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Lack of evidence of stimulatory autoantibodies to platelet-derived growth factor receptor in patients with systemic sclerosis.

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ARTHRITIS & RHEUMATISM
Vol. 60, No. 4, April 2009, pp 1137–1144
DOI 10.1002/art.24381
© 2009, American College of Rheumatology
Lack of Evidence of Stimulatory Autoantibodies to
Platelet-Derived Growth Factor Receptor in
Patients With Systemic Sclerosis
Jean-François Classen,1 Dan Henrohn,2 Fredrik Rorsman,2 Johan Lennartsson,3
Bernard R. Lauwerys,4 Gerhard Wikström,2 Charlotte Rorsman,3 Sandrine Lenglez,1
Karin Franck-Larsson,2 Jean-Paul Tomasi,4 Olle Kämpe,2 Marie Vanthuyne,4
Frédéric A. Houssiau,4 and Jean-Baptiste Demoulin1
Objective. Systemic sclerosis (SSc) is a severe
connective tissue disease of unknown etiology, characterized by fibrosis of the skin and multiple internal
organs. Recent findings suggested that the disease is
driven by stimulatory autoantibodies to platelet-derived
growth factor receptor (PDGFR), which stimulate the
production of reactive oxygen species (ROS) and collagen by fibroblasts. These results opened novel avenues
of research into the diagnosis and treatment of SSc. The
present study was undertaken to confirm the presence of
anti-PDGFR antibodies in patients with SSc.
Methods. Immunoglobulins from 37 patients with
SSc were purified by protein A/G chromatography.
PDGFR activation was tested using 4 different sensitive
bioassays, i.e., cell proliferation, ROS production, signal
transduction, and receptor phosphorylation; the latter
was also tested in a separate population of 7 patients
with SSc from a different research center.
Results. Purified IgG samples from patients with
SSc were positive when tested for antinuclear autoantibodies, but did not specifically activate PDGFR␣
or PDGFR␤ in any of the tests. Cell stimulation with
PDGF itself consistently produced a strong signal.
Conclusion. The present results raise questions
regarding the existence of agonistic autoantibodies to
PDGFR in SSc.
Systemic sclerosis (SSc; scleroderma) is a connective tissue disease characterized by autoimmunity, inflammation, blood vessel damage, and interstitial fibrosis of the skin, lungs, and other organs (1). Two distinct
subsets of the disease are commonly distinguished based
on skin involvement: diffuse cutaneous SSc and limited
cutaneous SSc (2). Serious complications, such as pulmonary arterial hypertension and lung fibrosis, remain
major treatment challenges (3). Scleroderma invariably
involves fibroblast activation and excessive extracellular
matrix production. Several cytokines and their receptors
are expressed in SSc lesions and may contribute to
fibroblast activation, including transforming growth factor ␤ (TGF␤), connective tissue growth factor, endothelin 1, and platelet-derived growth factor (PDGF) (1,4).
In particular, analysis of mice with experimental
scleroderma-like disease indicates that TGF␤ has a key
role in the development of fibrosis (1,5). However, these
mouse models do not share all of the features of human
scleroderma, and results from a phase I/II clinical trial of
recombinant anti-TGF␤1 antibody treatment in patients
with scleroderma were discouraging (6).
Supported by grants from the Foundation for Scientific
Research, Belgium.
1
Jean-François Classen, MSc Pharm, Sandrine Lenglez, JeanBaptiste Demoulin, MSc Pharm, PhD: de Duve Institute, Université
Catholique de Louvain, Brussels, Belgium; 2Dan Henrohn, MD, MSc
Pharm, Fredrik Rorsman, MD, PhD, Gerhard Wikström, MD, PhD,
Karin Franck-Larsson, MD, Olle Kämpe, MD, PhD: Uppsala University Hospital, Uppsala, Sweden; 3Johan Lennartsson, PhD, Charlotte
Rorsman: Ludwig Institute for Cancer Research, Uppsala University,
Uppsala, Sweden; 4Bernard R. Lauwerys, MD, PhD, Jean-Paul Tomasi, MD, PhD, Marie Vanthuyne, MD, Frédéric A. Houssiau, MD,
PhD: Saint-Luc University Hospital, Université Catholique de Louvain, Brussels, Belgium.
