Carboxylated Glycans Mediate Colitis through Activation
Geetha Srikrishna,2* Olga Turovskaya,†Raziya Shaikh,†Robbin Newlin,* Dirk Foell,‡
Simon Murch,3§Mitchell Kronenberg,†and Hudson H. Freeze2*
The role of carbohydrate modifications of glycoproteins in leukocyte trafficking is well established, but less is known concerning
how glycans influence pathogenesis of inflammation. We previously identified a carboxylate modification of N-linked glycans that
is recognized by S100A8, S100A9, and S100A12. The glycans are expressed on macrophages and dendritic cells of normal colonic
lamina propria, and in inflammatory infiltrates in colon tissues from Crohn’s disease patients. We assessed the contribution of
these glycans to the development of colitis induced by CD4?CD45RBhighT cell transfer to Rag1?/?mice. Administration of an
anti-carboxylate glycan Ab markedly reduced clinical and histological disease in preventive and early therapeutic protocols. Ab
treatment reduced accumulation of CD4?T cells in colon. This was accompanied by reduction in inflammatory cells, reduced
expression of proinflammatory cytokines and of S100A8, S100A9, and receptor for advanced glycation end products. In vitro, the
Ab inhibited expression of LPS-elicited cytokines and induced apoptosis of activated macrophages. It specifically blocked acti-
vation of NF-?B p65 in lamina propria cells of colitic mice and in activated macrophages. These results indicate that carboxylate-
glycan-dependent pathways contribute to the early onset of colitis. The Journal of Immunology, 2005, 175: 5412–5422.
involve a dysregulated mucosal immune response to gut-derived
bacterial Ags (reviewed in Refs. 1 and 2). In IBD, the normal
immune tolerance to bacterial Ags is lost, and in Crohn’s disease,
and in several animal models, it is replaced by a Th1-skewed cy-
tokine response (3). This response is characterized by the expres-
sion of proinflammatory cytokines such as TNF-?, IL-12, and
IFN-?, recruitment of macrophages and neutrophils, and tissue
damage. However, the initiating events and mechanisms that sus-
tain inflammation in IBD are not well defined.
The transfer of CD4?CD45RBhighT cells to immunodeficient
mice results in a colitis with features in common with Crohn’s
disease, including requirement for Th1 cytokine secretion (4, 5).
Professional APCs of mucosal tissues are key factors in the induc-
tion of both effector and regulatory responses in this and other
models of colitis. In the transfer model, expansion of CD4?T cells
rohn’s disease and ulcerative colitis are chronic, debili-
tating, and multifactorial inflammatory bowel diseases
(IBD).4The etiology of IBD is unknown but is thought to
in the colon of reconstituted mice, and subsequent colitis patho-
genesis require normal intestinal bacterial flora (6) and expression
of MHC class II molecules on APC of the host (7). In addition,
GALT APC are believed to exert a major influence on the polar-
ization of the T cell response (8–10). Activated macrophages also
produce cytokines and are effector cells in the tissue-destruction
phase of inflammation (11, 12). In fact, changes in phenotypically
distinct macrophage populations in IBD have been suggested to
promote development of chronic inflammation (13).
We earlier identified a new type of anionic modification on N-
linked sugar chains (glycans) from macrophages and endothelial
cells (14, 15). These glycans are distinct from selectin ligands.
Their key structural component is a carboxylate residue other than
sialic or uronic acids. We recently showed that some of the car-
boxylated N-glycans contain glutamic acid directly or indirectly
linked to the outer regions of the sugar chain (16). The carboxy-
lated glycans bind to annexin I, S100A8/A9, S100A12, and high
mobility group box-1 protein (HMGB-1) (17, 18), which have
been implicated in acute and chronic inflammation (19–23).
S100A12 and HMGB-1 both bind to receptor for advanced glyca-
tion end products (RAGE), a cell surface signaling receptor im-
plicated in the pathology of inflammation, cancer, diabetes, and
Alzheimer’s disease (24–28). Structurally diverse ligands bind
RAGE through its extracellular V-type domain, where two
N-linked glycosylation sites are located (29). A subpopulation of
RAGE molecules carries the carboxylated glycans, and deglyco-
sylation of the receptor significantly decreases binding of
S100A12 and HMGB-1, showing that ligand binding is glycan
dependent (Ref. 18; G. Srikrishna and H. H. Freeze, unpublished
observations). This is significant because several studies show that
blocking RAGE-ligand interactions alleviates progress of inflam-
matory pathologies (20, 30–32).
RAGE was believed to mediate colitis through binding to
S100A12 (20). Although mice do not have a functional S100A12
gene (33), structure-function studies suggest that murine S100A8
is a functional homolog of human S100A12 (34). In addition,
*The Burnham Institute, La Jolla, CA 92037;†La Jolla Institute for Allergy and
Immunology, San Diego, CA 92121;‡Department of Pediatrics, University of Mu ¨n-
ster, Mu ¨nster, Germany; and§Centre for Pediatric Gastroenterology, Royal Free and
University College Medical School, London, United Kingdom
Received for publication May 17, 2005. Accepted for publication August 9, 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 the Broad Medical Research Program of the Eli and
Edythe L. Broad Foundation and by National Institutes of Health Grants R01-
CA92608 (to H.H.F. and G.S.) and PO1 DK46763 (to M.K.).
2Address correspondence and reprint requests to Dr. Geetha Srikrishna or Dr. Hudson
H. Freeze, Glycobiology Program, The Burnham Institute, 10901 North Torrey Pines
Road, La Jolla, CA 92037. E-mail addresses: firstname.lastname@example.org and
3Current address: Clinical Sciences, Warwick Medical School, Coventry CV2 2DX,
4Abbreviations used in this paper: IBD, inflammatory bowel disease; HMGB-1, high
mobility group box-1 protein; RAGE, receptor for advanced glycation end products;
MLN, mesenteric lymph node; DC, dendritic cell; MAdCAM-1, mucosal addressin
cell adhesion molecule 1.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00
S100A12 is overexpressed in inflamed human colonic tissues, as is
S100A9 (11, 35–38).
The above studies, which suggest a role for RAGE, S100A12,
and/or S100A8/A9 in colitis, do not indicate that carbohydrate
modifications of RAGE or any other molecule are important for
the recognition events that mediate inflammation. Because the
aforementioned myeloid-related S100 proteins are carboxylate-
glycan binding lectins (17), and glycans on RAGE are important
for ligand binding, we hypothesized that carboxylated glycans ex-
pressed on RAGE, or other putative S100 protein receptors, may
have an in vivo role in the development of colitis. We showed
earlier that mAbGB3.1, a mAb raised against carboxylated gly-
cans, blocks acute peritoneal inflammation in mice (15). In the
present study, we evaluated the effect of this anti-glycan Ab on the
development of colitis in Rag1?/?mice that were transplanted
with CD4?CD45RBhighT cells. We demonstrate a potent blocking
effect of the Ab on colitis and provide evidence for an NF-?B-
mediated mechanism for the downstream effects of glycan
Materials and Methods
Colorectal tissue samples from patients with chronic active colitis were
obtained from the frozen tissue bank of Department of Gastroenterology at
the University of Mu ¨nster. Frozen normal colon tissues obtained at surgical
resections were also provided by the Cooperative Human Tissue Network
of National Cancer Institute. Five-micrometer frozen sections were cut
with a cryostat, mounted, and stored at ?80°C until analysis. Extra biopsy
samples for isolation of lamina propria cells were obtained during endos-
copy performed at the Royal Free and University College School of Med-
icine, London, after ethical approval and written informed consent from
Donor (C57BL/6 ? BALB/c)F1(CB6F1) and Rag1?/?mice were pur-
chased from The Jackson Laboratory and maintained under specific patho-
gen-free conditions. Donors were used between 6 and 12 wk of age. Re-
cipients received adoptive transfers between 7 and 12 wk of age. All mice
experiments were reviewed and approved by the Institutional Animal Care
and Use Committee of the La Jolla Institute of Allergy and Immunology.
