IL-7 activates LEC in vitro function and induces lymphangiogenesis in vivo. (A-G) Functional in vitro assays were performed to analyze the effect of IL-7 on LECs. (A) Overnight treatment with IL-7 significantly enhanced LEC adhesion to fibronectin-coated plates. This response could be reversed by concomitant treatment with CD132 or IL-7Rα blocking antibodies. Notably, the IL-7–induced response was comparable to the one induced by treatment with VEGF-A (positive control). (B) Overnight treatment of LECs with IL-7 led to an upregulation of the fibronectin-binding integrin α5β1, as seen in FACS. Data from 1 of 4 similar experiments are shown. (C-D) Tube formation assays were performed by covering confluent LEC monolayers with collagen type I containing IL-7 (50 ng/mL). (C) Representative images showing the IL-7–induced increase in LEC tube formation. (D) IL-7–induced tube formation could be reversed when performing the assay in presence of CD132 or IL-7Rα blocking antibodies. (E-G) Effect of siRNA-mediated knockdown of IL-7 in LECs. (E) Transfection with siRNA significantly reduced IL-7 mRNA levels in LECs (E) as well as the capacity of LECs (F) to form tube-like structures. (G) Moreover, knockdown of IL-7 levels in LECs reduced LEC migration, as measured in a scratch-wound assay. Data from 1 of 3 similar experiments are shown. (H-I) To analyze the effect of IL-7 on lymphangiogenesis in vivo, a cornea micropocket assay was performed. (H) Representative immunofluorescent micrographs demonstrating ingrowth of new LVs into the cornea 14 days after IL-7 pellet implantation. LYVE-1: red. Scale bar represents 50 μm. (I) A significant difference was observed in the area covered by lymphatics between the treatment and the control group (n = 9 mice per group). *P < .05; **P < .01; ***P < .001. ctr, control; IgG, immunoglobulin G.
