RESUMO
INTRODUCTION: Systemic lupus erythematosus (SLE) is characterized by a variety of autoantibodies and systemic clinical manifestations. A tolerogenic peptide, hCDR1, ameliorated lupus manifestations in mice models. The objectives of this study were to induce experimental SLE in pigs and to determine the ability of hCDR1 to immunomodulate the disease manifestations. RESULTS AND DISCUSSION: We report here the successful induction, by a monoclonal anti-DNA antibody, of an SLE-like disease in pigs, manifested by autoantibody production and glomerular immune complex deposits. Treatment of pigs with hCDR1 ameliorated the lupus-related manifestations. Furthermore, the treatment downregulated the gene expression of the pathogenic cytokines, interleukin (IL)-1beta, tumor necrosis factor alpha, interferon gamma, and IL-10, and upregulated the expression of the immunosuppressive cytokine transforming growth factor beta, the antiapoptotic molecule Bcl-xL, and the suppressive master gene, Foxp3, hence restoring the expression of the latter to normal levels. Thus, hCDR1 is capable of ameliorating lupus in large animals and is a potential candidate for the treatment of SLE patients.
Assuntos
Anticorpos Antinucleares/administração & dosagem , Regiões Determinantes de Complementaridade/administração & dosagem , Rim/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Fragmentos de Peptídeos/administração & dosagem , Animais , Anticorpos Antinucleares/química , Anticorpos Monoclonais/imunologia , Células Cultivadas , Regiões Determinantes de Complementaridade/química , Citocinas/biossíntese , Citocinas/genética , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Tolerância Imunológica , Imunomodulação , Rim/imunologia , Lúpus Eritematoso Sistêmico/fisiopatologia , Lúpus Eritematoso Sistêmico/terapia , Camundongos , Fragmentos de Peptídeos/química , Suínos/imunologia , Proteína bcl-X/biossíntese , Proteína bcl-X/genéticaRESUMO
The Golgi is a dynamic organelle whose correct assembly is crucial for cellular homeostasis. Perturbations in Golgi structure are associated with numerous disorders from neurodegeneration to cancer. However, whether and how dispersal of the Golgi apparatus is actively regulated under stress, and the consequences of Golgi dispersal, remain unknown. Here we demonstrate that 26S proteasomes are associated with the cytosolic surface of Golgi membranes to facilitate Golgi Apparatus-Related Degradation (GARD) and degradation of GM130 in response to Golgi stress. The degradation of GM130 is dependent on p97/VCP and 26S proteasomes, and required for Golgi dispersal. Finally, we show that perturbation of Golgi homeostasis induces cell death of multiple myeloma in vitro and in vivo, offering a therapeutic strategy for this malignancy. Taken together, this work reveals a mechanism of Golgi-localized proteasomal degradation, providing a functional link between proteostasis control and Golgi architecture, which may be critical in various secretion-related pathologies.
Assuntos
Complexo de Golgi/metabolismo , Ionóforos/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteostase/fisiologia , Animais , Apoptose/efeitos dos fármacos , Autoantígenos/metabolismo , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Complexo de Golgi/efeitos dos fármacos , Células HEK293 , Humanos , Membranas Intracelulares/metabolismo , Ionóforos/farmacologia , Proteínas de Membrana/metabolismo , Camundongos , Monensin/farmacologia , Monensin/uso terapêutico , Mieloma Múltiplo/patologia , Proteólise/efeitos dos fármacos , Proteostase/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Proteína com Valosina/metabolismoRESUMO
Systemic lupus erythematosus (SLE) is characterized in its early stages by the expansion of autoreactive T cells that trigger B-cell activation with subsequent multi-organ injury. Dendritic cells (DCs) in lupus were found to display an aberrant phenotype with higher expression of the maturation markers major histocompatibility complex (MHC) class II, CD80 and CD86, as well as higher production of proinflammatory cytokines including interleukin-12 (IL-12), resulting in an increased ability to activate T cells. A peptide (hCDR1) based on the complementarity determining region-1 of an anti-DNA antibody ameliorated SLE in both induced and spontaneous lupus models by downregulating T-cell functions. Our objectives were to determine whether DCs play a role in promoting the beneficial effects of hCDR1. We showed here that treatment with hCDR1 lowered the expression levels of MHC class II, CD80 and CD86 on DCs. The latter effect was associated with downregulation of messenger RNA expression and secretion of IL-12, a cytokine that upregulated T-cell proliferation and interferon-gamma (IFN-gamma) secretion. Moreover, DCs derived from hCDR1-treated mice downregulated proliferation and IFN-gamma secretion by T cells from untreated mice. Upregulation of transforming growth factor-beta (TGF-beta) secretion by T cells, following treatment with hCDR1, resulted in downregulation of IFN-gamma production and contributed to the phenotypic changes and magnitude of IL-12 secretion by DCs. The ameliorating effects of hCDR1 are therefore mediated at least partially by the upregulated secretion of TGF-beta by T cells that contribute to the induction of DCs with immature phenotype and suppressed functions. The resulting DCs further downregulate autoreactive T-cell functions.
