Binding of lymphocytes and cancer cells to lymphatic sinuses is compromised in MR^−/− mice. (A) Double staining of the WT ear with an anti-MR antibody (MR5D3, red color, left) and anti–LYVE-1 antibody (green color, middle) shows that some of the afferent lymphatics are positive for both markers (arrows pointing out yellow lymphatics in the merge, right). Many MR⁺LYVE⁻ macrophages are also visible. Negative control staining with class-matched negative control antibodies is shown in the inset. (B) Lymphatic sinuses (dark brown) are MR positive in WT mice (arrow, left), while MR^−/− mice completely lack the molecule (arrows, middle). Lymphocytes (small round cells) bind well to HEVs, whereas tumor cells (big cells) show efficient binding only to lymphatic sinuses. The basement membrane outlining an HEV is marked with … and a lymphatic sinus is marked with in this dark field microscopic micrograph. Three small lymphocytes binding with 9 tumor cells to the lymphatic sinus are pointed out by arrows. Because the adherent cells are lying on the top of the tissue section, the focus of the photograph is a compromise between the tissue and adherent cells. (C) Quantification of the binding of tumor cells and lymphocytes to lymphatic sinuses and HEVs in lymph nodes of MR^−/− and WT mice. The results are expressed as number of cells bound to HEVs and lymphatic sinusoids (means ± SEM, n = 6). Bars for panels A and B (right) represent 50 μm and for panel B (left and middle) 0.25 mm. Objectives: Olympus UPlanFl 20×/0.50 Ph 1 for panels A and B (right) and 4×/0.13 for panel B (left and middle). (D) FACS histograms of F9 and MC57G tumor cell lines stained with a negative control (huIg) and different MR-Fc chimeras.