Anna Bajnok

The effects of Kv1.3 and IKCa1 potassium channel inhibition on calcium influx of human peripheral T lymphocytes in rheumatoid arthritis

OBJECTIVE The transient increase of the cytoplasmic free calcium level plays a key role in the process of lymphocyte activation. Kv1.3 and IKCa1 potassium channels are important regulators of the maintenance of calcium influx during lymphocyte activation and present a possible target for selective immunomodulation. DESIGN Case-control study. SUBJECTS AND METHODS We took peripheral blood samples from 10 healthy individuals and 9 recently diagnosed rheumatoid arthritis (RA) patients receiving no anti-rheumatic treatment. We evaluated calcium influx kinetics following activation in CD4, Th1, Th2 and CD8 cells applying a novel flow cytometry approach. We also assessed the sensitivity of the above subsets to specific inhibition of the Kv1.3 and IKCa1 potassium channels. RESULTS The peak of calcium influx in lymphocytes isolated from RA patients is reached more rapidly, indicating that they respond more quickly to stimulation compared to controls. In healthy individuals, the inhibition of the IKCa1 channel decreased calcium influx in Th2 and CD4 cells to a lower extent than in Th1 and CD8 cells. On the contrary, the inhibition of Kv1.3 channels resulted in a larger decrease of calcium entry in Th2 and CD4 than in Th1 and CD8 cells. No difference was detected between Th1 and Th2 or CD4 and CD8 cells in the sensitivity to IKCa1 channel inhibition among lymphocytes of RA patients. However, specific inhibition of the Kv1.3 channel acts differentially on calcium influx kinetics in RA lymphocyte subsets. Th2 and particularly CD8 cells are inhibited more dominantly than Th1 and CD4 cells. CONCLUSION The inhibition of Kv1.3 channels does not seem to be specific enough in peripheral RA lymphocytes, since anti-inflammatory Th2 cells are also affected to a noteworthy extent.

Prevalence of Regulatory T-Cell Subtypes in Preeclampsia.

The prevalence of regulatory T cells (Tregs) is lower in preeclampsia (PE) compared with healthy pregnancy (HP). However, the proportion of recently described Treg subtypes has not been investigated.

Different calcium influx characteristics upon Kv1.3 and IKCa1 potassium channel inhibition in T helper subsets

Functional imbalance between T helper subsets plays important role in the pathogenesis of autoimmune disorders. Transient increase of cytoplasmic calcium level, and sustention of negative membrane potential by voltage sensitive Kv1.3 and calcium‐dependent IKCa1 potassium channels are essential for short‐term lymphocyte activation, thus present possible target for selective immunomodulation. We aimed to investigate calcium influx sensitivity to the inhibition of potassium channels in the main T helper subsets. Peripheral blood from 11 healthy individuals was drawn and calcium influx kinetics following activation with phytohemagglutinin in Th1, Th2, Th17, and Treg cells were evaluated. Alteration of calcium influx induced by specific inhibitors of Kv1.3 and IKCa1 potassium channels, and the expression of Kv1.3 channels were also assessed. Highest cytoplasmic calcium concentration was observed in stimulated Th1 cells, while the lowest level was measured in Treg cells. In Th1 and Th17 cells, inhibition of both investigated potassium channels decreased calcium influx. In Th2 cells only the inhibitor of Kv1.3 channels, while in Treg cells none of the inhibitors had significant effect. Upon the inhibition of IKCa1 channels, short‐term activation of proinflammatory cells was specifically decreased without affecting anti‐inflammatory subsets, indicating that selective immunomodulation is possible in healthy individuals.

B7 costimulation and intracellular indoleamine-2,3-dioxygenase (IDO) expression in peripheral blood of healthy pregnant and non-pregnant women

BackgroundB7 costimulatory molecules are expressed on antigen presenting cells (APCs) and are important regulators of T cell activation. We investigated the role of the B7 family of costimulatory molecules in the development of the systemic maternal immune tolerance during healthy pregnancy (HP). We also aimed to investigate the intracellular expression of indoleamine-2,3-dioxygenase (IDO) and plasma levels of tryptophane (TRP), kynurenine (KYN) and kynurenic acid (KYNA), important molecules with immunoregulatory properties, in order to describe their potential contribution to the pregnancy-specific maternal immune tolerance.MethodsWe determined the frequency of activated (CD11b+) monocytes expressing B7-1, B7-2, B7-H1, and B7-H2, and that of T cells and CD4+ T helper cells expressing CD28, CTLA-4, PD-1, and ICOS in peripheral blood samples of healthy pregnant (HP) and non-pregnant (NP) women using flow cytometry. We also examined the intracellular expression of IDO applying flow cytometry and plasma levels of TRP, KYN and KYNA using high-performance liquid chromatography.ResultsA significant increase in the prevalence of CD28+ T cells was observed in HP compared to NP women. At the same time a decrease was shown in the expression of CTLA-4 on these cells. The frequency of CD80+ monocytes was lower in HP women. The prevalence of IDO-expressing T cells and monocytes was higher in HP compared to NP women. Plasma KYN, KYNA and TRP levels were lower, while at the same time, the KYN/TRP ratio was higher in HP than in NP women.ConclusionsCostimulation via CD28 may not contribute to the immunosuppressive environment, at least in the third trimester of pregnancy. The development of the pregnancy-specific immune tolerance in the mechanism of B7 costimulation may be more related to the altered expression of B7 proteins on APCs rather than that of their receptors on T cells. The elevated intracellular IDO expression in monocytes and T cells, as well as higher plasma enzymatic IDO activity are likely to contribute to the systemic immunosuppressive environment in the third trimester characteristic for healthy gestation.

Modulation of T Lymphocyte Calcium Influx Patterns Via the Inhibition of Kv1.3 and Ikca1 Potassium Channels in Autoimmune Disorders

Currently available immunotherapies have improved the treatment of autoimmune diseases; however, these therapies are known to have considerable side-effects, such as increasing the susceptibility to infections. Therefore, there is an unmet need for novel immunosuppressive strategies with different mechanisms of action and higher specificity for disease-causing autoreactive T lymphocytes from existing immunomodulators. The increase of the cytoplasmic calcium concentration from intra- and extracellular sources (i.e., the endoplasmic reticulum and store-operated calcium entry through the plasma membrane) is the cornerstone of T lymphocyte activation and functionality. In the course of lymphocyte activation, potassium channels maintain the driving force for sustained calcium influx from the extracellular milieu as they grant the efflux of potassium from the cytoplasm, thus conserving an electrochemical potential gradient between the intra- and extracellular spaces. There are two major types of potassium channels in T cells: the voltage-gated Kv1.3 and the calcium-activated IKCa1 channels. The relation between the calcium currents through calcium release-activated calcium (CRAC) channels and the efflux of potassium 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 calcium influx in lymphocytes and a lower level of lymphocyte activation. Previous data suggest that selective modulation of lymphocyte activation through specific inhibition of potassium channels may be a possible therapeutic approach for the treatment of autoimmune disease (1–,6). Beeton et al. showed that terminally differentiated effector memory T (TEM) cells play a pivotal role in the pathogenesis of autoimmunity (4). Wulff et al. described that the characteristic potassium channel phenotype of TEM cells in multiple sclerosis (MS) is Kv1.3high IKCa1low, contrasting naive, and central memory T (TCM) cells, which exhibit a Kv1.3low IKCa1high channel phenotype (1). Therefore the therapeutic relevance of specific Kv1.3 channel inhibitors is of outstanding interest, as they may offer the possibility for selective modulation of pathogenic TEM cells, while naive and TCM cells (needed for physiological immune responses) would escape the inhibition through upregulation of IKCa1 channel expression. Beeton et al. demonstrated that the symptoms of experimental autoimmune encephalitis, a murine model of MS, significantly improved after treatment with selective Kv1.3 inhibitors (5). Although results from animal models are promising, limited data is available on the effects of potassium channel inhibition on T cell function in humans. Furthermore, besides naive and memory T cells, alterations in the activation pattern of effector (CD4+ helper and CD8+ cytotoxic) T lymphocytes have not been described upon Kv1.3 and IKCa1 inhibition. Although these cells might have a less-specific role in the maintenance of autoreactivity compared to TEM cells, their inhibition have important consequences on the overall immune response. Therefore, over the recent years, we have investigated calcium influx characteristics in effector T cell subsets in a number of autoimmune disorders. We isolated peripheral blood mononuclear cells from MS, rheumatoid arthritis (RA) and type 1 diabetes mellitus (T1DM) patients and applied a novel flow cytometry-based approach for the detection of calcium influx (7–10). Until the recent past, single-cell techniques were used for the investigation of calcium influx during lymphocyte activation. There has been no high-throughput method available to study the kinetics of lymphocyte activation in more subsets at the same time. Single-cell techniques are restricted by not being capable of characterizing this process in complex cellular systems, thus ignoring the interaction between the different lymphocyte subsets that may modulate the course of their activation. Therefore, over the recent years we have developed a novel algorithm that allows simultaneous monitoring of calcium influx in several lymphocyte subsets. Our software (FacsKin) fits functions to median values of the data of interest and calculates relevant parameters describing each function. By selecting the best fitting function, this approach provides an opportunity for the mathematical analysis and statistical comparison of kinetic flow cytometry measurements of distinct samples (more details available at www.facskin.com). Our findings indicate important differences in calcium influx kinetics in the studied autoimmune disorders compared to healthy controls.

