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*
Division of Renal and Inflammatory Disease, School of Medical and Surgical Sciences, University Hospital, Nottingham, United Kingdom;
Rayne Laboratory, Respiratory Medicine Unit, University of Edinburgh Medical School, Edinburgh, United Kingdom;
Katholieke Universiteit Leuven, Rega Institute, Leuven, Belgium; and
§
Molecular Medicine Centre, Western General Hospital, Edinburgh, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) for 24 h with
methylprednisolone, dexamethasone, and hydrocortisone, but not the
nonglucocorticoid steroids aldosterone, estradiol, and progesterone,
potentiated phagocytosis of apoptotic neutrophils. These effects were
specific in that the potentiated phagocytosis of apoptotic neutrophils
was completely blocked by the glucocorticoid receptor antagonist
RU38486, and glucocorticoids did not promote 5-day M ingestion of
opsonized erythrocytes. Similar glucocorticoid-mediated potentiation
was observed with 5-day M uptake of alternative apoptotic
"targets" (eosinophils and Jurkat T cells) and in uptake of
apoptotic neutrophils by alternative phagocytes (human glomerular
mesangial cells and murine M elicited into the peritoneum or derived
from bone marrow). Importantly, methylprednisolone-mediated enhancement
of the uptake of apoptotic neutrophils did not trigger the release of
the chemokines IL-8 and monocyte chemoattractant protein-1.
Furthermore, longer-term potentiation by methylprednisolone was
observed in maturing human monocyte-derived M, with greater
increases in 5-day M uptake of apoptotic cells being observed the
earlier glucocorticoids were added during monocyte maturation into
M. We conclude that potentiation of nonphlogistic clearance of
apoptotic leukocytes by phagocytes is a hitherto unrecognized property
of glucocorticoids that has potential implications for therapies aimed
at promoting the resolution of inflammatory
diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)4) and by
semiprofessional phagocytes (e.g., glomerular mesangial cells,
fibroblasts) without inciting proinflammatory secretory responses 6, 10, 11, 12 .
Although several molecular pathways by which phagocytes recognize
apoptotic cells have been identified (reviewed in Refs. 13 and 14), the
mechanisms that control phagocytic capacity for the clearance of
apoptotic cells are less well understood. The modulation of phagocyte
capacity for apoptotic cell clearance represents a potential
therapeutic target in the control of inflammatory disease. Although
CD44-mediated increases in the M uptake of apoptotic neutrophils may
hold some therapeutic promise 15 , it is unlikely that there is
clinical utility in the observation that pro-inflammatory cytokines
promote M ingestion of apoptotic cells 16 . Therefore, we sought to
investigate further the regulatory mechanisms that might control
phagocyte clearance of leukocytes undergoing apoptosis.
It is well established that glucocorticoids are powerful anti-inflammatory agents that suppress many phlogistic responses including inflammatory cell recruitment and activation 17, 18, 19, 20 . However, there have been relatively few studies of the effects of these steroids on the resolution phase of inflammation. We and others have shown that glucocorticoids delay constitutive apoptosis in neutrophils, whereas eosinophil apoptosis is accelerated 21, 22, 23 , providing an attractive explanation for the therapeutic efficacy of glucocorticoids in eosinophilic inflammation. However, implicit in this observation is the need for nonphlogistic clearance of an increased tissue load of apoptotic eosinophils, implying that glucocorticoids might also up-regulate the phagocytic capacity for apoptotic granulocytes so that greater numbers are safely removed. Moreover, although glucocorticoids delay neutrophil apoptosis, these agents are effective in suppressing inflammatory responses characterized by intense infiltration of tissues with neutrophils 24 that will eventually undergo apoptosis, implying that glucocorticoids may also up-regulate the phagocyte capacity to clear apoptotic neutrophils.
To test the hypothesis that glucocorticoids potentiate phagocyte capacity for the nonphlogistic clearance of apoptotic leukocytes, we have undertaken the first study of the effects of glucocorticoids upon this process, which is important in the resolution of inflammation. We report that glucocorticoids specifically promote safe clearance by various phagocyte types of apoptotic leukocytes from different lineages, establishing that glucocorticoids have hitherto unrecognized but beneficial regulatory effects upon phagocytes, which may promote the safe termination of inflammatory responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All reagents were obtained from Sigma (St. Louis, MO) unless otherwise stated. Culture media (Iscove’s modified DMEM, RPMI 1640, and HBSS) and supplements (100 U/ml penicillin, 100 58/ml streptomycin, 2 mM glutamine, and FCS) were obtained from Life Technologies Laboratories (Paisley, U.K.). Brewer’s thioglycollate medium was obtained from Difco (Detroit, MI). Methylprednisolone was obtained from Upjohn (Birmingham, U.K.); dexamethasone, hydrocortisone, aldosterone, progesterone, and estradiol were obtained from Sigma (Poole, U.K.). RU38486 was kindly provided by Dr. Ian Hall (University of Nottingham). Percoll was obtained from Pharmacia Fine Chemicals (Piscataway, NJ). Sterile tissue culture plasticware was obtained from Falcon Plastics (Cockeysville, MD).
