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Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2613-2621
Increased Fetal and Extramedullary Hematopoiesis in Fas-Deficient
C57BL/6-lpr/lpr Mice
By
Elke Schneider,
Géraldine Moreau,
Anne Arnould,
Florence Vasseur,
Naushad Khodabaccus,
Michel Dy, and
Sophie Ezine
From the Université René Descartes-Paris V, CNRS UMR
8603, Paris, France; INSERM U345, Paris, France; and Institut Necker,
Paris, France.
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ABSTRACT |
In this study, we examined the consequences of Fas deficiency on
hematopoiesis in C57BL/6-lpr/lpr mice. We found a striking extramedullary increase in hematopoietic progenitor cells, comprising erythroid and nonerythroid lineages alike. These modifications preceded
the lymphadenopathy, because early progenitors (colony-forming units-spleen [CFU-S] day 8) were already augmented in
day-18 fetal livers of the lpr phenotype. Three weeks after
birth, CFU-S increased in peripheral blood and spleen and
colony-forming cells (CFU-C) began to accumulate 1 to 3 weeks later.
Extramedullary myelopoiesis augmented progressively in Fas-deficient
mice, reaching a maximum within 6 months. By then, mature and immature
myeloid cells had infiltrated the spleen, the liver, and the peritoneal
cavity. Similar changes occurred in C57BL/6-gld/gld mice,
indicating that they resulted from Fas/FasL interactions. Medullary
hematopoiesis was not significantly modified in adult mice of either
strain. Yet, the incidence of CFU-S decreased after Fas cross-linking on normal bone marrow cells in the presence of interferon  ,
consistent with a regulatory function of Fas/FasL interactions in early
progenitor cell development. These data provide evidence that Fas
deficiency can affect hematopoiesis both during adult and fetal life
and that these modifications occur independently from other pathologies associated with the lpr phenotype.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MICE HOMOZYGOUS for the lpr gene
develop an age-dependent lupus-like autoimmune disease and a severe
lymphadenopathy characterized by the progressive accumulation of
TCR / +B220+CD4 CD8
double-negative (DN) T cells.1 They also suffer from
anomalies of the B-cell compartment, concerning both early and late
differentiation stages.2-4
It has been demonstrated that the lpr gene codes for a mutant
Fas/CD95/Apo-1, resulting from a retrotransposon insertion that leads
to abnormal transcription and greatly reduced expression of the Fas
receptor.5,6 A second murine autoimmune disease, called
gld, has been shown to be complementary to lpr, because it is caused by a point mutation of the Fas ligand (FasL), rendering the protein nonfunctional.7,8 Once Fas/FasL interactions had been identified as one of the mechanisms by which cytotoxic T
lymphocytes kill infected target cells and delete activated T
lymphocytes, these 2 murine mutations have been extremely useful in
exploring the Fas pathway of cell death.
Fas/CD95/Apo-1 is a transmembrane protein expressed by a variety of
tissues and several mature hematopoietic lineages, such as T
cells,9 B cells, monocytes, and
granulocytes.10-12 Its regulatory functions during the
immune response have been documented by a number of investigators who
emphasized the importance of the activation state for the
susceptibility of Fas+ cells to
apoptosis.9,13,14
The present study was based on the assumption that Fas/FasL
interactions might also exert some control on hematopoietic progenitor cell development. There is indeed increasing evidence in support of the
notion that normal hematopoietic growth involves not only cell division
and differentiation, but also programmed cell death or
apoptosis.15 It has been reported that the in vitro
deprivation of growth factors such as erythropoietin (Epo) or
interleukin-3 (IL-3) causes apoptosis of bone marrow cells and of
growth factor-dependent cell lines.16-19 In addition, there
is some recent evidence for functional Fas expression on primitive
hematopoietic progenitors freshly isolated from human fetal
liver20 and on human CD34+ progenitors after
exposure to interferon (IFN) and/or tumor necrosis factor (TNF).21 According to a recent report, Fas is also
displayed on murine hematopoietic progenitor cells after cytomegalovirus (CMV) infection.22
We have recently shown that IL-3-induced histamine and cytokine
production by myeloid precursors from murine spleen is greatly decreased after Fas cross-linking in the presence of
IFN.23 These data prompted us to evaluate the
consequences of nonfunctional Fas/FasL interactions on other
hematopoietic progenitor populations in Fas-deficient lpr and
FasL-deficient gld mice with a C57BL/6 genetic background.
