Perilymphatic myelomonocytic cell accumulation in D6-deficient mice. (A) Footpad injection of LPS leads to alterations in CD11b⁺Gr1^low and CD11b⁺Gr1^hi myelomonocytic cells in D6-deficient popliteal LNs. Representative FACS plots illustrating the R1 gate displaying the main increase in LN cell populations in LPS-treated, compared with PBS treated mice (first 2 panels). The remaining 4 panels show CD11b/Gr1 differences between WT (top) and D6-deficient (bottom) mice 24 hours after footpad injection of LPS, or control injection of PBS into the contralateral footpad. Numbers in the contour plots represent the percentage of the marked populations in each gate. Note that the 3.38% population in the D6-deficient, PBS, top right-hand quadrant, is not seen in other experiments and therefore does not represent a consistent cellular population in these mice. (B) Popliteal LNs from D6-deficient mice at 2 hours after LPS footpad injection were stained using anti–mouse Gr1 (green) and anti–mouse CD11b (red) monoclonal antibodies. In this representative confocal image, the CD11b⁺Gr1⁺ cells are denoted in yellow (merge). DAPI (blue) was used for nuclear staining. The isotype control staining is shown in the lower of the 3 insets (Iso). Note that both the anti–Gr1 and anti–CD11b antibodies are of the same isotype (rat IgG2b kappa), and so only one isotype control was needed. Images were acquired at ×63 magnification with a scan zoom at 1.0 on a Plan-Apochromat 63×/1.1 oil Ph3 objective. Scale bar represents 20 μm. (C) A dermal lymphatic vessel stained for Lyve 1 (green), CD11b (red), and Gr1 (blue). Separate CD1b⁺ (green arrow), Gr1⁺ (white arrow), and CD11b⁺Gr1⁺ (red arrow) cells are marked. Scale bar represents 50 μm. This image was cropped from a single confocal image, taken on a Zeiss LSM 510 confocal microscope. Original images were acquired at ×63 magnification with a scan zoom at 0.7 on a plan-apochromat 63×/1.4 oil Ph3 objective. (D) Perilymphatic accumulation of DCs in D6-deficient but not WT mice. DCs are labeled with CFSE/green (WT) or TAMRA/red (D6-deficient), and lymphatic vessels stained with Lyve1 (yellow). Nuclei are stained with DAPI (blue). Images were acquired using AxioVision Version 4.6, 12-2006 software on an Apotome fluorescence microscope (Zeiss AxioImager). Images were acquired at Xho magnification on an achroplan 1.0×/0.8w (water) objective. Scale bar represents 20 μm. (E) Mice were inoculated with MOG/CFA and skin examined by microscopy after 3 days. Sections from WT and D6-deficient mouse skins, at the site of inoculation, were stained for CD11c (red), Lyve-1 (yellow), and with DAPI (blue) staining for nuclei. Images were acquired using AxioVision software on an Apotome fluorescence microscope (Zeiss AxioImager). Images were acquired at Xho magnification on an achroplan 1.0×/0.8w (water) objective. Scale bar represents 50 μm. (A-B) Representative of at least 3 replicate experiments. (C-E) Representative of at least 2 replicate experiments. A minimum of 5 mice were used per experimental group for each of the experiments represented in this figure.