Biomedical subjects

H Walzel

Publications and source records attributed to H Walzel.

At least 19 recordsLinked to original sources

PubMed2001

The B-chain of mistletoe lectin I efficiently stimulates calcium signaling in human Jurkat T-cells.

Mistletoe lectin I (ML I), a heterodimeric disulfide-linked type II ribosome inactivating protein, exhibits immunomodulatory potency in stimulating the cytokine release in vitro and in vivo. However, data concerning early activation events in T-cells induced by ML I and its A and B chain preceding cytokine secretion and the receptors involved are of limited availability. Here we show by flow cytometric measurements that human T-lymphoblastoid Jurkat cells express surface glycoprotein receptors for ML I. One of which is shown to be the CD2 antigen involved in a variety of T-cell signaling events. The lectin induces in Jurkat T-cells an increase of the cytosolic calcium concentration ([Ca(2+)](i)) consisting of both, the transient release of Ca(2+) from internal stores and a sustained influx of extracellular Ca(2+). Studies with isolated A- and B-chains provided evidence that the lectin-induced increase in [Ca(2+)](i) is mediated by ML IB. The ML I and ML IB stimulated cellular calcium responses are inhibited by saccharidic competitors. In transiently transfected E6.1 cells ML IB stimulated the expression of the luciferase reporter construct pNFAT-TA-Luc that is activated through the nuclear factor of activated T-cells (NFAT). The ML IB stimulated expression of the reporter luciferase (Luc) is completely inhibited by cyclosporin A (0.2 microM) and by FK 506 at 0.05 microM. Pretreatment of Jurkat E6.1 cells with 1-deoxymannojirimycin (dMJ), an inhibitor of cis-Golgi alpha-mannosidase I, strongly reduced cell binding of ML IB-FITC and the ML IB induced calcium response. Benzyl-alpha-GalNAc, an inhibitor of O-linked glycosylation, has slightly decreasing effects in ML IB-FITC binding and was without effects on the lectin stimulated increase in [Ca(2+)](i). Inhibition of the lectin induced calcium responses by cholera toxin and by inhibitors of protein kinases as well as the absence of calcium responses in CD3- and CD45- Jurkat T-cell clones suggest that ML IB has the potency to induce early T-cell activation events.

Adjuvants, Immunologic↗

PubMed2000

High concentration of soluble HLA-DR in the synovial fluid: generation and significance in "rheumatoid-like" inflammatory joint diseases.

In the search for its role in inflammatory joint diseases, soluble HLA-DR (sHLA-DR) was quantitated in 72 synovial fluids (SF) by a newly established immunoenzyme assay. Unlike other soluble receptors which accumulated only moderately (sCD25, sCD4) or negligibly (sHLA class I, sCD8) in the SF, SF sHLA-DR levels exceeded serum levels by up to 3 orders of magnitude and varied disease dependently from "control" values (traumatic synovitis and osteoarthritis: 9.9 +/- 6.1 ng/ml). Clear-cut different SF sHLA-DR values in HLA-DR-associated "rheumatoid-like" (136.5 +/- 130.0 ng/ml) vs HLA-B27-associated "spondylarthropathy-like" arthritic forms (28.4 +/- 29.1 ng/ml) were most significant comparing oligoarticular juvenile chronic arthritis type I (147.6 +/- 112.6 ng/ml) and type II (3.3 +/- 1.1 ng/ml), thus offering a new classification marker. Also ex vivo, large amounts of sHLA-DR were released spontaneously by SF mononuclear cells and found to be related to the T-cell activation state. SF sHLA-DR may be shed in large complexes or micelles, as it eluted mainly at >450 kDa on gel filtration. Western blotting revealed that the majority of SF sHLA-DR consisted of full-length alpha- and beta-chains. Minor fractions of smaller sized antigens seemed to be generated by proteolytic cleavage rather than by alternative splicing, since only minute amounts of HLA-DRB mRNA lacking the transmembrane exon could be amplified by RT-PCR. Distinct forms of high-dose sHLA-DR, able to provoke rather than to suppress T-cell responses, are discussed as contributing to some HLA-DR disease association.

Adolescent↗

PubMed2000

Involvement of CD2 and CD3 in galectin-1 induced signaling in human Jurkat T-cells.