Induction of colitis and Ab treatment
CD4?CD45RBhighlymphocytes were isolated from spleens of donor mice
as described (6). Recipients were each injected i.v. with 4–5 ? 105sorted
CD4?CD45RBhighlymphocytes in 100 ?l of sterile PBS. In the “preven-
tive protocol,” mAbGB3.1 (10 ?g/g) was administered i.v. in 100 ?l of
PBS once in 10 days, starting 1 wk before T cell transfer, over a period of
7 wk for the long-term study, and 3 wk for the short-term study. Control
mice were administered an equivalent amount of isotype-matched control
Ab (TIB-132; American Type Culture Collection; mAb against an idio-
typic determinant on the P3X63Ag8 myeloma protein). The recipient mice
were weighed once every 5–6 days. They were constantly monitored for
clinical signs of illness, including general appearance, piloerection, diar-
rhea, and bloody stools. Diseased animals were sacrificed at 3 or 7 wk
posttransfer, or when they had lost 20% of their initial weight. In the
“therapeutic protocol,” mice received the Abs weekly starting either 10 or
21 days after T cell transfer and sacrificed 7 wk posttransfer. Segments of
colon and intestine were removed from all mice and fixed in PBS contain-
ing 4% formalin. Colon, spleen, and mesenteric lymph nodes (MLN) were
also embedded in optimal cutting temperature compound, frozen in dry
ice/methylbutane mixture (?65°C), and stored at ?80°C until analysis.
Sections from formalin-fixed colon tissues were stained with H&E. Tissues
were graded semiquantitatively according to an established scoring system
(6) by investigators blinded to the conditions: inflammatory infiltrate in the
lamina propria (score 0–3); mucin depletion (score 0–2); reactive epithe-
lial hyperplasia/atypia (score 0–3); and number of inflammatory foci per
10 high power fields (score 1–3).
Immunochemical analysis of tissue sections
Cryosections (6 ?m) were air-dried and fixed in cold acetone for 2 min at
room temperature. Sections were rehydrated in PBS; endogenous peroxi-
dases were neutralized with 1% hydrogen peroxide and blocked with avi-
din/biotin (Vector Laboratories). Samples were then incubated with the
respective primary Abs, followed by biotin-conjugated secondary Abs.
Binding was detected using streptavidin-peroxidase complex (Vectastain
mAbGB3.1 epitope in normal human colon on endothelial cells (single arrow) and macrophages (double arrows) (A), and in inflamed colonic tissue of a
patient with Crohn’s disease (B). Serial sections of the inflamed tissue (B and C) stained with anti-glycan Ab (B) and S100A12 (C) show that inflammatory
infiltrates (indicated by arrows) express both the glycan epitope and the ligand. Enlarged image of an inflammatory infiltrate (D), stained with S100A12,
shows the presence of large mononucleated macrophages. E-I represent analysis of non-reconstituted Rag1?/?mouse tissues and show anti-glycan Ab
epitope (E and F), RAGE (G), their colocalization (H), and S100A8 and S100A9 (I) in colonic lamina propria (LP) and spleen. A and E, Sections stained
with peroxidase-conjugated secondary reagent; B–D, sections stained with alkaline phosphatase-conjugated secondary reagent. Magnifications: A, ?200;
B and C, ?100; D, ?400; E, ?400.
Expression of carboxylated glycans, RAGE, and S100 proteins. Immunohistochemical staining shows expression of anti-glycan Ab
5413The Journal of Immunology
ABC (avidin/biotin complex) kit; Vector Laboratories) and diaminobenzi-
dine (DakoCytomation). Sections were then counterstained with hematox-
ylin. Abs used were mAbGB3.1 (15), and Abs against mouse Ags CD4,
CD11b, MAdCAM-1, CD86, TNF-?, IFN-?, F4/80, and CD11c (BD
Pharmingen). mAbGB3.1 was biotinylated in situ using DakoCytomation
Animal Research kit to reduce background due to anti-mouse Ig secondary
For colon sections from patients with Crohn’s disease, endogenous al-
kaline phosphatase was blocked with 20% acetic acid in PBS. After avidin-
biotin blocking, samples were incubated with the respective primary Abs,
followed by biotin-conjugated secondary Abs. Binding was detected using
ABC-alkaline phosphatase and alkaline phosphatase substrate (Vector Lab-
oratories). Abs used were mAbGB3.1, anti-human S100A12 (38), and anti-
human S100A9 (BMA Biomedicals).
Isolation of cells from colonic lamina propria, spleen, and MLN
Colonic mucosal immune cells were isolated as described earlier (6, 7).
Cell suspensions from spleen and MLN were obtained using standard
Flow cytometric analysis
Cells from spleen, MLN, or colonic lamina propria of mice were resus-
pended in HBSS staining buffer containing 1% BSA. After preincubation
with a blocking anti-FcR Ab (BD Pharmingen), cells were stained with
FITC or R-PE-conjugated Ab or with unlabeled Ab followed by labeled
secondary reagent. Cells were washed and analyzed immediately on a
FACScan flow cytometer (BD Biosciences). Abs used in this study include
mAbGB3.1, rabbit anti-RAGE (a kind gift of Novartis Tsukuba Research
Institute, Japan), and Abs to mouse CD4, CD11c, CD11b, ?4?7integrin
(BD Pharmingen), and CD205 (Serotec). Lamina propria cells from
Crohn’s patients were isolated, and flow-cytometric analysis was per-
formed as described previously (39).
Equivalent amounts of cell extract proteins from splenocytes or lamina
propria cells were electrophoresed on denaturing and reducing 12% poly-
acrylamide gels, and transferred to nitrocellulose membranes. The blots
were blocked with 10% dry skimmed milk, washed, and incubated with
primary Abs followed by alkaline phosphatase-conjugated secondary Abs.
Bound proteins were visualized using 5-bromo-4-chloro-3-indolyl phos-
phate/NBT (Sigma-Aldrich). Rabbit anti-mouse S100A8 and S100A9 were
generated as described earlier (40).
Cell culture and LPS stimulation
RAW 264.7 cells obtained from American Type Culture Collection were
maintained in DMEM (supplemented with glutamine, penicillin, and strep-
tomycin and 10% FBS) at 37°C in a humidified incubator containing 5%
CO2. For stimulation, cells were detached by vigorous pipetting and trans-
ferred to 12-well plates with fresh medium containing 2% serum at 1 ? 106
cells/ml. Cells were stimulated with 1 ?g/ml LPS from Escherichia coli
serotype 0111:B4 (Sigma-Aldrich) in the presence or absence of 10 ?g/ml
mAbGB3.1 or isotype control Ab. Culture supernatants and cells were
harvested at different time points after stimulation. Supernatants were
stored at ?80°C until analysis. RNA or nuclear extracts from cells
were prepared immediately after harvest and stored at ?80°C until
Measurement of cytokines and NO
TNF-?, IL-10, and IL-12 in culture supernatants or mice sera were mea-
sured using ELISA kits (BD Pharmingen). Total nitrite was measured using
Griess reagent after reduction of NO3
?by nitrate reductase (R&D
RNA extraction and real-time PCR
PCR primers and TaqMan probes for TNF-? and IL-23 p19 were designed
using PrimerSelect (DNAStar) and were obtained from BioSource Inter-
national or PE Applied Biosystems. Primers and probes for GAPDH were
obtained from PE Applied Biosystems. TaqMan probes contained a re-
porter dye (FAM) covalently attached to the 5? end and a quencher dye
(BHQ) at the 3? end. Forward and reverse primers and probes were as
follows: TNF-?: forward, 5?-CATCTTCTCAAAATTCGAGTGACAA-3?,
reverse, 5?-TGGGAGTAGACAAGGTACAACCC-3?, and probe, 5?-
forward, 5?-GTGCCCCGTATCCAGTGTGAAGA-3?, reverse, 5?-GT
GAAGTTGCTCCATGGGGCTATC, and probe, 5?-FAM-CCCACAAG
Table I. mAbGB3.1 reactivity is expressed on APCs of colonic lamina
Percentage of Colocalization of
71 ? 8
70 ? 10
50 ? 10
65 ? 15
40 ? 10
38 ? 12
21 ? 7
aTable shows the percentage of colocalization of mAbGB3.1 reactivity with den-
dritic cell and macrophage markers in colonic lamina propria cells from human
Crohn’s disease (CD45?cells) and from normal mice (mean ? SD of two
ventive protocol). Recipient mice were administered with anti-glycan Ab
mAbGB3.1 or a control Ab once every 10 days for 7 wk starting 1 wk
before CD4?CD45RBhighT cell transfer (arrow indicates cell transfer). A,
Change in body weight over time is expressed as the percentage of starting
weight (?, p ? 0.05). B, Colonic inflammation was scored at the end of the
experiment using an established scoring system (6). Each point represents
mean ? SE of six mice in each group. ?, p ? 0.05 between reconstituted,
untreated vs reconstituted, anti-glycan Ab treated.
