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C Dezutter-Dambuyant

Publications and source records attributed to C Dezutter-Dambuyant.

At least 73 records · Page 4Linked to original sources
PubMed1991

Expression and endocytosis of integrin VLA receptors for collagen, fibronectin and laminin by normal human keratinocytes.

The Very Late Activation (VLA) antigen family is involved in cell-extracellular matrix interactions and consists of six heterodimeric cell surface receptors with a common beta 1 and a variable alpha subunit. Using a panel of specific antibodies, we showed that human epidermal basal cells expressed VLA-2, VLA-3 and VLA-6 but failed to express VLA-4. Their functional roles were investigated and VLA-2 appeared as a specific receptor for type IV collagen and also as a laminin receptor. VLA-3 appeared as a receptor for fibronectin and laminin and to a lesser extent as a type I collagen receptor. VLA-6 appeared as a specific receptor for laminin. It also appeared that the VLA-alpha subunit specifically mediates the recognition of ligand but the beta 1 subunit is also involved in adhesion and that both subunits have a synergistic influence. Immunoprecipitation analyses confirmed that VLA-2, VLA-3 and VLA-6 were expressed by basal keratinocytes. Endocytosis of VLA-2 and VLA-3 was observed involving coated vesicles and endosomes that are structures characteristic of a receptor-mediated pathway. These findings provide first evidence that normal human basal keratinocytes are able of endocytosis mediated by receptors. Taken together, these results indicate that multiple VLA receptors function in combination to mediate epidermal basal cell adhesion to extracellular matrix.

Antibodies, Monoclonal↗
PubMed1991

Human epidermal Langerhans cells express integrins of the beta 1 subfamily.

The expression of VLA integrins on human epidermal Langerhans cells (LC) was investigated by indirect immunogold labeling on transmission electron microscopy, followed by a quantitative analysis. Labelings on suspensions enriched in freshly isolated LC were carried out with an antibody recognizing the beta 1 subunit, common to all members of the VLA family, and with antibodies specific for the six different alpha subunits, alpha 1 to alpha 6. Normal human epidermal LC were all beta 1 positive, of which 60% were highly positive. By labelings with different VLA-alpha-chain MoAb, there appeared two subpopulations of LC: one positive and one negative. On average 40% of LC bore small amounts of VLA-1 and VLA-3, 53% and 77% of LC expressed moderate amounts of VLA-2 and VLA-5, respectively, and VLA-4 and VLA-6 were expressed by 67% and 90% of LC, respectively. VLA proteins are mainly extracellular matrix protein receptors. VLA-6 (laminin receptor) and VLA-5 (fibronectin receptor) are expressed mainly by LC, and in this way could subserve LC to leave the epidermal compartment through the basement membrane, to migrate throughout the fibronectin network of the dermis before migrating via the afferent lymphatics to the regional lymph nodes, where they present antigen to T cells. VLA proteins such as VLA-4, VLA-3, or VLA-2, which are found involved in cell-cell contacts, could contribute to the promotion of T-cell activation by facilitating adherence between LC and T cells.

Humans↗
PubMed1991

Interaction of human epidermal Langerhans cells with HIV-1 viral envelope proteins (gp 120 and gp 160s) involves a receptor-mediated endocytosis independent of the CD4 T4A epitope.

The CD4 molecule is known to be the preferential receptor for the HIV-1 envelope glycoprotein. Epidermal Langerhans cells are dendritic cells which express several surface antigens, among them CD4 antigens. To clarify the exact role of CD4 molecules in Langerhans cell infection induced by HIV-1, we investigated the possible involvement of the interactions between HIV-1 gp 120 or HIV-1 gp 160s (soluble gp 160) and Langerhans cell surface. We also assessed the expression of CD4 molecules on Langerhans cell membranes dissociated by means of trypsin from their neighbouring keratinocytes. The cellular phenotype was monitored using flow cytometry and quantitative immunoelectron microscopy. We reported that human Langerhans cells can bind the viral envelope proteins (gp 120 or gp 160s), and that this binding does not depend on CD4 protein expression. This binding is not blocked by anti-CD4 monoclonal antibodies. We show that a proportion of gp 120/gp 160s-receptor complexes enters Langerhans cells by a process identified as a receptor-mediated endocytosis. The amount of surface bound gp 120/gp 160s is not consistent with the amount of CD4 antigens present on Langerhans cell membranes. Gp 120/gp 160s binding sites on Langerhans cell suspensions appeared to be trypsin resistant, while CD4 antigens (at least the epitopes known to bind the HIV-1) are trypsin sensitive. A burst of gp 120 receptor expression was detected on 1-day cultured Langerhans cells while CD4 antigens disappeared. These findings lead to the most logical conclusion that binding of gp 120/gp 160s is due to the presence of a Langerhans cell surface molecule different from CD4 antigens.

