THP-1 macrophage D/L-[14C]-Met uptake
茨城県立医療大学紀要
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Original Article

Volume 23

Basic Study on Atherosclerosis Imaging :The Effect of Foam-cell Formation on S-[methyl-14C]-D/L-methionine
Uptake by THP-1–derived Macrophages
Naoto Shikano1), Kentaro Kusanagi1), Masato Ogura1), Syuichi Nakajima1), Yuki Serizawa1), Yuko Hamai1),
Sanae Matsutani1), Masato Kobayashi2), Shohei Shinozaki3,4), Keiichi Kawai2), Kentaro Shimokado3)

1)

Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences
Division of Health Science, Graduate School of Health Sciences, Kanazawa University
3)
Department of Geriatrics and Vascular Medicine, Tokyo Medical and Dental University Graduate School of Medicine
4)
Department of Arteriosclerosis and Vascular Biology, Tokyo Medical and Dental University Graduate School of Medicine
2)

Abstract
Objective: Monocyte-derived macrophage accumulation and subsequent foam-cell formation are large contributors toatherosclerotic plaque formation. In the present study, we studied the uptake of the optical isomers of S-[methyl-14C]-D/
L-methionine (D/L-[14C]-Met) during differentiation of human-derived THP-1 monocytes to macrophage foam cells using
positron emission tomography (PET) with D/L-[11C]-Met as a qualitative diagnostic agent to assess plaque vulnerability. We also
investigated whether it is possible to augment D/L-[14C]-Met accumulation in THP-1–derived macrophages by stimulating an
uptake-associated amino acid exchange transporter.
Methods: THP-1 cells (1×106 cells/ml) were cultured in 35-mm-diameter dishes with 1 ml of Roswell Park Memorial Institutemedium containing fetal bovine serum and L-glutamine for 2 days. Phorbol 12-myristate 13-acetate (PMA) was added toeach dish at a concentration of 100 ng/ml and cells were cultured for 6, 12, 24, 48, and 72 hours to induce differentiation into
macrophages. Cells were cultured for 72 hours following PMA treatment to allow for differentiation and then treated withacetylated low-density lipoprotein (Ac-LDL, 50 μg/ml) and cultured for 6, 12, 24, 48, and 72 hours to induce macrophage foamcell formation. Cells were stained with oil Red O at different time points after PMA and Ac-LDL treatment, and the 10-min
radioactivity uptake of D/L-[14C]-Met (3.7 kBq) was measured. Cells were also treated with L-tyrosine ethyl ester (L-Tyr-OEt) tostimulate exchange transport, and the 60-min uptake of D/L-[14C]-Met (3.7 kBq) was compared with untreated cells.
Results: During PMA-induced macrophage differentiation, D/L-[14C]-Met uptake increased rapidly, reaching a maximum at 48
hours, with approximately 5.8% of the treatment dose in the L-form and approximately 2.2% in the D-form. Uptake of both forms
decreased thereafter. During foam-cell formation after Ac-LDL treatment, uptake decreased rapidly in the first 12 h and continued
decreasing up to 72 h. Uptake stimulation by L-Tyr-OEt resulted in an approximately 3-fold increase in D/L-[14C]-Met uptake
compared with the respective control treatments that did not contain L-Tyr-OEt (p<0.00005).
Conclusion: D/L-[14C]-Met was observed to accumulate in THP-1–derived macrophages, and this accumulation was enhanced by
L-Tyr-OEt in this study. In vivo application of this system in future PET basic studies using D/L-[11C]-Met could facilitate clearer
atherosclerosis imaging. Comparisons with other labeled compounds using animal models are anticipated.
Key words: atherosclerosis imaging, THP-1 cells, differentiation, macrophages, S-[methyl-14C]-D/L-methionine

Address: Naoto Shikano. Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences,
Ami4669-2, Ami, Inashiki, Ibaraki 300-0394, Japan
Phone: +81-29-840-2217, Fax: +81-29-840-2268, E-mail : sikano@ipu.ac.jp

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茨城県立医療大学紀要

1. Introduction

第23巻

with acute monocytic leukemia. One of the primarycharacteristics of this cell line is that the cells differentiate

