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J Biol Chem, Vol. 273, Issue 50, 33333-33341, December 11, 1998
From the Cytokine Research Laboratory, Department of Molecular
Oncology, University of Texas M. D. Anderson Cancer Center,
Houston, Texas 77030
The activity of tumor necrosis factor (TNF), a
proinflammatory cytokine, is regulated by a number of other cytokines,
including interleukin (IL)-4. How IL-4 regulates various activities of
TNF is not fully understood. In the present report, we investigated the
effect of IL-4 on the cell surface TNF receptors in human histiocytic
lymphoma U-937 cells. Pretreatment of cells with IL-4 down-regulated
TNF receptors in a dose- and time-dependent manner; an
almost 90% decrease occurred with 10 ng/ml IL-4 treatment for 24 h. Scatchard analysis revealed that the decrease was due to receptor
number and not affinity. IL-13, which shares a common receptor subunit
and various biological activities with IL-4, had no effect on TNF
receptors. IL-4's effect on TNF receptors was not cell type-specific,
since decreases also occurred on various epithelial and T cells. Both
the p60 and p80 forms of the TNF receptor were down-regulated to the
same extent. Western blot showed that IL-4 induced shedding of the TNF
receptors. The decrease of TNF receptors by IL-4 was accompanied by
down-regulation of TNF-induced activities, including cytotoxicity,
caspase-3 activation, NF- Extensive research within the last few years has revealed that the
immune system is regulated by soluble mediators, referred to as
cytokines, produced in part by T-helper-1 and -2 (Th1 and Th2)1 cell types. The central
dogma of immunology is that the cytokines produced by Th1 cells exhibit
immunostimulatory function, and those produced by Th2 display
immunosuppressive activities (for references see Ref. 1). Examples of
the Th1 type of cytokines include tumor necrosis factor (TNF),
lymphotoxin, and interferon- TNF, one of the Th1 cytokines, is also produced by macrophages and
plays an important role in growth modulation, inflammation, viral
replication, septic shock, and immune modulation (2). TNF transduces
its effects through two distinct receptors, viz. p60 and
p80, which have homologous extracellular domains but distinct cytoplasmic domains. The p60 receptors are expressed on all cell types,
whereas p80 is expressed only on myeloid, lymphoid, and endothelial
cells. Most of the signals are mediated through the p60 receptors. The
growth-modulatory effects of TNF are in part mediated through the
activation of caspases, and TNF-induced gene expression is partially
mediated through the activation of the nuclear transcription factors
NF- IL-4 is a cytokine produced by Th2 cells. It promotes growth of
preactivated B cells, induces germ line Several reports indicate that IL-4, as well as IL-13, can down-regulate
a wide variety of TNF-induced effects (13-20). There are also reports
demonstrating that IL-4 and TNF are synergistic in their action (21,
22). Just how IL-4 and IL-13 modulate the function of TNF is not
understood. In this report, we investigated the effect of IL-4 and
IL-13 on TNF receptors and the receptor-mediated early and late
cellular responses. The results indicate that IL-4, but not IL-13,
induces down-regulation and shedding of TNF receptors. This is
accompanied by suppression of TNF-induced NF- Materials--
Penicillin, streptomycin, neomycin, RPMI 1640 medium, and fetal calf serum were obtained from Life Technologies, Inc.
Glycine, 1-(5-isoquinolinyl sulfonyl)-2-methyl piperazine (H7),
3-(4,5-dihydro-6-(4-(3,4-dimethoxybenzoyl)-1-piperazinyl)-2(1H)-quinolinone (MTT), and wortmannin were obtained from Sigma. Rapamycin and PD098059 were kindly provided by Dr. Bernhard Ryffel (then from the
Institute for Toxicology, University of Zurich, Switzerland) and Dr.
Alan R. Saltiel (Parke Davis Research Division, Warner Lambert Company,
Ann Arbor, MI), respectively. Bacteria-derived recombinant highly
purified human IL-4, IL-13, and TNF were kindly provided by Dr. T. L. Nagabhushan, then of Schering-Plow Inc. (Bloomfield, NJ), by Dr.
Rene de Waal Malefyt of DNAX Inc. (Palo Alto, CA), and by Genentech,
Inc. (South San Francisco, CA), respectively. Double-stranded NF- Cell Lines--
The cell lines employed in this study were U-937
(human histiocytic lymphoma), ML-1a (myeloid lymphoma cells), MCF-7
(human breast cancer cells), H4 (glioma cells), and Jurkat (T-cells), obtained from the American Type Culture Collection (ATCC, Rockville, MD). We used U-937 cells for this study, because their cell surface is
known to express both forms of TNF receptors and transduce multiple
signals in response to TNF, including cytotoxicity, activation of
caspase-3, NF-
U-937, ML1a, Jurkat, and H4 cells were cultured in RPMI 1640 medium,
MCF-7 cells in Dulbecco's modified Eagle's medium, and HeLa cells in
minimum essential medium supplemented with 10% fetal bovine serum
(FBS), penicillin (100 units/ml), and streptomycin (100 µg/ml). HeLa
cells stably transfected with pCDM8 mammalian expression vector
containing the full-length TNF receptor p80 cDNA, as described
previously (see Ref. 23 and references therein), were used for our
studies. A plasmid carrying the hygromycin phosphotransferase gene was
used as a selection marker and was cotransfected with the pCDM18
plasmid. Transfected cells were routinely grown in minimum essential
medium containing 10% FBS, penicillin (100 units/ml), streptomycin
(100 µg/ml), and hygromycin (200 µg/ml). All of these cells were
free from mycoplasma as detected by Gen-Probe mycoplasma rapid
detection kit (Fisher).
