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*
Department of Medicine B,
Institute for Immunology, University of Münster, Münster, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß were measured by ELISA. GC receptor was blocked
with mifepristone. Caspase 3 was inhibited with caspase-3 inhibitor
(DEVD-CHO). Stimulation with different GC at therapeutic concentrations
resulted in monocyte apoptosis in a time- and dose-dependent manner.
Necrosis was excluded by propidium iodide staining. Proinflammatory
cytokines such as IL-1ß and TNF- were down-regulated by GC
treatment. Continuous treatment of monocytes with IL-1ß, but not with
TNF-, could almost completely prevent GC-induced cell death. The
addition of mifepristone or caspase-3 inhibitor could partially
abrogate GC-induced apoptosis as well as GC-induced inhibition of
IL-1ß. This is the first study to demonstrate induction of apoptosis
by GC in human monocytes. GC-induced monocyte apoptosis may be
partially mediated through effects on IL-1ß production. It is
conceivable that GC exert their anti-inflammatory capacity in
various diseases, at least in part, by the induction of apoptosis in
monocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IL-1ß, IL-6, IL-8, proteases, or free oxygen radicals
(3). In addition, they also produce anti-inflammatory
cytokines such as IL-10 (4, 5), IL-1Ra (6, 7), or soluble TNF receptors (8, 9). Peripheral
monocytes as well as differentiated tissue macrophages are known to
play a key role in the inflammatory process of various immunomediated
diseases such as inflammatory bowel disease (10, 11),
rheumatoid arthritis (12), or sepsis
(13).
Glucocorticoids (GC)3
are potent anti-inflammatory and immunosuppressive agents that act
on many cells including monocytes (14). They are potent
down-regulators of monocyte-secreted proinflammatory mediators
including TNF- (15, 16), IL-1ß (17), or
IL-6 (18), which mediate inflammatory activity in various
diseases (19, 20, 21). However, the exact cellular mechanisms
by which GC exert their anti-inflammatory capacity in monocytes are
only incompletely understood.
Apoptosis is a physiological self-destruction mechanism that is
regulated by an endogenous program and signals from other cells. It is
characterized by typical morphological features and leads to the
removal of the dying cell by macrophages in a way that prevents the
onset of inflammatory reactions (22, 23). Monocytes have
been shown to undergo rapid apoptosis in culture unless stimulated with
LPS or with proinflammatory cytokines such as TNF- or IL-1ß, which
enhance monocyte survival by omitting the initiation of apoptosis
(24, 25). Recent studies dealing with the role of
anti-inflammatory cytokines and their suppressive effects on
monocytes demonstrated that IL-4 as well as IL-10 is able to induce
apoptosis in monocytes (26, 27). Therefore, it was
postulated that the regulatory role of anti-inflammatory cytokines
in the development of inflammatory reactions also involves the
regulation of monocyte apoptosis.
In the present study, we investigated whether GC might evoke apoptosis in human monocytes. Using four different methods for the determination of apoptosis, we could clearly demonstrate that GC at therapeutic concentrations are strong inducers of apoptosis in human monocytes in a time- and dose-dependent manner. Our data suggest that, at least in part, the well-known anti-inflammatory and immunosuppressive effects of GC on monocytes and macrophages are mediated by the induction of apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PE-conjugated mouse anti-human Leu M3 mAb (anti-CD14, clone P9, IgG2b) and control mAb of appropriate Ig isotype were obtained from Becton Dickinson (Palo Alto, CA). FITC-labeled annexin-V (1/1 stoichiometric complex) was purchased from Bender Medsystems (Vienna, Austria). Caspase-3 inhibitor (28, 29, 30) was obtained from Calbiochem (65812 Bad Soden, Germany), and dexamethasone, prednisolone, hydrocortisone (water-soluble), mifepristone (31) (GC receptor antagonist), and all other reagents were obtained from Sigma (St. Louis, MO).
Isolation and culture of human monocytes
Human monocytes were isolated from 40 ml of EDTA-treated blood,
drawn from healthy volunteers, or from fresh leukocyte buffy coats. We
used a modification of the recently described hypotonic density
gradient centrifugation method with Ficoll and Percoll
(32). Briefly, mononuclear cells were collected from the
interphase after Ficoll separation and washed twice in PBS.
