RESUMO
The lectin pathway (LP) of complement activation is believed to contribute to brain inflammation. The study aims to identify the key components of the LP contributing to TBI outcome as possible novel pharmacological targets. We compared the long-term neurological deficits and neuropathology of wild-type mice (WT) to that of mice carrying gene deletions of key LP components after experimental TBI. WT or MASP-2 (Masp2-/-), ficolin-A (Fcna-/-), CL-11 (Colec11-/-), MASP-1/3 (Masp1-/-), MBL-C (Mbl2-/-), MBL-A (Mbl1-/-) or MBL-/- (Mbl1-/-/Mbl2-/-) deficient male C57BL/6J mice were used. Mice underwent sham surgery or TBI by controlled cortical impact. The sensorimotor response was evaluated by neuroscore and beam walk tests weekly for 4 weeks. To obtain a comparative analysis of the functional outcome each transgenic line was rated according to a health score calculated on sensorimotor performance. For selected genotypes, brains were harvested 6 weeks after injury for histopathological analysis. MASP-2-/-, MBL-/- and FCN-A-/- mice had better outcome scores compared to WT. Of these, MASP-2-/- mice had the best recovery after TBI, showing reduced sensorimotor deficits (by 33% at 3 weeks and by 36% at 4 weeks). They also showed higher neuronal density in the lesioned cortex with a 31.5% increase compared to WT. Measurement of LP functional activity in plasma from MASP-2-/- mice revealed the absence of LP functional activity using a C4b deposition assay. The LP critically contributes to the post-traumatic inflammatory pathology following TBI with the highest degree of protection achieved through the absence of the LP key enzyme MASP-2, underlining a therapeutic utility of MASP-2 targeting in TBI.
Assuntos
Lesões Encefálicas Traumáticas/genética , Lectina de Ligação a Manose da Via do Complemento/genética , Inflamação/genética , Recuperação de Função Fisiológica/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/patologia , Lesões Encefálicas Traumáticas/fisiopatologia , Colectinas/genética , Complemento C4b/metabolismo , Deleção de Genes , Inflamação/metabolismo , Lectinas/genética , Lectina de Ligação a Manose/genética , Serina Proteases Associadas a Proteína de Ligação a Manose/genética , Camundongos , Camundongos Knockout , Prognóstico , FicolinasRESUMO
Intestinal mucositis is a serious complication of chemotherapy that leads to significant morbidity that may require dose or drug adjustments. Specific mitigating strategies for mucositis are unavailable, due partly to an incomplete understanding of the pathogenic mechanisms. We have previously shown an effect of properdin, a positive regulator of complement activation, in models of colitis. Here we use properdin-deficient (PKO ) mice to interrogate the role of properdin and complement in small intestinal mucositis. Mucositis was induced by five daily injections of 5-fluorouracil (5-FU) in wild-type (WT), PKO , interleukin (IL)-10-/- and properdin/IL-10-/- double knock-out (DKO) mice. At the time of euthanasia their jejunum was collected for histology, immunohistochemistry and cytokine and complement activation measurements. Complement became activated in mice receiving 5-FU, indicated by increased intestinal levels of C3a and C5a. Compared to WT, PKO mice experienced significantly less mucositis, despite C3a levels as high as inflamed WT mice and slightly less C5a. Conversely, PKO mice had higher intestinal levels of IL-10. IL-10 expression was mainly by epithelial cells in both uninflamed and inflamed PKO mice. IL-10-/- mice proved to be highly susceptible to mucositis and DKO mice were equally susceptible, demonstrating that a lack of properdin does not protect mice lacking IL-10. We interpret our findings to indicate that, to a significant extent, the inflammation of mucositis is properdin-dependent but complement activation-independent. Additionally, the benefit achieved in the absence of properdin is associated with increased IL-10 levels, and IL-10 is important in limiting mucositis.