Assuntos
Autoantígenos/uso terapêutico , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Proteínas do Tecido Nervoso/uso terapêutico , Animais , Autoantígenos/farmacologia , Antígeno B7-1/efeitos dos fármacos , Antígeno B7-1/imunologia , Antígeno B7-2/efeitos dos fármacos , Antígeno B7-2/imunologia , Células Dendríticas/efeitos dos fármacos , Regulação para Baixo , Feminino , Antígenos de Histocompatibilidade Classe II/efeitos dos fármacos , Humanos , Interferon gama/agonistas , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-12/antagonistas & inibidores , Interleucina-12/imunologia , Interleucina-12/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Tecido Nervoso/farmacologia , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fator de Crescimento Transformador beta/farmacologia , Regulação para CimaRESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by dysregulation of cytokines, apoptosis, and B- and T-cell functions. The tolerogenic peptide, hCDR1 (Edratide), ameliorated the clinical manifestations of murine lupus via down-regulation of pro-inflammatory cytokines and apoptosis, up-regulation of the immunosuppressive cytokine TGF-beta, and the induction of regulatory T-cells. In the present study, gene expression was determined in peripheral blood mononuclear cells of 9 lupus patients that were treated for 26 weeks with either hCDR1 (five patients), or placebo (four patients). Disease activity was assessed by SLEDAI-2K and the BILAG scores. Treatment with hCDR1 significantly down-regulated the mRNA expression of the pathogenic cytokines IL-1beta, TNF-alpha, IFN-gamma, and IL-10, of BLyS (B-lymphocyte stimulator) and of the pro-apoptotic molecules caspase-3 and caspase-8. In contrast, the treatment up-regulated in vivo gene expression of both TGF-beta and FoxP3. Furthermore, hCDR1 treatment resulted in a significant decrease in SLEDAI-2K (from 8.0+/-2.45 to 4.4+/-1.67; P=0.02) and BILAG (from 8.2+/-2.7 to 3.6+/-2.9; P=0.03) scores. Thus, the tolerogenic peptide hCDR1, immunomodulates, in vivo, the expression of genes that play a role in SLE, consequently restoring the global immune dysregulation of lupus patients. Hence, hCDR1 has a potential role as a novel disease-specific treatment for lupus patients.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Fatores de Transcrição Forkhead/metabolismo , Imunoterapia , Leucócitos Mononucleares/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Fragmentos de Peptídeos/administração & dosagem , Adulto , Apoptose/efeitos dos fármacos , Caspases/genética , Caspases/imunologia , Caspases/metabolismo , Regiões Determinantes de Complementaridade/imunologia , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Progressão da Doença , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/fisiopatologia , Lúpus Eritematoso Sistêmico/terapia , Masculino , Pessoa de Meia-Idade , Tolerância a Antígenos Próprios/imunologia , Índice de Gravidade de Doença , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismo , Resultado do TratamentoRESUMO
Sexual preference for the opposite sex is a fundamental behavior underlying reproductive success, but the neural mechanisms remain unclear. Here, we examined the role of dopamine signaling in the nucleus accumbens core (NAcc) in governing chemosensory-mediated preference for females in TrpC2-/- and wild-type male mice. TrpC2-/- males, deficient in VNO-mediated signaling, do not display mating or olfactory preference toward females. We found that, during social interaction with females, TrpC2-/- males do not show increased NAcc dopamine levels, observed in wild-type males. Optogenetic stimulation of VTA-NAcc dopaminergic neurons in TrpC2-/- males during exposure to a female promoted preference response to female pheromones and elevated copulatory behavior toward females. Additionally, we found that signaling through the D1 receptor in the NAcc is necessary for the olfactory preference for female-soiled bedding. Our study establishes a critical role for the mesolimbic dopaminergic system in governing pheromone-mediated responses and mate choice in male mice.
Assuntos
Dopamina/metabolismo , Núcleo Accumbens/fisiologia , Receptores de Dopamina D1/genética , Comportamento Sexual Animal/fisiologia , Canais de Cátion TRPC/genética , Órgão Vomeronasal/fisiologia , Animais , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Núcleo Accumbens/citologia , Optogenética , Receptores de Dopamina D1/metabolismo , Atrativos Sexuais/biossíntese , Atrativos Sexuais/metabolismo , Transdução de Sinais , Olfato/fisiologia , Canais de Cátion TRPC/deficiência , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/fisiologia , Órgão Vomeronasal/citologiaRESUMO
The steroidogenic enzyme 21-hydroxylase (21OH) is the main autoantigen in autoimmune primary adrenal failure (Addison's disease). Autoantibodies against 21OH are immunological markers of an ongoing autoimmune process but are not directly involved in the tissue destruction. Autoreactive T cells are thought to mediate tissue damage, but the T cell antigen(s) has not been identified. To find out whether 21OH contains important immunodominant epitopes for T cells, we first immunized BALB/c and SJL inbred mouse strains with recombinant 21OH and showed that lymph node cells proliferated effectively following in vitro stimulation with recombinant 21OH (stimulation indices (SI) 20-40). We further synthesized a series of peptides based on 21OH with amino acid sequences with propensity to bind to major histocompatibility complex class II molecules. Only a few peptides could trigger lymphocytes of 21OH-primed mice to proliferate. One of these, 21OH (342-361), stimulated effectively 21OH-primed lymph node cells of SJL mice (SI = 4-8) and also, although to a lesser extent, of BALB/c mice (SI = 2.5). When SJL mice were immunized with 21OH (342-361), the immunizing peptide as well as peptide 21OH (346-361) triggered a significant proliferative response (SI = 24). A peptide from another part of 21OH, namely 21OH (191-202), did not stimulate the 21OH (342-361)-primed cells. Moreover, stimulation of lymph node cells of mice immunized with 21OH (342-361) with 21OH resulted in a significant proliferative response. We conclude that 21OH (342-361) is an immunodominant determinant for T cells in SJL and probably BALB/c mice. 21OH (342-361) corresponds to the substrate binding site of the enzyme. The p342-361 region may be involved in the pathogenesis of autoimmune adrenal failure in humans.
Assuntos
Doença de Addison/imunologia , Doenças Autoimunes/imunologia , Epitopos de Linfócito T/química , Esteroide 21-Hidroxilase/química , Animais , Anticorpos Monoclonais/química , Antígenos CD4/biossíntese , Antígenos CD8/biossíntese , Proliferação de Células , Relação Dose-Resposta a Droga , Epitopos/química , Feminino , Humanos , Epitopos Imunodominantes , Linfonodos/metabolismo , Linfonodos/patologia , Metástase Linfática , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Especificidade por Substrato , Linfócitos T/metabolismoRESUMO
IL-1 is one of the most pleiotropic pro-inflammatory and immunostimulatory cytokines. Overproduction of IL-1 has been shown to be involved in the pathogenicity of various autoimmune inflammatory diseases, including systemic lupus erythematosus (SLE). However, the different contributions that the IL-1 agonistic molecules make in their in vivo native milieu, IL-1beta which is mainly secreted against IL-1alpha which is mainly cell-associated, have not been established. Experimental SLE can be induced in mice by injection with monoclonal anti-DNA antibodies bearing a major idiotype designated, 16/6Id. In the present study, experimental SLE was induced in mice deficient in specific IL-1 molecules, i.e. IL-1alpha(-/-), IL-1beta(-/-), IL-1alpha/beta(-/-) (double KO) and in control BALB/c mice. Mice deficient in IL-1beta , i.e. IL-1beta(-/-) and IL-1alpha/beta(-/-) mice, developed lower levels of anti-dsDNA antibodies after immunization with 16/6Id, as compared to IL-1alpha(-/-) or control BALB/c mice. Disease manifestations were milder in mice deficient in IL-1beta expression. The representative cytokine cascade that is characteristic of overt experimental SLE was also shown to be reduced in groups of mice that lacked IL-1beta as compared to mice deficient in IL-1alpha, which is mainly cell-associated. Altogether, our results point to the importance of secretable IL-1beta, rather than cell-associated IL-1alpha, in the immunostimulatory and inflammatory phenomena that mediate the pathogenesis of experimental SLE.
