In recent years, there has been an increasing interest in non-HLA antigens as mediators of injury in solid organ transplantation, due to the fact that kidney transplants also undergo immunological rejections in the absence of detectable HLA antibodies [1, 2, 4]. As a non-HLA antigen, MICA expressed on endothelial and epithelial cells has been implicated in the pathogenesis of hyperacute, acute and chronic organ allograft rejections. Stastny has demonstrated that MICA might be a target during anti-graft immune responses in transplanted patients . In clinical studies, Sumitran-Holgersson demonstrated a close connection between the presence of MICA antibodies and early immunological responses in kidney allograft recipients, even though donor specific HLA antibodies were undetected . Moreover, Ozawa reported that the presence of MICA antibodies is closely related to chronic kidney allograft rejection [3, 30], while Mizutani reported that the frequency of anti-MICA antibodies is higher among recipients who undergo kidney allograft rejection compared with recipients who do not . Thus, MICA plays an prominent role in kidney allograft outcome.
Zwirner has demonstrated that antibodies against MICA are often produced after transplantation . In the latest review, Suarez-Alvarez mentioned that it is possible that the expression of MICA in human grafts could be induced by IRI , because it has been reported that IRI induces expression of retinoic acid early inducible 1 (RAE-1, MIC homolog that functions as a ligand for mouse NKG2D) on tubular epithelial cells in kidneys, contributing to acute allograft rejection .
In this study, we found that MICA expression was up-regulated by HIF-1alpha on human renal proximal tubular epithelial cells during hypoxia/reoxygenation. Using the HIF-1alphaDELTAODD-expressing adenovirus, HIF-1alpha was functionally and steadily expressed regardless of oxygen tension, providing a useful cell model for determining whether HIF-1alpha could influence MICA expression in normoxia. Our results showed that 96 h after transduction, Ad.CMV.HIF-1alpha and Ad.CMV.HIF-1alphaDELTAODD groups showed a substantial increase in MICA mRNA and surface expression compared with the Ad.CMV.LacZ and control groups. On the other hand, after 16 h of hypoxia and reoxygenation for 8 h, there was a decrease in MICA mRNA and surface expression levels in the HIF-1alpha siRNA treatment group compared with the hypoxia group. Taken together, these results demonstrated that the up-regulated surface expression of MICA on HK-2 cells during hypoxia/reoxygenation correlates with the over-expression of HIF-1alpha. These findings support our hypothesis that during organ IRI in kidney transplantation, the MICA surface expression on allografts is up-regulated by HIF-1alpha, inducing antibodies against MICA in the recipients' sera after transplantation, leading to poor kidney transplant outcomes [1, 3, 4, 10, 31].
NKG2 D ligands (such as MICA) are key targets of the immune response. NKG2 D is an activating receptor that is ubiquitously expressed by NK cells , a major component of the innate immune system . Binding of NKG2 D to its ligands, such as MICA, activates NK cells and promotes cytotoxic lysis of the cells expressing these molecules , which might lead to graft loss. Since adenovirus mediated HIF-1alpha/HIF-1alphaDELTAODD transduction could up-regulate MICA expression, we proposed that it may enhance NK cell cytotoxicity towards target cells, which over-express HIF-1alpha/HIF-1alphaDELTAODD. As expected, NK cells exhibited greater cytotoxicity towards the Ad.CMV.HIF-1alpha and Ad.CMV.HIF-1alphaDELTAODD groups compared with the Ad.CMV.LacZ and control groups 96 h after transduction. Moreover, an inhibition of HIF-1alpha expression by siRNA showed a great decrease in NK cell cytotoxicity compared with the hypoxia without RNAi treatment group 8 h after reoxygenation. Thus, NK cell cytotoxicity towards HK-2 cells positively correlated with HIF-1alpha expression, and blocking with a MICA antibody demonstrated that it is mediated by the surface expression of MICA, which is up-regulated by HIF-1alpha over-expression. Moreover, our ELISA results also demonstrated that co-culture of NK cells and HK-2 cells expressing MICA, which was up-regulated by over-expression of HIF-1alpha, could induce IFNgamma secretion by NK cells. Interestingly, although HIF-1alpha could induce many genes that protect cells from hypoxia stress, such as HO-1 and VEGF, it also up-regulates ligands of NKG2 D, such as MICA, enhancing NK cell cytotoxicity towards target cells, leading to their destruction. More studies are needed, to understand the balance of its dual role in helpful and harmful effects. In addition, although the inhibition of HIF-1alpha could induce an decrease NK cell cytotoxicity towards target cells, only 50% is inhibited compared with the hypoxia and hypoxia with negative control RNAi treatment groups, which may not correspond to the level of inhibition seen for MICA surface expression on HK-2 cells. This indicates that there are probably other ways, in addition to the HIF-1 pathway, which could also influence NK cell cytotoxic activity towards HK-2 cells under hypoxia conditions, which would require further study.
In addition, we also found that the potential hypoxia response elements to which HIF-1 binds during hypoxia contains a core sequence 5'-CGTG-3' 1000 base pairs upstream from the transcriptional start site of MICA (data not shown). However, in this study, we have not formally proved that MICA expression is up-regulated by HIF-1alpha directly or indirectly, it needs further studies because it might be important for developing strategies to reduce the harmful effect of MICA in kidney transplant outcome in the future.