Adjuvant effect of lipopolysaccharide on the induction of contacthypersensitivity to haptens in mice

Shoko Yokoi, Hironori Niizeki *, Hideyuki Iida, Hideo Asada, Sachiko Miyagawa

Department of Dermatology, Nara Medical University, Kashihara, Nara, Japan

Journal of Dermatological Science 53 (2009) 120–128

A R T I C L E I N F O

Article history:

Received 28 December 2007

Received in revised form 12 August 2008

Accepted 13 August 2008

Keywords:

Toll-like receptor 4

Langerhans cell

TNF-alpha

A B S T R A C T

Background: Toll-like receptor (TLR) 4 is a critical receptor and signal transducer for lipopolysaccharide

(LPS), a major component of Gram-negative bacteria. The MyD88-independent pathway downstream of

TLR4 leads to functional dendritic cell (DC) maturation, although LPS-induced cytokine production from

DCs is MyD88-dependent.

Objectives: We investigated whether intracutaneously injected LPS alters the functions of cutaneous DCs,

leading to enhanced contact hypersensitivity (CH).

Methods: The ear swelling response was measured to evaluate the magnitude of CH. Cell proliferation of

allogeneic splenocytes stimulated by DC-enriched draining lymph node (LN) cells was measured by

performing a [3H]-thymidine incorporation assay. Epidermal I-A+ cells were evaluated under an

epifluorescent microscope. I-A+ FITC-bearing cells from the draining LNs 24 h after FITC application were

analyzed on FACScan.

Results: LPS augmented CH induction in C3H/HeN (HeN) and MyD88-knockout (KO) mice but not in C3H/

HeJ (HeJ) and H-2Sd-bearing strains such as BALB/c mice. LPS failed to augment the allo-stimulatory

ability of DCs in the draining LNs after hapten applications. LPS altered the density and morphology of

epidermal I-A+ cell in HeN and BALB/c mice but not in TLR4-deficient HeJ mice. LPS increased the

proportion of I-A+ FITC-bearing cells in the LNs 24 h after FITC application in HeN, but not in BALB/c and

HeJ.

Conclusions: LPS augments the ability of DCs to migrate to the draining LNs, leading to enhanced CH via a

TLR4-dependent, MyD88-independent pathway. The different effects of LPS on CH in some strains of mice

may explain individual differences in the susceptibility to establish CH to daily antigen exposures in

clinical settings.

� 2008 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

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1. Introduction

Contact hypersensitivity (CH) is a T cell dependent immuneresponse, which is induced by the application of haptens on theskin. Dendritic cells (DCs) are the most potent antigen presentingcells and play major roles in the initiation and regulation ofadaptive immune responses to antigens [1]. Cutaneous DCs firstcapture antigens and then migrate to draining lymph nodes (LNs).During migration, cutaneous DCs undergo maturation, whichincludes the expression of costimulatory molecules and delivery ofmajor histocompatibility complex antigens to their surface. Themature migratory DCs then activate the naı̈ve T cells present in

* Corresponding author at: Present address: Department of Dermatology,

National Center for Child Health and Development, 2-10-1 Ohkura, Setagaya,

Tokyo, Japan. Tel.: +81 3 3416 0181; fax: +81 3 5494 7136.

E-mail address: niizeki-h@ncchd.go.jp (H. Niizeki).

0923-1811/$30.00 � 2008 Japanese Society for Investigative Dermatology. Published b

doi:10.1016/j.jdermsci.2008.08.009

draining LNs or transfer the antigens to resident DCs [2]. Therefore,cutaneous DCs are very important for the determination of thequality and quantity of CH.

Toll-like receptors (TLRs) are the main innate immune sensors.Each TLR responds to specific molecules of microbial origin.Stimulation of different TLRs induces distinct patterns of geneexpression, which not only leads to the activation of innateimmunity but also induces the development of antigen-specificacquired immunity [3].

Lipopolysaccharide (LPS) is a major component of the outermembranes of Gram-negative bacteria and induces a variety ofbiological responses including cytokine production from macro-phages, B cell proliferation, and endotoxin shock. TLR4 is essentialfor recognizing LPS because LPS-mediated cytokine release isabrogated in mice having natural mutation of TLR4 or its targetdisruption [4]. TLR4 is expressed on monocyte-derived DCs.Stimulation of TLR4 on DCs induces interleukin (IL)-12 production,enhances surface expression of costimulatory molecules, and

y Elsevier Ireland Ltd. All rights reserved.

S. Yokoi et al. / Journal of Dermatological Science 53 (2009) 120–128 121

finally, leads to DC maturation [5]. Moreover, Kaisho et al. [6]reported that the MyD88-independent pathway, downstream ofTLR4 could lead to functional DC maturation because LPS couldinduce functional maturation of the MyD88-deficient DCs,although LPS-induced cytokine production from DCs wasMyD88-dependent.

In this study, we determined whether intracutaneously injectedLPS could alter the functions of cutaneous DCs and lead toenhanced CH.

2. Materials and methods

2.1. Mice

We purchased adult mice aged 8–12 weeks of the BALB/c,B10.A, B10.D2, C3H/HeN, C3H/HeJ and C57BL/6 strains from JapanSLC, Inc. (Hamamatsu, Shizuoka, Japan). MyD88-knock out (KO)mice were kindly provided by Prof. Akira at the Research Institutefor Microbial Diseases, Osaka University [7]. They were maintainedin our animal facility with SPF environment. Experimentalprocedures were carried out with animals under general anesthe-sia achieved by intraperitoneal (i.p.) injections of ketamine(Sankyo, Tokyo, Japan) (80 mg/kg) and xylazine (Bayer Healthcare,Leverkusen, Germany) (16 mg/kg) or pentobarbitone (Dainippon-Sumitomo, Osaka, Japan) (50 mg/kg). Animal care was inaccordance with the guidelines of Nara Medical University. Eachcontrol or experimental panel consisted of 4–5 mice.

