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Immunology. Jan 2009; 126(1): 74–83.
PMCID: PMC2632697

Macrophage migration inhibitory factor stimulates interleukin-17 expression and production in lymph node cells

Abstract

Interleukin (IL)-17 is a pro-inflammatory cytokine produced by recently described T helper type 17 (Th17) cells, which have critical role in immunity to extracellular bacteria and the pathogenesis of several autoimmune disorders. IL-6 and transforming growth factor (TGF)-β are crucial for the generation of Th17 cells in mice, while the production of IL-17 is supported by various cytokines, including IL-23, IL-1β, IL-21, IL-15 and tumour necrosis factor (TNF)-α. In this study, the influence of a multifunctional cytokine, macrophage migration inhibitory factor (MIF), on IL-17 production in mice was investigated. Treatment of lymph node cells (LNCs) with recombinant MIF up-regulated mitogen-stimulated IL-17 expression and secretion. Additionally, LNCs from MIF knockout mice (mif−/−) had severely impaired production of IL-17, as well as of IL-1β, IL-6, IL-23 and TGF-β. When stimulated with recombinant IL-1β, IL-23 or TNF-α, mitogen-triggered mif−/− LNCs were fully able to achieve the IL-17 production seen in wild-type (WT) LNCs, while the addition of IL-6 and TGF-β had no effect. Finally, after injection of mice with complete Freund's adjuvant, secretion of IL-17 as well as the number of IL-17-positive cells was significantly lower in the draining lymph nodes of mif−/− mice in comparison with WT mice. The effect of MIF on IL-17 production was dependent on p38, extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) and Janus kinase 2/signal transducer and activator of transcription 3 (Jak2/STAT3), and not on nuclear factor (NF)-κB and nuclear factor of activated T cells (NFAT) signalling. Bearing in mind the contribution of MIF and IL-17 to the pathology of inflammatory and autoimmune disorders, from the results presented here it seems plausible that targeting MIF biological activity could be a valid therapeutic approach for the treatment of such diseases.

Keywords: complete Freund's adjuvant, concanavalin A, interleukin-17, inflammation, macrophage migration inhibitory factor (MIF)

Introduction

Inflammation is a complex defence reaction the aim of which is to neutralize an insult and restore normal tissue structure and function. Both macrophage migration inhibitory factor (MIF) and interleukin (IL)-17 have important roles in various immunoinflammatory disorders such as sepsis, acute respiratory distress syndrome, asthma, multiple sclerosis, diabetes mellitus type 1 and rheumatoid arthritis.13 Both MIF and IL-17 are expressed in a variety of cell types including lymphocytes, monocytes and other leucocytes,47 but a distinct lineage of CD4+ T cells [T helper type 17 (Th17)] have been designated as predominant producers of IL-17.7 Unlike most other cytokines, MIF is constitutively expressed and stored in intracellular pools and its secretion does not require de novo protein synthesis.1 MIF is also peculiar in its unique ability to directly regulate the immunosuppressive actions of glucocorticoids.8 MIF plays a major role in innate immunity against bacterial infections through enhancement of tumour necrosis factor (TNF)-α secretion,4 Toll-like receptor 4 (TLR4) expression,9 phagocytosis and intracellular killing mechanisms,10 and is equally efficiently involved in the adaptive immune response through favouring Th1 activation and differentiation.11,12 IL-17-producing cells differentiate from naïve T lymphocytes in the presence of IL-6 and transforming growth factor (TGF)-β, while the major promoting cytokines for sustained IL-17 generation are IL-23, IL-1β, IL-21 and IL-15.7,13 However, the development of these cells is antagonized by the cytokines and signalling pathways that govern the development of Th1 and Th2 cells and by IL-27.7,13 IL-17 participates in eradication of bacterial and fungal infections through amplification of inflammatory processes mediated by the induction of chemokines that are important in neutrophil recruitment, proliferation of myeloid cells or activation of fibroblasts to produce IL-6, IL-1β and prostaglandin E2.3,7,13

