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. 2013 Sep 12;140(2):250–258. doi: 10.1111/imm.12135

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

Yu Sun 1,*, Yu Wang 2,*, Jia-Hui Li 1,*, Shi-Hui Zhu 1, Hong-Tai Tang 1, Zhao-Fan Xia 1
PMCID: PMC3784171  PMID: 23777345

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

Introduction

Macrophage migration inhibitory factor (MIF) is a ubiquitously expressed pleiotropic cytokine that regulates the innate and adaptive immune responses. In the past, various studies repeatedly demonstrated that MIF could mediate disease-exacerbating effects and play a key role in the pathogenesis of chronic and acute inflammatory diseases, such as atherosclerosis, rheumatoid arthritis, sepsis, asthma and acute respiratory distress syndrome.1 Mechanisms underlying MIF target cell action were recently uncovered when CD74 was identified as a MIF receptor and when MIF co-receptors (CXC chemokine receptors, CXCR2 and CXCR4) were subsequently discovered.2–4 Further insights into MIF's role in inflammation were provided by the observations that its secretion by macrophages was induced by low-dose glucocorticoids (GCs) and that it counteracted the inhibitory effects of GCs on macrophage cytokine secretion,5 which opens the gate for investigating the interaction between MIF and GCs in inflammatory diseases.6

Glucocorticoids are well known as potent anti-inflammatory and immunosuppressive agents.7 In contrast to other pro-inflammatory cytokines that are generally suppressed by GCs, MIF expression and secretion are increased in response to physiological concentrations of GC. Synthesis and secretion of MIF are regulated by GCs in a bell-shaped, dose-dependent manner, such that the peak of MIF release is achieved by concentrations of GC from 10−14 to 10−12 m; and decreased at higher concentrations (10−8 to 10−6 m).5 Similar findings were reported in the human CEM T-cell line in addition to previously clarified monocytes/macrophages.8 Moreover, the pro-inflammatory effects of MIF have been shown to override the anti-inflammatory effects of GC, both in vitro and in vivo, probably causing GC resistance in the treatment of common inflammatory diseases. Recent studies demonstrated that MIF may play a role in the development of GC resistance in patients with rheumatoid arthritis, systemic lupus erythematosus9 and atherosclerosis,10 and that endogenous MIF can attenuate GC sensitivity through several signalling pathways, which involve the p38 mitogen-activated protein kinase (MAPK) pathway11,12 and the nuclear factor-κB/IκB cascade.9,13 However, how MIF exerts its counter-regulatory effects of GCs has not been fully elucidated.

The anti-inflammatory effects of GCs present with down-regulation of pro-inflammatory gene expression and/or up-regulation of certain gene products including annexin 1.14 Annexin 1 is a member of the membrane calcium protein family with molecular weight of 37 000.15 It is highly expressed in the cytoplasm of human and rat neutrophils, mononuclear cells and macrophages. Annexin 1 can be rapidly mobilized to the surface for secretion as cells are activated.16 Some research has shown that annexin 1 can inhibit the inflammatory response:17–20 arthritis has been found in annexin 1 knockout mice, accompanied by increased expression of interleukin-1 and interleukin-6; interleukin-1β secretion and leucocyte migration were significantly increased in an annexin 1 knockout mouse model of inflammation, suggesting that annexin 1 is closely related to the inflammatory response.21 It has been demonstrated that annexin 1 is responsible for the anti-inflammatory actions of GC;22,23 it functions as a negative regulator of cytosolic phospholipase A2α (cPLA2α) in cellular signal transduction. Inhibition of cPLA2α activity directly by annexin 1 blocks the release of arachidonic acid and its subsequent conversion to eicosanoids (i.e. prostaglandins, thromboxanes, prostacyclins and leukotrienes).24 Mice lacking annexin 1 have elevated levels of cPLA2α, an exaggerated inflammatory response, and partial resistance to the anti-inflammatory action of glucocorticoids.25–27 Previous findings reported by Mitchell et al. demonstrated that MIF could regulate cPLA2 activity via a protein kinase A and extracellular signal-regulated kinase-dependent pathway and that the GC suppression of cytokine-induced cPLA2 activity and arachidonic acid release could be reversed by the addition of recombinant MIF,28 indicating that MIF is also closely related to cPLA2 besides the interaction between annexin 1 and cPLA2.

Given the effects of MIF on GC and the effects of GC on annexin 1, in present study we focused on the role of annexin 1 in the MIF-induced counter-regulation of the GC anti-inflammatory effect. Our results indicate that MIF counter-regulates dexamethasone (Dex) -induced annexin 1 expression, further influencing the activation of cPLA2α and the release of eicosanoids in macrophages, which supports the possibility that over-expression of annexin 1 in cells could partially block the counter-regulatory effects of MIF on GCs, which may enhance the therapeutic effects of GCs on inflammation.