Assuntos
Modelos Animais de Doenças , Interleucina-1/genética , Lúpus Eritematoso Sistêmico/genética , Animais , Anticorpos/sangue , Feminino , Predisposição Genética para Doença , Imunidade Inata/genética , Interleucina-1/deficiência , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos KnockoutRESUMO
Myasthenia gravis (MG) is a T cell-dependent, antibody-mediated autoimmune disease. A dual altered peptide ligand (APL) that is composed of the tandemly arranged two single amino acid analogs of two myasthenogenic peptides was demonstrated to downregulate in vitro and in vivo murine MG associated autoreactive responses. Furthermore, treatment with the dual APL ameliorated the clinical manifestations of an established experimental autoimmune MG in mice. This study was undertaken in order to investigate the ability of the dual APL to immunomodulate MG-associated responses of peripheral blood lymphocytes (PBL) of patients with MG to the native autoantigen acetylcholine receptor (AChR). PBL of 22 of 27 patients with MG tested responded by proliferation to torpedo AChR. The proliferative responses of PBL of 21 of 22 responders were significantly inhibited by the dual APL. The inhibition was specific because a control peptide did not inhibit these proliferative responses. The dual APL also downregulated the levels of the secreted pathogenic cytokine IFN-gamma in supernatants of stimulated PBL of 80% of the tested patients. The latter inhibitions correlated with an upregulated production of the immunosuppressive cytokine, tumor growth factor beta. Thus, the results of our study demonstrate that the dual APL is capable of downregulating in vitro autoreactive responses of patients with MG and suggest that this peptide is a potential candidate for a novel specific treatment of patients with MG.
Assuntos
Ativação Linfocitária/efeitos dos fármacos , Miastenia Gravis/imunologia , Peptídeos/farmacologia , Receptores Colinérgicos/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Animais , Autoanticorpos/imunologia , Autoantígenos/imunologia , Autoimunidade , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/biossíntese , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Ligantes , Masculino , Camundongos , Pessoa de Meia-Idade , Peptídeos/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologiaRESUMO
IL-12 plays a crucial role in a variety of immune responses that are involved in immuno-senesence and autoimmune diseases. Experimental SLE can be induced in mice by the monoclonal anti-DNA autoantibody bearing the 16/6Id. Aging mice are less susceptible to disease induction. We evaluated the in vivo effects of IL-12 on several parameters related to aging and to experimental SLE. Young (2 months) and aging (10 months) mice that were or were not immunized with the 16/6Id were treated with IL-12. IL-12 treated unimmunized BALB/c and C3H.SW mice had significant elevated levels of anti-DNA antibodies. A high percentage of these mice had glomerular immune complex deposits (ICD). IL-12 treatment resulted in significant increase of IL-1 and IFN-gamma and a decrease of IL-10 production in unimmunized mice. The same IL-12 induced changes were observed in mice with experimental SLE but were less pronounced than in normal mice. In general, all the effects in SLE afflicted mice were more prominent in aging than in young mice. Treatment of mice with experimental SLE upregulated IL-1, IL-2, IFNgamma and downregulated IL-10. These changes were observed in the groups of young as well as old mice. In conclusion, administration of IL-12 to aging mice reversed their Th1/Th2 cytokine profile and thus rendered them vulnerable to the induction of experimental SLE.
Assuntos
Envelhecimento/imunologia , Interleucina-12/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Animais , Anticorpos Antinucleares/biossíntese , Complexo Antígeno-Anticorpo/metabolismo , Citocinas/biossíntese , DNA/imunologia , Feminino , Glomérulos Renais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Células Th1/imunologia , Células Th2/imunologiaRESUMO
The laboratory mouse serves as an important model system for studying gene, brain and behavioural interactions. Powerful methods of gene targeting have helped to decipher gene-function associations in human diseases. Yet, the laboratory mouse, obtained after decades of human-driven artificial selection, inbreeding, and adaptation to captivity, is of limited use for the study of fitness-driven behavioural responses that characterize the ancestral wild house mouse. Here, we demonstrate that the backcrossing of wild mice with knockout mutant laboratory mice retrieves behavioural traits exhibited exclusively by the wild house mouse, thereby unmasking gene functions inaccessible in the domesticated mutant model. Furthermore, we show that domestication had a much greater impact on females than on males, erasing many behavioural traits of the ancestral wild female. Hence, compared with laboratory mice, wild-derived mutant mice constitute an improved model system to gain insights into neuronal mechanisms underlying normal and pathological sexually dimorphic social behaviours.
