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Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 93-100
Interleukin-7 Influences the Development of Thymic Dendritic Cells
By
Alberto Varas,
Angeles Vicente,
Rosa Sacedón, and
Agustín G. Zapata
From the Department of Cell Biology, Faculty of Biology, Complutense
University of Madrid, Madrid, Spain.
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ABSTRACT |
Interleukin-7 (IL-7) has been shown to be a critical factor in B and
T lymphopoiesis, and to influence the differentiation of myeloid cell
lineages. In the present study we extend these results demonstrating
that IL-7 also plays an important role in the development of thymic
dendritic cells (DC). The addition of IL-7 to rat fetal thymus organ
cultures (FTOC) resulted in a drastic increase in the number of
CD3 CD4CD8 cells, which
mostly expressed typical DC markers, including major histocompatibility
complex class II, OX-62, CD11b, CD68, and CD54. These cells exhibited
morphological and ultrastructural features of DC, and were potent
stimulators of the allogeneic mixed leukocyte reaction. Although
increased numbers of DC were continuously generated throughout the
culture period in the presence of IL-7, they were not actively
dividing, indicating that DC in IL-7-treated cultures did not arise by
expansion of pre-existing cells. Reduced DC numbers obtained after the
addition of neutralizing anti-IL-7 antibodies to mouse FTOC confirmed
the relevance of endogenously produced IL-7 on thymic DC development.
Furthermore, the addition of IL-7 to FTOC derived from severe combined
immunodeficient mice also generated large numbers of DC in the absence
of thymocyte maturation.
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INTRODUCTION |
DENDRITIC CELLS (DC) are a heterogeneous
population comprising irregularly shaped, migratory cells of sparse but
wide distribution in both lymphoid and nonlymphoid tissues. In
functional terms, DC are potent stimulators of T-cell-mediated
immunity and, as such, have extraordinary antigen-presenting
capacity.1-3 The origin of DC and the growth factor
requirements for their development are not fully characterized. Several
reports have shown that substantial numbers of functional DC can be
generated from blood or bone marrow (BM) precursor cells under the
influence of granulocyte-macrophage colony-stimulating factor (GM-CSF)
alone or in combination with tumor necrosis factor- (TNF-) and
stem cell factor (SCF).4-10 Peripheral blood monocytes can
also differentiate into DC upon culture with GM-CSF and interleukin-4
(IL-4).11-13 However, although GM-CSF appears to be
necessary in vitro, recent in vivo studies have shown that increased
levels of GM-CSF or the lack of GM-CSF activity do not modify the
numbers of DC in the lymphoid tissues,14,15 suggesting that
other growth factors are important for DC generation. Additionally, DC
can arise from intrathymic progenitor cells also capable of forming T
lymphocytes16-18 and, interestingly, these DC develop in
vitro in the absence of GM-CSF.19 According to these
findings and those from previous reports suggesting that IL-7 can
enhance DC formation from thymic precursors,18,19 we
examined the effects of IL-7 on the development of thymic DC. IL-7 is a
unique and nonredundant cytokine known to play a crucial role in
thymocyte maturation,20-24 and has also been involved in the differentiation of B cells20,21,25,26 and other
hematopoietic cell lineages, including megakaryocytes and myeloid
cells.27-31 We now report that the addition of IL-7 to
fetal thymus organ cultures (FTOC) results in the continuous generation
of large numbers of functional DC, whereas deprivation of endogenously produced IL-7 drastically reduces the numbers of DC, implying a new
role of IL-7 in thymic DC maturation.
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MATERIALS AND METHODS |
Animals.
Wistar rats and Swiss mice were maintained in our animal facilities.
CB-17 severe combined immunodeficient (SCID) mice were obtained from
the specific pathogen-free breeding facilities at the Severo Ochoa
Center for Molecular Biology (Madrid, Spain). Rat fetuses at day 16 of
gestation and mouse fetuses at day 14 of gestation were obtained from
timed pregnancies. The day of finding a vaginal plug was designated day
0 of gestation.
Fetal thymus organ cultures.
