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 Previous Article | Table of Contents | Next Article 
Blood, 1 August 2001, Vol. 98, No. 3, pp. 736-742
IMMUNOBIOLOGY
Type I interferons in combination with bacterial stimuli induce
apoptosis of monocyte-derived dendritic cells
Manfred Lehner,
Thomas Felzmann,
Katharina Clodi, and
Wolfgang Holter
From the Children's Cancer Research Institute (CCRI),
St Anna Children's Hospital, Vienna, Austria.
 |
Abstract |
Both type I interferons (IFNs) as well as lipopolysaccharide (LPS)
individually compromise selected monocytic or dendritic cell (DC)
functions. This study investigates the influence of these agents on the
differentiation and the regulation of cell death of monocyte-derived
DCs generated in the presence of granulocyte-macrophage colony-stimulating factor plus interleukin-4 (IL-4). It is reported that excessive apoptosis occurred rapidly in monocyte-derived DC
cultures, if IFN- or IFN- was added in combination with LPS or
lipoteichoic acid (LTA). The small fraction of cells surviving in such
cultures displayed a mature DC phenotype with expression of CD83, CD80,
and CD86. IL-10 was found in the supernatants of monocyte-derived DC
cultures, if supplemented with LPS or IFN- plus LPS but not in
control cultures. When monocyte-derived DCs were generated in the
presence of IFN- without LPS, these cells displayed an immature DC
phenotype with a reduction of cell recovery but no overt apoptosis.
However, the addition of LPS, LTA, LPS plus IFN- , or tumor necrosis
factor (TNF-) plus prostaglandin E2 to such cells again resulted
in the rapid induction of apoptosis in the majority of cells, together
with a reduced production of IL-12 p70 and TNF-. Together, these
data indicate an exquisite sensitivity of monocyte-derived DCs to
activation-induced cell death if generated in the presence of IFN-,
indicating the existence of an important mechanism of immunosuppression
caused by IFN--inducing agents, such as viral or bacterial stimuli.
(Blood. 2001;98:736-742)
© 2001 by The American Society of Hematology.
| |
Introduction |
Evidence accumulating during many years has shown
that viral and bacterial infections can impair the function of many
cells of the immune system. Dendritic cells (DCs) are of central
relevance within the immune system because only these cells can
activate naive T cells and thereby initiate an adaptive immune
response. Thus, understanding their function during infections might
give insight into important immunopathologic mechanisms. For in vitro studies, DCs are commonly derived either from CD34-expressing precursors or from monocytes (reviewed by Banchereau and
Steinman1). With regard to monocyte-derived DCs several
effects of bacterial and viral stimuli in vitro and in vivo have
already been described.
For instance, interferon (IFN-), which is transiently released
during the first wave of innate immunity in response to bacteria and
viruses,2-10 has been reported to suppress interleukin-12 (IL-12) production of monocytes and DCs.11-14 The
superfamily of type I IFNs comprises at least 12 IFN- species and a
single IFN- that differentially bind to a common
receptor.15 Alpha IFNs are produced in modest amounts by
monocytes, macrophages, and B lymphocytes9 and in much
larger quantities by the precursors of lymphoid DCs
(pDC2),9,16,17 whereas IFN- is mainly produced by
fibroblasts.18 Despite their established protective role in innate immunity because of their antibacterial and antiviral activity,2,10,19-21 there is increasing evidence that
type I IFNs might also act negatively on immunity not only by
abrogating IL-12 production but also by impairing DC
differentiation22 and reducing the phagocytic and
oxidative activity of monocytes.23
Other studies showed that lipopolysaccharide (LPS) has suppressive
effects on monocytes and monocyte-derived DCs,4,10 possibly in part mediated by LPS-triggered type I IFN production. A
transient presence of endotoxin during the differentiation of monocyte-derived DCs abrogates IL-12 production of the resulting immature DCs on a second stimulation with LPS.24 If
permanently present during DC differentiation, it completely
desensitizes the cells to all further LPS-mediated maturation
signals.25 Monocytes from septic patients were found to be
hyporesponsive to LPS stimulation ex vivo, and this hyporesponsiveness
was reversed during IFN- treatment that also lead to clearance of
sepsis,26 suggesting that monocyte deactivation might be a
major mechanism of immunosuppression.
