KM+, a lectin from Artocarpus integrifolia, induces IL-12 p40 production by macrophages and switches from type 2 to type 1 cell-mediated immunity against Leishmania major antigens, resulting in BALB/c mice resistance to infection

Ademilson Panunto-Castelo1,3,5, Maria A. Souza1,5,6, Maria-Cristina Roque-Barreira2,3,5 and João S. Silva4,5

3Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, SP 14040-900, Brazil; 4Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, SP 14040-900, Brazil; 5Pós-graduação em Imunologia Básica e Aplicada, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, SP 14040-900, Brazil; and 6Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Uberlândia, MG 38400-902, Brazil

Received on June 18, 2001; revised on August 29, 2001; accepted on August 31, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
The outcome and severity of some diseases correlate with the dominance of either the T helper 1 (Th1) or Th2 immune response, which is stimulated by IL-12 or IL-4, respectively. In the present study we demonstrate that gamma interferon (IFN-{gamma}) secretion by murine spleen cells stimulated with KM+, a mannose-binding lectin from Artocarpus integrifolia, is due to IL-12 induction, because (1) macrophages from several sources (including cell lines) produced IL-12 p40 in response to KM+, and (2) lectin-free supernatants from J774 cell line cultures stimulated with KM+ induced the secretion of IFN-{gamma} by spleen cell cultures, an effect blocked by the supernatant pretreatment with anti-IL-12 antibody. The known pattern of susceptibility of BALB/c mice to infection with Leishmania major, attributed to high levels of IL-4 production leading to a Th2 nonprotective immune response, was modified by administration of KM+. Draining lymph node cells from these immunized BALB/c mice (in contrast to cells from animals immunized only with soluble leishmanial antigen [SLA]) secreted high levels of IFN-{gamma} and low levels of IL-4, which characterized a Th1 rather than a Th2 response pattern. The footpad thickness of BALB/c mice immunized with SLA plus KM+ and challenged with L. major was similar to that of uninfected mice. This beneficial effect against leishmanial infection was blocked by pretreatment of these mice with anti-IL-12 antibody. These observations indicate that KM+ induces IL-12 p40 in vivo and has a protective effect against L. major infection.

Key words: Artocarpus integrifolia/interleukin 12/KM+/lectin/Leishmania major


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
When experimentally infected with Leishmania major, an obligate intracellular parasite of macrophages in mammalian hosts, mice of several inbred strains (e.g., C57BL/6, C3H, CBA) are resistant to leishmaniasis. In contrast, BALB/c mice are susceptible, develop severe lesions, and do not become immune to reinfection. Murine resistance and susceptibility are genetically determined and clearly related to the development of the polarized CD4+ T helper 1 (Th1) and CD4+ T helper 2 (Th2) response, respectively (Reiner and Locksley, 1995Go). The differentiation of CD4+ T cells to Th1 and Th2 cells has been associated with production of interleukin-12 (IL-12) (Hsieh et al., 1993Go; Seder et al., 1993Go; Macatonia et al., 1995Go) and IL-4 (Le Gros et al., 1990Go; Hsieh et al., 1992Go; Seder et al., 1992Go), respectively. IL-12 is a heterodimeric protein composed of an induced 40-kDa (p40) subunit and a constitutive 35-kDa (p35) subunit. Early IL-12 production by antigen-presenting cells, such as macrophage and dendritic cells, in L. major infection is an absolute requirement for developing protective specific immunity (Sypek et al., 1993Go).

In the innate immune system, cytokine production is usually the result of cell activation, which can be induced by interactions of carbohydrate-recognition molecules with the glycoside part of a receptor on the cell surface. If the recognized receptor is involved in signal transduction pathways, the lectin binding can lead to specific cellular responses, including cytokine release (Villalobo and Gabius, 1998Go). Some animal, parasite, and plant lectins induce cytokine production, among them those related to the Th1 response, such as interferon-{gamma} (IFN-{gamma}) (Pryjma et al., 1991Go; Paul and Seder, 1994Go; Hostanska et al., 1995Go; Kishko et al., 1997Go) and IL-12 (Muraille et al., 1999Go; Campbell et al., 2000Go).

KM+ and jacalin are structurally related lectins extracted from jackfruit seeds (Artocarpus integrifolia) that present distinct sugar specificity and biological properties. Jacalin binds to D-galactose and is highly specific for glycoproteins having a terminal nonreducing {alpha}-D-galactosyl residue as well as for the disaccharide Galß1-3GalNAc{alpha}1-O-Ser/Thr (Hortin and Trimpe, 1990Go). KM+ binds to D-mannose and exhibits higher specificity for the trisaccharide present in the core of the N-linked oligosaccharide chains of glycoproteins (Man{alpha}1-3[Man{alpha}1-6]Man) (Rani et al., 1999Go). Very recently, the sugar specificity of A. integrifolia lectins was examined in molecular terms. Although KM+ and jacalin share 52% sequence identity and have common evolutionary origin, KM+, in contrast to jacalin, is not cleaved posttranslationally in two chains, conserving a glycine-rich linker, which sterically dictates the discrimination between mannose and galactose (Rosa et al., 1999Go). In terms of biological properties, jacalin induces IL-6 secretion by U937 monocytic cells (Taimi et al., 1994Go) and potentiates mouse humoral immune response to trinitrophenyl and Trypanosoma cruzi (Albuquerque et al., 1999Go). KM+, in turn, has been used as a tool to study the haptotactic mechanism of neutrophil migration in rats (Santos-de-Oliveira et al., 1994Go), made possible by the concomitant interaction of the lectin with appropriate glycans on both neutrophil surface (Pereira-da-Silva et al., in preparation) and extracellular matrix (Ganiko et al., 1998Go).

We now show that KM+ lectin induces macrophages to produce IL-12 p40, which then stimulates IFN-{gamma} secretion by lymphocytes. The injection of KM+ in BALB/c mice induced an inversion of cytokine pattern from Th2 to Th1 (from IL-4 to IFN-{gamma}). Following inoculation with KM+, BALB/c mice became resistant to L. major infection. These observations open perspectives concerning the use of the lectin KM+ for protection against intracellular pathogens.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
The lectin KM+ stimulates IFN-{gamma} production by mouse spleen cells
When stimulated with the lectin KM+, spleen cells from BALB/c mice produced IFN-{gamma}, drawing a bell shaped dose-response curve (Figure 1). The ascending limb was determined by the range of 0.5–1.0 µg/ml KM+ concentrations; maximal IFN-{gamma} production was 150.45 ± 0.91 U/ml. Higher KM+ concentrations determined a progressive reduction of IFN-{gamma} production, probably due to toxic effects exerted by the lectin on spleen cells. Responses to KM+ concentrations as low as 0.25 µg/ml were similar to the negative control.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1. KM+ induces IFN-{gamma} production by mouse spleen cells in a dose-response manner. Spleen cells (2 x 106/ml) from BALB/c mice were cultured in the presence of different concentrations of KM+ for 48 h. Results are the means of IFN-{gamma} concentration ± SD in triplicate wells. The results are representative of at least three experiments.

 
IFN-{gamma} release by mouse spleen cells stimulated with KM+ is due to lectin-induced IL-12 p40 production by macrophages
To determine if the observed IFN-{gamma} production by spleen cells was dependent on a soluble mediator induced in macrophages, the supernatant of J774 cells stimulated with KM+ was depleted of lectin by exhaustive adsorption to immobilized mannose and added to a nonstimulated culture of mouse spleen cells. Although this supernatant was 10 times diluted, it provoked an expressive response of IFN-{gamma} production (Figure 2A). In contrast, no response was induced by the supernatant of nonstimulated cells (Figure 2A) or by KM+ adsorbed on immobilized D-mannose to determine the efficiency of the lectin depletion procedure (data not shown). Positive controls were provided by the stimulus with optimal concentrations of KM+ or IL-12, all inducing high IFN-{gamma} release by spleen cells. We hypothesized that IL-12 could be the mediator released in the supernatant of KM+-stimulated macrophages able to induce IFN-{gamma} production by spleen cells. Supernatant samples of KM+-stimulated macrophages were then pretreated with anti-IL-12 monoclonal antibody (mAb), serially diluted, and assayed in terms of IFN-{gamma} induction on spleen cells. Taking as reference the response to macrophage supernatant pretreated with a nonrelevant antibody, a dose-dependent inhibition of IFN-{gamma} induction was clearly determined by the supernatant pretreated with anti-IL-12 antibody (Figure 2B). In addition, IL-12 p40 concentrations on the supernatants of J774 cells or peritoneal macrophages from BALB/c mice were proportional to the KM+ doses used to stimulate the cells (Figure 3). IL-12 induction by KM+ was not limited to J774 cells or peritoneal macrophages, because other macrophages derived from cell lines (macrophages 05 and 63) or spleen cells were also responsive to KM+ (Table I). The cytokine induction was not due to contamination of KM+ preparation with lipopolysaccharide from Gram-negative bacteria (LPS) because macrophages from C3H/HeJ mice (hyporesponsive to LPS) produced levels of IL-12 p40 very close to those released by peritoneal cells from LPS-responsive mice. In addition, the KM+ preparation contained less than 0.05 ng/ml of bacterial endotoxin, as determined by the Limulus amoebocyte lysate assay (data not shown). Concanavalin A (Con A), used as a control lectin, did not induce IL-12 p40 production (Table I).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2. The supernatant from KM+-stimulated J774 murine macrophage cell line induces IFN-{gamma} production by spleen cells, an effect inhibited by anti-IL-12 mAb. (A) J774 cells were cultured in the presence of KM+ (3 µg/ml). After 48 h, the supernatant was depleted of lectin by adsorption to D-mannose-agarose and used, 10 times diluted, to stimulate BALB/c spleen cells (2 x 106/ml) (Sup Mac KM+). Negative control: supernatant from nonstimulated J774 cells (Sup Mac Medium). Positive controls: KM+ (1 µg/ml) itself or rmIL-12 (2 ng/ml). Asterisk indicates significant difference (P < 0.0001) from value for negative control group (supernatant from nonstimulated J774 cells) as calculated by Student t-test. (B) Supernatants from KM+-stimulated J774 cell cultures were preincubated in the presence of different concentrations of (open circles) rat anti-IL-12 mAb or (closed circles) irrelevant rat mAb for 1 h before being added to the cultures. Results are the means of IFN-{gamma} concentration ± SD in triplicate wells. The results are representative of three experiments.

