Hermann Walzel

Galectin-1, a natural ligand for the receptor-type protein tyrosine phosphatase CD45.

Galectin-1 binds preferentially to N-acetyllactosamine residues on oligosaccharides associated with several cell surface glycoconjugates. In the present work, placental galectin-1 has been identified to be a natural ligand for the receptor-type protein tyrosine phosphatase CD45. The binding of galectin-1 to CD45 was detected by affinity chromatography of NP 40 solubilized Jurkat T cell membranes on galectin-1 agarose followed by immunoblotting of the galectin-1 agarose bound fraction applying monoclonal antibodies to CD45 isoforms. The PTPase activity of the galectin-1 agarose binding membrane fraction could be inhibited by sodium orthovanadate. Preincubation of Jurkat T cell membrane preparations with galectin-1 decreased the membrane-associated PTPase activity in a concentration-dependent manner. Incubation of Jurkat cells with galectin-1 suppressed the immunoprecipitated PTPase activity of CD45. Galectin-1 stimulates the cell surface expression of phosphatidylserine an early indicator of apoptosis. In CD45+ Jurkat T cells, galectin-1 induces higher levels of phosphatidylserine when compared with CD45- Jurkat cells. These observations indicate that galectin-1-mediated ligation of CD45 is involved in the induction of apoptosis in Jurkat T cells.

Binding of galectin-1 (gal-1) to the Thomsen-Friedenreich (TF) antigen on trophoblast cells and inhibition of proliferation of trophoblast tumor cells in vitro by gal-1 or an anti-TF antibody.

Galectin-1 (gal-1), a member of the mammalian β-galactoside-binding proteins, recognizes preferentially Galβ1-4GlcNAc sequences of several cell surface oligosaccharides. We demonstrate histochemically that the lectin recognizes appropriate glycotopes on the syncytiotrophoblast and extravillous trophoblast layer from second trimester human placenta and on BeWo chorion carcinoma cells. Gal-1 binding to BeWo cells was diminished by the Thomsen–Friedreich (TF)-disaccharide (Galβ1-3GalNAc-) conjugated to polyacrylamide (TF–PAA). Gal-1 also inhibited BeWo cell proliferation in a concentration-dependent manner. Similar antiproliferative effects were also observed with an anti-TF monoclonal antibody (mAb, A78-G/A7). Therefore, we conclude that ligation of Galβ1-4GlcNAc and Galβ1-3GalNAc epitopes on BeWo cells may have regulatory effects on cell proliferation.

Human monocyte activation induced by an anti-CD14 monoclonal antibody

An anti-CD14 mAb RoMo-1 rapidly induces in human monocytes a transient oxidative burst activity as detected by chemiluminescence assay. Pretreatment of these cells with the mAb markedly suppresses the monocyte chemiluminescence response to opsonized zymosan. In addition, the antibody induces a significant increase of IL-1 production and secretion by mononuclear cells, comparable to a similar effect of rIFN-gamma or LPS. Electron microscopy demonstrates internalization of the CD14 molecules after interaction with the mAb in a characteristic receptor-like manner.

Galectin-1 induced activation of the apoptotic death-receptor pathway in human Jurkat T lymphocytes

Galectin-1 (gal-1), a member of the family of β-galactoside binding proteins, participates in several biological processes such as immunomodulation, cell adhesion, regulation of cell growth and apoptosis. The aim of this study was to investigate whether gal-1 interferes with the Fas (Apo-1/CD95)-associated apoptosis cascade in the T-cell lines Jurkat and MOLT-4. Gal-1 and an Apo-1 monoclonal antibody (mAb) induced DNA-fragmentation in Jurkat T-cells whereas MOLT-4 cells were resistant. Gal-1 stimulated DNA-fragmentation could be efficiently inhibited by caspase-8 inhibitor II (Z-IETD-FMK) and a neutralizing Fas mAb. Fas could be identified as a target for gal-1 recognition as demonstrated by immunofluorescence staining, binding of the receptor glycoprotein to immobilized gal-1 and analyses by immunoblotting as well as by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gal-1 stimulates the activation and proteolytic processing of procaspase-8 and downstream procaspase-3 in Jurkat-T cells. Inhibition of gal-1 induced procaspase-8 activation by a neutralizing Fas mAb strongly suggests that gal-1 recognition of Fas is associated with caspase-8 activation. Our data provide the first experimental evidence for targeting of gal-1 to glycotopes on Fas and the subsequent activation of the apoptotic death-receptor pathway.

