Expression of the pro-inflammatory protein S100A12 (EN-RAGE) in rheumatoid and psoriatic arthritis

D. Foell1,2, D. Kane3, B. Bresnihan3, T. Vogl2, W. Nacken2, C. Sorg2, O. FitzGerald3 and J. Roth1,2

1Department of Paediatrics, 2Institute of Experimental Dermatology, University of Münster, Germany and 3Department of Rheumatology, St. Vincent's University Hospital, Dublin, Republic of Ireland.

Correspondence to: D. Foell, Department of Paediatrics, University of Münster, Albert-Schweitzer-Str. 33, D-48149 Münster, Germany. E-mail: dfoell{at}uni-muenster.de


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objectives. Infiltration of synovial tissue by neutrophils is crucial in rheumatoid arthritis (RA), psoriatic arthritis (PsA) and seronegative arthritis (SA). Altered vascular function and endothelial activation are important in PsA. S100A12 (EN-RAGE) is secreted by activated granulocytes and binds to the receptor for advanced glycation end products, which induces nuclear factor (NF)-{kappa}B-dependent activation of endothelium.

Methods. Immunohistochemical studies were performed to detect synovial S100A12 expression. We analysed serum and synovial fluid of 42 patients for S100A12 levels.

Results. S100A12 was strongly expressed in inflamed synovial tissue whereas it was nearly undetectable in synovia of controls or patients after successful treatment. Serum levels of S100A12 correlated with disease activity.

Conclusions. Local expression of S100A12 in inflamed tissue suggests a role in synovitis, especially in PsA. High serum concentrations of S100A12 in patients with active arthritis compared with healthy controls or patients in remission point to its usefulness as a serum marker.

KEY WORDS: S100A12, RAGE, Calgranulin C, Neutrophils, Synovitis, Psoriatic arthritis.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The different forms of chronic inflammatory arthritis comprise a heterogeneous group of autoimmune disorders affecting joints, resulting in cartilage and bone damage, and contributing to a large degree of disability among patients. Inflammation of the synovial tissue is a common feature of peripheral joint disease in rheumatoid arthritis (RA) and seronegative arthritis (SA). Synovial inflammation or ‘synovitis’ is characterized by hyperplasia of the lining layer and both cellular infiltration and hypervascularity of the sub-lining layer. In addition to T lymphocytes, phagocytes have a crucial role in the pathogenesis of synovial inflammation by secretion of various pro-inflammatory cytokines and metalloproteinases [1].

Psoriatic arthritis (PsA) is usually not as destructive as RA—this may be a result of less synovial macrophage infiltration with a subsequent lower production of pro-inflammatory cytokines [2, 3]. Nevertheless, neutrophils are frequently present in synovitis in PsA, and generalized up-regulation of neutrophil migration and secretion of lysosomal enzymes have been reported in PsA patients [46]. In addition, altered vascular growth and function, probably due to endothelial activation, seem to play a prominent role in PsA synovium [7, 8]. TH1 cytokines, monokines and vascular endothelial growth factor (VEGF) are present in PsA synovium and have been suggested to promote angiogenesis in psoriatic skin lesions [9, 10]. However, synovial expression of cytokines in PsA has been poorly characterized [11].

S100A12 (calgranulin C) belongs to the S100 family of calcium-binding proteins. The 20 members of this group share EF-hand domains which are involved in binding of calcium. Two S100 proteins expressed in phagocytes, S100A8 (myeloid-related protein 8, MRP8, calgranulin A) and S100A9 (MRP14, calgranulin B), have been shown to be secreted via a tubulin-dependent pathway [12]. S100A8 and S100A9 proteins accumulate at sites of inflammation, and high levels of these proteins are found in inflammatory diseases [1316].

S100A12 is expressed by granulocytes, whereas its expression by monocytes remains controversial [1720]. It is secreted by activated granulocytes [21]. Extracellular functions include potent chemotactic activity comparable with other strongly chemotactic agents [19, 22]. S100A12 is a ligand for the receptor for advanced glycation end products (RAGE) expressed on macrophages, lymphocytes and endothelium [18]. Intracellular signalling via protein kinases induces nuclear factor (NF)-{kappa}B-dependent secretion of different cytokines [23, 24]. NF-{kappa}B plays a central role in the pathogenesis of synovitis in RA and PsA [3]. The name EN-RAGE (for extracellular newly identified RAGE-binding protein) has been proposed to emphasize its central role for a receptor-mediated signalling pathway.

