(Received for publication, September 5, 1996, and in revised form, March 13, 1997)
From the Laboratoire de Chimie Biologique, Département de Biologie Moléculaire, Université Libre de Bruxelles, 1640 Rhode Saint Genèse, Brussels, Belgium and ¶ Sanofi Elf Biorecherches, 31676 Labège, Cedex, France
The production of tumor necrosis factor-
(TNF-
) by lipopolysaccharide (LPS)-stimulated macrophages can be
markedly inhibited by the two closely related cytokines, interleukin
(IL)-4 and IL-13. To investigate the molecular mechanism of this
inhibition, we analyzed the effect of the two cytokines on TNF-
production and TNF-
mRNA accumulation in the mouse macrophage
cell lines RAW 264.7 and J774 stimulated by LPS. Whereas LPS-induced
TNF-
production is strongly suppressed by both cytokines, TNF-
mRNA accumulation is not significantly affected, indicating that
IL-4 and IL-13 induce a translational repression of TNF-
mRNA.
Transfection of reporter gene constructs containing different regions
of the TNF-
gene revealed that the inhibitory action of IL-4 and
IL-13 is mediated by the UA-rich sequence present in the TNF-
mRNA 3
-untranslated region.
Tumor necrosis factor- (TNF-
)1
is a pleiotropic cytokine secreted by different cell types including
macrophages, mastocytes, T and B lymphocytes, and natural killer cells
in response to various stimuli (lipopolysaccharide (LPS), viruses,
parasites, etc.) (1). A wide variety of cell types express TNF-
receptors, and the pleiotropism of TNF-
results from the complexity
of the signal transduction pathways that are activated by its
receptors. TNF-
is characterized by cytostatic and cytolytic effects
on tumor cells of different origins (2), by antiviral properties (3), and by its important role in the activation of the immune system upon
host invasion (4). TNF-
has also been identified as a major mediator
of inflammatory processes, one of the most dramatic being Gram-negative
endotoxic shock (1). Indeed, upon exposure to LPS or other agents
simulating host invasion, macrophages produce large amounts of TNF-
that are released in the circulatory system. High levels of circulating
TNF-
trigger a state of shock and tissue injury that carries an
extremely high mortality rate (1).
The expression of the TNF- gene in mouse macrophages is regulated at
the transcriptional (5, 6) and translational (1) levels. In resting
macrophages, TNF-
synthesis is low because TNF-
gene
transcription is weak and TNF-
mRNA translation is severely
repressed. The translational repression is mediated by the UA-rich
sequence present in the TNF-
mRNA 3
-untranslated region (1).
Macrophage activation by LPS results in NF-
B-dependent activation of TNF-
gene transcription, derepression of TNF-
mRNA translation, and secretion of TNF-
protein. TNF-
production by macrophages can be down-regulated by various agents
including cytokines like IL-4 and IL-13 (7, 12). IL-4 and IL-13 are two
closely related cytokines that are synthesized mainly by activated T
lymphocytes (7, 8). Both cytokines share many biological activities
including the activation of B cell proliferation, IgE switching, and
inhibition of inflammatory cytokine production (9-11). TNF-
is one
of the major inflammatory cytokines being repressed by IL-4 and IL-13.
However, the molecular mechanism of this regulation has not been
elucidated. In this study, we investigated the mechanism by which IL-4
and IL-13 down-regulate TNF-
production in LPS-stimulated mouse RAW
264.7 and J774 macrophages. We show that in these cell types, IL-4 and
IL-13 inhibit LPS-induced TNF-
production mainly at the
translational level. Furthermore, down-regulation of TNF-
mRNA
translation by IL-4 and IL-13 is mediated by the UA-rich sequence
present in its 3
-untranslated region.
LPS from Escherichia coli (strain
0.127:B8) was obtained from Sigma. [-32P]UTP (800 Ci/mmol) and [14C]chloramphenicol (56 mCi/mmol) were
purchased from Amersham Life Science, Inc. Murine recombinant IL-4 and
TNF-
were purchased from Genzyme. Purified murine IL-13 was obtained
from Sanofi Elf Biorecherches, Labège, France.
Both reporter constructs used in this study
have been described elsewhere: CMV3TNF in Ref. 13 and
CMV3
TNFUA
in Ref. 14, referred to as construct 3.
