Mice
The α2AP deficient (α2AP−/−) mice were generated by homologous recombination using embryonic stem cells, as described previously (23). Wild type (α2AP+/+) and α2AP−/− mice were housed with a fixed 12 hours light and 12 hours dark cycle.
Induction of MAS in mice
MAS was induced in mice as described by Shimazu et al (22). The saline, or CpG (sequence: 5′-tccatgacgttcctgacgtt-3′ [bases are phosphorothioate]) (2.5 mg/kg) (Fasmac, Kanagawa, Japan) and D-(+)-galactosamine hydrochloride (DG) (0.25 g/kg) (Nacalai Tesque, Kyoto, Japan) were injected intraperitoneally on days 0, 2, 4, 7 9 in α2AP+/+ and α2AP−/− mice (10-week-old male mice). In other experiments, the saline or CpG and DG were injected intraperitoneally on days 0, 2, 4, 7 9 in C57BL/6J mice (10-week-old male). In addition, control IgG (100 µg/kg) or anti-α2AP antibodies (100 µg/kg) (R&D Systems, Minneapolis, MN) were injected intraperitoneally on days 4, 7 9 in C57BL/6J mice. On day 10, mice were euthanized, and organs were used for further experiments. Bone marrow cell density and fibrosis area (the blue area of Masson’s trichrome staining) were analyzed by using ImageJ.
Immunohistochemical staining
Immunohistochemical staining was measured as previously described (24). Paraffin sections were labeled with each protein antibodies, and then labeled with FITC-conjugated secondary antibodies (Thermo Fisher Scientific, MA, USA). The signals were detected using a laser-scanning microscope.
Measurement of serum IFN-γ
The collected blood samples were centrifuged to separate serum. The levels of serum IFN-γ was measured by ELISA (Diaclone, Besançon, France). The absorbance of samples was measured by using Multiskan JX (Thermo Labsystems, MA, USA).
Western blot analysis
We performed a Western blot analysis as previously described (25). The liver samples were homogenized and sonicated in lysis buffer (10 mM Tris-HCl buffer [pH 7.5], 1% SDS, 1% Triton X-100, and a protease inhibitor cocktail [Roche, Mannheim, Germany]). The cell samples were harvested and sonicated in the lysis buffer. The samples were separated by electrophoresis on SDS-polyacrylamide gels and transferred to a PVDF membrane. Then they detected by incubating with antibodies to each protein followed by incubation with horseradish peroxidase-conjugated anti-IgG antibodies.
Cell culture
Bone marrow macrophages were obtained as previously described (26). Bone marrow cells were obtained from tibia of 5- to 7-week-old adult mice as previously described (27), and were cultured for 16 hours in Minimum Essential Media a (MEMa) with 10% fetal calf serum (FCS) in the presence of macrophage colony-stimulating factor (M-CSF) (50 ng/ml) at 37 oC in a humidified atmosphere with 5% CO2 / 95% air. Nonadherent cells were harvested as hemopoietic cells and further cultured in MEMa with 10% FCS in the presence of M-CSF (50 ng/ml) for 3 days, the adherent cells were used as bone marrow-derived macrophages. RAW264.7 macrophages (RIKEN BioResource Center, Tsukuba, Japan) and UV♀2 vascular endothelial cells (ECs) (mouse cell line derived from UV-induced angioendothelioma-like tumor) (RIKEN BioResource Center, Tsukuba, Japan) were maintained in Dulbecco modified Eagle medium (DMEM) with 10% FCS at 37 oC in a humidified atmosphere with 5% CO2 / 95% air. Then, the cells were used for further experiments.
Phagocytosis assay
The phagocytosis of cells was evaluated using phagocytosis assay kit (FITC, Cayman chemical, MI, USA). The fluorescence of samples was measured by using M200PRO (ex/em 485/535 nm, Tecan, Männedorf, Switzerland).
Cell invasion assay
Cell invasion was measured using a Chemotaxicell chamber (Kurabo, Tokyo, Japan). Chemotaxicell chambers (5-µm pores) were inserted into wells of culture plates, and RAW264.7 cells were cultured in the presence or absence of IFN-γ, α2AP, and BEL for 24 hours. The invasion cells on the reverse side of the membrane and in the lower chamber were removed with trypsin and counted under a microscope.
Wound healing assay
RAW264.7 cells were cultured in a 24-well plate and allowed to form a confluent monolayer. Then the cells were scratched with a 1 mL pipette tip and then cultured in the presence or absence of IFN-γ, α2AP, and BEL for 24 hours. The migration of cells was analyzed according to the original and final wound zone by using ImageJ.
Cell proliferation assay
The proliferation of cells was evaluated using a cell count reagent SF (Nacalai Tesque, Kyoto, Japan) containing WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) according to the manufacturer’s instructions. The absorbance of samples was measured by using Multiskan JX (Thermo Labsystems, MA, USA).
Cell adhesion assay
Ninety-six-well plates were coated with 1 mg/ml fibrin and incubated at 37°C for 1 hour. The wells were then washed three times with PBS. Cells in DMEM were added to each well and allowed to adhere for 1 hour at 37°C. The wells were washed twice with PBS to remove any unbound cells. Adherent cells were fixed with formaldehyde and stained with Hematoxylin. Cell adhesion was measured at 560 nm using Multiskan JX (Thermo Labsystems, MA, USA).
Statistical analysis
All data were expressed as mean ± SEM. The statistical analysis was conducted with unpaired t-test for two-group comparison, with one‐way ANOVA followed by Tukey test for multiple comparison. Statistical significance was defined as a P value of < 0.05.