RESUMEN
OBJECTIVE: To explore prostaglandin (PG) E2 production by human articular chondrocytes induced by different chemokines. METHODS: Human chondrocytes were enzymatically isolated from the articular cartilage of patients with rheumatoid arthritis (RA), osteoarthritis (OA) or traumatic fracture (N) who underwent total joint replacement. They were cultured in vitro as monolayers and then exposed to MCP-1, RANTES or SDF-1 for 24 h. Levels of PGE2 and MMP-3 in the culture supernatant were then immunoassayed. RESULTS: PGE2 production was enhanced up to 2.7-fold in a subset of samples. Responses to different chemokines were heterogeneous even within the same disease groups. As previously reported, chemokines induced MMP-3 secretion by chondrocytes, but there was no significant correlation between levels of PGE2 and MMP-3. CONCLUSION: We here document the presence of "responders" among OA, RA and normal chondrocytes that produce enhanced levels of PGE2 upon stimulation by chemokines. The relationship between chemokines and prostaglandins could differentially influence the pathogenic network responsible for cartilage degradation in arthropathy.
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Artritis/inmunología , Quimiocinas/inmunología , Condrocitos/inmunología , Dinoprostona/inmunología , Metaloproteinasa 3 de la Matriz/inmunología , Anciano , Anciano de 80 o más Años , Artritis Reumatoide/inmunología , Artroplastia de Reemplazo , Cartílago Articular/inmunología , Quimiocina CCL2/inmunología , Quimiocina CCL5/inmunología , Quimiocina CXCL12 , Quimiocinas CXC/inmunología , Femenino , Fracturas Óseas/inmunología , Humanos , Masculino , Persona de Mediana Edad , Osteoartritis/inmunologíaRESUMEN
Allogeneic leukocyte immunotherapy is often used to improve fertility of patients with habitual abortion (HA), which probably acts through immune-mediated mechanisms. However, the involvement of T cells is not clear. This study examined the effect of allogeneic immunotherapy on T cells of patients with HA. Peripheral blood mononuclear cells were obtained from 5 healthy women and 14 women with HA. RNA was isolated from mononuclear blood cells. Reverse transcription-polymerase chain reaction (RT-PCR), followed by single-strand conformational polymorphism (SSCP) were used to analyze the gene segments of T-cell receptor beta chain (TCRbetaV) variable regions. Oligoclonal accumulation of T cells was identified in peripheral blood of nonpregnant patients with a history of HA. It was also revealed, however, that immunostimulation reduced the number of accumulating T-cell clones (p = 0.0004). The results, together with the clinical effectiveness of immunotherapy, suggest that accumulation of T-cell clonotypes, which probably resulted from antigenic stimulation, is involved in the pathogenesis of HA.
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Aborto Habitual/inmunología , Aborto Habitual/terapia , Traslado Adoptivo , Activación de Linfocitos , Transfusión de Linfocitos , Subgrupos de Linfocitos T/inmunología , Aborto Habitual/sangre , Adulto , Secuencia de Aminoácidos , Células Clonales , Femenino , Humanos , Inyecciones Subcutáneas , Masculino , Datos de Secuencia Molecular , Polimorfismo Conformacional Retorcido-Simple , Embarazo , Receptores de Antígenos de Linfocitos T alfa-beta/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Subgrupos de Linfocitos T/metabolismoRESUMEN
To identify the existence of antigen specific T cell responses and to follow the changes of these reactions, it is considered useful to evaluate whether certain T cells clonally accumulate in the lymphocyte population. For this purpose, we have established a novel method to analyze T cell clonality using a combination of reverse transcriptase-polymerase chain reaction of T cell receptor beta chain transcripts and single-strand conformation polymorphism (SSCP). Using this method, we obtained a smear-like pattern of electrophoresed DNA from the heterogeneous T cell population. On the other hand, a single T cell clone exhibits a band in the appropriate VP amplification and an accumulated T cell clone in a heterogeneous lymphocyte population is identified as a band in the background smear pattern. If a lymphocyte population was stimulated by an antigen either in vitro or in vivo, several distinct bands were found to be generated in the background smear. Thus, the dynamic changes of T cell clonal responses could be monitored with this method. Analyses of several immunological disorders, including autoimmune diseases, malignant disorders, and transplantations, revealed the involvement of antigen-specific T cell immune responses in these disorders. Furthermore, taking advantage of the reproducible mobility of a band of SSCP gel, we are now able to compare identities of the accumulated T cell clones in different samples without the need for nucleotide sequencing of each clone. Such information can thus elucidate the occurrence of uniform or stable immunological reactions in the host and also suggests that these reactions play an important role in vivo. Therefore, taken together, the above described novel T cell clonality analysis is considered to be useful in studying the T cell immune responses in various fields of immunology.
