Balance between survivin, a key member of the apoptosis inhibitor family, and its specific antibodies determines erosivity in rheumatoid arthritis
© Bokarewa et al., licensee BioMed Central Ltd. 2005
Received: 25 October 2004
Accepted: 20 December 2004
Published: 21 January 2005
Rheumatoid arthritis (RA) is a highly heterogeneous disease with respect to its joint destructivity. The reasons underlying this heterogeneity are unknown. Deficient apoptosis in rheumatoid synovial tissue has been recently demonstrated. We have therefore decided to study the synovial expression of survivin, a key member of the apoptosis inhibitor family. The levels of survivin and antibodies against survivin were assessed by an ELISA in matched blood and synovial fluid samples collected from 131 RA patients. Results were related to joint erosivity at the time of sampling. Monocytes were transfected with survivin anti-sense oligonucleotides and were assessed for their ability to produce inflammatory cytokines. Survivin levels were significantly higher in patients with destructive disease as compared with in RA patients displaying a non-erosive disease. High survivin levels were an independent prognostic parameter for erosive RA. In contrast, high levels of antibodies against survivin were found in patients with non-erosive RA, and were negatively related to erosivity. Survivin levels in RA patients were influenced by treatment, being significantly lower among patients treated with disease-modifying anti-rheumatic drugs. Specific suppression of survivin mRNA resulted in downregulation of IL-6 production. We conclude that survivin determines the erosive course of RA, whereas survivin antibodies lead to a less aggressive course of the disease. These findings together with decreased survivin levels upon disease-modifying anti-rheumatic drug treatment, and the downregulation of inflammatory response using survivin anti-sense oligonucleotides, suggest that extracellular survivin expression mediates the erosive course of joint disease whereas autoimmune responses to the same molecule, manifested as survivin targeting antibodies, mediate protection.
Keywordsapoptosis arthritis autoimmunity prognosis survivin
Rheumatoid arthritis (RA) is an inflammatory joint disease characterized by hyperplasia of synovial tissue and pannus formation growing invasively into the cartilage, followed by cartilage and bone destruction. Analyses of hyperplastic synovial tissues of patients with RA reveal features of transformed long-living cells such as the presence of somatic mutations, expression of oncogenes, and resistance to apoptosis [1–3]. Resistance to apoptosis further contributes to synovial hyperplasia and is closely linked to the invasive phenotype of synovial fibroblasts [4, 5].
Apoptosis is a tightly regulated process of elimination of aged cells without disrupting cellular integrity (reviewed in [6, 7]). Apoptosis may be initiated by extracellular stimuli through activation of death receptors on the cell surface, and intracellularly by the release of mitochondrial cytochrome c into the cytoplasm. Both pathways induce expression of apoptosis genes and activation of the caspase cascade, resulting in DNA fragmentation. The apoptosis signals are abrogated by the family of apoptosis-inhibiting proteins (IAPs).
A number of disturbances in the apoptosis machinery have been pointed out in RA patients. Fibroblasts from RA synovia are relatively resistant to apoptosis induced by extracellular Fas stimulation. Moreover, co-culture of synovial fibroblasts from RA joints with T cells and B cells induces anergy of lymphocytes. Increased levels of soluble Fas in RA synovial fluid have been suggested as one possible explanation for this fact . Indeed, administration of antagonistic anti-Fas antibodies or of Fas ligand has been shown effective in abrogation of arthritis in animal models [9, 10]. Resistance to Fas-induced apoptosis in RA synovium correlates with a markedly increased expression of sentrin-1 . Sentrin-1/SUMO is a molecule whose binding to a protein results in the prevention of ubiquitin-related processing and degradation of that protein. Sentrin-mediated protection has been shown for such proteins as p53 and IkBa. Upregulation of anti-apoptotic molecules belonging to the Bcl family and of the caspase-8 inhibitor FLIP has been repeatedly reported in RA . Inhibited apoptosis has been shown to contribute to the pathogenesis of experimental arthritis [13, 14].
