RESUMO
BACKGROUND: In Goodpasture's disease, circulating autoantibodies bind to the noncollagenous-1 (NC1) domain of type IV collagen in the glomerular basement membrane (GBM). The specificity and molecular architecture of epitopes of tissue-bound autoantibodies are unknown. Alport's post-transplantation nephritis, which is mediated by alloantibodies against the GBM, occurs after kidney transplantation in some patients with Alport's syndrome. We compared the conformations of the antibody epitopes in Goodpasture's disease and Alport's post-transplantation nephritis with the intention of finding clues to the pathogenesis of anti-GBM glomerulonephritis. METHODS: We used an enzyme-linked immunosorbent assay to determine the specificity of circulating autoantibodies and kidney-bound antibodies to NC1 domains. Circulating antibodies were analyzed in 57 patients with Goodpasture's disease, and kidney-bound antibodies were analyzed in 14 patients with Goodpasture's disease and 2 patients with Alport's post-transplantation nephritis. The molecular architecture of key epitope regions was deduced with the use of chimeric molecules and a three-dimensional model of the alpha345NC1 hexamer. RESULTS: In patients with Goodpasture's disease, both autoantibodies to the alpha3NC1 monomer and antibodies to the alpha5NC1 monomer (and fewer to the alpha4NC1 monomer) were bound in the kidneys and lungs, indicating roles for the alpha3NC1 and alpha5NC1 monomers as autoantigens. High antibody titers at diagnosis of anti-GBM disease were associated with ultimate loss of renal function. The antibodies bound to distinct epitopes encompassing region E(A) in the alpha5NC1 monomer and regions E(A) and E(B) in the alpha3NC1 monomer, but they did not bind to the native cross-linked alpha345NC1 hexamer. In contrast, in patients with Alport's post-transplantation nephritis, alloantibodies bound to the E(A) region of the alpha5NC1 subunit in the intact hexamer, and binding decreased on dissociation. CONCLUSIONS: The development of Goodpasture's disease may be considered an autoimmune "conformeropathy" that involves perturbation of the quaternary structure of the alpha345NC1 hexamer, inducing a pathogenic conformational change in the alpha3NC1 and alpha5NC1 subunits, which in turn elicits an autoimmune response. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases.)
Assuntos
Doença Antimembrana Basal Glomerular/imunologia , Autoanticorpos/química , Colágeno Tipo IV/química , Nefrite Hereditária/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos/sangue , Anticorpos/química , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Autoantígenos/sangue , Autoantígenos/química , Sítios de Ligação de Anticorpos , Colágeno Tipo IV/imunologia , Colágeno Tipo IV/metabolismo , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Epitopos/química , Epitopos/metabolismo , Feminino , Membrana Basal Glomerular/imunologia , Humanos , Isoanticorpos/química , Isoanticorpos/metabolismo , Glomérulos Renais/imunologia , Transplante de Rim/imunologia , Masculino , Pessoa de Meia-Idade , Nefrite/imunologia , Nefrite Hereditária/cirurgia , Complicações Pós-Operatórias/imunologia , Conformação Proteica , Isoformas de Proteínas , Estudos Retrospectivos , Adulto JovemRESUMO
Chemokines recruit and activate leukocytes during inflammation. CXCL16 is a recently discovered chemokine that is expressed as a transmembrane protein that is cleaved to form the active, soluble chemokine. We analyzed the role of CXCL16 in the development of inflammation and in the progression of the anti-glomerular basement membrane (GBM) antibody-induced experimental glomerulonephritis in Wistar-Kyoto rats. CXCL16 was expressed in glomerular endothelial cells and mediated adhesion of macrophages expressing CXCL16 and its cognate receptor, CXCR6. Glomerular infiltrates displayed a strong migratory response to soluble CXCL16. Soluble CXCL16 and its receptor CXCR6 were induced in nephritic glomeruli throughout the disease, and CXCL16 expression correlated with the up-regulation of ADAM10, suggesting that this disintegrin and metalloproteinase mediates the chemokine activity of CXCL16. Blocking CXCL16 in the acute inflammatory phase or progressive phase of established glomerulonephritis significantly attenuated monocyte/macrophage infiltration and glomerular injury; proteinuria also improved. We conclude that CXCL16/CXCR6 plays a critical role in stimulating leukocyte influx, which causes glomerular damage during anti-GBM glomerulonephritis. Blocking CXCL16 actions limits the progression of anti-GBM glomerulonephritis even when the disease is established.
