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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 879-885
HEMATOPOIESIS
PU.1 is required for myeloid-derived but not lymphoid-derived
dendritic cells
Anastasia Guerriero,
Peter B. Langmuir,
Lisa M. Spain, and
Edward W. Scott
From the Institute for Human Gene Therapy, University of
Pennsylvania, Philadelphia, PA, and the Wistar Institute, Philadelphia,
PA.
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Abstract |
The ets-family transcription factor PU.1 is
required for the proper development of both myeloid and lymphoid
progenitors. We used PU.1-deficient animals to examine the role of PU.1
during dendritic cell development. PU.1 /animals
produce lymphoid-derived dendritic cells (DC): low-density class II major histocompatibility complex
[MHC-II+] CD11c+ CD8 +
DEC-205+. But they lack myeloid-derived DC: low-density
MHC-II+ CD11c+ CD8
DEC-205. PU.1/ embryos also lack
progenitors capable of differentiating into myeloid DC in response to
granulocyte-macrophage colony-stimulating factor plus interleukin-4.
The appearance of lymphoid DC in developing PU.1/thymus was initially delayed, but this
population recovered to wild type (WT) levels upon organ culture of
isolated thymic lobes. PU.1/lymphoid DC were
functionally equivalent to WT DC for stimulating T-cell proliferation
in mixed lymphocyte reactions. These results demonstrate that
PU.1 is required for the development of myeloid DC but not lymphoid DC.
(Blood. 2000;95:879-885)
© 2000 by The American Society of Hematology.
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Introduction |
Considerable progress has been made recently in our
understanding of the developmental relationships within the dendritic cell family. All dendritic cells (DC) are derived from the
hematopoietic stem cell (HSC),1 but they subsequently
diverge into at least 2 distinct cell lineages. One lineage of DC has 2 subpopulations, Langerhans cells of the skin and
monocyte/macrophage-related DC found in most tissues, that are derived
from a common myeloid precursor.2 The primary role of
myeloid DC is "professional" antigen presentation, which is
essential for T-cell activation and the initiation of an immune
response.1 Myeloid DC can be cultured in response to
myeloid growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4
(IL-4).3 Recently, a second lineage of DC of
thymic origin has been described. This second lineage is derived from a
lymphoid-restricted progenitor. Lymphoid DC are found in the thymus,
spleen, and lymph nodes. Within the thymus, lymphoid DC are thought to
mediate negative selection and subsequent destruction of self-reactive
T cells. Lymphoid DC can also activate T cells, but they induce a
restricted T-cell cytokine profile and apoptosis.4,5
Dendritic cell lineages can be separated by differences in physical
properties and cell-surface phenotypes. The majority of DC are
low-density cells that express high levels of class II major
histocompatibility complex (MHC-II+) and the integrin
CD11c. In addition, myeloid DC in situ are CD11b+
DEC-205CD8 , whereas lymphoid
DC are CD11b DEC-205+
CD8+.6
Gene targeting experiments have demonstrated the importance of
transcription factors during hematopoietic
development.7 To date, 2 transcription
factors, Ikaros and RelB, have been shown to affect dendritic cell
development.8,9 Ikaros is a zinc-finger DNA-binding protein
whose expression is restricted to early hematopoietic and
lymphoid-derived cells.10 Two strains of Ikaros
mutant mice have been generated: a dominant negative mutation
(IkarosDN)11 and a null mutation
(Ikaros/).12 IkarosDN
mice have severe defects in B and T cells but not in erythroid or
myeloid development, whereas Ikaros/mice
retain limited T-cell development after birth. IkarosDN
mice lack all DC, but Ikaros/mice still
produce reduced numbers of lymphoid DC.8 RelB is a member
of the NF- B/Rel family, whose expression is restricted to lymphoid tissues.13 RelB/mice
lack medullary epithelium and have a defective thymic medullary structure.14,15 In addition, RelB is required for the
development of myeloid (CD8), but not lymphoid
(CD8+) DC.9 These studies demonstrate that
transcription factors have differential roles in the development of
myeloid and lymphoid DC.
The transcription factor PU.1, a member of the ets-family of
DNA-binding proteins, is only expressed in hematopoietic
cells.16,17 PU.1 is expressed in early hematopoietic
progenitors including the circulating monocytic progenitors of myeloid
DC17 and in early CD44+ thymic progenitors,
which are likely precursors of both T cells and lymphoid DC in the
thymus.18 Targeted mutagenesis of the PU.1 locus causes a late-gestational
embryonic19 or early neonatal lethality.20
Severe defects in both lymphoid and myeloid development are seen during
fetal development, characterized by the absence of B-lymphoid and
macrophage progenitors.19 Normal numbers of erythroid and
megakaryocyte progenitors as well as reduced numbers of T-lymphocyte
and aberrant neutrophil progenitors are present in the
mutants.18,20-22 Adoptive transfer of fetal liver cells and
the generation of embryonic stem-derived (ES-derived) chimeric mice,
established that the PU.1 mutation is cell intrinsic and cannot
be rescued by a wild type (WT) microenvironment.21
We examined the role of PU.1 in dendritic cell development and found
that PU.1/animals produce functional
CD8+ lymphoid DC but lack CD8
myeloid DC. PU.1/embryos are also missing any
progenitors that are capable of differentiating into DC in response to
GM-CSF plus IL-4. The appearance of lymphoid DC in developing
PU.1/thymus is initially delayed, but this
population recovers to WT levels upon organ culture of isolated thymic
lobes. These results demonstrate that PU.1 is required for the
development of myeloid DC but not lymphoid DC.
| |
Materials and methods |
Antibodies
We used purified biotin fluorescein isothiocyanate- or
phycoerythrin-conjugated monoclonal antibodies (biotin-, FITC- or
PE-conjugated mAbs) from several manufacturers (brand
names in parentheses): GR-1 (RB6-8C5), CD11b (M1/70), CD8 (53-6.7),
heat stable antigen (HSA) (30-F1), CD3 (7A2), CD4 (L3T4), B220
(RA3-6Bc), TER-119, isotype controls, and avidin-FITC/PE (PharMingen,
San Diego, CA); purified CD11c (N418; Serotec, Oxford, England); F4/80
(CalTag, CA); and DEC-205 (NLDC-145; Bioscience, King of
Prussia, PA). We also used FITC-conjugated goat-rat and
goat-hamster F(Ab)2 (Southern
Biotechnology, Birmingham, AL) and blocking sera (Sigma, St. Louis, MO).
Mouse strains
Wild type PU.1 embryos
(PU.1+/+ or
PU.1+/) and mutant embryos
(PU.1/) were generated and
genotyped as previously described.19 Rapid genotyping was
accomplished by flow cytometry, and single-cell suspensions of the
fetal liver were prepared by standard methodology. Cells were stained
with FITC-conjugated antibodies GR-1 and CD11b, and
an FACS scan (Becton Dickinson, San Jose, CA) was used to distinguish the WT embryos (PU.1+/+ and
PU.1+/) from
PU.1/embryos. Propidium iodide
uptake and scattergating for size were used to exclude dead cells from
analysis. All putative genotypes assigned by FACS analysis were
confirmed by Southern blotting.
Dendritic cell isolation and culture
With minor modifications, DC were isolated by the method of Vremec
et al.23 Briefly, E16.5 embryos were
genotyped by FACS (see below), and WT or
PU.1/embryos were pooled. Single-cell
suspensions were prepared with collagenase and
ethylenediaminetetraacetic acid (EDTA). Low-density cells
were isolated by density gradient centrifugation in Nycodenz (Nycomed
Pharma, Westbury, NY) media (308 mmol/kg H20). DC are greatly enriched in the low-density population of cells. For myeloid dendritic cell culture, the low-density fraction was plated
2 × 105 cells/well in 24-well tissue culture plates
in RPMI 1640 media (25 mmol/L HEPES, pH 7.2; 10-4
mol/L  -mercaptoethanol; and 10% fetal calf serum [FCS])
supplemented with GM-CSF (15 ng/mL) and IL-4 (5 ng/mL). Low-density
cells were cultured for 3 days, fixed with acetone, and stained for
dendritic cell-specific markers CD11c or DEC-205. For FACS analysis,
low-density cells were further fractionated into lineage-depleted
(lin) MHC-II+ cells by magnetic bead separation.
