Granzyme B (GzmB), a serine protease of cytotoxic T lymphocytes and natural killer (NK) cells, induces apoptosis by caspase activation after crossing the plasma membrane of target cells. The mechanism of this translocation during killer cell attack, however, is not understood. Killer cells release GzmB and the membrane-disturbing perforin at the contact site after target recognition. Receptor-mediated import of glycosylated GzmB and release from endosomes were suggested, but the role of the cation-independent mannose 6-phosphate receptor was recently refuted. Using recombinant nonglycosylated GzmB, we observed binding of GzmB to cellular membranes in a cell type-dependent manner. The basis and functional impact of surface binding were clarified. GzmB binding was correlated with the surface density of heparan sulfate chains, was eliminated on treatment of target cells with heparinase III or sodium chlorate, and was completely blocked by an excess of catalytically inactive GzmB or GzmK. Although heparan sulfate-bound GzmB was taken up rapidly into intracellular lysosomal compartments, neither of the treatments had an inhibitory influence on apoptosis induced by externally added streptolysin O and GzmB or by natural killer cells. We conclude that membrane receptors for GzmB on target cells are not crucial for killer cell-mediated apoptosis. IntroductionGranzymes are a family of granular serine proteases expressed by cytotoxic T lymphocytes (CTLs) and NK cells implicated in immune defense reactions. Granzyme B (GzmB), the most prominent member of this family, induces apoptosis of target cells after cytosolic delivery by caspase-dependent and -independent pathways, resulting in the activation of effector caspases and mitochondrial depolarization, respectively. 1,2 The mechanism of translocation across the plasma membrane, however, is poorly understood. In vivo, the apoptotic functions of granzymes strictly depend on the membrane-binding protein perforin. Mice without the functional perforin gene display a strong reduction of granular cytotoxicity. [3][4][5][6][7] Although perforin can form lytic pores across membranes at high concentrations, such as the structurally related C9 component of the terminal complement complex, 8 sublytic concentrations of perforin already synergize with granzymes in the absence of transmembrane pores. 9,10 In vitro, the sublytic activity of perforin can be replaced by other pore-forming proteins, such as streptolysin O (SLO) and pneumolysin. 9 The latter agents also cooperate in an unknown manner with GzmB at sublytic concentrations without generating pores for the delivery of proteins in the size range of granzymes. 9 Although membrane-bound perforin is able to generate nonspecific calcium channels but no open pores across the membranes of nucleated target cells, it is difficult to understand how macromolecules of 25 kDa, such as nonglycosylated GzmB, can reach the cytosol. Endocytosis and redistribution of externally added GzmB inside the target cell occurs rapidly, within 15 minutes, ...

There is increasing evidence that factors originally identified due to their neurotrophic activity also function within the immune system. This study focused on the related molecules glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) as well as their receptors. GDNF and NTN signaling is mediated by a two-component receptor: a signal-transducing component, RET, which is shared by both ligands, and a ligand-specific binding component, GFRα-1 (higher GDNF affinity) or GFRα-2 (higher NTN affinity). We report that human T cells, B cells, and monocytes produce NTN but not GDNF, as seen by RT-PCR and immunocytochemistry. RET was expressed by B cells, T cells, and monocytes. Exons 2–5 of RET encoding the cadherin-like domains 1–3 in the extracellular part and exons 16–19 encoding a section of the second tyrosine kinase domain were transcribed in CD4+ T cells, CD8+ T cells, B cells, and monocytes. Different splice variants encoding the C-terminal intracellular part (exons 19–21) of RET were detected. The ligand-binding receptors GFRα-1 and GFRα-2 were transcribed in all immune cell subsets. Quantitative PCR showed that GFRα-2 is by far the dominant ligand binding chain in T cells, B cells, and monocytes. Addition of GDNF or NTN to activated PBMCs reduced the amount of detectable TNF protein without altering its transcription. Together, this suggests that immune cells communicate with each other via NTN. Production of NTN by immune cells might also contribute to the neuroprotective immunity in the CNS observed in different model systems.

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