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Clonación Molecular/métodos , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Conformacional Retorcido-Simple , ARN Mensajero/análisis , Receptores de Antígenos de Linfocitos T/genética , Transducción de Señal/genética , Linfocitos T/inmunología , Enfermedades Autoinmunes/inmunología , Trasplante de Médula Ósea/inmunología , Células Clonales , Modelos Animales de Enfermedad , Femenino , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias Ováricas/inmunología , Embarazo/inmunología , ADN Polimerasa Dirigida por ARN , Neoplasias Uterinas/inmunologíaRESUMEN
The underlying mechanism of the degradation of articular cartilage is an imbalance between anabolic and catabolic pathways, which are under the control of cytokines. Chemokines are a novel class of small cytokines and have a wide range of effects in many different cell types, both inside and outside of the immune system. Their actions are mediated by a family of 7-transmembrane G-protein-coupled receptors. Recent studies have demonstrated that chondrocytes co-express chemokines and their receptors, and that the interaction of chemokines with their receptors results in the release of cartilage matrix-degrading proteinases, and affect proteoglycan metabolism in chondrocytes. These data reveal a catabolic pathway primed by chemokine/chemokine receptor system in articular cartilage, thus proposing a novel therapeutic approach against cartilage destruction in arthropathy.
RESUMEN
OBJECTIVE: To investigate the protective effect of interleukin-1 receptor antagonist (IL-1Ra) on bone and cartilage destruction in the induction phase of collagen-induced arthritis (CIA), an animal model of rheumatoid arthritis (RA). METHODS: DBA/1J mice were immunized with type II collagen for induction of collagen-induced arthritis (CIA) and simultaneously given different intraperitoneal doses of IL-1Ra daily, thrice weekly or once a week. Clinical symptoms of arthritis were noted daily and assessed using a scoring system during the course of disease. Bone and cartilage destruction in the mice was assessed by radiographic and histological methods respectively. RESULTS: Mice injected with IL-1Ra daily were completely protected from the occurrence of arthritis after immunization with type II collagen. Moreover, these mice were also protected against bone and cartilage destruction. However, weekly or thrice weekly treatment with IL-1Ra had no effect on arthritis and bone and cartilage destruction. CONCLUSION: Daily administration of recombinant IL-1Ra, injected at the same time as arthritis induction, is effective in blocking the occurrence of inflammatory as well as destructive changes in CIA. Daily bolus injections of IL-1Ra may therefore be useful for protection against joint damage following minor joint injury, whereas the maintenance of appropriate blood levels of the antagonist may be critical for its therapeutic effect on chronic inflammatory arthritis.
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Antirreumáticos/uso terapéutico , Artritis Experimental/prevención & control , Cartílago Articular/efectos de los fármacos , Sialoglicoproteínas/uso terapéutico , Animales , Antirreumáticos/administración & dosificación , Artritis Experimental/diagnóstico por imagen , Artritis Experimental/patología , Cartílago Articular/diagnóstico por imagen , Cartílago Articular/patología , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Miembro Posterior , Inyecciones Intraperitoneales , Proteína Antagonista del Receptor de Interleucina 1 , Articulaciones/efectos de los fármacos , Articulaciones/patología , Ratones , Ratones Endogámicos DBA , Radiografía , Índice de Severidad de la Enfermedad , Sialoglicoproteínas/administración & dosificaciónRESUMEN
OBJECTIVE: To determine whether YKL-39, a recently cloned secretory protein of articular chondrocytes, is arthritogenic in mice. METHODS: Recombinant YKL-39 (rYKL-39) was expressed and purified from E. coli. To induce arthritis in mice, rYKL-39 (1, 10 or 50 g in Freund's incomplete adjuvant) was injected into the right footpad of mice from four different strains (BALB/c, DBA/1J, C57BL/6 and ICR). The mice received a second immunization with rYKL-39 by intradermal injection into the root of the tail 10 days after the first immunization. Severity of arthritis was assessed by scoring each paw on a scale from 0 to 4. Sixty days after thefirst immunization, the mice were sacrificed and the joints were examined by immunohistochemistry and radiography. The anti-YKL-39 and anti type II-collagen (CII) antibody titres were also assayed using ELISA. RESULTS: Immunization with YKL-39 induced arthritis in all strains of mice tested, among which BALB/c was most susceptible. Histological examination showed synovial proliferation and irregularity of the cartilage surface in YKL-39-injected BALB/c mice. Moreover radiographic analysis revealed pathological changes in these mice. The YKL-39-immunised mice produced not only anti-YKL-39 antibody but also antibody against type II collagen, suggesting a spreading of autoimmunity after YKL-39. CONCLUSIONS: YKL-39, a cartilage-related protein, is found to induce arthritis accompanied by pathologic changes in bone and cartilage. A better understanding of the immune response against cartilage-related components including YKL-39 may help to elucidate the pathological processes of arthritic disorders.
