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National Jewish Medical and Research Center, Denver, CO 80206
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(but not that of
monocyte chemoattractant protein-1 (MCP-1)/JE), and increases TGF-ß
formation. Anti-TGF-ß neutralizing Abs largely reversed the
inhibitory effect of apoptotic cell uptake, and accordingly, exogenous
TGF-ß down-regulated the synthesis of the same mediators. Apoptotic
cell ingestion or TGF-ß also inhibited Mip-2 and Mip-1 gene
expression in LPS-treated J774 cells, whereas TNF- mRNA levels were
unaffected. Importantly, TGF-ß pretreatment of J774 cells did not
significantly alter chemokine and TNF mRNA stability. Finally, we found
that apoptotic cell uptake and TGF-ß did not modulate NF-
B or AP-1
DNA binding in J774 cells. We conclude that the decreased production of
chemokines and TNF resulting from apoptotic cell ingestion is largely
mediated by a common event, i.e., feedback inhibition by endogenous
TGF-ß, but involves different mechanisms. Whereas TNF- production
appears to be translationally down-regulated, the suppression of most
chemokines investigated appears to reflect transcriptional inhibition.
In a broader context, the impairment of chemokine and TNF generation by
apoptotic cell uptake might represent an important mechanism
contributing to the resolution of inflammation. An additional
consequence could be the selective recruitment of monocytes into
inflammatory sites, as MCP-1/JE production by mouse macrophages was
unaffected by apoptotic cell uptake, in contrast to other
chemokines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Whereas the identification of surface molecules involved in the
recognition of apoptotic cells by macrophages has been the subject of
sustained research efforts in recent years, much remains to be learned
about the effect of apoptotic cell phagocytosis on macrophage function.
This represents an intriguing issue, as phagocytosis of a variety of
targets normally triggers a battery of pro-inflammatory responses in
macrophages, including the generation of reactive oxygen-derived
intermediates, the release of proteolytic enzymes, the synthesis of
lipid mediators (such as leukotrienes and thromboxanes), and the
production of numerous inflammatory cytokines and growth factors. In
sharp contrast, ingestion of apoptotic cells by macrophages induces an
anti-inflammatory phenotype. For instance, it has been reported
that thromboxane B2, GM-CSF, or lysosomal enzymes
are no longer released under these conditions (5, 6). A
recent study from our laboratory significantly extended these findings
by showing that apoptotic cell uptake by human macrophages strongly
inhibits their ability to generate IL-1ß, IL-8, TNF-, and
leukotriene C4, in addition to thromboxane B2 and
GM-CSF (7). Conversely, human macrophages secreted
increased amounts of TGF-ß and PGE2 under the
same conditions, which repressed inflammatory cytokine release in an
autocrine fashion (7). Importantly, apoptotic cells
opsonized with Abs behaved as inflammatory phagocytic stimuli
(7), thereby emphasizing the unique effect of apoptotic
cell uptake on macrophage function. Collectively, these studies suggest
that macrophages not only contribute to the resolution of inflammation
through apoptotic cell removal, but also by actively suppressing
inflammatory mediator production. A similar process may conceivably
prevent the onset of inflammatory responses in situations such as
tissue remodeling.
Despite recent advances (outlined above) in our understanding of how apoptotic cell uptake modifies macrophage function, many essential questions remain to be elucidated. In particular, the prominent role played by endogenous TGF-ß in this context prompted us to investigate the molecular mechanisms involved in its action. For this purpose, we sought a cellular model in which TGF-ß would account for most of the effect of apoptotic cell uptake. In addition, our previous finding that IL-8 production was down-regulated following apoptotic cell ingestion led us to determine whether other chemokines might be similarly affected. We now report that in mouse J774 macrophages, apoptotic cell ingestion results in the early release of TGF-ß, and that the latter mainly accounts for the decreased production of TNF and several chemokines. More importantly, TGF-ß was found to exert this action by acting at the level of gene transcription or protein translation, depending on the inflammatory mediator. Finally, the sparing of the chemokine monocyte chemoattractant protein-1 (MCP-1)3 from this suppressive effect may selectively recruit monocytes into inflamed lesions, presumably to participate in the reparative process.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neutralizing anti-TGF-ß (pan TGF) and anti-mouse IL-10
Abs, as well as recombinant mouse TNF-, recombinant human TGF-ß1,
and recombinant human IL-10, were from R&D Systems (Minneapolis, MN). A
commercial kit for preparing F(ab')2 fragments
was from Pierce (Rockford, IL). LPS (LPS, from Escherichia
coli 0111:B4) was from List Biological Laboratories (Campbell,
CA); and PGE2 and indomethacin were from the
Cayman Chemical (Ann Arbor, MI). For EMSA analyses, an oligonucleotide
containing tandemly repeated NF-B sites identical to those of the
HIV promoter (5'-gatcaGGGACTTTCCgctgGGGACTTTCC-3') was synthesized,
whereas an oligonucleotide containing a consensus AP-1 sequence
(5'-cgcttgaTGAGTCAgccggaa-3') was from Promega (Madison, WI).
