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1.
Arthritis Rheum ; 46(2): 507-13, 2002 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11840454

RESUMEN

OBJECTIVE: To determine the involvement of mesenchymal progenitor cells in the induction of collagen-induced arthritis (CIA). METHODS: DBA/1 mice were immunized with type II collagen in adjuvant or adjuvant alone, and the presence of mesenchymal cells in the joints of prearthritic mice was studied by immunohistochemistry. RESULTS: An analysis of the joints on day 10 postimmunization (at least 10 days before the onset of arthritis) revealed synovial hyperplasia without leukocytic infiltration. Large, round cells expressing bone morphogenetic protein receptors (BMPRs), which serve as markers for primitive mesenchymal cells, were present in increased numbers in the bone marrow adjacent to the joint, in the synovium itself, and within enlarged bone canals that connect the bone marrow to the synovium. Similar changes were observed in mice given adjuvant without collagen. Adjuvant-induced infiltration of BMPR(+) cells and enlargement of bone canals were abrogated by anti-tumor necrosis factor (anti-TNF) treatment and were absent in TNFR p55/p75(-/-) mice. Increased numbers of bone marrow cells and enlarged bone canals were observed in nonimmunized TNF transgenic mice (which spontaneously develop arthritis). CONCLUSION: These findings suggest that in CIA, there is an antigen-independent (innate) prearthritic phase that prepares the joint for the subsequent immune-mediated arthritis. The induction phase involves marrow-derived mesenchymal cells and requires the presence of TNF.


Asunto(s)
Artritis Experimental/inmunología , Artritis Experimental/patología , Membrana Sinovial/patología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Antígenos CD/genética , Células de la Médula Ósea/química , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1 , Receptores de Proteínas Morfogenéticas Óseas de Tipo II , Femenino , Hiperplasia , Inmunohistoquímica , Articulaciones/patología , Masculino , Mesodermo/química , Mesodermo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Endogámicos DBA , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas/análisis , Receptores de Factores de Crecimiento/análisis , Receptores del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral , Receptores Tipo II del Factor de Necrosis Tumoral , Células Madre/patología , Membrana Sinovial/inmunología , Membrana Sinovial/metabolismo , Sinovitis/inmunología , Sinovitis/metabolismo , Sinovitis/patología
3.
J Clin Invest ; 107(3): 305-15, 2001 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11160154

RESUMEN

B-cell accumulation and formation of ectopic germinal centers are characteristic changes in the diseased joints of patients with rheumatoid arthritis (RA). Earlier studies suggested that interactions between B lymphocytes and specialized synovial "nurse-like" cells peculiar to the RA synovium may be responsible for the homing and sustained survival of B cells in the synovium. However, in this study, we found that B cells spontaneously migrate beneath ordinary fibroblast-like synoviocytes (FLSs) and then experience prolonged survival. FLSs isolated from joints of patients with osteoarthritis also supported this activity, termed B-cell pseudoemperipolesis. We found that FLSs constitutively expressed the chemokine stromal cell-derived factor-1 (SDF-1), and that pertussis toxin or antibodies to the SDF-1 receptor (CXCR4) could inhibit B-cell pseudoemperipolesis. However, expression of SDF-1 is not sufficient, as dermal fibroblasts also expressed this chemokine but were unable to support B-cell pseudoemperipolesis unless previously stimulated with IL-4 to express CD106 (VCAM-1), a ligand for the alpha(4)beta(1) integrin, very-late-antigen-4 (VLA-4 or CD49d). Furthermore, mAb's specific for CD49d and CD106, or the synthetic CS1 fibronectin peptide, could inhibit B-cell pseudoemperipolesis. We conclude that ordinary FLSs can support B-cell pseudoemperipolesis via a mechanism dependent upon fibroblast expression of SDF-1 and CD106.