2.2. Reagents

LPS from Escherichia coli 026:B6, fluorescein isothiocyanate(FITC), and 2,4-dinitro-l-fluorobenzene (DNFB) were purchasedfrom Sigma–Aldrich (St. Louis, MO). Anti-mouse tumor necrosisfactor (TNF)-a/TNFSF1A antibodies (Abs) and normal goat IgGwere purchased from Genzyme/Techne (Minneapolis, MN). Anti-I-Ak, I-Ad monoclonal antibodies (mAbs), and isotype-matchedcontrol Abs were purchased from BD Pharmingen (San Diego, CA).

2.3. Intradermal injection

Using a 1.0 ml syringe with a 30-gauge needle, the solution wasinjected into the intradermal (i.d.) space. Successful inoculationswere characterized by the appearance of a flat swelling withdefined lateral margins immediately beneath the epidermis [8].

2.4. Induction and expression of CH

On day 0, 25 ml of 0.5% DNFB (185 mg) in acetone was appliedon the dry-shaved abdominal cutaneous surface of mice. On day 5,CH was elicited by challenging one ear of each mouse with 20 ml of0.05% DNFB (15 mg). Ear swelling was measured using anengineer’s micrometer (Mitutoyo, Kawasaki, Kanagawa, Japan)24 and 48 h following the challenge and compared with thethickness of ear prior to the challenge [8–11].

2.5. Identification and enumeration of epidermal DCs

The epidermis was separated from the dermis by incubation inDispase II (Roche Diagnostics GmbH, Mannheim, Germany). DCs inthese epidermal sheets were stained with anti-I-A mAbs (BDPharmingen) and FITC-tagged goat anti-mouse Abs (Zymed, SouthSan Francisco, CA), as described previously [9], and evaluatedunder an epifluorescence microscope. With the aid of an eyepiecewith a 1 mm2 grid, a minimum of 10 fields were counted for eachsample to enumerate the number of positively stained cells

present, and the mean number of stained cells was expressed ascells/mm2.

2.6. Mixed lymphocyte reaction

The axillary and inguinal LNs were excised 5 days after applying25 ml of 0.5% DNFB on the abdominal skin of mice. The LNs weregently disrupted to yield a single cell suspension and filteredthrough 70-mM nylon meshes (BD FALCONTM). DC population wasenriched with the Mouse DC Enrichment Kit (DYNAL1) accordingto the manufacturer’s instructions. The purity of DCs was assessedunder an epifluorescent microscope after staining with anti-I-A-PEmAbs (BD Pharmingen) and estimated to be 50–70%. The enrichedDCs were X-irradiated (2.9 G) and washed three times withphosphate-buffered saline (PBS) containing 2% fetal bovine serum(FBS). Spleens were harvested from naı̈ve allogeneic mice (BALB/c,H-2d) and filtered through 100-mM nylon meshes. The erythro-cytes were lysed using the mouse erythrocyte lysing kit (R&DSystems). A total of 2 � 105 splenocytes were co-cultured withvarious numbers of prepared X-irradiated LN cells in RPMI mediumsupplemented with 10% heat-inactivated FBS (Sigma–Aldrich),0.1 mM non-essential amino acids (Gibco, Grand Island, NY), 2 mML-glutamine (Gibco), 25 mM HEPES buffer (Sigma–Aldrich), and 1%penicillin–streptomycin (Gibco) in round-bottomed 96-well platesat 37 8C. Five days after co-culture, all wells were pulsed with 1 mCiof [3H]-thymidine for an additional 16–18 h of culture. The cellswere then harvested, and [3H]-thymidine incorporation wasmeasured by liquid scintillation counting [12].

2.7. Immunofluorescent staining and flow cytometry

On the shaved abdominal skin of HeN and HeJ mice, we applied400 ml of 0.5% FITC in 1:1 acetone/dibutyl phthalate. Mice weresacrificed 48 h later and the axillary and inguinal LNs were excised.The LNs were gently disrupted to yield a single cell suspension andfiltered through 70-mM nylon meshes. The cells were collected,washed three times with PBS with 2% FBS. To enrich for DC, LN cellswere suspended in RPMI, and then 5 ml of the cell suspension wasgently underlaid with 4 ml of Nycoprep TM (Oslo, Norway),followed by centrifugation at 300 � g for 20 min. The cells at theinterface were collected, washed three times with PBS with 2% FBS.Cells were stained with phycoerythin (PE)-conjugated anti-I-Ak orI-Ad mAb, diluted 1/1000 or isotype-matched control Ab for 30 minon ice, and washed three times with PBS containing 1% bovineserum albumin (BSA) and 0.1% sodium azide. Propidium iodine (PI,1%) (Sigma–Aldrich) was used to identify the dead cells [13]. Thestained cells were analyzed on FACScan with the CellQuestsoftwareTM (Becton Dickinson Immunocytometry Systems, SanJose, CA).

2.8. Statistical analysis

The statistical significance of differences between the means ofthe each experimental group was determined using Student’s t

test. The mean differences were considered significant at P < 0.05.Each experiment was performed at least twice.

3. Results

3.1. LPS augments the induction of CH

We first examined the effect of LPS on the induction of CH. LPS(25 and 5 mg) or PBS was injected (i.d.) into panels of C3H/HeN(HeN), C3H/HeJ (HeJ), and BALB/c mice. Within 30 min, 185 mgDNFB was applied on the cutaneous surface of the injected sites.

