| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Production That Does Not Involve Inhibition of p38 Mitogen-Activated Protein Kinase or NF-
B in Primary Human Macrophages1
* Kennedy Institute of Rheumatology Division, Imperial College Faculty of Medicine, Charing Cross Campus, London, United Kingdom; and
University Department of Paediatrics, John Radcliffe Hospital, Oxford, United Kingdom
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production. The molecular pathways by which IL-10
inhibits TNF- production are obscure, with diverse mechanisms having
been published. In this study, a new approach has been taken for the
study of human cells. Adenovirus was used to deliver TNF-
promoter-based luciferase reporter genes to primary human monocytic
cells. The reporter genes were highly responsive to macrophage
activation and appeared to mirror the behavior of the endogenous
TNF- gene. When added, either with or after
the stimulus, IL-10 required the 3' untranslated region of the
TNF- gene to inhibit luciferase mRNA and protein
expression, indicating a posttranscriptional mechanism. However, if
macrophages were incubated with IL-10 before activation, inhibition of
gene expression was also mediated by the 5' promoter, suggesting a
transcriptional mechanism. To our knowledge, this is the first time
that a dual mechanism for IL-10 function has been demonstrated. Studies
to elucidate the mechanisms underlying the inhibition of TNF-
production addressed the effect of IL-10 on the activation of p38
mitogen-activated protein kinase and NF-
B. However, these studies
could demonstrate no requirement for the inhibition of p38
mitogen-activated protein kinase or NF-B activation as potential
mechanisms. Overall, these results may explain the diversity previously
ascribed to the complex mechanisms of IL-10 anti-inflammatory
activity. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and other proinflammatory mediators. Mice
defective in IL-10 expression develop an inflammatory Crohn’s-like
disease and produce enhanced amounts of TNF- in response to LPS
(2). Furthermore, in murine models, many inflammatory
diseases are ameliorated by administration of exogenous IL-10 (reviewed
by Donnelly et al. in Ref. 3).
Despite considerable scientific and clinical interest, the molecular
pathways underlying IL-10 inhibition of TNF- expression remain
obscure. In human PBMC, IL-10 has been reported to suppress
TNF- gene transcription (4, 5), possibly by
inhibiting the activation of the transcription factor NF-B (6, 7). However, unlike the inhibition of TNF- gene
transcription (4, 5), the blockade of NF-B activation
does not appear to require IL-10-induced de novo protein synthesis
(6, 7). In contrast, studies performed in murine
macrophages have claimed that IL-10 acts through the
posttranscriptional mechanisms by destabilizing TNF- mRNA
(8, 9), or, more recently, by inhibiting gene translation
via blocking the activation of p38 mitogen-activated protein kinase
(MAPK)5
(10). A role for IL-10-induced de novo protein synthesis
has also been described in murine macrophages (9).
Furthermore, Riley et al. (11) have demonstrated the
absolute requirement of the transcription factor STAT-3 in mediating
the anti-inflammatory effects of IL-10. There appears to be no
simple explanation for the variety of mechanisms ascribed to
IL-10-mediated inhibition of TNF- expression except for the
different systems used. Thus, it may be possible that there are major
differences between cell systems in how IL-10 exerts its effect.
For understanding human physiology and disease, a most
appropriate system for the study of IL-10 effects is primary human
monocyte-macrophages. However, a major drawback in using these cells
for signaling studies is the inability to transfect them and introduce
transgenes. Recently, we have successfully overcome this problem using
adenoviral vectors (12, 13). In this study, we used this
approach to study IL-10 regulation of TNF- at the gene
level in primary human macrophages. In particular, adenoviral vectors
were constructed, incorporating luciferase reporter genes, under the
control of the TNF- promoter, with or without the 3'
untranslated region (3'UTR). Once introduced into primary human
macrophages, these reporter genes gave high levels of induction in
response to LPS or zymosan. This, to our knowledge, is the first direct
examination of TNF- gene function in primary human cells.
Moreover, this study showed that, depending on the length of exposure
of cells to the cytokine, IL-10 could inhibit TNF-
expression by either the 5' promoter region or the 3'UTR, suggesting
that both transcriptional and posttranscriptional mechanisms could be
involved in this single cell type. However, unlike previous studies in
other systems, IL-10 was unable to inhibit LPS-induced activation of
p38 MAPK and had only a minor effect on NF-B-induced
transcription.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human mononuclear cells were isolated from single donor plateletphoresis residues obtained from the North London Blood Transfusion Center (London, U.K.) by Ficoll-Hypaque centrifugation preceding monocyte separation in a Beckman JE6 elutriator (Beckman, High Wycombe, U.K.). Monocyte purity was routinely >85% when assessed by flow cytometry (14). The elutriated human monocytes were cultured at 1 x 106/ml in RPMI 1640 (BioWhittaker, Verviers, Belgium) with 25 mM HEPES and 2 mM L-glutamine supplemented with 10% (v/v) heat-inactivated FCS and 10 U/ml penicillin/streptavidin. To optimize infection, macrophages were derived from the monocytes by culturing the cells with M-CSF at 100 ng/ml (Genetics Institute, Boston, MA) for 48 h (13).
