RESUMEN
Myasthenia gravis (MG) and its animal model experimental autoimmune MG (EAMG), are T-cell dependent, antibody-mediated autoimmune disorders. A dual altered peptide ligand (APL) composed of the tandemly arranged two single amino acids analogs of two myasthenogenic peptides, p195-212 and p259-271, was demonstrated to downregulate, in vitro and in vivo, MG-associated autoimmune responses. Upregulation of regulatory CD4(+)CD25(+) cells plays a key role in the mechanism of action of the dual APL. The objectives of the present study were to address the involvement of extracellular-regulated kinase (ERK)1,2 in the mechanisms by which the dual APL-induced CD4(+)CD25(+) cells suppress MG-associated autoimmune responses. We demonstrate here that administration of the dual APL increased activated ERK1,2 in the CD4(+)CD25(+)-enriched population. Further, inhibition of ERK1,2 by its inhibitor, U0126, in dual APL-induced CD4(+)CD25(+) cells, abrogated their ability to suppress interferon (IFN)-gamma secretion by lymph node (LN) cells of mice that were immunized with the myasthenogenic peptide. Moreover, inhibition of ERK1,2 in the dual APL-induced regulatory CD4(+)CD25(+) cells, resulted in downregulation of the forkhead box p3 (Foxp3) gene and protein expression levels, as well as in the downregulation of CD4(+)CD25(+) development, suggesting that the active suppression exerted by the dual APL via CD4(+)CD25(+) cells depends on ERK1,2 activity.
Asunto(s)
Autoinmunidad/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Miastenia Gravis Autoinmune Experimental/enzimología , Miastenia Gravis Autoinmune Experimental/inmunología , Péptidos/uso terapéutico , Secuencia de Aminoácidos , Animales , Autoinmunidad/inmunología , Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/inmunología , Terapia de Inmunosupresión/métodos , Subunidad alfa del Receptor de Interleucina-2/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Miastenia Gravis Autoinmune Experimental/tratamiento farmacológico , Miastenia Gravis Autoinmune Experimental/patología , Péptidos/inmunología , Fosforilación/efectos de los fármacos , Resultado del Tratamiento , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunologíaRESUMEN
(NZBxNZW)F1 mice spontaneously develop with age an autoimmune disease that resembles the human disease, systemic lupus erythematosus (SLE). Previous studies have demonstrated that susceptibility to experimentally induced SLE depended on the expression of MHC class I molecules: mice deficient in beta2-microglobulin did not express cell surface class I and were resistant to the induction of experimental SLE. Furthermore, the spontaneous SLE-like disease of (NZBxNZW)F1 mice was ameliorated by treatment with an agent that reduces MHC class I expression, methimazole (MMI). In the present study, the role of MHC class I has been examined in (NZBxNZW)F1 mice deficient in beta2-microglobulin expression. Homozygous (NZBxNZW)F1 beta2m-/- mice do not express class I or develop CD8+ T cells. Surprisingly, they show an increased susceptibility to disease. In sharp contrast, heterozygous (NZBxNZW)F1 beta2m+/- express class I, albeit at reduced levels, develop normal levels of CD8+ T cells and are less susceptible to autoimmune disease, relative to their wild-type litter mates. Taken together, these findings suggest that class I expression regulates the development of disease, both positively and negatively. We speculate that MHC class I expression itself confers susceptibility to disease through presentation of self-peptides, while also selecting for a CD8+ suppressor T cell population that mitigates disease.
Asunto(s)
Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Genes MHC Clase I , Animales , Anticuerpos Antinucleares/sangre , Presentación de Antígeno , Autoantígenos , Linfocitos T CD8-positivos/inmunología , Femenino , Expresión Génica , Heterocigoto , Homocigoto , Humanos , Hibridación Genética , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Masculino , Ratones , Ratones Endogámicos NZB , Ratones Noqueados , Microglobulina beta-2/deficiencia , Microglobulina beta-2/genéticaRESUMEN
OBJECTIVE: To determine the prevalence of autoantibodies directed against an epitope of the glutamate/N-methyl-D-aspartic acid (NMDA) receptor subunit NR2A (which is highly expressed in human brain) in the sera of lupus patients, and to investigate the possible correlation of these antibodies with clinical and serological manifestations of systemic lupus erythematosus (SLE). METHODS: Sera were obtained from 109 consecutive SLE patients. Controls were 65 patients with myasthenia gravis, 19 with autoimmune polyendocrine syndrome type I (APS I), and 65 healthy donors. A 15 amino acid long peptide based on a sequence within the NR2A subunit of the NMDA/glutamate receptor was synthesised. Antibodies to this peptide were determined by enzyme linked immunosorbent assay. Antibodies against double stranded DNA (dsDNA) were measured by Chrithidia luciliae assay. Disease activity was determined using the SLE disease activity index (SLEDAI). RESULTS: Sera of 34/109 SLE patients (31%) reacted specifically with the NR2A peptide compared with only 4/65 myasthenia gravis patients (6.1%, p<0.001), 1/19 APS I patients (5.3%, p<0.02), and 3/65 healthy controls (4.6%, p<0.001). No correlation was found between the presence of NR2A and dsDNA or anti-cardiolipin specific autoantibodies. In addition, no significant correlation was observed between the presence of NR2A specific antibodies and the SLEDAI score or any lupus related clinical manifestations. CONCLUSIONS: A significant number of SLE patients (31%) have NR2A specific antibodies that do not correlate with anti-dsDNA antibodies. Additional studies of lupus patients with neurological disorders should elucidate the role of NR2A specific antibodies in lupus related CNS manifestations.
