|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1299-1307
Interleukin-4 (IL-4) and IL-13 Enhance the Effect of IL-1 on
Production of IL-1 Receptor Antagonist by Human Primary Hepatocytes and
Hepatoma HepG2 Cells: Differential Effect on C-Reactive Protein
Production
By
Cem Gabay,
Brandon Porter,
Denis Guenette,
Bahri Billir, and
William P. Arend
From the Division of Rheumatology and the Division of
Gastroenterology, Department of Medicine, University of Colorado Health
Sciences Center, Denver, CO 80262.
 |
ABSTRACT |
Interleukin-1 receptor antagonist (IL-1Ra) is produced by
hepatocytes with characteristics of an acute-phase protein. To examine the role of IL-4 and IL-13 in production of IL-1Ra, human primary hepatocytes and HepG2 human hepatoma cells were cultured in the presence of IL-4 or IL-13 in combination with IL-1 and/or
IL-6. The results indicated that both IL-4 and IL-13 amplified the
stimulatory effect of IL-1 on production of IL-1Ra protein and
messenger RNA (mRNA) by both human primary hepatocytes and HepG2 cells. IL-1Ra refers to three different peptides, one secreted (sIL-1Ra) and
two intracellular (icIL-1RaI and icIL-1RaII), derived from the same
gene. sIL-1Ra and icIL-1RaI are the products of two different mRNA,
whereas icIL-1RaII is synthesized by alternative translation initiation
mainly from sIL-1Ra mRNA. Our results show that both sIL-1Ra and
icIL-1RaII, but not icIL-1RaI, are produced by HepG2 cells and human
hepatocytes. Transient transfection experiments as well as mRNA
stability studies indicated that IL-4 stimulated sIL-1Ra production
primarly at the level of transcription. Gel retardation assays showed
that IL-4 induced the formation of a STAT6-DNA complex with a STAT6
binding element within the sIL-1Ra promoter, but had no effect on
IL-1-induced NF- B binding activity. In contrast to IL-1Ra,
production of C-reactive protein by human primary hepatocytes was
stimulated by IL-6 and decreased by the addition of IL-4.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THE ACUTE-PHASE PROTEINS (APP) include
several plasma proteins, the levels of which are modified during
inflammatory conditions. These proteins have different biologic
functions and they contribute to both initiation and modulation of
inflammatory responses.1 Recently, we showed that
interleukin-1 (IL-1) receptor antagonist (IL-1Ra) is produced by
hepatocytes and regulated as an APP.2 IL-1Ra has been shown
to have antiinflammatory properties in several experimental animal
models of diseases, further emphasizing the role of APP as modulators
of the inflammatory response.
Although IL-6 is considered the major inducer of APP, other cytokines
such as IL-1, tumor necrosis factor (TNF)- , transforming growth
factor (TGF)-, interferon (IFN) , and IL-8 can also stimulate the
production of APP. In addition, changes in APP production by
hepatocytes are not always mediated by only one cytokine but are
influenced by a combination of cytokines, cytokine receptors, and
hormones.1
IL-4 is a cytokine that is produced primarily by CD4+ T
lymphocytes and mast cells.3,4 IL-4 inhibits the production
of IL-1, TNF-, IL-8, and prostaglandin E2,5-7 whereas it
increases the production of IL-1Ra by monocytes stimulated by
lipopolysaccharide (LPS).5 These observations suggest that
IL-4 could have antiinflammatory properties. IL-13, another
T-cell-derived cytokine, shares several biologic activities with IL-4.
However, IL-4 can induce unique responses in certain cells, such as T
lymphocytes, which do not respond to IL-13.8 IL-4 has been
reported to decrease the production of APP such as haptoglobin,
C-reactive protein (CRP), and albumin by human primary
hepatocytes, whereas it had no effect on other APP.9 In
addition, IL-4 was shown to decrease the effects of IL-1, TNF-,
and IL-6 on lipogenesis in mouse hepatocytes in vivo.10 The
effects of IL-13 on hepatocytes have not been reported.
The studies included herein show that both IL-4 and IL-13 amplified the
stimulatory effects of IL-1 on production of IL-1Ra by hepatocytes.
In contrast, IL-4 downregulated the IL-6-induced production of CRP by hepatocytes.
| |
MATERIALS AND METHODS |
Reagents.
IL-1, IL-6, and IL-4 were purchased from R&D Systems, Inc
(Minneapolis, MN); the LPS contamination in each cytokine preparation was less than 0.1 ng/mg protein according to the manufacturer. IL-13
was generously donated by DNAX (Palo Alto, CA). Rabbit antihuman CRP
antibodies were purchased from Dako (Glostrup, Denmark).
Cell cultures.
Human primary hepatocytes were isolated from livers obtained from 11 organ donors through a collaboration with the International Institute
for the Advancement of Medicine (Scranton, PA), a nonprofit tissue and
organ bank. Human livers used for hepatocyte isolation were obtained
under the United Network for Organ Sharing specifications and
guidelines but were determined unsuitable for orthotopic liver transplantation due to physical trauma to a portion of the organ, anoxia, or a high interstitial fat content. All livers had been serologically screened and found negative for human immunodeficiency virus (HIV), human T-cell lymphotrophic virus (HTLV),
hepatitis B, and hepatitis C before acceptance. Human hepatocytes were
isolated as previously described.11 Viable hepatocytes (200 × 103 cells/mL), as assessed by Trypan blue
exclusion, were seeded on 24-well culture Primaria plates (Becton
Dickinson, Franklin Lakes, NJ) in RPMI 1640 medium (Mediatech, Inc,
Herndon, VA) supplemented with penicillin, streptomycin, and 10% fetal
calf serum (FCS) (Summit Biotechnology, Greeley, CO). After 12-hour
culture, the cells were placed in medium supplemented with 5% FCS.
After 24-hour culture, the cells were placed in medium supplemented
with 0.2% bovine serum albumin (BSA). The cells were stimulated after
48 hours of culture. Supernatants were collected at 18 hours after stimulation and kept frozen at  20°C until assayed for IL-1Ra and CRP content.
HepG2 cells were purchased from the American Type Culture Collection
(Rockville, MD) and cultured in 24-well plates (Becton Dickinson) in
Dulbecco's modified Eagle's medium (DMEM) supplemented with
penicillin, streptomycin, and 10% FCS. After the cells reached confluence, the medium was renewed, and stimulants added. The supernatants were collected at 18 hours and kept frozen until assayed
for IL-1Ra content.
IL-1Ra enzyme-linked immunosorbent assay (ELISA).
IL-1Ra concentrations were measured in culture supernatants from human
primary hepatocytes and from HepG2 cells using a sandwich ELISA, as
previously described.2 The sensitivity of the assay was 78 pg/mL.
CRP ELISA.
CRP concentrations were measured in culture supernatants from human
primary hepatocytes using a sandwich ELISA, as previously described.12 The sensitivity of the assay was 0.4 ng/mL.
Western blot analysis.
Protein extracts were obtained from HepG2 cells, human primary
hepatocytes, and the human keratinocyte cell line A431 using Trizol
(Life Technologies, Gaithersburg, MD) as recently
described.13 Twenty µg total protein were heated in
reducing buffer and electrophoresed on 17.5% polyacrylamide gels
followed by electrophoretic transfer to polyvinylidene difluoride
(PVDF) membrane (DuPont-NEN, Boston, MA). Western blot analysis was
then performed as described.2
IL-1Ra messenger RNA (mRNA) analysis by ribonuclease protection
assay.
Total RNA was prepared from HepG2 cells and human primary hepatocytes
cultured in 100-mm plates. After 16 hours of stimulation, if not
otherwise mentioned, the cells were lysed in Trizol (Life Technologies) and RNA was prepared according to the
manufacturer's instructions.
A specific probe for human IL-1Ra isoforms mRNA was generated by
polymerase chain reaction (PCR) using the following primers (5'
to 3'): 5' icIL-1Ra, GCA TGA ATT CCA GGT ACT GCC CGG GTG
CTA CTT TAT; and 3' common IL-1Ra, GCA TAA GCT TTG CCT CCA GCT
GGA GTC TGG TCT C. The PCR products were cleaved with EcoRI and
HindIII and then cloned into Bluescript SK+ plasmid
(Stratagene, La Jolla, CA). Absence of mutation was verified by
sequencing. Generation of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was previously described.11 The RNase
protection assays were performed as recently described.2
Briefly, 10 µg total RNA was hybridized with the
32P-labeled riboprobe and RNase-treated. The RNA protected
fragments were resolved on denaturing 6% polyacrylamide-8 mol/L urea
gels. Autoradiography was immediately performed and quantitation of IL-1Ra and GAPDH mRNA fragments was performed by PhosphorImager (ImageQuant; Molecular Dynamics Inc, Sunnydale, CA).
HepG2 cell transfection studies.
The psRA-294Luc refers to the 294-bp of 5'-flanking DNA with
respect to the first exon of sIL-1Ra. Transfections were performed using a modification of the calcium phosphate method as
described.2 Briefly, 0.5 × 106 HepG2
cells were seeded in 60-mm dishes in complete medium. After 24 hours of
culture, 0.5 mL of calcium precipitate containing 7.5 µg plasmid DNA
was added to each plate. The cells then were incubated at 37°C for
4 hours and subjected to a glycerol shock for 2 minutes. After 24 hours
the cytokines were added. The cells were harvested after 24 hours and
luciferase activity was assessed as described previously.2
Luciferase activity was normalized to total protein concentration of
the cell lysate (protein assay kit; BioRad Laboratories, Richmond, CA).
Nuclear extracts.
Nuclear extracts were prepared from untreated HepG2 cells or cells
stimulated by IL-1, IL-4, or a combination of both cytokines for 30 minutes as previously described.2 Protease and phosphatase inhibitors were added to the buffers, including 1 µg/mL leupeptin, 1 µg/mL aprotonin, 1 µg/mL pepstatin, 10 mmol/L -glycerophosphate, and 1 mmol/L sodium vanadate. Protein concentrations were measured using a Bio-Rad protein assay kit. Nuclear proteins were kept at
80°C until used for electrophoretic mobility shift assay (EMSA).
EMSA.
For NF-B, 5 µg of nuclear extracts were incubated in 20 mL of
EMSA-binding reaction consisting of 10 mmol/L HEPES (pH 7.9), 80 mmol/L
KCl, 1 mmol/L EDTA (pH 8.0), 1 mmol/L ethyleneglycoltetracetic acid
(EGTA) (pH 8.0), 6% glycerol, and 4 µg poly (dI:dC) at
15°C for 15 minutes before adding 20,000 cpm of
32P-labeled oligonucleotide probe for an additional 15 minutes at 15°C. For STAT6, the binding reaction was performed with
10 µg of nuclear extracts in 20 mmol/L HEPES (pH 7.9), 50 mmol/L KCl, 0.1 mmol/L EDTA (pH 8.0), 1 mmol/L dithiothreitol (DDT),
5% glycerol, 200 µg/mL BSA, and 3 µg poly (dI:dC) at 4°C for
15 minutes before adding 50,000 cpm of 32P-labeled
oligonucleotide probe for an additional 15 minutes at room temperature
(RT). The reaction was analyzed on a 6% nondenaturing polyacrylamide
gel in 0.5× (0.25× for STAT6) Tris-borate/EDTA buffer.
After electrophoresis, the gel was fixed in 10% acetic acid, dried
under vacuum, and autoradiography was performed immediately.
For DNA competition studies, the binding reaction was conducted in the
presence of nonspecific competitor together with a 100-fold molar
excess of unlabeled probe. For antibody supershift assays, the extracts
were preincubated either with antibodies against the p65 (RelA) subunit
of NF-B, p50 subunit of NF-B, or STAT6. Rabbit IgG was used as
control. Both antibodies and consensus sequence oligonucleotides were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Oligonucleotides used in EMSA:
NF-B wild type (WT): ATG CGA GGA GGG TAT TTC CGC TTC T;
NF-B Mutant: ATG CGA GGA Gct cga g TC CGC TTC T;
Consensus NF-B: ATG TGA GGG GAC TTT CCC AGG;
SBE1 WT: GC TCT TCT TCC CAG GAA CTC AAT;
SBE1 Mutant: GC TCT TCT g CC CAG Gc A CTC AAT;
Consensus STAT6: GTA TTT CCC AGA AAA GGA AC; and
SBE2 WT: AT CTT AAT TTT GGG GAA ATT GCA C
(SBE = STAT6 binding element).
Double-stranded WT oligonucleotides were labeled with 32P
dNTPs by fill-in reaction of 5' overhangs using the
Klenow enzyme (Promega, Madison, WI) and isolated by two
passages over Sephadex G-50 spin columns (Pharmacia
Biotech, Uppsala, Sweden).
Statistical analysis.
Student's t-test (unpaired, two tailed) was used for
comparisons between specified different conditions.
| |
RESULTS |
Production of IL-1Ra by HepG2 cells stimulated in the presence of IL-4
and IL-13.
Confluent human hepatoma HepG2 cells were cultured in the presence of
IL-1 , IL-6, IL-4, and IL-13 alone or in combination. Culture
supernatants were collected after 18 hours and the IL-1Ra concentrations were determined by ELISA. As recently described, IL-1
alone or in combination with IL-6 was able to upregulate the production
of IL-1Ra (Fig 1). Concentrations of
IL-1 and IL-6 used in these experiments were previously shown to
maximally induce IL-1Ra.2 IL-4 alone or in the presence of
IL-6 did not stimulate the production of IL-1Ra (Fig 1A). In contrast,
IL-4 at concentrations of 1 ng/mL or 10 ng/mL significantly enhanced the effect of IL-1 or the combination of IL-1 and IL-6 on IL-1Ra production. Dose-response studies with IL-4 concentrations ranging from
0.1 to 100 ng/mL showed that a concentration of 10 ng/mL maximally
induced IL-1Ra production in the presence of IL-1 (data not shown).
The combination of IL-1 and IL-4 was almost as potent as the
combination of IL-1 and IL-6. In addition, IL-13 at a concentration
of 10 ng/mL also amplified the effect of IL-1 or the combination of
IL-1 and IL-6 on IL-1Ra production (Fig 1B).
View larger version (14K):
[in this window]
[in a new window]
| Fig 1.
Production of IL-1Ra by the HepG2 human hepatoma cell
line and by human primary hepatocytes. HepG2 cells were cultured in the
absence (control) or presence of IL-1, IL-6, and a combination of
IL-1 and IL-6 without ( ) or with the addition of 1 ng/mL ( ) or
10 ng/mL ( ) IL-4 (A). Similar experiments were performed with the
addition () of 10 ng/mL IL-13 in HepG2 cells (B) and of 10 ng/mL
IL-4 in human primary hepatocytes (C). After 18 hours of stimulation,
levels of IL-1Ra were measured by ELISA in culture supernatants. Values
represent the mean ± SEM of three experiments. *
P < 0.01 in comparison with cells cultured in the absence of
IL-4 or IL-13.
|
|
Production of IL-1Ra by human primary hepatocytes stimulated with
IL-4.
Human primary hepatocytes obtained from livers unsuitable for
transplantation were cultured in the presence of IL-1, IL-6, and
IL-4 alone or in combination. Culture supernatants were collected after
18-hour stimulation and the IL-1Ra concentrations were determined by
ELISA. A similar pattern of IL-1Ra production was found in hepatocytes
from three liver donors. However, due to large variations in basal and
induced IL-1Ra values between donors, the results are expressed as
fold-increase. As shown in Fig 1C, IL-4 significantly enhanced the
stimulatory effect of IL-1 on production of IL-1Ra by human primary
hepatocytes. IL-4 alone or in combination with IL-6 had a slight but
not significant enhancing effect on IL-1Ra production. The effect of
IL-13 on human primary hepatocytes was examined in one donor and the
results were similar to those observed with IL-4 (data not shown).
Western blot analysis.
The term IL-1Ra refers to three different proteins that are encoded by
the same gene, but are generated by alternate RNA splicing of two
different first exons. One form is secreted (17 kD sIL-1Ra) and one
form remains intracellular (now termed 18 kD icIL-1RaI). In addition,
our laboratory has recently described another intracellular smaller
molecular mass variant of IL-1Ra (16 kD icIL-1RaII).14 This
latter isoform is produced by alternative translation initiation mainly
from sIL-1Ra mRNA. In previous studies, we observed that HepG2 cells
expressed only the sIL-1Ra mRNA. In addition, by Western blot analysis,
we detected the presence of sIL-1Ra protein in culture supernatants and
of 16 kD icIL-1RaII in HepG2 lysates.2 To examine whether
the expression of this smaller molecular weight IL-1Ra isoform was also
present in normal human hepatocytes, protein extracts were prepared
from cell lysates and examined by Western blot analysis. As shown in
Fig 2, 16 kD icIL-1RaII, but not 18 kD
icIL-1RaI, was present in both HepG2 cells (lane 4) and human primary
hepatocytes (lane 5). In contrast, 18 kD icIL-1RaI was present in the
lysates of the human keratinocyte cell line A431 (lane 3).
View larger version (40K):
[in this window]
[in a new window]
| Fig 2.
IL-1Ra size isoforms in the lysates of HepG2 cells, human
primary hepatocytes, and the human keratinocyte cell line A431. Twenty
micrograms of lysate proteins from HepG2 cells and human primary
hepatocytes stimulated by IL-1 and IL-4 and from unstimulated A431
cells were electrophoresed on a 17.5% polyacrylamide gel, followed by
electroblotting onto a PVDF membrane. Western blot analysis was
performed using a mouse monoclonal antibody against human IL-1Ra that
recognized all the described isoforms. Lane 1, recombinant icIL-1RaI;
lane 2, recombinant sIL-1Ra; lane 3, A431 lysate; lane 4, HepG2 lysate;
lane 5, human primary hepatocyte lysate.
|
|
mRNA of IL-1Ra isoforms determined by RNase protection assay.
To further characterize the expression of the different IL-1Ra isoform
mRNAs in human primary hepatocytes, we created a riboprobe that
specifically recognized both IL-1Ra transcripts as two different protected size fragments after RNase digestion. The 5' end of the
riboprobe included the icIL-1RaI mRNA transcription start site and its
3' end finished 250 bases after the splice site, into the IL-1Ra
common region. As shown in Fig 3, both
HepG2 cells and human primary hepatocytes produced only sIL-1Ra mRNA
(lane 5 and 6). U937 cells were able to express both transcripts (lane 2), although sIL-1Ra mRNA was predominently produced. Interestingly, A431 cells also produced both IL-1Ra transcripts (lane 3) but, in
contrast to U937 cells, they predominently produced icIL-1RaI mRNA. In
addition, we also detected the presence of several icIL-1RaI mRNA size
products. The monocytic cell line U937 produced predominately a smaller
size transcript, whereas A431 cells produced mainly longer transcripts.
These differences are probably related to the use of different
transcription start sites.13,15 In KB cells, included as
negative controls, we did not detect any IL-1Ra isoform mRNA (lane 4).
View larger version (51K):
[in this window]
[in a new window]
| Fig 3.
IL-1Ra mRNA isoforms. Total RNA from HepG2 cells and
human primary hepatocytes stimulated with IL-1 and IL-4, from human
keratinocyte cell lines A431 and KB, and from the U937 human monocytic
cell line cultured with PMA and LPS were examined by RNase protection
assay using a riboprobe that recognized the different IL-1Ra mRNA
isoforms. Ten micrograms of total RNA were hybridized simultaneously
with 32P-labeled riboprobes complementary to IL-1Ra and
GAPDH mRNA, then digested with RNase A and RNase T1 (see Materials and
Methods). The protected fragments were analyzed in a 6% denaturing
polyacrylamide gel. Lane 1, undigested probes; lane 2, U937 cells; lane
3, A431 cells; lane 4, KB cells; lane 5, HepG2 cells; lane 6, human
primary hepatocytes.
|
|
Quantitation of sIL-1Ra mRNA production by HepG2 cells.
The production of sIL-1Ra mRNA by HepG2 cells cultured in the absence
or presence of cytokines was examined by RNase protection assay.
Time-course studies showed that sIL-1Ra mRNA production was increased 2 hours after stimulation by IL-1 and that elevated sIL-1Ra mRNA
levels were maintained for up to 48 hours (data not shown). In
addition, consistent with the results obtained in culture supernatants,
both IL-4 and IL-13 enhanced the effect of IL-1 on production of
sIL-1Ra mRNA, but neither cytokine exhibited any effect alone or in
combination with IL-6 (Fig 4A).