Dr. Kämpe has applications for patents on two novel autoantigens.
Address correspondence and reprint requests to JeanBaptiste Demoulin, MSc Pharm, PhD, de Duve Institute, Université
Catholique de Louvain, MEXP Unit, UCL 74.30, Avenue Hippocrate
74-75, B-1200 Brussels, Belgium. E-mail: JB.Demoulin@uclouvain.be.
Submitted for publication February 22, 2008; accepted in
revised form December 8, 2008.
1137
1138
CLASSEN ET AL
Table 1.
Characteristics of the systemic sclerosis patients*
Uppsala University
(Sweden)
(n ⫽ 7)
Université Catholique
de Louvain (Belgium)
(n ⫽ 37)
6/1
73 (64–76)
9 (2–28)
32/5
52 (26–77)
3 (0–25)
2
5
4
0
3
4
2
3
0
24
13
15
1
4
14
12
0
6
7
1
3
36
13
11
Female/male
Age, median (range) years
Disease duration, median
(range) years
Skin involvement
Diffuse
Limited
Interstitial lung fibrosis
Renal crisis
PAH
Treatment
Corticosteroids
NSAIDs
Cytotoxic drugs
Autoantibodies
Antinuclear
Anti–topoisomerase (Scl-70)
Anticentromere
* Except where indicated otherwise, values are the number of patients. PAH ⫽ pulmonary arterial
hypertension; NSAIDs ⫽ nonsteroidal antiinflammatory drugs.
Scleroderma is an autoimmune disorder, as illustrated by the presence of autoantibodies against nuclei
(ANAs), centromere (ACAs), topoisomerase I (Scl-70),
endothelial cells, and many other self antigens (7,8).
Some of these autoantibodies are useful diagnostic and
prognostic markers, but whether they play a role in the
pathogenesis of the disease is a matter of debate (7,8).
A recent report by Baroni et al suggested that
fibroblast activation in scleroderma may be caused by
stimulatory autoantibodies to the PDGF receptor
(PDGFR) ␣ and ␤ subunits, which are members of the
type III receptor tyrosine kinase family (9). These
autoantibodies have also been found in extensive
chronic graft-versus-host disease, which is characterized
by fibrotic lesions similar to those observed in scleroderma (10). In contrast to most serum biomarkers of
autoimmune disorders, these autoantibodies were described as being fully specific, since they were detected
in all patients with scleroderma or extensive chronic
graft-versus-host disease, but in none of the study patients with other autoimmune disorders and none of the
healthy controls. PDGFR antibodies are thought to
induce PDGFR ␣ chain and/or ␤ chain dimerization,
mimicking the effect of the natural dimeric ligands. IgG
isolated from SSc patients has been shown to induce
PDGFR tyrosine phosphorylation, to increase the production of reactive oxygen species (ROS), and to stimulate collagen production by fibroblasts in vitro. Based
on these data and on the demonstration that PDGF and
TGF␤ are important mediators of fibrosis in different
models, imatinib mesylate has been suggested as a
rational treatment of SSc (11–13). This potent tyrosine
kinase inhibitor is selective for PDGFR and Abl, which
may be involved in TGF␤ signaling and in fibrosis (14).
The above-described findings suggested that autoantibodies to PDGFR might have important implications with regard to the understanding of SSc as well as
for its diagnosis and for design of a rational treatment.
The present study was conducted to confirm this.
PATIENTS AND METHODS
Patients. A total of 44 patients with SSc were studied:
37 from Saint-Luc University Hospital and 7 from Uppsala
University Hospital (Table 1). All patients fulfilled the American College of Rheumatology (formerly, the American Rheumatism Association) criteria for the classification of SSc (15),
and their disease was further classified as diffuse cutaneous
SSc or limited cutaneous SSc according to the criteria of
LeRoy et al (2,16). The control group comprised 9 patients
with systemic lupus erythematosus (SLE) and 17 healthy
individuals from Saint-Luc University Hospital, and 5 healthy
individuals from Uppsala University Hospital. The study was
approved by the local ethics committees (Université
Catholique de Louvain and Uppsala University), and informed
consent was obtained from all study subjects.
