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Glucocorticoids Downregulate Gene Expression of GM-CSF, NAP-l/IL-S, and
IL-6, but not of M-CSF in Human Fibroblasts
By Andreas Tobler, Roland Meier, Michael Seitz, Beatrice Dewald, Marco Baggiolini, and Martin F. Fey
Cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage-CSF (M-CSF), neutrophilactivating peptide-1 /interleukin4 (NAP-1/IL-8), and interleukin-6 (IL-6) are pivotal in the regulation of hematopoiesis and
immune responses. In mesenchymal cells, their expression is
induced by tumor necrosis factor a (TNF) and other agents.
We now show that, while induction of cytokine expression
by TNF in human lung fibroblasts was parallel, glucocorticoid
hormones differentially affected their production. Dexamethasone (1 pmol/L) concordantly repressed expression of GMCSF, NAP-l/IL-8 and IL-6. RNA and protein levels were
reduced t o approximately 5%. 20%. and 30% of control cells,
respectively, as determined by Northern blot analyses and
immunoassays. A 50% reduction of RNA levels for all three
cytokines occurred in the range of 1 hour. In contrast,
dexamethasone (1 pmol/L) did not decrease M-CSF RNA
levels and protein release. M-CSF RNA and protein levels
were maintained even when dexamethasone (1 pmol/L) was
present for the whole duration of a 48-hour TNF stimulation.
Further experiments showed that dexamethasone downregulates expression of GM-CSF, NAP-I /IL-8, and IL-6 mainly by
decreasing the mRNA stability of these cytokines, and that
the dexamethasone-mediated repression of cytokine expression depends on ongoing protein and RNA syntheses. Our
study suggests that glucocorticoid hormones repress expression of a set of cytokine genes important in conditions of
stress. However, they seem not t o affect M-CSF expression,
which is likely t o be more crucial in maintaining long-term
functions of myeloid cells.
o 1992by The American Society of Hematology.
G
Ingelheim, Switzerland) and was diluted in McCoy’s medium
(GIBCO, Grand Island, NY) supplemented by 10% fetal bovine
serum (FBS; Nabi, Miami, FL). Dexamethasone (Sigma Chemical,
St Louis, MO) was dissolved in 95% ethanol to a stock concentration of 1 mmol1L and stored at -20°C. The final concentration of
ethanol did not exceed 0.1%. Actinomycin D (Fluka, Buchs,
Switzerland) was kept as stock solution (2 mg/mL) dissolved in
dimethyl sulfoxide. Its final concentration never exceeded 0.3%
vollvol, which did not change cytokine RNA levels in TNFstimulated fibroblasts. Cycloheximide (Sigma) was kept as stock
solution (1mg1mL) dissolved in phosphate-buffered saline.
Cells and cell culture. Normal human embryonic lung fibroblasts (WI38, American Type Tissue Culture Collection, Rockville,
MD) were cultured in McCoy’s medium supplemented with 10%
FBS in a humidified atmosphere containing 5% CO, at 37°C. They
were passaged by treatment with 0.5% trypsin, 0.02% EDTA
(wtlvol) in Hanks’ solution, and were cultured for different lengths
of time in the presence of TNF, dexamethasone, and/or actinomycin D and cycloheximide. Cell viability, as determined by trypan
blue exclusion, was not affected by the experimental conditions,
and dexamethasone did not alter the proliferative capacity of the
cells as determined by ’[Hlthymidine incorporation studies.14
Assessment of protein levels in the supematants of Fbroblasts.
GM-CSF was determined using a sandwich immunoassay with a
cocktail of three epitope-mapped monoclonal antibodies
RANULOCYTE-MACROPHAGE colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), interleukin-6 (IL-6) and neutrophil
activating peptide-1 /interleukin-8 (NAP-1 /IL-8) belong to
a complex cytokine network involved in the regulation of
hematopoiesis and the pathophysiology of inflammation.’-6
In the bone marrow GM-CSF, M-CSF, and IL-6, either
alone or in synergy with other factors, stimulate immature
myeloid progenitor cells to proliferate and differentiate.