Materials and methods

Materials

Lipopolysaccharide (Escherichia coli O111:B4, LPS) and Dex were obtained from Sigma-Aldrich Chemicals (St Louis, MO). Recombinant mouse MIF contained less than 1·0 EU per 1 μg of the cytokine, as determined by the limulus amoebocyte lysate method (R&D Systems, Inc., Minneapolis, MN). Rabbit polyclonal antibodies against MIF and annexin 1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antibodies against cPLA2α and phospho-cPLA2α were purchased from Cell Signaling Technology (Beverley, MA).

Removal of hormones by charcoal-dextran-treated fetal bovine serum

A 2% charcoal suspension in 0·2% dextran T70 of the same volume as fetal bovine serum (FBS) was centrifuged at 1000 g for 10 min. Supernatants were aspirated, and the FBS aliquot was mixed with the charcoal pellets. This charcoal–FBS mixture was maintained in suspension by continuous magnetic stirring for 30 min. This suspension was centrifuged twice at 1000 g for 15 min. The supernatant was filtered through a 0·2-μm cellulose-acetate filter. Cortisol in charcoal-dextran-treated FBS was not detectable by radioimmunology assay. The charcoal-dextran-treated FBS was used in all experiments associated with the treatment of Dex.

Cell culture

Mouse RAW 264.7 macrophages were cultured in RPMI-1640 medium containing 10% heat-inactivated FBS at 37° under 5% CO2 and subcultured every 2 days. For all experiments, logarithmically growing cells were used.

Transfection with siRNA

Using DNA vector-based small interfering (si) RNA technology, a small DNA insert (about 70 bp) encoding a short hairpin RNA targeting the gene of interest is cloned into a commercially available vector (Genscript, Nanjing, China). The insert-containing vector can be transfected into the cell, and it expresses the short hairpin RNA. The hairpin RNA is rapidly processed by the cellular machinery into 19–22 nucleotide double-stranded RNA (siRNA). The DNA insert for mouse MIF siRNA was 5′-GGATCCCGTTCATGTCGTAATAGTTGATGTTGATATCCGCATCAACTATTACGACATGAATTTTTTCCAAAAGCTT-3′, and the DNA insert for mouse control siRNA was 5′-GGATCCCATCTTGCCGATGCTGTGCAGGTTGATATCCGCCTGCACAGCATCGGCAAGATTTTTTTCCAAAAGCTT-3′. RAW 264.7 macrophages (106 cells/well in six-well plates) were transiently transfected with either DNA insert for the MIF siRNA or the control siRNA using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. After 48 hr, the culture medium was removed and the cells were cultured under serum-free conditions for 12 hr before stimulation.

RNA extraction and RT-PCR analysis

Total RNA was isolated using TRIzol reagent (Invitrogen), and 2 μg of total RNA was reverse transcribed using Reverse Transcription Reagents (Fermentas, Vilnius, Lithuania). Then, 3 μl of the double-strand product was mixed with 10 × Taq/RT buffer [1 × Taq/RT buffer: 10 mmol/l Tris–HCl (pH 8·3), 50 mmol/l KCl, 1·5 mmol/l MgCl2, 0·01% gelatine and 2·0 mmol/l dithiothreitol], 500 μmol/l deoxyoligonucleoside triphosphate mix, 25 mmol/l MgCl2, 500 μmol/l of each sense and antisense oligonucleotide, and 0·25 μl Taq polymerase (Promega, Madison, WI). The PCR primers for amplification of mouse MIF and β-actin were as follows: MIF (341 bp), sense primer 5′-ACACCAATGTTCCCCGC-3′, and antisense primer 5′-AAGCGAAGGTGGAACCGT-3′; β-actin (211 bp), sense primer 5′-CCTCTATGCCAACACAGTGC-3′, and antisense primer 5′-GTACTCCTGCTTGCTGATGC-3′. After a heating step at 94° for 5 min, PCR was performed for MIF and β-actin. Amplification was carried out under the following conditions: MIF, 94° for 2 min, 51° for 30 seconds and 72° for 45 seconds for 30 cycles; β-actin, 94° for 30 seconds, 58° for 30 seconds and 72° for 45 seconds for 34 cycles. Following these steps, a final extension at 72° for 10 min for these two samples used a thermal cycler (Perkin-Elmer, Norwalk, CT). The products were analysed after separation by gel electrophoresis (1·5% agarose) and analysed by scanning densitometry to produce a standard curve that determined the linear range of quantifiable reaction products. β-Actin mRNA expression was used as a loading control.