S. Yokoi et al. / Journal of Dermatological Science 53 (2009) 120–128122

Five days later, the ears of these mice were challenged with 15 mgDNFB and the ear swelling responses measured after 24 and 48 h.The results of a representative experiment are shown in Fig. 1A–C.When DNFB was applied on the PBS-injected skin (positivecontrol), intense CH was induced. However, when DNFB wasapplied on LPS-injected skin, the HeN recipients mounted furtherintense CH responses than the positive control mice upon earchallenge (Fig. 1A). The intensity of CH in the skin injectedpreviously with 25 mg LPS was not significantly enhancedcompared with that in the skin injected with 5 mg LPS. Thus, inHeN mice, injection of LPS into the skin significantly intensified theability of DNFB-applied skin to induce CH. In contrast, when LPSwas injected intradermally in the TLR4-sufficient BALB/c mice(Fig. 1B) and TLR4-deficient HeJ mice (Fig. 1C), the magnitude of CHwas similar to that in the positive control. Each experiment was

Fig. 1. LPS augments the induction of CH in C3H/HeN mice. Panels of C3H/HeN (A),

BALB/c (B), and C3H/HeJ mice (C) were injected intradermally with 25 mg LPS (a),

5 mg LPS (b), and PBS (c), respectively. Within 30 min, 185 mg DNFB was applied at

the injected sites. Five days later, the ears were challenged with DNFB. Unsensitized

animals were also challenged and used as negative controls (d). Ear swelling

responses at 24 h are presented as mean � S.E.M. (mm). *P < 0.05 versus PBS (positive

control).

repeated at least twice with very similar results. We thus concludethat TLR4 mediates the effect of LPS on the induction of CH in HeNmice. In addition, it is suggested that BALB/c mice, in which TLR4 issufficiently expressed, are a resistant strain with regard to theeffect of LPS on the CH induction. This indicates that not only TLR4but also another factor such as TNF-a may determine thesusceptibility to LPS (14).

3.2. H-2 linked to susceptibility to the effect of LPS on CH induction

We next determined whether susceptibility to the effect of LPSon CH induction is H-2 dependent. Previous studies in mice haveindicated that the UVB-susceptibility trait is related to the class IIIregion in the H-2 complex (14). Because BALB/c strain has an H-2d

genetic background, we chose B10.D2 and B10.A as B10-congenicinbred strains that carry H-2d haplotype (Table 1).

LPS (25 mg) or PBS was injected intradermally into panels ofmice. Within 30 min, 185 mg DNFB was applied on the cutaneoussurface of the injected sites. After 5 days, the ears of these micewere challenged with 15 mg DNFB, and the ear swelling responseswere measured after 24 and 48 h. The results of a representativeexperiment are shown in Fig. 2A and B. When DNFB was applied onthe PBS-injected skin (positive control), intense CH was induced.However, when LPS was injected intradermally in the H-2d-bearing B10.A mice (Fig. 2A) and B10.D2 mice (Fig. 2B), themagnitude of CH was similar to that in the positive control. Thus,the adjuvant effect of LPS on CH induction is H-2 dependent; k andb of H-2 are susceptible traits and d of H-2 is a resistant trait.

3.3. The augmentation of CH by LPS is MyD88-independent

We next determined whether the effect of LPS on CH is MyD88-dependent because TLR4 mediates the adjuvant effect of LPS on theinduction of CH.

Panels of MyD88-KO littermates and their wild-type counter-parts (B6 background) were administered (i.d.) LPS (25 mg/mouse)or PBS. Within 30 min, 185 mg DNFB was applied on the epidermisof the injected sites. Five days later, the ears of these mice werechallenged with 15 mg DNFB and the ear swelling responsemeasured after 24 and 48 h. The results of a representativeexperiment are shown in Fig. 3. Unsensitized animals were alsochallenged and used as negative controls (Fig. 3, groups c and f).When DNFB was applied on the skin on LPS-injected mice (groupd), the wild-type recipients (wild) mounted more intense CHresponses than the positive control mice (group e). Similarly, theMyD88-KO recipients (KO) in which LPS had been injectedpreviously (group a) mounted more intense CH responses thanthe positive control mice (group b). Thus, these data suggest that

Table 1Genetic background and LPS susceptibility in mice [14]

Gray box indicates LPS resistant mice and their H-2 alleles.

Fig. 2. LPS fails to augment the induction of CH in B10.A and B10.D2 mice. Panels of

mice of B10.A (A) and B10.D2 (B) received i.d. injections of 25 mg LPS (a) and PBS (b),

respectively. Within 30 min, the injected sites were painted with 185 mg DNFB.

After 5 days, the ears were challenged with DNFB. Unsensitized animals were also

challenged and used as negative controls (c). Ear swelling responses at 24 h are

presented as mean � S.E.M. (mm).

Fig. 3. LPS augments CH in MyD88-KO mice. Panels of MyD88-KO (a–c) and wild-

type mice (B6 background; d–f) were injected intradermally with 25 mg LPS (a and

d) or PBS (b and e). Within 30 min, 185 mg DNFB was applied on the injected sites.

After 5 days, the ears were challenged with 15 mg DNFB. Unsensitized animals were

also challenged and used as negative controls (c and f). Ear swelling responses at

24 h are presented as mean � S.E.M. (mm). *P < 0.05 versus PBS (positive control).

Fig. 4. LPS augments the allo-stimulatory ability of the draining LN cells. Mice

received i.d. injection of LPS (25 mg/mouse) or PBS alone. Within 30 min, 185 mg

DNFB was applied on the injected sites. After 5 days, the LN cells, the DC-enriched

LN cells and splenocytes were prepared. Allogeneic splenocytes as responders were

co-cultured with X-irradiated LN cells (Fig. 4A) or DC-enriched LN cells (Fig. 4B) as

stimulators for 5 days. Data are expressed as the mean cpm of triplicate

cultures � S.E.M. *P < 0.05 versus PBS (positive control). Empty and solid circles

indicate thymidine incorporation of stimulator cells in the LPS-treated group and the

PBS-treated group, respectively.

