Cutting Edge: Activation of Toll-Like Receptor 2 Induces
a Th2 Immune Response and Promotes Experimental
Vanessa Redecke,* Hans Ha ¨cker,†Sandip K. Datta,* Agnes Fermin,* Paula M. Pitha,‡
David H. Broide,* and Eyal Raz2*
Recognition of microbial components by APCs and their
activation through Toll-like receptors (TLR) leads to the
show that activation of TLR2 by its synthetic ligand
Pam3Cys, in contrast to activation of TLR9 by immuno-
resulted in the induction of Th2-associated effector mole-
cules like IL-13, and IL-1?, GM-CSF and up-regulation
12, IFN?, IL-18, IL-27). Accordingly, TLR2 ligands ag-
gravated experimental asthma. These data indicate that
the type of TLR stimulation during the initial phase of
immune activation determines the polarization of the
adaptive immune response and may play a role in the ini-
tiation of Th2-mediated immune disorders, such as
asthma. The Journal of Immunology, 2004, 172:
turally conserved components of pathogens. Probably with the
exception of TLR3, the receptor for dsRNA, cell activation by
all TLR family members is largely dependent on the adaptor
molecule myeloid differentiation factor 88 (MyD88). Stimula-
IL-1R-associated kinase and TNFR-associated factor 6 and ac-
This ultimately results in up-regulation of costimulatory mole-
cules, secretion of cytokines, and enhanced uptake and presen-
tation of Ag. Both TLR-dependent activation of APC and pro-
fficient immune responses depend on the interaction
between the innate and adaptive immune system. Im-
mune responses against invading pathogens are initi-
adaptive T and B cell responses. Polarization toward a Th1 or
Th2 phenotype is crucial for the defense against pathogens, but
can also be associated with the induction of autoimmune dis-
ease (Th1) or asthma (Th2). Although it is well known that all
TLR stimuli lead to activation of APCs, their particular influ-
TLR2 and TLR9 ligands elicit two disparate adaptive immune
Materials and Methods
(B&K Universal, East Yorkshire, U.K.) mice were 6- to 8-wk-old.
ISS-ODN (1668: TCCATGACGTTCCTGATGCT) was syn-
ington (Lakewood, NJ) and the synthetic lipopeptide Pam3Cys was obtained
from EMC Microcollections (Tu ¨bingen, Germany).
Cells were cultured in RPMI 1640 (Cellgro; Mediatech,
MD), 2 mM L-glutamine (Cellgro), and 100U/ml penicillin-100 ?g/ml strep-
tomycin (Pen/Strep; Cellgro). Mouse bone marrow-derived dendritic cells
(BMDCs) were cultured as previously described (3).
C57BL/6 (The Jackson Laboratory, Bar Harbor, ME) and 129/SvEv
C57BL/6 mice were immunized with 50 ?g of OVA alone or in combination
and total splenocytes were restimulated for secondary CTL and cytokine assays
as described (4, 5). Proliferation of splenocytes was determined by [3H]thymi-
129/SvEv were immunized s.c. with 50 ?g of OVA alone or in combination
with ISS-ODN (50 ?g) or Pam3Cys (50 ?g) on days 0 and 7. Mice were in-
21, the mice were tested for airway responsiveness to methacholine (3–24 mg/
ml; Sigma-Aldrich, St. Louis, MO) and a bronchoalveolar lavage for the differ-
ential lung cell count was performed as previously described (6, 7). Mediastinal
lymph nodes (LN) were digested with DNase I/collagenase VII (Boehringer
Mannheim/Roche, Indianapolis, IN/Sigma-Aldrich) and restimulated with
OVA for T cell cytokine analysis and used for FACS stains.
Departments of *Medicine and†Pharmacology, University of California, San Diego, La
Jolla, CA 92093; and‡Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins
University School of Medicine, Baltimore, MD 21231
Received for publication June 23, 2003. Accepted for publication December 30, 2003.
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 National Institutes of Health Grant AI 40682, Dynavax (to
(to H.H.), and National Institutes of Health Grant AI 052406 (to S.K.D.).
