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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 101-107
Involvement of Fas-Mediated Apoptosis in the Hematopoietic Progenitor
Cells of Graft-Versus-Host Reaction-Associated Myelosuppression
By
Takehiko Mori,
Takashi Nishimura,
Yasuo Ikeda,
Tomomitsu Hotta,
Hideo Yagita, and
Kiyoshi Ando
From the Division of Hematology, the Departments of
Internal Medicine and Immunology, Research Center for Genetic
Engineering and Cell Transplantation, Tokai University School of
Medicine, Kanagawa; the Division of Hematology, the Department of
Internal Medicine, Keio University School of Medicine, Tokyo; and the
Department of Immunology, Juntendo University School of Medicine,
Tokyo, Japan.
 |
ABSTRACT |
The influence of graft-versus-host (GVH) reaction on the host
hematopoietic cells clinically manifests itself both as adverse reactions in transfusion-associated GVH disease (GVHD) and as a
therapeutic graft-versus-leukemia (GVL) effect in either donor lymphocytes transfusion (DLT) or allogeneic bone marrow (BM)
transplantation. We examined the effect of GVH reaction on the host
hematopoiesis in the murine parent-into-F1 (P1  F1)
model of GVHD. The systemic transfer of 5 × 107 of
C57BL/6 (B6) splenocytes into (B6xDBA/2)F1 mice (BDF1), which results
in acute GVHD, reduced the peripheral blood cell counts, the number of
BM cells, and colony-forming unit-granulocyte macrophage (CFU-GM), whereas the injection of 108 of
DBA/2 cells into BDF1, which results in chronic GVHD, did not affect
hematopoiesis 2 weeks after the transfer. To clarify the mechanism of
such myelosuppression, we examined the Fas expression in both
hematopoietic progenitor cells as well as whole BM cells. The Fas
expressions in each fraction significantly increased in BDF1 mice 2 weeks after the induction of acute GVHD, whereas no such effects were
observed in the BDF1 mice with chronic GVHD. Furthermore, when such BM
cells were incubated with anti-Fas antibody (Jo2), which induces
apoptosis through Fas, the fraction of apoptotic cells increased and
the number of CFU-GM decreased significantly. The in vivo
administration of neutralizing anti-FasL antibody into BDF1 mice
receiving with B6 spleen cells thus protected the host mice from BM
failure. These results indicate that the functional expression of Fas
on hematopoietic cells plays an essential role in the myelosuppressive
effect of GVHD.
| |
INTRODUCTION |
IMMUNOLOGICALLY competent cells contained
in a graft can result in the immunologic recognition of the host major
histocompatability complex (MHC) in the opposite direction and thus
initiate a graft-versus-host (GVH) reaction.1-3 A GVH
reaction to the host hematopoietic cells manifests itself as either an
adverse effect or a therapeutic effect: the potential susceptibility of
the host hematopoietic system to immune attack by donor lymphocytes is
dramatically illustrated in the lethal marrow aplasia seen in
transfusion-associated GVH disease (TA-GVHD)4-6 and the
efficacy of donor lymphocyte transfusions (DLT) in irradicating large
leukemic cell loads when chronic myelocytic leukemia (CML)
relapse follows allogeneic bone marrow transplantation (BMT).7 Therefore, a clear understanding of the molecular
mechanism of the effect of the GVH reaction on the host hematopoietic
cells is of profound clinical importance. A better understanding and improved management of this GVH reaction should allow for the improved
prevention and treatment of GVHD and the development of new strategies
to enhance the graft-versus-leukemia (GVL) effect8,9 and to
also decrease the incidence of leukemic relapse after transplantation.