Western blotting analysis

Protein was extracted using cell lysis buffer (Cell Signaling, Beverly, MA). Protein concentration was measured by bicinchoninic acid protein assay (Pierce Chemical, Rockford, IL); 60–120 μg of protein (depending on the gel size) was separated on 10% SDS–polyacrylamide electrophoresis gels and transferred onto nitrocellulose membranes (Millipore, Bedford, MA). Membranes were incubated with antibodies against MIF, annexin 1, phospho-cPLA2α and cPLA2α. After washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Pierce Chemical, Rockford, IL). Signals were revealed using the ECL Western blotting analysis system. Western blots were scanned and densitometry ratios normalized to β-actin content were analysed using Quantity One (Bio-Rad Laboratories, Hercules, CA).

Prostaglandin E2 and leukotriene B4 analysis

Cell culture supernatants were collected 4 hr after stimulation. Prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) concentrations were measured by using a commercially available ELISA kit (R&D Systems, Inc.).

Statistical analysis

All statistical analyses were carried out using Student's t-test. P < 0·05 was considered statistically significant.

Results

MIF overrides the expression of annexin 1 induced by Dex

GCs could induce annexin 1 expression and decrease the release of arachidonic acid and its subsequent conversion to eicosanoids by inhibiting cPLA2α activation. The properties ascribed to annexin 1 may make it an intriguing candidate target of the MIF–GCs cross-talk. To prove this hypothesis we analysed the annexin 1 contents of resting and stimulated RAW 264.7 macrophages (Fig. 1a). Low levels of annexin 1 were constitutively expressed in resting, unstimulated RAW 264.7 cells. Annexin 1 was down-regulated (3·7-fold and 12-fold, respectively) after exposure to MIF (10 ng/ml) and LPS (100 ng/ml), but up-regulated (2·6-fold) after Dex treatment. Pre-incubation of cells with Dex before LPS exposure resulted in a more significant up-regulation of annexin 1 expression (fivefold), exogenous application of MIF given with Dex simultaneously counter-regulated the up-regulation of annexin 1 expression induced by Dex plus LPS (48% reduction) (Fig. 1a). Furthermore, exogenous recombinant MIF was found to fully override, in a dose-dependent manner, the annexin 1 up-regulation induced by Dex alone (Fig. 1b).

Figure 1.

Figure 1

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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).

Knockdown of intracellular MIF increases annexin 1 expression induced by Dex

As MIF is a constitutively expressed and released autocrine macrophage mediator,29 we hypothesize that endogenous depletion of MIF should cause an augmentation of annexin 1 level after treatment with Dex, so rendering macrophages more sensitive to the anti-inflammatory effects of GCs. To test this hypothesis, we first carried out a dose-dependent study in RAW 264.7 cells to assess the effects of MIF siRNA on MIF mRNA and protein expression by RT-PCR and Western blotting analysis, respectively. We found that MIF siRNA 2·0 μg/ml almost suppressed both MIF mRNA and protein expression (Fig. 2a,b). We next showed that RAW 264.7 macrophages transfected with MIF siRNA expressed higher amounts of annexin 1 than those transfected with control siRNA in response to different concentrations of Dex (Fig. 2c), indicating that endogenous knockdown of MIF may enhance the anti-inflammatory effects of GCs by increasing the expression of annexin 1.

Figure 2.

Figure 2

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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).

MIF overrides the inhibitory effect of Dex on release of PGE2 and LTB4

We examined the potential of MIF and LPS to promote the release of PGE2 and LTB4 by macrophages. As shown in Fig. 3(a,b), MIF and LPS both significantly stimulated PGE2 and LTB4 secretion from RAW 264.7 cells in a dose-dependent manner, whereas Dex showed no effects on these cells. The effect of MIF was obviously stronger than that of LPS, as ≥ 10 ng/ml of MIF led to a significant increase of PGE2 and LTB4 release (P < 0·01), whereas ≥ 1 μg/ml of LPS was needed for a similar effect. Dex inhibited PGE2 and LTB4 production by RAW 264.7 cells stimulated with LPS (0·1 μg/ml) (Fig. 3c,d), while treatment of RAW 264.7 cells with exogenous recombinant MIF overcame, in a dose-dependent manner, Dex inhibition of PGE2 and LTB4 secretion. A significant overriding effect occurred at a concentration of 10 ng/ml MIF, which is just within the range of circulating concentrations of MIF measured in humans with infectious or inflammatory diseases.30 These data suggest that MIF may counter-regulate Dex inhibition of PGE2 and LTB4 release by macrophages.

Figure 3.

Figure 3

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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).