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Artritis Experimental/inmunología , Glicoproteínas/inmunología , Adipoquinas , Animales , Anticuerpos/sangre , Especificidad de Anticuerpos , Artritis Experimental/diagnóstico por imagen , Artritis Experimental/patología , Cartílago , División Celular/efectos de los fármacos , División Celular/inmunología , Proteína 1 Similar a Quitinasa-3 , Colágeno Tipo II/inmunología , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Glicoproteínas/genética , Glicoproteínas/farmacología , Humanos , Inyecciones Intradérmicas , Lectinas , Leucocitos Mononucleares/citología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos ICR , Radiografía , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacologíaRESUMEN
OBJECTIVE: To clarify the presence of specific T cell immune response to autologous chondrocytes in patients with osteoarthritis (OA). METHODS: Peripheral blood mononuclear cells obtained from OA or post-traumatic patients were co-cultured with irradiated autologous chondrocytes, and their proliferative response was assessed using 3H-thymidine incorporation. Expression of HLA-class II molecules was also assessed on chondrocytes by immunohistochemistry or flow cytometry. RESULTS: T cell responses to autologous chondrocytes in OA yielded a significantly greater mean stimulation index (6.35 +/- 1.63) compared to controls (1.21 +/- 0.09, p < 0.01). This response was partially blocked by antibodies against HLA class I, class II, CD4 or CD8. Increased expression of HLA-DP, -DQ, and -DR was observed. CONCLUSION: This study showed the autologous T cell-stimulating property of OA chondrocytes in vitro. The elucidation of the autoimmune responses may contribute to the understanding of immune-mediated mechanisms in OA.
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Condrocitos/inmunología , Antígenos HLA-D/análisis , Osteoartritis/inmunología , Linfocitos T/inmunología , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/inmunología , Cartílago Articular/citología , Estudios de Casos y Controles , Células Cultivadas , Condrocitos/fisiología , Técnicas de Cultivo , Femenino , Humanos , Inmunohistoquímica , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Osteoartritis/fisiopatología , Probabilidad , Muestreo , Sensibilidad y Especificidad , Estadísticas no Paramétricas , Linfocitos T/fisiología , Trasplante AutólogoRESUMEN
OBJECTIVE: To investigate the modulation of expression of proteinase-activated receptor-2 (PAR-2) in articular chondrocytes by inflammatory cytokines. DESIGN: Articular synovium and cartilage tissues were collected from eight patients with osteoarthritis (OA), and three patients without arthropathy ("normal"). Chondrocytes were stimulated with interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha or transforming growth factor (TGF)-beta1. The expression of PAR-2 was detected using reverse transcriptase-polymerase chain reaction (PCR), Western blotting and immunofluorescence. Quantitative PCR was performed to assess the expression levels of PAR-2 messenger RNA (mRNA). RESULTS: The expression of PAR-2 mRNA was demonstrated in both OA and normal chondrocytes as well as in synovial fibroblasts. However, the level of PAR-2 in OA chondrocytes was much higher than in normal chondrocytes. Long-term culture revealed that PAR-2 mRNA expression was maintained up to three passages in OA but not in normal chondrocytes. IL-1beta and TNF-alpha both upregulated PAR-2 expression in normal and OA chondrocytes. In contrast, TGF-beta1 significantly decreased expression of PAR-2 in OA chondrocytes but increased PAR-2 in normal chondrocytes. CONCLUSIONS: Overexpression of PAR-2 in OA chondrocytes is upregulated by proinflammatory cytokines IL-1beta and TNF-alpha, and down-regulated by regulatory cytokine TGF-beta1. PAR-2 may be involved in the pathogenesis of OA.
