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Rheumatology Advance Access originally published online on August 27, 2007
Rheumatology 2007 46(10):1538-1546; doi:10.1093/rheumatology/kem198
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The expression of SOCS is altered in rheumatoid arthritis

P. Isomäki1,2, T. Alanärä1, P. Isohanni1, A. Lagerstedt3, M. Korpela2, T. Moilanen6, T. Visakorpi1,4 and O. Silvennoinen1,5

1Institute of Medical Technology, University of Tampere, 2Department of Internal Medicine, Division of Rheumatology, 3Department of Pathology, 4Laboratory Centre, 5Department of Clinical Microbiology, Tampere University Hospital and 6Coxa, Hospital for Joint Replacement, Tampere, Finland.

Correspondence to: P. Isomäki, MD PhD, Department of Molecular Immunology, Institute of Medical Technology, Biokatu 8, 33014 Tampere University, Tampere, Finland. E-mail: pia.isomaki{at}uta.fi


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. Cytokines play a key pathogenic role in rheumatoid arthritis (RA). Several cytokines signal through the JAK-STAT pathway, which is negatively regulated by the suppressors of cytokine signalling (SOCS) proteins. Since SOCS protein levels can profoundly modulate cellular responses to cytokines, we have investigated their expression in chronic RA.

Methods. The levels of SOCS1–3 and CIS1 mRNA in peripheral blood (PB) and synovial fluid (SF) mononuclear cells (MCs), purified T cells and monocytes from RA patients and healthy volunteers were studied using quantitative reverse transcriptase polymerase chain reaction (RT-PCR). SOCS mRNA and protein expression in synovial tissues were examined by RT-PCR and immunohistochemistry.

Results. The levels of SOCS1 and SOCS3 were significantly increased in PBMCs from RA patients when compared with healthy volunteers. These differences were mainly due to up-regulation of SOCS1 in PB T cells and of SOCS3 in PB monocytes. In addition, SOCS2 was up-regulated in PB T cells. Interestingly, SF T cells expressed lower and SF macrophages higher levels of SOCS molecules than their PB counterparts. Similarly, while a significant portion of macrophages in synovial tissues expressed SOCS1 and SOCS3 proteins, the majority of T cells remained SOCS negative. Finally, SOCS1 was up-regulated in the synovial membranes from patients with RA when compared with osteoarthritis.

Conclusions. SOCS expression levels are profoundly altered in RA, and the profile of SOCS expression is dependent on both the cell type as well as the cellular compartment.

KEY WORDS: Rheumatoid arthritis, Cytokines, Signal transduction, JAK-STAT pathway, SOCS proteins


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
Cytokines are thought to play a key pathogenic role in rheumatoid arthritis (RA) [1,2]. In order to identify cytokines that may be important in RA, the majority of studies have focused on investigating cytokine levels in the joints. According to these studies, cytokines produced by macrophages, such as tumour necrosis factor (TNF)-{alpha}, IL-1, IL-6, IL-15, IL-18 and granulocyte-macrophage colony-stimulating factor (GM-CSF), are abundant in rheumatoid synovium and function as proinflammatory mediators [1,2]. Significant quantities of anti-inflammatory cytokines such as IL-10 are also present in the joints [3]. However, in addition to the level of expression, the biological effects of cytokines are dependent on the activation of signal transduction pathways, and the balance between signal transduction molecules and their inhibitors can modulate cellular responsiveness to cytokines.

Several cytokines involved in RA, such as IL-6, IL-10, IL-12, IL-15, GM-CSF and interferons (IFNs), activate Janus kinases (JAKs) and signal transducer and activator of transcription (STAT) factors to exert their effects [4]. The activity of JAK–STAT pathway is tightly controlled by negative-feedback inhibition, mediated largely by the suppressors of cytokine signalling (SOCS) proteins [5–7]. SOCS family of proteins consists of SOCS1 to SOCS7 and CIS1, with SOCS1, SOCS2, SOCS3 and CIS1 being the best-characterized members of the family. In general, SOCS proteins are not constitutively expressed in cells, but their expression is induced by a range of cytokines via activated STATs [7]. Once expressed, they inhibit the JAK-STAT pathway by several mechanisms, including suppression of JAK catalytic activity, prevention of STAT recruitment to activated cytokine receptors and induction of substrate degradation [8,9].

Studies with gene-disrupted or transgenic mice have confirmed the role of SOCS proteins as important regulators of immune and inflammatory responses in vivo [8,9]. SOCS proteins also seem to regulate the development and course of arthritis. Using acute IL-1-dependent arthritis model, Wong et al. [10] recently demonstrated severe joint inflammation, characterized by increased activation of macrophages, T cells and osteoclasts, in mice lacking SOCS3 in haematopoietic and endothelial cell compartment. Previously, Egan et al. [11] have demonstrated increased arthritis severity in mice lacking SOCS1 and IFN-{gamma} [11]. Along the same lines, STAT1 gene-knockout mice show exacerbated zymosan-induced arthritis, possibly due to reduction in SOCS1 [12]. On the other hand, overexpression of SOCS has inhibitory effects on arthritis, as demonstrated by the ability of adenoviral-mediated induction of SOCS3 to drastically reduce the severity of collagen-induced arthritis [13].

Although in vivo animal studies suggest an important regulatory role for SOCS proteins in arthritis models, these results may not be directly translated to human arthritis. At present, data regarding the expression and possible function of SOCS proteins in RA are very limited. In patients with RA, certain cytokine-induced signal transduction responses seem to be perturbed [14,15]. Given the increasing number of cytokine-related therapies currently used to treat the patients with chronic, active RA, it is important to examine the factors that may lead to altered cytokine responses in these patients. In the present study, we provide new evidence indicating that SOCS expression is altered in multiple cellular compartments in chronic RA.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 Acknowledgements
 References
 
Patients' samples
Synovial tissue samples were obtained from patients with RA and osteoarthritis (OA) during orthopaedic surgery. For reverse transcriptase polymerase chain reaction (RT-PCR) analysis, the samples were immediately placed into RNAlater RNA stabilization reagent (Qiagen Inc., Valencia, CA, USA) and stored at –20°C. Alternatively, the samples were fixed in formalin and embedded in paraffin. Peripheral blood (PB) samples were collected from 22 patients with active RA. Synovial fluid (SF) samples were obtained from the same patients by needle aspiration from inflamed knee joints into heparinized tubes. PB from healthy blood donors (Finnish Red Cross Blood Transfusion Service, Tampere, Finland) were obtained as controls.

The demographic and clinical characteristics of the patients are presented inTable 1. Patients with RA met the 1987 ACR criteria for RA [16], and patients with OA were classified as having primary OA according to the ACR criteria [17,18]. Informed consent was obtained from all patients according to the Declaration of Helsinki, and this study was approved by the Medical Ethics Committee of Tampere University Hospital.


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  		TABLE 1. Demographic and clinical characteristics of patients with RA and OA

 
Cell preparations and culture conditions
PB mononuclear cells (MCs) and SFMCs were isolated by a Ficoll-Paque Plus (Amersham Biosciences, Buckinghamshire, UK) density gradient centrifugation and washed twice with phosphate-buffered saline (PBS) containing 2 mM EDTA at 4°C. T cells and monocytes/macrophages were further purified from PBMCs and SFMCs using magnetic cell sorting technology (MACS; Miltenyi Biotec) according to manufacturer's instructions. CD3+ T cells were purified by negative selection using a cocktail of hapten-conjugated antibodies (Abs), specific for non-T cells and anti-hapten-coated microbeads (Pan T Cell Isolation Kit; Miltenyi Biotec, Auburn, CA, USA). PB monocytes and SF macrophages were isolated by positive selection with anti-CD14-coated microbeads (Miltenyi Biotec). T cell and monocyte/macrophage populations contained >90% CD3+ cells and CD14+ cells, respectively, as assessed by flow cytometry.

To study the effects of SFs or cytokines on SOCS expression, PBMCs from healthy volunteers were cultured at 1 x 106 cells/ml/well in complete RPMI medium. SFs stored at –70°C were added at 1 : 5 dilution at the onset of culture. Alternatively, cells were cultured in the presence of 10 ng/ml of recombinant human IFN-{gamma} (Immunogenex, Los Angeles, CA, USA), IL-1ß, IL-6, IL-10 or TNF-{alpha} (all from PeproTech, London, UK) for 1 or 16 h.

