6-Methoxyflavanone abates cisplatin-induced neuropathic pain apropos anti-inflammatory mechanisms: A behavioral and molecular simulation study.

Cisplatin is used as a first line therapy in treating cancers. However, its use is often accompanied with the development of peripheral neuropathy. 6-Methoxyflavanone (6-MeOF) is a positive allosteric modulator at GABAA receptors and is known for attenuating diabetes-induced neuropathic pain. Neuropathy was induced in male Sprague-Dawley rats (150-250 g), via intraperitoneal injection of cisplatin (3 mg/kg) once a week for four consecutive weeks. 6-MeOF (25, 50 and 75 mg/kg, i.p) and gabapentin (75 mg/kg, i.p) were administered 30 min before each cisplatin injection. Static and dynamic allodynia were assessed using von Frey filaments and cotton buds. The anti-inflammatory activity was analyzed with plethysmometer. Body weights were also measured each week. The binding affinity of 6-MeOF with chloride channel, Cyclooxygenase-1 (COX-1) and Cyclooxygenase-2 (COX-2) was studied using docking approach. The in vitro COX-1 and COX-2 inhibitory effect of 6-MeOF was conducted with COX colorimetric assay. Administration of cisplatin for four consecutive weeks induced static (decreased paw withdrawal threshold; PWT) and dynamic allodynia (decreased paw withdrawal latency; PWL). Co-administration of 6-MeOF for four weeks significantly attenuated the cisplatin-induced expression of nocifensive behaviors observed as significant increase in PWT and PWL. Moreover, it also prevented the body weight loss induced by cisplatin administration. In silico studies depicted a good interaction of 6-MeOF with chloride ion channels and COX-1 and COX-2 enzymes. The in vitro study confirmed the inhibitory activity of 6-MeOF for COX-1 and COX-2. 6-MeOF may be effective in attenuating cisplatin-induced allodynia, probably through interaction with GABAergic receptors and reducing inflammation.

Towards dewetting monoclonal antibodies for therapeutical purposes.

Dewetting transition - a concept borrowed from fluid mechanics - is a physiological process that takes place inside the hydrophobic pores of ion channels. This transient phenomenon causes a metastable state that forbids water molecules to cross microscopic receptor cavities. This leads to a decreased conductance, a closure of the pore and, subsequently, severe impairment of cellular performance. We suggest that artificially-provoked dewetting transition in ion channel hydrophobic pores might stand for a molecular candidate to erase detrimental organisms, such as viruses, bacteria, and cancer cells. We describe a novel type of high-affinity monoclonal antibody, that: a) targets specific trans-membrane receptor structures of harmful or redundant cells; b) is equipped with lipophilic and/or hydrophobic fragments that prevent physiological water flow inside ion channels. Therefore, we achieve an artificial dewetting transition inside receptor cavities, that causes discontinuity within transmembrane ionic flows, channel blockage, and subsequent damage of morbid cells. As an example, we describe dewetting monoclonal antibodies that target the M2 channel of the Influenza A virus: they might prevent water from entering pores thus leading to virion impairment.

Suppression of adenosine monophosphate-activated protein kinase selectively triggers apoptosis in activated T cells and ameliorates immune diseases.

Deficient apoptosis of activated T cells can result in immunological disorders. Molecules associated with energy and metabolisms are suggested to be involved in pathogenesis of immune diseases, but remain uninvestigated. In the present study we reported that glibenclamide exerted a new pharmacological effect on inflammatory responses by selectively triggering apoptosis of activated T cells. Glibenclamide demonstrated an inhibition on activated T lymphocytes, whereas showed no toxicity in the naive cells. This effect was mainly related with its ability to facilitate apoptosis in activated T cells with an up-regulation of cleaved-caspases and cleaved-PARP. Glibenclamide enhanced Fas expression and suppressed the expression of antiapoptotic cellular FLICE-inhibitory protein. The underlying mechanism of glibenclamide was not associated with its classical inhibitory effect on ATP-sensitive potassium channels, but due to a unique suppression on the phosphorylation of 5' adenosine monophosphate-activated protein kinase, which was augmented during T cell activation. An in vivo experiment further demonstrated that glibenclamide ameliorated T-cell-mediated contact hypersensitivity in mice. Altogether, these results suggest that AMPK inhibition by glibenclamide can regulate the survival and death of T lymphocytes and be beneficial for the treatment of autoimmune diseases.