 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3. IL-12 production by (closed circles) J774 murine macrophage cell line or (open circles) BALB/c mouse peritoneal macrophages stimulated with the lectin KM+. J774 cells (2 x 106/well) or adherent cells (2 x 106/well) from thioglycollate-stimulated peritoneal cavity were cultured in the presence of different concentrations of KM+ for 48 h. Results are the means of IL-12 concentration ± SD in triplicate wells. The results are representative of three experiments.

 

View this table:
[in this window]
[in a new window]
 
Table I. In vitro IL-12 production by macrophages stimulated with KM+ lectin.
 
KM+ induces cytokine production through its carbohydrate-recognition property
The evidence that KM+ induces IL-12 p40 production suggested the obvious question concerning the dependency or not of the response on the carbohydrate-recognition domain of the lectin. To address this question, inhibition assays were carried out in the presence of sugars. The KM+-induced IL-12 p40 production by macrophages was remarkably inhibited by D-mannose (P < 0.0001) as compared to non–carbohydrate-treated or D-galactose–treated cells (Figure 4A). A similar pattern of monosaccharide inhibition was observed regarding IFN-{gamma} production by spleen cells stimulated with KM+ (data not shown). The carbohydrate inhibition effect was still more drastic when the mannotriose (Man{alpha}1-3[Man{alpha}1-6]Man) was used. As a structure recognized by KM+ with high affinity (Rani et al., 1999Go), mannotriose inhibited in a dose-dependent manner the IFN-{gamma} production by KM+-stimulated spleen cells, but not that directly induced by IL-12 (Figure 4B).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4. Cytokine production by KM+-stimulated cells is specifically inhibited by D-mannose. (A) J774 cells (2 x 106/well) were stimulated with KM+ (3 µg/ml) in the presence or absence of either D-mannose (0.1 M) or D-galactose (0.1 M) for 48 h. Results are the means of IL-12 concentration ± SD in triplicate wells. Asterisk indicates significant difference (P < 0.0001) from value for positive control group (KM+) and D-galactose-treated group as calculated by Student t-test. (B) Spleen cells (2 x 106/well) were stimulated with either (open circles) KM+ (1 µg/ml) or (closed circles) rmIL-12 (2 ng/ml) in the presence of different concentrations of mannotriose (Man{alpha}1-3[Man{alpha}1-6]Man). Results are the means of IFN-{gamma} concentration ± SD in triplicate wells.

 
KM+ administration modifies the cytokine pattern in mice, inducing resistance to L. major infection
Because KM+ induces macrophages to produce IL-12 p40, a major cytokine known to drive the immune response to a Th1 pattern, we hypothesized that KM+ could invert a cytokine pattern from Th2 to Th1 in vivo. To address this question we injected into the footpad of BALB/c mice 25 µg soluble leishmanial antigen (SLA), which typically induces a Th2 response, accompanied or not by 0.5 µg KM+. The animals of another group were injected with KM+ alone. The draining lymph node cells from all these mice were in vitro stimulated with SLA (50 µg/ml) and analyzed in terms of cytokine production. High concentrations of IL-4 (4.71 ± 0.4 ng/ml) and low concentrations of IFN-{gamma} (1.43 ± 0.28 ng/ml) were released by cells from the animals injected with SLA alone. In contrast, lower IL-4 (2.2 ± 0.57 ng/ml) and higher IFN-{gamma} (13.2 ± 0.31 ng/ml) concentrations were produced by draining lymph node cells from animals injected with SLA plus KM+ (Figure 5), corroborating the idea that a drive toward a Th1 antigen-specific response is stimulated in vivo by the lectin. This observation motivated us to investigate the course of experimental leishmaniasis in animals treated with KM+. The size of lesion present on the L. major inoculated pad of each mouse was used as a parameter to evaluate the disease for 6 weeks. Smaller lesions were observed in animals treated with KM+, accompanied or not by SLA, than in animals injected with SLA alone, which presented lesions of the same size as those of animals injected with phosphate buffered saline (PBS) alone. The beneficial effect of KM+ injection was especially detectable 5–6 weeks after infection (Figure 6A). To determine if the IL-12-inducing activity of KM+ was responsible for the in vivo inversion of secreted cytokines toward a Th1 pattern and for protection against leishmaniasis, animals pretreated with KM+ were injected with anti-IL-12 mAb and infected. As shown in Figure 6B, anti-IL-12 mAb abrogated the protection induced by the lectin according to the detected size of lesions. This effect was clearly manifested 4 weeks after L. major infection.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 5. Mouse immunization with SLA plus KM+ drives the cytokine production from Th2 to Th1 pattern. IFN-{gamma} (closed circles) and IL-4 (open circles) concentrations were determined in the culture supernatants from lymph node cells (5 x 106/ml) stimulated with SLA (50 µg/ml) for 72 h. These cells were obtained from mice immunized with SLA (500 µg/ml) in combination (SLA/KM+) or not (SLA) with the lectin KM+ (10 µg/ml). Assigned KM+ and PBS refer to cells obtained from mice injected with either lectin or PBS alone, respectively. The results are representative of three experiments.

