Effect of Redox Modulation on Xenogeneic Target Cells: The Combination of Nitric Oxide and Thiol Deprivation Protects Porcine Endothelial Cells from Lysis by IL-2-Activated Human NK Cells

Abstract

Evidence suggests that NK cells contribute to the pathogenesis of delayed rejection of vascularized xenografts, and NK cells have been suggested to participate in hyperacute xenograft rejection. Endothelial cells have been shown to be the primary target of the recipient’s immune responses that mediate both hyperacute and delayed xenograft rejection. Under conditions of oxidative stress induced by thiol deprivation, but not under normal conditions, pretreatment of porcine aortic endothelial cells (PAECs) with the NO donor, S-nitroso-N-acetyl-penicillamine, dramatically inhibited killing of PAEC target cells by IL-2-activated human NK cells. This same combined treatment reduced both surface expression and mRNA levels of E-selectin. Moreover, anti-E-selectin mAb, but not Ab to VCAM-1, protected PAEC from lysis by human IL-2-activated NK cells in a dose-dependent manner. These findings suggest that expression of porcine E-selectin is important for the cytotoxicity of PAEC mediated by activated human NK cells and may be involved in the redox-mediated modulation of that cytotoxicity. It is known that NF-κB activation is required for transcription of E-selectin, and the current data show that the suppression of E-selectin expression by S-nitroso-N-acetyl-penicillamine pretreatment and thiol deprivation was associated with reduced NF-κB DNA-binding activity in PAEC. These data suggest that the regulation of porcine E-selectin may be important for modulating delayed xenograft rejection and that manipulation of cellular redox systems may provide a means to protect xenogeneic endothelial cells from NK cell-mediated cytotoxicity.

Human NK cells can lyse porcine endothelial cells independent of their expression of Galα(1,3)‐Gal and killing is enhanced by activation of either effector or target cells

“…Because human TNF‐ α activates porcine EC [38] and enhances their susceptibility to lysis by human NK cells [39], and is likely to be produced in the context of a xenoresponse, we thought it valuable to test whether TNF‐ α activation differentially influenced susceptibility of the α Gal null EC to lysis by fresh human NK cells. Null and wild type EC were cultured with TNF‐ α at 10 ng/ml for 4 h prior to use as target cells, and a subset ( n = 3 to 4) of donors whose results are included in the data shown in Fig.…”

“…Consistent with the variation among human donors, it was necessary to test NK cells from a relatively large number of donors in order to appreciate the data fully. However, even the marginal difference did not occur when target cells were pretreated with human recombinant TNF‐ α , a cytokine that is known to activated porcine EC [38] and increase their susceptibility to lysis by human NK cells [39]. Importantly, NK cells produce TNF‐ α and it is likely to be produced in the context of a xenoresponse.…”

Linoleic acid induces proinflammatory events in vascular endothelial cells via activation of PI3K/Akt and ERK1/2 signaling

“…VCAM-1 gene expression was determined through reverse transcriptase-polymerase chain reaction (RT-PCR) as described earlier [12]. The oligonucleotide primers used to amplify the porcine VCAM-1 and the house keeping gene h-actin were as described earlier [15]. The sequences of the primer pairs in this experiment were as follows: porcine VCAM-1 (sense, 5V-ATGACATGCTT-GAGCCAGG-3V; antisense, 5V-GTGTCTCCTTCTTTGA-CACT-3V); porcine h-actin, was used as an internal control (sense, 5V-ATGTTTGAGACCTTCAACACGCCGG3V ; antisense, 5V-GCAGGACTCCATGCCCAGGAAGGAG-3V).…”

Linoleic acid-induced endothelial activation

“…The RNA was then reverse-transcribed to cDNA and amplified by PCR. The oligonucleotide primers used to amplify the porcine E-selectin and the housekeeping gene ␤ -actin were as described earlier (32). The sequences of the primer pairs in this experiment were as follows: porcine E-selectin (sense, 5 Ј -GACTCGGGCA-AGTGGAATGATGAG-3 Ј ; antisense, 5 Ј -CATCACCATTCTGAGGAT-GGCCGAC-3 Ј ); porcine ␤ -actin, used as an internal control (sense, 5 Ј -ATGTTTGAGACCTTCAACACGCCGG-3 Ј ; antisense, 5 Ј -GCA-GGACTCCATGCCCAGGAAGGAG-3 Ј ).…”