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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4616-4623
A New Strategy for Treatment of Autoimmune Diseases in Chimeric
Resistant MRL/lpr Mice
By
Kenji Takeuchi,
Muneo Inaba,
Shigeo Miyashima,
Ryokei Ogawa, and
Susumu Ikehara
From the First Department of Pathology and Department of Orthopedic
Surgery, Kansai Medical University, Moriguchi, Osaka, Japan.
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ABSTRACT |
A new strategy for the treatment of autoimmune diseases in chimeric
resistant MRL/lpr mice is established. The strategy includes injection
of cyclophosphamide (CY), fractionated irradiation (5 Gy × 2), bone
grafts (to recruit stromal cells), and two transplantations of whole
bone marrow cells (WBMCs) from allogeneic normal C57BL/6 mice
(CY/2X/Bone/2BMT). MRL/lpr mice, thus treated, survived more than 40 weeks (1 mouse survived for >40 weeks, 7 for >50 weeks, and 4 for
>60 weeks after these treatments). Immunohistological studies showed
that the mice were completely free from both lymphadenopathy and
autoimmune diseases such as systemic lupus erythematosis and rheumatoid
arthritis. The levels of autoantibodies (IgM/IgG rheumatoid factors and
IgM/IgG anti-ssDNA antibodies [Abs]) in the treated mice decreased to
those in the normal mice. In addition, successful cooperation among T
cells, B cells, and antigen-presenting cells (APCs) was observed.
Abnormal T cells with immunophenotypes of B220+/Thy-1+/CD3+/CD4 /CD8
present in untreated MRL/lpr mice disappeared, and the hematolymphoid cells of the treated mice were of donor origin. However, the mice that
had been irradiated with 8.5 Gy and then reconstituted with T-cell-depleted BMCs plus bone grafts died within 2 weeks due to the
side effect of irradiation. The depletion of CD8+ cells
(not CD4+ cells) from WBMCs resulted in graft failure;
60% of the recipient mice, thus treated, died within 2 weeks, and all
recipients died by 15 weeks. Furthermore, limiting dilution assays
showed that approximately more than 0.5% of T cells contained in the
BMCs are necessary not only for engraftment of BMCs but also for
long-term disease-free survival of the recipients. In contrast,
recipients that had received CD4-depleted BMCs with CY plus
fractionated irradiation (5Gy × 2) survived for more than 40 weeks
without showing graft-versus-host reaction (GVHR). This indicates that CD8+cells in the BMCs are essential for the successful
engraftment of the donor-type hematolymphoid cells.
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INTRODUCTION |
MRL/MP-lpr/lpr (MRL/lpr)
MICE spontaneously develop massive lymphadenopathy with the
accumulation of abnormal T cells with immunophenotypes of
B220+/Thy-1+/CD3+/CD4/CD8,1
which might be attributed to the mutation of the Fas gene encoding a
membrane receptor transducing apoptotic signals.2 The
MRL/lpr mouse is also known as an animal model for systemic lupus
erythematosis (SLE) and rheumatoid arthritis (RA). The mouse shows high levels of autoantibodies (rheumatoid factors and anti-DNA antibodies [Abs]).3-6 In our previous report, we
have shown that the combination of bone marrow transplantation
(BMT) with bone grafts has completely preventative effects on
autoimmune diseases in this strain; we have found that there is a
requirement for donor-derived stromal cells for successful allogeneic
BMT.7,8 However, we have also found that BMT with bone
grafts has no effect on the treatment of autoimmune diseases in MRL/lpr
mice. MRL/lpr mice seem to become more radiosensitive after the onset
of autoimmune disease (lupus nephritis), because they become
susceptible to uremic enterocolitis. To determine the optimal
radiation dose to treat autoimmune MRL/lpr mice, we have irradiated
MRL/lpr mice with 6 to 9.5 Gy in combination with BMT plus bone grafts.
Almost all MRL/lpr mice (after the onset) suffered from intestinal
death (due to infection from the intestine) if the doses were
greater than 8.5 Gy. Because irradiation doses less than 8.5Gy
cannot kill abnormal hematopoietic stem cells (HSCs) of MRL/lpr mice, as we previously described,9 we have performed fractionated total body irradiation (TBI) in the present study.
It has been shown that long-term injections of Abs against T cells
(anti-Thy1.2 or anti-CD4 Ab) to MRL/lpr mice retard both lymphadenopathy and the progression of autoimmune diseases, although autoimmune diseases recur unless the treatment is
continued.10,11 Furthermore, various nonspecific
anti-inflammatory and immunosuppressive agents have been used to treat
SLE.12,13 However, the long-term administration of these
drugs is required, which results in cumulative side effects. In the
present study, we show a new method for treating autoimmune diseases in
chimeric resistant MRL/lpr mice.