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6. KM+ induces protection against L. major infection in BALB/c mice by an IL-12-dependent mechanism. (A) BALB/c mice were immunized with SLA, in combination (KM+/SLA, closed triangles) or not (SLA, open triangles) with the lectin KM+. An experimental group was pretreated with the lectin only (KM+, closed circles). PBS was used as a negative control of the pretreatment (open circles). Three days after the last boosting, the animals were inoculated in one of the hind footpads with 1 x 106 infective-stage metacyclic promastigotes of L. major. (B) Animals pretreated with (closed circles) KM+ were concomitantly injected with either anti-IL-12 (open triangles) or irrelevant antibodies (closed triangles). A control group was pretreated with PBS alone (open circles). The evolution of the lesion was monitored by measuring footpad thickness. The results are representative of at least three experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
In the present article we demonstrated that the lectin KM+ induces macrophages to produce IL-12 p40, an ability that caused a protective Th1 response in BALB/c mice against infection with L. major.

Initially we demonstrated in vitro that KM+ promotes IFN-{gamma} production by murine spleen cells. This response was indirectly provoked by IL-12 p40 secretion induced by KM+, because isolated macrophages, including some from cell lines, produced IL-12 p40 when stimulated with KM+, and lectin-free culture supernatants from the KM+-stimulated J774 cell line has induced IFN-{gamma} production by spleen cells. This effect was blocked by pretreatment of the supernatant with anti-IL-12 antibody.

Substances able to induce IL-12 and/or IFN-{gamma} production are studied for their potential applicability as adjuvants in the vaccination against some parasites, whose survival depend on avoiding a host Th1 response. Recently, Muraille et al. (1999)Go have screened some plant lectins in terms of IL-12 inducing ability. Jacalin, the D-gal-binding lectin from A. integrifolia, was unable to induce IL-12, a fact also observed by us (data not shown), whereas Con A, used as a control lectin in our experiments, was reported by these authors as one of four lectins endowed with IL-12-inducing activity. Our observations concerning distinct activities of two mannose-binding lectins, KM+ and Con A, is probably related to their different fine specificities of sugar recognition. KM+ has a higher preference for mannopyranoside over glucopyranoside than Con A. Con A binds to mannobioses in the following affinity order, Man{alpha}1-2Man >> Man{alpha}1-6Man > Man{alpha}1-3Man, through a site that essentially accommodates a monosaccharide. Man{alpha}1-2Man, as compared to mannose itself, is poorly recognized by KM+. The mannobioses Man{alpha}1-3Man and Man{alpha}1-6Man have very different affinities for KM+, because they bind, respectively, 2- and 17-fold less to the lectin than the mannotriose (Man{alpha}1-3[Man{alpha}1-6]Man) does. This indicates that {alpha}1-3-linked mannose occupies the primary binding site, and the {alpha}1-6Man occupies the secondary subsite. These binding sites, specific for the {alpha}1,3 and {alpha}1,6 arms for the two oligosaccharides, together with the 3,6-disubstituted mannose residue, constitute the extended CRD of KM+ (Rani et al., 1999Go). The specificity of KM+ for Man{alpha}1-3(Man{alpha}1-6)Man was confirmed by our inhibition assays of cytokine induction, because concentrations as low as 5 or 10 mM mannotriose were able to inhibit by 50% or 95%, respectively, the IFN-{gamma} production by KM+-stimulated spleen cells. A similar KM+ specificity pattern has been observed in inhibition assays of neutrophil haptotaxis induced by KM+ (Ganiko, unpublished data). The carbohydrate inhibition pattern of the KM+ effects on cells suggests that membrane receptors recognized by the lectin and responsible by cell signaling contain glycan(s) with Man{alpha}1-3(Man{alpha}1-6)Man.

