Keratocyte Apoptosis and Failure of Corneal Allografts

Abstract

Murine models of high-risk and low-risk corneal transplantation were used to determine the role of keratocyte apoptosis in the failure of orthotopic allogeneic corneal transplants. Normal (low-risk, low-rejecting) and inflamed/vascularized (high-risk, high-rejecting) BALB/c recipient beds received fully mismatched C57BL/6 corneal allografts. Apoptosis was detected in the corneal stroma at various time points using an in situ terminal deoxynucleotide tranferase-mediated dUTP nick-end labeling assay, and ex vivo via Western analysis for active caspase-3. Apoptosis was also measured in a (donor-type) C57BL/6 keratocyte cell line after stimulation of Fas or via use of various pro-inflammatory cytokines. Significantly more apoptotic cells were present in the stroma of rapidly rejecting high-risk corneal allografts compared with low-risk grafts. Apoptotic cells were shown to be nearly uniformly CD45– and hence of a non-hematopoetic lineage. Apoptosis, however, was not present in highly inflamed but ungrafted corneas. Apoptosis was induced in keratocytes in vitro by dual stimulation with agonistic Fas mAb and either interleukin-1β or tumor necrosis factor-α. Apoptosis of resident non-bone marrow-derived fibroblastic cells of the corneal stroma is strongly correlated with the failure of corneal allografts, particularly in the highly inflamed microenvironment of the high-risk allograft.