Figure 1.
Identifying lymphatic sinuses in the human lymph node using specific markers of lymphatic endothelial cells.
Frozen LN sections were probed with an antibody to detect the transcription factor PROX1 to map the lymphatic sinuses in the human LN. The subcapsular and trabecular sinuses were PROX1+ (A). PROX1+ sinuses were also found in the paracortex (B), the medulla (C), and near the hilum (D). CD144 and VEGFR3 were expressed by lymphatic structures however they were not exclusive LEC markers as they were also expressed by BECs (E–G). Lymphatic sinuses in the medulla expressed varying levels of VEGFR3 (G). Similarly CD31 was also expressed by LECs and BECs throughout the LN (H). LECs lacked expression of CD105 (I) and CD59 (J), whilst these markers were clearly expressed by blood vessels. Blue represents DAPI staining of cell nuclei (A–E). C,capsule; T, trabecula; P, paracortex; M, medulla; H, hilum; S, sinus. Scale bars represent 100 µm (A–E) and 50 µm (F–J).
Figure 2.
Heterogeneous expression of LYVE1 and STAB2 by endothelial cells in lymphatic sinuses of human LNs.
Low magnification images demonstrated that LYVE1 expression is restricted to the paracortical and medullary sinuses whilst the other sinuses in the superficial areas lack expression of this marker (A). PROX1+CD31+ LECs of the paracortical and medullary sinuses express LYVE1 (B) and STAB2 (D), whereas the LECs in the subcapsular and trabecular sinuses are negative for LYVE1 (C) and STAB2 (E). Blue represents DAPI staining of cell nuclei (A). P, paracortex; M, medulla; C, capsule; T, trabecula; S, sinus. All scale bars represent 100 µm.
Table 1.
Contrasting phenotypes of the lymphatic endothelial cells and blood endothelial cells in human lymph nodes.
Figure 3.
DCN46+ LECs and APCs can be identified by their expression of PROX1 and CD169 respectively.
A dense body of cells staining for DCN46 was detected in the paracortical and medullary sinuses, which were located by their distinctive pattern of CD144 expression (A–B). In these regions, subsets of DCN46+ cells were found to co-express the exclusive lymphatic endothelial cell transcription factor PROX1 (C) and the APC marker CD169 (D). Simultaneous detection of DCN46, PROX1 and CD169 confirmed that the DCN46+ cells in the paracortical and medullary sinuses comprised two distinct populations: DCN46+PROX1+CD169− LECs and DCN46+PROX1−CD169+ APCs (E). Blue represents DAPI staining of cell nuclei (A). C,capsule; T, trabecula; P, paracortex; M, medulla; H,hilum; S, sinus. All scale bars 50 µm (A–E).
Figure 4.
Assessing the phenotype of the APCs and LECs in the lymphatic sinuses.
Within the sinuses in the paracortex and medulla, the PROX1+CD169− LECs expressed the lymphatic marker LYVE1. The majority of PROX1−CD169+ APCs in the paracortical and medullary sinuses lacked LYVE1 expression (A), although rare cells in these sinuses appeared to co-express CD169 and LYVE1 (B). CD169+ APCs in the subcapsular and trabecular sinuses lacked LYVE1 (B). LYVE1 was expressed by a subset of the DCN46+ cells in the medullary sinuses, which are likely to represent the LECs (C). CD169+STAB2− APCs were closely associated with the CD169−STAB2+ LECs in the paracortical and medullary sinuses (D–E). The subcapsular and trabecular sinuses did not express STAB2 (F), although CD169+ APCs were present. Blue represents DAPI staining of cell nuclei (D–F). C,capsule; T, trabecula; S, sinus. Scale bars represent 50 µm (A, C–E) and 100 µm (B, F).
Figure 5.
CD209 expression in lymphatic sinuses.
CD169+ APCs also expressed CD209 (A–D). Rare CD169+ CD209− APCs were detected in one of the LNs assessed (B: middle panel marked with asterisk). CD209+CD169− cells in the paracortical and medullary sinuses are likely to represent LECs (C–D). Serial LN sections stained with anti-CD209 (E) and DCN46 (F) showed that far fewer cells were stained by anti-CD209 antibody than by DCN46. Blue represents DAPI staining of cell nuclei (A–B, E–F). C,capsule; S, sinus. All scale bars represent 50 µm.