RESUMO
Purpose: Tofacitinib is a pan-Janus kinase (JAK) inhibitor that suppresses cytokine signaling and in turn, the cells that participate in inflammatory immunopathogenic processes. We examined the capacity of tofacitinib to inhibit the induction of experimental autoimmune uveitis (EAU) and related immune responses. Methods: EAU was induced in B10.A mice with immunization with bovine interphotoreceptor retinoid-binding protein (IRBP), emulsified in complete Freund's adjuvant (CFA), and a simultaneous injection of pertussis toxin. Tofacitinib, 25 mg/kg, was administered daily, and the vehicle was used for control. EAU development was assessed by histological analysis of the mouse eyes, and related immune responses were assessed by (i) the levels of interferon (IFN)-γ and interleukin (IL)-17, secreted by spleen cells cultured with IRBP; (ii) flow cytometric analysis of intracellular expression by spleen, or eye-infiltrating CD4 or CD8 cells of IFN-γ, IL-17, and their transcription factors, T-bet and RORγt. In addition, the inflammation-related cell markers CD44 and CD62L and Ki67, a proliferation marker, were tested. The proportions of T-regulatory cells expressing FoxP3 were determined by flow cytometric intracellular staining, while levels of antibody to IRBP were measured with enzyme-linked immunosorbent assay (ELISA). Results: Treatment with tofacitinib significantly suppressed the development of EAU and reduced the levels of secreted IFN-γ, but not of IL-17. Further, treatment with tofacitinib reduced in the spleen and eye-infiltrating cells the intracellular expression of IFN-γ and its transcription factor T-bet. In contrast, treatment with tofacitinib had essentially no effect on the intracellular expression of IL-17 and its transcription factor, RORγt. The selective effect of tofacitinib treatment was particularly evident in the CD8 population. Treatment with tofacitinib also increased the population of CD44, but reduced the populations of cells producing CD62L and Ki67. Treatment with tofacitinib had no effect on the proportion of FoxP3 producing regulatory cells and on the antibody production to IRBP. Conclusions: Treatment with tofacitinib inhibited the development of EAU, reduced the production of IFN-γ, but had essentially no effect on the production of IL-17.
Assuntos
Olho/metabolismo , Piperidinas/farmacologia , Pirimidinas/farmacologia , Células Th1/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Uveíte/tratamento farmacológico , Uveíte/imunologia , Animais , Antígenos CD4/sangue , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Antígenos CD8/sangue , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Olho/efeitos dos fármacos , Olho/patologia , Proteínas do Olho/farmacologia , Fatores de Transcrição Forkhead/sangue , Receptores de Hialuronatos/sangue , Terapia de Imunossupressão , Interferon gama/sangue , Interleucina-17/sangue , Antígeno Ki-67/sangue , Selectina L/sangue , Camundongos , Piperidinas/administração & dosagem , Pirimidinas/administração & dosagem , Proteínas de Ligação ao Retinol/farmacologia , Células Th1/imunologia , Células Th17/imunologiaRESUMO
Mutations in Serpinf1 gene which encodes pigment epithelium-derived factor (PEDF) lead to osteogenesis imperfecta type VI whose hallmark is defective matrix mineralization. We reported previously that PEDF reduced expression and synthesis of Sost/Sclerostin as well as other osteocytes genes encoding proteins that regulate matrix mineralization [1]. To determine whether PEDF had an effect on osteocyte gene expression in bone, we used bone explant cultures. First, osteocytes were isolated from surgical waste of bone fragments obtained from patients undergoing elective foot surgeries under approved IRB protocol by Penn State College of Medicine IRB committee. Primary osteocytes treated with PEDF reduced expression and synthesis of Sost/Sclerostin and matrix phosphoglycoprotein (MEPE) as well as dentin matrix protein (DMP-1). On the whole, PEDF reduced osteocyte protein synthesis by 50% and by 75% on mRNA levels. For bone explants, following collagenase digestion, bone fragments were incubated in alpha-MEM supplemented with 250 ng/ml of PEDF or BSA. After 7 days of incubation in a medium supplemented with PEDF, analysis of mRNA by PCR and protein by western blotting of encoded osteocyte proteins showed reduced Sclerostin synthesis by 39% and MEPE by 27% when compared to fragments incubated in medium supplemented with BSA. mRNA expression levels of osteocytes in bone fragments treated with PEDF were reduced by 50% for both SOST and MEPE when compared to BSA-treated bone fragments. Taken together, the data indicate that PEDF has an effect on osteocyte gene expression in bone and encourage further studies to examine effect of PEDF on bone formation indices in animal models and its effect on osteocyte gene expression in vivo following PEDF administration.