RESUMO
OBJECTIVE: To determine the Dynamin-related protein 1 (DRP1) regulation of mitochondrial fission in chondrocytes under pathological conditions, an area which is underexplored in osteoarthritis pathogenesis. DESIGN: DRP1 protein expression was determined by immunohistochemistry (IHC) or immunofluorescence (IF) staining of cartilage sections. IL-1ß-induced DRP1 mRNA expression in chondrocytes was quantified by qPCR and protein expression by immunoblotting. Mitochondrial fragmentation in chondrocytes was visualized by MitoTracker staining or IF staining of mitochondrial marker proteins or by transient expression of mitoDsRed. Mitochondrial reactive oxygen species (ROS) levels were determined by MitoSOX staining. Apoptosis was determined by lactate dehydrogenase (LDH) release assay, Caspase 3/7 activity assay, propidium iodide (PI), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and IF staining of cleaved caspase 3. Cytochrome c release was determined by confocal microscopy. Surgical destabilization of the medial meniscus (DMM) was used to induce osteoarthritis (OA) in mice. RESULTS: Expression of DRP1 and mitochondrial damage was high in human OA cartilage and in the joints of mice subjected to DMM surgery which also showed increased chondrocytes apoptosis. IL-1ß-induced mitochondrial network fragmentation and chondrocyte apoptosis via modulation of DRP1 expression and activity and induce apoptosis via Bax-mediated release of Cytochrome c. Pharmacological inhibition of DRP1 activity by Mdivi-1 blocked IL-1ß-induced mitochondrial damage and apoptosis in chondrocytes. Additionally, IL-1ß-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) is crucial for DRP1 activation and induction of mitochondrial network fragmentation in chondrocytes as these were blocked by inhibiting ERK1/2 activation. CONCLUSIONS: These findings demonstrate that ERK1/2 is a critical player in DRP1-mediated induction of mitochondrial fission and apoptosis in IL-1ß-stimulated chondrocytes.
Assuntos
Apoptose/fisiologia , Condrócitos/fisiologia , Dinaminas/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Dinâmica Mitocondrial/fisiologia , Idoso , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: Lysosomes are the major catabolic organelle of the cell and regulate the macromolecular and organelle turnover and programmed cell death. Here, we investigated the lysosome dysfunction in cartilage and its role in chondrocytes apoptosis and the associated mechanism. DESIGN: Lysosomal acidification in Osteoarthritis (OA) and aged cartilage was determined by LysoSensor staining. Lysosomal function in chondrocytes was blocked by siRNA mediated depletion of Lysosomal Associated Membrane Protein 2 (LAMP2) or with lysosome inhibitors. Chondrocyte apoptosis was determined by LDH release, Caspase-3/7 activation, TUNEL and PI uptake assays. Loss of mitochondrial membrane potential (MMP/ΔΨM) and mitochondrial superoxide level was determined by JC-1 and MitoSOX staining, respectively. Colocalization of mitochondria with BCL2 associated X (BAX) and Cytochrome c was determined by immunostaining. Destabilization of medial meniscus (DMM) was performed to induce OA in mice. RESULTS: Lysosomal acidification was found to be significantly decreased in aged mouse and human and mouse OA cartilage which also showed increased chondrocyte apoptosis. Inhibition of lysosomal function resulted in increased oxidative stress, accumulation of dysfunctional mitochondria and apoptosis in chondrocytes in monolayer and in cartilage explant cultures. Depletion of LAMP2 expression or treatment of chondrocytes with lysosomal function inhibitors increased the expression and mitochondrial translocation of BAX leading to Cytochrome c release. Lysosomal dysfunction-induced apoptosis in chondrocytes was not blocked by antioxidants MitoTempo or Diphenyleneiodonium (DPI) but was abrogated by inhibiting BAX. CONCLUSION: Lysosomal dysfunction induce apoptosis in chondrocytes through BAX-mediated mitochondrial damage and release of Cytochrome c. Our data points to lysosomal function restoration and/or BAX inhibition in chondrocytes as a therapeutic approach for OA.
