

Resident macrophages were isolated from DDY mice by peritoneal lavage with ice-cold PBS. Cells were isolated by centrifugation and prepared at 2× 106 cells/ ml in DMEM containing 10% FCS. The cells were dispensed as 2-ml portions into 35-mm diameter tissue culture wells and incubated at 37°C for 2 h. After washing three times with DMEM, adherent mouse peritoneal macrophages at a final concentration of 2 × 106 cells/well were cultured in 1 ml of DMEM, 50 mg/ml of b-VLDL and 0.2 mM [14C]oleate combined with bovine serum albumin (Sigma). LCM (50 mg/ml) with or without FK506 as described above was added into the macrophage culture. To provide controls, addition of 50 ml of the media of PBL (RPMI 1640+10% FCS) was made into the other culture. Next, 100 ng/ml of FK506 was added into the culture directly for examination of the direct effect of FK506 on macrophage esterified cholesterol synthesis. Each culture was incubated for 16 h at 37°C. After the incubation, the cellular cholesteryl[14C]oleate synthesized by the macrophage monolayers was measured as described by Brown et al. [17]. We added cholesteryl[3H]oleate into the lipid extraction mixture to correct for procedural losses. The concentration of cellular protein was measured with a Bio-Rad protein assay kit (Bio-Rad Laboratories, Richmond, CA). All data were expressed as nmol of [14C]oleate incorporated into cholesteryl[14C] oleate/mg cell protein, and represented the average of triplicate incubations.

2.11. Statistical analysis Statistical analysis was carried out using the Mann– Whitney U-test.

3. Results

3.1. Animal study 1 (Macroscopic and microscopic studies) Body weight gains were observed in both the control and FK506 treated groups during the experimental period and were not significantly different between the groups (Fig. 1). The plasma lipid levels are shown in Fig. 2. The TC, TG and PL levels increased after cholesterol loading in each group. No significant differences in levels of TC, TG and PL were noted between the three groups. At the end of the experiment, the average TC levels in the control, FK-low and FK-high groups were 17879 467, 15829426 and 19089443 mg/dl, respectively. The mean blood and plasma concentrations of FK506 in the FK-low and FK-high groups are listed in Table 1. These concentrations in the FK506 treated groups were within the immunosuppressive range. FK506 was not detected in the control group (data not shown). The results for the aortic surface involvement are summarized in Fig. 3A, and typical macroscopic atherosclerotic lesions in the thoracic aorta are illustrated in Fig. 3B. The extent of atherosclerosis was increased in both of the FK506 treated groups, and there appeared to be a dose-dependent response. The aortic surface affected by plaque in the FK-high group was significantly increased compared to the control group (46.59 17.3% vs. 29.39 12.4%, PB0.05). Although no significant differences were noted between the FK-low and control groups in terms of their aortic surface involvement, the lesions at the superior thoracic aorta and around the openings of branching vessels tended to be increased in the FK-low

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group macroscopically (Fig. 3B). Concerning the microscopic findings for the atherosclerotic lesions, the degree of intimal thickening and foam cell accumulation in the plaque were increased by FK506 treatment and the severity of atherosclerosis was associated with the FK506 concentration (Fig. 4).

Table 1 Whole blood and plasma concentrations of FK506 in FK506-treated groups

3.2. Animal study 2 (Immunohistochemical study)

FK-low group FK-high group

To evaluate the cellular components of the plaque, we employed the same animal model as above but the cholesterol loading was shortened to 8 weeks. Within this period, the atherosclerotic lesions were mild and the cell structure was well preserved, so that we could assess the number of T lymphocytes in the plaque easily by

2 weeks

8 weeks

Whole blood

Plasma

Whole blood

Plasma

4.53 9 1.67

0.15 90.01

3.13 90.17

0.32 9 0.14

5.50 91.73

0.16 90.05

5.30 9 0.40

0.36 90.12

Each group is n = 3; values given are ng/ml and are mean9 S.D.

immunostaining. In both the control and FK groups, foam cells occupied the thickening intima and most of them were positive for the macrophage antibody RAM11 (Fig. 5A, B). Small numbers of T lymphocyte positive mononuclear cells were found in the intima and adhering to the surface (Fig. 5C, D). The frequency of T lymphocyte positive cells in the plaque or adhering to the surface is shown in Fig. 6. We counted 300–1591 (mean: 834.9) cells expressing hematoxylin positive nuclei and 2–20 (means: 12 in the control group, 3.3 in the FK group) anti-T lymphocyte positive cells in the plaque per section. The FK506 treatment significantly (PB0.05) reduced the percentage of T lymphocyte positive cells to total cells in the plaque (Fig. 6B), but did not affect the cells adhering to the surface (Fig. 6C). The average total cell number in the plaque in the control and FK groups was 684.8 9 288.4 and 985.0 9 491.0, respectively (Fig. 6A). The total cell number in the plaque showed a tendency to increase in the FK group as compared to the control group, but the difference was not significant.

