Immunocytochemical study on the behavior and function of osteoclasis using monoclonal antibodies to osteoclasts

破骨细胞是负责骨吸收的主要细胞。然而，破骨细胞骨吸收的确切机制尚不清楚。因此，我们用免疫细胞化学方法研究了破骨细胞和成骨细胞在骨重塑中的行为和功能。破骨细胞中主要溶酶体膜涎糖蛋白（LGP 107）的免疫细胞化学定位：利用大鼠不同分化阶段的破骨细胞研究LGP 107的免疫细胞化学定位。LGP 107仅局限于活跃破骨细胞皱折边缘的顶质膜。蛋白质也集中在皱褶边缘膜附近的一些内吞液泡中。然而，术后和/或静止的破骨细胞完全没有LGP 107的膜定位。这些结果表明，该蛋白主要在活性破骨细胞中合成，并迅速转移到褶边膜上。LGP 107被认为对骨重组的特殊酸性环境的形成和维持有更大的贡献。LGP 107在成骨细胞系细胞中的表达和定位：在参与破骨细胞骨吸收的成骨细胞系细胞中研究了LGP 107的免疫细胞化学定位。在破骨细胞出现之前，LGP 107在成骨细胞和骨细胞的质膜上发生了强烈的免疫反应产物。此外，在活性破骨细胞附近的成骨细胞的质膜上也观察到强烈的反应。这些数据表明，成骨细胞和骨细胞中的LGP 107可能在细胞识别和/或细胞粘附中起重要作用，并且LGP 107可能参与类骨细胞的成骨降解以及骨表面暴露。体外免疫制备破骨细胞单克隆抗体：用免疫组织化学和生化方法研究了大鼠骨髓培养中形成的破骨细胞样多核细胞（MNC_S）单克隆抗体的特性。体外免疫后，获得单克隆抗体HOK1。该抗体与基质细胞反应弱，与MNC_S及其在培养皿上的推测迁移痕迹反应强烈。用纯化的大鼠骨桥蛋白进行Western blotting，证实HOK1识别的抗原为骨桥蛋白。在MNC_S培养形成的吸收腔隙中进一步观察到HOK1阳性免疫反应。目前的数据表明，骨桥蛋白优先存在于钙化基质的吸收腔隙中，破骨细胞可能将这种蛋白捕获在细胞表面。少

Osteoclasts are primary cells responsible for bone resorption. However, the precise mechanism as to osteoclastic bone resorption remains unclear. We therefore examined immunocytochemically the behavior and the function of osteoclasts and osteoblasts in bone rmodeling.1.Immunocytochemical localization of a major lysosomal membrane sialoglycoprotein (LGP 107) in osteoclasts : The immunocytochemical localization was investigated of LGP 107 using rat osteoclasts at various stages of differentiation. LGP 107 was exclusively confined to the apical plasma mambrane at the ruffled border of the active osteoclasts. The protein was also concentrated in a number of endocytic vacuoles near the ruffled border membrane. However, the post and/or resting osteoclasts were totally devoid of the membraneous localization of LGP 107. These results indicate that the protein is largely synthesized in the active osteoclast and rapidly translocated to the ruffled border membrane. LGP 107 is suggested to contrib … More ute to the formation and maintenance of the specialized acidic environment for bone reorption.2.Expression and localization of LGP 107 in osteoblastic lineage cells : The immunocytochemical localization of LGP 107 was investigated in osteoblast linage cells involved in osteoclastic bone resorption. Strong immunoreaction products for LGP 107 occurred on the plasma membranes in the osteoblasts and osteocytes prior to the appearance of osteoclasts. Furthermore, strong reactions were also observed on the plasma membranes in the osteoblastic cells adjacent to the active osteoclasts. These data suggest that LGP 107 in osteoblastic cells and osteocytes may play an important role in cell-recognition and/or cell-adhesion, and that LGP 107 may be involved in osteoblastic degradation of the osteoid as well as exposure of the bone surface.3.Production of monoclonal antibodies to osteoclasts by in vitro immunization : The characteristics of a monoclonal antibody produced against produced against osteoclast-like multinucleated cells (MNC_S) formed in rat bone marrow cultures were examined immunohistochemically and biochemically. After the in vitro immunization was performed, the monoclonal antibody HOK1 was obtained. This antibody reacted weakly with stromal cells and intensely with both MNC_S and their putative migratory traces on culture dishes. Western blotting using purified rat osteopontin verified that the antigen recognized by HOK1 was osteopontin. Postive HOK1 immunoreactivity was further observed in the resorption lacunae formed by a culture of MNC_S. The present data suggested that osteopontin is preferentially present on the resorption lacunae in resorbing calcified matrices and that osteoclasts might trap this protein on their cell surface. Less