Rheumatoid synovial fluid T cells are sensitive to APO2L/TRAIL
Introduction
Rheumatoid arthritis (RA) is one of the autoimmune diseases with higher prevalence. It is characterized by infiltration of lymphocytes and macrophages into the synovial fluid (SF), hyperplasia and decreased cell death of synovial cells, producing an abnormal immune response and eventually causing erosion of the affected joint [1]. Although the diagnostic of this disease is very reliable, its treatment is normally based in immunosuppressive agents with low specificity.
The resistance of synovial lymphocytes, macrophages and/or fibroblasts to apoptosis may be a contributing factor in the persistence of the disease. A special microenvironment that favors cell infiltration and proliferation of lymphocytes and the imbalance between cell proliferation and cell death appear to be relevant to pathological processes in SF of RA [2], [3]. In relation with pro-apoptotic receptors and ligands, previous studies demonstrated that the death receptor Fas/CD95 was expressed on fibroblast-like synoviocytes of the RA synovium [4], which were sensitive to Fas-induced apoptosis [4], [5]. Regarding T lymphocytes in SF of RA patients, it was initially described that they were spared from apoptosis, probably because of their elevated expression of the anti-apoptotic molecule Bcl-2 [6]. Other studies showed that synovial RA lymphocytes were defective in FasL expression [7]. However, in other study, important levels of FasL mRNA were detected in synovial lymphocytes of RA patients and soluble FasL was detected in the SF [8]. In addition, adenoviral expression of FasL was effective in the treatment of collagen-induced arthritis in mice [9], [10].
Another member of the tumor necrosis factor superfamily, APO2 ligand (APO2L)/tumor necrosis related apoptosis-inducing ligand (TRAIL), was described to induce apoptosis in a Fas/CD95-independent manner [11], [12]. APO2L/TRAIL interacts with five different receptors, of which only DR4 (TRAIL-R1) [13] and DR5 (TRAIL-R2) [14] are capable of transmitting a death signal in tumor cells [14], [15]. In addition, the soluble receptor osteoprotegerin is reported to block TRAIL-mediated apoptosis [16]. Our group proposed for the first time the possible implication of APO2L/TRAIL in the regulation of normal human T cell blast activation [17]. In agreement with this proposal, it was later demonstrated by other groups that mice treated with anti-APO2L/TRAIL blocking antibodies were more susceptible to the development of collagen-induced arthritis [18] or of experimental autoimmune encephalomyelitis [19]. This was later confirmed in the APO2L/TRAIL knockout mice, which were more sensitive than wild-type mice to the development of those experimental autoimmune diseases [20]. Recently, another group have demonstrated that APO2L/TRAIL receptor knockout mice are defective in the regulation of macrophage activation, producing large amounts of pro-inflammatory cytokines, a scenario also compatible with their greater sensitivity to autoimmune disease [21]. More recent studies from our group have demonstrated that the regulation of normal human T cell blast activation by APO2L/TRAIL is rather restricted to the regulation of CD8+ T cell blast expansion [22], [23]. Although we were performing our study using samples from RA patients, two studies were published using gene therapy based in APO2L/TRAIL for the treatment of experimental models of RA, with promising results [24], [25], [26].
We characterized previously that FasL and APO2L/TRAIL are stored inside human T cell blasts in cytoplasmic compartments with characteristics of multivesicular bodies [27] and that they were rapidly released to the supernatant in their bioactive form associated with the internal microvesicles/exosomes upon re-activation [27], [28]. The change in focusing that it represents our basic studies on the secretion of these molecules associated with exosomes, maintaining their bioactivity, could contribute to a better understanding of the ethiology of RA and other autoimmune diseases and to allow the development of more specific and efficient therapeutic interventions.
In this study, we have analyzed the expression of Fas, FasL or APO2L/TRAIL in CD3+ lymphocytes from SF of RA patients, together with their sensitivity to cytotoxic anti-Fas mAb or to rAPO2L/TRAIL, using as controls T lymphocytes 0present in SF of patients with traumatic arthritis and also PBL from healthy donors and T cell blasts generated from them. We describe that although T lymphocytes from SF of RA patients were resistant to anti-Fas mAb, they were sensitive to rAPO2L/TRAIL. Our data also indicate that the persistence of synovial lymphocytes in RA is associated with the extremely low amount of FasL and APO2L/TRAIL associated with exosomes in SF of RA patients if compared with SF of traumatic arthritis patients.
Section snippets
Patients
Sixty-two patients diagnosed as rheumatoid arthritis (RA) by the Rheumatology Service of the Clinical Hospital “Lozano Blesa” were included in the present study. All patients included in this study gave the pertinent informed consent. The study was approved by the Ethical Committee for Clinical Investigation of the Government of Aragón (CEICA).
The disease duration in RA patients was between 6 months and 10 years. We divided RA patients in three groups, depending on the stage of clinical disease
Results
In the present work, parameters were compared between CD3+ cells present in SF of RA patients, and, as controls, those in the SF of patients with traumatic arthritis. As additional controls, parameters were also compared with those observed in fresh PBL isolated from the blood of healthy donors (naive, non-activated T cells) and day 7 T cell blasts (normal activated T cells) generated in vitro from them, as indicated in our previous studies [17], [28]. As indicated in Methods, RA patients were
Discussion
In the present study we have characterized the phenotype of T lymphocytes present in the SF of RA patients, comparing it with that of T lymphocytes present in SF of traumatic arthritis patients, and also with PBL and T cell blasts obtained from healthy donors. Our data indicate that T lymphocytes present in SF of RA patients have a chronically activated phenotype, as determined by the elevated percentage of cells expressing the activation markers HLA-DR and CD69, clearly superior to that of
Acknowledgments
This work was supported by Grant PI020658 and C03/02 from Fondo de Investigaciones Sanitarias (Spain) and P1314 by Fundación Lair to MJML; by Grant SAF02001-1774 and SASF2004-03058 from Ministerio de Educación y Ciencia (MEC, Spain) to AA; and by Diputación General de Aragón/Fondo Social Europeo. MJML was supported by a contract form Fondo de Investigaciones Sanitarias. BSG and MRC were supported by fellowships from Fondo de Investigación Sanitarias adscribed to projects PI020658 and C03/02,
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2012, Biochemical PharmacologySelective elimination of pathogenic synovial fluid T-cells from Rheumatoid Arthritis and Juvenile Idiopathic Arthritis by targeted activation of Fas-apoptotic signaling
2011, Immunology LettersCitation Excerpt :Furthermore, since Treg may convert to TH17 in the inflammatory synovial milieu, the elimination of these Tregs by scFvCD7:sFasL might possibly contribute to reducing TH17 in the synovium. Recent reports indicate that treatment with the pro-apoptotic death ligand TRAIL ameliorates disease activity in animal models of RA [27–29]. In another study, TRAIL appeared to inhibit DNA synthesis and cell cycle progression in inflammatory cells, but lacked direct apoptotic activity [46].