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Effectiveness of antifolate receptor antibody conjugated with truncated Pseudomonas exotoxin in the targeting of rheumatoid arthritis synovial macrophages.

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ARTHRITIS & RHEUMATISM
Vol. 52, No. 9, September 2005, pp 2666–2675
DOI 10.1002/art.21228
© 2005, American College of Rheumatology
Effectiveness of Anti–Folate Receptor ␤ Antibody Conjugated
With Truncated Pseudomonas Exotoxin in the Targeting of
Rheumatoid Arthritis Synovial Macrophages
Ryusaku Nagayoshi,1 Taku Nagai,1 Kakushi Matsushita,1 Katsuaki Sato,1 Nobuhiko Sunahara,2
Takemasa Matsuda,2 Tadashi Nakamura,3 Setsuro Komiya,1 Masanori Onda,4
and Takami Matsuyama1
Objective. To define the distribution of folate
receptor ␤ (FR␤)–expressing cells in various tissues,
including rheumatoid arthritis (RA) synovial tissues,
and to verify the effects of an immunotoxin composed of
an anti-FR␤ monoclonal antibody (mAb) and truncated
Pseudomonas exotoxin A (PEA) on apoptosis and tumor
necrosis factor ␣ (TNF␣) production by adherent synovial mononuclear cells from RA patients.
Methods. Anti-FR␤ mAb were produced by immunizing mice with FR␤-transfected murine pre–B cells.
The distribution of the FR␤ antigen was examined by
immunohistochemical analysis using anti-FR␤ mAb
and macrophage-specific anti-CD163 mAb. Anti-FR␤
mAb was chemically crosslinked with truncated PEA.
FR␤-expressing macrophages were produced by the
transfection of adenovirus vector containing the FR␤
gene. Apoptotic cells were detected by staining with
propidium iodide. TNF␣ was measured by enzymelinked immunosorbent assay.
Results. FR␤-expressing cells were not present in
peripheral blood leukocytes and their activated cells. In
all of the tissues examined, most FR␤-expressing cells
were CD163ⴙ. The immunotoxin significantly induced
the apoptosis of FR␤-transfected macrophages and
adherent RA synovial mononuclear cells and inhibited
TNF␣ production by adherent RA synovial mononuclear cells.
Conclusion. We demonstrated the limited distribution of FR␤-expressing cells in various tissues.
The immunotoxin targeting FR␤-expressing cells will
provide a therapeutic tool for rheumatoid synovitis.
Activated synovial macrophages are thought to
play an important role in the pathogenesis of rheumatoid arthritis (RA) (1–3). These macrophages release
proinflammatory cytokines, proteinases, and other
chemical mediators that lead to the development of
synovitis and joint destruction. The removal of macrophages decreases the severity of joint disease in animal
models of RA (4,5). Several antirheumatic drugs, including D-penicillamine and gold salts, affect synovial
macrophages (6,7). Furthermore, one of the actions of
anti–tumor necrosis factor ␣ (anti-TNF␣) therapy may
be the result of antibody-dependent cellular cytotoxicity
of membranous TNF␣-expressing macrophages (8,9).
Thus, reagents that target synovial macrophages may be
very effective in the treatment of RA.
We have shown that folate receptor ␤ (FR␤)
messenger RNA (mRNA) and protein are highly expressed by synovial macrophages, but not by other
synovial cells, in the inflamed joints of patients with RA
(10). In a rat arthritis model, 99mTc-labeled folate was
found to specifically accumulate in affected joints (11).
Moreover, we have shown that treatment with a drug
that blocks FR␤ reduces disease activity in murine
collagen-induced arthritis (12).
Dr. Matsuyama’s work was supported by a grant-in-aid for
scientific research from the Japanese Ministry of Education, Culture,
Sports, Science, and Technology.
1
Ryusaku Nagayoshi, MD, Taku Nagai, PhD, Kakushi Matsushita, MD, PhD, Katsuaki Sato, PhD, Setsuro Komiya, MD, PhD,
Takami Matsuyama, MD, PhD: Graduate School of Medical and
Dental Sciences, Kagoshima University, Kagoshima, Japan; 2Nobuhiko Sunahara, MD, PhD, Takemasa Matsuda, MD, PhD: Kagoshima
Red Cross Hospital, Kagoshima, Japan; 3Tadashi Nakamura, MD,
PhD: Kumamoto Orthopaedic Hospital, Kumamoto, Japan; 4Masanori Onda, MD, PhD: Center for Cancer Research, National Cancer
Institute, Bethesda, Maryland.
