Article

Granzyme B is recovered by natural killer cells via clathrin-dependent endocytosis

Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
Cellular and Molecular Life Sciences CMLS (Impact Factor: 5.81). 05/2010; 67(18):3197-208. DOI: 10.1007/s00018-010-0377-8
Source: PubMed

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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    • "Indeed, a bidirectional trafficking of lytic granule proteins exposed at the plasma membrane on degranulation has been demonstrated; both lysosome-associated membrane protein-1 (LAMP-1, also known as CD107a) (84) and Munc13-4 (83) undergo a rapid endocytosis, leading to the hypothesis that granule exocytosis is coordinated with the retrieval of cytolytic machinery components. Additionally, cytolytic mediators are also recaptured into early endosomes of NK cells via a clathrin-dependent route after target cell stimulation thus contributing to the cytolytic potential (85). "
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    ABSTRACT: Target cell recognition by cytotoxic lymphocytes implies the simultaneous engagement and clustering of adhesion and activating receptors followed by the activation of an array of signal transduction pathways. The cytotoxic immune synapse represents the highly specialized dynamic interface formed between the cytolytic effector and its target that allows temporal and spatial integration of signals responsible for a defined sequence of processes culminating with the polarized secretion of lytic granules. Over the last decades, much attention has been given to the molecular signals coupling receptor ligation to the activation of cytolytic machinery. Moreover, in the last 10 years the discovery of genetic defects affecting cytotoxic responses greatly boosted our knowledge on the molecular effectors involved in the regulation of discrete phases of cytotoxic process at post-receptor levels. More recently, the use of super resolution and total internal reflection fluorescence imaging technologies added new insights on the dynamic reorganization of receptor and signaling molecules at lytic synapse as well as on the relationship between granule dynamics and cytoskeleton remodeling. To date we have a solid knowledge of the molecular mechanisms governing granule movement and secretion, being not yet fully unraveled the machinery that couples early receptor signaling to the late stage of synapse remodeling and granule dynamics. Here we highlight recent advances in our understanding of the molecular mechanisms acting in the activation of cytolytic machinery, also discussing similarities and differences between Natural killer cells and cytotoxic CD8(+) T cells.
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    • "In long-term cytotoxicity assays, increased loss of Pfn in NK cells resulted in impaired killing ability. In the process of reuse, the pathway of retrieved Pfn going to lytic granule could be similar to that of granzyme B recycling, from early endosome to LAMP (lysosome-associated membrane protein) 1 + granules (Li et al., 2010) that might take part in an unusual rapid and dramatic de novo formation of nascent secretory lysosomes from as yet unidentified compartments in NK cells after target-cell recognition (Liu et al., 2005). In this report, it was suggested that the nascent secretory lysosomes came from the cytoplasm instead of budding from the trans-Golgi network. "
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    ABSTRACT: 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.
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    ABSTRACT: The granzyme B-induced cell death has been traditionally viewed as a primary mechanism that is used by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells to eliminate harmful target cells including allogeneic, virally infected and tumour cells Granzyme B (GrB) is the most abundant senne protease which is stored in secretory granules of CTLs and NK cells After recognition of the target cell, the engaged CTLs and NK cells vectonally secrete GrB along with other granule proteins including perform into the immunological synapse From this submicroscopic intercellular cleft GrB translocates into the cytoplasm of the target cell Although several models have been proposed to explain the GrB delivery mechanism, conclusive understanding of this process remains still elusive Once in the cytoplasm, GrB cleaves and activates or inactivates, multiple protein substrates, resulting eventually into apoptotic demise of the target cell This review is focused on the gene structure and expression of GrB, its biosynthesis and activation, delivery mechanisms into the target cell cytoplasm, direct proteolytic involvement in activation of several pro-apoptotic pathways, and on regulation of its activity in cancer cells Moreover, emphasis is given to the GrB-mediated anticancer effects and future clinical applications of the GrB-based and tumour-targeted recombinant fusion constructs
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