The murine parent-into-F1 (P F1) GVHD, which results from the
injection of parental splenic lymphocytes into unirradiated, immune
competent F1 adult hosts, has been extensively used as an experimental
model of TA-GVHD.1 In murine models, the use of inbred
strains of defined genetic background has allowed for the development
of systems capable of reproducibly generating either acute or chronic
GVHD. In the case of (C57BL/6xDBA/2)F1 mice (BDF1), the transfer of
parental C57BL/6 (B6)(H-2b) spleen cells results in an
acute, life-threatening GVHD,10 whereas the transfer of
parental DBA/2 (D2)(H-2d) spleen cells results in a chronic
GVHD syndrome with symptoms similar to those seen in systemic lupus
erythematosus.11,12 Although the precise kinetics of Th1
and Th2 cytokine production during the early course of GVHD have been
analysed in this model, these effects on the hematopoietic cells have
not yet been fully studied.13,14
We herein examined the effect of GVHD on host hematopoiesis in this P1
F1 model. The systemic transfer of 5 × 107 of B6 splenocytes into BDF1 mice reduced the peripheral
blood (PB) cell counts, the number of BM cells, and colony-forming
unit-granulocyte macrophage (CFU-GM), whereas the
injection of 108 of D2 cells into BDF1 did not affect
hematopoiesis 2 weeks after the transfer. To clarify the mechanism of
such myelosuppression, we examined the Fas expression in both
hematopoietic progenitor cells as well as in whole BM cells. The Fas
expressions in each fraction significantly increased in the BDF1 mice 2 weeks after the induction of acute GVHD, whereas no such effect was
observed in the BDF1 mice with chronic GVHD. Furthermore, when such BM cells were incubated with anti-Fas antibody (Jo2), which is known to
induce apoptosis through Fas, the fraction of apoptotic cells increased
and the number of CFU-GM decreased significantly. Both the in vivo
administration of neutralizing anti-FasL antibody into BDF1 mice
receiving B6 spleen cells and the transfer of spleen cells from
FasL-defective (gld) B6 mice into BDF1 mice protected the host
mice from BM failure. Therefore, these results indicate that the
functional expression of Fas on hematopoietic cells thus plays an
essential role in the myelosuppressive effects of GVHD.
| |
MATERIALS AND METHODS |
Mice.
The female mice of an inbred C57BL/6 (B6) (H-2b), DBA/2
(D2) (H-2d), and B6 x DBA F1 (BDF1) strain were obtained
from Nippon Clea Inc (Tokyo, Japan). B6-gld (generalized
lymphoproliferative disease) (H-2b) mice were purchased
from Jackson Laboratory (Bar Harbor, ME). These mice were maintained in
specific pathogen-free conditions. Six- to 8-week-old mice were used
for all experiments. All animals were handled in accordance with the
guidelines established by the Animal Experimentation Committee of Tokai
University School of Medicine. Four or five mice per group were used
for each experiment.
Induction of GVHD.
Single-cell suspensions were prepared in an RPMI 1640 medium
(GIBCO-BRL, Grand Island, NY) from the spleens of B6 and D2 mice. The
cell suspensions were passed through sterile nylon mesh, then were
washed and diluted to a cell concentration of 25 × 107 and 50 × 107/mL for acute and chronic
GVHD induction, respectively. Acute and chronic GVHD were induced by
injecting 5 × 107 B6 spleen cells and 1 × 108 DBA spleen cells into the BDF1 mice.13,14
Age-matched uninjected BDF1 mice were used as control mice.
PB cell counts.
Blood samples were obtained by means of an intracardiac puncture after
anesthetization with ether. Samples were analyzed by an Automated
Hematology Analyzer (NE8000; Toa Medical Electronics Corp, Kobe,
Japan).
BM cell collection.
Suspensions of BM cells were obtained by flushing both the femurs and
tibias with phosphate-buffered saline (PBS).15 The suspensions were passed through nylon mesh and then were treated with
NH4Cl to lyse red blood cells. The cells were resuspended with PBS and the cell viability was assessed by trypan blue exclusion. All samples showed more than a 90% cell viability.
In vitro colony assay.
A CFU-GM assay was performed in a methylcellulose culture using the
METHOCULT M3430 kit (Stem Cell Technologies, Vancouver, Canada).
