van Lent P L E M, Blom A B, van der Kraan P, Holthuysen A E M, Vitters E, van Rooijen N, Smeets R L, Nabbe K C A M, van den Berg W B
University Medical Center, Nijmegen, The Netherlands.
Arthritis Rheum. 2004 Jan;50(1):103-11. doi: 10.1002/art.11422.
To investigate in vivo and in vitro whether macrophages have an intermediate role in transforming growth factor beta (TGFbeta)-induced osteophyte formation.
In vivo, synovial lining macrophages were selectively depleted by injection of clodronate-laden liposomes 7 days prior to injection of 20 ng or 200 ng of TGFbeta into murine knee joints 3 times, on alternate days. Total knee joint sections were obtained on day 7 after the last injection and stained with Safranin O. Production of bone morphogenetic protein 2 (BMP-2) and BMP-4 was determined by immunolocalization. The interaction between murine macrophages and mesenchymal cells (precursors with chondrogenic potential) was studied in vitro using a Transwell system in which RAW macrophages were cocultured with C3H10T1/2 mesenchymal cells. Spheroid neocartilage formation was quantified microscopically after staining with May-Grünwald-Giemsa.
Triple injections of 20 ng or 200 ng of TGFbeta into normal murine knee joints induced significant osteophyte formation at the lateral and medial sites of the patella and femur on day 7 after the last injection. Strikingly, removal of synovial lining macrophages prior to TGFbeta injection resulted in a drastic reduction of osteophyte formation (by 70% and 64% after injection of 20 ng and 200 ng of TGFbeta, respectively). Synovial lining cells produced BMP-2 and BMP-4 after TGFbeta stimulation, whereas BMP-2 and BMP-4 were absent in the synovial tissue after macrophage depletion. In vitro, clustering and spheroid formation of C3H10T1/2 was induced by TGFbeta concentrations of >1 ng/ml. However, in the Transwell system, in the presence of murine macrophages, 0.5 ng/ml of TGFbeta was very effective in generating large spheroids, suggestive of macrophage-derived (co)factors. In coculture supernatants, TGFbeta concentrations were not elevated in the presence of macrophages, indicating generation of other growth factors involved in spheroid formation.
These findings indicate that macrophages are crucial intermediate factors in osteophyte formation induced by TGFbeta, probably by inducing other chondrogenic signals.
研究巨噬细胞在转化生长因子β（TGFβ）诱导的骨赘形成过程中是否起中间作用，包括体内和体外研究。
在体内，于向小鼠膝关节注射20 ng或200 ng TGFβ前7天，通过注射载有氯膦酸盐的脂质体选择性清除滑膜衬里巨噬细胞，每隔一天注射一次，共注射3次。在最后一次注射后第7天获取全膝关节切片，并用番红O染色。通过免疫定位法测定骨形态发生蛋白2（BMP - 2）和BMP - 4的产生。在体外，使用Transwell系统研究小鼠巨噬细胞与间充质细胞（具有软骨形成潜能的前体细胞）之间的相互作用，其中RAW巨噬细胞与C3H10T1/2间充质细胞共培养。用May - Grünwald - Giemsa染色后，显微镜下对球状新软骨形成进行定量分析。
向正常小鼠膝关节三次注射20 ng或200 ng TGFβ后，在最后一次注射后第7天，在髌骨和股骨的外侧和内侧部位诱导出明显的骨赘形成。引人注目的是，在注射TGFβ之前清除滑膜衬里巨噬细胞会导致骨赘形成大幅减少（注射20 ng和200 ng TGFβ后分别减少70%和64%）。TGFβ刺激后，滑膜衬里细胞产生BMP - 2和BMP - 4，而巨噬细胞清除后滑膜组织中不存在BMP - 2和BMP - 4。在体外，浓度>1 ng/ml的TGFβ可诱导C3H10T1/2细胞聚集和球状形成。然而，在Transwell系统中，在存在小鼠巨噬细胞的情况下，0.5 ng/ml的TGFβ在产生大的球体方面非常有效，提示巨噬细胞衍生的（协同）因子。在共培养上清液中，存在巨噬细胞时TGFβ浓度未升高，表明产生了其他参与球状形成的生长因子。
这些发现表明巨噬细胞是TGFβ诱导的骨赘形成中的关键中间因子，可能是通过诱导其他软骨形成信号实现的。
