A validated in vitro system was used to investigate the nature of osteoclast-inducing growth factors (OGF) present in fetal rat calvarial conditioned medium (RCCM). Evidence is presented here that macrophage inflammatory protein-1alpha (MIP-1alpha), a member of the C-C chemokine family, is an essential factor for the induction of osteoclast differentiation in this system. Specific polyclonal antibodies against MIP-1alpha significantly inhibited development of TRAP-positive osteoclast precursors and multinucleated osteoclasts induced by RCCM. Anti-MIP-1alpha antibody treatment was accompanied by an increase in the number of macrophage-like cells, suggesting that bone-derived MIP-1alpha is involved in the direction of preosteoclast formation with an inhibitory action on progenitor cell proliferation. Reverse-phase HPLC of RCCM resolved multiple fractions with OGF activity. OGF fractions separated at low acetonitrile (AcN) concentrations (</=15%) did not bind heparin and were not blocked in their bioactivity by the anti-MIP-1alpha antibody. However, OGF fractions eluted at higher AcN concentrations (30-70%) showed heparin-binding activity and were inhibited in their bioactivity by the anti-MIP-1alpha antibody. Western blotting of RCCM with the anti-MIP-1alpha antibody revealed a distinct band with a molecular mass of around 8-14 kDa corresponding to MIP-1alpha. Recombinant rat MIP-1alpha dose dependently stimulated formation of mononuclear osteoclast precursors with maximum stimulation at 50 ng/ml, though it could not fully mimic RCCM activity. These results identify MIP-1alpha as a candidate responsible for bone-derived OGF bioactivity and confirm that chemokines play an important role in the process of osteoclast recruitment and differentiation.
Copyright 1999 Academic Press.