Microvascular activation and exocytosis after exposure to the serum from mismatched recipients by using donor microvascular cultures.

BACKGROUND: Microvascular injury resulting from activation and exocytosis are early signs of tissue damage caused by allografting. However, humoral anti-graft reactions are not easily detectable in transplant biopsies. The aim of this study was to establish a bioassay to recapitulate this process in a prospective approach.

METHODS: The study was executed by using our previously established protocol to isolate and freeze the donors' microvascular endothelial cells (MVEC) at the transplantation (34 living-related donors and 26 cadaver donors); and to collect sera from the recipients before the transplantation, one-, three- and six-months after transplantation. The activation and exocytosis of the MVEC were determined by incubating the donors' cultures with the recipients' sera. We determined if there was any endothelial activation by quantifying the releases of monocyte chemotactic protein-1 (MCP-1) and interleukin 8 (IL-8) in supernatants and the expressions of membrane intercellular adhesion molecule-1 (CD54) and intercellular adhesion molecule-1 (CD106) by flow cytometry. Endothelial exocytosis was determined by quantifying soluble E-selectin (CD62E) and cytoplasmic von Willebrand Factor (vWF) in supernatants. Endothelial activation or exocytosis was considered positive when the fold change (≧1.5) of post-transplantation to pre-transplantation was reached. We also monitored serum PRA and cytokines using Luminex multiple-plex and cytometric bead-based assay respectively.

RESULTS: We found 41.2% recipients (14 out of 34, ranging from 1.5 to 5.2 folds, p < 0.05) exhibited positive MVEC activation in the first month after transplantation as determined by IL-8 levels; 26.5% recipients (9 out of 34, ranging from 1.5 to 11.8 folds, p < 0.05) by MCP-1 levels. In the group of three months post-transplantation, 70.6% patients were positive (12 out of 17, ranging from 1.8 to 87.1 folds, p < 0.05) by IL-8 increased levels; 24% recipients (4 out of 17, ranging from 1.8 to 50.5 folds, p < 0.05) measured by MCP-1 levels. However, these changes disappeared six months after transplantation. Flow cytometric data showed that a time-dependent of CD54+ and CD106+ expressions existed over the course of six months. Most CD54+ and CD106+ cells were CD31- (platelet-endothelial cell adhesion molecule-1), though CD31+/CD106+ (37.5%, 3 out of 8) and CD31+/CD106+ (25%. 2 out of 8) were seen. When comparing donor MVEC activation to their recipient's proinflammatory cytokine levels or PRA status, we could not draw a conclusion regarding the connections between them. The sera collected from recipients at either one- or three-months after allografting did not significantly induce the release of either soluble CD62E or vWF (p > 0.05), indicating exocytosis was not significantly involved in the acute phase of allografting.

CONCLUSIONS: This bioassay enables us to detect the activation and exocytosis of donor MVEC elicited by respective sera from mismatched kidney recipients.