Low-density cells were stained with a cocktail of mAbs (CD3, Thy1.2,
Gr-1, F4/80, B220, and TER-119) and depleted of non-DC by magnetic bead
separation (Miltyni Biotechnology, Auburn, CA). Lin
cells were then stained with biotin-conjugated MHC-II+.
MHC-II+ cells were positively selected with
avidin-conjugated magnetic beads. Aliquots of the MHC-II+
cells were stained with avidin-FITC to check for purity. If the population contained less than 95% of the MHC-II+
expression, another round of positive selection was performed. Low-density MHC-II+ (greater than 95%) cells were used for
further FACS analysis of dendritic cell subsets. To prevent nonspecific
antibody uptake, purified dendritic cell populations were stained, in
the presence of 20% blocking serum, with either purified -CD11c
plus FITC-goat-hamster F(Ab)2; PE-CD8; purified
DEC-205 then FITC-goat-rat F(Ab)2; or isotype
controls. Propidium iodide exclusion and scattergating for size were
used to exclude dead cells from analysis. For the mixed leukocyte
reactions (MLRs), MHC-II could not be used for purification purposes since it is required for T-cell stimulation. Therefore, the isolation protocol was modified to use
CD11c-conjugated magnetic beads (Milteyni Biotech, Auburn, CA) to
select DC from the low-density cell populations.
Mixed leukocyte reactions for dendritic cell function
Varying numbers of enriched DC from either WT or
PU.1/embryos were incubated with 20 000
purified T cells from Balb/c mice. The T cells were purified from the
spleen using an enrichment column (#MTCC-5; R&D Systems, Minneapolis,
MN) per the manufacturer's instructions. Cells were cultured in
96-well plates with RPMI media, 10% serum for 3 days. We added
37 × 103 Bq (1 µCi)
3H-thymidine to each well, and the culture continued for 24 hours. Cells were harvested and counted (Wallac,
Gaithersburg, MD). T cells or DC alone served as the
background proliferation controls. Each assay test was performed a
total of 3 times in triplicate.
Fetal thymic organ culture
Fetal thymic organ cultures were performed as follows. Dissected
thymic lobes were cultured in RPMI (GibcoBRL Life Technologies, Grand
Island, NY) plus 10% FCS (Cansera, Ontario, Canada),
glutamine (GibcoBRL), and gentamycin. They were then suspended on rafts comprising nucleopore filters (Costar, Cambridge, MA) supported by
gelfoam (Upjohn, Kalamazoo, MI). Culture medium was partially replaced
every 3 days for a total of 10 days.
Histology
Whole E14.5 and E16.5 embryos or cultured thymic lobes were
embedded, and 10-µmol/L frozen sections were prepared (Cell
Morphology Core, Institute for Human Gene Therapy, University of
Pennsylvania, Philadelphia, PA). Immunohistochemistry was performed
with staining (Vecta-stain; Vector Labs, Burlingame, CA)
and the appropriate purified primary antibody according
to manufacturer's protocols. For visualization purposes, we used
alkaline phosphatase-conjugated secondary antibodies with a substrate
(Vector Black, Vector Labs). To allow quantification using an image
analysis system (Leica, Deerfield, IL), counterstain was
not used. To determine relative staining, equivalent WT and
PU.1/sections were stained in concert and
digitized (Leica Photomicroscopy system, Leica). Relative staining
intensities were sampled, and background was subtracted using image
analysis software (Leica and NIH Image). An average of 4 sections were
analyzed for each WT and PU.1/stain. The
staining of WT sections was taken as 100% for each marker analyzed.
