| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of
*
Medicine B and
Immunology and Cell Biology,
Institute for Immunology, University of Münster, Münster, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(3), IL-1
(2, 7), IL-6 (1, 8), or IL-8 (9) by GC has been reported. Recently,
we could demonstrate that GC induce monocyte apoptosis
(10), which may explain monocytopenia that is observed
during GC therapy (11, 12). It is conceivable that at
least some of the anti-inflammatory properties described for GC may
be attributed to the induction of monocyte apoptosis.
Under serum-free culture conditions monocytes will rapidly undergo
apoptosis (13). Stimulation with proinflammatory mediators
such as TNF-, IL-1, or LPS prevents monocyte apoptosis (14, 15). We have recently demonstrated that continuous treatment
with IL-1 almost completely abolished GC-induced monocyte apoptosis
(10). However, it remains still unclear whether the
observed down-regulation of proinflammatory cytokines follows
GC-induced monocyte apoptosis or is an initial proapoptotic signal.
Monocytes express detectable levels of death receptor CD95 and CD95 ligand (CD95L) on their membranes (13). Recently, it has been demonstrated that endogenous expression of CD95 and its ligand CD95L plays a role in spontaneous apoptosis, since blocking CD95 ligation prevented apoptosis of monocytes in culture (13). It was further shown that activated monocytes release CD95L (16, 17) suggesting that the CD95 pathway is involved in autocrine and paracrine monocyte apoptosis. The apoptotic pathway mediated by CD95 is well-defined (18, 19). Triggering of the receptor by its ligand or agonistic Abs induces a death-inducing signaling complex that consists of the adapter protein Fas-associated death domain protein and caspase-8 (20, 21). Caspase-8 is the most proximal element in the caspase cascade. Further downstream in the death pathway, caspase-8 triggers the proteolytic activation of other caspases and the cleavage of various cellular substrates, thereby mediating the apoptotic response (21, 22, 23).
As induction of apoptosis in monocytes seems to be an essential anti-inflammatory process, knowledge of its underlying mechanisms is of great importance. Especially in patients with immunological diseases, who are resistant to GC therapy or those who suffer from GC adverse effects, a better insight into the mechanisms of apoptosis may be of relevance for the development of therapeutic strategies.
In this study we investigated the signaling pathway of GC-induced monocyte apoptosis. We demonstrate that GC-induced apoptosis is associated with an enhanced expression of membrane-bound CD95 and CD95L as well as an increased release of both molecules from the cell surface. Treatment of monocytes with GC was followed by the proteolytic activation of caspase-8 and caspase-3. GC-induced apoptosis was prevented by caspase inhibition as well as by neutralizing CD95L Abs. Our data suggest that GC trigger monocyte apoptosis in an autocrine and paracrine manner involving a CD95-dependent signaling pathway.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
PE-conjugated mouse anti-human Leu M3 mAb (anti-CD14, clone P9, IgG2b) and control mAbs of appropriate isotypes were obtained from Becton Dickinson (Palo, Alto, CA). FITC-labeled annexin V was purchased from Bender Medsystems (Vienna, Austria). The broad caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) was obtained from Promega (Mannheim, Germany), and the caspase-3 inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO) from Calbiochem (Bad Soden, Germany). Anti-human CD95 mAb was purchased from R&D Systems (Wiesbaden, Germany), anti-human CD95-L was provided from PharMingen BD (Heidelberg, Germany) (65320C mouse IgG1 NOK-1) (24, 25) and anti-poly(ADP-ribose)polymerase (PARP) rabbit serum from Roche Molecular Research (Mannheim, Germany). Anti-caspase-8 mAb and a neutralizing anti-CD95L mAb (clone 5G51, mouse IgG1) were provided by BioCheck (Münster, Germany). A colorimetric caspase assay was obtained from Promega. A Western blot analysis system was received from Amersham Pharmacia Biotech (Freiburg, Germany). All other reagents were obtained from Sigma (St. Louis, MO).
Isolation and culture of human monocytes
Human monocytes were isolated from 40 ml EDTA-treated blood, drawn from healthy volunteers or from fresh leukocyte buffy coats. We used a modification of the recently described isotonic density gradient centrifugation method with Ficoll and Percoll (26). 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 an isotonic Percoll density gradient (1.129 g/ml). From the two interphases the upper interphase containing monocytes was collected and washed three times with PBS. The monocyte suspension was adjusted to 1 x 106 cells/ml and plated on 24-well plates (Greiner, Solingen, Germany). Monocytes were further enriched by 90 min of adherence to culture plates and washed twice in PBS. Enriched monocytes were incubated in RPMI 1640 medium supplemented with 1% heat-inactivated, pooled AB sera for 24–72 h in the presence or absence of dexamethasone (10-10–10-6 M). All culture reagents had endotoxin levels of less than 0.01 ng/ml LPS. Viability of the monocytes was more than 95% as determined by trypan blue exclusion and purity was at least 90% as assessed by flow cytometric analysis and nonspecific esterase staining.
Detection of annexin V staining and CD14 expression
Monocytes, prepared and treated as described above, were double-labeled with PE-conjugated Leu M3 mAb (anti-CD14) and annexin V-FITC in PBS for 1 h at room temperature in the dark. PE-conjugated murine IgG mAbs of unrelated specificity were always used as control. After staining, the cells were washed twice in PBS and measured by flow cytometry.
Determination of apoptosis by DNA electrophoresis
DNA extraction and electrophoresis were performed as described previously (10). In brief, 1 x 107 monocytes were first lysed in a hypotonic buffer (10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.2% Triton X-100). After centrifugation (14,000 x g, 30 min), supernatants containing cleaved chromatin were treated with 50 mg/ml RNase and 100 mg/ml proteinase K, and DNA was extracted by phenol/chloroform/isoamylalcohol. After precipitation with ethanol at -20°C and drying and heating of the samples, equal amounts of DNA were loaded on a 1.8% agarose gel and separated by electrophoresis for 2 h at 80 V.
Transmission electron microscopy
Morphological alterations indicative of apoptosis were evaluated by transmission electron microscopy. Cells were washed off the culture plates, centrifuged, fixed in 1% glutaraldehyde/0.1 M Na cacodylate (pH 7.4), and postfixed in 1% OsO4/0.15 M Na cacodylate (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 Electronic Instruments, Mahway, NJ) at an accelerating voltage of 60 kV.
Immunoblotting
The proteolytic activation of caspase-8 and cleavage of PARP were detected by immunoblotting. Following 24 h of culture, monocytes were lysed in 100 µl Laemmli sample buffer containing 2% SDS, 125 mM Tris-HCl (pH 6.8), 20% glycerol, and 1% 2-ME. After centrifugation, 50 µg total protein of each sample was separated under reducing conditions on a SDS-polyacrylamide gel. Protein transfer to Hybond-C nitrocellulose membranes (Amersham Pharmacia Biotech) was performed at 200 mA for 60 min using a Trans-Blot SemiDry Electrophoretic Transfer Cell (Bio-Rad, Richmond, CA). Membranes were blocked with 10% fat-free milk powder in TTBS buffer (0.01% Tween 20, 0.05 M Tris-HCl, 0.15 M NaCl, pH 7.5) for 120 min at room temperature and then incubated for 1 h with the primary Abs. Membranes were washed three times with TTBS and incubated with a peroxidase-conjugated secondary Ab for 1 h. After extensive washing the reaction was developed by enhanced chemiluminescent staining (Amersham Pharmacia Biotech).
Evaluation of necrosis
Necrotic membrane damage was determined by trypan blue exclusion or by propidium iodide (PI) uptake into nonpermeabilized cells and subsequent flow cytometry using standard protocols (14, 17).
Analysis of CD95 and CD95L surface expression
Monocytes were harvested from the culture plates by gentle pipetting on ice and labeled with anti-CD95 or anti-CD95L in FACS buffer (PBS containing 0.01% sodium azide and 15% FCS) for 1 h at room temperature. Cells were then washed in FACS buffer and stained with FITC-conjugated secondary Abs. After additional washings, cells were analyzed for CD95 and CD95L surface expression by flow cytometry.
Colorimetric caspase-3 and caspase-8 assays
After 24 h of culture, monocytes were lysed in 100 µl sample buffer containing 10 mM HEPES (pH 7.4), 220 mM mannitol, 68 mM sucrose, 2 mM NaCl, 2.5 mM KH2PO4, 0.5 mM EGTA, 2 mM MgCl2, 5 mM pyruvate, 1 mM PMSF, and 1 mM DTT. Total protein (50 µg) of each sample was then incubated with the colorimetric caspase-3 substrate Ac-DEVD-pNA. The release of the yellow chromophore pNA (p-nitroanilide) was measured in a spectrophotometer at 405 nm. For caspase-8 assay, monocytes were lysed in the same manner. Total protein (200 µg) of each sample was then incubated with the colorimetric caspase-8 substrate Ac-IETD-AFC. Ac-IETD-AFC is a synthetic tetrapeptide subtrate that is cleaved by active human caspase-8. This substrate is cleaved betwen D and AFC, releasing the fluorogenic AFC, which is detected by spectrophotometer at 490 nm.
