The immunosuppressant FK778 prevents adhesion molecule upregulation and inhibits endothelium-lymphocyte interaction in vitro

Objectives: In our recent studies, FK778 reduced endothelial adhesion molecule upregulation after rat cardiac transplantation, reduced mononuclear graft infiltration and significantly prolonged graft survival. In this study, the in-vitro effects of FK778 on endothelial cells (EC), lymphocytes and EC-lymphocyte interaction were investigated.

Material and Methods: Purified BN aortic endothelial cell cultures were incubated with FK778 in low (50mg/l) or high (200mg/l) concentration according to the FK778 plasma levels found in the in-vivo experiments. EC ICAM-1 and VCAM-1 expression was stimulated with TNF-α and adhesion molecule expression was quantified by immunofluorescence, FACS analysis and western-blotting. Purified Lewis lymphocytes were incubated with the same FK778 concentrations and were stimulated via TCR/CD28 signals. Lymphocyte CD25 expression was quantified by FACS analysis. Uridine addition was used in all assays to reverse the pyrimidine-synthesis blockade. Lymphocyte-EC interaction was assessed by cell culture adhesion assays and micromanipulator-assisted single-cell adhesion assays. TNF-α-receptor binding assays were performed using radio-labeled TNF.

Results: TNF-α stimulation significantly increased endothelial adhesion molecule expression. FK778 incubation dose-dependently inhibited TNF-stimulated upregulation of ICAM-1 and VCAM-1 in-vitro which was not affected by uridine addition. Lymphocyte CD25 surface expression was markedly induced by TCR/CD28 costimulation and was dose-dependently inhibited by FK778 incubation. The addition of uridine did not reverse this effect. FK778 dose-dependently attenuated Lew lymphocyte adhesion in both adhesion assays. FK778 did not interfere with TNF receptor binding.

Conclusions: FK778 directly reduces endothelial adhesion molecule upregulation, inhibits lymphocyte activation, and attenuates lymphocyte-endothelium interaction in-vitro. These effects can be separated from its blockade on pyrimidine synthesis and are not mediated via TNF-receptor blockade.