Asunto(s)
Linfocitos B/fisiología , Células del Estroma/metabolismo , Membrana Sinovial/fisiología , Artritis Reumatoide/metabolismo , Linfocitos B/metabolismo , Línea Celular , Permeabilidad de la Membrana Celular , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Quimiocinas CXC/farmacología , Fibroblastos/efectos de los fármacos , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Integrina alfa4beta1 , Integrinas/metabolismo , Interleucina-4/farmacología , Potenciales de la Membrana , Receptores Mensajeros de Linfocitos/metabolismo , Piel/metabolismo , Membrana Sinovial/citología , Molécula 1 de Adhesión Celular Vascular/biosíntesis
4.
Arthritis Res ; 2(6): 477-88, 2000.
Artículo en Inglés | MEDLINE | ID: mdl-11056678

RESUMEN

STATEMENT OF FINDINGS: Mesenchymal precursor cells found in the blood (BMPCs) of normal persons adhere to plastic and glass and proliferate logarithmically in DMEM-20% fetal calf serum (FCS) without growth factors. They form cells with fibroblast-like and stromal morphology, which is not affected by eliminating CD34, CD3, or CD14 cells. Osteogenic supplements (dexamethasone, ascorbic acid, and beta-glycerophosphate) added to the culture inhibited fibroblast formation, and BMPCs assumed the cuboidal shape of osteoblasts. After 5 days in supplemented medium, the elutriated cells displayed alkaline phosphatase (AP), and the addition of bone morphogenetic protein (BMP)2 (1 ng) doubled AP production (P < 0.04). Two weeks later, 30% of the cells were very large and reacted with anti-osteocalcin antibody. The same cultures also contained sudanophlic adipocytes and multinucleated giant cells that stained for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptors. Cultured BMPCs immunostain with antibodies to vimentin, type I collagen, and BMP receptors, heterodimeric structures expressed on mesenchymal lineage cells. In addition, BMPCs stain with anti-CD105 (endoglin), a putative marker for bone-marrow mesenchymal stem cells (MSCs).


Asunto(s)
Células Sanguíneas/citología , Mesodermo/citología , Células Madre/citología , Fosfatasa Alcalina/sangre , Antígenos CD34/análisis , Células de la Médula Ósea/citología , Recuento de Células , Separación Celular , Quimiocina CXCL12 , Quimiocinas CXC/genética , Humanos , Inmunohistoquímica , Microscopía por Video , Valores de Referencia
5.
Blood ; 96(8): 2655-63, 2000 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-11023495

RESUMEN

A subset of blood cells from patients with B-cell chronic lymphocytic leukemia (CLL) spontaneously differentiates in vitro into large, round, or fibroblast-like adherent cells that display stromal cell markers, namely vimentin and STRO-1. These cells also express stromal cell-derived factor-1 (SDF-1), a CXC chemokine that ordinarily is secreted by marrow stromal cells. Leukemia B cells attach to these blood-derived adherent cells, down-modulate their receptors for SDF-1 (CXCR4), and are protected from undergoing spontaneous apoptosis in vitro. Neutralizing antibodies to SDF-1 inhibit this effect. Moreover, the rapid deterioration in the survival of CLL B cells, when separated from such cells, is mitigated by exogenous SDF-1. This chemokine also results in the rapid down-modulation of CXCR4 and activation of p44/42 mitogen-activated protein-kinase (ERK 1/2) by CLL B cells in vitro. It is concluded that the blood of patients with CLL contains cells that can differentiate into adherent nurse-like cells that protect leukemia cells from undergoing spontaneous apoptosis through an SDF-1-dependent mechanism. In addition to its recently recognized role in CLL B-cell migration, SDF-1-mediated CLL B-cell activation has to be considered a new mechanism involved in the microenvironmental regulation of CLL B-cell survival. (Blood. 2000;96:2655-2663)