We analyzed hematopoietic changes in fetal liver, bone marrow, and
peripheral tissues in relation to age and progression of lymphadenopathy. Furthermore, we addressed the question whether Fas/FasL interactions were directly involved in the regulation of
hematopoietic progenitor frequencies by investigating the effect of in
vitro Fas cross-linking on these cells.
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MATERIALS AND METHODS |
Animals.
Specific pathogen-free, male or female C57BL/6-+/+,
C57BL/6-lpr/lpr, and C57BL/6-gld/gld mice were
purchased from the Jackson Laboratories (Bar Harbor, ME) and bred at
the CSEAL (Orléans, France). Implantation of
C57BL/6-lpr/lpr stage 2-cell embryos into hyper-ovulated
C57BL/6-+/+ females was also performed at the CSEAL. For the
generation of F2 embryos F1 (C57BL/6-+/+ × C57BL/6-lpr/lpr) mice were bred and intercrossed in our own
animal facility. The genotype of fetal tissues from F2 embryos was
determined as previously described.24
Cytokines and antibodies.
Murine recombinant (mr) IFN (specific activity, 5 × 106 U/mg; as measured by an antiviral assay using L-929
cells infected with EMC virus) was purchased from R&D Systems
(Abingdon, UK). The following antibodies were used: hamster
antimouse CD95(Fas, Apo-1) monoclonal antibody (MoAb; clone
Jo2; unlabeled or phycoerythrin [PE]-conjugated), control hamster
IgG, CD117(c-kit) (2B8), CD34 (RAM34), and anti-IgM (R6-60.2) MoAbs
(all from Pharmingen, San Diego, CA); TCR/ (H57-597), Ly-6G(Gr-1)
(RB6-8C5), CD11b (M1/70), CD45R(B220) (RA3-6B2), CD4 (H129.19), CD8
(53-6.7), CD19 (1D3), TER-119, and Sca-1 MoAbs were purified from
hybridoma supernatants, biotinylated, or conjugated with fluorescein
isothiocyanate (FITC) in our own laboratory.
Cell preparations.
Bone marrow cells were removed from femurs and tibias by flushing with
ice-cold Hanks' balanced salt solution (HBSS; GIBCO, Grand Island, NY). Axillary and mesenteric lymph node cell suspensions were prepared by disrupting the organs in a Potter-Elvehjem
homogenizer. Spleen cells were suspended in HBSS by gently teasing with
forceps. Peritoneal cells were recovered after repeated lavage of the
peritoneal cavity with HBSS. The liver was pressed on a 200-gauge
stainless steel mesh and suspended in HBSS. Hepatic mononuclear cells
were prepared as previously described.25 After washing, the
cells were resuspended in 30% to 35% Percoll solution containing 100 U/mL heparin and centrifuged at 2,000 rpm for 15 minutes at room temperature. The pellet was resuspended in red blood cell
(RBC) lysis solution and then washed twice with minimum
essential medium (MEM), supplemented with 1% sodium pyruvate
100×, 1% L-glutamine, 100 IU/mL penicillin, and 100 µg/mL
streptomycin culture (GIBCO) referred to as culture medium. Peripheral
blood cells obtained from cardiac puncture were used after RBC lysis.
Fetal livers were removed at day 18 of gestation and cells were
suspended in culture medium by gently teasing with forceps.