Survivin is a 142-amino-acid protein that belongs to the IAP family, and it inhibits the activity of caspase 3, caspase 7, and caspase 9, but not of the upstream initiator protease caspase 8. Survivin can thereby downregulate, directly or indirectly, both death-receptor-mediated and mitochondria-mediated pathways of apoptosis . Survivin has been also suggested to regulate cell division during mitosis. Indeed, survivin is the only one of IAPs that is tightly connected to the cell cycle being upregulated in the G2/M phase. Inside the dividing cell, survivin is found incorporated in centrosomes and mitotic spindles, and relocates to midbodies in the late telophase. Disruption of survivin function by negative mutation or by introduction of anti-sense oligonucleotides results in a cell-division defect [16, 17]. Survivin is abundantly expressed in all the most common human cancers and in transformed cell lines , while most normal differentiated adult tissues do not express this molecule. A few adult tissues reported to express survivin include the spleen, the testes, the thymi, the placentas, and the colonic crypts.
In the present study we demonstrate high levels of the anti-apoptotic protein survivin extracellularly in plasma and synovial fluid of patients with RA. In all the cases but one, high levels of survivin were associated with the erosive type of joint disease. Moreover, it is demonstrated that autoantibody responses to survivin led to a more benign (non-erosive) course of RA. The latter finding may have potential therapeutic consequences.
Plasma and synovial fluid samples were collected from 131 RA patients who attended the rheumatology clinics at Sahlgrenska University Hospital, Göteborg for acute joint effusion. RA was diagnosed according to the American College of Rheumatology criteria . At the time of synovial fluid and blood sampling all the patients received non-steroidal anti-inflammatory drugs.
Disease-modifying anti-rheumatic drugs (DMARDs) were used by 96 patients, 67 of which used methotrexate (MTX). Forty-two of these 67 patients combined medication of MTX with the inhibitors of tumour necrosis factor alpha (TNF-α), two other patients combined MTX with sulfasalazine, one patient combined it with cyklosporine A, and the remaining 22 patients were treated with MTX alone. DMARDs other then MTX were used by 14 patients, six patients were treated with sulfasalazine, five patients were treated with cyklosporine A (one patient in combination with azathioprine, one patient with leflunomide, two with sulfasalazine, and the remaining patient with infliximab), four patients used parenteral or oral gold salt compounds, one patient used leflunomide, and one patient used azathioprine. The inhibitors of TNF-α were used in 47 patients (42 patients in combination with MTX, three patients in combination with azathioprine, one patient in combination with cyklosporine, and the remaining patient in combination with cyclophosphamide). The remaining 35 of 131 patients had no DMARD treatment at the time of blood and synovial fluid sampling.
Recent radiographs of the hand and foot skeletons for all patients were studied. The presence of bone erosions, defined as the loss of cortical definition at the joint, was recorded in proximal interphalangeal joints, metacarpophalangeal joints, carpus joints, wrist joints, and metatarsophalangeal joints. The presence of one erosion was sufficient to fulfil the requirement of an erosive disease. We considered the presence of rheumatoid factor (RF) of any of the immunoglobulin isotypes as positive. Informed consent was obtained from the patients and the controls. The study was approved by the Ethics Committee of Sahlgrenska University Hospital.
Analyses of survivin and antibodies to survivin
Synovial fluid samples were obtained by arthrocentesis of knee joints. Synovial fluid was aspirated aseptically and transferred into tubes containing sodium citrate (0.129 mol/l; pH 7.4). We obtained blood samples simultaneously from the cubital vein and directly transferred them into sodium citrate medium. Blood samples from healthy individuals (n = 34; age range, 18–62 years; mean age, 42 ± 7 years) were used as controls. Collected blood and synovial fluid samples were centrifuged at 800 × g for 15 min, aliquoted, and stored frozen at -20°C until use.
Survivin levels were determined by a sandwich ELISA using a pair of matched antibodies (rabbit anti-human survivin; R&D Systems, Stockholm, Sweden). Briefly, 96-well polystyrene dishes (Nunc, Roskilde, Denmark) were coated with capture antibodies and were left overnight at room temperature. Following washing, plates were blocked with PBS–BSA containing 5% sucrose. Matched samples of plasma and synovial fluid were introduced into the parallel strips, at a dilution of one in 10 in PBS–BSA. Horseradish peroxidase-labelled detection antibodies and the corresponding substrate were used for colour development. Double-wave reading at 450 and 570 nm was used and the difference of absorbances was calculated. The obtained absorbance values were compared with the serial dilution of recombinant survivin and are presented as picograms per millitre.