Assuntos
Doença Antimembrana Basal Glomerular/imunologia , Doença Antimembrana Basal Glomerular/metabolismo , Quimiocinas CXC/metabolismo , Quimiotaxia de Leucócito/imunologia , Inflamação/metabolismo , Receptores Depuradores/metabolismo , Animais , Doença Antimembrana Basal Glomerular/patologia , Anticorpos , Western Blotting , Adesão Celular/imunologia , Citometria de Fluxo , Hibridização In Situ , Inflamação/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Microscopia Confocal , Ratos , Ratos WistarRESUMO
The chemokine monocyte chemoattractant protein (MCP)-1 has been implicated in the monocyte/macrophage infiltration that occurs during tubulointerstitial nephritis (TIN). We investigated the role of MCP-1 in rats with TIN by administering a neutralizing anti-MCP-1 antibody (Ab). We observed significantly reduced macrophage infiltration and delayed neutrophil clearance in the kidneys of TIN model rats treated with the anti-MCP-1 Ab. To exclude the possibility that an observed immune complex could affect the resolution of apoptotic neutrophils via the Fc receptor, TIN model rats were treated with a peptide-based MCP-1 receptor antagonist (RA). The MCP-1 RA had effects similar to those of the anti-MCP-1 Ab. In addition, MCP-1 did not affect macrophage-mediated phagocytosis of neutrophils in vitro. Deposition of the anti-MCP-1 Ab in rat kidneys resulted from its binding to heparan sulfate-immobilized MCP-1, as demonstrated by the detection of MCP-1 in both pull-down and immunoprecipitation assays. We conclude that induction of chemokines, specifically MCP-1, in TIN corresponds with leukocyte infiltration and that the anti-MCP-1 Ab formed an immune complex with heparan sulfate-immobilized MCP-1 in the kidney. Antagonism of MCP-1 in TIN by Ab or RA may alter the pathological process, most likely through delayed removal of apoptotic neutrophils in the inflammatory loci.
Assuntos
Quimiocina CCL2/antagonistas & inibidores , Nefrite Intersticial/metabolismo , Nefrite Intersticial/patologia , Neutrófilos/patologia , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Complexo Antígeno-Anticorpo , Apoptose , Quimiocina CCL2/análogos & derivados , Quimiocina CCL2/química , Quimiocina CCL2/imunologia , Quimiocinas , Quimiocinas CXC/metabolismo , Quimiotaxia/efeitos dos fármacos , Citocinas/metabolismo , Escherichia coli/metabolismo , Fibrose , Heparitina Sulfato/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Rim/imunologia , Rim/metabolismo , Macrófagos Peritoneais , Nefrite Intersticial/fisiopatologia , Infiltração de Neutrófilos , Neutrófilos/efeitos dos fármacos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/farmacologia , Fagocitose , Ratos , Ratos Endogâmicos BN , Receptores CCR2 , Receptores de Quimiocinas/antagonistas & inibidores , Fatores de TempoRESUMO
A basic conservation of cell migration guidance mechanisms in the nervous and immune systems was proposed when Slit, known for its role in axon guidance, was found to inhibit chemokine-induced leukocyte chemotaxis in vitro. These studies examined the role of Slit2 in modulating inflammation in vivo. In a rat model of glomerulonephritis, endogenous glomerular Slit2 expression fell after disease induction, and its inhibition during the early disease period accelerated inflammation. Ex vivo glomerular leukocytes showed decreased chemokine and chemoattractant-induced chemotaxis in response to Slit2, suggesting an anti-inflammatory role for glomerular Slit2. In contrast to the effect of inhibition, glomerulonephritis was ameliorated by systemic Slit2 administration. Slit2 treatment improved disease histologically and also improved renal function when given early in the disease course. Leukocytes harvested from rats receiving Slit2 showed decreased monocyte chemoattractant protein-1 (MCP)-1-mediated migration, consistent with a peripheral Slit2 effect. In keeping with this functional alteration, Slit2-mediated inhibition of RAW264.7 cell chemotaxis was associated with decreased levels of active cdc42 and Rac1, implicating GTPases in leukocyte Slit2 signaling. These findings suggest a role for endogenous Slit2 in the inhibition of chemoattractant-mediated signals, demonstrate a potentially important anti-inflammatory effect for Slit2 in vivo, and provide further evidence for conserved mechanisms guiding the process of migration in distinct cell types.