Anti-glycan Ab mAbGB3.1 treatment blocks colitis (pre-
5414CARBOXYLATED GLYCANS MEDIATE COLITIS
GACTCAAGGACAACAG-BHQ1-3?. External standard for TNF-? was
generated from total RNA extracted from mouse spleen by RT-PCR and
cloned as described (41). Reference standard for IL-23 was obtained by
various dilutions of RNA isolated from Con A-activated mouse spleen
Total RNA was extracted from RAW264.7 or spleen cells using TRIzol
reagent (Invitrogen Life Technologies). Reverse transcription and real-time
PCR were performed in a single step, using the LightCycler-RNA Master
Hybridization Probes kit (Roche Applied Science). Tth DNA polymerase,
reaction buffer and dNTPs, forward and reverse primers, TaqMan probes,
and purified RNA or standards at different dilutions in a total volume of 20
?l were directly added to LightCycler capillaries, which were inserted into
a Roche LightCycler instrument. After incubating for 20 min at 61°C to
allow mRNA reverse transcription, and an initial denaturation step at 95°C
for 30 s, the cDNAs were amplified as described (41).
Measurement of apoptosis
Apoptosis of RAW264.7 cells at different time points after activation was
measured by labeling with annexin V and propidium iodide (BD Pharm-
ingen) followed by flow cytometry. Cell growth was measured by manual
counting, and viability was assessed by trypan blue dye exclusion. Apo-
ptosis was examined in colonic lamina propria of mice by a TUNEL assay
using the ApopTag Fluorescein In Situ Apoptosis Detection kit (Chemicon
International). Apoptotic macrophages were identified by double staining
using rat anti-mouse F4/80 and Alexa Fluor 594-conjugated anti-rat IgG
(Invitrogen Life Technologies).
Measurement of NF-?B binding activity in nuclear extracts
Nuclear extracts from RAW264.7 or lamina propria cells of mice were
assayed for NF-?B binding activity using TransAM NF-?B assay kit (Ac-
tiveMotif) according to the manufacturer’s instructions. Mouse specific
anti-p50 was obtained from Santa Cruz Biotechnology.
Statistical comparisons were performed using one-way ANOVA or Stu-
dent’s t test. Differences were considered statistically significant when
p ? 0.05.
Endothelial cells and APC in human and mouse colon express
To begin to investigate a role of carboxylated glycans in the patho-
genesis of IBD, we first examined tissues from patients with es-
tablished IBD for glycan expression. In tissues from control sub-
jects, anti-glycan Ab mAbGB3.1 stained endothelial cells and
large mononucleated cells in the lamina propria (Fig. 1A). In tis-
sues from two Crohn’s disease patients examined, the Ab also
stained inflammatory infiltrates and serosal and submucosal aggre-
gates of macrophages (Fig. 1B). The cells were identified as mac-
rophages because they were large, mononucleated, and CD68 pos-
itive. Multiparameter analysis of lamina propria cells showed
predominant expression of anti-glycan Ab epitope on CD11c?,
CD80?, CD86?, and HLA-DR?lamina propria cells from in-
flamed tissues (Table I). The infiltrating macrophage lesions in
Crohn’s tissues also stained positive for S100A12 (Fig. 1, C and
D) and S100A9 (not shown), whereas the S100 proteins were not
detected in tissues from healthy controls, in agreement with other
studies (38). Because S100A12 and S100A9 bind to carboxylated
glycans recognized by mAbGB3.1 (henceforth referred to as anti-
glycan Ab), this suggested up-regulation and colocalization of the
ligand-receptor pair in inflamed regions of tissue in the patients.
Using immunohistochemistry and flow-cytometric analysis, we
also detected strong anti-glycan Ab reactivity in colonic lamina
propria cells of non-reconstituted Rag?/?mice (Figs. 1, E and F).
Soluble carboxylate-enriched glycopeptides decreased Ab binding
to lamina propria cells, consistent with specific binding (Fig. 1F).
Anti-glycan Ab reactivity was present on CD11c?and CD205?
dendritic cells (DC) and CD11b?macrophages (Table I). RAGE
was also expressed in colonic lamina propria cells (Fig. 1G) and
colocalized with anti-glycan Ab reactivity (Fig. 1H). Partial colo-
calization of anti-glycan Ab reactivity with RAGE suggested that
other proteins besides RAGE also express the glycans. Immuno-
blotting showed the presence of S100A9 in cells of colonic lamina
propria, and S100A8 and S100A9 in spleens of non-reconstituted
Rag?/?mice (Fig. 1I). We also found HMGB-1 in spleen, MLN,
and colonic lamina propria of these mice (not shown).
Anti-glycan Ab treatment blocks the onset of colitis in the
adoptive cell transfer model
To further explore the role of the glycans in IBD, we tested the
anti-carboxylate glycan Ab in the CD4?CD45RBhighT cell trans-
fer model of colitis. Animals were injected with a nonblocking
control Ab or anti-glycan Ab once every 10 days and monitored
for ?7 wk following cell transfer (half-life of the Abs in circula-
tion was ?7–8 days). Reconstituted mice untreated or treated with
the control Ab lost an average of 20–25% of their initial body
weight (Fig. 2A) and suffered from severe diarrhea. Mice treated
glycan Ab-treated mice. H&E staining of representative
proximal colon sections from Rag?/?mice 7 wk post-
transfer: non-reconstituted (A), CD4?CD45RBhighre-
constituted (B), reconstituted mice treated with anti-
glycan Ab (C), or reconstituted mice treated with control
Ab (D). Extensive inflammatory cell infiltration, hyper-
plasia, mucin depletion, and crypt abscesses were ob-
served in untreated and control Ab-treated mice,
whereas in four of six anti-glycan Ab-treated mice, co-
lonic architecture was intact with minimal or no inflam-
mation. Images are at the same magnification (?200).
Minimal colonic inflammation in anti-
5415The Journal of Immunology
with the anti-glycan Ab showed minimal weight loss, did not have
any diarrhea, and remained healthy throughout the treatment pe-
riod. Histopathological examination of the colons revealed marked
mucosal hyperplasia, goblet cell depletion, distortion of crypt ar-
chitecture, and extensive inflammatory cell infiltration with occa-
sional crypt abscesses in untreated or control Ab-treated mice,
whereas in two-thirds of anti-glycan Ab-treated mice, colonic ar-
chitecture was intact with minimal or no inflammatory changes
(Figs. 2B and 3). Splenomegaly observed in reconstituted, un-
treated or control Ab-treated mice was also absent in all reconsti-
tuted mice treated with anti-glycan Ab (not shown).
Anti-glycan Ab treatment initiated after cell transfer blocks
progress of disease
In the transfer model, CD4?T cell colonization in lamina propria
is apparent 8–11 days after reconstitution, along with an early
inflammatory infiltration (10). Severe colitis is established by 21
days posttransplantation. We therefore evaluated whether Ab ad-
ministration after the initiation of colitis would block or reverse the
disease. Mice received the Abs weekly starting either 10 or 21 days
after T cell transfer and sacrificed 7 wk posttransfer. At 10 days
posttransfer, we found evidence of inflammation in a parallel
group of reconstituted, untreated mice (average inflammation
scores of 2.5 in proximal and distal colons compared with 0.75 in
non-reconstituted mice; n ? 4). Anti-glycan Ab treatment started
10 days after cell transfer significantly impaired the progression of
disease. This recovery was accompanied by reduced or no weight
loss, good health, and reduced inflammation scores (Fig. 4).
Splenomegaly observed in reconstituted, untreated or control Ab-
treated mice was also absent in all reconstituted mice treated with
anti-glycan Ab. However, administration of the anti-glycan Ab
from 21 days after cell transfer did not prevent weight loss or
symptoms of disease (not shown). This suggested that the Ab
blocked an early step in colitis pathogenesis.
