RESUMO
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by progressive joint destruction associated with increased pro-inflammatory mediators. In inflammatory microenvironments, exogenous ATP (eATP) is hydrolyzed to adenosine, which exerts immunosuppressive effects, by the consecutive action of the ectonucleotidases CD39 and CD73. Mature B cells constitutively express both ectonucleotidases, converting these cells to potential suppressors. Here, we assessed CD39 and CD73 expression on B cells from treated or untreated patients with RA. Neither the frequency of CD73+CD39+ and CD73-CD39+ B cell subsets nor the levels of CD73 and CD39 expression on B cells from untreated or treated RA patients showed significant changes in comparison to healthy controls (HC). CpG+IL-2-stimulated B cells from HC or untreated RA patients increased their CD39 expression, and suppressed CD4+ and CD8+ T cell proliferation and intracellular TNF-production. A CD39 inhibitor significantly restored proliferation and TNF-producing capacity in CD4+ T cells, but not in CD8+ T cells, from HC and untreated RA patients, indicating that B cells from untreated RA patients conserved CD39-mediated regulatory function. Good responder patients to therapy (R-RA) exhibited an increased CD39 but not CD73 expression on B cells after treatment, while most of the non-responder (NR) patients showed a reduction in ectoenzyme expression. The positive changes of CD39 expression on B cells exhibited a negative correlation with disease activity and rheumatoid factor levels. Our results suggest modulating the ectoenzymes/ADO pathway as a potential therapy target for improving the course of RA.
Assuntos
Apirase/metabolismo , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Imunomodulação , Adenosina/metabolismo , Apirase/genética , Artrite Reumatoide/patologia , Artrite Reumatoide/terapia , Linfócitos B Reguladores/imunologia , Linfócitos B Reguladores/metabolismo , Estudos de Casos e Controles , Citocinas/biossíntese , Gerenciamento Clínico , Suscetibilidade a Doenças , Expressão Gênica , Humanos , Ativação Linfocitária , Contagem de Linfócitos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Resultado do TratamentoRESUMO
Solid tumors are infiltrated by immune cells where macrophages and senescent T cells are highly represented. Within the tumor microenvironment, a cross-talk between the infiltrating cells may occur conditioning the characteristic of the in situ immune response. Our previous work showed that tumors induce senescence of T cells, which are powerful suppressors of lympho-proliferation. In this study, we report that Tumor-Induced Senescent (TIS)-T cells may also modulate monocyte activation. To gain insight into this interaction, CD4+ or CD8+TIS-T or control-T cells were co-incubated with autologous monocytes under inflammatory conditions. After co-culture with CD4+ or CD8+TIS-T cells, CD14+ monocytes/macrophages (Mo/Ma) exhibit a higher expression of CD16+ cells and a reduced expression of CD206. These Mo/Ma produce nitric oxide and reactive oxygen species; however, TIS-T cells do not modify phagocyte capacity of Mo/Ma. TIS-T modulated-Mo/Ma show a higher production of pro-inflammatory cytokines (TNF, IL-1ß and IL-6) and angiogenic factors (MMP-9, VEGF-A and IL-8) and a lower IL-10 and IP-10 secretion than monocytes co-cultured with controls. The mediator(s) present in the supernatant of TIS-T cell/monocyte-macrophage co-cultures promote(s) tubulogenesis and tumor-cell survival. Monocyte-modulation induced by TIS-T cells requires cell-to-cell contact. Although CD4+ shows different behavior from CD8+TIS-T cells, blocking mAbs against T-cell immunoglobulin and mucin protein 3 and CD40 ligand reduce pro-inflammatory cytokines and angiogenic factors production, indicating that these molecules are involved in monocyte/macrophage modulation by TIS-T cells. Our results revealed a novel role for TIS-T cells in human monocyte/macrophage modulation, which may have deleterious consequences for tumor progression. This modulation should be considered to best tailor the immunotherapy against cancer.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Ligante de CD40/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Citocinas/biossíntese , Proteínas de Membrana/metabolismo , Monócitos/metabolismo , Linfócitos T CD4-Positivos/citologia , Ligante de CD40/genética , Linfócitos T CD8-Positivos/citologia , Comunicação Celular , Sobrevivência Celular , Senescência Celular , Técnicas de Cocultura , Citocinas/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação da Expressão Gênica , Células HeLa , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Receptor de Manose , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Proteínas de Membrana/genética , Monócitos/citologia , Óxido Nítrico/metabolismo , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Trypanosoma cruzi, the causative agent of Chagas' disease, may sabotage humoral response by affecting B cells at the different stages of its development. The present review highlights the contributions of our laboratory in understanding how T. cruzi hinders B-cell generation and B-cell expansion limiting host defence and favouring its chronic establishment. We discuss how homoeostatic mechanisms can be triggered to control exacerbated B-cell proliferation that favour T. cruzi infection by eliminating parasite-specific B cells. Specific targeting of evasion mechanisms displayed in T. cruzi infection, as in vivo Fas/FasL blockade or Gal-3 expression inhibition, allowed us to modulate B-cell responses enhancing the anti-parasite humoral immune response. A comprehensive understanding of the biology of the B cell in health and disease is strictly required to devise immunointervention strategies aimed at enhancing protective immune responses during infections.
Assuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/patologia , Doença de Chagas/imunologia , Doença de Chagas/parasitologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/imunologia , Animais , Subpopulações de Linfócitos B/parasitologia , Diferenciação Celular/imunologia , Doença de Chagas/patologia , HumanosRESUMO
During ageing, autoimmune disorders and the higher susceptibility to infectious have been associated with alterations in the humoral immune response. We report that splenic B lymphocytes from aged mice exhibit lower level of apoptosis induced by B-cell antigen receptor (BCR) ligation in vitro. Respect to B cells from young mice the anti-mu stimulated aged B cells show similar Bcl-2 and Bcl-xL expression but differential kinetic of A1 degradation and a higher level of cFLIP and FAIM. Even though B cells from aged mice show minor Fas expression they exhibit the same susceptibility to anti-Fas induced apoptosis. Aged B cells also present upon BCR stimulation, a higher proliferative response and similar level of activation markers expression than B cells from young mice. These data agree with the observation that aged mice exhibit an increment of T2 and mature B cell subset which rapidly enters cell cycle upon BCR engagement. The diminished apoptosis after activation in aged mice could compromise homeostatic mechanism allowing the persistence of self and non-self antigen specific B cells.
Assuntos
Envelhecimento/imunologia , Antígenos/imunologia , Subpopulações de Linfócitos B/patologia , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais , Regulação para Cima , Animais , Apoptose , Proteínas Reguladoras de Apoptose/análise , Biomarcadores/análise , Western Blotting/métodos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Sobrevivência Celular , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Feminino , Citometria de Fluxo , Proteínas Inibidoras de Apoptose/análise , Peptídeos e Proteínas de Sinalização Intracelular/análise , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor fas/imunologiaRESUMO
In the present work, we demonstrate that interleukin (IL)-4 is able to rescue B cells from Trypanosoma cruzi-infected mice, counteracting the strong apoptotic signals that these cells received in vivo. We have observed that IL-4 restrains the apoptosis of immunoglobulin (Ig)M(+) and IgG(+) B cells from infected and normal mice without inducing them to proliferate. In addition, IL-4 does not modify the quantity or quality of the antibodies secreted by B cells from infected mice, as it blocks their terminal differentiation to plasma cells and favors memory pathway. It is interesting that the protective effect of IL-4 over B cells from infected mice is mediated, at least partly, by the down-regulation of Fas ligand (FasL) expression, which leads to interference in the apoptosis executed by these B cells through the Fas/FasL death pathway. Accordingly, a marked up-regulation of the "FasL gene repressor" class II transactivator was observed, suggesting that this would be one mechanism underlying the IL-4-mediated FasL down-regulation.
Assuntos
Apoptose/efeitos dos fármacos , Linfócitos B/parasitologia , Interleucina-4/farmacologia , Proteínas Nucleares , Trypanosoma cruzi , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Doença de Chagas , Proteína Ligante Fas , Regulação da Expressão Gênica , Genes MHC da Classe II , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/fisiologia , Camundongos , Transativadores/biossíntese , Transativadores/efeitos dos fármacosRESUMO
It has been proposed that Trypanosoma cruzi, the aetiologic agent of Chagas' disease, produces mitogenic substances responsible for the polyclonal B-cell activation observed during the acute phase of the infection. Isolation and characterization of the molecules involved in the induction of polyclonal activation observed during infectious diseases have posed a great challenge for the immunologist over the last decade. In this work we report that a 33 kD protein obtained from an alkaline fraction of T. cruzi epimastigotes (FI) stimulates proliferation and promotes differentiation into antibody-secreting cells of normal murine B cells in a T-cell independent manner. By flow cytometry we also found that the 33 kDa protein induces an increase in the expression of MHC class II and B7.2 but not B7.1 molecules on the B-cell surface. Sequencing by mass spectrometry identified the T. cruzi 33 kD protein as hypothetical oxidoreductase, a member of the aldo/ketoreductase family. In this report we demonstrate that this protein is also present in the infective bloodstream trypomastigote form of the parasite and was identified as T. cruzi mitochondrial malate dehydrogenase (mMDH) by enzyme activity and by Western blotting using a specific mMDH polyclonal antiserum. The biologic relevance of mMDH-induced polyclonal activation concerning T. cruzi infection is discussed.
Assuntos
Antígenos de Protozoários/imunologia , Linfócitos B/imunologia , Doença de Chagas/imunologia , Ativação Linfocitária/imunologia , Malato Desidrogenase/imunologia , Trypanosoma cruzi/imunologia , Animais , Formação de Anticorpos , Linfócitos B/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/enzimologia , Mitocôndrias/imunologia , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/ultraestruturaRESUMO
Acute infection with Trypanosoma cruzi is characterized by multiple manifestations of immunosuppression of both cellular and humoral responses. B cells isolated at the acute stage of infection have shown marked impairment in their response to polyclonal activators in vitro. The present work aims at studying the B cell compartment in the context of acute T. cruzi infection to provide evidence for B cell activation, spontaneous apoptosis and arrest of the cell cycle upon mitogenic stimulation as a mechanism underlying B cell hyporesponse. We found that B cells from acutely infected mice, which fail to respond to the mitogen LPS, showed spontaneous proliferation and production of IgM, indicating a high level of B cell activation. Furthermore, these activated B cells also exhibited an increase in Fas expression and apoptosis in cultures without an exogenous stimulus. On the other hand, B cells from early acute and chronic infected mice did not present activation or apoptosis, and were able to respond properly to the mitogen. Upon in vitro stimulation with LPS, B cells from hyporesponder mice failed to progress through the cell cycle (G0/G1 arrest), nor did they increase the levels of apoptosis. These results indicate that B cell apoptosis and cell cycle arrest could be the mechanisms that control intense B cell expansion, but at the same time could be delaying the emergence of a specific immune response against the parasite.
Assuntos
Apoptose/imunologia , Linfócitos B/imunologia , Doença de Chagas/imunologia , Terapia de Imunossupressão , Lipopolissacarídeos/imunologia , Trypanosoma cruzi , Animais , Linfócitos B/patologia , Divisão Celular/imunologia , Doença de Chagas/patologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Several reports have described polyclonal activation in mice acutely infected with Trypanosoma cruzi. The aim of this work was to analyse the participation of one T. cruzi antigenic fraction in this immunological event. The antigen selected was FI, an antigenic fraction of pI 7-9 obtained from T. cruzi cytosol separated by isoelectricfocusing. FI is constituted by molecules with molecular weights of around 60 and 20 KDa. The authors assayed the ability of this antigenic fraction to induce polyclonal activation of spleen mononuclear cells from normal (NSMC) BALB/c mice. NSMC showed a marked lymphoproliferative response measured by 3H-thymidine incorporation after 3 days of culture in presence of FI. The values reached by FI-stimulated cells were 10 times higher than the controls (non-stimulated cells). This effect was dose-dependent. Furthermore, the authors observed that a purified T-cell population in the presence of adherent cells was unaffected by FI. Additionally, in a culture of NSMC, FI stimulated the proliferation of B cells as observed by the increase of the percentage of B220+ cells determined by FACS using FITC-conjugated anti-mouse B220. The authors noticed that the percentage of B220+Ly1+(CD5) populations in the presence of FI did not change with respect to the control (non-stimulated cells), indicating that FI expanded both conventional and CD5+ B cells. The isotypic pattern of the antibodies produced after 6 days of culture of NSMC in the presence of FI was predominantly IgM, which reacted with highly conserved antigens such as actin, myosin, myoglobin, thyroglobulin and carbonic anhydrase, but did not react with FI. A slight increase of IgG1 and IgG3 with respect to the control was observed but no changes on the levels of IgG2 was noticed. These results indicate that FI promotes activation, proliferation and differentiation in antibody-secreting cells of normal murine B lymphocytes.