Histochem Cell Biol (2008) 129:599–609 DOI 10.1007/s00418-008-0395-x

ORIGINAL PAPER

Galectin-1 induced activation of the apoptotic death-receptor pathway in human Jurkat T lymphocytes Bettina Brandt · Tom Büchse · Ehab Fathi Abou-Eladab · Markus Tiedge · Eberhard Krause · Udo Jeschke · Hermann Walzel

Accepted: 28 January 2008 / Published online: 21 February 2008 © Springer-Verlag 2008

Abstract Galectin-1 (gal-1), a member of the family of
-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 eYciently inhibited by caspase-8 inhibitor II (Z-IETD-FMK) and a neutralizing Fas mAb. Fas could be identiWed as a target for gal-1 recognition as demonstrated by immunoXuorescence 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

B. Brandt · T. Büchse · E. F. Abou-Eladab · M. Tiedge · H. Walzel Medical Faculty, Institute of Medical Biochemistry and Molecular Biology, University of Rostock, Schillingallee 70, 18057 Rostock, Germany E. F. Abou-Eladab Faculty of SpeciWc Education, Mansoura University, Mansoura, 35516 New Damietta City, Egypt E. Krause Leibniz Institute of Molecular Pharmacology, Robert-Rössle-Str. 10, 13125 Berlin, Germany U. Jeschke (&) First Department of Obstetrics and Gynaecology, Ludwig Maximilians University of Munich, Maistrasse 11, 80337 Munich, Germany e-mail: [email protected]

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 Wrst experimental evidence for targeting of gal-1 to glycotopes on Fas and the subsequent activation of the apoptotic death-receptor pathway. Keywords Galectin-1 · T cell apoptosis · Death receptor pathway Abbreviations Bp Base pair BSA Bovine serum albumin CD Cluster of diVerentiation Chaps 3-[(3-Cholamidopropyl)dimethylammonio]1-propanesulfonate DTT Dithiothreitol ECL Enhanced chemiluminescence EDTA Ethylene-diaminetetraacetic acid EGTA Ethylene glycol-bis(2-aminoethylether)N,N,N⬘,N⬘-tetraacetic acid FITC Fluorescein isothiocyanate gal-1 Galectin-1 HEPES N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid IgG Immunoglobulin G HRP Horseradish peroxidase kDa Kilo Dalton LC-MS/MS Liquid chromatography-tandem mass spectrometry mAb Monoclonal antibody NHS N-hydroxysuccinimide NP-40 Nonidet P-40 pAb Polyclonal antibody PIPES Piperazine-N,N´-bis(2-ethanesulfonic) acid

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PAGE PBS PI PtdSer SDS TBS Tris T TUNEL

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Polyacrylamide gel electrophoresis Phosphate-buVered saline Propidium iodide Phosphatidylserine Sodium dodecyl sulphate Tris-buVered saline Tris(hydroxymethyl) aminomethane Tween 20 Terminal deoxynucleotidyl transferase dUTP nick end labelling

Introduction Apoptosis in the immune system is a crucial process for the balance of immune homeostasis, regulating lymphocyte maturation, receptor repertoire selection and lymphocyte functions as growth and diVerentiation. Apoptosis in immune cells is a complex process mostly initiated by a variety of death signals and several downstream signalling pathways (Krammer 2000; Leist and Jaattela 2001). Therefore, a tight regulation of the cell death programme is required as inappropriate cell death contributes to autoimmune diseases, cancer, and the acquired immunodeWciency syndrom. Galectins, a family of evolutionary conserved
-galactoside binding proteins, have gained considerable attention because some members act as ampliWers of the inXammatory response (Rubinstein et al. 2004a), whereas others induce homeostatic signals by downregulation of immune eVector functions (Vespa et al. 1999; Chung et al. 2000). Gal-1, a member of this protein family, exerts immunoregulatory functions by induction of apoptosis in immature cortical thymocytes and in activated T-cells (Perillo et al. 1995, 1997; Pace et al. 2000). Gal-1 also sensitizes resting human T lymphocytes to Fas (Apo-1, CD95)-mediated cell death (Matarrese et al. 2005). When presented by thymic epithelial cells, endothelial cells, dendritic cells, macrophages, and Wbroblasts, gal-1 may be involved in regulation of apoptosis in the thymus during selection and in the periphery following an immune response (Hernandez and Baum 2002; Rabinovich et al. 2002; He and Baum 2004). Gal-1 is exported by an endoplasmic reticulum/Golgi-independent pathway and is then bound to appropriate cell surface glycoconjugates in an autocrine manner (Seelenmeyer et al. 2005). Gal-1 synthesis is strongly upregulated after peptide antigen-induced activation of murine T cells and inhibits antigen-induced proliferation of activated T cells (Blaser et al. 1998). This strongly suggests a potential autocrine suicide mechanism to achieve homeostasis during the termination of an immune response. Gal-1 has also been found in immune privileged tissues such as placenta (Hirabayashi and Kasai 1984), cornea (Allen et al. 1991), and

