Human T-cell clones with multiple and changing functions: indications of unexpected flexibility in immune response networks?

T-cell cloning' seems greatly to assist the reductionist approach to the dissection of cellular immune response networks. Each of the cell types involved can apparently be cloned, studied in isolation, and then recombined. The success of this approach depends, however, on the ability to maintain functionally active cells long enough to obtain sufficient working material; and the assumption that a T cell is committed irrevocably to a particular defined function, so that its properties do not change during clonal expansion. In this article Graham Pawelec and his colleagues discuss a restriction on the maintenance of clones and question the notion that a clone's function is rigidly fixed.     

Zebrafish and mouse TASK-2 K+ channels are inhibited by increased CO2 and intracellular acidification

TASK-2 is a K_2P K⁺ channel considered as a candidate to mediate CO₂ sensing in central chemosensory neurons in mouse. Neuroepithelial cells in zebrafish gills sense CO₂ levels through an unidentified K_2P K⁺ channel. We have now obtained zfTASK-2 from zebrafish gill tissue that is 49 % identical to mTASK-2. Like its mouse equivalent, it is gated both by extra- and intracellular pH being activated by alkalinization and inhibited by acidification. The pH_i dependence of zfTASK-2 is similar to that of mTASK-2, with pK _1/2 values of 7.9 and 8.0, respectively, but pH_o dependence occurs with a pK _1/2 of 8.8 (8.0 for mTASK-2) in line with the relatively alkaline plasma pH found in fish. Increasing CO₂ led to a rapid, concentration-dependent (IC₅₀ ~1.5 % CO₂) inhibition of mouse and zfTASK-2 that could be resolved into an inhibition by intracellular acidification and a CO₂ effect independent of pH_i change. Indeed a CO₂ effect persisted despite using strongly buffered intracellular solutions abolishing any change in pH_i, was present in TASK-2-K245A mutant insensitive to pH_i, and also under carbonic anhydrase inhibition. The mechanism by which TASK-2 senses CO₂ is unknown but requires the presence of the 245–273 stretch of amino acids in the C terminus that comprises numerous basic amino acids and is important in TASK-2 G protein subunit binding and regulation of the channel. The described CO₂ effect might be of importance in the eventual roles played by TASK-2 in chemoreception in mouse and zebrafish.     

Relation of exocytotic release of γ-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the -aminobutyric acid (GABA) carrier, NNC-711, {1-[(2-diphenylmethylene) amino]oxyethyl}-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride, blocks the Ca²⁺-independent release of [³H]GABA from rat brain synaptosomes induced by 50 mM K⁺ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca²⁺-dependent release of [³H]GABA in the total absence of carrier-mediated release. Reversal of the Na⁺/Ca²⁺ exchanger was used to increase the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) to test whether an increase in [Ca²⁺]_i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca²⁺]_i may rise to values above 400 nM, as a result of Na⁺/Ca²⁺ exchange, without inducing release of [³H]GABA, but subsequent K⁺ depolarization immediately induced [³H]GABA release. Thus, a rise of only a few nanomolar Ca²⁺ in the cytoplasm induced by 50 mM K⁺ depolarization, after loading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange, induced exocytotic [³H]GABA release, whereas the rise in cytoplasmic [Ca²⁺] caused by reversal of the Na⁺/Ca²⁺ exchanger was insufficient to induce exocytosis, although the value for [Ca²⁺]_i attained was higher than that required for exocytosis induced by K⁺ depolarization. The voltage-dependent Ca²⁺ entry due to K⁺ depolarization, after maximal Ca²⁺ loading of the synaptosomes by Na⁺/Ca²⁺ exchange, and the consequent [³H]GABA release could be blocked by 50 M verapamil. Although preloading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange did not cause [³H]GABA release under any conditions studied, the rise in cytoplasmic [Ca²⁺] due to Na⁺/Ca²⁺ exchange increased the sensitivity to external Ca²⁺ of the exocytotic release of [³H]GABA induced by subsequent K⁺ depolarization. Thus, our results show that the vesicular release of [³H]GABA is rather insensitive to bulk cytoplasmic [Ca²⁺] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca²⁺ entry through Ca²⁺ channels near the active zones.     

Activation of Ca2+-dependent K+ and Cl− currents by UTP and ATP in CFPAC-1 cells

Activation of Cl^– and K⁺ conductances by nucleotide receptor-operated mobilization of intracellular Ca²⁺ was investigated in CFPAC-1 cells with the perforated-patch technique. Adenosine 5-triphosphate (ATP) and uridine 5-triphosphate (UTP) caused a dose-dependent fast and transient membrane hyperpolarization. UTP was more effective than ATP. In voltageclamped cells, two currents with different ionic permeability and kinetics were activated by the nucleotides. The first one was carried by Cl^– ions, peaked in the first few seconds after addition of nucleotides, and lasted for 1±0.3 min. Its amplitude was about 2.7 nA at –100 mV with 100 mol/l of either ATP or UTP. The second current was carried by K⁺ ions and was blocked by Cs⁺. This current peaked more slowly and had a mean duration of 4.6±0.7 min. Its amplitude was 0.9 nA and 0.5 nA at –20 mV with 100 umol/l UTP and ATP, respectively. Activation of the nucleotide receptor caused a transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) that was similar in the presence or absence of extracellular Ca²⁺. The ED₅₀ for UTP was 24 umol/l and that for ATP was 94 mol/l. Depletion of the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ store by thapsigargin prevented both the nucleotide-induced [Ca²⁺]_i increase and the activation of membrane currents. Addition of 2 mmol/l Ca²⁺ to thapsigargin-treated cells produced a sustained increase of Cl^– and K⁺ currents, which was reversed by Ca²⁺ removal. The present study demonstrates that CFPAC-1 cells respond to nucleotide receptor activation with a transient increase in [Ca²⁺]_i that stimulates Ca²⁺-dependent Cl^– and K⁺ currents. This phenomenon is probably mediated by inositol 1,4,5-trisphosphate-dependent Ca²⁺ stores.     

Endoplasmic reticulum–mitochondria coupling: local Ca2+ signalling with functional consequences

Plasma membrane store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channels are a widespread and conserved Ca²⁺ influx pathway, driving activation of a range of spatially and temporally distinct cellular responses. Although CRAC channels are activated by the loss of Ca²⁺ from the endoplasmic reticulum, their gating is regulated by mitochondria. Through their ability to buffer cytoplasmic Ca²⁺, mitochondria take up Ca²⁺ released from the endoplasmic reticulum by InsP₃ receptors, leading to more extensive store depletion and stronger activation of CRAC channels. Mitochondria also buffer Ca²⁺ that enters through CRAC channels, reducing Ca²⁺-dependent slow inactivation of the channels. In addition, depolarised mitochondria impair movement of the CRAC channel activating protein STIM1 across the endoplasmic reticulum membrane. Because they regulate CRAC channel activity, particularly Ca²⁺-dependent slow inactivation, mitochondria influence CRAC channel-driven enzyme activation, secretion and gene expression. Mitochondrial regulation of CRAC channels therefore provides an important control element to the regulation of intracellular Ca²⁺ signalling.     

Functionally distinct IFN-γ+IL-17A+ Th cells in experimental autoimmune uveitis: T-cell heterogeneity,migration, and steroid response

Immunopathogenic roles for both Th1 (CD4⁺IFN-γ⁺) and Th17 (CD4⁺IL-17A⁺) cells have been demonstrated in experimental autoimmune uveitis (EAU). However, the role for Th17/Th1 (CD4⁺ T cells co-expressing IFN-γ and IL-17A) cells in EAU is not yet understood. Using interphotoreceptor retinoid-binding protein peptide-induced EAU in mice, we found increased levels of Th17/Th1 cells in EAU retinae (mean 9.6 ± 4.2%) and draining LNs (mean 8.4 ± 3.9%; p = 0.01) relative to controls. Topical dexamethasone treatment effectively reduced EAU severity and decreased retinal Th1 cells (p = 0.01), but had no impact on retinal Th17/Th1 or Th17 cells compared to saline controls. Using in vitro migration assays with mouse CNS endothelium, we demonstrated that Th17/Th1 cells were significantly increased within the migrated population relative to controls (mean 15.6 ± 9.5% vs. 1.9 ± 1.5%; p = 0.01). Chemokine receptor profiles of Th17/Th1 cells (CXCR3 and CCR6) did not change throughout the transendothelial migration process and were unaffected by dexamethasone treatment. These findings support a role for Th17/Th1 cells in EAU and their resistance to steroid inhibition suggests the importance of targeting both Th17 and Th17/Th1 cells for improving therapy.     

