Modifikation des Zelltodes in transplantierten Lungen nach Ischämie und Reperfusion durch adenoviral gesteuerten in vivo Interleukin 10 (IL-10) Gentransfer in Spenderlungen vor Organentnahme

Abstract

Background: We have previously shown that cell death is a pathophysiologic consequence of inflammatory processes during ischemia and reperfusion in transplanted lungs. Apoptosis is predominant in transplanted lungs after 6 to 12 h of preservation with satisfying lung function and necrosis in lungs after 18 to 24 h of preservation with poor graft function. The aim of this study was to determine whether donor lung transfection with the gene that encodes for the anti-inflammatory cytokine IL-10 ameliorates cell death in transplanted lungs. Methods: 15 Lewis rats were divided into 3 groups (n = 5). Group 1 received intratracheal administration of 5xl09 pfu Ad5ElRSVhIL-10 (IL-10), group 2 5x109 pfu ‘empty’vector (EV), and group 3 vector diluent (VD, 3% sucrose). After 24 h in vivo transfection, lungs were stored at 4°C for 24 h and then transplanted. After 2 h of reperfusion lungs were flushed with a trypan blue (TB) solution via the pulmonary artery in order to stain all dead cells in the lungs and then fixed in 10% formalin. For apoptosis detection the TUNEL technique was applied in combination with a propidium iodide stain, which stains all nucleated cells (dead + alive). TB + /TUNEL- cells were considered as necrotic cells, TB + /TUNEL+ cells were considered as apoptotic cells. This triple staining technique has previously been developed, validated and described by us. The total number of cells in study lungs and the number of apoptotic and necrotic cells were counted in random high power fields of tissue sections. Results: The total number of dead cells was similar in the EV, VD and IL-10 group with 32.1 ± 3.3, 30.2 ± 2.5 and 30.3 ± 3.8, respectively. The amount of apoptosis, however, was the highest in IL-10 lungs (9.7 ± 1.9) compared to 2 ± 1.9 and 1.8 ± 2 in VD and EV lungs. Opposite to that, the number of necrotic cells was the lowest in the IL-10 group with 20.6 ± 5.7 vs. 28.3 ± 3.1 and 30.3 ± 4.2 in the VD and EV group. The differences between apoptosis and necrosis within the EV and VD group were p < 0.001. In IL-10 lungs apoptosis differed from necrosis also significantly (p < 0.05). Apoptosis and necrosis in IL-10 lungs were significantly different from numbers in EVand VD lungs (all p < 0.05). Conclusions: Donor lung IL-10 gene transfection prior to transplantation ameliorates post-transplant cell death by decreasing the amount of necrotic cells and increasing the amount of apoptotic cells. It is possible that AdhlL-10, by decreasing pro-inflammatory cytokine production, leads to less overall injury which in turn preserves the ability of damaged cells to undergo the more quiescent and less tissue damaging mode of cell death — apoptosis — rather than necrosis. This study supports the role of gene therapy in lung transplantation and may help to make longer ischemic times and improved graft preservation possible in the future.