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Receptor for advanced glycation end products (RAGE) in a
dash to the rescue: inflammatory signals gone awry in the
primal response to stress
Kevan Herold,* Bernhard Moser,
†
Yali Chen,
†
Shan Zeng,
†
Shi Fang Yan,
†
Ravichandran Ramasamy,
†
Jean Emond,
†
Raphael Clynes,
‡
and Ann Marie Schmidt,
†,1
*Department of Medicine, Yale University School of Medicine, New Haven, Connecticut, USA; and Departments of
†
Surgery and
‡
Medicine, Columbia University Medical Center, New York, New York, USA
Abstract: The multiligand receptor for advanced
glycation end products (RAGE) of the Ig superfam-
ily transduces the biological impact of discrete
families of ligands, including advanced glycation
end products, certain members of the S100/cal-
granulin family, high mobility group box-1, mem-
brane-activated complex-1, and amyloid-peptide
and -sheet fibrils. Although structurally dissimi-
lar, at least at the monomeric level, recent evi-
dence suggests that oligomeric forms of these
RAGE ligands may be especially apt to activate the
receptor and up-regulate a program of inflamma-
tory and tissue injury-provoking genes. The chal-
lenge in probing the biology of RAGE and its im-
pact in acute responses to stress and the potential
development of chronic disease are to draw the line
between mechanisms that evoke repair versus
those that sustain inflammation and tissue damage.
In this review, we suggest the concept that the
ligands of RAGE comprise a primal program in the
acute response to stress. When up-regulated in en-
vironments laden with oxidative stress, inflamma-
tion, innate aging, or high glucose, as examples,
the function of these ligand families may be trans-
formed from ones linked to rapid repair to those
that drive chronic disease. Identification of the
threshold beyond which ligands of RAGE mediate
repair versus injury is a central component in de-
lineating optimal strategies to target RAGE in the
clinic. J. Leukoc. Biol. 82: 000 – 000; 2007.
Key Words: inflammation 䡠immunity
THE LIGAND FAMILIES OF RECEPTOR
FOR ADVANCED GLYCATION END
PRODUCTS (RAGE)
Advanced glycation end products (AGEs)
RAGE was first described as a receptor for AGEs, the products
of nonenzymatic glycation and oxidation, which form as post-
translational modifications of proteins and lipids, primarily on
lysine and arginine groups within the backbone protein. AGEs
may transform the configuration and function of the backbone
protein; depending on the site and context, AGEs may modify
long-lived protein species of the vessel wall and tissues, lead-
ing to extensive cross-linking and virtual insolubility [1–3].
AGEs, a heterogeneous group of structures, including such
specific species as carboxy methyl lysine (CML), pentosidine,
and pyralline AGEs, may form in settings such as aging,
hyperglycemia, oxidative stress, renal failure, and inflamma-
tion. AGE modifications may impart gain-of-function proper-
ties in their substrates. For example, AGE modification of
lipoproteins enhances their atherogenic potential [4, 5]. In
chronic disease, AGEs may beget further AGE formation; AGE
interaction with RAGE increases oxidative stress [6 – 8]. In
turn, oxidative stress increases AGE formation; mice deficient
in NADPH oxidase fail to generate the same amount of CML
AGE, as generated by wild-type mice in an inflammatory
milieu [9, 10]. Among the heterogeneous AGEs, CML AGEs
have been shown to be specific AGE ligands of RAGE [11]
(Fig. 1). In cultured endothelial cells (EC), smooth muscle
cells (SMC), and monocytes/macrophages, CML-AGE-RAGE
interaction activated NF-B and up-regulated genes linked to
inflammation. Upon infusion into wild-type mice, CML AGE
resulted in increased expression of VCAM-1 in lung tissue, a
process dependent on RAGE, as pretreatment of the animals
with anti-RAGE IgG suppressed CML AGE-mediated up-reg-
ulation of VCAM-1 [11].
AGEs are formed in diverse organisms, from bacteria, yeast,
and Drosophila to higher-order mammals as a consequence of
the inevitable production of a key pre-AGE methylglyoxal
through the metabolism of D-glucose [12–14]. AGEs are linked
integrally to damage, but are there salutary roles for AGEs?