In the transfer model, lymphocyte reconstitution is associated
with an initial homeostatic as well as Ag-driven expansion of
CD4?T cells in secondary lymphoid organs (7). This is followed
by generation of organ-tropic T cells with a Th1 cytokine profile.
Because APC play an important role in T cell maturation and ac-
tivation, the anti-glycan Ab may block one or more of APC-me-
diated pathways early in T cell pathology. To understand the
mechanisms underlying the protective effects of the anti-glycan
Ab, we therefore conducted systematic examination of tissues of
mice (7 or 3 wk after reconstitution) for cellular accumulation and
expression of cytokines and other molecules.
Leukocyte accumulation is reduced in the colons of Ab-treated
We first examined accumulation of T cells in different compart-
ments of reconstituted mice. At 7 wk posttransfer, CD4?T cell
infiltration was found throughout the intestine and colon in recip-
ient mice, and this was unaffected by control Ab treatment. In mice
treated with the anti-glycan Ab, there was a marked reduction in
the accumulation of CD4?T cells in the lamina propria of the
colon (Fig. 5A). This effect was site specific, because accumulation
of T cells in the small intestine (not shown) was unaffected by
anti-glycan Ab treatment. This suggested that the colonic micro-
environment, whose critical feature is the presence of a rich bac-
terial flora, contributes to the Ab-mediated effects.
We also examined CD4?T cell accumulation 3 wk after T cell
reconstitution. Compared with mice treated with the control Ab,
the anti-glycan Ab significantly reduced accumulation of CD4?T
cells in the colonic lamina propria, but it did not affect accumu-
lation in the spleen and MLN (Fig. 5B). This indicated that the
anti-glycan Ab treatment affected early as well as late accumula-
tion of T cells in the colon.
Next, we examined colon tissues of reconstituted mice for ac-
cumulation of inflammatory cells 7 wk posttransfer. In non-recon-
stituted mice, CD11b?and F4/80?myeloid cells were distributed
throughout the lamina propria of both the proximal and distal co-
lon (Fig. 6, shown for proximal colon). The expanded lamina pro-
pria of reconstituted mice untreated or treated with control Ab
contained significant inflammatory infiltrates consisting predomi-
nantly of CD11b?and F4/80?macrophages and a smaller number
progress of disease (therapeutic protocol). Reconstituted mice were treated
with anti-glycan Ab or a control Ab weekly for ?6 wk starting 10 days
(indicated by arrow) after T cell transfer. A, Change in body weight over
time is expressed as the percentage of starting weight (?, p ? 0.05). B,
Colonic inflammation was scored at the end of the experiment as indicated
in Fig. 2. Each point represents mean ? SE of four mice per group. ?, p ?
0.05 between reconstituted, untreated vs reconstituted and anti-glycan Ab
treated. Colons examined at 10 days posttransfer in a parallel group of
reconstituted, untreated mice showed evidence of mild inflammation (see
Anti-glycan Ab treatment after initiation of colitis blocks
5416CARBOXYLATED GLYCANS MEDIATE COLITIS
of neutrophils in crypt abscesses (Fig. 6). Clusters of activated
macrophages were confined to lamina propria, but in severe dis-
ease, they also extended to the submucosa. In reconstituted mice
treated with anti-glycan Ab, accumulation of CD11b?and F4/80?
cells was markedly reduced (Fig. 6).
Anti-glycan Ab reduces expression of mucosal addressin cell
adhesion molecule 1 (MAdCAM-1)
MAdCAM-1 and the gut-homing integrin ?4?7are important for
recruitment of T cells to the inflamed colonic tissue (42, 43). We
therefore examined whether reduced T cell accumulation in the
colons of anti-glycan Ab-treated mice was due to reduced fre-
quency of gut tropic T cells or reduced expression of MAdCAM-1.
Analysis of T cells from MLN of anti-glycan Ab-treated mice at 3
wk posttransfer showed no reduction in the frequency of ?4?7
cells (11.3 ? 1.5%; n ? 3), compared with the control Ab-treated
mice (12.5 ? 0.7%; n ? 3). However, markedly increased expres-
sion of MAdCAM-1 was seen in inflamed tissues from untreated
or control Ab-treated mice, whereas the addressin expression on
venules in colons of anti-glycan Ab-treated mice was similar to
constitutive expression seen in non-reconstituted mice (Fig. 6).
This suggested that the anti-glycan Ab could block the infiltration
of gut tropic T cells to the colon by decreasing MAdCAM-1
Proinflammatory cytokines are reduced in tissues and blood of
anti-glycan Ab-treated mice
TNF-? and IFN-? are also important mediators of disease in the
adoptive transfer model (4). Th1 cells produce IFN-?, whereas
TNF-? is a product of both Th1 cells and macrophages that are
matory cell infiltration and up-regulation of MAdCAM
in the colon of reconstituted mice. CD11b?F4/80?cel-
lular infiltration and MAdCAM expression in mice co-
lon 7 wk posttransfer was assessed by immunohisto-
chemical staining using specific Abs (six mice per
group). All images are at the same magnification
Anti-glycan Ab treatment blocks inflam-
assessed by immunochemical staining using anti-CD4, was reduced in colon (six mice per group). All images are at the same magnification (?200). B, At
3 wk posttransfer, the total number of CD4?cells was quantitated by flow-cytometric analysis of cells from spleen, MLN, and lamina propria of control
Ab- or anti-glycan Ab-treated mice. Each is the mean ? SE of three mice per group. ?, p ? 0.05.
Anti-glycan Ab-treated mice show reduced accumulation of CD4?T cells in colon. A, At 7 wk posttransfer, CD4?T cell accumulation,
5417The Journal of Immunology
activated by them. To investigate the role of the anti-glycan Ab in
blocking activation of T cells and effector functions of Th1 cells,
we analyzed expression of proinflammatory cytokines in tissues
and sera of mice.
At 7 wk posttransfer, in reconstituted and untreated or control
Ab-treated mice, increased IFN-? expression was associated with
the presence of increased CD4?lymphocytes in the lamina propria
and submucosa, whereas in the colon of anti-glycan Ab-treated
mice, IFN-? expression was minimal (Fig. 7). Anti-glycan Ab
treatment also reduced the frequency of IFN-?-positive lympho-
cytes in spleen examined 3 wk posttransfer (Table II).
At 7 wk posttransfer, TNF-? was highly expressed in colonic
lamina propria in reconstituted and untreated or control Ab-treated
mice (Fig. 7). TNF-? expression showed a temporal correlation
with macrophage (CD11b?F4/80?) infiltration, suggesting that
macrophages are the principal source of TNF-?. Expression of
TNF-? in colonic tissue was greatly reduced in anti-glycan Ab-
treated mice. We examined the efficacy of Ab therapeutic modality
on the production of TNF-? in colitis. TNF-? levels in blood were
elevated as early as 10 days posttransfer and were maximum 7 wk
posttransfer. When anti-glycan Ab treatment was initiated 10 days
after T cell transfer, TNF-? measured 7 wk posttransfer was re-
duced to the levels seen in non-reconstituted mice (Table III).
Anti-glycan Ab blocks proinflammatory cytokine production by
macrophages and induces their apoptosis
To further confirm that anti-glycan Ab treatment down-regulates
proinflammatory cytokine production in the colon, we analyzed the
effects of the Ab on LPS-elicited production of cytokines by mac-
rophages in vitro. LPS interacts with CD14 and the TLR4 complex
on macrophages to activate multiple signaling pathways (44) and
secretion of proinflammatory cytokines IL-12, TNF-?, and IL-1, as
well as anti-inflammatory cytokines, including IL-10 (45). Anti-
glycan Ab-reactive epitope is constitutively expressed on
RAW264.7 macrophages (not shown). We therefore activated
these macrophages in vitro with LPS in the presence or absence of
anti-glycan Ab or the isotype control Ab. LPS increased TNF-?
mRNA production in untreated cells by 3-fold (Fig. 8A). This was
followed by an enhanced secretion of TNF-? into the culture me-
dium. The anti-glycan Ab significantly reduced LPS-stimulated
TNF-?. It also inhibited LPS-induced production of IL-23 mRNA,
and secretion of IL-12, and NO, whereas it had no effect on the
production of IL-10 (Fig. 8A). In addition, treatment of LPS-acti-
vated cells with the anti-glycan led to increased apoptosis of the
macrophages, as determined by cellular growth (Fig. 8B) and an-
nexin V staining (not shown). This was activation dependent, be-
cause the Ab had no effect on the growth of unstimulated cells.