Thymic lobes were aseptically removed from 16-day-old rat embryos or
14-day-old mouse fetuses using a stereoscopic microscope, trimmed of
surrounding mesenchyme, and organ-cultured as follows. Four to six
individual thymic lobes were placed on the surface of 0.8-µm
polycarbonate filters (Millipore Ibérica, Madrid, Spain), which
rested on stainless steel screen pieces attached to the central well of
organ tissue culture dishes (Becton Dickinson, San Diego, CA). Lobes
were cultured in 1 mL of RPMI 1640 medium (2 mmol/L L-glutamine)
supplemented with sodium pyruvate (1 mmol/L), streptomycin (100 mg/mL), penicillin (100 U/mL) (all reagents: GIBCO-BRL, Eragny,
France), and 10% fetal calf serum (FCS) (Biosys, Compiègne,
France). The peripheral well of the culture dishes was filled with 5 mL
of sterile distilled water. Cultures were incubated at 37°C in a
humidified incubator containing 10% CO2 in air. Medium was
replaced daily. The IL-7-treated organ cultures were performed at a
concentration of 2,000 U/mL of recombinant human IL-7 (ampoule code
90/530; National Institute for Biological Standards and Control,
Hertfordshire, UK).
For neutralizing studies, mouse anti-human/mouse IL-7 (mouse IgG2b) was
used at a concentration of 75 µg/mL. In the presence of 5 ng/mL IL-7,
5 µg/mL antibody achieved 90% neutralization of bioactivity in the
2B clone proliferation assay (Genzyme, Cambridge, MA). In this case,
control cultures were supplemented with purified mouse IgG2b monoclonal
antibody (MoAb) from the MOPC 141 tumor line (ICN Biomedicals, Costa
Mesa, CA).
Antibodies.
Mouse anti-rat MoAbs of the following specificities were used in our
study: CD8 (OX-8 fluorescein isothiocyanate [FITC] or phycoerythrin
[PE]), CD4 (OX-38 FITC or PE), CD3 (G4.18 biotin), major
histocompatibility complex (MHC) class II (OX-6 FITC), CD11b (WT.5
FITC), CD54 (1A29 FITC), CD44 (OX-49 FITC), and CD25 (OX-39 FITC) were
obtained from Pharmingen (San Diego, CA). Antibodies recognizing an
integrin-like antigen specific for rat DC (OX-62) and CD68 (ED1 FITC)
were purchased from Serotec (Oxford, UK). The following rat anti-mouse
MoAbs were from Pharmingen: CD8 (53-6.7 FITC) and CD4 (H129.19 PE). The
biotinylated antibodies were revealed with second-step
streptavidin-CyChrome (Pharmingen), and unlabeled antibodies with
FITC-conjugated F(ab )2 fragment of rabbit anti-mouse IgG
(Serotec).
Immunofluorescence staining and flow cytometry analysis.
Single-cell suspensions were obtained by passing disrupted thymic lobes
through a 25-gauge hypodermic needle, and maintained on ice in
phosphate-buffered saline (PBS) containing 1% FCS and 0.1%
NaN3 before use. A total of 1 to 2 × 105
cells were incubated with saturating amounts of FITC- and PE-labeled MoAbs for 30 minutes at 4°C. After staining, cells were washed twice
and resuspended for analysis. Stainings with unlabeled antibodies were
followed, after washing, by incubation with FITC-conjugated F(ab)2 fragment of rabbit anti-mouse IgG. For
three-color immunofluorescent labeling, cells were stained with
anti-CD3-biotin, followed by anti-CD8-FITC, anti-CD4-PE, and
streptavidin-CyChrome. Stained cells were analyzed in a FACScan flow
cytometer (Becton Dickinson) from the Research Center, Faculty of
Biology, Complutense University of Madrid. Debris and dead cells were
excluded from the analysis by forward and side scatter and propidium
iodide gatings. The data were analyzed using PC-Lysis research software
(Becton Dickinson).
Cell-cycle analysis.
To determine the proportion of proliferating cells, 1 to 2 × 105 cells were stained with anti-CD8-FITC and anti-CD4-PE
for 30 minutes at 4°C. Cells were washed twice with PBS and fixed in 30% ethanol for a minimum of 30 minutes, but usually overnight at
4°C. The cells were then washed, resuspended in a solution of 25 µg/mL 7-AAD (Sigma España, Madrid, Spain) in PBS
with 0.025% Nonidet P-40 (Sigma), and incubated in the dark at 4°C
for 2 hours. Analysis was performed in a FACScan, using Cell Fit and
PC-Lysis softwares (Becton Dickinson).
