MHC antigen presentation on the surface of hepatocytes: modulation during and after hypoxic stress

Abstract

Presentation and recognition of MHC antigen on the surface of cells is the basic process of initiating rejection and distinguishing "self and not self". This is considered to begin when a transplanted organ is reperfused with the blood of the recipient. In this study, the modulation of MHC I antigen presentation during preservation was investigated in a cell culture model using primary hepatocyte cultures of male Wistar rats. By incubation with different preservation solutions used in clinical transplantation, expression of the MHC antigen was observed during cold hypoxia. Primary hepatocyte cultures were isolated from male Wistar rats by a modified Seglen technique and seeded to glass slides, thus obtaining monolayer cultures. After 1 day of resting the cultures were incubated under different conditions using Krebs Henseleit solution (KH), Euro-Collins solution (EC), HTK solution of Bretschneider (HTK) and University of Wisconsin solution (UW) as incubation media. The conditions of incubation were warm normoxia (37 degrees C, pO2 100 mm Hg) and cold hypoxia (4 degrees C, pO2 < 0.1 mm Hg), which is the main condition of organ preservation. Incubation time was 6 h. Before starting the incubation reference cultures were fixed and every 60 min several cultures were withdrawn from the experiment and fixed also. MHC expression was studied by staining the cultures with monoclonal antibodies against rat MHC class I and class II. As expected, MHC class II was not present on the surface of hepatocytes, while MHC class I was demonstrated on the controls as well as on the cultures that were incubated using KH and EC independent of temperature and hypoxia or normoxia. At the end of the incubation they were still positive but not as strong as in the beginning. During incubation using HTK and UW, MHC I was not detectable at all phases of the experiment. In conclusion, hypoxic stress did not completely eradicate MHC I expression in rat hepatocytes, while the composition of the preservation medium may result in a hepatocyte surface negative for MHC I. Hydroxyethylstarch may be the substance in UW that covers the MHC antigen, while in HTK, mannitol or the histidine complex may play the same role. MHC negativity of the cells of a preserved transplant is another reason for the benefit of UW or HTK.