RESUMO
ArtinM is a d-mannose-binding lectin found in the seeds of Artocarpus heterophyllus (jackfruit) that interacts with N-glycans, that is associated with receptors on the surface of phagocytic cells and induces the production of inflammatory mediators. Some of them are especially important because they may be required for antitumor immune response. This study aimed to evaluate the effect of ArtinM on hepatocellular preneoplastic foci. Wistar rats received 50 mg/kg of diethyl-nitrosamine (DEN) intraperitoneal weekly for 12 weeks. From the 14th week, the treated animals received 50 µg/kg of ArtinM subcutaneous every 2 weeks until the 18th week, whereas control animals were injected with vehicle alone. Preneoplastic-related factors were estimated using histological, western blotting and RT-PCR analysis. In comparison to the groups exposed to DEN, the ArtinM-treated rats showed diminution of preneoplastic foci, decreased expression of proliferating cell nuclear antigen (PCNA), increased number of nuclear p21 and p27 stained cells, augmented number of apoptotic cells, increased expression of p53, p42/44 MAPK and p21 proteins, reduced cyclin D1 (CCND1) protein levels and increased expression of TNFα and IFNγ genes. No difference was observed in interleukin 12 (IL12) protein levels. These findings indicate that ArtinM may provide protection against hepatocarcinogenesis as a result of the induction of cell-cycle blockage and pro-apoptotic mechanisms.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Hepáticas/prevenção & controle , Lectina de Ligação a Manose/farmacologia , Lesões Pré-Cancerosas/prevenção & controle , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Artocarpus/química , Western Blotting , Proliferação de Células/genética , Ciclina D1/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interferon gama/genética , Interleucina-12/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fitoterapia , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/genéticaRESUMO
ArtinM is a D-mannose-binding lectin extracted from the seeds of Artocarpus heterophyllus that interacts with TLR2 N-glycans and activates antigen-presenting cells (APCs), as manifested by IL-12 production. In vivo ArtinM administration induces Th1 immunity and confers protection against infection with several intracellular pathogens. In the murine model of Candida albicans infection, it was verified that, in addition to Th1, ArtinM induces Th17 immunity manifested by high IL-17 levels in the treated animals. Herein, we investigated the mechanisms accounting for the ArtinM-induced IL-17 production. We found that ArtinM stimulates the IL-17 production by spleen cells in BALB/c or C57BL/6 mice, a response that was significantly reduced in the absence of IL-23, MyD88, or IL-1R. Furthermore, we showed that ArtinM directly induced the IL-23 mRNA expression and the IL-1 production by macrophages. Consistently, in cell suspensions depleted of macrophages, the IL-17 production stimulated by ArtinM was reduced by 53% and the exogenous IL-23 acted synergistically with ArtinM in promoting IL-17 production by spleen cell suspensions. We verified that the absence of IL-23, IL-1R, or MyD88 inhibited, but did not block, the IL-17 production by ArtinM-stimulated spleen cells. Therefore, we investigated whether ArtinM exerts a direct effect on CD4+ T cells in promoting IL-17 production. Indeed, spleen cell suspensions depleted of CD4+ T cells responded to ArtinM with very low levels of IL-17 release. Likewise, isolated CD4+ T cells under ArtinM stimulus augmented the expression of TGF-ß mRNA and released high levels of IL-17. Considering the observed synergism between IL-23 and ArtinM, we used cells from IL-23 KO mice to assess the direct effect of lectin on CD4+ T cells. We verified that ArtinM increased the IL-17 production significantly, a response that was inhibited when the CD4+ T cells were pre-incubated with anti-CD3 antibody. In conclusion, ArtinM stimulates the production of IL-17 by CD4+ T cells in two major ways: (I) through the induction of IL-23 and IL-1 by APCs and (II) through the direct interaction with CD3 on the CD4+ T cells. This study contributes to elucidation of mechanisms accounting for the property of ArtinM in inducing Th17 immunity and opens new perspectives in designing strategies for modulating immunity by using carbohydrate recognition agents.
Assuntos
Artocarpus/química , Complexo CD3/imunologia , Interleucina-17/imunologia , Interleucina-1/imunologia , Interleucina-23/imunologia , Lectina de Ligação a Manose , Lectinas de Plantas , Células Th17/imunologia , Animais , Complexo CD3/genética , Candida albicans/imunologia , Candidíase/tratamento farmacológico , Candidíase/genética , Candidíase/imunologia , Interleucina-1/genética , Interleucina-17/genética , Interleucina-23/genética , Lectina de Ligação a Manose/química , Lectina de Ligação a Manose/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/imunologia , Células Th1/imunologiaRESUMO
Lectins are proteins able to recognize carbohydrates, without modifying their structure, via the carbohydrate-recognition domain (CRD). Here, the three-dimensional structure of the mannose-binding lectin isolated from Cymbosema roseum (CRLI) was determined with X-man molecule modeled into the carbohydrate recognition domain. CRLI relaxant activity in thoracic rat aorta was also investigated, and based on the results, a molecular docking of CRLI with heparan sulfate was performed to investigate the possible interaction with mechanoreceptors involved in vasorelaxation. CRLI (IC50=12.4 µg mL(-)(1)) elicited vasorelaxant response (96%) in endothelialized rat aorta contracted with phenylephrine. Endothelium-derived relaxant factors, extracellular calcium (Ca(2+)e) and muscarinic receptors were also evaluated as putative participants in the CRLI relaxant effect. CRLI relaxant effect was blocked by L-NAME, a nonselective inhibitor of nitric oxide synthase (NOS), and partially inhibited in a calcium-free solution (0Ca) and by atropine, but it remained unchanged in the presence of indomethacin and TEA. In summary, our data suggest interaction between CRLI and muscarinic receptors located in vascular endothelial cells leading to NOS activation triggered by a mechanism that involves Ca(2+)e along with the ability of CRLI to interact with heparan sulfate, a highly rated mechanoreceptor involved in eNOS activation.
Assuntos
Fabaceae/química , Lectina de Ligação a Manose/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas de Plantas/farmacologia , Receptores Muscarínicos/metabolismo , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Indometacina/farmacologia , Masculino , Lectina de Ligação a Manose/química , Músculo Liso Vascular/citologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Proteínas de Plantas/química , Ratos , Ratos WistarRESUMO
The aim of this work was to purify and partially characterize a mannose recognition lectin from Nile tilapia (Oreochromis niloticus) serum, named OniL. OniL was isolated through precipitation with ammonium sulfate and affinity chromatography (Concanavalin A-Sepharose 4B). In addition, we evaluated carbohydrate specificity, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profiles, and in vitro immunomodulatory activity on mice splenocyte experimental cultures through cytotoxic assays and cytokine production. The ammonium sulfate fraction F2 showed the highest specific hemagglutinating activity (331) and was applied to affinity matrix. Adsorbed proteins (OniL) were eluted with methyl-α-D: -mannopyranoside. OniL, a 17-kDa protein by SDS-PAGE constituted by subunits of 11 and 6.6 kDa, showed highest affinity for methyl-α-D: -mannopyranoside and D: -mannose. Immunological assays, in vitro, showed that OniL did not show cytotoxicity against splenocytes, induced higher IFN-γ production and lower IL-10 as well as nitrite release. In conclusion, OniL lectin was successfully purified and showed a preferential Th1 response in mice splenocytes.
Assuntos
Ciclídeos/sangue , Interferon gama/biossíntese , Lectina de Ligação a Manose/isolamento & purificação , Lectina de Ligação a Manose/farmacologia , Baço/efeitos dos fármacos , Baço/metabolismo , Animais , Cromatografia de Afinidade , Hemaglutinação/efeitos dos fármacos , Fatores Imunológicos/sangue , Fatores Imunológicos/isolamento & purificação , Fatores Imunológicos/metabolismo , Fatores Imunológicos/farmacologia , Masculino , Lectina de Ligação a Manose/sangue , Lectina de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Nitritos/metabolismo , Baço/imunologiaRESUMO
BACKGROUND: The D-mannose binding lectin ArtinM is known to recruit neutrophils, to degranulate mast cells and may have potential therapeutic applications. However, the effect of ArtinM on mast cell recruitment has not been investigated. METHODOLOGY: Male Wistar rats were injected i.p. with ArtinM or ConA (control). The ability of the lectin to degranulate peritoneal and mesenteric mast cells was examined. Recruitment of mast cells to the peritoneal cavity and mesentery after ArtinM injection was examined with or without depletion of peritoneal mast cells by distilled water. RESULTS: ArtinM degranulated both peritoneal and mesentery mast cells in vitro. Three days after i.p. injection of the lectin there were reduced numbers of mast cells in the peritoneal lavage, while at 7 days post injection of ArtinM, the number of peritoneal mast cells was close to control values. Since immature mast cells are recruited from the bone marrow, the effect of the lectin on bone marrow mast cells was examined. Injection of ArtinM resulted in an increased number of mast cells in the bone marrow. To determine if degranulation of mast cells in the peritoneal cavity was required for the increase in bone marrow mast cells, the peritoneal cavity was depleted of mast cells with ultrapure water. Exposure to ArtinM increased the number of mast cells in the bone marrow of rats depleted of peritoneal mast cells. CONCLUSIONS: The ArtinM induced recruitment of mast cells from the bone marrow to the peritoneal cavity may partially explain the therapeutic actions of ArtinM.
Assuntos
Células da Medula Óssea/citologia , Lectinas/química , Lectina de Ligação a Manose/química , Mastócitos/citologia , Neutrófilos/metabolismo , Cavidade Peritoneal , Animais , Medula Óssea/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Inflamação , Masculino , Manose/química , Lectina de Ligação a Manose/farmacologia , Mastócitos/efeitos dos fármacos , Modelos Biológicos , Ratos , Ratos WistarRESUMO
We demonstrate here that a mannose-binding protein from Schistosoma mansoni, termed Sm60, was recovered in the mannose-eluted fraction (Man(+)) upon affinity chromatography on immobilised mannose of the soluble antigen fraction from adult worm tegument and cercariae. Sm60 was detected in the Man(+) fraction as a prominent doublet with an apparent molecular mass of 60-66 kDa by SDS-PAGE and appeared as a single band with a pI of approximately 6.9 by isoelectrofocusing. Sm60 was also detected in preparations of schistosomula extract and soluble egg antigens using a mouse polyclonal anti-Sm60 serum on immunoblotting assay. This antiserum demonstrated that Sm60 was localised on the tegument of S. mansoni adult worm. In order to determine the role of Sm60 in host-parasite interactions, we showed that Sm60 induced in vitro migration of human neutrophil in a dose-dependent manner and in vitro mast cell degranulation. Sm60 triggered these activities through its carbohydrate-binding site, since these activities were selectively inhibited by 0.2 M D-mannose, but not by 0.2 M D-galactose. Furthermore, Sm60 induced in vivo neutrophil migration. In contrast, mast cell-depleted rats presented a significant reduction of the neutrophil migration induced by Sm60 as compared with non-depleted controls. These data suggest that in vivo neutrophil migration induced by Sm60 is modulated by mast cell-dependent mechanisms. Sm60 might play a key role in the host-parasite interaction, and its characterization opens perspective to examine the role of this molecule in the biology of S. mansoni.