D6 prevents association of inflammatory chemokines with LN endothelial cells. (A) ELISA measurements of hCCL3-L1 and hCXCL8 in inguinal LNs 1 hour after injection into subcutaneously air pouches of WT and D6-deficient mice. LNs were homogenized, on ice in 200 μL PBS and cellular debris pelleted before ELISA measurement using the supernatant. NS indicates not significant. (Bi) Two representative confocal images illustrating the association of AF647-CCL5 (injected into subcutaneously air pouches) with the subcapsular sinus of inguinal LNs in D6-deficient (KO), but not WT, mice. An arrow indicates the sites of AF647-CCL5 positivity (shown as a cropped image in the inset). Original magnifications ×20 with a scan zoom at 0.7 on a Plan-Neofcuar 20×/0.5 Ph2 objective. 4,6-Diamidino-2-phenylindole (DAPI, blue) was used as a nuclear stain for cells in the LN. Scale bar represents 100 μm. (Bii) Two representative confocal images demonstrating the lack of CXCL8 accumulation in the subcapsular sinus of WT or D6-deficient (KO) LNs. Original magnifications ×40 with a scan zoom at 0.7 on a Plan-Neofcuar 60×/1.3 oil objective. Scale bar represents 50 μm. (C) Frozen skin sections were stained with anti–mouse Lyve-1 monoclonal antibody (green) and anti–mouse CCL2 polyclonal antibody (red). Representative confocal images showing a CCL2-containing dermal lymphatic in inflamed skin of (i) a D6-deficient and (ii) a WT mouse 24 hours after TPA painting. Images were acquired at 63× magnification with a scan zoom at 1.7 on a Plan Apochromat 63×/1.4 oil Ph3 objective. Scale bar represents 20 μm. (D) The frequency of CCL2⁺ lymphatics in the dermis of inflamed WT and D6-deficient skin per 600 μm of each section. (E) Relative CCL2 transcript levels in the skins of WT and D6-deficient mice 24 hours after TPA administration. PCR used the following primer sets: GAPDH forward, 5′-TGAACGGGAAGCTCACTGGC-3′; reverse, 5′-TCCACCACCCTGTTGCTGTAG-3′; CCL2 forward, 5′-TGAGTAGGCTGGAGAGCTACA-3′; reverse, 5′-TCACTGTC- ACACTGGTCACTC-3′. PCR was performed as described previously.²¹  Data are from > 3 (A-B) and 2 (C-E) independent experiments with n > 3 mice per experimental group for each individual experiment.