influence of fk506 on experimental atherosclerosis in cholesterol-fed rabbits

Atherosclerosis 139 (1998) 95–106

Influence of FK506 on experimental atherosclerosis in cholesterol-fedrabbits

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, Japanb 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 ofFK506, an immunosuppressant, on the development of atherosclerosis in cholesterol-fed rabbits. New Zealand White rabbits fedon 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 causedan increase in the development of atherosclerotic lesions in a dose-dependent manner. Immunohistochemical analysis of the aortaafter 8 weeks on the diet revealed that the ratio of T lymphocytes to the total number of cells in the plaques decreased significantlyin the FK506 treated rabbits compared to the control rabbits. In culture, FK506 did not affect smooth muscle cell proliferationand cholesteryl ester formation in the macrophages. In contrast, culture medium from lymphocytes stimulated by concanavalinA decreased the accumulation of cholesteryl ester in the macrophages. This effect was inhibited by the culture medium in thepresence of FK506. These findings suggest that activated T lymphocytes may inhibit intracellular cholesterol accumulation inatherosclerotic plaque. © 1998 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: FK506; Atherosclerosis; T lymphocyte; Lymphokines

1. Introduction

T lymphocytes as well as monocyte-derivedmacrophages are observed in atherosclerotic lesions.About one-third of the T lymphocytes in humanatherosclerotic plaque are found to express the activa-tion markers, HLA-DR and very late activation anti-gen-1 (VLA-1) [1]. In addition, a high proportion ofsmooth muscle cells (SMCs) and macrophages in theplaque express class II MHC, one of the cell surfaceproteins that is induced from activated T lymphocytes

[2]. These observations suggest that lymphokines arereleased by activated T lymphocytes in atheroscleroticplaque and some of them influence plaque formation.Concerning the effect of activated T cells on atheroscle-rosis development, several studies have indicated that Tcell activation was suppressed by cyclosporine A (CsA)in animal models [3–7], but the data have been incon-sistent and frequently conflicting. This disparity in re-sults may reflect differences in the experimentalatherosclerotic models, such as cholesterol feeding orde-endothelialization, the dose of administration or thespecies of animal. On the other hand, the possibilityexists that the experimental findings using CsA do notreflect suppression of T cell activation in the models.

* Corresponding author. Tel: +81 3 39728111 (ext. 2413); fax:+81 3 39721098.

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

T. Matsumoto et al. / Atherosclerosis 139 (1998) 95–10696

For example, it has been reported that CsA increasesthe plasma total and LDL cholesterol at a therapeuticdose [8–10]. Apanay et al. [11] have demonstrated thatCsA increases the oxidizability of LDL in renal trans-plant recipients. These observations suggest that CsAcan affect the development of atherosclerosis as a resultof alteration of the lipid levels or lipoproteinmodification.

FK506 is a new immunosuppressant that is currentlyunder clinical investigation for baseline and rescue im-munosuppression in patients following organ transplan-tation. Although the chemical structure of FK506 isentirely different from that of CsA [12], FK506 inhibitsthe production of T cell-derived soluble mediators suchas IL-2, IL-3, and interferon-g (IFN-g) as does CsA. Ithas been reported that FK506 exerts its suppression atabout a 100 times lower concentration than CsA on theproduction of lymphokines and the expression of IL-2receptor [13]. In addition, FK506 therapy tends toinduce less hyperlipidemia when compared to CsA-based immunosuppression [14,15]. We inferred there-fore that FK506 might be more useful in elucidating theeffect of activated T lymphocytes on atheroscleroticplaque formation compared to CsA.

In this study, the extent of the atherosclerotic lesionsand the cellular composition of the intimal thickeningwere determined in cholesterol-fed rabbits undergoingFK506 treatment. We show that FK506 treatment canincrease the development of atherosclerotic lesions in-dependent of the plasma lipid levels. This finding sug-gests that activated T lymphocytes may contribute toinhibition 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) andRPMI 1640 medium were purchased from Gibco(Grand Island, NY). [Methyl-3H]thymidine was pur-chased from Amersham (UK). [1-14C]Oleic acid andcholesteryl-1,2,6,7-[3H](N)-oleate were purchased fromNew England Nuclear Company (Boston, MA). Mono-clonal anti-rabbit macrophage antibody (RAM-11) waspurchased from Dako (Copenhagen, Denmark), andmonoclonal anti-rabbit T cell antibody (CL8801) fromCedarlane Laboratories (Ontario, Canada). Con-canavalin A (Con A) was purchased from EY Labora-tories (San Metro, CA).