Poly(dI-dC) and T4 polynucleotide kinase were from Pharmacia (Uppsala,
Sweden); [
-32P]ATP and
[-32P]UTP were from ICN (Cleveland, OH).
Acetylated BSA, diisopropyl fluorophosphate (DFP), and
phenylethanesulfonyl fluoride (PMSF) were from Sigma-Aldrich (St-Louis,
MO). Aprotinin, leupeptin, pepstatin, 4-(2-aminomethyl)benzenesulfonyl
fluoride (AEBSF) and Nonidet P-40 (NP-40) were from Boehringer Mannheim
(Mannheim, Germany). Polystyrene flasks and plates for cell culture
were from Becton Dickinson (Lincoln Park, NJ). DMEM and RPMI 1640 were
from Life Technologies (Gaithersburg, MD), X-Vivo 10 medium was from
BioWhittaker (Walkersville, MD), and endotoxin-free FCS was from
HyClone (Logan, UT). All other reagents were molecular biology
grade.
Cell isolation and stimulation
Mouse J774A.1 macrophages (obtained from the American Type
Culture Collection (ATCC), Manassas, VA) were cultured in DMEM
supplemented with 10% heat-inactivated FCS, 2 mM
L-glutamine, 100 µg/ml streptomycin, and 100 U/ml
penicillin under a humidified 5% CO2 atmosphere
at 37°C. Human Jurkat cells (also from the ATCC) were cultured
(37°C, 5% CO2) in RPMI 1640 supplemented with
10% FCS, L-glutamine, and antibiotics and were used as a
source of apoptotic cells. Apoptosis was induced by irradiating Jurkat
cells under UV light (254 nm) for 10 min; cells were then further
cultured for 3.5 h. This typically resulted in >70% apoptotic
cells (as assessed morphologically), with 
5% necrotic cells (as
assessed by trypan blue staining). Apoptotic cells were used at a 5:1
ratio (Jurkat:J774 cells), as this optimally inhibited inflammatory
mediator production, as previously reported (7). Under
these conditions, a substantial proportion of J774 macrophages engulfed
apoptotic Jurkat cells or human neutrophils, whereas nonapoptotic
targets were not ingested (Fig. 1
). The
percentages of ingestion depicted in Fig. 1 are consistent with
previously published observations made in J774 cells (8
and do not reflect their general ability to ingest particles, as latex
beads are engulfed by virtually all macrophages (our unpublished
data).
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Mouse J774 cells were cultured in 12-well plates until nearly confluent, washed twice with PBS, and further cultured in X-Vivo 10 medium (without serum) in the presence or absence of the stimuli or apoptotic cells. The necessity to stimulate the cells in serum-free medium stems from the presence of high levels of latent TGF-ß in serum, which interferes with the detection of cell-derived TGF-ß by ELISA. Following stimulation, culture supernatants were collected at the indicated times, centrifuged (1500 x g for 5 min at 4°C) to pellet intact cells, and snap-frozen in liquid nitrogen before storage at -70°C. When cell-associated cytokines were measured, 0.5 ml of ice-cold PBS was added to the wells, and macrophages were gently scraped and combined with the small cellular pellet resulting from the centrifugation of culture supernatants. Pooled cells were then spun (2000 x g for 5 min at 4°C); the resulting pellets were snap-frozen in liquid nitrogen and stored at -70°C. Immediately before ELISA analysis, the cell pellets were resuspended in 1 ml of cold lysis buffer (PBS supplemented with 0.5% NP-40, 5 mM EDTA, 0.5 mM AEBSF, 1 mM PMSF, and 10 µg/ml each of aprotinin, leupeptin, and pepstatin A), vigorously mixed for 30 s, and centrifuged (15000 x g for 10 min at 4°C) to remove insoluble material. Cytokine concentrations in culture supernatants or in the corresponding cell lysates were analyzed by ELISA using commercially available kits, according to the manufacturer’s (R&D Systems) instructions. For TGF-ß1, samples were acid treated (to activate latent TGF-ß) and later neutralized before ELISA analysis, as recommended by the kit manufacturer (Genzyme, Cambridge, MA).
Ribonuclease protection assays (RPA)
Mouse J774 cells were cultured and stimulated as described above
for the ELISA experiments. After the desired incubation times, culture
medium was carefully removed, and cell lysates (prepared from 
3
x 106 cells) were directly analyzed in RPA using
the DirectProtect Lysate RPA kit (Ambion, Austin, TX) and multiprobe
templates mCK3b or mCK5 (PharMingen, San Diego, CA) that had been
transcribed with T7 polymerase and labeled with
[-32P]UTP using a Riboquant kit
(PharMingen). Samples were electrophoresed on 5% acrylamide sequencing
gels containing 8 M urea in 0.5x TBE; dried gels were exposed to
PhosphorScreens (Molecular Dynamics, Sunnyvale, CA) before quantitation
of RNA bands using a STORM 840 PhosphorImager and ImageQuant software
(Molecular Dynamics). For mRNA stability experiments, cells were
pretreated with or without 5 ng/ml TGF-ß for 60 min, stimulated with
1 ng/ml LPS, and actinomycin D was added at a final concentration of 5
µg/ml. Cells were then further cultured for the indicated times, and
processed for RPA analysis as described above.