GM-CSF also enhances activities of neutrophils, blood
monocytes, and tissue macrophages, and M-CSF augments
functional activities of macrophages. IL-6 induces the final
differentiation of B-lymphocytes into antibody-producing
cells and stimulates the release of major acute-phase
proteins. The recently identified NAP-l/IL-8 is a highly
selective neutrophil attractant and activator. Therefore,
these cytokines are of paramount importance in various
inflammatory processes.
Typically, all of these cytokines are produced by cells
widely distributed in various tissues, such as monocytes,
endothelial cells, and fibroblasts, and their expression is
rapidly induced by other cytokines. For example, tumor
necrosis factor a (TNF) stimulates human fibroblasts to
produce various cytokines such as GM-CSF, M-CSF, NAPl/IL-8, and IL-6.’.I3 If cytokine action is no longer necessary, counter-regulatory measures leading to their swift
downregulation must step into operation. Therefore, knowledge about possible downregulatory mechanisms and agents
is important.
We recently showed that glucocorticoids powerfully and
rapidly inhibit GM-CSF expression in TNF-stimulated
human WI38 fibroblast^.'^ In the present study we asked
whether glucocorticoids might concordantly affect the expression of a whole set of cytokine genes in human
fibroblasts; therefore, we examined their effects on IL-6,
NAP-l/IL-8, and M-CSF production in comparison with
GM-CSF. We found evidence for both a concordant and a
differential regulation of cytokine expression by giucocorticoids.
MATERIALS AND METHODS
Reagents. Recombinant human TNF, expressed in Escherichia
coli, was generously provided by Dr R. Lacher (Boehringer,
Blood, Vol79, No 1 (January 1). 1992:pp 45-51
From the Central Hematology Laboratory, Laboratory for Clinical
and Experimental Research, Department of Rheumatology, Inselspital
Beme, Theodor-Kocher-Institute,and Institute of Medical Oncology,
University of Beme, Switzerland.
Submitted March 19, 1991; accepted September 5, 1991.
Supported by grants from the Swiss National Science Foundation
(31.9141.87to A.T. and 31.28579.90 to M.F.F.), and in part by the
Roche Research Foundation, the Bemese Cancer Ligue, the Central
Laboratory of the Swiss Red Cross, and the Kurt & Senta Hermann
Foundation.
Address reprint requests to Andreas Tobler, MD, Central Hematology Laboratory of the Universiv of Beme, Inselspital, CH-3010 Beme,
Switzerland.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
O 1992 by The American Society of Hematology.
0006-49711921 7901-0025$3.00/0
45
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TOBLER ET AL
46
(MoAbs)." Samples and recombinant GM-CSF standards were
incubated in microtiter plates coated with three MoAbs (1.7
kg/mL of each MoAb). Bound rhuGM-CSF was detected by
incubation with a cocktail of three biotinylated GM-CSF MoAbs
(200 ng/mL of each MoAb) and streptavidin alkaline phosphatase
(1:7000). The detection limit was 0.2 ng/mL. IL-6 was determined
using a commercially available rhuIL-6 immunoassay (Research
and Diagnostic Systems, Minneapolis, MN) with a detection limit
of 4 pg/mL. M-CSF was determined by a radioimmunoassay that
uses rhuM-CSF as the standard, radioiodinated rhuM-CSF as
tracer, and a rabbit antiserum to rhuM-CSF.I6The detection limit
was 0.2 ng/mL. NAP-1/IL-8 was determined using a double-ligand
immunoassay method." Samples and recombinant NAP-1/IL-8
standards were incubated in microtiter plates coated with a mouse
anti-NAP-l/IL-b MoAb. After washing, a goat anti-NAP-l/IL-8
polyclonal antibody (pAb) conjugated to alkaline phosphatase was
added and the activity was measured with p-nitrophenylphosphate.
The detection limit was 0.2 ng/mL.
