The anti-inflammatory effects of interleukin-4 are not mediated by suppressor of cytokine signalling-1 (SOCS1)

Abstract

While it is known that the anti-inflammatory effects of interleukin (IL)-4 require new protein synthesis, the exact mechanisms by which IL-4 suppresses the production of pro-inflammatory cytokines by human monocytes and macrophages is unclear. IL-4 rapidly induced suppressor of cytokine signalling-1 (SOCS1) mRNA and protein, which peaked at 60 min, much earlier than lipopolysaccharide (LPS)-induced SOCS1 mRNA and protein which were consistently maximal 4 hr post-exposure. SOCS1 is a molecule generally considered to be induced for negative feedback of inflammatory processes. We investigated whether the early induction of SOCS1 by IL-4 was responsible for the suppression of LPS-induced tumour necrosis factor (TNF)-alpha production by IL-4. IL-4 suppressed LPS-induced TNF-alpha in freshly isolated monocytes at the level of transcription but acted by a different, possibly translational, mechanism in monocytes cultured overnight in macrophage colony-stimulating factor (M-CSF). Despite different modes of regulation by IL-4, the kinetics and magnitude of induction of SOCS1 mRNA and protein by IL-4 in the two cell types were identical. There was no significant difference in the suppression by IL-4 of LPS-induced TNF-alpha production by bone-marrow derived macrophages from wild-type mice, Ifngamma(-/-) mice and mice lacking SOCS1 (Socs1(-/-)Ifngamma(-/-)). These data suggest that SOCS1 is not involved in the suppression of LPS-induced TNF-alpha production by IL-4.

Figure 1

Suppression of lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF)-α production by interleukin (IL)-4 in freshly isolated monocytes (Fresh) and monocytes cultured overnight in macrophage colony-stimulating factor (M-CSF) (Overnight). (a) TNF-α protein in supernatants 24 hr after incubation with LPS (500 ng/ml) with or without IL-4 (10 ng/ml). Values are mean + standard error of the mean (SEM) and have been expressed as a ratio to the mRNA levels of ubiquitin-conjugating enzyme E2D2 (UBE2D2); Fresh, n = 12 donors; Overnight, n = 9 donors. (b) Suppression of LPS-induced TNF-α mRNA production by IL-4. Values are mean ± SEM; Fresh, n = 6 donors; Overnight, n = 3 donors. An asterisk indicates significant suppression by IL-4 (P < 0·05).

Figure 2

Effect of interleukin (IL)-4 on tumour necrosis factor (TNF)-α mRNA stability in freshly isolated monocytes. Actinomycin D (ActD; 20 μg/ml) was added 1 hr after stimulation with lipopolysaccharide (LPS) with or without IL-4 and the rate of TNF-α decay was measured using real-time polymerase chain reaction (PCR) [mean ± standard deviation (SD); triplicate samples for each time-point from one representative donor].

Figure 3

Effect of interleukin (IL)-4 on activation of signal transducer and activator of transcription 6 (STAT6) in freshly isolated monocytes (Fresh) and monocytes cultured overnight in macrophage colony-stimulating factor (M-CSF) (Overnight). (a) Activation of STAT6 by phosphorylation determined in the first 2 hr following IL-4 exposure using western blotting. A representative blot from one donor is shown. A quantitative analysis of phosphorylated STAT6 in protein lysates from three separate donors [mean ± standard error of the mean (SEM)], normalized to the amount of α-tubulin, was performed. Values shown are fold changes relative to phosphorylated STAT6 in untreated samples. (b) STAT6 electrophoretic mobility shift assay (EMSA). The effect of IL-4 on STAT6 DNA binding capacity in nuclear lysates to a radiolabelled STAT6 probe was investigated. A representative EMSA from one donor is shown as well as quantitative analysis of STAT6 DNA binding in nuclear extracts from two separate donors. Values shown are fold changes relative to DNA binding in untreated samples.

Figure 4

Induction of suppressor of cytokine signalling-1 (SOCS1) mRNA by interleukin (IL)-4, interferon (IFN)-γ and lipopolysaccharide (LPS). Monocytes were cultured overnight in macrophage colony-stimulating factor (M-CSF) and then treated with IL-4 (10 ng/ml), LPS (500 ng/ml) or IFN-γ (10 ng/ml) for up to 24 hr. SOCS1 mRNA levels were quantified using real-time polymerase chain reaction (PCR) and have been expressed as a ratio to the mRNA levels of ubiquitin-conjugating enzyme E2D2 (UBE2D2) (mean ± standard error of the mean; n = 4 donors). Arrows indicate the time-point of maximal induction.

Figure 5

Induction of suppressor of cytokine signalling-1 (SOCS1) protein by interleukin (IL)-4, interferon (IFN)-γ and lipopolysaccharide (LPS). Monocytes were cultured overnight in macrophage colony-stimulating factor (M-CSF) and then treated with IL-4 (10 ng/ml), LPS (500 ng/ml) or IFN-γ (10 ng/ml) for up 4 hr. SOCS1 and α-tubulin protein levels were measured using western blotting. Results of a quantitative analysis are shown upon normalization to the total amount of α-tubulin.

Figure 6

Comparison of the induction of suppressor of cytokine signalling-1 (SOCS1) mRNA and protein in freshly isolated monocytes (Fresh) and monocytes cultured overnight in macrophage colony-stimulating factor (M-CSF) (Overnight). (a) Kinetics of induction of SOCS1 mRNA by interleukin (IL)-4 (10 ng/ml), interferon (IFN)-γ (10 ng/ml) and lipopolysaccharide (LPS) (500 ng/ml) in fresh and overnight cultured monocytes. Values have been expressed as a ratio to the mRNA levels of ubiquitin-conjugating enzyme E2D2 (UBE2D2), (mean ± standard error of the mean; n = 4 donors). (b) Kinetics of induction of SOCS1 protein by IL-4 in fresh and overnight cultured monocytes. Shown is a representative blot from one donor.

Figure 7

Effect of interleukin (IL)-4 on lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF)-α production by bone marrow-derived macrophages (BMMs) from wild-type, Ifnγ^−/− and Socs1^−/−Ifnγ^−/− mice. BMMs were incubated for 24 hr with IL-4 (10 ng/ml), LPS (100 ng/ml) or LPS plus IL-4 (mean + standard error of the mean; n = 3 individual mice). An asterisk indicates significant suppression relative to LPS-induced levels (P < 0·05).

Figure 8

Nuclear factor (NF)-κB electrophoretic mobility shift assay (EMSA). The effect of interleukin (IL)-4 on lipopolysaccharide (LPS) activation of NF-κB was investigated. Nuclear lysates were prepared from freshly isolated monocytes (Fresh) and monocytes cultured overnight in macrophage colony-stimulating factor (M-CSF) (Overnight), cultured for up to 120 min with LPS (500 ng/ml) with or without IL-4 (10 ng/ml). (a) Representative EMSA. (b) Cumulative results from three donors (mean ± standard error of the mean).