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Imaging of activated macrophages in experimental osteoarthritis using folate-targeted animal single-photonemission computed tomographycomputed tomography.

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Vol. 63, No. 7, July 2011, pp 1898–1907
DOI 10.1002/art.30363
© 2011, American College of Rheumatology
Imaging of Activated Macrophages in
Experimental Osteoarthritis Using Folate-Targeted
Animal Single-Photon–Emission Computed
Tomography/Computed Tomography
Tom M. Piscaer,1 Cristina Müller,2 Thomas L. Mindt,3 Erik Lubberts,1 Jan A. N. Verhaar,1
Eric P. Krenning,1 Roger Schibli,2 Marjon De Jong,1 and Harrie Weinans1
Objective. Evaluation of macrophage activation
may provide essential information about the etiology
and progression rate of osteoarthritis (OA). Activated
macrophages abundantly express folate receptor ␤
(FR␤), which can be targeted using radioactive-labeled
folic acid. This study was undertaken to investigate
whether macrophage activation can be monitored in
small animal models of OA using a folate radiotracer
and to determine whether macrophage activation differs
in different models of OA with different OA progression.
Methods. Two rat models of OA were used: the
mono-iodoacetate (MIA) model, which is a fastprogressing biochemically induced model, and the anterior cruciate ligament transection (ACLT) model,
which induces OA at a slower pace. Images were obtained using high-resolution small animal singlephoton–emissioncomputedtomography/computedtomography. The specificity of the technique was tested by
eradicating macrophages using clodronate-laden liposomes and blockade of FR␤ by cold folic acid.
Results. The MIA model had high initial macrophage activation, with a peak after 2 weeks which
disappeared after 8 weeks. The ACLT model showed less
activation but was still active 12 weeks after induction.
The technique allowed monitoring of the disease process
over time, in which late stages of the disease showed less
macrophage activation than early stages, especially in
the fast-progressing MIA model of OA.
Conclusion. Our findings indicate that macrophage activation in experimental OA can clearly be
demonstrated and monitored by the folate radiotracer.
The high resolution, high sensitivity, and high specificity of the technique allow clear localization of macrophage activity in a disease model that is not known for
abundant macrophage involvement.
Macrophages might play a role in the development and progression of osteoarthritis (OA). Once
activated, macrophages can produce large quantities of
growth factors, enzymes, and proinflammatory cytokines. It has previously been shown that macrophages
enhance osteophyte growth in animal models of OA
(1,2). Furthermore, it is possible that macrophages play
a role in the maintenance and progression of the disease
by their contribution to synovial fibrosis, cartilage catabolism, and maintenance of an inflammatory state in the
joint (3,4) by production of growth factors (5,6), enzymes (7), and cytokines, respectively (8,9).
Currently, there are no validated measures of
disease activity for OA. Anatomic imaging techniques,
such as radiography and magnetic resonance imaging
(MRI), measure structural changes that are a result of
the disease process and do not visualize the activity of
the disease process itself. A number of studies have
demonstrated a possible association between synovitis
and OA inflammation and the formation of these structural changes (10). A recently introduced technique allows
monitoring of activated macrophages in vivo (11,12).
Supported by the Dutch Arthritis Association (LLP 11), the
Netherlands Ministry of Economic Affairs (Smart Mix Program), and
the Netherlands Ministry of Education, Culture, and Science.
Tom M. Piscaer, MD, Erik Lubberts, PhD, Jan A. N.
Verhaar, MD, PhD, Eric P. Krenning, MD, PhD, Marjon De Jong,
PhD, Harrie Weinans, PhD: Erasmus University Medical Center,
Rotterdam, The Netherlands; 2Cristina Müller, PhD, Roger Schibli,
PhD: Center for Radiopharmaceutical Sciences ETH-PSI-USZ, and
Paul Scherrer Institute, Villigen-PSI, Switzerland; 3Thomas L. Mindt,
PhD: University Hospital Basel, Basel, Switzerland.