Address correspondence and reprint requests to Takami
Matsuyama, MD, PhD, Department of Immunology, Graduate School
of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima City, Japan 890-854. E-mail: matuyama@m.kufm.
kagoshima-u.ac.jp.
Submitted for publication November 30, 2004; accepted in
revised form May 16, 2005.
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TARGETING OF RA SYNOVIAL MONONUCLEAR CELLS WITH ANTI-FR␤ AND TRUNCATED PEA
FR␤ is expressed by acute myelogenous leukemia cells (13,14). In vitro studies suggest that folate
liposomes that contain cytotoxic drugs might be useful
for the treatment of this disease (15). In addition,
immunotoxins, which are composed of specific antibodies combined with toxins, such as Pseudomonas
exotoxin A (PEA), diphtheria toxin, gelonin, saporin,
and ricin A, effectively bind to malignant cells that bear
the appropriate surface antigens (16,17) and monocyte/
macrophages (18–20). However, there are no reports of
immunotoxins that target FR␤. Before we develop and
test these immunotoxins, it is important to determine the
distribution of FR␤-expressing cells in normal and inflamed tissues from humans.
In the present study, we produced monoclonal
antibodies (mAb) against human FR␤. We used these
mAb to detect FR␤-expressing cells in a variety of
tissues and cells. We then prepared an immunotoxin
composed of an anti-FR␤ mAb and truncated PEA
(16,17) and verified that the immunotoxin selectively
targets RA synovial macrophages in vitro.
MATERIALS AND METHODS
Tissue samples. Synovial tissue was obtained from 10
RA patients who underwent total knee replacement surgery.
These patients fulfilled the American College of Rheumatology (formerly, the American Rheumatism Association) 1987
criteria for RA (21) and had previously been treated with
either methotrexate, D-penicillamine, gold salts, sulfasalazine,
bucillamine, or prednisone (⬍7.5 mg/day) or combinations of
these drugs. Adherent synovial mononuclear cells were prepared as previously described (22). Briefly, synovium was
digested with type V collagenase (Sigma-Aldrich, St. Louis,
MO) for 45 minutes at 37°C. Cell suspensions were filtered
through a stainless steel mesh. Mononuclear cells were isolated
on a Ficoll-Hypaque gradient, incubated in plastic plates for 1
hour at 37°C, and washed 10 times with phosphate buffered
saline. These steps for obtaining adherent cells were repeated.
Using the same procedures, adherent lung mononuclear cells
were prepared from lung tissues obtained by surgical resection
for lung carcinoma from 4 patients.
Samples of skin (n ⫽ 3), lung (n ⫽ 3), liver (n ⫽ 2),
spleen (n ⫽ 2), kidney (n ⫽ 1), intestine (n ⫽ 3), and lymph
node (n ⫽ 3) obtained by surgical resection for cancers were
used for immunohistochemistry analysis to detect FR␤expressing cells.
Peripheral blood was drawn from 4 healthy donors,
and synovial fluid was drawn from the swollen knee joints of 3
RA patients. Monocytes were prepared from the peripheral
blood of healthy donors and were stimulated with lipopolysaccharide (LPS; derived from Escherichia coli 0111:B4) (SigmaAldrich), macrophage colony-stimulating factor (M-CSF)
(Pierce, Rockford, IL), or interferon-␥ (IFN␥) (Pierce) as
described previously (22).
Informed consent was obtained from all donors in
2667
accordance with the requirements of the Human Investigations
Committee of Kagoshima University.
Production of mAb against the FR␤ antigen. Murine
FR␤-expressing pre–B cells were prepared for immunogens as
follows. FR␤ complementary DNA (cDNA) containing the
Kozak consensus and coding sequence was prepared from a
reverse transcription–polymerase chain reaction (RT-PCR)
product derived from RA synovial cells, as previously described (10). The primer sequences were 5⬘-AGAAAGACATGGTCTGGAAATGGATG-3⬘ (upstream) and 5⬘GACTGAACTCAGCCAAGGAGCCAGAGTT-3⬘ (downstream). The cDNA fragment was ligated with pCR2.1-TOPO
vector (Invitrogen, Carlsbad, CA), and its identity was confirmed by sequencing. The Eco RI–digested insert was then
ligated into pEF-BOS (23). This construct was transfected into
B300-19 cells (murine pre–B cell line) with the use of Lipofectamine (Invitrogen). After 48 hours, transfected cells were
selected in 1,000 ␮g/ml of G-418 disulfate (Nakarai Tesque,
Kyoto, Japan) in Dulbecco’s minimum essential medium
(DMEM) containing 10% fetal calf serum. Resistant colonies
were examined by RT-PCR using the above primers.
