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Production in Human Dendritic CellsChemokineFlagellin Induces Maturation and

The Toll-Like Receptor 5 Stimulus Bacterial

Aderem and Andrew D. LusterTerry K. Means, Fumitaka Hayashi, Kelly D. Smith, Alan

http://www.jimmunol.org/content/170/10/5165doi: 10.4049/jimmunol.170.10.5165

2003; 170:5165-5175; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2003 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Toll-Like Receptor 5 Stimulus Bacterial Flagellin InducesMaturation and Chemokine Production in HumanDendritic Cells1

Terry K. Means,2* Fumitaka Hayashi,2* Kelly D. Smith, †‡ Alan Aderem,† andAndrew D. Luster3*

Toll-like receptors (TLRs) are pattern recognition receptors that serve an important function in detecting pathogens and initiatinginflammatory responses. Upon encounter with foreign Ag, dendritic cells (DCs) go through a maturation process characterized byan increase in surface expression of MHC class II and costimulatory molecules, which leads to initiation of an effective immuneresponse in naive T cells. The innate immune response to bacterial flagellin is mediated by TLR5, which is expressed on humanDCs. Therefore, we sought to investigate whether flagellin could induce DC maturation. Immature DCs were cultured in theabsence or presence of flagellin and monitored for expression of cell surface maturation markers. Stimulation with flagellininduced increased surface expression of CD83, CD80, CD86, MHC class II, and the lymph node-homing chemokine receptorCCR7. Flagellin stimulated the expression of chemokines active on neutrophils (IL-8/CXC chemokine ligand (CXCL)8, GRO-�/CXCL1, GRO-�/CXCL2, GRO-�/CXCL3), monocytes (monocyte chemoattractant protein-1/CC chemokine ligand (CCL)2), andimmature DCs (macrophage-inflammatory protein-1�/CCL3, macrophage-inflammatory protein-1�/CCL4), but not chemokinesactive on effector T cells (IFN-inducible protein-10 kDa/CXCL10, monokine induced by IFN-�/CXCL9, IFN-inducible T cell �chemoattractant/CXCL11). However, stimulating DCs with both flagellin and IFN-inducible protein-10 kDa, monokine inducedby IFN-�, and IFN-inducible T cell � chemoattractant expression, whereas stimulation with IFN-� or flagellin alone failed toinduce these chemokines. In functional assays, flagellin-matured DCs displayed enhanced T cell stimulatory activity with aconcomitant decrease in endocytic activity. Finally, DCs isolated from mouse spleens or bone marrows were shown to not expressTLR5 and were not responsive to flagellin stimulation. These results demonstrate that flagellin can directly stimulate human butnot murine DC maturation, providing an additional mechanism by which motile bacteria can initiate an acquired immuneresponse. The Journal of Immunology, 2003, 170: 5165–5175.

D endritic cells (DCs)4 represent a class of professional APCsthat are critical in the initiation of a primary immune re-sponse. The proposed model for DC function is that the

DCs take up foreign Ag in the periphery, process the Ag, and migrateto the T cell area of lymph nodes, where they present the Ag in thecontext of MHC along with costimulatory receptors, thereby activat-

ing naive T cells (1). Furthermore, innate immunity is thought to betriggered by pattern recognition receptors on APCs. Toll-like recep-tors (TLRs), which recognize conserved motifs on microorganisms,are thought to be largely responsible for this innate recognition. TheTLR family consists of ten conserved germline-encoded type I trans-membrane receptors that are present on APCs and function in re-sponse to triacylated lipopeptides (TLR1/2 heterodimers), diacylatedlipopeptides (TLR2/6 heterodimers), dsRNA (TLR3), LPS (TLR4),flagellin (TLR5), imidazoquinolines (TLR7 and TLR8), and unmeth-ylated CpG DNA (TLR9) (reviewed in Refs. 2–8).

