RESUMO
BACKGROUND: Studies of neuromyelitis optica (NMO), an autoimmune disease of the central nervous system (CNS), have demonstrated that autoantibodies against the water channel aquaporin-4 (AQP4) induce astrocyte damage through complement-dependent cytotoxicity (CDC). In developing experimental models of NMO using cells, tissues or animals from mice, co-administration of AQP4-IgG and normal human serum, which serves as the source of human complement (HC), is required. The sensitivity of mouse CNS cells to HC and CDC in these models is not known. METHODS: We used HC and recombinant monoclonal antibodies (rAbs) against AQP4 to investigate CDC on mouse neurons, astrocytes, differentiated oligodendrocytes (OLs), and oligodendrocyte progenitors (OPCs) in the context of purified monocultures, neuroglial mixed cultures, and organotypic cerebellar slices. RESULTS: We found that murine neurons, OLs, and OPCs were sensitive to HC in monocultures. In mixed murine neuroglial cultures, HC-mediated toxicity to neurons and OLs was reduced; however, astrocyte damage induced by an AQP-specific rAb #53 and HC increased neuronal and oligodendroglial loss. OPCs were resistant to HC toxicity in neuroglial mixed cultures. In mouse cerebellar slices, damage to neurons and OLs following rAb #53-mediated CDC was further reduced, but in contrast to neuroglial mixed cultures, astrocyte damage sensitized OPCs to complement damage. Finally, we established that some injury to neurons, OLs, and OPCs in cell and slice cultures resulted from the activation of HC by anti-tissue antibodies to mouse cells. CONCLUSIONS: Murine neurons and oligodendroglia demonstrate variable sensitivity to activated complement based on their differentiation and culture conditions. In organotypic cultures, the protection of neurons, OLs, and OPCs against CDC is eliminated by targeted astrocyte destruction. The activation of human complement proteins on mouse CNS cells necessitates caution when interpreting the results of mouse experimental models of NMO using HC.
Assuntos
Ativação do Complemento/fisiologia , Proteínas do Sistema Complemento/toxicidade , Modelos Animais de Doenças , Neuromielite Óptica/induzido quimicamente , Neuromielite Óptica/metabolismo , Animais , Aquaporina 4/toxicidade , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neuromielite Óptica/imunologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-DawleyRESUMO
Endothelial cell activation and injury by the terminal pathway of complement is important in various pathobiological processes, including xenograft rejection. Protection against injury by human complement can be induced in porcine endothelial cells (ECs) with IL-4 and IL-13 through metabolic activation. However, despite this resistance, the complement-treated ECs were found to lose membrane permeability control assessed with the small molecule calcein. Therefore, to define the apparent discrepancy of permeability changes vis-à-vis the protection from killing, we now investigated whether IL-4 and IL-13 influence the release of the large cytoplasmic protein lactate dehydrogenase (LDH) in ECs incubated with complement or the pore-forming protein melittin. Primary cultures of ECs were pre-treated with IL-4 or IL-13 and then incubated with human serum as source of antibody and complement or melittin. Cell death was assessed using neutral red. Membrane permeability was quantitated measuring LDH release. We found that IL-4-/IL-13-induced protection of ECs from killing by complement or melittin despite loss of LDH in amounts similar to control ECs. However, the cytokine-treated ECs that were protected from killing rapidly regained effective control of membrane permeability. Moreover, the viability of the protected ECs was maintained for at least 2 days. We conclude that the protection induced by IL-4/IL-13 in ECs against lethal attack by complement or melittin is effective and durable despite severe initial impairment of membrane permeability. The metabolic changes responsible for protection allow the cells to repair the membrane injury caused by complement or melittin.