2.2. Preparation of rabbit b-VLDL

b-VLDL (dB1.006) was isolated from the plasma of1% cholesterol-fed rabbits by ultracentrifugation [16].

2.3. Animal model for macroscopic and microscopicstudies

Male New Zealand White rabbits (n=36) weighingabout 2.5 kg each were purchased from the TokyoAnimal Laboratory (Tokyo, Japan). They were fed onrabbit chow supplemented with 1.5% cholesterol duringthe period of the experiment. Two weeks after thefeeding, they were divided into three groups, adjustingthe plasma cholesterol levels and body weights to beessentially similar. They were then given 0.1 mg/kgbody weight (FK-high group, n=12) or 0.05 mg/kgbody weight (FK-low group, n=12) of FK506, orisotonic saline (control group, n=12) intramuscularlythree times a week for 12 weeks, respectively. Bodyweight was measured every week.

2.4. Measurement of plasma lipid and blood FK506concentrations

Fasting venous blood samples were collected in hep-arin from the marginal ear veins every 4 weeks. Theplasma was separated, and the total cholesterol (TC),triglyceride (TG) and phospholipid (PL) levels weremeasured by enzymatic methods. Blood samples fromthree rabbits each in the three groups were withdrawn24 h after the injection, and at 2 and 8 weeks after thebeginning of treatment. The whole blood and plasmaconcentrations of FK506 were measured byradioimmunoassay.

2.5. E6aluation of atherosclerotic lesions

At the end of the 12-week experimental period, therabbits were sacrificed by giving an overdose of intra-venous pentobarbital. The thoracic aortas were re-moved, and opened longitudinally along the posteriorside. 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 flattenthem and photographed. The aortic surface affected byplaque was assessed quantitatively by computerizedplanimetry (Olympus-Avio SP-500 color image ana-lyzer), and expressed as a percentage of the total aortacovered by the plaque involvement. Aortic arterial spec-imens for cross sectioning were taken from the aortasjust proximal to the carotid arteries. Each specimen wasembedded in paraffin and cut into 6-mm sections on amicrotome. The cross sections from each specimen werethen stained with hematoxylin and eosin, and examinedby light microscopy.

2.6. Animal model for immunohistochemical analysis

Eight male New Zealand White rabbits weighing

T. Matsumoto et al. / Atherosclerosis 139 (1998) 95–106 97

about 2.5 kg were fed on rabbit chow supplementedwith 1.5% cholesterol for 8 weeks. Two weeks after thefeeding, they were divided into two groups: an FKgroup (n=4) and a control group (n=4), withmatched plasma cholesterol levels and body weights.The FK group was given 0.1 mg/kg body weight ofFK506 and the control group received isotonic salineintramuscularly three times a week for 6 weeks. At theend of the experiment, the rabbits were perfused with2% paraformaldehyde (Wako, Osaka, Japan) in iso-tonic PBS under pentobarbital anesthesia. The thoracicaortas were removed, and tissue samples were obtainedfrom the aortas just proximal to the carotid arteries.They were rinsed and fixed with 4% paraformaldehydeand placed in OCT embedding compound (Miles,Elkhart, IN). The tissue samples were snap-frozen inliquid nitrogen, and cut into 3-mm sections on acryostat.

2.7. Immunohistochemistry

Sections were incubated with 3% hydrogen peroxidein PBS to block endogenous peroxidase activity. Afterrepeated rinsing with PBS, the sections were preincu-bated with 20% normal goat serum for 30 min atroom temperature. They were then incubated with ei-ther the monoclonal anti-rabbit macrophage antibody(RAM-11) or monoclonal anti-rabbit T lymphocyteantibody (CL8801) overnight at 4°C. The sectionswere next incubated with biotinylated anti-mouse IgG(DAKO) for 30 min and subsequently with peroxi-dase-conjugated streptavidin (DAKO) at room tem-perature. The peroxidase was visualized by incubationwith 3% 3-amino-9-ethylcarbazole (AEC, Sigma, St.Louis, MO) in N,N-dimethylformamide (Sigma) and0.01% hydrogen peroxide in 0.1 M acetate buffer (pH5.2). The materials were then counterstained with he-matoxylin and mounted. The cells existing in theatherosclerotic plaque and the T lymphocyte positivecells in the plaque or adhering to the surface werecounted under high-power magnification by light mi-croscopy. The total number of cells in the plaque wasdetermined by counting all the hematoxylin positivenuclei. The percentage of T lymphocyte positive cellsto total cells in the plaque and the percentage of Tlymphocyte positive cells to total cells adhering to thesurface of the intima were calculated and comparedbetween the FK group and control group.

