Presentation: DC-STAMP as a Surface Marker of Erosive vs Non-Erosive Arthritis (2007)

153 DC-STAMP as a Surface Marker of Erosive vs Non-Erosive Arthritis

PURPOSE: Inflammatory arthritis can be either erosive (rheumatoid and psoriatic arthritis) or non-erosive (Jaccoud arthritis). Previous studies identified alterations in monocyte differentiation potential into osteoclasts vs dendritic cells as a central component to the erosive vs non-erosive phenotype. In order to develop a rapid diagnostic of monocyte differentiation potential, and to better understand the mechanisms responsible, we investigated DC-STAMP expression during osteoclastogenesis and dendritic cell differentiation. DC-STAMP is a 53 kDa, seven-transmembrane protein whose cellular localization is differentially regulated in osteoclasts vs dendritic cells; plasma membrane vs ER membrane respectively. Here we report the generation of a monoclonal antibody that is specific for mouse and human DC-STAMP and its use to characterize this protein in osteoclast and dendritic cell differentiation.
METHODS: Anti-DC-STAMP mAb was prepared against an extracellular domain peptide homologous to both murine and human DC-STAMP. Purified anti-DC-STAMP mAb was used for immunoprecipitation and subsequent immunoblotting of membrane fraction proteins from RAW 264.7 cells under non-reducing and reducing conditions. Commercially available anti-DC-STAMP polyclonal antibody was also used to immunoblot the anti-DC-STAMP mAb-immunoprecipitated membrane fraction for comparison. Confocal microscopy using anti-DC-STAMP mAb was performed on RAW cells treated with RANKL. Directly conjugated anti-DC-STAMP mAb was used in flow cytometric analysis of surface DC-STAMP following RAW cell treatment with IL-4, RANKL, IL-4 + RANKL, and IFN-a.
RESULTS: In immunoprecipitation-immunoblotting, the DC-STAMP mAb recognized a single band at ~50 kDa under reducing conditions, and at ~100 kDa for non-reducing conditions. This demonstrates for the first time that DC-STAMP forms a homodimer. These results were confirmed with the commercial polyclonal DC-STAMP antibody. Confocal microscopy demonstrated that DC-STAMP localizes to the plasma membrane of RAW cells. FACS of RAW cells treated with RANKL over 4 days showed a decrease in the proportion of DC-STAMP+ cells from baseline (94% to 45%). While treatment with IL-4 showed no shift in DC-STAMP expression, IL-4 + RANKL showed a decrease in the proportion of DC-STAMP+ cells as seen with RANKL alone (94% to 46%). IFN-a treatment showed no shift in DC-STAMP. TRAP+ cells were seen only in RANKL +/- IL-4 conditions, and multinucleated TRAP+ cells (osteoclasts) were seen in RANKL-only treated wells.
CONCLUSIONS: The anti-DC-STAMP mAb described here allows characterization of a surface phenotype for osteoclast precursors. The ability of this mAb to identify cellular localization and changes in plasma membrane expression in the presence of different cytokines that promote or antagonize osteoclastogenesis shows promise for its use to delineate monocyte differentiation potential for diagnostic purposes in settings of elevated osteoclast precursors.

 K.A. Mensah, None; Y. Chiu, None; C. Hock, None; C.T. Ritchlin, None; E.M. Schwarz, None.