Fig. 1: IL-36 cytokines require proteolytic processing for activation.

a HeLa^IL-36R cells were either untreated, or were stimulated with full-length IL-36α, IL-36β, or IL-36γ, at the indicated concentrations. After 24 h, cytokine concentrations in the culture supernatants were determined by ELISA. Caspase-3-cleaved DEVD-IL-36γ (0.5 nM), where a caspase-3-processing motif (DEVD) was inserted into the IL-36 sequence N-terminal to the known processing sites^12,36, was used as a positive control. b HeLa^IL-36R cells were stimulated either alone, or in the presence of either IL36α, β, or γ (500 pM) that had been pre-incubated for 2 h at 37 °C with unstimulated neutrophil supernatant (Ctrl s/n), or 1:2 serial dilutions of PMA-activated neutrophil supernatants (PMA s/n). After 24 h, cytokine concentrations in the culture supernatants were determined by ELISA. c HeLa^IL-36R cells were stimulated either alone, or with IL36α, β, or γ (500 pM) that had been pre-incubated for 2 h at 37 °C with the indicated concentrations of purified human cathepsin G, elastase, or proteinase-3. After 24 h, cytokine concentrations in the culture supernatants were determined by ELISA. d Schematic representation of cathepsin G, elastase, and proteinase-3 cleavage motifs within IL-36 family cytokines. Results shown are representative of at least three independent experiments. Error bars represent the mean ±SEM of triplicate determinations from a representative experiment