Address correspondence to Tom M. Piscaer, MD, Erasmus
University Medical Center, Dr. Molenwaterplein 50, Box 1738, Rotterdam 3000 DR, The Netherlands. E-mail:
Submitted for publication September 9, 2009; accepted in
revised form March 15, 2011.
It is only after activation that macrophages express the functional form of folate receptor ␤ (FR␤)
(11,13). The vitamin folic acid binds to FR␤ with high
affinity (Kd ⬍ 10⫺9M). The presence of the functional
form of FR␤ on macrophages is specific for macrophages in an activated state (expression of CD80, CD86,
and Ly-6C/G), which are known to secrete tumor necrosis factor ␣ and reactive oxygen species (14). The
function of folate receptors on activated macrophages is
still unknown. Most cells use a folate carrier, such as the
reduced folate carrier and the proton-coupled folate
transporter, for folate uptake. On the other hand, the
high-affinity folate receptor, which internalizes folates
and folate conjugates via endocytosis, is expressed on
only a few cell types, including malignant tissue, with the
highest frequency on ovarian carcinoma cells (15). In
normal tissue and organs, the renal brush border membrane of the proximal tubule cells, the choroid plexus,
and the placenta are the only sites that express the folate
receptor (15).
By labeling a folic acid analog with a diagnostic
radionuclide, the presence of the folate receptor can be
traced with very high sensitivity using positron emission
tomography (PET) or single-photon–emission computed tomography (SPECT). This new molecular imaging technique has already been applied in animal and
human studies for the detection of ovarian carcinoma
and the specific detection of activated macrophages in
(experimental) rheumatoid arthritis (RA) (16–19). The
introduction of high-resolution multipinhole SPECT systems provided submillimeter resolution and up to 50
times increased sensitivity compared to clinical SPECT
and PET systems. This allows assessment and quantification of arthritic activity in small animals such as mice
and rats (20). When SPECT is used in combination with
microfocal computed tomography (micro-CT) as an
anatomic reference, the radioactive signal can be located
in the body in 3 dimensions.
Although it has been shown that activated macrophages play a role in experimental OA, this disease is not
considered to be immune mediated (characterized by
inflammation and the presence of an abundance of
activated macrophages), as is the case in RA. It will
therefore be challenging to image a relatively small
number of activated macrophages in OA, especially in
small animal models of OA, since very high sensitivity
and resolution are required to produce high-quality
images with proper visualization.
The aim of this study was to test the feasibility of
imaging activated macrophages in experimental OA in
vivo by targeting the functional form of FR␤ using a
radiolabeled folic acid conjugate in combination with
animal SPECT/CT. Furthermore, macrophage involvement in experimental OA was studied using 2 different
rat models of OA to detect potential differences and
exclude model-specific macrophage activation. The 2
models were the mono-iodoacetate (MIA) model, which
is a fast-progressing biochemically induced model of
OA, and the anterior cruciate ligament transection
(ACLT) model, which is a more slowly progressing,
surgically induced biomechanical model of OA.
Radiosynthesis. The synthesis of diethylenetriaminepentaacetic acid (DTPA)–folate has been described previously
(21). Radiolabeling was performed according to the following
procedure. A stock solution of DTPA-folate (10⫺3 moles/liter)
was mixed with phosphate buffered saline (PBS; pH 6.5) and
incubated with 111InCl3 (Mallinckrodt-Tyco), resulting in a
specific activity of 16 MBq/␮g of DTPA-folate. The reaction
mixture was incubated for 1 hour at room temperature.
Quality control was carried out by high-performance liquid
chromatography (Waters HPLC System, with a tunable absorbance detector and a UniSpec MCA ␥-detector [Canberra]
and a Symmetry C-18 reverse-phase column [5 ␮m, 25 cm ⫻
4.6 mm; Waters]). Elution was performed with aqueous 0.05M
triethylammonium phosphate buffer (pH 2.25) and methanol
with a linear gradient to 80% methanol over 30 minutes and a
flow rate of 1.3 ml/minute. After quality control, an excess of
0.1 ␮l/MBq of free DTPA was added for complexation of
potentially free 111In. The radiofolate was diluted and administered to the animals in PBS (pH 6.5).