S. Yokoi et al. / Journal of Dermatological Science 53 (2009) 120–128 123

LPS augments CH in MyD88-KO mice, as demonstrated in wild-type mice. We concluded that the effect of LPS upon CH isindependent of the MyD88 pathway.

3.4. LPS augments the allo-stimulatory ability of the draining LN cells

We next determined whether LPS could alter the allo-stimulatory ability of the draining LN cells. In this experiment,we used C57BL/6 mice as stimulators and BALB/c mice asresponders. LPS (25 mg/mouse) or PBS was injected (i.d.) intothe mice. Within 30 min, 185 mg DNFB was applied on thecutaneous surface of the injected sites. LN cells were obtained after5 days. In some experiments, DC population was enriched, X-irradiated, and cocultured with allogeneic splenocytes (2 � 105/well) from BALB/c for 5 days. The LN cells were cultured alone asthe negative control. Cell proliferation was measured by [3H]-thymidine uptake. The results of a representative experiment areshown in Fig. 4. LN cells from the LPS-treated mice showedsignificantly enhanced ability to stimulate allogeneic splenocytes(Fig. 4A). However, the ability of the DC-enriched LN cells from theLPS-treated mice to stimulate allogeneic splenocytes was compar-able with that in the PBS-treated mice (Fig. 4B). It is concluded thatLPS does not enhance the antigen-presenting function of migratoryDCs from skin when hapten is applied on the LPS-injected sites.

Table 3Anti-TNF-a antibodies restore the effect of LPS on epidermal dendritic cells

Number of I-A+ cellsa

Anti-TNF antibody + LPS 628.0 � 19.0

Normal goat IgG + LPS 484.8 � 17.9b

PBS 634.8 � 22.7

a Mean number of cells � S.E.M. per mm2.b P < 0.05 versus PBS.

S. Yokoi et al. / Journal of Dermatological Science 53 (2009) 120–128124

3.5. LPS alters the density and morphology of epidermal DCs

We next examined whether LPS can alter the density andmorphology of the epidermal DCs and Langerhans cells (LCs).Panels of mice belonging to 3 different inbred strains received i.d.injections of LPS (25 mg) or PBS alone as the control group. Theskins of these mice were excised 2 h later. LCs in these epidermalsheets were stained with anti-I-Ak or I-Ad mAb and then with FITC-tagged goat anti-mouse Ab and evaluated under an epifluorescencemicroscope. The results of a representative experiment arepresented in Table 2. The number of I-A+ epidermal cells wasclearly reduced in the skin excised from the LPS-injected sites inHeN and BALB/c mice. In contrast, the number of I-A+ epidermalcells in the LPS-injected HeJ mice was similar to that of I-A+epidermal cells in the PBS-injected control mice. We conclude thatthe intracutaneously injected LPS can reduce the density ofepidermal I-A+ cells. The lack of an effect of LPS on epidermal DCsin the TLR4-deficient HeJ mice indicates that the effect of LPS isTLR4-dependent.

The changes in the epidermal I-A+ cells after i.d. injections ofLPS included not only a reduction in the density of these cells butalso an extensive alteration in the morphology of HeN (Fig. 5A) andBALB/c (Fig. 5C) mice. In mice in which i.d. injections of LPS wereadministered, the I-A+ cells no longer displayed the dendrites, andthe cell bodies appeared plump and round, rather than slender,which is the morphology of I-A+ cells after PBS injection (Fig. 5Dand F). The morphology of I-A+ cells in C3H/HeJ mice (Fig. 5B),however, was similar to that of I-A+ cells in the PBS-injectedcontrol mice (Fig. 5E); this indicates that TLR4 signaling mediatesthe morphological alteration of epidermal DCs by LPS injections.Therefore, LPS appears to have two histologically distinct effects onepidermal DCs: reduction in the number of I-A+ cells and loss ofdendrites.

3.6. Anti-TNF-a Abs restore the effects of intracutaneously injected

LPS on the density of epidermal DCs

We next examined the possibility that the inhibitory effects ofLPS on the density and the morphology of epidermal LCs might bereversed by anti-TNF-a Abs. LPS was injected intradermally intoBALB/c mice 6 h after i.p. injections of anti-TNF-a polyclonal Abs(200 mg) or normal goat IgG Abs. The skins of these mice wereexcised 2 h later. The density of epidermal DCs was assessed by afluorescence microscope, using anti-I-Ad mAbs (Table 3). Intra-dermal injections of LPS after i.p. administration of normal goat IgGAbs reduced the number of I-A+ cells. In contrast, i.p. adminis-tration of anti-TNF-a Abs restored the number of I-A+ cells in LPS-injected skin, which was comparable with the number of I-A+ cellsin PBS-injected skin. This indicates that TNF-a mediates the effectof LPS on the density of epidermal DCs.

However, the morphology of the epidermal I-A+ cells remainedmore or less plump and round even after anti-TNF-a Ab

Table 2LPS alters the density of epidermal I-A+ cells

Mice Treatment Number of I-A+ cellsa

C3H/HeN PBS 610.0 � 15.4

LPS 525.6 � 14.2b

BALB/c PBS 599.2 � 17.4

LPS 484. � 15.6b

C3H/HeJ PBS 622.0 � 15.1

LPS 631.2 � 23.2

a Mean number of cells � S.E.M. per mm2.b P < 0.05 versus PBS.

pretreatment (Fig. 6B) and was almost comparable with themorphology after normal goat IgG Ab pretreatment (Fig. 6A).Although anti-TNF-a Ab pretreatment largely inhibited thereduction in the number of I-A+ cells induced by LPS, it was ableto only partially restore the morphology of epidermal cells.

