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* Department of Infectious Diseases and
Hartwell Center for Biotechnology and Bioinformatics, St. Jude Children’s Research Hospital, Memphis, TN 38015
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6, and IL-12, chemokines, and
prostaglandins via blocking cyclooxygenase 2 expression
(1, 2). Because of these broad effects, IL-10 has elicited
considerable clinical interest to treat chronic inflammatory conditions
such as Crohn’s disease (3) and hepatitis C-induced
fibrosis (4).
No consensus has emerged as to how IL-10 inhibits production of
cytokines and chemokines from macrophages. Inhibition of TNF-
production by IL-10, for example, has been attributed to effects on
NF-
B activation (5), mitogen-activated protein
kinase (MAPK)4
signaling pathways (6), rate of transcription
(7), mRNA stability (8), translational
efficiency (6), cleavage from the membrane, and uptake via
TNF receptors (9) (for reviews, see Refs. 1
and 2). Also, conflicting results have been reported for
most of these effects. In contrast, genetic and biochemical analysis
has elucidated the membrane proximal events of IL-10 signaling. The
functional IL-10R consists of the ligand binding IL-10R1 and the
accessory subunit IL-10R2 (1, 2). Although IL-10R2 is
expressed on most cells and tissues, IL-10R1 is expressed on
hemopoietic cells and up-regulated on macrophages upon activation
(1, 2). This fact, combined with genetic evidence from a
variety of animal model systems, supports the notion that macrophages
are the primary target of IL-10 (10, 11). Binding of IL-10
to its receptor initiates signaling via the Janus kinase-STAT
pathway. Limited studies using fetal liver-derived Janus kinase
1-deficient macrophages (12) suggest this kinase is
essential for early IL-10 signaling. STAT3 plays a pivotal role because
the conditional inactivation of STAT3 in myeloid lineage cells results
in abrogated IL-10 responses of macrophages and development of chronic
enterocolitis similar to IL-10-deficient
(IL-10-/-) mice (11).
It is not known how IL-10 attenuates macrophage activation downstream
of STAT3. Experimental evidence suggests that new protein synthesis is
required for IL-10 to inhibit LPS-induced cytokine production, since
IL-10 does not appear to inhibit IL-12 p40 or TNF- expression in
LPS-stimulated macrophages when cycloheximide is added (8, 13). To date, few genes have been described to be induced by
IL-10. We reasoned that a gene expression screen for
IL-10-induced genes in macrophages should identify a range of targets
potentially involved in deactivation of macrophages. We took advantage
of macrophages from mice deficient in IL-10 or STAT3 and microarray
techniques to identify and validate genes induced and repressed by
IL-10 in resting and activated macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-10 and IL-4 were purchased from BD PharMingen (San Diego,
CA), LPS was obtained from Sigma-Aldrich (St. Louis, MO).
IL-10-/- mice (14) on a C57BL/6
background and controls were purchased from The Jackson Laboratory (Bar
Harbor, ME). STAT3flox/- and LysMcre breeding
pairs were a gift from I. Förster (Technical University of
Munich, Munich, Germany). Peritoneal-derived macrophages (PDM) and bone
marrow-derived macrophages (BMDM) were isolated as previously described
(10). Detection of IL-10 and TNF- in culture
supernatants was by ELISA using Ab pairs from BD PharMingen.
Affymetrix gene chip analysis
IL-10-/- BMDM were stimulated with IL-10
(10 ng/ml), LPS (100 ng/ml), or IL-10 + LPS for 45 min or 3 h. For
both timepoints, two completely independent experiments were performed.
Total RNA was prepared using TRIzol (Life Technologies, Gaithersburg,
MD), processed, and hybridized to MG-U74Av2 gene chips according to
Affymetrix protocols (Santa Clara, CA). Chips were scanned and
analyzed using Affymetrix Microarray Suitev4.0 software. Sample loading
and variations in staining were standardized by scaling the average of
the fluorescent intensities of all genes on an array to constant target
intensity (2500) for all arrays used. The signal intensity for each
gene was calculated as the average intensity difference, represented by
(
(PM-MM)/(number of probe pairs)), where PM and MM denote
perfect-match and mismatch probes.
