Macrophage migration inhibitory factor and CD74 regulate macrophage chemotactic responses via MAPK and Rho GTPase.

Animals

The generation of MIF −/− and CD74 −/− mice has been described elsewhere ( 39 , 40 ). Mice were maintained on the C57BL/6 background and wild-type (WT) C57BL/6 mice were used as controls. All animal experiments were performed in accordance with the regulations of Monash University Animal Ethics Committee. Cytokine/chemokines Recombinant human MIF was produced in an Escherichia coli expression system ( 32 ). Recombinant murine CCL2 was purchased from Peprotech Inc (NJ, USA). Recombinant murine CSF-1 was purchased from R&D Systems (Minneapolis, MN, USA).

Intravital microscopy

Intravital microscopy of the cremaster muscle was performed as previously described ( 25 ). Briefly, the cremaster muscle of anesthetized (ketamine/xylazine) mice was exteriorised onto an optically-clear viewing pedestal and the cremasteric microcirculation was visualized using an intravital microscope (Axioplan 2 Imaging; Carl Zeiss, Australia). Three postcapillary venules (25–40 μm in diameter) were examined for each experiment. Images were visualized using a video camera and recorded on video-tape for subsequent playback analysis. Leukocyte-endothelial cell interactions (rolling, adhesion and emigration) were assessed as described previously ( 25 ). CCL2 (345 ng) or MIF (1 μg) were injected intrascrotally in 200 μL saline 4 hrs prior to examination via intravital microscopy, as previously described ( 25 , 41 ).

Cell isolation

Bone marrow-derived macrophages were generated via differentiation from bone marrow precursors by culturing bone marrow cells for 5 days in RPMI (Sigma-Aldrich, Castle Hill, NSW, Australia) supplemented with 10% FCS, 50 U/mL penicillin, 50 ug/mL streptomycin (Gibco, Invitrogen, Carlsbad, CA) in the presence of CSF-1 (1 ng/mL). Peritoneal macrophages were isolated from mice using a modification of a published technique ( 42 ). Briefly, mice were injected i.p. with 4% thioglycollate (2 mL), and cells harvested 4 d later via peritoneal lavage. Cells were incubated for 1 hr and non-adherent cells discarded. Cells harvested were routinely > 90% macrophages as defined by expression of F4/80 and CD115. In vitro macrophage migration assays In vitro macrophage migration assays were performed using a chemotaxis chamber (Neuro Probe; MD, USA) as described previously ( 43 ). Briefly, CCL2 (10–200 ng/mL in RPMI/0.1% BSA) was added to the bottom chamber, on top of which was placed a 5 μm pore size membrane filter followed by a silicone gasket and the top chamber. Thioglycollate-elicited peritoneal macrophages (1×10 5 in 50 μL RPMI/1 % FCS) were applied to the top chamber then incubated for 2.5 hrs at 37°C, 5% CO 2 . Macrophage migration was determined according to the manufacturer’s instructions. In brief, data were generated for wells without chemokine, in addition to wells incubated with CCL2 (10–200 ng/mL). Unless otherwise stated, data for each strain are shown as cells/field of view (fov) for treated cells following subtraction of cells/fov in untreated wells. Chemokinesis controls were performed with equal amounts of CCL2 in the top and bottom wells.

Leukocyte adhesion molecule and chemokine receptor expression

Bone marrow derived-macrophages were examined for expression of adhesion molecules and chemokine receptors using flow cytometry. Alternatively, whole blood from various mouse strains was lysed using NH 4 Cl, and monocytes identified via staining for CD115 and F4/80, as previously described ( 44 ). The following antibodies were used purchased from BD Biosciences (San Diego, CA) unless stated otherwise: anti-CD45-APC, anti-CD11b-FITC, anti-CD115-PE (eBiosciences, San Diego, CA), anti-CXCR2-APC (R&D Systems, Minneapolis, MN), anti-LFA-1-Alexa 647 (BioLegend, San Diego, CA), and sheep anti-rabbit IgG-FITC (Silenus Laboratories, Heidelberg, Vic., Australia). Rabbit anti-mouse CCR2 was a generous gift from Prof. Shaun McColl (University of Adelaide). PS/2 (anti-α4 integrin) was grown from hybridoma and conjugated to Alexa 488 (Invitrogen, Carlsbad, CA) in-house. Cells were labelled with appropriate antibody cocktails then analysed on a MoFlo flow cytometer (Dako-Cytomation, Fort Collins, CO). Cells were defined as positive relative to the staining levels achieved in cells stained with isotype control antibodies.

