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
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Abstract |
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Key words: galectin-3 / glycocytochemistry / neoglycoconjugate / spleen murine dendritic cells / Trypanosoma cruzi
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Introduction |
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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., 2002; Reuter and Gabius, 1999
). 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, 2002
; Gabius, 1997
; Kasai and Hirabayashi, 1996
). 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, 2002
; Lahm et al., 2001
; Nagy et al., 2003
; Timoshenko et al., 2003
). 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., 2000
; Higashi et al., 2002
). 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, 2002; Gabius, 1997
; Kasai and Hirabayashi, 1996
). Its designation as MAC-2 antigen is popular in immunology, and it is localized in various tissues and immunocompetent/inflammatory cells (Flotte et al., 1983
; Rabinovich et al., 2002
). Gal-3 is involved in many immunoregulatory processes, such as DC/T lymphocyte adhesion (Swarte et al., 1998
), cellcell adhesion and adhesion of cells to matrix glycoproteins (Kaltner and Stierstorfer, 1998
), inflammatory responses and cell migration toward inflammatory foci and cell proliferation (Rabinovich et al., 2002
), and is also known as a new chemoattractant (Sano and Liu, 2001
). Gal-3 participates in myelin phagocytosis in Schwann cells (Reichert et al., 1994
) 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., 2001
). 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., 2001
; Brewer, 2002
; Gabius, 2001
; Vrasidas et al., 2003
). 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, 2002
) 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, 1999). 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., 2001
; Zuniga et al., 2001
). However, although a recent report describes Gal-3-depending binding of T. cruzi trypomastigotes to laminin (Moody et al., 2000
), 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., 1999
; Olivares-Fontt et al., 1996
). We studied the impact of this infection on splenic DCs (sDCs) and a DC line (D2SC-1) (Lutz et al., 1994
; O'Rourke et al., 2000
) 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.
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Results |
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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 /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 - 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. cruziconditioned 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).
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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., 1998). 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. cruziinfected and TCM-treated D2SC-1 cells (Figure 5).
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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., 1994; Moody et al., 2000
). 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.
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Discussion |
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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., 2000; Smetana et al., 1999
) 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., 2002
; Reuter and Gabius, 1999
). 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
-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, 1997
; Steinman, 2000
) and a C-type lectin first found on macrophages (Iida et al., 1999
). Remarkably, this macrophage C-type lectin failed to bind the Forssman disaccharide despite its activity for
-GalNAc (Iida et al., 1999
) 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., 1985; Buckner et al., 1999
; de Diego et al., 1991
; Deutschlander et al., 1978
; Melo and Brener, 1978
; Tarleton and Kuhn, 1983
). 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., 1999
, 2002
). 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., 2002
). Furthermore, Brodskyn et al. (2002)
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 cellderived 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, 1999) 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 DCT 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., 1998
). However, Gal-3 has also an anti-adhesion property because it decreases thymocytes interaction with the thymic microenvironment (Villa-Verde et al., 1999
, 2002
), a property also found for tumor cells (Andre et al., 1999
). 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. cruziinfected 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. cruziinfected D2SC-1 cells (and not TCM-treated D2SC-1 cells; Figure 4) showed an up-regulation of Gal-3 expression. Both T. cruziinfected 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, 1988).
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., 2003; Planelles et al., 2003
). 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., 1999
, 2002
). 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. cruziinfected mice (Chaussabel et al., 2003
). 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., 2003
). 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, 2001) involves several recognition/adhesion systems leading to host cell invasion followed by an obligate intracellular multiplication step (for review see Vray, 2002
). 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., 1994
). 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
1-3Gal determinants on T. cruzi has been reported (Bretana et al., 1992
) and also further types of lectincarbohydrate interactions participate in the interactions between trypanosomatids and host's cells, for example, for glycoaminoglycans (Kock et al., 1997
). 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.
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Materials and methods |
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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., 1999). 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) 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., 1999
).
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., 1999a).
The D2SC-1 cells (Lutz et al., 1994; O'Rourke et al., 2000
) 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. cruziinfected 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., 1999b).
Semi-quantitative RT-PCR analysis
Semi-quantitative RT-PCR analysis were performed as previously described (Belot et al., 2001). 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., 2003).
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., 2001a,b
). 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., 2001b). 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., 2002). The protein Arefined 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., 1999
, 2001
).
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 - and ß-anomers of N-acetyl-D-galactosamine (
/ß-D-GalNAc), ß-D-glucose,
-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(
1-4)-D-Glc). The chemical structures of these seven sugar moieties are illustrated and detailed elsewhere (Camby et al., 2001b
).
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., 2001a,b
). 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., 2001). 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., 1989) and for laminin (Stroeken et al., 1998
). 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., 1998; Hittelet et al., 2003
). 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., 1998
; Hittelet et al., 2003
). 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 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).
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