RESUMO
To develop an animal model for antimyeloperoxidase (MPO)-associated necrotizing crescentic glomerulonephritis (NCGN), we immunized Brown Norway rats with MPO and localized a neutrophil lysosomal enzyme extract, primarily consisting of MPO and elastinolytic enzymes, plus H2O2, the substrate of MPO, to the glomerular basement membrane (GBM). Upon immunization rats developed antibodies and positive skin tests to MPO. After unilateral perfusion of the left kidney with the lysosomal enzyme extract and H2O2, MPO and immunoglobulin (Ig)G localized transiently along the GMB. At the time of maximal inflammation, at 4 and 10 d after perfusion, MPO, IgG, and C3 could not be detected anymore. MPO-immunized rats perfused with the lysosomal enzyme extract and H2O2, in contrast to control-immunized and/or control-perfused rats, developed a proliferative GN characterized by intra- and extracapillary cell proliferation, ruptured Bowman's capsule, periglomerular granulomatous inflammation, and formation of giant cells. Monocytes, polymorphonuclear leukocytes (PMN), and to a far lesser extent T cells were found in the glomeruli. Interstitial infiltrates consisted of monocytes, PMN, and T cells. Granulomatous vasculitis of small vessels was found at 10 d after perfusion. The proliferative NCGN in this rat model closely resembles human anti-MPO-associated pauci-immune NCGN, and enables the study of the pathophysiology of anti-MPO-associated NCGN.
Assuntos
Modelos Animais de Doenças , Glomerulonefrite/enzimologia , Peroxidase/metabolismo , Animais , Anticorpos/imunologia , Membrana Basal/imunologia , Imunofluorescência , Glomerulonefrite/imunologia , Humanos , Peróxido de Hidrogênio/metabolismo , Imunofenotipagem , Rim/enzimologia , Rim/imunologia , Rim/metabolismo , Microscopia Eletrônica , Perfusão , Peroxidase/imunologia , Testes de Precipitina , Ratos , Linfócitos T/imunologiaRESUMO
Human CD4+FoxP3+T-cells are heterogeneous in function and include not only suppressive cells (Tregs), but also effector cells that transiently express FoxP3 upon activation. Previous studies in Granulomatosis with Polyangiitis (GPA-)patients have demonstrated an increase in FoxP3+T-cells with impaired suppressive capacity and an increase in Th17 cells. We hypothesized that the increase in FoxP3+T-cells results from an increase in non-suppressive effector-like cells. The frequency of circulating CD4+FoxP3+T-cell subsets were determined by flow cytometry in 46 GPA-patients in remission and 22 matched healthy controls (HCs). Expression levels of FoxP3 and CD45RO were used to distinguish between CD45RO- FoxP3low resting Tregs (rTreg), CD45RO+FoxP3high activated Tregs (aTreg) and CD45RO+FoxP3low proinflammatory non-suppressive T-cells (nonTreg). Intracellular expression of IFNγ, IL-17, and IL-21 was compared within these subsets. We found a significant increase in the frequency of nonTreg cells in GPA-patients as compared with HCs. Importantly, within the nonTreg subset, antineutrophil cytoplasmic autoantibody (ANCA-)positive patients demonstrated a significantly higher percentage of IL-17+ and IL-21+ cells when compared with ANCA-negative patients and HCs. Moreover, expanded nonTregs from ANCA-positive patients induced excessive proliferation of responder cells in vitro and exhibited higher IL-21 production. Production of IL-17 and IL-21 in non-suppressive FoxP3+T-cells may point toward a pathogenic role in ANCA formation.
Assuntos
Fatores de Transcrição Forkhead/genética , Granulomatose com Poliangiite/genética , Memória Imunológica/genética , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Anticorpos Anticitoplasma de Neutrófilos , Linfócitos T CD4-Positivos/imunologia , Feminino , Citometria de Fluxo , Granulomatose com Poliangiite/imunologia , Granulomatose com Poliangiite/patologia , Humanos , Memória Imunológica/imunologia , Interferon gama/genética , Interleucina-17/genética , Interleucinas/genética , Antígenos Comuns de Leucócito/genética , Masculino , Pessoa de Meia-Idade , Células Th17/imunologiaRESUMO
Buruli ulcer disease (BUD) is an emerging predominantly tropical disease caused by Mycobacterium ulcerans. The initial pre-ulcerative skin lesion often breaks down into an ulcer with undermined edges. Healing is common but may require considerable time, and scarring often results in functional limitations. Considerable evidence has now emerged that patients with early BUD cannot mount a sufficient protective T helper 1 (Th1) cell response to M. ulcerans, but uncertainty remains as to whether immune protection is restored over time. This study investigates the Th1 cell response of patients with various stages of BUD on mycobacterial antigens. We measured interferon (IFN)-gamma levels after ex vivo whole blood stimulation with tuberculin purified protein derivative (PPD), and compared the Th1 cell response of individuals with pre-ulcerative, ulcerative and healed BUD as well as healthy controls. Moreover, the systemic Th1 cell response was related to histopathological features in the various stages of surgically resected BUD lesions. We show that patients with ulcerative and healed BUD produce significantly higher IFN-gamma levels after mycobacterial ex vivo whole blood stimulation than healthy controls, and that patients with a granulomatous tissue response produce higher IFN-gamma levels than individuals without. We therefore suggest that the mounted Th1 cell response in ulcerative BUD patients might be related to their histopathological tissue response.
