ATGs modulate the endothelial activity and the immune cell adhesion

Introduction: ATG-Fresenius, a highly purified rabbit polyclonal anti-human T-lymphocyte immunoglobulin resulting from immunisation of rabbits with the Jurkat T-Lymphoblast cell line is currently used for the prevention of acute rejection in patients receiving solid organ-transplants. The aim of this study was to investigate the in vitro activity of ATG-Fresenius that underlies its activity in prevention of ischemia-reperfusion injury in solid organ transplantation.

Material and methods: Human vascular endothelial cells (HUVEC) and peripheral blood mononuclear cells (PBMCs) were isolated from umbilical vein or peripheral blood, incubated 20 to 24h before analysis of adhesion molecule expression and use in the ATG Fresenius-binding or immune cell adhesion assays. The cells were cultured alone or activated with TNF-alpha (HUVECs) or phytohaemagglutinin (PBMCs) for 20h. HUVEC were incubated for 30 min at 2 – 8 °C with 10 and 100 mg/mL ATG-Fresenius (Fresenius Biotech GmbH) or reference rabbit IgG (Fresenius Biotech GmbH). Analysis of adhesion of immune cells to endothelial cells used co-cultures of peripheral blood mononuclear cells (PBMC) and adherent HUVEC. Endothelial cell expression of the adhesion molecules CD62E and CD54 was determined by flow-cytometry. The relative amounts of T-, B- and NK-cells attached to HUVEC were also determined by flow cytometry (FACSCalibur). T cells were identified by CD3 expression, B-cells by CD19 expression, NK-cells by CD16 expression, and HUVEC by CD31 expression. Groups were compared using one-way analysis of variance (ANOVA) and Tukey-Kramer multiple comparison test.

Results: We show that ATG-Fresenius binds to endothelial cells, with a higher level of binding to activated endothelial cells that express increased levels of E-selectin and ICAM-1. The increased binding of ATG-Fresenius to cytokine-activated endothelial cells is consistent with its known binding to ICAM-1 and selectins. We also show that ATG-Fresenius inhibits adhesion of prestimulated immune cells to activated endothelium. Binding of ATGs to endothelial cells could be also demonstrated in vivo by means of immunhistochemistry.

Conclusion: Our in vitro results provide a rationale supporting of pre-reperfusion administration of ATG-Fresenius in thoracic organ transplantation.