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Originally published online as doi:10.1189/jlb.0507287 on October 15, 2007

Published online before print October 15, 2007
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(Journal of Leukocyte Biology. 2008;83:149-155.)
© 2008 by Society for Leukocyte Biology

Extracellular survivin up-regulates adhesion molecules on the surface of leukocytes changing their reactivity pattern

Simona Mera, Mattias Magnusson, Andrej Tarkowski and Maria Bokarewa1

Department of Rheumatology and Inflammation Research, Sahlgrenska University Hospital, Göteborg, Sweden

1 Correspondence: Department of Rheumatology and Inflammation Research, Guldhedsgatan 10, S-413 46 Göteborg, Sweden. E-mail: maria.bokarewa{at}rheuma.gu.se


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ABSTRACT
 
Rheumatoid arthritis (RA) is an autoimmune disease with joints as a principal target of inflammation. We have shown recently that the extracellular expression of the antiapoptotic protein survivin is associated with a destructive course of RA. Here, we address the potential impact of extracellular survivin on peripheral blood leukocytes (PBL). The binding of survivin to the surface of human PBL as well as the expression of adhesion molecules were assessed by FACS. The expression of adhesion molecules on leukocytes as a function of circulating survivin was analyzed in blood of 24 patients with RA and compared with eight healthy individuals. We show that extracellular survivin expresses immunomodulatory properties. It binds to the surface of the majority of granulocytes and a significant part of lymphocytes and monocytes inducing the activation of {alpha}-chains of β-integrins and their ligand ICAM-1. Survivin-induced expression of {alpha}-chains of β2-integrins is regulated by p38 MAPK and PI-3K but not by the NF-{kappa}B signaling pathway. Clinical relevance of our findings is supported by the in vivo association of high circulating survivin levels with an increased expression of CD11c on monocytes and granulocytes in RA patients. The results of our study demonstrate that extracellular survivin affects the phenotype of leukocytes having a possible impact on homing of inflammatory cells during arthritis.

Key Words: β2-integrins • survivin • p38 • inflammation • rheumatoid arthritis


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INTRODUCTION
 
Survivin is a 142-amino acid-long protein described originally as a member of the inhibitors of apoptosis family of proteins containing a baculovirus repeat. Indeed, survivin blocks death receptor-mediated and mitochondria-mediated pathways of apoptosis [1 ]. A direct, antiapoptotic effect of survivin is mediated by binding to caspases or to a second mitochondrial activator of caspases [2 ]. Intracellular functions of survivin are not limited to its role in apoptosis. Indeed, survivin also functions as a regulator of cell division. During mitosis, it shuttles inside the nucleus and binds to aurora kinases, coordinating the chromosomal and cytoskeletal events [3 ]. Until recently, survivin expression was tightly connected to the G2/M phase of cell cycle. This has been challenged by the observation of cell cycle-independent survivin expression in mature neutrophils [4 ] and in T lymphocytes [5 ]. Recently, survivin attracts increasing attention as a supervisor of cellular immunity by regulation of T cell development and function [6 , 7 ]. In addition, survivin has been shown to act as a central mediator of antigen-induced expansion of differentiated T cells [5 ]. Survivin is considered as an important driving force in neoplasms, abundantly expressed in most of human cancers and in transformed cell lines [1 ]. This makes survivin attractive as a target for the induction of tumor-specific cytotoxicity [8 ].

We have shown previously that high levels of survivin are present extracellularly in a significant fraction of patients with rheumatoid arthritis (RA). Moreover, extracellular survivin was related to the erosive course of joint disease, whereas autoimmune responses to the same molecule, manifested as survivin-targeting antibodies, correlated to protection from joint destruction [9 ]. Extracellular properties of survivin with respect to cell-to-cell interactions and inflammation are largely unknown. In the present study, we demonstrate that the antiapoptotic protein survivin is expressed continuously by peripheral blood leukocytes (PBL) and mediates immunomodulatory action when exposed extracellularly. It binds to neutrophils by means of a yet-unknown receptor inducing the expression of {alpha}-chains of β-integrins (CD11b, CD11c, and CD49d). These in vitro-obtained data are supported by the increased CD11c expression in RA patients expressing high extracellular levels of survivin.