Assuntos
Ativação do Complemento/imunologia , Fluoruracila/efeitos adversos , Interleucina-10/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Mucosite/etiologia , Mucosite/metabolismo , Properdina/deficiência , Animais , Complemento C5a/imunologia , Modelos Animais de Doenças , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Camundongos , Camundongos Knockout , Mucosite/patologia , FenótipoRESUMO
OBJECTIVE: Patients undergoing abdominal aortic aneurysm (AAA) repair are exposed to an ischaemia-reperfusion injury (IRI), which is in part mediated by complement activation. We investigated the role of the novel lectin pathway of complement during IRI in patients undergoing AAA repair. METHODS: Patients undergoing elective open infrarenal AAA repair had systemic blood samples taken at induction of anaesthesia, prior to aortic clamping, prior to aortic declamping and at reperfusion. Control patients undergoing major abdominal surgery were also included. Plasma was assayed for levels of mannan-binding lectin (MBL) using ELISA techniques. Consumption of plasma MBL was used as a measure of lectin pathway activation. RESULTS: Twenty-three patients undergoing AAA repair and eight control patients were recruited. No lectin pathway activation could be demonstrated in the control patients. AAA patients experienced a mean decrease in plasma MBL levels of 41% representing significant lectin pathway activation (p = 0.003). CONCLUSION: Consumption of MBL occurs during AAA repair, suggesting an important role for the lectin pathway in IRI. Specific transient inhibition of lectin pathway activity could be of significant therapeutic value in patients undergoing open surgical AAA repair.
Assuntos
Aneurisma da Aorta Abdominal/fisiopatologia , Aneurisma da Aorta Abdominal/cirurgia , Ativação do Complemento/fisiologia , Lectina de Ligação a Manose/sangue , Idoso , Aneurisma da Aorta Abdominal/sangue , Feminino , Humanos , Masculino , Traumatismo por ReperfusãoRESUMO
Activation of the lectin pathway of complement is initiated by the binding to microbial carbohydrate structures of a multimolecular fluid-phase complex composed of a carbohydrate recognition subcomponent that associates with three specific serine proteases and an enzymatically inert protein of 19 kDa. The first carbohydrate recognition subcomponent of the lectin pathway identified was mannan-binding lectin (MBL), hence the serine proteases were named MBL-associated serine proteases (MASPs) and numbered according to the sequence of their discovery. Here we describe the primary structures of the two distinct serine proteases MASP-1 and MASP-3 in the rat (and of MASP-3 in the mouse), show their association with plasma MBL complexes, and demonstrate that in rat and mouse, as in man, MASP-1 and MASP-3 are encoded by a single structural gene. For both species, we present the genomic region and regulatory elements responsible for the processing of either MASP-1 or MASP-3 mRNA by alternative splicing/alternative polyadenylation. Furthermore, we demonstrate the evolutionary conservation of MASP-3 mRNA in cDNA transcripts from guinea pig, rabbit, pufferfish, and cow.
Assuntos
Lectina de Ligação a Manose da Via do Complemento/genética , Camundongos/genética , Ratos/genética , Serina Endopeptidases/genética , Processamento Alternativo/genética , Sequência de Aminoácidos , Animais , Sequência Conservada/genética , Primers do DNA , DNA Complementar/genética , Serina Proteases Associadas a Proteína de Ligação a Manose , Técnicas de Sonda Molecular , Dados de Sequência Molecular , Poliadenilação/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Serina Endopeptidases/metabolismoAssuntos
Antígenos CD , Antígenos de Diferenciação Mielomonocítica/genética , Cromossomos Humanos Par 12/genética , Hibridização in Situ Fluorescente , Proteínas de Membrana , Mapeamento de Híbridos Radioativos , Receptores de Superfície Celular/genética , Receptores Imunológicos/genética , Receptores de Lipoproteínas , Duplicação Gênica , Genes Duplicados/genética , Ligação Genética/genética , Humanos , Dados de Sequência Molecular , Receptores Depuradores , Receptores Depuradores Classe BRESUMO
Mannan-binding lectin (MBL) and C1q activate the complement cascade via attached serine proteases. The proteases C1r and C1s were initially discovered in a complex with C1q, whereas the MBL-associated serine proteases 1 and 2 (MASP-1 and -2) were discovered in a complex with MBL. There is controversy as to whether MBL can utilize C1r and C1s or, inversely, whether C1q can utilize MASP-1 and 2. Serum deficient in C1r produced no complement activation in IgG-coated microwells, whereas activation was seen in mannan-coated microwells. In serum, C1r and C1s were found to be associated only with C1q, whereas MASP-1, MASP-2, and a third protein, MAp19 (19-kDa MBL-associated protein), were found to be associated only with MBL. The bulk of MASP-1 and MAp19 was found in association with each other and was not bound to MBL or MASP-2. The interactions of MASP-1, MASP-2, and MAp19 with MBL differ from those of C1r and C1s with C1q in that both high salt concentrations and calcium chelation (EDTA) are required to fully dissociate the MASPs or MAp19 from MBL. In the presence of calcium, most of the MASP-1, MASP-2, and MAp19 emerged on gel-permeation chromatography as large complexes that were not associated with MBL, whereas in the presence of EDTA most of these components formed smaller complexes. Over 95% of the total MASPs and MAp19 found in serum are not complexed with MBL.