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MATERIALS AND METHODS |
Mice.
MRL/lpr, C57BL/6 (B6), DBA/2, and C3H/HeN mice were obtained from SLC
(Shizuoka, Japan) and maintained in our animal facility under specific
pathogen-free conditions.
Antibodies and surface marker analyses.
Purified rat monoclonal antibodies (MoAbs) against Thy-1 (30-H-12), CD4
(GK1.5), and CD8 (53-6.72) were purchased from PharMingen (San Diego,
CA). These MoAbs were used to deplete CD4+,
CD8+, or T cells in combination with magnetic beads
conjugated with sheep antirat IgG Ab (Dynabeads M-450; Dynal A.S.,
Oslo, Norway). MoAbs against B cells (B220, RA3-6B2), macrophages
(Mac-1, M1/70), granulocytes (Gr-1, RB6), and erythroid-lineage cells
(TER119) were also from PharMingen. These were used to enrich T cells
from the peripheral blood. Fluorescein isothiocyanate (FITC)-coupled anti-H-2Kb and phycoerythrin (PE)-coupled
anti-H-2Kk MoAb from PharMingen were used for H-2 typing.
FITC- or PE-coupled MoAbs against Thy-1, B220, CD4, and CD8 were also
from PharMingen to analyze the cell surface phenotype. Lymph node and
spleen cells were prepared from recipient mice, and the cells were
stained with appropriate FITC- or PE-conjugated MoAbs to detect
abnormal T cells with the immunophenotypes of
B220+/Thy-1+/CD3+/CD4/CD8
or donor-derived cells. Cells were analyzed by a FACScan (Becton Dickinson & Co, Mountain View, CA).
Depletion of CD4+, CD8+, or whole
T cells.
Bone marrow cells (BMCs) were treated with appropriately diluted MoAbs
against CD4, CD8, or Thy-1.2, followed by antirat IgG-conjugated magnetic beads to deplete the cells bearing the respective marker. Residual CD4+, CD8+, or T cells after the
treatment were less than 0.05% when stained with FITC- or PE-anti-CD4,
CD8, or Thy-1.2, and examined by a FACScan.
Enrichment of T cells from peripheral blood mononuclear cells.
Peripheral blood was collected, and mononuclear cells were enriched by
centrifugation on a cushion of Lympholyte-M (Cedarlane Laboratories
Ltd, Hornby, Ontario, Canada). Mononuclear cells were treated with a
mixture of MoAbs (anti-B220, antimacrophage [Mac-1], anti-granulocyte
[Gr-1], and erythroid-lineage cells) and then incubated with antirat
IgG-conjugated magnetic beads. After depleting cells with these
markers, more than 98% of the residual cells were positive for Thy-1.2
and used as highly enriched T cells.
Treatment of MRL/lpr mice.
Experimetal procedures are depicted in Fig
1. The onset of autoimmune diseases in MRL/lpr mice was monitored by
proteinuria (>2.5+) and lymphadenopathy. Female MRL/lpr mice (3 to 4 months of age) with autoimmune diseases were intraperitoneally injected with 100 mg/kg cyclophosphamide (CY) and, 1 day later, lethally irradiated (fractionated radiation: 5 Gy × 2 = 10 Gy; 4-hour
interval). Four hours after the second irradiation, the bones of B6
mice, from which BMCs had been flushed out, were engrafted under the subcutis of the MRL/lpr mice (2 femurs and 2 tibias per mouse), and the
mice received 5 × 107 whole BMCs from B6 mice through
a tail vein. One day after the first BMT plus bone graft, the mice were
further transplanted with B6 whole BMCs (WBMCs; 5 × 107 cells; experimental protocol 2 in Fig 1). MRL/lpr mice
that had been irradiated (8.5 Gy) and transplanted with
T-cell-depleted BMCs plus bone graft of B6 mice were also prepared.
This protocol was previously used for the prevention of autoimmune
diseases in MRL/lpr (experimental protocol 1 in Fig 1).7,8
Furthermore, the following groups were prepared: (1) CY/2X/Bone/BMT
(without first BMT); (2) 2X/Bone/2BMT (without CY injection); (3)
CY/2X/Bone/2BMT( CD4) (CD4-depleted BMCs were used for both BMT),
(4) CY/2X/Bone/2BMT(CD8) (CD8-depleted BMCs were used for both
BMT), and (5) CY/2X/Bone/2BMT (T cell) (T-cell-depleted BMCs were
used for both BMTs). In some experiments, various percentages (0.05, 0.1, 0.5, and 1.0) of mature T cells enriched from the peripheral blood
(the purity of T cells was >98%) were added to T-cell-depleted BMCs
(5 × 107 cells) and transplanted to MRL/lpr mice to
provide quantitative information as to the number of T cells being
coadministered to conditioned recipients.