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Osso e Ossos/metabolismo , Proteínas do Olho/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores de Crescimento Neural/farmacologia , Osteócitos/metabolismo , Serpinas/farmacologia , Técnicas de Cultura de Tecidos , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Morfogenéticas Ósseas/genética , Células Cultivadas , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Marcadores Genéticos/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Pessoa de Meia-Idade , Osteócitos/efeitos dos fármacos , Fosfoproteínas/genética , Fosfoproteínas/metabolismoRESUMO
Oxidative stress has been implicated in neurodegenerative diseases, such as age-related macular degeneration. Hydrogen peroxide and sodium iodate can mediate oxidative injury. Sodium iodate induces a selective retinal degeneration targeting the RPE. We describe a method of chronic sodium iodate-mediated injury on RPE cells that may serve to evaluate protective factors against oxidative stress. Cytotoxicity and cell viability curves of ARPE-19 cells with sodium iodate were generated. The antioxidant pigment epithelium-derived factor decreased sodium iodate-mediated cytotoxicity without affecting ARPE-19 cell viability. A cell culture system to evaluate protection against oxidative stress injury with PEDF is discussed.
Assuntos
Antioxidantes/farmacologia , Proteínas do Olho/farmacologia , Fatores de Crescimento Neural/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Serpinas/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Células Epiteliais/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/toxicidade , Iodatos/toxicidade , Degeneração Macular/patologia , Estresse Oxidativo , Proteínas Recombinantes/farmacologia , Epitélio Pigmentado da Retina/citologiaRESUMO
BACKGROUND AND AIMS: Pigment epithelium-derived factor (PEDF) has been shown to be a potent inhibitor of inflammation through its anti-oxidative property. Since oxidative response is considered to play the pivotal role of the development and progression of nonalcoholic steatohepatitis (NASH), it is conceivable that PEDF may play a protective role against NASH. In this study, we examined whether administration of PEDF slowed the progression of NASH in mice models. METHODS: Mice were fed methionine- and choline-deficient (MCD) diet with or without intramuscular administration of adenovirus-expressing PEDF (Ad-PEDF). Effects of PEDF administration on NASH were histologically and biochemically evaluated. RESULTS: Administration of Ad-PEDF significantly decreased hepatic fat storage as well as serum levels of ALT in MCD diet-fed mice. Dihydroethidium staining showed that MCD diet-triggered oxidative stress was reduced in the liver of Ad-PEDF-administered mice compared to that of PBS- or Ad-LacZ-administered mice. Activation of Kupffer cells and hepatic fibrosis was also inhibited by Ad-PEDF administration. Quantitative real-time RT-PCR revealed that MCD diet up-regulated expressions of TNF-α, IL-1ß, IL-6, TGF-ß, collagen-1, and collagen-3 mRNA, which were also attenuated with Ad-PEDF administration, whereas MCD diet-induced down-regulation of expressions of PPAR-γ mRNA was restored with Ad-PEDF administration. Furthermore, immunoblotting analysis showed that MCD diet-induced up-regulation of NADPH oxidase components was significantly decreased in Ad-PEDF-administered mice. CONCLUSIONS: The present results demonstrated for the first time that PEDF could slow the development and progression of steatohepatitis through the suppression of steatosis and inflammatory response in MCD diet-fed mice. Our study suggests that PEDF supplementation may be a novel therapeutic strategy for the treatment of NASH.
