Title

 

Authors

D. Lattuada, C. Casnici, K. Crotta and O. Marelli

Dept. of Pharmacology, Università degli Studi di Milano, Milan, Italy

 

Abstract

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease primarily attacking synovial joints. Synovial hyperplasia, cell activation, articular inflammation and invasion of the synovium into the adjacent bone and cartilage represent the characteristic features of the disease. Synovial hyperplasia and in addition to the infiltration of inflammatory cells attributed to the proliferation of RA-fibroblast-like cells (RA-FLS) on the one hand and to the resistance of RA-FLS against apoptosis on the other. Have been shown that defective regulation of apoptosis in cells present in synovial tissue, synovial fluid (SF), and circulating immune response cells may contribute to the pathogenesis of rheumatoid diseases. Inhibited apoptosis of T cells, correlated with impaired Fas signaling pathway, was also found in RA synovial tissue. Those abnormalities can be responsible for ineffective clearance of activated T cells in RA joints and persistent infiltration of rheumatoid synovium. Additionally, RA synoviocytes express elevated levels of tumor necrosis factor-a (TNF-a), inducing nuclear factor-kB (NF-kB) prosurvival signaling pathway. Moreover, the inhibitor of apoptosis (IAP) family of proteins, cross-linked IAP (XIAP) and cellular inhibitor of apoptosis 2 (cIAP2), were found to be expressed in unstimulated synovial cells from RA synovial tissue. XIAP is a TNF-a-inducible specific inhibitor of apoptosis of the RA synovial fibroblast cell line, whereas in ex vivo RA synovial cells the expression of both XIAP and cIAP2 was found to be down regulated by TNF-alfa.A promising approach is the development of small therapeutic compounds, referred to as Smac mimetics, that are designed to block the function of IAP protein family, and these are currently in clinical trials for the treatment of cancer. Aim of our research is to assess the activity of SMAC mimetic compounds in the states of lymphocytes hyper-activation such as that which occurs in autoimmune diseases characterized by apoptosis down regulation. Weusedfor ourstudiesthe new monomeric Smac-mimetic molecule SMAC 066 compared them with a “standard” compound reported in literature, the monomeric Smac 060 on the proliferative response of PBL activated by phytohemagglutinin(PHA). Different doses of SMAC were administered at the beginning of culture. The SMAC molecules significantly inhibit the proliferation of human PBL stimulated by PHA.In parallel to exclude a toxic effect of SMAC lymphocytes, PBL cultured with only culture medium were treated with SMAC for 72 hours, does not show any toxicity.Smac066 is more effective in inhibiting the proliferation of Smac 060. To determine the mechanisms of Smac-mimetic-induced cell death, we first examined the effect of the two selected compounds on cell cycle. Flow cytometric DNA analysis was performed to investigate if Smac mimetic molecules could induce an alteration on the progression of cell cycle. Were observed a consistent sub G1 apoptotic peak in the lymphocytes activated by PHA and treated with Smac. To assess if there was a prevalent involvement of a particular subpopulation of T lymphocytes activated by PHA with SMAC treatment with we performed the test of annexin V  in populations CD3 ⁺, CD4⁺ and CD8 ⁺. While there is a variability between individual donors, apoptosis is induced by more than 066 SMAC monomer control, but we have not shown prevalent action on a particular lymphocyte subpopulation. Preliminary analysis of cell cycle of cells derived from synoviocytes of patients with rheumatoid arthritis treated with Smac molecules, showed that an increase of peak sub-G1.We are currently evaluating the effects of Smac molecules on anti-apoptotic protein IAP that are over expressed in patients affected by rheumatoid arthritis.