Potential hints to innate functions for AGEs in modifying
immune responses may be inferred from findings in in vitro
analyses. AGEs may be found on the surface of aging lympho-
cytes [15, 16]. Further, findings in dendritic cells (DC) suggest
that DC glycation may promote their development but impair
1
Correspondence: Division of Surgical Science, Department of Surgery,
Columbia University Medical Center, 630 West 168th Street, P&S 17-501,
New York, NY 10032, USA. E-mail: ams11@columbia.edu
Received December 22, 2006; revised April 14, 2007; accepted April 17,
2007.
doi: 10.1189/jlb.1206751
0741-5400/07/0082-0001 © Society for Leukocyte Biology Journal of Leukocyte Biology Volume 82, August 2007 1
Uncorrected Version. Published on May 18, 2007 as DOI:10.1189/jlb.1206751
Copyright 2007 by The Society for Leukocyte Biology.
their ability to stimulate primary T cell responses [17]. Al-
though potentially injurious in impeding the organism’s re-
sponse to antigen stress, it is conceivable that in certain
settings, T cell or DC glycation may suppress untoward im-
mune responses.
S100/calgranulins
The family of S100/calgranulins is composed of multiple mem-
bers. Although not all members of the family likely bind
RAGE, increasing evidence suggests that S100/calgranulins
beyond S100A12 and S100b bind this receptor. S100/cal-
granulins may be expressed by a wide variety of cell types,
including cells linked to rapid and sustained inflammatory
responses, such as neutrophils, monocytes/macrophages, lym-
phocytes, and DC [18]. The expression of S100/calgranulins by
EC, neurons, and transformed cells suggests that a diverse
array of responses may be elicited by release of these species.
S100/calgranulins are primarily intracellular molecules; how-
ever, upon their release into the extracellular space by auto-
crine and/or paracrine interactions, they may gain new func-
tions via their ability to bind cell surface receptors such as
RAGE [19].
In vitro analyses in cultured neurons suggested that one
measure potentially distinguishing the adaptive versus injuri-
ous impact of S100b was the “dose” to which the cells were
exposed. Whereas low (nM) concentrations of S100 mediated
survival, exposure of cultured neuronal and glioma cells re-
sulted in recruitment of proinjury pathways [20].
Thus, these concepts suggest that far from being inert,
intracellular molecules, linked solely to calcium binding and
its consequences inside the cell, S100/calgranulins possess
distinct functions and importance in the biology of the cell
through homeostasis to crisis.
High mobility group box-1 (HMGB1)
Akin to S100/calgranulins, HMGB1 usually is expressed in the
intracellular space, specifically in the nucleus, where these
molecules play roles as nonhistone, DNA-binding molecules.
Like S100/calgranulins, “activation” stimuli may trigger re-
lease of HMGB1 onto the surface of highly activated cells or
directly into the extracellular space [21, 22]. It is in that
context, we propose, that HMGB1 may be freed to interact with
RAGE.
Studies reported in the mid-1990s uncovered for the first
time that HMGB1, or amphoterin, was a signal transduction
ligand of RAGE. In the first studies, RAGE and HMGB1 were
found to colocalize in the developing cerebral cortex of embry-
onic rats; cell culture analyses suggested that the HMGB1-
RAGE interaction contributed to outgrowth of neurites in neu-
rons from prenatal rat brain [23]. It is intriguing that the
HMGB1-RAGE interaction exerted its influence in migrating
cells; specifically, HMGB1 and RAGE are expressed by trans-
formed cells, and their interaction is linked to activation of cell
migration and possibly, mechanisms linked to tumor metasta-
sis, such as activation of matrix metalloproteinases (MMPs).
Studies in vivo in murine tumors indicated that administration
of a soluble receptor decoy of RAGE, sRAGE, sharply limited
local tumor growth and particularly, metastases in vulnerable
mice [24]. In parallel, activity of MMPs and MAPK activation
was reduced greatly by RAGE blockade [24].
The studies of Tracey and colleagues [25] elucidated for the
first time the provocative possibility that HGMB1 was linked to
amplification of inflammatory mechanisms as a late mediator of
the impact of endotoxin. Treatment of RAW 264.7 murine
macrophages with LPS evoked up-regulation and release of
HMGB1 8 h after incubation. In vivo, blocking antibodies to
HMGB1 protected rodents against the impact of overwhelming
sepsis [25]. Recent studies suggest the possibility that HMGB1
may not only interact with RAGE but as well, certain Toll
receptors, such as TLR2 and TLR4 [26].
DC express HMGB1 and RAGE; release of HMGB1 by these
cells provokes clonal expansion, survival, and functional po-
larization of differentiating T cells, at least in part through
activation of MAPKs and NF-B [27]. Further experimentation
reveals that at least in vitro,HMGB1 stimulates up-regulation
of CCR7 and CXCR4 chemokine receptors in DC and their
migratory ability [22]. Although these studies were limited to
the in vitro milieu, they nevertheless suggest the possibility
that the RAGE axis plays critical roles in the adaptive immune
response. These implications from cell culture analyses require
rigorous validation in vivo.