Relative staining percentages were then calculated for the
PU.1/sections and assigned to the nearest
5%. Staining for HSA in the fetal thymus was equivalent between WT and
PU.1/embryos and served as a positive control.
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Results |
Histology and culture of myeloid dendritic cell progenitors
Previous studies of PU.1/mice have
demonstrated severe myeloid defects. PU.1 is expressed at high levels
in the myeloid lineage and is absolutely required for
monocyte/macrophage differentiation.19,20 Very low levels
of myeloid progenitors do exist in
PU.1/animals, however, and these progenitors
can differentiate into aberrant neutrophil-like cells in
vitro22 or postnatally.20 To investigate the
effects of the PU.1 mutation on myeloid DC in tissues, E16.5 embryos
were sectioned and stained for DC CD11b or CD11c
expression. No positive staining was seen for CD11b in PU.1/embryo sections (data not shown). The
thymus was positive for the CD11c dendritic cell (Figure
1) and negative elsewhere in the embryo,
except for background staining in the gut due to
endogenous alkaline phosphatase activity (data not shown). These
staining patterns suggested that myeloid DC, in addition to monocytic
cells, were absent in PU.1/mutants. Given the
rarity of DC in whole animals, we decided to concentrate and expand
myeloid DC by culture from WT and
PU.1/embryos.
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| Fig 1.
Myeloid dendritic cell culture.
Low-density cells ( = 1.077 g/cm3) were isolated from
WT and PU.1/E16.5 embryos. The cells were
cultured (2 × 105 cells/well) in media containing
GM-CSF and IL-4 for 3 days. Cells were then fixed and stained for the
DC markers CD11c or DEC-205. (A) Photomicrograph of CD11c stained DC
from WT or PU.1/embryos. (B) Stained cells
were counted, and the results were tabulated as DC per 105
low-density cells plated in the original culture. Results are given in
mean ± SEM (standard error of the mean) per number of embryos
tested (n).
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Several investigators have shown that myeloid DC can be cultured in the
presence of GM-CSF and IL-4.3 In addition,
PU.1/embryos still express
GM-CSFR, which suggests that GM-CSF responsive hematopoietic progenitors may still be present.24
Therefore, DC were enriched from whole E16.5 WT and
PU.1/embryos via collagenase treatment and
density gradient centrifugation (as described earlier). The enriched
cell populations were then cultured for 3 days in media containing
GM-CSF and IL-4. In these culture conditions, myeloid DC express CD11c
and up-regulate expression of DEC-205. Cultured cells were fixed and
immunohistochemically stained for CD11c or DEC-205 expression. Figure
1A shows the characteristic staining observed for CD11c, a myeloid DC
with clear dendritic processes. The results of triplicate cultures were
quantified and expressed as the number of myeloid DC per
105 cells plated in the original culture (Figure 1B). WT
cultures contained numerous easily distinguishable DC that were
positive for CD11c or DEC-205. PU.1/cultures,
however, exhibited only background levels of staining for these markers.
Histology of thymic dendritic cells
Thymic DC are thought to be derived from a separate lymphoid-related
progenitor than myeloid DC.25 Both myeloid and lymphoid DC
express the integrin CD11c and high levels of MHC-II. The majority of
lymphoid DC, however, express DEC-205 in situ (without the need for
culture) and the lymphoid-specific marker CD8. To examine lymphoid
DC in the thymus, frozen sections were prepared from WT or
PU.1/E14.5 and E16.5 embryos as well as from
cultured thymic lobes. Staining for CD11c, DEC-205, or HSA
expression was done in concert to allow the assessment of
relative staining intensity between sections. To facilitate their
quantification, the sections were not counterstained. Figure
2 shows the staining pattern of equivalent sections of WT or PU.1/thymus stained for
CD11c cells. Initial expression of CD11c is delayed in
PU.1/embryos, as evidenced by the low-level
staining of E14.5 thymus. By E16.5 staining,
CD11c+ cells in PU.1/thymus
recovered to near WT levels. When thymic lobes are placed into organ
culture for 10 days, the staining intensity for WT and
PU.1/lobes becomes equivalent. A nearly
identical pattern is observed for DEC-205 expression (Figure
3). The WT staining pattern for both CD11c
and DEC-205 expression was mainly restricted to the subcapsular cortex
of the developing thymus in the E14.5 and E16.5 sections. The staining
patterns of PU.1/thymus were less organized
due to their general hypocellularity.
