| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
The Children’s Research Center, Our Lady’s Hospital for Sick Children, and
The Conway Institute of Biomolecular and Biomedical Research, University College, Dublin, Ireland
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production and only minimal
TNF-
and IL-1 cytokine production. Time-kinetic studies
demonstrated that IL-10 production was markedly accelerated in the
presence of apoptotic neutrophils, whereas there was a sustained
reduction in the production of TNF- and IL-1. This suppression of
proinflammatory production was not reversible by depletion of IL-10 or
TGF- or by addition of exogenous IFN-
. It was demonstrated, using
Transwell experiments, that monocyte-apoptotic cell contact was
required for induction of the immunosuppressive monocyte response. The
response of monocytes contrasted with that of human monocyte-derived
macrophages in which there was a reduction in IL-10 production. We
conclude from these data that interaction between activated monocytes
and apoptotic neutrophils creates a unique response, which changes an
activated monocyte from being a promoter of the inflammatory cascade
into a cell primed to deactivate itself and other
cells. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(3). This proinflammatory cytokine is the principal
mediator of the inflammatory response. TNF- induces the release of
other cytokines (IL-1, IL-6), eicosanoids, reactive oxygen species,
and the activation of the complement and coagulation cascades
(4). The second phase of the monocyte response involves
the production of IL-10, a potent immunosuppressive cytokine that is
detectable within 8 h of exposure to LPS (5). IL-10
down-regulates the production of the proinflammatory cytokines
(6) and plays a role in the development of LPS tolerance
(7) whereby, after initial stimulation with LPS, monocytes
become tolerant to further LPS challenges (8). TGF- is
also released by activated monocytes. It is a pleiotropic cytokine with
both pro- and anti-inflammatory properties (9). Its
actions on activated cells of the monocyte-macrophage lineage are
generally suppressive, including the down-regulation of TNF-,
IFN-, and monocyte chemoattractant protein-1
(MCP-1)3
(10). The balance between the production of pro- and
anti-inflammatory cytokines by the monocyte determines the
effectiveness of the inflammatory response (11). The
cytokine profile of monocytes is recognized as a critical indicator of
survival in septic patients, where a high IL-10/TNF- ratio in
febrile patients is associated with increased mortality
(12).
Monocytes are also central to the pathogenesis of chronic inflammation
(3, 13). Rheumatoid arthritis and inflammatory bowel
disease are characterized by heightened expression, at the site
of inflammation, of MCP-1 and MCP-3 (14, 15). This results
in enhanced recruitment of monocytes from the bloodstream to the site
of inflammation that generate a sustained inflammatory cascade, giving
rise to tissue destruction (16). Inhibitors of
monocyte-derived cytokines such as TNF-, using blocking Abs, has
been effective in the treatment of these conditions (17, 18).
Neutrophil apoptosis has been shown to impact on the inflammatory
response in a number of ways (19). It regulates
inflammation by reducing the number of cells capable of fuelling an
inflammatory reaction. Apoptotic cells maintain their membrane
integrity and are rapidly ingested by macrophages, thus preventing the
release of toxic granules (20). Their uptake by phagocytes
does not provoke the release of proinflammatory cytokines
(21). In fact, uptake of apoptotic neutrophils by
LPS-stimulated human monocyte-derived macrophages (HMDM) inhibits the
production of TNF-, IL-1, and IL-10 through the induction of
TGF- (22). Although the effect of
apoptotic neutrophils on HMDM has been explored extensively (19, 20, 23), their impact on cytokine production by activated blood
monocytes has not as yet been defined. However, apoptotic
neutrophils have been shown to produce a monocyte-specific chemotactic
factor, thereby contributing to the recruitment of these
proinflammatory cells to sites of inflammation (24).
Monocytes differ from mature macrophages in that they are unable to phagocytose apoptotic cells (25). Apoptotic lymphocytes have been shown to alter cytokine production in LPS-activated monocytes, including an augmentation of IL-10 (26). However, as apoptotic neutrophils and monocytes are considered to play such critical and yet opposing roles in determining the outcome of the inflammatory response, it was essential that the relationship between these two cell types be defined.
In this study we report that, through cell-cell contact, apoptotic
neutrophils interact with monocytes, altering their primary response to
LPS. This altered response is immunosuppressive in nature, with an
accelerated and exaggerated release of IL-10, enhanced production of
TGF-, and only minimal production of the proinflammatory cytokines
TNF- and IL-1-.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
IMDM and HBSS were purchased from BioWhittaker (Wokingham, U.K.). Human AB serum was purchased from Quest Biomedical (West Midlands, U.K.).