Membrane-activated complex-1 (Mac-1)
RAGE is an endothelial adhesion receptor, which mediates
direct interaction with the 2 integrin Mac-1. RAGE-Mac-1
interaction is enhanced by incubation with the proinflammatory
RAGE ligand, S100B [28]. Recent studies indicated that
HMGB1-mediated recruitment of neutrophils was dependent
on Mac-1 but not on LFA-1. In bone marrow chimera experi-
ments, Mac-1-dependent neutrophil recruitment induced by
HMGB1 required the presence of RAGE on neutrophils but not
on EC [29]. Thus, a HMGB1-dependent pathway for inflam-
Fig. 1. CML structure. Previous studies indicated that CML AGEs are
prevalent products, which accumulate in the tissues in such settings as
inflammation, hyperglycemia, and renal failure. CML adducts are specific
ligands of RAGE. The structure of this prevalent species is depicted in the
figure.
2 Journal of Leukocyte Biology Volume 82, August 2007 http://www.jleukbio.org
matory cell recruitment and activation requires the interplay
between RAGE and Mac-1. These findings establish mecha-
nisms by which RAGE and Mac-1, via HMGB1, may be linked
to acute inflammatory responses initiated by neutrophil recruit-
ment and activation.
Amyloid-peptide (A) and -sheet fibrils
In addition to AGEs, S100/calgranulins, HMGB1, and Mac-1,
RAGE is also a signal transduction receptor for Aand
-sheet fibrils [30, 31]. An emerging concept is that although
seemingly dissimilar, common features envelop many of the
ligand families of RAGE, perhaps leading to recognition by
identical or at least closely neighboring sites within the extra-
cellular domain of RAGE, specifically, but possibly not exclu-
sively, in the V-type Ig domain of RAGE [19]. Many of ligands
of RAGE are found in monomeric and oligomeric forms. Al-
though soluble, monomeric ligands interacted with RAGE in
ARPE-19 cells, their ability to stimulate signal transduction
and modulation of gene expression via RAGE was enhanced
significantly in the oligomeric state [32].
Furthermore, the ligands of RAGE may potentiate each
other’s formation and aggregation; AGE precursor species
methylglyoxal and glyoxal may increase the aggregation and
cytotoxicity of intracellular Acarboxy-terminal fragments
[33]. In addition, it has been suggested that glycation stimu-
lates amyloid formation [34, 35]. These concepts suggest that
the ligands of RAGE may indeed supermodify each other and
that the ligands may be more similar than distinct. Studies are
gaining first insights into the structure of the receptor and
providing physical evidence for the basis of ligand-RAGE
interactions [36].
SIGNAL TRANSDUCTION—CENTRAL TO THE
BIOLOGICAL IMPACT OF LIGAND-RAGE
INTERACTION
The ligands of RAGE share common properties upon their
binding to the receptor. First, among the known ligands, such
as AGEs, S100A12 and S100b, and HMGB1, Aand -sheet
fibrils, there is cross-competition in radioligand-binding assays
[11, 19]. Second, RAGE ligands bind to the V-type Ig domain,
as elucidated by radioligand-binding assays to recombinant V
domain [11]. Although we were unable to show that ligands
bound C-type domains directly, recent studies suggested hex-
americ forms of S100A12 bound the C-type Ig domain of
RAGE [36]. Third, the binding affinities, as established in
radioligand-binding assays for ligand binding to RAGE, are
quite similar in the nM range (⬃50 nM). Fourth, extensive
evidence indicates that each of the RAGE ligands exerts its
impact as a consequence of RAGE-mediated signal transduc-
tion. The cytoplasmic domain of RAGE is essential for RAGE-
mediated changes in gene expression and cellular properties
[11, 19]. These studies suggested that the interaction of ligands
with RAGE activates signaling pathways and thus, the platform
to alter patterns of gene expression in the cell.
RAGE is expressed in multiple, distinct cell types; thus, it
is not surprising that diverse signal transduction pathways may
be impacted by RAGE. An important but not sole means by
which RAGE exerts effects on gene expression is via activation
of NF-B [7, 37], which impacts proinflammatory/prodeath and
prosurvival pathways depending on the cell type and context.
Multiple experiments have suggested that the ligand-RAGE
interaction in cells such as EC, SMC, monocytes/macrophages,
and neurons activates NF-B. Generation of reactive oxygen
species (ROS) is a key intermediate step, at least in certain
cases, as pretreatment of EC, for example, with antioxidants,
suppresses AGE-RAGE-mediated activation of NF-B [38].
RAGE-mediated activation of NADPH oxidase may account, at
least in part, for these observations [6, 8].
Multiple members of the MAPK family have been shown to
be activated by RAGE; the ligand-RAGE interaction activates
p44/p42 (ERK) MAPK, p38 MAPK, and JNK MAPK [24, 39,
40]. In addition, other studies have illustrated that AGE-
mediated activation of ras and src kinase via RAGE in SMC is
a key step in activation of NF-B [37, 41]. The specific
signaling pathways triggered by RAGE are influenced by the
context of stimulatory signals; in the setting of arterial injury,
for example, RAGE-mediated activation of JAK/STAT path-
ways critically impacts on SMC proliferation and migration
[42]. In monocytes/macrophages, NF-B is a central target of
ligand-RAGE. Recent studies suggested that in cultured mi-
croglial cells, RAGE-mediated up-regulation of cyclooxygen-
ase-2 required recruitment of cdc42/rac and JNK MAPK signal
transduction [43].
Consistent with the concept that in distinct cell types and
forms of stress, diverse signaling may be impacted by RAGE,
it has been shown that in cultured mesangial cells, AGE-
RAGE-mediated generation of ROS triggered TGF-/Smad
signaling [44]. In other cell types, cultured, primary, sensory
neurons exposed to S100 displayed increased caspase-3 activ-
ity and nuclear DNA degradation, at least in part via activation
of PI-3K signaling [45].
Under intense investigation at this time is the precise means
by which the short, highly charged, cytoplasmic domain of
RAGE signals. Earlier reports suggested that this domain
interacted with ERK MAPK [46]; however, it is unlikely that
such findings explain or underlie the diverse signal transduc-
tion repertoire of RAGE. Further, essential to establish will be
if and how distinct ligands of RAGE may stimulate specific (or
not) signaling pathways. In addition, the complexity of this
system is enhanced by the concept that the ligands of RAGE
may interact with distinct binding molecules themselves. For
example, HMGB1 may interact with TLR2 and -4; thus, it is
possible that RAGE-distinct signaling may be characteristic of
those ligands [26]. In other settings, it was suggested that
S100b activation of myotubes was independent of RAGE,
although the specific, distinct receptors were not identified
[47]. Furthermore, it has been reported that AGEs may bind
CD36 and other scavenger receptors (SRs) [48]. Taken to-
gether, these observations suggest that specific tools, such as
RAGE antagonism and RAGE null mice, would be essential in
dissecting the specific role for RAGE in biological responses.
Based on the striking ability of RAGE ligands to activate
signal transduction and thereby, alter gene expression patterns,
it was reasonable to test these concepts in vivo. Integral to the
biology of RAGE and its ligands is their up-regulation and
Herold et al. RAGE, primal responses to stress and chronic disease 3
increased accumulation in multiple biological and disease
settings. In the sections to follow, we present evidence linking
ligand-RAGE signaling to fundamental mechanisms in the
inflammatory response.
TESTING THE ROLE OF RAGE IN
INFLAMMATION—FIRST STUDIES TESTING
PHARMACOLOGICAL ANTAGONISM OF THE
LIGAND-RAGE AXIS
Our earliest work suggested that the biological repertoire of the
AGE-RAGE interaction contributed to the pathogenesis of
diabetic complications. The discovery of S100/calgranulins
and HMGB1 as putative ligands of RAGE compelled us to
consider that RAGE played roles in inflammatory responses,
even in the absence of hyperglycemia. Using sRAGE, an
extracellular ligand-binding decoy of RAGE, prepared and
purified in a baculovirus expression system, and F(ab⬘)
2
frag-
ments of anti-RAGE IgG or anti-S100A12 IgG, experimenta-
tion revealed that blockade of ligand-RAGE suppressed the
challenge phase of footpad edema in which infiltration of
inflammatory cells and granuloma formation developed in mice
sensitized and challenged with methylated BSA. In parallel,
nuclear extracts retrieved from RAGE-antagonized mouse
foodpads revealed strikingly diminished activation of the
proinflammatory transcription factor NF-B [19]. In mice
highly vulnerable to colitis mediated by genetic deficiency of
IL-10, chronic administration of sRAGE reduced gut inflam-
mation and activation of NF-B [19]. In parallel, gene expres-
sion was altered in sRAGE-treated mice, as animals treated
with sRAGE displayed decreased levels of TNF-␣in plasma
[19].
