Functional links between bone remodeling and the immune system in chronic inflammatory arthritis are mediated, in part, by the ligand of receptor activator of nuclear factor-kappa-B (RANK-L). Because neutrophils play a crucial role in chronic inflammation, the goal of this study was to determine whether proteins of the RANK/RANK-L pathway are expressed by synovial fluid (SF) neutrophils from patients with rheumatoid arthritis (RA) and to characterize this pathway in normal human blood neutrophils. The expression of RANK-L, osteoprotegerin (OPG), RANK, and tumor necrosis factor receptor-associated factor 6 (TRAF6) was determined by polymerase chain reaction, enzyme-linked immunosorbent assay, Western blotting, and cytofluorometry. RANK signaling was analyzed by the degradation of inhibitor of kappaB-alpha (I-κB-α). SF neutrophils from patients with RA express and release OPG and express the membrane-associated forms of RANK-L and RANK. In contrast, normal blood neutrophils express only the membrane-associated form of RANK-L. They do not express the mRNAs encoding OPG and RANK. SF neutrophils from RA patients and normal blood neutrophils release no soluble RANK-L. They express the mRNA for TRAF6. The expression of OPG and RANK by normal human blood neutrophils, however, can be induced by interleukin-4 + tumor necrosis factor-alpha and by SFs from patients with RA. In contrast, SFs from patients with osteoarthritis do not induce the expression of OPG and RANK. Moreover, the addition of RANK-L to normal blood neutrophils pretreated by SF from patients with RA decreased I-κB-α, indicating that RANK signaling by neutrophils stimulated with SF is associated with nuclear factor-kappa-B activation. In summary, RANK-L is expressed by inflammatory and normal neutrophils, unlike OPG and RANK, which are expressed only by neutrophils exposed to an inflammatory environment. Taken together, these results suggest that neutrophils may contribute to bone remodeling at inflammatory sites where they are present in significantly large numbers.

Osteoprotegerin (OPG) acts as a decoy receptor for receptor activator of nuclear factor-kB ligand (RANKL) and TNF-related apoptosis-inducing ligand (TRAIL). OPG regulates bone remodeling and the immune response. The primary objective was to decipher, among human peripheral blood mononuclear leukocytes (PBML) that produce OPG, the subset(s) responsible for this synthesis and its regulation. To this end, normal human PBML and CD4-, 8-, 19-, 14-enriched subpopulations were studied in vitro for OPG synthesis. PBML were subjected to adherence and immunomagnetic separation, and OPG expression was analyzed by PCR, northern and western blotting, and ELISA. The antiapoptotic effects of OPG were studied on TRAIL-stimulated RPMI 8226 myeloma cells. OPG was time-dependently produced by primary CD4 þ T lymphocytes exclusively. OPG secretion was upregulated by anti-CD3 antibody stimulation or incubation with interleukin (IL)-4, IL-1b, TNF-a, GM-CSF, and vitamin D 3 . In contrast, IL-10 inhibited the basal and IL-4-induced production of OPG by T cells. Conditioned media from activated T lymphocytes decreased TRAIL-induced apoptosis of RPMI 8226 cells. This effect was reversed by addition of RANKL to the T-cell conditioned media. As human immunodeficiency virus-1 (HIV-1) targets CD4 þ T cells, we evaluated the effects of recombinant HIV-1 gp120 proteins on OPG synthesis. The gp120 from three different HIV-1 strains significantly reduced the basal output of OPG from T cells. Furthermore, all four protease inhibitors (PIs) used in highly active antiretroviral therapy decreased OPG synthesis by human blood T cells, nelfinavir being the most efficient PI. The simultaneous presence of an HIV-1 gp120 and a PI abrogated the basal output of OPG. In conclusion, these results highlight a new role for T lymphocytes involved in pathologies. Activated CD4 þ T cells could, through OPG release, have a paracrine effect on adjacent cells and contribute to reduce the local process of bone remodeling and cellular apoptosis.

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