RESUMO
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
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
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
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
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
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
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
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.