Assuntos
Proteínas do Sistema Complemento/toxicidade , Células Endoteliais/imunologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Interleucina-13/administração & dosagem , Interleucina-4/administração & dosagem , Meliteno/toxicidade , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/imunologia , Citoplasma/metabolismo , Citotoxicidade Imunológica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Suínos , Transplante Heterólogo/efeitos adversos , Transplante Heterólogo/métodosRESUMO
Injury to endothelial cells (ECs) often results in cell retraction and gap formation. When caused by antigen aggregation or complement, this injury can be prevented by pretreatment of the ECs with IL-4, suggesting that IL-4 modifies the intercellular junction. Therefore, we investigated the effects of IL-4 on expression of intercellular junction proteins and whether such effects are required for IL-4-induced resistance of ECs against complement-mediated injury. We found that IL-4 induces upregulation of the junction protein claudin-5 in porcine ECs through activation of Jak/STAT6 and phosphorylation and translocation of FoxO1 from the nucleus to the cytoplasm. Increased claudin-5 expression resulted in increased transmembrane electrical resistance of the endothelial monolayer and participated in IL-4-induced protection of the ECs from complement injury. Down-regulation of FoxO1 using siRNA by itself caused up-regulation of claudin-5 expression and partial protection from cytotoxicity. This protection was enhanced by stimulation with IL-4. We previously reported that increased phospholipid synthesis and mitochondrial protection were required for IL-4-induced resistance of ECs against complement injury and now we demonstrate a contribution of claudin-5 expression in IL-4-induced protection.
Assuntos
Claudina-5/metabolismo , Células Endoteliais/efeitos dos fármacos , Fatores de Transcrição Forkhead/metabolismo , Interleucina-4/farmacologia , Regulação para Cima/efeitos dos fármacos , Animais , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteínas do Sistema Complemento/toxicidade , Citoplasma/metabolismo , Células Endoteliais/metabolismo , Fatores de Transcrição Forkhead/genética , Humanos , Immunoblotting , Janus Quinase 3/antagonistas & inibidores , Janus Quinase 3/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Pirimidinas/farmacologia , Pirróis/farmacologia , Interferência de RNA , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismo , SuínosRESUMO
Accommodation has been termed as a condition without graft rejection even in the presence of antidonor antibody. We previously reported an in vitro accommodation model, which demonstrated that preincubation of A/B antigen-expressing endothelial cells with anti-A/B antibody resulted in ERK inactivation followed by resistance to complement-mediated cytotoxicity through the induction of complement regulatory genes. However, under the in vivo condition, the effects of complement and coagulation system cannot be ignored. The purpose of this study is to find effective ways to navigate accommodation by exploring the relevant signal transduction. Preincubation with a low level of complement or thrombin failed to induce resistance to complement-mediated cytotoxicity. AMP-activated protein kinase (AMPK) activators such as resveratrol, AICAR and metformin protected endothelial cells against complement-mediated cytotoxicity through the increase in CD55, CD59, haem oxygenase-1 (HO-1) and ferritin heavy chain (ferritin H) genes, all of which were attenuated by AMPKα knock-down. Resveratrol counteracted the inhibitory effect of pretreated complement and thrombin on acquisition of resistance to complement-mediated cytotoxicity through AMPKα. AMPK regulation in endothelial cells could become the potential strategy to induce accommodation in clinical pro-inflammation and pro-coagulation.
Assuntos
Proteínas Quinases Ativadas por AMP/farmacologia , Proteínas do Sistema Complemento/toxicidade , Citoproteção/efeitos dos fármacos , Trombina/farmacologia , Imunologia de Transplantes , Aminoimidazol Carboxamida/análogos & derivados , Antígenos CD55/biossíntese , Antígenos CD59/biossíntese , Linhagem Celular , Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica/imunologia , Rejeição de Enxerto/prevenção & controle , Humanos , Resveratrol , Ribonucleotídeos , Transdução de Sinais/fisiologia , Estilbenos/farmacologiaRESUMO
This paper presents an optimized procedure for assessing an immune-mediated cytotoxicity, produced after the addition of human and baboon serum to transgenic porcine fibroblasts. This procedure is performed with the xCELLigence Real-Time Cell Analyzer (RTCA). The xCELLigence system measures the impedance variations in the culture media of a 96-well microelectronic plate, and shows the changes in cell number and morphology in a real-time plot. However, different factors need to be optimized before developing an RTCA assay. Thus, we studied the influence of several variables, such as the number of cells seeded, the time the cells were allowed to grow before the tests, the serum concentration and the addition of rabbit complement. The findings were confirmed by the WST-1 classical cytotoxicity test. The results showed that 7.5 × 10(3) cells seeded per well produced the adequate CI in 10 h. The area under the curve and the CImin versus concentration values showed a very high correlation index (r(2) = 0.966 and r(2) = 0.92 for the first 50 h after challenge, respectively), proving that CI variations are directly proportional to the quantity of serum added. The addition of complement resulted in lower CImin values. Therefore, both the cytolysis level with and without exogenous complement addition had to be assessed. There was a high correlation between the relative cytotoxicity assessed by WST-1 and the CI obtained by RTCA when exogenous complement was not added (r(2) = 0.827; p < 0.001). The correlation was average when rabbit complement was added (r(2) = 0.523; p = 0.046). In conclusion, culture conditions have an important influence on RTCA cytotoxicity assays.