Assuntos
Proteínas de Transporte/metabolismo , Complemento C1/metabolismo , Serina Endopeptidases/metabolismo , Cálcio/química , Proteínas de Transporte/sangue , Proteínas de Transporte/imunologia , Centrifugação com Gradiente de Concentração , Cromatografia em Gel , Colectinas , Complemento C1q/imunologia , Complemento C1q/metabolismo , Complemento C1r/metabolismo , Complemento C1s/metabolismo , Complemento C4b/metabolismo , Ácido Edético/química , Humanos , Soros Imunes/química , Imunoglobulina G/metabolismo , Lectinas/metabolismo , Mananas/metabolismo , Serina Proteases Associadas a Proteína de Ligação a Manose , Concentração Osmolar , Ligação Proteica/imunologia , Serina Endopeptidases/isolamento & purificaçãoRESUMO
Recent evidence suggests that the pathophysiology of neurodegenerative and inflammatory neurological diseases has a neuroimmunological component involving complement, an innate humoral immune defense system. The present study demonstrates the effects of experimentally induced global ischemia on the biosynthesis of C1q, the recognition subcomponent of the classical complement activation pathway, in the CNS. Using semiquantitative in situ hybridization, immunohistochemistry, and confocal laser scanning microscopy, a dramatic and widespread increase of C1q biosynthesis in rat brain microglia (but not in astrocytes or neurons) within 24 h after the ischemic insult was observed. A marked increase of C1q functional activity in cerebrospinal fluid taken 1, 24, and 72 h after the ischemic insult was determined by C1q-dependent hemolytic assay. In the light of the well-established role of complement and complement activation products in the initiation and maintenance of inflammation, the ischemia-induced increase of cerebral C1q biosynthesis and of C1q functional activity in the cerebrospinal fluid implies that the proinflammatory activities of locally produced complement are likely to contribute to the pathophysiology of cerebral ischemia. Pharmacological modulation of complement activation in the brain may be a therapeutic target in the treatment of stroke.
Assuntos
Encéfalo/imunologia , Complemento C1q/biossíntese , Ataque Isquêmico Transitório/imunologia , Microglia/imunologia , Microglia/metabolismo , Regulação para Cima/imunologia , Animais , Encéfalo/patologia , Complemento C1q/líquido cefalorraquidiano , Complemento C1q/genética , Digoxigenina , Imuno-Histoquímica , Hibridização In Situ , Ataque Isquêmico Transitório/líquido cefalorraquidiano , Ataque Isquêmico Transitório/patologia , Masculino , Microglia/patologia , Sondas RNA , RNA Complementar , Ratos , Ratos Wistar , Radioisótopos de Enxofre , Regulação para Cima/genéticaRESUMO
Recently, we described two novel constituents of the multimolecular initiation complex of the mannan-binding lectin (MBL) pathway of complement activation, a serine protease of 76 kDa, termed MASP-2, and a MASP-2 related plasma protein of 19 kDa, termed MAp19. Upon activation of the MBL/MASPs/MAp19 complex, MASP-2 cleaves the fourth complement component C4, while the role of MAp19 within the MBL/MASP-1/MASP-2/MAp19 complex remains to be clarified. In humans, the mRNA species encoding MASP-2 (2.6 kb) and MAp19 (1.0 kb) arise by an alternative polyadenylation/splicing mechanism from a single structural MASP-2 gene. Here, we report the complete primary structures of the rat homologue of MASP-2 and of rat and mouse MAp19. We show that both MASP-2 and MAp19 are part of the rat MBL pathway activation complex and demonstrate their exclusively hepatic biosynthesis. Southern blot and PCR analyses of rat genomic DNA indicate that as in humans, rat MASP-2 and MAp19 are encoded by a single structural gene.
