The CCR7 ligand elc (CCL19) is transcytosed in high endothelial venules and mediates T cell recruitment

Abstract

Lymphocyte homing to secondary lymphoid tissue is defined by a multistep sequence of interactions between lymphocytes and endothelial cells in high endothelial venules (HEVs). After initial selectin-mediated tethering and rolling, firm adhesion of lymphocytes requires rapid upregulation of lymphocyte integrin adhesiveness. This step is mediated in part by the HEV-derived chemokine SLC (secondary lymphoid-tissue chemokine, or CCL21) that binds to the CC chemokine receptor (CCR)7 on lymphocytes. However, the CC chemokine ELC (Epstein-Barr virus-induced molecule 1 ligand chemokine, or CCL19) shares the same receptor, and ELC transcripts have been observed in the T cell areas of lymphoid organs. Here, we show that perivascular ELC is transcytosed to the luminal surfaces of HEVs and enables efficient T cell homing to lymph nodes. In situ hybridization on sections of human tonsil showed no ELC mRNA in HEVs, but immunostaining revealed ELC protein in cytoplasmic vesicles of HEV cells. Furthermore, ELC injected into the footpads of mice entered the draining lymph nodes and was presented by HEVs. Finally, intracutaneous injections of ELC in mice lacking functionally relevant ELC and SLC (plt/plt mice) restored T cell trafficking to draining lymph nodes as efficiently as SLC. We conclude that perivascular ELC is transcytosed to the luminal surfaces of HEVs and participates in CCR7-mediated triggering of lymphocyte arrest.

Figure 1

ELC protein is accumulated within HEVs, whereas ELC transcripts are found perivascularly. In situ hybridization with an RNA probe complementary to ELC mRNA. (a) Overview of tonsillar section showing positive cells (black) confined to T cell areas. (b) Higher magnification shows strong signals from perivascular cells partly surrounding the HEV (border delineated), whereas endothelial cells are negative. Single exposures of two-color immunofluorescence staining identifying ELC by means of MAB361 (c and e) and HEVs by means of mAb MECA-79 (d, f, and h). Most MECA79⁺ HEVs contain ELC, whereas MECA-79⁺ELC⁻ vessels are also seen (c and d, arrows). MECA-79⁻ELC⁺ vessels are also found in the T cell zone (c and d, arrowheads). (e) HEV-associated ELC staining is distinctly luminal (arrowheads) and remarkably granular intra-cellularly (arrows), in contrast to the MECA-79 antigen distribution (f ). Staining with an irrelevant control mAb (g) produces no signal together with MECA-79 (h). Scale bars: b and e–h, 15 μm; c and d, 25 μm.

Figure 2

ELC protein is found in cytoplasmic vesicles of HEVs and at the HEV–lymphocyte interface. Silver-enhanced immunogold staining with goat anti–human ELC on sections of Unicryl-embedded tonsillar tissue. (a) Electron micrograph of interface between an HEV cell and lymphocyte shows vesicle-associated gold particles near the luminal plasma membrane of endothelial cells (arrows and high magnification insets). Note also membrane-proximal cytoplasmic vesicles of adjacent lymphocyte (arrowheads). (b and c) ELC labeling in intracellular vesicles (arrows and high magnification inset). Note absence of markers in the interendothelial cleft (b, arrowheads). (d) Staining with irrelevant control antibody. EC, endothelial cell; LC, lymphocyte; PC, pericyte. Scale bars: a and d, 500 nm; b and c, 200 nm.

Figure 3

HEVs possess binding sites for ELC. Autoradiography of tissue sections made after tonsillar organ culture with radioiodinated ELC. (a and b) Strong signals are predominately associated with HEV cells and the HEV lumen. In contrast, flat-walled vessels (c) show no signal. (d) Addition of excess cold ELC abolishes HEV-associated signals. Scale bars: 25 μm.

Figure 4

ELC injected into mice footpads is taken up by HEVs and presented at their luminal surfaces. ¹²⁵I–ELC injected into BALB/c mice is detected by autoradiography in the afferent lymphatics (a, arrow) as well as in the subcapsular sinus (arrowheads) of draining lymph nodes after 90 min. In the T zone, ELC signals are predominately found in the HEVs (b, arrow) and lymphatic sinus (arrowheads). Higher magnification of areas outlined in c and e reveals radiolabel both inside HEV cells and on their luminal surfaces (d and f, arrows), as well as in the surrounding sinus (arrowheads). Injected ¹²⁵I–MIP-1α is preferentially sequestered within the B zone, but HEV cells in the T zone are not labeled (g). Endothelial cells in contralateral lymph nodes contain no radiolabel (ELC-injected mouse shown in h). Scale bars: 25 μm.

Figure 5

Intracutaneously injected ELC reconstitutes T cell homing to lymph nodes in plt/plt mice. plt/plt mice received intracutaneous injections of ELC, SLC (2.5 nmol), or PBS vehicle (50 μl) in the flank skin, followed by transfer of green fluorescent T cells (T–GFP) through the tail vein. (a) Total number of T–GFP cells homed to lymph nodes after 90 min. Significantly more cells homed to the ipsilateral than the contralateral lymph nodes. (b) There was no difference in the circulating level of T–GFP cells (*P < 0.005, **P < 0.003). Data are shown as mean ± SEM. n = 4 animals per chemokine.