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| Fig 2.
Thymic lobes stained for CD11c expression.
WT or PU.1/embryos or cultured thymic lobes
were fixed, and cryosections were prepared. Individual sections were
stained with CD11c antibodies followed by an APC secondary antibody.
A substrate kit (Vector Black, Vector) was used for visualization. All
staining was done in concert to allow relative staining intensities to
be compared. No counterstain was used. E14.5 and E16.5 indicate the
gestational age of the embryo. FTOC indicates E16.5
thymic lobes that have been placed in organ culture for an additional
10 days prior to sectioning.
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| Fig 3.
Thymic lobes stained for DEC-205 expression.
WT or PU.1/embryos or cultured thymic lobes
were fixed, and cryosections were prepared. Individual sections were
stained with DEC-205 antibodies (Figure 2). E14.5 and E16.5 indicate
the gestational age of the embryo. FTOC indicates E16.5
thymic lobes that have been placed in organ culture for an additional
10 days prior to sectioning.
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Individual sections from an average of 4 embryos were digitized (Leica
Photomicroscopy system, Leica) and analyzed for staining intensity with image analysis software. Background staining from muscle
was subtracted, and WT sections were assigned a value of 100%. Figure
4 depicts the relative staining intensity
of the PU.1/sections. HSA expression in the
thymus was equivalent between WT and
PU.1/embryos. At the E14.5 staining,
PU.1/embryos had fewer than 10% of WT levels
for both CD11c+ and DEC-205+ cells. By the
E16.5 section, staining for both markers was approximately 70% of WT
levels. Individual thymic lobes were also cultured for 10 days, then
sectioned and stained. Staining for the lymphoid DC markers CD11c and
DEC-205 was equivalent for both genotypes after culture. These staining
patterns suggest that lymphoid DC development was initially delayed in
PU.1/embryos, but then recovered to WT
levels. However, both DEC-205 and CD11c are expressed on cells other
than DC, which could complicate interpretation of the staining
patterns. Therefore, we performed a more stringent flow cytometric
analysis for DC subsets.
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| Fig 4.
Quantification of dendritic cell staining.
Immunohistochemical staining of cryosections was performed
simultaneously in batches for both WT and
PU.1/embryos or cultured thymic
lobes. Sections were stained for HSA, CD11c, or DEC-205 expressions. A
minimum of 3 sections (average of 5 sections) from different embryos
were digitized (Leica Photomicroscopy system, Leica) for each
gestational age and stain. Staining intensity was quantified with
analysis software (NIH-Image). Background staining from muscle was
subtracted, and WT sections were assigned a value of 100%. Mean values
and standard deviations for relative staining intensity are shown to
the nearest 5%.
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Specific block to myeloid but not lymphoid dendritic cells
To further characterize dendritic cell populations in
PU.1/embryos, we directly examined both
myeloid and lymphoid DC isolated from whole embryos. All DC are
low-density cells that are highly positive for MHC-II+ and
CD11c expression. Within this population, lymphoid DC also express
CD8 and DEC-205, whereas myeloid DC do not.6 Therefore, the major lymphoid DC population comprises low-density
MHC-II+, CD11c+, CD8+, and
DEC-205+ cells, while the major myeloid DC population
comprises low-density MHC-II+, CD11c+,
CD8, and DEC-205cells. To
examine these populations in E16.5 embryos, the enrichment scheme
depicted in Figure 5A was followed.
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| Fig 5.
Dendritic cell isolation and flow cytometry analysis for
myeloid and lymphoid DC.
(A) Isolation scheme for low-density MHC-II+ cells.
Low-density cells are isolated by density gradient centrifugation
followed by magnetic bead enrichment for MHC-II+ cells.