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| Fig 1.
Experimental protocols. Experimental protocol 1: Female
MRL/lpr mice (3 to 4 months of age) with autoimmune diseases were irradiated (8.5 Gy) and transplanted with T-cell-depleted BMCs plus
bone graft of B6 mice. Experimental protocol 2: Female MRL/lpr mice
with autoimmune diseases were intraperitoneally injected with 100 mg/kg
CY and then lethally irradiated (5 Gy × 2 = 10 Gy for 4-hour
interval) 1 day later. Four hours after the second irradiation, the
bones of the B6 mice, from which the BMCs had been flushed out, were
engrafted under the subcutis of the MRL/lpr mice, and the mice received
5 × 107 WBMCs from B6 mice. One day after the first BMT
plus bone graft, the mice were further transplanted with B6 WBMCs (5 × 107 cells).
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Immunological assays.
Recipient mice were killed, and their spleens were removed. The
immunological functions of the mice were assayed as follows: (1)
antibody production against sheep red blood cells
(SRBCs) and (2) mixed leukocyte reaction (MLR). In the
anti-SRBC antibody response, 4 × 106 spleen cells
were cultured with the same number of SRBCs in 24-well flat-bottom
culture plates for 5 days, and anti-SRBC antibody production was
measured by the modified Jerne's plaque-forming cell (PFC) assay, as
previously described.14 MLR was performed as follows.
Triplicate cultures were set up in 96-well round-bottom microwell trays
(Corning Glass Works, Corning, NY). Each well contained 2 × 105 responder T cells and 3 × 105
irradiated (15 Gy) spleen cells in a total of 0.2 mL of RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, and
50 µmol/L 2-mercaptoethanol (2-ME; Wako, Osaka, Japan). The culture
was incubated for 72 hours and pulsed with 0.5 µCi of
3[H]-thymidine for the last 16 hours of the culturing
period.
Proteinuria.
Proteinuria was measured using testing papers (Albustix; Miles-Sankyo
Ltd Co, Tokyo, Japan).
Measurement of autoantibodies.
RF (IgG and IgM) and anti-ssDNA Abs (IgG and IgM) in the sera of the
recipient mice were determined by a standard enzyme-linked immunosorbent assay (ELISA), based on the method
described by Izui and Eisenberg.15 Relative quantities of
autoantibodies were measured by absorbance of OD405, which
is the maximum absorbance using phosphatase substrate tablet (Sigma
104; Sigma Diagnostics, St Louis, MO).
Pathological findings.
The kidneys or joints of the recipient mice were removed and fixed in
10% phosphate-buffered formalin. For the immunofluorescence study, the
specimens were frozen in dry-ice/acetone, as previously described.16 Briefly, 3-µm sections were incubated at
room temperature with FITC-conjugated rabbit antimouse IgG or
FITC-conjugated antimouse C3 (Medical and Biological Laboratories,
Nagoya, Japan) and then washed three times with phosphate-buffered
saline. The samples were also embedded in paraffin, sectioned, and then
stained with hematoxilin and eosin (H-E).
Statistical analyses.
Statistical analyses in the survival rate of recipient mice were
performed using a logrank test.
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RESULTS |
Prolonged survival of MRL/lpr mice treated with CY/2X/Bone/2BMT.
In our previous report, MRL/lpr mice (before the onset of diseases)
treated with BMT (T-cell-depleted BMCs) plus bone grafts after 8.5 Gy
irradiation survived more than 1 year without showing any symptoms of
autoimmune diseases, indicating that this protocol is useful for the
prevention of the diseases. However, MRL/lpr mice at the age of 3 to 4 months (after the onset of the diseases) treated with this protocol
died within 2 weeks due to the side effects of radiation (8.5 Gy) or
graft rejection (Fig 2). Therefore, we have
devised a new method that reduces the side effects of radiation and
prevents graft rejection. As shown in Fig 1, MRL/lpr mice that had
shown symptoms of autoimmune diseases (proteinuria and massive
lymphadenopathy) were treated with CY and fractionated radiation (5 Gy × 2), followed by two transplantations of WBMCs plus bone grafts
from normal B6 mice (CY/2X/Bone/2BMT). MRL/lpr mice that had received
such treatments survived more than 40 weeks after the treatment (1 mouse survived for >40 weeks, 7 for >50 weeks, and 4 for >60
weeks; Fig 2). Furthermore, more than 70% of the recipients of
CY/2X/Bone/BMT (without the first BMT) survived more than 25 weeks.
However, 67% of the mice treated with 2X/Bone/2BMT (without CY
injection) died within 6 weeks. Furthermore, all the recipients that
had received T-cell-depleted BMCs [CY/2X/Bone/2BMT(T cell)]
died within 10 weeks without the reconstitution of donor-derived cells,
indicating that transplantation with T-cell-containing BMCs is
essential to the engraftment and treatment. The mice treated with
CY/2X/Bone/2BMT(CD8) (CD8-depleted BMCs were used for both BMT)
died within 15 weeks due to graft rejection and lupus nephritis; cells
recovered from these mice had H-2 of the recipient type when examined
by a FACScan (H-2k+ cells were 97.5% ± 0.5%).
Furthermore, recipients treated with 2X/Bone/2BMT (without CY
injection), CY/2X/Bone/2BMT(T cell), or
CY/2X/Bone/2BMT(CD8) showed proteinuria; histological
examinations showed the presence of lupus nephritis. In contrast, 80%
of the MRL/lpr mice treated with CY/2X/Bone/2BMT(CD4)
(CD4-depleted BMCs were used for both BMT) survived more than 40 weeks
(8 of 10 mice), suggesting that CD8+cells in the BMCs are
neccessary for the engraftment of donor cells. As mentioned above, the
presence of T cells in the transplanted BMCs is a critical factor in
the long-term, disease-free survival of the recipient mice (mouse BMCs
usually contain approximately 1% of T cells). Therefore, various
ratios (0.05%, 0.1%, 0.5%, and 1.0%) of mature T cells enriched
from the peripheral blood (the purity of T cells was >98% as
determined by a FACScan) were added to T-cell-depleted BMCs (5 × 107) to provide the quantitative information as to the
number of mature T cells necessary for the treatment. As shown in
Table 1, the recipients that received BMCs
containing 0.05% and 0.1% of mature T cells showed hematolymphoid
cells with the recipient H-2 phenotype, and no donor-derived cells were
detected in these recipients when tested 6 weeks after the treatment as
observed in those treated with CY/2X/Bone/2BMT (T cell).
However, the hematolymphoid cells of the recipients that had received
BMCs containing greater than 0.5% of T cells were almost all of donor origin (>98%). Thus, the presence of small numbers of T cells in
BMCs (~0.5% of BMCs that contain 0.3% of CD8+ T cells)
seems to be necessary for the engraftment of donor BMCs and for
long-term survival.
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| Fig 2.
Survival rate in 6 groups. Numbers in parentheses are
numbers of mice in each group. Treatment of mice is shown in the
figure. Statistical analyses were performed by a logrank test, and
asterisks (**) represent the P values of treated
(CY/2X/Bone/2BMT) versus other groups; **P < .01, *statistical insignificance.
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Serum autoantibody levels of MRL/lpr mice treated with
CY/2X/Bone/2BMT.
As previously reported, untreated MRL/lpr mice and MRL/lpr mice
reconstituted with syngeneic BMCs showed increased RFs (IgM and IgG)
and anti-ssDNA Abs (IgM and IgG) at the age of 18 weeks, whereas
MRL/lpr mice treated with CY/2X/Bone/2BMT showed normal levels in these
parameters even 48 weeks after the treatment, with the levels being
comparable with those of 20-week-old B6 mice
(Fig 3).
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| Fig 3.
Autoantibodies in MRL/lpr mice treated with
CY/2X/Bone/2BMT (Experimental protocol 2). RFs and anti-ss DNA Abs were
measured at 48 weeks after the treatment (64 weeks of age).
Autoantibodies in normal C57BL/6 and untreated MRL/lpr mice were
measured at 18 weeks of age. The results are expressed as the mean ± SD at 405 nm from 5 mice. Asterisks (* and **) represent P
values of treated versus untreated MRL/lpr mice; *P < .005 and **P < .001.
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Immunological findings of MRL/lpr mice treated with CY/2X/Bone/2BMT.
Untreated MRL/lpr mice showed an extremely low anti-SRBC response
(number of PFC/culture, 130 ± 8), whereas MRL/lpr mice treated with
CY/2X/Bone/2BMT showed a high anti-SRBC response even 48 weeks after
the treatment (number of PFC/culture, 440 ± 29), although the level
was not completely restored to the normal level as seen in B6 or DBA/2
mice (numbers of PFC/culture, 737 ± 46 and 980 ± 141, respectively). These findings indicate that cooperation is achieved
among the T cells, B cells, and antigen-presenting cells (APCs) of the
treated MRL/lpr mice.
