Jpn. J. Infect. Dis., 54, 209‑219, 2001

Invited Review

Regulation of Innate Immune Responses by Toll‑Like Receptors
Kiyoshi Takedal･2 and Shizuo Akiral･2*
JDepartment of Host Defense, Resewch lnstitutefor Microbial Diseases. Osaka Universio'and
Zsolutionl0riented Research for Science and Technology, Japan Science and花chnology Corporation,

Yamada‑oka 3‑I, Suita, Osaka 565‑0871, Japan(Received October 1 1, 2001)

CONTENTS:
1. Introduction

2. Innate immune responses in Drosophila
3. Innate immune activation by Tb1日ike receptors (TLRs)

3‑ I. Identification ofTLRs in mammals
3‑2. TLR4 is a signaling component of the lipopolysaccharide (LPS) receptor
3‑3. The TLR family is responsible for recognltlOn Of microbial components
4. Signaling pathway via TLRs

4‑1 ･ Comparison of signaling pathways with Dnosophila Toll and mammalian TLR
4‑2. MyD88 is a critical component in TLR signaling
413. MyD881independent TLR signaling
5･ Suppression of innate immune cell activity by microbial components

5‑1. Mechanism for LPS tolerance
5‑2. Microbial components induce LPS tolerance
6. Future prospects

SUMMARY: 1mmate irrmune response in DT10SOPhila is mediated by signaling through Toll receptors･ In marrmals,
Tol1‑like receptors (TLRs), comprising a large family, recognize a specific pattem of microbial components･ So

far,the roles ofTLR2, TLR4, TLR5, TLR6, and TLR9 have been revealed. The recognition ofmicrobial components

by TLRs leads to activation of innate immunity, which provokes inflammatory responses and finally the developmentof adaptlVe immunity. The inflammatory response depends on a TLR‑mediated MyD88‑dependent cascade･
However, there seems to exist additional cascades in TLR signaling. ln the case ofTLR4 signaling, an MyD88‑
independent pa血way lS now being characterized. In addition to the activation of imate immune responses, TLR一

mediated signaling leads to suppression of the activity or innate immune cells, represented by Hlipopolysaccharide(LPS) tolerance". Progress in elucidating the molecular mechanisms fわr LPS tolerance has been made throughthe analysis ofTLR‑mediated signaling pathways. Thus, the activity for innate immune responses isknown to befinely regulated by TLRs.

Thus, a血ptive immunity lS a highly sophisticated system to

1. IntroductiorL

combat microorganisms. In contrast, activation or innate

types of immunity, innate and adaptive (acquired) immunity

immunityis dependent on germline.lenCOded receptors torecognize microorganisms, and innate Impunity seems to be a

( 1 ). Imate in皿unity is phylogenetically conseⅣed and present

primitive system compared with adaptive Immunity. However,in almost all multicellular organisms, whereas adaptlVe

irmate immunlty Plays an important role not only in the first

Host defense agalnSt invasion by pathogens relies on twoline of host defense against invasion by microorganisms but

immunity is not found in invertebratesI Adaptive immunitylS a System Whereby a foreignantlgen is recognized by antlgen

also in the instruction of adaptive immunity (2,3). T cell

receptors expressed on the surface of B and T lymphocytes.

receptors recognlZe PePtide antlgenS that are processed in and

In order to cope with a variety of antigens, B and T cells

presented with major histocompatibilitycomplex (MHC) classreamnge genes fb∫ immunoglobulin and the T cell receptor,

I and class ll on antigen‑presentlng Cells. Furthemore, activa‑

to produce over 1 0 I I types of antigen receptors. Lymphocytes

tion and differentiation of naive T cells into type I helper T

bearing receptors that have suitable affinity to a specific antigen

cells is mediated by co‑stimulatory molecules expressed onshow clonal expansion when stimulated with the antigen.

the antlgen‑PreSentlng Cells, and cytokines such as interleukin‑
1 2 (lL 1 2) produced by the antigen‑presenting cells (4). These
antigen‑presenting cells include dendritic cells (DCs) and*Corresponding author: Tel: +81‑6‑6879‑8303, Fax: +8 I‑6‑6879‑

macrophages, both orwhich play an important role in innate

8305, E‑mail: sakira@biken.osaka‑u.ac.jp

immunity by recognlZlng and up‑taking pathogens.