Assuntos
Citotoxinas/toxicidade , Testes de Toxicidade/métodos , Animais , Adesão Celular , Proliferação de Células/efeitos dos fármacos , Proteínas do Sistema Complemento/toxicidade , Impedância Elétrica , Feminino , Humanos , Masculino , Papio , Suínos , Fatores de TempoRESUMO
Neuromyelitis optica is an antibody-mediated autoimmune inflammatory disease of the central nervous system. Reports have suggested that interferon beta which is beneficial for multiple sclerosis, exacerbates neuromyelitis optica. Our aim was to determine whether type I interferon plays a role in the formation of neuromyelitis optica lesions. Immunoglobulin G from a neuromyelitis optica patient was injected intracerebrally with human complement to type I interferon receptor deficient and wildtype mice. Loss of aquaporin-4 and glial fibrillary acidic protein was reduced in type I interferon receptor deficient mice brain. Our findings suggest that type I interferon signaling contributes to neuromyelitis optica pathogenesis.
Assuntos
Regulação da Expressão Gênica/genética , Neuromielite Óptica/patologia , Receptor de Interferon alfa e beta/metabolismo , Animais , Aquaporina 4/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Proteínas do Sistema Complemento/toxicidade , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Imunoglobulina G/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Neuromielite Óptica/induzido quimicamente , Neuromielite Óptica/imunologia , Receptor de Interferon alfa e beta/deficiência , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
The specificity and potential pathogenicity of autoantibodies vary between neurological diseases. It is often unclear whether their detection in cerebrospinal fluid (CSF) is a consequence or a cause of pathology. The goal was to test whether administration of brain-specific antibodies into CSF would be sufficient for pathology. Purified immunoglobulin G from a neuromyelitis optica patient was injected intrathecally with complement to naïve mice. Histopathological analysis at 7 days revealed damage to the ependyma, disruption of the CSF parenchymal barrier and pathologic lesions, distant from the site of injection. In the absence of complement there was no pathology. Autoantibody and complement in CSF are thus sufficient to initiate a pathologic cascade.
Assuntos
Aquaporina 4/imunologia , Encéfalo/patologia , Proteínas do Sistema Complemento/líquido cefalorraquidiano , Imunoglobulina G/líquido cefalorraquidiano , Animais , Encéfalo/imunologia , Encéfalo/metabolismo , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/toxicidade , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/imunologia , Doenças Desmielinizantes/metabolismo , Epêndima/patologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Células HEK293 , Humanos , Imunoglobulina G/toxicidade , Injeções Espinhais , Camundongos , Camundongos Endogâmicos C57BL , Neuromielite Óptica/sangue , Neuromielite Óptica/imunologia , TransfecçãoRESUMO
Both the neural and glial components of the neuromuscular junction (NMJ) have been identified as potential sites for anti-ganglioside antibody (Ab) binding and complement-mediated injury in murine models for the human peripheral nerve disorder Guillain-Barré syndrome (GBS). Some patients suffering from the acute motor axonal neuropathy (AMAN) forms of GBS recover very rapidly from paralysis; it has been proposed that in these cases the injury was restricted to the distal motor axons and nerve terminals (NTs) which are able to regenerate over a very short time-frame. To test this hypothesis, the ventral neck muscles of mice (n=45) expressing cytosolic fluorescent proteins in their axons (CFP) and Schwann cells (GFP) were subjected to a single topical application of anti-ganglioside Ab followed by a source of complement. Group A (n=15) received Ab that selectively bound to the NTs, group B (n=15) received Abs that bound both to the NTs and the perisynaptic Schwann cells (pSCs) and group C (control animals; n=15) only received complement. Evolution of the injury was documented by in vivo imaging, and following euthanasia the muscles were reimaged ex vivo both quantitatively and qualitatively, either immediately, or after 1, 2, 3 or 5 days of regeneration (each n=3 per group). Within 15 minutes of complement application, a rapid loss of CFP overlying the NMJ could be seen; in group A, the GFP signal remained unchanged, whereas in group B the GFP signal was also lost. In group C no changes to either CFP or GFP were observed. At 24 h, 6% of the superficial NMJs in group A and 12% of the NMJs in group B exhibited CFP. In both groups, CFP returned within the next five days (group A: 93.5%, group B: 94%; p=0.739), with the recovery of CFP being preceded by a return of GFP-positive cells overlying the NMJ in group B. Auxiliary investigations revealed that the loss of CFP at the NMJ correlated with a loss of NT neurofilament immuno-reactivity and a return of CFP at the NMJ was accompanied by a return of neurofilament. In ultrastructural investigations, injured NTs were electron lucent and exhibited damaged mitochondria, a loss of filaments and a loss of synaptic vesicles. The examination of muscles after five days of regeneration revealed physiological NT-profiles. The results described above indicate that following a single anti-ganglioside Ab-mediated and complement-mediated attack, independent of whether there are healthy and mature perisynaptic Schwann cells overlying the NMJ, the murine NT is capable of recovering both its architectural and axolemmal integrity very rapidly. This data supports the notion that an equivalent mechanism may account for the rapid recovery seen in some clinical cases of AMAN.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Autoanticorpos/administração & dosagem , Proteínas do Sistema Complemento/toxicidade , Gangliosídeos/imunologia , Neurônios Motores/patologia , Junção Neuromuscular/lesões , Terminações Pré-Sinápticas/imunologia , Regeneração/fisiologia , Animais , Anticorpos Monoclonais/toxicidade , Autoanticorpos/toxicidade , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/imunologia , Músculos do Pescoço/imunologia , Músculos do Pescoço/patologia , Junção Neuromuscular/imunologia , Junção Neuromuscular/patologia , Regeneração/imunologiaRESUMO
The complement system functions as an immune surveillance system that rapidly responds to infection. Activation of the complement system by specific recognition pathways triggers a protease cascade, generating cleavage products that function to eliminate pathogens, regulate inflammatory responses, and shape adaptive immune responses. However, when dysregulated, these powerful functions can become destructive and the complement system has been implicated as a pathogenic effector in numerous diseases, including infectious diseases. This review highlights recent discoveries that have identified critical roles for the complement system in the pathogenesis of viral infection.
Assuntos
Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/toxicidade , Viroses/patologia , Vírus/imunologia , Vírus/patogenicidade , HumanosRESUMO
Neuromyelitis optica (NMO)-immunoglobulin G (IgG) is a clinically validated serum biomarker that distinguishes relapsing central nervous system (CNS) inflammatory demyelinating disorders related to NMO from multiple sclerosis. This autoantibody targets astrocytic aquaporin-4 (AQP4) water channels. Clinical, radiological, and immunopathological data suggest that NMO-IgG might be pathogenic. Characteristic CNS lesions exhibit selective depletion of AQP4, with and without associated myelin loss; focal vasculocentric deposits of IgG, IgM, and complement; prominent edema; and inflammation. The effect of NMO-IgG on astrocytes has not been studied. In this study, we demonstrate that exposure to NMO patient serum and active complement compromises the membrane integrity of CNS-derived astrocytes. Without complement, astrocytic membranes remain intact, but AQP4 is endocytosed with concomitant loss of Na(+)-dependent glutamate transport and loss of the excitatory amino acid transporter 2 (EAAT2) . Our data suggest that EAAT2 and AQP4 exist in astrocytic membranes as a macromolecular complex. Transport-competent EAAT2 protein is up-regulated in differentiating astrocyte progenitors and in nonneural cells expressing AQP4 transgenically. Marked reduction of EAAT2 in AQP4-deficient regions of NMO patient spinal cord lesions supports our immunocytochemical and immunoprecipitation data. Thus, binding of NMO-IgG to astrocytic AQP4 initiates several potentially neuropathogenic mechanisms: complement activation, AQP4 and EAAT2 down-regulation, and disruption of glutamate homeostasis.
Assuntos
Aquaporina 4/imunologia , Astrócitos/metabolismo , Autoanticorpos/metabolismo , Regulação da Expressão Gênica/imunologia , Proteínas de Transporte de Glutamato da Membrana Plasmática/metabolismo , Ácido Glutâmico/metabolismo , Neuromielite Óptica/imunologia , Aquaporina 4/metabolismo , Astrócitos/citologia , Autoanticorpos/imunologia , Transporte Biológico/imunologia , Western Blotting , Membrana Celular/efeitos dos fármacos , Proteínas do Sistema Complemento/toxicidade , Primers do DNA/genética , Transportador 2 de Aminoácido Excitatório , Humanos , Imunoglobulina G/metabolismo , Imunoprecipitação , Neuromielite Óptica/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/metabolismoRESUMO
Infusion reactions are a major side effect of the administration of therapeutic Abs and are the result of a complex immune reaction. In this study, we report that substitutions of Fc amino acids in the anti-HLA-DR Ab HD8 reduce its ability to induce infusion reactions in rats and monkeys. We first showed that i.v. administration of IgG1- and IgG2-subclass HD8 Abs induces severe infusion reactions in monkeys. These Abs express strong complement-dependent cytotoxicity (CDC), and in vivo depletion of complement in rats by pretreatment with cobra venom factor abrogated the lethal infusion reactions generated by HD8-IgG1. Thus, the infusion reactions appear to be largely driven by the complement system. To reduce the CDC function of HD8-IgG1, its Fc region was modified by two amino acid substitutions at Pro(331)Ser and Lys(322)Ala. The modified Ab was incapable of expressing CDC in vitro and did not induce severe infusion reactions in rats and monkeys, even at extremely high doses. The modified Ab retained its Ab-dependent cellular cytotoxicity function as well as its antitumor activity in a tumor-bearing mouse model. In summary, complement appears to drive infusion reactions, and modifications that eliminate the CDC activity of an Ab also reduce its ability to induce infusion reactions.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/imunologia , Ativação do Complemento/imunologia , Isoanticorpos/toxicidade , Animais , Animais Congênicos , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Proteínas do Sistema Complemento/toxicidade , Cricetinae , Cricetulus , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Infusões Intravenosas/efeitos adversos , Isoanticorpos/administração & dosagem , Isoanticorpos/uso terapêutico , Linfoma/imunologia , Linfoma/mortalidade , Linfoma/terapia , Macaca fascicularis , Masculino , Camundongos , Camundongos SCID , Ratos , Transplante HeterólogoRESUMO
Complement activation is a central component of inflammation and sepsis and can lead to significant tissue injury. Complement factors are serum proteins that work through a cascade of proteolytic reactions to amplify proinflammatory signals. Inter-alpha-trypsin inhibitor (IaI) is an abundant serum protease inhibitor that contains potential complement-binding domains, and has been shown to improve survival in animal sepsis models. We hypothesized that IaI can bind complement and inhibit complement activation, thus ameliorating complement-dependent inflammation. We evaluated this hypothesis with in vitro complement activation assays and in vivo in a murine model of complement-dependent lung injury. We found that IaI inhibited complement activation through the classical and alternative pathways, inhibited complement-dependent phagocytosis in vitro, and reduced complement-dependent lung injury in vivo. This novel function of IaI provides a mechanistic explanation for its observed salutary effects in sepsis and opens new possibilities for its use as a treatment agent in inflammatory diseases.
