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Biomedical subjects

D Hanau

Publications and source records attributed to D Hanau.

At least 55 records · Page 3Linked to original sources
PubMed1995

Release of Fc gamma RIIa2 by activated platelets and inhibition of anti-CD9-mediated platelet aggregation by recombinant Fc gamma RIIa2.

Thrombin-activated human platelets and megakaryocyte cell lines release soluble Fc gamma RII (Fc gamma RIIa2) containing the extracellular and intracellular regions of Fc gamma RIIa1, but lacking the transmembrane domain. Use of polyclonal antibodies directed either against the entire intracytoplasmic tail, or against a peptide located near the C-terminal part of the intracellular region of Fc gamma RIIa2, showed the presence of both a complete form of Fc gamma RIIa2 and a C-terminal truncated form in supernatants of platelets after release of their alpha granule contents and in culture supernatants of megakaryocyte cell lines. Furthermore, recombinant Fc gamma RIIa2 inhibited in a dose-dependent manner Fc-dependent anti-CD9 antibody-induced platelet aggregation. Thus, release of Fc gamma RIIa2 by activated platelets could play an important role in the regulation of platelet activation by immune complexes.

Antibodies, Monoclonal↗
PubMed1995

Characterization of the protein tyrosine phosphatase CD45 on human epidermal Langerhans cells.

Human Langerhans cells (LC) express CD45, but clear data about the isoform(s) and their function(s) are lacking. In the present study, double labeling experiments reveal that freshly isolated LC from normal skin are CD45RO+/RA-/RB-. However, after isolation and short-time culture where LC undergo an in vitro maturation resembling that to lymphoid dendritic cells, CD45RB emerges whereas CD45RO expression decreases. This evolution results from dynamic alternative RNA splicing. Addition of granulocyte-macrophage colony-stimulating factor or tumor necrosis factor-alpha to short-time cultures has no significant effect on CD45RB, but both cytokines accelerate the loss of CD45RO. LC isolated from lesional skin of atopic eczema highly express CD45RO and CD45RB. Cross-linking of CD45 on LC isolated from atopic individuals inhibits the calcium mobilization in response to activation via Fc epsilon receptor type I (Fc epsilon RI). Hence, the protein tyrosine phosphatase CD45 from human LC is subjected to a splicing phenomenon related to the differentiation and activation stage of these cells and regulates their Fc epsilon RI-mediated activation.

Calcium↗
PubMed1995

Role of B cell receptor Ig alpha and Ig beta subunits in MHC class II-restricted antigen presentation.

The ability of the B cell antigen receptors (BCRs) to enhance MHC class II-restricted antigen presentation was ascribed to mig-associated Ig alpha/Ig beta heterodimers. The relative role of Ig alpha and Ig beta subunits in antigen presentation was investigated by fusing their cytoplasmic tails to the extracellular and transmembrane domains of Fc receptors. Ig alpha and Ig beta chimera mediate antigen internalization and increase the efficiency of antigen presentation, but they drive antigens to different endosomal compartments. Furthermore, antigens internalized by either chimera are degraded and presented with different kinetics. The cytoplasmic tail of Ig alpha targets antigen towards a major population of newly synthesized MHC class II located in class II-rich compartments. In contrast, Ig beta targets antigen towards a minor population of recycling MHC class II molecules, located in transferrin receptor-containing endosomes. Altogether, our data indicate that the composition of BCR could be therefore an important way to modulate the immune response.

Animals↗
PubMed1995

Association of HLA class I antigen deficiency related to a TAP2 gene mutation with familial bronchiectasis.

Two siblings with pansinusitis, nasal polyps, and bronchiectasis were found to have histocompatibility lymphocyte antigen (HLA) class I antigen deficiency ("bare lymphocyte syndrome") and dysfunction of natural killer cells. Reduced class I cell surface expression resulted from a single mutation in the TAP2 gene, which is located in the class II region of the major histocompatibility complex and encodes subunit 2 of the class I peptide transporter. The defect was transmitted in an autosomal recessive manner. This deficiency did not lead to severe viral infections but was apparently associated with susceptibility to bacterial infections of the respiratory mucosae. We suggest that class I HLA typing should be systematically performed in children with unexplained bronchiectasis.