Antibody purification. Immunoglobulins were purified
from serum using Ultralink immobilized protein A/G, according to the instructions of the manufacturer (Pierce, Rockford,
IL). After extensive washing, elution was performed in the
presence of 50 mM glycine (pH 2.8). The samples were quickly
neutralized, diluted with phosphate buffered saline (PBS), and
concentrated in a Centricon Plus 20 (Millipore, Bedford, MA).
ABSENCE OF ANTI-PDGFR ANTIBODIES IN SSc
Figure 1. Results of proliferation assays. A, Cell surface expression of
platelet-derived growth factor receptor ␣ (PDGFR␣) and PDGFR␤
on 32D␣/␤ cells was monitored by flow cytometry using specific
monoclonal antibodies (white) or control reagents (gray). B, 32D and
32D␣/␤ cells were incubated for 24 hours in the presence of increasing
concentrations of PDGF-AA (circles) or PDGF-BB (squares). 3Hthymidine was added 4 hours before the end of the incubation period.
Cells were harvested to measure radioactivity incorporated into DNA.
Values are the mean and SD. C, Using the same protocol, cells were
stimulated with purified IgG (0.2 mg/ml) from 11 healthy controls, 36
systemic sclerosis (SSc) patients, and 9 systemic lupus erythematosus
(SLE) patients. Results were expressed as a percentage of the response
obtained with IgG compared with PDGF-BB (50 ng/ml), after subtraction of background incorporation measured in the absence of stimulation. Bars show the means. D, 32D␣/␤ cells were stimulated with
control medium (⫺), PDGF, or IgG from controls or SSc patients (0.2
mg/ml [left bars] or 0.6 mg/ml [right bars]), and radioactivity was
measured as 3H-thymidine incorporation. Values are the mean and SD.
1139
Alternatively, immunoglobulin enrichment was performed by precipitation in the presence of ammonium sulfate,
as follows. One volume of saturated ammonium sulfate was
added to diluted serum, followed by incubation overnight at
4°C. Precipitates were dissolved in PBS and dialyzed extensively against PBS. IgG samples from Swedish patients were
purified using the Melon Gel IgG Spin Purification Kit
(Pierce).
Concentrations of all samples were calculated based on
absorbance at 280 nm, and IgG purity was confirmed using
sodium dodecyl sulfate–polyacrylamide gels. ANAs were detected using HEp-2 cells (Bio-Rad, Richmond, CA). Anti–
Scl-70 and ACAs were quantified by EliA (Sweden Diagnostics, Freiburg, Germany). Levels of human PDGF-AB were
measured by enzyme-linked immunosorbent assay (ELISA;
R&D Systems, Minneapolis, MN).
Measurements of PDGFR activity. After culture of
32D cells in medium supplemented with 10% fetal calf serum
(FCS) and interleukin-3 (IL-3) as previously described (17),
32D␣/␤ cells were generated by sequentially electroporating
PDGFR␣ complementary DNA (cDNA) cloned in the pEFMYC-Cyto vector, and PDGFR␤ cDNA inserted into pEFBOS-Puro (18). Cells were selected in the presence of Geneticin and Puromycin as described (17). Homogeneous cell
populations expressing both receptors were sorted by flow
cytometry using specific antibodies against PDGFR␣ (R&D
Systems) and PDGFR␤ (19). Parental 32D cells were used as
a control. For proliferation assays, cells were washed 3 times
with medium and seeded in a 96-well plate at 104 cells per well
in medium containing 10% FCS in the presence of PDGF
(PeproTech, Rocky Hill, NJ), IL-3 (positive control), or purified IgG. After 20 hours, 3H-thymidine (0.5 ␮Ci) was added to
each well. Four hours later, cells were harvested and radioactivity incorporated into DNA was counted using a TopCount
platform (PerkinElmer, Zaventem, Belgium) as previously
described (20). Results were expressed as a percentage, i.e., ([I
– C]/[P – C]) ⫻ 100, where I, P, and C represent the average
thymidine incorporation in cells stimulated with IgG, PDGF,
and control medium, respectively.
