Adenosine deaminase (ADA; EC 188.8.131.52) 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, kinetic 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 complexed 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 properties (4). In contrast, ADA2 has a lower affinity for adenosine and lower catalytic activity with deoxyadenosine than ADA1 (5).
Elevated serum ADA activities have been reported in patients with diseases in which cellular immunity is stimulated. For example, ADA1 activity is elevated 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 serum is ADA2 (9, 10). Most human cells contain only small amounts of ADA2, and its tissue source is not yet completely clear, although the monocyte/macrophage cell system is likely a major source (10, 11). It is also known that total ADA activity is significantly higher in the synovial fluid (SF) of rheumatoid arthritis (RA) patients than in the SF of osteoarthritis (OA) patients (12, 13).
Methotrexate (MTX), one of the most effective antirheumatic drugs, reportedly increases extracellular adenosine concentrations at sites of inflammation and represses the infiltration of inflammatory cells (14). In addition, the nonselective adenosine receptor antagonists theophylline and caffeine are capable of reversing the antiinflammatory effects of MTX in rat adjuvant arthritis (15). Taken together, these findings suggest that adenosine is an effector molecule mediating the antirheumatic effects of MTX via adenosine receptor signaling, and that reduction of the local concentration of adenosine by ADA may contribute to the joint inflammation of RA. In the present study, therefore, we examined the activities and cellular sources of 2 ADA isozymes, ADA1 and ADA2, in RA, with the aim of gaining better understanding of the pathophysiologic role of ADA in rheumatoid inflammation.
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- MATERIALS AND METHODS
Intracellular adenosine is involved in cellular energy production and purine metabolism, whereas extracellular adenosine acts as an autocrine and/or paracrine factor via specific cell surface receptors, exerting multiple physiologic actions in a variety of systems, including the immune system (25–28). Consider, for example, the case of inherited ADA deficiency. The resultant accumulation of extracellular and intracellular adenosine leads to severe combined immunodeficiency. The mechanism by which this occurs is currently thought to involve direct lymphotoxicity of intracellular adenosine as well as A2A adenosine receptor–mediated signaling (29). In that regard, investigators at our laboratory previously showed that adenosine signaling via A2A receptors represses T cell function by competing with T cell receptor–mediated signaling (30).
The antiinflammatory effects of MTX are reportedly due in large part to its capacity to enhance extracellular adenosine at sites of inflammation (14). Since several lines of evidence suggest that T cells, especially Th1-type cells, are responsible for the pathogenesis of RA (31), extracellular adenosine likely mediates the antiarthritic effects of MTX by negatively regulating T cell function. Supporting this notion is the finding that the nonselective adenosine receptor antagonists theophylline and caffeine are capable of reversing the antiinflammatory effects of MTX in rat adjuvant arthritis (15). In that context, an ADA-catalyzed reduction in the local concentration of adenosine would be expected to contribute to the joint inflammation of RA.
In addition to the cytosol, ADA is found on the surface of many cells (ecto-ADA) (32), and 2 cell surface receptors for ADA have been identified: CD26 (dipeptidyl peptidase IV) and A1 adenosine receptors (32). Ecto-ADA interacting with CD26 is reportedly a costimulatory molecule that facilitates a variety of signaling events in different cell types (32), including T cells (33). ADA may thus play a regulatory role either by catalyzing the irreversible deamination of adenosine to inosine and/or by transmitting signals when interacting with CD26 or A1 receptors.
When enzyme activities were assayed in vitro, the adenosine concentration used (10 mM) was sufficient for both ADA1 and ADA2 to function at maximum velocity. Under these conditions, we found that ADA2 activity was greater than ADA1 activity in both RA SF and OA SF (Figures 1A and B), which is consistent with an earlier report that the level of ADA2 activity in tuberculous pleural effusions is much higher than that of ADA1 activity (34). However, all of these measurements were obtained in vitro and may not be relevant to in vivo conditions. Indeed, the fact that ADA2 has a Km for adenosine of 2,000 μM, while that of ADA1 is 52 μM (4, 35), means that ADA1 should be the functionally predominant isozyme in RA SF (and OA SF), given the range of adenosine concentrations found there (Figure 1C). In contrast, considering that the half-life of adenosine in whole blood is ∼1 minute (36) due to cellular uptake via nucleoside transporters and degradation to inosine by ecto-ADA, the adenosine concentration measured in SF might underestimate the true concentration inside the joints, even with addition of both dipyridamole (a nucleoside transporter inhibitor) and coformycin (an ADA inhibitor) to the SF immediately after its withdrawal from the joint. Still, ADA1 should be the predominant ADA isozyme in RA joints at adenosine concentrations in the range of ≤1 mM.