In Drosophila, which has irmate immunitybut not adaptive
This article is an Invited Review based on a lecture presented at

immunity, slgnaling pathways via Tわll receptors have been

the 1 lth Symposium of the NationaHnstitute of Infectious

shown to play Important roles in the host defense agalnSt

Diseases, Tbkyo, 21 May 2001.

invasion by microorganisms. Tわll‑like receptors (TLRs) were209

subsequently identified in mammals. Evidence is now accumul

transmembrane receptor, Toll･ An adaptor Tube associates withlating that TLRs are key receptors for innate immune recognl‑

the cytoplasmic portion of Tわll and transduces the signal totion. In this review, we will focus on recent findings on TLRs,

activate the serine/threonine kinase Pelle in response towhich are responsible f♭r the recognltlOn Or pathogens andSpatzle. In 1996,the Toll signaling pathway was shown to be
involvedinthe induction ofanantifungal peptide, Drosomycln.

regulation of innate immune activation.

Accordingly, mutant flies lacking Toll were found to be highly
susceptible to fungal infection (6). Subsequently, genetic

2. Innate immune responses in Dyosophl'ta

studies with mutant nies that show high susceptibility toln Drosophila, host defense is elicited by virtue of thesynthesis ofpq)tidesinresponse tofimgalinvasion (Drosomycin)

microbial infections have established that DIF and Dorsal

or bacterial invasion (Diptericin, Drosocin, Cecropin, Attacin,

whereas Relish controls antibacterial responses (7‑ 1 0). Relish

Defensin). The genes encoding these anti‑microbial peptides

mutant flies do not induce the antibacterial peptide Diptericin

mediate ant汎Ingal responses in the Tわll signaling pathway,

possess, in the promoter reglOnS, binding motifs analogous toand unlike the Toll mutant nies, show high susceptibility tothose of mammalian NF‑ fCB, the Rel family of transcrlptlOnbacterial infection. Toll is a large family comprlSlng at least

factors responsible for the gene induction of an innammatorynine members (Toll, 1 8‑wheeler, Toll 3‑9) in Dy10SOPhila ( I 1 ).

response (5). 1n Drosophila, three Rel types of transcription
factors, Dorsal, Dorsal‑type irrmune factor (DIF), and Relish,
have been identified. Dorsal was initially identified as the

Other Toll family members may therefore be responsible formorphogen de丘ning dorso‑ventral pattemlng during embryo一

to be sensitive to bacterial infection; however, these mutants

genesis. Dorsal is sustained inthe inactive stateinthe cytoplasm

did not exhibit a slgnificant reduction in bacteria‑induced

the bacteria‑induced activation or Relish One candidate is
I 8‑wheeler. Mutant flies lacking 1 81Wheeler have been shown

through interaction with the ankyrin‑repeat protein, Cactus･

expression of Diptericin (1 2). Therefore, there may be other

Degradation of Cactus and translocation of Dorsal into thereceptors which recognize bacteria in addition to 1 8‑wheeler,

nucleus are trlggered by an association of Spatzle with theas pointed out in a recent review (13). Genetic studies have

Fungal infection

Bacterial i nfectionlISpatzle/β＼
1 8‑wheelerI?

1＼,⊂⊃ d'AKJI
DmlKKγ＼■l函Dredd

Dorsal ○○ DIF
DriOSOmyCln

DI'p ter7'cin

Fig. 1. Antimicrobial signaling pathways in DTDSOPhiLa,
In Drosophz'la, fungal infection induces clcavage of pro‑Sp凱zlc. Spatzlc associateswith Toll and activates Pelle. This signaling

pathway leads to the degradation of Cactus, then RcI‑type transcription factors, Dorsal and Dorsal‑type immune factor (DIP),
translocatc intothc nucleus and induce the expression of Drosomycln. ln the case ofbactcrial infection, the immune deficiency(lMD) pathway is activated to produce an antibacterial peptide, Diptcricin. nLC Celt surface receptor inthe lMD pathway is ycHo
bc identirICd. ln this pathway, dTAKl regulatesthe activation of an I FCB kinase complex composed of DmiKK β and DmIKK γ ,and finally activates Relish throughcleavage. Drcdd is also involved inthe activation of Relish･ 1 81Wheeler, a mcmbcr ofthc
Toll family, lS also implicated in the antibactcrialrcsponsc; however, it does not seem to be involved in the IIMD pathway･

210

LPS recognltlOn.

identiBed several molecules involved inantibacterial responses.