Adolescent↗
PubMed1995

A three-base deletion removing a leucine residue in a leucine-rich repeat of platelet glycoprotein Ib alpha associated with a variant of Bernard-Soulier syndrome (Nancy I).

Leucine-rich repeats are conserved structural motifs present in the four components of the human platelet glycoprotein Ib/IX/V complex receptor for the adhesive protein von Willebrand factor. The absence or abnormality of this complex is responsible for Bernard-Soulier disease, an autosomal recessive bleeding disorder. We report a deletion of leucine 179, located in a highly conserved position of the seventh leucine-rich repeat of GPIb alpha, found in a variant form of Bernard-Soulier disease (Bernard-Soulier Nancy I). Three affected siblings of a family were characterized by absence of ristocetin-induced platelet agglutination, although ADP aggregation was normal. Flow cytometry studies showed detectable amounts of all four members of the GPIb/IX/V complex on the surface of the patients' platelets. Western blotting revealed normal levels of GPIX, decreased levels of GPIb beta and GPV, and < 1% of GPIb alpha. RT-PCR studies showed the presence of mRNA coding for GPIb alpha, GPIb beta, GPIX and GPV. Sequencing showed a three-base deletion which results in the absence of a leucine residue, highly conserved across the seven leucine-rich repeats of GPIb alpha and also within the other members of the leucine-rich glycoprotein family. The absence of the leucine 179 in a patient's GPIb alpha is believed to cause a conformational change in the protein which would account for the lack of binding of most of the MoAbs tested and would be responsible for the absence of von Willebrand factor binding. These results point to the leucine-rich region of GPIb alpha as being required for the correct exposure of the von Willebrand binding site as well as for the correct assembly and stability of the GPIb/IX/V complex on the platelet surface.

Amino Acid Sequence↗
PubMed1995

Presence of functional P2T and P2U purinoceptors on the human megakaryoblastic cell line, Meg-01 characterization by functional and binding studies.

The platelet receptor for ADP has been classified as a P2 purinoceptor of the P2T type where ADP is the natural agonist and ATP a competitive antagonist. Since the P2T receptor seems to be specific to platelets, we investigated the possibility that the human megakaryoblastic cell line Meg-01 might express the platelet ADP receptor, using functional studies and the ADP analogue [33P]2MeSADP as a specific P2T radioligand. ADP and 2MeSADP were able to induce shape change and pseudopod formation in Meg-01 cells. [33P]2MeSADP binding to membrane preparations was saturable and specific, binding sites being of high affinity (Kd = 13 +/- 3 nM) and present at a concentration of 13 +/- 7 fmoles/mu g protein. In contrast to UTP and GTP, ADP and ATP were able to displace the specific [33P]2MeSADP binding. ADP, 2MeSADP, UTP and ATP induced a dose dependent increase in intracellular calcium concentration. Desensitization experiments demonstrated that UTP, and ADP share a common P2U purinoceptor and that Meg-01 cells also express a P2T purinoceptor for which ADP and 2MeSADP are agonists and ATP a competitive antagonist. It was concluded that the human megakaryoblastic cell line Meg-01 expresses two distinct types of functional P2 receptor; a P2U and a P2T purinoceptor. This cell line could therefore provide the necessary material to clone the as yet unidentified platelet P2T purinoceptor.

Binding, Competitive↗
PubMed1994

Homozygous human TAP peptide transporter mutation in HLA class I deficiency.