2.8. Lymphocyte proliferation assay

Male New Zealand White rabbits were givenFK506 at 0.1 mg/kg body weight (FK-high, n=1) or0.05 mg/kg body weight (FK-low, n=1) or isotonicsaline (control, n=1) intramuscularly three times aweek for 1 week, respectively. Fresh heparinized

blood was withdrawn from the rabbits 1 h after thelast injection. Peripheral blood lymphocytes (PBL)were isolated by gradient centrifugation over Ficoll-Paque (Pharmacia LKB Biotechnology, Piscataway,NJ). The cells were washed three times with PBS, andthen seeded at 2×106 cells/ml into 96-well plates(Corning, NY) in 200 m l of RPMI 1640 containing10% fetal calf serum (FCS). The cells were maintainedin the culture medium in the presence of 5 mg/ml ofCon A for 72 h at 37°C in 5% CO2 in air. Subse-quently, 0.25 mCi of [3H]thymidine was added to eachwell, and the cells were incubated for an additional 16h. The cells were harvested onto filter papers and theradioactivities were counted in a liquid scintillationcounter. All results were calculated from triplicatedishes.

2.9. SMC proliferation assay

Rabbit aortic medial SMCs were isolated by theexplant method. The cells (passages 4 to 7) were sus-pended at 6×104 cells/ml in DMEM containing 10%FCS, 100 mg/ml of streptomycin, and 100 U/ml ofpenicillin. The cells were dispensed as 200-m l portionsinto 96-well plates, and incubated for 24 h at 37°C in5% CO2 in air. The medium was changed to DMEMcontaining 0.4% FCS, and the cells were incubated for48 h to synchronize with the G0 stage. The cells weremaintained in culture medium (DMEM+10% FCS)alone or in the presence of various concentrations ofFK506 ranging from 1 pg/ml to 100 mg/ml for 24 hat 37°C. Then, 0.4 mCi of [3H]thymidine was added toeach well, and the cells were incubated for an addi-tional 24 h. After the cells had been washed withPBS, 100 m l of 0.02% EDTA and 100 m l of trypsin(Gibco) were added to each well. The suspended cellswere harvested onto filter papers and the radioactivi-ties were counted in a liquid scintillation counter. Theresults were calculated from triplicate dishes. The dataare presented as percentage changes based on the re-sponse in the control wells cultured in the absence ofFK506.

2.10. Esterified cholesterol synthesis

Rabbit PBL were isolated from heparinized bloodby gradient centrifugation over Ficoll-Paque. Cellswere washed three times with PBS, and incubated in a60-mm diameter dish in RPMI 1640 containing 10%FCS for 2 h. The non-adherent mononuclear cells, ata final concentration of 2×106 cells/ml, were thencultured in RPMI 1640 medium containing 10% FCSwith 20 mg/ml of Con A. Next, with or without addi-tion of 100 ng/ml of FK506, they were incubated at37°C for 72 h. After centrifugation, the media werecollected as lymphocyte conditioned media (LCM).


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% cholesteroldiet 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 for12 weeks. Body weight was measured every week after the administration. Values at −2 and 0 weeks indicate the values before the cholesterolfeeding and before the administration, respectively. Values are means9S.D. (n=12 per group).

