Induction of CAWS-induced vasculitis
CAWS was prepared from C. albicans strain NRBC1385 using a previously described method . Six-week-old male C57/BL6 mice were purchased from Oriental Yeast. The original lines of LPA1-deficient mice  were backcrossed to the inbred C57BL/6 strain for at least 15 generations. We then used these mice as the C57BL/6 background. In order to induce vasculitis, CAWS (1 mg/mouse) was injected intraperitoneally into mice in a volume of 0.2 ml once daily from days 1 to 5. On day 28, the heart was harvested and examined. The experimental protocol for animal experiments was approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University.
Real-time reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was prepared from tissues including the aorta and coronary artery of CAWS-induced vasculitis and normal mice, and first-strand cDNA was synthesized. Quantitative real-time PCR was performed as described previously . cDNA was amplified with primers for LPA1–6 and 18S ribosomal RNA (rRNA) as previously described . 18S rRNA was used as an internal control in order to standardize the amount of sample mRNA, and the relative expression of real-time PCR products was determined.
Paraffin-embedded skin tissues (4-μm-thick sections) from CAWS-induced vasculitis mice, vasculitis patients, and healthy donors were deparaffinized, immersed in 1 mM EDTA at 99–100 °C for 20 min, removed from the heat, and left to stand at room temperature for 20 min, followed by rinsing with a mixture of Tris-buffered saline with Tween 20. Endogenous peroxidase activity was blocked by an incubation in 0.3% H2O2 for 30 min. Sections were then blocked with 1% skim milk for 45 min and stained with a rabbit anti-ATX polyclonal antibody (pAb) (2 μg/ml; Cayman Chemical), -LPA1 pAb (10 μg/ml: Lifespan Biosciences), or normal rabbit IgG (Sigma Aldrich) as an isotype control at room temperature for 45 min. Antibody binding was detected using the Envision kit (DakoCytomation) as described previously .
Treatment of CAWS-induced vasculitis with an LPA1 antagonist
Mice injected with CAWS were treated with an LPA1 antagonist (LA-01 [11, 12] provided by Ono Pharmacological; 200, 60 mg/kg/day, or vehicle) by oral gavage twice a day from days 0 to 28. On day 28, the fixed hearts were embedded in paraffin and sectioned. In order to observe histological changes in the coronary arteries and aorta in detail, step sections in a horizontal direction were prepared every 20 μm. Sections were stained with hematoxylin and eosin (H&E). In order to quantitatively evaluate vascular inflammation, each of the 5 areas (3 aortic root areas and both coronary arteries) was scored on a scale of 0–3 according to the classification system for the areas of cellular infiltration: (a) aortic root (score 0 for no inflammation; 1, cell infiltration < 100 μm in diameter; 2, 100–199 μm in diameter; 3, ≥ 200 μm in diameter) and (b) coronary arteries (score 0 for no inflammation; 1, cell infiltration < 50 μm in diameter; 2, 50–99 μm in diameter; 3, ≥ 100 μm in diameter). The severity of arteritis in each mouse was defined as the sum of the scores of the 5 segments (maximum possible score of 15).
In vitro chemotaxis assay of neutrophils
The Ly-6G-positive neutrophils of wild-type (WT) or LPA1-deficient mice were purified from spleen using MACS microbead-coupled mAbs and magnetic cell separation columns (Miltenyi Biotec) as previously described . The purity of Ly-6G-positive cells was more than 95%. The purified neutrophils from WT mice were incubated with LA-01 (0, 1, and 10 nM) at 37 °C for 30 min in RPMI 1640 medium (Sigma-Aldrich). After being incubated, neutrophils were washed twice with RPMI 1640. Neutrophils (1 × 106 cells/well) were added to the upper well of the transwell with a 3.0-μm pore polycarbonate membrane insert (Corning), while LPA [1-Oleoyl LPA (Cayman Chemical)] (10 μM) was added to the lower wells, and then incubated at 37 °C for 2 h. The number of cells that migrated into the lower well was counted using the Accuri C6 Flow Cytometer (Accuri Cytometers). Triplicates of three independent experiments were performed.
Migration of neutrophils into inflamed vascular walls
Murine Ly6G-positive splenocytes were labeled with Cell Tracker Green CMFDA (5-chloromethylfluorescein diacetate) (Molecular Probes), according to the protocol supplied by the manufacturer, and previously described . The labeled cells (1.0 × 107) were transferred into the tail vein of CAWS-induced vasculitis mice on day 21. Recipient mice were treated with LA-01 (100 mg/kg) or saline 12 h and 30 min before and 12 h after the transfer. Twenty-four hours after the transfer, the number of labeled cells in the synovium was counted under a fluorescent microscope (Biozero) as previously described .
Enzyme-linked immunosorbent assay (ELISA)
Human umbilical vein endothelial cells (HUVECs) were cultured overnight in 48-well plates (1 × 105 cells/well), then incubated with LA-01 (0, 0.2, 1, 10 nM) for 30 min before being stimulated with LPA (10 μM) in FCS-free endothelial growth basal medium-2 at 37 °C for 24 h. CXC chemokine ligand 1 (CXCL1) and IL-8 protein levels in the culture supernatant were assessed using ELISA kits (R&D Systems) according to the instructions supplied by the manufacturer.
The skin tissues of affected lesions were obtained from active vasculitis patients [microscopic polyangiitis (MPA), n = 5; polyangiitis nodosa (PN), n = 3; eosinophilic granulomatosis with polyangiitis (EGPA), n = 1] who fulfilled diagnostic criteria by the Research Group of Intractable Vasculitis, Ministry of Health, Labor, and Welfare of Japan  as well as from healthy controls [n = 3]. Tissues were stained with ATX and LPA1 as described above. The experimental protocol was approved by the Ethics Committee of the Tokyo Medical University, and all subjects provided informed consent according to the Declaration of Helsinki principles.
Data are expressed as the mean ± SEM. A comparison of data from the two groups was conducted by the Student t test. P values less than 0.05 were considered to be significant.