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PREDICTIVE ROLE OF EXTRACELLULAR SURVIVIN FOR THE
DEVELOPMENT OF JOINT DESTRUCTIONS IN RHEUMATOID
ARTHRITIS
Maria BOKAREWA, Department of Rheumatology and Inflammation Research, Göteborg University
In collaboration with
Lennart Bertilsson, Rheumatology Clinics, Sahlgrenska University Hospital
Göran Kvist, Rheumatology Unit, Southern Alfsborg Hospital, Borås
Christopher Schaufelberger, Rheumatology Unit, Mölndal Hospital,
Andrey Shestakov, Department of Rheumatology, Göteborg University
Tomas Torstenson, Rheumatology Unit, Uddevalla Hospital
Specific aims
1) To prospectively analyse the role of survivin for the development of destructive joint disease in
patients with RA
2) To assess the role of survivin in differentiation and phenotypical changes of synovial and bone cells
facilitating joint destruction
3) To evaluate an efficient and safe mean of disrupting survivin signalling preventing development of RA
Background
Chronic inflammation followed by progressive destruction of the joints is a hallmark of RA. It is
generally accepted that the inflammatory process in RA synovial tissue is coordinated by cytokines.
Uncontrolled, tumour-like growth of synovial tissue followed by invasion and destruction of cartilage
is a classical feature of RA. Synovial fibroblasts of RA patients demonstrate features of transformed
cells with multiple oncogene mutations and impaired apoptosis, having the ability to proliferate, to
invade the surrounding tissues in a tumor-like fashion. Apoptosis is a programmed cell death that may
be initiated by disruption of mitochondrial integrity inside the cell or by stimulation with TNF or Fasligand of the cell surface Fas/APO-1.
We were first to show that activity of apoptosis inhibitor survivin is dramatically increased in RA
patients. Moreover, high levels of extracellular survivin are associated with 16-fold increased
frequency of a destructive joint disease (2). In contrast, circulating antibodies against survivin in RA
patients are associated with less aggressive, non-erosive course of arthritis. These findings indicate
survivin to be an important participant in pathogenesis of RA. Little is known about regulation of
survivin expression. Signalling through Wnt glycoproteins followed by nuclear translocation of catenin has been suggested as one of mechanisms upregulating expression of survivin. Members of
Wnt family are overexpressed in RA synovium. Transfection of normal synovial fibroblasts with Wnt
expressing construct results in RA-like cytokine production pattern and activated invasive cell
phenotype. Nuclear hormone receptor, peroxisome proliferator-activated receptor (PPAR)- is shown
to antagonise Wnt signalling. We have shown that PPAR- agonists downregulate expression of both
resistin and survivin. In turn, incubation of human leukocytes with resistin results in suppression of
PPAR- transcription. Agonists of PPAR- (glitazones) are used as anti-diabetic drugs, but other
applications as anti-tumour and anti-atherosclerotic agents are being presently tested in clinical trials.
In the mouse model of collagen II-induced arthritis, PPAR- agonists are shown to alleviate arthritis.
We suggest resistin-survivin axis to be a novel inflammatory pathway in the pathogenesis of RA.
Working hypothesis
We suggest resistin-survivin axis as a novel pathway in the pathogenesis of RA. Uncontrolled
expression of resistin in the inflamed synovial tissues during RA down-regulates PPAR-gamma and
facilitates activation of Wnt signalling system followed by intracellular accumulation of survivin.
Extracellular survivin regulates cell migration and differentiation facilitating their invasive and
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osteoclastic phenotype. This would support destruction of cartilage and subchondral bone. Resistin
orchestrates cytokine-driven synovial inflammation while survivin contributes to destructive arthritis.
Materials and Methods
1. Extracellular functions of survivin
Which cells respond to stimulation with extracellular survivin? How does stimulation with
survivin change function of leukocytes?