Assuntos
Tecido Adiposo/patologia , Proteínas do Olho/farmacologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/prevenção & controle , Fatores de Crescimento Neural/farmacologia , Inibidores de Proteases/farmacologia , RNA Mensageiro/metabolismo , Serpinas/farmacologia , Adenoviridae/genética , Alanina Transaminase/sangue , Animais , Deficiência de Colina/complicações , Colágeno Tipo I/genética , Colágeno Tipo III/genética , Dieta , Modelos Animais de Doenças , Regulação para Baixo , Proteínas do Olho/genética , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Injeções Intramusculares , Interleucina-1beta/genética , Interleucina-6/genética , Células de Kupffer , Cirrose Hepática/prevenção & controle , Masculino , Metionina/administração & dosagem , Metionina/deficiência , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidases/metabolismo , Fatores de Crescimento Neural/genética , Estresse Oxidativo , PPAR gama/genética , Serpinas/genética , Fator de Necrose Tumoral alfa/genética , Regulação para CimaRESUMO
Pigment epithelium-derived factor (PEDF) encoded by serpinf1 is a potent antiangiogenic factor found in a wide variety of fetal and adult tissues. Several reports have shown that lack of PEDF leads to osteogenesis imperfecta (OI) type VI whose hallmark is a defect in mineralization that leads to excessive osteoid build up that fails to mineralize. Because PEDF is antiangiogenic factor it would pose serious consequences on bone development and healing of fractures. To understand possible mechanisms by which PEDF plays a role in bone development and regulation of matrix mineralization, we determined the effects of exogenous PEDF on vascular endothelial growth factor (VEGF) expression by human mesenchymal stem cells (hMSCs) and mechanisms of its regulation by PEDF. Human MSCs incubated in normal medium supplemented with exogenous PEDF increased VEGF expression; this increase was also seen when PEDF was added to hMSCs undergoing osteogenic differentiation. MSCs maintained in osteogenic medium increased synthesis of both VEGF and PEDF but both factors were maintained relatively in balance during differentiation. To understand mechanisms by which exogenous PEDF regulated VEGF expression, hMSCs exposed to PEDF activated Erk signaling pathway in MSCs; inhibition of Erk signaling reduced VEGF mRNA expression as well as protein production suggesting that PEDF regulates VEGF expression in MSCs via Erk signaling pathway. In conclusion, PEDF increases VEGF expression by MSCs suggesting that regulation of VEGF by PEDF may be part of the mechanisms by which PEDF regulates osteoblastic mineralization.
Assuntos
Matriz Óssea/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Proteínas do Olho/farmacologia , Células-Tronco Mesenquimais/metabolismo , Fatores de Crescimento Neural/farmacologia , Serpinas/farmacologia , Regulação para Cima/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Idoso , Matriz Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Meios de Cultura/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Pessoa de Meia-Idade , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismoRESUMO
Gliotoxic responses complicate human eye diseases, the causes of which often remain obscure. Here, we activated Müller cells (MCs) by the gliotoxin DL-α-aminoadipate (AAA) and assayed possible protective effects by pigment epithelium-derived factor (PEDF) in RPE-free retinal explants of the E6 chick embryo. These models are suited to analyze gliotoxic reactions in vitro, since the avian retina contains only Müller cells (MCs) as glial components, and the RPE-free explants are devoid of a major PEDF source. ChAT- and AChE-immunohistochemistry (IHC) revealed that AAA treatment disrupted the differentiation of cholinergic amacrine cells in the inner plexiform layer. At the applied concentration of 1 mM AAA, apoptosis of MCs was slightly increased, as shown by TUNEL and caspase-3 activity assays. Concomitantly, cell-free gaps emerged in the middle of the retina, where MCs were swollen and amassed glutamine synthetase (shown by GS and Vimentin IHC). AAA treatment strongly activated MCs, as shown by GFAP IHC, and by an increase of stress-related catalase activity. Remarkably, nearly all effects of AAA on MCs were effectively counter-balanced by 50 ng/ml PEDF co-treatment, as also shown by RT-PCR. These findings suggest that supplementation with PEDF can protect the retina against gliotoxic attacks. Further studies should establish whether PEDF similarly protects a gliotoxic human retina.