Radiolabeling of TNF and Receptor Binding Assay--
Human TNF
was iodinated with [125I]Na by the IODO-GEN method,
purified, and examined for cell surface receptors as described previously (23). Briefly, cells (2 × 106/ml) were
incubated for 1 h on ice in an Eppendorf tube with radiolabeled TNF (2 × 105 cpm/ml) in the presence and absence of a
50-fold excess of unlabeled TNF. Thereafter, cells were centrifuged
(10,000 × g) at 4 °C for 1 min, resuspended in cold
medium, layered on 800 µl of ice-cold 5% sucrose in Dulbecco's
phosphate-buffered saline, and centrifuged (10,000 × g) for 2 min. The upper medium containing free radioactive TNF was then removed, the bottom of the tube containing the cell pellet
was dissected with a razor, and the disintegrations were counted in a
Assay for NF-
For the AP-1 assay, the EMSA was similar to that for NF- Western Blot for TNF Receptor--
For assay of TNF receptor by
Western blot, the cell extract protein and culture supernatant were
used. Cells were extracted with 10 mM Tris buffer, pH 7.9, and 1% Nonidet P-40 plus protease inhibitors for 30 min on ice. The
supernatant was concentrated by filtration through 10,000 Mr cut-off membrane. A 50-µg aliquot of cell
extract protein and 60 µg of concentrated supernatant were resolved
on 12% SDS-polyacrylamide gels. After electrophoresis, the proteins
were electrotransferred to nitrocellulose filters, probed with a rabbit
polyclonal antibody against p80 TNF receptor, and then detected by
chemiluminescence (ECL, Amersham Pharmacia Biotech) (26).
Immunoblot Analysis of Poly(ADP) Ribose Polymerase (PARP)
Degradation--
TNF-induced apoptosis was examined by proteolytic
cleavage of PARP (23). Briefly, cells (2 × 106/ml)
were treated with cycloheximide (2 µg/ml) and TNF (1 nM) for 2 h at 37 °C. After treatment, cell extracts were prepared by incubating the cells for 30 min on ice in 0.05 ml of buffer containing 20 mM HEPES, pH 7.4, 2 mM EDTA, 250 mM NaCl, 0.1% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml
aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml
benzamidine, and 1 mM dithiothreitol. The lysate was
centrifuged, and the supernatant was collected. Cell extract protein
(50 µg) was resolved in 7.5% SDS-polyacrylamide gel electrophoresis,
electrotransferred onto a nitrocellulose membrane, blotted with mouse
anti-PARP antibody, and then detected by chemiluminescence (ECL;
Amersham Pharmacia Biotech). Apoptosis was represented by the cleavage
of 116-kDa PARP into an 85-kDa peptide product.
TNF Cytotoxicity Assay--
The cytotoxic effect of TNF on cells
was determined by the modified tetrazolium salt
3-(4,5-dimethylthiozol-2-yl) 2-5-diphenyl-tetrazolium bromide (MTT)
assay (27). Briefly, cells (10,000 cells/well) were incubated in the
presence or absence of the indicated test sample in a final volume of
0.1 ml for 24 h at 37 °C. Thereafter, 0.025 ml of MTT solution
(5 mg/ml in phosphate-buffered saline) was added to each well. After a
2-h incubation at 37 °C, 0.1 ml of the extraction buffer (20% SDS,
50% dimethyl formamide) was added. After an overnight incubation at
37 °C, the optical densities at 590 nm were measured using a 96-well
multiscanner autoreader (Dynatech MR 5000), with the extraction buffer
as a blank. Percentage of cytotoxicity was determined as follows: % Cytotoxicity = (1 c-Jun Kinase Assay--
The c-Jun kinase assay was performed by
a modification of the method described earlier (23). Briefly, after
treatment of cells (3 × 106/ml) with TNF for 10 min,
cell extracts were prepared by lysing cells in buffer containing 20 mM HEPES, pH 7.4, 2 mM EDTA, 250 mM
NaCl, 1% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml benzamidine,
and 1 mM dithiothreitol. Cell extracts (150-250
µg/sample) were subjected to immunoprecipitation with 0.3 µg of
anti-JNK antibody for 60 min at 4 °C. Immune complex was collected
by incubation with protein A/G-Sepharose beads for 45 min at 4 °C.
