Up-regulation of galectin-3 and its ligands by Trypanosoma cruzi infection with modulation of adhesion and migration of murine dendritic cells

Bernard Vray1,2, Isabelle Camby3, Vincent Vercruysse2, Tatjana Mijatovic3, Nicolai V. Bovin4, Paola Ricciardi-Castagnoli5, Herbert Kaltner6, Isabelle Salmon7, Hans-Joachim Gabius6 and Robert Kiss3

2 Laboratoire d'Immunologie Expérimentale, Faculté de Médecine, 808 Route de Lennik, 1070 Brussels, and Laboratoire de Parasitologie, Département de Biologie des Organismes, Faculté des Sciences, Université Libre de Bruxelles, Brussels, Belgium; 3 Laboratoire de Toxicologie, Institut de Pharmacie, Université Libre de Bruxelles, Brussels, Belgium; 4 Shemyakin Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia; 5 Department of Biotechnology and Biosciences, Plaza della Scienza, University of Milano-Bicocca, Milano, Italy; 6 Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University, Munich, Germany; and 7 Service d'Anatomie Pathologique, Hopital Erasme, Université Libre de Bruxelles, Brussels, Belgium

Received on January 25, 2004; revised on February 11, 2004; accepted on February 24, 2004


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The impact of a pathogen-induced inflammatory response on dendritic cells (DCs) and on their expression of galectin-3 (Gal-3) was studied on splenic DCs (sDCs) from Trypanosoma cruzi–infected mice. We determined the lectin expression and also presentation of ligands using the labeled galectin as probe. By reverse transcriptase polymerase chain reaction, western blot analysis, quantitative glycocytochemistry, and computer-assisted quantitative microscopy, we demonstrate that, in sDCs from infected mice, expression of Gal-3 and Gal-3-specific ligands were markedly up-regulated and adhesiveness was increased with Gal-3-coated substratum. Gal-3 expression was also enhanced in T. cruzi–infected D2SC-1 cells. To assess influence on migration, we had to work exclusively with D2SC-1 cells because sDCs rapidly lost their capacity to adhere to substratum. Migration of infected- and TCM-treated D2SC-1 cells were reduced when substratum was coated with Gal-3. Expression of Gal-3 by D2SC-1 was reduced when they were incubated with anti-Gal-3 antisense oligonucleotide without effect on cell invasion by the parasite. By using seven neoglycoconjugates, we probed the cellular capacity to specifically bind carbohydrate ligands. Similar to Gal-3, an up-regulation was noted with respect to sites specific for Man and {alpha}-GalNAc, respectively, revealing that infection-dependent changes are not confined to Gal-3-dependent parameters. Considered together, these data document for the first time that a parasitic infection can modulate both in vivo and in vitro the expression of Gal-3 and of ligands for this lectin in DCs with functional consequences on their capacities of adhesion and migration. These results suggest a new immunomodulatory property of T. cruzi.

Key words: galectin-3 / glycocytochemistry / neoglycoconjugate / spleen murine dendritic cells / Trypanosoma cruzi


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Infection control is driven by the development of efficient immune responses initiated by dendritic cells (DCs). Present in most tissues and organs, immature DCs act as roving sentinels of the immune system. They capture and process infectious microorganisms and/or microorganism-derived antigenic molecules. Then, on activation, they migrate toward T lymphocyte–rich areas of lymph nodes and spleen. At the same time, they undergo a process of maturation, losing their capacity to internalize antigens while increasing the level of expression of various proteins and differentiating into fully potent antigen-presenting cells. Once having entered the lymphoid organs, they prime the rare naive T lymphocytes specific for the relevant antigen and initiate the adaptative immune response (Banchereau et al., 2000Go; Lanzavecchia and Sallusto, 2000Go; Sousa et al., 1999Go). Whereas these processes are well described at the cellular level, the ways how distinct molecules participate in the host defense are less well defined. Thus we focus on one assumed effector in this report.

Because interactions at the cell surface are of prime importance for activity and routing of DCs, attention is paid to the role of the glycan chains of cellular glycoconjugates to act as ligands in information transfer for lectins (Gabius et al., 2002Go; Reuter and Gabius, 1999Go). Due to their spatially strategic position at branch termini, ß-galactosides play a prominent role in this respect, and, for example, the members of the lectin family of galectins home in on such epitopes to affect cell growth and adhesion (Cooper, 2002Go; Gabius, 1997Go; Kasai and Hirabayashi, 1996Go). Interestingly, a complex network of family members is expressed in various cell types with a characteristic profile of activities that can engender differential prognostic relevance in cancer (Cooper, 2002Go; Lahm et al., 2001Go; Nagy et al., 2003Go; Timoshenko et al., 2003Go). Of note in our context is the significant maturation-dependent down-regulation of expression of mRNA for a galectin, that is, galectin-3 (Gal-3), in DCs (Dietz et al., 2000Go; Higashi et al., 2002Go). The observed regulation intimates a functional correlation, warranting study on this lectin's expression and aspects of function more closely in our model.

Gal-3, a multifunctional 35-kDa protein, is the only known member of the chimera-type group constituted of a nonlectin domain connected to a typical carbohydrate-recognition domain (Cooper, 2002Go; Gabius, 1997Go; Kasai and Hirabayashi, 1996Go). Its designation as MAC-2 antigen is popular in immunology, and it is localized in various tissues and immunocompetent/inflammatory cells (Flotte et al., 1983Go; Rabinovich et al., 2002Go). Gal-3 is involved in many immunoregulatory processes, such as DC/T lymphocyte adhesion (Swarte et al., 1998Go), cell–cell adhesion and adhesion of cells to matrix glycoproteins (Kaltner and Stierstorfer, 1998Go), inflammatory responses and cell migration toward inflammatory foci and cell proliferation (Rabinovich et al., 2002Go), and is also known as a new chemoattractant (Sano and Liu, 2001Go). Gal-3 participates in myelin phagocytosis in Schwann cells (Reichert et al., 1994Go) and contributes to the phagocytosis of microorganisms and apoptotic cells by macrophages. Regarding cell growth control, it can block the negative growth regulation in neuroblastoma cells by galectin-1 revealing functional divergence (Kopitz et al., 2001Go). In contrast to the homodimeric cross-linking galectin-1, Gal-3 is monomeric in solution but can aggregate when bound to a surface with polyvalent ligands, this difference affording a route to design galectin-type-specific inhibitors (Andre et al., 2001Go; Brewer, 2002Go; Gabius, 2001Go; Vrasidas et al., 2003Go). Notably for protozoan infection, Gal-3 has been described as a host factor capable to distinguish different species of Leishmania via their polygalactose profile, implicating this lectin in infection and host response in this case (Pelletier and Sato, 2002Go) prompting work on other types of protozoan infection.

Trypanosoma cruzi is a parasitic protozoa that is the etiological agent of Chagas' disease, a major public health problem in Latin America (Schofield and Dias, 1999Go). Regarding the relation between galectins and infection, focus has so far been given to galectin-1, testing macrophages from infected mice for responsiveness to this lectin and detecting its up-regulation in J774 cells and macrophages in vivo after infection (Giordanengo et al., 2001Go; Zuniga et al., 2001Go). However, although a recent report describes Gal-3-depending binding of T. cruzi trypomastigotes to laminin (Moody et al., 2000Go), no data are currently available on the expression and functionality of Gal-3 and Gal-3-binding sites in DCs confronted with T. cruzi infection. This present lack of available evidence prompted us to perform this study. For this, we used a murine model of T. cruzi infection (Chaussabel et al., 1999Go; Olivares-Fontt et al., 1996Go). We studied the impact of this infection on splenic DCs (sDCs) and a DC line (D2SC-1) (Lutz et al., 1994Go; O'Rourke et al., 2000Go) with focus on the expression of Gal-3. A further aspect concerns the application of the mammalian galectin after labeling as tool to map presence of binding sites.

In this report, using a panel of various techniques such as semi-quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR), western blot analysis, and glyco- and immunohistochemical staining quantification by computer-assisted microscopy, we show that T. cruzi infection can modulate both in vivo and in vitro expression of Gal-3 and also several carbohydrate-binding sites in sDCs with functional consequences on activities in regulating adhesion and migration.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Characterization of expression of Gal-3 and Gal-3-binding sites in sDCs from infected mice
To assess whether Gal-3 was expressed by sDCs and, if positive, whether its expression was modulated on T. cruzi infection, three independent methods were used. First, as shown by RT-PCR (Figure 1A), the accumulation of Gal-3-specific mRNA copies in sDCs isolated from infected mice was sharply enhanced in comparison with that of sDCs from noninfected mice. Second, increased presence of Gal-3 at the protein level was ascertained by western blot analysis (Figure 1B). Finally, the morphological pattern of expression of Gal-3 was studied by glycohistochemistry. It can be seen that, in comparison with sDCs from noninfected mice (Figure 1C), Gal-3 was fairly abundantly present and the intensity of Gal-3-dependent staining increased in sDCs harvested from infected mice at day 21 postinfection (Figure 1D). Furthermore, the quantification by computer-assisted microscopy of the level of expression of Gal-3 (G3 in Figure 2) revealed a statistically significant increase in both the labeling index (LI) and the mean optical density (MOD) variables on infection.



View larger version (93K):
[in this window]
[in a new window]
 
Fig. 1. Characterization of Gal-3 expression in sDC from T. cruzi–infected mice. (A) Accumulation of Gal-3-specific mRNA copies in sDCs isolated from infected mice in comparison with that of sDC from noninfected mice. Open bars: T. cruzi–infected sDCs; closed bars: noninfected sDC. Data from two independent experiments (exp1 and exp2) are shown. (B) Western blot analysis of expression of the Gal-3 protein obtained from sDCs harvested from noninfected mice (DC-NI lane) and infected mice (DC-I lane). (C, D) Morphological pattern of increased expression of Gal-3 in sDC harvested from noninfected (C) or from T. cruzi–infected mice (D) (G x 500). Gal-3 immunopositive sDCs are stained in brown. Representative microscopic fields from at least 50 examined fields.

 


View larger version (65K):
[in this window]
[in a new window]
 
Fig. 2. Quantitative evaluation of the percentage of stained tissue area and the staining intensity in sDCs from infected mice for Gal-3, Gal-3-binding sites, and binding sites of carrier-immobilized carbohydrate. Quantitative evaluation (by computer-assisted microscopy) of (A) the percentage of stained tissue area (referred to as the LI variable) and (B) the staining intensity (expressed as the MOD variable) for Gal-3 (G3) and binding sites with specificity to Gal-3 (G3BS) and to {alpha}-D-GalNAc (aGal), ß-D-GalNAc (bGal), Gal-ß(1-4)-D-Glc (Lac), D-GalNAc-{alpha}(1-3)-D-GalNAcb (bFs), ß-D-Glc (bGlc), {alpha}-D-Man (Man), and D-Glc-{alpha}(1-4)-D-Glc (Malt) in sDCs (black bars) from T. cruzi–infected mice compared to noninfected mice (open bars). Data are expressed as means ± SE from three independent experiments.

 
Next we addressed the issue as to whether presentation of ligand sites for Gal-3 was also subject to regulation. For this purpose, the mammalian galectin is the reagent of choice because commonly used probes from plants will differ in their fine-specificity profiles against carbohydrates despite showing identical monosaccharide specificity (Rüdiger and Gabius, 2001Go). Interestingly, the expression of Gal-3-binding sites was also enhanced on infection (G3BS in Figure 2A and B). sDCs from infected mice were devoid of any amastigotes as shown by both light microscopy and transmission electron microscopy (data not shown). Together these data demonstrate that T. cruzi infection positively affects Gal-3 expression in sDCs. To further determine whether the binding capacity of the cells toward carbohydrates relevant for adhesion/immune regulation is also subject to regulation or not, we tested carrier-immobilized carbohydrate ligands.

Characterization of sites with affinity for carbohydrate ligands expressed in sDCs from infected mice
The panel of the glycohistochemical probes was deliberately prepared to keep all chemical parameters constant except for the nature of the carbohydrate group to avoid an impact of any other factor. Thus differences in staining can be attributed to this parameter. Controls included demonstration that ligand-free carrier failed to yield staining, and binding was shown to be dependent on the carbohydrate revealed by competitive inhibition. As can be seen from Figure 2, specific staining was detectable and a nonuniform pattern of alteration occurred with infection. Notably, even the parameter of the anomeric position made a significant difference (please see {alpha}/ß-GalNAc data). Lactose as ligand showed a similar increase relative to Gal-3, and lactose in fact is a tool to visualize galectins in tissue sections. The up-regulation of binding sites for mannose is suggestive for increased presence of one (or more) C-type lectin(s) of DCs, such as DC-SIGN, DCIR, or another lectin, and this observation warrants further study.

In summary, infection by T. cruzi is associated with up-regulation of several carbohydrate-binding activities, among them those for {alpha}- and ß-D-GalNAc, D-mannose, maltose, or lactose moieties as evidenced by the glycohistochemical approach (Figure 2). As mentioned, the binding site for lactose was likely to be connected to galectins with its marked activity toward lactose. These data further reveal that T. cruzi infection leads to obvious modifications in the presentation of a distinct group of receptor activities and also glycan epitopes reactive with Gal-3. To our knowledge, this has never been reported before, at least on sDCs. To correlate these observations with functional activities, we investigated the influence of T. cruzi infection on adhesion and migration of DCs.

Gal-3-mediated adhesiveness of sDCs from infected mice and infected- or T. cruzi–conditioned medium (TCM)–treated D2SC-1 cells
Our observation on enhanced expression of Gal-3 and of binding sites in sDCs from infected mice prompted us to investigate selected functional consequences of these modifications, starting with cell adhesiveness. To assess consequences of Gal-3-enhanced expression on T. cruzi infection on cell adhesiveness, we tested the ability of cells to adhere to substratum coated with Gal-3. In fact, because galectin secretion in situ by inflammatory cells, a common process for these effectors, can establish a specific environment, those conditions are attempted to be mimicked by coating culture dishes with Gal-3. Our results on Gal-3-binding sites would predict increased adhesiveness. However, it should not be extrapolated that presence of binding sites will necessarily translate to adhesion. As revealed by our experimental series, substratum coated with Gal-3 significantly increased the adhesiveness of the sDCs harvested from infected mice in the two different samples analyzed when compared to plastic as control (Figure 3A and B). Gal-3-coated substratum enhanced only slightly (or not at all) the adhesiveness of sDCs from noninfected mice (Figure 3A and B).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 3. Determination of the adhesiveness of sDCs from infected mice and infected or TCM-treated D2SC-1 cells. Determination of the adhesiveness to plastic (Plast) or to substratum coated with Gal-3 of sDCs from infected (black bars) or noninfected mice (open bars) (A and B) and of D2SC-1 cells (C) cultured in conventional culture medium (open bars), or after infection with T. cruzi (black bars) or in TCM containing culture medium (hatched bars). Data are expressed as means ± SE from three independent experiments.

 
D2SC-1 cells are murine immature DC easily cultured in vitro and presenting most of the usual features of DCs (Lutz et al., 1994Go; O'Rourke et al., 2000Go). The adhesiveness of TCM-treated D2SC-1 cells to Gal-3-coated substratum was also enhanced but not that of T. cruzi–infected cells (Figure 3C). These results must likely reflect the up-regulation of galectin-binding sites measured with biotinylated Gal-3 (Figure 2) and increased availability of galectins in the environment has thus primarily an effect on TCM-treated D2SC-1 cells.

Gal-3 expression and migration of infected D2SC-1 cells
The effects of infection on cell adhesiveness led us to investigate the impact of infection on cell migration. Of note, it is well known that sDCs adhering to plastic undergo a maturation process: They lost their adherence to substratum rapidly and were found free in the culture medium after 4 h. This period is too short to evaluate cell migration as shown in preliminary experiments (data not shown). As a consequence, to investigate the effect of T. cruzi infection on the migratory behavior of DCs, we went to work with D2SC-1 cells instead of sDCs. D2SC-1 cells adhere to the substratum for more then 24 h and to a higher extent to culture supports than sDCs (note different scales on y-axis in Figure 3). Such a level of adhesion was required for the cells to remain in focus for the computer-assisted phase-contrast microscopy analyses, allowing us to quantify the course of migration accurately. In fact, each living cell in the culture under study was automatically tracked. The maximum relative distance to the origin (the quantitative MRDO variable) was calculated for each cell from these trajectories (De Hauwer et al., 1998Go). In addition, when compared to sDCs, Gal-3 expression followed the same pattern when the D2SC-1 cells were infected as shown with the changes of LI and MOD variables except that the addition of TCM to the culture medium failed to up-regulate the density of Gal-3 in D2SC-1 cells (Figure 4). Interestingly, contrary to sDCs from infected mice that contain no amastigotes (the intracellular forms of parasite multiplication), some D2SC-1 cells were readily infected (57.3% ± 26.0 infected cells; n = 3) and contained amastigotes (3.0 ± 1.6 amastigotes per infected cell; n = 3). It is obvious that on Gal-3-coated substratum, cell migration was inhibited both with T. cruzi–infected and TCM-treated D2SC-1 cells (Figure 5).



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4. Quantitative evaluation of the percentage of stained tissue area and the staining intensity in infected D2SC-1 cells for immunopositivity against Gal-3. Quantitative evaluation (by computer-assisted microscopy) of the percentage of stained tissue area (referred to as the LI variable, left y-axis) and the staining intensity (referred to as the MOD variable, right y-axis) for immunopositivity against Gal-3 in sections of D2SC-1 cell pellets from cells maintained in culture with conventional culture medium (open bars) or after infection with T. cruzi (black bars) or TCM-containing culture medium (hatched bars). Data are expressed as means ± SE from three independent experiments.

 


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 5. Determination of the migration capacity of infected or TCM-treated D2SC-1 cells. Determination of the maximum relative distance from the origin (the quantitative MRDO variable) migrated by D2SC-1 cells cultured in conventional culture medium (open bars), or after infection with T. cruzi (black bars) or in TCM-containing culture medium (hatched bars) plated on a plastic substratum precoated or not (Ct) with Gal-3. The trajectory of each cell was quantitatively tracked by computer-assisted phase contrast videomicroscopy. Data are expressed as means ± SE. A minimum of 654 and a maximum of 1002 cells were analyzed for each experimental condition.

 
In brief, adhesion counteracts migration. An environment with exogenous supplementation of Gal-3 thus negatively affects migration of D2SC-1 cells. Furthermore, the presence of Gal-3, as seen for sDCs, contributes to support the reasoning that the D2SC-1 cell line is an appropriate model for murine DCs in the experiments that we carried out to monitor epitope-specific adhesion and migration. With this model at hand, the role of Gal-3 in the infection could be investigated.

Gal-3 and invasion of D2SC-1 cells by T. cruzi
Gal-3 enhances binding of T. cruzi trypomastigotes to laminin through Gal-3 binding sites expressed on parasite membrane which is inhibited by lactose (Giordano et al., 1994Go; Moody et al., 2000Go). In an effort to get an insight into the role of Gal-3 expression on cell invasion, experiments were performed by blocking the synthesis of endeogenous Gal-3. For this, D2SC-1 cells were incubated with anti- Gal-3 antisense oligonucleotides for 12 h. As shown by measurement of fluorescence intensity, expression of Gal-3 was sharply inhibited with anti-Gal-3 antisense oligonucleotides at 0.01 and 0.1 µM but not with scrambled nucleotide used as control (Figure 6A). Then, D2SC-1 cells were first treated with anti-Gal-3 antisense oligonucleotide for 12 h and, after washing, T. cruzi trypomastigotes were added to treated cells in a 10-to-1 parasite-to-cell ratio. As shown in Figure 6B, the percentage of infected D2SC-1 cells and the mean number of amastigotes per infected cell were not modified, indicating that endogenous Gal-3 was probably not involved in the cell invasion process under these conditions.



View larger version (71K):
[in this window]
[in a new window]
 
Fig. 6. Effect of anti-Gal-3 antisense oligonucleotide on Gal-3 expression and cell invasion. (A) D2SC-1 cells were incubated for 12 h in culture medium alone (control, CT) or in culture medium containing either anti-Gal-3 antisense nucleotide (AS) or a scrambled control oligonucleotide (SC) at two different concentrations (0.01 µM or 0.1 µM). Specific Gal-3 immunofluorescence (arbitrary unit: au) was measured. (B) D2SC-1 cells were incubated in the presence of 0.01 µM anti-Gal-3 antisense nucleotide for 12 h, and then T. cruzi trypomastigotes were added to cells in a 10-to-1 parasite-to-cell ratio. The percentage of infected D2SC-1 cells (hatched bars, left y-axis) and the mean number of amastigotes per infected cell (squared-hatched bars, right y-axis) were measured 24 h later. Data are expressed as means ± SE from one out of two independent experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
During the acute phase, T. cruzi infection is known to induce a strong inflammatory response. Also, Gal-3 belongs to a set of proteins undergoing regulation of expression on maturation of DCs (Dietz et al., 2000Go; Higashi et al., 2002Go). This prompted us to investigate the expression of Gal-3 and its binding sites known to be implicated in inflammatory processes. In this report, we show that T. cruzi infection can modulate both in vivo and in vitro the expression of this galectin and also binding sites for it in DCs with functional consequences on their capacities of adhesion and migration. The results commend the use of the labeled mammalian lectin in studies on regulation of lectin expression to obtain information on the ligand side, too.

The occurrence of Gal-3 and Gal-3-binding sites in sDCs is evocative of Gal-3 shown in Birbeck granules and Gal-3 ligands in human Langerhans cells (or immature DCs of the epidermis and mucosal tissues) (Holikova et al., 2000Go; Smetana et al., 1999Go) and in human neutrophils. Clear evidence for up-regulation seen for other classes of carbohydrate-binding sites (e.g., lectins), too, fits in nicely with the concept of glycan/lectin functionality, as explained in the introduction (Gabius et al., 2002Go; Reuter and Gabius, 1999Go). The nonuniform changes that we observed in LI and MOD variables served as inherent control because probe synthesis was deliberately kept constant to yield products with identical chemical properties except for the structure of the carbohydrate ligand. D-mannose and also {alpha}-N-acetyl-D-galactosamine were potent ligands, which are known to bind to the tandem-repeat-type mannose receptor and the DC-specific ICAM-3-grabbing nonintegrin (Gabius, 1997Go; Steinman, 2000Go) and a C-type lectin first found on macrophages (Iida et al., 1999Go). Remarkably, this macrophage C-type lectin failed to bind the Forssman disaccharide despite its activity for {alpha}-GalNAc (Iida et al., 1999Go) in agreement with our glycohistochemical results.

In our experimental model of infection, sDCs were harvested from infected mice. They were devoid of parasites. This observation fits well with other reports showing that spleen is usually weakly or not at all infected (Andrade et al., 1985Go; Buckner et al., 1999Go; de Diego et al., 1991Go; Deutschlander et al., 1978Go; Melo and Brener, 1978Go; Tarleton and Kuhn, 1983Go). This does not mean that factors originating from a parasite are not active on the sDC functions. On the contrary, such a mechanism is strongly suggested by the effect of TCM on D2SC-1 cell functions. As shown previously on TCM-treated human DCs, synthesis of cytokines, up-regulation of CD40 and major histocompatibility complex class I and II molecules, and antigen-presenting functions were significantly reduced (Van Overtvelt et al., 1999Go, 2002Go). In addition, when human DCs were cocultured with T. cruzi, both infected and noninfected DCs showed a down-regulation of major histocompatibility complex class I expression (Van Overtvelt et al., 2002Go). Furthermore, Brodskyn et al. (2002)Go reported recently that glycosylinositolphospholipids derived from T. cruzi epimastigotes (the vector form of T. cruzi) interfere with production of proinflammatory cytokines and immunocompetent molecules expressed by macrophages and DCs. Thought we cannot rule out an indirect effect of host cell–derived factors that are in turn activated by the parasites, these data strongly suggest that parasite-derived molecules could act in a systemic way, alter functions of immunocompetent cells, and thereby favor the spreading of infection.

