Adenoviral-mediated overexpression of I(kappa)B(alpha) in endothelial cells inhibits natural killer cell-mediated endothelial cell activation.

Abstract

Activated natural killer (NK) cells have been found in rejecting discordant xenografts and may contribute to endothelial cell (EC) activation and damage. The transcription of genes associated with EC activation, such as E-selectin and interleukin (IL)-8, is regulated by the transcription factor NF-kappaB. In resting EC, NF-kappaB is complexed within the cytoplasm to I(kappa)B(alpha), and EC activation leads to dissociation of the I(kappa)B(alpha)-NF-kappaB complex and nuclear translocation of NF-kappaB. We investigated whether overexpression of I(kappa)B(alpha) in EC, using adenoviral gene transfer, could block NF-kappaB translocation, thereby inhibiting NK cell-mediated EC activation. Co-culture of human NK cells with porcine EC resulted in a threefold increase in E-selectin expression after 4 hr and secretion of greater than 650 pg/ml porcine IL-8 over 24 hr. Overexpression of I(kappa)B(alpha) inhibited the NK cell-mediated induction of E-selectin expression and IL-8 secretion, whereas overexpression of P-galactosidase did not. The inhibition of EC activation was not due to variation in NK-EC adhesion, as the level of adhesion was similar between adenovirally infected and noninfected EC over 4 hr. The level of NK cell-mediated EC cytotoxicity was not significantly different after 4 hr of co-culture, but after 24 hr, cytotoxicity was increased in virally infected cells. Cytotoxicity was more marked in cells overexpressing I(kappa)B(alpha) than cells overexpressing beta-galactosidase. SLA class I and the induction of SLA class II antigen in response to interferon-gamma treatment were reduced in cells infected with adeno-I(kappa)B(alpha) and empty adenovirus, demonstrating that viral infection alone can influence EC biology. Overexpression of I(kappa)B(alpha) using adenovirus provides a novel approach to inhibiting NK cell-mediated EC activation, but additional strategies will be required to inhibit NK cell-mediated cytotoxicity.