[Show abstract][Hide abstract] ABSTRACT: Adenosine, a purine nucleoside, is present at high concentrations in tumors, where it contributes to the failure of immune cells to eliminate cancer cells. The mechanisms responsible for the immunosuppressive properties of adenosine are not fully understood. We tested the hypothesis that adenosine's immunosuppressive functions in human T lymphocytes are in part mediated via modulation of ion channels. The activity of T lymphocytes relies on ion channels. KCa3.1 and Kv1.3 channels control cytokine release and, together with TRPM7, regulate T cell motility. Adenosine selectively inhibited KCa3.1, but not Kv1.3 and TRPM7, in activated human T cells. This effect of adenosine was mainly mediated by A2A receptors, as KCa3.1 inhibition was reversed by SCH58261 (selective A2A receptor antagonist), but not by MRS1754 (A2B receptor antagonist), and it was mimicked by the A2A receptor agonist CGS21680. Furthermore, it was mediated by the cAMP/protein kinase A isoform (PKAI) signaling pathway, as adenylyl-cyclase and PKAI inhibition prevented adenosine effect on KCa3.1. The functional implication of the effect of adenosine on KCa3.1 was determined by measuring T cell motility on ICAM-1 surfaces. Adenosine and CGS21680 inhibited T cell migration. Comparable effects were obtained by KCa3.1 blockade with TRAM-34. Furthermore, the effect of adenosine on cell migration was abolished by pre-exposure to TRAM-34. Additionally, adenosine suppresses IL-2 secretion via KCa3.1 inhibition. Our data indicate that adenosine inhibits KCa3.1 in human T cells via A2A receptor and PKAI, thereby resulting in decreased T cell motility and cytokine release. This mechanism is likely to contribute to decreased immune surveillance in solid tumors.
Preview · Article · Nov 2013 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Effector memory T cells (TM) play a key role in the pathology of certain autoimmune disorders. The activity of effector TM cells is under the control of Kv1.3 ion channels, which facilitate the Ca(2+) influx necessary for T cell activation and function, i.e. cytokine release and proliferation. Consequently, the knock-down of Kv1.3 expression in effector TM's may be utilized as a therapy for the treatment of autoimmune diseases. In this study we synthesized lipid unilamellar nanoparticles (NPs) that can selectively deliver Kv1.3 siRNAs into TM cells in vitro. NPs made from a mixture of phosphatidylcholine, pegylated/biotinylated phosphoethanolamine and cholesterol were functionalized with biotinylated-CD45RO (cell surface marker of TM's) antibodies via fluorophore-conjugated streptavidin (CD45RO-NPs). Incubation of T cells with CD45RO-NPs resulted into the selective attachment and endocytosis of the NPs into TM's. Furthermore, the siRNA against Kv1.3, encapsulated into the CD45RO-NPs, was released into the cytosol. Consequently, the expression of Kv1.3 channels decreased significantly in TM's, which led to a remarkable decrease in Ca(2+) influx. Our results can form the basis of an innovative therapeutic approach in autoimmunity.
[Show abstract][Hide abstract] ABSTRACT: The migration of T lymphocytes is an essential part of the adaptive immune response as T cells circulate around the body to carry out immune surveillance. During the migration process T cells polarize, forming a leading edge at the cell front and a uropod at the cell rear. Our interest was in studying the involvement of ion channels in the migration of activated human T lymphocytes as they modulate intracellular Ca(2+) levels. Ca(2+) is a key regulator of cellular motility. To this purpose, we created protein surfaces made of the bio-polymer PNMP and coated with ICAM-1, ligand of LFA-1. The LFA-1 and ICAM-1 interaction facilitates T cell movement from blood into tissues and it is critical in immune surveillance and inflammation. Activated human T lymphocytes polarized and migrated on ICAM-1 surfaces by random walk with a mean velocity of ∼6 µm/min. Confocal microscopy indicated that Kv1.3, CRAC, and TRPM4 channels positioned in the leading-edge, whereas KCa3.1 and TRPM7 channels accumulated in the uropod. The localization of KCa3.1 and TRPM7 at the uropod was associated with oscillations in intracellular Ca(2+) levels that we measured in this cell compartment. Further studies with blockers against Kv1.3 (ShK), KCa3.1 (TRAM-34), CRAC (SKF-96365), TRPM7 (2-APB), and TRPM4 (glibenclamide) indicated that blockade of KCa3.1 and TRPM7, and not Kv1.3, CRAC or TRPM4, inhibits the T cell migration. The involvement of TRPM7 in cell migration was confirmed with siRNAs against TRPM7. Downregulation of TRPM7 significantly reduced the number of migrating T cells and the mean velocity of the migrating T cells. These results indicate that KCa3.1 and TRPM7 selectively localize at the uropod of migrating T lymphocytes and are key components of the T cell migration machinery.
