Biomedical subjects

R C Monteiro

Publications and source records attributed to R C Monteiro.

At least 19 recordsLinked to original sources

PubMed2005

New insights in the pathogenesis of IgA nephropathy.

IgA nephropathy (N) or Berger's disease is the most common form of primary glomerulonephritis worldwide and one of the first cause of end-stage renal failure. The disease is characterized by the accumulation in mesangial areas of complexes containing polymeric IgA1. The mechanisms involved in the pathogenesis of IgAN is only now emerging. We discussed here three essential points: (i) the generation of abnormal IgA1 and formation of IgA1 complexes; (ii) the generation of mesangial injury mediated by interaction of IgA1 complexes with mesangial IgA1 receptors, and (iii) the progression of IgA-mediated mesangial injury towards renal failure. In summary, our data reveal that quantitative and structural changes of IgA1 play a key role on the onset of the disease due to functional abnormalities of two IgA receptors: the Fc alphaRI (CD89) expressed by blood myeloid cells and the transferrin receptor (CD71) on mesangial cells. Abnormal IgA induces release of soluble CD89 soluble leading to the formation of circulating IgA complexes, which in turn may be trapped by CD71 that is overexpressed on mesangial cells in IgAN patients allowing formation of IgA1 deposits. The elucidation of IgA-receptor interactions may open new avenues for drug design and treatment of IgAN.

Antigen-Antibody Complex↗

PubMed2001

Identification of the transferrin receptor as a novel immunoglobulin (Ig)A1 receptor and its enhanced expression on mesangial cells in IgA nephropathy.

The biological functions of immunoglobulin (Ig)A antibodies depend primarily on their interaction with cell surface receptors. Four IgA receptors are presently characterized. The FcalphaRI (CD89) expressed by myeloid cells selectively binds IgA1 and IgA2 antibodies, whereas the poly-IgR, Fcalpha/muR, and asialoglycoprotein receptors bind other ligands in addition to IgA. IgA binding by mesangial cells, epithelial cells, and proliferating lymphocytes is also well documented, but the nature of the IgA receptors on these cells remains elusive. A monoclonal antibody (A24) is described here that specifically blocks IgA binding to epithelial and B lymphocyte cell lines. Both the A24 antibody and IgA1 myelomas bind a cell surface protein that is identified as the transferrin receptor (CD71). The transferrin receptor selectively binds IgA1 antibodies, monomeric better than polymeric forms, and the IgA1 binding is inhibitable by transferrin. Transferrin receptor expression is upregulated on cultured mesangial cells as well as on glomerular mesangial cells in patients with IgA nephropathy. The characterization of transferrin receptor as a novel IgA1 receptor on renal mesangial cells suggests its potential involvement in the pathogenesis of IgA nephropathy.

Animals↗

PubMed2001

IgE receptor type I-dependent tyrosine phosphorylation of phospholipid scramblase.

To identify new effectors of IgE receptor (FcepsilonRI) signaling, we purified proteins from FcepsilonRI-stimulated RBL-2H3 rat mast cells on anti-phosphotyrosine beads and generated mouse monoclonal antibodies (mAb) against these proteins. Two mAbs bound to a protein that was identified as a new isoform of phospholipid scramblase (PLSCR) after screening an RBL-2H3 cDNA expression library. This isoform differed from PLSCR1 by the absence of an exon 3-encoded sequence and by an insert coding six QGPY(P/A)GP repeats. The PLSCR family of proteins is responsible for a redistribution of phospholipids across the plasma membrane. Although rat PLSCR is a 37-kDa protein, anti-phosphotyrosine immunoblots revealed the presence of 37-49 kDa phosphoproteins in the material immunoprecipitated with either anti-PLSCR mAb but not with unrelated monoclonal or polyclonal antibodies. Depletion of PLSCR resulted in the absence of these phosphoproteins. Additional experiments led to the identification of these phosphoproteins as phospho-PLSCR itself. Stimulation of RBL-2H3 cells upon FcepsilonRI engagement resulted in a dramatic increase in PLSCR tyrosine phosphorylation. A comparison of the relative amounts of phospho-PLSCR and nonphosphorylated PLSCR demonstrated that only a tiny fraction was thus modified, indicating a finely targeted involvement of PLSCR in FcepsilonRI signaling. Thus, this study reports the cloning of a new isoform of PLSCR, as well as the first observation that a member of the PLSCR family is a target for tyrosine kinases and is involved in signaling by an immune receptor. These findings open new perspectives on the role of phospholipid scramblases and to the mechanisms involved in their regulation.