Time resolved kinetics of the guinea pig Na–Ca exchanger (NCX1) expressed in Xenopus oocytes: voltage and Ca2+ dependence of pre-steady-state current investigated by photolytic Ca2+concentration jumps

Kinetic properties of the Na–Ca exchanger (guinea pig NCX1) expressed in Xenopus oocytes were investigated by patch clamp techniques and photolytic Ca²⁺ concentration jumps. Current measured in oocyte membranes expressing NCX1 is almost indistinguishable from current measured in patches derived from cardiac myocytes. In the Ca–Ca exchange mode, a transient inward current is observed, whereas in the Na–Ca exchange mode, current either rises to a plateau, or at higher Ca²⁺ concentration jumps, an initial transient is followed by a plateau. No comparable current was observed in membrane patches not expressing NCX1, indicating that photolytic Ca²⁺ concentrations jumps activate Na–Ca exchange current. Electrical currents generated by NCX1 expressed in Xenopus oocytes are about four times larger than those obtained from cardiac myocyte membranes enabling current recording with smaller concentration jumps and/or higher time resolution. The apparent affinity for Ca²⁺ of nonstationary exchange currents (0.1 mM) is much lower than that of stationary currents (6 μM). Measurement of the Ca²⁺ dependence of the rising phase provides direct evidence that the association rate constant for Ca²⁺ is about 5 × 10⁸ M⁻¹ s⁻¹ and voltage independent. In both transport modes, the transient current decays with a voltage independent but Ca²⁺-dependent rate constant, which is about 9,000 s⁻¹ at saturating Ca²⁺ concentrations. The voltage independence of this relaxation is maintained for Ca²⁺ concentrations far below saturation. In the Ca–Ca exchange mode, the amount of charge translocated after a concentration jump is independent of the magnitude of the jump but voltage dependent, increasing at negative voltages. The slope of the charge–voltage relation is independent of the Ca²⁺ concentration. Major conclusions are: (1) Photolytic Ca²⁺ concentration jumps generate current related to NCX1. (2) The dissociation constant for Ca²⁺ at the cytoplasmic transport binding site is about 0.1 mM. (3) The association rate constant of Ca²⁺ at the cytoplasmic transport sites is high (5 × 10⁻⁸ M⁻¹s⁻¹) and voltage independent. (4) The minimal five-state model (voltage independent binding reactions, one voltage independent conformational transition and one very fast voltage dependent conformational transition) used before to describe Ca²⁺ translocation at saturating Ca²⁺ concentrations is valid for Ca²⁺ concentrations far below saturation.     

Blood lymphocyte blastogenesis in patients with thyroid dysfunction: ex?vivo response to mitogen activation and cyclosporin?A

Objective  

To evaluate lymphocyte activation following mitogen and cyclosporin A (CsA) administration in peripheral blood of hyperthyroxinaemic and hypothyroid patients.     