Granulocyte isolation and induction of apoptosis
Human neutrophils (>98% pure on May-Giemsa-stained cytopreparations) were isolated from fresh, citrated blood of healthy volunteers by dextran sedimentation and discontinuous plasma-Percoll (Pharmacia Fine Chemicals) density gradient centrifugation 2, 25 . The neutrophils were aged in tissue culture at 37°C and 5% CO2 for 24 h in Iscove’s DMEM with 10% autologous platelet-rich, plasma-derived serum to undergo apoptosis verified by typical morphology 2 . Only aged neutrophils with a viability (assessed by trypan blue dye exclusion) >98% were used.
Human eosinophils (95–98% pure on May-Giemsa-stained cytopreparations) were isolated from the blood of mildly eosinophilic but healthy human volunteers by using a combination of discontinuous plasma-Percoll density gradient centrifugation to obtain eosinophil-enriched granulocyte populations followed by immunomagnetic depletion of neutrophils using 3G8 CD16 mAb, essentially as described 6, 26 . Purified eosinophils were then cultured for up to 72 h in conditions identical to those for neutrophils to undergo apoptosis verified by typical morphology 26 . Only aged eosinophils with viability >98% by trypan blue dye exclusion were used.
Preparation of human monocyte-derived M
Human monocytes were prepared by well-established methods
from the mixed mononuclear cell band of the discontinuous
plasma-Percoll gradients used to prepare neutrophils 2 . Mononuclear
cells were suspended in Iscove’s DMEM at 4 x 106/ml,
and 100 µl was added to each well of a 96-well plate, which was then
incubated at 37° for 1 h. Nonadherent cells, including
contaminating lymphocytes, were then washed off, and adherent monocytes
were cultured in Iscove’s DMEM with 10% autologous platelet-rich,
plasma-derived serum for 5 days to mature into monocyte-derived
M.
Preparation of murine M populations
Ten-week-old BALB/c mice were purchased from the University of
Nottingham Biomedical Facility and were housed and treated under U.K.
Home Office-approved conditions. Thioglycollate-elicited inflammatory
peritoneal M and bone marrow-derived M were prepared as described
27 . Briefly, inflammatory peritoneal exudate M were elicited into
the peritoneal cavity of 10-wk-old female BALB/c mice by injection of
3% Brewer’s thioglycollate and harvested 5 days later after humane
killing by peritoneal lavage with DMEM medium alone. Exudate cells were
adhered to 96-well plates for 1 h at 0.1 x 106
cell/well, and nonadherent cells were washed off and replaced with DMEM
containing 10% FCS plus supplements as above. Peritoneal M were
used within 48 h after isolation. Bone marrow-derived M were
isolated from femurs of humanely killed mice, cut at both ends, and the
bone marrow expelled by flushing with a 25-gauge needle with DMEM with
10% FCS supplements as above and 10% conditioned medium from L929
cells as a source of monocyte-CSF. M were employed after 7–10 days
culture.
Culture of human mesangial cells
Mesangial cells were prepared using standard methods of serial
culture/trypsinization in tissue culture flasks of adherent outgrowth
cells from glomeruli obtained by sieving diced human normal renal
cortex as described previously 3, 11 . Cells were cultured in RPMI
1640 medium with 10% FCS and 5 ml/500 ml of
insulin/selenium/transferrin growth supplement from Life Technologies
Laboratories (Grand Island, NY) and were used between passages four and
six after subculture into 96-well plates. Great care was taken to
verify the purity and phenotype of mesangial cells, as previously
described 3, 11 . Cells were uniformly smooth muscle actin-positive
and CD45-negative (by immunofluorescence). Cells did not take up
acetylated low-density lipoprotein or opsonized zymosan particles
(excluding M contamination) and were cytokeratin and factor
VIII-related Ag-negative (by immunofluorescence), excluding
contamination with glomerular epithelial or endothelial cells.
Culture of Jurkat T cells and induction of apoptosis
Cells of the Jurkat T cell line (a gift from C. Gregory,
University of Nottingham) were grown in suspension culture in RPMI 1640
with 5% FCS and supplements. To induce morphologically verified
apoptosis, cells were deprived of serum with cycloheximide at 50
µg/ml for 4 h before being washed and employed in interaction
assays with M.
Interaction assays
A microscopically quantified phagocytic assay of M
phagocytosis of aged polymorphonuclear leukocytes (PMNs) was used,
which has been described and illustrated in detail before 2, 28 .
