Expression of K2P5.1 potassium channels on CD4+ T lymphocytes correlates with disease activity in rheumatoid arthritis patients

Abstract

Introduction: CD4+ T cells express K(2P)5.1 (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 K(2P)5.1 is upregulated upon (autoimmune) T cell stimulation. The aim of this study was to correlate expression levels of K(2P)5.1 on T cells from patients with rheumatoid arthritis (RA) to disease activity in these patients.

Methods: Expression levels of K(2P)5.1 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 K(2P)5.1.

Results: K(2P)5.1 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, K(2P)5.1 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 K(2P)5.1 expression to disease activity parameters during a longitudinal follow-up for six months.

Conclusions: Disease activity in RA patients correlates strongly with K(2P)5.1 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 K(2P)5.1 as a potential biomarker for disease activity and differential diagnosis.

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 K_2P5.1 and β-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) K_2P5.1 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).

Figure 2

K_2P5.1 expression on CD4⁺ T lymphocytes correlates with disease activity parameters. A) Expression levels of K_2P5.1 (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 K_2P5.1 and ESR rates (mm/h) are depicted for CD4⁺ (left side) and CD8⁺ (right side) T lymphocytes. C) Δct values for K_2P5.1 and CRP levels (mg/dl) are shown for CD4⁺ (left side) and CD8⁺ (right side) T lymphocytes. D) The correlation coefficient for K_2P5.1 and DAS28, ESR or CRP is shown for CD4⁺ and CD8⁺ T lymphocytes. E) K_2P5.1 expression levels on CD4⁺ T cells are shown on clinically defined patient subgroups as stated in the Material and Methods section.

Figure 3

A longitudinal follow-up study of RA patients undergoing therapy change. Comparison of A) DAS28, B) CRP values, C) ESR values and D) relative K_2P5.1 expression levels at therapy change (t = 0), after three months (t = 3) and six months (t = 6) of follow-up. Left panel shows individual values for all patients while mean values ± SEM are depicted on the right side. Note that the y-axis in D) is split for better clarity.

Figure 4

A longitudinal study of RA patients receiving tocilizumab. A) DAS28 B) CRP values, C) ESR values and D) relative K_2P5.1 expression levels are compared for patients with therapy switch to tocilizumab over six months. Note that the y-axis in D) is split for better clarity and that two patients are marked in red.