Monocyte Scintigraphy in Rheumatoid Arthritis: The
Dynamics of Monocyte Migration in Immune-Mediated
Rogier M. Thurlings1, Carla A. Wijbrandts1, Roelof J. Bennink2, Serge E. Dohmen3, Carlijn Voermans3,
Diana Wouters4, Elena S. Izmailova5, Danielle M. Gerlag1, Berthe L. F. van Eck-Smit2, Paul P. Tak1*
1Department of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Amsterdam, Noord Holland, The Netherlands,
2Department of Nuclear Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, Noord Holland, The Netherlands, 3Landsteiner Laboratory,
Department of Experimental Immunohematology, Sanquin Research, Amsterdam, Noord Holland, The Netherlands, 4Department of Immunopathology, Sanquin
Research, Amsterdam, Noord Holland, The Netherlands, 5Millennium Pharmaceuticals, Inc, Department of Research and Development, Cambridge, Massachusetts, United
States of America
Background: Macrophages are principal drivers of synovial inflammation in rheumatoid arthritis (RA), a prototype immune-
mediated inflammatory disease. Conceivably, synovial macrophages are continuously replaced by circulating monocytes in
RA. Animal studies from the 1960s suggested that macrophage replacement by monocytes is a slow process in chronic
inflammatory lesions. Translation of these data into the human condition has been hampered by the lack of available
techniques to analyze monocyte migration in man.
Methods/Principal Findings: We developed a technique that enabled us to analyze the migration of labelled autologous
monocytes in RA patients using single photon emission computer tomography (SPECT). We isolated CD14+ monocytes by
CliniMACS in 8 patients and labeled these with technetium-99m (99mTc-HMPAO). Monocytes were re-infused into the same
patient. Using SPECT we calculated that a very small but specific fraction of 3.461023(0.9525.161023) % of re-infused
monocytes migrated to the inflamed joints, being detectable within one hour after re-infusion.
Conclusions/Significance: The results indicate monocytes migrate continuously into the inflamed synovial tissue of RA
patients, but at a slow macrophage-replacement rate. This suggests that the rapid decrease in synovial macrophages that
occurs after antirheumatic treatment might rather be explained by an alteration in macrophage retention than in monocyte
influx and that RA might be particularly sensitive to treatments targeting inflammatory cell retention.
Citation: Thurlings RM, Wijbrandts CA, Bennink RJ, Dohmen SE, Voermans C, et al. (2009) Monocyte Scintigraphy in Rheumatoid Arthritis: The Dynamics of
Monocyte Migration in Immune-Mediated Inflammatory Disease. PLoS ONE 4(11): e7865. doi:10.1371/journal.pone.0007865
Editor: Andrew Boswell, Genentech, United States of America
Received April 22, 2009; Accepted July 31, 2009; Published November 17, 2009
Copyright: ? 2009 Thurlings et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by Millenium Pharmaceuticals, Inc. This company participated in the design of the study, performed the pre-clinical in vitro
experiments concerning monocyte activation, monitored the clinical study and approved the manuscript. Millenium Pharmaceuticals, Inc. participated in the
analysis and interpretation of the clinical study and in the preparation of the manuscript. All authors were involved in the study design, had access to all data,
were responsible for the decision to publish, and prepared the manuscript.
Competing Interests: Millennium Pharmaceuticals Inc. (Cambridge, Massachusetts) was involved in the study design and supported the study. Millennium does
not hold any IP (patent) applications with claims relevant to the materials or assays described in the manuscript. Millennium might consider using the monocyte
scan described in the manuscript for the evaluation of novel antirheumatic drugs in the future.
* E-mail: firstname.lastname@example.org
Macrophages in the inflamed synovial tissue of rheumatoid
arthritis (RA) patients play a central role in the sustenance of
synovial inflammation and promotion of tissue destruction [1–3].
Conceivably they are continuously replaced by circulating
monocytes . The dynamics of this replacement is a matter of
controversy. Data on the effects of anti-rheumatic treatments
suggest this might be a highly dynamic process [5–11], while
animal studies from the 1960s suggested it might occur at a slow
Newly developed imaging techniques, such as Single Photon
emission Computed Tomography (SPECT), Positron Emission
Tomography (PET) and more recently bioluminescence and
fluorescence reflectance imaging, offer the possibility to portray
the in vivo dynamics of cell migration in patients . The
application of these imaging modalities to analyze the behavior of
monocytes is hampered by the relative scarcity of these cells in the
peripheral blood and the technical difficulties of specific cell
isolation at the GMP level and efficient labeling to result in an
adequate detection signal. These problems might be addressed by
the combination of scintigraphic imaging with sophisticated cell
isolation procedures, such as immunomagnetic cell selection .
We recently developed a procedure using a combination of
immunomagnetic cell selection with CD14 coated beads and an
improved labeling procedure with technetium-99m (99mTc)-
hexamethylpropylene-amino-oxime (HMPAO) and SPECT to
visualize the migratory behavior of autologous monocytes [18,19].
PLoS ONE | www.plosone.org1 November 2009 | Volume 4 | Issue 11 | e7865
We applied this method in patients with active RA to test the
hypothesis that synovial inflammation is maintained by a
continuous influx of monocytes into the synovial compartment
and to analyze the dynamics of such influx.
Eight RA patients (4 male and 4 female) were included into the
study. The median age of the patients was 52 years (range 39 to 59
years) and the mean disease duration was 19 (range 10–38) years.
Erosions were present in all patients. Two patients had nodular
disease. Four patients were seropositive for IgM rheumatoid
factor. The mean (6SD) disease activity score evaluated in 28
joints (DAS28) at screening was 5.860.8. All patients were treated
with stable dosages of methotrexate.
Applying immunomagnetic cell selection with CD14 labeled
beads, on average 19.96106(10.4236.96106) monocytes were
isolated, with a mean recovery of 40.8% (24–69%) CD14 positive
cells. This resulted in a cell suspension with a purity of 90.4% (79–
96%) CD14 positive cells as determined by FACS analysis.
Labeling with99mTc-HMPAO resulted in a mean radioactivity of
211 (43–393) MBq. Having shown that CD62L expression on
monocytes did not change after the bead isolation procedure and
migratory capacity in vitro (unpublished observations), we decided
to re-infuse labeled monocytes in RA patients. Re-infusion was
well tolerated in all patients. No signs of increased complement
activation could be demonstrated one hour after re-infusion of
radioactively labeled monocytes: C3b/c (mean6SD): 26.4613.5
99mTc-HMPAO labeling did not affect the monocyte
and C4b/c 8.361.5 before treatment versus 26.0612.3 and
16.2610.0 1 hour after re-infusion, respectively).
Migration of labeled monocytes was visualized using scintigraphy.
by redistribution in liver, spleen and bone marrow (Figure 1),
following the pattern of labeled leukocytes . As expected, renal
activity with visualization of the urinary bladder was seen in all
patients. Furthermore, physiological bowel uptake could be detected
from one hour post infusion. Significant uptake of radioactivity in
stomach and/or thyroid was not observed. In 2 patients whole-body
imaging was feasible up to 20 hours post infusion.
Small but distinct uptake was found in the joints of all patients,
with a mean of 9 (range 1–25) positive joints (Figures 1 and 2).
There was an increased signal in di-arthrodial joints in all patients
at all time points, with a maximal signal at one hour post re-
infusion (Figure 3). We calculated that a median of 4827
(interquartile range [IQR] 2094–8370) labeled monocytes migrat-
ed into the biopsied joints that were analyzed in more detail,
representing 3.461023(0.9525.161023) % of re-infused mono-
cytes. The results were confirmed after 2 weeks, when the scans
were repeated: there was no change in clinical parameters, the
number of joints with increased signal on the scintigraphic images,
and joint signal intensity. Using these numbers, an estimate was
made about the extent of monocyte influx into the biopsied joints.
The median monocyte concentration in peripheral blood was
(1.622.36109) at the time of blood withdrawal.
Assuming an average blood volume of 5 liters, a median of
323.000 (272.000–1.150.000) monocytes entered the biopsied
joints (3.461023% of total circulating monocytes).
Figure 1. Scintigraphic images of labeled autologous monocytes in a patient with rheumatoid arthritis. Anterior and posterior (A,B)
whole body images of a patient 2 hours after infusion of monocytes labeled with 283 MBq99mTc-HMPAO. Transient pulmonary accumulation occurs,
with an equivalently increased uptake in liver, spleen and bone marrow (A,B). A reference source is placed just below the knees. Panel C and D show
the same images but with masking of the pulmonary, bladder and source signal. Increased articular uptake is observed in di-arthrodial joints as the
shoulders, elbows, knees and small hand joints.
Monocyte Scintigraphy in RA
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Since macrophages are the dominant synovial inflammatory cell
population and the extent of their tissue infiltration correlates with
disease activity [1–3,20], we compared scintigraphic signal intensity
with synovial macrophage infiltration and disease activity param-
eters. Synovial macrophage infiltration was assessed by immuno-
histochemical staining of arthroscopic biopsies from selected joints.