3.7. LPS alters the ability of antigen-bearing DCs to migrate into

draining LNs

We next determined whether LPS could alter the ability ofantigen-bearing DCs to migrate into draining LNs. Within 30 minafter the i.d. injections of LPS (25 mg/mouse) or PBS, 400 ml of 0.5%FITC was epicutaneously applied on the HeN (I-Ak), HeJ (I-Ak), orBALB/c (I-Ad). Forty-eight hours after applying FITC, draining LNswere collected. A suspension of LN cells was prepared andimmunolabeled with PE-conjugated anti-I-Ak, or I-Ad mAb. Thedata for two-color analysis for FITC+ and I-A+ cells are shown inFig. 7A. The proportion of I-A+ FITC-bearing cells in the LNs of HeNmice was significantly higher in the LPS-treated mice (0.49%) than inthe PBS-treated mice (0.21%). However, those of BALB/c (Fig. 7A) orHeJ (data not shown) were approximately identical between the 2groups. The expression intensity of I-A in the LPS-treated mice wascomparable with that in the PBS-injected mice (n = 2 for each group)(Fig. 7B). The proportion of dead cells in the FITC-bearing cells iscomparable between the LPS- and PBS-treated groups (data notshown). These data suggest that LPS alters the ability of antigen-bearing DCs to migrate into draining LNs. There was no significantdifference in the proportion of I-A+ FITC-bearing cells in the TLR4-deficient HeJ mice, indicating that the effect of LPS on the ability ofantigen-bearing DCs to migrate to draining LNs is TLR4-dependent.

3.8. TNF-a itself abrogates but in synergy with LPS augments the

induction of CH

Because anti-TNF-a antibodies block the migration of epider-mal DCs (Table 3), we assessed the role of TNF-a in the induction ofCH. As reported previously [15], TNF-a itself impairs CH induction.We tested the dose of TNF-a that is capable of impairing CHinduction in different strains of mice. Panels of B6 or BALB/c micereceived TNF-a (50 ng), or vehicle (PBS with 0.1% BSA) on theabdominal skin. Within 30 min, 185 mg DNFB was applied on theinjected sites. After 5 days, the ears of these mice were challengedwith 15 mg DNFB and the ear swelling response was measuredafter 24 and 48 h. The results of a representative experiment areshown in Fig. 8A. This dose of TNF-a is solely effective on the LPS-susceptible strain, HeN mice, as reported previously [15,16]. Thus,we chose this dose for the subsequent experiment.

We next applied LPS (25 mg), TNF-a (50 ng), or both reagents onthe abdominal skin of BALB/c mice. Within 30 min, 185 mg DNFBwas applied on the injected sites. After 5 days, the ears of thesemice were challenged with 15 mg DNFB, and the ear swellingresponse was measured after 24 and 48 h. The results of arepresentative experiment are shown in Fig. 8B. Surprisingly, micethat received both reagents showed enhanced CH (Fig. 8B, group a).In contrast, mice that received either LPS or TNF-a showed

Fig. 5. LPS alters the density and morphology of epidermal DCs. Panels of C3H/HeN (A and D), C3H/HeJ (B and E), and BALB/c (C and F) mice received i.d. injections of LPS

(25 mg) (upper row; A–C) or PBS (lower row; D–F). The original magnification: �200.

S. Yokoi et al. / Journal of Dermatological Science 53 (2009) 120–128 125

comparable to that of the positive control (Fig. 8B, groups b and c).Thus, we concluded that TNF-a is capable of enhancing CHinduction.

4. Discussion

The proposed role of TNF-a in CH has been controversial. Micedeficient for p55 TNF-a receptor exhibit an enhanced CH reaction,suggesting an immunosuppressive role of this receptor in CH [17].TNF-a-deficient mice show a reduced CH reaction. This suggeststhat TNF-a is necessary for optimal CH and establishes aphysiological role of TNF-a in CH [18]. Moreover, TNF-a stimulatesmurine LCs to migrate from the skin into draining LNs afterallergen application [19]. We showed that LPS augmented theinduction of CH in TLR4-sufficient HeN mice, but not in TLR4-deficient HeJ mice. Anti-TNF-a Abs prevented the decrease in thedensity of epidermal DCs induced by intracutaneous injection ofLPS, which increases the number of I-A+ cells in the draining LNs inHeN but not in HeJ mice. Furthermore, we showed that TNF-a iscapable of enhancing the effect of LPS on CH induction (Fig. 8B).These data indicate that DC migration via TLR4 is TNF-a-

dependent and that TNF-a has the ability to modulate themagnitude of CH.

In contrast, LPS could not enhance CH in BALB/c mice despitethe presence of TLR4, indicating that BALB/c mice respond onlyslightly to LPS in an allergic response against DNFB. A study of thegenetic effect on the in vivo production of TNF-a showed that TNF-a production is genetically controlled by H-2D, to which the TNFlocus is closely linked [20,21]. The quantitative difference in TNF-aproduced in response to ultraviolet (UV) B radiation has beenreported to account for the phenotypic traits of UVB-susceptibilityboth in humans and mice [14,22,23]. Acute low-dose protocol ofUV exposure of the skin impairs CH induction via the TLR4-dependent pathway [15]. Furthermore, we showed that TNF-a iscapable of enhancing the effect of LPS on CH induction (Fig. 8B).These results indicate that the quantitative difference in TNF-aproduced via TLR4 in response to the LPS pathway may also berelated to the phenotypic traits of LPS responsiveness.

The source of TNF-a production in the induction phase of CHremains unclear. Sugita et al. [24] reported an interestingexperiment showing that Langerhans cell (LC) function can beup-regulated indirectly by cytokines that are released by

Fig. 6. Anti-TNF-a Abs restores LPS-altered epidermal DCs. BALB/c mice received i.d. injection of LPS 6 h after intraperitoneal injections of normal goat IgG Abs (A) or anti-TNF-

a Abs (B). The original magnification is �200.

S. Yokoi et al. / Journal of Dermatological Science 53 (2009) 120–128126

keratinocytes stimulated with CpG, a TLR9 ligand. TLR9 ligand wascapable of enhancing the hapten-presenting ability of LCs whenLC-enriched epidermal cells, but not purified LCs, were used as theLC source; this suggests that bystander keratinocytes play a role inthe enhancement of LC function. The addition of a cocktail ofneutralizing antibodies against keratinocytes-induced cytokines,including TNF-a, abrogated the CpG-promoted, antigen-present-ing ability of LC-enriched epidermal cells.

Fig. 7. LPS alters an ability of antigen-bearing DCs to migrate to draining LNs. Panels o

injection of LPS (25 mg/mouse) or PBS. Twenty-four hour later, the draining LN cells wer

with PE-conjugated I-Ak monoclonal Ab. A total of 5 � 104 cells were analyzed using FA

FITC+ and I-Ak+ cells were shown (A). The intensity of expression of I-A in LPS-treated

group). The dotted line indicates the intensity of isotype-matched antibody in LPS-trea

In this study, another candidate of TNF-a production wasdermal mast cells. Marshall et al. [25] reported that LPS enhance LCmigration in mast cell-deficient mice, indicating that mast cells arenot associated with LPS-dependent enhanced LC migration.

In TNF-a-deficient mice, however, nickel chloride (Ni) con-comitant with LPS induced a Ni allergy to a similar degree to that inthe respective control mice [26]. In this model, interestingly,Ni + LPS induced a Ni allergy only weakly in IL-1-deficient and

f mice of C3H/HeN and BALB/c received application of 0.5% FITC (400 ml) after i.d.

e collected, the DC populations were enriched with Nycoprep, then immunolabeled

CScaliber for I-Ak+ FITC-bearing cells. Representative data of two-color analysis for

mice (solid line) was comparable with PBS-injected mice (gray box) (n = 2 for each

ted mice (B).

Fig. 8. TNF-a itself abrogates in synergy with LPS augments the induction of CH.

Panels of B6 (a–c) or BALB/c (d–f) mice received TNF-a (50 ng: a and d), or vehicle

(PBS with 0.1% BSA: b and e) on the abdominal skin. Within 30 min, 185 mg DNFB

was applied on the injected sites. After 5 days, the ears of these mice were

challenged with 15 mg DNFB and the ear swelling response was measured after 24

and 48 h. Unsensitized animals were also challenged and used as negative controls

(c and f). Panels of BALB/c mice received vehicle (group d), LPS (25 mg: group c),

TNF-a (50 ng: group b), or both reagents (group a) on the abdominal skin of BALB/c

mice. Within 30 min, 185 mg DNFB was applied on the injected sites. After 5 days,

the ears of these mice were challenged with 15 mg DNFB and the ear swelling

response was measured after 24 and 48 h. The results of a representative

experiment at 24 h are shown. Ear swelling responses at 24 h are presented as

mean � S.E.M. (mm). *P < 0.05 versus positive control.

S. Yokoi et al. / Journal of Dermatological Science 53 (2009) 120–128 127

macrophage-depleted mice but not in mast cell-deficient mice andeven in nude (T cell-deficient) mice. The authors suggest that thepromotion and augmentation of metal allergies by LPS in mice aredependent on innate immunity [26]. Taken together with ourresults, the mechanism by which LPS alters the magnitude of CHdepends on antigens. They suggested some similarities anddissimilarities between the 2 types of CH responses: metal-induced and classical hapten-induced [26]. Moreover, similarpromoting effects of TLR ligands (such as TLR7 and TLR9 ligands)have been reported in models entailing CH induction by the

classical haptens, and in these models, TLR ligands enhance theantigen-presenting functions of DCs [24,27–29].

IL-1 is an indispensable cytokine for CH induction. Antono-poulos et al. [30] reported that TNF-a failed to induce LC migrationin caspase-1-deficient mice. Caspase-1 is necessary to cleave theproprotein form of IL-1 into the active form of IL-1 protein.Intradermal injection of IL-1 beta (50 ng) but not TNF-a (50 ng)resulted in a similar reduction in epidermal LCs in both wild-typeand caspase-1-deficient mice, indicating that, after receiving anappropriate signal, caspase-1-deficient epidermal LCs are capableof migration and that IL-1 and IL-1 signal are downstream of TNF-ato induce LC migration.

We wanted to determine whether MyD88 is required toconduct CH response, because MyD88 is also an adaptor protein ofIL-1 receptor [5]. Mice deficient for MyD88 showed a vigorous CHcomparable to that in wild-type mice (Fig. 3, groups b and e),indicating that CH response to DNFB is independent of the IL-1-MyD88 pathway. Fig. 3 also shows that LPS can augment CHinduction in MyD88-KO mice, indicating that augmented CH byLPS is independent of the (TLR4-) MyD88 pathway. This result isconsistent with the fact that caspase-1 activation induced by IL-1signaling is independent of the MyD88 pathway [3]. We doubt thatLPS is capable of enhancing CH induction in IL-1R1-KO mice sinceno MyD88-independent IL-1 pathway is blocked in these mice.

TLR4 stimulation facilitates the activation of 2 pathways: theMyD88-dependent and Toll/IL-1 receptor domain-containingadaptor-inducing IFN-b (TRIF)-dependent pathways [3,5]. TheMyD88-dependent pathway involves the early phase of nuclearfactor-kB (NF-kB) activation, which leads to the production ofinflammatory cytokines. The TRIF-dependent pathway activatesinterferon (IFN)-regulatory factor (IRF) 3 and involves the latephase of NF-kB activation followed by late-phase TNF-a produc-tion [3,5]. In the present study, we showed that LPS augmented CHin both MyD88-KO mice and wild-type mice and that the effect ofLPS upon CH was independent of the MyD88-dependent pathway.Kaisho et al. [6] reported that the TLR4-dependent effects of LPS onthe antigen-presenting ability of DCs are mediated mainly via theMyD88-independent pathway. We suspect that the TRIF pathwayis associated with LPS with regard to CH induction [3,5]. Furtherstudies are required to address this issue.