Antigens, Surface↗
PubMed1991

Eosinophilic granuloma of bone and biochemical demonstration of 49-kDa CD1a molecule expression by Langerhans-cell histiocytosis.

Histiocytic cells infiltrating the lesions in eosinophilic granuloma of bone as well as in cutaneous histiocytosis X were studied using a murine monoclonal antibody (MA) produced with proliferating cells from an eosinophilic granuloma of bone. This MA reacts with Langerhans cells (LC) of normal human skin or mucous membranes and with proliferating cells of eosinophilic granuloma of bone and skin lesions of Letter-Siwe disease, as shown by immunohistochemistry and immunogold labelling. As other murine MA's obtained after immunization with human cortical thymocytes, this MA immunoprecipitates the 49-kDa CD1a antigen found on human LC and thymic-cell surfaces but not its breakdown product after treatment with trypsin, as demonstrated by analysis of immunoelectron labelling, cytofluorometry and gel electrophoresis. This first production of a CD1a MA from an eosinophilic granuloma supports the concept of Langerhans-cell histiocytosis.

Antigens, CD↗
PubMed1990

Human epidermal Langerhans cells express only the 40-kilodalton Fc gamma receptor (FcRII).

In man, three distinct classes of receptors for the Fc fragments of IgG (FcRI, II, III) have been defined. The FcRI has a Mr of about 72 kDa, binds human IgG-coated E, and is recognized by mAb such as 32. The FcRII has a Mr of 40 kDa, binds murine IgG1-coated E, and reacts with the mAb IV.3 and CIKM5, which recognize CDw32 moieties. Lastly, the FcRIII has a Mr of 50 to 70 kDa and is recognized by anti-CD16 mAb. In the present study we have shown that i) only murine IgG1-coated E form rosettes with 49 +/- 1.5% (mean +/- SEM, n = 9) of CD1a+ epidermal cells (EC) (which represent Langerhans and indeterminate cells) and that ii) the mAb anti-FcRII CIKM5 prevents this rosette formation. Among the mAb reacting with the three different types of FcR, only those recognizing FcRII i) stain about 55 +/- 1.5% (mean +/- SEM, n = 9) of the CD1a+ EC and ii) reveal the presence of dendritic cells in epidermal sheets obtained by suction blister. Under the electron microscope i) apparently all the cells forming rosettes or reacting with the gold-labeled anti-FcRII mAb (CIKM5 or the F(ab) fragment of IV.3) contained Birbeck granules and ii) the gold-labeled mAb were internalized in unfixed Langerhans cells by receptor-mediated endocytosis and accumulated in lysosomes. Labeling by the anti-FcRII mAb of the CD1a+ cells in suspension disappears after 48 h of culture. All these observations strongly suggest that CD1a+ EC express only the FcRII. This conclusion was confirmed by immunoprecipitation experiments, whereas no specific immunoprecipitate was noted with the anti-FcRI or anti-FcRIII mAb, the anti-FcRII mAb immunoprecipitated a protein of Mr 40 kDa.

Antibodies, Monoclonal↗
PubMed1990

Identification of specific human epithelial cell integrin receptors as VLA proteins.

Cell adhesion to extracellular matrix is mediated by a set of heterodimeric cell surface receptors called integrins. We have examined the expression of the very late antigens or alpha beta 1 group of integrins in human epithelial cells. The six known members of this group share a common beta 1 subunit but have distinct alpha subunits that confer selective affinity toward collagen, fibronectin, and laminin essentially. Using a panel of specific antibodies we showed that freshly harvested human epidermal basal cells express VLA-2 and VLA-3 receptors, a low amount of VLA-5, but fail to express VLA-4. The findings reveal that these receptors are characterized by the alpha subunits which associate with a beta subunit different in weight (Mr 110,000 reduced) from that normally seen (Mr 130,000). Moreover, immunoprecipitates of VLA-2 contained additional proteins of Mr 80,000 and Mr 40,000 and immunoprecipitates of VLA-3 contained an additional protein of Mr 90,000. Experiments carried out to investigate the functional roles of these receptors in mediating cell adhesion to extracellular matrix revealed that cell attachment to type IV collagen was completely inhibited by antibodies to VLA-2 alpha chain, that antibody to VLA-3 alpha chain significantly blocked attachment to fibronectin while antibodies to both VLA-2 and VLA-3 partially inhibited attachment to type I collagen. Cell attachment to types I and IV collagen and to fibronectin was not affected by antibodies to VLA-4 and VLA-6. These results show that multiple VLA receptors function in combination to mediate epidermal basal cell adhesion to extracellular matrix. This cooperation function of multiple VLA receptors and their differential expression could be considered to be one of the controlling points in the localization of epithelial basal cells in the epidermis.