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testis (Wollina et al. 1999). Thus, gal-1 might contribute to the immune-privileged status by inducing apoptosis in inWltrating lymphocytes to delay the rejection of allografts. Expression of gal-1 has also been well documented in diVerent tumor types where it acts as a negative regulator of T cell activation and survival, a scenario responsible for the immune escape of tumor cells. Notably, blockade of gal-1 expression in vivo promotes tumor rejection and stimulates the generation of a tumor-speciWc T cell-mediated response in syngeneic mice (Rubinstein et al. 2004b). The gal-1 death pathway in T cells is not fully understood but apparently distinct from that mediated by Fas or glucocorticoids (Perillo et al. 1995,1997). It has been demonstrated that gal-1 induces T cell death in a caspase- and cytochrome c-independent manner by nuclear translocation of endonuclease G (Hahn et al. 2004), involves hyperpolarization of mitochondria (Matarrese et al. 2005), downregulation of Bcl-2, and activation of the transcription factor AP-1 (Rabinovich et al. 2000). Gal-1 induced activation of the TCR
/Lck/ZAP-70 pathway was found to be essential to stimulate ceramide release and to trigger the mitochondrial pathway of apoptosis (Ion et al. 2005,2006). In the present work we demonstrate the targeting of gal-1 to glycotopes on Fas and the induction of the apoptotic caspase-dependent death-receptor pathway in human Jurkat T lymphocytes. Gal-1 induced apoptotic DNA fragmentation is preceded by proteolytic processing and activation of initiator caspase-8 and of downstream executor caspase-3.

Materials and methods Materials Annexin V FITC, bovine serum albumin, cellobiose, CHAPS, DTT, EDTA, EGTA, isopropyl-
-D-thiogalactopyranoside,
-lactose, anti-rabbit IgG-HRP, NP-40, HEPES, PIPES, propidium iodide, ribonuclease A, and Tris were from Sigma (Deisenhofen, Germany). Antipain, aprotinin, DNA molecular weight marker 100 bp ladder, leupeptin, pepstatin, pefabloc, proteinase K, and trypsin (sequencing grade) were from Roche Molecular Biochemicals (Mannheim, Germany). FCS, kanamycin, penicillin, streptomycin, RPMI 1640 medium were from Gibco-BRL (Eggenstein, Germany) and ECL detection reagents, Hybond ECL nitrocellulose membranes, NHS-activated Sepharose from GE Healthcare Europe (Freiburg, Germany). The Fas (C-20) pAb and the Fas (C-20) blocking peptide were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and staurosporine, caspase inhibitor VI (Z-VAD-FMK), caspase-3 inhibitor II (Z-DEVD-FMK)), caspase-8 inhibitor II (Z-IETD-FMK), the pET22b(+) expression vector, the TUNEL assay (Apo-BrdU) kit, and NHS-Biotin were from

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Merck Biosciences (Schwalbach, Germany). Caspase-3 pAb, cleaved caspase-3 (Asp175) pAb, cleaved caspase-3 blocking peptide, caspase-8 mAb, the restriction enzymes Nde I and Xho I were ordered from New England Biolabs (Frankfurt, Germany). The neutralizing Fas mAb (clone ZB4, IgG1 isotype) was purchased from Upstate Biotechnology (Lake Placid, NY), the human Fas mAb (clone APO-1-1) from ALEXIS Biochemicals (Lörrach, Germany), and the Fas mAb (clone LOB3/11) was ordered from Serotec (Düsseldorf, Germany). Caspase-Glo 3/7 and caspase-Glo 8 assays, were purchased from Promega (Madison, USA) and Trizol reagent, M-MLV reverse transcriptase, the pCR2.1TOPO vector from Invitrogen (Karlsruhe, Germany). PfuTurbo Cx Hotstart DNA polymerase was ordered from Stratagene-Europe (Amsterdam, The Netherlands) and Rosetta2 (DE3) competent cells were from Merck KGaA (Darmstadt, Germany). Cells The human leukemic T cell line Jurkat (clone E6.1, European Collection of Cell Cultures, Salisbury, UK) was maintained at 37°C and 5% CO2 in RPMI 1640 medium supplemented with 10% FCS and 10 g/ml kanamycin. The human leukemic T cell line MOLT-4 (German Collection of Microorganisms and Cell Cultures, Braunschweig) was cultured in RPMI 1640 medium containing 20% FCS, penicillin (100 milliunits/ml), and streptomycin (50 g/ml). Preparation, immobilization, and biotinylation of recombinant human gal-1 Total RNA was isolated from human placental sections using TRIzol reagent according to the manufacturer`s protocol (Invitrogen). Total RNA (1 g) was reverse transcribed into cDNA using oligo(dT)18 primer and M-MLV reverse transcriptase according to the manufacturer’s instructions (Invitrogen). PCR was performed using PfuTurbo Cx Hotstart DNA polymerase (Stratagene-Europe) with the following primers 5⬘-CTAGTTGTCATATGGCTTGTGGTCTGGTCG-3⬘ (forward) and 5⬘- ACGCGCGA CTCGAGATGGGCTGGCTGATTTCAGTC-3⬘ (reverse) generating restriction sites for Nde I and Xho I. PCR products were initially subcloned into the pCR2.1-TOPO vector using the TOPO-TA cloning technology (Invitrogen). The restriction fragment produced by Nde I and Xho I was further ligated into the isopropyl-
-D-thiogalactopyranoside (IPTG)-inducible expression vector pET22b(+) cutted by the same restriction enzymes. Gal-1 cDNA was veriWed by sequence analysis. Rosetta2 (DE3) competent cells (Merck KGaA) were transformed with the construct. Overexpression of gal-1 was induced with 1 mM IPTG at 37°C. Cells were harvested, washed with PBS, and lysed in