RNA isolation and quantitative RT-PCR analysis
Total RNAs were isolated from the cells and synovial tissues using RNeasy MiniKit (Qiagen Inc.) according to manufacturer's instructions. An additional 15 min incubation step in the presence of 27 Units DNAse I (Qiagen Inc.) was added to the protocol to digest any contaminating DNA. Total RNA of quantity 1 µg was reverse transcribed in a reaction volume of 20 µl using M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and random hexamers (Amersham Biosciences) as a primer. For preparing the standard curve, we isolated RNA from PBMCs, activated with phycohaemagglutinin for 3 days and stimulated with human IFN-{gamma} for 1 h. Serial dilutions (one to five) of the standard cDNA were made to correspond to cDNA transcribed from 375, 75, 15, 3, 0.6 and 0.16 ng of total RNA.

The forward and reverse primers recognizing separate exons of SOCS genes were designed using the Primer3 program (available at http://primer3.sourceforge.net/). The following primers were used: 5'-CTGGGATGCCGTGTTATTTT-3' and 5'-TAGGAGGTGCGAGTTCAGGT-3' for SOCS1, 5'-CAGGGAATGGCAGAGACACT-3' and 5'-TGGCAGAGAGAGAAGGGATG-3' for SOCS2, 5'-GCCACCTACTGAACCCTCCT-3' and 5'-ACGGTCTTCCGACAGAGATG-3' for SOCS3, 5'-AGCCCAGACAGAGAGTGAGC-3' and 5'-TGACAGCGTGAACAGGTAGC-3' for CIS1 and 5'-GAATATAATCCCAAGCGGTTTG-3' and 5'-ACTTCACATCACAGCTCCCC-3' for TBP.

The 15 µl real-time PCRs were performed in the LightCycler apparatus (Roche Diagnostics, Mannheim, Germany) using QuantiTect SYBR Green PCR kit (Qiagen Inc.) or LC-FastStart DNA Master SYBR Green I Kit (Roche Diagnostics) for SOCS1. Reactions contained 1.5 µl aliquot of sample or standard cDNA, 0.5 µM of the relevant primers and 1x ready-to-use reaction mixture containing Taq DNA polymerase, SYBR Green I fluorescent dye, dNTPs, 2.5 mM MgCl2 and reaction buffer. PCR amplification consisted of initial 15 min enzyme activation step at 95°C, followed by 45 cycles of denaturation (15 s at 95°C), annealing (20 s at 58°C) and extension (15 s at 72°C). The mean SOCS expression values from duplicate samples were normalized by dividing them by the mean values obtained for TBP house-keeping gene.

Immunohistochemical stainings
Formalin-fixed paraffin-embedded synovial tissue sections were deparaffinized, and the antigen epitopes were revealed by heating in 0.05 M Tris/HCl–0.001 M EDTA (pH 9) at 105°C for 10 min. Next, endogenous peroxidase activity was blocked by incubating the slides in 1% H2O2 for 10 min. The sections were stained with 2 µg/ml of rabbit anti-SOCS1 Ab H-93 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or 0.5 µg/ml of anti-SOCS3 mAb 1B2 (a gift from Douglas Hilton, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) for 1 h at RT in blocking solution. The antibodies were visualized with PowerVision+TM Poly-HRP IHC Detection Kit (ImmunoVision Technologies Corporation, Brisbane, CA, USA) and diaminobenzidine as a chromogen (ImmunoVision Technologies Co.), using Lab Vision Autostainer (Lab Vision Corporation, Fremont, CA, USA). Sections were counterstained with haematoxylin.

The specificity of SOCS Abs was confirmed by immunostainings of HeLa cells transfected with expression plasmids, containing either SOCS1 or SOCS3 cDNA (obtained from Douglas Hilton).

