Complement inactivation or depletion of α-Gal XNA does not abrogate the transcription of early growth response factor-1 (Egr-1) in a model of pig-to-human liver xenotransplantation
Introduction Egr-1 is an inducible pluripotent transcription factor capable of up-regulating cytokines, adhesion molecules and thrombotic factors relevant to xenotransplantation. The purpose of this study is to identify whether hepatic sinusoidal microvascular endothelial cells (PHSEC) up-regulate Egr-1 in an in vitro model of pig-to-human liver xenotransplantation and whether this up-regulation is attenuated by depletion of complement or anti-α-Gal XNA.
Materials and methods PHSEC were cultured in serum-free medium and incubated with 20% normal human serum (NHS) for up to 8 h. Complement was inactivated by heating human serum (HHS). Matrix comprised of silicon beads conjugated to αGal(1,3)βGal(1,4)Glc was used to remove the anti-α-Gal XNA from human serum. Semi-quantitative assaying of the transcription of Egr-1 was performed by rt-PCR. Experimental conditioned were: LPS 10 μg/mL; dexamethasone 1 μM; pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, 100 μM; melittin, a transmembrane pore-forming protein to simulate the membrane attack complex (MAC), 0.5 μM.
Results The CH50 of HHS was 0 and 90-98% of anti-α-Gal XNA were depleted from human serum. The transcription of Egr-1 by PHSEC in response to NHS peaked at 1 h and returned to basal levels by 6 h. Exposure to LPS failed to up-regulate Egr-1 above basal levels. Simulating the MAC with melittin induced Egr-1 transcription, but only for 2 h. The transcription of Egr-1 was not decreased by inhibiting protein synthesis, by inhibiting NF-κB or by dexamethasone. Inactivating complement or depleting anti-α-Gal XNA both decreased Egr-1 transcription by 33%. The combination of inactivating complement and depleting anti-α-Gal XNA did not further attenuate the transcription of Egr-1.
Conclusions Egr-1 is transiently up-regulated in this in vitro model of pig-to-human liver xenotransplantation. Egr-1 is not a ubiquitous mediator of endothelial cell activation in PHSEC as it occurs with xenogeneic stimuli (human serum) and the MAC (melittin) but not under septic conditions (LPS). The up-regulation of Egr-1 is dependent on both complement and xenoreactive antibodies. Low levels of anti-α-Gal antibodies and minor XNA are sufficient to maintain Egr-1 transcription.