[Show abstract][Hide abstract] ABSTRACT: Optimization of the polymer layer. A. Light microscopy images of Si/SiO2 wafer chips coated with 3% PNMP solution, developed in pH 7.4 with two different time points of UV irradiation using TedPella mask. B. Effect of varying pH levels on the thickness of the PNMP polymer layer. All PNMP layers were coated on using optimized uniform specifications in different % PNMP solution (Table S1) with a UV irradiation for 45 min. C. Light microscope images of Si/SiO2 wafer chips coated with 1% PNMP solution and 12 min UV irradiation using TedPella mask and developed in different pH levels. D. Scanning Electron Microscopy (SEM) images of PNMP-pattern.
[Show abstract][Hide abstract] ABSTRACT: Native TRPM4 channels are localized in the leading-edge and have no migratory role in activated CD3+ T cells. A. Confocal images of migrating activated CD3+ T cells (left) stained for TRPM4 (green) together with anti-CD44 (uropod; red) and anti-CXCR-4 (leading-edge; red) antibodies. Bright-field (bf) images are in the left panels and merge images are in the right panels. Yellow areas indicate colocalization. Scale bar = 5 µm. The correlation coefficient (right) indicates that TRPM4 channels are localized in the leading-edge (n = 18) and not in the uropod (n = 31). B. The effect of 100 uM glibenclamide was obtained by following single cells by time-lapse bright-field microscopy before and after treatment with the blocker. The mean velocity shows no significant change in cell migration after inhibition of TRPM4 channels (n = 16).
[Show abstract][Hide abstract] ABSTRACT: HA-KCa3.1 channels accumulation at the uropod. A. T cells from one healthy donor were transiently transfected with HA-KCa3.1. After 2 h pre-incubation on the array, cells were fixed and stained with anti-HA (green) and anti-CD44 (red) antibodies. Images were obtained by confocal microscopy as described under materials and methods. The polarized cell is marked by the arrow in the brightfield image. Yellow color indicates colocalization of the HA-KCa3.1 and CD44 signals. Scale bar = 5 µm. B. Membrane localization of the KCa3.1 channels was conformed by analyzing the merged image in Panel A in X–Z and Y–Z planes. The X–Z and Y–Z scans of the images show that KCa3.1 is present at the cell periphery along with CD44, thereby confirming that in the migrating cells membrane KCa3.1 channels accumulate at the uropod of the polarized migrating T cell.
[Show abstract][Hide abstract] ABSTRACT: Kv1.3 channels do not affect the cell migration in primary activated CD3+ T cells but KCa3.1 channels decreased significantly the velocity. A. Migration of a representative activated CD3+ T cells on ICAM-1, recorded by time-lapse bright-field microscopy, before and after application of ShK (10 nM). Illustrated are snapshots of different time points. The asterisk represents the starting point, and arrow the initial direction. The dotted line is the coming track, and the continuous line is the covered distance. The time-lapse bright-field microscopy was recorded continuously with a gap of 1.5–2 min to add ShK indicated by the arrow at 22 min. B. Representative migrating CD3+ T cells before and after application of TRAM-34 (250 nM) in regarding to the time recorded by time-lapse bright-field microscopy. The recording was continuous with a gap of 1.5–2 min for TRAM-34 application at 22 min. Illustrated are snapshots of different time points. The asterisk shows the starting point, whereas the arrow represents the initial direction. The dotted line is the coming track, and the continuous line is the covered distance. Scale bar = 5 µm.
[Show abstract][Hide abstract] ABSTRACT: Specificity of Orai1, TRPM4 and TRPM7 antibodies. Activated primary T cells were fixed and stained with Orai1 (top), TRPM4 (middle) and TRPM7 (bottom) antibodies (ab). The corresponding images of cells treated with secondary antibodies only (2° only), no antibodies (cells only) or the antibody preadsorbed to the corresponding antigen (preadsorbed) are shown as right side panels. The corresponding DIC micrographs for each set are shown in the bottom of the florescence images.