Although it is known that deletion or neutralization of MIF severely impairs TNF-α, IL-1β, IL-6 and IL-23 production,14,15 all of which are important for the generation of IL-17, the possibility that MIF supports IL-17 production has not been investigated to date. The aim of this study was to determine the contribution of MIF to IL-17 expression in murine lymph node cells (LNCs) in various experimental settings. Our results suggest that MIF potently stimulates IL-17 production in LNCs, through utilization of mitogen-activated protein (MAP) kinases and Janus kinase 2/signal transducer and activator of transcription 3 (Jak2/STAT3) signalling.

Materials and methods

Animals

Breeder knockout mice lacking the functional gene encoding MIF (mif−/−) on a C57BL/6 background were a kind gift from Dr Christine Metz (Laboratory of Medicinal Biochemistry, The Feinstein Institute for Medical Research, North Shore LIJ Health System, NY) and their wild-type (WT) counterparts (C57BL/6) were purchased from The Jackson Laboratory, Bar Harbor, ME. Animals were bred and maintained under standard laboratory conditions in the Animal Facility at the Institute for Biological Research ‘Siniša Stanković’. All experiments were approved by the local Ethical Committee (IBISS, No. 10/2006).

In vitro studies

Cervical lymph nodes collected from mice killed by cervical dislocation were dispersed through nylon mesh in RPMI-1640 + 2% fetal calf serum (FCS) (Sigma, St Louis, MO), pooled, filtered through the conical mesh and centrifuged at 500 g for 5 min. Cell pellets were re-suspended in RPMI-1640 + 5% FCS. The number of viable LNCs was determined by trypan blue exclusion. LNCs were either left unstimulated or stimulated with concanavalin A (Con A) (5 μg/ml) in the presence or absence of 10 ng/ml of recombinant murine MIF (rMIF), TGF-β (R&D Systems, Minneapolis, MN), IL-1β, TNF-α (BD Pharmingen, San Diego, CA), IL-6, IL-23 (eBioscience, San Diego, CA) or inhibitors [SB20358016, 1 μm; SP60012516, 1 μm; cyclosporin A17, 0·1 μm; AG49018, 6 μm (Calbiochem, Darmstadt, Germany); MG13216, 0·1 μm; PD9805916, 5 μm (Sigma)].

Induction of local inflammation: ex vivo studies

Local inflammation was induced by injection of 50 μl of emulsified complete Freund's adjuvant (CFA) (Difco Laboratories, Detroit, MI) in phosphate-buffered saline (PBS) (Sigma) into the plantar surface of both hind paws of mice (four experiments; five mice per group). The obvious signs of ongoing inflammation (redness and swelling of the paws) were visible in both strains as early as 2 days after the injection. Both left and right popliteal lymph nodes (PLNs) were collected 2, 5, 7 and 10 days after CFA injection. PLNs were processed identically to the cervical lymph nodes. PLN cells (PLNCs) obtained from each animal were pooled and incubated either in culture medium alone or in the presence of 5 μg/ml Con A (Pharmacia, Uppsala, Sweden) or 10 ng/ml purified protein derivative (PPD; Staten Serum Institute, Copenhagen, Denmark).