Scanning procedure. Twenty hours prior to scanning,
rats were injected with 60 MBq of 111In-DTPA-folate (15 MBq
in mice), diluted with sterile saline into a volume of 600 ␮l, into
the tail vein. During injection, the animals were under isoflurane anesthesia. All animal SPECT/CT scans were performed
with a 4-head multiplexing multipinhole dedicated small animal SPECT/CT camera (NanoSPECT/CT; Bioscan). Each
detector head was fitted with a tungsten-based collimator of
nine 1.5-mm diameter pinholes, with a field of view of 350 mm
in width and 28 mm in length. The NanoSPECT/CT is fitted
with a fast (200 ␮m) helical micro-CT system for anatomic
reference of the radioactivity. Before scanning, the animals
were placed in a dedicated, temperature-controlled animal
bed, and the hind limbs were stretched and fixed in a custommade Perspex frame to reduce noise from radioactivity accumulation in the kidneys and the intestinal tract in the field of
view. Energy peaks were set at 170 keV and 240 keV (⫾10%).
All animals were scanned from the midtibia to the pelvis.
Acquisition time was 67 minutes per scan for the rats (60
minutes for SPECT and 7 minutes for CT) and 35 minutes for
the mice (30 minutes for SPECT and 4 minutes for CT, with
1 minute for the transition between SPECT and CT.)
Animal SPECT/CT data reconstruction and analysis.
After scanning, SPECT data were reconstructed iteratively
with HiSPECT software (Scivis) at an isotropic voxel size of
600 ␮m. SPECT and CT data were automatically coregistered,
Figure 1. Timelines for the different experiments, indicating the time points at which experimental osteoarthritis (OA) or rheumatoid arthritis (RA)
was induced (by anterior cruciate ligament transection [ACLT] or mono-iodoacetate [MIA] injection for OA or by antigen-induced arthritis for RA),
the time points at which folate-enhanced imaging (single-photon–emission computed tomography/computed tomography [SPECT/CT]) was
performed, and the species and number of animals used. FR ⫽ folate receptor.
since both modalities used the same axis of rotation. All of the
acquired images were analyzed using InVivoScope processing
software (Bioscan). The fused images were reoriented to
achieve the same orientation for all images. A region of
interest (ROI) was determined for quantification of the radioactivity. The ROI was placed using only the micro-CT scan,
such that the researcher was not influenced by the radioactive
signal. The ROI was a cylindrical shape around the knee area;
the epiphysis of the distal femur was used as the proximal limit,
and the epiphysis of the proximal tibia was used as the distal
limit. The ROI contained a volume of 1 cm3 for the rats and 0.2
cm3 for the mice.
Animals and experimental animal models. All procedures were approved by the animal ethics committee of the
Erasmus Medical Center (EUR 1456). The rats (used in the
MIA, ACLT, naive, and macrophage depletion groups) were
all male Wistar rats (obtained from Harlan) that were 14 weeks
of age at the beginning of the experiment. The mice (used in
the antigen-induced arthritis [AIA] group) were 6-week-old
C57BL/6 mice (obtained from Harlan). Because a normal
rodent diet is highly enriched with folic acid, the animals were
fed a folate-deficient rodent diet starting 7 days prior to the
experiments. We chose to test 2 commonly used models of OA,
namely, the MIA model and the ACLT model. Figure 1 shows
the planning scheme for the experiments.
MIA-induced OA in rats. The MIA model is a biochemically induced OA model that initiates direct, fast-progressing
cartilage degeneration and is used primarily for studying
OA-related pain (22). MIA inhibits GAPDH activity, and
thereby diminishes chondrocyte metabolism, resulting in chon-
drocyte death and lesion formation (22). Within 6 weeks, the
model develops to complete OA, characterized by cartilage
cracks, cell death, bone erosion, and partial denudation.