Amino Acid Sequence↗

PubMed2001

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.

Antigen-Antibody Complex↗

PubMed2001

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.

APACHE↗

PubMed2001

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.

Adolescent↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Adult↗

PubMed1999

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.

Antigens, CD↗

PubMed1999

Elevation of serum IgA in spondyloarthropathies and IgA nephropathy and its pathogenic role.

Ankylosing spondylitis and IgA nephropathy share some immunologic features, eg, elevated serum IgA and IgA-immune complex levels. These entities are frequently found as being associated. IgA and IgA immune complex catabolism involves asialoglycoprotein receptors and specific IgA Fc receptors (FcalphaR or CD89) on tissue and blood cells. Recent studies revealed impaired CD89 expression in both diseases. These abnormalities, which are associated with receptor saturation, might generate the increase in serum IgA and IgA immune complex levels by either altered recycling or failure of degradation. This article reviews the literature on IgA abnormalities and discusses the potential role of FcalphaR in IgA nephropathy and AS and the consequences of its similar defect in the two diseases.

Glomerulonephritis, IGA↗

PubMed1998

Overexpression of natural killer T cells protects Valpha14- Jalpha281 transgenic nonobese diabetic mice against diabetes.

Progression to destructive insulitis in nonobese diabetic (NOD) mice is linked to the failure of regulatory cells, possibly involving T helper type 2 (Th2) cells. Natural killer (NK) T cells might be involved in diabetes, given their deficiency in NOD mice and the prevention of diabetes by adoptive transfer of alpha/beta double-negative thymocytes. Here, we evaluated the role of NK T cells in diabetes by using transgenic NOD mice expressing the T cell antigen receptor (TCR) alpha chain Valpha14-Jalpha281 characteristic of NK T cells. Precise identification of NK1.1(+) T cells was based on out-cross with congenic NK1.1 NOD mice. All six transgenic lines showed, to various degrees, elevated numbers of NK1.1(+) T cells, enhanced production of interleukin (IL)-4, and increased levels of serum immunoglobulin E. Only the transgenic lines with the largest numbers of NK T cells and the most vigorous burst of IL-4 production were protected from diabetes. Transfer and cotransfer experiments with transgenic splenocytes demonstrated that Valpha14-Jalpha281 transgenic NOD mice, although protected from overt diabetes, developed a diabetogenic T cell repertoire, and that NK T cells actively inhibited the pathogenic action of T cells. These results indicate that the number of NK T cells strongly influences the development of diabetes.

Animals↗

PubMed1998

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.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1998

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.

Base Sequence↗

PubMed1996

Defective Fc gamma RII gene expression in macrophages of NOD mice: genetic linkage with up-regulation of IgG1 and IgG2b in serum.

A quantitative trait locus for increased IgG serum levels in the NOD mouse strain was mapped to distal chromosome 1, close to the fcgr2 locus encoding the low-affinity type II receptor for the Fc portion of IgG (Fc gamma RII). Expression of membrane-inserted (b2) and soluble (b3) isoforms of Fc gamma RII was strongly decreased in macrophages of NOD compared with C57BL/6 (B6) mice. In contrast, B cell-specific (Fc gamma RIIb1) isoform was only slightly decreased and Fc gamma RIII was not altered. This Fc gamma RII regulatory defect was cis-encoded by fcgr2 or by a closely linked locus, occurred at the mRNA level, and was associated with multiple mutations in the fcgr2 gene promoter. In relation with this defect, binding of IgG1- and IgG2b- but not IgG2a-opsonized RBC by macrophages of NOD and congenic B6.NOD-fcgr2 mice was severely impaired, but was normal in macrophages of NOD.B6-fcgr2 congenic mice, indicating that Fc gamma RII plays a nondispensable role in binding of IgG1 and IgG2b isotypes. Likewise, serum levels of IgG1 and IgG2b but not IgG2a were up-regulated in NOD compared with NOD.B6-fcgr2 congenic mice. These findings indicate that macrophage Fc gamma RII may regulate serum IgG1 and IgG2b through their catabolism, and validate the NOD strain as a model to investigate the functions of Fc gamma RII isoforms.

Animals↗

PubMed1996

Identification of Fc alpha receptor (CD89) isoforms generated by alternative splicing that are differentially expressed between blood monocytes and alveolar macrophages.

