Rheumatoid arthritis (RA) is a complex inflammatory disorder characterized by at least 3 major events: chronic synovial inflammation, cartilage destruction, and bone erosion. These 3 events are probably closely linked to each other; however, the underlying cellular and molecular processes may differ considerably. Given the complex nature of joint destruction in RA, it was highly surprising that selective blockade of a single proinflammatory cytokine, tumor necrosis factor (TNF), effectively inhibited the arthritic process (1, 2). These findings revised the assumption that proinflammatory cytokine networks are highly complex and therefore virtually impossible to target by a selective pharmacologic intervention.
As a consequence of these findings, the existence of “master players” within these proinflammatory cytokine networks, such as TNF, were postulated to essentially trigger joint inflammation and destruction (3). The critical number of such molecules that are necessary to promote the development of disease is not known. Studies in animals, such as the human TNF–transgenic (hTNFtg) mouse model, however, suggest that deregulated expression of a single proinflammatory cytokine is sufficient to trigger a complex, progressive, and destructive inflammatory process that closely resembles RA in humans (4). Thus, hTNFtg mice develop an arthritis that shows all 3 pathophysiologic processes that are found in human disease: chronic synovial inflammation, cartilage destruction, and bone erosion (4, 5).
It is still unclear why pharmacologic blockade of TNF does not fully stop the signs and symptoms of RA. Indeed, complete remission of signs and symptoms of RA is rarely achieved by any disease-modifying antirheumatic drug, including TNF blockers (1, 6–10). The most obvious explanation may be the complex nature of the disease, suggesting that several proinflammatory pathways act independently of TNF. Thus, the degree to which TNF fuels the RA disease process has not been completely clarified. Although, clinical studies have quantitatively assessed the efficacy of TNF blockers in RA (1, 6–9), it remains to be determined whether the lack of complete clinical response is due to a failure of TNF blockers to neutralize the entire biologic activity of TNF or is due to activation of TNF-independent pathways of inflammation.
Interestingly, the inhibitory potential of TNF blockade varies in the individual patient, depending on which pathophysiologic processes are addressed. TNF blockade has been shown to arrest bone erosion in a large number of patients whose clinical signs of inflammation show no response (6). The relative role of TNF in joint inflammation, bone erosion, and cartilage destruction may therefore differ and may depend on differences in the molecular mechanisms that underlie each of these 3 events. Two major target molecules of TNF, interleukin-1 (IL-1), and RANKL, may have at least a predilection for 1 of these 3 mechanisms. Blockade of IL-1 has been shown to have its strongest effects on the reduction of joint space narrowing, which is considered a surrogate marker for cartilage damage (11). Experimental blockade of RANKL has been shown to be highly effective in preventing local bone erosion, but not synovial inflammation, in animal models of arthritis (12–15).
In this study, we used hTNFtg mice as a model of erosive arthritis to gain new insights into the mechanisms of TNF-driven synovial inflammation, cartilage destruction, and bone erosion. We addressed the specific potential of TNF blockade to interfere with each of these pathologic features, and we compared it with the effects of blocking 2 of the central downstream mediators, IL-1 and RANKL. We also examined potential additive effects of TNF, IL-1, and RANKL blockade on these 3 features.
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- MATERIALS AND METHODS
In the present study, we analyzed the efficacy of TNF, IL-1, and RANKL blockade, either as monotherapy or as combined therapy, in inhibiting synovial inflammation, bone erosion, and cartilage destruction in TNF-driven arthritis. Comparison of the effects of 8 different therapeutic regimens yielded 3 major conclusions: complete remission of disease is hardly achieved by any of the monotherapies (18), not even by TNF inhibition; combined therapies are highly superior to monotherapy in inhibiting inflammation, bone erosion, and cartilage damage; and these therapies vary in their ability to affect these 3 pathologic processes. The effects of the monotherapies and the double-combination therapies on the 3 pathologic processes are summarized in Figure 9.
Figure 9. Qualitative and quantitative effects of single and combined inhibition of TNF (aTNF), IL-1 (IL-1 ra), and RANKL (OPG). The abilities of A, single inhibition and B, combined inhibition of TNF, IL-1, and RANKL to reduce synovial inflammation, bone erosion, and cartilage damage are shown as percentages of the respective features in untreated human TNF–transgenic mice. No effect would be indicated as a point in the center of the circle; complete inhibition of all 3 features would be indicated as filling of the entire circle. A, Effects of IL-1Ra (open area), OPG (shaded area), and anti-TNF (solid area). B, Effects of IL-1Ra plus OPG (open area), anti-TNF plus OPG (shaded area), and anti-TNF plus IL-1Ra (solid area). See Figure 1 for definitions.
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Systemic overexpression of TNF is sufficient to trigger a molecular process that leads to chronic synovitis, bone erosion, and cartilage destruction. The clinical and histopathologic changes induced by TNF in hTNFtg mice therefore closely resemble the changes found in human RA. Although they are considered to be highly complex, the pathophysiologic events in RA in humans are crucially influenced by TNF, as has been demonstrated by the clinical efficacy of TNF blockers. Assessments of surrogate markers for all 3 events, synovial inflammation (swelling and tenderness seen clinically), bone erosion (erosions seen on radiographs), and cartilage destruction (joint space narrowing seen on radiographs), have unraveled 2 interesting features of TNF blockade in human RA. First, attempts to block TNF alone barely achieve complete remission of disease (1, 6–9). Second, the efficacy of TNF blockade varies among the 3 pathologic features, as illustrated by the fact that in many patients, the effects of TNF on bone are independent from a clinical response in the signs and symptoms of disease (6). Why are these effects so different? Are inflammation, bone erosion, and cartilage destruction governed by different pathophysiologic mechanisms? Is TNF not a “master switch” in these events?
In the present studies, we used the hTNFtg mouse model to ensure that all pathophysiologic processes were based on the overexpression of the single cytokine TNF. This enabled us to selectively study how TNF-driven synovial inflammation, bone erosion, and cartilage destruction were influenced by blockade of TNF itself, by IL-1 and/or RANKL as 2 essential downstream mediators of TNF, or by a combination of these approaches. To yield optimal effects, we used very high doses of anti-TNF antibody, IL-1Ra, and OPG. Despite these high doses and early initiation of treatment (at the onset of clinical arthritis), anti-TNF treatment did not completely overcome synovial inflammation. The same results were observed with cartilage damage, which was inhibited, but not completely blocked, by anti-TNF treatment. These observations are in contrast with the fact that selective TNF blockade almost completely blocked bone erosion. Moreover, the findings are astonishing, since TNF is the only primary pathogenic factor in this animal model.
Thus, it appears that complete inhibition of synovial inflammation and cartilage damage is hardly achievable by just blocking TNF alone. The reason for this is not entirely clear; however, 2 mechanisms should be considered.
First, the effects of TNF may be reduced, but not completely abrogated, by anti-TNF antibody. This is supported by a) the presence of histologic signs of inflammation and cartilage damage and b) increased levels of molecules downstream of TNF, such as MMPs 3, 9, and 13 in synovial tissue and articular cartilage as well as IL-1 in serum, in hTNFtg mice treated with anti-TNF. It therefore appears that for synovial inflammation and cartilage destruction, even minimal local amounts of TNF are sufficient.
Second, a 2-phase model can be assumed whereby in the first phase, transgene-driven production of TNF before the appearance of clinical signs of arthritis might lead to synovial cell activation, increased cytokine production, and increased cytokine receptor levels. In the second phase, even low local levels of TNF (i.e., despite anti-TNF antibody treatment) might still trigger membrane-bound TNF receptors on activated cells to induce the production of high levels of IL-1. This idea is strengthened by the presence of inflammatory infiltrates in the synovium of hTNFtg mice at baseline, which had a cellular composition similar to that found in later stages of disease. In addition, MMP expression by synovial cells and chondrocytes and systemic levels of human TNF, mouse sTNFRI, and IL-1 were elevated at baseline, suggesting that the first phase of human TNF overexpression already entailed profound molecular and cellular changes in the synovial tissue before the arthritis became clinically apparent. Molecules induced by TNF, such as IL-1, could then perpetuate arthritis even more efficiently and require only low levels of TNF to fuel the disease. For example, IL-1, but not TNF, can stably up-regulate factors important for MMP synthesis, such as the Ets family of transcription factors (19).
Taken together, these data may explain the partial therapeutic resistance to TNF blockers in RA. In fact, the observation that TNF blockade is a far more efficient blocker of bone erosion than synovial inflammation is consistent with clinical results showing radiologic responses to TNF blockade even in patients who have small clinical responses. Indeed, RANKL levels and osteoclast numbers were most effectively influenced by anti-TNF treatment in our experiments; moreover, these parameters were not increased in mice at the baseline assessment.
TNF is a potent inducer of IL-1 (20), which itself is considered to be a crucial mediator in the pathogenesis of destructive arthritis (21, 22). Hypothesizing that TNF-mediated up-regulation of IL-1 is responsible for at least part of the effects of TNF, it was surprising that even high doses of IL-1Ra administered by continuous infusion were not sufficient to significantly block synovial inflammation, bone erosion, or cartilage damage in hTNFtg mice. This weak response to IL-1Ra may be the result of TNF-mediated effects bypassing IL-1, or it may be due to the possibility that complete blockade of TNF-activated IL-1 is difficult to achieve in this model, even when a continuous infusion of IL-1Ra is used. In fact, the observation that the combination of anti-TNF/IL-1Ra completely blocked synovial inflammation, bone erosion, and cartilage damage points to the latter effect. It favors the concept that IL-1 is mediating a major part, if not most, of the TNF-induced pathophysiologic processes in the synovial membrane and its neighboring structures. However, with regard to the blockade of IL-1 by IL-1Ra, these data indicate a higher therapeutic efficacy of IL-1Ra if concentrations of TNF are low. Thus, at least in conditions where IL-1 expression is driven by TNF, a combined approach of reducing TNF and blocking IL-1 is much more efficient than a single therapeutic approach.
Data from clinical studies support these concepts. First, monotherapy with IL-1Ra, although of proven clinical benefit, does not eliminate the clinical signs and symptoms of arthritis in a majority of patients (23, 24). Second, the effects of IL-1Ra on surrogate markers of bone erosion and cartilage damage appear to be superior to its effects on inflammation (11). This observation is consistent with the findings of our quantitative histologic assessment of inflammation, bone erosion, and cartilage damage in hTNFtg mice, which showed a predilection for IL-1Ra to inhibit the latter 2 pathophysiologic processes. Third, preliminary results of combined blockade of TNF and IL-1 suggest that such approaches may have additive effects, at least in experimental models (25, 26).
Blockade of RANKL-mediated osteoclast differentiation and function by OPG treatment (12–14) or by genetic deletion of RANKL (15) has proved to be powerful tool for blocking arthritic bone erosion without affecting joint inflammation. Data from our previous studies have shown that OPG was effective in hTNFtg mice (13) and suggested that RANKL, as key downstream mediator of TNF, is crucially involved in the formation of local bone erosion in the course of TNF-driven arthritis. TNF itself is not capable of triggering osteoclastogenesis on its own (27, 28), but it induces profound osteoclastogenesis in the presence of only minimal amounts of RANKL; the similar efficacy of anti-TNF and OPG in reducing the number of osteoclasts and bone erosions is consistent with these observations. Synovial inflammation and cartilage destruction, however, were not affected by OPG treatment, which suggests a selective role in arthritic bone erosion.
The effects of OPG, and thus blockade of RANKL–RANK interactions, may be more complex under conditions of TNF blockade: Whereas OPG was ineffective as monotherapy, the combination of OPG and anti-TNF was highly effective in reducing synovial inflammation. Thus, RANKL–RANK interactions regulated by TNF may also exert a certain proinflammatory role in the synovium. Blocking these interactions by OPG alone, however, seems difficult to accomplish and requires an additional reduction of TNF, whereas blocking bone erosion is at least partly achievable by OPG monotherapy. The cellular basis of this process remains to be elucidated, but may involve the interaction of RANKL-expressing T cells and fibroblasts with RANK-positive cells (16, 29), such as cells of the monocyte/macrophage lineage or dendritic cells (30). Mature osteoclasts, however, are unlikely to have a major impact on synovial inflammation, since TNF-mediated synovial inflammation is unaffected by the lack of osteoclasts (17). The main target of OPG, however, remains bone erosion. This is underlined by its lack of effect on cartilage degradation, although RANKL and its receptor are expressed by chondrocytes (31).
In summary, we have shown that IL-1 and RANKL are key downstream mediators of TNF-driven arthritis. Blockade of TNF combined with inhibition of either molecules, preferentially IL-1 and to a lesser extent RANKL, may prove to be a suitable strategy for achieving complete remission of symptoms in a majority of patients with RA.