Analysis of CD95 and CD95L secretion
Cell culture supernatants were collected after 24 h. CD95 and CD95L were determined in an equal volume of each sample. For immunoblot analysis a standard procedure was used as described above.
Statistical analysis
Results are given as means ± SD. For statistical analysis Student’s unpaired t test was used. Statistical significance was considered if p < 0.05. All experiments were performed at least ten times with different blood donors, unless otherwise indicated.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Monocytes were cultured for 48 h and treated with different
doses of dexamethasone (10-10,
10-8, and 10-6 M).
Apoptosis could be demonstrated by three independent methods including
annexin V staining, DNA laddering, and transmission electron microscopy
(Fig. 1
). GC treatment of the monocytes
increased the number of annexin V-positive cells in a dose-dependent
fashion. After incubation with 10-6 M
dexamethasone, the percentage of apoptotic cells increased from
15.7 ± 4.1% in the medium control to 53.7 ± 11.4% in
GC-treated monocytes (Fig. 1A). Agarose gel electrophoresis
showed that marked DNA laddering typical for apoptosis was already
detectable in monocytes treated with a dose of
10-8 M dexamethasone (Fig. 1B).
|
C shows a healthy
control monocyte with a normal relation of the nucleus and cytoplasm as
well as intact organelles. In contrast, cells treated with different
concentrations of dexamethasone revealed typical features of apoptosis
including cytoplasmic and chromatin condensation and a marked
vacuolization of the cytoplasm (Fig. 1, D–F).
To differentiate between apoptotic and necrotic cells, we performed
double staining with annexin V-FITC and the DNA dye PI. As shown in
Fig. 2, monocytes treated with different
doses of dexamethasone revealed increased annexin V staining, but no
membrane damage. After 48 h of cell culture, we found 10.0%
single-positive monocytes in the medium control (Fig. 2A).
GC treatment increased the number of annexin V-positive and PI-negative
cells to 17.7%, 19.9% and 42.9% at concentrations of
10-10 M, 10-8 M and
10-6 M dexamethasone, respectively (Fig. 2, B–D). In contrast, 94.5% of the cells
permeabilized with saponin became double-positive for PI and annexin V
(Fig. 2E). Therefore, the data show that dexamethasone
induces apoptotic, but not necrotic cell death in human monocytes.
|
To investigate the specificity of the observed effects, we
performed experiments with the GC receptor antagonist mifepristone
(27). Fig. 3 shows that
simultaneous treatment with GC and mifepristone blocked monocyte
apoptosis, as the number of annexin V-positive cells decreased from
47.3% to 20.7%. We further investigated the involvement of caspases
in dexamethasone-induced apoptosis. Pretreatment of cells with
DEVD-CHO, a peptide inhibitor more selective for caspase-3,
significantly attenuated apoptosis, and the number of annexin
V-positive cells was reduced almost 2-fold (Fig. 3). Using a
colorimetric substrate assay we further determined whether incubation
with GC induces caspase-3 activity. Indeed, dexamethasone treatment
strongly increased the proteolytic activity. The increase in caspase-3
activity was markedly prevented following combined stimulation by GC
together with either mifepristone or the broad caspase inhibitor
zVAD-fmk (Fig. 4A).
|
|
Caspase-3 is a key effector caspase in various forms of apoptosis
and degrades several cellular proteins including the DNA repair enzyme
PARP and DNA fragmentation factor (28, 29, 30). Using
immunoblot analysis we found that treatment of monocytes with different
concentrations of dexamethasone induced the cleavage of the 116-kDa
full-length form of PARP into the characteristic p85 fragment (Fig. 4B). However, in control monocytes, the p85 fragment was
only weakly detectable.
We further studied the activation of caspase-8/Fas-associated death
domain protein-like IL-1-converting enzyme, which is the most
proximal regulatory caspase during death receptor-mediated cell death.
