9-phenanthrol inhibits human TRPM4 but not TRPM5 cationic channels.

BACKGROUND AND PURPOSE: TRPM4 and TRPM5 are calcium-activated non-selective cation channels with almost identical characteristics. TRPM4 is detected in several tissues including heart, kidney, brainstem, cerebral artery and immune system whereas TRPM5 expression is more restricted. Determination of their roles in physiological processes requires specific pharmacological tools. TRPM4 is inhibited by glibenclamide, a modulator of ATP binding cassette proteins (ABC transporters), such as the cystic fibrosis transmembrane conductance regulator (CFTR). We took advantage of this similarity to investigate the effect of hydroxytricyclic compounds shown to modulate ABC transporters, on TRPM4 and TRPM5. EXPERIMENTAL APPROACH: Experiments were conducted using HEK-293 cells permanently transfected to express human TRPM4 or TRPM5. Currents were recorded using the whole-cell and inside-out variants of the patch-clamp technique. KEY RESULTS: The CFTR channel activator benzo[c]quinolizinium MPB-104 inhibited TRPM4 current with an IC(50) in the range of 2 x 10(-5) M, with no effect on single-channel conductance. In addition, 9-phenanthrol, lacking the chemical groups necessary for CFTR activation, also reversibly inhibited TRPM4 with a similar IC(50). Channel inhibition was voltage independent. The IC(50) determined in the whole-cell and inside-out experiments were similar, suggesting a direct effect of the molecule. However, 9-phenanthrol was ineffective on TRPM5, the most closely related channel within the TRP protein family. CONCLUSIONS AND IMPLICATIONS: We identify 9-phenanthrol as a TRPM4 inhibitor, without effects on TRPM5. It could be valuable in investigating the physiological functions of TRPM4, as distinct from those of TRPM5.

Enhanced expression of Fc alpha receptor I on blood phagocytes of patients with gram-negative bacteremia is associated with tyrosine phosphorylation of the FcR-gamma subunit.

Sepsis caused by gram-negative bacteria is a common finding having high incidence and mortality. Fc alpha RI (CD89), a receptor for immunoglobulin A (IgA), has been shown to mediate bacterial phagocytosis, which might play a role in the pathogenesis of sepsis. In this study the expression and function of Fc alpha RI were analyzed on blood monocytes and neutrophils of patients with bacteremia. We found a marked increased in expression of the alpha- and gamma-subunits of the Fc alpha RI on both types of cells in patients with gram-negative bacteremia, but not in patients with gram-positive bacteremia. This increase was independent of serum IgA levels. Fc alpha RI M(r) was lower on cells from gram-negative patients than on cells from controls (50-65 kDa versus 55-75 kDa), despite a similar 32-kDa backbone, indicating altered glycosylation. Increased levels of Fc alpha RI on blood phagocytes correlated with enhanced serum IL-6 levels, but not with IFN gamma or TNF-alpha. FcR-gamma chain associated with Fc alpha RI was phosphorylated in patients neutrophils, indicating functional engagement of this receptor during gram-negative sepsis. Increased expression and activation of Fc alpha RI-gamma 2 complexes following gram-negative infections suggests its involvement in host defense against bacteria.

ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology.

Free ADP-ribose (ADPR), a product of NAD hydrolysis and a breakdown product of the calcium-release second messenger cyclic ADPR (cADPR), has no defined role as an intracellular signalling molecule in vertebrate systems. Here we show that a 350-amino-acid protein (designated NUDT9) and a homologous domain (NUDT9 homology domain) near the carboxy terminus of the LTRPC2/TrpC7 putative cation channel both function as specific ADPR pyrophosphatases. Whole-cell and single-channel analysis of HEK-293 cells expressing LTRPC2 show that LTRPC2 functions as a calcium-permeable cation channel that is specifically gated by free ADPR. The expression of native LTRPC2 transcripts is detectable in many tissues including the U937 monocyte cell line, in which ADPR induces large cation currents (designated IADPR) that closely match those mediated by recombinant LTRPC2. These results indicate that intracellular ADPR regulates calcium entry into cells that express LTRPC2.

Colostral neutrophils express Fc alpha receptors (CD89) lacking gamma chain association and mediate noninflammatory properties of secretory IgA.