Antibodies of IgG and IgM class specific for survivin were measured in blood and synovial fluid samples by an ELISA. Briefly, 96-well polystyrene dishes (Nunc) were coated with human recombinant survivin (R&D Systems). Reconstituted survivin (0.5 μμg/ml) was introduced in each well and left overnight at room temperature. Following washing with PBS containing 0.1% Tween-20, plates were blocked with 1% ovalbumin (Sigma, St Louis, MO, USA) in PBS for 2 hours at room temperature. Matched samples of plasma and synovial fluid were introduced into the parallel strips, in a dilution of one in 100 using PBS–1% ovalbumin. This dilution was established as being on a linear scale in preliminary titration experiments. Horseradish peroxidase-labelled detection antibodies (rabbit F(ab')2-anti-human IgG and IgM; Dako, Glostrup, Denmark), ExtrAvidin peroxidase conjugate (Sigma), and the corresponding substrate were used for colour development. The absorbance at 405 nm was registered. Absorbances of the patient samples were compared with the mean values obtained in the control group of healthy individuals.
Interaction with survivin transcription
Peripheral blood mononuclear cells (PBMC) were prepared from heparinized blood of healthy individuals by separation on a Lymphoprep density gradient. We washed the cells, and resuspended in complete medium (Iscoves medium containing 1% l-glutamine, 5 × 10-5 M β-mercaptoethanol, 50 μg/ml gentamycin sulphate, and 10% heat-inactivated FCS). We cultured PBMC in 24-well plates in a humidified atmosphere of 5% CO2 at 37°C. In addition, we cultured the human monocytic cell line THP-1 (American Type Culture Collection, Manassas, VA, USA) in 10-ml culture flasks (Nunc) in RPMI 1649 medium supplemented with 10% FCS, 1% sodium pyruvate, gentamycin, and 2.5% Hepes in a humidified atmosphere of 5% CO2 at 37°C. For the experiments, 4-day-old THP-1 cells were harvested, washed, and adjusted to 1 × 106 cells/ml.
For the transfection experiments, phosphorothioated oligonucleotides containing the anti-sense-targeting human survivin gene  were synthesized by MWG Oligo (Ebersberg, Germany). The following anti-sense sequences were used: aSur 1, 5'-CCCAGCCTTCCAGCTCCTTG-3' ; and aSur 2, 5'-GCACCTAGTCTCCCTGCACC-3'. Irrelevant non-sense sequences were used as controls: non-sense 1, 5'-GTCCTCCACTGGCCTCACTC-3' ; and non-sense 2, 5'-CCCCGATTCACCTCGTCCGT-3'. Oligonucleotides were delivered to THP-1 cells using oligofectamine reagent (Invitrogen, Carlsbad, CA, USA). Before the transfection procedure we seeded THP-1 cells in 96-well tissue culture plates and cultured them overnight in RPMI medium free of antibiotics and FCS. Transfection was performed in RPMI medium supplemented with 2.5% Hepes and 100 mg/ml CaCl2. We mixed 0.6 μl oligofectamine with diluted oligonucletides and added it to the washed THP-1 cells. Following 4 hours of incubation at 37°C in a CO2 incubator, the transfection procedure was discontinued by adding RPMI medium containing a threefold excess of FCS. At this time point, we also stimulated the cells with phytohaemagglutinine (PHA) (1.5 μg/ml) if required. Following 48 hours of stimulation, THP-1 cultures were aseptically collected, centrifuged at 1000 × g for 5 min, and kept frozen at -20°C until analysis. We prepared cell lysates by incubating the cell pellet for 1 hour in 1 mM EDTA buffer containing 6 M urea and proteinase inhibitors (Complete MiniTab; Boehringers, Ingelheim, Germany). These preparations were assessed for proliferation, survivin expression, and IL-6 levels.
Cell survival and apoptosis in the transfected cultures were assessed by surface expression of annexin V and propidium iodide intake. Following transfection and stimulation for 48 hours, THP-1 cells were washed and stained with FITC-marked anti-annexin V antibodies and were subjected to flow cytometry (FACSort; Becton Dickinson, San Jose, CA, USA). The results were analysed using the CELLQuest software (Becton Dickinson).
Proliferation of THP-1 cells was assessed by incubating the cell suspension with the test substance for 48 hours. The cells were then pulsed for 12 hours with 1 μCi [3H]thymidine (specific activity, 42 Ci/mmol; Amersham, Bucks, UK). Cells were collected onto glass fibre filters. Thymidine incorporation was measured in a beta-counter. We compared the counts obtained in cells transfected with survivin anti-sense oligonucleotides and those incubated with oligofectamine alone. The results were expressed as a percentage.
The level of IL-6 in supernatants was assessed by a bioassay. The effect of test samples on proliferation of the IL-6-dependent cell line B13.29  was assessed following 72 hours of culturing. The results were analysed by incorporation of [3H]thymidine (Amersham) during the last 4 hours of incubation at 37°C. Cells were collected onto a glass fibre filter. Proliferation in the presence of test samples was compared with that induced by standard dilutions of recombinant IL-6 (Genzyme, Cambridge, MA, USA). The results were further recalculated as in the proliferation assay.
We expressed the level of survivin and antibodies against survivin in the blood, in synovial fluid samples, as well as in cell lysates as the mean ± standard error of the mean. The survivin levels in the matched blood and synovial fluid samples were analysed by the paired Student t test. We further performed a comparison of survivin levels between the patient blood samples and the healthy controls using the Mann–Whitney U test.
We stratified the patient material according to radiological findings (erosive RA versus non-erosive RA) and calculated the difference in survivin levels between the groups employing the Mann–Whitney U test. An arbitrary level of survivin corresponding to three standard deviations of the control group (300 pg/ml) was chosen as a cut-off. The RA patients were further stratified as having 'high' (>300 pg/ml) or 'low' (<300 pg/ml) levels of survivin. We performed the evaluation of survivin as a prognostic factor for the development of joint destruction, comparing the group having 'high' and 'low' survivin levels in a multivariate analysis. In order to control for the role of other prognostic factors (RF, disease duration, age, presence of antibodies against survivin), a multivariate logistic regression was performed. Odds ratios (with 95% confidence interval) are given for descriptive purposes. All tests were two-tailed and conducted at the 5% significance level.
We evaluated a possible influence of the ongoing treatment on the survivin levels, and we stratified patient material according to DMARD treatment (treated versus untreated). For the simultaneous comparison of the survivin levels in more than two groups the equality of variance F test was employed. The inter-relation between the survivin levels and duration of the joint disease, age, white blood cell (WBC) count, and C-reactive protein was calculated employing the Spearman correlation coefficient. For all the statistical evaluation of the results, P < 0.05 was considered significant. All statistical evaluations were performed using StatView PowerPC software.
Clinical and demographic characteristics of patients with rheumatoid arthritis (RA) and of healthy controls
Erosive RA (n = 88)
Non-erosive RA (n = 43)
Controls (n = 34)
Age (years) [mean ± standard deviation (range)]
63 ± 2 (28–85)
53 ± 3 (19–83)
42 ± 1.8 (18–67)
Duration of the disease (years)
12.7 ± 1.2
8 ± 1.4
Rheumatoid factor (+/-)
Treatment with DMARDs
Methotrexate (n = 25)
Other DMARDs (n = 13)
TNF-α blockade (n = 47)
None (n = 45)
Extracellular survivin determines the erosive course of RA
Clinical comparison of patients with rheumatoid arthritis (RA) expressing high* and low levels of survivin
Survivin high, erosive RA (n = 27)
Survivin low, erosive RA (n = 61)
Survivin low, non-erosive RA (n = 42)†
Survivin levels (pg/ml)
1180 ± 309
97 ± 9
127 ± 5
1039 ± 523
132 ± 4
124 ± 2
Disease duration (years)
15.5 ± 2.4
13.6 ± 1.2
8.3 ± 1.4
58 ± 3
60 ± 2
53 ± 3
Rheumatoid factor-positive (n)
C-reactive protein (mg/l)
45 ± 9
29 ± 5
39 ± 7
White blood cell count (× 109/ml)
8.7 ± 0.6
7.2 ± 0.3
7.1 ± 0.3
10.8 ± 1.9
11.2 ± 2.9
13.1 ± 2.8
The level of survivin was also studied in RA synovial fluid samples separated with respect to the cell pellet and the supernatant by centrifugation (n = 9). Survivin levels found in supernatants and in the lysates of synovial fluid cells obtained from the same sample revealed a strong correlation (r = 0.87, P < 0.0001). These data indicate that survivin is produced and secreted locally in the joints of RA patients.
To evaluate the predictive value of high survivin levels for the development of destructive joint disease, a logistic regression model was constructed, taking erosive changes at radiological examination of the hand and foot skeletons as a dependent variable. We found that high levels of survivin were significantly associated with erosive changes (odds ratio, 18.76; 95% confidence interval, 2.45–143.65; P = 0.0048). To determine whether survivin was independently associated with erosive RA, we developed a multivariate logistic regression model with radiological changes as the dependent variable and with RF, duration of RA, gender, and the survivin level as independent variables. After adjusting for the presence of RF, gender, and the duration of RA, a high level of survivin was significantly associated with erosive RA (odds ratio, 16.02; 95% confidence interval, 2.02–127.19; P = 0.013). Our data thus demonstrate that RA patients having high levels of survivin are 16 times more likely to develop erosive joint disease compared with those with low levels of survivin.
Autoantibodies specific for survivin relate to the non-erosive course of RA
Influence of survivin expression on inflammatory responses
PBMC from healthy individuals and from RA patients were stimulated with various B-cell and T-cell mitogens, superantigen, and TNF-α (10–100 ng/ml lipopolysaccharide, 0.5–5 μg/ml Concanavalin A, 10–100 ng/ml TNF-α, 10–100 ng/ml TSST-1, 0.5–5 μg/ml PHA) for 6–48 hours. Supernatants and cell lysates were evaluated for survivin expression by an ELISA. Detectable levels of survivin were not found in supernatants. In the cell lysates, levels of survivin varied in response to the aforementioned stimuli (Fig. 4). In the tested panel, the T-cell mitogen PHA was found to be a potent inducer of survivin expression both by PBMC originating from RA patients (n = 3) and by PBMC from healthy controls (n = 6)
Stimulation of THP-1 with PHA was therefore used in the subsequent transfection experiments. To assess the role of survivin in the inflammatory process, the human mononuclear cell line THP-1 was transfected with oligonucleotides targeting different regions of survivin mRNA. Oligonucleotides were delivered in complex with oligofectamine as described in Materials and methods. Successful transfection with the inhibitory sequence was confirmed by a downregulation of survivin expression in THP-1 lysates as assessed by ELISA. THP-1 cells displayed, as expected, high spontaneous intracellular expression of survivin, which correlated well with their proliferative activity.
Suppression of apoptosis has been suggested as a key mechanism supporting selection and accumulation of distinct lymphocyte subsets in chronically inflamed joint tissues . Indeed, synovial T cells in RA are highly differentiated and would not normally be expected to survive for a prolonged time within inflamed joints unless their death was actively inhibited . In the present study we demonstrate that high expression of survivin, a member of the IAP family, is a new and potentially important mechanism of apoptosis suppression in patients with RA. Survivin is known as a multipotent inhibitor of apoptosis, neutralizing several caspases at the final steps of the apoptosis cascade, thus abrogating signals from both the death-receptor-dependent and mitochondrial pathways of apoptosis. Together with previous findings of upregulation of other caspase inhibitors (Bcl and FLIP) [12, 13], high levels of survivin give new insights in numerous alterations of the apoptosis machinery during the course of RA.
We observed that survivin levels were clearly increased in synovial fluid and plasma of RA patients compared with the healthy controls. Survivin expression was originally considered a reflection of cell proliferation. Indeed, survivin is continuously overexpressed in cancer cells . Survivin gene transcription is repressed by wild-type p53 [24–26]. Multiple mutations and functional dysregulation of p53 have been demonstrated in the synovial tissue of RA patients [3, 27] and constitute one of the possible reasons for increased survivin production in this non-malignant condition. Notably, high survivin levels (over three standard deviations of the mean of healthy blood donors) were registered exclusively in patients with erosive joint disease and were associated with markers of inflammation such as WBC count and C-reactive protein levels, as well as with the absence of immunosuppressive treatment. This category of RA patients typically displays chronic joint inflammation, progressive joint destruction, and early mortality [28, 29].