Human IFN-, human IL-10, anti-human IL-10 Ab, and anti-human
TGF- Ab were all purchased from R&D Systems (Oxon, U.K.). LPS
(Escherichia coli serotype 055:B5) was obtained from
Sigma-Aldrich (Dublin, Ireland.) Anti-human CD16(FITC) and CD14(FITC)
CD45(PE) Abs and mouse anti-human CD45 (clone 2D1H130) IgG1 Ab were
purchased from BD Biosciences (Oxford, U.K.). Rabbit anti-mouse IgG
was purchased from Dako (Cambridgeshire, U.K.).
Every effort was made to ensure minimal contamination with LPS in our system. All steps in the neutrophil and monocyte purification protocols and culturing were conducted using certified nonpyrogenic plastics (Corning/Costar, Bucks, U.K.) and media, certified to have an endotoxin level of 0.005 endotoxin U/ml (BioWhittaker). All solutions were made up with endotoxin-free water (Nanopure, Barnstead, U.K.).
Monocyte and macrophage isolation
Blood was obtained from healthy adult volunteers and placed in sterile tubes containing PBS/0.1 M EDTA (4 ml/20 ml blood). It was diluted 1/1 with PBS. PBMC were separated by density centrifugation through Ficoll-Hypaque (Lymphoprep, Nycomed, Oslo, Sweden), washed twice in cold IMDM, and resuspended in IMDM/10% human AB serum (3 x 106/ml). The mononuclear cell suspension was plated (1 ml/well) in 24-well plates (Corning/Costar, Corning, NY). After incubation for 45 min at 37°C, the nonadherent cells were removed by washing with warm HBSS containing Ca2+ and Mg2+ (BioWhittaker). After a 15-min incubation at 4°C, monocytes were retrieved by vigorous washing of the plates with cold PBS. They were washed and resuspended in IMDM without serum (0.5 x 106/ml). Cell viability, by ethidium bromide and acridine orange staining, was >90%. Monocyte purity, determined using two-color flow cytometric analysis (CD14/CD45), was >80%. Human monocyte-derived macrophages were generated from adherence-purified monocytes (1 x 106/ml) cultured for 7 days in IMEM, 10% human AB serum, and 2 mM glutamine in 24-well culture plates. The medium was changed on day 3.
Induction of apoptosis in purified human neutrophils
Neutrophils were purified from whole blood by dextran sedimentation and centrifugation on a Percoll gradient as previously described (27). The purity of the neutrophil population, as assessed by single-color flow cytometric analysis of CD15 expression, was >90%. In some experiments fresh neutrophils were suspended in IMDM (5 x 106/ml) in the absence of serum directly after isolation and added to monocyte cultures (10/1).
For the purpose of inducing apoptosis, the neutrophils (2 x 106/ml) were suspended in IMDM (BioWhittaker) with 10% autologous serum and incubated for 24 h at 37°C in 5% CO2 to induce apoptosis. They were then harvested and washed twice with HBSS without Ca2+ and Mg2+ (BioWhittaker) and resuspended in IMDM (5 x 106/ml) in the absence of 10% serum. Apoptosis at this time was >80% as determined by Annexin V positivity and loss of cell surface expression of CD16 (28). Cell viability, under UV illumination using ethidium bromide and acridine orange staining, was >90%. To opsonize apoptotic neutrophils, the apoptotic neutrophils were rotated end-to-end at 4°C with mouse anti-human CD45 Ab for 30 min. After three washes, the secondary Ab, rabbit anti-mouse IgG, was added for an additional 30 min. The cells were washed again and resuspended in IMDM (5 x 106/ml) without serum (22).
Coculture of monocytes and macrophages with apoptotic neutrophils
Purified monocytes in IMDM (0.25 x
106/ml) or HMDM were stimulated with LPS (10
µg/ml) in the presence or the absence of apoptotic neutrophils or
opsonized apoptotic neutrophils (2.5 x
106/ml) for 18 h in 24-well plates (Costar,
Bucks, U.K.). Apoptotic cells were added at the same time as the
stimulus. Supernatants were then recovered and centrifuged at 500
x g for 10 min at 4°C to remove particulate debris and
were stored in aliquots at -70°C. In time-course experiments,
supernatants were harvested at 2, 4, 6, 8, 10, and 18 h. In
experiments comparing the effects of freshly isolated and apoptotic
neutrophils, supernatants were harvested at 8 h. In some
experiments, monocytes (0.25 x 106 in 1 ml)
were separated from apoptotic neutrophils (2.5 x
106 in 0.5 ml) using a 0.4-µm pore size
Transwell filter (Costar). To investigate the role played by IL-10 and
TGF- in this assay, anti-human IL-10 Ab (10 µg/ml) and
anti-human TGF- Ab (100 µg/ml) were added to cultures. In
other experiments, IFN- was added (1–500 ng/ml) to the
cultures.
Collection of supernatants from apoptotic neutrophil cultures
After 24 h in culture at 37°C, aged apoptotic neutrophils (3 x 106/ml) were incubated in serum-free IMEM with LPS (10 µg/ml) for 18 h at 37°C. The supernatants were then recovered, centrifuged at 500 x g for 10 min at 4°C, and then filtered through a 0.2-µm pore size filter to remove all particulate matter. They were then used to resuspend freshly isolated monocytes.
These fresh monocytes (2 x 106/ml) were, in turn, incubated for 18 h at 37°C in the presence or the absence of LPS (10 µg/ml). Supernatants were harvested and stored as before.
Analysis of cytokines
Cytokine concentrations in the culture supernatants were
determined by ELISA, using TNF--matched Ab pairs (R&D Systems),
human TGF-, and IL-1 (Quantikine kits; R&D Systems), and OptEIA
human IL-10 sets (BD PharMingen, Oxford, U.K.) Assays were
performed according to the instructions provided. Color development was
assessed using a VERSAmax microplate reader (Biosciences,
Dublin, Ireland).
Statistical analysis
The Mann-Whitney nonparametric test was preformed using the software SPSS for Windows.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
There is growing evidence that apoptotic neutrophils have an
active role to play in the regulation and resolution of inflammation
following phagocytosis by macrophages and dendritic cells (19, 29). However, their influence on activated blood monocytes,
freshly recruited to sites of inflammation, has not been defined.
Freshly isolated human monocytes (0.25 x
106/ml) were incubated with LPS (10 µg/ml) for
18 h in the presence or the absence of aged neutrophils (10/1),
and the effect on cytokine production was measured. In the presence of
apoptotic neutrophils, monocyte production of TNF- and IL-1- was
reduced by 88 ± 7 and 86 ± 11%, respectively (Fig. 1
, A and B). This
inhibitory effect was not dependent on the concentration of LPS used
(Fig. 1E). The production of IL-10 was increased 4-fold
(Fig. 1C), and TGF- production was increased 2-fold (Fig. 1D). It has been previously reported that apoptotic
lymphocytes enhance IL-10 production in monocytes (26) and
produce IL-10 themselves in response to LPS (30). In our
system, neither IL-10 nor TGF- was produced by the apoptotic
neutrophils in either the presence or the absence of LPS (10 µg/ml),
as measured by ELISA. In the absence of LPS stimulation, monocytes
cultured in the presence of apoptotic neutrophils produced none of the
four cytokines measured (Fig. 1, A–D).
|
The kinetics of cytokine production by LPS-stimulated monocytes in
the presence of apoptotic neutrophils over 18 h were studied. In
the presence of LPS (10 µg/ml), TNF- and IL-1 were detectable
(78 ± 26 and 479 ± 99 pg/ml) by ELISA 4 h
poststimulation (Fig. 2). These levels
increased to 1585 ± 163 and 2854 ± 720 pg/ml, respectively,
by 18 h. In the presence of apoptotic neutrophils, TNF- was
also detectable (39 ± 17 pg/ml) at 4 h poststimulation (Fig. 2A). However, at each time point after 6 h there was
significantly less TNF- produced than in cells incubated with LPS
alone, with a maximum level of only 316 ± 100 pg/ml present at
10 h poststimulation. In the presence of apoptotic neutrophils
there was a significant reduction in IL-1 levels at each time point
(4–18 h inclusive), with a maximum level of 1070 ± 287 pg/ml
recorded at 18 h poststimulation (Fig. 2B).
|
C). The effect on cytokine production is specific to apoptotic neutrophils
To confirm that the pattern of cytokine change seen was the result
of exposure to apoptotic neutrophils and not neutrophils, the effects
of fresh and opsonized neutrophils on LPS-stimulated monocytes were
examined. Monocytes (0.25 x 106/ml) were
stimulated with LPS (10 µg/ml) and incubated with freshly isolated or
apoptotic neutrophils (2.5 x 106/ml) for
8 h. This time point was chosen to minimize neutrophil death in
the fresh neutrophil cultures. At 8 h the fresh neutrophil
population was <15% Annexin positive. It had been established that
the effects of apoptoticneutrophils on cytokine production are
apparent at this time point (Fig. 2). In the presence of apoptotic
neutrophils the productions of IL-1, TNF-, and IL-10 were altered
as before. In contrast, incubation with fresh neutrophils had no impact
on their output (Table I).
|
and IL-1 production were unaffected by the
presence of opsonized apoptotic neutrophils. IL-10 production was
increased from 342 ± 10 to 696 ± 203 pg/ml in the presence
of apoptotic neutrophils and further increased to 1101 ± 203
pg/ml in the presence of opsonized neutrophils (Table II). This up-regulation of IL-10 by
opsonized cells is consistent with published data (22, 31). These data collectively demonstrate that the induction of
reduced proinflammatory cytokine production by monocytes coupled with
the promotion of augmented IL-10 production are specific to apoptotic
neutrophils.
|
The effect of apoptotic neutrophils on macrophage cytokine
production occurs in association with phagocytosis by the macrophages.
