ISOLATION, PHENOTYPE, AND ALLOSTIMULATORY ACTIVITY OF MOUSE-LIVER DENDRITIC CELLS

Donor liver-derived dendritic cells (DC) have recently been identified within various lymphoid and nonlymphoid tissues of organ allograft re cipients, including non,immunosuppressed mice transplanted with and pe rmanently accepting major histocompatibility complex (MHC)-disparate h epatic allografts. These findings have raised questions about the basi s of the tolerogenicity of the Liver-and, in particular, about the pro perties of liver-derived DC. To study further the structure, immunophe notype and allostimulatory activity of leukocytes resident in normal m ouse (B10.BR; H-2(k), I-E(k)) Liver, a procedure was developed to maxi mize the yield of viable, nonparenchymal cells (NPC) obtained followin g collagenase digestion of perfused liver fragments and density centri fugation (Percoll). These cells comprised populations expressing lymph oid and myeloid cell surface antigens. As compared with spleen cells, they proved good allostimulators of naive (B10; H-2(b), I-E(-)) spleni c T cells when tested in primary mixed leukocyte reactions (MLR). Afte r overnight (18-hr) incubation of the NPC, enrichment for transiently adherent, low-density (LD) cells on metrizamide gradients permitted th e recovery of low numbers of cells (approx. 25 x 10(5) per liver), man y of which displayed distinct DC morphology. Flow cytometric analysis revealed that these cells were CD3(-); CD4(-); CD8(-), and B220(-), bu t strongly expressed CD45 (leukocyte-common antigen), and mild-to-mode rate levels of CD11b, heat-stable antigen, and CD44. The cells also ex pressed moderate intensity of NLDC 145 but not 33D1, DC restricted mar kers which have been shown to be differentially expressed on mouse DC isolated from various organs. This DC-enriched population was more str ongly MHC class II(I-E(k))(+) than NPC, as determined by immunocytoche mistry and flow cytometry and exhibited much more potent allostimulato ry activity for naive T cells. These findings demonstrate that freshly isolated murine liver NPC, and perhaps their counterparts in situ, ex hibit allostimulatory activity that is enhanced in the nonadherent, lo w-density (DC-enriched) fraction after overnight culture. They further suggest that the maturation of liver DC may play a key role in determ ining the immunogenicity and or tolerogenicity of hepatic allografts.