Effects of Pseudomonas aeruginosa on CFTR chloride secretion and the host immune response.

In the healthy lung the opportunistic pathogen, Pseudomonas aeruginosa, is rapidly eliminated by mucociliary clearance, a process that is dependent on the activity of the CFTR anion channel that, in concert with a number of other transport proteins, regulates the volume and composition of the periciliary surface liquid. This fluid layer is essential to enable cilia to clear pathogens from the lungs. However, in cystic fibrosis (CF), mutations in the CFTR gene reduce Cl- and [Formula: see text] secretion, thereby decreasing periciliary surface liquid volume and mucociliary clearance of bacteria. In CF this leads to persistent infection with the opportunistic pathogen, P. aeruginosa, which is the cause of reduced lung function and death in ~95% of CF patients. Others and we have conducted studies to elucidate the effects of P. aeruginosa on wild-type and Phe508del-CFTR Cl- secretion as well as on the host immune response. These studies have demonstrated that Cif (CFTR inhibitory factor), a virulence factor secreted by P. aeruginosa, is associated with reduced lung function in CF and induces the ubiquitination and degradation of wt-CFTR as well as TAP1, which plays a key role in viral and bacterial antigen presentation. Cif also enhances the degradation of Phe508del-CFTR that has been rescued by ORKAMBI, a drug approved for CF patients homozygous for the Phe508del-CFTR mutation, thereby reducing drug efficacy. This review is based on the Hans Ussing Distinguished Lecture at the 2016 Experimental Biology Meeting given by the author.

CHRFAM7A, a human-specific and partially duplicated α7-nicotinic acetylcholine receptor gene with the potential to specify a human-specific inflammatory response to injury.

Conventional wisdom presumes that the α7nAChR product of CHRNA7 expression mediates the ability of the vagus nerve to regulate the inflammatory response to injury and infection. Yet, 15 years ago, a 2nd structurally distinct and human-specific α7nAChR gene was discovered that has largely escaped attention of the inflammation research community. The gene, originally called dupα7nAChR but now known as CHRFAM7A, has been studied exhaustively in psychiatric research because of its association with mental illness. However, dupα7nAChR/CHRFAM7A expression is relatively low in human brain but elevated in human leukocytes. Furthermore, α7nAChR research in human tissues has been confounded by cross-reacting antibodies and nonspecific oligonucleotide primers that crossreact in immunoblotting, immunohistochemistry, and RT-PCR. Yet, 3 independent reports show the human-specific CHRFAM7A changes cell responsiveness to the canonical α7nAChR/CHRNA7 ion-gated channel. Because of its potential for the injury research community, its possible significance to human leukocyte biology, and its relevance to human inflammation, we review the discovery and structure of the dupα7nAChR/CHRFAM7A gene, the distribution of its mRNA, and its biologic activities and then discuss its possible role(s) in specifying human inflammation and injury. In light of emerging concepts that point to a role for human-specific genes in complex human disease, the existence of a human-specific α7nAChR regulating inflammatory responses in injury underscores the need for caution in extrapolating findings in the α7nAChR literature to man. To this end, we discuss the translational implications of a uniquely human α7nAChR-like gene on new drug target discovery and therapeutics development for injury, infection, and inflammation.

Amelioration of Muc5b mucin hypersecretion is enhanced by IL-33 after 2-APB administration in a murine model of allergic rhinitis.

We attempted to clarify whether hypersecretion of Muc5b mucin from mouse nasal submucosal glands that is enhanced by interleukin (IL)-33 under allergic conditions can be ameliorated by administration of 2-APB. Immunohistochemistry was used to examine both the distribution of T cells in the nasal mucosa of an allergic rhinitis mouse model and expressions of IL-33 receptor ST2 and Muc5b protein in mouse submucosal gland cells. The amounts of protein and mRNA of Orai1, Muc5b, IL-4, IL-5, IL-13 and IL-33 in mouse nasal lavage fluid (NLF) and nasal mucosa were determined using enzyme-linked immunosorbent assay and real-time reverse transcription-polymerase chain reaction. Expressions of Orai1, Muc5b, IL-4, IL-5, IL-13 and IL-33 were up-regulated in the allergic state and IL-33 increased the levels of Muc5b, IL-4, IL-5 and IL-13, but did not influence proliferation of T cells; however, ST2 was diminished in nasal submucosal gland cells. 2-APB reduced proliferation of T cells and the Orai1 level in the nasal mucosa. It also reduced the concentrations of IL-4, IL-5 and IL-13 in NLF and nasal mucosa, and hypersecretion of Muc5b from glandular cells that was enhanced by IL-33, but did not affect IL-33 production. 2-APB decreased Muc5b mucin hypersecretion from submucosal gland that was enhanced by IL-33 in allergic mice by limiting Ca(2+) release-activated Ca(2+) channel activity in which Orai1 plays a crucial role in the gland cells and/or by controlling channel activation in T cells and proliferation of these cells.

