The Maintenance of Lymphatic Vessels in the Cornea Is Dependent on the Presence of Macrophages

Abstract

Purpose.: It has been shown previously that the presence in the cornea of antigen-presenting cells (APC), such as macrophages (MPS) and lymphangiogenesis, is a risk for corneal transplantation. We sought to determine whether the existence of lymphatic vessels in the non-inflamed cornea is associated with the presence of MPS. Methods.: Flat mounts were prepared from corneas of untreated C57BL/6, CD11b−/−, F4/80−/−, and BALB/c mice, and after suture placement or corneal transplantation, observed by immunofluorescence for the presence of lymphatic vessels using LYVE-1 as a marker of lymphatic endothelium. Innate immune cells were detected in normal mouse corneas using CD11b, F4/80, CD40, as well as MHC-class II. Digital images of the flat mounts were taken using a spot image analysis system, and the area covered by lymphatic vessels was measured using NIH Image software. Results.: The number of spontaneous lymphatic vessels in C57BL/6 corneas was significantly greater than in BALB/c corneas (P = 0.03). There were more CD11b+ (P < 0.01) and CD40+, MHC-class II (+) cells in the C57BL/6 corneas than in BALB/c mouse corneas. MPS depletion via clodronate liposome in C57BL/6 mice led to fewer spontaneous lymphatic vessels and reduced inflammation-induced lymphangiogenesis relative to control mice. Mice deficient in CD11b or F4/80 had fewer spontaneous lymphatic vessels and less lymphangiogenesis than control C57BL/6 mice. Conclusions.: C57BL/6 mouse corneas have more endogenous CD11b+ cells and lymphatic vessels. The endogenous lymphatic vessels, along with pro-inflammatory MPS, account for the high risk of corneal graft rejection in C57BL/6 mice. CD11b+ and F4/80+ MPS appear to have an important role in of the formation of new lymphatic vessels.