Asunto(s)
Autoanticuerpos/sangre , Lupus Eritematoso Sistémico/inmunología , Receptores de N-Metil-D-Aspartato/inmunología , Adulto , Anticuerpos Antinucleares/sangre , ADN/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Miastenia Gravis/inmunología , Poliendocrinopatías Autoinmunes/inmunología , Índice de Severidad de la EnfermedadRESUMEN
A peptide based on the complementarity determining region (CDR)1 of a human monoclonal anti-DNA autoantibody (hCDR1) was shown to either prevent or treat an already established murine lupus in systemic lupus erythematosus (SLE)-prone mice or in mice with induced experimental SLE. The present study was undertaken to determine the therapeutic potential of hCDR1 in a model of lupus in severe combined immunodeficient (SCID) mice engrafted with peripheral blood lymphocytes (PBL) of patients with SLE. To this end, PBL obtained from lupus patients were injected intraperitoneally into two equal groups of SCID mice that were treated either with the hCDR1 (50 micro g/mouse) once a week for 8 weeks, or with a control peptide. Mice were tested for human IgG levels, anti-dsDNA autoantibodies, anti-tetanus toxoid antibodies and proteinuria. At sacrifice, the kidneys of the successfully engrafted mice were assessed for human IgG and murine complement C3 deposits. Of the 58 mice transplanted with PBL of SLE patients, 38 (66%) were engrafted successfully. The mice that were treated with the control peptide developed human dsDNA-specific antibodies. Treatment with hCDR1 down-regulated the latter significantly. No significant effect of the treatment on the levels of anti-tetanus toxoid antibodies could be observed. Treatment with hCDR1 resulted in a significant amelioration of the clinical features manifested by proteinuria, human IgG complex deposits as well as deposits of murine complement C3. Thus, the hCDR1 peptide is a potential candidate for a novel specific treatment of SLE patients.
Asunto(s)
Traslado Adoptivo/métodos , Autoanticuerpos/inmunología , Regiones Determinantes de Complementariedad/administración & dosificación , Lupus Eritematoso Sistémico/terapia , Animales , Complemento C3/análisis , Regiones Determinantes de Complementariedad/inmunología , Citocinas/inmunología , Femenino , Humanos , Inmunoglobulina G/análisis , Inyecciones Intraperitoneales , Riñón/inmunología , Linfocitos , Ratones , Ratones SCID , Proteinuria , Toxoide Tetánico/inmunologíaRESUMEN
BACKGROUND: Sex hormones have been shown to influence the immune system and to modify the course of autoimmune disorders. OBJECTIVE: To examine the effects of the oestrogen antagonist tamoxifen on the course of systemic lupus erythematosus (SLE) in (NZBxNZW)F1 mice. METHODS: Groups of 8 week old (NZBxNZW)F1 female mice were treated with tamoxifen (800 micro g/mouse; twice a week) or with double distilled water for four months. Mice were evaluated monthly for the presence of autoantibodies directed against DNA and nuclear extract (NE) by enzyme linked immunosorbent assay (ELISA). White blood cells and thrombocytes were quantified by a cell counter and proteinuria by combistix kit. At 6 months of age, all mice that did not die spontaneously were killed and evaluated for the presence of glomerular immune deposits by indirect immunofluorescence assay. IgG isotypes of autoantibodies in the mouse sera and glomeruli were determined by gamma chain specific antibodies. RESULTS: Tamoxifen treatment significantly reduced autoantibody production directed against either NE or DNA. The latter reduction was mainly in autoantibodies of the IgG3 isotype. Furthermore, tamoxifen had significant beneficial effects on the course of SLE in (NZBxNZW)F1 mice. At 6 months of age, 40% of the untreated mice died spontaneously, whereas all the tamoxifen treated mice were still alive. All untreated mice showed severe thrombocytopenia and persistent proteinuria, with diffuse glomerular immune deposits of IgG2a and IgG3 isotypes in their kidneys. In contrast, the tamoxifen treated mice had a normal number of thrombocytes and only minimal proteinuria. Moreover, glomerular immune deposits were detected in <40% of the tamoxifen treated mice. The latter were mainly of the IgG2a but not of the IgG3 isotype. CONCLUSION: The results clearly show the remarkable therapeutic effects of tamoxifen on SLE of (NZBxNZW)F1 female mice and suggest that these beneficial effects are related to the specific reduction of IgG3 autoantibodies.
