Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 May 1;180(9):6346-53.
doi: 10.4049/jimmunol.180.9.6346.

Cytokine Secretion Depends on Galalpha(1,3)Gal Expression in a Pig-To-Human Whole Blood Model

Affiliations
Free PMC article

Cytokine Secretion Depends on Galalpha(1,3)Gal Expression in a Pig-To-Human Whole Blood Model

Marit Saethre et al. J Immunol. .
Free PMC article

Abstract

Transplants from alpha1,3-galactosyltransferase (Gal) gene-knockout pigs to nonhuman primates are largely protected from hyperacute but not acute humoral xenograft rejection. The present study investigates the role of Gal in cytokine responses using a novel pig-to-human whole blood in vitro model, developed for species-specific analysis of porcine and human cytokines. Porcine (n = 7) and human (n = 27) cytokines were measured using ELISA or multiplex technology, respectively. Porcine aortic endothelial cells from control (Gal(+/+)) and Gal-deficient (Gal(-/-)) pigs were incubated with human lepirudin anticoagulated whole blood from healthy donors. E-selectin expression was measured by flow cytometry. The C3 inhibitor compstatin and a C5aR antagonist were used to study the role of complement. Cytokine species specificity was documented, enabling detection of 2 of 7 porcine cytokines and 13 of 27 human cytokines in one single sample. Gal(+/+) porcine aortic endothelial cells incubated with human whole blood showed a marked complement C5b-9 dependent up-regulation of E-selectin and secretion of porcine IL-6 and IL-8. In contrast, Gal(-/-) cells responded with E-selectin and cytokine expression which was so weak that the role of complement could not be determined. Human IL-6, IL-8, IFN-gamma, MIP-1alpha, MIP-1beta, eotaxin, and RANTES were detected in the Gal(+/+) system, but virtually no responses were seen in the Gal(-/-) system (p = 0.03). The increase in human cytokine release was largely complement dependent and, in contrast to the porcine response, mediated through C5a. Species-specific analysis of cytokine release revealed a marked, complement-dependent response when Gal(+/+) pig cells were incubated with human whole blood, compared with Gal(-/-) cells which induced virtually no cytokine release.

Conflict of interest statement

Disclosures

The authors have no financial conflict of interest.

Figures

FIGURE 1
FIGURE 1
E-selectin expression on immortalized Gal+/+ and Gal−/− PAECs. PAECs were incubated 4 h with TNF-α, 50% human serum (HS), and 50% human whole blood (HWB). E-selectin cell surface expression was measured by flow cytometry. Data represent MFI in one representative experiment.
FIGURE 2
FIGURE 2
Effect of complement blocking on E-selectin expression on immortalized Gal+/+ and Gal−/− PAECs. Gal+/+ (●) and Gal−/− (○) PAECs were incubated with human whole blood and increasing concentrations of the complement-inhibitors compstatin (left panel) and a C5aR antagonist (middle panel) as well as a control peptide (right panel). E-selectin expression on incubated PAEC was determined by flow cytometry. Representative data from one blood donor are presented.
FIGURE 3
FIGURE 3
Effect of complement blocking on porcine and human cytokine production. Immortalized Gal+/+ (●) and Gal−/− (○) PAECs were incubated with human whole blood to measure porcine (A) or human (B) IL-8 production. Complement inhibitors compstatin (left panel) and a C5aR antagonist (middle panel) as well as a control peptide (right panel) were added in increasing concentrations (x-axis). Representative data from one blood donor are presented. Cytokine data are presented as concentration (nanograms per milliliter).
FIGURE 4
FIGURE 4
Detection of porcine cytokines in cell supernatants. Porcine IL-8 (left panel) and IL-6 (right panel) concentrations were analyzed in supernatants from immortalized Gal+/+ and Gal−/− PAECs incubated with human whole blood (HWB) alone, HWB with complement inhibitor C5aR (C5aRa, 14 µM), or HWB with complement inhibitor compstatin (comp., 100 µM). Both IL-8 and IL-6 concentration was significantly higher from Gal+/+ compared with Gal−/− porcine PAECs (*, p = 0.03). Data are presented as box plots showing median, quartiles, and minimum and maximum values from five experiments, each using a different donor.
FIGURE 5
FIGURE 5
Detection of human cytokines in cell supernatants. Cytokine production was detected in cell supernatants when immortalized Gal+/+ and Gal−/− PAECs were incubated with human whole blood (HWB) alone or with complement inhibitors C5aR antagonist (C5aRa, 14 µM) or compstatin (comp., 100 µM). A, Human cytokines (IL-6, IL-8, IFN-γ, and TNF-α) for which cross-reactivity to porcine cytokines was excluded in the human multiplex system (Table II). B, Human cytokines (MIP-1α, MIP-β, eotaxin, and IP-10) for which cross-reactivity with porcine cytokines could not be determined. Significant differences were found between human cytokines detected in the Gal+/+ compared with Gal−/− PAECs for all cytokines (*, p = 0.03). Data are presented as box plots showing median, quartiles, and minimum and maximum values from five experiments, each using a different donor.
FIGURE 6
FIGURE 6
Comparison of immortalized and primary Gal+/+ and Gal−/− PAECs. Primary (A) and immortalized PAECs (B) were compared with respect to E-selectin expression and human and porcine cytokines in cell supernatants (here shown as IL-6) after incubation with human whole blood (HWB), HWB with compstatin (Comp.), or HWB with a C5aR antagonist (C5Ra). E-selectin expression data represents percent median fluorescent intensity of Gal+/+ PAECs incubated with HWB (marked with an asterisk (*)). Cytokine data are presented as concentration (nanograms per milliliter).

Similar articles

See all similar articles

Cited by 10 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback