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Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 745-755
RAPID COMMUNICATION
Neutralization of Tumor Necrosis Factor Activity Shortly After the
Onset of Dendritic Cell Hematopoiesis Reveals a Novel Mechanism
for the Selective Expansion of the CD14-Dependent Dendritic Cell
Pathway
By
Frances Santiago-Schwarz,
Marguerite McCarthy,
John Tucci, and
Steven E. Carsons
From the Division of Rheumatology, Allergy and Immunology, Winthrop
University Hospital, Mineola, NY; and the Department of Medicine, State
University of New York at Stony Brook.
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ABSTRACT |
The CD14-dependent and -independent dendritic cell (DC) pathways are
instituted simultaneously when CD34+ progenitor cells are
treated with granulocyte-macrophage colony-stimulating factor
(GM-CSF)/tumor necrosis factor (TNF) ± stem cell factor (SCF) (GTS).
If TNF activity is neutralized within 48 hours of cytokine exposure, DC
development is halted and myelogranulocytic hematopoiesis takes place.
In this study, we show that disruption of TNF activity at a later time
point produced a distinct alteration within the DC system. Instead of
downregulating DC development, treatment of GTS cultures with
antibodies to TNF (anti-TNF) on day 3 provoked the selective expansion
of the CD14-dependent (monocyte) DC pathway from progenitor cell
populations lacking CD14 and CD1a. After an initial decrease in
proliferation, anti-TNF produced a rebound in cell growth that yielded
intermediate myeloid progenitors exhibiting CD14-dependent DC
differentiation potential and CD14+CD1a+ DC
precursors. Cultures enriched in CD14-dependent DCs were more potent
stimulators of a mixed leukocyte reaction, compared with control
GTS cultures containing both types of DCs. The intermediate progenitors
expanded in the presence of anti-TNF were
CD115+CD33+DR+, long-lived,
and displayed clonogenic potential in methylcellulose. When
exposed to the appropriate cytokine combinations, these cells yielded
granulocytes, monocytes, and CD14-dependent DCs. Antigen-presenting function was acquired only when DC maturation was induced from these
myelodendritic progenitors with GM-CSF + interleukin-4 or GTS. These studies show a novel mechanism by which TNF regulates the DC
system, as well as providing a strategy for the amplification of the
CD14-dependent DC pathway from immature progenitors. Although TNF is
required to ensure the institution of DC hematopoiesis from
CD34+ progenitor cells, its activity on a later
progenitor appears to limit the development of CD14-dependent DCs.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
DENDRITIC CELLS (DCs) are
antigen-presenting cells (APC) that are functionally characterized by
their exceptional capacity to stimulate naive T-cell responses. They
initiate both TH1- and TH2-driven immune responses, as well as
CD8+ T-cell responses.1 Recent advances have
shown that DCs do not represent a single cell type and that distinct
hematopoietic lineages may give rise to DC subtypes.1-6 In
humans, cells with DC phenotype and function may develop from lymphoid
progenitors that also have the potential to develop into T and natural
killer (NK) cells.1,6-9 These lymphoid progenitors may home
to the thymus and lack myeloid cell markers.6,8 Their
development into mature, lymphoid-derived DCs appears to be
interleukin-3 (IL-3) dependent and granulocyte-macrophage
colony-stimulating factor (GM-CSF) independent.6,9
Studies with multipotent CD34+ progenitor cells and
peripheral blood mononuclear cells describe two other DC subtypes that are associated with the myeloid lineage.1-6,10 One DC
subtype is represented by a CD14+CD1a+
intermediate, while the other stems from a
CD14 precursor.2-6,10 Both
CD14-dependent and -independent pathways produce mature DCs exhibiting
the capacity to stimulate naive T cells in the mixed leukocyte reaction
(MLR) and expressing high levels of class II major histocompatability
complex (MHC) antigens and costimulatory molecules required to deliver
secondary signals for T-cell activation. Immature cells of the
CD14-dependent DC pathway exhibit monocyte (mono)-associated markers
such as nonspecific esterase (NSE) activity, complement receptors
(CD11b), M-CSF receptors (CD115), and phagocytic potential, whereas
cells of the CD14-independent DC pathway exhibit low or absent levels
of these markers.4,10 It has been suggested that
CD14-dependent DCs correspond to interstitial and/or
circulating blood DCs and that CD1a+-derived DCs are
related to epidermal DCs (Langerhans cells).4,5
Both CD14-dependent and -independent DC pathways develop from
CD34+ hematopoietic progenitors when these cells are
treated with GM-CSF/tumor necrosis factor (TNF) ± stem cell factor
(SCF).4,5,10 Thus far, the earliest identifiable precursors
for CD14-dependent DCs are nondividing
CD14+CD1a+cells. For the CD1a+ DC
pathway, the earliest precursors are CD14
CD1a+, which are also nonproliferating.