Binding of galectin-1 (gal-1) on trophoblast cells and inhibition of hormone production of trophoblast tumor cells in vitro by gal-1

Galectin-1 (gal-1), a member of the mammalian β-galactoside-binding proteins, recognizes preferentially Galβ1–4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. As demonstrated histochemically, the lectin recognizes appropriate glycoepitopes on the syncytiotrophoblast and on chorionic carcinoma cells (BeWo). Freshly isolated trophoblast cells and trophoblast tumor cells Jeg3 did not bind gal-1. BeWo cells in contrast to Jeg3 form a syncytium in vitro and synthesize progesterone as well as hCG. BeWo cells were used as an approach to study the effects of gal-1 on hormone production. The lectin decreased cellular hCG and progesterone production as well as hCGβ gene transcription as measured by real-time RT-PCR. Gal-1 mediated inhibition of cellular progesterone production was reduced in the presence of a Thomsen-Friedenreich (TF)–polyacrylamide conjugate. Inhibition of cellular hCG and progesterone production was also induced by anti-TF monoclonal antibodies. The results demonstrate that ligation of Galβ1–4GlcNAc and Galβ1–3GalNAc (TF) epitopes on BeWo cells may have regulatory effects on hCG and progesterone production.

Galectin-induced activation of the transcription factors NFAT and AP-1 in human Jurkat T-lymphocytes.

Galectin-mediated ligation of glycoepitopes on T-cell activation markers induces an increase in the cytosolic calcium concentration ([Ca(2+)](i)) originating from a transient Ca(2+) release of internal stores as well as a sustained influx across the plasma membrane. In transiently transfected Jurkat T-lymphocytes, galectins [galectin-1 (gal-1), recombinant human galectin-1 (rec gal-1), nematode 32-kDa galectin (LEC-1), nematode 16-kDa galectin (LEC-6)] differentially stimulate the expression of the luciferase reporter gene constructs pNFAT-TA-Luc and pAP1(PMA)-TA-Luc, which are activated by the nuclear factor of activated T-cells (NFAT) or the transcription factor, activator protein 1 (AP-1), respectively. The galectin-stimulated expression of the reporter constructs is completely inhibited by lactose (30 mM) and asialofetuin (30 microM) carrying Galbeta1-4GlcNAc sequences. Preincubation of pNFAT-TA-Luc-transfected cells with cyclosporine A (0.1 microM) and FK506 (0.01 microM) abrogated the gal-1-induced expression of the reporter luciferase (Luc). Electrophoretic mobility shift assays (EMSAs) provided evidence for gal-1-stimulated increase in the binding of nuclear extracts to a synthetic oligonucleotide with an AP-1 consensus sequence.

Stimulation of Syncytium Formation in vitro in Human Trophoblast Cells by Galectin-1

BACKGROUND Galectin-1 (gal-1), a member of the mammalian beta-galactoside-binding proteins, exerts biological effects by recognition of glycan ligands, including those involved in cell adhesion and growth regulation. In trophoblast cells, gal-1 binds to cell surface glycoproteins (e.g., Mucin1). It has been demonstrated that gal-1 recognizes appropriate glycotopes on the syncytiotrophoblast and extravillous trophoblast from second trimester human placenta and choriocarcinoma cells BeWo, which reveal two coexisting phenotypes, the cytotrophoblast-like and the syncytiotrophoblast-like phenotype. So the aim of this study was to investigate the effect of gal-1 on syncytium formation in BeWo and human villous trophoblasts (HVT) cells. MATERIALS AND METHODS The effect of gal-1 on syncytium formation was investigated with immunocytochemical and double immunofluorescence stainings, cell-labelling and Real-time RT-PCR. BeWo choriocarcinoma and HVT cells were incubated in vitro for 24 and 48 h in the absence (controls) and presence of gal-1 and forskolin and stained with antibodies against Ki67, beta-catenin, E-cadherin and syncytin. BeWo and HVT cells were incubated with 60 microg/ml gal-1 for 48 h (BeWo) or 96 h (HVT) and cell fusion was detected by fluorescent cell-labelling solution. Finally, BeWo cells were incubated for 1 h or 48 h in the absence and presence of 60 microg/ml gal-1 and Real-time RT-PCR was performed. RESULTS We showed with immunocytochemical staining a downregulation of beta-catenin expression in the 24 h BeWo cell culture and with double immunofluorescence staining an inhibition of the beta-catenin and E-cadherin expression in the 48 h BeWo cell culture stimulated with gal-1 or forskolin. The inhibition of E-cadherin was demonstrated on mRNA level in the 1 h BeWo cell culture too. Increased cell fusion was also showed with DiO and DiI fluorescent cell-labelling solution in the 48 h BeWo cell culture. In addition, we demonstrated the downregulation of Ki67 protein expression in the 24 h BeWo cell culture and on mRNA level in the 1 h BeWo cell culture. We also showed the upregulation of syncytin protein and mRNA expression after incubation of the 48 h BeWo cell culture with gal-1 or forskolin. Similar results were obtained with HVT cells: the amount of cell fusion was significantly increased in the gal-1 treated 48 h HVT cell culture in vitro compared to untreated cells as demonstrated with beta-catenin and E-cadherin double immunofluorescence staining. This increase was also shown by fluorescent cell-labelling with DiO and DiI in the 96 h HVT cell culture compared to untreated cells. CONCLUSION Our data suggest that gal-1 stimulates the syncytium formation in choriocarcinoma cells BeWo and HVT cells in vitro and inhibits the expression of beta-catenin, E-cadherin and in addition Ki67 in BeWo cells. Therefore gal-1 may be a major trigger for the process of trophoblast cell fusion.