Cytotoxic T cells expressing the co-stimulatory receptor NKG2 D are increased in cigarette smoking and COPD.

BACKGROUND: A suggested role for T cells in COPD pathogenesis is based on associations between increased lung cytotoxic T lymphocyte (CD8+) numbers and airflow limitation. CD69 is an early T cell activation marker. Natural Killer cell group 2 D (NKG2D) receptors are co-stimulatory molecules induced on CD8+ T cells upon activation. The activating function of NKG2 D is triggered by binding to MHC class 1 chain-related (MIC) molecules A and B, expressed on surface of stressed epithelial cells. The aim of this study was to evaluate the expression of MIC A and B in the bronchial epithelium and NKG2 D and CD69 on BAL lymphocytes in subjects with COPD, compared to smokers with normal lung function and healthy never-smokers. METHODS: Bronchoscopy with airway lavages and endobronchial mucosal biopsy sampling was performed in 35 patients with COPD, 21 healthy never-smokers and 16 smokers with normal lung function. Biopsies were immunohistochemically stained and BAL lymphocyte subsets were determined using flow cytometry. RESULTS: Epithelial CD3+ lymphocytes in bronchial biopsies were increased in both smokers with normal lung function and in COPD patients, compared to never-smokers. Epithelial CD8+ lymphocyte numbers were higher in the COPD group compared to never-smoking controls. Among gated CD3+cells in BAL, the percentage of CD8+ NKG2D+ cells was enhanced in patients with COPD and smokers with normal lung function, compared to never-smokers. The percentage of CD8+ CD69+ cells and cell surface expression of CD69 were enhanced in patients with COPD and smokers with normal lung function, compared to never-smokers. No changes in the expression of MIC A or MIC B in the airway epithelium could be detected between the groups, whereas significantly decreased soluble MICB was detected in bronchial wash from smokers with normal lung function, compared to never-smokers. CONCLUSIONS: In COPD, we found increased numbers of cytotoxic T cells in both bronchial epithelium and airway lumen. Further, the proportions of CD69- and NKG2D-expressing cytotoxic T cells in BAL fluid were enhanced in both subjects with COPD and smokers with normal lung function and increased expression of CD69 was found on CD8+ cells, indicating the cigarette smoke exposure-induced expansion of activated cytotoxic T cells, which potentially can respond to stressed epithelial cells.

Developmental switch of the expression of ion channels in human dendritic cells.

Modulation of the expression and activity of plasma membrane ion channels is one of the mechanisms by which immune cells can regulate their intracellular Ca(2+) signaling pathways required for proliferation and/or differentiation. Voltage-gated K+ channels, inwardly rectifying K+ channels, and Ca(2+)-activated K+ channels have been described to play a major role in controlling the membrane potential in lymphocytes and professional APCs, such as monocytes, macrophages, and dendritic cells (DCs). Our study aimed at the characterization and identification of ion channels expressed in the course of human DC differentiation from monocytes. We report in this study for the first time that immature monocyte-derived DCs express voltage-gated Na+ channels in their plasma membrane. The analysis of the biophysical and pharmacological properties of the current and PCR-based cloning revealed the presence of Nav1.7 channels in immature DCs. Transition from the immature to a mature differentiation state, however, was accompanied by the down-regulation of Nav1.7 expression concomitant with the up-regulation of voltage-gated Kv1.3 K+ channel expression. The presence of Kv1.3 channels seems to be common for immune cells; hence, selective Kv1.3 blockers may emerge as candidates for inhibiting various functions of mature DCs that involve their migratory, cytokine-secreting, and T cell-activating potential.

