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Deficient expression of interleukin-10 receptor ╨Ю┬▒ chain in rheumatoid arthritis synoviumLimitation of animal models of inflammation.

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ARTHRITIS & RHEUMATISM
Vol. 52, No. 10, October 2005, pp 3315–3321
© 2005, American College of Rheumatology
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antibodies (clone 37607.11; R&D Systems, Minneapolis, MN).
Five-micrometer paraffin sections were fixed overnight at
50°C, dewaxed in xylol, and rehydrated in graded ethanol
baths. After microwave pretreatment in the presence of 1 mM
EDTA (pH 8) and proteolysis digestion using trypsin (Sigma,
St. Louis, MO), the slides were blocked for 1 hour with a Tris
NaCl solution containing 4% weight/volume milk powder and
2% volume/volume horse serum. The slides were incubated
overnight with anti–IL-10R␣ monoclonal antibodies. Primary
antibodies were detected by biotin-conjugated AffiniPure
F(ab⬘)2 fragment goat anti-mouse IgG (H⫹L) (Jackson ImmunoResearch, West Grove, PA), avidin–biotin–peroxidase complex reagent for alkaline phosphatase, and fast red (Dako, Zug,
Switzerland). Negative controls were performed using murine
IgG as primary antibodies. Expression of IL-10R␣ protein in
the synovial tissues was assessed by 2 independent observers,
using a semiquantitative score, where 0 ⫽ no staining, 0.5 ⫽
very weak staining, 1.0 ⫽ weak staining, 2.0 ⫽ strong staining,
and 3.0 ⫽ very strong staining, and differentiating between 3
compartments (lining, sublining, and vessels).
The accumulation of IL-10R␣ messenger RNA
(mRNA) in synovial tissues was quantified by real-time polymerase chain reaction (PCR) after isolation of RNA with the
RNeasy kit (Qiagen, Zurich, Switzerland), including DNase
digestion, and 2-step reverse transcription (RT)–PCR (reagents were obtained from PE Applied Biosystems, Warrington, UK). PCR was conducted on a GeneAmp PCR
System 9700, using the predeveloped IL-10R␣ TaqMan Gene
Expression Assay (PE Applied Biosystems) and 18S ribosomal
RNA as an internal standard. DNA contamination of all
samples was evaluated by using RNA as reaction template (RT
negative control).
Tissue specimens were washed with PBS, minced, and
digested enzymatically with 1.5 ␮g/ml Dispase II (Boehringer
Mannheim, Rotkruez, Switzerland) in PBS. The released cells
were grown for 6 passages in Dulbecco’s modified Eagle’s
medium (DMEM; Life Technologies, Basel, Switzerland) supplemented with 10% fetal calf serum (FCS; PAA Laboratories,
Linz, Austria), 50 IU/ml penicillin, 50 ␮g/ml streptomycin, 2
mM L-glutamine, 0.5 ␮g/ml amphotericin B, and 10 mM
HEPES (all from Life Technologies). The cells were incubated
with medium alone or with medium containing a physiologic
amount of endotoxin-free proinflammatory cytokines (i.e., 10
mg/ml TNF␣ and 1 ng/ml IL-1␤ [both from Invitrogen, Paisley,
UK]), or cyclodextrin-encapsulated dexamethasone (10 ng/ml;
Sigma).
The cultures were treated with collagenases (Accutase;
Omnilab, Breda, The Netherlands) to obtain single-cell suspensions, resuspended in DMEM with 10% FCS, and kept at
37°C for 30 minutes in a tube rotator before staining. The
purity of the fibroblast cultures was analyzed using the fibroblast marker CD90 (Thy-1, clone AS02; Dianova, Hamburg,
Germany) and CD45 (Hle-1; Becton Dickinson, Basel, Switzerland). The expression of IL-10R␣, intracellularly or on the
cell surface, was detected using murine monoclonal antibodies
(clone 37606.11; R&D Systems) and a fluorescein-conjugated
AffiniPure F(ab⬘)2 fragment goat anti-mouse IgG (H⫹L)
(Jackson ImmunoResearch). Intracellular staining was per-
DOI 10.1002/art.21274
Deficient expression of interleukin-10 receptor ␣ chain
in rheumatoid arthritis synovium: limitation of animal
models of inflammation
Rheumatoid arthritis (RA) is a chronic systemic disorder of unknown etiology, in which cytokines are thought to be
key modulators. Thus, controlling the production and activity
of proinflammatory cytokines such as tumor necrosis factor ␣
(TNF␣) represented a major therapeutic breakthrough. Although the first data from clinical trials showed efficacy, they
also revealed that blockade of these cytokines did not fully
control arthritis in all patients (1). Alternatively, the antiinflammatory cytokines interleukin-4 (IL-4) and IL-10 were
considered to be promising inhibitors of the pathologic immune response in RA (2). Consequently, gene therapy in
animals models targeted mainly the proinflammatory cytokines, resulting in overexpression of antiinflammatory cytokines.
