Human complement-activating immunoglobulin (Ig)G3 antibodies are essential for porcine endothelial cell activation.
BACKGROUND
Complement-activating naturally occurring anti-porcine endothelial cell antibodies (Abs) are responsible for hyperacute rejection in porcine-to-primate transplantation, whereas the role of complement in acute vascular rejection, characterized by type II endothelial cell activation, is less well understood. We previously demonstrated a correlation between porcine type II endothelial cell activation, as detected by E-selectin expression, and human immunoglobulin (Ig)G3 anti-Gal alpha1-3Gal (Gal) Abs, which was not seen for IgG1, IgG2 or IgG4. The present study was undertaken to investigate whether there is a causal relationship between human anti-porcine IgG3 Abs and porcine endothelial cell activation.


METHODS
IgG3 was isolated employing a Protein A column to 98.3% purity. Porcine endothelial cells were incubated with isolated human IgG3 or the combination of IgG1, IgG2 and IgG4. E-selectin expression and complement activation were investigated by flow cytometry and Western blotting, respectively.


RESULTS
Purified IgG3, in contrast to the other IgG subclasses, induced a substantial increase in E-selectin expression. This activation was accompanied by complement activation as detected by C3 cleavage, and was abolished by heat inactivation or by adding the complement inhibitor FUT-175. Depletion of anti-Gal Abs reduced E-selectin expression by 60%, consistent with the presence of complement-activating anti-porcine non-Gal Abs of the IgG3 subclass.


CONCLUSIONS
Collectively, these data strengthen the hypothesis that human anti-porcine endothelial cell Abs of the IgG3 subclass are essential for endothelial cell activation in porcine-to-human species grafts and demonstrate such activation to be partly independent of Gal epitopes.
Saethre
Marit
M
Institute of Immunology, Rikshospitalet University Hospital, University of Oslo, Norway. marit.sathre@medisin.uio.no
Lea
Tor
T
Borgen
Merethe S
MS
Fiane
Arnt E
AE
Michaelsen
Terje E
TE
Thorsby
Erik
E
Haraldsen
Guttorm
G
Mollnes
Tom E
TE
D000818:Animals; D001011:Aorta; D001550:Benzamidines; D018929:Cell Culture Techniques; D003167:Complement Activation / Q000276:immunology*; D051056:Complement Inactivating Agents / Q000494:pharmacology; D004187:Disaccharides / Q000276:immunology; D019040:E-Selectin / Q000187:drug effects / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology / Q000276:immunology / Q000502:physiology* / Q000637:transplantation; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006084:Graft Rejection / Q000517:prevention & control; D006146:Guanidines / Q000494:pharmacology; D006168:Guinea Pigs; D006801:Humans; D007074:Immunoglobulin G / Q000145:classification / Q000276:immunology* / Q000302:isolation & purification; D011323:Primates; D013205:Staphylococcal Protein A; D013552:Swine; D014183:Transplantation, Heterologous / Q000502:physiology
D001550:Benzamidines; D051056:Complement Inactivating Agents; D004187:Disaccharides; D019040:E-Selectin; D006146:Guanidines; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A; C055075:galactosyl-(1-3)galactose; C032855:nafamostat