One milligram of MIA (Sigma-Aldrich) was dissolved
in 50 ␮l of sterile saline. We injected the MIA solution into one
rat knee joint (n ⫽ 5 rats); the contralateral knee joint was
used as a control condition and was injected with an equal
amount of sterile saline. The knees of the rats were shaved and
disinfected with iodine. The injections were performed under
sterile conditions using a 27-gauge needle, while the rats were
under isoflurane anesthesia. These animals were imaged 1, 2,
4, and 8 weeks after intraarticular injection of MIA (Figure 1).
Ratios of radioactivity within each scan were determined using
the saline-injected knee as a reference (experimental OA side
versus control side). The results were analyzed using a paired
t-test, and P values less than 0.05 were considered significant.
ACLT model of OA in rats. The ACLT model is a
surgically induced, biomechanical model of OA, which progresses more slowly than the MIA model. It is characterized by
mild superficial fibrillation of the cartilage in the first weeks to
more pronounced fibrillation throughout the whole cartilage
surface and proteoglycan loss after 10 weeks (23).
After shaving the knee joint (n ⫽ 5 rats), the skin was
disinfected with iodine and an incision was made through the
patellar tendon in the longitudinal direction to obtain access to
the knee joint. The patellar tendon was spread using 2 surgical
hooks. Once the ACL was visible, it was transected using a von
Greaffe knife (Fine Science Tools) with the knee in a semiflexed position of ⬃30°. A positive anterior drawer test ensured complete transection of the ligament. Sham operations
were performed in the contralateral knee joint and consisted of
opening the joint space without transection of the ACL. The
procedures were all performed under sterile conditions. This
procedure was modified from the protocol described by Stoop
et al (23). These animals were imaged at 2, 4, 9, and 12 weeks
after transection of the ACL (Figure 1). The time points for
scanning in this group differed from those in the MIA group,
since experimental OA is known to progress much more slowly
in the ACLT model. Ratios of radioactivity within each scan
were determined, using the sham-operated knee as a reference
(experimental OA side versus control side). The results were
analyzed using a paired t-test, and P values less than 0.05 were
considered significant.
AIA model of RA (positive control) in mice. To ensure
that the radioactivity uptake indicated joint macrophage activity, an animal model in which activated macrophages are
known to be involved abundantly, namely, the AIA model of
RA (24), was used as a positive control condition.
Methylated bovine serum albumin (mBSA; 8 mg/ml)
(Sigma) was emulsified in an equal volume of Freund’s complete adjuvant (CFA) containing 1 mg/ml heat-killed Mycobacterium tuberculosis (strain H37Ra; Difco). On day ⫺8, mice
(n ⫽ 2) were immunized by intradermal injection of 100 ␮l
mBSA/CFA emulsion into the base of the tail. Thereafter, on
day 0, arthritis was induced by unilaterally injecting mice knee
joints with 60 ␮g of mBSA in 0.9% NaCl in 6 ␮l. The
contralateral side was injected with an equal amount of sterile
saline and was used as a control condition. These animals were
imaged 10 days after intraarticular injection of mBSA (Figure
1). The mean amount of radioactivity uptake in the mouse
knees with AIA was compared to the uptake in the contralateral saline-injected mouse knees within each scan. Student’s
paired t-test was used for analysis.
Determination of the systemic effect of experimental OA
on the contralateral control knee in naive animals. To test the
possible systemic effect of experimental OA of one knee on
macrophage activation in the contralateral knee, rats (n ⫽ 4)
that did not receive any treatment in either knee (naive)
were assessed. These animals were imaged once at the age of
14 weeks. The mean radioactivity uptake in the knees of these
rats was compared to the mean radioactivity uptake in the
saline-injected knees of the animals in the MIA group 2 weeks
after injection and in the sham-operated knees of the ACLT
animals 4 weeks after operation (2 weeks later than MIA
animals to rule out any effect of sham operation). Additionally,
the radioactivity of the knees of the naive animals was compared to that in MIA-injected rat knees 2 weeks after MIA
injection. Student’s unpaired t-tests were used for statistical
Macrophage depletion to determine macrophage specificity. To test the specificity of the folate tracer for macrophage
targeting, clodronate-laden liposomes were used for macrophage depletion. Clodronate-laden liposomes are selectively
ingested by phagocytosing cells. After uptake, the clodronate is
released, and subsequently, the cell undergoes apoptosis (25).