Briefly, 1 mL culture medium contained 105 of BM cells,
0.9% methylcellulose, 1% bovine serum albumin, 30% fetal bovine
serum, 0.1 mmol/L 2-mercaptoethanol, 2 mmol/L L-glutamine, 3 U
erythropoietin, and 2% pokeweed mitogen-stimulated spleen cell-conditioned medium. The culture medium was prepared in a 35-mm
petri dish (Becton Dickinson, San Jose, CA) and incubated at 37°C in a 5% CO2 atmosphere. The number of colonies
was counted after 14 days of culture using an inverted microscope.
Cell staining for flow cytometry.
Briefly, BM cells were suspended with PBS containing 2% fetal calf
serum (FCS; GIBCO-BRL) and 0.1% NaN3, and incubation for cell staining was performed on ice for 30 minutes. Regarding the detection of lineage markers, rat monoclonal antibodies (MoAbs) recognizing Mac-1 (M1/70), Gr-1 (RB6-8C5), B220 (RA3-6B2), and an
erythroid marker (TER119) and fluorescein isothiocyanate
(FITC)-conjugated MoAb for murine CD3 (145-2C11, hamster IgG) were
used. Antibodies for Mac-1, Gr-1, and B220 were purchased from
Pharmingen (San Diego, CA), and TER119 was kindly provided by Dr Tatsuo
Kina (Kyoto University, Kyoto, Japan). FITC-conjugated anti-rat IgG
polyclonal antibody (Jackson Immuno Research Laboratories Inc, West
Grove, PA) was used as a secondary antibody for the detection of
lineage markers. To assess the expression of Fas, hamster anti-Fas MoAb (Jo2; Pharmingen, San Diego, CA) conjugated with
phycoerythrin (PE) was used. To detect hematopoietic progenitor cells,
biotinylated anti-c-kit (2B8; Pharmingen) was used. To assess
the expression of Fas on lineage marker negative
(Lin ) c-kit positive
(c-kit+) cells, the cells stained with anti-lineage
markers were further stained with a biotinylated anti-c-kit
and PE-conjugated anti-Fas antibodies followed by the incubation with
streptavidin-RED670 (GIBCO-BRL). After the Lin
population was gated, the expressions of c-kit and Fas were
observed by detecting RED670 and PE, respectively.
Staining for the detection of apoptosis.
To assess the cells undergoing apoptosis, the relative DNA content was
observed as previously described, with some
modifications.15 After permeabilization with 0.1% NP-40
(Sigma, St Louis, MO), the cells were treated with 0.05 mg/mL RNaseA
(Sigma) for 30 minutes at 37°C. After washing, the cells were
suspended in 0.5 mL of propidium iodide (PI) solution (50 mg/mL of PI
with 0.1% Na citrate) and then were incubated for 30 minutes at
4°C. The cells in a discrete subpopulation of signals under the
G0/G1 cell cycle region (subdiploid cells) were designated as cells
undergoing apoptosis.
Flow cytometry analysis.
Flow cytometry was performed using a FACScan (Becton Dickinson)
equipped with three filters including a 530-nm band-pass (for FITC), a
585-nm band-pass (for PE and PI), and a 650-nm long-pass filter (for
RED670). The data were analyzed on the LYSIS II program (Becton
Dickinson). To analyze the cells stained with PI, the coefficient
variance of PI-stained thymocytes from an uninfected BDF1 mouse was
confirmed to be under 2% on a linear scale before the experiments.
Treatment of BM cells in vitro with anti-Fas MoAb.
BM cells were suspended in an RPMI 1640 medium (GIBCO-BRL) supplemented
with 30% FCS in a 12-well plate (Iwaki Glass, Tokyo, Japan) at a
density of 1 × 106 cells/mL (2 mL/well) and incubated
for 18 hours with hamster anti-mouse Fas antigen MoAb (Jo2; Pharmingen)
at a concentration of 1 µg/mL.16 Incubation was performed
under a humidified 5% CO2 atmosphere at 37°C. The
ratio of the cells undergoing apoptosis was then determined by flow
cytometry as described above.
Ultrastructural studies.