To determine whether the anti-glycan treatment induced apo-
ptosis of activated (infiltrating) macrophages in vivo, we per-
formed an immunochemical analysis of lamina propria macro-
phages 7 wk posttransfer in the therapeutic protocol. We identified
apoptotic macrophages by F4/80 and TUNEL double staining. In
untreated and treated mice, ?90% of apoptotic cells were T cells.
In the untreated mice and in the control Ab-treated mice, the
TUNEL?non-T cells did not show any overlap with F4/80-stained
cells. In the anti-glycan Ab-treated mice, ?50% of TUNEL?
non-T cells were macrophages, because they were also positive for
F4/80 (not shown). These results suggested that the anti-glycan Ab
might specificallyblock proinflammatory
inflammatory cytokine production and may also promote apoptosis
of infiltrating macrophages in colitis. Further confirmation would
require more kinetic examination of apoptotic infiltrating macro-
phages at different time points after reconstitution in the untreated
and treated mice.
Table II. IFN-?-positive T cells in spleen
Number of IFN-
Reconstituted and untreated
Reconstituted and control Ab treated
Reconstituted and anti-glycan Ab treated
22.3 ? 9.1
83.2 ? 11.7
75.7 ? 17.4
25.6 ? 7.8
aAverage number of IFN-?-positive T cells from three high-power fields, exam-
ined 3 wk after transfer. Mean ? SD; n ? 3 in each group.
Table III. Levels of TNF-? in sera of reconstituted mice (therapeutic
Treatment and Time of Sampling TNF-? (ng/ml)
Reconstituted, 10 days posttransfer,
Reconstituted, 7 wk posttransfer, untreated
Reconstituted, 7 wk posttransfer, control
Ab treatment initiated at 10 days
Reconstituted, 7 wk posttransfer, anti-
glycan Ab treatment initiated at 10 days
0.139 ? 0
0.501 ? 0.208
1.317 ? 0.378
1.294 ? 0.357
0.093 ? 0.014
aMean ? SD, n ? 4 in each group.
bLevels in non-reconstituted mice remained steady through the test period.
pression is reduced in tissues of anti-
glycan Ab-treated mice. IFN-?- and
TNF-?-positive cells in tissues of non-
reconstituted and reconstituted mice at 7
wk posttransfer were detected using spe-
cific Abs (six mice per group). Arrows
mark IFN-??cells. All images are the
same magnification (?200).
IFN-? and TNF-? ex-
5418CARBOXYLATED GLYCANS MEDIATE COLITIS
RAGE and carboxylated glycan binding proteins S100A8,
S100A9, and HMGB-1 are up-regulated in colitis
Both IFN-? and TNF-? stimulate macrophage expression of
S100A8 (46), and S100A9 expression is up-regulated in activated
spleen cells (47). S100A8 is chemotactic in mice (48, 49) and
S100A9 promotes integrin-mediated adhesion of phagocytes (50).
Together, they may provide a strong stimulus for early infiltration
of neutrophils and monocytes. We therefore examined whether
S100A8 and S100A9 are up-regulated in inflamed tissues of re-
constituted mice. Immunoblot analysis showed that S100A8 and
S100A9 are strongly up-regulated in spleen and colonic lamina
propria of reconstituted and untreated or control Ab-treated mice
examined 3 wk posttransfer, but not in anti-glycan Ab-treated mice
(Fig. 9). We also found up-regulation of RAGE in colonic lamina
propria and spleens of untreated and control Ab-treated mice, but
not in anti-glycan Ab-treated mice (Fig. 9). The two forms of
tokine expression and induces apoptosis of RAW264.7
macrophages. A, RAW264.7 macrophages in culture
were activated with LPS in the presence or absence of
anti-glycan Ab or an equivalent amount of isotype con-
trol Ab. Cellular cytokine mRNA (1 h after activation)
was measured by real-time PCR. Secreted cytokines (in
supernatants collected 20 h after activation), and NO
were measured as described in Materials and Methods.
Each point is the mean ? SE of two experiments, with
duplicate measurements for each assay. ?, p ? 0.05 be-
tween activated, untreated vs activated, anti-glycan Ab
treated. B, Treatment of LPS-activated cells with the
anti-glycan Ab led to increased apoptosis, as determined
by cellular growth (expressed as percentage of cells in
unactivated cultures for each time point) and annexin V
staining and flow cytometry (not shown). The Ab had no
effect on the growth of unstimulated cells. Each point is
the mean of duplicate measurements.
Anti-glycan Ab inhibits LPS-elicited cy-
5419The Journal of Immunology
RAGE seen in spleen may represent proteins encoded by alterna-
tively spliced mRNAs (51). In addition, we also detected HMGB-1
in the sera of mice with inflammation, whereas it was undetectable
in unreconstituted mice (not shown).
Because anti-glycan Ab treatment started 10 days after cell
transfer significantly impaired progression of disease, we next ex-
amined whether S100A8, S100A9, and RAGE are up-regulated in
tissues at 10 days posttransfer. ELISA quantitation showed that
S100A9 was moderately up-regulated in spleen (?2-fold; p ?
0.005), but not in colonic lamina propria, compared with non-
reconstituted mice (n ? 4 each group; data not shown). Expres-
sions of RAGE and S100A8 in spleen and colonic lamina propria
were unaffected 10 days posttransfer in both spleen and lamina
propria. This suggests that S100A9 up-regulation in spleen may be
an early event during initiation of colitis, and may play an impor-
tant role in the anti-glycan Ab-mediated effects.
Anti-glycan Ab reduces activation of NF-?B p65
Because HMGB-1, S100A8, S100A9, and S100A12 are all strong
inducers of NF-?B (20, 52), the above results suggested that
NF-?B activation could play a role in carboxylated glycan-medi-
ated effects. RAGE promoter has two NF-?B binding sites and
activation of NF-?B by RAGE ligation results in up-regulation of
the receptor, thus amplifying the signal and initiating a patholog-
ical cycle of cellular perturbation.
At 3 wk posttransfer, NF-?B p65 was highly up-regulated in
lamina propria cells of recipients untreated or treated with control
Ab, but was significantly reduced in cells from anti-glycan Ab-
treated mice (Fig. 10). In vitro, LPS-stimulated RAW264.7 mac-
rophages showed significant activation of NF-?B, which was
blocked by anti-glycan Ab treatment. We observed inhibition of
activation of NF-?B p65, but not that of other NF-?B/Rel family
members (Fig. 10). This finding is interesting because NF-?B p65
is increased in colonic lamina propria of Crohn’s disease patients
(53–55), and administration of p65 antisense oligonucleotides re-
duces clinical and histological signs of trinitrobenzene sulfonic
acid- or dextran sulfate sodium-induced colitis (56–58). We also
found a mild but statistically significant up-regulation of NF-?B
p65 in the spleen (?1.5-fold; p ? 0.05; not shown), but not in
colonic lamina propria of mice 10 days posttransfer, compared
with non-reconstituted mice (n ? 4 each group), concomitant with
an increase in S100A9 in spleen, and elevated TNF-? in serum
(Table III). Because S100A9 binds carboxylated glycans (17), and
S100A9 can induce NF-?B activation (52), inhibition of NF-?B
activation could be a crucial factor in the disease-blocking effects
of the anti-glycan Ab.
Oligosaccharides are increasingly being recognized as important
mediators of signaling in innate and adaptive immune responses
(59–63). Considerable diversity of oligosaccharide structures pro-
vides enormous potential for information display on cell surfaces
and specific recognition by different lectins. Glycans that have the
same structure can also have different functions depending upon
the proteins and cell types that carry them. Examples are the se-
lectin ligands that mediate both inflammation-initiated leukocyte
rolling as well as physiological lymphocyte homing and recircu-
lation. However, not all glycan structures in mammals have been
proven, much less functionally characterized. We earlier identified
a family of novel carboxylated glycans on endothelial cells and
macrophages that mediate inflammation. Here, we show that in-
terfering with the interaction between these glycans and their pu-
tative lectin partners using a monoclonal anti-glycan Ab prevents
the pathogenic process in a mouse model of IBD.