2Address correspondence and reprint requests to Dr. Eyal Raz, Department of Internal
Medicine and The Sam and Rose Stein Institute for Research on Aging, University of Cal-
ifornia, San Diego, La Jolla, CA 92093-0663. E-mail address: email@example.com
3Abbreviations used in this paper: TLR, Toll-like receptor; MyD88, myeloid differentia-
tion factor 88; BMDC, bone marrow-derived dendritic cell; LN, lymph node; AHR, air-
Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00
OVA-specific IgG2a, IgG1, and IgE levels were measured from serum samples
collected by retro-orbital eye bleeds (8). IFN-?, IL-5 (BD PharMingen, San
Diego, CA), and IL-13 (RD Biosystems, Minneapolis, MN) were determined
from supernatants of splenocytes that were restimulated with OVA in vitro (5).
The levels of IL-12p40, IL-12p70, IL-10, and IL-6 (BD PharMingen) and
IL-13 (RD Biosystems) were determined by ELISA.
Bioassay (type I IFN)
Type I IFN levels in supernatants from BMDC 16 h after stimulation were
measured using an antiviral protection assay as described (9).
(San Diego, CA). Surface marker expression was analyzed on a FACSCalibur
flow cytometer using CellQuest (BD Biosciences, Franklin Lakes, NJ) and
FlowJo software (Tree Star, San Carlos, CA).
Quantitative real-time PCR was performed using the ABI Prism 7700 (Ap-
plied Biosystems, Foster City, CA). Primers were generated using the
Primer3 software (Ref. 10; www-genome.wi.mit.edu/genome_software/other/
TLR2 ligands bias the adaptive immune response toward a Th2
phenotype and can lead to aggravation of asthma
To determine the role of particular TLRs in the generation of
adaptive immune responses, mice were immunized with differ-
duction of Ig subclasses (IgG2a, IgG1, and IgE), secretion of
cytokines from in vitro-restimulated splenocytes, CTL re-
sponse, and the effect on a murine experimental model of
asthma were analyzed. Because our preliminary results indi-
cated that the TLR2 ligand (Pam3Cys) and TLR9 ligand (ISS-
ODN) lead to the most distinctive immune responses, we con-
centrated on these two ligands in our current investigations. As
shown in Fig. 1, A–C, ISS-ODN and Pam3Cys induced differ-
ent Ab profiles. Immunization with ISS-ODN/OVA resulted
in an Ag-specific IgG2a response, whereas immunization with
Pam3Cys/OVA resulted in a pronounced IgG1 response and
induction of IgE (Fig. 1C). While ISS-ODN primed CD4 T
cells to produce IFN-?, Pam3Cys induced IL-13 production
(Fig. 1, D–E). Restimulation with medium alone did not in-
duce any production of IFN-? or IL-13. The IgG isotype bias
and the production of IgE and IL-13 induced by Pam3Cys/
OVA were abrogated in TLR2- deficient mice, proving that
TLR2 is a critical receptor for Pam3Cys (data not shown). Im-
munization with peptidoglycan (100 ?g) as an adjuvant in
combination with OVA was less potent than immunizations
with Pam3Cys/OVA in regard to cytokine production and in-
duction of Ab response, but also showed a preferential induc-
tion of IgG1 (26,547 ? 14,455 U/ml for peptidoglycan/OVA
vs 299,082 ? 73,142 U/ml for Pam3Cys/OVA, 3 wk after im-
munization) over IgG2a (not detectable for peptidoglycan/
OVA vs 1473 ? 925 U/ml for Pam3Cys/OVA, 3 wk after
Both stimuli, ISS and Pam3Cys, led to induction of CTL
activity (Fig. 1F); however, the induction of CTL activity by
ISS-ODN was significantly more prominent than that induced
by Pam3Cys. Titrating the amount of Pam3Cys over three or-
ders of magnitude did not significantly change this low CTL
Immunization with ISS-ODN/OVA and Pam3Cys/OVA
induced a similar proliferative response in OVA-restimulated
splenocyte cultures (Fig. 1G).