Enzyme-linked immunosorbent assay (ELISA) and cell-based ELISA

To collect supernatants for ELISA, LNCs (5 × 105) were incubated in 500 μl of culture medium at 37° in a humidified atmosphere with 5% CO2. Cell-free supernatants were collected after 3, 6, 12, 24 and 48 hr for studies of IL-17 production kinetics, while for the rest of the experiments supernatants were removed after 24 hr of incubation and stored at −20° until determination of cytokine concentration. Remaining cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (0·5 mg/ml) (Sigma) in order to determine cell viability and non-cytotoxic concentrations of inhibitors. The secretion of IL-17, IL-1β (BD Pharmingen), IL-6, IL-23 (eBioscience) and TGF-β (R&D Systems, Minneapolis, MN) was determined by sandwich ELISA according to the manufacturer's instructions. Values for duplicate wells were calculated using a standard curve which was obtained on the basis of known concentrations of applied recombinant cytokines. The expression of STAT3 (eBioscience), c-Fos, phosphorylated forms of p38, extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), IκB and Jun in LNCs from WT or mif−/− mice was determined in triplicate by cell-based ELISA using specific antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA) according to a previously described protocol.19 Briefly, LNCs (3 × 105) were seeded on poly-l-lysin (1 μg/ml) (Sigma) coated 96-well plates. All primary antibodies were used at 1 : 200 dilutions, while appropriate horseradish peroxidase (HRP)-conjugated detecting antibodies (US Biochemical Corporation, Cleveland, OH) were used at 1 : 2000 dilutions. After the addition of 3,3′,5,5′-tetramethylbenzidinine (Sigma) and 0·1 m HCl to stop the reaction, absorbance was measured at 450 nm (A450). After the measurement of A450, the wells were stained with crystal violet to determine relative cell numbers. The obtained A450 values were corrected for differences in cell number. Results are shown as mean ± standard deviation (SD) of three experiments.

Fluorescence-activated cell sorter (FACS) analysis

LNCs or PLNCs (3 × 105) were pelleted by centrifugation at 500 g for 2 min in 500 μl of PBS + 2% FCS (Sigma). For surface staining, cells were immediately incubated for 30 min at 4° with 0·5 μg of phycoerythrin (PE) Armenian hamster anti-mouse CD3e (clone 145-2C11), 0·5 μg of fluorescein isothiocyanate (FITC) rat anti-mouse B220 (clone RA3-6B2), 0·5 μg of FITC rat anti-mouse CD11b (clone M1/70), 0·25 μg of FITC Armenian hamster anti-mouse CD11c, 0·5 μg of FITC rat anti-mouse pan-NK (clone DX5) or appropriate isotype controls (eBioscience). For intracellular detection of IL-17, cells were stimulated with either Con A (5 μg/ml) or phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) and ionomycin (500 ng/ml) for 1·5 hr, and subsequently cultured in the presence of monensin (2 μm) for an additional 5·5 hr. Cells were then collected by centrifugation and incubated for 15 min at room temperature in 200 μl of PBS with 10% of normal rat serum, fixed with 2% paraformaldehyde for 10 min, washed in PBS + 2% FCS and then permeabilized in 100 μl of 0·1% Triton-X100 (Sigma) in PBS + 2% FCS. Cells were then incubated with 0·5 μg of rat anti-mouse IL-17-PE (clone eBio17B7) or the same amount of isotype control rat immunoglobulin G2a, κ(eBioscience) for 30 min at 4°. After washing two times in 500 μl of PBS + 2% FCS at 500 g for 2 min, cells were re-suspended in 500 μl of PBS. Stained cells were detected on FACSCalibur (BD Biosciences, Oxford, UK) and analysed using cellquestpro software (BD Biosciences). Results are shown as mean ± SD of three to five experiments (five mice per group).

RNA isolation and semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) of cytokine mRNA

