RESUMO
Brucella spp. have been associated with abortion in humans and animals. Although the mechanisms involved are not well established, it is known that placental Brucella infection is accompanied by inflammatory phenomena. The ability of Brucella abortus to infect and survive in human endometrial stromal cells (T-HESC cell line) and the cytokine response elicited were evaluated. B. abortus was able to infect and proliferate in both non-decidualized and decidualized T-HESC cells. Intracellular proliferation depended on the expression of a functional virB operon in the pathogen. B. abortus internalization was inhibited by cytochalasin D and to a lower extent by colchicine, but was not affected by monodansylcadaverine. The infection did not induce cytotoxicity and did not alter the decidualization status of cells. B. abortus infection elicited the secretion of IL-8 and MCP-1 in either decidualized or non-decidualized T-HESC, a response also induced by heat-killed B. abortus and outer membrane vesicles derived from this bacterium. The stimulation of T-HESC with conditioned media from Brucella-infected macrophages induced the production of IL-6, MCP-1 and IL-8 in a dose-dependent manner, and this effect was shown to depend on IL-1ß and TNF-α. The proinflammatory responses of T-HESC to B. abortus and to factors produced by infected macrophages may contribute to the gestational complications of brucellosis.
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ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of Smilax campestris Griseb (Smilacaceae) have been employed in the treatment of several inflammatory diseases as a traditional herbal medicine. However, the cellular and molecular mechanisms involved in the observed effects remain elusive. Macrophages are known to play a central role in inflammatory responses. These cells are activated in response to a diversity of danger signals and produce several mediators of inflammation that eventually regulate the immune response. For all the above mentioned, scientific evidence is required to support the popular use of S. campestris. AIM OF THE STUDY: We aimed to investigate the anti-inflammatory effect of S. campestris aqueous extract (SME) in activated THP-1 human macrophages, on the production of some mediators of inflammation and oxidative stress in order to provide scientific support for its popular use. MATERIALS AND METHODS: The characterization of SME was assessed by HPLC-MS/MS. The production of the pro-inflammatory cytokines and chemokines was evaluated by ELISA. The activity of metalloproteases was evaluated by zymography. The subcellular localization of the NF-κB transcription factor was analysed by Western blot. The superoxide anion and glutathione levels were assessed by flow cytometry. The cytotoxicity induced by SME in THP-1 macrophages was also investigated by the LDH release test. RESULTS: In the present study, we have identified catechin and glycosylated derivatives of quercetin (quercetin-3-O-glucoside, quercetin-3-O-galactoside, rutin and quercetin-3-rhamnoside) as major components of the aqueous SME. We found that SME significantly decreased the production of the pro-inflammatory cytokines tumour necrosis factor (TNF)- α, interleukin (IL)-1ß, IL-6, IL-8 and monocyte chemoattractant protein (MCP)-1 and the activity of the metalloproteinase (MMP)-9, in lipopolysaccharide-activated macrophages derived from the monocytic cell line THP-1. Furthermore, SME diminished the expression of NF-κB p65 subunit in the nuclear fraction. In addition, SME decreased the production of superoxide anion in THP-1 macrophages, without altering the levels of reduced glutathione. CONCLUSION: These results suggest that SME exerts its anti-inflammatory effects in human activated macrophages by inhibiting the production of pro-inflammatory cytokines, matrix metalloproteinases and the NF-κB transcription factor pathway along with a reduction of oxidative stress mediators. Moreover, catechin and glycosylated derivatives of were identified by HPLC-MS/MS in SME. Our findings provide scientific support for the traditional use of the S. campestris extracts.