Assuntos
Glomerulonefrite/metabolismo , Inflamação/tratamento farmacológico , Proteínas do Tecido Nervoso/administração & dosagem , Proteínas do Tecido Nervoso/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Animais , Antígenos CD/metabolismo , Western Blotting , Linhagem Celular , Quimiocina CCL2/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Modelos Animais de Doenças , Regulação para Baixo , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Glomerulonefrite/etiologia , Glomerulonefrite/patologia , Glomerulonefrite/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos WKY , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Interleukin-18 (IL-18), a potent inducer of interferon gamma (IFN-gamma) production, is a cytokine involved in the cell-mediated immune response that is expressed during inflammatory and pathologic conditions. IFN-gamma plays a role in the development of some models of glomerulonephritis (GN); however, the role of IL-18 in the production of IFN-gamma during these pathologies has not been studied. METHODS: Rat IL-18 cDNA was isolated and the regulation of IL-18 gene expression was studied. IFN-gamma and IL-18 expression were determined in anti-glomerular basement membrane (GBM) antibody (Ab)-induced GN. Recombinant active IL-18 (rIL-18) was used to further identify its effect on IFN-gamma production during this GN. Glomerular injury and levels of IFN-gamma were assayed in Wistar Kyoto (WKY) rats with anti-GBM GN in the presence or absence of rIL-18. RESULTS: Rat IL-18, similar to the mouse clone, requires processing by the IL-1beta converting enzyme to become activated. A rat IL-18 5'-untranslated region (UTR) translational inhibitor was identified that strongly inhibited the synthesis of IL-18. This translational inhibitor with different lengths (180 and 130 bp) was highly expressed during GN and correlated with minimal IFN-gamma mRNA expression. Injection of recombinant active IL-18 in WKY rats with anti-GBM GN was associated with an increase of glomerular IFN-gamma levels, proliferating cell nuclear antigen (PCNA)-ED1+ cells, and PCNA-CD8+ cells, with worsening of glomerular injury. CONCLUSION: These data suggest that the translational control of IL-18 expression by its 5'-UTR limits the production of IL-18, resulting in restricted expression of mRNA and protein IFN-gamma in this model of GN. Furthermore, it was suggested that possible IL-18/IFN-gamma induction of local proliferation of macrophages and CD8+ cells might be an important mechanism for amplifying CD8+-mediated macrophage-dependent GN.
Assuntos
Doença Antimembrana Basal Glomerular/metabolismo , Doença Antimembrana Basal Glomerular/patologia , Interferon gama/antagonistas & inibidores , Interleucina-18/antagonistas & inibidores , Interleucina-18/genética , Biossíntese de Proteínas , Regiões 5' não Traduzidas/genética , Regiões 5' não Traduzidas/metabolismo , Regiões 5' não Traduzidas/fisiologia , Sequência de Aminoácidos/genética , Animais , Sequência de Bases/genética , Células COS , Caspase 1/genética , Chlorocebus aethiops , Clonagem Molecular , Interferon gama/genética , Interleucina-18/farmacologia , Masculino , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos WKY , Proteínas Recombinantes/farmacologiaRESUMO
Significant glomerular vasoconstriction and production of reactive oxygen species has been known to occur with exposure to anti-glomerular basement membrane antibody (AGBM-Ab) in the rat model. Previously published studies have demonstrated that such effects can be reduced by therapy with phentolamine, an alpha-adrenergic antagonist. It was hypothesized that antioxidant pretreatment with water-soluble probucol would improve glomerular hemodynamics 60 to 90 min after the administration of AGBM-Ab. These relationships were examined with both in vivo renal micropuncture and in vitro studies in rats. Single-nephron GFR (SNGFR) decreased markedly in untreated rats after AGBM-Ab as a result of afferent and efferent arteriolar vasoconstriction with consequent reductions in nephron plasma flow (SNPF) and decreases in the glomerular ultrafiltration coefficient (LpA). Basal SNGFR was increased, and SNGFR was significantly higher after AGBM-Ab in probucol-treated versus untreated rats. This finding was due solely to higher values for SNPF and prevention of afferent arteriolar constriction. A reduction in LpA after AGBM-Ab was not prevented by probucol treatment. In vitro analyses of glomeruli revealed reduced myeloperoxidase activity in antioxidant-treated rats. Lipoxygenase activity and leukotriene products, however, were not changed by antioxidant therapy, yet vasoconstriction was prevented. H(2)O(2) generation before and after formyl-methionyl-leucyl-phenylalanine stimulation was significantly reduced before and after AGBM-Ab in glomeruli harvested from rats that were treated with the antioxidant. Antioxidant therapy in this model of AGBM-Ab injury did not prevent reductions in LpA, an index of glomerular membrane damage, but did prevent afferent arteriolar vasoconstriction. Reactive oxygen species generation was reduced by probucol. The specific mechanisms whereby antioxidant therapy ameliorates glomerular hemodynamic effects will be defined in additional studies and is likely to involve either enhanced vasodilator or diminished vasoconstrictor activity.
