We have recently established a new bone marrow transplantation (BMT) method for the treatment of intractable autoimmune diseases in MRL/lpr mice; the method consists of fractionated irradiation (5.5 Gy × 2), followed by BMT of whole bone marrow cells (BMCs) from allogeneic C57BL/6 mice via the portal vein (abbreviated as 5.5 Gy × 2 + PV). In the present study, we investigate the mechanisms underlying the early engraftment of donor-derived cells in MRL/lpr mice by this method. In the mice treated with this method, the number of donor-derived cells possessing the mature lineage (Lin) markers rapidly increased in the BM, spleen, and liver; almost 100% were donor-derived cells by 14 days after the treatment. The number of donor-derived hemopoietic progenitor cells (defined as c-kit+/Lin− cells) increased in the BMCs, hepatic mononuclear cells, and especially spleen cells by 14 days after the treatment. Simultaneously, hemopoietic foci adjoining donor-derived stromal cells were observed in the liver when injected via the PV, but not via the peripheral vein (i.v.). When adherent cell-depleted BMCs were injected via the PV, recipients showed a marked reduction in the survival rate. However, when mice were transplanted with adherent cell-depleted BMCs with cultured stromal cells, all the recipients survived.
These findings suggest that not only donor hematopoietic stem cells (HSCs) but also donor stromal cells administered via the PV were trapped in the liver, resulting in the early engraftment of donor HSCs in cooperation with donor-derived stromal cells. This new strategy to facilitate the early recovery of hemopoiesis would therefore be of great advantage in human application.
MRL/MP-lpr/lpr (MRL/lpr) mice, which spontaneously develop massive lymphadenopathy, are well known as an animal model for systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). The onset of autoimmune diseases in MRL/lpr mice has previously been retarded by various treatments such as the injection of monoclonal antibodies (mAbs) against T cells or immunosuppressants [1, 2]. We have previously proposed that autoimmune diseases are “stem cell disorders” [3-5], and shown that allogeneic bone marrow transplantation (BMT) can be used to prevent and treat autoimmune diseases in (NZB × NZW)F1(B/WF1), BXSB, (NZW × BXSB)F1(W/BF1), and nonobese diabetic mice [6-9]. However, MRL/lpr mice were found to be radiosensitive because, after the onset, the mice usually died due to infection from the intestine if the dose is greater than 8.5 Gy. Furthermore, irradiation doses less than 8.5 Gy cannot completely eliminate abnormal hemopoietic stem cells (HSCs) of MRL/lpr mice. Conventional BMT (8.5 Gy irradiation plus allogeneic BMT) therefore had no effect on the prevention and treatment of autoimmune diseases in MRL/lpr mice [6, 9]. We have recently found that donor-derived stromal cells are necessary for successful allogeneic BMT [10-12], since major histocompatibility complex (MHC) restriction exists between HSCs and stromal cells . Indeed, the combination of BMT plus bone grafts (to recruit donor stromal cells) was found to completely prevent the recurrence of autoimmune diseases in MRL/lpr mice . However, we have found that this combination has no effect on the treatment of autoimmune diseases in MRL/lpr mice, because MRL/lpr mice become more radiosensitive after the onset of lupus nephritis due to the development of uremic enterocolitis. Therefore, we have carried out fractionated irradiation and devised a new strategy, which includes the injection of cyclophosphamide (CY), fractionated irradiation (5 Gy × 2), bone grafts (to recruit stromal cells), and two transplantations of whole bone marrow cells (BMCs) from B6 mice . However, this strategy is not applicable to humans, since it involves bone grafts to recruit donor stromal cells.
We have previously found that most donor HSCs are trapped and retained in the liver when they are injected either via the portal vein (PV) or even i.v. , and that the HSCs induce clonal anergy to host CD8+ T cells . In addition, we have recently found that PV plus i.v. injections of donor BMCs can induce persistent donor-specific tolerance in the skin allograft system [17, 18]. Taking these findings into consideration, we have very recently established a new strategy for the treatment of severe autoimmune diseases in chimeric-resistant MRL/lpr mice; MRL/lpr mice receive BMCs via the PV after the fractionated irradiation (5.5 Gy × 2 + PV), and no immunosuppressants are used . The survival rate in the mice thus treated was significantly higher than that in the mice treated with (5.5 Gy × 2 + i.v.). In this report, we analyze the mechanism(s) underlying the successful hemopoietic recovery after this treatment, and show that the donor stromal cells play a key role in the early engraftment of donor hemopoiesis.