For optimal IL-17 mRNA expression, RNA was isolated with RNA Isolator (Fermentas, Vilnius, Lithuania) from LNCs (5 × 106/ml) after 6 hr of incubation. RNA (1 μg) was reverse transcribed using Moloney leukemia virus reverse transcriptase and random primers (Fermentas). PCR amplification of cDNA (1 μl per 20 μl of PCR reaction) was carried out in a real-time PCR machine (Applied Biosystems, Woolston, UK) using SYBRGreen PCR master mix (Applied Biosystems) as follows: 10 min at 50° for dUTP activation and 10 min at 95° for initial denaturation of cDNA, followed by 40 cycles (15 seconds of denaturation at 95° and 60 seconds of the primer annealing and chain extension step at 60°). Primer pairs were 5′-GGGAGAGCTTCATCTGT-3′ and 5′-GACCCTGAAAGTGAAGGG-3′ for IL-17 (accession number NM_010552.3), 5′-CCGCTGAGAGGGCTTCAC-3′ and 5′-TGCAGGAGTAGGCCACATTACA-3′ for retinoic-acid-related orphan receptor (Ror)-γt (NM_011281.1; described elsewhere)20 and 5′-GGACCTGACAGACTACC-3′ and 5′-GGCATAGAGGTCTTTACGG-3′ for β-actin (NM_007393.2). The expression of these genes was calculated according to the formula 2−(Cti – Cta), where Cti is the cycle threshold of the gene of interest and Cta is the cycle threshold value of β-actin, and is given as the mean ± SD of three experiments.

Statistical analysis

To determine the significance of the differences between MIF-treated and control WT LNCs, or WT and mif−/− LNCs, analysis of variance (ANOVA) followed by the Student–Newman–Keuls test was used. Results from a representative experiment of at least three are presented as mean ± SD of duplicate or triplicate measurements, as stated previously for each method. A P-value less than 0·05 was considered to be significant.

Results

The effect of MIF on IL-17 production and expression in LNCs

In order to investigate the importance of MIF for IL-17 production, LNCs from WT or mif−/− C57BL/6 mice were used. The accumulation of IL-17 in supernatants from cultures of either non-stimulated WT LNCs or WT LNCs treated with rMIF (0·1–100 ng/ml) did not reach detectable levels (data not shown), thus suggesting that MIF alone was not able to induce IL-17 expression and production in LNCs. However, rMIF had a potent, dose-dependent (Fig. 1a) and sustained (Fig. 1b) stimulatory effect on Con A-triggered IL-17 production in WT LNCs. Accordingly, the expression of IL-17 mRNA in Con A + MIF-treated WT LNCs was almost sevenfold higher in comparison with Con A-stimulated cultures (Fig. 1c). In addition, expression of the IL-17-promoting transcription factor Ror-γt was also up-regulated in WT LNCs treated with MIF (Fig. 1e). Thus, it was clear that exogenous MIF contributed to IL-17 production in mitogen-stimulated LNCs. Furthermore, Con A-stimulated LNCs from mif−/− mice showed significantly lower levels of IL-17 expression and production (Fig. 1c,d), as well as lower percentages of IL-17-expressing cells (Fig. 1f) in comparison with Con A-treated WT LNCs. These results suggested that endogenous MIF significantly contributed to Con A-induced IL-17 production in LNCs. The addition of rMIF to Con A-stimulated mif−/− LNCs elevated both the secretion and expression of IL-17 to the levels seen in WT LNCs (Fig. 1c,d). Surprisingly, the expression of Ror-γt was up-regulated in Con A-treated mif−/− LNCs (Fig. 1e) and remained unchanged after the addition of rMIF, hence suggesting that the observed effect of endogenous MIF on Con A-triggered IL-17 generation was independent of Ror-γt expression.

Figure 1
The effect of macrophage migration inhibitory factor (MIF) on interleukin (IL)-17 secretion and expression, and retinoic-acid-related orphan receptor (Ror)-γt expression. Lymph node cells (LNCs) isolated from wild-type (WT) (a–f) or mif ...