BALB/c mice were immunized with the FR ␤ transfected B300-19 cells. Their spleen cells were fused with
NS-1 myeloma cells. Hybridomas were screened for reactivity
with FR␤-transfected B300-19 cells by flow cytometric and
Western blot analyses. Two anti-FR␤ mAb (36b [IgG2a isotype] and 94b [IgG1 isotype]) were selected for further evaluation.
Preparation of truncated PEA. Truncated PEA was
purified as previously described (24). Briefly, the plasmid
pMS8-38-402, which encodes the truncated LysPE38QQR
protein, was transfected into BL21 (DE3) cells (Stratagene,
La Jolla, CA). After induction with IPTG, periplasm was
collected. Truncated PEA was purified through Poros HQ
(Applied Biosystems, Foster City, CA) and TSK 3000 SW
(Tosoh, Tokyo, Japan) columns using a Vision Workstation
Liquid Chromatography system (Japan Perceptive, Tokyo,
Japan). The activity of truncated PEA was evaluated by the
Carroll method (25). Briefly, the ADP ribosylation of truncated PEA was measured using wheat germ extract containing
ADP ribosyltransferase (Promega, Tokyo, Japan) and 14Clabeled nicotinamide adenine dinucleotide (Daiichikagaku,
Tokyo, Japan).
Preparation of immunotoxin. Anti-FR␤ immunotoxin
was prepared according to a slight modification of Hassan’s
procedure (24). Briefly, 1 ml of anti-FR␤ mAb (3 mg/ml) was
incubated with 100 ␮ g of succinimidyl trans-4(maleimidomethyl) cyclohexane-1-carboxylate (SMCC; SigmaAldrich) for 1 hour at room temperature. One milliliter of
truncated PEA (10 mg/ml) was incubated with 400 ␮g of
succinimidyl 3-(2-pyridyldithio)propionate (SPDP; SigmaAldrich) for 12 hours at 4°C. Excess SMCC and SPDP were
removed using a PD-10 column (Amersham Pharmacia, Tokyo, Japan). Truncated PEA coupled with SPDP was activated
with tris(2-carboxyethyl)phosphine (Molecular Probes, Eugene, OR) and mixed with anti-FR␤ mAb–SMCC. This mixture was purified using PD-10, Poros HQ, and TSK 3000 SW
columns.
Determination of the binding capacity of immunotoxin
to the FR␤ antigen. A total of 2 ⫻ 105 FR␤-transfected
B300-19 cells, FR␤-transfected B300-19 cells pretreated with
2668
NAGAYOSHI ET AL
Figure 1. Reactivity of anti–folate receptor ␤ (FR␤) monoclonal antibody (mAb). A, In these studies, a,
FR␤-transfected B300-19 cells, b, B300-19 cells, and d, KB nasopharyngeal epidermoid carcinoma cells
were reacted with anti-FR␤ mAb, and c, KB nasopharyngeal epidermoid carcinoma cells were reacted
with anti-FR␣ mAb. Gray histograms show the control (IgG2a) mAb; black histograms show the anti-FR␤
(36b) or anti-FR␣ mAb. FITC ⫽ fluorescein isothiocyanate. B, FR␤-transfected B300-19 cells, B300-19
cells, and adherent rheumatoid arthritis (RA) synovial mononuclear cells (SMC) were biotinylated, and
the lysates were immunoprecipitated with anti-FR␤ mAb 36b (IgG2a isotype; lanes a, c, and e), 94b (IgG1
isotype; lanes b, d, and f), or control antibody (IgG2a [G2a]; lane g). The immunoprecipitates were
analyzed on sodium dodecyl sulfate–12% polyacrylamide gels under reducing conditions and detected as
described in Materials and Methods. Molecular weight markers are shown at the left.
anti-FR␤ mAb (10 ␮g/ml for 15 minutes), or nontransfected
B300-19 cells were sequentially incubated with the immunotoxin (1 ␮g/ml) or with the control mAb (1 ␮g/ml) and then
with rabbit anti-PEA antibody, followed by fluorescein
isothiocyanate–conjugated goat anti-rabbit antibody (Zymed,
South San Francisco, CA). Cells were then analyzed by flow
cytometry.
