Independent roles of macrophage migration inhibitory factor and endogenous, but not exogenous glucocorticoids in regulating leukocyte trafficking

Abstract

Objectives: Macrophage migration inhibitory factor (MIF) promotes leukocyte recruitment and antagonizes the anti-inflammatory effects of glucocorticoids (GC). The aim of this study was to examine whether interaction between MIF and GC underlies the ability of MIF to promote leukocyte-endothelial cell (EC) interactions.

Methods: Intravital microscopy was used to assess leukocyte-EC interactions in wild-type and MIF(-/-) mice following treatment with lipopolysaccharide (LPS), the GC dexamethasone, and inhibition of endogenous GC, using the GC-receptor antagonist, RU486.

Results: Dexamethasone reduced LPS-induced leukocyte interactions in wild-type mice to levels similar to those observed in MIF(-/-) mice not treated with dexamethasone, whereas in MIF(-/-) mice, leukocyte interactions were not further inhibited by dexamethasone. RU486 increased LPS-induced leukocyte adhesion and emigration to a similar extent in both wild-type and MIF(-/-) mice, indicating that endogenous GC exert a similar inhibitory effect on leukocyte trafficking in wild-type and MIF(-/-) mice. Both MIF deficiency and RU486 treatment reduced VCAM-1 expression, while neither treatment modulated expression of ICAM-1 or chemokines CCL2, KC, and MIP-2.

Conclusions: These results suggest that endogenous MIF and GC regulate leukocyte-EC interactions in vivo reciprocally but through predominantly independent mechanisms, and that the anti-inflammatory effect of MIF deficiency is comparable to that of exogenous GC.

Figure 1. Exogenous MIF reverses effect of MIF-deficiency on leukocyte trafficking

Leukocyte adhesion (A) and emigration (B) in cremasteric post-capillary venules of LPS-treated (10 ng) wild-type mice, MIF^−/− mice and MIF^−/− mice which received rMIF (1 ug). Adhesion data were collected 4.5 hrs post LPS whereas emigration data were collected 5 hours post-LPS. N=6 mice/gp. * p < 0.05 vs LPS-treated wild-type mice.

Figure 2. Differential effect of dexamethasone on leukocyte trafficking in wild-type and MIF^−/− mice

Effect of DEX (0.2 mg/kg, i.p.) on LPS-induced leukocyte rolling flux, adhesion and emigration in cremasteric post-capillary venules of wild-type and MIF^−/− mice. Responses were examined at two doses of LPS: A, B & C show parameters from wild-type and MIF^−/− mice treated with 10 ng LPS, in the presence or absence of DEX pretreatment. D, E & F show data from similarly-treated wild-type and MIF^−/− mice treated with 100 ng LPS. N=6 mice/gp. * p < 0.05 for mice treated with LPS & DEX vs. mice treated with LPS alone in the corresponding strain.

Figure 3. GC receptor inhibition increases recruitment in both wild-type and MIF^−/− mice

Effect of GC receptor inhibition via RU486 on leukocyte adhesion (A, C) and emigration (B, D) in LPS-treated wild-type mice (A, B) and LPS and MIF^−/− mice (C, D). Mice were treated with LPS (10 ng) treatment then adhesion data were collected 4.5 hrs post LPS whereas emigration data were collected 5 hours post-LPS. N=6–8/gp. * p < 0.05 for mice treated with LPS & RU486 vs. mice treated with LPS alone in the corresponding strain.

Figure 4. MIF deficiency and GC receptor inhibition do not alter chemokine expression

A, C, E, F: Effect of GC inhibition on LPS-induced chemokine mRNA expression in muscle in the presence and absence of endogenous MIF. Wild-type and MIF^−/− mice were treated with vehicle alone, LPS (10 ng, 4 hrs) + vehicle, or with the GC receptor antagonist, RU486 prior to LPS treatment. Four hrs later, mRNA for CCL2 (A), KC (C), MIP-2 (E) and RANTES (F) were quantitated using real-time PCR and expressed relative to 18s mRNA. Data are shown as mean ± sem of n=4–8 separate experiments. * denotes p < 0.05 vs vehicle. B, D. Effect of GC inhibition on LPS-induced CCL2 (B) and KC (D) protein expression in muscle in the presence and absence of endogenous MIF. Wild-type and MIF^−/− mice were treated with LPS (10 ng, 4 hrs) + vehicle, or with RU486 prior to LPS treatment. CCL2 and KC protein expression were quantitated in homogenised muscle samples using ELISA, and expressed as pg chemokine/mg total protein. Data from individual experiments are shown and horizontal line indicates group mean (n=9−12 separate experiments).

Figure 5. ICAM-1 and VCAM expression in wild-type and MIF^−/− mice, and in the presence and absence of RU486

A, B. Roles of endogenous MIF and GC in regulating LPS-induced expression of mRNA for ICAM-1 (A) and VCAM-1 (B) in muscle. Wild-type and MIF^−/− mice were treated with vehicle alone, LPS (10 ng, 4 hrs) + vehicle, or with RU486 prior to LPS treatment. ICAM-1 & VCAM-1 mRNA were quantitated using real-time PCR and expressed relative to 18s mRNA. Data are shown as mean ± sem of n = 5–8. * denotes p<0.05 vs vehicle. C. Roles of endogenous MIF and GC in regulating LPS-induced VCAM-1 expression in the cremasteric microvasculature. Wild-type and MIF^−/− mice were treated with LPS (10 ng) following pretreatment with RU486, or vehicle. Four hrs later, VCAM-1 expression was assessed using fluorescence microscopy to quantitate deposition of an Alexa 488-conjugated anti-VCAM-1 Ab. Data are shown for wild-type and MIF^−/− mice following treatment with vehicle or RU486 (n=6/group). * p = 0.013 for MIF^−/− vs wild-type vehicle-treated mice. ** p = 0.018 for effect of RU486 in LPS-treated wild-type mice.