Data on S100A12 in inflammation have so far only been published for the murine system. This study is the first to demonstrate the up-regulation of local S100A12 expression in synovial tissue resulting in elevated concentrations in serum and synovial fluid of patients with chronic active arthritis. Analyses of synovial tissue of patients with PsA before and after initiation of methotrexate (MTX) therapy revealed a clear decrease of S100A12 expression with improving disease activity which was reflected by subsiding S100A12 serum concentrations.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
We investigated S100A12 concentrations in a total of 42 patients with chronic inflammatory arthritis. Serum was obtained from 14 patients with PsA (mean disease duration 14.6 ± 8.6 months), who were treated with methotrexate (mean dose 12.9 ± 4.8 mg). No other medications apart from non-steroidal anti-inflammatory drugs (NSAIDs) were taken. Serum was obtained before and after successful treatment (mean follow up interval 6.4 ± 1.3 months). In addition, paired serum and synovial fluid samples were available from 28 patients who underwent arthroscopy (eight patients with PsA, nine patients with RA, 11 patients with SA). Synovial biopsies were performed in four patients with PsA before and after therapy with methotrexate. In addition, biopsies were obtained from five patients with PsA not receiving methotrexate, two patients with RA and two patients with SA. Two biopsies of patients without synovial inflammation served as negative controls. All patients gave their informed consent. The study was approved by the Research Ethics Committee of the University College Dublin. Data of patients are summarized in Table 1.


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TABLE 1. Data of patients and healthy controls

 
Clinical and laboratory assessment
All patients were examined by the same physician. Clinical status of patients was documented by recording early morning stiffness (EMS), pain score, Ritchie articular index (RI) [25] and swollen joint count (SJC). In addition to S100A12 levels, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were documented.

Estimation of normal S100A12 serum levels
Normal levels of S100A12 were determined in the serum of 30 healthy adults without signs of inflammation, who either underwent routine blood tests at the University Hospital Münster or volunteered in our laboratories.

Purification of S100A12 protein and antibodies
S100A12 was isolated from human granulocytes as described in detail previously [17, 26]. Polyclonal affinity-purified rabbit antisera against human S100A12 (a-S100A12) were prepared as reported before [17]. Briefly, rabbits were immunized with purified human S100A12. Monospecificity of rabbit anti-human S100A12 antibody was analysed by immunoreactivity against purified human S100A12 and Western blot analysis of lysates of granulocytes. No cross-reactivity of anti-S100A12 antibody against the most homologous proteins S100A8 or S100A9 was detected.

Determination of S100A12 concentrations by sandwich ELISA
Concentrations of S100A12 were determined using a double-sandwich enzyme-linked immunosorbent assay (ELISA) system established in our laboratory. Flat-bottom 96-well microtitre plates (Maxisorp; Nunc, Hamburg, Germany) were coated at 50 µl/well with 10 µg/well of anti-S100A12 in 0.1 M sodium carbonate buffer, pH 9.6; incubated overnight at 4°C; washed three times with phosphate-buffered saline and 0.1% Tween 20, pH 7.4 (wash buffer); and blocked with wash buffer containing 0.25% bovine serum albumin (block buffer) for 1 h at 37°C. After washing once with wash buffer, 50 µl of samples with varying dilutions in block buffer were added for 2 h at room temperature. The ELISA was calibrated with purified S100A12 in concentrations ranging from 0.016–125 ng/ml. The assay has a linear range between 0.5 and 20 ng/ml and a sensitivity of <0.5 ng/ml. After three washes, biotinylated rabbit anti-human S100A12 (10 µg/well) was added and incubated for 30 min at 37°C. Plates were washed and incubated with streptavidin–horseradish peroxidase conjugate (Pierce, Rockford, Illinois, USA) for 30 min at 37°C. After washing three times, plates were incubated with ABTS [2,2'-azinobis(3-ethylbenzthiazoline sulfonic acid); Roche Diagnostics, Mannheim, Germany] and H2O2 in 0.05 M citrate buffer, pH 4.0 for 20 min at room temperature. Absorbency at 405 nm was measured using an automatic ELISA reader (MRX microplate reader, Dynatech Laboratories, Denkendorf, Germany).

