Kobara Miyuki, Sunagawa Nahoko, Abe Masaki, Tanaka Nana, Toba Hiroe, Hayashi Hironori, Keira Natsuya, Tatsumi Tetsuya, Matsubara Hiroaki, Nakata Tetsuo
Dept. of Clinical Pharmacology, Kyoto Pharmaceutical University, 5 Misasagi Nakauchi-cho, Yamashina-ku, Kyoto, Japan.
J Appl Physiol (1985). 2008 Mar;104(3):601-9. doi: 10.1152/japplphysiol.00254.2007. Epub 2007 Nov 29.
The mechanisms by which apoptotic myocytes are removed by macrophages have not been fully elucidated. This study examined whether apoptotic myocytes actively recruit macrophages by generating monocyte chemoattractant protein-1 (MCP-1) in experiments in vitro and in vivo. Neonatal rat cardiac myocytes were incubated for 4 h in the presence or absence of staurosporine (STS, 0.2-1 mumol/l), an apoptosis inducer. Nuclear staining with DAPI showed that STS induced apoptosis in a dose-dependent fashion. STS (1 mumol/l) caused extensive DNA fragmentation and increased caspase-3 activity compared with a serum-deprived control. MCP-1 mRNA and protein levels in myocytes increased twofold and fourfold, respectively, on STS treatment, and immunochemical staining revealed that apoptotic myocytes expressed MCP-1. To elucidate the role of MCP-1 expressed in apoptotic myocytes to recruit macrophages/monocytes, rat monocytes were incubated in the supernatant of STS-treated myocytes using a trans-well system. The culture medium of STS-treated myocytes recruited monocytes in a MCP-1-dependent fashion. In addition, experiments were performed in vivo using ischemia-reperfused rat hearts. Rats were subjected to 30 min of ligation of the left coronary artery followed by 24 h of reperfusion. After the reperfusion, in the ischemic border myocardium, 17.1 +/- 1.1% of myocytes were terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) positive. Moreover, double staining using the TUNEL technique and immunohistochemistry with MCP-1 antibody showed that 69.8 +/- 3.9% of TUNEL-positive myocytes expressed MCP-1 protein. Concomitantly, activated macrophages infiltrated the areas of apoptosis remarkably. These results suggest that apoptotic myocytes produce MCP-1, which have a critical role in the active recruitment of macrophages.
巨噬细胞清除凋亡心肌细胞的机制尚未完全阐明。本研究在体外和体内实验中检测凋亡心肌细胞是否通过产生单核细胞趋化蛋白-1（MCP-1）来主动招募巨噬细胞。将新生大鼠心肌细胞在存在或不存在凋亡诱导剂星形孢菌素（STS，0.2 - 1μmol/L）的情况下孵育4小时。用DAPI进行核染色显示，STS以剂量依赖的方式诱导凋亡。与血清剥夺对照相比，STS（1μmol/L）导致广泛的DNA片段化并增加了caspase-3活性。STS处理后，心肌细胞中MCP-1的mRNA和蛋白质水平分别增加了两倍和四倍，免疫化学染色显示凋亡心肌细胞表达MCP-1。为了阐明凋亡心肌细胞中表达的MCP-1在招募巨噬细胞/单核细胞中的作用，使用Trans-well系统将大鼠单核细胞在STS处理的心肌细胞的上清液中孵育。STS处理的心肌细胞的培养基以MCP-1依赖的方式招募单核细胞。此外，使用缺血再灌注大鼠心脏进行体内实验。大鼠左冠状动脉结扎30分钟，然后再灌注24小时。再灌注后，在缺血边缘心肌中，17.1±1.1%的心肌细胞末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）呈阳性。此外，使用TUNEL技术和MCP-1抗体免疫组织化学的双重染色显示，69.8±3.9%的TUNEL阳性心肌细胞表达MCP-1蛋白。同时，活化的巨噬细胞显著浸润凋亡区域。这些结果表明，凋亡心肌细胞产生MCP-1，其在巨噬细胞的主动招募中起关键作用。