These biopsy samples had been obtained one day after the second
scintigraphy. Six out of 8 synovial biopsies passed synovial tissue
quality control. The number of CD163 positive macrophages in the
synovium correlated significantly with the number of scintigraphy
positive joints one, two and three hours after re-infusion (r=0.89,
P=0.019; r=0.89, P=0.019; r=0.94, P,0.01, respectively).
Furthermore, there was a significant correlation between the
number of CD163 positive macrophages and the percentage of
monocytes shown by scintigraphy two hours after re-infusion
(r=0.89, P=0.019). The other immunohistologic markers were not
significantly correlated with scintigraphic data (data not shown).
Subsequently, the relationship between scintigraphic signal and
disease activity parameters was analyzed. Of interest, the swollen
in the biopsied joints selected for detailed quantification on the
images that were taken one and two hours after re-infusion (day 1:
r=0.97 and r=0.73, respectively; P,0.01, day 14: r=0.78 and
r=0.90; P,0.01). There was also a positive correlation between the
number of swollen joints and the number of positive joints shown by
scintigraphic scans at day 14 (r=0.81, r=0.76 and r=0.81; all
P,0.01, at respectively 1, 2 and 3 hours after infusion).
In the present study we used a recently developed procedure,
that visualizes the migratory behavior of monocytes [18,19], to test
the hypothesis that synovial inflammation in RA is maintained by
influx of monocytes into the synovial compartment. The results
suggest that while there is indeed a continuous influx of circulating
monocytes into the synovial compartment, their numbers are
small, indicating that only a relatively small fraction of synovial
macrophages is replaced per day.
We found that a median of 3.461023(0.9525.161023) % of
labelled monocytes entered the synovial compartment after re-
Figure 2. Scintigraphic detail images of hands and feet of labeled autologous monocytes in a patient with rheumatoid arthritis.
Scintigraphic detail images of hands in palmar position (A–C) and feet in plantar position (D,E) and anterior position (F) of RA patients 2 h after
infusion of monocytes labeled with 99mTc-HMPAO. Images of the hands show increased uptake in the wrists, MCP and IP joints (A–C). Images of the
feet show increased uptake of the ankle, tarsus, MTP and IP joints (D–F).
Monocyte Scintigraphy in RA
PLoS ONE | www.plosone.org3November 2009 | Volume 4 | Issue 11 | e7865
infusion. Since we found no indication that the monocytes were
activated by the labelling or isolation procedures, this suggests that
similar percentages of unlabeled circulating monocytes enter the
synovial compartment, being a median of 323.000 cells. Since the
number of synovial macrophages in large inflamed joints exceeds
such numbers considerably, these data indicate that in RA patients
the rate of synovial macrophage renewal by circulating monocytes
It was recently shown that a range of anti-rheumatic treatments
induces a significant decrease in synovial macrophages associated
with clinical improvement within hours to weeks after initiation of
therapy [5,6]. It was hypothesized that these treatments ultimately
affect migration of monocytes and/or retention of macrophages
. In studies on infliximab, an effective biological treatment for
RA which blocks TNFa , a marked decrease in synovial
macrophage numbers occurred already 24 hours after initiation of
treatment, which could not be explained by induction of apoptosis
[8–11]. The results of the current study suggest that this effect may
mostly explained by an effect of TNFa blockade on macrophage
retention. Of interest, a decrease in VCAM-1 and ICAM was
found shortly after infliximab treatment [8,10]. While these are
important molecules involved in adhesion of monocytes to the
vascular endothelium, they are in RA patients also abundantly
expressed by cells in the intimal lining layer and the synovial
sublining, where they play a pivotal role in inflammatory cell
retention and survival .
Thus, the data indicate that in line with animal models on
chronic inflammatory lesions, macrophage renewal by monocytes
is slow in chronic synovitis [12–15]. Together with the previous
studies showing a rapid decrease in cellularity after successful
antirheumatic therapy (even in the absence of apoptosis), these
data support the notion that therapeutic strategies aimed at
interfering with retention of inflammatory cells at the site of
inflammation might be capable of inducing clinical improvement
in immune-mediated inflammatory disease. Accordingly, recent
studies have shown benefit of interfering with adhesion molecules:
anti-function associated antigen (LFA)-1 antibody (efalizumab)
treatment in psoriasis and anti-a4b1-integrin (VLA-4) antibody
(natalizumab) treatment in multiple sclerosis [24,25].
In conclusion, we developed monocyte scintigraphy, which allowed
us to demonstrate the dynamic influx of monocytes into the synovial
compartment of RA patients. This approach provides insight into the
pathogenesis of this immune-mediated inflammatory disease and
supports the notion that blocking only the influx of inflammatory cells
may be insufficient to induce clinical improvement.
Materials and Methods
Patients fulfilling the American College of Rheumatology (ACR)
1987 revised classification criteria for RA  were included into
the study. All patients had active RA, as defined by a disease
activity score evaluated in 28 joints (DAS28) .3.2 . Patients
were on stable disease-modifying antirheumatic drug (DMARD)
treatment at inclusion.
Approval was granted by the Medical Ethics Committee of the
Academic Medical Center/University of Amsterdam (AMC).
Each patient gave written informed consent prior to participation.
Isolation of Monocytes
Hundred milliliters of peripheral blood was taken from each
patient. CD14+ monocytes were isolated using a positive selection
procedure with magnetic-activated cell sorting according to the
manufacturer’s protocol (MACSHMiltenyi Biotech, Bergisch Glad-
bach, Germany). After selection, the percentage of CD14, CD3,
and CD66 positive cells was determined by fluorescence-activated
cell sorting (FACS) analysis. The CD14+ enriched cells were
resuspended in 10 ml buffer containing 0.9% (w/v) NaCl, 20% (w/
v) human serum albumin (Sanquin Blood Supply Foundation
division of Plasma Products, Amsterdam, the Netherlands) and
3.8% (w/v) TNC (NVI, Bilthoven, the Netherlands) for labeling.
Exametazime (CeretecTM, RVG16226) was supplied as a ready-
for-labeling kit (GE Healthcare B.V., Amersham, Cygne Centre,
Eindhoven, the Netherlands).99mTc-pertechnetate was obtained
4329, Tyco Healthcare, Mallinckrodt Medical, Petten, the
Netherlands) and was eluted in accordance with the instructions
of the manufacturer. Radiochemical purity control (RPC) assays
were done by means of chromatography on ITLC-SG strips, using
a mobile phase of 0.9% sodium chloride (NaCl) . Radiolabel-
ing of cells was performed as described earlier . Briefly, the
cells were centrifuged and freshly prepared
very high specific activity in a low volume was added to the
monocyte cell pellet. After incubation the excess of
HMPAO was diluted and subsequently removed from the cell
pellet after centrifugation. The labeled monocytes were resus-
pended in 0.9% NaCl and re-infused into the same patient.
99Mo-carrying UltratechnekowH FM generator (DRN
An average of 206106monocytes labeled with 200 MBq99mTc-
HMPAO was injected intravenously within 15 minutes after
radiolabeling. Whole body imaging was performed at 15 minutes
and 1, 2, 3, and 20 hours post infusion using a dual head
gammacamera (140 keV, window 15%, 25661024 matrix,
10 cm/min) fitted with low energy all purpose collimators
(Siemens Ecam). Detail images of the hands (palmar) and feet
(plantar) were acquired in a 2566256 matrix for 5 minutes. This
procedure was repeated two weeks after the baseline scintigraphy.
Figure 3. Percentage of re-infused monocytes in a joint in time.
Detailed signal intensity calculation of percentage of re-infused
monocytes in a selected joint of the individual patients in time after
infusion. A stable presence of monocytes in the joints is visible.
Monocyte Scintigraphy in RA
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The scintigraphic scans were analyzed for signal intensity in
joints and other tissues. The number of positive joints and the
exact signal intensity of the biopsied joint was selected for more
detailed quantification. The signal intensity was calculated in
counts per region of interest, subtracting the background signal
from the joint signal. A correction was made for the number of re-
infused monocytes and the injected dose, using a standard dose
source, leading to a deduction of the percentage of re-infused
monocytes per ROI.
In Vitro Assays to Determine the Influence of Isolation
and Labeling on Monocyte Migratory Function and
Monocytes were isolated from whole blood of subjects and
migratory function was assessed by in vitro chemotaxis assay
comparing radioactively labeled and non-labeled cells. Briefly,
chemotaxis assay was done using 24-well chemotaxis plates
(Corning Costar, Corning, NY) with inserts containing pre-grown
ECV304 cells. Purified monocytes were re-suspended in 600 ul of
pre-warmed RPMI medium containing 3% fetal calf serum
(GIBCO, BRL) and added to the upper chamber of the transwell
plate. Chemotaxis was performed for 2 hours at 37uC against
various concentrations of recombinant MCP-1 (R&D Systems,
Minneapolis, MN). Cell migration rate was quantified by flow
cytometry for non-labeled monocytes and scintillation gamma
counter for labeled monocytes. Comparison of migrating cell
percentage for non-labeled and Tc- HMPAO labeled monocytes
did not demonstrate an impairment of monocyte migratory
capacity in the in vitro chemotaxis assay (data not shown).
Monocyte activation was tested by assessing CD62L expression
after the bead isolation procedure using CD62L-PE conjugated
antibody (BD Biosciences). Antibody concentrations were used
according the manufacturer’s protocol. Antibody staining was
performed using 50 ml of whole blood. Blood cells were incubated
with antibodies for 15 min and washed twice with PBS containing
1% bovine serum albumin (Sigma-Aldrich, MO). Red cells were
lysed by washing cells twice with BD FACSTMlysis solution (BD
Biosciences, CA). Purified monocytes were resuspended in PBS
containing 1% bovine serum albumin (Sigma-Aldrich, MO),
incubated with antibody and fixed with FACSTMlysis solution
(BD Biosciences, CA). Samples were analyzed by flow cytometry
using a FACS Calibur (Becton Dickinson, NJ). Frequency of
CD62L expression on monocytes did not change after the bead
isolation procedure (data not shown).
In Vivo Assessment of Complement Activation after
To exclude the possibility that re-infusion of labeled monocytes
induced complement activation, complement activation products
were measured in the serum before and one hour after re-infusion.
Activation of C3 (C3b/c) and C4 (C4b/c) was assessed with an
ELISA as described before in detail . In brief, monoclonal
antibodies recognizing neo-epitopes on activated C3 and C4 were
used as capturing antibodies. Biotinylated polyclonal rabbit anti-
human C3 and polyclonal sheep anti-human C4 antibodies were
used as detecting antibodies.
Arthroscopy and Synovial Biopsy
The day after the second set of scans, all patients underwent a
mini-arthroscopy under local anesthesia from an actively inflamed
knee, ankle or wrist, to obtain synovial tissue samples . Biopsies
were taken with a 2.3-mm grasping forceps (Storz, Tuttlingen,
Germany) from 6 or more sites within the joint to minimize sampling
error. The tissue samples were snap frozen en bloc in Tissue Tek
OCT (Miles, Elkhart, IN) after collection. Frozen blocks were stored
in liquid nitrogen until sectioning. Sections of 5 mm were cut using a
cryostat and mounted on Star Frost adhesive glass slides (Knittelgla ¨-
ser, Braunschweig, Germany). Sealed slides were stored at 280uC
until immunohistochemical staining was performed.
Synovial tissue sections were stained using the following
monoclonal antibodies to analyze the cell infiltrate: anti-CD55
(67:Serotec, Oxford, UK) to detect fibroblast-like synoviocytes
(FLS), anti-CD68 (EBM11: DAKO, Glostrup, Denmark) to detect
macrophages and anti-CD3 (SK7, Becton Dickinson, San Jose, CA)
for T-cells as described previously . The scavenger receptor
CD163 (Ber-MAC3; DAKO) was stained to detect alternatively
activated tissue macrophages. Staining of cellular markers was
performed using a three-step immunoperoxidase method . For
control sections the primary antibody was omitted or irrelevant
immunoglobulins were applied. Tissue quality was assessed by
analyzing the presence of an intimal lining layer.
Digital Image Analysis
All sections were analyzed at random by trained technicians
who were blinded for clinical and scintigraphic data. The analysis
was done by computer-assisted image analysis as previously
described in detail (323). In short, images were acquired and
analyzed using a Syndia algorithm on a Qwin-based analysis
system (Leica, Cambridge, UK). For all markers 18 high-power
fields were analyzed. Positive staining of cellular markers was
expressed as positive cells per mm2(counts/mm2). CD68+
macrophages were analyzed separately for the intimal lining layer
and the synovial sublining.
Associations between joint signal parameters and swollen joint
count, tender joint count, DAS28, ESR, CRP and immunohisto-
chemical markers were expressed by Spearman’s correlation
coefficients. The changes in joint signal intensity and clinical
parameters after two weeks were tested with the Wilcoxon signed
ranks test for paired non-parametric data.
We thank the clinical team of the Department of Clinical Immunology and
Rheumatology, and Nuclear Medicine and the staff of the Laboratory for
Stem Cell Transplantation at Sanquin Research for their assistance.
Conceived and designed the experiments: RMT CAW RJB SED CV DW
ESI DMG BLFvES PPT. Performed the experiments: RMT CAW RJB
SED CV DW. Analyzed the data: RMT CAW RJB SED DW ESI PPT.
Contributed reagents/materials/analysis tools: RJB SED CV DW ESI
BLFvES PPT. Wrote the paper: RMT CAW RJB SED CV DW ESI
DMG BLFvES PPT.
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