Entry and trafficking of granzyme B in target cells during granzyme B-perforin-mediated apoptosis

Blood. 1998 Aug 1;92(3):1044-54.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenoviridae / physiology
  • Animals
  • Apoptosis / physiology*
  • Biological Transport
  • COS Cells / drug effects
  • COS Cells / metabolism
  • Cell Compartmentation
  • Cell Nucleus / metabolism
  • Cytoplasm / metabolism
  • Endocytosis*
  • Endosomes / metabolism
  • GTP-Binding Proteins / physiology
  • Granzymes
  • HeLa Cells / drug effects
  • HeLa Cells / metabolism
  • Humans
  • Jurkat Cells / cytology
  • Jurkat Cells / metabolism
  • Membrane Glycoproteins / pharmacology
  • Membrane Glycoproteins / physiology*
  • Microinjections
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Models, Biological
  • Perforin
  • Pore Forming Cytotoxic Proteins
  • Recombinant Proteins / metabolism
  • Serine Endopeptidases / pharmacology
  • Serine Endopeptidases / physiology*
  • T-Lymphocytes, Cytotoxic / cytology
  • T-Lymphocytes, Cytotoxic / metabolism*
  • Transfection
  • Tumor Cells, Cultured
  • rab5 GTP-Binding Proteins

Substances

  • Membrane Glycoproteins
  • Pore Forming Cytotoxic Proteins
  • Recombinant Proteins
  • Perforin
  • GZMB protein, human
  • Granzymes
  • Serine Endopeptidases
  • GTP-Binding Proteins
  • rab5 GTP-Binding Proteins