Abstract
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.
Highlights
Neutrophils, which are among the first cells to arrive in inflamed tissues, are activated during their margination and diapedesis across blood vessels and by cytokines at the site of inflammation [1]
SF neutrophils obtained from patients with RA and incubated for up to 4 days in control medium (CM) or in survival medium (SM) did not release bEyxpnroersmsiaolnbolofoRdAcNeKlls-L, OPG, receptor activator of nuclear factor-kappa-B (RANK), and tumor necrosis factor receptor-associated factor 6 (TRAF6) mRNAs by synovial fluid (SF) neutrophils isolated from patients with rheumatoid arthritis (RA) and by normal blood cells. (a) Purified neutrophils of SF from four patients with RA and of blood from seven normal donors were evaluated by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. (b) Purified peripheral blood mononuclear leukocytes (PBMLs) and platelets of blood from seven normal donors were evaluated by semi-quantitative Reverse Transcriptase (RT)-PCR analyses
SF from patients with RA activates the expression of RANK-L, OPG, and RANK in normal blood neutrophils Having established that inflammatory cytokines can stimulate healthy blood neutrophils to express OPG and RANK (Figure 3) and that SF neutrophils from patients with RA spontaneously expressed RANK-L, OPG, and RANK proteins (Figure 2), we investigated the effect of SF on the expression of these genes by incubating healthy human blood neutrophils in the presence of SF from patients with RA (Figure 4)
Summary
Neutrophils, which are among the first cells to arrive in inflamed tissues, are activated during their margination and diapedesis across blood vessels and by cytokines at the site of inflammation [1]. They are involved in various chronic inflammatory diseases such as arthritis, active autoimmune colitis, and skin lesions of psoriasis [2,3]. Joints in RA was confirmed by the K/BxN mouse model of RA [6] Besides their role in innate immunity, neutrophils act as antigen-presenting cells and regulate the adaptive immune response [7]. The same inflammatory conditions that induce neutrophils to differentiate into dendritic-like cells have the capacity to delay the apoptosis of neutrophils, which are cells that are constitutively programmed for apoptotic cell death [14]
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