Assuntos
Úlcera de Buruli/imunologia , Interferon gama/biossíntese , Adolescente , Adulto , Antígenos de Bactérias/imunologia , Úlcera de Buruli/patologia , Células Cultivadas , Criança , Progressão da Doença , Feminino , Granuloma/imunologia , Granuloma/patologia , Humanos , Interleucina-10/biossíntese , Masculino , Fito-Hemaglutininas/imunologia , Células Th1/imunologia , Tuberculina/imunologia , Cicatrização/imunologiaRESUMO
Proteinase 3 (PR3) is the major autoantigen for anti-neutrophil cytoplasmic antibodies (ANCA) in patients with Wegener's granulomatosis. Little is known about the major antigenic sites on PR3. To facilitate epitope mapping, PR3 was cloned in insect cells using a baculovirus expression system. Four different sequences of the PR3 cDNA were amplified by PCR: two clones containing the pro-peptide of PR3 with or without a His-tag (rproPR3-his and rproPR3, respectively) and two clones without the pro-peptide and with or without a His-tag (rPR3-his and rPR3, respectively). The PR3 sequences were cloned behind the polyhedrin promoter and the honeybee melittin signal peptide enabling secretion of rPR3. Plasmids were transposed into the genome of baculovirus, and wild types as well as PR3-containing virus genomes were transfected into Sf21 insect cells. All four rPR3 variants were secreted into the medium and were recognized by anti-neutrophil PR3 rabbit serum and by at least two anti-PR3 monoclonal antibodies. Mature forms of PR3 were recognized by almost all patient sera, whereas the pro-forms of PR3 were recognized by 14 of 18 PR3-ANCA sera tested. On SDS-PAGE, the four rPR3 forms migrated at approximately 32 kDa. RPR3-his and rproPR3-his could be purified by means of this His-tag. In conclusion, especially the mature rPR3s are well recognized by PR3-ANCA sera. The presence of a C-terminal His-tag facilitated purification of His-tagged rPR3. Thus, rPR3 expressed in insect cells can be used as a tool for diagnostic tests as well as for epitope mapping studies.
Assuntos
Autoantígenos/biossíntese , Autoantígenos/genética , Granulomatose com Poliangiite/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Spodoptera/genética , Animais , Autoantígenos/imunologia , Autoantígenos/isolamento & purificação , Baculoviridae/genética , Sítios de Ligação de Anticorpos/genética , Western Blotting , Linhagem Celular , Células Clonais , Clonagem Molecular/métodos , Ensaio de Imunoadsorção Enzimática , Histidina/genética , Humanos , Soros Imunes/metabolismo , Mieloblastina , Fragmentos de Peptídeos/análise , Precursores de Proteínas/análise , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , Precursores de Proteínas/isolamento & purificação , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de Proteína , Serina Endopeptidases/imunologia , Serina Endopeptidases/isolamento & purificação , Spodoptera/virologia , Fatores de TempoRESUMO
PURPOSE: Assessment of the value of determination of antineutrophil cytoplasmic antibodies (ANCA) and its specificities for classification of patients with biopsy-proven necrotizing arteritis. PATIENTS AND METHODS: The serum samples of 28 consecutive patients with biopsy-proven vasculitis involving medium- and/or small-sized arteries were tested for ANCA by an indirect immunofluorescence technique, by neutrophil extract enzyme-linked immunosorbent assay (ELISA), and by catching ELISA. RESULTS: Eight patients had Churg-Strauss syndrome; six had myeloperoxidase (MPO) antibodies, and in the other two patients, ANCA were not detected. Six patients had polyarteritis nodosa (PAN) limited to the skin and the musculoskeletal system; ANCA were not detected in these patients. Two patients had systemic PAN and both had MPO antibodies. The remaining 12 patients had overlapping clinical features of the different forms of vasculitis. Five patients had polyarteritis in combination with chronic nasal inflammation and glomerulonephritis compatible with Wegener's granulomatosis (WG) but without granulomas in the respiratory tract. All five patients had 29-kd serine protease antibodies. Two patients had polyarteritis in combination with nasal polyposis and asthma compatible with Churg-Strauss syndrome, but eosinophilia was not detected. Both patients had MPO antibodies. Three patients with unclassified granulomatous arteritis had either elastase antibodies or ANCA of unknown specificity. One patient with unclassified systemic vasculitis had 29-kd serine protease antibodies, and one patient with necrotizing arteritis of the bowel in combination with Schönlein-Henoch purpura was negative for ANCA. CONCLUSION: Determination of ANCA and its specificities is a useful adjunct to the classification of patients with biopsy-proven necrotizing arteritis. Within the spectrum of idiopathic vasculitides, 29-kd serine protease antibodies are associated with WG, MPO antibodies are associated with Churg-Strauss syndrome and systemic PAN, and PAN limited to the skin and the musculoskeletal system is not associated with ANCA.