NF-?B/Rel family of plieotropic transcription factors play a pivotal
role in host immune and inflammatory responses (64–66) and are
believed to be important in the pathophysiology of IBD. They are
are up-regulated in tissues from colitic mice. Twenty micrograms of extract
proteins were prepared from splenocytes or colonic lamina propria cells of
mice 3 wk posttransfer, separated on SDS-PAGE gels, and probed using
specific Abs after transfer. Lanes: 1, non-reconstituted; 2, reconstituted,
untreated; 3, reconstituted, anti-glycan Ab treated; 4, reconstituted, control
Ab treated. The two forms of RAGE at 45 and 32 kDa in spleen may
represent splice variants.
Myeloid-related proteins S100A8 and S100A9 and RAGE
in RAW264.7 macrophages. Nuclear extracts were prepared from lamina
propria cells from reconstituted mice 3 wk posttransfer (A) and RAW264.7
macrophages treated with 1 ?g/ml LPS (with and without priming with
IFN-?) for 60 min in the presence of anti-glycan Ab or a control Ab (B).
Twenty micrograms of nuclear lysate protein was incubated in wells coated
with consensus NF-?B binding oligonucleotide sequences, and bound pro-
tein was measured using anti-p65 or anti-RelB. ?, p ? 0.05 between re-
constituted, untreated vs reconstituted, anti-glycan Ab-treated mice (n ? 3)
(A) or LPS-activated, untreated vs LPS-activated, anti-glycan Ab-treated
Anti-glycan Ab treatment inhibits activation of NF-?Bp65
5420CARBOXYLATED GLYCANS MEDIATE COLITIS
flammatory cytokines, and stress. They regulate expression of proin-
flammatory cytokines, adhesion molecules, and inducible NO syn-
thase, and play an antiapoptotic role in many systems (67). Our study
shows that carboxylated glycans may play a critical role in early
events of colitis pathology by blocking NF-?B activation. However,
the specific events and mediators remain unknown. The Ab may
block either a single step or multiple steps during the initiation or
recurrence of disease. Likely early targets include DC maturation,
DC-T cell interactions, proliferation and polarization of T cells, hom-
ing, and infiltration and survival of inflammatory cells into the colon.
The sugar chains may mediate multiple interactions involving differ-
and a network, rather than a linear sequence in the activation of
Although the effects of the Ab are predominantly localized to
the colon, peripheral effects such as blocking initial Ag-driven ex-
pansion and activation of T cells in spleen cannot be ruled out.
CD4?T cells in colon and spleen have an activated phenotype at
early time points following cell transfer (7). DCs from colon mi-
grate to draining lymph nodes and peripheral sites providing a
mechanism for systemic activation of T cells to mucosal Ags (68).
The anti-glycan Ab may have an initial inhibitory effect on sys-
temic expansion and activation of T cells. Reduced IFN-?-positive
T cells in spleen of anti-glycan Ab-treated mice, associated with
reduced expression of S100A8 and S100A9 and RAGE in spleen
3 wk posttransfer support this view. In addition, we also found
up-regulation of S100A9 and NF-?B in the spleen of mice 10 days
posttransfer. These findings underscore the importance of NF-?B-
mediated initial events in secondary lymphoid organs in the onset
Possible roles of S100 proteins and RAGE in the early onset of
colitis and in mediating the protective effects of the anti-glycan Ab
need further exploration. We showed earlier that RAGE from bo-
vine and mouse lung express the glycans (18), and the glycans
mediate RAGE-ligand binding (18). RAGE ligation leads to long-
term activation of NF-?B and inflammation (20, 69). S100 proteins
and HMGB-1 released from activated inflammatory cells provide
a potent positive feedback for sustained pathology by activating
RAGE-mediated signaling pathways, activation of NF-?B and up-
regulation of RAGE expression (25). Sustained RAGE activation
combined with an exaggerated host response in the absence of
regulatory mechanisms such as seen in IBD and experimental co-
litis could lead to irreversible complications of disease. The anti-
glycan Ab could block this cycle by interfering with RAGE-ligand
interaction. However, RAGE knockout mice have normal adaptive
immune responses (70). In preliminary studies, we found that
CD4CD45RBhighcells from RAGE?/?mice were as effective as
wild-type donor T cells in eliciting disease (not shown). Also, the
presence of RAGE?/?GB3.1?/?immune cells in wild-type mice
(Fig. 1; G. Srikrishna and H. H. Freeze, unpublished observations)
brings up the question whether the glycans mediate their effects
through RAGE, or independent of RAGE, or both. In addition, the
cellular receptors for S100A8/A9 have not been identified. Our
future studies will address these questions.
In summary, our findings indicate that carboxylated glycans ex-
pressed on APC and other cells may play critical roles in the ini-
tiation and progression of colitis. Because colon tissues from
Crohn’s disease patients express increased levels of the glycan
epitope, further characterizing and targeting of the carboxylated
glycan-dependent pathway(s) may be a promising new approach to
the treatment of human IBD.
We are grateful to Dr. Nissi Varki (University of California, San Diego, La
Jolla, CA) for expert histology advice and for critical reading of the
manuscript. We thank Charles DeRossi (The Burnham Institute),
Dr. Patrick Stordeur (Erasme University Hospital, Brussels, Belgium), and
David Boyle and Dr. Sanna Rosengren (University of California, San
Diego) for help and advice with cytokine real-time PCR assays. We also
thank Dr. Paul Ashwood (Royal Free and University College Medical
School, London) for flow-cytometric analysis of human Crohn’s samples,
Jonamani Nayak (The Burnham Institute) for excellent technical help, and
Douglas Haynes for help with illustrations. Dr. Angelika Bierhaus (Uni-
versity of Heidelberg, Heidelberg, Germany) kindly provided the RAGE
knockout mice used in initial studies mentioned in Discussion.
The authors have no financial conflict of interest.
1. Strober, W., I. J. Fuss, and R. S. Blumberg. 2002. The immunology of mucosal
models of inflammation. Annu. Rev. Immunol. 20: 495–549.
2. Bouma, G., and W. Strober. 2003. The immunological and genetic basis of in-
flammatory bowel disease. Nat. Rev. Immunol. 3: 521–533.
3. Blumberg, R. S., L. J. Saubermann, and W. Strober. 1999. Animal models of
mucosal inflammation and their relation to human inflammatory bowel disease.
Curr. Opin. Immunol. 11: 648–656.
4. Powrie, F., M. W. Leach, S. Mauze, S. Menon, L. B. Caddle, and R. L. Coffman.
1994. Inhibition of Th1 responses prevents inflammatory bowel disease in scid
mice reconstituted with CD45RBhiCD4?T cells. Immunity 1: 553–562.
5. Leach, M. W., A. G. Bean, S. Mauze, R. L. Coffman, and F. Powrie. 1996.
Inflammatory bowel disease in C.B-17 scid mice reconstituted with the
CD45RBhighsubset of CD4?T cells. Am. J. Pathol. 148: 1503–1515.
6. Aranda, R., B. C. Sydora, P. L. McAllister, S. W. Binder, H. Y. Yang,
S. R. Targan, and M. Kronenberg. 1997. Analysis of intestinal lymphocytes in
mouse colitis mediated by transfer of CD4?, CD45RBhighT cells to SCID re-
cipients. J. Immunol. 158: 3464–3473.
7. Matsuda, J. L., L. Gapin, B. C. Sydora, F. Byrne, S. Binder, M. Kronenberg, and
R. Aranda. 2000. Systemic activation and antigen-driven oligoclonal expansion
of T cells in a mouse model of colitis. J. Immunol. 164: 2797–2806.
8. Iwasaki, A., and B. L. Kelsall. 1999. Mucosal immunity and inflammation. I.
Mucosal dendritic cells: their specialized role in initiating T cell responses.
Am. J. Physiol. 276: G1074–G1078.
9. Malmstrom, V., D. Shipton, B. Singh, A. Al-Shamkhani, M. J. Puklavec,
A. N. Barclay, and F. Powrie. 2001. CD134L expression on dendritic cells in the
mesenteric lymph nodes drives colitis in T cell-restored SCID mice. J. Immunol.
