Apoptosis induced by a corneal-endothelium-derived cytokine.

ABSTRACT

PURPOSE: The purpose of this study was to isolate and characterize cDNA clones encoding target proteins for autoantibodies in patients at high risk for transplant rejection. METHODS: A pool of 10 sera from patients at high risk for rejection who had undergone corneal transplantation was used for immunoscreening of an endothelial cDNA library, and the cDNA fragments were subcloned into prokaryotic expression vectors to generate recombinant fusion proteins. Cytotoxicity of recombinant protein was determined by a modified 51Cr-release assay. Apoptosis induced by recombinant protein was determined by fluorescent dye-chromatin fragmentation assay and by TdT-dUTP terminal nick-end labeling (TUNEL) assay. An enzyme-linked immunosorbent assay was used to detect the presence of antibodies to recombinant protein in the sera of high-risk patients undergoing corneal transplantation and of control subjects. RESULTS: Screening of 500,000 plaques identified six positive clones, one of which demonstrated extensive homology with a novel tumor-derived cytokine termed endothelial monocyte-activating polypeptide (EMAP). EMAP was synthesized as a 39-kDa precursor that was proteolytically cleaved to generate an active 22-kDa cytokine. The mature peptide of EMAP alone was capable of inducing the death of cultured endothelial cells, whereas the propeptide was inactive. The protein synthesis inhibitor cycloheximide potentiated EMAP-induced apoptosis in endothelial cells. Cell death by apoptosis was evidenced by DNA fragmentation, extensive surface bleb formation, and chromatin condensation. A statistically significant difference was found in the level of antibodies specific to EMAP between patients at high risk for corneal transplant rejection and control subjects (P<0.001). The antibody levels were elevated in patients with severe graft reaction when compared with patients with no graft reaction (P<0.001). CONCLUSIONS: These studies demonstrated that EMAP is a novel protein in corneal endothelial cells that is capable of inducing programmed cell death. Overexpression of this cytokine could initiate endothelial cell damage leading to stromal edema and corneal decompensation.