Resident macrophages were isolated from DDY miceby peritoneal lavage with ice-cold PBS. Cells wereisolated by centrifugation and prepared at 2×106 cells/ml in DMEM containing 10% FCS. The cells weredispensed as 2-ml portions into 35-mm diameter tissueculture wells and incubated at 37°C for 2 h. Afterwashing three times with DMEM, adherent mouseperitoneal macrophages at a final concentration of 2×106 cells/well were cultured in 1 ml of DMEM, 50mg/ml of b-VLDL and 0.2 mM [14C]oleate combinedwith bovine serum albumin (Sigma). LCM (50 mg/ml)with or without FK506 as described above was addedinto the macrophage culture. To provide controls, addi-tion of 50 m l of the media of PBL (RPMI 1640+10%FCS) was made into the other culture. Next, 100 ng/mlof FK506 was added into the culture directly for exam-ination of the direct effect of FK506 on macrophageesterified cholesterol synthesis. Each culture was incu-bated for 16 h at 37°C. After the incubation, thecellular cholesteryl[14C]oleate synthesized by themacrophage monolayers was measured as described byBrown et al. [17]. We added cholesteryl[3H]oleate intothe lipid extraction mixture to correct for procedurallosses. The concentration of cellular protein was mea-sured with a Bio-Rad protein assay kit (Bio-Rad Labo-ratories, Richmond, CA). All data were expressed asnmol of [14C]oleate incorporated into cholesteryl[14C]oleate/mg cell protein, and represented the average oftriplicate 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 microscopicstudies)

Body weight gains were observed in both the controland FK506 treated groups during the experimentalperiod and were not significantly different between thegroups (Fig. 1). The plasma lipid levels are shown inFig. 2. The TC, TG and PL levels increased aftercholesterol loading in each group. No significant differ-ences in levels of TC, TG and PL were noted betweenthe three groups. At the end of the experiment, theaverage TC levels in the control, FK-low and FK-highgroups were 17879467, 15829426 and 19089443mg/dl, respectively. The mean blood and plasma con-centrations of FK506 in the FK-low and FK-highgroups are listed in Table 1. These concentrations in theFK506 treated groups were within the immunosuppres-sive range. FK506 was not detected in the controlgroup (data not shown). The results for the aorticsurface involvement are summarized in Fig. 3A, andtypical macroscopic atherosclerotic lesions in the tho-racic aorta are illustrated in Fig. 3B. The extent ofatherosclerosis was increased in both of the FK506treated groups, and there appeared to be a dose-depen-dent response. The aortic surface affected by plaque inthe FK-high group was significantly increased com-pared to the control group (46.5917.3% vs. 29.3912.4%, PB0.05). Although no significant differenceswere noted between the FK-low and control groups interms of their aortic surface involvement, the lesions atthe superior thoracic aorta and around the openings ofbranching vessels tended to be increased in the FK-low


group macroscopically (Fig. 3B). Concerning the micro-scopic findings for the atherosclerotic lesions, the degreeof intimal thickening and foam cell accumulation in theplaque were increased by FK506 treatment and theseverity of atherosclerosis was associated with the FK506concentration (Fig. 4).

3.2. Animal study 2 (Immunohistochemical study)

To evaluate the cellular components of the plaque, weemployed the same animal model as above but thecholesterol loading was shortened to 8 weeks. Within thisperiod, the atherosclerotic lesions were mild and the cellstructure was well preserved, so that we could assess thenumber of T lymphocytes in the plaque easily by

Table 1Whole blood and plasma concentrations of FK506 in FK506-treatedgroups

2 weeks 8 weeks

Plasma Whole PlasmaWholebloodblood

0.1590.01 3.1390.17FK-low 0.3290.144.5391.67group

0.1690.05 5.3090.40 0.3690.125.5091.73FK-highgroup

Each group is n=3; values given are ng/ml and are mean9S.D.

immunostaining. In both the control and FK groups,foam cells occupied the thickening intima and most ofthem were positive for the macrophage antibody RAM-11 (Fig. 5A, B). Small numbers of T lymphocyte positivemononuclear cells were found in the intima and adheringto the surface (Fig. 5C, D). The frequency of Tlymphocyte positive cells in the plaque or adhering to thesurface is shown in Fig. 6. We counted 300–1591 (mean:834.9) cells expressing hematoxylin positive nuclei and2–20 (means: 12 in the control group, 3.3 in the FKgroup) anti-T lymphocyte positive cells in the plaque persection. The FK506 treatment significantly (PB0.05)reduced the percentage of T lymphocyte positive cells tototal cells in the plaque (Fig. 6B), but did not affect thecells adhering to the surface (Fig. 6C). The average totalcell number in the plaque in the control and FK groupswas 684.89288.4 and 985.09491.0, respectively (Fig.6A). The total cell number in the plaque showed atendency to increase in the FK group as compared to thecontrol group, but the difference was not significant.