Assuntos
Autoanticorpos/análise , Biomarcadores/química , Imunoglobulina G/análise , Neutrófilos/imunologia , Poliarterite Nodosa/imunologia , Adulto , Idoso , Anticorpos Anticitoplasma de Neutrófilos , Artérias , Biópsia , Síndrome de Churg-Strauss/imunologia , Síndrome de Churg-Strauss/patologia , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Granulomatose com Poliangiite/imunologia , Granulomatose com Poliangiite/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Poliarterite Nodosa/patologia , Vasculite/imunologia , Vasculite/patologiaRESUMO
Levels of soluble IL-2 receptor (sIL-2R), soluble CD4 (sCD4) and CD8 (sCD8) were measured by sandwich ELISA as markers for T-cell activation in serial serum samples drawn monthly from 16 patients showing 18 histologically proven relapses of Wegener's granulomatosis (WG). Levels of sIL-2R increased from 1162 U/ml (median, 95% CI 843 to 1814 U/ml) at three months before the relapse to 1684 U/ml (95% CI 1254 to 2202 U/ml) at the time of relapse for the whole group (P = 0.10). The 8 major relapses showed a profound rise in sIL-2R levels (P < 0.01). The level of sIL-2R at the moment of relapse correlated with the level of C-reactive protein (r = 0.547, P < 0.05) and with the disease activity score (r = 0.814, P < 0.001). There were no significant changes in levels of sCD4 or sCD8.
Assuntos
Granulomatose com Poliangiite/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Anticorpos Anticitoplasma de Neutrófilos , Autoanticorpos/sangue , Antígenos CD4/sangue , Antígenos CD8/sangue , Granulomatose com Poliangiite/sangue , Humanos , Imunoglobulina G/sangue , Receptores de Interleucina-2/metabolismo , RecidivaRESUMO
Anti-MPO and anti-proteinase 3 antibodies are strongly associated with certain forms of vasculitis and glomerulonephritis and a pathophysiological role for the antibodies has been hypothesized (Kallenberg et al. 1991). To test this hypothesis WKY, Lewis and BN rats were immunized with human MPO. BN rats developed a strong humoral response, cross reacting with autologous MPO, and Lewis rats a strong cellular response to human MPO.
