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Human Galectin-2: Novel Inducer of T Cell Apoptosis with Distinct Profile of Caspase Activation¹

Andreas Sturm^2,*, Martin Lensch, Sabine André, Herbert Kaltner, Bertram Wiedenmann^*, Stefan Rosewicz^*, Axel U. Dignass^* and Hans-Joachim Gabius

^* Medizinische Klinik mit Sektion Hepatologie und Gastroenterologie, Charité-Universitätsmedizin Berlin, Campus Virchow Klinikum, Berlin, Germany; and Institut für Physiologische Chemie, Tierärztliche Fakultät, Ludwig-Maximilians-Universität München, Munich, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Galectin-2 is structurally closely related to galectin-1, but has a distinct expression profile primarily confined to the gastrointestinal tract. Prominent differences in the proximal promoter regions between galectins-2 and -1 concern Sp1-, hepatocyte NF-3, and T cell-specific factor-1 binding sites. Of note, these sequence elements are positioned equally in the respective regions for human and rat galectins-2. Labeled galectin-2 binds to T cells in a -galactoside-specific manner. In contrast to galectin-1, the glycoproteins CD3 and CD7 are not ligands, while the shared affinity to ₁ integrin (or a closely associated glycoprotein) accounts for a substantial extent of cell surface binding. The carbohydrate-dependent binding of galectin-2 induces apoptosis in activated T cells. Fluorogenic substrate and inhibitor assays reveal involvement of caspases-3 and -9, in accordance with cleavage of the DNA fragmentation factor. Enhanced cytochrome c release, disruption of the mitochondrial membrane potential, and an increase of the Bax/Bcl-2 ratio by opposite regulation of expression of both proteins add to the evidence that the intrinsic apoptotic pathway is triggered. Cell cycle distribution and expression of regulatory proteins remained unaffected. Notably, galectins-1 and -7 reduce cyclin B1 expression, defining functional differences between the structurally closely related galectins. Cytokine secretion of activated T cells was significantly shifted to the Th2 profile. Our study thus classifies galectin-2 as proapoptotic effector for activated T cells, raising a therapeutic perspective. Of importance for understanding the complex galectin network, it teaches the lesson that selection of cell surface ligands, route of signaling, and effects on regulators of cell cycle progression are markedly different between structurally closely related galectins.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The increasing appreciation of cell surface glycans to serve as versatile hardware for storage of biological information has led to invigorate work on endogenous lectins (1, 2). They decode the sugar-encoded signals and trigger ensuing responses relevant for diverse cell activities such as proliferation. A hallmark in this research area is the work on the selectins mediating initial contacts between leukocytes and endothelial cells en route to leukocyte recruitment in inflammation (3). The strategic positioning of the sialylated/sulfated Lewis epitopes that act as lectin ligands is matched by a different class of -galactosides that are docking points for another group of tissue lectins, i.e., the galectins (4, 5, 6, 7). Moving from initial in vivo studies in which a homodimeric (prototype) galectin effectively suppressed symptoms of autoimmune disease in two models to observations on the cellular level delineating this lectin as inhibitory (apoptotic) effector for activated T cells (8, 9, 10, 11), galectin-1 (Gal-1)³ has solidly proven its potency to keep inflammatory and autoimmune responses in check in various systems (for review, see Refs. 6 and 12). Its selection of distinct glycoprotein ligands, including CD3, CD7, CD43, and CD45, and its cross-linking ability appear to be crucial for the induction of T cell apoptosis (10, 13, 14, 15, 16, 17). Clinically, susceptibility to Gal-1-induced cell death has recently been pinpointed to contribute to explain selection of CD7-negative T cell clones during progression of the Sézary syndrome and loss of T cells in AIDS (18, 19). Although further questions on the role of Gal-1 in T cell homeostasis are being answered, the emerging insights into the intrafamily diversity of galectins prompt us to address another intriguing issue.

Despite the status of Gal-1 as role model for homodimeric (prototype) galectins, it should not be overlooked that this subgroup is constituted by more than one member (20). In fact, Gal-2 and -7 are also prototype proteins and are expressed by human tumor cells with cell type specificity (21). A recent study on Gal-7 intimated its potential to reduce cell proliferation after carbohydrate-dependent surface binding in neuroblastoma cells, in this system acting as functional homologue of Gal-1 (22). Gal-2 has to date not been tested functionally as a lectin. This present lack of investigations with Gal-2 prompted us to perform this study, using T lymphocytes as target cells. Structurally, Gal-2 shares 43% amino acid sequence identity with Gal-1 (Fig. 1), and the analysis of expression in rat tissues and human tumor cell lines had revealed its presence to be confined to the gastrointestinal tract (21, 23, 24, 25). Thus, Gal-2 is likely to encounter T cells, especially in inflammatory bowel disease, posing the clinically relevant question concerning an immunomodulatory capacity. After all, no previous report had to date provided information on functional overlap with or divergence from Gal-1. In our study, we answer the questions as to whether Gal-2 can bind to T cells and trigger apoptosis. Effects on parameters of mitochondrial integrity and caspase activation within the intrinsic pathway, cell cycle distribution, and expression of proteins regulating cell cycle progression as well as cytokine secretion, integrin expression, and cell adhesion of T cells were analyzed. Moreover, we also initiate analysis of Gal-7 as additional effector of classical T cell apoptosis and, to start with, scrutinize the proximal promoter regions for clues to explain the distinct expression profiles of Gal-1 and -2.

View larger version (34K): [in this window] [in a new window]   FIGURE 1. Intergalectin and interspecies sequence comparison of the subgroup of mammalian prototype galectins including Gal-1, -2, and -7. Amino acid sequences of human Gal-1, -2, and -7 as well as rat Gal-2 were aligned using the program Multalin (http://prodes.toulouse.inra.fr/multalin/multalin.html; version 5.4.1). Identical residues found in all four galectins are indicated as white letters on black background, whereas residues that are identical or similar between at least two of the sequences are in black letters on gray background. A consensus sequence calculated from the four galectin sequences is added to the alignment; consensus symbols represent: !, I or V; and #, N, D, Q, or E (A). The same program was used to calculate the phylogenic relationships between these galectins and to visualize them in a family tree diagram (B).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

CD3 mAb (OKT3; Janssen-Cilag, Neuss, Germany), PMA (Sigma-Aldrich, Taufkirchen, Germany), and PHA (Invitrogen Life Technologies, Karlsruhe, Germany) were used for T cell activation. The broad-spectrum caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) was purchased from BIOMOL (Hamburg, Germany), and caspase-1 inhibitor Z-Tyr-Val-Ala-Asp(Ome)-Ch₂F (zYVAD-fmk), caspase-3 inhibitor Z-Asp-Glu-Val-Asp(OMe)-fluoromethylketone (zDEVD-fmk), caspase-8 inhibitor Z-Ile-Glu-Thr-Asp(OMe)-fluoromethylketone (zIETD-fmk), and caspase-9 inhibitor Z-Leu-Glu(Ome)-His-Asp-(Ome)-Ch₂F (zLEHD-fmk) were obtained from Calbiochem (Schwalbach, Germany). FITC-labeled anti-cyclin B1 and PE-labeled anti-active caspase-3 were purchased from BD Pharmingen (Heidelberg, Germany), and CD3 PE-, CD4 PE-, CD8 FITC-, CD3 PE-, FITC-, and PE-labeled polyclonal anti-mouse IgG preparations from DakoCytomation (Hamburg, Germany). Secondary FITC-labeled goat anti-mouse IgG was purchased from BioSource International (Solingen, Germany), and allophycocyanin-labeled streptavidin was obtained from Caltag Laboratories (Burlingame, CA). The carboxyfluorescein (FAM) caspase detection kits, measuring caspase activity, were obtained from Biocarta (San Diego, CA). Lactose, sucrose, cycloheximide, and rhodamine 123 were purchased from Sigma-Aldrich, and propidium iodide (PI) from Calbiochem. All protease- and phosphatase-specific inhibitors used in Western blotting were obtained from Sigma-Aldrich. The Abs against human caspase-3, DNA fragmentation factor (DFF), poly(ADP-ribose) polymerase, retinoblastoma (Rb) protein, cyclin A, p21, p27, and p53 were purchased from BD Pharmingen. Abs against human Bax, Bcl-2, and cytochrome c were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The cytofluorometric bead array kit was purchased from BD Pharmingen, and IFN-, IL-10, and IL-2 ELISA from R&D Systems (Wiesbaden, Germany). For flow cytometry and blocking experiments, anti-human ₁ integrin mAb (clone FB12), anti-human ₂ integrin mAb (clone P1E6), anti-human ₃ integrin mAb (clone P1B5), anti-human ₄ integrin (clone P1H4), anti-human ₅ integrin (clone P1D6), anti-human ₁ integrin (clone P4C10), anti-human ₂ integrin (clone 3E1), and anti-human ₄ integrin (clone ASC-9) were used (Chemicon International, Temecula, CA).

