ARTHRITIS & RHEUMATISM Vol. 48, No. 3, March 2003, pp 668–674 DOI 10.1002/art.10956 © 2003, American College of Rheumatology Specific Increase in Enzymatic Activity of Adenosine Deaminase 1 in Rheumatoid Synovial Fibroblasts Yuji Nakamachi, 1 Masahiro Koshiba, 1 Takashi Nakazawa, 1 Saori Hatachi, 1 Ryuichi Saura, 2 Masahiro Kurosaka, 1 Hideaki Kusaka, 3 and Shunichi Kumagai 1 Objective. Adenosine deaminase (ADA; EC 3.5.4.4) activity is elevated in the synovial fluid (SF) of patients with rheumatoid arthritis (RA). Since the an- tiinflammatory effect of methotrexate is reportedly as- sociated with increased levels of extracellular adeno- sine, the present study was undertaken to clarify the role of 2 ADA isozymes, ADA1 and ADA2, in the pathogen- esis of RA. Methods. The activities of ADA1 and ADA2 were measured in SF from RA and osteoarthritis (OA) pa- tients, in sera from RA patients, and in lysates prepared from mononuclear and polymorphonuclear cells from SF from RA patients, peripheral blood from RA pa- tients, and fibroblast-like synoviocytes (FLS) from RA and OA patients. Also measured were the effects of proinflammatory cytokines on ADA1 activity and ADA messenger RNA (mRNA) expression in RA FLS, as determined using real-time polymerase chain reaction. The adenosine concentration in RA SF was measured by radioimmunoassay. Results. The adenosine concentration in RA SF ranged from 0.027 M to 0.508 M (mean  SD 0.156  0.132 M). At those concentrations, ADA1 would be expected to be functionally dominant due to its higher affinity for adenosine. ADA1 activity in RA SF (mean  SD 14.4  8.5 IU/liter) was significantly higher than that in OA SF (3.0  1.1 IU/liter) or RA sera (3.0  0.6 IU/liter); moreover, ADA1 activity in RA FLS lysate was the highest among the cell lysates tested. Proinflamma- tory cytokines did not affect ADA1 activity or ADA mRNA expression in RA FLS. Conclusion. Elevated ADA1 activity is an intrinsic characteristic of RA FLS, which likely contributes to the pathogenesis of RA by neutralizing the antirheumatic properties of endogenous adenosine. Adenosine deaminase (ADA; EC 3.5.4.4) is a key enzyme in purine metabolism that catalyzes irreversible deamination of adenosine and 2'-deoxyadenosine to inosine and 2'-deoxyinosine, respectively (1). In humans, 3 ADA isozymes with differing molecular weights, ki- netic properties, and tissue distributions have been identified (2): 1) a 35-kd enzyme (ADA1); 2) a 280-kd enzyme comprising two 35-kd ADA1 enzymes com- plexed with a nonenzymatic 200-kd combining protein that was recently shown to be identical to CD26 (ADA1 + combining protein) (3); and 3) a 100-kd enzyme (ADA2) (4). The first 2, which share the same catalytic subunit, do not differ significantly in their kinetic prop- erties (4). In contrast, ADA2 has a lower affinity for adenosine and lower catalytic activity with deoxyade- nosine than ADA1 (5). Elevated serum ADA activities have been re- ported in patients with diseases in which cellular immu- nity is stimulated. For example, ADA1 activity is ele- vated in patients with acute lymphoblastic leukemia (6) or acute hepatitis (7), and ADA2 activity is elevated in patients with human immunodeficiency virus infection (8). In human tissues and cells the majority of ADA activity is derived from ADA1, but the prevalent form in Supported in part by a grant-in-aid for scientific research from the Japan Society for the Promotion of Science. 1 Yuji Nakamachi, BSc, Masahiro Koshiba, MD, PhD, Takashi Nakazawa, MD, Saori Hatachi, MD, Masahiro Kurosaka, MD, PhD, Shunichi Kumagai, MD: Kobe University Graduate School of Medi- cine, Kobe, Japan; 2 Ryuichi Saura, MD: Kobe University School of Medicine, Kobe, Japan; 3 Hideaki Kusaka, PhD: Pharmaceutical Re- search Institute, Kyowa Hakko Kogyo Co., Ltd., Shizuoka, Japan. Address correspondence and reprint requests to Masahiro Koshiba, MD, PhD, Clinical Pathology and Immunology, Department of Biomedical Informatics, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E- mail: mkoshiba@med.kobe-u.ac.jp. Submitted for publication July 26, 2002; accepted in revised form November 13, 2002. 668