Adenovirus-mediated FR␤ gene transfer into monocyte/
macrophages. FR␤ cDNA was excised from the pCR2.1
vector by Xba I digestion, blunted using a DNA blunting kit
(Takara Shuzo, Kyoto, Japan), and subcloned into the Swa I
site of pAxCAwt, which had been generated using an adenovirus expression vector kit (Takara Shuzo). Following the
manufacturer’s instructions, human embryonic kidney 293
cells (RIKEN Cell Bank, Tsukuba, Japan) were transfected
with this cosmid vector and adenovirus genomic DNA–
terminal protein complex to produce replication-incompetent
E1- and E3-deficient adenoviruses expressing FR␤. Titers
of recombinant adenovirus were determined by plaque assays
of 293 cells (26). To obtain high titers of adenovirus, infection
of the virus into 293 cells was performed 4 times. The
adenoviruses were suspended in culture medium, adjusted to
2 ⫻ 108 plaque-forming units/ml, and stored at –80°C until they
were used.
Monocyte/macrophages were transfected with the adenovirus as previously described (27). Briefly, monocytes
were incubated with 20 ng/ml of M-CSF for 24 hours, transfected with the adenovirus vector at a multiplicity of infection
of 50, and centrifuged at 2,000g for 2 hours at 37°C.
Measurement of apoptotic cells. FR␤-expressing
B300-19 cells (2 ⫻ 105/ml), B300-19 cells (2 ⫻ 105/ml),
macrophages (1 ⫻ 106/ml), adherent RA synovial mononuclear cells (1 ⫻ 106/ml), and adherent lung mononuclear
cells (1 ⫻ 106/ml) in DMEM (Nikken, Kyoto, Japan) contain-
TARGETING OF RA SYNOVIAL MONONUCLEAR CELLS WITH ANTI-FR␤ AND TRUNCATED PEA
2669
Figure 2. Distribution of folate receptor ␤ (FR␤)–expressing cells in rheumatoid arthritis synovial tissues. Cryosectioned synovial tissues were
stained with A, Alexa Fluor 488–conjugated anti-CD163 monoclonal antibody (mAb) (green), B, biotinylated anti-FR␤ mAb (red), or C, both of
these mAb (yellow), followed by rhodamine–avidin D. Sections were examined by confocal laser microscopy. LL ⫽ lining layer; SL ⫽ sublining layer.
ing 10% human AB serum were placed into 24-well plates and
incubated at 37°C in an atmosphere of 5% CO2 for various
times (see below) in the presence of either immunotoxin or a
mixture of equimolar amounts of anti-FR␤ mAb and truncated
PEA at the concentrations indicated below. Propidium iodide
(Molecular Probes) staining was performed as described by
Nicoletti et al (28). Briefly, cells were stained with propidium
iodide (50 ␮g/ml) in 0.1% sodium citrate plus 0.1% Triton
X-100 for 20 minutes at 4°C. Apoptotic cells were detected by
flow cytometry.
Immunoprecipitation and Western blot analyses.
FR␤-expressing B300-19 cells, B300-19 cells, and adherent RA
synovial mononuclear cells were biotinylated with Sulfo-NHSLC-Biotin (Pierce) and lysed in buffer containing 1% Triton X
and proteinase inhibitors. The lysates were immunoprecipitated with either anti-FR␤ mAb or control mAb bound to
protein G–agarose (Santa Cruz Biotechnology, Santa Cruz,
CA). The immunoprecipitates were run on sodium dodecyl
sulfate (SDS)–polyacrylamide 12% gels under reducing conditions, transferred to Hybond ECL nitrocellulose membranes
(Amersham Pharmacia), and stained with streptavidin–
peroxidase (Zymed). The membranes were incubated in enhanced chemiluminescent reagents (Amersham Pharmacia).
Chemiluminescence was detected on Hyperfilm ECL (Amersham Pharmacia).
The immunotoxin, anti-FR␤ mAb, and truncated PEA
were run on SDS–polyacrylamide 10% gels under reducing
conditions, transferred to Hybond ECL nitrocellulose membranes, and reacted with rabbit anti-PEA antibody (a gift of
Dr. Ira Pastan, Laboratory of Molecular Biology, National
Cancer Institute, National Institutes of Health, Bethesda, MD)
and either horseradish peroxidase (HRP)–conjugated goat
anti-rabbit antibody or HRP-conjugated goat anti-mouse IgG
antibody (both from Bio-Rad, Hercules, CA). The blots were
then developed as described above.
Immunohistochemical analysis. Serial frozen sections
were stained using biotinylated anti-FR␤ mAb or biotinylated
anti-CD163 mAb (Maine Biotechnology Service, Portland,
ME), streptavidin–peroxidase, and aminoethylcarbazole reagent (Nichirei, Tokyo, Japan). Cryosectioned RA synovial
tissues were stained using biotinylated anti-FR␤ mAb followed
by rhodamine–avidin D (Vector, Burlingame, CA) and Alexa
Fluor 488–conjugated anti-CD163 mAb (Molecular Probes).