Subsequently, cells were separated into lymphocytes and monocytes on a
hypotonic Percoll density gradient (1.129 g/ml). From the two
interphases, the upper interphase containing the monocytes was
collected and washed three times with PBS. The monocyte suspension was
adjusted to 106 monocytes/ml and plated on
24-well plates (Greiner, Solingen, Germany). Monocytes were further
enriched by a 90-min adherence to culture plates and washed two times
with PBS. Enriched monocytes were incubated in RPMI 1640 medium
supplemented with 1% (v/v) heat-inactivated, pooled AB sera (complete
culture medium) for 24–96 h in the presence or absence of
dexamethasone (10-9 to
10-3 M), prednisolone (4 x
10-6 to 4 x 10-3
M), and hydrocortisone (3 x 10-5 to 3
x 10-2 M). All culture reagents used had
endotoxin levels of <0.01 ng/ml LPS. The viability of the monocytes
was >95% as determined by trypan blue exclusion; purity was >90% as
assessed by flow cytometric analysis (Fig. 1
A) and nonspecific esterase
staining.
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Monocytes, prepared and treated as described above, were double-labeled with PE-conjugated Leu M3 mAb (anti-CD14) and annexin-V FITC in PBS (with 100 mg/l CaCl2 and 100 mg/l MgCl2) for 1 h at room temperature in the dark. PE- and FITC-conjugated murine IgG mAbs of unrelated specificities were always used as controls. After staining, the cells were washed twice with PBS and measured by flow cytometry.
Determination of apoptosis by DNA electrophoresis
DNA extraction and electrophoresis were performed as described previously (33). In brief, 1 x 107 monocytes were first lysed by a hypotonic lysing buffer (10 mM Tris-HCl, 1 mM EDTA, and 0.2% Triton X-100, pH 7.4). After centrifugation (14,000 x g, 30 min) supernatants containing cleaved chromatin were treated with RNase (50 mg/ml) and proteinase K (100 mg/ml); DNA was extracted by phenol/chloroform/isoamylalcohol. After precipitation with conc. ethanol (99.6%) at -20°C and drying and heating of samples, equal amounts of DNA were loaded on a 1.8% agarose gel and separated by electrophoresis for 2 h at 80 V. The lower detection limit for visualization of oligonucleosomal bands was 1.0 µg of DNA.
TUNEL procedure
DNA strand breaks were identified by labeling free 3'-OH termini with modified nucleotides in an enzymatic reaction. TdT catalyzes the polymerization of nucleotides to free 3'-OH DNA ends in a template-independent manner. First, monocytes were fixed in 4% paraformaldehyde at room temperature for 30 min. Subsequently, the cytoplasma membrane was permeabilized with 0.1% Triton X-100 for 2 min on ice. In the next step, DNA was labeled with FITC-conjugated NTP in the presence of TdT for 1 h at 37°C. After labeling, cells were washed twice with PBS and analyzed by flow cytometry and immunofluorescence microscopy. Cells labeled in absence of TdT were always used as negative controls, whereas cells pretreated with DNase after permeabilization were used as positive controls.
Transmission electron microscopy (TEM)
Typical morphological alterations indicative of apoptosis were evaluated by TEM. Cells were washed off the culture plates, centrifuged, fixed in 1% glutaraldehyde/0.1 M sodium cacodylate-HCl (pH 7.4), and postfixed in 1% OsO4/0.15 M sodium cacodylate-HCl (pH 7.4). Samples were dehydrated in ascending ethanol series and embedded in epoxy resin (Epon 812). Ultrathin sections were mounted on 150 mesh Formvar-coated copper grids and poststained with aqueous saturated uranyl acetate and 2% lead citrate before being examined on a Philips CM 10 electron microscope (Philips Electronics, Mahway, NJ) at an accelerating voltage of 60 kV by one of the authors (M.S.).
Evaluation of cell necrosis
The viability of monocytes treated with GC was determined by trypan blue exclusion or by flow cytometry analysis of propidium iodide (PI) uptake from nonpermeabilized cells using standard protocols (24, 34).
Measurement of cytokines
IL-1ß and TNF- were determined in supernatants of cultured
monocytes using sandwich ELISAs developed by Immunotech (Marseilles,
France). IL-1ß and TNF- ELISAs had a sensitivity of 5 pg/ml. A
standard cytokine preparation (recombinant cytokine with defined
concentration) was used as internal control in all tests. All assays
were tested for cross-reaction by adding several cytokines and soluble
receptors to different cytokine standards.
Blocking of GC receptor and caspase 3
Mifepristone was added to monocytes that were cultured for 48 h in the presence of different concentrations of dexamethasone. (Mifepristone was used at a concentration 10 times higher than the GC to block the GC receptor) (31). To inhibit caspase activity, we added a caspase-3 inhibitor to GC-treated monocytes (DEVD-CHO, 10-6 M) (28, 29, 30). Apoptosis was determined by staining with annexin-V as described above.