[Show abstract][Hide abstract] ABSTRACT: Biomaterials, especially those based on nanomaterials, have emerged as critical tools in biomedical applications. The applications encompass a wide range such as implantable devices, tissue regeneration, drug delivery, diagnostic systems, and molecular printing. The type of materials used also covers a wide range: metals (permanent and degradable), polymers (permanent and degradable), carbon nanotubes, and lipid nanoparticles. This paper explores the use of microfluidic platforms as a high-throughput research tool for the evaluation of nanobiomaterials. Typical screening of such materials involves cell/tissue cultures to determine attributes such as cell adhesion, proliferation, differentiation, as well as biocompatibility. In addition to this, other areas such as drug delivery and toxicity can also be evaluated via microfluidics. Traditional approach for screening of such materials is very time-consuming, and a lot of animals should be sacrificed since it involves one material and a single composition or concentration for a single test. The microfluidics approach has the advantage of using multiple types of drugs and their concentration gradients to simultaneously study the effect on the nanobiomaterial and its interaction with cell/tissue. In addition to this, microfluidics provides a unique environment to study the effect of cell-to-extracellular interaction and cell-to-cell communication in the presence of the nanobiomaterials.
Full-text · Article · Jun 2012 · Journal of Nanomaterials
[Show abstract][Hide abstract] ABSTRACT: The NH(2) terminus of the sodium-bicarbonate cotransporter 1 (NBCe1) plays an important role in its targeting to the plasma membrane. To identify the amino acid residues that contribute to the targeting of NBCe1 to the plasma membrane, polarized MDCK cells were transfected with expression constructs coding for green fluorescent protein (GFP)-tagged NBCe1 NH(2)-terminal deletion mutants, and the localization of GFP-tagged proteins was analyzed by confocal microscopy. Our results indicate that the amino acids between residues 399 and 424 of NBCe1A contain important sequences that contribute to its localization to the plasma membrane. Site-directed mutagenesis studies showed that GFP-NBCe1A mutants D405A and D416A are retained in the cytoplasm of the polarized MDCK epithelial cells. Examination of functional activities of D405A and D416A reveals that their activities are reduced compared with the wild-type NBCe1A. Similarly, aspartic acid residues 449 and 460 of pancreatic NBCe1 (NBCe1B), which correspond to residues 405 and 416 of NBCe1A, are also required for its full functional activity and accurate targeting to the plasma membrane. In addition, while replacement of D416 with glutamic acid did not affect the targeting or functional activity of NBCe1A, substitution of D405 with glutamic acid led to the retention of the mutated protein in the intracellular compartment and impaired functional activity. These studies demonstrate that aspartic acid residues 405 and 416 in the NH(2) terminus of NBCe1A are important in its accurate targeting to the plasma membrane.
No preview · Article · Mar 2012 · AJP Cell Physiology
[Show abstract][Hide abstract] ABSTRACT: The cAMP/PKA signaling system constitutes an inhibitory pathway in T cells and, although its biochemistry has been thoroughly investigated, its possible effects on ion channels are still not fully understood. K(V)1.3 channels play an important role in T-cell activation, and their inhibition suppresses T-cell function. It has been reported that PKA modulates K(V)1.3 activity. Two PKA isoforms are expressed in human T cells: PKAI and PKAII. PKAI has been shown to inhibit T-cell activation via suppression of the tyrosine kinase Lck. The aim of this study was to determine the PKA isoform modulating K(V)1.3 and the signaling pathway underneath. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), a nonselective activator of PKA, inhibited K(V)1.3 currents both in primary human T and in Jurkat cells. This inhibition was prevented by the PKA blocker PKI(6-22). Selective knockdown of PKAI, but not PKAII, with siRNAs abolished the response to 8-BrcAMP. Additional studies were performed to determine the signaling pathway mediating PKAI effect on K(V)1.3. Overexpression of a constitutively active mutant of Lck reduced the response of K(V)1.3 to 8-Br-cAMP. Moreover, knockdown of the scaffolding protein disc large 1 (Dlg1), which binds K(V)1.3 to Lck, abolished PKA modulation of K(V)1.3 channels. Immunohistochemistry studies showed that PKAI, but not PKAII, colocalizes with K(V)1.3 and Dlg1 indicating a close proximity between these proteins. These results indicate that PKAI selectively regulates K(V)1.3 channels in human T lymphocytes. This effect is mediated by Lck and Dlg1. We thus propose that the K(V)1.3/Dlg1/Lck complex is part of the membrane pathway that cAMP utilizes to regulate T-cell function.