Assuntos
Peroxidase/imunologia , Animais , Anticorpos/sangue , Formação de Anticorpos , Humanos , Imunidade Celular , Imunização , Linfócitos/imunologia , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Ratos Endogâmicos WKYRESUMO
OBJECTIVE: Wegener's granulomatosis (WG) is strongly associated with antineutrophil cytoplasmic autoantibodies (ANCAs) directed against proteinase 3 (PR3). Recent studies have shown that membrane-bound PR3 (mPR3) is differentially expressed and colocalizes with CD177/NB1 on circulating neutrophils. We undertook this study to assess the differential expression of CD177 on neutrophils from patients with ANCA-associated systemic vasculitis (ASV) in comparison with patients with systemic lupus erythematosus (SLE), patients with rheumatoid arthritis (RA), and healthy individuals, and to investigate whether colocalization of mPR3 and CD177 affects anti-PR3-mediated neutrophil activation. METHODS: Expression of CD177 and mPR3 was analyzed by flow cytometry on isolated neutrophils from patients with ASV (n=53), those with SLE (n=30), those with RA (n=26), and healthy controls (n=31). Neutrophil activation mediated by anti-PR3 antibodies was assessed by measuring the oxidative burst with a dihydrorhodamine assay. RESULTS: Percentages of CD177-expressing neutrophils were significantly higher in patients with ASV and those with SLE than in healthy controls. In 3 healthy donors, CD177 expression was not detected. After priming with tumor necrosis factor alpha, neutrophils remained negative for CD177 while mPR3 expression was induced. Neutrophils from CD177-negative donors or CD177- neutrophils sorted from donors with bimodal expression were susceptible to anti-PR3-mediated oxidative burst. Variation in the extent of anti-PR3-mediated neutrophil activation among different donors occurred independent of the percentage of CD177-expressing neutrophils. CONCLUSION: Membrane expression of CD177 on circulating neutrophils is increased in patients with ASV and in those with SLE, but not in RA patients. However, primed neutrophils from CD177-negative individuals also express mPR3 and are susceptible to anti-PR3-mediated oxidative burst, suggesting that recruitment of CD177-independent mPR3 is involved in anti-PR3-induced neutrophil activation.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/análise , Isoantígenos/análise , Glicoproteínas de Membrana/análise , Mieloblastina/análise , Ativação de Neutrófilo/fisiologia , Neutrófilos/química , Neutrófilos/imunologia , Receptores de Superfície Celular/análise , Vasculite/imunologia , Anticorpos Anticitoplasma de Neutrófilos/imunologia , Artrite Reumatoide/imunologia , Membrana Celular/enzimologia , Citometria de Fluxo , Proteínas Ligadas por GPI , Granulomatose com Poliangiite/imunologia , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Mieloblastina/imunologia , Mieloblastina/fisiologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Active SLE is characterized by immune deposits and subsequent vascular inflammation in many organs. Expression and up-regulation of adhesion molecules is basic to migration of inflammatory cells into the tissues. Recently, soluble isoforms of these molecules have been described which might be an expression of their up-regulation in the tissues and, as such, of disease activity. The purpose of this study was to evaluate whether changes in levels of soluble adhesion molecules reflect disease activity. We analysed serial sera in a 6-month period preceding 22 consecutive exacerbations of SLE for levels of soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1), and sE-selectin. Levels were related to clinical disease activity (SLEDAI), and levels of anti-dsDNA and complement. At the time of maximal disease activity, levels of sVCAM-1 in patients with SLE were higher than those in controls (P < 0.0001), levels in patients with renal involvement being higher than in those without (P < 0.02). Levels of sVCAM-1 correlated with SLEDAI scores (P < 0.05) and, inversely, with levels of C3 (P = 0.01). In addition, in the presence of anti-dsDNA, levels of sVCAM-1 tended to correlate with levels of these autoantibodies (P < 0.1). Levels of sICAM-1 were normal and sE-selectin levels even decreased compared with controls. Levels of sVCAM-1 were higher at the moment of relapse (P = 0.001) than at 6 months before this time point. This rise correlated with the rise in SLEDAI score (P < 0.02). Levels of sICAM-1 and sE-selectin did not rise, and remained in the normal range in all exacerbations studied. In conclusion, in contrast to sICAM-1 and sE-selectin, levels of sVCAM-1 are increased, rise parallel to disease activity during exacerbations in SLE, and are associated with decreasing levels of complement factors. This favours the hypothesis of immune deposit formation, activation of the complement cascade and activation of endothelial cells. Concurrent up-regulation of vascular adhesion molecules may thus result in transmigration of activated inflammatory cells inducing tissue damage.