Asunto(s)
Anticuerpos Antinucleares/sangre , Antagonistas de Estrógenos/uso terapéutico , Inmunoglobulina G/sangre , Lupus Eritematoso Sistémico/tratamiento farmacológico , Tamoxifeno/uso terapéutico , Animales , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/inmunología , Núcleo Celular/inmunología , ADN/inmunología , Femenino , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/mortalidad , Nefritis Lúpica/prevención & control , Ratones , Ratones Endogámicos NZB , Tasa de SupervivenciaRESUMEN
Two peptides, based on the sequences of the complementarity-determining regions (CDR) 1 and 3 of a pathogenic murine monoclonal anti-DNA autoatibody that bears the 16/6 idiotype (Id), were shown to either prevent or treat an already established systemic lupus erythematosus (SLE) in two murine models of lupus. Two additional peptides based on the human monoclonal anti-DNA, 16/6 Id were synthesized. This study was undertaken in order to investigate the ability of the CDR-based peptides to immunomodulate SLE-associated responses of peripheral blood lymphocytes (PBL) of SLE patients. PBL of 24 of the 62 SLE patients tested proliferated in vitro following stimulation with the human 16/6 Id. Peptides based on the CDRs of both the human and murine anti-DNA autoantibodies inhibited efficiently and specifically the 16/6 Id-induced proliferation and IL-2 production. The latter inhibitions correlated with an up-regulated production (by 2.5-3.5-fold) of the immunosuppressive cytokine, TGF-beta. Overall, the results of our study demonstrate that the CDR-based peptides are capable of down-regulating in vitro autoreactive T cell responses of PBL of SLE patients. Thus, these peptides are potential candidates for a novel specific treatment of SLE patients.
Asunto(s)
Anticuerpos Antinucleares/inmunología , Regiones Determinantes de Complementariedad/inmunología , Lupus Eritematoso Sistémico/inmunología , Activación de Linfocitos/inmunología , Fragmentos de Péptidos/inmunología , Adolescente , Adulto , Secuencia de Aminoácidos , Animales , División Celular/inmunología , Células Cultivadas , Niño , Regiones Determinantes de Complementariedad/química , Femenino , Humanos , Tolerancia Inmunológica , Interleucina-2/biosíntesis , Masculino , Ratones , Persona de Mediana Edad , Datos de Secuencia Molecular , Factor de Crecimiento Transformador beta/biosíntesis , Regulación hacia ArribaRESUMEN
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the increased production of autoantibodies and by systemic clinical manifestations and damage to multiple organs. The aim of the present study was to analyse matrix metalloproteinase (MMP)-9 activity in sera of patients with active and inactive SLE in order to evaluate its role in the pathogenesis and course of the disease, as well as its diagnostic value. We measured activity levels of MMP-9 and MMP-2, using both gel zymography and activity assay kits, in sera of 40 SLE patients and of 25 healthy controls. We found that MMP-9 activity, but not MMP-2 activity, is significantly elevated in the sera of SLE patients compared with sera samples of healthy controls. High activity levels of MMP-9 were determined in sera of 68% of the SLE patients. Elevated levels of MMP-9 were correlated with the presence of discoid rash, Raynaud phenomenon, pneumonitis, mucosal ulcers and anti-phospholipid antibodies. Changes in activity levels of MMP-9, but not of MMP-2, were observed in sera of the same patient at different periods of the disease course. High levels of MMP-9 did not correlate with disease activity index (SLEDAI, BILAG) in female patients, but correlated with SLE activity in the group of male patients. The results of the present study suggest that MMP-9 plays a role in the pathogenesis of SLE.