3.3. Effect of FK506 on lymphocyte proliferation ex6i6o

In order to confirm that T lymphocyte activity wassuppressed by FK506 administration, we analyzed thePBL proliferation in FK506 treated rabbits and com-pared the results with control rabbits. As shown in Fig.7, [3H]thymidine incorporation in the rabbit PBL wassignificantly (PB0.05) suppressed in both of the FK506treated groups. The inhibitory rates for the FK-low andFK-high rabbits were 49.9 and 84.3%, respectively.Suppression of the PBL proliferation reaction was corre-lated with the development of atherosclerosis.

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 asignificant (PB0.05) reduction in the DNA synthesis ofthe SMCs. This concentration of FK506 was thought tobe toxic because the viability of the SMCs decreased

Fig. 2. Time course of lipid levels in FK506 treated and untreatedcontrol 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 threetimes a week for 12 weeks. Blood samples were taken before thecholesterol loading (−2 weeks), before the administration (0 weeks)and 4, 8 and 12 weeks after the administration. The plasma concen-trations of total cholesterol (TC), triglyceride (TG), and phospholipid(PL) were then determined. Values are means9S.D. (n=12 pergroup).


significantly as assessed by the trypan blue exclusiontest (data not shown). The above findings indicated thatFK506 had no direct effect on rabbit SMC prolifera-tion in culture.

3.5. Cholesteryl ester formation in macrophages

To elucidate whether or not FK506 directly enhancedfoam cell formation, the cholesteryl ester formation byb-VLDL was examined in cultured mouse peritonealmacrophages. As shown in Fig. 9, the incorporation of

[14C]oleate into cholesteryl ester in the macrophageswas significantly (PB0.05) reduced by the addition ofCon A stimulated PBL conditioned medium[LCM(FK− )]. When the conditioned medium ob-tained from Con A stimulated PBL in the presence ofFK506 [LCM(FK+ )] was added, the ability of LCMto inhibit cholesteryl ester formation in themacrophages was reduced. In contrast, FK506 exertedno direct effect on the incorporation of [14C]oleate intocholesteryl ester.

4. Discussion

The present data show that FK506 can increase theaccumulation of macrophage-derived foam cells in theaortic intima of cholesterol-fed rabbits independent ofthe plasma lipid levels. In previous experiments using aballoon catheter injury model, we found that FK506did not affect the intimal thickening, but increased theappearance of foam cells in the intima [18]. FK506 mayenhance intracellular cholesterol accumulation andfoam cell formation, but not affect SMC proliferationin the atherogenic process. It has been reported that theserum trough level of an immunosuppressive dose ofFK506 ranged from 0.1 to 0.4 ng/ml in beagle dogs[19]. The concentration of FK506 in the FK506 treatedgroups employed in the present study was thereforeconsidered sufficient to reach the range for suppressionof T cell-mediated immunity and prolongation of organallograft survival. Furthermore, as shown in Fig. 7, theadministration of low (0.05 mg/kg) and high (0.1 mg/kg) doses of FK506 could significantly inhibit the pro-liferative response of rabbit lymphocytes to mitogenstimulation. A loss of body weight has been reported asan early toxic effect of the administration of an over-dose of FK506 [20]. However, all of our animals treatedwith FK506 gained in body weight as well as thecontrols during the course of treatment. We also foundthat FK506 had no direct effect on rabbit vascularSMC proliferation and cholesterol ester formation inmouse peritoneal macrophages in vitro. We infer there-fore that the accelerating effect of FK506 on the devel-opment of atherosclerotic lesions was due, at least inpart, to an inhibition of T lymphocyte activation.

Several investigators have evaluated the role of Tlymphocytes or cell-mediated immunity in the develop-ment of atherosclerosis using various animal models invivo. To suppress the cell-mediated immunity, athymicanimal models or immunosuppressant agent treatedanimal models have been developed, but have yieldedconflicting results. A few studies have examined theeffect of CsA, a potent suppressor of T cell activation,on experimental atherosclerosis. Jonasson et al. [3]demonstrated that CsA suppressed the intimal prolifer-ative lesions in a balloon catheter injury rat model. In

Fig. 3. Effect of FK506 on aortic atherosclerotic lesions in choles-terol-fed rabbits. NZW rabbits fed on a 1.5% cholesterol diet wereadministered FK506 at 0.05 mg/kg (FK-low), 0.1 mg/kg (FK-high) orisotonic saline (control) intramuscularly three times a week for 12weeks. The thoracic aortas were removed and stained with Sudan IV.The aortic surface involvement was determined by computerizedplanimetry of the Sudan IV positive areas. (A) Percentage of aorticintimal area covered by atherosclerotic lesions in each group. Points() indicate mean values (n=12 per group). Lines within the boxesindicate the 50th percentile (median). The lower and upper ends ofthe boxes indicate the 25th and 75th percentiles, respectively. Hori-zontal lines below and above the boxes indicate the 10th and 90thpercentiles, respectively; *PB0.05 vs. control. (B) Typical macro-scopic atherosclerotic lesions in aortas of each group. The aortasshown revealed median values for the aortic surface involvement. Thedark areas in the aorta indicate Sudan IV positive atheroscleroticlesions.


Fig. 4. Light micrographs of aortic atherosclerotic lesions in FK506 treated and untreated control rabbits on a cholesterol diet. NZW rabbits fedon 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 weekfor 12 weeks. Sections were obtained from the aortas just proximal to the carotid arteries, and were stained with hematoxylin and eosin (originalmagnification, ×80).


Fig. 5. Immunohistochemical detection of macrophages and T lymphocytes in atherosclerotic lesions of FK506 treated and untreated controlrabbits 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. SectionsA and B were immunostained with monoclonal antibody RAM-11 specific for macrophages (original magnification, ×80). Sections C and D wereimmunostained with monoclonal antibody CL8801 specific for T lymphocytes (original magnification, ×320). Arrows indicate CL8801 positiveT lymphocytes.


Fig. 6. Effect of FK506 on the density of T lymphocytes in atherosclerotic lesions of cholesterol-fed rabbits. The experimental design was the sameas described in Fig. 5. (A) Total cell number in the plaque, which was determined by counting all hematoxylin positive nuclei. (B) Percentage ofT lymphocyte positive cells to total cells in the plaque. (C) Percentage of T lymphocyte positive cells to total cells adhering to the surface of theintima. Bars represent means9S.D. (n=3 per group); *PB0.05 vs. control.

contrast, Ferns et al. [4] found that CsA was associatedwith the development of intimal macrophage-derivedfoam 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 ofatherosclerotic lesions in rabbits, and similar resultswere obtained by Emeson and Shen [5] in C57BL/6mice. On the other hand, Drew and Tipping [6] re-ported that CsA reduced the extent of early atheroscle-rotic lesions in rabbits. CsA can influence the lipidmetabolism [8–10], blood pressure [21], proliferation ofvascular SMC [22] and endothelial function [23] notthrough suppression of T cell activation. CsA couldthus affect atherogenesis outside of the suppression ofT cell activation and so give rise to the discrepancies of

results observed under various experimental conditions.It has been found that FK506 therapy induced lesshyperlipidemia [14,15] or oxidizability of LDL [11]compared to CsA-based immunosuppression in organtransplant recipients. The incidence of hypertension hasbeen reported to be less frequent in FK506 treatedpatients than in those treated with CsA [24,25]. Re-cently, CsA has been suggested to inhibit the inductionof nitric oxide synthase in vascular SMC, while FK506has a weaker or no effect on the induction of nitrite/ni-trate production [26,27]. Dogan et al. [28] demonstratedthat CsA enhances the induction of E-selectin by en-dothelial 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 ofmonocytes to vascular endothelial cells and infiltrationinto plaque. We consider therefore that FK506 treat-ment of animals is a more appropriate model for exam-ining the role of T lymphocytes in the atherogenicprocess. Cellular immunity deficient animals, such asathymic rats [29], thymectomized guinea pigs [30] andclass I MHC deficient mice [31], have demonstrated anincrease in the development of atherosclerosis. Thesefindings are consistent with our results.

In the present study, we showed that conditionedmedia from lymphocytes activated by Con A signifi-cantly reduced the accumulation of esterified choles-terol in mouse peritoneal macrophages stimulated byincubation with b-VLDL (Fig. 9). This inhibitory effectwas reduced by inactivated conditioned media incu-bated with FK506. Such findings are in agreement withthe data of Fogelman et al. [32]. This suggests thatlymphokines from activated T lymphocytes may beuseful in protecting macrophages from foam cell forma-tion in atherosclerotic plaque.

