A nongenomic mechanism for progesterone-mediated immunosuppression: Inhibition of K+ channels, Ca2+ signaling, and gene expression in T lymphocytes

The mechanism by which progesterone causes localized suppression of the immune response during pregnancy has remained elusive. Using human T lymphocytes and T cell lines, we show that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K+ channels (KV and KCa, respectively), resulting in depolarization of the membrane potential. As a result, Ca2+ signaling and nuclear factor of activated T cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca2+ signals after thapsigargin stimulation, as well as oscillatory Ca2+ signals, but not the Ca2+ transient after TCR stimulation. K+ channel blockade by progesterone is specific; other steroid hormones had little or no effect, although the progesterone antagonist RU 486 also blocked KV and KCa channels. Progesterone effectively blocked a broad spectrum of K+ channels, reducing both Kv1.3 and charybdotoxin-resistant components of KV current and KCa current in T cells, as well as blocking several cloned KV channels expressed in cell lines. Progesterone had little or no effect on a cloned voltage-gated Na+ channel, an inward rectifier K+ channel, or on lymphocyte Ca2+ and Cl- channels. We propose that direct inhibition of K+ channels in T cells by progesterone contributes to progesterone-induced immunosuppression

The effect of cattle slurry in combination with nitrate and the nitrification inhibitor dicyandiamide on in situ nitrous oxide and dinitrogen emissions

peer-reviewedA field study was conducted to determine the effect of the nitrification inhibitor dicyandiamide (DCD) on N2O and N2 emissions after cattle slurry (CS) application in the presence of nitrate (NO3) fertiliser on seven different occasions (between March 2009 and March 2011). N2O emissions from CS in the presence of NO3 fertiliser were very high (0.4–8.7% of applied N) over a 20-day period, under mild moist conditions. Emissions were significantly larger from the CS treatment compared to an NH4+-N source, supplying the same rate of N as in the slurry. This study supports the view that organic fertilisers should not be applied at the same time as nitrate-based fertilisers, as significant increases in N2O emissions occur. The average N2O mole fraction (N2O/(N2O + N2)) over all seven application dates was 0.34 for CSNO3 compared to 0.24 for the NH4ClNO3 treatment, indicating the dominance of N2 emissions. The rate of nitrification in CSNO3 was slower than in NH4ClNO3, and DCD was found to be an effective nitrification inhibitor in both treatments. However, as N2O emissions were found to be predominantly associated with the NO3 pool, the effect of DCD in lowering N2O emissions is limited in the presence of a NO3 fertiliser. To obtain the maximum cost-benefit of DCD in lowering N2O emissions, under mild moist conditions, it should not be applied to a nitrate containing fertiliser (e.g. ammonium nitrate or calcium ammonium nitrate), and therefore the application of DCD should be restricted to ammonium-based organic or synthetic fertilisers.This research was funded by the Irish National Development Plan, through the Research Stimulus Fund (RSF 07 519), administered by the Irish Department of Agriculture, Food and the Marine

Charge Screening by Internal pH and Polyvalent Cations as a Mechanism for Activation, Inhibition, and Rundown of TRPM7/MIC Channels

The Mg2+-inhibited cation (MIC) current, believed to represent activity of TRPM7 channels, is found in lymphocytes and mast cells, cardiac and smooth muscle, and several other eukaryotic cell types. MIC current is activated during whole-cell dialysis with divalent-free internal solutions. Millimolar concentrations of intracellular Mg2+ (or other divalent metal cations) inhibit the channels in a voltage-independent manner. The nature of divalent inhibition and the mechanism of channel activation in an intact cell remain unknown. We show that the polyamines (spermine, spermidine, and putrescine) inhibit the MIC current, also in a voltage-independent manner, with a potency that parallels the number of charges. Neomycin and poly-lysine also potently inhibited MIC current in the absence of Mg2+. These same positively charged ions inhibited IRK1 current in parallel with MIC current, suggesting that they probably act by screening the head group phosphates on PIP2 and other membrane phospholipids. In agreement with this hypothesis, internal protons also inhibited MIC current. By contrast, tetramethylammonium, tetraethylammonium, and hexamethonium produced voltage-dependent block but no inhibition. We show that inhibition by internal polyvalent cations can be relieved by alkalinizing the cytosol using externally applied ammonium or by increasing pH in inside-out patches. Furthermore, in perforated-patch and cell-attached recordings, when intracellular Mg2+ is not depleted, endogenous MIC or recombinant TRPM7 currents are activated by cytosolic alkalinization and inhibited by acidification; and they can be reactivated by PIP2 following rundown in inside-out patches. We propose that MIC (TRPM7) channels are regulated by a charge screening mechanism and may function as sensors of intracellular pH