Following the initial detection of regulation of the two Gal-3 parameters, selected functional tests were performed to probe cell adhesion and migration on substratum coated with Gal-3. Due to the constitutive secretion of galectins (Hughes, 1999Go) and the relevance of extracellular galectins as effectors in the environment, adhesion and migration effects were monitored. Our quantitative measurements revealed increased adhesiveness and decreased migrating behavior in an environment rich in Gal-3. Interestingly, Gal-3 is involved in DC–T lymphocyte binding, especially after triggering by L-selectin, a process that increases density of Gal-3 binding sites on T cell membrane surfaces (Swarte et al., 1998Go). However, Gal-3 has also an anti-adhesion property because it decreases thymocytes interaction with the thymic microenvironment (Villa-Verde et al., 1999Go, 2002Go), a property also found for tumor cells (Andre et al., 1999Go). The level of Gal-3 expression in D2SC-1 cells is already on a rather high level compared with sDCs, rendering it difficult to observe conspicuous regulatory mechanisms.

Only TCM-treated D2SC-1 cells (and not T. cruzi–infected D2SC-1 cells; Figure 3) had an enhanced adhesiveness to Gal-3-coated substratum, indicating an enhanced expression of Gal-3-binding site, whereas only T. cruzi–infected D2SC-1 cells (and not TCM-treated D2SC-1 cells; Figure 4) showed an up-regulation of Gal-3 expression. Both T. cruzi–infected and TCM-treated D2SC-1 maintained a reduced capacity of migration. This result is indicative for a complex relationship between expression of Gal-3/Gal-3 binding sites and parasite-derived factors in a not yet defined manner, as is also true for Gal-3 expression in vitro and in vivo for macrophage-like P388D1 cells (Gabius and Vehmeyer, 1988Go).

DCs play a pivotal role in the initiation of immune responses, but so far there are few reports on their response to T. cruzi infection. Two recent reports underline the dramatic consequences of T. cruzi infection on biological activity of murine sDCs (Alba Soto et al., 2003Go; Planelles et al., 2003Go). These data are in line with our previous reports showing that human monocyte-derived DCs can be infected in vitro by T. cruzi, whereafter intracellular multiplication proceeds. This infection triggers functional consequences as already mentioned (Van Overtvelt et al., 1999Go, 2002Go). Because effects are also induced by TCM, at least these responses appeared to be mediated by soluble factors released by T. cruzi. The fact that T. cruzi modifies adhesion and migration capacities of DCs is of importance and fits well with a recent immunohistochemical study showing that the lipopolysaccharide-mediated migration/maturation process of sDCs is inhibited in T. cruzi–infected mice (Chaussabel et al., 2003Go). In addition, T. cruzi infection triggers a sharp increase in both the number and the percentage of CD11c+ sDCs, mainly at day 21 postinoculation (Chaussabel et al., 2003Go). It was also the case in the experiments reported here (data not shown). Thus the number of sDCs isolated from infected and noninfected mice was accurately adjusted to 10 x 106 cells before the preparation of the cell pellets and Gal-3-dependent immunohistochemical staining. This CD11c marker is expressed on myeloid and lymphoid DCs, and flow cytometry analysis confirmed that sDC samples contained at least 90% CD11c+ cells, so that a counterstaining to identify sDCs was not necessary. Finally, these data indicate that T. cruzi infection triggers not only increases in the number and the percentage of CD11c+ as previously described but also up-regulates the level of Gal-3 expression in these sDCs. Because these modifications could alter in vivo the migration step of DCs, which is a pivotal feature in the process of antigen presentation, it could thus constitute a new immunomodulatory property of T. cruzi.

The complex life cycle of T. cruzi (Tyler and Engman, 2001Go) involves several recognition/adhesion systems leading to host cell invasion followed by an obligate intracellular multiplication step (for review see Vray, 2002Go). Prior to invasion of host cells, T. cruzi has to move through the extracellular matrix and to bind to components such as laminin (Giordano et al., 1994Go). At this stage, the interaction between Gal-3, so far not yet characterized parasitic ligands, and laminin might provide a versatile point for modulation. In addition, the presence of abundant Gal{alpha}1-3Gal determinants on T. cruzi has been reported (Bretana et al., 1992Go) and also further types of lectin–carbohydrate interactions participate in the interactions between trypanosomatids and host's cells, for example, for glycoaminoglycans (Kock et al., 1997Go). From these results, one could assume that endogenous Gal-3 could participate in cell invasion. However, our data indicate that inhibition of endogenous Gal-3 synthesis by anti-Gal-3 antisense oligonucleotide did not modify cell invasion, suggesting that cell surface Gal-3 did not appear to be involved in this process and that up-regulation of Gal-3 in DCs by T. cruzi infection might be directed toward alterations of other systems, such as cell adhesion and migration or also regulation of immune activities. However, it should be kept in mind that despite the activity of anti-Gal-3 antisense oligonucleotide, other factors, such as a neosynthesis of Gal-3 during the incubation time of D2SC-1 with parasites, might skew the invasion of D2SC-1 cells by T. cruzi.

In conclusion, our article clearly shows that Gal-3 and ligand density for this lectin undergo significant up-regulation. Also, the capacity of glycan binding beyond lactose is subject to regulation on T. cruzi infection of DCs with an epitope-specific pattern of change. The change in Gal-3 parameters has implications for cell adhesiveness and migration in culture supports coated with the galectin. Overall, our data suggest that on inflammation consecutive to infection, the migratory behavior of DCs could be modulated, a newly described immunomodulatory property of T. cruzi.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
T. cruzi trypomastigotes and infection of mice
T. cruzi trypomastigotes (Tehuantepec strain, Mexico) were maintained by weekly intraperitoneal inoculations into male BALB/c mice (6–8 weeks old) (Bantin & Kingman Universal, Hull, Humberside, United Kingdom) with 100 blood-form trypomastigotes in 0.2 ml phosphate buffered saline (PBS) on day 0. Parasitemia was monitored by counting the trypomastigotes in blood samples collected by tail incision every week. Survival rates were determined daily. Spleens were harvested from the mice, which were sacrificed by cervical dislocation on day 21 postinfection at the peak of parasitemia.

To obtain large quantities of parasites, trypomastigotes (2.5 x 105 parasites/rat) were inoculated into 7 Gy X-ray irradiated F344 Fischer rats (Iffa Credo, Brussels; Mark I-68A irradiation; J. L. Shepherd and Associates, San Fernando, CA). Trypomastigotes were obtained from the blood (containing 10 U heparin/ml) of the infected rats by ion exchange chromatography on DEAE-cellulose (Whatman DE 52) equilibrated with PBS-glucose (pH 7.4). The trypomastigotes were centrifuged (15 min, 1800 x g, 4°C) and resuspended in endotoxin-free PBS, as detailed previously (Chaussabel et al., 1999Go). The maintenance and care of mice and rats complied with the guidelines of the Free University of Brussels Ethics Committee for the human use of laboratory animals.

TCM
TCM was prepared according to a previously described method in Kierszenbaum et al. (1998)Go to obtain the trypanosomal immunosuppressive factor. Briefly, suspensions of T. cruzi (2 x 107 trypomastigotes/ml in RPMI 1640 medium) were incubated at 37°C and in a 5% CO2 atmosphere for 24 h. The parasites were then removed by filtration through a sterile 0.22 µm pore-size filter (Millipore, Bedford, MA). TCM was aliquoted and stored at –20°C until used. When necessary, it was diluted in culture medium to obtain a final concentration of 75% (Van Overtvelt et al., 1999Go).

Cells
To obtain sDCs, spleens were treated with collagenase, further dissociated in Ca2+- and Mg2+-free Hank's balanced salt solution in the presence of ethylene diamine tetraacetic acid, and cells were separated into low- and high-density fractions on a Nycodenz (Torshov, Norway) gradient. Low-density-fraction cells were then incubated with monoclonal antibody (mAb) N418 (anti-integrin CD11c antibody)-coupled microbeads and fractionated over a MACS column (Miltenyi Biotec, Bergschgladbach, Germany). Flow cytometry (see later description) using fluorescein isothiocyanate (FITC)-labeled N418 mAb showed that the samples contained at least 90% of positive cells (Maldonado-Lopez et al., 1999aGo).

The D2SC-1 cells (Lutz et al., 1994Go; O'Rourke et al., 2000Go) were cultured in Iscove MDM medium (Sigma, St. Louis, MO) supplemented with 5% fetal calf serum, penicillin (100 IU/ml), streptomycin (100 µg/ml), and 2-mercaptoethanol (0.05 mM) (Gibco, Grand Island, NY) and incubated at 37°C CO2 atmosphere. To stop their proliferation, they were irradiated with 30 Gy X-rays.