The beads were extensively washed with lysis buffer (4 × 400 µl) and kinase buffer (2 × 400 µl; 20 mM HEPES,
pH 7.4, 1 mM dithiothreitol, 25 mM NaCl).
Kinase assays were performed for 15 min at 30 °C with
GST-Jun-(1-79) in 20 mM HEPES, pH 7.4, 10 mM
MgCl2, 1 mM dithiothreitol, and 10 µCi
[ The aim of this study was to examine the effect of IL-4 on the TNF
receptors and receptor-mediated cellular responses of U-937 cells.
Treatment of these cells with either cytokine under the experimental
conditions had no effect on the viability of these cells.
IL-4 but Not IL-13 Inhibits TNF Binding--
U-937 cells (2 × 106/ml) were treated with different concentration of
either IL-4 or IL-13 for 24 h at 37 °C in a CO2
incubator. Then cells were suspended in fresh medium and examined for
specific binding to TNF. The results in Fig.
1A indicate that IL-4
inhibited TNF binding in a dose-response fashion. A greater than 80%
decrease in TNF binding occurred with 10 ng/ml of IL-4. IL-13, however, had no effect on TNF binding. The lack of effect of IL-13 was not due
to either lack of receptors or lack of biological activity, because it
was quite active in down-regulating TNF-induced NF-
To determine the time needed to down-regulate TNF receptors by IL-4,
U-937 (2 × 106/ml) cells were pretreated with 10 ng/ml IL-4 for 1, 2, 4, 6, 9, 12, 18, and 24 h at 37 °C and
then washed with fresh ice-cold medium, cooled on ice, and then
examined for TNF binding. The results in Fig. 1B indicate
that TNF binding was inhibited with increase in time of exposure to
IL-4; an almost 80% decrease in binding occurred with a 24-h pretreatment.
IL-4 Decreases TNF Receptor Number but Not the Affinity--
To
determine whether IL-4 down-regulates TNF receptor number or its
affinity or both, ligand-competitive displacement experiments were
performed. U-937 cells (1 × 106/ml) were treated with
0, 2, and 10 ng/ml IL-4 for 24 h at 37 °C in a CO2
incubator. Then cells (1.2 × 106/ml) were washed,
suspended in fresh cold medium, and examined for TNF binding; 1.2 × 106 cells/ml were incubated at 4 °C with different
concentrations of 125I-TNF in the presence and absence of
unlabeled TNF (50-fold excess). The results in Fig.
2A indicate that specific TNF
binding increased with increases in labeled TNF. Pretreatment of cells
with IL-4, however, decreased the binding. Scatchard analysis of the
data (Fig. 2B) showed that treatment of cells with 2 and 10 ng/ml IL-4 decreased TNF receptor number from 7142/cell to 4761/cell
and 1039/cell, respectively, whereas receptor affinities
(Kd) remained unchanged (0.21, 0.28, and 0.35 nM for untreated cells and 2 and 10 ng/ml IL-4-treated
cells, respectively). These results suggest that IL-4 decreased the TNF
receptor number and not the affinity.
IL-4 Decreases TNF Receptors on Myeloid, Epithelial, and T
Cells--
We examined the effect of IL-4 on other myeloid (ML1a),
epithelial (HeLa and MCF-7), T cell (Jurkat), and glioma (H4) cells. For this, 2 × 106 cells/ml were pretreated with IL-4
(10 ng/ml) for 24 h at 37 °C in a CO2 incubator and
then examined for specific TNF binding at 4 °C. IL-4 decreased the
cell surface expression of TNF receptors on all different cell types
(Fig. 3), thus suggesting that the effects of IL-4 are not cell type-specific.
IL-4 Down-regulates both p60 and p80 Forms of TNF
Receptors--
Previously, it has been shown that myeloid and T cells
express both the p60 and p80 forms of the TNF receptor, whereas all other cell types express only the p60 form (2). Since the effects of
IL-4 are not cell type-specific, most likely both types of TNF
receptors are down-regulated. To ascertain more directly the effect of
IL-4 on each type of receptor, we used receptor-specific antibodies.
U-937 cells were pretreated with IL-4 (10 ng/ml) for 24 h at
37 °C in a CO2 incubator and then incubated with
antibodies to either the p60 or p80 receptor for 1 h at 37 °C
and thereafter examined for TNF receptors as described earlier. In
untreated cells, IL-4 down-regulated TNF receptor by almost 80% (Fig.
4A). Anti-p80 antibody
incubation led to approximately 70% inhibition of TNF binding,
indicating that the remaining 30% of surface TNF receptors were p60.
In contrast, anti-p60 antibody incubation led to about 30% inhibition
of TNF binding, indicating that about 70% of surface TNF receptors
were p80. IL-4 down-regulated both p60 and p80 receptors to the same
extent. In contrast, IL-13 (10 ng/ml for 24 h) had no effect
either on total, p60, or p80 forms of the receptor. Thus, these results
affirm the conclusion that IL-4 down-regulates both types of TNF
receptor.