Seven days after injection of clodronate-laden liposomes,
optimal depletion of macrophages is established (26).
The animal model used was similar to the MIA model
described above. These animals (n ⫽ 4) were injected intraarticularly with 50 ␮l of clodronate-laden liposomes 1 week
after injection of MIA. The animals were imaged 2 weeks after
Figure 2. Frontal, sagittal, and coronal images of a rat with MIAinduced OA obtained using microfocal computed tomography (microCT), microfocal single-photon–emission computed tomography
(micro-SPECT), and micro-SPECT/CT. Micro-CT shows bone (high
attenuation), muscle (intermediate attenuation), fat (low attenuation),
and skin/fur (intermediate attenuation), as indicated by the arrows.
Boxed and encircled areas are the regions of interest. Micro-SPECT
images show radioactivity accumulation. Micro-SPECT/CT images
show radioactivity accumulation and its anatomic localization. The
contralateral untreated control knee served as control. See Figure 1 for
other definitions. Color figure can be viewed in the online issue,
which is available at
MIA injection and 1 week after injection of clodronate-laden
liposomes (Figure 1). The mean radioactivity uptake levels in
the MIA-injected/macrophage-depleted rat knees were compared to the contralateral saline-injected/non–macrophagedepleted control rat knees within each scan. Student’s paired
t-test was used for statistical analysis.
Folate receptor blocking to determine receptor specificity.
In order to determine whether the compound 111In-DTPAfolate was receptor specific, we performed one blocking experiment. Two animals from the MIA group were assessed 3
weeks after intraarticular injection of MIA for folate receptor–
specific radioactivity accumulation after blockade (Figure 1).
Fifteen minutes prior to intravenous injection of 111In-DTPAfolate, the animals were injected with a 500-fold excess of free
folic acid (Sigma-Aldrich) to obtain folate receptor blocking
on folate receptors in both the experimental OA knees and the
contralateral control knees. Because folate receptor blocking
by intravenous injection of folic acid affects the MIA-injected
knee as well as the contralateral control knee in the same
Figure 3. Representative small animal SPECT/CT images of macrophage activity at different time points in
3-dimensional (3-D) projections (frontal view) and 2-dimensional (2-D) transections (sagittal view). Left, Images of a
representative rat injected with MIA. Right, Images of a representative rat subjected to ACLT. One fixed threshold was
used for all images. The contralateral untreated control knee served as control. See Figure 1 for other definitions.
animal, the amount of radioactivity in each scanned knee
(MIA and saline injected) after blocking was compared to the
amount of radioactivity uptake 2 weeks and 4 weeks after
induction of OA in the same knee and not to the contralateral
knee at the same time point.
In vivo folate receptor–targeted imaging. The
high-resolution SPECT/CT scans allowed accurate localization of the distribution of radioactivity within each
hind limb. Activity was localized in the joint areas
(especially the affected OA knees) and the fatty tissue:
inguinal fat, popliteal fat, and anterior and posterior
subdermal fat (intermediate attenuation). Most likely,
this activation was located in the lymph nodes, since the
draining lymph nodes are embedded in this fatty tissue.
(Figure 2 shows an example of a scan obtained 4 weeks
after MIA injection.) Radioactivity was also located in
the gastrointestinal tract (results not shown) (15). Areas
of folate receptor–specific radioactivity accumulation,
such as the kidneys, choroid plexus, or salivary glands,
were not imaged. All of the animals had a mean activity
level between 250 and 850 kBq per voxel in the selected
ROI (the knee).
