POSTMITOTIC OSTEOCLAST PRECURSORS ARE MONONUCLEAR-CELLS WHICH EXPRESSMACROPHAGE-ASSOCIATED PHENOTYPES

We previously demonstrated that osteoclast-like multinucleated cells w ere formed within 6 days in cocultures of mouse osteoblastic cells and spleen cells in response to 1 alpha,25-dihydroxyvitamin D-3[1 alpha,2 5(OH)(2)D-3] which was added together with hydroxyurea on Days 4-6 (fi nal 2 days of the 6-day coculture period). Using this coculture system , chronological changes of macrophage-associated phenotypes such as no nspecific esterase (NSE) and antigens to Mac-1, Mac-2, and F4/80 were examined in postmitotic osteoclast precursors during differentiation i nto osteoclasts induced by 1 alpha,25(OH)(2)D-3 (10 nM) added on Day 4 . Osteoclast differentiation was assessed by examining expression of c alcitonin receptors (CTRs) by autoradiography using I-125-labeled salm on CT. CTRs were first detected on small mononuclear cells within 12 h r after adding 1 alpha,25(OH)(2)D-3. The number of CTR-positive mononu clear cells attained a maximum at 24 hr and decreased thereafter. CTR- positive multinucleated cells were first observed at 24 hr and reached a maximum population at 48 hr. All CTR-positive cells showed tartrate -resistant acid phosphatase activity (a marker enzyme of osteoclasts). Most of the CTR-positive mononuclear cells which appeared at 12 hr we re positive for NSE and antigens to Mac-1 and Mac-2, but negative for F4/80 antigen. The proportion of CTR-positive cells expressing NSE and Mac-1 to total CTR-positive mononuclear cells decreased time dependen tly. Like authentic osteoclasts, CTR-positive multinucleated cells wer e negative for NSE and antigens to Mac-1 and F4/80, but positive for M ac-2. These results indicate that postmitotic osteoclast precursors ar e mononuclear phagocytes with macrophage-associated phenotypes, some o f which disappear rapidly during their differentiation into osteoclast s. (C) 1994 Academic Press, Inc.