Preparation of galectins

cDNAs for human and rat Gal-2 were cloned from mRNA pools of HT-29 colon carcinoma cells or rat duodenum, respectively. The introduction of a NcoI restriction site resulted in a Thr(Ser)²Ala substitution in the corresponding protein sequences. For recombinant expression, the pQE-60 vector system (Qiagen, Hilden, Germany) was used, and lectin purification was performed with affinity chromatography on lactosylated Sepharose 4B, obtained by ligand coupling after divinyl sulfone activation, as crucial step (26, 27). Homogeneity and quaternary structure were ascertained by gel filtration and one- and two-dimensional gel electrophoresis. Gal-1 and -7 were prepared, as described previously, and the galectins were biotinylated under activity-preserving conditions by an optimized procedure, label incorporation quantitated by two-dimensional gel electrophoresis, and maintenance of carbohydrate-binding activity ascertained by solid-phase assays (28, 29, 30). Polyclonal Abs were raised in rabbits, and cross-reactivity to related galectins was excluded by ELISA and Western blotting (31, 32).

Sequence alignment and search for transcription elements

Genomic sequences of human Gal-1, human Gal-2, and rat Gal-2 were taken from the National Center for Biotechnology Information GenBank database (www.ncbi.nlm.nih.gov/GenBank/index.html), reformatted, and edited using Genetics Computer Group (Accelrys Sequence Analysis Software Package, San Diego, CA) programs available under the Heidelberg Unix Sequence Analysis Resources biocomputing service of the German Cancer Center (http://genius.embnet.dkfz-heidelberg.de/). A search for potential transcription factor binding sites was performed using the factor algorithm based on a selection of 429 consensus transcription factors from the Transfac database version 3.5 (hereby enabling interspecies comparisons). Amino acid sequences were aligned using the program Multalin (http://prodes.toulouse.inra.fr/multalin/multalin.html; version 5.4.1).

Preparation of T lymphocytes

PBMC from healthy volunteers were isolated from heparinized venous blood using Ficoll-Hypaque density gradients. For isolation of peripheral blood T lymphocytes (PBT), PBMC cells were incubated for 30 min at 4°C with magnetically labeled CD19, CD14, and CD16 Ab directed against B lymphocytes, monocytes, and neutrophils, respectively (Miltenyi Biotec, Bergisch-Gladbach, Germany). T cells were then collected using a magnetic cell-sorting system (MACS; Miltenyi Biotec). Lamina propria T cells (LPT) were isolated from surgical specimen obtained from patients subjected to bowel resection for malignant and nonmalignant conditions of the large bowel, including colon cancer and benign polyps, as previously described (33). Briefly, the dissected intestinal mucosa was freed of mucus and epithelial cells in sequential washing steps with DTT and EDTA, and digested overnight at 37°C with collagenase and DNase. Mononuclear cells were separated from the cell suspension by running a Ficoll-Hypaque density gradient centrifugation. For LPT purification, macrophage-depleted lamina propria-derived mononuclear cells were incubated for 30 min at 4°C with magnetically labeled beads, as described above, and collected by negative selection using the MACS system. As assessed by flow cytometry, the purified PBT and LPT populations contained >99% and >92% CD3⁺ cells, respectively.

Polymerase chain reaction

Total RNA was isolated using the Advantage RT-PCR kit (BD Clontech, Heidelberg, Germany), according to the manufacturer’s instructions. Aliquots (2.5 µg) of total RNA were reverse transcribed, essentially as previously described (21), and Gal-2-specific mRNA was amplified using the following set of primers: sense, 5'-ATGACGGGGGAACTTGAGGTT-3' and antisense, 5'-TTACGCTCAGGTAGCTCAGGT-3'. The thermal cycle commenced with a hot start at 94°C for 4 min, followed by 36 cycles, each consisting of 94°C for 60 s, annealing for 60 s at 60°C, and extension at 72°C for 120 s, and terminated after a final 10-min period at 72°C. The products were separated on a 1% Tris-acetate/EDTA agarose gel and visualized by ethidium bromide staining under UV light.

Western blotting

For immunoblotting, cells were washed in PBS and lysed in cell lysis buffer (1% Triton X-100, 0.5% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 5 mM EDTA, 50 mM protease and phosphatase inhibitor mixtures, 1 mM PMSF, 100 µg/ml trypsin-chymotrypsin inhibitor, and 100 µg/ml chymostatin in PBS). The concentration of proteins in each lysate was measured using the Bio-Rad protein assay (Bio-Rad, München, Germany). Equivalent amounts of protein (10 µg) were fractionated on a 10–20% Tris-glycine gel and electrotransferred to a 0.2-µm nitrocellulose membrane (Invitrogen Life Technologies). Membranes were blocked with 5% milk in 0.1% Tween 20/PBS (Fisher Scientific, Schwerte, Germany), followed by incubation for 60 min at room temperature with the indicated primary Ab. The membranes were washed six times with 0.1% Tween 20/PBS and then incubated for 1 h with the appropriate HRP-conjugated secondary Ab (Santa Cruz Biotechnology), washed, and incubated with the chemiluminescent substrate (PerkinElmer Life Sciences, Rodgau-Jügesheim, Germany) for 5 min. The membranes were then exposed to an x-ray film (Amersham, Freiburg im Breisgau, Germany).

Flow cytometric analyses

For surface staining, cells were washed twice in ice-cold PBS, and 1 x 10⁶ cells were resuspended in flow buffer (HBSS containing 1% BSA and 0.1% sodium azide). Cells were incubated with the respective mAb at predetermined saturating concentrations or with isotype-matched nonspecific mouse mAb (DakoCytomation) for 30 min at 4°C, washed twice with flow buffer, and incubated with FITC-labeled goat anti-mouse IgG for 30 min at 4°C. The cells were again washed twice with flow buffer, fixed in 1% paraformaldehyde, and analyzed by a single laser flow cytometer (FACSCalibur), using CellQuest software (BD Pharmingen). Cells stained with a negative control Ab were gated to contain <2% positive cells. To perform analysis of intracellular proteins, cells were washed twice with PBS, adjusted to 1 x 10⁶ cells per sample, and fixed in 90% methanol at –20°C. After fixation, cells were washed twice with PBS and incubated with a monoclonal mouse anti-Bax, Bcl-2, Rb, cyclin A, p21, p27, or anti-p53 Ab for 45 min at 4°C, followed by washing and an incubation step with a goat anti-mouse FITC-labeled mAb (BioSource International) for 45 min at 4°C. Thereafter, cells were washed and analyzed by flow cytometry, as described above. The background level of immunofluorescence was determined by incubating cells with FITC-labeled mouse IgG (isotype control).