We showed that LPS increased the proportion of I-Ak+ FITC-bearing cells in draining LNs 24 h after the hapten painting (Fig. 7).A majority of migratory DCs in the LNs at this time point arederived from the dermis but not from the epidermis [31]. DermalDCs migrate into the draining LNs earlier than epidermal DCs. Wedid not observe that LPS increased the proportion of I-Ak+ FITC-bearing cells in draining LNs 48 h after painting (data not shown).These data imply that LPS is capable of enhancing the migratoryability of dermal DCs.

We also showed that LPS failed to increase the proportion of I-Ak+ FITC-bearing cells in draining LNs 24 h after FITC painting inBALB/c mice (Fig. 7). LPS has no effect on CH induction in this strainof mice (Fig. 1C). However, the same dose of LPS did alter thedensity and morphology of epidermal DCs (Fig. 5). This isconsistent with a recent report showing that dermal DCs areindispensable for CH induction [31]. This report also shows thatelimination of epidermal DCs does not alter CH response to hapten.Therefore, we conclude that the dose of LPS we chose does alter thedensity and morphology of epidermal DCs, however, this dose maybe insufficient to augment the migration of dermal DCs, leading tono effect on CH response.

A small amount of TNF-a is capable of alteration in the numberand morphology of LCs in C3H/HeN mice rather than in BALB/cmice [15]. Simultaneous administration of LPS and TNF-asucceeded in enhancing CH response in BALB/c (Fig. 8B). This

S. Yokoi et al. / Journal of Dermatological Science 53 (2009) 120–128128

indicates that BALB/c strain needs more amount of TNF-a toenhance conventional CH response.

We showed that LPS responsiveness with regard to CH varies insome strains of mice. The effect of LPS on CH induction is lower inBALB/c mice than in HeN and C57BL/6 mice. All low-responsivestrains carry H-2d or H-2a background (Table 2).

In studies on humans, a common mutation in TLR4 is associatedwith the difference in LPS responsiveness and alters the ability ofthe host to respond to environmental stress [32]. Moreover, in astudy on Swedish children, decreased LPS-induced IL-12 and IL-10responses were found to be associated with TLR4 polymorphismand were independently associated with asthma [33]. In a studyconducted on the Japanese, TNF-a gene polymorphism was foundto be associated with an increased production of TNF-a protein[34]. These results imply a possibility to set up standard protocol inclinical settings for the application of bacterial components inhuman vaccines. Moreover, not only antigenicity of environmentalfactors but also LPS responsiveness as an adjuvant may explain inpart the individual difference in the susceptibility to establish CHfor daily antigen exposures.

Acknowledgements

We thank Professor Shizuo Akira for kindly providing MyD88-KO mice. We also thank Dr. Kenji Kabashima for technical advicesin Flow Cytometry analyses and Dr. Yasuyuki Sumikawa fortechnical assistance. This study was supported in part by a Grant-in-Aid for Scientific Research 16591112 from the Ministry ofEducation, Culture, Sports, Science, Technology of Japan, and LydiaO’Leary Memorial Foundation (to H. Niizeki), and the Departmentof Indoor Environmental Medicine, Nara Medical University (to H.Asada).

References

[1] Asahina A, Tamaki K. Role of Langerhans cells in cutaneous protective immu-nity: is the reappraisal necessary? J Dermatol Sci 2006;44:1–9.

[2] Allan RS, Waithman J, Bedoui S, Jones CM, Villadangos JA, Zhan Y, et al.Migratory dendritic cells transfer antigen to a lymph node-residentdendritic cell population for efficient CTL priming. Immunity 2006;25:153–62.

[3] Takeda K, Akira S. Microbial recognition by Toll-like receptors. J Dermatol Sci2004;34:73–82.

[4] Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, et al. Cuttingedge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipo-polysaccharide: evidence for TLR4 as the Lps gene product. J Immunol1999;162:3749–52.

[5] Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innateand acquired immunity. Nat Immunol 2001;2:675–80.

[6] Kaisho T, Takeuchi O, Kawai T, Hoshino K, Akira S. Endotoxin-induced matura-tion of MyD88-deficient dendritic cells. J Immunol 2001;166:5688–94.

[7] Kawai T, Adachi O, Ogawa T, Takeda K, Akira S. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 1999;11:115–22.

[8] Niizeki H, Streilein JW. Hapten-specific tolerance induced by acute, low-doseultraviolet B radiation of skin is mediated via interleukin-10. J Invest Dermatol1997;109:25–30.

[9] Bacci S, Nakamura T, Streilein JW. Failed antigen presentation after UVBradiation correlates with modifications of Langerhans cell cytoskeleton. JInvest Dermatol 1996;107:838–43.

[10] Niizeki H, Alard P, Streilein JW. Calcitonin gene-related peptide is necessary forultraviolet B-impaired induction of contact hypersensitivity. J Immunol1997;159:5183–6.

[11] Niizeki H, Kurimoto I, Streilein JW. A substance P agonist acts as an adjuvantto promote hapten-specific skin immunity. J Invest Dermatol 1999;112:437–42.

[12] Ding W, Beissert S, Deng L, Miranda E, Cassetty C, Seiffert K, et al. Alteredcutaneous immune parameters in transgenic mice overexpressing viral IL-10in the epidermis. J Clin Invest 2003;111:1923–31.

[13] Kawamura T, Azuma M, Kayagaki N, Shimada S, Yagita H, Okumura K. Fas/Fasligand-mediated elimination of antigen-bearing Langerhans cells in draininglymph nodes. Br J Dermatol 1999;141:201–5.

