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S. M. Nicholls, A. D. Dick; Evidence of Synthesis of Inducible Nitric Oxide Synthase (NOS-2) Protein by Macrophages During Corneal Graft Rejection but Not of Nitric Oxide (NO) Production. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1921.
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To elucidate the role of macrophages in the effector process of corneal graft rejection.
Corneas from LEW or PVG strain rats were transplanted to PVG strain recipients. Animals were killed at early (day 10) and mid (day 15) stages of graft rejection, eyes were removed and snap frozen. Sections were cut and labelled by the immunoperoxidase method with antibodies to NOS-2 or nitrotyrosine (a reaction product of NO). At day 15, further sections were co-labelled with fluorescent antibodies to NOS-2 or nitrotyrosine and CD68 (monocytes/macrophages), CD163 (tissue macrophages), CD11b/c, CD11c, MHC class II or CD161 (NK cells). Other sections were co-labelled with antibodies against NOS-2 or nitrotyrosine and DAF-FM diacetate (to detect NO). Sections were examined by immunofluorescence microscopy with appropriate staining controls.
On day 10, cells expressing NOS-2 were few and restricted to the peripheral stroma of the allograft. By day 15 (extensive corneal endothelial damage evident) such cells were numerous in stroma of 5/5 corneas examined and adhering to the endothelium of 4/5 corneas, again restricted to the donor. NOS-2 was not found in isografts (n=3) or non-transplanted corneas (n=4). NOS-2 was associated primarily with cells expressing CD11b/c and CD11c (although not all such cells expressed NOS-2) and to a lesser extent with cells expressing CD68, CD163 and MHC class II. NK cells did not express NOS-2. Surprisingly, nitrotyrosine and NO were not co-located with NOS-2, being found sporadically around sutures, very occasionally in the peripheral cornea, in scattered conjunctival cells and in a conjunctival leukocyte aggregate. Where found, nitrotyrosine and NO were usually co-localised.
Although NOS-2 protein is expressed by macrophages infiltrating a corneal allograft with endothelial damage, NO is not produced by such cells. NO producing cells are found in conjunctiva and cornea, but not in association with donor cells. Therefore corneal cell cytotoxicity is unlikely to be mediated by NO.
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