Recombinant human survivin is expressed in E.coli using a pHIS8 expression vector provided
by Mark A. Verdecia, The Salk Institute, LaJolla, CA (5). The survivin sequence in the plasmid was
verified by DNA sequencing using T7 and SP6 primers. Following expression, bacterial pellet was
submitted to lyzosyme cleavage and sonication. Survivin was purified through His-tag on a Ni2+-NTA
column. His-tag was removed by thrombin cleavage. Survivin was depleted from endotoxin (using
Detoxy gel) and dialysed. The presence of survivin in the eluate was tested by Western blot and its
concentration was detected by anti-survivin ELISA. Mouse monoclonal antibodies to human survivin
are raised in collaboration with Prof.Pietro Speziale, University of Pavia, Italy.
Peripheral blood granulocytes and mononuclear cells (lymphocytes and monocytes) from
healthy controls and patients with RA will be stimulated with recombinant survivin. The effects of
extracellular survivin will be assessed with respect to phenotypical and functional changes –
expression of adhesion molecules (integrins, selectins, immunoglobulin superfamily), co-stimulatory
molecules (CD25, CD28, CD40, CD80), and cytokine production (IL1, IL2, IL4, IL6, IL10, IFN-).
To evaluate the capacity of survivin to induce apoptosis, leukocytes from healthy subjects and
RA patients treated with recombinant survivin will be assessed for the expression of annexin V
(FACS), TUNEL assay, activation state of mitochondrial membrane (ApoAlert), and cytochrome c
release (ELISA). Cell cycle response to survivin treatment will be assessed using FACS and
employing bromodeoxyuridine incorporation for the assessment of S-phase cells and propidium iodide
incorporation for the assessment of total DNA content.
To assess the role of survivin in lymphocyte response, PBMC will be stimulated with tetanus
toxoid (antigen specific response), phytohaemagglutinin (mitogen-induced response), TSST-1
(superantigen induced response), or submitted to mixed leukocyte response (allogenic response).
Binding of survivin to different types of leukocytes will be visualised by FACS.
Samples of rheumatoid synovia will be cultured in survivin-rich medium. Additionally,
explants of synovial tissues will be treated with antibodies against survivin. Synovial tissues will be
assessed for a) the invasive growth and proliferation capacity, b) expression of adhesion molecules and
apoptosis markers, c) expression of proteases and their specific inhibitors (uPA/PAI, cathepsin
K/cystatin c, metalloproteinases/TIMPs).
2. Role of extracellular survivin in cell differentiation
What is the role of survivin in maturation and differentiation of mononuclear cells? What is the
role of extracellular survivin in osteogenesis and bone remodelling? Which intracellular
mechanisms are activated by survivin?
In a cross-sectional study of RA patients we observed high correlation between the presence of
extracellular circulating survivin and joint destructions (2). However, survivin had no direct correlation
to the markers of inflammation, as serum amyloid A protein or IL-6 in vivo (6) and induced no
cytokine expression in vitro. In contrast, stimulation of human leukocytes with survivin resulted in
significant activation of p38 MAP-kinase (5), a potent regulator of osteogenesis.
We aim to assess the role of survivin in differentiation of osteoclasts/osteoblasts. Mononuclear
cell lines THP-1 and HL60 will be used to assess further differentiation of monocytes mediated by
survivin. THP-1 will be driven to a macrophage and fibroblast differentiation using PMA and all-trans
retinoic acid (7). HL60 will be differentiated to osteoclasts using DMSO and 1, 25(OH)2dihydroxyvitamin D3 (8). Cells will be cultured in M-CSF/RANKL rich medium in the presence and
absence of survivin. The development of osteoclasts will be followed by the formation of multinuclear
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TRAP-positive cells. The osteoblastic differentiation will be followed by mineralization (cresolphthalein) and alkaline phosphatase (ALP) activity. Expression of RANK and osteoprotegrin
will be assessed immunohistologically.
Additionally, cells will be cultured in the presence of constant levels of anti-survivin
antibodies. Alternatively, intracellular survivin will be blocked using siRNA. The role of p38 MAPK
in osteoclast differentiation will be tested using synthetic inhibitor of p38 (SB203580). For monitoring
the effect of p38 deficiency, the resorbtion capacity of differentiated osteoclasts will be employed.
PPAR-gamma is known as a potent regulator of both PI3-kinase and Wnt signalling pathways.
Role of PPAR-gamma in survivin-induced cell differentiation will be assessed using PPAR-gamma
agonist (rosiglitazone) and PPAR-gamma antagonist (T0070907) supplementation in PBMC cultures.