In vitro colony-forming assay.
Total colony-forming units-cells (CFU-C) were quantified
in Complete Methylcellulose Medium with Recombinant Cytokines and Erythropoietin (MethoCult M3434; StemCell Technologies Inc, Vancouver, British Columbia, Canada) or in MethoCult M3230 supplemented routinely with optimal concentrations of murine recombinant IL-3 (1 ng/mL), stem
cell factor (SCF; 100 ng/mL), IL-6 (100 U/mL), and Epo (2 U/mL). In
some experiments, these growth factors were serially diluted to compare
the sensitivity of normal and Fas-deficient spleen cell progenitors. To
evaluate the effect of DN T cells on colony formation, 106
lymph node cells from C57BL/6-lpr/lpr mice suffering from
severe lymphadenopathy (~50% DN lymphocytes) were cocultured with 2 × 105 normal spleen cells in the methylcellulose
assay. Furthermore, 107 lymph node cells/mL from
Fas-deficient mice were incubated for 24 hours in culture medium
supplemented with 10% fetal calf serum (FCS). Supernatants were then
collected and assayed at a 5-fold dilution with or without the standard
combination of growth factors for their effect on colony formation from
control spleen cells. Sera from control and Fas-deficient C57BL/6 mice
were tested for their colony-stimulating activity at a 10-fold final dilution.
Total and sorted cell populations were plated in a final volume of
1 mL at concentrations ranging from 5 to 500 × 103 cells per culture dish (FALCON 1008). Colonies were
scored on day 7 to 8. They were analyzed in some experiments for the
presence of erythroid cells.
Colony-forming units-spleen (CFU-S) assay.
The spleen colony assay of Till and McCulloch26 was used to
determine the number of CFU-S in total peripheral blood, spleen, and
bone marrow cell suspensions. In brief, syngeneic C57BL/6 hosts were
exposed to a lethal dose (9.5 Gy, 0.87 Gy/min) of whole body
irradiation from a 137Cs source. Amounts of 1 to 5 × 104 bone marrow cells, 1 to 5 × 105
splenocytes, and 0.1 to 1 × 106 peripheral blood
cells in 200 µL of MEM were then injected per mouse via the
retro-orbital sinus. At least 5 mice were used in each experimental
group. After 8 or 12 days, recipient spleens were excised and surface
colonies were counted after fixation in Bouin's solution. No
endogenous colonies were detected in these conditions.
Flow cytometry analysis and cell sorting.
Cell suspensions were incubated on ice in the presence of rat antimouse
CD16/CD32 MoAb (1µg/106 cells; Pharmingen) to block Fc
receptor functions before specific staining. Subsequently, cells were
washed, pelleted, and labeled with the appropriate antibodies, using
3-color immunofluorescence. Biotinylated antibodies were revealed with
Streptavin-TRI-COLOR (Caltag, Tebu, Le Perray-en-Yvelines, France).
Cells were analyzed in a FACScan cytofluorometer (Becton Dickinson,
Mountain View, CA), using the LYSYS II software. RBCs and debris were
excluded on the basis of forward and side scatter parameters, and dead cells were gated out by propidium iodide staining. At least 15,000 cells were acquired within the live gate. Lin cells
designate a progenitor-enriched population expressing neither myeloid
nor lymphoid lineage markers (negative for CD11b, Gr-1, CD45R, TER 119, CD4, CD8, and TCR/).
Before fluorescence sorting, spleen cell suspensions from C57BL/6 and
C57BL/6-lpr/lpr mice were partially depleted for B and T
lymphocytes using FITC-conjugated anti-CD4, anti-CD8, and
anti-CD45R(B220) MoAbs. After 30 minutes of staining on ice,
splenocytes were washed twice and incubated for a further 30 minutes
with sheep antirat IgG-coated magnetic beads (Dynabeads M-450; Dynal
A.S., Oslo, Norway) according to the manufacturer's instructions.