Galectin-1 (gal-1) a member of the mammalian beta-galactoside-binding proteins recognizes preferentially Galbeta1-4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. In the present work, gal-1 has been identified to be a ligand for the CD3-complex as well as for CD2 as detected by affinity chromatography of Jurkat T-cell lysates on gal-1 agarose and by binding of the biotinylated lectin to CD3 and CD2 immunoprecipitates on blots. In CD45(+)Jurkat E6.1 cells, the lectin stimulates a sustained increase in the intracytoplasmic calcium concentration ([Ca(2+)](i)) consisting of both the release of calcium from intracellular stores and the calcium influx from the extracellular space. This effect of gal-1 on [Ca(2+)](i)is completely inhibited by lactose at 10 mM and was absent in CD45(-)Jurkat J45.01 cells. Preincubation of Jurkat E6.1 cells with cholera toxin or with the protein tyrosine kinase inhibitor herbimycin A reduced the gal-1 induced calcium response whereas the increase in [Ca(2+)](i)stimulated by CD2 or CD3 monoclonal antibodies (mAbs) was completely inhibited. Depolarization of E6.1 cells in a high-potassium buffer, a standard method to activate voltage-operated calcium channels, was without effect on [Ca(2+)](i). Membrane depolarization with gramicidin or by a high-potassium buffer was without effects on the lectin-mediated calcium release from intracellular stores but inhibited the gal-1 induced receptor-operated calcium influx. In Jurkat E6.1 cells the lectin stimulates the transient generation of inositol-1,4,5-trisphosphate and the tyrosine phosphorylation of phospholipase Cgamma1. The results suggest that the ligation of CD2 and CD3 by gal-1 induces early events in T-cell activation comparable with that elicited by CD2 or CD3 mAbs.

Antibodies, Monoclonal↗

PubMed1999

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.

Apoptosis↗

PubMed1999

Influence of inhibitors on increase in intracellular free calcium and proliferation induced by platelet-activating factor in bovine oviductal cells.

Oviductal endosalpingeal cells were isolated mechanically from heifers and cultured until there was 100% confluency. The cells were loaded with the Ca(2+)-sensitive fluorochrome, fura-2/acetoxymethylester, and cytosolic free calcium ([Ca2+]i) was monitored by spectrofluorimetry. Platelet-activating factor, at a concentration of 30 nmol l-1, induced an intracellular Ca2+ increase in cultured bovine oviductal cells, mainly via influx from the extracellular space. In fura-2-loaded oviductal cells, different Ca2+ channel blockers were investigated to characterize the pathways responsible for the Ca2+ influx. The negative effects of Ni(2+)-, La(3+)-activated K+ channel blockers, such as apamin and charybdotoxin, and Ca2+ channel blockers, such as dotarizine, on the platelet-activating factor-induced [Ca2+]i increase indicate the minor participation of the voltage-gated Ca2+ channels. TMB-8 and flufenamic acid blocked the platelet-activating factor-induced Ca2+ increase directly on non-selective cationic channels or acted via a Ca2+ release-triggered Ca2+ influx. Platelet-activating factor, at concentrations of 1.25 mumol l-1 and 2.5 mumol l-1, significantly stimulated the proliferation and depolarization of oviductal cells, but 10 mumol l-1 significantly decreased both parameters and exerted a cytotoxic effect on cells. After incubation with TMB-8 or flufenamic acid, the cell proliferation was inhibited in a concentration-dependent manner, with IC50 values of 26.57 mumol l-1 and 95.29 mumol l-1, respectively. The depolarization was significantly inhibited at 50 mumol l-1 for both TMB-8 and flufenamic acid. The results of the present study may contribute to further understanding of the mechanism behind the actions of platelet-activating factor on oviductal cells.

Analysis of Variance↗

PubMed1998

Detection and functional characterisation of the transcription factor peroxisome proliferator-activated receptor gamma in lutein cells.

A prominent functional change during differentiation of lutein cells from follicular thecal and granulosa cells is an enhanced production and secretion of progestins. The regulation of this process is not fully understood but may be associated with the expression of transcription factors which activate genes, products of which are involved in pathways of the cholesterol and lipid metabolism. As peroxisome proliferator-activated receptors (PPARs) play a role in both pathways, we were interested in the expression of PPARgamma, a PPAR form which is involved in adipogenic differentiation. First, we were able to show the expression of PPARgamma in bovine lutein cells (day 12 of the ovarian cycle) at the mRNA and protein level by imaging, flow cytometry and blot analysis, and secondly a role of PPARgamma in the secretion of progesterone. The cells (24 h culture) responded dose dependently by increasing progesterone secretion (up to 1.5-fold of the basal level) to an endogenous ligand of PPARgamma, 15-deoxy-delta12,14 prostaglandin J2 (15-dPGJ2) and to the thiazolidinedione ciglitizone. Aurintricarboxylic acid (ATA) was found to reduce the intracellular PPARgamma level and to promote cell cycle progress, indicating that ATA can be used as a tool for experimental changes of PPARgamma proteins in intact cells and for studying the physiological consequences. The ATA-mediated decrease of PPARgamma was accompanied by reduced progesterone production and a progression of the cell cycle, suggesting a function of PPARgamma in both processes. The response to ATA was abrogated by a high dose (>490 nM) of 15-dPGJ2, suggesting that 15-dPGJ2 exerts its effect on steroidogenic activity via PPARgamma and that the 15-dPGJ2-PPARgamma system plays a role in the maintenance of a differentiated quiescent stage in lutein cells.

Analysis of Variance↗

PubMed1996

Cell calcium signalling induced by endogenous lectin carbohydrate interaction in the Jurkat T cell line.

The effects of the beta-galactoside-binding lectin from human placenta (HPL14) on intracellular calcium concentration ([Ca2+]i) were examined in the human Jurkat T cell line. The lectin induces a concentration dependent increase in [Ca2+]i. This calcium signalling effect is clearly mediated through complementary cell surface galactoglycoconjugates because it can be blocked by beta-galactosides. The observed Ca2+ - response involves both the release of calcium from intracellular stores and a calcium influx from the extracellular space. It is sustained in the presence of 1 mM extracellular calcium whereas it becomes transient when the influx of extracellular calcium was blocked by calcium chelation to EGTA. Voltage-sensitive calcium channel blockers like verapamil and prenylamine were without effect on the action of HPL14. Protection of the sugar binding activity of HPL14 in the absence of a thiol-reducing reagent by carboxamidomethylation (CM-HPL14) or by substitution Cys2 with serine (C2S) results in lectin proteins with considerably decreased calcium signalling efficiency. The recombinant lectin (Rec H) and the mutant protein obtained by substitution of highly conservative Trp68 with tyrosine (W68Y) induce lower levels of [Ca2+]i compared to wild type lectin.

Binding Sites↗

PubMed1996

Effect of ATP and platelet-activating factor on intracellular calcium concentrations of cultured oviductal cells from cows.

Oviductal endosalpingeal cells were mechanically isolated from heifers at different reproductive stages (cyclic: days 8-14, at oestrus: day 0 and pregnant: day 7) and cultured until 100% confluent. The cells were loaded with the Ca(2+)-sensitive fluorochrome fura-2/AM, and cytosolic free calcium ([Ca2+]i) was monitored spectrofluorimetrically and by use of a microscope image analysis system. ATP (400 mumol l-1) evoked a prompt increase in [Ca2+]i in all cell preparations in both the presence or absence of extracellular Ca2+ when measured with the cuvette method. Single cell measurements using oviductal cells from cyclic heifers revealed a heterogeneous [Ca2+]i pattern in response to ATP, with some cells either failing to respond or reacting very slowly. Platelet-activating factor (PAF, 30 nmol l-1) induced a rapid increase in [Ca2+]i especially in cells derived from cyclic and pregnant animals, but the effect was significantly less in cells from heifers at oestrus. The increase in [Ca2+]i in bovine cells induced by PAF was reduced when extracellular calcium was depleted, indicating that the effect of PAF on [Ca2+]i involves an influx from the extracellular space. Voltage-sensitive calcium channels do not appear to be involved in the influx of extracellular Ca2+ since verapamil had no effect on the PAF-induced increase in [Ca2+]i. The PAF receptor antagonist WEB 2086 inhibited the PAF-mediated effects on [Ca2+]i.

Adenosine Triphosphate↗

PubMed1995

Short-term culture of surface-biotinylated cells: application in non-radioactive analysis of surface protein shedding.

As an alternative to radiolabelling, cell surface biotinylation followed by immunoprecipitation and enhanced chemiluminescence detection can be used to characterize membrane-bound surface proteins. Since many membrane proteins are known to be shed from the surface, we tested whether surface biotinylation can also be used to characterize these soluble proteins released into culture supernatant. First, we found that biotinylated cells could be cultured for up to 7 days without any influence on viability and 3H-thymidine incorporation. Moreover, the PHA response (3H-thymidine incorporation) of peripheral blood mononuclear cells was affected only by high concentrations of the biotinylation reagent N-hydroxysuccinimidobiotin and the LPS response (cytokine release) was unaffected by surface biotinylation. Second, we were able to monitor the shedding of biotinylated HLA class I antigens from Balm 1 cells by immunoprecipitation, SDS-PAGE, and enhanced chemiluminescence detection on blots. Membrane-bound HLA class I antigens disappeared from the cell surface during 5-day culture and, simultaneously, two forms of soluble HLA class I heavy chains (36 and 44 kDa) accumulated in the culture supernatant. Thus, short-term culture of surface biotinylated cells can be used in the analysis of the shedding processes of leukocyte antigens.