Ex vivo gene transfer has been performed with synovial
cells, fibroblasts, T cells, dendritic cells, and different cells of
xenogeneic origin (3). For instance, primary cells transduced
with retroviral vectors to drive expression of IL-4 and IL-10,
delivering these immunoregulatory proteins to the inflamed
lesions, were found to represent efficient therapies in experimental models of autoimmune disease such as type II
collagen–induced arthritis in mice (4). Recombinant human
IL-10 has been produced and tested in clinical trials involving
diseases such as RA, inflammatory bowel disease, psoriasis,
organ transplantation, and chronic hepatitis C (5). However, in
contrast to the promising results observed in animal models,
several clinical trials performed with human recombinant
IL-10 in patients with RA showed little efficacy.
The IL-10 receptor (IL-10R) is composed of at least 2
subunits that are members of the interferon receptor family.
The unresponsiveness of keratinocytes to IL-10 results from
the absence of clear IL-10R␣ expression, whereas IL-10R␤ is
strongly expressed (6). Because any stimulus activating IL10R␣ expression suffices to render most cells responsive to
IL-10 (7), we compared the expression of this subunit in
synovial tissue obtained from patients with RA and patients
with osteoarthritis (OA).
Synovial tissue specimens were obtained during smalljoint arthroplasty from 6 patients with RA and 6 patients with
OA. All of the patients with RA fulfilled the 1987 revised
criteria of the American College of Rheumatology (formerly,
the American Rheumatism Association) for a diagnosis of RA
(8). All patients provided informed consent, and the study was
approved by the ethics committee of the University Hospital
Zurich. The specimens were divided into 3 parts. The first part
was embedded in paraffin for immunohistochemical analysis,
the second part was frozen in liquid nitrogen for RNA
extraction, and the third part was kept in phosphate buffered
saline (PBS) until cell culture was performed.
The expression of IL-10R␣ was determined by immunohistochemical analysis, using specific murine monoclonal
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CONCISE COMMUNICATIONS
Figure 1. Expression of interleukin-10 receptor ␣ (IL-10R␣) in synovial tissue from a patient with rheumatoid arthritis (RA)
and a patient with osteoarthritis (OA). No or only weak expression of IL-10R␣ was detected in RA synovial tissue. In contrast,
OA tissue showed high to very high expression of IL-10R␣, particularly in the sublining layer.
formed using Cytofix/Cytoperm buffer (Becton Dickinson).
Purified murine IgG1 (R&D Systems) was used as a negative
primary antibody control. The fluorescence-positive marker
was set at ⬍2%, using the negative control antibodies. Firstlevel supply/side scatter gating was set on single cells, without
debris or large cell aggregates. The Mann-Whitney U test was
used for comparison of median values.
Immunohistochemical analysis revealed that those OA
synovial tissues that have important inflammatory components
also have a high expression of IL-10R␣. This protein was
detected in all OA samples (2 of 5 samples had high expression, and 3 of 5 samples had very high expression, particularly
in the sublining layer). Figure 1 illustrates the difference
between expression in RA and OA synovial tissue. In RA
synovial tissue, only very weak staining of IL-10R␣ (2 of 5
samples) could be observed, and the staining was restricted
mostly to the perivascular region.
Semiquantification of IL-10R␣ protein expression in
the synovial tissue by 2 independent observers confirmed the
difference between OA and RA in the sublining layer: the
median score for 6 OA samples was 1.50, compared with a
median score of 0.37 for 6 RA samples (P ⬍ 0.005). Regarding
the lining layer (median scores 0.50 and 0.12, respectively; P ⫽
0.09), the difference did not reach the level of significance,
whereas the vessels expressed even more IL-10R␣ in RA
samples than in OA samples (median scores 0 and 0.75,
respectively; P ⬍ 0.02).
Quantification of IL-10R␣ mRNA by real-time PCR in
whole synovial tissue showed no significant difference between
6 OA samples and 6 RA samples (for OA, median ⌬Ct ⫽ 9.05
[range 4.96–12.11]; for RA, median ⌬Ct ⫽ 6.57 [range 5.04–
9.56]; P ⫽ 0.18).
Using flow cytometry, we were able to show differences in the constitutive expression of IL-10R␣ on the cell
surface and in the response to stimulation with proinflammatory cytokines. Figure 2A shows representative examples. RA
synovial fibroblasts had a deficiency in the constitutive expression of IL-10R␣. The prevalence of positive cells varied
between 1% and 3% (n ⫽ 5). In contrast, OA synovial
Figure 2. Expression of interleukin-10 receptor ␣ (IL-10R␣) in osteoarthritis synovial fibroblasts (OASF) and rheumatoid arthritis synovial
fibroblasts (RASF), and the effect of proinflammatory cytokines.
Up-regulation of IL-10R␣ in the presence of proinflammatory cytokines occurred only in OA synovial fibroblasts, not in RA synovial
fibroblasts. TNF ⫽ tumor necrosis factor.
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fibroblasts showed 2 subpopulations, the first with low levels of
IL-10R␣, and the second with high levels of IL-10R␣. The
prevalence of cells with high levels of IL-10R␣ varied from
29% to 48% (n ⫽ 3). Figure 2B shows the percent of cells
positive for IL-10R␣ before and after stimulation, and Figure
2C shows the average expression of IL-10R␣ as reflected by
the median fluorescence intensity. The differences between
RA and OA samples were significant, both before and after
stimulation (P ⱕ 0.02). The intracellular expression of IL10R␣ in permeabilized cells, however, was not significantly
different (data not shown).
Dexamethasone did not decrease the basal or cytokinestimulated expression of IL-10R␣ in OA or RA synovial
fibroblasts (data not shown). Thus, the altered expression of
IL-10R␣ is not the consequence of previous treatment with
glucocorticoids.
Our results indicate a deficiency of IL-10R␣ protein in
RA synovial tissue, particularly in the sublining layer, and on
the cell surface of RA synovial fibroblasts. In contrast to OA
synovial fibroblasts, RA synovial fibroblasts are unable to
up-regulate the expression of IL-10R␣ in response to the
inflammatory microenvironment.
A previous study (8) demonstrated that dendritic cells
from the synovial fluid of patients with RA did not constitutively express IL-10R␣ mRNA or detectable surface protein.
This deficiency appears to be specific for the RA synovial
microenvironment, because the expression of IL-10R␣ in RA
peripheral blood leukocytes has been found to be within the
normal range (10).
The expression of IL-10R␣ protein on the cell surface
does not necessarily correlate with the accumulation of IL10R␣ mRNA, because in cells deficient at the cell surface, it
can be stored intracellularly (9). It has been hypothesized that
the capacity to transport intracellular IL-10R␣ to the cell
surface or to signal through IL-10R␣ is modulated by combined or sequential exposure to IL-10 and certain proinflammatory cytokines.
IL-10 is a pleiotropic cytokine produced by activated T
lymphocytes, B cells, monocytes, and macrophages, inhibiting
a broad array of immune parameters, including the production
of other cytokines, and even antigen presentation and antigenspecific T cell proliferation. Normal mesenchymal lining cells
(11) and OA synovial fibroblasts express IL-10R␣. In RA
synovium, we observed that the expression of IL-10R␣ (if any)
was mainly restricted to the perivascular region, in spite of the
fact that a previous study (12) demonstrated that normal
vascular tissue does not show IL-10R␣. Already this difference
reflects a pathologic alteration and could also explain the lack
of quantitative difference in IL-10R␣ mRNA using whole
synovial tissues (i.e., neoangiogenesis being an important
pathophysiologic process in RA).
Although RA synovial fibroblasts are shown to be
activated and to produce a variety of enzymes and to be the
major factors mediating cartilage destruction in RA (13), they
are deficient in the expression of IL-10R␣, both in vitro and in
situ. Moreover, in contrast to OA synovial fibroblasts, RA
synovial fibroblasts did not increase the expression of IL-10R␣
in response to physiologic doses of proinflammatory cytokines.
