Hepatic parenchymal replacement in mice by transplanted allogeneic hepatocytes is facilitated by bone marrow transplantation and mediated by CD4 cells

Authors


Abstract

The lack of adequate donor organs is a major limitation to the successful widespread use of liver transplantation for numerous human hepatic diseases. A desirable alternative therapeutic option is hepatocyte transplantation (HT), but this approach is similarly restricted by a shortage of donor cells and by immunological barriers. Therefore, in vivo expansion of tolerized transplanted cells is emerging as a novel and clinically relevant potential alternative cellular therapy. Toward this aim, in the present study we established a new mouse model that combines HT with prior bone marrow transplantation (BMT). Donor hepatocytes were derived from human alpha(1)-antitrypsin (hAAT) transgenic mice of the FVB strain. Serial serum enzyme-linked immunosorbent assays for hAAT protein were used to monitor hepatocyte engraftment and expansion. In control recipient mice lacking BMT, we observed long-term yet modest hepatocyte engraftment. In contrast, animals undergoing additional syngeneic BMT prior to HT showed a 3- to 5-fold increase in serum hAAT levels after 24 weeks. Moreover, complete liver repopulation was observed in hepatocyte-transplanted Balb/C mice that had been transplanted with allogeneic FVB-derived bone marrow. These findings were validated by a comparison of hAAT levels between donor and recipient mice and by hAAT-specific immunostaining. Taken together, these findings suggest a synergistic effect of BMT on transplanted hepatocytes for expansion and tolerance induction. Livers of repopulated animals displayed substantial mononuclear infiltrates, consisting predominantly of CD4(+) cells. Blocking the latter prior to HT abrogated proliferation of transplanted hepatocytes, and this implied an essential role played by CD4(+) cells for in vivo hepatocyte selection following allogeneic BMT. Conclusion: The present mouse model provides a versatile platform for investigation of the mechanisms governing HT with direct relevance to the development of clinical strategies for the treatment of human hepatic failure. (HEPATOLOGY 2008;47:706–718.)

To date, orthotopic liver transplantation remains the only therapeutic option for many acute and chronic end-stage liver diseases. However, donor organs are limited and can be used to treat only one patient at a time. Thus, great efforts are underway to explore new strategies for parenchymal liver cell replacement. Hepatocyte transplantation (HT) is currently the most promising option as hepatocytes have been proven to possess tremendous in vivo expansion capability, require little pretransplant manipulation, and may permit the distribution of cells from a single donor organ to multiple recipients. However, because of technical restraints, only a limited number of hepatocytes can be transplanted at a time; moreover, donor cells may be subject to immune surveillance. This raises an urgent need to develop novel strategies to induce tolerance and to select donor cells for in vivo expansion.

Recent studies in the field of HT have concentrated on animal models for liver repopulation and on the determination of intrinsic factors driving the in vivo expansion of transplanted cells. This has led to the development of several mouse models based on the genetic12 or chemical inhibition3 of endogenous liver regeneration. These approaches have allowed for the selection not only of transplanted hepatocytes but also of bone marrow (BM)–derived4 and even human hepatocytes5 within the recipient mouse liver. More recently, the concept of focused liver irradiation combined with additional hepatocyte proliferative stimuli, such as partial hepatectomy (PH)6 and injection of Fas-activating antibodies,7 has emerged as an effective strategy to mitotically arrest endogenous hepatocytes, allowing for efficient liver repopulation with transplanted hepatocytes.

Another factor to consider is bone marrow transplantation (BMT), which could contribute to successful HT by providing tolerance against the allogeneic cells.8 Transplanted rodent hepatocytes can engraft permanently in syngeneic hosts,9, 10 whereas following allogeneic transplantation, hepatocytes are usually quickly rejected by T cell–dependent mechanisms.11 Lethal irradiation of recipient mice is known to provide the appropriate conditioning for successful engraftment of allogeneic BM, and hematopoietic chimerism following BMT has been shown to provide central tolerance in various models of organ transplantation.12

In the present study, we sought to investigate the potential synergistic role of BMT on hepatocyte engraftment, expansion, and tolerance induction during allogeneic HT. We established a murine hepatocyte transplant model with allo-disparity by deriving donor hepatocytes from FVB mice transgenic for human alpha(1)-antitrypsin (hAAT)9 and transplanting them into Balb/C mice following various pretreatments aimed at enhancing cell engraftment. This strategy allowed us to progressively monitor the implantation and expansion status of the cell transplant in individual mice via simple quantitation of serum hAAT protein levels.

The key finding in these experiments was the selective achievement of complete liver repopulation in hepatocyte-transplanted mice tolerized by allogeneic BMT. Moreover, our studies of the underlying mechanisms identified an essential role of CD4(+) cells.

Abbreviations

BM, bone marrow; BMT, bone marrow transplantation; BrdU, bromodeoxyuridine; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorting; GvHD, graft versus host disease; hAAT, human alpha(1)-antitrypsin; HSC, hematopoietic stem cell; HT, hepatocyte transplantation; IFNγ, interferon γ; IL-6, interleukin-6; Lin, lineage panel; MHC, major histocompatibility complex; PH, partial hepatectomy; SGPT, serum glutamate pyrovate aminotransferase; TBS, trishydroxymethylaminomethane-buffered saline; TNFα, tumor necrosis factor α; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

Materials and Methods

Animals.

Mice transgenic for hAAT under transcriptional control of the hAAT promoter were used for donor hepatocytes, as described previously.9 Recipient animals were BM-transplanted at the age of 6 to 8 weeks. HTs were performed another 6 to 8 weeks later via splenic injections of 1 × 106 freshly purified hepatocytes. FVB recipients were either derived from the hAAT breeding colony (negative littermates) or purchased (Taconic Farms, Germantown, NY). Recipient Balb/C mice were derived from the Stanford in-house breeding colony. Mice were housed in 12-hour light/dark cycles, with free access to food and water, and were cared for in accordance with Stanford University's Administrative Panel on Laboratory Animal Care. All experiments implemented 4 to 6 animals per experimental group and analyzed time point.

