1. Art and Diseño

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Type-I Interferons Are Potent Inhibitors of Interleukin-8 Production in
Hematopoietic and Bone Marrow Stromal Cells
By M. Javad Aman, Gregor Rudolf, Jutta Goldschmitt, Walter E. Aulitzky, Charles Lam, Christoph Huber,
and Christian Peschel
Interleukin-8 (IL-8) is produced by many cell types upon
stimulation with bacterial products or inflammation-associated cytokines such as tumor necrosis factor-a and IL-I
Interferons (IFNs) represent another group of cytokines
that are induced by similar stimuli in inflammatory reactions. We show now that type-I IFNs are potent inhibitors
of IL-8 expression in vitro and in vivo. A significant reduction of both secretion of IL-8 protein and accumulation of
IL-8 mRNA in vitro was observed in several cell types comprising peripheral blood mononuclear cells (PBMNC) from
healthy donors and from patients with chronic myelogenous leukemia (CML), the myelomonocytic cell line THP-1,
and bone marrow (BM) stromal cells as a representative
model for BM microenvironment. By contrast, in lipopoly-
.
saccharide-stimulated polymorphonuclear phagocytes IFN
failed to suppress IL-8 expression. In untreated patients
with CML, a constitutive expression of IL-8 mRNA was
detected in freshly isolated PBMNC that was markedly reduced 5 hours after therapeutic application of IFN-a. The
mechanism of IL-8 downregulation was studied more in
detail in the THP-1 cell line. The experiments showed that
de novo protein synthesis was not required for the inhibitory effect. RNA decay analysis and nuclear run-on assays
suggest that in THP-1 cell line the inhibitionof IL-8 expression is predominantly regulated at the posttranscriptional
level.
0 1993 by The American Society of Hematology.
L
OCAL ACCUMULATION of neutrophils in inflammareported in fibroblast^.^^ We have studied the role of type-I
tory disease states is predominantly regulated by reIFNs on expression of IL-8 in hematopoietic and bone
lease of chemotactic substances and a recently recognized
marrow (BM) stromal cells in vitro and after in vivo applicagroup of cytokines, the interleukin-8 (IL-B)/monocyte chetion of IFN-a to patients with chronic myelogenous leukemotactic and activating factor (MCAF) family of chemomia (CML). Our results show that IFNs downregulate IL-8
kines.I4 Chemotactic activity induced by IL-8 is associated
production in vivo and in vitro in hematopoietic cells and
with the induction of numerous additional functions in neusuggest that IFNs may have antiinflammatory effects by limtrophils. IL-8 increases adhesion of neutrophils to unstimuiting accumulation of neutrophils by inhibiting the crucial
lated endothelial cells in vitro, whereas binding of neutrochemotactic signal to neutrophils.
phils to endothelial cells preactivated by IL-1,tumor
necrosis factor (TNF), or lipopolysaccharide (LPS) is inhibMATERIALS AND METHODS
ited.5,6IL-8 induces expression of adhesion molecules and
Materials. Recombinant human-IFN-a2b (rh-IFN-a2b) with a
complement receptor type-I' and induces the release of sevspecific activity of 1.8 x lo8 U/mg was purchased from Essex
eral neutrophilic proteins.* Secretion of IL-8 has been dePharma (Miinchen, Germany). rh-IFN-8 (3.0 X 10' U/mg) was
tected in many cell types, including monocytes/macrokindly provided by Asta Pharma (Frankfurt, Germany), rh-granuphages, T lymphocytes, fibroblasts, endothelial cells,
locyte-macrophage colony-stimulating factor (rh-GM-CSF) by
Sandoz AG (Niimberg, Germany), rh-IL-4 by Shering Plough
granulocytes, and
In most of these cells,
(Kenilworth, NJ), and rh-TNF-a (6.6 X lo6 U/mg) by Knoll AG
only small amounts of IL-8 are produced constitutively.
(Ludwigshafen,Germany). LPS was purchased from Sigma ChemiHowever, bacterial products, viruses, mitogens, phorbol escals (Deisenhofen, Germany), actinomycin-D and cycloheximide
ters, and the proinflammatory cytokines IL-1 and TNF-a
from Merck Inc (Darmstadt, Germany), and ~u~~P-labeled
nucleoare potent inducers of IL-8."3'2,L4Thus, in inflammatory
tides from Amersham Buchler (Braunschweig,Germany).
responses, IL-8 production can be induced either directly by
Isolation of blood cells. Peripheral blood mononuclear cells
microbial components or indirectly by cytokines that are
secreted in response to microbial stimuli.