However, the part played by surface contact and actual phagocytosis has
not been fully elucidated. Monocytes do not phagocytose apoptotic
neutrophils (25), but do express a number of receptors
capable of recognizing and binding them, including CD36
(32) and CD14 (33). We hypothesized that
cell-cell contact was necessary and sufficient to generate the cytokine
changes inducible in monocytes by apoptotic neutrophils. After
overnight aging, apoptotic neutrophils (3 x
106/ml) were cultured in the presence of LPS (10
µg/ml) for 18 h. Supernatants were harvested from these cultures
and added to monocytes (0.25 x 106/ml) for
a further 18 h in the presence of LPS (10 µg/ml). No change in
cytokine production by the monocytes was observed (Fig. 3A). To confirm the need for
cell-cell contact, we used 0.4-µm pore size Transwell filters to
separate LPS-activated monocytes from apoptotic neutrophils. In the
presence of the filters there was no change in pro- and
anti-inflammatory cytokine profiles (Fig. 3B). These
results demonstrate that the cytokine regulatory effect of apoptotic
neutrophils on activated monocytes is cell-cell contact dependent.
|
or IL-10
IL-10 is a major inhibitor of TNF- and IL-1 production
(5). However, it has been previously shown in other models
that down-regulation of TNF- can be independent of a simultaneous
increase in IL-10 production (34, 35). To explore whether
the augmented monocyte-derived IL-10 output was responsible for the
down-regulation of their proinflammatory cytokine production, monocytes
were incubated in the presence or the absence of apoptotic neutrophils
with anti-IL-10 (10 µg/ml).
In response to LPS stimulation, IL-1 production was 1132 ± 165
pg/ml (Fig. 4A). TNF-
production was 263 ± 55 pg/ml (Fig. 4B). In the
presence of apoptotic neutrophils, IL-1 production was reduced to
162 ± 100 pg/ml (Fig. 4A). TNF- production was
reduced to 16 ± 3 pg/ml (Fig. 4B). These figures were
only minimally increased with the addition of anti-IL-10 to
328 ± 101 and 61 ± 40 pg/ml. It has previously been shown
that inhibition of proinflammatory cytokine production in the
macrophage can be reversed using anti-TGF- Ab (22).
We explored whether this could be responsible for the reduction in
proinflammatory cytokine output in the monocyte system. Monocytes were
incubated in the presence or the absence of apoptotic neutrophils with
anti-TGF- Ab (100 µg/ml). In the presence of Ab, LPS induced
production of TNF- and IL-1 and increased from 310 ± 60 and
1444 ± 100 pg/ml to 527 ± 60 and 1998 ± 120 pg/ml,
respectively. In the presence of apoptotic neutrophils, IL-1
production was minimally increased by the addition of anti TGF- Ab
from 250 ± 20 to 541 ± 30 pg/ml. TNF- production in the
presence of apoptotic neutrophils was 58 ± 18 pg/ml, and this was
unchanged by the addition of anti-TGF- Ab (58.5 ± 22
pg/ml). These results indicate that the down-regulation of
proinflammatory cytokines enforced by apoptotic neutrophils is
independent of the induction of IL-10 or TGF-.
|
Exposure to endotoxin leads to the secondary down-regulation of a
number of LPS-driven responses, including the production of TNF-.
This phenomenon is called LPS tolerance. This repression of
proinflammatory cytokine production can be reversed by the addition of
IFN- (36). To examine the effect of IFN- on the
down-regulation of TNF- production induced by apoptotic neutrophils,
monocytes were incubated with IFN- (1–500 ng/ml) alone or in
combination with LPS (10 µg/ml) in the presence or the absence of
apoptotic neutrophils. In the absence of apoptotic neutrophils, IFN-
enhanced TNF- production in a dose-dependent fashion, both alone
(Fig. 5B) and in combination
with LPS (Fig. 5A) to maximum levels of 1961 ±
180 and 2184 ± 388 pg/ml, respectively. This was in accordance
with previous reports. However, IFN- was unable to reverse the
suppression of LPS-driven TNF- production induced by apoptotic
neutrophils (Fig. 5A). Monocytes activated by IFN- alone
also down-regulate their TNF- production in the presence of
apoptotic neutrophils (Fig. 5B). There was a small,
dose-dependent increase in output when IFN- was added in combination
with LPS (10 µg/ml), but only to a maximum of 376 ± 65 pg/ml.
This was consistent with the small, dose-dependent increase in TNF-
production stimulated by IFN- alone in the presence of apoptotic
neutrophils to a maximum level of 245 ± 75 pg/ml.
|
It has been suggested that contrasting reports of cytokine
profiles in the presence of apoptotic neutrophils were due to
differences in the models used (37). Following the same
model system as before, apoptotic neutrophils (2.5 x
106/ml) were incubated with 7-day-old
monocyte-derived macrophages (0.25 x
106/ml) and stimulated with LPS (10 µg/ml) for
18 h. Phagocytosis of the apoptotic cells by macrophages was
confirmed by staining for myeloperoxidase activity (38).