Imaging of activated macrophages in the MIA
and ACLT models of OA. In the MIA-treated rats, the
MIA-injected knees showed a mean increase in radioactivity accumulation 1, 2, and 4 weeks after injection
compared to the contralateral control knees (Figure 3,
left panels and Figure 4A). At the first scan, the mean ⫾
SD difference in radioactivity in the MIA-injected knees
of the rats was 24 ⫾ 12% compared to the contralateral
saline-injected knees (P ⬍ 0.005). Two weeks after
injection, the mean difference increased to 47 ⫾ 17%
(P ⬍ 0.001), and 4 weeks after injection, the difference
returned to a mean ⫾ SD of 24 ⫾ 13% (P ⬍ 0.01). Eight
weeks after the injection of MIA (i.e., at the last scan),
there was no longer any difference noted between the
MIA-injected and the contralateral saline-injected sides
(0 ⫾ 17%; P ⬎ 0.74) (Figure 4A).
The first scan of the ACLT animals was performed 2 weeks after induction of ACLT to overcome
the bias of macrophage activation due to wound healing
of the ACLT procedure. At all time points measured,
Figure 4. Percentage differences in mean radioactivity uptake in OA knees versus contralateral control knees at the
indicated time points in rats injected with MIA (A) and rats subjected to ACLT (B). Data are presented as box plots,
where the boxes represent the 95% confidence interval, the lines within the boxes represent the median, and the lines
outside the boxes represent the minimum and maximum values. ⴱ ⫽ P ⬍ 0.05. See Figure 1 for definitions.
the uptake of radioactivity in the affected knees was
higher than that in the sham-operated knees (Figure 3,
right panels, and Figure 4B). At 2 weeks, the uptake of
activity in the affected knees was 37 ⫾ 34% higher than
that in the unaffected sides (P ⬍ 0.05). The level of
activation remained high until the end of the experiment, with a mean ⫾ SD difference of 31 ⫾ 11% at 4
weeks (P ⬍ 0.05), 34 ⫾ 12% at 9 weeks (P ⬍ 0.005), and
Figure 5. Small animal single-photon–emission computed tomography/computed tomography images of the antigen-induced arthritis model of
rheumatoid arthritis (RA). Three-dimensional (3-D) projections (frontal view) of the contralateral control paw and methylated bovine serum
albumin (mBSA)–injected paw and 2-dimensional (2-D) transections (sagittal and coronal views) of the affected hind limb of a representative mouse
are shown. The images show uptake of radioactivity in the knees as well as in the lymph drainage of the knees (arrowheads) and the popliteal lymph
node (arrows). Color figure can be viewed in the online issue, which is available at
Figure 6. A, Mean radioactivity accumulation in the knees of untreated (naive) animals, saline-injected knees of animals in the MIA group 2 weeks
after injection, sham-operated knees of animals in the ACLT group 4 weeks after transection of the ACL, and MIA-injected knees of animals in
the MIA group 2 weeks after injection. P values were determined by Student’s unpaired t-test. B, Percentage difference in mean radioactivity uptake
in the macrophage-depleted MIA-injected knees of rats compared to their saline-injected non–macrophage-depleted knees. C, Percentage difference
in mean radioactivity uptake in MIA-injected and control rat knees with folate receptor blocking (performed 3 weeks after MIA injection) compared
to the same MIA-injected and control rat knees without folate receptor blocking scanned 2 weeks and 4 weeks after MIA injection (1 week before
and 1 week after blocking). Data are presented as box plots, where the boxes represent the 95% confidence interval, the lines within the boxes
represent the median, and the lines outside the boxes represent the minimum and maximum values. ⴱ ⫽ P ⬍ 0.05. See Figure 1 for definitions.
24 ⫾ 10% at 12 weeks (P ⬍ 0.01) (Figure 4B). Besides
joint (synovial) radioactivity uptake, a clear pattern of
radioactivity uptake was seen at sites outside the knee
region, probably the lymphoid system, in most animals in
both the MIA and the ACLT groups.
Imaging of mice in the AIA model of RA (positive
control) group. Ten days after induction of arthritis, the
mice with AIA had a mean ⫾ SD of 330 ⫾ 81% higher
radioactivity uptake on the affected side than on the
contralateral control side. As expected, this was far
higher than the lesions in the rats with OA (ACLT and
MIA groups). The mice with AIA also showed a clear
activation pattern from the knee joint up to the inguinal
regions through the fat, as well as clearly activated spots
in the popliteal fat. This might indicate macrophage
activation in the whole lymph draining passage of the
joint, including the popliteal lymph node as well as the
subdermal lymph draining passage to the inguinal lymph
nodes (Figure 5).