Assessment of RNA levels. Total cellular RNA was extracted by
the acid guanidinium thiocyanate phenol-chloroform method.'*
RNA samples (10 pg) were size separated by an agarose formaldehyde gel (1% wt/vol) and transferred to a nylon membrane
(Hybond-N, Amersham, Amersham, England). Hybridization with
random primer [32P]-labeled probes (1 X lo6 cpm/mL hybridization solution) was for 16 to 24 hours at 42°C as de~cribed.'~.'~
Filters
were washed to a stringency of 0.1% SSC at 65T, and exposed for 6
hours to 4 days at -70°C to XM-films (3M, Trimax, Ferrania,
Italy). Autoradiographs were scanned by laser densitometry (LKB
2202 Ultrascan, Bromma, Sweden). The density readings of the
signals corresponding to the cytokine RNA levels were normalized
to the respective beta-actin RNA levels, as described elsewhere.'*
DNA probes. Purified inserts were used as DNA probes. The
human GM-CSF cDNA probe (0.8 kb, EcoRI-BamHI) was derived
from plasmid pCSF-1:''
M-CSF cDNA was purified from the
pcCSF-12 plasmid (1.6 kb, XhoI-EcoRI)?' IL-6 cDNA was from
plasmid pXM (0.5 kb, PstI) (Genetics Institute, Cambridge, MA),
and NAP-1/IL-8 cDNA was derived from plasmid p(NAP)6T3
(0.85 kb, BamHI).U The beta-actin DNA probe (0.7 kb, EcoRIBamHI) was from plasmid pHF A-3'ut.=
RESULTS
Cytokine expression and release in TNF-stimulated fibroblasts. Fibroblasts were cultured for 48 hours in the
presence of TNF alone at concentrations ranging from
0.125 to 12.5 ng/mL (Table l), and the release of four
cytokines was monitored in the same culture supernatants.
The conditioned media of unstimulated cells contained
readily detectable levels of M-CSF, very low levels of IL-6,
but no measurable GM-CSF or NAP-l/IL-8. Stimulation
by TNF markedly enhanced the release of all four cytokines, and maximum levels were observed in the presence of
1.25 to 12.5 ng/mL TNF. The levels of protein released at
the highest T N F concentration were as follows
(means ? SD, see also footnote Table 1): GM-CSF 1.0 &
0.4 ng/mL; IL-6 19.8 f 0.1 ng/mL; NAP-l/IL-8 126 f 26
ng/mL; M-CSF 4.3 f 0.4 ng/mL. Thus, with GM-CSF,
M-CSF, and IL-6, near maximal responses were seen at
1.25 ng/mL TNF, whereas 12.5 ng/mL TNF still led to an
increase of NAP-1DL-8 protein levels.
Similar experiments were performed to assess steadystate RNA levels in fibroblasts cultured for 8 hours with
TNF (0.125 to 25 ng/mL) (Fig 1). A low constitutive
production was observed for M-CSF, as opposed to GMCSF, IL-6, or NAF'-l/IL-8, and the inducing effect of TNF
was concentration dependent. The TNF effect was also
rapid; maximum RNA levels were reached after 2 hours for
IL-6, 4 hours for M-CSF, and 8 hours for GM-CSF and
NAP-l/IL-8 (Fig 2). Maximum RNA levels of GM-CSF,
NAP-l/IL-8, and M-CSF were maintained for 48 hours of
TNF (12.5 ng/mL) exposure (data not shown).
Taken together, these results show that the expression of
all four cytokines was induced more or less in parallel by
TNF.
Effects of dexamethasone. As shown on Table 1, addition of dexamethasone (1 kmol/L) almost completely
reduced GM-CSF protein levels, and markedly decreased
those of IL-6 and NAP-l/IL-8 (to 7% to 20% and 28% to
59%, respectively), almost irrespective of the TNF concentrations (0.125 to 25 ng/mL). In parallel to the inhibition of
protein production, dexamethasone (1 kmol/L) decreased
RNA levels of GM-CSF, IL-6, and NAP-l/IL-8 to 0% to
5%, 5% to 20%, and 20% to 40% (100% values = RNA
levels from cells stimulated with 25 ng/mL TNF alone) (Fig
1). However, the drug neither reduced M-CSF protein nor
RNA levels, which rather showed a slight increase in its
presence. These results show that dexamethasone inhibits
expression of GM-CSF, IL-6, and NAP-l/IL-8, but not of
M-CSF.
