MTX is an effective drug in the management of RA. Its ability to suppress inflammation results, at least in part, from its ability to increase extracellular concentrations of adenosine which stimulates adenosine A2A
]. The primary site of inflammation in RA is the synovium. Suppression of the inflammatory process that drives this inflammation in the synovium and formation of the tissue destructive pannus is critical to the control of inflammatory joint symptoms and prevention of joint erosion in RA.
Most attention with respect to the anti-inflammatory mechanism of action of MTX has focused on the folate pathway with less attention on the adenosine pathway. ADORA3
has been reported to be expressed in PBMCs [6
] and ADORA1
in synovial fluid cells from patients with RA [18
] and isolated synoviocytes [19
]. Both the adenosine A2A
receptors, but not ADORA1
, have been reported to be up-regulated in lymphocyte and neutrophil membranes from patients with RA as compared to healthy controls [20
]. However, there have been no previous studies examining expression of the adenosine receptors within synovial tissue from patients with RA. Herein we have demonstrated that the genes for all four adenosine receptors are expressed at varying levels in rheumatoid synovium.
was expressed significantly more than the other ADOR genes. This may be the result of the inflammatory process occurring within the synovium, with previous reports suggesting that expression of ADORA3
is up-regulated by TNF-α via activation of NFκB [17
, through which MTX is also thought to exert its anti-inflammatory effects, was expressed in the lowest amounts of all the ADOR subtypes [7
Several possibilities could explain the difference in expression of the various adenosine receptor subtypes. These include the cellular constituents of the tissue examined. Consistent with this possibility, ADORA3 protein expression was more widespread in synovial tissue. In contrast ADORA2B protein was prominent in vascular ECs but also present in fibroblasts and some T cells, albeit in much reduced amounts; ADORA2A protein was restricted to cells with a dendritic appearance. Overall, the patterns of ADOR protein distribution were consistent with the hierarchical order we observed for expression of the corresponding ADOR genes. For all ADOR subtypes, we did not observe significant protein associated with the infiltrating inflammatory cells. We are not able to exclude that these patterns of protein expression and the differences in gene expression could also be affected by the inflammatory cytokine milieu within the synovium. However, we further considered the effect of anti-rheumatic therapy and the possible influence on ADORA expression.
Both MTX and anti-TNF therapy have been reported to have effects on adenosine receptor expression and affinity. MTX therapy results in up-regulation of the ADORA2A
, but not ADORA1
expression in lymphocyte and neutrophil membranes from RA patients as compared to healthy controls [20
]. The up-regulation of ADORA3
expression on PBMCs has been observed after only ten weeks of MTX therapy [5
]. In addition, the affinity of the ADORA2A
was lower in patients receiving MTX compared to healthy controls [20
]. In comparison, in RA patients receiving anti-TNF therapy the expression and affinity of ADORA2A
was similar to that observed in healthy controls [20
]. This is consistent with an effect of TNF or the indirect effect of inflammation in general. In our cohort, expression of ADORA2A
but not ADORA3
, was increased in those patients receiving MTX compared to those not receiving MTX. While we observed no effect of anti-TNF therapy, the number of patients was small. The presence of the adenosine receptors in the synovial membrane provides evidence that MTX has the potential to exert important anti-inflammatory effects at the primary site of the inflammatory process in RA.
It is important to note that the specimens used in our study were from RA patients with late stage disease and these patients differed from the cohort used for the genotyping studies. There remains a possibility that changes in ADOR subtype expression related to MTX therapy could be different at earlier stages of disease. However, given that expression of ADORA2A
is regulated by inflammatory cytokines such as TNF-α [6
] it would be reasonable to assume there may be similar findings. This will need to be confirmed in studies of patients with early RA.
Signalling through ADOR depends on a variety of factors including receptor expression, receptor sensitivity and extra-cellular concentration of ligand. Our data show greatest gene expression of ADOR3
and the ADOR3VAR
in rheumatoid synovia and that expression of a designated ADOR subtype is not necessarily exclusive to a particular cell type. Furthermore, there are known variations in the kinetic properties of the different ADOR subtypes, with ADORA1
several orders of magnitude more sensitive to adenosine than ADORA2B
]. It remains to be determined whether variation in expression compensates for the different kinetics attributed to the different ADOR subtypes and what consequences this has for individual synovial cell types.
The importance of ADORA3
in the anti-inflammatory process has also been highlighted by the finding in activated PBMCs that addition of an adenosine agonist (IB-MECA, CF101) resulted in reduced ADORA3
expression and down-regulation of TNF-α, thus breaking the autocrine loop and inhibiting the inflammatory process [17
]. The clinical efficacy of an oral adenosine A3
receptor agonist, CF101, in patients with RA has been examined in a small 12-week study. The level of expression of ADORA3
in PBMCs at baseline correlated with ACR50 and ACR70 responses (P
= 0.036) [22
]. We have now demonstrated high levels of ADORA3
expression in the joint tissues targeted by the inflammation. These data provide an encouraging line of evidence for further investigation of adenosine A3
agonists as therapeutic agents in RA, particularly in patients with high levels of ADORA3
MTX has also been suggested to have a beneficial effect on cardiovascular mortality in RA, an effect that is not observed with other disease modifying anti-rheumatic drugs (DMARDs) [23
]. This effect appears to be mediated through ADORA2A
activation mediating reverse cholesterol transport and limiting foam cell formation [25
]. One of the key challenges in the management of RA is rapid and effective disease control without provoking adverse effects. Currently there is no reliable means to predict which patients will respond to MTX, nor who will experience adverse effects. In this regard efforts have focused on the ability of polymorphisms of genes involved in the folate pathway to predict efficacy and toxicity of MTX. To date no SNP or combination of SNPs within the folate pathway has been identified that will reliably predict response or adverse effects associated with MTX.
Polymorphisms of adenosine monophosphate deaminase 1 (AMPD1)
, aminoimidazole carboxamide ribonucleotide transformylase (ATIC)
, and inosine triphosphate pyrophosphatase (ITPA)
, which are involved in the adenosine pathway, have been associated with good clinical response to MTX in patients with RA in some but not all studies [26
]. Genetic variations in the ADOR genes have received less attention but have been reported to be associated with good clinical response to MTX [26
] or have no effect [27
]. Polymorphisms within the ADORA2A
gene have been associated with adverse gastrointestinal events (nausea, vomiting or diarrhea) associated with MTX in patients with RA [28
]. Herein we have investigated polymorphisms within the ADORA3
gene. We have shown that the SNP rs1544224
is not associated with disease activity in patients with RA receiving MTX. Whether this SNP can predict MTX efficacy will need to be formally examined in prospective clinical studies. The cross-sectional design of this study resulted in the cohort of patients being enriched for patients who tolerated MTX. Patients with more severe adverse effects such as hepatotoxicity or myelotoxicity would have discontinued MTX and not entered this study. Nevertheless, there are a number of other adverse effects associated with MTX for which patients elect not to discontinue therapy because the benefits of therapy outweigh the adverse effects. We have shown a weak association between the ADORA3 rs1544224
SNP and forgetfulness and hair loss in our cohort. Confirmation of this will be required in other cohorts.