Colostrum plays an important role in protecting newborn infants against acute gastrointestinal and respiratory infections. IgA antibodies have been considered the major effector component; however, the role of their receptors on colostral phagocytes, especially neutrophils, has not been studied. Here, we demonstrate that CD15+ colostrum neutrophils express IgA Fc receptors (Fc alphaR, CD89) at levels similar to those of blood neutrophils. Most colostral cells (70%) bear secretory IgA (SIgA) on their surface (and intracellularly), whereas blood cells do not. The Fc alphaR on colostral neutrophils was identified as the a.1 isoform with a similar molecular mass (55-75 kDa) as that identified for blood neutrophils. Removal of N-linked carbohydrates revealed a major protein core of 32 kDa for both cell types. In contrast, co-immunoprecipitation and immunoblot experiments using a mild detergent, digitonin, revealed a lack of gamma chain association with Fc alphaR (gamma-less) exclusively on colostral neutrophils. The functional role of these gamma-less Fc alphaR cells was evaluated by measuring superoxide release and killing of SIgA-coated enteropathogenic E. coli. No increase in superoxide release was observed in colostral cells compared with blood neutrophils, whereas optimal release was obtained with PMA stimulation. Furthermore, despite similar bacterial phagocytosis index between both cell types, IgA-mediated bacterial-killing was not detectable with colostral neutrophils, whereas killing was detectable on blood cells. These results reveal exclusive expression of gamma-less Fc alphaR on colostral neutrophils associated with receptor hyperoccupation by IgA and with low, bacterial-killing activity, which suggest that this receptor may mediate noninflammatory effects of SIgA.

A subset of human dendritic cells expresses IgA Fc receptor (CD89), which mediates internalization and activation upon cross-linking by IgA complexes.

Immature dendritic cells (DC) sample Ags within nonlymphoid tissues and acquire exogenous proteins/pathogens via scavenger receptors or Ig FcR such as Fc gamma R and Fc epsilon R. IgA is present in a significant proportion among serum Ig and is the main isotype in mucosae, where DC are numerous. We found that a functional Fc alpha R (CD89) was expressed in situ and in vitro on interstitial-type DC but not on Langerhans cell-type DC. Interstitial-type DC expressed CD89 as a 50- to 75-kDa glycoprotein with a 32-kDa protein core, which was down-regulated upon addition of TGF-beta 1. DC, Fc alpha R specifically, bound IgA1 and IgA2. Cross-linking of CD89 on DC triggered endocytosis in time-dependent manner. In addition, internalization of polymeric IgA complexes induced the production of IL-10 and DC activation, as reflected by up-regulation of CD86 costimulatory molecules, class II MHC expression, and increased allostimulatory activity. Therefore, interstitial-type DC may use Fc alpha R-mediated Ag sampling in the subepithelium to check tissue integrity while Langerhans cells inside epithelial layers may neglect IgA immune complexes.

Fcalpha receptor (CD89) mediates the development of immunoglobulin A (IgA) nephropathy (Berger's disease). Evidence for pathogenic soluble receptor-Iga complexes in patients and CD89 transgenic mice.

The pathogenesis of immunoglobulin A (IgA) nephropathy (IgAN), the most prevalent form of glomerulonephritis worldwide, involves circulating macromolecular IgA1 complexes. However, the molecular mechanism(s) of the disease remain poorly understood. We report here the presence of circulating soluble FcalphaR (CD89)-IgA complexes in patients with IgAN. Soluble CD89 was identified as a glycoprotein with a 24-kD backbone that corresponds to the expected size of CD89 extracellular domains. To demonstrate their pathogenic role, we generated transgenic (Tg) mice expressing human CD89 on macrophage/monocytes, as no CD89 homologue is found in mice. These mice spontaneously developed massive mesangial IgA deposition, glomerular and interstitial macrophage infiltration, mesangial matrix expansion, hematuria, and mild proteinuria. The molecular mechanism was shown to involve soluble CD89 released after interaction with IgA. This release was independent of CD89 association with the FcRgamma chain. The disease was induced in recombination activating gene (RAG)2(-/-) mice by injection of serum from Tg mice, and in severe combined immunodeficiency (SCID)-Tg mice by injection of patients' IgA. Depletion of soluble CD89 from serum abolished this effect. These results reveal the key role of soluble CD89 in the pathogenesis of IgAN and provide an in vivo model that will be useful for developing new treatments.

Impaired expression of IgA Fc receptors (CD89) by blood phagocytic cells in ankylosing spondylitis.

OBJECTIVE: Expression of IgA Fc receptors (CD89, FcalphaR) and their occupancy by endogenous IgA were studied on blood monocytes and neutrophits to determine if FcalphaR defects could account for enhanced serum IgA and IgA-IC commonly found in patients with ankylosing spondylitis (AS). METHODS: Peripheral blood samples were obtained from 34 patients with AS, 15 patients with rheumatoid arthritis, and 34 healthy individuals. Cell surface FcalphaR was analyzed using a quantitative flow cytometry method in which blood cells were stained with anti-FcalphaR monoclonal antibodies recognizing epitopes outside the IgA binding site and with F(ab')2 fragments of anti-IgA antibodies. Modulation of cell surface FcalphaR was evaluated after incubation of blood cells at 37 degrees C in absence of plasma. Biochemical characterization of iodinated FcalphaR molecules was determined by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: FcaR expression was significantly decreased on monocytes and neutrophils in patients with AS compared to control groups. FcalphaR levels were inversely correlated with serum IgA, suggesting its negative regulatory role. Modulation experiments resulted in rapid and higher FcalphaR upregulation in AS than in controls, indicating that these molecules were downregulated only at the cell surface. Moreover, analysis of the surface iodinated FcalphaR molecules by SDS-PAGE revealed higher Mr (60-90 kDa) in AS than controls (55-75 kDa), also suggesting an altered glycosylation. Analysis of receptor occupancy revealed high levels of endogenous IgA bound to monocytes and neutrophils in patients with AS, pointing to a saturation of IgA Fc receptors. CONCLUSION: We observed impaired expression of FcalphaR in patients with AS that is characterized by a downregulation process associated with post-translational alterations and enhanced binding of endogenous IgA. These alterations might lead to a defective blood clearance by FcalphaR resulting in the enhancement of IgA and IgA-IC in AS patients. Decreased FcalphaR expression represents a new marker for this disease.

Alternative endocytic pathway for immunoglobulin A Fc receptors (CD89) depends on the lack of FcRgamma association and protects against degradation of bound ligand.

IgA is the most abundant immunoglobulin in mucosal areas but is only the second most common antibody isotype in serum because it is catabolized faster than IgG. IgA exists in monomeric and polymeric forms that function through receptors expressed on effector cells. Here, we show that IgA Fc receptor(s) (FcalphaR) are expressed with or without the gamma chain on monocytes and neutrophils. gamma-less FcalphaR represent a significant fraction of surface FcalphaR molecules even on cells overexpressing the gamma chain. The FcalphaR-gamma2 association is up-regulated by phorbol esters and interferon-gamma. To characterize gamma-less FcalphaR functionally, we generated mast cell transfectants expressing wild-type human FcalphaR or a receptor with a point mutation (Arg --> Leu at position 209) which was unable to associate with the gamma chain. Mutant gamma-less FcalphaR bound monomeric and polymeric human IgA1 or IgA2 but failed to induce exocytosis after receptor clustering. The two types of transfectant showed similar kinetics of FcalphaR-mediated endocytosis; however, the endocytosis pathways of the two types of receptor differed. Whereas mutant FcalphaR were localized mainly in early endosomes, those containing FcalphaR-gamma2 were found in endo-lysosomal compartments. Mutant gamma-less FcalphaR recycled the internalized IgA toward the cell surface and protected against IgA degradation. Cells expressing the two forms of FcalphaR, associated or unassociated with gamma chains, may thus have differential functions either by degrading IgA antibody complexes or by recycling serum IgA.

IgA Fc receptor (CD89) activation enables coupling to syk and Btk tyrosine kinase pathways: differential signaling after IFN-gamma or phorbol ester stimulation.

IgA Fc receptors (Fc alphaR) can mediate a variety of inflammatory responses. It has been demonstrated that the FcRgamma subunit is critical in mediating signaling through Fc alphaR. We show that aggregation of Fc alphaR on U937 cells and blood neutrophils results in tyrosine phosphorylation of several intracellular proteins, including the FcR gamma subunit, p72syk, and Bruton tyrosine kinase (Btk). Syk was found to be associated with Fc alphaR and its phosphorylation was increased in phorbol myristate acetate (PMA)- and interferon-gamma (IFN-gamma)-treated U937 cells. In contrast, phosphorylation of Btk was only detected after cell treatment with PMA but not IFN-gamma. These data indicate that signaling through Fc alphaR gamma2 involves at least two subfamilies of tyrosine kinases, syk and Btk. Our results also suggest that activation of tyrosine kinase pathways through Fc alphaR depends on the activation state of the cell. This may be an important regulatory mechanism in IgA-mediated responses at inflammatory sites.

Down-regulation of Fc alpha receptors on blood cells of IgA nephropathy patients: evidence for a negative regulatory role of serum IgA.