To study the role of Wnt signalling, human wnt5a cDNA will be subcloned into the expression vector
pcDNA3 (Invitrogen), and synovial fibroblasts will be transfected with wnt5a expressing DNA
plasmid. Expression of wnt protein (wnt2 and wnt3a) will be assessed by Western blot. Intracellular
expression of p38, PI3-kinase and PPAR-gamma will be assessed at different phases of cell
differentiation by Fast Activated Cell-based ELISA (FACE, Active Motif).
3. Predictive role of survivin for the development of destructive rheumatoid arthritis
What is the frequency of patients with early RA displaying high circulating survivin levels? May
survivin level be used to identify patients with unfavourable course of RA and to predict the
development of joint destruction?
To assess the frequency and prognostic significance of circulating survivin in early RA,
patients with RA duration below 3 years attending Rheumatology Units at the hospitals of the Western
region of Sweden (Västra Götaland) will be studied. At inclusion, RA patients will be clinically
assessed, perform radiological examination of hand and foot skeleton, and leave blood samples.
Clinical characteristics of disease activity (Disease Activity Score, DAS28) and functional disability
(Health Assessment Questionarie, HAQ) will be registered. Blood samples will be used for the
measurement of survivin level (ELISA) and determination of established predictive factors of RA
including rheumatoid factors and antibodies against citrullinated peptides will be performed. Three
years later the same cohort of patients will be evaluated clinically, radiologically and for the presence
of circulating survivin. To assess the predictive value of survivin on the course of RA, the patients will
be stratified according to the presence of circulating survivin and other predictive markers in the first
sample. The outcome will be evaluated with respect to joint destruction (radiologically evaluated as
erosivity, Larsen score (4)), and the requirement of treatment intensity. Biochemical markers of
cartilage and bone metabolism in the patients with high and low levels of extracellular survivin will be
compared including the levels of bone sialoprotein, C-terminal telopeptide of type I collagen (ICTP),
type II collagen C-telopeptide (CTX-II), cartilage oligometric matrix protein (COMP).
We expect to include 400 patients during a three-year period with a drop out from the study
between 25-30% before the second examination. At present about 150 RA patients with the disease
duration less than 3 years are enrolled in the study and they left their first blood sample. In the
preliminary assessment of these 150 samples, circulating survivin is found in more than 40% of these
patiens.
Significance
The recognition of survivin as an important prognostic factor for the development of joint destructions
helps identification of patients with poor prognosis of RA and monitoring of therapeutic efficacy.
Clinical relevance
Rheumatoid arthritis (RA) is a common disease with the prevalence of 0.5-1% in the entire population.
Pathogenetic mechanisms and prognostic features of RA are not completely understood. My research
group suggests a novel set of molecular events initiating inflammation in the joints and leading to joint
destruction. We demonstrated that anti-apoptotic molecule, survivin, may be used as a potential
predictive marker of destructive course of arthritis. Our hypothesis is based on the results obtained in
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patient material and is supported by studies on cell level and in animal models. We plan to expand our
observations preparing a prospective clinical study including a cohort of patients from Västra Götaland
region. We will also continue our studies on the molecular events initiated by survivin on the cell
surface and inside the cells. We believe that better understanding of survivin-mediated processes is an
important part of joint inflammation. Targeting these processes may become a new efficient treatment
modality of patients with RA.
Ethical considerations
Synovial fluid and blood samples will be obtained after the patient is informed and gives an agreement
to participate in the study. Patient identity will be protected by an immediate coding of all the samples.
Analyses of the samples are performed using coded tubes. Study is approved by the Ethic Commettee
of Sahlgrenska University Hospital (S-441-01, 385-04). Animal studies will be performed with an
agreement and according to the guidelines of Helsinki Convention. Animal models of collageninduced arthritis (332-2003), staphylococcal arthritis (326-2003), and resistin-induced arthritis (2712003) are all approved by the Committee for Animal Experiments, Göteborg University.
Preliminary results
1. Extracellular expression of survivin during rheumatoid arthritis
We have shown that high extracellular levels of survivin may be detected in about 30% of RA patients.