Functional consequences of Kv1.3 ion channel rearrangement into the immunological synapse.

Formation of immunological synapse (IS), the interface between T cells and antigen presenting cells, is a crucial step in T cell activation. This conjugation formation results in the rearrangement and segregation of a set of membrane bound and cytosolic proteins, including that of the T cell receptor, into membrane domains. It was showed earlier that Kv1.3, the dominant voltage-gated potassium channel of T cells redistributes into the IS on interaction with its specific APC. In the present experiments we investigated the functional consequences of the translocation of Kv1.3 channels into the IS formed between mouse helper T (T(h)2) and B cells. Biophysical characteristics of whole-cell Kv1.3 current in standalone cells (c) or ones in IS (IS) were determined using voltage-clamp configuration of standard whole-cell patch-clamp technique. Patch-clamp recordings showed that the activation of Kv1.3 current slowed (tau(a,IS)=2.36+/-0.13 ms (n=7); tau(a,c)=1.36+/-0.06 ms (n=18)) whereas the inactivation rate increased (tau(i,IS)=263+/-29 ms (n=7); tau(i,c)=365+/-27 ms (n=17)) in cells being in IS compared to the standalone cells. The equilibrium distribution between the open and the closed states of Kv1.3 (voltage-dependence of steady-state activation) was shifted toward the depolarizing potentials in T cells engaged into IS (V(1/2,IS)=-20.9+/-2 mV (n=7), V(1/2,c)=-26.4+/-1.5 mV (n=12)). Thus, segregation of Kv1.3 channels into the IS modifies the gating properties of the channels. Application of protein kinase (PK) inhibitors (PKC: GF109203X, PKA: H89, p56Lck: damnacanthal) demonstrated that increase in the inactivation rate can be explained by the dephosphorylation of the channel protein. However, the slower activation kinetics of Kv1.3 in IS is likely to be the consequence of the redistribution of the channels into distinct membrane domains.

IgM anti-GQ1b monoclonal antibody inhibits voltage-dependent calcium current in cerebellar granule cells.

Miller-Fisher syndrome (MFS), which is known to be associated with anti-GQ1b antibodies and to cause ataxia, is a variant of an acute inflammatory neuropathy. However, the pathogenic role of anti-GQ1b antibodies remains unclear. In this study, we investigated the effects of mouse IgM anti-GQ1b monoclonal antibody (IgM anti-GQ1b mAb) on the spontaneous muscle action potential of a rat spinal cord-muscle co-culture system and on the voltage-dependent calcium channel (VDCC) current in cerebellar granule cells and Purkinje cells using the whole-cell patch clamp technique. The frequency of spontaneous muscle action potential of the innervated muscle cells was transiently increased by IgM anti-GQ1b mAb and then was blocked completely, which was the same finding as reported previously. Moreover, the cerebellar granule cell VDCC current was decreased by 30.76+/-7.60% by 5 microg/mL IgM anti-GQ1b mAb, whereas IgM anti-GQ1b mAb did not affect the VDCC current in cerebellar Purkinje cells. In immunocytochemistry, IgM anti-GQ1b mAb stained the whole cell surface of cerebellar granule cells, but not that of Purkinje cells. Therefore, the clinical symptoms of Miller-Fisher syndrome, such as cerebellar-like ataxia, may be explained by the inhibitory effects of anti-GQ1b antibodies on VDCC current in cerebellar granule cells.

Inhibition of Kv1.3 potassium current by phosphoinositides and stromal-derived factor-1alpha in Jurkat T cells.