Asunto(s)
Enfermedades Autoinmunes/enzimología , Lupus Eritematoso Sistémico/enzimología , Metaloproteinasa 9 de la Matriz/sangre , Adolescente , Adulto , Anticuerpos Antifosfolípidos/sangre , Artritis/sangre , Artritis/enzimología , Artritis/etiología , Enfermedades Autoinmunes/sangre , Enfermedades Autoinmunes/etiología , Femenino , Estudios de Seguimiento , Humanos , Enfermedades Renales/sangre , Enfermedades Renales/enzimología , Enfermedades Renales/etiología , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/etiología , Masculino , Metaloproteinasa 2 de la Matriz/sangre , Persona de Mediana Edad , Úlceras Bucales/sangre , Úlceras Bucales/enzimología , Úlceras Bucales/etiología , Trastornos por Fotosensibilidad/sangre , Trastornos por Fotosensibilidad/enzimología , Trastornos por Fotosensibilidad/etiología , Neumonía/sangre , Neumonía/enzimología , Neumonía/etiología , Enfermedad de Raynaud/sangre , Enfermedad de Raynaud/enzimología , Enfermedad de Raynaud/etiología , Índice de Severidad de la Enfermedad , Factores Sexuales , Vasculitis/sangre , Vasculitis/enzimología , Vasculitis/etiologíaRESUMEN
Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are T-cell regulated, antibody-mediated diseases. Peptides p195-212 and p259-271 of the human acetylcholine receptor (AChR) alpha-subunit, were previously shown to be immunodominant T cell epitopes in MG patients as well as in SJL and BALB/c mice, respectively. A dual altered peptide ligand (APL) composed of the two single amino acid analogs of the myasthenogenic peptides was shown to inhibit, in vitro and in vivo, MG-associated autoimmune responses. Furthermore, the dual APL was shown to down-regulate the clinical manifestations of an established EAMG in C57BL/6 mice injected with Torpedo AChR (TAChR). In the present study we attempted the elucidation of the mechanism(s) by which the dual APL down-regulates EAMG-associated responses. It is shown here that the dual APL acts by actively suppressing, in a specific manner, myasthenogenic T cell responses. The active suppression is mediated, at least partially, by the up-regulation of the secretion of TGF-beta following administration of the dual APL. The up-regulated secretion of TGF-beta is accompanied by down-regulation of IFN-gamma and IL-2 [T helper (Th) 1-type cytokine] secretion and by an up-regulation of IL-10 secretion (Th2-type cytokine). Furthermore, the inhibitory effect of the dual APL could be adoptively transferred to p195-212 or TAChR-immunized mice. The down-regulation of IL-2 secretion and the ability of recombinant IL-2 to rescue lymph node cells of mice treated with the dual APL from a state of unresponsiveness suggests that the dual APL acts also, at least partially, by causing the cells to undergo anergy.
Asunto(s)
Miastenia Gravis/inmunología , Fragmentos de Péptidos/administración & dosificación , Receptores Colinérgicos/inmunología , Linfocitos T/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Citocinas/biosíntesis , Femenino , Inmunización , Interleucina-2/farmacología , Interleucina-4/fisiología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Linfocitos T/inmunología , Torpedo , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
We have previously shown that the clinical manifestations of experimental systemic lupus erythematosus (SLE) correlate with an early increased secretion of TNFalpha and IL-1. In the present study, we examined the efficacy of two therapeutic modalities which lower TNFalpha production or activity, on the clinical manifestations of the disease. Experimental SLE was induced in naive C3H.SW mice by injection of the human anti-DNA monoclonal antibody (mAb) bearing the common idiotype, 16/6 Id. Two weeks after booster injections, treatment with either an anti-TNFalpha mAb, or pentoxiphylline (PTX) was started, for a period of 6 weeks. Production of TNFalpha (by splenocytes) and IL-1 (by peritoneal macrophages) was determined 3 and 7 months after disease induction. The experimental mice were also followed for disease manifestations. Both treatment protocols, with anti-TNFalpha mAb and with PTX, reduced the production of the two pro-inflammatory cytokines. TNFalpha and IL-1, in mice with experimental SLE. Anti-DNA antibodies were significantly lower in the mice treated with either protocol. In addition, a significantly lower rate of leukopenia, proteinuria and immune complex deposition was observed in treated mice. Abrogation of TNFalpha and IL-1 production in the early stages of experimental SLE by an anti-TNFalpha mAb or by PTX improves the clinical status of mice afflicted with this autoimmune disease.
Asunto(s)
Lupus Eritematoso Sistémico/inmunología , Pentoxifilina/uso terapéutico , Factor de Necrosis Tumoral alfa/inmunología , Vasodilatadores/uso terapéutico , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Humanos , Inmunoterapia , Lupus Eritematoso Sistémico/tratamiento farmacológico , Ratones , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
A peptide based on complementarity-determining region (CDR)-1 of a monoclonal murine anti-DNA Ab that bears the common idiotype, 16/6Id, was synthesized and characterized. The peptide, designated pCDR1, was found to be an immunodominant T-cell epitope in BALB/c mice. The CDR1-based peptide was shown to be capable of inhibiting the in vivo priming of BALB/c mice immunized with the peptide or with the whole anti-DNA 16/6Id(+) mAbs of either mouse or human origin. We show here that administration of pCDR1 (weekly, i.v., 100 microgram/mouse) in aqueous solution for 5 weeks starting at the time of disease induction with the human 16/6Id prevented the development of clinical manifestations of experimental systemic lupus erythematosus (SLE). Further, 10 weekly injections of pCDR1 to BALB/c mice with an established experimental SLE down-regulated clinical manifestations of SLE (e.g., anti-DNA auto-Abs, leukopenia, proteinuria, immune complex deposits in the kidneys) in the treated mice. Prevention of SLE induction was shown to be associated mainly with a decrease in the levels of IL-2, INFgamma, and the proinflammatory cytokine TNFalpha. On the other hand, the secretion of the immunosuppressive cytokine TGFbeta was elevated. Amelioration of the clinical manifestations of an already established experimental SLE correlated with a dramatic decrease in TNFalpha secretion, elevated levels of TGFbeta, and immunomodulation of the Th1 and Th2 type cytokines to levels close to those observed in healthy mice.
Asunto(s)
Anticuerpos Antinucleares/inmunología , Anticuerpos Monoclonales/inmunología , Fragmentos de Inmunoglobulinas/uso terapéutico , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/prevención & control , Secuencia de Aminoácidos , Animales , Anticuerpos Antinucleares/sangre , Anticuerpos Antinucleares/química , Anticuerpos Monoclonales/química , Células Cultivadas , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Esquema de Medicación , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Humanos , Fragmentos de Inmunoglobulinas/administración & dosificación , Fragmentos de Inmunoglobulinas/química , Inyecciones Intravenosas , Inyecciones Subcutáneas , Lupus Eritematoso Sistémico/patología , Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Bazo/inmunología , Linfocitos T/inmunologíaRESUMEN
Myasthenia gravis (MG) is a T cell-regulated, antibody-mediated autoimmune disease. Immunization with two myasthenogenic peptides, p195-212 and p259-271, which are sequences of the human acetylcholine receptor, resulted in MG-associated immune responses. A dual altered peptide ligand (APL) composed of the two APLs of the myasthenogenic peptides inhibited, in vitro and in vivo, those responses. This study was aimed at understanding the mechanism(s) underlying the in vivo inhibitory properties of the dual APL. To this end, we analyzed T cells of mice that were immunized with p259-271 for their adhesiveness toward vascular cell adhesion molecule 1, for the activity of their secreted matrix metalloproteinases (MMPs), and for their intracellular phospholipase C (PLC) activity. Immunization with p259-271 triggered the above three activities and in vivo administration of the dual APL inhibited the latter. Thus, treatment of mice with the dual APL interferes with functions required for T cells to migrate and interact with the self-AChR. This is the first indication that very late antigen 4, MMP-9, and PLC are targets for immunomodulation of autoreactive T cells by altered peptide ligands.
Asunto(s)
Epítopos de Linfocito T/inmunología , Inhibidores de la Metaloproteinasa de la Matriz , Miastenia Gravis/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Fosfolipasas de Tipo C/antagonistas & inhibidores , Molécula 1 de Adhesión Celular Vascular/metabolismo , Secuencia de Aminoácidos , Animales , Adhesión Celular/efectos de los fármacos , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/metabolismo , Citometría de Flujo , Humanos , Inmunización , Fosfatos de Inositol/metabolismo , Cinética , Ligandos , Ganglios Linfáticos/citología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/enzimología , Ganglios Linfáticos/inmunología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos BALB C , Péptidos/química , Péptidos/inmunología , Péptidos/metabolismo , Péptidos/farmacología , Receptores Colinérgicos/química , Receptores Colinérgicos/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimología , Fosfolipasas de Tipo C/metabolismoRESUMEN
In this report we summarize evidence to support a model for the development of Graves' disease. The model suggests that Graves' disease is initiated by an insult to the thyrocyte in an individual with a normal immune system. The insult, infectious or otherwise, causes double strand DNA or RNA to enter the cytoplasm of the cell. This causes abnormal expression of major histocompatibility (MHC) class I as a dominant feature, but also aberrant expression of MHC class II, as well as changes in genes or gene products needed for the thyrocyte to become an antigen presenting cell (APC). These include increased expression of proteasome processing proteins (LMP2), transporters of antigen peptides (TAP), invariant chain (Ii), HLA-DM, and the co-stimulatory molecule, B7, as well as STAT and NF-kappaB activation. A critical factor in these changes is the loss of normal negative regulation of MHC class I, class II, and thyrotropin receptor (TSHR) gene expression, which is necessary to maintain self-tolerance during the normal changes in gene expression involved in hormonally-increased growth and function of the cell. Self-tolerance to the TSHR is maintained in normals because there is a population of CD8- cells which normally suppresses a population of CD4+ cells that can interact with the TSHR if thyrocytes become APCs. This is a host self-defense mechanism that we hypothesize leads to autoimmune disease in persons, for example, with a specific viral infection, a genetic predisposition, or even, possibly, a TSHR polymorphism. The model is suggested to be important to explain the development of other autoimmune diseases including systemic lupus or diabetes.
