Calcitonin receptor antibodies in the identification of osteoclasts

Osteoclasts are the cells responsible for bone resorption, and their number and rate of formation are critical in determining bone mass, To identify a nd quantify osteoclasts, as well as to study their formation in bone and in osteoclastogenic cultures, osteoclast-specific cell markers are required. Only the calcitonin receptor (CTR) expression unambiguously identifies oste oclasts and distinguishes them from macrophage polykaryons, However, presen t autoradiographic methods for CTR detection are cumbersome and time consum ing. We have developed rabbit polyclonal antibodies specific for the C-term inal intracellular domain of the mouse and rat C1a CTR. These antibodies la beled HEK-293 cells stably transfected with CTR (but not untransfected HEK- 293 cells), This labeling is abrogated by preabsorbing the antibodies with the recombinant antigen, The antibodies immunostained primary mouse and rat osteoclasts as well as osteoclasts in sections of mouse bone. Osteoclasts (both mononuclear and multinucleated) formed from mouse bone marrow or sple en cells cocultured with osteoblasts in the presence of 1,25 dihydroxyvitam in D-3 and prostaglandin E-2 a ere also specifically immunostained by the C TR antibodies. Cocultures incubated under conditions that did not allow ost eoclastogenesis (i.e., omission of mediators or osteoblasts, or culture for less than 4 days) were not immunostained by CTR antibodies. Autoradiograph ic detection of I-125-labeled salmon calcitonin combined with CTR immunohis tochemistry showed that both methods labeled the same cells, A CTR polyclon al antibody and monoclonal antibody F4/80 were used in combination to show immunofluorescence labeling of murine osteoclasts and macrophage population s, respectively, in marrow/osteoblast cocultures. These results indicate th at simple and rapid CTR antibody-based methods can be used to identify oste oclasts, and can be used to characterize the antigenic profile of osteoclas ts by using double immunofluorescence analysis. (C) 1999 by Elsevier Scienc e Inc. All rights reserved.