RESUMO
Secondary infections due to post-sepsis immunosuppression are a major cause of death in patients with sepsis. Repetitive inoculation of increasing doses of lipopolysaccharide (LPS) into mice mimics the immunosuppression associated with sepsis. Myeloid-derived suppressor cells (MDSCs, Gr-1(+) CD11b(+)) are considered a major component of the immunosuppressive network, interfering with T-cell responses in many pathological conditions. We used LPS-immunosuppressed (IS) mice to address whether MDSCs acquired their suppressive ability in the bone marrow (BM) and whether they could migrate to lymph nodes (LNs) to exert their suppressive function. Our results showed that Gr-1(+) CD11b(+) cells of IS mice already had the potential to inhibit T-cell proliferation in the BM. Moreover, soluble factors present in the BM from IS mice were responsible for inducing this inhibitory ability in control BM cells. In addition, migration of Gr-1(+) CD11b(+) to LNs in vivo was maximal when cells obtained from the BM of IS mice were inoculated into an IS context. In this regard, we found chemoattractant activity in cell-free LN extracts (LNEs) from IS mice and an increased expression of the LN-homing chemokine receptor C-C chemokine receptor type 7 (CCR7) in IS BM Gr-1(+) CD11b(+) cells. These results indicate that Gr-1(+) CD11b(+) cells found in BM from IS mice acquire their suppressive activity in the same niche where they are generated, and migrate to LNs to exert their inhibitory role. A better understanding of MDSC generation and/or regulation of factors able to induce their inhibitory function may provide new and more effective tools for the treatment of sepsis-associated immunosuppression.
Assuntos
Antígenos Ly/imunologia , Células da Medula Óssea/imunologia , Antígeno CD11b/imunologia , Quimiotaxia/efeitos dos fármacos , Hospedeiro Imunocomprometido , Lipopolissacarídeos , Linfonodos/imunologia , Células Mieloides/imunologia , Sepse/imunologia , Animais , Antígenos Ly/metabolismo , Células da Medula Óssea/metabolismo , Antígeno CD11b/metabolismo , Células Cultivadas , Microambiente Celular , Técnicas de Cocultura , Modelos Animais de Doenças , Linfonodos/metabolismo , Ativação Linfocitária , Camundongos Endogâmicos BALB C , Células Mieloides/metabolismo , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Sepse/induzido quimicamente , Sepse/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
The hemolytic uremic syndrome (HUS) is characterized by hemolytic anemia, thrombocytopenia and renal dysfunction. The typical form of HUS is generally associated with infections by Gram-negative Shiga toxin (Stx)-producing Escherichia coli (STEC). Endothelial dysfunction induced by Stx is central, but bacterial lipopolysaccharide (LPS) and neutrophils (PMN) contribute to the pathophysiology. Although renal failure is characteristic of this syndrome, neurological complications occur in severe cases and is usually associated with death. Impaired blood-brain barrier (BBB) is associated with damage to cerebral endothelial cells (ECs) that comprise the BBB. Astrocytes (ASTs) are inflammatory cells in the brain and determine the BBB function. ASTs are in close proximity to ECs, hence the study of the effects of Stx1 and LPS on ASTs, and the influence of their response on ECs is essential. We have previously demonstrated that Stx1 and LPS induced activation of rat ASTs and the release of inflammatory factors such as TNF-α, nitric oxide and chemokines. Here, we demonstrate that rat ASTs-derived factors alter permeability of ECs with brain properties (HUVECd); suggesting that functional properties of BBB could also be affected. Additionally, these factors activate HUVECd and render them into a proagregant state promoting PMN and platelets adhesion. Moreover, these effects were dependent on ASTs secreted-TNF-α. Stx1 and LPS-induced ASTs response could influence brain ECs integrity and BBB function once Stx and factors associated to the STEC infection reach the brain parenchyma and therefore contribute to the development of the neuropathology observed in HUS.