Asunto(s)
Apoptosis/fisiología , Linfocitos B/citología , Células Sanguíneas/fisiología , Comunicación Celular , Quimiocinas CXC/fisiología , Regulación hacia Abajo , Leucemia Linfocítica Crónica de Células B/patología , Proteínas de Neoplasias/fisiología , Células Madre Neoplásicas/citología , Receptores CXCR4/biosíntesis , Linfocitos B/efectos de los fármacos , Células Sanguíneas/química , Diferenciación Celular , Supervivencia Celular , Quimiocina CXCL12 , Quimiocinas CXC/antagonistas & inhibidores , Quimiocinas CXC/biosíntesis , Quimiocinas CXC/genética , Quimiocinas CXC/inmunología , Cromosomas Humanos Par 13 , Técnicas de Cocultivo , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/farmacología , Inmunofenotipificación , Hibridación Fluorescente in Situ , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/efectos de los fármacos , ARN Mensajero/sangre , ARN Neoplásico/sangre , Células del Estroma/química , Células del Estroma/fisiología , Trisomía , Células Tumorales Cultivadas
6.
Arthritis Rheum ; 43(9): 2046-55, 2000 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-11014356

RESUMEN

OBJECTIVE: To evaluate the presence of cells of an early mesenchymal lineage, as judged by the expression of bone morphogenetic protein receptors (BMPRs), in the joints of normal individuals and patients with rheumatoid arthritis (RA). METHODS: Synovial fluids, single cell suspensions of cultured fibroblast-like synoviocytes (FLS), and synovial tissues were examined by immunohistology with antibodies to BMPR type IA (BMPRIA), BMPRIB, and BMPRII and then quantified using computerized image analysis. Other antibodies were evaluated by cytofluorography. RESULTS: In primary cultures of joint effusions from patients with RA and other forms of inflammatory arthritis, there were large adherent cells with the appearance of either fibroblasts or stromal cells that stained with antibodies to mesenchymal elements-CD44, type I collagen, alpha-actin, and vimentin-but not with antibodies to hematopoietic markers. These cells proliferated rapidly, expressed BMPRIA and BMPRII, and soon became the predominant cells in culture. They were retained through multiple passages and persistently displayed surface vascular cell adhesion molecule 1. Immunohistochemical analysis of cultured RA FLS (passages 3, 4, and 6; n = 6) revealed that 11.6% were BMPR-positive, while only 2.0% of osteoarthritis FLS (passage 4; n = 3) were BMPR-positive, and 1 normal synovial culture had no BMPR-positive cells. In all RA synovial membranes examined (n = 9), BMPRI- and BMPRII-expressing cells were identified in the intimal lining and were also scattered in the subintima. These cells constituted approximately 25% and approximately 7% of the cells in each area, respectively. Double immunostaining showed no coexpression of BMPR-positive cells with CD68, CD34, or CD3. Cells expressing BMPR were not seen in any normal synovial samples (n = 4). Strong staining for BMPR was identified on cells at the invasive front of the pannus and at sites of cartilage erosion. CONCLUSION: The inflamed RA joint contains BMPR-positive mesenchymal cells. Their origin is still speculative, but since their counterparts in the bone marrow are essential for osteoclastogenesis, support lymphocyte development and maturation, and protect T cells and B cells from programmed cell death, the BMPR-positive cells may be essential elements in the pathogenesis of RA and other inflammatory forms of chronic synovitis.


Asunto(s)
Artritis Reumatoide/metabolismo , Articulación de la Rodilla/metabolismo , Mesodermo/citología , Receptores de Superficie Celular/biosíntesis , Receptores de Factores de Crecimiento , Receptores de Proteínas Morfogenéticas Óseas , Cartílago Articular/citología , Humanos , Fenotipo , Líquido Sinovial/citología , Membrana Sinovial/citología
7.
Immunol Today ; 21(2): 78-82, 2000 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-10652465

RESUMEN

Oxidative stress at sites of chronic inflammation can cause permanent genetic changes. The development of mutations in the p53 tumor suppressor gene and other key regulatory genes could help convert inflammation into chronic disease in rheumatoid arthritis and other inflammatory disorders.