The importance of the KM+ property of inducing IL-12 p40 for driving the immune response to a Th1 pattern has been substantiated in vivo using a murine model of infection with L. major. Mice were of the BALB/c strain, known to be highly susceptible to the infection, a fact attributed to the polarization of the immune response toward a Th2 pattern. In fact, this preferential driving is critically associated with IL-4 production, which, by occurring in the initial phase of infection, will be sufficient to instruct Th2 cell development and to establish progressive disease (Himmelrich et al., 2000Go). The early burst of IL-4 expression in BALB/c mice occurs in a restricted population of Vß4-V{alpha}8 CD4+ T cells following cognate recognition of a single epitope of the Leishmania homologous to mammalian RACK1 (LACK). The role of these LACK-reactive cells in the development of an aberrant Th2 response in BALB/c mice has already been demonstrated to be critical. When depleted of Vß4 T cells, BALB/c mice did not generate early IL-4 transcripts in CD4+ T cells on the first day of infection, a Th1 response was developed and the mice became resistant to L. major infection (Launois et al., 1997Go). In addition, BALB/c mice tolerant to LACK were resistant to the infection (Julia et al., 1996Go). Recently, Julia et al. (2000)Go have demonstrated that lymphoid organs of naive BALB/c mice contain T cells with a memory/effector phenotype and specificity for a microbial antigen from the intestinal flora, which cross-react with LACK. These LACK-reactive T cells secrete IL-4 shortly after L. major infection. In our experimental model, the draining lymph node cells from BALB/c mice that received only SLA released the expected Th2 pattern of cytokines, with high IL-4 and low IFN-{gamma} levels. This was successfully reversed by KM+ administration, with production of low IL-4 and high IFN-{gamma} levels, validating the hypothesis that the IL-12 p40–inducing property of the lectin could be important to drive immunity to a Th1 response, able to protect an organism against Leishmania infection.

The immunized mice were challenged with L. major, and their footpad thickness was observed, a usual parameter to evaluate the course of L. major infection. The mice that received KM+ with or without the antigen had a footpad thickness similar to that of uninfected mice. This beneficial effect was blocked by pretreatment with anti-IL-12 antibody before the challenge, because in this experimental condition the animals’ footpads were as thick as that of nonimmunized mice challenged with L. major. Although antigen is required for the IFN-{gamma} production by lymph node cells from immunized mice, the beneficial effect of KM+ on the manifestation of the infection was independent of antigen administration. These data indicate that KM+ is an IL-12 p40–inducing lectin, which acts in vivo by promoting a protective Th1 immune response against L. major infection.

Even though KM+ appears to induce protective effect against infections whose pathogens are susceptible to Th1-mediated immune response, some questions about the mechanism of KM+ action remain to be answered. The macrophage receptor to which KM+ binds to induce IL-12 release is still under investigation. Targets of KM+ recognition on the surface of other leukocytes are also under study in our laboratory. Human neutrophil receptor for KM+, implicated on the lectin attractant property, belongs to the family of heptahelical receptors coupled to heterotrimeric G proteins (Pereira-da-Silva et al., in preparation), whereas rat mast cells are degranulated by KM+ as a response to its direct interaction with the IgE receptors on cell surface (Moreno, unpublished data). The protective effect of KM+ in other infection models, in which Th1 response is protective, is currently under examination, opening perspectives about the use of KM+ as an immunoregulator.


    Material and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
Mice and reagents
Female BALB/c and C3H/HeJ mice, 6–8 weeks old, were bred and maintained under standard conditions in the animal house of the School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil. Lectin KM+ was purified as previously described (Santos-de-Oliveira et al., 1994Go). Briefly, dried seeds from A. integrifolia were ground and suspended, 10% (w/v), in 10 mM PBS, pH 7.2, for 24 h at 37°C. After centrifugation, supernatant fraction (crude extract) was dialyzed and depleted of jacalin by at least three suscessive adsorption procedures on a 5-ml settled bed of D-galactose-agarose. The absence of jacalin was checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The jacalin-depleted extract was submitted to affinity chromatography on a 5-ml bed of D-mannose-agarose, previously equilibrated at 4°C with PBS containing 0.5 M NaCl. After washing with equilibrating buffer, the adsorbed material was eluted with 0.1 M D-mannose in equilibrating buffer, concentrated, and dialyzed against PBS in an ultradiafiltration system using YM10 membrane (Amicon Division, W.R. Grace, Beverly, MA). KM+ preparation contained less than 0.05 ng/ml of bacterial endotoxin, as determined by the Limulus amoebocyte lysate assay (Sigma-Aldrich, St. Louis, MO).

Con A was purchased from ICN Pharmaceuticals (Costa Mesa, CA). Recombinant murine IL-2 (rmIL-2) (200 µg/ml) and rmIL-4 (500 µg/ml) were obtained from Genzyme (Cambridge, MA). rmIFN-{gamma} (2 x 105 U/ml) and rmIL-12 (500 µg/ml) were obtained from Sigma-Aldrich and Genetics Institute (Boston, MA), respectively. mAb to the following murine molecules were obtained as generous gifts: IL-12 (C17.8, C15.8, and C15.1) (G. Trinchieri, Wistar Institute, Philadelphia, PA), IFN-{gamma} (XMG1.2) (DNAX, Palo Alto, CA), and IL-4 (DVD6 and 11B11) (T. R. Mosmann, University of Alberta, Edmonton, Canada).

Parasites and preparation of SLA
Promastigotes of L. major LV39 strain (MRHO/SU/59/P) were maintained at 28°C in Schneider’s Drosophila medium (Sigma-Aldrich) supplemented with 2 mM of L-glutamine, and 20% heat-inactivated fetal calf serum (Gibco BRL, Life Technologies, Gaithersburg, MD), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM HEPES (Sigma-Aldrich). SLA was prepared from log-phase L. major by sonication and ultracentrifugation as previously described (Scott et al., 1987Go).