3.3. Effect of FK506 on lymphocyte proliferation ex 6i6o In order to confirm that T lymphocyte activity was suppressed by FK506 administration, we analyzed the PBL proliferation in FK506 treated rabbits and compared the results with control rabbits. As shown in Fig. 7, [3H]thymidine incorporation in the rabbit PBL was significantly (P B0.05) suppressed in both of the FK506 treated groups. The inhibitory rates for the FK-low and FK-high rabbits were 49.9 and 84.3%, respectively. Suppression of the PBL proliferation reaction was correlated with the development of atherosclerosis. Fig. 2. Time course of lipid levels in FK506 treated and untreated control rabbits on a cholesterol diet. NZW rabbits fed on a 1.5% cholesterol diet were administered FK506 at 0.05 mg/kg (FK-low), 0.1 mg/kg (FK-high) or isotonic saline (control) intramuscularly three times a week for 12 weeks. Blood samples were taken before the cholesterol loading ( −2 weeks), before the administration (0 weeks) and 4, 8 and 12 weeks after the administration. The plasma concentrations of total cholesterol (TC), triglyceride (TG), and phospholipid (PL) were then determined. Values are means 9 S.D. (n= 12 per group).

3.4. Effect of FK506 on SMC proliferation in 6itro FK506 at less than 10 mg/ml did not affect the [3H]thymidine incorporation in rabbit vascular SMCs (Fig. 8). At a concentration of 100 mg/ml, there was a significant (PB 0.05) reduction in the DNA synthesis of the SMCs. This concentration of FK506 was thought to be toxic because the viability of the SMCs decreased

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significantly as assessed by the trypan blue exclusion test (data not shown). The above findings indicated that FK506 had no direct effect on rabbit SMC proliferation in culture.

3.5. Cholesteryl ester formation in macrophages To elucidate whether or not FK506 directly enhanced foam cell formation, the cholesteryl ester formation by b-VLDL was examined in cultured mouse peritoneal macrophages. As shown in Fig. 9, the incorporation of

[14C]oleate into cholesteryl ester in the macrophages was significantly (P B0.05) reduced by the addition of Con A stimulated PBL conditioned medium [LCM(FK− )]. When the conditioned medium obtained from Con A stimulated PBL in the presence of FK506 [LCM(FK+)] was added, the ability of LCM to inhibit cholesteryl ester formation in the macrophages was reduced. In contrast, FK506 exerted no direct effect on the incorporation of [14C]oleate into cholesteryl ester.

4. Discussion

Fig. 3. Effect of FK506 on aortic atherosclerotic lesions in cholesterol-fed rabbits. NZW rabbits fed on a 1.5% cholesterol diet were administered FK506 at 0.05 mg/kg (FK-low), 0.1 mg/kg (FK-high) or isotonic saline (control) intramuscularly three times a week for 12 weeks. The thoracic aortas were removed and stained with Sudan IV. The aortic surface involvement was determined by computerized planimetry of the Sudan IV positive areas. (A) Percentage of aortic intimal area covered by atherosclerotic lesions in each group. Points ( ) indicate mean values (n= 12 per group). Lines within the boxes indicate the 50th percentile (median). The lower and upper ends of the boxes indicate the 25th and 75th percentiles, respectively. Horizontal lines below and above the boxes indicate the 10th and 90th percentiles, respectively; *P B0.05 vs. control. (B) Typical macroscopic atherosclerotic lesions in aortas of each group. The aortas shown revealed median values for the aortic surface involvement. The dark areas in the aorta indicate Sudan IV positive atherosclerotic lesions.

The present data show that FK506 can increase the accumulation of macrophage-derived foam cells in the aortic intima of cholesterol-fed rabbits independent of the plasma lipid levels. In previous experiments using a balloon catheter injury model, we found that FK506 did not affect the intimal thickening, but increased the appearance of foam cells in the intima [18]. FK506 may enhance intracellular cholesterol accumulation and foam cell formation, but not affect SMC proliferation in the atherogenic process. It has been reported that the serum trough level of an immunosuppressive dose of FK506 ranged from 0.1 to 0.4 ng/ml in beagle dogs [19]. The concentration of FK506 in the FK506 treated groups employed in the present study was therefore considered sufficient to reach the range for suppression of T cell-mediated immunity and prolongation of organ allograft survival. Furthermore, as shown in Fig. 7, the administration of low (0.05 mg/kg) and high (0.1 mg/ kg) doses of FK506 could significantly inhibit the proliferative response of rabbit lymphocytes to mitogen stimulation. A loss of body weight has been reported as an early toxic effect of the administration of an overdose of FK506 [20]. However, all of our animals treated with FK506 gained in body weight as well as the controls during the course of treatment. We also found that FK506 had no direct effect on rabbit vascular SMC proliferation and cholesterol ester formation in mouse peritoneal macrophages in vitro. We infer therefore that the accelerating effect of FK506 on the development of atherosclerotic lesions was due, at least in part, to an inhibition of T lymphocyte activation. Several investigators have evaluated the role of T lymphocytes or cell-mediated immunity in the development of atherosclerosis using various animal models in vivo. To suppress the cell-mediated immunity, athymic animal models or immunosuppressant agent treated animal models have been developed, but have yielded conflicting results. A few studies have examined the effect of CsA, a potent suppressor of T cell activation, on experimental atherosclerosis. Jonasson et al. [3] demonstrated that CsA suppressed the intimal proliferative lesions in a balloon catheter injury rat model. In

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Fig. 4. Light micrographs of aortic atherosclerotic lesions in FK506 treated and untreated control rabbits on a cholesterol diet. NZW rabbits fed on a 1.5% cholesterol diet were administered FK506 at 0.05 mg/kg (B), 0.1 mg/kg (C) or isotonic saline (A) intramuscularly three times a week for 12 weeks. Sections were obtained from the aortas just proximal to the carotid arteries, and were stained with hematoxylin and eosin (original magnification, ×80).

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Fig. 5. Immunohistochemical detection of macrophages and T lymphocytes in atherosclerotic lesions of FK506 treated and untreated control rabbits on a cholesterol diet. NZW rabbits fed on a 1.5% cholesterol diet were administered with FK506 at 0.1 mg/kg (A and C) or isotonic saline (B and D) intramuscularly three times a week for 8 weeks. Sections were obtained from the aortas just proximal to the carotid arteries. Sections A and B were immunostained with monoclonal antibody RAM-11 specific for macrophages (original magnification, ×80). Sections C and D were immunostained with monoclonal antibody CL8801 specific for T lymphocytes (original magnification, ×320). Arrows indicate CL8801 positive T lymphocytes.

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Fig. 6. Effect of FK506 on the density of T lymphocytes in atherosclerotic lesions of cholesterol-fed rabbits. The experimental design was the same as described in Fig. 5. (A) Total cell number in the plaque, which was determined by counting all hematoxylin positive nuclei. (B) Percentage of T lymphocyte positive cells to total cells in the plaque. (C) Percentage of T lymphocyte positive cells to total cells adhering to the surface of the intima. Bars represent means 9S.D. (n= 3 per group); *P B0.05 vs. control.

contrast, Ferns et al. [4] found that CsA was associated with the development of intimal macrophage-derived foam cells after arterial de-endothelialization in rabbits. In studies on cholesterol-fed animals, Roselaar et al. [7] showed that CsA treatment led to the development of atherosclerotic lesions in rabbits, and similar results were obtained by Emeson and Shen [5] in C57BL/6 mice. On the other hand, Drew and Tipping [6] reported that CsA reduced the extent of early atherosclerotic lesions in rabbits. CsA can influence the lipid metabolism [8–10], blood pressure [21], proliferation of vascular SMC [22] and endothelial function [23] not through suppression of T cell activation. CsA could thus affect atherogenesis outside of the suppression of T cell activation and so give rise to the discrepancies of

Fig. 7. Effect of FK506 on the proliferation of T lymphocytes ex vivo. NZW rabbits were administered with FK506 at 0.05 mg/kg (FK-low), 0.1 mg/kg (FK-high) or isotonic saline (control) intramuscularly three times a week for 1 week. Fresh heparinized blood from the rabbits was withdrawn and peripheral blood lymphocytes (PBL) were isolated by gradient centrifugation. The cells were seeded and maintained in RPMI 1640 containing 10% FCS in the presence of 5 mg/ml of Con A for 72 h at 37°C in 5% CO2 in air. Then 0.25 mCi of [3H]thymidine was added and the cells were incubated for an additional 16 h. The cells were harvested onto filter papers and the radioactivities were counted in a liquid scintillation counter. Bars represent means 9 S.D.; *PB0.05 vs. control.