Asunto(s)
Artritis Reumatoide/metabolismo , Estrés Oxidativo/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Bolsa Sinovial/citología , Bolsa Sinovial/metabolismo , Genes p53/efectos de los fármacos , Genes p53/genética , Humanos , Mutación , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/genética
8.
Arthritis Rheum ; 42(5): 948-53, 1999 May.
Artículo en Inglés | MEDLINE | ID: mdl-10323450

RESUMEN

OBJECTIVE: The p53 tumor suppressor gene is overexpressed in synovial tissue (ST) from patients with longstanding rheumatoid arthritis (RA), and may contain somatic mutations. The aim of this study was to determine p53 expression in ST from RA patients in different stages of the disease, compared with disease controls. METHODS: ST biopsy specimens were obtained from the knee joints of 31 RA patients in varying disease phases, 8 patients with reactive arthritis (ReA), 10 patients with inflammatory osteoarthritis (OA), and 6 control patients (4 with meniscus pathology, 2 with vascular insufficiency). ST was also obtained from the clinically uninvolved knee joints of 9 RA patients. Expression of p53 was determined by immunohistology with DO1 monoclonal antibody (mAb) in all patients and by Western blot analysis with DO7 mAb in a subgroup of the patients. RESULTS: The p53 protein was detected by immunohistology in 10 of the 13 patients with early RA (duration <6 months) and in 12 of the 14 patients with longstanding RA (duration >5 years). The p53 protein was also demonstrated in clinically uninvolved knee joints. Western blots revealed immunoreactive p53 in ST extracts from all RA patients. Expression of p53 was about twice as high in ST from patients with longstanding RA as in early RA samples, but the difference did not reach statistical significance. Small amounts of p53 were also detected in ST from ReA and OA patients, although the expression in RA synovium was significantly higher. Immunohistologic analysis of normal ST gave negative results for p53. CONCLUSION: This study shows that p53 overexpression is specific for RA, compared with OA and ReA. This phenomenon is probably secondary to increased production of wild-type p53 protein in response to DNA damage and secondary to somatic mutations caused by the genotoxic local environment in inflamed ST. Of interest, p53 overexpression can also be found in the earliest stages of RA and in clinically uninvolved joints.


Asunto(s)
Artritis Reactiva/genética , Artritis Reumatoide/genética , Osteoartritis de la Rodilla/genética , Membrana Sinovial/química , Proteína p53 Supresora de Tumor/genética , Anciano , Western Blotting , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Prohibitinas , Factores de Tiempo
9.
Am J Pathol ; 152(4): 1091-8, 1998 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-9546370

RESUMEN

Recent studies show that 1) the p53 tumor suppressor protein is overexpressed by rheumatoid arthritis (RA) synovium and fibroblast-like synoviocytes (FLS) and 2) somatic mutations previously identified in human tumors are present in RA synovium and FLS. We have hypothesized that abnormalities in p53 can contribute to chronic destructive RA synovitis. To understand the functional consequences of p53 abnormalities in FLS, RA and normal FLS expressing wild-type p53 were transduced with a retroviral vector encoding the human papilloma virus 18 E6 gene, which inactivates endogenous p53 protein. Three RA and one normal FLS lines were infected with recombinant retrovirus encoding the neomycin resistance gene (neo) or E6+neo. FLS proliferation, apoptosis, and invasion was studied in E6, neo, and uninfected parental strains (PS). The growth rate for E6 was significantly increased with a sixfold increase in cell number after 7 days compared with a twofold to threefold increase in neo and PS. When FLS were treated with cytokines, proliferative response of E6, neo, and PS to interleukin-1 and transforming growth factor-beta were similar. However, response to platelet-derived growth factor was significantly greater in E6 FLS compared with neo or PS. Apoptosis was studied by incubating FLS with sodium nitroprusside as a source of nitric oxide or hydrogen peroxide for 8 hours and examining DNA fragmentation and E6 cells were significantly less susceptible to cell death. In addition, E6 FLS were more invasive into cartilage extracts than neo or PS using an in vitro cell invasion assay. These data suggest that p53 is a critical regulator of FLS proliferation, apoptosis, and invasiveness. Abnormalities of p53 function might contribute to synovial lining expansion and joint destruction in RA.