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Cartílago Articular/inmunología , Condrocitos/inmunología , Citocinas/inmunología , Osteoartritis/inmunología , Receptor PAR-2/análisis , Anciano , Anciano de 80 o más Años , Línea Celular , Membrana Celular/inmunología , Células Cultivadas , Citoplasma/inmunología , Relación Dosis-Respuesta Inmunológica , Femenino , Fibroblastos/inmunología , Humanos , Interleucina-1beta/inmunología , Masculino , Persona de Mediana Edad , ARN Mensajero/análisis , Membrana Sinovial/inmunología , Factores de Tiempo , Factor de Crecimiento Transformador beta/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
OBJECTIVE: The vascular invasion of bone marrow tissue into the subchondral plate is often observed in articular cartilage and we named it the subchondral bone absorption pit; however, its implication in the pathogenesis of osteoarthritis (OA) has been poorly understood. The purpose of this study was to evaluate its characteristics and roles in osteoarthritic conditions. METHODS: Articular cartilage specimens from 11 patients with medial type knee OA and 7 non-arthritic cadavers were analyzed with HE staining. OA sections were stained with safranin-O, TRAP (tartrate resistant acid phosphatase) and immunostained with anti-MMP-1, MMP-3, MMP-13, vitronectin receptor (VNR)-alpha chain, vimentin and bone morphogenic protein (BMP) 2/4 antibodies. RESULTS: Subchondral bone resorption pits were classified according to the extent of invasion: pits with bone marrow tissue were located within uncalcified cartilage below the tidemark in grade I and invaded beyond the tidemark in grade II, while no invasion was seen in grade 0. Grade II pits were dominant in OA compared to non-arthritic joints, especially medial condyles. Proteoglycan detected with safranin-O staining was lost around the tip of grade II pits and the density of pits was related to the modified Mankin Score. Cells in pits expressed vimentin, MMP-1, MMP-3 and MMP-13. Some polynuclear cells co-expressed VNR-alpha chain and MMP-13, whereas pits showed reparative features expressing BMP. CONCLUSION: These results suggest that subchondral bone resorption pits contribute to cartilage degradation by expressing matrix metalloproteinases in OA.
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Células de la Médula Ósea/enzimología , Resorción Ósea/fisiopatología , Metaloproteinasas de la Matriz/análisis , Osteoartritis de la Rodilla/fisiopatología , Anciano , Proteínas Morfogenéticas Óseas/análisis , Remodelación Ósea/fisiología , Resorción Ósea/metabolismo , Cadáver , Cartílago Articular/irrigación sanguínea , Cartílago Articular/metabolismo , Cartílago Articular/fisiopatología , Colagenasas/análisis , Humanos , Inmunohistoquímica/métodos , Metaloproteinasa 1 de la Matriz/análisis , Metaloproteinasa 13 de la Matriz , Metaloproteinasa 3 de la Matriz/análisis , Neovascularización Patológica/fisiopatología , Osteoartritis de la Rodilla/metabolismo , Proteoglicanos/metabolismo , Vimentina/análisis , Vitronectina/análisisRESUMEN
OBJECTIVES: To characterise cartilage intermediate layer protein (CILP)-induced arthropathy in mice. METHODS: The first and second halves of the nucleotide triphosphate pyrophosphohydrolase (NTPPHase) non-homologous region of human CILP were prepared as recombinant proteins (C1 and C2, respectively), including three overlapping fragments of C2 (C2F1, C2F2, and C2F3). C57BL/6 mice were immunised with these proteins to induce arthritis. In addition, a separate group of mice were immunised repeatedly with the mixture of C1 and C2 to see the effect of chronic immunisation. Arthritis developed in the mice, and cellular and humoral immune responses against CILP were analysed. RESULTS: Immunisation with C2 and with the mixture C2F1/C2F2/C2F3 caused the severest arthritis to develop in mice. Immunisation with one of C1, C2F1, C2F2, or C2F3 caused milder arthritis, even though each of the fragments carried T cell epitopes. Immunisation either with C1 or C2 alone evoked cellular and humoral immune responses to both the C1 and C2 proteins. Further, the repeated immunisation with the C1/C2 mixture caused tendon calcification and bone irregularity, together with decreased NTPPH activity. CONCLUSIONS: The results show that multiple T cell epitopes are needed for the development of CILP-induced arthritis, and present the characteristic new model of mild arthropathy accompanied by extra-articular calcifications. An immune response to putative murine CILP/NTPPH may be involved in the ectopic calcifications in the arthritic mice.
