Mechanism of TNF-α–induced leukocyte adhesion and transmigration in vivo. (A) WT or TNFR^−/− mice were given intrascrotal injections of IL-1β (50 ng), TNF-α (300 ng), or saline; and 4 hours later, leukocyte responses of adhesion and transmigration were quantified in cremaster muscle by intravital microscopy as detailed in “Intravital microscopy.” (B) To investigate the contribution of TNF-α receptors on leukocytes or EC in responses to locally administered TNF-α, a cell-transfer technique was used. Bone marrow leukocytes were isolated from donor WT or TNFR^−/− mice, labeled with calcein-AM, and injected intravenously into WT or TNFR^−/− recipient mice. The mice were then injected intrascrotally with saline or TNF-α; and 4 hours later, the cremaster muscle was exteriorized and leukocyte responses of adhesion and transmigration of fluorescently labeled leukocytes were quantified. (C) As in panel B, WT or TNFR^−/− cells were fluorescently labeled and injected intravenously into WT recipients that had received saline or TNF-α intrascrotally. The cremaster muscle was then exteriorized 120 minutes later, and adhesion and transmigration of fluorescent cells were quantified every 30 minutes for a further 120 minutes. Data are corrected for the small levels of responses detected in mice injected with intrascrotal saline and presented as mean ± SEM for n = 3 to 10 mice per group. Statistically significant differences between control and stimulated groups are indicated as follows: ***P < .001. Additional statistical comparisons are indicated as follows: #P < .05, ##P < .01, ###P < .001.