Alternatively, double immunofluorescence stainings were performed following the revealing of antigen epitopes as described above. The synovial tissue sections were simultaneously stained with 1 : 10 dilution of anti-CD3 mAb PS1 (IgG2a; Novocastra Laboratories, Newcastle upon Tyne, UK) or 1 : 100 dilution of anti-CD68 mAb PG-M1 (IgG3; Dako, Glostrup, Denmark) and with 20 µg/ml of rabbit anti-SOCS1 Ab H-93 or 10µg/ml of anti-SOCS3 mAb 1B2 (IgG1) for 1 h at RT in blocking solution. Following three washes in PBS, 0.025% Tween, the slides were incubated with the relevant isotype-specific, fluorescent secondary Abs at 1 : 200 dilution for 30 min at RT. The following secondary Abs were used: Alexa Fluor® 488—conjugated anti-mouse IgG2a and anti-mouse IgG3, Texas Red—conjugated anti-rabbit IgG and Alexa Fluor® 555—conjugated anti-mouse IgG1 (all from Molecular Probes, Inc., Eugene, OR, USA).

Statistical analysis
Statistical analysis was performed using a non-parametric Mann–Whitney U-test. Correlations were calculated using Spearman's rank correlation method.


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 Acknowledgements
 References
 
Increased expression of SOCS1 and SOCS3 and decreased expression of CIS1 in PBMCs from patients with RA
In order to identify the factors that may alter cellular responsiveness to cytokines in chronic RA, we have examined the expression of SOCS molecules. First, SOCS1–3 and CIS1 mRNA levels in PBMCs from patients with active RA (for details, seeTable 1) were studied using quantitative RT-PCR. The results were compared with those obtained for PBMCs from healthy volunteers.

Marked differences in the expression of SOCS molecules between RA patients and healthy volunteers were observed. The expression of SOCS1 and SOCS3 were significantly increased in RA PBMCs when compared with normal PBMCs (Fig. 1). SOCS2 mRNA levels were also clearly increased in 4 out of 15 patients, but in the rest of the patients SOCS2 levels were comparable with healthy volunteers. In contrast, the expression of CIS1 was down-regulated in RA patients when compared with healthy volunteers, indicating that up-regulation of SOCS molecules in PBMCs is not a general characteristic of RA patients (Fig. 1).


Figure 1
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  		FIG. 1. Levels of SOCS1–3 and CIS1 mRNA in PBMCs derived from 10 healthy volunteers and 15 patients with RA studied using quantitative real-time RT-PCR. Data are presented as relative SOCS expression divided by TBP levels, and horizontal lines represent the mean expression levels in each group. Significant differences between the study groups are marked with an asterisk: **P < 0.01, ***P < 0.001.

 
Within the RA group, the expression levels of various SOCS molecules differed considerably. To test whether this would be due to the differences in disease activity, possible correlations between C-reactive protein and erythrocyte sedimentation rate values and SOCS expression levels were examined. However, no significant correlations between these parameters were observed (data not shown). In addition, SOCS expression levels were not significantly different in five patients treated with TNF-{alpha} blocking agents when compared with patients receiving conventional therapy.

Increased levels of SOCS1 and SOCS2 in PB T cells and SOCS3 in PB monocytes
The MC fractions from PB consist mainly of T and B cells, NK cells and monocytes. Both T cells and monocytes/macrophages are thought to play an important role in the pathogenesis of RA [2]. In order to examine which of these cell types contributed to the altered expression of SOCS molecules in RA PBMCs, T cells and monocytes were purified from the PB of RA patients and healthy volunteers.

As shown inFig. 2, SOCS1 expression was increased in both PB T cells (left panel) and monocytes (right panel) from patients with RA when compared with healthy volunteers. However, SOCS1 expression level in RA PB T cells was approximately six times higher than in monocytes, thus suggesting that the increased expression of SOCS1 in unfractionated PBMC from RA patients is mainly due to up-regulation of SOCS1 in PB T cells. The levels of SOCS2 were also increased in PB T cells from patients with RA, whereas PB monocytes from patients and controls exhibited negligible SOCS2 expression. Similar to SOCS1, SOCS3 was significantly up-regulated in both PB T cell and monocyte fractions derived from RA patients (Fig. 2). However, in contrast to SOCS1, SOCS3 was primarily expressed by PB monocytes. Finally, although CIS1 was down-regulated in PBMCs from patients with RA, the levels of CIS1 were not clearly different either in PB T cells or monocytes between RA patients and controls.


