Elevated CXCL1 expression in EHCgp130^−/−venules impairs transendothelial migration of neutrophils to superfused chemoattractants. (A) Whole-mount confocal images of unstimulated cremaster muscle. Mice expressing GFP in myeloid cells (control LysMCre⁺ or EHCgp130^−/−/LysMCre⁺) were injected intravenously with biotin-conjugated rat anti-CD31 mAb and then sacrificed. The cremaster muscle was rapidly isolated, fixed in situ, permeabilized, and stained with secondary Alexa 546–conjugated streptavidin. GFP-positive neutrophils show as green and CD31-positive endothelial cells show as red. The bar represents 50 µm. (B) Three-dimensional reconstructions of confocal microscopic images of trauma-stimulated venules of cremaster muscle from WT and EHCgp130^−/− mice. Neutrophils (green) were stained with anti–neutrophil elastase antibody, and endothelial cells (red) were stained with anti-CD31 antibody. The bar represents 10 µm. (C-D) Recombinant CXCL1 at the indicated concentration was superfused over the cremaster muscle of WT or EHCgp130^−/− mice immediately after exteriorization. After 30 minutes, the number of firmly adherent leukocytes (C) or emigrated extravascular leukocytes (D) was measured. (E) Synthetic peptide WKYMVm at the indicated concentration was superfused over the cremaster muscle of WT or EHCgp130^−/− mice immediately after exteriorization. After 30 minutes, the number of emigrated extravascular leukocytes was measured. (C-E) Some EHCgp130^−/− mice were injected intravenously with heparinase or anti-CXCL1 mAb 30 minutes before cremaster muscle exteriorization; for clarity, some of these data symbols are offset to the right or left of the symbols for untreated EHCgp130^−/− mice. (F-G) WT bone marrow neutrophils in buffer with or without the indicated concentration of CXCL1 were placed in the upper chamber of transwell membranes coated with or without the indicated concentration of ICAM-1 or CXCL1. The bottom chamber contained buffer with or without the indicated concentration of WKYMVm peptide. After 60 minutes, the number of neutrophils migrating into the lower chamber was measured. The data in (A) are representative of 3 independent experiments. The data in (C-E) represent the mean ± SEM from 15 to 20 venules from 4 to 5 mice in each group. The data in (F-G) represent the mean ± SEM from 3 independent experiments. **P < .01.