Cells are subjected to successive rounds of positive selection until
more than 95% are MHC II+ cells, as determined by flow
cytometry. (B) Low-density MHC-II+ cells (panel 1) are then
stained for DC markers CD11c (panel 2), CD8 (panel 3), or DEC-205
(last panels) and analyzed by flow cytometry. Representative staining
histograms are presented with isotype controls provided. Dead cells
were excluded from analysis by propidium iodide exclusion and
scattergating for size.
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Briefly, E16.5 PU.1/embryos and WT
(PU.1+/+ and PU.1+/) embryos were
pooled, minced, and treated with collagenase to form single-cell suspensions. Low-density cells were then isolated by density gradient centrifugation. With adult lymphoid tissues, such as spleen and thymus,
density separation alone results in greater than 100-fold enrichment
for DC, and 20%-40% of cells express high-density
MHC-II+. For the whole E16.5 embryos, however, less than
2% of the low-density cells were MHC-II+, thus
necessitating further enrichment steps. Low-density cells were
therefore stained with mAbs to MHC-II+ and positively
selected with magnetic beads. Flow cytometry was used to monitor the
purification until the cell population was greater than 95%
MHC-II+ cells (Figure 5B, first panels).
Approximately 3 × 103 low-density MHC-II+ cells were isolated per WT embryo, and
1 × 103 low-density MHC-II+ cells were
isolated per PU.1/embryo. Purified
low-density MHC-II+ cells were then analyzed for either
CD11c, CD8, or DEC-205 expression. Single-color flow cytometric
analyses were performed due to the extreme difficulty of isolating the
small initial populations and the limitations imposed by the available
antibody reagents.
Figure 5B shows the results of flow cytometric analysis for dendritic
cell subsets. The first panel shows the MHC-II staining for the input populations. More than 95% of the purified cells were
positive for MHC-II. The intensity of MHC-II staining is somewhat
reduced due to the presence of avidin-conjugated magnetic beads, which
were used for purification. The beads occupy a portion of the available
binding sites on the biotinylated antibody, thus reducing the amount of
avidin-FITC that can bind. The low-density MHC-II+ cells
from both genotypes stained positive for CD11c expression, which
indicates that the enriched cells are almost pure DC. WT cells had a
bimodal staining profile for both CD8 and DEC-205 expression, and
approximately 65% of the DC were negative for both markers, and 35%
were positive. Thus WT embryos have both myeloid DC (low-density
MHC-II+ CD11c+ CD8
DEC-205) and lymphoid DC (low-density
MHC-II+ CD11c+ CD8+
DEC-205+). In contrast,
PU.1/embryos have lymphoid DC
(CD8+ DEC-205+), but lack myeloid DC
(CD8 DEC-205 ).
PU.1/lymphoid dendritic cells are functional
To test the ability of PU.1/lymphoid DC to
present antigen and activate T cells, mixed leukocyte reactions were
performed. Various numbers of WT or PU.1/DC
from a mixed C57BL/6 and 129-SV strain background were
cocultured with splenic T cells from Balb/c mice, and the proliferative
response was measured. PU.1/lymphoid DC
demonstrated an equivalent or better ability to stimulate T- cell
proliferation when compared to wild type DC (Figure
6). Due to limitations in our ability to
isolate embryonic DC, peak or saturation levels of T-cell proliferation
were not reached, as evidenced by the lack of plateau on the graphs.
However, as few as 1000 PU.1/DC were able to
induce a 10-fold T-cell proliferative response in the mixed leukocyte
reaction. This demonstrates that the function of
PU.1/lymphoid DC is not impaired by the
mutation.
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| Fig 6.
Mixed leukocyte cultures to measure dendritic cell
function.
Varying numbers of purified DC from WT embryos ( ) or
PU.1/embryos ( ) were cocultured with
purified splenic T cells from Balb/c mice. The proliferative response
was measured at day 4 by 3H-thymidine incorporation and is
presented as total CPM per well. The results represent 3 independent
assays done in triplicate, with standard deviations indicated by the
error bars.