Immunization and infection of mice
Using previously described immunization protocols (Afonso et al., 1994Go), BALB/c mice (five per group) were injected with 50 µl PBS, KM+ (10 µg/ml), SLA (500 µg/ml), or KM+ plus SLA (10 µg/ml and 500 µg/ml) in the hind footpad. After 10 and 22 days, mice were boosted subcutaneously and intraperitoneally, respectively, with the same preparations. In some experiments, in addition to being injected with KM+, the mice were concomitantly treated intraperitoneally with rat anti-mouse IL-12 IgG (mAb C17.8) (1 mg) or irrelevant rat IgG (control isotype) (1 mg). Three days after the last boosting, the animals were inoculated in one of the hind footpads with 1 x 106 infective-stage metacyclic promastigotes of L. major, isolated as previously described (Afonso et al., 1994Go), from stationary culture (4–5 days old) by negative selection using peanut agglutinin (Vector Laboratories, Burlingame, CA). The evolution of the lesion was monitored by measuring footpad thickness using a metric caliper.

Lymph node cell cultures
Popliteal lymph node cells from immunized mice were washed three times in Hank’s balanced salt solution (HBSS) and adjusted to 5 x 106 cells/ml in RPMI-C (RPMI 1640 [Flow Laboratories, McLean, VA] containing 2 mM L-glutamine, 50 µM 2-mercaptoethanol, 100 U/ml penicillin, 100 µg/ml streptomycin [Sigma-Aldrich], and 5% heat-inactivated fetal calf serum [Hyclone, Logan, UT]). The cell suspension was distributed in 24-well cell culture plates (Corning, Corning, NY), 1 ml per well, and cultured at 37°C in a humidified 5% CO2 atmosphere in the presence or in the absence of SLA (50 µg/ml). After 72 h incubation, the supernatants were harvested by centrifugation and stored at –20°C until enzyme-linked immunosorbent assay (ELISA)–based cytokine measurements were performed.

Macrophage and cell line cultures
BALB/c or C3H/HeJ mouse macrophages harvested from peritoneal cavities 3 days after the injection of 1 ml of 3% sodium thioglycollate (Sigma-Aldrich), or adherent spleen cells were washed in HBSS, resuspended in RPMI-C, and dispensed in 24-well cell culture plates (2 x 106 cells/well). After 2–4 h incubation at 37°C, the nonadherent cells were removed by exhaustive washing with HBSS, and the adherent cells incubated with KM+ (1 µg/ml) or Con A (2 µg/ml) in RPMI-C. After 48 h incubation, the supernatants were harvested by centrifugation and stored at –20°C until ELISA-based cytokine measurements were performed.

The 05 and 63 macrophage hybridomas (kindly provided by M. E. Dorf, Harvard Medical School, Boston, MA) were resuspended in RPMI-C and cultured in 25-cm2 tissue culture flask at 37°C in a humidified 5% CO2 atmosphere. After 48 h, the confluent cell monolayer was scraped, washed in Hanks’ medium, resuspended in RPMI-C, and dispensed in 24-well cell culture plates (2 x 106/well). After adherence, cell monolayers were washed in HBSS and incubated with KM+ or Con A, as described above. In some experiments, KM+ (3 µg/ml) was preincubated or not with 0.1 M D-mannose or 0.1 M D-galactose for 1 h before being added to the cultures. After 48 h incubation, the supernatants were harvested by centrifugation and stored at –20°C until ELISA-based cytokine measurements were performed.

Spleen cell cultures
Suspensions of spleen cells from normal and infected mice were washed in HBSS and treated with lysing buffer (nine parts of 0.16 M ammonium chloride and one part of 0.17 M Tris–HCl, pH 7.5) for 4 min. The erythrocyte-free cells were then washed three times in HBSS and adjusted to 2 x 106 cells/ml in RPMI-C. The cell suspensions were distributed in 24-well cell culture plates (Corning), 1 ml per well, and cultured for 48 h at 37°C in a humidified 5% CO2 atmosphere in the presence or absence of KM+ (0.25 to 10 µg/ml) or rmIL-12 (2 ng/ml). In some experiments, KM+ was preincubated with different doses of D-mannose or mannotriose (Man{alpha}1-3[Man{alpha}1-6]Man) (Dextra Laboratories, Reading, UK) for 1 h before being added to the cultures. After 48 h incubation, the supernatants were harvested by centrifugation and stored at –20°C until ELISA-based cytokine measurements were performed.

Supernatants from the J774 macrophage cell line stimulated or not with 3 µg/ml KM+ for 48 h were depleted of lectin by adsorption on D-mannose-agarose beads. Adsorption of a KM+ preparation (same volume of the supernatant, 3 µg/ml) on D-mannose-agarose column was used as a control of lectin depletion procedure. The lectin-free supernatants (100 µl) were added to a spleen cell culture (900 µl) and cultured at 37°C in a humidified 5% CO2 atmosphere. In some experiments, macrophage supernatants were preincubated with 5–100 µg/ml anti-IL-12 IgG (mAb C17.8) or rat irrelevant IgG (control isotype) for 1 h before being added to the cultures. After 48 h incubation, the supernatants were harvested by centrifugation and stored –20°C until ELISA-based cytokine measurements were performed.