To test whether the Th1 (by ISS-ODN) and Th2 (by
Pam3Cys) polarization observed above alters the propensity of
the immunized animals to develop experimental asthma,
challenged with OVA intranasally at two occasions and airway
and cytokine release of in vitro-restimulated bronchial LN cells
decreased the number of eosinophils in the lung and induced
IFN-?, whereas priming with Pam3Cys/OVA aggravated the
AHR, increased the number of eosinophils and led to the pro-
duction of Th2 cytokines (Fig. 1, H–J).
Taken together, these data show that both ISS-ODN and
Pam3Cys induce significant Ag-dependent immune responses.
However, ISS-ODN polarizes the immune response toward a
Th1 phenotype, whereas Pam3Cys leads to Th2-specific cyto-
kine and Ig production and only a modest CTL response. The
data further suggests that the opposing Th1/Th2 polarization
induced by ISS-ODN and Pam3Cys can have an effect on the
development of Th2-associated diseases such as experimental
TLR2 ligands differentially induce Th2-associated cytokines and B7RP-1
Costimulatory molecules and the cytokine production by DC
explore whether these factors may explain the differential Th
stimulated with ISS-ODN or Pam3Cys and the expression of
costimulatory molecules and the production of cytokines were
determined. As shown in Fig. 2A, both ISS-ODN and
Pam3Cys induced up-regulation of the costimulatory mole-
cules CD40, B7-1 and B7-2, whereas only Pam3Cys led to an
up-regulation of B7RP-1. The up-regulation of B7RP-1 was
even more pronounced in mature DC from mediastinal LN af-
ter immunization with OVA and Pam3Cys (Fig. 2B), whereas
ISS-ODN showed less effect.
We next analyzed production of IFN??, IL-12 p40, IL-12
p70, IL-6, IL-10, and IL-13 (Fig. 2C), and a panel of mRNAs
known to be involved in Th1/Th2 polarization (Fig. 2D) by
ciated with a Th1 phenotype like IL-12, IL-18, IL-27, and
IFN??, Pam3Cys preferentially induced Th2-associated cyto-
and I?B? were comparably induced. A similar pattern of cyto-
kine production with Pam3Cys inducing Th2-associated cyto-
kines was observed in primary CD11c?DC isolated from the
spleen. Again, the cytokine response and gene induction by
Pam3Cys in BMDC was dependent on TLR2 (data not
In this study, we analyzed the effect of specific TLR stimuli on
Th polarization and experimental asthma. Preliminary experi-
ments indicated that ISS-ODN (TLR9) and Pam3Cys (TLR2)
an adjuvant, Pam3Cys induced a Th2 polarization as reflected
2740CUTTING EDGE: DIFFERENTIAL EFFECTS OF TLR2 AND 9 LIGANDS
in the cytokine profile and the production of specific IgG sub-
As expected, the effector functions induced by Pam3Cys were
ferent TLR ligands induce distinct Th cell responses in human
Porphyromonas gingivalis, which in contrast to LPS from other
species activates cells via TLR2, has also been described to in-
duce T cell cytokines associated with a Th2 phenotype (12). In
a different study, it was shown that the Th1/Th2 polarizing ef-
fect of LPS from Escherichia coli, which triggers cells via TLR4,
was concentration-dependent (13). At low concentrations LPS
induced a Th2 bias, whereas at higher concentrations a Th1
phenotype was reported. In contrast, Pam3Cys-induced Th2
a 10-fold higher amount of Pam3Cys than required for maxi-
mum induction of IL-13 and IgG1 production did not induce
Th1-specific parameters like IFN-? or IgG2a production.
