Transendothelial Migration of Leukocytes after Methylene Blue Treatment: Influence on Adhesion Molecules

Abstract

Purpose Patients undergoing heart transplantation can develop vasoplegic syndrome, which is associated with high mortality and morbidity, especially if refractory to conventional treatment. Methylene blue (MB) has been used in the management of this entity. Our aim was to investigate the effect of MB on transendothelial migration of lymphocytes and leukocytes and on the expression of endothelial adhesion molecules. Methods and Materials Monolayers of human microvascular endothelial cells (HMECs) were cultured on fibronectin coated transwell filters. Once a confluent monolayer was observed, the cells were stimulated with either 100ng/ml LPS for 1h to mimic acute inflammation, with 60μM MB for 2h, or with 100ng/ml LPS for 1h followed by 60μM MB for 2h and vice versa. For positive control 50ng/ml CXCL12 was added below the transwell filter as chemoattractant. Jurkat cells, or fresh isolated PBMCs were placed on top of the HMEC monolayer after stimulation. The transmigrated cells were evaluated after 2 h. FACS analyses of the expression of ICAM-1, VCAM-1, CD62-L and CD62-E were performed after co-cultures of HMECs and Jurkat/PBMCs. For statistical analysis One-Way Anova was performed. Results Prophylactic administration of MB significantly reduces the transendothelial migration of lymohocytes after inflammation. However, these effect could not be confirmed on PBMCs. The co-incubation of HMECs with freshly isolated PBMCs caused an upregulation of CD62-E (6-fold), ICAM-1 (5-fold) and VCAM-1 (3-fold). Though CD62-L is expressed at a very low level on HMECs, the contact with PBMCs led to a 33-fold upregulation. These effects were not observed on HMEC when co-cultured with T-lymphocytes. Conclusions Methylene blue is able to modulate the endothelial activity through immunomodulatory mechanisms. Transendothelial migration of lymphocytes is blocked by the prophylactic administration of MB. This effect is not seen in PBMCs. The expression of adhesion molecules after MB suggests that MB exerts a T-lymphocyte mediated modulation of the inflammatory response.