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

K/BxN serum transfer arthritis was induced in wild-type (WT), MIF -/-, and MCP1 (CCL2)-deficient mice, and in WT mice treated with anti-KC (CXCL1) mAb. In vivo leukocyte trafficking was examined using intravital microscopy, and in vitro neutrophil function was examined using migration chambers and MAP kinase activation.

MATERIALS AND METHODS Animals

Animal experiments were approved by the Monash University Animal Ethics Committee. MIF -/- and MCP-1(CCL2) -/- mice on the C57BL/6 background were used ( 45 , 46 ). Wild-type (WT) C57BL/6 mice were used as controls. K/BxN serum transfer arthritis: induction and disease assessment Serum-induced arthritis was induced by intraperitoneal (IP) injections of mice with pooled K/BxN sera (days 0 & 2, either 8 μl/g body weight – high dose, or 4 μl/g – low dose) terminating on day 8 ( 47 ). In selected studies, anti-KC antibody (R&D Systems) or control Ig (both 50 μg, IP) were given every 2 days from day 0 and the final injection was given on day 6. Ankle thickness was measured with a caliper (Mitutoyo, Kawasaki-shi, Japan) and results expressed as mean change in ankle thickness in mm (thickness on day 8 – thickness on day 0) ( 48 ). Each limb was scored daily on a scale of 0 (no observable redness or swelling) to 5 (severe redness and swelling). The scores of 4 limbs were added together to obtain the clinical index (maximum score = 20). For histological assessment, ankle joint tissues were decalcified and processed. Four μm-thick sagittal ankle sections were stained with safranin-O and counter-stained with fast green/iron hematoxylin. Histological sections were scored 0-3 for each of four parameters: synovitis, joint space exudate, cartilage degradation, and bone damage, and total histological score calculated from the sum of these data as described elsewhere ( 44 ).

Whole joint extract processing

Whole joint extracts were processed using a previously described protocol ( 49 ). Briefly, whole ankle joints were frozen, pulverized with a mortar and pestle and tissue powder shredded using a QIAshredder kit following manufacturer's instructions (Qiagen). RNA and protein were extracted using RNeasy kit (Qiagen). Samples were stored at -80 °C until required for ELISA and real-time PCR analysis. Chemokine ELISA ELISA was performed using a commercially available Quantikine kit (KC; R & D systems) or paired antibodies (MCP-1; R&D systems) following manufacturer's protocols. The detection limits were 15 pg/ml and 31 pg/ml for KC and MCP-1, respectively. Real-time PCR Complementary DNA was synthesized from total RNA (0.5 μg) using Superscript III reverse transcriptase (Invitrogen) and random primers (Invitrogen). PCR amplification was performed on a LightCycler Rotor-Gene 3000 (Corbett Research) using SYBR Green I (Invitrogen). The following primer-specific nucleotide sequences of murine IL-1 (5’-CCCAAGCAATACCCAAAGAA-3’ and 5’-CATCAGAGGCAAGGAGGAAA-3’), IL-1R (5’TGCGGGACACTAAGGAGAAA-3’ and 5’CTCTTCCCAATCCAGTTCCA-3’), TNF (5’-GCCTCTTCTCATTCCTGCTT-3’ and 5’-CACTTGGTGGTTTGCTACGA-3’), IL-6 (5’-TTCCATCCAGTTGCCTTCTT-3’ and 5’-ATTTCCACGATTTCCCAGAG-3), MCP-1 (F5’-CCCCAAGAAGGAATGGGTCC-3’ and 5’GGTTGTGGAAAAGGTAGTGG-3’), and KC (5’GGGTGTTGTGCGAAAAGAAGTG-3’ and 5’-CAAAATGTCCAAGGGAAGCGTC-3’) were used. 18S (5’-GTAACCCGTTGAACCCCATTC-3’ and 5’-GCCTCACTAAACCATCCAATCG-3’) expression was used to normalize expression of respective mRNA species.

Intravital microscopy

Intravital microscopy of the cremaster muscle was performed as previously described ( 32 ). 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). Images were visualized using a video camera and recorded on video-tape for subsequent playback analysis. Leukocyte-endothelial cell interactions (adhesion and emigration) were assessed as described ( 32 ). To assess neutrophil recruitment, the exteriorized cremaster muscle was superfused with recombinant KC (CXCL1) (Peprotech Inc (NJ, USA)) (10 nM, in superfusion buffer), and leukocyte interactions were assessed 0, 30 and 60 mins after commencing KC superfusion ( 50 ).

Neutrophil in vitro migration assays

To isolate mouse neutrophils, bone marrow was extracted from mouse femurs and tibias by lavage, and neutrophils purified by flow cytometry-based cell sorting. Neutrophils were gated by their characteristic size and granularity. The sorted population was > 90% neutrophils based on identification via high Gr1 and M1/70 staining. In vitro neutrophil migration assays were performed using a chemotaxis chamber (Neuro Probe; MD, USA) as described previously ( 51 ). Briefly, 25 μl KC (100 ng/ml in RPMI/1% FCS) 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. Neutrophil suspensions (1×10 5 in 50 μl RPMI/1 % FCS) were applied to the top chamber then incubated for various durations (10-90 mins) in a 37°C incubator in 5% CO 2 . The number of cells that migrated to the lower chamber was counted using a hemocytometer.

Leukocyte adhesion molecule and chemokine receptor expression

Bone marrow derived neutrophils were examined for expression of adhesion molecules and chemokine receptors using flow cytometry. The following antibodies were used (from BD Biosciences, San Diego, CA, unless stated otherwise): anti-CD45 (clone 30-F11), anti-Gr1 (RB6-8C5), anti-CD62L (MEL-14), anti-CD11a (M17/4 – BioLegend), San Diego, CA), anti-CD11b (M1/70), and anti-CXCR2 (242216) (R&D Systems, Minneapolis, MN). FITC-conjugated sheep anti-rabbit IgG was purchased from Silenus. Cells were labelled with appropriate antibody cocktails then analysed on a MoFlo flow cytometer (Dako-Cytomation, Fort Collins, CO). Data were compared to cells labelled in an identical fashion with an isotype control antibody.

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 KC (100 ng/ml). PMA (30 ng/ml, 10 mins) was used as a positive control. Cells were lysed in cell lysis buffer (Cell Signaling Technology, Beverly, MA) containing phosphatase and protease inhibitors. Immunoblotting was performed using antibodies directed against phosphorylated (P) and total (T) p38 and ERK1/2 ( 27 ). 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 performed using the Odyssey system (Li-Cor Biotechnology, Lincoln, NE), and data normalized to total protein content.

Statistical analysis

Data were analysed using the Mann-Whitney test for comparisons of group means of clinical and histological scores, or Student's t-test for comparisons of continuous variables. Results are expressed as the mean ± SEM. For each test, values of p < 0.05 were regarded as statistically significant.