Despite these data it is unclear how DCs respond to a combination of
type I IFNs and bacterial products. With the use of the culture of
monocyte-derived DCs, our study revealed a strong synergistic effect
between type I IFNs and products from gram-negative as well as
gram-positive bacteria for the induction of apoptosis. This effect
occurs if both stimuli are present together during the whole culture
but also when the bacterial stimulus is added later to immature
DCs generated in the presence of type I IFNs.
| |
Materials and methods |
Monocyte isolation and DC culture
Human mononuclear cells were obtained from blood containing
citrate as anticoagulant from healthy donors by density gradient centrifugation using endotoxin-free Ficoll-Paque-PLUS (Pharmacia, Uppsala, Sweden). Monocytes were isolated from these mononuclear cells
by a second density gradient centrifugation (M.L. and W.H., manuscript
submitted, March 2001) using Ficoll-Paque-PLUS adjusted to 1.068 g/mL
by dilution with phosphate-buffered saline (BioWhittaker, Walkersville,
MD). The remaining cells, containing 80% to 90% monocytes as assessed
by counting on a Sysmex F820 instrument, were frozen in RPMI 1640 (Life
Technologies, Grand Island, NY) supplemented with 10% dimethyl
sulfoxide (Sigma, St Louis, MO) and 20% fetal calf serum
containing less than 100 pg/mL endotoxin (PAA Laboratories, Linz,
Austria). For DC differentiation monocytes were cultured in 24-well
culture plates (Iwaki, Japan) at 0.5 × 106/mL in RPMI
1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine
(Life Technologies), 1000 U/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; Leucomax; Roche, Basel,
Switzerland), and 1000 U/mL recombinant human IL-4 (Strathmann Biotec,
Hamburg, Germany). Half of the medium (including all supplements) was
exchanged every 2 days. IFN-2c (Berofor; Boehringer Ingelheim,
Vienna, Austria), recombinant human IFN- (Sigma), LPS from
Escherichia coli 055:B5 (Sigma) and lipoteichoic acid (LTA)
from Bacillus subtilis (Sigma) were added in different
concentrations and combinations at the indicated time points. Further,
some cultures were additionally supplemented with 1000 U/mL recombinant
human tumor necrosing factor (TNF-; Bender MedSystems, Vienna,
Austria), 1 µg/mL prostaglandin E2 (PGE2; Sigma), 1000 U/mL IFN-
(Bender MedSystems), or 40 000/24-well irradiated (6000 rad) SNJB7
neuroblastoma cells transfected with CD40 ligand (CD40L).46
Flow cytometric analysis
For immunophenotypic analysis the following monoclonal
antibodies were used: anti-CD80-phycoerythrin (PE; clone MAB104;
Immunotech, Marseille, France), anti-CD83-PE (clone HB15A;
Immunotech), anti-CD86-fluorescein isothiocyanate (FITC; clone 2331;
Pharmingen, San Diego, CA), anti-CD1a-FITC (clone HI149; Pharmingen),
and anti-CD14-allophycocyanin (clone MoP9; Becton Dickinson,
Erembodegem, Belgium). Apoptosis was detected by staining with Annexin
V-FITC (Alexis, San Diego, CA) according to the manufacturer's
protocol. Cells were analyzed on a FACScan flow cytometer (Becton
Dickinson, Mountain View, CA), and data analysis was performed with
CellQuest software (Becton Dickinson).
Cytokine measurements
On the basis of standard sandwich enzyme-linked immunosorbent
assay methodology, commercially available pairs of monoclonal antibodies (Pharmingen) were used to quantify human IL-12 p70, IL-10,
and TNF-.
| |
Results |
Combination of type I IFNs with LPS or LTA induces extensive
apoptosis in cultures of monocyte-derived DCs
IFN- and LPS each are described to affect the differentiation
of monocyte-derived DCs. To investigate potential synergistic effects
between type I IFNs and bacterial stimuli on monocyte differentiation
toward DCs, we first analyzed the effect of IFN- and LPS present
during the whole culture period. IFN- (1000 U/mL) as well as 1 ng/mL
LPS were added on day 0 alone or in combination to monocytes in medium
containing IL-4 and GM-CSF. On day 6, light microscopy indicated
extensive cell death in the cultures containing both LPS and IFN-
(not shown).