Members of the TNF superfamily are critical in controlling DC
differentiation and survival. The presence of TNF during the earliest
phases of DC maturation is an absolute requirement for the in vitro
generation of DC hematopoiesis from multipotent CD34+
progenitors.11,12 The addition of TNF to CD34+
progenitors must occur within the first 48 hours of culture in order
for DC development to proceed.11,12 If anti-TNF antibodies are added to GM-CSF/TNF/SCF (GTS)-treated CD34+ progenitor
cell cultures during this period, DC hematopoiesis is blocked and the
granulocyte pathway is favored.11 The mechanism of action
involving TNF is emerging as a complex balance between its positive and
negative regulatory functions. TNF induces the upregulation of GM-CSF
receptors on DC intermediates, inhibits the granulocytic pathway, and
induces specific apoptotic events.11-13 More recently, it
has been shown that CD40 ligand, a member of the TNF receptor family,
also drives a subset of CD34+ progenitors to mature into
CD1a DCs.14 Because both TNF and CD40
ligand result in NF- B activation, there may be common mediators
involving TNF/CD40L signaling of DC differentiation. RANK/TRANCE-R are
novel members of the TNF receptor family that mediate the survival of
mature DCs by preventing apoptosis.15,16 RelB, a member of
the NF-B/Rel family of transcription factors, has also been shown to
be present selectively on mature DCs.17 This factor is
increased upon DC maturation in the CD14+ pathway, and
activates genes required for APC function when translocated to the
nucleus.17
In this study we show that, despite the absolute requirement for TNF
during the earliest phases of DC hematopoiesis from CD34+
progenitors, neutralization of TNF activity at a later time point produces a distinct deviation within the myeloid-associated DC system.
Instead of downregulating the DC pathway and favoring granulocyte
development, anti-TNF treatment provoked the selective expansion of the
CD14-dependent DC pathway from
CD14CD1a progenitor cells present
in GTS-treated cultures. After an initial decrease in cell
proliferation, a rebound in cell growth yielded the expansion of
CD14+CD1a+ DC precursors and the persistence of
myelodendritic progenitors expressing CD115, CD33, DR (class II MHC
proteins), and colony-forming unit (CFU) potential. The myelodendritic
progenitors exhibited trilineage potential and differentiated along the
granulocytic, monocytic, and mono-DC lineages when cultured with the
appropriate cytokine combinations. However, potent MLR stimulatory
activity was achieved only with cytokines known to produce mature DC
progeny.
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MATERIALS AND METHODS |
Enrichment of neonatal cord blood-derived progenitor cells.
Cord blood was collected from healthy full-term infants into sterile
heparinized containers during repeat caesarean sections, according to
institutional guidelines. Mononuclear cells (MNC) were prepared by
density centrifugation on Lymphoprep gradients (endotoxin poor;
Nycomed, Oslo, Norway) and placed on nylon wool columns
for the isolation of nonadherent cells, as previously described by
us.11,18,19 Separation of CD34+ progenitor
cells from the nonadherent population employed positive immunoselection
using immunomagnetic beads (Dynabeads; Dynal Inc, Oslo,
Norway) and yielded cell populations that were greater
than 85% CD34+, as described
elsewhere.11,18,19
Liquid cultures.
CD34+ cells were adjusted to 0.4 × 105
cells/mL in RPMI 1640 medium (GIBCO-BRL, Grand Island, NY) containing 2 mmol/L L-glutamine, 10 mmol/L HEPES, 50 IU/mL penicillin, 50 µg/mL
streptomycin, 5% pooled normal human serum (NHS/RPMI), and incubated
in Teflon vials (Scientific Specialties Service, Randalstown, MD)
at 37°C in a 5% CO2 humidified incubator. For
induction of DC hematopoiesis, cultures were supplemented with GTS at
the onset of the culture period.19 DC growth peaks around
day 10-11 under these conditions, as previously
described.11,13,18,19
Recombinant human (rh) SCF (Genzyme, Boston, MA) was used
at 50 ng/mL, human rTNF- (Knoll Pharmaceuticals, Whippany, NJ) at
500 U/mL, human rGM-CSF (Genzyme) at 100 U/mL, human rIL-4 (Genzyme) at
200 U/mL, human rM-CSF (Genzyme) at 50 ng/mL, and human rG-CSF
(GIBCO-BRL) at 500 U/mL. The optimal concentration for each factor was
calculated after dose analysis in prior studies.11,13,18,19 The cytokine combinations used do not support the development of
lymphocytes or erythrocytes.12,18,20 For in situ analyses and examination under phase microscopy, cultures were also established in either 24-well plates or Lab-Tek chamber slides (Nunc, Naperville, IL). Because of the profound effects of endotoxin on cell development and function,2,21-23 cultures were maintained under strict
endotoxin-poor conditions, as previously described by us.18
On day 3, GTS cultures were split and a portion of the cells were
treated with 15 µg/mL of rabbit polyclonal anti-TNF (Genzyme) or
nonimmune rabbit Ig (Vector). Thereafter, the cultures were supplemented with fresh NHS/RPMI media without exogenous cytokines, on
a weekly basis. As indicated, untreated and anti-TNF-treated GTS
cultures received M-CSF, GM-CSF + G-CSF, GTS, or GM-CSF + IL-4.