To obtain T. cruzi–infected cells, D2SC-1 cells were incubated for 2 h with a suspension of T. cruzi trypomastigotes in a 10-to-1 parasite-to-cell ratio. After washing, the cells were further incubated for 12 h. Cell samples were centrifuged (Cytospin Shandon, London), stained with Giemsa stain, and examined under a light microscope to determine the infection level by quantifying the percentage of infected cells and the mean number of amastigotes per infected cells.

Flow cytometry
For immunophenotyping, the sDCs were washed in PBS supplemented with 0.1% bovine serum albumin (Sigma) and 10 mM NaN3. The cells were incubated with a solution containing 2.4G2 mAb (a rat anti-mouse FcR mAb, 500 µg/mL, Pharmingen, San Diego, CA) for 15 min at 4°C before staining to prevent antibody binding to FcR. They were then incubated with buffer containing FITC-coupled N418 mAb (Maldonado-Lopez et al., 1999bGo).

Semi-quantitative RT-PCR analysis
Semi-quantitative RT-PCR analysis were performed as previously described (Belot et al., 2001Go). Briefly, total RNAs from sDCs obtained after the sacrifice (at day 21 postinfection) of infected and noninfected mice were prepared with the TRIzol isolation reagent (GibcoBRL, Life Technologies, Invitrogen SA, Merelbeke, Belgium) according to the manufacturer's recommendation after a purification step allowing the removal of any other cell type. One microgram total RNA was used on a thermal cycler (iCycler, BioRad, Nazareth, Belgium) as a template for cDNA synthesis. The analysis of Gal-3 mRNA expression by semi-quantitative RT-PCR was performed after establishing a standard curve via serial dilutions (108 to 10 copies/µl) of the PCR product generated with external primers (Gal-3 sense = 5'-ATGTTGCCTTCCACTTTAACC-3', Gal-3 antisense = 5'-AGATCATGGCGTGGTTAG-3'). The primers used were designed on the basis of HYBSIMULATOR software (Advanced Gene Computing Technology, Irvine, CA) and were purchased from Invitrogen SA (Life Technologies). The purification of the PCR products was carried out by the High Pure PCR Product Purification Kit (Roche Diagnostics) in accordance with the manufacturer's instructions. The semi-quantitative PCR reactions took place in capillaries with 20 µl reaction medium (LC-Fastart DNA Master SYBR Green 1, Roche Diagnostics), 20 ng purified cDNA, and 0.5 µM of both sense and antisense internal primers (Gal-3 sense 5'-ACGAAGCAGGACAATAACTG-3', Gal-3 antisense 5'-GGTTATGTCACCACTGATCC-3', Invitrogen). The reactions were performed in a Lightcycler thermocycler instrument (Roche Diagnostics). After amplification, data analysis was carried out using the fit points algorithm of the Lightcycler quantification software. The standard curve enabled the quantification of the samples to take place.

Western blot analysis
Protein extracts were prepared with the TRIzol isolation reagent (GibcoBRL, Life Technologies) according to the manufacturer's recommendation from the same sDCs as those used for total RNA extraction described, that is, DCs obtained after the sacrifice of noninfected mice and infected mice at day 21 postinfection. The presence of Gal-3 in these extracts was then visualized by the western blot procedure as previously described (Hittelet et al., 2003Go).

Quantitative histochemistry
When working with the DCs, we systematically used histochemical procedures to avoid any of the problems of membrane permeabilization likely to occur with cytochemical procedures. Thus in the case of both sDCs and D2SC-1 cells, we obtained cell pellets by centrifuging 10 x 106 cells from each cell type for 10 min at 800 x g. These pellets were then fixed for 4 h in buffered formalin (4%), dehydrated, and embedded in paraffin wax. Three pellets were prepared for each cell type. The histochemical procedures were carried out, as detailed previously (Camby et al., 2001aGo,bGo). Briefly, 5-µm-thick sections were taken from each cell pellet, and incubation with the various probes was carried out at 25 ± 1°C for 60 min under conditions involving minimum background staining and the working dilution (see later description) for specific anti-Gal-3 antibody.

The extent of specifically bound antibody or probes was visualized by avidin-biotin-peroxidase complex kit reagents (Vector Labs, Burlingame, CA), with diaminobenzidine/H2O2 as the chromogenic substratums. The control reactions for the anti-Gal-3 antibody under study omitted of the incubation step in the case of the primary antibody and also the preincubation of the antibody with the antigen. The specificity of this antibody was checked by western blot using purified Gal-3 and other galectins (i.e., galectins -1, -2, -4, -7, and -8) and cell extracts (data not shown).

The neoglycoconjugates were synthesized and biotinylated as previously described (Camby et al., 2001bGo). The control reactions for the application of the biotinylated probes included competitive inhibitions to ascertain sugar specificity. The omission of the incubation step with a labeled marker served to exclude any staining by the binding of kit reagents, such as the mannose-rich glycoproteins horseradish peroxidase and avidin. Also, processing of a ligand-free but labeled carrier excluded any affinity of the carrier to the sections. Counterstaining was carried out with hematoxilin.

To obtain the anti-Gal-3 antibody, murine Gal-3 was first expressed as a recombinant protein from plasmid prCBP35 s, kindly provided by Dr. J. L. Wang (Michigan State University, East Lansing). It was purified by affinity chromatography on lactosylated Sepharose 4B, and the polyclonal anti-Gal-3 antibody was raised in rabbits, the lack of cross-reactivity to other galectins was rigorously tested by blotting and enzyme-linked immunosorbent assay tests (Kaltner et al., 2002Go). The protein A–refined fraction was used at 2.5 µg/ml. Biotinylation of the lectin was performed under activity-preserving conditions, and the lack of a harmful influence on activity was checked by solid-phase and cell assays (Andre et al., 1999Go, 2001Go).

In addition to this antibody, eight biotinylated probes were tested. Biotinylated Gal-3 was used to localize Gal-3-binding sites, and seven neoglycoconjugates were used to localize accessible binding sites for four distinct monosaccharide epitopes including {alpha}- and ß-anomers of N-acetyl-D-galactosamine ({alpha}/ß-D-GalNAc), ß-D-glucose, {alpha}-D-mannose, and three disaccharides, that is, the ß-anomer of the Forssman epitope, D-lactose (D-Gal(ß1-4)- D-Glc) and D-maltose (D-Glc({alpha}1-4)-D-Glc). The chemical structures of these seven sugar moieties are illustrated and detailed elsewhere (Camby et al., 2001bGo).

To characterize the staining with the markers in the two cell types quantitatively, two variables were measured by a SAMBA 2005 computer-assisted microscope system (Samba Technologies, Grenoble, France) incorporating a 40x magnification lens (Olympus BX50 microscope, aperture 0.65). The LI refers to the percentage of tissue area specifically stained by a given histochemical marker. The MOD denotes staining intensity. The way we used the computer-assisted system to quantify the histochemical staining and also the standardization procedures how we used computer-assisted microscopy have been explained in detail elsewhere (Camby et al., 2001aGo,bGo). A negative histological control slide (from which the incubation step with the primary antibody was omitted) was analyzed for each specimen under study. The software used on the computer-assisted microscope automatically subtracted the LI and MOD values of the negative control sample from each corresponding positive one.

Inhibition of Gal-3 expression
The inhibition of Gal-3 expression was carried out by incubating cells in a serum-free culture medium, with a Gal-3 antisense phosphorothioate oligonucleotide (5'-FLUO-GTTGACCGCAACCTT-3', Eurogentec, Liège, Belgium). The antisense oligonucleotide sequence used here was optimized on the basis of HYBSIMULATOR software to avoid any cross-hybridization with the mRNAs from the other mammalian galectins described so far.

The control cells were incubated with the solvent only or with a scrambled (5'-FLUO-GCCCAAAGCTCTTGT-3') Gal-3 antisense phosphorothioate oligonucleotide. The influence exerted on Gal-3 expression in the DCs by either 0.01 or 0.1 µM of the scrambled as opposed to the actual Gal-3 antisense oligonucleotides was quantitatively determined by a computer-assisted fluorescence microscope as previously described (Nagy et al., 2001Go). By test series and with the data obtained (Figure 6A) an incubation period of 12 h with the 0.01 µM dose of Gal-3 antisense oligonucleotide and its scrambled counterpart was found to be optimal for use in further experiments.

