Effect of dual blockade of JAM-A–dependent and PECAM-1–dependent pathways on IL-1β–induced leukocyte transmigration through mouse cremasteric venules as observed by IVM. (A) The expression profiles of JAM-A and PECAM-1 in representative cremasteric venules of WT mice. Unstimulated cremaster muscles were immunostained for the expressions of JAM-A and PECAM-1, as detailed in “Immunofluorescence labeling and analysis of cremaster muscle tissues by confocal microscopy.” Samples were observed using a Zeiss LSM 5 PASCAL confocal laser-scanning microscope. Scale bar equals 50 μm. (B,C) WT, PECAM-1^−/−, or JAM-A^−/− mice were injected intrascrotally with saline or IL-1β (50 ng). Additional groups of mice were pretreated with the anti–JAM-A mAb BV-11, the anti–PECAM-1 mAb Mec13.3, or appropriate control mAbs (all at 3 mg/kg intravenously), as indicated. At 4 hours after the administration of IL-1β, cremaster muscles were exteriorized, and leukocyte transmigration was quantified by IVM, as detailed in “Materials and methods, Intravital microscopy.” (D) Leukocytes were isolated from the bone marrow of WT, JAM-A^−/−, or PECAM-1^−/− mice, labeled with calcein-AM, and injected intravenously into WT, JAM-A^−/−, or PECAM-1^−/− recipient mice. The mice were then injected intrascrotally with saline or IL-1β (50 ng); 4 hours later, the cremaster muscle was exteriorized, and transmigration of fluorescent leukocytes was quantified by IVM. (E) WT, JAM^−/−, or JAM-A^−/−/PECAM-1^−/− double-deficient mice were injected intrascrotally with saline or IL-1β (50 ng); 4 hours later, leukocyte transmigration was quantified. The data represent means (± SEM) of responses quantified from n = 3–12 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).