This in vitro observation is consistent with the in situ situation,
as shown by immunohistochemical analysis. Probably, the
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synovial tissue in OA responds adequately to the inflammatory
microenvironment, whereas in RA the response is strongly
reduced. There is little information on factors—besides proinflammatory cytokines—that modulate the expression of IL10R␣ (e.g., lipopolysaccharides increase expression of IL10R␣ in rat liver and hypothalamus) (14).
Regarding the lack of efficiency of IL-10 in human
RA, an additional mechanism has been considered by other
investigators (15). Thus, up-regulation of Fc␥ receptor
expression in RA during IL-10 treatment may counteract
the otherwise antiinflammatory effects of this cytokine by
potentiating immune complex–mediated proinflammatory responses.
Taken together, our data clearly indicate a deficiency
of IL-10R␣ in RA synovial tissue, particularly in the sublining
and synovial fibroblasts. In fact, the different cell types within
RA synovial tissue can be responsive to IL-10 at varying
degrees, as suggested by the increased IL-10R␣ expression in
small vessels. The lack of IL-10R␣ up-regulation upon stimulation with proinflammatory cytokines could explain why clinical studies performed with recombinant IL-10 in patients with
RA showed little efficacy.
Michel Neidhart, PhD
Astrid Jüngel, PhD
Caroline Ospelt, MD
Beat A. Michel, MD
Renate E. Gay, MD
Steffen Gay, MD
University Hospital Zurich
Zurich, Switzerland
1. Taylor PC. Anti-cytokines and cytokines in the treatment of
rheumatoid arthritis. Curr Pharm Des 2003;9:1095–106.
2. Lubberts E, van den Berg WB. Potential of modulatory cytokines
in the rheumatoid arthritis process. Drug News Perspect 2001;14:
517–22.
3. Bessis N, Doucet C, Cottard V, Douar AM, Firat H, Jorgensen C,
et al. Gene therapy for rheumatoid arthritis. J Gene Med 2002;4:
581–91.
4. Slavin AJ, Tarner IH, Nakajima A, Urbanek-Ruiz I, McBride J,
Contag CH, et al. Adoptive cellular gene therapy of autoimmune
disease. Autoimmun Rev 2002;1:213–9.
5. Asadullah K, Sterry W, Volk HD. Interleukin-10 therapy: review
of a new approach. Pharmacol Rev 2003;55:241–69.
6. Seifert M, Gruenberg BH, Sabat R, Donner P, Gruetz G, Volk
HD, et al. Keratinocyte unresponsiveness towards interleukin-10:
lack of specific binding due to deficient IL-10 receptor 1 expression. Exp Dermatol 2003;12:137–44.
7. Moore KW, de Waal Malefyt R, Coffman RL, O’Garra A.
Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 2001;19:683–765.
8. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF,
Cooper, NS, et al. The American Rheumatism Association 1987
revised criteria for the classification of rheumatoid arthritis.
Arthritis Rheum 1988;31:315–24.
9. McDonald KP, Pettit AR, Quinn C, Thomas GJ, Thomas R.
Resistance of rheumatoid synovial dendritic cells to the immunosuppressive effects of IL-10. J Immunol 1999;163:5599–607.
10. Cairns AP, Crockard AD, Bell AL. Interleukin-10 receptor expression in systemic lupus erythematosus and rheumatoid arthritis.
Clin Exp Rheumatol 2003;21:83–6.
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11. Ritchlin C, Haas-Smith SA. Expression of interleukin 10 mRNA
and protein by synovial fibroblastoid cells. J Rheumatol 2001;28:
698–705.
12. Tedgui A, Mallat Z. Anti-inflammatory mechanisms in the vascular wall [review]. Circ Res 2001;88:877–87.
13. Ospelt C, Neidhart M, Gay RE, Gay S. Synovial activation in
rheumatoid arthritis. Front Biosci 2004;9:2323–34.
14. Ledeboer A, Binnekade R, Breve JJ, Bol JG, Tilders FJ, van Dam
AM. Site-specific modulation of LPS-induced fever and interleukin-1␤ expression in rats by interleukin-10. Am J Physiol Regul
Integr Comp Physiol 2002;282:R1762–72.
