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Apoptosis induced by a corneal-endothelium-derived cytokine
1Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-9142, USA.
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.
Insights
Researchers identified a novel cytokine, endothelial monocyte-activating polypeptide (EMAP), in corneal endothelial cells. Elevated EMAP antibodies in high-risk transplant patients correlate with graft rejection, suggesting EMAP’s role in transplant rejection.
Area of Science:
- Immunology
- Cell Biology
- Transplantation Science
Background:
- Autoantibodies play a role in transplant rejection.
- Identifying target antigens for these autoantibodies is crucial for understanding rejection mechanisms.
Purpose of the Study:
- To isolate and characterize cDNA clones encoding target proteins for autoantibodies in patients at high risk for transplant rejection.
- To investigate the role of a novel cytokine, endothelial monocyte-activating polypeptide (EMAP), in corneal endothelial cells and its association with transplant rejection.
Main Methods:
- Immunoscreening of an endothelial cDNA library using sera from high-risk corneal transplant patients.
- Subcloning of cDNA fragments and expression of recombinant fusion proteins.
- Assays for cytotoxicity, apoptosis induction (TUNEL, DNA fragmentation), and antibody detection (ELISA).
Main Results:
- Identification of a novel cytokine, endothelial monocyte-activating polypeptide (EMAP), homologous to a tumor-derived cytokine.
- Mature EMAP induced apoptosis in cultured endothelial cells, evidenced by DNA fragmentation and chromatin condensation.
- Significantly higher levels of antibodies specific to EMAP were found in high-risk corneal transplant rejection patients compared to controls and correlated with graft severity.
Conclusions:
- Endothelial monocyte-activating polypeptide (EMAP) is a novel protein in corneal endothelial cells that induces programmed cell death.
- Overexpression of EMAP can lead to endothelial cell damage, potentially causing stromal edema and corneal decompensation.
- EMAP and its specific antibodies are implicated as potential biomarkers and contributors to corneal transplant rejection.
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