Double-stained sections were imaged by confocal laser microscopy (Leica Microsystems, Wetzlar, Germany) using a 488-nm
argon ion laser and a band-pass 515–545-nm emission filter for
Alexa Fluor 488 and a 568-nm argon ion laser and a band-pass
570–630-nm emission filter for rhodamine. Large field of view
images were acquired using a 20⫻ objective lens with detector
pinholes completely opened to give pseudoconventional images. The resulting images were 12 bit, and 1,024 ⫻ 1,024
pixels, giving a pixel resolution of 0.45 ␮m pixel size. Contrast
and brightness were set using bright samples to ensure that
there was no saturation of the pixels.
Measurement of TNF␣ production by adherent RA
synovial mononuclear cells. RA synovial mononuclear cells
(1 ⫻ 106/ml) were suspended in DMEM containing 10%
human AB serum, placed in 24-well plates with either the
immunotoxin or a mixture of equimolar amounts of antiFR␤ mAb and truncated PEA, and incubated for 12 hours
at 37°C in an atmosphere of 5% CO2. After 2 washes with
phosphate buffered saline, the cells were incubated for 12
hours with 10 ␮g/ml of LPS. TNF␣ in culture supernatants was
measured in duplicate by an enzyme-linked immunosorbent
assay using an anti-TNF␣ antibody and a biotinylated antiTNF␣ antibody according to the manufacturer’s protocol
(Pierce). Serial dilutions of recombinant TNF␣ (Pierce) were
used as the standard for the determination of TNF␣ concentrations.
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NAGAYOSHI ET AL
Figure 3. Characterization of the immunotoxin composed of anti–folate receptor ␤ (antiFR␤) monoclonal antibody (mAb) and truncated Pseudomonas exotoxin A (PEA). A, The
immunotoxin (lane a), anti-FR␤ mAb (lane b), and truncated PEA (lane c) were transferred
by Western blotting and were reacted with anti-PEA antibody (anti-PE) or anti-mouse IgG
antibody (anti-mouse Ig) as described in Materials and Methods. Arrowheads show the
bands reacting with both the anti-PEA antibody and the anti-mouse IgG antibody.
Molecular weight markers are shown at the left. B, To examine the binding of the
immunotoxin to the FR␤ antigen, a, FR␤-transfected B300-19 cells, b, FR␤-transfected
B300-19 cells pretreated with anti-FR␤ antibody, and c, B300-19 cells were reacted with the
immunotoxin (black histograms) or with the control antibody (gray histograms) and were
then reacted with rabbit anti-PEA antibody, followed by fluorescein isothiocyanate (FITC)–
conjugated goat anti-rabbit antibody. Cells were then analyzed by flow cytometry.
Statistical analysis. The nonparametric MannWhitney U test was used to test for differences. P values less
than 0.05 were considered significant.
RESULTS
Characterization of FR␤-specific mAb. The 36b
and 94b mAb reacted with FR␤-transfected B300-19
cells but not with B300-19 cells or with KB nasopharyngeal epidermoid carcinoma cells expressing the FR␣
antigen (Figure 1A). Moreover, these antibodies immunoprecipitated a 40-kd protein from FR␤-transfected
B300-19 cells and adherent RA synovial mononuclear
cells (Figure 1B), findings consistent with previously
reported results using a polyclonal FR␤-specific mAb
(10).
We used the mAb to evaluate the expression of
FR␤ on peripheral blood leukocytes and in various
tissues. The antibodies did not react with freshly ob-
TARGETING OF RA SYNOVIAL MONONUCLEAR CELLS WITH ANTI-FR␤ AND TRUNCATED PEA
Figure 4. Apoptosis of folate receptor ␤ (FR␤)–transfected B300-19
cells induced by an immunotoxin composed of anti-FR␤ monoclonal
antibody (mAb) and truncated Pseudomonas exotoxin A (PEA).
FR␤-transfected B300-19 cells (F) or B300-19 cells (E) were cultured
in the presence of the indicated concentrations of immunotoxin for 24,
36, or 48 hours. FR␤-transfected B300-19 cells were also cultured in
the presence of the indicated concentrations of a mixture of anti-FR␤
mAb and truncated PEA (‚) for 48 hours. The number of apoptotic
cells was measured by propidium iodide staining and flow cytometric
analysis. Values are the mean ⫾ SEM percentage of apoptotic cells
induced by the immunotoxin (n ⫽ 4 experiments). ⴱ ⫽ P ⬍ 0.05 versus
B300-19 cells and versus FR␤-transfected B300-19 cells cultured with
the mixture of anti-FR␤ mAb and truncated PEA.