In conclusion, extracellular survivin is bound by human leukocytes and participates in several steps of leukocyte activation during inflammation. These findings together with our previous observation of autoimmune response to survivin in patients with RA favor the regulatory role of this molecule in autoimmune arthritis.


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MATERIALS AND METHODS
 
Expression and purification of recombinant human survivin
The human survivin gene inserted into the pHIS8 expression vector was kindly provided by Mark A. Verdecia (The Salk Institute, La Jolla, CA, USA). The survivin sequence in the plasmid was verified by DNA sequencing using T7 and SP6 primers. Constructs of pHIS8-CHS were transformed into Escherichia coli BL21(DE3). Transformed E. coli were grown at 37°C in Terrific broth containing 50 µg/mL kanamycin, until absorbance at 600 nm = 0.5. After induction with 0.375 mM isopropyl 1-thio-D-galactopyranoside, the cultures were grown at 18°C for 4 h. Cells were pelleted, harvested, and resuspended in 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 20 mM imidazole (pH 8.0), 20 mM β-ME, 10% (v/v) glycerol, and 1% (v/v) Tween-20 (lysing buffer) containing 0.5 mg/ml lysozyme and 0.1 M PMSF. After sonication and centrifugation, the supernatant was passed over a Ni2+- nitrilotriacetic acid (NTA) column, which was washed with 10 bed vol lysis buffer and 10 bed vol lysis buffer without Tween-20, and then, the His8-tagged protein was eluted with lysis buffer without Tween-20 but containing 250 mM imidazole (pH 8.0). The presence of a 16.5-kDa survivin + Histag/protein in the eluate was verified by SDS-PAGE. Incubation with thrombin during dialysis for 24 h at 4°C against the lysis buffer without Tween-20 removed the amino-terminal His8-tag. Dialyzed protein was reloaded on a Ni2+-NTA column, and the flow-through was depleted of thrombin using a benzamidine-Sepharose column. Endotoxin concentrations were determined in each preparation following absorption with endotoxin-removing gel (Detoxi-gel, Pierce Biotechnology, Rockford, IL, USA). The survivin solution was dialyzed against PBS, and in all cell experiments, endotoxin levels were equalized to 1 ng/ml.

Patients
Blood samples were collected from 24 patients with RA who attended the Rheumatology Clinics at Sahlgrenska University Hospital (Göteborg, Sweden). RA was diagnosed according to the American College of Rheumatology criteria [10 ]. At the time of blood sampling, all of the patients had an advanced erosive joint disease, and all received disease-modifying antirheumatic drugs (methotrexate was used by 20 patients, cyclophosphamide by four patients). For the binding studies and FACS analyses, the blood samples were obtained from healthy individuals. The Ethics Committee of the University of Göteborg (Sweden) approved the study. All of the patients gave their informed consent before the blood sampling, obtained from the cubital vein in a vacuum tube covered with heparin. The blood volume needed for the FACS analyses was withdrawn, and the remaining blood sample was submitted to centrifugation at 800 g for10 min. The obtained plasma was aliquoted and stored frozen at –20°C until use.

Survivin levels
The survivin level in the recombinant preparations and in the plasma of RA patients and control individuals was determined by a sandwich ELISA using a matched antibody pair (R&D Systems, Abingdon, UK) as described previously [9 ].

Intracellular staining for survivin
Whole blood (200 µl) of RA patients and healthy controls was incubated with the antibodies for surface staining or isotype controls (see above). At the end of incubation, erytrocytes were lysed using 3 ml lysing buffer (Becton Dickinson, Erembodegum, Belgium) for an additional 15 min. Leukocytes were sedimented by centrifugation at 800 g x 5 min, and supernatant was discharged. The cell membrane was permeabilized with Cytofix/Cytoperm solution containing paraformaldehyde and saponin, washed with Perm/Wash (Becton Dickinson), and sedimented by centrifugation. The cells were incubated with 20 µl antisurvivin antibodies (clone #91630, R&D Systems) or mouse IgG1 isotype control (both PE-conjugated) for 60 min in the dark at room temperature.

Preparation of human leukocytes
For the binding studies and FACS analysis, human leukocytes were prepared from 200 µl heparinized blood by lysing erythrocytes with NH4Cl and washing with PBS containing 1% of FCS and 0.5 mM EDTA. The leukocyte pellet was obtained by centrifugation at 200 g x 5 min.

For the Western blot analysis and determination of MAPK-regulated transcription factors, cultures of human PBMC were prepared. PBMC were separated from heparinized blood on a Lymphoprep density gradient. Following washing, cells were resuspended in Iscove’s medium (containing 1% L-glutamine, 5x10–5 M β-ME, 50 µg/ml gentamycin sulfate, and 10% FCS) at 2 x 106/ml and stimulated with recombinant survivin (10 µg/ml). PBMC were cultured in 24-well plates in a humidified atmosphere containing 5% CO2 at 37°C.

Survivin stimulation
To assess the binding of survivin to the cell surface, leukocytes were fixed with 1% formaldehyde and washed with PBS buffer containing FCS, 1% prior to incubation with survivin. Following sedimentation, cells were stained with antisurvivin antibodies (clone #91630, R&D Systems) or mouse IgG1 isotype control (PE-conjugated) and subjected to FACS analyses.

To study the effect of survivin on the expression of integrins, leukocytes were incubated with increasing concentrations of recombinant survivin (0.1–1–10 µg/ml) for 45 min at room temperature, washed thoroughly with PBS buffer containing 1% FCS, and fixed with 1% formaldehyde for 15 min at room temperature and stained for the expression of integrins as described below. To study the role of intracellular pathways for survivin effects, leukocytes were incubated with the specific pathway inhibitors prior to stimulation with survivin for 60 min at 37°C. The inhibitors used in the study are parthenolide (25 µM, Sigma Chemical Co., St. Louis, MO, USA) for the NF-{kappa}B pathway; LY294002 (50 µM, BioSource, Nivelles, Belgium) for PI-3K; SB203580 (50 µM, Alexis Biochemicals, Lausen, Switzerland) for MAPK p38; as well as PD98059 (50 µM, BioSource, Nivelles, Belgium) for p44/42 (MEK1). The inhibitors were removed by washing, and leukocyte cultures were incubated with recombinant survivin (10 µg/ml) for 60 min at room temperature. The stimulation was discontinued by another cycle of washing. The cells were stained for the expression of integrins and submitted to FACS analyses.

Flow cytometric analysis (FACS)
Human leukocytes were stimulated as above, washed, and incubated with optimal concentrations of mouse mAb and the isotype IgG-matched controls. Phenotypic analysis of cells was performed using 10 µl anti-CD3 (SK7, PerCp-conjugated), anti-CD8 [SK1, allophycocyanin (APC)-conjugated], anti-CD4 (SK3, FITC-conjugated), anti-CD20 (H1, FITC-conjugated), and anti-CD14 (MoP9, APC-conjugated) antibodies or control mouse isotype IgG for 20 min in the dark at room temperature. Surface expression of the adhesion molecules was assessed by anti-CD11b (D12, PE-conjugated), anti-CD11c (B-ly6, APC-conjugated), anti-CD54 (HA58, APC-conjugated), anti-CD62 ligand (CD62L; SK11, PE-conjugated), and anti-CD49d (L25, FITC-conjugated). All of the antibodies and isotype controls were purchased from Becton Dickinson. Following 20 min incubation at room temperature, staining was discontinued by washing, and the cell pellet was resuspended in the PBS-FACS buffer containing FCS 1%, 0.5 mM EDTA, and 0.1% NaN3. We analyzed 50,000–100,000 cells with a FACSCalibur equipped with CellQuest software. The percentage of positive cells and the geometric mean fluorescence intensity were recorded.

Determination of p38 MAPK-regulated transcription factors
For the determination of MAPK-regulated transcription factors, PBMC were stimulated with survivin (10 µg/ml) for 15 min and 60 min, respectively. Following stimulation, leukocytes were washed with ice-cold PBS and lysed using the buffer containing Hepes 50 mM, sucrose 250 mM, NaF 5 mM, MgSO4 5 mM, EDTA 1 mM, Na3VO4 5 mM, DTT 1 mM, and a cocktail of protease inhibitors (Complete Mini, Roche Diagnostics, Mannheim, Germany). The amount of protein in the lysates was measured using Bradford’s protein reagent. The equalized lysates from PBMC stimulated with survivin and not stimulated were tested for the presence of STAT1 and myocyte enhancer-binding factor 2 (MEF2) using the TransAM MAPK family kit (Active Motif, Carlsbad, CA, USA), following the manufacturer’s instructions.

Statistical analysis
The frequency of integrin-expressing cells in survivin-stimulated and nonstimulated cell cultures is presented in percent [median (range interval)]. The comparisons were performed using the Mann-Whitney test. All tests were two-tailed and conducted at 5% significance level. For all of the statistical evaluations of the results, P values below 0.05 were considered significant.


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RESULTS
 
Survivin binds to the PBL
PBL were incubated with different amounts of recombinant survivin (0–0.1–1.0–10 µg/ml). Nonspecific binding of survivin to the leukocyte surface was prevented by 1% FCS, added to the washing buffer. Surface staining for survivin was assessed using FACS within the lymphocyte, monocyte, and granulocyte gates in four healthy individuals. Figure 1 shows binding of survivin to the surface of leukocytes in a representative experiment. As presented in Figure 1 , the intensity of survivin binding was dose-dependent and could be detected in all three main leukocyte populations. The survivin binding was detected on 21% (range 18–27%, n=4) of lymphocytes, 46% (range 38–52%, n=4) of granulocytes, and 5% of the monocyte population (range 3–8%, n=4). To exclude the possible influence of heparin sulfate used as an anticoagulant at the time of blood sampling, the binding experiments were also performed in EDTA anticoagulated blood and yielded similar results (data not shown).


Figure 1
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  		Figure 1. Survivin binding to the PBL, which when obtained from healthy individuals, were prepared as described in Materials and Methods and incubated with recombinant survivin (0–1–10 µg/ml) for 1 h at room temperature, washed thoroughly, and stained with antisurvivin (Sur) antibodies (PE-conjugated). FACS analysis showed the presence of survivin on the surface of neutrophils and to a lower extent, monocytes. Representative histogram plots of four independent experiments show staining of the cells incubated with 1 µg/ml (open graph, bold) and 10 µg/ml survivin (filled gray) and without survivin (open graph, dash line) within the lymphocyte, monocyte, and granulocyte cell populations.

Extracellular survivin induces integrin expression
Integrins are adhesion receptors mediating cell interactions with the surrounding tissues and numerous physiological processes inside the cell, including hyperplasia, apoptosis, and proliferation. The expression of adhesion molecules on the PBL of healthy individuals was assessed following incubation with recombinant survivin (10 µg/ml). As a control, cells were incubated with the appropriate concentration of LPS (1 ng/ml), identical to that found in survivin preparation. The expression of the {alpha}-chains of β2-integrins (CD11b, CD11c) and their ligand (CD54/ICAM), as well as {alpha}-chains of β1-integrins (CD49d) and L-selectin (CD62L) was analyzed. The incubation of leukocytes with survivin induced a pronounced expression of CD11b and CD11c molecules on neutrophils and monocytes. Incubation with survivin resulted in the increased amount of cells expressing integrins and their intensity of staining (measured as geometric mean; Fig. 2a and 2b ). The expression of CD11b following incubation with survivin was prominent on granulocytes shifting from 52% (n=6, intensity 17.4±11.6) on the surface of nonstimulated cells to 98% (n=9, intensity 168±35, P=0.0001) and on monocytes from 56% (n=6, intensity 8.6±2.4) to 80% (n=9, intensity 69±12, P=0.002). The expression of CD11b on lymphocytes was only up-regulated marginally by survivin, changing from 19% (n=6, intensity 6.5±2.1) to 28% (n=9, intensity 12.3±1.7, not significant). The expression of CD11c showed a similar pattern being changed, predominantly on granulocytes from 48% (n=6, intensity 5.2±2.2) to 66% (n=9, intensity 12.1±2.4, P=0.03) and monocytes [intensity from 4.2±1.6 (n=6) to 27.3±12.0 (n=9, P=0.001)]. In contrast, the lymphocyte expression of CD11c was not changed following incubation with survivin. The expression of β1-integrin {alpha}-chain (CD49d) was not increased in the leukocyte populations (Fig. 2c) . Following incubation with survivin, the expression pattern of CD54 was also increased with respect to frequency and intensity (Fig. 2d) . Its expression was increased on lymphocytes from 19% (n=6, intensity 4.7±1.1) to 46% (n=8, intensity 9.9±2.1, P=0.046) and on granulocytes from 11% (n=6, intensity 5.4±3.1) to 22% (n=8, intensity 7.9±4.5, P=0.04). The expression of CD62L was increased selectively on lymphocytes from 3% (n=4, intensity 2.1±0.3) to 54% (n=4, intensity 12.5±2.8, P=0.001; Fig. 2e ) but not on monocytes or granulocytes.