Cell lysate preparation and Western blot analysis

Cells were cultured at various timepoints in RPMI/0.1% FCS at 37°C, 5% CO 2 in the presence or absence of CCL2 (100 ng/mL). LPS (1 μg/mL, 30 mins, Sigma-Aldrich) was used as a positive control. Cells were lysed in cell lysis buffer (Cell Signaling Technology, Beverly, MA) containing phosphatase and protease inhibitors (Roche Australia) ( 45 ). Immunoblotting was performed using antibodies directed against phosphorylated (P) and total (T) p38, ERK1/2, and Akt (Cell Signaling Technology, Danvers, MA) as described elsewhere ( 45 ). Briefly, equal amounts of cellular proteins were fractionated on 10% SDS-polyacrylamide electrophoresis gels and transferred to Hybond-C extra nitrocellulose membranes (Millipore, Bedford, MA). Membranes were blocked with blocking buffer then incubated sequentially with appropriate primary and fluorescence-conjugated secondary antibodies. Membrane blot densitometry was measured by scanning using the Odyssey system (Li-Cor Biotechnology, Lincoln, NE). Densitometry ratios were normalized to appropriate total protein content and results expressed as relative density.

RhoA GTPase activity assay

The activity of RhoA GTPase in cell lysates from control and CCL2-treated bone macrophages was assessed using a G-LISA® assay to measure the GTP-bound form of RhoA (Cytoskeleton Inc, Denver, CO). Briefly, bone marrow-derived macrophages from WT, MIF −/− and CD74 −/− mice were incubated with CCL2 (100 ng/mL, 0–30 mins). Cell lysates were incubated on RhoA GTPase affinity plates and colour developed using HRP detection reagent mixture. Samples were read on a DYNATECH MR7000 plate reader (Primera Scientific, Princeton, NJ) and results expressed as a ratio of optical density of treated cells to that of untreated cells. Staining of F-actin fibre formation using Rhodamine-phalloidin Macrophages plated on eight-well chamber slides (Lab-Tek, Brendale, QLD, Australia) were grown for 24 h and then cultured in serum-free RPMI-1640 medium for 16 h. After incubating with CCL2 (100 ng/mL, 30 min) or 10% FCS (10 min), cells were washed once with phosphate-buffered saline (PBS) and fixed with 4% formaldehyde in PBS for 15 min. The fixed cells were washed twice with PBS and permeabilized with 0.5% Triton X-100 in PBS. Cells were then stained with rhodamine-phalloidin (Cytoskeleton Inc., Denver, CO) in PBS and cell nuclei were counterstained with DAPI (Vector Laboratories Inc, Burlingame, CA). After washing three times with PBS, the stained cells were examined using fluorescence microscopy. In some experiments, the role of Rho in F-actin fibre formation was assessed by 4h pretreatment of the cells with the cell permeable Rho inhibitor CT04 (1 μg/mL) (Cytoskeleton Inc) ( 46 ).

Measurement of MKP-1 mRNA via quantitative RT-PCR Total

RNA was extracted from cells using RNeasy mini kit (Qiagen, Doncaster, Vic., Australia) and 0.5 μg RNA was reverse transcribed using oligo(dT) 20 and Superscript III reverse transcriptase (Invitrogen). PCR amplification was performed on a Rotor-Gene 3000 (Corbett Research, Qiagen). Primers used were as follows: MKP-1 (5′-TGTCGATAACTGAAAGCTACAA-3′ and 5′-AACTTCATGCTTGACACACC-3′), and β-actin (5′-TGTCCCTGTATGCCTCTGGT-3′ and 5′-GATGTCACGCACGATTTCC-3′) ( 47 , 48 ). Standard and sample cDNAs were diluted 1:20 and added to individual capillary tubes. Amplification (40 cycles) was conducted in a total volume of 20 μL containing primer concentrations of SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Melting curve analysis was performed at the end of PCR. Relative quantification of target mRNA expression was calculated and normalized to β-actin. The results are presented as the fold induction of mRNA expression relative to the amount present in control samples.

Statistical analysis

Results are expressed as the mean ± SEM. Data were analysed using either Student’s t-test or one-way ANOVA when multiple comparisons were required. For each test, values of p < 0.05 were regarded as statistically significant.