Biotin↗

PubMed1995

Immunohistochemical and glycohistochemical localization of the beta-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.

Animals↗

PubMed1994

In vitro effects of solubilized HLA-DR--role in immunoregulation?

To test the hypothesis that soluble HLA-DR antigens (sHLA-DR), binding to the T-cell receptor (TCR) and/or CD4 structures, compete with and abrogate functions of their cell-bound counterparts, we studied effects of detergent-solubilized, affinity-purified HLA-DR molecules on the DNA synthesis, IL-2, and IL-1 secretion by human peripheral blood mononuclear cells (PBMC). While resting T cells did not show any response, there was a dose-dependent suppression of T-cell responses induced by mitogen (phytohemagglutinin, PHA), recall antigen (purified protein derivative of tuberculin, PPD), or HLA class II alloantigens (Daudi cells). In the PHA system, sHLA-DR affected DR-identical and DR-disparate PBMC with equal efficiency, suggesting a nonspecific interference with accessory functions of cell-bound HLA class II molecules. A competitive ligation of lymphocyte (rather than of monocyte) CD4 is suggested based on the failure of sHLA-DR (i) to potentiate inhibitory effects of anti-CD4 mAb, (ii) to suppress mitogen responses after depletion of CD4+ lymphocytes and also following separate monocyte (vs lymphocyte) pretreatment, and (iii) to induce a reproducible IL-1 secretion inhibition. In the PPD system, suppressive sHLA-DR effects on autologous or DR-identical PBMC significantly exceeded that on DR-disparate PBMC, and in the MLR, third party allogeneic sHLA-DR was exceeded in its suppressive potency both by sHLA-DA pertinent to responder cells and by sHLA-DR pertinent to stimulator cells. These additional specific effects may result from competition (with cell-bound class II restriction and recognition determinants) at the TCR level rather than from peptide competition at the antigen-presenting cell level. Interference by sHLA-DR with the primary and/or accessory signaling may offer new therapeutic strategies in allotransplantation and autoimmunity.

Dose-Response Relationship, Drug↗

PubMed1993

Fluorescence microscopic studies and flow cytometric measurements of lectin and hormone binding to isolated rat pancreatic acinar cells.

The binding of fluorescence-labelled lectins and a fluorescence-marked hormone to the cell surface of isolated rat pancreatic acinar cells was studied by light microscopy and flow cytometric measurements. The pancreatic acinar cells were prepared by collagenase digestion. The fluorescence of cells was studied after binding of FITC-labelled WGA or UEA I as well as of FITC-marked pancreocymin/cholecystokinin (CCK-FITC) in a fluorescence microscope or FACScan. The strong binding of lectins was inhibited by preabsorption with the specific sugars. In comparison to the lectins the binding of CCK-FITC was low. There were two populations of acinar cells with different CCK-FITC binding capacity as detected by flow cytometry. The CCK-FITC cell surface fluorescence was significantly decreased by preincubation with unmarked hormone as well as with the non-labelled lectins. The inhibition of CCK-FITC binding by lectins is discussed in respect to a possible competition of the lectins and CCK for the CCK receptor.

Animals↗

PubMed1993

Lectin binding studies with FITC-marked WGA and UEA I and flowcytometric measurements on isolated rat pancreatic acinar cells.

Lectin binding to the glycocalyx of isolated rat pancreatic acinar cells was studied by flowcytometric measurements. The pancreatic exocrine cells were prepared after collagenase digestion and cleaned in a density gradient. The fluorescence of cells was measured in a FACScan after binding of FITC marked WGA or UEA I. The binding of lectins was inhibited by preabsorption of WGA-FITC with N-acetyl-glucosamine, sialic acid or chitinous and by preabsorption of UEA-FITC with alpha-L-fucose, respectively. Furthermore, we were able to measure a decreased WGA-FITC and UEA-FITC binding after a short preincubation of isolated cells with the peptide hormone cholecystokinin and its agonists (caerulein, pentagastrin).

Animals↗

PubMed1992

Comparative studies on internalization of gold labelled mistletoe lectin I (MLI), its subunits, and of an immunotoxin into mouse L 1210V leukemia cells.