As with the monocyte model, we observed a reduction in TNF- (71
± 13%; Fig. 6A) and a 2-fold
increase in TGF- release (Fig. 6C) in the presence of
apoptotic neutrophils. However, in contrast to the monocyte model, we
observed a reduction in IL-10 production (69 ± 18%) by the
macrophages, as had been reported previously (Fig. 6B)
(22).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and IL-1
throughout that time.
The induction kinetics of proinflammatory genes differ from those of
the IL-10 gene due to fundamental differences in molecular regulation
(40, 41). As a result, LPS stimulation results in rapid
release of TNF- and IL-1, whereas release of IL-10 is delayed
(5). In the past, LPS-activated monocytes have been
manipulated through the use of -agonists (42),
antioxidants (43), and adenosine receptor agonists
(44, 45), resulting in reduced production of
proinflammatory cytokines and augmented IL-10 production. We have shown
that contact with apoptotic neutrophils has a similar quantitative
effect (Fig. 1). However, unlike previous models of monocyte modulation
(5, 42, 44), apoptotic neutrophils also induce an altered
kinetic response to LPS stimulation. After stimulation with LPS in our
model, monocytes produce IL-10, detectable by ELISA, after 8–10 h.
However, in the presence of apoptotic neutrophils. IL-10 is detectable
as early as 4 h after stimulation (Fig. 2C). Monocytes
are traditionally seen a promoters of the inflammatory cascade
(46, 47). We show that by becoming primarily
IL-10-producing cells, through contact with apoptotic neutrophils, they
also have a role as suppressors of inflammation.
We have also shown that in this model activated monocytes augment their
release of TGF-. This extends the immunosuppressive phenotype that
they achieve through interaction with apoptotic neutrophils. TGF-
not only inhibits release of proinflammatory cytokines, but also
promotes tissue repair through recruitment of fibroblasts and the
promotion of matrix deposition, including fibronectin and collagen
(10, 48).
It might be argued that these monocytes could be described as endotoxin tolerant due to their suppressed proinflammatory response to LPS stimulation. Endotoxin-tolerant cells produce only minimal levels of proinflammatory cytokines upon LPS stimulation and normal or increased amounts of IL-10 (36, 49). However, tolerance is defined as a failure to respond to secondary challenge after primary stimulation with LPS (50). In our model apoptotic cells and monocytes are added to the culture plates simultaneously and then stimulated with LPS. Therefore, this work is not a model of LPS tolerance, but instead demonstrates that in the presence of apoptotic neutrophils, monocytes on exposure to LPS become IL-10-producing, immunosuppressive cells.
LPS-tolerant cells can be triggered to produce TNF- when IFN- is
added to the culture (36, 51). Our work shows that, in
contrast, the suppressive effect of apoptotic neutrophils on TNF-
production cannot be reversed by IFN- (Fig. 5A). IFN-
induction of TNF- was also inhibited by the presence of apoptotic
neutrophils, demonstrating that the inhibitory mechanism is not
specific to LPS stimulation alone (Fig. 5B).
IL-10 is recognized as a major deactivating cytokine, capable of
inhibiting the production of TNF- and IL-1 (6). Here
we report that the inhibition of TNF- and IL-1 is not reversed by
the depletion of IL-10 (Fig. 4). This pattern of down-regulation of
TNF-, independent of IL-10, has been demonstrated before using
acetylcholine to alter cytokine production (52).
Similarly, the inhibitory effect of cAMP-elevating drugs on TNF-
production is independent of their elevating effect on IL-10 levels
(53).
TGF- is a pleiotropic cytokine that has a contrasting effect on
resting and activated cells of the innate immune system (48, 54). We have shown that LPS-induced TNF- and IL-1
production can be augmented in the presence of anti-TGF- Ab, but
that, in contrast, the inhibitory effect of apoptotic neutrophils on
these cytokines is not reversed (Fig. 4, C and
D). The dampening of TNF- production is mediated through
numerous, mechanically distinct, mechanisms involving diverse,
intracellular signaling pathways (55, 56, 57). The particular
mechanism in our model remains to be elucidated.
We applied our model to HMDM and showed that, in contrast to monocytes,
these cells down-regulate their production not only of TNF- and
IL-1 but also of IL-10, while increasing their production of TGF-
(Fig. 6). This is consistent with previous work by Fadok et al.
(22). Therefore, using the same experimental conditions we
have shown that monocytes and macrophages differentially alter their
cytokine profile in the presence of apoptotic neutrophils. This
demonstrates that monocytes are not just precursor macrophages, but
that they have an independent role in inflammation. The differential
cytokine output from monocytes and macrophages in the presence of
apoptotic neutrophils may be due to an alternative signaling mechanism.
We hypothesize that signaling occurs downstream of a receptor ligand
mechanism. We have shown that contact between monocytes and apoptotic
neutrophils is necessary for a change in cytokine profile to occur. In
the macrophage, ligation of the phosphatidylserine receptor using
mab217 creates a similar cytokine response to apoptotic neutrophils
(58). Monocytes do not express the phosphatidylserine
receptor (58). There are, however, a multitude of other
receptors involved in the tethering of apoptotic neutrophils by
macrophages (CD36 (32), CD14 (33),
vitronectin (38), SR-BI, scavenger receptor A
(59), CD91 (60), and CD68 (19))
with an undefined role in cytokine regulation in the presence of
apoptotic neutrophils. A number of these are expressed on the monocyte,
including CD36, CD14, CD68, and CD91. Interestingly, both ourselves and
Voll (26) have shown that a reduction in TNF- and a
simultaneous increase in IL-10 can be achieved through the ligation of
the CD36 receptor (data not shown), while Fadok (37)
reports that this is not the case in the macrophage. Monocytes do not
phagocytose apoptotic cells, unlike macrophages (25, 61).
However, an actual role for phagocytosis of apoptotic neutrophils,
independent of surface ligation, has not been defined. In the
macrophage model it has been shown that the critical structure required
from an apoptotic neutrophil to generate an anti-inflammatory
signal is phosphatidylserine-containing membrane (62).
We have shown that in the presence of fresh neutrophils, which do not express phosphatidylserine on their surface, there is no inhibition of proinflammatory cytokine production. Similarly, in the presence of opsonized apoptotic neutrophils, which would trigger the CD16 receptor, we show that LPS stimulation drives a proinflammatory response. This is a previously reported phenomenon of CD16 ligation (22, 31).
This is the first report to define the interaction between apoptotic neutrophils and blood monocytes. Voll et al. (26) showed that in the presence of apoptotic lymphocytes, monocytes also enhanced production of IL-10 at 18 h coupled with reduced proinflammatory cytokine production. Interestingly, it has been shown that in sepsis, apoptosis of lymphocytes is greatly enhanced (63, 64), whereas apoptosis of neutrophils is delayed (65, 66). This may reflect a mechanism for maintaining a balance between the two cell types as a way of regulating cytokine production (67). It has been suggested that inhibition of lymphocyte apoptosis might be an effective therapeutic target in sepsis (68, 69). However, it would now appear that a fuller understanding is needed of the specific role played by each of these two apoptotic cell types in the regulation of IL-10 and proinflammatory cytokine production before targeting one alone (70).
Sepsis occurs when there is an overwhelming inflammatory response to microbial invasion (4). Sepsis is now known to have two stages (11). The first stage is dominated by a proinflammatory drive. The second phase is associated with high levels of IL-10. This is the time when patients are particularly vulnerable to opportunistic infection (71). IL-10 levels have been demonstrated as sensitive indicators of high mortality risk (39).
To date, apoptotic cells have been described as playing a role in the resolution of inflammation (22). Our work suggests that their role is more complex and that it is possible that in sepsis, through their effects on monocytes, they may contribute to the high levels of IL-10 described, which often lead to life-threatening immunosuppression.
The modulation of activated monocytes into IL-10-producing cells by apoptotic neutrophils may also have significance to the treatment of chronic inflammatory conditions such as rheumatoid arthritis. This condition is characterized by a heightened influx of monocytes (16), dysregulated proinflammatory drive (72), reduced availability of IL-10 (73), and delayed neutrophil apoptosis (74).
In conclusion, we have shown that after contact with apoptotic
neutrophils, monocytes no longer act as promoters of the inflammatory
cascade. Instead, they generate a unique anti-inflammatory response
to LPS stimulation characterized by accelerated release of IL-10 and
enhanced production of TGF-. It is likely that this is evidence of a
previously unrecognized mechanism through which inflammation is
regulated that has implications for the treatment of both acute and
chronic inflammatory conditions.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Aideen Byrne, The Children’s Research Center, Our Lady’s Hospital for Sick Children, Crumlin, Dublin 12, Ireland. E-mail address: byrneaideen{at}ireland.com
3 Abbreviations used in this paper: MCP-1, monocyte chemoattractant protein-1; HMDM, human monocyte-derived macrophages.
Received for publication August 13, 2001. Accepted for publication December 14, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. J. Exp. Med. 181:1887.. Microbes Infect. 1:1275.[Medline]
. Annu. Rev. Immunol. 16:137.[Medline]
blockade in patients with rheumatoid arthritis. Arthritis Rheum. 43:38.[Medline]
, PGE2, and PAF. J. Clin. Invest. 101:890.[Medline]
RIII and acquire annexin V binding sites during apoptosis in vitro. Blood 85:532. receptor type I. J. Exp. Med. 188:217.v3). J. Immunol. 161:6250. generation from human peripheral blood monocytes. Cell Biochem. Biophys. 29:179.[Medline]
B into human macrophages: lipopolysaccharide-induced, but not zymosan-induced, proinflammatory cytokines are inhibited, but IL-10 is nuclear factor-B independent. J. Immunol. 162:2939.-adrenergic receptors on macrophages in modulation of LPS-induced cytokine release. J. Recept. Signal Transduct. Res. 19:191.[Medline]
. Eur. J. Immunol. 29:1890.[Medline]
, and nitric oxide production in RAW 264.7 macrophages. Brain Res. Bull. 45:183.[Medline]
, and nitric oxide production in RAW 264.7 macrophages and in endotoxemic mice. J. Immunol. 157:4634.[Abstract]
: a matter of life and death. J. Leukocyte Biol. 55:401.[Abstract]
. Eur. J. Immunol. 20:1181.[Medline]
: a balancing act. Int. Rev. Immunol. 16:553.[Medline]
production of monocytes. Shock 15:101.[Medline]
, and heat induce programmed cell death in activated neutrophils. Shock 5:47.[Medline]
This article has been cited by other articles:
|
|
 |
|
  R. Rizzo, M. Stignani, L. Melchiorri, and O. R. Baricordi Comment on "Experimental Extracorporeal Photopheresis Inhibits the Sensitization and Effector Phases of Contact Hypersensitivity via Two Mechanisms: Generation of IL-10 and Induction of Regulatory T Cells" J. Immunol., April 15, 2009; 182(8): 4497 - 4497. [Full Text] [PDF]
|
|
|
|
|
|
T. Baranek, R. Debret, F. Antonicelli, B. Lamkhioued, A. Belaaouaj, W. Hornebeck, P. Bernard, M. Guenounou, and R. Le Naour Elastin Receptor (Spliced Galactosidase) Occupancy by Elastin Peptides Counteracts Proinflammatory Cytokine Expression in Lipopolysaccharide-Stimulated Human Monocytes through NF-{kappa}B Down-Regulation J. Immunol., November 1, 2007; 179(9): 6184 - 6192. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Chandra, T. S. McCormick, Y. Imamura, P. K. Mukherjee, and M. A. Ghannoum Interaction of Candida albicans with Adherent Human Peripheral Blood Mononuclear Cells Increases C. albicans Biofilm Formation and Results in Differential Expression of Pro- and Anti-Inflammatory Cytokines Infect. Immun., May 1, 2007; 75(5): 2612 - 2620. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Miyake, H. Kaise, K.-i. Isono, H. Koseki, K. Kohno, and M. Tanaka Protective Role of Macrophages in Noninflammatory Lung Injury Caused by Selective Ablation of Alveolar Epithelial Type II Cells J. Immunol., April 15, 2007; 178(8): 5001 - 5009. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kirsch, A. Woywodt, M. Beese, K. Wyss, J.-K. Park, U. Erdbruegger, B. Hertel, H. Haller, and M. Haubitz Engulfment of apoptotic cells by microvascular endothelial cells induces proinflammatory responses Blood, April 1, 2007; 109(7): 2854 - 2862. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Verstappen and J.W. Von den Hoff Tissue Inhibitors of Metalloproteinases (TIMPs): Their Biological Functions and Involvement in Oral Disease Journal of Dental Research, December 1, 2006; 85(12): 1074 - 1084. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Lucas, L. M. Stuart, A. Zhang, K. Hodivala-Dilke, M. Febbraio, R. Silverstein, J. Savill, and A. Lacy-Hulbert Requirements for Apoptotic Cell Contact in Regulation of Macrophage Responses J. Immunol., September 15, 2006; 177(6): 4047 - 4054. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Cvetanovic, J. E. Mitchell, V. Patel, B. S. Avner, Y. Su, P. T. van der Saag, P. L. Witte, S. Fiore, J. S. Levine, and D. S. Ucker Specific Recognition of Apoptotic Cells Reveals a Ubiquitous and Unconventional Innate Immunity J. Biol. Chem., July 21, 2006; 281(29): 20055 - 20067. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. A. Fraser, S. S. Bohlson, N. Jasinskiene, N. Rawal, G. Palmarini, S. Ruiz, R. Rochford, and A. J. Tenner C1q and MBL, components of the innate immune system, influence monocyte cytokine expression J. Leukoc. Biol., July 1, 2006; 80(1): 107 - 116. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Carrero, B. Calderon, and E. R. Unanue Lymphocytes are detrimental during the early innate immune response against Listeria monocytogenes J. Exp. Med., April 17, 2006; 203(4): 933 - 940. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Desnues, M. Ihrig, D. Raoult, and J.-L. Mege Whipple's Disease: a Macrophage Disease Clin. Vaccine Immunol., February 1, 2006; 13(2): 170 - 178. [Full Text] [PDF]