In recent years, atherosclerosis has become
recognized as a chronic inflammatory disease in which

and mature into macrophages following stimulation withphorbol 12-myristate 13-acetate (PMA).8)

inflammation is known to be involved in both the onset

The transporter in system L, which transports D/L-

and development of the lesion, with eventual plaque[14C]-Met, is a Na+-independent exchanger that transports

rupture. Plaques that are prone to rupture are termed

neutral amino acids with large, neutral side chains,“vulnerable” and are pathologically characterized by aincluding tyrosine, by exchanging one extracellularlipid-rich core, a thin fibrous cap that covers the area, andamino acid with one intracellular amino acid. Basedinfiltration by inflammatory cells such as lymphocytes

on our previous study, 9) we hypothesized that the ethyl

and macrophages.1) Macrophage accumulation following

esters of neutral amino acids such as tyrosine passvascular endothelial damage is the most significant

through the lipid bilayer of the cell membrane due tohistopathological finding of an atherosclerotic plaquetheir fat-soluble nature, undergo enzymatic hydrolysis in

and is considered to be profoundly involved not only inthe cell, and are then converted to neutral amino acids.

plaque formation but also in plaque destabilization. In the

These neutral amino acids then serve as substrates forlipid core of the plaque, macrophages take up low-density

intracellular system L exchange transport, resulting inlipoproteins (LDLs) such as oxidized LDL and acetylated

the augmentation of amino acid uptake. Based on thisLDL via scavenger receptors and subsequently become

principle, we investigated the augmented accumulation offoam cells in which cholesterol esters accumulate. An

D/L-[14C]-Met in THP-1–derived macrophages.

increased number of foam cells in the intima of the bloodvessels promotes intimal thickening and plaque formation,
2. Materials and methods

and this induces the formation of a necrotic core due to thecontinuous death of foam cells, leading to weakening of
the atherosclerotic plaque. 2)

2.1. Materials

Some researchers have reported that fluorine-18

D/L-[14C]-Met was purchased from American

emissionRadiolabeled Chemicals Inc. THP-1 (human acutetomography (PET) is a promising tool for detecting

monocytic leukemia, JCRB0112) cells were purchased

vulnerable atherosclerotic plaques, depending on thefrom JCRB. Plastic tissue culture dishes (diameter 35

extent of macrophage infiltration into the atherosclerotic

mm, Cat. no. 3000-035) were purchased from IWAKI.

lesion. 3-5) In evaluating glucose derivatives for PET

Other chemicals of reagent grade were purchased fromanalyses of macrophages and foam cells in atherosclerotic

Funakoshi or Kanto Chemical Co., Inc.

fluorodeoxyglucose([18F]-FDG)

positronlesions, Ogawa et al. reported that [ F]-FDG enables
18

visualization of the early stage of foam-cell formation

2.2. Maintenance of THP-1 cells in culture

in vulnerable plaques. Changes in hexokinase activity

THP-1 cells were plated onto 35-mm diameter dishes intended to accompany changes in [ F]-FDG uptake.

1 ml of Roswell Park Memorial Institute (RPMI) medium

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6,7)

In the present study, we examined the accumulation

containing fetal bovine serum (FBS) and L-glutamine atof amino acid PET agents in macrophages and foama density of 1×105 cells/ml and cultured at 37°C, pH 7.4,

cells of atherosclerotic lesions. Using the radiolabeled

and 5% CO2 for 6 days. Cells were counted every 24 h.

compounds [ C]-L-methionine and [ C]-D-methionine
11

11(D/L-[11C]-Met) as PET agents, we investigated the2.3. Induction of THP-1 cell differentiation

uptake of these [ C]-labeled amino acids during theUsing the culture system described in section 2.2 above,differentiation process of THP-1 cells, which are human

THP-1 cells were plated at a density of 1×106 cells/ml andmonocytes established in 1980 from a 1-year-old childcultured at 37°C, pH 7.4, and 5% CO2 for 2 days. PMA

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THP-1 macrophage D/L-[14C]-Met uptake

3(100 ng/ml) was added to the dishes, and the cells wereof each culture sample using a liquid scintillation countercultured for 6, 12, 24, 48, and 72 h to induce differentiation(PerkinElmer Inc., Tri-Carb 2910TR).

into macrophages. Cells were cultured for 72 h after
PMA treatment to ensure sufficient differentiation into

2.5. Uptake of D/L-[14C]-Met by THP-1 cells during

macrophages and then treated with acetylated low-density

differentiation and foam-cell formation

lipoprotein (Ac-LDL; 50 μg/ml) and cultured for 6, 12, 24,Theuptakeofboth

D/L-[14C]-Met

optical48, and 72 h to induce macrophage foam-cell formation.

isomers was evaluated in each plot. After removing

To confirm the progression of macrophage foam-

1 ml of the culture medium from the dish, the cells werecell formation, oil red O lipid staining was performed as

incubated in 1 ml of PBS for 10 min. After removing thedescribed below. First, 30 mg of oil red O powder was