The murine macrophage cell lines RAW 264.7 and J774 were obtained from the American Type Culture Collection. The cells were maintained in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum (FBS Myoclone Super Plus, Life Technologies, Inc.) and 1% penicillin/streptomycin.
RAW 264.7 cells were cotransfected with each of the described constructs and pSV2Neo as described (15). Cells were first selected for 1 day with 0.3 mg/ml G418 sulfate, for 1 day with 0.6 mg/ml, and for 2 weeks with 1 mg/ml. The resistant clones were then pooled. The cells were stimulated simultaneously with LPS (10 ng/ml) and IL-4 (5 ng/ml) or IL-13 (5 ng/ml) unless otherwise specified.
Measurement of TNF-TNF- levels were
measured in cell supernatants by sandwich enzyme-linked immunosorbent
assay using a polyclonal rabbit anti-mouse TNF-
antibody for coating
and the same biotinylated polyclonal antibody for detection (generously
provided by Dr. W. Buurman, University of Limburg Maastricht, The
Netherlands).
CAT assays were performed as described (17), and quantification of CAT activity was performed with a Molecular Dynamics PhosphorImager.
Northern Blot AnalysisNorthern blot analysis was performed
as described (16) using 10 and 20 µg of total cytoplasmic RNA for
TNF- and CAT mRNA detection, respectively. Blots were hybridized
with antisense riboprobes for TNF-
and CAT mRNAs (16, 17). For
quantification, the blots were rehybridized with a
glyceraldehyde-3-phosphate dehydrogenase antisense riboprobe. RNA
quantification was achieved by normalizing the radioactive signal
corresponding to TNF-
mRNA to that of glyceraldehyde-3-phosphate
dehydrogenase in the same RNA sample.
RAW 264.7 mouse macrophages
produce large amounts of TNF- in response to LPS. Moreover, the
transcriptional and post-transcriptional regulation of TNF-
biosynthesis by LPS is well characterized in this cell line. Therefore,
RAW 264.7 cells provided an appropriate system to analyze the mechanism
by which IL-4 and IL-13 suppress LPS-induced TNF-
production. We
also analyzed the mechanism by which these cytokines down-regulate
LPS-induced TNF-
production in J774 cells, which produce high levels
of TNF-
upon LPS stimulation.
A recent study has shown that depending on the time of exposure to IL-4
or IL-13 relative to LPS stimulation, IL-4 and IL-13 have either
inhibitory or stimulatory effects on LPS-induced TNF- production in
human peripheral blood mononuclear cells (18). When added to the cell
culture 20 h or more before LPS stimulation, IL-4 and IL-13 prime
LPS-induced TNF-
production, whereas when added simultaneously or a
few hours before LPS stimulation, IL-4 and IL-13 exert a marked
inhibitory effect on the production of this cytokine (18). Therefore,
before analyzing the mechanism by which these cytokines inhibit
LPS-induced TNF-
production, we verified that IL-4 and IL-13, when
added to the cell culture at the same time as LPS, could inhibit
TNF-
production in RAW 264.7 and J774 cells. We performed
experiments in which increasing concentrations of each cytokine were
added to RAW 264.7 or J774 cell cultures simultaneously with LPS.
TNF-
production was assayed by enzyme-linked immunosorbent assay
using a polyclonal anti-mouse TNF-
antibody to measure the totality
of TNF-
produced in response to the various treatments. As shown in
Fig. 1, LPS-induced TNF-
production in RAW 264.7 cells was inhibited by both cytokines in a dose-dependent
manner, reaching 72 and 63% inhibition at the highest doses of IL-4
and IL-13 tested, respectively (IL-4, 5 ng/ml; and IL-13, 5 ng/ml).
Similarly, the highest doses of IL-4 and IL-13 inhibited TNF-
production in LPS-stimulated J774 cells by 65 and 67%,
respectively.