DmIKKB, DmlKKT, dTAKl and the immune dejiciency

In addition to TLR4, now established as an essential(imd) gene product (Drosophila homologues of mammalian

receptor fわr the recognition of LPS, several molecules are

IKK β , IKK T, TAKland Rip, respectively) have been shown

involved in the fbmation of the LPS receptor complex. CD 1 4, a

to be involved in the expression of antibacterial peptides andglycosylphosphatidylinositol (GPI)‑anchored molecule prefer‑resistance to Gram‑negative bacteria ( 14‑ 1 7). Other genetic
screens identified Dredd, a homologue ofmammalian caspase,entially expressed in monocytes/macrophages and neutrophils,that is involved in antibacterial peptide expression throughin response to LPS (41). Miyake and colleagues identified
MD‑2 as a molecule that associates with the extracellular

has recently been shown to physically associate with TLR4

activation of Relish (1 8‑20). In mammals, there have been
no reports ofcaspases involvement in the activation of NF‑

portion of TLR4 and enhances LPS responsivenePs (42,43)･
Genetic analysts Of CHO cell lines hypo‑responsIVe tO LPS

fCB, and it remains unclear how Dredd regulates Relish

led to the identification ofMD‑2 as a critical component in the
LPS response (44). Miyake and colleagues further identified

activlty. It has been established that DflOSOPhila discriminates
between pathogens and elicits a specific immune response

RP 1 05, which bears leucine‑rich repeats that are structurally
related to TLRs in the extracellular portion (45). B cells from
RPIO5‑deficient mice showed a severely reduced response
to LPS. They also showed an association between TLR4 andvia two slgnaling pathways leading to the expression or anti‑

fungal and antibacterial peptides (Fig. 1) (for a review, seereferences 13, 21, 22).

RP105, indicatlng that RPIO5 is closely lnVOIved in the

3. 1mmate immune activation by TLRs

recognition of LPS together with TLR4 in 良 cells (46). Thus,

3‑1. IdentirlCation ofTLRs in mammals

several components have been implicated in the recognltlOn

Followlng the identification ofToll as a key receptor of hostof LPS, indicatlng that the functional LPS receptor consists

defense response in Drosophila, a mammalian homologue ofof a large complex of several molecules.

3‑31 The TLR family is responsible for recognition of

Tわll was identi丘ed as hTbll (now temed TLR4) by Medzhitov

microbial components

and colleagues. Enforced expression of TLR4 induced theactivation of NF‑ FC B and the expression of several inflamma‑
tory genes (23)A Subsequent studies identified several proteins

Although it remains controversial whether or not TLR2
recognlZeS LPS, there is evidence that TLR2 is involved inthat are structurally related to TLR4. The TLR family now

the recognltlOn Of a variety or microbial components. These

consists of ten members (TLRトTLRI 0), and is expected toinclude lipoproteins from Gram‑negative bacteria, Gram‑

expand (24‑28)i The involvement ofTLRs in the recognition

positive bactena, mycoplasma, mycobacteria and splrOChetesof microorganisms has thus been well established.

(47‑53), peptidoglycan and lipoteichoic acid (LTA) from

3‑2･ TLR4 is a slgnaling component of the LPS receptor

Gram‑positive bacteria (36,54‑57), lipoarabinomannan丘･om

Lipopolysaccharide (LPS) is a major component of the outermycobacteria (58,59), and zymosanfrom fungi(34)･ In v享vomembrane of Gram‑negative bacteria. It was first demon‑
strated that over‑expression of TLR2 in human embryonicroles ofTLR2 have also been established in a study uslng
TLR2 knockout mice, which show high susceptibility tokidney 293 cells conferred an LPS response (29,30). It is wellinfection by Staphylococcus aureus (60).

known that two mouse strains, C3H/HeJ and C57BLIO/ScCr,

One of the mechanisms by which TLR2 recognlZeS micro‑

are hypo‑responsive to LPS ･ Two independent groupsanalyzed

bial components has been eluci血ted. TLR2 fbms heterodimers

the gene responsible fわr hypo‑responsiveness to LPS, andwith other TLRs to discriminate among bacterial components.

found mutations in Tlr4 in these strains (31,32). In the C3H/

A study on the ectoplC expression ofTLRl and TLR2 in HeLa

HeJ mouse strain, a polnt mutation in the cytoplasmic reglOncells revealed that TLR2 functionally cooperateswith TLRlof the Tlr4 gene resulted in an amino acid change from proline
to histidine. This mutation has been shownto result in defective
TLR4‑mediated signaling, and to have a dominant negative
effect on LPS‑dependent responses (33,34). The other LPS

to modulate the response to factors released from Neisseria
menfngitidis (61)･ Aderem and colleagues introduced thehypo‑responsive strain, C57BL 1 0/ScCr, was shown to be nul1‑

with TLR6 to detect the specific pattem of the peptidoglycan

mutated in the Tlr4 gene (31,32). The generation ofTLR4

or.modulin secreted from S･ aureus (62,63)･ TLR6knOckout

knockout mice confirmed the essential role of TLR4 in LPS

mlCe did not show any response to mycoplasma‑derived

rePognition (33)･ Subsequent studies with TLR2 knockout
mlCe Or Chinese hamster ovary (CHO) fibroblasts geneticallylacking TLR2 demonstrated that TLR2 is not involved in the