Data analysis
Data sets of 12,488 probe sets per array were compared using Microsoft Excel (Microsoft, Redmond, WA) and Spotfire software. To avoid negative ratios, average intensity differences < 5 were first set to 5 (15). Data were normalized by mean using the untreated sample as the baseline within each experiment. To identify differentially expressed genes, we excluded all genes from the analysis that were scored absent in the test sample (for up-regulated genes) or absent in the baseline sample (for down-regulated genes) in one or both experiments. Fold changes were calculated separately for both experiments as the ratio of normalized average intensity difference (test sample) divided by normalized average intensity difference (baseline sample). Thresholds were set for fold change (2-fold and greater unless otherwise indicated) and absolute difference (at least 500) between normalized average intensity differences. Consistency between experiments varied between samples depending on treatment and timepoint. For example, of the genes induced >3-fold by IL-10 in the first experiment, 50.0% for the 45 min and 55.6% for the 3 h timepoint were up-regulated at least 2-fold in the second experiment. When LPS treatment was compared with untreated, the corresponding numbers were 87.7 and 78.0%, respectively. To minimize the number of false-positives, only those genes that reproducibly met all the thresholds described above in both independent experiments were considered differentially expressed.
Northern blotting and real-time quantitative RT-PCR
For Northern analysis, 10–15 µg total RNA were separated on
1% formaldehyde-agarose gels and blotted onto Hybond N (Amersham,
Piscataway, NJ). Probes were prepared from plasmids containing either
full-length cDNAs (IL-12p40, GAPDH, IL-1R antagonist (IL-1ra),
suppressor of cytokine signaling (SOCS)3, junB, growth arrest and DNA
damage (GADD)45
, GADD45) or expressed sequence tags (ESTs)
(NFIL-3, JE/monocyte chemoattractant protein (MCP)-1, tumor progression
locus (Tpl)-2). For real-time quantitative RT-PCR, 1 µg of
total RNA was reverse-transcribed using Superscript II (Life
Technologies) and a mix of random hexamer and oligo(dT) primers.
Primers were designed using PrimerExpress software (Applied Biosystems,
Foster City, CA). For 
-actin, TNF-, arginase-1 and arginase-2,
internal TaqMan probes were designed and included in the PCR. Sequences
of primers and probes are available from the authors upon request. For
all other target genes, the SYBR-green master mix was used to detect
accumulation of PCR product during cycling on the SDS7700 (Applied
Biosystems). Expression of target genes was normalized to -actin and
displayed as fold-change relative to the untreated 45-min sample used
as the calibrator (set to 1).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We reasoned that experimental parameters, especially the choice of
macrophage type and timepoints after stimulation, would be critical
determinants of the results obtained by gene expression profiling. We
analyzed the influence of these variables on inhibition of TNF-
production by IL-10 to establish conditions optimal to identify
IL-10-induced genes and investigate its overall impact on the
transcriptional response to LPS. In response to LPS, both BMDM and PDM
rapidly produced TNF- that was inhibited by IL-10 as expected (Fig. 1
). However, PDM made more TNF- and
showed a stronger inhibitory effect of IL-10. This difference was
inversely correlated with much higher production of IL-10 in BMDM,
suggesting that endogenous IL-10 blunted TNF- production. We
concluded that a system devoid of endogenous IL-10 would be
advantageous for uncovering the full spectrum of IL-10-induced changes
in gene expression. BMDM from IL-10-/- mice
produced high amounts of TNF- and were fully responsive to
inhibition by IL-10. TNF- production was down-regulated by IL-10 as
early as 1 h after stimulation, but this effect was increased at
2–4 h. Therefore, we chose two timepoints for global expression
profiling. After 45 min, mRNAs encoding IL-10-induced inhibitors of
inflammatory responses should be present at detectable levels. A
timepoint of 3 h was chosen to visualize later IL-10-induced
differences in the transcriptional response to LPS. This later
timepoint is expected to contain both directly and indirectly
IL-10-induced genes that may play a role in the anti-inflammatory
effects of this cytokine.
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Stimulation with LPS induced a >2-fold induction of 149 genes
after 45 min, and of 402 genes after 3 h, whereas 40 and 752 genes
were repressed >2-fold by LPS at these timepoints (Fig. 2). This substantial reprogramming of
gene expression is consistent with other studies examining the impact
of Toll-like receptor (TLR) ligands and pathogens on the macrophage
transcriptome (16, 17, 18). In contrast, treatment with IL-10
induced 26 and 37 and repressed 25 and 22 genes >2-fold after 45 min
and 3 h, respectively (Fig. 2). A comparison of gene expression
profiles in macrophages treated with LPS or IL-10 + LPS showed few
differences after 45 min, but after 3 h the expression of 194
genes differed >2-fold (125 induced, 69 repressed by addition of
IL-10) (Fig. 2).
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). As
expected, a number of cytokines and chemokines previously described to
be down-regulated by IL-10 (1, 2) were repressed in the
microarray analysis confirming the robustness of this system. In
addition, many transcripts belonging to various functional categories
were identified as suppressed by IL-10 for the first time (Fig. 3). It
should be noted that 104 of the 259 LPS-induced genes were not
regulated by IL-10 in both independent experiments.