Knockdown of intracellular MIF may enhance the inhibitory effect of Dex on release of PGE2 and LTB4

MIF counter-regulated Dex inhibition of PGE2 and LTB4 secretion, so we further investigated whether endogenous depletion of MIF could enhance the inhibitory effect of Dex on release of PGE2 and LTB4. As shown in Fig. 4(a), 100 nm Dex was needed in control cells (transfected with control siRNA), whereas 10 nm Dex was needed in MIF siRNA cells (transfected with MIF siRNA) to inhibit 30% PGE2 secretion. Likewise, 10 nm Dex inhibited 20% LTB4 production in MIF siRNA cells, whereas 100 nm Dex was needed to achieve the same effect in control cells (Fig. 4b), indicating that macrophages with knockdown of intracellular MIF were substantially more sensitive than control macrophages to GCs, as reflected by an approximately tenfold reduction of the concentration of Dex needed to achieve comparable inhibition of PGE2 and LTB4 secretion.

Figure 4.

Figure 4

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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.

cPLA2α phosphorylation is involved in MIF counter-regulation of Dex

Annexin 1 is an anti-inflammatory protein that physically interacts with and inhibits activation of cPLA2α. The GC-induced annexin 1 could inhibit phosphorylation of cPLA2α, and then decreased the release of arachidonic acid and its subsequent conversion to eicosanoids. For the investigation of whether counter-regulated annexin 1 by MIF would further enhance cPLA2α activation, we analysed the cPLA2α phosphorylation level by Western blotting. Under basal conditions, exogenous application of MIF significantly increased cPLA2α phosphorylation, while knockdown of intracellular MIF by MIF siRNA decreased it (Fig. 5a). The tendency for cPLA2α phosphorylation level to be increased by exogenous MIF and decreased by MIF siRNA was similar after LPS stimulation, regardless of the presence (Fig. 5b) or absence of Dex (Fig. 5c). Additionally, Dex uniformly suppressed cPLA2α phosphorylation independence on LPS/MIF stimulation (Fig. 5c), possibly as a result of the high concentration of Dex tested. As shown in Fig. 5(d), Dex reduced LPS-induced cPLA2α phosphorylation levels, but exogenous MIF application counter-regulated Dex inhibition of cPLA2α phosphorylation. In contrast, endogenous depletion of MIF could amplify the inhibitory effect of Dex on cPLA2α phosphorylation. No matter whether cells were stimulated with LPS or LPS plus Dex, RAW 264.7 macrophages treated with exogenous recombinant MIF exhibited higher cPLA2α phosphorylation levels, while MIF siRNA transfected cells showed lower levels when compared with the control siRNA transfected cells (Fig. 5e). These results indicate that MIF counter-regulated Dex inhibition of LPS-induced cPLA2α phosphorylation, and knockdown of intracellular MIF could enhance the anti-inflammatory effect of Dex at least partly via reduction of cPLA2α phosphorylation.

Figure 5.

Figure 5

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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).

Discussion

The data described in the present study demonstrated that Dex inhibited eicosanoid secretion (PGE2 and LTB4) by LPS-stimulated RAW 264.7 macrophages. However, MIF counter-regulated inhibition of Dex on release of PGE2 and LTB4 from RAW 264.7 macrophage treated with LPS. Consistent with our findings, data from Mitchell et al. showed that MIF overrode GC-induced inhibition on arachidonic acid production by tumour necrosis factor-activated NIH-3T3 cells.28 Further experiments, to our knowledge, are the first to show that the molecular mechanism by which MIF counter-regulated the anti-inflammatory effect of GCs could be via decreasing annexin 1 expression. Although annexin 1 is not the only element of the signalling pathway involved in eicosanoid regulation, our results still indicate that it is the crucial one, which could not be compensated by others.

Another protein induced by GCs is MAPK phosphatase 1 (MKP-1), which is the archetypal member of a family of dual specificity phosphatases that inactivate MAPK.31 It has also been proved to be a critical target of MIF–GC cross-talk based on the fact that MIF could suppress GC-induced expression of MKP-1.11 Another pathway through which MIF and GCs may interact with each other involves the activation of the transcriptional factor nuclear factor-κB and down-regulation of IκBα.9 In view of the similar action of annexin 1 with MKP-1 and IκBα induced by GCs, based on present findings, we believe that annexin 1 does participate in the counter-regulation of MIF on GCs. Another finding in our study was that endogenous depletion of MIF could amplify the therapeutic response to GCs in macrophages through enhancing annexin 1 expression. Other research also supported this opinion based on the findings that increased GC sensitivity in the absence of endogenous MIF was associated with an increase of MKP-1 expression12 and interactions with IκB molecules.9 Previous studies have demonstrated that neutralization of MIF by anti-MIF antibodies or a MIF DNA vaccine attenuated disease severity in multifactor-induced arthritis.32 Our findings also indicate that anti-MIF strategies may increase GC sensitivity and improve the anti-inflammatory effects of GC by decreasing the secretion of eicosanoids.