Assuntos
Apoptose , Artrite Experimental/metabolismo , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Citocromos c/metabolismo , Lisossomos/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Osteoartrite do Joelho/metabolismo , Envelhecimento/metabolismo , Animais , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/genética , Meniscos Tibiais/cirurgia , Camundongos , Superóxidos/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
OBJECTIVES: Recent studies have shown that tRNA-derived RNA fragments (tRFs) are novel regulators of post-transcriptional gene expression. However, the expression profiles and their role in post-transcriptional gene regulation in chondrocytes is unknown. Here, we determined tRFs expression profile and explored tRF-3003a role in post-transcriptional gene regulation in IL-1ß stimulated chondrocytes. METHODS: We used qPCR arrays to determine tRNAs and tRFs expression in age- and sex-matched primary human OA chondrocytes and TC28/I2 cells stimulated with IL-1ß. Chondrocytes were transfected with tRNA-CysGCA overexpression plasmid or tRF-3003a mimic and 3'UTR luciferase reporter plasmids of mRNAs harboring predicted tRF target "seed sequence". The AGO-RNA-induced silencing complex (AGO-RISC)-dependent repressive activity of tRF-3003a was determined by siRNA-mediated knockdown of AGO2. RESULTS: IL-1ß increased the expression levels of specific tRNAs and of tRF-3003a, a type 3 tRF produced by the cleavage of tRNA-CysGCA. tRF-3003a "seed sequence" was identified in the 3'UTR of JAK3 mRNA and tRNA-CysGCA overexpression or transfection of a tRF-3003a mimic in chondrocytes downregulated JAK3 expression and significantly reduced the activity of the 3'UTR reporter. RIP assay showed enrichment of tRF-3003a into AGO2/RISC in IL-1ß treated chondrocytes. The suppressive effect of tRF-3003a on JAK3 3'UTR reporter was abrogated with siRNA-mediated depletion of AGO2. CONCLUSIONS: We demonstrate that under pathological conditions chondrocytes display perturbations in the expression profile of specific tRNAs and tRFs. Furthermore, a specific tRF namely tRF-3003a can post-transcriptionally regulate JAK3 expression via AGO/RISC formation in chondrocytes. Identification of this novel mechanism may be of value in the design of precision therapies for OA.
Assuntos
Condrócitos/metabolismo , Regulação da Expressão Gênica , Osteoartrite/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/genética , RNA de Transferência de Cisteína/genética , Regiões 3' não Traduzidas , Proteínas Argonautas , Linhagem Celular , Condrócitos/efeitos dos fármacos , Humanos , Interleucina-1beta/farmacologia , Janus Quinase 3/genética , Osteoartrite/metabolismo , Cultura Primária de Células , RNA Mensageiro/efeitos dos fármacos , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA de Transferência de Cisteína/metabolismoRESUMO
OBJECTIVE: Mitochondrial dysfunction, oxidative stress and chondrocyte death are important contributors to the development and pathogenesis of osteoarthritis (OA). In this study, we determined the expression and role of Parkin in the clearance of damaged/dysfunctional mitochondria, regulation of reactive oxygen species (ROS) levels and chondrocyte survival under pathological conditions. METHODS: Human chondrocytes were from the unaffected area of knee OA cartilage (n = 12) and were stimulated with IL-1ß to mimic pathological conditions. Mitochondrial membrane depolarization and ROS levels were determined using specific dyes and flow cytometry. Autophagy was determined by Western blotting for ATG5, Beclin1, immunofluorescence staining and confocal microscopy. Gene expression was determined by RT-qPCR. siRNA, wild-type and mutant Parkin plasmids were transfected using Amaxa system. Apoptosis was determined by PI staining of chondrocytes and TUNEL assay. RESULTS: IL-1ß-stimulated OA chondrocytes showed high levels of ROS generation, mitochondrial membrane damage, accumulation of damaged mitochondria and higher incidence of apoptosis. IL-1ß stimulation of chondrocytes with depleted Parkin expression resulted in sustained high levels of ROS, accumulation of damaged/dysfunctional mitochondria and enhanced apoptosis. Parkin translocation to depolarized/damaged mitochondria and recruitment of p62/SQSTM1 was required for the elimination of damaged/dysfunctional mitochondria in IL-1ß-stimulated OA chondrocytes. Importantly we demonstrate that Parkin elimination of depolarized/damaged mitochondria required the Parkin ubiquitin ligase activity and resulted in reduced ROS levels and inhibition of apoptosis in OA chondrocytes under pathological conditions. CONCLUSIONS: Our data demonstrates that Parkin functions to eliminate depolarized/damaged mitochondria in chondrocytes which is necessary for mitochondrial quality control, regulation of ROS levels and chondrocyte survival under pathological conditions.
