RESUMO
Environmental factors including drugs, mineral oils and heavy metals such as lead, gold and mercury are triggers of autoimmune diseases in animal models or even in occupationally exposed humans. After exposure to subtoxic levels of mercury (Hg), genetically susceptible strains of mice develop an autoimmune disease characterized by the production of highly specific anti-nucleolar autoantibodies, hyperglobulinemia and nephritis. However, mice can be tolerized to the disease by a single low dose administration of Hg. Lymphocyte Activation Gene-3 (LAG-3) is a CD4-related, MHC-class II binding molecule expressed on activated T cells and NK cells which maintains lymphocyte homeostatic balance via various inhibitory mechanisms. In our model, administration of anti-LAG-3 monoclonal antibody broke tolerance to Hg resulting in autoantibody production and an increase in serum IgE level. In addition, LAG-3-deficient B6.SJL mice not only had increased susceptibility to Hg-induced autoimmunity but were also unresponsive to tolerance induction. Conversely, adoptive transfer of wild-type CD4(+) T cells was able to partially rescue LAG-3-deficient mice from the autoimmune disease. Further, in LAG-3-deficient mice, mercury elicited higher amounts of IL-6, IL-4 and IFN-γ, cytokines known to play a critical role in mercury-induced autoimmunity. Therefore, we conclude that LAG-3 exerts an important regulatory effect on autoimmunity elicited by a common environmental pollutant.
Assuntos
Antígenos CD/imunologia , Autoimunidade/imunologia , Poluentes Ambientais/imunologia , Ativação Linfocitária/imunologia , Animais , Anticorpos Antinucleares/imunologia , Autoanticorpos/imunologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Tolerância Imunológica/imunologia , Imunoglobulina E/imunologia , Interferon gama/imunologia , Interleucina-4/imunologia , Interleucina-6/imunologia , Células Matadoras Naturais/imunologia , Mercúrio/efeitos adversos , Mercúrio/imunologia , Camundongos , Exposição Ocupacional , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
Mer tyrosine kinase (MerTK) is a major macrophage apoptotic cell (AC) receptor. Its functional impairment promotes autoimmunity and atherosclerosis, whereas overexpression correlates with poor prognosis in cancer. However, little is known about mechanisms regulating MerTK expression in humans. We found that MerTK expression is heterogenous among macrophage subsets, being mostly restricted to anti-inflammatory M2c (CD14(+)CD16(+)CD163(+)CD204(+)CD206(+)CD209(-)) cells, differentiated by M-CSF or glucocorticoids. Small numbers of MerTK(+) "M2c-like" cells are also detectable among circulating CD14(bright)CD16(+) monocytes. MerTK expression levels adapt to changing immunologic environment, being suppressed in M1 and M2a macrophages and in dendritic cells. Remarkably, although glucocorticoid-induced differentiation is IL-10 independent, M-CSF-driven M2c polarization and related MerTK upregulation require IL-10. However, neither IL-10 alone nor TGF-ß are sufficient to fully differentiate M2c (CD16(+)CD163(+)MerTK(+)) macrophages. M-CSF and IL-10, both released by T lymphocytes, may thus be required together to promote regulatory T cell-mediated induction of anti-inflammatory monocytes-macrophages. MerTK enables M2c macrophages to clear early ACs more efficiently than other macrophage subsets, and it mediates AC clearance by CD14(bright)CD16(+) monocytes. Moreover, M2c cells release Gas6, which in turn amplifies IL-10 secretion via MerTK. IL-10-dependent induction of the Gas6/MerTK pathway may, therefore, constitute a positive loop for M2c macrophage homeostasis and a critical checkpoint for maintenance of anti-inflammatory conditions. Our findings give new insight into human macrophage polarization and favor a central role for MerTK in regulation of macrophage functions. Eliciting M2c polarization can have therapeutic utility for diseases such as lupus, in which a defective AC clearance contributes to initiate and perpetuate the pathological process.
Assuntos
Apoptose/imunologia , Movimento Celular/imunologia , Polaridade Celular/imunologia , Macrófagos/enzimologia , Macrófagos/imunologia , Proteínas Proto-Oncogênicas/biossíntese , Receptores Proteína Tirosina Quinases/biossíntese , Apoptose/genética , Comunicação Autócrina/genética , Comunicação Autócrina/imunologia , Diferenciação Celular/imunologia , Movimento Celular/genética , Polaridade Celular/genética , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Humanos , Imunofenotipagem , Macrófagos/citologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/fisiologia , Ativação Transcricional/imunologia , c-Mer Tirosina QuinaseRESUMO
The mechanisms leading to autoimmune diseases remain largely unknown despite numerous lines of experimental inquiry and epidemiological evidence. The growing number of genome-wide association studies and the largely incomplete concordance for autoimmune diseases in monozygotic twins support the role of the environment (including infectious agents and chemicals) in the breakdown of tolerance leading to autoimmunity via numerous mechanisms. The present article reviews the major theories on the mechanisms of the environmental influence on autoimmunity by addressing the different degrees of confidence that characterize our knowledge. The theories discussed herein include (i) the role of innate immunity mediated by toll-like receptors in triggering the autoimmune adaptive response characterizing the observed pathology; (ii) changes in spleen marginal zone B cells in autoantibody production with particular focus on the B10 subpopulation; (iii) Th17 cell differentiation and T regulatory cells in the aryl hydrocarbon receptor model; (iv) self antigen changes induced by chemical and infectious agents which could break tolerance by post-translational modifications and molecular mimicry; and finally (v) epigenetic changes, particularly DNA methylation, that are induced by environmental stimuli and may contribute to autoimmunity initiation. We are convinced that these working hypotheses, in most cases supported by solid evidence, should be viewed in parallel with animal models and epidemiological observations to provide a comprehensive picture of the environmental causes of autoimmune diseases.
Assuntos
Autoanticorpos/imunologia , Doenças Autoimunes/etiologia , Autoimunidade , Produtos Biológicos/toxicidade , Poluentes Ambientais/toxicidade , Imunidade Adaptativa/efeitos dos fármacos , Autoanticorpos/genética , Autoantígenos/genética , Autoantígenos/imunologia , Doenças Autoimunes/genética , Autoimunidade/efeitos dos fármacos , Congressos como Assunto , Epigênese Genética/imunologia , Interação Gene-Ambiente , Humanos , Imunidade Inata/efeitos dos fármacos , Modelos Imunológicos , Baço/efeitos dos fármacos , Baço/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologiaRESUMO
In AKI, dying renal cells release intracellular molecules that stimulate immune cells to secrete proinflammatory cytokines, which trigger leukocyte recruitment and renal inflammation. Whether the release of histones, specifically, from dying cells contributes to the inflammation of AKI is unknown. In this study, we found that dying tubular epithelial cells released histones into the extracellular space, which directly interacted with Toll-like receptor (TLR)-2 (TLR2) and TLR4 to induce MyD88, NF-κB, and mitogen activated protein kinase signaling. Extracellular histones also had directly toxic effects on renal endothelial cells and tubular epithelial cells in vitro. In addition, direct injection of histones into the renal arteries of mice demonstrated that histones induce leukocyte recruitment, microvascular vascular leakage, renal inflammation, and structural features of AKI in a TLR2/TLR4-dependent manner. Antihistone IgG, which neutralizes the immunostimulatory effects of histones, suppressed intrarenal inflammation, neutrophil infiltration, and tubular cell necrosis and improved excretory renal function. In summary, the release of histones from dying cells aggravates AKI via both its direct toxicity to renal cells and its proinflammatory effects. Because the induction of proinflammatory cytokines in dendritic cells requires TLR2 and TLR4, these results support the concept that renal damage triggers an innate immune response, which contributes to the pathogenesis of AKI.
Assuntos
Injúria Renal Aguda/metabolismo , Histonas/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Injúria Renal Aguda/imunologia , Animais , Permeabilidade Capilar , Citocinas/metabolismo , Células Endoteliais/fisiologia , Células Epiteliais/metabolismo , Injeções Intra-Arteriais , Rim/patologia , Túbulos Renais/metabolismo , Leucócitos/fisiologia , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Necrose , Artéria Renal , Traumatismo por Reperfusão/prevenção & controleRESUMO
There is emerging evidence that platelets are major contributors to inflammatory processes through intimate associations with innate immune cells. Here, we report that activated platelets induce the formation of neutrophil extracellular traps (NETs) in transfusion-related acute lung injury (TRALI), which is the leading cause of death after transfusion therapy. NETs are composed of decondensed chromatin decorated with granular proteins that function to trap extracellular pathogens; their formation requires the activation of neutrophils and release of their DNA in a process that may or may not result in neutrophil death. In a mouse model of TRALI that is neutrophil and platelet dependent, NETs appeared in the lung microvasculature and NET components increased in the plasma. We detected NETs in the lungs and plasma of human TRALI and in the plasma of patients with acute lung injury. In the experimental TRALI model, targeting platelet activation with either aspirin or a glycoprotein IIb/IIIa inhibitor decreased NET formation and lung injury. We then directly targeted NET components with a histone blocking antibody and DNase1, both of which protected mice from TRALI. These data suggest that NETs contribute to lung endothelial injury and that targeting NET formation may be a promising new direction for the treatment of acute lung injury.