As a result of its induction by GC and anti-inflammatory effects, a role for annexin 1 in the regulation of GC sensitivity has been reported in models of acute and chronic inflammation.18,20,33 Within a few minutes of contact with GCs, existing annexin 1 in some cell types (e.g. the macrophage) is phosphorylated on Ser27 (and possibly other residues), followed by a secretion into the external medium. This phase, which begins within 5–10 min of contact with GCs, may persist for 30–90 min or until the internal pool of annexin 1 is depleted. Interestingly, macrophages activated at this point, are resistant to the eicosanoid-blocking action of GCs. After approximately 1 hr, an up-regulation of annexin 1 mRNA occurs, which leads to the synthesis of new annexin 1 protein.34,35 However, this process is cell specific. Some cells, for example the macrophage and the mast cell, respond in a positive sense to GC signals with an enhanced synthesis of the protein.35 Based on the above information, in the present study, we only investigated the new annexin 1 protein synthesis induced by GC. Another possibility that MIF also interferes with the rapid secretion of annexin 1 caused by GC could not be excluded and needs further exploration.

The ability of GCs to inhibit eicosanoid production can be traced to two main mechanisms. Acutely, these drugs prevent the phosphorylation/activation of cPLA2 through an annexin 1-dependent mechanism36 whereas more chronic exposure to GCs down-regulates Cox-2 mRNA through an annexin 1-independent mechanism.37 Our present study only focused on the inhibitory effect of GCs on phosphorylation/activation of cPLA2 at acute phase via modulation of annexin 1. An increasing number of reports have suggested that cPLA2 is a key enzyme responsible for signal transduction in inflammation, cytotoxicity and mitogenesis. Annexin 1 suppresses cPLA2 activity not only in vivo, but also in cultured cells. Hence, annexin 1 may function as an endogenous negative regulator of cPLA2 by specific interaction mechanism.24 However, previous findings reported that MIF could regulate cPLA2 activity via a protein kinase A and extracellular signal-regulated kinase-dependent pathway in the NIH/3T3 fibroblast cell line28 and recombinant human MIF was also found to up-regulate cPLA2 mRNA expression in cultured fibroblast-like synoviocytes from rheumatoid arthritis.38 In view of this action of MIF on cPLA2, MIF overriding the anti-inflammatory effect of GCs partly through direct regulation of cPLA2 is also possible. This hypothesis needs more investigation.

A substantial body of evidence suggests that many effects of annexin 1 are exerted through a cell surface receptor-mediated mechanism. The scientific popularity of annexin 1 received a significant boost after the discovery of its receptor, formyl peptide receptor type 2 (FPR2), a G-protein coupled receptor.39–41 If we take into account a large number of biochemical and cellular studies, it is clear that the binding of annexin 1 to FPR2 initiates a cascade of signalling events, including PLA2, phospholipase D and MAPK.42,43 Activation of these multifunctional pathways has different outcomes in different cell types.44 While considering the inhibitory effects of the new synthesis of GC-induced annexin 1 protein on eicosanoids, in the present study we paid attention to the intercellular newly produced annexin 1, not to the extracellular rapidly secreted annexin 1, and so ignored the role of annexin 1 receptor, FPR2, under these conditions. Our next step is to confirm whether FPR2 is involved in the MIF counter-regulation of GCs.

In conclusion, we provide evidence that annexin 1 is a crucial target of MIF–GC cross-talk. The identification of annexin 1 in MIF counter-regulation of GCs may provide a novel molecular basis for further investigation. More importantly, these observations hold great promise for developing annexin 1-based therapeutic strategies for the management of patients with GC-resistant inflammatory and autoimmune diseases, so avoiding the side effects of GC with large concentrations.

Competing interests

The authors declare that they have no competing interests.

Contributions to authorship

Yu Sun and Yu Wang were involved in the performance of the experiments and wrote the manuscript. Jia-Hui Li was responsible for siRNA and Western blotting experiments. Shi-Hui Zhu and Hong-Tai Tang were responsible for the design of the experiments and interpretation of the data. Zhao-Fan Xia reviewed all phases of analysis and finalized the writing of the manuscript. All of the authors have read and approved the manuscript.

Acknowledgments

This work was supported by grants from the National Nature Science Foundation of China (No. 81000825) to Yu Sun, (No. 81000831) to Yu Wang and (No. 81000826) to Jia-Hui Li, and was supported by Projects of International Cooperation and Exchanges NSFC (No. 81120108015) to Zhao-Fan Xia.