lipopeptides (64). In contrast, TLR2 knockout mice showedno response to either type of lipopeptides. Reconstitution ofrecognition of LPS (35,36). Thus, genetic叩prOaChes havethe chimeric constructs ofTLR2 and TLR6 in TLR2/TLR6

clearly demonstrated that TLR4, not TLR2 is the LPS receptor･

double knockout cells revealed that the requlrement Of both

domlnant negative丘)m of TLR2 and TLR6 into the RAW264

macrophage cell line and demonstrated that TLR2 associates

lipopeptides, but showed a normal response to bacteria‑derived

Overexpression of TLR2 in 293 cells seems to confer a
response to the TLR2 1igand contaminatlng the preparation

TLR2 and TLR6 f♭r the recognltlOn Of mycoplasma‑derived

lipopeptides. Thus, TLR2 associates with other TLRs toof LPS used. Indeed, re‑purification of LPS demonstrated that

discriminate among microbial components･

TLR4, but not TLR2, is a receptor for LPS (37,38). However,In addition to the cell wall components of pathogens,recent studies have indicated that TLR2 recognlZeS the LPS

bacterialDNA isknown to activate immune cells. The immuno̲

extracted from Leptospira interrogans or Porphyromonas

stimulatory activlty Of bacterial DNA is attributed to thegingivalis, indicating that TLR4 recognlZeS LPS from entero‑bacteria such as Escherichia colt and Salmonella, whereas

presence of unmethylated CpG motifs. CpG motifs in vertebrate
genomic DNA are observed at a reduced frequency and highly

TLR2 may recogni.ze LPS froヮOther bacteria (39,40)･ Even

methylated, which leads to no immunostimulatory activlty･

in these studies, mlnOr COntamlnation with the TLR2 1igand

Synthetic oligodeoxynucleotides (ODNs) containing unmethyl‑in the LPS preparation cannot be 和led out･ lt will requlre

ated CpG motifs also activate immune cells. The involve‑
ment ofTLR9 in the recognltlOn OfCpG DNA has been shown

further investigation to clarify the involvement of TLR2 in211

Table 1. TLR family members and their ligands

TLR family microbial components
TLR]

TLR2

?

peptidoglycan, 1ipopeptides

TLR3

?

TLR4

LPS

TLR5

flagdlin

TLR6

mycoplasma‑dcrivcd lipopeptidcs

TLR7

?

TLR8

?

TLR9

CpG DNA

TLRl0

?

↓

〔子TAKl'through the generation of TLR9 knockout mice (65).

Most recently, TLR5 has been shownto recognizeflagellin,a monomeric component of bacterialflagella (66). A subse‑
quent study indicated that TLR5 basolaterally expressed on
intestinal epithehal cells confersthe ability to recognizeflagellinlNB kinase complex

aom pathogenic bacteria (67).

Thus, it has now been established that TLRs recognlZe a
specific pattem of microbial components (Table I). The

recognition of microbial components by TLRs trlggerS theactivation of innate immunlty.

4. Signaling pathway via TLRs

411. Comparison of signaling pathwayswith Drosophl'ta
Tou and mammalian TLR
The signaling pathway via Drosophila Toll has been shown
to be highly homologous to the mammalian IL‑1 signaling

pathway (Fig･ 2)･ The cytoplasmic region ofDnosophila TolllS Very Similar to that of the mammalian ILI receptor. Thisregion is now called the Tわll/lL‑ 1 receptor (TIR) domain. ln

Fig. 2. Signaling pathwayvia the TL‑I receptor and TLRs.

the lL‑ 1 signaling pathway, the MyD88 adaptor molecule, afunctional homologue of QylOS9Phil̲a Tube, associates withtheToll‑like rc.Ceptors (TLRs) and the intcrlcukin‑ I (IL‑ ) ) rcccptor utilize a

corrunon slgnaling pathway. An adaptor molcculc, MyD88, associateswith the cytoplasmic rcgIOn Ofbothreceptors, and recmits IL‑I receptor

IL‑ 1 receptor, and recnlits the Pelle‑related serine/threonine

associated kinasc (IRAK) tothe reccptor upon reccptor activationJRAK

kinase, lL‑ 1 receptor associated kinase (IRAK), to the receptor

then activates tumor necrosis factor receptor‑associated factor 6

upon stimulation with lL‑1 (68‑70). This leads to activation(TRAF6)I leading tothc activation of Ire B kina苧c cOmPlex･ TransfomIingof IRAK through phosphorylation and the association of
IRAK with TRAF6. Although Drosophila homologues of
TRAF protein (dTRAF 1 13) have been identified, it remains
unclear whether dTRAF is involved in innate immune

