RESUMO
Phosphatase activity of Leishmania spp. has been shown to deregulate the signalling pathways of the host cell. We here show that Leishmania mexicana promastigotes and amastigotes secrete proteins with phosphatase activity to the culture medium, which was higher in the Promastigote Secretion Medium (PSM) as compared with the Amastigote Secretion Medium (ASM) and was not due to cell lysis, since parasite viability was not affected by the secretion process. The biochemical characterization showed that the phosphatase activity present in PSM was higher in dephosphorylating the peptide END (pY) INASL as compared with the peptide RRA (pT)VA. In contrast, the phosphatase activity in ASM showed little dephosphorylating capacity for both peptides. Inhibition assays demonstrated that the phosphatase activity of both PSM and ASM was sensible only to protein tyrosine phosphatases inhibitors. An antibody against a protein phosphatase 2C (PP2C) of Leishmania major cross-reacted with a 44·9 kDa molecule in different cellular fractions of L. mexicana promastigotes and amastigotes, however, in PSM and ASM, the antibody recognized a protein about 70 kDa. By electron microscopy, the PP2C was localized in the flagellar pocket of amastigotes. PSM and ASM induced the production of tumor necrosis factor alpha, IL-1ß, IL-12p70 and IL-10 in human macrophages.
Assuntos
Citocinas/imunologia , Interações Hospedeiro-Parasita , Leishmania mexicana/enzimologia , Macrófagos/imunologia , Proteína Fosfatase 2C/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Transporte Biológico , Meios de Cultura/química , Citocinas/biossíntese , Humanos , Interleucina-10/biossíntese , Interleucina-10/imunologia , Leishmania mexicana/genética , Leishmania mexicana/imunologia , Leishmania mexicana/ultraestrutura , Camundongos , Microscopia Eletrônica , Proteína Fosfatase 2C/imunologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas de Protozoários/imunologia , Transdução de SinaisRESUMO
Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. Leishmania promastigotes primarily infect macrophages in the host, where they transform into amastigotes and multiply. Lipophosphoglycan (LPG), the most abundant surface molecule of the parasite, is a virulence determinant that regulates the host immune response. Promastigotes are able to modulate this effect through LPG, creating a favourable environment for parasite survival, although the mechanisms underlying this modulation remain unknown. We analysed the participation of TLR2 and TLR4 in the production of cytokines and explored the possible phosphorylation of ERK and/or p38 MAP kinase signalling cascades in human macrophages stimulated with Leishmania mexicana LPG. The results show that LPG induced the production of TNF-α, IL-1ß, IL-12p40, IL-12p70 and IL-10 and led to phosphorylation of ERK and p38 MAP kinase. Specific inhibitors of ERK or p38 MAP kinases and mAbs against TLR2 and TLR4 reduced cytokine production and phosphorylation of both kinases. Our results suggest that L. mexicana LPG binds TLR2 and TLR4 receptors in human macrophages, leading to ERK and MAP kinase phosphorylation and production of pro-inflammatory cytokines.
Assuntos
Citocinas/metabolismo , Regulação da Expressão Gênica , Leishmania mexicana/imunologia , Leishmaniose/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glicoesfingolipídeos , Humanos , Leishmania mexicana/patogenicidade , Leishmaniose/parasitologia , Macrófagos/imunologia , Macrófagos/metabolismo , Fosforilação , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Leishmania are protozoan parasites that infect macrophages and their survival is partially achieved through inhibition of the cellular oxidative burst by parasite lipophosphoglycan (LPG). PKCalpha is the predominant PKC isoenzyme required for macrophage oxidative burst, yet it is not known if different susceptibility of BALB/c and C57BL/6 mice to Leishmania mexicana could be related to PKCalpha. We analysed the effect of L. mexicana promastigotes and parasite LPG on expression of PKCalpha and on its activity in macrophages of both mouse strains. Our data show that expression of the isoenzyme was not altered either by LPG or by L. mexicana promastigotes. Yet LPG exerted opposing effects on PKCalpha activity of macrophages between both strains: in susceptible BALB/c cells, it inhibited PKCalpha activity, whereas in the more resistant strain it augmented enzymatic activity 2.8 times. In addition, LPG inhibited oxidative burst only in susceptible BALB/c macrophages and the degree of inhibition correlated with parasite survival. Promastigotes also inhibited PKCalpha activity and oxidative burst in macrophages of BALB/c mice, whereas in C57BL/6, they enhanced PKCalpha activity and oxidative burst inhibition was less severe. Our data indicate that control of PKCalpha-induced oxidative burst by L. mexicana LPG relates with its success to infect murine macrophages.
