The Nuclear I?B Protein I?BNS Selectively Inhibits
Lipopolysaccharide-Induced IL-6 Production in Macrophages
of the Colonic Lamina Propria1
Tomonori Hirotani,*†Pui Y. Lee,*¶Hirotaka Kuwata,§Masahiro Yamamoto,*‡
Makoto Matsumoto,§Ichiro Kawase,†Shizuo Akira,*‡and Kiyoshi Takeda2§
Macrophages play an important role in the pathogenesis of chronic colitis. However, it remains unknown how macrophages
residing in the colonic lamina propria are regulated. We characterized colonic lamina proprial CD11b-positive cells (CLPM?).
CLPM? of wild-type mice, but not IL-10-deficient mice, displayed hyporesponsiveness to TLR stimulation in terms of cytokine
production and costimulatory molecule expression. We compared CLPM? gene expression profiles of wild-type mice with IL-
10-deficient mice, and identified genes that are selectively expressed in wild-type CLPM?. These genes included nuclear I?B
proteins such as Bcl-3 and I?BNS. Because Bcl-3 has been shown to specifically inhibit LPS-induced TNF-? production, we
analyzed the role of I?BNS in macrophages. Lentiviral introduction of I?BNS resulted in impaired LPS-induced IL-6 production,
but not TNF-? production in the murine macrophage cell line RAW264.7. I?BNS expression led to constitutive and intense DNA
binding of NF-?B p50/p50 homodimers. I?BNS was recruited to the IL-6 promoter, but not to the TNF-? promoter, together with
p50. Furthermore, small interference RNA-mediated reduction in I?BNS expression in RAW264.7 cells resulted in increased
LPS-induced production of IL-6, but not TNF-?. Thus, I?BNS selectively suppresses LPS-induced IL-6 production in macro-
phages. This study established that nuclear I?B proteins differentially regulate LPS-induced inflammatory cytokine production in
macrophages. The Journal of Immunology, 2005, 174: 3650–3657.
stood (1). A number of animal models of mucosal inflammation
have been developed to analyze the pathogenesis of IBD (2, 3). In
the course of analyzing these models, many types of cells includ-
ing T cells, B cells, and epithelial cells have been shown to con-
tribute to the pathogenesis of colitis. Among these cell popula-
tions, T cells have been shown to possess effector and regulatory
functions in the development of chronic colitis. Both CD4?Th1
and Th2 cells are critically involved in mucosal immunity as ef-
fector cells, and disrupting the balance of Th1/Th2 polarization
leads to the development of chronic mucosal inflammation (4).
Crohn’s disease is considered to be a Th1-dependent inflammatory
disease, whereas ulcerative colitis is a Th2-dependent disease (3).
nflammatory bowel diseases (IBD)3including Crohn’s dis-
ease and ulcerative colitis are chronic immune-mediated dis-
orders for which pathogenesis and etiology are poorly under-
In addition, regulatory T cells, such as CD25?CD4?T cells, TGF-
?-producing Th3 cells, and IL-10-producing type 1 regulatory T
cells, all have regulatory functions in prevention of chronic mu-
cosal inflammation and even in amelioration of established colitis
(5, 6). B cells also have regulatory functions in mucosal inflam-
mation observed in TCR-?-deficient mice (7). Thus, important
roles of adaptive immunity comprising T cells and B cells in mu-
cosal inflammation are well characterized.
However, recent accumulating evidence demonstrates that in-
nate immunity plays crucial roles in controlling Ag-specific adap-
tive immunity (8–11). Accordingly, the involvement of innate im-
munity in the development of chronic colitis has been proposed.
Abnormal activation of innate immune cells has been shown to
initiate the development of chronic colitis in mice lacking Stat3
specifically in macrophages and neutrophils (12). The phenotype
observed in the Stat3 mutant mice was very reminiscent of that
observed in IL-10-deficient mice, indicating that major target cells
of IL-10 in suppressing chronic mucosal inflammation are cells of
macrophage lineage (12–14). The mechanisms by which abnormal
activity of innate immune cells leads to the development of chronic
colitis were further analyzed. In the absence of Stat3, innate im-
mune cells showed increased levels of inflammatory cytokine pro-
duction through TLR, which are essential for the recognition of
microbial components in innate immune cells. Among these cyto-
kines, IL-12p40 is responsible for the exaggerated Th1 cell devel-
opment and thereby induces Th1-dependent chronic colitis (15).
Thus, critical involvement of TLR-dependent activation of innate
immunity has clearly been shown in triggering chronic mucosal
inflammation. In addition to TLR-mediated activation of innate
immunity, NOD2, which is responsible for TLR-independent rec-
ognition of microbial components, has been implicated in the
pathogenesis of Crohn’s disease in human (16–18). Thus, mole-
cules critically involved in innate immune responses, such as TLRs
*Department of Host Defense, Research Institute for Microbial Diseases and†De-
partment of Molecular Medicine, Graduate School of Medicine, Osaka University,
and‡Exploratory Research for Advanced Technology, Japan Science and Technology
Agency, Osaka, Japan;§Department of Molecular Genetics, Medical Institute of Bio-
regulation, Kyushu University, Fukuoka, Japan; and¶Department of Medicine, Di-
vision of Rheumatology and Clinical Immunology, University of Florida, Gainesville,
Received for publication October 5, 2004. Accepted for publication January 11, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by grants from the Special Coordination Funds of the
Ministry of Education, Culture, Sports, Science and Technology.
