ARTHRITIS & RHEUMATISM
Vol. 65, No. 3, March 2013, pp 599–607
DOI 10.1002/art.37787
© 2013, American College of Rheumatology
Abatacept (CTLA-4Ig) Treatment Reduces the
Migratory Capacity of Monocytes in
Patients With Rheumatoid Arthritis
M. Bonelli, E. Ferner, L. Göschl, S. Blüml, A. Hladik, T. Karonitsch, H. P. Kiener, R. Byrne,
B. Niederreiter, C. W. Steiner, E. Rath, M. Bergmann, J. S. Smolen, and C. Scheinecker
treated with CTLA-4Ig. The expression of several adhesion molecules was significantly diminished. In addition, monocytes displayed a significant reduction in
their endothelial adhesion and transendothelial migratory capacity upon treatment with CTLA-4Ig. Likewise,
isolated monocytes from healthy controls revealed a
significant reduction in their migratory and spreading
activity after preincubation with CTLA-4Ig or antiCD80 and anti-CD86 antibodies.
Conclusion. We describe direct effects of CTLA4Ig therapy on phenotype and functional characteristics
of monocytes in RA patients that might interfere with
the migration of monocytes to the synovial tissue. This
additional mechanism of CTLA-4Ig might contribute to
the beneficial effects of CTLA-4Ig treatment in RA
patients.
Objective. The binding of abatacept (CTLA-4Ig)
to the B7 ligands CD80 and CD86 prevents the engagement of CD28 on T cells and thereby prevents effector T
cell activation. In addition, a direct effect of CTLA-4Ig
on antigen-presenting cells (APCs) could contribute to
the therapeutic effect. To further elucidate the mechanism of CTLA-4Ig, we performed phenotype and functional analyses of APCs in patients with rheumatoid
arthritis (RA) before and after the initiation of CTLA4Ig therapy.
Methods. Peripheral blood mononuclear cells
were analyzed before and at 2 and 4 weeks after the
initiation of CTLA-4Ig therapy. Proportions of APCs
were determined by flow cytometry. CD14ⴙ monocytes
were further analyzed for the expression of costimulatory and adhesion molecules and for their transendothelial migratory capacity in vitro. In addition, CD14ⴙ
monocytes from healthy controls were analyzed for their
migratory and spreading capacity.
Results. Proportions and absolute numbers of
monocytes were significantly increased in RA patients
Rheumatoid arthritis (RA) is a chronic debilitating systemic autoimmune disease characterized by inflammation and destruction of the joints. While activation of fibroblast-like synoviocytes, macrophages,
dendritic cells (DCs), and B cells, including production
of various cytokines and autoantibodies, is essential in
RA, activated T cells play a central role in upstream
parts of the inflammatory cascade (1,2). The growing
understanding of the pathophysiologic mechanisms underlying RA at the molecular and cellular levels has
triggered an avalanche of new and highly efficient
biologic therapies targeting proinflammatory cytokines
or their receptors (e.g., infliximab, etanercept, adalimumab, tocilizumab, anakinra) as well as cell surface or
costimulatory molecules (e.g., rituximab, abatacept).
Abatacept is a soluble recombinant human
CTLA-4Ig fusion protein comprising the extracellular
domain of human CTLA-4 and a fragment of the Fc
domain of human IgG1. It has been proposed that
Supported by the Ö sterreichische Nationalbank—
Jubiläumsfond (grant 12754), the European Union Seventh Framework Programme (project Masterswitch; HEALTH-F2-2008-223404),
and the Innovative Medicines Initiative Joint Undertaking under grant
agreement 115142 (BTCure).
M. Bonelli, MD, E. Ferner, MD, L. Göschl, MD, S. Blüml,
MD, A. Hladik, T. Karonitsch, MD, H. P. Kiener, MD, R. Byrne, PhD,
B. Niederreiter, C. W. Steiner, E. Rath, MD, M. Bergmann, MD, J. S.
Smolen, MD, C. Scheinecker, MD: Medical University of Vienna,
Vienna, Austria.
Dr. Smolen has received consulting fees, speaking fees, and/or
honoraria from Bristol-Myers Squibb (less than $10,000) and a research grant from Bristol-Myers Squibb.
Address correspondence to C. Scheinecker, MD, Division of
Rheumatology, Internal Medicine III, General Hospital of Vienna,
Medical University of Vienna, Waehringer Guertel 18-20, A-1090
Vienna, Austria. E-mail: [email protected].
Submitted for publication February 2, 2012; accepted in
revised form November 1, 2012.
