THU0259 Induction of Apoptosis and Autophagy in Circulating PBMC by Microparticles from Patients with Systemic Lupus Erytematosus

• Published in: Annals of the rheumatic diseases Vol. 75; no. Suppl 2; pp. 281 - 282
• Main Authors: Miranda, F., Barbati, C., Alessandri, C., Spinelli, F.R., Ceccarelli, F., Truglia, S., Valesini, G., Conti, F.
• Format: Journal Article
• Language: English
• Published: London BMJ Publishing Group LTD 01.06.2016
• Subjects: Actin; Animal models; Annexin V; Apoptosis; Arachidonic acid; Autoimmune diseases; Autophagy; Caspase-8; Cell activation; Cell culture; Centrifugation; Ceramide; Flow cytometry; Inflammation; Leukocytes (mononuclear); Lupus; Lymphocytes; Microparticles; Peripheral blood mononuclear cells; Phagocytosis; Propidium iodide; Protein turnover; Systemic lupus erythematosus
• ISSN: 0003-4967; 1468-2060
• DOI: 10.1136/annrheumdis-2016-eular.5030

BackgroundSystemic Lupus Erythematosus (SLE) is a systemic autoimmune disease characterized by heterogeneous clinical manifestation and a complex pathogenesis. Lymphocytes from patients with SLE display multiple alterations, including increased cell activation, abnormal apoptosis and impairment of autophagic pathway. Recently, evidence from genetic, cell biology and animal models suggested that autophagy, a major pathway for organelle and protein turnover, may play a pivotal role in the occurrence and development of SLE. Some studies showed altered profile of circulating microparticles (MPs) in SLE patients. These MPs are released constitutively and may increase as a result of cellular activation and apoptosis. Originally considered as inert debris, MPs are now known to display diverse pro-inflammatory and pro-thrombotic activities, and they are implicated in the intercellular communications. MPs can influence the course of rheumatic and other immune-mediated diseases.ObjectivesThe aim of our study was to evaluate the possible role of MPs purified from SLE patients on the modulation of apoptosis and autophagy in peripheral blood mononuclear cells (PBMCs) isolated from healthy donors.MethodsPBMCs from healthy donors were isolated by Ficoll-Hypaque density-gradient centrifugation, cultured and treated with 0.5,2.5,5 *106 MPs purified by 10 sera of active SLE for 16 h. Apoptosis was measured using FITC-conjugated annexin V (AV) and a propidium iodide (PI) apoptosis detection kit. Acquisition was performed on a FACS Calibur. Autophagy was measured by Western blot for LC3II.ResultsMPs appeared capable of inducing apoptosis. Indeed, PBMCs showed significantly higher apoptosis if treated with MPs compared with untreated cells (percentage of apoptotic cells treated with 5, 2.5 and 0.5*106 MPs: 15, 10, 9 vs. 7%, P=0.0001, P=ns, P=ns, with respectively). Moreover, MPs increased autophagy in a dose response manner (LC3-II/β-actin ratio: 1.2, 1, 0,6 vs. 0.5, with 5, 2.5 and 0.5*106 MPs, respectively P=0.0001).ConclusionsWe demonstrated that MPs from SLE patients induce apoptosis in PBMCs, thereby possibly decreasing the number of circulating leucocytes. The mechanisms underlying such phenomena are not known. Some authors suggested that MPs, through the arachidonic acid pathway, may induce apoptosis. Indeed, this pathway, together with the sphingomyelinic, increase the concentration of ceramide, thus leading into apoptosis mediated by the activation of caspase 8 and by the release of downstream pro-apoptotic factors. Moreover, available data do not exclude the possibility that components of MPs other than arachidonic acid, may also contribute to the ceramide-mediated apoptosis. Finally, this is the first study addressing the relations between MPs and autophagy, suggesting an increase of this cellular event after exposure to MPs. Since an augmented autophagy has been implicated in autoimmunity, it could be of interest to target MPs in order to re-balance the autophagic processes.Disclosure of InterestNone declared