2Address correspondence and reprint requests to Dr. Kiyoshi Takeda, Department of
Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, 3-1-1
3Abbreviations used in this paper: IBD, inflammatory bowel disease; CLPM?, co-
lonic lamina proprial CD11b-positive cell; ChIP, chromatin immunoprecipitation;
siRNA, small interference RNA.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00
and NOD2, are associated with the pathogenesis of colitis. How-
ever, it remains unclear how activities of these innate immune cell
populations are regulated in the intestinal mucosa.
In this study, we isolated CD11b-positive cells from the colonic
lamina propria (CLPM?), and analyzed their responsiveness to
TLR ligands. CLPM? of wild-type mice, but not IL-10-deficient
mice, showed hyporesponsiveness to TLR ligands. Therefore, we
compared CLPM? gene expression profiles in wild-type mice with
IL-10-deficient mice, which led to identification of genes that are
specifically expressed in CLPM? of wild-type mice, but not in
CLPM? of IL-10-deficient mice or wild-type peritoneal macro-
phages. We further analyzed whether these gene products are ca-
pable of inhibiting TLR-mediated responses in macrophages, and
found that a member of the I?B family of proteins, I?BNS, inhibits
LPS-induced IL-6 production in macrophages.
Materials and Methods
Reagents and cell culture
LPS from Escherichia coli (O55:B5) was purchased from Sigma-Aldrich.
The mouse macrophage cell line (RAW264.7) and human embryonic kid-
ney 293T cells were cultured in DMEM supplemented with 10% FBS, 100
?g/ml streptomycin, and 10 U/ml penicillin G. Mouse peritoneal macro-
phages were collected by peritoneal lavage with HBSS at 3 days after i.p.
injection of 2 ml of 4% sterile thioglycolate into 8- to 12-wk-old mice.
Peritoneal macrophages were cultured in RPMI 1640 medium with 10%
FBS, 100 ?g/ml streptomycin, and 10 U/ml penicillin G.
C57BL/6 mice were purchased from the Central Laboratory of Experimen-
tal Animals (Tokyo, Japan). IL-10-deficient mice were purchased from The
Jackson Laboratory. All experiments using these mice were approved by
and performed according to the guidelines of the animal ethics committee
of Kyushu University and Osaka University.
Isolation of CLPM?
CLPM? was isolated using a protocol modified from an EDTA perfusion
method (19). Mice were anesthetized and their peritoneal and pleural cav-
ities were opened for systemic perfusion from left ventricle with 15 ml of
HBSS containing 20 mM EDTA. Following perfusion, colons were re-
moved, cut into pieces of 2?3 cm in length, resuspended in HBSS, and
shaken with a microbead beater (Biospec Products) at 5000 rpm for 50 s to
remove epithelial cells. The colon pieces were then washed with RPMI
1640, mechanically minced and resuspended in RPMI 1640 supplemented
with 10% FBS, 2 mg/ml collagenase type II (Invitrogen Life Technolo-
gies), 1 mg/ml dispase (Invitrogen Life Technologies), 15 mg/ml DNase
(Boehringer), 100 ?g/ml streptomycin, and 10 U/ml penicillin G for 30
min at 37°C in a shaking incubator. After filtration of digested tissue with
40-?m nylon mesh, isolated cells were washed with PBS and CD11b-
positive cells were purified using MACS selection system using CD11b
MicroBeads (Miltenyi Biotec) following manufacturer’s instructions.
Measurement of inflammatory cytokines
The cells (5 ? 104) were cultured in 96-well plates with 10 or 100 ng/ml
LPS for 24 h. The concentrations of TNF-?, IL-6, and IL-12p40 in the
culture supernatants were determined by ELISA according to the manu-
facturer’s instructions (Genzyme Techne).
Single cell suspension of colonic lamina propria was stained with PE-
conjugated anti-CD11b Ab (BD Pharmingen) and biotin-conjugated anti-
TLR4/MD-2 Ab (eBioscience), followed by FITC-conjugated streptavidin.
Stained cells were analyzed on a FACSCalibur (BD Biosciences).
Total RNA from wild-type or IL-10-deficient lamina proprial CD11b-pos-
itive cells was extracted with an RNeasy kit (Qiagen), followed by mRNA
purification with an Oligotex mRNA kit (Amersham Pharmacia Biotech).
Double-stranded cDNA was synthesized from 1 ?g of mRNA with the
SuperScript Choice System (Invitrogen Life Technologies) primed with
T7-oligo(dT) 24 primer. These cDNA were used to prepare biotin-labeled
cRNA by an in vitro transcription reaction using T7 RNA polymerase in
the presence of biotinylated ribonucleotides, according to the manufactur-
er’s protocol (Enzo Diagnostics). The cRNA products were purified using
an RNeasy kit (Qiagen), fragmented, and hybridized to Affymetrix Murine
Genome U74Av2, Bv2 and Cv2 microarray chips, according to the man-
ufacturer’s protocol (Affymetrix). The hybridized chips were stained,
washed, and scanned with a GeneArray scanner (Affymetrix).
Total RNA (1 ?g) was primed with random hexamers, followed by reverse
transcription with Superscript II (Invitrogen Life Technologies). PCR anal-
ysis was performed using recombinant TaqDNA polymerase (Takara
Shuzo). Conditions for the reactions were 30 s of denaturation step at 94°C,
30 s of annealing step at 60°C, and 1 min of elongation step at 72°C for
25–30 cycles. Specific primers used were: I?BNS, sense 5?-GCTGTATC
CTGAGCCTTCCCTGTC-3? and antisense 5?-GCTCAGCAGGTCTTC
CACAATCAG-3?; I?B?, sense 5?-GCTCAACCTGGCTTACTTCTAC
GG-3? and antisense 5?-CGGAAGCCTTCTGCTTGTTGCTTC-3?; Bcl-3,
sense 5?-GATGCCCATTTACTCTACCCCGAC-3? and antisense 5?-GC
GTGCTGGATCTCCTG-3? and antisense 5?-GCTCCCTCTAAGCAAAT
CACACCG-3?; macrophage scavenger receptor 2, sense 5?GGTGCTGG
AAACAGCTCTTGGAC-3? and antisense 5?-GCTCAGCAGGTCTTCCA
CAATCAG-3?; ?-actin, sense 5?-CTATGTGGGTGACGAGGCCCAGAG-3?
and antisense 5?-GGGTACATGGTGGTACCACCAGAC-3?.
