(A–D) TNF-α–KO or control WT macrophage chimeras were generated to test whether macrophage-derived TNF-α promotes PMN TEM. (A) Representative whole-mount confocal microscopy images show near-complete loss of host CX3CR1 macrophages and repopulation by donor (nonfluorescent, F4/80⁺) macrophages 8 weeks following grafting. Scale bar: 25 μm. (B) Quantification of total (F4/80⁺) repopulating donor macrophages, (C) VAMs per vessel length, and (D) reduction in host CX3CR1 macrophages 8 weeks following grafting. (E) Representative whole-mount confocal microscopy images and (F) quantification, showing reduced ICAM-1 expression (in situ fluorescence labeling, red) relative to PECAM-1 (CD31, blue) in TNF-α–KO macrophage chimeras. Scale bar: 20 µm. (G) Quantification of ICAM-1 hot spots per vessel length in TNF-α–KO and WT macrophage chimeras. (H) Representative time lapse intravital microscopy (IVM) images show decreased rolling (yellow arrows) and increased PMN adhesion (green arrows) in WT chimeras, whereas PMN adhesion was substantially reduced in TNF-α–KO macrophage chimeras. Scale bar: 20 μm. (I) Quantification of PMN adhesion from IVM, (J) PMN rolling (per 30 seconds recordings), (K) rolling velocity, and (L) extravasated tissue PMNs (white arrows in H). (M–O) WT mice were pretreated with IgG control or neutralizing Abs against TNF-α (400 μg, i.p.), TNFR2, or ICAM-1 (400 μg, i.v.). (M) Quantification of ICAM-1 hot spots per vessel length using in situ fluorescence labeling. (N) Representative whole-mount confocal microscopy images and (O) quantification, showing a reduced number of extravasated PMNs with TNF-α/TNFR2/ICAM-1 neutralization (PMNs stained for Ly6G, red). Scale bar: 25 μm. For whole-mount preparations, images are representative of n = 6 independent experiments. For IVM, n = 3–5 mice per condition. *P < 0.05, **P < 0.01, ***P < 0.001. Two-sided Student’s t test and 1-way ANOVA with Tukey’s multiple comparison test. Data are presented as mean ± SEM.