Assuntos
Lesão Pulmonar Aguda/etiologia , Plaquetas/fisiologia , Neutrófilos/fisiologia , Reação Transfusional , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/patologia , Animais , Anticorpos Monoclonais/uso terapêutico , Aspirina/uso terapêutico , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Agregação Celular , Células Cultivadas , Desoxirribonuclease I/uso terapêutico , Histonas/imunologia , Histonas/metabolismo , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Pulmão/imunologia , Pulmão/patologia , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Permeabilidade , Peroxidase/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/uso terapêutico , Tromboxanos/metabolismoRESUMO
Cerebrovascular inflammation contributes to diverse CNS disorders through mechanisms that are incompletely understood. The recruitment of neutrophils to the brain can contribute to neurotoxicity, particularly during acute brain injuries, such as cerebral ischemia, trauma, and seizures. However, the regulatory and effector mechanisms that underlie neutrophil-mediated neurotoxicity are poorly understood. In this study, we show that mouse neutrophils are not inherently toxic to neurons but that transendothelial migration across IL-1-stimulated brain endothelium triggers neutrophils to acquire a neurotoxic phenotype that causes the rapid death of cultured neurons. Neurotoxicity was induced by the addition of transmigrated neutrophils or conditioned medium, taken from transmigrated neutrophils, to neurons and was partially mediated by excitotoxic mechanisms and soluble proteins. Transmigrated neutrophils also released decondensed DNA associated with proteases, which are known as neutrophil extracellular traps. The blockade of histone-DNA complexes attenuated transmigrated neutrophil-induced neuronal death, whereas the inhibition of key neutrophil proteases in the presence of transmigrated neutrophils rescued neuronal viability. We also show that neutrophil recruitment in the brain is IL-1 dependent, and release of proteases and decondensed DNA from recruited neutrophils in the brain occurs in several in vivo experimental models of neuroinflammation. These data reveal new regulatory and effector mechanisms of neutrophil-mediated neurotoxicity (i.e., the release of proteases and decondensed DNA triggered by phenotypic transformation during cerebrovascular transmigration). Such mechanisms have important implications for neuroinflammatory disorders, notably in the development of antileukocyte therapies.
Assuntos
Circulação Cerebrovascular/imunologia , DNA Mitocondrial/antagonistas & inibidores , Neurônios/enzimologia , Neurônios/patologia , Infiltração de Neutrófilos/imunologia , Peptídeo Hidrolases/metabolismo , Animais , Células Cultivadas , Circulação Cerebrovascular/genética , Meios de Cultivo Condicionados/farmacologia , DNA Mitocondrial/imunologia , DNA Mitocondrial/metabolismo , Endotélio Vascular/enzimologia , Endotélio Vascular/imunologia , Endotélio Vascular/patologia , Espaço Extracelular/enzimologia , Espaço Extracelular/genética , Espaço Extracelular/imunologia , Imunofenotipagem , Interleucina-1alfa/deficiência , Interleucina-1alfa/fisiologia , Interleucina-1beta/deficiência , Interleucina-1beta/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/imunologia , Infiltração de Neutrófilos/genética , Peptídeo Hidrolases/genética , Cultura Primária de Células , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: Recently, a growing number of studies have revealed a prothrombotic and cytotoxic role for extracellular chromatin. Cerebral ischemia/reperfusion injury is characterized by a significant amount of cell death and neutrophil activation, both of which may result in the release of chromatin. The goal of this study was to assess the effect of extracellular chromatin in ischemic stroke using a mouse model of transient middle cerebral artery occlusion. METHODS AND RESULTS: Similar to reports in stroke patients, we observed increased levels of circulating nucleosomes and DNA after ischemic stroke in mice. In addition, we observed that general hypoxia also augmented extracellular chromatin. We hypothesized that targeting extracellular chromatin components would be protective in ischemic stroke. Indeed, treatment with recombinant human DNase 1 significantly improved stroke outcome. Neutralization of histones using an antihistone antibody was also protective as evidenced by smaller infarct volumes, whereas increasing levels of extracellular histones via histone infusion exacerbated stroke outcome by increasing infarct size and worsening functional outcome. CONCLUSIONS: Our results indicate that extracellular chromatin is generated and is detrimental during cerebral ischemia/reperfusion in mice. Targeting DNA and histones may be a new therapeutic strategy to limit injury resulting from ischemic stroke.