View larger version (19K):
[in this window]
[in a new window]
View larger version (41K):
[in this window]
[in a new window]
| Fig 4.
Determination of IL-1Ra mRNA levels in HepG2 cells and in
human primary hepatocytes by RNase protection assay. HepG2 cells were
cultured in the absence (control) or presence of IL-1 , IL-6, and a
combination of IL-1 and IL-6 both without () and with the
addition of 10 ng/mL IL-4 () or 10 ng/mL IL-13 () for 16 hours
(A). Ten micrograms of total RNA from the HepG2 cells were hybridized
simultaneously with 32P-labeled riboprobes complementary to
IL-1Ra and GAPDH mRNA, then digested with RNase A and RNase T1. The
protected fragments were analyzed in a 6% denaturing polyacrylamide
gel. The intensity of the protected RNA fragments was analyzed by
phosphorImager. The results represent IL-1Ra/GAPDH ratio × 103. Values represent the mean ± SEM of three
experiments. *P < .05 in comparison
with cells cultured in the absence of IL-4 or IL-13. Human primary
hepatocytes were cultured in the absence (control) or presence of
IL-1, IL-6, and a combination of IL-1 and IL-6, both without and
with the addition of 10 ng/mL IL-4 or 10 ng/mL IL-13 for 16 hours (B).
Ten micrograms of total RNA from the hepatocytes were examined by RNase
protection assay as described above. These results are representative
of three different experiments.
|
|
sIL-1Ra mRNA production by human primary hepatocytes.
To examine the effect of IL-4 and IL-13 on production of sIL-1Ra mRNA
by human hepatocytes, the cells were cultured 16 hours in the absence
or presence of cytokines. Consistent with the results obtained in
culture supernatants, both IL-4 and IL-13 increased the effect of
IL-1 on sIL-1Ra mRNA levels but had no effect alone or when used in
combination with IL-6 (Fig 4B).
IL-4 enhanced the effect of IL-1 on sIL-1Ra promoter
activity.
We recently showed that the sIL-1Ra promoter was active in HepG2 cells
and that this promoter contained response elements for transcription
factors NF-B and C/EBP that play important role in the response to
IL- and IL-6.2 To examine whether IL-4 is able to
stimulate the activity of the sIL-1Ra promoter, HepG2 cells were
transfected with a plasmid containing the 294-bp sIL-1Ra promoter
fragment coupled with the luciferase reporter gene and cultured in the
presence or absence of IL-4 and/or IL-1. The results showed
that luciferase activity was increased by IL-1 alone and that,
consistent with results observed at the endogenous gene level, IL-4
significantly enhanced the stimulatory effect of IL-1
(Fig 5).
View larger version (9K):
[in this window]
[in a new window]
| Fig 5.
Secreted IL-1Ra promoter transfections. HepG2 cells were
transfected with a construct containing the 294-bp sIL-1Ra promoter
fragment coupled with the luciferase reporter gene. The cells were then
cultured in the absence (control) or presence of IL-1 and/or
IL-4. After 24 hours of stimulation, the cell lysates were assayed for
luciferase activity (see Materials and Methods). The data are expressed
as fold increase over unstimulated cells. The results represent the
mean ± SEM of three experiments. * P < .05 in
comparison with cells cultured in the presence of IL-1 alone.
|
|
To examine whether the effect of IL-4 on sIL-1Ra production was also
mediated at a posttranscriptional level, HepG2 cells and human primary
hepatocytes were cultured in the presence of IL-1 alone or with a
combination of IL-1 and IL-4. After 4 hours of stimulation, the
medium was changed and -amanitin was added to block transcription.
The results of these experiments indicated that IL-4 did not increase
sIL-1Ra mRNA half-life (data not shown).
IL-4 induces STAT6 binding activity.
The data described above indicated that IL-4 regulated IL-1Ra gene
expression at the level of transcription. The effects of IL-4 and IL-13
on transcriptional regulation of several genes are mediated through
STAT6.16-18 In addition, some investigators recently
described the presence of two potential STAT6 binding sites within the
sIL-1Ra promoter, STAT6 binding element 1 and 2 (SBE1 and SBE2),
located between bases 247 and 238 and bases 208 and 200,
respectively.19 They showed that SBE1, but not SBE2, was
involved in the regulation of IL-1Ra production in response to IL-4 in
macrophages.19 To examine the effect of IL-4 on STAT6 binding activity, gel retardation assays were performed using nuclear
extracts from HepG2 cells, cultured in the presence or absence of IL-4
and/or IL-1, and a 32P-labeled oligonucleotide
containing SBE1. As shown in Fig 6, treatment with IL-4 markedly enhanced the STAT6 binding activity (lane 3). Stimulation with both IL-4 and IL-1 produced a
pattern similar to that observed with IL-4 alone (lane 4). In contrast, we did not detect any STAT6 binding activity in nuclear extracts from
both unstimulated and IL-1-stimulated HepG2 cells (lanes 1 and 2).
To further characterize the protein binding to our oligonucleotide, competition studies were performed using nuclear extracts from IL-4-stimulated HepG2 cells. The formation of the DNA-binding complex
was completely competed away by unlabeled wild-type SBE1 and by an
oligonucleotide containing the STAT6 consensus region (lanes 5 and 8),
but not by a mutated oligonucleotide (lane 6).
View larger version (45K):
[in this window]
[in a new window]
| Fig 6.
EMSA of STAT6 binding activity. The DNA binding activity
of nuclear extracts from HepG2 cells cultured in the presence or
absence of IL-1 and/or IL-4 for 30 minutes were examined
using a 32P-labeled oligonucleotide from the sIL-1Ra
promoter region containing the recently characterized STAT6 binding
element (SBE1). Competition studies were performed with nuclear
extracts from HepG2 cells stimulated with IL-4 (see Materials and
Methods). Lane 1, unstimulated; lane 2, IL-1; lane 3, IL-4; lane 4, IL-1 and IL-4; lane 5, competition with the cold probe; lane 6, competition with the cold probe mutated in the potential STAT6 binding
site; lane 7, competition with the cold oligonucleotide containing
another potential STAT6 binding site within the sIL-1Ra promoter
(SBE2); lane 8, competition with the cold oligonucleotide containing
the STAT6 consensus element; lane 9, preincubation of nuclear extracts
with antibodies against STAT6 before the addition of the
32P-labeled SBE1 probe; lane 10, preincubation of nuclear
extracts with control IgG. Lanes 11 and 12 contain the SBE1 probe and
the antibodies against STAT6 or control IgG, respectively, without
nuclear extracts. The dark arrow shows the STAT6-DNA complex. The open
arrow represents the supershifted complex. NS = nonspecific
protein-DNA binding. These results are representative of three
different experiments.
|
|
As previously reported,19 SBE2 only partially competed for
the STAT6-DNA complex (lane 7), indicating that SBE2 bound STAT6 with a
lower affinity than SBE1. In addition, when gel retardation assays were
performed using SBE2 as a 32P-labeled probe, we did not
detect the presence of the STAT6-DNA complex (data not shown). To
examine whether the binding activity induced by IL-4 was due to STAT6,
immunological reactivity of the complexed protein was tested using
antibodies raised against STAT6. The results showed that the STAT6-DNA
complex was largely competed away with the presence of a higher
molecular weight band (supershift) when nuclear extracts were
pretreated in the presence of anti-STAT6 antibodies (lane 9). In
contrast, control IgG had no effect (lane 10).