Nuclear extract preparation and EMSA
Mouse J774 cells were cultured in 6-well plates until nearly
confluent; cells were washed twice with PBS and further cultured in
X-Vivo 10 medium (without serum) in the presence or absence of the
stimuli or apoptotic cells. Incubations were stopped by adding an equal
volume of ice-cold X-Vivo 10 medium to the wells, and nuclear extracts
were then prepared by a modified Dignam procedure (9), as
follows. Cells were collected by gentle scraping and centrifuged at
1000 x g for 3 min at 4°C. The resulting cell
pellets were resuspended in ice-cold lysis buffer (10 mM HEPES (pH
7.90), 10 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 0.5
mM EGTA, and 0.5 mM DTT) containing an antiprotease mixture (0.5 mM
DFP, 0.5 mM AEBSF, 1 mM PMSF, and 10 µg/ml each of aprotinin,
leupeptin, and pepstatin A, final concentrations). After a 10-min
incubation on ice, an equal volume of lysis buffer containing the
antiprotease mixture as well as 0.2% NP-40 was added (to yield a final
concentration of 0.1% NP-40). Samples were immediately vortex mixed
for 15 s before centrifugation at 1200 x g (5 min
at 4°C). The resulting nuclear pellets were washed once with lysis
buffer containing the antiprotease mixture before being resuspended in
ice-cold nuclear extraction buffer (20 mM HEPES (pH 7.90), 400 mM NaCl,
1.5 mM MgCl2, 1 mM EDTA, 0.5 mM DTT, and 10%
(v/v) glycerol) containing the antiprotease mixture. After a 20-min
incubation on ice (with frequent mixing), samples were spun
(15,000 x g for 15 min at 4°C), and supernatants
(the nuclear extracts) were snap-frozen in liquid nitrogen and stored
at -70°C. Aliquots of the extracts were routinely processed for
protein content determination. Nuclear extracts (amounts used are
specified in the figure legends) were analyzed in EMSA as follows. For
NF-B binding, the extracts were processed exactly as described
previously (10) using 30,000 cpm of oligonucleotide probe
(end-labeled with T4 kinase). For the analysis of AP-1 DNA binding,
nuclear extracts were incubated in a modified binding buffer (20 mM
HEPES (pH 7.50), 50 mM KCl, 1 mM EDTA, 5 mM DTT, 0.1% NP-40, and 6%
glycerol) supplemented with 0.4 µg poly(dI-dC) and 8 µg acetylated
BSA before the addition of 30,000 cpm of labeled oligonucleotide probe.
For supershift experiments, binding reactions were conducted in the
presence of specific antisera to individual NF-B/Rel, Jun, or Fos
proteins (30 min at 4°C), before the addition of
32P-labeled probes. Samples were electrophoresed
on 6% acrylamide gels at 4°C in 0.5x TBE; dried gels were then
exposed to PhosphorScreens (Molecular Dynamics).
Statistical analyses
Where mentioned, statistical significance was assessed using the Student’s t test for paired data (one-tailed).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
chemokines in mouse macrophages
Ingestion of apoptotic cells by human monocyte-derived macrophages
(HMDM) profoundly alters their ability to generate pro-inflammatory
cytokines and IL-8 (6, 7). We first examined whether
apoptotic cell uptake by murine macrophage-like J774 cells similarly
affects the production of a cytokine (TNF-), and of C-X-C chemokines
(Mip-2, KC) and C-C chemokines (Mip-1, MCP-1). As shown in Fig. 2, apoptotic cell uptake markedly
decreased the ability of LPS-treated J774 cells to secrete TNF-,
Mip-2, KC, and Mip-1, whereas the release of MCP-1/JE was not
significantly affected. By comparison, the LPS-elicited release of
IL-10 was only slightly suppressed following apoptotic cell uptake
(Fig. 2). Finally, the secretion of TGF-ß1 was
substantially increased in J774 cells that had ingested apoptotic
cells, both in the presence and absence of LPS (Fig. 2). Similar
results were obtained when J774 cells were stimulated with 10 ng/ml LPS
instead of 1 ng/ml, or when apoptotic cell uptake took place 3 h
before LPS stimulation (data not shown). Table I summarizes the effect of apoptotic cell
uptake on chemokine and TNF- release in J774 cells.