Cell adhesion experiments
sDCs from noninfected (controls) versus T. cruzi infected animals were plated at a cell density of 2 x 105 cells/ml culture medium (RPMI 1640, Gibco) on plastic substratum precoated with Gal-3 and one plastic substratum without prior coating step (control). We used coating procedures similar to those previously described for fibronectin (Cheresh et al., 1989Go) and for laminin (Stroeken et al., 1998Go). Briefly, 1 ml containing 3.75 µg of each compound was exposed on the surface of a 25 cm2 Falcon dish for 48 h up to total evaporation. Thereby, a coating density of 0.15 µg of each compound per cm2 of culture support was reached. The DCs were seeded on 6 cm diameter plastic dishes (Falcon, Life Technologies) 2 h prior to cell counting. Each experiment included 10 dishes per experimental condition and was repeated three times independently. The number of adhering DCs per mm2 at a G x 200 magnification (phase-contrast Zeiss Axiovert-135 microscope) was recorded for each dish. The same experiments were carried out with the D2SC-1 cells.

Cell migration experiments
The level of D2SC-1 cell motility was measured by instrumentation detailed elsewhere (De Hauwer et al., 1998Go; Hittelet et al., 2003Go). Briefly, this equipment enabled each living cell in the culture under study to be automatically tracked. From these trajectories, the quantitative MRDO variable was calculated for each cell under analysis. This variable thus described the greatest linear distance between the original and subsequent positions of the cell divided by the observation time (De Hauwer et al., 1998Go; Hittelet et al., 2003Go). All experiments were performed in triplicates over 24 h with a cell concentration of 4 x 104 cells/ml culture medium and one image recorded every 4 min. At the beginning of the experiments, the minimal number of cells in a given experimental condition was 67, and the maximum number at the end of the experiments was 312. As the result of these analyses was carried out in triplicate, a minimum of 654 and a maximum of 1002 cells were analyzed for each experimental condition.

Statistical analysis
The statistical comparisons of the data were carried out by the Student t-test (for two groups) after a check of the equality of variance by means of the Levene test, and the normal distribution fitting of the data by means of the {chi}2 test of quality of fit. When these parametric conditions were not satisfied, the nonparametric Mann-Whitney (for two groups) test was performed. The statistical analyses were carried out using Statistica (Statsoft, Tulsa, OK).


    Acknowledgements
 
We are very grateful to Dr. J. L. Wang (East Lansing, MI) for kindly providing us with the expression vector for murine Gal-3. We thank I. Mazza for help in preparing the manuscript. I.C. is a scientific research worker associated with the Fonds National de la Recherche Scientifique (FNRS, Belgium). R.K. is a director of research with FNRS; T.M. is a holder of a grant from the Fondation Yvonne Boël (Brussels). This work was supported by grants from the Fondation Yvonne Boël, the Fondation Emile Defay, and the Centre de Recherche Interuniversitaire en Vaccinologie. Travel grant from NATO and financial support for EC high-level conferences were helpful to organize the international study group for this investigation.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: bvray{at}ulb.ac.be


    Abbreviations
 
DC, dendritic cell; FITC, fluorescein isothiocyanate; LI, labeling index; mAb, monoclonal antibody; MOD, mean optical density; MRDO, maximum relative distance to the origin; PBS, phosphate buffered saline; RT-PCR, reverse transcriptase-polymerase chain reaction; sDC, splenic dendritic cell; TCM, T. cruzi–conditioned medium


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Alba Soto, C.D., Mirkin, G.A., Solana, M.E., and Gonzalez Cappa, S.M. (2003) Trypanosoma cruzi infection modulates in vivo expression of major histocompatibility complex class II molecules on antigen-presenting cells and T-cell stimulatory activity of dendritic cells in a strain-dependent manner. Infect. Immun., 71, 1194–1199.[Abstract/Free Full Text]

Andrade, S.G., Andrade, V., Brodskyn, C., Magalhaes, J.B., and Netto, M.B. (1985) Immunological response of Swiss mice to infection with three different strains of Trypanosoma cruzi. Ann. Trop. Med. Parasitol., 79, 397–407.[ISI][Medline]

Andre, S., Kojima, S., Yamazaki, N., Fink, C., Kaltner, H., Kayser, K., and Gabius, H.J. (1999) Galectins-1 and -3 and their ligands in tumor biology. Non-uniform properties in cell-surface presentation and modulation of adhesion to matrix glycoproteins for various tumor cell lines, in biodistribution of free and liposome-bound galectins and in their expression by breast and colorectal carcinomas with/without metastatic propensity. J. Cancer Res. Clin. Oncol., 125, 461–474.[CrossRef][ISI][Medline]

Andre, S., Pieters, R. J., Vrasidas, I., Kaltner, H., Kuwabara, I., Liu, F.T., Liskamp, R.M., and Gabius, H.J. (2001) Wedgelike glycodendrimers as inhibitors of binding of mammalian galectins to glycoproteins, lactose maxiclusters, and cell surface glycoconjugates. ChemBiochem., 2, 822–830.[CrossRef][ISI][Medline]

Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y.J., Pulendran, B., and Palucka, K. (2000) Immunobiology of dendritic cells. Annu. Rev. Immunol., 18, 767–811.[CrossRef][ISI][Medline]

Belot, N., Rorive, S., Doyen, I., Lefranc, F., Bruyneel, E., DeDecker, R., Micik, S., Brotchi, J., Decaestecker, C., Salmon, I., and others. (2001) Molecular characterization of cell substratum attachments in human glial tumors relates to prognostic features. Glia, 36, 375–390.[CrossRef][ISI][Medline]

Bretana, A., Avila, J.L., Contreras-Bretana, M., and Tapia, F.J. (1992) American Leishmania spp. and Trypanosoma cruzi: galactosyl alpha(1-3) galactose epitope localization by colloidal gold immunocytochemistry and lectin cytochemistry. Exp. Parasitol., 74, 27–37.[CrossRef][ISI][Medline]

Brewer, C.F. (2002) Binding and cross-linking properties of galectins. Biochim. Biophys. Acta, 1572, 255–262.[ISI][Medline]

Brodskyn, C., Patricio, J., Oliveira, R., Lobo, L., Arnholdt, A., Mendonca-Previato, L., Barral, A., and Barral-Netto, M. (2002) Glycoinositolphospholipids from Trypanosoma cruzi interfere with macrophages and dendritic cell responses. Infect. Immun., 70, 3736–3743.[Abstract/Free Full Text]

Buckner, F.S., Wilson, A.J., and Van Voorhis, W.C. (1999) Detection of live Trypanosoma cruzi in tissues of infected mice by using histochemical stain for beta-galactosidase. Infect. Immun., 67, 403–409.[Abstract/Free Full Text]

Camby, I., Belot, N., Rorive, S., Lefranc, F., Maurage, C.A., Lahm, H., Kaltner, H., Hadari, Y., Ruchoux, M.M., Brotchi, J., and others. (2001a) Galectins are differentially expressed in supratentorial pilocytic astrocytomas, astrocytomas, anaplastic astrocytomas and glioblastomas, and significantly modulate tumor astrocyte migration. Brain. Pathol., 11, 12–26.[ISI][Medline]

Camby, I., Decaestecker, C., Gordower, L., DeDecker, R., Kacem, Y., Lemmers, A., Siebert, H.C., Bovin, N.V., Wesseling, P., Danguy, A., and others. (2001b) Distinct differences in binding capacity to saccharide epitopes in supratentorial pilocytic astrocytomas, astrocytomas, anaplastic astrocytomas, and glioblastomas. J. Neuropathol. Exp. Neurol., 60, 75–84.[ISI][Medline]

Chaussabel, D., Jacobs, F., de Jonge, J., de Veerman, M., Carlier, Y., Thielemans, K., Goldman, M., and Vray, B. (1999) CD40 ligation prevents Trypanosoma cruzi infection through interleukin-12 upregulation. Infect. Immun., 67, 1929–1934.[Abstract/Free Full Text]

Chaussabel, D., Pajak, B., Vercruysse, V., Bisseye, C., Garze, V., Habib, M., Goldman, M., Moser, M., and Vray, B. (2003) Alteration of migration and maturation of dendritic cells and T-cell depletion in the course of experimental Trypanosoma cruzi infection. Lab. Invest., 83, 1373–1382.[CrossRef][ISI][Medline]