The activation of Kv1.3 potassium channel has obligatory roles in immune responses of T lymphocytes. Stromal cell-derived factor-1alpha (SDF-1alpha) binds to C-X-C chemokine receptor type 4, activates phosphoinositide 3-kinase, and plays essential roles in cell migration of T lymphocytes. In this study, the effects of phosphoinositides and SDF-1alpha on Kv1.3 current activity were examined in the Jurkat T cell line using whole cell patch-clamp techniques. The internal application of 10 microM phosphatidylinositol 4,5-bisphosphate (PIP(2)) or 10 microM phosphatidylinositol-3,4,5-trisphosphate (PIP(3)) significantly reduced Kv1.3 current, but that of 10 microM phosphatidylinositol-4-monophosphate (PIP) did not. The coapplication of 10 microg/ml anti-PIP(3) antibody with PIP(2) from the pipette did not change the reduction of Kv1.3 current by PIP(2), but the coapplication of the antibody with PIP(3) eliminated the reduction. The heat-inactivated anti-PIP(3) antibody had no effect on PIP(3)-induced inhibition. These results suggest that PIP(2) per se can reduce Kv1.3 current as well as PIP(3). External application of 1 muM Akt-kinase inhibitor VIII did not reverse the effect of intracellular PIP(3). External application of 10 and 30 ng/ml SDF-1alpha significantly reduced Kv1.3 current. Internal application of anti-PIP(3) antibody reversed the SDF-1alpha-induced reduction. These results suggest that, in Jurkat T cells, PIP(2), PIP(3), and SDF-1alpha reduce Kv1.3 channel activity and that the reduction by SDF-1alpha may be mediated by the enhancement of PIP(3) production. These novel inhibitory effects of phosphoinositides and SDF-1alpha on Kv1.3 current may have a significant function as a downregulation mechanism of Kv1.3 activity for the maintenance of T lymphocyte activation in immune responses.

Attenuated P2X7 pore function as a risk factor for virus-induced loss of asthma control.

RATIONALE: Upper respiratory tract infection is a guideline accepted risk domain for the loss of asthma control. The ionotrophic nucleotide receptor P2X(7) regulates compartmentalized acute inflammation and the immune response to airway pathogens. OBJECTIVES: We hypothesized that variability in P2X(7) function contributes to neutrophilic airway inflammation during a cold and thereby is linked to acute asthma. METHODS: Research volunteers with asthma were enrolled at the onset of a naturally occurring cold and monitored through convalescence, assessing symptoms, lung function, and airway inflammation. P2X(7) pore activity in whole blood samples was measured using a genomically validated flow cytometric assay. MEASUREMENTS AND MAIN RESULTS: Thirty-five participants with mild to moderate allergic asthma were enrolled and 31 completed all visits. P2X(7) pore function correlated with the change in nasal lavage neutrophil counts during the cold (R(s) = 0.514, P = 0.004) and was inversely related to the change in asthma symptoms (R(s) = -0.486, P = 0.009). The change in peak expiratory flow recordings, precold use of inhaled corticosteroids, and P2X(7) pore function were multivariate predictors of asthma symptoms (P = 0.001, < 0.001 and = 0.003 respectively). Attenuated P2X(7) activity was associated with the risk of losing asthma control (crude odds ratio, 11.0; 95% confidence interval, 1.1-106.4) even after adjustment for inhaled corticosteroids and rhinovirus (odds ratio, 15.0). CONCLUSIONS: A whole blood P2X(7) pore assay robustly identifies participants with loss-of-function genotypes. Using this assay as an epidemiologic tool, attenuated P2X(7) pore activity may be a novel biomarker of virus-induced loss of asthma control.

A key role for redox signaling in rapid P2X7 receptor-induced IL-1 beta processing in human monocytes.

P2X(7) receptors (P2X(7)Rs) are ATP-gated ion channels that trigger caspase-1 activation in the presence of TLR ligands. Inflammatory caspase-1 is responsible for the proteolytic activation of IL-1beta. However, the signaling events that couple P2X(7)Rs to caspase-1 activation remain undefined. In this study we demonstrate that ATP-induced cellular oxidation is critical for caspase-1 activation and subsequent IL-1beta processing. Purinergic receptor stimulation, including P2X(7)Rs, of endotoxin-primed human monocytes augments NADPH oxidase activity whereas concurrent purinergic receptor stimulation triggers protein denitroyslation, leading to the formation of peroxynitrite. IL-1beta cleavage is blocked under conditions where superoxide anion formation is blocked or monocytes are treated with antioxidants or a peroxynitrite scavenger. Nigericin, a K(+)/H(+) antiporter, also increases NADPH oxidase activity, leading to IL-1beta and caspase-1 processing that is blocked by a peroxynitrite scavenger or inhibition of NADPH oxidase. These data demonstrate that signaling via NADPH oxidase activity is fundamental for the processing of mature IL-1beta induced by P2X(7)R stimulation.

Characterization of ATP-gated P2X7 receptors in fish provides new insights into the mechanism of release of the leaderless cytokine interleukin-1 beta.

Mammalian interleukin-1beta (IL-1beta) is produced as a biologically inactive precursor molecule, which is proteolytically cleaved to an active form by IL-1beta-converting enzyme (ICE) after the activation of P2X(7) receptor by extracellular ATP. The mechanism of IL-1beta release in non-mammalian vertebrates is largely unknown, although most of the IL-1beta gene sequences lack a conserved ICE recognition site. Here we have cloned the P2X(7) receptor from the bony fish seabream and compared agonist and antagonist profiles at this and other non-mammalian P2X(7) receptors expressed in HEK cells, as well in seabream SAF-1 cells expressing endogenous P2X(7) receptors. We used this information to further investigate the mechanisms of IL-1beta release induced by mammalian and fish P2X(7) receptors. Despite phosphatidylserine externalization and cell permeabilization in seabream leukocytes after the addition of high BzATP concentrations, IL-1beta remained unprocessed within the cell. However, activation of rat P2X(7) receptors ectopically expressed in HEK293 together with human ICE led to the specific secretion of unprocessed seabream IL-1beta. In contrast, neither seabream nor zebrafish P2X(7) receptors induced the secretion of mammalian or fish IL-1beta when expressed in HEK293, while a chimeric receptor harboring the ATP-binding domain of seabream P2X(7) and the intracellular region of its rat counterpart did so. These findings indicate that P2X(7) receptor-mediated activation of ICE and release of IL-1beta result from different downstream signaling pathways and suggest that although the mechanisms involved in IL-1beta secretion are conserved throughout evolution, distinct inflammatory signals have been selected for the secretion of this cytokine in different vertebrates.

Immunosuppressive effects of a Kv1.3 inhibitor.

The voltage gated potassium channel (Kv1.3) has been shown to play a role in immune responsiveness. Blockade of the channel led to diminution of T cell activation and delayed type hypersensitivity. Previous in vitro studies of the blockade were focused on T cell activation and proliferation. In this study we examined other T and monocytic cell mediated events to glean the extent of the immunosuppressive effects of a Kv1.3 specific inhibitor, Margatoxin (MgTX). We found that MgTX inhibited the intracellular production of Th-1 as well as Th-2 cytokines. MgTX can also inhibit IL-2 production and proliferation of T cells upon stimulation with anti-CD3 and VCAM-1. Furthermore, a redirected cytolytic activity was also inhibited by MgTX. However, MgTX did not inhibit generation of CTL to EBV transformed lymphoma cells or antibody-dependent cellular cytolysis mediated by monocytes. It appears that a Kv1.3 blockade does not affect all immune responses, particularly those of innate immunity.

A pyrazole derivative, YM-58483, potently inhibits store-operated sustained Ca2+ influx and IL-2 production in T lymphocytes.

In nonexcitable cells, Ca(2+) entry is mediated predominantly through the store depletion-dependent Ca(2+) channels called store-operated Ca(2+) (SOC) or Ca(2+) release-activated Ca(2+) channels. YM-58483, a pyrazole derivative, inhibited an anti-CD3 mAb-induced sustained Ca(2+) influx in acute T cell leukemia, Jurkat cells. But it did not affect an anti-CD3 mAb-induced transient intracellular Ca(2+) increase in Ca(2+)-free medium, nor anti-CD3 mAb-induced phosphorylation of phospholipase Cgamma1. It was suggested that YM-58483 inhibited Ca(2+) influx through SOC channels without affecting the TCR signal transduction cascade. Furthermore, YM-58483 inhibited thapsigargin-induced sustained Ca(2+) influx with an IC(50) value of 100 nM without affecting membrane potential. YM-58483 inhibited by 30-fold the Ca(2+) influx through SOC channels compared with voltage-operated Ca(2+) channels, while econazole inhibited both SOC channels and voltage-operated Ca(2+) channels with an equivalent range of IC(50) values. YM-58483 potently inhibited IL-2 production and NF-AT-driven promoter activity, but not AP-1-driven promoter activity in Jurkat cells. Moreover, this compound inhibited delayed-type hypersensitivity in mice with an ED(50) of 1.1 mg/kg. Therefore, we concluded that YM-58483 was a novel store-operated Ca(2+) entry blocker and a potent immunomodulator, and could be useful for the treatment of autoimmune diseases and chronic inflammation. Furthermore, YM-58483 would be a candidate for the study of capacitative Ca(2+) entry mechanisms through SOC/CRAC channels and for identification of putative Ca(2+) channel genes.