Mixed leukocyte reaction (MLR) assay.
Cells recovered from 12-day control and IL-7-treated FTOC were
incubated with a cocktail of anti-CD4, anti-CD8, and anti-CD3 MoAbs
followed by two rounds of immunomagnetic bead depletion (Dynabeads;
Dynal, Oslo, Norway). The purity of the recovered CD3CD4CD8 cells was 90%
to 95%. Thymic DC were isolated from adult rat thymus as
described.32 Subsequently, both
CD3CD4CD8 cells and DC
were treated with 25 µg/mL mitomycin C (Sigma) for 30 minutes at
37°C, extensively washed, and used at different numbers (1 cell to
2.5 to 5 × 104 cells) as stimulators for allogeneic T
cells (2 × 105) isolated from rat lymph nodes. The
cultures were performed in 96-well round-bottom culture plates, using
0.1 mL RPMI 1640/10% FCS. After 5 days at 37°C in 10%
CO2-in-air incubator, the cultures were pulsed for 4 hours
with 10 µmol/L 5-bromo-2-deoxy-uridine (BrdU). A specific kit from
Boehringer Mannheim (BrdU Labeling and Detection Kit III; Mannheim,
Germany) was used to measure BrdU incorporation into newly
synthesized DNA. Briefly, the labeling medium was removed and cells
were dried (2 hours at 60°C), fixed in 70% ethanol in HCl (0.5 mol/L) for 30 minutes at  20°C, treated with nucleases (30 minutes
at 37°C), and then incubated with peroxidase-conjugated Fab fragments
of mouse anti-BrdU (30 minutes at 37°C). The peroxidase reaction was
developed with ABTS-substrate (Boehringer Mannheim), and
the sample absorbance was measured using an enzyme-linked immunosorbent
assay (ELISA) reader at 405 nm with a reference wavelength at 492 nm.
Electron microscopy.
Organ-cultured thymic lobes were fixed by immersion in 4%
glutaraldehyde, buffered to pH 7.3 with Millonig's fluid,
postfixed in 1% osmium tetroxide in the same buffer, and dehydrated in
acetone for embedding in Araldite (Fluka, Buchs, Switzerland). Sections were obtained with a Reichert OM-U3 ultramicrotome
(Reichert-Jung, Wien, Austria). Semithin sections stained with an
alkaline solution of toluidine blue were used in the histological
analysis, and ultrathin sections were double-stained with uranyl
acetate and lead citrate, and examined in a JEOL 10.10 electron microscope (Jeol, Tokyo, Japan).
Immunocytochemistry.
Air-dried cytospin preparations were fixed for 5 minutes in acetone at
20°C. Slides were incubated with anti-rat (or mouse) MHC class II
antibodies for 60 minutes. Endogenous peroxidase activity was inhibited
with 0.33% H2O2 in methanol for 5 minutes, and
preparations were then incubated for 45 minutes with a 1/100 solution
of peroxidase-conjugated rabbit anti-mouse (or rat) Igs in PBS (Dako,
Glostrup, Denmark). The peroxidase reaction was developed with 0.05%
3, 3-diaminobenzidine (Sigma) in PBS with 0.1%
H2O2 for 10 minutes. The reaction was stopped
and slides were counterstained with methylene blue and mounted.
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RESULTS |
Treatment of rat FTOC with IL-7.
Rat FTOC were performed using day 16 fetal thymus lobes, containing
90% to 95% of triple-negative (TN) CD3CD4
CD8 cells,33 and were cultured for 12 days in the continuous presence of recombinant human IL-7 (2,000 U/mL
based on previous titration in FTOC, data not shown). Under these
conditions, IL-7 decreased the total cell yield of these lobes compared
with control cultures (Table 1). The
recovered cells were triple-labeled for flow cytometric analysis and
the expression of CD3, CD4, and CD8 was examined. Two major effects
were observed: there was a reduction in the proportion of
CD4+CD8+ thymocytes, and an accumulation of
mature CD4CD8+ thymocytes and TN cells (Fig
1). Despite the reduced cellularity, the
absolute numbers of mature CD8+ thymocytes and TN cells
were significantly higher after IL-7 treatment, showing a fourfold and
a threefold increase, respectively, when compared with the control
cultures (Table 1).
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| Fig 1.
Phenotypic analysis of cells recovered from FTOC treated
with IL-7. Cells were recovered from fetal thymic lobes after 12 days
of culture, and triple-labeled for flow cytometry analysis as described
in Materials and Methods. CD4 versus CD8 expression is represented in
the dot plots with percentages of cells included in the corner of each
quadrant. The cells within each quadrant were also examined for
expression of CD3. The percentages in the corner of each histogram
represent the proportion of cells expressing high levels of CD3. The
profiles shown are representative of six independent experiments.
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The specificity of the IL-7 effect could be shown by the absence of
these effects, when FTOC were grown in the continuous presence of IL-7
with neutralizing anti-human IL-7 MoAb for 12 days of culture (data not
shown).
IL-7 treatment generates large numbers of dendritic cells.
The expression of different cell markers was analyzed on
double-negative (DN) CD4CD8 cells raised
after 12 days of culture with IL-7, to better characterize this thymic
cell subset. As shown in Fig 2, the
proportion of DN cells expressing MHC class II, CD11b, and CD68
increased after culture with IL-7. Similarly, the MoAb OX-62, which
recognizes rat dendritic cells,34 reacted with the majority
of the DN cell subset from IL-7-treated FTOC, but only with a small
percentage of control DN cells (Fig 2). The frequency of
CD54+ DN cells also increased in the presence of IL-7. In
addition, most DN cells expressed CD44 in both control and
IL-7-treated lobes, although with a higher intensity after IL-7
administration, and the proportion of CD25+ DN cells was
three to four times higher in IL-7-treated FTOC than in control
cultures (Fig 2).
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| Fig 2.
Phenotype of CD4CD8 cells
raised after 12 days of culture in the absence (gray histograms) or
presence of IL-7 (black histograms). These
CD4CD8 cells were determined by gating on
negative fluorescence of thymic cells stained with PE-conjugated
anti-CD4 and anti-CD8. A minimum of 5,000 cells was analyzed for the
expression of MHC class II, CD11b, CD68, OX-62, CD54, CD44, and CD25.
Black lines represent the background fluorescence using isotype-matched
irrelevant FITC-conjugated MoAb, or omitting the particular MoAb under
study. The results are representative of a series of three independent
experiments.
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Accordingly, the DN cell subset seemed to include a large number of
nonlymphoid cells after 12 days of treatment with IL-7. A histological
and ultrastructural study of IL-7-treated and control FTOC was then
performed to confirm the nature of these cells. A peripheral band of
pale cells, morphologically identifiable as DC, was clearly present in
IL-7-treated lobes (Fig 3). In contrast, thymocytes largely
predominated in control FTOC (Fig 3). The
ultrastructural analysis confirmed the existence of a large number of
mature DC in IL-7-treated cultures. They were large, irregular,
electron-lucent elements, with lobulated nuclei and cytoplasmic
organelles arranged close to the nucleus (Fig
4A). Moreover, immature DC, which had shorter cell processes, a less irregular nucleus, and few membranous organelles scattered throughout the cytoplasm, were also observed in
these lobes (Fig 4B).
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| Fig 3.
Large numbers of DC develop in the presence of IL-7.
Thymic lobes were organ-cultured under control conditions (original
magnifications: A, 130×; B, 650×) or in the presence of IL-7
(original magnifications: C, 130×; D, 650×) for 12 days. Note the
peripheral band of pale cells in IL-7-treated lobes (C). These cells
mostly correspond to DC (asterisks; D). In contrast, thymocytes
(arrows) predominate in control lobes (B).
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| Fig 4.
Ultrastructure of DC. DC appear as large, irregular
elements showing a lobulated nucleus (N), and an electron-lucent
cytoplasm with few organelles (stars) arranged close to the nucleus.
Note the interdigitating cell processes in close contact (arrows) with the neighboring lymphocytes (L) (A; original magnification ×3,400). Immature DC exhibit shorter cell processes, a less irregular but usually indented nucleus, and membranous organelles scattered throughout the cytoplasm (B; original magnification ×7,900).
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T-cell stimulatory activity of DC generated in IL-7-treated FTOC.