Figure 2
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  		Figure 2. Induction of adhesion molecules by extracellular survivin. PBL prepared as described in Materials and Methods were incubated with 10 µg/ml (2 µg/sample, open graph, bold) or without (open graph) recombinant survivin, washed, and stained for the surface expression of adhesion molecules. Representative histograms of individual expression within the lymphocyte, monocyte, and granulocyte populations are shown regarding (a) CD11b, (b) CD11c, (c) CD49d, (d) CD54, and (e) CD62L expression.

The up-regulation of integrins is mediated through p38 MAPK and PI-3K signaling
To identify intracellular pathways participating in the up-regulation of integrins in response to survivin, leukocyte cultures were incubated with the optimal concentration of specific inhibitors. Parthenolide was used for interrupting NF-{kappa}B signaling, LY294002, for inhibition of PI-3K, SB203580, for inhibition of p38, and PD98059 was used for inhibition of p44/42 MAPKs. Following incubation with these inhibitors, survivin (10 µg/ml) was added to the leukocyte cultures. We observed that survivin-induced expression of CD11b, CD11c, and their ligand ICAM (CD54) was dependent on the p38 MAPK pathway and could be diminished significantly by its inhibitor SB203580 (Fig. 3a ). Indeed, significant changes in the CD11b (n=5, intensity change 6.2±4.1, P=0.046), CD11c (n=4, intensity change 3.2±0.9, P=0.044), but not CD54 (n=5, intensity change 3.1±4.1, not significant) could be observed by treatment of leukocytes with the p38 inhibitor SB203580 prior to stimulation with survivin (Fig. 3a) . A reduction of CD11b expression was also achieved by incubation of leukocytes with LY294002 (n=3, intensity change 2.7±1.7, P=0.047), indicating the requirement of PI-3K for the expression of this molecule (Fig. 3b) . Treatment of leukocyte cultures with the p44/42 kinase inhibitor PD98059 resulted as well in a minor reduction of β-integrin expression (Fig. 3c) . A tendency to the change in the CD11b expression following treatment of leukocytes with PD98059 was observed, however did not reach a level of significance (n=4, intensity change 1.9±2.2). In contrast, the NF-{kappa}B pathway and its inhibitor parthenolide had neither influence on the expression of CD11b and CD11c (Fig. 3d) nor on the expression of CD54. These observations indicate that the p38 MAPK signaling pathway is important for the survivin-induced expression of {alpha}-chains of β-integrins.


Figure 3
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  		Figure 3. Reduction of survivin-induced β-integrin expression on granulocytes by blocking p38 MAPK and PI-3K pathways. PBL were treated with specific intracellular pathway inhibitors as described in Materials and Methods. Following washing, leukocytes were incubated with recombinant survivin (10 µg/ml) for an additional 60 min. Stimulation was discontinued by washing and fixation with 1% formaldehyde. The expression of integrins was assessed in the cells treated with the inhibitors (bold line) and nontreated (filled gray) by FACS analyses. Control isotype antibodies are shown as dashed lines. Histograms show the changes in the expression of CD11b, CD11c, and CD54 on granulocytes following survivin stimulation in the presence of (a) SB203580 (p38 MAPK inhibitor), (b) LY294002 (PI-3K inhibitor), (c) PD98059 (p44/42 MAPK inhibitor), and (d) parthenolide (NF-{kappa}B inhibitor).