Counterbalance of exogeneously induced cytokine secreFrom the Division of Hematology, III. Department of Medicine,
tion represents a crucial prerequisite for the regulation of
Johannes-Gutenberg University, Mainz, Germany: and the Sandoz
many biologic responses to prevent harmful local or sysResearch Institute, Vienna, Austria.
temic damage. Thus, downregulation of IL-8 production
Submitted January 26, 1993: accepted June 21, 1993.
results in termination of neutrophil attraction and limits
Supported by research grants from the Max Planck Institut fur
local inflammatory responses. IL-8 gene expression and the
Biochemie, project no. 54, Martinsried, Germany; and the Sandoz
release of biologically active IL-8 have been inhibited with
Research Foundation, Vienna,Austria. This work was performed as
part of the thesis of M.J.A. for a doctoratefrom the Department of
glucocorticoids,'5 IL-4,16 and IL-10,'' but little is known
Biology of the University of Mainz.
about the mechanisms of these effects.
Address reprint requests to Christian Peschel, MD, Division of
Interferons (IFNs) induce a broad spectrum of activities
Hematology, III. Department of Medicine, Johannes-Gutenberg
in many cell types comprising activating and inhibitory sigUniversity Mainz, Langenbeckstr I , 0-55131 Mainz, Germany.
nals." They have been found to activate macrophages and
The publication costs of this article were defrayed in part by page
enhance the production of inflammatory ~ y t o k i n e s . ' ~ , ~charge
~
payment. This article must therefore be hereby marked
However, IFNs-a/P have also been reported to inhibit in"advertisement" in accordance with 18 U.S.C. section I734 solely to
flammation after local LPS
The antiinflammaindicate this fact.
tory activities of IFN may depend on increased expression
0 I993 by The American Society of Hematology.
of IL-1 -receptor antagonist22and downregulation of IL-8 as
0006-497I/93/S208-0002$3.00/0
Blood, Vol82, No 8 (October 15). 1993: pp 237 1-2378
2371
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AMAN ET AL
2372
20
IL-8
10
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lFNa IOOOUlml
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Med
LPS
LPS+IFN-a
C
Fig 1. Effect of IFN-n on IL-8-mRNA expression and release in normal PBMNC. (A) Downregulation of 118 mRNA after incubation of
PBMNC with 3,000 U/mL IFN-u for 3 hours. The lower part of the panel shows the 28s rRNA from ethidium bromide-stained gel as loading
control. (B) Inhibitory effect of IFN-u on IL-8 production. PBMNC (1 X loa cells/mL) from 8 healthy donors were incubated for 24 hours in
medium with or without 1,000 U/mL IFN-U,and IL-8 w a s determined in the supematants. Columns represent mean f SEM. (C) Inhibition
of LPS-induced IL-8 secretion in normal PBMNC by IFN-a. Three experiments were performed in triplicates, and data from one representative experiment are presented a s mean f SEM. Concentrations used were LPS a t 10 pg/mL and IFN-u a t 1,000 U/mL.
(PBMNC) were isolated from the peripheral blood (PB) of healthy
donors and CML patients in chronic phase after informed consent
by centrifugation over Ficoll-Hypaque density gradient (Biochrom,
Berlin, Germany). Polymorphonuclear phagocytes (PMN) were
isolated from the PB of healthy donors by centrifugation over 3%
dextrane as described e l s e ~ h e r e .The
' ~ purity of PMN was shown to
be greater than 98% by differential morphologic analysis of cytospin
preparations. BM samples were obtained after informed consent
from normal BM transplant harvests.
Cell ciihire. PBMNC, PMN, or myelomonocytic THP-I cells
were incubated in RPMl 1640 medium (Biochrom, Berlin, Germany) containing 10% fetal calf serum (FCS) (Biochrom), penicil-
- 1
I
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0
A
.
dc
.d
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o
A o. d o. d o . Ao . do
n
o
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Fig 2. Effect of various concentrations of IFN on 11-8production. Noma1 PBMNC (1 X 10"/mL) were cultured in triplicates
with increasing concentrations of IFN-n for 24 hours, and IL-8 concentration w a s determined in the supematants. Three experiments
were performed, and data from one representative experiment are
presented a s mean f SEM.
!in 50 IU/mL, streptomycin 50 IU/mL, Na-Pyruvate 1 mmol/L,
L-glutamine 2 mmol/L, L-asparagine 20 pglmL, 2-mercaptoethan01 0.05 mmol/L, HEPES IO mmol/L, and nonessential amino
acids 0.7 X (Biochrom) at 37°C.and 5% COzwith factors and for
the times indicated in results. PBMNC and THP-I cells were cultured at IO6 cells/mL and PMN at IO' cells/mL.
Stromal cell ciilrrire. BM stromal cell cultures were essentially
obtained as described previously2' with some modifications. Briefly
BM mononuclear cells separated by centrifugation over Ficoll-Hypaque were incubated at a cell density of I X IO6/mL in 25 cm2
tissue flasks at 33°C in culture medium consisting of RPMl 1640
supplemented with 10% FCS, 10%horse serum, I .O pmol/L hydrocortison (Sigma), and the additives as indicated above. In weekly
intervals, cultures were fed by replacing 75% of culture medium.
When cultures were covered more than 80% by adherent cell. primary cultures were treated with trypsin-EDTA (Biochrom); detached stromal cells were pooled and expanded in new culture
flasks at a ratio of 1 5 . The adherent cells were incubated under the
same culture conditions until cultures became confluent again. By
this culture method, homogenous stromal cell layers were obtained
that were morphologically and functionally comparable in different
culture flasks. Hydrocortison was removed from these cultures a
few days before use for RNA and protein analysis. 1L-8 protein was
measured in the culture supernatants of stromal cells after incubation for 24 hours with various factors as indicated in the Results
section. For RNA analysis, the factors as indicated were added to
parallel culture flasks from the same donor. After 6 hours. cells were
lysed directly in the culture flask with guanidinium-isothiocyanate
and processed as described below.
Northern blot ana/wis. Total cytoplasmic RNA was purified
from PBMC, PMN, or THP-I cells using the single step method of
guanidinium/phenol-chloroform extraction as described previo u s l ~ ?A~total of I5 pg RNA was subjected to electrophoresis on a
I ?h agarose-formaldehyde gel and transferred onto nylon membrane (Hybond-N: Amersham Buchler). Blots were hybridized t o
~'~P-labeled
cDNA probes using random primer DNA labeling kit
(Boehringer, Mannheim, Germany) and after washing were ex-
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2373
DOWNREGULATION OF IL-8 BY IFNS
p
A
0
$3
S
Q
C
3h
3h
3h
B
B
Q
$ 8
B
8 sQ
IL-8
Fig 3. Effect of IFNs in vivo and in vitro on 11-8
mRNA expression in CML-PBMNC. (A) Downregulation of IL-8 mRNA in CML-PBMNC after in vitro
incubation with 3,000 U/mL IFN-a or -B for 3 hours
(representative of 6 experiments). The filter was
rehybridired to a cDNA probe for IFN-inducible
gene MxA. (B) 11-8 mRNA expression in freshly isolated PBMNC from 3 CML-patients before and 4 to
6 hours after subcutaneous application of 5.0 X
10’ IU IFN-a2b.
IL4
Mx-A
I
posed to Cronex-4 autoradiography films (DuPont, Bad-Homburg,
Germany) at -70°C. h-IL-8 cDNA, a 350-bp EcoRI fragment
cloned into PUCl9, was kindly provided by Dr 1. Lindley (Sandoz
Forschungsinstitut, Vienna, Austria).Chicken &actin cDNA probe
(1.8-kb fragment cloned in P-GEM3) was obtained from Paul Ehrlich lnstitut (Langen, Germany). h-MxA cDNA (2.3-Kb Sma-I
fragment)was a generous gift of Dr P. Staeheli (Institut fur Molekularbiologie, Zurich, Switzerland).”
Nuclear nm-on rranscriprion assay. Nuclear run-on transcription assay was performed using a modification ofa previously published protocol.**Briefly, nuclei were isolated from NP-40-lysed
cells by centrifugation at 5 0 0 ~then
.
incubated for 30 minutes with
I
28s
Pat.1
Pat.2
d
Q
8
E
Pat.3
100 pCi a-’*uridine triphosphate in a transcription buffer (IO
mmol/L Tris, pH 8.0. 5 mmol/L MgCI,, 100 mmol/L KCI, cold
nucleotides I mmol/L, and 5 mmol/Ldithiothreitol) at 26°C. After
treatment with DNase-I and proteinase-K, RNA was purified as
described above and used as a probe to hybridize to linearized plasmid immobilized on nylon membrane in a slot blot apparatus ( 5
pgldot). Within each experiment the same level of counts per minute of RNA was applied to each blot strip.
IL-8 ELISA. Cells were cultured in medium for 24 hours with
or without factors as indicated in Results section, and IL-8 concentration was measured using Quantikine Human IL-8 Immunoassay
(R&D Systems, Minneapolis, MN) according to the manufacturer’s
instructions.
RESULTS
IL-8
post therapy
I
28s
Fig 4. Inhibition of IL-8 expression in vitro after therapy with
IFN. PBMNC were obtained before and 5 hours after administration
of 5.0 X 10” IU IFN-u2b. RNA samples were prepared immediately (constitutive) and after incubation of the cells without or with
IFN-a 1,000 U/mL.
Effect of IFN-a on e,vpre.wion and secretion of IL-8 in
normal h-PBMNC. Expression of IL-8 mRNA was studied in PBMNC from healthy donors immediately after isolation and after incubation with IFN-a in vitro. Examination of RNA samples separated directly from PBMNC of 5
normal donors showed no constitutive expression of IL-8
(as shown in one representative experiment in Fig IA).
After short-term incubation of these cells in culture medium alone, a significant enhancement of IL-8 mRNA was
observed, as has been described recently by ~ t h e r s . ‘How~
ever, when IFN-a(3,000 U/mL) was added to the cultures,
the induction of IL-8 transcripts was markedly inhibited
(Fig IA).
