Rheumatoid arthritis (RA) is a chronic inflammatory disease in which inflammation of the synovium leads to joint destruction (1). Interactions between RANK, expressed by osteoclasts, and its ligand RANKL, expressed by osteoblasts, have major effects on bone. RANKL-deficient mice lack osteoclasts and exhibit osteopetrosis, whereas transgenic mice show severe osteoporosis (2, 3).
In addition to its well-studied contribution to bone metabolism, this RANKL/RANK system is involved in dendritic cell (DC)–T cell interactions (4). These are critical in lymph node (LN) formation, since RANKL- and RANK-deficient mice have a complete lack of LNs with the related immune defects (3, 5). However, this important phenomenon has not been given much attention in humans, chiefly because of limited access to LNs. Since RA synovium has several but not all of the characteristics of a lymphoid organ (6), in the present study we compared the RANKL/RANK expression pattern in RA synovium with that in LN samples. Although a study of paired samples would be easy to perform in the mouse, such a study in human RA would be much more difficult if not impossible to perform. However, when we examined the pathology department database, we were able to select 11 paired samples that had been obtained ∼20 years ago when control of disease was difficult. At the time of joint surgery, LN biopsies had been performed to eliminate lymphomas in patients with reactive LNs, which are often observed in active, uncontrolled RA. Using this unique set of samples, we used immunohistochemistry to investigate RANK and RANKL expression by immature CD1a+ and mature DC-LAMP+ DC subsets and by subsets of CD3+ and CD4+ T cells expressing the Th1 cytokines interleukin-17 (IL-17) and interferon-γ (IFNγ).
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Expression of RANK and RANKL in paired RA synovium and LN sections. In RA synovium sections, RANKL was expressed in the lining layer but also in the sublining layer by fibroblast-like synoviocytes and cells in the lymphocytic infiltrates (Figure 1A), while RANK+ cells were detected exclusively in the lymphocytic perivascular infiltrates and not in the lining layer (Figure 1B). As observed in activated PBMCs, some RANK+ cells were identified as CD14+ cells (results not shown). In LN sections from RA patients, RANKL+ and RANK+ cells were diffusely expressed both in the T cell zone and in germinal centers (Figures 1C and D, respectively).
Figure 1. RANK and RANKL expression in paired samples of synovium and lymph nodes (LNs) obtained from patients with rheumatoid arthritis (RA). Immunostaining of paraffin-embedded sections using anti-RANKL or anti-RANK antibodies showed that RANKL-producing cells were detected in both the lining and sublining layers (A), whereas RANK+ cells were restricted to the lymphocytic perivascular infiltrates (B). In paired LN sections, RANKL+ and RANK+ cells were diffusely expressed both in the T cell zone and in germinal centers (C and D, respectively). (Original magnification × 250.)
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Coexpression of RANK/RANKL and DC/T cell markers in paired RA synovium and LN sections. To further clarify the phenotype of RANKL+ and RANK+ cells, double staining was performed with anti-RANKL or anti-RANK antibodies and DC markers (CD1a, DC-LAMP) or T cell markers (CD3, CD4, IL-17, IFNγ) both in RA synovium and in LN sections. Some immature CD1a+ and mature DC-LAMP+ DCs expressed RANK (Figures 2A and B, respectively). Double staining with anti-RANK and anti-CD3, anti-CD4, anti-IFNγ, or anti–IL-17 antibodies showed that none of the CD3+ (Figure 2C), CD4+ (Figure 2D), IL-17+ (Figure 2E), or IFNγ+ (Figure 2F) T cells expressed RANK. Double staining with anti-RANKL and anti-CD1a or anti–DC-LAMP antibodies showed that some RANKL+ cells expressed the immature DC marker CD1a (Figure 2G). However, none of the mature DC-LAMP+ DCs expressed RANKL (Figure 2H). Double staining with the T cell markers showed that some CD3+ (Figure 2I), CD4+ (Figure 2J), IL-17+ (Figure 2K), or IFNγ+ (Figure 2L) T cells expressed RANKL. These results indicated an association between RANK expression and T cell interactions, since such expression was specific to the perivascular infiltrates of the synovium. The same picture was observed both in RA synovium and in LN sections.
Figure 2. Phenotype of RANK+ and RANKL+ cells in paired samples of synovium and LNs obtained from RA patients. In RA synovium (A, B, E–H, K, L) and LN (C, D, I, J) sections, double staining with anti-RANK and anti-CD1a or anti–DC-LAMP antibodies revealed that RANK was expressed by some immature CD1a+ (arrows in A) and mature DC-LAMP+ (arrows in B) dendritic cell (DC) subsets. However, none of the CD3+ (C), CD4+ (D), interleukin-17+ (IL-17+) (E), or interferon-γ+ (IFNγ+) (F) cells expressed RANK. Double staining with anti-RANKL and anti-CD1a or anti–DC-LAMP antibodies revealed that some immature CD1a+ DCs expressed RANKL (arrows in G), whereas none of the mature DC-LAMP+ DCs was RANKL+ (H). Double staining showed that some CD3+ (arrows in I), CD4+ (arrow in J), IL-17+ (arrow in K), or IFNγ+ (arrows in L) cells were RANKL+. See Figure 1 for other definitions. (Original magnification × 250.)