Fundamental Ca2+ signaling mechanisms in mouse dendritic cells: CRAC is the major Ca2+ entry pathway.

Although Ca(2+)-signaling processes are thought to underlie many dendritic cell (DC) functions, the Ca(2+) entry pathways are unknown. Therefore, we investigated Ca(2+)-signaling in mouse myeloid DC using Ca(2+) imaging and electrophysiological techniques. Neither Ca(2+) currents nor changes in intracellular Ca(2+) were detected following membrane depolarization, ruling out the presence of functional voltage-dependent Ca(2+) channels. ATP, a purinergic receptor ligand, and 1-4 dihydropyridines, previously suggested to activate a plasma membrane Ca(2+) channel in human myeloid DC, both elicited Ca(2+) rises in murine DC. However, in this study these responses were found to be due to mobilization from intracellular stores rather than by Ca(2+) entry. In contrast, Ca(2+) influx was activated by depletion of intracellular Ca(2+) stores with thapsigargin, or inositol trisphosphate. This Ca(2+) influx was enhanced by membrane hyperpolarization, inhibited by SKF 96365, and exhibited a cation permeability similar to the Ca(2+) release-activated Ca(2+) channel (CRAC) found in T lymphocytes. Furthermore, ATP, a putative DC chemotactic and maturation factor, induced a delayed Ca(2+) entry with a voltage dependence similar to CRAC. Moreover, the level of phenotypic DC maturation was correlated with the extracellular Ca(2+) concentration and enhanced by thapsigargin treatment. These results suggest that CRAC is a major pathway for Ca(2+) entry in mouse myeloid DC and support the proposal that CRAC participates in DC maturation and migration.

Selective blocking of voltage-gated K+ channels improves experimental autoimmune encephalomyelitis and inhibits T cell activation.

Kaliotoxin (KTX), a blocker of voltage-gated potassium channels (Kv), is highly selective for Kv1.1 and Kv1.3. First, Kv1.3 is expressed by T lymphocytes. Blockers of Kv1.3 inhibit T lymphocyte activation. Second, Kv1.1 is found in paranodal regions of axons in the central nervous system. Kv blockers improve the impaired neuronal conduction of demyelinated axons in vitro and potentiate the synaptic transmission. Therefore, we investigated the therapeutic properties of KTX via its immunosuppressive and symptomatic neurological effects, using experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. The T line cells used to induce adoptive EAE were myelin basic protein (MBP)-specific, constitutively contained mRNA for Kv1.3. and expressed Kv1.3. These channels were shown to be blocked by KTX. Activation is a crucial step for MBP T cells to become encephalitogenic. The addition of KTX during Ag-T cell activation led to a great reduction in the MBP T cell proliferative response, in the production of IL-2 and TNF, and in Ca(2+) influx. Furthermore, the addition of KTX during T cell activation in vitro led a decreased encephalitogenicity of MBP T cells. Moreover, KTX injected into Lewis rats impaired T cell function such as the delayed-type hypersensitivity. Lastly, the administration of this blocker of neuronal and lymphocyte channels to Lewis rats improved the symptoms of EAE. We conclude that KTX is a potent immunosuppressive agent with beneficial effects on the neurological symptoms of EAE.

New insights into endotoxin-induced activation of macrophages: involvement of a K+ channel in transmembrane signaling.

LPS (endotoxins) activate cells of the human immune system, among which are monocytes and macrophages, to produce endogenous mediators. These regulate the immune response, but may also cause severe harm leading to septic shock. The activation of monocytes/macrophages by LPS is mediated by a membrane-bound LPS receptor, mCD14. As mCD14 lacks a transmembrane domain, a further protein is required for the signal transducing step to the cell interior. Here we show, using excised outside-out membrane patches, that activation of a high-conductance Ca(2+)- and voltage-dependent potassium channel is an early step in the transmembrane signal transduction in macrophages. The channel is activated by endotoxically active LPS in a dose-dependent manner. Channel activation can be completely inhibited by LPS antagonists and by anti-CD14 Abs. Activation of the channel is essential for LPS-induced cytokine production as shown by its inhibition by selective K(+) channel blockers.