Macrophage migration inhibitory factor drives neutrophil accumulation by facilitating IL-1β production in a murine model of acute gout

Abstract

This study evaluated the role of macrophage migration inhibitory factor in inflammation caused by monosodium urate crystals. The concentration of macrophage migration inhibitory factor was increased in synovial fluid of patients with acute gout, and there was a positive correlation between intra-articular macrophage migration inhibitory factor and IL-1β concentrations. In mice, the injection of monosodium urate crystals into the knee joint increased the levels of macrophage migration inhibitory factor in macrophages and in inflamed tissue. The injection of recombinant macrophage migration inhibitory factor into the joint of mice reproduced the inflammatory response observed in acute gout, including histologic changes, the recruitment of neutrophils, and increased levels of IL-1β and CXCL1. Importantly, the accumulation of neutrophils and the amount IL-1β in the joints were reduced in macrophage migration inhibitory factor-deficient mice when injected with monosodium urate crystals. We observed a similar effect when we blocked macrophage migration inhibitory factor with (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid or anti-macrophage migration inhibitory factor. In addition, the blockade of IL-1R and CXCR2 reduced recombinant macrophage migration inhibitory factor-induced neutrophil recruitment. Mechanistically, recombinant macrophage migration inhibitory factor is important for the synthesis of il1β mRNA in vivo and in isolated macrophages. Altogether, macrophage migration inhibitory factor promotes neutrophil accumulation and is important for IL-1β production, which are 2 crucial events contributing to the pathogenesis of acute gout.

Keywords: MIF; arthritis; chemokine; inflammasome.

Figure 1.. Patients with gout present an increase level of MIF.

SF was obtained from patients with acute gout. MIF and IL-1β were quantified by ELISA (A). Pearson correlation analysis shows the association of levels of MIF with IL-1β: r = 0.636; P = 0.026 (B).

Figure 2.. MSU crystals increase the production of MIF in a murine model of gout.

Mice received intra-articular injection of MSU crystals (100 µg/cavity), and periarticular tissue was collected in different times to quantify MIF by ELISA (A). Synovial tissue was collected 1 h after the injection of MSU crystals to quantify MIF by quantitative PCR. V, Vehicle group (B). Control groups received saline (Sal) injection (10 µl). Macrophages recovered from the synovial cavity, 6 h after the injection of saline or MSU crystals, were intracellularly stained for MIF, and the images were obtained by confocal microscopy (C). The mean fluorescence intensity (MFI) of MIF along the cell was measured off-line using Volocity software 6.3 (PerkinElmer), and the fluorescence profile was assessed using ImageJ (NIH; D). BMDMs (5 × 10⁵/well) were stimulated with MSU crystals (300 μg/ml, and MIF proteins were analyzed in the supernatant by ELISA (E). Bars show the means ± sem results in 5 mice/group. *P < 0.05 versus saline group; **P < 0.01 versus saline group and ***P < 0.001 versus saline group.

Figure 3.. rMIF induces neutrophil recruitment and cytokine production in vivo.

Mice received intra-articular injection of rMIF (100 ng/cavity), and total cell and neutrophil accumulation was evaluated 6 and 15 h after the injection (A). Levels of the cytokine IL-1β and CXCL1 into the articular cavity (B) and periarticular tissue (C). The histopathological evaluation of tissue damage (D) and histologic score (E) was investigated 6 h after the challenge with MSU crystals or rMIF. (D) The arrowheads indicate the inflammatory infiltrate, and the asterisk shows hyperplasia of synovial membrane. Control groups received saline injection (10 µl). Bars show the means ± sem results in 5 mice/group. *P < 0.05 versus saline group (ANOVA followed by Newman-Keuls post-test).

Figure 4.. Mif^−/− mice have reduced inflammatory response induced by MSU crystals.

Mif^−/− mice were injected with MSU crystals (100 µg/cavity), and leukocyte recruitment to articular cavity was evaluated 15 h after injection (A and B). Levels of IL-1β were measured in periarticular tissue (C). Control groups received saline injection (10 µl). Bars show the means ± sem results in 5 mice/group. *P < 0.05 versus saline group; #P < 0.05 versus MSU group (ANOVA followed by Newman-Keuls post-test).

Figure 5.. The blockage of MIF reduces inflammatory response induced by MSU crystals.

Mice were treated with ISO-1 (50 µg, intra-articularly), 1 h before the injection of MSU crystals, and the analyses were evaluated 15 h after the injection of MSU crystals. Accumulation of neutrophils into the joint (A). The levels of the cytokine IL-1β in periarticular tissue were measured by ELISA (B). Paw withdrawal threshold assessment (C). Control groups received saline injection (10 µl). Bars show the means ± sem results in 5 mice/group. *P < 0.05 versus saline group; #P < 0.05 versus vehicle group (mice only challenged with MSU crystals; ANOVA followed by Newman-Keuls post-test).

Figure 6.. Accumulation of neutrophils in the joint is dependent on CXCL1 and IL-1β.

Mice were treated with Reparixin (RPTX; 30 mg/kg, s.c.) or IL-1Ra (5 mg/kg, i.p.), 30 min before the injection of MSU crystals (100 µg/cavity) or rMIF (100 ng/cavity), and accumulation of leukocytes (A and C), mainly neutrophils (B and D), in cavity was evaluated. Control groups received saline injection (10 µl). Bars show the means ± sem results in 5 mice/group. *P < 0.05 versus saline group; #P < 0.05 versus MSU group; §P < 0.05 versus rMIF group (ANOVA followed by Newman-Keuls post-test).

Figure 7.. MIF contributes to gouty inflammation through IL-1β synthesis.

Mice received injection of rMIF (100 ng/cavity) to evaluate levels of il1β mRNA in synovial tissue, 1 h later (A). Mice was treated with ISO-1 (50 µg/cavity), 10 min before the injection of MSU crystals (100 µg/cavity), and il1β mRNA levels, 3 h after the challenge was assessed (B). BMDMs were treated with rMIF (200 ng/ml) or LPS (1 µg/ml) for 1 h. The mRNA was extracted from cells for IL-1β measurement (C). Control groups received saline injection (10 µl). Bars show the means ± sem results in 5 mice/group. *P < 0.05 versus saline group; ***P < 0.001 versus saline group; #P < 0.05 versus vehicle group (mice only challenged with MSU crystal; ANOVA followed by Newman-Keuls post-test and Student’s t test).