甲OWth factor β‑activated kinase (TAKl) is Implicated in the actlVa‑

tlOn OflfCB kinasc; however, an in vivo role ofTAKl has yet to bc
revealed. I FC B is phosphorylatcd by I JC B killaSC and degraded, leading to

the nuclear localization ofnuclear factor‑KB (NF‑fCB) transcription
factors.

slgnaling･ The IL‑ 1 signaling pathway finally induces phos‑
phorylation of inhibitory protein I fC B, a homologue of Cactus,
and nuclear translocation of transcriptlOn factor nuclear factor‑

produce a detectable level of lL‑6 in response to nagellin

fCB (NF‑FCB), a homologue ofDorsal and DIF･

(66)･ These results demonstrate that MyD88 is critical tothe
slgnaling pathway via the TLR family. Indeed, actlVation of

4‑2. MyD88 is a critical component in TLR signaling
Mammalian TLRs also possess TIR domains in their

NF‑fCB and c‑Jun N‑terminal kinase (JNK) in response tocytoplasmic reglOn and utilize MyD88 as an adaptor fわrpeptidoglycan, 1ipoprotein and CpG DNA was not observed

signaling (7 1 ,72). The generation of MyD88knOckout micein MyD88 knockout cells. llowever, stimulation with LPS of
MyD88 knockout macrophages induced activation of NF‑

revealed an essential role ofMyD88 in the signaling pathway

via the lL‑ 1 receptor family (73). Further, it has been demon‑

KB and JNK, although delayed (74). This indicates that,strated that MyD88 knockout mice show no inflammatory

although MyD88 is indispensable for LPS‑TLR41mediatedresponse to LPS (74). Similarly, mice deficient in IRAK or
TRAF6 exhibit impaired responses to both IL‑1 and LPS,
indicatlng that IRAK and TRAF6 are critical components ofproduction of innammatory cytokines, there exists an MyD88‑
independent component in the LPS signaling pathway.

both the ILl receptor‑ and TLR4‑mediated signaling

4‑3･ MyD88‑independent TLR signaling
The roles or LPS‑induced activation in the MyD88‑

pathways (75‑79). 1n other studies, macrophagesfrom MyD88

independent slgnaling pathway are now being clarified. The

knockout mice were shown to produce no innammatory

stimulation with LPS of MyD88 knockout macrophages hascytokines in response to peptidoglycan, 1ipoproteins and CpG

been shoⅥm to induce expression or IFN‑inducible genes such

DNA (52,80‑82). And MyD88 knockout mice failed toas IP‑10 and GARG16 through activation of IRF3 (83).

212

Similarly, the stimulation of DCs with LPS has been shownbreaks caused by stress‑induced damage from ionized radiation

to induce the expression of distinct types of cytokines andand by programmed DNA rearrangement (called VDJ recombi‑

chemokinesfrom that with TLR2 agonist (84). Kupffer cells

nation) during the development ofTand B cells. Macrophages
from DNA‑PKcs‑deficient mice were shown to be severely
impaired in CpG DNA‑induced production of innammatory
cytokines. Further, CpG DNA‑induced activation of DNA‑
PKcs led to activation ofNF‑fCB. Thus, several moleculesfrom MyD88 knockout mice produced IL‑1 8 in response to

LPS (85). Further, DCs from MyD88knOckout mice matured
in response to LPS (86, 87). Thus, several LPS responses were

demonstrated in MyD88 knockout mice.
Analysis of MyD8 8‑independent activation of LPS signaling

appear to be involved in the signaling pathways of the TLR

has recently led to identification of a novel adaptor moleculefamily. These molecules might be responsible for the distinct

named TIR domain‑containing adaptor protein (TIRAP)/

biologlC responses Of different TLRs.

MyD88‑adaptor‑like (Mat) (88, 89)I TIRAP/Mal po.Ssesses a
TIR domain similar to MyD88, and speciflCally assoclateS With
TLR4. The dominant negative fbm orTIRAP/Mal inhibited

5･ Suppression of innate immune cell activity by

microbial components

the LPS‑induced activation of NF‑ fCB, but not TLR2 or TLR9‑

5‑I. Mechanism for LPS tolerance

mediated activation. Furthermore, the blockade or TIRAP一

mediated signaling by addition of a cell permeable TIRAP

lt has now been established that the stimulation ofTLRs

peptide led to impaired LPS‑induced maturation of both wild‑

by microbial components activates innate immune cells such

type and MyD88‑deficient DCs. These findings indicate that
TIRAP/Mal is an adaptor molecule involved in the LPS‑
induced MyD88‑independent pathways (Fig. 3).

as macrophages and DCs. However, an exposure to microbial

Similarly to TLR4‑mediated signaling, which has a unlquechallenge of LPS. This phenomenon was flrSt described over

TIRAP/Ma1‑mediated component in addition to the common
MyD88‑dependent pathway, each TLR seems to have its own

injections of LPS showed a decrease in febrile response (92),components such as LPS results in an inability amongexperimental animals and macrophages to respond to a second

50 years ago in a study in which rabbits treated with repeatedsignaling pathway. In TLR2 signaling, stimulation withand is now known as endotoxin (or LPS) tolerance. Althoughheat‑killed S. aureus has been shown to cause the recruitment

LPS tolerance is considered a host response to prevent theof active Racl and phosphatidyトinositoト3 (PI3K) to theexcessive production of inflammatory cytokines and shock

cytoplasmic portion of TLR2. This induced the activation

syndrome, it is a major clinical problem in the treatment ofofAkt followed by activation of the p65 subunit ofNF‑FCB

patients with Gram一megative sepsis. 1n these patients, LPS

independently oflrcBα degradation (90). In the case ofCpG

from Gram‑negative bacteria causes not only fatal septic shock

DNA recognition, the involvement of the catalytic subunit or

but also LPS tolerance･ Some patients that recover from septlCDNA‑dependent protein kinqse (DNA‑PKcs) in the CpG

shock become tolerant to LPS, and have a severely reduced

DNA‑induced immune cell actlVation has been demonstrated

host defense response against OPPOrtunistic infections leading(91)I DNA‑PKcs is a member of the PI3K family, and was
orlglnally implicated in the repalr Or DNA double‑stranded

to high mortality(93). Therefore, the molecular mechanism
for LPS tolerance has long been a subject of investlgation(94)･ LPS tolerance in macrophages/monocytes is character‑ized by a reduced production of inflammatory cytokines,

TLR4

including tumor necrosis factor‑α (TNF‑α), lL‑6 and IL‑ 12(94‑96), and alterat.Ions tO the LPS‑induced activation of

slgnaling cascades, Including protein kinase C, PI3K, MAP
kinases, and IfCB kinases (97‑101). Accumulation of the

p50 subunit ofNF‑fCB, which has no transactivating activity,
has also been reported (102, 103). However, the precise
mechanism responsible fわr LPS tolerance remains unclear.

The丘nding that TLRs are involved in the recognltlOn Or

microbial components has contributed to our understandingor LPS tolerance･ The utilization ormonoclonal antibody thatrecognlZeS the TLR4‑MD‑2 complex explained one possible
mechanism (104). Pre‑exposure to LPS induced a time‑dependent reduction in the production of inflammatory

cytokines andthe activation oflRAK and NF‑fCB in mouse′一プ一一ノ｢‑ー

peritoneal macrophages. LPS gradually reduced the surface
expression of血e TLR4‑MD‑2 complex on macrophages･ Thiseffect was welllCOrrelated with the time‑dependent onset of
肝N‑inducible genes

DC maturationinfla 蒙ﾖF'友LPS tolerance･ This study indicates that down‑regulation of

ｶ匁U2

the TLR4‑MD‑2 complex (high airlmity LPS receptor) is?neof the major mechanisms for LPS tolerance. 1ntemalizat10n

Fig･ 3･ Signaling pathwayvia TLR4.
The signaling pathway of TLR4 consists of MyD88‑dependent andor the TLR4‑MD‑2 Complex together with LPS might‑indepcndcnt components･ me MyD88‑dependent pathway is indispens‑

account f♭r the down‑regulation; however, the mechanism

able for lipopolysaccharide (LPS)‑induced expression of innammatoryby which it is down‑regulated remains unknown.

cytokincs･ In contrast, the MyD88‑independent pathway is rcsponsiblc

In THP‑ 1 human promonocytic cell lines, it has been shownfor LPS‑induced expression of interferon (lFN)‑inducible genes andthat LPS pre‑treatment induces down‑regulation OHRAK,

maturation ofdendritic cells (DCs). An adaptor molecule that is Involved

and that IRAK is no longer activated in response to a secondin the MyD881independent pathway, has recently been identified as

challenge of LPS (105)･ This is also one orthe possible mech‑Toll/IL‑1 receptor domain‑containing adaptor protein (TIRAP) or
MyD88‑adaptor‑like (Mat).