Assuntos
Anti-Inflamatórios/farmacologia , Flavonoides/farmacologia , Macrófagos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Smilax/química , Anti-Inflamatórios/análise , Anti-Inflamatórios/isolamento & purificação , Argentina , Linhagem Celular , Citocinas/imunologia , Citocinas/metabolismo , Etnofarmacologia , Flavonoides/análise , Flavonoides/isolamento & purificação , Glutationa/metabolismo , Humanos , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Medicina Tradicional/métodos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/análise , Extratos Vegetais/isolamento & purificação , Superóxidos/metabolismo , Testes de Toxicidade , Água/químicaRESUMO
OBJECTIVE: Dopamine is an immunomodulatory neurotransmitter. In the skin, keratinocytes and macrophages produce proinflammatory cytokines and metalloproteinases (MMPs) which participate in wound healing. These cells have a catecholaminergic system that modulates skin pathophysiologic processes. We have demonstrated that dopamine modulates cytokine production in keratinocytes via dopaminergic and adrenergic receptors (ARs). The aim of this study was to evaluate the effect of dopamine and its interaction with ß-ARs in human HaCaT keratinocytes and THP-1 macrophages. We evaluated the production of inflammatory mediators implicated in wound healing. METHODS: Cells were stimulated with dopamine in the absence or presence of the ß-adrenergic antagonist propranolol. Wound closure, MMP activity, and the production of IL-8, IL-1ß, and IκB/NFκB pathway activation were determined in stimulated cells. RESULTS: Dopamine did not affect the wound closure in human keratinocytes, but diminished the propranolol stimulatory effect, thus delaying cell migration. Similarly, dopamine significantly decreased MMP-9 activity and the propranolol-induced MMP activity. Dopamine significantly increased the p65-NFκB subunit levels in the nuclear extracts, which were reduced in the presence of propranolol in keratinocytes. On the other hand, dopamine significantly increased MMP-9 activity in THP-1 macrophages, but did not modify the propranolol-increased enzymatic activity. Dopamine significantly increased IL-8 production in human macrophages, an effect that was partially reduced by propranolol. Dopamine did not modify the p65-NFκB levels in the nuclear extracts in THP-1 macrophages. CONCLUSION: We suggest that the effect of dopamine via ß-ARs depends on the physiological condition and the cell type involved, thus contributing to either improve or interfere with the healing process.
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Dopamina/farmacologia , Queratinócitos/fisiologia , Macrófagos/fisiologia , Receptores Adrenérgicos beta/fisiologia , Cicatrização/fisiologia , Antagonistas Adrenérgicos beta/farmacologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Relação Dose-Resposta a Droga , Humanos , Queratinócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Cicatrização/efeitos dos fármacosRESUMO
Primary tumor excision is one of the most widely used therapies of cancer. However, the risk of metastases development still exists following tumor resection. The liver is a common site of metastatic disease for numerous cancers. Breast cancer is one of the most frequent sources of metastases to the liver. The aim of this work was to evaluate the efficacy of the orally administered Salmonella Typhi vaccine strain CVD 915 on the development of liver metastases in a mouse model of breast cancer. To this end, one group of BALB/c mice was orogastrically immunized with CVD 915, while another received PBS as a control. After 24 h, mice were injected with LM3 mammary adenocarcinoma cells into the spleen and subjected to splenectomy. This oral Salmonella-based vaccine produced an antitumor effect, leading to a decrease in the number and volume of liver metastases. Immunization with Salmonella induced an early cellular immune response in mice. This innate stimulation rendered a large production of IFN-γ by intrahepatic immune cells (IHIC) detected within 24 h. An antitumor adaptive immunity was found in the liver and celiac and portal lymph nodes (LDLN) 21 days after oral bacterial inoculation. The antitumor immune response inside the liver was associated with increased CD4(+) and dendritic cell populations as well as with an inflammatory infiltrate located around liver metastatic nodules. Enlarged levels of inflammatory cytokines (IFN-γ and TNF) were also detected in IHIC. Furthermore, a tumor-specific production of IFN-γ and TNF as well as tumor-specific IFN-γ-producing CD8 T cells (CD8(+)IFN-γ(+)) were found in the celiac and portal lymph nodes of Salmonella-treated mice. This study provides first evidence for the involvement of LDLN in the development of an efficient cellular immune response against hepatic tumors, which resulted in the elimination of liver metastases after oral Salmonella-based vaccination.
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Probiotics can modulate the immune system, conferring beneficial effects on the host. Understanding how these microorganisms contribute to improve the health status is still a challenge. Previously, we have demonstrated that Enterococcus faecalis CECT7121 implants itself and persists in the murine gastrointestinal tract, and enhances and skews the profile of cytokines towards the Th1 phenotype in several biological models. Given the importance of dendritic cells (DCs) in the orchestration of immunity, the aim of this work was to elucidate the influence of E. faecalis CECT7121 on DCs and the outcome of the immune responses. In this work we show that E. faecalis CECT7121 induces a strong dose-dependent activation of DCs and secretion of high levels of IL-12, IL-6, TNFα, and IL-10. This stimulation is dependent on TLR signaling, and skews the activation of T cells towards the production of IFNγ. The influence of this activation in the establishment of Th responses in vivo shows the accumulation of specific IFNγ-producing cells. Our findings indicate that the activation exerted by E. faecalis CECT7121 on DCs and its consequence on the cellular adaptive immune response may have broad therapeutic implications in immunomodulation.
