Ccl11

Chemotaxis of BMEos was performed in 24-well chemotaxis chambers containing polycarbonate filters (pore size: 5 µm, Kurabo, Osaka, Japan). The wells of the lower chamber were filled with 5% bovine serum albumin (Wako)-RPMI1640 medium (Sigma-Aldrich) with LPS (Escherichia coli O111:B4; Sigma-Aldrich) and CCL11 (R&D Systems) and incubated for 2 h at 37°C. Eosinophils were then applied to the wells of the upper chambers and incubated for a further 2.5 h. The number of cells migrating from the upper chamber to the lower chamber was counted via the trypan blue-exclusion test. An invasion assay was performed with a BioCoat invasion system (BD Bioscience) in accordance with the manufacturer’s instructions. The insert membrane was coated with a basement membrane extract. Eosinophils (5 × 10⁵ cells) were applied to the wells of the upper chambers. The lower chambers of the 24-well plate were filled with 750 µl of serum-free RPMI1640 medium with 1 µg/ml LPS and 10 nM CCL11 and then incubated for 22 h at 37°C. The number of cells migrating from the upper chamber to the lower chamber was again counted using the trypan blue-exclusion test. The percent of invasion was calculated as the number of cells invading through the Matrigel insert membrane divided by the number of cells migrating through the control insert membrane.

Biopsies specimens were taken from lymph nodes and liver of acute PCM patients before the beginning of treatment. Samples were fixed in 4% formaldehyde and embedded in paraffin. Immunohistochemical analysis was performed using antibodies for MBP, CCL5 (Santa Cruz, CA, USA) and CCL11 (R&D Systems, MN, USA). A polymer-based method was used (MACH 4 Free biotin-Detection, Biocare Medical, USA), according to the manufacturer´s instructions. Brown staining indicated positive reaction.