Materials and Methods
Female MRL/Mp-lpr/lpr (MRL/lpr, H-2k) and C57BL/6 (B6, H-2b) mice were obtained from SLC (Shizuoka, Japan) and maintained until use in our animal facilities under specific pathogen-free conditions.
Preparation of Allogeneic Whole BMCs, Adherent Cell-Depleted BMCs, and Cultured Stromal Cells
BMCs were collected from the femurs and tibias of B6 mice. To remove adherent cells, the whole BMCs were passed through a Sephadex G-10 column (Pharmacia Fine Chemicals; Uppsala, Sweden; http://www.pnu.com) as previously described . Cultured stromal cells were obtained as follows: the femurs, tibias, and humeri, from which the BMCs had been extensively washed out, were cut into pieces, and then cultured in a flask. The medium in the flask was replaced weekly with the same volume of fresh medium . Three weeks later, nonadherent cells were extensively removed, and the adherent cells were then collected from the surface of the flasks by trypsin-EDTA treatment.
Injection Via PV
The BMCs were injected via the PV as described previously . In brief, a midline abdominal incision was made to expose the viscera. BMCs in 0.3 ml of RPMI were injected via the superior mesenteric vein using a 27-gauge needle.
The onset of autoimmune diseases in MRL/lpr mice was monitored by proteinuria (>2.5+) and lymphadenopathy. The mice (4-5 months of age) with autoimmune diseases were irradiated in fractionated irradiations (5.5 Gy × 2 = 11 Gy; 4-hour interval). One day after the irradiation, the mice were transplanted with whole BMCs (3 × 107) via the PV (5.5 Gy × 2 + PV), and the mice that had been treated with (5.5 Gy × 2 + PV) were further injected i.v. with whole BMCs (3 × 107) 5 days after the PV injection (5.5 Gy × 2 + PV + i.v.). Two further experimental groups were also prepared; the first was transplanted via the PV with the adherent cell-depleted BMCs (3 × 107), which were passed through a Sephadex G-10 column (5.5 Gy × 2 + PV [G10]), and the second was transplanted with adherent cell-depleted BMCs (2.7 × 107) plus cultured stromal cells (0.3 × 107) via the PV (5.5 Gy × 2 + PV [G10 + stromal cells]). The stromal cells used were ones cultured for more than 3 weeks, and they showed positive staining for anti-PA-6 mAb but negative staining anti-CD11b mAb. Mice that were irradiated (5.5 Gy × 2) and transplanted i.v. with whole BMCs (3 × 107) (5.5 Gy × 2 + i.v.), and mice that were irradiated with 8.5 Gy and transplanted i.v. with whole BMCs (3 × 107) (8.5 Gy + i.v.) served as controls.
Preparation of Hepatic Mononuclear Cells (HMNCs)
Mice were systematically perfused with 4 ml of heparinized (10 units/ml) phosphate-buffered saline (PBS) (0.01 M pH 7.2) to eliminate blood. The liver was further perfused with 5 ml of PBS containing collagenase (Type IV, 400 units/ml, Sigma Chemical Co.; St. Louis, MO; http://www.sigma-aldrich.com) via the PV. After perfusion, the liver was suspended in 5 ml of the collagenase-PBS and incubated at 37°C for 40 min. A single-cell suspension was then obtained by simply cutting and flushing. After washing twice with PBS, the cells were suspended in 10 ml of PBS containing 1% fetal bovine serum and placed on 10 ml of Lympholyte-Mammal density solution (1.0860 g/ml, Cedarlane Laboratories Ltd.; Hornby, Ontario, Canada; http://www.cedarlanelabs.com). After centrifugation for 30 minutes at 2,800 rpm at room temperature, the HMNCs were collected from the defined layer at the interface.