Analysis by flow cytometry of calcium influx kinetics in peripheral lymphocytes of patients with rheumatoid arthritis

The transient increase of the cytoplasmic free calcium level in T lymphocytes plays a key role in initiating and maintaining the autoimmune reaction in rheumatoid arthritis (RA). Kv1.3 and IKCa1 potassium channels are important regulators of the maintenance of calcium influx during lymphocyte activation and present a possible target for selective immunomodulation. We aimed to compare peripheral T lymphocyte calcium influx kinetics upon activation in patients with recently diagnosed and established RA, and to demonstrate the differences in analysis of kinetic flow cytometry data when using two different algorithms. We took peripheral blood samples from nine patients with recently diagnosed and six patients with established RA. We evaluated calcium influx kinetics following activation in CD4, Th1, Th2, and CD8 cells applying an approach based on smoothing of median fluorescence values (FlowJo) and an algorithm based on function fitting (FacsKin). We assessed the sensitivity of the above subsets to specific inhibition of the Kv1.3 and IKCa1 potassium channels. Th2 cells of patients with established RA react slower to activating stimuli, whereas CD8 cells show a faster reaction than in patients with recently diagnosed RA. While initially Th1 cells are less sensitive to the inhibition of Kv1.3 and IKCa1 channels in RA, their sensitivity increases along with the duration of the disease. With the algorithm of function fitting instead of smoothing, more statistically significant differences of potassium channel inhibition between the two RA groups could be demonstrated. The function fitting algorithm applied by FacsKin is suitable to provide a common basis for evaluating and comparing flow cytometry kinetic data.

Calcium influx kinetics, and the features of potassium channels of peripheral lymphocytes in primary Sjögren’s syndrome

OBJECTIVE The transient increase of the cytoplasmic free calcium level plays a key role in the process of lymphocyte activation. Kv1.3 and IKCa1 potassium channels are important regulators of the maintenance of calcium influx and present a possible target for selective immunomodulation. DESIGN Case-control study. SUBJECTS AND METHODS We took peripheral blood samples from 8 healthy individuals and 15 primary Sjögrens syndrome (pSS) patients. We evaluated calcium influx kinetics following activation in peripheral T lymphocytes. We also assessed the sensitivity of T lymphocytes to specific inhibition of the Kv1.3 and IKCa1 potassium channels, and the Kv1.3 channel expression. RESULTS The basal cytoplasmatic calcium levels were lower in both Th1 and Th2 lymphocytes in pSS compared to controls. The peak of calcium influx in lymphocytes isolated from pSS patients is reached later, indicating that they respond more slowly to stimulation compared to controls. In healthy individuals, the inhibition of the IKCa1 channel decreased calcium influx in Th2 and CD4 cells to a lower extent than in Th1 and CD8 cells. On the contrary, the inhibition of Kv1.3 channels resulted in a larger decrease of calcium entry in Th2 and CD4 than in Th1 and CD8 cells. In the pSS group, neither of the inhibitors induced alteration in calcium influx. Expression of Kv1.3 channels on CD4, Th2 and CD8 lymphocytes in pSS was significantly higher compared to controls. CONCLUSION The altered expression and specific inhibition of potassium channels seem to be related to altered calcium influx kinetics in pSS which distinguish pSS either from healthy controls or other systemic autoimmune diseases.

