Figure 3. Granzyme B activity does not directly promote inflammatory cytokine production.

(A) Recombinant IL-1α was incubated with granzyme B (200 nM) for 3 h at 37°C. Residual granzyme B activity was then measured after further incubation for 30 min either alone, or in the presence of the granzyme B inhibitor PI-9 (1 mM). Granzyme B activity was measured by monitoring hydrolysis of the synthetic granzyme B substrate IETD-AFC by fluorimetry.

(B) ³⁵S-labeled Bid was incubated, either alone, or with the indicated concentrations of active or PI-9-treated granzyme B for 2 h at 37°C. Reactions were analysed by SDS-PAGE followed by fluorography.

(C) HeLa cells were incubated for 8 h with the indicated concentrations of full-length or granzyme B-cleaved IL-1α, where residual granzyme B activity after proteolysis of IL-1α was inhibited by addition of saturating amounts of PI-9. IL-6 and IL-8 levels in culture supernatants were determined by ELISA.

(D) HeLa cells were incubated for 8 h with the indicated concentrations of IL-1α^FL or IL-1α^104-271. IL-6 and IL-8 levels were determined by ELISA.

(E) HeLa cells were incubated for 8 h with the indicated concentrations of IL-1α^FL, granzyme B-treated IL-1α^FL, IL-1α^104-271, or granzyme B-treated IL-1α^104-271, as indicated. IL-6 and IL-8 levels were determined by ELISA.

Results are representative of at least three independent experiments. Error bars represent the mean ±SEM of determinations from three independent experiments.