10. Leithauser, F., Z. Trobonjaca, P. Moller, and J. Reimann. 2001. Clustering of
colonic lamina propria CD4?T cells to subepithelial dendritic cell aggregates
precedes the development of colitis in a murine adoptive transfer model. Lab.
Invest. 81: 1339–1349.
11. Lugering, N., T. Kucharzik, R. Stoll, and W. Domschke. 1998. Current concept
of the role of monocytes/macrophages in inflammatory bowel disease—balance
of proinflammatory and immunosuppressive mediators. Ital. J. Gastroenterol.
Hepatol. 30: 338–344.
12. Grip, O., S. Janciauskiene, and S. Lindgren. 2003. Macrophages in inflammatory
bowel disease. Curr. Drug Targets Inflamm. Allergy 2: 155–160.
13. Allison, M. C., and L. W. Poulter. 1991. Changes in phenotypically distinct
mucosal macrophage populations may be a prerequisite for the development of
inflammatory bowel disease. Clin. Exp. Immunol. 85: 504–509.
14. Norgard-Sumnicht, K. E., L. Roux, D. K. Toomre, A. Manzi, H. H. Freeze, and
A. Varki. 1995. Unusual anionic N-linked oligosaccharides from bovine lung.
J. Biol. Chem. 270: 27634–27645.
15. Srikrishna, G., D. Toomre, A. Manzi, K. Panneerselvam, H. Freeze, A. Varki, and
N. Varki. 2001. A novel anionic modification of N-glycans on mammalian en-
dothelial cells is recognized by activated neutrophils and modulates acute inflam-
matory responses. J. Immunol. 166: 624–632.
16. Srikrishna, G., L. Brive, and H. Freeze. 2005. Novel carboxylated N-glycans
contain oligosaccharide-linked glutamic acid. Biochem. Biophys. Res. Comm.
17. Srikrishna, G., K. Panneerselvam, V. Westphal, V. Abraham, A. Varki, and
H. H. Freeze. 2001. Two proteins modulating transendothelial migration of leu-
kocytes recognize novel carboxylated glycans on endothelial cells. J. Immunol.
18. Srikrishna, G., H. J. Huttunen, L. Johansson, B. Weigle, Y. Yamaguchi,
H. Rauvala, and H. H. Freeze. 2002. N-glycans on the receptor for advanced
glycation end products influence amphoterin binding and neurite outgrowth.
J. Neurochem. 80: 998–1008.
19. Perretti, M. 1998. Lipocortin 1 and chemokine modulation of granulocyte and
monocyte accumulation in experimental inflammation. Gen. Pharmacol. 31:
20. Hofmann, M. A., S. Drury, C. Fu, W. Qu, A. Taguchi, Y. Lu, C. Avila,
N. Kambham, A. Bierhaus, P. Nawroth, et al. 1999. RAGE mediates a novel
5421The Journal of Immunology
proinflammatory axis: a central cell surface receptor for S100/calgranulin Download full-text
polypeptides. Cell 97: 889–901.
21. Abraham, E., J. Arcaroli, A. Carmody, H. Wang, and K. Tracey. 2000. Cutting
edge: HMG-1 as a mediator of acute lung inflammation. J. Immunol. 165:
22. Roth, J., M. Goebeler, and C. Sorg. 2001. S100A8 and S100A9 in inflammatory
diseases. Lancet 357: 1041.
23. Donato, R. 2001. S100: a multigenic family of calcium-modulated proteins of the
EF-hand type with intracellular and extracellular functional roles. Int. J. Biochem.
Cell Biol. 33: 637–668.
24. Schmidt, A. M., M. Hofmann, A. Taguchi, S. D. Yan, and D. Stern. 2000. RAGE:
a multiligand receptor contributing to the cellular response in diabetic vasculopa-
thy and inflammation. Semin. Thromb. Hemost. 26: 485–493.
25. Schmidt, A. M., S. D. Yan, S. F. Yan, and D. M. Stern. 2001. The multiligand
receptor RAGE as a progression factor amplifying immune and inflammatory
responses. J. Clin. Invest. 108: 949–955.
26. Bucciarelli, L. G., T. Wendt, L. Rong, E. Lalla, M. A. Hofmann, M. T. Goova,
A. Taguchi, S. F. Yan, S. D. Yan, D. M. Stern, and A. M. Schmidt. 2002. RAGE
is a multiligand receptor of the immunoglobulin superfamily: implications for
homeostasis and chronic disease. Cell. Mol. Life Sci. 59: 1117–1128.
27. Naka, Y., L. G. Bucciarelli, T. Wendt, L. K. Lee, L. L. Rong, R. Ramasamy,
S. F. Yan, and A. M. Schmidt. 2004. RAGE axis: animal models and novel
insights into the vascular complications of diabetes. Arterioscler. Thromb. Vasc.
Biol. 24: 1342–1349.
28. Chavakis, T., A. Bierhaus, and P. P. Nawroth. 2004. RAGE (receptor for ad-
vanced glycation end products): a central player in the inflammatory response.
Microbes Infect. 6: 1219–1225.
29. Neeper, M., A. M. Schmidt, J. Brett, S. D. Yan, F. Wang, Y. C. Pan, K. Elliston,
D. Stern, and A. Shaw. 1992. Cloning and expression of a cell surface receptor
for advanced glycosylation end products of proteins. J. Biol. Chem. 267:
30. Hofmann, M. A., S. Drury, B. I. Hudson, M. R. Gleason, W. Qu, Y. Lu, E. Lalla,
S. Chitnis, J. Monteiro, M. H. Stickland, et al. 2002. RAGE and arthritis: the
G82S polymorphism amplifies the inflammatory response. Genes Immun. 3:
31. Chavakis, T., A. Bierhaus, N. Al-Fakhri, D. Schneider, S. Witte, T. Linn,
M. Nagashima, J. Morser, B. Arnold, K. T. Preissner, and P. P. Nawroth. 2003.
The pattern recognition receptor (RAGE) is a counterreceptor for leukocyte in-
tegrins: a novel pathway for inflammatory cell recruitment. J. Exp. Med. 198:
32. Hudson, B. I., L. G. Bucciarelli, T. Wendt, T. Sakaguchi, E. Lalla, W. Qu, Y. Lu,
L. Lee, D. M. Stern, Y. Naka, et al. 2003. Blockade of receptor for advanced
glycation endproducts: a new target for therapeutic intervention in diabetic com-
plications and inflammatory disorders. Arch. Biochem. Biophys. 419: 80–88.
33. Fuellen, G., D. Foell, W. Nacken, C. Sorg, and C. Kerkhoff. 2003. Absence of
S100A12 in mouse: implications for RAGE-S100A12 interaction. Trends Immu-
nol. 24: 622–624.
34. Ravasi, T., K. Hsu, J. Goyette, K. Schroder, Z. Yang, F. Rahimi, L. P. Miranda,
P. F. Alewood, D. A. Hume, and C. Geczy. 2004. Probing the S100 protein
family through genomic and functional analysis. Genomics 84: 10–22.
35. Schmid, K. W., N. Lugering, R. Stoll, P. Brinkbaumer, G. Winde, W. Domschke,
W. Bocker, and C. Sorg. 1995. Immunohistochemical demonstration of the cal-
cium-binding proteins MRP8 and MRP14 and their heterodimer (27E10 antigen)
in Crohn’s disease. Hum. Pathol. 26: 334–337.
36. Lawrance, I. C., C. Fiocchi, and S. Chakravarti. 2001. Ulcerative colitis and
Crohn’s disease: distinctive gene expression profiles and novel susceptibility can-
didate genes. Hum. Mol. Genet. 10: 445–456.
37. Robinson, M. J., P. Tessier, R. Poulsom, and N. Hogg. 2002. The S100 family
heterodimer, MRP-8/14, binds with high affinity to heparin and heparan sulfate
glycosaminoglycans on endothelial cells. J. Biol. Chem. 277: 3658–3665.
38. Foell, D., T. Kucharzik, M. Kraft, T. Vogl, C. Sorg, W. Domschke, and J. Roth.
2003. Neutrophil derived human S100A12 (EN-RAGE) is strongly expressed
during chronic active inflammatory bowel disease. Gut 52: 847–853.
