Isolation, phenotype, and allostimulatory activity of mouse liver dendritic cells

Abstract

Donor liver-derived dendritic cells (DC) have recently been identified within various lymphoid and nonlymphoid tissues of organ allograft recipients, including nonimmunosuppressed mice transplanted with and permanently accepting major histocompatibility complex (MHC)-disparate hepatic allografts. These findings have raised questions about the basis of the tolerogenicity of the liver--and, in particular, about the properties of liver-derived DC. To study further the structure, immunophenotype and allostimulatory activity of leukocytes resident in normal mouse (B10.BR;H-2k, I-Ek) liver, a procedure was developed to maximize the yield of viable, nonparenchymal cells (NPC) obtained following collagenase digestion of perfused liver fragments and density centrifugation (Percoll). These cells comprised populations expressing lymphoid and myeloid cell surface antigens. As compared with spleen cells, they proved good allostimulators of naive (B10; H-2b, I-E-) splenic T cells when tested in primary mixed leukocyte reactions (MLR). After overnight (18-hr) incubation of the NPC, enrichment for transiently adherent, low-density (LD) cells on metrizamide gradients permitted the recovery of low numbers of cells (approx. 2-5 x 10(5) per liver), many of which displayed distinct DC morphology. Flow cytometric analysis revealed that these cells were CD3-, CD4-, CD8-, and B220-, but strongly expressed CD45 (leukocyte-common antigen), and mild-to-moderate levels of CD11b, heat-stable antigen, and CD44. The cells also expressed moderate intensity of NLDC 145 but not 33D1, DC restricted markers which have been shown to be differentially expressed on mouse DC isolated from various organs. This DC-enriched population was more strongly MHC class II(I-Ek)+ than NPC, as determined by immunocytochemistry and flow cytometry and exhibited much more potent allostimulatory activity for naive T cells. These findings demonstrate that freshly isolated murine liver NPC, and perhaps their counterparts in situ, exhibit allostimulatory activity that is enhanced in the non-adherent, low-density (DC-enriched) fraction after overnight culture. They further suggest that the maturation of liver DC may play a key role in determining the immunogenicity and or tolerogenicity of hepatic allografts.

Figure 1

Flow plan for the isolation of nonparenchymal cells from normal mouse liver and for the preparation of DC-enriched suspensions.

Figure 2

Cryostat section of normal B10.BR mouse (H-2K^k1-E⁺) liver, showing strongly MHC class II–positive cells in a portal area. All positive cells appear to be associated with blood vessel components. There is no staining of bile duct epithelium, hepatocytes, or endothelial cells (avidin-biotin peroxidase, counterstained with hematoxylin; ×400).

Figure 3

Representative flow cytometric analysis of cell surface markers expressed on the NPC population freshly isolated from normal (B10.BR) mouse liver. The cells were stained by direct or indirect immunofluorescence and five thousand gated events were acquired for each mAb.

Figure 4

Allostimulatory activity for 2.10⁵ naive B.10 (H-2^b) mouse splenic T cells of variable numbers of γ-irradiated, freshly isolated NPC prepared from normal B10.BR (H-2^k) mouse liver. Cells were cultured for 72 hr; [³H]TdR was added to the cultures 18 hr before harvesting. Results are mean cpm ± 1 SD and representative of 3 separate experiments. (△) freshly isolated NPC; (●) freshly isolated B10.BR spleen cells; (○) unstimulated B10 splenic T cells. Syngeneic (B10) liver NPC stimulators gave results almost identifical to those obtained with unstimulated T cells.

Figure 5

Giemsa-stained cytocentrifuge preparations of nonadherent, low-density, liver-derived cells recovered from metrizamide gradients after overnight culture of NPC from normal B10.BR mouse livers. The cells are agranular, with variable degrees of cytoplasmic vacuolation and irregularly shaped, eccentric nuclei. Some cells display distinct cytoplasmic projections. One of the cells (right panel) exhibits prominent nucleoli (arrows) ×1000.

Figure 6

Strongly MHC class II⁺ cells with distinct dendritic morphology present in the nonadherent, LD fraction recovered from overnight-cultured B10.BR mouse liver NPC. Cells were stained using a mAb to I-E^k and the avidin-biotin-peroxidase procedure (counter-stained with hematoxylin, ×1000).

Figure 7

Cell surface markers on normal mouse liver DC-enriched cell populations stained by direct or indirect immunofluorescence and analyzed by flow cytometry. Nonadherent LD cells were harvested from metrizamide gradients after overnight culture of NPC from normal B10.BR mouse livers. Five thousand gated events were acquired for each mAb (detailed in Table 1). The results are representative of 3 separate experiments.

Figure 8

Allostimulatory activity for 2.10⁵ naive B.10 (H-2^b) mouse splenic T cells, of variable numbers of γ-irradiated, overnight-cultured NPC or DC-enriched stimulator cell suspensions prepared from normal B10.BR (H-2^k) mouse liver. Cells were cultured for 72 hr; [³H]TdR was added to the cultures 18 hr before harvesting. Results are mean cpm ± 1 SD and representative of 3 separate experiments. (■) DC-enriched cell population (nonadherent LD NPC population after overnight culture); (□) HD NPC population after overnight culture; (△) bulk NPC; (●) fresh B10.BR spleen cells; (○) unstimulated splenic T cells. Syngeneic (B10) liver NPC stimulators gave results almost identifical to those obtained with unstimulated T cells.