Labeled cells were then withdrawn against the inner wall of the test
tube using a strong magnet, and unbound cells were collected. This prepurified population was resuspended in culture medium supplemented with 2% FCS and TCR/ IgM
cells were sorted on a FACS Vantage cell sorter (Becton Dickinson) at a
flow rate of 5,000 cells per second.
Fas cross-linking.
Bone marrow and spleen cell suspensions were adjusted to a final
concentration of 2.5 × 106 and 10 × 106 nucleated cells per milliliter, respectively, in
culture medium with 10% horse serum (GIBCO). They were plated into
Falcon 3047 multiwell plates (2 mL/well) and incubated for 24 hours in the presence of control hamster IgG (5 µg/mL), IFN (100 U/mL), anti-CD95(Fas) MoAb (5 µg/mL), or IFN + anti-CD95(Fas) MoAb in a humidified atmosphere of 95% air and 5%
CO2. Cells were then recovered and assayed for clonogenic progenitors.
Histomorphological examination.
Spleen and bone marrow cell suspensions were cytocentrifuged on glass
slides and imprints were made from liver slices. All preparations were
stained with May-Grünwald-Giemsa.
Statistical analyses.
The standard Student's t-test was used to establish
statistical significance.
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RESULTS |
Hematopoietic changes in spleen and bone marrow from Fas-deficient
C57BL/6-lpr/lpr mice.
We set out to evaluate total colony-forming cells (CFU-C) in spleen and
bone marrow cell suspensions from 6-month-old Fas-deficient mice. As
shown in Table 1, total CFU-C per spleen
were more than 30-fold increased at this age. The number of cells per
spleen attained 384.4 ± 75.8 × 106 versus 109.4 ± 3.8 × 106 in control mice with the same genetic
background (means ± SEM from 3 separate experiments). In contrast,
the bone marrow of Fas-deficient mice was affected neither in terms of
progenitor frequencies nor of cellularity (cells per femur + tibia:
39.4 ± 7.8 × 106 in lpr v 40.4 ± 3.8 × 106 in normal mice; means ± SEM from
3 experiments). The gld mutation caused a similar increase in
spleen progenitor cells, indicating that both Fas and FasL are involved
in this effect (Table 1).
Knowing that DN T cells accumulate in spleens from adult
C57BL/6-lpr/lpr mice, we verified whether this population could
eventually enhance colony formation by providing additional
hematopoietic growth factors. To this end, we performed the clonogenic
assay with or without lymph node cells of the lpr phenotype
(106/mL) composed of approximately 50% DN T
lymphocytes. We found no significant difference between the 2 conditions (Table 2). Also, 5-fold diluted
supernatant from these cells (107/mL), removed after 24 hours of incubation, did not support colony growth or change the number
of CFU-C obtained in response to the currently used growth factors.
Conversely, depletion of the DN T-cell compartment resulted in a
similar enrichment of CFU-C, whether it was performed on C57BL/6 or
C57BL/6-lpr/lpr spleens (Table 2).
In the same line of evidence, we could rule out a higher sensitivity to
growth factors as an explanation for enhanced colony formation, because
splenocytes from lpr and control mice displayed a similar
dose-response curve to serial dilutions of the growth factor cocktail
(data not shown). Furthermore, we detected no colony stimulating
activity in sera of Fas-deficient mice as compared with controls (data
not shown).
Once we had established that the number of clonogenic progenitors was
increased in the spleen of Fas-deficient mice rather than their cloning
efficiency, we examined whether a particular subset of CFU-C was
affected in preference to another. Table 3 shows that this is not the case, because erythroid and nonerythroid colonies were similarly enhanced, suggesting the expansion of common
precursor cell.
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Table 3.