39. Perez-Machado, M. A., P. Ashwood, M. A. Thomson, F. Latcham, R. Sim,
J. A. Walker-Smith, and S. H. Murch. 2003. Reduced transforming growth factor-
?1-producing T cells in the duodenal mucosa of children with food allergy. Eur.
J. Immunol. 33: 2307–2315.
40. Goebeler, M., J. Roth, U. Henseleit, C. Sunderkotter, and C. Sorg. 1993. Expres-
sion and complex assembly of calcium-binding proteins MRP8 and MRP14 dur-
ing differentiation of murine myelomonocytic cells. J. Leukocyte Biol. 53: 11–18.
41. Stordeur, P., L. F. Poulin, L. Craciun, L. Zhou, L. Schandene, A. de Lavareille,
S. Goriely, and M. Goldman. 2002. Cytokine mRNA quantification by real-time
PCR. J. Immunol. Methods 259: 55–64.
42. Souza, H. S., C. C. Elia, J. Spencer, and T. T. MacDonald. 1999. Expression of
lymphocyte-endothelial receptor-ligand pairs, ?4?7/MAdCAM-1 and OX40/
OX40 ligand in the colon and jejunum of patients with inflammatory bowel
disease. Gut 45: 856–863.
43. Hokari, R., S. Kato, K. Matsuzaki, A. Iwai, A. Kawaguchi, S. Nagao,
T. Miyahara, K. Itoh, E. Sekizuka, H. Nagata, et al. 2001. Involvement of mu-
cosal addressin cell adhesion molecule-1 (MAdCAM-1) in the pathogenesis of
granulomatous colitis in rats. Clin. Exp. Immunol. 126: 259–265.
44. Beutler, B. 2002. TLR4 as the mammalian endotoxin sensor. Curr. Top. Micro-
biol. Immunol. 270: 109–120.
45. Gordon, S. 2003. Alternative activation of macrophages. Nat. Rev. Immunol. 3:
46. Xu, K., and C. L. Geczy. 2000. IFN-? and TNF regulate macrophage expression
of the chemotactic S100 protein S100A8. J. Immunol. 164: 4916–4923.
47. Raftery, M. J., C. A. Harrison, P. Alewood, A. Jones, and C. L. Geczy. 1996.
Isolation of the murine S100 protein MRP14 (14 kDa migration-inhibitory-factor-
related protein) from activated spleen cells: characterization of post-translational
modifications and zinc binding. Biochem. J. 316: 285–293.
48. Lackmann, M., P. Rajasekariah, S. E. Iismaa, G. Jones, C. J. Cornish, S. Hu,
R. J. Simpson, R. L. Moritz, and C. L. Geczy. 1993. Identification of a chemo-
tactic domain of the pro-inflammatory S100 protein CP-10. J. Immunol. 150:
49. Cornish, C. J., J. M. Devery, P. Poronnik, M. Lackmann, D. I. Cook, and
C. L. Geczy. 1996. S100 protein CP-10 stimulates myeloid cell chemotaxis with-
out activation. J. Cell. Physiol. 166: 427–437.
50. Newton, R. A., and N. Hogg. 1998. The human S100 protein MRP-14 is a novel
activator of the ?2integrin Mac-1 on neutrophils. J. Immunol. 160: 1427–1435.
51. Schlueter, C., S. Hauke, A. M. Flohr, P. Rogalla, and J. Bullerdiek. 2003. Tissue-
specific expression patterns of the RAGE receptor and its soluble forms—a result
of regulated alternative splicing? Biochim. Biophys. Acta 1630: 1–6.
52. Ryckman, C., G. A. Robichaud, J. Roy, R. Cantin, M. J. Tremblay, and
P. A. Tessier. 2002. HIV-1 transcription and virus production are both accentu-
ated by the proinflammatory myeloid-related proteins in human CD4?T lym-
phocytes. J. Immunol. 169: 3307–3313.
53. Neurath, M. F., C. Becker, and K. Barbulescu. 1998. Role of NF-?B in immune
and inflammatory responses in the gut. Gut 43: 856–860.
54. Rogler, G., K. Brand, D. Vogl, S. Page, R. Hofmeister, T. Andus, R. Knuechel,
P. A. Baeuerle, J. Scholmerich, and V. Gross. 1998. Nuclear factor ?B is acti-
vated in macrophages and epithelial cells of inflamed intestinal mucosa. Gastro-
enterology 115: 357–369.
55. Schreiber, S., S. Nikolaus, and J. Hampe. 1998. Activation of nuclear factor ?B
inflammatory bowel disease. Gut 42: 477–484.
56. Neurath, M. F.,I.Fuss, G.Schurmann,
H. Muller-Lobeck, W. Strober, C. Herfarth, and K. H. Buschenfelde. 1998. Cy-
tokine gene transcription by NF-?B family members in patients with inflamma-
tory bowel disease. Ann. NY Acad. Sci. 859: 149–159.
57. Lawrance, I. C., F. Wu, A. Z. Leite, J. Willis, G. A. West, C. Fiocchi, and
S. Chakravarti. 2003. A murine model of chronic inflammation-induced intestinal
fibrosis down-regulated by antisense NF-?B. Gastroenterology 125: 1750–1761.
58. Murano, M., K. Maemura, I. Hirata, K. Toshina, T. Nishikawa, N. Hamamoto,
S. Sasaki, O. Saitoh, and K. Katsu. 2000. Therapeutic effect of intracolonically
administered nuclear factor ?B (p65) antisense oligonucleotide on mouse dextran
sulphate sodium (DSS)-induced colitis. Clin. Exp. Immunol. 120: 51–58.
59. Feizi, T. 2000. Carbohydrate-mediated recognition systems in innate immunity.
Immunol. Rev. 173: 79–88.
60. Dennis, J. W., C. E. Warren, M. Granovsky, and M. Demetriou. 2001. Genetic
defects in N-glycosylation and cellular diversity in mammals. Curr. Opin. Struct.
Biol. 11: 601–607.
61. Lowe, J. B. 2001. Glycosylation, immunity, and autoimmunity. Cell 104:
62. Rudd, P. M., T. Elliott, P. Cresswell, I. A. Wilson, and R. A. Dwek. 2001.
Glycosylation and the immune system. Science 291: 2370–2376.
63. Daniels, M. A., K. A. Hogquist, and S. C. Jameson. 2002. Sweet ‘n’ sour: the
impact of differential glycosylation on T cell responses. Nat. Immunol. 3:
64. Grossmann, M., Y. Nakamura, R. Grumont, and S. Gerondakis. 1999. New in-
sights into the roles of ReL/NF-?B transcription factors in immune function,
hemopoiesis and human disease. Int. J. Biochem. Cell Biol. 31: 1209–1219.
65. Tak, P. P., and G. S. Firestein. 2001. NF-?B; a key role in inflammatory diseases.
J. Clin. Invest. 107: 7–11.
66. Li, Q., and I. M. Verma. 2002. NF-?B regulation in the immune system. Nat. Rev.
Immunol. 2: 725–734.
67. Pagliari, L. J., H. Perlman, H. Liu, and R. M. Pope. 2000. Macrophages require
constitutive NF-?B activation to maintain A1 expression and mitochondrial ho-
meostasis. Mol. Cell. Biol. 20: 8855–8865.
68. Lanzavecchia, A., and F. Sallusto. 2001. Regulation of T cell immunity by den-
dritic cells. Cell 106: 263–266.
69. Bierhaus, A., S. Schiekofer, M. Schwaninger, M. Andrassy, P. M. Humpert,
J. Chen, M. Hong, T. Luther, T. Henle, I. Kloting, et al. 2001. Diabetes-associated
sustained activation of the transcription factor nuclear factor-?B. Diabetes 50:
70. Liliensiek, B., M. A. Weigand, A. Bierhaus, W. Nicklas, M. Kasper, S. Hofer,
J. Plachky, H. J. Grone, F. C. Kurschus, A. M. Schmidt, et al. 2004. Receptor for
advanced glycation end products (RAGE) regulates sepsis but not the adaptive
immune response. J. Clin. Invest. 113: 1641–1650.
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