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and
chemokine release resulting from apoptotic cell uptake
Our previous work showed that the effect of apoptotic cell uptake
on cytokine production by human macrophages involved several endogenous
mediators, foremost among which was TGF-ß
(7). For this reason, and because TGF-ß1 is released
early following apoptotic cell uptake by J774 cells, we first examined
whether neutralizing anti-TGF-ß F(ab')2 Abs
would affect inflammatory cytokine release under these conditions. The
need to use F(ab')2 fragments stems from the fact
that whole Abs (as well as irrelevant rabbit IgG) potently induced the
release of TNF and chemokines by themselves. As shown in Fig. 3A, the apoptotic
cell-mediated inhibition of chemokine and TNF- release was
substantially reversed by the anti-TGF-ß
F(ab')2 Abs both in unstimulated and LPS-treated
J774 cells. Neither the constitutive nor the LPS-induced secretion of
these mediators was affected by the anti-TGF-ß
F(ab')2 Abs alone (Fig. 3A),
consistent with the fact that little or no TGF-ß is detected in J774
macrophages that did not ingest apoptotic cells (Fig. 2
).
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in J774 cells that had ingested
apoptotic cells (Fig. 3B). Finally, neutralizing
anti-IL-10 Abs only marginally affected the release of these
mediators by LPS-treated J774 cells, whether or not the macrophages had
ingested apoptotic targets (Fig. 3C). This is consistent
with the fact that J774 cells release very little IL-10 under these
conditions (Fig. 2). Taken together, these results clearly show that in
J774 cells, the repression of TNF- and chemokine release resulting
from apoptotic cell uptake is mainly mediated by endogenous TGF-ß (as
opposed to IL-10, PAF, or prostanoids). Thus, the interaction between
TGF-ß and J774 cells is likely to represent a useful model to
investigate the mechanisms whereby apoptotic cell ingestion regulates
inflammatory mediator production.
Exogenous TGF-ß mimics the effect of apoptotic cell uptake on the
release of TNF- and chemokines by macrophages
The above findings led us to investigate the effect of exogenous
TGF-ß on the secretion of TNF- and chemokines. As shown in Fig. 4, preincubation of J774 cells with
TGF-ß1 markedly inhibited the LPS-induced release of TNF-, Mip-2,
KC, and Mip-1, whereas MCP-1/JE release was only marginally
decreased. This TGF-ß-mediated inhibition was already evident at
early time points (Fig. 4) and was found to be dose-dependent, a
threshold effect being observed using as little as 100 pg/ml TGF-ß1
(data not shown). Table II summarizes the
effect of exogenous TGF-ß on chemokine and TNF- release in J774
cells. In addition, we found that pretreatment of J774 cells with
TGF-ß followed by washing and subsequent LPS stimulation in the
absence of TGF-ß was just as effective in inhibiting inflammatory
mediator release as when J774 cells were stimulated with LPS in the
continued presence of TGF-ß (Fig. 5A). Thus, mere exposure of
the macrophages to TGF-ß is sufficient to down-regulate TNF- and
chemokine secretion. Finally, pretreatment of J774 macrophages with
TGF-ß for up to 20 h still resulted in a marked inhibition of
chemokine and TNF secretion (Fig. 5B); addition of TGF-ß
1 h after LPS stimulation also led to the same result (Fig. 5B).
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by J774 macrophages (Fig. 6); at
a concentration of 100 U/ml, IL-10 was about as effective as 5 ng/ml
TGF-ß1. This is consistent with previous reports showing that IL-10
inhibits LPS-induced cytokine/chemokine secretion in macrophages
(11, 12, 13, 14). A notable exception was MCP-1/JE, whose
secretion was dramatically augmented by IL-10, even in LPS-stimulated
cells (Fig. 6). That IL-10 can increase MCP-1 production has already
been reported in monocytes/macrophages (15, 16, 17), as well
as in fibroblasts and endothelial cells (18, 19). Thus,
while endogenous IL-10 does not appear to mediate the inhibitory effect
of apoptotic cells in our system, this cannot be attributed to a poor
responsiveness of J774 cells to IL-10.
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We next investigated whether the inhibition of inflammatory
mediator release by either apoptotic cell ingestion or exogenous
TGF-ß occurs at the level of secretion and/or protein synthesis. To
this end, cell-associated levels of chemokines and TNF- were
analyzed by ELISA. Fig. 7 shows that the
bulk of the chemokines being synthesized is secreted, and that
chemokines (Mip-2, KC, Mip-1) are more efficiently secreted than
TNF-. More importantly, both TGF-ß and apoptotic cell uptake
inhibited the cell-associated levels of chemokines and TNF- to a
similar extent as when secretion was examined (Fig. 7). Thus, apoptotic
cells and TGF-ß appear to inhibit inflammatory mediator production by
acting at the level of, or upstream from, translation.
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gene expression
To determine whether the inhibition of inflammatory mediator
synthesis is paralleled by similar changes in mRNA levels, RPA analyses
were performed using J774 cells that had either ingested apoptotic
Jurkat cells or been pretreated with TGF-ß. In the former instance, a
human GAPDH band (originating from the ingested Jurkat cells)
hybridized with our RNA probes, which precluded a reliable quantitation
of the mouse cytokine mRNA bands in initial experiments. However, this
difficulty was overcome by deliberately overrunning the gels, which
effectively resolved the human and mouse GAPDH bands (Fig. 8A). As shown in Fig. 8B, the constitutive gene expression of the inflammatory
mediators was generally unaltered by TGF-ß or apoptotic cell uptake,
with the exception of TNF- and Mip-2 mRNA, which were modestly
down-regulated. In LPS-stimulated cells, however, TGF-ß pretreatment
and apoptotic cell uptake markedly inhibited the accumulation of Mip-2
and Mip-1 mRNA, whereas that of TNF- mRNA was not significantly
affected (Fig. 8B). In contrast, the LPS-elicited
accumulation of MCP-1/JE mRNA was enhanced following TGF-ß
pretreatment (Fig. 8B). Finally, TGF-ß1 mRNA levels were
unaffected by exogenous TGF-ß, apoptotic cell uptake, and/or LPS
stimulation (Fig. 8B). Noteworthy is that only the ß1
isoform of TGF was consistently detected in J774 cells, as mRNA
encoding the ß2 and ß3 isoforms were either weak or undetectable
(Fig. 8A). In a final set of experiments, we also determined
that chemokine and TNF- mRNA stability remained essentially the
same, whether or not J774 cells were pretreated with TGF-ß before LPS
stimulation (Fig. 9). Thus, it appears
that apoptotic cell uptake and TGF-ß inhibit stimulated TNF-
production via a translational effect, whereas the decreased production
of Mip-1 and Mip-2 probably involves transcriptional events.
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B or AP-1 activation
We finally investigated whether TGF-ß or apoptotic cell uptake
alter the LPS-induced activation of transcription factors NF-B and
AP-1 in J774 cells. The rationale for these experiments is that the
LPS-inducible gene expression of the inflammatory mediators under
investigation is largely dependent upon the activation of NF-B or
AP-1, or both (20, 21, 22, 23, 24, 25, 26). As shown in Fig. 10A, TGF-ß did not
markedly affect the constitutive NF-B DNA-binding activity detected
in nuclear extracts from J774 cells, although a small increase (up to
2-fold) was observed in three of nine independent experiments. By
contrast, stimulation of J774 cells with LPS consistently resulted in a
dramatic induction of NF-B activity (Fig. 10A).
Supershift analyses revealed that both the constitutive and
LPS-inducible NF-B complexes contain RelA and p50 NF-B1 (data not
shown). Pretreatment of J774 cells with TGF-ß or
PGE2 before LPS stimulation did not alter the
intensity of the NF-B signal (Fig. 10A), regardless of
the time of addition of TGF-ß or PGE2 (from 90
min before to 30 min after LPS), and regardless of the time elapsed
after LPS stimulation (from 15 to 180 min). Similar results were
obtained when HMDM or mouse RAW 264.7 cells were used instead of J774
cells. When the same samples were analyzed in EMSA using an AP-1 probe,
constitutive AP-1 binding was somewhat decreased by TGF-ß and
moderately enhanced by PGE2, whereas it was
potently increased by LPS (Fig. 10B); the AP-1 complex was
found to contain c-Jun, JunD, and c-Fos (data not shown). Pretreatment
of J774 cells with TGF-ß moderately enhanced AP-1 activation by LPS,
whereas PGE2 pretreatment had no effect (Fig. 10B). Thus, it does not appear that TGF-ß inhibits
inflammatory mediator production at the level of NF-B or AP-1
binding in macrophages.
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C, apoptotic cell uptake by itself moderately enhanced
constitutive NF-B binding, whereas it had little or no effect on
NF-B activation by LPS; similar results were obtained using an AP-1
probe (data not shown). Thus, the modulation of inflammatory mediator
production resulting from apoptotic cell uptake is not likely to
reflect altered binding of these transcription factor in
macrophages. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and in LPS- or zymosan-stimulated HMDM
(7). Thus, in macrophages that have ingested apoptotic
cells, the suppression of inflammatory mediator production (and
induction of TGF-ß) appears to be a general phenomenon that does not
depend on the nature of the apoptotic target, the macrophage activation
state, or the type of stimulus used. The importance of this phenomenon
is illustrated by the fact that chemokine production by J774 cells
remained inhibited for 20 h after exposure to TGF-ß (Fig. 5). We also report that the inhibitory effect of apoptotic cell uptake is largely mediated by endogenous factors, among which TGF-ß appears to play a paramount role. First, TGF-ß is the only cytokine examined, whose release was up-regulated following ingestion of apoptotic cells, both in unstimulated and LPS-treated J774 cells. More compelling evidence is that anti-TGF-ß neutralizing Abs largely reversed the inhibitory effects of apoptotic cell uptake. Finally, exogenously added TGF-ß closely mimicked the suppressive action of apoptotic cell ingestion. These observations, together with the fact that endogenous TGF-ß plays a similar role in primary human macrophages (7) and murine bone marrow-derived macrophages, suggest that TGF-ß represents the main endogenous factor mediating the inhibitory effect of apoptotic cells. Although this phenomenon may also involve other endogenous factors (namely, PGE2 and PAF) in HMDM (7), both products proved to be potent inducers TGF-ß release in these cells (7). In J774 macrophages, however, the suppressive effect of apoptotic cell uptake was unaffected by a PAF receptor antagonist and a cyclooxygenase inhibitor. Accordingly, J774 cells fail to synthesize detectable amounts of PAF in response to 1 ng/ml LPS (P. Henson and R. Murphy, unpublished data), and only release very small amounts of PGD2 and E2, amounting to <140 pM after 2–4 h (C. Amura and P. Henson, unpublished data). Such PGE2 levels are in keeping with results obtained in previous studies performed in J774 cells (27). Therefore, our data indicate that endogenous TGF-ß can effectively mediate the action of apoptotic cells without an additional requirement for prostanoids or PAF. Such a central role for TGF-ß emphasizes the necessity to elucidate the mechanisms regulating TGF-ß1 induction in macrophages undergoing phagocytosis of apoptotic cells. In this regard, we showed herein that these mechanisms are probably posttranscriptional because TGF-ß1 mRNA levels were unaffected by apoptotic cell uptake. Whether the regulation involves mRNA stabilization, increased translation or secretion, or activation of latent TGF-ß currently represents an area of ongoing investigation in our laboratory.