BM cells were fixed with 2.5% glutaraldehyde in sodium phosphate
buffer, pH 7.2, for 1 hour, postfixed in 1% osmium tetroxide, dehydrated, and embedded in Quetol-512 (Nisshin EM, Tokyo, Japan) according to routine techniques. Thin sections were mounted on copper
grids and examined by transmission electron microscope (JEOL 1200 EX;
JEOL, Tokyo, Japan) after staining with uranyl acetate and lead
solution.
In vivo administration of anti-Fas ligand antibodies.
Intraperitoneally, five mice received 500 µg/body of MoAb against Fas
ligand (MFL1) on day 0, 1, 4, 7, and 10 after spleen cell
transfer.17 As a control, the same amounts of rat IgG1 (Pharmingen) were intraperitoneally injected into mice.
Enzyme-linked immunosorbent assay (ELISA).
Mouse tumor necrosis factor- (TNF-) and interferon- (IFN-)
in the sera were evaluated with commercial ELISA (Amersham Life
Science, Arlington Heights, IL) according to the manufacturer's instruction. The results from four mice were expressed as the mean ± SEM.
Statistical analysis.
A comparison of the mean values of the parameters was done using the
two-group paired t-test.
| |
RESULTS |
PB cell counts, BM cellularity, and CFU-GM after GVHD induction.
On day 14 after acute GVHD induction by B6 spleen cells, peripheral
blood cells significantly decreased (Table
1). The reduction of blood cells was not obvious on day 7 (data not
shown). In contrast, the induction of chronic GVHD by D2 spleen cells
had no such a pancytopenic effect on day 14. BM cellularity also
decreased on day 14 in the BDF1 mice receiving B6 spleen cells but not
D2 spleen cells. To further assess the status of functional progenitor
cells, we did a CFU-GM assay using BM cells on day 14 and found the
reduction of CFU-GM in BDF1 mice receiving B6 spleen cells but not D2
spleen cells. This reduction of BM cellularity and the number of GFU-GM was not obvious on day 7 (data not shown). These findings indicated that the myelosuppression evolved in the second week of acute GVHD but
not chronic GVHD in this model.
Induction of apoptosis and Fas expression on the BM cells by GVHD.
Fas-mediated apoptosis has been reported to be associated with
myelosuppression in various pathological
conditions.15,18-20 Apoptosis was examined by staining with
PI and the percentage of cells undergoing apoptosis significantly
increased in the BM cells from mice with acute GVHD, from 3.21% ± 1.02% to 10.26% ± 2.02% (P < .05, Fig 1). The expression of Fas on
hematopoietic progenitor cells as well as whole BM cells and cells of
each lineages were thus examined on days 7, 10, and 14. The
representative patterns of Fas expression on day 14 are shown in
Fig 2. The Fas expressions on whole BM
cells significantly increased by acute GVHD, whereas no such effects
were observed in chronic GVHD (Fig 2A). The Fas expressions on
Linc-kit+ fraction of BM cells,
which represent hematopoietic progenitor cells,21 was also
upregulated by acute GVHD (Fig 2B). As summarized in
Table 2, upregulation of Fas expression on
hematopoietic progenitor cells was significant on days 10 and 14. Induction of Fas expression was accompanied with the reduction of the
total number of hematopoietic progenitor cells. Both upregulation of
Fas and the reduction of progenitor cells were not significant on day 7 (data not shown). The Fas expressions in Gr-1+,
Mac-1+, B220+, and Ter-119+
fractions were also increased by acute GVHD (data not shown). These
data suggest that the Fas-mediated apoptosis of hematopoietic cells
including progenitor cells was thus involved in the myelosuppression induced by acute GVHD.
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| Fig 1.
The detection of cells undergoing apoptosis in BM. The
mice were injected with 5 × 107 of B6 spleen cells. On
day 14 after transfer, the mice were killed to remove their BM cells.