The influence of MIF deletion on production of IL-17-inducing cytokines in LNCs

The data presented so far clearly show that Con A-stimulated IL-17 expression in LNCs is impaired in the absence of MIF. In order to investigate the possibility of an indirect influence of MIF on IL-17 production in T cells, we compared Con A-stimulated production of several known inducers of IL-17 in WT and mif−/− LNCs. We found that Con A-triggered mif−/− LNCs secreted markedly lower amounts of IL-1β, IL-6, IL-23 and TGF-β, and higher amounts of TNF-α, in comparison with WT cells (Fig. 2a). These results suggested that endogenous MIF contributes to IL-17 production indirectly. Accordingly, if recombinant IL-1β or IL-23 was added to Con A-stimulated mif−/− LNCs, both IL-17 production (Fig. 2b) and expression (Fig. 2c) reached the levels obtained with WT cells (dashed line in Fig. 2). In contrast, IL-6 and TGF-β had no noticeable effect on IL-17 production (Fig. 2b), while the expression of IL-17 was slightly elevated (Fig. 2c). As for TNF-α, its stimulatory effect on IL-17 production was evident (Fig. 2c), although the expression of IL-17 remained unchanged (Fig. 2c). These results indicate that MIF could stimulate IL-17 production in LNCs indirectly, through up-regulation of IL-1β and IL-23.

Figure 2
Concanavalin A (Con A)-stimulated cytokine production in wild-type (WT) and mif−/− lymph node cell (LNC) and their effect on interleukin (IL)-17 expression and production in mif−/− LNC. LNCs (5 × 105/well) from ...

Signalling pathways involved in IL-17 expression in WT and mif−/− LNCs

Our next goal was to explore the contribution of MAP kinases, NF-kB and Jak2/STAT3 signalling pathways to the stimulatory effect of MIF on IL-17 generation in LNCs. In our experiments, Con A-triggered IL-17 secretion from WT LNCs was markedly down-regulated (by 43%) in the presence of SP600125, a specific inhibitor of JNK MAP kinase (Fig. 3a). Accordingly, the activation of JNK in mif−/− LNCs was reduced in comparison to WT cells (Fig. 3b), thus suggesting that impaired JNK activation in the absence of MIF could be at least partly responsible for the observed lower capacity to produce IL-17. However, in both WT and mif−/− LNCs, the level of phosphorylation of JNK substrate Jun (Fig. 3c) or the expression of c-Fos (Fig. 3d) upon Con A stimulation remained unchanged. However, inhibitors of MAP kinases p38 (SB203080) and ERK (PD98059) had only mild effects on IL-17 production in Con A-stimulated WT LNCs (Fig. 3a), although there was an obvious difference in the level of activation of both kinases in LNCs of WT and mif−/− mice (Fig. 3e,f). Additionally, inhibitors of NF-κB (MG132), STAT3 (AG490) and NFAT [cyclosporin A (CsA)] signalling also inhibited IL-17 production in Con A-stimulated WT LNCs (Fig. 3a). These findings were consistent with the results obtained from cell-based ELISA, which showed clear differences in the level of activation of NF-κB (deduced from the level of the NF-κB inhibitor IκB) and STAT3 concentration upon Con A-stimulation of LNCs of WT and mif−/− mice (Fig. 3g,h). Importantly, the treatment of mif−/− LNCs with the MAP kinase inhibitors and Jak2/STAT3 signalling blocker had minor effects on IL-17 production (Fig. 3a), suggesting that MAP kinases and Jak2/STAT3 signalling made a limited contribution to IL-17 production in mif−/− LNCs. However, NF-κB or NFAT inhibition significantly abrogated IL-17 generation in mif−/− LNCs (Fig. 3a), indicating that these pathways remained highly operative in the absence of MIF. Taken together, these results suggest that the effect of MIF on IL-17 production in Con A-stimulated LNCs is, at least partly, dependent on the MAP kinase and Jak2/STAT3 signalling pathways.

Figure 3
Signalling molecules involved in interleukin (IL)-17 production in wild-type (WT) or mif−/− lymph node cell (LNC). (a) LNCs (5 × 105/well) isolated from WT or mif−/− were cultured with concanavalin A (Con A) (5 ...