The fact that neutralizing anti-TGF Abs did not completely block
the effect of apoptotic cells raises the possibility that additional
endogenous factors may also act as minor participants. Although the
anti-inflammatory cytokine, IL-10, would theoretically represent an
attractive candidate, its secretion was not increased following
apoptotic cell uptake by J774 cells (this paper) or HMDM
(7). Accordingly, we observed that although MCP-1/JE
release was dramatically up-regulated by IL-10, no increase in MCP-1/JE
secretion occurred in J774 cells that had ingested apoptotic targets.
Finally, anti-IL-10-neutralizing Abs only marginally increased the
production of TNF-, KC, Mip-2, and Mip-1 in LPS-stimulated J774
cells, whether apoptotic cells had been engulfed or not. That similar
observations were made in HMDM (7), further argues against
a significant role for endogenous IL-10. In contrast to macrophages,
human monocytes have been reported to release more IL-10 following
coincubation with apoptotic lymphocytes and to secrete less TNF-,
IL-1ß, and IL-12 under these conditions (28). Despite
this, no evidence for an autocrine role of IL-10 was presented in that
study. This being said, it remains likely that IL-10 would contribute
to inhibit macrophage function in vivo, should the cytokine be produced
by other cells in the microenvironment.
Our investigation of the mechanisms underlying the suppressive effect
of apoptotic cell uptake and endogenous TGF-ß on macrophage function
revealed that the decreased TNF- synthesis and release by
LPS-stimulated J774 cells was not accompanied by changes in gene
expression or mRNA stability, which points to a translational
inhibition. This conclusion is consistent with studies performed in
mouse peritoneal macrophages, in which TGF-ß was found to reduce the
LPS-elicited production of TNF- by acting at a posttranscriptional
step (29, 30). By contrast, we reported that apoptotic
cell uptake down-regulated TNF- mRNA levels in HMDM
(7). This may be related to the concurrent increase
PGE2 synthesis observed under these conditions,
as PGE2 has long been known to inhibit TNF gene
expression in LPS-stimulated mouse macrophages and HMDM (31, 32). In contrast to TNF-, the secretion, total protein
synthesis, and mRNA accumulation of Mip-2, Mip-1 and KC were all
down-regulated to a similar extent in LPS-treated J774 cells following
TGF-ß exposure or apoptotic cell uptake, whereas no significant
changes in mRNA stability were observed. This indicates that
transcriptional inhibition is the mechanism whereby TGF-ß represses
inflammatory chemokine production. It must be stressed, however, that
these results do not completely rule out an additional inhibitory
mechanism at the level of translation, especially in view of the fact
that addition of TGF-ß 1 h after LPS inhibited chemokine
production to a similar extent as when TGF-ß was added before LPS
(Fig. 5B). This being said, the exact mechanism by which
transcription is inhibited remains elusive, in view of the fact that
TGF-ß pretreatment and apoptotic cell uptake did not alter NF-B or
AP-1 DNA binding in LPS-stimulated J774 cells or HMDM. One possibility
is that TGF-ß and apoptotic cell uptake somehow impair the ability of
NF-B to transactivate chemokine promoters, despite normal binding.
However, this implies that the expression of other B-dependent genes
(such as the one encoding TNF-) would be similarly decreased, and we
have shown that this is not the case. Moreover, alterations of the
transactivating potential of NF-B complexes would not explain the
decreased activity of the Mip-1 gene, which does not contain B or
AP-1 motifs in its LPS response region (22).
Alternatively, TGF-ß could promote the binding of factors to
repressor elements within chemokine gene promoters. Studies are in
progress to further delineate the nature of the transcriptional events
being affected by apoptotic cell uptake and TGF-ß in activated
macrophages.