It has been established that FK506 inhibits the pro-duction of lymphokines such as IL-2, IL-3, and IFN-g

Fig. 7. Effect of FK506 on the proliferation of T lymphocytes exvivo. NZW rabbits were administered with FK506 at 0.05 mg/kg(FK-low), 0.1 mg/kg (FK-high) or isotonic saline (control) intramus-cularly three times a week for 1 week. Fresh heparinized blood fromthe rabbits was withdrawn and peripheral blood lymphocytes (PBL)were isolated by gradient centrifugation. The cells were seeded andmaintained in RPMI 1640 containing 10% FCS in the presence of 5mg/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 addi-tional 16 h. The cells were harvested onto filter papers and theradioactivities were counted in a liquid scintillation counter. Barsrepresent means9S.D.; *PB0.05 vs. control.


Fig. 8. Effect of FK506 on proliferation of smooth muscle cells (SMCs) in vitro. After 48 h of starvation, rabbit aortic SMCs were incubated inDMEM 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 addedto all wells 24 h before the end of the incubation. The cells were then washed, trypsinized, and harvested for scintillation counting. The resultswere calculated from triplicate dishes. The data are presented as percentage changes based on the response in the control wells cultured in theabsence of FK506. The values represent means9S.D.; *PB0.05 vs. control wells.

induced by antigens and lectins. Among theselymphokines, IFN-g can inhibit vascular SMC prolifer-ation [33,34]. Recent reports [35] have demonstratedthat IFN-g inhibits the expression of scavenger receptorand the foam cell formation of macrophages. On theother hand, evidence of local IFN-g secretion in humanatherosclerotic plaque has been obtained by Hansson etal. [1] employing immunohistochemistry. The induction

of HLA-DR antigen expression on SMC in plaque [2]also supports the evidence indirectly, since HLA-DR isinduced by IFN-g. Based on these findings, the acceler-ative effect on plaque formation exhibited by FK506would appear to result from an inhibition of IFN-gsecretion from T lymphocytes, but the precise mecha-nism of the in vivo effect of IFN-g remains to beelucidated. In the case of exogenous administration ofIFN-g, inhibitory effects have been observed in a ratballoon angioplasty model [36] and in a cholesterol-fedrabbit model [37].

In the present experiments, we found that the T cellnumber per lesion area was significantly smaller inFK506 treated rabbits compared to the control rabbits(Fig. 6). The present findings are in agreement withthose of a previous study [6] employing CsA. In con-trast, Roselaar et al. [7] have shown that CsA adminis-tration did not affect the number of T cells in thelesions. To resolve this discrepancy, further experimentsare needed on changes in the characterization of T cells,including cell surface markers, in plaque with adminis-tration of immunosuppressants.

In summary, suppression of T cell activation byadministration of FK506 increased the severity ofmacrophage-rich atherosclerotic lesions in cholesterol-fed rabbits. Furthermore, we demonstrated thatlymphocyte-conditioned medium stimulated by Con Aprotected mouse peritoneal macrophages fromcholesteryl ester accumulation in vitro. These findingssuggest that activated T lymphocytes in plaque mayinhibit intracellular cholesterol accumulation and foamcell formation in atherosclerotic plaque through a spe-cific lymphokine-mediated mechanism.

Fig. 9. Effect of lymphocyte conditioned medium (LCM) and FK506on cholesteryl ester formation by b-VLDL in mouse peritonealmacrophages. Mouse peritoneal macrophages were incubated withDMEM medium containing 0.2 mM [14C]oleate in the absence (con-trol) or presence of 100 ng/ml of FK506 (FK506) or 50 mg/ml ofLCM 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 Afor 72 h, and the media were then collected after centrifugation. LCM(FK+ ): Rabbit PBL were incubated with 20 mg/ml of Con A in thepresence of 100 ng/ml of FK506 for 72 h, and the media were thencollected after centrifugation. Following the incubation, the incorpo-ration of [14C]oleate into cholesteryl[14C]oleate was measured asdescribed by Brown et al. [17]. Bars represent means9S.D.; *PB0.05 vs. control.


Acknowledgements

We thank Akiko Tsunemi for her technicalassistance.

References

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influence of fk506 on experimental atherosclerosis in cholesterol-fed rabbits

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