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RESEARCH ARTICLE Open Access
Expression of K2P5.1 potassium channels on CD4
+
T lymphocytes correlates with disease activity in
rheumatoid arthritis patients
Stefan Bittner1†, Nicole Bobak2†, Martin Feuchtenberger3, Alexander M Herrmann2, Kerstin Göbel2,
Raimund W Kinne4, Anker J Hansen5, Thomas Budde6, Christoph Kleinschnitz1, Oliver Frey7, Hans-Peter Tony3,
Heinz Wiendl2, Sven G Meuth1,2,6*
Abstract
Introduction: 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.
Methods: 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.
Results: 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.
Conclusions: 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.
Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory
disease which is characterized by pain, swelling and pro-
gressive destruction of multiple joints. The systemic nat-
ure of RA causes, next to loss of joint function,
substantially decreased quality of life and increased mor-
tality of patients. Current treatments are mainly based
on immunosuppressive disease-modifying antirheumatic
drugs, among them the rapidly expanding family of
biologic agents [1]. Close monitoring of disease activity
is mandatory for the evaluation of treatment efficacy as
a substantial percentage of patients do not respond ade-
quately to first-line therapy. In these cases, as well as in
patients with disease exacerbations, a change in treat-
ment strategy is required [2]. Monitoring of disease
activity includes patient history, clinical examination,
blood values (C-reactive protein (CRP) levels and ery-
throcyte 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].
* Correspondence: sven.meuth@ukmuenster.de
† Contributed equally
1Department of Neurology, University of Wuerzburg, Josef-Schneider-Str. 11,
Wuerzburg, 97080, Germany
Full list of author information is available at the end of the article
Bittner et al. Arthritis Research & Therapy 2011, 13:R21
http://arthritis-research.com/content/13/1/R21
© 2011 Bittner et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
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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 [7] as it is hypothesized that the
counterbalancing efflux of potassium channels is manda-
tory for a longer lasting elevation of the intracellular
Ca2+ levels during T cell stimulation [7]. 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 patho-
genic 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 [8]. The
therapeutic effects of blockade of T cell costimulation
by abatacept provides more direct evidence [9].
Therefore, we investigated the correlation of K2P5.1
expression levels on T lymphocytes from RA with differ-
ent disease activity parameters. The influence of differ-
ent therapies was taken into account as they might
potentially influence K2P5.1 expression. Finally, a longi-
tudinal 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
T lymphocytes were isolated from the peripheral blood
of 73 RA patients (cross-sectional-study: 58 patients,
longitudinal study: 20 patients, included in both studies:
5 patients) and 10 age- and sex-matched healthy donors.
The patients were seen between April 2009 and August
2010 in the outpatient RA center at the University of
Wuerzburg. We differentiated the patients according to
EULAR criteria in patients in remission (DAS28 ≤2.6),
patients with low disease activity (2.6 <DAS28 ≤3.2),
with moderate disease activity (3.2 <DAS28 ≤5.1) and
high disease activity (DAS28 >5.1). The DAS28 specifies
the disease activity measuring the number of tender and
swollen joints, the ESR and VAS of general health. Con-
ventional therapy with disease modifying anti-rheumatic
drugs (DMARDs) included leflunomide, hydroxychloro-
quine, sulfasalazine, methotrexate and glucocorticoids.
Treatment with biologic agents included tumor necrosis
factor alpha (TNFa) blockers (etanercept, adalimumab,
infliximab and certolizumab) and anti-CD20 treatment
(rituximab). Patients receiving interleukin (IL)-6 recep-
tor blocking antibodies (tocilizumab) were excluded
from the primary analysis because of its direct influence
on CRP and ESR levels. However, they were included
into the longitudinal study due to their influence on
CRP and ESR values. In additional sets of experiments,
the synovial fluid of patients who underwent joint punc-
tures for diagnostic or therapeutic purposes was investi-
gated. See Table 1 for details on the RA patients. All
patients gave informed consent in accordance with the
Declaration of Helsinki and a protocol approved by the
Ethics Committee of the University of Wuerzburg Medi-
cal School (No. 109/10).
Cell isolation
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 sepa-
rated 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 indir-
ect cell isolation (data not shown). Synovial fluid from
patients undergoing joint puncture for therapeutic or
diagnostic purposes was processed accordingly.
Real-time RT-PCR
For analysis of K2P5.1-mRNA expression, RNA was puri-
fied using Trizol reagent (Invitrogen, Carlsbad, CA, USA)
and cDNA synthesis was performed using a standard pro-
tocol with random hexamer primers (all reagents were
purchased from Applied Biosystems, Foster City, CA,
Table 1 Characteristics of RA patients
Patients (male/female) 19/54
Age 56.6 (23 to 79)
DAS28 3.59 (1.10 to 7.24)
ESR 19.3 (1.0 to 69.0)
CRP 1.06 (0.02 to 4.68)
All of the probands had been diagnosed with RA by a board-certified
rheumatologist. Values are depicted as mean and range. Abbrevations: CRP,
C-reactive protein; DAS28, disease activity score in 28 joints; ESR, erythrocyte
sedimentation rate.
Bittner et al. Arthritis Research & Therapy 2011, 13:R21
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USA). This cDNA was used in a RT-PCR assay with speci-
fic 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 manu-
facturer’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), certoli-
zumab (Ucb S.A. Brussels, Belgium), tocilizumab
(Roche, Welwyn Garden City, UK) or hydroxychloro-
quine (Sanofi-Aventis, Frankfurt am Main, Germany)
over 24 hours before PCR analysis.