PBS, the cells were incubated with 1 ml of PBS containing

dissolved in 10 ml of 100% isopropyl alcohol (2-propanol)

18.5 kBq of D/L-[14C]-Met for 10 min. After removing

and mixed well to make a stock solution. To prepare a

the PBS, the surface of the cell layer was washed twice

working solution, 3 ml of the stock solution and 2 ml ofwith 1 ml of PBS (4°C) and incubated with 1 ml of NaOH

distilled water (3:2 ratio) were mixed well, left to standfor 1 day to lyse the cells. Radioactivity was measured infor 10 min, and subsequently filtered. After removing 1

200 μl of each culture sample using a liquid scintillation

ml of medium from the dish, the cells were washed twice

counter (PerkinElmer Inc., Tri-Carb 2910TR).

with 1 ml of phosphate-buffered saline (PBS; NaCl, 137
mM; KCl, 2.7 mM; Na2HPO•12H2O, 8 mM; KH2PO4,

2.6. Uptake augmentation study using an amino acid ethyl

1.5 mM; CaCl•2H2O, 1.8 mM; MgCl•6H2O, 1.0 mM;

ester

37°C; pH 7.4) and fixed with 1 ml of 10% formalin for

The augmentation of uptake stimulated by an amino

10 min. After removing the formalin, the cells wereacid ethyl ester exchange transport mechanism that utilizes

washed again twice with 1 ml of PBS and incubated withthe system L exchange transporter was investigated at 24

1 ml of 60% isopropanol for 1 min. After removing theh after PMA addition to the dishes, as described above.

isopropanol, filtered oil red O working solution was added

After removing 1 ml of the culture medium from the dish,and incubated for 15 min for staining. Oil red O working

the cells were incubated in 1 ml of PBS (37°C, pH 7.4) for

solution was removed after staining was completed, and10 min. After removing the PBS, the cells were incubatedthe cells were washed once with 1 ml of 60% isopropanolin 1 ml of PBS containing 3.7 kBq of D/L-[14C]-Met andand twice with 1 ml of PBS. The cells were then examined

1 mM L-tyrosine ethyl ester (L-Tyr-OEt) for 60 min.

under a microscope and imaged using a digital microscope

After removing the PBS, the surface of the cell layer wascamera.

washed twice with 1 ml of PBS (4°C) and incubated in 1
ml of NaOH for 1 day to lyse the cells. Radioactivity wasmeasured in 200 μl of each culture sample using a liquid

2.4. Time course of D/L-[ C]-Met uptake
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The uptake of both [ C]-Met optical isomers was
14

scintillation counter.

measured at different time points in macrophages derived
from THP-1 cells following a 48-h PMA treatment. After

2.7. Statistical analysis

removing 1 ml of culture medium from the dish, 1 ml of

Data were collated as the mean ± standard deviation

PBS was added and the cells were incubated for 10 min.

of five measurements, and each experiment was performed

After removing the PBS, the cells were incubated with 1

in duplicate. Results were analyzed using Student’s t test;

ml of PBS containing 3.7 kBq of D/L-[ C]-Met for 5, 15,p-values <0.01 were considered to statistically significant.

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30, and 60 min. After removing the PBS, the surface ofthe cell layer was washed twice with 1 ml of PBS (4°C)
and subsequently incubated with 1 ml of NaOH for 1 day
to lyse the cells. Radioactivity was measured in 200 μl

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茨城県立医療大学紀要

3. Results

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3.3. Time course of D/L-[14C]-Met uptake
The time course of D/L-[14C]-Met uptake bymacrophages derived from THP-1 cells following a 72-h

3.1. Maintenance of THP-1 cells in culture
The growth curve of THP-1 cells is shown in Figure

PMA treatment is shown in Figure 3. At 15 min, uptake

1. Cell counts for plating and culture duration wereof the D-form was 3.6% ± 0.24(%) and that of the L-form

determined from the growth curve.

was 4.15 ± 0.11(%), and at 30 min, uptake of the D-form
was 4.04 ± 0.16(%) and that of the L-form was 4.78 ±0.15(%). A statistically significant difference in uptake
between isomers was observed at both 15 and 30 min(p<0.02, p<0.004, respectively).

Figure 1. Growth curve of THP-1 cells.