To examine the inhibitory effects of dexamethasone in
more detail, fibroblasts were cultured for 8 hours in the
presence of 12.5 ng/mL TNF and increasing concentrations
of dexamethasone (0.1 nmol/L, 10 nmol/L, 1 pmol/L). As
shown in Fig 3, the drug markedly decreased RNA levels
for GM-CSF and NAP-l/IL-8 without affecting those of
Table 1. Induction of Cytokine Productionby Cultured W138 Fibroblasts Stimulated With TNF: Effect of Dexamethasone
0.125 ng1mLTNF
1.25nglmLTNF
Cytokines
No TNF or Dexa
0.125 nglmL TNF
GM-CSF
IL-6
NAP-1IIL-8
M-CSF
< 0.2
0.2 f 0.01
<0.2
0.9 f 0.2
< 0.2
1.9 f 0.04
5.7 -c 0.8
2.2 f 0.2
12.5 ng1mLTNF
+
+
+
1 pmollL Dexa
1.25 nglmLTNF
1 pmol1L Dexa
12.5 nglmL TNF
< 0.2
0.9 k 0.4
17.9 f 0.8
35.0 f 2.2
4.4 f 0.7
<0.2
1.2 f 0.1
15.1 f 1.2
4.4 f 0.4
1.0 2 0.4
19.8 0.1
126.0 f 26.0
4.3 I0.4
0.5
3.3
3.2
k
k
k
0.5
0.8
0.5
*
1 pmol1L Dexa
<0.2
1.4
35.1
5.5
* 0.01
2
k
4.3
0.3
Abbreviation: Dexa, dexamethasone.
Values represent concentrations of cytokine in the culture medium (ng protein/mL) obtained in one representative experiment. Two additional
series of identical experiments were performed with similar results. Data are expressed as means f SD of triplicate (IL-6, NAP-1/IL-8) or
quadruplicate (M-CSF, GM-CSF)measurements.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
47
REGULATION OF CYTOKINES BY GLUCOCORTICOIDS
1 2 3 4 5 6 7 8 9
TNFa - + + + + + + +
D E X A - - + - + - + - +
+
kb
Probe
0.9 -
GM-CSF
% 0 25 0 80 0 40
0 100 5
4.0 -
M-CSF
1.3 -
IL-6
sone was present for the whole 48-hour duration of the
experiment (data not shown).
Effects of actinomycin D and cycloheximideon the downregulatoty action of glucocorticoids. To analyze whether dexamethasone decreases RNA levels of GM-CSF, NAP-1, and
IL-6 independently of ongoing protein synthesis, experiments were performed with cycloheximide, which inhibits
translation (Fig 4).Cycloheximide (20 kg/mL) alone led to
a superinduction of all four cytokines studied. In cells
preincubated for 1 hour with cycloheximide, dexamethasone (1 p ” l / L ) was unable to decrease RNA levels of any
of the cytokines. Similarly, cycloheximide abolished the
repressive effect of dexamethasone in fibroblasts stimulated
with TNF. These experiments demonstrate that the suppressive effect of dexamethasone on GM-CSF, NAP-l/IL-8,
and IL-6 expression depends on ongoing protein synthesis,
C
TNFa TNFU+DEXA
m
-
1 2 3 4 5 6 7 8
% 10 50 100 110 120 85 140 100 150
% 0 60
1.8
5 95 10 100 15 100 20
-
NAP- 1/IL-8
kb
Probe
0.9 -
GM-CSF
O h 0 75 75 100 35
% 0 45 20 80 25 80 40 100 30
2.1 -
S-actin
Fig 1. Effect of dexamethasone on cytokine RNA levels in fibroblasts stimulated with TNF. Cells were exposed for 8 hours t o TNF
(0.125-25 ng/mL) alone or in combination with 1pmol/L dexamethasone (Northern blot). The TNF concentrations in lanes 1 t o 9 were 0,
0.125,0.125,1.25,1.25,12.5,12.5,25.0, and 25.0ng/mL.The presence
of dexamethasone is indicated by
Percent values represent signal
intensity as assessed by densitometry of autoradiographs. The 100%
values are from cells stimulated with 25 ng/mL TNF alone (lane 8).
The Northern blot was hybridized sequentially with GM-CSF, NAP-1/
IL-8 and beta-actin probes, and simultaneously with M-CSF and IL-6.
5
5
5
4.0 -
M-CSF
1.8 -
NAP- 1/IL-8
% 30 80 115 100 115 120 120 120
+.
