Weak response of porcine C5a receptor towards human C5a in miniature pig endothelial cells and PMNs.
BACKGROUND
The anaphylatoxin C5a is a potent inflammatory molecule generated during complement activation. Although some reports have implicated C5a in xenograft rejection, to date, the molecular compatibility between human C5a and porcine C5a receptor (C5aR) has been little studied. To examine the need for pC5aR-deficient pig in xenotransplantaion, we aimed to look at the degree of direct interaction between human C5a (recipient side) and porcine endothelial cells (PECs) and porcine polymorphonuclear neutrophils (PMNs) (donor side).


METHODS
Following the treatment of human C5a to isolated porcine PMNs, transmigration of PMNs was measured by Transwell system and superoxide generation by cytochrome c reduction assay. Next, the effects of human C5a on several intracellular signaling pathways were further checked; actin cytoskeletal change was observed under a confocal microscope after staining with Alexa Fluor-546-phalloidin, intracellular calcium mobilization was measured by spectrofluorophotometer. The degree of direct effect of human C5a on porcine PMNs was compared with that in human PMNs. Finally, microarray was performed to monitor the effect of human C5a on gene expression of PEC and the expression of several candidate proteins was checked by flow cytometry.


RESULTS
We found that human C5a was able to induce chemotaxis, superoxide generation, actin cytoskeletal change, and intracellular calcium mobilization in porcine PMNs. However, higher concentration of human C5a was required to stimulate porcine PMNs in comparison with activating human PMNs. The amino acid sequences of porcine C5aR with those of human C5aR showed a sequence homology of only 67%. To elucidate the effect of human C5a to PECs, microarray analysis following the treatment of PECs with human C5a was performed. These data showed that human C5a did not significantly affect gene transcription patterns in PECs. Additionally, treatment of PECs with human C5a also did not induce protein expression of several cell adhesion molecules, including vascular cell adhesion molecule-1, intercellular adhesion molecule-1, P-selectin, and E-selectin, or secretion of interleukin-8 from PECs.


CONCLUSIONS
These results suggest that human C5a may play a minor role on PEC activation possibly due to molecular incompatibility across the species barrier.
Yi
Kye Sook
KS
Cancer Research Institute, Seoul National University College of Medicine, Seoul.
Lee
Sukmook
S
Kang
Yoon-Ho
YH
Bae
Yoe-Sik
YS
Hwang
Seung Yong
SY
Ha
Insu
I
Kim
Hyori
H
Kim
Min Soo
MS
Cho
Bumrae
B
Kang
Hee Jung
HJ
Bang
Ki Tae
KT
Kim
Jae Yong
JY
Yang
Jaeseok
J
Chung
Junho
J
Ahn
Curie
C
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D002460:Cell Line; D015936:Complement C5a / Q000235:genetics / Q000502:physiology*; D004730:Endothelium, Vascular / Q000502:physiology*; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000502:physiology* / Q000637:transplantation; D044087:Receptor, Anaphylatoxin C5a / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D013556:Swine, Miniature; D014183:Transplantation, Heterologous
D044087:Receptor, Anaphylatoxin C5a; D011994:Recombinant Proteins; D015936:Complement C5a