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Table 1. Demographic and clinical characteristics of the patients with rheumatoid arthritis at baseline
and during CTLA-4Ig treatment*
Characteristic
Baseline
(week 0)
Week 2
Week 4
Age, mean years
Prednisolone, %
Prednisolone, mg/day
MTX, %
Leflunomide, %
CRP, mg/dl
SDAI score (range 0–86)
CDAI score (range 0–17)
DAS28-CRP
56
58
5.3 ⫾ 6.8
58
25
1 ⫾ 1.2 (0.02–4.5)
25 ⫾ 8.5 (10.1–36.4)
26 ⫾ 8.9 (9.7–40.4)
3.7 ⫾ 0.6 (2.4–4.5)
–
–
–
–
–
1.4 ⫾ 1.5 (0.01–5.4)
24 ⫾ 8.9 (9.4–36.9)
24 ⫾ 8.3 (7.7–35.9)
3.7 ⫾ 0.6 (2.7–4.7)
–
–
–
–
–
4.6 ⫾ 0.7 (0.01–7.5)
24 ⫾ 7.2 (13.5–35)
21 ⫾ 7.5 (9.2–35.4)
3.8 ⫾ 0.6 (2.9–4.8)
* Except where indicated otherwise, values are the mean ⫾ SD (range). MTX ⫽ methotrexate; CRP ⫽
C-reactive protein; SDAI ⫽ Simplified Disease Activity Index; CDAI ⫽ Clinical Disease Activity Index;
DAS28-CRP ⫽ Disease Activity Score in 28 joints using the CRP level.
CTLA-4Ig decreases T cell responses by competing for
B7 ligand (CD80/CD86) access to CD28 and limiting
the CD28 signaling that is required for T cell activation
(3). In addition, however, CTLA-4Ig therapy might exert
effects beyond T cells. For example, reverse signaling to
antigen-presenting cells (APCs) upon binding of CTLA4Ig to CD80/CD86 has been described that might interfere with APC activation and function (4,5). Whether
such signaling or other CTLA-4Ig–mediated effects contribute to a beneficial or adverse outcome, in particular
in the therapeutic setting of RA patients, is not entirely
clear so far. We therefore assessed the effects of CTLA4Ig treatment on APCs in RA patients, and indeed we
observed a profound effect of CTLA-4Ig on the phenotype and function of monocytes.
PATIENTS AND METHODS
Patients and healthy volunteers. Twelve patients with
RA according to the 1987 revised criteria of the American
College of Rheumatology (ACR) (6) who were eligible for
CTLA-4Ig therapy were consecutively selected from our outpatient clinic. All patients were treated with 10 mg/kg CTLA4Ig at baseline, week 2, and week 4. No premedication with
glucocorticoids or antihistamines was given before the patients
received CTLA-4Ig infusions. Heparinized whole blood samples were obtained at baseline and at weeks 2 and 4 during
treatment with CTLA-4Ig.
In addition, heparinized whole blood samples were
obtained from healthy volunteers. We obtained Ethics Committee approval for this study as well as informed consent from
the patients. Detailed demographic and clinical characteristics
of the RA patients are provided in Table 1. We assessed
clinical disease activity with the Simplified Disease Activity
Index (SDAI) and the Clinical Disease Activity Index (CDAI)
(7) as well as with the Disease Activity Score in 28 joints using
the C-reactive protein level (DAS28-CRP) (8).
Antibodies. Monoclonal antibodies (mAb) targeting a
variety of molecules were used unlabeled or as fluorescein
isothiocyanate (FITC), phycoerythrin (PE), PerCP, allophycocyanin, PE–Cy5.5, PE–Cy7, or allophycocyanin–Cy7 conjugates. Monoclonal antibodies against CD80 (L307.4), CD86
(2331 [FUN-1]), CD19 (SJ25C1), and HLA–DR (B8.12.2)
were obtained from Becton Dickinson, mAb against CD14
(RMO52) was obtained from Beckman Coulter, mAb against
CD40 (L0D7/6), CD50 (MEM-171), and CD54 (84H10) were
obtained from Serotec, and mAb against CD15 (HI98), CD58
(HCD58), CD62E (HCD62E), CD106 (STA), CD80 (1610A1), and CD86 (GL-1) were obtained from BioLegend.
Anti-CD1c (AD5-8E-7) and anti-CD303 (AC144) were obtained from Miltenyi Biotec. Alexa Fluor 488 phalloidin was
obtained from Invitrogen, and antivinculin mAb was obtained
from Sigma. In all experiments, appropriate Ig isotypematched control mAb served as negative controls.
