RESUMO
AIM: In this study, we employed chimeric human/mouse Proteinase 3 (PR3) proteins as tools to induce an autoantibody response to PR3 in rats and mice. METHOD: Rats and mice were immunised with recombinant human PR3 (HPR3), recombinant murine PR3 (mPR3), single chimeric human/mouse PR3 (HHm, HmH, mHH, mmH, mHm, Hmm) or pools of chimeric proteins. Antibodies to mPR3 and HPR3 were measured by ELISA. Antibodies to rat PR3 were determined by indirect immunofluorescence (IIF) on rat white blood cells. Urinalysis was performed by dipstick analysis. Kidney and lung tissue was obtained for pathological examination. RESULTS: In mice, immunisation with the chimeric human/mouse PR3 Hmm led to an autoantibody response to mPR3. Rats immunised with the chimeric human/mouse PR3 Hmm, HmH and mmH, or a pool of the chimeric human/mouse PR3 proteins, produced antibodies selectively binding to rat granulocytes as detected by IIF. No gross pathological abnormalities could be detected in kidney or lungs of mice or rats immunised with chimeric human/mouse PR3. CONCLUSION: Immunisation with chimeric human/mouse proteins induces autoantibodies to PR3 in rats and mice. Chimeric proteins can be instrumental in developing experimental models for autoimmune diseases.
Assuntos
Autoanticorpos/sangue , Quimera , Mieloblastina/administração & dosagem , Animais , Doenças Autoimunes/imunologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Granulócitos/imunologia , Humanos , Imunização , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos WKY , Proteínas Recombinantes/administração & dosagem , Especificidade da EspécieRESUMO
OBJECTIVE: Accumulating data support the role of regulatory T cells, a subset of CD4+ T cells that expresses CD25(high) and the transcription factor forkhead box P3 (FoxP3), in controlling and preventing autoimmunity. In Wegener's granulomatosis (WG), an autoimmune vasculitis, up-regulation of CD25 on circulating CD4+ T cells has been observed, even in patients in remission. The objective of this study was to test whether the frequency and/or function of Treg cells from WG patients in remission are disturbed. METHODS: Peripheral blood mononuclear cells were freshly isolated from 52 WG patients in remission and from 27 age- and sex-matched healthy control subjects. The proportion of circulating Treg cells was assessed by flow cytometry using CD4, CD25, FoxP3, and CD45RO markers. Anergy and suppressive function of CD25(high),CD4+ T cells were determined using polyclonal stimulants and coculture assay in 10 WG patients in remission and in 10 age- and sex-matched healthy controls. RESULTS: In WG patients, a significant increase was observed in the percentage of circulating CD25(high),CD4+ and CD25(low),CD4+ T cells, whereas CD25-,CD4+ T cells were decreased, as compared with healthy controls. Among circulating CD4+ T cells, an expanded percentage of Treg cells (CD25(high),FoxP3+) with memory phenotype was present in WG patients. However, when the suppressive function of CD25(high),CD4+ T cells was tested, CD25(high),CD4+ T cells from WG patients showed diminished or absent suppression of responder T cell proliferation. The impaired suppression was not due to responder cell resistance (as shown by crisscross experiments with T cells from healthy controls) or altered survival of Treg cells. CONCLUSION: These data indicate that WG patients in remission have an expanded proportion of Treg cells that are functionally defective. This observation may be relevant to the development and relapsing course of this autoimmune vasculitis.
Assuntos
Granulomatose com Poliangiite/imunologia , Granulomatose com Poliangiite/fisiopatologia , Linfócitos T Reguladores/patologia , Linfócitos T Reguladores/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Autoimunidade/imunologia , Antígenos CD4/metabolismo , Estudos de Casos e Controles , Proliferação de Células , Feminino , Fatores de Transcrição Forkhead/metabolismo , Granulomatose com Poliangiite/terapia , Humanos , Imunossupressores/uso terapêutico , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Masculino , Pessoa de Meia-Idade , Indução de Remissão , Linfócitos T Reguladores/imunologiaRESUMO
BACKGROUND: Recently, the in vivo pathogenic role of anti-neutrophil cytoplasm autoantibodies (ANCA) in ANCA-associated vasculitis has been challenged by Abdel-Salam et al. In their report, they observed that ANCA directed against proteinase 3 (PR3) cannot bind to their target autoantigen PR3 on circulating neutrophils (PMN). Here we present evidence that human PR3-ANCA do specifically bind to PMN that express PR3 on their membrane. METHODS: PMN were isolated from donors showing bimodal membrane PR3 expression on their PMN (N= 3). TNFalpha-primed PMN or PMA-stimulated PMN were incubated with serum or plasma from PR3-ANCA-positive patients with Wegener's granulomatosis (WG) (N= 8) or healthy controls (N= 8). Binding of IgG in serum or plasma samples to PMN was assessed by indirect immunofluorescence. RESULTS: Binding of IgG in undiluted plasma or serum from PR3-ANCA-positive WG-patients to PMN was significantly increased compared to plasma or serum from healthy controls. Dilution of plasma and serum showed concentration-dependent binding of IgG. Double staining for PR3 and IgG demonstrated that IgG in plasma or serum from PR3-ANCA-positive patients only bound to those PMN that expressed PR3, and not to PMN that lacked PR3 expression on their membrane. CONCLUSION: PR3-ANCA in undiluted serum or plasma from PR3-ANCA-positive WG patients bind to TNFalpha- primed and PMA-stimulated PMN that express PR3 on their membrane. Therefore, the hypothesis that PR3-ANCA can bind and activate primed PMN is still the most attractive explanation for the contribution of PR3-ANCA to the pathogenesis of Wegener's granulomatosis.