Assuntos
Ácido 2-Aminoadípico/toxicidade , Células Ependimogliais/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/toxicidade , Proteínas do Olho/farmacologia , Gliose/prevenção & controle , Fatores de Crescimento Neural/farmacologia , Retina/embriologia , Serpinas/farmacologia , Acetilcolina/metabolismo , Acetilcolinesterase/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Embrião de Galinha , Células Ependimogliais/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/metabolismo , Gliose/patologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Técnicas de Cultura de Órgãos , Estresse Oxidativo , Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/citologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: To investigate if topical treatment of neuroprotectin D1 (NPD1) increases regeneration of functional nerves after lamellar keratectomy. METHODS: An 8-mm stromal dissection was performed in the left eye of each rabbit. The rabbits were treated with NPD1, pigment epithelial-derived factor (PEDF) in combination with docosahexaenoic acid (DHA) or vehicle for 6 weeks, and corneas were obtained at 8 weeks. After fixation, corneal wholemounts were stained with mouse monoclonal anti-ßIII-tubulin antibody and double stained with chicken anti-calcitonin gene-related peptide (CGRP) antibody. Corneal sensitivity and tear secretion were measured using the Cochet-Bonnet esthesiometer and the Schirmer's test, respectively. Additional rabbits were treated with NPD1, PEDF+DHA, or vehicle, and corneal sections were stained with a rat monoclonal anti-neutrophil antibody. Cultures of trigeminal ganglia from 5-day-old mice were treated with NPD1, PEDF+DHA, lipoxin A4 (LXA4), 12- or 15-hydroxyeicosatetraenoic acid (12[S] or 15[S]-HETE), and nerve growth factor (NGF) as positive control. RESULTS: NPD1 increased subepithelial corneal nerve area three times compared with vehicle-treated rabbits. The effect was similar to PEDF+DHA-treated animals. There was recovery of CGRP-positive neurons and an increase in corneal sensitivity and tear secretion in NPD1-treated animals. NPD1 decreased neutrophil infiltration after 2 and 4 days of treatment. In the in vitro cultures, NPD1 and PEDF+DHA induced a 3-fold increase in neurite outgrowth compared with cultures without supplementation. Treatments with LXA4, 12(S)-, and 15(S)- HETE did not stimulate neurite outgrowth. CONCLUSIONS: NPD1 has anti-inflammatory and nerve regenerative properties. This study demonstrates that NPD1 may offer an effective treatment for neurotrophic corneas.
Assuntos
Córnea/inervação , Ácidos Docosa-Hexaenoicos/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Nervo Oftálmico/fisiologia , Animais , Axônios/fisiologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Células Cultivadas , Combinação de Medicamentos , Proteínas do Olho/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Camundongos , Fatores de Crescimento Neural/farmacologia , Neuritos/fisiologia , Coelhos , Ratos , Serpinas/farmacologia , Lágrimas/fisiologia , Gânglio Trigeminal/citologia , Gânglio Trigeminal/efeitos dos fármacos , Tubulina (Proteína)/metabolismoRESUMO
PURPOSE: To investigate the involvement of δ-like ligand (Dll)4 in the development of experimental autoimmune uveoretinitis (EAU) in B10.RIII mice. METHODS: B10.RIII mice were immunized with interphotoreceptor retinoid binding protein (IRBP) peptide 161-180 in complete Freund's adjuvant together with intraperitoneal injection of Bordetella pertussis toxin. mRNA expressions of Notch receptors and their ligands in the eye were evaluated. To investigate the involvement of Dll in EAU, anti-Dll1, anti-Dll4, or control antibody (Ab) was intraperitoneally injected during both the induction and the effector phases or only the effector phase. Alternatively, mice were intraperitoneally injected with γ-secretase inhibitor (GSI) or the control vehicle during the induction phase. Fourteen days after immunization, the eyes and spleens were harvested. The eyes were used for histologic and/or cytokine mRNA expression analysis, whereas the spleens were used for flow cytometric analysis, and antigen-recall proliferation and cytokine assays. RESULTS: Expression of Notch1, 2, 4, and Dll4 in the eye were upregulated by EAU induction. Anti-Dll4 Ab treatment during both the induction and effector phases, but not only the effector phase, significantly reduced the severity of EAU. IFN-γ, IL-12p35, IL-17A, and TGF-ß mRNA expression in the eye were significantly attenuated by treatment with anti-Dll4 Ab. Splenocytes from anti-Dll4 Ab-treated mice showed significantly less proliferation and IL-17 production on antigen stimulation. Also, the severity of EAU was significantly reduced by γ-secretase inhibitor treatment during the induction phase. CONCLUSIONS; Dll4-mediated Notch signaling during the sensitization is critical for the development of EAU. This can be a novel prophylactic target for autoimmune uveitis.