Assuntos
Proteínas de Transporte/química , Ativação do Complemento , Lectinas/imunologia , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Proteínas de Transporte/biossíntese , Proteínas de Transporte/sangue , Proteínas de Transporte/genética , Clonagem Molecular , Colectinas , Sondas de DNA , DNA Complementar/análise , Éxons , Íntrons , Mananas/imunologia , Serina Proteases Associadas a Proteína de Ligação a Manose , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , RNA Mensageiro/química , RNA Mensageiro/isolamento & purificação , Ratos , Ratos Wistar , Serina Endopeptidases/biossíntese , Serina Endopeptidases/sangue , Serina Endopeptidases/genéticaRESUMO
Mannan-binding lectin (MBL) forms a multimolecular complex with at least two MBL-associated serine proteases, MASP-1 and MASP-2. This complex initiates the MBL pathway of complement activation by binding to carbohydrate structures present on bacteria, yeast, and viruses. MASP-1 and MASP-2 are composed of modular structural motifs similar to those of the C1q-associated serine proteases C1r and C1s. Another protein of 19 kDa with the same N-terminal sequence as the 76-kDa MASP-2 protein is consistently detected as part of the MBL/MASP complex. In this study, we present the primary structure of this novel MBL-associated plasma protein of 19 kDa, MAp19, and demonstrate that MAp19 and MASP-2 are encoded by two different mRNA species generated by alternative splicing/polyadenylation from one structural gene.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Ativação do Complemento/genética , Genes/imunologia , Lectinas/metabolismo , Mananas/metabolismo , Serina Endopeptidases/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Sanguíneas/química , Proteínas de Transporte/química , Colectinas , Éxons , Humanos , Íntrons , Serina Proteases Associadas a Proteína de Ligação a Manose , Dados de Sequência Molecular , Peso Molecular , RNA Mensageiro/química , Ratos , Análise de Sequência de DNA , Serina Endopeptidases/química , Serina Endopeptidases/imunologia , Transcrição Gênica/imunologiaAssuntos
Formação de Anticorpos , Via Alternativa do Complemento , Imunidade Celular , Properdina/fisiologia , Animais , Infecções Bacterianas/etiologia , Complemento C3/fisiologia , Grânulos Citoplasmáticos/metabolismo , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Síndromes de Imunodeficiência/complicações , Síndromes de Imunodeficiência/genética , Interferon gama/fisiologia , Neutrófilos/fisiologia , Fagócitos/imunologia , Fagócitos/metabolismo , Properdina/química , Properdina/deficiência , Properdina/genética , Relação Estrutura-Atividade , Linfócitos T/imunologia , Linfócitos T/metabolismo , Cromossomo X/genéticaRESUMO
Fractalkine is the only as yet known member of a novel class of chemokines. Besides its novel Cys-X-X-X-Cys motif, fractalkine exhibits features which have not been described for any other member of the chemokine family, including its unusual size (397 amino acids human, 395 mouse) and the possession of a transmembrane anchor, from which a soluble form may be released by extracellular cleavage. This report demonstrates the abundant mRNA and fractalkine protein expression in neuronal cells. The neuronal expression of fractalkine mRNA is unaffected by experimentally induced inflammation of central nervous tissue.
Assuntos
Quimiocinas CX3C , Quimiocinas CXC/biossíntese , Encefalomielite Autoimune Experimental/metabolismo , Proteínas de Membrana/biossíntese , Neurônios/metabolismo , Animais , Células Cultivadas , Quimiocina CX3CL1 , Quimiocinas CXC/análise , Quimiocinas CXC/genética , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Imuno-Histoquímica , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Camundongos , Neurônios/patologia , RNA Mensageiro/metabolismo , RatosRESUMO
Several proteins have been identified as candidate cell-surface receptors for the complement protein C1q. Some of these also interact with the structurally-related collectin proteins. Previous descriptions of C1q-binding properties of cells, and information on the cellular distribution of candidate receptors suggest that there is more than one physiologically relevant receptor for C1q. Two such candidate receptors, cell-surface calreticulin (also referred to as cC1qR or collectin receptor) and megalin are discussed in this review.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Complemento C1q/metabolismo , Receptores de Hialuronatos , Glicoproteínas de Membrana/metabolismo , Receptores de Complemento/metabolismo , Ribonucleoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/isolamento & purificação , Calreticulina , Proteínas de Transporte/isolamento & purificação , Bovinos , Colectinas , Dimerização , Endocitose , Complexo Antigênico da Nefrite de Heymann , Humanos , Proteínas Mitocondriais , Dados de Sequência Molecular , Receptores de Complemento/isolamento & purificação , Ribonucleoproteínas/isolamento & purificaçãoRESUMO
gC1qBP is a 33 kDa glycoprotein that binds to the globular 'heads' of C1q. We have cloned cDNAs encoding the rat and mouse homologues of gC1qBP. Comparison of the cDNA-derived amino acid sequences of gC1qBP reveals that either of the rodent sequences is 89.9% identical to the reported human sequence. Recombinant rat gC1qBP binds avidly to human C1q. gC1qBP mRNA is abundantly expressed in every rat and mouse tissue analysed. Rat mesangial cells synthesise gC1qBP, but do not express gC1qBP on the cell surface. In rat serum, gC1qBP is present at low levels.