Assuntos
Comportamento Animal , Camundongos Knockout , Animais , Cruzamentos Genéticos , Feminino , Técnicas de Inativação de Genes , Genótipo , Hormônios/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fenótipo , Polimorfismo de Nucleotídeo Único , Olfato/genética , Comportamento Social , Canais de Cátion TRPC/genéticaRESUMO
Social behaviour has a key role in animal survival across species, ranging from insects to primates and humans. However, the biological mechanisms driving natural interactions between multiple animals, over long-term periods, are poorly studied and remain elusive. Rigorous and objective quantification of behavioural parameters within a group poses a major challenge as it requires simultaneous monitoring of the positions of several individuals and comprehensive consideration of many complex factors. Automatic tracking and phenotyping of interacting animals could thus overcome the limitations of manual tracking methods. Here we report a broadly applicable system that automatically tracks the locations of multiple, uniquely identified animals, such as mice, within a semi-natural setting. The system combines video and radio frequency identified tracking data to obtain detailed behavioural profiles of both individuals and groups. We demonstrate the usefulness of these data in characterizing individual phenotypes, interactions between pairs and the collective social organization of groups.
Assuntos
Comportamento Animal/fisiologia , Meio Ambiente , Dispositivo de Identificação por Radiofrequência/métodos , Comportamento Social , Animais , Automação , Cruzamentos Genéticos , Coleta de Dados , Feminino , Hierarquia Social , Locomoção , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Fatores de Tempo , Gravação em VídeoRESUMO
A tolerogenic peptide, hCDR1, ameliorated murine lupus via the upregulation of functional regulatory cells and by immunomodulating cytokine production. In the present study we analyzed the ability of hCDR1 to similarly affect gene expression and regulatory T cells when incubated with peripheral blood mononuclear cells (PBMC) of lupus patients. To this end, peripheral blood mononuclear cells (PBMC) of 11 lupus patients and five gender- and age-matched healthy controls were cultured with hCDR1 or a control peptide. Gene expression and regulatory T-cells were assessed. hCDR1 significantly downregulated interleukin (IL)-1beta, interferon (IFN)-gamma, and IL-10 gene expression. Furthermore, hCDR1 upregulated the expression of the anti-apoptotic Bcl-xL molecule and downregulated the pro-apoptotic caspase-3, resulting in reduced rates of apoptosis. hCDR1 increased the expression of transforming growth factor (TGF)-beta, FoxP3 and the negative regulators Foxj1 and Foxo3a. No significant effects were observed using a control peptide or when PBMC of healthy donors were incubated with hCDR1. The elevated gene expression of FoxP3 was due to hCDR1-induced upregulation of TGF-beta, resulting in an increase of CD4+CD25+FoxP3+ functional, regulatory cells. The ability of the regulatory cells to diminish IFN-gamma expression and to upregulate TGF-beta was abrogated after the addition of a neutralizing anti-CD25 antibody, confirming their role in the beneficial effects of hCDR1.
Assuntos
Regiões Determinantes de Complementaridade/imunologia , Citocinas/metabolismo , Leucócitos Mononucleares/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Apoptose/imunologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/farmacologia , Citocinas/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Tolerância Imunológica , Terapia de Imunossupressão , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Lúpus Eritematoso Sistêmico/sangue , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Peptídeos/química , Peptídeos/imunologia , Peptídeos/farmacologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia , Regulação para Cima/efeitos dos fármacosRESUMO
Expansion of autoreactive T cells and their resistance to anergy was demonstrated in systemic lupus erythematosus (SLE). A pair of transcription factors, early growth response 2 (Egr-2) and 3 (Egr-3), are negative regulators of T cell activation that were shown to be important in anergy. A peptide (designated hCDR1 for human CDR1) based on the CDR-1 of an anti-DNA Ab ameliorated SLE in both induced and spontaneous lupus models. Our objectives were to determine the expression levels of Egr-2 and Egr-3 in autoreactive T cells following immunization with the lupus-inducing anti-DNA Ab that bears a common Id designated 16/6Id and also in a full-blown SLE and to determine the effect of hCDR1 on these transcription factors. We demonstrated diminished expression levels of Egr-2 and Egr-3 mRNA both early after immunization with the 16/6Id and in SLE-afflicted (NZB x NZW)F1 (New Zealand Black and New Zealand White) mice. Furthermore, by down-regulating Akt phosphorylation and up-regulating TGFbeta secretion, treatment with hCDR1 significantly up-regulated Egr-2 and Egr-3 expression. This was associated with an increased expression of the E3 ligase Cbl-b. Inhibition of Akt in T cells of immunized mice decreased, whereas silencing of the Egr-2 and Egr-3 in T cells of hCDR1-treated mice increased IFN-gamma secretion. Thus, hCDR1 down-regulates Akt phosphorylation, which leads to up-regulated expression of T cell Egr-2 and Egr-3, resulting in the inhibition of IFN-gamma secretion that is required for the maintenance of SLE.