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Artritis Reactiva , Proteínas de la Matriz Extracelular/genética , Modelos Animales , Pirofosfatasas/genética , Animales , Anticuerpos Monoclonales/análisis , Artritis Reactiva/diagnóstico por imagen , Artritis Reactiva/inmunología , División Celular , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas de la Matriz Extracelular/inmunología , Femenino , Inyecciones , Articulaciones/inmunología , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Pirofosfatasas/sangre , Pirofosfatasas/inmunología , Radiografía , Proteínas Recombinantes de Fusión/administración & dosificación , Linfocitos T/inmunologíaRESUMEN
OBJECTIVE: To investigate whether cartilage intermediate layer protein (CILP), a protein recently cloned from human articular cartilage, is recognized as an autoantigen in patients with osteoarthritis (OA) and rheumatoid arthritis (RA), and whether the immune response against CILP is involved in disease pathogenesis. METHODS: Recombinant fusion proteins, which contain the first half (C1), second half (C2), or 3 fragments within the C2 region (designated C2F1, C2F2, and C2F3) of the non-porcine nucleotide pyrophosphohydrolase-homologous region of CILP, were prepared using Escherichia coli. Autoantibodies to these proteins in serum samples from patients with OA or RA and from age-matched healthy individuals were detected by enzyme-linked immunosorbent assay and Western blotting. In addition, mice were immunized with a mixture of the C1 and C2 fusion proteins to assess the arthrogenicity of CILP. RESULTS: Production of antibodies to the C2 region was detected in 10.5% (11 of 105) of the tested OA patients and in 8.0% (7 of 88) of the tested RA patients, although antibodies to the C1 region were rarely detected in either patient group. All C2F1, C2F2, and C2F3 fragments were found to carry autoepitopes. The C2F2 fusion protein was recognized most frequently in the tested OA patients, whereas the C2F3 fusion protein was dominantly recognized in the tested RA patients. All 4 mice strains, DBA/1J, ICR, C57BL/6, and BALB/c, immunized with the CILP fusion proteins developed chronic arthritis; in particular, the ICR mice developed polyarthritis that was characterized by infiltration of mononuclear cells in the synovium and exfoliation of the surface of cartilage. CONCLUSION: The immune response to CILP may play a role in the pathogenesis of inflammatory joint destruction. Our results support the role of an immune-mediated process in the joint destruction present in chronic arthropathies such as OA and RA. The results suggest that suppression of immune responses to various components of the cartilage, such as CILP, might be therapeutically beneficial in these chronic arthropathies.
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Artritis Reumatoide/inmunología , Cartílago Articular/inmunología , Proteínas de la Matriz Extracelular/inmunología , Glicoproteínas/inmunología , Osteoartritis/inmunología , Pirofosfatasas , Adulto , Anciano , Anciano de 80 o más Años , Animales , Artritis Experimental/etiología , Artritis Experimental/inmunología , Artritis Experimental/patología , Artritis Reumatoide/patología , Cartílago Articular/patología , Cartilla de ADN/química , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Articulaciones/patología , Masculino , Ratones , Ratones Endogámicos , Persona de Mediana Edad , Osteoartritis/patología , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: Recent studies have suggested that interleukin (IL) 15 induces T cell accumulation in synovial lesions of rheumatoid arthritis (RA). This study aimed at determining whether this cytokine could explain in vivo T cell clonality in RA. METHODS: Peripheral blood mononuclear cells (PBMC) from patients with RA were stimulated in vitro with IL15 or IL2. After isolation of mRNA from stimulated cells and synovial T cells, genes coding the V-D(N)-J (CDR3) region of T cell receptor beta chains were amplified by a reverse transcriptase polymerase chain reaction. A single strand conformation polymorphism analysis was used to detect the clonotype(s) of accumulating T cells. Nucleotide and amino acid sequencing was also performed. RESULTS: Stimulation of PBMC with IL15 resulted in oligoclonal expansion of T cells. However, IL15 induced clones from PBMC were mostly different from the dominantly expanding T cell clones in synovial fluid. Furthermore, IL15 and IL2 responding clones were only partially identical. CONCLUSIONS: Although IL15 results in clonal accumulation of T cells, T cell clonality in rheumatoid joints could not be explained by the effect of IL15 alone. The results indicated the requirement of other factor(s), in addition to IL15, in the pathological process affecting RA joints. The results also suggested different responses by each T cell clone to IL15 or IL2.
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Artritis Reumatoide/inmunología , Enfermedades Autoinmunes/inmunología , Interleucina-15/farmacología , Líquido Sinovial/inmunología , Linfocitos T/efectos de los fármacos , Anciano , Secuencia de Aminoácidos , Secuencia de Bases , Técnicas de Cultivo de Célula , División Celular/efectos de los fármacos , División Celular/inmunología , Células Clonales/efectos de los fármacos , Células Clonales/inmunología , Femenino , Humanos , Interleucina-15/inmunología , Interleucina-2/inmunología , Activación de Linfocitos , Persona de Mediana Edad , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Linfocitos T/inmunologíaRESUMEN
T cell receptors, which recognize antigen peptides on MHC molecules, are essential probes for the analysis of T cell antigen specificity. The identification of paired T cell receptor (TCR) chains, alpha/beta or gamma/delta, usually requires the establishment of T cell clones, which is not always available. In this study, we tried, as an alternative method, the paired cloning of TCR alpha/beta genes directly from a single T cell. T cells were sorted as a single cell from which RNA was extracted. Then, TCR alpha/beta CDR3 regions were amplified from the single cell-derived cDNA by reverse transcriptase-polymerase chain reaction to determine their sequences. We successfully identified pairs of TCR alpha/beta genes, and reconstructed the TCR molecule by a bacterial expression system. This strategy makes it possible to obtain recombinant TCR molecules from a single T cell without cellular cloning and promotes the investigation of T cell antigen specificity.
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Clonación Molecular/métodos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Linfocitos T/inmunología , Diferenciación Celular , Células Clonales , Humanos , Reacción en Cadena de la Polimerasa/métodos , Receptores de Antígenos de Linfocitos T alfa-beta/inmunologíaRESUMEN
OBJECTIVE: To investigate whether autoimmunity to YKL-39, a recently cloned cartilage protein, occurs in patients with rheumatoid arthritis (RA). METHODS: Autoantibody to YKL-39 was assayed by enzyme linked immunosorbent assay (ELISA) and western blotting in serum samples from patients with RA, systemic lupus erythematosus (SLE), and healthy donors, using recombinant YKL-39 protein. This reactivity was compared with that against a YKL-39 homologue, YKL-40 (human cartilage gp-39/chondrex), which has been reported to be an autoantigen in RA. RESULTS: Autoantibody to YKL-39 was detected in seven of 87 patients with RA (8%), but not in serum samples from patients with SLE or healthy donors. YKL-40 reactivity was found in only one of 87 RA serum samples (1%), with no cross reactivity to YKL-39. CONCLUSION: The existence of anti-YKL-39 antibody in a subset of patients with RA is reported here for the first time. Further, it was shown that the immune response to YKL-39 was independent of that to YKL-40. Clarification of the antibody and T cell responses to autoantigens derived from chondrocyte, cartilage, or other joint components may lead to a better understanding of the pathophysiology of joint destruction in patients with RA.
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Artritis Reumatoide/inmunología , Autoanticuerpos/sangre , Autoantígenos/inmunología , Cartílago Articular/inmunología , Glicoproteínas/inmunología , Adipoquinas , Adulto , Anciano , Especificidad de Anticuerpos , Autoinmunidad , Western Blotting , Proteína 1 Similar a Quitinasa-3 , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Lectinas , Lupus Eritematoso Sistémico/inmunología , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/inmunologíaRESUMEN
OBJECTIVE: To identify the characteristics of cells isolated from pannus-like soft tissue on osteoarthritic cartilage (OA pannus cells), and to evaluate the role of this tissue in osteoarthritis (OA). METHODS: OA pannus cells were isolated from pannus-like tissues in five joints obtained during arthroplasty. The phenotypic features of the isolated cells were characterized by safranin-O staining and immunohistochemical studies. Expression of MMP-1, MMP-3 and MMP-13 was also assessed using reverse transcriptase-polymerase chain reactions (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry. RESULTS: Foci and plaque formation of pannus-like tissue over cartilage surface were found in 15 of 21 (71.4%) OA joints macroscopically, and among them, only five samples had enough tissue to be isolated. OA pannus cells were positive for type I collagen and vimentin, besides they also expressed type II collagen and aggrecan mRNA. Spontaneous expression of MMP-1, MMP-3 and MMP-13 was detected in OA pannus cells. Similar or higher levels of MMPs were detected in the supernatant of cultured OA pannus cells compared to OA chondrocytes, and among these MMP-3 levels were relatively higher in OA pannus cells. Immunohistochemically, MMP-3 positive cells located preferentially in pannus-like tissue on the border of original hyaline cartilage. CONCLUSION: Our results showed that OA pannus cells shared the property of mesenchymal cells and chondrocytes; however, their origin seemed different from chondrocytes or synoviocytes. The spontaneous expression of MMPs suggests that they are involved in the articular degradation in OA.
