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Prepublished online as a Blood First Edition Paper on August 15, 2002; DOI 10.1182/blood-2002-04-1164.
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Blood, 1 January 2003, Vol. 101, No. 1, pp. 143-150
HEMATOPOIESIS
Interferon- switches monocyte differentiation from
dendritic cells to macrophages
Yves Delneste,
Peggy Charbonnier,
Nathalie Herbault,
Giovanni Magistrelli,
Gersende Caron,
Jean-Yves Bonnefoy, and
Pascale Jeannin
From the Department of Biology, Centre d'Immunologie
Pierre Fabre, Saint Julien en Genevois, France.
 |
Abstract |
Human monocytes differentiate into dendritic cells (DCs) or
macrophages according to the nature of environmental signals. Monocytes stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin 4 (IL-4) yield DCs. We tested here
whether interferon- (IFN-), a potent activator of macrophages, may modulate monocyte differentiation. Addition of IFN- to IL-4 plus
GM-CSF-stimulated monocytes switches their differentiation from DCs to
CD14 CD64+ macrophages. IFN- increases
macrophage colony-stimulating factor (M-CSF) and IL-6 production by
IL-4 plus GM-CSF-stimulated monocytes by acting at the transcriptional
level and acts together with IL-4 to up-regulate M-CSF but not IL-6
production. IFN- also increases M-CSF receptor internalization.
Results from neutralizing experiments show that both M-CSF and IL-6 are
involved in the ability of IFN- to skew monocyte differentiation
from DCs to macrophages. Finally, this effect of IFN- is limited to
early stages of differentiation. When added to immature DCs, IFN-
up-regulates IL-6 but not M-CSF production and does not convert them to
macrophages, even in the presence of exogenous M-CSF. In conclusion,
IFN- shifts monocyte differentiation to macrophages rather than DCs through autocrine M-CSF and IL-6 production. These data show that IFN- controls the differentiation of antigen-presenting cells and
thereby reveals a new mechanism by which IFN- orchestrates the
outcome of specific immune responses.
(Blood. 2003;101:143-150)
© 2003 by The American Society of Hematology.
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Introduction |
Peripheral blood monocytes can differentiate into
dendritic cells (DCs) or macrophages depending on environmental factors encountered during their migration from blood to peripheral
tissues.1-4 Transendothelial trafficking5 and
culture in the presence of serum from systemic lupus erythematosus
(through the presence of interferon  [IFN-]) induce monocyte
differentiation into immature DCs.6 On contact with
interleukin 4 (IL-4) plus granulocyte-macrophage colony-stimulating
factor (GM-CSF; cytokines that could be produced by tissue mast cells),
monocytes also differentiate into immature DCs.2-4
Addition of transforming growth factor  (TGF-) or exposition of
monocytes to GM-CSF plus IL-15 led to DCs with features of Langerhans
cells.7,8 In contrast, macrophage colony-stimulating factor (M-CSF) is a potent macrophage differentiation
factor.1 IL-69,10 and IL-1011
also shift monocyte differentiation from DCs to macrophages. Tumor
cells produce IL-6 and M-CSF that shift the differentiation of
CD34+ progenitors from DCs to macrophages.9
Fibroblasts, via IL-6 production, up-regulate functional M-CSF receptor
(CD115) expression and autocrine M-CSF consummation by monocytes,
thereby switching their differentiation from DCs to
macrophages.10
DCs are the most potent antigen-presenting cells
(APCs).12 In the periphery, immature DCs capture antigens
and, on contact with stress factors (such as microbial components),
migrate to the lymphoid organs and undergo a maturation process. They
express high levels of costimulatory and accessory molecules,
up-regulate major histocompatibility complex (MHC) class I and II
molecules, and neoexpress CD83. In the lymph nodes, mature DCs prime
naive antigen-specific T cells.12 In contrast to DCs,
macrophages are effector cells that produce various mediators and have
evolved to ingest as many pathogens as possible. Although they present antigens, macrophages are less efficient than DCs and unable to prime
naive T cells.13
IFN-, released during early and late stages of the immune
response by natural killer (NK) cells and activated T cells,
respectively, regulates several aspects of the immune
response.14 In addition to direct antiviral activity,
IFN- orchestrates leukocyte-endothelium interaction14
and plays a crucial role in vivo in "cancer
immunosurveillance."15 IFN- is a potent activator of
macrophages. On contact with IFN-, monocyte-macrophages undergo
biochemical and morphologic modifications that allow them to perform
their functional activities.16 INF- stimulates
macrophage antimicrobial and tumoricidal activities and accessory cell
functions and modulates proteasome gene expression.16 IFN- also acts on uncommitted myeloid immature DCs to polarize them
into TH1 cell-promoting effector cells that produce high levels of IL-12 on stimulation.17 We tested here whether
IFN- could be involved in monocyte differentiation and report that IFN- switches monocyte differentiation from DCs to macrophages, at
least partly via an autocrine production of M-CSF and IL-6.
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Materials and methods |
Cytokines
All human and murine recombinant cytokines were from R & D
Systems (Abingdon, United Kingdom).