RAW264.7 cells and murine peritoneal macrophages were treated with 10
ng/ml IL-10 (Genzyme) for 1, 2, 4, or 6 h. Total RNA was isolated with
TRIzol (Invitrogen Life Technologies) and treated with DNaseI (Promega).
Reverse transcription was performed using MMLV Reverse Transcriptase
(Promega) and oligo(dT) primers (Promega). Finally these solutions were
directly used as templates for PCR. Quantitative real-time PCR was per-
formed on an ABI 7000 sequence detection system (Applied Biosystems)
using TaqMan universal PCR Master Mix (Applied Biosystems), as pre-
viously described (20). TaqMan probes mix for I?BNS was purchased
from Applied Biosystems. All data were normalized to EF1-? expression
in the same cDNA set.
Lentiviral introduction of I?BNS into macrophages
The lentiviral vector, CSII-EF-MCS-IRES-hrGFP (cPPT-containing SIN
vector plasmid with multiple cloning sites for cDNA insertion followed by
the IRES-GFP sequence under the control of the EF-1? promoter), was
used to generate CSII-EF-I?BNS. Woodchuck hepatitis virus posttransla-
tional regulatory element was ligated at the 3? end of GFP. The lentiviral
vectors were cotransfected into 293T cells with pMDLg/pRRE (packaging
plasmid), pRSV-Rev (Rev expression plasmid), and pMD.G (VSV-G ex-
pression plasmid). Infectious lentiviruses in the culture supernatants were
harvested at 48 h after transfection. RAW cells (5 ? 105) were cultured
with the lentiviruses for 24 h, and then the culture medium was replaced.
After 48 h, the cells expressing human recombinant GFP were sorted by
FACSVantage SE (BD Biosciences).
Northern blot analysis
The cells were stimulated with 100 ng/ml LPS. Total RNA was extracted
using TRIzol reagent (Invitrogen Life Technologies), electrophoresed,
transferred to a nylon membrane, and hybridized with cDNA probe.
Western blot analysis
Cells (2 ? 106) were lysed with lysis buffer containing with 20 mM Tris-
HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, and Complete Mini
(Roche). The lysates were separated on SDS-PAGE and transferred to
polyvinylidene fluoride membrane. The membranes were incubated with
anti-Flag M2 Ab (Sigma-Aldrich), anti-I?B? Ab, anti-ERK Ab, anti-p38
Ab (Santa Cruz Biotechnology), anti-phospho-p38 Ab, and anti-phospho-
ERK Ab (Cell Signaling Technology). Bound Abs were detected with an
ECL system (PerkinElmer).
The cell lysates were precleared with protein G-Sepharose beads (Amer-
sham Pharmacia Biotech) and then incubated with protein G-Sepharose
beads together with anti-Flag M2 Ab, anti-p50 Ab, and anti-p65 Ab (Santa
Cruz Biotechnology). Immunoprecipitates were separated on SDS-PAGE,
transferred to polyvinylidene fluoride membrane, and incubated anti-Flag
M2 Ab, anti-p50 Ab, or anti-p65 Ab. Bound Abs were visualized with an
ECL system (PerkinElmer).
3651 The Journal of Immunology
The cells were stimulated with 100 ng/ml LPS for 30 or 60 min. Then,
nuclear proteins were extracted and incubated with an end-labeled, double-
stranded oligonucleotide containing a NF-?B binding site on the IL-6 pro-
moter in 25 ?l of binding buffer (10 mM HEPES-KOH, (pH 7.8), 50 mM
KCl, 1 mM EDTA, (pH 8.0), 5 mM MgCl2, and 10% glycerol) for 20 min
at room temperature and loaded onto a native 5% polyacrylamide gel. The
DNA-protein complexes were visualized by autoradiography. The speci-
ficities of the shifted bands were determined by adding Abs specific for p65
and p50 (Santa Cruz Biotechnology).
RAW264.7 cells (1 ? 105) were transiently transfected with a total 0.5 ?g
of expression vector, and 100 ng of IL-6 promoter-luciferase construct (21)
or TNF-? promoter-luciferase construct (22) using a Superfect transfection
reagent (Qiagen). After 24 h, cells were treated with or without 10 ng/ml
LPS for 6 h. The luciferase activity was measured using the dual-luciferase
reporter assay system (Promega). The Renilla-luciferase reporter gene (20
ng) was used as an internal control.
Chromatin immunoprecipitation (ChIP) was performed essentially with a
described protocol (Upstate Biotechnology). In brief, RAW cells were
stimulated with 100 ng/ml LPS for 1 or 2 h, and then fixed with formal-
dehyde for 10 min. The cells were lysed, sheared by sonication, and in-
cubated overnight with specific Ab followed by incubation with protein
A-agarose saturated with salmon sperm DNA (Upstate Biotechnology).
