Interleukin-36 (IL-36) ligands require processing for full agonist (IL-36α, IL-36β, and IL-36γ) or antagonist (IL-36Ra) activity

Abstract

IL-36α, IL-36β, and IL-36γ (formerly IL-1F6, IL-1F8, and IL-1F9) are IL-1 family members that signal through the IL-1 receptor family members IL-1Rrp2 (IL-1RL2) and IL-1RAcP. IL-36Ra (formerly IL-1F5) has been reported to antagonize IL-36γ. However, our previous attempts to demonstrate IL-36Ra antagonism were unsuccessful. Here, we demonstrate that IL-36Ra antagonist activity is dependent upon removal of its N-terminal methionine. IL-36Ra starting at Val-2 is fully active and capable of inhibiting not only IL-36γ but also IL-36α and IL-36β. Val-2 of IL-36Ra lies 9 amino acids N-terminal to an A-X-Asp motif conserved in all IL-1 family members. In further experiments, we show that truncation of IL-36α, IL-36β, and IL-36γ to this same point increased their specific activity by ∼10(3)-10(4)-fold (from EC(50) 1 μg/ml to EC(50) 1 ng/ml). Inhibition of truncated IL-36β activity required ∼10(2)-10(3)-fold excess IL-36Ra, similar to the ratio required for IL-1Ra to inhibit IL-1β. Chimeric receptor experiments demonstrated that the extracellular (but not cytoplasmic) domain of IL-1Rrp2 or IL-1R1 is required for inhibition by their respective natural antagonists. IL-36Ra bound to IL-1Rrp2, and pretreatment of IL-1Rrp2-expressing cells with IL-36Ra prevented IL-36β-mediated co-immunoprecipitation of IL-1Rrp2 with IL-1RAcP. Taken together, these results suggest that the mechanism of IL-36Ra antagonism is analogous to that of IL-1Ra, such that IL-36Ra binds to IL-1Rrp2 and prevents IL-1RAcP recruitment and the formation of a functional signaling complex. In addition, truncation of IL-36α, IL-36β, and IL-36γ dramatically enhances their activity, suggesting that post-translational processing is required for full activity.

FIGURE 1.

Antagonistic activity of IL-36Ra requires removal of its N-terminal methionine. A, Jurkat reporter cells were incubated with a dose titration of huIL-36Ra (starting at 1.18 μm, 1:5 dilutions) expressed in E. coli or COS cells before stimulation with huIL-36γ (used at EC₉₀ ∼ 0.138 μm). B, cells were incubated with a dose titration of huIL-36Ra beginning with Met-1 (M1) or Val-2 (V2) (starting at 1.18 μm, 1:5 dilutions) before stimulation with huIL-36γ (used at EC₉₀ ∼ 0.138 μm). C, cells were incubated with a dose titration of huIL-36Ra(V2) (starting at 2.94 μm, 1:5 dilutions) before stimulation with huIL-36α (used at EC₉₀ ∼ 0.576 μm), huIL-36β (used at EC₉₀ ∼ 0.209 μm), or huIL-36γ (used at EC₉₀ ∼ 0.435 μm). In A–C, all cells were treated with the huIL-36Ra proteins for 15 min before the addition of agonist for 5 h, although in other experiments, we have shown that simultaneous addition of IL-36Ra with the IL-36 agonist is equally effective as preincubation with IL-36Ra prior to agonist addition. IL-8p-luciferase activity was measured and is expressed as a percentage of the luciferase activity in the absence of IL-36Ra (set at 100%). IL-36Ra(V2) IC₅₀ values were similar and ranged from 0.001 to 0.006 μm. Data are means ± S.D. from one experiment (in duplicate or triplicate) that is representative of at least three similar experiments with comparable results.

FIGURE 2.

Truncating IL-36α, IL-36β, and IL-36γ enhances their activity. A, alignment of the protein sequences of human IL-36Ra, IL-36α, IL-36β, and IL-36γ highlighting the A-X-Asp (@XD) motif, the position 9 amino acids N-terminal to it, and the huIL-36γ truncations (positions −8, −9, and −10) generated for activity testing. B, Jurkat reporter cells were stimulated with a dose titration of full-length (FL) huIL-36γ (starting at 2.94 μm, 1:5 dilutions) or huIL-36γ truncated at position −8, −9, or −10 (with respect to the A-X-Asp motif) (huIL-36γ(Q17) starting at 2.94 μm, huIL-36γ(S18) starting at 0.005 μm, and huIL-36γ(M19) starting at 2.94 μm; 1:5 dilutions). C, Jurkat reporter cells were stimulated with titrations of full-length (starting at 2.35 μm) or appropriately truncated (starting at 0.002 μm) huIL-36α, IL-36β, and IL-36γ (1:5 dilutions) for comparison. In B and C, all cells were stimulated with the IL-36 proteins for 5 h before measurement of luciferase activity. Data are expressed as relative light units (R.L.U) and are means ± S.D. from one experiment (in duplicate) that is representative of at least three similar experiments with comparable results.