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EDTA-MEPBS (20 mM Na2HPO4, pH 7.2, 150 mM NaCl, 4 mM 2-mercaptoethanol, 2 mM EDTA) by sonication on ice. Gal-1 was puriWed by aYnity chromatography on lactosyl Sepharose 4B as previously described (Walzel et al. 2002). The gal-1 protein was veriWed as a 14 kDa band in silver-stained SDS-PAGE gels. Immobilization of gal-1 to NHS-activated Sepharose and biotinylation with NHS-Biotin were performed as previously described (Walzel et al. 2000). ImmunoXuorescence staining Cytospin preparations from MOLT-4 and Jurkat T-cells were Wxed in acetone (5 min.), dried, wrapped, and stored at ¡80°C. Then the slides were washed and incubated overnight at 4°C with biotinylated gal-1 at 0.3 g/ml PBS, pH 7.4, and with anti-CD95 mouse mAb (clone LOB3/11) at 10 g/ml. After washing, slides were incubated with Cy2labelled rabbit anti-mouse antibody (1:200 dilution) and Cy3-labelled streptavidin. Then the slides were washed and Wnally embedded in mounting buVer (Jeppesen and Nielsen 1998) and examined with an Axiophot photomicroscope (Zeiss, Jena, Germany). For Cy2-labelled antibodies Wlter set 38 (Zeiss) was used with excitation: BP470/40, beamsplitter: FT495 and emission: BP525/50. For Cy3-labelled antibodies Wlter set 20 (Zeiss) was used with excitation: BP546/12, beamsplitter: FT560 and emission: BP57-640. Images were obtained with a digital camera system (Axiocam; Zeiss, Jena, Germany). Separation of cell lysates on gal-1 Sepharose Cells (1.1 £ 108) were sonicated in 0.5 ml EDTA-MEPBS supplemented with 1% NP-40, 1 mM pefabloc, 1 mM DTT, aprotinin, leupeptin, pepstatin, and antipain (each at 10 g/ ml) and incubated on ice for 1 h. The supernatants obtained by centrifugation at 10,000g for 10 min at 2°C were incubated with 200 l gal-1 Sepharose for 2 h in a 0.22 m centrifuge Wlter unit (Costar). Then the beads were washed six times with cell lysis buVer. Bound glycoproteins were eluted from the gel beads with 200 l 0.2 M lactose in cell lysis buVer and the fraction was treated with 100 l of 3-fold concentrated electrophoresis sample buVer for 5 min at 100°C. Analysis of gal-1 Sepharose-bound cell lysate glycoproteins for Fas on blots The blots were blocked with 5% nonfat dry milk in TBST (20 mM Tris/HCl, pH 7.6, 140 mM NaCl, 0.1% Tween-20) for 1 h at room temperature followed by incubation with a Fas (C-20) pAb (1:1,000 dilution) in TBST with 5% BSA for 16 h at 6°C. After washing in TBST, the blots were

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incubated with anti-rabbit IgG-HRP (1:2,000 dilution) in TBST with 5% BSA for 2 h. Bands were visualized by enhanced chemiluminescence (ECL). Protein identiWcation by liquid chromatography-tandem mass spectrometry (LC-MS/MS) Gal-1 Sepharose-bound cell lysate glycoproteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After Coomassie staining, gel lanes between 45 and 50 kDa were cut into nine pieces of equal size. In-gel digestion with trypsin, peptide extraction, and peptide separation were performed as described previously (Czupalla et al. 2006). In brief, LC-MS/MS analysis was performed on a Micromass CapLC liquid chromatography system and a quadrupole orthogonal acceleration timeof-Xight mass spectrometer Q-TOF Ultima (Micromass, Manchester, UK) equipped with a Z-spray nanoelectrospray source. LC-separations were performed on a capillary column (PepMap C18, 3 m, 100 Å, 150 mm £ 75 m i.d., Dionex, Idstein, Germany). Data were acquired in a datadependent mode using one MS scan followed by MS/MS scans of the most abundant peak. The processed MS/MS spectra (MassLynx version 4.0 software) and the MASCOT server version 1.9 (Matrix Science Ltd., London, UK) were used to search against the Swiss-Prot database (Release 53.2). The maximum of two missed cleavages was allowed and the mass tolerance of precursor and sequence ions was set to 0.1 and 0.2, respectively. A protein was accepted as identiWed if the total MASCOT score was greater than the signiWcance threshold and at least 2 peptides appeared the Wrst time in the report and were the Wrst ranking peptides. Measurement of cell associated caspase-3/7 and caspase-8 activity