Determination of binding of biotinylated Gal-2

To measure Gal-2 binding to T lymphocytes, aliquots of cell suspensions were kept as controls or stimulated for 1, 6, 12, 24, 48, and 72 h with anti-CD3 mAb or PMA and PHA and incubated in the presence of 5 µg/ml biotin-labeled Gal-2. Cells were then harvested, washed, and stained with anti-CD3 FITC mAb and allophycocyanin-labeled streptavidin (BioSource International), followed by flow cytofluorometric analysis. Cells cultured in the absence of biotin-labeled Gal-2 and stained with FITC-labeled mouse IgG (BD Pharmingen) or allophycocyanin-labeled streptavidin served as isotype controls. Each analysis was performed on at least 20,000 events.

Magnetic separation of Gal-2-containing complexes

For separation of Gal-2-associated complexes, 1 x 10⁸ tosyl-activated supermagnetic polystyrene beads (Dynalbeads; Dynal Biotech, Oslo, Norway) were coated with either 350 µg of BSA, Gal-1, Gal-2, CD7, ₁ integrin, or OKT3 overnight at 37°C under tilt rotation. After incubation, the beads were washed in 0.2 M Tris buffer (pH 8.5 containing 0.1% BSA) and then incubated with 2 x 10⁶ PBT for 1 h at 37°C. Free cells were removed by extensive washing, and cells bound to the beads were subsequently treated with homogenization buffer (20 mM Tris-HCl, pH 7.6, 10 mM MgCl₂, 0.05% Triton X-100, and 50 mM phosphatase and protease inhibitor mixtures). The extract was resuspended in SDS/DTT protein loading buffer, heated to 95°C for 5 min, and subjected to SDS-PAGE electrophoresis.

Determination of extent of apoptosis and necrosis

Apoptosis was determined by monitoring fragmentation of nuclear DNA and access of annexin V to cell surface phosphatidylserine. To detect fragmentation of nuclear DNA, T cells were cultured in the presence and absence of the respective galectins, with or without blocking Abs or further activation for 48 h. Thereafter, cells were fixed in 0.2 ml of 37% formaldehyde for 10 min at room temperature. The cells were then treated with 1 ml of PBS containing 0.2% Nonidet P-40 (Sigma-Aldrich) for 2 min at room temperature and then rinsed once in PBS. The apoptotic cells were detected by staining of nuclear DNA with 4',6'-diamidino-2-phenylindole (DAPI) (Calbiochem). DAPI was added at a concentration of 0.2 ng/ml PBS, and cells were incubated at room temperature for 20 min in this solution. Cells were rinsed twice in PBS, and the coverslips were maintained cell side down on a microscope slide and analyzed using a Zeiss Axiovert 135M microscope (Carl Zeiss, Oberkochen, Germany). To detect early phases of apoptosis, access to phosphatidylserine was measured. Cells were cultured as described, harvested at the respective time points, stained with FITC-labeled annexin V and PI, and analyzed by flow cytometry using the CellQuest software program (BD Pharmingen). A minimum of 15,000 cells was monitored in each case.

Determination of caspase activity

To measure caspase activity, 5 x 10⁵ PBT were incubated in the presence or absence of 50 µg/ml Gal-2 together with the PE-labeled affinity-purified anti-caspase-3 Ab (BD Pharmingen), the carboxyfluorescein-labeled caspase-8 inhibitor FAM-LETD-fmk, or caspase-9 inhibitor FAM-LEHD-fmk (Biocarta), all binding irreversibly to activated caspases-3, -8, or -9, respectively. Cells were then analyzed by flow cytometry (FACSCalibur), and the increase of caspase activity was determined after proper gating in comparison with untreated cells.

Assessment of mitochondrial membrane potential

Because accumulation of rhodamine 123 is proportional to and de-energizing of the mitochondria decreases its fluorescence (34), we accordingly measured the mitochondrial membrane potential of PBT. Cells were stimulated in the presence or absence of Gal-2 with CD3 for 24 h, harvested, washed, and resuspended in 1 ml of rhodamine 123 (10 µg/ml; Sigma-Aldrich) for 30 min at 37°C in the dark. The samples were washed twice in cold PBS, and fluorescence analysis by flow cytometry using an argon ion laser with an emission filter at 530 nm was performed immediately without fixation (FACSCalibur; Beckman Coulter, Fullerton, CA). PBT without stimulation and unstained samples were used as controls.

Analysis of T cell cycling

Flow cytometry was performed after staining for DNA content and cyclin B1, essentially as previously described (33). Briefly, cells were washed twice with PBS, adjusted to 1 x 10⁶ cells/sample, and fixed in 90% methanol at –20°C. After fixation, cells were washed twice with PBS and incubated for 45 min at 4°C with a cyclin B1-specific FITC-labeled mAb. Following washes to remove free Ab, cells were resuspended in PBS containing 5 µl of RNase (0.6 µg/ml, 30–60 Kunitz U; Sigma-Aldrich), incubated at 37°C for 15 min, and then chilled on ice. A total of 125 µl of PI (200 µg/ml) was added before analysis by flow cytometry. Each analysis was performed on at least 25,000 events.

Measurement of cytokine secretion

To determine cytokine secretion, PBT were cultured for 48 h with or without anti-CD3 mAb and incubated in the presence or absence of 0, 10, or 50 µg/ml Gal-2. The supernatant was then collected, and cytokine secretion was determined by a cytometric bead array (CBA), performed according to the manufacturer’s instruction (BD Pharmingen). Briefly, six bead populations with distinct fluorescence intensities, coated with capture Abs specific for TNF-, IFN-, IL-10, IL-5, IL-4, and IL-2 proteins, were mixed with PE-conjugated detection Abs and incubated with recombinant standards or test sample to form sandwich complexes. Following acquisition of sample data using flow cytometry, the cytokine concentrations were calculated using the BD CBA analysis software. A control was added to examine whether Gal-2 may have cytokine-binding properties beyond its recently proven affinity for lymphotoxin- (35).

Adhesion assay

Ninety-six-well flat-bottom plates were precoated overnight at 4°C with 12 µg/well fibronectin (Chemicon International) in Dulbecco’s PBS (DPBS), 40 µg/well collagen type I (Sigma-Aldrich) in 0.1 M acetic acid, or 3% BSA (Sigma-Aldrich) in DPBS as a control, and then washed three times with DPBS. Freshly isolated T cells were made fluorescent by incubation with 5 µM calcein-AM (Molecular Probes Europe BV, Leiden, The Netherlands) for 30 min at 37°C in 5% CO₂ at 5 x 10⁶ cells/ml in RPMI 1640 medium. The cells were then washed three times with RPMI 1640 medium containing 5% FCS. Calcein-labeled T cells were resuspended in RPMI 1640, and 5 x 10⁵ T cells/well were added to 96-well plates containing purified extracellular matrix components or BSA for the indicated periods of time. Saturating concentrations of integrin-blocking mAbs (predetermined by flow cytometry) were preincubated with T cells for 30 min at 37°C before adding T cells to the wells for the adhesion assay. Nonadherent cells were removed by a standardized washing technique that was developed to minimize background binding, which includes both an orbital and a rocking motion repeated three times with galectin-free RPMI 1640 medium. Extent of adhesion was quantified with a multiwell fluorescent spectrophotometer (Tecan, Groedig, Austria). For each experimental group, the results were expressed as the mean percentage ± SD of bound T cells from triplicate wells.

