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.
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Abstract |
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Key words: Artocarpus integrifolia/interleukin 12/KM+/lectin/Leishmania major
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Introduction |
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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, 1998). Some animal, parasite, and plant lectins induce cytokine production, among them those related to the Th1 response, such as interferon-
(IFN-
) (Pryjma et al., 1991
; Paul and Seder, 1994
; Hostanska et al., 1995
; Kishko et al., 1997
) and IL-12 (Muraille et al., 1999
; Campbell et al., 2000
).
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 -D-galactosyl residue as well as for the disaccharide Galß1-3GalNAc
1-O-Ser/Thr (Hortin and Trimpe, 1990
). 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
1-3[Man
1-6]Man) (Rani et al., 1999
). 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., 1999
). In terms of biological properties, jacalin induces IL-6 secretion by U937 monocytic cells (Taimi et al., 1994
) and potentiates mouse humoral immune response to trinitrophenyl and Trypanosoma cruzi (Albuquerque et al., 1999
). KM+, in turn, has been used as a tool to study the haptotactic mechanism of neutrophil migration in rats (Santos-de-Oliveira et al., 1994
), 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., 1998
).
We now show that KM+ lectin induces macrophages to produce IL-12 p40, which then stimulates IFN- 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-
). 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.
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Results |
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Discussion |
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Initially we demonstrated in vitro that KM+ promotes IFN- 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-
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- 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)
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
1-2Man >> Man
1-6Man > Man
1-3Man, through a site that essentially accommodates a monosaccharide. Man
1-2Man, as compared to mannose itself, is poorly recognized by KM+. The mannobioses Man
1-3Man and Man
1-6Man have very different affinities for KM+, because they bind, respectively, 2- and 17-fold less to the lectin than the mannotriose (Man
1-3[Man
1-6]Man) does. This indicates that
1-3-linked mannose occupies the primary binding site, and the
1-6Man occupies the secondary subsite. These binding sites, specific for the
1,3 and
1,6 arms for the two oligosaccharides, together with the 3,6-disubstituted mannose residue, constitute the extended CRD of KM+ (Rani et al., 1999
). The specificity of KM+ for Man
1-3(Man
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-
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
1-3(Man
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., 2000). The early burst of IL-4 expression in BALB/c mice occurs in a restricted population of Vß4-V
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., 1997
). In addition, BALB/c mice tolerant to LACK were resistant to the infection (Julia et al., 1996
). Recently, Julia et al. (2000)
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-
levels. This was successfully reversed by KM+ administration, with production of low IL-4 and high IFN-
levels, validating the hypothesis that the IL-12 p40inducing 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- 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 p40inducing 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.
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Material and methods |
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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- (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-
(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 Schneiders 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., 1987).
Immunization and infection of mice
Using previously described immunization protocols (Afonso et al., 1994), 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., 1994
), from stationary culture (45 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 Hanks 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 24 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 TrisHCl, 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 (Man1-3[Man
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 5100 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-), 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-
, 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.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed
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References |
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