Hydrolase Participation in Allograft Rejection in Rat Penetrating Keratoplasty

Background: A rat model of orthotopic corneal graft rejection was used to investigate the alterations in hydrolase activity within the corneal graft or within cellular infiltrates during acute rejection.

Methods: The distribution of the lysosomal enzymes [acid phosphatase (AP), N-acetyl-beta-D-glucosaminidase (NAG), beta-glucuronidase (beta-Gluc), beta-galactosidase (beta-Gal), dipeptidylpeptidase II (DPPII)] and of the membrane-bound proteases [aminopeptidase M (APM), aminopeptidase A (APA), gamma-glutamyltransferase (GGT), alkaline phosphatase (ALP), dipeptidylpeptidase IV (DP-PIV)] were investigated by histochemical methods in the grafts at 3, 5, 8, 10 and 12 days following allogeneic transplantation. Serial sections of the grafts were also examined for RT1b, CD4, CD4+, CD8, CD11b/c and CD45, in order to determine hydrolase activity within infiltrating cells.

Results: Allogeneic grafts were invaded by macrophages, CD4- and CD8-positive lymphocytes. In contrast, syngeneic grafts, performed as a control, contained occasional lymphocytes and focal aggregations of macrophages around suture sites. The allogeneic cellular infiltrate stained intensely for AP and ALP; moderately for beta-Gluc, NAG and beta-Gal; and mildly for GGT, DPPII and APM in grafts at all postoperative times. Serial sectioning indicated that the majority of the lysosomal hydrolases were located in macrophages; AP, APM and GGT were, however, observed in lymphocytes. Vessel ingrowth could be observed with enzyme staining for AP, beta-Gluc, NAG, ALP, APA and APM. Hydrolase activity in the corneal endothelium served as an indicator of endothelial function during the rejection process.

Conclusion: Changes in normal hydrolase activities in corneal grafts in the rat model indicate decreasing corneal function during the rejection process. Hydrolases released from infiltrating cells contribute to the morphological disruption and, possibly, to graft rejection.