The Interleukin-1α Precursor is Biologically Active and is Likely a Key Alarmin in the IL-1 Family of Cytokines

Abstract

Among the 11 members of the IL-1 family cytokines, the precursors of IL-1α, IL-1β, and IL-33 have relatively long N-terminal pro-sequences of approximately 100 amino acid residues prior to the N-terminus of the mature forms. Compared to the mature forms secreted from the cell, 80-90% of the primary translation product is in the intracellular compartment in the precursor form. However, the precursors are readily released from cells during infections but also with non-infectious conditions such a hypoxia and trauma. In this setting, the precursors act rapidly as "alarmins" in the absence of a processing mechanism to remove the pro-sequence and generate a mature form. In the case of IL-1α, the release of the precursor activates adjacent cells via receptor-mediated signaling. However, there are no data comparing the specific activity of the IL-1α precursor to the mature form. In the present study, we compared the precursor and mature forms of recombinant human IL-1α, IL-1β, and IL-33 proteins on the induction of cytokines from A549 cells as well as from human peripheral blood mononuclear cells (PBMC). Similar to the mature form, the IL-1α precursor was active in inducing IL-6 and TNFα, whereas the precursor forms of IL-1β and IL-33 were not active. On PBMC, precursor and mature IL-1α at 0.04 and 0.2 nM were equally active in inducing IL-6. Given the fact that during necrotic cell death, the IL-1α precursor is released intact and triggers IL-1 receptors on tissue macrophages, these data identify the precursor form of IL-1α as a key player in sterile inflammation.

Keywords: DAMPs; IL-1 family cytokine; precursor IL-1α; recombinant protein; sterile inflammation.

Figure 1

Expression and purification of recombinant cytokines. Six different recombinant cytokine proteins were expressed in E. coli as indicated. Precursor (pro) IL-1α and mature (mat) IL-1α (200 ng/lane) in left panel, proIL-1β and mature IL-1β (100 ng/lane) in middle panel, and proIL-33 and mature IL-33 (500 ng/lane) were purified by affinity chromatography and then subjected to 10% SDS-PAGE. The purity of each recombinant protein was visualized by silver staining. The data represents one of three independent experiments.

Figure 2

Comparison of precursor and mature forms of IL-1α and IL-1β on IL-6 production. (A) Mean ± SEM IL-6 supernatant levels induced in A549 cells after 24 h of incubated with IL-1α precursor or mature forms and (B) by IL-1β precursor or mature forms at concentrations indicated under the horizontal axis. Inset in (B) shows the RT-PCR product of IL-1R1 and IL-1R3 in A549 cells. (C) Mean ± SEM IL-6 supernatant levels from PBMCs after 24 h of incubation with IL-1α forms and (D) by IL-1β at concentrations indicated under the horizontal axis. Inset in (D) shows PCR product of IL-1R1 and IL-1R3 in PBMCs. *p < 0.05; **p < 0.01; ***p < 0.001 compared to control (cont). RT-PCR products shown are representative of one of two independent experiments. IL-6 data from A549 cells shown are one of two independent experiments performed in triplicate. PBMC data are representative of one of two donors performed in triplicate.

Figure 3

Inhibition of IL-1 induced IL-6 by precursor and mature forms by anti-IL-1α antibody. Anti-IL-1α antibody was first incubated with either precursor or mature form of IL-1α or IL-1β before being added to cells. The molar ratio (MR) of antibody to ligand is indicated under the horizontal axis. (A) Mean ± SEM IL-6 supernatant levels induced in A549 cells after 24 h of incubated with IL-1α forms and (B) by IL-1β forms. (C) Mean ± SEM IL-6 supernatant levels from PBMCs after 24 h of incubation with IL-1α forms and (D) with IL-1β forms. *p < 0.05; **p < 0.01; ***p < 0.001 compared to control (cont). IL-6 data from A549 cells shown are one of two independent experiments performed in triplicate. PBMC data are representative of one of two donors performed in triplicate.

Figure 4

IL-1α and IL-1β-mediated regulation of human TNFα in PBMCs. (A) The cell culture supernatants of PBMCs treated with precursor or mature recombinant IL-1α from Figure 2C and (B) precursor or mature recombinant IL-1β from Figure 2D were used for TNFα levels. Concentrations of the recombinant cytokines are indicated under the horizontal axis. (C) The inhibition of anti-IL-1α in precursor or mature recombinant IL-1α-induced TNFα levels (D) that of precursor or mature recombinant IL-1β-induced TNFα levels were measured in supernatants from Figures 3C,D, respectively. Data are mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001 compared to control (cont). PBMC data are representative of one of two donors performed in triplicate.

Figure 5

IL-1Ra inhibition of precursor and mature forms of IL-1α and IL-1β activity. Prior to stimulation with IL-1 (0.5 nM), increasing concentrations of IL-1Ra were added to cells and are indicated as molar excess of IL-1Ra to IL-1 under the horizontal axis as molar ratio (MR). A549 cells were stimulated with 0.5 nM of precursor or mature recombinant IL-1α (A) and 0.5 nM of precursor or mature recombinant IL-1β (B) in the presence of absence of IL-1Ra. Similar experiments of human PBMCs were performed with 0.5 nM of precursor or mature recombinant IL-1α (C) and 0.5 nM of precursor or mature recombinant IL-1β (D). IL-6 levels in the supernatants represent mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001) compared to control (cont). IL-6 data from A549 cells shown are one of two independent experiments performed in triplicate. PBMC data are representative of one of two donors performed in triplicate.

Figure 6

Inhibition of IL-1α or IL-1β induced TNFα by IL-1Ra. Prior to stimulation with IL-1α or IL-1β, increasing concentrations of IL-1Ra were added to cells indicated as molar excess of IL-1Ra to IL-1 under the horizontal axis as molar ratio (MR). PBMC cells were stimulated with 0.5 nM of precursor or mature recombinant IL-1α (A) and in (B) 1.0 nM of precursor or mature recombinant IL-1β. TNFα levels in the supernatants represent mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001) compared to control (cont). PBMC data are representative of one of two donors performed in triplicate.

Figure 7

Mean ± SEM levels of IL-8 released from human HMC-1 cells. HMC-1 cells were stimulated for 24 h with increasing concentrations of either mature or precursor form of IL-33 as indicated under the horizontal axis (*p < 0.05; ***p < 0.001). Representative data from one of two independent experiments performed in triplicate are shown.