Western blotting
Whole cell lysates from unstimulated and CD3/CD28-
bead stimulated MACS-isolated CD4+ T lymphocytes
were analyzed as described previously [6] using rabbit
anti-K2P5.1 and HRP-donkey anti-rabbit (Amersham,
Freiburg, Germany). HRP was inactivated with 2% NaN3
and blots were stained with b-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
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).
Statistical analysis
All results are presented as mean ± standard error of
measure (SEM). Statistical analysis was performed using
a modified Student’s t-test [10] in case of normally dis-
tributed data, or a Mann-Whitney test otherwise. Spear-
man’s rank correlation was used for correlation analysis.
P-values < 0.05 were considered statistically significant.
Results
The potassium channel K2P5.1 has been previously
shown to regulate T cell function in vitro and in vivo. In
a first set of experiments, freshly isolated CD4+ T cells
from healthy donors were either left unstimulated or sti-
mulated with CD3/CD28 beads for two days. A clear
upregulation of K2P5.1 could be shown on protein level
(one representative example is shown in Figure 1A, left
panel; quantification of three independent experiments -
right panel). In MS, putatively pathogenic T cells
derived from the CSF - reflecting the site of inflamma-
tion - showed significantly higher levels of K2P5.1 than
T cells from the peripheral blood of the same patients
[7]. Based on these findings, we next analyzed the
K2P5.1 expression on activated CD4
+ T lymphocytes out
of the synovial fluid of RA patients. Synovial fluid
derived T lymphocytes showed a slight upregulation of
the activation marker CD69, but not of CD25 when
compared to cells from the peripheral blood (Figure 1B).
In patients with RA, K2P5.1 is upregulated both on RNA
level (Figure 1C, left panel, black lines) and on protein
level (Figure 1C, middle panel, black lines) as assessed
by real time RT-PCR and flow cytometry, respectively.
A representative flow cytometry staining can be found
in Figure 1C, right panel. In contrast, no difference
could be observed in one patient with reactive arthritis
(Figure 1C, left and middle panel, grey lines). These
findings point towards a shared pathophysiological motif
of both T cell-mediated disorders - namely MS (CD8+
T cells) and RA (CD4+ T cells). Finally, TASK2 expres-
sing T cells could be identified immunohistochemically
within human synovial tissue sections. Exemplary cost-
ainings for the T cell marker CD3 and TASK2 can be
found in Figure 1D.
Activity of CD4+ and CD8+ T lymphocytes critically
depend on constitutively expressed K2P5.1 potassium
channels. Chronic stimulation as it may occur under
autoimmune conditions leads to an upregulation of
these channels on RNA and protein level. In a first set
of experiments, MACS-isolated T cells from the periph-
eral blood of 58 RA patients were analyzed for expres-
sion of K2P5.1 by RT-PCR. We compared the
correlation of K2P5.1 channel expression on both CD4
+
and CD8+ T lymphocytes with different disease activity
parameters. We found a positive correlation between
K2P5.1 expression levels measured as Δct values and
DAS28 on CD4+ T lymphocytes (R = 0.63; Figure 2A
left panel; Figure 2D). Moreover, a weaker correlation
could be found for ESR (R = 0.39; Figure 2B left panel;
Figure 2D) and CRP levels in the peripheral blood (R =
0.28; Figure 2C left panel; Figure 2D). In contrast,
K2P5.1 expression on CD8
+ T lymphocytes and three
disease activity parameters were found to be only weakly
Bittner et al. Arthritis Research & Therapy 2011, 13:R21
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AM
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Figure 1 Comparison of CD4+ T lymphocytes derived from peripheral blood and synovial fluid. A) Western blotting of unstimulated and
stimulated CD4+ T cells for K2P5.1 and b-actin as loading control (left panel). Quantification of three independent Western blotting experiments
(right panel). B) Exemplary flow cytometry stainings for the activation markers CD69 (left panel) and CD25 (right panel) are shown for cells
derived from the peripheral blood (grey) and synovial fluid (black). C) K2P5.1 expression levels on CD4
+ cells were compared by RT-PCR (left
panel) and flow cytometry staining (middle panel). Black lines: RA; grey lines: reactive arthritis. One representative example for RA is depicted on
the right side (white: isotyp, grey: peripheral blood, black: synovial fluid). D) Immunohistochemical staining of human synovial tissue sections for
CD3 (left panel), TASK2 (middle panel) and overlay (right panel).
Bittner et al. Arthritis Research & Therapy 2011, 13:R21
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AB
C
21 22 23 24 25
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K2P5.1 expression (ǻct)
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1.0 CD4
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Figure 2 K2P5.1 expression on CD4
+ T lymphocytes correlates with disease activity parameters. A) Expression levels of K2P5.1 (Δct values)
and DAS28 scores are shown for 58 individual patients for CD4+ (left side) and CD8+ (right side) T lymphocytes. Note that the x-axis (Δct values)
is logarithmic scale and that lower Δct values mean higher gene expression. B) Δct values for K2P5.1 and ESR rates (mm/h) are depicted for CD4
+
(left side) and CD8+ (right side) T lymphocytes. C) Δct values for K2P5.1 and CRP levels (mg/dl) are shown for CD4
+ (left side) and CD8+ (right
side) T lymphocytes. D) The correlation coefficient for K2P5.1 and DAS28, ESR or CRP is shown for CD4
+ and CD8+ T lymphocytes. E) K2P5.1
expression levels on CD4+ T cells are shown on clinically defined patient subgroups as stated in the Material and Methods section.
Bittner et al. Arthritis Research & Therapy 2011, 13:R21
http://arthritis-research.com/content/13/1/R21
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