RESUMO
Anti-neutrophil cytoplasmic antibodies (ANCA) appear to play an important role in the pathogenesis of ANCA-associated vasculitis (AAV). However, ANCA alone are not sufficient to generate disease, and some evidence suggests that infectious triggers may serve as inciting events for AAV disease activity. Antibodies of the immunoglobulin (Ig)M isotype often serve as markers of recent infection, and IgM ANCA have been identified previously in patients with AAV, although the frequency and clinical relevance of IgM ANCA is not well established. We sought to characterize IgM ANCA more clearly by creating a novel enzyme-linked immunosorbent assay (ELISA) for IgM antibodies to proteinase 3 [IgM proteinase 3 (PR3)-ANCA], which we applied to two large, clinically well-characterized trial cohorts of patients with granulomatosis with polyangiitis and microscopic polyangiitis. In the first cohort, IgM PR3-ANCA occurred with a frequency of 15·0%, and were associated with a higher degree of disease severity and a trend towards a higher rate of alveolar haemorrhage (29·6 versus 15·7%, P = 0·10). Analysis of follow-up samples in this cohort showed that the presence of IgM PR3-ANCA was transient, but could recur. In the second cohort, IgM PR3-ANCA occurred with a frequency of 41·1%, and were also associated with a higher degree of disease severity. A higher rate of alveolar haemorrhage was observed among those with IgM PR3-ANCA (45·3 versus 15·8%; P < 0·001). The association of transient IgM PR3-ANCA with an acute respiratory manifestation of AAV suggests a possible link between an infectious trigger and AAV disease activity.
Assuntos
Autoanticorpos/imunologia , Granulomatose com Poliangiite/imunologia , Imunoglobulina M/imunologia , Poliangiite Microscópica/imunologia , Mieloblastina/imunologia , Adulto , Idoso , Anticorpos Anticitoplasma de Neutrófilos/imunologia , Biomarcadores , Feminino , Granulomatose com Poliangiite/diagnóstico , Humanos , Imunoglobulina G/imunologia , Masculino , Poliangiite Microscópica/diagnóstico , Pessoa de Meia-Idade , Índice de Gravidade de DoençaRESUMO
We present the first evidence that adhesion mediated by a member of the cadherin gene family can be regulated by a G protein-coupled receptor. We show that activating the M3 muscarinic acetylcholine receptor (mAChR) rapidly induces E-cadherin-mediated adhesion in a small cell lung carcinoma (SCLC) cell line. This response is inhibited by E-cadherin antibodies, and does not occur in another SCLC cell line which expresses functional mAChR but reduced levels of E-cadherin. Protein kinase C may be involved, since phorbol 12-myristate 13-acetate also induces E-cadherin-mediated aggregation. Immunofluorescence analyses indicate that mAChR activation does not grossly alter E-cadherin surface expression or localization at areas of cell-cell contact, suggesting mAChR activation may increase E-cadherin binding activity. Our findings suggest that G protein-coupled receptors may regulate processes involving cadherin-mediated adhesion, such as embryonic development, neurogenesis, and cancer metastasis.
Assuntos
Caderinas/metabolismo , Carcinoma de Células Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores Muscarínicos/fisiologia , Adesão Celular/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Humanos , Metástase Neoplásica , Ésteres de Forbol/farmacologia , Proteína Quinase C/metabolismo , Receptores Muscarínicos/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
OBJECTIVE: The glycosylation status of autoantigens appears to be crucial for the pathogenesis of some autoimmune diseases, since carbohydrates play a crucial role in the distinction of self from non-self. Proteinase 3 (PR3), the main target antigen for anti-neutrophil cytoplasmic antibodies (ANCA) in patients with Wegener's granulomatosis (WG), contains two Asn-linked glycosylation sites. The present study explores the influence of the glycosylation status of PR3 on the PR3 recognition by ANCA in a well characterized population of patients with WG. METHODS: Forty-four patients with WG (459 serum samples) who participated in a multicenter randomized trial, were tested by capture ELISA for ANCA against PR3 and deglycosylated recombinant variants of PR3. RESULTS: The patients were followed for a median of 27 months, and the median number of serum samples per patient was 10. At baseline, the correlation between the levels of ANCA against PR3 and against all the deglycosylated recombinant variants of PR3 were greater than 0.94 (?<0.001 for all the comparisons). Longitudinal analyses comparing the levels of ANCA against PR3 versus all the deglycosylated recombinant variants of PR3, using linear mixed models, showed no significant statistical differences (rho >or=0.90 in all cases). CONCLUSION: The glycosylation status of PR3 has no impact on its recognition by ANCA in WG.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Granulomatose com Poliangiite/imunologia , Mieloblastina/imunologia , Adulto , Anticorpos Anticitoplasma de Neutrófilos/metabolismo , Reações Antígeno-Anticorpo , Linhagem Celular Transformada , Feminino , Glicosilação , Granulomatose com Poliangiite/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Mieloblastina/metabolismoRESUMO