Evaluation of Erythroid and Nonerythroid Progenitors in
Spleens From Normal and Fas-Deficient C57BL/6-lpr/lpr Mice
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We tested this assumption by measuring the number of both CFU-C and
CFU-S (day 8) in spleens from Fas-deficient and control mice at
different ages. It is clear from Fig 1 that
CFU-S were markedly increased in spleens of the lpr phenotype.
This modification appeared at the age of 3 to 4 weeks, before the
augmentation of CFU-C that was not significant at that time point. It
is noteworthy that, in spleens from 3-week-old C57BL/6-lpr/lpr
mice, the percentage of DN T lymphocytes was not yet enhanced versus
control cells (1.6% ± 0.4% v 2.3% ± 0.1%;
means ± SEM from 3 separate analyses). The size of the colonies
generated in irradiated recipients was similar, whether donor cells
were from mutated or wild-type mice.
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| Fig 1.
Age-dependent changes in the number of CFU-S and CFU-C in
spleens from C57BL/6-lpr/lpr mice ( ), as compared with
C57BL/6 controls ( ). Data are means ± SEM established from 3 individuals of the same age. CFU-S and CFU-C were determined as
described in Materials and Methods.
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Extramedullary increase of myeloid cells in C57BL/6-lpr/lpr
mice.
The early increase in spleen progenitor cells was followed by a
striking extramedullary expansion of myeloid cells, as shown in
Fig 2. The cytospin preparations of spleen
cell suspensions (Fig 2A and B) and liver imprints (Fig 2C and D)
demonstrate the marked myeloid infiltration in these organs at the age
of 6 months. Hematopoietic foci comprise several differentiation
stages, from precursor cells to mature monocytes and segmented
granulocytes. The myeloproliferative rather than lymphoproliferative
aspect of this pathology is further emphasized by differential spleen cell counts represented in Table 4.
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| Fig 2.
Histomorphological changes in spleen and liver from
6-month-old Fas-deficient mice. Cytospin preparations and liver
imprints were stained with May-Grünwald-Giemsa. (A) Normal
splenocytes; (B) splenocytes from Fas-deficient mice; (C) normal liver
imprints; (D) liver imprints from Fas-deficient mice. Original
magnification × 300.
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These morphological data were validated by the phenotypic analysis of
C57BL/6-lpr/lpr splenocytes (Fig
3). In comparison with age-matched controls, we observed a clear-cut
expansion of granulocytes and monocytes, as assessed by the expression
of Gr-1 and CD11b. Erythroid cells, recognized by the TER-119 MoAb,
were also increased. The accumulation of hematopoietic progenitors in
the spleen was confirmed by the higher number of cells displaying c-kit
or CD34 and of more immature progenitors identified by Sca-1 expression on cells lacking both lymphoid and myeloid lineage markers
(lin). B cells, evaluated by CD19 expression, were
not modified, in contrast with DN T cells, which had accumulated at
this age.
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| Fig 3.
Phenotypic analysis of spleen cells from 6-month-old
C57BL/6-lpr/lpr mice (), relative to age-matched controls
(). Freshly isolated cells were stained with appropriate
antibodies and analyzed in a FACScan cytofluorometer, using LYSYS II
software. One representative experiment is shown, with 3 mice per
group.
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Origin of extramedullary hematopoiesis in C57BL/6-lpr/lpr
mice.