Taken together these findings indicate that the IL-4-induced binding
activity in HepG2 cell nuclear extracts was due to the presence of
STAT6 and confirmed the presence of a STAT6 binding site (SBE1) within
the sIL-1Ra promoter.
IL-4 does not alter the IL-1-induced NF-B binding
activity in HepG2 cells.
IL-4 has been shown to decrease the NF-B binding activity in
macrophages.20,21 In contrast, nothing is known regarding the effect of this cytokine on NF-B activation in hepatocytes. We
recently showed that NF-B was involved in the regulation of production sIL-1Ra by HepG2 cells after IL-1 stimulation and the
results presented herein showed that IL-4 enhanced the stimulatory effect of IL-1 on IL-1Ra production. To examine the effect of IL-4
on NF-B activity in HepG2 cells, gel retardation assays were
performed using nuclear extracts from HepG2 cells as described above
and a 32P-labeled oligonucleotide containing the previously
described NF-B binding site within the sIL-1Ra
promoter.2 As shown in Fig 7A,
IL-1 induced the formation of the NF-B-DNA complex (lane 2) and
addition of IL-4 did not modify the NF-B activity (lane 4). In
contrast, we did not detect any NF-B binding activity in the nuclear
extracts from unstimulated or IL-4-stimulated HepG2 cells (lanes 1 and
3). To ensure that the binding activity was due to NF-B, competition
studies were performed with unlabeled oligonucleotides. The presence of
the complex was competed away by the wild-type nucleotide (lane
5) and by an oligonucleotide containing the consensus NF-B
binding site (lane 7), but not by a mutated oligonucleotide (lane 6).
In addition, as shown in Fig 7B, the complex was totally supershifted
when nuclear extracts were incubated with antibodies against the p65
(RelA) subunit of NF-B (lane 3), whereas control immunoglobulins had
no effect (lane 5). Interestingly, antibodies against the p50 subunit
of NF-B had also little effect (lane 4), indicating that p65 was the
most abundant protein in this complex.
View larger version (49K):
[in this window]
[in a new window]
View larger version (64K):
[in this window]
[in a new window]
| Fig 7.
EMSA of NF-B binding activity. The DNA binding
activity of nuclear extracts from HepG2 cells cultured in the presence
or absence of IL-1 and/or IL-4 for 30 minutes were examined
using a 32P-labeled oligonucleotide from the sIL-1Ra
promoter containing the NF-B binding site (A). Competition studies
were performed with nuclear extracts from HepG2 cells stimulated with
IL-1 (see Materials and Methods). Lane 1, unstimulated; lane 2, IL-1; lane 3, IL-4; lane 4, IL-1 and IL-4; lane 5, competition
with the cold probe; lane 6, competition with the cold probe mutated in
the NF-B binding site; lane 7, competition with the cold
oligonucleotide containing the NF-B consensus region.
Characterization of the protein present in the NF-B-DNA complex was
performed using specific antibodies against p65(RelA) and p50 (B). Lane
1, unstimulated; lane 2, IL-1; lane 3, preincubation of nuclear
extracts of IL-1-stimulated HepG2 cells with antibodies against
NF-B p65 (RelA) before the addition of the 32P-labeled
NF-B probe; lane 4, preincubation with antibodies against p50; lane
5, preincubation with control IgG; lanes 6 to 8 contain the NF-B
probe and the antibodies against the p65 subunit, p50 subunit, and
control IgG, without nuclear extracts. The dark arrow shows the
NF-B-DNA complex. The open arrow represents the supershifted
complex. NS = nonspecific protein-DNA binding. These results are
representative of three different experiments.
|
|
Effect of IL-4 on the production of CRP by human primary hepatocytes.
Because HepG2 cells do not produce CRP, we determined the effects of
IL-4 on CRP production by human primary hepatocytes. Supernatants were
obtained from hepatocytes cultured in the absence or presence of
cytokines for 18 hours and were analyzed by ELISA. As shown in
Fig 8, the regulation of CRP and IL-1Ra
production was clearly different. CRP was only stimulated by IL-6,
whereas IL-1 was devoid of any effect. Interestingly, IL-4
significantly downregulated the inducing effect of IL-6. Similar
results were obtained with IL-13 (data not shown).
View larger version (13K):
[in this window]
[in a new window]
| Fig 8.
Production of CRP by human primary hepatocytes. Human
primary hepatocytes were isolated from livers unsuitable for
transplantation and cultured in the absence (control) or presence of
IL-1, IL-6, and a combination of IL-1 and IL-6 without () or
with () the addition of 10 ng/mL IL-4 for 18 hours. Levels of CRP
were measured by ELISA in culture supernatants. Results are expressed
as fold increase over unstimulated cells. Values represent the mean ± SEM of experiments performed with three donors. * P < .05 in comparison with cells cultured in the absence of IL-4.
|
|
| |
DISCUSSION |
In this study, we confirmed that both sIL-1Ra and 16 kD icIL-1RaII, but
not 18 kD icIL-1RaI, are produced by HepG2 and human primary
hepatocytes. In addition, we showed that both IL-4 and IL-13
significantly amplified the stimulatory effects of IL-1 on
production of IL-1Ra by both human hepatoma HepG2 cells and human
primary hepatocytes. The effect of IL-4 was not mediated through the
induction of another cytokine such as IL-1 or IL-6, because in
contrast to IL-1Ra, IL-1 was not produced by either HepG2 cells or
human primary hepatocytes (C. Gabay, unpublished results).
In addition, we previously showed that human primary hepatocytes do not
produce IL-6.11 The results of transfection studies with a
construct containing the sIL-1Ra promoter as well as mRNA stability
studies indicated that IL-4 primarily stimulated sIL-1Ra transcription.
In addition, gel retardation assays showed that IL-4 induced the
formation of a STAT6-DNA complex with a recently characterized STAT6
binding element within the sIL-1Ra promoter. We confirmed that IL-6 is
the main inducer of CRP and showed that in contrast to IL-1Ra, IL-4
downregulated the effect of IL-6 on production of CRP by hepatocytes.