Minor adaptations for M cultured in 96-well plates have been
described 16 . Briefly, PMNs aged for 24 h in culture, to undergo
apoptosis, were washed once in HBSS, suspended in Iscove’s DMEM, and
0.5 x 106 aged PMNs in 50 µl of medium were added
to each washed well of M. After interaction for 30 min at 37°C in
5% CO2 atmosphere, the wells were washed in saline at
4°C to remove noningested apoptotic PMNs, fixed with 2%
glutaraldehyde in 0.9% saline, stained for myeloperoxidase (MPO;
present in PMNs but not M) using hydrogen peroxide and
dimethoxybenzidine as substrate as previously described 2, 29 , and
then the proportion of M ingesting neutrophils were counted by
inverted light microscopy, exactly as described 2, 28 . Because it was
also important to determine whether the phagocytic capacity of
individual M had been increased, the number of PMNs within 100
randomly selected M in each well was counted in some experiments.
These counts were possible because the duration of the phagocytic assay
was sufficiently short for ingested PMNs to remain intact 2, 28, 29 .
The M uptake of aged apoptotic eosinophils, 0.1 x
106 in 50 µl medium for each well of M or of
IgG-opsonized human erythrocytes (EIgG), 0.1 x 106 in
50 µl medium for each well of M, was determined by similar means,
as described 26, 28 , because these cell types also stain for
peroxidase. The uptake of apoptotic Jurkat T cells was assessed by
staining with Haemalum (BDH, Poole, U.K.), as described 4 .
To quantify mesangial cell phagocytosis of apoptotic PMNs, we used a previously described, reproducible, microscopically quantified assay in which the uptake of apoptotic PMN was shown by electron microscopy and susceptibility to inhibition by colchicine to be a result of active phagocytosis 3 . Culture medium was aspirated from the mesangial cells, and 50 µl of a 20 x 106/ml suspension of neutrophils in RPMI 1640 with 10% FCS was added to each well together with 50 µl of Iscove’s DMEM. The two cell types were cocultured in 5% CO2 at 37°C for 3 h, and the interaction was then stopped by the addition of cold normal saline and the removal of nonadherent cells by manual washing with a Pasteur pipette. The mesangial cell monolayer was then trypsinized, and a separate cytocentrifuge preparation was prepared for each well. These were fixed with 2% glutaraldehyde and stained for MPO and finally counterstained with Haemalum. The proportion of mesangial cells containing brown MPO-positive globules wholly within the outline of the cell was then counted by light microscopy with a minimum of 500 cells per slide being counted.
Effect of steroid hormones
Before interaction with apoptotic leukocytes, M or mesangial
cells were preincubated for varying durations of time in medium plus
supplements/serum, as described above, containing the desired final
concentration of steroid hormone. Control wells were left undisturbed
in medium plus supplements/serum. However, preliminary experiments (not
shown) demonstrated that the replacement of medium after aspiration at
the preincubation times used did not, in itself, alter the phagocytic
signal.
Effect of glucocorticoid receptor antagonist RU38486
The effect of glucocorticoid receptor antagonist RU38486 was assessed using final concentrations of RU38486 or ethanol-based vehicle control >10-fold those employed for glucocorticoids. Medium was aspirated from phagocytes 24 h before assay of phagocytosis and was replaced by 180 µl of RU38486 at 11.1-fold final steroid concentration in medium plus supplements/serum as above. Twenty minutes later, 20 µl of medium plus supplements/serum containing no steroid (as a control) or steroid at 10-fold final concentration was added, and the phagocytes were incubated for the desired period until interaction with apoptotic leukocytes.
Effect of cycloheximide
The protein synthesis inhibitor cycloheximide was included at 2.5 µM for 12 h in medium plus supplements/serum ± steroid before assay of phagocytosis of apoptotic leukocytes.
Assessment of M and mesangial cell response following the
ingestion of apoptotic PMNs
Monocyte-derived M in 24-well plates were either incubated in
Iscove’s DMEM alone or interacted with apoptotic PMNs or opsonized
zymosan. After 30 min, M were carefully washed, and 250 µl
Iscove’s DMEM was added to each well. After 24 h coculture with
M, the medium was aspirated, centrifuged, and stored at -80°C.
M phagocytosis of neutrophils or zymosan was quantified by fixing
three of the wells on each plate with 2% glutaraldehyde and, in the
case of neutrophils, by staining for MPO as outlined previously. The
proportion of M ingesting apoptotic neutrophils or opsonized zymosan
was counted by inversion light microscopy 28 .
Mesangial cells in 96-well plates were either cultured in the presence of RPMI 1640 with 10% FCS with or without 10 ng/ml IL-1 for 6 h or interacted for 3 h with apoptotic PMNs as previously outlined. Mesangial cells were then carefully washed with normal saline, and the medium was replaced with 50 µl of RPMI 1640 without FCS and incubated at 37°C. The medium was aspirated after 24 h, centrifuged, and stored at -80°C. Cytocentrifuge preparations from 4 wells on each plate were used to quantify mesangial cell phagocytosis of apoptotic PMNs.
Measurement of IL-8 and monocyte chemoattractant protein-1 (MCP-1) levels in tissue culture supernatants
IL-8 and MCP-1 levels were measured by ELISA using a peroxidase
conjugate for detection. Briefly, assay plates were coated with protein
A-purified goat polyclonal Ab against pure natural IL-8 30 or rabbit
polyclonal Ab against recombinant MCP-1 31 . Samples were tested at
1/10 dilution. Immunoreactivity was specifically measured using mAbs
against IL-8 and MCP-1 (R&D Systems, Abingdon, U.K.), respectively, as
secondary Abs. Detection was obtained by peroxidase-conjugated,
affinity-purified goat anti-mouse IgG. The ELISA for IL-8 and MCP-1
were specific because natural forms of other chemokines including
MCP-2, MCP-3 32 , granulocyte chemotactic protein-2,
ENA-78, GROa, inflammatory protein-10,
NAP-2 33 , or cytokines (IL-1, IFN-
, IFN-ß) were not
detectable. The sensitivity of the MCP-1 and IL-8 ELISA were <1 ng/ml
and <100 pg/ml, respectively.
Statistical methods
All values are expressed as mean ± SEM of the indicated number of experiments. Statistical significance (defined as p < 0.05) was evaluated using Student’s t test and where appropriate by one-way ANOVA with comparison between groups using the Newman-Keuls procedure.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To facilitate the detection of possible potentiating effects upon
M phagocytosis of apoptotic neutrophils 16 , we studied
"semimature" cultures of human monocyte-derived M matured for 5
days in adherent culture on plastic because maximal capacity for the
uptake of apoptotic cells generally requires maturation for around 7
days 29 . Pretreatment of 5-day M with the glucocorticoid
methylprednisolone (Fig. 1
) or
dexamethasone (data not shown) for different periods of time up to
24 h caused a progressive increase in the proportion of M that
were capable of phagocytosing apoptotic neutrophils and in the number
of apoptotic neutrophils ingested per M. For example,
methylprednisolone at 200 nM for 24 h augmented the number of
apoptotic neutrophils ingested per 100 phagocytically active M from
138.9 ± 4.1 to 209.6 ± 5.1 (p <
0.01; n = 9). Short preincubation times (<3 h) were
without effect (Fig. 1), excluding the remote possibility that there
was a "carry over" effect of glucocorticoid treatment of M upon
added apoptotic neutrophils. The specificity of glucocorticoid effects
upon M phagocytosis was further investigated by comparison of the
effects of pretreatment of M for 24 h with a number of
different steroids. These studies demonstrated that methylprednisolone,
dexamethasone, and hydrocortisone acted in a concentration-dependent
manner to augment phagocytosis (Fig. 2),
whereas the mineralocorticoid aldosterone at 5 nM, a concentration
10-fold above that inducing maximal effects on sodium transport in
vitro 34 , and the sex steroids progesterone (1 µM) and estradiol (1
µM) were without effect (Table I). The
specificity of the observed augmentation of M phagocytosis of
apoptotic neutrophils by glucocorticoids was further demonstrated by
the failure of glucocorticoid pretreatment to induce either binding or
phagocytosis of nonapoptotic neutrophils (data not shown) and the lack
of effect of methylprednisolone on the phagocytosis of EIgG; in a
series of nine experiments, pretreatment for 24 h with
methylprednisolone at 200 nM increased the proportion of 5-day M
ingesting apoptotic neutrophils from 14.1 ± 1.5% to 29.6 ±
3.2% (mean ± SE, p < 0.001) but had no
significant effect on the proportion of 5-day M ingesting EIgG
(untreated 5-day M 52.7 ± 6.4% vs methylprednisolone-treated
5-day M 54.3 ± 7.1%).
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uptake of apoptotic neutrophils is
specific for the glucocorticoid receptor and inhibitable by
cycloheximide
To confirm that glucocorticoids were acting via M steroid
receptors, we assessed the effect of the specific steroid receptor
antagonist RU38486 upon glucocorticoid-promoted 5-day M phagocytosis
of apoptotic neutrophils (Fig. 3).
RU38486 used at a 10-fold greater concentration than the
glucocorticoids completely blocked increased phagocytosis stimulated by
24-h treatment with methylprednisolone, dexamethasone, and
hydrocortisone without exhibiting any independent inhibitory effect
upon M phagocytosis of apoptotic cells (Fig. 3). Together with the
lack of effect upon M phagocytosis of EIgG cited above, these data
argue strongly against a nonspecific "membrane" effect on M.
Furthermore, methylprednisolone failed to promote phagocytosis of
apoptotic neutrophils by M pretreated for 12 h with the protein
synthesis inhibitor cycloheximide (a reversible blocker of mRNA
translation) at 2.5 µM, a concentration previously shown to inhibit
>95% of protein synthesis in myeloid cells and to inhibit M
phagocytosis of apoptotic cells without toxic effects on the M, as
evidenced by retained ability to ingest opsonized erythrocytes 35, 36 . Thus, in a series of seven experiments in which 15.7 ± 1.7%
(mean ± SE) of 5-day M ingested apoptotic neutrophils under
control conditions and 12-h pretreatment with methylprednisolone at 200
nM increased recognition to 38.3 ± 4.2%
(p < 0.001 vs control), such glucocorticoid
pretreatment was unable to reverse the inhibitory effect of concomitant
12-h pretreatment with cycloheximide at 2.5 µM, which significantly
reduced the proportion of M ingesting apoptotic neutrophils whether
M had been treated with methylprednisolone (4.9 ± 0.9%,
p < 0.001) or not (5.7 ± 1.4%,
p < 0.001).
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In view of the observation that ligation of M CD44 specifically
promotes phagocytosis of apoptotic neutrophils, having little effect
upon uptake of apoptotic cells of other lineages 15 , it was of
interest to determine whether the prophagocytic effect of
glucocorticoids upon M was restricted to uptake of apoptotic
neutrophils. This proved not to be the case in that methylprednisolone
also promoted phagocytosis of apoptotic Jurkat cells from 13.4 ±
1.4% of M under control conditions to 24.2 ± 1.5% after 24-h
culture with 200 nM methylprednisolone (mean ± SE,
n = 6, p < 0.001). Furthermore, 24-h
incubation of M with 1 µM dexamethasone increased M
phagocytosis of apoptotic eosinophils from 49.0 ± 3.9% to
67.8 ± 5.1% (mean ± SE, n = 6,
p < 0.001).
We were also concerned to establish whether the prophagocytic effect of
glucocorticoids was limited to the human monocyte-derived M, because
previous work has established that other phagocyte populations
recognize apoptotic cells by mechanisms distinct from the
Vß3 vitronectin
receptor/thrombospondin/CD36 system characterized in the human
monocyte-derived M (reviewed in 13 . Human mesangial cells,
"semiprofessional" glomerular phagocytes, which usually recognize
apoptotic neutrophils by a CD36-independent,
Vß3-mediated mechanism 11 also exhibited
increased phagocytosis of apoptotic neutrophils when treated with
glucocorticoids (Fig. 4). Furthermore,
this effect was not limited by phagocyte species in that
glucocorticoids exhibited a similar promotion of phagocytosis of
apoptotic neutrophils by murine bone marrow-derived M populations
and murine thioglycollate-elicited peritoneal inflammatory M (Fig. 5), which normally employ
Vß3-independent phosphatidylserine
receptors in the uptake of apoptotic cells 27 .
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Growing evidence supports the concept that phagocyte clearance of
leukocytes undergoing apoptosis is an injury-limiting leukocyte
disposal mechanism likely to promote the resolution of inflammation
(reviewed in 37 . Strong support is provided by data that
demonstrate that M and mesangial cells phagocytosing apoptotic cells
are not stimulated to secrete proinflammatory eicosanoids, granule
enzymes, or cytokines 6, 10, 11 . The wide-ranging
anti-inflammatory effects of glucocorticoids suggested that the
promotion of phagocyte clearance of apoptotic cells would be unlikely
to provoke proinflammatory responses from ingesting phagocytes, but it
was clearly necessary to test this assumption. We found that increases
in M uptake of apoptotic neutrophils induced by methylprednisolone
pretreatment did not invoke the release of the proinflammatory
chemokines IL-8 and MCP-1 by M (Table II) or mesangial cells (Table III). However, methylprednisolone at 200
nM failed to abrogate IL-8 and MCP-1 secretion triggered by M
phagocytosis of opsonized zymosan particles (Table III), indicating
that glucocorticoids were not acting to suppress production of these
chemokines by M.
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phagocytic
capacity at 5 days
Although human monocyte-derived M are able to ingest senescent
neutrophils undergoing apoptosis, freshly isolated human peripheral
blood monocytes lack this capacity, which is progressively acquired
over several days as adherent monocytes cultured with autologous serum
differentiate into M 29 . Because the mechanisms responsible are
very poorly understood, we considered it possible that early exposure
of maturing monocytes to glucocorticoids might undesirably disrupt
acquisition of the capacity to ingest apoptotic neutrophils. In fact,
the earlier maturing monocytes were exposed to 200 nM prednisolone
throughout subsequent culture, the greater (compared with control) the
potentiation of the uptake of apoptotic neutrophils at 5 days (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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treated for 24
h from 4 days’ maturation it was mediated via the RU48686-inhibitable
steroid receptor, not induced by the mineralocorticoid aldosterone or
by sex steroids, and not observed for FcR-mediated phagocytosis.
Furthermore, there was evidence of a generally relevant promotion of
phagocyte clearance of apoptotic cells in that glucocorticoids promoted
M phagocytosis of apoptotic Jurkat T lymphocytes and apoptotic
eosinophils as well as promoting the uptake of apoptotic neutrophils by
both human glomerular mesangial cells and two different populations of
murine M. Finally, the promoting effect of glucocorticoids was not
limited to "semimature" 4-day M because earlier exposure to
glucocorticoid during M differentiation resulted in a still greater
capacity for phagocytosis of apoptotic cells at 5 days.