Disappearance of abnormal T cells in MRL/lpr mice treated with
CY/2X/Bone/2BMT.
It has been shown that the numbers of abnormal T cells with
immunophenotypes of
B220+/Thy-1+/CD3+/CD4/CD8
increase in the spleen and lymph nodes of MRL/lpr mice with age (Table 2).1 We have found that
this is due to the presence of abnormal HSCs of MRL/lpr
mice.9 However, as shown in Table 2, these abnormal T cells
did not appear in the MRL/lpr mice treated with CY/2X/Bone/2BMT at 48 weeks (32 weeks after the treatment) and 72 weeks (56 weeks after the
treatment). It should be noted that almost all the cells in the spleen
and lymph nodes are of donor origin (H-2b but not
H-2k; Table 2), indicating that the hematolymphoid cells
are completely reconstituted with donor cells. In MLR, newly developed
T cells were tolerant to both host (MRL/lpr)-type and donor (B6)-type MHC determinants, but they showed a normal responsiveness to the third
party (DBA/2) cells (data not shown). It should also be noted that the
hematolymphoid cells in the mice treated with 2X/Bone/2BMT (without CY
treatment) are of MRL/lpr origin (data not shown), indicating that CY
injection is essential for the engraftment of donor hematolymphoid
cells; CY seems to eliminate host-derived activated T cells, as
previously reported.17
Histopathological findings of MRL/lpr mice treated with
CY/2X/Bone/2BMT.
Neither lymphadenopathy nor autoimmune diseases such as lupus nephritis
(Fig 4B) and RA
(Fig 5B) were observed in the MRL/lpr mice
treated with CY/2X/Bone/2BMT, even at 72 weeks of age, whereas untreated MRL/lpr mice clearly showed typical lupus nephritis (Fig 4A)
and RA-like lesions (Fig 5A) with the infiltration of lymphocytes
and pannus formation at 18 weeks of age. It is noted that MRL/lpr mice,
thus treated, did not show the recurrence of autoimmune manifestations.
This was the case when the recipients were reconstituted with
CD4-depleted BMCs [CY/2X/Bone/2BMT(CD4)]; these mice survived
more than 40 weeks without any evidence of graft-versus-host disease
(GVHD), as shown in Fig 6.
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| Fig 4.
Immunofluorescence microscopical findings of glomerular
IgG deposits in the kidneys of (A) untreated MRL/lpr at 18 weeks of age
and (B) MRL/lpr mice treated with CY/2X/Bone/2BMT (56 weeks after the
treatment [72 weeks of age]). The glomerulus of an untreated MRL/lpr
mouse shows the IgG deposit, whereas the glomerulus of the treated
mouse shows no IgG deposit.
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| Fig 5.
Histopathologic findings in the hindpaw joint of (A)
untreated MRL/lpr at 18 weeks of age and (B) MRL/lpr mice treated with CY/2X/Bone/2BMT (56 weeks after the treatment [72 weeks of age]). The
joint of the untreated MRL/lpr mouse shows marked lymphoid cell
infiltration and pannus formation, whereas the joint of the treated
MRL/lpr mouse shows neither lymphoid cell infiltration nor pannus
formation.
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| Fig 6.
Histopathologic findings in the spleen (A), bone marrow
(B), liver (C), colon (D), skin (E), and lung (F) of mice reconstituted with CD4-depleted BMCs [CY/2X/Bone/2BMT(CD4)]. Histopathologic examination was performed 40 weeks after the treatment. The spleen and
bone marrow show normal architecture with normal hematopoiesis, and
there is no remarkable lymphocyte infiltration in the liver, colon,
skin, or lung, indicating no GVHD.
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DISCUSSION |
We have previously found that allogeneic BMT plus bone grafts (to
recruit donor stomal cells) has completely preventative effects on
autoimmune diseases in MRL/lpr mice.7 However, this strategy was found to have no effect on the treatment of autoimmune diseases in MRL/lpr mice after the onset of the diseases (Fig 2;
8.5Gy/Bone/BMT), because MRL/lpr mice are radiosensitive (8.5 Gy is the
maximum) and MRL/lpr mice become more sensitive to radiation due to
renal failure after the onset of autoimmune diseases and are resistant
to allogeneic engraftment when irradiated at lower doses.9
Chu et al18 have reported that the massive
upregulation of Fas-ligand is observed on T cells (particularly on
CD4/CD8 double-negative T cells) from enlarged lymph nodes of old (4 to
5 months) MRL/lpr mice. The GVH-like reaction is thought to be due to
the increased production of Fas-ligand in MRL/lpr donor cells, which
may induce apoptosis of MRL/+ recipient cells in [MRL/lpr  MRL/+] chimeras.18,19 In our system, the excessive
production of Fas-ligand in old MRL/lpr mice probably leads to the
Fas/Fas-ligand-induced death of the donor cells, resulting in the
resistance to allogeneic engraftment.