Surface Marker Analyses
Spleen cells, HMNCs, and BMCs were prepared from recipient mice. To detect donor- or residual recipient-derived cells, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-H-2Db and phycoerythrin (PE)-conjugated anti-H-2Kk mAbs (PharMingen; San Diego, CA; http://www.pharmingen.com). FITC- or PE-conjugated mAbs against B220, CD4, CD8, CD11b, and Gr-1 (PharMingen) were used to analyze the cell surface phenotypes. Furthermore, to detect donor-derived c-kit+ progenitor cells, the cells were analyzed with three colors. The cells were stained with biotinylated mAbs against lineage (Lin) markers (anti-CD4, anti-CD8 [Caltag; Burlingame, CA; http://www.caltag.com], anti-B220, anti-Gr-1, and anti-CD11b [PharMingen] mAbs), followed by streptavidin-RED670 (GIBCO BRL; Rockville, MD), then further stained with PE-anti-c-kit mAb and FITC-anti-H-2Db or anti-H-2Kk (PharMingen). The cells with the immunophenotype of c-kit+/ Lin–/H-2Db+ analyzed by a FACScan® (Becton Dickinson & Co; Mountain View, CA; http://www.bd.com) were categorized as hemopoietic progenitors.
Analyses of Donor-Derived Stromal Cells
The BM-derived stromal cells from the humeri were prepared according to the method described above, and they were collected from the surface of the flask using Cell Dissociation Solution® (Sigma). To detect donor-derived stromal cells, the cultured cells were stained with anti-PA6 mAb , which was reactive to the stromal cells, followed by staining with PE-anti-rat IgM (PharMingen). After blocking with normal rat IgM, the cells were further stained with FITC-anti-H-2Db or anti-H-2Kk, and analyzed by a FACScan®. The cultured stromal cells were also stained with PE-anti-CD11b and FITC-anti-H-2b mAbs. Cells stained with isotype-matched Ig were prepared as negative controls.
The livers and spleens of the recipient mice were removed and fixed in 10% formalin, and the sections were stained with hematoxylin and eosin. For the immunofluorescence study, the specimens were frozen in dry-ice/acetone, as previously described . The 3-μm sections were stained with anti-PA6 mAb, followed by PE-anti-rat IgM and then blocked by normal rat IgM. These sections were further stained with FITC-anti-H-2Db or anti-H-2Kk. The stained samples were examined on a confocal laser scanning microscope (LSM-GB200®, Olympus; Tokyo, Japan; http://www.olympus.com) equipped with a 20× objective lens. The samples were visualized using a band pass F490-560 filter after excitation at 488 nm for FITC and a high-pass TR 610 filter after excitation at 568 nm for PE.
Statistical analyses of the survival rate of recipient mice were performed using a log rank test.
MRL/lpr mice (16 to 20 weeks of age) with autoimmune diseases such as massive lymphadenopathy and proteinuria were treated using various strategies. As shown in Figure 1, more than 70% of the MRL/lpr mice treated with (5.5 Gy × 2 + i.v.) died within 40 days of the treatment, and the rest died within 180 days. All the recipients treated with (8.5 Gy + i.v.) (conventional i.v. BMT) died within 28 days due to the side effects of radiation. In contrast, 70% of the MRL/lpr mice treated with (5.5 Gy × 2 + PV) survived more than 1 year, and all the recipients treated with (5.5 Gy × 2 + PV + i.v.) survived more than 1 year. To confirm that BMT via the PV is effective in successful engraftment of donor cells, the mice were injected with a small number (3 × 106) of whole BMCs via the PV. Consequently, 60% (6/10) of the recipients also survived more than 1 year after the treatment (data not shown), indicating an advantage of BMT via the PV. To confirm the necessity of stromal cells for the engraftment of donor-derived cells (differentiation and proliferation of donor HSCs), two additional groups were prepared: A) adherent cell-depleted BMCs (3 × 107) were injected via the PV (5.5 Gy × 2 + PV [G10]), and B) adherent cell-depleted BMCs (3 × 107) with cultured stromal cells (0.3 × 107) were injected via the PV (5.5 Gy × 2 + PV [G10 + stromal cells]). Although 75% of the mice treated with (5.5 Gy × 2 + PV [G10]) died within 90 days, all of the mice treated with (5.5 Gy × 2 + PV [G10 + stromal cells]) survived more than 80 days. These findings indicate that BMT via the PV is more effective in prolonging survival than i.v., and that donor-derived stromal cells are important for the donor cell engraftment.