Human monocyte differentiation
Peripheral blood mononuclear cells (PBMCs) were isolated by
Ficoll-Paque (Life Technologies, Cergy Pontoise, France) density gradient centrifugation. Monocytes were purified from PBMCs by positive
selection using a magnetic cell separator (MACS; Miltenyi Biotec,
Bergisch Gladbach, Germany). Purity, assessed by fluorescence-activated cell sorting (FACS) analysis using a fluorescein isothiocyanate (FITC)-labeled anti-CD13 monoclonal antibody (mAb), was more than 98%. Monocytes were differentiated into DCs by 5 days of culture in
complete medium (CM) consisting of RPMI 1640 medium supplemented with
10% fetal calf serum (FCS), 2 mM L-glutamine, 50 U/mL
penicillin, 50 µg/mL streptomycin, 10 mM HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and 0.1 mM nonessential amino acids (all from Life Technologies) at
5 × 106 cells/5 mL/well in 6-well tissue culture plates
(Corning Costar, Cambridge, MA) with 20 ng/mL IL-4 and 20 ng/mL
GM-CSF. Macrophages were obtained by culturing monocytes for 5 days in
CM with 2 ng/mL GM-CSF and 20 ng/mL M-CSF.18 In some
experiments, monocytes were cultured for 5 days in CM with 20 ng/mL
GM-CSF. All these cultures were also performed in the presence of
different concentrations of IFN- (2-50 ng/mL) added at different
time points, from day 0 to day 5. In neutralization experiments,
monocytes in GM-CSF plus IL-4 containing or not 25 ng/mL IFN- were
treated at day 0 and day 2 with 30 µg/mL neutralizing antihuman M-CSF
mAb (R & D Systems) plus 30 µg/mL neutralizing antihuman IL-6 mAb
(Diaclone, Besancon, France) or with 30 µg/mL mouse isotype control
mAbs (BD Pharmingen, San Diego, CA). In other experiments, day 5 immature DCs were recultured in GM-CSF plus IL-4 without or with 25 ng/mL IFN- alone, 25 ng/mL IFN- plus 100 ng/mL M-CSF, or 100 ng/mL M-CSF plus 100 ng/mL IL-6 or were recultured in CM without
cytokine. Finally, in some experiments cells were stimulated with 20 ng/mL lipopolysaccharide (LPS; Sigma, St Louis, MO).
MLRs with human cells
DCs and macrophages, obtained as described above, were washed,
irradiated (3000 rad), and cultured in quintuplicate at
4 × 102, 2 × 103, or
2 × 104 cells/200 µL/well in 96-well
flat-bottomed plates with 105 allogenic T cells purified
from PBMCs by rosetting with sheep red blood cells (the purity,
assessed by FACS analysis using a FITC-labeled anti-CD3 mAb, was
> 95%). In some experiments, day-5 immature DCs were treated or not
with 25 ng/mL IFN-. After 48 hours, cells were or were not
stimulated with 2 ng/mL LPS for 24 hours and used in mixed lymphocyte
reaction (MLR) assays at 2 × 104 cells/mL with
106 allogenic purified T cells. After 5 days, cells were
pulsed during the last 16 hours with 0.25 µCi/well (0.00925 MBq) 3H-thymidine (Amersham, Uppsala, Sweden).
Thymidine incorporation was measured by standard liquid scintillation
counting. Results are expressed in counts per minute (mean of
quintuplicate values).
Murine cells
C57BL/6 (IAb) and Balb/c (IAd) mice were
from Harlan (Gannat, France). Murine DCs were generated as
described19 by culturing bone marrow cells from C57BL/6
mice in CM supplemented with 50 µM -mercaptoethanol (-ME) and
containing 3 ng/mL GM-CSF. In some experiments, 25 ng/mL murine IFN-
was added at the beginning of the culture. After 5 days, the phenotype
of the cells was analyzed by FACS and MLRs were performed as follows.
Briefly, allogenic CD4+ T cells from Balb/c mice were
purified by incubation with a FITC-labeled antimurine CD4 mAb (BD
Pharmingen) followed by positive selection using anti-FITC mAb-coated
microbeads (Miltenyi Biotec). After 5 days of culture in GM-CSF,
myeloid APCs were depleted in Gr1+ cells by incubation with
an anti-Gr1 mAb (Caltag, Burlingame, CA) followed by antimouse Ig
mAb-coated beads (Dynal, Oslo, Norway). In 96-well flat-bottomed
culture plates (Corning Costar), 4 × 105
CD4+ T cells plus 5 × 104 APCs were cultured
in triplicate for 72 hours. During the last 16 hours,
3H-thymidine was added. Results are expressed in counts per
minute × 103 as mean ± SD (n = 3).