Antibodies, Monoclonal↗
PubMed1990

Ontogeny of Langerhans cells: phenotypic differentiation from the bone marrow to the skin.

We have performed double immunolabelings for cytofluorometric analysis and electron microscopy to investigate the coexpression of the CD1a (OKT6 and DMC1 monoclonal antibodies) antigen and the promonocyte/monocyte differentiation antigens CD14 (My4) or CD33 (My9) on putative bone marrow and umbilical cord blood precursors of the Langerhans cells (LC) and the epidermal LC. By cytofluorometric analysis, the percentage of CD1a+ cells which coexpressed the CD33 antigen was different from the bone marrow (5% of CD33+ cells are CD1a+), to the cord blood (3% of the CD33+ are CD1a+) and to the epidermis (the whole population of CD33+ LC are CD1a+). The ultrastructural morphology of the CD1a-expressing bone marrow, cord blood cells closely approximated that of a promonocyte/monocyte. Only LC epidermal were specifically recognized by the intracytoplasmic Birbeck granules. These CD1a+/CD33+ or CD14+ subpopulations found in three different locations (epidermis, bone marrow, cord blood) display a similar quantitative expression of the CD14 and CD33 antigens.

Antigens, CD↗
PubMed1990

The effect of trypsin on CD1a molecule of human thymocytes.

The cortical thymocytes expressed at least three distinct cell-surface differentiation antigens. CD1a (Mr 49,000), CD1b (Mr 45,000) and CD1c (Mr 43,000) which are non-covalently attached to beta 2-microglobulin. In the present study, we confirm the presence of two out of the three CD1 molecules on epidermal Langerhans cells by biochemical analysis. Furthermore some CD1a monoclonal antibodies immunoprecipitated an additional molecule with an apparent relative mass of 27,000 from Langerhans cell-enriched epidermal cell lysates and not from fresh iodinated thymocyte lysates. From trypsin-treated thymocyte lysates, this low molecular weight protein was considered as a cleavage product of Mr 49,000 molecule (CD1a molecule) by this enzyme which is used to obtain epidermal cell suspensions. This Mr 27,000 was found to content one N-linked oligosaccharide residue by endoglycosidase F treatment. On CD1-expressing cells (thymocytes and Langerhans cells) it would be tempting to take advantage of the sensitivity of CD1a molecule to trypsin in order to precise the structure/function relationship of CD1a antigen.

Antibodies, Monoclonal↗
PubMed1989

A combined method for detection of cell surface marker expression and bromodeoxyuridine (BrdU) uptake by epidermal cells in suspension.

A rapid and convenient immunocytochemical method is presented for the simultaneous detection of surface antigen expression and S-phase cells using monoclonal antibodies and cells in suspension. The first reagent permits the nature of the cells to be determined while the second immunostaining stage reveals which of these cells are proliferating (BrdU-positive) cells. Staining and fixation conditions have been defined for the detection of cell surface markers by red fluorescence and BrdU labelling by green fluorescence. All the steps were carried out on cells in suspension and the membrane antigen distribution was not affected by the BrdU staining protocol. The method has been applied to two human epidermal cell subpopulations: Langerhans' cells and basal keratinocytes. This technique should have general applicability to the study of the cytokinetic characteristics of phenotypically defined cells in heterogeneous populations.

Antigens, Surface↗
PubMed1989

Cleavage of Langerhans cell surface CD1a molecule by trypsin.