Human lymphocyte antigen (HLA) class I proteins of the major histocompatibility complex are largely dependent for expression on small peptides supplied to them by transporter associated with antigen processing (TAP) protein. An inherited human deficiency in the TAP transporter was identified in two siblings suffering from recurrent respiratory bacterial infections. The expression on the cell surface of class I proteins was very low, whereas that of CD1a was normal, and the cytotoxicity of natural killer cells was affected. In addition, CD8+ alpha beta T cells were present in low but significant numbers and were cytotoxic in the most severely affected sibling, who also showed an increase in CD4+CD8+ T cells and gamma delta T cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Human epidermal Langerhans cells secrete a soluble receptor for IgG (Fc gamma RII/CD32) that inhibits the binding of immune complexes to Fc gamma R+ cells.

Langerhans cells (LC) express Fc gamma RII on their cell surface. In this paper, we demonstrate that these cells also release soluble Fc gamma RII (sFc gamma RII) molecules. LC express transcripts encoding a membrane-associated receptor and a transmembrane-deleted Fc gamma RIIA. The latter form was identified in LC culture supernatants using specific antibodies. CHO cells, transfected with LC-derived cDNA encoding the transmembrane-deleted Fc gamma RIIA, secrete sFc gamma RIIA that include the intracellular domain and exhibit the same backbone as the protein identified in LC supernatants. Secreted sFc gamma RIIA exhibits the same pattern of binding to human and mouse IgG subclasses as do membrane Fc gamma RII and inhibits the binding of immune complexes to Fc gamma RII+ cells. In addition, CHO cells expressing the membrane-associated Fc gamma RIIA release truncated and unstable Fc gamma RIIA molecules that lack the intracellular domain. Thus, sFc gamma RII can result from shedding of membrane molecules and/or from secretion of soluble receptors lacking the transmembrane domain.

Alternative Splicing↗
PubMed1994

CD1 expression is not affected by human peptide transporter deficiency.

Conventional major histocompatibility complex class I molecules are highly polymorphic and present peptides to cytotoxic T cells. These peptides derive from the proteolytic degradation of endogenous proteins in the cytosol and are translocated into the endoplasmic reticulum by a peptide transporter consisting of two transporter associated with antigen processing (TAP) molecules. Absence of this transporter leads to the synthesis of unstable peptide free class I molecules that are weakly expressed on the cell surface. Mouse nonconventional class I molecules (class Ib) may also present TAP-dependent peptides. In humans, CD1 antigens are nonconventional class I molecules. Recently, we characterized a human HLA class I deficiency resulting from a homozygous TAP deficiency. We show here that CD1a and -c are normally expressed on epidermal Langerhans cells of the TAP-deficient patients, as are CD1a, -b, and -c on dendritic cells differentiated in vitro from monocytes. Moreover, the CD1a antigens present on the surface of the dendritic cells are functional, since they internalize by receptor-mediated endocytosis gold-labeled F(ab')2 fragments of an anti-CD1a mAb. This suggests either that CD1 molecules are empty molecules, that they are more stable than empty conventional class I proteins, or that CD1 molecules present TAP-independent peptides.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Detection and quantification of secreted soluble Fc gamma RIIA in human sera by an enzyme-linked immunosorbent assay.

Fc gamma RIIA can be produced in a soluble form that contains both the extracellular and intracellular regions of the receptor, due to an alternative splicing of the transmembrane domain-coding exon. We have developed an enzyme-linked immunosorbent assay (ELISA) that permits the specific detection and quantification in human sera of this secreted soluble Fc gamma RIIA. It uses the monoclonal antibody (MAb) IV.3 as capture antibody and rabbit polyclonal IgG directed against the intracellular region of Fc gamma RIIA as detector antibodies. The enzymatic reaction was amplified using an NADH/NAD+ amplification system. As little as 0.8-1.5 ng/ml (20-38 pM) of purified recombinant secreted Fc gamma RIIA could be detected. The serum levels of secreted sFc gamma RIIA ranged from 0 to 30 ng/ml in sera from 51 healthy donors. The mean value was 11.9 ng/ml +/- 6.55 (297 pM +/- 163) and the median value was 10.6 ng/ml (265 pM) (range: 0-764 pM).

