Lymphocyte calcium influx characteristics and its modulation by Kv1.3 and IKCa1 potassium channel inhibitors in multiple sclerosis

Abstract

Objective: Kv1.3 and IKCa1 potassium channels are known to be involved in the regulation of calcium influx during activation of lymphocytes. Our aim was to compare the calcium influx characteristics of T lymphocytes in multiple sclerosis (MS) patients and healthy individuals and its modulation by Kv1.3 and IKCa1 channel inhibitors. Subjects and methods: We used flow cytometry to evaluate calcium influx in Th1, Th2, CD4 and CD8 subsets of lymphocytes isolated from 10 healthy individuals, 11 MS patients without and 6 MS patients with interferon beta therapy. Lymphocytes were treated with specific channel inhibitors and phytohemagglutinin was applied to activate cells. Results: Calcium influx kinetics and potassium channel function in lymphocytes were in some aspects different in MS patients without interferon beta compared to healthy individuals. The reactivity of lymphocytes is increased in MS patients, however the amount of calcium entering the cells during activation is not different from that of healthy individuals. Potassium channels function more actively in lymphocytes of MS patients. Inhibition of Kv1.3 potassium channels in MS patients may be used to decrease the activation of the CD8 subset, but as this effect is of limited specifity and also involves the anti-inlammatory Th2 cells, the therapeutic application of Kv1.3 channel inhibitors should be further investigated and characterized in MS. In MS patients treated with interferon beta, calcium influx kinetics and potassium channel function are more similar to those in healthy individials, but these observations are limited to the Th1 subset. Conclusion: Our results suggest that interferon beta treatment may have a selective effect on calcium influx in Th1 cells, and Th2 cells are less affected. INTRODUCTION T lymphocytes play a crucial role in the pathogenesis of multiple sclerosis (MS). The increase of the cytoplasmic free Ca2+ level ([Ca]cyt) plays an essential role in the process of lymphocyte activation. The engagement of the TCR/CD3 complex leads to Ca2+ release from intracellular stores that is followed by further Ca2+ entry from the extracellular space through calcium release activated calcium (CRAC) channels. In the course of lymphocyte activation, K+ channels maintain the driving force for sustained Ca2+ influx as they grant the efflux of K+ from the cytoplasm, thus conserving an electrochemical potential gradient. There are two major types of K+ channels in T cells: the voltage-gated Kv1.3 and the Ca2+-activated IKCa1 channels (Figure 1). The relation between the Ca2+ currents through CRAC channels and K+ efflux makes the proliferation and activation of lymphocytes sensitive to pharmacological modulation of Kv1.3 and IKCa1 channels, and provides an opportunity for targeted intervention. Specific inhibition of these channels results in a diminished Ca2+ influx in lymphocytes and a lower level of lymphocyte activation. In the present study, we took peripheral blood samples from MS patients receiving no immunomodulatory treatment, MS patients treated with IFN beta and healthy individuals. We used a recently developed flow cytometry approach to characterize Ca2+ influx kinetics of major lymphocyte subsets (CD4, Th1, Th2 and CD8 cells) and their sensitivity to the inhibition of Kv1.3 and IKCa1 lymphocyte K+ channels in MS. RESULTS Lymphocytes isolated from MS patients without IFN beta are characterized by increased reactivity as reflected by lower tmax values compared with healthy individuals, i.e. the peak of the Ca influx is reached more rapidly in the CD4, Th1 and Th2 subsets. Interestingly, these alterations were restricted to CD4 cells and were not present in the CD8 subset (Figure 4). The investigated lymphocyte subsets show altered sensitivity to the inhibition of the Kv1.3 and IKCa1 channels in MS patients without IFN beta. In this group, the inhibition of the IKCa1 channel results in a similar decrease of Ca influx in all investigated subsets (Figure 3). However, MGTX, the specific blocker of the Kv1.3 channel decreased the AUC value to a higher extent in CD8 cells than in CD4 cells. The specificity of Kv1.3 inhibition is limited to CD8 cells since other cell types, including Th1 and Th2 cells, behave in a similar manner in MS patients without IFN beta and healthy subjects upon Kv1.3 channel inhibition. Th1 and Th2 cells are similarly suppressed upon the inhibition of Kv1.3 channels. Since the cytokine balance