Blocking antibody to JAM-C increases number of L-selectin⁻ inflammatory monocytes in the blood of mice with peritonitis. (A) L-selectin expression on purified human monocytes was lost after transmigration. Transmigrated monocytes were rescued by trypsinisation and labeled with anti–L-selectin FITC. As a control, analysis by flow cytometry of monocyte suspensions treated with trypsin at the same concentration for the same time remained L-selectin⁺ (top panel), but rescued monocytes from trypsinized HUVEC-monocyte suspensions were L-selectin⁻ (bottom panel). (B) L-selectin expression on peripheral blood mouse inflammatory monocytes at 30 minutes after thioglycollate-induced peritonitis is shown. Example profiles are shown of L-selectin expression analysis on peripheral blood inflammatory monocytes (Mac-1⁺/F4/80⁺/Gr1⁺) from an individual mouse treated with either a control antibody (top panel) or a blocking antibody to JAM-C (bottom panel). L-selectin⁻ monocytes are gated on R1 and L-selectin⁺ monocytes are gated on R2. Numbers represent percentage of total gated population. (C) Mice treated with the JAM-C–blocking mAb H33 (n = 5) showed a significant increase in L-selectin⁻ inflammatory monocytes in the blood (median, 3.61%) at 30 minutes compared with an isotype control antibody (median, 1.24%; n = 6). The median value for each experimental group is marked with a horizontal bar. Statistical analysis was conducted using the Mann-Whitney U test (*P = .03).