Impaired IL-1R1 signaling leads to Inhibition, Nuclear factor kappa B (NF-kB)

The initial step in IL-1 signal transduction is a ligand-induced conformational change in the first extracellular domain of the IL-1RI that facilitates recruitment of IL-1RacP (Cavalli et al., 2015). Through conserved cytosolic regions called Toll- and IL-1R–like (TIR) domains (Radons et al., 2003), the trimeric complex rapidly assembles two intracellular signaling proteins, myeloid differentiation primary response gene 88 (MYD88) and interleukin-1 receptor–activated protein kinase (IRAK) 4 (Brikos et al., 2007; Li et al., 2002). IL-1, IL-1RI, IL-RAcP, MYD88, and IRAK4 form a stable IL-1–induced first signaling module. The binding of MyD88 triggers a cascade of kinases that produce a strong pro-inflammatory signal leading to activation of NF-κB reviewed by (Brikos et al., 2007; Weber, Wasiliew and Kracht, 2010).

IL-1Ra blocks IL-1 signaling:

  IL-1Ra down modulation of EGF receptor (3 nM of ED50) (Dripps et al., 1991)

  IL-1Ra suppression of IL-1-induced endothelial cell-leukocyte adhesion (approximately 10 ng/ml of ED50) (Dripps et al., 1991)

IL-1Ra suppresses rhIL-1a-induced mouse thymocytes proliferation (ED50 almost 3 mg/mL) (Arend et al., 1990)

IL-1Ra competed for binding of ¹²⁵I-IL-1a to type I IL-1R present on EL4 thymoma cells, 3T3 fibroblasts, hepatocytes, and Chinese hamster ovary cells expressing recombinant mouse type I IL-1R. The IC50 values for IL-1ra binding (ranging from 2 to 4 ng/ml) were similar to those of IL-1a. (McIntyre et al., 1991)

Recombinant mIL-1Ra competitively inhibited ¹²⁵I-labeled IL-1 alpha binding to murine type I IL-1R present on EL4 6.1 cells (Ki value of 0.21 nM) and antagonized IL-1-stimulated co-mitogenesis in murine thymocytes (0.7 x 10(6)-1.1 x 10(6) units/mg). (Shuck et al., 1991)

Peripheral blood mononuclear cells (PBMC) obtained after completion of the IL-lra infusion synthesized significantly less interleukin 6 ex vivo than PBMC from saline-injected controls. (Granowitz et al., 1992)

Canakinumab (ACZ885, Ilaris):

Canakinumab binds to human IL-1β with high affinity; the antibody-antigen dissociation equilibrium constant is approximately 35–40 pM(Dhimolea, 2010).

The antibody binds to human IL-1β with high affinity (about 40 pmol/l). The antibody was found to neutralize the bioactivity of human IL-1β on primary human fibroblasts in vitro 44.6 pmol/l (7.1 ± 0.56 ng/ml; n = 6) of ED50. Application of Canakinumab intraperitoneally 2 hours before injecting the IL-1β producing cells completely suppressed joint swelling in mouse models of arthritis (0.06 mg/kg of EC50) (Alten et al., 2008).

Primary human fibroblasts are stimulated with recombinant IL-1b or conditioned medium obtained from LPS-stimulated human PBMCs in the presence of various concentrations of Canakinumab or IL-1RA ranging from 6 to 18,000 pM. Supernatant is taken after 16 h stimulation and assayed for IL-6 by ELISA. Canakinumab typically have 1 nM or less of EC50 for inhibition of IL-6 production (Canakinumab Patent Application WO02/16436.)

Rilonacept (IL-1 Trap, Arcalyst):

Incubation of the human MRC5 fibroblastic cell line with IL-1β induces secretion of IL-6. At a constant amount of IL-1β (4 pM), the IC50 of the IL-1 trap is ∼2 pM. Another unique property of the IL-1 trap is that it not only blocks IL-1β, but also blocks IL-1α with high affinity (KD = ∼3 pM; data not shown). The titration curve of IL-1 trap in the presence of 10 pM IL-1β shows an IC50 of 6.5 pM, which corresponds to a calculated KD of 1.5 pM (This affinity is 100 times higher than that of the soluble single component receptor IL-1RI (Economides et al., 2003).

IRAK4 inhibitor:

By reconstituting IRAK-4-deficient cells with wild type or kinase-inactive IRAK-4, it is demonstrated that the kinase activity of IRAK-4 is required for the optimal transduction of IL-1-induced signals, including the activation of IRAK-1, NF-κB, and JNK, and the maximal induction of inflammatory cytokines (Lye et al., 2008).

Various concentrations of kinase-active or kinase-inactive IRAK-4 were transiently overexpressed in IRAK-4-deficient cells that were also transiently transfected with an NF-κB-dependent luciferase reporter and α-galactosidase expression vector.  IRAK-4 is recruited to the IL-1R-associated complex 1 min after IL-1β treatment (10 ng/mL). Transfected cells were left untreated or treated with IL-1β (10 ng/ml) for 6 h before luciferase and α-galactosidase activities were measured. The luciferase activity was divided by the α-galactosidase activity, and fold activation was calculated compared with the activity of untreated cells carrying an empty α-vector (normalized as 1). The results demonstrated that kinase-active IRAK-4 dose dependently activates IL-1-mediated NF-κB. Kinase-inactive IRAK-4 expression resulted in severely reduced IL-1 responses and defective NF-κB and JNK activation induced by IL-1 (Lye et al., 2004).

IL-1Ra blocks IL-1 signaling:

Suppression of IL-1-induced IL-1, TNFa, or IL-6 synthesis was dose-dependent (P ≦ .0001). At a twofold molar excess, IL-lra inhibited IL-1-induced IL-1 or TNFa synthesis by 50% (P < .01); an equimolar concentration of IL-lra inhibited synthesis of these two cytokines by over 20% (P < .05). A 10-fold molar excess of IL-lra over IL-lb reduced IL-lb-induced IL-la by 95% (P = .01) and IL-la-induced IL-1b by 73% (P < .01). In elutriated monocytes, a 10-fold molar excess of IL-lra reduced IL-lb-induced IL-la by 82% (P < .05), TNFa by 64% (P = .05), and IL-6 by 47% (P < .05). (Granowitz et al., 1992)

Rilonacept (IL-1 Trap, Arcalyst) blocks IL-1 signaling:

The titration curve of IL-1 trap in the presence of 10 pM IL-1β shows an IC50 of 6.5 pM, which corresponds to a calculated KD of 1.5 pM (This affinity is 100 times higher than that of the soluble single component receptor IL-1RI (Economides et al., 2003).