Caspase-8 is synthesized as an inactive precursor of 54 kDa and,
following formation of 43- and 41-kDa intermediate cleavage products,
processed to a p18 and p10 heterodimer. Treatment of monocytes with
dexamethasone resulted in the conversion of procaspase-8 to the 43- and
41-kDa intermediate fragments as well as to the p18 and p10 subunits
(Fig. 5A). The results
indicate that activation of caspase-8 is involved in GC-induced
monocyte apoptosis. Upon simultaneous treatment with dexamethasone and
mifepristone, the p43 and p41 lanes as well as the p18 and p10 bands
were only weakly expressed. Caspase-8 activation could also be
demonstrated by a colorimetric assay that was increased in monocytes
after treatment with GC. Addition of mifepristone as well as zVAD-fmk
almost completely abolished the effect of GC on monocytes (Fig. 5B).
|
Dexamethasone-induced caspase-8 activation suggesting the possible
involvement of CD95 and CD95L in GC-induced apoptosis. Monocytes and
macrophages are known to express both CD95 and CD95L on their membrane
surface (13). To investigate a potential involvement of
the CD95 system in GC-induced apoptosis, we determined CD95 and CD95L
expression on monocyte surface by FACS analysis. Treatment of monocytes
with dexamethasone led to increase of the surface expression of both
CD95 and CD95L as demonstrated by changes in the mean fluorescence
(Fig. 6). Cotreatment with the GC
receptor antagonist mifepristone inhibited the inducible and also the
constitutive expression of CD95 and CD95L. In contrast, simultaneous
treatment of monocytes with dexamethasone and the caspase inhibitor
DEVD-CHO diminished monocyte apoptosis (see Fig. 2), but did not block
the up-regulation of membrane-bound CD95 and CD95L (Fig. 6).
|
).
Cotreatment with mifepristone inhibited the release of CD95 and CD95L
into supernatants of GC-treated monocytes.
|
To examine the functional relevance of the CD95 system in
GC-induced monocyte apoptosis, we treated monocytes simultaneously with
various doses of dexamethasone together with either agonistic
anti-CD95 mAb or neutralizing anti-CD95L mAb. After 48 h,
monocytes were stained with annexin V-FITC and measured by FACS
analysis. As expected, anti-CD95-mAb significantly enhanced
monocyte apoptosis in the medium control as well as in GC-treated
cultures (Fig. 8). In contrast,
neutralizing CD95L by anti-CD95L mAb decreased GC-induced monocyte
apoptosis; incubation with 10-6 M dexamethasone
resulted in 38.4 ± 6,1% annexin V-positive cells, while
cotreatment with anti-CD95L reduced the amount of apoptotic cells
to 24.3 ± 4.9%. These results suggest that CD95/CD95L
interaction is functionally involved in GC-induced cell death of
monocytes.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
GC exert immunosuppressive and anti-inflammatory effects on
different cell types including monocytes and macrophages. Some of the
anti-inflammatory actions induced in monocytes are caused by the
down-regulation of proinflammatory cytokines, such as TNF-, IL-1,
and IL-6 (1, 2, 3, 7, 8), as well as by repression of NF-
B
activation (34, 35). Induction of apoptosis in various
cell populations, such as lymphocytes, as a potential immunomodulating
mechanism of GC action has been described earlier
(36, 37, 38). Recently, we have demonstrated that GC are
capable of inducing apoptosis also in human monocytes
(10). However, the signaling pathway responsible for
GC-induced monocyte apoptosis remained unknown so far.
As spontaneous monocyte apoptosis was reported to be mediated at least in part by a mechanism requiring CD95 and CD95L (39, 40), we determined the potential involvement of those molecules in GC-induced apoptosis. Monocytes express CD95 and CD95L on their membrane surfaces (13). Under serum-free conditions, monocytes increase membrane-bound CD95 and CD95L, thereby inducing autocrine and paracrine cell death (13). This process can be abolished by blocking CD95L. It has also been shown that monocytes contain high levels of intracellular CD95L, which can be released or expressed on the surface membrane after cellular activation (16, 17, 41). Therefore, it was postulated that CD95 ligation plays an important role in monocyte apoptosis. In our study we found a significant enhancement of CD95 and CD95L expression on monocyte surface membranes as well as an increased release into cell culture supernatants after treatment with GC. This mechanism seemed to be directly induced by GC, as blocking of the GC receptor with mifepristone abolished up-regulation of CD95 and CD95L expression. By contrast, caspase inhibition did not alter CD95 and CD95L up-regulation. Moreover, treatment with agonistic anti-CD95-enhanced apoptosis, whereas a neutralizing Ab directed against CD95L abrogated GC-induced apoptosis. Therefore, we suggest that GC-induced monocyte apoptosis is at least in part mediated by an autocrine and paracrine mechanism involving the CD95 pathway. CD95-triggered apoptosis of "bystander" monocytes and other immune cells at inflamed sites has been shown recently (17, 42) and might explain anti-inflammatory activities of GC in vitro and in vivo (43, 44).