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Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Enterococcus faecalis/imunologia , Imunomodulação/efeitos dos fármacos , Imunomodulação/imunologia , Interferon gama/imunologia , Probióticos/administração & dosagem , Animais , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Imunidade/efeitos dos fármacos , Imunidade/imunologia , Interleucina-10/imunologia , Interleucina-12/imunologia , Interleucina-6/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
AIM: Catecholamines regulate functions of the nervous, neuroendocrine and immune systems. Dopamine may modulate the activity of keratinocytes, which play a role in secreting cytokines and chemokines. The aim of this study was to evaluate the effect of dopaminergic agonists on the production of IL-6 and IL-8 by a non-tumoral human keratinocyte cell line (HaCaT). METHODS: Cells were stimulated with dopamine and the D(2) dopamine receptor agonist cabergoline. Levels of IL-6 and IL-8 in culture supernatants were then determined. Cell proliferation was also assessed. Assays were carried out in the presence or absence of the dopaminergic and ß-adrenergic receptor antagonists (sulpiride and propranolol, respectively) and ascorbic acid. RESULTS: Dopamine stimulated the production of IL-6 and IL-8 in a concentration-dependent manner. The effects observed on the secretion of IL-6 were more potent than those corresponding to IL-8 and were reduced by ascorbic acid. The dopamine-induced IL-6 secretion was partially reduced by sulpiride and abrogated by propranolol. The latter drug was able to block the effect of dopamine on the secretion of IL-8. The cabergoline-induced IL-6 release was reduced by sulpiride. Cell viability was not affected by any of the drugs. CONCLUSIONS: Dopaminergic agonists can stimulate keratinocytes to produce IL-6 and IL-8 which are related to inflammatory cutaneous processes. These effects are mediated by dopaminergic and ß-adrenergic receptors and by receptor-independent oxidative mechanisms.
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Agonistas de Dopamina/farmacologia , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Queratinócitos/imunologia , Queratinócitos/metabolismo , Regulação para Cima/imunologia , Linhagem Celular , Relação Dose-Resposta Imunológica , Humanos , Queratinócitos/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Cyclooxygenase (COX)-2, the inducible isoform of cyclooxygenase, plays a role in the process of uterine decidualization and blastocyst attachment. On the other hand, overexpression of COX-2 is involved in the proliferation of the endometrial tissue during endometriosis. Deregulation of the renin-angiotensin-system plays a role in the pathophysiology of endometriosis and pre-eclampsia. Angiotensin II increases intracellular Ca(2+) concentration by targeting phospholypase C-gamma in endometrial stromal cells (ESC). A key element of the cellular response to Ca(2+) signals is the activity of the Ca(2+)- and calmodulin-dependent phosphatase calcineurin. Our first aim was to study whether angiotensin II stimulated Cox-2 gene expression in rat ESC and to analyze whether calcineurin activity was involved. In cells isolated from non-pregnant uteri, COX-2 expression--both mRNA and protein--was induced by co-stimulation with phorbol ester and calcium ionophore (PIo), as well as by angiotensin II. Pretreatment with the calcineurin inhibitor cyclosporin A inhibited this induction. We further analyzed the role of the calcineurin/NFAT signaling pathway in the induction of Cox-2 gene expression in non-pregnant rat ESC. Cyclosporin A abolished NFATc1 dephosphorylation and translocation to the nucleus. Cyclosporin A also inhibited the transcriptional activity driven by the Cox-2 promoter. Exogenous expression of the peptide VIVIT -specific inhibitor of calcineurin/NFAT binding- blocked the activation of Cox-2 promoter and the up-regulation of COX-2 protein in these cells. Finally we analyzed Cox-2 gene expression in ESC of early-pregnant rats. COX-2 expression--both mRNA and protein--was induced by stimulation with PIo as well as by angiotensin II. This induction appears to be calcineurin independent, since it was not abrogated by cyclosporin A. In conclusion, angiotensin II induced Cox-2 gene expression by activating the calcineurin/NFAT signaling pathway in endometrial stromal cells of non-pregnant but not of early-pregnant rats. These results might be related to differential roles that COX-2 plays in the endometrium.
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Angiotensina II/farmacologia , Calcineurina/metabolismo , Ciclo-Oxigenase 2/genética , Endométrio/metabolismo , Fatores de Transcrição NFATC/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Inibidores de Calcineurina , Sinalização do Cálcio/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Fatores de Transcrição NFATC/genética , Fosforilação , Gravidez , Ligação Proteica , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Regulation of the immune response is necessary to allow successful pregnancy. Asymmetric IgG antibodies are involved in fetal maintenance. We have previously demonstrated that estrogen (E2) and progesterone (P4) modulate the synthesis of asymmetric antibodies but the underlying mechanisms remain unclear. Since IL-6 and a progesterone-induced blocking factor (PIBF) were shown to regulate asymmetric antibody synthesis, in this work we analyzed whether E2 and P4 were able to modulate IL-6 signal transduction pathways and the ability of P4 to induce PIBF synthesis, in hybridoma B cells was also evaluated. We found that the IL-6 treatment induced an increase in the expression of gp130 and JAK1 by the hybridoma. E2 and P4 diminished the IL-6-induced gp130 expression in a dose-dependent manner, whereas the expression of JAK1 was not significantly affected. At 10(-6)M concentration, the steroids inhibited the phosphorylation of gp130 and diminished the IL-6-induced STAT3 phosphorylation and translocation to the nucleus. Maximal PIBF expression was observed when the hybridoma was cultured with 10(-10)M P4, compared to the control (p<0.05). Results demonstrate two molecular mechanisms, the modulation of the IL-6R signal transduction pathway and PIBF induction, which could be involved in the immunoregulatory role of sexual steroids during pregnancy.
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Células Produtoras de Anticorpos/imunologia , Estrogênios/metabolismo , Interleucina-6/imunologia , Progesterona/metabolismo , Transdução de Sinais/fisiologia , Animais , Células Produtoras de Anticorpos/citologia , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Feminino , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Camundongos , Gravidez , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismoRESUMO
Pregnancy induces collagen-induced arthritis (CIA) remission in rats. Placental hormones, cytokines and growth factors can regulate immune cell activity at the feto-maternal interface as well as at the systemic level. We assessed the effect of placental culture supernatants (PS) in CIA developed in rats after the inoculation of collagen type II (CII) in complete Freund's adjuvant. After the onset of CIA, animals were injected by ip route with seven doses of PS. On the 18th day of treatment with PS, serum anti-CII antibody (total IgG, IgG(1), IgG(2a), IgG(2b), IgG asymmetric molecules) and cytokine levels were determined by ELISA. An arthritic index was used by daily measure of joint swelling and visual signs of arthritis. Our results demonstrated that the PS treatment diminished CIA symptoms, reduced TNF-alpha, INF-gamma and anti-CII antibody serum levels, increased the proportion of asymmetric IgG anti-CII antibodies and affected IgG(1)/IgG(2a) and IgG(1)/IgG(2b) ratio. Two weeks after the last PS inoculation there was a recurrence of arthritis, a rise in IgG anti-CII and, simultaneously, the percentage of asymmetric IgG anti-CII fell. We concluded that PS have an effective CIA suppressor activity partly due to the modulation of humoral immune response and may be closely related to an inhibitory effect on TNF-alpha and INF-gamma production.
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Formação de Anticorpos/imunologia , Artrite Experimental/imunologia , Colágeno Tipo II/imunologia , Imunoglobulina G/sangue , Placenta/metabolismo , Animais , Anticorpos/imunologia , Formação de Anticorpos/efeitos dos fármacos , Artrite Experimental/sangue , Artrite Experimental/fisiopatologia , Artrite Experimental/terapia , Técnicas de Cultura de Células , Células Cultivadas , Colágeno Tipo II/administração & dosagem , Meios de Cultivo Condicionados/farmacologia , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Feminino , Imunoglobulina G/imunologia , Gravidez , Ratos , Ratos Wistar , Índice de Gravidade de DoençaRESUMO
We have previously demonstrated that 10-20% of the IgG isolated from non-immune sera is asymmetrically glycosylated, in such a way that it fails to trigger immune effector mechanisms. As a result, a major portion of the non-immune asymmetric IgG molecules of the host could be self-specific, acting as auto-protective antibodies. In order to test this hypothesis, we investigated whether asymmetric IgG molecules are capable of recognizing self-antigens. About 40% of F(ab')2 fragment from normal rat IgG was able to react specifically with autologous rat cells. Moreover, upon being purified from normal rat sera, 78% of the asymmetric IgG sub-population showed self-reactivity. We demonstrated that about 14% of rat asymmetric IgG-F(ab')2 fragments was able to react with bacteria isolated from the intestine of uninfected rats. Lastly, in order to test whether there is a correlation between the decline of immune responses during ageing and asymmetric antibody production, we assayed IgG isolated from sera of young and old rats. There was an increase in the asymmetric:symmetric IgG ratio with ageing. We therefore suggest that asymmetric antibodies may exert a beneficial action by protecting self-antigens as well as normal intestinal flora from a deleterious immune response.