Phenotype analysis. After informed consent was obtained, blood was withdrawn during routine screening laboratory testing. Peripheral blood mononuclear cells (PBMCs)
were isolated from heparinized blood by LSM (PAA Laboratories) density-gradient centrifugation. PBMCs were resuspended in phosphate buffered saline (PBS)/3% human IgG
(Baxter International) in order to block Fc receptors and
prevent nonspecific antibody binding and were incubated for
15 minutes at 4°C in the dark with combinations of FITC-, PE-,
PE–Cy5.5–, PE–Cy7–, allophycocyanin-, and allophycocyanin–
Cy7–conjugated mAb. Afterward, the cells were washed with
PBS/1% bovine serum albumin. Background fluorescence was
assessed using appropriate isotype- and fluorochromematched control mAb. After staining with the indicated antibodies, cells were analyzed by flow cytometry (FACSCanto II,
FACSDiva software; BD Biosciences). Absolute numbers of
cells were calculated from whole blood counts obtained from
routine laboratory testing.
Isolation of monocytes. PBMCs were isolated as described above. CD14⫹ monocytes were purified by fluorescenceactivated cell sorting (FACS) on a FACSAria (BD Immunocytometry Systems). Forward scatter area versus pulse width
discrimination of doublets and cell aggregates was logically
REDUCED MIGRATORY AND SPREADING CAPACITY OF MONOCYTES UPON ABATACEPT TREATMENT
combined with the identification of monocytes by means of a
mouse mAb against human CD14 to trigger a positive sort
decision. Post-sort reanalyses revealed the achievement of
purities ⬎98%.
Transendothelial migration assay. Human umbilical
vein endothelial cells (HUVECs) were isolated from individual
human umbilical cord veins by collagenase digestion as described previously (9) and maintained in Clonetics EBM-2
Medium (CC-3156; Lonza) supplemented with SingleQuots
Supplements and Growth Factors (CC-4147; Lonza) until
grown to confluence. Monocyte interaction with HUVECs was
examined by their capacity to migrate into a hydrated, neutralized bovine collagen gel (PureCol; Inamed) coated with a
confluent monolayer of endothelial cells prepared 24 hours
prior to the analyses, as described previously (10). Monocytes
were applied to the gels at 2 ⫻ 105 per well and incubated for
36 hours at 37°C in an atmosphere of 5% CO2, 95% relative
humidity. Nonadherent cells were collected by rinsing 3 times
with warm EBM-2 Medium. Monocytes that had actively
migrated into the collagen gels were recovered by gently
mincing and digesting the gels with 0.1% collagenase solution
in Hanks’ balanced salt solution for 1 hour at 37°C. Each
fraction was sedimented at 200g, resuspended in medium, and
enumerated using Neubauer hemocytometer slides. Trypan
blue exclusion provided information on viability of monocytes,
which could be distinguished from HUVECs in the collagenase-digested fraction by size and morphology.
Spreading assay. CD14⫹ monocytes from the PBMCs
of healthy volunteers were purified by high-gradient magnetic
cell sorting using the MACS Monocyte Isolation Kit II (Miltenyi Biotec) according to the manufacturer’s instructions.
Monocytes were incubated overnight with 100 ␮g/ml CTLA4Ig or medium alone. Next, CD14⫹ monocytes were placed
onto fibronectin-coated coverslips. After 90 minutes, the cells
were fixed and permeabilized with 2% paraformaldehyde and
0.3% Triton X-100 (Sigma) in PBS, respectively. In pilot
experiments, 100 ␮g/ml of CTLA-4Ig and 90 minutes of
incubation were found to be optimal for the detection of
differences in cell morphology (data not shown).
Subsequently, cells were processed for confocal microscopy using phalloidin to label F-actin and an antibody to
vinculin to label focal adhesions. Nuclear counterstaining
was performed using DAPI (Invitrogen). For confocal microscopy, images were collected on an upright Leica TCS SP5
microscope (Leica Microsystems) with a 63⫻ oil immersion
objective (Leica Microsystems), zoom 2. Fluorochromes were
excited with a Multi-Photon MaiTai Ti:Sapphire laser
(Spectra-Physics; Newport Corporation) tuned at 800 nm for
DAPI, an argon laser at 488 nm for phalloidin, and a HeNe
laser at 568 nm and at 633 nm for vinculin detection. Detector
slits were configured to minimize any cross-talk between the
channels. Z-stacks (optical sections) of the images were collected with an optical thickness of 0.2 ␮m. Images were
processed using the Leica LAS software and Adobe Photoshop
8.0.1 (Adobe). Cell dimension and cell morphology were
analyzed using the TissueQuest software program (TissueGnostics).
Statistical analysis. Values are shown throughout as
the mean ⫾ SEM, except for patient characteristics, where
mean ⫾ SD values are shown. All values from the FACS
analyses of week 0 were normalized to 100% to calculate
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percent increase or decrease after treatment with CTLA-4Ig.
