Blood monocytes differentiate into effector cells with distinct phenotypes. One of these cells, the OC, a uniquely polarized polykaryon with bone-resorbing activity, is directly involved in the pathogenesis of a spectrum of disorders. Identification of a specific surface marker on OC progenitors is of great importance, because it would assist in the evaluation of metabolic, inflammatory and neoplastic disorders, serve as a marker of treatment response, and potentially provide a novel therapeutic target. We selected DC-STAMP as a strong candidate for an OCP biomarker based on its surface localization and critical role in the regulation of cell-to-cell fusion during OC differentiation. In this study, we generated a DC-STAMP-specific antibody, examined the potential of DC-STAMP as a biomarker of OCP, characterized the expression of DC-STAMP during OC differentiation, identified an important motif ITIM on the cytoplasmic tail of DC-STAMP, and elucidated the involvement of DC-STAMP in signaling during osteoclastogenesis.
Collectively, our data indicate that DC-STAMP is a potential OCP marker. First, DC-STAMP had the highest expression on monocytes, the progenitor population of human OCP.(3,36)
Second, monocytes with a higher surface DC-STAMP expression generated more OC and formed deeper and more numerous resorption pits in vitro
compared to monocytes that expressed low surface DC-STAMP. Third, blockade of DC-STAMP with an anti-DC-STAMP antibody potently inhibited OC formation in vitro
, a finding that underscores the essential role of DC-STAMP in OCP differentiation. Fourth, a positive correlation between CD16 and DC-STAMP surface expression as well as physical interaction of these two molecules indicate that CD16 and DC-STAMP may form a receptor complex. The association of DC-STAMP with CD16 is of particular relevance given that OCP arise from the CD16+ monocyte population in PsA.(3)
Lastly, four distinct DC-STAMP expression patterns were observed on human PBMC. These patterns differed in PsA and controls and increased expression of DC-STAMP was associated with a higher frequency of OC formation in vitro
. Thus it is plausible that increased DC-STAMP expression on monocytes may facilitate the identification of Ps patients at risk for arthritis.
The finding that human OCP arise from DC-STAMPhigh
monocytes contrasts with our previous findings in the murine RAW cell line where DC-STAMPlo
cells display the master fusogen phenotype.(25)
It is well known that analysis of osteoclastogenesis is greatly influenced by the context of the culture conditions, timing and the source of myeloid cells.(25,37)
Differences in the induction of monocyte fusion in murine and human cells have been previously reported.(37)
Of note, we used freshly isolated monocytes in contrast to serially cultured RAW cells analyzed in the previous study. It is known that many cell surface markers, including CD16 and DC-STAMP (),(3,25)
manifest significant dynamic alterations during culture. Another explanation for the discrepancies in the murine and human studies is lineage heterogeneity in the sorted human cells. Multiple OC populations have been reported in murine bone marrow cells using a serial sorting procedure,(38)
but this approach is not practical due to the low percentage of DC-STAMP+CD14+ monocytes in human PBMC. We plan to perform serial sorting on a larger population of human monocytes available from leukopheresis patients in the future to further address this potential technical issue.
Osteoclast formation and activation is regulated by a complex interplay of signaling mechanisms that involve protein-tyrosine phosphorylation.(39)
Indeed, a number of immunoreceptors act in concert with RANKL and M-CSF to promote osteoclastogenesis,(13,14)
including triggering receptor expressed in myeloid cells-2 (TREM-2) and os
eceptor (OSCAR). Both TREM-2 and OSCAR are ITAM-bearing molecules that activate calcium signaling. Analogous to T cell activation which requires both signal 1 (TCR and MHC engagement) and signal 2 (co-stimulation molecule recognition), OC differentiation requires an early activation signal generated by RANKL/MCSF, followed by a second activation signal triggered by ligand engagement on the ITAM-bearing receptors.(12,40,41)
Signaling molecules and cascades, including ITAM and ITIM adaptor molecules, comprise a collaborative network of interactions to regulate cell responses.(42)
Indeed, ITIM and ITAM interactions take place at an early stage of immune regulation and OC formation.(20)
The activation signals mediated through receptors that contain ITAM associated subunits are counterbalanced by inhibitory signals triggered by stimulation of ITIM adaptor molecules.
CD16 is considered as an ITAM-bearing protein due to its association with FcRγ (43,44)
The presence of an ITAM on CD16 and an ITIM on DC-STAMP, the positive correlation between CD16 and DC-STAMP expression on fresh monocytes (), the reciprocal regulation of CD16 and DC-STAMP during osteoclastogenesis ( and Chiu et al.,(3)
), as well as protein-protein interactions between DC-STAMP and CD16 (), raised the possibility that motifs on CD16 and DC-STAMP deliver counterbalancing signals. It is well documented that coaggregation of ITAM- and ITIM-bearing receptors by an extracellular ligand is required to trigger ITIM-mediated inhibition of cellular signaling responses. Following coaggregation, the cytoplasmic ITIMs undergo tyrosine phosphorylation and recruit SH2-containing proteins such as SHP-1.(35)
Our data suggest that analogous to the two ITIM-bearing proteins PIRB and LILRB, the ITIM on DC-STAMP, delivers a negative signal based on the following observations. Osteoclastogenesis but not cell fusion was blocked when DC-STAMP was over-expressed in transfected RAW264.7 cells (). We also demonstrated a physical interaction between DC-STAMP and SHP-1 (). The finding that exposure of monocytes to 1A2 in the presence of RANKL and M-CSF decreased phosphorylation of SHP-1 (lane 5 in ) but increased PLC-γ2 phosporylation () is consistent with the inhibitory effect of DC-STAMP in signaling. Signaling molecules downstream of SHP-1 undergo dephosphorylation in a particular temporal sequence and at specific cellular locations to allow for efficient signaling. In particular, the phosphorylation status of SHP-1 directly determines its cellular localization and phosphatase activity.(45)
The absence of detectable phosphorylated SHP-1 in 1A2-treated monocytes () which were unable to differentiate into OC in the presence of RANKL+M-CSF () suggests that binding of 1A2 to surface DC-STAMP might block phosphorylation of tyrosine on ITIM, and inhibit the recruitment of SHP-1 to DC-STAMP and its subsequent phosphorylation. As a result, the binding of 1A2 to DC- STAMP blocks inhibitory signaling delivered by ITIM on DC-STAMP, thus activation signals from ITAM-bearing molecules are relatively strengthened, which causes an increase in the level of phosphorylated PLC-γ2. Although an elevated PLC-γ2 level may favor osteoclastogenesis, monocytes fail to form OC in the presence of 1A2 due to the inhibition of cell-to-cell fusion and a possible alteration of downstream calcium oscillations, critical for the later stages of OC differentiation. (37,46,47)
An alternative explanation for the increased PLC-γ2 level is that DC-STAMP, an ITIM-bearing molecule, acts as a positive regulator of osteoclastogenesis. Parallel to the observations with the NK receptor Ly49Q,(21)
DC-STAMP may compete with other ITIM-bearing receptors for SHP-1 and thus limit recruitment of SHP-1 to inhibitory ITIM-bearing proteins such as PIR-B.(21)
Based on our results and two models previously proposed by Humphrey et al.,(14)
and Nimmerjahn & Ravetch,(40,41)
we proposed a modifed view of the OC signaling cascades that takes DC-STAMP and CD16 into consideration as shown in . Following activation, the ITAM in the cytoplasmic domain of CD16 is phosphorylated by a Src family kinase, that facilitates docking to SH2 sites and activation of Syk kinases, and in turn triggers Ras kinase pathway signaling through Sos. Ligand induction also triggers inhibitory signaling by the ITIM motif on DC-STAMP which attenuates Ras activation by recruitment of the SH2-domain containing phosphatase (SHP-1).(48)
Factors that may activate CD16 are shown in the top box but the ligand of DC-STAMP has not been identified. Of note, our simplified model does not include all ITAM- and ITIM bearing molecules involved in osteoclastogenesis. ITAM-bearing molecules such as TREM-2 and OSCAR(18),(14)
and ITIM-bearing molecules such as LILRB & PIR-B(20)
must be considered as well given their important actions during osteoclast formation.
A model of signaling cascades induced by DC-STAMP, CD16, and RANK during osteoclast differentiation
A comprehensive model of signaling that includes DC-STAMP must incorporate the findings that both DC-STAMP mRNA and protein levels fall rapidly in monocytes following exposure to RANKL. One potential explanation is that DC-STAMP expression is required to initiate the regulation of cell fusion but removal of the inhibitory signal mediated by the ITIM and SHP-1 on later stage ITAM mediated calcium signaling is also required for the OC differentiation program to fully progress. In this paradigm, temporal and spatial (migration from cell surface to cytoplasm) regulation of DC-STAMP is required for OC formation. Of note, treatment of monocytes with TNFα alone can promote polykaryon formation in a subset of monocytes.(37)
In addition, TNFα triggered a rise in DC-STAMP mRNA on day 6 after TNFα exposure. Experiments are underway to better understand the interactive effect of RANKL and TNFα on DC-STAMP kinetics and function.
In conclusion, we developed a novel anti-DC-STAMP monoclonal antibody 1A2 to characterize DC-STAMP expression on human PBMC. DC-STAMP is primarily expressed on the surface of monocytes and a subset of CD3+ cells and expression levels on monocytes correlated with the level of osteoclastogenesis in vitro.
Moreover, PBMC isolated from a subset of subjects with PsA expressed significantly higher levels of DC-STAMP by flow cytometry than controls and these findings raise the possibility that DC-STAMP is a marker for OCP in inflammatory arthritis. The declining surface expression of DC-STAMP contrasts directly with the gradual increase of CD16 expression observed during osteoclastogenesis(49)
. In addition, the interaction of DC-STAMP with CD16 coupled with the identification of an ITIM in the cytoplasmic tail of DC-STAMP that binds SHP-1 provides new insights into the molecular mechanisms that underlie OC formation. Lastly, inhibition of osteoclastogenesis by 1A2 supports the concept that DC-STAMP is a potential therapeutic target for the treatment of inflammatory bone disorders.