Granzyme B is recovered by natural killer cells via clathrin-dependent endocytosis.
ABSTRACT When they recognize a target cell, natural killer (NK) cells mount an attack to kill the target by exerting their cytotoxicity via the exocytosis of cytotoxic granules. Although the details of this process (which includes the movement of cytotoxic granules in the immune synapse and their fusion with the plasma membrane, releasing granzymes and perforin into the synaptic cleft) are relatively better understood, the post-exocytosis regulation of the process is still largely unknown. Here we show that a clathrin-dependent endocytosis stimulated by target cell occurs in NK92 cell line, which is closely correlated with granzyme B recovery. Inhibition of the endocytosis significantly attenuates the cytotoxicity of NK92 cells. The NK cell recovery of its released effector molecules, in turn, suggests that endocytosis may well play a key role in the post exocytosis regulation of immune cells.
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ABSTRACT: Cytotoxic T lymphocyte and natural killer cell-initiated cell death is one of the primary mechanisms used by higher organisms to eliminate viruses and transformed cells. In this context, target cell death is rapid and efficient and initiated via two main pathways, involving either the ligation of death receptors or through the granule-exocytosis pathway. The granule-exocytosis pathway has attracted much attention over the past 10 years and consequently, a mechanism for granule-dependent killing has become reasonably well established. In the granule-dependent pathway, several proteolytic enzymes called granzymes are delivered to the target cell, promoting the activation of a family of death-inducing proteases called caspases. If caspases are inhibited by viral proteins or are inactivated through mutation, granzyme-mediated proteolysis of other cellular substrates ensures the timely death of infected or transformed cells. Here, we examine the findings that have shaped our current understanding of the mechanics of granule-dependent killing and discuss recent insights that have clarified some long-standing discrepancies in the granzyme literature.Cell Death and Differentiation 03/2008; 15(2):251-62. · 8.37 Impact Factor
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ABSTRACT: We measured synaptic vesicle mobility using fluorescence recovery after photobleaching of FM 1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] stained mouse motor nerve terminals obtained from wild-type (WT) and synapsin triple knock-out (TKO) mice at room temperature and physiological temperature. Vesicles were mobile in resting terminals at physiological temperature but virtually immobile at room temperature. Mobility was increased at both temperatures by blocking phosphatases with okadaic acid, decreased at physiological temperature by blocking kinases with staurosporine, and unaffected by disrupting actin filaments with latrunculin A or reducing intracellular calcium concentration with BAPTA-AM. Synapsin TKO mice showed reduced numbers of synaptic vesicles and reduced FM 1-43 staining intensity. Synaptic transmission, however, was indistinguishable from WT, as was synaptic vesicle mobility under all conditions tested. Thus, in TKO mice, and perhaps WT mice, a phospho-protein different from synapsin but otherwise of unknown identity is the primary regulator of synaptic vesicle mobility.Journal of Neuroscience 01/2008; 27(50):13691-700. · 6.91 Impact Factor
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ABSTRACT: The granule exocytosis cytotoxicity pathway is the major molecular mechanism for cytotoxic T lymphocyte (CTL) and natural killer (NK) cytotoxicity, but the question of how these cytotoxic lymphocytes avoid self-destruction after secreting perforin has remained unresolved. We show that CTL and NK cells die within a few hours if they are triggered to degranulate in the presence of nontoxic thiol cathepsin protease inhibitors. The potent activity of the impermeant, highly cathepsin B-specific membrane inhibitors CA074 and NS-196 strongly implicates extracellular cathepsin B. CTL suicide in the presence of cathepsin inhibitors requires the granule exocytosis cytotoxicity pathway, as it is normal with CTLs from gld mice, but does not occur in CTLs from perforin knockout mice. Flow cytometry shows that CTLs express low to undetectable levels of cathepsin B on their surface before degranulation, with a substantial rapid increase after T cell receptor triggering. Surface cathepsin B eluted from live CTL after degranulation by calcium chelation is the single chain processed form of active cathepsin B. Degranulated CTLs are surface biotinylated by the cathepsin B-specific affinity reagent NS-196, which exclusively labels immunoreactive cathepsin B. These experiments support a model in which granule-derived surface cathepsin B provides self-protection for degranulating cytotoxic lymphocytes.Journal of Experimental Medicine 09/2002; 196(4):493-503. · 13.21 Impact Factor