Apoptotic cell death is characterized by the binding of Annexin V and
by cell shrinkage (in contrast to necrosis). Figure 1 shows the flow-activated cell sorter
(FACS) analysis of a DC culture treated with IFN- plus LPS compared
with an untreated culture after staining with Annexin V-FITC and
propidium iodide. Figure 1A illustrates that in the control cultures
the majority of cells are contained within a relatively homogenous DC
population with regard to cell size (estimated by forward scatter
[FSC]) and granularity (estimated by the side scatter [SSC]), both
parameters are decreased if IFN- plus LPS had been added to the
culture (Figure 1B). Most of the IFN--plus-LPS-treated cells
acquired Annexin V positivity, indicating the presence of extensive
apoptosis, with a subpopulation being also positive for propidium
iodide (Figure 1D). Double-positive cells represent cells that have
already lost integrity of their membranes during the late apoptotic
process. In this representation, however, a clear demarcation of the
apoptotic cell population was not possible. For the quantification of
apoptosis we, therefore, used the observed reduction in the forward
scatter together with increased binding of Annexin V to achieve a
sufficient discrimination of apoptotic cells (Figure 1E,F). The same
mode of representation was used for the quantification of apoptosis in
all further experiments. We could also detect the typical condensation of apoptotic nuclei in cytospins of cells stained with Annexin V and
4,6-diamidino-2-phenylindole (not shown).
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| Figure 1.
Quantification of apoptosis by FACS analysis after
staining with Annexin V-FITC and propidium iodide.
Density gradient-purified monocytes were cultured for 6 days with
GM-CSF plus IL-4 in the absence (A,C,E) or presence (B,D,F) of 1000 U/mL IFN- plus 1 ng/mL LPS. Shown are the results of a single
experiment by 3 alternative modes of representations displaying the
forward/side scatter profile (A-B), Annexin V-FITC versus propidium
iodide (PI) (C-D), and forward scatter versus Annexin V-FITC (E-F).
Gates G1 (apoptotic cells) and G2 (viable cells) were drawn arbitrarily
in the representation forward scatter versus Annexin (E-F), because in
this representation both populations could best be discriminated. In
this experiment cells in gate G1 (apoptotic cells) amounted to 12.2%
of total cells in panel E (control culture) and 64.8% in panel F
(culture with IFN- plus LPS), whereas the cells in gate G2 were
considered as viable cells (75% of total cells in panel E and 22% in
panel F). The small population of lymphocytes (best visible in panel A)
amounted to 8.3% of total events in this experiment.
|
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In the following experiments we investigated if the observed synergy in
induction of apoptosis also exists between IFN- as an alternative to
type I IFN and LTA, a cell wall component of gram-positive bacteria.
Figure 2 compares the fraction of
apoptotic cells on day 6 in cultures containing IFN- or IFN- and
LPS or LTA alone or in combination. In contrast to the effect of the single factors, all combinations of a type I IFN with a bacterial stimulus induced strong apoptosis.
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| Figure 2.
Type I IFNs in combination with LPS or LTA induce
apoptosis.
Different combinations of 1000 U/mL IFN-, 1000 U/mL IFN-, 1 ng/mL
LPS, and 1 µg/mL LTA were added on day 0 to cultures supplemented
with IL-4 and GM-CSF. The percentage of Annexin-positive cells was
determined by FACS analysis on day 6 as detailed in Figure 1. The data
were obtained from 4 different donors, each represented by an
individual symbol. Two donors ( ,  ) were tested twice in
independent experiments.
|
|
Titration experiments with IFN- plus LPS (Figure
3A) and IFN- plus LTA (Figure 3B)
revealed strong synergistic effects of 100 to 1000 U/mL IFN- with
both bacterial components at low concentrations, and in kinetic studies
it was evident that apoptosis occurred rapidly following the addition
of IFN- plus LPS (Figure 4).