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Discussion |
Previous studies have shown that transcription factor PU.1 plays a
role in the earliest stages of both lymphoid and myeloid development.26 PU.1 appears to be absolutely required in
order for myelomonocytic and B-lymphoid progenitors to develop. In
addition, PU.1 is required for efficient commitment to and/or
differentiation of T lymphocytes. PU.1 is expressed in uncommitted
CD44+, CD25 thymocytes,
but it is not expressed by the time thymocytes manifest CD4 or CD8
expression.18 Since PU.1 plays a role in both myeloid and
lymphoid lineages, we were interested in determining its role in the
development of DC.
DC are rare but widely distributed migratory antigen presenting cells
(APC) of the immune system.1 The major role associated with
DC is the initiation of primary immune responses by T
lymphocytes.27 Until recently, DC were known to be derived
from the hematopoietic stem cell, but their lineage relationships were
unknown. It is now clear, however, that DC can arise from 2 separate
hematopoietic lineages and are referred to as myeloid DC or lymphoid
DC.28 Most DC are derived from myelomonocytic precursors
and express myeloid related markers such as CD11b. These precursors can
be matured into myeloid DC in culture in the presence of GM-CSF. At
peripheral sites, myeloid DC phagocytose antigens process the antigen
and migrate to the lymph nodes, where they mature into cells capable of
presenting antigen to T cells in order to initiate an immune
response.1 Indeed, myeloid DC are vastly more potent at
inducing T-cell responses than any other APC. This fact, coupled with
the myeloid dendritic cell migratory nature and APC, makes the cells
likely to be the predominate APC in initiating immune responses.
Thymic DC have a different function and origin. Thymic DC are thought
to mediate negative selection of developing T lymphocytes. They do so
by presenting self-antigens and initiating the death of self-reactive T
cells within the thymus. The earliest hematopoietic progenitors found
in the thymus have given rise to B cells, T cells, and thymic DC but
not myeloid cells.29,30 CD44+ CD25+
CD4CD8thymic progenitors can give
rise to T cells and thymic DC, but not B or myeloid cells, whereas
CD25+
CD44CD4CD8cells
only give rise to T cells.31 Thymic DC are characterized in
part by their expression of CD8. Similar CD8+ DC are
found in the spleen, and they also have negative regulatory effects on
T-cell function.4,5 The CD8+ splenic DC are
postulated to arise from the same progenitor as thymic
DC,31-34 hence the designation of lymphoid DC used in this paper. PU.1 is expressed in the multipotent thymic progenitors that
give rise to lymphoid DC, but PU.1 is down-regulated in committed T-cell progenitors, which suggests that it may play a role in lymphoid
dendritic cell development.18
PU.1/embryos were sectioned and stained for a
variety of markers for both myeloid and lymphoid DC. In any tissue
tested, the PU.1/embryos did not contain
cells that stained for the myeloid-specific marker CD11b (data not
shown). This is in accord with previous flow cytometry
data.19 Indeed, the lack of CD11b+ cells is
used to genotype the embryos. This suggests the absence of all
myelomonocytic lineage cells, including myeloid DC, in PU.1/embryos. One major caveat, however, is
the fact that expression of CD11b is regulated in part by
PU.1.35 Thus, the lack of staining may represent only a
lack of CD11b expression and not a lack of myeloid cells. To address
this concern, we attempted to grow myeloid DC from
PU.1/embryos via culture in media with GM-CSF
and IL-4. While WT embryos yielded numerous CD11c+ and
DEC-205+ myeloid DC, myeloid DC could not be cultured from
PU.1/embryos (Figure 1).
Sections from multiple WT and PU.1/embryos
were also stained either for CD11c, DEC-205, or HSA expression.
Comparison of staining intensities between WT and
PU.1/embryos suggested a delay in the
development of lymphoid DC in the thymus of
PU.1/embryos (Figures 2-4). At the E14.5
staining, very few stained cells were present in mutant thymus. After
culture of thymic lobes, however, both WT and
PU.1/lobes stained with equal intensity for
both lymphoid dendritic cell markers. It still remains to be determined
if the delay is due to the direct role of PU.1 in lymphoid dendritic
cell differentiation or to a secondary effect from the general
hypocellularity of PU.1/thymus.