IL-4 and IL-13 Inhibit LPS-induced TNF-
TNF- production by LPS-stimulated mouse
macrophages results from a combination of TNF-
gene transcriptional
activation and TNF-
mRNA translational derepression. To
determine at which level IL-4 and IL-13 inhibit LPS-induced TNF-
production, we analyzed LPS-induced TNF-
mRNA accumulation in
the presence or absence of each cytokine. Since TNF-
mRNA
accumulation peaks 2 h after LPS addition to the cell culture
(19), we studied the effect of IL-4 and IL-13 on TNF-
mRNA
accumulation at this time point after LPS induction. We observed that
whereas both cytokines inhibited LPS-induced TNF-
production,
TNF-
mRNA accumulation was not affected by IL-4 and IL-13 in RAW
264.7 cells (Fig. 2). In J774 cells, both IL-4 and IL-13
markedly suppressed LPS-induced TNF-
production, as previously
observed. Northern blot analysis of TNF-
mRNA revealed that
LPS-induced TNF-
mRNA accumulation was slightly reduced by IL-4
and IL-13 (Fig. 3). However, this 13% decrease cannot
account by itself for the reduction of TNF-
production, which
reached 60 and 80% in response to IL-4 and IL-13, respectively. Altogether, these results indicate that the suppressive effect of IL-4
and IL-13 on TNF-
biosynthesis is mostly if not completely exerted
at the translational level in both cell lines.
Down-regulation of TNF-
As the 3-untranslated region of the TNF-
mRNA
plays a central role in the translational control of TNF-
gene
expression, we analyzed the effect of IL-4 and IL-13 on the expression
of a stably transfected CAT reporter gene containing this sequence (CMV3
TNF; Fig. 4) in RAW 264.7 cells. Indeed, former
studies showed that in RAW 264.7 cells transfected with this construct, LPS stimulates the expression of the reporter gene by acting only at
the translational level (13). In unstimulated RAW 264.7 cells, the
presence of the CMV promoter led to the constitutive accumulation of
CMV3
TNF mRNA as evaluated by Northern blotting, but this mRNA was poorly translated in CAT protein (Fig.
5A). Upon stimulation with LPS, translational
derepression resulted in CAT protein accumulation without significant
variation of the CMV3
TNF mRNA. Exposure of the
CMV3
TNF-transfected cells to LPS in combination with IL-4 or IL-13
markedly reduced the accumulation of CAT activity (Fig. 5A)
without affecting the level of CMV3
TNF mRNA. In contrast, IL-4 and
IL-13 had no effect on the expression of the CMV3
TNFUA
construct, in which the UA-rich sequence is specifically deleted (see
Figs. 4 and 5B). As a control, we verified that the
inhibition of LPS-induced TNF-
production by IL-4 and IL-13 in both
transfected cell lines was equivalent to that observed in untransfected
RAW 264.7 cells (data not shown). These results indicate that IL-4 and
IL-13 suppress the expression of the CMV3
TNF reporter gene at the
translation level and require the presence of the UA-rich sequence to
exert their inhibitory action on TNF-
mRNA translation.
It is now well established that in monocytes/macrophages, IL-4 and
IL-13 mediate several similar functions, among which one of the major
is the inhibition of proinflammatory cytokine production. Indeed, both
IL-4 and IL-13 inhibit the production of several cytokines including
IL-1, IL-1
, IL-6, IL-12, macrophage inflammatory protein-
,
granulocyte/macrophage colony-stimulating factor, granulocyte colony-stimulating factor, interferon-
, and TNF-
by
monocytes/macrophages activated with LPS (7, 10, 12, 20). The
inhibitory effect of IL-4 and IL-13 on the production of TNF-
has
been demonstrated in human peripheral blood monocytes (7, 10-12, 21,
22), human alveolar macrophages (23), murine bone marrow macrophages (11, 20) and murine peritoneal macrophages (24). The mechanism by which
IL-4 and IL-13 inhibit LPS-induced TNF-
production in monocytes/macrophages is still controversial. For instance, several studies show that IL-4 and IL-13 decrease LPS-induced TNF-
mRNA accumulation in human peripheral blood mononuclear cells (7, 21, 22).
However, one report describes that in mouse peritoneal macrophages,
IL-4 does not modify TNF-
mRNA accumulation in response to LPS
(24). The expression of the TNF-
gene in macrophages is regulated
both at the transcriptional and post-transcriptional levels (1). In
unstimulated mouse macrophages, TNF-
production is severely
repressed both at the transcriptional and translational levels. Indeed,
under these conditions, TNF-
gene transcription is poor, and TNF-
mRNA translation is blocked. This translational repression is
mediated by the UA-rich sequence located in the 3
-untranslated region
of TNF-
mRNA. Macrophage activation by LPS leads to a 50-fold
increase in TNF-
gene transcription and a 200-fold increase in
TNF-
mRNA translation, leading to an overall 10,000-fold
increase in TNF-
biosynthesis (1). Translational regulation of
TNF-
mRNA also plays an important role under other circumstances. Indeed, LPS tolerance, which is characterized by an
impaired TNF-
production in response to a secondary LPS challenge, is mainly due to a defective translation of TNF-
mRNA (14). Furthermore, production of TNF-
in response to infection by several viruses (Sendai virus, Newcastle disease virus, vesicular stomatitis virus, vaccinia virus, and Mengo virus) results to a great extent from
TNF-
mRNA translational activation (25).
