ENDOTOXINS MODULATE THE AUTOCRINE FUNCTION OF ORGAN-CULTURED DONOR CORNEAS AND INCREASE THE INCIDENCE OF ENDOTHELIAL-CELL DEATH

Background/aims-Bacterial endotoxin is a potent inflammatory stimulato r, the local and systemic responses thereby elicited being mediated vi a the release of cytokines from diverse cell types. Under physiologica l conditions, the corneal endothelium is protected from these toxins b y the epithelial and vascular barriers, but in organ culture these saf eguards are no longer operative, and such substances will therefore ha ve ready access to this cell layer. The consequences of such exposure may take the form of overt damage to the endothelium and/or a more dis creet influence on the cornea's immunological status, the effects of w hich may be realised only after transplantation, by its poor performan ce. The media bathing organ cultured donor corneas were monitored for the presence of various cytokine mediators of the inflammatory respons e before and after incubation with endotoxin, and these data compared with those pertaining to endothelial cell morphology and numerical den sity. Methods-Six pairs of fellow donor corneas were cultured for an i nitial equilibration period of 10 days and then transferred to fresh m edium; thereafter, one of each pair was incubated in the absence, and the other in the presence, of endotoxin (50 mu g/ml = 25 000 units/ml) , and culturing continued for a further 10 days. Samples of medium wer e withdrawn at regular intervals throughout the 20 days and Institut d es screened for the cytokines IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-1 0, GM-CSF, and TNF by ELISA; endothelial cell morphology and area dens ity were assessed on days 0, 10, and 20. Results-Spiking of organ cult ure media with endotoxin led to a substantial increase in the level of IL-8, and a smaller one in that of IL-6, but none of the other cytoki nes were detected. In five of the six stimulated corneas, these change s coincided with an increased incidence of endothelial cell loss, comp ared with that incurred hy the fellow control, and the surviving popul ation also evinced signs of degeneration not seen in the latter. Concl usion-Endotoxin induced increases in the levels of IL-6 and IL-8 appea r to be correlated with endothelial cell loss. Since no adverse effect s of this toxin on long term cultured monolayers of human corneal endo thelial cells have been previously observed, the damage incurred in co rneal organ culture may well be attributable to the influence of cytok ines produced by other corneal cells or a non-intrinsic (passenger) ce ll population, such as macrophages, Langerhans phocytes present under conditions.