RESUMO
×Ö¹ndoleamine-2,3-dioxygenase (IDO) plays a role in immune regulation. Increased IDO activity was reported in systemic lupus erythematosus (SLE). We investigated the effects of the tolerogenic peptide hCDR1, shown to ameliorate lupus manifestations, on IDO gene expression. mRNA was prepared from splenocytes of hCDR1- treated SLE-afflicted (NZBxNZW)F1 mice, from blood samples of lupus patients, collected before and after their in vivo treatment with hCDR1 and from peripheral blood mononuclear cells (PBMC) of patients incubated with hCDR1. IDO gene expression was determined by real-time RT-PCR. hCDR1 significantly down-regulated IDO expression in SLE-affected mice and in lupus patients (treated in vivo and in vitro). No effects were observed in healthy donors or following treatment with a control peptide. Diminished IDO gene expression was associated with hCDR1 beneficial effects. Our results suggest that the hCDR1-induced FOXP3 expressing regulatory T cells in lupus are not driven by IDO but rather by other hCDR1 regulated pathways.
Assuntos
Anticorpos Monoclonais/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Lúpus Eritematoso Sistêmico/genética , Fragmentos de Peptídeos/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Adulto , Idoso , Animais , Anticorpos Monoclonais/uso terapêutico , Ensaios Clínicos Fase II como Assunto , Regulação para Baixo , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Fragmentos de Peptídeos/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Baço/citologia , Linfócitos T Reguladores/metabolismoRESUMO
Primary Sjogren's syndrome (pSS) is an autoimmune disease characterized by lymphocytic infiltration of exocrine glands. We investigated whether the tolerogenic peptide, hCDR1, that ameliorates lupus manifestations would have beneficial effects on pSS as well. The in vitro effects of hCDR1 on gene expression of pro-inflammatory cytokines and regulatory molecules were tested in peripheral blood mononuclear cells (PBMC) of 16 pSS patients. hCDR1, but not a control peptide, significantly reduced gene expression of IL-1ß, TNF-α, MX-1 and BlyS and up-regulated immunosuppressive (TGF-ß, FOXP3) molecules in PBMC of pSS patients. hCDR1 did not affect gene expression in patients with rheumatoid arthritis and anti-phospholipid syndrome. Further, hCDR1 up-regulated the expression of Indoleamine 2,3-dioxygenase (IDO) via elevation of TGF-ß. IDO inhibition led to a significant decrease in the expression of FOXP3 which is crucial for the induction of T regulatory cells. Thus, hCDR1 is potential candidate for the specific treatment of pSS patients.
Assuntos
Citocinas/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Peptídeos/farmacologia , Síndrome de Sjogren/imunologia , Adulto , Idoso , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Citocinas/genética , Citocinas/metabolismo , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/imunologia , Humanos , Fatores Imunológicos/farmacologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Peptídeos/genética , Síndrome de Sjogren/sangue , Síndrome de Sjogren/genética , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Systemic lupus erythematosus (SLE) is a chronic multisystem disease in which various cell types and immunological pathways are dysregulated. Current therapies for SLE are based mainly on the use of non-specific immunosuppressive drugs that cause serious side effects. There is, therefore, an unmet need for novel therapeutic means with improved efficacy and lower toxicity. Based on recent better understanding of the pathogenesis of SLE, targeted biological therapies are under different stages of development. The latter include B-cell targeted treatments, agents directed against the B lymphocyte stimulator (BLyS), inhibitors of T cell activation as well as cytokine blocking means. Out of the latter, Belimumab was the first drug approved by the FDA for the treatment of SLE patients. In addition to the non-antigen specific agents that may affect the normal immune system as well, SLE-specific therapeutic means are under development. These are synthetic peptides (e.g. pConsensus, nucleosomal peptides, P140 and hCDR1) that are sequences of conserved regions of molecules involved in the pathogenesis of lupus. The peptides are tolerogenic T-cell epitopes that immunomodulate only cell types and pathways that play a role in the pathogenesis of SLE without interfering with normal immune functions. Two of the peptides (P140 and hCDR1) were tested in clinical trials and were reported to be safe and well tolerated. Thus, synthetic peptides are attractive potential means for the specific treatment of lupus patients. In this review we discuss the various biological treatments that have been developed for lupus with a special focus on the tolerogenic peptides.