Assuntos
Glicoesfingolipídeos/metabolismo , Leishmania mexicana/patogenicidade , Macrófagos/imunologia , Macrófagos/parasitologia , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/biossíntese , Explosão Respiratória , Animais , Suscetibilidade a Doenças/imunologia , Perfilação da Expressão Gênica , Leishmaniose Tegumentar Difusa/imunologia , Leishmaniose Tegumentar Difusa/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Análise de SobrevidaRESUMO
The regulatory effect of mast cells on the pathogenesis of leishmaniasis is unclear. We report a comparative analysis of TLR2 membrane expression, TNF-α, IL-10 and MIP-1α production, and granule release of bone marrow-derived mast cells (BMMCs) from susceptible BALB/c and resistant C57BL/6 mice, stimulated in vitro with Leishmania mexicana lipophosphoglycan (LPG). We studied the kinetics of mast cell degranulation and parasite numbers in lesions of both mouse strains infected with L. mexicana. We found that BMMCs of C57BL/6 mice expressed more TLR2 and produced higher levels of both cytokines and MIP-1α, whereas BALB/c BMMCs significantly augmented their granule release. Lesions of BALB/c mice showed higher levels of degranulated mast cells at 3 h of infection, whereas after 3 days of infection, the number of degranulated mast cells in C57BL/6 was higher than in BALB/c lesions. Throughout infection, BALB/c mice harboured more parasites. The regulatory effect of mast cells seems to depend on the genetic background of the host: mast cells of BALB/c mice facilitate disease progression due to an augmented inflammatory response early in the infection, whereas mast cells of C57BL/6 mice produce cytokines that regulate inflammation and maintain an elevated number of immune cells in the lesions, promoting disease control.
Assuntos
Glicoesfingolipídeos/imunologia , Leishmania mexicana/imunologia , Leishmania mexicana/patogenicidade , Leishmaniose Cutânea/genética , Leishmaniose Cutânea/imunologia , Mastócitos/imunologia , Animais , Contagem de Células , Degranulação Celular , Quimiocina CCL3/biossíntese , Modelos Animais de Doenças , Interleucina-10/biossíntese , Leishmaniose Cutânea/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Doenças dos Roedores/genética , Doenças dos Roedores/imunologia , Doenças dos Roedores/patologia , Pele/parasitologia , Pele/patologia , Receptor 2 Toll-Like/biossíntese , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Leishmania mexicana causes localized and diffuse cutaneous leishmaniasis. Patients with localized cutaneous leishmaniasis (LCL) develop a benign disease, whereas patients with diffuse cutaneous leishmaniasis (DCL) suffer from a progressive disease associated with anergy of the cellular response towards Leishmania antigens. We evaluated the production of the interleukins (IL) IL-12, IL-15, IL-18 and tumour necrosis factor-alpha (TNF-alpha) and the expression of the costimulatory molecules CD40, B7-1 and B7-2 in monocytes from LCL and DCL patients, stimulated in vitro with Leishmania mexicana lipophosphoglycan (LPG) for 18 h. LCL monocytes significantly increased TNF-alpha, IL-15 and IL-18 production, and this increase was associated with reduced amounts of IL-12. DCL monocytes produced no IL-15 or IL-18 and showed a decreasing tendency of TNF-alpha and IL-12 production as the severity of the disease increased. No difference was observed in the expression of CD40 and B7-1 between both groups of patients, yet B7-2 expression was significantly augmented in DCL patients. It remains to be established if this elevated B7-2 expression in DCL patients is cause or consequence of the Th2-type immune response that characterizes these patients. These data suggest that the diminished ability of the monocytes from DCL patients to produce cell-activating innate proinflammatory cytokines when stimulated with LPG is a possible cause for disease progression.
Assuntos
Citocinas/metabolismo , Leishmania mexicana/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Tegumentar Difusa/imunologia , Monócitos/metabolismo , Adolescente , Adulto , Animais , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Antígenos CD40/metabolismo , Citocinas/biossíntese , Feminino , Glicoesfingolipídeos/administração & dosagem , Humanos , Interleucina-12/biossíntese , Interleucina-15/biossíntese , Interleucina-18/biossíntese , Masculino , México , Pessoa de Meia-Idade , Monócitos/imunologia , Estatísticas não Paramétricas , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Activation of procollagenase constitutes a crucial event in collagenolytic activity regulation. In this study we have purified by DEAE-cellulose, Ultrogel AcA-44, and zinc chelate sepharose chromatographies, a procollagenase-activator from the culture medium of the guinea pig carrageenin granuloma model. On SDS-PAGE, the activator migrates as a principal band of Mr approximately 44,000. The molecule activates procollagenase from human lung fibroblasts in a concentration dependent manner and an enhancement of collagenase activity of trypsin-treated crude culture medium was observed. A loss of about 50% of its activity occurs after heating. In addition, this activator degrades gelatin and casein. All these data suggest that this procollagenase-activator might be stromelysin. The activator was found in both phases of the granuloma, at 7 days when collagen is actively deposited and an important proportion of the collagenolytic activity remains in latent form; and at 14 days, when this enzymatic activity is fully expressed.