Assuntos
Condrócitos/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Apoptose , Western Blotting , Sobrevivência Celular , Células Cultivadas , Humanos , Potencial da Membrana Mitocondrial , Osteoartrite do Joelho/metabolismo , Reação em Cadeia da Polimerase , Ubiquitina-Proteína Ligases/metabolismoRESUMO
OBJECTIVE: Collagen induced arthritis (CIA) in mice is mediated by synergistic T cell and humoral immune responses specific for type II collagen (CII). We have previously shown that in arthritic joints of BUB mice (TCR Vbetaa, H-2q) the TCR repertoire is enrichedfor Vbeta10 expressing T cells, and that immunization with a Vbeta10 peptide (Vbeta10p) prevents the phenotypic expression of disease. The objective of the present study was to understand how immunization with a synthetic TCR Vbeta peptide affected the development of the pathogenic CII-specific immune response in BUB mice. METHODS: Arthritic and protected animals were tested for Vbeta10p- and CII-specific cytokine production by a highly specific and sensitive ELISA spot assay, andfor CII-specific antibody production by standard ELISA. In adoptive transfer experiments, Vbeta10p-specific LN cells (INF-gamma producing) were injected into naive mice prior to immunization with type-II collagen/CFA. RESULTS: Immune cells from arthritic animals produced IFN-gamma and IL-2, without IL-4 and IL-5 in response to CII and an immunodominant epitope, A2, derivedfrom CII. Serum from these mice contained anti-CII antibodies of both IgGI and IgG2a subtypes. Our results show for thefirst time that immunization with Vbeta10p resulted in Vbeta10p-specific IFN-gamma and IL-2 production that was restricted to the CD4+ T cell subset. Emergence of this Vbeta10p-specific immune response was associated with a dramatic decrease in the frequency of CII and A2-specific, cytokine producing T cells in arthritis protected mice. Protective immunity was cell mediated and could be adoptively transferred. In contrast, the protective immunization had only a marginal effect on the anti-CII antibody response indicating that the CII specific humoral immune response was not significantly affected. CONCLUSION: Immunization with TCR Vbeta10p leads to expansion of a population of Vbeta10p- specific CD4+ Tcells. This anti-TCR Vbeta10p specific type 1 cytokine producing immune response was protective in adoptive transfer studies and appears to inhibit the expansion of the pathogenic anti-CII cellular immunity. Additionally, the anti-TCR Vbeta10p-specific cellular immune response was mediated by CD4+ T cells and these T cells did not produce IL-4 or IL-5. Thus, our results suggest that protection against CIA in mice immunized with synthetic TCR Vbeta10p was achieved by a specific down-regulation of the CII-specific Thl type cellular immune response and not via immune deviation.
Assuntos
Artrite Experimental/imunologia , Artrite Experimental/prevenção & controle , Colágeno Tipo II/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Células Th1/imunologia , Vacinação , Animais , Artrite Experimental/fisiopatologia , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática , Articulações/patologia , Articulações/fisiopatologia , Linfonodos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Amplitude de Movimento Articular , Organismos Livres de Patógenos EspecíficosRESUMO
OBJECTIVE: To determine the expression profile of protein kinase (PK) and protein tyrosine kinase (PTK) genes in human primary osteoarthritis (OA) chondrocytes and to compare it with that of immortalized human chondrocytes T/C 28a4 with a view to learning whether T/C 28a4 cells can be used for elucidating signal transduction pathways in human chondrocytes. DESIGN: We used the Atlas Human cDNA Array and a method based on PCR with degenerate primers to analyse the expression profile of protein kinase genes in primary human OA chondrocytes and compared it with that of immortalized human chondrocyte cell line T/C 28a4 using RT-PCR and Western blotting. RESULTS: A total of 21 PTK genes were identified and several of these have never been shown to be expressed in human OA chondrocytes. Comparative expression analysis of some selected kinase genes showed that the mRNA expression pattern of many protein kinase genes in OA chondrocytes was identical to that of T/C 28a4 cells. However, there were differences in the level of protein expression of selected protein kinases in these cells. For example, mRNA expression of the novel kinase HCK was detected in OA chondrocytes and in the cell lines analysed but by Western blotting HCK protein was not detected in OA chondrocytes. In these studies, we also identified a novel mutant form of the discoidin domain receptor 2 (DDR2) transcript from chondrocyte-like cell line HTB-94. CONCLUSIONS: Our results provide novel information about protein kinase gene expression in OA chondrocytes and indicate that the transformed chondrocyte cell line T/C 28a4 may be suitable for elucidating signal transduction pathways in chondrocytes and to investigate how they regulate chondrocyte function in inflammatory and degenerative joint diseases.
