The role of Robo4 molecule in leukocyte traffic

Leukocyte traffic is an essential part of the immune system as lymphocytes continuously migrate to lymph nodes in search of foreign antigens. Lymphocytes can access the lymph node either via blood vasculature or afferent lymphatics. A potential molecule in regulating these pathways is the Roundabout Guidance Receptor 4 (Robo4). Robo4 is expressed in blood vessels, the medullary sinus of the lymph nodes and Peyer’s patch (PP) lymphatics. Peyer’s patches are located in the small intestine and merely have efferent lymphatics. In this study Robo4 knockout (KO) and wild type mouse models were used to investigate the effects of Robo4 on leukocyte migration. A monoclonal Robo4 murine antibody was generated to explore the Robo4 expression and localization with imaging. Furthermore, the function of Robo4 in the leucocyte migration and B cell turnover in Peyer’s patches was examined. The localization of Robo4 expression was limited specifically to the medullary sinus of the mouse lymph node and lymphatics of the PPs. Robo4 had a small effect on the migration of lymphocytes into the lymphoid tissues via blood vessels in vivo, and it controlled the adhesion between lymphocytes and the medullary sinus in vitro. Moreover, Robo4 contributed specifically to naïve B cell turnover in PPs because the percentage of naïve B cells was increased in the Robo4 KO mice. Interestingly, concomitant increase in PP size or increased apoptosis was not observed as a compensatory mechanism. In conclusion, Robo4 is a new regulator of lymphocyte migration in the lymphoid tissues

Robo4 contributes to the turnover of Peyer’s patch B cells

All leukocytes can get entrance into the draining lymph nodes via the afferent lymphatics but only lymphoid cells can leave the nodes. The molecular mechanisms behind this phenomenon have remained unknown. We employed genome wide microarray analyses of the subcapsular sinus and lymphatic sinus (LS) endothelial cells and found Robo4 to be selectively expressed on LS lymphatics. Further analyses showed high Robo4 expression in lymphatic vessels of Peyer’s patches, which only have efferent lymphatic vessels. In functional assays, Robo4-deficient animals showed accumulation of naïve  B cells (CD19+/CD62Lhi/CD44lo) in Peyer’s patches, whereas no difference was seen within other lymphocyte subtypes. Short-term lymphocyte homing via high endothelial venules to peripheral and mesenteric lymph nodes and Peyer’s patches was also slightly impaired in Robo4 knockout animals. These results show for the first time, selective expression of Robo4 in the efferent arm of the lymphatics and its role in controlling the turnover of a subset of B lymphocytes from Peyer’s patches.</p