Cd162

Human epithelial cells were cultivated on inserts of a Transwell system (Costar, MA, USA) until reaching confluence. Thereby, cells were seeded on the bottom side of the inserts, so that cells migrate—comparable to the in vivo migration—from the basolateral side through the epithelial monolayer. PMNs from whole human blood migrated through a monolayer of epithelial cells along a chemotactic gradient (fMLP; 20 ng/ml; Sigma-Aldrich, Steinheim, Germany) (Supplemental Figure 3). After 4 h, PMNs in the upper chamber and also migrated PMNs in the lower chamber were collected. To determine PMNs in the epithelial layer, wells were treated with 800 μl Accutase and incubated and washed with 2 ml medium. Absolute cell counts were determined. PMNs were identified CD45 (B183057, BioLegend) and CD66b (E16847-109 eBioscience) positive, adhesion molecules by: CD11a (catalog number 3189709, BD), CD11b (54342, BD), CD31 (4065855, BD), CD54 (4301917, BD), CD162 (61514, BD), CD47 (E15361-103, eBioscience), CD172a (3290897, BD), CD44 (B180554, BioLegend), and CD29 (4262918, BD). As corresponding IgG controls were IgG1 (16651, BD) and IgM (E13114-104, eBioscience) used.

Recombinant proteins and antibodies. Human glycosylated recombinant proteins: C1-INH, CX3CL, CD44, CD45, CD55, CD97, CD99, CD162, TIM1, TIM3, TIM4, fibronectin, E-selectin and human Integrin alpha M beta 2 were obtained from R&D systems Inc. (Minneapolis, MN 55413), while the human glycosylated recombinant proteins: CD34, CD43, CD93, and CD164 were obtained from Sino Biologicals (Beijing 100176 P. R. China). Antibodies used in this study were dye conjugated mouse anti human CD3, CD4, CD8, CD14, CD16, CD56, CD43, CD44, CD45 and CD162 from BD and Invitrogen). Rabbit anti-sialic acid antibody was purchased from Cloud Clone Corp, Houston, TX.