RESUMO
Entamoeba histolityca produces the monocyte locomotion inhibitory factor (MLIF), a pentapeptide with powerful anti-inflammatory properties. MLIF may regulate trauma-induced inflammation through the effects it exerts directly or indirectly on immune cells, modulating the production and/or expression of the cytokines involved in the inflammatory processes that occur after damage. The aim of the present study was to evaluate the effect of MLIF on production of pro/anti-inflammatory cytokines after contusion in the rat tibia. Fifty-four Wistar rats were subjected to controlled contusion with a special guillotine-type device, and 36 rats were injected with MLIF or tenoxicam into the tibia. Eighteen animals received saline; the animals were sacrificed 24 or 48 hours after injection. Cytokine mRNA and protein production were determined by reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence, and hematoxylin-eosin staining was performed to visualize cellular infiltration in the rats' injured tissue. Expression levels of the cytokines interferon gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and transforming growth factor-beta (TGF-ß) mRNA were inhibited significantly by MLIF at 24 hours post-contusion. MLIF significantly increased the expression levels of IL-10 at 24 hours compared with tenoxicam or the control group. These changes were associated with a significant decrease in protein production levels of TNF-α, IFN-γ, IL-6 and TGF-ß at 24 hours. Histological evaluation showed the presence of infiltration by neutrophils, monocytes and leucocytes in control tissues. This infiltration was decreased after MLIF administration, and intense infiltration was observed in tenoxicam-treated group. MLIF inhibited the expression of pro-inflammatory cytokines and increased the expression of anti-inflammatory cytokine IL-10.
Assuntos
Anti-Inflamatórios/farmacologia , Contusões/tratamento farmacológico , Citocinas/metabolismo , Inflamação/tratamento farmacológico , Oligopeptídeos/farmacologia , Tíbia/efeitos dos fármacos , Animais , Contusões/metabolismo , Inflamação/metabolismo , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Masculino , Piroxicam/análogos & derivados , Piroxicam/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Tíbia/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Asthma studies suggest that alteration in the inflammation pattern may be associated with the severity of asthma. The aim of this study was to compare in vitro the expression of chemokines, chemokine receptors and cytokine production from CD4+ T human lymphocytes of asthmatic, both obese and non-obese patients with different severity levels of asthma. Lymphocytes were labeled with monoclonal anti-human CXCR3/IP-10, MIP-1a/CCR5 antibodies and were analyzed by flow cytometry. Cell culture supernatants were used to measure production of interleukin IL-6 and resistin by ELISA. CXCR3/IP-10 expression increased in non-obese patients with mild persistent asthma (2.2%, p<0.05), moderate persistent asthma (3%, p<0.003) and severe persistent asthma (4%, p<0.004); this effect was stronger in obese patients with severe persistent asthma (35%, p<0.004). MIP-1 α / CCR5 increased in non-obese patients with intermittent asthma (0.65%, p<0.05) and severe asthma (1.4%, p<0.03); in obese patients, this expression was greater in intermittent asthma (8%, p<0.05) and severe persistent asthma (12%, p<0.04). Resistin production strongly increased in obese patients with intermittent (976 ng/ml) and severe persistent asthma (795 ng/ml). IL-6 increased in both lean and obese persons; however, the highest value was registered in the group of severe persistent obese asthmatics (992 pg/ml). Obesity per se increased the inflammatory profile of chemokines / cytokines secreted by cells of the blood, increasing the inflammatory status in asthmatic patients. Resistin showed characteristics of a pro-inflammatory cytokine mainly in severely obese asthmatics.
Assuntos
Asma/sangue , Quimiocina CCL3/sangue , Quimiocina CXCL10/sangue , Obesidade/sangue , Receptores de Quimiocinas/sangue , Resistina/sangue , Asma/complicações , Índice de Massa Corporal , Linfócitos T CD4-Positivos/fisiologia , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Interleucina-6/sangue , Masculino , Obesidade/complicações , Cultura Primária de Células , Receptores CCR5/sangue , Receptores CXCR3/sangue , Índice de Gravidade de Doença , Estatísticas não ParamétricasRESUMO
Entamoeba histolytica produces Monocyte Locomotion Inhibitory Factor (MLIF), which may contribute to the delayed inflammation observed in amoebic hepatic abscesses. Leukocytes are affected through the modulation of cytokine expression and/or production. We evaluated the effects of MLIF on the activation and production of intracellular cytokines in human CD4+ T lymphocytes by flow cytometry. Cells were stimulated for 24 h with PMA, MLIF, or PMA+MLIF. Cellular activation was measured using anti-CD69. Th1/Th2 production was studied by the expression of intracellular cytokines and cytokine/chemokine receptors. MLIF increased CD69 and induced the over-expression of the IL-lss, IFN-gamma, IL-2, IL-4, and IL-10 intracellular cytokines; PMA+MLIF inhibited Th1 cytokine (IFN-gamma) and increased Th2 cytokines (IL-4 and IL-10). The co-expression of the cytokine and chemokine receptors IFN-gamma/CCR5 and IL-1ss/CCR5 was inhibited by PMA+MLIF and Th2 co-expression was increased. MLIF effects varied depending on the conditions. MLIF alone activated the Th1 and Th2 cytokines and cytokine/receptor expression; however, PMA+MLIF increased the expression of Th2 but inhibited it in Th1.
