Osteoclast-associated intracellular ITAM signalling molecules in human peri-implant osteolysis and rheumatoid arthritis.

Abstract

Peri-implant osteolysis (PO) and rheumatoid arthritis (RA) are examples of local inflammation-mediated bone loss, in which osteoclasts are believed to mediate the osteolysis. Besides the well-established OPG/RANK/RANKL system, ITAM-mediated signalling pathway has been found to be the co-stimulatory intracellular pathway mediating osteoclast differentiation and activity. TREM2, DAP12, FcRγ and OSCAR are components of the ITAM-mediated signalling pathway identified in osteoclasts. Another important molecule in the osteoclasts regulation is NFATc1, the key transcriptional factor mediating osteoclastogesis. Despite their known importance in the regulation of osteoclast and bone resorption, little is known if there any alteration in the expression of these molecules could be associated with the progression of bone loss in PO and RA. In relation to study in context of PO, the expression of ITAM-related molecules, TREM2, DAP12, OSCAR and FcRγ, along with NFATc1 and osteoclast cell marker cathepsin K in PO tissues in comparison to OA tissues was examined at protein level through immunohistochemistry as well as at mRNA level using qRT-PCR. The effects of PE particles, a common PO-induced wear particles, on osteoclast formation and resorption activity as well as mRNA expression of NFATc1 and ITAM-associated molecules were studied in vitro using a novel collagen gel PBMC assay. As for studies on RA, the expression of all those molecules in RA (active and inactive) tissues was compared to OA and normal tissues. The levels of soluble OSCAR in synovial fluids from RA and OA patients was also measured through ELISA and compared. Following observation on immunostaining of RA tissues, the regulation on the expression of OSCAR in endothelial cells following TNFα and IL-1β stimulation was studied in BMEC culture in vitro. OSCAR protein expression was analysed through immunofluoresence and ELISA on the cell culture supernatants meanwhile mRNA level was measured using qRT-PCR. Higher level of protein and mRNA for all those ITAM-associated molecules and cathepsin K was found in PO compared to OA tissues. Closer examination on tissue immunostaining found presence of PE particles inside and close to some cells positive for ITAM-related molecules. Investigation on the effect of PE in culture of PBMC-derived osteoclast cells found that the particles promote more osteoclasts formed and higher resoprtion activity. The PE particles also appeared to stimulate the mRNA expression of cathepsin K and all ITAMassociated molecules studied. Examination on the immunostaining indicated that highest number of cells positive for NFATc1, TREM2, DAP12, OSCAR and FcRγ in active RA tissues compared to inactive RA, OA and normal tissues. High concentration of soluble OSCAR was found in synovial fluids of both RA and OA groups. Study on the expression OSCAR in BMEC demonstrated that TNFα and IL-1β could upregulate the expression of mRNA and protein in secreted form. In general the expression of NFATc1, TREM2, DAP12, OSCAR and FcRγ was found high in PO and RA. Induction in expression of ITAM-associated molecules by PE particles and stimulation of OSCAR expression in endothelial cells by pro-inflammatory cytokines may suggest that these molecules may have role in the progression of PO and OA.