Assuntos
Anticorpos Monoclonais/farmacologia , Doenças Autoimunes/metabolismo , Modelos Animais de Doenças , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas de Membrana/imunologia , Retinite/metabolismo , Uveíte/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Proteínas de Ligação ao Cálcio , Proliferação de Células/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Proteínas do Olho/farmacologia , Citometria de Fluxo , Injeções Intraperitoneais , Camundongos , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor Notch1/genética , Receptor Notch2/genética , Receptor Notch4 , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas de Ligação ao Retinol/farmacologiaRESUMO
Oxidative stress is associated with the development of retinopathy in diabetes; dietary supplementations of multi-antioxidants have no beneficial effects clinically. An antioxidant which could specifically target pathogenesis of diabetic retinopathy is the need of the hour. Pigment epithelium-derived factor is a potent, endogenously produced, multifunctional factor (neurotrophic, anti-angiogenic, anti-inflammatory etc.,) in the eye which recently was also shown to possess anti-oxidative action. However, its anti-oxidative effect against high glucose-induced oxidative stress in retinal endothelial cells has not been investigated. Here, we examined its anti-oxidative effect on cell morphology, survival, reactive oxygen species generation, lipid peroxidation, antioxidant status and caspase-3 activation under high glucose conditions in bovine retinal endothelial cells (BRECs). Cells grown at 33 mM glucose in the presence of PEDF at concentrations 10-50 nM did not exhibit shrinkage. Pigment epithelium-derived factor inhibited the high glucose-induced rise in reactive oxygen species generation and lipid peroxidation. In these cells, reduced glutathione levels and mitochondrial and superoxide dismutase activities increased markedly while reactive oxygen species generation decreased significantly in presence of PEDF as compared with cells grown in the absence of PEDF under high glucose conditions (10-20 nM, *p < 0.01&**p < 0.001; 30-50 nM, ***p < 0.0001). Our results suggest that pigment epithelium-derived factor has an anti-oxidant effect in bovine retinal endothelial cells at a high glucose level. The action of pigment epithelium-derived factor not only varies with the cell type but also depends on its concentration and environmental conditions. Therefore, further studies are required to determine if pigment epithelium-derived factor might constitute a preventive and/or a curative treatment for retinal neovascularization.
Assuntos
Antioxidantes/farmacologia , Endotélio Vascular/efeitos dos fármacos , Proteínas do Olho/farmacologia , Glucose/farmacologia , Fatores de Crescimento Neural/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Vasos Retinianos/citologia , Serpinas/farmacologia , Animais , Apoptose , Caspase 3/metabolismo , Bovinos , Permeabilidade da Membrana Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Ativação Enzimática , Glutationa/metabolismo , L-Lactato Desidrogenase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Superóxido Dismutase/metabolismoRESUMO
All-trans retinol generated in rod photoreceptors upon the bleaching of rhodopsin is known to move from the rods to the retinal pigment epithelium (RPE), where it is enzymatically converted to 11-cis retinal in the retinoid visual cycle. Interphotoreceptor retinoid-binding protein (IRBP) contained in the extracellular compartment (interphotoreceptor matrix) that separates the retina and RPE has been hypothesized to facilitate this movement of all-trans retinol, but the precise role of IRBP in this process remains unclear. To examine the activity of IRBP in the release of all-trans retinol from the rods, initially dark-adapted isolated retinas obtained from toad (Bufo marinus) eyes were bleached and then incubated in darkness for defined periods (5-180 min) in physiological saline (Ringer solution) supplemented with IRBP (here termed 'IRBP I') at defined concentrations (2-90 microm). Retinoids present in the retina and extracellular medium were then determined by extraction and HPLC analysis. Preparations incubated with > or =10 microm IRBP I showed a pronounced release of all-trans retinol with increasing period of incubation. As determined with 25 microm IRBP I, the increase of all-trans retinol in the extracellular medium was accompanied by a significant decrease in the combined amount of all-trans retinal and all-trans retinol contained in the retina. This effect was not mimicked by unsupplemented Ringer solution or by Ringer solution containing 25 or 90 microm bovine serum albumin. However, incubation with 'IRBP II', a previously described variant of IRBP with altered lectin-binding properties, led to the appearance of substantial all-trans retinol in the extracellular medium. The results suggest that in vivo, IRBP plays a direct role in the release of all-trans retinol from the rods during operation of the visual cycle.