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. Results from the 3 h timepoints
are listed in Table II.
Of the known target genes of IL-10 (1, 2), a significant
fraction was confirmed in the microarray analysis including IL-1ra,
CD32, CCR5, CCR1, scavenger receptor, and arginase-2. Some genes
reported to be induced by IL-10 were not confirmed because they were
not represented on the microarray (tissue inhibitor of
metalloproteinase 1), not regulated (CD14, TNFR2, CD16, and
CD64), or did not pass one or more of the thresholds in the stringent
analysis criteria (p19INK4D, fMLPR). One of the
genes most strongly induced by IL-10 was SOCS3, which has been reported
as a target gene common to IL-10 and LPS, and implicated in inhibition
of macrophage responses to IFN- (19, 20, 21). Around half
of the genes induced by IL-10 were independently up-regulated in
response to LPS (Table I). For some of these genes, the combination of
IL-10 and LPS resulted in synergistic induction (IL-1ra, Bcl-3,
metallothionein-2, NFIL-3). Some genes were up-regulated specifically
by IL-10 (e.g., GADD45, connexin 43, CCAAT/enhancer
binding protein (C/EBP) 
, IL-4R) or the combination of IL-10 +
LPS, but not by LPS (e.g., B-ATF) (Table I). Independent confirmation
of the results was obtained by Northern analysis or real-time
quantitative RT-PCR for a subset of 15 genes scored as induced by the
microarray analysis (Fig. 4).
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B and MAPK pathways,
both essential for the initiation and propagation of proinflammatory
gene expression (5, 6). We found several genes regulated
by IL-10 that have been implicated in controlling NF-B activation,
such as metallothionein-2 (22) and Bcl-3
(23). Other IL-10 targets are involved in MAPK pathway
regulation, e.g., GADD45 (24) and Tpl-2, a MAPK kinase
kinase (25) that can also activate IB kinases
(26). Mice deficient in Tpl-2 have a selective defect in
TNF- production due to impaired nucleocytoplasmic transport of the
TNF- mRNA (27). Because IL-10 inhibits TNF-
production, it is not clear whether induction of Tpl-2 by IL-10 is
related to the anti-inflammatory effect of IL-10. To resolve this
question, it will be important to test macrophages from mice lacking
Tpl-2 for IL-10-induced deactivation. Further, several genes for
transcriptional regulators were also induced by IL-10. One example of
this group is the basic region leucine zipper transcription
factor NFIL-3 that can function as transcriptional activator
(28) or repressor (29). STAT3 dependence of gene induction and repression by IL-10
Macrophages deficient in STAT3 are hyperresponsive to LPS and fail
to respond to IL-10 with down-regulation of cytokines and inhibition of
proliferation (11). To determine whether STAT3 is required
for the induction and repression of the IL-10-dependent target genes
described in this study, we used BMDM deficient in STAT3
(11). Expression of selected genes was analyzed by
Northern blotting (Fig. 5A) or
real-time quantitative RT-PCR (Fig. 5B). Inhibition by IL-10
of LPS-induced expression of IL-12 p40, TNF-, and JE/MCP-1 was
dependent on functional STAT3. The reduced levels of these mRNAs in the
LPS-stimulated wild-type BMDM compared with
IL-10-/- and STAT3-/-
BMDM illustrates the attenuation of inflammatory responses by
endogenous IL-10. For the IL-10-induced targets identified in this
study including Tpl-2, NFIL-3, GADD45, IL-1ra,
1-microglobulin/bikunin precursor (AMBP), protein C
receptor, MT-2, and B-ATF, the absence of STAT3 completely
abrogated inducibility by IL-10 and reduced induction of IL-4R,
Bcl-3, and connexin 43 mRNAs (Fig. 5). This side-by-side comparison of
wild-type macrophages with cells incapable of producing or responding
to IL-10 also revealed that the expression of Tpl-2, AMBP, IL-4R,
and B-ATF after stimulation with LPS is mediated indirectly by IL-10 or
other factors signaling via STAT3 (Fig. 5).
