Galectin-3-mediated xenoactivation of human monocytes.
BACKGROUND
Delayed xenograft rejection (DXR) remains a roadblock to successful xenotransplantation. A feature of DXR is early recruitment of monocytes to the xenograft. Naïve human monocytes can recognize and adhere to unstimulated porcine aortic endothelial cells (PAEC) more than human aortic endothelial cells, partly due to endothelial expression of the xenoantigen galactose-alpha(1,3)galactose-beta(1,4)GlcNAc-R (alpha-gal). Previous work from our laboratory has implicated galectin-3 as a candidate molecule on monocytes involved in initial recognition and adhesion of human monocytes to PAEC.


METHODS
Flow cytometry was used to analyze monocyte activation and galectin-3 accumulation in PAEC. Reactive oxygen intermediate production was analyzed using dihydrorhodamine measured in a fluorescence plate reader. Western blotting was performed to determine galectin-3 secretion and expression by human monocytes. Immunofluorescence staining for the tight junction protein zona occludens-1 was used as a measure of PAEC monolayer integrity.


RESULTS
We demonstrate that galectin-3 can be secreted from monocyte intracellular stores on contact with alpha-gal. Soluble galectin-3 binds PAEC partly by expression of alpha-gal. Binding is reduced on endothelium derived from alpha-gal knockout animals, but not completely. Competing terminal sugars expressed on human aortic endothelial cells such as sialic acid, may block galectin-3 binding. Furthermore, soluble galectin-3 activates monocytes in an autocrine/paracrine manner. Blocking galectin-3 reduces the activation of human monocytes. Finally, the inhibition of galectin-3 reduces monocyte-mediated endothelial injury on co-culture with PAEC.


CONCLUSIONS
Galectin-3 plays a role in human monocyte activation and adhesion in the presence of PAEC, which may contribute to DXR. Additional transgenic strategies targeting galectin-3 ligands on porcine endothelium may be required to achieve optimal xenograft survival.
Greenwald
Allen G
AG
Division of Thoracic Surgery, Department of Surgery, Toronto General Hospital Research Institute, University Health Network, University of Toronto, Toronto, ON, Canada.
Jin
Rongyu
R
Waddell
Thomas K
TK
D015478:Antigens, Heterophile / Q000276:immunology*; D002478:Cells, Cultured; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000378:metabolism; D004727:Endothelium; D005110:Extracellular Space / Q000276:immunology / Q000378:metabolism; D037502:Galectin 3 / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D009000:Monocytes / Q000276:immunology* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D012995:Solubility
D015478:Antigens, Heterophile; D037502:Galectin 3; D017382:Reactive Oxygen Species