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in the Wgure legends. Cells were lysed on ice for 1 h in PBS, pH 7.4, supplemented with 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM Na3VO4, 10 g/ml leupeptin, 10 g/ml pepstatin for immunodetection caspase-8 and in 50 mM PIPES/NaOH, pH 6.5, 2 mM EDTA, 0.1% CHAPS, 5 mM DTT, 1 mM pefabloc, 20 g/ml leupeptin, 10 g/ml pepstatin, 10 g/ml aprotinin for immunodetection of caspases-3. After centrifugation of the lysates at 10,000g for 10 min at 2°C, protein concentrations of the supernatants were quantiWed by the Bradford assay using BSA as standard (Bradford 1976). Blots were blocked by incubation with 5% nonfat dry milk in TBST for 1 h followed by incubation with the primary antibody (1:1,000 dilution) in TBST with 5% nonfat dry milk for 16 h at 8°C. After washing, blots were incubated with IgG-HRP conjugates (1:2,000 dilution) in TBST with 5% nonfat dry milk for 2 h. Bands were visualized luminographically on X-ray Wlms using the ECL-Plus detection system. Flow cytometric measurement of phosphatidylserine (PtdSer) expression Gal-1 treated cells and untreated control cells were suspended at 1 £ 106 cells/ml binding buVer (10 mM HEPES/ NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) and double stained by the addition of FITC-annexin V and propidium iodide (PI) each at 1 g/ml (Vermes et al. 1995). The cell suspension was incubated for 10 min in the dark and analyzed on FACSCalibur cytoXuorimeter (Becton Dickinson). FITC and PI Xuorescence were excited by a 488 nm argon laser and detected using the FL1-height and FL3-height dye channels in log mode, respectively. For analysis FITC positive apoptotic cells were distinguished from FITC/PI double positive necrotic cells. DNA extraction and analysis by agarose gel electrophoresis

Caspase-3/7 and caspase-8 activities were measured in cell based assays using luminogenic substrates with tetrapeptide sequences speciWc for caspase-8 (LETD-) and selective for caspase-3/7 (DEVD-). The cells were treated in 96-well plates (2 £ 104/100 l RPMI 1640 medium) with gal-1 or cultured in medium alone for 6 h at 37°C. Then the luminogenic substrates were added in a buVer system optimized for cell lysis, caspase activity, and luciferase activity (Promega). Assay plates were incubated at 22°C for 1 h before recording the luminescence with a microplate luminometer LB 96V (Berthold, Bad Wildbad, Germany). Immunodetection of full length and cleaved caspase-3 and -8 on blots The cells (2 £ 106/ml RPMI 1640 medium) were stimulated with gal-1 at 37°C for the periods of time as indicated

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Degraded low molecular-weight DNA from apoptotic cells was selectively extracted with phosphate-citrate (PC) buVer (Gong et al. 1994). The cells were collected by centrifugation, Wxed in 70% ethanol and were stored at ¡25°C for 16 h. Then the cells were pelleted at 800g for 5 min. Cell pellets (2 £ 106 cells) were suspended in 40 l of PC buVer (0.2 M Na2HPO4 adjusted with 0.1 M citric acid to pH 7.8) at room temperature for 30 min. After centrifugation at 1,000g for 5 min, supernatants were evaporated in a vacuum concentrator 5301 (Eppendorf, Hamburg, Germany) for 15 min. Then 3 l 0.25% NP-40 were added to the cell extract followed by 3 l of a solution of RNase A (1 mg/ ml). After 30 min incubation at 37°C, 3 l of a solution of proteinase K (1 mg/ml) were added and the extract was incubated for additional 30 min at 37°C. Then the extracts were mixed with 12 l loading buVer (0.25% bromophenol

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blue, 0.25% xylene cyanol FF, 30% glycerol) and subjected to 1.5% agarose gel electrophoresis. DNA ladders were visualized by ethidium bromide staining under UV light. TUNEL assay Labelling of DNA breaks with bromolated deoxyuridine triphosphate (Br-dUTP) and identiWcation of Br-dUTP sites by a Xuorescein labelled mAb were performed according to the manufacturer´s protocol (Merck Biosciences). Cells were analyzed on a FACSCalibur Xow cytometer employing a 488 nm argon laser for excitation. FITC Xuorescence was measured using the FL1-height dye channel in log mode. Clumped cells were excluded from analysis by PI staining of the DNA and doublet discrimination based on plotting the FL3-area versus FL3-widht in linear mode.