Glossary

CD-FBS

charcoal-dextran-treated FBS

cPLA2α

cytosolic phospholipase A2α

Dex

dexamethasone

FBS

fetal bovine serum

GCs

glucocorticoids

LPS

lipoplysaccharides

LTB4

leukotriene B4

MIF

macrophage migration inhibitory factor

MKP-1

mitogen-acitvated protein kinase phosphatase 1

PGE2

prostaglandin E2

RNAi

ribonucleic acid interference

Disclosures

The authors declare no financial or commercial conflict of interests.

References

  • 1.Calandra T, Roger T. Macrophage migration inhibitory factor: a regulator of innate immunity. Nat Rev Immunol. 2003;3:791–800. doi: 10.1038/nri1200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Leng L, Metz CN, Fang Y, et al. MIF signal transduction initiated by binding to CD74. J Exp Med. 2003;197:1467–76. doi: 10.1084/jem.20030286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Shi X, Leng L, Wang T, et al. CD44 is the signaling component of the macrophage migration inhibitory factor–CD74 receptor complex. Immunity. 2006;25:595–606. doi: 10.1016/j.immuni.2006.08.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Bernhagen J, Krohn R, Lue H, et al. MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment. Nat Med. 2007;13:587–96. doi: 10.1038/nm1567. [DOI] [PubMed] [Google Scholar]
  • 5.Calandra T, Bernhagen J, Metz CN, Spiegel LA, Bacher M, Donnelly T, Cerami A, Bucala R. MIF as a glucocorticoid-induced modulator of cytokine production. Nature. 1995;377:68–71. doi: 10.1038/377068a0. [DOI] [PubMed] [Google Scholar]
  • 6.Flaster H, Bernhagen J, Calandra T, Bucala R. The macrophage migration inhibitory factor–glucocorticoid dyad: regulation of inflammation and immunity. Mol Endocrinol. 2007;21:1267–80. doi: 10.1210/me.2007-0065. [DOI] [PubMed] [Google Scholar]
  • 7.Rhen T, Cidlowski JA. Anti inflammatory action of glucocorticoids – new mechanisms for old drugs. N Engl J Med. 2005;353:1711–23. doi: 10.1056/NEJMra050541. [DOI] [PubMed] [Google Scholar]
  • 8.Leng L, Wang W, Roger T, Merk M, Wuttke M, Calandra T, Bucala R. Glucocorticoid-induced MIF expression by human CEM T cells. Cytokine. 2009;48:177–85. doi: 10.1016/j.cyto.2009.07.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Wang FF, Zhu LA, Zou YQ, et al. New insights into the role and mechanism of macrophage migration inhibitory factor in steroid-resistant patients with systemic lupus erythematosus. Arthritis Res Ther. 2012;14:R103. doi: 10.1186/ar3828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Ayoub S, Hickey MJ, Morand EF. Mechanisms of disease: macrophage migration inhibitory factor in SLE, RA and atherosclerosis. Nat Clin Pract Rheumatol. 2008;4:98–105. doi: 10.1038/ncprheum0701. [DOI] [PubMed] [Google Scholar]
  • 11.Roger T, Chanson AL, Knaup-Reymond M, Calandra T. Macrophage migration inhibitory factor promotes innate immune responses by suppressing glucocorticoid-induced expression of mitogen-activated protein kinase phosphatase-1. Eur J Immunol. 2005;35:3405–13. doi: 10.1002/eji.200535413. [DOI] [PubMed] [Google Scholar]
  • 12.Aeberli D, Yang Y, Mansell A, Santos L, Leech M, Morand EF. Endogenous macrophage migration inhibitory factor modulates glucocorticoid sensitivity in macrophages via effects on MAP kinase phosphatase-1 and p38 MAP kinase. FEBS Lett. 2006;580:974–81. doi: 10.1016/j.febslet.2006.01.027. [DOI] [PubMed] [Google Scholar]
  • 13.Daun JM, Cannon JG. Macrophage migration inhibitory factor antagonizes hydrocortisone-induced increases in cytosolic IκBα. Am J Physiol Regul Integr Comp Physiol. 2000;279:R1043–9. doi: 10.1152/ajpregu.2000.279.3.R1043. [DOI] [PubMed] [Google Scholar]
  • 14.McLeod JD, Goodall A, Jelic P, Bolton C. Changes in the cellular distribution of lipocortin-1 (Annexin-1) in C6 glioma cells after exposure to dexamethasone. Biochem Pharmacol. 1995;50:1103–7. doi: 10.1016/0006-2952(95)00234-q. [DOI] [PubMed] [Google Scholar]