Asunto(s)
Enfermedad de Graves/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Regulación de la Expresión Génica , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Sistema Inmunológico/inmunología , Autotolerancia/inmunología , Timo/citología , Tiroiditis Autoinmune/inmunología , Tirotropina/inmunologíaRESUMEN
A peptide based on the sequence of the complementarity determining regions 1 (pCDR1) of a pathogenic murine monoclonal anti-DNA antibody (5G12) that bears the 16/6 Id, was synthesized. This peptide was shown to be immunodominant in BALB/c mice, and induced a mild lupus-like disease upon immunization. Furthermore, the pCDR1 when injected in a soluble form was capable of inhibiting the proliferation of lymph node cells primed to either the peptide or the anti-DNA, 16/6 Id antibodies of either murine (5C12) or human (16/6 Id) origin. We have designed and synthesized 39 analogs based on pCDRI with single amino acid substitutions. Out of the above, two analogs, namely, Asp14 and Ser16 inhibited the proliferative responses of a pCDR1-specific T cell line to its stimulating peptide by more than 50%. These two analogs were therefore further studied. Administration of analog Ser16 concomitant with the immunization with pCDR1 inhibited efficiently the proliferative responses of lymph node cells to pCDR1, although pCDR1 was more efficient in its inhibitory capacity. Neither of the analogs were capable of inhibiting significantly the proliferative responses to the human monoclonal anti-DNA antibody with the 16/6 Id whereas pCDR1 did so efficiently. Thus, pCDR1 is more efficient than all its tested analogs in immunomodulating SLE associated immune responses.
Asunto(s)
Anticuerpos Antinucleares/inmunología , Regiones Determinantes de Complementariedad/inmunología , Péptidos/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , División Celular , Línea Celular , Femenino , Humanos , Lupus Eritematoso Sistémico , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Péptidos/síntesis químicaRESUMEN
Myasthenia gravis (MG) is a T cell-regulated antibody-mediated autoimmune disease. Immunization with two myasthenogenic peptides, p195-212 and p259-271, that are sequences of the human acetylcholine receptor alpha subunit was shown to induce experimental autoimmune MG (EAMG)-associated immune responses. A peptide composed of the two altered peptide ligands (APL) of the myasthenogenic peptides (designated as dual APL) inhibited, in vitro and in vivo, those responses. The objectives of this study were to examine (i) whether in vivo T cell activation by p259-271 affects the cytokine profile and the T cell migration ability, and (ii) whether the latter are immunomodulated by in vivo administration of the dual APL. Our results showed that immunization of mice with p259-271 enriched the population of lymph node and spleen cells with subsets of T cells with strong adhesiveness towards E- and P-selectins. This enrichment was associated with an acquisition of a T(h)1-type cytokine profile. Treatment of the immunized mice with the dual APL interfered with both the migratory potential of the autoreactive T cells, and the production of the T(h)1-type cytokines IL-2 and IFN-gamma (known to play a pathogenic role in MG and EAMG). T cells derived from APL-treated mice acquired a T(h)3-type cytokine profile, characterized by the secretion of the immunosuppresive cytokine transforming growth factor-ss. Thus, our results suggest that T cell selectin ligands and T cell-derived cytokines are involved in the induction and immunomodulation of EAMG- and MG-associated T cell responses.
Asunto(s)
Adhesión Celular , Citocinas/análisis , Endotelio/metabolismo , Miastenia Gravis Autoinmune Experimental/inmunología , Selectinas/metabolismo , Linfocitos T/inmunología , Animales , Movimiento Celular , Células Cultivadas , Humanos , Inmunización , Interferón gamma/análisis , Interleucina-2/análisis , Ganglios Linfáticos/inmunología , Linfotoxina-alfa/análisis , Ratones , Péptidos/inmunología , Receptores Colinérgicos/inmunología , Bazo/inmunologíaRESUMEN
The parent-into-immunocompetent-F(1) model of graft-vs-host disease (GVHD) induces immune dysregulation, resulting in acute or chronic GVHD. The disease outcome is thought to be determined by the number of parental anti-F(1) CTL precursor cells present in the inoculum. Injection of C57BL/6 (B6) splenocytes into (B6 x DBA/2)F(1) (B6D2F(1)) mice (acute model) leads to extensive parental cell engraftment and early death, whereas injection of DBA/2 cells (chronic model) results in little parental cell engraftment and a lupus-like disease. This study demonstrated that injection of BALB/c splenocytes into (BALB/c x B6)F(1) (CB6F(1)) mice resulted in little engraftment of parental lymphocytes and the development of lupus as expected. Injection of B6 splenocytes into CB6F(1) initiated an initial burst of parental cell engraftment similar to that of B6 into B6D2F(1). However, the acute disease resolved, and the CB6F(1) mice went on to develop chronic GVHD with detectable Abs to ssDNA, dsDNA, and extractable nuclear Ags. Limiting dilution CTL assays determined that B6 splenocytes have CTL precursor frequencies of 1/1000 against both CB6F(1) and B6D2F(1), whereas DBA/2 and BALB/c splenocytes have a CTL precursor frequency of 1/20,000 for their respective F(1)s. The Th cell precursor frequency for B6 anti-DBA/2 was 3-fold higher than that for B6 anti-BALB/c determined by limiting dilution proliferation assays. These results indicate the importance of adequate allospecific helper as well as effector T cells for the induction and maintenance of acute GVHD in this model, and presents an unexpected model in which initial acute GVHD is replaced by the chronic form of disease.