Statistical analysis

Statistical analysis was performed using paired Student’s t test. Results are expressed as mean ± SEM, and significance was inferred at p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sequence comparisons

Alignment of amino acid sequences illustrates the close similarity between these prototype galectins (Fig. 1A). When looking at the calculated family tree, the identity scores that reach 67% between human and rat Gal-2 and 43% between human Gal-1 and -2 translate into two subgroups separating Gal-1 and -2 vs Gal-7 (Fig. 1B). These data argue in favor of a close structural and, presumably, functional relationship between Gal-1 and -2, an issue to be addressed by our experiments. To provide evidence for presence of putative regulatory elements responsible for the marked difference in the expression profiles of Gal-1 and -2, which adds to the analysis performed previously (24), we used a database with 429 consensus transcription factors for scrutinizing the proximal promoter regions. The shared presence of two consecutive TATA boxes and equally located Sp1, hepatocyte NF (HNF)-3, and T cell-specific factor (TCF-1) binding sites in the proximal promoter regions of rat and human Gal-2 relative to human Gal-1 are prominent features (Fig. 2). The detected differences in presence or positioning of defined regulatory elements have potential relevance to contribute to explain the differential expression profiles. They also underscore an elaboration within the promoter region most likely to be leading to a histologically strictly defined production of Gal-2, seen in rat tissues in the gastrointestinal tract (25). It should be mentioned that our applied strategy failed to detect the AP-1 (positions 267–272) and the Sp1 (positions 180–186) binding sites in the upstream region of human Gal-2, inferred previously (24). Applying a list of 1115 human transcription factors instead of the consensus table (of note, there is sequence variation in the definition of an Sp1 site between the two data listings), presence of that Sp1 site could be confirmed. However, the proposed AP-1 site could not be delineated by either strategy. Regarding the promoter comparison, the search using the human database raised evidence for presence of three additional Sp1 sites in the proximal promoter region of human Gal-1. They are located at positions 55–65 (two overlapping sites) and 143–150 (identical with the GCF binding site found with the consensus approach; see Fig. 2). Again, these sites were not found in the Gal-2-specific sequences. To address the question as to whether human Gal-2 might be present in T cells, we performed RT-PCR and Western blotting.

View larger version (35K): [in this window] [in a new window]   FIGURE 2. Intergalectin and interspecies comparison of proximal promoter regions of mammalian Gal-1 and -2. Respective sequence information of human Gal-1 (hGal-1; accession, Homo sapiens chromosome 22 contig NT_011520; region, 17380467–17380777) and -2 (hGal-2; accession, H. sapiens chromosome 22 contig NT_011520; region, complement of 17284952–17285292), as well as rat Gal-2 (rGal-2; accession, Rattus norvegicus chromosome 7 supercontig NW_047780; region, complement of 11420257–11420597) was processed. Translation start codons are given in italics and underlined. CAAT and TATA boxes are in bold letters. Putative binding sites for transcription factors are marked according to their occurrence: , indicate sites that occur in all three sequences; , indicate sites that occur only in two sequences; and framed , indicate sites that are unique for the actual sequence. The depicted sites for transcription factor binding are: : hGal-1, Sp1 binding site (positions 196–205), TCF-1 binding site (236–240)/hGal-2, Sp1 binding site (72–82), TCF-1 binding sites (206–212 and 290–294)/rGal-2, Sp1 binding site (78–86), TCF-1 binding site (208–212). : hGal-1, PuF binding site (240–246)/hGal-2, C/EBP binding site (3–7), PuF binding site (117–123), HNF-3 binding site (151–157), Brn-2 binding site (239–248)/rGal-2, C/EBP binding sites (17–24 and 91–95), HNF-3 binding site (155- 161), Brn-2 binding site (239–248). : hGal-1, GCF binding sites (143–150 and 200–206; note that the latter overlaps almost completely with the Sp1 binding site), AP-2 binding site (157–165), E2A box (289–298)/hGal-2, CCCTC-motif-binding factor binding site (40–44), c-Ets-1 binding site (99–106)/rGal-2, AP-3 binding site (16–23), GATA-1, -2, -3 binding site (197–202), c-Myb binding site (306–311).

As inherent quality control in these experiments, we performed the analysis of Gal-2 in parallel with Gal-1 and used the intestinal epithelial cell line IEC-6 in addition to T lymphocytes. The loading control with a housekeeping gene, i.e., GAPDH, ascertained that the experimental requirements were fulfilled, and Gal-1-specific mRNA was detectable at the calculated molecular size (Fig. 3A). Protein expression was pronounced in the epithelial cells and especially in stimulated T lymphocytes (Fig. 3B). In contrast, Gal-2 was not expressed in T cells, IEC-6 cells serving as positive control (Fig. 3). These experiments revealed a qualitative difference in the expression of the two homodimeric prototype galectins. To proceed to define functional characteristics, we first addressed the question as to whether Gal-2 will bind to T cells, a prerequisite to elicit any regulatory role.

View larger version (81K): [in this window] [in a new window]   FIGURE 3. Differential patterns of expression of Gal-1 and -2 in T cells. Gal-1 is consistently detected in resting and activated PBT on the level of mRNA (A) and protein (B), while Gal-2 expression was not detectable in PBT or LPT (A and B). Cells were cultured in the absence and presence of cross-linked anti-CD3 mAb for 72 h. mRNA expression was determined by PCR analysis, and protein presence by Western blotting. The nontransformed intestinal epithelial cell line IEC-6 served as positive control. The graphs are representative of three individual series of experiments.

For this purpose, we biotinylated the lectin under activity-preserving conditions and verified maintenance of the carbohydrate-binding specificity by solid-phase assays (data not shown). Labeled Gal-2 bound to unstimulated T cells in a -galactoside-specific manner, because presence of 50 mM lactose as pan-galectin inhibitor reduced binding by >70% (Fig. 4). Sucrose tested at the same concentration failed to affect the staining intensity in flow cytometry, excluding nonspecific effects (data not shown). To figure out whether and how stimulation of T cells might modify reactivity of Gal-2, we used either PMA/PHA or anti-CD3 Ab. In both cases, we found an increase in cell staining (Fig. 4). Evidently, Gal-2, which is not expressed in T lymphocytes, can bind to carbohydrate ligand(s) on the T cell surface. The extent of binding is increased by cell stimulation. Compared with Gal-1, binding of Gal-2 appeared to be slightly stronger. To gain insight into the biochemical nature of cell surface targets for Gal-2, we used the Ab-blocking approach targeting defined cell surface glycoproteins and used Gal-1 as internal control.

View larger version (49K): [in this window] [in a new window]   FIGURE 4. Binding of Gal-2 to T cells without and with stimulation. PBT were cultured in the presence of cross-linked anti-CD3 mAb or PMA/PHA for 24 h, and biotinylated Gal-2 was used to monitor ligand presentation. An isotype control was added to exclude nonspecific triggering, and the haptenic sugar lactose was added to ascertain carbohydrate-dependent binding. Binding of Gal-2 to T cells was determined by two-color flow cytometry using allophycocyanin-labeled streptavidin as marker. A comparison using control cells and CD3 with biotinylated Gal-1 as marker is shown in the right part of the bottom section. The graph is representative for three to five individual series of experiments.

Gal-2 associates with ₁ integrin, but not CD3 or CD7

Any interference on galectin binding to stimulated T cells by the presence of epitope-specific Abs was quantitated by flow cytometry. Cell surface binding of Gal-1 was expectably reduced by Abs against CD3, CD7, and ₁ integrin, respectively, intimating an interaction with the Ag concerned or a determinant in close vicinity (data not shown). Gal-2 binding, however, was not significantly influenced by the presence of reagents specific for CD3 and CD7 (Fig. 5A). Despite the high sequence alignment score, Gal-1 and -2 actually have distinct preferences for glycoproteins as ligands. In contrast, the Ab against ₁ integrin was a potent inhibitor, markedly reducing cell staining (Fig. 5A). As control, the Ab against ₂ integrin failed to inhibit Gal-2 binding to T cells (data not shown). Relative to presence of lactose, the comparison of the two profiles intimates that ₁ integrin appreciably contributes to the overall ligand panel. Thus, these experiments add a clear difference in ligand selection to the differences in the expression profile. In addition to the blocking approach, we ran Western blotting on the fraction of extract glycoproteins with ligand capacity for galectins. In the case of Gal-1, presence of CD3, CD7, and ₁ integrin was predicted by the cytofluorometric results. Indeed, we could detect these glycoproteins after an affinity fractionation of extract with immobilized Gal-1 (Fig. 5B). Remarkably, a complete correlation for cytofluorometric and biochemical results was also found for Gal-2: no trace of CD3 or CD7, but the presence of ₁ integrin could be visualized by the Western blots in the fraction of Gal-2-binding glycoproteins (Fig. 5B). A BSA control ascertained the absence of nonspecific protein-protein interactions (data not shown). Despite their conspicuous structural relationship, the two galectins bind distinct glycoproteins, and Gal-2 homes in on ₁ integrin or a tightly associated glycoprotein as a major target. Having described the cell-binding capacity of Gal-2 and detected absence of reactivity to CD3 and CD7 by two independent approaches, we next asked the question as to whether cell binding might induce apoptosis.