Using flow cytometry, the signals of PI, which represent the DNA
contents, were examined. The G0/G1 peak was adjusted at channel 100 and
subdiploid cells were designated as cells undergoing apoptosis. The
numbers in the figure represent the percentage of cells undergoing
apoptosis. Four mice were examined in each group and the representative
data were shown. *Significant increases versus uninjected control
(P < .05).
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| Fig 2.
(A) The expression level of Fas antigen on whole BM
cells. The mice were injected with 5 × 107 of B6 spleen
cells (B6/BDF1) or 1 × 108 of D2 cells (D2/BDF1). On day
14 after transfer, the mice were killed to remove their BM cells. Using
flow cytometry, the expression level of Fas on these cells was
examined. BDF1 represents the uninjected control mice. Four mice were
examined in each group and the representative data were shown. (B) The
expression level of Fas on hematopoietic progenitor cells. The
expression level of c-kit and Fas on the BM cells obtained on
day 14 after the transfer of B6 cells (B6/BDF1) was examined after the
Lin populations were gated. The numbers in the figure
represent the percentage of
c-kit+Fas+ cells in the
Lin cell populations. BDF1 represents the uninjected
control mice. Twelve mice were examined in both groups and the
representative data were shown.
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Induction of apoptosis by anti-Fas MoAb in vitro.
To confirm whether the expressed Fas on hematopoietic progenitor cells
is functional or not, we examined the effects of apoptosis-inducing anti-Fas antibody (Jo2) on BM cells in vitro.16 On day 14 after GVHD induction, BM cells were obtained and incubated with 1 µg/mL of Jo2 in vitro. After 18 hours of incubation, the percentage of cells undergoing apoptosis significantly increased in the BM cells
from BDF1 mice receiving B6 spleen cells, from 7.25% ± 0.63% to
14.83% ± 2.22% (P < .05) . In contrast, no such an
increase was observed in the BM cells from the normal control mice
(data not shown). Apoptosis was further confirmed by the electron
microscopic feature of cells with chromatin condensation in the
presence of plasma membrane integrity (Fig
3). To assess the number of functional progenitor cells, a CFU-GM assay
was performed using BM cells incubated with Jo2 for 18 hours in vitro
(Fig 4). CFU-GM significantly decreased
after treatment with Jo2 in the mice with acute GVHD, but no such
effect was observed in the control BDF1 mice or mice with chronic GVHD.
These data confirmed that the induced expression of Fas in the
hematopoietic cells in acute GVHD was functional in vitro.
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| Fig 3.
Electron microscopic appearance of BM cells after
treatment with anti-Fas antibody (Jo2) . Cells were cultured for 18 hours in the presence of Jo2. (Original magnification × 13,500.)
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| Fig 4.
The effects of anti-Fas antibody (Jo2) treatment on
CFU-GM. The mice were injected with 5 × 107 of B6 spleen
cells (B6/BDF1) or 1 × 108 of D2 cells (D2/BDF1). On day
14 after transfer, the mice were killed to remove their BM cells. The
cells were treated with 1 mg/mL of Jo2 antibody for 18 hours. A CFU-GM
assay was performed in methylcellulose culture containing
105 BM cells. The number of colonies was then
counted after 14 days of culture. The results from four mice were
expressed as the mean ± SEM. ( ), Fas( ); (), Fas(+).
*Significant decrease versus the cells incubated without anti-Fas
antibody (P < .01).
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In vivo administration of anti-FasL antibody or transfer of spleen
cells from FasL-defective (gld) B6 mice protect the recipients from
myelosuppression induced by GVHD.
To further confirm the role of Fas-mediated apoptosis in vivo, we
examined the effects of the administration of the neutralizing anti-FasL antibody (MFL1) on the myelosuppression induced by GVHD. On
day 14 after acute GVHD induction, the total number of BM cells in the
mice treated with MFL1 was significantly higher than that in the
control IgG-treated mice with GVHD, although the number of cells
significantly decreased in comparison to the normal control mice
(Fig 5). Furthermore, spleen cells from
FasL-defective (gld) B6 mice of 5 week of age, which induced
severe GVHD-associated cachexia but only minimal signs of hepatic and
cutaneous GVHD pathology in recipient mice,22 were
transferred into the BDF1 mice and myelosuppression was found to be
completely protected in these mice (Fig 5). Thus, these data confirmed
the essential role of Fas-mediated apoptosis in BM aplasia associated
with GVHD.
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| Fig 5.
The effects of anti-Fas ligand antibody administration in
vivo and the transfer of Fas ligand defective B6 spleen cells. The mice
were received 500 µg/body of MoAb against Fas ligand on days 0, 1, 4, 7, and 10 after B6 spleen cell transfer (B6/BDF1 Fas-L). The mice
were injected with 5 × 107 of FasL-defective (gld) B6
spleen cells (gld/BDF1) or B6 cells (B6/BDF1). On day 14 after
transfer, the mice were killed to remove their BM cells. The total cell
numbers from five mice were expressed as the mean ± SEM. BDF1
represents the control mice injected with the same amounts of rat IgG1
intraperitoneally on the same schedule as mentioned above. *Significant
increase versus B6/BDF1 (P < .01).
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Serum levels of IFN- and TNF-.
Recently, the Fas expression on hematopoietic progenitor cells was
reported to be induced by cytokines such as IFN- and
TNF-.18,19 Both cytokines have also been shown to play
pivotal roles in the pathogenesis of acute GVHD.13,14,23
In this study, elevated levels of both IFN-(23.0 ± 1.5 ng/mL) and TNF- (66.0 ± 10.3 pg/mL) were observed in the
sera from acute GVHD mice on day 10. In contrast, serum IFN- and
TNF- were not detectable (<10 pg/mL) in control mice. These data
suggested that IFN- or TNF- might mediate the Fas expression on
hematopoietic progenitor cells in acute GVHD.
| |
DISCUSSION |
We showed myelosuppression characterized by pancytopenia and decreased
BM cellurarity and CFU-GM in the BDF1 mice receiving B6 spleen cells
but not those of D2 at 2 weeks after parental cell transfer. The
injection of B6 lymphocytes into immunocompetent BDF1 hosts results in
acute GVHD characterized by anti-host cytotoxic T-lymphocyte
(CTL) development.1,10 In contrast, the
injection of lymphocytes from the opposite parent, D2, into the same F1 host results in chronic GVHD characterized by B-cell hyperactivity, autoantibody formation, and a lupuslike disease.11,12
Therefore, the myelosuppression was characteristic feature of acute
GVHD in this model. Although acute and chronic GVHD are strikingly different entities at 2 weeks of disease, both forms of GVHD evolve from a common starting point, ie, the donor CD4+ T-cell
recognition of host alloantigen and interleukin-2 (IL-2) production.1 A precise analysis of the kinetics of cytokine production shows that both forms of GVHD are initially characterized by
increased T-helper 2 (Th2) cytokines (IL-4 and IL-10) production and
the earliest distinguishing features of acute GVHD are detectable at
days 5 through 7 of the disease at which time no myelosuppression was
yet detected.13,14 Therefore, the development of
myelosuppression closely paralleled the evolution of acute GVHD.
The recipient hematopoietic system is reported to be the most sensitive
target of GVHD in that it is affected by spleen cell doses that do not
apparently damage other target organs in the P F1 GVHD
model with class I and II disparities.24-26 In particular, Sprent et al27 reported that the transfer of small doses of purified unprimed CD4+ cells from a donor was able to cause
marrow aplasia in lightly irradiated (600 cGy) recipients expressing
major histocompatibility complex class II differences. When class
II-deficient knockout mice were used as recipients in this experiment,
no atrophy of the host marrow occurred, thus demonstrating the class
II-mediated inhibition of hematopoiesis.27 Therefore, the
allospecific CD4+ CTL induced from the B6 graft but not
from the D2 graft would undoubtedly be the prime effector attacking the
BM cells in the recipient BDF1 mice. Apoptosis was reported to be
involved in class II-mediated inhibition of hematopoiesis in long-term
marrow cultures.28 In addition to these, however, we also
showed that the induced expression of functional Fas on the
hematopoietic cells was also associated with the effect of GVH reaction
on hematopoiesis.