In our study, we aimed at determining the mechanism by which IL-4 and
IL-13 inhibit LPS-induced TNF- production in the mouse macrophage
cell line RAW 264.7, in which the regulation of TNF-
biosynthesis is
well characterized. Furthermore, we also investigated this regulatory
mechanism in another mouse macrophage cell line, J774, which also
produces high levels of TNF-
in response to LPS. We first verified
that IL-4 and IL-13 down-regulate TNF-
production in both cell types
when added to the cell culture at the same time as LPS. We then
demonstrated that under these conditions, both cytokines inhibit
LPS-induced TNF-
production without significantly affecting TNF-
mRNA accumulation. Therefore, we conclude that IL-4 and IL-13
down-regulate TNF-
gene expression at the translational level. By
analyzing the effect of IL-4 and IL-13 on the expression of CAT
reporter DNA constructs with or without the TNF-
mRNA UA-rich
sequence in RAW 264.7 cells, we show that both IL-4 and IL-13 exert
their inhibitory action on mRNA translation through the
intermediate of this UA-rich sequence. Recently, we have identified in
RAW 264.7 cells a protein complex that binds the TNF-
mRNA UA-rich sequence upon stimulation with LPS (26). The LPS-induced binding of this complex to the UA-rich sequence requires tyrosine phosphorylation since it is blocked by the protein tyrosine
phosphorylation inhibitor herbimycin A. The observation that IL-4 and
IL-13 affect TNF-
mRNA translation suggests that IL-4 and IL-13
might interfere with the binding of this protein complex to the UA-rich
sequence upon LPS stimulation. The mechanism by which this phenomenon
might occur is still speculative. However, it is well known that
stimulation of macrophages by LPS promotes tyrosine phosphorylation of
various members of the serine/threonine mitogen-activated protein
kinase family, including mitogen-activated protein kinases,
mitogen-activated protein kinase-like p38, and its human homologs CSBP1
and CSBP2, resulting in an increase in their kinase activity (27-32).
Interestingly, inhibitors of CSBP1 and CSBP2 kinase activity
specifically impair LPS-induced TNF-
mRNA translation by
preventing TNF-
mRNA recruitment into polysomes upon LPS
stimulation (33). These observations indicate that p38 and CSBPs are
the LPS-signaling intermediates that mediate TNF-
mRNA
translational activation most probably by triggering the binding of the
protein complex to the UA-rich sequence.
How do IL-4 and IL-13 down-regulate LPS-induced TNF- mRNA
translation? Both cytokines share a common receptor subunit, IL-4 receptor-
, which is essential for intracellular signaling (34). Upon
IL-4 or IL-13 stimulation, IL-4 receptor-
becomes phosphorylated on
tyrosine residues and recruits several signaling proteins including 4PS
(also called IRS-2). 4PS, which also becomes phosphorylated on tyrosine
residues, forms a signaling complex containing phosphatidylinositol 3-kinase, the adapter molecules GRB/Sos and Nck, and the tyrosine phosphatase PTP1D (also known as SH-PTP2 or Syp) (35, 36). So far, no
downstream signals originating from GRB/Sos, Nck, and PTP1D have been
observed upon IL-4/IL-13 stimulation, and the significance of their
recruitment by 4PS remains unclear. We can speculate that upon IL-4 or
IL-13 stimulation, the tyrosine phosphatase activity of PTP1D might
down-regulate the kinase activity of p38 or CSBP1 and CSBP2 by
controlling their phosphorylation status. Consequently, the LPS-induced
binding of the protein complex to the UA-rich sequence and TNF-
mRNA translation would be inhibited.
We thank Dr. W. Buurman for providing the
polyclonal rabbit anti-mouse TNF- antibody used in the mouse TNF-
enzyme-linked immunosorbent assay. We thank Drs. B. Beutler, F. Bazzoni, and J. Han for providing the DNA constructs. We thank Dr.
Louis Droogmans for critical reading of the manuscript.