To determine whether IL-7 produced functional DC, we evaluated the
allostimulatory capacity of TN cells generated in 12-day IL-7-treated
FTOC, using purified T cells as responder cells. As shown in Fig
5, these TN cells were found to be as
efficient as freshly isolated thymic DC at stimulating the
proliferation of allogeneic T cells. In contrast, TN cells from control
cultures gave a poor proliferative response, even when used at high
numbers, which agrees with the low incidence of DC among control TN
cells. This indicates that TN cell subset raised in IL-7-treated
cultures is not only composed of cells that appear morphologically like DC and express cell-surface markers characteristic of DC, but are also
efficient antigen-presenting cells.
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| Fig 5.
MLR-stimulatory capacity of TN cells generated in the
presence of IL-7. TN cells derived from 12-day IL-7-treated FTOC
(including 75% to 85% of OX-62+ cells;  ) and control
FTOC (including 15% to 25% of OX-62+ cells;  ) were
used at different numbers as stimulators for allogeneic T cells
(2 × 105) isolated from lymph nodes. Those were
compared with normal thymic DC isolated from rat thymus ( ). After 5 days the cultures were pulsed for 4 hours with BrdU. A specific kit was
used to measure BrdU incorporation into newly synthesized DNA. Full
details are given in Materials and Methods. Results are the means of
the pooled data from two to three independent experiments, each with
five cultures per point. Standard deviations represented less than 10%
of the mean values.
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Kinetics of DC production in IL-7-treated FTOC.
To know whether the increased DC numbers were due to a gradual process
occurring throughout the culture period, we comparatively analyzed the
frequency and morphology of MHC class II+ cells present in
both control and IL-7-treated FTOC after 7 and 12 days of culture. The
proportion of MHC class II-expressing cells had almost doubled after 1 week of culture in the presence of IL-7 (C: 2.7%; IL-7: 4.7%),
whereas a 10-fold increase was obtained at day 12 of culture (C: 1.4%;
IL-7: 14.7%). Furthermore, on both days 7 and 12, most MHC class
II+ cells (60% to 75%) exhibited a typical DC morphology,
while under control conditions around 70% of these MHC class
II+ cells corresponded to macrophages (data not shown). The
gradual production of DC throughout the culture period was clearly
evidenced by comparing the absolute numbers of DC in both culture
conditions (Fig 6A). After 7 days in culture, DC numbers increased
three times under control conditions, whereas a ninefold increment
occurred in the presence of IL-7. In the following days, DC production practically stopped in control FTOC, increasing 1.1 and 1.4 times between days 7-12 and 12-18, respectively, but new DC were generated with IL-7, which experimented a fivefold increase between
days 7-12 (Fig 6A).
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| Fig 6.
Kinetics of DC generation in the presence of IL-7. Cells
recovered from control and IL-7-treated FTOC were cytospun onto
slides, and stained with anti-MHC class II MoAbs. Percentages of MHC
class II+ cells exhibiting DC morphology were determined
at days 0, 7, 12, and 18. Five thousand cells were scored in 5 to 10 cytospin preparations. At each time point, the absolute numbers of
total DC (A) and their distinct subpopulations (B) were obtained by multiplying the total number of cells per lobe by the corresponding percent for each population. Values represent the mean ± SEM of two
to three independent experiments. Asterisks refer to the statistical significance differences between control and IL-7-treated FTOC: *P  .05, **P .01, ***P .001. ND, not
determined.
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Also supporting the continuous production of DC in IL-7-treated FTOC
were the changes undergone by the proportion of distinct DC subsets,
previously defined in rat thymus according to their morphology and MHC
class II expression.35 Briefly, type I DC were considered
as immature DC because they constituted the first DC subpopulation
appearing during thymus development, and because they showed short,
thin or bulbous-like cell processes, and a weak expression of MHC class
II antigens. Type II DC corresponded to elements exhibiting the
features of typically mature DC, including irregular shape and a strong
reactivity for MHC class II molecules, whereas among the type III DC,
which transiently appeared during thymus ontogeny, there were some very
irregular ones with long, thin cell processes, and a stronger
expression of MHC class II molecules than that found in mature type II
DC. The three DC subsets appeared in both control and IL-7-treated
lobes, but their relative proportions were substantially modified by
the presence of IL-7. As described in vivo,35 in control
cultures mature type II DC predominated (70% to 80%), although there
was an important proportion of type III DC (13%) in 7-day FTOC, which
decreased after 12 days of culture (2%). In contrast, in IL-7-treated
FTOC the frequency of immature, type I DC was always significantly
higher (C: 14% to 18%; IL-7: 35% to 45%). Accordingly, while the
absolute numbers of type I DC gradually decreased throughout the
culture period in control FTOC, a continuous increase was observed
after culture with IL-7 (Fig 6B). The cell numbers of types II and III
DC also increased in IL-7-treated cultures, whereas in control lobes
the numbers of type II DC remained unchanged and those of type III DC
even decreased (Fig 6B).
