Figure 4
Figure 4. The role of leukocyte and/or endothelial JAM-A in leukocyte responses induced by IL-1β and I/R injury in mouse cremasteric venules. To investigate the potential roles of leukocyte and endothelial-cell JAM-A in leukocyte transmigration in vivo, a cell-transfer technique was used. (A) Bone marrow leukocytes were isolated from WT or JAM-A−/− mice, labeled with calcein-AM, and injected intravenously into WT recipient mice, as detailed in “Materials and methods, Quantification of fluorescent leukocyte responses.” The mice were then injected intrascrotally with saline or IL-1β (50 ng); 4 hours later, the cremaster muscle was exteriorized, and responses of fluorescent leukocytes, firm adhesion (left panel), and transmigration (right panel) were quantified by IVM. (B) In the reverse experiment, leukocytes were isolated from the bone marrow of WT mice, labeled with calcein-AM, and injected intravenously into WT and JAM-A−/− mice. With respect to the latter, to investigate the potential role of leukocyte JAM-A in the noted residual transmigration response, the experiment was repeated in an additional group of mice where the recipient mice were pretreated with the anti–JAM-A mAb BV-11 (3 mg/kg, intravenously) 10 minutes prior to the injection of WT leukocytes (▨). The mice were then injected intrascrotally with saline or IL-1β (50 ng); 4 hours later, the cremaster muscle was exteriorized, and responses of fluorescent leukocytes, firm adhesion (left panel), and transmigration (right panel) were quantified by IVM. (C) WT or eJAM−/− mice were injected intrascrotally with saline or IL-1β (50 ng) or subjected to sham-operation or I/R injury, as detailed in “Materials and methods, Quantification of fluorescent leukocyte responses.” Leukocyte responses of firm adhesion (left panel) and transmigration (right panel) were quantified at 4 hours after intrascrotal injection of saline or IL-1β, and at 120 minutes after initiation of reperfusion for the I/R studies. The data represent means (± SEM) of responses quantified from n = 3–8 mice/group. Statistically significant differences between control and stimulated groups are shown by asterisks (*P < .05, **P < .01, and ***P < .001). Additional statistical comparisons are indicated by lines and number signs (#P < .05, ##P < .01).

The role of leukocyte and/or endothelial JAM-A in leukocyte responses induced by IL-1β and I/R injury in mouse cremasteric venules. To investigate the potential roles of leukocyte and endothelial-cell JAM-A in leukocyte transmigration in vivo, a cell-transfer technique was used. (A) Bone marrow leukocytes were isolated from WT or JAM-A−/− mice, labeled with calcein-AM, and injected intravenously into WT recipient mice, as detailed in “Materials and methods, Quantification of fluorescent leukocyte responses.” The mice were then injected intrascrotally with saline or IL-1β (50 ng); 4 hours later, the cremaster muscle was exteriorized, and responses of fluorescent leukocytes, firm adhesion (left panel), and transmigration (right panel) were quantified by IVM. (B) In the reverse experiment, leukocytes were isolated from the bone marrow of WT mice, labeled with calcein-AM, and injected intravenously into WT and JAM-A−/− mice. With respect to the latter, to investigate the potential role of leukocyte JAM-A in the noted residual transmigration response, the experiment was repeated in an additional group of mice where the recipient mice were pretreated with the anti–JAM-A mAb BV-11 (3 mg/kg, intravenously) 10 minutes prior to the injection of WT leukocytes (▨). The mice were then injected intrascrotally with saline or IL-1β (50 ng); 4 hours later, the cremaster muscle was exteriorized, and responses of fluorescent leukocytes, firm adhesion (left panel), and transmigration (right panel) were quantified by IVM. (C) WT or eJAM−/− mice were injected intrascrotally with saline or IL-1β (50 ng) or subjected to sham-operation or I/R injury, as detailed in “Materials and methods, Quantification of fluorescent leukocyte responses.” Leukocyte responses of firm adhesion (left panel) and transmigration (right panel) were quantified at 4 hours after intrascrotal injection of saline or IL-1β, and at 120 minutes after initiation of reperfusion for the I/R studies. The data represent means (± SEM) of responses quantified from n = 3–8 mice/group. Statistically significant differences between control and stimulated groups are shown by asterisks (*P < .05, **P < .01, and ***P < .001). Additional statistical comparisons are indicated by lines and number signs (#P < .05, ##P < .01).

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