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. 2011 Apr;63(4):960-70.
doi: 10.1002/art.30203.

Macrophage migration inhibitory factor regulates neutrophil chemotactic responses in inflammatory arthritis in mice

Leilani L Santos  1 , Huapeng Fan, Pam Hall, Devi Ngo, Charles R Mackay, Gunter Fingerle-Rowson, Richard Bucala, Michael J Hickey, Eric F Morand
Affiliations

Macrophage migration inhibitory factor regulates neutrophil chemotactic responses in inflammatory arthritis in mice

Leilani L Santos et al. Arthritis Rheum. 2011 Apr.

Abstract

Objective: Macrophage migration inhibitory factor (MIF) facilitates multiple aspects of inflammatory arthritis, the pathogenesis of which has been significantly linked to the activity of neutrophils. The effects of MIF on neutrophil recruitment are unknown. This study was undertaken to investigate the contribution of MIF to the regulation of neutrophil chemotactic responses.

Methods: K/BxN serum-transfer arthritis was induced in wild-type (WT), MIF(-/-) , and monocyte chemotactic protein 1 (MCP-1; CCL2)-deficient mice as well as in WT mice treated with monoclonal antibodies to cytokine-induced neutrophil chemoattractant (anti-KC). Leukocyte trafficking in vivo was examined using intravital microscopy, and neutrophil function in vitro was examined using migration chambers and assessment of MAP kinase activation.

Results: K/BxN serum-transfer arthritis was markedly attenuated in MIF(-/-) mice, with reductions in the clinical and histologic severity of arthritis and the synovial expression of KC and interleukin-1. Arthritis was also reduced by anti-KC antibody treatment, but not in MCP-1-deficient mice. In vivo, neutrophil recruitment responses to KC were reduced in MIF(-/-) mice. Similarly, MIF(-/-) mouse neutrophils exhibited reduced chemotactic responses to KC in vitro, despite displaying unaltered chemokine receptor expression. Reduced chemotactic responses of MIF(-/-) mouse neutrophils were associated with reduced phosphorylation of p38 and ERK MAP kinases.

Conclusion: These findings suggest that MIF promotes neutrophil trafficking in inflammatory arthritis via facilitation of chemokine-induced migratory responses and MAP kinase activation. Therapeutic MIF inhibition could limit synovial neutrophil recruitment.

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Figures

Figure 1
Figure 1. Effect of MIF deficiency on K/BxN serum transfer arthritis
WT and MIF-/-mice were injected with K/BxN sera (days 0 & 2) and arthritis severity assessed daily for 8 days. A, B: Representative photomicrographs of ankles of WT (A) and MIF-/- (B) mice at the completion of the experiment (day 8). Clinical index over the 8 day experimental time course (C), and change in ankle thickness (D) as assessed on day 8 in the three strains of mice. Data represent mean ± sem of n=4/group. *, p < 0.05 for WT mice versus MIF-/- mice. E-H: Histological assessment of K/BxN arthritis in safranin-O-stained sections of ankle joints. E, F: Representative joint sections (X200) of WT (E) and MIF-/- (F) mice demonstrating the reduction in histological manifestations of arthritis in MIF-/- mice. G: Scoring (0-3) of synovial sections for synovitis, joint space exudate, cartilage destruction and bone damage, and (H) total histological score (max = 12) for WT and MIF-/- mice. * p < 0.05 and ** p < 0.01 versus WT mice.
Figure 2
Figure 2. Effect of MIF deficiency on synovial chemokine and cytokine expression in K/BxN serum transfer arthritis
Synovial tissue was collected from WT and MIF-/- mice on day 8, and mRNA and protein levels of various molecules analysed by real-time PCR and ELISA, respectively. Shown are mRNA and protein levels of KC (A, B) & MCP-1 (C, D), and mRNA levels of IL-1 (E), TNF (F), IL-1R (G) and IL-6 (H). mRNA data are expressed relative to 18s expression. Chemokine protein levels are expressed relative to total protein. N=4 mice/group. * denotes p < 0.05 versus WT mice.
Figure 3
Figure 3. Effect of KC inhibition on arthritis severity in the low dose K/BxN serum transfer arthritis model
A: Clinical index of arthritis severity in the high dose K/BxN serum transfer protocol in anti-KC and control Ig-treated mice (n=4/gp). B-G: Results of analysis of mice undergoing the low dose serum transfer protocol and treatment with either control Ig or anti-KC (n=4/gp). Clinical index (B) and mean ankle thickness (C) are shown. D: Histological scoring (0-3) of synovial sections for synovitis, joint space exudate, cartilage destruction and bone damage, and total histological score (E) in mice which underwent the low dose serum transfer protocol and anti-KC treatment. F, G: Representative joint sections (X200) of mice treated with control Ig (F) or anti-KC (G) demonstrating the histological manifestations of arthritis in the low dose serum transfer model (F), and their reduction in anti-KC-treated mice (G). N=4 mice/gp. * denotes p < 0.05 versus control Ig-treated mice.
Figure 4
Figure 4. Effect of MIF on leukocyte responses to chemokines in the intact microvasculature
A & B: Leukocyte adhesion (A), and emigration (B) in cremasteric postcapillary venules of WT mice induced by superfusion with KC (10 nM), as determined by intravital microscopy (n=6/gp for each) after 0, 30 & 60 mins of KC superfusion. *, p < 0.05 vs WT; **, p < 0.01 vs WT. C. Comparison of in vitro migratory function of neutrophils from WT and MIF-/- mice. Neutrophils were isolated from bone marrow and migratory responses to KC (100 ng/ml) quantitated after 30, 45 and 60 min. Also shown are 60 min data for controls performed in the absence of KC (No Rx), and with addition of KC (100 ng/ml) to upper and lower wells (Chemokinesis control – ‘Chemo Control’). Data represent mean ± sem of at least six individual assays. *, p < 0.05 vs WT.
Figure 5
Figure 5. Comparison of MAP kinase activation in neutrophils from WT and MIF-/- mice
A-D: Western blot analysis of ERK and p38 phosphorylation in neutrophils from WT, and MIF-/- mice. Neutrophils were exposed to KC (100 ng/mL) for 0-15 min. As a positive control, additional cells were exposed to PMA (30 ng/ml, 10 min; samples shown in separate gel due to space limitations). Subsequently, cell lysates were examined for total p38 MAPK (T-p38) and phosphorylated p38 MAPK (p-p38) (A, B) or total ERK (T-ERK1) and phosphorylated ERK (p-ERK) (C, D) via Western blot. A, C: Representative blots from n=3 independent experiments. B, D: Densitometric analysis showing ratio of p-p38 to total p38 (B) or p-ERK to total ERK (D). Data are shown as mean ± sem. #, p < 0.05 vs untreated WT cells. *, p < 0.05 vs WT at time point shown.
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