Type 1 diabetes can be cured by transplantation of isolated pancreatic islets. Because of the shortage of human donor tissue, adult porcine islets (APIs) constitute a possible alternative tissue source. Upon intraportal injection, islets are subjected to an instant blood-mediated inflammatory reaction (IBMIR) leading to blood clotting, leukocyte islet-infiltration, islet damage and insulin release. Xenogeneic islets surviving IBMIR are rejected in a cellular process involving CD4(+) T lymphocytes and macrophages. We have investigated whether APIs themselves produce and secrete chemokines and/or inflammatory cytokines that may contribute to IBMIR and/or cell-mediated rejection. APIs, cultured for 1, 4, 8 and 11 days post-isolation, expressed mRNA for monocyte chemoattractant protein-1 (MCP-1), IL-1beta and TNF-alpha. API culture supernatants induced migration of human monocytes, which was significantly blocked by an anti-human MCP-1 antibody (Ab). Immunohistochemistry revealed MCP-1 in the cytoplasm of alpha- and beta-cells in isolated islets and in islets in situ. However, APIs or their supernatants were not able to activate human aortic endothelial cells (HAECs) in vitro, and neither IL-1beta nor TNF-alpha were detected by enzyme-linked immunosorbent assay (ELISA) in API culture supernatants. Both recombinant porcine IL-1beta and TNF-alpha were able to activate human endothelial cells (ECs) inducing CD62E and CD106 expression as analyzed by flow cytometry. In conclusion, MCP-1 secreted by APIs may contribute to both IBMIR and rejection by attracting monocytes into the islet; monocytes which upon transformation into macrophages will potentiate antigen presentation and execute islet rejection.
Extracellular adherence protein (Eap) from Staphylococcus aureus inhibits the adherence of neutrophils to nonstimulated and tumor necrosis factor alpha-stimulated endothelial cells in both static adhesion assays and flow adhesion assays. Consequently, Eap also impaired their transendothelial migration. During an S. aureus infection, Eap may thus serve to reduce inflammation by inhibiting neutrophil adhesion and extravasation.
The contribution of Gal § 1,3Gal ( § -Gal) to cell-mediated organ xenograft rejection is controversial. We have used recombinant lentiviruses encoding a porcine § 1,3 galactosyltransferase ( § 1,3GalT) to obtain § -Gal-expressing primary human aortic endothelial cells (HAEC) at a frequency of 70-90%. These cells were compared to non-transduced and mock-transduced HAEC with regard to their susceptibility to human NK cell-mediated lysis, ability to stimulate IFN-+ production by NK cells, and support of NK cell adhesion under static and dynamic conditions. Using green fluorescent protein (GFP) as a reporter gene, it was shown that the frequency of green fluorescent HAEC increased until day 5 posttransduction, and at a multiplicity of infection of 2.5, it reached 98%. Lentiviral transduction did not result in activation of HAEC, and transduced HAEC responded as expected to TNF- § and IFN-+ stimulation. No differences were detected between non- § -Gal-and § -Galexpressing HAEC in terms of their susceptibility to NK cell-mediated lysis, ability to stimulate IFN-+ production by NK cells, or ability to support NK cell adhesion under static and dynamic conditions. In conclusion, these data argue against an important role for the § -Gal epitope in the direct interaction between endothelium and NK cells and prove that recombinant lentiviruses are efficient gene carriers for primary human endothelial cells.
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