Session Type: Abstract Submissions (ACR)
Presence of anti-citrullinated protein antibodies (ACPA) is a major risk factor for bone erosion in rheumatoid arthritis (RA) and antibodies against modified citrullinated vimentin induce osteoclast (OC) formation from monocytes. Besides monocytes, dendritic cells (DC) are potent osteoclast precursors. We aimed to further characterize DC derived osteoclastogenesis and to explore the effect of pooled and individual monoclonal ACPAs on this pathway.
ACPA positive and negative IgGs were isolated from synovial fluid (SF, n=26) and peripheral blood (PB, n=38) samples of RA patients. CD14+ monocytes from PB of ACPA+ RA patients and healthy individuals were first cultured in the presence of GM-CSF and IL-4 to generate DC or M-CSF to generate MΦ, and then further differentiated to OC in the presence of RANKL and M-CSF. In parallel, cells were grown on osteoassay surfaces and bone resorption area was quantified by computer assisted image analysis. In house generated anti-citrullinated monoclonal antibodies obtained from SF B-cells were also tested. Cytokines were measured by cytometric bead arrays in cultures supernatants. Mass spectrometry analysis was performed on different stages of differentiation of DC derived OC. Immunohistochemistry (IHC) was used to stain the OCs with biotinylated ACPA IgG and monoclonal anti-citrullinated proteins antibodies. The effect of PAD inhibition (C.I.amidine) and IL-8 inhibition was tested in the osteoclasts cultures.
SF derived ACPAs enhanced osteoclastogenesis and bone resorption from both DC (fold increase of 1.6±0.2 for osteoclasts number and 2.0±0.3 for bone resorption area) and MΦ (fold increase of 1.6±0.4 for osteoclasts number and 2.0±0.6 for bone resorption area) of healthy donors. Similar effect was observed when the precursor cells were derived from ACPA+ RA patients in both DC (fold increase of 2.3±0.9 for osteoclasts number and 2.6±0.1 for bone resorption area) and MΦ (fold increase of 1.8±0.6 for osteoclasts number and 2.3±0.7 for bone resorption) assays. PB derived ACPAs were equally effective with SF ACPAs. Principal component analysis confirmed distinct proteomic profiles during osteoclasts maturation from DC and MΦ, respectively. Vimentin significantly increased during DC-OC maturation, with citrullinated vimentin peptides detectable in matured osteoclasts. Increased osteoclastogenesis was associated with significantly higher levels of IL-8 levels in cultures supernatants of both DC (fold increase of 2.4±0.5) and MΦ (fold increase of 2.0±0.5). Two of the tested anti-citrullinated monoclonal antibodies had similar effects, while a third one had no such effect. Fab fragments of these monoclonal antibodies retained similar effects. Binding of these antibodies on OC was confirmed using IHC. Both PAD activity and IL-8 appear to be required for ACPAs effects.
SF and PB derived ACPA IgGs with broad specificities enhanced osteoclastogenesis from both DC and MΦ. This effect appears to be restricted to certain ACPAs specificities and at least partially mediated through a Fab mediated mechanism.
A. I. Catrina,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/anti-citrullinated-proteins-antibodies-promotes-osteoclastogenesis-and-bone-destruction-in-rheumatoid-arthritis/