Systemic effects of experimental OA in naive
animals. We scanned 4 animals that were not affected by
experimental OA to assess the normal radioactivity
uptake pattern in the hind limbs. These hind limbs
showed a base level of radioactivity uptake in both knee
joints, as well as in regions that were probably the
draining lymph node areas. The radioactivity pattern
resembled the pattern of radioactivity seen in the contralateral control (saline-injected or sham-operated)
knees of all animals with experimental OA (Figure 6A).
There was no difference detected between the mean
radioactivity level of this control condition and the
contralateral saline-injected knees of the MIA-injected
rats 2 weeks after induction of OA (P ⬎ 0.35) or the
contralateral sham-operated knees of the animals with
ACLT 4 weeks after transection (P ⬎ 0.47) (Figure 6).
As expected, there was a difference detected between
the radioactivity uptake in the MIA-injected knees 2
weeks after injection and the radioactivity uptake in the
knees of the naive animals (P ⬍ 0.025).
Macrophage specificity of DTPA-folate determined by clodronate-induced depletion of macrophages.
To test cell-specific targeting of 111In-DTPA-folate,
macrophages were depleted from the OA knee joint of
rats by injection of clodronate-laden liposomes. The
liposomes were injected 1 week after MIA injection. The
contralateral side was injected with sterile saline and was
used as a control condition. The signal in the
macrophage-depleted knees of rats 2 weeks after MIA
injection decreased and was a mean ⫾ SD of 67 ⫾ 18%
less than in the contralateral, saline-injected, non–
macrophage-depleted sides (P ⬍ 0.009) (Figure 6B). At
this time point after MIA injection, rats showed a 47%
higher uptake in MIA-injected knees than in contralateral control knees. This confirms the macrophagespecific targeting of 111In-DTPA-folate.
Folate receptor specificity of DTPA-folate determined by folate receptor blocking. An excess amount of
folic acid injected intravenously 15 minutes prior to
injection of the 111In-DTPA-folate almost completely
blocked the radioactivity uptake seen in the knees of the
animals with MIA-induced OA, confirming the folate
receptor specificity of the compound. The mean decrease in uptake of radioactivity under blocking conditions was 93% compared to the mean radioactivity
uptake in the same knees 1 week prior to blocking as
well as 1 week after blocking (Figure 6C).
The findings of this study indicate that macrophage activation in experimental OA can be monitored
using dedicated animal SPECT/CT with a folate radiotracer for targeting the folate receptor that is expressed
on activated, but not resting, macrophages. This technique allows the localization of macrophage activity in a
disease model that is not known for abundant macrophage involvement.
As expected, we found increased macrophage
activity around the knee area in the animals. Extraarticular accumulation of radioactivity was also visible,
mostly in the subdermal and inguinal fat regions, but
because of the inconsistency of this signal and the small
number of animals, we cannot draw conclusions based
on these findings.
In the MIA model, the increased activation of
macrophages was reduced in all animals at the latest
time point in the experiment. The MIA model is known
to progress quickly, even in the first weeks (27,28). The
ACLT model is known to progress more slowly (23). The
fast progression of the MIA model in the first weeks
matches the relatively high uptake of radioactivity, and
the progressive state of OA at 6–8 weeks might be the
reason that fewer macromolecular complexes end up in
the synovial fluid, diminishing the macrophage response
and corresponding uptake of radiofolate tracer. Care
was taken to exclude model-specific influences regarding
macrophage activation by sham operation on and injection into the contralateral hind limb. However, we
cannot exclude the possibility that naive triggers, such as
pain, direct activation by MIA, and wound healing,
resulted in macrophage activation. Although these animal models of OA cannot be compared with human OA
directly, these results are consistent with previous findings in human OA, in which increased mononuclear cell
infiltration and overexpression of mediators of inflammation are much more pronounced in early than in late
disease (29,30).
The positive control condition (RA model)
clearly proved that it is possible to trace inflammation
using a folate-based imaging agent, as previously shown
(11,31). The presence of activated macrophages in the
popliteal lymph node and other efferent lymph nodes
has never been imaged before using folate-based imaging. The RA model also showed that the activity in the
popliteal and inguinal fat region is most likely an enhancement of the lymphoid system, since the whole
draining pathway could clearly be visualized (Figure 5).
The relatively small increase in macrophage activation in the OA knees of the animals compared to the
contralateral knees and the knees of naive animals
suggests that the amount of activation in OA models is
only slightly enhanced compared to the normal state,
even in the faster progressing MIA models. However, we
cannot exclude the possibility that species-dependent
differences exist between mice and rats.
The cell specificity of folate-based imaging has
been thoroughly investigated in multiple previous studies, using flow cytometry, confocal microscopy, and in
vivo macrophage elimination using clodronate-laden
liposomes as determinants for cell phenotyping (11,14,
32–34). In the present study, an almost total absence of
radioactivity in the knee joint after elimination of macrophages using clodronate-laden liposomes was observed.
This indicates that macrophages are indeed the target of
the radioactive folate. Furthermore, images obtained
after receptor blockade with free folic acid showed that
highly receptor-specific targeting of the folate receptor
by 111 In-DTPA-folate occurred. Several 99m Tcradiofolates have already been developed and studied,
and the radiofolate EC20 has been used in clinical
trials (35).
Other options for imaging macrophages in vivo
include the use of ultrasmall paramagnetic iron oxide
particles in MRI (36) or CD11b/CD14 and anti-CD14⫹
antibody–labeled tracers (37). However, these tech-
niques are by far not as sensitive (MRI) and specific
(CD14⫹) for activated macrophage targeting.
OA is a heterogeneous disease. Its etiology and
progression involve biomechanics, hormones, and a diverse spectrum of cytokines, enzymes, and growth factors that can all affect joint tissue. Cartilage disruption
leads to leakage of large molecular complexes, such as
collagens and proteoglycans, into the synovial fluid.
Chondrocytes generate a repair response characterized
by increased syntheses of inferior types of collagen (e.g.,
type I and type IX) and proteoglycans. In OA, catabolic
processes appear to increase faster in relation to anabolic processes in the cartilage. This results in the
presence of various macromolecules in the joint fluid
that will end up in the synovial tissue (38). Furthermore,
it is clear that multiple signaling molecules produced
either by chondrocytes or by neighboring fibroblasts or
macrophages can also cause an activity response, as was
recently shown by Blom and colleagues (39). Although
we investigated the possibility of imaging activated macrophages in experimental OA through FR␤ and demonstrated its presence in experimental OA, this might also
create the opportunity to test the effect of specific folate
antagonists such as BCG 495 in OA (13).
This study clearly demonstrates that in vivo imaging and quantification of macrophage activity by radiolabeled folate conjugate in small animal models of
OA is feasible. The technique allows monitoring of the
disease process over time, in which late-stage disease
showed less macrophage activation than early stages.
Although the technique was tested on small animal
models of OA, it can easily be translated to humans with
OA, thereby possibly providing a new imaging biomarker for the activity and potential progression of the
We thank Dr. N. van Rooijen (Department of Molecular Cell Biology, Vrije Universiteit, VUMC, Amsterdam, The
Netherlands) for kindly providing the clodronate-laden liposomes.
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Piscaer had full access to all of
the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Study conception and design. Piscaer, Müller, Mindt, Verhaar, Krenning, Schibli, De Jong, Weinans.
Acquisition of data. Piscaer, Müller, Lubberts, Verhaar, Schibli.
Analysis and interpretation of data. Piscaer, Müller, Verhaar, Krenning, Schibli, Weinans.
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using, tomography, osteoarthritis, folate, experimentov, macrophage, tomographycomputed, photonemission, animals, single, imagine, targeted, computer, activated
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