View larger version (37K): [in this window] [in a new window]   FIGURE 5. Gal-2 binds to 1 integrin, but not CD3 or CD7. PBT were preincubated with CD3, CD7, and 1 integrin-specific mAb (1/100 dilution) for 1 h at room temperature and then cultured for additional 24 h in the presence of 5 µg/ml biotinylated Gal-2. PBT were then harvested, and binding of Gal-2 to T cells was determined by two-color flow cytometry using allophycocyanin-labeled streptavidin as marker (A). For biochemical analysis, PBT were incubated with Gal-1- or -2-coated tosyl-activated beads, pelleted, and subsequently lysed. Bead-bound cell fragments were separated magnetically and immunoblotted for detection of CD3, CD7, or the 1 integrin. Gal-2 shows no evidence for interaction with CD3 and CD7 by coimmunoprecipitation, but forms a complex with 1 integrin, whereas Gal-1 coimmunoprecipitates with CD3, CD7, and 1 integrin (B). The illustrated blots are representative for four individual experiments yielding very similar results.

To control our assay system with standard annexin V staining and the level of reactivity of cells, we took advantage of the well-described activity of Gal-1 as internal control. Activated (but not resting) T cells underwent galectin-dependent induction of apoptosis. Having validated the experimental conditions, Gal-2 was tested. It turned out to be an effector that was stronger than Gal-1 (Fig. 6A). PI staining quantitated the extent of necrotic cells, which reached 10% of the cell population, whereas annexin V positivity was triggered in close to 60% of the cells (Fig. 6A). The rat homologue was in this respect effective in an extent similar to human Gal-2 (data not shown), substantiating their vicinity in the calculated family tree (Fig. 1B). The time course of the reaction with Gal-2 followed a gradual increase over 72 h (Fig. 6B). As with Gal-1, resting T cells were not responsive. Regarding CD4/CD8 characteristics, there was no difference in the reactivity pattern (data not shown). Because 50 mM lactose was effective to impair binding of the lectin, we reasoned that cell viability should remain unaffected when blocking carbohydrate-dependent binding. This was indeed the case, while sucrose presence proved inert (data not shown). Treatment with 10 nM and 1 µM cycloheximide additionally indicated that abolishment of de novo protein synthesis did not impair induction of apoptosis. Of note, presence of the ₁ integrin-specific Ab, which reduced cell binding of Gal-2, did not significantly reduce the extent of apoptosis. Having documented the activity of Gal-2 as proapoptotic effector, we set forth to address the issue as to whether caspases are involved and, if positive, which enzymes are crucial.

View larger version (19K): [in this window] [in a new window]   FIGURE 6. Induction of T cell apoptosis by Gal-2 and comparison with the respective activity of Gal-1. Analysis of annexin V binding and PI staining revealed that Gal-2 is more potent to induce T cell apoptosis and necrosis than Gal-1, and that cell activation was required to induce Gal-2-mediated cell death (A). PBT were cultured with 0, 10, 25, 50, or 100 µg/ml Gal-1 or -2, respectively, in the presence or absence of cross-linked anti-CD3 mAb for 24 h. Induction of T cell apoptosis by Gal-2 is time dependent (B). PBT were cultured with 50 µg/ml Gal-2 in the presence of cross-linked anti-CD3 mAb for the indicated periods of time. Extents of apoptosis and necrosis were assessed by annexin V and PI staining, respectively. Data represent mean ± SEM of six to seven individual experiments. *, p 0.05 for increase vs control (no presence of galectin); +, p < 0.05 for effect of Gal-2 vs that of Gal-1.

We determined caspase activities in Gal-2-treated, anti-CD3-activated PBT using fluorogenic substrate assays. The fluorescence profiles given in Fig. 7A show that there indeed is an increase in the activity of certain caspases. It was specific in the test panel for caspases-3 and -9, whereas no signal alteration was seen in caspase-8 assays. To confirm and extend this observation and pinpoint functional relevance, we systematically studied the effect of caspase inhibition by broad-spectrum and specific caspase inhibitors (tested at a constant concentration of 50 µM). The broad-range inhibitor zVAD proved to strongly interfere with the proapoptotic capacity of Gal-2 on stimulated T cells. Participation of caspases was thus independently confirmed. Neither the caspase-8 (zIETD)- nor the caspase-1 (zYVAD)-directed substances affected Gal-2-dependent apoptosis induction (Fig. 7B). Regarding caspase-8, the complete agreement between both assays is noteworthy. The fluorometric assays had given reason to expect caspases-3 and -9 to be important. Fittingly, the respective inhibitors were effective to reduce cellular annexin V positioning to a level close to that reached in the presence of the pan-caspase inhibitor. Having documented the pattern of caspase activation by Gal-2, we proceeded to include Gal-1 in aliquots of our cell preparations comparatively. The ability of zVAD to reduce Gal-1-induced cell death provided evidence that caspases are involved in Gal-1-mediated apoptosis (Fig. 7B). A conspicuous difference was the marked effect of the caspase-8 inhibitor (Fig. 7B). Gal-1-induced apoptosis thus has its own profile with involvement of caspases-3, -8, and -9. Assays with Gal-7, another homodimeric prototype galectin, similarly revealed proapoptotic activity of the stimulated T cells. Inhibition of caspases-1, -3, and -8, but not of caspase-9, reduced its extent. Evidently, caspase involvement is different among the prototype subgroup of galectins, warranting further study. In this study, we focus on the pathway of inducing apoptosis for Gal-2. Given the involvement of caspase-9 in mediating the apoptotic response to Gal-2 exposure, it appeared likely that Gal-2 makes use of the intrinsic pathway, a suggestion testable by looking at the mitochondrial membrane potential. Thus, we next proceeded to analyze this parameter exploiting the redistribution of rhodamine 123.

View larger version (34K): [in this window] [in a new window]   FIGURE 7. Gal-2-mediated apoptosis is dependent on caspases-3 and -9. Gal-2 induces activity of caspases-3 and -9, but not of caspase-8 (A). PBT were incubated in the presence or absence of 50 µg/ml Gal-2 for 24 h, and caspase activity was assessed by flow cytometry, as described in Materials and Methods. Illustrated plots are representative for four independent experiments, which yielded very comparable results. The pan-caspase inhibitor zVAD and the caspase-3 inhibitor (zDEVD) impaired the activity of Gal-2, -1, and -7 to induce cell death. The caspase-1 inhibitor zYVAD blocked only Gal-7-induced cell death, whereas the caspase-9 inhibitor zLEHD blocked Gal-2- and -1-induced apoptosis. The caspase-8 inhibitor zIETD blocked Gal-7- and -1-induced cell death (B). Data represent mean ± SEM of four independent series of experiments. *, p < 0.05 for increase vs control (no presence of galectin); +, p < 0.05 for decrease vs galectin activity (100%).