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Sistemas de Liberação de Medicamentos , Epitopos de Linfócito T/uso terapêutico , Imunossupressores/uso terapêutico , Lúpus Eritematoso Sistêmico , Peptídeos/uso terapêutico , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Ensaios Clínicos como Assunto , Epitopos de Linfócito T/imunologia , Humanos , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
BACKGROUND: The 16/6-idiotype (16/6-Id) of the human anti-DNA antibody was found to induce experimental lupus in naïve mice, manifested by production of autoantibodies, leukopenia and elevated inflammatory markers, as well as kidney and brain involvement. We assessed behavior and brain pathology of naive mice injected intra-cerebra-ventricularly (ICV) with the 16/6-Id antibody. METHODS: C3H female mice were injected ICV to the right hemisphere with the human 16/6-Id antibody or commercial human IgG antibodies (control). The mice were tested for depression by the forced swimming test (FST), locomotor and explorative activity by the staircase test, and cognitive functions were examined by the novel object recognition and Y-maze tests. Brain slices were stained for inflammatory processes. RESULTS: 16/6-Id injected mice were cognitively impaired as shown by significant differences in the preference for a new object in the novel object recognition test compared to controls (P = 0.012). Similarly, the preference for spatial novelty in the Y-maze test was significantly higher in the control group compared to the 16/6-Id-injected mice (42% vs. 9%, respectively, P = 0.065). Depression-like behavior and locomotor activity were not significantly different between the16/6-Id-injected and the control mice. Immunohistochemistry analysis revealed an increase in astrocytes and microglial activation in the hippocampus and amygdala, in the 16/6-Id injected group compared to the control. CONCLUSIONS: Passive transfer of 16/6-Id antibodies directly into mice brain resulted in cognitive impairments and histological evidence for brain inflammation. These findings shed additional light on the diverse mosaic pathophysiology of neuropsychiatric lupus.See related Commentary article: http://www.biomedcentral.com/1741-7015/11/91.
Assuntos
Anticorpos/administração & dosagem , Anticorpos/toxicidade , Disfunção Cognitiva/induzido quimicamente , Encefalite/induzido quimicamente , Animais , Encéfalo/patologia , Feminino , Histocitoquímica , Humanos , Camundongos , Camundongos Endogâmicos C3HRESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disease that involves dysregulation of B and T cells. A tolerogenic peptide, designated hCDR1, ameliorates disease manifestations in SLE-afflicted mice. In the present study, the effect of treatment with hCDR1 on the CD74/macrophage migration inhibitory factor (MIF) pathway was studied. We report here that B lymphocytes from SLE-afflicted mice express relatively elevated levels of CD74, compared with B cells from healthy mice. CD74 is a receptor found in complex with CD44, and it binds the pro-inflammatory cytokine MIF. The latter components were also up-regulated in B cells from the diseased mice, and treatment with hCDR1 resulted in their down-regulation and in reduced B-cell survival. Furthermore, up-regulation of CD74 and CD44 expression was detected in brain hippocampi and kidneys, two target organs in SLE. Treatment with hCDR1 diminished the expression of those molecules to the levels determined for young healthy mice. These results suggest that the CD74/MIF pathway plays an important role in lupus pathology.
Assuntos
Antígenos de Diferenciação de Linfócitos B/imunologia , Autoantígenos/imunologia , Linfócitos B/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Oxirredutases Intramoleculares/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Fatores Inibidores da Migração de Macrófagos/imunologia , Proteínas do Tecido Nervoso/imunologia , Peptídeos/imunologia , Animais , Antígenos de Diferenciação de Linfócitos B/biossíntese , Antígenos de Diferenciação de Linfócitos B/genética , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Autoantígenos/química , Linfócitos B/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/biossíntese , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Imunomodulação , Oxirredutases Intramoleculares/biossíntese , Oxirredutases Intramoleculares/genética , Lúpus Eritematoso Sistêmico/patologia , Fatores Inibidores da Migração de Macrófagos/biossíntese , Fatores Inibidores da Migração de Macrófagos/genética , Camundongos , Camundongos Endogâmicos NZB , Proteínas do Tecido Nervoso/química , Peptídeos/química , Peptídeos/farmacologiaRESUMO
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
Dysregulated expression of Bcl-xL and Bcl-2 may initiate the development of autoimmune diseases including systemic lupus erythematosus (SLE). A tolerogenic peptide designated hCDR1 was shown to ameliorate manifestations of spontaneous and induced murine SLE. Recently, we demonstrated that Bcl-xL plays a critical role in the modulating effects of hCDR1, as manifested by reducing the state of activation of lymphocytes and by down-regulating the secretion of the pathogenic cytokines, IFN-gamma and IL-10. Here we studied the role of Bcl-xL in the development and function of CD4 regulatory T-cells (Treg) from hCDR1-treated, SLE-afflicted (New-Zealand-Black x New-Zealand-White) F1 mice. We report that Bcl-xL was up-regulated in CD4 Treg of tolerized mice, where it played a role in inducing the regulatory/inhibitory molecules Foxp3, CTLA-4, and TGF-beta and in repressing PD-1. Further, Bcl-xL mediated the induction of CTLA-4 and TGF-beta in effector T cells (Teff) by CD4 Treg of the tolerized mice. The induction of Bcl-xL in Teff by Treg was TGF-beta dependent and CTLA-4 independent, leading to inhibition of proliferation and to a decrease in activated Teff. We conclude that Bcl-xL is required for the development and function of CD4 Treg, which ameliorate lupus following treatment with a tolerogenic peptide.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Lúpus Eritematoso Sistêmico/imunologia , Fragmentos de Peptídeos/administração & dosagem , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Proteína bcl-X/metabolismo , Animais , Antígenos CD/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Fatores de Transcrição Forkhead/biossíntese , Humanos , Tolerância Imunológica , Interferon gama/metabolismo , Interleucina-10/metabolismo , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Lúpus Eritematoso Sistêmico/terapia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima , Proteína bcl-X/genéticaRESUMO
A peptide, designated human CDR1 (hCDR1), that is based on the CDR1 of an anti-DNA Ab ameliorates systemic lupus erythematosus (SLE) in murine models via the induction of CD4(+)CD25(+) regulatory T cells (Tregs). In the present study, the involvement of CD8 Tregs in the mode of action of hCDR1 was investigated in SLE-afflicted (NZB x NZW)F1 mice and in SJL mice following immunization with the lupus-inducing anti-DNA mAb that bears a common Id, 16/6Id. Treatment with hCDR1 up-regulated Foxp3-expressing CD8(+)CD28(-) Tregs in association with clinical amelioration of lupus manifestations. Furthermore, the in vivo depletion of the latter cells diminished the clinical improvement and the inhibitory effects of hCDR1 on the secretion of IFN-gamma and resulted in the up-regulation of IL-10. However, the stimulatory effect of hCDR1 on the secretion of TGF-beta was not affected by the CD8 Tregs. In the absence of CD8 Tregs, CD4(+)CD25(+) Tregs were unable to expand in the hCDR1-treated mice, and the expression of Foxp3 was reduced, thereby interfering further with the suppressive function of CD4(+)CD25(+) Tregs as determined in the in vitro assays. However, CD8 cells from hCDR1-treated mice that were adoptively transferred into SLE-afflicted mice led to up-regulation of CD4(+)CD25(+) cells with intensified Foxp3 expression in the recipient mice. Thus, a functional link between two subsets of Tregs is demonstrated in which CD8(+)CD28(-) Tregs are required for both the optimal expansion and function of lupus ameliorating hCDR1-induced CD4(+)CD25(+) Tregs.