Proportions of lymphocyte subpopulations were compared
using Student’s t-test for normally distributed values and the
Mann-Whitney test for values without Gaussian distribution.
Relationships between different results were examined using
Pearson’s correlation coefficient and Spearman’s rank correlation tests, as appropriate. P values less than 0.05 were
considered significant in all statistical tests. All statistical
analyses were performed using GraphPad Prism software,
version 4.0 and SPSS software, version 12.0.
RESULTS
Baseline demographic and clinical characteristics. Demographic and clinical characteristics at baseline
and during treatment of RA patients with CTLA-4Ig
are shown in Table 1. After 2 weeks and 4 weeks of
treatment with CTLA-4Ig, no significant changes were
observed in the CRP, SDAI, CDAI, or DAS28-CRP
values compared with baseline.
CTLA-4Ig treatment increases proportions of
CD14ⴙ monocytes in RA patients. We analyzed freshly
isolated PBMCs from patients with RA to determine the
proportions of CD14⫹ monocytes, CD19⫹ B cells,
CD1c⫹ myeloid DCs, and CD303⫹ plasmacytoid DCs
before and after treatment with CTLA-4Ig. As shown in
Figure 1a, proportions of CD14⫹ cells increased significantly during treatment with CTLA-4Ig (P ⫽ 0.02 at
week 2, P ⫽ 0.01 at week 4). In contrast, no significant
change was observed in the proportions of B cells or
DCs. Consistent with this, analysis of total cell numbers
from PBMCs revealed a significant increase only for
CD14⫹ monocytes (P ⫽ 0.03 at week 2, P ⫽ 0.04 at
week 4). No difference was observed for CD19⫹ B cells,
CD1c⫹ myeloid DCs, or CD303⫹ plasmacytoid DCs
(Figure 1b).
Down-regulation of adhesion molecules on
monocytes upon treatment with CTLA-4Ig. The observed effect of CTLA-4Ig on monocyte numbers encouraged us to further study the effect of CTLA-4Ig on
this cell population.
CTLA-4Ig treatment does not affect the expression
of costimulatory molecules on CD14⫹ monocytes. For
this purpose, CD14⫹ cells were analyzed for the expression of the costimulatory molecules CD80, CD86, HLA–
DR, and CD40. As shown in Figure 2, however, no
significant changes were observed in the percentage or
in the mean fluorescence intensity (MFI) of costimulatory molecules expressed on CD14⫹ cells after treatment with CTLA-4Ig.
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patients treated with CTLA-4Ig. Interestingly, a significant decrease was observed in the percentage of
CD106⫹ cells (P ⫽ 0.03 after 4 weeks), CD62E⫹ cells
(P ⫽ 0.002 after 2 weeks, P ⫽ 0.04 after 4 weeks), and
CD15⫹ cells (P ⫽ 0.001 after 2 weeks, P ⫽ 0.04 after 4
weeks) among CD14⫹ monocytes. In addition, a significant decrease was observed in the MFI for expression of
CD50 (P ⫽ 0.04), CD54 (P ⫽ 0.03), CD58 (P ⫽ 0.03),
and CD15 (P ⫽ 0.04) on CD14⫹ monocytes after 2
weeks and for expression of CD58 (P ⫽ 0.02) after 4
weeks of treatment with CTLA-4Ig (Figure 3).
CD80 and CD86 expression on CD14ⴙ monocytes. In order to test the expression levels of CD80 and
CD86, CD14⫹ monocytes from healthy controls were
isolated by cell sorting and analyzed for the expression
Figure 1. CTLA-4Ig treatment increases proportions and absolute
numbers of CD14⫹ monocytes in patients with rheumatoid arthritis
(RA). Proportions of antigen-presenting cells were analyzed by flow
cytometry before and at different time points during CTLA-4Ig
therapy. Absolute numbers of cells were calculated from whole blood
counts obtained from routine laboratory testing. Values are the
mean ⫾ SEM. Significant differences are marked with an asterisk. a, A
significant increase in the percentage of CD14⫹ cells was observed in
RA patients (n ⫽ 12) 2 weeks (P ⫽ 0.02) and 4 weeks (P ⫽ 0.01) after
the initiation of CTLA-4Ig therapy. b, Likewise, a significant increase
was observed for absolute numbers of CD14⫹ monocytes at week 2
(P ⫽ 0.03) and week 4 (P ⫽ 0.04) of CTLA-4Ig therapy. G/l ⫽
⫻109/liter.
CTLA-4Ig treatment decreases the expression of
adhesion molecules on CD14⫹ monocytes. Next, we
analyzed the expression of the adhesion molecules
CD15, CD50 (intercellular adhesion molecule 3 [ICAM3]), CD54 (ICAM-1), CD58 (lymphocyte function–
associated antigen 3), CD62E (E-selectin), and CD106
(vascular cell adhesion molecule 1) on CD14⫹ cells in
Figure 2. Influence of CTLA-4Ig treatment on the expression of
costimulatory molecules on CD14⫹ monocytes during CTLA-4Ig
treatment. CD14⫹ monocytes were analyzed by flow cytometry for
costimulatory molecules before and at different time points during
CTLA-4Ig therapy. Values are the mean ⫾ SEM (n ⫽ 12 patients). No
significant difference was observed in the percentage or mean fluorescence intensity (MFI) of costimulatory molecules expressed on
CD14⫹ cells.
REDUCED MIGRATORY AND SPREADING CAPACITY OF MONOCYTES UPON ABATACEPT TREATMENT
Figure 3. Down-regulation of adhesion molecules on CD14⫹ monocytes during CTLA-4Ig treatment. CD14⫹ cells were analyzed by flow
cytometry for adhesion molecules before and at different time points
during CTLA-4Ig therapy. Values are the mean ⫾ SEM (n ⫽ 5
patients). Significant differences are marked with an asterisk. A
significant decrease was observed in the percentage of CD15⫹ cells
(P ⫽ 0.001 after 2 weeks of treatment with CTLA-4Ig, P ⫽ 0.04 after
4 weeks), CD62E⫹ cells (P ⫽ 0.002 after 2 weeks, P ⫽ 0.04 after 4
weeks), and CD106⫹ cells (P ⫽ 0.03 after 4 weeks) among CD14⫹
monocytes. A significant decrease was observed in the mean fluorescence intensity (MFI) for the expression of CD15 (P ⫽ 0.04), CD50
(P ⫽ 0.04), CD54 (P ⫽ 0.03), and CD58 (P ⫽ 0.03) on CD14⫹
monocytes after 2 weeks of treatment with CTLA-4Ig, and for the
expression of CD58 (P ⫽ 0.02) after 4 weeks of treatment with
CTLA-4Ig.
of CD80 and CD86 by flow cytometry after overnight
culture. Overnight culture of monocytes in medium
alone was found to be sufficient to induce the expression
of CD80 and CD86. (Representative histograms for
CD80 and CD86 expression on human monocytes are
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available online at http://www.meduniwien.ac.at/user/
clemens.scheinecker/.)
Treatment with CTLA-4Ig reduces the migratory
capacity of CD14ⴙ monocytes from patients with RA.
To further examine whether the observed downregulation of adhesion molecules on monocytes after
CTLA-4Ig treatment also affects the migratory behavior
of monocytes, we isolated CD14⫹ cells by FACS before
and after treatment with CTLA-4Ig and analyzed the
cells using an in vitro assay for transendothelial migration. In fact, counting the migrated CD14⫹ cells in
24-well plates after transmigration revealed that monocytes from RA patients had a decreased capacity to
migrate through the transendothelial cell layer after 2
weeks of treatment with CTLA-4Ig (P ⫽ 0.03), and this
decreased capacity was even more pronounced after 4
weeks of treatment with CTLA-4Ig (P ⫽ 0.001). Likewise, a significant increase in the nonadherent cell
fraction was observed after 2 weeks (P ⫽ 0.01) and 4
weeks (P ⫽ 0.001) of treatment with CTLA-4Ig (Figure
4a). This indicates that monocytes from RA patients
exhibit a significantly decreased capacity to adhere to
and to migrate through the endothelial cell layer after
treatment with CTLA-4Ig, as compared to before treatment with CTLA-4Ig. Thus, the rise in circulating monocytes may be due to a decreased capacity to migrate into
the tissues.
In vitro incubation with CTLA-4Ig reduces the
migratory capacity of CD14ⴙ monocytes from healthy
controls. To see whether the results of the migration
assay from RA patients could also be reproduced in
vitro, CD14⫹ cells from healthy controls were isolated
and incubated with various concentrations of CTLA-4Ig
overnight. Indeed, preincubated monocytes showed a
dose-dependent decrease of transendothelial migration
after incubation with 50 ␮g/ml CTLA-4Ig (P ⫽ 0.02) and
100 ␮g/ml CTLA-4Ig (P ⫽ 0.001). Corresponding results
were observed for the nonadherent cell fraction. Incubation of monocytes from healthy controls with 50 ␮g/ml
and 100 ␮g/ml CTLA-4Ig led to a significant increase in
the nonadherent cell fraction (P ⫽ 0.02 for both) (Figure
4b).
CD80/CD86-dependent reduced migratory capacity of CTLA-4Ig–treated monocytes. To further elucidate whether the observed effects of CTLA-4Ig were
CD80/CD86 dependent, CD14⫹ cells from healthy controls were isolated and incubated with anti-CD80 plus
anti-CD86 antibodies or CTLA-4Ig (100 ␮g/ml) overnight. Indeed, a significant and comparable decrease in
transendothelial migration was observed after preincubation of monocytes with anti-CD80 plus anti-CD86
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Figure 4. Influence of CTLA-4Ig on adhesion and transendothelial migration of CD14⫹ cells in patients with rheumatoid arthritis (RA) and in
healthy controls. Values are the mean ⫾ SEM. Significant differences are marked with an asterisk. a, The adhesive and migratory capacity of
monocytes from RA patients was assessed by a transendothelial migration assay before and at different time points during CTLA-4Ig treatment. The
nonadherent cell fraction was significantly increased after 2 weeks (P ⫽ 0.01) and 4 weeks (P ⫽ 0.001) of treatment with CTLA-4Ig. In contrast,
the percentage of migrated CD14⫹ cells was significantly decreased after 2 weeks (P ⫽ 0.03) and 4 weeks (P ⫽ 0.001) of treatment with CTLA-4Ig
(n ⫽ 5 patients). b, Incubation of fluorescence-activated cell sorting–isolated CD14⫹ cells from healthy controls with 50 ␮g/ml and 100 ␮g/ml
CTLA-4Ig led to a significant increment of the nonadherent cell fraction (P ⫽ 0.02 for both). The migrated cell population was significantly
decreased after incubation with 50 ␮g/ml (P ⫽ 0.02) and 100 ␮g/ml (P ⫽ 0.001) CTLA-4Ig (n ⫽ 3 experiments). c, CD14⫹ cells from healthy controls
were isolated and incubated with anti-CD80 plus anti-CD86 antibodies or 100 ␮g/ml CTLA-4Ig overnight. Incubation with 100 ␮g/ml CTLA-4Ig led
to a significant increase of the nonadherent cell fraction (P ⫽ 0.02). A similar result was observed after incubation with anti-CD80 and anti-CD86
as compared to appropriate isotype control monoclonal antibodies (P ⫽ 0.008). The migrated cell population was significantly decreased after
incubation with 100 ␮g/ml CTLA-4Ig (P ⫽ 0.0003) as well as after incubation with anti-CD80 and anti-CD86 (P ⫽ 0.02) (n ⫽ 3 experiments).
mAb as compared to appropriate isotype control mAb
(P ⫽ 0.02) as well as with CTLA-4Ig as compared to
medium control (P ⫽ 0.0003). Similar results were
observed for the nonadherent cell fraction. Incubation
of monocytes from healthy controls with anti-CD80 plus
anti-CD86 mAb (P ⫽ 0.008) as well as with CTLA-4Ig
(P ⫽ 0.02) led to a significant increase in the nonadherent cell fraction as compared to isotype control mAb or
medium control, respectively (Figure 4c).
CD14ⴙ monocytes from healthy controls treated
with CTLA-4Ig display diminished spreading capacity.
In order to analyze the influence of CTLA-4Ig on actin
cytoskeletal dynamics of cell-to-matrix adhesion in vitro,
cell spreading assays were performed. We isolated
CD14⫹ cells from the PBMCs of healthy controls by
magnetic-activated cell sorting and incubated the cells
overnight with or without 100 ␮g/ml CTLA-4Ig. Fibronectin was used as an adhesion substrate. The cells
were placed on fibronectin-coated coverslips for various
periods of time; their capacity to spread as a function of
actin reorganization was assessed by staining of polymerized actin with phalloidin. Focal adhesions were
visualized with an antibody to vinculin.
Fluorescence imaging showed that cells cultured
with CTLA-4Ig exhibited diminished spreading when
compared to untreated cells. CTLA-4Ig–treated CD14⫹
REDUCED MIGRATORY AND SPREADING CAPACITY OF MONOCYTES UPON ABATACEPT TREATMENT
605
DISCUSSION
Figure 5. CTLA-4Ig leads to decreased spreading of monocytes.
Magnetic-activated cell sorting–isolated CD14⫹ cells from healthy
controls were incubated with 100 ␮g/ml CTLA-4Ig and then seeded
onto fibronectin-coated coverslips. Cell spreading was analyzed after
90 minutes. Polymerized actin was stained with phalloidin; focal
adhesions were visualized with an antibody to vinculin. Top, Immunofluorescence analysis revealed reduced cell spreading as well as
impaired formation of focal adhesions in CTLA-4Ig–treated as compared to untreated monocytes. Bottom, A significant difference (ⴱ ⫽
P ⬍ 0.0001) was observed for the mean cell area in CTLA-4Ig–treated
monocytes (n ⫽ 1,545) as compared to untreated monocytes (n ⫽
3,093). Values are the mean ⫾ SEM. One of 2 similar experiments is
shown.
monocytes were mostly round shaped with few cellular
processes and poor assembly of focal adhesions. In
contrast, the untreated monocytes displayed a more
polygonal cell shape with a rim of cortical actin, prominent cellular processes emanating from the cell body,
and multiple focal adhesions at the periphery of the cells
(Figure 5a). Cell dimensions were further analyzed using
TissueQuest software. The total cell area for CD14⫹
cells that were cultured with CTLA-4Ig was significantly
reduced compared to the total cell area for cells cultured
without CTLA-4Ig (mean ⫾ SEM 397 ⫾ 3 ␮m2 versus
424 ⫾ 2 ␮m2; P ⬍ 0.0001) (Figure 5b). (Representative
examples of images that were analyzed using the TissueQuest program are available online at http://www.
meduniwien.ac.at/user/clemens.scheinecker/.)
We show that treatment of RA patients with
CTLA-4Ig increased the proportions of peripheral blood
CD14⫹ monocytes and diminished their expression profile of adhesion molecules. The phenotype changes were
accompanied by a reduced adhesion and transendothelial migratory capacity of isolated monocytes in vitro.
These results were strengthened by similar in vitro
observations with CD14⫹ monocytes from healthy controls, suggesting that CTLA-4Ig treatment exerts important effects beyond the inhibition of T cell activation.
CTLA-4Ig is a selective costimulation modulator
that avidly binds to the CD80/CD86 ligands on an APC.
This results in the inability of these ligands to engage the
CD28 receptor on the T cell that is required for T cell
activation. CTLA-4Ig was shown to dose-dependently
reduce T cell proliferation, serum concentrations of
acute-phase reactants, and other markers of inflammation, including rheumatoid factor produced by B cells
(11).
Besides its effect on T cells, CTLA-4Ig has been
suspected to affect other cell types, in particular, APCs.
Several studies have shown that CTLA-4Ig induces
reverse signaling in APCs, although partially conflicting
results have been reported so far (4,5).
For example, intracellular signaling events have
been described that are induced by ligation of CD80 and
CD86 in a B cell lymphoma, and signaling via CD86 in B
cells has been reported to increase immunoglobulin
production (12). Alternatively, CD80/CD86 engagement
has been suggested to activate B7 molecules and intracellular signaling events, including the recruitment of
p38 MAPK and NF-␬B. This has been shown to induce
an increase in the production of the enzyme indoleamine 2,3-dioxygenase and induction of tryptophan catabolism in murine (13) and human (5) DCs, ultimately
leading to the inhibition of T cell proliferation. On the
other hand, no or only minimal changes in gene expression have been observed upon in vitro treatment of
APCs with abatacept in vitro (14). However, effects on
the gene expression levels of CD14⫹ monocytes have
not been analyzed so far.
We did not observe significant changes in clinical
disease activity scores upon treatment with CTLA-4Ig.
This, however, is not surprising since our study was not
powered for clinical efficacy. Moreover, even in large
clinical trials, a 20% improvement in disease activity
according to the ACR criteria (an ACR20 response)
(15) or an ACR50 response was only observed beginning
on day 30 or day 60, respectively, of CTLA-4Ig treat-
606
ment. It is therefore conceivable that early detectable
CTLA-4Ig–mediated effects on monocytes might well
contribute to the overall effect of CTLA-4Ig. The main
mechanism of action, however, is still the modulation of
CD28 signaling on T cells. This might explain the longer
time period that is required for clinical effects to become
apparent.
Our data show a significant increase in the percentage as well as absolute numbers of CD14⫹ monocytes in RA patients upon CTLA-4Ig treatment, an
observation that, to our knowledge, has not previously
been reported. Whether these changes affect recently
identified subsets of monocytes (16,17) in different ways
was not the aim of the present study, but might justify
future analysis. Our subsequent analyses did not reveal
substantial changes in the expression profile of costimulatory molecules on monocytes, but rather—and most
striking—a reduction in the expression of certain adhesion molecules that are required for the adhesion to, and
active transmigration of, monocytes through endothelial
barriers. Moreover, and consistent with the phenotype
analysis, the functional assessment of isolated CD14⫹
monocytes revealed that abatacept treatment led to a
reduced adhesion of monocytes to endothelial cells and
a reduced capacity of monocytes to migrate through an
endothelial cell layer in vitro. In addition, we were able
to show that the observed effect of CTLA-4Ig on
monocyte migration and adhesion was CD80/CD86 dependent, since in vitro experiments with antibodies
against CD80 and CD86 led to similar results in migration assays as compared to CTLA-4Ig.
The migratory capacity of cells depends on several mechanisms, including the protrusion in the front of
the cell, adhesion at focal contacts, concentration of the
cytoplasm, and breaking of older adhesions at the rear of
the cell. The reorganization of actin fibers, however,
represents the main mechanism operative in this process
(18). Indeed, spreading assays revealed a substantial
influence of CTLA-4Ig on actin reorganization as well as
formation of focal contacts. Thus, the down-regulation
of adhesion molecules by abatacept appears to mediate
its effects via reduced actin dynamics. The decreased
adhesion and migratory capacity of the monocytes is
likely responsible for the increase in CD14⫹ monocytes
in the peripheral blood of abatacept-treated RA patients.
One of the key elements of inflammation is the
accumulation of cells at sites of inflammation, and
monocytes are a major proinflammatory cell population that accumulates at sites such as the RA synovial
membrane, contributing to the dramatic amplification of
BONELLI ET AL
the inflammatory process. A reduction in the migratory
capacity of monocytes might contribute importantly to
the decrease in inflammation and thereby help to ameliorate the disease (19). Indeed, besides efforts to interfere with lymphocyte activation, modulation of the recruitment of leukocytes to sites of inflammation is a
promising evolving concept of targeted therapies for RA
(20).
Moreover, putting a brake on monocyte migration may also reduce the number of osteoclast precursors, a cell population that is importantly involved in
joint destruction, in the synovial tissue (19,21). Indeed,
it is conceivable that the direct inhibitory effects of
CTLA-4Ig on osteoclastogenesis and its benefit on joint
damage in a tumor necrosis factor–dependent arthritis
model (22) are consequences of the changes described
here.
Thus, CTLA-4Ig seems to possess the capacity to
interfere with RA pathogenesis at 2 important pathogenetic sites, namely, at the site of the T cells, by inhibiting
their activation, and at the site of the monocytes, by
interfering with their migration into the joint. Accordingly, CTLA-4Ig appears to target RA pathogenesis in a
dual way.
There are several limitations to our study. First,
the number of patients assessed was relatively small;
however, the results obtained were consistent and were
seen ex vivo in RA patients as well as in vitro in healthy
controls. Second, these data have not been reported
previously despite many attempts to characterize additional pathways of the mode of action of CTLA-4Ig; one
reason might be that previous studies on CTLA-4Ig–
mediated effects have so far mainly focused on T cells
and T cell subsets where no substantial shifts in the
distribution were found (23), whereas detailed analysis
of monocyte phenotype and function was not previously
addressed. Finally, we performed our analyses within the
very first weeks of abatacept therapy, and we therefore
did not relate them to clinical outcomes. In this context,
however, it is of lesser importance whether the effect on
monocytes is accompanied by a clinical effect or not,
since clinical responses are quite variable, and many
patients respond differently to various agents despite
a clearcut pharmacologic effect of these drugs, thus
highlighting the heterogeneity of the pathways leading
to RA.
It is noteworthy that most of the changes in
monocyte phenotype and function were already seen 2
weeks after the first infusion of CTLA-4Ig and continued to be seen at week 4 (2 weeks after the second
infusion). This indicated that the effect of CTLA-4Ig on
REDUCED MIGRATORY AND SPREADING CAPACITY OF MONOCYTES UPON ABATACEPT TREATMENT
monocytes came about quite rapidly and was maintained.
In summary, our data indicate that CTLA-4Ig
treatment exerts effects on cells beyond T cells in RA
patients. CTLA-4Ig was found to modulate monocytes
with regard to phenotype characteristics and their migratory capacity, which might exert another beneficial
effect in the treatment of RA.
8.
9.
10.
ACKNOWLEDGMENTS
We would like to thank the Cell Sorting Core Unit of
the Medical University of Vienna and Mr. Günther Hofbauer
for expert flow cytometry assistance, as well as Mrs. Anneliese
Nigisch and Karolina von Dalwigk for the preparation and
maintenance of cells for the migration and spreading assays.
11.
12.
AUTHOR CONTRIBUTIONS
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. Scheinecker 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. Bonelli, Ferner, Göschl, Blüml, Hladik,
Kiener, Byrne, Niederreiter, Bergmann, Smolen, Scheinecker.
Acquisition of data. Bonelli, Ferner, Göschl, Blüml, Hladik,
Karonitsch, Niederreiter, Steiner, Rath, Scheinecker.
Analysis and interpretation of data. Bonelli, Ferner, Göschl, Blüml,
Hladik, Niederreiter, Steiner, Scheinecker.
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