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| Figure 3.
Dose dependency of apoptosis in response to IFN- plus
LPS or LTA.
IFN- and LPS (A) or LTA (B) was added at the indicated
concentrations alone ( ) or together with 10 U/mL ( ), 100 U/mL
( ), or 1000 U/mL IFN- ( ) on day 0 to cultures of monocytes
supplemented with IL-4 and GM-CSF. The induction of apoptosis was
quantified by FACS analysis on day 6 as detailed in Figure
1.
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| Figure 4.
Rapid induction of apoptosis in cultures containing
IFN- and LPS.
On day 0, 1000 U/mL IFN- ± 1 ng/mL LPS was added to monocyte
cultures supplemented with GM-CSF plus IL-4. Annexin V positivity was
quantified on days 2, 4, and 6. , control;  , IFN-;
, IFN- plus LPS. Shown are the data obtained with monocytes from
3 different donors (mean ± SD).
|
|
Surviving cells in cultures containing IFN- plus LPS are
phenotypically mature DCs
The extensive apoptosis in the presence of IFN- and LPS
strongly reduced the yield of viable cells. The phenotype of the remaining cells was compared with that of control cultures of immature
DCs differentiated in the absence of IFN- plus LPS and with cells
derived from such control cultures but matured by the addition of LPS
for the last 3 days of the culture. FACS analysis for selected DC
markers for all cultures was done on day 8 (Figure 5A). The permanent presence of IFN-
plus LPS resulted in an increased expression of CD83, CD80, and CD86 in
the surviving cells, indicating the presence of mature DCs. These
increases were not seen in the presence of IFN- or LPS alone (not
shown). When these cells were additionally exposed to the strong
maturation stimulus IFN- plus CD40L from day 5 on, there was almost
no further response (Figure 5B).
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| Figure 5.
Surviving DCs generated in the presence of IFN- plus
LPS have a mature phenotype.
Monocyte-derived DCs were generated with GM-CSF plus IL-4 in the
absence (control cultures, dotted lines, A) or presence of IFN- plus
LPS (bold lines, A-B). During the last 3 days, a part of both cultures
was subjected to a maturation step employing 100 ng/mL LPS (control
cultures, thin line, A) or CD40L plus IFN- (cultures containing
IFN- plus LPS, thin line, B). Cells were stained on day 8 of total
culture for the presence of CD83, CD80, and CD86; only large viable
cells were analyzed.
|
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Although the remaining viable cells of the cultures containing IFN-
and LPS thus resembled mature DCs, there were peculiarities. Despite a
homogeneous expression of CD83, CD80, and CD86 (Figure 5A), further
analysis revealed CD14 expression in a subpopulation of cells (Figure
6A). To some extent, varying between
individuals, some CD14 expression was also seen with IFN- alone.
Because IL-10 was described to induce differentiation of immature
monocyte-derived DCs into CD14+ macrophagelike
cells,27 we tested if the observed CD14 expression was
correlated with elevated IL-10 production. In the cultures containing
both IFN- and LPS approximately 2 ng/mL IL-10 was found on day 4 (Figure 6B). Although there was no CD14 expression at all with LPS
alone, it also induced some IL-10 (300 pg/mL) as previously
reported,28 whereas IFN- alone induced some CD14 but
not any IL-10.
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| Figure 6.
CD14 expression and IL-10 production of cells
differentiated in presence of IFN- plus LPS.
The dot plots in panel A were obtained by FACS analysis on day 6 from
cultures containing 1000 U/mL IFN- and/or 1 ng/mL LPS as indicated.
Despite homogeneous expression of the DC maturation markers CD83, CD80,
and CD86 (not displayed), the expression of CD14 is retained in a
subpopulation of cells cultured in the presence of IFN- plus LPS.
Shown are the data from one representative experiment; similar results
were obtained in 3 further experiments. Panel B displays the amount of
IL-10 found in the supernatants on day 4 (mean ± SD of 3 independent experiments).