Assuntos
Antioxidantes/farmacologia , Glomérulos Renais/imunologia , Glomérulos Renais/lesões , Animais , Anticorpos/farmacologia , Araquidonato 5-Lipoxigenase/metabolismo , Membrana Basal/imunologia , Taxa de Filtração Glomerular/efeitos dos fármacos , Hemodinâmica , Peróxido de Hidrogênio/farmacologia , Rim/efeitos dos fármacos , Rim/metabolismo , Lipoxigenase/metabolismo , Masculino , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Néfrons/metabolismo , Peroxidase/metabolismo , Probucol/farmacologia , Ratos , Espécies Reativas de Oxigênio , Água/farmacologiaRESUMO
Glomerular monocyte/macrophage (Mo/M phi) infiltrates play a role in many forms of glomerulonephritis (GN), and the intensity of Mo/M phi trafficking correlates with the loss of renal function and histological damage. We analyzed the functional role of macrophage-derived chemokine (MDC), a potent mononuclear cell chemoattractant, during the progression of anti-glomerular basement membrane (GBM) antibody (Ab) GN, a model of crescentic GN in the WKY rat, and whether the effects of MDC were dependent on its receptor CCR4. MDC mRNA and protein expression were markedly induced in nephritic glomeruli throughout the disease. Blocking the function of MDC did not affect the developing of the disease from days 2 to 7, but it dramatically blocked M omicron/M phi infiltration in the glomeruli, prevented crescent formation, and reversed renal function impairment during days 7 to 14 of the anti-GBM GN. In this study, we also found that MDC activity on M omicron/M phi in this GN was at least partly dependent on a new variant of CCR4. These results suggest that MDC is critically involved in the development of anti-GBM GN from acute glomerular injury to irreversible tissue damage. In addition, an antagonist to MDC may represent a prime drug target for therapeutic application to intervene in the progression of anti-GBM GN and in other M omicron/M phi-dominant GN.
Assuntos
Quimiocinas CC/genética , Quimiocinas/antagonistas & inibidores , Glomerulonefrite/imunologia , Leucócitos Mononucleares/patologia , Macrófagos/imunologia , Receptores de Quimiocinas/genética , Baço/imunologia , Sequência de Aminoácidos , Animais , Quimiocina CCL22 , Quimiocinas CC/química , Primers do DNA , Progressão da Doença , Biblioteca Gênica , Glomerulonefrite/genética , Glomerulonefrite/prevenção & controle , Humanos , Glomérulos Renais/imunologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos WKY , Receptores CCR4 , Receptores de Quimiocinas/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Gênica , TransfecçãoRESUMO
To study an involvement of glomerular endothelial cells in the development of anti-Thy-1 nephritis, we examined the expression of endothelial cell adhesion molecules during the course of this model. Ribonuclease protection assay elucidated that expression of mRNA for intercellular adhesion molecule-1 (ICAM-1) was markedly enhanced in the glomeruli with a peak at 2 h (6.5-fold, p < 0.05) after the anti-Thy-1 antibody injection when mesangial cell lysis was recognized and IL-1beta mRNA expression was induced in the glomeruli. The glomerular ICAM-1 was predominantly localized in the endothelial cells and was intensely immunostained at day 1 in the glomerular endothelial cells. In contrast, platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial-cadherin mRNA expression increased gradually with a peak at day 6 (2.6-fold (p < 0.05) and 4.2-fold (p < 0.05), respectively) in the glomeruli with mesangial proliferative lesion. PECAM-1 was also immunolocalized in the glomerular endothelial cells and the immunoreactivity was greatly enhanced at day 6. Glomerular expression of vascular cell adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 (E-selectin) was unchanged at a low level during the course of anti-Thy-1 nephritis. Blocking of ICAM-1 by administration of anti-ICAM-1 antibody showed significant decrease in the number of polymorphonuclear leukocytes accumulating in the glomeruli by 45.7% (9.4 +/- 0.2 vs. 5.1 +/- 0.1 per glomerular cross section, p < 0.01) at 2 h. These results suggest a significant involvement of glomerular endothelial cells in the development and repair of anti-Thy-1 nephritis via direct or indirect intercellular interactions between mesangial cells and glomerular endothelial cells.