Plasmids
Human TNF- promoter (-1173 bp) with 3'UTR of the
human TNF- gene (pGL3-TNF--3'UTR), or without the
3'UTR (pGL3-TNF-) (15), were subcloned into
the pAdTrack vector (16) to generate pAdTrack-p5'3'UTR and
pAdTrack-p5'. KpnI/SalI fragments containing the
human TNF- promoter, the luciferase reporter gene, and the SV40 late
poly(A) signal were derived from pGL3-TNF- inserted into
KpnI/SalI sites of the AdTrack vector.
pAdTrack-p5'3'UTR was obtained by substituting a
XbaI/BamHI fragment containing the SV40 late
poly(A) signal in the pGL3-TNF- plasmid for 
1 kbp of
3'UTR amplified by PCR with corresponding primers: 3'UTR-F
(XbaI), aattctagaGGAGGACGAACATCCAAC; and
3'UTR-R(BamHI), aatGgATcCCCAGAGTTGGAAATTC. The
KpnI/SalI fragments were subsequently cloned into
the pAdTrack vector.
Adenoviral vectors and their propagation
The pAdEasy-1 adenoviral plasmid was provided by Prof. B.
Vogelstein (Howard Hughes Medical Institute, Baltimore, MD).
Recombinant viruses were generated by homologous recombination in
BJ5183 Escherichia coli transformed by heat-shock with 1
µg of each of the linearized PmeI pAdTrack
constructs and 100 ng of pAdEasy-1. Kanamycin-resistant positive
recombinant clones were selected and confirmed by restriction enzyme
digestion. Viral DNA was transfected into HEK 293 cells. Viruses were
purified by ultracentrifugation through two cesium chloride gradients,
as described in He et al. (16). Plaque assays were
performed by HEK 293 cells, exposing the cells to each virus for 1
h in serum-free DMEM (Life Technologies, Paisley, U.K.) and,
subsequently, overlaying the cells with an agarose mixture (1.5%
agarose, 2x DMEM with 4% FCS; v/v, 1/1) and incubated for 10–14 days
to determine viral titer (16). The IB encoding virus
was kindly provided by Dr. R. de Martin (University of Vienna,
Vienna, Austria) and adeno-NF-B luciferase reporter virus
(AdvNF-B-luc), previously described (17), was kindly
provided by Dr. P. McCray (University of Iowa, Iowa City, IA).
Infection and IL-10 treatment
Human macrophages were plated at a density of 2 x
105 cells/well in 96-well plates and exposed to
virus at the optimal multiplicity of infection (m.o.i.; 40:1 for Advp5'
and Advp5'3'UTR; 100:1 for Ad0 AdvIB; 200:1 for
AdvNK-B-luc) for 1 h in serum-free medium, followed by washing
and reculturing in growth medium with 2% (v/v) FCS for 24 h.
Infected cells were then stimulated with 10 ng/ml LPS (Salmonella
typhimurium; Sigma, Poole, U.K.) or 30 µg/ml zymosan (Sigma) for
4 h, unless otherwise stated, in the presence or absence of
IL-10.
Measurement of human TNF- production
TNF- levels were measured in cell supernatants by sandwich
ELISA as previously reported (18).
Luciferase assay
After stimulation, cells were washed once in PBS and lysed with 100 µl of chloramphenicol acetyltransferase lysis buffer (0.65% (v/v) Nonidet P-40, 10 mM Tris-HCL (pH 8), 0.1 mM EDTA (pH 8), 150 mM NaCl). Cell lysate (50 µl) was transferred to a luminometer cuvette strip and luciferase assay buffer (220 µl) was added. Luciferase activity was measured with a luminometer (Labsystems, Chicago, IL) by dispensing 30 µl luciferin (1.5 mM; Sigma) per assay point. Cell lysates were assayed for protein concentration by Bradford assay and luciferase activity was adjusted accordingly.
RNase protection assay
After M-CSF treatment, cells were plated at 2 x
106/well in a 12-well plate and infected, as
described above. In Advp5'- and Advp5'3'UTR-infected cells, luciferase
and GAPDH mRNAs were detected by RNase protection assay (RPA) by using
luciferase and GAPDH riboprobes, respectively. In parallel, TNF- and
GAPDH mRNAs were detected in Adv0-infected cells. Riboprobe vectors
were constructed as follows. A 352-bp HincII-XbaI
luciferase fragment was cloned from pGL3c (Promega, Madison, WI) into
pBluescript KS-digested EcoRV and XbaI. A 268-bp
TNF- gene fragment was amplified by PCR from human genomic DNA and
subcloned into the SpeI site of pBluescript
KS+ (kindly provided by Dr. A. Clark, Kennedy
Institute, London, U.K.). Riboprobe template constructs were linearized
by appropriate restriction enzyme and purified by phenol-chloroform
extraction and ethanol precipitation. Luciferase and GAPDH riboprobes
were synthesized using T7 RNA polymerase and TNF- riboprobe by using
T3 RNA polymerase (Boehringer Mannheim, Indianapolis, IN) in the
presence of 50 µCi of [-32P]UTP (800
mCi/mmol; Amersham Pharmacia Biotech, Little Chalfont, U.K.). The final
concentration of unlabeled UTP in the in vitro transcription reactions
was 12 µM, except in the case of luciferase, where it was 2.4 µM.
RPAs were conducted using the Direct Protect kit (Ambion, Austin, TX).
Under the hybridization conditions DNA-RNA heteroduplexes are not
detected. Protected RNA fragments were resolved by electrophoresis on
denaturing 6% polyacrylamide gels, quantified by phosphor imaging
(Fuji FLA-2000; Raytek Scientific, Sheffield, U.K.) and visualized by
autoradiography. Each experiment was performed twice and serial
dilutions of lysates were used to check that quantitations were within
the linear range of the assay.
Immunoprecipitation and in vitro kinase assays
MAPKs were immunoprecipitated from cleared cell lysates, as
described previously (19). In vitro kinase assays for p38
MAPK were performed using either His6-MAPKAPK-2 or
GST-ATF-2 as a substrate, c-Jun N-terminal kinase (JNK) assays were
performed using GST-ATF-2 as a substrate, and p42 MAPK assays
were performed using myelin basic protein (Sigma) as a substrate.
Immunoprecipitates were incubated with 30 µl kinase assay buffer (25
mM Tris (pH 7.5), 25 mM MgCl2, 25 mM
-glycerophosphate) containing 20 µM ATP and 0.5 µCi
[
-32P]ATP (Amersham Pharmacia Biotech) with
50 µg/ml appropriate substrate protein for 25 min at room
temperature. Reactions were terminated by the addition of gel sample
buffer and boiling for 5 min. All substrates were separated by
SDS-PAGE. Gels were dried and phosphorylated substrates were visualized
using a Fuji FLA-2000 phosphor imager and by autoradiography at
-70°C.
NF-B EMSAs
Following stimulation, cells were scraped into ice-cold PBS and
lysed in hypotonic lysis buffer (0.125% Nonidet P-40, 5 mM HEPES (pH
7.9), 10 mM KCl, 1.5 mM MgCl2), and nuclei were
harvested by centrifugation (13,000 x g for 30
s). Nuclear protein extracts were prepared by incubating the nuclei in
hypertonic extraction buffer (5 mM HEPES (pH 7.9), 25% glycerol, 500
mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA) for 2 h
with constant agitation. NF-B DNA binding activities were determined
by incubating 1–3 µg of each extract with
[-32P]ATP-labeled double-stranded NF-B
consensus oligonucleotide (Promega), followed by resolution on a 5%
(w/v) nondenaturing polyacrylamide gel. Gels were dried on to filter
paper and retarded DNA; protein complexes were visualized using
Hyperfilm MP (Amersham Pharmacia Biotech).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
reporter genes delivered
by adenoviral infection into primary human macrophages and demonstrate
the involvement of the 3'UTR in mRNA stability
The 5' promoter and 5' promoter-3'UTR constructs, described
previously (15), were incorporated into recombinant
adenoviruses (reporter viruses, Advp5' and Advp5'-3'UTR, respectively),
as previously described by He et al. (16). Primary human
macrophages were infected with these adenoviral constructs at a m.o.i.
of 40:1, as previous studies had indicated that this concentration
resulted in the successful infection of >90% of cells (Ref.
13 and data not shown). LPS activation of the reporter
gene resulted in a potent stimulation of both the 5' and 5'3'UTR
constructs, respectively (Fig. 1
). The
reporter gene also responded equally well to the yeast product, zymosan
(Fig. 1), an alternative stimulus of TNF- production with a similar
potency to LPS (20). A consistent finding in all
experiments was the lower absolute response of constructs containing
the 3'UTR. These data support the view, obtained from previous studies
in murine macrophage cell lines, that the 3'UTR is generally
suppressive to TNF- expression (21).
|
production reached a maximum 4 h after stimulation, and thereafter
decreased slowly with a t1/2 of
18.5 h (Fig. 2A). The
kinetics of luciferase activity from Advp5'- or Advp5'3'UTR-infected
LPS-stimulated human macrophages followed a similar profile to TNF-,
with maximum expression at 4 h and apparent
t1/2 estimated to be 23.5 and 12
h, respectively (Fig. 2, B and C). The shorter
half-life of luciferase, when under the additional control of the
3'UTR, might be expected from the overall destabilizing effect of this
element on TNF- mRNA (21, 22) and explain
why the absolute signals produced in the presence of the 3'UTR are
consistently lower (Fig. 1
). To confirm this, studies on luciferase
mRNA stability were performed and showed that the presence of the 3'UTR
does indeed increase the rate of decay of luciferase mRNA (Fig. 2D). In the presence of the 3'UTR the mRNA had a
t1/2 of 56 min; however, in the
absence of this element there was little decay (<15%) within the same
timeframe. Similar studies on TNF- mRNA indicate a
t1/2 of 30 min, similar to
luciferase mRNA from the 3'UTR constructs (Fig. 2D). One
might have expected that the half-life of the endogenous TNF-
protein would have been comparable to the 5'3'UTR rather than the 5'
construct. However, these data do not take into account potential
differences in the biological half-lives of the TNF- and luciferase
proteins, and one can only assume that the half-life of the endogenous
TNF- would be longer in the absence of the 3'UTR in humans, as shown
previously in mouse cells with deletions of the 3'UTR on the AU-rich
region of the 3'UTR (23).
|
production but has no effect on p38 MAPK activation
Adenovirus delivery of TNF--based reporter gene constructs to
primary human macrophages was then used to investigate what role the 5'
and 3' regions may play in the IL-10 inhibition of TNF- expression.
Adeno-reporter virus-infected cells were simultaneously treated with
LPS and various concentrations of IL-10 for 4 h, after which time
TNF- production and luciferase activities were assayed (Fig. 3). IL-10 inhibited TNF- expression to
a maximum of 80% at 10 ng/ml (Fig. 3). However, the responses of the
two reporter constructs were quite distinct. The 5' construct was only
weakly inhibited by IL-10 (10%), whereas the 5'3'UTR construct
showed a dose response profile similar to the endogenous TNF-,
although the maximum inhibition attained was less (60% at 10 ng/ml).
The IC50 for IL-10 on TNF- protein expression
was 0.2–0.3 ng/ml, compared with 2–3 ng/ml for the reporter gene.
