Expression of K2P5.1 potassium channels on CD4+T lymphocytes correlates with disease activity in rheumatoid arthritis patients
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CD4+ T cells express K2P5.1 (TWIK-related acid-sensitive potassium channel 2 (TASK2); KCNK5), a member of the two-pore domain potassium channel family, which has been shown to influence T cell effector functions. Recently, it was shown that K2P5.1 is upregulated upon (autoimmune) T cell stimulation. The aim of this study was to correlate expression levels of K2P5.1 on T cells from patients with rheumatoid arthritis (RA) to disease activity in these patients.
Expression levels of K2P5.1 were measured by RT-PCR in the peripheral blood of 58 patients with RA and correlated with disease activity parameters (C-reactive protein levels, erythrocyte sedimentation rates, disease activity score (DAS28) scores). Twenty patients undergoing therapy change were followed-up for six months. Additionally, synovial fluid and synovial biopsies were investigated for T lymphocytes expressing K2P5.1.
K2P5.1 expression levels in CD4+ T cells show a strong correlation to DAS28 scores in RA patients. Similar correlations were found for serological inflammatory parameters (erythrocyte sedimentation rate, C-reactive protein). In addition, K2P5.1 expression levels of synovial fluid-derived T cells are higher compared to peripheral blood T cells. Prospective data in individual patients show a parallel behaviour of K2P5.1 expression to disease activity parameters during a longitudinal follow-up for six months.
Disease activity in RA patients correlates strongly with K2P5.1 expression levels in CD4+ T lymphocytes in the peripheral blood in cross-sectional as well as in longitudinal observations. Further studies are needed to investigate the exact pathophysiological mechanisms and to evaluate the possible use of K2P5.1 as a potential biomarker for disease activity and differential diagnosis.
KeywordsRheumatoid Arthritis Rheumatoid Arthritis Patient Synovial Fluid Etanercept Tocilizumab
disease activity score of 28 joints
disease modifying anti-rheumatic drugs
erythrocyte sedimentation rate
magnetic cell sorting
peripheral blood mononuclear cells
TWIK-related acid-sensitive potassium channel 2
visual analogy scale.
Rheumatoid arthritis (RA) is a chronic inflammatory disease which is characterized by pain, swelling and progressive destruction of multiple joints. The systemic nature of RA causes, next to loss of joint function, substantially decreased quality of life and increased mortality of patients. Current treatments are mainly based on immunosuppressive disease-modifying antirheumatic drugs, among them the rapidly expanding family of biologic agents . Close monitoring of disease activity is mandatory for the evaluation of treatment efficacy as a substantial percentage of patients do not respond adequately to first-line therapy. In these cases, as well as in patients with disease exacerbations, a change in treatment strategy is required . Monitoring of disease activity includes patient history, clinical examination, blood values (C-reactive protein (CRP) levels and erythrocyte sedimentation rate (ESR)) and composite scores such as the widely used disease activity score (DAS28). The DAS28 score includes the number of swollen and painful joints, the ESR rate and the patient's subjective evaluation on a visual analogy scale (VAS) [3, 4].
The potassium channel K2P5.1 (TWIK-related acid-sensitive potassium channel 2 (TASK2); KCNK5) belongs to the family of two-pore domain potassium channels (K2P channels) which has recently been shown to be expressed on T lymphocytes [5, 6]. K2P5.1 is important for T cell functions such as proliferation or cytokine production  as it is hypothesized that the counterbalancing efflux of potassium channels is mandatory for a longer lasting elevation of the intracellular Ca2+ levels during T cell stimulation . Moreover, chronic repetitive stimulation leads to an upregulation of K2P5.1 channel expression whereas pharmacological blockade or siRNA-induced gene silencing of K2P5.1 results in a reduction of T cell effector functions. It has additionally been shown that expression levels of this channel are strongly increased on T lymphocytes from the peripheral blood from clinically active relapsing-remitting multiple sclerosis (MS) patients. Interestingly, expression and MS-specific upregulation were found predominantly on CD8+ T cells rather than on CD4+ T cells which may be due to a disease-specific pathogenic role of cytotoxic T lymphocytes. Expression of K2P5.1 was even higher on cerebrospinal fluid (CSF)-derived T lymphocytes than in the peripheral blood and K2P5.1-positive T lymphocytes can be found within inflammatory lesions from MS patients. So far, it was not known whether these findings are MS-specific or can be similarly found in other autoimmune disorders. CD4+ T helper cells play an important role in the pathogenesis of RA. This is suggested by its association with certain MHC II loci, especially HLA-DRB1, and PTPN22, which is relevant for T cell function . The therapeutic effects of blockade of T cell costimulation by abatacept provides more direct evidence .