For ROS detection, cells (106/ml) were washed 3 times,
loaded with dichlorofluorescein diacetate (10 ␮M; Invitrogen,
San Diego, CA) in serum-free medium for 30 minutes at 37°C,
and then stimulated with PDGF (25 ng/ml), imatinib mesylate
(100 nM; Novartis, Basel, Switzerland), or IgG (0.2–1 mg/ml)
for 15 minutes in duplicate. Cells were washed twice with PBS
and analyzed by flow cytometry (Becton Dickinson, Mountain
View, CA). BJ human fibroblasts immortalized with telomerase (kindly provided by Dr. F. d’Adda di Fagagna, IFOM
Foundation, Milan, Italy) were treated similarly after starvation for 24 hours. Cells were trypsinized before analysis by flow
cytometry.
F␣ mouse embryonic fibroblasts transfected with human PDGFR␣ (a kind gift from Dr. Andrius Kazlauskas,
Harvard Medical School, Boston, MA) were cultured as previously described (9,21). To remove cells that did not express
the receptor, cells were sorted by flow cytometry after staining
with anti-PDGFR␣ antibodies. Porcine aortic endothelial
(PAE) cells stably transfected with PDGFR␣ or PDGFR␤
were cultured as described (19,22). F␣ and PAE cells were
starved overnight in medium containing 0.1% FCS and incubated for 15 minutes in the presence of PDGF-BB or purified
patient IgG (0.3 mg/ml). Cells were lysed in ice-cold radioim-
1140
CLASSEN ET AL
Figure 2. Production of reactive oxygen species. A, 32D␣/␤ cells were loaded with dichlorofluorescein acetate and then incubated for 15 minutes
in the presence of control medium, PDGF-BB, imatinib, or PDGF-BB plus imatinib. Cells were analyzed by flow cytometry. B, Using the same
protocol, 32D␣/␤ cells or BJ human fibroblasts were treated for 15 minutes with patient IgG (0.2 mg/ml). A total of 35 samples (24 SSc, 5 SLE, and
6 healthy controls) were tested with 32D␣/␤ cells and 5 with BJ cells, with similar results. Results of 1 representative experiment with IgG from 2
SSc patients are shown. C, 32D␣/␤ cells were treated for 15 minutes with increasing concentrations of IgG from controls or SSc patients. D, Cells
were treated for 15–60 minutes with IgG from SSc patients (0.2 mg/ml). Values in B–D are the mean and SD. See Figure 1 for definitions.
munoprecipitation buffer (1% Triton X-100, 5 mM EDTA, 140
mM NaCl, 50 mM Tris [pH 8], 0.1% sodium deoxycholate, and
10% glycerol) supplemented with 1% Trasylol, 1 mM Pefabloc
(Roche, Basel, Switzerland), and 1 mM sodium orthovanadate.
After centrifugation, ERK phosphorylation in cell lysates was
analyzed by immunoblotting with anti–phospho–T202/Y204–
ERK-1/2 antibodies (Cell Signaling Technology, Beverly, MA)
and anti–ERK-1/2 antibodies (19). Receptors were immunoprecipitated from lysate with antibodies recognizing PDGFR␣
or PDGFR␤ and immunoblotted against phosphotyrosine
(PY99; Santa Cruz Biotechnology, Santa Cruz, CA) or the
receptor, as previously described (19,22).
RESULTS
Features of SSc in the patients included in this
study varied in terms of disease type, treatment, complications, and autoantibodies (Table 1). In the majority of
patients, the disease was in an active stage.
IgG was purified by standard protein A/G affinity
chromatography, which had been used previously to
isolate anti-PDGFR autoantibodies (9,10). As a control
for antibody integrity, we compared levels of ANAs,
ACAs, and anti–Scl-70 in the serum and in the purified
immunoglobulins of patients with scleroderma (8). Results matched perfectly for most patients, indicating that
the purification process did not significantly affect the
autoantibody profile (data available online at www.
icp.be/mexp/pdgf/ssc).