  • 15.Traverso V, Morris JF, Flower RJ, Buckingham J. Lipocortin 1 (annexin 1) in patches associated with the membrane of a lung adenocarcinoma cell line and in the cell cytoplasm. J Cell Sci. 1998;111:1405–18. doi: 10.1242/jcs.111.10.1405. [DOI] [PubMed] [Google Scholar]
  • 16.Perretti M, D'Acquisto F. Annexin A1 and glucocorticoids as effectors of the resolution of inflammation. Nat Rev Immunol. 2009;9:62–70. doi: 10.1038/nri2470. [DOI] [PubMed] [Google Scholar]
  • 17.Perretti M, Christian H, Wheller SK, Aiello I, Mugridge KG, Morris JF, Flower RJ, Goulding NJ. Annexin I is stored within gelatinase granules of human neutrophil and mobilized on the cell surface upon adhesion but not phagocytosis. Cell Biol Int. 2000;24:163–74. doi: 10.1006/cbir.1999.0468. [DOI] [PubMed] [Google Scholar]
  • 18.Yang YH, Morand EF, Getting SJ, et al. Modulation of inflammation and response to dexamethasone by Annexin 1 in antigen-induced arthritis. Arthritis Rheum. 2004;50:976–84. doi: 10.1002/art.20201. [DOI] [PubMed] [Google Scholar]
  • 19.Renshaw D, Montero-Melendez T, Dalli J, Kamal A, Brancaleone V, D'Acquisto F, Cirino G, Perretti M. Downstream gene activation of the receptor ALX by the agonist annexin A1. PLoS One. 2010;5:e12771. doi: 10.1371/journal.pone.0012771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Hannon R, Croxtall JD, Getting SJ, et al. Aberrant inflammation and resistance to glucocorticoids in annexin 1–/– mouse. FASEB J. 2003;17:253–5. doi: 10.1096/fj.02-0239fje. [DOI] [PubMed] [Google Scholar]
  • 21.Gerke V, Creutz CE, Moss SE. Annexins: linking Ca2+ signalling to membrane dynamics. Nat Rev Mol Cell Biol. 2005;6:449–61. doi: 10.1038/nrm1661. [DOI] [PubMed] [Google Scholar]
  • 22.Liu NK, Zhang YP, Han S, Pei J, Xu LY, Lu PH, Shields CB, Xu XM. Annexin A1 reduces inflammatory reaction and tissue damage through inhibition of phospholipase A2 activation in adult rats following spinal cord injury. J Neuropathol Exp Neurol. 2007;66:932–43. doi: 10.1097/nen.0b013e3181567d59. [DOI] [PubMed] [Google Scholar]
  • 23.Lim LH, Pervaiz S. Annexin 1: the new face of an old molecule. Faseb J. 2007;21:968–75. doi: 10.1096/fj.06-7464rev. [DOI] [PubMed] [Google Scholar]
  • 24.Kim SW, Rhee HJ, Ko J, Kim YJ, Kim HG, Yang JM, Choi EC, Na DS. Inhibition of cytosolic phospholipase A2 by annexin I. Specific interaction model and mapping of the interaction site. J Biol Chem. 2001;276:15712–9. doi: 10.1074/jbc.M009905200. [DOI] [PubMed] [Google Scholar]
  • 25.Roviezzo F, Getting SJ, Paul-Clark MJ, et al. The annexin-1 knockout mouse: what it tells us about the inflammatory response. J Physiol Pharmacol. 2002;53:541–53. [PubMed] [Google Scholar]
  • 26.Lim LH, Solito E, Russo-Marie F, Flower RJ, Perretti M. Promoting detachment of neutrophils adherent to murine postcapillary venules to control inflammation: effect of lipocortin 1. Proc Natl Acad Sci USA. 1998;95:14535–9. doi: 10.1073/pnas.95.24.14535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Pitzalis C, Pipitone N, Perretti M. Regulation of leukocyte–endothelial interactions by glucocorticoids. Ann N Y Acad Sci. 2002;966:108–18. doi: 10.1111/j.1749-6632.2002.tb04208.x. [DOI] [PubMed] [Google Scholar]
  • 28.Mitchell RA, Metz CN, Peng T, Bucala R. Sustained mitogen-activated protein kinase (MAPK) and cytoplasmic phospholipase A2 activation by macrophage migration inhibitory factor (MIF). Regulatory role in cell proliferation and glucocorticoid action. J Biol Chem. 1999;274:18100–6. doi: 10.1074/jbc.274.25.18100. [DOI] [PubMed] [Google Scholar]
  • 29.Calandra T, Bernhagen J, Mitchell RA, Bucala R. The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor. J Exp Med. 1994;179:1895–902. doi: 10.1084/jem.179.6.1895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Calandra T, Echtenacher B, Roy DL, et al. Protection from septic shock by neutralization of macrophage migration inhibitory factor. Nat Med. 2000;6:164–70. doi: 10.1038/72262. [DOI] [PubMed] [Google Scholar]