Statistical analysis
Results are given as the mean ± SD. For statistical analysis, the Student’s unpaired t test was used. Statistical significance was considered if p was <0.05. All experiments were performed at least 10 times with different blood donors, unless otherwise indicated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Treatment with GC induced apoptosis in human monocytes. We were
able to demonstrate monocyte apoptosis with four independent methods.
By measuring annexin-V/CD14 binding on cell surfaces and TUNEL, we were
able to identify the early stages of apoptosis. Agarose gel
electrophoresis and TEM were performed to demonstrate the late stages
of apoptosis. Using FACS analysis, we determined a significant increase
of annexin-V-positive cells from 14% up to 71% after incubation with
dexamethasone at different concentrations and a decrease of
CD14+ cells from 52% to 12% (Fig. 1
B). Using the TUNEL method, we were able to demonstrate
GC-induced apoptosis in monocytes by fluorescence staining as well as
by FACS analysis (Fig. 2). Although the
control population showed only few TUNEL-positive cells with a
homogenous fluorescent nucleus, almost all cells of the
dexamethasone-treated population were TUNEL-positive. Stimulation with
a high dexamethasone concentration (10-4 M
dexamethasone) induced nucleus fragmentation (Fig. 2). Apoptosis was
quantified in parallel by fluorescence microscopy and FACS analysis. We
were able to demonstrate an enhancement of TUNEL-positive cells after
stimulation with dexamethasone from 30% up to 95% (Fig. 2). In
addition, we were able to demonstrate apoptosis by characteristic
DNA-laddering using agarose gel electrophoresis (Fig. 3). In the time kinetics, specific
DNA-laddering could be shown after dexamethasone
(10-6 M) incubation lasting for >72 h.
Apoptosis-specific laddering was dose-dependent at dexamethasone
concentrations of 10-6 M to
10-4 M after an incubation period of 72 h
(Fig. 3B). Using low concentrations of dexamethasone, only
intact genomic DNA could be identified. Morphological alterations of
GC-treated monocytes were determined by TEM. Fig. 4, A–C shows normal, typical
control monocytes, whereas Fig. 4, D–F demonstrates three
morphologically altered monocytes stimulated with
10-6 M dexamethasone. Fig. 4, A–C
represents a normal nucleus-plasma-relation with a predominance of the
cytoplasm. We also observed heterochromatic DNA in the nucleus and
intact cytoplasmatic organelles in control monocytes. In Fig. 4, D–F, the relation between nucleus and cytoplasm is shifted
to the nucleus. In GC-treated monocytes, we show shrunk cells, a
condensed nucleus, and increased vacuolization of the cytoplasm. These
signs are typical of apoptotic cells.
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GC induced apoptosis in human monocytes in a dose- and
time-dependent manner. We were able to demonstrate a significant
increase of annexin-V-positive cells at rising dexamethasone
concentrations (10-8 M to
10-3 M) from 30.3% (p
< 0.05) to 53.7% (p < 0.01) (medium: 15.6%)
(Fig. 5A). After an incubation
period of 48 h, we noticed a significant increase (and a strongly
significant increase after 72 h) of annexin-V-positive monocytes.
Annexin-V-positive monocytes in the medium were 21.7% after a 72-h
incubation, whereas an increase was found in the dexamethasone-treated
group of up to 50.1% at 10-8 M
(p < 0.01), 65.05% at
10-6 M (p < 0.01), and
69.0% at a concentration of 10-4 M
dexamethasone (p < 0.01) (Fig. 5B).
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Monocytes were incubated with mifepristone (10-fold higher
concentration than the GC) (31) to block the GC receptor
and with dexamethasone (10-8 M and
10-6 M). We determined a diminished rate of
apoptosis in monocytes that were cotreated with GC and mifepristone
(30.2% vs 19.7%; 10-8 M dexamethasone vs
dexamethasone plus mifepristone, p < 0.05) (Fig. 6).
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Monocytes were costimulated with different concentrations of
dexamethasone (10-8 M,
10-6 M, and 10-4 M) and
with caspase-3 inhibitor (10-6 M DEVD-CHO)
(28, 29, 30). After 48 h of cell culture, apoptosis was
measured by annexin-V staining. We were able to demonstrate that
GC-induced apoptosis was diminished in monocytes that were treated with
caspase-3 inhibitor (30.2% stimulated with 10-8
M dexamethasone vs 16.4% treated with 10-8 M
dexamethasone plus DEVD-CHO; p < 0.05) (Fig. 7).