anisms which may be responsible f♭r LPS tolerance. The

213

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匡重璽

廿感は

匡室

岡

〔亘亘)
Fig. 4. 1mduction orLPS tolerance.

ln mouseperitonea] macrophages, lipopolysaccharide (LPS) treatment induces down‑regulation of the surface expression of the
TLR4‑MD‑2 complex. This may be one of the mechanisms for LPS‑induced LPS tolerance. Lipoproteln, Which is recognized
by TLR2, induces tolerance to lipoprotein and LPS. Lipoprotein pretreatmcnt may affectthe corrmOn Slgnaling pathway via
TLRs. TlluS, LPS tolerance is induced through distinct mcchanisms.

mechanism by which expression OHRAK is down‑regulated

to IL‑1 , which shares its signaling.pathway with TLRs (101)･

in LPS‑tolerant cells remains unclear.

These丘ndings indicate that activatlOn Of the signaling pathway

5‑2. Microbial components induce LPS tolerance

through the entire TLR/IL‑lR family induces tolerance toln addition to LPS, microbial components such as bacterial

LPS. LPS is a most potent activator of the immune system,

lipopeptides, peptidoglycan, muramyl dipeptide from Gram‑and exposure to an excess of LPS often induces multi‑Organ

positive bacteria, and CpG DNA have been shown to induce

failure with a high mortality rate. Therefore, the host may

tolerance tp subsequent stimulation ( 1 06‑ 1 09)･ The mechanism

acqulre a mechanism to limit inflammatory responses by

for induction Of tolerance in macrophages by lipopeptides or

inducing tolerance to LPS through several ligands which share

LTA, both of which are recognized by TLR2, has been investi‑

a common slgnaling pathway. A recent publication described

gated (57,1 10). Both LTA and lipopeptides were shown tothat LPS‑induced tolerance was induced even in cells over‑induce tolerance to themselves in macrophages and mice･

expressing TLR4 (I 12). One can imagine that TLR4 and other

Further, both LTA and lipopeptides induced cross‑tolerance to

TLRs use a common signaling pathway via MyD88. There‑

LPS. Unlike LPS, Iipopeptides did not induce down‑regulation

fore, when TLR4 is constantly expressed, LPS‑inducedof the TLR4‑MD‑2 complex in macrophages, indicatlng thattolerance is achieved by the same mechanism as TLR2

lipopeptides‑induced tolerance occurred through a mechanism

agonisトinduced tolerance. In nomal macrophages, stimulation

distinct from LPS‑induced tolerance (1 10). Additionally,

with LPS rapidly reduces the number of high amnity LPS

induction of lipopeptides‑induced LPS cross‑tolerance wasreceptors composed of the TLR4‑MD‑2 complex to minimize

observed in IL‑ 1 0‑deficient mice ( 1 10). And it has been shownthat LTA‑induced cross‑tolerance to LPS is not transferred to

LPS‑induced inflammation, as demonstrated in a study with

TLR2‑deficient macrophages co‑cultured with wild‑type cells(104). Ir an antibody that detects TLR4 or MD‑2 alone is(57)･ These results indicate that soluble factors induced by
microbial stlmulation do not mediate induction of cross‑

However, the stimulation of macrophages with LPS actuallya monoclonal antibody detectlng the TLR4‑MD‑2 complex
used, down‑regulation of both proteins might not be obseⅣed.

tolerance, and that a common pathway via TLR2 and TLR4

induces conformational changes in the LPS receptor, leading

is affected in macrophages pre‑treated with TLR2 agonists.
Thus, the tolerances induced by TLR4 and TLR2 agonists

to the destruction of the high afrlnity receptor. We speculatethat under physiological conditions, LPS (TLR4 agonist)

occur through quite different mechanisms (Fig. 4). Like

induces tolerance by down‑regulating the high affinity LPS

lipopeptides, CpG DNA (TLR9 agonist) induces cross‑tolerance

receptor, and other TLR agonists induce tolerance to LPS

to LPS in mouse peritoneal macrophages (1 1 I ). Furthermore,through activation of a common slgnaling pathway. Elucidation

tolerance to LPS has been shown to be induced in responseof the mechanism f♭r induction of cross‑tolerance to LPS by214

Other TLR ligands will shed fdher light on our understand‑983.

7. Manfruelli, P., Reichhart, J. M., Steward, R., Hoffmann,
J. A. and Lemaitre, B. (1999): A mosaic analysis in

1ng Or LPS tolerance.

Drosophila fat body cells of the control of antimicrobial

6. Future prospects

peptide genes by the Rel proteins Dorsal and DIF.

EMBOJ., 18, 3380‑3391.