However, if the concentration of half-maximal inhibition is calculated,
then the activity of IL-10 is similar for the endogenous gene (0.1
ng/ml) and the 5'3'UTR construct (0.2–0.3 ng/ml). This suggests that
aspects of the inhibitory activity of IL-10 on the reporter gene and
the endogenous gene are similar. The kinetics of IL-10 inhibition of
TNF- expression and luciferase expression were also compared using
the optimal concentration of 10 ng/ml (Fig. 4). Over the 2- to 24-h period
poststimulation, the expression of TNF- and the 5'3'UTR construct
gave very similar profiles with very little activation detected in the
presence of IL-10 (Fig. 4, A and B). The effect
of IL-10 on the 5' construct was again much weaker, with no significant
inhibition of luciferase activity over the time course (Fig. 4C). These data suggest that the major inhibitory effect of
IL-10 is mediated via the 3'UTR and that there appears to be little
effect on the transcription of the gene when IL-10 is administered at
the same time as LPS. To gain a further insight into the mechanism of
IL-10 activity, luciferase mRNA levels were analyzed by RPA. As shown
in Fig. 5, simultaneous addition of IL-10
caused a marked reduction of luciferase mRNA from the 5'3'UTR
construct, whereas there was only a marginal effect on mRNA from the 5'
construct. These data indicate that IL-10 causes a reduction in mRNA
levels via the 3'UTR. Data obtained with TNF- mRNA showed
similar results to the 5'3'UTR construct (Fig. 5).
|
|
|
expression, the data so far suggest that IL-10 mediates its activity at
this level. If this is so, IL-10 should still be able to inhibit
TNF- production, at least for a period, if added after LPS. Reporter
virus-infected macrophages were stimulated with LPS, IL-10 was added
for periods of up to 2 h postactivation, and the cells were
harvested at 4 h for assay. As expected, IL-10 had little effect
on the activity of the 5' construct, regardless of when it was added
(Fig. 6). The inhibitory effect of IL-10
on TNF- and the expression of the 5'3'UTR construct was maintained,
even if IL-10 was added 1 h after LPS activation, but was greatly
reduced if the cytokine was added 2 h postactivation (Fig. 6).
These data suggest that IL-10 does not inhibit the early events that
are involved in TNF- production (e.g., transcription) and instead
targets later events (e.g., posttranscriptional). These data support
the hypothesis that posttranscriptional control mediated via the 3'UTR
is the target of IL-10.
|
expression (24, 25) and has very
recently been shown to be inhibited by IL-10 in murine macrophages
(10), we investigated whether this kinase was a target for
IL-10 in human macrophages. As shown in Fig. 7, IL-10 was unable to inhibit
LPS-induced p38 MAPK activation. As the other related p42/44
extracellular signal-regulated kinase, MAPKs, and p54 JNK are also
involved in regulating TNF- expression, the effect of
IL-10 on these kinases was also studied. However, like p38 MAPK, there
was no inhibitory effect on the activation of these kinases (Fig. 7).
|
production
through the 5' promoter
As time of exposure to IL-10 could obviously have a bearing on its
function, this study was extended to investigate the effect of adding
IL-10 before LPS for periods of up to 24 h (Fig. 8). Preincubating macrophages with IL-10
for 12 h produced a modest increase in the inhibition of
endogenous TNF- production or luciferase activity from the 5'3'UTR
reporter gene, when compared with the effect of simultaneous addition
of LPS and IL-10 (Fig. 8). Further periods of preincubation, up to
24 h, did not elicit any major additional effect. However,
pre-exposure of cells to IL-10 had a profound effect on the expression
of the 5' reporter. Preincubation of macrophages with IL-10 for 12
h before LPS stimulus resulted in 50% inhibition of the 5' construct
that increased to nearly 70% when the preincubation period was
extended to 24 h (Fig. 8). This was compared with the 80%
inhibition of endogenous TNF- production. The effect of
preincubating macrophages for 24 h with different concentrations
of IL-10 was also examined. As shown in Fig. 9, LPS-induced TNF- protein expression
was inhibited to a maximum of 90% (10 ng/ml IL-10) with an
IC50 of 0.1 ng/ml, regardless of which
reporter construct had been infected into the macrophages. IL-10 also
inhibited the expression of the 5'3'UTR reporter to an identical degree
to the endogenous gene (Fig. 9B). However, in contrast to
data in Fig. 3A, preincubation for 24 h with IL-10 now
produced a dose-dependent inhibition of the 5' construct that showed a
maximum inhibition of 60% at 10 ng/ml (Fig. 9A). The
IC50 for IL-10 on the 5' construct was 5 ng/ml,
but this reduced to 0.5 ng/ml if the half-maximal inhibition was again
calculated. These data suggest that, in addition to posttranscriptional
regulation of the TNF- gene, IL-10 can also inhibit
transcription of the TNF- gene if cells are exposed to
this inhibitory factor for a sufficient period. Indeed, studies on mRNA
levels showed that preincubation for 24 h with IL-10 resulted in a
decrease in luciferase mRNA, regardless of the presence of the 3'UTR
(Fig. 5). However, we were unable to perform nuclear run-on experiments
to confirm an effect on transcription, as we cannot obtain sufficient
cells from a single donor to perform fully controlled experiments.