IL-4 and IL-13 are T-cell-derived cytokines that exibit a broad range
of activities in the regulation of inflammatory and immune responses
and are thought to play important roles during allergic
responses.22 However, the effects of these cytokines are
primarily antiinflammatory.23 Both cytokines reduce
production of IL-1, TNF-, and other proinflammatory mediators,
whereas they increase production of IL-1Ra by monocytes and
macrophages. In addition, IL-4 has been shown to downregulate the
production of prostaglandin E2 by synovial macrophage-type cells
through the inhibition of cyclooxygenase 2.24 Both IL-4 and
IL-13 reduced inflammation in different animal models of
arthritis.25-27 The observation that both IL-4 and IL-13
enhanced IL-1-induced production of IL-1Ra by hepatocytes further
emphasizes the antiinflammatory role of these cytokines during the
acute-phase response. Interestingly, IL-4 has been shown to amplify the
stimulatory effect of IL-1 on expression of monocyte chemotactic
protein by endothelial cells28 and of IL-6 by endothelial
cells and fibroblasts.21,28 IL-4 has also been reported to
enhance the inducing effects of TNF- on production of IL-1Ra by
neutrophils29 and on VCAM-1 expression by endothelial
cells.22 However, we did not observe any effect of the
combination of TNF- and IL-4 on production of IL-1Ra by HepG2 cells
(C. Gabay, unpublished data).
Some of the antiinflammatory effects of IL-4 and IL-13 have been shown
to be mediated through inhibition of NF-B activation in
macrophages.20,21 However, the effect of IL-4 on NF-B
activation has not been examined in hepatocytes. We recently showed
that NF-B plays an important role in the regulation of sIL-1Ra
production in HepG2 cells after IL-1 stimulation.2 Our
results showed that in HepG2 cells, IL-4 does not modify the
IL-1-induced NF-B binding activity. This observation is consistent
with similar findings in dermal and synovial fibroblasts,21
indicating that the effects of IL-4 on NF-B activation varies in
different cells.
The term IL-1Ra refers to three different proteins derived from the
same gene. One isoform includes a leader sequence and is glycosylated
and secreted, 17 kD sIL-1Ra, and two intracellular18kD icIL-1RaI and
16kD icIL-1RaII.14 Our results indicated that both HepG2
cells and primary hepatocytes produced only sIL-1Ra mRNA. In addition,
the presence of IL-1Ra in the culture of both HepG2 cells and human
primary hepatocytes is related to production and secretion of sIL-1Ra
protein. By Western blot analysis, we observed that only icIL-1RaII was
detected in the cell lysate of both cell types, whereas sIL-1Ra was
present in the supernatant of HepG2 cells.2 These
observations are consistent with the results obtained in mouse liver in
vivo after the injection of LPS.13
Our results showed that CRP and IL-1Ra are regulated differently in
human primary hepatocytes. IL-1, which is the main inducer of
IL-1Ra, exhibited no effect on the induction of CRP. Consistent with
the results of previous studies in human primary hepatocytes, IL-1
reduced the stimulatory effects of IL-6.12 Another major finding of this study is that IL-4 significantly downregulated the
effect of IL-6 in inducing CRP production by hepatocytes. Other
investigators observed that IL-4 alone, but not in the presence of IL-6
significantly decreased the production of CRP.9 Differences between their results and ours may be due to variations in culture conditions.
The differential effects of IL-4 and IL-13 on regulation of CRP and
IL-1Ra in vitro may have some clinical relevance. Although both CRP and
IL-1Ra react as APP in some inflammatory conditions such as infectious
diseases, several clinical observations indicate that CRP and IL-1Ra
may be differently regulated in other inflammatory conditions. In
systemic lupus erythematosus and dermatomyositis, circulating levels of
IL-1Ra are generally elevated and correlate with disease activity
whereas serum levels of CRP remain usually normal.30,31 In
contrast, in patients with spondylarthrtopathies and rheumatoid
arthritis, a reverse pattern has been observed.32 It can be
hypothesized that these variations may reflect a difference in the
balance of cytokines produced by T helper 1 (Th1) and Th2 lymphocytes.
IL-4 and IL-13 are both considered as Th2 cytokines and evidence from
experimental animal models suggests that the features of systemic lupus
erythematosus and other connective tissue diseases are consistent with
a prominent Th2 response.33-36 In addition, elevated
circulating levels of IL-4 and IL-13 were observed in patients with
scleroderma,37,38 another connective tissue disease
generally associated with normal CRP levels. It is therefore possible
that IL-4 and IL-13 contribute to the pattern of APP observed in these
inflammatory diseases.
In conclusion, our results showed that both IL-4 and IL-13 amplified
the inducing effect of IL-1 on production of IL-1Ra by hepatocytes,
suggesting another possible mechanism through which IL-4 and IL-13 may
exert their antiinflammatory effects in vivo. In addition, the
observation that IL-4 and IL-13 downregulated the production of CRP by
hepatocytes indicates that expression of different APP genes to common
extracellular signals may vary.
| |
FOOTNOTES |
Submitted April 9, 1998; accepted October 14, 1998.
Supported by National Institutes of Health grant AR40135 (W.P.A), by
the Rocky Mountain Chapter of the Arthritis Foundation (W.P.A), and by
a postdoctoral fellowship grant from the Swiss National Science
Foundation and from la Fondation Suisse de Bourse de Medecine et
Biologie (C.G.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address correspondence to Dr Cem Gabay, Division of Rheumatology,
University of Colorado Health Sciences Center, Box B-115, 4200 East
Ninth Avenue, Denver, CO 80262; e-mail: gabay_c{at}defiance.uchsc.edu.
| |
REFERENCES |
1. Gabay C, Kushner I: The acute-phase proteins and other systemic
responses to inflammation. N Engl J Med 1998 (in press)
2.
Gabay C, Smith MF Jr, Eidlen D, Arend WP:
Interleukin-1 receptor antagonist is an acute-phase protein.
J Clin Invest
99:2930, 1997[Medline]
[Order article via Infotrieve]
3.
Howard M, Farrar J, Hilfiker M, Johnson B, Takatsu K, Hamoka T, Paul WE:
Identification of a T cell-derived B cell growth factor distinct from interleukin-2.
J Exp Med
155:914, 1982[Abstract/Free Full Text]
4.
Brown MA, Pierce JH, Watson CJ, Fiaco J, Ihle JN, Paul WE:
B cell stimulatory factor-1/interleukin-4 mRNA is expressed by normal and transformed mast cells.
Cell
50:809, 1987[Medline]
[Order article via Infotrieve]
5.
Vannier E, Miller LC, Dinarello CA:
Coordinated antiinflammatory effects of interleukin 4: Interleukin 4 suppresses interleukin 1 production but upregulates gene expression and synthesis of interleukin 1 receptor antagonist.
Proc Natl Acad Sci USA
89:4076, 1992[Abstract/Free Full Text]
6.
Hart PH, Vitti GF, Burgess DR, Whitty GA, Piccoli DS, Hamilton JA:
Potential anti-inflammatory effects of interleukin 4: Suppression of human monocyte tumor necrosis factor , interleukin 1, and prostaglandin E2.
Proc Natl Acad Sci USA
86:3803, 1989[Abstract/Free Full Text]
7.
Standiford TJ, Strieter RM, Chensue SW, Westwick J, Kasahara K, Kunkel SL:
IL-4 inhibits the expression of IL-8 from stimulated human monocytes.
J Immunol
145:1435, 1990[Abstract]
8.
Zurawski G, De Vries JE:
Interleukin 13, an interleukin 4-like cytokine that acts on monocytes and B cells, but not on T cells.
Immunol Today
15:19, 1994[Medline]
[Order article via Infotrieve]
9.
Loyer P, Ilyin G, Razzak ZA, Banchereau J, Dezier J-F, Campion J-P, Guguen-Guillouzo C, Guillouzo A:
Interleukin 4 inhibits the production of some acute-phase proteins by human hepatocytes in primary culture.
FEBS Lett
336:215, 1993[Medline]
[Order article via Infotrieve]
10.
Grunfeld C, Soued M, Adi S, Moser AH, Fiers W, Dinarello CA, Feingold KR:
Interleukin 4 inhibits stimulation of hepatic lipogenesis by tumor necrosis factor , interleukin 1, and interleukin 6 but not by interferon-1.
Cancer Res
51:2803, 1991[Abstract/Free Full Text]
11.
Gabay C, Silacci P, Genin B, Mentha G, Le Coultre C, Guerne P-A:
Soluble interleukin-6 receptor strongly increases the production of acute-phase proteins by hepatoma cells but exerts minimal changes on human primary hepatocytes.
Eur J Immunol
25:2378, 1995[Medline]
[Order article via Infotrieve]
12.
Gabay C, Genin B, Mentha G, Iynedjian PB, Roux-Lombard P, Guerne P-A:
IL-1 receptor antagonist (IL-1Ra) does not inhibit the production of C-reactive protein or serum amyloid A protein by human primary hepatocytes. Differential regulation in normal and tumour cells.
Clin Exp Immunol
100:306, 1995[Medline]
[Order article via Infotrieve]
13.
Gabay C, Porter B, Fantuzzi G, Arend WP:
Mouse interleukin-1 receptor antagonist (IL-1Ra) isoforms. cDNA cloning and protein expression of intracellular isoform and tissue distribution of secreted and intracellular IL-1Ra in vivo.
J Immunol
159:5905, 1997[Abstract]
14.
Malyak M, Guthridge JM, Hance KR, Dower SK, Freed JH, Arend WP:
Characterization of a low molecular weight isoform of interleukin-1 receptor antagonist.
J Immunol
161:1997, 1998[Abstract/Free Full Text]
15.
Butcher C, Steinkasserer A, Tejura S, Lennard AC:
Comparison of two promoters controlling expression of secreted or intracellular IL-1 receptor antagonist.
J Immunol
153:701, 1996[Abstract]
16.
Mikita T, Campbell D, Wu P, Williamson K, Schindler U:
Requirements for interleukin-4-induced gene expression and functional characterization of Stat6.
Mol Cell Biol
16:5811, 1996[Abstract]
17.
Koehler L, Alliger P, Minty A, Caput D, Ferrara P, Hoell-Neugebauer B, Rank G, Rieber EP:
Human interleukin-13 activates the interleukin-4-dependent transcription factor NF-IL4 sharing DNA binding motif with an interferon--induced nuclear binding factor.
FEBS Lett
345:187, 1994[Medline]
[Order article via Infotrieve]
18.
Izuhara K, Heike T, Otsuka T, Yamaoka K, Mayumi M, Imamura T, Niho Y, Harada N:
Signal transduction pathway of interleukin-4 and interleukin-13 in human B cells derived from X-linked severe combined immunodeficiency patients.
J Biol Chem
271:619, 1996[Abstract/Free Full Text]
19.
Ohmori Y, Smith MF Jr, Hamilton TA:
IL-4-induced expression of the IL-1 receptor antagonist gene is mediated by STAT6.
J Immunol
157:2058, 1996[Abstract]
20.
Lentsch AB, Shanley TP, Sarma V, Ward PA:
In vivo suppression of NF-B and preservation of IB by interleukin-10 and interleukin-13.
J Clin Invest
100:2443, 1997[Medline]
[Order article via Infotrieve]
21.
Donnelly RP, Crofford LJ, Freeman SL, Buras J, Remmers E, Wilder RL, Fenton MJ:
Tissue-specific regulation of IL-6 production by IL-4. Differential effects of IL-4 on nuclear factor-B activity in monocytes and fibroblasts.
J Immunol
151:5603, 1993[Abstract]
22.
Iademarco MF, Barks JL, Dean DC:
Regulation of vascular cell adesion molecule-1 expression by IL-4 and TNF- in cultured endothelial cells.
J Clin Invest
95:264, 1995
23.
Miossec P, Van den Berg W:
Th1/Th2 cytokine balance in arthritis.
Arthritis Rheum
40:2105, 1997[Medline]
[Order article via Infotrieve]
24.
Sugiyama E, Taki H, Kuroda A, Mino T, Yamashita N, Kobayashi M:
Interleukin-4 inhibits prostaglandin E2 production by freshly prepared adherent rheumatoid synovial cells via inhibition of biosynthesis and gene expression of cyclo-oxygenase II but not of cyclo-oxygenase I.
Ann Rheum Dis
55:375, 1996[Abstract/Free Full Text]
25.
Allen JB, Wong HL, Costa GL, Bienkowski MJ, Wahl SM:
Suppression of monocyte function and differential regulation of IL-1 and IL-1Ra by IL-4 contribute to resolution of experimental arthritis.
J Immunol
151:4344, 1993[Abstract]
26.
Bessis N, Boissier MC, Ferrara P, Blankenstein T, Fradelizi D:
Attenuation of collagen-induced arthritis in mice by treatment with vector cells engineered to secrete interleukin-13.
Eur J Immunol
26:2399, 1996[Medline]
[Order article via Infotrieve]
27.
Joosten LAB, Lubberts E, Durez P, Helsen MMA, Jacobs MJM, Goldman M, Van den Berg WB:
Role of interleukin-4 and interleukin-10 in murine collagen-induced arthritis.
Arthritis Rheum
40:249, 1997[Medline]
[Order article via Infotrieve]
28.
Colotta F, Sironi M, Borre A, Luini W, Maddalena F, Mantovani A:
Interleukin 4 amplifies monocyte chemotactic protein and interleukin 6 production by endothelial cells.
Cytokine
4:24, 1992[Medline]
[Order article via Infotrieve]
29.
Marie C, Pitton C, Fitting C, Cavaillon J-M:
IL-10 and IL-4 synergize with TNF- to induce IL-1ra production by human neutrophils.
Cytokine
8:147, 1996[Medline]
[Order article via Infotrieve]
30.
Gabay C, Gay-Crosier F, Roux-Lombard P, Meyer O, Guerne P-A, Vischer T, Dayer J-M:
Elevated serum levels of interleukin-1 receptor antagonist in polymyositis-dermatomyositis: A biological marker of disease activity with a possible role in the lack of acute-phase protein response.
Arthritis Rheum
37:1744, 1994[Medline]
[Order article via Infotrieve]
31.
Suzuki H, Takemura H, Kashiwagi H:
Interleukin-1 receptor antagonist in patients with active systemic lupus erythematosus.
Arthritis Rheum
38:1055, 1996
32.
Gabay C, Cakir N, Moral F, Roux-Lombard P, Meyer O, Dayer J-M, Vischer T, Yazici H, Guerne P-A:
Circulating levels of tumor necrosis factor soluble receptors in systemic lupus erythematosus are significantly higher than in other rheumatic diseases and correlate with disease activity.
J Rheumatol
24:303, 1997[Medline]
[Order article via Infotrieve]
33.
Ishida H, Muchamuel T, Sakaguchi S, Andrade S, Menon S, Howard M:
Continuous administration of anti-interleukin 10 antibodies delays onset of autoimmunity in NZB/W F1 mice.
J Exp Med
179:395, 1994[Abstract/Free Full Text]
34.
Hagiwara E, Adams EM, Plotz PH, Klinman DM:
Abnormal numbers of cytokine producing cells in patients with polymyositis and dermatomyositis.
Clin Exp Rheum
14:485, 1996[Medline]
[Order article via Infotrieve]
35.
Kalechman Y, Gafter U, Da JP, Albeck M, Alarcon-Segovia D, Sredni B:
Delay in the onset of systemic lupus erythematosus following treatment with the immunomodulator AS101. Association with IL-10 inhibition and increase in TNF- levels.
J Immunol
159:2658, 1997[Abstract]
36.
Erb KJ, Rüger B, Von Brevern M, Ryffel B, Schimpl A, Rivett K:
Constitutive expression of interleukin (IL)-4 in vivo causes autoimmune-type disorders in mice.
J Exp Med
185:329, 1997[Abstract/Free Full Text]
37.
Needleman BW, Wigley FM, Stair RW:
Interleukin-1, interleukin-2, interleukin-4, interleukin-6, tumor necrosis factor alpha, and interferon gamma levels in sera from patients with scleroderma.
Arthritis Rheum
35:67, 1992[Medline]
[Order article via Infotrieve]
38.
Hasegawa M, Fujimoto M, Kikuchi K, Takehara K:
Elevated serum levels of interleukin 4 (IL-4), IL-10, and IL-13 in patients with systemic sclerosis.
J Rheumatol
24:328, 1997[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
C. C. Caldwell, J. Tschoep, and A. B. Lentsch
Lymphocyte function during hepatic ischemia/reperfusion injury
J. Leukoc. Biol.,
September 1, 2007;
82(3):
457 - 464.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. C. O'Connor, C. L. Sherry, C. B. Guest, and G. G. Freund
Type 2 Diabetes Impairs Insulin Receptor Substrate-2-Mediated Phosphatidylinositol 3-Kinase Activity in Primary Macrophages to Induce a State of Cytokine Resistance to IL-4 in Association with Overexpression of Suppressor of Cytokine Signaling-3
J. Immunol.,
June 1, 2007;
178(11):
6886 - 6893.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R.-O. Casasnovas, N. Mounier, P. Brice, M. Divine, F. Morschhauser, J. Gabarre, J.-Y. Blay, L. Voillat, P. Lederlin, A. Stamatoullas, et al.
Plasma Cytokine and Soluble Receptor Signature Predicts Outcome of Patients With Classical Hodgkin's Lymphoma: A Study From the Groupe d'Etude des Lymphomes de l'Adulte
J. Clin. Oncol.,
May 1, 2007;
25(13):
1732 - 1740.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. K. Banda, C. Guthridge, D. Sheppard, K. S. Cairns, M. Muggli, D. Bech-Otschir, W. Dubiel, and W. P. Arend
Intracellular IL-1 Receptor Antagonist Type 1 Inhibits IL-1-Induced Cytokine Production in Keratinocytes through Binding to the Third Component of the COP9 Signalosome
J. Immunol.,
March 15, 2005;
174(6):
3608 - 3616.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Odamaki, A. Kato, H. Kumagai, and A. Hishida
Counter-regulatory effects of procalcitonin and indoxyl sulphate on net albumin secretion by cultured rat hepatocytes
Nephrol. Dial. Transplant.,
April 1, 2004;
19(4):
797 - 804.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Bas, B. R. Gauthier, U. Spenato, S. Stingelin, and C. Gabay
CD14 Is an Acute-Phase Protein
J. Immunol.,
April 1, 2004;
172(7):
4470 - 4479.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S.-J. Lin, P.-Y. Shu, C. Chang, A.-K. Ng, and C.-p. Hu
IL-4 Suppresses the Expression and the Replication of Hepatitis B Virus in the Hepatocellular Carcinoma Cell Line Hep3B
J. Immunol.,
November 1, 2003;
171(9):
4708 - 4716.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Chen, R. Lund, T. Aittokallio, M. Kosonen, O. Nevalainen, and R. Lahesmaa
Identification of Novel IL-4/Stat6-Regulated Genes in T Lymphocytes
J. Immunol.,
October 1, 2003;
171(7):
3627 - 3635.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. J. Hirst, M. P. Hallsworth, Q. Peng, and T. H. Lee
Selective Induction of Eotaxin Release by Interleukin-13 or Interleukin-4 in Human Airway Smooth Muscle Cells Is Synergistic with Interleukin-1beta and Is Mediated by the Interleukin-4 Receptor alpha -Chain
Am. J. Respir. Crit. Care Med.,
April 15, 2002;
165(8):
1161 - 1171.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. La and S. M. Fischer
Transcriptional Regulation of Intracellular IL-1 Receptor Antagonist Gene by IL-1{{alpha}} in Primary Mouse Keratinocytes
J. Immunol.,
May 15, 2001;
166(10):
6149 - 6155.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Gabay, M. G. Dreyer, N. Pellegrinelli, R. Chicheportiche, and C. A. Meier
Leptin Directly Induces the Secretion of Interleukin 1 Receptor Antagonist in Human Monocytes
J. Clin. Endocrinol. Metab.,
February 1, 2001;
86(2):
783 - 791.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
S. Gupta, M. Jiang, A. Anthony, and A. B. Pernis
Lineage-Specific Modulation of Interleukin 4 Signaling by Interferon Regulatory Factor 4
J. Exp. Med.,
December 20, 1999;
190(12):
1837 - 1848.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Yoshidome, A. Kato, M. Miyazaki, M. J. Edwards, and A. B. Lentsch
IL-13 Activates STAT6 and Inhibits Liver Injury Induced by Ischemia/Reperfusion
Am. J. Pathol.,
October 1, 1999;
155(4):
1059 - 1064.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction