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a First Department of Pathology,
b Transplantation Center, Kansai Medical University, Osaka, Japan
Key Words. Bone marrow transplantation • Hemopoietic stem cells • Bone marrow stromal cells • Portal vein
Susumu Ikehara, M.D., Ph.D., First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan. Telephone: 81-6-6-6993-9429; Fax: 81-6-6994-8283; e-mail: ikehara{at}takii.kmu.ac.jp
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ABSTRACT |
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These findings suggest that the interaction of HSCs with stromal cells in the liver is the first crucial step for successful engraftment of allogeneic HSCs. It is likely that donor stromal cells and HSCs trapped in the liver migrate into the recipient BM and spleen, where they form CFU-BM and CFU-S, respectively.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6(B6)] and [MRL/MP-lpr/lpr B6] [7, 8]. In addition, we have shown that donor-type BM stromal cells are found in the recipient BM after BMT plus bone graft [7, 8]; the stromal cells in the engrafted bones were found to migrate into the recipient BM where they support hemopoiesis. These findings indicate that the stromal cells contribute to successful allogeneic BMT, and that major histocompatibility complex (MHC) barriers can be overcome by cografting BMCs with donor bones containing stromal cells. We have also found that an MHC restriction exists between hemopoietic stem cells (HSCs) and stromal cells not only in vivo [9, 10] but also in vitro [11]; day-12 colony-forming unit-spleen (CFU-S) and CFU-granulocyte/macrophage counts were found to increase in the MHC-compatible microenvironment (stromal cells) [9, 10]. It has been reported that the portal venous (PV) administration of antigens is more effective in inducing tolerance than i.v. injection [12]. Since the liver is the largest organ in the reticuloendothelial system, it is conceivable that the liver plays a crucial role in modifying immunogenic alloantigens into tolerogenic forms [13]. We have also demonstrated that allogeneic tolerance can be induced when donor BMCs are injected via the PV, but not i.v. [14, 15], and that hemopoietic foci are formed in the recipient liver [14]; when injected via the PV, most of the allogeneic cells are trapped in the recipient liver [14]. In addition, we have found that one of the mechanisms behind the PV-induced tolerance is the absence of costimulatory signals on donor HSCs that are trapped in the recipient liver; the donor HSCs induce clonal anergy to CD8+ T cells [16].
In the present study, we examine whether BM stromal cells injected via the PV play an important role in reconstituting the recipient mice with donor hemopoietic cells in comparison with the i.v. We also show that donor-derived stromal cells trapped in the liver facilitate the engraftment of allogeneic BMCs.
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MATERIALS AND METHODS |
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Cell Preparation and i.v. or PV Injection
BMCs were flushed out using a 23-gauge needle attached to a 2.5-ml syringe from the femurs, tibias, and humeruses, and suspended in phosphate-buffered saline containing 2% fetal calf serum. In some experiments, BMCs were injected into lethally irradiated (9.5 Gy) recipient mice without any purification. Low-density (LD) BMCs were purified by discontinuous density gradient centrifugation using Percoll (Pharmacia; Uppsala, Sweden; http://www.pnu.com). The LD cells (1.060 < 
< 1.074 g/cm3) were incubated with monoclonal antibody (mAb) (rat IgG class) cocktails against lineage markers (Mac-1, Gr-1, B220, CD4, CD8, CD71, and TER119) for 30 min on ice, and then incubated twice with sheep anti-rat IgG-conjugated immunobeads (Ibs) (Dynal Inc.; Oslo, Norway; http://www.dynal.no) at 4°C for 20 min with gentle agitation at a 2:1 bead/cell ratio. Ibs-rosetted cells were removed using a magnetic particle concentrator. Nonrosetted cells were recovered and reincubated with the same number of beads mentioned above. The remaining nonrosetted cells were considered as HSCs.