results observed under various experimental conditions. It has been found that FK506 therapy induced less hyperlipidemia [14,15] or oxidizability of LDL [11] compared to CsA-based immunosuppression in organ transplant recipients. The incidence of hypertension has been reported to be less frequent in FK506 treated patients than in those treated with CsA [24,25]. Recently, CsA has been suggested to inhibit the induction of nitric oxide synthase in vascular SMC, while FK506 has a weaker or no effect on the induction of nitrite/nitrate production [26,27]. Dogan et al. [28] demonstrated that CsA enhances the induction of E-selectin by endothelial cells in a fetal intestinal organ culture system, but E-selectin expression was not enhanced by FK506. This effect may have a potential to elicit adhesion of monocytes to vascular endothelial cells and infiltration into plaque. We consider therefore that FK506 treatment of animals is a more appropriate model for examining the role of T lymphocytes in the atherogenic process. Cellular immunity deficient animals, such as athymic rats [29], thymectomized guinea pigs [30] and class I MHC deficient mice [31], have demonstrated an increase in the development of atherosclerosis. These findings are consistent with our results. In the present study, we showed that conditioned media from lymphocytes activated by Con A significantly reduced the accumulation of esterified cholesterol in mouse peritoneal macrophages stimulated by incubation with b-VLDL (Fig. 9). This inhibitory effect was reduced by inactivated conditioned media incubated with FK506. Such findings are in agreement with the data of Fogelman et al. [32]. This suggests that lymphokines from activated T lymphocytes may be useful in protecting macrophages from foam cell formation in atherosclerotic plaque. It has been established that FK506 inhibits the production of lymphokines such as IL-2, IL-3, and IFN-g

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Fig. 8. Effect of FK506 on proliferation of smooth muscle cells (SMCs) in vitro. After 48 h of starvation, rabbit aortic SMCs were incubated in DMEM with 10% FCS, and with or without FK506 ranging from 1 pg/ml to 100 mg/ml for 48 h at 37°C. [3H]Thymidine (0.4 mCi) was added to all wells 24 h before the end of the incubation. The cells were then washed, trypsinized, and harvested for scintillation counting. The results were calculated from triplicate dishes. The data are presented as percentage changes based on the response in the control wells cultured in the absence of FK506. The values represent means 9 S.D.; *PB 0.05 vs. control wells.

induced by antigens and lectins. Among these lymphokines, IFN-g can inhibit vascular SMC proliferation [33,34]. Recent reports [35] have demonstrated that IFN-g inhibits the expression of scavenger receptor and the foam cell formation of macrophages. On the other hand, evidence of local IFN-g secretion in human atherosclerotic plaque has been obtained by Hansson et al. [1] employing immunohistochemistry. The induction

Fig. 9. Effect of lymphocyte conditioned medium (LCM) and FK506 on cholesteryl ester formation by b-VLDL in mouse peritoneal macrophages. Mouse peritoneal macrophages were incubated with DMEM medium containing 0.2 mM [14C]oleate in the absence (control) or presence of 100 ng/ml of FK506 (FK506) or 50 mg/ml of LCM for 16 h at 37°C. Two kinds of LCM were prepared as follows. LCM (FK−): Rabbit PBL were stimulated with 20 mg/ml of Con A for 72 h, and the media were then collected after centrifugation. LCM (FK+): Rabbit PBL were incubated with 20 mg/ml of Con A in the presence of 100 ng/ml of FK506 for 72 h, and the media were then collected after centrifugation. Following the incubation, the incorporation of [14C]oleate into cholesteryl[14C]oleate was measured as described by Brown et al. [17]. Bars represent means 9 S.D.; *PB 0.05 vs. control.

of HLA-DR antigen expression on SMC in plaque [2] also supports the evidence indirectly, since HLA-DR is induced by IFN-g. Based on these findings, the accelerative effect on plaque formation exhibited by FK506 would appear to result from an inhibition of IFN-g secretion from T lymphocytes, but the precise mechanism of the in vivo effect of IFN-g remains to be elucidated. In the case of exogenous administration of IFN-g, inhibitory effects have been observed in a rat balloon angioplasty model [36] and in a cholesterol-fed rabbit model [37]. In the present experiments, we found that the T cell number per lesion area was significantly smaller in FK506 treated rabbits compared to the control rabbits (Fig. 6). The present findings are in agreement with those of a previous study [6] employing CsA. In contrast, Roselaar et al. [7] have shown that CsA administration did not affect the number of T cells in the lesions. To resolve this discrepancy, further experiments are needed on changes in the characterization of T cells, including cell surface markers, in plaque with administration of immunosuppressants. In summary, suppression of T cell activation by administration of FK506 increased the severity of macrophage-rich atherosclerotic lesions in cholesterolfed rabbits. Furthermore, we demonstrated that lymphocyte-conditioned medium stimulated by Con A protected mouse peritoneal macrophages from cholesteryl ester accumulation in vitro. These findings suggest that activated T lymphocytes in plaque may inhibit intracellular cholesterol accumulation and foam cell formation in atherosclerotic plaque through a specific lymphokine-mediated mechanism.

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Acknowledgements We thank assistance.

Akiko

Tsunemi

for

her

technical

[18]

[19]

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