Precipitated DNA was analyzed by quantitative PCR (35 cycles) using
primers 5?-ACTAGCCAGGAGGGAGAACAGAAACTC-3? and 5?-CA
CAAGCAGGAATGAGAAGAGGCTGAG-3? for the TNF-? promoter
and 5?-TAGCAGCAGGTCCAACTGTGCTATCTG-3? and 5?-AAGC
CTCCGACTTGTGAAGTGGTATAG-3? for the IL-6 promoter.
In another experiment, peritoneal macrophages from wild-type mice and
Stat3 mutant mice were pretreated with 10 ng/ml IL-10 for 18 h, then
stimulated with 100 ng/ml LPS for 1 h, and used for ChIP assay.
RAW cells (4 ? 106) were transfected with 500 pmol of dsRNA using
Nucleofector (Amaxa). The target small interference RNA (siRNA), 5?-
GUGCAGAUGUUACUGCAAAA-3?, was designed and produced by
Dharmacon. The control siRNA was purchased from Dharmacon (catalog
Characterization of CD11b-positive cells in the colonic lamina
To analyze the function of colonic macrophages, we first isolated
CLPM? according to procedures described in Materials and Meth-
ods. Flow cytometric analysis showed that 30–40% of CLPM?
also expressed CD11c, indicating the presence of both macroph-
age-lineage cells and dendritic cell-lineage cells in the population.
Using highly purified (over 97% purity) CLPM?, we analyzed
their response to TLR ligands such as LPS and CpG DNA. We first
stimulated CD11b-positive cells from the spleen and colonic lam-
ina propria with LPS or CpG DNA, and analyzed for inflammatory
cytokine production (Fig. 1A). CD11b-positive cells from the
spleen produced significant amounts of TNF-?, IL-6, IL-12p40,
and IL-10 in response to LPS or CpG DNA. However, TLR li-
gand-induced increase in production of TNF-?, IL-6, and IL-
12p40 was not observed in CLPM?, although IL-6 was produced
in the absence of stimulation. In addition, IL-10 production was
constitutively observed in CLPM?. We next analyzed TLR ligand-
induced augmentation of surface molecules such as CD40, CD80,
CD86, and MHC class II (Fig. 1B). These surface molecules were
not up-regulated in response to LPS or CpG DNA in CLPM?.
the colonic lamina propria, then stimulated with 100 ng/ml LPS or 10 nM CpG DNA for 24 h. Concentrations of TNF-?, IL-6, IL-12p40, and IL-10 in
the culture supernatants were measured by ELISA. N.D., Not detected. B, The cells were also analyzed for surface expression of CD40, CD80, CD86, and
MHC class II by flow cytometry. C, Colonic lamina proprial cells were stained with anti-CD11b and anti-TLR4/MD-2 Abs. D, CLPM? were isolated from
IL-10-deficient mice, in which chronic colitis was already developed, and were analyzed for LPS-induced production of TNF-?, IL-6, and IL-12p40 by
ELISA. E, CLPM? were isolated from 4- to 5-wk-old Stat3 mutant mice, in which chronic colitis was not developed yet, and analyzed for LPS-induced
production of TNF-?, IL-6, and IL-12p40 by ELISA. F, CLPM? isolated from 4- to 5-wk-old wild-type and Stat3 mutant mice were analyzed for surface
expression of CD40, CD80, CD86, and MHC class II by flow cytometry.
Characterization of CD11b-positive cells in the colonic lamina propria (CLPM?). A, CD11b-positive cells were isolated from the spleen and
3652I?BNS INHIBITS IL-6 PRODUCTION IN MACROPHAGES
Thus, CLPM? were refractory to TLR ligands in terms of inflamma-
tory cytokine production and costimulatory molecule expression.
Surface expression of TLR4-MD-2 complex on CLPM? was ob-
served (Fig. 1C). Therefore, the hyporesponsiveness to TLR ligands
was not due to the lack of TLR4 expression in CLPM? (Fig. 1C).
We next isolated CLPM? from IL-10-deficient mice, in which
chronic colitis has already developed, and analyzed for inflamma-
tory cytokine production in response to TLR ligands (Fig. 1D).
Although CLPM? from wild-type mice did not show LPS-induced
production of TNF-? and IL-6, CLPM? from IL-10-deficient mice
produced small amounts of TNF-? and IL-6 even when cultured
with media alone, and the production was robustly enhanced in
response to LPS. CLPM? from mice lacking Stat3 in macrophage
(Stat3 mutant mice) also showed increased TNF-? and IL-6 pro-
duction in response to LPS (data not shown). Even in CLPM?
from young (4- to 5-wk-old) IL-10-deficient or Stat3 mutant mice,
which have not developed colitis yet, LPS stimulation resulted in
increased production of TNF-? and IL-6, indicating that enhanced
production of inflammatory cytokines was not due to environmen-
tal effects, but intrinsic to CLPM? themselves (Fig. 1E). Surface
expression of CD40, CD80, CD86, and MHC class II was up-
regulated in CLPM? from young Stat3 mutant mice (Fig. 1F).
Thus, CLPM? from IL-10-deficient or Stat3 mutant mice showed
enhanced inflammatory response even before colitis was devel-
oped. These findings suggest that under normal conditions,
CLPM? become tolerant to TLR ligand stimulation, and failure to
establish tolerance correlates with the development of chronic
Identification of genes that are specifically expressed in CLPM?