FACS analysis
The phenotype of cells was analyzed by cytofluorometry using a
FACSvantage cytofluorometer (BD Biosciences, Erembodegem, Belgium). For
human cells, the following mAbs were used: FITC-labeled anti-CD1a (Immunoquality Products, Groningen, The Netherlands), anti-CD14 (Dako,
Glostrup, Denmark), anti-CD64 (Caltag), anti-CD86, and anti-HLA-DR
(both from BD Pharmingen) mAbs; unlabeled anti-mannose receptor (MR;
Research Diagnostic, Flanders, NJ), -MHC I, -CD83 (both from Beckman
Coulter, Villepinte, France) revealed by FITC-labeled antimouse IgG
antibody (Silenus, Melbourne, Australia) and goat anti-CD115 antibody
(R & D Systems) revealed by FITC-labeled antigoat IgG antibody
(Silenus). To analyze intracellular expression of CD115 and RFD7, cells
were fixed and permeabilized using the Intrastain kit (Dako) before
staining with anti-CD115 or anti-RFD7 mAbs (Serotec, Oxford, United
Kingdom) revealed by FITC-labeled antigoat IgG antibody or antimouse
IgG antibody, respectively. Murine cells were phenotyped using
FITC-labeled anti-CD11b, anti-CD11c, anti-CD86, anti-IAb
(all from BD Pharmingen), biotin-labeled anti-F4/80 mAb (Caltag) revealed by FITC-labeled streptavidin (Molecular Probes, Eugene, OR)
and phycoerythrin (PE)-labeled anti-CD11c (BD Pharmingen) and anti-Gr1
mAbs. Isotype control mAbs were from BD Pharmingen. Results are
expressed as a percentage of positive cells or in mean fluorescence
intensity (MFI) values after subtraction of the MFI obtained
with the control mAb.
Analysis of mRNA expression by RT-PCR
In freshly purified human monocytes and in monocytes cultured in
GM-CSF plus IL-4 in the absence or presence of 25 ng/mL IFN- for 2, 4, 8, or 16 hours, the expression of the mRNA encoding IL-6, IL-10,
M-CSF, and CD115 was determined by reverse transcription-polymerase chain reaction (RT-PCR). Briefly, RNA was extracted using Trizol reagent (Life Technologies) and the single-strand cDNA was synthesized using 2 µg total RNA by reverse transcription using an oligo-dT primer (Amersham). PCRs were performed with cDNA corresponding to 50 ng
total RNA and primers designed to amplify the coding sequence of the
cytokines and cytokine receptor. PCR was as follows: 94°C for 5 minutes, 30 cycles 94°C for 30 seconds, 60°C for 30 seconds, and
72°C for 1 minute followed by a final extension at 72°C for 5 minutes. RNA integrity and cDNA synthesis were verified by amplifying
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. The amplified
fragments were size-separated on a 1% agarose gel and visualized by
ethidium bromide.
Quantification of human cytokines and soluble IL-6
receptor
IL-6, M-CSF, and glycoprotein 80 (gp80; soluble IL-6 receptor)
were quantified in the cell-free culture supernatants by enzyme-linked immunosorbent assay (ELISA; R & D Systems; sensitivity of 0.7, 9, and
6.5 pg/mL, respectively). Results are expressed in nanograms per
milliliter or micrograms per milliliter (as mean ± SD,
n = 4).
Phagocytosis and cytochemistry
To analyze endocytic properties, cells were incubated for 20 minutes at 37°C with FITC-labeled dextran (40 000 molecular weight), Staphylococcus aureus, or latex beads (2 µm diameter; all
from Molecular Probes). After extensive washings, cells were analyzed by FACS. Results are expressed in MFI or as a percentage of fluorescent cells. Nonspecific esterase activity was analyzed using the naphthyl acetate esterase kit (Sigma) following the manufacturer's instructions. Cell staining was analyzed by light microscopy.
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Results |
IFN- shifts monocyte differentiation from DCs to
activated macrophages
CD1aCD14+ monocytes cultured with GM-CSF
plus IL-4 differentiate after 5 days into
CD1a+CD14 immature DCs2,3 (Table
1). When cultured in GM-CSF plus IL-4
containing IFN-, monocytes differentiate into
CD1alowCD14 cells (Table 1). Compared to
monocyte-derived immature DCs, cells treated with IFN- express CD64
and CD86, express higher levels of MHC class I and class II molecules,
express lower levels of MR (Table 1), and present reduced T-cell
costimulatory properties (Figure 1A, left
panel). Microscopic observation shows that, whereas immature DCs are
mostly nonadherent round cells, IFN--treated cells form a network
of adherent elongated cells (Table 1 and Figure 1B). In contrast to
macrophages, LPS-stimulated immature DCs undergo a maturation process.
They present morphologic changes associated with veils (Table 1 and
Figure 1B); neoexpress CD83 (Table 1); up-regulate MHC class II, CD40,
CD80, and CD86 expression (data not shown); and acquire potent
costimulatory properties (Figure 1A, right panel). On LPS stimulation,
IFN--treated cells are adherent, do not present veils (Table 1 and
Figure 1B), and do not acquire CD83 expression (Table 1); in addition,
the expression of MHC class II and costimulatory molecules (CD40, CD80,
and CD86) is not modulated (data not shown). Moreover, these cells have limited costimulatory properties compared to mature DCs (Figure 1A,
right panel). Lastly, in response to 2 ng/mL LPS, IFN--treated cells produce undetectable levels of bioactive IL-12, in contrast to
immature DCs (835 ± 160 pg/mL; data not shown). IFN--treated cells do not express Langerhans cell-associated markers (langerhin and
E-cadherin; data not shown). Together, these data suggest that GM-CSF
plus IL-4 plus IFN--treated monocytes present a macrophage phenotype.
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Table 1.
Characteristics of monocytes cultured for 5 days with
GM-CSF plus IL-4, GM-CSF, or M-CSF in the absence or presence of
IFN-
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| Figure 1.
IFN- shifts monocyte differentiation from DCs to
macrophages.
(A) Monocytes were cultured in medium containing M-CSF (circles) or
GM-CSF plus IL-4 (triangles) in the absence (filled symbols) or
presence (open symbols) of 25 ng/mL IFN-. After 5 days, cells were
(right panel) or were not (left panel) stimulated for 24 hours with 20 ng/mL LPS. Cells were then irradiated and used to stimulate allogenic T
cells. Results are expressed in counts per minute × 103
as means ± SDs of quintuplicate values. Results shown are of 1 experiment representative of 3. Results show mean SD of
quintuplicate values of 1 experiment representative of 3. (B) Monocytes
were cultured in GM-CSF plus IL-4 in the absence (left) or presence of
25 ng/mL IFN- (right). After 5 days, cells were (top) or were not
(bottom) stimulated for 24 hours with 20 ng/mL LPS and observed by
microscopy (original magnification, × 100).
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Some macrophage subsets, such as monocytes differentiated into
macrophages in the presence of M-CSF, are characterized by CD14, CD64,
RFD7, and MR expression and by potent phagocytic and nonspecific
esterase activities (Table 1). IFN- modulates macrophage phenotype
and functions; it up-regulates MHC class I, HLA-DR, and CD64
expression20 and down-regulates CD14,21
RFD7,22 and MR23 expression as well as
phagocytic properties14,24 and nonspecific esterase
activity (Table 1). In agreement with these data, monocytes cultured in
GM-CSF plus IL-4 plus IFN- express CD86 and CD64 but not RFD7 and
CD14, and present reduced endocytic and nonspecific esterase activities
(Table 1). In parallel, IFN- poorly modulates macrophage accessory
cell function25 (Figure 1A, right panel).
Together, these data show that IFN- skews monocyte differentiation
from immature DCs to IFN--stimulated macrophages.
By comparing the respective properties of IL-4 and IFN- on
GM-CSF-treated monocytes (Table 1, left and middle panels), it appears
that IL-4 and IFN- have an additive effect on the up-regulation of
MHC class I and MHC class II molecule expression.26 This additive effect was observed at any time point analyzed during the
differentiation process (from day 1 to day 5; data not shown). Moreover, IL-4 and IFN- have also an additive effect in
up-regulating CD64 and CD86 expression on day 5-differentiated
macrophages. Together, these observations suggest a more complex
dialogue between IL-4 and IFN- than just a decrease in
monocyte-differentiating cell sensitivity to IL-4 mediated by
IFN-.27
IFN- prevents murine bone marrow progenitor differentiation
into DCs
We therefore tested whether the ability of IFN- to skew myeloid
cell differentiation from DCs to macrophages could be extended to an
IL-4-independent model of DC generation. In the presence of GM-CSF,
murine bone marrow-derived progenitors differentiate into
DCs.19 Bone marrow progenitors were incubated for 5 days with GM-CSF in the absence or presence of IFN-. Under the aegis of
GM-CSF, MHC class II progenitors give rise to a major
population of DCs (75%-90% of the cells according to the experiments,
n = 5) characterized by MHC class II, CD11c, CD86, and F4/80
expression and potent T-cell stimulatory properties (Table
2). In addition to DCs, macrophages (adherent, CD11c, Gr1, CD11b+,
and low or no MHC class II) and granulocytes (nonadherent,
Gr1+ and MHC class II) are also
present19 (data not shown).
When progenitors are cultured in the presence of GM-CSF plus IFN-
for 5 days, no CD11c+ cells are generated (Table 2). In
addition to a minor proportion of Gr1+ granulocytes
(15%-32%), the Gr1 and CD11c cells
express low or undetectable levels of MHC class II, but express CD86,
F4/80, and CD11b (Table 2). They do not express B, T, or NK cell
markers (data not shown). Finally, in contrast to DCs, these
CD11cCD11b+ cells present poor
MLR-stimulating activity (Table 2). These data show that IFN- skews
murine bone marrow progenitor differentiation from DCs to
macrophagelike cells. They also support a direct effect of
IFN- on DC precursors.