BM and Hematopoietic Stem Cell (HSC) Isolation.

BM isolation was performed as described.13 Briefly, donor mice were euthanized by carbon dioxide asphyxiation followed by cervical dislocation. The lower extremities were prepared under sterile conditions, and the soft tissue was removed from both femurs. The femurs were then excised and placed into an ice-cold betadine solution. Under a laminar flow hood, the femurs were washed once in betadine and transferred into ice-cold Hank's balanced salt solution supplemented with 2% fetal bovine serum. The ends of the femurs were removed, and the BM was flushed with a 25-gauge needle and ice-cold media. The cells were triturated, centrifuged, and suspended in a dilution of 1 or 10 × 106 unfractionated BM cells per 200 μL in ice-cold media.

For HSC isolation, BM cells from FVB mice were stained with a lineage panel (Lin; mixture of biotinylated antibodies to Ter119, B220, CD3, Gr1, and CD11b; BD Biosciences, San Jose, CA). Lin cells were separated by magnetic depletion with streptavidin coupled to magnetic beads (Miltenyi Biotec, Auburn, CA) and counterstained with streptavidin Texas red (Pharmingen, San Jose, CA). C-kit (2B8)–positive, stem cell antigen-1 (D7)–positive, and CD45 (30F11)–positive cells were then fractionated twice by flow cytometry (DIVA-Van, Becton Dickinson, San Jose, CA) prior to intravenous injection of 3 × 104 cells via the tail vein.

Hepatocyte Isolation.

Hepatocytes were harvested and purified as described previously.14 Briefly, transgenic hAAT mice were anesthetized with ketamine, xylazine, and acepromazine. A laparotomy was then performed, and the inferior vena cava was cannulated. A calcium-free buffer was infused at 2 mL/minute, and the portal vein was cut once the liver had blanched completely. After perfusion with 25 mL of Ca-free buffer, 0.1% collagenase with 5 mM CaCl2 was infused for an additional 5 minutes at 2 mL/minute. The liver was excised, and the cells were dispersed in William's Eagle media supplemented with glutamine and antibiotics. The suspension was filtered through 80-μm gauze and centrifuged at 50g for 5 minutes. Cells were washed three times in ice-cold media, subsequently counted, checked for viability via trypan blue exclusion, and finally resuspended to a concentration of 1 × 106 cells per 100 μL.

BMT.

BMT was performed as described.13 Briefly, recipient mice were exposed to a lethal irradiation dose of 9.6 Gy, which was split up over two sessions separated by 3 hours. Unfractionated BM cells (1 × 106 syngeneic and 1 × 107 allogeneic) or 3 × 104 HSCs diluted into 200 μL of media were injected via the tail vein within 2 hours of the second irradiation dose. After BMT, animals were housed in autoclaved cages with antibiotic-supplemented water for 4 weeks.

Fluorescence-Activated Cell Sorting (FACS) Analysis.

Animals previously lethally irradiated and allogeneically BM-reconstituted were bled retro-orbitally 6 to 8 weeks after BMT into phosphate-buffered saline/ethylene glycol tetraacetic acid. Following red blood cell lysis, the remaining cells were centrifuged and subsequently incubated with antibodies against anti-CD45.1 (A20) or anti-CD45.2 (104) and/or against hematopoietic lineage markers: Mac-1 (M1/70), Gr-1 (RB6-8C5), B220 (RA3-6B2), CD3 (145-2C11), CD4 (GK1.5), CD8 (53–6.7), NK1.1 (pk136), and pan-NK (DX5; Pharmingen/eBioscience, San Diego, CA). The cells were subsequently analyzed with a FACS machine (FACSCalibur, BD, Mountain View, CA).

HT.

HT was performed as described previously.13 Briefly, recipient mice were anesthetized via isoflurane inhalation with an appropriate vaporator. A lateral abdominal incision was made, the spleen was localized, and 1 × 106 hAAT hepatocytes in a total volume of 100 μL of media were injected intrasplenically. Sutured spleens were returned carefully, and the skin was closed.

In Vivo Cell Depletion.

To deplete CD4 (GK1.5), CD8 (53-6.7), and natural killer (anti-asialo monosialotetrahexosylganglioside; Wako, Oakland, CA) cells in vivo, 100 mg of respective antibodies was applied intravenously over the course of the experiment (shown later in Fig. 5D). Efficiency of cell depletion was validated by FACS analysis after the second antibody injection. Anti-CD4 and anti-CD8 antibodies were a kind gift of Dr. Shizuru at Stanford University.

Figure 5.

Determination of the hepatic proliferative capacity of BM-transplanted mice in the PH model. (A) Syngeneically BM-transplanted FVB (FVBFVB-BM) and Balb/C (Balb/CBalb/C-BM) mice were partially hepatectomized 12 weeks after BMT. BrdU was injected 2 hours before sacrifice. Displayed are BrdU stainings of liver sections over a time course of 72 hours after PH. (B) Graphical depiction of the numbers of BrdU(+) hepatocytes after PH (see panel A). *P = 0.08, **P < 0.05. (C) Hepatocyte proliferation was determined in hAAT(+)-hepatocyte–transplanted Balb/CFVB-BM. Therefore, BrdU was injected into mice with ongoing liver repopulation 16 weeks after HT. One group underwent an additional PH. Displayed are anti-hAAT (red)/BrdU (green) costaining experiments. (D) Relative numbers of BrdU(+) hepatocytes in the fractions of hAAT(+) (transplanted) and hAAT(−) cells (host parenchyma) are displayed. *P = 0.09, **P < 0.05. (E) Selection of hepatocytes from irradiated mice: Donor hepatocytes were isolated from BM-transplanted hAAT(+) mice (8 weeks after BMT). Recipient animals were either left untreated or BM-transplanted (Balb/CFVB-BM and FVBFVB-BM). Displayed are hAAT-serum levels in recipient mice at indicated time points. P < 0.001.