Proliferation.
Proliferative events were measured by the uptake of [3H]
thymidine and manual hemacytometer-assisted cell counts (Improved Neubauer, Fisher Scientific, Pittsburgh, PA). For
thymidine uptake, 0.5 µCi of [3H] thymidine (specific
activity, 25 Ci/mmol; Amersham, Arlington Heights, IL) was added to
100-µL aliquots taken from Teflon cultures and placed in 96-well
microtiter plates. After 5 hours of incubation, cells were obtained
using an automated sample harvester and counted in a liquid
scintillation counter. Results are expressed as the mean of triplicate
samples, and the SE was  20% in all experiments.
Immunofluorescence analysis.
Antibodies to DR, CD11b, CD13, CD33, CD34, CD38, and CD80 were obtained
from Becton Dickinson (San Jose, CA). Anti-CD1a was obtained from
Biosource (Camarillo, CA), anti-CD4 from the American Type Culture
Collection (Rockville, MD), anti-CD14 from Sigma (St Louis, MO),
anti-CD83 from Immunotech (Marseille, France), anti-CD86 from
Pharmingen (San Diego, CA), and anti-CD115 from Serotec (Oxford, UK).
In indirect assays, reactivity was detected with either fluorescein
(FITC)- or phycoerythrin (PE)-linked anti-mouse Ig or anti-rabbit Ig
(Fab )2 fragments (Boehringer Manneheim Corp, Indianapolis, IN; Cappel, Westchester, PA; Jackson Immunoresearch, West
Grove, PA). Matched nonimmune mouse or rabbit Ig isotypes were used as
negative controls (Becton Dickinson; Coulter Immunology, Hialeah, FL).
Cells were analyzed on a FACScan flow cytometer (Becton Dickinson)
calibrated with Calbrite beads (Becton Dickinson) for FITC and PE. The
distribution of debris, dead cells, and any contaminating red blood
cells was assessed on the basis of forward and right-angle scatter
before proceeding with the analysis. A total of 10,000 events were
examined using a 488-nm wavelength excitation. Acquired events were
analyzed using Cell Quest Software (Becton Dickinson). Results are
expressed as percent positive cells after subtracting negative control
values.
Allogeneic mixed leukocyte reaction.
Cells grown under various conditions were removed from Teflon cultures,
centrifuged twice in RPMI, adjusted to equal concentrations in 5%
NHS/RPMI, and irradiated with a 60Co source (for a total of
2,000 rads). Varying numbers of these stimulator cells were then added
to 96-well microtiter plates containing 5 × 104
responder cells/well (nylon wool enriched T-cell populations obtained
from normal peripheral blood). A proliferative response was measured
after 7 days by adding 0.5 µCi of [3H] thymidine to
each well and harvesting the cells as described above. Thymidine uptake
in control cultures containing irradiated stimulator cells or responder
cells cultured alone was less than 200 cpm.
Colony-forming assays.
Cells from untreated GTS cultures and GTS cultures receiving anti-TNF
on day 3 were removed from culture on the day indicated and seeded in
duplicate in 24-well plates at 2 × 104 cells/mL.
Colony growth was assessed in semisolid cultures containing: RPMI 1640 medium (GIBCO), 2 mmol/L L-glutamine, 10 mmol/L HEPES, 50 IU/mL
penicillin, 50 µg/mL streptomycin, 5% pooled NHS, 15% fetal bovine
serum, 1% StemPro methylcellulose (GIBCO), and cytokine combinations,
as specifically indicated.
Cytochemistry.
Cells cultured in suspension (Teflon) were prepared for Wright stain
(Hemacolor; EM Diagnostic Systems, Gibbstown, NJ) and NSE
analysis by depositing them onto slides by means of cytocentrifugation (Shandon, Pittsburgh, PA). No fewer than 500 cells were analyzed.
Statistics.
Where indicated, Student's t-test was used to analyze data
using Sigma Stat (Jandel Scientific, San Rafael, CA).
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RESULTS |
Phenotypic characteristics of GTS cultures on day 3.
In a previous study, we showed that by day 3, progenitor cell cultures
treated with GTS contain CD33+CD13+ cells that
undergo subsequent expansion into the DC pathway.13 In
Fig 1 we provide an extended cell-surface
profile of the day 3 cultures. Cells were positive for CD33, CD13,
and DR, and negative or mostly negative for molecules associated
with later stages of DC development, including CD86, CD80,
CD14, CD1a, CD83, CD4, and CD11b. The anti-TNF-treated GTS cells on
day 3 exhibited decreased levels of CD34 (10%), did not exhibit NSE
activity, and were nonphagocytic (data not shown).
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| Fig 1.
Flow-assisted characterization of GTS cultures on day 3. Each histogram depicts a representative experiment (N = 5 to 8 for each marker). The numbers inside the panels depict positive reactivity for the experiment shown, after assessing negative controls.
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Anti-TNF treatment on day 3 alters the proliferation pattern associated
with DC development in GTS cultures. Thymidine uptake confirmed that,
as previously described, proliferation in GTS cultures is marked by day
5, peaks on day 7, and declines sharply after day 10 (Fig 2A).19 Anti-TNF treatment
of GTS cultures on day 3 produced decreases in proliferation that were
evident on day 5. The decreases between the two cultures were
significant (P = .0012) by day 7, with anti-TNF treatment
reducing proliferative potential by about 60%. Notwithstanding, after
day 7, the proliferative capacity of the anti-TNF-treated GTS cultures
remained stable whereas that of the GTS cultures decreased sharply. By
day 10, proliferation in the two groups was virtually identical
(P = .760).
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| Fig 2.
Anti-TNF treatment on day 3 alters the growth pattern
associated with DC development in GTS cultures. GTS and
anti-TNF-treated GTS cultures were compared in parallel. Results
represent the mean of multiple experiments. (A) Proliferation was
measured by thymidine uptake, N = 3 to 8 for all days. (B) Cell
content was assessed by hemacytometer-assisted cell counts, N = 7 to
16 for all days. The addition of nonimmune rabbit IgG to GTS cultures on day 3 did not alter the growth pattern.
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A kinetic analysis of cell content (Fig 2B) showed significant
decreases in the absolute number of cells present in the
anti-TNF-treated GTS cultures on days 5, 7, and 10, compared with the
untreated GTS cultures (P < .006 for all days). Despite this
effect, cell content steadily increased in the anti-TNF-treated GTS
cultures to the point that on day 13 differences between the control
GTS cultures and the anti-TNF treated GTS cultures were no longer significant (P = .4). On day 18, in fact, the anti-TNF-treated GTS cultures contained a significantly higher (P = .005) number of cells than the GTS cultures. Thymidine uptake showed the persistence of proliferative events (>3,500 cpm) in the anti-TNF GTS cultures on
days 13 and 18 and almost no proliferation in the untreated GTS
cultures (<300 cpm, P .03, Fig 2A). These results imply that progenitor/precursor cells with proliferative capacity endure in
the anti-TNF-treated GTS cultures.
Anti-TNF treatment alters the phenotypic characteristics of progeny
on day 10.
We previously noted that the period between days 3 and 7 in GTS
cultures marks a critical time for DC development, as judged by the
tremendous expansion (>80-fold) of
DR+CD13+CD33+ DC intermediates and
the distribution of CD14 and CD1a antigens.13 Later
assessment on day 10 (peak DC development) showed that further maturation of DCs occurred, as revealed by reactivity with antibodies to DR, CD86, CD1a, CD80, and DR. In agreement with prior reports, we
observed the presence of both CD14+CD1a+ and
CD1a+ DC intermediates before peak DC development on day
10.4,5,10 The percentage of
CD14+CD1a+ cells did not exceed 10%,
supporting the implication that the CD14+-dependent DC
pathway exists as a secondary DC pathway when hematopoiesis is
instituted from CD34+ progenitors with GTS.10
The data depicted in Fig 3 show that the
cellular rebound observed in anti-TNF-treated GTS cultures produced a
distinctive cell-surface profile that is associated with the
development of the CD14+-dependent DC
pathway.4,5,10 On day 10, the percentage of cells
coexpressing CD14 and CD1a was increased sevenfold versus GTS cultures
(P = .0007, N = 9). Anti-TNF also decreased the total percentage of CD14+ cells and the fluorescence intensity of
CD14, while increasing the percentage of CD1a+ cells
(approximately threefold, P = .0001, N = 9). These
changes are similar to those previously reported to accompany the
development of mono-derived DCs from CD14+ precursors
isolated from either peripheral blood or cord blood GTS
cultures.1-3 However, because CD14+ cells were
less than 0.5% at the time we added anti-TNF (day 3, Fig 1), our
results signify that the amplification of the CD14-dependent DC pathway
must stem from an earlier, CD14 myeloid progenitor.
Additional evidence in favor of the expansion of the CD14-dependent DC
pathway in the anti-TNF-treated GTS cultures includes increases in
cells which coexpress CD11b and CD86 (approximately fourfold, P = .02, N = 3) and NSE (Table
1).4,5,10 Interestingly, despite the increases in markers
denoting the CD14-dependent DC pathway, anti-TNF treatment did not
significantly alter the distribution of
CD13+CD33+ or CD83+ cells.
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| Fig 3.
Flow-assisted characterization of anti-TNF-treated GTS
cultures on day 10 revealed the expansion of the CD14-dependent DC pathway. A representative experiment for each group is shown. For CD14
versus CD1a, N = 9, for all other combinations, N  3. The numbers
in the upper right quadrants depict percent positive cells for the
experiment shown, after subtracting negative controls. With the
exception of the CD80 versus CD1a combination, all comparisons yielded
statistically significant differences (P .03). The addition of nonimmune rabbit IgG to control GTS cultures did not alter the
distribution of any of the markers.
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Anti-TNF treatment of GTS cultures on day 3 produces distinct progeny
in methylcellulose and liquid cultures.