Assuntos
Proteínas do Olho/farmacologia , Retina/metabolismo , Proteínas de Ligação ao Retinol/farmacologia , Rodopsina/metabolismo , Vitamina A/metabolismo , Animais , Bufo marinus , Cromatografia Líquida de Alta Pressão , Meios de Cultura , Adaptação à Escuridão/fisiologia , Relação Dose-Resposta a Droga , Proteínas do Olho/fisiologia , Estimulação Luminosa , Epitélio Pigmentado Ocular/efeitos dos fármacos , Epitélio Pigmentado Ocular/metabolismo , Retina/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Proteínas de Ligação ao Retinol/fisiologia , Técnicas de Cultura de TecidosRESUMO
PURPOSE: The purpose of this study was to isolate and characterize cDNA clones encoding target proteins for autoantibodies in patients at high risk for transplant rejection. METHODS: A pool of 10 sera from patients at high risk for rejection who had undergone corneal transplantation was used for immunoscreening of an endothelial cDNA library, and the cDNA fragments were subcloned into prokaryotic expression vectors to generate recombinant fusion proteins. Cytotoxicity of recombinant protein was determined by a modified 51Cr-release assay. Apoptosis induced by recombinant protein was determined by fluorescent dye-chromatin fragmentation assay and by TdT-dUTP terminal nick-end labeling (TUNEL) assay. An enzyme-linked immunosorbent assay was used to detect the presence of antibodies to recombinant protein in the sera of high-risk patients undergoing corneal transplantation and of control subjects. RESULTS: Screening of 500,000 plaques identified six positive clones, one of which demonstrated extensive homology with a novel tumor-derived cytokine termed endothelial monocyte-activating polypeptide (EMAP). EMAP was synthesized as a 39-kDa precursor that was proteolytically cleaved to generate an active 22-kDa cytokine. The mature peptide of EMAP alone was capable of inducing the death of cultured endothelial cells, whereas the propeptide was inactive. The protein synthesis inhibitor cycloheximide potentiated EMAP-induced apoptosis in endothelial cells. Cell death by apoptosis was evidenced by DNA fragmentation, extensive surface bleb formation, and chromatin condensation. A statistically significant difference was found in the level of antibodies specific to EMAP between patients at high risk for corneal transplant rejection and control subjects (P<0.001). The antibody levels were elevated in patients with severe graft reaction when compared with patients with no graft reaction (P<0.001). CONCLUSIONS: These studies demonstrated that EMAP is a novel protein in corneal endothelial cells that is capable of inducing programmed cell death. Overexpression of this cytokine could initiate endothelial cell damage leading to stromal edema and corneal decompensation.