Using a preembedding electron microscopic technique, the binding and internalization of gold labelled mistletoe lectin I (MLI.Au), its 2 A subunits (MLI-A.Au) and of the B subunit (MLI-B.Au) in murine L 1210V leukemia cells was analysed. Furthermore, the endocytosis of a gold marked immunotoxin (MoAb-16-MLI-A.Au), consisting of a monoclonal antibody (MoAb-16) reacting with L 1210V cells and the cytotoxic A subunits (MLI-A) was detected. The cells were incubated with MLI.Au, MLI-A.Au, MLI-B.Au, or MoAb-16-MLI-A.Au at 37 degrees C for 1, 3, 5, 10, 20 or 30 min, respectively. Remarkable differences were found in the endocytotic pathway and internalization kinetics. The endocytosis of MLI, its subunits and of the immunotoxin has been compared to that of the other ligands in various systems.

Animals↗

PubMed1992

The activity of two immunotoxins composed of monoclonal antibody MoAb-16 and A-chain of ricin (MoAb-16-RTA) or A-chain of mistletoe lectin I (MoAb-16-MLIA).

The A-chains of ricin obtained from Ricinus communis or mistletoe lectin I from Viscum album were coupled to the monoclonal, anti-L1210V antibody MoAb-16, using SPDP as a cross linking agent. The cytotoxic activity in vitro of these immunotoxins was compared. Each of two immunotoxins tested, applied in vitro for 1 h in appropriate doses, caused irreversible inhibition of leukemic L1210 cells proliferation. Unexpectedly, MoAb-16-MLIA immunotoxin appeared to be cytotoxic to normal bone marrow progenitor cells, as observed in NCFUS tests. Moreover, this immunotoxin revealed cytotoxic effect to the P388 leukemia cells which do not share the antigen, common within L1210 leukemia cells, detected by MoAb-16 antibody.

Animals↗

PubMed1991

Internalization and action of an immunotoxin containing mistletoe lectin A-chain.

An immunotoxin consisting of the enzymatically active A-chain of mistletoe lectin I and a monoclonal antibody against a surface protein on mouse leukemia L1210V cells was found to inhibit protein synthesis in these cells as efficiently as the native mistletoe toxin. The immunotoxin was somewhat more slowly endocytosed than the native toxin, but in both cases the endocytic uptake continued under conditions in which uptake from clathrin-coated pits was inhibited by mild acidification of the cytosol. This indicates that the toxin and the immunotoxin were at least partially internalized by a non-clathrin-dependent uptake mechanism and that uptake by this pathway is responsible for most of the toxic effect on the cells. The results indicate that efficient immunotoxins can be made with antibodies against cell surface epitopes that are endocytosed by a mechanism not involving clathrin-coated pits.

Animals↗

PubMed1991

Binding studies of gold labelled lectins on carbohydrate compounds of the flask cells in claw-frog kidney.

The carbohydrate compounds of the mucus of flask cells in the kidney of claw-frogs (Xenopus laevis) were studied by gold marked lectins (WGA, RCA, L, LCA, HPA, PNA). We used a post-embedding technique. Seminthin or ultrathin sections of Lowieryl K H M-embedded kidney tissue were incubated. For light microscopy, a gold-silver technique was used. The mucus of the flask cells reacted strongly with WGA, RCA L and HPA, whereas LCA and PNA showed no binding. The Golgi apparatus and small cytoplasmatic vesicles reacted also positively with WGA, RCA L and HPA. The autoradiographically detected secretion routes of the glycosaminoglycan-rich secretion of flask cells are also demonstrable by lectins.

Animals↗

PubMed1991

Comparative studies on internalization of gold-labelled mistletoe lectin I, its subunits, as well as of an immunotoxin in murine L 1210 leukemia cells.

The binding and internalization of gold-labelled mistletoe lectin I (ML I), its A (ML I-A) and B (ML I-B) subunits, as well as of an immunotoxin consisting of an monoclonal anti L 1210 antibody and the cytotoxic A chains of ML I, were studied on murine L 1210 leukemia cells by a preembedding electron microscopic technique. We found that receptor-mediated endocytosis differs remarkably between the whole lectin, its subunits, and the immunotoxin. Whereas ML I, its A chain, as well as the immunotoxin are internalized by coated pits/coated vesicles or in combination with uncoated membranes, the B chain is exclusively endocytosed via uncoated deep invaginations of the cell membrane. The endocytosis via clathrin-coated or uncoated vesicles is discussed taking into account the binding and internalization kinetics of ligand-receptor complexes related with the movement by the cytoskeleton.

Animals↗