Assuntos
Condrócitos/fisiologia , Osteoartrite/genética , Proteínas Tirosina Quinases/genética , Western Blotting , Linhagem Celular Transformada , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Sondas de Oligonucleotídeos , Osteoartrite/patologia , Proteínas Quinases/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologiaRESUMO
Green tea polyphenol-(-)epigallocatechin-3-gallate (EGCG)-is a potent chemopreventive agent in many test systems and has been shown to inhibit tumor promotion and induce apoptosis. In this study we describe a novel observation that EGCG displayed strong inhibitory effects on the proliferation and viability of HTB-94 human chondrosarcoma cells in a dose-dependent manner and induced apoptosis. Investigation of the mechanism of EGCG-induced apoptosis revealed that treatment with EGCG resulted in DNA fragmentation, induction of caspase-3/CPP32 activity, and cleavage of the death substrate poly(ADP-ribose)polymerase (PARP). Pretreatment of cells with a synthetic pan-caspase inhibitor (Z-VAD-FMK) and a caspase-3-specific inhibitor (DEVD-CHO) prevented EGCG-induced PARP cleavage. The induction of apoptosis by EGCG via activation of caspase-3/CPP32-like proteases may provide a mechanistic explanation for its antitumor effects.
Assuntos
Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Catequina/análogos & derivados , Apoptose/fisiologia , Neoplasias Ósseas , Caspase 3 , Catequina/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Condrossarcoma , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Cinética , Células Tumorais CultivadasRESUMO
Tumour necrosis factor alpha (TNF-alpha) is a cytokine with pleiotropic effects on cells ranging from proliferation to apoptosis. These biological effects of TNF-alpha are believed to be elicited by the induction or enhancement of the expression of TNF-alpha responsive genes in the target cells. TNF-alpha is pro-inflammatory and a principal mediator in the pathogenesis of arthritis. The activation of an inflammatory cascade by TNF-alpha in arthritis results in the degradation of cartilage, joint destruction and loss of function. Because TNF-alpha is an important mediator in the pathogenesis of arthritis, the present study addresses the identification of novel TNF-alpha responsive genes in HTB-94 cell line which is of human origin and maintains a chondrocytic phenotype. The three identified cDNAs were previously not known to be induced or upregulated by TNF-alpha in chondrocytes or cells of chondrocytic lineage. One of the identified cDNAs had sequence similarity to human hydroxyl lyase mRNA (PLOD), an enzyme involved in collagen biosynthesis and its metabolism; the second cDNA had sequence similarity to the human cytoplasmic anti-proteinase-2 mRNA (CAP-2), a member of a group of proteins shown to be associated with protecting cells from TNF-alpha-induced apoptosis; and the third cDNA had sequence similarity to a dual specificity kinase, TTK, which is associated with cell proliferation. Relative gene expression level analysis by PCR and by Northern blotting revealed that treatment with TNF-alpha enhanced the expression of PLOD, CAP2 and TTK transcripts which confirmed the results obtained with display gels. Furthermore, TTK mRNA expression was also induced in human articular chondrocytes treated with TNF-alpha but not in untreated chondrocytes. Our results suggest that these genes may play a role in chondrocytic responses to TNF-alpha-mediated stimuli affecting the cartilage homeostasis.