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by IL-10 correlates with increased
IL-4-dependent expression of arginase-1 (Fig. 6)
The finding of increased IL-4R expression in macrophages
treated with IL-10 (Tables I
and II, Figs. 4 and 5), suggested an
enhanced sensitivity to IL-4 as a functional consequence of exposure to
IL-10. Importantly, both cytokines are known to promote "alternative
activation" of macrophages, a functional state characterized by high
phagocytic capacity but a reduced ability to kill pathogens
(30). A hallmark of "alternative activation" is high
arginase activity, which competes with inducible NO synthase for
L-arginine, the common substrate of both enzymes (31, 32), and can be due to the expression of either one of two
isoforms. Arginase-2 was shown to be induced by LPS (33),
and we found in this study that IL-10 synergized with LPS in increasing
arginase-2 expression (Fig. 4, Table II). Expression of arginase-1 is
induced by the Th2 cytokines IL-4 and IL-13 (34) in a
STAT6-dependent manner (32). IL-10 strongly synergizes
with IL-4 to induce arginase-1 (35), but the mechanistic
basis for this effect has been unknown. Therefore, we
evaluated whether the magnitude of arginase-1 induction after IL-4
stimulation is linked to the IL-10-mediated increase in IL-4R
expression we observed in the array analysis. IL-4 strongly induced
arginase-1 expression, which was further increased 10-fold by addition
of IL-10 (Fig. 6), correlating with the increased levels of IL-4R in
macrophages treated with IL-10 alone or in combination with IL-4. In
contrast, LPS down-regulated IL-4R expression and potently inhibited
expression of arginase-1 in response to IL-4 (Fig. 6). Combined
addition of IL-10 and LPS restored high level IL-4R expression and
brought back synergistic induction of arginase-1 in macrophages exposed
also to IL-4. Taken together, increased expression of the IL-4R may
represent the basis for the synergistic effect of IL-10 on IL-4-induced
arginase-1 expression in macrophages.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Genes induced by IL-10
We found that IL-10 induced a highly restricted number of genes in resting macrophages. In LPS-activated macrophages, IL-10 induced a different set of genes. It is tempting to speculate that key anti-inflammatory genes may be represented in this latter group because the effects of IL-10 on macrophages are most clearly demonstrated with concomitant activation. Two independent signals from the IL-10R and a TLR may be necessary to engender the full range of anti-inflammatory mechanisms.
Several genes regulated by IL-10 with or without concomitant LPS
stimulation warrant further investigation for their potential
role in IL-10 signaling. One obvious candidate is SOCS3, a member
of the SOCS family that inhibits cytokine receptor signaling by
binding phosphotyrosine residues on key signaling molecules and
targeting them for destruction via its ubiquitin E3 ligase activity
(36). SOCS3 binds to gp130 (37, 38), the
signaling component of IL-6 family cytokine receptors
(39), but may have additional targets, as overexpression
studies have shown that it can inhibit IFN- signaling
(20). It has even been speculated that IL-10-induced SOCS3
might inhibit LPS-induced p38 MAPK signaling and thereby interfere with
TNF- mRNA translation (6). The question of whether
SOCS3 indeed plays a role in the control of macrophage activation by
IL-10 could best be answered using SOCS3-deficient macrophages. Because
SOCS3-deficient embryos die in mid-gestation (40), we are
in the process of generating radiation chimeras to perform such
experiments in the near future. Other IL-10 targets also give potential
clues to the anti-inflammatory effects of IL-10. Among these, three
genes encoding proteins involved in MAPK and related pathways,
GADD45, GADD45, and Tpl-2, suggest that a focus of IL-10
signaling research should be on these pathways. The role of IL-10 in
regulating MAPK and related pathways is presently a controversial topic
and requires clarification at the molecular level (1, 6, 41).
Global effects of IL-10 on proinflammatory gene expression induced by LPS
We found that IL-10 regulates a large number of LPS-induced genes.
Our study confirmed the inhibition by IL-10 of many previously reported
proinflammatory gene products (Fig. 3) and extended this finding at the
genomic level with the identification of numerous new IL-10-repressed
genes. Although IL-10 repressed the expression of a large fraction of
LPS-induced genes, a further fraction (
40%) remained unchanged.
This result suggests two important interpretations that contribute to
understanding the anti-inflammatory effects of IL-10. First, the
large number of LPS-induced genes inhibited by IL-10 suggests a common
mechanism is operative. It is unlikely that IL-10 induces a different
mediator for each inflammatory target. We favor the interpretation that
IL-10 regulates a limited group of gene products (transcriptionally
and/or posttranscriptionally) that subsequently regulate the
inflammatory targets. Second, it is unlikely that IL-10 targets more
global cell processes such as transcriptional initiation, because of
the large fraction of LPS-induced genes unaffected by the addition of
IL-10. Therefore, the IL-10-induced anti-inflammatory mechanism is
specific enough to target a fraction of LPS-induced genes while leaving
others unaffected.
STAT3 is essential for all observed effects of IL-10
Previous work has suggested that STAT3 is crucial for IL-10
signaling (11, 42). This is most clearly shown in mice
lacking STAT3 in macrophages and neutrophils which have a strikingly
similar phenotype to IL-10-deficient mice (11).