Asunto(s)
Cruzamientos Genéticos , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Aguda , Animales , Enfermedades Autoinmunes/genética , Enfermedad Crónica , Pruebas Inmunológicas de Citotoxicidad , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Enfermedad Injerto contra Huésped/patología , Antígenos H-2/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Especificidad de la Especie , Bazo/inmunología , Bazo/patología , Bazo/trasplante , Células Madre/inmunología , Células Madre/patología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/patología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
A peptide based on the complementarity determining region (CDR) 3 of a pathogenic anti-DNA 16/6 Id(+) monoclonal antibody was previously shown to be a dominant T-cell epitope in experimental SLE, and to be capable of inhibiting SLE-associated proliferative responses. Single amino acid-substituted analogs of pCDR3 were designed and analyzed for their ability to stimulate or inhibit the proliferation of a pCDR3-specific T-cell line. Alterations in positions 9 and 10 neutralized the proliferative potential of pCDR3, whereas alterations in positions 6-8 and 11-15 retained the proliferative potential of the peptides. Similar to pCDR3, its analogs Ala11 and Nle13 inhibited efficiently the in vivo priming of lymph node cells either to pCDR3 or to the human monoclonal anti-DNA 16/6 Id(+) antibody. Substituting both positions 11 (Tyr --> Ala) and 13 (Met --> Nle) reduced this inhibitory capacity compared to the single substituted analogs. Also, truncation of pCDR3 at the C- and/or N-terminus obliterated the inhibitory activities of the peptide. Analogs Ala11 and Nle13 immunomodulated serological and clinical smanifestations of experimental SLE. Nevertheless, the original pCDR3 was a more efficient modulator of the disease.
Asunto(s)
Autoanticuerpos/inmunología , Regiones Determinantes de Complementariedad/inmunología , ADN/inmunología , Lupus Eritematoso Sistémico/inducido químicamente , Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Femenino , Idiotipos de Inmunoglobulinas , Lupus Eritematoso Sistémico/inmunología , Ratones , Ratones Endogámicos , Datos de Secuencia MolecularRESUMEN
We have examined the humoral and cellular responses of SLE patients to peptides based on the complementarity-determining regions (CDR) of a monoclonal anti-DNA antibody with a major idiotype- 16/6 Id, in comparison to their responses to the whole 16/6 Id-bearing antibody. Sera of 63% of the SLE patients had antibodies that bound the 16/6 Id, 80% had antibodies to one of the CDR-based peptides, and 40% of the patients reacted with both CDRs. Sera of only a few controls reacted with either the 16/6 Id (6%) or the CDR based peptides (4%) (P < 0.01). Peripheral blood lymphocytes (PBL) of 39% of the patients proliferated in response to the 16/6 Id or to one of the CDR-based peptides (37%), while in the control group the proliferation rates were 66% to the 16/6 Id and 59% to one of the CDR-based peptides (P < 0.05). The correlation between (both) the humoral and cellular immune responses to the CDR-based peptides and to the 16/6 Id suggests the relevance of these peptides to the 16/6 Id and provides additional information on the pathogenic moiety of the latter antibody.
Asunto(s)
Anticuerpos Antinucleares/genética , Anticuerpos Monoclonales/genética , Regiones Determinantes de Complementariedad/genética , Lupus Eritematoso Sistémico/inmunología , Péptidos/genética , Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiidiotipos/sangre , Estudios de Casos y Controles , Humanos , Inmunidad Celular , Idiotipos de Inmunoglobulinas/genética , Técnicas In Vitro , Activación de Linfocitos , Ratones , Datos de Secuencia MolecularRESUMEN
Two peptides based on the complementarity-determining regions (CDR) of a pathogenic murine anti-DNA antibody were employed in an attempt to prevent the spontaneous systemic lupus erythematosus (SLE)-like disease of (NZBxNZW)F1 mice. Female mice, at the age of 2 months, were injected with either the CDR1- or the CDR3-based peptides (pCDR1, pCDR3) subcutaneously or intravenously in aqueous solution for a total of 8-10 treatments. A reduction was observed in the total and pathogenic IgG2a and IgG3 anti-DNA antibody titers in the CDR-treated groups. Treatment reduced the number of mice that developed proteinuria and immune complex deposits in their kidneys. The severity of renal pathology was significantly reduced in the pCDR3 (P<0.02) and pCDR1 (P< or = 0.05) treated mice. Thus, both CDR-based peptides administered in aqueous solution were capable of preventing the SLE-like disease in (NZBxNZW)F1 mice, although the beneficial effects of pCDR3 appeared to be more pronounced than those of pCDR1 in the treated mice.
