NADPH-OXIDASE EXPRESSION AND IN-SITU PRODUCTION OF SUPEROXIDE BY OSTEOCLASTS ACTIVELY RESORBING BONE

Recent reports have suggested that production of superoxide or other r eactive oxygen species by activated osteoclasts may play a role in the complex process of bone resorption; however, the enzyme responsible f or production of superoxide by osteoclasts has not been characterized. To determine if osteoclasts express NADPH-oxidase, a superoxide-gener ating enzyme found in phagocytic leukocytes, immunohistochemical studi es were performed on tibia from 1-5-d-old rats using mAbs 449 and 48 a nd an antiserum specific for p47-phox. These antibodies recognize epit opes on the alpha and beta subunits of cytochrome b(558), respectively , and the p47 cytosolic component of NADPH-oxidase. We found that oste oclasts attached to bone surfaces in tibia expressed all three compone nts, as did mature polymorphonuclear and some mononuclear leukocytes i n the bone marrow. In many adherent osteoclasts, the cytochrome b(558) subunits were localized to the ruffled-border and bone interfaces. St udies were also performed on mature rat tibia that had undergone contr olled fracture. By two weeks the healing fractures develop a callus ri ch in actively resorbing osteoclasts. Osteoclasts within the calluses, and attached to bone surface, also expressed the cytochrome b(558) pr oteins. In addition to demonstrating the expression of NADPH-oxidase, the active production of superoxide by osteoclasts was also demonstrat ed in situ in freshly isolated tibia using 3,3'-diaminobenzidine (DAB) -Mn2+, a histochemical method specific for superoxide localization. Os teoclasts attached to bone surfaces contained deposits of oxidized DAB which were observed by light microscopy. Nonstimulated polymorphonucl ear and mononuclear leukocytes in the bone marrow did not contain DAB deposits unless stimulated with phorbol myristate acetate, a known act ivator of NADPH-oxidase. These findings indicate that osetoclasts cont ain NADPH-oxidase, and during the process of resorbing bone, are activ ely producing superoxide.