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Granulomatose com Poliangiite/imunologia , Neutrófilos/imunologia , Serina Endopeptidases/imunologia , Anticorpos Anticitoplasma de Neutrófilos/sangue , Especificidade de Anticorpos , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Mieloblastina , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Ligação Proteica/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
We have previously reported diminished immunohistochemical staining of decorin in lung tissue from patients with severe emphysema. The aim of this study is to investigate whether this diminished staining is due to a quantitative abnormal production of decorin by pulmonary fibroblasts in vitro. Therefore, we measured decorin (Western blot), collagen type I (ELISA), and fibronectin (ELISA) production by fibroblasts obtained from lung tissue of patients with severe and mild emphysema at basal culture conditions and after modulation with transforming growth factor-beta1, basic fibroblast growth factor, and interferon-gamma. Decorin production at basal culture conditions was significantly higher in fibroblast cultures from patients with severe emphysema compared to fibroblasts from mild emphysema. After stimulation with transforming growth factor-beta1 and basic fibroblast growth factor, decorin production was significantly more reduced in fibroblast cultures from patients with severe emphysema whereas collagen type I and fibronectin production were not affected. We conclude that decorin production by lung fibroblasts of patients with severe emphysema is dysregulated after modulation with cytokines known to be important in smoking associated inflammation. This dysregulation of decorin production may contribute to the impaired lung tissue repair, present in patients with emphysema, since these alterations in the extracellular matrix may cause diminished cytokine binding and neutralization.
Assuntos
Proteínas da Matriz Extracelular/biossíntese , Fibroblastos/metabolismo , Proteoglicanas/biossíntese , Enfisema Pulmonar/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Decorina , Feminino , Humanos , Pulmão/citologia , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de DoençaRESUMO
The presence of antineutrophil cytoplasmic autoantibodies with specificity for proteinase 3 (PR3-ANCA) usually is detected by enzyme-linked immunosorbent assay (ELISA) with purified PR3 as a substrate. We studied the technical performance of direct and capture ELISA using a recombinant proteolytically inactive form of PR3 produced in the baculovirus expression system for the detection of PR3-ANCA in 114 patients with systemic vasculitis at diagnosis. We found that ELISA using recombinant PR3 produced in insect cells is a promising alternative for ELISA with native PR3. We found a correlation between tests using recombinant or native PR3, as well as correlation of the ELISA results with ANCA titers measured by the indirect immunofluorescence technique. However, the specificity for ANCA-associated vasculitis of ELISA with recombinant PR3 was lower than ELISA using native PR3. Compared with the direct assay, capture ELISA is a more sensitive method for PR3-ANCA detection, with both native and recombinant PR3, and its results depend on the monoclonal antibody used to capture the antigen.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas Recombinantes/imunologia , Serina Endopeptidases/imunologia , Vasculite/imunologia , Animais , Anticorpos Monoclonais , Células Cultivadas , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Vetores Genéticos , Humanos , Masculino , Mieloblastina , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , Serina Endopeptidases/metabolismo , Spodoptera/genética , Spodoptera/metabolismo , Vasculite/diagnóstico , Vasculite/enzimologia , Proteínas Virais/imunologia , Proteínas Virais/metabolismoRESUMO
Wegener's granulomatosis is a systemic disease characterized by the presence of antineutrophil cytoplasm autoantibodies specific for proteinase 3 (PR3-ANCA). The functional characteristics of PR3-ANCA differ between quiescent and active disease, suggesting changes in the properties of the autoantibodies in time. Using biosensor technology, we found that PR3-ANCA of different patients (n = 8) recognize a limited number of overlapping regions on PR3 at the time of diagnosis of Wegener's granulomatosis. This area might cover an immunodominant epitope, common for PR3-ANCA from all patients, irrespective of the size of the total area recognized by an individual autoantibody. Experiments with sera (n = 4) collected at the moment of diagnosis and at the time of relapse showed that the individual epitope specificities of PR3-ANCA change during the course of the disease. These changes in epitope specificity of PR3-ANCA may be responsible for the differences in functional properties of these autoantibodies between various stages of the disease.