The data provide new insights into the anti-inflammatory effects of
glucocorticoids, demonstrating a hitherto unrecognized mechanism by
which these agents may ameliorate inflammatory injury (the promotion of
safe phagocytic clearance of leukocytes being deleted by apoptosis).
Data from in vitro studies emphasize that glucocorticoids may
coordinately promote the deletion of certain leukocyte types by
engaging apoptosis 22, 23, 38 , while at the same time up-regulating
the capacity for phagocytic clearance (this study). Recently published
observations that steroid treatment of asthma is associated with the
resolution of eosinophilic inflammation and increased evidence of
eosinophil phagocytosis by broncho-alveolar M provide evidence that
similar mechanisms may operate in vivo in man 8 . Furthermore,
although glucocorticoids slow constitutive apoptosis in neutrophils
21, 23 , glucocorticoid-treated neutrophils eventually undergo
apoptosis and therefore require phagocytic clearance in vivo to prevent
secondary necrosis with the resultant release of toxic granule
contents. Further studies will be required to establish whether these
findings can be extrapolated to inflammatory conditions in different
tissues in vivo. However, it appears likely that the observed increases
in both the proportion of M ingesting apoptotic neutrophils and in
the number of ingested cells per phagocytically active M will equate
to a significant increase in clearance capacity in vivo.
Nevertheless, our observation that glucocorticoid-mediated enhancement
of M phagocytosis of apoptotic cells was not achieved by a costly
loss of the teleogically appropriate lack of proinflammatory response
by the phagocyte is of fundamental importance.
Methylprednisolone-enhanced phagocytosis of apoptotic cells failed to
stimulate IL-8 and MCP-1 release from either M or mesangial cells.
By contrast, under the conditions employed, methylprednisolone did not
suppress M release of IL-8 and MCP-1 after phagocytosis of opsonized
zymosan. These findings were underscored by comparable observations
when mesangial cells were employed as the phagocyte, where IL-1 was
employed as a positive control stimulus 11, 30 instead of opsonized
zymosan. Therefore, failure to elicit a proinflammatory response did
not merely reflect a general suppression of chemokine synthesis by
glucocorticoids, which has been reported in other in vitro systems
(reviewed in 24 .
The modulation of phagocyte capacity for uptake of apoptotic cells by
glucocorticoids exhibits interesting differences from previous reports
on the regulation of phagocytosis of these and other particles. In
contrast to CD44-mediated enhancement of M uptake of apoptotic
neutrophils 15 , the prophagocytic effect of glucocorticoids required
several hours of treatment and was unable to reverse inhibition by
cycloheximide, consistent with a requirement for new protein synthesis.
Moreover, unlike CD44 ligation, prophagocytic effects were not
restricted to apoptotic target cells of the neutrophil lineage.
Furthermore, in other phagocytic systems glucocorticoids have been
reported to inhibit rat alveolar M ingestion of carbon particles
39 and murine M phagocytosis of heat-killed Saccharomyces
cerevisiae 40, 41, 42, 43 . Nevertheless, freshly isolated human monocyte
phagocytosis of ß-glucan particles prepared from the same species of
yeast was enhanced by glucocorticoids such as dexamethasone at 200 nM
44 , and there are reports that glucocorticoids up-regulate M
receptors for cytokines, including those for granulocyte-M CSF 45 ,
which can promote M uptake of apoptotic cells 16 . Consequently,
the current data are not necessarily inconsistent with previous studies
of the glucocorticoid regulation of phagocytosis.
Further studies will be needed to define mechanisms by which
glucocorticoids potentiate phagocytosis of apoptotic cells. Our
preliminary immunofluorescence flow cytometry studies (data not shown)
on 5-day human monocyte-derived M treated with 200 nM
methylprednisolone for 24 h have shown no detectable difference in
M surface expression of components of the
vß3 vitronectin
receptor/thrombospondin/CD36 recognition mechanism 35 and a modest
down-regulation of CD14 consistent with previous reports 46 ,
suggesting that up-regulated expression of these surface receptors is
unlikely to account for the potentiating effects of glucocorticoids.
This work on surface receptor expression will need to be expanded, and
possible effects of glucocorticoids on cytoskeletal elements 47
should also be considered.
Lastly, although hydrocortisone and dexamethasone might appear to be equipotent in our studies, comparisons of concentrations at which different glucocorticoids exert a similar effect in vitro should be made with considerable caution because ABC transporters such as MDR1A can expel synthetic glucocorticoid substrates such as dexamethasone from within leucocytes 48 , with the result that the effective concentration of glucocorticoid at the intracellular receptor is lower than the extracellular concentration applied to the cell.
In conclusion, we report that glucocorticoids specifically enhance the
nonphlogistic phagocytic uptake of apoptotic leukocytes of differing
lineages by human and murine M populations and also
"semiprofessional" phagocytes, glomerular mesangial cells. The data
add new emphasis to the anti-inflammatory properties of
glucocorticoids and suggest that further work could identify novel
therapeutic approaches toward promoting the safe resolution of
inflammatory conditions.
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Acknowledgments |
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Footnotes |
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2 Y.L. and J.M.C. contributed equally to this work and are joint first authors.
3 Address correspondence and reprint requests to Prof. John Savill, Department of Medicine, Royal Infirmary of Edinburgh, Lauriston Place, Edinburgh HE3 9YW, U.K. E-mail address:
4 Abbreviations used in this paper: M, macrophage(s); EIgG, IgG-opsonized E; MPO, myeloperoxidase; PMNs, polymorphonuclear leukocytes; MCP, monocyte chemoattractant protein.
Received for publication August 5, 1998. Accepted for publication December 15, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3/CD36/thrombospondin recognition mechanism and lack of phlogistic response. Am. J. Pathol. 149:911.[Abstract]
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 B. R Walker Glucocorticoids and Cardiovascular Disease Eur. J. Endocrinol., November 1, 2007; 157(5): 545 - 559. [Abstract] [Full Text] [PDF]
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C. N. Serhan, S. D. Brain, C. D. Buckley, D. W. Gilroy, C. Haslett, L. A. J. O'Neill, M. Perretti, A. G. Rossi, and J. L. Wallace Resolution of inflammation: state of the art, definitions and terms FASEB J, February 1, 2007; 21(2): 325 - 332. [Abstract] [Full Text] [PDF]
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 J. S. Gilmour, A. E. Coutinho, J.-F. Cailhier, T. Y. Man, M. Clay, G. Thomas, H. J. Harris, J. J. Mullins, J. R. Seckl, J. S. Savill, et al. Local amplification of glucocorticoids by 11beta-hydroxysteroid dehydrogenase type 1 promotes macrophage phagocytosis of apoptotic leukocytes. J. Immunol., June 15, 2006; 176(12): 7605 - 7611. [Abstract] [Full Text] [PDF]
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 S W Tas, P Quartier, M Botto, and L Fossati-Jimack Macrophages from patients with SLE and rheumatoid arthritis have defective adhesion in vitro, while only SLE macrophages have impaired uptake of apoptotic cells Ann Rheum Dis, February 1, 2006; 65(2): 216 - 221. [Abstract] [Full Text] [PDF]
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 G. R. Small, P. W. F. Hadoke, I. Sharif, A. R. Dover, D. Armour, C. J. Kenyon, G. A. Gray, and B. R. Walker Preventing local regeneration of glucocorticoids by 11{beta}-hydroxysteroid dehydrogenase type 1 enhances angiogenesis PNAS, August 23, 2005; 102(34): 12165 - 12170. [Abstract] [Full Text] [PDF]
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 A V Kamath, I D Pavord, P R Ruparelia, and E R Chilvers Is the neutrophil the key effector cell in severe asthma? Thorax, July 1, 2005; 60(7): 529 - 530. [Full Text] [PDF]
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P. Maderna, S. Yona, M. Perretti, and C. Godson Modulation of Phagocytosis of Apoptotic Neutrophils by Supernatant from Dexamethasone-Treated Macrophages and Annexin-Derived Peptide Ac2-26 J. Immunol., March 15, 2005; 174(6): 3727 - 3733. [Abstract] [Full Text] [PDF]
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N. Jiang and D. S. Pisetsky The effect of inflammation on the generation of plasma DNA from dead and dying cells in the peritoneum J. Leukoc. Biol., March 1, 2005; 77(3): 296 - 302. [Abstract] [Full Text] [PDF]
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C. J. G. de Almeida, L. B. Chiarini, J. P. da Silva, P. M. R. e Silva, M. A. Martins, and R. Linden The cellular prion protein modulates phagocytosis and inflammatory response J. Leukoc. Biol., February 1, 2005; 77(2): 238 - 246. [Abstract] [Full Text] [PDF]
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 R. P. Schleimer Glucocorticoids Suppress Inflammation but Spare Innate Immune Responses in Airway Epithelium Proceedings of the ATS, November 1, 2004; 1(3): 222 - 230. [Abstract] [Full Text] [PDF]
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M. Takahashi, K. Kurosaka, and Y. Kobayashi Immature dendritic cells reduce proinflammatory cytokine production by a coculture of macrophages and apoptotic cells in a cell-to-cell contact-dependent manner J. Leukoc. Biol., May 1, 2004; 75(5): 865 - 873. [Abstract] [Full Text] [PDF]
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 N. Jiang and D. S. Pisetsky The Effect of Dexamethasone on the Generation of Plasma DNA from Dead and Dying Cells Am. J. Pathol., May 1, 2004; 164(5): 1751 - 1759. [Abstract] [Full Text] [PDF]
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 B. McMahon and C. Godson Lipoxins: endogenous regulators of inflammation Am J Physiol Renal Physiol, February 1, 2004; 286(2): F189 - F201. [Abstract] [Full Text] [PDF]
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 J Oei, K Lui, H Wang, and R Henry Decreased neutrophil apoptosis in tracheal fluids of preterm infants at risk of chronic lung disease Arch. Dis. Child. Fetal Neonatal Ed., May 1, 2003; 88(3): F245 - F249. [Abstract] [Full Text] [PDF]
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 T. Yamaryo, K. Oishi, H. Yoshimine, Y. Tsuchihashi, K. Matsushima, and T. Nagatake Fourteen-Member Macrolides Promote the Phosphatidylserine Receptor-Dependent Phagocytosis of Apoptotic Neutrophils by Alveolar Macrophages Antimicrob. Agents Chemother., January 1, 2003; 47(1): 48 - 53. [Abstract] [Full Text] [PDF]
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 V. L. M. Esnault Apoptosis: the central actor in the three hits that trigger anti-neutrophil cytoplasmic antibody-related systemic vasculitis Nephrol. Dial. Transplant., October 1, 2002; 17(10): 1725 - 1728. [Full Text] [PDF]
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C. Ward, I. Dransfield, J. Murray, S. N. Farrow, C. Haslett, and A. G. Rossi Prostaglandin D2 and Its Metabolites Induce Caspase-Dependent Granulocyte Apoptosis That Is Mediated Via Inhibition of I{kappa}B{alpha} Degradation Using a Peroxisome Proliferator-Activated Receptor-{gamma}-Independent Mechanism J. Immunol., June 15, 2002; 168(12): 6232 - 6243. [Abstract] [Full Text] [PDF]
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M. B. Hampton, M. C. M. Vissers, J. I. Keenan, and C. C. Winterbourn Oxidant-mediated phosphatidylserine exposure and macrophage uptake of activated neutrophils: possible impairment in chronic granulomatous disease J. Leukoc. Biol., May 1, 2002; 71(5): 775 - 781. [Abstract] [Full Text] [PDF]
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K. M. Giles, K. Ross, A. G. Rossi, N. A. Hotchin, C. Haslett, and I. Dransfield Glucocorticoid Augmentation of Macrophage Capacity for Phagocytosis of Apoptotic Cells Is Associated with Reduced p130Cas Expression, Loss of Paxillin/pyk2 Phosphorylation, and High Levels of Active Rac J. Immunol., July 15, 2001; 167(2): 976 - 986. [Abstract] [Full Text] [PDF]
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 M. Beigier-Bompadre, P. Barrionuevo, F. Alves-Rosa, C. J. Rubel, M. S. Palermo, and M. A. Isturiz N-Formyl-Methionyl-Leucyl-Phenylalanine Inhibits both Gamma Interferon- and Interleukin-10-Induced Expression of Fc{gamma}RI on Human Monocytes Clin. Vaccine Immunol., March 1, 2001; 8(2): 402 - 408. [Abstract] [Full Text] [PDF]
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M. Schmidt, N. Lugering, A. Lugering, H.-G. Pauels, K. Schulze-Osthoff, W. Domschke, and T. Kucharzik Role of the CD95/CD95 Ligand System in Glucocorticoid-Induced Monocyte Apoptosis J. Immunol., January 15, 2001; 166(2): 1344 - 1351. [Abstract] [Full Text] [PDF]
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U. A. Hirt, F. Gantner, and M. Leist Phagocytosis of Nonapoptotic Cells Dying by Caspase- Independent Mechanisms J. Immunol., June 15, 2000; 164(12): 6520 - 6529. [Abstract] [Full Text] [PDF]
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C. Godson, S. Mitchell, K. Harvey, N. A. Petasis, N. Hogg, and H. R. Brady Cutting Edge: Lipoxins Rapidly Stimulate Nonphlogistic Phagocytosis of Apoptotic Neutrophils by Monocyte-Derived Macrophages J. Immunol., February 15, 2000; 164(4): 1663 - 1667. [Abstract] [Full Text] [PDF]
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C. HASLETT Granulocyte Apoptosis and Its Role in the Resolution and Control of Lung Inflammation Am. J. Respir. Crit. Care Med., November 1, 1999; 160(5): S5 - 11. [Abstract] [Full Text] [PDF]
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M. Schmidt, H.-G. Pauels, N. Lugering, A. Lugering, W. Domschke, and T. Kucharzik Glucocorticoids Induce Apoptosis in Human Monocytes: Potential Role of IL-1{beta} J. Immunol., September 15, 1999; 163(6): 3484 - 3490. [Abstract] [Full Text] [PDF]
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 M. C. Martin, I. Dransfield, C. Haslett, and A. G. Rossi Cyclic AMP Regulation of Neutrophil Apoptosis Occurs via a Novel Protein Kinase A-independent Signaling Pathway J. Biol. Chem., November 21, 2001; 276(48): 45041 - 45050. [Abstract] [Full Text] [PDF]
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