Assuntos
Moléculas de Adesão Celular/sangue , Lúpus Eritematoso Sistêmico/imunologia , Adulto , Anticorpos Antinucleares/análise , Autoanticorpos/imunologia , Biomarcadores , Proteínas do Sistema Complemento/análise , DNA/imunologia , Selectina E , Feminino , Humanos , Molécula 1 de Adesão Intercelular , Estudos Longitudinais , Lúpus Eritematoso Sistêmico/patologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Solubilidade , Regulação para Cima , Molécula 1 de Adesão de Célula VascularRESUMO
T cell-mediated immunity is hypothesized to play an important role in the pathogenesis of granulomatous inflammation and vasculitis as found in patients with WG. The antigenic specificities of those T cells remain, however, unknown. Anti-neutrophil cytoplasmic antibodies (ANCA) present in patients with WG are directed to proteinase 3 (PR3) and myeloperoxidase (MPO). In the present study we investigated the proliferative capacity of peripheral blood mononuclear cells (PBMC) from patients with WG and age- and sex-matched controls in response to the WG autoantigens PR3 and MPO. Possible mitogenic effects of active PR3 and toxic effects of active MPO were excluded by using heat-inactivated PR3 and MPO. Antigen-specific stimulation induced by these autoantigens was studied by using processed PR3 and MPO in the lymphocyte stimulation test (LST). Proliferation induced by processed antigen correlated with that by heat-inactivated free antigen. The general capacity to proliferate in response to mitogens and recall antigens did not differ between patients and controls. However, patients with WG who were or had been positive for PR3-ANCA (n = 17) responded more strongly to PR3 than to MPO and showed higher responses to PR3 compared with controls (n = 13). Within the PR3-ANCA group T cell proliferation did not correlate with ANCA titre. In a small group of patients with MPO-ANCA (n = 5) no differences were observed compared with controls for MPO-specific proliferation. The data presented demonstrate that autoreactive PR3-specific T cells are present in patients with WG. Their fine specificity and possible role in the pathogenesis of WG have to be defined in further studies.
Assuntos
Autoanticorpos/farmacologia , Granulomatose com Poliangiite/imunologia , Peroxidase/farmacologia , Serina Endopeptidases/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Anticitoplasma de Neutrófilos , Especificidade de Anticorpos , Células Apresentadoras de Antígenos/enzimologia , Células Apresentadoras de Antígenos/imunologia , Antígenos/farmacologia , Autoanticorpos/análise , Autoanticorpos/biossíntese , Feminino , Granulomatose com Poliangiite/sangue , Granulomatose com Poliangiite/enzimologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Mitógenos/farmacologia , Mieloblastina , Peroxidase/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Serina Endopeptidases/imunologia , Estimulação Química , Linfócitos T/enzimologiaRESUMO
The occurrence of focal fibrinoid necrosis of capillary loops in the very early stages of ANCA-associated necrotizing crescentic glomerulonephritis (NCGN) and the increased prevalence of this disease at older age suggest that renal ischemia may play an additional role in its pathophysiology. In the present study we investigated the contribution of renal ischemia to the induction of anti-myeloperoxidase (MPO) associated NCGN in a previously described rat model of this disease. The development of renal lesions is dependent on the presence of an anti-MPO immune response and the localization of a lysosomal extract containing lytic enzymes and MPO in combination with hydrogen peroxide (H2O2) along the glomerular basement membrane (GBM). The hypothesis tested whether perfusion of hydrogen peroxide (H2O2) could be replaced by ischemia/reperfusion (I/R) injury, as I/R injury activates endothelial cells to produce oxygen metabolites. I/R was induced by clamping the renal artery for 20 minutes in kidneys in which the circulation had been restored several minutes after perfusion with the lysosomal extract in MPO immunized rats. Rats developed lesions characterized by intra- and extracapillary cell proliferation, periglomerular infiltration, ruptures in Bowman's capsule, ischemic tubuli, and interstitial mononuclear infiltrate. Immune deposits, however, persisted for a longer time along the GBM after perfusion of lytic enzymes followed by I/R injury compared to previous studies in which H2O2 in conjunction with lytic enzymes were perfused in MPO-immunized rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Complemento C3/metabolismo , Glomerulonefrite Membranoproliferativa/etiologia , Imunoglobulina G/metabolismo , Traumatismo por Reperfusão/complicações , Animais , Glomerulonefrite Membranoproliferativa/metabolismo , Glomerulonefrite Membranoproliferativa/patologia , Imunização , Imunoglobulina G/imunologia , Imunofenotipagem , Molécula 1 de Adesão Intercelular/metabolismo , Peroxidase/administração & dosagem , Peroxidase/imunologia , Peroxidase/metabolismo , Ratos , Ratos Endogâmicos BN , Traumatismo por Reperfusão/metabolismo , Aderências Teciduais , Regulação para CimaRESUMO
Levels of soluble IL-2 receptor (sIL-2R), soluble CD4 (sCD4) and CD8 (sCD8) were measured by sandwich ELISA as markers for T cell activation in serial serum samples from 16 patients showing 18 histologically proven relapses of Wegener's granulomatosis (WG). Levels of sIL-2R increased from 1065 U/ml (median, range 373-2345 U/ml) 6 months before the relapse to 1684 U/ml (median, range 486-3404 U/ml) at the moment of relapse for the whole group (P = 0.10). The eight major relapses showed a profound rise in sIL-2R levels, from 1008 U/ml (median, range 686-1553 U/ml) 6 months before the relapse, to 1994 U/ml (median, range 1469-3404 U/ml) at the moment of relapse (P < 0.01). The levels of sIL-2R at the moment of relapse were significantly higher at the eight major relapses than at the time of the 10 minor relapses (P < 0.05). Minor relapses were not accompanied by a significant rise in sIL-2R levels. Titres of antineutrophil cytoplasmic antibodies (ANCA) rose by two or more titresteps or from negative to positive in 15/18 patients during the 6 months period before the relapse. In all seven cases with both a rise of the ANCA titre and an at least 25% increase in sIL-2R levels, the rise in ANCA preceded the rise in sIL-2R by at least 1 month. The level of sIL-2R at the moment of relapse correlated with the level of C-reactive protein (r = 0.488, P < 0.05) and with the disease activity score (r = 0.824, P < 0.002). There were no significant changes in levels of sCD4 or sCD8, although the levels of sCD4 tended to be higher at the time of major relapses. We conclude that major relapses of Wegener's granulomatosis are accompanied by systemic T cell activation. T cell activation, however, does not appear to precede the rise in ANCA titre.