These proinflammatory effects of RAGE signaling were
probed further in a murine model of bovine Type II collagen-
induced arthritis in DBA/1 mice; significant reduction in joint
swelling and erythema was noted when mice were treated with
sRAGE. In the context of inflammatory arthritis, a possible
genetic link to RAGE was uncovered by the observation that a
genetic variant of RAGE, the G82S polymorphism, was in
linkage disequilibrium with HLA-DR4. In in vitro analyses,
transfected cells expressing G82S displayed increased binding
affinity to RAGE ligand S100A12 and enhanced generation of
cytokines and MMPs upon transfection with G82S versus the
wild-type allele in the presence of S100A12 [49]. Studies in
human subjects with rheumatoid arthritis (RA) supported this
genetic link. However, based on association studies in cell
culture, future experiments are required to probe if the pres-
ence of this variant is linked to the extent or temporal appear-
ance of joint and bone destruction in RA.
RAGE AND IMMUNE RESPONSES: STUDIES
PROBING CELLULAR CONTRIBUTIONS OF
LIGAND-RAGE AXIS
The next step in probing the mechanisms linked to ligand-
RAGE signaling in immune/inflammatory consequences was to
specifically dissect the cellular mechanisms underlying these
observations. Administration of sRAGE and studies in homozy-
gous RAGE null mice revealed that the impact of sepsis
induced by cecal ligation and puncture was attenuated signif-
icantly by antagonism or deletion of RAGE [50]. When ho-
mozygous RAGE null animals were reconstituted with endo-
thelial or hematopoietic cell expression of the receptor, the
protective impact of the global deletion mutant was reversed
[50]. One interpretation of these findings was that RAGE was
linked to amplification of inflammation in sepsis but not to the
adaptive immune response. Studies using distinct, cell-specific
mutants of RAGE, however, suggest that in specific settings,
RAGE may play roles in adaptive immune mechanisms.
RAGE and the pathogenesis of Type 1 diabetes:
studies in NOD mice
Roles for RAGE in inflammatory signaling were further studies
in NOD/scid mice subjected to adoptive transfer of diabeto-
genic spleen cells. Compared with baseline, pancreata from
diabetic NOD/scid mice, which had received a transfer of
splenocytes, revealed marked up-regulation of RAGE and
S100 on islet cells containing an inflammatory infiltrate. In
parallel, it was shown that RAGE was also expressed in a
population of T cells (CD4⫹and CD8⫹) and B cells [51].
To test the potential role of RAGE in mediating autoimmune
diabetes, NOD/scid mice receiving a transfer of splenocytes
from a diabetic NOD donor were treated with sRAGE or
vehicle, murine serum albumin. Animals treated with sRAGE
displayed significant reduction in the rate of transfer of diabe-
tes. By Day 36 after transfer, 92% of control animals but only
10% of mice treated with sRAGE became diabetic. In parallel,
gene expression patterns were altered when RAGE signaling
was impacted; levels of cytokines IL-1and TNF-␣were
reduced significantly in the sRAGE-treated islets compared
with murine serum albumin-treated animals. The expression of
IL-10 was increased in sRAGE-treated mice islets, along with
increased TGF-, compared with vehicle-treated animals [51].
In contrast to injection of diabetogenic splenocytes, when
preactivated, diabetogenic BDC2.5 cells were injected into
mice, sRAGE displayed no effect on prevention of diabetes
[51]. These investigations suggested that the ligand-RAGE axis
contributed, in part, to T lymphocyte priming or cognate DC-T
lymphocyte interactions. Studies are underway to address these
concepts; first studies in cultured T cells suggest that blockade
of RAGE attenuates T lymphocyte proliferation in response to
allostimulation [52].
Roles for RAGE in T lymphocyte responses in
the adaptive immune response
Prompted by these findings in vitro, we probed roles for RAGE
in T lymphocytes in an established model of heterotopic,
allogeneic heart transplantation, wherein fully mismatched
grafts (donor, CD1 strain, H2
q
) were transplanted into C57BL/6
recipients (H2
b
). Mice were treated with sRAGE, 100 or 200
g/day, beginning 1 day prior to transplantation and continued
once daily until sacrifice. Control animals received equal vol-
umes of PBS. The mean graft survival time in vehicle (PBS)-
treated mice was 7.3 ⫾0.7 days. Mice treated with sRAGE,
100 g/day, displayed significantly increased graft survival,
4 Journal of Leukocyte Biology Volume 82, August 2007 http://www.jleukbio.org
11.7 ⫾1.7 days. In animals treated with the higher dose of
sRAGE, 200 g/day, graft survival time was even greater, at
19.5 ⫾2.8 days [52].
Immunofluorescence microscopy revealed a significant re-
duction in cells expressing RAGE in sRAGE-treated graft
recipients. In parallel, S100 and HMGB1-expressing cell
staining was reduced significantly in hosts that received
sRAGE. Inflammation was, in turn, reduced significantly in the
sRAGE-treated grafts. Compared with PBS-treated animals,
mice treated with sRAGE displayed significantly less edema
and inflammatory cell infiltration, including reduced numbers
of T lymphocytes [52].
The finding that significantly less T cells were present in the
sRAGE-treated allografts led us to test the hypothesis that
RAGE modulated alloimmune responses directly. We used
purified T cells and MHC Class II
⫹
APC retrieved from MHC-
mismatched mice and used sRAGE and blocking antibodies to
the receptor. Incubation with sRAGE resulted in a statistically
significant, dose-dependent decrease in lymphocyte prolifera-
tion versus IgG control-treated cultures. To substantiate the
specific role of RAGE, cells in the mouse allogeneic mixed
lymphocyte culture were incubated with blocking antibodies to
RAGE. Compared with nonimmune IgG, incubation with
monoclonal anti-RAGE IgG resulted in a statistically signifi-
cant, dose-dependent decrease in lymphocyte proliferation
[52].
These findings suggested that sRAGE suppressed donor-
reactive, T cell-priming responses and led us to test the effect
of RAGE antagonism on human lymphocyte proliferation trig-
gered by alloresponses. Incubation with sRAGE resulted in a
statistically significant decrease in lymphocyte proliferation
versus control-treated cultures. Similar effects were noted with
anti-RAGE IgG. We propose that ligands released from or
presented on the surface of the irradiated cells provided the
stimulus for proliferation and activation of lymphocytes [52].
Studies are underway at this time to elucidate the precise
signaling mechanisms impacted by RAGE in T cell-prolifera-
tive and cytokine responses.
Further studies suggested the importance of RAGE in mod-
ulating T cell infiltration into immune/inflammatory sites. In a
murine model of multiple sclerosis, experimental autoimmune
encephalomyelitis (EAE), RAGE, and its inflammatory ligand
S100 were overexpressed. Blockade of RAGE, using sRAGE,
suppressed EAE disease induced by myelin basic protein
(MBP) peptide or encephalitogenic T cells or when EAE oc-
curred spontaneously in the TCR-transgenic mice devoid of
endogenous TCR-␣and TCR-chains. In these studies, the
striking impact of RAGE antagonism was evident by markedly
decreased infiltration of the spinal cord by immune and in-
flammatory cells. In parallel, nuclear extracts retrieved from
spleen tissue revealed marked reduction in activation of NF-
B, thereby suggesting that RAGE-mediated signaling ac-
counted, at least in part, for genes in gene expression and
cellular properties [53].
Thus, to address the role of CD4 T cell RAGE signaling
directly in these processes, transgenic mice were generated
with targeted overexpression of dominant-negative (DN) RAGE
in CD4⫹T cells. DN RAGE consists of the extracellular and
membrane-spanning domain of RAGE; solely, the RAGE cy-
tosolic domain is deleted. Thus, although RAGE ligands may
bind the truncated receptor, they are unable to affect signal
transduction via RAGE. Compared with wild-type littermate
mice, transgenic CD4 DN RAGE mice were resistant to MBP-
induced EAE [53]. These data reinforced the significant impact
of CD4⫹T cell signaling in RAGE-dependent, adaptive im-
mune responses and underscored roles specifically for RAGE
in mediating migration of effector cells into immune foci.
To further probe the specific mechanisms linking RAGE to
T cell responses, we recently used OT-II T cells reactive with
OVA. Preliminary studies using RAGE-expressing versus
RAGE null OT-II cells suggest that RAGE is required for
effective T cell priming. Experiments are underway to delin-
eate the specific signal transduction mechanisms underlying
these findings [54].
Roles for RAGE in DC/macrophage responses in
the adaptive immune response
Extensive studies, particularly in vitro, highlighted roles for
RAGE signaling in monocyte/macrophage migration and acti-
vation, the latter as defined by up-regulation of proinflamma-
tory factors such as cytokines and MMPs. These concepts were
probed in vivo in a murine model of massive liver injury. The
ability of the liver to regenerate is finite; in experimental
systems, 70% resection of the liver triggers a fully effective
regeneration program in which liver mass is restored in parallel
with function. However, in contrast, when 20% more liver
tissue is removed (85% resection), the threshold for recruit-
ment of effective regeneration programs is exceeded in rodents,
such that overwhelming inflammation and apoptosis of the
remnant ensue. Given the strong link to inflammatory mecha-
nisms in this setting, we probed the role of the RAGE axis.
The first suggestion that RAGE might be implicated in
massive liver injury was the observation that consequent to
resections, RAGE mRNA transcripts were up-regulated selec-
tively in the 85% but not 70% resection setting. Immunohis-
tochemistry using anti-RAGE IgG revealed the intriguing find-
ing that the principal site of RAGE expression in the remnant
after massive resection was in cells expressing CD11c and
CD68, thus suggesting that RAGE was expressed largely in
mononuclear phagocyte (MP)-derived DC (MPDDC) [55].
These observations led us to posit that MPDDC, in part via
RAGE, modulated inflammatory responses in the liver rem-
nant, which contributed to massive apoptosis and failure of
regeneration.
To address these concepts, we first administered sRAGE to
the animals undergoing massive resection. Compared with
vehicle-treated mice, a significant increase in survival of wild-
type C57BL/6 mice was noted in sRAGE-treated mice; the
effects of sRAGE were dose-dependent. To target RAGE and
its inflammatory ligands directly, we prepared F(ab⬘)
2
frag-
ments of anti-RAGE IgG, anti-S100 IgG, and anti-HMGB1
IgG. Compared with administration of nonimmune F(ab⬘)
2
frag-
ments, mice treated with anti-RAGE, anti-S100, or anti-
HMGB1 F(ab⬘)
2
fragments displayed significantly increased
survival. Consistent with the concept that sRAGE trapped
RAGE ligands, we found that when plasma of sRAGE-treated
mice was subjected to immunoprecipitation with anti-RAGE
IgG, followed by immunoblotting of bound material with anti-
Herold et al. RAGE, primal responses to stress and chronic disease 5
bodies to S100/calgranulin, S100/calgranulin epitopes were
revealed. Thus, studies using sRAGE and these antiligand
antibody fragments strongly suggested that the ligand-RAGE
axis contributed to impaired survival and failure of regenera-
tion in these remnants [55].
In liver resection, it is well-established that inflammation
must be tempered appropriately, such that proregenerative and
not prodeath pathways would be recruited selectively. Our
findings revealed that administration of sRAGE modulated
cytokine expression in the remnant and drove early up-regu-
lation of NF-B activity, in parallel, with reduction in TUNEL-
expressing cells and decreased activation of caspase-3. As
sRAGE and anti-RAGE F(ab⬘)
2
fragments would be expected
to target the remnant globally, we prepared transgenic mice in
which RAGE signaling would be mutated in cells of MP
lineage, including cells expressing CD11c and CD68, as driven
by the macrophage SR Type A (SR-A) promoter, referred to as
SR DN RAGE mice. Compared with wild-type mice, we found
that transgenic SR DN RAGE mice displayed significantly
improved survival, regeneration of the hepatic remnant, mod-
ulation of proregenerative cytokines, and early up-regulation of
NF-B activity. It is important to note that by using this
promoter, it was not possible to dissect the specific impact of
infiltrating monocytes/macrophages, Kupffer cells, or MPDDC.
However, it was evident that blunting RAGE impact in cells of
MP lineage restored effective regeneration, even in the face of
massive resection of the liver [55].
Thus, in these studies, blockade of RAGE in massive liver
resection restored beneficial, inflammatory responses and ac-
tivation of NF-B, suggesting that RAGE played key roles in
modulation of these central pathways in inflammatory mecha-
nisms. These considerations prompted us to probe the hypoth-
esis that at least in certain settings, RAGE might contribute to
beneficial, inflammatory mechanisms.
RAGE and inflammatory mechanisms linked to
nerve regeneration
The specific hypothesis that RAGE-dependent mechanisms
might mediate adaptive repair as a consequence of inflamma-
tory signaling was addressed in a murine model of unilateral
crush of the sciatic nerve. Upon crush of the nerve, rapid
recruitment of proinflammatory mechanisms ensues, which
contributes to adaptive remodeling in the crushed nerve seg-
ments, along with up-regulation of regenerative pathways. Our
studies revealed that RAGE and its ligands, particularly S100/
calgranulins and HMGB1, were up-regulated rapidly within
hours of sciatic nerve crush. RAGE expression was up-regu-
lated in macrophages and in axonal elements within the
crushed nerve segment [56]. As these experiments placed
RAGE and its ligands at the site of peripheral nerve crush, it
was logical to explore the outcome of RAGE blockade in this
context. Did RAGE contribute to repair versus failure of re-
generation?