Results Galectin-1 induces in Jurkat T lymphocytes caspase-dependent DNA-fragmentation Exposure of human Jurkat T lymphocytes to gal-1 induced cell death as demonstrated by a rapid and time-dependent translocation of phosphatidylserine (PtdSer) from the inner to the outer leaXet of the plasma membrane and nuclear DNA-fragmentation (Fig. 1). Flow cytometric measurements of cells stained with FITC-labelled annexin V plus propidium iodide (PI) allowed to discriminate apoptotic cells before loss of cell membrane integrity (FITC+/PI¡), necrotic (FITC+/PI+), and intact cells (FITC¡/PI¡) (Vermes et al. 1995). Treatment of the cells with 40 g/ml gal-1 increased the FITC+/PI¡ apoptotic cell population from 2.8% (control incubation, Fig. 1a, I) to 10.9% after 6 h (II) and to 25.9% after 10 h (III). Cell stimulation with gal-1 in the presence of the disaccharidic competitor lactose (30 mM) decreased the apoptotic cell rate from 10.9% (II) to 3.8% (IV). Staining of these cells with PI did not exceed 1.7% throughout the time course of the experiments. To characterize the functional role of caspases in gal-1 induced T-cell death we investigated the eVects of caspase inhibitors on DNA-fragmentation as PtdSer externalization may occur independently of caspase activation (Leist and Jaattela 2001). In Jurkat E6.1 cells gal-1 and a Fas mAb (clone APO-1-1, IgG1 isotype), (Huang et al. 1999) strongly induced DNA-cleavage into oligonucleosomal-sized fragments when compared to control cells cultured in RPMI 1640 medium alone (Fig. 1b). MOLT-4 cells were resistant to gal-1 and Fas mAb induced DNA-fragmentation. When Jurkat E6.1 cells were pretreated with caspase-8 inhibitor II (Z-IETD-FMK) for 30 min gal-1 stimulated DNA-cleavage was largely attenuated. Caspase-3 inhibitor II (Z-DEVD-

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FMK, 40 M) also inhibited gal-1 induced DNA-ladder formation (data not shown). Gal-1 induced DNA-fragmentation is attenuated by a neutralizing Fas mAb To test whether gal-1 interferes with the Fas death pathway, Jurkat E6.1 cells were exposed to gal-1 in the presence of a neutralizing Fas mAb (clone ZB4, IgG1 isotype) (Zhang et al. 2003). Gal-1-induced DNA-fragmentation was markedly decreased by the Fas mAb, whereas the IgG1 mAb (isotype control) displayed slightly decreasing eVects (Fig. 2a). We further analyzed the inhibitory eVect of the neutralizing Fas mAb on gal-1 induced DNA-breaks by TUNEL assay. As measured by Xow cytometry, the apoptotic rate of control cells (1.9%) increased to 14.5% after gal-1 treatment and decreased to 3.4% in the presence of the neutralizing Fas mAb (Fig. 2b). The apoptotic rate in the presence of the IgG1 mAb (isotype control) was 14.1%. Gal-1 binds to Fas in a carbohydrate-dependent manner The inhibitory eVects of the neutralizing Fas mAb on gal-1induced DNA-fragmentation prompted us to study by immunoXuorescence staining whether gal-1 and a Fas mAb bind simultaneously to Fas (Apo-1/CD95). On Jurkat E6.1 cells (Fig. 3a–c) and MOLT-4 cells (Fig. 3d–f) expression of Fas (panels a and d) and intense binding of gal-1 to the same cells is demonstrated (panels b and e). Triple Wlter exitation clearly demonstrates staining for Fas and gal-1 simultaneously on Jurkat T-cells (Fig. 3c), but not for MOLT-4 cells (Fig. 3f). We further separated whole cell lysates from Jurkat E6.1 and MOLT-4 cells on gal-1 Sepharose. After extensive washing of the beads, the bound fractions were eluted with cell lysis buVer containing 200 mM lactose. Analysis of the gal-1 agarose bound cell lysate fractions on blots with the Fas (C20) pAb revealed a strong band of approximately 45 kDa for Jurkat T-cells (Fig. 3g). When the gal-1 agarose bound fraction from MOLT-4 cells was analyzed a weak band at 47 kDa was generated (Fig. 3g). SpeciWcity for Fas was veriWed by the Fas (C-20) blocking peptide (4 g/ml) which completely inhibited Fas recognition on blots (data not shown). In the next step, the gal-1 Sepharose-bound cell lysate glycoprotein fraction from Jurkat E6.1 cells was separated by SDS-PAGE and the gel slices between 45 and 50 kDa were digested with trypsin, followed by LC-MS/MS. The mass spectrometric analysis conWrmed the presence of Fas (Apo-1/CD95, SwissProt P25445) by MS/MS fragment ions of two tryptic peptides (310IQTIILK316 and 289EAYDTLIK296). Thus, binding of Fas (Apo-1/CD95) to immobilized gal-1 indicates the presentation of appropriate glycoepitopes for gal-1 recognition.