As already stated above for CFU-C, the incidence of CFU-S did not
significantly change in the bone marrow of C57BL/6-lpr/lpr mice, relative to age-matched controls. However, a slight transient increase in day-8 CFU-S occurred shortly after birth (1,810.3 ± 164.4 CFU-S/femur + tibia in 2-week-old Fas-deficient mice v 1,040.0 ± 116.4 in aged-matched controls; means ± SEM from 4 separate experiments; P < .001). More primitive day-12 CFU-S
were also not significantly increased above normal in adult bone marrow of Fas-deficient mice, whereas they were in the spleen (6,548.0 ± 187.9 CFU-S/spleen in 2-month-old C57BL/6-lpr/lpr mice
v 1,900.4 ± 237.4 CFU-S/spleen in age-matched
controls; means ± SEM from 3 separate experiments; P < .01). We postulated that hematopoietic changes might nevertheless occur
in the bone marrow but remain inconspicuous because of a prompt
emigration of these cells into the periphery. In accordance with this
hypothesis, we found that CFU-S day 8 were increased in peripheral
blood of 3-week-old C57BL/6-lpr/lpr mice, as compared with
controls of the same age (14.5 ± 2.7 v 4.1 ± 0.8 CFU-S
per 106 white blood cells [WBCs]; expressed as
means ± SEM from 3 separate experiments; P < .05).
The notion that extramedullary hematopoietic changes in Fas-deficient
mice might originate from the bone marrow is also consistent with the
striking accumulation of hematopoietic progenitors at several
peripheral sites. Indeed, as shown in Table
5, the increase in CFU-C frequencies was also observed in the
peritoneal cavity, the liver, and peripheral blood of 6-month-old mice.
Furthermore, it already took place in younger mice, concomitantly with
changes in spleen (data not shown).
Hematopoietic changes during fetal hematopoiesis.
The early hematopoietic modifications in Fas-deficient mice prompted us
to examine the frequency of progenitor cells in fetal livers. As shown
in Table 6, the incidence of CFU-S was
already clearly above normal in this organ at day 18 of gestation, as compared with age-matched controls. To exclude a possible contribution of maternal growth factors to the enhanced fetal hematopoiesis, we
implanted stage 2-cell embryos homozygous for the lpr gene into
wild-type C57BL/6 females. Table 6 shows that the difference between
Fas-deficient and normal fetal livers in terms of CFU-S frequencies
persisted in these conditions. A similar increase occurred in
homozygous lpr/lpr fetuses versus +/+ siblings of the
same litter generated in heterozygous C57BL/6-lpr/+ females (Table 6). These observations indicate clearly that the maternal environment is not responsible for the increased CFU-S frequencies in
Fas-deficient fetal liver. We did not observe any significant difference between fetal livers from age-matched Fas-deficient and
wild-type mice in terms of cellularity (data not shown).
Effect of Fas cross-linking on hematopoietic progenitor frequencies.
Previous reports on the expression of Fas on human hematopoietic
progenitor cells20,21,27 prompted us to test the expression and functionality of the Fas antigen by cross-linking with anti-Fas MoAb. This 24-hour treatment was performed with or without IFN, which has been reported to increase the expression of Fas or facilitate its activation.20,21,27 As shown in
Table 7, CFU-C frequencies were drastically
diminished after treatment of total spleen cells with anti-Fas MoAb,
provided that IFN was present during the incubation. In these
conditions, Fas was effectively expressed on progenitor-enriched
lin spleen cells (Fig
4B), as compared with freshly isolated cells, which exhibited only low
background expression (Fig 4A).
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| Fig 4.
IFN-induced Fas expression on lin
spleen and bone marrow cells. Flow cytometry analysis was performed on
at least 20,000 events gated from the population without lineage
markers. Splenocytes (A and B) and bone marrow cells (C and D) were
stained directly or after 24 hours of incubation with 100 U/mL of
mrIFN. The broken line represents control PE-conjugated hamster
MoAb.
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Finally, we verified the effect of Fas cross-linking on medullary CFU-S
and CFU-C. The results in Table 8
demonstrate that CFU-S frequencies were diminished by the ligation of
Fas that required the presence of IFN to become effective.
Interestingly, the same treatment affected bone marrow CFU-C
frequencies less than their splenic counterpart (Table 7). Once again,
Fas was clearly expressed on medullary lin cells in
these conditions, whereas it was not detected before incubation (Fig 4C
and D).
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DISCUSSION |
In the present study, we addressed the question whether, in addition to
its acknowledged immunoregulatory functions, the Fas-mediated death
pathway could influence early stages of hematopoietic development. For
this purpose, we evaluated the effect of Fas or FasL deficiency in mice
homozygous for the lpr or the gld gene, respectively, on the incidence of various progenitor subsets.
We found a marked increase in peripheral CFU-C in 6-month-old
C57BL/6-lpr/lpr mice. At this age, lymphadenopathy and
splenomegaly were well established and DN T lymphocytes had accumulated
in spleen and lymph nodes. Taking into account the increase of
mononuclear cells, the total number of colony-forming cells was around
30 times higher in mutant than in age-matched wild-type mice. A
comparable increase in CFU-C took place in spleens from FasL-deficient
C57BL/6 mice, proving the involvement of the Fas/FasL system in these hematopoietic changes.
As expected from the similar increase in all progenitor subsets growing
in methylcellulose, the incidence of their common CFU-S precursors was
also higher in Fas-deficient than in control mice. This augmentation
appeared as early as 3 weeks after birth, when CFU-C were not yet modified.
Because the accumulation of DN T lymphocytes in lymph nodes and spleen
is one of the most prominent features of Fas or FasL deficiency, we
addressed the question of the role played by this population in the
hematopoietic changes described here. DN T cells share several features
of activated lymphocytes and could be a source of hematopoietic growth
factors. Yet, previous reports have provided no evidence for their
capacity to produce IL-3 or granulocyte-macrophage colony-stimulating
factor (GM-CSF),28 and in our hands, the transcripts for
these cytokines were not increased in Fas-deficient spleen cells
relative to controls, as assessed by reverse
transcriptase-polymerase chain reaction (RT-PCR) analysis (G. Moreau,
personal communication).
Several lines of evidence contributed to rule out the participation of
DN T lymphocytes in the increased colony formation from spleen cells
lacking a functional Fas receptor. First, the difference between
C57BL/6 and C57BL/6-lpr/lpr mice in terms of colony formation
remained the same after T- and B-cell depletion of the spleen, proving
that these cells do not enhance to cloning efficiency by providing
supplementary growth factors. Second, the addition of DN T cells from
mice with severe lymphadenopathy (or their supernatants) during the
clonogenic assay did not increase the number of colonies formed by
normal spleen cells in response to optimal or suboptimal concentrations
of growth factors and had no effect by itself. In this context it
should also be noted that hematopoietic progenitors from Fas-deficient
and control mice displayed the same sensitivity to growth factors.
Further evidence against the participation of DN T cells in the
hematopoietic modifications resulting from Fas deficiency was provided
by the increased CFU-S in day-18 fetal livers of the lpr
phenotype. We could exclude the maternal environment as a possible
cause of this augmentation, which was observed in all Fas-deficient
fetal livers, whether the fetuses were removed from females homozygous
or heterozygous for the lpr gene or after development of
homozygous lpr/lpr fetuses in C57BL/6 females.
Surprisingly, none of the modifications described so far took place in
the bone marrow. It might be argued that medullary progenitors are
protected from Fas/FasL interactions. Several mechanisms could account
for such an effect, namely the expression of antiapoptotic molecules,
such as Bcl-2,29 or the cell cycle status of hematopoietic
progenitors.30 It is also possible that stromal cells
prevent the activation of the Fas pathway by providing protective
cytokines, such as IL-1,17 or by increasing cellular adhesion.31
Yet, in the light of our present data, we favor the hypothesis that
bone marrow progenitors are actually increased in Fas- and
FasL-deficient mice, but that they promptly emigrate into the
periphery, because there are not enough hematopoietic niches to
maintain them. Indeed, we observed a transient increase in medullary
CFU-S day 8 shortly after birth of Fas-deficient mice, in accordance
with the recent report of Traver et al,32 although it was
less striking and less persistent. It is noteworthy that these
investigators found no significant extramedullary modifications, possibly because the Fas mutation occurred on a different genetic background.
The appearance of hematopoietic progenitors at peripheral sites, such
as liver, peritoneal cavity, and peripheral blood, that are not
normally involved in hematopoiesis could be interpreted as a
consequence of their emigration from the bone marrow. The fact that
CFU-S and, to a lesser degree, CFU-C were actually diminished after Fas
cross-linking on bone marrow cells incubated in the presence of IFN
provides another argument against an entirely protective influence of
the medullary environment. It is therefore plausible that hematopoietic
changes in peripheral organs originate from the bone marrow, although
an independent expansion of progenitors from Fas-deficient spleens
cannot be excluded. In this context, it should also be noted that
increased endotoxin levels in lpr mice are not a likely cause for the
shift towards extramedullary hematopoiesis, because colony-stimulating
factors that would be induced in these conditions33 could
not be detected in sera.
The expression of Fas on hematopoietic progenitors and their decrease
after Fas cross-linking suggest that the Fas pathway might have a
direct regulatory effect on these cells. It has indeed been reported
that human CD34+ cells express Fas after stimulation with
IFN and/or TNF or even spontaneously in the case of primitive
fetal liver cells.20,21,26 This requirement for IFN was
also demonstrated in our study, because cross-linking of Fas alone
induced no significant decrease in CFU-C or CFU-S. It is in accordance
with a recent report showing that, in addition to its upregulatory
function on Fas expression,21 IFN is also capable of
stimulating caspase activity.34
So far, we can only speculate on the identity of the FasL+
cells in this particular context. Indeed, until recently, FasL
expression seemed to be much more restricted to lymphoid cells than
that of Fas antigen.35 Natural killer (NK) cells, present
both in bone marrow and spleen, express FasL
spontaneously36 and have been implicated in the regulation
of CFU-S.37 They disappear in Fas-deficient mice at about
the same time, as the lymphadenopathy becomes
significant.38 There is also some recent evidence for FasL
display on nonlymphoid cells, such as monocytes and neutrophils, which
express also Fas, making them capable of regulating their own
survival.39,40 Erythroid precursors have been reported to
express FasL spontaneously,41 a feature that may also apply to other immature cells. In our hands, FasL expression could be detected in freshly isolated bone marrow cells by RT-PCR analysis (G. Moreau, personal communication).
Bone marrow stroma might contribute to Fas-mediated apoptosis by
providing IFN/42 that can replace IFN- during
cross-linking (data not shown). According to a recent report, Fas
expression on hematopoietic progenitors can also be increased by
hematopoietic growth factors,43 which might eventually be
secreted by medullary stroma.
In 6-month-old Fas-deficient mice, myeloid infiltration in the spleen,
the liver, and the peritoneal cavity is particularly striking. The
proportion of granulocytes is particularly elevated, as assessed both
by morphological features and lineage markers. Because neutrophil cell
death seems to be directly modulated by Fas/FasL interactions, their
increased survival in the absence of functional Fas might explain this
preferential expansion.39
Whatever the exact mechanisms accounting for extramedullary
myelopoiesis in C57BL/6-lpr/lpr mice, our data provide the
first evidence that Fas deficiency affects the development of
hematopoietic progenitors before the development of other
abnormalities. Further exploration of this experimental model might
eventually yield new insights into the regulation of these cells by the
Fas pathway and their role in the development of the autoimmune disease.
| |
FOOTNOTES |
Submitted February 19, 1998; accepted June 6, 1999.
Supported in part by Grants No. 1827 and 1167, respectively, from the
"Association pour la Recherche contre le Cancer" (ARC) and the
"Ligue National contre le Cancer."
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Elke Schneider, PhD, CNRS UMR 8603, Hôpital Necker, 161, rue de Sèvres, 75743 Paris, Cedex 15, France; e-mail: schneider{at}necker.fr.
| |
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ABSTRACT
INTRODUCTION