ELISA-based cytokine detection assay
All cytokines were detected as secreted protein products in culture supernatants using cytokine-specific ELISA. Microtiter plates were coated with capture mAb C17.8 (IL-12 p40), XMG-1.2 (IFN-{gamma}), or BVD6 (IL-4). Detection was carried out with a cytokine-specific polyclonal rabbit antibody, a goat anti-rabbit IgG biotin conjugate, and streptoavidin peroxidase. The reaction was developed using OPD as substrate (Abbott Diagnostics). Standard curves were prepared with rmIL-12, rmIFN-{gamma}, or rmIL-4.

Statistical analysis
Statistical determinations of the difference between means of experimental groups were performed using a two-tailed Student t-test for unpaired data. Differences which provided P < 0.01 were considered to be statistically significant. All experiments were performed at least three times.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Mrs. Sandra M.O. Thomaz, Ms. Imaculada C. Bragheto, Ms. Patrícia E. Vendruscolo, and Mr. Wander C.R. Silva for technical assistance and Mrs. Elettra Greene for revising the English of the text. A. Panunto-Castelo received a postdoctoral research fellowship from FAPESP (98/00701-7). We are grateful to Dr. Lewis J. Greene for a critical reading of the manuscript.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
Con A, concanavalin A; ELISA, enzyme-linked immunosorbent assay; HBSS, Hank’s balanced salt solution; IFN-{gamma}, interferon-{gamma}; IL, interleukin; LACK, Leishmania homologous to mammalian RACK1; LPS, lipopolysaccharide from Gram-negative bacteria; mAb, monoclonal antibody; SLA, soluble leishmanial antigen; PBS, phosphate buffered saline; Th1, CD4+ T helper 1 cells; Th2, CD4+ T helper 2 cells.


    Footnotes
 
1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
Afonso, L.C., Scharton, T.M., Vieira, L.Q., Wysocka, M., Trinchieri, G., and Scott, P. (1994) The adjuvant effect of interleukin-12 in a vaccine against Leishmania major. Science, 263, 235–237.[ISI][Medline]

Albuquerque, D.A., Martins, G.A., Campos-Neto, A., and Silva, J.S. (1999) The adjuvant effect of jacalin on the mouse humoral immune response to trinitrophenyl and Trypanosoma cruzi. Immunol. Lett., 68, 375–3781.[ISI][Medline]

Campbell, D., Mann, B.J., and Chadee, K. (2000) A subunit vaccine candidate region of the Entamoeba histolytica galactose-adherence lectin promotes interleukin-12 gene transcription and protein production in human macrophages. Eur. J. Immunol., 30, 423–430.[ISI][Medline]

Ganiko, L., Martins, A.R., Espreafico, E.M., and Roque-Barreira, M.-C. (1998) Neutrophil haptotaxis induced by the lectin KM+. Glycoconj. J., 15, 527–530.[ISI][Medline]

Himmelrich, H., Launois, P., Maillard, I., Biedermann, T., Tacchini-Cottier, F., Locksley, R.M., Rocken, M., and Louis, J.A. (2000) In BALB/c mice, IL-4 production during the initial phase of infection with Leishmania major is necessary and sufficient to instruct Th2 cell development resulting in progressive disease. J. Immunol., 164, 4819–4825.[Abstract/Free Full Text]

Hortin, G.L. and Trimpe, B.L. (1990) Lectin affinity chromatography of proteins bearing O-linked oligosaccharides: application of jacalin agarose. Anal. Biochem., 188, 271–277.[ISI][Medline]

Hostanska, K., Hajto, T., Spagnoli, G.C., Fischer, J., Lentzen, H., and Herrmann, R. (1995) A plant lectin derived from Viscum album induces cytokine gene expression and protein production in cultures of human peripheral blood mononuclear cells. Nat. Immun., 14, 295–304.[ISI][Medline]

Hsieh, C.S., Heimberger, A.B., Gold, J.S., O’Garra, A., and Murphy, K.M. (1992) Differential regulation of T helper phenotype development by interleukins 4 and 10 in an alpha beta T-cell-receptor transgenic system. Proc. Natl Acad. Sci. USA, 89, 6065–6069.[Abstract]

Hsieh, C.S., Macatonia, S.E., Tripp, C.S., Wolf, S.F., O’Garra, A., and Murphy, K.M. (1993) Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science, 260, 547–549.[ISI][Medline]

Julia, V., McSorley, S.S., Malherbe, L., Breittmayer, J.P., Girard-Pipau, F., Beck, A., and Glaichenhaus, N. (2000) Priming by microbial antigens from the intestinal flora determines the ability of CD4(+) T cells to rapidly secrete IL-4 in BALB/c mice infected with Leishmania major. J. Immunol., 165, 5637–5645.[Abstract/Free Full Text]

Julia, V., Rassoulzadegan, M., and Glaichenhaus, N. (1996) Resistance to Leishmania major induced by tolerance to a single antigen. Science, 274, 421–423.[Abstract/Free Full Text]

Kishko, I.H., Vasylenko, M.I., Pidhorsqkyi, V.S., and Kovalenko, E.O. (1997) Lectin of Bacillus subtilis sp. as overinducer of gamma-interferonogenesis. Mikrobiol. Z., 59, 20–26.