Cell surface IL-1α trafficking is specifically inhibited by interferon-γ, and associates with the membrane via IL-1R2 and GPI anchors

IL-1 is a powerful cytokine that drives inflammation and modulates adaptive immunity. Both IL-1α and IL-1β are translated as proforms that require cleavage for full cytokine activity and release, while IL-1α is reported to occur as an alternative plasma membrane-associated form on many cell types. However, the existence of cell surface IL-1α (csIL-1α) is contested, how IL-1α tethers to the membrane is unknown, and signaling pathways controlling trafficking are not specified. Using a robust and fully validated system, we show that macrophages present bona fide csIL-1α after ligation of TLRs. Pro-IL-1α tethers to the plasma membrane in part through IL-1R2 or via association with a glycosylphosphatidylinositol-anchored protein, and can be cleaved, activated, and released by proteases. csIL-1α requires de novo protein synthesis and its trafficking to the plasma membrane is exquisitely sensitive to inhibition by IFN-γ, independent of expression level. We also reveal how prior csIL-1α detection could occur through inadvertent cell permeabilisation, and that senescent cells do not drive the senescent-associated secretory phenotype via csIL-1α, but rather via soluble IL-1α. We believe these data are important for determining the local or systemic context in which IL-1α can contribute to disease and/or physiological processes.     

Increase in intracellular Cl− concentration by cAMP- and Ca2+-dependent stimulation of M1 collecting duct cells

In the lungs of cystic fibrosis (CF) patients, mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) lead to defective Cl^– secretion and hyperabsorption of electrolytes. This may be a an important cause for the defective mucociliary clearance in CF lungs. Previous studies have suggested that inhibition of ENaC during activation of CFTR or by purinergic stimulation could be related to an increase in the intracellular [Cl^–]_i. This was examined in the present study using cultured mouse M1 collecting duct cells transfected with the chloride-sensitive enhanced yellow fluorescent protein YFP_V163S. Calibration experiments showed a linear decrease of YFP fluorescence intensity with increasing [Cl^–]_i (0–100 mM). Activation of CFTR by isobutyl-1-methylxanthine (IBMX, 100 µM) and forskolin (2 µM) increased [Cl^–]_i by 9.6±1.5 mM (n=35). Similarly, ATP (100 µM) increased [Cl^–]_i transiently by 9.5±2.2 mM (n=17). The increase in [Cl^–]_i was reduced by the Na⁺/K⁺/2 Cl^–-cortransporter-1 (NKCC1) blocker azosemide (100 µM), the CFTR blocker SP-303 (50 µM), the blocker of Ca²⁺-activated Cl^– channels DIDS (100 µM) or the ENaC blocker amiloride (10 µM). Changes in YFP_V163S fluorescence were not due to changes in cell volume or intracellular pH. The present data thus demonstrate an increase in [Cl^–]_i following stimulation with secretagogues, which could participate in the inhibition of ENaC.     

Voltage-operated Ca2+ channels and the acrosome reaction: which channels are present and what do they do?

Evidence from pharmacological studies suggests that induction of the acrosome reaction of mammalian spermatozoa by solubilized zona pellucida, and possibly by progesterone, is dependent upon Ca2+ influx through voltage-operated Ca2+ channels. Studies on Ca2+ accumulation and membrane potential in ligand-stimulated or artificially depolarized spermatozoa support such a conclusion. Electrophysiological studies on rodent spermatogenic cells have revealed the presence of a 'T' type voltage-operated Ca2+ current. This current has pharmacological attributes consistent with those of the putative channel responsible for Ca2+ influx mediating the acrosome reaction. However, use of molecular techniques to study human and rodent testis and spermatogenic cells has detected the presence of three different voltage-operated Ca2+ channel subunits. One of these (alpha lE) may generate T-currents, though this is currently disputed. Voltage-operated Ca2+ channel structure and the relationship between channel subunit expression and the characteristics of consequent Ca2+ currents is briefly reviewed. The nature and function of T-channel-mediated Ca2+ influx is examined in the context of the time-course of ligand- and depolarization-induced elevation of [Ca2+]i in mammalian spermatozoa. It is likely that a secondary Ca2+ response (mobilization of stored Ca2+ or activation of a second Ca(2+)-influx pathway) is required for the acrosome reaction. Evidence for the existence and participation of various candidates is discussed (including voltage-operated Ca2+ channels, which may be functionally expressed only in mature spermatozoa), the available evidence favouring a secondary Ca(2+)-influx pathway. Immediate priorities for future research in this area are proposed.     

