Pinkoski M J, Hobman M, Heibein J A, Tomaselli K, Li F, Seth P, Froelich C J, Bleackley R C
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
Blood. 1998 Aug 1;92(3):1044-54.
In the widely accepted model of granule-mediated killing by cytotoxic lymphocytes, granzyme B entry into the target cell is facilitated by the pore forming molecule, perforin. Using indirect immunofluorescence and also direct visualization of fluorescein isothiocyanate (FITC)-conjugated granzyme B, we demonstrate internalization in the absence of perforin. Induction of the lytic pathway, however, required a second signal that was provided by perforin or adenovirus (Ad2). The combination of agents also resulted in a dramatic relocalization of the granzyme. Microinjection of granzyme B directly into the cytoplasm of target cells resulted in apoptosis without the necessity of a second stimulus. This suggested that the key event is the presence of granzyme B in the cytoplasm, and that when the enzyme is internalized by a target cell, it trafficks to an intracellular compartment and accumulates until release is stimulated by the addition of perforin. We found that the proteinase passed through rab5-positive vesicles and then accumulated within a novel compartment. On the basis of these results, we propose a new model for granzyme-perforin-induced target cell lysis in which granzyme B is subjected to trafficking events in the target cell that control and contribute to cell death.
在细胞毒性淋巴细胞通过颗粒介导杀伤的广泛接受的模型中，颗粒酶B通过成孔分子穿孔素进入靶细胞。我们使用间接免疫荧光以及异硫氰酸荧光素（FITC）偶联的颗粒酶B的直接可视化，证明了在没有穿孔素的情况下颗粒酶B的内化。然而，裂解途径的诱导需要由穿孔素或腺病毒（Ad2）提供的第二个信号。这些试剂的组合还导致颗粒酶的显著重新定位。将颗粒酶B直接显微注射到靶细胞的细胞质中导致凋亡，而无需第二个刺激。这表明关键事件是颗粒酶B在细胞质中的存在，并且当该酶被靶细胞内化时，它转运到细胞内区室并积累，直到通过添加穿孔素刺激释放。我们发现蛋白酶穿过rab5阳性囊泡，然后在一个新的区室中积累。基于这些结果，我们提出了一种颗粒酶 - 穿孔素诱导的靶细胞裂解的新模型，其中颗粒酶B在靶细胞中经历控制并导致细胞死亡的转运事件。