The T-cell surface differentiation antigens expressed on cortical thymocytes are composed of 3 molecules, CD1a (Mr 49,000), CD1b (Mr 45,000), and CD1c (Mr 43,000), which are non-covalently attached to beta 2-microglobulin. In the present study, differences in quantitative binding (immunogold labelling) were observed with four CD1a monoclonal antibodies (mAb), Na1/34, L544, Vit6 and OKT6, on epidermal Langerhans cells obtained through trypsinization and Ficoll-Hypaque sedimentation. These cells were surface-labelled with 125I and then lysed. Immunoprecipitation was carried out with five CD1a mAb, BL6, 10D12.2, L404, L544 and OKT6, and immunoprecipitates were electrophoretically run. All CD1a mAb except OKT6 immunoprecipitated an additional molecule with an apparent relative mass of 27,000, under reducing conditions. CD1a antigen (Mr 49,000) was borne by the same chain of Mr 49,000 on cortical thymocytes and Langerhans cells, whereas the Mr 27,000 molecule was never found on thymic cells. On two-dimensional gel analysis, the Mr 27,000 molecule showed a pattern with 3 major spots with pI of 5.6, 5.9 and 6.2. This Mr 27,000 protein was found to contain one N-linked oligosaccharide residue by endoglycosidase-F treatment. By sequential immunoprecipitation, this Mr 27,000 molecule was shown to be different from the major histocompatibility complex class II beta-chains (DR, DP). As the Mr 27,000 molecule was not precipitated with OKT6, sequential immunoprecipitation confirmed specific recognition of this low molecular weight protein by other CD1a mAb. The protein of apparent molecular mass 27,000 was considered to be a breakdown product of Mr 49,000 (CD1a) antigen. These results suggested that the CD1a molecule was sensitive to trypsin.

Antibodies, Monoclonal↗
PubMed1989

DMC1: a monoclonal antibody produced from histiocytosis X cells which reacts with the native CD1a molecule of human epidermal Langerhans cells.

Human epidermal Langerhans cells express two (CD1a and CD1c) of the three human thymic cell surface differentiation antigens (CD1a, CD1b, and CD1c). The first cluster of differentiation antigens (CD1) is defined by a group of monoclonal antibodies (MCA). All these MCA were obtained after immunization of mice or rats with human cortical thymocytes. OKT6 MCA (a CD1a MCA) was the first to be described as reactive with human epidermal Langerhans cells. We produced a murine MCA, called DMC1, after immunization with proliferating Langerhans cells of Eosinophilic Granuloma of the bone (Histiocytosis X). In tissues DMC1 MCA reacted with epidermal dendritic cells (Langerhans cells) in the skin and cortical thymocytes in the thymus as observed on indirect immunofluorescence. At the ultrastructural level, DMC1 MCA was specific for Birbeck granule-containing Langerhans cells and did not react with melanocyte and keratinocyte populations. The quantitative analysis of immunoelectron labeling and the cytofluorometric study showed that the intensity of labeling was inversely correlated with the concentration of trypsin used in the preparation of epidermal cell from skin samples. DMC1 MCA precipitated a protein with a relative mass of 49,000 (CD1a molecule) from lysates of iodinated epidermal Langerhans cells under reducing conditions. It recognized the original CD1a molecule (Mr 49,000) but not the membrane breakdown product of CD1a (Mr 27,000) brought about by trypsin.

Animals↗
PubMed1989

Reappearance of CD1a antigenic sites after endocytosis on human Langerhans cells evidenced by immunogoldrelabeling.

We show evidence of the reappearance of CD1a antigenic sites on the surface of human isolated Langerhans cells after internalization of CD1a antigen/CD1a monoclonal antibody (BL6) complexes. The internalization was visualized by immunogoldlabeling, and the reappearance of CD1a binding sites was shown by immunogoldrelabeling. The relabeling was distinguished from the labeling either by using two sizes of gold granules (15 and 5 nm) or by quantitative estimation with one size of gold granules, before and after the relabeling. This reappearance of sites is cycloheximide insensitive, and is evidenced, even if the transfer of gold particles to lysosomes is blocked by the monensin. These results suggest that the reexpression of CD1a antigens is due to antigens stored in the cytoplasm or to recycling of internalized sites. Some immunolabeled Birbeck granules were observed in continuity with the plasma membrane, which demonstrates their membrane origin and their involvement in the endocytosis process. However, the weak labeling of these organelles makes us believe that they are not specialized CD1a endocytosis structures.

Antigens↗
PubMed1989

A surface glycoprotein complex related to the adhesive receptors of the VLA family, shared by epidermal Langerhans cells and basal keratinocytes.