Assuntos
Apoptose/efeitos dos fármacos , Citocinas , Endotélio Corneano/efeitos dos fármacos , Proteínas do Olho/farmacologia , Proteínas de Neoplasias/farmacologia , Proteínas de Ligação a RNA/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Sequência de Aminoácidos , Animais , Autoanticorpos/análise , Sequência de Bases , Bovinos , Células Cultivadas , Transplante de Córnea , DNA Complementar/análise , Endotélio Corneano/química , Endotélio Corneano/patologia , Ensaio de Imunoadsorção Enzimática , Proteínas do Olho/genética , Proteínas do Olho/imunologia , Rejeição de Enxerto/imunologia , Humanos , Marcação In Situ das Extremidades Cortadas , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
PURPOSE: Experimental autoimmune uveoretinitis (EAU) in mice is an important model for elucidating basic mechanisms in autoimmune eye disease. The need for pertussis toxin (PTX) as an additional adjuvant to elicit EAU has limited the usefulness of this model in some types of studies by introducing a pleiotropic factor with confounding effects on the immune response. METHODS: In the present study the authors examined the ability of B10.RIII mice, the most susceptible strain known so far, to develop EAU in response to the retinal antigen, interphotoreceptor retinoid-binding protein (IRBP), and to a major uveitogenic epitope of IRBP, peptide (p)161-180, in the absence of PTX treatment. RESULTS: The data indicate that high disease scores in response to IRBP and p161-180 were found in B10.RIII mice, without the need for PTX as part of the immunization protocol. Unlike the B10.A strain in which appreciable disease did not develop without PTX, B10.RIII mice mounted a high IFN-gamma response to IRBP in the absence of PTX treatment. Interestingly, and unlike the effect with IRBP, in vitro recall response to p161-180 was low in IFN-gamma, despite good EAU scores. CONCLUSIONS: The data indicate that an important mechanism through which PTX facilitates induction of cell-mediated autoimmunity is by promoting a Th1 polarization of the immune response. The propensity of B10.RIII mice to mount a more polarized Th1 response to IRBP than other strains may contribute to their ability to develop EAU without pertussis adjuvant. Nevertheless, the induction of EAU by p161-180 in the context of a relatively limited IFN-gamma production indicates that non-Th1- and Th-related mechanisms are likely to act in concert to determine the outcome of disease.
Assuntos
Adjuvantes Imunológicos/farmacologia , Doenças Autoimunes/induzido quimicamente , Toxina Pertussis , Retinite/induzido quimicamente , Proteínas de Ligação ao Retinol/farmacologia , Células Th1/imunologia , Uveíte/induzido quimicamente , Fatores de Virulência de Bordetella/farmacologia , Transferência Adotiva , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Autoimunidade/efeitos dos fármacos , Autoimunidade/imunologia , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Proteínas do Olho/farmacologia , Hipersensibilidade Tardia/etiologia , Hipersensibilidade Tardia/imunologia , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/imunologia , Ativação Linfocitária , Camundongos , Camundongos Mutantes , Fragmentos de Peptídeos/farmacologia , Reprodutibilidade dos Testes , Retinite/genética , Retinite/imunologia , Uveíte/genética , Uveíte/imunologia , VacinaçãoRESUMO
PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP), an extracellular protein believed to support the exchange of retinoids between the neural retina and retinal pigment epithelium (RPE) in the vertebrate eye, exhibits a modular, i.e., repeat, structure. The present study was undertaken to determine whether an individual module of IRBP has activity in retinoid transfer between the RPE and rod photoreceptors. METHODS: The retinoid transfer activity of a recombinant protein corresponding to the fourth module of Xenopus laevis IRBP (X4IRBP) was examined in two ways. First, X4IRBP was tested for its ability to support the regeneration of porphyropsin in detached/reattached Xenopus retina/RPE-eyecups. Following illumination and removal of native IRBP, Xenopus eyecups supplemented with 42 microM X4IRBP or (as a control) Ringer's solution were incubated in darkness and then analyzed for regenerated porphyropsin. Second, toad (Bufo marinus) RPE-eyecup preparations were used to evaluate X4IRBP's ability to promote the release of 11-cis retinal from the RPE. RESULTS: The regeneration of porphyropsin in X4IRBP-supplemented Xenopus retina/RPE-eyecups (0.45 +/- 0.04 nmol; mean +/- SEM, n = 11) exceeded that in controls (0.13 +/- 0.02 nmol, n = 11). For promoting the release of 11-cis retinal from the toad RPE, 42 microM X4IRBP was more effective than equimolar bovine serum albumin although considerably less than that of 26 microM native bovine IRBP. CONCLUSIONS: The results indicate a low but significant activity of IRBP's fourth module in reactions relevant to retinoid exchange.