Involvement of CD95 in GC-induced apoptosis is supported by the observed cleavage of caspase-8 into its intermediate products and the active p18 subunit as well as demonstration of caspase-8 activity by a fluorometric assay. Caspase-8, the proximal element in death receptor-mediated apoptosis, triggers the proteolytic activation of other downstream caspases and subsequent cleavage of a variety of cellular substrates (22). A key apoptotic process is the activation of the effector caspase-3, which is also activated during spontaneous monocyte apoptosis (45). We observed an activation of caspase-3 in GC-treated monocytes, whereas inhibition of the GC receptor abolished caspase-3 activation. There is ample evidence that caspase-3 is responsible for the observed cleavage of PARP and other manifestations of apoptosis. For instance, DNA fragmentation and exposure of phosphatidylserine to the outer membrane leaflet have been shown to be directly mediated by caspase-3 activation (46).
The mechanisms by which GC induce apoptosis are presumably controlled by cell type-specific processes. In thymocytes and T cell hybridomas, for instance, GC induce apoptosis by mechanisms which are independent of CD95. Paradoxically, in these cells dexamethasone protects against activation-induced cell death by suppressing the CD95 and CD95L expression (47). An inhibition of the inducible expression of these molecules has also been observed for ligands of related nuclear receptors, such as retinoids, which, similarly to GC, protect against activation-induced cell death of T-cells, but induce apoptosis in several other cell types (47). It is known that CD95L and other cytokines of the TNF family are transcriptionally regulated in a cell type- and context-specific manner (48). Future experiments have to elucidate which transcriptional activators or control elements regulate CD95 and CD95L expression in response to GC in monocytes. It is interesting to note that the effects of GC are strikingly opposite in different liver cell types (49). While dexamethasone diminished LPS-induced stimulation of CD95L expression in nonparenchymal liver cells, it markedly stimulated CD95L expression in parenchymal cells.
Apoptosis induced in monocytes by immunosuppressive steroids may be an
important mechanism in the treatment of chronic inflammatory diseases.
Systemic monocytopenia, which is observed following steroid treatment,
may be a consequence of an increased rate of monocyte apoptosis
(11, 12). While our data clearly demonstrate that GC
induce apoptosis by a CD95-dependent pathway, our results do not
exclude that additional mechanisms may exist. GC are known as potent
monocyte deactivators that down-regulate proinflammatory cytokine
expression. As TNF- and IL-1 exert anti-apoptotic effects on
human monocytes (14, 15), down-regulation of these
mediators by GC may exert proapoptotic effects. We could recently
demonstrate that IL-1 may abolish GC-induced monocyte apoptosis
(10). It is conceivable that inhibition of IL-1
synthesis contributes to GC-induced apoptosis. It has also been shown
that NF-B, an essential transcription factor for proinflammatory
cytokine synthesis, controls the transcription of survival genes
(50, 51) and, therefore, may play a role in monocyte
apoptosis induced by GC. Inhibition of anti-apoptotic mechanisms
such as the production of members of the Bcl-2 family, activation of
protein kinase B/Akt, or the STAT pathway might also be involved in
GC-induced apoptosis. Therefore, further studies will have to clarify
whether mechanisms other than CD95 may additionally contribute to
GC-induced apoptosis in monocytes.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Torsten Kucharzik, Department of Medicine B, University of Münster, Albert-Schweitzer-Strasse 33, D-48129 Münster, Germany.
3 Abbreviations used in this paper: GC, glucocorticoids; CD95L, CD95 ligand; DEVD-CHO, N-acetyl-Asp-Glu-Val-Asp-aldehyde; PARP, poly(ADP-ribose)polymerase; PI, propidium iodide; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; Ac-IETD-AFC, N-acetyl-Ile-Glu-Thr-Asp-7-amino-4-trifluoromethyl coumarin.