In PBMNC obtained from 8 healthy donors (Fig 1 B), the
release of IL-8 protein was significantly inhibited by IFN-a.
IFN-a (1,OOO U/mL) decreased the level of IL-8 from 17.01
f 3.18 ng/mL in control cultures to 4.59 f 1.69 ng/mL
(mean 5 SEM, P c .01). thus representing a reduction of
73% 5 5.46% (95% confidence interval, 60.1% to 85.9%).
Exogenously induced IL-8 secretion was also inhibited by
IFN-a,as shown in Fig IC, in cultures of PBMNC that were
stimulated with LPS. The effect of IFN-a was concentrationdependent between 30 and 1,000 U/mL, and substantial inhibition of more than 60% was already observed at
100 U/mL (Fig 2).
Regulation of IL-8 mRNA expression bv IFN-a in
C M L . Regulation of IL-8 mRNA expression by IFN was
also examined in blood samples from 6 untreated patients
with CML in stable chronic disease phase. Addition of IFNa and IFN-/3 (3,000 U/mL) to cultures of PBMNC of CML
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AMAN ET AL
2374
1
11-8
,
IMx-A
28s
-I
I
1
L
ished by the IFN treatment. PBMNC of IFN-treated patients retained the capacity to produce IL-8 in vitro. This de
novo induction of IL-8 could again be downregulated by
IFN (Fig 4). suggesting that IFN exerts a direct regulatory
effect on IL-8 production rather than causing a depletion of
IL-8-producing cells from circulation. These ex vivo results
show that the inhibitory effect on IL-8 expression can be
achieved by systemicadministration of moderate, therapeutic doses of IFN-a.
Regirlation qf'lL-8production by BM stromal cells. BM
stromal cells were incubated with culture medium, LPS ( 2 5
pg/mL) or TNF-a (500 U/mL) with or without IFN-a
(3,000 U/mL), respectively. After 6 hours. RNA was prepared and Northern blot analysis was performed. As shown
in Fig 5A, stromal cells expressed low levels of IL-8 mRNA
constitutively, which could be further enhanced by TNF
and LPS. IFN-a significantly inhibited the constitutive expression of IL-8 and reduced IL-8 expression induced by
LPS or TNF. Inhibition of mRNA expression wasaccompanied by a decreased release of IL-8 into the culture supematants (Fig 5B).
on lL-8 erprmsion in PMN. IL-8 production was
also observed in PMN upon stimulation with LPS. Accumulation of IL-8 mRNA was readily detectable but was not
inhibited by IFN-a under conditions that nearly blocked
expression in PBMNC and BM stromal cells (Fig 6). IL-8
production by PMN stimulated with granulocyte-CSF or
LPS was also unaffected by IFN-a (data not shown). In
these experiments. PMN could be shown to respond to IFNa. as indicated by expression of the IFN-responsive gene.
MxA (Fig 6).
cffcxv
Med
lFNa
TNFa
TNFa+IFNa
Fig 5. Inhibitory effect of IFN-a on IL-8 production and mRNA
expression in normal BM stromal cells. (A) Stromal layers were incubated for 6 hours in medium alone or with the indicated substances in the following concentrations: IFN-a, 3,000 U/mL; TNFa, 500 U/mL; and LPS, 25 pg/mL. They were then subjected to
RNA analysis. The first two lanes were exposed for a longer period
of time. The middle part of the panel shows the same filter hybridized to MxA cDNA. (B) IL-8 production by normal BM stromal cells
cultured for 24 hours with or without TNF-a (200 U/mL) and IFN-a
(3,000 U/mL). Three experiments were performed in triplicates,
and data from one representative experiment are shown as mean
2 SEM.
patients in chronic phase inhibited the spontaneous expression of IL-8 mRNA as observed in control cultures (Fig 3A).
Rehybridization of the filters with a cDNA probe for the
IFN-inducible protein MxA showed that IFN-awas active
in the cultures as shown by strong induction of MxA-gene
expression.
Therapeutic administration of IFN-a to CML patients
caused a comparable decrease of mRNA expression as seen
in vitro. RNA was prepared from PBMNC of CML patients
before and 4 to 6 hours after subcutaneous injection of a
single dose of IFN-cu2b (5 X IO6 IU). In contrast to normal
controls, freshly isolated PBMNC from 7 of 12 CML patients showed significant expression of IL-8 mRNA. As
shown in Fig 3B. for three typical experiments. the constitutive expression of IL-8 mRNA was almost completely abol-
IL-8
Mx-A
28s
Fig 6. IFN-a does not inhibit LPS-induced IL-8 mRNA expression in PMN. Cells were cultured for 3 hours in medium alone or
with the indicated additions and hybridized with cDNA probes for
IL-8 or MxA.