Asunto(s)
Anticuerpos Antinucleares/química , Anticuerpos Monoclonales/química , Enfermedades Autoinmunes/prevención & control , Fragmentos de Inmunoglobulinas/uso terapéutico , Inmunoterapia , Lupus Eritematoso Sistémico/prevención & control , Secuencia de Aminoácidos , Animales , Anticuerpos Antinucleares/sangre , Anticuerpos Antinucleares/inmunología , Anticuerpos Monoclonales/inmunología , Enfermedades Autoinmunes/inmunología , Modelos Animales de Enfermedad , Femenino , Inmunización Pasiva , Fragmentos de Inmunoglobulinas/administración & dosificación , Fragmentos de Inmunoglobulinas/inmunología , Inmunoglobulina G/sangre , Inyecciones Intravenosas , Inyecciones Subcutáneas , Riñón/patología , Lupus Eritematoso Sistémico/inmunología , Nefritis Lúpica/patología , Nefritis Lúpica/prevención & control , Ratones , Ratones Endogámicos NZB , Ratones Endogámicos , Datos de Secuencia MolecularRESUMEN
A molecular homology has been demonstrated between sequences of the heavy chain variable regions of the anti-DNA, anti-cardiolipin monoclonal antibody, 2C4C2, isolated from C3H.SW mice with induced systemic lupus erythematosus, and sequences of the anti-DNA monoclonal antibody BW16 originating in the lupus-prone (NZBXNZW)F1 mice. It was of interest to determine whether these homologous sequences function also as immunodominant T-cell epitopes, in order to establish a connection between spontaneous and induced experimental models. Therefore, three peptides were designed and synthesized based on the complementarity determining region (CDR)1, CDR2 and CDR3 of the heavy chain of the monoclonal antibody 2C4C2. In the present study, we compare these peptides with the CDR1- and CDR3-based peptides of another murine anti-DNA antibody; namely, 5G12. The comparison was carried out by analyzing the ability of the peptides to induce T-cell activation in (NZBXNZW)F1 lupus-prone mice and in mouse strains susceptible to induction of experimental systemic lupus erythematosus. Immunization of (NZBXNZW)F1 mice with the 2C4C2 mAb or with its CDR-based peptides, as well as immunization with the 5G12-based CDR peptides, induced significant lymph node proliferation to the pCDR3 of the 5G12 mAb. Naive (NZBXNZW)F1 splenocytes exhibited activation to the same peptide. It is also shown that MHC class II molecules of (NZBXNZW)F1 macrophages bind preferentially the 5G12-based pCDR3. It is proposed that the CDR3-based peptide of 5G12 mAb of experimental lupus is also a dominant and relevant epitope in the (NZBXNZW)F1 lupus-prone mice.
Asunto(s)
Anticuerpos Antinucleares/inmunología , Regiones Determinantes de Complementariedad , Epítopos de Linfocito T/inmunología , Epítopos Inmunodominantes/inmunología , Región Variable de Inmunoglobulina/inmunología , Lupus Eritematoso Sistémico/inmunología , Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , ADN/inmunología , Femenino , Inmunización , Región Variable de Inmunoglobulina/química , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Linfocitos T/inmunologíaRESUMEN
Aging mice of strains susceptible to the induction of experimental systemic lupus erythematosus (SLE) develop a milder disease than young animals. To find out whether the decrease in susceptibility to disease is due to age-associated changes in cytokine profile, we first examined the secretion of cytokines by healthy mice aged 2-15 months. A gradual age-related decline in the levels of interleukin (IL)-2 and interferon (IFN) gamma, and an increase in IL-4, IL-10, IL-1, and tumor necrosis factor (TNF) alpha were observed. Experimental SLE was induced in 2- and 10-month-old mice by immunization with the monoclonal anti-DNA antibody bearing the 16/6 Id. Early increased production of pro-inflammatory cytokines (TNFalpha and IL-1), followed by a peak of the Th1-type cytokines (IL-2, IFNgamma) were observed in young mice. The Th2-type cytokines (IL-4, IL-10) peaked later. In contrast, only a mild increase in all of the above cytokines was determined in 10-month immunized mice. It thus appears that the decline in susceptibility to SLE induction in older mice may be related to changes in the capacity to produce cytokines.