Monocyte Adhesion to Xenogeneic Endothelium during Laminar Flow Is Dependent on α-Gal-Mediated Monocyte Activation

Abstract

Monocytes are the predominant inflammatory cell recruited to xenografts and participate in delayed xenograft rejection. In contrast to allogeneic leukocytes that require up-regulation of endothelial adhesion molecules to adhere and emigrate into effector tissues, we demonstrate that human monocytes adhere rapidly to unstimulated xenogeneic endothelial cells. The major xenoantigen galactoseα(1,3)galactoseβ(1,4)GlcNAc-R (α-gal) is abundantly expressed on xenogeneic endothelium. We have identified a putative receptor for α-gal on human monocytes that is a member of the C-type family of lectin receptors. Monocyte arrest under physiological flow conditions is regulated by α-gal, because cleavage or blockade results in a dramatic reduction in monocyte adhesion. Recruitment of human monocytes to unactivated xenogeneic endothelial cells requires both α4 and β2 integrins on the monocyte; binding of α-gal to monocytes results in rapid activation of β2, but not α4, integrins. Thus, activation of monocyte β2 integrins by α-gal expressed on xenogeneic endothelium provides a mechanism that may explain the dramatic accumulation of monocytes in vivo.

Human Monocytes Recognize Porcine Endothelium via the Interaction of Galectin 3 and α-GAL

“…In the previous study, we found that the fluorescent signal after incubation of polymeric ␣-gal and human monocytes was significantly decreased in the absence of Ca 2ϩ /Mg 2ϩ . This led us to the assumption of the involvement of C-type lectins (15). Conversely, we demonstrated in the current study that the binding of Galectin-3 to its ligand is calcium independent.…”

“…Indeed, we have previously demonstrated specific and saturable binding of a soluble Gal-␣(1,3)Gal-␤(1,4)GlcNAc-R fluorescent probe to monocytes in suspension (15). Interestingly, monocytes obtained from different donors showed a variation in MFI, while there was essentially no binding to lymphocytes ( Fig.…”

“…1B). Moreover, previous studies from our group have documented that the high expression level of Gal-␣(1,3)Gal-␤(1,4)GlcNAc-R on porcine endothelial cells contributes to the elevated levels of firm adhesion to xenogeneic endothelial cells by human monocytes (15).…”

“…Detailed methods for isolating porcine aortic endothelial cells (PAEC) were described in our previous study (15). In brief, freshly harvested porcine aortas (from a local slaughterhouse) were treated with 0.1% collagenase (Sigma-Aldrich) at 37°C to liberate endothelial cells.…”

“…For example, studies in a guinea pig-to-rat xenograft model have demonstrated that host monocyte infiltration persists in the absence of complement or a chemokine-dependent mechanism, and that 80% of the accumulated monocytes were positively detected by a galactose/ N-acetylgalactosamine-specific mAb (14). Our previous study has also demonstrated that human monocytes have the capacity to bind to the major xenoantigen ␣-gal (15). The evidence underpinning the existence of a lectin-dependent xeno-Ag recognition pathway is considerable; however, the exact receptor that mediates this binding has not yet been identified.…”