PV injection was carried out according to the slightly modified version of the protocol described previously [12]. Briefly, recipient mice were exposed to 9.5 Gy of gamma irradiation from a 137Cs source (Gammacell 40 Exactor; Nordion International Inc.; Kanata, Ontario, Canada; http://www.mdsnordion.com) at a dosage of 1.140 Gy/min 20-24 h before PV injection. Mice to be injected via the PV were anesthetized with pentobarbital. Using sterile procedures, a midline abdominal incision was made to expose the viscera. Donor cells (0.3 ml) were injected through the PV using a 27-gauge needle. On completion of the injection, the needle was rapidly withdrawn, and homeostasis without hematoma formation was usually secured by gentle pressure with a cotton wool swab. Intravenous injection was performed through the tail vein using a 27-gauge needle.
BM Adherent Cells
To obtain adherent cells, BMCs from three- to six-week-old B6 mice were prepared as described above and cultured in Collagen type 1 Cellware flasks (Becton Dickinson Labware; Franklin, NJ; http://www.bd.com) containing 
-minimal essential medium (GIBCO; Grand Island, NY) supplemented with 10% horse serum (NDH-1128; Nikken Bio-Medical Lab.; Tokyo, Japan), 10–6 M hydrocortisone, and antibiotics at 37°C in 5% CO2 in air [17]. The medium in each culture flask was replaced with the same volume of fresh medium weekly. After three to four weeks, nonadherent hematopoietic cells had disappeared from the culture flasks and the adherent cells were then collected from the surface of the flasks by trypsin-EDTA treatment. The cell suspensions were prepared by repeated aspirations in a pipette, and cell clumps were removed by filtration through nylon wool mesh (70 µm). These single cells were transplanted into the recipient mice via i.v. or PV injection with or without HSCs.
Analysis of Chimerism
Eight weeks after the PV injection, the peripheral blood mononuclear cells (PBMCs) were phenotyped for recipient/ donor cells by flow cytometry. In brief, the PB of the recipient mice was collected and the PBMCs isolated by discontinuous density gradient centrifugation using Lymphocyte-mammal (Cedarlane; Ontario, Canada; http://www.cedarlanelabs.com). The PBCs were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-H-2K Ab and, together with anti- Mac-1, Gr-1, B220, CD4, or CD8 mAbs, then incubated with biotinylated Ab. The stained cells were then quantified with a FACStar (Becton Dickinson).
Confocal Microscopy
At multiple time points, a part of the recipient liver was taken and frozen using Optimal Cutting Temperature compound (Tissue-Tek; Sakura Finetek, Inc.; Elkhart, IN; http:www.sakuraus.com). The liver sections (3 µm, without any fixation) were incubated for 20 min at room temperature with anti-PA6 Ab (rat IgM class) [18] and FITC-conjugated mouse anti-rat 
chain antibody (Cosmo Bio; Tokyo, Japan). Previously, we showed that the anti-PA6 Ab can react with BM stromal cells in particular, but not with peritoneal macrophages or thymic dendritic cells. The Ab also does not show any positivity for any cells in the spleen, lymph node (LN), thymus or liver. The thus-incubated liver sections were further stained using phycoerythrin (PE)-labeled anti-H-2b or H-2k Abs (PharMingen; San Diego, CA; http://www pharmingen.com). The negative control was stained with anti-H-2d and normal rat immunoglobulins. The stained samples were examined on a confocal laser scanning microscope (LSM-GB200, Olympus; Tokyo, Japan; http://www.olympus.com) equipped with a 20x objective lens. The samples were visualized using a band pass PB535 filter after excitation at 488 nm for FITC and a high pass over 590 after excitation at 488 nm for PE.
CFU-S Assay
C3H or B6 mice (eight to nine weeks of age) were 9.5 Gy-irradiated and divided into four groups the next day. In the first group, 1 x 104 HSCs and 5 x 105 BM adherent cells from donor mice were injected into the irradiated mice via PV pathway. In the second group, the same number of cells were injected via the i.v. In the third group, only the HSCs were injected via the PV. In the fourth group, the HSCs were injected via the i.v. The recipient mice were sacrificed on day 5, 7, 14, or 16 after the transplantation, and the spleens were removed and fixed with Bouin's fixing liquid. The colonies of each spleen were counted using a microscope.