Other work using dominant-negative versions of STAT3 in macrophage cell
lines suggested that IL-10 signals via STAT3-dependent and -independent
pathways (43). Using STAT3-deficient macrophages, we show
that all IL-10-induced genes tested require STAT3 and that inhibition
of gene expression of proinflammatory targets also requires STAT3
signaling (Fig. 5). Although we cannot rule out some STAT3-independent
effects for IL-10, our results suggest that STAT3 is essential for
most, if not all, IL-10 signaling.
IL-10 controls macrophage arginase expression in response to IL-4 and LPS
In addition to identifying candidate mediators of IL-10's
deactivating effects, we also expected to find genes affecting
macrophage function in other ways. The up-regulation of IL-4R
expression by IL-10 in a STAT3-dependent manner caught our attention,
because it implied a possibly enhanced responsiveness to IL-4 as a
functional consequence of exposure to IL-10. In fact, we observed that
opposing changes in IL-4R expression induced by IL-10 or LPS in
IL-10-deficient macrophages were linked to corresponding changes in the
expression level of arginase-1 in response to IL-4 (Fig. 6). These
observations offer a mechanistic explanation for the previously
described synergistic induction of arginase-1 expression by IL-4 and
IL-10 (35). Further, the microarray experiments also
showed that expression of the extrahepatic isoform arginase-2 in
LPS-stimulated macrophages (33) is controlled by IL-10
(Table II, Fig. 4). Because the ability to make IL-10 also determines
expression of arginase-1 in response to LPS (Fig. 6) or the combination
of TNF- and IFN- (34), IL-10 increases total
arginase levels in macrophages in multiple ways.
Concluding remarks
Certain caveats are evident in a microarray study of this nature.
The most significant are the timepoints chosen for data analysis. In
this study, the two timepoints chosen were based upon the
well-recognized effects of IL-10 on proinflammatory mediator
production. Thus, we focused on 45 min and 3 h as a representative
window where the expression of TNF- and several other cytokines and
chemokines is substantially reduced in in vitro macrophage culture and
in in vivo models where mice are challenged with TLR agonists.
However, it is clear that IL-10 can have later effects that may be
mediated by distinct mechanisms (41) and even have
proinflammatory effects (44). A second caveat is
that we can only observe changes in mRNA levels and IL-10 may induce a
plethora of cellular changes at the proteome level that also contribute
to its anti-inflammatory effects. Despite these limitations, our
study has revealed several aspects of IL-10 function not previously
appreciated. Understanding the rules that govern the IL-10-mediated
shaping of the macrophage transcriptome and its subsequent influence on
the proteome will provide insights into the endogenous
anti-inflammatory response.
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Acknowledgments |
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Footnotes |
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2 Current address: Boling Center, 711 Jefferson Avenue, No. 415, Memphis, TN 38163.
3 Address correspondence and reprint requests to Dr. Peter J. Murray, Department of Infectious Diseases, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105. E-mail address: peter.murray{at}stjude.org
4 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; PDM, peritoneal-derived macrophages; BMDM, bone marrow-derived macrophages; IL-1ra, IL-1R antagonist; SOCS, suppressor of cytokine signaling; GADD, growth arrest and DNA damage; EST, expressed sequence tag; JE/MCP, JE/monocyte chemoattractant protein; Tpl, tumor progression locus; TLR, Toll-like receptor; C/EBP, CCAAT/enhancer binding protein; AMBP, 1-microglobulin/bikunin precursor.
Received for publication April 8, 2002. Accepted for publication June 18, 2002.