Figure 2
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  		FIG. 2. Expression of SOCS1–3 and CIS1 mRNA in PB and SF T cells (left panel) and monocytes/macrophages (right panel) derived from healthy volunteers (n = 8) or patients with RA (n = 5–8). Data are presented as relative SOCS expression divided by TBP levels, and horizontal lines represent the mean expression levels in each group. Significant differences between the study groups are marked with an asterisk: *P < 0.05, **P < 0.01, ***P < 0.001.

 
Together, these results demonstrate up-regulation of SOCS1 and SOCS2 in PB T cells and up-regulation of SOCS3 primarily in PB monocytes from patients with RA.

Differential SOCS expression between PB and SF T cells and monocytes/macrophages
Synovial T cells and macrophages are thought to be derived from the peripheral circulation. However, in the joints these cells are exposed to a wide variety of inflammatory mediators, and show both phenotypical and functional alterations when compared with T cells and monocytes in the PB [19]. Therefore, it was relevant to compare SOCS expression levels between SF T cells and macrophages and their PB counterparts.

Both SF T cells and macrophages demonstrated SOCS expression profiles that were different from those in PB T cells and monocytes from RA patients. In SF T cells, SOCS2 and CIS1 levels were significantly decreased when compared with PB T cells from RA patients, CIS1 levels being even lower than in T cells from healthy controls (Fig. 2, left panel). The mean levels of SOCS1 and SOCS3 in SF T cells were also slightly lower than in PB T cells from RA patients, although they were still elevated compared with control T cells. SF macrophages, in turn, expressed higher levels of SOCS mRNA than their PB counterparts. In addition to up-regulation of SOCS3, SF macrophages demonstrated increased expression of SOCS1 and SOCS2 in comparison with PB monocytes derived either from RA patients or healthy controls (Fig. 2, right panel).

Thus, SF T cells express generally lower and SF macrophages higher levels of SOCS mRNA than their PB counterparts, indicating that SOCS molecules are differentially expressed in various cellular compartments.

Regulation of SOCS expression by inflammatory SFs
The observed differences in SOCS expression between PB and SF T cells and monocytes/macrophages suggest that there may be factors in the SF that directly affect SOCS expression. To test this possibility, PBMCs from healthy volunteers were cultured in the presence of inflammatory SFs derived from 17 patients with active RA, and SOCS mRNA levels were determined by quantitative RT-PCR following 1 and 16 h culture.

Stimulation indexes of individual SF samples shown inFig. 3 represent SOCS levels in cells cultured in the presence of SF divided by those in cells cultured in the presence of medium alone. Following 1 h culture, inflammatory SFs induced strong up-regulation of SOCS mRNA, and the strongest induction was observed with SOCS3 (mean stimulation index 6.9). The results obtained with cells cultured for 16 h in the presence of SFs, were somewhat different. Upon longer culture, the increase in SOCS1 and SOCS3 levels induced by SFs was more modest and a decrease in SOCS2 and CIS1 expression was observed in 11 and 10 out of 17 samples, respectively (Fig. 3). Thus, these results suggest that although short exposure of MCs to inflammatory SFs clearly results in up-regulation of SOCS levels, prolonged exposure may lead to decreased SOCS expression.


Figure 3
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  		FIG. 3. Regulation of SOCS mRNA expression by inflammatory SFs. PBMCs derived from healthy volunteers were cultured in the presence or absence of SFs (1 : 5 dilution) derived from 17 patients with RA for 1 or 16 h. Stimulation indexes represent the relative SOCS mRNA levels (SOCS/TBP ratio) in PBMCs stimulated by SFs divided by the relative SOCS mRNA levels in cells cultured in the presence of medium alone. Each dot represents the mean of two separate experiments.

 
Up-regulation of SOCS mRNA by cytokines
Given the current knowledge on regulation of SOCS expression, it seems likely that cytokines are involved in the up-regulation of SOCS mRNA levels by inflammatory SFs. For example, IFN-{gamma}, IL-6 and IL-10 are present at increased concentrations in rheumatoid SFs [1] and can induce SOCS expression in several cell types [5–9]. In addition, data indicates that IL-1 and TNF-{alpha}, that do not generally activate the JAK-STAT pathway, can up-regulate SOCS1 and SOCS3 in certain cell types [10,20–22]. To examine which of these cytokines is most potent in inducing SOCS expression in PBMCs, the cells were cultured in the presence or absence of cytokines for 1 or 16 h, and SOCS mRNA levels were determined.

As expected, IFN-{gamma}, IL-6 and IL-10 all increased SOCS1 and SOCS3 expression in PBMCs; IFN-{gamma} being the best inducer of SOCS1, while IL-10 induced highest levels of SOCS3 (Fig. 4). IFN-{gamma} also up-regulated CIS1 and IL-10 increased SOCS2 and CIS1 levels in PBMCs. In general, up-regulation of SOCS mRNA by cytokines was more pronounced following 1 h culture, which is in accordance with the findings obtained with cells stimulated by SFs. IL-1ß or TNF-{alpha} did not significantly affect SOCS mRNA levels following 1 h culture. However, in cells cultured in the presence of these cytokines for 16 h, IL-1ß induced a modest but statistically significant increase in SOCS3 levels (relative expression 7.2 vs 3.1;Fig. 4). Importantly, none of the cytokines tested decreased SOCS levels, suggesting that the exposure to these cytokines may not be involved in the down-regulation of SOCS expression observed in SF T cells.


Figure 4
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  		FIG. 4. Expression of SOCS mRNA in PBMCs derived from healthy volunteers and stimulated by 10 ng/ml of various cytokines for 1 or 16 h. The bars represent the mean (±S.D.) SOCS/TBP ratio from four (left panel) or five (right panel) experiments. Significant differences in comparison with cells cultured in the presence of medium alone (control) are marked with an asterisk: *P < 0.05, **P < 0.01.

 
Together, these results suggest that elevated levels of cytokines, such as IFN-{gamma}, IL-1, IL-6 and IL-10, may lead to persistently increased SOCS expression in patients with RA.

Increased expression of SOCS1 in rheumatoid synovial tissues
The results so far demonstrated primarily up-regulated SOCS expression in patients with active RA when compared with healthy controls and suggested that the elevated levels of cytokines may contribute to this finding. In order to investigate SOCS levels in RA patients with long-standing, less-active disease, synovial tissues from patients with RA were obtained during orthopaedic operations (seeTable 1 for details). Synovial tissues obtained from patients with OA were studied as controls.

The expression of SOCS1 mRNA was significantly increased in synovial tissues from patients with RA when compared with those from patients with OA (Fig. 5). A similar tendency was observed with SOCS3 and CIS1, but these differences did not reach statistical significance (P = 0.0628 and 0.0508, respectively). The expression of SOCS2 was not significantly different between the two patient groups (Fig. 5). Similar to the results obtained with PBMCs, SOCS levels between individual RA patients varied considerably. Some patients expressed particularly high levels of SOCS molecules, and the highest levels of both SOCS2 and SOCS3 mRNA were detected in the same patient. Finally, no significant correlations were observed between CRP and SOCS mRNA levels in RA patients (data not shown).


Figure 5
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  		FIG. 5. Expression of SOCS1–3 and CIS1 mRNA in synovial tissues from 10 patients with OA and 13 patients with RA. Data are presented as relative SOCS expression divided by TBP levels, and horizontal lines represent the mean expression levels in each group. An asterisk denotes a significant difference between the patient groups: *P < 0.05.

 
Taken together, these results indicate that the expression of SOCS1 is up-regulated in the synovial membranes of patients with long-standing RA.

Expression of SOCS1 and SOCS3 proteins in the synovial membranes
In addition to transcriptional control, SOCS1 and SOCS3 levels are regulated by post-transcriptional mechanisms [23,24]. To verify the expression of SOCS1 and SOCS3 proteins in the RA synovium, and to gain insight into the cell types expressing these proteins, immunohistochemical stainings of five rheumatoid synovial membranes were performed with Abs specific for SOCS1 and SOCS3.

Significant proportion of synovial tissue cells expressed both SOCS1 and SOCS3 proteins. In particular, the vast majority of cells in the synovial lining layer showed intense staining with both SOCS1 and SOCS3 Abs (Fig. 6B, C, E and F). Adjacent tissue sections stained with unspecific rabbit or mouse IgG remained negative (Figs 6A and D). In the sublining stroma, SOCS1-positive cells were rather abundant, whereas SOCS3 immunostaining was observed only in localized areas. In addition, blood vessel endothelium also demonstrated prominent expression of SOCS proteins.


Figure 6
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  		FIG. 6. Immunohistochemical stainings of RA synovial tissue with unspecific rabbit IgG (A) or mouse IgG (D) and Abs specific for SOCS1 (B-C) or SOCS3 (E–F). Stainings from one out of five RA patients are shown (magnification x200).

 
To investigate SOCS1 and SOCS3 protein expression in synovial tissue T cells and macrophages, double immunofluorescence stainings were performed. Results from five RA patients revealed that only occasional CD3+ cells were shown to express SOCS1 or SOCS3, whereas the vast majority of T cells remained SOCS-negative (Fig. 7). In contrast, a significant proportion of CD68+ cells were shown to express both SOCS1 and SOCS3. However, not all SOCS1 and SOCS3-positive cells in the lining and sublining layers were macrophages, suggesting that fibroblast-like synoviocytes also express SOCS proteins.


Figure 7
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  		FIG. 7. Double immunofluorescence stainings of RA synovial tissue with Abs specific for CD3 or CD68 (green) and SOCS1 or SOCS3 (red). CD3 is located on the cell surface, CD68 in cytoplasmic granuli and SOCS1 and SOCS3 in the cytoplasm. Colocalization is seen as yellow. Stainings from one out of five RA patients are shown.

 
Together, these results show that in the synovial membranes SOCS1 and SOCS3 proteins are primarily expressed in the lining layer and in blood vessel endothelium. In addition, while synovial tissue macrophages show prominent expression of SOCS1 and SOCS3 proteins, T cells in the synovium are mainly SOCS-negative.


   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
Acknowledgements
 References
 
In order to characterize factors that may alter cellular responsiveness to cytokines in chronic RA, we have investigated the expression of SOCS molecules. SOCS proteins are at the crossroads of multiple immunological and inflammatory pathways, and several factors such as altered cytokine milieu and cellular activation status may affect SOCS expression profiles in RA. Therefore, we considered it necessary to perform a detailed analysis of SOCS expression at different cellular compartments. SOCS levels were clearly altered in patients with RA, and the profile of SOCS expression was highly dependent on the cell type and cellular localization studied. While PB T cells and monocytes and synovial macrophages generally exhibited elevated levels of SOCS molecules, SOCS expression in synovial T cells was more restricted. This conclusion was supported by the findings in T cells derived from either SF or synovial tissues and observed both at mRNA and protein expression levels. In contrast, SF macrophages exhibited increased levels of SOCS mRNA when compared with PB monocytes, and a significant proportion of synovial tissue macrophages also expressed SOCS1 and SOCS3 proteins. Together, the differential SOCS levels in PB and SF cells suggest that alterations in SOCS expression may arise secondary to the changes in the cellular environment and/or cellular differentiation status.

Previous data regarding SOCS expression in RA are limited. Yamana et al. [15] reported increased SOCS1 but decreased SOCS3 expression in PB CD4+ T cells from RA patients. While the present study confirms the results on elevated levels of SOCS1 in PB T cells, we also demonstrated clear up-regulation of SOCS2 and SOCS3 in these cells. A recent study indicated SOCS3 to be consistently overexpressed in PBMCs from rats with collagen-induced arthritis, and similar results have previously been observed in mouse arthritis models [25]. In addition, Shouda et al. [13] have demonstrated increased SOCS3 mRNA expression in synovial tissues from three patients with RA when compared with those with OA, and there was a similar tendency in our study. Thus, there is considerable evidence to suggest that, in addition to SOCS1, SOCS3 is generally up-regulated in arthritis, at least in certain cell types.

Our results demonstrate several cytokines as possible candidates for driving increased SOCS expression in RA. IFN-{gamma}, IL-1ß, IL-6 and IL-10 are up-regulated in the joints and/or peripheral circulation of patients with RA [1]. These cytokines induced SOCS expression in PBMCs at variable kinetics, and are likely to contribute to increased SOCS levels observed in PB T cells and monocytes, as well as in synovial macrophages. However, it was somewhat surprising that synovial T cells, that are similarly exposed to these cytokines, express lower levels of SOCS molecules than circulating T cells. It is possible that other factors than cytokines present in the inflamed joints may lead to down-regulation of SOCS levels in these cells, as suggested by decreased SOCS2 and CIS1 expression in PBMC-cultured O/N in the presence of inflammatory SFs. For example, chronic exposure of synovial T-cells to reactive oxygen species has previously been shown to cause alterations in the expression and function of signal transduction molecules involved in T-cell receptor signalling [26,27]. On the other hand, differential SOCS expression in synovial T cells may merely reflect changes in the activation and differentiation status of these cells when compared with PB T cells.

Overexpression of SOCS has also been demonstrated in other human autoimmune diseases and in allergy. For example, SOCS3 is increased in the intestinal mucosa derived from patients with Crohn's disease and ulcerative colitis [28]. The levels of SOCS1, SOCS2 and SOCS3 are also elevated in the skin of patients with psoriasis or allergic contact dermatitis [29]. Moreover, Seki et al. [30] showed up-regulated levels of SOCS3 in PB T cells from patients with asthma and atopic dermatitis, and correlation between SOCS3 expression and serum IgE levels. They also demonstrated preferential expression of SOCS3 in human Th2 cells and enhanced Th2 development in SOCS3 transgenic mice, concluding that SOCS3 is involved in Th2-mediated allergic diseases [30]. However, RA, Crohn's disease and psoriasis are considered to be Th1-mediated autoimmune diseases [31–33]. Therefore, it seems that SOCS molecules can be up-regulated in human diseases associated with both Th1 and Th2 pathology.

Gene disruption and transgenic mouse models of individual SOCS molecules show highly specific phenotypes, which also depend on the cell types that are targeted [8,9]. Thus, the functional consequences of altered SOCS expression in autoimmune and inflammatory diseases are also likely to depend on the SOCS molecules as well as the cell types or tissues affected. In patients with long-standing RA, increased levels of SOCS1 and SOCS3 could suppress cytokine signalling and potentially prevent the deleterious actions of proinflammatory cytokines, such as IL-6, IL-12, IL-15, GM-CSF and IFN-{gamma}. On the other hand, high levels of these SOCS proteins could also inhibit the beneficial effects of anti-inflammatory cytokines, and thus lead to the failure of immunoregulatory and protective mechanisms in RA. Indeed, it was recently demonstrated that IL-10 has lost its ability to down-regulate IFN-{gamma} production by PB CD4+ T cells from RA patients. This phenomenon is due to the impaired induction of STAT3 by IL-10, and is likely to be mediated via increased expression of SOCS1 in these cells [15]. In addition, synovial macrophages from RA patients show attenuated signal transduction responses following IL-10 receptor ligation [14]. This finding is particularly interesting given our present results on increased SOCS1 and SOCS3 expression in SF macrophages. Finally, recent results from studies with T-cell specific SOCS3 conditional knockout mice suggest that SOCS3 negatively regulates the generation of immunoregulatory T cells, producing transforming growth factor-ß1 and IL-10 [34].

Together, we show that SOCS levels are profoundly altered in RA, with each SOCS molecule showing an individual expression pattern depending on the cell type and localization studied. Up-regulation of SOCS expression may significantly affect cellular responsiveness to cytokines in chronic RA, and could be involved in disease progression by causing unresponsiveness to anti-inflammatory cytokines. These findings establish the need for analysing SOCS functions at specific cellular localizations in autoimmune diseases.


   Acknowledgements
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
References
 
We thank Paula Kosonen, Merja Lehtinen and Mariitta Vakkuri for excellent technical assistance, and Teemu Honkanen for his assistance with double immunofluorescence stainings. This study was supported by grants from the Academy of Finland (grant 200861), the Medical Research Fund of Tampere University Hospital and the Scandinavian Rheumatology Research Foundation.

Formula


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Submitted 4 February 2007; revised version accepted 18 June 2007.
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Regulators of cytokine signalling in rheumatoid arthritis
Rheumatology, December 1, 2007; 46(12): 1745 - 1746.
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