Assuntos
Astrócitos/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Toxina Shiga I/toxicidade , Fator de Necrose Tumoral alfa/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Barreira Hematoencefálica , Encéfalo/irrigação sanguínea , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Endotélio Vascular/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , RatosRESUMO
Secondary infections due to post-sepsis immunosuppression are a major cause of death in patients with sepsis. Strategies aimed at restoring immune functions offer a new perspective in the treatment of sepsis. In the present study, we used LPS (lipopolysaccharide)-immunosuppressed mice to analyse the effects of ATRA (all-trans retinoic acid) on different immune parameters. The IS (immunocompromised) group had decreased lymphocyte and increased MDSC (myeloid-derived suppressor cell) counts in lymph nodes. They also had an impaired in vitro T-cell proliferation, mediated by MDSCs. ATRA administration restored T-cell proliferation, which was associated with a decreased number of live MDSCs. The IS group treated with ATRA had an increased number of CD4+ and CD8+ T-cells. ATRA partially improved the primary humoral immune response, even when immunosuppression was established first and ATRA was administered subsequently. Our results demonstrate that ATRA restores immunocompetence by modulating the number of leucocytes and the survival of MDSCs, and thus represents an additional potential strategy in the treatment of the immunosuppressive state of sepsis.
Assuntos
Imunocompetência/efeitos dos fármacos , Terapia de Imunossupressão , Lipopolissacarídeos/farmacologia , Modelos Animais , Tretinoína/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Large amounts of anti-inflammatory mediators, such as interleukin (IL)-10, are produced and found early in the course of sepsis. We explore the role of IL-10 on neutrophil (PMN) activation/function using an in vitro model. Isolated human PMN were pre-incubated with lipopolysaccharide (LPS) and/or IL-10 for 18h. Subsequently, a second LPS exposure was performed and CD11b and CD66b up-regulation, and the reactive oxygen species (ROS) generation were measured 2h later. We found that IL-10 prevented PMN activation and the secretion of TNF-α and IL-8 induced by the first LPS contact. In the absence of IL-10, a second LPS exposure induced additive effects that were prevented by IL-10. Only ROS generation was highly affected by the blockade of PMN-secreted TNF-α or IL-8. Additionally, IL-10 prevented other possible mechanisms of LPS priming. Therefore, IL-10 modulates PMN activation preventing autocrine activating loops and priming mechanisms, rendering PMN less responsive to a second LPS exposure.
Assuntos
Interleucina-10/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Comunicação Autócrina/efeitos dos fármacos , Degranulação Celular/efeitos dos fármacos , Separação Celular , Humanos , Interleucina-8/metabolismo , Neutrófilos/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Ácidos Teicoicos/farmacologiaRESUMO
PURPOSE: The interaction of Shiga toxin (Stx) and/or lipopolysaccharide (LPS) with monocytes (Mo) may be central to the pathogenesis of hemolytic uremic syndrome (HUS), providing the cytokines necessary to sensitize endothelial cells to Stx action. We have previously demonstrated phenotypical alterations in Mo from HUS patients, including increased number of CD16+ Mo. Our aim was to investigate cytokine production in Mo from HUS patients. METHODS: We evaluated TNF-α and IL-10 intracellular contents and secretion in the different Mo subsets in mild (HUS 1) and moderate/severe (HUS 2 + 3) patients. As controls, we studied healthy (HC) and infected children (IC). We also studied Mo responsive capacity towards LPS, measuring the modulation of Mo surface molecules and cytokine production. RESULTS: In basal conditions, the intracellular measurement of TNF-α and IL-10 revealed that the highest number of cytokine-producing Mo was found in HUS 2 + 3 and IC, whereas LPS caused a similar increase in TNF-α and IL-10-producing Mo for all groups. However, when evaluating the release of TNF-α and IL-10, we found a diminished secretion capacity in the entire HUS group and IC compared to HC in basal and LPS conditions. Similarly, a lower Mo response to LPS in HUS 2 + 3 and IC groups was observed when surface markers were studied. CONCLUSION: These results indicate that Mo from severe cases of HUS, similar to IC but different to mild HUS cases, present functional changes in Mo subpopulations and abnormal responses to LPS.