IFN- up-regulates M-CSF production by monocytes
M-CSF is the most potent macrophage differentiation factor
described.1 We then tested whether IFN- may affect
M-CSF production by monocytes. Human monocytes were cultured in GM-CSF
plus IL-4 containing or not IFN-, and M-CSF was quantified in the
supernatants at different time points during the differentiation
process. Monocytes cultured in GM-CSF plus IL-4 produce
M-CSF28 (Figure 2A). M-CSF production is maximal at day 1 and declines during the 5-day period of
culture (Figure 2A). Surprisingly, the presence of IFN- together with GM-CSF plus IL-4 results in a sustained (Figure 2A) and
dose-dependent up-regulation of M-CSF production (maximal increase of
300% ± 42% using 25 ng/mL IFN-; mean ± SD, n = 4;
Figure 2B). M-CSF mRNA expression is induced by culturing monocytes in
GM-CSF plus IL-428 and is up-regulated by IFN- as early
as 2 hours after stimulation (Figure
3).
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| Figure 2.
IFN- up-regulates M-CSF and IL-6 production by IL-4
plus GM-CSF-stimulated monocytes.
(A,C) Monocytes in GM-CSF plus IL-4 were ( ) or were not ( )
stimulated with 25 ng/mL IFN- at day 0 and M-CSF (A) and IL-6 (C)
were quantified in the cell-free supernatants from day 1 to day 5. (B,D) Monocytes were cultured in GM-CSF ( ) or GM-CSF plus IL-4 ()
in the absence or presence of 1 to 50 ng/mL IFN- added at day 0 and
M-CSF (B) and IL-6 (D) were quantified in the 24-hour supernatants (day
1). As control, monocytes were cultured in medium without cytokine
(dotted bars). (A-D) Results are expressed in micrograms per milliliter
as means ± SDs of 4 experiments. (E) Monocytes in GM-CSF plus
IL-4 were () or were not () stimulated with 25 ng/mL IFN-. At
days 1, 3, and 5, membrane CD115 expression was analyzed by FACS. At
day 1, intracellular CD115 expression was analyzed after cell
permeabilization. Results are expressed in MFI (after subtraction of
the MFI obtained with the control mAb) as means ± SDs of 4 separate experiments. (F) Monocytes in GM-CSF plus IL-4 were not ()
or were () stimulated with 25 ng/mL IFN- in the absence or
presence of neutralizing anti-M-CSF plus anti-IL-6 mAbs or of isotype
control mAbs. After 5 days, cells were stimulated with 20 ng/mL LPS and
CD83 expression was analyzed by FACS. Results are expressed in MFI
values as means ± SDs, n = 3.
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| Figure 3.
IFN- enhances M-CSF and IL-6 mRNA expression by
monocytes in GM-CSF plus IL-4.
Freshly isolated monocytes were not (none) or were cultured in GM-CSF
plus IL-4 in the absence or presence of 25 ng/mL IFN-. After 2, 4, 8, and 16 hours, the expression of the mRNA encoding M-CSF, IL-6,
IL-10, CD115, and GAPDH was analyzed by RT-PCR.
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We next evaluated the respective roles of IL-4 and IFN- on M-CSF
production (Figure 2B). Monocytes were exposed to GM-CSF in the absence
or presence of IL-4 and different concentrations of IFN-. M-CSF was
quantified in the 24-hour supernatants. GM-CSF induces M-CSF
production28 (Figure 2B) in a dose-dependent manner (data
not shown). IL-4 and IFN- have antagonistic effects on GM-CSF-induced M-CSF production as they down-regulate28
and up-regulate M-CSF production, respectively (Figure 2B).
Surprisingly, when added together, IFN- and IL-4 have an additive
effect on the up-regulation of M-CSF production (Figure 2B). Monocytes
in GM-CSF produce higher levels of M-CSF when stimulated with IFN- plus IL-4 than with IFN- alone (increase of 300% ± 42% and
80% ± 19% using 25 ng/mL IFN-, respectively; mean ± SD,
n = 4; Figure 2B). Finally, IFN- also up-regulates murine M-CSF
production by bone marrow progenitors (data not shown). Thus, we report
for the first time that IFN- is a potent inducer of M-CSF production by human and murine myeloid cells.
IFN- up-regulates IL-6 production by monocytes
IL-6 shifts monocyte differentiation from DCs to macrophages
by enhancing M-CSF consummation.9,10 We then evaluated
whether IFN- may control the production of IL-6 by
monocyte-differentiating DCs. Human monocytes were maintained in GM-CSF
plus IL-4 containing or not IFN-, and IL-6 was quantified in the
supernatants during the differentiation process. Monocytes in GM-CSF
plus IL-4 produce IL-6 (Figure 2C). Maximal IL-6 levels are obtained at
day 1 and then decline time dependently (Figure 2C). Addition of
IFN- up-regulates IL-6 production in a time- (Figure 2C) and
dose-dependent manner (with a maximum obtained using 50 ng/mL; Figure
2D). IL-6 mRNA expression is induced by culturing monocytes in GM-CSF
plus IL-4 and is up-regulated by IFN- (with an effect detectable 2 hours after stimulation; Figure 3). Analysis of the respective roles of
IL-4 and IFN- on IL-6 production shows that GM-CSF induces IL-6
production by monocytes and that IL-4 and IFN- down- and up-regulate
GM-CSF-induced IL-6 production, respectively (Figure 2D). In contrast
to M-CSF, no additive effect of IL-4 plus IFN- on IL-6 production is
observed as IL-4 partly prevents IFN--induced IL-6 production
(Figure 2D). A previous study reported that soluble IL-6 receptor
(gp80) cooperates with IL-6 and M-CSF in switching monocyte
differentiation to macrophages.10 We observed that IFN-
only slightly up-regulated gp80 production with a maximum occurring at
day 3 (0.26 ± 0.05 and 0.35 ± 0.06 ng/mL, in the absence or
presence of IFN-, respectively; mean ± SD, n = 4). Lastly,
IL-10 has also been shown to shift monocyte differentiation into
macrophages.11 We failed in detecting IL-10 production (data not shown) or an up-regulation of IL-10 mRNA expression on
treatment with IFN- (Figure 3).
Together, these data show that IFN- up-regulates IL-6 production by
monocytes in GM-CSF plus IL-4 by acting, at least partly, at the
transcriptional level.
IFN- down-regulates CD115 cell surface expression on
monocytes cultured in GM-CSF plus IL-4
M-CSF consummation is associated with CD115
internalization.29 We then evaluated whether the addition
of IFN- to monocytes cultured in GM-CSF plus IL-4 results in a
modulation of CD115 expression. Monocytes in GM-CSF plus IL-4 express
CD115 and this expression remains stable during the differentiation
process10,28 (Figure 2E). The presence of IFN- in GM-CSF
plus IL-4 results in a decrease in membrane CD115 expression, observed
at day 1 and maximal at day 3 (Figure 2E). CD115 expression returns to basal level at day 5 (Figure 2E). Analysis of CD115 expression in
permeabilized cells shows similar levels of expression on monocytes cultured in GM-CSF plus IL-4 with or without IFN- (Figure 2E), thereby suggesting an internalization of CD115 by IFN--treated cells. In accordance with these data, a decrease in M-CSF
concentrations in the day 3 supernatants of monocytes in GM-CSF plus
IL-4 plus IFN- is observed (Figure 2A). Moreover, IFN- slightly
enhances CD115 mRNA expression on monocytes in GM-CSF plus IL-4 (Figure 3). Together, these data suggest that the up-regulation of M-CSF and
IL-6 production by monocytes induced by IFN- is associated to an
autocrine enhancement of M-CSF consummation.
Both M-CSF and IL-6 are involved in the effect of IFN- on
monocyte differentiation into DCs
In neutralizing experiments, we evaluated whether autocrine IL-6
and M-CSF production induced by IFN- was involved in its ability to
skew monocyte differentiation from DCs to macrophages. Monocytes in
GM-CSF plus IL-4 were treated or not with IFN- in the absence or
presence of neutralizing anti-M-CSF or anti-IL-6 mAbs. At day 5, cells were stimulated with LPS for 24 hours before to analyze CD83
expression. Results show that IFN--treated cells partly recover the
ability to acquire CD83 expression in the presence of neutralizing mAbs
(61% ± 10%; mean ± SD, n = 4; Figure 2F). When anti-M-CSF
mAb or anti-IL-6 mAb was used alone, CD83 expression was also slightly
recovered (35% ± 8% and 29% ± 5%, respectively; Figure 2F), thereby suggesting that both cytokines participate to the
effect of IFN- on monocyte differentiation. No significant effect
was observed with control mAbs (Figure 2F). As control, the
neutralizing mAbs do not affect IL-4 plus GM-CSF-induced monocyte differentiation into DCs (Figure 2F). Together, these data show that
IFN- shifts differentiating DCs toward macrophages at least partly
by up-regulating autocrine consummation of M-CSF and IL-6.
Immature DCs do not produce M-CSF nor differentiate into
macrophages in response to IFN-
We then evaluated whether IFN- may reconvert immature DCs
toward macrophages. IFN- has been reported to polarize immature DCs
into DC1s that produce high levels of IL-12 and present potent costimulatory properties.17 In accordance with these data,
we show that immature DCs exposed to IFN- retain a phenotype of CD1a+CD14 DC (Table
3). Moreover, on LPS stimulation,
immature DCs exposed to IFN- acquire veils (data not shown),
neoexpress CD83, and present potent T-cell costimulatory properties
(Table 3).
We tested whether IFN- may control M-CSF and IL-6 production by
immature DCs. IFN- does not modulate M-CSF production (Figure 4A) nor mRNA expression (data not shown)
by immature DCs. Kinetic experiments show that the ability of monocytes
cultured in GM-CSF plus IL-4 to produce M-CSF in response to IFN- is
maximal at day 1 and decreases time dependently during the
differentiation process (Figure 4A). In contrast, immature DCs retain
the ability to produce IL-6 in response to IFN- (Figure 4B). These
data show that the regulation of M-CSF production in response to
IFN- is tightly regulated during the differentiation process from
monocytes to DCs.
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| Figure 4.
IFN- does not modulate M-CSF production by immature
DCs.
(A-B) Day 5 immature DCs were maintained in GM-CSF plus IL-4 and 25 ng/mL IFN- was not () or was () added at day 0, 2, or 4. After
24 hours, M-CSF (A) and IL-6 (B) were quantified in the supernatants.
Results are expressed in micrograms per milliliter (means ± SDs, n = 4).
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We evaluated whether IFN- may reconvert immature DCs to macrophages
when exogenous M-CSF is added. In the presence of a high concentration
of M-CSF (100 ng/mL), IFN--treated immature DCs in GM-CSF plus IL-4
retain a DC phenotype; they express CD1a but not CD14 (Table 3),
present a morphology of immature DCs (data not shown), and, on LPS
stimulation, acquire CD83 expression (Table 3) and veils (data not
shown). In agreement with others,10 they also present a
decrease in cell surface CD115 expression (Table 3), thereby suggesting
a consummation of M-CSF by immature DCs. Finally, even in the presence
of exogenous M-CSF and IL-6, immature DCs in GM-CSF plus IL-4 retain a
DC phenotype (Table 3). As a positive control,30 on GM-CSF
and IL-4 removal, immature DCs acquire CD14 expression and convert
toward adherent macrophages (Table 3). Thus, these data suggest that,
in the presence of GM-CSF plus IL-4, immature DCs may express
functional CD115 but are resistant to IFN--, IL-6-, and
M-CSF-induced macrophage differentiation.
| |
Discussion |
IFN- produced by NK or activated T cells participates in the
control of the innate and adaptive phases of the immune
response.14 IFN- exerts most of its effects on APCs; it
activates macrophages and polarizes immature DCs into Th1
cell-promoting effector DCs.14,16,17 We show here for the
first time that IFN- also participates to the control of APC
differentiation; IFN- switches monocyte differentiation to activated
macrophages at least in part via autocrine activation by IL-6 and
M-CSF.
M-CSF is a major macrophage differentiation factor1 that
skews CD34+ progenitor and monocyte differentiation from
DCs to macrophages.9,10,30 By enhancing functional M-CSF
receptor expression and M-CSF consummation,10 IL-6
cooperates with M-CSF in this process.9,10 In agreement with these data, we report that IFN- switches monocyte
differentiation into macrophages via an up-regulation of autocrine IL-6
and M-CSF production.
GM-CSF induces M-CSF production by monocytes10,28 in a
dose-dependent manner with a maximum at 100 ng/mL (data not shown). In
our experimental conditions, the concentrations of M-CSF present in the
supernatants of monocytes in GM-CSF plus IL-4 were lower than those
reported by others.10,28 The use of 20 ng/mL instead of
100 ng/mL GM-CSF in our study may explain this difference.
We also show that monocytes treated with IL-4 plus GM-CSF produce IL-6.
Levels of IL-6 induced by IL-4 plus GM-CSF were lower than those
induced by GM-CSF alone. Thus, it appears that IL-4 inhibits
GM-CSF-induced M-CSF28 and IL-6 production. It is
therefore tempting to speculate that the inhibitory effect of IL-4 on
M-CSF and IL-6 production may contribute to explain its DC
differentiation factor property.
Although IFN- antagonizes many IL-4-mediated
responses,27 it has been also previously observed that
IL-4 and IFN- act synergistically to enhance MR-dependent
phagocytosis31 and CD23 expression on
macrophages.32 We report that whereas IFN- up-regulates IL-6 and M-CSF production, IL-4 down-regulates their production. However, when used together, they mutually inhibit their respective effects on IL-6 production and have an additive effect on the up-regulation of M-CSF production. Together, these observations suggest
a complex dialogue between IL-4 and IFN- not limited to antagonistic effects.
IFN- antagonizes many physiologic responses mediated by IL-4, by
acting at posttranscriptional27 or transcriptional levels. IFN- and IL-4 activate signal transducer and activator of
transcription (Stat)1 and Stat6, respectively.33,34
IFN- inhibits IL-4-mediated Stat6 activation by inducing the
expression of suppressors of cytokine signaling (SOCS)1.35
The antagonist effect of IFN- on IL-4-induced inhibition
of IL-6 production could be related to the SOCS1-mediated Stat6
inhibition. Moreover, expression of IL6 gene is
under the control of transcription factors, including AP-1, nuclear
factor- B (NF-B), and NF-IL-6.36 Whereas NF-IL-6 is
strongly inhibited by IL-4,37 it is poorly affected by
IFN-.38 Consequently, the ability of IL-4 to
down-regulate IFN--induced IL-6 production could be related to the
strong inhibition of NF-IL-6 which is mediated by IL-4 and not
counteracted by IFN-.
In accordance with data showing a differential regulation of M-CSF and
IL-6 gene expression in monocytes,39 we report that IFN- plus IL-4 have an additive effect of M-CSF production. The role
of members of the Stat family in the regulation of M-CSF expression is
poorly documented. Nevertheless, we could hypothesize that the
down-regulation of IL-4-induced activation of some members of the Stat
family mediated by IFN- may contribute to explain this effect. In
addition, the increase of M-CSF production induced by IL-4 plus IFN-
could be also indirect. M-CSF increases M-CSF mRNA expression by
GM-CSF-activated monocytes.40 M-CSF receptor gene
expression is controlled by transcription factors including NF-B,
AP-1, and PU-1. PU-1 is activated by IFN-41 and
interacts with the interferon regulatory factor 4 (IRF4) which
expression is increased by IL-4.42 Thus, the additive
effect of IFN- and IL-4 on M-CSF production could be consecutive to
an increase of M-CSF receptor transcription. Additional experiments are
required to determine the effects of IL-4 plus IFN- on the
expression of the transcription factors involved in IL-6 and M-CSF production.
DCs and macrophages have different roles in the immune response.
Whereas DCs initiate specific immune responses, macrophages and
especially IFN--activated macrophages exhibit potent bactericidal and antitumoral activities.14 When added to differentiated
uncommitted immature DCs, IFN- polarizes them into Th1
cell-promoting effector DCs.17 Th1 cells produce IFN-,
IL-2, and tumor necrosis factor (TNF-) and evoke cell-mediated
immunity and phagocyte-dependent inflammation.
In addition to acting on mature APC functions, IFN- may also act on
DC/macrophage precursors to favor their differentiation into
macrophages. Interestingly, IFN- acts mainly at the initial stages
of monocyte differentiation. The ability of cells to produce M-CSF in
response to IFN- varies with the status of differentiation. In
contrast to monocytes, immature DCs express CD115 but do not revert to
macrophages on contact with IL-6, M-CSF, or IFN-. The mechanisms
underlying this resistance remain to be identified. These data also
suggest that the effects of IFN- are tightly controlled according to
the status of cell differentiation. Lastly, in agreement with data
showing that environmental cytokines tightly control monocyte
differentiation into immature DCs and macrophages and the
interconversion into one another,4,30 we observed that the
removal of IFN- from the culture at the early stages of
differentiation partly prevents its blocking effect on DC
differentiation. IL-10 also shifts monocyte differentiation from DCs to
macrophages.11 IL-10 and IFN- give rise to
macrophagelike cells with divergent antigen presentation and
endocytosis properties.43 These observations suggest that
monocyte differentiation and the function of the cells generated are
also tightly controlled by the nature of cytokines encountered.
DCs are the only APCs that prime naive T cells and initiate specific
immune responses.12 Consequently, they have a central role
in vaccine strategies and especially in antitumor
immunotherapies.44,45 Tumor-specific vaccination using
antigen-loaded autologous DCs has reached the stage of human clinical
trials. These studies have been made possible by the development of
methods for obtaining large numbers of DCs. Most of these studies have
been carried out with ex vivo generated monocyte-derived
DCs.44,45 Thus, to identify factors that control monocyte
differentiation is crucial in order to optimize DC generation. Our
results show that IFN- skews monocyte differentiation from DCs to
macrophages, suggesting that the presence of IFN--producing cells
together with monocytes could interfere with the differentiation
process into DCs. Finally, in agreement with others, we report that
IFN--stimulated macrophages present undetectable or low levels of
markers that are usually considered as macrophage markers (RFD7, CD14,
and MR). This observation points out that the expression of CD1a or
CD14 (or both) is sometimes not sufficient to clearly distinguish
between monocyte-derived macrophages or DCs and that the study of
additive morphologic, phenotypic, and functional parameters is required.
In conclusion, our data show that, in addition to a direct effect on
APC functions, IFN- skews monocyte differentiation from DCs to
macrophages and thereby reveals a new mechanism by which IFN- may
control the outcome of the immune response.
| |
Acknowledgments |
We sincerely acknowledge Ms R. Vellaidom for manuscript handling.
| |
Footnotes |
Submitted April 17, 2002; accepted August 6, 2002.
Prepublished online
as Blood First Edition Paper, August 15, 2002; DOI
10.1182/blood-2002-04-1164.
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.
Reprints: Pascale Jeannin, Centre d'Immunologie
Pierre Fabre, 5, Avenue Napoléon III, F-74160 Saint-Julien en
Genevois, France; e-mail:
pascale.jeannin{at}pierre-fabre.com.
| |
References |
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Becker S, Warren MK, Haskill S.
Colony-stimulating factor-induced monocyte survival and differentiation into macrophages in serum-free culture.
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Sallusto P, Lanzavecchia A.
Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor .
J Exp Med.
1994;179:1109-1118[Abstract/Free Full Text].
3.
Zhou LJ, Tedder TF.
CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells.
Proc Natl Acad Sci U S A.
1996;93:2588-2592[Abstract/Free Full Text].
4.
Chapuis F, Rosenzwajg M, Yagello M, Ekman M, Biberfeld P, Gluckman JC.
Differentiation of human dendritic cells from monocytes in vitro.
Eur J Immunol.
1997;27:431-441[Medline]
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Abstract
Introduction