ICAM-1^high Cav-1^low vessels mediate the majority of neutrophil recruitment in response to injection of rhIL-8 in murine dermis. Wild-type murine skin injected with 2.5 μg of rhIL-8 was removed and processed for immunofluorescence at 1.5 hours. Vessels containing aggregates of four or more neutrophils were analyzed for surface expression of PV-1, caveolin-1, ICAM-1, and PECAM-1 (see Materials and Methods). A: Sections of injected skin co-stained with anti-caveolin-1 (green) and a rat anti-mouse neutrophil antibody (Serotec) recognizing a polymorphic 40-kDa antigen expressed by polymorphonuclear cells, but absent on resident tissue macrophages (red) (top left). Arrow depicts a cav-1^low vessel enclosing neutrophils recruited in response to IL-8.Graph depicting intensity of caveolin-1 expression per vessel counted (top right). Sections stained with hamster anti-mouse ICAM-1 (red) and rat anti-mouse neutrophil antibody (Serotec; green, middle left). Graph depicting intensity of ICAM-1 expression per vessel counted (upper middle right). Sections stained with rat anti-mouse PECAM-1 (red) and rabbit anti-neutrophil myeloperoxidase (green, middle left) Graph depicting intensity of PECAM-1 expression per vessel counted (lower middle right). Sections stained with anti-neutrophil myeloperoxidase (green) and anti-PV-1 (red, bottom left). Graph depicting intensity of PV-1 expression per vessel counted (bottom right). Images captured at original magnification ×40. Histograms depict pooled values of vessels counted from at least 24 skin sections from three experiments. B: IL-8 injected murine skin at 1.5 hours co-stained with anti-caveolin-1 (blue), anti-ICAM-1 (red) and anti-neutrophil (Serotec, green) depicting an ICAM-1^high cav-1^low vessel. Arrow depicts a cav-1^low vessel enclosing neutrophils recruited in response to IL-8. Image captured at 60X.

Cav-1^high and cav-1^low expressing HEVs in the murine lymph node. Untreated wild-type murine inguinal lymph nodes were excised and co-stained with an HEV-specific antibody to peripheral node addressin-PNAd (red) and caveolin-1 (green). A: Section from a wild-type inguinal lymph node co-stained with anti-PNAd and anti-caveolin-1 depicting a mixed population of cav^high and cav^low expressing HEVs (arrows) Images captured at original magnification ×10. B: Section from a wild-type lymph node stained with anti-PNAd (red), anti-caveolin-1 (green) and Dapi (blue) depicting an HEV with relatively low levels of caveolin-1 as compared with the level of expression in the surrounding vessels. Images captured at original magnification ×40. C: A cav-1^low HEV (arrow) next to a cav-1^high HEV. Images captured at original magnification ×20. D: Graph depicting the distribution of cav-1^high and cav-1^low in PNAd positive HEVs in the murine inguinal lymph node. E: Inguinal lymph nodes were excised and processed for electron microscopy and HEVs were identified by their distinct cuboidal cellular morphology and rounded appearance. An HEV in the murine inguinal lymph node (scale bar = 2 μm) (top). An endothelial cell from an HEV with few visible caveolae. Arrows indicate coated pits (middle) (scale bar = 200 nm). An endothelial cell from another HEV taken from the same lymph node sample, a large number of caveolae can be detected (bottom) (scale bar = 200 nm).

Knockdown of caveolin-1 in HDMVECs decreases transcellular neutrophil migration. Neutrophils were added to primary HDMVECs transfected with caveolin-1 siRNA and luciferase control siRNA and allowed to migrate (see Materials and Methods). Fixed coverslips containing neutrophils and endothelial cells were stained with antibodies to caveolin-1 (green), VE-cadherin (blue), and CD45 (red). Neutrophils in the process of diapedesis could be distinguished by their perpendicular orientation to the monolayer and ring-like cluster of intense CD45 expression, which marked the point of penetration through the endothelium. Active migration events were scored as paracellular or transcellular based on the presence or absence of colocalization with the junctional marker VE-cadherin, respectively. A: Representative confocal images of HDMVECs transfected with luciferase control siRNA (left) and caveolin-1 siRNA (right) stained with caveolin-1 (green) and VE-cadherin (blue). B: Immunoblot analysis of protein levels of caveolin-1 and ICAM-1 in control and siRNA transfected HDMVECs. β-actin was used to normalize protein loading. C: Top (top) and side (bottom) views of representative confocal images depicting a single neutrophil migrating in a transcellular manner in control siRNA transfected HDMVEC (left) and another neutrophil migrating along a paracellular route (right) through caveolin-1 siRNA transfected HDMVECs. D: Graphs quantifying the percentage of neutrophils migrating in a transcellular and paracellular manner in control and caveolin-1 siRNA transfected HDMVECs. Values represent the mean ± SD from multiple replicate coverslips from two independent experiments. *P < 0.001; Student’s t-test.

Variable levels of ICAM-1 and caveolin-1 in dermal microvasculature. A: PV-1 shows a restricted pattern of expression identifying small vessels in the murine dermis. Sections of wild-type mouse skin were stained with antibodies to PECAM-1, vascular endothelial growth factor receptor 2 and PV-1 (red). Images captured at original magnification ×20. B: Representative confocal images of PV-1 (red) expressing vessels (arrows) colocalizing with vessels (arrows) alternately expressing low (top) or high levels (bottom) of caveolin-1 (green). C: Representative confocal images of PV-1 (green) expressing vessels colocalizing with vessels alternately expressing low (top) or high levels (bottom) of ICAM-1 (red). Images captured at original magnification ×40. D: Graphs depicting the intensity of expression of the endothelial proteins caveolin-1 (left) and ICAM-1 (right) per PV-1 positive vessel counted (see Materials and Methods). Histograms depict pooled values of vessels counted from at least 24 skin sections from three experiments.

ICAM-1^high, cav-1^low, P-selectin-expressing dermal venules mediate the majority of neutrophil recruitment in the skin. A: Untreated wild-type murine skin was excised and stained with rat anti-murine P-selectin (red) and rabbit anti-caveolin-1 (green). A cav^high, P-selectin expressing dermal venule (left). A cav^low (arrows), P-selectin expressing dermal venule (right). Graph depicting the intensity of expression of caveolin-1 per P-selectin positive vessel counted. B: Untreated wild-type skin stained with antibodies to P-selectin (green) and ICAM-1 (red). All P-selectin positive vessels were observed to express high levels of ICAM-1, whereas not all ICAM-1 expressing vessels were positive for P-selectin (unpublished data). C: Wild-type skin stained 1.5 hours postinjection of 2.5 μg rhIL-8. Vessels containing aggregates of neutrophils, as identified by a rabbit anti-murine myeloperoxidase antibody (green) were analyzed for expression of P-selectin (red). All vessels were identified as positive for P-selectin expression. Images captured at original magnification ×40.

Normal neutrophil recruitment and extravasation in the absence of caveolin-1 and caveolae. Wild-type and Cav-1^−/− mice were shaved on their flanks and injected with PBS on one side (top), and 2.5 μg of rhIL-8 on the other (bottom). Three hours after injection, skin was removed, fixed and stained with rat-anti-mouse neutrophil antibody (Serotec) (red) and rabbit anti-caveolin-1 (green). Figure shows a similar number of extravasated neutrophils (red) in the dermis of both wild-type and Cav-1^−/− mice. Images captured at original magnification ×20.