Analyses of Donor-Derived Hemopoietic Cells
We have recently found that most donor cells (particularly HSCs) are trapped and retained in the liver after BMT either via the PV or even the i.v.; however, the percentage of the donor HSCs in the liver administered via the PV was found to be significantly higher than that via the i.v. Therefore, to investigate the process where the donor-derived cells proliferate in the hemopoietic organs treated with (5.5 Gy × 2 + PV) or (5.5 Gy × 2 + i.v.), we examined the percentage and number of the donor-derived cells in the BM, spleen, and liver on days 3, 7, 10, and 14 of the treatment. Surprisingly, when treated with (5.5 Gy × 2 + PV), the percentage of donor-derived cells rapidly increased by 10 days and reached almost 100% on day 14 of the treatment (Fig. 2A left). Furthermore, the number of donor-derived cells in the BMCs, spleen cells, and HMNCs reached the normal level on day 14 of the treatment (Fig. 2B left). In contrast, when treated with (5.5 Gy × 2 + i.v.), the percentage of donor-derived cells in the hemopoietic organs increased until day 10, but then gradually decreased after this point (Fig. 2A right). The absolute number of donor-derived cells actually decreased in the hemopoietic organs (Fig. 2B right).
The donor-derived cells with mature lineage markers (B220+, CD4+, CD8+, CD11b+, or Gr-1+) were next analyzed in the hematolymphoid organs. As shown in Table 1, on day 14 of the treatment with (5.5 Gy × 2 + PV), the donor-derived cells bearing lineage markers increased in the bone marrow, spleen, and liver. Furthermore, the donor-derived cells with mature lineage markers were found to be at normal levels 1 year after the treatment (data not shown). In contrast, on day 14 of the treatment with (5.5 Gy × 2 + i.v.), hardly any donor-derived cells with lineage markers were detected in the hemopoietic organs. These findings indicate that BMT via the PV significantly facilitates the early engraftment and continuous proliferation and differentiation of donor cells.
Table Table 1.. Analyses of surface antigens on the donor-derived cells in MRL/lpr mice treated with (5.5 Gy × 2 + PV) or (5.5 Gy × 2 + i.v.)
We next examined whether the donor-derived progenitor cells, defined as c-kit+/Lin– cells, exist and proliferate in the hemopoietic organs. In the recipients treated with (5.5 Gy × 2 + PV), the percentage of donor-derived progenitor cells in the spleen cells and HMNCs were more than 2% on day 14 of the treatment (Fig. 3B left). The absolute number of donor-derived progenitor cells had increased to more than 3 × 105 in the liver and BM, and more than 2 × 106 in the spleen 10 days after the treatment (Fig. 3C left). Furthermore, the percentage of donor-derived progenitor cells in the BMCs was found to be more than 2% 1 year after the treatment (data not shown). In contrast, when treated with (5.5 Gy × 2 + i.v.), less than 1% of the donor-derived progenitor cells detected in these organs 14 days after the treatment were donor-derived progenitor cells (Fig. 3B right), and hardly any donor-derived progenitor cells were detected in these hemopoietic organs on day 14 of the treatment (Fig. 3C right).
Analyses of Donor-Derived Stromal Cells
We have recently found that an MHC restriction exists between HSCs and stromal cells , and that donor-derived stromal cells are required for successful allogeneic BMT [10, 11]. To examine whether the donor-derived stromal cells are generated from the recipient bone, the bone pieces without BMCs from MRL/lpr mice treated with (5.5 Gy × 2 + PV) or (5.5 Gy × 2 + i.v.) were cultured for 3 weeks, and the cultured stromal cells then collected. Consequently, the number of cultured stromal cells from the recipient bones treated with (5.5 Gy × 2 + PV) was 4.2 ± 1.2 × 105 cells, whereas that from the recipient bones treated with (5.5 Gy × 2 + i.v.) was 1.5 ± 1.5 × 104 cells (the mean ± SD of five mice). Furthermore, as shown in Figure 4, these cultured stromal cells were stained by anti-H-2Db and anti-PA6 mAbs indicating that these cells were stromal cells of donor-origin. Therefore, the donor-derived stromal cells are not only trapped efficiently in the recipient liver by the PV injection, but also migrate into the bones and spleens, where they construct the hemopoietic microenvironments. Furthermore, it is noted that the cultured stromal cells were not stained by macrophage-specific anti-CD11b mAb (Fig. 4D), indicating that macrophages are not involved in the donor cell engraftment.