Cell-cycle analysis.
A cell-cycle analysis was performed using 7-AAD in combination with
anti-CD4 and anti-CD8 MoAbs to examine the proliferative status of
CD4CD8 cell subset. Comparison of the
percentage of cycling cells within DN cell subpopulation did not show
significant differences between control and IL-7-treated FTOC after 7 and 12 days of culture (Table 2). The
frequency of proliferating cells in the DN MHC class II+
cell subset did not change either (data not shown). Therefore, these
results suggest that DC in IL-7-treated cultures did not arise by
expansion of pre-existing cells.
IL-7 deprivation with neutralizing anti-IL-7 antibodies results in a
drastic reduction in the numbers of DC.
To determine whether in the absence of added IL-7 DC development
involves endogenous IL-7, we analyze the generation of DC after the
addition of a neutralizing anti-human/mouse IL-7 MoAb to mouse FTOC,
because this antibody did not neutralize rat IL-7. A maximal effect was
obtained with an MoAb concentration of 75 µg/mL (data not shown).
After a 12-day culture period in the presence of the antibody the total
cell recovery from thymic lobes was 66% reduced as compared with the
control cultures (Table 3), whereas a
higher reduction (78%) in DC numbers was observed (Table 3).
Therefore, these results indicate that DC development is highly
dependent of endogenously produced IL-7.
IL-7-induced DC generation in the absence of maturing thymocytes.
As previously shown in Fig 1, IL-7 treatment increased the numbers of
mature CD8+ thymocytes, as a consequence of the ability of
IL-7 to promote the differentiation of TcR cells, selectively to
the CD8 cell lineage.36 It has also been demonstrated that
the treatment of mature T cells with IL-7 upregulates the expression
and secretion of GM-CSF and IL-4,37,38 two cytokines that
have been involved in the maturation pathway of DC.4-13 To
avoid the possibility that accumulating mature thymocytes in
IL-7-treated FTOC could be influencing the development of DC, fetal
thymus lobes derived from SCID mice were organ-cultured in the presence
of IL-7 for 12 days. Treatment with IL-7 resulted in a threefold
increase in the total number of cells
(Table 4), but did not promote T-cell maturation, development being arrested at the
CD4CD8 stage in both control and
IL-7-treated FTOC (data not shown). In contrast, IL-7 administration
increased the proportion of DC, the number of which increased ninefold
(Table 4).
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DISCUSSION |
Several reports have shown that the in vitro differentiation of the
so-called myeloid-derived DC,1-3 generated from peripheral blood or BM CD34+ hematopoietic progenitors, or from blood
monocytes, is induced principally by GM-CSF, usually in combination
with other cytokines including TNF-, IL-4, and SCF.4-13
On the contrary, the named lymphoid-related DC, coming from the
earliest intrathymic precursor cell subsets
CD44+CD25 and
CD44+CD25+,16,17 develop in the
absence of GM-CSF.19 In addition, it has been recently
shown that there are no important variations in the number of DC in
spleen and thymus from GM-CSF overproducing transgenic
mice,15 or after in vivo administration of GM-CSF or GM-CSF
plus IL-4.14 Likewise, mice lacking expression of GM-CSF
receptor and GM-CSF-null mice do not have decreased numbers of
DC.15 These results indicate that GM-CSF does not appear to
be a major growth factor for the differentiation of the known DC
subsets and, therefore, other cytokines would be involved in the
process. In this regard, it has been said that transforming growth
factor 1 is essential for the development of epidermal Langerhans
cells,39,40 and that the injection of Flt3 ligand into mice
results in increased numbers of DC in several organs.14
We show here that IL-7 plays an important role in the development of
thymic dendritic cells. Addition of IL-7 to FTOC results in a drastic
numerical increase in cells expressing characteristic DC markers and
exhibiting typical morphological and ultrastructural features of DC, as
well as the capacity to stimulate the proliferation of allo-reactive T
cells.
The phenotype of DC developing in the presence of IL-7 is similar to
that of freshly isolated rat thymic DC, including the lack of CD4 and
CD8 surface expression.32,35,41 In contrast, DC from the
human thymus express CD4, whereas mouse thymic DC predominantly express
CD8.42 The relevance of CD4 and CD8 expression for the
biology of DC is still unknown, although the distinct expression in
different species argues against an important and specific role for
these T-cell markers in DC function. In agreement, a recent study with
CD8-null mice has shown that neither the development nor the
functionality of DC is affected by the lack of CD8
molecule.43
On the other hand, increased numbers of both immature and mature DC are
continuously generated in the presence of IL-7, whereas in control
cultures the numbers of immature DC progressively decrease, and those
of mature DC remain practically unchanged. Because no differences are
found in the proportion of proliferating cells in this thymic cell
subset, these results indicate that the higher DC production is the
result of a continuous and enhanced DC differentiation throughout the
culture period, rather than the product of the IL-7-induced
proliferation of the pre-existing DC population. Furthermore,
endogenous IL-7 deprivation with a neutralizing anti-IL-7 MoAb results
in a drastic inhibition of DC formation. In agreement with our
findings, Márquez et al18 reported that human
intrathymic CD34+ precursors cultured in the presence of
IL-7 were able to develop simultaneously into both T and non-T (DC and
monocytes)-lineage cells, while Saunders et al19 described
that murine CD4low thymic precursors proliferated and
differentiated to DC, but not to T-lineage cells, after a 4-day culture
period with a mixture of five to seven cytokines including IL-7, the
omission of which had a marked effect on DC generation. Likewise, it
has been described that the differentiation of DC from a
human BM cell precursor population in liquid cultures supplemented with
a cocktail of nine cytokines, including IL-7.44 The
addition of IL-7 to mouse FTOC has also been reported to result in the
accumulation of TN cells.45 However, these cells were
identified as early T-cell precursors on the basis of the expression of
CD44, CD25, and Sca-1, cell markers that have been repeatedly reported
on DC.18,32,46,47 Although no morphological study was
performed, it is highly likely that these cells were DC, as is borne
out by our results. Increased numbers of DN/TN thymic cells are also
seen in IL-7-transgenic mice48 and after hematopoietic
reconstitution with BM infected with IL-7-producing
retrovirus,49 but the real nature of those cells was not
investigated.
The development of large numbers of DC in IL-7-treated FTOC
established from SCID mice shows that IL-7-induced DC maturation does
not depend on growth factors produced by maturing thymocytes in
response to IL-7. An indirect effect through other thymic cell components can be also discarded because thymic epithelial cells do not
express IL-7 receptors,50 and because IL-7 concentrations required to stimulate the secretion of cytokines (including IL-1, IL-1, IL-6, and TNF-) from monocytes are higher than those used in our study.51 To our knowledge there is no information
available related to a possible IL-7-induced cytokine secretion from
thymic progenitor cells. Therefore, we favor a direct role for IL-7 in DC development, which is also supported by the evidence that the highest levels of IL-7 receptor expression are found in the
CD44+CD25 and
CD44+CD25+ murine progenitor
populations,52 which constitute the intrathymic precursor
cell subsets able to give rise to both thymic DC and to the thymic
T-lineages.17 Moreover, the expression of IL-7 receptor on
DC has been recently shown.53 Other factors, such as IL-1
and TNF-, have been pointed out to be essential for the in vitro
generation of DC from intrathymic precursor cells.19 However, the highest proportion of thymic DC is found very early in
ontogeny (R.S., unpublished results, May 1995), when the
expression of IL-7 is maximal,54 and that of IL-1 or
TNF- undetectable in the thymus.55-57
Therefore, we can conclude that IL-7 is another cytokine to incorporate
in the ever-increasing list of growth factors that affect the
development of DC.
| |
FOOTNOTES |
Submitted August 19, 1997;
accepted February 24, 1998.
Supported in part by CAYCIT Grants No. PB91-0374 and PB94-0332 from the
Spanish Ministry of Education and Culture. R.S. is a fellow of the
Spanish Ministry of Education and Culture.
Address reprint requests to Agustín G. Zapata, PhD, Department
of Cell Biology, Faculty of Biology, Complutense University, 28040 Madrid, Spain.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract