| I IntroductionNatural killer cells (NK cells) are crucial for immune surveillance against virus-infected cells and transformed autologous cells. When NK cells encounter target cells, they discharge lytic molecules (perforin, granzymes and etc.) to induce target cells apoptosis. During the killing process, NK cell can directly eliminate target cells without prior sensitization through exocytosis of preformed secretory lysosomes (SLs). Moreover, NK cell has the potential to kill multiple target cells in a sequential manner. Nevertheless, there is little information about the quantity of released lytic molecules at every exocytosis after stimulation. In some instances, not a few of them are exocytosed after stimulation. So, how NK cell constantly replenishes its granular content during sustained killing process is unknown. Though NK cells can synthesis new lytic molecules to fullfil its lytic pool, the process of newly synthesed proteins to acquire mature form and enter into lytic granule needs several hours. So, it is important to investigate the mechanisms of NK cells about preserving granular stock. And it will provide clues to uncover mechanism of cytotocicity of NK cells in killing ability especially in sustained killing ability.At neurological synapse, there is an efficient compensatory endocytic process following exocytosis, which contributes to the rapid restoration of the functionality of the presynapse. The process is not only necessary for sustaining membrane surface area but also for membrane proteins'participation in the forming of new secretory vesicles. So neuron cells re-internalize and reuse excessive secretory granule membrane proteins even soluble component as a means of replenishing vesicle pools. Immunological synapse is of great similarity with that at neuronal synapse. And the observation of a bidirectional vesicular traffic within the cSMAC of NK cells suggests some similar endocytosis process at immunological synapse. So it is useful to explore that whether an endocytosis occurred when NK cell kill target cells and the relative biological significance. In our former study, one of the granule contents, granzyme B, was elucidated to be recovered after target cell stimulation. And the recovery contributed to the cytotoxicity of NK cells. It is known that granzyme B induced apoptosis of target cell is entirely dependent on perforin, and whether perforin is recovered as granzyme B is unknown. So the objective of this study is to explore whether perforin is recovered and whether the recovery plays a role in the killing ability of NK cells.II NK92 cells endocytose perforin after target cells stimulationEarly endosomal antigen-1(EEA-1)-enriched endosome represents the first endocytotic compartment to which recently internalized material is delivered. Appearance of EEA-1+gigantosomes was once viewed as accepting of materials in the early step of endocytosis. In this study, fluorescence labeled transferrin acted as an indicator and the localization of endocytosed transferrin was observed. The endocytosed transferrin entered into EEA-1+endosome. And the EEA-1+endosomes that co-localized with transferrin were significantly larger than the other EEA-1+ endosomes which were not co-localized with transferrin. It illustrats that enlarged EEA-1+endosome is due to accepting internalized materials. To explore whether NK92 cells endocytose simultaneously when confronted with target cells, the changes of EEA-1+vesicles in target cell stimulated NK92 cells were observed. By 15 minutes, the number of EEA-1+staining large vesicle increased compared with that of resting NK92 cells indicating NK92 cells endocytosed after target cell stimulation. Next it was investigated that whether perforin would be retrieved into those EEA-1+ endosomes. By 15 minutes, perforin was found co-localized with EEA-1+endosomes in target cell stimulated NK92 cells. Furthermore, Ca2+chelator EGTA which inhibits the exocytosis of NK92 cells abolished the enlargement of EEA-1+endosomes and the clo-colization of perforin and EEA-1+endosomes. The results indicate that endocytosis occurred in exocytosed NK92 cells after target cell stimulation and perforin is involved in the endocytosis process.â…¢Perforin is endocytosed via a clathrin dependent pathwayIn order to examine the pathway involved in perforin uptake, NK92 cells were treated with a variety of endocytosis inhibitors and were harvested for the assay of remained amount of perforin after stimulated by target cells PEC. Treatment with cycloheximide (CHX) which inhibits the synthesis of new proteins was used. So the perforin analysed in immunoblotting was due to the stored perforin. In CPZ (clathrin-dependent pathway specific inhibitor) treated NK92 cells, perforin was less than that in untreated cells after stimulation. Furthermore, both of nystatin, specific inhibitor of lipid raft-dependent endocytosis, and amiloride, specific inhibitor of macropinocytic-dependent endocytosis did not reduce the amount of perforin in treated NK92 cells compared with that in untreated cells. To further verify perforin is retrieved into stimulated NK92 cells via a clathrin-dependent pathway, the effect of CPZ on the co-localization of perforin and EEA-1 was observed. The co-localization of perforin and EEA-1 was hardly observed in CPZ treated NK92 cells. The same result was found in clathrin deficient NK92 cells which the clathrin heavy chain has been knocked down by siRNA for 48 h. While in scrambled siRNA-transfected NK92 cells, the co-localization of perforin and EEA-1 was observed. These data suggest that endocytosis of perforin is clathrin-mediated, while lipid raft-and macropinocytic-dependent pathways are not involved in the retrieval of perforin.IV Inhibition of clathrin-dependent endocytosis attenuates the cytotoxicity of NK92 cellsTo test whether the endocytosis plays a role in the cytotoxicity of NK92 cells, NK92 cells were pretreated with CPZ and the cytotoxicity was measured. In NK:target cell (E:T) ratio of 10:1 and 5:1, NK92 cells displayed attenuated killing ability in CPZ treatment compared to non-treated NK92 cells. The same result was found in clathrin deficient NK92 cells which the clathrin heavy chain has been knocked down by siRNA for 48 h. To explore whether the endocytosis contributes to the sustained killing ability of NK92 cells, NK:target cell (E:T) ratio was adjusted to 1:2 and 1:5 and the killing ability was measured at different time point. Coincident with the former test, clathrin dependent pathway inhibited NK92 cells no matter in CPZ treatment or clathrin knock down treatment both displayed attenuated killing ability compared with relevant control respectively. It shows that the endocytosis in NK92 cell plays a role in its killing ability.V Human peripheral blood NK cells endocytose perforin after target cells stimulationMoreover, the primary NK cells were observed either. Freshly isolated human NK cells were tested for purity around 95%. After stimulated by target cells, the co-localization of perforin and EEA-1 was found by 15 min. While in unstimulated NK cells, the co-localization was not found. And when clathrin dependent pathway was inhibited, the killing ability of NK cells was attenuated. It shows that the clathrin dependent endocytosis in NK cell plays a role in its killing ability, and the recovery of perforin was suggested to play a role in the killing ability for its recovery was associated with the clathrin dependent endocytosis.â…¥ConclusionIn this study, it was demonstrated that part of perforin in NK92 cells were retrieved into EEA-1 positive early endosomes via a clathrin-mediated endocytosis pathway. Inhibiting clathrin-dependent endocytosis resulted in decreased remaining content of perforin and attenuated cytotoxicity of NK92 cells. Furthermore, freshly isolated human NK cells were found the same results. It suggests that the clathrin-dependent endocytosis and internalization of cytotoxic molecules play roles in the killing ability of NK cells.
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