We performed fractionated radiation (5Gy × 2) to reduce the acute
radiation injury, and CY was used to eliminate host-derived activated T
cells.17,20,21 The mechanisms underlying tolerance induction by CY have been reported: CY induces tolerance by (1) clonal
deletion of reactive T cells,22 (2) clonal anergy in MHC
class I and/or class II disparate combinations,23
(3) the involvement of donor-derived veto cells in the recipient mice after the injection of CY in MHC class I disparate
transplantation,24 and (4) the induction of suppressor T
cells in CY-induced tolerance to the non-H-2-encoded
alloantigens.25 In the present study, the newly developed T
cells were tolerant to both host (MRL/lpr)-type and donor (B6)-type MHC
determinants, but they showed a normal responsiveness to the third
party (DBA/2) cells (Fig 5). Furthermore, the hematolymphoid cells in
the mice without CY treatment were of MRL/lpr origin. Therefore, the
tolerant state in the recipient mice may be maintained by the
combination of these mechanisms after the injection of CY.
To facilitate the engraftment of donor hematopoietic cells, we
performed BMT twice. Although there was no statistically significant difference between the groups treated with CY/2X/Bone/2BMT and CY/2X/Bone/BMT, the former group had a higher survival rate,
particularly at 3 to 4 weeks after the treatment (100% v 70%
to 80%; Fig 2). To further prevent graft rejection, WBMCs (not
T-cell-depleted BMCs) were used. MRL/lpr mice, thus treated
(CY/2X/Bone/2BMT), showed a good survival rate (>90%) for 50 weeks
after the treatment without showing any symptoms of autoimmune diseases
through their whole life (Fig 2) without a reappearance of abnormal T
cells.
To elucidate which T-cell subsets (CD4 or CD8) are necessary for the
engraftment, we compared the effects of CD4- or CD8-depleted BMCs on
the survival rates. As shown in Fig 2, MRL/lpr mice with CD4-depleted
BMT [CY/2X/Bone/2BMT (CD4)] showed a good survival rate (80%)
for more than 40 weeks after the treatment, whereas MRL/lpr mice with
CD8-depleted BMT [CY/2x/Bone/2BMT (CD8)] died within 20 weeks
of the treatment. Thus, it seems likely that donor-derived CD8+ T cells are necessary for the engraftment. This was in
accordance with the report by Martin26: the addition of a
small number of donor CD8+ T cells (not CD4+ T
cells) to T-cell-depleted donor BMCs was capable of reconstituting recipients with donor hematopoietic cells. The graft-enhancing effect
of CD8+ T cells in the BM might be attributed to their
cytotoxic or suppressive activity against host CD8+
and/or CD4+ T cells responsible for causing graft
rejection.25
In the last few decades, remarkable advances have been made in treating
autoimuune diseases, which include anti-inflammatory drugs (such as
steroid hormones), immunosuppressants, and cytotoxic drugs. However,
long-term administration is necessary in these treatments, which
results in cumulative side effects. Recently, human data have also
accumulated indicating that allogeneic BMT27 (not
autologous BMT28) can be used to treat various autoimmune
diseases; it has been reported that no autoimmune diseases have been
seen to recur after allogeneic BMT in 13 patients with autoimmune
diseases plus leukemia or aplastic anemia during long-term observations
(range, 7 to 20 years).29 However, there have recently been
reports on the rapid recurrence or persistence of autoimmune diseases
after autologous BMT,28 as we9,16 and Karussis
et al30 have previously reported in MRL/lpr mice. Based on
these findings, we would like to propose here a new safe method for
allogeneic BMT using chimeric resistant MRL/lpr mice: (1) fractionated
radiation (5 Gy × 2) rather than one shot of a high dose of
radiation (8.5 Gy), (2) CY for the induction of tolerance, and (3) two
injections of WBMCs or CD4-depleted BMCs.
In conclusion, we believe that chimeric resistant mice such as MRL/lpr
mice provide a useful tool for not only discovering a new safe method
for allogeneic BMT but also analyzing the mechanism behind tolerance
induction.
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FOOTNOTES |
Submitted August 27, 1997;
accepted January 30, 1998.
K.T. and M.I. contributed equally to this work.
Supported by a grant for Experimental Models for Intractable Diseases
from the Ministry of Health and Welfare of Japan and a grant from the
Japanese Private School Promotion Foundation.
Address reprint requests to Susumu Ikehara, MD, PhD, First Department
of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi
City, Osaka 570, Japan.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
The authors thank Fujio Ishida and Eiichi Ohtsuki (Research Center of
Kansai Medical University) for flow cytometry studies and Keiko Ando
for preparing the manuscript.
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REFERENCES |
1.
Morse HC III,
Davidson WF,
Yetter RE,
Murphy ED,
Roths JB,
Coffman R:
Abnormalities induced by the mutant gene lpr: Expansion of a unique lymphocyte subset.
J Immunol
129:2612,
1982[Abstract]
2.
Watanabe-Fukunaga R,
Brannan CI,
Copeland NG,
Jenkins NA,
Nagata S:
Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis.
Nature
356:314,
1992[Medline]
[Order article via Infotrieve]
3.
Roths JB:
Autoimmunity and lymphoproliferation. Induction by mutant gene lpr, and acceleration by a male-associated factor in strain BXSB mice
, in Rose NR,
Bigazzi PE,
Werner NL
(eds):
Genetic Control of Autoimmune Disease.
New York, NY, Elsevier North Holland
, 1978
, p 207
4.
Andrews BS,
Eisenberg RA,
Theofilopoulos AN,
Izui S,
Wilson CB,
McConahey PJ,
Murphy ED,
Roths JB,
Dixon FJ:
Spontaneous murine lupus-like syndrome.
J Exp Med
148:1198,
1978[Abstract/Free Full Text]
5.
Theofilopoulos AN,
Dixon FJ:
Murine models of systemic lupus erythematosus.
Adv Immunol
37:269,
1985[Medline]
[Order article via Infotrieve]
6.
Davidson WF,
Dumont FJ,
Bedigan HG,
Fowlkes BJ,
Morse HC III:
Phenotypic, functional, and molecular genetic comparisons of the abnormal lymphoid cells of C3H-lpr/lpr and C3H-gld/gld mice.
J Immunol
136:4075,
1986[Abstract]
7.
Ishida T,
Inaba M,
Hisha H,
Sugiura K,
Adachi Y,
Nagata N,
Ogawa R,
Good RA,
Ikehara S:
Requirement of donor-derived stromal cells in the bone marrow for successful allogeneic bone marrow transplantation.
J Immunol
152:3119,
1994[Abstract]
8.
Miyashima S,
Nagata N,
Nakagawa T,
Hosaka N,
Takeuchi K,
Ogawa R,
Ikehara S:
Prevention of lpr-graft-versus host disease and transfer of autoimmune diseases in normal C57BL/6 mice by transplantation of bone marrow cells plus bones (stromal cells) from MRL/lpr mice.
J Immunol
156:79,
1996[Abstract]
9.
Ikehara S,
Yasumizu R,
Inaba M,
Izui S,
Hayakawa K,
Sekita K,
Toki J,
Sugiura K,
Iwai H,
Nakamura T,
Muso E,
Hamashima Y,
Good RA:
Long-term observations of autoimmune-prone mice treated for autoimmune disease by allogeneic bone marrow transplantation.
Proc Natl Acad Sci USA
86:3306,
1989[Abstract/Free Full Text]
10.
Wofsy D,
Ledbetter JA,
Hendler PL,
Seaman WE:
Treatment of murine lupus with monoclonal anti-T cell antibody.
J Immunol
134:852,
1985[Abstract]
11.
Santoro TJ,
Portanova JP,
Kotzin BL:
The contribution of L3T4+ T cells to lymphoproliferation and autoantibody production in MRL-lpr/lpr mice.
J Exp Med
167:1713,
1988[Abstract/Free Full Text]
12.
Ginzler EM,
Bollet AJ,
Friedman EA:
The natural history and response to therapy of lupus nephritis.
Annu Rev Med
31:463,
1980[Medline]
[Order article via Infotrieve]
13.
Steinberg AD,
Steinberg SC:
Long-term preservation of renal function in patients with lupus nephritis receiving treatment that includes cyclophosphamide versus those treated with predonisone alone.
Arthritis Rheum
34:945,
1991[Medline]
[Order article via Infotrieve]
14.
Jerne NK,
Nordin AA:
Plaque formation in agar by single antibody-producing cells.
Science
140:405,
1963[Abstract/Free Full Text]
15.
Izui S,
Eisenberg RA:
Circulating anti-DNA-rheumatiod factor complexes in MRL/l mice.