IL-4 Induces Shedding of TNF Receptors--
To determine if the
decrease in receptor number is due to the shedding of TNF receptors, we
examined the supernatants from IL-4-treated cells by Western blot
analysis. The number of TNF receptors expressed on most cells were too
low to be detected by the Western blot analysis. Therefore, we used
HeLa cells that were stably transfected with a plasmid containing the
human p80 TNF receptor gene. These cells have been previously
characterized for the expression of the TNF receptor (23). HeLa
p80-transfected cells were treated with IL-4 (2 and 10 ng/ml) for
24 h at 37 °C in a CO2 incubator, and then the cell
extract was prepared and the supernatants were concentrated and
analyzed by Western blot for p80 receptor by using polyclonal anti-p80
receptor antibodies. The band for p80 receptor disappears from the cell
extract fraction with increasing concentrations of IL-4 and reappears
in the supernatant fraction (Fig. 4B). The band in the cell
pellet fraction was at the 70-80-kDa location, whereas that in the
supernatant fraction was at 35-40 kDa. This location is consistent
with published reports, which indicate that shedding of TNF receptors
involves only the extracellular domain (28). Thus, it is clear that
IL-4 induces the shedding of the TNF receptors.
IL-4 Inhibits TNF-induced Apoptosis--
How down-regulation of
TNF receptors by IL-4 affects TNF-induced cellular responses was also
investigated. TNF is cytotoxic to a wide variety of cells, including
U-937 cells. To test the effect of IL-4 on TNF-induced cytotoxicity,
these cells were pretreated with IL-4 (10 ng/ml) for 24 h,
stimulated with different concentrations of TNF for 24 h at
37 °C in a CO2 incubator, and then examined for
viability by the MTT dye uptake assay. IL-4 inhibited TNF-induced cytotoxicity in a dose-dependent manner (Fig.
5A). At 8 ng/ml IL-4, almost
all of the cells were protected from the cytotoxicity.
Several reports within the last few years have indicated that TNF
induces apoptosis of cells by activating a protease, caspase-3. This
protease, when activated, cleaves the substrate PARP from 120 to 80 and
40 kDa (29). To determine if IL-4 pretreatment affects the TNF-induced
cleavage of PARP, U-937 cells were pretreated with IL-4 (10 ng/ml) for
24 h and stimulated with different concentrations of TNF for
2 h in the presence of cycloheximide (2 µg/ml); then the
extracts were analyzed for PARP by Western blot. The results in Fig.
5B indicate that 1000 pM TNF induced complete
cleavage of PARP in untreated cells, but in IL-4-treated cells
TNF-induced PARP cleavage was completely inhibited. These results
suggest that IL-4 also blocks TNF-induced apoptosis.
IL-4 Inhibits TNF-dependent NF-
Besides NF-
TNF is a potent activator of JNK (31). This kinase plays a role in
TNF-induced AP-1 activation and apoptosis (31). Thus, we examined the
effect of IL-4 on TNF-induced JNK activation. The U-937 cells were
pretreated with different concentrations of IL-4 for 24 h and
stimulated with TNF (1000 pM) for 10 min. Activation of JNK
was then examined. About a 17-fold activation of JNK occurred with TNF;
the activation gradually decreased with increasing concentrations of
IL-4, until at 10 ng/ml IL-4, the activation of JNK by TNF was totally
inhibited (Fig. 6C).
Wortmannin Blocks the Down-regulatory Effects of IL-4 on TNF
Receptors--
There are some recent reports that IL-4 may mediate its
effects through activation of phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C (32, 33). The Wright et al. report (33) was in part based on inhibition of phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C by wortmannin, H7, and
neomycin, respectively. We examined the effect of these inhibitors on
IL-4-induced down-regulation of TNF receptors. Cells were pretreated
with neomycin (10 ng/ml), wortmannin (100 nM), or H7 (1 µM) for 1 h, exposed to IL-4 for 24 h at
37 °C in a CO2 incubator, cooled on ice, and examined
for TNF receptors. Wortmannin inhibited IL-4-induced down-regulation of
TNF receptors by almost 80% and H7 by 40%, whereas neomycin had no
effect (Fig. 7A). These results suggest that phosphatidylinositol 3-kinase and protein kinase C
may play an important role in the down-regulation of TNF receptors by
IL-4. Concurrently, wortmannin also reversed the suppressive effects of
IL-4 on TNF-induced PARP cleavage (Fig. 7B), NF- Rapamycin Blocks the Down-regulatory Effects of IL-4 on TNF
Receptors--
It is known that phosphatidylinositol 3-kinase plays an
important role in the activation of p70 S6 kinase (34). Therefore, we
examined the effect of rapamycin, a potent inhibitor of p70 S6 kinase,
on IL-4-induced down-regulation of TNF receptors. As shown in Fig.
8A, rapamycin alone had no
effect on TNF receptors, but it blocked the IL-4-induced
down-regulation of the receptors. This effect of rapamycin was found to
be dose-dependent (Fig. 8B). These results thus
suggest that p70 S6 kinase is involved in IL-4-induced regulation of
TNF receptors. Under these conditions, PD098059 had no effect on the
IL-4-induced down-regulation of TNF receptors (Fig. 8A),
suggesting the lack of a role for mitogen-activated protein kinase
pathway (35).
The purpose of this study was to investigate the effect of IL-4
and IL-13 on TNF cellular signaling. In our experiments, IL-4 induced
down-regulation of both forms of cell surface TNF receptors. In
contrast, IL-13 had no effect on TNF receptors. IL-4 reduced TNF
receptor number but not affinity and induced receptor-shedding, and the
effect was not cell type-specific. IL-4 also blocked TNF-induced apoptosis and NF- Several previous reports indicate that IL-4 antagonizes the effects of
TNF in vitro (13-19) and in vivo (15, 20). Among in vitro effects, IL-4 inhibits TNF-induced activation of
endothelial cells (13), blocks TNF-induced expression of tissue factor
and plasminogen activator inhibitor-1 from endothelial cells (17), inhibits TNF-induced E-selectin expression (19), and blocks TNF-induced
proliferation of B cells (16). In vivo, IL-4 blocks TNF-induced hepatic lipogenesis (15) and cachexia and death (20). It is
quite possible that all of these suppressive effects of IL-4 in
vitro and in vivo on TNF-induced responses are mediated through the down-regulation of TNF receptors as described here.
Our results show that IL-4 down-regulates TNF receptors by inducing
receptor shedding. These results suggest that IL-4 must activate a
protease involved in the cleavage of the extracellular domain of the
receptor. The soluble forms of TNF receptors have been found in the
urine, synovial fluids, serum, and ovarian ascites (36). In
vitro, a wide variety of agents are known to induce TNF receptor
shedding, including TNF itself (2). The precise protease involved in
TNF receptor cleavage is not known, but its suppression by metabolic
inhibitors has suggested a metalloprotease (37). In the case of the p80
receptor, mutation of proline 211, which resides in the extracellular
domain, reduces the phorbol 12-myristate 13-acetate-induced shedding of
the receptor (41), suggesting that this hydrophobic residue is critical
for shedding. While IL-4 down-regulated both types of TNF receptors,
IL-10, which like IL-4 is known to suppress TNF signaling, has been
shown to up-regulate the p80 form of the TNF receptor (40).
Although there has been a report that IL-4 can inhibit TNF-induced
NF- How IL-4 can suppress the effects of TNF in one system and enhance in
another is not clear. It is unlikely that this difference is due to the
selective down-regulation of one TNF receptor type over another or to
differences in cell type, because we found that both the p60 and p80
forms of the TNF receptor are down-regulated and that IL-4 decreases
TNF receptors on a wide variety of different cell types. It is
possible, however, that the cell surface TNF receptor density
requirement for different signals transduced by TNF is different and
thus some signals are more affected by IL-4 than others. It is also
possible that the synergistic effects between TNF and IL-4 occur at the
postreceptor level. For instance, TNF-induced NF- We found that IL-4 but not IL-13 down-regulated TNF receptors. Thus,
the mechanism by which IL-13 transduces its signal must differ
significantly from that of IL-4. These two cytokines induce many of the
same biological responses, including class switching to IgE and
induction of major histocompatibility complex class II antigens and
CD23 on human B cells. IL-4 induces the tyrosine phosphorylation of a
170-kDa protein, a substrate called 4PS, and of the Janus kinase family
members JAK1 and JAK3. Although both induce the tyrosine
phosphorylation of 4PS and JAK1 (10), only IL-4 induces the tyrosine
phosphorylation of JAK3. In addition to common Like IL-4, other cytokines are also known to down-regulate TNF-induced
NF- *
This research was supported by The Clayton Foundation for
Research.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
Th1 and Th2, T-helper-1 and -2 cells, respectively; TNF, tumor necrosis factor; NF-
Interleukin-4 Down-regulates Both Forms of Tumor Necrosis Factor
Receptor and Receptor-mediated Apoptosis, NF-
B, AP-1, and c-Jun
N-Terminal Kinase
COMPARISON WITH INTERLEUKIN-13*
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
B and AP-1 activation, and c-Jun N-terminal
kinase induction. Wortmannin reversed the IL-4-induced TNF receptor
down-regulation and all other measured cellular responses, indicating a
critical role of phosphatidylinositol 3-kinase. Rapamycin also blocked the effect of IL-4-induced regulation, thus suggesting the role of p70
S6 kinase. Overall, our results suggest that TNF receptor down-regulation by IL-4 plays a critical role in the antagonistic effects of IL-4 on TNF-induced cellular responses and that this mechanism differs from that of IL-13.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
; those of the Th2 type include
interleukin (IL)-4, IL-10, and IL-13. Several studies have also
suggested that cytokines produced by Th2 cells regulate the activities
of those produced by Th1 cells and vice versa. The exact
mechanism of this regulation is less clear, but cytokine production and
signal transduction may both be involved.
B and AP-1. In addition, TNF is a potent activator of the
serine/threonine protein kinases of the mitogen-activated protein
kinase family, including c-Jun N-terminal kinase (JNK)/stress-activated
protein kinase.
transcripts, directs naive
B cells to switch to IgE and IgG4 synthesis, and induces expression of
low affinity IgE receptors (3). It down-regulates Fc receptor
expression and inhibits the synthesis of proinflammatory cytokines
including TNF (4-8). IL-4 also increases the expression of vascular
cell adhesion molecule 1 on endothelial cells (9). IL-4 interacts with
cells through a receptor consisting of a distinct 
-chain and of a
c-chain that is shared by several other interleukins. The
interaction of IL-4 with the -chain and c-chain of the receptor activates protein tyrosine kinases JAK1 and JAK3, respectively, leading
to the activation of the transcription factor STAT6 (3, 10). IL-13 is
another cytokine produced by the Th2 cells. It shares 30% amino acid
identity with IL-4. IL-13 has been assigned most of the biological
effects characteristic of IL-4 and shares certain receptor components
(11, 12). In addition to common -chain and c-chain, IL-13 binds
to two additional components, which include IL-13R1 (low affinity;
2-10 nM) and IL-13R2 (high affinity; 0.25 nM) subunits. The latter subunits do not bind IL-4 and thus
form the basis for differences between the two cytokines in certain systems.
B and AP-1 activation,
inhibition of TNF-induced cytotoxicity and activation of caspase-3, and
abrogation of TNF-induced JNK activation. These suppressive effects of
IL-4 on TNF appear to be mediated through activation of
phosphatidylinositol 3-kinase and of p70 S6 kinase.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
B
and AP-1 oligonucleotides and single-stranded NF-B oligonucleotide
were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Antibody against TNF p60 and p80 receptors raised in rabbit were
polyclonal but affinity-purified. Anti-poly(ADP)-ribose polymerase
antibody was obtained from Pharmingen (San Diego, CA).
B, AP-1, and JNK (2). In addition, U-937 cells express
receptors for both IL-4 and IL-13 (12).
-counter. To calculate the specific binding, labeled TNF binding
obtained in the presence of unlabeled TNF (nonspecific) was subtracted
from labeled TNF binding in the absence of unlabeled TNF (total).
B and AP-1--
NF-B was assayed by
electrophoretic mobility shift assay (EMSA) following the method
described by Chaturvedi et al. (24). Briefly, nuclear
extracts were prepared by the method of Schreiber et al.
(25). EMSAs were performed by incubating 5 µg of nuclear extract with
16 fmol of 32P-end-labeled 45-mer double-stranded NF-B
oligonucleotide from the human immunodeficiency virus long terminal
repeat,
5'-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG- 3' (boldface letters represent the NF-B binding site). A
double-stranded mutated oligonucleotide,
5'-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3', was used to examine the specificity of binding of NF-B to the DNA.
The specificity of binding was also examined by competition with the
unlabeled oligonucleotide.
B but used a
32P-end-labeled double-stranded oligonucleotide of AP-1
(5'-CGCTTGATGACTCAGCCGGAA-3'; 3'-GCGAACTACTGAGTCGGCCTT-5'), and the nuclear extract
containing 6 µg of protein was analyzed on a 6.6% native gel.
Specificity of binding was determined routinely by using an excess of
unlabeled oligonucleotide.
(A570 of test
sample)/(A570 of control sample)) × 100%.
-32P]ATP. Reactions were stopped by the addition of
15 µl of 2× SDS sample buffer, boiled for 5 min, and subjected to
SDS-polyacrylamide gel electrophoresis (9%). GST-Jun-(1-79) was
visualized by staining with Coomassie Blue, and the dried gel was
analyzed with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
B activation in
U-937 cells (data not shown). Because IL-13 lacked effect, it was not
tested any further.
View larger version (14K):
[in a new window]
Fig. 1.
A, effect of IL-13 and IL-4 on
125I-TNF binding. U-937 (2 × 106) cells
were treated with different concentrations of IL-13 and IL-4 separately
for 24 h at 37 °C in a CO2 incubator. Then cells
were suspended in fresh medium, cooled on ice, and subjected to binding
assay in which 125I-TNF (2 × 105 cpm) was
used per tube; free radiolabeled TNF was then separated with 5%
sucrose gradient and counted in a -counter. B, IL-4
inhibits TNF binding in a time-dependent manner. U-937
(2 × 106) cells were pretreated with 10 ng/ml IL-4
for different times as indicated in the figure, at 37 °C
in a CO2 incubator. Then cells were washed with fresh
medium and cooled in ice. Then 125I-TNF (2 × 105 cpm/tube) was used for the binding assay.
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Fig. 2.
Scatchard analysis of IL-4-treated and
untreated cells. U-937 cells (1 × 106/ml) were
treated with 0, 2, and 10 ng/ml IL-4 for 24 h at 37 °C. Then
cells were washed, suspended in fresh cold medium, and aliquoted in
tubes containing 1.4 × 106 cells/tube; different
concentrations of 125I-TNF were added. In one set of tubes
of all combinations a 50-fold excess of unlabeled TNF was added 30 min
before the addition of labeled TNF at 4 °C. Then TNF binding was
assayed as described earlier. Specific TNF binding was detected after
deduction of cold competitive binding from total binding. A
indicates the specific binding of TNF in untreated and IL-4 (2 and 10 ng/ml)-treated cells. From the specific binding, we calculated the
bound value in fmol and the bound/free value. B, a Scatchard
plot of untreated and IL-4 (2 and 10 ng/ml)-treated cells.
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Fig. 3.
Effect of IL-4 on other cell types.
MCF-7, ML1a, HeLa, Jurkat, and H4 cells (2 × 106)
were treated with IL-4 (10 ng/ml) for 24 h at 37 °C. Then cells
were examined for specific TNF binding at 4 °C.
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Fig. 4.
A, effect of IL-4 and IL-13 on
down-regulation of p60 and p80 TNF receptors. U-937 cells were treated
with IL-4 (10 ng/ml) and IL-13 (10 ng/ml) for 24 h at 37 °C.
Then cells were washed with fresh medium and incubated with anti-TNF
receptor antibody (both against p60 and p80) separately for 1 h at
37 °C. After the cells were washed with fresh cold medium,
125I-TNF binding was assayed as described earlier.
B, effect of IL-4 on shedding of TNF receptor in
p80-overexpressing cells. HeLa cells were stably transfected with human
TNF p80 receptor. These cells were taken to show the shedding of TNF
receptor. HeLa p80 cells were treated with IL-4 (2 and 10 ng/ml) for
24 h at 37 °C. Then cells and supernatants were collected from
untreated and IL-4 (2 and 10 ng/ml)-treated cells. Supernatants were
concentrated with an Mr 10,000 cut-off filter.
Then both cell extract and concentrated supernatant were analyzed in
12% SDS-polyacrylamide gel electrophoresis, and Western blot was
developed against polyclonal anti-p80 TNF receptor antibody raised in
rabbit (1:3000).
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Fig. 5.
A, IL-4 inhibits TNF-mediated cell
viability. U-937 cells were pretreated with IL-4 (10 ng/ml) for 24 h and stimulated with different concentrations of TNF for 24 h at
37 °C. Then the cell viability was determined by the MTT dye uptake
method. B, IL-4 inhibits TNF-mediated PARP degradation.
U-937 cells were pretreated with IL-4 (10 ng/ml) for 24 h and
stimulated with different concentrations of TNF for 2 h in the
presence of cycloheximide (2 µg/ml), and PARP cleavage was then
detected.
B, AP-1, and JNK
Activation--
One of the earliest responses induced by TNF is the
activation of nuclear transcription factor NF-B. To determine if
IL-4 affects TNF-induced NF-B activation, U-937 cells were
preincubated for 24 h with different concentrations of IL-4,
activated with TNF (100 pM) for 30 min at 37 °C, and
then examined for NF-B activation by EMSA. The results (Fig.
6A) indicate that IL-4 blocked TNF-induced NF-B activation in a dose-dependent manner,
maximum inhibition occurring at 5-10 ng/ml IL-4. Results also show
that IL-4 by itself did not activate NF-B.
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Fig. 6.
A, IL-4 inhibits
TNF-dependent NF- B activation. U-937 cells were
preincubated for 24 h with different concentrations of IL-4
followed by treatment with TNF (100 pM) for 30 min at
37 °C. They were then examined for NF-B activation by
electrophoretic mobility shift assay. B, IL-4 inhibits
TNF-induced AP-1 activation. U-937 cells were preincubated for 24 h with different concentrations of IL-4 followed by treatment with TNF
(1 nM) for 30 min at 37 °C. They were then examined for
AP-1 activation by electrophoretic mobility shift assay. C,
effect of IL-4 on TNF-induced c-Jun kinase activation. The U-937 cells
were pretreated with different concentrations of IL-4 for 24 h and
then stimulated with TNF (1000 pM) for 10 min; activation
of c-Jun kinase was then detected.
B, TNF is also a potent activator of another
transcription factor, AP-1, and the pathway leading to AP-1 activation by TNF may differ from that of AP-1 (30). Thus, we examined the effect
of IL-4 on TNF-induced activation of AP-1. Treatment of cells with 1 nM TNF induced AP-1 expression 7-fold in U-937 cells (Fig.
6B). This activation was inhibited by IL-4 in a
dose-dependent manner (Fig. 6B). IL-4 by itself
did not activate AP-1.
B
activation (Fig. 7C), and AP-1 activation (Fig.
7D). These results suggest that down-regulation of TNF
receptor and TNF-mediated responses by IL-4 occurs through activation
of phosphatidylinositol 3-kinase.
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Fig. 7.
A, effect of IL-4 on
125I-TNF binding and detection of role of neomycin,
wortmannin, and H7. Cells pretreated with neomycin (10 ng/ml),
wortmannin (100 nM), and H7 (1 µM) for 1 h were incubated with different concentrations of IL-4 for 24 h at
37 °C. Then cells were washed with fresh medium and cooled on ice.
Then 125I-TNF binding was examined as described under
"Experimental Procedures." B, wortmannin protects
IL-4-mediated protection of PARP cleavage induced by TNF. U-937 cells
were pretreated with 100 nM wortmannin for 1 h and
then treated with IL-4 (10 ng/ml) for 24 h at 37 °C. Then the
cells were stimulated with 1 nM TNF in combination with
cycloheximide (2 µg/ml) for 2 h, and protein was assayed for
PARP cleavage. C and D, wortmannin protects
IL-4-mediated inhibition of NF- B and AP-1 activation induced by TNF.
U-937 cells were pretreated with 100 nM wortmannin for
1 h and then treated with IL-4 (10 ng/ml) for 24 h at
37 °C. Then the cells were stimulated with 100 pM TNF
for NF-B assay (C) or 1 nM TNF for AP-1 assay
(D).
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Fig. 8.
A, effect of PD098059 and rapamycin on
IL-4-mediated down-regulation of TNF receptor. U-937 cells (0.5 × 106/ml) were suspended in RPMI 1640 medium supplemented
with 10% FBS. Cells were pretreated with PD098059 (50 µM) and rapamycin (10 ng/ml) for 30 min and incubated
with IL-4 (10 ng/ml) for 24 h at 37°C in a CO2
incubator. Then cells were washed and suspended in fresh ice-cold
medium at 3 × 106/ml and examined for TNF binding as
described under "Experimental Procedures." The results indicated
are as specific count in cpm ± S.D. of triplicate assays.
B, effect of different concentrations of rapamycin on
IL-4-mediated down-regulation of TNF receptor. U-937 cells (0.5 × 106/ml) were suspended in RPMI 1640 medium supplemented
with 10% FBS, pretreated with different concentrations of rapamycin
for 30 min, and then treated with IL-4 (10 ng/ml) for 24 h at
37°C in a CO2 incubator. Cells were then washed,
suspended in fresh ice-cold medium at a concentration of 2 × 106/ml, and examined for TNF binding. The results are mean
cpm ± S.D. of duplicate assays.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
B, AP-1, and JNK activation. All of these effects of IL-4 could be reversed by wortmannin. IL-4-induced down-regulation TNF receptors was also reversed by rapamycin.
B activation (19), its ability to block TNF-induced apoptosis,
AP-1 activation, and JNK activation has not previously been reported.
Our results may explain the suppression of various other effects as
indicated above induced by TNF. Other reports show that IL-4
potentiates the biological effects of TNF, including cytotoxicity,
monocytic differentiation, and induction of vascular cell adhesion
molecule 1 in vascular smooth muscle cells (9, 21, 22), but none of
these studies examined the effect of IL-4 on TNF receptors.
B activation is
known to synergize with STAT6 activation induced by IL-4 (39).
-chain and
c-chain, IL-13 binds to two additional components, which include
IL-13R1 (low affinity; 2-10 nM) and IL-13R2 (high
affinity; 0.25 nM) subunits. The latter subunits do not
bind IL-4 and thus may explain why IL-4 and not IL-13 down-regulates TNF receptors.
B activation, but their effects are not known to correlate with
decreases in TNF receptors (42-44). How IL-4 transduces the signal to
down-regulate TNF receptors is not clear. The effects of IL-4 were not
through inhibition of glycogen synthase kinase-3, since lithium
chloride, an inhibitor of this kinase (38), had no effect on TNF
receptors (data not shown). Our results do indicate, however, that the
down-regulation of TNF receptors by IL-4 may be mediated through
phosphatidylinositol 3-kinase. This is consistent with a recent report
that showed the role of phosphatidylinositol 3-kinase in IL-4 signaling
(32). Since phosphatidylinositol 3-kinase is known to activate p70 S6
kinase (34), we found that rapamycin, a potent inhibitor of p70 S6
kinase, blocked the IL-4-induced down-regulation of the receptors, thus
suggesting that p70 S6 kinase is involved in IL-4-induced regulation of
TNF receptors. Overall, our results provide the molecular basis for the
suppression of TNF-mediated cellular responses by IL-4 and suggest that
this mechanism of action differs from that of IL-13.
FOOTNOTES
To whom correspondence should be addressed. Tel.: 713-792-3503 or
713-792-6459; Fax: 713-794-1613.
B, nuclear transcription factor-B; EMSA, electrophoretic
mobility shift assay; IL, interleukin; JNK, c-Jun kinase; FBS, fetal
bovine serum; H7, 1-(5-isoquinolinyl sulfonyl)-2-methylpiperazine; PARP, poly(ADP) ribose polymerase; MTT, 3-(4,5-dihydro-6-(4-(3,4-dimethoxybenzoyl)-1-piperazinyl)-2(1H)-quinolinone.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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