Assuntos
Granulomatose com Poliangiite/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Anticorpos Anticitoplasma de Neutrófilos , Autoanticorpos/análise , Biomarcadores , Antígenos CD4/análise , Antígenos CD8/análise , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/análise , Receptores de Interleucina-2/análiseRESUMO
Systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) are characterized by B-cell hyperactivity as manifested by spontaneous in vitro production of immunoglobulins (Ig), but pokeweed mitogen (PWM)-induced Ig synthesis of peripheral blood (PB) mononuclear cells, a T-cell dependent process, is markedly decreased. We analyzed the defective capacity to produce Ig in vitro in 11 patients with SLE and 11 with MCTD. PWM-activated PB T-cells, both from SLE- and MCTD-patients, did not differ from control T-cells in their capacity to produce B-cell growth factors (BCGF) and B-cell differentiation factors (BCDF). However, B-cell proliferative responsiveness to control T-cell factors was decreased compared to control B-cells (p less than 0.01). In contrast to PB B-cells splenic B-cells from a patient with MCTD who underwent splenectomy were highly responsive to T-cell factors in a dose dependent way. It is concluded that the defective PWM-induced Ig production of PB lymphocytes in SLE/MCTD is due to decreased responsiveness of PB B cells to T-cell factors. However, analysis of splenic lymphocytes may be more representative for evaluation of the immunoregulatory disturbances in these disorders.
Assuntos
Linfócitos B/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Doença Mista do Tecido Conjuntivo/imunologia , Adulto , Idoso , Linfócitos B/patologia , Diferenciação Celular , Feminino , Humanos , Interleucina-4 , Interleucinas/imunologia , Lúpus Eritematoso Sistêmico/patologia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Baço/imunologia , Baço/patologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
OBJECTIVE: To assess the value of measuring serum levels of soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1 (sVCAMP-1), and soluble E-selectin for monitoring disease activity in Wegener's granulomatosis (WG). METHODS: A sandwich enzyme-linked immunosorbent assay was used to measure levels of soluble adhesion molecules at the time of diagnosis in 22 consecutive patients with WG, in 12 WG patients studied serially prior to disease relapse, at the time of upper airways infection in 18 patients with inactive WG, and in 57 controls. Disease activity was assessed by disease activity score and C-reactive protein levels. RESULTS: At diagnosis of WG, sICAM-1 and sVCAM-1 levels were significantly elevated and correlated with disease activity. At the time of relapse, a significant increase in all 3 soluble adhesion molecules was found compared with levels at 6 months prior to relapse, but only sVCAM-1 levels were significantly elevated compared with those in controls. Levels of soluble adhesion molecules at the time of relapse did not differ from those measured during an upper airways infection without disease activity. CONCLUSION: Elevated serum levels of sICAM-1 and sVCAM-1 can be found in active WG and correlate with disease activity. However, their clinical relevance for followup is limited due to lack of sensitivity and specificity for WG disease activity.
