Neutrophils present in synovial fluids and tissues of rheumatoid arthritis are a major source of RANKL
In a paper recently published online (Ann Rheum Dis, doi: 10.1136/ard.2011.153312), the authors found that B lymphocytes from rheumatoid arthritis (RA) synovial fluids and tissues expressed RANKL.
They stated that B cells were a major source of this cytokine in RA without considering that certain other synovial fluid cells and tissular cells have been reported to express RANKL in RA. In fact, they did not introduce nor discuss previous studies that demonstrated RANKL expression by macrophages in synovial tissue from RA,1 and by neutrophils in RA synovial fluids.2, 3 In addition, among RA patients the authors showed in figure 2, 4/9 and 2/8 had synovial fluid RANKL-expressing macrophages and neutrophils, respectively. Considering the high number of neutrophils in synovial fluids of active RA and their presence in synovium of early and late RA,4 it is impossible to neglect RANKL expressed by these inflammatory cells (Figure 1A, B, C). Moreover, neutrophils present in RA synovial tissues also express RANKL (Figure 1D, E). It is also important to stress that, although multiple antibodies against RANKL are presently available, much attention must be paid to their efficiency in recognizing RANKL depending on the types of experiments, as recently reported by Vandooren et al.5 As a corollary, convincing data based on anti-RANKL evaluation require the demonstration of similar results by using different antibodies in similar experiments with or without appropriate competition data if needed. In addition, these data will be comforted by showing converging results of the presence of RANKL using different methodologies, i.e PCR, cytofluorometry, histochemistry, western blotting (whole cell lysates, cell membranes, or specific organelles). We showed, by using these various methods, the presence of RANKL in neutrophils from RA synovial fluids, in neutrophils (from healthy donors) incubated in cell-free RA synovial fluids, and in murine neutrophils from air pouches.2, 3
Interestingly, most of the non-inflammatory cells do not express RANKL (or have a very low RANKL expression); they require a pathological condition and the presence of inflammatory stimuli to become RANKL-expressing cells.
In conclusion, several cell types like cells of myeloid origin (i.e. T and B lymphocytes, NK cells,6 monocytes/macrophages, neutrophils), cells of mesenchymal origin (i.e. osteoblasts, fibroblasts, synoviocytes...) and tumor cells can acquire the capacity to express large amounts of RANKL, and thus be implicated in disease pathogenesis and be efficiently targeted by an anti-RANKL therapy.
1. Crotti TN, Smith MD, Weedon H, et al. Receptor activator NF-kappaB ligand (RANKL) expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathy, osteoarthritis, and from normal patients: semiquantitative and quantitative analysis. Ann Rheum Dis 2002;61:1047-54.
2. Poubelle PE, Chakravarti A, Fernandes MJ, et al. Differential expression of RANK, RANK-L, and osteoprotegerin by synovial fluid neutrophils from patients with rheumatoid arthritis and by healthy human blood neutrophils. Arthritis Res Ther 2007;9:R25.
3. Chakravarti A, Raquil MA, Tessier P, et al. Surface RANKL of Toll-like receptor 4-stimulated human neutrophils activates osteoclastic bone resorption. Blood 2009;114:1633-44.
4. Tak PP, Smeets TJ, Daha MR, et al. Analysis of the synovial cell infiltrate in early rheumatoid synovial tissue in relation to local disease activity. Arthritis Rheum 1997;40:217-25.
5. Vandooren B, Cantaert T, Noordenbos T, et al. The abundant synovial expression of the RANK/RANKL/Osteoprotegerin system in peripheral spondylarthritis is partially disconnected from inflammation. Arthritis Rheum 2008;58:718-29.
6. Soderstrom K, Stein E, Colmenero P, et al. Natural killer cells trigger osteoclastogenesis and bone destruction in arthritis. Proc Natl Acad Sci U S A 2010;107:13028-33.
Figure 1 Expression of RANKL by rheumatoid synovial fluid (SF) and tissue neutrophils. (A) Purified SF neutrophils from a patient with active RA were stained with a mouse anti-human CD66b monoclonal antibody followed by a goat anti-mouse Alexa 488 antibody (Invitrogen; A11001). (B) Purified SF neutrophils were stained for human RANKL by a polyclonal goat anti- human RANKL (santa cruz; sc-7627) antibody followed by a rabbit anti-goat Alexa 568 (Invitrogen; A11079) antibody. (C) Merge of neutrophil staining with both anti-CD66b (green) and anti-RANKL (red) that shows SF neutrophils expressing RANKL. (D-F) Paraffin-embedded RA synovial tissue was deparaffinized, rehydrated and serial 3 ?m sections were stained for human RANKL using a polyclonal rabbit anti-human RANKL (Peprotech; 500- P133) antibody followed by an HRP-conjugated anti-rabbit antibody and diaminobenzidine; sections were counterstained with haematoxylin. (D) magnification: x100. (E) RA synovial tissue neutrophils (segmented nucleus) express RANKL (brown); magnification: x600. (F) Negative control with isotype (rabbit IgG)-matched staining; magnification: x600. Image
Conflict of Interest: