Gene expression, synthesis, and secretion of interleukin 18 and interleukin 1beta are differentially regulated in human blood mononuclear cells and mouse spleen cells

Abstract

Interleukin (IL)-18, formerly called interferon gamma (IFN-gamma)-inducing factor, is biologically and structurally related to IL-1beta. A comparison of gene expression, synthesis, and processing of IL-18 with that of IL-1beta was made in human peripheral blood mononuclear cells (PBMCs) and in human whole blood. Similar to IL-1beta, the precursor for IL-18 requires processing by caspase 1. In PBMCs, mature but not precursor IL-18 induces IFN-gamma; in whole human blood stimulated with endotoxin, inhibition of caspase 1 reduces IFN-gamma production by an IL-1beta-independent mechanism. Unlike the precursor for IL-1beta, precursor for IL-18 was expressed constitutively in PBMCs and in fresh whole blood from healthy human donors. Western blotting of endotoxin-stimulated PBMCs revealed processed IL-1beta in the supernatants via an caspase 1-dependent pathway. However, in the same supernatants, only unprocessed precursor IL-18 was found. Unexpectedly, precursor IL-18 was found in freshly obtained PBMCs and constitutive IL-18 gene expression was present in whole blood of healthy donors, whereas constitutive IL-1beta gene expression is absent. Similar to human PBMCs, mouse spleen cells also constitutively contained the preformed precursor for IL-18 and expressed steady-state IL-18 mRNA, but there was no IL-1beta protein and no spontaneous gene expression for IL-1beta in these same preparations. We conclude that although IL-18 and IL-1beta are likely members of the same family, constitutive gene expression, synthesis, and processing are different for the two cytokines.

Figure 1

Effect of IL-18 on IFN-γ induction. (A) Enhancing effect of mature IL-18 (10 nM) on phytohemagglutinin- (10 μg/ml) induced production of IFN-γ in human PBMCs. ProIL-18 was also added at 10nM. PBMCs were stimulated for 72 h, and IFN-γ was assayed on supernatants by ECL assay. Data are the mean ± SEM (n = 3). (B) Effect of reversible ICE inhibitor (ICEi) and IL-1Ra on LPS-induced IFN-γ production in whole human blood. Whole blood was stimulated with LPS (10 ng/ml) in the presence of ICEi (10 μM) or IL-1Ra (10 μg/ml) and after 24 h, the concentration of IFN-γ was measured in the whole blood after lysis in 0.5% Triton X-100. Data are the mean ± SEM (n = 3). A paired Student’s t test was used to assess significance between LPS and LPS + ICEi.

Figure 2

Effect of ICE inhibitor on IL-1β and IL-18 processing and release. PBMCs from two separate donors were stimulated with LPS (100 ng/ml) in the presence or absence of 10 μM ICE inhibitor (ICEi). The letter C above the lanes indicates the unstimulated control PBMCs. After 24 h at 37°C, the supernatants were removed and concentrated in a Centricon device, electrophoresed in two separate gels, and blotted (in duplicate). (A) Western blotting with anti-human IL-1β. (B) Duplicate blot to that in A but developed with anti-human IL-18. (C) Cell lysates from the same cultures used in A but developed with anti-human IL-1β. (D) Same cultures as used in A but developed with anti-human IL-18. Molecular mass makers (in kDa) are indicated by arrows on left side of figure. r-pIL-18 and r-mIL-18 indicate recombinant pro- and mature IL-18, respectively.

Figure 3

Detection of IL-18 in freshly isolated human PBMCs. Whole blood from three donors was obtained and PBMCs were isolated. Without further processing, cells were counted and centrifuged at 250 × g, and lysis buffer was immediately added. The lysates were subjected to SDS/PAGE with 15% gels and Western blotting with horseradish peroxidase-conjugated anti-rabbit IgG. Recombinant human pro- and mature IL-18 are indicated as in Fig. 2. Each lane represents a different human donor.

Figure 4

Differential presence of steady-state IL-18 and IL-1β mRNA by reverse transcription-coupled PCR. Whole blood and PBMCs were obtained from two donors. Lanes 1 and 2 depict the IL-18 PCR product in unstimulated PBMCs immediately after separation from the whole blood of two donors. Lanes 3 and 4 show the IL-18 PCR product in whole blood from the same donors. Lanes 5 and 6 show the IL-1β PCR product in unstimulated PBMCs immediately after separation from the whole blood of two donors. Lanes 7 and 8 show the IL-1β PCR product in whole blood from the same donors. Lane 9 is the water control.

Figure 5

Differential expression of IL-18 and IL-1β in mouse spleen. Spleens from three C57BL6 mice were removed. (A) IL-18 as assessed by Western blotting. The molecular mass markers (in kDa) of murine proIL-18 (pIL-18) or mature (mIL-18) are shown. Under the bracket labeled control, each lane represents spleen cells from a different mouse. Under the bracket labeled LPS, the mice were injected 2 h previously with LPS (100 μg). (B) Gene expression for IL-1β and IL-18 under conditions identical to those in A.

Figure 6

Effect of IFN-γ on time course of LPS-induced expression of IL-12 and IL-18 (A) IL-12. PBMCs were stimulated with LPS (100 ng/ml) with and without IFN-γ (250 units/ml). Lanes: 1, unstimulated PBMCs; 2, IFN-γ; 3 and 6, after 1 h; 4 and 7, after 3 h; 5 and 8, after 6 h. (B) IL-18. Lanes: 1, unstimulated PBMCs; 2 and 6, after 1 h; 3 and 7, after 3 h; 4 and 8, after 6 h; 5 and 9, after 24 h.