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Cartílago Articular/patología , Metaloproteinasas de la Matriz/análisis , Osteoartritis/patología , Anciano , Anciano de 80 o más Años , Cartílago Articular/metabolismo , Células Cultivadas , Colágeno/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Inmunohistoquímica/métodos , Masculino , Persona de Mediana Edad , Osteoartritis/metabolismo , Osteoartritis de la Cadera/metabolismo , Osteoartritis de la Cadera/patología , Osteoartritis de la Rodilla/metabolismo , Osteoartritis de la Rodilla/patología , Proteoglicanos/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Vimentina/análisisRESUMEN
OBJECTIVE: Osteopontin (OPN), secreted mainly from chondrocytes, is suggested to be involved in the ossification and remodeling of bone and also in regulation of cytokine profiles. We investigated whether patients with osteoarthritis (OA) and rheumatoid arthritis (RA) display autoimmunity against OPN. METHODS: Recombinant human OPN (rhOPN) was prepared as a fusion protein with beta-galactosidase using E. coli. Serum samples from patients with OA or RA and from age matched healthy donors were tested for autoantibodies to rhOPN using ELISA and Western blotting. Reactivity of the same samples to purified native human OPN (nhOPN) was investigated by ELISA separately, to evaluate conformational epitopes. RESULTS: By ELISA, autoantibodies to rhOPN were found in one (0.95%) of 105 patients with OA and 2 (2.3%) of 88 patients with RA. These autoantibodies to rhOPN were confirmed by Western blotting. In contrast, 11 (9.5%) of 105 OA serum and 13 (15%) of 88 RA serum samples reacted to nhOPN. The anti-OPN positive RA patients showed high serum levels of rheumatoid factor and C-reactive protein and accelerated erythrocyte sedimentation rate compared to the anti-OPN negative group, although the differences did not achieve statistical significance. CONCLUSION: Our data showed that OPN is one of the autoantigens in OA and RA. Preferential recognition of nhOPN to rhOPN indicates that major epitope(s) of OPN would be conformational. Clinically, existence of the anti-OPN antibodies may be linked to disease severity in RA.
Asunto(s)
Artritis Reumatoide/inmunología , Autoanticuerpos/sangre , Osteoartritis/inmunología , Sialoglicoproteínas/inmunología , Anciano , Anciano de 80 o más Años , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteopontina , Proteínas Recombinantes/inmunologíaRESUMEN
Although chondrocyte apoptosis has been noted in arthritic joints, the mechanism is not clear. To investigate whether Fas-mediated apoptosis has a role in this process, the presence of Fas mRNA and expression of cell surface Fas protein in monolayer-cultured human articular chondrocytes was analyzed. Fas mRNA was found in all chondrocyte samples analyzed; moreover, the majority of cells in chondrocyte populations expressed cell-surface Fas (12-90%, average 49%). Nevertheless, treatment with an agonistic anti-Fas antibody did not induce significant apoptosis in these chondrocytes in vitro. However, it was also found that chondrocytes express Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (FLIP), a molecule which blocks Fas-mediated apoptosis. Correspondingly, activation of caspase-8 was minimal in these cultured chondrocytes. In conclusion, although human articular chondrocytes do express cell-surface Fas, this receptor did not fully mediate death-inducing signals in vitro. This resistance to Fas may be partly due to the constitutive expression of FLIP.