Immunohistochemical study
Cryofixed and paraffin-embedded sections were prepared as described elsewhere. Rabbit anti-human S100A12 antibody was used to detect S100A12 expression. Mouse anti-human CD15, a granulocyte-associated antigen, was used to detect granulocytes in infiltrates. Mouse anti-human CD163 antibody (clone RM3/1, detecting a macrophage-specific scavenger receptor) was employed to characterize macrophages in infiltrates. Species-matching control antibodies of irrelevant specificity were used as negative controls. Finally the sections were counterstained with Mayer's haematoxylin. Secondary antibodies and substrates for colour reactions were used as described before [15].

Immunofluorescence microscopy
For double-labelling experiments on synovial sections, anti-S100A12 antibody was followed by anti-CD15 antibody. We used affinity-purified goat anti-mouse or goat anti-rabbit secondary antibodies conjugated with either Texas Red or FITC (Dianova, Hamburg, Germany). Fluorescence was analysed using a Zeiss Axioskop connected to an Axiocam camera supplied with Axiovision 3.0 for windows (Zeiss, Göttingen, Germany). No cross-reactivity or spillover was detected in control experiments after omitting specific antibodies or replacing them by isotype-matched control antibodies of irrelevant specificity.

Statistical analysis
The Mann–Whitney U-test (for unpaired values without normal distribution) and Wilcoxon test (for paired variables) were performed to determine significant differences between distinct categories. SPSS for windows version 11.0 was used to determine correlation of S100A12 with other parameters. Data are expressed as mean ± standard error of the mean (SEM). P values less than 0.05 were considered to be significant.


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients and controls did not differ in age or gender distribution. Patients with RA had significantly more affected joints according to the SJC and RI than those with SA. Patients with PsA were in between these groups. Patients with PsA and RA had higher acute-phase responses as estimated by ESR and CRP (Table 1). S100A12 serum levels were highest in RA (mean 340 ± 90 ng/ml), markedly elevated in PsA (mean 260 ± 60 ng/ml) and less but still significantly elevated in SA (190 ± 20 ng/ml) compared with healthy controls (60 ± 20 ng/ml).

Concentrations of S100A12 in serum and synovial fluid
In paired samples of serum and synovial fluid we found 5- to 10-fold higher S100A12 levels in synovial fluid than in serum in all patients. Synovial fluid levels of S100A12 were higher in SA (4920 ± 1680 ng/ml) than in RA and PsA (1870 ± 1160 ng/ml and 1720 ± 425 ng/ml, respectively). Data are summarized in Fig. 1. Serum levels of S100A12 correlated well with synovial fluid concentrations in PsA (r = 0.37, P < 0.01), RA (r = 0.41, P < 0.01) and SA (r = 0.32, P < 0.05). Serum concentrations of S100A12 also showed a strong correlation with other parameters used to determine disease activity, most significantly with ESR (r = 0.47, P < 0.01) and RI (r = 0.36, P < 0.01). In contrast, concentrations of S100A12 in synovial fluid showed no correlation with neutrophil counts in aspirates (r = 0.19, P = 0.51), thus indicating that S100A12 is not just a waste product of neutrophil breakdown within the intra-articular space.



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FIG. 1. S100A12 levels were determined in parallel by sandwich ELISA in serum (A) and synovial fluid (B) of patients with PsA, RA and SA. In addition, serum levels of S100A12 were determined in 30 healthy controls. Box plots show 25th and 75th percentiles. Horizontal lines within boxes show medians, bold horizontal lines indicate means. Vertical bars indicate 5th and 95th percentiles. *P < 0.05 (serum levels of patients compared with controls; synovial fluid levels of RA patients compared with PsA and SA).

 
Local expression of S100A12 in synovial tissue
To confirm local expression of S100A12 at sites of inflammation we performed immunohistochemical studies. No S100A12 was found in synovial tissue of controls without arthritis. We found expression of S100A12 in inflamed synovial tissue of patients with RA, SA and PsA. In RA and SA, we found S100A12-positive cells in infiltrates and the sub-lining layer. There was a diffuse staining for S100A12 in association with infiltrates, indicating extracellular S100A12 after secretion by infiltrating granulocytes. There was a distinct expression pattern of S100A12 in PsA compared with RA and SA, with a strong association of S100A12 expression with small blood vessels. S100A12 was expressed by granulocytes that adhered to the endothelium of synovial vessels and in perivascular infiltrates. S100A12 seemed to be released by cells at the endothelium in inflamed synovia of PsA as well. Co-staining with CD15 revealed that mainly granulocytes expressed S100A12. We proved co-expression of S100A12 and CD15 in double-labelling experiments using immunofluorescence microscopy. Staining for CD163 clearly revealed a different pattern for macrophages, which contributed to the majority of cells in inflamed synovial tissue (Fig. 2).



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FIG. 2. Immunohistochemical studies were performed on sections of synovial biopsies. Virtually no S100A12 was found in synovial tissue of controls without arthritis (A), whereas S100A12 was extensively expressed in inflamed synovial tissue of patients with RA (B). Expression pattern in SA was similar to RA (not shown). CD163-positive macrophages were the most abundant cell type in infiltrates, but showed a different distribution than S100A12-positive cells (C). Immunofluorescence microscopy of double-labelling studies clearly proved expression of S100A12 by infiltrating CD15-positive neutrophils. Double-labelled cells appear yellow owing to the summation of colours (D). The inserted small images in D show emission at a single wavelength for anti-S100A12-Texas Red (red; upper image) and anti-CD15-FITC (green; lower image). In PsA, S100A12 was expressed predominantly in the sub-lining layer with a perivascular pattern. The expression of S100A12 was most impressive around small blood vessels and in perivascular neutrophilic infiltrates (E). S100A12 was expressed by granulocytes adherent to vascular endothelium and infiltrating the interstitial tissue. S100A12 seemed to be released upon contact of neutrophils with the endothelium (F). Strong S100A12 expression was found in synovial tissue of patients with PsA before methotrexate treatment (G), while it was nearly undetectable in synovia of the same patients after effective methotrexate treatment (H). Scale bars, 100 µm.

 
Correlation of S100A12 with disease activity in response to treatment
We analysed the effects of methotrexate treatment on S100A12 expression in serum of 14 patients and in synovial membrane of four patients with PsA. Before treatment, extensive expression of S100A12 was found in synovial tissue, predominantly in the sub-lining layer and in perivascular infiltrates. S100A12 expression was almost undetectable in synovial biopsies of the same patients after effective methotrexate treatment (Table 2; Fig. 2G, H). All patients improved significantly in clinical scores according to RI, pain score, SJC and EMS. CRP and ESR levels also decreased. Response to therapy was paralleled by a marked decrease of S100A12 serum levels after methotrexate treatment (mean 240 ± 265ng/ml prior to methotrexate compared with 100 ± 80 ng/ml after methotrexate; P < 0.05). S100A12 levels correlated well with improving EMS, pain score, RI and SJC. Data are summarized in Fig. 3 and Table 3.


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TABLE 2. Immunohistochemical analysis of S100A12 expression in synovial tissue

 


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FIG. 3. S100A12 was analysed using sandwich ELISA in serum of 14 patients with PsA in active disease and after methotrexate therapy. We found significant differences between mean S100A12 serum levels prior to and after successful methotrexate treatment (P < 0.05). Bold horizontal lines indicate mean levels. S100A12 serum levels correlated well with clinical improvement (see Table 3).