Assuntos
Complemento C1q/química , Complemento C1q/metabolismo , Receptores de Hialuronatos , Glicoproteínas de Membrana/química , Receptores de Complemento/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte , Córtex Cerebral/metabolismo , Biblioteca Gênica , Humanos , Fígado/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/biossíntese , Camundongos , Proteínas Mitocondriais , Dados de Sequência Molecular , Peso Molecular , Ratos , Receptores de Complemento/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Properdin is an important regulatory constituent of the complement system. In contrast to most other components of complement, biosynthesis of properdin is restricted to a few cell types only, i.e., monocytes/macrophages and peripheral blood T cells. This report demonstrates the presence of properdin mRNA in peripheral blood granulocytes and shows that properdin is stored in the granules of human neutrophils and secreted upon stimulation with TNF-alpha, C5a, IL-8, or FMLP. Subcellular fractionation using Percoll density gradients and Western blot analyses revealed that the bulk of properdin is contained in the secondary granules. Moreover, flow cytometric analyses indicated that properdin is present on the surface of neutrophils. In contrast to alternative pathway components, components of the classical pathway of complement activation, such as C2 and C4, were not detected. Our findings suggest that neutrophils can actively stabilize and amplify the alternative activation pathway of complement by secretion of properdin as part of the innate defense against microorganisms.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Neutrófilos/imunologia , Properdina/metabolismo , Compartimento Celular , Membrana Celular/metabolismo , Quimiotaxia de Leucócito , Fator B do Complemento/metabolismo , Expressão Gênica , Humanos , Inflamação/fisiopatologia , Properdina/genética , RNA Mensageiro/genéticaRESUMO
C1q receptor (C1qR/collectin receptor/cC1qR) has an almost complete amino acid sequence identity with calreticulin (CRT). C1qR/CRT is located on the surface of many cell types. Binding of C1q to C1q receptor elicits a range of immunological responses. C1qR also interacts with the collectins SP-A, MBL, CL43 and conglutinin via a cluster of charged residues on the collagen tails of the ligands. In order to localise C1q and collectin binding activity within C1qR/CRT, recombinant C1qR/CRT domains [N (residues 18-196), P (197-308) and C (309-417)] were produced. Both the N- and P-domains bound to C1q, demonstrating that the binding site spans the intersection of these domains. Amino acid alignment analysis identified a putative CUB module within this region. This S-domain (residues 160-283) was expressed and showed concentration-dependent binding to immobilised C1q, demonstrating that it contains the C1q binding site. Competitive inhibition studies of the S-domain-C1q interaction revealed that the S-domain binds to C1q collagen tails and to the collectin proteins, SP-A, MBL, CL43 and conglutinin. The C1q and collection binding site on C1qR/CRT has therefore been localised to the S-domain.
Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Transporte/química , Complemento C1q/química , Receptores de Hialuronatos , Receptores de Complemento/química , Ribonucleoproteínas/química , Sequência de Aminoácidos , Sítios de Ligação , Ligação Competitiva , Proteínas de Ligação ao Cálcio/genética , Calreticulina , Proteínas de Transporte/genética , Linhagem Celular , Colágeno/química , Colectinas , Complemento C1q/genética , Complemento C1q/isolamento & purificação , Humanos , Radioisótopos do Iodo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Proteínas Mitocondriais , Dados de Sequência Molecular , Receptores de Complemento/genética , Proteínas Recombinantes/química , Ribonucleoproteínas/genética , Alinhamento de SequênciaRESUMO
C1q receptor (C1qR/collectin receptor) is located on many cell types. Binding of C1q to these cells elicits numerous responses. Protein sequencing has shown that C1qR is almost identical to calreticulin (CaR), an abundant multifunctional protein. Radioiodinated C1qR and CaR bind to C1q with identical characteristics. Three recombinant C1qR/CaR domains (N-, C-terminal domains and central P-domain) were expressed using the Thiofusion system, and used to study the interaction with C1q. Both the N- and P-domains were implicated in C1q binding. A region, termed the S-domain, spanning the N and P intersection was expressed, and showed concentration-dependent binding to C1q, demonstrating that the C1q binding site lies within this region.