Figure 5.
Figure 5. Influence of surface heparan sulfate on GzmB binding and apoptosis induction. (A-C) K562 cells were treated with heparinase III and analyzed for cell surface HS, GzmB-Bio binding, and susceptibility to apoptosis induction. (A) Binding of anti–HS-FITC is reduced to background levels after heparinase III treatment. Buffer-treated (▦) or heparinase-treated (▪) cells were stained in triplicate with the FITC-labeled anti-HS monoclonal antibody (anti HS-FITC) or with an FITC-labeled IgM (immunoglobulin M)–isotype control antibody (Iso-FITC). (B) Heparinase III treatment drastically reduces GzmB binding to K562 cells. Bar shading indicates same treatments as in panel A. (C) In vitro apoptosis induction is not impaired by heparinase III treatment. K562 cells, treated as described for panel A, were subjected to GzmB-SLO–induced apoptosis. The cells were treated as indicated either with buffer or with heparinase III and thereafter were incubated with the indicated concentrations of recombinant GzmB without SLO or with sublytic concentrations of SLO. After 5-hour treatment, apoptosis was determined by FACS after staining with annexin V–FITC and PI. Shown is the mean ± SD of triplicates of the percentage of annexin V–positive cells encompassing early apoptotic cells (PI-negative) and late-stage apoptotic cells (PI-positive). (D) K562 cells were treated as described in panel C, but cells were analyzed after consecutive time points. Shown is the percentage of apoptotic cells (annexin V–positive and PI-negative). Similarly when necrotic cells were included, no significant differences between buffer and heparinase-treated cells were observed (data not shown). ▦ indicates SLO alone; ▪, SLO with heparinase. (E) Inhibition of GzmB-Bio binding to K562 (▦) or HL-60 (▪) cells by enzymatically inactive GzmK (GzmK-FPR-cmk). Active GzmK and chemical irreversibly inactivated GzmK-FPR-cmk (both 25 μg/mL) totally abolished GzmB-Bio (10 μg/mL) binding. (F) Apoptosis induced by GzmB delivered by SLO is not inhibited when GzmB-binding is blocked. The experiment was performed as described for panel C, with K562 cells preincubated with GzmK-FPR-cmk. ▪ indicates presence, ▦ absence of SLO. N.d. indicates not determined.

Influence of surface heparan sulfate on GzmB binding and apoptosis induction. (A-C) K562 cells were treated with heparinase III and analyzed for cell surface HS, GzmB-Bio binding, and susceptibility to apoptosis induction. (A) Binding of anti–HS-FITC is reduced to background levels after heparinase III treatment. Buffer-treated (▦) or heparinase-treated (▪) cells were stained in triplicate with the FITC-labeled anti-HS monoclonal antibody (anti HS-FITC) or with an FITC-labeled IgM (immunoglobulin M)–isotype control antibody (Iso-FITC). (B) Heparinase III treatment drastically reduces GzmB binding to K562 cells. Bar shading indicates same treatments as in panel A. (C) In vitro apoptosis induction is not impaired by heparinase III treatment. K562 cells, treated as described for panel A, were subjected to GzmB-SLO–induced apoptosis. The cells were treated as indicated either with buffer or with heparinase III and thereafter were incubated with the indicated concentrations of recombinant GzmB without SLO or with sublytic concentrations of SLO. After 5-hour treatment, apoptosis was determined by FACS after staining with annexin V–FITC and PI. Shown is the mean ± SD of triplicates of the percentage of annexin V–positive cells encompassing early apoptotic cells (PI-negative) and late-stage apoptotic cells (PI-positive). (D) K562 cells were treated as described in panel C, but cells were analyzed after consecutive time points. Shown is the percentage of apoptotic cells (annexin V–positive and PI-negative). Similarly when necrotic cells were included, no significant differences between buffer and heparinase-treated cells were observed (data not shown). ▦ indicates SLO alone; ▪, SLO with heparinase. (E) Inhibition of GzmB-Bio binding to K562 (▦) or HL-60 (▪) cells by enzymatically inactive GzmK (GzmK-FPR-cmk). Active GzmK and chemical irreversibly inactivated GzmK-FPR-cmk (both 25 μg/mL) totally abolished GzmB-Bio (10 μg/mL) binding. (F) Apoptosis induced by GzmB delivered by SLO is not inhibited when GzmB-binding is blocked. The experiment was performed as described for panel C, with K562 cells preincubated with GzmK-FPR-cmk. ▪ indicates presence, ▦ absence of SLO. N.d. indicates not determined.

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