Assuntos
Moléculas de Adesão Celular/sangue , Granulomatose com Poliangiite/sangue , Adolescente , Adulto , Idoso , Infecções Bacterianas/sangue , Selectina E , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Seguimentos , Granulomatose com Poliangiite/diagnóstico , Granulomatose com Poliangiite/fisiopatologia , Humanos , Molécula 1 de Adesão Intercelular , Masculino , Glicoproteínas de Membrana/sangue , Pessoa de Meia-Idade , Infecções Respiratórias/sangue , Solubilidade , Molécula 1 de Adesão de Célula Vascular , Viroses/sangueRESUMO
OBJECTIVES: To assess serial activation of T-cell subsets in relation to auto-antibody production and the occurrence of disease exacerbations in patients with systemic lupus erythematosus (SLE). METHODS: To study the possible role of T-cells in the pathophysiology of the disease, 16 consecutive exacerbations were prospectively studied in a cohort of patients with SLE, and serial plasma levels of sIL-2R, sCD4, and sCD8 preceding and during these exacerbations were determined. Levels of these molecules were related to total IgM and IgG, and anti-dsDNA. RESULTS: During major disease exacerbations (n = 6), levels of sIL-2R increased significantly (p < 0.001). Levels of sCD4 were predominantly in the normal range, whereas levels of sCD8 were frequently increased. No change in levels of both molecules could be detected in the period before the exacerbation. During minor exacerbations (n = 10), levels of sIL-2R remained stable. Levels of sCD4, however, tended to drop, whereas levels of sCD8 tended to rise. No correlations were found between sIL-2R, sCD4 or sCD8 on the one hand, and total IgM, IgG, or anti-dsDNA on the other. CONCLUSIONS: Levels of sIL-2R are increased, and rise before major exacerbations of SLE. Levels of sCD4 and sCD8, however, are not related to levels of sIL-2R, and do not reflect B-cell activation, nor disease activity during exacerbations of SLE. Thus for the clinical follow up of SLE measurement of levels of sCD4 or sCD8 is of limited value.
Assuntos
Antígenos CD4/sangue , Antígenos CD8/sangue , Lúpus Eritematoso Sistêmico/imunologia , Receptores de Interleucina-2/metabolismo , Linfócitos T/imunologia , Adulto , Anticorpos Antinucleares/sangue , DNA/imunologia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , SolubilidadeRESUMO
We describe a solid-phase enzyme-linked immunosorbent assay (ELISA) for quantitation of antibodies to nucleoribonucleoprotein (nRNP/Sm). nRNP/Sm was purified from rabbit thymus acetone powder by immunoaffinity chromatography and characterized by counterimmunoelectrophoresis (CIE) and immunoblotting using sera with well-known specificities. The purified antigen was used in ELISA. Positive results in ELISA were obtained only in sera with anti-nRNP or anti-Sm specificity as determined in CIE. Levels of anti-nRNP/Sm as quantitated by ELISA were higher in the sera of patients with active connective tissue disease (n = 7) than in those with inactive disease (n = 6) (P less than 0.01). Differences in anti-nRNP/Sm levels were also found between patients with mildly active disease (n = 19) and those with active disease (P less than 0.01). Fluctuations of anti-nRNP/Sm levels related to disease activity were seen in longitudinal observation. Although anti-nRNP/Sm levels as quantitated by ELISA correlated with titres of antinuclear antibodies as determined by immunofluorescence (r = 0.46, P less than 0.05), quantitation of anti-nRNP/Sm by ELISA is superior since the assay is antigen-specific and its quantitation independent of titration-related inaccuracies.
Assuntos
Anticorpos Antinucleares/análise , Ensaio de Imunoadsorção Enzimática , Doença Mista do Tecido Conjuntivo/imunologia , Ribonucleoproteínas/imunologia , Adulto , Idoso , Contraimunoeletroforese , Eletroforese em Gel de Poliacrilamida , Feminino , Imunofluorescência , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-IdadeRESUMO
The mechanisms underlying glomerular capillary wall injury in Wegener's granulomatosis (WG) are not well understood. Anti-neutrophil cytoplasmic antibodies (ANCA), present in sera from patients with WG, are known to stimulate respiratory burst and degranulation of primed polymorphonuclear neutrophils (PMN) in vitro. Experimental studies have shown that oxygen radical production and lysosomal enzymes are important mediators of glomerular capillary wall injury. In the present study we investigated the presence of activated PMN and the extracellular localization of lysosomal enzymes in 28 consecutive renal biopsies from patients with WG. The presence of activated PMN within the renal biopsies was compared with the capacity of ANCA, isolated from simultaneously drawn serum samples, to activate primed PMN obtained from a normal donor. Both parameters were also related to renal function. Renal biopsies were obtained from newly diagnosed WG patients before therapy had started. Activation of PMN in the biopsies was assessed by measuring hydrogen peroxide production in situ. The number of activated PMN in the biopsy correlated with the extent of impairment of renal function. Proteinase 3, myeloperoxidase, and elastase, all targets of ANCA, were localized extracellularly in renal tissue and were also found within tubular epithelial cells. All ANCA positive samples were capable of activating primed PMN. The amount of activation correlated with the ANCA titer in those samples. No correlation, however, was found between the in vitro capacity of ANCA-positive IgG fractions to activate primed PMN and the number of activated PMN present in the renal biopsy. We conclude that activated PMN producing toxic oxygen metabolites and releasing lysosomal enzymes, are present in renal biopsies from patients with WG. The amount of activated PMN present within the kidney, and not the capacity of the corresponding ANCA to activate PMN, correlates with renal tissue damage as assessed by serum creatinine levels.