3.2. Induction of THP-1 cell differentiation
Oil red O staining results are shown in Figure 2. At
12 h after Ac-LDL treatment, the cell count reached amaximum, and the greatest intensity in red-orange staining

Figure 3. Time course of D/L-[14C]-Met uptake by THP1–derived macrophages 72 h after PMA treatment. The
upper line represents L-[14C]-Met uptake, and the underline represents D-[14C]-Met uptake.

was observed in the dish as a whole. However, cells withextremely dark red-orange staining were observed after

3.4. Uptake of D/L-[14C]-Met by THP-1 cells during

12 h, even though the cell count had decreased. These

differentiation and foam-cell formation

cellular changes were consistent with those reported byVia et al.8)

The uptake of D/L-[14C]-Met by THP-1 cells after
PMA treatment is shown as a curve in Figure 4A. D/L[14C]-Met uptake by macrophages after Ac-LDL treatment
is shown as a curve in Figure 4B. As shown in Figure 4A,
uptake increased rapidly after PMA treatment. Uptakeof both the L- and D-forms decreased after 48 h. Figure
4B clearly shows that uptake continued to decrease after
Ac-LDL treatment. In all conditions and time points
examined in this study, uptake of the L-form of [14C]-Met
was greater than that of the D-form.

Figure 2. Oil red O staining of THP-1–derived
macrophages at 6 (A), 12 (B), 24 (C), 48 (D), and 72 (E)h after PMA treatment.

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THP-1 macrophage D/L-[14C]-Met uptake

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OEt–treated cells, and this difference was also statisticallyAsignificant (p<0.00008).

Figure 5. Uptake augmentation by amino acid ethyl
esters (D/L-[14C]-Met uptake by macrophages treatedsimultaneously with L-Tyr-OEt is shown. Values are
expressed as a ratio relative to each control 1.0).

B

4. Discussion
Diseases such as myocardial infarction and cerebral
infarction are caused by atherosclerosis, a pathologic
condition that can occur systemically. These diseases are a
critical issue in our aging society. The quality of the plaqueis the greatest concern when atherosclerosis is diagnosed,and determining the location and degree of vulnerable
plaques (which have a greater risk of rupturing thanstable plaques) is critical. Nuclear medicine tests provide
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Figure 4. D/L-[ C]-Met uptake curve after PMA
treatment (A), D/L-[14C]-Met uptake curve after Ac-LDL
treatment (B). The upper line represents L-[ 14C]-Met
uptake, and the under line represents D-[14C]-Met uptake.
Cells were incubated for 10 min with D/L-[14C]-Met.

specific and non-invasive in vivo visualization data at themolecular and cellular levels through the administrationof radioisotope-labeled probes. These tests are used forthe qualitative diagnosis of atherosclerotic plaques. In
addition, single-photon emission computed tomography

3.5. Uptake augmentation study using an amino acid ethyl(SPECT) and especially PET allow for quantitative

ester

imaging of factors that facilitate determinations of plaqueFigure 5 shows D-[14C]-Met and L-[14C]-Met

vulnerability based on morphologic information as well

uptake by control and L-Tyr-OEt–treated cells. The

as cellular-level and gross biological changes in the lesion

data are expressed as the ratio relative to each control.

area.

The D-[ C]-Met uptake ratio was 1.00 ± 0.28 in control

Risk factors of atherosclerosis include hypertension,

cells and 3.31 ± 0.10 in L-Tyr-OEt–treated cells, and this

hyperlipidemia, and diabetes. When vascular endothelial

difference was statistically significant (p<0.00005). The

damage and intimal swelling caused by these conditions

L-[ C]-Met uptake ratio was 1.00 ± 0.03 in control cells

trigger an inflammatory response, monocytes infiltrate(uptake level: 6.90% ± 0.18%) and 3.27 ± 0.20 in L-Tyr-through the vascular wall and differentiate into macrophages.

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茨城県立医療大学紀要

第23巻

In hyperlipidemia, excess LDL particles infiltrate throughwould be expected to play a pathologic role. However,the vascular wall and undergo oxidative modification,

recent studies have indicated three new classifications

leading to endothelial cell damage. Macrophages thatof macrophage activation (e.g., a host defense system,

become activated exacerbate the inflammation while

wound healing system, and immunoregulatory system),proliferating, take up these oxidized LDL particles, andand there is ongoing discussion regarding the multifaceted

eventually become foam cells. Atheromatous plaques are

and varied properties of macrophage activation.1, 2)

generated through such a process and contribute to theBased on the above, even if activated macrophages canprogression of atherosclerosis. Myocardial infarction andbe captured as an image, further analysis would still be

cerebral infarction are caused by a decrease in blood flow

necessary in order to determine the type of macrophagecaused by a narrowing of the blood vessel caliber due toand more specifically, to determine whether or not theincreased atheromatous plaques as well as the generation

accumulation of such activated macrophages is involvedof thrombi due to plaque rupture.10, 11)

in atherosclerotic plaque formation.