Therefore, we investigated the correlation of K2P5.1 expression levels on T lymphocytes from RA with different disease activity parameters. The influence of different therapies was taken into account as they might potentially influence K2P5.1 expression. Finally, a longitudinal study was conducted in a subset of patients who underwent therapy change due to disease exacerbation and these patients were followed up for six months.
Materials and methods
Material from RA patients
Characteristics of RA patients
56.6 (23 to 79)
3.59 (1.10 to 7.24)
19.3 (1.0 to 69.0)
1.06 (0.02 to 4.68)
Peripheral blood mononuclear cells (PBMCs) from RA patients and healthy donors were isolated out of fresh blood samples by density gradient centrifugation using a lymphocyte separation medium (PAA Laboratories, Pasching, Austria). CD4+ and CD8+ T cells were separated by magnetic cell sorting (MACS) according to the manufacturer's instruction (Miltenyi, Bergisch Gladbach, Germany) and purity was >95%. Direct cell isolation had no effect on K2P5.1 expression when compared to indirect cell isolation (data not shown). Synovial fluid from patients undergoing joint puncture for therapeutic or diagnostic purposes was processed accordingly.
For analysis of K2P5.1-mRNA expression, RNA was purified using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and cDNA synthesis was performed using a standard protocol with random hexamer primers (all reagents were purchased from Applied Biosystems, Foster City, CA, USA). This cDNA was used in a RT-PCR assay with specific primers for KCNK5 (Hs00186652_m1; FAM-labeled; Applied Biosystems) and endogen control primers for 18sRNA (Hs_4319413E; VIC-labeled; Applied Biosystems). Real time RT-PCR was performed according to the manufacturer's protocol.
In one set of experiments cultured CD4+ T cells from healthy donors were treated with methotrexate (Medac, Hamburg, Germany), etanercept (Wyeth Europa Ltd., Maidenhead, Berkshire, UK), adalinumab (Abbott Laboratories Ltd., Maidenhead, Berkshire, UK), certolizumab (Ucb S.A. Brussels, Belgium), tocilizumab (Roche, Welwyn Garden City, UK) or hydroxychloroquine (Sanofi-Aventis, Frankfurt am Main, Germany) over 24 hours before PCR analysis.
Whole cell lysates from unstimulated and CD3/CD28-bead stimulated MACS-isolated CD4+ T lymphocytes were analyzed as described previously  using rabbit anti-K2P5.1 and HRP-donkey anti-rabbit (Amersham, Freiburg, Germany). HRP was inactivated with 2% NaN3 and blots were stained with β-actin antibody for protein loading control. Quantification of Western blot results was performed using Image J.
Flow cytometry analysis
The following antibodies were used: rabbit anti-K2P5.1 (Sigma, St. Louis, MO, USA) and goat anti-rabbit Cy2 (Dianova, Hamburg, Germany; intracellular staining), CD4-FITC (RPA-T4), CD69-PerCP (BD Pharmingen, Franklin Lakes, NJ, USA), and CD25-PE (Miltenyi). Flow cytometry was done using a FACSCalibur system (BD Bioscience, Heidelberg, Germany) and CellQuest Pro Software (BD Bioscience).
