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Cell Biology International (2012) 36, 223–228 (Printed in Great Britain)

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Perforin is recaptured by natural killer cells following target cells stimulation for cytotoxicity

Lei Wang*†, Rulin Sun*, Pan Li*, Yang Han*, Ping Xiong*, Yong Xu*, Min Fang*, Zheng Tan*, Fang Zheng*¹ and Feili Gong*¹

*Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Peoples Republic of China, and †Institute of Laboratory Medicine, Hubei University of Chinese Medicine, No. 1, Huangjia Lake West Road, Wuhan, 430065, Peoples Republic of China

When encountering target cells, NK (natural killer) cells exocytose Pfn (perforin) and granzyme B to kill challengers. We previously reported that granzyme B is recycled and reused by NK cells via clathrin-dependent endocytosis. However, whether Pfn, a main secretory vesicle content, indispensible to granzyme B killing, undergoes endocytosis remains unknown. We demonstrate that Pfn is recaptured by early endosomes of NK cells via a clathrin-dependent endocytosis after target cell stimulation. Inhibition of clathrin-dependent endocytosis significantly attenuated the cytotoxicity of NK cells. The data suggest that the recovery of Pfn contributes to the cytotoxicity of NK cells. The assay of endocytosis of lytic molecule presents a particular focus for exploring the mechanism of abnormal cytotoxicity of NK cells.

Key words: clathrin, early endosome, endocytosis, natural killer cells, perforin

Abbreviations: Ami, amiloride, CFSE, carboxyfluorescein succinimidyl ester, CHC, clathrin heavy chain, CHX, cycloheximide, CPZ, chlorpromazine hydrochloride, E:T, effector:target, EEA1, early endosome antigen 1, FCS, fetal calf serum, HRP, horseradish peroxidase, NK, natural killer, Nys, nystatin, PEC, porcine endothelial cell, Pfn, perforin, PI, propidium iodide, siRNA, small interfering RNA

¹Correspondence may be addressed to either of the authors (email zhengfangtj@yahoo.com or flgong@163.com).

Received 2 May 2011/11 September 2011; accepted 10 October 2011

Published as Cell Biology International Immediate Publication 10 October 2011, doi:10.1042/CBI20110242

© The Author(s) Journal compilation © 2012 Portland Press Limited