Assuntos
Síndrome Hemolítico-Urêmica/imunologia , Interleucina-10/imunologia , Monócitos/imunologia , Fator de Necrose Tumoral alfa/imunologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Interleucina-10/sangue , Lipopolissacarídeos/imunologia , Masculino , Fator de Necrose Tumoral alfa/sangueRESUMO
Hemolytic-uremic syndrome (HUS) is generally caused by Shiga toxin (Stx)-producing Escherichia coli. Endothelial dysfunction mediated by Stx is a central aspect in HUS development. However, inflammatory mediators such as bacterial lipopolysaccharide (LPS) and polymorphonuclear neutrophils (PMN) contribute to HUS pathophysiology by potentiating Stx effects. Acute renal failure is the main feature of HUS, but in severe cases, patients can develop neurological complications, which are usually associated with death. Although the mechanisms of neurological damage remain uncertain, alterations of the blood-brain barrier associated with brain endothelial injury is clear. Astrocytes (ASTs) are the most abundant inflammatory cells of the brain that modulate the normal function of brain endothelium and neurons. The aim of this study was to evaluate the effects of Stx type 1 (Stx1) alone or in combination with LPS in ASTs. Although Stx1 induced a weak inflammatory response, pretreatment with LPS sensitized ASTs to Stx1-mediated effects. Moreover, LPS increased the level of expression of the Stx receptor and its internalization. An early inflammatory response, characterized by the release of tumor necrosis factor alpha (TNF-alpha) and nitric oxide and PMN-chemoattractant activity, was induced by Stx1 in LPS-sensitized ASTs, whereas activation, evidenced by higher levels of glial fibrillary acid protein and cell death, was induced later. Furthermore, increased adhesion and PMN-mediated cytotoxicity were observed after Stx1 treatment in LPS-sensitized ASTs. These effects were dependent on NF-kappaB activation or AST-derived TNF-alpha. Our results suggest that TNF-alpha is a pivotal effector molecule that amplifies Stx1 effects on LPS-sensitized ASTs, contributing to brain inflammation and leading to endothelial and neuronal injury.
Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/imunologia , Lipopolissacarídeos/imunologia , Toxina Shiga I/imunologia , Toxina Shiga I/toxicidade , Fator de Necrose Tumoral alfa/imunologia , Animais , Células Cultivadas , Escherichia coli/patogenicidade , Mediadores da Inflamação/metabolismo , Óxido Nítrico/metabolismo , RatosRESUMO
The membrane-anchored form of the chemokine fractalkine (CX(3)CL1) has been identified as a novel adhesion molecule that interacts with its specific receptor (CX(3)CR1) expressed in monocytes, T cells and natural killer cells to induce adhesion. In addition, CX(3)CL1 can be cleaved from the cell membrane to induce chemotaxis of CX(3)CR1-expressing leucocytes. Recently, marked variations in CX(3)CR1 monocyte expression have been observed during several pathological conditions. Regulation of CX(3)CR1 in monocytes during basal or inflammatory/anti-inflammatory conditions is poorly understood. The aim of this study was therefore to examine CX(3)CR1 expression during monocyte maturation and the effect of soluble mediators on this process. We found that basal expression of CX(3)CR1 in fresh monocytes was reduced during culture, and that lipopolysacchairde accelerated this effect. In contrast, interleukin-10 and interferon-gamma treatment abrogated CX(3)CR1 down-modulation, through a phosphatidylinositol 3 kinase-dependent pathway. Most importantly, CX(3)CR1 membrane expression correlated with monocyte CX(3)CL1-dependent function. Taken together, our data demonstrate that CX(3)CR1 expression in monocytes can be modulated, and suggest that alterations in their environment are able to influence CX(3)CL1-dependent functions, such as chemotaxis and adhesion, leading to changes in the kinetics, composition and/or functional status of the leucocyte infiltrate.
Assuntos
Interferon gama/farmacologia , Interleucina-10/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de Quimiocinas/biossíntese , Receptor 1 de Quimiocina CX3C , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Interferon gama/imunologia , Interleucina-10/imunologia , Monócitos/imunologia , Receptores de Quimiocinas/imunologia , Fatores de TempoRESUMO
Sepsis constitutes one of the major causes of death in ICUs. In sepsis induced by gram-negative, although lipopolysaccharide (LPS) initially induces an exacerbated secretion of proinflammatory cytokines leading to endotoxic shock and death resembling a septic shock, it is also capable of inducing refractoriness to subsequent challenge with LPS, a state known as endotoxin tolerance, which is considered the initial step of the immunosuppression found in septic patients. As we previously demonstrated the importance of glucocorticoids in endotoxin tolerance, the aim of this study was to evaluate the contribution of Interleukin-10 (IL-10) both in the endotoxic shock and in the development of the tolerance and its relationship with glucocorticoids. Our results show that, upon LPS challenge, IL-10 knockout mice (KO) mice had an enhanced LPS sensitivity, along with elevated levels of proinflammatory cytokines as tumor necrosis factor-α, IL-12 and interferon-γ, and enhanced tissue damage, despite the high levels of glucocorticoids. This effect may be because, in part, of the higher expression of tumor necrosis factor receptors in IL-10 KO mice. Further, the injection of dexamethasone did not protect IL-10 KO mice from a LPS lethal challenge. Although tolerance was achieved in the absence of IL-10, it was weaker and the elevated levels of glucocorticoids were not able to reverse the high sensitivity of IL-10 KO mice to LPS. Nevertheless, glucocorticoids would play a pivotal role in the establishment and maintenance of this partial tolerance in IL-10 KO mice. Finally, our results show that IL-10 and glucocorticoids could act in a bidirectional way influencing the inflammatory and anti-inflammatory periods.
Assuntos
Endotoxinas/toxicidade , Glucocorticoides/farmacologia , Interleucina-10/deficiência , Choque Séptico/tratamento farmacológico , Animais , Citocinas/metabolismo , Dexametasona/farmacologia , Tolerância Imunológica/efeitos dos fármacos , Interleucina-10/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Mifepristona/uso terapêutico , Choque Séptico/induzido quimicamente , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Monocytes (Mo) mediate central functions in inflammation and immunity. Different subpopulations of Mo with distinct phenotype and functional properties have been described. Here, we investigate the phenotype and function of peripheral Mo from children with hemolytic uremic syndrome (HUS). For this purpose, blood samples from patients in the acute period of HUS (HUS AP) were obtained on admission before dialysis and/or transfusion. The Mo phenotypic characterization was performed on whole blood by flow cytometry, and markers associated to biological functions were selected: CD14 accounting for lipopolysaccharide (LPS) responsiveness, CD11b for adhesion, Fc receptor for immunoglobulin G type I (FcgammaRI)/CD64 for phagocytosis and cytotoxicity, and human leukocyte antigen (HLA)-DR for antigen presentation. Some of these functions were also determined. Moreover, the percentage of CD14+ CD16+ Mo was evaluated. We found that the entire HUS AP Mo population exhibited reduced CD14, CD64, and CD11b expression and decreased LPS-induced tumor necrosis factor production and Fcgamma-dependent cytotoxicity. HUS AP showed an increased percentage of CD14+ CD16+ Mo with higher CD16 and lower CD14 levels compared with the same subset from healthy children. Moreover, the CD14++ CD16- Mo subpopulation of HUS AP had a decreased HLA-DR expression, which correlated with severity. In conclusion, the Mo population from HUS AP patients presents phenotypic and functional alterations. The contribution to the pathogenesis and the possible scenarios that led to these changes are discussed.
Assuntos
Antígenos de Diferenciação/imunologia , Sangue Fetal/imunologia , Síndrome Hemolítico-Urêmica/imunologia , Síndrome Hemolítico-Urêmica/fisiopatologia , Monócitos/imunologia , Contagem de Células , Pré-Escolar , Citotoxicidade Imunológica , Citometria de Fluxo , Síndrome Hemolítico-Urêmica/diagnóstico , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Lactente , Lipopolissacarídeos/farmacologia , Fenótipo , Fatores de Necrose Tumoral/imunologiaRESUMO
The central role of the immune system is the preservation of the health against several pathogenic microbes and injury agents. However, on special conditions defensive mechanisms triggered towards the foreign agent can damage the host. Clinical and experimental evidence indicate that inflammatory reaction triggered by the main components of Shiga toxin (Stx)-producing Escherichia coil (STEC), participate in the evolution to the complete form of HUS. When children are diagnosed of HUS, they present evidence that have suffered a very strong and early inflammatory response. These features include: the presence of a marked neutrophilia, the polymorfonuclear leucocytes (PMN) are "deactivated or exhausted" and the monocytes are differentiated towards an inflammatory phenotype (CD14-reduced and CD16-enhanced membrane expression). In addition, HUS-patients show a marked reduction in the absolute and relative number of leucocytes carrying the receptor (CX3CR1) for the chemokine "Fractalkine" (FKN, CX3CL1), which are the classic monocytes and Natural Killer cells (NK). All these cells express a high cytotoxic potencial. The chemokine FKN is expressed in endothelial and epithelial renal cells, and is involved in the pathogenic mechanism of different nephropathies. Noteworthy, we found a significant correlation between the severity of the renal damage (as days of anuria) and the alterations described above. Finally, the protective role of specific immune response, mainly through the antibody production with Stx-neutralizing capacity, is discussed.
Assuntos
Síndrome Hemolítico-Urêmica/imunologia , Imunidade Inata/imunologia , Ativação de Neutrófilo/imunologia , Toxina Shiga/toxicidade , Animais , Antígenos CD/imunologia , Quimiocina CX3CL1/imunologia , Citocinas/imunologia , Modelos Animais de Doenças , Escherichia coli/imunologia , Escherichia coli/patogenicidade , Infecções por Escherichia coli/imunologia , Fatores de Crescimento de Fibroblastos/imunologia , Síndrome Hemolítico-Urêmica/terapia , Humanos , Células Matadoras Naturais/imunologia , Murinae , Neutrófilos/imunologia , Ratos , Diálise Renal , Toxina Shiga/antagonistas & inibidores , Toxina Shiga/imunologiaRESUMO
A small primary or secondary tumor load can occasionally induce more deleterious effects than a histologically identical larger one. In the four murine models studied herein this enhanced tumor aggressiveness could not be attributed to NRAS mutations or other hereditary changes, differential vascularization of live tumor tissues, or necrosis content. Instead, the main tumor feature associated with a more aggressive behavior was the presence of a high number of vessels, sometimes filled with inflammatory cells, inside a tumor area, which we have identified and designated as the transition zone between the live and the necrotic zones. Our experiments suggest that during tumor growth, different cachectic factors are produced within the transition and necrotic zones by dying tumor cells and by tumor infiltrating macrophages only reaching the general circulation through the vessels present in the transition zone. Therefore, a small tumor displaying high vascularization of its transition area could be harmful to its host, while, in contrast, a large tumor could behave as a relatively benign one if its transition zone exhibited little or no vascularization, and in consequence its cachectic factors remained "trapped." Similar histological images to those observed in mice were seen in a significant percentage of human cancer biopsies, raising the possibility that such images might have a prognostic value.
Assuntos
Neoplasias/patologia , Neovascularização Patológica/patologia , Animais , Biomarcadores Tumorais/análise , Linhagem Celular Tumoral , DNA de Neoplasias/análise , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Necrose , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias/irrigação sanguínea , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/genética , RNA Neoplásico/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Regulation of inflammation is a crucial event since its alteration, such as in sepsis and chronic autoimmune (i.e. rheumatoid arthritis, lupus erythematosus) or infectious diseases (i.e. tuberculosis, leprosy), determines severe tissue damage. Although there is a general consensus that regulation of inflammation results from a balance between proinflammatory and antiinflammatory pathways, we arrived at the conclusion that well known chemoattractants/proinflammatory molecules such as bacterial formyl peptides or immune complexes (IC), could induce, paradoxically, strong antiinflammatory effects. Thus, we demonstrated that N-formyl-methionyl-leucyl-phenylalanine (FMLP) exerted a drastic antiinflammatory effect, inhibiting the secretion of tumor necrosis alpha (TNF-alpha) induced by lipopolysaccharides, a potent TNF-alpha inducer. We also determined that in human neutrophils FMLP and IC induced the downregulation of receptors for the Fc portion of IgG (FcgammaRII and FcgammaRIIIB). Moreover, FMLP inhibited interferon gamma (IFN-gamma)-induced FcgammaRI expression and IC downregulate class II molecules of the major histocompatibility complex on monocytes. Part of these effects were mediated by the release of aspartic-, serin-, or metalloproteases. All these results favor the postulation of a new concept on the regulation of inflammation carried out through an alternative and non conventional pathway, in which a chemoattractant/proinflammatory agent could, under certain circumstances, act as an antiinflammatory molecule.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Inflamação/imunologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/imunologia , Receptores de IgG/imunologia , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Regulação da Expressão Gênica/fisiologia , Humanos , Inflamação/fisiopatologia , Interferon gama/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Neutrófilos/metabolismo , Receptores de IgG/metabolismo , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Prior exposure to endotoxins renders the host temporarily refractory to subsequent endotoxin challenge (endotoxin tolerance). Clinically, this state has also been pointed out as the initial cause of the non-specific humoral and cellular immunosuppression described in these patients. We recently demonstrated the restoration of immune response with mifepristone (RU486), a receptor antagonist of glucocorticoids. Here we report the treatment with other modulators of glucocorticoids, i.e. dehydroepiandrosterone (DHEA), a hormone with anti-glucocorticoid properties, or metyrapone (MET) an inhibitor of corticosterone synthesis. These drugs were able to partially, but significantly, restore the humoral immune response in immunosuppressed mice. A significant recovery of proliferative responsiveness was also observed when splenocytes were obtained from DHEA- or MET-treated immunosuppressed mice. In addition, these treatments restored the hypersensitivity response in immunosuppressed mice. Finally, although neither DHEA nor MET improved the reduced CD4 lymphocyte count in spleen from immunosuppressed mice, both treatments promoted spleen architecture reorganization, partially restoring the distinct cellular components and their localization in the spleen. The results from this study indicate that DHEA and MET could play an important role in the restoration of both adaptive humoral and cellular immune response in LPS-immunosuppressed mice, reinforcing the concept of a central involvement of endogenous glucocorticoids on this phenomenon.
Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Desidroepiandrosterona/farmacologia , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Terapia de Imunossupressão , Lipopolissacarídeos/farmacologia , Metirapona/farmacologia , Animais , Contagem de Linfócito CD4 , Proliferação de Células , Glucocorticoides/antagonistas & inibidores , Hipersensibilidade Tardia/imunologia , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose/efeitos dos fármacos , Receptores de Glucocorticoides/antagonistas & inibidores , Explosão Respiratória/efeitos dos fármacos , Baço/citologia , Baço/imunologiaRESUMO
Concomitant tumor resistance (CR) is a phenomenon originally described in 1906 in which a tumor-bearing host is resistant to the growth of secondary tumor implants and metastasis. Although recent studies have indicated that T-cell-dependent processes mediate CR in hosts bearing immunogenic small tumors, manifestations of CR induced by immunogenic and nonimmunogenic large tumors have been associated with an elusive serum factor. In this study, we identify this serum factor as tyrosine in its meta and ortho isoforms. In three different murine models of cancer that generate CR, both meta-tyrosine and ortho-tyrosine inhibited tumor growth. In addition, we showed that both isoforms of tyrosine blocked metastasis in a fourth model that does not generate CR but is sensitive to CR induced by other tumors. Mechanistic studies showed that the antitumor effects of the tyrosine isoforms were mediated, in part, by early inhibition of mitogen-activated protein/extracellular signal-regulated kinase pathway and inactivation of STAT3, potentially driving tumor cells into a state of dormancy. By revealing a molecular basis for the classical phenomenon of CR, our findings may stimulate new generalized approaches to limit the development of metastases that arise after resection of primary tumors, an issue of pivotal importance to oncologists and their patients.
Assuntos
Transplante de Neoplasias/imunologia , Neoplasias Experimentais/imunologia , Tirosina/fisiologia , Animais , Cromatografia Líquida de Alta Pressão , Resistência à Doença , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Feminino , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/patologia , Fenilalanina/farmacologia , Fator de Transcrição STAT3/fisiologiaRESUMO
Shiga toxin (Stx)-producing Escherichia coli (STEC) infection is associated with a broad spectrum of clinical manifestations that include diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Systemic Stx toxemia is considered to be central to the genesis of HUS. Distinct methods have been used to evaluate anti-Stx response for immunodiagnostic or epidemiological analysis of HUS cases. The development of enzyme-linked immunosorbent assay (ELISA) and western blot (WB) assay to detect the presence of specific antibodies to Stx has introduced important advantages for serodiagnosis of HUS. However, application of these methods for seroepidemiological studies in Argentina has been limited. The aim of this work was to develop an ELISA to detect antibodies against the B subunit of Stx2, and a WB to evaluate antibodies against both subunits of Stx2 and Stx1, in order to analyze the pertinence and effectiveness of these techniques in the Argentinean population. We studied 72 normal healthy children (NHC) and 105 HUS patients of the urban pediatric population from the surrounding area of Buenos Aires city. Using the WB method we detected 67% of plasma from NHC reactive for Stx2, but only 8% for Stx1. These results are in agreement with the broad circulation of Stx2-expressing STEC in Argentina and the endemic behavior of HUS in this country. Moreover, the simultaneous evaluation by the two methods allowed us to differentiate acute HUS patients from NHC with a great specificity and accuracy, in order to confirm the HUS etiology when pathogenic bacteria were not isolated from stools.
Assuntos
Síndrome Hemolítico-Urêmica/imunologia , Síndrome Hemolítico-Urêmica/microbiologia , Toxinas Shiga/imunologia , Anticorpos/imunologia , Argentina , Estudos de Casos e Controles , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Seguimentos , Síndrome Hemolítico-Urêmica/tratamento farmacológico , Humanos , Masculino , Testes Sorológicos , Toxinas Shiga/química , Fatores de Tempo , Resultado do TratamentoRESUMO
Although animals can be immunized against the growth of some tumor implants, most of the attempts to use immunotherapy to cause the regression of animal and human tumors once they have become established have been disappointing even when strongly immunogenic tumors were used as target. In this paper, we demonstrate that the failure to achieve an efficient immunological treatment against an established strongly immunogenic murine fibrosarcoma was paralleled with the emergence of a state of immunological unresponsiveness (immunological eclipse) against tumor antigens observed when the tumor surpassed the critical size of 500 mm(3). In turn, the onset of the immunological eclipse was coincidental with the onset of a systemic inflammatory condition characterized by a high number of circulating and splenic polymorphonucleated neutrophils (PMN) displaying activation and Gr1(+)Mac1(+) phenotype and an increasing serum concentration of the pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6 cytokines and C-reactive protein (CRP) and serum A amyloid (SAA) phase acute proteins. Treatment of tumor-bearing mice with a single low dose (0.75 mg/kg) of the synthetic corticoid dexamethasone (DX) significantly reduced all the systemic inflammatory parameters and simultaneously reversed the immunological eclipse, as evidenced by the restoration of specific T-cell-dependent concomitant immunity, ability of spleen cells to transfer anti-tumor activity and recovery of T-cell signal transduction molecules. Two other anti-inflammatory treatments by using indomethacin or dimeric TNF-alpha receptor, also partially reversed the immunological eclipse although the effect was not as striking as that observed with DX. The reversion of the immunological eclipse was not enough on its own to inhibit the primary growing tumor. However, when we used the two-step strategy of inoculating DX to reverse the eclipse and then dendritic cells loaded with tumor antigens (DC) as an immunization booster, a significant inhibition of the growth of both established tumors and remnant tumor cells after excision of large established tumors was observed, despite the fact that the vaccination alone (DC) had no effect or even enhanced tumor growth in certain circumstances. The two-step strategy of tumor immunotherapy that we present is based on the rationale that it is necessary to eliminate or ameliorate the immunological eclipse as a precondition to allow an otherwise ineffective anti-tumor immunological therapy to have a chance to be successful.
Assuntos
Anti-Inflamatórios/uso terapêutico , Células Dendríticas/transplante , Tolerância Imunológica/efeitos dos fármacos , Imunoterapia/métodos , Inflamação/tratamento farmacológico , Neoplasias Experimentais/terapia , Animais , Western Blotting , Vacinas Anticâncer/uso terapêutico , Células Dendríticas/imunologia , Dexametasona/uso terapêutico , Feminino , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/imunologiaRESUMO
Several environmental factors can differentially regulate monocyte and macrophage response patterns, resulting in the display of distinct functional phenotypes. Galectin-1, an endogenous lectin found at peripheral lymphoid organs and inflammatory sites, has shown immunoregulatory activity in vivo in experimental models of autoimmunity and cancer. Whereas compelling evidence has been accumulated regarding the effects of galectin-1 on T cell fate, limited information is available on how galectin-1 may impact other immune cell types. In the present study, we report a novel role for galectin-1 in the regulation of monocyte and macrophage physiology. Treatment with galectin-1 in vitro differentially regulates constitutive and inducible FcgammaRI expression on human monocytes and FcgammaRI-dependent phagocytosis. In addition, galectin-1 inhibits IFN-gamma-induced MHC class II (MHC-II) expression and MHC-II-dependent Ag presentation in a dose-dependent manner. These regulatory effects were also evident in mouse macrophages recruited in response to inflammatory stimuli following treatment with recombinant galectin-1 and further confirmed in galectin-1-deficient mice. Investigation of the mechanisms involved in these functions showed that galectin-1 does not affect survival of human monocytes, but rather influences FcgammaRI- and MHC-II-dependent functions through active mechanisms involving modulation of an ERK1/2-dependent pathway. Our results provide evidence of a novel unrecognized role for galectin-1 in the control of monocyte/macrophage physiology with potential implications at the crossroad of innate and adaptive immunity.
Assuntos
Galectina 1/fisiologia , Macrófagos/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Monócitos/imunologia , Receptores de IgG/metabolismo , Animais , Apresentação de Antígeno , Apoptose , Sobrevivência Celular , Galectina 1/farmacologia , Antígenos de Histocompatibilidade Classe II/análise , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imunidade Inata , Ativação de Macrófagos , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Monócitos/efeitos dos fármacos , Fagocitose , Receptores de IgG/análiseRESUMO
The interaction between immune complexes (IC) and the receptors for the Fc portion of IgG (FcgammaRs) triggers regulatory and effector functions in the immune system. In this study, we investigated the effects of IC on differentiation, maturation, and functions of human monocyte-derived dendritic cells (DC). When IC were added on day 0, DC generated on day 6 (IC-DC) showed lower levels of CD1a and increased expression of CD14, MHC class II, and the macrophage marker CD68, as compared with normally differentiated DC. The use of specific blocking FcgammaR mAbs indicated that the effect of IC was exerted mainly through their interaction with FcgammaRI and to a lesser extend with FcgammaRII. Immature IC-DC also expressed higher levels of CD83, CD86, and CD40 and the expression of these maturation markers was not further regulated by LPS. The apparent lack of maturation following TLR stimulation was associated with a decreased production of IL-12, normal secretion of IL-10 and CCL22, and increased production of CXCL8 and CCL2. IC-DC displayed low endocytic activity and a reduced ability to induce allogeneic T cell proliferation both at basal and LPS-stimulated conditions. Altogether, these data reveal that IC strongly affect DC differentiation and maturation. Skewing of DC function from Ag presentation to a proinflammatory phenotype by IC resembles the state of activation observed in DC obtained from patients with chronic inflammatory autoimmune disorders, such as systemic lupus erythematosus disease and arthritis. Therefore, the altered maturation of DC induced by IC may be involved in the pathogenesis of autoimmune diseases.
Assuntos
Complexo Antígeno-Anticorpo/fisiologia , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Inibidores do Crescimento/fisiologia , Monócitos/citologia , Monócitos/imunologia , Animais , Complexo Antígeno-Anticorpo/sangue , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Relação Dose-Resposta Imunológica , Inibidores do Crescimento/sangue , Humanos , Imunoglobulina G/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Ativação Linfocitária/imunologia , Teste de Cultura Mista de Linfócitos , Monócitos/metabolismo , Monócitos/patologia , Coelhos , Receptores de IgG/antagonistas & inibidores , Receptores de IgG/biossíntese , Receptores de IgG/fisiologia , Linfócitos T/imunologia , Fatores de TempoRESUMO
Hemolytic Uremic Syndrome (HUS) is the main cause of acute renal failure in children. The high percentage of patients who develop long-term sequelae constitutes an important medical concern. The identification of parameters that correlate with the degree of renal failure may be useful to plan the best treatment soon after hospitalization. Here, we investigated the functional state of neutrophils (PMN) from HUS patients on admission, before dialysis and/or transfusion, in relation to the severity of renal impairment reached during the acute period (AP). We found that all PMN activation parameters measured in severe cases of HUS (HUS AP3) were statistically lower comparing to children with mild cases of HUS (HUS AP1). As HUS PMN phenotype and dysfunction is compatible with that of cells undergoing cell death, we also studied spontaneous apoptosis. Not only were HUS PMN not apoptotic, but HUS AP3 PMN showed an increased survival. Almost all phenotypic and functional parameters measured on PMN correlated with severity. Our results revealed a marked deactivation of PMN in severe cases of HUS, and suggest that studying the functional state of PMN could be of prognostic value.
Assuntos
Síndrome Hemolítico-Urêmica/diagnóstico , Síndrome Hemolítico-Urêmica/fisiopatologia , Neutrófilos/fisiologia , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , PrognósticoRESUMO
Experimental and clinical evidence suggest that activated neutrophils (PMN) could contribute to endothelial damage in Hemolytic Uremic Syndrome (D+HUS). Additionally, while PMN-activating cytokines and PMN-derived products have been found in D+HUS sera, we have demonstrated phenotypic alterations in D+HUS PMN compatible with a deactivation state. Here, we investigated whether D+HUS PMN were actually hyporesponsive, and explored some of the mechanisms probably involved in their derangement. Twenty-two D+HUS children were bled in the acute period, and blood samples from healthy, acute uremic and neutrophilic children were obtained as controls. We evaluated degranulation markers in response to cytokines, intracellular granule content, and reactive oxygen species (ROS) generation in circulating D+HUS and control PMN. The influence of D+HUS-derived plasma and the direct effects of Stx in vitro were evaluated on healthy donors' PMN. We found that D+HUS PMN presented reduced degranulatory capacity in response to cytokines and intracellular granule content, and decreased ROS generation. D+HUS plasma or Stx did not affect the phenotype and function of healthy donors' PMN. These results suggest that upon hospitalization D+HUS PMN are functionally impaired and show features of previous degranulation, indicating a preceding process of activation with release of ROS and proteases involved in endothelial damage.