2671
tained peripheral blood leukocytes or with monocyte/
macrophages that had been stimulated with LPS,
M-CSF, or IFN␥ (data not shown). In contrast, FR␤expressing cells were observed in samples of skin, lung,
liver, spleen, kidney, intestine, and lymph node (data not
shown), as well as in RA synovium (Figure 2). FR␤ was
expressed at high levels in RA synovial tissues and at
much lower levels in the other tissues.
To determine which cells expressed FR␤, we
compared anti-FR␤ mAb–stained sections with serial
sections stained with macrophage-specific anti-CD163
mAb. In all tissues, most FR␤⫹ cells also expressed
CD163. FR␤-expressing cells in RA synovial tissues
were distributed mainly in the sublining layer, while
CD163⫹ cells were observed in the lining and sublining
layers. Interestingly, alveolar macrophages did not stain
with the anti-FR␤ mAb, and Kupffer cells stained
weakly with the anti-FR␤ mAb.
Effectiveness of the immunotoxin composed of
anti-FR␤ mAb and truncated PEA in targeting RA
synovial macrophages. This limited distribution of FR␤expressing cells led us to hypothesize that toxinconjugated anti-FR␤ mAb (immunotoxins) would specifically inhibit the activity of RA synovial macrophages
and be useful for the treatment of RA. To test this
Figure 5. Apoptosis of folate receptor ␤ (FR␤)–transfected macrophages induced by an immunotoxin composed of anti-FR␤ monoclonal
antibody (mAb) and truncated Pseudomonas exotoxin A (PEA). A, Macrophages were transfected with an adenovirus vector containing
either a, the sense FR␤ gene or b, the antisense FR␤ gene. On day 4 after transfection, macrophages were stained with anti-FR␤ mAb.
Gray histograms show the control (IgG2a) mAb; black histograms show the anti-FR␤ mAb. Results are representative of 4 separate
experiments using macrophages from different donors. FITC ⫽ fluorescein isothiocyanate. B, On day 1 after transfection, macrophages
transfected with the adenovirus vector containing the sense (F) or antisense (E) FR␤ gene were cultured for 3 days in the presence of
either the immunotoxin or a mixture of anti-FR␤ antibody and truncated PEA. Apoptotic cells were measured by propidium iodide
staining and flow cytometric analysis. The percentage of apoptotic cells in each sample was determined by subtracting the percentage of
apoptotic cells induced by the mixture of anti-FR␤ mAb and truncated PEA from that induced by the immunotoxin. Values are the
mean ⫾ SEM of 4 separate experiments using macrophages from different donors. ⴱ ⫽ P ⬍ 0.05.
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NAGAYOSHI ET AL
Figure 6. Apoptosis and inhibition of tumor necrosis factor ␣ (TNF␣) production by adherent rheumatoid arthritis (RA) synovial mononuclear cells
induced by the immunotoxin composed of anti–folate receptor ␤ (anti-FR␤) monoclonal antibody (mAb) and truncated Pseudomonas exotoxin A
(PEA). A, Adherent RA synovial mononuclear cells (F) or adherent lung mononuclear cells (E) were cultured in the presence of the immunotoxin
or a mixture of anti-FR␤ mAb and truncated PEA at an equimolar ratio for 3 days. Apoptotic cells were measured by propidium iodide staining
and flow cytometric analysis. The percentage of apoptotic cells in each sample was determined by subtracting the percentage of apoptotic cells
induced by the mixture of anti-FR␤ mAb and truncated PEA from that induced by the immunotoxin. Values are the mean ⫾ SEM of 4 separate
experiments using different RA synovial tissues and lung tissues. ⴱ ⫽ P ⬍ 0.05. B, Adherent RA synovial mononuclear cells were incubated with
various concentrations of immunotoxin (F) or a mixture of anti-FR␤ mAb and truncated PEA (E) at an equimolar ratio for 12 hours. Cells were
then stimulated with lipopolysaccharide for 12 hours. Tumor necrosis factor ␣ (TNF␣) in the culture supernatants was measured by enzyme-linked
immunosorbent assay. Results are expressed as the percentage of TNF␣ in the absence of the immunotoxin or the mixture of anti-FR␤ antibody
and truncated PEA. Values are the mean ⫾ SEM of 4 separate experiments using different adherent RA synovial mononuclear cell preparations.