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2375
DOWNREGULATION OF IL-8 BY IFNS
Fig 7. Effect of IFN on IL-8 mRNA expression in
THP-1 cells. Cells were cultured for 3 hours in medium alone or with indicated additions and hybridized with cDNA probes for 11-8 or MxA. Concentrations were used as follows: IFN-aIB, 3,000 U/ml;
GM-CSF, 500 U/mL; LPS, 1 0 pg/mL; and IL-4.250
U/mL.
Mx-A
I
Mechanism qfIL-8 downregtilation. The mechanism of
action by IFN on IL-8 production was studied in more detail using the myelomonocytic cell line, THP- l. The constitutive or stimulated expression of IL-8 mRNA (LPS or GMCSF) was inhibited to similar extents by IFN-aand IFN-@.
Significant inhibition was also obtained with IL-4, and IL-8
mRNA was completely blocked when IFN-aand IL-4 were
combined (Fig 7). Hybridization of the blots to the MxA
probe again confirmed the specific activity oftype-I lFNs in
this cell population.
The possibility ofa posttranscriptional downregulation of
IL-8 expression was evaluated using actinomycin-D. As
shown in Fig 8, in IFN-treated cells IL-8 mRNA decayed
more rapidly than in the controls suggesting the involvement of a posttranscriptional mechanism.
Nuclear run-on assays were performed in THP-l cells
treated with IFN-a and TNF alone or in combination (Fig
9). TNF enhanced the transcription of IL-8 mRNA, and
this effect was not modified by IFN-a, indicating that the
inhibition of IL-8 expression by IFN occurs at a posttranscriptional level. The nuclear RNA probes obtained were
also hybridized to the @-actincDNA and to the plasmid
PUC-19, and no effect of either cytokine was observed.
Transcription of the MxA gene was clearly induced in presence of IFN-a,confirming the IFN-specific activity in this
assay.
Act-D
0
IL-8
28s
The potential role of proteins induced de novo by IFN in
the downregulation of IL-8 expression was studied in THPI cells and PBMNC using cycloheximide (CHX) as a protein synthesis inhibitor (Fig IO). CHX superinduced IL-8
mRNA expression. The addition of CHX had no apparent
effect on the inhibition of IL-8 expression by IFN, indicating a direct effect by IFN rather than the induction of IFNresponsive genes mediating the suppression of IL-8 expression.
DISCUSSION
IL-8 and its analogs are the most selective chemoattractants for neutrophils. The activation processes in
neutrophils induced by IL-8 are of crucial importance in
establishing an inflammatory response against invading microorganisms.'.2 Thus, IL-8 represents a key cytokine in
host defence and inflammation. Secretion of IL-8 is induced
in phagocytes and several other cell types as a reaction to
microbial substances and by other proinflammatory cytokines. Counterbalance of inflammatory processes is of paramount importance in limiting reactions directed against invading microorganisms to an appropriate level to avoid
irreversible damage of host tissue. Therefore, the study of
mechanisms responsible for downregulation of expression
of the IL-8 gene as a means to inhibit inflammatory pro-
ACt-D+IFN-a
7
7
15' 30' l h 2h 3h 4h 5h 15' 30' l h 2h 3h 4h 5h
Fig 8. Reduction of 11-8
mRNA half-life in THP-1 cells
by IFN-U. THP-1 cells werestimulated with 1 0 pg/mL LPS for 4
hours and subsequently incubated with 1 0 pg/mL actinomycin-D with or without 3,000 U/
mL IFN-a. RNA was isolated
from each culture at indicated
times and hybridized with
cDNA probes for IL-8.
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AMAN ET AL
2376
TNF-a
IFN-a
m
I
m
+
+
I
+
+
I L-8
P-Actin
Fig 9. Nuclear run-on analysis of 11-8transcription. Nuclei were isolated 2 hours after incubation
of THP-1 cells with medium or TNF-a (200 U/mL)
withorwithout 1,000 U/mLIFN-u,andinvitroelongation of transcription and hybridization were performed. PUC-19 served as negative control and a
P-GEM3 plasmid containing chicken 8-actin cDNA
and MxA as positive control. The data depicted are
representative for three experiments performed.
PUC-19
MxA
cesses has substantial pathophysiologicand therapeutic implications.
Our observation that IL-8 is downregulated by IFN-a/fi
in several cell types in vitro and in vivo extends our understanding of the regulation of this protein and defines a novel
role for type-I IFNs. Our finding suggests that the inflammation-associated cytokines TNF, IL-6, and IL-8 are differentially regulated by another factor that by itself is a result of
the host response to microorganisms. Whereas production
of TNF-a or IL-6 is enhanced by IFN-cr (Kohase et all9
Jourdon et al:'
and Aman et al [manuscript in preparation]), IFNs at the same time specificallyinhibit production
of the key cytokine for neutrophil activation. Differential
regulation of IL-l expression by IFNs, as described re~ently,'~
is another example of a complex regulatory role for
IFNs in cytokine networks. Very recently, the downregulation of IL-8 gene expression by IFN-fi has been described in
human fibroblasts; Oliveira et aIz3 concentrated on the
PBMNC
TH P-1
Fig 10. Downregulation of 11-8 mRNA in presence of CHX.