The identification ofTLRs involved in innate immunlty lS

a hot topic in immunology. A role for the TLR family in the

8. Meng, X., Khanuja, B. S. and lp, Y. T. (1999): Toll

recognltlOn Of microbial components has been elucidated.

receptor‑mediated DylDSOPhila immune response requlreS

However, it remainsunknOwn how TLRs recognlZe invading

Dif, an NF‑FCB factor. Genes Dev., 13, 792‑797.

pathogens under physiological conditions. Phagocytosis plays

9. Rutschmann, S., Jung, A. C., Hetru, C., Reichhart, ∫.

a major role in innate immunity. TLR9 isknown to be c0‑M., Ho蝕Iann, J. A. and Ferrandon, D. (2000): The Rel

localized with CpG DNA in endosomes (113). TLR2 andprotein DIP mediates the antifungal but not the anti‑
bacterial host defbnse in Drosophila. Immunity, 1 2, 5691

TLR6 have been shown to be recmited to phagosomesfrom

580.

the cell surface a鮎r stimulation (34,62)･ From these finding.S,

We speculate that TLRs recognlZe microbial components ln

10. Hedengren, M., Asling, 良., Dushay, M. S., Ando, 1.,

the phag0‑1ysosome, where pathogens are phagocytosed,

Ekengren, S., Wihlborg, M. and Hultmark, D. (1999):

digested and degraded, and microbial components are exposed.

Relish, a central factor in the control of humoral but

However,flow cytometric analyses with monoclonal anti‑
bodies have demonstrated that some TLRs, such as TLRl , 2,

not cellular immunlty in Drosophila. Mol. Cell, 4, 827‑

and 4, are actually expressed on the cell surface (42,43,1 14,

ll. Tauszig, S., Jouanguy, E., Hoffmann, J. A. and lmler,

1 1 5). In this respect, we suspect that some TLRs recognize

J･‑L･ (2000): Toll‑related rece.ptors and the control of837.

microbial components that are secreted from pathogens on

antimicrobial peptide expresslOn in Drosophila. Proc.

the cell surface in addition to those in phagollysosomes.

Natl. Ac礼d. S°i. USA, 97, 10520‑10525.

12. Williams, M. J., Rodriguez, A., Kimbrell, D. A. andⅥsualization of the interaction between TLRs and pathogens

Eldon, E. D. (1997): The 18‑wheeler mutation reveals
complex antibacterial gene regulation in Drosophila

will be important to understand how innate immune responses∬e trlggered. Elucidation of the pathway or signaling via eachhost defense. EMBO J., 16, 6120‑6130.

TLR will also be or interest. This will reveal why activation oreach TLR evolves a distinct response,and how innate immune

13. Khush, R. S., Leulier, F. andLemaitre, B. (2001): DfDSO‑

responsesare positively (activation of irmate irrmuTityleading

phila immunity: two paths to NF‑rcB. Trends. Immu‑

to development of adaptive ipmunity) and negatlVely (LPS

nology, 22, 260‑264.

tolerance) regulated. Analysts Of the molecular mechanism
behind innate immunlty has only Just restarted with theidentification of TLRs, and many questions and mysteries

14. Silverman, N., Zhou, R., Stoven, S., Pandey, N.,
Hultmark, D. and Maniatis, T. (2000): A Dy10SOPhila

remaln.

antibacterial immunity. Genes Dev., 14, 2461 ‑247 1.
15･ Lu, Y･, Wu, L P･ andAnderson, K. V. (2001): The anti‑

lrcB kinase complex required for Relish cleavage andbacterial armofthe Drosophila innate immune response
requlreS an lfCB kinase. Genes Dev., 15, 104‑1 10.
16. Vidal, S., Khush, R. S., Leulier, F., Tzou, P., Nakamura,

ACKNOWLEJ)GMENTS
We thank E. Horita for her secretarial assistance.

M･ and Lemaitre, B･ (200 1 ): Mutationsinthe DTIOSOPhiladTAKl gene reveal aconserved function for MAPKRKs

This work was supported by grants from Special Coordi‑

nation Funds of the Ministry of Education, Culture, Sports,
Science and Technology, the Naito Foundation, the Osakain the control of rel/NF‑ rcB‑dependent innate immune

Foundation fb∫ Promotion or Clinical lmmunology, and the

responses. Genes Dev., 15, 1900‑1912.

Japan Research Foundation for Clinical Pharmacology.

17. Rutschmann, S., Jung, A. C., Zhou, 良., Silveman, N.,

Hoffmann, J. A. and Ferrandon, D. (2000): Role ofDrosophila IJuT in a Toll‑independent antibacterial

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