In the next set of experiments, we tried to reveal the mechanisms
for differential responses to TLR ligands seen in CLPM? of wild-
type and IL-10-deficient mice. DNA microarray analysis using
mRNA purified from CLPM? of wild-type mice and IL-10-defi-
cient mice led to identification of several genes that are selectively
expressed in wild-type CLPM?, but not in IL-10-deficient
CLPM? (data not shown). These genes include I?BNS, Bcl-3,
macrophage scavenger receptor 2, and CD163. RT-PCR analysis
confirmed that these genes were expressed in wild-type CLPM?,
but not in IL-10-deficient CLPM? or wild-type peritoneal CD11b-
positive cells (Fig. 2A). CD163 is a member of the scavenger re-
ceptor cysteine-rich superfamily, and was shown to be an IL-10-
inducible gene in monocytes/macrophages (23–25). Bcl-3 has been
shown to be induced by IL-10 in macrophages and is responsible
for suppression of LPS-induced TNF-? production (22). In addi-
tion to Bcl-3, I?BNS was selectively expressed in wild-type
CLPM?. Like Bcl-3, I?BNS is a member of the nucleus-localized
I?B family proteins bearing ankyrin-repeats (26). We analyzed
whether I?BNS expression is induced by IL-10 in the RAW mac-
rophage cell line and peritoneal macrophages. Real-time RT-PCR
analysis showed that I?BNS mRNA was induced within 1 h of
IL-10 treatment in both RAW cells and peritoneal macrophages,
indicating that like Bcl-3, I?BNS is an IL-10-inducible gene in
these cells (Fig. 2, B and C). Because Bcl-3 was shown to inhibit
LPS-induced TNF-? production and I?BNS is structurally related
to Bcl-3, we decided to analyze the role of I?BNS in macrophages.
I?BNS inhibits IL-6 production in macrophages
To analyze the role of I?BNS in macrophages, we introduced
I?BNS together with GFP into RAW264.7 cells using a lentiviral
vector system (22, 27). A lentiviral vector containing GFP alone
was used as control in all experiments. RAW264.7 cells were in-
fected with lentivirus, and after 2 days of culture, GFP-positive
cells were isolated by FACS sorting. Following stimulation with
LPS, the production of TNF-? and IL-6 in the culture supernatants
was analyzed (Fig. 3A). RAW cells expressing GFP alone secreted
significant amounts of IL-6 in response to LPS. However, LPS-
induced secretion of IL-6 was severely reduced in cells expressing
I?BNS/GFP. Similar amounts of TNF-? were produced by RAW
cells expressing GFP alone and I?BNS/GFP in response to LPS.
We also analyzed the LPS-induced mRNA expression of IL-6,
TNF-?, and IL-1? (Fig. 3B). Introduction of I?BNS/GFP resulted
in severely impaired LPS-induced IL-6 mRNA expression. How-
ever, even in cells expressing I?BNS/GFP, LPS-induced mRNA
expression of TNF-? and IL-1? was not impaired. Thus, lentiviral
expression of I?BNS in macrophages resulted in specific inhibition
of LPS-induced IL-6 production.
I?BNS associates with NF-?B p50 and enhances its DNA-
Because Bcl-3 has a regulatory function on NF-?B activity, we
next examined LPS-induced NF-?B activation in cells expressing
I?BNS/GFP. We first analyzed LPS-induced degradation of I?B?
by Western blot analysis (Fig. 4A). LPS stimulation induced deg-
radation of I?B? in cells expressing I?BNS/GFP as well as cells
expressing GFP alone. LPS-induced phosphorylation of ERK1/2
and p38 was not impaired in cells expressing I?BNS/GFP, indi-
cating that expression of I?BNS did not affect LPS signaling path-
way in the cytoplasmic compartment (Fig. 4B). We next analyzed
whether the DNA-binding activity of NF-?B was altered in cells
expressing I?BNS (Fig. 4C). LPS stimulation predominantly en-
hanced DNA-binding activity of p50/p65 heterodimers in cells
transduced with GFP alone. In cells expressing I?BNS/GFP, the
DNA-binding activity of p50/p50 homodimers became evident
even before LPS stimulation, and the DNA-binding activity of
mice, and peritoneal CD11b-positive cells (M?), and then analyzed for expression of I?BNS, I?B?, Bcl-3, macrophage scavenger receptor 2 (Msr2), and
CD163 by RT-PCR. B and C, RAW264.7 cells (B) and peritoneal macrophages (C) were treated with 10 ng/ml IL-10 for the indicated periods. I?BNS and
EF1-? mRNA were measured by quantitative real-time PCR. Expression of I?BNS was normalized to housekeeping gene EF1-?. Data were expressed as
relative fold induction of I?BNS compared with nontreated condition.
Identification of genes that are selectively expressed in CLPM?. A, Total RNA was purified from CLPM? of wild-type or IL-10-deficient
3653The Journal of Immunology
p50/p50 homodimers remained dominant even after LPS stimula-
tion. The specificity of the bands was confirmed by supershifts
using anti-p50 and anti-p65 Abs. Thus, cells expressing I?BNS
showed altered DNA-binding activity of NF-?B. Previous studies
showed that Bcl-3 preferentially interacted with p50 subunit of
NF-?B (28). Therefore, we next analyzed whether I?BNS associ-
ates with NF-?B. RAW cells were lentivirally introduced with
Flag-tagged I?BNS and subjected to coimmunoprecipitation anal-
ysis using Abs that detect endogenous p50 or p65 subunit (Fig.
4D). Flag-tagged I?BNS that coimmunoprecipitated with p50, but
not p65, was detected by anti-Flag Ab (Fig. 4D, left). Conversely,
p50, but not p65, coimmunoprecipitated with I?BNS (Fig. 4D,
right). Thus, these findings indicate that like Bcl-3, I?BNS spe-
cifically associates with p50 subunit of NF-?B in macrophages.
I?BNS inhibits LPS-induced activation of the IL-6 promoter
We next analyzed the mechanism by which I?BNS specifically
inhibits IL-6 production in macrophages. We first examined the
effect of transient overexpression of I?BNS on LPS-induced acti-
vation of the IL-6 and TNF-? promoters using a reporter gene
assay. Ectopic expression of I?BNS suppressed LPS-induced tran-
scriptional activity of the IL-6 promoter in RAW cells in a dose-
dependent manner (Fig. 5A). In contrast, I?BNS expression had no
effect on LPS-induced transactivation of the TNF-? promoter (Fig.
5B). Thus, I?BNS has an inhibitory effect on the LPS-induced
activation of the IL-6 promoter, but not the TNF-? promoter.
We next performed ChIP assays to further investigate how
I?BNS specifically regulates IL-6 promoter activity. RAW cells
constitutively expressing Flag-I?BNS were stimulated with LPS
for 1 or 2 h, and ChIP assays were performed using Abs that detect
Flag or endogenous p50 and p65 (Fig. 5C). In RAW cells express-
ing I?BNS, both I?BNS and p50 were recruited to the IL-6 pro-
moter before LPS stimulation. The recruitment of I?BNS and p50
to the IL-6 promoter was stably observed even after LPS stimu-
lation. Furthermore, LPS-induced recruitment of p65 to the IL-6
promoter was reduced in RAW cells expressing I?BNS. In con-
trast, I?BNS was not recruited to the TNF-? promoter, and LPS-
induced recruitment of p50 and p65 was normally observed at the
TNF-? promoter, indicating that I?BNS expression did not have
any effect on the TNF-? promoter. Taken together, these results
suggest that I?BNS suppresses the IL-6 promoter activity by se-
lective recruitment to the IL-6 promoter with NF-?B p50.
We addressed whether the altered recruitment of p50 and p65 to
the IL-6 promoter was observed in IL-10-pretreated primary mac-
rophages, in which I?BNS expression was induced. Peritoneal
macrophages from wild-type mice were pretreated with IL-10 for
18 h, then stimulated with LPS for 1 h, and analyzed by ChIP assay
(Fig. 6). In nonpretreated macrophages, recruitment of p50 and
p65 to the IL-6 promoter was observed only after LPS stimulation.
However, in IL-10 pretreated cells, p50 was recruited to the IL-6
promoter even before LPS stimulation and LPS-induced recruit-
ment of p65 was severely impaired. In addition, IL-10-mediated
alteration of NF-?B recruitment to the IL-6 promoter was not ob-
served in Stat3-deficient macrophages, in which IL-10 signaling
was abolished. Thus, I?BNS-mediated alteration of NF-?B recruit-
ment to the IL-6 promoter correlates with changes mediated by
IL-10 in macrophages, indicating that I?BNS mediates IL-10-in-
duced inhibition of IL-6 production.
Inhibition of I?BNS expression results in increased IL-6
production in macrophages
To further clarify the involvement of I?BNS in suppression of
LPS-induced IL-6 production in macrophages, we used siRNA to
block expression of I?BNS in RAW cells. RAW cells were trans-
fected with control (nonspecific) siRNA or I?BNS siRNA. After
transfection, the cells were stimulated with LPS and analyzed for
expression of I?BNS, IL-6, and TNF-?. I?BNS mRNA expression
was induced by LPS as well as IL-10 in RAW cells (Fig. 7A).
Introduction of I?BNS siRNA resulted in reduced I?BNS mRNA
expression to ?30% (Fig. 7A). In these cells, LPS-induced TNF-?
mRNA expression was not significantly altered. However, LPS-
induced IL-6 mRNA expression was increased to ?200% of the
level found in control cells. LPS-induced production of TNF-? and
IL-6 was also analyzed by ELISA (Fig. 7B). I?BNS knockdown in
RAW cells had no effect on LPS-induced TNF-? production.
However, in cells transfected with I?BNS siRNA, LPS-induced
IL-6 production was increased by about 2-fold compared with cells
transfected with control siRNA. We also analyzed activity of the
IL-6 promoter in cells transfected with I?BNS siRNA (Fig. 7C).
LPS-induced activation of the IL-6 promoter, but not the TNF-?
promoter, was increased in cells with reduced I?BNS expression.
Thus, siRNA-mediated reduction of I?BNS expression in macro-
phages enhanced LPS-induced activation of the IL-6 promoter and
production of IL-6. Taken together, these data demonstrate that
I?BNS negatively regulates LPS-induced IL-6 production in
In this study, we first characterized CLPM?. Several studies in
animal models of IBD and human IBD patients indicate that cells
of macrophage lineage play an important role in intestinal mucosal
immune responses. Aberrant activation of macrophages due to the
absence of Stat3 led to the development of chronic colitis (12).
Increased CD40L-induced production of IL-12 in mucosal den-
dritic cells was also demonstrated in mice with colitis (29). In
humans, mucosal macrophages from IBD patients showed higher
expression of several surface molecules such as CD14, CD16, and
HLA-DR (30–32). Furthermore, mucosal macrophages from IBD
patients showed enhanced activity, such as the release of oxygen
induced IL-6 production in macrophages. A, RAW cells were infected with
lentivirus expressing I?BNS/GFP or GFP alone. After infection for 24 h,
RAW cells were washed and additionally incubated for 2 days. Then, GFP-
positive cells were purified by FACS sorting, and stimulated with 100
ng/ml LPS for 24 h. Concentrations of TNF-? and IL-6 in the culture
supernatants were determined by ELISA. B, GFP-positive cells (GFP alone
and I?BNS/GFP) were purified, stimulated with LPS (100 ng/ml) for the
indicated period, and then total RNA extracts were analyzed for the mRNA
expression of IL-6, TNF-?, and IL-1?. Hybridization with the ?-actin
probe confirmed even loading of RNA in each lane.
Lentiviral introduction of I?BNS results in impaired LPS-
3654I?BNS INHIBITS IL-6 PRODUCTION IN MACROPHAGES
radicals and mature IL-1?, and presentation of Ag (33–35). Thus,
enhanced activity of mucosal macrophages is associated with the
pathogenesis of colitis.
Recently, TLRs have been shown to be essential for activation
of innate immune cells, such as macrophages and dendritic cells,
through the recognition of microbial components (8–11). Involve-
ment of TLR-dependent responses in the pathogenesis of IBD has
further been postulated because chronic colitis was greatly reduced
in mice lacking Stat3 and TLR4 (15). In the present study, we
demonstrated that CLPM? in normal mice, unlike CD11b-positive
cells from the spleen, are refractory to TLR stimulation in terms of
inflammatory cytokine production and costimulatory molecule ex-
pression. Furthermore, CLPM? from IL-10-deficient or Stat3 mu-
tant mice, even in the absence of colitis, showed significant re-
sponses to TLR stimulation, indicating that the responsiveness of
CLPM? to TLR ligands correlates with the pathogenesis of
chronic colitis in these mutant mice.
We further analyzed the molecular basis for the hyporesponsive-
ness of CLPM? by comparing gene expression profiles of CLPM?
from wild-type mice and IL-10-deficient mice. DNA microarray anal-
ysis led to the identification of several genes that are selectively ex-
pressed in CLPM? of wild-type mice, but not in peritoneal macro-
phages or CLPM? of IL-10-deficient mice. Among these genes,
CD163 and Bcl-3 were reported to be induced by IL-10 in macro-
phages or monocytes (22, 23, 36). In addition, we found that I?BNS
is selectively expressed in CLPM? of wild-type mice, and further is
induced by IL-10 in peritoneal macrophages and RAW cells. I?BNS
was originally identified as a gene that is induced upon TCR stimu-
suggested to be involved in negative selection of thymocytes, the role
of I?BNS in macrophages remained unclear. In addition, although
I?BNS was shown to be localized in the nucleus and preferentially
associates with p50 subunit of NF-?B, the mechanism by which
I?BNS inhibits NF-?B activity remains unknown. Because I?BNS is
structurally related to Bcl-3, we postulated that I?BNS has a regula-
tory role in macrophages, and analyzed the effect of I?BNS expres-
sion in the RAW macrophage cell line. Lentiviral expression and
siRNA-mediated knockdown of I?BNS in RAW cells demonstrated
B, RAW cells were transiently cotransfected with the IL-6 promoter-
luciferase or TNF-? promoter-luciferase construct (10 ng), together with an
I?BNS expression vector (0, 0.05, 0.25, or 0.5 ?g) as indicated. After 24 h of
transfection, cells were treated with or without 10 ng/ml LPS for 6 h and then
the luciferase activities were measured. The data are representative of three
independent experiments yielding similar results. Data are expressed as rela-
tive fold activation compared with the nonstimulated (?) set. C, RAW cells
that constitutively express Flag-I?BNS were stimulated with 100 ng/ml LPS
for 1 or 2 h, and ChIP assays were performed with anti-Flag, anti-p50, or
anti-p65 Abs. The immunoprecipitated IL-6 promoter (left) or TNF-? pro-
moter (right) was detected by PCR with promoter-specific primers. Data are
representative of three independent experiments. The expression of Flag-
I?BNS by Western blot analysis is shown at bottom.
I?BNS is specifically recruited to the IL-6 promoter. A and
with LPS (100 ng/ml) for the indicated period (min), and the total cell lysates were analyzed for expression of Flag-I?BNS, I?B?, and ERK by Western
blot analysis. B, Western blots showing the extent of phosphorylation of p38 and ERK MAPKs at the indicated time period after LPS stimulation in RAW cells
expressing GFP alone or I?BNS/GFP. C, RAW cells introduced with GFP alone or I?BNS/GFP were stimulated with 100 ng/ml LPS for the indicated time period.
Nuclear extracts were subjected to EMSA using the NF-?B binding site of the IL-6 promoter as a probe. The specificities of the shifted bands were determined
by adding specific Abs to p50 and p65. Two types of NF-?B bindings to the probe, p50/p65 heterodimer (?) and p50/p50 homodimer (??), are indicated. D, RAW
cells introduced with GFP alone or I?BNS/GFP were lysed and immunoprecipitated (IP) with anti-p50 or anti-p65 Abs (left). The immunoprecipitated lysates were
subsequently immunoblotted with anti-Flag Ab. The same lysates were immunoprecipitated with anti-Flag Ab, and blotted with anti-p50 or anti-p65 Abs (right).
Impaired NF-?B activity in I?BNS-introduced macrophages. A, GFP-positive cells (GFP alone and I?BNS/GFP) were purified, stimulated
3655 The Journal of Immunology
the case of Bcl-3, which is specifically recruited to the TNF-? pro-
induced TNF-? production (22), I?BNS suppressed LPS-induced ac-
tivation of the IL-6 promoter through constitutive recruitment to the
promoter together with p50. Thus, nuclear I?B proteins Bcl-3 and
I?BNS differentially modulate TLR-mediated gene induction in
I?BNS and Bcl-3 are differentially recruited to specific promot-
ers through association with p50 homodimers. But, it remains un-
clear how the specific recruitment of these nuclear I?B proteins are
induced. Nuclear I?B proteins do not seem to have the ability to
directly bind the promoters. Therefore, association with p50 may
cause modulation of the DNA binding specificity through un-
known mechanisms. Similar specific recruitment was also demon-
strated in I?B?, which is structurally related to I?BNS and Bcl-3
(21, 37–39). In the case of I?B?, the specific recruitment to the
IL-6 promoter led to activation of the promoter (21, 37). Thus,
several lines of evidence demonstrate that nuclear I?B proteins
specifically regulate expression of NF-?B target genes.