Assuntos
Proteínas de Ciclo Celular , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Proteínas Quinases/genética , Serpinas/genética , Fator de Necrose Tumoral alfa/farmacologia , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , DNA Complementar/genética , Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 3 da Matriz/genética , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , RNA Mensageiro/genética , RNA Mensageiro/metabolismoAssuntos
Artrite Experimental/etiologia , Artrite Reumatoide/etiologia , Modelos Animais de Doenças , Animais , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Colágeno , Humanos , Receptores de Hialuronatos/imunologia , Camundongos , Camundongos Endogâmicos DBA , Linfócitos T/imunologiaRESUMO
Identification of common dietary substances capable of affording protection or modulating the onset and severity of arthritis may have important human health implications. An antioxidant-rich polyphenolic fraction isolated from green tea (green tea polyphenols, GTPs) has been shown to possess anti-inflammatory and anticarcinogenic properties in experimental animals. In this study we determined the effect of oral consumption of GTP on collagen-induced arthritis in mice. In three independent experiments mice given GTP in water exhibited significantly reduced incidence of arthritis (33% to 50%) as compared with mice not given GTP in water (84% to 100%). The arthritis index also was significantly lower in GTP-fed animals. Western blot analysis showed a marked reduction in the expression of inflammatory mediators such as cyclooxygenase 2, IFN-gamma, and tumor necrosis factor alpha in arthritic joints of GTP-fed mice. Histologic and immunohistochemical analysis of the arthritic joints in GTP-fed mice demonstrated only marginal joint infiltration by IFN-gamma and tumor necrosis factor alpha-producing cells as opposed to massive cellular infiltration and fully developed pannus in arthritic joints of non-GTP-fed mice. The neutral endopeptidase activity was approximately 7-fold higher in arthritic joints of non-GTP-fed mice in comparison to nonarthritic joints of unimmunized mice whereas it was only 2-fold higher in the arthritic joints of GTP-fed mice. Additionally, total IgG and type II collagen-specific IgG levels were lower in serum and arthritic joints of GTP-fed mice. Taken together our studies suggest that a polyphenolic fraction from green tea that is rich in antioxidants may be useful in the prevention of onset and severity of arthritis.
Assuntos
Artrite Experimental/prevenção & controle , Flavonoides , Fenóis/uso terapêutico , Polímeros/uso terapêutico , Chá , Animais , Formação de Anticorpos , Artrite Experimental/imunologia , Artrite Experimental/patologia , Galinhas , Colágeno/imunologia , Humanos , Imunoglobulina G/análise , Imunoglobulina G/sangue , Interferon gama/análise , Interferon gama/genética , Articulações/efeitos dos fármacos , Articulações/imunologia , Articulações/patologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Fenóis/isolamento & purificação , Polímeros/isolamento & purificação , Polifenóis , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/genéticaRESUMO
Alloreactive T lymphocytes can respond to foreign MHC complexed with foreign peptides through the direct pathway of allorecognition and can additionally recognize allopeptides expressed in the context of recipient (self) MHC through the indirect pathway. To better elucidate how indirect pathway-responsive CD4(+) T cells mediate allograft rejection, we isolated and characterized a TH1 T cell line from BALB/c recipients of B10.A skin that responds to a defined immunodominant, self-restricted allopeptide, I-Abetak58-71. When transferred into BALB/c severe combined immunodeficiency recipients of B10.A skin allografts, this cell line specifically induced a form of skin graft rejection characterized by the presence of TH1 cytokines, macrophage infiltration, and extensive fibrosis. Recall immune responses and immunofluorescence of the rejecting skin revealed only the presence of the peptide-specific T cells within the recipient animals, with no evidence of a direct pathway alloresponse. These studies demonstrate that T cells reactive to a single self-restricted allopeptide can mediate a form of allogeneic skin graft rejection that exhibits characteristics of a chronic, fibrosing process.
Assuntos
Rejeição de Enxerto/imunologia , Epitopos Imunodominantes/imunologia , Peptídeos/imunologia , Transplante de Pele/imunologia , Células Th1/imunologia , Transplante Homólogo/imunologia , Animais , Antígenos CD/imunologia , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Testes Imunológicos de Citotoxicidade , Feminino , Fibrose/patologia , Citometria de Fluxo , Técnica Direta de Fluorescência para Anticorpo , Rejeição de Enxerto/patologia , Hipersensibilidade Tardia/imunologia , Imunidade Celular , Memória Imunológica , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID , Reação em Cadeia da Polimerase , RNA/genética , Pele/patologia , Transplante de Pele/patologia , Células Th1/metabolismo , Transplante Homólogo/patologiaRESUMO
Multiple TCRBV genes have been implicated in experimental autoimmune myasthenia gravis (EAMG) pathogenesis in susceptible H-2(b) strains of mice. We studied the contribution of specific TCRBV and AV genes in EAMG pathogenesis using B10.BV8S2 transgenic mice (H-2[b]). The TCR transgenic mice predominantly have TCRBV8S2 transgene, but can use any of the endogenous AV gene repertoire. The transgenic mice were immunized with acetylcholine receptor (AChR) in CFA and evaluated for EAMG pathogenesis. Although the lymphocyte responses to AChR in B10.BV8S2 transgenic and nontransgenic TCR wild-type mice were equivalent, a marked reduction in lymphocyte response to the dominant AChR alpha chain peptide 146-162 was observed in the TCR transgenic mice. After boosting with AChR in CFA, anti-AChR Abs were detected in the serum, and 14 of 42 (33%) of the TCR transgenic mice developed clinical EAMG. Furthermore, EAMG in TCR transgenic mice was prevented by treatment with mAb to TCRBV8, which depleted BV8-expressing T cells. Cloning and sequencing of TCRAV genes from AChR-reactive T cells from B10.BV8S2 transgenic mice revealed a pattern of restricted TCRAV gene usage. The majority (60%) of the clones sequenced showed a sequence identical with that of the TCRAV1S8 gene. In the normal spleen cells of TCR transgenic mice, AV gene usage was more random. Thus, despite the presence of a complete endogenous TCRAV repertoire in B10.BV8S2 transgenic mice, T cells responding to AChR preferentially used a single endogenous TCRAV gene, thus implicating the involvement of the TCRAV1S8 gene in EAMG pathogenesis.
Assuntos
Genes , Miastenia Gravis/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores Colinérgicos/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular , Imunização , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miastenia Gravis/prevenção & controleRESUMO
OBJECTIVE: To define the genetic basis of a family with an autosomal, dominantly inherited form of spondyloepiphyseal dysplasia (SED) associated with tall stature. METHODS: A 6 generation family with early onset osteoarthritis (OA) associated with mild SED was studied. 14 individuals were examined clinically and radiologically, and DNA analysis was performed on 5. As the clinical pattern of joint involvement and tall stature of affected individuals resembled a family recently reported with an exon 11 mutation in COL2A1, this same mutation was specifically sought. In 2 clinically affected and 3 unaffected family members, exon 11 was amplified by polymerase chain reaction (PCR) followed by restriction enzyme digestion with Asp H1, the enzyme recognition sequence of which is altered by the mutation. The PCR product containing exon 11 was then directly sequenced. RESULTS: OA with widespread involvement of peripheral joints, in addition to spondylodysplasia, was seen in 14 members of the kindred. Affected family members had brachydactyly and were of average to above average height. Asp H1 digestion of the PCR product containing exon 11 in those with clinical disease was consistent with the presence of a mutation. Direct sequencing of this PCR product conclusively showed that a single base substitution was present in those with clinical disease, resulting in an arginine 75-cysteine (Arg75-Cys) mutation. CONCLUSION: We describe a 3rd family with an Arg75-Cys mutation with precocious generalized OA and mild SED. This finding supports the concept of mutational hot spots on COL2A1 related to the hypermutability of the cytosine-guanine doublet.