15. Van Roon J, Wijngaarden S, Lafeber FP, Damen C, van de Winkel
J, Bijlsma JW. Interleukin 10 treatment of patients with rheumatoid arthritis enhances Fc ␥ receptor expression on monocytes and
responsiveness to immune complex stimulation. J Rheumatol
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DOI 10.1002/art.21371
Molecular evidence of HLA–B27 in a historical case of
ankylosing spondylitis
The association between HLA–B27 and ankylosing
spondylitis (AS) is the strongest known association of any HLA
antigen with human disease (1). AS belongs to the family of
spondylarthropathies (SpA), a group of related chronic inflammatory rheumatic diseases sharing several clinical features (2).
The most common one is their association with HLA–B27 (3).
Despite the strong linkage between HLA–B27 and AS,
no clear evidence of B27 in ancient and historical samples has
been reported to date. The present study was conducted to
confirm a morphologic diagnosis and to describe the application of a method that may be used to expand the capacity of
paleopathologic research.
The remains of a male individual (age 62 years [⫾5
years] at the time of death) who had been buried at Blanche
Eglise Church in La Neuveville, Switzerland has been subjected to macroscopic, radiologic, and genetic analysis. The
individual died between the 14th and 18th centuries AD. We
sought evidence of an HLA–B27 allele in this individual, who
was assumed to have had AS based on findings described
below.
We extracted DNA from 2 distal parts of the femur
and carried out HLA–B27 sequence-specific polymerase chain
reaction (PCR) studies. The extraction and testing of ancient
DNA was performed under recommended conditions (4). The
B27 sequence-specific primers have been described previously
(5). Negative control procedures were conducted for all extractions and all PCRs, and, as expected, showed no positive
results. Two of 3 PCR products from each extraction from the
“La Neuveville” individual were sequenced directly, and 1
product from each extraction was cloned (10 clones and 12
clones, respectively, were sequenced) (results not shown). In
addition, genetic typing of the historical individual and of all
researchers involved in this study was performed, using the
AmpFLSTR Profiler Plus PCR Amplification Kit according to
the instructions of the manufacturer (Applied Biosystems,
Foster City, CA).
Based on the spinal remains, the “La Neuveville”
individual was presumed to have AS rather than a degenerative
disease or another type of SpA. Macroscopic and radiologic
pathodiagnostic findings included extensive syndesmophytes,
ossified interspinous ligaments, ankylosed facet joints, and
mild left convex scoliosis (Figure 1). B27 sequence-specific
Figure 1. Lateral view of a gross specimen (A) and anterposterior
radiograph (B) of the osseous lower thoracic/lumbar spine, suggesting
ankylosing spondylitis, in a man who died between the 14th and 18th
centuries AD, at the age of ⬃62 years.
PCR of DNA extracted from 2 parts of the femur showed
positive results matching those in recently obtained positive
clinical control samples from a rheumatology outpatient unit
(Johannes Gutenberg University Mainz). The retrieved sequences (GenBank accession nos. AY829003–AY829007) represent one of several known HLA–B27 alleles. Additional
genetic typing of all researchers involved in this study supported the authenticity of the ancient DNA cloning and
sequencing results (Table 1); no evidence of contamination
was found, despite the application of rigorous authentication
criteria (4).
In addition to the fact that the authentication criteria
for analysis of ancient DNA were fulfilled, there was further
evidence that the material tested was endogenous DNA from
the “La Neuveville” individual in that 1) none of the study
researchers was typed as B27 positive and 2) B27-positive and
B27-negative controls were not brought into the building until
after the extractions and PCR studies of the “La Neuveville”
individual had been performed.
Although it is clear that HLA–B27 is the predominant
predisposing genetic factor in AS, other genetic factors (within
and outside the major histocompatibility complex) and environmental factors are likely involved as well. It should be noted
that AS can also occur in the absence of this allele. In addition,
the contribution of B27 to the genetic susceptibility to AS has
been estimated to be ⬃20–30%. Thus, B27 testing alone is not
clinically helpful. However, in combination with positive results from the case history, the physical examination, and the
radiologic diagnosis, a positive result on B27 testing increases
the probability that the patient has AS (2).
This represents the first successful attempt to link
allele HLA–B27 and its pathogenic effects in historical human
remains. Future studies may focus on the episodic historical
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expressions, model, inflammation, chains, deficiency, animals, arthritis, interleukin, receptov, synoviumlimitation, rheumatoid
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