Analysis of Macrophage Phenotype in Rejected Corneal Allografts

Abstract

PURPOSE. We investigated the phenotype of macrophages infiltrating rejected corneal allografts. METHODS. We performed allogeneic or syngeneic corneal transplantation in mice, and humanely killed animals at day 28 during allograft rejection when 60% of corneal allografts were rejected. We divided allografts into two groups: grafts with rejection as rejectors and grafts without rejection as nonrejectors, and analyzed for macrophage infiltration and their phenotype using immunohistochemistry. In addition, we investigated the time course of proinflammatory cytokines and chemokines by analyzing corneal grafts at days 7, 28, and 42 using real-time RT-PCR. Also, we assayed human corneal allografts with chronic graft failure. RESULTS. We found that a large number of CD11b(+), F4/80(+), or inducible nitrous oxide synthase cells (iNOS(+)) infiltrated corneal allografts during rejection in mice, while the cells were found rarely in syngeneic or allogeneic grafts that were not rejected. There were rare CD11c(+) cells in rejectors and nonrejectors. Many mannose receptor cells (MRC+) were present in nonrejectors, but not in rejectors. The levels of Th1 cytokines, IFN-gamma, and IL-2 were highly increased in rejectors at day 28, indicating immune rejection. Also, the levels of IL-12a, IL-1 beta, TNF-alpha, CCL3, and iNOS that are produced by activated macrophages were markedly increased in rejectors at day 28, compared to syngeneic grafts or nonrejectors. Similarly, human corneal allografts with chronic graft failure had higher levels of IL-12a, IL-1 beta, CCL3, and iNOS than controls. CONCLUSIONS. Increased numbers of macrophages in rejected corneal allografts implicate that these cells might contribute to the immunopathogenesis of corneal graft rejection.