The THP-1 cells used in the present study wereAs shown in Figure 4B, uptake decreased rapidly

from a human monocytic cell line derived from acuteafter Ac-LDL treatment. Since obvious morphologicalmonocytic leukemia. THP-1 cells differentiate and maturechanges in the cells and a decrease in radioactivity uptake

into macrophages following PMA stimulation. As shown

were observed after Ac-LDL treatment, it is likely thatin Figure 1, these monocytic THP-1 cells proliferated

there was a progression in foam-cell formation. We

normally in the culture system used in this study.

hypothesize that this had occurred because the expression

8)

During the induction of macrophage differentiation,
as shown in Figure 2, red-orange staining was observed at

or function of the neutral amino acid transporter attenuatedprogression of foam-cell formation.

the highest intensity in macrophages at 12 h after Ac-LDL

Previously identified pathways for the uptake oftreatment. Up to this time point, there was a trend toward

neutral amino acids by cells include transport systems

macrophage proliferation. However, at 12 h and later,

L, A, and ASC. Systems A and ASC are Na+ dependent

macrophage foam cells detached from the surface of theand can be distinguished by the pH-dependence of thedish, leading to a decrease in the cell count. Nonetheless,system and specific blockade by non-natural amino acids.

as shown in Figure 4A, D/L-[ C]-Met uptake continued

System A is Na+ dependent, and uptake can be blocked

to increase up to 48 h. This was postulated to be becausewith α-(methylamino)-isobutyric acid (MeAIB). System

the monocytes differentiated into macrophages, which

ASC is also Na+ dependent, but blockade by MeAIB isthen became activated, and this consequently led to

not observed in this system. System L is Na+ independent

the upregulation in the amino acid transporter that is

and is blocked specifically by the non-natural amino acidresponsible for D/L-[ C]-Met uptake. As a result, greater

2-aminobicyclo[2.2.1]heptane-2-carboxylic acid.12-14) In

uptake was observed. In the present experimental system,

our experimental system, it was revealed that the majority

it appears that expression of the amino acid transporterof D/L-[14C]-Met uptake occurs through system L (dataresponsible for D/L-[14C]-Met uptake by macrophages

not shown). Regarding [14C]-Met, a large difference inpeaked around 48 h after PMA treatment.

uptake between the L-form and its optical isomer D-form

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14

Activated macrophages can be classified based

has been reported.15-17) Even during THP-1 differentiation,

on function, and at least two types have been identified[14C]-L-Met uptake in the present study was 2- to 3-foldto date. M1 macrophages are immune-responsive andgreater than that of [14C]-D-Met (Figure 3).

exhibit antibacterial and antiviral activities. In contrast,

As shown in Figure 5, uptake of both the D- andM2 macrophages are immunosuppressive and are

L- forms of [14C]-Met increased with the simultaneous

responsible for tissue repair and angiogenesis.1) Sinceadministration of L-Tyr-OEt. In accordance with theatherosclerosis is an inflammatory disease triggered by an

expected mechanism shown in Figure 6, it appears that

immune response, the involvement of M1 macrophages

our observation was a result of L-Tyr-OEt–induced

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THP-1 macrophage D/L-[14C]-Met uptake

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和文抄録
単球由来マクロファージ集積とその後の泡沫化は，アテローム性動脈硬化性病変の形成早期における中心的病態である。ヒト由来 THP-1 単球のマクロファージ泡沫細胞化過程での S -[methyl-14C]-D/L-methionine(D/L-[14C]-Met) の各光学異性体の取り込み，プラークの脆弱性評価目的の定性的診断薬としての S-[methyl11C]-D/L-methionine (D/L-[11C]-Met) 集積，およびアミノ酸交換輸送体を刺激することによって，THP-1 由来マクロファージにおける D/L-[14C]-Met 集積増大が可能かどうかを調べた。細胞を L- チロシンエチルエス
テルで処理して交換輸送を刺激し，D/L-[14C]-Met の 60 分間の取り込みを未処理細胞と比較した。D/L-[14C]Met は THP-1 由来のマクロファージに集積し，それは L-Tyr-OEt を用いると増加した。今後の課題として，

in vivo モデル動物実験で positron emission tomography (PET) による画像的評価を行い，臨床で用いられている fluorine-18 fluorodeoxyglucose ([18F]-FDG) や他のアミノ酸標識化合物との比較・検討を基礎的に行うことが期待される。
キーワード : 動脈硬化イメージング , THP-1 細胞 , 分化 , マクロファージ , S-[methyl-14C]-D/L-methionine

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