The role of the Fas-FasL pathway to induce apoptosis is established in
the primary target organs of acute GVHD skin and liverwhere Fas is
constitutively expressed: T cells from FasL-defective (gld) and perforin-deficient donor mice were transplanted into lethally irradiated MHC-mismatched allogeneic recipients mice to determine the
role of these cytotoxic pathways in acute GVHD, and Fas-mediated cytotoxicity was thus shown to play an essential role in the
pathophysiology of hepatic and cutaneous GVHD.22,29
Both liver and skin tissue is known to express Fas
constitutively,30 and mice injected with the anti-Fas MoAb
Jo2 rapidly develop fulminant lethal hepatitis.16 But
hematopoietic progenitor cells do not express Fas constitutively and
are not sensitive to FasL mimicking stimulation by anti-Fas antibody,
Jo2, as shown in Fig 4.31 Therefore, the induced expression of Fas on the hematopoietic cells including progenitor cells is considered to play an essential role in the GVH-associated
myelosuppression in this model.
Recently, the Fas expression on hematopoietic progenitor cells and its
role in apoptosis in vitro has been reported.18,19 We
already reported the significance of Fas-mediated apoptosis of
hematopoietic progenitor cells in myelosuppression induced by a murine
CMV (MCMV) infection in vivo.15 We showed herein that the
induction of Fas on the hematopoietic progenitors is also required in
the effect of GVHD on hematopoiesis.
The precise mechanism for the induction of Fas on hematopoietic
progenitor cells still needs to be determined, whereas we showed
elevated levels of IFN- and TNF- in mice with acute GVHD. The
predominant cytokines produced in acute GVHD are Th1 cytokines, IL-2
and IFN-, in response to host antigens, whereas those in chronic
GVHD are Th2.13,14,32 IFN- production was reported to
increase in the B6 BDF1 mice but not in the D2 BDF1
mice.13,14 On the other hand, we reported that D2 BDF1
mice with the systemic administration of IL-12 developed acute GVHD
with the generation of antihost CTL.33 We examined the
effects of the systemic administration of anti-IL-12 or anti-IFN-
antibody into BDF1 mice with acute GVHD (manuscript in
preparation). Both antibodies showed a decreased expression of Fas on hematopoietic cells and also protect the suppression of hematopoiesis. These results indicate that IL-12 and
IFN- were associated with the functional induction of Fas on
hematopoietic cells in acute GVHD. TNF- is another candidate for the
mediater of Fas induction because it has been implicated in the
pathogenesis of GVHD.23 Further studies including in vivo
administration of anti-TNF- neutralizing antibody are needed to
determine the precise mechanism for the induction of Fas on hematopoietic progenitor cells.
These findings might have important clinical implications, especially
regarding the treatment of CML. IFN- was shown to enhance the
expression of Fas on CML progenitor cells and also rendered them more
susceptible to apoptosis induced by the agonistic anti-Fas antibody.34 On the other hand, CML is the most susceptible
type of hematologic neoplasm when donor lymphocytes are
administered.7 Further studies to elucidate the mechanism
of inducing Fas in hematopoietic cells and its difference between
normal cells and leukemic cells is thus expected to produce tremendous
benefits in the field of leukemia and lymphoma therapy.
| |
FOOTNOTES |
Submitted September 17, 1997;
accepted March 2, 1998.
Supported in part by Grant No. JSPS-RFTF97I00201 from the Japan Society
for the Promotion of Science and the Science Frontier Program of MESSC.
Address reprint requests to Kiyoshi Ando, MD, Division of Hematology,
Department of Internal Medicine, Tokai University School of Medicine,
Bohseidai, Isehara, Kanagawa, 259-1193 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.
| |
ACKNOWLEDGMENT |
We thank Drs A. Akatsuka and M. Tokunaga for expert techinical
assistance in ultrastructural studies and A. Kanemura for preparation of mice.
| |
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Abstract
Introduction
Abstract