|
|
|
|
|
|
A. Maeda, A. Schwarz, K. Kernebeck, N. Gross, Y. Aragane, D. Peritt, and T. Schwarz Intravenous Infusion of Syngeneic Apoptotic Cells by Photopheresis Induces Antigen-Specific Regulatory T Cells J. Immunol., May 15, 2005; 174(10): 5968 - 5976. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Zheng, M. He, M. Long, R. Blomgran, and O. Stendahl Pathogen-Induced Apoptotic Neutrophils Express Heat Shock Proteins and Elicit Activation of Human Macrophages J. Immunol., November 15, 2004; 173(10): 6319 - 6326. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Asano, M. Miwa, K. Miwa, R. Hanayama, H. Nagase, S. Nagata, and M. Tanaka Masking of Phosphatidylserine Inhibits Apoptotic Cell Engulfment and Induces Autoantibody Production in Mice J. Exp. Med., August 16, 2004; 200(4): 459 - 467. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Hu, J. H. Jennings, J. Sonstein, J. Floros, J. C. Todt, T. Polak, and J. L. Curtis Resident Murine Alveolar and Peritoneal Macrophages Differ in Adhesion of Apoptotic Thymocytes Am. J. Respir. Cell Mol. Biol., May 1, 2004; 30(5): 687 - 693. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Shumilla, V. Lacaille, T. M. C. Hornell, J. Huang, S. Narasimhan, D. A. Relman, and E. D. Mellins Bordetella pertussis Infection of Primary Human Monocytes Alters HLA-DR Expression Infect. Immun., March 1, 2004; 72(3): 1450 - 1462. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Yamaguchi, C. Kubo, Y. Masuhiro, E. T. Lally, T. Koga, and S. Hanazawa Tumor Necrosis Factor Alpha Enhances Actinobacillus actinomycetemcomitans Leukotoxin-Induced HL-60 Cell Apoptosis by Stimulating Lymphocyte Function-Associated Antigen 1 Expression{dagger} Infect. Immun., January 1, 2004; 72(1): 269 - 276. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. I. Ojielo, K. Cooke, P. Mancuso, T. J. Standiford, K. M. Olkiewicz, S. Clouthier, L. Corrion, M. N. Ballinger, G. B. Toews, R. Paine III, et al. Defective Phagocytosis and Clearance of Pseudomonas aeruginosa in the Lung Following Bone Marrow Transplantation J. Immunol., October 15, 2003; 171(8): 4416 - 4424. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Lucas, L. M. Stuart, J. Savill, and A. Lacy-Hulbert Apoptotic Cells and Innate Immune Stimuli Combine to Regulate Macrophage Cytokine Secretion J. Immunol., September 1, 2003; 171(5): 2610 - 2615. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Odaka, T. Mizuochi, J. Yang, and A. Ding Murine Macrophages Produce Secretory Leukocyte Protease Inhibitor During Clearance of Apoptotic Cells: Implications for Resolution of the Inflammatory Response J. Immunol., August 1, 2003; 171(3): 1507 - 1514. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. A. S. H. Dalm, P. M. van Hagen, P. M. van Koetsveld, S. Achilefu, A. B. Houtsmuller, D. H. J. Pols, A.-J. van der Lely, S. W. J. Lamberts, and L. J. Hofland Expression of somatostatin, cortistatin, and somatostatin receptors in human monocytes, macrophages, and dendritic cells Am J Physiol Endocrinol Metab, August 1, 2003; 285(2): E344 - E353. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Emoto, Y. Emoto, V. Brinkmann, M. Miyamoto, I. Yoshizawa, M. Staber, N. van Rooijen, A. Hamann, and S. H. E. Kaufmann Increased Resistance of LFA-1-Deficient Mice to Lipopolysaccharide-Induced Shock/Liver Injury in the Presence of TNF-{alpha} and IL-12 Is Mediated by IL-10: A Novel Role for LFA-1 in the Regulation of the Proinflammatory and Anti-Inflammatory Cytokine Balance J. Immunol., July 15, 2003; 171(2): 584 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Brown, D. Goldblatt, J. Buddle, L. Morton, and A. J. Thrasher Diminished production of anti-inflammatory mediators during neutrophil apoptosis and macrophage phagocytosis in chronic granulomatous disease (CGD) J. Leukoc. Biol., May 1, 2003; 73(5): 591 - 599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Garn, A. Siese, S. Stumpf, P. J. Barth, B. Muller, and D. Gemsa Shift Toward an Alternatively Activated Macrophage Response in Lungs of NO2-Exposed Rats Am. J. Respir. Cell Mol. Biol., March 1, 2003; 28(3): 386 - 396. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