Asunto(s)
Apoptosis/fisiología , Artritis Reumatoide/metabolismo , Proteínas Portadoras/metabolismo , Condrocitos/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Osteoartritis/metabolismo , Proteínas/fisiología , Proteínas Reguladoras de la Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Células Cultivadas , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: To characterise the type and kinetics of T cell clones in synovial lesions of patients with rheumatoid arthritis (RA). METHODS: Mononuclear cells from serial samples of synovial fluid (SF) and peripheral blood from nine RA patients were separated phenotypically using antibody coated magnetic beads. After mRNA preparation, reverse transcription-polymerase chain reaction (RT-PCR) was performed to amplify V-D(N)-J (that is, the third complementarity determining, CDR3) regions of their T cell receptor beta chain genes. This was followed by single strand conformation polymorphism (SSCP) analysis to detect the clonotypes of accumulating T cells. Amino acid sequences of the dominant clones were also determined. RESULTS: Although peripheral T cells were heterogeneous, accumulation of oligoclonal T cells was detected in SF. The predominant accumulating clone was the CD8 subset, which was persistently present in serial samples obtained over almost one year of follow up. A proportion of these cells expressed CD25 or CD45RO, or both, suggesting they are 'memory' clones. CONCLUSION: The persistent presence of CD8+ T cell clones in RA joints indicates that they may be involved in the perpetuation of the chronic inflammatory process in RA joints.
Asunto(s)
Artritis Reumatoide/inmunología , Linfocitos T CD8-positivos/inmunología , Líquido Sinovial/inmunología , Adulto , Anciano , Secuencia de Aminoácidos , Enfermedad Crónica , Femenino , Humanos , Inmunofenotipificación , Antígenos Comunes de Leucocito/análisis , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Complejo Receptor-CD3 del Antígeno de Linfocito T/genética , Receptores de Interleucina-2/análisis , Análisis de Secuencia de ADN , Factores de TiempoRESUMEN
Recently, oligoclonal T cell accumulation has been reported in affected joints of patients with rheumatoid arthritis. To characterize such clonally accumulating T cells, we quantitatively monitored their frequency by analyzing TCR B chains. As a result, despite a similar TCR BV usage between synovial fluid (SF) and PBL, obviously skewed TCR BJ usage was detected in SF. Subsequent DNA sequencing demonstrated that the skewed BJ gene usage in SF resulted from clonal T cell accumulation. The complementarity-determining region 3 of TCR B chains of the detected clones appeared to have homologous amino acid sequences. Further, one of the predominant TCR B chains of the clones was identical with that reported previously. In the follow-up study, most of the accumulated clones persisted; however, in some, proportions were drastically decreased or increased during the time course. In particular, one of the clones expanded sixfold, from 11 to 67%, in the BV8-BJ2S3 TCR population of SF, even though the clone was not detected in PBL. In summary, oligoclonal T cell accumulation in SF was persistent, but it appeared to fluctuate independently of the clonality in PBL. The expansion of the clones in SF may occur by antigenic stimuli within the joint.
Asunto(s)
Artritis Reumatoide/inmunología , Líquido Sinovial/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Secuencia de Aminoácidos , Artritis Reumatoide/genética , Artritis Reumatoide/patología , Células Clonales , Femenino , Humanos , Persona de Mediana Edad , Datos de Secuencia Molecular , Familia de Multigenes/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/aislamiento & purificación , Análisis de Secuencia de ADN , Subgrupos de Linfocitos T/metabolismo , Factores de TiempoRESUMEN
OBJECTIVE: To investigate and characterize pannus-like tissue which is often present on osteoarthritic articular cartilage. DESIGN: Cartilage specimens from 15 knee and five hip joints of patients with osteoarthritis (OA) undergoing arthroplasty were stained for HE and Safranin-O. They were also immunostained by antitype I collagen, type II collagen, CD68, IL-1beta and MMP3 antibodies. RESULTS: Ninety percent of joints have pannus-like tissue on the articular surface, preferentially in a marginal area. The articular cartilage was divided into three regions according to the location: the marginal zone, the intermediate zone and the paraeburnated zone. Pannuslike tissue in OA knee joint occurred 45.9%, 27.5% and 11.1% of the surface of each region respectively. Histologically, pannus-like tissue could be classified into the vascular type and the fibrous type. Extracellular matrix of both types of tissues were negative for Safranin-O and type II collagen, but positive for type I collagen. IL-1beta and MMP3 expressing cells are predominant in pannus-like tissue, whereas CD68 positive cells were infiltrated in only a few samples. Vascular type tissue kept continuity with bone marrow suggesting mesenchymal origin. CONCLUSION: Pannus-like tissue exists in advanced OA cartilage, preferentially in the marginal zone. It expressed IL-1beta and MMP3, which strongly suggests that it contributes to cartilage degradation.