Journal: Immunity

Article Title: Intracellular Interleukin-1 Receptor 2 Binding Prevents Cleavage and Activity of Interleukin-1α, Controlling Necrosis-Induced Sterile Inflammation

doi: 10.1016/j.immuni.2013.01.008

Figure Lengend Snippet: Necrosis-Induced Sterile Inflammation Is Cell Type Specific (A) IL-6 concentrations in conditioned media of macrophages (Macs), Jurkat cells, or VSMCs incubated with lysates from their respective necrotic cells or with IL-1α. (B and C) Immunoblots of IL-1α content and processing in whole cell (WC) or necrotic lysates (NL) (B) or in necrotic cell lysates pretreated with protease inhibitors as indicated (C). (D and E) IL-6 and MCP-1 concentrations in conditioned media of VSMCs incubated with necrotic VSMC lysates made in the presence of protease inhibitors (D), or with IL-1α alone, or with calpeptin or EGTA (E). Data represent mean ± SD; ∗ p ≤ 0.007 versus control, n = 3; ∗∗ p ≤ 0.03 (MCP-1), p ≤ 0.002 (IL-6), n ≥ 4. NS, not significant. See also Figure S1 .

Article Snippet: Cells were treated as indicated with Calpeptin (30 μM), Lactacystin (10 μM; both Biomol), IL-1α pAb (1 μg/ml), IL-1α and IL-1β (all PeproTech), IL-1RA (Amgen), IL-1R2 (250 ng; R&D), Z-YVAD, Z-VAD-fmk (both 10 μm; Bachem), LPS (1 μg/ml), EGTA (5 mM), and BB-94 (1 μg/ml; Tocris).

Techniques: Sterility, Incubation, Western Blot, Control

Journal: Immunity

Article Title: Intracellular Interleukin-1 Receptor 2 Binding Prevents Cleavage and Activity of Interleukin-1α, Controlling Necrosis-Induced Sterile Inflammation

doi: 10.1016/j.immuni.2013.01.008

Figure Lengend Snippet: Silencing of IL-1R2 Enables IL-1α Processing and Restores Necrotic Cell-Induced Inflammation (A–C) siRNA-mediated silencing reduces IL-1R2 mRNA (A) and protein in whole cell (WC) and necrotic lysates (NL) (B), which leads to calpain-dependent IL-1α cleavage upon necrosis (C) that is inhibited with calpeptin (+C). (D and E) IL-2 concentrations in conditioned media of EL4 cells incubated with necrotic lysates from HeLa cells treated with siRNA to IL-1R2 or scrambled, either alone (D) or with IL-1β treatment (E), ± α pAb. Data represent mean ± SD; ∗ p = 0.0007, n = 3 independent silencings and treatments. NS, not significant. (F) GR1 + cells recruited intraperitoneally in wild-type or Il1r1 −/− mice injected with saline or 8.3 × 10 5 control or IL-1R2-silenced necrotic HeLa cells. Data represent mean ± SEM; ∗∗ p ≤ 0.006, n ≥ 4. NS, not significant. (G) Immunoblot for IL-1R2 and IL-1α in necrotic lysates of empty vector or soluble IL-1R2-transfected VSMCs. See also Figure S4 .

Article Snippet: Cells were treated as indicated with Calpeptin (30 μM), Lactacystin (10 μM; both Biomol), IL-1α pAb (1 μg/ml), IL-1α and IL-1β (all PeproTech), IL-1RA (Amgen), IL-1R2 (250 ng; R&D), Z-YVAD, Z-VAD-fmk (both 10 μm; Bachem), LPS (1 μg/ml), EGTA (5 mM), and BB-94 (1 μg/ml; Tocris).

Techniques: Incubation, Injection, Saline, Control, Western Blot, Plasmid Preparation, Transfection

Journal: Immunity

Article Title: Intracellular Interleukin-1 Receptor 2 Binding Prevents Cleavage and Activity of Interleukin-1α, Controlling Necrosis-Induced Sterile Inflammation

doi: 10.1016/j.immuni.2013.01.008

Figure Lengend Snippet: Caspase-1 Cleavage of IL-1R2 Restores Necrotic Cell-Induced IL-1α-Dependent Inflammation (A) Immunoblot for IL-1α in necrotic lysates from LPS- and ATP-treated macrophages ± calpeptin (during lysis) or ± caspase inhibitor Z-VAD (during LPS and ATP treatment). (B) IL-2 concentrations in conditioned media of EL4 cells incubated with necrotic lysates from control or LPS- and ATP-treated macrophages ± α and/or β pAb, or ± Z-VAD (during LPS and ATP treatment). Data represent mean ± SEM; ∗ p < 0.00004, ∗∗ p ≤ 0.02, n ≥ 3. (C and D) Immunoblot for IL-1R2 cleavage after incubation with active caspase-1 ± Z-YVAD (C) or a panel of active caspases (D). (E) Edman degradation of cleaved IL-1R2 detected two sequences (italic underlined) corresponding to processing at two tetrapeptide sites (large bold). (F) Immunoblot for cleaved IL-1R2 after mutation of both caspase-1 sites. (G) IL-2 concentrations in conditioned media of EL4 cells incubated with IL-1α, and IL-1R2 ± caspase-1 cleavage. Data represent mean ± SD; ∗ p = 0.009, n = 3. (H) IL-1α concentration in conditioned media of activated THP-1 cells transfected with caspase site mutant IL-1R2, or as indicated. Data representative of mean ± SD from n = 2. See also Figure S5 .

Article Snippet: Cells were treated as indicated with Calpeptin (30 μM), Lactacystin (10 μM; both Biomol), IL-1α pAb (1 μg/ml), IL-1α and IL-1β (all PeproTech), IL-1RA (Amgen), IL-1R2 (250 ng; R&D), Z-YVAD, Z-VAD-fmk (both 10 μm; Bachem), LPS (1 μg/ml), EGTA (5 mM), and BB-94 (1 μg/ml; Tocris).

Techniques: Western Blot, Lysis, Incubation, Control, Mutagenesis, Concentration Assay, Transfection