In the present study we investigated the effect of the active metabolite of leflunomide – A77 1726 – on the production of IL-1Ra by human joint cells. We observed that A77 1726, while having no effect alone, markedly enhanced the secretion of IL-1Ra in the presence of IL-1β or TNF-α in synovial fibroblasts and articular chondrocytes. The effect of A77 1726 was maximal at 100 μmol/l – a dose that lies within the range of plasma concentrations that may be observed in leflunomide-treated patients [6
]. Because IL-1Ra has been shown to exert chondroprotective effects, our observations suggest that in the presence of proinflammatory cytokines, which are present in significant amounts in inflamed joints, A77 1726 might exert a beneficial effect by increasing the local production of this anti-inflammatory mediator by joint cells.
IL-1Ra, which was initially discovered for impeding the binding of IL-1 to lymphoma cells, is produced in four different isoforms, one secreted and three intracellular, which are derived from the same gene [24
]. The exact biological functions of the different IL-1Ra isoforms are still not clear [25
]. The major role of secreted IL-1Ra is to block the effects of IL-1 by binding competitively to IL-1 receptor I without inducing signal transduction. The intracellular isoforms may be released from cells under certain circumstances, but they have also been suggested to perform important regulatory roles within cells. Synovial fibroblasts and de-differentiated chondrocytes produce both secreted and intracellular IL-1Ra [28
], and in these cells IL-1β-induced IL-1Ra production was enhanced in culture supernatants and in cell lysates in response to A77 1726. In contrast, cell lysates of freshly isolated chondrocytes contained no significant amounts of IL-1Ra, even after stimulation with IL-1β and A77 1726, which is consistent with our previous observations [28
In a recent study we observed that over-expression of either the secreted or the type I intracellular IL-1Ra isoform similarly protected mice from collagen-induced arthritis, blocking inflammation and joint damage [30
]. In RA, IL-1Ra has been shown to be one of the most potent agents available to decrease the progression of joint destruction [31
], although its effects on inflammation and symptoms are frequently considered disappointing. It is generally considered that a 10- to 100-fold molar excess of IL-1Ra over IL-1 is required to suppress completely the biological effects of IL-1, although lower amounts of IL-1Ra can significantly inhibit IL-1-induced responses [34
]. In the present study, the levels of IL-1Ra produced by synovial fibroblasts and de-differentiated chondrocytes on stimulation with A77 1726 and IL-1β usually ranged between equimolar concentrations and a twofold molar excess of IL-1Ra over IL-1. Even higher molar ratios of IL-1Ra:IL-1 were obtained when IL-1 was combined with TNF-α. Although large amounts of IL-1 are needed to obtain maximal catabolic effects in vitro
, multiple lines of evidence (for example [35
]) indicate that even very low levels of catabolic cytokines, including IL-1, can synergize to induce substantial effects. In vivo
, it is likely that multiple cytokines present in low amounts act in synergy to induce proinflammatory and catabolic effects. Thus, the blockade of low amounts of IL-1 might be sufficient to decrease such a synergistic effect in vivo
. The increased production of both secreted and intracellular IL-1Ra, which was observed in joint cells in response to A77 1726, might therefore be potentially beneficial by contributing to prevent joint damage in inflammatory arthropathies such as RA. In this regard, administration of leflunomide has been shown to limit joint destruction and improve function scores significantly, and to a greater degree than with methotrexate, according to at least two studies [1
]. The mechanisms that result in this protection are likely to be multiple. However, a stimulatory effect on IL-1Ra synthesis might be particularly relevant, given the important role of IL-1 in joint destruction [37
We investigated putative pathways involved in mediating the stimulatory effect of A77 1726 on IL-1Ra production, first focusing on the known effect of A77 1726 on pyrimidine synthesis. Addition of exogenous uridine did not significantly modulate the effect of A77 1726, suggesting that it was unlikely to be related to the inhibition of pyrimidine synthesis.
A77 1726 was previously reported to inhibit COX-2 activity [11
]. Also, the findings reported here confirm a previous report that 100 μmol/l A77 1726 completely blocked IL-1β-induced PGE2
production in synovial fibroblasts [12
]. Similarly, we observed that A77 1726 inhibited IL-1β-induced PGE2
production in chondrocytes. Interestingly, IL-1β triggered production of lower amounts of PGE2
in freshly isolated chondrocytes than in de-differentiated chondrocytes, suggesting low levels of expression and/or activity of COX-2 in these primary cells. This observation substantiates a recent report, which described a similar, differentiation stage-dependent regulation of COX-2 expression and PGE2
production in rabbit articular chondrocytes [38
]. Indomethacin increased IL-1β-induced IL-1Ra production in synovial fibroblasts and de-differentiated chondrocytes, suggesting that inhibition of COX-2 may indeed enhance IL-1Ra production in the presence of IL-1β in these cells. However, the stimulatory effect of indomethacin was repeatedly less potent than that of A77 1726. In addition, indomethacin did not affect IL-1Ra production in primary chondrocytes. This observation is consistent with the low levels of PGE2
produced in these cells, which are suggestive of low COX-2 expression/activity. It is also in agreement with a previous study that described a lack of effect of indomethacin on IL-1Ra production in IL-1-stimulated OA chondrocytes [39
]. Moreover, both in synovial fibroblasts and in chondrocytes, we observed that IL-1β-induced PGE2
production was strongly inhibited at relatively low concentrations (10–50 μmol/l) of A77 1726, as compared with the higher doses (50–100 μmol/l) that were required to enhance IL-1Ra production efficiently (data not shown). The dose dependency of these two effects thus appeared to be slightly different. Taken together, these observations strongly suggest that, in addition to the inhibitory effect of A77 1726 on COX-2 activity and PGE2
production, other mechanisms contribute to its stimulatory effect on IL-1Ra secretion.
Because high doses of A77 1726 have been reported to inhibit different types of receptor and nonreceptor tyrosine kinases [13
], we assessed whether tyrosine kinase inhibitors would affect IL-1β-induced IL-1Ra production in fibroblasts and chondrocytes. Two types of inhibitors were tested: genistein, a broad range tyrosine kinase inhibitor; and PP1, a more specific inhibitor of the src family of tyrosine kinases, the members of which have been reported to mediate IL-1 signalling in various cell types [40
]. Both inhibitors decreased IL-1β-induced IL-1Ra expression in synovial fibroblasts and chondrocytes (data not shown). These observations suggest that the increase in IL-1Ra production observed in the presence of A77 1726 is unlikely to be due to the inhibition of tyrosine kinases. Thus, although our findings suggest that part of the effect of A77 1726 on IL-1Ra production occurs through the inhibition of COX-2 activity, other unknown mechanisms, which remain to be characterized, are likely to be involved.