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Fig. 1 Gal-1 induced apoptosis and eVects of caspase inhibitors on Jurkat E6.1 and MOLT-4 cells. FITC-annexin V/PI Xow cytometry (panel A), DNA-fragmentation (panel B). Panel A: Jurkat cells were cultured at 2 £ 106 in RPMI 1640 medium alone (I) or with 40 g/ml gal-1 for 6 h (II), 10 h (III), and with 40 g/ml gal-1 plus 30 mM lactose for 6 h (IV). Cells were stained with FITC-annexin V and PI and subjected to Xow cytometry. The lower left quadrants of each dot plot show the percentage of viable cells, lower right quadrants apoptotic cells, and upper right quadrants necrotic cells. Panel B: Jurkat E6.1 and MOLT-4 cells were incubated in medium alone or preincubated with 40 M caspase-8 inhibitor II (Z-IETD-FMK) for 30 min at 37°C. Then the cells (2 £ 106/well) were stimulated for 6 h with a Fas mAb (clone APO-1-1) or gal-1 as indicated. DNA-extraction and separation in agarose gels was performed as described in Materials and methods. Molecular weight standards (100-bp DNA-ladder) are shown on the left. Shown are representative data from three independent experiments

Gal-1 induces initiator procaspase-8 activation in Jurkat E6.1 cells The interaction of gal-1 with glycoepitopes on Fas raises the question whether gal-1 signalling is mediated by the death receptor forming a death-inducing signalling complex (DISC) (Peter and Krammer 2003). In the DISC, receptors and adaptor proteins associate to signalling protein oligomeric transduction structures (SPOTS) leading to caspase-8 oligomerization and activation. Therefore, we measured the eVects of T-cell stimulation with gal-1 on caspase-8 activity using the speciWc luminogenic substrate

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Z-LETD-aminoluciferin (Thornberry et al. 2000). To reduce nonspeciWc background signals, cell lysis and substrate cleavage were performed in the presence of the peptide aldehyde Z-LLL-CHO (60 M), a potent inhibitor of the proteasome as well as calpains and cathepsins (Wiertz et al. 1996). The eVects of gal-1 on caspase-8 activity of Jurkat E6.1 and MOLT-4 cells are expressed as relative luminescence units (RLU) for the control reactions determining the basal protease activity and for stimulated cultures (Fig. 4a). Gal-1 induced in Jurkat E6.1 cells a concentration-dependent increase of caspase-8 activity. The gal-1 eVect was blocked by lactose, but not by cellobiose

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Gal-1 induced activation of procaspase-8 in Jurkat T-cells is blocked by a neutralizing Fas mAb To get evidence whether gal-1 binding to Fas (Apo-1/ CD95) induces procaspase-8 activation, the cells were incubated with a neutralizing Fas mAb (clone ZB4, IgG1 isotype) or with an IgG1 mAb (isotype control) prior to stimulation with gal-1. Then the cells were analyzed for proteolytic procaspase-8 processing by immunoblotting (Fig. 5a) and caspase-8 activity using Z-LETD-aminoluciferin as a substrate (Fig. 5b). Gal-1 eYciently induced caspase-8 activation as demonstrated by the increase of proteolytic caspase-8 processing and substrate cleavage. The neutralizing Fas mAb reduced the stimulating eVects of gal-1 on caspase-8 activation to the level of control cells in the absence of gal-1. The gal-1 eVect on caspase-8 activation was not blocked by the IgG1 mAb. The experiments strongly suggest that gal-1 recognition of Fas on Jurkat T lymphocytes is associated with caspase-8 activation. Galectin-1 stimulates the processing of eVector procaspase-3

Fig. 2 Inhibition of gal-1-induced DNA-fragmentation in Jurkat E6.1 cells by a neutralizing Fas mAb (clone ZB4) by ethidium bromide staining in agarose gels (panel A) and by TUNEL assay (panel B). Panels A and B: 2 £ 106 cells/well were preincubated with the Fas mAb and an IgG mAb (isotype control) for 20 min at 37°C and then cultured for 6 h with gal-1 or in medium alone (control). Panel A: DNA-extraction and separation in agarose gels was performed as described in Materials and methods. Molecular weight standards (100-bp DNA-ladder) are shown on the left. Panel B: DNA breaks of Jurkat E6.1 cells were labeled with Br-dUTP, stained with a Xuorescein labeled BrdU mAb and analyzed by Xow cytometry. Shown are means § SD from three independent experiments

(Fig. 4a). In MOLT-4 cells, however, gal-1 failed to stimulate substrate cleavage. We also investigated the kinetics of gal-1 induced procaspase-8 activation by immunoblotting. Jurkat T cells were incubated with gal-1 for up to 6 h and analyzed for cleaved and full length caspase-8 (Fig. 4b). After 4 h of gal-1 exposure, caspase-8 p43/41 cleavage products were clearly detectable in cell lysates with a further increase after 6 h. Full length caspase-8 protein expression decreased after 6 h of gal-1 exposure corresponding to the increase of the cleavage products. Notably, the increase of p43/41 occurred prior to the onset of DNA-fragmentation (Walzel et al. 2006). Furthermore, procaspase-8 processing was blocked by caspase-8 inhibitor II (Z-IETDFMK, 40 M) and by lactose at 30 mM (Fig. 4c). Gal-1 failed to induce the proteolytic procaspase-8 processing in MOLT-4 cells.