Animals↗
PubMed1993

Human keratinocytes release the endogenous beta-galactoside-binding soluble lectin immunoglobulin E (IgE-binding protein) which binds to Langerhans cells where it modulates their binding capacity for IgE glycoforms.

A better understanding of the pathophysiological role of Langerhans cells (LC) in atopic diseases is dictated by the characterization of the structures involved in immunoglobulin (IgE)-binding on their cell surface. We previously reported that human LC express the high affinity receptor for IgE (Fc epsilon RI), as well as the low affinity receptor for IgE (Fc epsilon RII/CD23). In the present study, we document the presence of a third IgE-binding structure on human LC, the IgE-binding protein (epsilon BP), an endogenous soluble beta-galactoside binding lectin. Immunohistochemical studies performed on normal human skin revealed an anti-epsilon BP reactivity in the cytoplasm of keratinocytes and in that of acinous cells of eccrine sweat glands. epsilon BP was also found on the cell surface of LC, as shown by anti-epsilon BP/anti-CD1a double labeling and flow cytometric analysis. Anti-epsilon BP binding to the surface of LC was completely abolished by preincubation with lactose and restored by addition of recombinant human epsilon BP, indicating that epsilon BP binds to LC surface by virtue of its lectin property. Immunoblot analysis of anti-epsilon BP-reactive material in keratinocytes and purified LC disclosed a protein with an apparent molecular weight of 33,000 consistent with epsilon BP. Interestingly, mRNA transcripts for epsilon BP were detected only in keratinocytes but not in purified LC isolated from normal skin. epsilon BP was found to be released in culture supernatants of keratinocytes. Incubation of LC with these supernatants resulted in epsilon BP-binding to LC surface via protein-carbohydrate interaction. Most importantly, we could show that binding of human myeloma IgE to LC was inhibited by epsilon BP. In contrast, neuraminidase-treated human myeloma IgE binds to LC only in the presence of epsilon BP. In situ binding studies revealed that keratinocytes, although containing epsilon BP intracytoplasmatically, failed to exhibit any IgE-binding properties. Collectively, our results suggest that human keratinocytes produce the beta-galactoside-binding lectin epsilon BP, which subsequently binds to the surface of LC where it is functional in modulating their binding capacity for IgE glycoforms.

Antigens, Differentiation↗
PubMed1993

A peptide ligand of the human thrombin receptor antagonizes alpha-thrombin and partially activates platelets.

The peptide YFLLRNP antagonizes the aggregation of human platelets when induced by low concentrations of alpha-thrombin or the thrombin receptor agonist peptide (SFLLRNP), demonstrating that it interacts specifically with the thrombin receptor. Platelets exposed to YFLLRNP show immediate shape change (pseudopod formation) and potentiation of the ADP and platelet-activating factor response, but no Ca2+ mobilization, P47 (pleckstrin) phosphorylation, secretion, or aggregation. Thus, YFLLRNP induces a state of partial activation of the platelets. Furthermore, with platelets prestimulated with adrenalin (10 microM), YFLLRNP induces aggregation, but no secretion, and only in the presence of added fibrinogen. We also found that prostacyclin inhibits the YFLLRNP-induced shape change; but EDTA, aspirin, and apyrase (ADP scavenger) do not. Thus, the thrombin receptor in platelets may communicate, independently of Ca2+ mobilization and P47 phosphorylation (protein kinase C activation), with intracellular signaling mechanisms that 1) modulate the cytoskeleton structure, 2) potentiate other platelet responses, and 3) stimulate coupling between the thrombin receptor and fibrinogen binding (the glycoprotein IIb-IIIa complex). YFLLRNP may be useful for differentiating between several possible activation states of the platelet thrombin receptor.

Amino Acid Sequence↗
PubMed1993

Alpha IIb beta 3 integrin dissociation induced by EDTA results in morphological changes of the platelet surface-connected canalicular system with differential location of the two separate subunits.

Treatment of human platelets by EDTA (5 mM at 37 degrees C and pH 7.4 for 30 min) induces ultrastructural morphological changes of the surface-connected canalicular system (SCCS). The first consists in dilations of some portions of the channels, whereas the second is represented by collapse of parts of the canaliculi. The collapsed elements of the EDTA treated SCCS are made up of two parallel limiting membranes and a central striated zone. Some of the EDTA treated platelets form microaggregates, the cohesion of which is apparently due to the appearance of pentalaminar interplatelet structures. EDTA treatment is known to induce an irreversible loss of platelet aggregability which is due to irreversible dissociation of the membrane GPIIb-IIIa complexes. In the present study, we looked for involvement of GPIIb-IIIa in the formation of these pentalaminar structures, and were able to demonstrate that the morphological changes described are in fact directly dependent on the EDTA induced dissociation of GPIIb-IIIa complexes. Indeed, we observed that these changes (a) cannot be induced in type I Glanzmann's thrombasthenia, where GPIIb-IIIa complexes are absent, (b) do not appear when human platelets are preincubated with monoclonal anti-GPIIb-IIIa complex-dependent (CD41a) antibodies, which protect the complex from EDTA induced dissociation, (c) appear only at alkaline pH and at 37 degrees C, which corresponds to the range of pH and temperature where EDTA can dissociate GPIIb-IIIa complexes, (d) are accompanied by the disappearance in fluorescence flow cytometry of the heterodimer complex-dependent epitopes, when using anti-CD41a antibodies and (e) do not appear in rat platelets, where GPIIb-IIIa does not dissociate after EDTA treatment. Furthermore, using gold-labeled mAbs concomitantly with the addition of EDTA, we observed that almost only GPIIb was present in the collapsed regions of the canaliculi. Using double labeling studies with polyclonal anti-GPIIb antibodies coupled to 10 nm gold particles and polyclonal anti-GPIIIa antibodies coupled to 20 nm gold particles, we observed that while both 10 and 20 nm particles were present in the dilated portions of the canaliculi almost only the small particles, coupled to the anti-GPIIb antibodies, labeled the collapsed portions of the SCCS. On Lowicryl thin sections, polyclonal antibodies against GPIIb labeled the central striated zone while both GPIIb and GPIIIa were found in the dilated portions of the SCCS. All these observations lead us to suggest that homopolymers of GPIIb could be responsible for "zipping" of the SCCS.

Blood Platelets↗
PubMed1993

Identification, in mouse macrophages and in serum, of a soluble receptor for the Fc portion of IgG (Fc gamma R) encoded by an alternatively spliced transcript of the Fc gamma RII gene.

Low affinity Fc gamma R are a heterogeneous group of glycoproteins which exist in transmembrane (TM) as well as in soluble forms. Two membrane isoforms of the murine type II Fc gamma R, Fc gamma RIIb1 and Fc gamma RIIb2, have been described. They result from the translation of alternatively spliced pre-mRNA, Fc gamma RIIb2 lacking sequences of the first intracytoplasmic domain (IC1). Soluble forms of Fc gamma R (sFc gamma R) have previously been shown to result from proteolysis of membrane receptors. We report here the identification, in macrophages, of a mRNA derived from the Fc gamma RII gene by splicing exons encoding the TM and IC1 domains, i.e. corresponding to a TM-deleted Fc gamma RIIb2 mRNA. A soluble protein possibly encoded by this mRNA was identified in macrophage supernatants. In accordance with Fc gamma R nomenclature, we propose to name this new Fc gamma RII isoform Fc gamma RIIb3. It is the most abundant sFc gamma R present in serum, as compared with sFc gamma R resulting from cleavage of membrane Fc gamma R.

Alternative Splicing↗
PubMed1992

Antibodies to thrombomodulin induce receptor-mediated endocytosis in human saphenous vein endothelial cells.

The membrane glycoprotein thrombomodulin (TM) is an essential endothelial cell (EC) cofactor, which forms a 1:1 stoichiometric complex with thrombin. Binding of thrombin to the high affinity TM receptor transforms its procoagulant activity into an anticoagulant potential, by activating protein C. The fate of TM in the presence of thrombin is still unclear: some authors claim that the thrombin-TM complex is internalized in EC, while others find this complex to be stable for at least 2 h at 37 degrees C on the EC surface. In the present study, we investigated the interactions of thrombin and Fab-fragments of anti-TM antibodies, coupled to 5 or 15 nm gold particles with saphenous vein endothelial cells. Our results demonstrate that TM can be observed both on the plasma membrane and in coated structures only in the presence of anti-TM antibodies. Addition of thrombin decreased the extent of this labeling, while in double labeling experiments, where cells were incubated with 5 nm gold coupled thrombin and 15 nm gold coupled Fab fragments of anti-TM antibodies, thrombin was cointernalized only when anti-TM antibodies were present. These results show that thrombin-TM complex is not significantly internalized in EC. The internalization of this complex induced by anti-TM antibodies could play an important role in the thrombotic complications induced by anti-EC autoantibodies.

Amino Acid Sequence↗
PubMed1992

Human epidermal Langerhans cells express the high affinity receptor for immunoglobulin E (Fc epsilon RI).

It has been suggested that epidermal Langerhans cells (LC) bearing immunoglobulin E (IgE) may be involved in the genesis of atopic disease. The identity of the IgE receptor(s) on LC remained unclear, although it represents a crucial point in understanding cellular events linked to the binding of allergens to LC via IgE. In this report, we demonstrate that epidermal LC express the high affinity receptor for the Fc fragment of IgE (Fc epsilon RI) which has, so far, only been described on mast cells and basophils. Epidermal LC react with antibodies specific for the alpha subunit of the tetrameric (alpha, beta, 2 gamma) Fc epsilon RI. Specific transcripts for Fc epsilon RI alpha and Fc epsilon RI gamma were detected in LC and correspond to those of human basophils and of the human basophil cell line KU812. Furthermore, human basophils, KU812 cells, and LC express the putative beta subunit. Thus human LC express the complete structure of Fc epsilon RI. This finding opens new perspectives in the putative functional role of this structure on antigen-presenting cells.

Amino Acid Sequence↗
PubMed1992

Fc gamma RII/CD32-negative human Langerhans cells may be responsible for the immunostimulatory activity of freshly isolated epidermal cells.

Cultured murine and human epidermal Langerhans cells (LC) undergo a phenotypical and functional maturation process. In fact, they loose Fc gamma RII and Birbeck granules, increase HLA-DR expression, and become potent accessory cells for allogeneic MLR. However, resident/freshly isolated human epidermal LC represent a phenotypically heterogeneous cell population. Indeed, a subset of CD1a+ LC lacks Birbeck granules, is Fc gamma RII/CD32-, and strongly expresses HLA-DR and the RFD1 antigen that is considered to be specific for interdigitating cells. In the present study the functional capacity of this Fc gamma RII/CD32- CD1a+ LC subset was investigated in MLR assays by comparing the stimulatory activity of freshly isolated crude epidermal cells (EC) with that of freshly isolated EC depleted in CD1a+ or in Fc gamma RII+ cells. Thereby, we observed that crude EC stimulated allogeneic PBMC while the removal of CD1a+ cells abrogated this stimulation. However, crude EC depleted in Fc gamma RII/CD32+ cells still exhibited a stimulatory capacity that was at least equal to that of crude EC. Taken together, these data suggest that among resident human epidermal LC there exists a subset of phenotypically and functionally more differentiated cells that may be solely responsible for the stimulatory capacity of freshly isolated crude EC.

Antibodies, Monoclonal↗