  • 31.Liu Y, Shepherd EG, Nelin LD. MAPK phosphatases – regulating the immune response. Nat Rev Immunol. 2007;7:202–12. doi: 10.1038/nri2035. [DOI] [PubMed] [Google Scholar]
  • 32.Onodera S, Ohshima S, Tohyama H, et al. A novel DNA vaccine targeting macrophage migration inhibitory factor protects joints from inflammation and destruction in murine models of arthritis. Arthritis Rheum. 2007;56:521–30. doi: 10.1002/art.22407. [DOI] [PubMed] [Google Scholar]
  • 33.Damazo AS, Yona S, Flower RJ, Perretti M, Oliani SM. Spatial and temporal profiles for anti-inflammatory gene expression in leukocytes during a resolving model of peritonitis. J Immunol. 2006;176:4410–8. doi: 10.4049/jimmunol.176.7.4410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Carnuccio R, Di Rosa M, Flower RJ, Pinto A. The inhibition by hydrocortisone of prostaglandin biosynthesis in rat peritoneal leucocytes is correlated with intracellular macrocortin levels. Br J Pharmacol. 1981;74:322–4. doi: 10.1111/j.1476-5381.1981.tb09974.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Yazid S, Norling LV, Flower RJ. Anti-inflammatory drugs, eicosanoids and the annexin A1/FPR2 anti-inflammatory system. Prostaglandins Other Lipid Mediat. 2012;98:94–100. doi: 10.1016/j.prostaglandins.2011.11.005. [DOI] [PubMed] [Google Scholar]
  • 36.Croxtall JD, Choudhury Q, Newman S, Flower RJ. Lipocortin 1 and the control of cPLA2 activity in A549 cells. Glucocorticoids block EGF stimulation of cPLA2 phosphorylation. Biochem Pharmacol. 1996;52:351–6. doi: 10.1016/0006-2952(95)02442-5. [DOI] [PubMed] [Google Scholar]
  • 37.Masferrer JL, Seibert K, Zweifel B, Needleman P. Endogenous glucocorticoids regulate an inducible cyclooxygenase enzyme. Proc Natl Acad Sci USA. 1992;89:3917–21. doi: 10.1073/pnas.89.9.3917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Sampey AV, Hall PH, Mitchell RA, Metz CN, Morand EF. Regulation of synoviocyte phospholipase A2 and cyclooxygenase 2 by macrophage migration inhibitory factor. Arthritis Rheum. 2001;44:1273–80. doi: 10.1002/1529-0131(200106)44:6<1273::AID-ART219>3.0.CO;2-8. [DOI] [PubMed] [Google Scholar]
  • 39.Hayhoe RP, Kamal AM, Solito E, Flower RJ, Cooper D, Perretti M. Annexin 1 and its bioactive peptide inhibit neutrophil–endothelium interactions under flow: indication of distinct receptor involvement. Blood. 2006;107:2123–30. doi: 10.1182/blood-2005-08-3099. [DOI] [PubMed] [Google Scholar]
  • 40.Walther A, Riehemann K, Gerke V. A novel ligand of the formyl peptide receptor: annexin I regulates neutrophil extravasation by interacting with the FPR. Mol Cell. 2000;5:831–40. doi: 10.1016/s1097-2765(00)80323-8. [DOI] [PubMed] [Google Scholar]
  • 41.Perretti M, Chiang N, La M, Fierro IM, Marullo S, Getting SJ, Solito E, Serhan CN. Endogenous lipid- and peptide-derived anti-inflammatory pathways generated with glucocorticoid and aspirin treatment activate the lipoxin A4 receptor. Nat Med. 2002;8:1296–302. doi: 10.1038/nm786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Chiang N, Serhan CN, Dahlén SE, et al. The lipoxin receptor ALX: potent ligand-specific and stereoselective actions in vivo. Pharmacol Rev. 2006;58:463–87. doi: 10.1124/pr.58.3.4. [DOI] [PubMed] [Google Scholar]
  • 43.Ye RD, Boulay F, Wang JM, Dahlgren C, Gerard C, Parmentier M, Serhan CN, Murphy PM. International Union of Basic and Clinical Pharmacology. LXXIII. Nomenclature for the formyl peptide receptor (FPR) family. Pharmacol Rev. 2009;61:119–61. doi: 10.1124/pr.109.001578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.D'Acquisto F, Piras G, Rattazzi L. Pro-inflammatory and pathogenic properties of Annexin-A1: the whole is greater than the sum of its parts. Biochem Pharmacol. 2013;85:1213–8. doi: 10.1016/j.bcp.2013.02.011. [DOI] [PubMed] [Google Scholar]

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