The Distribution of Activation Markers and Selectins on Peripheral T Lymphocytes in Preeclampsia

Introduction Impaired maternal immune tolerance resulting in systemic inflammation plays a pivotal role in the pathogenesis of preeclampsia. Phenotypical changes of monocytes and neutrophil granulocytes have already been studied in preeclampsia, and some studies also included T lymphocyte activation markers; however, the results are controversial and a comprehensive analysis of activation markers is lacking. The characteristics of cellular adhesion molecules in preeclampsia are yet to be described. Material and Methods Peripheral blood samples of 18 preeclamptic patients and 20 healthy pregnant women in the third trimester were evaluated using flow cytometry to characterize the cell surface expression of T lymphocyte activation markers and selectins. Results We found an elevated ratio of HLA-DR and CD122-, CD62E-, and CD62L-expressing cells among the CD4+ T lymphocytes in PE in comparison to healthy pregnancy. No alterations were found in the prevalence of CD69-, CD25-, and CD62P-expressing lymphocytes and CD11c-expressing monocytes. Conclusions Our findings support the role of activated T lymphocytes and specific cell adhesion molecules in the pathogenesis of preeclampsia.

Altered calcium influx of peripheral Th2 cells in pediatric Crohn’s disease: infliximab may normalize activation patterns

Objective Crohns disease is a chronic inflammation of the gastrointestinal tract with an abnormal immune phenotype. We investigated how intracellular calcium kinetics of Th1 and Th2 lymphocytes alter upon specific inhibition of Kv1.3 and IKCa1 channels in pediatric Crohns disease. Study design Blood was taken from 12 healthy and 29 Crohns disease children. Of those, 6 were switched to infliximab and re-sampled after the 4th infliximab treatment. Intracellular calcium levels were monitored using flow cytometry in the presence or absence of specific inhibitors of Kv1.3 and IKCa1 potassium channels. Results In Crohns disease treated with standard therapy, calcium response during activation was higher than normal in Th2 cells. This was normalized in vitro by inhibition of Kv1.3 or IKCa1 potassium channels. After the switch to infliximab, potassium channel function and expression in Th2 lymphocytes were comparable to those in Th1 cells. Conclusion These results may indicate that potassium channels are potential immune modulatory targets in Crohns disease.

Real time kinetic flow cytometry measurements of cellular parameter changes evoked by nanosecond pulsed electric field.

Nanosecond pulsed electric field (nsPEF) is a novel method to increase cell proliferation rate. The phenomenon is based on the microporation of cellular organelles and membranes. However, we have limited information on the effects of nsPEF on cell physiology. Several studies have attempted to describe the effects of this process, however no real time measurements have been conducted to date. In this study we designed a model system which allows the measurement of cellular processes before, during and after nsPEF treatment in real time. The system employs a Vabrema MitoplicatorTM nsPEF field generating instrument connected to a BD Accuri C6 cytometer with a silicon tube led through a peristaltic pump. This model system was applied to observe the effects of nsPEF in mammalian C6 glioblastoma (C6 glioma) and HEK‐293 cell lines. Viability (using DRAQ7 dye), intracellular calcium levels (using Fluo‐4 dye) and scatter characteristics were measured in a kinetic manner. Data were analyzed using the FACSKin software. The viability and morphology of the investigated cells was not altered upon nsPEF treatment. The response of HEK‐293 cells to ionomycin as positive control was significantly lower in the nsPEF treated samples compared to non‐treated cells. This difference was not observed in C6 cells. FSC and SSC values were not altered significantly by the nsPEF treatment. Our results indicate that this model system is capable of reliably investigating the effects of nsPEF on cellular processes in real time.