Fig. 2. RAGE and key roles in acute stress versus pathways linked to chronic disease— hypotheses and unifying concepts. We hypothesize that the ligands of
RAGE possess adaptive roles in primal responses to short and self-limiting stresses, which accompany host existence within their environment. In uncomplicated
environments, such stresses may promote release of RAGE ligands, largely in monomeric forms, in a manner linked to their rapid engagement of primarily innate
immune receptors and to a degree, RAGE. Rapid detoxification and removal of these ligands after the burst of release and response to stress ensure repair and
return to homeostasis. In contrast, in complex settings, chronic inflammation, hyperglycemia, and innate aging prime these ligands to undergo supermodification,
in which oligomeric forms may predominate. We predict that under such conditions, these ligand configurations recognize and activate RAGE preferentially and
chronically. In turn, the consequences of such interactions favor long-term tissue stress. Identification of strategies to retain primal RAGE responses to stress, yet
derail amplification pathways, which damage tissues irrevocably, holds great promise in harnessing lessons learned from the biology of RAGE to clinical trials.
6 Journal of Leukocyte Biology Volume 82, August 2007 http://www.jleukbio.org
To address these questions, we administered sRAGE or
blocking F(ab⬘)
2
fragments derived from anti-RAGE IgG to
wild-type mice subjected to acute crush to the sciatic nerve.
Motor and sensory conduction velocities, walking tract analy-
ses, and regeneration, as assessed by myelinated fiber densi-
ties, were impaired in RAGE-blocked mice. Histological and
molecular analysis revealed that macrophage infiltration and
thus, Wallerian degeneration were reduced in RAGE-blocked
mice [56]. To determine if macrophage and/or neuronal RAGE
signaling participated critically in the response to nerve crush,
transgenic mice expressing DN RAGE in macrophages or
peripheral neurons, driven by the SR-A or thy-1 promoter,
respectively, were used. Compared with littermate controls,
regeneration was delayed in the transgenic mouse but espe-
cially, in double-transgenic mice expressing DN RAGE in
macrophages and peripheral neurons [57].
These studies were the first to demonstrate that recruitment
of RAGE facilitated beneficial inflammatory mechanisms. Cer-
tainly, however, the biology of tissue repair in the context of
RAGE is complex, as administration of sRAGE to diabetic
mice subjected to full-thickness excisional wounds accelerated
wound healing, largely by suppression of exaggerated inflam-
matory mechanisms and blunting of excessive MMP activity. In
contrast, when sRAGE was administered to nondiabetic mice,
no impact, beneficial or deleterious, was observed on wound
closure, perhaps as the wounds close quite rapidly [58]. Fur-
ther, in bacterially challenged mice, periodontal wound healing
was improved in diabetic mice treated with sRAGE; in parallel,
cytokine generation and MMP activity in gingival tissue were
reduced significantly [59].
PERSPECTIVES AND HYPOTHESES
In the full context of the biological response to stress, exquisite
control of inflammatory mechanisms is critical to executing the
best balance between rapid resolution of injury versus ampli-
fication of proinjury pathways. Thus, it is not surprising that
multiple receptors and pathways and their cross-talk must be
involved in the primal response to acute stress (Fig. 2). In the
biology of RAGE, we propose that the ligand families, which
may mediate rapid repair in such responses, are likely one and
the same, sustaining and mediating chronic tissue perturbation
and injury. We predict that the distinction will lie in the
specific environments and forms of presentation of RAGE
ligands. Common to many of the ligand families of RAGE is the
ability to form oligomers, rendering them more apt to bind and
activate RAGE. Environmental modulation of the ligand fam-
ilies, such as in chronic diseases beset by hyperglycemia,
aging, obesity, chronic inflammation, or autoimmunity, as ex-
amples, may supermodify these ligands, thus tipping the bal-
ance between “forms” likely to mediate rapid repair versus
those that engage the receptor chronically (Fig. 2). Indeed, we
predict that in simpler, monomeric forms, such as in uncom-
plicated and rapidly resolving acute stresses, these ligands
may activate RAGE and other innate receptors but that the
environment facilitates rapid removal of these ligands as repair
ensues. However, in chronically stressed environments, we
propose that these ligands may be more apt to achieve higher
order forms that cross the threshold to recognize RAGE
strongly and perhaps selectively. It is possible that aggregation
mechanisms may overwhelm natural clearance systems and
over-run the chronically injured environment and lead to sus-
tained recruitment and activity of RAGE.
In conclusion, striking the optimal balance in therapeutic
targeting of RAGE will require crossing a fine line of resolution
and repair between acute and chronic stimulation and thus,
resolution and repair or irreversible tissue injury. If our pre-
dictions are correct, then a chief challenge of ongoing work is
to identify the threshold for the ability of ligands to recruit
RAGE in distinct stresses. Once so identified, the optimal
pathways to targeting the receptor selectively in maladaptive
stress are likely to be uncovered.
ACKNOWLEDGMENTS
This work was supported by grants from the United States
Public Health Service and the Juvenile Diabetes Research
Foundation.
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