Our next goal was to directly assess which dendritic cell populations
are present in PU.1/ embryos. Given the small
size of E16.5 embryos, it was impractical to isolate DC from dissected
hematopoietic tissues. Therefore, we took advantage of the high levels
of MHC-II+ expressed by all DC to purify them from total
low-density cells isolated from whole embryos. It was then possible to
analyze low-density MHC-II+ populations by flow cytometry
for dendritic cell subsets. The majority of DC found in WT embryos were
myeloid DC, MHC-II+ CD11c+
CD8 DEC-205cells.6
WT embryos also contained a smaller population of MHC-II+
CD11c+ CD8+ DEC-205+ lymphoid DC
(Figure 5). In contrast, PU.1/embryos only
contained MHC-II+ CD11c+ CD8+
DEC-205+ lymphoid DC. Myeloid DC were clearly absent from
the low-density MHC-II+ population of
PU.1/cells (Figure 5). This result is
supported by the dendritic cell culture results and histology and the
fewer numbers of low-density MHC-II+ cells isolated from
the mutants (1 × 103 versus
3 × 103 per embryo). Previous transplantation and
ES cell chimera experiments have demonstrated that the
hematopoietic defects seen in
PU.1/animals are cell
intrinsic.21 The functional proliferative assays demonstrate that PU.1/lymphoid DC present
antigen and stimulated T-cell activation as well as, if not better
than, wild type DC. Therefore, PU.1 is required for myeloid dendritic
cell development but not for lymphoid dendritic cell development.
The dendritic cell phenotype of PU.1/mice is
similar to that seen with RelB/mice
and Ikaros/mice. All 3 mutations lack
CD8 myeloid DC. For both RelB and Ikaros mutants,
myeloid development is robust, and RelB/mice
actually exhibit myeloid hyperplasia. Why both mutations lack myeloid
DC remains uncertain. In the case of PU.1, however, the lack of myeloid
DC may be directly linked to effects on myeloid progenitors. PU.1 is
required for the expression of a host of myeloid-specific
genes,36 some of which may be crucial for myeloid dendritic
cell development. Another common feature of all 3 mutations is the
retention of some T-cell development potential, and all mutations are
capable of producing CD8+ lymphoid DC.
PU.1/embryos have exceedingly hypocellular
thymuses seeded with early hematopoietic progenitors
(CD44+, HSABright, c-kitint,
Thy1, CD25,
Sca1, CD4, and
CD8). Corresponding progenitors with respect to
number and cell-surface phenotype are found in WT fetal
thymus.18 Development of lymphoid DC in
PU.1/thymus is delayed to a much lesser
extent than development in T cells (2-fold down at E16.5 versus greater
than 100-fold down). This suggests that PU.1 may play a more direct
role in the T-cell lineage. An equally attractive alternative is that
lymphoid DC must develop within the thymus in order to induce efficient
T-cell commitment and/or development. PU.1-deficient mice may provide the experimental means to dissect the requirements for lymphoid dendritic cell development and their function during T-cell differentiation.
| |
Acknowledgments |
The authors would like to thank Ken Shortman (WEHI,
Melbourne, Australia) for a detailed isolation protocol for DC. We also thank Robert Fisher (University of Pennsylvania) for a critical review
of the manuscript.
| |
Footnotes |
Submitted May 6, 1999; accepted October 5, 1999.
Supported by grants PO1 DK52558 (L.M.S. and E.W.S.), CA72769, and
HL58716 (E.W.S.) from the National Institutes of Health, Bethesda, MD.
E.W.S. is a Leukemia Society of America Scholar.
Reprints: Edward W. Scott, Institute for Human Gene Therapy,
Biomedical Research Building II, Room 513, 421 Curie Boulevard, University of Pennsylvania, Philadelphia, PA 19104-6140; e-mail: scotte{at}mail.med.upenn.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction