Macrophage migration inhibitory factor counter-regulates dexamethasone-induced annexin 1 expression and influences the release of eicosanoids in murine macrophages

Abstract

Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine and glucocorticoid (GC) counter-regulator, has emerged as an important modulator of inflammatory responses. However, the molecular mechanisms of MIF counter-regulation of GC still remain incomplete. In the present study, we investigated whether MIF mediated the counter-regulation of the anti-inflammatory effect of GC by affecting annexin 1 in RAW 264.7 macrophages. We found that stimulation of RAW 264.7 macrophages with lipopolysaccharide (LPS) resulted in down-regulation of annexin 1, while GC dexamethasone (Dex) or Dex plus LPS led to significant up-regulation of annexin 1 expression. RNA interference-mediated knockdown of intracellular MIF increased annexin 1 expression with or without incubation of Dex, whereas Dex-induced annexin 1 expression was counter-regulated by the exogenous application of recombinant MIF. Moreover, recombinant MIF counter-regulated, in a dose-dependent manner, inhibition of cytosolic phospholipase A2α (cPLA2α) activation and prostaglandin E2 (PGE2 ) and leukotriene B4 (LTB4 ) release by Dex in RAW 264.7 macrophages stimulated with LPS. Endogenous depletion of MIF enhanced the effects of Dex, reflected by further decease of cPLA2α expression and lower PGE2 and LTB4 release in RAW 264.7 macrophages. Based on these data, we suggest that MIF counter-regulates Dex-induced annexin 1 expression, further influencing the activation of cPLA2α and the release of eicosanoids. These findings will add new insights into the mechanisms of MIF counter-regulation of GC.

Keywords: annexin 1; glucocorticoids; leukotriene B4; macrophage migration inhibitory factor; prostaglandin E2.

Figure 1

Macrophage migration inhibitory factor (MIF) overrides the expression of annexin 1 induced by dexamethasone (Dex). (a) RAW 264.7 macrophages were pre-incubated for 2 hr with Dex (100 nm) or with Dex (100 nm) plus MIF (10 ng/ml) before stimulation with lipopolysaccharide (LPS; 100 ng/ml) for 30 min. Expression of annexin 1 was assayed by SDS–PAGE and Western blotting. (b) RAW 264.7 macrophages were incubated for 2 hr with Dex (100 nm) or with Dex (100 nm) plus different concentrations of MIF (10, 100, 1000 nm). Annexin 1 expression of total cell lysates was analysed by SDS–PAGE and Western blotting. Data are representative of three independent experiments (**P < 0·01).

Figure 2

Knockdown of intracellular macrophage migration inhibitory factor (MIF) increases annexin 1 expression induced by dexamethasone (Dex). RAW 264.7 macrophages were transfected with 2·0, 1·0, 0·5 μg/ml vector-based MIF small interfering (si) RNA and control siRNA (Control). MIF mRNA (a) and protein (b) expression were determined by RT-PCR and Western blotting. (c) RAW 264.7 macrophages transfected with MIF siRNA (2·0 μg/ml) and control siRNA were incubated for 2 hr with different concentrations of Dex (10, 100, 1000 nm). Annexin 1 expression of total cell lysates was analysed by Western blotting. Data are representative of three independent experiments (**P < 0·01).

Figure 3

Macrophage migration inhibitory factor (MIF) overrides the inhibitory effect of dexamethasone (Dex) on release of prostaglandin E₂ (PGE₂) and leukotriene B₄ (LTB₄). (a, b) Cells were stimulated with different concentrations of MIF, lipopolysaccharide (LPS) or Dex. (c, d) Cells were pre-incubated for 2 hr with Dex or with Dex plus MIF at the indicated concentrations before stimulation with LPS (100 ng/ml). Cell supernatants were harvested and concentrations of PGE₂ (a, c) and LTB₄ (b, d) were quantified. Data are mean ± SD of three separate experiments. *P < 0·05, **P < 0·01 when compared with stimulation of MIF (0 ng/ml) (a, b). *P < 0·05 when compared with stimulation of LPS after pre-incubation with Dex without MIF (c, d).

Figure 4

Knockdown of intracellular macrophage migration inhibitory factor (MIF) may enhance the inhibitory effect of dexamethasone (Dex) on release of prostaglandin E₂ (PGE₂) and leukotriene B₄ (LTB₄). RAW 264.7 macrophages transfected with MIF small interfering (si) RNA (2·0 μg/ml) and control siRNA were pre-incubated for 2 hr with different concentrations of Dex (1, 10, 100, 1000 nm) before stimulation with LPS (100 ng/ml) for 4 hr. PGE₂ (a) and LTB₄ (b) concentrations in cell culture supernatants were expressed as the relative per cent inhibition of PGE₂ and LTB₄ eicosanoid production, respectively, calculated by the following formula: % inhibition = ([LPS-induced eicosanoid] – [Dex plus LPS-induced eicosanoid])/(LPS-induced eicosanoid) × 100; where LPS is lipopolysaccharide. Closed squares: macrophages transfected with control siRNA. Open circles: macrophages transfected with MIF siRNA (2·0 μg/ml). (c) The efficiency of the MIF siRNA (2·0 μg/ml) knockdown in RAW 264.7 macrophages. Results are mean ± SD of six determinations from three independent experiments. *P < 0·05, ** P < 0·01 when comparing PGE₂ and LTB₄ secretion by MIF siRNA versus control siRNA. con siRNA: control siRNA.

Figure 5

Cytosolic phospholipase A2α (cPLA2α) phosphorylation is involved in macrophage migration inhibitory factor (MIF) counter-regulation of dexamethasone (Dex). (a) RAW 264.7 macrophages were transfected with MIF small interfering (si) RNA (2·0 μg/ml) or control siRNA (con siRNA), or were stimulated with recombinant mouse MIF (10 ng/ml). (b) RAW 264.7 macrophages transfected with MIF siRNA and con siRNA or stimulated with recombinant mouse MIF were stimulated with lipopolysaccharide (LPS; 100 ng/ml) for 30 min. (c) RAW 264.7 macrophages transfected with MIF siRNA and con siRNA or stimulated with recombinant mouse MIF were pre-incubated for 2 hr with Dex (100 nm) before stimulation with LPS (100 ng/ml) for 30 min. MIF, cPLA2α and phospho-cPLA2α (p-cPLA2α) expression of total cell lysates were analysed by Western blotting. Data are representative of three independent experiments. (d, e) Histograms of densitometric analysis (mean ± SD) of p-cPLA2α/ cPLA2α are shown (**P < 0·01).