Altogether these findings place survivin at the centre of attention as a potential prognostic factor for the destructive course of disease in RA. Indeed, using logistic regression analysis, we demonstrated that RA patients having high levels of survivin had a 16 times higher risk to develop destructive joint disease as compared with the patients with low levels of survivin. Moreover, in a multivariant model we showed that the role of survivin is independent of the presence of RF, the duration of the rheumatic disease, and gender. Interestingly, survivin expression has been shown to be an important prognostic factor in acute leukaemia [30, 31], and a predictor of recurrence in soft-tissue sarcomas  and urinary bladder cancer [33, 34]. In the latter case, extracellular urinary survivin levels were used for the evaluation of treatment and recurrence of cell carcinoma.
Survivin expression determined locally in the inflamed joints and also systemically in circulation of patients with RA was measured extracellularly. Whether survivin found extracellularly originates from dead cells or is a subject of active secretion is presently unknown. The number of in vitro leukocyte-activating stimuli (e.g. lipopolysaccharide, PHA, TSST-1, Concanavalin A) will not induce secretion of survivin. This observation suggests, but does not prove, that extracellular survivin found in synovial fluid originates from dead cells. Alternatively, some other cells (e.g. fibroblasts) or endogenous stimuli give rise to secretion of this molecule. Little is known about extracellular functions of survivin. Survivin has been suggested to function as a self-antigen in patients with haematologic malignancies and solid tumours. In our patient material we demonstrate the presence of antibodies to survivin in the plasma and synovial fluid of patients with RA. Interestingly, reactivity against survivin was significantly higher in the patient group with non-erosive RA. Notably, patients with non-erosive RA have extracellular survivin levels undistinguishable from these of the healthy controls. The association of a high level of antibodies against survivin with non-erosive joint disease may be a reflection of a protective autoimmune mechanism existing in these patients.
To assess the role of survivin in the inflammatory process, we first studied its inducibility in differentiated mature human PBMC. Most of the pro-inflammatory stimuli including lipopolysaccharide, Concanavalin A, TSST-1, and TNF-α leading to a significant release of inflammatory cytokines and chemokines, failed to induce survivin expression by PBMC. In contrast, downregulation of survivin expression using specific anti-sense oligonucleotides resulted in the decrease of IL-6 production by human monocytes. These two observations suggest that the regulatory role of survivin in inflammation is mediated by an increase of cytokine production. The connection between survivin expression and production of IL-6 deserves special attention in the view of recent success of the neutralization of IL-6 for alleviation of RA . These observations support the regulatory role of survivin in the pathogenesis of arthritis.
Studying the variability of survivin levels in patients with RA, we observed that in most cases survivin levels were inclined to decrease in survivin-positive patients and almost never converted from absent to high in survivin-negative cases (data not shown). We also showed that the decrease of survivin levels could be mediated by treatment with DMARDs. This suggests survivin to be a transient phenomenon in the course of RA and may explain a relatively low frequency of patients having high survivin levels (21%) in the cohort tested. However, the results of our study may be affected by the fact that most of the patients were treated with DMARDs at the time of sampling, and even those without ongoing DMARD therapy might have received immunosuppressive treatment previously.
Our study suggests that survivin regulates the inflammatory and destructive process inside the joints of patients with RA. Indeed, high levels of extracellular survivin are associated with chronic erosive arthritis, indicating poor prognosis. In contrast, antibodies against survivin are characteristic of the patients with the non-erosive, benign course of RA. Our findings on survivin expression and autoimmunity to this molecule provide new insight regarding the role of apoptosis in RA.
= bovine serum albumin
= disease-modifying anti-rheumatic drug
= enzyme-linked immunosorbent assay
= erosive rheumatoid arthritis group
= fluorescence-activated cell sorting
= foetal calf serum
= fluorescein isothiocyanate
= inhibitor of apoptosis proteins
= non-erosive rheumatoid arthritis group
= peripheral blood mononuclear cells
= phosphate-buffered saline
= rheumatoid arthritis
= rheumatoid factor
= tumour necrosis factor alpha
= white blood cell.
The work was supported by the Göteborg Medical Society, the Swedish Association against Rheumatism, King Gustaf V:s Foundation, the Swedish Medical Research Council, the Nanna Svartz' Foundation, Börje Dahlin's Foundation, the National Inflammation Network, the Lundberg Foundation, Åke Wiber's Foundation, and the University of Göteborg.
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