% 0 40 85 100 50 35 40 30
1.3 M-CSF. At 10 nmol/L and 1 p ” l / L , RNA levels for
GM-CSF were reduced to 30% and 20%, and those for
NAP-1 /IL-8 to 85% and 30%, respectively.
The time course of these effects was determined in
fibroblasts cultured for 8 hours in the presence of 12.5
ng/mL TNF by adding dexamethasone (1 p ” l / L ) at the
beginning and 6, 4, and 2 hours before completion of the
experiment (Fig 2). Dexamethasone rapidly reduced RNA
levels of GM-CSF, IL-6, and NAP-l/IL-8; a decrease to
approximately 50% and below was observed when dexamethasone was added 2 hours before termination of the
experiment. Again, dexamethasone (1 pmol/L) did not
reduce, but even slightly enhanced, M-CSF RNA levels.
Also, RNA levels of GM-CSF and NAP-l/IL-8 were
reduced well below 50% when dexamethasone was added,
even 24 hours after the start of TNF stimulation, and
M-CSF RNA levels were maintained even when dexametha-
IL-6
% 0 120 80 100 40 40 35 20
2.1 -
p-act in
Fig 2. Time-dependent effect of dexamethasone on levels of
cytokine RNA in TNF-stimulated fibroblasts. Cells were exposed t o
TNF alone or in combination with dexamethasone for 0 t o 8 hours
(Northern blot). Lane 1, untreated cells; lanes 2 through 4, cells
exposed t o TNF (12.5 ng/mL) alone for 2, 4, and 8 hours; lanes 5
through 8, addition of dexamethasone (1pmol/L) at time points 6,4,
2, and 0 hours of an 8-hour TNF exposure. The Northern blot was
hybridized sequentially with GM-CSF, IL-6, and beta-actin probes,
and simultaneously with M-CSF and NAP-1/IL-8 probes. The percent
values refer t o signal intensity as assessed by densitometry of
autoradiographs. The 100% values are from cells stimulated by TNF
alone for 8 hours (lane 4).
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TOBLER ET AL
TNF,
TNF,+DEXA
n-
1 2 3 4
kb
Probe
0.9 -
GM-CSF
% 100 100 30 20
1.8 -
NAP- 1/IL-8
% 100 100 85 30
that in the same experiment dexamethasone strikingly
reduced GM-CSF, NAP-l/IL-8, and IL-6 mRNA levels in
the absence of actinomycin D (Fig SA, lane 10). Similar
results were obtained in two independent experiments. In
contrast, the T/2 of GM-CSF, NAP-l/IL-8, and IL-6
mRNA was in the range of 1 hour in the presence of
dexamethasone (1 pmol/L) alone, as assessed by experiments in which the fibroblasts were pretreated with TNF
(12.5 ng/mL) for 4 hours and subsequently exposed for 1 to
4 hours to dexamethasone (Northern blots not shown,
densitometry readings of autoradiographs shown in Fig
SB). Our results demonstrate that the T/2 of GM-CSF,
NAP-l/IL-8, and IL-6 mRNA was markedly longer when
overall transcription was blocked than in the presence of
dexamethasone, and that ongoing RNA synthesis is necessary for the repressive effects of dexamethasone.
TNF,
CHX
DEXA
+-+++- + - - + + - +
---+-+++
-
-
1 2 3 4 5 6 7 8
4.0
-
M-CSF
kb
Probe
0.9 -
GM-CSF
% 100 100 100 100
2.1
-
w-
we--
4.0
-
1.8
-
NAP- 1 /IL-8
1.3
-
IL-6
2.1
-
P-actin
M-CSF
p-actin
Fig 3. Concentration-dependent effect of dexamethasoneon cytokine RNA levels in TNF-stimulated fibroblasts. Fibroblasts were exposed for 8 hours t o TNF (12.5 ng/mL) alone or in combination with
increasing concentrations of dexamethasone (Northern blot). Lane 1,
cells exposed t o TNF alone; lanes 2 through 4, TNF in combination
with 0.1 nmol/L, 10 nmol/L, and 1 pmol/L dexamethasone. Percent
values refer t o signal intensity as assessed by densitometry of
autoradiographs. The 100% values are from cells stimulated with TNF
alone (lane 1). The Northern blot was hybridized sequentially with
GM-CSF, NAP-1/IL-8, M-CSF, and beta-actin probes.
suggesting that an indirect regulatory mechanism is involved in the glucocorticoid effects.
To examine whether glucocorticoids might downregulate
GM-CSF, NAP-1 /IL-8, and IL-6 expression by decreasing
mRNA stability, experiments were performed with actinomycin D, which blocks overall transcription (Fig SA and B).
The half-lifes (T/2) of the four cytokine mRNAs were more
than 5 hours when fibroblasts were pretreated for 4 hours
by TNF and subsequently exposed to actinomycin D (6
p,g/mL) for 1 to 8 hours. When dexamethasone was added
simultaneously with actinomycin D for 1 to 8 hours to cells
prestimulated for 4 hours with TNF, cytokine mRNA levels
remained more or less unchanged. It is important to note
Fig 4. Effects of cycloheximide (CHX) on the dexamethasonemediated suppression of cytokine RNA levels. Fibroblasts were
exposed t o either CHX (20 pg/mL) or TNF (12.5 ng/mL) alone, or they
were exposed t o CHX for 1hour, and subsequently TNF or dexamethasone (1pmol/L) alone or in combination were added (Northern blot).
Lane 1, untreated cells; lane 2, exposure t o CHX alone for 5 hours;
lanes 3 and 4, exposure t o either TNF or dexamethasone alone for 4
hours; lane 5, pretreatment with CHX and subsequent 4-hour exposure t o TNF; lane 6, pretreatment with CHX and then 4 hours
exposure t o both TNF and dexamethasone; lane 7, concurrent 4 hours
exposure t o TNF and dexamethasone; lane 8,pretreatment with CHX
and subsequent 4 hours exposure t o dexamethasone. The Northern
blot was hybridized sequentially with GM-CSF, M-CSF, NAP-1/lL-8,
IL-6, and beta-actin probes.
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REGULATION OF CYTOKINES BY GLUCOCORTICOIDS
+Actin0
+Actin0
+DEXA +DEXA
n
n
1 2 3 4 5 6 7 8 9 1 0
TNF,
A
kb
Probe
0.9
-
GM-CSF
4.0
-
M-CSF
1.8
-
NAP- 1/IL-8
Dexa
+
ActinoA
Dexa
+ Actino
Actino
Actino
GM-CSF
z
M-CSF
Z
20
Dexa
0
I
L"
2
4
hours
Actino
6
+
8
10
0
2
6
4
s
10
hours
Dexa
0 6 0
NAP-1IIL-8
z
=
20
0
2
6
4
hours
Fig 5. Effects of actinomycin D on the dexamethasone-mediated suppression of cytokine RNA levels in TNF-stimulated fibroblasts. (A)
Fibroblasts were exposed for 4 hours t o TNF (12.5 ng/ mL) and for the subsequent 1t o 8 hours t o either actinomycin D (6 pg/mL) or actinomycin D
and dexamethasone (1 pmol/L) (Northern blot). Lane 1, cells exposed for 4 hours t o TNF alone; lanes 2 through 5, cells prestimulatedfor 4 hours
with TNF and exposed for the next 1,2,5, and 8 hours t o actinomycin D; lanes 6 through 9, cells prestimulated for 4 hours with TNF and exposed
for the next 1,2,5, and 8 hours t o actinomycin D and dexamethasone; lane 10, cells prestimulatedfor 4 hours with TNF and exposed for the next 8
hours t o dexamethasone. The Northern blot was hybridized sequentially with GM-CSF, M-CSF, NAP-1/IL-8, IL-6, and beta-actin probes. (B) Plots
of relative mRNA levels of GM-CSF, M-CSF, NAP-1/IL-8, and IL-6 in TNF-stimulated fibroblasts in the presence of dexamethasone (Dexa), as
described in Results and of actinomycin D (Actino), or actinomycin D and dexamethasone, as shown in A. Intensities of hybridization signals were
determined by densitometry readings of several different exposures of the autoradiographs. The 100% values are from cells stimulated for 4
hours with TNF. Note the markedly shorter T / 2 of the respective transcripts in TNF-stimulated cells treated with dexamethasone alone as
compared with those treated with actinomycin D or with actinomycin D and dexamethasone.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
TOBLER ET AL
50
DISCUSSION
The present study shows that the synthetic glucocorticoid
dexamethasone concordantly represses TNF-induced expression of GM-CSF, NAP-l/IL-8, and IL-6 in human lung
fibroblasts. Inhibition by dexamethasone was observed at
the level of RNA and protein. It occurred rapidly, and it
was most effective for GM-CSF.
