Lack of IFN-gamma synthesis in aqueous humor during corneal graft rejection correlates with suppressed nitric oxide production by macrophages.
OBJECTIVE
To investigate cytokine production by leukocytes in aqueous humor (AH) during corneal graft rejection and nitric oxide (NO) production by macrophages as a potential mediator of graft damage.


METHODS
Rats received corneal allotransplants and were killed during acute rejection. Leukocytes in AH that expressed tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin (IL)-10 were quantified by flow cytometry. Isograft and further allograft recipients were killed, and sectioned corneas with conjunctivae were examined by histology for production of inducible nitric oxide synthase (iNOS), NO, and nitrotyrosine (NT).


RESULTS
Between 80% and 90% of T cells, NK cells, and macrophages in AH expressed TNF-alpha, and at least 20% expressed IL-10. However, IFN-gamma was undetectable unless cells were first stimulated in vitro with PMA and ionomycin, which yielded IFN-gamma in 25% of cells. iNOS(+) macrophages were identified in donor cornea and AH, correlating precisely with rejection. Cells producing low levels of NO (NO(dim) cells) were found in donor stroma, but NT(+) cells were rare. Both NT(+) and NO(+) cells were rare in the anterior chamber (AC) or attached to corneal endothelium. NT(+) macrophages that were also NO(bright) were associated with sutures in allograft and isograft recipients and within conjunctivae, either scattered or in leukocyte aggregates.


CONCLUSIONS
IFN-gamma synthesis is lacking in the AC during rejection, correlating with lack of NO but not of iNOS expression. NO does not appear to mediate endothelial cell death. NT and high levels of NO production are associated with nonspecific inflammatory cells.
Department of Clinical Science, Academic Unit of Ophthalmology, School of Medical Sciences, South Bristol, Bristol, United Kingdom. s.m.nicholls@bris.ac.uk
Susan M
SM
Nicholls
Andrew D
AD
Dick
D000818:Animals; D001082:Aqueous Humor / Q000166:cytology / Q000378:metabolism*; D016039:Corneal Transplantation / Q000276:immunology* / Q000473:pathology; D004195:Disease Models, Animal; D005260:Female; D005434:Flow Cytometry; D006084:Graft Rejection / Q000378:metabolism* / Q000473:pathology; D007371:Interferon-gamma / Q000096:biosynthesis*; D016753:Interleukin-10 / Q000096:biosynthesis; D008264:Macrophages / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis; D051381:Rats; D011917:Rats, Inbred Lew; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D014443:Tyrosine / Q000031:analogs & derivatives / Q000096:biosynthesis
D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II