ARTHRITIS & RHEUMATISM Vol. 44, No. 1, January 2001, pp 21–30 © 2001, American College of Rheumatology Rheumatoid Arthritis Synovial Macrophages Express the Fas-Associated Death Domain–Like Interleukin-1–Converting Enzyme–Inhibitory Protein and Are Refractory to Fas-Mediated Apoptosis Harris Perlman, 1 Lisa J. Pagliari, 1 Hongtao Liu, 1 Alisa E. Koch, 1 G. Kenneth Haines, III, 2 and Richard M. Pope 1 Objective. The chronic inflammation and progres- sive joint destruction observed in rheumatoid arthritis (RA) are mediated in part by macrophages. A paucity of apoptosis has been observed in RA synovial tissues, yet the mechanism remains unknown. The present study sought to characterize the expression of Fas, Fas ligand (FasL), and Fas-associated death domain–like interleukin-1–converting enzyme–inhibitory protein (FLIP), and to quantify the apoptosis induced by ago- nistic anti-Fas antibody, using mononuclear cells (MNC) isolated from the peripheral blood (PB) and synovial fluid (SF) of RA patients. Methods. The expression of Fas, FasL, and FLIP and apoptosis induced by agonistic anti-Fas antibody in MNC from the PB and SF of RA patients were deter- mined by flow cytometry. Immunohistochemistry em- ploying a monospecific anti-FLIP antibody was per- formed on RA and osteoarthritis (OA) synovial tissue. Results. CD14-positive monocyte/macrophages from normal and RA PB and from RA SF expressed equivalent levels of Fas and FasL. Furthermore, unlike the CD14-positive PB monocytes, RA SF monocyte/ macrophages were resistant to the addition of agonistic anti-Fas antibody. In contrast, both CD14-positive PB and SF monocyte/macrophages were sensitive to apopto- sis mediated by a phosphatidylinositol 3-kinase inhibi- tor. Intracellular staining of the caspase 8 inhibitor, FLIP, in CD14-positive SF monocyte/macrophages re- vealed a significant up-regulation of FLIP compared with normal and RA PB monocytes. Immunohistochem- ical analysis of synovial tissue from RA and OA patients revealed increased FLIP expression in the RA synovial lining compared with the OA synovial lining. Further- more, FLIP expression was observed in the CD68- positive population in the RA synovial lining. Forced reduction of FLIP by a chemical inhibitor resulted in RA SF macrophage apoptosis that was enhanced by agonistic anti-Fas antibody, indicating that FLIP is necessary for SF macrophage survival. Conclusion. These data suggest that up- regulation of FLIP in RA macrophages may account for their persistence in the disease. Thus, the targeted suppression of FLIP may be a potential therapeutic strategy for the amelioration of RA. Rheumatoid arthritis (RA) is an autoimmune disease characterized by infiltration of lymphocytes and macrophages into the synovium, hyperplasia of the synovial lining, and destruction of cartilage and bone. Macrophage-like and fibroblast-like synoviocytes com- prise the normal synovial lining, which consists of 1–2 cell layers but increases to as much as 10 or more cell layers in active RA (1). Synovial macrophages express high levels of collagenase, stromelysin, interleukin-1 (IL-1), tumor necrosis factor  (TNF), granulocyte– Dr. Perlman’s work was supported by grants from the North- western Memorial Foundation and the NIH (AR-02147). Dr. Pope’s work was supported by grants from the NIH (AR-43642 and AR- 30692) and the Arthritis Foundation. Dr. Koch’s work was supported by grants from the NIH (AR-41492 and AI-40987), funds from the VA Research Service, and a Gallagher Professorship for Arthritis Re- search. 1 Harris Perlman, PhD, Lisa J. Pagliari, BS, Hongtao Liu, MD, PhD, Alisa E. Koch, MD, Richard M. Pope, MD: Northwestern University Medical School, and the Veterans Administration Chicago Healthcare System, Lakeside Division, Chicago, Illinois; 2 G. Kenneth Haines, III, MD: Northwestern University Medical School, Chicago, Illinois. Address correspondence and reprint requests to Richard M. Pope, MD, Northwestern University Medical School, Department of Medicine, Division of Rheumatology, 303 East Chicago Avenue, Ward 3-315, Chicago, IL 60611. Submitted for publication July 18, 2000; accepted in revised form September 6, 2000. 21