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Addition of IFN- plus LPS to immature DCs induces maturation
without apoptosis
In the experiments described above, type I IFNs and bacterial
stimuli were added to monocytes on day 0. To investigate the response
of immature DCs to a combination of these stimuli, immature DCs were
challenged with LPS or IFN- alone or with a combination of both. LPS
plus IFN- as well as LPS alone triggered maturation with induction
of CD83 (not shown) and a very high expression of CD80 and CD86 3 days
after stimulation (Figure 7A). IFN-
alone did not induce maturation. Importantly, no increased apoptosis was observed in the cultures containing IFN- plus LPS
(Figure 7B).
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| Figure 7.
The addition of IFN- plus LPS to immature DCs on day 7 triggers
maturation without apoptosis.
Panel A shows the phenotype of monocyte-derived DCs on day 10 (ie, 3 days after addition of the indicated stimuli). The percentage of
apoptotic cells on day 10 is shown in panel B (mean ± SD of 3 independent experiments).
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Immature DCs generated in the presence of IFN- undergo apoptosis
in response to a variety of activation stimuli
The above experiments had investigated the effects of type I IFNs
and bacterial stimuli when added together at the start of the culture
to monocytes or to monocyte-derived immature DCs. Next we asked whether
bacterial products or other activating signals would induce apoptosis
if added later to immature DCs, which had been differentiated in the
presence of IFN-. Therefore, immature DCs generated in the presence
or absence of 1000 U/mL IFN- were stimulated with LTA, LPS, LPS plus
IFN-, and TNF- plus PGE2 on day 5. Three days later, extensive
apoptosis was present in all cultures that had been differentiated in
the presence of IFN- (Figure 8A).
Importantly, apoptosis was induced to the same extent by activation of
the IFN- precultured DCs with TNF- plus PGE2 as with LPS or LTA,
suggesting that IFN- had sensitized the immature DCs to
activation-induced cell death in general. To some extent 1000 U/mL
IFN- alone also increased apoptosis on day 8, but addition of the
maturation stimuli dramatically raised the percentage of Annexin
V-binding cells. The expression of CD80, CD86, and CD83 of the
surviving cells in these cultures was completely identical to that of
the respective activated DCs differentiated without IFN- (data not
shown). Although quantitatively reduced, IFN- primed immature DCs
were also competent for production of IL-12 p70 and TNF- in response
to LPS plus IFN-, whereas IL-10 was not produced (Figure
9).
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| Figure 8.
Immature DCs generated by GM-CSF plus IL-4 in the
presence of IFN- undergo apoptosis in response to a variety of
activation stimuli.
The response of immature DCs generated in the permanent presence of
1000 U/mL IFN- () to the indicated stimuli was compared with that
of immature DCs generated in the absence of IFN- (). Apoptosis
was quantified on day 8 (ie, 3 days after activation with 1 µg/mL
LTA, 100 ng/mL LPS, 100 ng/mL LPS plus 1000 U/mL IFN-, and 1000 U/mL
TNF- plus 1 µg/mL PGE2 as indicated [mean ± SD of 3 independent experiments]).
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| Figure 9.
Cytokine production of DCs generated in the presence of
IFN- in response to LPS plus IFN-.
The production of IL-12 p70, TNF-, and IL-10 was determined on day 8 (ie, 3 days after activation with 100 ng/mL LPS plus 1000 U/mL
IFN-). The response of immature DCs generated in the permanent
presence of 1000 U/mL IFN- () was compared with that of immature
DCs generated in the absence of IFN- () (mean ± SD of 3 independent experiments).
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| |
Discussion |
In this study we describe the effects of the combined action of
type I IFNs and microbial products on the differentiation and survival
of monocyte-derived DCs. We found that IL-4/GM-CSF-stimulated cultures
of monocytes differentiating toward DCs in the presence of IFN- or
IFN- underwent strong apoptosis if microbial products such as LPS or
LTA were additionally added. Provided that type I IFNs were present
from the initiation of the cultures, bacterial products also induced
apoptosis when added later to immature DCs. Immature DCs differentiated
in the presence of IFN- died also in response to TNF- plus PGE2.
No apoptosis was seen, however, when type I IFNs alone or together with
a bacterial stimulus were added to already differentiated immature DCs.
Cells surviving IFN- plus LPS treatment developed into cells
displaying a mature DC phenotype with an expression of CD83, CD80, and
CD86 identical to that displayed by normal mature DCs, grown in the
absence of IFN- and further matured by an appropriate stimulus.
Our results fit well with a recent report,22 in which a
reduced yield of monocyte-derived DCs grown in the presence of type I
IFNs was described. We show that this reduction is due to apoptosis and
is dramatically enhanced by the addition of a bacterial stimulus even
at a low dose (30-100 pg/mL LPS). The kinetics of the apoptosis induction through the combined action of IFN- and LPS was very rapid. Only 2 days after culture initiation a predominant fraction of
cells underwent apoptosis. A similar synergy has been reported for the
bovine system, in which macrophages rapidly undergo LPS-induced apoptosis after priming with IFN-.29 Interestingly,
however, murine macrophages go in apoptosis in response to LPS plus
IFN- but are protected by pretreatment with IFN- or
.30
Somehow contrasting the detrimental effects of type I IFNs on DC
differentiation seen in our study, 2 recent reports suggested IFN-
as an adjuvant supporting DC development with GM-CSF in the absence of
IL-4.31,32 Interestingly, Santini et al31 described also an increase of Annexin binding cells in their cultures containing IFN- plus GM-CSF, although in their study a beneficial effect of IFN- on the generation and maturation of DCs prevailed. In
our system it was mainly the addition of a second stimulus, such as
LPS, LTA, or TNF- plus PGE2, that resulted in the apoptosis of the
majority of newly generated DCs. Our data thus imply that such
IFN--pretreated immature DCs in general are sensitized to activation-induced cell death.
The phenotype of the surviving cells differed depending on the time
point of the addition of the bacterial stimulus. In the presence of
IFN-, GM-CSF, and IL-4, the cells developed into viable
CD14 and CD1a+ immature DCs (Figure 6A).
Addition of bacterial stimuli to the immature DCs precultured with
IFN- induced strong apoptosis, but the surviving cells expressed
CD83, CD80, and CD86 completely identically to mature control DCs
(Figure 8 and not shown). In the cultures in which both stimuli were
present from the initiation of the culture, the few viable cells also
displayed a mature DC phenotype without further activation (Figure 5A)
and hardly responded to further stimulation with IFN- plus CD40L
(Figure 5B). These cells also stimulated allogeneic T cells in a mixed
lymphocyte reaction in accordance with their mature DC phenotype (not shown).
The mature population resulting from the early presence of IFN- plus
LPS further contained a varying but substantial fraction of
CD14+ cells (Figure 6A). Because IL-10 was described to
shift differentiation of DC cultures toward macrophagelike cells by
raising the expression of the receptor for the endogenously produced
macrophage colony-stimulating factor,27 we analyzed the
culture supernatants for the presence of IL-10. In accordance with
earlier reports,28,33 we found little amounts of IL-10 in
cultures containing a low dose of LPS and markedly raised amounts in
cultures supplemented with LPS plus IFN- (Figure 6B). Because the
addition of IL-10 to cultures treated with IFN- alone strongly
increased CD14 expression and a neutralizing anti-IL-10 monoclonal
antibody added to IFN- plus LPS-stimulated cultures decreased CD14
expression (data not shown), endogenously produced IL-10 indeed
appeared to influence the phenotype of the cells in our system.
With regard to the underlying mechanism of apoptosis induction,
endogenously produced nitric oxide (NO) could be a possible candidate
mediating cell death. In murine macrophages NO is induced by LPS in
synergy with a type I IFN or IFN-,34 and in murine dendritic cells NO production in response to LPS plus IFN- was reported to cause apoptosis.35 In bovine macrophages
IFN- could prime for LPS-induced apoptosis, but this effect did not
correlate with enhanced NO production, instead LPS-induced NO
production was strongly reduced by IFN-.29 In the human
system controversy exists as to whether and under which conditions
monocytic cells can be activated for increased NO
production.36-39 Sharara et al38 found a
modestly increased NO production of human monocytes treated with
IFN-, and Weinberg et al39 reported that a basal NO
production by human monocytes could not be increased by treatment with
LPS plus IFN-. Together these published data suggest that
significant NO production by human DCs is unlikely to occur. In
accordance with such an interpretation of the published literature, we
found no inhibition of IFN- plus LPS induced apoptosis by the NO
synthase inhibitor L-NMMA
(NG-monomethyl-L-arginine, monoacetate) (data not shown).