IgA nephropathy (IgAN) is associated with increased serum IgA1 and IgA1-immune complexes (IC). As Fc alpha receptors (Fc alpha R) are candidate molecules to regulate IgA levels, increased receptor occupation by IgA1 prompted us to study the expression of Fc alpha R on blood cells of IgAN patients. Surface and cytoplasmic Fc alpha R expression were markedly decreased on monocytes, despite normal levels of transcripts. Fc alpha R expression on patients' neutrophils was slightly decreased, exclusively at the cell surface. However, when autologous plasma was removed from the cells Fc alpha R was up-regulated. This observation led us to search for circulating regulatory factors. In vitro experiments revealed that Fc alpha R was down-regulated on normal monocytes following long-term culture with control or patient purified serum IgA at high concentrations (5 mg/ml). Moreover, polymeric myeloma IgA1 induced stronger down-regulation than monomeric IgA1. These results point to a negative regulatory role of serum IgA on surface Fc alpha R expression. This is also supported by a negative correlation between levels of Fc alpha F on blood cells and serum IgA. On the other hand, endogenous IgA bound to IgAN cells was significantly higher than IgA bound to control cells pre-incubated with patients' plasma, suggesting abnormalities in the receptor-ligand interaction. Patient Fc alpha R had a higher Mr (60 to 85 kDa) than those of controls (55 to 75 kDa) and a decreased binding to a sialic acid-specific lectin on blots, indicating post-translational modifications with impaired sialylation of surface Fc alpha R molecules that might be involved in enhanced IgA binding. Continuous Fc alpha R occupation by IgA, associated with receptor down-regulation, might contribute to the enhancement of circulating IgA1 and IgA1-IC by impairing their binding and degradation. Finally, increased receptor occupation by IgA on monocytes was linked to mesangial proliferation and glomerular sclerosis, suggesting a role for IgA-bound cells in the pathogenesis of mesangial damage.

Altered expression of monocyte IgA Fc receptors is associated with defective endocytosis in patients with alcoholic cirrhosis. Potential role for IFN-gamma.

Expression, saturation, and endocytosis of IgA Fc receptors (Fc alpha R) were analyzed in blood phagocytic cells of patients with alcoholic liver cirrhosis (ALC). Surface Fc alpha R expression was decreased in monocytes but not in neutrophils, as evaluated by IgA binding and anti-Fc alpha R mAb. The Fc alpha R of ALC patients were saturated by IgA1 and IgA2. ALC Fc alpha R had a higher M(r) (60 to 90 kDa) than those of controls (55 to 75 kDa) with a similar 32-kDa protein core after N-glycanase treatment, suggesting the expression of Fc alpha R molecules with altered carbohydrate moieties. Treatment of U937 cells with IFN-gamma induced a decrease of surface Fc alpha R expression in a dose-dependent manner, with a similar M(r) as observed for ALC patient Fc alpha R (60 to 90 kDa). Fc alpha R endocytosis was induced by anti-Fc alpha R or IgA. Neutrophils internalized Fc alpha R molecules faster than did monocytes. Endocytosed Fc alpha R co-localized with cathepsin D, suggesting an endolysosomal compartment pathway. In ALC monocytes, Fc alpha R endocytosis was defective, with nearly 50 to 60% of receptors detected on the cell surface even after 90 min at 37 degrees C. Similarly, delayed Fc alpha R endocytosis was observed on IFN-gamma-treated U937 cells as compared with PMA-activated cells. Defective internalization of surface-bound IgA with reflux of IgA to cell surface was also observed on ALC monocytes, but not on normal cells preincubated with patients' plasma, ruling out direct effects of IgA. The inverse correlation between monocyte Fc alpha R levels and serum IgA levels associated with defective endocytosis suggest that altered Fc alpha R expression might contribute to receptor saturation and generation of increased plasma levels of IgA and IgA-immune complexes in ALC patients.

[Value of parathyroid scintigraphy in preoperative localization of parathyroid adenoma. Study apropos of 138 tests]. / Valeur de la scintigraphie parathyroïdienne dans le repérage pré-opératoire des adénomes parathyroïdiens. Etude à propos de 138 examens.

The authors have studied, about a total of 138 exams, the value of parathyroid nuclide scan. All the patients, who had primary hyper parathyroidism, have been operated upon. The results of parathyroid scintigraphy have been compared to the results of surgical exploration. Overall sensibility of the exam has been 80%, better with the MIBI (89%) than with the Thallium (78%). The specificity has also been better with the MIBI (95%) than with the Thallium (87%) with a global result of 88%). Computerized treatment of images with automatized subtraction especially studied in Institut Jean-Godinot, Rheims, explain the quality of these results.

[Herpes gestationis. Apropos of 2 cases]. / L'herpès gestationis. A propos de deux cas.

Herpes gestationis is an autoimmune disorder specific of pregnancy. The authors report two cases of this condition with distinctive clinical and immunological features. Both the pathogenesis and the nosologic connections between Herpes gestationis and Bullous pemphigoid are reviewed and discussed.