Assuntos
Osteoartrite/genética , Osteocondrodisplasias/genética , Pró-Colágeno/genética , Adolescente , Adulto , Mapeamento Cromossômico , Feminino , Humanos , Masculino , Mutação , Osteoartrite/diagnóstico por imagem , Linhagem , Reação em Cadeia da Polimerase , RadiografiaRESUMO
Collagen-induced arthritis (CIA) in susceptible strains of mice is an animal model of T cell-mediated inflammatory polyarthritis. Analysis of T cell receptor (TCR) V beta gene usage in cells isolated from arthritic joints of BUB/BnJ (BUB) mice (H-2q, TCR V beta a) showed that TCR V beta chain gene usage was limited to TCR V beta 3 and V beta 10 gene families. All of the BUB mice immunized with a mixture of TCR V beta 3 and TCR V beta 10 peptides, but not with control TCR V beta 14 peptide, were refractory to the induction of CIA. Immunization with TCR V beta 3 and V beta 10 peptides completely blocked the development of clinical and subclinical inflammation, formation of pannus and synovial hyperplasia, and the erosion of cartilage and bone. Further studies revealed that preimmunization of BUB mice with V beta 10 peptide alone was sufficient to render the mice resistant to CIA. Analysis of TCR V beta chain gene expression in lymph node cells from arthritic and arthritis-protected mice showed the expression of TCR V beta 10 subfamily in all of the arthritic mice, but not in arthritis-protected mice. Immunization with TCR V beta peptides did not diminish the humoral responses to chicken type-II collagen and also elicited significant levels of anti-V beta 3 and anti-V beta 10 peptide antibodies. Antibodies cross-reactive with mouse chicken type-II collagen were detected in both the arthritic and arthritis-protected mice. Adoptive transfer of serum from arthritis-protected BUB mice significantly delayed the onset (P < 0.005) of arthritis in recipient BUB mice. In contrast, mice injected with serum from arthritic mice had early onset of arthritis. These results demonstrate that immunization of BUB mice with TCR V beta chain peptides elicited antibodies reactive with the self-TCR and prevented the induction of collagen-induced arthritis by eliminating or downregulating pathogenic T cells and consequently blocking the development of humoral immune response. These findings may have clinical applications in treating human autoimmune diseases characterized by common TCR gene usage.
Assuntos
Artrite/prevenção & controle , Colágeno/imunologia , Depleção Linfocítica , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Imunização , Imunoterapia Adotiva , Masculino , Camundongos , Dados de Sequência MolecularRESUMO
The proteoglycans synthesized by human osteoarthritic femoral head cartilage and nonarthritic articular cartilage age-matched to the osteoarthritic cartilage specimens was studied in explant cultures and in chondrocytes generated by explant outgrowth from the cartilages. Twenty-four hours after explanation, both nonarthritic articular cartilage and osteoarthritic cartilage synthesized principally one large proteoglycan core protein that migrated on 3-5% acrylamide gels with an apparent molecular mass (M(r)) of approximately 520 kDa after enzymatic digestion with chondroitinase ABC and keratanase. The proteoglycan was found in both the explant itself and in the medium compartment of the culture as well. This proteoglycan contained chondroitin-6-sulfate, keratan sulfate and the hyaluronan binding region as evidenced by immunoblotting with murine anti-proteoglycan monoclonal antibodies indicating that the proteoglycan was aggrecan. To a much lesser extent two additional proteoglycan core proteins were also found in the explant but were not seen in the culture medium compartment. These proteoglycans possessed apparent M(r)'s of approximately 480 kDa and approximately 390 kDa on 3-5% acrylamide gels after chondroitinase ABC and keratanase digestion. The medium compartment contained principally the approximately 520 kDa proteoglycan core protein. In osteoarthritic cartilage explants, the pattern of newly synthesized proteoglycans recovered from the tissue as assessed on 3-16% polyacrylamide gradient gels remained relatively the same from day 1 after explantation up to 36 days of culture. By contrast, the proteoglycans recovered from the culture medium contained chondroitin sulfate and keratan sulfate after 1, 7, and 21 days in culture but by 36 days appeared to contain only chondroitin sulfate. Chondrocytes generated from osteoarthritic cartilage and age-matched nonarthritic articular cartilage synthesized different patterns of large (greater than 200 kDa) proteoglycan. Whereas chondrocytes derived from osteoarthritic cartilage continued to synthesize principally the approximately 520 kDa proteoglycan core protein, the chondrocytes derived from nonarthritic cartilage synthesized in addition to this proteoglycan, abundant amounts of the other two proteoglycan core proteins as well.
Assuntos
Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Proteoglicanas/biossíntese , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Articulação do Quadril , Humanos , Prótese Articular , Pessoa de Meia-Idade , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoartrite/cirurgia , Fenótipo , Proteoglicanas/genéticaRESUMO
We have earlier shown that T-cells in arthritic joints and LNs of B10.Q mice (H-2q, TCR V beta b) use a restricted number of TCR V beta chain genes (V beta 6, 8, 9). In the present study, we have investigated the TCR V beta chain gene expression in arthritic joints and LN of BUB/BnJ mice (H-2q, TCR V beta a). Mice were immunized with [table: see text] chicken type-II collagen, and arthritic joints and draining LNs were removed at the onset of arthritis and the TCR V beta chain gene expression was studied by PCR. A restricted usage of TCR V beta was observed in both the tissues. A dominant usage of TCR V beta 4, 7, and 15 was found in the LNs while TCR V beta 3 and 10 were predominantly expressed in arthritic joints in the majority of the arthritic mice (5/7). Our results indicate that (a) in H-2q mice with CIA there is a restricted usage of TCR V beta chain genes regardless of the TCR V beta genotype; and (b) in the absence of TCR V beta 8 and 9, TCR V beta 3 and 10 are predominantly used by joint-infiltrating T-cells.
Assuntos
Artrite Experimental/imunologia , Doenças Autoimunes/imunologia , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Subpopulações de Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Artrite Experimental/patologia , Antígenos H-2/imunologia , Camundongos , Dados de Sequência Molecular , Subpopulações de Linfócitos T/patologiaRESUMO
OBJECTIVE: To define the clinical, pathological and molecular genetic characteristics of a family with mild spondyloepiphyseal dysplasia (SED) and precocious osteoarthritis. METHODS: The proband was a 46-year-old man with precocious generalized OA, tall stature, mild chondrodysplasia and moderate deafness. His daughter, aged 21, showed similar clinical features. Electron microscopic (EM) analysis of collagen from an affected joint of the proband was performed. DNA was extracted from whole blood on the proband, his affected daughter, unaffected wife and second daughter, to look for a mutation in exons 31 or 11, sites where point mutations have been previously described in mild forms of SED. After finding no mutation in exon 31, exon 11 of COL2A1 was further analyzed. Exon 11 was amplified using polymerase chain reaction (PCR), and screening for the mutation was undertaken using a restriction enzyme digestion, the recognition sequence of which is altered by this point mutation. Sequence analysis was then performed. RESULTS: Electron microscopic (EM) analysis of cartilage from the proband showed thin appearing collagen fibrils organized into parallel lamellar structures. DNA studies revealed a single base change in one allele of exon 11 which produced an arginine to cysteine mutation at position 75 of the triple helix of type II collagen in the proband and his affected daughter. CONCLUSION: This is the 2nd example of an Arginine75-Cysteine mutation associated with SED; in our case, however, contrasting clinical features were present. Recurrent mutations at a few specific sites of COL2A1 suggest the possibility of susceptibility "hot spots" for mutational events.
Assuntos
Estatura , Éxons , Genes , Osteoartrite/genética , Osteocondrodisplasias/genética , Mutação Puntual , Pró-Colágeno/genética , Adulto , Alelos , Sequência de Bases , Cartilagem Articular/ultraestrutura , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sondas Moleculares/genética , Dados de Sequência Molecular , Osteocondrodisplasias/diagnóstico por imagem , Osteocondrodisplasias/patologia , Pró-Colágeno/classificação , RadiografiaRESUMO
There are increasing numbers of mutations described in the gene for type II collagen (COL2A1). Recently, COL2A1 mutations were shown to be associated with milder forms of chondrodysplasia, which may present with precocious generalized osteoarthritis (OA). The arginine519-cysteine and the arginine75-cysteine mutations are 2 such sites on COL2A1 where multiple unrelated families have been reported presenting with early onset, generalized OA and chondrodysplasia. The observation of multiple sites where recurrent mutations occur suggests that certain areas of COL2A1 are more prone to mutational events.