Influence of hypothermia and subsequent rewarming upon leukocyte-endothelial interactions and expression of Junctional-Adhesion-Molecules A and B

Abstract

Patients with risks of ischemic injury, e.g. during circulatory arrest in cardiac surgery, or after resuscitation are subjected to therapeutic hypothermia. For aortic surgery, the body is traditionally cooled down to 18 °C and then rewarmed to body temperature. The role of hypothermia and the subsequent rewarming process on leukocyte-endothelial interactions and expression of junctional-adhesion-molecules is not clarified yet. Thus, we investigated in an in-vitro model the influence of temperature modulation during activation and transendothelial migration of leukocytes through human endothelial cells. Additionally, we investigated the expression of JAMs in the rewarming phase. Exposure to low temperatures alone during transmigration scarcely affects leukocyte extravasation, whereas hypothermia during treatment and transendothelial migration improves leukocyte-endothelial interactions. Rewarming causes a significant up-regulation of transmigration with falling temperatures. JAM-A is significantly modulated during rewarming. Our data suggest that transendothelial migration of leukocytes is not only modulated by cell-activation itself. Activation temperatures and the rewarming process are essential. Continued hypothermia significantly inhibits transendothelial migration, whereas the rewarming process enhances transmigration strongly. The expression of JAMs, especially JAM-A, is strongly modulated during the rewarming process. Endothelial protection prior to warm reperfusion and mild hypothermic conditions reducing the difference between hypothermia and rewarming temperatures should be considered.

Effect of Rewarming on Leukocyte-Endothelial Interaction After Deep Hypothermic Preservation

“…Other studies observed similar effects consisting of an increase in leukocyte infiltration after transplantation following prolonged cold storage and described concomitant destruction of the endothelial integrity [2]. This is not only in keeping with the results of a previous study of our group, which showed a similar effect during rewarming out of mild and moderate hypothermia [9], but also with the results of our current study, showing modulation of the endothelial barrier after deep hypothermia.…”

“…Nevertheless, in the present study, we observed a temperature-dependent modulation of JAM-A in that a significant downregulation of JAM-A occurred during post-hypothermic rewarming out of deep hypothermic conditions. This finding is of particular interest, as we demonstrated, in a previous study, that downregulation of JAM-A correlates with an enhanced leukocyte-endothelial interaction with increased TEM [9]. Considering that early leukocyte infiltration can result in higher rejection rates [17]; it may be concluded that a rewarming process resulting in augmented TEM and downregulation of JAM-A may be associated with a higher likelihood of subsequent graft dysfunction.…”

“…HuMEC-1 and PBL were activated with LPS as previously described at 4°C. PBL and HuMEC-1 were co-cultured at 37°C for 120 minutes to simulate a rewarming process [9]. After removal of the PBL and washing of the HuMEC-1, the endothelial monolayer was fixed with 4% paraformaldehyde (AppliChem), followed by blocking with PBS supplemented with 10% donkey serum (Jackson ImmunoResearch).…”

“…Rabbit anti-JAM-A-IgG (1: 200; Santa Cruz Biotechnology) and goat anti-JAM-B-IgG (1: 100; Santa Cruz Biotechnology), donkey anti-rabbit-IgG-PE (1: 100; Santa Cruz Biotechnology) and donkey anti-goat-IgG-FITC (1: 100; Santa Cruz Biotechnology) in PBS containing 1% donkey serum were used for primary and secondary staining of the HuMEC-1, as previously described [9]. JAM surface protein expression was measured using a microplate reader at 485/535 nm and 530/590 nm, and the fluorescence intensity (FI) was calculated as a ratio to control.…”

“…Beyond this, rewarming was shown to be associated with the induction of pro-inflammatory processes such as enhanced leukocyte infiltration [8,9], which may also impair graft function [10]. The interactions between leukocytes and the endothelium are strongly associated with the development of acute cellular rejection [11,12], which is important because attenuating early graft rejection helps preventing late graft loss and/or dysfunction [13,14].…”