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RESULTS |
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shows the kinetics of the donor-type cells in the various organs. The time course in the reconstitution of donor-type cells shows different patterns in the various hematolymphoid organs. On the third day, about 11% of donor-type hepatic mononuclear cells (HMNCs) were detected in the liver in the PV group. In contrast, only 3% of HMNCs were found in the i.v. group. Similarly, donor-type cells in the BM and thymus in the PV group reached 47% and 35% on day 7 after BMT, respectively. In contrast, lower percentages of donor-type cells were detected in the i.v. group; 40% in the BM and 24% in the thymus on day 7 after BMT. There was no significant difference between the groups in organs other than the thymus on day 14 after BMT. These findings suggest that many donor HSCs and/or progenitor cells can be trapped in the liver when injected via the PV, and that they then migrate into the BM, LN, and thymus within seven days. The percentages of donor cells in the recipient organs increased dose (the number of injected BMCs)-dependently (data not shown).
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). The dose (1 x 104 cells/mouse) of HSCs was determined so as to form countable CFU-S in the recipient's spleen (less than 40/spleen). The spleens of the recipients were taken on days 5, 7, 14, and 16 after BMT, and the number of CFU-S were counted. The results are shown in Figure 2. On days 5 and 7, there was no significant difference between the PV and i.v. groups. On day 14, however, CFU-S counts in the HSCs + stromal cells PV group were significantly higher than the other groups. Interestingly, the number of CFU-S was higher in the group injected with HSCs alone via the PV than the group injected i.v. with HSCs + BM stromal cells (Fig. 2). This suggests that the supply of stromal cells is effective only in the PV injection but not i.v. injection. On day 16, more than half of the CFU-S were confused, and no differences were observed among the four groups. Similar results were observed in different donor-host mouse combinations (data not shown). As a control, the BM stromal cells alone were injected into lethally irradiated recipients, but no colony was found either in the i.v. or PV group (data not shown). No CD45+ (leukocyte common antigen) cells were detected in the BM stromal cells (data not shown), indicating that the population did not contain any hemopoietic cells.
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Histology of Livers in Recipients
These results show that donor-derived BM stromal cells contribute greatly to the CFU-S formation. This phenomenon may be attributed to accelerated hemopoiesis in the recipient liver induced by the PV injection, since the hemopoietic colonies are found in the livers of recipient mice administered BMCs via the PV, as described in our previous paper [14]. To examine whether the HSCs that have been trapped in the recipient liver proliferate in collaboration with donor BM stromal cells also trapped in the liver, frozen sections of the recipient livers were prepared on days 7 to 14 after BMT and stained by hematoxilin and eosin. As shown in the photographs of light microscopy in Figure 3, hemopoietic colonies were found in the group injected with HSCs + BM stomal cells via the PV on day 14 after BMT (four to six colonies/section), particularly around the blood vessels. No distinct hemopoietic colony was detected earlier than on day 14 after BMT. Moreover, confocal microscopic analyses revealed the coexistence of both donor-derived hematopoietic cells (red) and stromal cells (yellow) in the colonies (Fig. 3). Significantly lower numbers of hemopoietic colonies were found in the liver in the PV group injected with HSCs alone, and no colony was found in the i.v. group injected with either HSCs alone or HSCs + BM stromal cells. Again, these results indicate that the donor-derived stromal cells play a crucial role in the CFU-S formation via the PV injection.
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shows the survival rate of C3H mice that received 1 x 104 HSCs and/or 5 x 105 BM stromal cells of B6 mice. All the mice in which the cells were administered via the i.v. died within 23 days, even if donor BM stromal cells were injected along with the HSCs. In contrast, 67% of the recipient mice survived when HSCs and 5 x 105 BM stromal cells were injected via the PV. However, such prolonged survival was not seen in the mice injected with HSCs + 3 x 105 BM stromal cells, suggesting that more than 5 x 105 BM stromal cells are necessary to restore hemopoiesis.
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). All of the lympoid and myeloid lineage cells were donor-type in all the mice that received HSCs + BM stromal cells via the PV. However, recipient-type cells were predominant in PBMCs of some mice injected with HSCs alone.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously reported that day-12 CFU-S counts markedly increase in MHC-matched recipients when donor BMCs are i.v.-injected [10], indicating that the MHC-compatible microenvironment is essential to the differentiation and proliferation of HSCs. In the present study, we have investigated the mechanisms underlying the enhancement in both day-14 CFU-S formation (Fig. 2
) and survival (Fig. 4), which is obtained by cografting donor-derived stromal cells via the PV. The histological examination revealed that the higher number of hepatic hemopoietic foci is observed in the mice receiving HSCs and stromal cells via the PV than in other groups (Fig. 3). These findings indicate that primitive HSCs and stromal cells, which have been first trapped in the liver, proliferate, then migrate into the spleen to form many CFU-S. Since day-14 CFU-S (not day-7 CFU-S) counts are considered to represent the number of multipotent HSCs, the enhancement in day-14 CFU-S counts indicates that primitive HSCs really proliferate in the recipient mice. Although it is difficult, at present, to elucidate which kinds of cells in the liver can trap the donor HSCs and stromal cells, hepatic sinusoid endothelial cells and Kupffer cells are possible candidates, since there is a report that the hepatic sinusoid plays an important role in the traffic of rat dendritic cells from the blood [22]. We are now investigating the hepatic cells responsible for trapping HSCs and stromal cells.
To date, several laboratories have reported that the PV administration of antigens is more effective than the i.v. injection; donor-specific tolerance across major, minor and xeno histocompatibility complex barriers can be induced by the administration via the PV [23-25]. We have also found that the PV injection of allogeneic cells resulted in the persistent tolerance specific for alloantigens used for the PV inoculation, although the i.v. administration of allogeneic cells induces transient tolerance [14, 15]. Fujiwara et al. have also found that the PV inoculation of allogeneic cells generates serum factor(s) able to transfer in H-2 and nonH-2-unrestricted manners [26]. Very recently, we have found that the combination of PV preimmunization of allogeneic cells with cyclosphosphamide treatment induces permanent acceptance (potent and persistent tolerance) of organ allografts [27].
There is a possibility that, after the antigens are taken up, alloantigens on the allogeneic cells are changed into other form(s) or associated with some components inappropriate for triggering the corresponding clones before alloantigens enter the inferior vena cava. This view is associated with the hypothesis that the liver plays a crucial role in changing immunogenic antigens into tolerogenic antigens [13]. We have also reported that HSCs trapped in the liver after the PV administration can induce clonal anergy to CD8+ T cells [16]. Although many mechanisms can be considered, the present study shows that donor stromal cells injected via the PV are trapped in the recipient liver, and that they then support the hemopoietic colony formation of donor HSCs. The stromal cells also seem to protect the HSCs from attack by recipient T cells, macrophages, and natural killer cells that are radioresistant. As a result, CFU-S counts markedly increase and, finally, a higher survival rate is achieved. This study shows that the simultaneous transplantation of stromal cells with the MHC-matched HSCs contributes greatly to the reconstitution of hemopoiesis, and provides an important new insight for achieving successful allogenic BMT via the PV without long-term usage of immunosuppressive agents.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the Japanese Private School Foundation, a grant from the "Haiteku Research Center," a grants-in-aid for scientific research (B) 11470062, and grants-in-aid for scientific research on priority areas (A) 10181225 and (A) 1116221 of the Ministry of Education.
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REFERENCES |
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ß T cells of mice immunized with allogeneic cells via the portal vein in vitro. Cell Immunol 1993;149:107-116.[CrossRef][Medline]
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