Activation of p38-dependent transcription factors in PBMC stimulated with survivin
The activity of transcription factors downstream of p38 MAPK was assessed in PBMC of healthy subjects stimulated with survivin (n=2). The cell lysates were prepared following 15 and 60 min of stimulation, respectively. The protein concentration was equalized, and the presence of MEF2 and STAT1 in the survivin-stimulated and nonstimulated lysates was assessed, as described in Materials and Methods. MEF2 and STAT1 were up-regulated in the lysates of survivin-stimulated (138% and 118%, respectively) compared with nonstimulated cells (Fig. 4 ). The up-regulation of MEF2 was more pronounced and showed a time-dependent pattern (15 min, 114%; 60 min, 138%). The activation of MEF2 occurs by a direct association with p38 [11 ]. This further supports our observations of the importance of p38 MAPK signaling for the mediation of survivin effects intracellularly.


Figure 4
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  		Figure 4. Intracellular activity of p38-dependent transcription factors in human PBMC following stimulation with recombinant survivin. PBMC (4x106, n=2) were stimulated with recombinant survivin (10 µg/ml) at 37ºC. Lysates of PBMC were prepared following 15 and 60 min of incubation, as described in Materials and Methods. Protein content of the lysates was assessed by the Bradford reagent. The activity of STAT1 and MEF2 trascription factors was assessed in the lysates equalized with respect to their protein concentration. The change of transcription factor activity in the presence of survivn was expressed in percent of the base level in nonstimulated PBMC.

Increased expression of integrins in RA patients with high extracellular levels of survivin
We have recently identified extracellular survivin in synovial fluid and in circulation of a significant proportion of RA patients. We have also shown that circulating survivin was associated with a destructive joint disease [9 ]. In this study, we analyzed if the circulating survivin originated from the intracellular depots of circulating leukocytes. Peripheral leukocytes were permeabilized with saponin and stained with antisurvivin antibodies labeled with PE. Detectable survivin was observed in lymphocytes and monocytes of RA patients and of healthy individuals (Table 1 ). In contrast, intracellular survivin was not found in the granulocytes. The intracellular expression of survivin was similar in the healthy controls (n=8) and in the patients with RA (n=24) with respect to the prevalence and intensity of staining [lymphocytes, 97% (range 94–99) vs. 87% (range 18–99), not significant; monocytes, 55% (range 34–74) vs. 63% (range 39–90), not significant].


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  		Table 1. Intracellular Expression of Survivin (%) in Lymphocytes, Monocytes and Granulocytes of RA Patients and of Healthy Controls

To assess a possible connection between the intracellular survivin and its extracellular circulating levels, plasma concentration of survivin was measured in RA patients and in controls. Eight of 24 RA patients had high levels of survivin in plasma (varying between 340 and 35,000 pg/ml), and the remaining 16 RA patients and the controls had low levels of circulating survivin. For further analyses, the patient group was stratified by the presence of extracellular survivin into survivinhigh (n=8) and survivinlow (n=16; Table 1 ). The survivinhigh RA patients had a significantly higher number of survivin-expressing monocytes [74% (range 56–90)], as compared with the healthy controls [55% (range 34–74), P<0.05] and to the survivinlow RA patients [59% (range 15–95), not significant]. This also applied to CD11c-expressing monocytes (P<0.05). In contrast, the survivinlow RA patients had a tendency to a decreased number of survivin-expressing lymphocytes as compared with the healthy controls [81% (range 18–98) vs. 97% (range 94–99), not significant] as well as within the CD3+ population [59% (range 13–84) vs. 69% (range 58–75), P=0.057].

To assess the impact of extracellular survivin on the in vivo integrin expression in RA patients, the frequencies of CD11c+ cells in the leukocyte populations were assessed. To exclude the influence of culturing on integrin expression, the staining procedures were performed using fresh whole blood samples. We observed that survivinhigh RA patients had a significantly higher amount of CD11c+ cells in the monocyte and in the granulocyte populations as compared with healthy controls (Fig. 5 ).


Figure 5
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  		Figure 5. The expression of CD11c on the surface of PBL of RA patients as a function of circulating levels of survivin. Leukocytes were obtained from 200 µl whole blood of RA patients and of healthy controls as described in Materials and Methods and stained for the surface expression of CD11c. Cells were submitted to FACS analyses and gated for lymphocyte, monocyte, and granulocyte cell populations. Levels of circulating survivin in plasma were assessed by an ELISA. RA patients were stratified by the presence of circulating survivin into survivinhigh (n=8) and survivinlow (n=16). The controls (n=8) had low levels of survivin in plasma. n.s., Not significant.