The impact of MIF deficiency on IL-17 production during inflammation

Local inflammation was induced in mice by intraplantar injection of CFA. After 2 days, signs of inflammation (redness and swelling) were evident in both WT and mif−/− mice and remained visible throughout the observation period. Following isolation of cells from draining PLNs at day 7 after CFA injection, cellular composition was determined and similar percentages of all cell subsets were found in PLNs of both strains. In more detail, WT and mif−/− PLNs contained 32·6 ± 5·4% versus 34·8 ± 11·4% CD3+ cells (T lymphocytes), 44·2 ± 6·5% versus 42·1 ± 8·5% B220+ cells (B lymphocytes), 5·8 ± 2·3% versus 5·2 ± 3·3% CD11b+ cells (macrophages), 3·1 ± 0·8% versus 3·2 ± 0·9% CD11c+ cells (dendritic cells) and 2·7 ± 0·6% versus 2·9 ± 1·1% pan-NK cells [natural killer (NK) cells], respectively. At different time-points post injection, PLNCs were isolated and cultivated for 24 hr without any stimulation or in the presence of Con A or PPD, an antigen derived from Mycobacterium tuberculosis. The data obtained clearly showed that IL-17 secretion from PLNCs isolated from mif−/− mice was markedly lower in comparison to PLNCs isolated from WT mice, irrespective of the presence and type of in vitro stimuli (Fig. 4a–c). Furthermore, the proportion of IL-17-secreting cells was also reduced in mif−/− PLNs (Fig. 4d). Thus, the ex vivo results confirmed the in vitro observations, and strongly suggested the importance of MIF for IL-17 production in murine LNCs.

Figure 4
Interleukin (IL)-17 production from lymph node cell (LNC) and IL-17+ cells during complete Freund's adjuvant (CFA)-induced inflammation in wild-type (WT) and mif−/− mice. LNCs (5 × 105) isolated from popliteal lymph nodes at indicated ...

Discussion

In the present study we have identified MIF as a stimulator of IL-17 expression and production in activated lymphocytes. The stimulatory effect of MIF is, at least partly, dependent on IL-1β and IL-23 production, and involves the signalling pathways of MAP kinase and Jak2/STAT3. The effect of MIF on IL-17 production was evident in mitogen-stimulated and antigen-stimulated lymphocytes.

In order to differentiate from naïve T cells, the Th17 lineage requires the presence of IL-6 and TGF-β7,13 or IL-21 according to the latest findings.21 In contrast, up-regulation of IL-17 production in already established Th17 cells could be achieved through the action of several cytokines, including IL-1β, TNF-α or IL-23.22,23 Interestingly, none of the aforementioned cytokines is able to act alone in vitro and promote IL-17 production. Each of them requires the co-operation of T-cell activators (anti-CD3 and anti-CD28) or other cytokines (IL-2 or IL-15) for optimal induction of IL-17 secretion.2428 Similarly, our results show that MIF alone is unable to stimulate IL-17 production from WT LNCs. However, the presence of Con A provides the necessary support for MIF action, resulting in a dose-dependent and sustained up-regulation of IL-17 production. These data indicate that MIF presumably acts on already differentiated and activated IL-17-producing cells. The importance of MIF for IL-17 production is confirmed in mif−/− mice, as these animals produce less IL-17 upon Con A stimulation. The possibility that MIF is also involved in differentiation of Th17 cells is supported by the observed lower number of IL-17-producing cells within in vitro Con A-stimulated LNCs from mif−/− mice when compared with WT LNCs. In addition, the number of these cells was also markedly decreased in mif−/− LNCs during progression of inflammation induced by CFA, suggesting that inherent MIF deficiency impairs the development of Th17 cells.

Apart from secretion of IL-17, TNF-α, IL-6 and IL-22,7,13 Th17 cells are characterized by expression of the essential transcription factor Ror-γt,20 which can be up-regulated under the influence of IL-1β, IL-6, TGF-β or IL-21, and, as shown in our study, by the action of MIF. However, the expression of this transcription factor was unexpectedly up-regulated in mif−/− LNCs upon Con A stimulation. The observed discrepancy between elevated expression of Ror-γt and low expression of IL-17 mRNA in mif−/− LNCs suggests that other factors essential for IL-17 generation are impaired in MIF-deficient cells. Hypothetically, Ror-γt expression could be negatively regulated by IL-17 itself, meaning that lack of IL-17 would deregulate such negative feedback and allow up-regulation of Ror-γt expression. In addition to Ror-γt, optimal IL-17 expression in T lymphocytes involves several transcription factors including NFAT, STAT3 and NF-κB, as well as activation of MAP kinases.2931 Our results indeed confirm previous findings, as inhibition of all these pathways clearly affects IL-17 production in WT LNCs. However, IL-17 production is only slightly impaired in mif−/− LNCs if MAP kinase (ERK, p38 and JNK) signalling or Jak2/STAT3 signalling is inhibited. Hence, only weak activation of these pathways in mif−/− LNCs was seen in response to Con A stimulation. These data suggest that impaired IL-17 production in mif−/− LNCs is a consequence of the lower activity of MAP kinases (ERK, p38 and JNK) and the Jak2/STAT3 pathway, thus indirectly suggesting that MIF uses these pathways when stimulating IL-17 expression. Usually, JNK activation leads to Jun phosphorylation and subsequent activation of the activator protein 1 (AP-1) transcription factor (containing Jun and c-Fos subunits). The fact that AP-1 activation was absent in WT LNCs suggests that this transcription factor is not involved in IL-17 production and that JNK could exert its effects through activation of other transcription factors such as activating transcription factor-2 (ATF-2), Elk-1, NFAT and p53.32 However, significant inhibition of IL-17 in the presence of MG132 or CsA (blockers of NF-κB and NFAT, respectively) as well as intact activation of NF-κB upon Con A stimulation in mif−/− mice suggests that these pathways mediate IL-17 production in the absence of MIF. Hypothetically, this could mean that MIF stimulates IL-17 expression without utilization of these transcription factors in WT LNCs.

As previously stated, a number of cytokines are involved in differentiation of Th17 cells and enhancement of IL-17 production. According to our results, MIF could also stimulate IL-17 production through regulation of IL-17 inducers, as LNCs from mif−/− mice produced less IL-1β, IL-6, IL-23 and TGF-β in comparison with WT cells. This is consistent with previous results in which either MIF blockade or deletion decreased the production of these cytokines in animal models of experimental myocarditis, sepsis, Trypanosoma cruzi infection or experimental IgA nephropathy.14,15,33,34 One of the main characteristics of MIF is its ability to stimulate TNF-α production in macrophages, thereby potentiating the lethal outcome during septic shock. In contrast, our results suggest that lack of MIF increases TNF-α secretion from LNCs. This could be a consequence of the redundant nature of cytokines; that is, in mif−/− mice TNF-α takes over the missing functions mediated by MIF. As the absence of MIF affects the production of IL-17 inducers, it could be assumed that MIF is involved in both differentiation of Th17 cells and regulation of IL-17 production. Furthermore, the addition of IL-1β, IL-23 and TNF-α up-regulates IL-17 production and expression in mif−/− LNCs to the levels seen in WT cells. In contrast, IL-6 and TGF-β did not affect IL-17 production in mif−/− LNCs. This is consistent with previous reports suggesting that in vitro these cytokines are able to act only on naïve T cells, promoting their differentiation into Th17 after several days of incubation and in the presence of inhibitors of the Th1 lineage.7,13 Therefore, the observed lack of activity could be explained by their inability to operate either in our experimental conditions or on differentiated Th17 cells.

It was recently discovered that, during inflammatory processes induced by M. tuberculosis, production of IL-17 is highly up-regulated.3436 Our results confirm the presence of IL-17+ cells in draining lymph nodes of WT mice treated with CFA, as well as detectable amounts of IL-17 secreted from unstimulated cells. In vitro challenge with either mitogen Con A or Mycobacterium-related antigen PPD resulted in up-regulation of IL-17 production. The fact that the cellular compositions of inflamed PLNs from WT and mif−/− mice are similar regarding the percentages of T and B lymphocytes, macrophages, dendritic cells and NK cells suggests the existence of regular immune cell recruitment to the site of inflammation in the circumstance of MIF absence. Therefore, the observed lower number of IL-17-producing cells in PLNs from mif−/− mice cannot be attributed to impaired homing of these cells to the inflamed nodes. During inflammation triggered by M. tuberculosis, IL-23 has been identified as an inducer of IL-17 production,3436 but it may be hypothesized that several cytokines could contribute to amplification of IL-17 at the site of inflammation. MIF may be one of these cytokines, as mif−/− mice during inflammatory processes triggered by CFA had lower number of IL-17-secreting cells in PLNCs and impaired IL-17 production after in vitro challenge of PLNCs with Con A or PPD.

Macrophage migration inhibitory factor MIF is the key molecule in the pathogenesis of several inflammatory and autoimmune disorders, including delayed-type hypersensitivity, Gram-negative and Gram-positive sepsis, leishmaniasis, glomerulonephritis, colitis, experimental autoimmune myocarditis, type 1 diabetes, rheumatoid arthritis and experimental autoimmune encephalomyelitis (EAE).8,37 MIF actually promotes inflammation by recruiting cells of both innate and acquired immunity and amplifying the production of various pro-inflammatory mediators [nitric oxide, TNF-α, IL-1β and interferon (IFN)-γ]. Similar activity at sites of inflammation in rheumatoid arthritis and EAE has been attributed to IL-17.3,7 Furthermore, instead of Th1, Th17 cells are now denoted as ‘pathogenic’ in the initiation and promotion of various autoimmune disorders.3,7,13 Our demonstration that MIF acts upstream of IL-17 suggests that MIF has an important position at the pinnacle of the inflammatory cascade, promoting pathogenic Th17 cells that mediate autoimmunity.

Inhibition of MIF either by neutralization with specific antibodies or by the use of chemical inhibitors has proved effective for treating inflammatory and autoimmune diseases.37 Apart from diminishing inflammation through down-regulation of innate and Th1 responses, MIF inhibition hypothetically could also interfere with progression of inflammation through impairment of IL-17 production. As neutralization of the first cytokine in the chain reaction has generally proved more effective than inhibition of downstream effects of the cytokine in question, neutralization of MIF has become even more attractive in terms of inhibiting action of Th17 cells. However, given the fact that MIF has such a broad effect on a range of cytokines, MIF neutralization could carry a considerable risk of higher susceptibility to infections, as already shown for other anti-cytokine therapies.38 Nevertheless, the inhibition of MIF expression and/or activities could be a potent means of medical treatment and/or prevention of inflammatory and autoimmune disorders.

Acknowledgments

This work was supported by the Serbian Ministry of Science (Grant No. 143029B). The authors are grateful to Drs Ferdinando Nicoletti (University of Catania, Italy), Yousef Al-Abed and Christine Metz (The Feinstein Institute for Medical Research, USA) for providing mif−/− mice and Dr Marija Mostarica Stojkovic for helpful discussion.

Abbreviations

CFA
complete Freund's adjuvant
Con A
concanavalin A
CsA
cyclosporine A
EAE
experimental autoimmune encephalomyelitis
IFN
interferon
IL
interleukin
LNC
lymph node cell
MIF
macrophage migration inhibitory factor
mif−/−
MIF knockout mouse
PLNCs
popliteal lymph node cells
TGF
transforming growth factor
TNF
tumour necrosis factor
WT
wild type

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