Nanoscale patterning of STIM1 and Orai1 during store-operated Ca2+ entry

Stromal interacting molecule (STIM) and Orai proteins constitute the core machinery of store-operated calcium entry. We used transmission and freeze-fracture electron microscopy to visualize STIM1 and Orai1 at endoplasmic reticulum (ER)-plasma membrane (PM) junctions in HEK 293 cells. Compared with control cells, thin sections of STIM1-transfected cells possessed far more ER elements, which took the form of complex stackable cisternae and labyrinthine structures adjoining the PM at junctional couplings (JCs). JC formation required STIM1 expression but not store depletion, induced here by thapsigargin (TG). Extended molecules, indicative of STIM1, decorated the cytoplasmic surface of ER, bridged a 12-nm ER-PM gap, and showed clear rearrangement into small clusters following TG treatment. Freeze-fracture replicas of the PM of Orai1-transfected cells showed extensive domains packed with characteristic "particles"; TG treatment led to aggregation of these particles into sharply delimited "puncta" positioned upon raised membrane subdomains. The size and spacing of Orai1 channels were consistent with the Orai crystal structure, and stoichiometry was unchanged by store depletion, coexpression with STIM1, or an Orai1 mutation (L273D) affecting STIM1 association. Although the arrangement of Orai1 channels in puncta was substantially unstructured, a portion of channels were spaced at ?15 nm. Monte Carlo analysis supported a nonrandom distribution for a portion of channels spaced at ∼15 nm. These images offer dramatic, direct views of STIM1 aggregation and Orai1 clustering in store-depleted cells and provide evidence for the interaction of a single Orai1 channel with small clusters of STIM1 molecules

Drug hypersensitivity caused by alteration of the MHC-presented self-peptide repertoire

Idiosyncratic adverse drug reactions are unpredictable, dose independent and potentially life threatening; this makes them a major factor contributing to the cost and uncertainty of drug development. Clinical data suggest that many such reactions involve immune mechanisms, and genetic association studies have identified strong linkage between drug hypersensitivity reactions to several drugs and specific HLA alleles. One of the strongest such genetic associations found has been for the antiviral drug abacavir, which causes severe adverse reactions exclusively in patients expressing the HLA molecular variant B*57:01. Abacavir adverse reactions were recently shown to be driven by drug-specific activation of cytokine-producing, cytotoxic CD8+ T cells that required HLA-B*57:01 molecules for their function. However, the mechanism by which abacavir induces this pathologic T cell response remains unclear. Here we show that abacavir can bind within the F-pocket of the peptide-binding groove of HLA-B*57:01 thereby altering its specificity. This supports a novel explanation for HLA-linked idiosyncratic adverse drug reactions; namely that drugs can alter the repertoire of self-peptides presented to T cells thus causing the equivalent of an alloreactive T cell response. Indeed, we identified specific self-peptides that are presented only in the presence of abacavir, and that were recognized by T cells of hypersensitive patients. The assays we have established can be applied to test additional compounds with suspected HLA linked hypersensitivities in vitro. Where successful, these assays could speed up the discovery and mechanistic understanding of HLA linked hypersensitivities as well as guide the development of safer drugs

POLDER observations of cloud bidirectional reflectances compared to a plane-parallel model using the International Satellite Cloud Climatology Project cloud phase functions

International audienceThis study investigates the validity of the plane-parallel cloud model and in addition the suitability of water droplet and ice polycrystal phase functions for stratocumulus and cirrus clouds, respectively. To do that, we take advantage of the multidirectional viewing capability of the Polarization and Directionality of the Earth's Reflectances (POLDER) instrument which allows us to characterize the anisotropy of the reflected radiation field. We focus on the analysis of airborne-POLDER data acquired over stratocumulus and cirrus clouds during two selected flights (on April 17 and April 18, 1994) of the European Cloud and Radiation Experiment (EUCREX'94) campaign. The bidirectional reflectances measured in the 0.86 μm channel are compared to plane-parallel cloud simulations computed with the microphysical models used by the International Satellite Cloud Climatology Project (ISCCP). Although clouds are not homogeneous plane-parallel layers, the extended cloud layers under study appear to act, on average, as a homogeneous plane-parallel layer. The standard water droplet model (with an effective radius of 10 μm) used in the ISCCP analysis seems to be suitable for stratocumulus clouds. The relative root-mean-square difference between the observed bidirectional reflectances and the model is only 2%. For cirrus clouds, the water droplet cloud model is definitely inadequate since the rms difference rises to 9%; when the ice polycrystal model chosen for the reanalysis of ISCCP data is used instead, the rms difference is reduced to 3%

In vivo imaging and quantitative analysis of leukocyte directional migration and polarization in inflamed tissue

Directional migration of transmigrated leukocytes to the site of injury is a central event in the inflammatory response. Here, we present an in vivo chemotaxis assay enabling the visualization and quantitative analysis of subtype-specific directional motility and polarization of leukocytes in their natural 3D microenvironment. Our technique comprises the combination of i) semi-automated in situ microinjection of chemoattractants or bacteria as local chemotactic stimulus, ii) in vivo near-infrared reflected-light oblique transillumination (RLOT) microscopy for the visualization of leukocyte motility and morphology, and iii) in vivo fluorescence microscopy for the visualization of different leukocyte subpopulations or fluorescence-labeled bacteria. Leukocyte motility parameters are quantified off-line in digitized video sequences using computer-assisted single cell tracking. Here, we show that perivenular microinjection of chemoattractants [macrophage inflammatory protein-1alpha (MIP-1alpha/Ccl3), platelet-activating factor (PAF)] or E. coli into the murine cremaster muscle induces target-oriented intravascular adhesion and transmigration as well as polarization and directional interstitial migration of leukocytes towards the locally administered stimuli. Moreover, we describe a crucial role of Rho kinase for the regulation of directional motility and polarization of transmigrated leukocytes in vivo. Finally, combining in vivo RLOT and fluorescence microscopy in Cx3CR1(gfp/gfp) mice (mice exhibiting green fluorescent protein-labeled monocytes), we are able to demonstrate differences in the migratory behavior of monocytes and neutrophils.Taken together, we propose a novel approach for investigating the mechanisms and spatiotemporal dynamics of subtype-specific motility and polarization of leukocytes during their directional interstitial migration in vivo