A proteolytic signalling cascade is extensively used by cells for the activation of the short prodomain eVector caspases, including caspase-3 (Hengartner 2000). For the measurement of caspase-3/7 activity in Jurkat T lymphocytes a luminogenic substrate was applied containing the DEVDsequence, which has been shown to be selective for caspase-3 and -7 (Bayascas et al. 2002). Stimulation of Jurkat E6.1 cells with 20, 40, and 80 g/ml gal-1 resulted in 2.5-, 3.3-, and 4.0-fold increases of substrate cleavage relative to nonstimulated controls (Fig. 6a). Gal-1 induced stimulation of substrate cleavage could be blocked by lactose, but not by cellobiose. Procaspase-3 processing was detectable 4 h after stimulation with gal-1 as demonstrated by 17/19 kDa cleavage products with further increase up to 8 h (Fig. 6b). Caspase-3 cleavage was also initiated in Jurkat T cells by a 2 h stimulation with 1 M staurosporine (data not shown). In gal-1 stimulated cells caspase-3 cleavage products gradually increased with exposure time and expression of full length caspase-3 protein concomitantly decreased when cell stimulation was extended to 6 and 8 h (Fig. 6b). Preincubation of Jurkat E6.1 cells with caspase inhibitor VI (Z-VAD-FMK, 50 M), caspase-3 inhibitor II (Z-DEVD-FMK, 50 M) or cell stimulation with gal-1 in the presence of the disaccharidic competitor lactose (30 mM) strongly attenuated the generation of caspase-3 cleavage products (Fig. 6c). SpeciWcity for caspase-3 cleavage products was veriWed by inhibition of the 17/19 kDa bands in the presence of cleaved caspase-3 blocking peptide (Fig. 6c).

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Fig. 3 ImmunoXuorescence staining of Jurkat E6.1 cells (panels a–c), MOLT-4 cells (panels d–f) and binding of Fas to gal-1 Sepharose as detected by immunoblotting (g). Cells were incubated with a Fas mAb (clone LOB3/11), (sections a and d) or with gal-1-biotin (sections b and e) followed by incubation with a Cy-2 labelled secondary antibody or with Cy-3 conjugated streptavidin. Triple Wlter excitation (sections c and f) for simultaneous staining of Fas and gal-1 binding (yellow col-

our) is indicated by arrows. Panel g: Gal-1 Sepharose bound extract glycoproteins from Jurkat E6.1 and MOLT-4 cells were separated by SDS-PAGE (60 l/lane) and transferred to Hybond ECL membranes. Then the blots were probed with a Fas (C-20) pAb (0.2 g/ml) followed by incubation with an IgG-HRP conjugate. Bands were detected with the ECL-system. Shown are representative data from three independent experiments

Discussion

caspases in gal-1 induced cell death has been identiWed (Rabinovich et al. 2002; Sturm et al. 2004; Matarrese et al. 2005), gal-1 also promotes T-cell death in a caspase- and cytochrome c-independent process (Hahn et al. 2004). It has been shown that MOLT-4 T-cells, resistant to Fas killing (Su et al. 1995), undergo apoptosis from gal-1 treatment (Perillo et al. 1995). Therefore, it was concluded that gal-1 triggered cell death is not mediated by the interaction with Fas (Apo-1/CD95). Although gal-1 induced phosphatidylserine exposure on MOLT-4 cells, no DNA-fragmentation was recorded and the cells continued to grow normally (Dias-BaruY et al. 2003). We also demonstrate that gal-1 treated MOLT-4 cells do not undergo apoptosis and there is no detectable DNA-fragmentation. This contradiction may arise from the very high concentrations of gal-1 (20 M) used for induction of apoptosis when compared to the gal-1 concentrations (1.4–2.7 M) in our experiments. It is conceivable that gal-1 at higher concentrations binds to further receptors and induces diVerent signalling routes. The intracellular pathways of gal-1 induced T cell death also involve hyperpolarization of mitochondria associated with cytochrome c release and procaspase-9 activation (Matarrese et al. 2005), Bcl-2 down-regulation, and AP-1 activation (Rabinovich et al. 2000,2002). Although increased caspase8 activity was measured, caspase-8 activation was not inhibited to prove its essential role (Matarrese et al. 2005).

Although several glycosylated receptors for gal-1 recognition have been identiWed on T cells (Walzel et al. 2000; Elola et al. 2005), the precise roles of each receptor in cell death signalling are unknown. In particular, no direct data were available so far demonstrating Fas as a target structure for gal-1 binding. In the present study we could show an eYcient attenuation of gal-1 induced DNA-fragmentation by a neutralizing Fas mAb and caspase-8 inhibitor II in Jurkat T lymphocytes. The data provide cumulative evidence for the involvement of the Fas-death receptor and of activated caspases in gal-1 signalling. Binding of Fas to immobilized gal-1 strongly argues for gal-1 mediated activation of Fas in a carbohydrate-dependent manner. Therefore, we evaluated whether binding of the homodimeric (proto-type) gal-1 cross-links the Fas receptor and delivers an eVective signal leading to caspase-8 oligomerization and cleavage (Krammer 2000). Gal-1 eYciently induced in human Jurkat T lymphocytes the activation and proteolytic processing of initiator procaspase-8 and downstream procaspases-3. The inhibition of gal-1 induced procaspase-8 activation by a neutralizing Fas mAb strongly suggests that gal-1 induces the activation of the apoptotic death receptor pathway. Thus, gal-1 is able to induce the type I cell death pathway in Jurkat T cells (ScaYdi et al. 1998). Although the role of

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Fig. 4 EVects of gal-1 on procaspase-8 activation and processing in Jurkat E6.1 and MOLT-4 cells. Panel A: 2 £ 104 cells/well were exposed for 6 h at 37°C to gal-1 in the presence or absence of lactose or cellobiose. Caspase-8 activation was measured using the substrate LETD-aminoluciferin (Promega). Data are means § SD from four experiments. Panel B: Jurkat E6.1 cells (2 £ 106/ml) were stimulated with gal-1 for the indicated time periods. For immunoblot analysis cell extract proteins (45 g/lane) were separated by SDS-PAGE. Blots were probed for full length and cleaved caspase-8 with a caspase-8 (1C12) mAb and developed using the ECL-Plus detection system. Panel C: Cells (2 £ 106/ml) were incubated with caspase-8 inhibitor II (ZIETD-FMK) at 37°C for 30 min followed by stimulation with gal-1 or with gal-1 plus lactose for 7 h as indicated. Analysis for caspase-8 cleavage products by immunoblotting was performed as described above

The death mechanisms by gal-1 are highly dependent on the cell type, the cell activation status, and might also be inXuenced by the relative distribution of intracellular versus extracellular gal-1 (Camby et al. 2006). Susceptibility to gal-1 induced cell death is modulated by the expression of speciWc glycosyltransferases and depends upon the glycosylation status of the cells (Amano et al. 2003; Walzel et al. 2006). Addition of 2,6-linked sialic acid to Gal
1-4GlcNAc sequences by ST6Gal I sialyltransferase inhibits gal-1

607

Fig. 5 Inhibition of gal-1 induced proteolytic procaspase-8 processing (a) and activation (b) by a neutralizing Fas mAb. Panel A: Jurkat E6.1 cells (2 £ 106/ml RPMI 1640 medium) and 2 £ 104 cells/0.1 ml (Panel B) were incubated with the Fas mAb (clone ZB4) or an IgG1 mAb (isotype control) for 20 min at 37°C prior to stimulation with gal-1 as indicated. Analysis for caspase-8 cleavage and measurement of caspase-8 activation using the luminogenic substrate LETD-aminoluciferin were performed as described in the legend to Fig. 4. Data are means § SD from four experiments

binding to its preferred disaccharidic recognition structure. Expression of the ST6Gal I enzyme in a gal-1 sensitive murine T cell line resulted in increased sialylation of N-glycans on the CD45 protein tyrosine phosphatase (PTPase), abrogated the reduction in CD45 PTPase activity that results from gal-1 binding (Walzel et al. 1999), and reduced the susceptibility of the cells to gal-1 induced cell death (Amano et al. 2003). Furthermore, deWciency in Lck tyrosine kinase and ZAP-70 kinase abolishes gal-1 induced T cell death, and restoration of enzyme expression restored apoptosis (Ion et al. 2005). Gal-1 induces the acid sphingomyelinase mediated release of ceramide in activated peripheral T-cells and leukemic T-cell lines. Elevated ceramide levels were found to be essential to induce the mitochondrial pathway of apoptosis characterized by decreased Bcl-2 activity, depolarization of mitochondria as well as activation of caspase-9 and caspase-3 (Ion et al. 2006). For triggering of these events Lck and ZAP70 are essentially involved.

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of activated T cells comprise a synergistic action of tumorimmune escape. Acknowledgements This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG, WA 1771/1-1 and JE 181/71). We gratefully acknowledge the excellent technical assistance of Mrs. G. Gaede. We thank M. Schümann for help with the MS measurements.

References

Fig. 6 EVects of gal-1 on procaspase-3/7 activation and procaspase-3 processing in Jurkat E6.1 cells. Panel A: Jurkat E6.1. cells (2 £ 104/ 0.1 ml) were stimulated with gal-1 for 6 h at 37°C in the presence or absence of lactose or cellobiose. Caspase-3/7 activation was measured by a luminometric assay using the substrate DEVD-aminoluciferin (Promega). Data are means § SD from four experiments. Panel B: Cells (2 £ 106/ml) were stimulated at 37°C with gal-1 for the time periods as indicated followed by immunoblot analysis of cell lysates (40 g/lane) for full length and cleaved caspase-3. Blots were developed with ECL-Plus reagents. Shown is a representative blot from three independent experiments. Panel C: Cells (2 £ 106/ml) were preincubated at 37°C for 30 min with caspase inhibitor VI (Z-VAD-FMK) or caspase-3 inhibitor II (Z-DEVD-FMK) followed by stimulation with gal-1 for 7 h in the presence and absence of lactose. Cleaved caspase-3 was detected by immunoblot analysis. SpeciWcity was veriWed by a cleaved caspase-3 blocking peptide

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