Dynamics of Macrophage Infiltration during Corneal Allograft Rejection

Abstract

Purpose: : Corneal allotransplantation is the main treatment to cure blindness caused by diseases of the cornea that result in scarring and opacification. Although corneal allografts enjoy a remarkable degree of immune privilege, immune rejection remains the leading cause of keratoplasty failure. Massive infiltration of macrophages has been observed during corneal graft rejection. Understanding the behavior of macrophages will be important for a more complete elucidation of the mechanisms behind corneal graft rejection. In this project we use MaFia mice (with GFP-labeled macrophages) and in vivo confocal microscopy to investigate macrophage dynamics during allografts rejection in live animals.

Methods: : Orthotopic complete MHC mismatched corneal allotransplants (BALB/c → C57BL/6 Tg MaFia) as well as MHC matched syngeneic transplants (C57BL/6 → C57BL/6 Tg MaFia) were performed. MaFia are EGFP knock-in mice in which macrophages and dendritic cells are GFP+ and engineered to undergo apoptosis upon ligation with a specific dimerizer. Fluorescence confocal longitudinal non-invasive imaging was performed at post operative day (POD)1, POD3, POD7 and POD14 using Leica DMLFSA microscope. Z-stack images, quantitative analyses and 3-D time-lapse movies were generated with Velocity software.

Results: : Many GFP+ cells were seen in both allogeneic and syngeneic grafts at POD1, with increased infiltration at POD3 and POD7. At POD7 all the sutures were removed and the macrophage dynamics changed between allografts and syngeneic grafts. In the allogeneic grafts, the infiltration of macrophages continued to progress until the end of the experiment at POD35 when the grafts were rejected and fibrotic. In contrast, the number of GFP+ cells in the syngeneic grafts gradually reduced once the sutures were removed. By POD28 almost no GFP+ cells could be seen in these grafts. The cells might be left or apoptotic.

Conclusions: : The different dynamics of macrophage infiltration in allografts and syngeneic grafts suggest that macrophages may play an important role during corneal graft rejection. It will be important to characterize the interaction of macrophages with alloreactive T cells that are known to be involved in allograft rejection.

Keywords: cornea: basic science • transplantation • immunomodulation/immunoregulation