In normal condition, both I?BNS and I?B? have regulatory roles
in the production of IL-6 in macrophages. I?BNS suppresses LPS-
induced IL-6 production, whereas I?B? promotes IL-6 production
(21). Thus, although the ankyrin-repeats of both molecules are
highly conserved, they have opposite functions. I?BNS consists of
327 amino acids and the ankyrin-repeat covers almost the entire
protein. In contrast, I?B? is a protein with 629 amino acids and
possesses an N-terminal region of ?300 amino acids in addition to
the C-terminal ankyrin-repeat. The full-length I?B? has no inhib-
itory effect on NF-?B activity, but introduction of the C-terminal
ankyrin-repeat of I?B? resulted in impaired NF-?B activation (37).
Thus, the ankyrin-repeat of I?B? has an inhibitory effect on NF-?B
activity, but the N-terminal region may activate NF-?B with un-
I?BNS and Bcl-3 are expressed in CLPM? of wild-type mice,
but not the CLPM? of IL-10-deificient mice. In addition, expres-
sion of I?BNS and Bcl-3 is induced by IL-10 treatment in perito-
neal macrophages and RAW cells. Therefore, expression of
I?BNS and Bcl-3 in CLPM? of wild-type mice might be induced
by IL-10. Indeed, constitutive production of IL-10 was observed in
CLPM? of wild-type mice. Additionally, IL-10 might be provided
from other types of cells such as IL-10-producing type 1 regulatory
T cells or epithelial cells. There might be other explanations of the
characteristics of CLPM? of wild-type mice. I?BNS and Bcl-3
were also found to be induced by LPS stimulation, but their ex-
pression was not observed in CLPM? of IL-10-deficient or Stat3
mutant mice. Therefore, CLPM? of wild-type mice might be a
distinct cell population from macrophages residing in other tissues,
and IL-10 might be involved in the development of these unique
CLPM? or in the recruitment of CLPM? to the colonic lamina
propria. Constitutive expression of both I?BNS and Bcl-3 in
CLPM? may suppress exaggerated inflammatory responses in the
intestinal mucosal surface. A more precise characterization of
the IL-6 promoter. Peritoneal macrophages from wild-type or Stat3 mutant
mice were cultured for 18 h in the presence or absence of 10 ng/ml IL-10,
then stimulated with 100 ng/ml LPS for 1 h, and ChIP assays were per-
formed with anti-p50, anti-p65 Abs, or control rabbit Ig. The immunopre-
cipitated IL-6 promoter was detected by PCR. Representative of three in-
IL-10-induced alteration in recruitment of p50 and p65 to
siRNA as indicated. Three hours later, the cells were stimulated with 100 ng/ml LPS for 1 or 2 h. Total RNA was prepared and analyzed for expression
of I?BNS, IL-6, TNF-?, and ?-actin by Northern blotting. Data are representative of three independent experiments. B, After transfection of I?BNS siRNA
or control siRNA, RAW cells were stimulated with 100 ng/ml LPS for 24 h. Concentrations of TNF-? and IL-6 in the culture supernatants were determined
by ELISA. C, RAW cells were transfected with 500 pmol I?BNS siRNA or control siRNA together with 100 ng of IL-6 or TNF-? promoter reporter
plasmid. Three hours after transfection, the cells were stimulated with 100 ng/ml LPS for 6 h, and the luciferase activities were measured. Representative
of three independent experiments yielding similar results. Data are expressed as relative fold activation compared with the nonstimulated (?) set.
I?BNS is required for suppression of LPS-induced IL-6 expression. A, RAW cells were transfected with 500 pmol I?BNS siRNA or control
3656I?BNS INHIBITS IL-6 PRODUCTION IN MACROPHAGES
CLPM? of wild-type mice will be required to answer to this
CLPM? are tolerant to TLR stimulation and show no inflam-
matory cytokine production. However, Bcl-3 and I?BNS are likely
to be involved in suppression of the specific cytokine such as
TNF-? and IL-6, respectively. Therefore, there might be other
mechanisms that suppress TLR responses. NOD2, a member of the
NOD family of proteins, might be involved in one of such mech-
anisms. NOD2 is implicated in the negative regulation of TLR2-
mediated NF-?B activation (40). Mutations in NOD2 have been
shown to be associated with Crohn’s diseases (16, 17).
In this study, we showed that nuclear I?B proteins, such as
Bcl-3 and I?BNS, differentially modulate LPS-induced inflamma-
tory cytokine production in macrophages. CLPM? in wild-type
mice are tolerant to TLR stimulation, which may contribute to
prevention of chronic intestinal inflammation. Several mechanisms
are involved in the hyporesponsiveness of CLPM?, and selective
inhibition of LPS responses by nuclear I?B proteins may explain
a part of these mechanisms. Further studies will be required to
understand regulations of CLPM? activity, which will definitely
be useful for the development of an effective cure for IBD.
We thank H. Miyoshi for providing us a lentivirus vector, N. Okita and
N. Kitagaki for technical assistance, and M. Hashimoto and M. Kurata for
The authors have no financial conflict of interest.
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3657The Journal of Immunology