In contrast to the other chemokines, MCP-1 release was only marginally
inhibited by TGF-ß, and this was accompanied by a moderate
up-regulation at the mRNA level. Although the protein and mRNA data are
in apparent contradiction in the latter instance, this may reflect a
translational inhibition by TGF-ß (as observed with TNF-, and
possibly with the other chemokines as well), especially because no
difference in MCP-1/JE mRNA stability was noted between J774 cells
cultured in the presence or absence of TGF-ß. The latter observation
also indicates that an increase in MCP-1 mRNA levels is likely to
result from an enhanced transcription of the MCP-1/JE gene. In this
regard, several studies have established that MCP-1/JE gene
transcription is principally AP-1 driven (24, 25, 26) and
accordingly, we found that TGF-ß pretreatment moderately enhanced
AP-1 activation in LPS-treated J774 cells. Thus, TGF-ß appears to
regulate the LPS-elicited production of MCP-1/JE by acting on at least
two discrete steps. By comparison, MCP-1/JE production and gene
expression were unaltered by apoptotic cell uptake. This may stem from
the fact that exogenous TGF-ß generally exerts more pronounced
effects than apoptotic cell ingestion, presumably because J774 cells
are exposed to less TGF-ß in the latter instance. From a more general
standpoint, our data are consistent with the fact that MCP-1 mRNA
levels are also enhanced by TGF-ß in other cell types, such as
astrocytes, osteoblasts, and bone marrow stromal cells
(33, 34, 35). However, our observations are in contrast with a
recent report (36) in which similar concentrations of
TGF-ß inhibited the gene expression and release of MCP-1/JE in mouse
primary macrophages and murine macrophage cell lines. Although cells
were pretreated with TGF-ß for 6 h in that study, we only
observed a slight decrease in MCP-1/JE release under these conditions
(our unpublished data). Macrophages were also stimulated with a LPS
concentration 1000-fold higher than the one used herein, but we find it
unlikely that this would explain our divergent results. Thus, unless
the latter are related to the different J774 sublines used (i.e.,
J774.2 vs J774A.1 herein), we can offer no explanation for this
discrepancy.
In a broader context, the cytokine production profile of macrophages
that have ingested apoptotic cells could have profound implications in
an inflammatory setting. In addition to producing lesser amounts of
pro-inflammatory cytokines (such as TNF- and IL-1ß), macrophages
are likely to exert an overall immunosuppressive action in the
microenvironment through the production of TGF-ß. Similarly,
macrophages are believed to represent a major source of neutrophil
chemoattractants; therefore, the decreased production of chemokines
such as Mip-2 and KC by macrophages is likely to limit neutrophil
recruitment into inflammatory sites, and the related tissue damage.
Finally, the fact that MCP-1/JE production is not attenuated under the
same conditions might result in the selective recruitment of monocytes
into inflammatory sites. This could further contribute to the
resolution of the inflammatory reaction, in view of the reported
ability of monocytes to release anti-inflammatory cytokines such as
IL-10 once they come in contact with apoptotic cells (28).
Importantly, all of the effects reported herein were achieved despite
the fact that only one quarter of the J774 macrophages actually
ingested apoptotic targets, which further emphasizes the
anti-inflammatory potential of this process. Collectively, the
consequences of apoptotic cell uptake by macrophages outlined above
lend support to the notion that apoptotic cell clearance from inflamed
sites must constitute an important mechanism for the resolution of
inflammation. Conversely, dysfunctions in the ability of macrophages to
ingest apoptotic cells (and to release TGF-ß) might represent an
important component of several inflammatory pathologies, as illustrated
by the fact that TGF-ß1 knockout mice are afflicted by severe and
generalized inflammation (37, 38).
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Acknowledgments |
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Footnotes |
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2 Address correspondence to Dr. Patrick P. McDonald, Centre de Recherche Clinique, CUSE-Fleurimont, 3001 12th Avenue North, Sherbrooke, Quebec, Canada J1H 5N4. E-mail address:
3 Abbreviations used in this paper: MCP-1, monocyte chemoattractant protein-1; DFP, diisopropyl fluorophosphate; PMSF, phenylethanesulfonyl fluoride; AEBSF, 4-(2-aminomethyl)benzenesulfonyl fluoride; HMDM, human monocyte-derived macrophages; Mip, macrophage inflammatory protein; NP-40, Nonidet P-40; PAF, platelet-activating factor; RPA, ribonuclease protection assay.
Accepted for publication September 16, 1999.
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R. M. Clancy, A. D. Askanase, R. P. Kapur, E. Chiopelas, N. Azar, M. E. Miranda-Carus, and J. P. Buyon Transdifferentiation of Cardiac Fibroblasts, a Fetal Factor in Anti-SSA/Ro-SSB/La Antibody-Mediated Congenital Heart Block J. Immunol., August 15, 2002; 169(4): 2156 - 2163. [Abstract] [Full Text] [PDF]
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F. Bucchieri, S. M. Puddicombe, J. L. Lordan, A. Richter, D. Buchanan, S. J. Wilson, J. Ward, G. Zummo, P. H. Howarth, R. Djukanovic, et al. Asthmatic Bronchial Epithelium Is More Susceptible to Oxidant-Induced Apoptosis Am. J. Respir. Cell Mol. Biol., August 1, 2002; 27(2): 179 - 185. [Abstract] [Full Text] [PDF]
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S. D. Kobayashi, J. M. Voyich, C. L. Buhl, R. M. Stahl, and F. R. DeLeo Global changes in gene expression by human polymorphonuclear leukocytes during receptor-mediated phagocytosis: Cell fate is regulated at the level of gene expression PNAS, May 14, 2002; 99(10): 6901 - 6906. [Abstract] [Full Text] [PDF]
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B. Hu, A. Punturieri, J. Todt, J. Sonstein, T. Polak, and J. L. Curtis Recognition and phagocytosis of apoptotic T cells by resident murine tissue macrophages require multiple signal transduction events J. Leukoc. Biol., May 1, 2002; 71(5): 881 - 889. [Abstract] [Full Text] [PDF]
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Y. Q. Xiao, K. Malcolm, G. S. Worthen, S. Gardai, W. P. Schiemann, V. A. Fadok, D. L. Bratton, and P. M. Henson Cross-talk between ERK and p38 MAPK Mediates Selective Suppression of Pro-inflammatory Cytokines by Transforming Growth Factor-beta J. Biol. Chem., April 19, 2002; 277(17): 14884 - 14893. [Abstract] [Full Text] [PDF]
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B. Endlich, D. Armstrong, J. Brodsky, M. Novotny, and T. A. Hamilton Distinct Temporal Patterns of Macrophage-Inflammatory Protein-2 and KC Chemokine Gene Expression in Surgical Injury J. Immunol., April 1, 2002; 168(7): 3586 - 3594. [Abstract] [Full Text] [PDF]
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A. M. Hohlbaum, M. S. Gregory, S.-T. Ju, and A. Marshak-Rothstein Fas Ligand Engagement of Resident Peritoneal Macrophages In Vivo Induces Apoptosis and the Production of Neutrophil Chemotactic Factors J. Immunol., December 1, 2001; 167(11): 6217 - 6224. [Abstract] [Full Text] [PDF]
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P.L.E.M. van Lent, R. Licht, H. Dijkman, A.E.M. Holthuysen, J.H.M. Berden, and W.B. van den Berg Uptake of apoptotic leukocytes by synovial lining macrophages inhibits immune complex-mediated arthritis J. Leukoc. Biol., November 1, 2001; 70(5): 708 - 714. [Abstract] [Full Text] [PDF]
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T. Magnus, A. Chan, O. Grauer, K. V. Toyka, and R. Gold Microglial Phagocytosis of Apoptotic Inflammatory T Cells Leads to Down-Regulation of Microglial Immune Activation J. Immunol., November 1, 2001; 167(9): 5004 - 5010. [Abstract] [Full Text] [PDF]
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L. Tourneur, B. Malassagne, F. Batteux, M. Fabre, S. Mistou, E. Lallemand, P. Lores, and G. Chiocchia Transgenic Expression of CD95 Ligand on Thyroid Follicular Cells Confers Immune Privilege upon Thyroid Allografts J. Immunol., August 1, 2001; 167(3): 1338 - 1346. [Abstract] [Full Text] [PDF]
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M. Adib-Conquy, K. Asehnoune, P. Moine, and J.-M. Cavaillon Long-term-impaired expression of nuclear factor-{kappa}B and I{kappa}B{alpha} in peripheral blood mononuclear cells of trauma patients J. Leukoc. Biol., July 1, 2001; 70(1): 30 - 38. [Abstract] [Full Text] [PDF]
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 G. Wang, Y. L. Siow, and K. O Homocysteine induces monocyte chemoattractant protein-1 expression by activating NF-{kappa}B in THP-1 macrophages Am J Physiol Heart Circ Physiol, June 1, 2001; 280(6): H2840 - H2847. [Abstract] [Full Text] [PDF]
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V. A. Fadok, D. L. Bratton, L. Guthrie, and P. M. Henson Differential Effects of Apoptotic Versus Lysed Cells on Macrophage Production of Cytokines: Role of Proteases J. Immunol., June 1, 2001; 166(11): 6847 - 6854. [Abstract] [Full Text] [PDF]
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 R. E. Cocco and D. S. Ucker Distinct Modes of Macrophage Recognition for Apoptotic and Necrotic Cells Are Not Specified Exclusively by Phosphatidylserine Exposure Mol. Biol. Cell, April 1, 2001; 12(4): 919 - 930. [Abstract] [Full Text]
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D. Shiffman, T. Mikita, J. T. N. Tai, D. P. Wade, J. G. Porter, J. J. Seilhamer, R. Somogyi, S. Liang, and R. M. Lawn Large Scale Gene Expression Analysis of Cholesterol-loaded Macrophages J. Biol. Chem., November 22, 2000; 275(48): 37324 - 37332. [Abstract] [Full Text] [PDF]
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) or diluent control
(
), before stimulation with 1 ng/ml LPS for 60–90 min. Actinomycin
D was then added, and cells were collected at the indicated times;
cytokine mRNA levels were assessed by RPA, as described in the legend
to Fig. 8