Steinbeck M J, Appel W H, Verhoeven A J, Karnovsky M J
Harvard Medical School, Boston, Massachusetts 02115.
J Cell Biol. 1994 Aug;126(3):765-72. doi: 10.1083/jcb.126.3.765.
Recent reports have suggested that production of superoxide or other reactive oxygen species by activated osteoclasts may play a role in the complex process of bone resorption; however, the enzyme responsible for production of superoxide by osteoclasts has not been characterized. To determine if osteoclasts express NADPH-oxidase, a superoxide-generating enzyme found in phagocytic leukocytes, immunohistochemical studies were performed on tibia from 1-5-d-old rats using mAbs 449 and 48 and an antiserum specific for p47-phox. These antibodies recognize epitopes on the alpha and beta subunits of cytochrome b558, respectively, and the p47 cytosolic component of NADPH-oxidase. We found that osteoclasts attached to bone surfaces in tibia expressed all three components, as did mature polymorphonuclear and some mononuclear leukocytes in the bone marrow. In many adherent osteoclasts, the cytochrome b558 subunits were localized to the ruffled-border and bone interfaces. Studies were also performed on mature rat tibia that had undergone controlled fracture. By two weeks the healing fractures develop a callus rich in actively resorbing osteoclasts. Osteoclasts within the calluses, and attached to bone surface, also expressed the cytochrome b558 proteins. In addition to demonstrating the expression of NADPH-oxidase, the active production of superoxide by osteoclasts was also demonstrated in situ in freshly isolated tibia using 3,3'-diaminobenzidine (DAB)-Mn2+, a histochemical method specific for superoxide localization. Osteoclasts attached to bone surfaces contained deposits of oxidized DAB which were observed by light microscopy. Nonstimulated polymorphonuclear and mononuclear leukocytes in the bone marrow did not contain DAB deposits unless stimulated with phorbol myristate acetate, a known activator of NADPH-oxidase. These findings indicate that osteoclasts contain NADPH-oxidase, and during the process of resorbing bone, are actively producing superoxide.
最近的报告表明，活化的破骨细胞产生超氧化物或其他活性氧物质可能在复杂的骨吸收过程中起作用；然而，负责破骨细胞产生超氧化物的酶尚未得到鉴定。为了确定破骨细胞是否表达NADPH氧化酶（一种在吞噬性白细胞中发现的超氧化物生成酶），使用单克隆抗体449和48以及对p47-phox特异的抗血清，对1至5日龄大鼠的胫骨进行了免疫组织化学研究。这些抗体分别识别细胞色素b558的α和β亚基上的表位以及NADPH氧化酶的p47胞质成分。我们发现，附着在胫骨骨表面的破骨细胞表达所有这三种成分，骨髓中的成熟多形核白细胞和一些单核白细胞也表达。在许多贴壁破骨细胞中，细胞色素b558亚基定位于皱褶边缘和骨界面。还对经历了控制性骨折的成年大鼠胫骨进行了研究。到两周时，愈合的骨折处形成富含活跃吸收破骨细胞的骨痂。骨痂内以及附着在骨表面的破骨细胞也表达细胞色素b558蛋白。除了证明NADPH氧化酶的表达外，还使用3,3'-二氨基联苯胺（DAB）-Mn2+（一种超氧化物定位的组织化学方法）在新鲜分离的胫骨中原位证明了破骨细胞超氧化物的活性产生。附着在骨表面的破骨细胞含有氧化DAB沉积物，通过光学显微镜观察到。骨髓中未受刺激的多形核白细胞和单核白细胞除非用佛波酯肉豆蔻酸酯（一种已知的NADPH氧化酶激活剂）刺激，否则不含有DAB沉积物。这些发现表明破骨细胞含有NADPH氧化酶，并且在骨吸收过程中，正在活跃地产生超氧化物。