Anti-neutrophil cytoplasmic autoantibodies recognizing conformational epitopes (c-ANCA) of proteinase 3 (PR3) from azurophil granules are a diagnostic hallmark in Wegener's granulomatosis (WG). Because a functional PR3 homologue has not been identified in rodents, it is difficult to assess immunopathological responses in rats or mice immunized with patients' derived c-ANCA or human PR3. Here we report the full length cDNA cloning and functional expression of murine PR3 in HMC-1 cells. Recombinant murine PR3 shows highly similar substrate specificities towards synthetic peptides and is inhibited by human alpha1-proteinase inhibitor like human PR3. However, neither human c-ANCA, rabbit sera nor mouse monoclonal antibodies to human PR3 recognize the murine homologue. Consequently, it is unlikely that disease observed in mice after immunization with c-ANCA or human PR3 is caused by pathogenic antibodies directed against mouse PR3. Recombinant human-mouse chimaeric variants will be a valuable new tool to localize the disease-specific immunodominant epitopes in human PR3.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Serina Endopeptidases/imunologia , Vasculite/imunologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Modelos Animais de Doenças , Epitopos/química , Epitopos/imunologia , Humanos , Mastócitos/enzimologia , Camundongos , Dados de Sequência Molecular , Mieloblastina , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , alfa 1-Antitripsina/farmacologiaRESUMO
We developed a stable expression system for conformationally intact recombinant human PR3 (rPR3) using the human mast cell line HMC-1. Like in U937 cells, the rPR3 is processed from a 34 kDa precursor to the 29 kDa mature form, primarily as the result of oligosaccharide trimming. The rPR3 binds [3H]DFP and hydrolyzes the substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-pNA. The enzymatic activity is inhibited by greater than 95% by alpha 1-PI. The rPR3 and the enzymatically inactive mutant rPR3-S176A are both packaged in granules. Thus, proteolytic autoprocessing is not required for PR3's targeting to granules. This rPR3 is the first to be recognized by most c-ANCA from WG patients and all anti-PR3 ANCA that were detected by standard anti-PR3 specific ELISA. This expression system for rPR3 represents a versatile tool for the analysis of its intracellular processing, structure-function relationships and interaction with autoantibodies.
Assuntos
Autoanticorpos/imunologia , Mastócitos/enzimologia , Serina Endopeptidases/imunologia , Serina Endopeptidases/metabolismo , Anticorpos Anticitoplasma de Neutrófilos , Sequência de Bases , Linhagem Celular , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Granulomatose com Poliangiite , Humanos , Hidrólise , Isoflurofato/metabolismo , Mastócitos/metabolismo , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Dados de Sequência Molecular , Mieloblastina , Oligopeptídeos/metabolismo , Fenótipo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , TransfecçãoRESUMO
Serum and plasma are used interchangeably to measure anti-neutrophil cytoplasmic antibodies (ANCA), even though the release of ANCA target antigens during the preparation of serum could affect ANCA assays and cause discrepancies between the results obtained from serum and plasma. To what extent ANCA test results obtained from serum agree and correlate with results from plasma remains unknown. Therefore, a comprehensive comparison was performed using serum and plasma samples which were collected in 175 patients with active Wegener's granulomatosis at enrollment of a recent randomized trial. These paired serum and plasma samples were subjected to parallel ANCA testing by standard indirect immunofluorescence on ethanol-fixed neutrophils, a direct enzyme-linked immunoassay (ELISA) for proteinase 3 (PR3)-ANCA and myeloperoxidase (MPO)-ANCA, and two different capture ELISAs for PR3-ANCA. The concordance of categorical serum and plasma ANCA results was assessed using kappa-coefficients. These were > 0.8 for all assays, indicating a very good concordance between positive and negative serum and plasma results. Spearman's correlation coefficients for serum and plasma PR3-ANCA values obtained by direct ELISA and both capture ELISAs were > or = 0.95 (P < 0.0001). Our study shows that serum and plasma samples can be used interchangeably for measuring ANCA.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/sangue , Granulomatose com Poliangiite/diagnóstico , Plasma/imunologia , Soro/imunologia , Biomarcadores/sangue , Coleta de Amostras Sanguíneas/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Técnica Indireta de Fluorescência para Anticorpo/métodos , Humanos , Reprodutibilidade dos TestesRESUMO
ANCA directed against PR3 are highly specific for Wegener's granulomatosis and microscopic polyangiitis, and have been implicated in the pathogenesis of small vessel vasculitis. Most PR3-ANCA are directed against conformational epitopes on PR3. This study was designed to determine whether the cleavage of the N-terminal activation dipeptide of PR3 is required for the binding of PR3-ANCA. Recombinant PR3 (rPR3) variants were expressed in the epithelial cell line, 293. As confirmed by radiosequencing, the rPR3 secreted into the 293 cell culture supernatant is N-terminally unprocessed. Two enzymatically inactive rPR3 mutants were expressed in 293 cells: rPR3-S176A and delta-rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, delta-rPR3-S176A does not. Culture supernatants of rPR3-S176A and delta-rPR3-S176A expressing 293 cells were used as sources of target antigen for PR3-ANCA testing by capture ELISA. Forty unselected consecutive PR3-ANCA+ sera were tested. With delta-rPR3-S176A as antigen all 40 were recognized, compared with only 34 of 40 when rPR3-S176A served as target antigen. The majority of the serum samples contained a mixture of antibodies reacting with epitopes accessible on the mature and on the proform of PR3. In conclusion, the cleavage of the N-terminal activation dipeptide of PR3 is not an absolute requirement for recognition by all PR3-ANCA. However, a substantial proportion of PR3-ANCA recognize (a) target antigen(s) exposed only after the conformational change of PR3 associated with the N-terminal processing. In 15% of sera this PR3-ANCA subset occurred exclusively. PR3-ANCA subtypes can be differentiated using specifically designed rPR3 variants as target antigens, and non-haematopoietic mammalian cells without regulated secretory pathway can be used for their expression.