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DISCUSSION
 
We have demonstrated recently that a high extracellular level of survivin is present in circulation and in synovial fluid of a subset of RA patients associated with a destructive course of joint disease [9 ]. In the present study, we demonstrate that extracellular survivin is biologically active. It binds to human leukocytes, inducing p38 MAPK-dependent expression of {alpha}-chains of β2-integrins. Clinical relevance of these findings is proven by an increased frequency of CD11b expression on the leukocytes of RA patients having high levels of survivin in circulation.

Adhesion molecules on leukocytes are instrumental in recruitment, transmigration, and homing, playing an important role in various processes, including inflammation, tumor growth, and angiogenesis [12 , 13 ]. In our in vitro experiments, the effect of survivin was assessed regarding the expression of three different groups of adhesion molecules: {alpha}-chains of β-integrins (β1, CD49d; β2, CD11b and CD11c); β2-integrin ligand, ICAM (CD54); and L-selectin (CD62L). We observed that survivin binds to the leukocyte surface inducing expression of {alpha}-chains of β2-integrins (CD11b, CD11c). Further studies of the intracellular events mediated by survivin indicted the vital role of p38-MAPK. Indeed, we demonstrated that stimulation of leukocytes with survivin resulted in the activation of MEF2, a family of transcription factors directly dependent on p38 MAPK [11 ]. Moreover, a p38-MAPK inhibitor efficiently down-regulated survivin-induced expression of CD11b and CD11c (see Fig. 3 ). The expression of {alpha}-chains of β2-integrins induced by survivin was by far most prominent on the neutrophils. In addition, survivin supported activation of β2-integrins indirectly by simultaneous up-regulation of their ligand, ICAM-1 (CD54), on the surface of lymphocytes. The cross-linking of β2-integrins results in their clustering, followed by the intracellular activation of integrin-linked kinases and the PI-3K/Akt pathway [14 15 16 ]. This may partly explain our findings supporting the abrogation of β2-integrin expression by the PI-3K inhibitor (Fig. 3) . Taken together, we demonstrate that extracellular survivin activates PI-3K and p38-MAPK-related processes, thereby regulating adhesion molecule-dependent processes, such as cell survival, activation, and differentiation [17 , 18 ] during RA.

In the clinical setting, the present study shows that the expression of integrins on the surface of leukocytes in RA patients is related directly to the presence of circulating survivin (Fig. 5) . Moreover, the intracellular expression of survivin in CD3+ and CD11c+ cells in RA patients was associated with the increased levels of survivin in blood circulation (Table 1) , suggesting that these cells are a source of extracellular survivin. Integrins are known as important participants of joint inflammation. Indeed, the abundant expression of integrins has been demonstrated repeatedly in the RA synovia. Moreover, the expression of integrins on the hyperplasive synovia and pannus contributes to the invasive behavior of synovial fibroblasts, leading to the destruction of joint cartilage in RA patients [19 , 20 ].

Our study demonstrates that antiapoptotic survivin signals extracellularly through the p38 MAPK, leading to the expression of intergins. Thus, extracellular expression of survivin found in a significant population of patients with RA may be of significance for the pathogenesis of inflammation.


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ACKNOWLEDGEMENTS
 
The Ethic Committee of Sahlgrenska Hospital approved this study. The work has been supported by Göteborg Medical Society, Swedish Association Against Rheumatism, King Gustaf V:s Foundation, Swedish Medical Research Council, Nanna Svartz’ Foundation, National Inflammation Network, and the University of Göteborg. S. M. performed binding assays and FACS analysis of the patient material. M. M. prepared recombinant survivin. A. T. contributed to the conception of the study and preparation of the manuscript. M. B. contributed to the study design, methodological development of the concept, clinical, laboratory, and statistical evaluation of material from RA patients, and preparation of the manuscript. All authors read and approved the final manuscript. The author(s) declare that they have no competing interests.

Received May 7, 2007; revised July 31, 2007; accepted August 2, 2007.


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A. MALAMITSI-PUCHNER, S. BAKA, M. BOUTSIKOU, S. LIOSI, D. GOURGIOTIS, E. PROTONOTARIOU, D. HASSIAKOS, and D. D. BRIANA
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