Assuntos
Anticorpos Monoclonais/farmacologia , Anergia Clonal/efeitos dos fármacos , Proteína 2 de Resposta de Crescimento Precoce/antagonistas & inibidores , Proteína 3 de Resposta de Crescimento Precoce/antagonistas & inibidores , Lúpus Eritematoso Sistêmico/imunologia , Fragmentos de Peptídeos/farmacologia , Animais , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Proteína 3 de Resposta de Crescimento Precoce/genética , Proteína 3 de Resposta de Crescimento Precoce/metabolismo , Humanos , Interferon gama/metabolismo , Interleucina-2/farmacologia , Camundongos , Camundongos Endogâmicos , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fator de Crescimento Transformador beta1/metabolismo , Regulação para CimaRESUMO
OBJECTIVE: To identify genes that are differently expressed in (NZB x NZW)F(1) mice with established lupus compared with healthy controls, and to determine how gene expression is affected by treatment with hCDR1 (Edratide), a peptide synthesized on the basis of the sequence of the first complementarity-determining region (CDR1) of an autoantibody. METHODS: RNA was extracted from spleen cells of young, disease-free mice and of older mice with systemic lupus erythematosus (SLE) that were treated with hCDR1 or with vehicle alone. Gene expression was assessed using the DNA microarray technique and verified by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: In mice with SLE, numerous genes showed increased or decreased expression relative to that in the disease-free controls. Treatment with hCDR1 restored the expression of many of these genes to control levels. Real-time RT-PCR verified that in diseased mice RNA transcripts of Tnfsf4, Il5ra, Zbtb20, and Nid1 were up-regulated, while transcripts of Tfpi and S100a8 were down-regulated, and confirmed the effects of hCDR1 on the expression of those genes. Kidney immunostaining demonstrated that the up-regulated expression of OX40 ligand, which is a protein product of the gene tumor necrosis factor (ligand) superfamily member 4, in diseased mice was reduced by hCDR1. CONCLUSION: Expression of numerous genes in mice with SLE differs from that in young, disease-free control mice. Treatment with hCDR1 restores the expression of 22% of these genes to levels similar to those in controls. Thus, one of the mechanisms by which hCDR1 exerts its beneficial effects on the clinical symptoms of SLE is through regulation of gene expression.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/genética , Fragmentos de Peptídeos/uso terapêutico , Sequência de Aminoácidos , Animais , Primers do DNA , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Fragmentos de Peptídeos/química , RNA/isolamento & purificação , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/patologiaRESUMO
Experimental systemic lupus erythematosus (SLE) can be induced in mice following immunization with an anti-DNA mAb expressing a major Id, 16/6Id. Treatment with a peptide, designated human CDR1 (hCDR1; Edratide), that is based on the sequence of CDR1 of the 16/6Id ameliorated disease manifestations. In the present study, we investigated the roles of apoptosis and related molecules in BALB/c mice with induced experimental SLE following treatment with hCDR1. A higher state of activation and increased rate of apoptosis were found in lymphocytes of SLE-afflicted mice as compared with healthy controls. The latter effects were associated with up-regulated caspase-8 and caspase-3, and down-regulated Bcl-x(L). The ameliorative effects of hCDR1 were associated with down-regulation of caspase-8 and caspase-3, up-regulation of Bcl-x(L), and a reduced rate of apoptosis. Treatment of diseased mice with an apoptosis-reducing compound that inhibited caspases down-regulated the secretion of the pathogenic cytokine IFN-gamma and lowered the intensity of glomerular immune complex deposits and the levels of proteinuria. Furthermore, coincubation of Bcl-x(L) inhibitors with hCDR1-treated cells abrogated the ability of hCDR1 to reduce the activation state of lymphocytes and to down-regulate the secretion of IL-10 and IFN-gamma. Moreover, the Bcl-x(L)-expressing CD4(+)CD25(+) cells from hCDR1-treated mice induced the expression of Bcl-x(L) in CFSE-labeled CD4(+)CD25(-) cells of the SLE-afflicted mice. Thus, the reduction of apoptosis and the up-regulation of Bcl-x(L), which plays an apparent role in tolerance induction, contribute to at least part of the beneficial effects of hCDR1 on lupus manifestations.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/prevenção & controle , Proteínas do Tecido Nervoso/fisiologia , Peptídeos/fisiologia , Sequência de Aminoácidos , Animais , Caspase 3/biossíntese , Caspase 8/biossíntese , Inibidores de Caspase , Modelos Animais de Doenças , Regulação para Baixo/imunologia , Feminino , Humanos , Lúpus Eritematoso Sistêmico/enzimologia , Lúpus Eritematoso Sistêmico/patologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Regulação para Cima/imunologia , Proteína bcl-X/biossínteseRESUMO
Myasthenia gravis (MG) and experimental autoimmune MG are T cell-dependent antibody-mediated autoimmune diseases. A dual altered peptide ligand (APL), composed of the tandemly arranged two single amino acid analogs of two myasthenogenic peptides, p195-212 and p259-271, down-regulated in vitro and in vivo MG-associated T cell responses. In the present study, we investigated the role of CD8(+)CD28(-) regulatory cells in the mechanism of action of the dual APL. We demonstrated that treatment of mice with the dual APL concomitant with immunization with a myasthenogenic peptide resulted in an increased population of CD8(+)CD28(-) cells that express forkhead box P3 (Foxp3). The dual APL inhibited the proliferation of lymph node (LN) cells of the Torpedo acetylcholine receptor-immunized WT C57BL/6 mice, whereas the inhibition was abrogated in CD8(-/-) knockout mice. Moreover, the dual APL did not inhibit the secretion of IFN-gamma by LN cells from CD8(-/-) mice immunized with Torpedo acetylcholine receptor. However, the mRNA expression of IL-10 and TGF-beta by LN cells from CD8(-/-) mice was up-regulated similarly to that of the WT mice. Furthermore, the dual APL elevated the proapoptotic markers caspases 3 and caspase 8, whereas it down-regulated the antiapoptotic marker Bcl-xL in both CD8(-/-) and WT mice. Finally, the dual APL-induced CD4(+)CD25(+)Foxp3(+) cells were up-regulated in CD8(-/-) mice to a similar extent to that observed in the WT mice. Thus, we suggest that CD8(+)CD28(-) regulatory cells play a partial role in the mechanism of action by which the dual APL suppresses experimental autoimmune MG-associated T cell responses.
Assuntos
Antígenos CD28/metabolismo , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Miastenia Gravis/metabolismo , Peptídeos/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD8/genética , Caspase 3/metabolismo , Caspase 8/metabolismo , Proliferação de Células , Células Cultivadas , Regulação para Baixo , Feminino , Fatores de Transcrição Forkhead/metabolismo , Imunização , Interleucina-10/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Ligantes , Camundongos , Camundongos Knockout , Miastenia Gravis/imunologia , Receptores Nicotínicos/imunologia , Torpedo/imunologia , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima , Proteína bcl-X/metabolismoRESUMO
A peptide (hCDR1) based on the sequence of the complementarity-determining region-1 of an anti-DNA autoantibody ameliorates clinical manifestations of lupus. We analyzed the beneficial effects of hCDR1 when given alone or in combination with dexamethasone, while comparing the mechanisms of action of the latter. Treatment with either hCDR1 or dexamethasone, or a combination of the latter significantly reduced titers of dsDNA-specific autoantibodies, levels of proteinuria, and intensity of glomerular immune complex deposits. Both drugs down-regulated the secretion and expression of IFN-gamma and IL-10, but only treatment with hCDR1 up-regulated TGF-beta. While both drugs reduced the expression of Fas ligand (FasL) and caspase 8, treatment with hCDR1 resulted in reduced whereas dexamethasone administration resulted in increased rate of apoptosis. Furthermore, down-regulation of FasL appeared to play a role in cytokine modulation. We conclude that specific treatment with hCDR1 ameliorates murine lupus via distinct mechanisms of action than those of dexamethasone.
Assuntos
Apoptose/imunologia , Autoanticorpos/fisiologia , Regiões Determinantes de Complementaridade/fisiologia , Citocinas/biossíntese , Dexametasona/farmacologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Fragmentos de Peptídeos/fisiologia , Animais , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Citocinas/metabolismo , Proteína Ligante Fas , Feminino , Humanos , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NZB , Fatores de Necrose Tumoral/metabolismoRESUMO
A peptide (hCDR1) based on the complementarity determining region-1 of an anti-DNA antibody ameliorates systemic lupus erythematosus (SLE) in induced and spontaneous lupus models. Our objectives were to determine the effects of hCDR1 on TCR signaling and on its negative regulators, Foxj1 and Foxo3a. BALB/c mice were immunized with the SLE-inducing anti-DNA antibody, designated 16/6Id, and treated with hCDR1. hCDR1 treatment specifically inhibited IFN-gamma secretion by T cells in association with down-regulated T-bet expression and NF-kappaB activation; however, GATA-3 expression was not affected. Furthermore, TCR signaling (ZAP-70 phosphorylation) was inhibited, and the mRNA expression of the two modulators of Th1 activation, Foxj1 and Foxo3a, was significantly up-regulated. The latter were also elevated in SLE-afflicted (NZBxNZW)F1 mice that were treated with hCDR1. Addition of TGF-beta, which was elevated following treatment with hCDR1, to T cells from 16/6Id immunized mice, up-regulated Foxj1 and Foxo3a mRNA expression, similarly to hCDR1. In contrast, anti-TGF-beta antibodies added to hCDR1-treated T cells abrogated its effect. Thus, hCDR1 elevates TGF-beta, which contributes to the up-regulation of T cell Foxj1 and Foxo3a expression, leading to inhibition of NF-kappaB activation and IFN-gamma secretion, which is required for the maintenance of SLE.
Assuntos
Anticorpos Monoclonais/farmacologia , Fatores de Transcrição Forkhead/metabolismo , Interferon gama/antagonistas & inibidores , Lúpus Eritematoso Sistêmico/imunologia , Fragmentos de Peptídeos/farmacologia , Linfócitos T/efeitos dos fármacos , Animais , Feminino , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas com Domínio T/metabolismo , Linfócitos T/imunologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Regulação para Cima , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
Treatment with peptides based on the complementarity determining regions (CDR) of murine and human monoclonal anti-DNA antibodies that bear the common idiotype, 16/6 Id, ameliorates disease manifestations of mice with either induced or spontaneous SLE. Aberrant expression and function of the p21Ras/MAP kinase pathway are associated with active SLE. Therefore, we examined the effect of treatment with a CDR1-based peptide of a human autoantibody (hCDR1) on the p21Ras pathway and SLE manifestations of SLE-prone (NZBxNZW)F1 mice. Untreated SLE-afflicted mice demonstrated increased expression of p21Ras and the phosphorylated active form of its down-stream element JNK kinase in conjunction with reduced hSOS and unchanged p120GAP, as compared to healthy controls. Amelioration of SLE manifestations following treatment with hCDR1 was associated with a diminished expression of phosphorylated JNK kinase, mainly in the T cell population that also exhibited reduced rates of apoptosis. Thus, hCDR1 therapy ameliorates SLE, at least in part, via down-regulation of the activity of the pro-apoptotic JNK kinase.
Assuntos
Autoanticorpos/efeitos dos fármacos , Doenças Autoimunes/imunologia , Regiões Determinantes de Complementaridade/uso terapêutico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , MAP Quinase Quinase 4/metabolismo , Fragmentos de Peptídeos/uso terapêutico , Animais , Apoptose/imunologia , Autoanticorpos/imunologia , Doenças Autoimunes/genética , Cruzamentos Genéticos , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Imunização , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Endogâmicos NZB , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
Systemic lupus erythematosus (SLE), which is characterized by the increased production of autoantibodies and defective T cell responses, can be induced in mice by immunization with a human anti-DNA mAb that expresses a major Id, designated 16/6Id. A peptide based on the sequence of the CDR1 of the 16/6Id (human CDR1 (hCDR1)) ameliorated the clinical manifestations of SLE and down-regulated, ex vivo, the 16/6Id-induced T cell proliferation. In this study, we examined the mechanism responsible for the hCDR1-induced modulation of T cell functions related to the pathogenesis of SLE. We found that injection of hCDR1 into BALB/c mice concomitant with their immunization with 16/6Id resulted in a marked elevation of TGF-beta secretion 10 days later. Addition of TGF-beta suppressed the 16/6Id-stimulated T cell proliferation similarly to hCDR1. In addition, we provide evidence that one possible mechanism underlying the hCDR1- and TGFbeta-induced inhibition of T cell proliferation is by down-regulating the expression, and therefore the functions, of a pair of key cell adhesion receptors, LFA-1 (alphaLbeta2) and CD44, which operate as accessory molecules in mediating APC-T cell interactions. Indeed, T cells of mice treated with hCDR1 showed a TGF-beta-induced suppression of adhesion to the LFA-1 and CD44 ligands, hyaluronic acid and ICAM-1, respectively, induced by stromal cell-derived factor-1alpha and PMA. The latter suppression is through the inhibition of ERK phosphorylation. Thus, the down-regulation of SLE-associated responses by hCDR1 treatment may be due to the effect of the up-regulated TGF-beta on the expression and function of T cell adhesion receptors and, consequently, on T cell stimulation, adhesion, and proliferation.