FIGURE 3.

Large molar excess of IL-36Ra is required to block truncated IL-36 agonist activity. A, Jurkat reporter cells were treated with a dose titration of huIL-36Ra(V2) (starting at 2.94 μm, 1:5 dilutions) for 15 min before the addition of human IL-36α(K6), IL-36β(R5), and IL-36γ(S18) at their EC₉₀ concentrations (IL-36α(K6), ∼0.118 nm; IL-36β(R5), ∼0.019 nm; and IL-36γ(S18), ∼0.294 nm) for 5 h. IL-8p-luciferase activity was measured and is expressed as a percentage of the luciferase activity in the absence of IL-36Ra (set at 100%). IL-36Ra IC₅₀ values were similar for inhibition of IL-36α, IL-36β, and IL-36γ and ranged from 0.003 to 0.006 μm. B, Jurkat cells were transiently transfected with huIL-1R1 and the IL-8p-luciferase reporter. Cells were treated with a dose titration of huIL-1Ra (starting at 0.294 μm, 1:5 dilutions) for 15 min before the addition of huIL-1β at the EC₉₀ concentration (IL-1β, ∼0.647 pm). IL-8p-luciferase activity was measured and is expressed as a percentage of the luciferase activity in the absence of inhibitor. The ratios of IL-1Ra to IL-1β and of IL-36Ra to IL-36 agonist were calculated and are plotted versus the percentage of IL-8p-luciferase activity. The ratios of IL-36Ra(V2) to full-length (FL) and truncated IL-36 agonists were calculated from Fig. 1C and panel A. Data are means ± S.D. from one experiment (in duplicate) that is representative of at least three similar experiments with comparable results.

FIGURE 4.

IL-36Ra blocks IL-36 ligand binding to the IL-1Rrp2 receptor and the subsequent recruitment of IL-1RAcP. A, Jurkat cells were transfected with the IL-8p-luciferase reporter along with various receptor constructs. Cells were treated with 1.18 μm huIL-1Ra or huIL-36Ra(V2) for 15 min before the addition of full-length (FL) huIL-36α (0.059 μm), truncated (Trunc.) huIL-36α (0.006 μm), or huIL-1β (0.059 nm) for 5 h. Luciferase activity was measured and is expressed as relative light units (R.L.U). Data are means ± S.D. from one experiment (in quadruplicate) that is representative of at least three similar experiments with comparable results. B, murine BaF3 cells stably overexpressing muIL-1Rrp2 were pretreated with 0.2, 0.5, 5, or 50 μg/ml muIL-36Ra(V2) or the BSA control for 1 min before the addition of 0.1 μg/ml muIL-36β(S4) for 4 min at 37 °C. Control samples receiving no muIL-36Ra(V2) or BSA were treated with 50 μg/ml full-length muIL-36β, 0.1 μg/ml muIL-36β(S4), or medium alone. muIL-1RAcP was immunoprecipitated from cell lysates overnight with an anti-muIL-1RAcP antibody. Co-immunoprecipitation of IL-1Rrp2 was detected with an anti-muIL-1Rrp2 antibody by Western blotting. Data are representative of at least three similar experiments with comparable results.

FIGURE 5.

IL-36Ra(V2) and IL-36α(K6) can compete with anti-IL-1Rrp2 antibody binding. A, Jurkat reporter cells were preincubated with or without human IL-36α(K6), IL-36Ra(V2), or IL-1Ra for 15 min before staining with anti-huIL-1Rrp2 antibody or anti-FLAG control antibody. B, Jurkat reporter cells were preincubated with or without human IL-36α(K6), IL-36Ra(V2), or IL-1Ra for 15 min before staining with anti-huIL-1RAcP antibody or anti-FLAG control antibody. Data are representative of at least two similar experiments with comparable results. PE, phycoerythrin.