One of the hallmarks of mucosal-host defense is the clearance of inhaled Ags by alveolar macrophages (AM) through interactions of IgA Abs and IgA Fc receptors (Fc alpha R). AM constitutively expressed Fc alpha R at lower levels than freshly isolated and in vitro-differentiated monocytes as determined by immunofluorescence using four anti-Fc alpha R mAb. SDS-PAGE analysis of iodinated cell surface proteins revealed that Fc alpha R on AM has an Mr of 50 to 65 kDa, slightly lower than that on monocytes (55-75 kDa). Treatment of AM Fc alpha R by N-glycanase gave rise to a protein core of 28 KDa, smaller than the 32-kDa backbone of blood monocytes. AM Fc alpha R molecules were unaffected by phosphatidylinositol-phospholipase C treatment. Fc alpha R transcripts were analyzed by reverse transcription-PCR using primers in the 5' and 3' regions of a U937 Fc alpha R cDNA. Three transcripts were amplified, cloned, and sequenced from AM and/or monocyte mRNA, the full length Fc alpha R and two alternatively spliced products corresponding to deletions of 66 and 288 nucleotides in the portion coding for the extracellular domain; they were named Fc alpha R a.1, a.2, and a.3, respectively. These PCR products were transcribed and translated in vitro into three proteins (Mr 32, 30, and 22 kDa, respectively), in which the 32- and 30-kDa species were immunoprecipitated by an anti-Fc alpha R mAb. The predicted size of the protein encoded by the Fc alpha R a.2 transcript without the leader peptide is Mr approximately 27,400, a value that is consistent with the Mr of AM Fc alpha R backbone. These results indicate that AM express at their surfaces a protein product of an alternatively spliced Fc alpha R transcript, the Fc alpha R a.2 isoform, that might have physiologic relevance in IgA-mediated host defense at mucosal sites.

Alternative Splicing↗

PubMed1995

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.

Antibodies, Monoclonal↗

PubMed1995

Detection of antilineage specific leucocyte antibodies by a quantitative immunocytometry method in sera from candidates for renal allografts.

A sensitive and quantitative flow cytometry method for detecting antileucocyte antibodies was developed to study retrospectively samples from candidates using frozen donor cells and frozen recipient sera. This immunofluorescence method was used to compare levels of reactivities of serum antibodies before and after donor specific blood transfusion treatment and after renal transplantation. The results demonstrate that the flow cytometry crossmatch is a sensitive and accurate method which should be used prospectively before precluding transplantation in the presence of a positive B cell standard crossmatch. Antibodies detected by flow cytometry before transplantation could be responsible for an early acute rejection episode. Finally, combination of flow cytometry crossmatch and standard crossmatch assays might thus be useful before precluding transplantation in living related donors.

Adolescent↗

PubMed1995

T cell activation through Thy-1 is associated with the expression of a surface protein (p100) on a subset of CD4 cells.

Thy-1 molecules, which lack a transmembrane domain, can nonetheless induce T cell activation; it has thus been suggested that a separate transmembrane molecule associated with Thy-1 is required for signal transduction. We have previously characterized a transmembrane protein with an Mr of 100,000 (p100), which is non-covalently bound to two glycosyl-phosphatidylinositol (GPI)-linked molecules, Thy-1 and ThB. p100 is selectively expressed on the T cell surface and divides peripheral CD4 cells into two subpopulations. This differential expression on CD4 cells allowed us to investigate the role of p100 in signal transduction through Thy-1 molecules. Here we report that only p100+ CD4 cells proliferate and release cytokines in response to cross-linkage of Thy-1, although both p100+ and p100- CD4 cells strongly express Thy-1 on their surfaces. Control stimulation by anti-CD3 antibodies or concanavalin A induces identical thymidine uptake by the two CD4 cell populations. Interestingly, these two populations of CD4 cells had different cytokine release profiles after activation through CD3: only p100+ CD4 cells released high amounts of IL-2 and IFN-gamma, whereas both populations released IL-4. p100 expression correlates with the induction of homotypic aggregation of T cells after Thy-1 triggering. p100 is associated with kinase activity (fyn and lck), and phosphorylated proteins of 90, 59, 57 and 33 kDa co-precipitate with Thy-1 only in p100+ CD4 cells. Altogether, these data suggest that p100 is involved in signal transduction through Thy-1. p100 expression by activated CD4 cells in vivo may be relevant to the proposed function of Thy-1 as an accessory signaling molecule in cell activation.

Animals↗