Immunfluorescence staining was performed on human synovial tissue sections (n = 5). For double labelling, slices were postfixated in 4% paraformaldehyde (PFA) and incubated in blocking solution. Slices were then incubated consecutively with anti-CD3 (1:100, Dako, Glostrup, Denmark) and TASK2 (Sigma). Secondary antibodies were Alexa goat anti-mouse Fluor 488 and goat anti-rabbit Cy3 (Dianova, Hamburg, Germany).
All results are presented as mean ± standard error of measure (SEM). Statistical analysis was performed using a modified Student's t-test  in case of normally distributed data, or a Mann-Whitney test otherwise. Spearman's rank correlation was used for correlation analysis. P-values < 0.05 were considered statistically significant.
In vitro effects of methotrexate, etanercept, adalinumab, certolizumab, tocilizumab and hydroxychloroquine on K2P5.1 expression levels
CD4 T cells, unstimulated
CD4 T cells, stimulated
1.08 ± 0.18
0.93 ± 0.08
0.97 ± 0.07
1.01 ± 0.04
0.96 ± 0.14
1.12 ± 0.05
0.99 ± 0.09
1.14 ± 0.08
1.12 ± 0.22
1.24 ± 0.15
0.82 ± 0.3
0.84 ± 0.35
Disease activity in RA patients was found to correlate strongly with K2P5.1 expression levels in CD4+ T lymphocytes in the peripheral blood in a cross-sectional study in 58 patients. Furthermore, longitudinal observations in individual patients showed comparable changes in all disease surrogate markers with K2P5.1 expression. It seems plausible that K2P5.1 expression reflects the activation status of chronically stimulated autoimmune CD4+ T lymphocytes.
However, a number of questions remain to be addressed which are beyond the focus of this initial study, for example: Can K2P5.1 expression serve as a biomarker for disease activity in RA? Current serum markers (CRP and ESR levels) which are also part of composite scores like DAS28 or DAS28-CRP reflect rather the level of systemic inflammation than the specific activation status of pathogenic immune cells. Moreover, their use seems to be limited especially in the case of tocilizumab which inhibits the systemic acute phase reaction at least in part independently from its well-proven anti-rheumatic effects . This problem has, for example, been addressed by Matsui et al. who evaluated neutrophil CD64 as a biomarker for otherwise masked infection under tocilizumab therapy . We observed that in contrast to our cross-sectional study and our longitudinal study with other medications, K2P5.1 expression changes behaved differently in tocilizumab-treated patients. Two out of nine patients even showed an opposite upregulation of K2P5.1 at three months preceding a relapse about three months later. At the present, it seems too early and patient numbers are too low to state that K2P5.1 upregulation generally precedes clinical relapses in RA, whether it reveals otherwise masked clinical developments under tolizumab therapy or whether its validity is limited under these circumstances. Therefore, in light of the current results further patient studies need to assess the prognostic value, time course and clinical validity of monitoring K2P5.1 expression in RA patients. Furthermore, it has to be proven whether K2P5.1 can be used for differential diagnosis.
Additionally, research efforts are needed concerning the mechanisms underlying K2P5.1 function and regulation in the pathophysiology of RA. Especially the use of animal models for RA may help to provide insight in this context.
We show here for the first time a correlation of K2P5.1 expression levels in CD4+ T lymphocytes and disease activity in patients suffering from RA. Since other studies already showed a functional role of K2P5.1 for T cell effector function this member of the two-pore domain potassium channel family might represent an interesting molecular target for diagnostic and/or therapeutic applications. However, further studies from independent cohorts are warranted to confirm and extend the presented findings. Furthermore, the use of animal models for RA might help to shed more light on the functional role of K2P5.1 in RA pathogenesis.
We thank Barbara Reuter for excellent technical assistance. This work was supported by Interdisziplinäres Zentrum für klinische Forschung (Interdisciplinary Center for Clinical Research A-54-1, S.G.M., H.W.), the Deutsche Forschungsgemeinschaft (SFB 581, TP A10, S.G.M.), and the Bundesministerium für Bildung und Forschung (Kompetenznetzwerk Multiple Sclerosis, Consortium UNDERSTAND MS, H.W.).
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