Assuntos
Citocinas/biossíntese , Oligopeptídeos/farmacologia , Receptores CCR4/efeitos dos fármacos , Receptores CCR5/efeitos dos fármacos , Células Th1/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Células Cultivadas , Entamoeba histolytica/imunologia , Feminino , Citometria de Fluxo , Humanos , Masculino , Oligopeptídeos/biossíntese , Receptores CCR4/imunologia , Receptores CCR5/imunologia , Acetato de Tetradecanoilforbol/farmacologia , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Chemotaxis is defined as the ability of living cells to determine the direction of locomotion through a concentration gradient of attractant and repellent substances. Biological substances such as complement and its components, as well as their relationship with the disease are described.
Assuntos
Quimiotaxia , Doença , Quimiocinas/fisiologia , Proteínas do Sistema Complemento/fisiologia , Humanos , Imunidade , Inflamação/imunologiaRESUMO
The monocyte locomotion inhibitory factor (MLIF) is an anti-inflammatory oligopeptide produced by Entamoeba histolytica. Among its different effects, it inhibits locomotion of human monocytes, hence its original name. Previous experimental studies have shown that the anti-inflammatory properties of MLIF (Met-Gln-Cys-Asn-Ser) remained when aminoacid glutamine was substituted by a proline in the second position (pMLIF: Met-Pro-Cys-Asn-Ser). By changing the order of MLIF amino acids, the resulting scrambled oligopeptide (sMLIF: Gln-Cys-Met-Ser-Asn) has failed activity. By means of ab initio study at the Hartree-Fock and Density Functional Theory levels, it was found that MLIF and pMLIF peptides maintain a great structural similarity among the last three amino acids (...Cys-Asn-Ser) predicting a pharmacophore. The objective of this work was to experimentally verify in vivo and in vitro the existence of the pharmacophore group in MLIF. We assayed three tripeptides by respiratory burst and delayed hypersensitivity skin reactions. The tripeptide Cys-Asn-Ser carboxyl-terminal end group maintained 100% of its biological properties, as well as the anti-inflammatory activity of MLIF, while the other tripeptides tested did not do that.
Assuntos
Entamoeba histolytica/imunologia , Oligopeptídeos/metabolismo , Animais , Células Cultivadas , Cobaias , Humanos , Hipersensibilidade Tardia/imunologia , Fatores Imunológicos/farmacologia , Masculino , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/farmacologia , Explosão Respiratória/imunologiaRESUMO
Entamoeba histolytica produces, in axenic culture, the monocytes locomotion inhibitory factor (MLIF), a oligopeptide with selective anti-inflammatory properties. We evaluated the effect of MLIF on the expression of pro- and anti-inflammatory cytokines in CD4+ T lymphocytes from children with asthma and allergic rhinitis. Twelve children with severe asthma, 12 children with allergic rhinitis and 6 healthy controls were recruited for this study between May and December 2016. CD4+ T cells were cultured for 24 h at 37°C, 5% CO2 in the presence of MLIF, 1-phorbol 12-myristate 13-acetate (PMA), MLIF+PMA or RPMI. Interleukin-10 (IL-10), IL-4, interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) expression levels were measured in the supernatants of T-cell cultures using the enzyme-linked immunosorbent assay (ELISA). Pro- and anti-inflammatory cytokines were inhibited by MLIF (IFN-γ p=0.0036, TNF-α p<0.001, IL-4 p=0.0082) in asthmatic patients, however IFN-γ was not significantly inhibited (NS) in patients with allergic rhinitis when compared to the RPMI group. In CD4+ T cells treated with PMA+MLIF, the expression levels of IFN-γ, TNF-α and IL-4 were strongly inhibited (p<0.001, p<0.001 and p<0.0094), compared to PMA treatment alone, for both, rhinitis and asthma. IL-10 expression was not affected by MLIF in neither of the two diseases. We conclude that MLIF alters the pro/anti-inflammatory balance and induces inhibition of IL-4, IFN-γ and TNF-α, but does not affect IL-10.
Assuntos
Asma/imunologia , Linfócitos T CD4-Positivos/imunologia , Entamoeba histolytica/fisiologia , Oligopeptídeos/metabolismo , Proteínas de Protozoários/metabolismo , Rinite Alérgica/imunologia , Adolescente , Alérgenos/imunologia , Células Cultivadas , Criança , Pré-Escolar , Citocinas/metabolismo , Feminino , Humanos , Imunomodulação , Lactente , Mediadores da Inflamação/metabolismo , MasculinoRESUMO
Asthma studies suggest that alteration in the inflammation pattern may be associated with the severity of asthma. The aim of this study was to compare in vitro the expression of chemokines, chemokine receptors and cytokine production from CD4+ T human lymphocytes of asthmatic, both obese and non-obese patients with different severity levels of asthma. Lymphocytes were labeled with monoclonal anti-human CXCR3/IP-10, MIP-1a/CCR5 antibodies and were analyzed by flow cytometry. Cell culture supernatants were used to measure production of interleukin IL-6 and resistin by ELISA. CXCR3/IP-10 expression increased in non-obese patients with mild persistent asthma (2.2%, p<0.05), moderate persistent asthma (3%, p<0.003) and severe persistent asthma (4%, p<0.004); this effect was stronger in obese patients with severe persistent asthma (35%, p<0.004). MIP-1 α / CCR5 increased in non-obese patients with intermittent asthma (0.65%, p<0.05) and severe asthma (1.4%, p<0.03); in obese patients, this expression was greater in intermittent asthma (8%, p<0.05) and severe persistent asthma (12%, p<0.04). Resistin production strongly increased in obese patients with intermittent (976 ng/ml) and severe persistent asthma (795 ng/ml). IL-6 increased in both lean and obese persons; however, the highest value was registered in the group of severe persistent obese asthmatics (992 pg/ml). Obesity per se increased the inflammatory profile of chemokines / cytokines secreted by cells of the blood, increasing the inflammatory status in asthmatic patients. Resistin showed characteristics of a pro-inflammatory cytokine mainly in severely obese asthmatics.
Assuntos
Feminino , Humanos , Masculino , Asma/sangue , /sangue , /sangue , Obesidade/sangue , Receptores de Quimiocinas/sangue , Resistina/sangue , Asma/complicações , Índice de Massa Corporal , Estudos de Casos e Controles , /fisiologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , /sangue , Obesidade/complicações , Cultura Primária de Células , /sangue , /sangue , Índice de Gravidade de Doença , Estatísticas não ParamétricasRESUMO
Entamoeba histolytica produces monocyte locomotion inhibitory factor (MLIF), a pentapeptide with in vitro and in vivo anti-inflammatory properties. MLIF may interfere with leukocyte migration, disturbing the balance of pro- and anti-inflammatory cytokines secreted by CD4(+) T lymphocytes. We evaluated the effect of MLIF on expression of pro- and anti-inflammatory cytokines in human CD4(+) T lymphocytes. Regulatory cytokines [interleukin-1 beta (IL-1beta), IL-2, interferon gamma (IFN-gamma), IL-5, IL-6, and IL-10] were studied by enzyme-linked immunosorbent assay method in CD4(+)-cell supernatant fluids. Proinflammatory cytokines were produced per se by MLIF (IL-1beta, IL-2, and IFN-gamma) and also anti-inflammatory cytokines (IL-5, IL-6, and IL-10) with 1-phorbol-12 myristate-13 acetate + MLIF; the IL-1beta, IFN-gamma, IL-5 and IL-6 production was inhibited but not that of IL-10 which disclosed increase in its expression. MLIF disturbs the pro- and anti-inflammatory balance, and it induces inhibition of IL-1beta (principal proinflammatory cytokine) and increases IL-10 (prototype of an anti-inflammatory cytokine).
Assuntos
Linfócitos T CD4-Positivos/imunologia , Citocinas/biossíntese , Entamoeba histolytica/imunologia , Oligopeptídeos/imunologia , Proteínas de Protozoários/imunologia , Animais , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , MasculinoRESUMO
Entamoeba histolytica produces Monocyte Locomotion Inhibitory Factor (MLIF), which may contribute to the delayed inflammation observed in amoebic hepatic abscesses. Leukocytes are affected through the modulation of cytokine expression and/or production. We evaluated the effects of MLIF on the activation and production of intracellular cytokines in human CD4+ T lymphocytes by flow cytometry. Cells were stimulated for 24 h with PMA, MLIF, or PMA+MLIF. Cellular activation was measured using anti-CD69. Th1/Th2 production was studied by the expression of intracellular cytokines and cytokine/chemokine receptors. MLIF increased CD69 and induced the over-expression of the IL-l©¬, IFN-¥ã, IL-2, IL-4, and IL-10 intracellular cytokines; PMA+MLIF inhibited Th1 cytokine (IFN-¥ã) and increased Th2 cytokines (IL-4 and IL-10). The co-expression of the cytokine and chemokine receptors IFN-¥ã/CCR5 and IL-1©¬/CCR5 was inhibited by PMA+MLIF and Th2 co-expression was increased. MLIF effects varied depending on the conditions. MLIF alone activated the Th1 and Th2 cytokines and cytokine/receptor expression; however, PMA+MLIF increased the expression of Th2 but inhibited it in Th1.