Launois, P., Maillard, I., Pingel, S., Swihart, K.G., Xenarios, I., Acha-Orbea, H., Diggelmann, H., Locksley, R.M., MacDonald, H.R., and Louis, J.A. (1997) IL-4 rapidly produced by V beta 4 V alpha 8 CD4+ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice. Immunity, 6, 541–549.[ISI][Medline]

Le Gros, G., Ben-Sasson, S.Z., Seder, R., Finkelman, F.D., and Paul, W.E. (1990) Generation of interleukin 4 (IL-4)-producing cells in vivo and in vitro: IL-2 and IL-4 are required for in vitro generation of IL-4- producing cells. J. Exp. Med., 172, 921–929.[Abstract]

Macatonia, S.E., Hosken, N.A., Litton, M., Vieira, P., Hsieh, C.S., Culpepper, J.A., Wysocka, M., Trinchieri, G., Murphy, K.M., and O’Garra, A. (1995) Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells. J. Immunol., 154, 5071–5079.[Abstract/Free Full Text]

Muraille, E., Pajak, B., Urbain, J., and Leo, O. (1999) Carbohydrate-bearing cell surface receptors involved in innate immunity: interleukin-12 induction by mitogenic and nonmitogenic lectins. Cell. Immunol., 191, 1–9.[ISI][Medline]

Paul, W.E. and Seder, R.A. (1994) Lymphocyte responses and cytokines. Cell, 76, 241–251.[ISI][Medline]

Pryjma, J., Ernst, M., Fetting, R., Woloszyn, M., Zembala, M., and Flad, H.D. (1991) The role of monocytes in the induction and regulation of IFN-gamma production by lectin-activated human T lymphocytes. Eur. Cytokine Netw., 2, 273–279.[Medline]

Rani, P.G., Bachhawat, K., Misquith, S., and Surolia, A. (1999) Thermodynamic studies of saccharide binding to artocarpin, a B-cell mitogen, reveals the extended nature of its interaction with mannotriose [3, 6-Di-O-(alpha-D-mannopyranosyl)-D-mannose]. J. Biol. Chem., 274, 29694–29698.[Abstract/Free Full Text]

Reiner, S.L. and Locksley, R.M. (1995) The regulation of immunity to Leishmania major. Annu. Rev. Immunol., 13, 151–177.[ISI][Medline]

Rosa, J.C., Oliveira, P.S.L., Garratt, R., Beltramini, L., Resing, K., Roque-Barreira, M.-C., and Greene, L.J. (1999) KM+, a mannose-specific lectin from Artocarpus integrifolia: amino acid sequence, predicted tertiary structure, carbohydrate recognition and analysis of the beta-prism fold. Protein Sci., 8, 13–24.[Abstract]

Santos-de-Oliveira, R., Dias-Baruffi, M., Thomaz, S.M., Beltramini, L.M., and Roque-Barreira, M.C. (1994) A neutrophil migration-inducing lectin from Artocarpus integrifolia. J. Immunol., 153, 1798–1807.[Abstract/Free Full Text]

Scott, P., Pearce, E., Natovitz, P., and Sher, A. (1987) Vaccination against cutaneous leishmaniasis in a murine model. I. Induction of protective immunity with a soluble extract of promastigotes. J. Immunol., 139, 221–227.[Abstract/Free Full Text]

Seder, R.A., Gazzinelli, R., Sher, A., and Paul, W.E. (1993) Interleukin 12 acts directly on CD4+ T cells to enhance priming for interferon gamma production and diminishes interleukin 4 inhibition of such priming. Proc. Natl Acad. Sci. USA, 90, 10188–10192.[Abstract]

Seder, R.A., Paul, W.E., Davis, M.M., and Fazekas de St. Groth, B. (1992) The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4+ T cells from T cell receptor transgenic mice. J. Exp. Med., 176, 1091–1098.[Abstract]

Sypek, J.P., Chung, C.L., Mayor, S.E., Subramanyam, J.M., Goldman, S.J., Sieburth, D.S., Wolf, S.F., and Schaub, R.G. (1993) Resolution of cutaneous leishmaniasis: interleukin 12 initiates a protective T helper type 1 immune response. J. Exp. Med., 177, 1797–1802.[Abstract]

Taimi, M., Dornand, J., Nicolas, M., Marti, J., and Favero, J. (1994) Involvement of CD4 in interleukin-6 secretion by U937 monocytic cells stimulated with the lectin jacalin. J. Leukoc. Biol., 55, 214–220.[Abstract]

Villalobo, A. and Gabius, H. (1998) Signaling pathways for transduction of the initial message of the glycocode into cellular responses. Acta Anat., 161, 110–129.[ISI][Medline]