Apart from being induced directly in the DCs, alternatively, apoptosis
might be mediated indirectly, for instance, through the action of T
cells expressing Fas ligand. Both sensitivity as well as resistance of
human monocyte-derived DCs to Fas-mediated apoptosis has been
described.40-42 In preliminary experiments, cross-linked
Fas ligand neither induced nor a blocking CD95 Fas antibody reduced
apoptosis in our system (not shown). Most of our DC cell preparations
based on density gradient centrifugation purified monocytes contained
between 5% and 15% T cells. However, further purification and
depletion of these T cells by FACS sorting did not have any influence
on the apoptosis induction in response to IFN- plus LPS (not shown),
making an involvement of contaminating T cells unlikely.
Regardless of the intracellular mechanism responsible for the observed
apoptosis, what is the biological relevance of our finding? It is
conceivable that bacterial products and sufficiently high
concentrations of type I IFNs could coincide during bacterial infections, because bacteria and their products have been shown to
induce type I IFNs in monocyte and macrophage cells at least under
certain circumstances.2-4,10 Although LPS directly induces IFN- in GM-CSF-primed human monocytes4 and human
DCs,10 we have not seen marked apoptosis in response to
LPS alone nor has such a phenomenon been described to our knowledge.
This phenomenon could be due to a requirement of IFN priming before LPS
can subsequently induce apoptosis, which would not occur if the type I
IFN accumulation is delayed during culture. Indeed, such a critical
timing of both stimuli was described with bovine
macrophages.29 However, in vivo LPS-induced IFN- could
make other monocytic cells or DCs not yet in contact with the LPS
susceptible to subsequent activation-induced cell death. Monocytes from
septic patients might be primed by type I IFNs by such a mechanism and
indeed have been reported to display a predilection to undergo
apoptosis in response to LPS.43
Type I IFNs are produced in large amounts by the rare type 2 DCs9,16,17 and at much lower levels by other more frequent cells such as monocytes or B cells.9 Usually, this
synthesis of type I IFN by activated cells appears to be only
transient,44 resulting in elevated type I IFN serum levels
in acute but not in chronic viral infections.5-7 Thus,
cell death by coincidence of IFN- priming and bacterial activation
seems less likely in chronic viral infections but still could occur,
for instance in bacterially colonized tissues (ie, the upper
respiratory and the gastrointestinal tract during an acute viral illness).
Apart from infectious situations, a prolonged and more systemic
presence of type I IFNs might also be achieved during treatment with
type I IFNs, as currently employed in hepatitis C, multiple sclerosis,
or selected malignancies. This treatment could result in the permanent
priming of monocytes and immature DCs for activation-induced apoptosis
in response to any infection or other activating stimuli. Interestingly, a transient decrease in monocytes has been reported after IFN- application to healthy humans.45
IFN- and endotoxin have already been individually known to influence
the function of monocytes and derived DCs. Our study reveals that both
agents in combination are potent inducers of apoptosis in these cells,
pointing to a new and potentially relevant mechanism of
immunosuppression in infectious disease.
| |
Acknowledgments |
We thank Dr G. Mehes (CCRI, Vienna, Austria) for technical
assistance in fluorescence microscopy and Dr H. Kovar (CCRI) for expert
advice in detection of apoptosis.
| |
Footnotes |
Submitted January 3, 2001; accepted April 3, 2001.
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: Wolfgang Holter, Children's Cancer Research
Institute, Kinderspitalgasse 6, A-1090, Vienna, Austria; e-mail:
holter{at}ccri.univie.ac.at.
| |
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Abstract
Introduction