Clin Immunol Immunopathol
15:536,
1980[Medline]
[Order article via Infotrieve]
16.
Ikehara S,
Good RA,
Nakamura T,
Sekita K,
Inoue S,
Oo MM,
Muso E,
Ogawa K,
Hamashima Y:
Rationale for bone marrow transplantation in the treatment of autoimmune diseases.
Proc Natl Acad Sci USA
82:2483,
1985[Abstract/Free Full Text]
17.
Mayumi H,
Good RA:
Long-lasting skin allograft tolerance in adult mice induced across fully allogeneic (multimajor H-2 plus multiminor histocompatibility) antigen barriers by a tolerance-inducing method using cyclophosphamide.
J Exp Med
169:213,
1989[Abstract/Free Full Text]
18.
Chu JL,
Ramos P,
Rosendorff A,
Nikolic-Zugic J,
Lacy E,
Matsuzawa A,
Elkon KB:
Massive upregulation of the Fas ligand in lpr and gld mice: Implications for Fas regulation and the graft-versus-host disease-like wasting syndrome.
J Exp Med
181:393,
1995[Abstract/Free Full Text]
19.
Ito MR,
Terasaki S,
Itoh J,
Katoh H,
Yonehara S,
Nose M:
Rheumatic diseases in an MRL strain of mice with a deficit in the functional Fas ligand.
Arthritis Rheum
40:1054,
1997[Medline]
[Order article via Infotrieve]
20.
Shin T,
Himeno K,
Mayuni H,
Nomoto K:
Drug-induced tolerance to allografts in mice. III. Prolongation of skin graft survival by tolerance induction in congenic mice disparate in the major H-2 antigens.
Transplantation
39:333,
1985[Medline]
[Order article via Infotrieve]
21.
Mayumi H,
Kayashima K,
Shin T,
Nomoto K:
Drug-induced tolerance to allografts in mice. v. Prolongation of skin graft survival in tolerant mice with combined immunosuppressive treatment.
Transplantation
39:335,
1985[Medline]
[Order article via Infotrieve]
22.
Tomita Y,
Ayukawa K,
Yoshikai Y,
Nomoto K:
Mechanism of cyclophosphamide-induced tolerance to IE-encoded alloantigens: Evidence of clonal deletion in effector cells for skin allograft rejection.
Transplantation
53:602,
1992[Medline]
[Order article via Infotrieve]
23.
Tomita Y,
Nishimura Y,
Harada N,
Eto M,
Ayukawa K,
Yoshikai Y,
Nomoto K:
Evidence for involvement of clonal anergy in MHC class I and class II disparate skin allograft tolerance after the termination of intrathymic clonal deletion.
J Immunol
145:4026,
1990[Abstract]
24.
Tomita Y,
Nomoto K:
Prevention of induction of unresponsiveness to class I antigens by veto activity of donor marrow in cyclophosphamide-treated mice.
Transplantation
56:1473,
1993[Medline]
[Order article via Infotrieve]
25.
Tomita Y,
Mayumi H,
Eto M,
Nomoto K:
Importance of suppressor T cells in cyclophosphamide-induced tolerance to the non-H-2-encoded alloantigens.
J Immunol
144:463,
1990[Abstract]
26.
Martin PJ:
Donor CD8 cells prevent allogeneic marrow graft rejection in mice: Potential implications for marrow transplantation in humans.
J Exp Med
178:703,
1993[Abstract/Free Full Text]
27.
Marmont AM:
Immune ablation followed by allogeneic or autologous bone marrow transplantation: A new treatment for severe autoimmune diseases?
Stem Cells
12:125,
1994[Medline]
[Order article via Infotrieve]
28.
Euler HH,
Marmont AM,
Bacigalupo A,
Fastenrath S,
Dreger P,
Hoffknecht M,
Zander AR,
Schalke B,
Hahn U,
Haas R,
Schmitz N:
Early recurrence of persistence of autoimmune diseases after unmanipulated autologous stem cell transplantation.
Blood
88:3621,
1996[Abstract/Free Full Text]
29.
Nelson JL,
Torrez R,
Louie FM,
Choe OS,
Storb R,
Sullivan KM:
Pre-existing autoimmune diseases in patients with longterm survival after allogeneic bone marrow transplantation.
J Rhematol
24:23,
1997
30.
Karussis DM,
Vourka-Karussis U,
Lehmann D,
Abramsky O,
Ben-Nun A,
Slavin S:
Immunomodulation of autoimmunity in MRL/lpr mice with syngeneic bone marrow transplantation.
Clin Exp Immunol
100:111,
1995[Medline]
[Order article via Infotrieve]
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Abstract
Introduction
Abstract