[14] Vincek V, Kurimoto I, Medema JP, Prieto E, Streilein JW. Tumor necrosis factoralpha polymorphism correlates with deleterious effects of ultraviolet B lighton cutaneous immunity. Cancer Res 1993;53:728–32.

[15] Yoshikawa T, Streilein JW. Genetic basis of the effects of ultraviolet light B oncutaneous immunity. Evidence that polymorphism at the Tnfa and Lps locigoverns susceptibility. Immunogenetics 1990;32:398–405.

[16] Vermeer M, Streilein JW. Ultraviolet B light-induced alterations in epidermalLangerhans cells are mediated in part by tumor necrosis factor-alpha. Photo-dermatol Photoimmunol Photomed 1990;7:258–65.

[17] Kondo S, Wang B, Fujisawa H, Shivji GM, Echtenacher B, Mak TW, et al. Effect ofgene-targeted mutation in TNF receptor (p55) on contact hypersensitivity andultraviolet B-induced immunosuppression. J Immunol 1995;155:3801–5.

[18] Pasparakis M, Alexopoulou L, Episkopou V, Kollias G. Immune and inflamma-tory responses in TNF alpha-deficient mice: a critical requirement for TNFalpha in the formation of primary B cell follicles, follicular dendritic cellnetworks and germinal centers, and in the maturation of the humoral immuneresponse. J Exp Med 1996;184:1397–411.

[19] Cumberbatch M, Kimber I. Tumour necrosis factor-alpha is required foraccumulation of dendritic cells in draining lymph nodes and for optimalcontact sensitization. Immunology 1995;84:31–5.

[20] Muller KM, Lisby S, Arrighi JF, Grau GE, Saurat JH, Hauser C. H-2D haplotype-linked expression and involvement of TNF-alpha in Th2 cell-mediated tissueinflammation. J Immunol 1994;153:316–24.

[21] Freund YR, Sgarlato G, Jacob CO, Suzuki Y, Remington JS. Polymorphisms in thetumor necrosis factor alpha (TNF-alpha) gene correlate with murine resistanceto development of toxoplasmic encephalitis and with levels of TNF-alphamRNA in infected brain tissue. J Exp Med 1992;175:683–8.

[22] Niizeki H, Naruse T, Hecker KH, Taylor JR, Kurimoto I, Shimizu T, et al.Polymorphisms in the tumor necrosis factor (TNF) genes are associated withsusceptibility to effects of ultraviolet-B radiation on induction of contacthypersensitivity. Tissue Antigens 2001;58:369–78.

[23] Alard P, Niizeki H, Hanninen L, Streilein JW. Local ultraviolet B irradiation impairscontact hypersensitivity induction by triggering release of tumor necrosis factor-alpha from mast cells Involvement of mast cells and Langerhans cells insusceptibility to ultraviolet B. J Invest Dermatol 1999;113:983–90.

[24] Sugita K, Kabashima K, Atarashi K, Shimauchi T, Kobayashi M, Tokura Y. Innateimmunity mediated by epidermal keratinocytes promotes acquired immunityinvolving Langerhans cells and T cells in the skin. Clin Exp Immunol2007;147:176–83.

[25] Jawdat DM, Rowden G, Marshall JS. Mast cells have a pivotal role in TNF-independent lymph node hypertrophy and the mobilization of Langerhanscells in response to bacterial peptidoglycan. J Immunol 2006;773:1755–62.

[26] Sato N, Kinbara M, Kuroishi T, Kimura K, Iwakura Y, Ohtsu H, et al. Lipopoly-saccharide promotes and augments metal allergies in mice, dependent on innateimmunity and histidine decarboxylase. Clin Exp Allergy 2007;37:743–51.

[27] Akiba H, Satoh M, Iwatsuki K, Kaiserlian D, Nicolas JF, Kaneko F. CpG immu-nostimulatory sequences enhance contact hypersensitivity responses in mice.J Invest Dermatol 2004;123:488–93.

[28] Gunzer M, Riemann H, Basoglu Y, Hillmer A, Weishaupt C, Balkow S, et al.Systemic administration of a TLR7 ligand leads to transient immune incompe-tence due to peripheral-blood leukocyte depletion. Blood 2005;106:2424–32.

[29] Thatcher TH, Luzina I, Fishelevich R, Tomai MA, Miller RL, Gaspari AA. Topicalimiquimod treatment prevents UV-light induced loss of contact hypersensi-tivity and immune tolerance. J Invest Dermatol 2006;126:821–31.

[30] Antonopoulos C, Cumberbatch M, Dearman RJ, Daniel RJ, Kimber I, Groves RW.Functional caspase-1 is required for Langerhans cell migration and optimalcontact sensitization in mice. J Immunol 2001;166:3672–7.

[31] Bursch LS, Wang L, Igyarto B, Kissenpfennig A, Malissen B, Kaplan DH, et al.Identification of a novel population of Langerin+ dendritic cells. J Exp Med2007;204:3147–56.

[32] Arbour NC, Lorenz E, Schutte BC, Zabner J, Kline JN, Jones M, et al. TLR4mutations are associated with endotoxin hyporesponsiveness in humans. NatGenet 2000;5:187–91.

[33] Fageras Bottcher M, Hmani-Aifa M, Lindstrom A, Jenmalm MC, Mai XM,Nilsson L, et al. A TLR4 polymorphism is associated with asthma and reducedlipopolysaccharide-induced interleukin-12 (p70) responses in Swedish chil-dren. J Allergy Clin Immunol 2004;114:561–7.

[34] Higuchi T, Seki N, Kamizono S, Yamada A, Kimura A, Kato H, et al. Polymorph-ism of the 50-flanking region of the human tumor necrosis factor (TNF)-alphagene in Japanese. Tissue Antigens 1998;51:605–12.