Received for publication May 1, 2000. Accepted for publication October 17, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and IL-1 production in human peripheral blood mononuclear adherent cells by a glucocorticoid hormone. J. Immunol. 135:3191.[Abstract]
secretion. Inflamm. Res. 46:447.[Medline]
production by glucocorticoids in human monocytes: the mechanism of action depends on the activation signal. Biochem. Biophys. Res. Commun. 180:1383.[Medline]
. J. Immunol. 163:3484., and IL-1 prevent programmed cell death (apoptosis) in human peripheral blood monocytes. J. Immunol. 146:1541.[Abstract]
, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell 81:801.[Medline]
converting enzyme-like protease involved in Fas-mediated apoptosis. J. Biol. Chem. 271:1841.B in mediation of immunosuppression by glucocorticoids. Science 270:283.B activity through induction of IB synthesis. Science 270:286.B in preventing TNF-induced cell death. Science 274:782.B or die?. Curr. Biol. 7:R94.[Medline]This article has been cited by other articles:
|
|
 |
|
  D. Lang, S. Reuter, T. Buzescu, C. August, and S. Heidenreich Heme-induced heme oxygenase-1 (HO-1) in human monocytes inhibits apoptosis despite caspase-3 up-regulation Int. Immunol., February 1, 2005; 17(2): 155 - 165. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Y. Almawi, O. K. Melemedjian, and M. M. A. Jaoude On the link between Bcl-2 family proteins and glucocorticoid-induced apoptosis J. Leukoc. Biol., July 1, 2004; 76(1): 7 - 14. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Xu, R. Lucas, M. Schuchmann, S. Kuhnle, T. Meergans, A. P. Barreiros, A. W. Lohse, G. Otto, and A. Wendel GM-CSF Restores Innate, But Not Adaptive, Immune Responses in Glucocorticoid-Immunosuppressed Human Blood In Vitro J. Immunol., July 15, 2003; 171(2): 938 - 947. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Herr, E. Ucur, K. Herzer, S. Okouoyo, R. Ridder, P. H. Krammer, M. von Knebel Doeberitz, and K.-M. Debatin Glucocorticoid Cotreatment Induces Apoptosis Resistance toward Cancer Therapy in Carcinomas Cancer Res., June 15, 2003; 63(12): 3112 - 3120. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. L. Tamma and R. F. Coico IgD-receptor (IgD-R) cross-linking partially protects murine T cells from dexamethasone-induced apoptosis J. Leukoc. Biol., June 1, 2003; 73(6): 764 - 770. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. Marchetti, B. Di Marco, G. Cifone, G. Migliorati, and C. Riccardi Dexamethasone-induced apoptosis of thymocytes: role of glucocorticoid receptor-associated Src kinase and caspase-8 activation Blood, January 15, 2003; 101(2): 585 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Cipollone, A. Ganci, A. Greco, M. R. Panara, M. Pasquale, D. Di Gregorio, E. Porreca, A. Mezzetti, F. Cuccurullo, and P. Patrignani Modulation of Aspirin-Insensitive Eicosanoid Biosynthesis by 6-Methylprednisolone in Unstable Angina Circulation, January 7, 2003; 107(1): 55 - 61. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Joashi, S. M. Tibby, C. Turner, A. Mayer, C. Austin, D. Anderson, A. Durward, and I. A. Murdoch Soluble Fas may be a proinflammatory marker after cardiopulmonary bypass in children J. Thorac. Cardiovasc. Surg., January 1, 2002; 123(1): 137 - 144. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
).
Typical apoptotic DNA laddering was detected in GC-treated monocytes
(
). A molecular size marker of a 123-bp DNA ladder is indicated.
C–F, Morphological features of monocytes
treated with dexamethasone as detected by electron microscopy; in the
control population (C), cells revealed an intact
morphology of the nucleus and cytoplasm. After treatment with
dexamethasone (D–F), monocytes exhibited
typical signs of apoptosis with nuclear condensation, degeneration of
organelles, cytoplasmic hypervacuolization and loss of plasma membrane
microvilli. Magnification, x7200.
) were detected. By contrast, after
simultaneous treatment with dexamethasone and mifepristone, the
intermediate cleavage products of 43 and 41 kDa as well as the bands of
18 and 10 kDa were only weakly expressed. Data reveal representative
results of four different experiments that show similar results.
B, Monocytes were treated with 10-6 M
dexamethasone for 48 h. Caspase-8 activity was measured by a
colorimetric caspase assay. Caspase-8 was activated by stimulation with
GC and inhibited by cotreatment with 10-5 M mifepristone
as well as with the caspase-inhibitor zVAD-fmk (50 µM). Data
represent means ± SD from four independent experiments. *,
p < 0.05 compared with dexamethasone; 