 

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TABLE 3. Improvement of disease activity in patients with PsA after initiation of MTX treatment

 

    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
This study demonstrates for the first time a strong expression of human S100A12 in synovial inflammation in RA, SA and particularly PsA. Analyses of S100A12 in synovial fluid and serum indicate that this protein is expressed and secreted at local sites of inflammation in synovitis. S100A12 is expressed by infiltrating granulocytes that initiate inflammatory processes in the synovium resulting in chronic arthritis. Upon secretion by activated granulocytes it exhibits pro-inflammatory functions via NF-{kappa}B activation after binding to RAGE on myelomonocytic cells and endothelium [19]. Blocking NF-{kappa}B in human rheumatoid synovial cultures reduced local production of TNF{alpha} [27], and inhibitors of NF-{kappa}B kinases are important modulators of synovial inflammation [28, 29]. Thus, modulation of S100A12 expression is a novel potential mechanism of NF-{kappa}B inhibition which subsequently may reduce synovial inflammation. On the other hand, allelic variations within key domains of RAGE have been postulated to up-regulate inflammatory responses upon ligation of S100A12, thus promoting susceptibility for chronic inflammatory arthritis [30]. Data from models of inflammation in mice also prove the important role of S100A12 as a key mediator of a novel pro-inflammatory axis [18]. Reports from murine models of collagen-induced arthritis indicate the ability of S100A12 to trigger synovial inflammation [31].

MRP8 (S100A8) and MRP14 (S100A9) have been demonstrated to be overexpressed in the lining layer of inflamed synovial tissue in RA [32]. In contrast, S100A12 was found predominantly in the sub-lining associated with infiltrating granulocytes. Moreover, we found a clear difference in the distribution of S100A12 in PsA compared with RA and SA. We found a distinct perivascular distribution of S100A12. This difference is interesting regarding the specific processes believed to be involved in the pathogenesis of PsA. Angiogenesis and altered function of microvascular endothelium has been reported to be an important phenomenon of synovial inflammation in PsA. Activation of endothelial cells by S100A12 has already been demonstrated [18]. Enhanced angiogenesis in models of diabetic mice can be inhibited by blocking the interaction of S100A12 with RAGE [3336]. RAGE mediates an up-regulation of the connective tissue growth factor IGFBP-rP2 (insulin-like growth factor binding protein-related protein 2), which is a potent inducer of angiogenesis [37]. In addition, RAGE increases adhesion of granulocytes to stimulated endothelial cells [38]. Thus, the perivascular expression pattern in PsA points to a possible role for S100A12 in angiogenesis associated with this form of arthritis.

Our study indicates that S100A12 serum levels might serve as a marker for local disease activity in different forms of arthritis. Patients with active arthritis revealed significantly higher S100A12 levels than healthy controls. We found about 10-fold higher concentrations of S100A12 in synovial fluid of patients. The high local expression of S100A12 at the site of inflammation seems to be responsible for the correlating levels that are detected in serum. In this context, the higher levels in synovial fluid of patients with SA in comparison with serum levels correlated with the smaller numbers of affected joints. In PsA, and especially RA, the greater number of inflamed joints with secretion of S100A12 is likely to result in the higher concentrations of S100A12 found in serum. In PsA, S100A12 levels reflected successful immunosuppressive treatment with methotrexate. S100A12 was a reliable marker of the effects of methotrexate therapy in serum and synovium. The profound effect of methotrexate on S100A12 expression in the synovia of PsA patients might be due to the reduction of pro-inflammatory cytokines that activate neutrophils and induce S100A12 expression [39]. On the other hand there is a direct effect of methotrexate on neutrophil chemotaxis that might inhibit the migration of neutrophils into synovial tissue [40].

The expression of S100A12 in human arthritis provokes the question whether this protein and its interaction with RAGE might be a target for novel therapies. In different mouse models of inflammation including arthritis, blocking this interaction with soluble RAGE (sRAGE) and anti-S100A12 antibodies revealed clear anti-inflammatory effects [18, 31]. Further studies on the functional role of S100A12 in human arthritis have to prove the usefulness of new biological therapies that focus on pro-inflammatory activities of human S100A12.


    Acknowledgments
 
We thank S. Oberfeld and K. Fischer for excellent technical assistance. This study was supported by grants from the Interdisciplinary Centre of Clinical Research (IZKF; Project C16), University of Münster, Germany.

Conflict of interest

The authors have declared no conflicts of interest.


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 Introduction
 Patients and methods
 Results
 Discussion
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Submitted 13 November 2002; Accepted 5 March 2003