The staining profile with the dye that accumulates in the mitochondrial membrane was monitored. It was markedly affected by Gal-2 exposure of activated T cells (Fig. 8A). The mitochondrial polarization potential was significantly lowered compared with control cells. Fittingly, cytochrome c release was enhanced, exclusively observed in activated, but not resting cells (data not shown). Thus, mitochondrial parameters characteristic of the intrinsic pathway and ensuing caspase-9 activation were positively modulated by Gal-2. To further describe the range of Gal-2 effects in this pathway, we included an analysis on Bax expression, a protein capable of forming ion-conducting channels through the lipid bilayer of the inner mitochondrial membrane. To enable delineation of a shift in the proportions between proapoptotic activities such as Bax and antiapoptotic proteins, we also looked at Bcl-2 expression. Illustrated by flow cytometry and Western blotting, Gal-2 treatment of activated T cells actually had a strong effect on these two parameters, in perfect accord with our data on mitochondrial parameters and caspase involvement (Fig. 8, B and C). Bax expression was increased; Bcl-2 expression was reduced. The overall Bax/Bcl-2 ratio was increased, favoring proapoptotic functionality. Because DAPI staining visualized strong nuclear DNA fragmentation, an ensuing monitoring of the regulation of caspase-3-dependent proteolytic cleavage of the DFF was included. Gal-2 treatment indeed led to increased presence of the product of proteolytic activation (Fig. 8C), adding further support to Gal-2 exploiting this pathway. To extend the analysis of Gal-2-dependent effects on cell growth, it is notable that resting T cells, despite binding capacity, were not responsive. The activity profile of Gal-2 on cycling cells may thus also include modulation of cell cycle parameters.

View larger version (31K): [in this window] [in a new window]   FIGURE 8. Gal-2 disrupts the mitochondrial membrane potential, decreases the level of Bcl-2, but increases the level of Bax expression and induces cleavage of the DFF. PBT were cultured for 48 h in the presence of 0, 25, or 50 µg/ml Gal-2, respectively. Rhodamine 123 staining demonstrates a reduction of the mitochondrial membrane potential by Gal-2 (A). Flow cytometry (B) and Western blotting (C) show that Gal-2 profoundly reduced antiapoptotic Bcl-2, while increasing proapoptotic Bax and inducing cleavage of the DFF. Gel loading was controlled by monitoring a housekeeping gene product (GAPDH). The given plots are representatives of three individual series of experiments.

Using DNA staining, we performed an analysis of the cell cycle parameters of PBT in response to Gal-2. When gated on the viable cell fraction, cell cycle phase distribution was comparable in anti-CD3-activated PBT cultured in the absence or presence of 25 and 50 µg/ml Gal-2 (Fig. 9A). Fittingly, independent analysis of a set of five key regulatory proteins that are crucial for cell cycle progression by Western blotting revealed no alterations. In detail, protein levels neither of the cell cycle promoters cyclin D2, Rb protein, or cyclin A, nor the cell cycle inhibitors p21, p27, and p53 were changed (Fig. 9B). Instead of [³H]thymidine incorporation as a means to study DNA synthesis, we checked presence of cyclin B1, a salient effector for cycle progression. Its cytofluorometric determination, shown in Fig. 10, gave no clue for a Gal-2-dependent up- or down-regulation, underscoring a lack of effect of this particular galectin in this respect.

View larger version (45K): [in this window] [in a new window]   FIGURE 9. Gal-2 does not alter T cell cycling. The number of PBT in S or G2/M phases is comparable regardless of the presence of Gal-2 (A). PBT were cultured without or with 25 or 50 µg/ml Gal-2 and activated by cross-linked anti-CD3 mAb for 72 h. Cell cycle phases were assessed by measuring the DNA content by PI staining, followed by flow cytometry. Each illustrated panel is representative of five individual series of experiments. The extent of presence of cell cycle regulators is not altered by the presence of Gal-2 (B). PBT were cultured without or in the presence of 25 or 50 µg/ml Gal-2 and activated with cross-linked anti-CD3 mAb for 72 h, after which the level of protein expression was assessed by Western blotting. Each given series of blots is representative of at least three individual series of experiments.

View larger version (38K): [in this window] [in a new window]   FIGURE 10. Gal-1 and -7, but not -2, inhibit G2/M cell cycle phase progression. Flow cytofluorometric analysis revealed that Gal-1 and -7 decrease cyclin B1 expression in the G2/M phase of activated PBT in contrast to Gal-2. PBT were cultured in the presence of cross-linked CD3 mAb for 72 h, after which cyclin B1 expression and DNA content were quantitatively examined by flow cytometry. The given plots are representative for four different experiments.

Gal-2 distinctively modulates cytokine secretion

Toward this end, we performed a CBA using cytokine capture beads, followed by flow cytometry. Gal-2 failed to modulate cytokine production of resting T cells. Activation of T cells with anti-CD3 mAb profoundly up-regulated IFN-, TNF-, IL-10, IL-5, and IL-2 production (Fig. 11). In activated T cells, Gal-2 down-regulated significantly and dose dependently IFN- and TNF- production, while increasing IL-5 and IL-10 secretion (Fig. 11). Due to the recently documented interaction of Gal-2 with the TNF superfamily member lymphotoxin- (35), we performed controls to test whether this galectin might also interact with other cytokines, especially TNF-, hereby affecting the accuracy of the measurements. Using the series of standard dilutions in the absence and in the presence of up to 50 µg of Gal-2/ml, we obtained no evidence for interference in determining TNF- and IFN-, supporting the validity of this assay for cytokine assessment in the presence of Gal-2 (data not shown). IL-4 secretion did not change upon T cell activation and was not altered by presence of Gal-2. In contrast, IL-2 secretion was up-regulated by cell activation, but, as could be expected in view of the lack of a Gal-2-dependent effect on T cell cycling, it was not influenced by presence of Gal-2 (Fig. 11). Having hereby examined cell cycle and cytokine parameters, we finally turned to analyze Gal-2-dependent effects on the capacity of the treated cells to recruit integrins as mediators of adhesion and outside-in signaling.

View larger version (17K): [in this window] [in a new window]   FIGURE 11. Gal-2 modulates profile of cytokine secretion by activated T cells. The effect of Gal-2 on cytokine secretion of resting and stimulated PBT was determined for IFN-, TNF-, and IL-10, -5, -4, and -2 by a commercially available CBA using standards. Data represent mean ± SEM of three independent experiments. +, p < 0.05 for data sets with stimulated T cells vs resting T cells treated with 50 µg/ml Gal-2; *, p < 0.05 for data sets for Gal-2 treatment on stimulated T cells vs T cells treated with CD3.

Treatment of the activated T cells had a bearing on integrin reactivity to Abs, a measure of accessibility. ₁ integrin staining by the Ab was reduced, an effect interestingly not seen for LPT, a source for memory T cells (Fig. 12), although Gal-2 binding to LPT can be reduced by 30% in the blocking approach, as seen with PBT. The surface accessibility of ₅ integrin was also diminished, while ₄ integrin reactivity was only slightly changed and the ₁ integrin-dependent signal even up-regulated (Fig. 12). For comparison, identical experiments were performed with Gal-1, which revealed quantitative differences (Fig. 12). The question as to whether this galectin-dependent effect with an Ab as tool to measure integrin accessibility might modulate the capacity of the cells to interact with integrin-binding partners of the extracellular matrix was next addressed. We studied cell adhesion in an assay with collagen type I and fibronectin as matrix. Besides blocking crucial binding sites, the bivalent Gal-2 might also bridge glycan chains of the matrix and surface glycoproteins, rendering ambivalent effects on adhesion feasible.

View larger version (25K): [in this window] [in a new window]   FIGURE 12. Galectin presence modulates cell surface presentation of integrins. Presence of Gal-1 and -2 (bold line) reduces cell surface presentation of integrin subunits 1 and 5 comparably, does not affect 4, and slightly increases 1 cell surface presence in PBT. In LPT, Gal-1 and -2 fail to modulate cell surface presence of 1 integrin. The dotted line indicates staining by an isotype Ab as control. PBT and LPT were incubated in the presence or absence of 50 µg/ml Gal-1 or -2, respectively, and cell surface presence of integrins was assessed by flow cytometry. Negative control cells were gated to contain <3% positive cells. Representative histograms of three individual series of experiments are shown.

View larger version (31K): [in this window] [in a new window]   FIGURE 13. Gal-2 affects T cell adhesion to collagen type I and fibronectin in a characteristic manner compared with Gal-1 and -7. Gal-2 inhibits PBT and LPT adhesion to collagen type I, but increases T cell attachment to fibronectin. Gal-1 is generally inhibitory, and Gal-7 does not significantly alter T cell adhesion to collagen or fibronectin (A). Blocking mAb for integrins inhibit Gal-2-mediated modulation of cell adhesion to collagen type I and fibronectin (denoted as integrin + gal) (B). Preincubation of T cells with Gal-2 before adding 1 integrin-specific mAb (denoted as gal + 1) reversed the inhibitory effect of the 1 integrin-specific mAb. PBT and LPT were labeled with calcein, and 5 x 105 cells/well were allowed to adhere for 2 h to a BSA-coated plastic surface as a control and to collagen type 1 or fibronectin as substrata. Nonadherent cells were removed by washing. The fluorescence intensity of adherent T cells was quantified using a fluorescence spectrophotometer. Data represent mean ± SEM of five independent experiments. *, p < 0.05 for data sets with galectins vs control (0 µg/ml Gal-2); +, p < 0.05 for data sets vs control (25 µg/ml Gal-2).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Starting from its detection in leukocytes and malignant T cells and the 10-fold up-regulation of production of its mRNA in a T leukemic line (CEM) during glucocorticoid-induced apoptosis (36, 37, 38), Gal-1 has become a role model for investigating effects of human lectins on T cell homeostasis by carbohydrate-dependent cell binding. The strategic positioning readily accessible for contact on the cell surface and the potential for swift remodeling of carbohydrate determinants, e.g., by reversible 2,6-sialylation acting as molecular switch to turn off ligand capacity toward galectins, convey attractivity to glycan chains for use in cell communication. Because today recombinant production ensures access to human lectins, this broad hypothesis can be thoroughly tested for individual cases step by step. Three motives prompted this study: first, the accruing insights into the complex network of galectins make it likely that T cells will encounter more family members in the body than Gal-1. Thus, this interaction system is furnished with the hardware for fine-tuning. Second, the analysis of negative growth regulation of SK-N-MC neuroblastoma cells has indicated overlapping and divergent functionalities of Gal-1, -3, and -7 with direct competition for a ligand, i.e., the pentasaccharide of ganglioside GM₁ (22, 39, 40). Last, but not least, any galectin capable of restoring T cell homeostasis offers to let patients benefit from turning potent endogenous regulators in inflammation and autoimmune disease into a new class of therapeuticals. In view of frequent occurrence of inflammatory bowel diseases and the fact that no functional study has to date been performed on gastrointestinal Gal-2, we set out to address pertinent issues in Gal-2 expression, binding properties, and immunological activity in a stepwise manner. The lessons drawn from our analysis are listed in the following paragraphs.

The establishment of the family tree for the homodimeric prototype Gal-1, -2, and -7 reflected close sequence relationship between human and rat Gal-2 and the high degree of sequence similarity to Gal-1. At an increased distance and in its own branch, Gal-7 was found. What has been known to be conspicuously different about Gal-1 and -2 were their expression profiles with rather ubiquitous presence for Gal-1, also known as galaptin (41), vs a pattern confined to a gastrointestinal location for Gal-2. Our sequence alignments of the proximal promoter regions and the database-supported search for occurrence of binding sites for transcription factors spotted several clues toward explaining this regional disparity in protein presence. To ascribe relevance of the detected differences, we added analysis of rat Gal-2 in this respect, because it shares restricted tissue expression with human Gal-2 relative to human Gal-1. We also excluded presence of Gal-2 in peripheral blood and tissue-residing T cells (PBT, LPT) in contrast to Gal-1. Cells of an intestinal epithelial line served as positive control to preclude a false-negative result.

Because galectins are commonly secreted to make them available to cells in the microenvironment and plant lectins such as PHA prove that cells can veritably be reactive with agglutinins without synthesizing them, we next tested T cell binding of Gal-2. Resting and mitogen-stimulated T cell populations were positive, a prerequisite for triggering any cell biological effects. Mitogenic activation influenced cell binding only to a small extent. Notably, competition with lactose, but not sucrose, was not only possible, but even nearly complete, attributing binding to specific lectin-carbohydrate recognition. The ensuing delineation of distinct glycoprotein targets on the cell surface was performed by two independent techniques, i.e., competition of surface binding of Gal-2 by target-specific Abs, as successfully tested previously (14), and Western blotting of the galectin-binding fraction. Besides CD3 and CD7, which had been indicated to be ligands for Gal-1 with implications for inhibiting proliferation and inducing apoptosis of human and murine T cells (13, 14, 42, 43), we also delineated ₁ integrin as potential target. Owing to identification of ₁₁ and ₇₁ integrins and the ₁ chain itself as Gal-1 ligands in smooth muscle cells, a role of Gal-1 in modulation of crucial matrix interactions had been postulated (44, 45, 46). Further analysis strengthened the connection between galectins and integrins. The binding of ₃₁ and _M (CD11b) integrins by the tandem-repeat-type Gal-8 on human nonsmall cell lung cancer cells and neutrophils and the ensuing regulation of cell adhesion and survival or superoxide generation, respectively, attest the inherent specificity in the interplay between integrins and galectins (47, 48). Because there is the possibility for special, cell type-associated integrin glycosylation, the cited results should not automatically be extrapolated. Thus, it is necessary to run parallel assays with the tested galectins in the same cell system to reach a firm conclusion on target specificity. Evidently, Gal-1 retained ₁ integrin on coated beads, and, corroborating the close structural similarity, Gal-2 assays yielded the same results. Exploiting a ₁ integrin-specific Ab to block access for Gal-2, this glycoprotein (and/or a spatially closely neighboring binding partner) was found to be a major site on the cell surface for interacting with Gal-2. When proceeding in our analysis, Gal-2 was found to harbor distinctive features. Of notable interest, CD3 and CD7 were not reactive. The remarkably nonrandom association to only a distinct set of suitable -galactosides was underscored by our parallel experiments with Gal-1. These positive controls and the application of two independent assays reliably excluded a false-negative result. Thus, despite very similar sequences, especially around the carbohydrate binding site with its central Trp residue and conservation of the set of essential residues, the ligand fine specificities are qualitatively different for these two targets.

To offer explanations for this difference, it is instructive to consider the current notion about levels of specificity for galectins when interacting with natural ligands. First, the essential set of hydrogen bonds/stacking and interaction profile with an amphiphilic surface is provided by the -galactoside lactose (or N-acetyllactosamine), the pan-galectin ligand. As our results intimate, the natural variety of -galactoside derivatives with substitutions on the core structure appears to matter for galectins when selecting endogenous binding partners. By inhibition assays, isothermal titration calorimetry, or frontal affinity chromatography, it became evident that naturally occurring oligosaccharides indeed differ in ligand capacity for human galectins (49, 50, 51). A combined nuclear magnetic resonance-spectroscopical and modeling study provided the first insights into the way a complex ligand, in this study the pentasaccharide of ganglioside GM₁, makes contacts to the binding pocket beyond the primary site accommodating the galactose moiety (52, 53). Having established this experimental strategy, it will eventually be possible to relate distinct sequence differences in galectins that have a bearing on the architecture of the binding site to affinity alterations. A further factor to be reckoned with for avidity is the density of ligand presentation, either in bi- up to penta-antennary N-glycans, in branched O-glycans, which come about in core 2 structures, or in mucins with repeated display, e.g., of core 1 antennae. By analogy, the intriguing target specificity of selectins is similarly reasoned to hinge on a combination of structural and topological features (3). In our field, assays with naturally occurring glycoproteins are beginning to deliver the desired information (54, 55, 56). Finally, the density of presentation can also affect the conformational equilibrium of carbohydrate determinants and thereby their affinity, as carbohydrates are endowed with capacity to fluctuate between bioactive and bioinert shapes (2, 53). Equally important, conformational aspects of a galectin can be affected by ligand binding, documented for Gal-1 in solution by small angle neutron scattering (57), and this factor’s influence on the cross-linking activity of a galectin will have to be defined rigorously, too. Our result thus gives further analysis of the distinctive binding properties of Gal-2 reason and direction. It also has an immunologic meaning. The lack of reactivity to CD7 (and also CD3) not only revealed different fine specificities between Gal-2 and Gal-1. Due to the role of CD7 in Gal-1-induced apoptosis of activated T cells and Sézary cells (13, 18, 58), this result casted doubt on Gal-2 being an inducer of T cell apoptosis. Nonetheless, we tested Gal-2.

Our respective assays documented a strong activity of Gal-2 (human and rat) on mitogen-treated T cells, even surpassing the activity of Gal-1. Of course, the actual primary target glycan(s) and the effector glycoconjugate(s) will now have to be figured out. The current debate on this biochemical aspect in activated murine T cells for Gal-1 as effector renders it likely that this issue will not be resolved readily (59). In our report, we next focused on the intracellular chain of events. Caspases-3 and -9 were singled out to work in the effector branch by fluorogenic substrate assays and testing of caspase inhibitors. Fittingly, the Bax/Bcl-2 ratio was increased (by the way, Crohn’s disease is characterized by a Bax/Bcl-2 imbalance that makes autoreactive T cells resistant to apoptotic stimuli (60)), the mitochondrial membrane potential disturbed, cytochrome c released, and the DFF proteolytically activated. These sets of internally consistent results, successfully probing the intrinsic pathway at different sites, delimit Gal-2-mediated signaling from that of Gal-1 and -7 in our assays. Of note, Gal-1 activated caspases-8 and -9, while Gal-7 activity was abolished by inhibiting caspase-1. Beyond prototype galectins, the reaction pathways of Gal-2 and also Gal-1 are clearly different from tandem-repeat-type Gal-9, which activates the calcium-calpain-caspase-1 pathway (61). Lack of preference toward CD4 or CD8 subpopulations and of effect of cycloheximide treatment further separated Gal-2- vs Gal-9-dependent effects. The ongoing discussion about requirement for caspase involvement in the course of Gal-1-induced apoptosis in human T cells (17, 19) substantiates the merit of running parallel assays with different galectins to comparatively define their activities. Our results confirm the potency of the pan-caspase inhibitor to prevent apoptosis (19). They clearly argue in favor of involvement of caspases-3, -8, and -9 for Gal-1 to induce apoptosis in human PBT. Experiments with murine LPT and human Gal-1 recently likewise demonstrated a critical role of caspases-8 and -9 (62). That this in vitro activity should not necessarily be expected to lead to induction of T cell apoptosis in a treatment model was shown for Gal-1 in rat nephrotoxic serum nephritis (63).

A further clear difference in cellular responses to Gal-2 relative to Gal-1 was uncovered by measuring cell cycling. Compared with Gal-1 and, newly described in this work, Gal-7, Gal-2 did not influence this parameter. We determined this result, in line with absence of Gal-2 reactivity with CD3, by Western blotting of regulatory proteins and cytofluorometric detection of cyclin B1, the two sets of data being in full accord. It is noteworthy that inhibitory Gal-1 effects on proliferation were also seen in murine T cells (42). At nanomolar concentrations and without dependence on sugar binding, a monomeric form of Gal-1 had been reported to cause a S/G₂ growth arrest with progression to apoptosis exclusively for leukemic, but not normal T cells (64). We have to date no evidence for a similar conversion of Gal-2 to a monomeric form by oxidation.

Regarding the potential for a clinical perspective, it was further of interest to analyze whether and to what extent Gal-2 treatment might influence the balance of Th1- vs Th2-derived cytokines. Monitoring the murine BI-141 hybridoma, Gal-1, in line with its CD3-binding capacity, impaired IL-2-secretion, and murine LPS-treated spleen cells and human IL-2-activated T cells responded to Gal-1 treatment with reduced secretion of the proinflammatory Th1-derived cytokines TNF- and IFN- (65, 66). Gal-2 clearly shifted the secretion profile to Th2-derived cytokines. The down-regulation of secretion of TNF- and IFN- was accompanied by a marked increase of IL-5 secretion. Of note in this respect is the recently documented capacity of Gal-2 to interact with the TNF superfamily member lymphotoxin-, with whom it is coexpressed in smooth muscle cells and macrophages in the intima of human atherosclerotic plaques, and to regulate its secretion (35). In this work, we show that Gal-2 presence does not interfere with the applied technique for TNF-/IFN- quantitation. These results broaden our knowledge on Gal-2-dependent effects and contribute to ascribing clinical relevance to the presented data.

Upon cell binding, the bivalent prototype galectins cannot only be engaged in cross-linking cis interactions, visible by receptor segregation in confocal immunofluorescence microscopy (14), but also in trans interactions. They bridge T cells and other cells or matrix compounds, for example, fibronectin. In this context, an integrin with the ₁ subunit can offer a suitable glycan chain to form a galectin-dependent contact with matrix compounds, as inferred for a set of carcinoma cells (27). Remarkably, this functionality of glycans of integrins establishes them as truly bifunctional molecules with sensor epitopes residing in the protein and the carbohydrate parts. Presence of Gal-2 on the T cell surface enhanced adhesion to fibronectin, whereas Gal-1 under the same conditions reduced this parameter. A similar result for T cells had been reported in the case of Gal-1 previously (65), adding weight to the validity of our experimental conditions and the detected difference. Rat Gal-2 showed the same efficacy as the human protein, so that the sequence deviations from interspecies comparison (human and rat Gal-2) appear less relevant than those between human Gal-1 and -2. Involvement of the ₁ integrin was rendered likely by Ab-blocking studies, pointing to a functional relevance of this interaction for modulation of T cell adhesion.

In summary, we have initiated functional analysis of gastrointestinal Gal-2. It is an inducer of apoptosis of activated T cells, although it lacks reactivity to CD3 and CD7 characteristic for Gal-1. The intrinsic apoptotic pathway is triggered, as shown by a series of internally consistent assay results. Gal-1 and -7 likewise critically depend on caspases to switch on programmed cell death. Importantly (and somewhat surprisingly), the profile of caspase activation has unique features in each case. Our analysis of the signaling pathways with their conspicuous differences among closely related subfamily members will thus serve as basis to launch systematic dissection of their activities. The detected divergence may also have relevance for tumor development and progression (67). Further differences to Gal-1 reported in this work are the lack of influence on cell cycling and the increased cell adhesion to fibronectin. The way Gal-2 skews the balance from Th1- to Th2-derived cytokines warrants further study in animal models. Thus, despite the close structural similarity to Gal-1, gastrointestinal Gal-2 has an obviously distinct functionality on T cell homeostasis with its own potential clinical perspective.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the Mizutani Foundation for Glycoscience (Tokyo, Japan).

² Address correspondence and reprint requests to Dr. Andreas Sturm, Charité-Universitätsmedizin Berlin, Campus Virchow Clinic, Department of Hepatology and Gastroenterology, Augustenburger Platz 1, 13353 Berlin/Germany. E-mail address: andreas.sturm{at}charite.de

³ Abbreviations used in this paper: Gal, galectin; DAPI, 4',6'-diamidino-2-phenylindole; DFF, DNA fragmentation factor; DPBS, Dulbecco’s PBS; HNF, hepatocyte NF; LPT, lamina propria T cell; PBT, peripheral blood T lymphocyte; PI, propidium iodide; Rb, retinoblastoma; TCF, T cell-specific factor; CBA, cytometric bead array.

Received for publication April 20, 2004. Accepted for publication July 9, 2004.

	References

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