Coupling of exocytosis to depolarization in rat pancreatic islet β-cells: effects of Ca2+, Sr2+ and Ba2+-containing extracellular solutions

Summary Using rat -cells we present evidence that Sr²⁺ and Ba²⁺, like Ca²⁺, support depolarization-induced increases in membrane capacitance which reflect insulin granule exocytosis. Even with identical total charge entry, Sr²⁺ and Ba²⁺ are 3–5 and 20-fold less effective than Ca²⁺ in supporting release. While exocytosis supported by Sr²⁺ is graded with cation entry and complete within 250ms of depolarization, exocytosis supported by Ba²⁺ begins abrupty after a threshold of charge entry and continues for many seconds. Ba²⁺-supported release continues in the presence of greatly enhanced cytosolic Ca²⁺ buffering, arguing against release of Ca²⁺ from stores as its principal action. These results suggest that Sr²⁺ and Ba²⁺ support exocytosis largely by binding to Ca²⁺-dependent release-activating sites, though with less affinity than Ca²⁺.     

The release of intracellular Ca2+ in lacrimal acinar cells by α-, β-adrenergic and muscarinic cholinergic stimulation: the roles of inositol trisphosphate and cyclic ADP-ribose

Stimulation of rat lacrimal acinar cells with acetylcholine (ACh) and the -adrenergic agonist isoprenaline causes a rapid increase in inositol phosphates with 1–4 phosphate groups, resulting in release of Ca²⁺ from intracellular stores. Stimulation with the -adrenergic agonist phenylephrine, however, causes a release of Ca²⁺ from internal stores which is 36% of that observed with ACh stimulation, but without inositol phosphate production. This Ca²⁺ rise was completely inhibited by 100 M ryanodine. Adrenaline (causing activation of both - and -adrenergic receptors) induces a Ca²⁺ release with inositol phosphate synthesis identical to that occuring in the -adrenergic response. Thus, the signalling pathway for -adrenergic stimulation occurs via a path different from that which releases Ca²⁺ via muscarinic cholinergic and -adrenergic stimulation. In permeabilized lacrimal acinar cells cyclic adenosine 5-diphosphoribose (cADP-ribose) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃] cause release of Ca²⁺ from intracellular stores. The Ca²⁺ release evoked by cADP-ribose, but not by Ins(1,4,5)P ₃, was abolished by 100 M ryanodine, implicating a possible involvement of cADP-ribose in phenylephrine-induced Ca²⁺ signalling. When the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) is raised by application of ionomycin, inositol phosphates are synthesized with a half-maximal effect seen at 425 nM. In contrast, loading cells with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) reduced the adrenaline-induced inositol phosphate synthesis by 27%. The stimulation-induced rise in [Ca²⁺]_i, therefore, appears to cause further synthesis of inositol phosphates, thereby amplifying the receptor-mediated response.     

Voltage-dependent Na+ and Ca2+ currents in human pancreatic islet β-cells: evidence for roles in the generation of action potentials and insulin secretion

We describe three voltage-dependent inward currents in human pancreatic -cells. First, a rapidly inactivating Na⁺ current, blocked by tetrodotoxin (TTX) is seen upon brief depolarization to or beyond –40 mV. Second, a transient, low-voltage-activated (LVA), amiloride-blockable Ca²⁺ current is seen upon depolarization to or beyond –55 mV; it inactivates within less than 1s of sustained depolarization to –40 mV. Third, a more sustained, high-voltage-activated (HVA) Ca²⁺ current, which shows variable sensitivity to dihydropyridines is seen upon depolarization to or beyond –40 mV, and thereafter slowly inactivates over a time course of many seconds. Our pharmacological evidence suggests that all three currents contribute to action potential initiation and upstroke when the background membrane potential (V _m) is equal or negative to –45 to –40 mV, a situation often induced by glucose concentrations (5–6 mM) in the range of those seen post-prandially. Consistent with this, TTX drastically reduces both transient and sustained insulin secretion in the presence of 5–6 mM glucose, but has little effect in 10 mM glucose, at which concentration cells rapidly depolarize to –35 mV, a V _m sufficient to rapidly inactivate Na⁺ and LVA Ca²⁺ currents.