Journal: Nature immunology

Article Title: Dendritic cells enter lymph vessels by hyaluronan-mediated docking to the endothelial receptor LYVE-1.

doi: 10.1038/ni.3750

Figure Lengend Snippet: Figure 3 LYVE-1-specific mAbs inhibit in vivo trafficking of DCs through dermal lymphatics. (a,b) The recovery of endogenous DCs in draining LNs in BALB/c mice after injection of rat IgG or anti-LYVE-1 mAb B1/10, C1/8 or mAb2125, during either sensitization (a) or challenge (b) by topical application of oxazolone and FITC. Data show numbers of CD45+FITC+CD11c+ DCs (n = 5). (c) The effect of anti-LYVE-1 mAbs on DC trafficking in C57BL/6 mice 24 h after skin painting with oxazolone and FITC. The anti-ICAM-1 mAb YN1-1 was used as a positive control (n = 4). (d) The recovery of DCs from draining LNs 6–48 h after topical application of oxazolone and FITC in BALB/c mice injected with rat IgG2a or C1/8 (n = 4). (e,f) The entry of CMFDA-labeled BMDCs into dermal afferent lymphatics immunostained with rabbit anti-LYVE-1 after oxazolone skin painting. Magnification, 630×; scale bars, 20 µm (rat IgG and B1/10) or 50 µm (C1/8 and mAb2125) (e). Arrows indicate BMDCs within lymphatic vessel lumens; arrowheads indicate BMDCs restricted to basolateral surfaces of vessels. Data in f are expressed as a percentage of lymphatic-vessel-associated BMDCs (n = 5). (g) The recovery of BMDCs from draining LNs 24 h after rat IgG or mAb injection. (h,i) Results of ex vivo skin crawl-out assays from ear tissue, showing numbers of egressed DCs (h) and remaining dermal DCs (i) (n = 5). Data in plots represent the mean ± s.e.m. n.s., not significant; *P < 0.05, **P < 0.01, Mann-Whitney U-test. Data are from one experiment representative of three (a–c,e–i) or two (d) different experiments.

Article Snippet: Rat anti-mouse LYVE-1 (mAb2125) was from R&D Systems; mAbs B1/10 and C1/8 were generated previously, with mouse LYVE-1 Fc used as the immunogen28,29, as was polyclonal rabbit anti-LYVE-1 (ref. 20).

Techniques: In Vivo, Injection, Positive Control, Labeling, Ex Vivo, MANN-WHITNEY

Journal: Nature immunology

Article Title: Dendritic cells enter lymph vessels by hyaluronan-mediated docking to the endothelial receptor LYVE-1.

doi: 10.1038/ni.3750

Figure Lengend Snippet: Figure 7 DCs adhere to LECs in an HA- and LYVE-1-dependent manner via LYVE-1-enriched transmigratory cups. (a,b) Adhesion of LPS-matured, CMFDA-labeled BMDCs to primary mLEC monolayers after 3 h of incubation in the presence of rat IgG or anti-LYVE-1 mAbs (a), or after 2 h of preincubation with HAase (b), as assessed by fluorescence plate reader. Data are the mean and s.e.m.; n = 4. (c–e) Basolateral-to-luminal transmigration of LPS-matured, fluorescently labeled BMDCs across mLEC monolayers grown on the undersurface of Transwell filters, quantitated over time by fluorescence plate reader in the presence of rat IgG, B1/10 (c), C1/8 (d) or mAb2125 (e). Data are the mean ± s.e.m.; n = 4. (f–h) Confocal microscopy images of cultured primary mLEC monolayers immunostained with rabbit anti-LYVE-1 (f), viewed 3 h after the addition of fluorescently labeled BMDCs (CMFDA), and counterstained with DAPI (g,h). LYVE-1-enriched cups surrounding individual DCs (asterisks) are indicated by arrows (g). A digital zoomed-in view of an orthogonal view is shown in h (magnification, 630×; scale bars, 20 µm). *P < 0.05, Mann-Whitney U-test. Data and images are from one experiment representative of three separate experiments (a–h).

Article Snippet: Rat anti-mouse LYVE-1 (mAb2125) was from R&D Systems; mAbs B1/10 and C1/8 were generated previously, with mouse LYVE-1 Fc used as the immunogen28,29, as was polyclonal rabbit anti-LYVE-1 (ref. 20).

Techniques: Labeling, Incubation, Fluorescence, Transmigration Assay, Confocal Microscopy, Cell Culture, MANN-WHITNEY