Moreover, survivin expression has been found to be an independent prognostic marker of erosive
disease increasing risk of joint destructions in RA 16-folds. In contrast, antibodies against survivin
were associated with less aggressive non-erosive course of joint disease (2).
P<0.0019
P<0.05
600
P<0.035
Plasma
Synovial fluid
450
300
150
0
RA, erosive
n=88
RA, non-erosive
n=43
Healthy controls
n=34
Increased frequency of circulating survivin was also observed in patients with juvenile chronic arthritis
(about 20%). Circulating survivin was associated with systemic and oligoarticular type of JCA and the
presence of RF. Indicating a possible predictive value of circulating survivin for identification of
patients with unfavourable course of JCA (6).
2. Recombinant survivin and monoclonal antibodies
For the evaluation of extracellular properties of survivin we a) successfully expressed recombinant
human survivin, b) obtained significant amount of monoclonal antibodies to survivin for our further
studies.
3. Properties of extracellular survivin
We have identified two leukocyte populations sensitive to survivin. Survivin binds to lymphocytes and
neutrophils. Stimulation with extracellular survivin leads to the expression of 2-integrins on the
surface of human neutrophils. Survivin-dependent upregulation of 2-integrins was independent on
NF-kappaB pathway and was mediated by p38 MAP-kinase and PI3-kinase/Akt signalling. The
clinical relevance of this observation is supported by studies of RA patients showing that the
neutrophils of patients with high extracellular survivin levels express higher proportion of 2-integrins
(5). In human lymphocyte survivin prevented CD3-dependent proliferation and production of IL2 and
INF-gamma cytokines.
5
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Pr
oli
fer
ati
on
,
%
P<0.05
P<0.05
n.s.
150
survivin, 0 microg/ml
survivin, 0.05 microg/ml
survivin, 5 microg/ml
survivin, 25 microg/ml
100
50
0
TSST-1
n=6
anti-CD3
ConA
n=4
n=6
4. Intracellular signalling initiated by survivin
Our recent in vitro experiments revealed that intracellular effects of survivin are mediated by PI3kinase and p38 MAP-kinase, potent regulators of osteogenesis. The effects are abrogated by specific
inhibitors of transcription factors (SB203580 for p38, and LY294002 for PI3-kinase). These two
findings support our version of survivin being an important participant of bone remodelling during
arthritis.
References
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properties. J Immunology 2005, 173:5789-95.
2. Bokarewa MI, Lindblad S, Bokarew D, Tarkowski A. Balance between survivin, a key member of apoptosis inhibitor
family, and its specific antibodies determines erosivity in rheumatoid arthritis. Arth Res&Ther 2005, 7:R349-R358.
3. Pullerits R, Jonsson IM, Verdrengh M, Bokarewa MI, Andersson U, Erlandssson-Harris H, et al. High mobility group
box chromosomal protein (HMGB-1) is a proinflammatory molecule inducing arthritis. Arthritis Reum 2003,
48:1693-700.
4. Larsen A. How to apply Larsen score in evaluating radiographs of rheumatoid arthritis in long-term studies? J Rheumatol
1995, 22:1974–5.
5. Mera, S., Levshin, N., Magnusson, M., Tarkowski, A., and Bokarewa, M.I. Extracellular survivin activates adhesion
molecules on the surface of human granulocytes supporting leukocyte interactions. 2006, submitted.
6. Galeotti, L., Adrian, K., Berg, S., Tarkowski, A., and Bokarewa, M.I. Circulating survivin is a marker of severe juvenile
chronic arthritis. 2006, submitted.
7. Jakob F, Siggelkow H, Homann D, Kohrle J, Adamski J, Schutze N. Local estradiol metabolism in osteoblast- and
osteoclast-like cells. J Steroid Biochem Mol Biol 1997, 61:167-74.
8. Zhou Z, Immel D, Xi CX, Bierhaus A, Feng X, Mei L, Nawroth P, Stern DM, Xiong WC. Regulation of osteoclast
function and bone mass by RAGE. J Exp Med 2006, 203:1067-80.
9. Shestakov A, Fu H, Dahlgren C, Tarkowski A, and Bokarewa MI. Regulatory mechanisms of resistin release from
specific granules of neutrophils. Manuscript.