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Monocytes were cultured for 48 h and stimulated with
different concentrations of dexamethasone (10-8
M, 10-6 M, and 10-4 M).
Cells were further treated with proinflammatory cytokines (50 U/ml
TNF-, 50 U/ml IL-1ß, and 50 U/ml TNF- plus 50 U/ml IL-1ß)
every 8 h. Apoptosis was determined by flow cytometry. We were
able to demonstrate that continuous stimulation with IL-1ß almost
completely prevented GC-induced monocyte apoptosis. The monocyte
apoptosis induced by different concentrations of GC decreased after
treatment with IL-1ß (Fig. 8). In
contrast, treatment with TNF- induced no changes or even an increase
of apoptosis.
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We examined different GC with regard to their capacity to induce
apoptosis in human monocytes. Prednisolone, hydrocortisone, and
dexamethasone were tested by the same relative concentrations according
to their biological strength in human compartments. The cell culture
incubation period for these experiments was 48 h. All drugs
induced apoptosis in human monocytes in a dose-dependent manner (Fig. 9). After an incubation period of 48
h, we determined the following annexin-V-positive monocytes: medium:
15.6%; concentration B: 10-6 M dexamethasone at
29.3%, 4 x 10-6 M prednisolone at 25.8%,
and 3 x 10-5 M hydrocortisone at 36.0%
(p < 0.05) (compared with medium control).
After stimulation with higher concentrations of GC (concentration C:
10-4 M dexamethasone, 4 x
10-4 M prednisolone, and 3 x
10-3 M hydrocortisone), there was also a
strongly significant induction of apoptosis (p
< 0.01).
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The exclusion of necrotic cell death is essential for the
demonstration of programmed cell death (PCD). For this purpose,
monocytes were incubated with different dexamethasone concentrations
for 48 h in cell culture and stained with PI. Untreated monocytes
with permeable cytoplasma membrane served as positive controls. As
shown in Fig. 10 treatment with various
doses of dexamethasone failed to increase PI staining compared with
controls (filled histogram). After a 48-h incubation, we found 90%
PI-negative monocytes in the control medium, whereas the ratio was 91%
in monocytes treated with 10-8 M dexamethasone,
88% in monocytes treated with 10-6 M
dexamethasone, and 89% in cells stimulated with
10-4 M dexamethasone.
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and IL-1ß after
treatment with GC
To examine the influence of GC on cytokine production, we
performed ELISAs of TNF- and IL-1ß in cell culture supernatants.
Monocytes were incubated with different concentrations of dexamethasone
as well as with mifepristone and caspase-3 inhibitor for 48 h. We
noticed a down-regulation of cytokine production depending upon GC
concentrations (Table I). After an
incubation period of 48 h, a strong decrease of TNF- (64.0
± 15.1% to 29.2 ± 13.4%; control vs
10-4 M dexamethasone; p < 0.01)
and of IL-1ß (327.6 ± 38.2 to 126.1 ± 20.3; control vs
10-4 M dexamethasone; p < 0.01)
was evident. Incubation with mifepristone (10-5
M) partially diminished the effects of GC on IL-1ß production by
monocytes compared with cells treated with dexamethasone
(10-6 M) alone (299.7 pg/ml IL-1ß vs 172.1
pg/ml IL-1ß). Caspase-3 inhibition (caspase-3 inhibitor,
10-6 M) also partially abrogated the effect of
GC on IL-1ß secretion in monocytes compared with dexamethasone
(10-6 M)-treated cells alone (274.2 pg/ml vs
172.1 pg/ml IL-1ß).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß, which contribute to the
immune and inflammatory activities of monocytes and macrophages.
Induction of apoptosis in monocytes as a potential immunomodulating
mechanism of GC has not yet been described. In the present study, the
apoptotic effects of GC on highly purified populations of human
monocytes have been determined by four different methods. As marker for
early apoptosis, we performed binding studies with annexin-V, which was
bound on apoptotic monocytes. A significant increase of
annexin-V-positive monocytes after stimulation with dexamethasone was
observed. TUNEL also became strongly positive in monocytes treated with
GC in a dose-dependent manner. Later on by means of TEM, GC-treated
monocytes revealed typical morphological signs of apoptosis, such as
cytoplasmic hypervacuolization and chromatin condensation of the
nucleus. Apoptosis was further confirmed by characteristic ladder
patterns in DNA electrophoresis. Monocyte necrosis could be excluded
using PI staining. In our study, we used GC concentrations from
10-9 M to 10-6 M, which
are reached in the treatment of acute or chronic inflammatory diseases.
To exclude the different properties of various types of GC,
dexamethasone, prednisolone, and hydrocortisone were examined. When we
compared the relative concentrations of these GC depending upon their
biological strength, we could not find any significant difference
concerning their apoptogenic capacity.
We could further demonstrate a diminished induction of apoptosis after
cotreatment with the GC receptor antagonist mifepristone. Therefore, we
conclude that there is a direct receptor-mediated effect of GC on
monocyte apoptosis. GC-induced monocyte apoptosis could also be
partially blocked by treatment with caspase-3 inhibitor. Caspase-3 is a
main enzyme in the apoptosis-signaling pathway that deactivates
poly(ADP-ribose)-polymerase and therefore seems to be involved in
GC-induced monocyte apoptosis. Until now, the exact mechanisms of the
immunosuppressive activity of GC were unknown. It is likely that
multiple modes of action exist (35). High-affinity GC
receptors have been identified in earlier studies (36).
Monocytopenia (37) as well as diminished chemotaxis and
decreased antimicrobial activity have been described elsewhere. In
addition, it could be demonstrated that monocyte-derived
proinflammatory cytokines such as IL-1, IL-6, and TNF- are
suppressed by GC (9, 17, 38). Down-regulation of the
proinflammatory cytokines TNF- and IL-1ß by GC has also been
determined in our monocyte culture system. Recently, it has also been
suggested that proinflammatory cytokines such as IL-1ß and TNF-
exert anti-apoptotic effects as they rescue monocytes from
undergoing apoptosis and prolong survival of monocytes (24, 25). Down-regulation of anti-apoptotic mediators such as
TNF- or IL-1ß as determined in our study may promote apoptosis in
monocytes. We were able to demonstrate that continuous treatment with
IL-1ß, in contrast to TNF-, almost completely prevents GC-induced
apoptosis. Therefore, IL-1ß seems to have important antiapoptotic
properties, and it is conceivable that the observed effects of
GC-induced monocyte apoptosis are mediated through effects of GC
inhibition of IL-1ß production. However, the partial reversal of
GC-induced IL-1ß inhibition of caspase-3 antagonism implies that
apoptosis is required for full inhibition of IL-1ß. This suggests a
direct effect of apoptosis on IL-1ß production rather than these
effects being exclusively in the reverse direction.
In activated immune cells, the induction of apoptosis may be an additional important mechanism by which GC to exert their anti-inflammatory properties. There are several reports demonstrating that immunosuppressive agents, including GC, induce apoptosis in T lymphocytes (39, 40), whereas no study deals with GC-induced apoptosis in monocytes.
The induction of PCD by immunosuppressive or anti-inflammatory cytokines has recently been described by others. IL-4, a T cell-derived cytokine that acts on monocytes, as well as IL-10, have been shown to induce apoptosis in peripheral monocytes (26, 27, 33). PCD as evoked in monocytes by immunosuppressive agents such as steroids or anti-inflammatory cytokines may therefore be an important mechanism in the treatment of immunomediated diseases, in addition to the well known monocyte-deactivating effects of these mediators. Following steroid treatment, systemic monocytopenia may be a consequence of an increased rate of monocyte apoptosis.
The induction of apoptosis in monocytes by GC may have two contrasting effects. During the state of inflammation, the induction of PCD in highly activated monocytes, which secrete a variety of mediators maintaining inflammation, may have therapeutic benefit. The disadvantage of monocyte apoptosis, however, may be that potent immune cells belonging to the mononuclear phagocyte system will be eliminated. Consequently, an increased risk of infectious diseases may develop in patients treated with GC.
All in all, our data suggest that induction of apoptosis in human monocytes appears to be an important way for GC to exert their anti-inflammatory and immunosuppressive properties. The monocyte apoptosis induced by immunosuppressive agents such as GC may regulate cell number at the inflammatory site and also reduce release of toxic mediators into systemic circulation.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Torsten Kucharzik, Department of Medicine B, University of Münster, Albert-Schweitzer-Str. 33, D-48129 Münster, Germany. E-mail address:
3 Abbreviations used in this paper: GC, glucocorticoid(s); PCD, programmed cell death; PI, propidium iodide; TEM, transmission electron microscopy.
Received for publication February 24, 1999. Accepted for publication July 13, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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suppression. Immunol. Cell Biol. 75:345.[Medline]
secretion. Inflamm. Res. 46:447.[Medline]
and IL-1ß production in human peripheral blood mononuclear adherent cells by a glucocorticoid hormone. J. Immunol. 140:1895., and IL-1ß prevent programmed cell death (apoptosis) in human peripheral blood monocytes. J. Immunol. 146:1541.[Abstract]
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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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