|
|
production is independent
of NF-B
An inhibitory mechanism that involves the 5' promoter region of
the TNF- gene suggest that IL-10 may be interfering with
the function of a transcription factor. As NF-B has been implicated
previously as a target for IL-10, the effect of the cytokine on the
activation of this transcription factor was investigated. As shown in
Fig. 10A, IL-10 had no
effect on the activation of NF-B by LPS, as measured by EMSA,
regardless of the length of the exposure to the cytokine. As would be
expected from this result, we also observed no effect of IL-10 on
IB degradation (data not shown). Next, using an adenovirus
encoding an NF-B-driven luciferase reporter gene previously
described by Sanlioglu et al. (17), the effect of IL-10 on
the transactivating activity of the transcription factor was also
examined. As shown in Fig. 10B, preincubation with IL-10 for
24 h had no effect on the NF-B transcriptional activity,
although there was a slight inhibition of the NF-B reporter gene
(20%) when IL-10 was added simultaneously with LPS. As expected,
the coinfection of macrophages with the IB-encoding adenovirus
(AdvIB) inhibited gene expression by >90%, whereas a control
virus had no effect. We extended this study further by addressing
whether a role for NF-B in TNF- expression was
essential for IL-10 inhibition. We have previously shown using
AdvIB that NF-B is not a requirement for zymosan-induced
TNF- production (20). In this study, similar data were
obtained with the reporter genes that showed that coinfection of the
cells with the reporter viruses and AdvIB resulted in an 80–90%
inhibition of the response of both constructs to LPS. In contrast, we
observed no significant inhibition in response to zymosan (Fig. 10C). A control virus Ad0 had no effect on responses to
either stimulus. However, IL-10 showed the same inhibitory profile to
zymosan-induced reporter gene activity as seen above with LPS, namely
inhibition via the 3'UTR, when cytokine and stimulus were added
simultaneously, and an effect on the 5' region only when cells were
preincubated with IL-10 (Fig. 10D). IL-10 inhibited
zymosan-induced production of TNF- protein to levels similar to
those obtained with the reporter genes, except, of course, in the case
of the t0 time point and the 5'
construct (Fig. 10D). These data support the conclusion that
IL-10-induced inhibition of TNF- expression is NF-B
independent.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
gene mediate the suppressive effect of IL-10. A
particular aspect to this study was the harnessing of our previously
successful experience of using adenovirus to deliver transgenes into
primary human monocytic cells (12, 13, 20, 26). Using this
approach, we were able to deliver, for the first time to our knowledge,
TNF- gene-based reporter constructs into primary human
macrophages and investigate TNF- gene regulation in a
system highly relevant to human pathology. Unlike previous studies, the
data showed that IL-10 apparently uses two independent mechanisms for
inhibiting TNF- expression, by targeting either the 5' promoter or
the 3'UTR.
The description of a potential posttranscriptional mechanism via the
3'UTR for the IL-10-mediated inhibition of TNF- production in human
cells is novel, as previous studies in human cells have implicated a
transcriptional target (4, 5, 7, 27). However, none of
these studies used the approach of analyzing gene function. These data
are in agreement with studies in murine macrophages, where a
posttranscriptional mechanism has been proposed (9, 28).
Previous studies on the 3'UTR have shown that this region has an
overall suppressive effect (21) on TNF-
expression (which this study has now confirmed in human macrophages).
However, although we were able to show that IL-10 was able to decrease
mRNA levels via the 3'UTR, it was not possible to confirm previous
findings in murine macrophages (9) that this effect was
mediated by destabilizing TNF- mRNA (data not shown). A
potential target for IL-10 in this context would be p38 MAPK, as this
kinase is involved in the posttranscriptional control of
TNF- expression (24). However, we were
unable to show any effect of IL-10 on the activation of p38 MAPK, the
related p54/JNK, or p42/44 MAPK. These results agree with previous
findings that IL-10 did not inhibit LPS-induced phosphorylation of p38
MAPK in human monocytes (3). A recent study has suggested
that cytokines and growth factors could affect mRNA splicing
(29). The luciferase reporter gene used in this work is,
of course, devoid of introns; however, the apparent similarity of the
responses of the endogenous TNF- and reporter genes to
IL-10 indicate that introns and mRNA splicing are not targets for IL-10
activity. Therefore, the mechanism by which IL-10 may inhibit
TNF- expression posttranscriptionally is still unclear. A
potential target is the AU-rich ARE regions found in the 3'UTRs of
TNF- and many other cytokine genes. These regions have
been implicated in the regulation of mRNA stability and turnover
(30), as well as in mRNA translation
(31, 32, 33, 34). The recent study in murine macrophages by
Kontoyiannis et al. (10) has identified this region as a
target for IL-10-mediated suppression of TNF- gene
translation by a mechanism involving inhibition of p38 MAPK. However,
IL-10 inhibition of TNF- in human macrophages appears to be grossly
different, as there is no inhibition of p38 MAPK. Also, as
TNF- mRNA levels are inhibited, this precludes any major
role for an effect on TNF- gene translation. The full
understanding of the mechanism for the posttranscriptional regulation
of TNF- by IL-10 in human cells will obviously require a more
extensive study and may require the discovery of as yet unknown
pathways involved in the general posttranscriptional control of
this gene.
The second inhibitory mechanism of IL-10 in primary human macrophages
required the 5' promoter region, suggesting an inhibition of gene
transcription. However, the amount of material available from
individual donors prevented any meaningful run-on experiments being
performed to confirm this. In contrast to the posttranscriptional
mechanisms, this inhibitory mechanism required a prolonged pre-exposure
to IL-10. The inference from these data would be that there is a
requirement for gradual changes in cell physiology, e.g., protein
synthesis, to mediate this effect of IL-10. A requirement for
IL-10-directed protein synthesis for the inhibition of LPS-induced
TNF- gene transcription is supported by previous studies
in human cells, showing that the protein synthesis inhibitor
cycloheximide inhibits IL-10 function (4, 5). However, so
far our own studies with cycloheximide have proved inconclusive,
possibly because the general inhibition of protein synthesis leads to
toxicity. It has been reported in human PBMC that IL-10 can suppress
the activation of the key proinflammatory transcription factor, NF-B
(7), possibly by inhibiting IB kinase activity
(6) or NF-B DNA binding (6). Previously,
we have also observed IL-10-mediated inhibition of LPS-induced NF-B
activity in the murine cell lines RAW 264.7 and the pre-B cell line
70Z/3. However, the concentrations required to achieve this effect were
high (an IC50 of 100 ng/ml), far in excess of
that required to inhibit TNF- expression (an
IC50 of 0.3 ng/ml) (28). However,
our studies in human macrophages have failed to show any effect of
IL-10 (10 ng/ml) on NF-B activation, as judged by EMSA. Additional
studies using an adeno-NF-B reporter virus showed that preincubation
with IL-10 had no effect on transcriptional function. The only effect
of IL-10 on NF-B function we observed was a 20% decrease in
LPS-induced NF-B reporter gene activity when IL-10 was added
simultaneously with LPS. This does not appear to account for a
mechanism that operates via the 3'UTR or the potent suppression of
IL-10 on TNF- expression. Moreover, our studies with zymosan show
that a NF-B-dependent mechanism of gene induction is not an
essential requirement for the IL-10 inhibition of TNF- expression.