A murine monoclonal antibody, designated K20, was raised by immunization with a human malignant T-cell line. It reacted specifically with membrane glycoprotein complexes on early haematopoietic cells, T cells, and monocytes. In epidermis, K20 specifically reacted with Langerhans cells and basal keratinocytes, as demonstrated by double labeling experiments. Membrane immunoprecipitation analysis demonstrated that the antigen identified by K20 on lymphoid cells and epidermal cells was different. While on lymphoid cells, K20 recognized glycoprotein complexes made of a constant 130-kD subunit associated with subunits of higher molecular weight ranging from 150 to 200 kD, a complex of 105-145 kD was precipitated from Langerhans and basal cells. Metabolic labeling studies demonstrated that these proteins were synthesized by the basal cells. The antigen identified by K20 was thought to belong to the integrins, a family of cell surface receptors that play a role in cell adhesion, cell interactions, wound healing, and immune defense mechanisms. K20 is the first monoclonal antibody that specifically recognizes a membrane antigen common to Langerhans and basal cells. Additionally, K20 is the first of five reported monoclonal antibodies to have been characterized on the epidermal cells that detect antigens shared by lymphoid subpopulations and normal basal keratinocytes.

Antibodies, Monoclonal↗
PubMed1989

Human epidermal basal keratinocytes express CDw29 antigens.

Two monoclonal antibodies, K20 and 4B4, assigned to the CDw29 cluster of differentiation antigens, were shown to react with basal keratinocytes (BK). The aim of this study was to identify the antigens recognized by K20 and 4B4 on epidermal cells, and to determine whether they were identical to those found on lymphocytes. Basal keratinocyte-enriched cell suspensions were labelled with 125I and then 1% NP40 cell lysates were used for immunoprecipitation. Under reducing conditions, K20 and 4B4 immunoprecipitated from basal keratinocytes a broad MW 105,000 band and proteins of MW 145,000, 90,000 and 80,000. Under non-reducing conditions, each band was shifted down by approximately 5000 MW. Metabolic labelling studies demonstrated that the MW 145,000 and 105,000 subunits were synthesized by basal keratinocytes. On lymphoid cells, K20 and 4B4 are known to precipitate glycoprotein complexes made of a broad MW 130,000 protein band (beta subunit) associated with a protein of MW 150,000 (alpha subunit) and proteins of MW 90,000 and 80,000 expressed in very low amounts. The MW 145,000 and 105,000 bands immunoprecipitated by K20 from basal keratinocytes correspond to the alpha and beta subunits present on lymphoid cells. It has recently been demonstrated that K20 recognizes the common beta subunit of the very late antigens family (VLA) and that 4B4 defines the helper-inducer subset of T lymphocytes. The present investigation provides evidence that basal keratinocytes share antigens of the VLA family with lymphoid cells and that they play an important role in the immune response in the skin-immune system.

Antigens, CD↗
PubMed1989

[HIV envelope proteins are bound by human epidermal Langerhans cells by a binding site which differs from the site on the CD4 molecule, and are internalized by receptor endocytosis].

Langerhans cells (LC) are epidermal dendritic cells which express several surface antigens among them the CD4 antigens. We investigated the fate of HIV envelope glycoproteins (gp 120 and gp 160) incubated with healthy human trypsinized LC in suspension. After trypsin treatment only the epitope for OKT4 appeared to be resistant. In absence of antigenic sites identified by OKT4A, Leu 3a or BL4, LC fixed and internalized gp 120 or gp 160 recombinant HIV proteins. This finding support the hypothesis that there exists at the surface of LC a second molecule which may act as a HIV receptor.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

Langerhans cells in S-phase in normal skin detected by simultaneous analysis of cell surface antigen and BrdU incorporation.

We report on a double immunofluorescence staining for the detection of the Langerhans Cell (LC) population in S-phase. After trypsinization, the epidermal cell suspensions were enriched for LC and exposed to 10 microM BrdU for 2 h. Studies of BrdU labeled cells included the determination of their cell surface phenotype. Both membrane labeling and incorporated BrdU as revealed using anti-BrdU demonstrated the BrdU as revealed using anti-BrdU demonstrated the presence of LC in S-phase. We observed 6% of the epidermal LC in S-phase. This is a proof that LC are able to proliferate in the epidermal microenvironment. This technique, besides being rapid and free of radioactivity, allows the cytokinetic study of phenotypically defined LC in heterogeneous epidermal cell populations.

Antigens, Surface↗