Asunto(s)
Apoptosis/genética , Artritis Reumatoide/patología , Membrana Sinovial/patología , Proteína p53 Supresora de Tumor/fisiología , Artritis Reumatoide/genética , Northern Blotting , División Celular/genética , Movimiento Celular/genética , Células Cultivadas , Colagenasas/metabolismo , Citometría de Flujo , Humanos , Peróxido de Hidrógeno/farmacología , Interleucina-1/farmacología , Nitroprusiato/farmacología , Proteínas Oncogénicas Virales/genética , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Represoras/genética , Transfección , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/farmacología
10.
Proc Natl Acad Sci U S A ; 94(20): 10895-900, 1997 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-9380731

RESUMEN

The factors that regulate the perpetuation and invasiveness of rheumatoid synovitis have been the subject of considerable inquiry, and the possibility that nonimmunologic defects can contribute to the disease has not been rigorously addressed. Using a mismatch detection system, we report that synovial tissue from the joints of severe chronic rheumatoid arthritis patients contain mutant p53 transcripts, which were not found in skin samples from the same patients or in joints of patients with osteoarthritis. Mutant p53 transcripts also were identified in synoviocytes cultured from rheumatoid joints. The predicted amino acid substitutions in p53 were identical or similar to those commonly observed in a variety of tumors and might influence growth and survival of rheumatoid synoviocytes. Thus, mutations in p53 and subsequent selection of the mutant cells may occur in the joints of patients as a consequence of inflammation and contribute to the pathogenesis of the disease.


Asunto(s)
Artritis Reumatoide/genética , Genes p53 , Mutación , Membrana Sinovial/patología , Células Cultivadas , Clonación Molecular , ADN Complementario , Humanos , Fenotipo
11.
Arthritis Rheum ; 40(9): 1644-52, 1997 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-9324019

RESUMEN

OBJECTIVE: To examine the expression and function of CD95 (Fas) and its ligand in rheumatoid arthritis (RA). METHODS: We used flow cytometry and reverse transcriptase-polymerase chain reaction methods to assess lymphocyte expression of CD95 and its ligand. We also examined whether lymphocytes could undergo Fas-mediated apoptosis with anti-CD95 monoclonal antibody (MAb) or human Fas ligand-expressing fibroblasts, and if synovial fluid contained a soluble factor(s) that could inhibit such interactions. Finally, we determined whether anti-CD3 MAb could induce synovial T cells to express the Fas ligand in vitro. RESULTS: Nearly all RA synovial fluid or synovial tissue lymphocytes expressed CD95 and could be induced to undergo apoptosis by CD95 crosslinking. We did not detect a soluble inhibitor in RA synovial fluid that could account for the survival of CD95+ synovial cells in vivo. Instead, we detected little or no expression of Fas ligand by RA synovial lymphocytes. However, we could induce such cells to express Fas ligand with anti-CD3 MAb or phorbol ester and ionomycin in vitro. CONCLUSION: There is ineffective clearance of activated cells in the RA joint due to deficient expression of the Fas ligand.


Asunto(s)
Artritis Reumatoide/metabolismo , Linfocitos B/metabolismo , Líquido Sinovial/citología , Membrana Sinovial/citología , Linfocitos T/metabolismo , Receptor fas/biosíntesis , Apoptosis , Cartilla de ADN/química , Citometría de Flujo , Humanos , Recuento de Leucocitos , Ligandos , Reacción en Cadena de la Polimerasa , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Receptor fas/inmunología
13.
Am J Pathol ; 150(3): 1125-38, 1997 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-9060847