CD34+cells treated with GTS yielded numerous typical DC
colonies in methylcellulose after  12 days, as expected
(Fig 4a).19 GTS cultures
treated with anti-TNF also yielded numerous CFUs. However, most of the
colonies consisted of small round cells (Fig 4b) and few, if any,
typical DC colonies were observed. In comparison to liquid GTS cultures
which contained many adherent DC clusters (Fig 4c), the liquid
anti-TNF-treated GTS cultures contained predominantly nonadherent
cells around day 10 (Fig 4d). Examination of these nonadherent cells
under higher magnification showed that they were either small round
cells or veiled cells.
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| Fig 4.
Anti-TNF-treated GTS cultures produce distinct progeny
in methylcellulose and liquid cultures, compared with GTS cultures. GTS
cultures yielded typical DC colonies around day 12 in methylcellulose (a) and adherent DC clusters in liquid cultures (c). In
methylcellulose, anti-TNF-treated GTS cultures produced large CFUs
containing small round cells and lacking typical DCs (b). The liquid
anti-TNF-treated cultures (d) contained predominantly nonadherent
cells. Original magnification × 20.
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The increased APC functions of anti-TNF-treated GTS cultures
correlate with increases in DR and costimulatory molecules.
The coexpression of class II MHC antigens (DR) and costimulatory
molecules (CD80/CD86) with CD1a was greater in the anti-TNF-treated GTS cultures, compared with control GTS cultures by at least twofold on
day 10. In Fig 5, we investigated the
functional outcome of these differences using the allogeneic MLR.
T-cell activation was increased when stimulators were derived from the
anti-TNF-treated GTS cultures. In wells containing only one stimulator
cell per 200 responder cells (.015:1), anti-TNF-treated GTS cultures
produced T-cell responses that were at least three times greater than
those produced by control GTS cultures. Thus, there was a positive
correlation between the increased expression of class II MHC
molecules/costimulatory molecules with CD1a and the increased MLR
stimulatory capacity of the anti-TNF-treated GTS cultures.
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| Fig 5.
Anti-TNF-treated GTS cultures exhibit increased MLR
capacity on day 10, when compared with untreated GTS cultures. Results represent the mean ± the SE of triplicate samples. One of three representative experiments is shown.
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Anti-TNF-treated GTS cultures, but not control GTS cultures, contain
myeloid progenitors with CFU potential on day 10.
The results shown in Fig 3 demonstrate that in addition to promoting
increases in precursors associated with the CD14-dependent DC pathway,
anti-TNF treatment of GTS cultures produced approximately a threefold
increase in CD33+CD115+ cells on day 10 (P = .008, N = 4). CD115, the receptor for M-CSF, is
distributed throughout the myelomonocytic lineage on cells both lacking
and exhibiting proliferative and CFU potential.24-27 CD115
may also exist on nonproliferating mono-DC precursors, but is promptly
lost during the DC maturation process.27-29
Because of the persistence of late proliferative events and the pattern
of CD115 reactivity, we speculated that myeloid progenitors with CFU
potential were sustained in the anti-TNF-treated GTS cultures on day
10. Figure 6 depicts the CFU capacity of
cells that were removed from the anti-TNF-treated GTS cultures on day 10 and recultured with cytokines promoting myeloid colony formation (GM-CSF + SCF) in methlycellulose. Two principal types of CFUs developed under these conditions. One resembled a mono-CFU (Fig 6a),
the other a granulocytic colony (Fig 6b). NSE and Wright stain analysis
of cells obtained from these methylcellulose cultures confirmed the
presence of both monos and granulocytes (Fig 6c and d). Cells obtained
from control GTS cultures on day 10 had lost their capacity to yield
CFUs in methylcellulose, which is consistent with their low
proliferative potential in liquid cultures (Fig 2A).
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| Fig 6.
Anti-TNF-treated GTS cultures contain myeloid
progenitors with CFU potential on day 10. Cells removed from
anti-TNF-treated GTS cultures on day 10 and placed in methylcellulose
with GM-CSF+SCF yielded colonies resembling mono CFU (a) and
granulocyte CFU (b). Nonspecific esterase (c) and Wright stain (d)
analysis confirmed the presence of both mono-macrophages and
granulocytes. Original magnification for (a), (b), and (d) = 40×,
for (c) = 60×. Cells removed from untreated GTS cultures on day 10 and cultured in parallel failed to yield CFU.
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To further characterize the progenitors present on day 10, developmental potential was also tested in liquid cultures in the presence of lineage-restricted myeloid cytokines. M-CSF treatment of
cells removed from anti-TNF-treated GTS cultures produced greater than
90% mono-macrophages, whereas GM-CSF + G-CSF treatment yielded predominantly granulocytes. Marked proliferation accompanied the differentiation process with both cytokine treatments (data not shown).
The addition of M-CSF or GM-CSF + G-CSF to control GTS cultures failed
to produce any mono/granulocyte progeny or proliferative events. Thus,
on day 10, myeloid progenitors cells that are responsive to both
lineage- and nonlineage-restricted myeloid cytokines persist in the
anti-TNF-treated GTS cultures, but not in the control GTS cultures.