ⴱ ⫽ P ⬍ 0.05.
hypothesis, we conjugated an anti-FR␤ mAb (36b) with
truncated PEA. Western blot analysis under reducing
conditions showed that most of the immunotoxin molecules were of higher molecular weight than the anti-FR␤
mAb heavy chain. Several immunotoxin bands reacted
with both anti-PEA antibody and anti-mouse IgG antibody, indicating that the mAb chains were conjugated
with truncated PEA (Figure 3A). Furthermore, flow
cytometry studies indicated that the immunotoxin bound
to the FR␤ antigen (Figure 3B). Specifically, the immunotoxin reacted with FR␤-expressing B300-19 cells but
not with B300-19 cells. Moreover, the binding of immunotoxin to FR␤-expressing B300-19 cells was inhibited
by an excess of anti-FR␤ mAb.
Next, we examined whether the immunotoxin
could induce apoptosis of FR␤-expressing cells. As
expected, the immunotoxin induced the apoptosis of
FR␤-transfected B300-19 cells, but not B300-19 cells. In
addition, a mixture of anti-FR␤ mAb and truncated
PEA did not induce apoptosis of FR␤-transfected
B300-19 cells (Figure 4). These findings indicate the
specific effect of the immunotoxin on FR␤-expressing
cells.
The ability of the immunotoxin to induce apoptosis of FR␤-expressing macrophages was then evaluated.
As described above, peripheral blood monocytes and
cultured or in vitro–activated macrophages do not express FR␤. Thus, we transfected monocyte/macrophages
with a recombinant adenovirus vector encoding the FR␤
gene. The percentages of FR␤-expressing cells after
transfection ranged from 30% to 40% (Figure 5A).
When the immunotoxin was added to cultures of FR␤expressing macrophages or FR␤-nonexpressing macrophages, the immunotoxin induced significant apoptosis
of the FR␤-expressing macrophages as compared with
the FR␤-nonexpressing macrophages. However, at the
highest concentration tested (5 ␮g/ml), the mixture of
anti-FR␤ antibody and truncated PEA induced apoptosis of the FR␤-expressing and FR␤-nonexpressing macrophages (Figure 5B).
We then studied the effects of the immunotoxin
on RA synovial macrophages. Adherent RA synovial
TARGETING OF RA SYNOVIAL MONONUCLEAR CELLS WITH ANTI-FR␤ AND TRUNCATED PEA
mononuclear cells contained 40–50% FR␤-expressing
cells (or CD14⫹ cells). Many freshly obtained RA
synovial macrophages are already apoptotic (29). Thus,
the maximum percentage of adherent RA synovial
mononuclear cells that could be induced to undergo
apoptosis by the immunotoxin would be 40–50%. We
found that the immunotoxin induced apoptosis in fewer
than 25% of adherent RA synovial mononuclear cells
(Figure 6A). In contrast, the immunotoxin did not
induce apoptosis in RA synovial fibroblast-like cells
from long-term cultures (data not shown).
Adherent lung mononuclear cells contain FR␤–
alveolar macrophages and FR␤⫹ tissue macrophages.
As with adherent RA synovial mononuclear cells, ⬃40–
50% of adherent lung mononuclear cells expressed
CD14. We found that the concentration of immunotoxin
needed to induce apoptosis of adherent lung mononuclear cells was higher than that needed to induce
apoptosis of adherent RA synovial mononuclear cells.
These results suggest that RA synovial macrophages are
more sensitive to the immunotoxin than are lung macrophages. Next, we examined the effects of the immunotoxin on TNF␣ production by LPS-stimulated adherent
RA synovial mononuclear cells. At low concentrations,
the immunotoxin significantly inhibited TNF␣ production, as compared with the mixture of anti-FR␤ mAb
and truncated PEA (Figure 6B).
DISCUSSION
We produced 2 mAb that react with FR␤ but not
with FR␣. Immunohistochemical analysis revealed FR␤expressing cells in a wide variety of human tissues; most
of the FR␤-expressing cells also expressed the macrophage marker CD163. However, FR␤ was not detected
on peripheral blood leukocytes, even after in vitro
stimulation with LPS, M-CSF, or IFN␥. Previous studies
using a polyclonal antibody and Northern blotting techniques indicated that myeloid cells and activated macrophages express FR␤ antigen and mRNA; however, FR␤
on the myeloid cells was not functional (14). It is
intriguing that epitopes recognized by our mAb are
absent on myeloid cells. This suggests that these
epitopes might be associated with the function of FR␤.
It has been reported that mice with the Folbp-2
deletion, which is equivalent to FR␤, showed normal
growth (30). Furthermore, folic acid has a higher affinity
for FR␣ than for FR␤. At present, the physiologic role
of FR␤ on hematopoietic cells is not well defined. We
recently showed that peroxynitrite generated from nitric
oxide and reactive oxide induced the nitration of folic
2673
acid; one of the products, 10-nitro-folic acid, has a
higher affinity for FR␤ than for FR␣ (31). These
findings, combined with those from the current study,
suggest that FR␤ on tissue macrophages might be
required for the incorporation of folate derivatives for
the synthesis of proinflammatory proteins.
We found FR␤-expressing macrophages in the
sublining layer of RA synovial tissues. In a previous
study by Cauli et al (32), many 27E10⫹ early macrophages were found in the sublining layer, whereas
25F9⫹ mature macrophages were more abundant in the
lining layers. Thus, the majority of FR␤-expressing
macrophages in RA synovial tissues seem to be acute
inflammatory macrophages. Further examinations using
various pathologic tissues should help to clarify the
nature of FR␤-expressing macrophages.
There are several reports about folate-containing
reagents that target FR␤-expressing leukemia cells and
macrophages (33,34). However, folic acid binds more
avidly to FR␣ than to FR␤. In addition, cells have routes
for folic acid uptake other than via folate receptors.
Thus, reagents with higher affinity for FR␤ than for
FR␣ should be used for targeting FR␤-expressing cells.
Anti-FR␤ mAb has many advantages over other methods of drug delivery via FR␤ in terms of specificity. In
the present study, we produced an immunotoxin composed of an anti-FR␤ mAb and a truncated PEA
(LysPE38QQR), which lacks the cell-binding domain
but retains the translocation and the adenosine diphosphate ribosylation domains (24). After internalization
and proteolytic processing, domain II functions to translocate the toxin to the cytosol; the domain catalytically
ADP-ribosylates elongation factor 2 in the cytosol, leading to the arrest of protein synthesis and the induction of
apoptosis (16,17).
Although most malignant cells are actively proliferating, RA synovial macrophages in the cultures we
used are nondividing. We assumed that monocyte/macrophages might be resistant to PEA, as compared with
dividing tumor cells. However, in the present culture
system, our immunotoxin induced apoptosis and inhibited the production of TNF␣ by adherent RA synovial
mononuclear cells. FR␤ was found only on macrophages
in the adherent RA synovial mononuclear cell cultures.
Moreover, macrophages are the main producers of
TNF␣ in adherent RA synovial mononuclear cell cultures (1,2). The immunotoxin did not induce apoptosis
of cultured RA synovial fibroblast-like cells. Taken
together, these findings support the notion that the
immunotoxin mainly damaged RA synovial macrophages. The action of the immunotoxin is not mediated
2674
NAGAYOSHI ET AL
through macrophages Fc␥ receptors, since the immunotoxin induced more apoptosis of the FR␤ gene–
transfected macrophages than of the antisense gene–
transfected macrophages.
The data from the present study suggest that the
immunotoxin might ameliorate joint inflammation in
patients with RA by inhibiting the activity of synovial
macrophages. However, improvements in the immunotoxin, such as using recombinant monomeric or dimeric
Fv immunotoxins, would cause less immunogenicity and
more accessibility to lesions (35,36).
The toxic side effects of immunotoxins are of at
least 2 types. One type results from damage to normal
cells that express the target antigen. Another type is
nonspecific and is usually characterized by damage to
liver cells (37). A recent study indicated that an antiCD64 mAb conjugated with ricin A induced apoptosis of
RA synovial fluid macrophages and inhibited the production of TNF␣ and interleukin-1␤ in synovial tissue
explants (20). However, the CD64 antigen is expressed
on monocytes and activated neutrophils in addition to
tissue macrophages. Thus, immunotoxins using an antiFR␤ mAb have advantages over those using an antiCD64 mAb because of the limited distribution of FR␤
and the low levels of FR␤ expression in normal tissues
(38). Severe systemic toxicity might be observed with the
in vivo use of immunotoxins. Thus, we would prefer to
develop strategies to target activated macrophages in the
joints by local injections of the immunotoxin during the
first clinical trial.
We have recently observed that our immunotoxin
inhibited the growth of FR␤-expressing HL-60 cells
(promyelocytic leukemia cells) implanted in SCID mice
(Nagayoshi R, et al: unpublished observations). In addition to reducing the activity of RA and FR␤-expressing
leukemias, the immunotoxin should reduce the activity
of other diseases in which macrophage activation is
involved in the pathogenesis (39).
ACKNOWLEDGMENTS
The authors would like to thank Dr. Ira Pastan (Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD) and Dr. S. A.
Michie (Department of Pathology, Stanford University School
of Medicine, Palo Alto, CA) for critical review of the manuscript.
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