RESUMEN

A distinctive cell was identified from sites of rheumatoid arthritis cartilage injury. Similar cells are not found in lesions of osteoarthritis cartilage. We have designated them as pannocytes (PCs). Their rhomboid morphology differs from the bipolar shape of fibroblast-like synoviocytes or the spherical configuration of primary human articular chondrocytes. Chondrocytes are short-lived, whereas the original PC line grew for 25 passages before becoming senescent. Features in common with cultured primary chondrocytes include maximal proliferation in response to transforming growth factor-beta a catabolic response to interleukin-1 beta, collagenase production, and mRNA for the induced lymphocyte antigen and inducible nitric oxide synthase. Despite the presence of the inducible nitric oxide synthase message, PCs do not produce NO either constitutively or when cytokine stimulated. Each of the mesenchymal cells, fibroblast-like synoviocytes, primary chondrocytes, and PCs have the gene for type I collagen, but the type II collagen gene is detected only in primary chondrocytes. PCs can be distinguished from fibroblast-like synoviocytes and primary chondrocytes by their morphology, bright VCAM-1 staining, and growth response to cytokines and growth factors. Their prolonged life span in vitro suggests that PCs might represent an earlier stage of mesenchymal cell differentiation, and they could have a heretofore unrecognized role in rheumatoid arthritis joint destruction.


Asunto(s)
Artritis Reumatoide/patología , Cartílago Articular/patología , Fibroblastos/patología , Membrana Sinovial/patología , Antígenos de Superficie/análisis , Artritis Reumatoide/metabolismo , Cartílago Articular/metabolismo , División Celular , Membrana Celular/ultraestructura , Células Cultivadas , Colagenasas/biosíntesis , Citoplasma/ultraestructura , Fibroblastos/metabolismo , Genotipo , Humanos , Microscopía Electrónica , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/metabolismo , Receptores de Citocinas/biosíntesis , Membrana Sinovial/metabolismo
15.
Am J Pathol ; 149(6): 2143-51, 1996 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-8952546

RESUMEN

DNA damage induces p53 tumor suppressor gene expression and protein production, which in turn facilitates DNA repair or apoptosis. Wild-type p53 protein has a short half-life, so it is rarely detected in non-neoplastic tissue. Because DNA fragmentation is abundant in the intimal lining in rheumatoid arthritis (RA) synovial tissue (ST) using in situ end-labeling (Firestein GS, Yeo M, Zvaifler NJ: Apoptosis in rheumatoid arthritis synovium. J Clin Invest 1995, 96:1631-1638), we assessed ST p53 expression. Immunohistochemical analysis of fixed RA synovium using antibody PAb 1801 showed prominent p53 staining in the cytoplasm and nuclei of intimal lining cells. Noninflammatory and osteoarthritis (OA) ST had significantly less p53 in the lining. These data were confirmed by Western blot analysis of ST extracts, with abundant p53 found in RA compared with OA. p53 expression in cultured fibroblast-like synoviocytes (FLS) was then examined. Flow cytometry on permeabilized cells showed that RA FLS constitutively express p53 protein. Western blots showed that RA FLS expressed significantly more p53 than either OA FLS or dermal fibroblasts. Immunohistochemistry of FLS cultured in chamber slides localized the p53 to the cytoplasm of most resting FLS, with nuclear staining in only 10.7 +/- 2.4%. Exposure to hydrogen peroxide for increased nuclear staining to 70.7 +/- 12.8% after 8 hours (P = 0.003). These data indicate that p53 is overexpressed in RA ST in the intimal lining, which is the primary site of DNA damage, and is constitutively expressed by FLS.


Asunto(s)
Apoptosis/genética , Artritis Reumatoide/genética , Artritis Reumatoide/metabolismo , Regulación de la Expresión Génica , Genes p53 , Membrana Sinovial/metabolismo , Anciano , Apoptosis/inmunología , Artritis Reumatoide/patología , Western Blotting , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Membrana Sinovial/patología , Proteína p53 Supresora de Tumor/genética
16.
J Clin Invest ; 98(3): 815-25, 1996 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-8698874