Cheresh, D.A., Berliner, S.A., Vicente, V., and Ruggeri, Z.M. (1989) Recognition of distinct adhesive sites on fibrinogen by related integrins on platelets and endothelial cells. Cell, 58, 945–953.[ISI][Medline]

Cooper, D.N. (2002) Galectinomics: finding themes in complexity. Biochim. Biophys. Acta, 1572, 209–231.[ISI][Medline]

de Diego, J.A., Penin, P., del Rey, J., Mayer, R., and Gamallo, C. (1991) A comparative pathological study of three strains of Trypanosoma cruzi in an experimental model. Histol. Histopathol., 6, 199–206.[ISI][Medline]

De Hauwer, C., Camby, I., Darro, F., Migeotte, I., Decaestecker, C., Verbeek, C., Danguy, A., Pasteels, J.L., Brotchi, J., Salmon, I., and others. (1998) Gastrin inhibits motility, decreases cell death levels and increases proliferation in human glioblastoma cell lines. J. Neurobiol., 37, 373–382.[CrossRef][ISI][Medline]

Deutschlander, N., Vollerthun, R., and Hungerer, K.D. (1978) Histopathology of experimental Chagas disease in NMRI-mice. A long term study following paw infection. Tropenmed. Parasitol., 29, 323–329.[ISI][Medline]

Dietz, A.B., Bulur, P.A., Knutson, G.J., Matasic, R., and Vuk-Pavlovic, S. (2000) Maturation of human monocyte-derived dendritic cells studied by microarray hybridization. Biochem. Biophys. Res. Commun., 275, 731–738.[CrossRef][ISI][Medline]

Flotte, T.J., Springer, T.A., and Thorbecke, G.J. (1983) Dendritic cell and macrophage staining by monoclonal antibodies in tissue sections and epidermal sheets. Am. J. Pathol., 111, 112–124.[Abstract]

Gabius, H.J. (1997) Animal lectins. Eur. J. Biochem., 243, 543–576.[Abstract]

Gabius, H. (2001) Probing the cons and pros of lectin-induced immunomodulation: case studies for the mistletoe lectin and galectin-1. Biochimie, 83, 659–666.[CrossRef][ISI][Medline]

Gabius, H.J. and Vehmeyer, K. (1988) Effect of microenvironment and cell-line type on carbohydrate-binding proteins of macrophage-like cells. Biochem. Cell. Biol., 66, 1169–1176.[ISI][Medline]

Gabius, H.J., Andre, S., Kaltner, H., and Siebert, H.C. (2002) The sugar code: functional lectinomics. Biochim. Biophys. Acta, 1572, 165–177.[ISI][Medline]

Giordanengo, L., Gea, S., Barbieri, G., and Rabinovich, G.A. (2001) Anti-galectin-1 autoantibodies in human Trypanosoma cruzi infection: differential expression of this beta-galactoside-binding protein in cardiac Chagas' disease. Clin. Exp. Immunol., 124, 266–273.[CrossRef][ISI][Medline]

Giordano, R., Chammas, R., Veiga, S.S., Colli, W., and Alves, M.J. (1994) An acidic component of the heterogeneous Tc-85 protein family from the surface of Trypanosoma cruzi is a laminin binding glycoprotein. Mol. Biochem. Parasitol., 65, 85–94.[CrossRef][ISI][Medline]

Higashi, N., Fujioka, K., Denda-Nagai, K., Hashimoto, S., Nagai, S., Sato, T., Fujita, Y., Morikawa, A., Tsuiji, M., Miyata-Takeuchi, M., and others. (2002) The macrophage C-type lectin specific for galactose/N-acetylgalactosamine is an endocytic receptor expressed on monocyte-derived immature dendritic cells. J. Biol. Chem., 277, 20686–20693.[Abstract/Free Full Text]

Hittelet, A., Camby, I., Nagy, N., Legendre, H., Bronckart, Y., Decaestecker, C., Kaltner, H., Nifant'ev, N.E., Bovin, N.V., Pector, J.C., and others. (2003) Binding sites for Lewis antigens are expressed by human colon cancer cells and negatively affect their migration. Lab. Invest., 83, 777–787.[ISI][Medline]

Holikova, Z., Smetana, K., Bartunkova, J., Dvorankova, B., Kaltner, H., and Gabius, H.J. (2000) Human epidermal Langerhans cells are selectively recognized by galectin-3 but not by galectin-1. Folia Biol. (Praha), 46, 195–198.[Medline]

Hughes, R.C. (1999) Secretion of the galectin family of mammalian carbohydrate-binding proteins. Biochim. Biophys. Acta, 1473, 172–185.[ISI][Medline]

Iida, S., Yamamoto, K., and Irimura, T. (1999) Interaction of human macrophage C-type lectin with O-linked N-acetylgalactosamine residues on mucin glycopeptides. J. Biol. Chem., 274, 10697–10705.[Abstract/Free Full Text]

Kaltner, H., Seyrek, K., Heck, A., Sinowatz, F., and Gabius, H.J. (2002). Galectin-1 and galectin-3 in fetal development of bovine respiratory and digestive tracts. Comparison of cell type-specific express profiles and subcellular localization. Cell Tissue Res., 307, 35–46.[CrossRef][ISI][Medline]

Kaltner, H. and Stierstorfer, B. (1998) Animal lectins as cell adhesion molecules. Acta Anat. (Basel), 161, 162–179.[CrossRef][Medline]

Kasai, K. and Hirabayashi, J. (1996) Galectins: a family of animal lectins that decipher glycocodes. J. Biochem. (Tokyo), 119, 1–8.[Abstract]

Kierszenbaum, F., Majumder, S., Paredes, P., Tanner, M.K., and Sztein, M.B. (1998) The Trypanosoma cruzi immunosuppressive factor (TIF) targets a lymphocyte activation event subsequent to increased intracellular calcium ion concentration and translocation of protein kinase C but previous to cyclin D2 and cdk4 mRNA accumulation. Mol. Biochem. Parasitol., 92, 133–145.[CrossRef][ISI][Medline]

Kock, N.P., Gabius, H.J., Schmitz, J., and Schottelius, J. (1997) Receptors for carbohydrate ligands including heparin on the cell surface of Leishmania and other trypanosomatids. Trop. Med. Int. Health, 2, 863–874.[ISI][Medline]

Kopitz, J., von Reitzenstein, C., Andre, S., Kaltner, H., Uhl, J., Ehemann, V., Cantz, M., and Gabius, H.J. (2001) Negative regulation of neuroblastoma cell growth by carbohydrate-dependent surface binding of galectin-1 and functional divergence from galectin-3. J. Biol. Chem., 276, 35917–35923.[Abstract/Free Full Text]

Lahm, H., Andre, S., Hoeflich, A., Fischer, J.R., Sordat, B., Kaltner, H., Wolf, E., and Gabius, H.J. (2001) Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures. J. Cancer Res. Clin. Oncol., 127, 375–386.[CrossRef][ISI][Medline]

Lanzavecchia, A. and Sallusto, F. (2000) Dynamics of T lymphocyte responses: intermediates, effectors, and memory cells. Science, 290, 92–97.[Abstract/Free Full Text]

Lutz, M.B., Granucci, F., Winzler, C., Marconi, G., Paglia, P., Foti, M., Assmann, C.U., Cairns, L., Rescigno, M., and Ricciardi-Castagnoli, P. (1994) Retroviral immortalization of phagocytic and dendritic cell clones as a tool to investigate functional heterogeneity. J. Immunol. Methods, 174, 269–279.[CrossRef][ISI][Medline]

Maldonado-Lopez, R., De Smedt, T., Michel, P., Godfroid, J., Pajak, B., Heirman, C., Thielemans, K., Leo, O., Urbain, J., and Moser, M. (1999a) CD8alpha+ and CD8alpha- subclasses of dendritic cells direct the development of distinct T helper cells in vivo. J. Exp. Med., 189, 587–592.[Abstract/Free Full Text]

Maldonado-Lopez, R., De Smedt, T., Pajak, B., Heirman, C., Thieleman, K., Leo, O., Urbain, J., Maliszewski, C.R., and Moser, M (1999b) Role of CD8alpha+ and CD8alpha– dendritic cells in the induction of primary immune responses in vivo. J. Leukoc. Biol., 66, 242–246.[Abstract]

Melo, R.C. and Brener, Z. (1978) Tissue tropism of different Trypanosoma cruzi strains. J. Parasitol., 64, 475–482.[ISI][Medline]

Moody, T.N., Ochieng, J., and Villalta, F. (2000) Novel mechanism that Trypanosoma cruzi uses to adhere to the extracellular matrix mediated by human galectin-3. FEBS Lett., 470, 305–308.[CrossRef][ISI][Medline]

Nagy, N., Brenner, C., Markadieu, N., Chaboteaux, C., Camby, I., Schafer, B.W., Pochet, R., Heizmann, C.W., Salmon, I., Kiss, R., and Decaestecker, C. (2001) S100A2, a putative tumor suppressor gene, regulates in vitro squamous cell carcinoma migration. Lab. Invest., 81, 599–612.[ISI][Medline]

Nagy, N., Legendre, H., Engels,O., Andre, S., Kaltner, H., Wasano, K., Zick, Y., Pector, J.C., Decaestecker, C., Gabius, H.J., and others. (2003) Refined prognostic evaluation in colon carcinoma using immunohistochemical galectin fingerprinting. Cancer, 97, 1849–1858.[CrossRef][ISI][Medline]

Olivares-Fontt, E., Heirman. C., Thielemans, K., and Vray, B. (1996) Granulocyte-macrophage colony-stimulating factor: involvement in control of Trypanosoma cruzi infection in mice. Infect. Immun., 64, 3429–3434.[Abstract]

O'Rourke, R.W., Kang, S.M., Lower, J.A., Feng, S., Ascher, N.L., Baekkeskov, S., and Stock, P.G. (2000) A dendritic cell line genetically modified to express CTLA4-IG as a means to prolong islet allograft survival. Transplantation, 69, 1440–1446.[CrossRef][ISI][Medline]

Pelletier, I. and Sato, S. (2002) Specific recognition and cleavage of galectin-3 by Leishmania major through species-specific polygalactose epitope. J. Biol. Chem., 277, 17663–17670.[Abstract/Free Full Text]

Planelles, L., Thomas, M.C., Maranon, C., Morell, M., and Lopez, M.C. (2003) Differential CD86 and CD40 co-stimulatory molecules and cytokine expression pattern induced by Trypanosoma cruzi in APCs from resistant or susceptible mice. Clin. Exp. Immunol., 131, 41–47.[CrossRef][ISI][Medline]

Rabinovich, G.A., Rubinstein, N., and Toscano, M.A. (2002) Role of galectins in inflammatory and immunomodulatory processes. Biochim. Biophys. Acta, 1572, 274–284.[ISI][Medline]

Reichert, F., Saada, A., and Rotshenker, S. (1994) Peripheral nerve injury induces Schwann cells to express two macrophage phenotypes: phagocytosis and the galactose-specific lectin MAC-2. J. Neurosci., 14, 3231–3245.[Abstract]

Reuter, G. and Gabius, H.J. (1999) Eukaryotic glycosylation: whim of nature or multipurpose tool? Cell. Mol. Life Sci., 55, 368–422.[CrossRef][ISI][Medline]

Rüdiger, H. and Gabius, H.J. (2001) Plant lectins: occurrence, biochemistry, functions and applications. Glycoconj. J., 18, 589–613.[CrossRef][ISI][Medline]

Sano, H. and Liu, F.T. (2001) Galectins: another family of chemoattractants? Mod. Asp. Immunobiol., 2, 4–6.

Schofield, C.J. and Dias, J.C. (1999) The Southern Cone Initiative against Chagas disease. Adv. Parasitol., 42, 1–27.[ISI][Medline]

Smetana, K., Holikova, Z., Klubal, R., Bovin, N.V., Dvorankova, B., Bartunkova, J., Liu, F.T., and Gabius, H.J. (1999) Coexpression of binding sites for A(B) histo-blood group trisaccharides with galectin-3 and Lag antigen in human Langerhans cells. J. Leukoc. Biol., 66, 644–649.[Abstract]

Sousa, C., Sher, A., and Kaye, P. (1999) The role of dendritic cells in the induction and regulation of immunity to microbial infection. Curr. Opin. Immunol., 11, 392–399.[CrossRef][ISI][Medline]

Steinman, R.M. (2000) DC-SIGN: a guide to some mysteries of dendritic cells. Cell, 100, 491–494.[ISI][Medline]

Stroeken, P.J., van Rijthoven, E.A., van der Valk, M.A., and Roos, E. (1998) Targeted disruption of the beta-1 integrin gene in a lymphoma cell line greatly reduces metastatic capacity. Cancer Res., 58, 1569–1577.[Abstract]

Swarte, V.V., Mebius, R.E., Joziasse, D.H., Van den Eijnden, D.H., and Kraal, G. (1998) Lymphocyte triggering via L-selectin leads to enhanced galectin-3-mediated binding to dendritic cells. Eur. J. Immunol., 28, 2864–2871.[ISI][Medline]

Tarleton, R.L. and Kuhn, R.E. (1983) Changes in cell populations and immunoglobulin-producing cells in the spleens of mice infected with Trypanosoma cruzi: correlations with parasite-specific antibody response. Cell. Immunol., 80, 392–404.[ISI][Medline]

Timoshenko, A.V., Gorudko, I.V., Maslakova, O.V., Andre, S., Kuwabara, I., Liu, F.T., Kaltner, H., and Gabius, H.J. (2003) Analysis of selected blood and immune cell responses to carbohydrate-dependent surface binding of proto- and chimera-type galectins. Mol. Cell. Biochem., 250, 139–149.[CrossRef][ISI][Medline]

Tyler, K.M. and Engman, D.M. (2001) The life cycle of Trypanosoma cruzi revisited. Int. J. Parasitol., 31, 472–480.[CrossRef][ISI][Medline]

Van Overtvelt, L., Vanderheyde, N., Verhasselt, V., Ismaili, J., De Vos, L., Goldman, M., Willems, F., and Vray, B. (1999) Trypanosoma cruzi infects human dendritic cells and prevents their maturation: inhibition of cytokines, HLA-DR, and costimulatory molecules. Infect. Immun., 67, 4033–4040.[Abstract/Free Full Text]

Van Overtvelt, L., Andrieu, M., Verhasselt, V., Connan, F., Choppin, J., Vercruysse, V., Goldman, M., Hosmalin, A., and Vray, B. (2002) Trypanosoma cruzi down-regulates lipopolysaccharide-induced MHC class I on human dendritic cells and impairs antigen presentation to specific CD8(+) T lymphocytes. Int. Immunol., 14, 1135–1144.[Abstract/Free Full Text]

Villa-Verde, D., Calado, T.C., Ocampo, J., Silva-Monteiro, E., and Savino, W. (1999) The conveyor belt hypothesis for thymocyte migration: participation of adhesion and de-adhesion molecules. Braz. J. Med. Biol. Res., 32, 569–572.[ISI][Medline]

Villa-Verde, D.M., Silva-Monteiro, E., Jasiulionis, M.G., Farias-De-Oliveira, D.A., Brentani, R.R., Savino, W., and Chammas, R. (2002) Galectin-3 modulates carbohydrate-dependent thymocyte interactions with the thymic microenvironment. Eur. J. Immunol., 32, 1434–1444.[CrossRef][ISI][Medline]

Vrasidas, I., Andre, S., Valentini, P., Bock, C., Lensch, M., Kaltner, H., Liskamp, R.M., Gabius, H.J., and Pieters, R.J. (2003) Rigidified multivalent lactose molecules and their interactions with mammalian galectins: a route to selective inhibitors. Org. Biomol. Chem., 1, 803–810.[CrossRef][ISI][Medline]

Vray, B. (2002) Macrophages in parasitic infection. In Bernard Burke and Claire E. Lewis (Eds.), The Macrophage. Oxford University Press, Oxford, pp. 253–304.

Zuniga, E., Gruppi, A., Hirabayashi, J., Kasai, K.I., and Rabinovich, G.A. (2001) Regulated expression and effect of galectin-1 on Trypanosoma cruzi-infected macrophages: modulation of microbicidal activity and survival. Infect. Immun., 69, 6804–6812.[Abstract/Free Full Text]