PBMNC f" healthy individuals (4 experiments) and THP-1 cells
(2 experiments) were incubated for 3 hours with 1 0 jtg/mL CHX
with or without 3,000 U/mL IFN-a and hybridized with a cDNA
probe for IL-8.
mechanism of inhibition by IFN in this cell type. In the
present report, we focused on the regulation of IL-8 production in various hematopoietic cells and in stromal cells as a
major component of the BM microenvironment. Downregulation of an inflammation-associated cytokine in hemopoietic cells that are capable of invading inflammatory lesions
suggests that local production of IFN in such lesions can
contribute to confining the tissue reaction to an appropriate
level. The influence on cytokine production by BM stromal
cells implicates an indirect role of IFN in the regulation of
hematopoiesis.
In PBMNC from normal donors and CML patients and
in other cellular sources, IL-8 mRNA expression and protein secretion induced by in vitro cultivation were clearly
reduced by IFN-a in a dosedependent manner. In THP-I
cells the action of IFN was compared with IL-4 which has
been previously shown to inhibit IL-8 expression in human
monocytes.'6 These experiments showed a comparable inhibitory effect of both cytokines. Furthermore, IFN-a and
IL-4 seemed to synergize in downregulation of the IL-8 expression. The effect of I F N was independent of the type of
stimulation used for IL-8 induction. Marked downregulation of IL-8 expression was observed in spontaneous cytokine production in vitro in PBMNC and was also observed
after stimulation of the different cell types tested with LPS
or cytokines such as TNF-a or GM-CSF.
In contrast to normal donors, a constitutive expression of
IL-8 mRNA was observed in PBMNC of patients with
CML. Overexpression of cytokines by CML cells has been
observed by several groups, and autocrine or paracrine secretion ofgrowth factors has been suggested to contribute to
the uncontrolled expansion of the leukemic cell populat i ~ n . ~ ' .However,
~'
whether constitutive expression of IL-8
plays a pathophysiologic role in CML development remains
to be determined. Downregulation of IL-8 expression has
also been observed in RNA samples obtained from patients
after the first dose of IFN-ain a clinical trial, thus confirming an in vivo relevance of this mechanism.
In purified PMN, IFN-a failed to suppress IL-8 expression at both the RNA and protein levels. This was not due to
a lack of sensitivity by PMN to IFN-a,because the antiviral
gene, MxA, characteristic for type-I IFNs, could clearly be
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2377
DOWNREGULATION OF 11-8 BY IFNS
induced. Therefore, the mechanisms of IL-8 production appear to be differentially regulated depending on the source
and on the state of differentiation of the expressing cells.
Regulation of IL-8 expression by IFN has also been observed in stromal cell cultures as a representative model for
an essential nonhematopoietic constituent of the BM microenvironment. BM stromal cells stimulate proliferation
and differentiation of hematopoietic cells in direct cellular
~ontact.~’
Furthermore, homing of hematopoietic stem cells
and release of mature hematopoietic elements into the PB
are regulated in the BM microenvironment by yet poorly
defined mechanism^.'^.^^ Stromal cells can be induced to
produce cytokines by LPS and by factors such as IL-1 and
TNF.35IL-8 release by BM stromal cells is paralleled by its
production in related cell types such as fibroblasts and endothelial cells. However, a function of IL-8 in the regulation of
hematopoiesis remains to be elucidated. IFN-a has been
described to exert functional activitiesin BM microenvironment by influencing adherence of hematopoietic cells to
stromal cells in CML in vitro.36Regulation of cytokine release by stromal cells represents a new feature of the functional spectrum of IFNs.
The mechanism of IFN action in suppressingIL-8 production remains unclear. In presence of the RNA synthesis inhibitor actinomycin-D, an accelerated degradation of IG8
mRNA in THP-1 cells was observed, suggesting a posttranscriptional mechanism. The data from nuclear run-on experiments are in keeping with this point of interaction with
IL-8 synthesis. In contrast, Oliveira et a123showed that the
inhibition of IL-8 production is most pronounced at the
transcriptional level. This discrepancy could be explained
by the different cellular systems that were used by both
groups. However, it has been discussed by these investigators that IFN might, in addition, block IL-8 synthesis at
another level. A differential regulation of IL-8-mRNA expression in different experimental systems is not unprecedented and has been described for induction of IL-8 by
proinflammatory cytokines at both the transcriptional3’
and posttranscriptional level.3s Thus, control of IL-8 expression by enhancing and suppressing cytokines may be
regulated by more than one mechanism and might take
place at a transcriptional as well as a posttranscriptional
level. Interestingly, de novo protein synthesis is not required
for IFN-induced downregulation in hematopoietic cells,
confirming the results described in human fibroblast^.'^
The present data show a profound inhibitory effect of
type-I IFNs on IL-8 production in hematopoietic cells in
vivo and in vitro and in BM stromal cell cultures. IFNs
appear to differentially regulate the expression of various
cytokines associated with inflammatory responses. The
downregulation of IL-8, a prototypic inflammatory chemokine, by IFN, which by itself is induced by microbial stimuli, suggests a physiologically regulatory role of IFNs in limiting accumulation and activation of neutrophils; therefore,
IL-8 downregulationby IFN may inhibit inflammatory processes.
ACKNOWLEDGMENT
We are indebted to T. Horn, K. Hiibner, and U. Petrat for excellent technical assistance.
REFERENCES
1. Baggiolini M, Clark-LewisI: Interleukin-8, a chemotactic and
inflammatory cytokine. FEBS Lett 307:97, 1992
2. Baggioloni M, Dewald B, Walz A: Interleukin 8 and related
chemotactic cytokines, in Gallin JI, Goldstein IM, Snyderman R
(eds): Inflammation: Basic Principles and Clinical Correlates. New
York, NY, Raven, 1992, p 247
3. Oppenheim JJ, Zachariae COC, Mukaida N, Matsushima K
Properties of the novel proinflammatory supergene “intercrine”
cytokine family. Annu Rev Immunol9:6 17, I99 1
4. Matsushima K, Baldwin ET, Mukaida N: Interleukin-8 and
MCAF Novel leukocyte recruitment and activating cytokines, in
Kishimoto T (ed): Interleukins: Molecular Biology and Immunology. Chem Immunol51:236, 1992
5. Detmers PA, Lo SK, Olsen-Egbert E, Walz A, Baggiolini M,
Cohn ZA: Neutrophil activating protein l/interleukin 8 stimulates
the binding activity of the leucocyte adhesion receptor CDI lb/
CD I8 on human neutrophils. J Exp Med 17 1 :1 155, 1990
6. Huber A, Kunkel SL, Todd RF 111, Weiss SJ: Regulation of
transendothelial neutrophil migration by endogenous interleukin8. Science 254:99, 1991
7. Paccaud JP, Shifferli JA, Baggiolini M: NAP-I/IL-8 induces
upregulation of CRI receptors in human neutrophil leukocytes.
Biochem Biophys Res Commun 166:187, 1990
8. Baggiolini M, Walz A, Kunkel SL: Neutrophil-activatingpeptide- ]/interleukin 8, a novel cytokine that activates neutrophils. J
Clin Invest 84:1045, 1989
9. Peveri P, Wlaz A, Dewald B, Baggiolini M: A novel neutrophil-activating factor produced by human mononuclear phagocytes. J Exp Med 167:1547, 1988
10. Smyth JM, Zachariae COC, Norihisa Y, Ortaldo JR, Hishinuma A, Matsushima K IL-8 gene expression and production in
human peripheral blood lymphocytes. J Immunol 146:3815, 1991
I 1. Strieter RM, Kunkel SL, Showell HJ, Remick DG,Phan SH,
Ward PA, Marks RM: Endothelial cell gene expression of a neutrophil chemotactic factor by TNF-a, LPS, and IL-la. Science
243:1467, 1989
12. Strieter RM, Phan SH, Showell HJ, Remick DG, Lynch JP,
Genord M, Raiford C, Eskandari M, Marks RM, Kunkel S L Monokine-induced neutrophil chemotactic factor gene expression in human fibroblasts. J Biochem 264: 10621, I989
13. Bazzoni F, Cassatella MA, Rossi F, Ceska M, Dewald B,
Baggiolini M: Phagocytosing neutrophils produce and release high
amounts of the neutrophil-activating peptide/interleukin 8. J Exp
Med 173:771, 1991
14. Matsushima K, Morishita K, Yoshimura T, Lavu S,
Kobayashi Y, Lew W, Appella E, Kung HF, Leonard EJ, Oppenheim JJ: Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF
mRNA by interleukin 1 and tumor necrosis factor. J Exp Med
167:1883, 1988
15. Tobler A, Meier R, Seitz M, Dewald B, Baggiolini M, Fey
M F Glucocorticoids downregulate gene expression of GM-CSF,
NAP-l/IL-8, and IL-6, but not of M-CSF in human fibroblasts.
Blood 79:45, 1992
16. Standiford TJ, Strieter RM, Chensue SW, Westwick J, Kasahara K, Kunkel S L IL-4 inhibits the expression of IL-8 from stimulated human monocytes. J Immunol 145:1435, 1990
17. de Waal Malefyt R, Abrams J, Bennett B, Fidgor CG, de
Vnes JE: Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: An autoregulatory role of IL-10 produced by
monocytes. J Exp Med 174: 1209, I99 1
18. Vilcek J: Interferons, in Sporn MB, Roberts AB (eds): Peptide Growth Factors and Their Receptors 11. New York, NY,
Springer-Verlag, 1990
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
2378
19. Kohase M, May LT, Tamm I, Vilcek J, Sehgal PB: A cytokine network in human diploid fibroblasts: Interactions of &interferons, tumor necrosis factor, platelet derived growth factor, and
interleukin 1. Mol Cell Biol 7:273, 1987
20. Jourdan M, Zhang XG, Portier M, Boiron JM, Bataille R,
Klein B IFN-a induces autocrine production of IL-6 in myeloma
cell lines. J lmmunol 147:4402, 1991
21. Heremans H, Dijkmans R, Sobis H, Vandekerckhove F,
Billiau A: Regulation by interferons of the local inflammatory response to bacterial lipopolysaccharide. J Immunol 138:4175, 1987
22. Tilg H, Mier JW, Vogel W, Aulitzky WE, Wiedermann CJ,
Huber C, Dinarello CA: Induction of circulating interleukin- 1 receptor antagonist by interferon-alpha treatment. J Immunol
150:4687, 1993
23. Oliveira IC, Sciavolino PJ, Lee TH, Vilcek J: Downregulation of interleukin 8 gene expression in human fibroblasts: Mechanism of transcriptional inhibition by interferon. Proc Natl Acad Sci
USA 89:9049, 1992
24. Colligan JE, Kruisbeek AM, Margulies DH, Shevach EM,
Strober W Isolation and functional analysis of neutrophils, in
Current Protocols in Immunology, vol 1. New York, NY, Wiley,
1991, p 7.23.1
25. Cashman J, Eaves AC, Eaves CJ: Regulated proliferation of
primitive hemopoietic progenitor cells in long-term human
marrow cultures. Blood 66:1002, 1985
26. Asubel FM, Brent R, Kingston RE, Moore DD, Seidman
JG, Smith JA, Struhl K Guanidinium methods for total RNA preparation, in Current Protocols in Molecular Biology, vol 1. New
York, NY, Wiley, 1987, p 4.2.4
27. Aebi M, Fah J, Hurt N, Samuel CE, Thomas D, Bazzigher L,
Pavlovic J, Haller 0,Staeheli P: cDNA structures and regulation of
two interferon-induced human Mx proteins. Mol Cell Biol95062,
1989
28. Asubel FM, Brent R, Kingston RE, Moore DD, Seidman
JG, Smith JA, Struhl K Identification of newly transcribed RNA,
in Current Protocols in Molecular Biology, vol 1. New York, NY,
Wiley, 1987, p 4.10.1
AMAN ET AL
29. Schindler R, Ghezzi P, Dinarello CA: IL-I induces IL-I . IV.
IF"-gamma suppresses IL- 1 but not lipopolysaccharide-induced
transcription of IL- I . J lmmunol 144:2216, 1990
30. Wetzler M, Kurzrock R, Lowe DG,Kantajian H, Gutterman JU, Talpaz M: Alteration in bone marrow adherent layer
growth factor expression: A novel mechanism of chronic myelogenous leukemia progression. Blood 78:2400, 1991
31. Herrmann F, Helfrich SG, Lindemann A, Schleiermacher E,
Huber C, Mertelsmann R: Elevated circulating levels of tumor necrosis factor predict unresponsiveness to treatment with interferon
alpha-2b in chronic myelogenous leukemia. J Clin Oncol 10:631,
I992
32. Eaves CJ, Sutherland HJ, Cashman JD, Otsuka T, Lansdorp
PM, Humphries RK, Eaves AC, Hogge D E Regulation of human
hematopoietic cells in long-term marrow culture. Semin Hematol
28:126, 1991
33. Tavassoli M, Hardy CL: Molecular basis of homing of intravenously transplanted stem cells to the marrow. Blood 76:1059,
I990
34. Petrides PE, Dittmann KH: How do normal and leukemic
white blood cells egress from the bone marrow? Morphological facts
and biochemical riddles. Blut 61:3, 1990
35. Rennick D, Yang G, Gemmell L, Lee F: Control of hemopoiesis by a bone marrow stromal cell clone: Lipopolysaccharide- and interleukin- I-inducible production of colony-stimulating factors. Blood 69:682, 1987
36. Dowding C, Guo AP, Osterholz J, Sickowski M, Goldman J,
Gordon M: Interferon-alpha overrides the deficient adhesion of
chronic myeloid leukemia primitive progenitor cells to bone
marrow stromal cells. Blood 78:499, 199 1
37. Mukaida N, Mahe Y , Matsushima K Cooperative interaction of nuclear factor-kB and cis-regulatory enhancer binding protein-like factor binding elements in activating the interleukin-8
gene by proinflammatory cytokines. J Biol Chem 265:21128, 1990
38. Stoeckle MY: Post-transcriptional regulation of gro-alpha,
beta, gamma and IL-8 mRNAs by IL-16. Nucleic Acids Res 19:917,
1991
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1993 82: 2371-2378
Type-I interferons are potent inhibitors of interleukin-8 production in
hematopoietic and bone marrow stromal cells
MJ Aman, G Rudolf, J Goldschmitt, WE Aulitzky, C Lam, C Huber and C Peschel
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