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Effect of the proinflammatory cytokines IL-1β, TNFα, and IL-17 on the expression of RANKL by RA synoviocytes. Following the study of RANK and RANKL expression by DC or T cell subsets in samples of inflamed tissue, we switched to an in vitro model to reproduce some of the interactions observed in vivo. In particular, we looked at the contribution of the proinflammatory cytokines produced by monocytes (IL-1β, TNFα) and by T cells (IL-17) to RANKL expression by RA synoviocytes (9). Immunostaining of stimulated synoviocytes for RANKL showed that staining intensity of RANKL was enhanced in IL-1β–stimulated synoviocytes compared with that in TNFα- or IL-17–stimulated synoviocytes, and that combinations of these cytokines were more effective (Figure 3). These results indicate that the increased expression of RANKL, particularly in the lining layer and the perivascular infiltrates, reflects the local effect of inflammatory cytokines.
Figure 3. Effects of cytokines and their combinations on RANKL protein expression by synoviocytes. Synoviocytes from patients with rheumatoid arthritis were incubated with cytokines alone and in combination for 48 hours and stained with an anti-RANKL antibody (brown). CT = control (no added cytokine); TNFα = tumor necrosis factor α; IL-17 = interleukin-17. (Original magnification × 400.)
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The involvement of RANK–RANKL interactions has been clearly identified in bone destruction. Although deficient mice show major LN and immune defects (3, 5), these observations have not been given much attention. RA is the perfect context in which to study these phenomena, given the combination of local inflammation and bone destruction (10). In addition, LN hypertrophy is a common feature of RA disease activity.
The active RA synovium and LNs have many similarities as well as differences (6). Planning a study to compare the two sites would be impossible today. In addition, better therapy for RA with the treatments used today may modify the spontaneous picture observed in active disease. Accordingly, we used a set of paired synovium and LN samples to investigate the anatomic localization of RANK+ and RANKL+ cells in untreated (or at best poorly treated) RA patients. This choice has obvious limitations that we had to consider, including limited access to patient information, limited quality of fixation, and reduced amount of material left for new sections.
Using immunohistochemistry with paraffin sections, we focused on RANK and RANKL expression by DC and T cell subsets. In RA synovium, RANKL+ cells were detected in the lining layer and the lymphocytic infiltrates, whereas RANK expression was restricted to the perivascular infiltrates, in accordance with previous reports (11, 12). In LN sections, in which this had not been studied before, RANK+ and RANKL+ cells were diffusely expressed both in the T cell zone and in germinal centers. Thus, RANK expression appears to be associated with an active immune reaction.
Accordingly, we next focused on DC and T cell interactions, using markers previously tested in this context (8). Double staining showed that some immature CD1a+ DCs expressed RANK and RANKL, while some mature DC-LAMP+ DCs expressed only RANK. Although RANK and RANKL expression are not specific, it appears interesting that immature DCs can be distinguished by their expression of both RANK and RANKL from mature DCs, which express only RANK.
When we examined T cells, double staining showed that some CD3+, CD4+, IFNγ+, and IL-17+ cells expressed RANKL, while none of them expressed RANK. Accordingly, these subsets of T cells can interact directly with RANK+ cells such as DCs, as first observed between osteoclasts and osteoblasts. In addition, such interaction can be mediated through the production of Th1 cytokines (13). This can take place both in the synovium, leading to increased inflammation, and in juxtaarticular bone, leading to bone destruction, as well as in LNs.
To reproduce some of these interactions observed in vivo, we investigated the contribution of the proinflammatory cytokines produced by monocytes (IL-1β, TNFα) and by T cells (IL-17) to RANKL expression by RA synoviocytes. IL-1β was the most potent of the 3 cytokines when tested alone. Treatment with TNFα or IL-1β in combination with IL-17 was particularly potent at inducing RANKL expression, indicating the enhancing contribution of IL-17–producing cells and the role of synoviocytes in osteoclastogenesis (14). Indeed, blocking of IL-17 with an antibody and a soluble receptor in cultures of RA juxtaarticular bone explants led to a reduction in bone destruction, indicating the contribution of IL-17 to osteoclast activation (15). This effect was increased when inhibitors of IL-1, TNFα, and IL-17 were combined, indicating that these factors were more potent when interacting than when acting separately.
The down-regulation of RANKL expression through the actions of antiinflammatory cytokines or cytokine inhibitors (antibodies or soluble receptors) could be a strategy for reducing the involvement of RANK/RANKL, as observed in the effect of such treatment on joint destruction. There are drawbacks to such an effect, however, as can be seen with the side effects of anti-TNF treatment, mostly involving opportunistic infections. Such adverse effects must be considered when evaluating inhibition of the RANK/RANKL pathway for treatment of patients with RA.
In conclusion, in chronic inflammatory diseases such as RA, RANK expression appears to be restricted to mature DCs. In turn, these DCs can interact with RANKL+ T cells, some of which concomitantly express the Th1 cytokines involved in cell-mediated immunity and bone destruction. Inhibition of proinflammatory cytokines has been associated with positive effects on inflammation and destruction. Controlling the RANK/RANKL pathway may allow an improved potent result by acting on cell interactions present at synovium and bone sites. Conversely, induction of defects in LN function may have adverse consequences.