Assuntos
Granulomatose com Poliangiite/imunologia , Neutrófilos/imunologia , Anticorpos Anticitoplasma de Neutrófilos , Autoanticorpos/imunologia , Autoantígenos/metabolismo , Biomarcadores , Proteínas do Sistema Complemento/imunologia , Granulomatose com Poliangiite/enzimologia , Humanos , Imunoglobulina G/imunologia , Rim/enzimologia , Rim/imunologia , Mieloblastina , Neutrófilos/enzimologia , Elastase Pancreática/metabolismo , Peroxidase/metabolismo , Espécies Reativas de Oxigênio , Serina Endopeptidases/metabolismo , Superóxidos/metabolismoRESUMO
T cell-mediated immunity is thought to play an important role in the pathogenesis of WG. In previous studies a minority of WG patients as well as some healthy controls showed in vitro proliferation of their peripheral blood mononuclear cells (PBMC) to PR3, the main autoantigen in WG. The relevant peptides responsible for this in vitro proliferation have not been identified. In order to define immunogenic peptides, PBMC of 13 WG patients in remission and 10 healthy controls were tested for proliferation to linear peptides of PR3 and to whole PR3. Fifty overlapping peptides spanning the whole PR3 sequence were synthesized. Peptides were tested in pools of five peptides and as single peptide. PBMC of two WG patients and one healthy control proliferated to whole PR3 and to peptide pools. In addition, 10 WG patients and eight healthy controls that did not proliferate to whole PR3 did proliferate to pools of PR3 peptides. Although more WG patients tended to react to particular peptide pools, no significant difference was seen between lymphocyte proliferation to PR3 peptides of WG patients and that of healthy controls. The pools of peptides recognized were mainly located at the N- and C-terminus of PR3. No correlation was observed between HLA type and proliferation on particular peptide pools. No proliferation of PBMC was observed to single peptides. In conclusion, T cells of WG patients proliferate in vitro more frequently to PR3 peptides than to the whole PR3 protein. Peptides derived from the signal sequence, the propeptide or peptides located at the C-terminus of PR3 induce highest levels of proliferation. No specific PR3 sequence could be identified that was preferentially recognized by PBMC of WG patients compared with controls.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Autoantígenos/imunologia , Granulomatose com Poliangiite/imunologia , Oligopeptídeos/imunologia , Serina Endopeptidases/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Sequência de Aminoácidos , Animais , Autoantígenos/farmacologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Divisão Celular , Feminino , Granulomatose com Poliangiite/sangue , Antígenos HLA-A/imunologia , Antígenos HLA-B/imunologia , Antígenos HLA-DQ/imunologia , Antígenos HLA-DR/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mieloblastina , Oligopeptídeos/farmacologia , Peroxidase/imunologia , Coelhos , Serina Endopeptidases/farmacologiaRESUMO
58 patients with biopsy-proven Wegener's granulomatosis (WG) were prospectively screened for clinical evidence of the disease 3-monthly, with antineutrophil cytoplasmic antibody (ANCA) measurements every month. Over 24 months, ANCA rose in 20 patients, 9 of whom were randomly assigned to receive combined 9 and 3 month courses of cyclophosphamide and prednisolone, respectively, at the time of ANCA rise; and 11 patients who were untreated except if there was a clinical relapse. 6 of 11 untreated patients relapsed within 3 months of ANCA rise. 3 of the remaining 5 patients relapsed after 3 months. There were no early or late relapses in patients randomised to treatment. Patients receiving no treatment at the time of ANCA rise took more cyclophosphamide and prednisolone than patients who were treated. Side-effects did not significantly differ between the two groups.