Histological Findings in the Spleen and Liver of Treated MRL/lpr Mice
The frequencies of donor-derived progenitor cells and stromal cells were higher in the recipients treated with (5.5 Gy × 2 + PV) than those treated with (5.5 Gy × 2 + i.v.). Therefore, we next carried out in vivo histological analyses. As shown in Figure 5A, hemopoietic foci consisting of a mixture of myeloid and erythroid lineage cells were observed around the vein in the liver, and various lineage cells such as mononuclear leukocytes, granulocytes, erythrocytes, and megakaryocytes were also generated in the spleen (Fig. 5E). Furthermore, confocal laser scanning microscopy confirmed the presence of donor-derived cells in these foci. Figure 5B (C superimposed on D) clearly shows that the hemopoietic focus consists of the donor-derived cells stained with FITC-anti-H-2b mAb. In addition, it is of interest that the stromal cells detected by anti-PA6 mAb (visualized by PE-anti-rat IgM) were observed in association with or close to the hemopoietic foci around the vein in the liver (donor-derived cells; green-colored, stromal cells; orange-colored in Fig. 5B). Furthermore, many stromal cells appeared among the donor-derived cells in the spleen (Fig. 5F; G superimposed on H). These findings indicate that the donor BMCs administered via the PV are trapped in the liver and proliferate in cooperation with the donor-derived stromal cells.
Patients with leukemia or aplastic anemia have successfully been treated with allogeneic BMT, and various hemopoietic factors such as GM-CSF or G-CSF have been generally used to rapidly restore donor hemopoiesis. However, these colony-stimulating factors have been reported not only to exacerbate inflammatory diseases such as vasculitis [23, 24] but also to cause the flare-up of autoimmune diseases such as RA and Felty's syndrome [25-27]. These colony-stimulating factors activate both donor and recipient HSCs. Therefore, it is important to rapidly generate only the donor-derived cells. We have previously found that the donor-derived stromal cells play a crucial role in the donor cell engraftment because of the existence of an MHC restriction between the HSCs and stromal cells [10-14]. To recruit the stromal cells in the hemopoietic organs, we have previously performed the co-transplantation of bone and BMCs from the same donor. Consequently, we have shown that the combination of BMT plus bone grafts after TBI (8.5 Gy) can completely prevent autoimmune diseases in MRL/lpr mice , and that double injections of BMCs plus bone grafts after the injection of CY and TBI (5 Gy × 2) can be used to treat autoimmune diseases in chimeric resistant MRL/lpr mice after the onset . However, it is difficult to apply these co-transplantations to humans, since we have to carry out bone grafts to recruit donor stromal cells. Considering this problem, we have devised a new strategy to treat intractable autoimmune diseases in MRL/lpr mice. The method consists of BMT via the PV without any immunosuppressants or bone grafts.
In the present study, more than 80% of MRL/lpr mice treated with (5.5 × 2 + PV) survived more than 100 days without any symptoms of recurrence of autoimmune diseases, whereas more than 70% of the mice treated with (5.5 × 2 + i.v.) died within 40 days (Fig. 1). These findings indicate that allogeneic BMT via the PV is able to cure autoimmune diseases in MRL/lpr mice even after their onset. We have previously shown that significant numbers of the allogeneic HSCs injected via the PV are trapped and retained in the recipient liver. Actually, the number of HSCs detected in the liver after the PV injection of BMCs is three times more than when injected i.v. . This is the case in the present study, where the donor-derived hemopoietic progenitor cells (defined as c-kit+/Lin– cells) in the liver, BM, and spleen had increased remarkably by 14 days after the treatment. Furthermore, we have found that donor HSCs can induce CD8+ T cells to clonal anergy, since HSCs express MHC class Ihigh but not costimulatory molecules such as B7 . Therefore, the donor cell engraftment has been enhanced after the PV injection of BMCs by the retention of donor hemopoietic progenitors, leading to the formation of hemopoietic foci in the liver (Fig. 5A and B) and the donor-specific tolerance to the residual (if any, even after the irradiation) donor-specific T cells.