Institution of the mono-DC pathway from progenitors present on day
10.
Because of the switch to the mono-DC pathway in the anti-TNF-treated
GTS cultures, we investigated whether the myeloid progenitors present
on day 10 also exhibited DC developmental potential. In these
experiments cells were removed from anti-TNF-treated GTS cultures on
day 10 and recultured with a combination of DC-restricted cytokines
(GM-CSF + IL-4 + SCF or GTS). Expansion into the CD14-dependent DC
pathway occurred with this treatment, as determined by morphological, cell surface, and functional characterization. A distinct cellular progression was noted, as shown in Fig 7. Three to five days after the
addition of GTS or GM-CSF + IL-4 + SCF (days 13-15), twofold to
threefold increases in CD14+CD1a+ DC precursors
were observed, when compared with the parent anti-TNF GTS cultures.
Upon further differentiation on days 17-18, CD1a expression and CD14
CD1a coexpression decreased dramatically, suggesting the
differentiation of the CD14+ CD1a+ and
CD1a+ cells into mature DCs. As early as day 13 there was a
dramatic decrease in CD14 single expression with GM-CSF + IL-4 + SCF
treatment, further indicating the development of CD14-dependent DC
pathway (data not shown).2,27-30 Morphological assessment
showed typical DC clusters and veiled cells in anti-TNF-treated GTS
cultures receiving cytokines inducing DC development between days 15-18 (data not shown). In contrast, DC-associated differentiation events were rare when anti-TNF-treated GTS cultures were untreated or when
anti-TNF-treated GTS cells were removed from culture and exposed to
NHS/RPMI only (data not shown).
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| Fig 7.
Institution of the CD14-dependent DC pathway from
progenitors present on day 10 in anti-TNF-treated GTS cultures. ( ),
Anti-TNF-treated GTS cultures; ( ), anti-TNF-treated GTS cells
exposed to GTS on day 10; ( ), anti-TNF-treated GTS cells exposed to
GM-CSF + SCF + IL-4 on day 10. Results represent one of two similar
experiments.
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Assessment of T-cell stimulatory activity revealed the development of
potent APC functions consistent with DC development, as shown in Fig
8A. Only anti-TNF-treated GTS cells that had been subjected to the
differentiating effects of DC-restricted cytokines on day 10 induced a
potent MLR on day 18; untreated anti-TNF GTS cells were weak
stimulators of a MLR on day 18, as were anti-TNF GTS cells
treated with either M-CSF or G-CSF. Control GTS cultures also exhibited
poor MLR potential on day 18, consistent with the loss of
DCs.13
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| Fig 8.
Potent MLR stimulatory function is achieved only
after mature DCs develop from myelodendritic progenitors present in the
anti-TNF-treated GTS cultures. (A) On day 18, cells that had been
treated with GM-CSF + SCF + IL-4 on day 10 produced potent T-cell
responses. Cells that were left untreated (GTS, anti-TNF on day 3),
control GTS cultures, or cells that were treated on day 10 with GM-CSF + G-CSF or M-CSF showed weak stimulatory potential on day 18. Results
represent one of three typical experiments. (B) Progenitor cells
exhibiting DC differential potential are preserved in anti-TNF-treated GTS cultures beyond day 18. Cells removed from anti-TNF-treated cultures on day 18 and exposed to GM-CSF + IL-4 acquired MLR
stimulatory capacity by day 26. Cells obtained from untreated anti-TNF
GTS cultures and control GTS cultures on day 26 produced weak T-cell responses, as did anti-TNF GTS cells exposed to GM-CSF + G-CSF or
M-CSF on day 18. Results represent one of two experiments.
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These data show that the progenitor cell population expanded by
anti-TNF treatment of GTS exhibits the capacity to yield three distinct
lineages, namely, monos, granulocytes, and CD14-dependent DCs.
As expected, only the CD14-dependent DCs exhibited potent APC
functions. The increased stimulatory capacity expresssed by these cells
always correlated with increased levels of cells coexpressing DR
and CD86 antigens (data not shown).
Induction of mature DCs from progenitors preserved in
anti-TNF-treated GTS cultures beyond day 18.
The advancement of the CD14-dependent DC pathway from the progenitors
present in the anti-TNF-treated GTS cultures on day 10 was strictly
regulated by DC cytokines. Notwithstanding, significant proliferation
was observed in the anti-TNF GTS cultures on days 13 and 18 (P .03; Fig 2), even in the absence of additional cytokine
supplementation. Although the distribution of
CD14+CD1a+ and
CD14CD1a+ DC precursors had decreased in
the anti-TNF-treated GTS cultures by day 18 (Fig 7), CD115/CD33
coexpression was still increased approximately seven
times in these cultures, compared with control GTS
cultures. These observations led to the speculation that the capability
to exhibit very late DC differentiating potential might be associated
with the long-term preservation of the myeloid progenitors. The data in
Figs 8B and 9 support this assumption.
Although cells derived from day 26 anti-TNF-treated GTS cultures were
incapable of stimulating an MLR, cells previously exposed to GM-CSF + IL-4 + SCF on day 18 demonstrated increased MLR capacity on day 26 (Fig
8B). Even later (day 35), treatment of cells obtained from anti-TNF-treated GTS cultures with GM-CSF + IL-4 or GTS produced CD14-dependent DCs, as revealed by the downregulation of CD115 and the
distribution of DR, CD1a, CD14, and CD86 antigens (Fig 9).
View larger version (47K):
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| Fig 9.
Cells present in anti-TNF-treated GTS cultures on day 35 developed into DCs when exposed to GTS or GM-CSF + SCF + IL-4, whereas untreated anti-TNF GTS cells remained in an
undifferentiated state. Phenotype analysis was performed on day 40. The
numbers in the quadrants depict percent positive cells, after
subtracting negative controls. Results are representative of four
separate experiments.
|
|
The equivalent of the anti-TNF-treated GTS progenitors could not be
shown in the control GTS cultures. This is not unexpected because after
day 18, viability is greatly decreased and greater than 40% of the
cells are undergoing apoptosis.13 By day 35, few viable
cells remained in the control GTS cultures. These were large,
long-lived macrophages, as revealed by Wright stain, NSE, and flow
analysis (data not shown). Although control GTS cells expressed DR
antigens on day 35, they were unresponsive to DC restricted cytokines.
| |
DISCUSSION |
In this study we reveal a new role for TNF in DC development and a
novel strategy for obtaining large numbers of CD14-dependent DCs from
immature myeloid progenitor cells. Our findings indicate that while TNF
is required to ensure the onset of DC hematopoiesis from
CD34+ progenitor cells, its effect on a later progenitor
serves to limit the development of the CD14-dependent DC pathway.
Three days after exposure of CD34+ progenitors to GTS,
cultures contain committed myeloid progenitors expressing CD13, CD33, and DR, as previously noted.13,30,31 If GTS cultures
progress without interruption, these committed myeloid progenitors
undergo tremendous expansion into both CD14-dependent and -independent DC pathways between days 3 and 7, as judged by the greater than 80-fold
increase of DR+CD13+CD33+ DC
intermediates and the distribution of CD14 and CD1a antigens during
this period.4,13 Although both pathways were induced, the
percentage of CD14+CD1a+ precursors did not
exceed 10%, implying that the CD14-dependent DC pathway exists as a
secondary DC pathway when hematopoiesis is instituted from
CD34+ progenitors with GTS.10
Treatment of GTS cultures with anti-TNF on day 3 promoted approximately
sevenfold increases in CD14+CD1a+
mono-DC precursors by day 10, indicating the selective amplification of
the CD14-dependent DC pathway. When compared with day 10 untreated GTS
cultures, the anti-TNF-treated GTS cultures exhibited a superior capacity to stimulate T cells, which correlated with increases in class
II MHC antigens (DR) and costimulatory molecules (CD86). Anti-TNF also
decreased the percentage of CD14+cells and the fluorescence
intensity of CD14, while increasing the percentage of CD1a+
cells. These changes are similar to those previously reported to
accompany the development of mono-derived DCs from CD14+
precursors isolated from either peripheral blood or cord blood GTS
cultures.2-5,10 However, because CD14+ cells
were nearly absent (<0.5%) at the time we added anti-TNF (day 3, Fig
1), our results indicate that the amplification of the CD14-dependent
DC pathway can be achieved from an earlier, CD14
progenitor.
Although distinct nonproliferating precursors for the CD14-dependent
and -independent DC pathways can be detected in GTS cultures on days
5-7, the inability to clearly identify an earlier progenitor that is
common to both DC pathways has led some investigators to speculate that
the CD14-independent and -dependent DC pathways stem from separate
CD34+ progenitors.1,32 We cannot discount the
existence of separate progenitors in this study. However, because both
DC pathways arose from day 3 cell populations that lacked CD14 and
CD1a, and CD13, CD33 expression was not altered by
anti-TNF, the possibility that common progenitors exist must also be
considered. Strategies for manipulating the selective
amplification of both DC pathways from putative common DC (CDC)
progenitors are currently being investigated in our laboratory.
In addition to expanding CD14+CD1a+ DC
precursors, the neutralization of TNF activity on day 3 produced a
rebound growth effect that was related to increases in
CD115+ CD33+ cells. Although CD115 may be
present on nonproliferating mono-DC and mono
precursors,27-29 the persistence of proliferation in the anti-TNF GTS cultures beyond day 10 suggested the preservation of an
immature CD115+ myeloid progenitor cell committed to the
granulomonocytic lineages.24-26 Assessment of
differentiation potential in methylcellulose and liquid cultures showed
that these progenitors in fact expressed multilineage potential. When
exposed to the appropriate cytokine combination, cells obtained from
anti-TNF GTS cultures on day 10 exhibited the capacity to yield monos,
granulocytes, and CD14-dependent DCs. Of the lineages produced, only
the CD14-dependent DCs exhibited the capacity to yield potent APC
function. This function was strictly dependent on the induction of
mature DCs from the myeloid progenitors with either GM-CSF + TNF + SCF
or GM-CSF + SCF + IL-4. Thus, anti-TNF supports the expansion of an
intermediate myeloid progenitor characterized by CD115, CD33, DR
positivity, and CD14-dependent DC differential potential. We refer to
these progenitors as "myelodendritic," so as to reflect their
trilineage potential.
Other investigators have recently described the distribution of DC
progenitors of myeloid origin in bone marrow and lymphoid tissue that
share several features with the myelodendritic progenitors we
describe.33 The progenitors studied by these investigators exhibited CD115 and DR antigens and did not require TNF to proliferate. When subjected to the appropriate cytokine treatment, these progenitors differentiated into monos, granulocytes, and mono-derived DCs.
The undifferentiated myelodendritic progenitors provoked by
neutralization of anti-TNF activity on day 3 were remarkably long-lived and persisted for weeks, even in the absence of additional cytokine supplementation. Although anti-TNF-treated GTS cultures contained few
DC precursors on day 18, as judged by the distribution of CD14 and CD1a
antigens and poor MLR stimulatory potential (Figs 7 and 8), significant
increases in CD115+CD33+ cells were still noted
in these cultures. When removed from culture on day 18 and exposed to
the appropriate cytokine treatment, these cells were still capable of
differentiating into monos, granulocytes, and CD14-dependent DCs.
Again, only cells treated with DC restricted cytokines on day 18 developed into mature DCs that showed MLR stimulatory activity on day
26. Even as late as day 35, anti-TNF-treated GTS cultures were still
capable of yielding DC progeny.
Although the mechanisms of TNF action are clearly complex, we have
developed a hypothesis to explain how TNF may be involved in regulating
the CD14-dependent and -independent DC pathways. Although TNF and
GM-CSF are both required for DC hematopoiesis to proceed from
CD34+ progenitors, we and others have determined that TNF
signaling must occur first.11,12 TNF rapidly upregulates
GM-CSF receptors on the developing progenitors, which in
turn optimizes responsiveness to GM-CSF.11 In light of the
fact that TNF also downregulates CD115 and GM-CSF upregulates
CD115,27,34 it is conceivable that the priority in TNF
signaling also preferentially yields progenitor cells lacking CD115.
Subsequent differentiation events would yield predominantly CD14 (mono)
independent DCs, such as those developing in GTS cultures. When TNF
activity is neutralized, GM-CSF signaling would dominate to yield
CD115+ progenitors exhibiting the capacity to produce
CD14-dependent DCs. The downregulation of CD115 would then be mediated
by TNF during the process of DC maturation, as previously
shown.27-29 In support of this hypothesis, we noted that
GTS cultures exhibited decreased expression of CD115+ on
day 7, as well as day 10, when compared with the anti-TNF-treated GTS
cultures (data not shown). Our previous correlations between TNF-mediated apoptosis and downregulation of the myelogranulocytic pathway during the critical day 3-7 period may reflect yet another consequence of CD115 inhibition during the development of the CD14-independent DC pathway.13
The addition of anti-TNF to GTS cultures on days 5 and day 10 produced
only a modest expansion of the CD14-dependent DC pathway, as shown by
increases in proliferation and the distribution of CD14+
CD1a+ DC precursors (data not shown). Therefore,
TNF-sensitive progenitors for the CD14-dependent DC pathway were still
present on these days, but as a minority population.
The recent realization that distinct DC subsets exist has prompted much
scientific and clinical interest. It has been suggested that DC
subtypes are restricted to activating either TH1 or TH2 subsets and
that local and systemic DC/T-cell abnormalities associated with
specific diseases such as psoriasis and atopic asthma may reflect the
predominance of a particular DC type.35,36 In line with our
current study, we have recently described that synovial fluid and serum
from patients with rheumatoid arthritis (RA) also yield the
preferential expansion of the CD14-dependent DC pathway from
CD34+ progenitors.37 These effects were
correlated with high levels of soluble TNF receptors, which are
biologically related to anti-TNF. The function of such naturally
occurring TNF antagonists in vivo would conceivably include
neutralization of TNF activity that is generated during the
inflammatory process in RA, including regulation of DC subtypes.
Further elucidation of such regulatory mechanisms would no doubt be
useful in the advancement of therapeutic TNF antagonists that are
currently in development for the treatment of RA and other inflammatory
diseases.
| |
FOOTNOTES |
Submitted February 24, 1998;
accepted May 12, 1998.
Address reprint requests to Frances Santiago-Schwarz, PhD, Division of
Rheumatology, Allergy and Immunology, 222 Station Plaza North Suite
430, Winthrop University Hospital, Mineola, NY 11501.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors gratefully acknowledge the cooperation of the staff in
Labor and Delivery, Department of Obstetrics and Gynecology at Winthrop
University Hospital, in the collection of cord blood, in particular
Joanne Pastore. We also thank Dr Donald Coppock for review of the
manuscript and the Department of Radiation Oncology at
Winthrop-University Hospital for assistance with cell irradiation.
| |
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Abstract
Introduction
Abstract