A STUDY ON MECHANISMS OF NEUTROPHIL APOPTOSIS IN RAT CARRAGEENIN-INDUCED PLEURISY

粒细胞的凋亡和随后吞噬细胞的清除是炎症消退的关键。然而，还没有研究解决如何在炎症部位进行决议。本文研究了角叉菜胶致大鼠胸膜炎中性粒细胞凋亡和单核细胞摄取的时程变化，用TUNEL法、吖啶橙染色法和DNA凝胶电泳法检测了中性粒细胞的DNA断裂情况。中性粒细胞聚集在角叉菜胶注射后3-5小时开始，然后维持在一个平台期至24小时。中性粒细胞在第1~3天急剧下降，单核白细胞在第5小时开始聚集，第2天达到高峰，TUNEL阳性小体和丫啶橙阳性小体分别在24小时和9小时开始出现在单核白细胞的胞浆中。两种方法均显示，含DNA片段化的单个核细胞在第1、2天迅速增加，在第3天达到高峰。中性粒细胞DNA从5h开始出现特征性的梯状条带。此外，在角叉菜胶注射后9小时，渗出液中的白细胞中可检测到一种与细胞凋亡相关的蛋白Bad。这些结果表明，中性粒细胞刚开始在炎症部位聚集后就开始发生凋亡。因此，急性炎症的演变和消退过程可能是同时进行的。随着炎症的进展，副淋巴结伴增大。肿大的淋巴结内出现大量中性粒细胞和单核白细胞。这些细胞表达诱导型一氧化氮合酶。此外，肿大的淋巴结中具有树突状突起的细胞表达环氧合酶-2。这些结果表明，部分中性粒细胞转移到邻近的引流淋巴结。

Granulocyte apoptosis and subsequent clearance by phagocytes is critical for the resolution of inflammation. However, no studies have addressed how the resolution proceed in the inflammatory site. We studied the time course of neutrophil apoptosis and the following ingestion by mononuclear leukocytes in rat carrageenin-induced pleurisy, detecting DNA fragmentation by the TUNEL method, by acridine orange staining and from the DNA ladder pattern on electrophoresis. Neutrophil accumulation started 3-5 hrs after carrageenin injection, then maintained a plateau until 24 hr. Neutrophils decreased steeply between days 1 and 3. Mononuclear leukocytes started to accumulate at 5 hr, and reached a peak at day 2. TUNEL-positive bodies and acridine orange-positive bodies first became detectable in the cytoplasm of the mononuclear leukocytes from 24 hr and 9 hr, respectively. Both methods indicated that mononuclear leukocytes containing fragmented DNA increased rapidly on days 1 and 2, and reached a peak at day 3. The characteristic ladder pattern of neutrophil DNA was observed from 5 hr. Furthermore, an apoptosis related protein, Bad, became detectable in the exudate leukocytes from 9 hr after carrageenin injection. These results indicate that neutrophils start to undergo apoptosis just after the beginning of their accumulation in the inflammation site. Thus, evolution and resolution processes may proceed concurrently in acute inflammation.Parathymic lymph nodes enlarged with progression of inflammation. A large number of neutrophils and mononuclear leukocytes increasingly appeared in the enlarged lymph nodes. These cells expressed inducible nitric oxide synthase. In addition, cells possessing dendritic processes in the enlarged lymph nodes expressed cyclooxygenase-2. These results suggest that a part of neutrophils transmigrate into adjacent draining lymph nodes.