Whether the differences in concentration dependence between
LPS and Pam3Cys are due to different signaling pathways or
either alone or in combination with ISS-ODN (50 ?g) or Pam3Cys (500, 50, or 5 ?g) at days 0 and 14 and rechallenged i.v. with 20 ?g of OVA 3 days before
response of OVA-restimulated splenocytes was determined by [3H]thymidine uptake. H–J, Mice were immunized s.c. with OVA (50 ?g) either alone or in com-
bination with ISS-ODN (50 ?g) or Pam3Cys (50 ?g) twice and rechallenged intranasally with 5 ?g of OVA 7 days and 1 day before testing. H, Airway respon-
siveness to aerosolized methacholine was tested. I, Percentage of macrophages (macro.), lymphocytes (lympho.), neutrophils (neutro.), and eosinophils (eos.) from
the BAL was determined. J, OVA-specific cytokine secretion of mediastinal LN cells was measured. Data are shown as mean ? SEM, n ? 5 per group (n ? 8 per
group for the AHR and eosinophil count), ISS ? ISS-ODN, Pam ? Pam3Cys, n.d. ? not detectable.
Differential immune responses induced by ISS-ODN- and Pam3Cys-based immunizations. A–G, Mice were immunized s.c. with OVA (50 ?g)
2741 The Journal of Immunology
whether different cell types contribute to the phenomenon ob-
served needs to be investigated.
At the cellular level, stimulation of BMDCs with ISS-ODN
or Pam3Cys showed distinct activation profiles. Up-regulation
of costimulatory molecules like CD40, B7-1 and B7-2 was
comparable, whereas up-regulation of B7RP-1, which was
shown to support Th2 responses (14), seemed more pro-
IL-1?, and GM-CSF, which support Th2 differentiation or al-
lergy (15–18), were preferentially induced by Pam3Cys. In
contrast, Th1-associated cytokines like IL-12, IFN?, IL-18,
and IL-27 (17, 19) were greatly diminished in comparison to
dependent genes (data not shown).
The signaling pathways of TLR2 and TLR9 share common
molecules. It was shown that signaling through the adaptor
molecule MyD88 plays an important role in the induction of
believed to be completely dependent on MyD88, was shown to
induce a strong Th1 bias (21). In contrast, TLR2 uses at least
one additional molecule called Toll-IL-1R domain-containing
ecules might explain the diversity seen with those two TLR li-
gands remains to be elucidated.
TLR ligands can play an important role in the initiation or pre-
vention of Th2-associated diseases. Immunization with Ag in
of TLR9, can result in experimental asthma. There have been
reports suggesting that bacterial infections can be associated
with asthma. Interestingly, chlamydia and mycoplasma, bacte-
ria that are strongly associated with the onset of asthma, as well
as some viral infections and air pollution particles were shown
to elicit some of their effects via TLR2-mediated mechanisms
(24–27). Our study demonstrates that the type of TLR stimu-
adaptive immune responses. Therefore, it seems reasonable to
consider TLR2 and TLR2-dependent signaling pathways as
lines: ISS-ODN or Pam3Cys (Pam) stimulated cells. B, Expression of B7RP-1 on DC from mediastinal LN cells in vivo. Mice were immunized as described in Fig.
1, G–H. Mediastinal LN were harvested and surface expression of B7RP-1 determined by flow cytometry. Data shown are pooled cell preparations from three mice
per group and are gated and the live CD11c?/MHC class II high population. C, Concentration of IL-12p40, IL-12-p70, IL-13, IL-10, and IL-6 were measured by
ELISA from the culture supernatant of BMDC after stimulation with ISS-ODN (1 ?M) or Pam3Cys (5 ?g/ml) for 16 h. IFN-? was determined by bioassay. Data
GMCSF, and IL-10 mRNA were determined by quantitative real-time PCR. BMDC were cultured in the absence (control) or presence of ISS-ODN (1 ?M) and
Pam3Cys (5 ?g/ml) for 6 h. Data from one representative experiment of five experiments normalized to the expression of CPH are shown and expressed as fold
increase over control. ISS ? ISS-ODN, Pam ? Pam3Cys.
2742 CUTTING EDGE: DIFFERENTIAL EFFECTS OF TLR2 AND 9 LIGANDS
possible inducers of the immune deviation that results in Download full-text
asthma in humans.
We thank Dr. Shizuo Akira for providing the TLR2?/?mice, M. Corr and P.
Charos for their help in the care of the mice, and T. Hayashi, C. Rosetto, and
C. Tran for technical assistance.
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