is of utmost importance in the regulation of the autoimmune reaction, the inhibition of the Th2 subset would probably result in a setback of therapeutic efforts in MS. In MS patients with IFN beta, the characteristics of Ca influx regarding the Slope value resembles again to that of healthy subjects (reflecting a decreased rapidity of lymphocyte activation), but only in the Th1 subset (the Th2 subset is not affected). Furthermore, the tmax value is elevated in MS patients with IFN beta compared with MS patients without IFN beta, and is comparable to that in healthy controls, reflecting a decrease in lymphocyte reactivity upon IFN beta treatment. Again, this finding was observed only in the Th1, but not in the Th2 subset (Figure 5). Consequently, IFN beta treatment seems to decrease lymphocyte activation and modulate Ca influx kinetics specifically in the Th1 subset, but not in Th2 cells. This is beneficial in the treatment of MS, since it decreases the secretion of pro-inflammatory cytokines, but does not affect anti-inflammatory cytokine production by the Th2 subset, which compensates the ongoing inflammation. Additionally, our results suggest that IFN beta influences Kv1.3 and IKCa1 channel functions preferentially in the Th1 subset, and only modestly in Th2 cells. While IFN beta decreases the function of both channels among Th1 cells (decreasing IKCa1 function to a higher extent than that of Kv1.3), IKCa1 channel function remains unaffected, and Kv1.3 channel function is increased in Th2 cells upon IFN beta treatment compared with MS patients without IFN beta (Figure 5). These effects probably also contribute to the suppression of cytokine production by IFN beta preferentially in the Th1 subset. CONCLUSIONS We have demonstrated important differences in Ca2+ influx kinetics and lymphocyte K+ channel function in MS patients without IFN beta compared with healthy individuals. Specific immunomodulation of the CD8 effector lymphocytes can be reached through the inhibition of the Kv1.3 channel in MS patients without IFN beta. This effect does not seem to be specific enough concerning all lymphocyte subsets playing a role in the development of the autoimmune response, since it also affects anti-inflammatory cytokine producing Th2 cells. IFN beta therapy induces compensatory changes in Ca2+ influx kinetics and lymphocyte K+ channel function in MS, shaping these properties more similar to those of healthy individuals. The results of our pilot study suggest that IFN beta treatment modulates Ca2+ influx kinetics selectively in Th1 cells. Therefore, its suppressive effect on lymphocyte activation is seen in Th1 cells, while Th2 function is less affected. METHODS Peripheral blood samples were taken from 10 healthy individuals, 11 relapsing-remitting MS patients receiving only supportive therapy, but no immunomodulatory treatment and 6 relapsing-remitting MS patients receiving regular dose IFN beta treatment. Peripheral blood mononuclear cells (PBMCs) were separated. Cell surface staining was applied to distinguish CD4, Th1 (CD4+ CXCR3+), Th2 (CD4+ CCR4+) and CD8 cells. Cells were loaded with Ca2+ binding dyes (Fluo-3, FuraRed). Cells were treated with inhibitors (MGTX, TRAM, 60 nM). Cells were activated with phytohemagglutinin. Samples were measured on flow cytometer for 10 minutes in a kinetic manner. Analysis was performed using special software and different parameters were calculated (Figure 2). This work was supported by the Hungarian Scientific Research Fund [OTKA; grant number 76316]; and the Social Renewal Operational Programme [grant number TAMOP-4.2.2.08/1/KMR-2008-0004]. Characteristics Healthy individuals n = 11 MS patients without IFN beta n = 11 MS patients with IFN beta n = 6 Age (years) 36 [23-46] 44 [24-51] 39 [32-56] Gender (male/female) 5/6 4/7 2/4 MS duration (years) 4 [1-20] 7 [1-18] EDSS score 2 [1-3] 4.5* [2-5] Clinical characteristics of study participants The tmax value was increased in MS patients with IFN beta when compared with MS patients without IFN beta in the Th1 lymphocyte subset. The Slope value was higher in MS patients without IFN beta compared with healthy individuals supporting a more rapid increase of [Ca]cyt. This parameter decreased in the CD4 and the Th1 subsets in MS patients with IFN beta compared with MS patients without IFN beta. MGTX and TRAM treatment decreased AUC values of all investigated lymphocyte subsets in samples of all three study groups. The extent of the decrease in parameter values was characteristic for the investigated study group and the inhibited K+ channel in each subset.