Assuntos
alfa-Globulinas/fisiologia , Ativação do Complemento/imunologia , Proteínas Inativadoras do Complemento/fisiologia , Proteínas do Sistema Complemento/toxicidade , Pulmão/imunologia , Pulmão/patologia , alfa-Globulinas/deficiência , alfa-Globulinas/genética , alfa-Globulinas/metabolismo , Animais , Ativação do Complemento/genética , Proteínas Inativadoras do Complemento/deficiência , Proteínas Inativadoras do Complemento/genética , Proteínas Inativadoras do Complemento/metabolismo , Proteínas do Sistema Complemento/metabolismo , Feminino , Doenças do Complexo Imune/imunologia , Doenças do Complexo Imune/metabolismo , Doenças do Complexo Imune/patologia , Doenças do Complexo Imune/prevenção & controle , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose/imunologia , Ligação Proteica/imunologia , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Subunidades Proteicas/fisiologiaRESUMO
Vascular endothelial cells (ECs) can be injured in a variety of pathologic processes that involve activated complement. We reported previously that porcine ECs incubated with exogenous IL-4 or IL-13 are protected from cytotoxicity by human complement and also from apoptosis by TNF-alpha. The resistance to complement consists of an intrinsic mechanism that is lost a few days after cytokine removal. In our current study, we investigated whether transfer of the IL-4 gene into porcine ECs in vitro and into porcine vascular tissues in vivo would induce efficient and durable protection from human complement. We found that ECs transduced with adenoIL-4 or adenoIL-13 exhibited continuous production of the cytokine and prolonged protection from complement-mediated killing. IL-4 also protected ECs from activation: ECs incubated with IL-4 did not develop cell retraction and intercellular gaps upon stimulation with sublytic complement. The endothelium and subendothelium of pig iliac arteries that were transduced with the IL-4 gene were effectively protected from complement-dependent immediate injury after perfusion with human blood. However, after similar perfusion, the endothelium was immediately lost from arteries that were transduced with a control adenovirus. The protection was not due to up-regulation of the complement regulators decay accelerating factor, membrane cofactor protein, and CD59, or to reduced complement activation, but required the participation of Akt. Although our studies model protection in pig-to-primate xenotransplantation, our findings of IL-4 induction of Akt-mediated protection may be more broadly applicable to EC injury as manifested in ischemia-reperfusion, allotransplantation, and various vascular diseases.
Assuntos
Proteínas do Sistema Complemento/toxicidade , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Artéria Ilíaca/imunologia , Artéria Ilíaca/metabolismo , Interleucina-4/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução Genética , Adenoviridae/genética , Animais , Sangue/imunologia , Células Cultivadas , Proteínas do Sistema Complemento/metabolismo , Citotoxicidade Imunológica/genética , Endotélio Vascular/citologia , Líquido Extracelular/imunologia , Líquido Extracelular/metabolismo , Técnicas de Transferência de Genes , Humanos , Artéria Ilíaca/citologia , Imunidade Inata/genética , Interleucina-13/biossíntese , Interleucina-13/genética , Interleucina-4/biossíntese , Interleucina-4/fisiologia , Perfusão , Proteínas Proto-Oncogênicas c-akt/fisiologia , SuínosRESUMO
Previous work demonstrated that a brief, sublethal excitotoxic insult strikingly increased the sensitivity of cortical neurons to the cytotoxic effects of the terminal pathway of complement, a process termed "excitotoxic sensitization." Here, we sought to elucidate the cellular mechanism of excitotoxic sensitization in embryonic rat cortical neurons in vitro. Excitotoxic sensitization did not increase membrane attack complex deposition on cortical neurons and produced only a small reduction of membrane attack complex removal, because of a selective decrease of endocytic elimination. Membrane attack complexes and other osmotic stressors, namely hypotonic stress and glutamate, induced transient swelling of cortical neurons, followed by return to normal volume despite persistence of the stressor, a homeostatic response termed regulatory volume decrease (RVD). A minimal excitotoxic insult impaired this homeostatic response and sensitized neurons to cytotoxic effects of diverse osmotic stressors. Structurally distinct membrane-impermeable osmolytes, dextran and polyethylene glycol, prevented excitotoxic sensitization to diverse osmotic stressors including membrane attack complexes. Paraquat, a reactive oxygen species generator, alone was sufficient to impair RVD, and MnTBAP [Mn(III)tetrakis(4-benzoic acid)porphyrin chloride], a reactive oxygen species scavenger, prevented excitotoxin-mediated impairment of RVD. Together, these findings demonstrate that impairment of RVD is the mechanism of excitotoxic sensitization, that reactive oxygen species alone are sufficient to impair RVD, and that reactive oxygen species are necessary for excitotoxic sensitization-mediated impairment of RVD.
Assuntos
Proteínas do Sistema Complemento/toxicidade , Agonistas de Aminoácidos Excitatórios/farmacologia , Neurônios/fisiologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Complexo de Ataque à Membrana do Sistema Complemento , Relação Dose-Resposta a Droga , Ácido Glutâmico/farmacologia , Neurônios/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/farmacologiaRESUMO
When activated on or in the vicinity of cells, complement usually causes loss of function and sometimes cell death. Yet the liver, which produces large amounts of complement proteins, clears activators of complement and activated complexes from portal blood without obvious injury or impaired function. We asked whether and to what extent hepatocytes resist injury and loss of function mediated by exposure to complement. Using cells isolated from porcine livers as a model system, we found that, in contrast to endothelial cells, hepatocytes profoundly resist complement-mediated lysis and exhibit normal synthetic and conjugative functions when complement is activated on their surface. The resistance of hepatocytes to complement-mediated injury was not a function of cell surface control of the complement cascade but rather an intrinsic resistance of the cells dependent on the PI3K/Akt pathway. The resistance of hepatocytes to complement might be exploited in developing approaches to the treatment of hepatic failure or more broadly to the treatment of complement-mediated disease.
Assuntos
Proteínas Inativadoras do Complemento/fisiologia , Proteínas do Sistema Complemento/toxicidade , Citotoxicidade Imunológica/imunologia , Hepatócitos/imunologia , Hepatócitos/patologia , Animais , Anticorpos Heterófilos/metabolismo , Sítios de Ligação de Anticorpos , Células Cultivadas , Ativação do Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Hepatócitos/metabolismo , Humanos , Imunidade Inata , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , Transdução de Sinais/imunologia , SuínosRESUMO
Complement-mediated cytotoxicity for porcine islet cells (PICs) was evaluated using sera of six animal species. Then soluble complement receptor type-1 (sCR1) as an anti-complement agent was added to those sera, and the changes in 50% hemolytic unit of complement serum (CH50) and cytotoxic effect of those sera on PICs were examined. All the sera except for that of pig showed cytotoxicity. However, the extent of toxicity was considerably different between species. In the rat and human serum, sCR1 significantly reduced CH50 and cytotoxicity, however in the dog serum, sCR1 had no suppressive effects. These results may suggest that complement contribute to humoral cytotoxicity for PICs as a main factor, and the compatibility of complement with PICs differs between animal species.
Assuntos
Proteínas do Sistema Complemento/toxicidade , Ilhotas Pancreáticas/patologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Cães , Cobaias , Humanos , Ilhotas Pancreáticas/efeitos dos fármacos , Ratos , Especificidade da Espécie , SuínosRESUMO
We have developed "Cytomedicine," which consists of functional cells entrapped in semipermeable polymer, and previously reported that APA microcapsules could protect the entrapped cells from injury by cellular immune system. However, microencapsulated cells were not protected from humoral immune system. Here, we developed a novel APA microcapsule, in which APA microbeads (APA(Ba) microbeads) were modified to contain a barium alginate hydrogel within their centers in an attempt to make it more difficult for antibody and complement to permeate the microcapsules. The permeability of APA(Ba) microbeads was clearly less than that of APA microcapsules, presumably due to the presence of barium alginate hydrogel. Cells encapsulated within APA(Ba) microbeads were protected against treatment with xenogeneic anti-serum. Furthermore, murine pancreatic beta-cells encapsulated in APA(Ba) microbeads remained viable and continued to secrete insulin in response to glucose. Therefore, APA(Ba) microbeads may be a useful carrier for developing anti-complement device for cytomedical therapy.
Assuntos
Alginatos/química , Proteínas do Sistema Complemento/toxicidade , Hidrogéis , Polilisina/análogos & derivados , Polilisina/química , Animais , Anticorpos Heterófilos/toxicidade , Bário/química , Proteínas Inativadoras do Complemento/química , Proteínas do Sistema Complemento/metabolismo , Citoproteção , Hidrogéis/química , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Membranas Artificiais , Camundongos , Microesferas , Permeabilidade , Células Tumorais CultivadasRESUMO
Bordetella pertussis must survive the defenses of the human respiratory tract including the complement system. The BrkA (Bordetella resistance to killing) protein prevents killing by the antibody-dependent classical pathway. In this study, the ability of B. pertussis to activate the human complement cascade by other pathways was examined. B. pertussis was not killed in serum depleted of C2, however serum depleted for factor B killed B. pertussis as efficiently as intact serum, suggesting complement activation occurred exclusively by the classical pathway. B. pertussis was not killed by serum depleted of antibody, suggesting the bacteria fail to activate the antibody-independent branches of the classical pathway, including the mannose binding lectin pathway. Mutants lacking the terminal trisaccharide of lipopolysaccharide retained the complement-resistant phenotype, suggesting this structure does not influence activation of complement.
Assuntos
Bordetella pertussis/patogenicidade , Via Clássica do Complemento , Bordetella pertussis/crescimento & desenvolvimento , Complemento C1q/imunologia , Proteínas do Sistema Complemento/metabolismo , Proteínas do Sistema Complemento/toxicidade , Imunidade Inata , Lipopolissacarídeos/metabolismo , Lectina de Ligação a Manose/metabolismo , Modelos Biológicos , Mutação , Coqueluche/sangue , Coqueluche/imunologiaRESUMO
Borrelia burgdorferi, the etiological agent of Lyme disease, comprises three genospecies, Borrelia garinii, afzelii, and burgdorferi sensu strictu, that exhibit different pathogenicity and differ in the susceptibility to C-mediated killing. We examined C-sensitive and C-resistant strains of B. burgdorferi for deposition of C3 and late C components by fluorescence microscope and flow cytometry. Despite comparable deposition of C3 on the two strains, the resistant strain exhibited reduced staining for C6 and C7, barely detectable C9, and undetectable poly C9. Based on these findings, we searched for a protein that inhibits assembly of C membrane attack complex and documented an anti-human CD59-reactive molecule on the surface of C-resistant spirochetes by flow cytometry and electron microscopy. A molecule of 80 kDa recognized by polyclonal and monoclonal anti-CD59 Abs was identified in the membrane extract of C-resistant strains by SDS-PAGE and Western blot analysis. The molecule was released from the bacterial wall using deoxycholate and trypsin, suggesting its insertion into the bacterial membrane. The CD59-like molecule acts as C inhibitor on Borrelia because incubation with F(ab')(2) anti-CD59 renders the serum-resistant strain exquisitely susceptible to C-mediated killing and guinea pig erythrocytes bearing C5b-8, unlike the RBC coated with C5b-7, are protected from reactive lysis by the bacterial extract. Western blot analysis revealed preferential binding of the C inhibitory molecule to C9 and weak interaction with C8 beta.
Assuntos
Proteínas de Bactérias/biossíntese , Atividade Bactericida do Sangue/imunologia , Borrelia burgdorferi/crescimento & desenvolvimento , Borrelia burgdorferi/imunologia , Antígenos CD59/biossíntese , Proteínas Inativadoras do Complemento/biossíntese , Proteínas do Sistema Complemento/toxicidade , Citotoxicidade Imunológica , Anticorpos Bloqueadores/metabolismo , Anticorpos Bloqueadores/farmacologia , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação de Anticorpos , Borrelia burgdorferi/metabolismo , Borrelia burgdorferi/ultraestrutura , Antígenos CD59/imunologia , Antígenos CD59/metabolismo , Antígenos CD59/ultraestrutura , Membrana Celular/imunologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Complemento C3/metabolismo , Complemento C7/metabolismo , Complemento C8/metabolismo , Complemento C9/metabolismo , Proteínas Inativadoras do Complemento/imunologia , Proteínas Inativadoras do Complemento/metabolismo , Proteínas Inativadoras do Complemento/ultraestrutura , Complexo de Ataque à Membrana do Sistema Complemento/antagonistas & inibidores , Técnica Direta de Fluorescência para Anticorpo , Humanos , Imunidade Inata , Especificidade da Espécie , Tripsina/farmacologiaRESUMO
The phenomenon of fast death of mice after parenteral administration of mink serum was explained by high activity of mink complement in particular by unusually high activity of its alternative pathway of activation. The presence of antibodies to mouse erythrocytes in mink serum was necessary precondition for their lysis under action of mink complement by classical and alternative pathways. However, removal of these antibodies resulting in cancellation of hemolysis did not effect toxicity of mink serum for nice in vivo. Partial decomplementization of mink serum zymosan completely prevented death of animals.