Assuntos
Isquemia Encefálica/etiologia , Cromatina/fisiologia , Acidente Vascular Cerebral/etiologia , Animais , Isquemia Encefálica/tratamento farmacológico , Desoxirribonuclease I/uso terapêutico , Histonas/antagonistas & inibidores , Hipóxia/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nucleossomos/fisiologia , Acidente Vascular Cerebral/tratamento farmacológicoRESUMO
PURPOSE: Mechanisms underlying transition of a thrombus susceptible to tissue plasminogen activator (TPA) fibrinolysis to one that is resistant is unclear. Demonstration of a new possible thrombus scaffold may open new avenues of research in thrombolysis and may provide mechanistic insight into thrombus remodeling. MATERIALS AND METHODS: Ten human thrombus samples were collected during cases of thrombectomy and open surgical repair of abdominal aortic aneurysms (five samples < 3 d old and five samples > 1 y old). Additionally, an acute murine hindlimb ischemia model was created to evaluate thrombus samples in mice. Human sections were immunostained for the H2A/H2B/DNA complex, myeloperoxidase, fibrinogen, and von Willebrand factor. Mouse sections were immunostained with the H2A antibody. All samples were further evaluated after hematoxylin and eosin and Masson trichrome staining. RESULTS: An extensive network of extracellular histone/DNA complex was demonstrated in the matrix of human ex vivo thrombus. This network is present throughout the highly cellular acute thrombus. However, in chronic thrombi, detection of the histone/DNA network was predominantly in regions of low collagen content and high cell density, which were mostly near the lumen. These regions of high cell density contained neutrophils and monocytes. Similarly, sections from the acute murine hindlimb ischemia model also exhibited extensive immunoreactivity to the histone antibody in the extracellular space within murine thrombi. CONCLUSIONS: Extensive detection of genomic DNA associated with histones in the extracellular matrix of human and mouse thrombi suggest the presence of a new thrombus-associated scaffold.
Assuntos
DNA/metabolismo , Desoxirribonucleases/uso terapêutico , Fibrinolíticos/uso terapêutico , Histonas/metabolismo , Músculo Esquelético/irrigação sanguínea , Terapia Trombolítica/métodos , Trombose/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Angiografia Digital , Animais , Aneurisma da Aorta Abdominal/complicações , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/metabolismo , Feminino , Membro Posterior , Humanos , Imuno-Histoquímica , Isquemia/complicações , Fluxometria por Laser-Doppler , Masculino , Camundongos , Pessoa de Meia-Idade , Monócitos/metabolismo , Neutrófilos/metabolismo , Trombose/diagnóstico por imagem , Trombose/etiologia , Trombose/genética , Trombose/metabolismo , Fatores de Tempo , Tomografia Computadorizada por Raios XRESUMO
Plasmodium falciparum is a protozoan parasite of human erythrocytes that causes the most severe form of malaria. Severe P. falciparum infection is associated with endothelial activation and permeability, which are important determinants of the outcome of the infection. How endothelial cells become activated is not fully understood, particularly with regard to the effects of parasite subcomponents. We demonstrated that P. falciparum histones extracted from merozoites (HeH) directly stimulated the production of IL-8 and other inflammatory mediators by primary human dermal microvascular endothelial cells through a signaling pathway that involves Src family kinases and p38 MAPK. The stimulatory effect of HeH and recombinant P. falciparum H3 (PfH3) was abrogated by histone-specific antibodies. The release of nuclear contents on rupture of infected erythrocytes was captured by live cell imaging and confirmed by detecting nucleosomes in the supernatants of parasite cultures. HeH and recombinant parasite histones also induced endothelial permeability through a charge-dependent mechanism that resulted in disruption of junctional protein expression and cell death. Recombinant human activated protein C cleaved HeH and PfH3 and abrogated their proinflammatory effects. Circulating nucleosomes of both human and parasite origin were detected in the plasma of patients with falciparum malaria and correlated positively with disease severity. These results support a pathogenic role for both host- and pathogen-derived histones in P. falciparum-caused malaria.
Assuntos
Endotélio Vascular/metabolismo , Histonas/farmacologia , Interleucina-8/biossíntese , Merozoítos , Plasmodium falciparum , Animais , Permeabilidade Capilar/fisiologia , Células Cultivadas , Endotélio Vascular/parasitologia , Humanos , Estágios do Ciclo de Vida , Pulmão/irrigação sanguínea , Pulmão/parasitologia , Sistema de Sinalização das MAP Quinases/fisiologia , Malária Falciparum/parasitologia , Microvasos , Proteína C/farmacologia , Proteínas Recombinantes , Pele/irrigação sanguínea , Pele/parasitologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Quinases da Família src/fisiologiaRESUMO
OBJECTIVE: Caspase-activated DNase (CAD) is an endonuclease that is activated by active caspase 3 during apoptosis and is responsible for degradation of chromatin into nucleosomal units. These nucleosomal units are then included in apoptotic bodies. The presence of apoptotic bodies is considered important for the generation of autoantigen in autoimmune diseases, such as systemic lupus erythematosus (SLE), that are characterized by the presence of antinuclear antibodies. The present study was carried out to determine the role of CAD in SLE and to investigate the ability of lupus autoantibodies to bind to CAD-deficient or CAD-sufficient apoptotic cells. METHODS: The Sle1, Sle123, and 3H9 mouse models of SLE, in which autoimmunity is genetically predetermined, were used. To determine the role of chromatin fragmentation in SLE, CAD deficiency was introduced in these mouse models. RESULTS: Deficiency of CAD resulted in increased anti-double-stranded DNA antibody titers in lupus-prone mice. Surprisingly, the absence of CAD exacerbated only genetically predetermined autoimmune responses. To further determine whether nuclear modifications are needed in order to maintain tolerance to nuclear autoantigens, we used the 3H9 mouse, an anti-DNA heavy chain knockin; in this model, the autoreactive B cells are tolerized by anergy. In accordance with findings in the CAD-mutant Sle1 and Sle123 mice, CAD-deficient 3H9 mice spontaneously generated anti-DNA antibodies. Finally, we showed that autoantibodies with specificities toward histone-DNA complexes bind more to CAD-deficient apoptotic cells than to CAD-sufficient apoptotic cells. CONCLUSION: We propose that in mice that are genetically predisposed to lupus development, nuclear apoptotic modifications are needed to maintain tolerance. In the absence of these modifications, apoptotic chromatin is abnormally exposed, facilitating the autoimmune response.
Assuntos
Anticorpos Antinucleares/imunologia , Apoptose/imunologia , Autoantígenos/imunologia , Desoxirribonucleases/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Animais , Núcleo Celular/imunologia , Modelos Animais de Doenças , Tolerância Imunológica/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , CamundongosRESUMO
We previously reported that extracellular histones are major mediators of death in sepsis. Infusion of extracellular histones leads to increased cytokine levels. Histones activate TLR2 and TLR4 in a process that is enhanced by binding to DNA. Activation of TLR4 is responsible for the histone-dependent increase in cytokine levels. To study the impact of histone release on pathology we used two models: a Con A-triggered activation of T cells to mimic sterile inflammation, and acetaminophen to model drug-induced tissue toxicity. Histones were released in both models and anti-histone Abs were protective. TLR2- or TLR4-null mice were also protected. These studies imply that histone release contributes to death in inflammatory injury and in chemical-induced cellular injury, both of which are mediated in part through the TLRs.
Assuntos
Histonas/metabolismo , Fígado/lesões , Fígado/metabolismo , Síndrome de Resposta Inflamatória Sistêmica/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Western Blotting , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Interphase nuclear architecture is disrupted and rapidly reformed with each cell division cycle. Successive cell generations exhibit a "memory" of this nuclear architecture, as well as for gene expression. Furthermore, many features of nuclear and mitotic chromosome structure are recognizably species and tissue specific. We wish to know what properties of the underlying chromatin structure may determine these conserved features of nuclear architecture. Employing a particular mouse autoimmune anti-nucleosome monoclonal antibody (PL2-6), combined with deconvolution immunofluorescence microscopy, we present evidence for a unique epitope (involving a ternary complex of histones H2A and H2B and DNA) which is localized only at the exterior chromatin surface of interphase nuclei and mitotic chromosomes in mammalian, invertebrate and plant systems. As only the surface chromatin region is identified with antibody PL2-6, we have assigned it the name "epichromatin". We describe an "epichromatin hypothesis", suggesting that epichromatin may have a unique evolutionary conserved conformation which facilitates interaction with the reforming post-mitotic nuclear envelope and a rapid return of interphase nuclear architecture.