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 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]
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K. J. Staples, T. Smallie, L. M. Williams, A. Foey, B. Burke, B. M. J. Foxwell, and L. Ziegler-Heitbrock IL-10 Induces IL-10 in Primary Human Monocyte-Derived Macrophages via the Transcription Factor Stat3 J. Immunol., April 15, 2007; 178(8): 4779 - 4785. [Abstract] [Full Text] [PDF]
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 G. Trinchieri Interleukin-10 production by effector T cells: Th1 cells show self control J. Exp. Med., February 19, 2007; 204(2): 239 - 243. [Abstract] [Full Text] [PDF]
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J. Li, D. K. Pritchard, X. Wang, D. R. Park, R. E. Bumgarner, S. M. Schwartz, and W. C. Liles cDNA microarray analysis reveals fundamental differences in the expression profiles of primary human monocytes, monocyte-derived macrophages, and alveolar macrophages J. Leukoc. Biol., January 1, 2007; 81(1): 328 - 335. [Abstract] [Full Text] [PDF]
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J. P. Edwards, X. Zhang, K. A. Frauwirth, and D. M. Mosser Biochemical and functional characterization of three activated macrophage populations J. Leukoc. Biol., December 1, 2006; 80(6): 1298 - 1307. [Abstract] [Full Text] [PDF]
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R. Lang, M. Hammer, and J. Mages DUSP Meet Immunology: Dual Specificity MAPK Phosphatases in Control of the Inflammatory Response J. Immunol., December 1, 2006; 177(11): 7497 - 7504. [Abstract] [Full Text] [PDF]
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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]
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K. C. El Kasmi, J. Holst, M. Coffre, L. Mielke, A. de Pauw, N. Lhocine, A. M. Smith, R. Rutschman, D. Kaushal, Y. Shen, et al. General Nature of the STAT3-Activated Anti-Inflammatory Response J. Immunol., December 1, 2006; 177(11): 7880 - 7888. [Abstract] [Full Text] [PDF]
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A. D. Panopoulos, L. Zhang, J. W. Snow, D. M. Jones, A. M. Smith, K. C. El Kasmi, F. Liu, M. A. Goldsmith, D. C. Link, P. J. Murray, et al. STAT3 governs distinct pathways in emergency granulopoiesis and mature neutrophils Blood, December 1, 2006; 108(12): 3682 - 3690. [Abstract] [Full Text] [PDF]
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M. F. Tomczak, S. E. Erdman, A. Davidson, Y. Y. Wang, P. R. Nambiar, A. B. Rogers, B. Rickman, D. Luchetti, J. G. Fox, and B. H. Horwitz Inhibition of Helicobacter hepaticus-Induced Colitis by IL-10 Requires the p50/p105 Subunit of NF-{kappa}B J. Immunol., November 15, 2006; 177(10): 7332 - 7339. [Abstract] [Full Text] [PDF]
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 M. Groeneweg, E. Kanters, M. N. Vergouwe, H. Duerink, G. Kraal, M. H. Hofker, and M. P. J. de Winther Lipopolysaccharide-induced gene expression in murine macrophages is enhanced by prior exposure to oxLDL J. Lipid Res., October 1, 2006; 47(10): 2259 - 2267. [Abstract] [Full Text] [PDF]
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X. Zhu, M. S. Chang, R. C. Hsueh, R. Taussig, K. D. Smith, M. I. Simon, and S. Choi Dual Ligand Stimulation of RAW 264.7 Cells Uncovers Feedback Mechanisms That Regulate TLR-Mediated Gene Expression J. Immunol., October 1, 2006; 177(7): 4299 - 4310. [Abstract] [Full Text] [PDF]
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D. P. Bailey, M. Kashyap, L. A. Bouton, P. J. Murray, and J. J. Ryan Interleukin-10 induces apoptosis in developing mast cells and macrophages J. Leukoc. Biol., September 1, 2006; 80(3): 581 - 589. [Abstract] [Full Text] [PDF]
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 C. J. Orihuela, S. Fillon, S. H. Smith-Sielicki, K. C. El Kasmi, G. Gao, K. Soulis, A. Patil, P. J. Murray, and E. I. Tuomanen Cell Wall-Mediated Neuronal Damage in Early Sepsis Infect. Immun., July 1, 2006; 74(7): 3783 - 3789. [Abstract] [Full Text] [PDF]
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 K. B. Holven, B. Halvorsen, V. Bjerkeli, J. K. Damas, K. Retterstol, L. Morkrid, L. Ose, P. Aukrust, and M. S. Nenseter Impaired Inhibitory Effect of Interleukin-10 on the Balance Between Matrix Metalloproteinase-9 and Its Inhibitor in Mononuclear Cells From Hyperhomocysteinemic Subjects Stroke, July 1, 2006; 37(7): 1731 - 1736. [Abstract] [Full Text] [PDF]
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R. L. Chelvarajan, Y. Liu, D. Popa, M. L. Getchell, T. V. Getchell, A. J. Stromberg, and S. Bondada Molecular basis of age-associated cytokine dysregulation in LPS-stimulated macrophages J. Leukoc. Biol., June 1, 2006; 79(6): 1314 - 1327. [Abstract] [Full Text] [PDF]
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A. Doni, M. Michela, B. Bottazzi, G. Peri, S. Valentino, N. Polentarutti, C. Garlanda, and A. Mantovani Regulation of PTX3, a key component of humoral innate immunity in human dendritic cells: stimulation by IL-10 and inhibition by IFN-{gamma} J. Leukoc. Biol., April 1, 2006; 79(4): 797 - 802. [Abstract] [Full Text] [PDF]
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M. Hammer, J. Mages, H. Dietrich, A. Servatius, N. Howells, A. C.B. Cato, and R. Lang Dual specificity phosphatase 1 (DUSP1) regulates a subset of LPS-induced genes and protects mice from lethal endotoxin shock J. Exp. Med., January 23, 2006; 203(1): 15 - 20. [Abstract] [Full Text] [PDF]
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M. F. Tomczak, M. Gadjeva, Y. Y. Wang, K. Brown, I. Maroulakou, P. N. Tsichlis, S. E. Erdman, J. G. Fox, and B. H. Horwitz Defective Activation of ERK in Macrophages Lacking the p50/p105 Subunit of NF-{kappa}B Is Responsible for Elevated Expression of IL-12 p40 Observed after Challenge with Helicobacter hepaticus J. Immunol., January 15, 2006; 176(2): 1244 - 1251. [Abstract] [Full Text] [PDF]
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L. Liu, E. Tran, Y. Zhao, Y. Huang, R. Flavell, and B. Lu Gadd45{beta} and Gadd45{gamma} are critical for regulating autoimmunity J. Exp. Med., November 21, 2005; 202(10): 1341 - 1348. [Abstract] [Full Text] [PDF]
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 Y. Zhang, X.-W. Wang, D. Jelovac, T. Nakanishi, M.-h. Yu, D. Akinmade, O. Goloubeva, D. D. Ross, A. Brodie, and A. W. Hamburger The ErbB3-binding protein Ebp1 suppresses androgen receptor-mediated gene transcription and tumorigenesis of prostate cancer cells PNAS, July 12, 2005; 102(28): 9890 - 9895. [Abstract] [Full Text] [PDF]
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R. D. Stout, C. Jiang, B. Matta, I. Tietzel, S. K. Watkins, and J. Suttles Macrophages Sequentially Change Their Functional Phenotype in Response to Changes in Microenvironmental Influences J. Immunol., July 1, 2005; 175(1): 342 - 349. [Abstract] [Full Text] [PDF]
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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]
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B. A. Butcher, L. Kim, A. D. Panopoulos, S. S. Watowich, P. J. Murray, and E. Y. Denkers Cutting Edge: IL-10-Independent STAT3 Activation by Toxoplasma gondii Mediates Suppression of IL-12 and TNF-{alpha} in Host Macrophages J. Immunol., March 15, 2005; 174(6): 3148 - 3152. [Abstract] [Full Text] [PDF]
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T. Hirotani, P. Y. Lee, H. Kuwata, M. Yamamoto, M. Matsumoto, I. Kawase, S. Akira, and K. Takeda The Nuclear I{kappa}B Protein I{kappa}BNS Selectively Inhibits Lipopolysaccharide-Induced IL-6 Production in Macrophages of the Colonic Lamina Propria J. Immunol., March 15, 2005; 174(6): 3650 - 3657. [Abstract] [Full Text] [PDF]
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C. A. Ogden, J. D. Pound, B. K. Batth, S. Owens, I. Johannessen, K. Wood, and C. D. Gregory Enhanced Apoptotic Cell Clearance Capacity and B Cell Survival Factor Production by IL-10-Activated Macrophages: Implications for Burkitt's Lymphoma J. Immunol., March 1, 2005; 174(5): 3015 - 3023. [Abstract] [Full Text] [PDF]
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S. L. Pull, J. M. Doherty, J. C. Mills, J. I. Gordon, and T. S. Stappenbeck Activated macrophages are an adaptive element of the colonic epithelial progenitor niche necessary for regenerative responses to injury PNAS, January 4, 2005; 102(1): 99 - 104. [Abstract] [Full Text] [PDF]
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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]
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 S. Gingras, E. Parganas, A. de Pauw, J. N. Ihle, and P. J. Murray Re-examination of the Role of Suppressor of Cytokine Signaling 1 (SOCS1) in the Regulation of Toll-like Receptor Signaling J. Biol. Chem., December 24, 2004; 279(52): 54702 - 54707. [Abstract] [Full Text] [PDF]
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R. D. Stout and J. Suttles Functional plasticity of macrophages: reversible adaptation to changing microenvironments J. Leukoc. Biol., September 1, 2004; 76(3): 509 - 513. [Abstract] [Full Text] [PDF]
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V. S. Carl, J. K. Gautam, L. D. Comeau, and M. F. Smith Jr Role of endogenous IL-10 in LPS-induced STAT3 activation and IL-1 receptor antagonist gene expression J. Leukoc. Biol., September 1, 2004; 76(3): 735 - 742. [Abstract] [Full Text] [PDF]