The studies showing inhibition of NF-B reported that IL-10 inhibited
activation of the factor with either minimal (5 min) or no pre-exposure
of the cells to the cytokine before stimulus. This would not fit with
the observations in this study and the studies of others (4, 5) showing the requirement for prolonged exposure to IL-10
(4, 5), which indicated de novo protein synthesis was
required for IL-10 inhibition of TNF- gene transcription.
In summary, our data using primary human macrophages do not agree with
previous studies that IL-10 has a profound effect on NF-B activation
and that this is a mechanism by which IL-10 inhibits TNF-
expression. We can only conclude that, to some extent, the differences
in these results may be due to our use of primary cells rather than
cell lines and that changes in signaling mechanisms can occur between
such systems. Such a conclusion is supported by recent studies on
NF-B-inducing kinase. This kinase was proposed to play an essential
role in NF-B activation by many stimuli, including LPS, TNF-, and
IL-1, in cell lines (35). However, this kinase has
subsequently been shown to play no such role in response to these
stimuli in primary human or murine cells (36, 37, 38). Our
data indicate that another transcriptional mechanism is the target of
IL-10 and that the mechanism involved is more likely to be indirect,
probably requiring the expression of IL-10-induced proteins. The
identification of the transcriptional target for IL-10 may be dependent
on a greater understanding of the general mechanisms controlling
TNF- gene transcription.
The previous studies in human cells focusing on transcriptional
regulation have overlooked posttranscriptional mechanisms operating via
the 3'UTR. The advantage of this study is that, by correlating the
effect of IL-10 with different regions of the TNF-
promoter, it was possible to identify and delineate both mechanisms and
show, for the first time, that both mechanisms could coexist. Also, by
using the adenoviral system to undertake this study in primary cells,
any potential problems with performing studies in transformed cell
lines could be avoided. This report provides some key insights as to
why there has been much disagreement in the field of IL-10 function, by
showing that multiple mechanisms can coexist and that the nature of the
system studied has a major impact on the result.
|
Acknowledgments |
|---|
B virus, and Dr. Paul
McCray for the AdvNF-B-luc. |
Footnotes |
|---|
2 Current address: Laboratoire de Chimie Biologique, Unité Mixte de Recherche 8576 du Center National de Recherche Scientifique, Université des Sciences et Technologies de Lille, Villeneuve d’Ascq, France.
3 Current address: University of California Stein Clinical Research, La Jolla, CA 92093.
4 Address correspondence and reprint requests to Dr. Brian M. J. Foxwell, Kennedy Institute of Rheumatology Division, Imperial College Faculty of Medicine, 1 Aspenlea Road, London W6 8LH, U.K. E-mail address: b.foxwell{at}ic.ac.uk
5 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; 3'UTR, 3' untranslated region; m.o.i., multiplicity of infection; AdvNF-B-luc, adeno-NF-B luciferase reporter virus; JNK, c-Jun N-terminal kinase; RPA, RNase protection assay.
Received for publication October 4, 2001. Accepted for publication March 5, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B kinase activity and nuclear factor B DNA binding. J. Biol. Chem. 274:31868.B (NF B) activation in human monocytes: IL-10 and IL-4 suppress cytokine synthesis by different mechanisms. J. Biol. Chem. 270:9558. and interleukin-10. J. Biol. Chem. 267:23301.B inhibits both inflammatory and destructive mechanisms in rheumatoid synovium but spares anti-inflammatory mediators. Proc. Natl. Acad. Sci. USA 96:5668.B reveals that macrophage tumor necrosis factor production in rheumatoid arthritis is NF-B dependent. Proc. Natl. Acad. Sci. USA 95:8211. processing in RA synovial membrane cell cultures. J. Clin. Invest. 97:2833.[Medline]
B interactions at the distal tumor necrosis factor promoter region in human monocytes. J. Biol. Chem. 273:21178. secretion through IKK regulation of NF-B. J. Biol. Chem. 276:30188.B into human macrophages: lipopolysaccharide-induced, but not zymosan- induced, proinflammatory cytokines are inhibited, but IL-10 is NF-B independent. J. Immunol. 162:2939. mRNA stability by the mitogen-activated protein kinase p38 signalling cascade. FEBS Lett. 483:57.[Medline]
production in monocytes is phosphatidylinositol 3-kinase-dependent and nuclear factor-B-independent. J. Biol. Chem. 274:33455.B- in human monocytes. Shock 10:389.[Medline]
production is independent of its ability to inhibit NF B activity. Eur. J. Immunol. 28:1719.[Medline]
mRNA has an inhibitory effect on translation. Proc. Natl. Acad. Sci. USA 84:6030. mRNA. Gene 72:191.[Medline]
B induction by TNF, CD95 and IL-1. Nature 385:540.[Medline]
B-inducing kinase is dispensable for activation of NF-B in inflammatory settings but essential for lymphotoxin receptor activation of NF-B in primary human fibroblasts. J. Immunol. 167:5895. receptor-induced NF-B transcriptional activity in NIK-deficient mice. Science 291:2162.B-inducing kinase. Nat. Genet. 22:74.[Medline]This article has been cited by other articles:
|
|
 |
|
  B. Schaljo, F. Kratochvill, N. Gratz, I. Sadzak, I. Sauer, M. Hammer, C. Vogl, B. Strobl, M. Muller, P. J. Blackshear, et al. Tristetraprolin Is Required for Full Anti-Inflammatory Response of Murine Macrophages to IL-10 J. Immunol., July 15, 2009; 183(2): 1197 - 1206. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Michlewska, I. Dransfield, I. L. Megson, and A. G. Rossi Macrophage phagocytosis of apoptotic neutrophils is critically regulated by the opposing actions of pro-inflammatory and anti-inflammatory agents: key role for TNF-{alpha} FASEB J, March 1, 2009; 23(3): 844 - 854. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. E. Simmonds, F. V. Lali, T. Smallie, P. L. C. Small, and B. M. Foxwell Mycolactone Inhibits Monocyte Cytokine Production by a Posttranscriptional Mechanism J. Immunol., February 15, 2009; 182(4): 2194 - 2202. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Dagvadorj, Y. Naiki, G. Tumurkhuu, F. Hassan, S. Islam, N. Koide, I. Mori, T. Yoshida, and T. Yokochi Interleukin-10 inhibits tumor necrosis factor-{alpha} production in lipopolysaccharide-stimulated RAW 264.7 cells through reduced MyD88 expression Innate Immunity, April 1, 2008; 14(2): 109 - 115. [Abstract] [PDF]
|
|
|
|
 |
|
 C. D. Palmer, B. E. Mutch, S. Workman, J. P. McDaid, N. J. Horwood, and B. M. J. Foxwell Bmx tyrosine kinase regulates TLR4-induced IL-6 production in human macrophages independently of p38 MAPK and NF{kappa}B activity Blood, February 15, 2008; 111(4): 1781 - 1788. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. K. Weaver, E. Bohn, B. A. Judd, M. P. Gil, and R. D. Schreiber ABIN-3: a Molecular Basis for Species Divergence in Interleukin-10-Induced Anti-Inflammatory Actions Mol. Cell. Biol., July 1, 2007; 27(13): 4603 - 4616. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. M. Williams, U. Sarma, K. Willets, T. Smallie, F. Brennan, and B. M. J. Foxwell Expression of Constitutively Active STAT3 Can Replicate the Cytokine-suppressive Activity of Interleukin-10 in Human Primary Macrophages J. Biol. Chem., March 9, 2007; 282(10): 6965 - 6975. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Qin, C. A. Wilson, K. L. Roberts, B. J. Baker, X. Zhao, and E. N. Benveniste IL-10 Inhibits Lipopolysaccharide-Induced CD40 Gene Expression through Induction of Suppressor of Cytokine Signaling-3 J. Immunol., December 1, 2006; 177(11): 7761 - 7771. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Shahrara, C. C. Park, V. Temkin, J. W. Jarvis, M. V. Volin, and R. M. Pope RANTES Modulates TLR4-Induced Cytokine Secretion in Human Peripheral Blood Monocytes J. Immunol., October 15, 2006; 177(8): 5077 - 5087. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Rajasingh, E. Bord, C. Luedemann, J. Asai, H. Hamada, T. Thorne, G. Qin, D. Goukassian, Y. Zhu, D. W. Losordo, et al. IL-10-induced TNF-alpha mRNA destabilization is mediated via IL-10 suppression of p38 MAP kinase activation and inhibition of HuR expression FASEB J, October 1, 2006; 20(12): 2112 - 2114. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Drechsler, A. Dolganiuc, O. Norkina, L. Romics, W. Li, K. Kodys, F. H. Bach, P. Mandrekar, and G. Szabo Heme Oxygenase-1 Mediates the Anti-Inflammatory Effects of Acute Alcohol on IL-10 Induction Involving p38 MAPK Activation in Monocytes J. Immunol., August 15, 2006; 177(4): 2592 - 2600. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Sud, C. Bigbee, J. L. Flynn, and D. E. Kirschner Contribution of CD8+ T Cells to Control of Mycobacterium tuberculosis Infection J. Immunol., April 1, 2006; 176(7): 4296 - 4314. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Qasimi, A. Ming-Lum, A. Ghanipour, C. J. Ong, M. E. Cox, J. Ihle, N. Cacalano, A. Yoshimura, and A. L-F. Mui Divergent Mechanisms Utilized by SOCS3 to Mediate Interleukin-10 Inhibition of Tumor Necrosis Factor {alpha} and Nitric Oxide Production by Macrophages J. Biol. Chem., March 10, 2006; 281(10): 6316 - 6324. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kamimura, C. Viedt, A. Dalpke, M. E. Rosenfeld, N. Mackman, D. M. Cohen, E. Blessing, M. Preusch, C. M. Weber, J. Kreuzer, et al. Interleukin-10 Suppresses Tissue Factor Expression in Lipopolysaccharide-Stimulated Macrophages via Inhibition of Egr-1 and a Serum Response Element/MEK-ERK1/2 Pathway Circ. Res., August 19, 2005; 97(4): 305 - 313. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. J. Murray The primary mechanism of the IL-10-regulated antiinflammatory response is to selectively inhibit transcription PNAS, June 14, 2005; 102(24): 8686 - 8691. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Hoentjen, R. B. Sartor, M. Ozaki, and C. Jobin STAT3 regulates NF-{kappa}B recruitment to the IL-12p40 promoter in dendritic cells Blood, January 15, 2005; 105(2): 689 - 696. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Grutz New insights into the molecular mechanism of interleukin-10-mediated immunosuppression J. Leukoc. Biol., January 1, 2005; 77(1): 3 - 15. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Campbell, C. J. Ciesielski, A. E. Hunt, N. J. Horwood, J. T. Beech, L. A. Hayes, A. Denys, M. Feldmann, F. M. Brennan, and B. M. J. Foxwell A Novel Mechanism for TNF-{alpha} Regulation by p38 MAPK: Involvement of NF-{kappa}B with Implications for Therapy in Rheumatoid Arthritis J. Immunol., December 1, 2004; 173(11): 6928 - 6937. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. A. Ricchetti, L. M. Williams, and B. M. J. Foxwell Heme oxygenase 1 expression induced by IL-10 requires STAT-3 and phosphoinositol-3 kinase and is inhibited by lipopolysaccharide J. Leukoc. Biol., September 1, 2004; 76(3): 719 - 726. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Bhattacharyya, P. Sen, M. Wallet, B. Long, A. S. Baldwin Jr, and R. Tisch Immunoregulation of dendritic cells by IL-10 is mediated through suppression of the PI3K/Akt pathway and of I{kappa}B kinase activity Blood, August 15, 2004; 104(4): 1100 - 1109. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Veal, C.-L. Hsieh, S. Xiong, J. M. Mato, S. Lu, and H. Tsukamoto Inhibition of lipopolysaccharide-stimulated TNF-{alpha} promoter activity by S-adenosylmethionine and 5'-methylthioadenosine Am J Physiol Gastrointest Liver Physiol, August 1, 2004; 287(2): G352 - G362. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Nishiki, F. Hato, N. Kamata, E. Sakamoto, T. Hasegawa, A. Kimura-Eto, M. Hino, and S. Kitagawa Selective activation of STAT3 in human monocytes stimulated by G-CSF: implication in inhibition of LPS-induced TNF-{alpha} production Am J Physiol Cell Physiol, June 1, 2004; 286(6): C1302 - C1311. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Zhou, A. A. Nazarian, and S. T. Smale Interleukin-10 Inhibits Interleukin-12 p40 Gene Transcription by Targeting a Late Event in the Activation Pathway Mol. Cell. Biol., March 15, 2004; 24(6): 2385 - 2396. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Herrero, X. Hu, W. P. Li, S. Samuels, M. N. Sharif, S. Kotenko, and L. B. Ivashkiv Reprogramming of IL-10 Activity and Signaling by IFN-{gamma} J. Immunol., November 15, 2003; 171(10): 5034 - 5041. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Dai, S. Datta, M. Novotny, and T. A. Hamilton TGF{beta} inhibits LPS-induced chemokine mRNA stabilization Blood, August 15, 2003; 102(4): 1178 - 1185. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. J. Horwood, T. Mahon, J. P. McDaid, J. Campbell, H. Mano, F. M. Brennan, D. Webster, and B. M.J. Foxwell Bruton's Tyrosine Kinase Is Required For Lipopolysaccharide-induced Tumor Necrosis Factor {alpha} Production J. Exp. Med., June 16, 2003; 197(12): 1603 - 1611. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Biswas, S. Datta, J. D. Gupta, M. Novotny, J. Tebo, and T. A. Hamilton Regulation of Chemokine mRNA Stability by Lipopolysaccharide and IL-10 J. Immunol., June 15, 2003; 170(12): 6202 - 6208. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Gasche, P. Grundtner, P. Zwirn, W. Reinisch, S. H. Shaw, A. Zdanov, U. Sarma, L. M. Williams, B. M. Foxwell, and A. Gangl Novel Variants of the IL-10 Receptor 1 Affect Inhibition of Monocyte TNF-{alpha} Production J. Immunol., June 1, 2003; 170(11): 5578 - 5582. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Dolganiuc, K. Kodys, A. Kopasz, C. Marshall, T. Do, L. Romics Jr., P. Mandrekar, M. Zapp, and G. Szabo Hepatitis C Virus Core and Nonstructural Protein 3 Proteins Induce Pro- and Anti-inflammatory Cytokines and Inhibit Dendritic Cell Differentiation J. Immunol., June 1, 2003; 170(11): 5615 - 5624. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kiriakidis, E. Andreakos, C. Monaco, B. Foxwell, M. Feldmann, and E. Paleolog VEGF expression in human macrophages is NF-{kappa}B-dependent: studies using adenoviruses expressing the endogenous NF-{kappa}B inhibitor I{kappa}B{alpha} and a kinase-defective form of the I{kappa}B kinase 2 J. Cell Sci., February 15, 2003; 116(4): 665 - 674. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Blanquicett, G. Y. Gillespie, L. B. Nabors, C. R. Miller, S. Bharara, D. J. Buchsbaum, Robert. B. Diasio, and M. R. Johnson Induction of Thymidine Phosphorylase in Both Irradiated and Shielded, Contralateral Human U87MG Glioma Xenografts: Implications for a Dual Modality Treatment Using Capecitabine and Irradiation Mol. Cancer Ther., October 1, 2002; 1(12): 1139 - 1145. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Lang, D. Patel, J. J. Morris, R. L. Rutschman, and P. J. Murray Shaping Gene Expression in Activated and Resting Primary Macrophages by IL-10 J. Immunol., September 1, 2002; 169(5): 2253 - 2263. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