A sensitive bioassay for detecting PDGFR activation was developed using the 32D mouse cell line,
which proliferates in the presence of IL-3. Cells were
transfected with the human PDGFR ␣ and ␤ subunits
and selected to obtain 32D␣/␤ cells. Cell surface expression of each receptor was tested by flow cytometry
(Figure 1A). In accordance with the results of previous
studies (23), we observed that 32D␣/␤ cells proliferated
in the presence of PDGF-AA or -BB, as shown in Figure
1B. PDGF-AA binds to PDGFR ␣-chain only, while
ABSENCE OF ANTI-PDGFR ANTIBODIES IN SSc
PDGF-BB binds to both receptor subunits. A significant
signal was consistently observed with PDGF-BB at concentrations of ⬍0.5 ng/ml. Nontranfected 32D cells were
used as control. We used this sensitive assay for PDGFR
activation to analyze the presence of stimulatory autoantibodies in IgG fractions isolated from the serum of
SSc patients from Saint-Luc University Hospital. At a
concentration of 0.2 mg/ml, used in the reports that
described anti-PDGFR antibodies (9,10), IgG samples
stimulated 32D␣/␤ cell growth very weakly (Figure 1C).
At a higher IgG concentration (0.6 mg/ml), proliferation
of 32D␣/␤ cells was increased, but SSc IgG had no
specific effect (Figure 1D). We obtained similar results
using antibodies purified by ammonium sulfate precipitation (data available online at www.icp.be/mexp/pdgf/
ssc). Overall, no difference between SSc and control
samples was observed under any of the experimental
conditions tested.
Autoantibodies to PDGFR were initially detected by monitoring the production of cellular ROS,
measured after loading mouse fibroblasts with dichlorofluorescein diacetate, which is oxidized into highly
fluorescent dichlorofluorescein in the presence of ROS
(9). We first observed that PDGF-BB stimulated ROS
production in 32D␣/␤ cells (Figure 2A). This effect
could be blocked by imatinib and was absent in nontransfected 32D cells, demonstrating that it was mediated by PDGFR. Incubation of the cells with IgG from
SSc patients did not increase ROS production (Figure
2B). We obtained the same results with IgG concentrations of up to 2 mg/ml and cell stimulation for up to 1
hour (Figures 2C and D). We also tested ROS production in human BJ fibroblasts, which express endogenous
PDGFR. PDGF weakly but reproducibly increased the
ROS content of these cells; again, no effect was observed
with incubation of the cells with patient IgG (Figure 2B).
ROS production has been linked to the Ras/MAP
kinase pathway, which, according to Baroni et al, is
activated by SSc autoantibodies (9). To investigate this,
we used F␣ mouse embryonic fibroblasts, which express
human PDGFR␣ (data available online at www.icp.be/
mexp/pdgf/ssc) and have been shown to respond to SSc
IgG (9). We observed a highly variable increase in ERK
phosphorylation upon stimulation of F␣ cells with any
type of purified IgG. To measure average ERK phosphorylation in the presence of immunoglobulins, we
incubated fibroblasts with pooled IgG from groups of 4
SSc patients, 4 SLE patients, and 4 healthy controls. As
shown by the immunoblotting results presented in Figure 3A, IgG from all sources (SSc, SLE, and controls)
1141
Figure 3. PDGFR␣ phosphorylation and signal transduction in F␣
fibroblasts. A, F␣ cells expressing human PDGFR␣ were starved for 24
hours in medium containing 0.1% fetal calf serum, and then stimulated
for 15 minutes with PDGF-BB (50 ng/ml) or with pooled IgG isolated
from healthy controls, patients with SLE, or patients with SSc (all at
0.4 mg/ml). ERK phosphorylation in cell lysates was detected by
immunoblotting (IB) with anti–phospho-ERK antibodies. Membranes
were reprobed with anti-ERK antibodies as a loading control. B,
PDGFR␣ was immunoprecipitated (IP) from F␣ cells stimulated with
PDGF-BB (50 ng/ml) or IgG (0.6 mg/ml) and processed as described
above. Receptor phosphorylation was detected by immunoblotting
with antiphosphotyrosine antibodies (pTyr). See Figure 1 for other
definitions.
had a similar weak stimulatory effect, compared with the
effect observed with PDGF.
We next tested whether IgG activated PDGFR␣
in these cells, by performing antiphosphotyrosine immunoblot analysis on immunoprecipitated PDGFR. Again,
a weak but significant increase in PDGFR phosphorylation was observed in cells incubated with IgG. As in the
ERK phosphorylation studies, there was no significant
difference in stimulatory effect between SSc and control
IgG (Figure 3B), suggesting that the effect on ERK and
PDGFR phosphorylation was mediated by factors contaminating the IgG preparations, rather than by autoan-
1142
CLASSEN ET AL
Figure 4. PDGFR␣ and PDGFR␤ activation in porcine aortic endothelial (PAE) cells. A, PAE cells stably transfected with PDGFR␣ or PDGFR␤
were incubated for various amounts of time in the presence of PDGF-BB (10 ng/ml) or purified IgG from 2 SSc patients (300 ␮g/ml). Receptors
were immunoprecipitated from cell lysates and immunoblotted (IB) against phosphotyrosine (pTyr) or PDGFR. B, Cells were stimulated for 15
minutes with increasing concentrations of SSc IgG. C, In samples from 7 SSc patients and 5 controls, the ratio between the signals obtained with
the antiphosphotyrosine antibody and those obtained with the anti-PDGFR antibody (arbitrary units [AU]) was determined. Each experiment was
performed twice, with identical results. See Figure 1 for other definitions.
tibodies. PDGF-AB is the most abundant PDGF isoform in serum. Using a specific ELISA, we were able to
detect traces of PDGF-AB in our IgG samples, which
may partially explain the nonspecific effect of antibody
preparations at high concentrations.
Sera were collected from a separate population
of 7 SSc patients at Uppsala University Hospital and
analyzed at an independent laboratory (Uppsala branch
of the Ludwig Institute for Cancer Research). Two sera
from the Belgian cohort were also included in these
analyses. IgG was purified using Melon Gel (see Patients
and Methods) and tested using PAE cells transfected
with PDGFR. In this well-characterized model for analysis of PDGFR activation, PDGF induces strong receptor phosphorylation, followed by the activation of numerous signal transduction pathways, chemotaxis, and
cell division (19,22,24). Addition of SSc IgG (20–600
␮g/ml) to these cells for various amounts of time did not
induce any detectable increase in PDGFR␣ or PDGFR␤
phosphorylation, as shown by Western blotting (Figure
4). These observations confirmed that purified IgG from
SSc patients does not stimulate PDGFR␣ or PDGFR␤.
DISCUSSION
Using 4 different methods for assessing PDGFR
activation, i.e., receptor phosphorylation, MAP kinase
signaling, ROS production, and cell proliferation, we
were unable to detect any specific stimulatory activity in
purified IgG from patients with scleroderma. In each
experiment, PDGF, used as a positive control, produced a strong signal. We used 32D and PAE cells that
were highly sensitive to PDGF, due to high levels of
expression of transfected human PDGFR␣ and/or
PDGFR␤. Flow cytometry experiments using mouse
anti-PDGFR antibodies showed that the receptors were
accessible to antibodies at the cell surface (Figure 1A).
However, it is conceivable that anti-PDGFR activity is
sensitive to cell type. For instance, a cell-specific posttranslational modification of the receptor could affect
antibody binding. For this reason, we also tested human
fibroblasts expressing endogenous receptors, as well as
F␣ mouse fibroblasts, which had initially been used to
detect activating antibodies to PDGFR (9,10). We conclude that our IgG preparations did not contain detect-
ABSENCE OF ANTI-PDGFR ANTIBODIES IN SSc
able amounts of antibodies able to activate human
PDGFR.
The study patients had either diffuse or limited
cutaneous SSc. In many of the patients SSc was at an
early progressive stage, when pathologic autoantibodies
are more likely to be detected; however, patients with
SSc at later stages, presenting with complications, were
also included. Patients were from different regions,
including Western, Northern, and Southern Europe. At
the time samples were obtained for this study, many
patients had not yet been treated, whereas others had
received corticosteroids, cytotoxic drugs, or angiotensinconverting enzyme inhibitors. It is not likely that differences in patient cohorts explain the discrepancy between
our findings and previously published results regarding
the role of PDGFR in SSc (9).
We studied immunoglobulins that were purified
from serum, using the well-established protein A/G
affinity purification method, which was also used by
Baroni and colleagues (9). By assessing the level of
ANAs in the samples produced by this method compared with the corresponding sera, we were able to
confirm that IgG activity was not affected by the purification procedure. In addition, we obtained similar negative results with immunoglobulins produced by ammonium sulfate precipitation, which contained more serum
contaminants but included IgM. An independent set of
sera from 7 SSc patients was purified using Melon Gel
technology, which is based on removal of most abundant
serum proteins by affinity chromatography. One advantage of this method is that it does not involve an acidic
elution step, which may sometimes alter antibody activity. It is unlikely that specific human IgG could not be
isolated by at least one of these purification protocols,
unless it displays very peculiar biochemical properties.
One-step purification methods do not produce
pure antibodies. In F␣ and 32D␣/␤ cells, we observed a
nonspecific effect of IgG preparations produced by
protein A/G chromatography. This is likely due to
contamination of the antibodies with PDGF, which is
released in serum in massive amounts by activated
platelets, and with other molecules that are able to
indirectly activate PDGFRs. This process, known as
receptor transactivation, can be triggered by angiotensin
II, inflammatory cytokines, drugs, and many other factors (25–28). Such contaminants are difficult to trace
because they are highly variable in terms of chemical
structure and are active at low concentrations. Of note,
levels of PDGF and angiotensin II have been found to
be elevated in patients with SSc (29–31).
Recently, Balada et al described antibodies that
1143
bind to the cytoplasmic domain of PDGFR␣, produced
as a tagged recombinant protein (32). These antibodies
were found in the serum of healthy individuals. Because
these antibodies are not expected to bind to intact cells,
they would not be detectable with the assays used in the
present study and in the study by Baroni et al (9).
In conclusion, using well-established methods, we
were unable to find evidence of stimulatory antiPDGFR autoantibodies in 2 groups of patients with
scleroderma. PDGF ligands and receptors, which are
highly expressed in SSc lesions, may play a role in the
disease independently of the presence of autoantibodies.
However, determining which of the multiple cytokines
and receptors expressed in SSc is the best target for
therapy will require further careful studies.
ACKNOWLEDGMENTS
We are very grateful to Dr. Carl-Henrik Heldin (Uppsala University) and Dr. Pedro Buc-Calderon (Université
Catholique de Louvain) for helpful discussions, and to Mrs.
Hayat Bardani (Saint-Luc University Hospital) for technical
assistance. We thank Dr. Andrius Kazlauskas (Harvard Medical School, Boston, MA), Dr. Anabelle Decottignies (de Duve
Institute), and Dr. F. d’Adda di Fagagna (IFOM Foundation,
Milan, Italy) for generous donation of reagents, and Dr. Stefan
Constantinescu (de Duve Institute) for critical reading of the
manuscript.
AUTHOR CONTRIBUTIONS
Dr. Demoulin 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 design. Henrohn, F. Rorsman, Lennartsson, Wikström, C.
Rorsman, Kämpe, Demoulin.
Acquisition of data. Classen, Henrohn, F. Rorsman, Lennartsson,
Wikström, C. Rorsman, Lenglez, Franck-Larsson, Tomasi, Demoulin.
Analysis and interpretation of data. Classen, Henrohn, Lennartsson,
Wikström, C. Rorsman, Lenglez, Franck-Larsson, Tomasi, Kämpe,
Vanthuyne, Houssiau, Demoulin.
Manuscript preparation. Classen, Henrohn, F. Rorsman, Lennartsson, Lauwerys, Wikström, Franck-Larsson, Vanthuyne, Houssiau,
Demoulin.
Statistical analysis. Classen, Demoulin.
Sample collection. Henrohn, F. Rorsman, Lauwerys, Franck-Larsson.
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factors, platelet, patients, lack, autoantibodies, growth, evidence, systemic, sclerosis, receptov, derived, stimulators
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