The Human Endometrium Expresses the Glycoprotein Mucin-1 and Shows Positive Correlation for Thomsen-Friedenreich Epitope Expression and Galectin-1 Binding

Mucin 1 (MUC1) is a glycoprotein in human endometrium and is abundant at the luminal epithelial surface in the receptive phase. It has a highly glycosylated ecto-domain that contains keratan sulfate chains, that disappears at the time of implantation. In addition, the glycoforms on MUC1 differ in fertile and infertile women. Therefore the aims of this study were investigations on glycosylation of MUC1 with the Thomsen-Friedenreich (TF) epitope on normal human endometrium throughout the menstrual cycle and binding of galectin-1 on the TF epitope in the endometrium and the expression of galectin-1 on the human oocyte. Human endometrial tissue was obtained from 54 premenopausal patients and was immunohistochemically analyzed with monoclonal antibodies against MUC1, TF epitope, galectin-1, and biotinylated galectin-1. In addition, human oocytes were analyzed for TF, galectin-1 expression, and galectin-1 binding. We identified a significant upregulation of MUC1 and TF epitope and, in addition, galectin-1 binding in glandular epithelium and epithelial apical surface tissue from proliferative to secretory phase. With double staining experiments, we identified a coexpression of TF and MUC1 in the early secretory phase and galectin-1 binding to TF during the same period of time. In addition we identified TF epitope and galectin-1 expression plus binding on the human oocyte and irregularly fertilized oocytes. Upregulation of TF epitope on the glandular epithelium and epithelial apical surface tissue in the secretory phase and binding of galectin-1 at the same time show the possibility of galectin-1–mediated trophectoderm binding to the endometrium within the window of implantation.

Immunohistochemical and glycohistochemical localization of the β-galactoside-binding S-type lectin in human placenta

The localization of the beta-galactoside binding lectin was studied immunohistochemically on acetone-fixed cryostat sections of full-term placental tissue using a biotinylated monoclonal antibody and glycohistochemically applying biotinylated asialofetuin and lactosylated bovine serum albumin. On blots of placental tissue lysates the lectin is recognized by the biotinylated lactosylated bovine serum albumin. The glycoconjugate recognition of the lectin on blots was inhibited in the presence of 0.1 M lactose showing the specificity of the interactions. The anti-lectin monoclonal antibody stained syncytiotrophoblast and trophoblastic cells. Both reagents applied for glycohistochemistry stained syncytiotrophoblast and trophoblastic cells of placental villi and the trophoblastic layer of extraplacental membranes. A strong uniform cytoplasmic staining was characteristic for syncytiotrophoblast and to a lower extent for cytotrophoblastic cells. The localization of the lectin is discussed with respect to a possible immunosuppressive function.

Are soluble monocyte-derived HLA class II molecules candidates for immunosuppressive activity?

Supernatants of human blood monocyte cultures suppressed PHA responses (IL-2 synthesis, IL-2R expression, DNA synthesis) of autologous and allogeneic lymphocytes. The main suppressive activity was found in the 65-kDa (and 23-kDa) range. It could be incompletely neutralized by mAb specific for a non-polymorphic HLA DR determinant and could also be adsorbed to and eluted from an anti-DR immunoabsorbent column. On blots of monocyte lysates and monocyte culture supernatants, the mAb RoDR recognized antigens of nearly the same Mr. The hypothesis that soluble HLA DR alpha beta heterodimers or beta chains are likely candidates for the suppressor factor was confirmed by analogous effects of purified HLA DR molecules. We favor a model in which soluble MHC class II molecules (in contrast to surface-bound ones) may interfere with the association and cross-linking processes necessary for T cell activation by competing for CD4 binding sites.