Macrophage migration inhibitory factor enhances lipopolysaccharide-induced fibroblast proliferation by inducing toll-like receptor 4

In order to unveil the mechanism of the co-stimulatory effect of MIF on LPS-induced fibroblast proliferation, the following hypothesis was proposed (Fig. 1).

LPS induced fibroblast proliferation via TLR4, and MIF enhanced TLR4 expression. Together, MIF has co-stimulatory function to enhance LPS-induced fibroblast proliferation via Toll-like receptor 4 induction. Blank and solid stars indicated naturally expressed and MIF-induced TLR4, respectively.

L-929 cells were cultured with DMEM containing 10 % FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere with 5 % CO₂ (Forma Series II CO2 incubator, Thermo Electron Co.). Mouse fibroblast suspension was adjusted to 5 × 10⁴/ml, and 200 μl were seeded per well in 96-well plates. After 1 h incubation (to allow cells to adhere to plates), cells were treated with LPS at various concentrations for 48 h. Then, cell proliferation was assesses by adding 20 μl CCK-8 reagent in each well, followed by incubation at 37 °C for 2 h. Signals were spectrophotometrically measured at 450 nm on a full wavelength microplate analyzer (Molecular Device); data were recorded as optical density (OD). The procedure described above was also used to evaluate the effects of various concentrations of MIF in the presence or absence of 25 μg LPS.

A total of 1 × 10⁶ L-929 cells were seeded on sterile coverslips (0.13 mm thickness) in each 6-well plates, in 1.5 ml of medium. 40 h later, coverslips were gently washed 3 times with PBS and fixed with 4 % paraformaldehyde at room temperature for 20 min. After PBS wash, blocking was carried out with donkey serum (1:10) for 2 h at 37 °C; then, the coverslips were sequentially incubated with rabbit anti-mouse TLR4 antibody (1:50 dilution; 4 °C overnight) and FITC-conjugated donkey anti-rabbit IgG secondary antibody (1:100; 37 °C 1 h). The coverslips were mounted with sealing medium, followed by analysis on a laser confocal scanning microscope (Zeiss LSM-710).

L-929 cells were seeded 5 × 10⁵ per well in 6-well plate treated with MIF at three concentrations, and two replicates per concentration were applied. 48 h later, the medium in each well was aspirated, the attached cells were washed with PBS; after addition of 1 ml Trizopure, total RNA was extracted. RNA purity and concentration were assessed by OD measurement at 260/280 nm on spectrophotometer (Thermo Nanodrop 2000). cDNA was obtained with reverse transcription kit (ReverTra Ace-alpha), and real time PCR was carried out with Toyobo thunderbird SYBR qPCR Mix (quantitative PCR amplification kit). Specific primers were designed and synthesized by Invitrogen Biotechnology (China): TLR4 [NCBI(GenBank):NM_021297], forward, 5'- AGTTTAGAGAATCTCTGGTGGCTGTG-3', and reverse, 5'- TTCCCTGAAAGGCTTGGTCT-3'); β-actin [NCBI(GenBank):NM_007393.3], forward, 5'- CTGAGAGGGAAATCGTGCGT-3', and reverse, 5'-CCACAGGATTCCATACCCAAGA-3'. Thermal cycling conditions included: initial denaturing step at 95 °C for 1 min; 40 cycles of 95 °C for 15 s, 58 °C for 20 s and 72 °C for 20 s; final extension at 72 °C for 5 min. All samples were tested in triplicate on 96-well PCR plate. Relative expression of the TLR4 gene was calculated using the △△Ct method.

L-929 cells were seeded at a density of 5 × 10⁵ per well in 6-well plate, with MIF added at 375 ng/ml for 48 h. Then, cells were pelleted centrifugation at 300× g for 10 min, and resuspended in 100 μl buffer solution. Afterwards, 10 μl PE-conjugated TLR4-MD2 (CD284) antibody was added for 10 min at 4 °C, and cells were analyzed on flow cytometer (BD Accuri C6).

Statistical analyses were performed with SPSS software (SPSS 19, Inc, Chicago, IL, USA). Data are mean ± SEM, and one-way ANOVA with dunnett's test was used for group comparisons. *P < 0.05 was considered statistically significant.

After LPS 48 h of incubation, OD450 value were significantly higher in the two LPS-treated groups (5 and 25 μg/ml) compared with control group (P < 0.05), indicating that LPS induced cell proliferation in mouse fibroblasts. Importantly, cell proliferation was induced by LPS in a dose-dependent manner (Fig. 2).

TLR4 expression in the macrophages is downregulated by MIF depletion, leading to the hypo-response of macrophage to LPS[5]. To assess the regulatory role of MIF in fibroblast TLR4 expression, we first examined the expression profile of TLR4 in the mouse fibroblast cell line L-929. Under a confocal microscope, no FITC signal was observed, although clear cell outline was present (Fig. 3, left panel); meanwhile, a spindle-shaped fibroblast outline was high-lighted by FITC signal. The overt staining of cellular membrane suggested that TLR4 was widely expressed and uniformly distributed on the cellular membrane of mouse fibroblasts (Fig. 3).

Mouse fibroblast cells were stimulated by MIF at 375 ng/ml. Real-time fluorescence quantitative PCR showed that TLR4 mRNA levels were increased by about 33 %. This was further confirmed by flow cytometry, which showed about 20 % increase in TLR4 fluorescence signals after treatment with 375 ng/ml MIF in comparison with control cells (Fig. 4).

After treatment with 25 μg/ml LPS, fibroblast proliferation was induced. Meanwhile, MIF could boost LPS-induced fibroblast proliferation. Interestingly, fibroblast cell proliferation increased with MIF dose. These data indicated that LPS alone directly induced fibroblast proliferation, which was further enhanced by MIF. At the concentrations used in these experiments, MIF had no direct stimulating effect on fibroblast proliferation. The stimulatory effect of MIF on fibroblast proliferation was dependent on LPS. These findings strongly support our hypothesis (Fig. 5).