Comparison of ADA isozyme activities in FLS, MNCs, and PMNs revealed that RA FLS possessed the highest ADA1 activity, >10-fold greater than that in the other types of cells obtained from RA patients. This could reflect in part the higher protein content of FLS since the size of the individual cells is significantly larger than MNCs or PMNs. This is not a complete explanation, however, because ADA1 activity in RA FLS was also 5-fold greater than in OA FLS, even though the respective sizes of the 2 cell types are similar. In addition, no significant difference in ADA2 activities in RA FLS and OA FLS was found.
Conversely, it was previously reported that there is a significant positive correlation between matrix metalloproteinase 9 (MMP-9) and ADA1 levels in RA SF (37), and a positive correlation between PMN count and MMP-9 (38). In that context, the fact that PMNs are usually the most abundant cell type in RA SF raises the possibility that they are the major source of ADA1, even though each cell contains little ADA1. Unfortunately, RA FLS, which constitutively express MMP-9 (39), were not investigated as a possible source of MMP-9 in those studies. In the present study, moreover, ADA1 activity was not significantly correlated with PMN count or with the total cell number in RA SF. Finally, proliferation of FLS in joint tissue, not SF, is characteristic of RA. Thus, the highly elevated ADA1 activity seen in RA SF would appear to be a characteristic feature of RA FLS, which suggests that these cells are the major source of ADA1 in RA joints.
It also seems possible that the apparently higher deaminase activity in RA FLS reflects an abundance of ecto-ADA bound to RA FLS via CD26 and/or A1 receptors. In fact, RA FLS express all 4 adenosine receptors, including A1 receptor (40), and the ecto-ADA from HEp-2 cells and from lymphocytes obtained from patients with chronic lymphocytic leukemia was identified as ADA1 (41). To further address the question of whether the increased ADA1 activity is an intrinsic characteristic of RA FLS, we compared levels of ADA gene expression in RA FLS and OA FLS. The fact that human ADA genes (GenBank accession nos. K02567, BC007678, NM_000022, X02994, or XM_029810) have essentially the same coding sequences suggests that ADA isozymes are derived from a single gene through mechanisms such as alternative splicing, posttranscriptional modification, and/or posttranslational modification (42). Whatever the case, the level of ADA gene expression appears higher in RA FLS than in OA FLS; certainly the amount of transcript was significantly higher in RA FLS (Figure 2, inset). This is in accordance with our conclusion that the increase in cellular ADA is an intrinsic characteristic of RA FLS. That the difference in the level of ADA mRNA expression in RA FLS and OA FLS is less marked than the difference in the ADA activities is consistent with the notion that ADA isozymes are derived from posttranscriptional modification of a single gene product rather than from different individual gene products (43).
Proinflammatory cytokines such as IL-1, TNFα, and IL-6 are generally believed to contribute to the pathogenesis of RA (22). This prompted us to test whether these cytokines might be responsible for the induction of ADA in RA FLS. Under the in vitro conditions in our study, however, none of the cytokines tested significantly affected ADA1 activity or ADA mRNA expression in either RA FLS or OA FLS (Figure 3).
We found a significant correlation between ADA1 activity in RA SF and CRP levels in RA sera, which raises the possibility that the elevated ADA1 activity in RA SF may be secondary to the systemic inflammation. Our other results provide evidence against this idea, however. For instance, in RA patients, serum CRP levels correlated significantly with ADA1 activity but not with ADA2 activity, even though ADA2 is the predominant ADA isozyme in serum. Moreover, the proinflammatory cytokine IL-6, which is responsible for increases in CRP production in the liver, had no effect on ADA activity or expression in RA FLS. We therefore conclude that the observed increases in ADA1 activity and ADA gene expression in RA FLS reflect an intrinsic abnormality of RA FLS and are not an inflammation-induced secondary effect.
Finally, our findings raise the possibility that novel approaches to the treatment of RA might be developed based on a strategy of increasing extracellular adenosine and normalizing the intrinsic abnormality of RA FLS. Such an approach has the potential to be highly efficacious and would seem to warrant further investigation.