Assuntos
Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Fatores de Transcrição Forkhead , Tolerância Imunológica , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/imunologia , Proteínas do Tecido Nervoso/imunologia , Transferência Adotiva , Animais , Autoantígenos/farmacologia , Autoantígenos/uso terapêutico , Linfócitos T CD8-Positivos/transplante , Proliferação de Células , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Proteínas do Tecido Nervoso/farmacologia , Proteínas do Tecido Nervoso/uso terapêutico , Linfócitos T Reguladores/citologiaRESUMO
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 disease mediated by T and B cells. It is characterized by a variety of autoantibodies and systemic clinical manifestations. A tolerogenic peptide, designated hCDR1, ameliorated the serological and clinical manifestations of SLE in both spontaneous and induced models of lupus. In the present study, we evaluated the status of mature B cells in the bone marrow (BM) of SLE-afflicted mice, and determined the effect of treatment with the tolerogenic peptide hCDR1 on these cells. We demonstrate herein that mature B cells of the BM of SLE-afflicted (New Zealand Black x New Zealand White)F(1) mice were largely expanded, and that treatment with hCDR1 down-regulated this population. Moreover, treatment with hCDR1 inhibited the expression of the pathogenic cytokines [interferon-gamma and interleukin (IL)-10], whereas it up-regulated the expression of transforming growth factor-beta in the BM. Treatment with hCDR1 up-regulated the rates of apoptosis of mature B cells. The latter was associated with inhibited expression of the survival Bcl-xL gene and of IL-7 by BM cells. Furthermore, the addition of recombinant IL-7 abrogated the suppressive effects of hCDR1 on Bcl-xL in the BM cells and resulted in elevated levels of apoptosis. Hence, the down-regulated production of IL-7 contributes to the hCDR1-mediated apoptosis of mature B cells in the BM of SLE-afflicted mice.
Assuntos
Anticorpos Monoclonais/imunologia , Subpopulações de Linfócitos B/imunologia , Células da Medula Óssea/imunologia , Interleucina-7/antagonistas & inibidores , Lúpus Eritematoso Sistêmico/imunologia , Fragmentos de Peptídeos/imunologia , Animais , Apoptose/imunologia , Modelos Animais de Doenças , Regulação para Baixo/imunologia , Feminino , Tolerância Imunológica/imunologia , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-7/imunologia , Camundongos , Camundongos Endogâmicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Baço/imunologia , Fator de Crescimento Transformador beta/biossíntese , Regulação para Cima/imunologiaRESUMO
Interferon-gamma (IFN-gamma) plays a pathogenic role in systemic lupus erythematosus (SLE). Uncontrolled IFN-gamma signaling may result from a deficiency in the negative regulator, namely, suppressor of cytokine signaling-1 (SOCS-1). We investigated the activation status of IFN-gamma signaling pathway in SLE-afflicted (New-Zealand-BlackxNew-Zealand-White)F1 mice and determined its responsiveness when treating with a tolerogenic peptide, hCDR1, which ameliorates SLE. SOCS-1 was suppressed and pSTAT1 was enhanced in spleen-derived cells from SLE-affected mice as compared with healthy controls. Treatment with hCDR1 reversed the expression of these two molecules in association with clinical amelioration. In vitro stimulation with IFN-gamma resulted in elevated levels of SOCS-1 in cells from both vehicle and hCDR1-treated mice but this effect reached significance only in cells of the latter group, which also exhibited reduced levels of pSTAT1. Thus, SOCS-1 is diminished in SLE-affected mice, and treatment with hCDR1 results in its up-regulation thereby restoring control of IFN-gamma signaling pathway.
Assuntos
Autoantígenos/uso terapêutico , Terapia de Imunossupressão , Interferon gama/imunologia , Lúpus Eritematoso Sistêmico/terapia , Proteínas do Tecido Nervoso/uso terapêutico , Fator de Transcrição STAT1/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Modelos Animais de Doenças , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Feminino , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Peptídeos/uso terapêutico , Fator de Transcrição STAT1/agonistas , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/agonistas , Proteínas Supressoras da Sinalização de Citocina/antagonistas & inibidores , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by dysregulated immune responses mediated by T and B cells. A tolerogenic peptide, designated hCDR1, ameliorated the serological and clinical manifestations of SLE in mouse models of lupus. We investigated the role of B-cell activating factor (BAFF) in the beneficial effects of hCDR1. BAFF production was reduced in hCDR1-treated mice in association with diminished production of dsDNA-specific autoantibodies and proteinuria levels. In addition, IFN-gamma and IL-10, which induce BAFF secretion, were down-regulated in hCDR1-treated mice. The reduced levels of BAFF correlated with a lower rate of maturation and differentiation of B cells, and with a decrease in integrin expression and anti-apoptotic gene expression by B cells. Moreover, BAFF signaling through the NF-kB pathways was inhibited in hCDR1-treated mice. Thus, down-regulation of BAFF plays a role in the mechanism of action by which hCDR1 ameliorates lupus manifestations.
Assuntos
Autoantígenos/farmacologia , Fator Ativador de Células B/metabolismo , Lúpus Eritematoso Sistêmico/fisiopatologia , Proteínas do Tecido Nervoso/farmacologia , Peptídeos/farmacologia , Animais , Autoantígenos/genética , Fator Ativador de Células B/sangue , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Feminino , Integrinas/metabolismo , Interferon gama/metabolismo , Interleucina-10/metabolismo , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Tecido Nervoso/genética , Peptídeos/genética , Peptídeos/uso terapêutico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Baço/efeitos dos fármacos , Baço/metabolismo , Análise de SobrevidaRESUMO
In primary adrenocortical failure (Addison's disease) caused by autoimmunity, autoantibodies to the steroidogenic cytochrome P450 enzyme 21-hydroxylase (21OH) are detected in the majority of patients. It is currently uncertain whether the autoantibodies themselves participate in the pathogenesis, or if they merely reflect an on-going T cell mediated response. The identification of T cells reactive with 21OH, if any, has been hampered by the lack of a high-quality antigen. In the current study recombinant human 21OH has been expressed in Spodoptera frugiperda insect cells using a baculovirus expression system. Recombinant enzymatically active 21OH was purified to apparent homogeneity by immobilized metal ion affinity chromatography. The purified enzyme was highly immunogenic in immunized SJL/J mice, and immune responses to 21OH-derived peptides assayed as T cell proliferation and interferon gamma production could be invoked after priming with the recombinant protein. Furthermore, purified 21OH was recognized by sera from patients with autoimmune Addison's disease, and it could block the binding of radiolabeled in vitro translated 21OH in a sensitive fluid-phase radioimmunoassay. We conclude that the recombinant preparation of 21OH presented here is of sufficient purity and quality to be used for studies of cellular and humoral immunity in autoimmune Addison's disease.
Assuntos
Doença de Addison/imunologia , Autoantígenos/metabolismo , Peptídeos/metabolismo , Esteroide 21-Hidroxilase/metabolismo , Linfócitos T/metabolismo , Doença de Addison/sangue , Doença de Addison/diagnóstico , Doença de Addison/patologia , Animais , Autoanticorpos/sangue , Autoantígenos/química , Autoantígenos/genética , Autoantígenos/imunologia , Baculoviridae/genética , Linhagem Celular , Proliferação de Células , Cromatografia de Afinidade , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Soros Imunes , Imunização , Interferon gama/metabolismo , Ativação Linfocitária , Camundongos , Peptídeos/química , Peptídeos/genética , Peptídeos/imunologia , Spodoptera/enzimologia , Spodoptera/genética , Esteroide 21-Hidroxilase/química , Esteroide 21-Hidroxilase/genética , Esteroide 21-Hidroxilase/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Transgenes/imunologiaRESUMO
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
OBJECTIVE: Anticitrullinated protein antibodies (ACPA) have major diagnostic significance in rheumatoid arthritis (RA). ACPA are directed against different citrullinated antigens, including filaggrin, fibrinogen, vimentin, and collagen. The presence of ACPA is associated with joint damage and extraarticular manifestations, suggesting that ACPA may have a significant role in the pathogenesis of RA. METHODS: To verify the effect of ACPA on RA-immune cells, peripheral blood mononuclear cells (PBMC) from cyclic citrullinated peptide (CCP)-positive patients with RA and healthy controls were cocultured in vitro with ACPA. ACPA-positive stained cells were analyzed by flow cytometry and the effect of ACPA on mRNA expression levels was evaluated by real-time PCR. We tested whether the stimulatory effects induced by ACPA could be inhibited by the addition of a new multiepitope citrullinated peptide (Cit-ME). RESULTS: We found that ACPA bind specifically to PBMC from CCP-positive patients with RA through the Fab portion. ACPA induce upregulation of pathogenic cytokine expression (4- to 13-fold increase) in PBMC derived from CCP-positive patients with RA. Moreover, ACPA upregulated IL-1ß and IL-6 mRNA expression levels by 10- and 6-fold, respectively, compared to control IgG. Cit-ME, a genuine ligand of ACPA, inhibited the ACPA-induced upregulation of IL-1ß and IL-6 by 30%. CONCLUSION: ACPA bind to a limited percentage of PBMC and upregulate inflammatory cytokine expression, suggesting that ACPA is involved in RA pathogenesis. Targeting ACPA to decrease their pathogenic effects might provide a novel direction in developing therapeutic strategies for RA.
Assuntos
Artrite Reumatoide/sangue , Autoanticorpos/sangue , Citrulina/imunologia , Leucócitos Mononucleares/metabolismo , Peptídeos Cíclicos/metabolismo , Transcriptoma/imunologia , Afinidade de Anticorpos/imunologia , Citrulina/síntese química , Proteínas Filagrinas , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Estatísticas não ParamétricasRESUMO
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
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 was demonstrated to down-regulate in vitro and in vivo myasthenia gravis (MG) associated autoreactive responses. In this study, we demonstrate the suppressive properties of the dual APL following immunization with the whole Torpedo AChR (TAChR) and in mice with established experimental autoimmune MG (EAMG). The dual APL acts by up-regulating CD4+ CD25+ cells expressing characteristic regulatory markers along with an associated increase in levels of IL-10 and TGF-beta. The latter cytokine plays a key role in the ameliorating effects of the dual APL.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Terapia de Imunossupressão/métodos , Miastenia Gravis Autoimune Experimental/imunologia , Miastenia Gravis Autoimune Experimental/terapia , Peptídeos/farmacologia , Receptores Nicotínicos/imunologia , Animais , Autoimunidade/efeitos dos fármacos , Autoimunidade/imunologia , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Feminino , Interleucina-10/imunologia , Interleucina-10/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Miastenia Gravis Autoimune Experimental/fisiopatologia , Receptores de Interleucina-2/biossíntese , Frações Subcelulares , Torpedo , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismo , Resultado do Tratamento , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
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
OBJECTIVE: The objective of this study was to define the prevalence of systemic lupus erythematosus (SLE) in patients with myasthenia gravis (MG). METHODS: Seventy-eight MG patients recruited unselectively from Israeli MG database were evaluated by medical history, physical examination and serology (ANA at 1:100 and anti-ds-DNA at 1:10 dilution) for the presence of SLE, which was defined by the presence of four or more American College of Rheumatology diagnostic criteria. RESULTS: Thirty-one (40%) of our patients were males and 47 (60%) were females. Their mean age at time of the study was 51.5 +/- 14.5 years. Forty patients (51%) had an early-onset disease (<40 years); 90% had generalized and 10% had limited ophthalmic MG. Significant titers of ANA and ds-DNA autoantibodies were observed in 38.5% and 19.2% of the patients. In six (7.7%), a definitive diagnosis of SLE was established (MG was first diagnosed; there was no association with previous thymectomy), three of them revealed lupus-related neurologic manifestations. All six patients were females with an early onset generalized MG. CONCLUSION: High prevalence of SLE and lupus-related autoantibodies exist in female MG patients. Thus, MG patients should be evaluated for the coexistence of SLE, and assessment for MG is suggested in lupus patients with unexplained muscular weakness.