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M. J. Raftery, D. Wieland, S. Gronewald, A. A. Kraus, T. Giese, and G. Schonrich Shaping Phenotype, Function, and Survival of Dendritic Cells by Cytomegalovirus-Encoded IL-10 J. Immunol., September 1, 2004; 173(5): 3383 - 3391. [Abstract] [Full Text] [PDF]
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R. P. Donnelly, F. Sheikh, S. V. Kotenko, and H. Dickensheets The expanded family of class II cytokines that share the IL-10 receptor-2 (IL-10R2) chain J. Leukoc. Biol., August 1, 2004; 76(2): 314 - 321. [Abstract] [Full Text] [PDF]
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A.-L. Pauleau, R. Rutschman, R. Lang, A. Pernis, S. S. Watowich, and P. J. Murray Enhancer-Mediated Control of Macrophage-Specific Arginase I Expression J. Immunol., June 15, 2004; 172(12): 7565 - 7573. [Abstract] [Full Text] [PDF]
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P. H. Correll, A. C. Morrison, and M. A. Lutz Receptor tyrosine kinases and the regulation of macrophage activation J. Leukoc. Biol., May 1, 2004; 75(5): 731 - 737. [Full Text] [PDF]
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P. Kropf, M. A. Freudenberg, M. Modolell, H. P. Price, S. Herath, S. Antoniazi, C. Galanos, D. F. Smith, and I. Muller Toll-Like Receptor 4 Contributes to Efficient Control of Infection with the Protozoan Parasite Leishmania major Infect. Immun., April 1, 2004; 72(4): 1920 - 1928. [Abstract] [Full Text] [PDF]
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N. E. Rodriguez, H. K. Chang, and M. E. Wilson Novel Program of Macrophage Gene Expression Induced by Phagocytosis of Leishmania chagasi Infect. Immun., April 1, 2004; 72(4): 2111 - 2122. [Abstract] [Full Text] [PDF]
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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]
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S. Fernandez, P. Jose, M. G. Avdiushko, A. M. Kaplan, and D. A. Cohen Inhibition of IL-10 Receptor Function in Alveolar Macrophages by Toll-Like Receptor Agonists J. Immunol., February 15, 2004; 172(4): 2613 - 2620. [Abstract] [Full Text] [PDF]
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A. C. Morrison, C. B. Wilson, M. Ray, and P. H. Correll Macrophage-Stimulating Protein, the Ligand for the Stem Cell-Derived Tyrosine Kinase/RON Receptor Tyrosine Kinase, Inhibits IL-12 Production by Primary Peritoneal Macrophages Stimulated with IFN-{gamma} and Lipopolysaccharide J. Immunol., February 1, 2004; 172(3): 1825 - 1832. [Abstract] [Full Text] [PDF]
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L. Williams, L. Bradley, A. Smith, and B. Foxwell Signal Transducer and Activator of Transcription 3 Is the Dominant Mediator of the Anti-Inflammatory Effects of IL-10 in Human Macrophages J. Immunol., January 1, 2004; 172(1): 567 - 576. [Abstract] [Full Text] [PDF]
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H. Kuwata, Y. Watanabe, H. Miyoshi, M. Yamamoto, T. Kaisho, K. Takeda, and S. Akira IL-10-inducible Bcl-3 negatively regulates LPS-induced TNF-{alpha} production in macrophages Blood, December 1, 2003; 102(12): 4123 - 4129. [Abstract] [Full Text] [PDF]
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A.-L. Pauleau and P. J. Murray Role of Nod2 in the Response of Macrophages to Toll-Like Receptor Agonists Mol. Cell. Biol., November 1, 2003; 23(21): 7531 - 7539. [Abstract] [Full Text] [PDF]
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J. A. Willment, H.-H. Lin, D. M. Reid, P. R. Taylor, D. L. Williams, S. Y. C. Wong, S. Gordon, and G. D. Brown Dectin-1 Expression and Function Are Enhanced on Alternatively Activated and GM-CSF-Treated Macrophages and Are Negatively Regulated by IL-10, Dexamethasone, and Lipopolysaccharide J. Immunol., November 1, 2003; 171(9): 4569 - 4573. [Abstract] [Full Text] [PDF]
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 J. H. Von der Thusen, J. Kuiper, T. J. C. Van Berkel, and E. A. L. Biessen Interleukins in Atherosclerosis: Molecular Pathways and Therapeutic Potential Pharmacol. Rev., March 1, 2003; 55(1): 133 - 166. [Abstract] [Full Text] [PDF]
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) or not (
) to macrophages,
followed by stimulation with LPS (100 ng/ml) for the indicated time.
Cytokine levels in the supernatant were measured by ELISA. In the case
of TNF-
). Total RNA was prepared
3 h and 8 h after addition of IL-4. After reverse
transcription, cDNA was subjected to real-time quantitative RT-PCR for