SummaryAlthough several investigators have attempted to identify the site of synthesis of factor VIII (FVIII), the cellular species responsible for maintenance of plasma FVIII has not been clearly defined. Indications point at hepatocytes and certain endothelial cells. The present study investigated the FVIII coagulant antigen (VIII : Ag) of hepatocytes obtained by two-step collagenase digests of human liver pieces. Following Percoll gradient centrifugation, less than 1% of cells harvested were non-parenchymal. Lysates of freshly isolated and purified hepatocytes contained 165–250 mU of VIII: Ag/106 cells as defined by a two-site ELISA employing a haemophilic antibody against human FVIII. This material contained a single peak of VIII: Ag polypeptides as jugded from the VIII: Ag ELISA profile of Mono-Q fast protein liquid chromatography fractions. A haemophilic antibody specific for epitopes of the light chain of FVIII, employed in immunoisolation of VIII : Ag in lysate of human hepatocytes, extracted a polypeptide pattern that was studied in a reduced SDS-PAGE electrophoresis gel and compared to that of immunoisolate from normal plasma. After electroblotting onto nitrocellulose and reaction with a monoclonal antibody towards the light chain of FVIII, the appearance of a doublet at 78–79 kDa in both these materials indicated the presence of the light chain of FVIII in human hepatocyte lysate. During culture, human hepatocytes secreted 20–80 mU of VIII: Ag per 1 × 106 cells per 24 hours. Further, a significant secretion of VIII: Ag was found in media of cultured human hepatoma cells, Hep-G2, whereas human blood monocytes and human fibroblasts did not secrete detectable VIII: Ag. In all of these cell cultures, vWf : Ag was indetectable or present as trace. Our results suggest that the human hepatocyte is a production site of FVIII.

The effect of in vitro administration of Cyclosporin A (CsA) during mitogen, antigen and alloantigen activation of human T-lymphocytes on high affinity interleukin-2 (IL-2) receptor expression and -turnover and IL-2 production was investigated. The presence of CsA reduced 3H-thymidine incorporation and binding of radiolabelled human recombinant (ala125) IL-2 to high-affinity receptors in a dose-dependent fashion, although a pronounced inter- and intra-individual variation in sensitivity to CsA mediated immunosuppression was observed. Maximum inhibition was obtained when antigen and CsA were added to culture medium simultaneously. Preincubation with CsA did not influence the response. Although the number of IL-2 receptors was reduced, the turnover of the remaining high affinity IL-2 receptors on CsA treated cells was unaffected. Thus, binding, internalization and degradation were qualitatively unaltered by CsA administration. Finally, T cell activation in the presence of CsA reduced radioimmuno detectable IL-2 in cell culture supernatants to about 20%. CsA, added during antigen activation, reduced the number of Tac antigen presenting cells, but anti-Tac was unable to detect variations in the expression of high affinity IL-2 receptors. The present data indicate that CsA mediates immunosuppression by affecting early events during T-cell activation, and that variations in high affinity IL-2 receptor expression and IL-2 production are secondary to this affection.

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