Preview · Article · Feb 2012 · AJP Cell Physiology
[Show abstract][Hide abstract] ABSTRACT: The response of T cells to antigens (T cell activation) is marked by an increase in intracellular Ca²⁺ levels. Voltage-gated and Ca²⁺-dependent K⁺ channels control the membrane potential of human T cells and regulate Ca²⁺ influx. This regulation is dependent on proper accumulation of K⁺ channels at the immunological synapse (IS) a signaling zone that forms between a T cell and antigen presenting cell. It is believed that the IS provides a site for regulation of the activation response and that K⁺ channel inhibition occurs at the IS, but the underlying mechanisms are unknown. A mathematical model was developed to test whether K⁺ efflux through K⁺ channels leads to an accumulation of K⁺ in the IS cleft, ultimately reducing K⁺ channel function and intracellular Ca²⁺ concentration ([Ca²⁺](i)). Simulations were conducted in models of resting and activated T cell subsets, which express different levels of K⁺ channels, by varying the K⁺ diffusion constant and the spatial localization of K⁺ channels at the IS. K⁺ accumulation in the IS cleft was calculated to increase K⁺ concentration ([K⁺]) from its normal value of 5.0 mM to 5.2-10.0 mM. Including K⁺ accumulation in the model of the IS reduced calculated K⁺ current by 1-12% and consequently, reduced calculated [Ca²⁺](i) by 1-28%. Significant reductions in K⁺ current and [Ca²⁺](i) only occurred in activated T cell simulations when most K⁺ channels were centrally clustered at the IS. The results presented show that the localization of K⁺ channels at the IS can produce a rise in [K⁺] in the IS cleft and lead to a substantial decrease in K⁺ currents and [Ca²⁺](i) in activated T cells thus providing a feedback inhibitory mechanism during T cell activation.
Full-text · Article · Jan 2012 · Journal of Theoretical Biology
[Show abstract][Hide abstract] ABSTRACT: Hypoxia in solid tumors contributes to decreased immunosurveillance via down-regulation of Kv1.3 channels in T lymphocytes
and associated T cell function inhibition. However, the mechanisms responsible for Kv1.3 down-regulation are not understood.
We hypothesized that chronic hypoxia reduces Kv1.3 surface expression via alterations in membrane trafficking. Chronic hypoxia
decreased Kv1.3 surface expression and current density in Jurkat T cells. Inhibition of either protein synthesis or degradation
and endocytosis did not prevent this effect. Instead, blockade of clathrin-coated vesicle formation and forward trafficking
prevented the Kv1.3 surface expression decrease in hypoxia. Confocal microscopy revealed an increased retention of Kv1.3 in
the trans-Golgi during hypoxia. Expression of adaptor protein-1 (AP1), responsible for clathrin-coated vesicle formation at the trans-Golgi, was selectively down-regulated by hypoxia. Furthermore, AP1 down-regulation increased Kv1.3 retention in the trans-Golgi and reduced Kv1.3 currents. Our results indicate that hypoxia disrupts AP1/clathrin-mediated forward trafficking of
Kv1.3 from the trans-Golgi to the plasma membrane thus contributing to decreased Kv1.3 surface expression in T lymphocytes.
Preview · Article · Nov 2011 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Systemic lupus erythematosus (SLE) T cells exhibit several activation signaling anomalies including defective Ca(2+) response and increased NF-AT nuclear translocation. The duration of the Ca(2+) signal is critical in the activation of specific transcription factors and a sustained Ca(2+) response activates NF-AT. Yet, the distribution of Ca(2+) responses in SLE T cells is not known. Furthermore, the mechanisms responsible for Ca(2+) alterations are not fully understood. Kv1.3 channels control Ca(2+) homeostasis in T cells. We reported a defect in Kv1.3 trafficking to the immunological synapse (IS) of SLE T cells that might contribute to the Ca(2+) defect. The present study compares single T cell quantitative Ca(2+) responses upon formation of the IS in SLE, normal, and rheumatoid arthritis (RA) donors. Also, we correlated cytosolic Ca(2+) concentrations and Kv1.3 trafficking in the IS by two-photon microscopy. We found that sustained [Ca(2+)](i) elevations constitute the predominant response to antigen stimulation of SLE T cells. This defect is selective to SLE as it was not observed in RA T cells. Further, we observed that in normal T cells termination of Ca(2+) influx is accompanied by Kv1.3 permanence in the IS, while Kv1.3 premature exit from the IS correlates with sustained Ca(2+) responses in SLE T cells. Thus, we propose that Kv1.3 trafficking abnormalities contribute to the altered distribution in Ca(2+) signaling in SLE T cells. Overall these defects may explain in part the T cell hyperactivity and dysfunction documented in SLE patients.