There have recently been reports indicating that stromal stem cells (mesenchymal stem cells) are essential for the proliferation and differentiation of HSCs [28-31]. As shown in Figure 4, when injected via the PV (but not i.v.), donor-derived stromal cells could be generated in the recipient bone pieces. Furthermore, in the recipient liver and spleen, the donor-derived stromal cells appeared close to the donor-derived cells (Fig. 5B and F). These findings suggest that the donor stromal cells or their precursor cells are more efficiently engrafted by the PV injection, and some are trapped in the recipient liver, while some migrate into the recipient spleen and BM.
In the present study, the percentage of donor-derived cells (and those possessing the mature lineage markers) rapidly increased in the hemopoietic organs such as the BM, spleen, and liver; the number of donor-derived cells in these organs regained normal levels 14 days after the treatment with (5.5 Gy × 2 + PV). Hemopoietic foci, consisting of a mixture of myeloid and erythroid lineage cells, were observed around the vein in the liver, and various lineage cells such as granulocytes, erythrocytes, and megakaryocytes were generated also in the spleen (Fig. 5A and E). These findings strongly suggest that the donor whole BMCs including the stromal cells (or their precursors) construct a suitable microenvironment in the hemopoietic organs when injected via the PV, and this thereby facilitates the proliferation and differentiation of donor HSCs in cooperation with MHC-matched donor stromal cells in the liver. Indeed, as shown in Figure 1, a marked reduction in the survival rate was observed in the recipients that received adherent cell-depleted BMCs, even though the BMCs were injected via the PV. Furthermore, when the recipients had been transplanted PV with adherent cell-depleted BMCs plus cultured stromal cells, all recipients survived more than 80 days. These findings suggest that the stromal cells play a significant role in the donor cell engraftment.
The long-term administration of reagents such as any anti-inflammatory drugs, immunosuppressants, and cytotoxic drugs that have been used to treat SLE and RA results in cumulative side effects and a large burden on patients. Furthermore, intractable autoimmune diseases such as RA with HLA-DRB1*04 subtypes develop into severe autoimmune diseases, including multiple bone destruction [32-34], and seldom show prolonged remission in spite of their being treated with various disease-modifying anti-rheumatic drugs. Because no suitable strategy has been established for autoimmune diseases, patients with autoimmune diseases are severely handicapped in daily life. In humans, it has recently been reported that autoimmune diseases such as RA, SLE, multiple sclerosis, psoriasis, and Crohn's disease are ameliorated after allogeneic BMT [35-41]. No recurrence has been reported after allogeneic BMT for autoimmune diseases plus leukemia or aplastic anemia during long-term observation (range 7-20 years) [42, 43], suggesting that allogeneic BMT is an ideal strategy for the treatment of intractable autoimmune diseases.
In the present study, we have shown that not only donor HSCs but also donor stromal cells administered via the PV are trapped in the liver, and that the early engraftment of the HSCs has occurred in cooperation with the donor-derived stromal cells in the hemopoietic organs. This new treatment facilitates the early recovery of hemopoiesis; therefore, the long-term use of immunosuppressants is not necessary, freeing the patients from the burden of the severe side effects. We believe that this new strategy for allogeneic BMT is useful for human application, since laparoscope-guided administration via the PV is a well-established and safe technique.
We thank Ms. Y. Tokuyama, Ms. M. Shinkawa, and Ms. S. Miura (First Department of Pathology) and Mr. K. Kobayashi (Central Research Center) for their expert technical assistance, and Mr. Hilary Eastwick-Field and Ms. K. Ando for their help in the preparation of the manuscript.
This work was supported by a grant from the Japanese Private School Foundation, a grant from “Haiteku Research Center” of the Ministry of Education, grants-in-aid for Scientific Research (B) 11470062 and grant-in-aid for Scientific Research on Priority Areas (A) 10181225, 1116221.