PH.

FVB and Balb/C mice, 6 to 8 weeks old, were BM-transplanted and 12 weeks later subjected to PH. Briefly, the mouse abdomen was opened following anesthesia with isoflurane. The median and bilobulated right liver lobe were ligated and removed completely before the abdomen was sutured in layers. At the indicated time points after PH, bromodeoxyuridine (BrdU; 4 mg/mouse) was administered intraperitoneally, and mice were sacrificed 2 hours later.

Bleeding.

Mice were anesthetized with isoflurane and bled retro-orbitally at specific time intervals, as outlined previously.13 The samples were spun at 10,000 rpm for 10 minutes to collect serum. The samples were aliquoted and stored at −20°C until processing via enzyme-linked immunosorbent assay (ELISA).

Serum ELISA.

Serum was analyzed for hAAT expression with a standard sandwich ELISA, as described.13 Briefly, plates were coated with anti-hAAT antibodies (DiaSorin, Stillwater, MN). The samples were incubated overnight at 4°C after blocking with 5% milk powder in trishydroxymethylaminomethane-buffered saline (TBS)–Tween 20. An antigen-specific indicator antibody (Research Diagnostics, Inc., Flanders, NJ) linked to horseradish peroxidase was used to detect bound antigen. After the application of the substrate tetramethyl-1,3-butanediamine (TMBD; Sigma, Saint Louis, MO) and termination of the substrate reaction with sulfuric acid, the absorbance was measured in a fluorescent plate reader at a wavelength of 450 nm. Absorbance values were converted to nanograms per milliliter by comparison with a standard curve made from human serum. For ELISA-based cytokine analyses, samples were treated according to the manufacturer's instructions [interferon γ (IFNγ) and tumor necrosis factor α (TNFα), eBioscience; interleukin-6 (IL-6), BD, San Diego, CA].

Immunohistochemical Analyses and BrdU Detection.

For immunofluorescence analysis, cryosections (7 μm) were air-dried and fixed with ice-cold acetone (or ethanol). After rehydration, samples were treated with 2 N HCl for 30 minutes and neutralized with 0.1 M sodium borate (pH 8.0) for 10 minutes. Samples were washed with TBS–Tween 20. Antibodies were incubated in 0.2% bovine serum albumin/TBS (anti-hAAT, 1:200, RDI, Falmouth, MA; anti-BrdU, 1:40, Becton Dickinson; anti-CD4, clone GK1.5, 1:100, eBioscience). Alexa Fluor 488–conjugated or Alexa Fluor 594–conjugated secondary antibodies (Molecular Probes, Boston, MA) were used for immunofluorescence detection. Sections were analyzed with a fluorescence microscope (Zeiss, Stuttgart, Germany).

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL) Assay.

Cryosections were air-dried and fixed with 4% paraformaldehyde at room temperature. After being washed with phosphate-buffered saline, slides were incubated for 10 minutes with 3% H2O2 in methanol followed by sodium citrate (150 mM) for 2 minutes. After washing, the substrate mixture (terminal transferase) was applied according to the instructions of the manufacturer (Roche, Mannheim, Germany).

Western Blot Analysis.

Whole cell liver extracts were separated on 12% sodium dodecyl sulfate–polyacrylamide gels and blotted onto nitrocellulose membranes (Bio-Rad, Richmond, VA). Membranes were blocked (6% TBS–Tween 20) and incubated with antibodies (p21; Santa Cruz, Santa Cruz, CA; αTubulin, RDI). Antigen-antibody complexes were visualized with the enhanced chemiluminescence detection system as recommended by the manufacturer (Amersham, Boston, MA).

Statistical Analyses.

Data were compared at each time point with the Student t test. Statistical significance was validated by a P value below 0.05.

Results

Allogeneic BM Transplantation Provides Tolerance Against Major Histocompatibility Complex–Disparate Hepatocytes.

To test the hypothesis that BMT can provide tolerance across highly disparate major histocompatibility complex (MHC) mouse strains, lethally irradiated (9.6 Gy) Balb/C recipient mice were immune-reconstituted with 1 × 107 unfractionated BM cells derived from FVB mice (Fig. 1A). Successful BM engraftment and reconstitution were analyzed by the determination of the percentage of CD45 chimerism in peripheral blood mononuclear cells via FACS analyses (Balb/C CD45.2, FVB CD45.1; data not shown). FVB (donor) BM-derived cells typically accounted for over 99% of peripheral blood mononuclear cells (recipient mice with lower chimerism were excluded from further analyses) within 6 weeks following BMT. Full hematopoietic chimerism persisted throughout the lifetime of the BM-transplanted animals.

Figure 1.

Complete liver repopulation in allogeneically BM-transplanted and hepatocyte-transplanted mice. (A) Experimental design. Adult female mice (6–8 weeks of age) were lethally irradiated and immune-reconstituted with either syngeneic or allogeneic unfractionated BM. CD45 disparate Balb/C mice were used as allogeneic recipients for FVB-BM (to distinguish between donor-derived and host-derived BM cells). Six to eight weeks after BMT, successfully BM-reconstituted mice (defined by CD45.1/2 FACS analysis of peripheral Mac1/Gr1-positive and CD3/B220-positive cells) were transplanted intrasplenically with 1 × 106 hAAT(+) hepatocytes. Serum hAAT levels were determined at indicated time points. (B) Serum hAAT levels in BM-transplanted mice after HT. FVB and Balb/C mice (BM-transplanted and untreated controls) served as hepatocyte recipients. FVB mice underwent syngeneic BMT (FVBFVB-BM), and Balb/C mice underwent syngeneic BMT (Balb/CBalb/C-BM) and allogeneic BMT (Balb/CFVB-BM). *P < 0.001. (C) hAAT immunofluorescence analyses in hepatocyte-transplanted mice. Animals were sacrificed at 12 weeks of age (time of HT for the 0-week time point) and at 8 and 28 weeks post-HT. Liver cryosections were immunostained for hAAT protein (red fluorescent Alexa 594 secondary antibody). (D) Untreated, focally liver-irradiated or syngeneically BM-transplanted FVB mice were hepatocyte-transplanted and analyzed via hAAT ELISA. *P < 0.001.

Six to eight weeks after successful BM reconstitution, 1 × 106 hepatocytes derived from hAAT(+) transgenic FVB mice were transplanted intrasplenically. Because the hAAT protein has a short half-life and is secreted by the liver into the blood, sequential analysis of changes in serum hAAT levels in individual mice over time serves as an indirect measure of successful donor hepatocyte engraftment and expansion.9 As positive controls, we initially transplanted FVB hAAT(+) hepatocytes into untreated wild-type hAAT(−) FVB or syngeneically BM-transplanted littermates (FVB). In both scenarios, we observed the expected stable hAAT expression over the time course of the experiment (FVB and FVBFVB-BM; Fig. 1B). However, when FVB hAAT(+) hepatocytes were transplanted into untreated Balb/C (data not shown) or syngeneically BM-transplanted recipients (Balb/CBalb/C-BM), serum hAAT protein declined to undetectable levels after 2 weeks (Balb/CBalb/C-BM; Fig. 1B). In striking contrast, persistent serum hAAT protein levels were detected at the 2-week time point in Balb/C recipients previously transplanted and reconstituted with allogeneic FVB-BM (Balb/CFVB-BM). Moreover, serum hAAT levels in this experimental group continued to escalate throughout the experiment until a serum level equivalent to that of the donor hAAT(+) mice (∼5 × 103 μg/mL) was achieved (Balb/CFVB-BM; Fig. 1B).

To provide in situ evidence for successful hAAT(+) hepatocyte engraftment, immunostaining for the hAAT protein was performed on sectioned livers. As shown in Fig. 1C, these analyses revealed abundant clusters of hAAT(+) cells at early time points in allogeneically transplanted Balb/CFVB-BM mice. Impressively, 28 weeks after HT, that is, at a time when the serum hAAT levels had reached that of donor mice (Fig. 1B), the transgenic hepatocytes had completely replaced the host parenchymal cells in the recipient mice. Substantially fewer clusters were seen at similar time points in syngeneic FVBFVB-BM transplanted mice.

Irradiation Alone Does Not Promote Proliferation of Transplanted Hepatocytes.

Next, in order to determine the specific individual effect of hepatic irradiation alone on donor cell repopulation, that is, in the absence of BMT, the following experiments were performed. We compared recipient mice treated with syngeneic BMT (FVBFVB-BM) to mice whose livers had been focally irradiated (while the remaining mouse body was shielded) prior to HT (FVB9.6Gy). Liver-directed irradiation was administered at the same dose (9.6 Gy) normally used for BMT. Interestingly, although the initial hepatocyte engraftment in irradiated or BM-transplanted mice was similar to that in nonirradiated animals, analyses at later time points revealed significant differences among the experimental subgroups. Mice subject to focal liver irradiation displayed a modest 20% increase in hAAT levels 24 weeks after HT (FVB9.6Gy; Fig. 1D). In contrast, hAAT levels in syngeneically BM-transplanted recipients (FVBFVB-BM) rose 3- to 5-fold (300%-500%) during the same time period compared to the immediate posttransplant level (Fig. 1D). These results corroborated our findings from the allogeneic transplant experiments (Fig. 1B) and together strongly suggest that BMT, not irradiation alone, is crucial for the successful expansion of engrafting hepatocytes.

Distinct Inflammatory Response and Enhanced Apoptosis in Allogeneically BM-Transplanted Balb/C Mice.

We next analyzed liver histomorphology in BM-transplanted mice. Widespread mononuclear infiltrates were present at all time points (10 and 40 weeks after BMT) in allogeneically BM-transplanted Balb/C mice (Balb/CFVB-BM; Fig. 2C,I). Such infiltrates were substantially less prevalent in syngeneically BM-transplanted Balb/C or FVB mice (Balb/CBalb/C-BM or FVBFVB-BM, data not shown).

Figure 2.

Mononuclear infiltration after BMT. (A-H) BM-transplanted animals were sacrificed 10 and 40 weeks after BMT to unravel possible BMT-related changes in the microscopic liver architecture. Photomicrographs of hematoxylin-eosin–stained cryosections were taken at (A-D) ×100 and (E-H) ×400 magnifications. Shown are (A,E) nontreated (Balb/C), (B,F) syngeneically BM-transplanted (Balb/CBalb/C-BM), and (C,D) allogeneically BM-transplanted (Balb/CFVB-BM) mice. Panel H [40-week Balb/CFVB-BM + FVB hAAT(+) hepatocytes] was taken from a mouse at the time point of complete liver repopulation by transplanted hepatocytes, as opposed to the non–hepatocyte-transplanted (40-week Balb/CFVB-BM) liver in panel G from a Balb/CFVB-BM mouse. (I) Infiltrating mononuclear cells were counted in individual groups 10 and 40 weeks after BMT and graphically depicted as cells per view field (×100 magnification). *P < 0.05, **P < 0.01.

Histological analysis 40 weeks after BMT revealed further changes in the liver architecture of allogeneically BM-transplanted Balb/C mice (Balb/CFVB-BM; Fig. 2D,G). These included abundant areas containing condensed small hepatocytes (next to larger parenchymal cells) indicative of liver regeneration. Allogeneically BM-transplanted mice also showed significant mononuclear infiltrates as well as early signs of portoportal bridging. In contrast, Balb/CFVB-BM recipients that were fully repopulated by transplanted hepatocytes displayed a homogeneous normal liver architecture containing expanded donor hepatocytes uniform in size (Fig. 2H). In addition, the previously recognized mononuclear cell infiltrates were substantially less prevalent in fully repopulated mice 40 weeks post-BMT in comparison with the 10-week time point (Fig. 2I).

To establish the correlation between the observed liver mononuclear infiltrates and a general inflammatory response, we performed total peripheral white blood cell counts. Allogeneically BM-transplanted Balb/C (Balb/CFVB-BM) mice showed the highest numbers of circulating white blood cells, despite minor variations between individual animals in all groups (Fig. 3A).

Figure 3.

Inflammatory response. (A) White blood cell counts in peripheral blood were performed in the indicated groups 10 weeks after BMT. Displayed are average numbers of cells per animal. Differences between groups were not significant. (B) SGPT levels were determined in experimental mice before and 10 weeks after BMT. **P < 0.001. (C) Analysis of IL-6 plasma levels in peripheral blood determined via ELISA before and 10 weeks after BMT. **P < 0.001. (D) Displayed is a TUNEL analysis (terminal transferase-mediated labeling of DNA breaks) of apoptotic cells in hepatocyte-transplanted Balb/CFVB-BM mice (8 weeks after transplantation versus untreated Balb/C mice). To determine the occurrence and rates of apoptosis, hAAT (red fluorescence) and TUNEL stainings (green fluorescence) were performed on serial tissue sections (blue fluorescence: 4′,6-diamidino-2-phenylindole). (E) Apoptotic cells were counted and graphically displayed in relation to the overall cell number. P < 0.01.

For an exact calculation of the degree of liver damage, we analyzed serum hepatocyte aminotransferase [serum glutamate pyrovate aminotransferase (SGPT)] levels in the different experimental groups. The highest aminotransferase levels were found in the allogeneically BM-transplanted Balb/C mice (Balb/CFVB-BM; Fig. 3B). Next, we measured several candidate proinflammatory cytokines and found that TNFα and IFNγ were detectable at variable levels in the peripheral blood among experimental groups, whereas low IL-6 levels were found in both allogeneically and syngeneically BM-transplanted Balb/C mice (Balb/CFVB-BM and Balb/CBalb/C-BM; Fig. 3C).

To determine whether the previously described inflammatory process contributed to increased programmed cell death of hepatocytes, we analyzed recipient liver tissues for the presence of indicators of apoptosis by TUNEL assay. Serial stainings for apoptosis and hAAT were undertaken in BM-transplanted and hepatocyte-transplanted Balb/C mice (Fig. 3D). Quantitative analysis revealed that the vast majority of the apoptotic cells were found among hAAT(−) host cells (0.95%) and that the areas covered by hAAT-positive cells had significantly fewer apoptotic cells (0.15%; Fig. 3E).

CD4(+) Cells Promote Selection of Transplanted Allogeneic Hepatocytes.

In order to further characterize the observed mononuclear infiltrates in the livers of allogeneically BM-transplanted recipients, we performed liver immunostainings for CD4 (Fig. 4A) and CD8 (Fig. 4B) antigens. The majority of the infiltrating cells appeared to be CD4-positive. Thus, we next sought to resolve the spatial relationship between the infiltrating CD4(+) cells and proliferating transplanted allogeneic hepatocytes by performing hAAT/CD4 coimmunostainings of Balb/CFVB-BM livers transplanted with hAAT(+) hepatocytes. We found that the CD4(+) infiltrates were present in the areas of host (Balb/C) parenchymal liver tissue but strikingly absent from the foci of engrafted hAAT(+) donor (FVB) hepatocytes (Fig. 4C).

Figure 4.

CD4(+) cells promote selection of transplanted allogeneic hepatocytes. (A,B) Hepatic cryosections of BM-transplanted FVB and Balb/C mice were stained with fluorescein isothiocyanate–conjugated (A) anti-CD4 or (B) anti-CD8 antibodies. Displayed are ×40 photomicrographs taken from untreated or syngeneically or allogeneically BM-transplanted FVB and Balb/C mice. Various numbers of CD4(+) cells (green) were observed within the livers of recipient animals and spread throughout the parenchyma. (C) Cryosections of previously BM-transplanted and hepatocyte-transplanted Balb/C mice were costained with anti-CD4 (green fluorescence) and anti-hAAT (red fluorescence) antibodies. Tissue samples were taken 8 weeks after HT. (D) Experimental outline for the antibody blocking experiments. Balb/C mice were first allogeneically BM-transplanted (Balb/CFVB-BM). Intravenous injections of 150 mg of anti-CD4, 100 mg of anti-CD8, and 100 mg of anti-NK were started 5 weeks after BMT and then repeated at the indicated time points. Antibody recipient mice underwent an hAAT(+) HT 1 day after the second antibody injection. Mice were then bled as usual after HT at the reported time points to determine serum hAAT levels. (E,F) Relative rise in hAAT levels from antibody-depleted mice. Relative increases in hAAT serum levels were calculated from the ratios of hAAT levels at the 4-week or 20-week and 3-day post-HT time points. *P = 0.05, **P < 0.05, ***P < 0.01. (G) Role of HSCs in the selection of transplanted hepatocytes. Animals that had received unfractionated BM cells (1 × 107) were compared to mice treated with purified HSCs (3 × 104) as the source for immune reconstitution. Six weeks after allogeneic BM/HSC transplantation, reconstituted animals were transplanted with 1 × 106 hAAT(+) hepatocytes, and hAAT levels were determined over time.

Next, in order to determine which subset of mononuclear cells was critically mediating the proliferative response of transplanted hepatocytes, we pretreated Balb/CFVB-BM mice with antibodies blocking CD4, CD8, and natural killer cells (Fig. 4D). The antibodies were administered at doses that completely depleted the specified cells15, 16 and in weekly intervals prior to, and up to 3 weeks after, HT. Effective cell depletion was confirmed for at least 4 weeks after HT by FACS analysis of peripheral blood (data not shown). Serum hAAT levels were analyzed 3 days, 4 weeks, and 20 weeks after HT. The relative increase in serum hAAT concentrations over the levels at day 3 were used to evaluate the relative amounts of hepatocyte engraftment (Fig. 4E,F). Interestingly, 4 and 20 weeks after HT and antibody treatment, mice that had received anti-CD4 or, to a lesser degree, anti-CD8 antibodies displayed a significantly attenuated increase in serum hAAT levels in comparison with controls.

In order to elucidate whether the observed inflammation and repopulation was due to graft versus host disease (GvHD), we compared Balb/C mice reconstituted with 1 × 107 unfractionated FVB-BM cells to a cohort of mice reconstituted with purified HSCs (FVB, HSC, 3 × 104). Both groups underwent subsequent HT 6 weeks after hematopoietic reconstitution. Serum hAAT measurements revealed comparable levels of hepatocyte engraftment and expansion in both groups. Hepatocyte engraftment was delayed at earlier time points in the HSC cohort, but both experimental groups were similarly repopulated after 28 weeks (Fig. 4G).

Analysis of the Cellular Proliferation Capacity in BM-Transplanted Mice.

Irradiation is well known to inhibit cell cycle progression.17 In contrast, only a few studies have investigated the impact of BMT (which requires lethal host irradiation for its conditioning) on liver physiology thus far. Therefore, it is unclear whether and how BMT affects the proliferative capacity of hepatocytes at late time points after treatment, when the transplanted hepatocytes are still undergoing cell division. To establish the regenerative potential of BM-reconstituted mice, we performed PHs in syngeneically BM-transplanted Balb/C (Balb/CBalb/C-BM) and FVB (FVBFVB-BM) mice 12 weeks after BMT and compared the results to non–BM-transplanted mice. Hepatocyte proliferation was determined by the counting of BrdU-positive proliferating hepatocytes 36, 48, and 72 hours post-PH.

Notably, at 36 and 48 hours after PH, livers of mice from both BM-transplanted groups showed fewer BrdU-positive cells compared to their nonirradiated counterparts. This was particularly obvious for BM-transplanted Balb/C mice, which displayed the weakest BrdU staining of all groups 48 hours after PH (Fig. 5A,B). However, at 72 hours post-PH, the numbers of proliferating cells were comparable between the groups.

These results prompted us to investigate the hepatic proliferative response in allogeneic Balb/CFVB-BM hepatocyte-transplanted recipients directly. BrdU was injected into normal hepatocyte-repopulating mice and additionally into mice that had undergone a PH 16 weeks after HT. Double staining for hAAT and BrdU revealed proliferating cells in endogenous hAAT(−) host parenchyma and in engrafted hAAT(+) donor hepatocyte clusters (Fig. 5C). Quantitative analysis demonstrated a significantly higher proliferation ratio for the hAAT(+) cells (FVB, 0.95%) compared to surrounding host cells (Balb/C, 0.65%, P < 0.05; Fig. 5C) in nonpartially hepatectomized mice. There was also a higher BrdU uptake in hAAT(+) hepatocytes after PH, although the difference was not statistically significant.

Next, we sought to determine whether irradiation of donor animals affected the selective repopulation capability of donor hepatocytes in our mouse model. We therefore transplanted hepatocytes that had been isolated from previously irradiated and syngeneically BM-transplanted hAAT(+) mice. Three days post-PH, cell viability and primary engraftment (Fig. 5E) of hepatocytes derived from those BM-transplanted donor mice were similar to those of the nonirradiated hAAT(+) donor animals used before. However, long-term analysis revealed a rapid decline in serum hAAT levels following the initial engraftment in both untreated and BM-transplanted syngeneic recipients (FVBFVB-BM; Fig. 5E). In contrast, hepatocytes from BM-transplanted hAAT(+) donor mice remained able to repopulate the livers of previously allogeneically BM-transplanted Balb/C recipients (Balb/CFVB-BM). This indicated that the selection process as a direct result of allo-disparity was more robust in comparison with BMT-induced hepatocyte damage.

Prolonged p21 Activation in BM-Transplanted Balb/C Mice.

We next sought to determine the molecular mechanism underlying the observed reduced hepatocyte proliferation rate in BM-transplanted Balb/C mice. We analyzed expression of the cyclin-kinase inhibitor p21, a p53-dependent protein known to be up-regulated in conditions of cell cycle arrest in the liver.18 Western blot analysis of whole liver cell extracts (Fig. 6A) revealed that p21 was up-regulated in BM-transplanted mice at 10 weeks after BMT. The p21 up-regulation was even more pronounced in BM-transplanted Balb/c mice compared to the FVB counterparts, whereas no p21 signal was evident in both naïve strains. Forty weeks after BMT, the p21 signal declined in all mice. It was still detectable in the BM-transplanted Balb/C recipients but not in the long-term BM-transplanted FVB mice. Moreover, mice found to have complete donor cell liver repopulation had no detectable p21 expression as determined by western blot analysis. To evaluate whether p21 expression solely depends on irradiation-mediated effects, we compared liver only–irradiated mice with fully BM-transplanted recipients 10 weeks after treatment. There was no detectable difference between those treatment conditions (Fig. 6B).

Figure 6.

Hepatocyte selection correlates to the p21 status. Total liver proteins were isolated from different mouse groups at the indicated time points. Each sample was pooled from at least three mice. Seventy milligrams of protein extract per sample was analyzed by western blot and with an anti-p21 antibody. Cell extracts obtained from mice 36 hours post-PH were used as controls. (A) Displayed is a western blot showing p21 induction after BMT in hepatocyte recipient mice. BMT induced comparable levels of p21 in recipient mice. However, in fully hepatocyte-repopulated (40 weeks after HT) mice, the p21 signal completely vanished, whereas it could still be detected in nonrepopulated Balb/CFVB-BM mice. (B) p21 levels were investigated in BM-transplanted and liver only–irradiated FVB mice, and this revealed equal expression of the protein in both groups.

Discussion

Numerous previous studies have investigated the phenomenon of liver repopulation by transplanted hepatocytes with the hope that expanded knowledge of the underlying biological processes will lead to improved clinical transplantation efficiency. Here, we have presented an advanced model of liver repopulation allowing us to define several previously unrecognized but critical parameters explaining the observed in situ selection of transplanted hepatocytes. Our findings address several relevant questions regarding allogeneic immune tolerance and survival of engrafting hepatocytes. Moreover, these findings permit us to conclude that selection and proliferation of transplanted hepatocytes across and within MHC are related to the processing of engrafting cells by the hematopoietic system. Finally, we have established that the resulting tolerance is directly related to BM transfer and inflammation.

Previous studies have suggested that BMT can provide efficient central tolerance to transplanted allogeneic cells and whole organs in both mice and humans.19–21 However, there are unique biologic considerations that differ between cellular and whole organ transplant, where tissue integrity and total organ function including antigen presentation and immune-related functions are preserved.19

Successful HT requires that injected cells survive circulating modulators of immunity while trafficking to the hepatic sinusoids for possible engraftment. Once in the liver, engrafting cells must traverse several cell layers and a basement membrane in order to functionally integrate into the remaining liver parenchyma. During this process, more than 80% of the transplanted cells are lost prior to engraftment.22 Thus, providing adequate tolerance for the few engrafting cells is paramount. More cells surviving the initial transplant procedure provide a more substantial base for the designated therapeutic effect of the graft. In the various experiments presented here, the average initial engraftment rate of transplanted hepatocytes was very similar among the recipient mice. However, important variations were found in the level of long-term engraftment of transplanted hepatocytes. Therefore, our studies suggest that in addition to optimal initial engrafting conditions and immune recognition, other previously undefined factors influence long-term cell survival and expansion.

Previous work has shown that transplanted syngeneic hepatocytes can engraft long-term10 and survive for the lifetime of the recipient mouse. Moreover, it has been demonstrated that transplanted hepatocytes can efficiently repopulate the host liver under conditions of impaired host cell proliferation.1–3, 14 Previous work by Roy-Chowdhury's group6 suggested that liver irradiation combined with PH or other forms of liver injury7 can provide the needed environment to achieve efficient repopulation rates by transplanted hepatocytes in rodents. However, the irradiation used in those studies was focally directed at the liver and given in very large doses that were nonmyeloablative (because BM of recipients was not exposed to the irradiation). Additionally, in our model, there is an important time delay between the event of irradiation during BMT and the observed selective effects on transplanted hepatocytes.

Recently, it has been demonstrated that BM cells can also contribute to hepatocellular gene expression via fusion with existing host hepatocytes.4 In our previous work,13 we showed that the frequency of fusion events of hAAT(+) BM cells with hepatocytes was observed in a range of 1 to 3 × 104. This corresponds to an hAAT level around 50–100 ng/mL,13 which is 100–200 times lower than the initial engraftment level after transplantation of adult hepatocytes (8–10 × 103 ng/mL; see Fig. 1 B). Selection of such BM-derived hepatocytes occurs at a significant level only in the FAH−/− model23 or under other strong selection conditions.24 As we use hAAT(−) mice as donors for BMT and because such fusion events have to be considered extremely rare, it is very unlikely that this mechanism is prominent in our observations.

Taken together, our data suggest that liver irradiation itself may prime the liver for successful HT. It provides an environment in which engrafting hepatocytes can be selected for expansion in preference to the endogenous cells. However, the underlying mechanisms have not yet been fully defined. It is known that ionizing radiation causes oxidative injury and double-stranded DNA breaks. Both result in an impairment of liver cell proliferation in a p53-dependent manner.25 However, the irradiation alone cannot explain the robust hepatocyte repopulation that we observed after BMT. This is because the cell selection was 3- to 5-fold greater in mice that underwent a syngeneic BMT (FVBFVB-BM) compared to mice that did not have BMT but were treated by selective liver irradiation (FVB9.6Gy; Fig. 1D) only. This finding is further supported by recent studies that combined focal liver irradiation with additional liver injuring treatments.7, 26 Hepatocytes have been shown to initiate cell cycle progression in response to a variety of well-characterized growth factors (such as hepatocyte growth factor27) and cytokines (such as IL-628 and TNFα29). The coordinated activation of these factors can then result in cell proliferation. Detailed mechanisms leading to hepatocellular proliferation have been mostly investigated in numerous PH studies30 and are therefore not part of the presented study.

For our model of combined BMT and HT, we speculate the following scenario. Irradiation results in some degree of liver tissue damage. The following course of immune reconstitution initiated by BMT itself can be characterized as an inflammatory process.31 Over time, a weak subclinical inflammatory response32 occurs, and in addition, immunological mechanisms, caused by the introduction of the transplanted BM, could further result in the release (for example, by activated Kupffer cells33) of proliferation-inducing mediators. This might be partly reflected by the observed elevation of serum IL-6 in BM-transplanted Balb/C mice (Fig. 3C). The measured IL-6 levels, however, are substantially lower compared to situations of severe infections or liver injury. They do not exclude the existence of cytokine activities on a paracrine level resulting from direct cell-cell interactions. However, it still remains to be elucidated whether the hepatocyte proliferation observed in our model is a result of a BMT-related liver process or a more generalized systemic late response.31 In contrast to the syngeneic situation, the experimental outcome was quite dramatic in allogeneically BM-transplanted Balb/C mice (Balb/CFVB-BM). Surprisingly, these animals repopulated nearly the entire liver with transplanted allogeneic hepatocytes.

Thus, underlying mechanism(s) for the observed repopulation were delineated further. The most prominent histological finding in Balb/CFVB-BM animals, which displayed hair loss and poor feeding, was the observed infiltration of mononuclear cells, reminiscent of GvHD. These cells appeared to be mainly CD4(+). The significance of the CD4(+) cells for hepatocyte repopulation was further established by cell-specific blocking experiments (Fig. 4D-F), which revealed a remarkable delay in the onset of detectable hAAT protein in the serum of CD4(+) cell-depleted animals. Notably, the hepatocyte repopulation did not begin until the time when the antibodies were no longer effective at depleting CD4(+) cells (about 4 weeks post-HT, equivalent to 1 week after the last antibody injection).

Taken together, these data suggest that in our model the presence of donor-derived CD4(+) cells modulates liver repopulation by transplanted hepatocytes. Arguably, the underlying mechanisms can be primary (cytotoxic) effects on host liver cells and/or stimulation of the graft. It is likely that the immune cells derived from the BM (FVB) act in part like GvHD. Thus, the inflammatory response impairs survival and proliferation of the endogenous (host) hepatocytes, which already have been injured by irradiation at the time of BMT. This hypothesis was supported by our finding that the CD4(+) infiltrates spared the areas of expanding hAAT(+) graft hepatocytes (Fig. 4C). Therefore, the transplanted cells were not affected by any direct harmful effects generated from the CD4(+) cells and could additionally benefit from the release of the aforementioned cytokines related to the liver inflammation, which enabled them to proliferate.

A contribution of CD4(+) cells for the modulation of liver damage under conditions of GvHD has been reported.34 Despite this report, the role of CD4(+) cells in hepatocyte allograft rejection is still controversial.35, 36 Nonetheless, the CD4(+)-dominated inflammatory response observed in our experiments did not correlate with a classical GvHD.37–39 The allogeneic response observed in a typical GvHD reaction is usually based on donor-educated lymphocytes [usually CD8(+) cells40], which are transferred during BMT41 together with other precursors and mature cells of the BM. If there had been an effect from the transferred donor lymphocytes in our model, we would have expected a vastly diminished selection of the transplanted hepatocytes in HSC-only reconstituted animals compared to recipients of unfractionated BM.42 Thus, the observed infiltrating inflammatory cells are most likely generated after the engraftment of the allogeneic BM.

Inflammation and cellular infiltration thus promote tissue damage in host livers resulting in programmed cell death, as demonstrated in Fig. 4D. Interestingly, hAAT-positive transplanted hepatocytes were only modestly affected by apoptosis, as most of the apoptotic cells were found outside the hAAT(+) areas, thus providing an explanation for the observed donor cell selection. Enhanced apoptosis of cells can be caused by the so-called streaming effects described recently by Oertel et al.43 In this case, apoptosis would occur as a secondary event in response to the proliferation of engrafted hAAT(+) cells. Alternatively, apoptosis of surrounding host hepatocytes and other cells, caused by infiltrating immune cells and inflammatory cytokines, could contribute to the selection of the transplanted hepatocytes.

We also considered the relevance of other mechanisms that may have contributed to the observed high hepatocyte repopulation rate in Balb/CFVB-BM mice. This mouse strain is known to have a greater susceptibility to different forms of injury, a fact that has been established in the CCl4 model of chronic liver injury.44 A molecular correlate of the proposed greater injury in Balb/C mice was found in the prolonged activation of p21,45 a p53-dependent cyclin kinase inhibitor. The association between irradiation-induced p53 activation of p21 and cell cycle arrest46 has been previously described. However, inflammatory stress via signal transducer and activator of transcription 3 activation47 can result in enhanced p21 expression as well. Therefore, different pathways may trigger higher p21 expression in our model and might be involved in blocking proliferation of host hepatocytes.

In summary, our data demonstrate that BMT can induce tolerance after HT and is involved in triggering the selection of transplanted hepatocytes. Therefore, this study lends insight to several governing principles that may facilitate the application of clinically relevant HT. BMT and the accompanying irradiation synergistically improve the engraftment of transplanted hepatocytes6 and promote hepatocyte proliferation under syngeneic conditions. Additionally, CD4(+) cells are responsible for triggering massive cell selection under allogeneic conditions. Concepts to induce tolerance in human organ transplantation48, 49 based on nonmyeloablative BM transfer are currently being introduced into the clinic. These results therefore provide further motivation that adequate protocols can be developed, which may lead to tolerance induction for transplanted hepatocytes. The ultimate goal would be to use such a strategy for liver repopulation as a primary treatment of numerous liver diseases.

Ancillary