Assuntos
Cromatina/química , Evolução Molecular , Animais , Anticorpos Monoclonais/imunologia , Arabidopsis , Autoanticorpos/imunologia , Caenorhabditis elegans , Linhagem Celular Tumoral , Cromatina/metabolismo , Drosophila , Histonas/química , Histonas/metabolismo , Humanos , Interfase , Camundongos , Microscopia de Fluorescência , Nucleossomos/imunologiaRESUMO
Systemic lupus erythematosus is a prototypic autoimmune disease characterized by antibodies to DNA and other nuclear molecules. While these antibodies can form immune complexes, the mechanisms generating the bound nuclear antigens are not known. These studies investigated whether microparticles can form complexes with anti-DNA and other anti-nucleosomal antibodies. Microparticles are small membrane-bound vesicles released from dead and dying cells; these particles contain a variety of cellular components, including DNA. To assess antigenicity, microparticles generated in vitro from apoptotic cell lines were tested using murine monoclonal anti-DNA and anti-nucleosomal antibodies as well as plasma from lupus patients. Antibody binding was assessed by flow cytometry. As these studies showed, some but not all of the monoclonal antibodies bound to microparticles prepared from apoptotic HL-60, THP-1 and Jurkat cells. For HL-60 cells, both staurosporine and UV radiation led to the production of antigenically active particles. For the anti-DNA antibody with high particle binding, prior treatment of DNase reduced activity. With plasma from patients with SLE, antibody binding to microparticles was present although a clear relationship with anti-DNA antibody levels was not observed. To determine whether lupus plasma contains immune complexes with particle properties, particle preparations were tested for bound IgG by flow cytometry. These studies indicated that lupus plasma contains particles with IgG binding, with numbers correlated with anti-DNA levels. Together, these findings indicate that microparticles display DNA and nucleosomal molecules in an antigenic form and could represent a source of immune complexes in SLE.
Assuntos
Anticorpos Antinucleares/imunologia , Micropartículas Derivadas de Células/imunologia , DNA/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Nucleossomos/imunologia , Apoptose , Linhagem Celular , Humanos , Imunoglobulina G/imunologiaRESUMO
Receptor editing is the process of ongoing antibody gene rearrangement in a lymphocyte that already has a functional antigen receptor. The expression of a functional antigen receptor will normally terminate further rearrangement (allelic exclusion). However, lymphocytes with autoreactive receptors have a chance at escaping negative regulation by "editing" the specificities of their receptors with additional antibody gene rearrangements. As such, editing complicates the Clonal Selection Hypothesis because edited cells are not simply endowed for life with a single, invariant antigen receptor. Furthermore, if the initial immunoglobulin gene is not inactivated during the editing process, allelic exclusion is violated and the B cell can exhibit two specificities. Here, we describe the discovery of editing, the pathways of receptor editing at the heavy (H) and light (L) chain loci, and current evidence regarding how and where editing happens and what effects it has on the antibody repertoire.
Assuntos
Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Rearranjo Gênico do Linfócito B/fisiologia , Tolerância Imunológica , Receptores de Antígenos de Linfócitos B/fisiologia , Animais , Doenças Autoimunes/patologia , Humanos , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Statins are inhibitors of 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis. Recent clinico-epidemiologic studies correlate patients receiving statin therapy with having reduced mortality associated with severe bacterial infection. Investigating the effect of statins on the innate immune capacity of phagocytic cells against the human pathogen Staphylococcus aureus, we uncovered a beneficial effect of statins on bacterial clearance by phagocytes, although, paradoxically, both phagocytosis and oxidative burst were inhibited. Probing instead for an extracellular mechanism of killing, we found that statins boosted the production of antibacterial DNA-based extracellular traps (ETs) by human and murine neutrophils and also monocytes/macrophages. The effect of statins to induce phagocyte ETs was linked to sterol pathway inhibition. We conclude that a drug therapy taken chronically by millions alters the functional behavior of phagocytic cells, which could have ramifications for susceptibility and response to bacterial infections in these patients.
Assuntos
Espaço Extracelular/microbiologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Macrófagos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Pneumonia Estafilocócica/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Acil Coenzima A/antagonistas & inibidores , Animais , Células Cultivadas , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/imunologia , Espaço Extracelular/imunologia , Espaço Extracelular/metabolismo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CFTR , Neutrófilos/imunologia , Neutrófilos/microbiologia , Fagócitos/efeitos dos fármacos , Fagócitos/imunologia , Fagócitos/microbiologia , Pneumonia Estafilocócica/imunologia , Pneumonia Estafilocócica/microbiologiaRESUMO
Neutrophils are key effectors of the host innate immune response against bacterial infection. Staphylococcus aureus is a preeminent human pathogen, with an ability to produce systemic infections even in previously healthy individuals, thereby reflecting a resistance to effective neutrophil clearance. The recent discovery of neutrophil extracellular traps (NETs) has opened a novel dimension in our understanding of how these specialized leukocytes kill pathogens. NETs consist of a nuclear DNA backbone associated with antimicrobial peptides, histones and proteases that provide a matrix to entrap and kill various microbes. Here, we used targeted mutagenesis to examine a potential role of S. aureus nuclease in NET degradation and virulence in a murine respiratory tract infection model. In vitro assays using fluorescence microscopy showed the isogenic nuclease-deficient (nuc-deficient) mutant to be significantly impaired in its ability to degrade NETs compared with the wild-type parent strain USA 300 LAC. Consequently, the nuc-deficient mutant strain was significantly more susceptible to extracellular killing by activated neutrophils. Moreover, S. aureus nuclease production was associated with delayed bacterial clearance in the lung and increased mortality after intranasal infection. In conclusion, this study shows that S. aureus nuclease promotes resistance against NET-mediated antimicrobial activity of neutrophils and contributes to disease pathogenesis in vivo.
Assuntos
Staphylococcus aureus Resistente à Meticilina/fisiologia , Nuclease do Micrococo/metabolismo , Neutrófilos/metabolismo , Infecções Respiratórias/imunologia , Infecções Estafilocócicas/imunologia , Animais , Carga Bacteriana/genética , Bacteriólise/genética , Bacteriólise/imunologia , Micropartículas Derivadas de Células/imunologia , Micropartículas Derivadas de Células/microbiologia , Células Cultivadas , Espaço Extracelular/imunologia , Espaço Extracelular/microbiologia , Regulação Bacteriana da Expressão Gênica , Humanos , Evasão da Resposta Imune/genética , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , Camundongos Endogâmicos , Nuclease do Micrococo/genética , Mutagênese Sítio-Dirigida , Neutrófilos/imunologia , Neutrófilos/microbiologia , Neutrófilos/patologia , Infecções Respiratórias/microbiologia , Infecções Respiratórias/fisiopatologia , Deleção de Sequência/genética , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/fisiopatologiaRESUMO
Neutrophil extracellular traps (NETs) are part of the innate immune response to infections. NETs are a meshwork of DNA fibers comprising histones and antimicrobial proteins. Microbes are immobilized in NETs and encounter a locally high and lethal concentration of effector proteins. Recent studies show that NETs are formed inside the vasculature in infections and noninfectious diseases. Here we report that NETs provide a heretofore unrecognized scaffold and stimulus for thrombus formation. NETs perfused with blood caused platelet adhesion, activation, and aggregation. DNase or the anticoagulant heparin dismantled the NET scaffold and prevented thrombus formation. Stimulation of platelets with purified histones was sufficient for aggregation. NETs recruited red blood cells, promoted fibrin deposition, and induced a red thrombus, such as that found in veins. Markers of extracellular DNA traps were detected in a thrombus and plasma of baboons subjected to deep vein thrombosis, an example of inflammation-enhanced thrombosis. Our observations indicate that NETs are a previously unrecognized link between inflammation and thrombosis and may further explain the epidemiological association of infection with thrombosis.
Assuntos
DNA/isolamento & purificação , Trombose/etiologia , Animais , HumanosRESUMO
Systemic lupus erythematosus is a chronic autoimmune disorder that predominantly affects women of childbearing age. Lupus-associated glomerulonephritis is a major cause of mortality in these patients. Current treatment protocols for systemic lupus erythematosus include cyclophosphamide, prednisolone, azathioprine, and mycophenolate mofetil. However, in mice none of these agents alone or in combination were shown to reverse established proteinuria. Using New Zealand Black x New Zealand White F1 mice, we report that administration of the topoisomerase I inhibitor irinotecan from week 13 completely prevented the onset of proteinuria and prolonged survival up to at least 90 wk without detectable side effects. Furthermore, application of irinotecan to mice with established lupus nephritis, as indicated by grade 3+ (> or =300 mg/dl) and grade 4+ (> or =2000 mg/dl) proteinuria and, according to a median age of 35 wk, resulted in remission rates of 75% and 55%, respectively. Survival was significantly prolonged with 73 wk (grade 3+ and 4+ combined) versus 40 wk for control animals. Although total IgG and anti-dsDNA Abs in the serum and mesangial IgG deposits in the kidneys were not reduced in irinotecan-treated mice, subendothelial immune deposits were considerably diminished, suggesting a prevention of glomerular basement membrane disruption. This effect was accompanied by increased rates of ssDNA breaks and inhibition of renal cell apoptosis being different to what is known about irinotecan in anticancer therapy. In conclusion, our data provide evidence that irinotecan might represent an entirely new strategy for the treatment of systemic lupus erythematosus.
Assuntos
Camptotecina/análogos & derivados , Cruzamentos Genéticos , Nefrite Lúpica/mortalidade , Nefrite Lúpica/prevenção & controle , Inibidores da Topoisomerase I , Animais , Doenças Autoimunes/enzimologia , Doenças Autoimunes/mortalidade , Doenças Autoimunes/prevenção & controle , Camptotecina/administração & dosagem , DNA Topoisomerases Tipo I/fisiologia , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/uso terapêutico , Feminino , Irinotecano , Nefrite Lúpica/enzimologia , Nefrite Lúpica/imunologia , Camundongos , Camundongos Endogâmicos NZB , Proteinúria/enzimologia , Proteinúria/imunologia , Proteinúria/mortalidade , Proteinúria/prevenção & controle , Distribuição Aleatória , Análise de Sobrevida , Fatores de TempoRESUMO
Hyperinflammatory responses can lead to a variety of diseases, including sepsis. We now report that extracellular histones released in response to inflammatory challenge contribute to endothelial dysfunction, organ failure and death during sepsis. They can be targeted pharmacologically by antibody to histone or by activated protein C (APC). Antibody to histone reduced the mortality of mice in lipopolysaccharide (LPS), tumor necrosis factor (TNF) or cecal ligation and puncture models of sepsis. Extracellular histones are cytotoxic toward endothelium in vitro and are lethal in mice. In vivo, histone administration resulted in neutrophil margination, vacuolated endothelium, intra-alveolar hemorrhage and macro- and microvascular thrombosis. We detected histone in the circulation of baboons challenged with Escherichia coli, and the increase in histone levels was accompanied by the onset of renal dysfunction. APC cleaves histones and reduces their cytotoxicity. Co-infusion of APC with E. coli in baboons or histones in mice prevented lethality. Blockade of protein C activation exacerbated sublethal LPS challenge into lethality, which was reversed by treatment with antibody to histone. We conclude that extracellular histones are potential molecular targets for therapeutics for sepsis and other inflammatory diseases.
Assuntos
Líquido Extracelular/metabolismo , Histonas/metabolismo , Sepse/mortalidade , Sepse/patologia , Animais , Anticorpos/farmacologia , Anticorpos/uso terapêutico , Bovinos , Linhagem Celular Transformada , Modelos Animais de Doenças , Endotélio/efeitos dos fármacos , Endotélio/patologia , Endotélio/ultraestrutura , Escherichia coli/fisiologia , Líquido Extracelular/efeitos dos fármacos , Citometria de Fluxo , Hemorragia/etiologia , Hemorragia/patologia , Histonas/efeitos dos fármacos , Histonas/imunologia , Histonas/farmacologia , Nefropatias/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão/métodos , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêutico , Papio , Polissacarídeos/efeitos adversos , Sepse/tratamento farmacológico , Sepse/etiologia , Fator de Necrose Tumoral alfa/efeitos adversosRESUMO
OBJECTIVE: Nucleosomes have been identified as a key autoantigen in systemic lupus erythematosus (SLE). Nucleosomes are present in the circulation due to a disturbed apoptosis and/or an insufficient clearance in SLE. During apoptosis, histones can be modified, thereby making them more immunogenic. Recently, we showed the importance of apoptosis-induced acetylation of histone H4 in the pathogenesis of SLE. The lupus-derived antibody LG11-2 was previously shown to react with the N-terminal tail of histone H2B, which contains amino acid residues that can be modified including phosphorylation of serine 14, known to occur during apoptosis. Here, we evaluate whether apoptosis-induced histone modifications on H2B exist that are targeted by LG11-2 or lupus-derived plasmas. METHODS: Immunofluorescence staining and western immunoblot analysis of control, apoptotic and trichostatin A-treated cells/chromatin were performed with monoclonal antibody LG11-2. Reactivity of LG11-2 and plasmas from lupus mice and SLE patients with acetylated and/or phosphorylated H2B peptides was determined in competition ELISA. RESULTS: LG11-2 showed enhanced reactivity with apoptotic and hyperacetylated H2B compared to normal H2B. This enhanced reactivity was due to the acetylation of lysine 12 in H2B. This modification was also recognized by autoantibodies from pre-diseased lupus mice, but to a lesser extent by plasmas of diseased lupus mice and lupus patients. CONCLUSIONS: The apoptosis-induced acetylation on H2BK12 is a target for autoantibodies in SLE. Since the anti-H2BK12ac reactivity was mainly found in pre-diseased lupus mice, this epitope seems important in the early phase of the anti-chromatin autoimmune response with subsequent epitope spreading to unmodified H2B.