The regulation of M-CSF expression was found to differ
from that of the other cytokines. First, a constitutive
expression of M-CSF was easily detected in primary fibroblasts. Second, dexamethasone did not reduce the expression of M-CSF in cells stimulated by TNF. It is important to
note that the same supernatants of fibroblasts were used to
determine protein release by specific immunoassays, and
that the same Northern blots were sequentially hybridized
with the specific cDNA probes. Therefore, the effects
observed in our studies, particularly the differences between M-CSF and the other cytokines, are directly comparable.
Our data are in keeping with other studies that have
demonstrated differences in the regulation of M-CSF gene
expression compared with that of the other cytokines. For
example, in human dermal fibroblasts parental cells did not
produce GM-CSF, G-CSF, and IL-1, but constitutively
expressed M-CSF.Z4Introduction of a mutant Val” H-ras
oncogene into these fibroblasts resulted in a constitutive
expression of GM-CSF, G-CSF, and IL-1, whereas levels of
M-CSF production remained unchanged. Another study
has shown that M-CSF and G-CSF transcripts are differentially regulated in human peripheral blood monocytes.*’
G-CSF expression can be induced by endotoxin, but not
IL-3 and GM-CSF, whereas M-CSF expression is increased
by GM-CSF and IL-3 but not by endotoxin.
The T/2s of GM-CSF, NAP-l/IL-8, and IL-6 mRNA in
TNF-stimulated fibroblasts were much shorter in the presence of dexamethasone than when overall transcription was
blocked by actinomycin D. This indicates that glucocorticoids exert their action mainly by decreasing the stability of
GM-CSF, NAP-l/IL-8, and IL-6 mRNA. If the preferential effect of dexamethasone were to interfere directly with
transcription, one would expect that dexamethasone and
actinomycin D would similarly affect cytokine mRNA
levels. Cytokines prone to rapid up- and downregulation,
such as GM-CSF, IL-6, and NAP-l/IL-8, possess repeated
AUUUA sequences in the 3’ untranslated region of the
mRNAs, whereas M-CSF mRNA does
Earlier
studies have shown that these sequences are critical for
mRNA stability because they may represent recognition
sites for RNases.” Therefore, one possible mechanism by
which glucocorticoids destabilize RNAs of cytokines may
be through the synthesis of certain ribonucleases that may
bind to defined mRNA sequences, for example, to AUUUArich motifs, leading to the rapid degradation of the target
mRNA. Our findings that ongoing protein and RNA
syntheses are necessary for the action of dexamethasone
supports the notion of an indirect effect of glucocorticoids.
Recently, an AUUUA-specific mRNA binding protein was
identified? and another study showed that AUUUA-rich
sequences in the 3’ untranslated region mediate the increased turnover of interferon-beta mRNA induced by
dexamethas0ne.j’
Our presented in vitro evidence that glucocorticoid
hormones inhibit expression of GM-CSF, IL-6, and NAP-1 /
IL-8 suggests that they coordinately regulate a whole set of
cytokine genes that are crucial in conditions of stress, such
as inflammatory reactions. On the other hand, glucocorticoid hormones do not affect expression of M-CSF, which is
likely to be more critical in sustaining long-term functions
of myeloid cells, for instance, the survival of macrophages.
ACKNOWLEDGMENT
We thank E. Ischi, G. Niklaus, and L. Theilkaes for excellent
technical assistance, and Dr. G. Zenke (Sandoz-AG, Basel, Switzerland) for performing the GM-CSF enzyme immunoassay. We are
indebted to Dr. P. Ralph, Cetus Corporation, Emeryville, CA, for
performing M-CSF radioimmunoassays.
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1992 79: 45-51
Glucocorticoids downregulate gene expression of GM-CSF,
NAP-1/IL-8, and IL-6, but not of M-CSF in human fibroblasts
A Tobler, R Meier, M Seitz, B Dewald, M Baggiolini and MF Fey
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