IL-18 binding protein increases spontaneous and IL-1-induced prostaglandin production via inhibition of IFN-gamma

Abstract

IL-18 shares with IL-1 the same family of receptors and several identical signal transduction pathways. Because of these similarities, IL-18 was investigated for its ability to induce prostaglandin E(2) (PGE(2)) synthesis in human peripheral blood mononuclear cells (PBMC), a prominent, proinflammatory property of IL-1. IL-18 was highly active in PBMC by inducing the synthesis of the chemokine IL-8; however, no induction of PGE(2) synthesis nor cyclooxygenase type-2 gene expression was observed in PBMC stimulated with IL-18. In the same cultures, IL-1beta induced a 12-fold increase in PGE(2). Although IL-1beta-induced IL-8 synthesis was augmented 3-fold by IL-18, IL-18 suppressed IL-1beta-induced PGE(2) production by 40%. The suppressive effect of IL-18 on PGE(2) production was mediated by interferon (IFN)-gamma because anti-human IFN-gamma-antibody prevented IL-18-induced reduction in PGE(2). Consistent with these observations, IL-12, a known inducer of IFN-gamma, augmented IL-1beta-induced IFN-gamma but suppressed IL-1beta-induced PGE(2) by 75%. IL-18 binding protein (IL-18BP) is a naturally occurring and specific inhibitor of IL-18. When recombinant IL-18BP was added to PBMC cultures, unexpectedly, spontaneous PGE(2) production increased. PGE(2) production was also increased by the addition of IL-18BP to PBMC stimulated with either IL-1beta or IL-12 and also in whole blood cultures stimulated with Staphylococcus epidermidis. These studies demonstrate that IL-18BP decreases endogenous IL-18 activity by reducing IFN-gamma-mediated responses.

Figure 1

Production of PGE₂ by PBMC in response to IL-1β or IL-18. PBMC were cultured at 5 × 10⁶ cells/ml in 1 ml of RPMI containing IL-12 (5 ng/ml) and were incubated for 48 hr in the presence or absence of IL-1β (100 ng/ml) or IL-18 (5 nM). The data represent the fold-change ±SEM over the level of spontaneous PGE₂ production in unstimulated cells (set at 1). n = 3 donors (*, P < 0.01). Inset depicts the RT-PCR-amplified mRNA of COX-2 and GAPDH from PBMC stimulated with IL-1β (100 ng/ml) or IL-18 (5 nM) and rotated at 7 rpm (Rollerdrum, New Brunswick Scientific) for 6 hr at 37°C. RT-PCR data represent one of three similar experiments.

Figure 2

The effect of IL-18 and IL-18BP on IL-1β-induced PGE₂ and IFN-γ production in PBMC. PBMC were cultured in flat bottom wells at 5 × 10⁶ cells/ml in RPMI containing IL-12 (5 ng/ml) in the presence or absence of IL-1β (100 ng/ml). In addition, PBMC were simultaneously stimulated with either IL-18 (5 nM) or IL-18BP (100 ng/ml). After 48 hr, levels of IFN-γ (A) and PGE₂ (B) were measured. The data represent the fold-increase ±SEM over that of the spontaneous production in unstimulated cells (set at 1). n = 8 donors. (A) *, P < 0.05 compared with IL-1β only. (B) *, P < 0.05 compared with control; **, P < 0.01 compared with the combination of IL-1β plus IL-18.

Figure 3

Effect of anti-IFN-γ antibody on suppression of IL-1β-induced PGE₂ production by IL-18. (A) PBMC from three donors were cultured in flat bottom wells at 5 × 10⁶ cells/ml in RPMI containing IL-12 (5 ng/ml) in the absence or presence of IL-1β (100 ng/ml), IL-18 (5 nM), or the combination of these plus anti-IFN-γ antibody (1:1,000). (B) PBMC from the same donors were incubated with IL-1β (100 ng/ml), IFN-γ (30 units/ml), or the combination of these plus anti-IFN-γ antibody (1:1,000). PGE₂ data represent the fold-change ±SEM over that of the production in IL-1β-stimulated cells (set at 1). *, P < 0.05 as calculated by paired t test compared with levels from unstimulated cells.

Figure 4

The effect of IL-12 on IL-1β-induced PGE₂, IL-8, and IFN-γ production in PBMC. PBMC from five donors were cultured in flat bottom wells at 5 × 10⁶ cells/ml in RPMI and were incubated with either IL-1β (100 ng/ml), IL-12 (5 ng/ml), or the combination of these. After 48 hr, IFN-γ (A), PGE₂ (B), and IL-8 (C) were measured. The data represent the fold-change ±SEM over that of the production in IL-1β-stimulated cells (set at 1). Statistical significance was calculated by paired t test.

Figure 5

Effect of IL-18BP on IL-12-induced IFN-γ production in PBMC. PBMC from three donors were cultured in flat bottom wells at 5 × 10⁶ cells/ml in RPMI and were incubated in the absence or presence of 0, 10, 50, 100, or 200 ng/ml of IL-12 (unbroken line). The broken line represents cultures incubated with IL-12 plus 100 ng/ml of IL-18BP. IFN-γ levels represent the fold-change ±SEM over that of the production in unstimulated cells (control, set at 1).

Figure 6

Effect of IL-12 and IL-18BP on spontaneous production of PGE₂. PBMC from 4 donors were cultured in flat bottom wells at 5 × 10⁶ cells/ml in RPMI and were incubated in the absence or presence of IL-12 (5 ng/ml) or IL-18BP (100 ng/ml). PGE₂ production represents the fold-change ±SEM over that in unstimulated cells (set at 1). *, P < 0.05 as calculated by paired t test.

Figure 7

Effect of IL-18BP on PGE₂ production in whole blood cultures. Whole blood was obtained from three donors, and 0.5 ml was aliquoted into 75-mm polypropylene tubes to which was added a suspension of S. epidermidis (0.5 ml of RPMI). After 24 hr at 37°C, the cultures were lysed in Triton X-100 (0.5%) and were assayed for PGE₂. IL-18BPa His6-tag was added at the time of stimulation with S. epidermidis, and the concentrations (ng/ml) are indicated under the horizontal axis.