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Abstract

Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) recognize virus-infected or malignant transformed cells and destroy them through two independent mechanisms. Apoptotic target-cell death can be triggered by the engagement of death-receptors or by the release of cytotoxic granules. Cytotoxic granules gather at the site of contact between the effector cell (NK cell or CTL) and the target cell where the immunological synapse is formed. At the immunological synapse, the cytotoxic granules membrane fuses with the cell membrane and releases its contents. Cytotoxic granules have secretory lysosome characteristics, including an electron-dense core found in secretory organelles and a vesicular surrounding region typical of lysosomes. They contain perforin and serine proteases; granzymes, as well as several lysosomal proteins (Lamp 1 and 2, CD63, cathepsins B, D and L).
Perforin, a calcium-dependent pore forming protein, facilitates entry of granzymes into the cytoplasm of a target cell, where they access their substrates to trigger cell death. Peforin-deficient mice had decreased ability to clear bacterial and viral infections, fail to reject transplanted tumors and as they age, spontaneously develop B cell lymphoma.
In the secretory granules the 12–20 residues from the C-terminal are cleaved from the perforin. The cleavage is inhibited by incubation with inhibitor of cysteine proteases E-64, inhibitor of cysteine and serine proteases leupeptin and ammonium chloride indicating a possible role of cysteine cathepsins in this process. The low pH of cytotoxic granules (pH 5,1- 5,5) favor the interaction of perforin with the proteoglycans.
The aim of our study was to elucidate the role of cysteine cathepsins in regulation of perforin processing and degradation. Our initial studies showed that in vitro cathepsins B, K, L and S cleave and deactivate perforin. With the use of specific antibodies for the C-terminal of perforin we showed that in vitro cathepsin L preferentially cleaves C-terminal part of perforin.
Incubations of human NK cells (NK-92, YT, YT 5, KHGY1) with inhibitors of cysteine cathepsins E-64d and L1 showed that inhibitors of cysteine cathepsins blocks processing of perforin. With the use of inhibitors of cysteine cathepsins E-64d and L1 and inhibitor of N-glycosylation tunicamycin we showed cathepsins processed the C-terminal part, which is glycosylated. Further, we showed that inhibition of cysteine cathepsins in human NK cells and primary mouse CTLs decrease significantly killing of the target cells. With co-localization studies in YT NK cell and NK-92 cells we confirmed that perforin is mainly localized in secretory granules and not in endoplasmic reticulum. We also showed that perforin partially co-localize with cathepsins L, but does not co-localize with cathepsin B or cathepsin S. We wanted to confirm the role of cathepsin L with the experiments on NK cells and CTL isolated from cathepsin L knockout mice. We showed differences in the processing of perforin in cathepsin L knockout mice and wild type. Inhibitors of cysteine cathepsins blocked processing of perforin. But killng of the target cells by CTLs and NK cells from mouse deficient in cathepsins L was normal, comparable to CTL and NK cells from wild type. Inhibiton of cysteine cathepsins with E-64d and L1 decrease target cell killing of CTL of cathepsin L knockout mice, but at the same time did not affect target cell killing of NK cells from catthepsin L knockout mice. We concluded that granule-bound cathepsins are indeed essential for processing perforin to its active form, and that cathepsin L is important, but not exclusive participant in this process.
Contrary to our results, NK cells and CTL from cathepsin L knockout mouse killed target cells more efficiently as NK cells and CTL from wild type mouse first days of the differentiations. Since cathepsin L has also many functions in the nucleus of the cells, we conclude that this effect is connected with the function of cathepsin L in the nucleus. We showed, that activity of cathepsin L is regulated with endogenous inhibitors: stefin B (Čeru et al., 2010) and serpin A3G (Konjar et al., 2010).