RESUMEN

The optimal clinical management of patients with antiphospholipid antibody syndrome (APS) is uncertain because of a lack of an underlying hypothesis to explain why antiphospholipid autoantibodies (aPL) form to such ubiquitous compounds as phospholipids (PL). In this paper, we demonstrate that many, if not most, aPL are actually directed at neoepitopes of oxidized PL, or neoepitopes generated by adduct formation between breakdown products of oxidized PL and associated proteins. Each cardiolipin (CL) molecule contains four unsaturated fatty acids and is highly susceptible to oxidation, particularly upon exposure to air. Yet, standard anticardiolipin antibodies (aCL) immunoassays routinely bind CL to microtiter wells by evaporation of the ethanol solvent overnight at 4 degrees C. Using a variety of techniques, we demonstrated that rapid oxidation occurs when CL is plated and exposed to air. Sera from apo E-deficient mice, which have high autoantibody titers to oxidized low density lipoprotein, showed a striking time-dependent increase in binding to CL that was exposed to air for increasing periods of time. Monoclonal antibodies to oxidized LDL, cloned from the apo E-deficient mice, also bound to oxidized CL. Both sera and affinity-purified aCL-IgG from APS patients bound to CL progressively as it was oxidized. However, the monoclonal antibodies from apo E-deficient mice, or sera or aCL-IgG from APS patients did not bind to a reduced CL analog that was unable to undergo peroxidation. These data demonstrate that many aPL are directed at neoepitopes of oxidized phospholipids, and suggest that oxidative events may be important in the pathophysiology of APS. In turn, this suggests new therapeutic strategies, possibly including intensive antioxidant therapy.


Asunto(s)
Anticuerpos Anticardiolipina/inmunología , Anticuerpos Monoclonales/inmunología , Cardiolipinas/análisis , Epítopos , Lipoproteínas LDL/inmunología , Animales , Síndrome Antifosfolípido/sangre , Síndrome Antifosfolípido/etiología , Apolipoproteínas E/deficiencia , Cardiolipinas/inmunología , Cardiolipinas/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Oxidación-Reducción
17.
J Clin Invest ; 96(3): 1631-8, 1995 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-7657832

RESUMEN

RA synovial tissue (ST) was studied to determine if and where apoptosis occurs in situ. Genomic DNA was extracted from 5 RA and 1 osteoarthritis ST samples. Agarose gel electrophoresis demonstrated DNA ladders characteristic for apoptosis from each tissue. In situ and labeling (ISEL) was used to identify DNA strand breaks consistent with apoptosis in frozen sections. 12 RA and 4 osteoarthritis ST were studied by ISEL and all were positive, but only 2 of 4 normal tissues were positive. The primary location of apopotic cells was the synovial lining. Some sublining cells were also positive, but lymphoid aggregate staining was conspicuously absent. Immunohistochemistry and ISEL were combined and showed that the lining cells with DNA strand breaks were mainly macrophages, although some fibroblastlike cells were also labeled. Sublining cells with fragmented DNA included macrophages and fibroblasts, but T cells in lymphoid aggregates, which expressed large amounts of bcl-2, were spared. DNA strand breaks in cultured fibroblastlike synoviocytes was assessed using ISEL. Apoptosis could be induced by actinomycin D, anti-fas antibody, IL-1, and TNF-alpha but not by IFN-gamma. Fas expression was also detected on fibroblast-like synoviocytes using flow cytometry. Therefore, DNA strand breaks occur in synovium of patients with arthritis. Cytokines regulate this process, and the cytokine profile in RA (high IL-1/TNF; low IFN-gamma) along with local oxidant injury might favor induction of apoptosis.


Asunto(s)
Apoptosis , Artritis Reumatoide/patología , Citocinas/farmacología , Osteoartritis/patología , Membrana Sinovial/patología , Apoptosis/efectos de los fármacos , Línea Celular , Células Cultivadas , ADN/análisis , ADN/aislamiento & purificación , Dactinomicina/farmacología , Electroforesis en Gel de Agar , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Interferón gamma/farmacología , Interleucina-1/farmacología , Osteoartritis/cirugía , Proteínas Recombinantes , Membrana Sinovial/efectos de los fármacos , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacología
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