Introduction

Polyclonal antithymocyte globulins (ATGs) are immunosuppressive drugs widely used in induction of immunosuppression and treatment of acute rejection after solid organ transplantation. The aim of this study was to investigate the in vitro influence of ATGs on the leukocyte transmigration and the extent of endothelial response in naÏve and adult endothelial cells, as well as to investigate the underlying mechanisms.

Material and Methods

HUVECs and adult endothelial cells from an immortalized human microvascular line (HMEC-1) were cultured. The cells were activated for 20 h (HUVECs) or 2 h (HMEC-1) prior to the migration assay. PBMC were cultured alone or activated with PHA (10 Μg/mL) for 20 h. Both PBMC and HUVEC /HMEC-1 were each incubated with ATG (ATG-Fresenius©, Fresenius Biotech, Germany) or control rabbit IgG (100 Μg/mL) for 30-120 minutes. PBMCs and endothelial cells were incubated together for 5 hours. Endothelial transmigration of PMBCs was assessed in different wells (n=30 in 6 independent experiments) using trypan blue staining. Negative controls were performed. For the determination of maximal transendothelial migration of activated and naive PBMC, lymphocyte migration was stimulated by adding CX3C to the well. Expression of adhesion molecules (e.g. ICAM-1 and MHC class I) on endothelial cells was studied by flow-cytometry. Groups were compared using one-way analysis of variance (ANOVA) and Tukey-Kramer multiple comparison test.

Results

NaÏve PBMC showed a higher level of migration: naÏve PBMC plus activated HUVEC (7.71 x 10⁴ cells [1.95-9.03]); naÏve PBMC plus naÏve HUVEC (3.48 x 10⁴ cells [3.10-3.99]); activated PBMC plus activated HUVEC (2.08 x 10⁴ cells [1.71-3.72]). ATG decreased migration of naÏve PBMC through a barrier of activated HUVEC. Migration of naÏve PBMC through a layer of naÏve HUVEC was slightly reduced by ATG. PMBC migration was significantly reduced after treatment of HMEC as compared to untreated controls (1.55E+05±1.00E+04 vs. 6.85E+05±1.87E+05; p<0.05). Incubation of EC with ATG significantly reduced the surface expression of ICAM-1 and MHC class I on adult endothelial cells.

Conclusion

Our in vitro results provide a rationale supporting preconditioning with ATG in solid organ transplantation, as a down-regulation of adhesion molecules by ATG may decrease graft cell infiltration after solid organ transplantation.

Beiras-Fernandez, A.: Speaker’s Bureau, Fresenius Biotech.

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