In vitro studies of DC maturation have been conducted usingcells derived from peripheral blood monocytes cultured in thepresence of GM-CSF and IL-4. Such cells have an immature phe-notype, having a high rate of endocytosis and expressing low lev-els of MHC class II, CD83, and the costimulatory molecules CD80and CD86. Upon maturation, DCs down-regulate mechanisms ofAg capture, including endocytic activity and expression of Fc re-ceptors, but they increase expression of costimulatory, adhesion,and MHC class II molecules (1, 9). Moreover, monocyte-derivedDCs pulsed with tumor Ags ex vivo are a promising new tool inthe treatment of malignant diseases (10, 11).

Recent work has shown DC maturation after exposure to mi-crobial lipopeptides, LPS, viruses, and parasites (9, 12–20). Eachof these microbes or their components has been found to activatecells through TLRs. This report demonstrates that human mono-cyte-derived DCs express TLR5, flagellin induces human DC mat-uration in vitro, and that this maturation is accompanied by che-mokine and cytokine production.

*Center for Immunology and Inflammatory Diseases and Division of Rheumatology,Allergy and Immunology, Massachusetts General Hospital and Harvard MedicalSchool, Boston, MA 02129; †Institute for Systems Biology, Seattle, WA 98103; and‡Department of Pathology, University of Washington, Seattle, WA 98195

Received for publication November 25, 2002. Accepted for publication March4, 2003.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grants T32-HL07874 (toT.K.M.), P01-DK50305 and R01-AI40618 (to A.D.L.), T32-AR07258 (to F.H.), K08-AI01751 (to K.D.S.), and R37-AI025032, R01-AI052286, and R01-AI32972 (toA.A.).2 T.K.M. and F.H. contributed equally to this work.3 Address correspondence and reprint requests to Dr. Andrew D. Luster, Massachu-setts General Hospital, 149 13th Street, Charlestown, MA 02129. E-mail address:aluster@partners.org4 Abbreviations used in this paper: DC, dendritic cell; TLR, Toll-like receptor; fliC,flagellin purified from S. typhimurium; QPCR, quantitative PCR; iDC, immature DC;MIP, macrophage-inflammatory protein; CCL, CC chemokine ligand; IP-10, IFN-�-inducible protein 10 kDa; CXCL, CXC chemokine ligand; MFI, mean fluorescenceintensity; fliC-DC, fliC-stimulated DC; LPS-DC, LPS-stimulated DC; GRO, growth-related oncogene; MCP, monocyte chemoattractant protein; MIG, monokineinduced by IFN-�; I-TAC, IFN-inducible T cell � chemoattractant; IRF3, IFN-regulatory factor 3.

The Journal of Immunology

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Materials and MethodsReagents

Human and murine IL-4 and GM-CSF were purchased from PeproTech(Rocky Hill, NJ). Human IFN-� was purchased from R&D Systems (Minne-apolis, MN). LPS (Escherichia coli K12) was from Sigma-Aldrich (St. Louis,MO). LPS was purified from contaminant lipoproteins normally found in com-mercially available LPS preparations by double phenol extraction, exactly asdescribed (21). Synthetic lipopeptide palmitoylcysteine[(RS)-2,3-di(palmitoy-loxy)propyl]serine-lysine-OH is derived from an E. coli lipoprotein and waspurchased from Bachem (Torrance, CA). Flagellin was purified from Salmo-nella typhimurium (fliC) as previously described (22). The flagellin preparationcontained �0.2 pg of LPS per microgram of protein as tested by Limulusamebocyte assay. This purified flagellin activates NF-�B in Chinese hamsterovary cells transfected with TLR5, but not with TLR2 or TLR4 (22). All otherreagents were purchased from Sigma-Aldrich unless stated otherwise.

Generation and maturation of DCs

Buffy coats were obtained from healthy volunteers and fractionated overHistopaque-1077. The PBMC layer was recovered and erythrocyte de-pleted by incubation in RBC lysis buffer for 5 min at room temperature.PBMCs were cultured in complete medium (RPMI 1640, 1% L-glutamine,1% penicillin/streptomycin, and 10% low-endotoxin FCS) for 2 h in T75flasks (Corning Glass, Corning, NY). After incubation, nonadherent cellswere removed by three washes with 1� PBS (Invitrogen, Carlsbad, CA).The remaining adherent cells were then cultured in complete medium sup-plemented with GM-CSF and IL-4 (50 ng/ml each). On days 2 and 4, theDC cultures received an additional dose of GM-CSF and IL-4 (50 ng/mleach). On day 5, nonadherent DCs were harvested by gentle pipetting,counted, and plated in fresh medium containing GM-CSF and IL-4 (50ng/ml each). On day 6, some DCs were matured by addition of 100 ng/mlLPS or 0.2–60 nM fliC for 1–48 h.

Primary human blood DCs were purified from human peripheral bloodby immunomagentic depletion of CD3-, CD11b-, and CD16-expressingcells followed by positive selection of CD4� cells (Miltenyi Biotec,Auburn, CA).

Murine splenic DCs were isolated from spleens of C57BL/6 mice aspreviously described (23). Briefly, spleens were dissected into small pieces(1 mm3) and incubated at 37°C in complete RPMI 1640 supplemented with2 mg/ml collagenase D for 45 min. Cell suspension was obtained by vig-orous pipetting and passage through a 70-�m nylon mesh filter and waswashed with complete medium. After lysis of RBCs, CD11c� DCs wereisolated using CD11c microbeads according to the manufacturer’s instruc-tions (Miltenyi Biotec). The cells isolated were 90% CD11c� as measuredby FACS analysis.

Murine bone marrow-derived DCs were isolated from the marrow cav-ities of femurs and tibiae and were incubated in culture medium with 50ng/ml GM-CSF and 50 ng/ml IL-4 at 106 cells/ml. On day 5, DCs wereharvested by gentle pipetting and were enriched by 14.5% (by weight)metrizamide density gradient separation. After centrifugation (15 min, 4°C,2000 rpm), the low-density interface containing the DCs was collected bygentle pipette aspiration. The DCs were washed twice with culture me-dium, enumerated (purity �90% by positive-expression CD11c by FACS),and placed in culture medium with added cytokines for further studies.

Quantitative PCR (QPCR)

Total RNA was extracted using the RNeasy kit according to the manufac-turer’s protocol (Qiagen, Valencia, CA). Briefly, after DNase I (Invitrogen)treatment, 1 �g of total RNA from each sample was used as template forthe reverse transcription reaction. One hundred microliters of cDNA weresynthesized using oligo(dT)15, random hexamers, and multiscribe reversetranscriptase (Applied Biosystems, Foster City, CA). All samples werereverse transcribed under the same conditions (25°C for 10 min, 48°C for30 min) and from the same reverse transcription master mix to minimizedifferences in reverse transcription efficiency. All oligonucleotide primersfor QPCR were designed using Primer Express software 1.0 (PE Biosys-tems, Foster City, CA) and synthesized by Invitrogen. The 25-�l QPCRcontains 2 �l of cDNA, 12.5 �l of 2� SYBR Green master mix (Strat-agene, La Jolla, CA), and 250 nmol of sense and anti-sense primer. Thereaction conditions were as follows: 50°C for 2 min, 95°C for 10 min, then40 cycles of 95°C for 15 s and 60°C for 1 min. Emitted fluorescence foreach reaction was measured during the annealing/extension phase, and am-plification plots were analyzed using the MX4000 software version 3.0(Stratagene). A series of standards was prepared by performing 10-foldserial dilutions of full-length cDNAs in the range of 20 million copies totwo copies per QPCR. Subsequent analysis was performed on the dataoutput from the MX4000 software using Excel XP (Microsoft, Redmond,

WA). The threshold cycle, i.e., the cycle number at which the amount ofamplified gene of interest reaches threshold fluorescence, was determinedby using the adaptive baseline algorithm in the MX4000 analysis software.This algorithm sets the threshold cycle at 10 SDs above the backgroundfluorescence between cycles 5 through 9. Quantity values (i.e., copies) forgene expression were generated by comparison of the fluorescence gener-ated by each sample with standard curves of known quantities. Next, thecalculated number of copies was divided by the number of copies of the house-keeping gene GAPDH. In addition, we saw no significant changes in theQPCR results when the data were normalized using another constitutivelyactive gene, �2-microglobulin.

T cell proliferation assay

Immature DCs (iDCs), LPS- or fliC-treated DCs, were washed three times,diluted in fresh complete medium, and used as allogenic stimulators. Cellswere seeded in 96-well round-bottom culture plates with APC serial dilu-tions ranging from 10,000 to 500 DCs/well and were mixed together withfreshly purified CD3� T cells (100,000/well). After 5 days of incubation,cells were pulsed with 1 �Ci of [3H]thymidine per well for 18 h and wereharvested on filter paper. Proliferative responses were measured as[3H]thymidine incorporation by an automatic beta counter. Tests were per-formed in triplicates, and results were expressed as the mean cpm.

Endocytic activity

Endocytic activity of DCs was measured by the uptake of FITC-conjugateddextran (molecular mass, 40,000 kDa; Molecular Probes, Eugene, OR) aspreviously described (9). Briefly, DCs stimulated for 24 h in the absence orpresence of LPS or flagellin were incubated in complete medium plus 10%FCS plus 1 mg/ml FITC-conjugated dextran for 45 min at 4°C to measurenonspecific binding or at 37°C to measure specific uptake. Cells were thenwashed extensively and analyzed by flow cytometry.

Flow cytometry

Surface expression of various markers was assessed using CellQuest anal-ysis software on a FACSCalibur (BD Biosciences, Mountain View, CA)flow cytometer. Surface expression was determined using the followingFITC- and PE-conjugated Abs: CD86-FITC (Research Diagnostics,Flanders, NJ), CCR7-PE (R&D Systems), CD80-FITC, CD83-FITC, HLA-DR-FITC, and CD11c-PE (BD PharMingen, San Diego, CA). The isotypecontrol Abs were used accordingly in all experiments and were purchasedfrom BD PharMingen. Human DCs were incubated in 1% human AB se-rum/PBS, and murine DCs were incubated with rat anti-CD16/CD32 (BDPharMingen) to block nonspecific binding. Murine CCR7 was detectedusing macrophage-inflammatory protein 3� (MIP-3�)/CC chemokine li-gand (CCL) 19-Fc fusion protein provided by Jason Cyster (University ofCalifornia, San Francisco, CA) (24). MIP-3�-Fc was detected by additionof biotin-conjugated goat anti-human Fc, followed by PE-conjugatedstreptavidin (Caltag Laboratories, Burlingame, CA).

Quantitation of chemokines by ELISA

Human IL-8/CXC chemokine ligand (CXCL) 8 (BioSource International,Camarillo, CA), IFN-�-inducible protein 10 kDa (IP-10)/CX chemokineligand (CXCL) 10, IL-10, and IL-12 protein levels in the DC culture su-pernatants were measured by sandwich ELISA (R&D Systems).

ResultsPhenotype of iDCs

It has been previously shown that LPS, lipopeptides, dsRNA, andunmethylated CpG DNA induce cellular activation via TLRs andthat these pathogen-associated substances can induce DC matura-tion (9, 13, 15, 25–30). Flagellin, a conserved protein monomerthat makes up the bacterial flagellar filament, is recognized throughTLR5. Therefore, we decided to test whether flagellin could alsoinduce maturation of DCs (22, 31–34). We used a common in vitroculture system for the production of human monocyte-derived DCsin which adherent PBMCs are cultured in IL-4 and GM-CSF for 5days. As shown in Fig. 1A, day 0 adherent cells have surface ex-pression of CD14, CD11c, and class II MHC, but not CD83, asurface phenotype consistent with these cells being monocytes(35). After culture of these cells in IL-4 and GM-CSF for 3 and 5days, surface expression of CD14 decreases, whereas surface ex-pression of CD11c increases, a change also reflected at the mRNA

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level (Fig. 1B). This phenotype is consistent with the differentia-tion of blood monocytes into iDCs (36). In agreement with otherreports, these monocyte-derived human DCs gain expression ofTLR3, reduce expression of TLRs 1, 2, 4, 5, and 8, and lose ex-pression of TLR9 (Fig. 1C) (37). This is consistent with the pre-viously described ability of these cells to respond to dsRNA, butnot unmethylated CpG DNA (38).

As a comparison to in vitro monocyte-derived DCs, we alsoanalyzed TLR expression on primary blood DCs. As shown in Fig.1D, primary DCs express a TLR expression profile similar to thatof monocyte-derived DCs, with the exception of a higher level ofTLR10.

Bacterial flagellin matures human DCs

Analysis of TLR expression in iDCs by QPCR revealed TLR5expression in these cells (Fig. 1C). To determine whether TLR5was functional in iDCs, we treated iDCs with (fliC). After 24 h, weobserved an increase in the surface expression of CD83, CD86,and CCR7 in fliC-stimulated DCs (fliC-DCs) (Fig. 2, A and B),demonstrating TLR5 function in iDCs. This increase is also evi-dent at the mRNA level (Fig. 2C). LPS-stimulated DCs (LPS-DCs;used as positive controls in this study) also have a mature pheno-type, consistent with previous reports (9, 38–41). Finally, the TLRagonists LPS and flagellin induced expression of maturation mark-ers (CD83, CD80, CD86, CCR7), the neutrophil chemoattractant(IL-8), and proinflammatory cytokine (TNF) from primary bloodDCs in a manner similar to that of monocyte-derived DCs (Fig. 2D).

To determine whether this maturation is accompanied by otherhallmarks of DC maturation, we conducted two additional exper-iments. We measured endocytic uptake in monocyte-derived DCsusing fluorescently labeled dextran. Whereas iDCs are proficient atendocytic uptake, mature DCs reduce their fluid phase uptake inpreparation for Ag presentation to T cells (9). As shown in Fig. 3,we observed a significant reduction in endocytosis in fliC-DCs,similar to that seen in LPS-DCs.

Another feature of DC maturation is an enhanced ability to stim-ulate T cells due to increased MHC and coreceptor surface expres-sion (42, 43). To determine whether fliC-DCs have enhanced abil-ity to stimulate T cells, a T cell proliferation assay was performed.Immature DCs, LPS-DCs, and fliC-DCs were cocultured with Tcells from a separate donor for 6 days, and allospecific T cellproliferation was measured by radiolabeled thymidine incorpora-tion. T cell proliferation in cells cocultured with fliC-DCs wassimilar to that seen in T cells cocultured with LPS-DCs, and weobserved a consistent, dose-dependent increase compared withiDCs (Fig. 4A). We also assessed the T cell stimulatory activity offliC-DCs by measuring the production of IFN-� during the T cellproliferation assay. As shown in Fig. 4B, T cells cultured withfliC-DCs produced 3- to 12-fold greater levels of IFN-� relative toiDC cocultures (Fig. 4B).

Chemokine and cytokine expression in DC maturation

At the junction between innate and acquired immunity, DCs playan important role in initiating an immune response. The chemo-taxis of effector cells to sites of infection and secondary lymphoidorgans is an important aspect of immunity. To examine the poten-tial role of DCs in trafficking of immune effector cells, we mea-sured expression of chemokines (as well as cytokines) in fliC-DCs.We observed increased mRNA levels for a number of chemokines,notably growth-related oncogene-� (GRO-�)/CXCL1, GRO-�/CXCL2, GRO-�/CXCL3, IL-8/CXCL8, monocyte chemoattrac-tant protein-1 (MCP-1)/CCL2, MIP-1�/CCL3, and MIP-1�/CCL4, which are expressed quite early after stimulation (�1–3 h)at high levels. Interestingly, the T cell chemoattractants IP-10/

FIGURE 1. Phenotype of human monocyte-derived iDCs. A, Adherentmonocytes (day 0) and DCs cultured in vitro with 50 ng/ml IL-4 and 50ng/ml GM-CSF for 3 and 5 days were stained with fluorescent-conjugatedAbs and examined by flow cytometry to measure the surface markers in-dicated (open histograms) or an isotype control Ab (filled histograms).Histograms depict the level of surface expression and the value indicatedon each histogram is the mean fluorescence intensity (MFI) of the marker-specific Ab. B and C, Portions of cells (3 � 105 cells each) used in A wereharvested for RNA. D, Human primary CD4� blood DCs (3 � 105 cells)were isolated and harvested for RNA. CD14, CD11c, CD83, and TLR1–10mRNA expression was determined by QPCR and is depicted as the numberof copies of the gene of interest per copy of the housekeeping geneGAPDH. The results shown are from a single experiment with a singledonor and are representative of four similar experiments.

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CXCL10, monokine induced by IFN-� (MIG)/CXCL9, and IFN-inducible T cell � chemoattractant (I-TAC)/CXCL11, which areexpressed in LPS-DCs at later time points (�8 h), were not ex-pressed in fliC-DCs (Fig. 5, A and B).

As shown in Fig. 5C, fliC stimulated the expression of the proin-flammatory cytokines IL-1� and TNF, as well as the Th1 cytokinesIFN-� and IL-12 p40. In contrast with LPS-DCs, fliC-DCs failedto induce expression of IFN-� and IFN-�. The inability of fliC toinduce IFN-�/� expression may explain why the IFN-induciblechemokines IP-10, MIG, and I-TAC were not expressed by fliCstimulation. Recently, Vogel and coworkers (44) reported thatTLR4, but not TLR2, agonists could induce IFN-� expression andsubsequently STAT1�/� phosphorylation. In mice, LPS-inducedIP-10 expression has been shown to be STAT1-dependent, as dem-onstrated by the failure of STAT1�/��/� macrophages to expressIP-10 mRNA in response to LPS (45). To determine whether other

non-IFN-�-inducing stimuli also fail to induce IP-10, MIG, andI-TAC, we stimulated iDCs with synthetic lipopeptide (palmitoylcys-teine[(RS)-2,3-di(palmitoyloxy)propyl]serine-lysine-OH, a TLR1/2stimulus). Lipopeptide-stimulated DCs failed to express IFN-�/� aspreviously described and also failed to express IP-10, MIG, and I-TAC (data not shown). Thus, TLR5 (fliC) and TLR1/2 (lipopeptide)agonists appear to similarly activate only a subset of TLR4 (LPS)-inducible proinflammatory genes.

To determine whether the increase in chemokine and cytokinemessage level is accompanied by protein secretion, ELISA wasperformed on the supernatants of fliC-DCs. We observed increasedIL-8 in the supernatant of fliC-DCs compared with that of iDCs,whereas IP-10 protein production was not detected in fliC-DCs(Fig. 5, E and F). This pattern of chemokine secretion induced byflagellin is similar to what has been previously described for mac-rophages stimulated with lipopeptide (44).

FIGURE 2. Dose-dependent induction of DC maturation by flagellin. A and B, Adherent cells from peripheral blood were cultured in 50 ng/ml IL-4 and50 ng/ml GM-CSF for 5 days. On day 6, fliC or LPS was added at the concentrations indicated for 24 h. Cells were stained with a FITC- or PE-conjugatedmAb specific for CD83, CD80, CD86, or CCR7 (open histograms) or isotype control (filled histograms) and were examined by flow cytometry. Histogramsdepict the level of surface expression, and the value indicated on each histogram is the MFI of the marker-specific Ab. C, Portions of cells (3 � 105 cellseach) used in A were harvested for RNA. D, Human primary CD4� blood DCs (3 � 105 cells) were stimulated with LPS (100 ng/ml) or flagellin (20 nM)and harvested for RNA at 3 h (IL-8 and TNF) or 24 h (CD83, CD80, CD86, and CCR7). CD83, CD80, CD86, CCR7, TLR5, TNF, and IL-8 mRNAexpression was determined by QPCR and is depicted as the number of copies of the gene of interest per copy of the housekeeping gene GAPDH. The resultsshown are from a single experiment with a single donor and are representative of three similar experiments.

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Upon encounter with microbial pathogens, DCs produceimmunostimulatory cytokines, such as IFN-� and IL-12, whichare important for selective activation of T cells into the Th1phenotype. Interestingly, LPS-stimulated DCs, but not flagel-lin-stimulated DCs, produced bioactive IL-12 p70 (Fig. 5G).Meanwhile, neither LPS nor flagellin induced IL-10 proteinproduction (Fig. 5H). These data fit with previously publisheddata by Thoma-Uszynski et al. (12), which demonstrate thatLPS and lipopeptide induced IL-12, but not IL-10, in monocyte-derived DCs (10). These data suggest that fliC-DCs may not beas potent at generating a Th1 cytokine response as LPS-DCs orlipopeptide DCs.

Exogenous human IFN-� restores TLR5-mediated geneexpression

LPS, but not flagellin, induced the expression of IP-10, MIG,and I-TAC in a time-dependent manner (Fig. 5, A and B).Treatment of DCs with flagellin (20 nM) in combination withrIFN-� (50, 25, and 10 U/ml) induces expression of IP-10,MIG, and I-TAC mRNA, whereas stimulation with IFN-� orflagellin alone failed to induce these chemokines (Fig. 6, A–C).This suggests that IFN-� production after LPS stimulationprovides an additional signal required for IP-10, MIG, andI-TAC expression. Meanwhile, addition of IFN-� had no effecton flagellin-induced IL-8 mRNA expression (Fig. 6D). Todetermine whether the increase in IP-10 message level wasaccompanied by protein secretion, ELISA was performed on thesupernatants of flagellin and IFN-�-stimulated DCs. As shownin Fig. 6E, treatment of DCs with flagellin in combination withrIFN-� induced IP-10 protein production.

Bacterial flagellin fails to mature murine DCs

To demonstrate TLR5 function in murine DCs, we isolated murinesplenic DCs and stimulated them with fliC. Surprisingly, purifiedbacterial flagellin was unable to mature murine splenic DCs invitro as determined by cell surface and mRNA expression ofCD80, CD86, and CCR7 (Fig. 6, A and B). As shown in Fig. 6C,QPCR analysis of murine DCs revealed no expression of TLR5,consistent with their inability to respond to flagellin. Interestingly,murine bone marrow-derived DCs, peritoneal macrophages, andthe murine macrophage cell line RAW 264.7 fail to express TLR5mRNA (Fig. 6D). We were only able to demonstrate positiveTLR5 expression in murine skin epithelia (Fig. 6D). These resultsare in agreement with a recent report by Applequist et al. (46),which also used mouse skin cDNA as a positive control and dem-onstrated little to no expression of TLR5 in several murine mono-cyte, macrophage, and DC cell lines. Also, we detected little or noTLR5 mRNA expression in resident and thioglycollate-elicited

FIGURE 3. DC maturation with flagellin down-regulates endocytic ac-tivity. Day 5 iDCs were stimulated with 20 nM fliC or 100 ng/ml LPS for24 h. Cells were incubated with FITC-conjugated dextran (1 mg/ml) for 45min at 4°C (filled histograms) or at 37°C (open histograms) and wereexamined by flow cytometry to measure nonspecific and specific binding,respectively. Histograms depict the level of FITC-conjugated dextran up-take, and the value indicated is the MFI of the cells cultured at 37°C. Theresults shown are from a single experiment with a single donor and arerepresentative of three similar experiments.

FIGURE 4. Flagellin-matured DCs have enhanced T cell stimulatoryactivity in vitro. Day 5 iDCs were stimulated with 20 nM fliC or 100 ng/mlLPS for 24 h. Purified T cells were then added to DCs at the ratio indicatedand were allowed to incubate for an additional 5 days. A, On day 6, 1�Ci/well [3H]thymidine was added and allowed to incubate for an addi-tional 18 h. T cell proliferation was measured by [3H]thymidine uptake(cpm). B, The concentrations of IFN-� from culture supernatants takenfrom 1:10 T:DC ratio wells with DCs stimulated with 6.5–60 nM fliC weremeasured by ELISA. The error bars represent the SD of triplicatedeterminations.

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peritoneal macrophages from C57BL and BALB/c mice. Further-more, these macrophages, as well as purified splenic DCs, failed toproduce TNF or IL-8 protein production in response to flagellinstimulation (data not shown).

Interestingly, human DCs express TLR5, but not TLR9 (Fig.1C), whereas mouse DCs express TLR9, but not TLR5 (Fig. 6C).The biological consequence of this species-specific difference re-mains unclear and will be discussed below.

FIGURE 5. Chemokine and cytokine expression during flagellin-induced DC maturation. Total RNA was isolated from day 5 iDCs (1 � 106 cells)stimulated with 20 nM fliC or 100 ng/ml LPS for 1, 3, 8, 24, and 48 h. Expression of chemokines (A and B) and cytokines (C and D) was quantified byQPCR. The number of transcripts is normalized to the number of copies of GAPDH. Data shown are representative of three experiments. E–H, Theconcentrations of the chemokines and cytokines IL-8, IP-10, IL-12 p70, and IL-10 in the above cultured supernatants were measured by ELISA. The resultsshown are from a single experiment with a single donor and are representative of three similar experiments.

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DiscussionIn this report, we demonstrate maturation of human monocyte-derived DCs to the TLR5 stimulus bacterial flagellin. This obser-vation helps explain previous reports regarding the high antigenic-ity and adjuvant activity of bacterial flagellin (47). Our resultsdemonstrate that TLR5 is expressed on human iDCs and that thesecells are sensitive to flagellin stimulation, consistent with otherreports demonstrating TLR5 expression in human monocytes andiDCs by Northern blot and RT-PCR analysis (37, 38, 48).

We demonstrated a dose-dependent induction of DC maturationusing 0.2–60 nM fliC. In subsequent experiments we chose topresent data using 20 nM � 1 �g/ml fliC, because it was the lowestconcentration that consistently gave a robust response and wasequivalent to the potent maturation inducer LPS. Flagellin (20 nM)and LPS (100 ng/ml) consistently induced DC activation as mea-sured by T cell stimulatory activity, induction of maturation andcostimulatory molecules, reduction in endocytic activity, andequivalent expression of TNF and IL-1� (Fig. 5B). However, wehave consistently found that LPS induces higher levels of CD86 on

LPS-DCs, which may explain the slightly higher T cell stimulatoryactivity of LPS-DCs compared with fliC-DCs (Figs. 2B and 4).

Interestingly, our observations suggest that there are differencesbetween DCs matured with LPS and DCs matured with flagellin.fliC-DCs do not express the T cell chemokines MIG, IP-10, andI-TAC or IFN-�/�, which are all expressed by LPS-DCs (Fig. 7).This suggests that there are qualitative differences between thesignals generated by TLR5 compared with that provided by TLR4,a notion consistent with recent reports demonstrating the role ofIFN regulatory factor 3 (IRF3) in mediating TLR3- and TLR4-specific transcriptional responses, but not TLR2 and TLR9 re-sponses (49, 50). The simplest explanation for the chemokine dis-parity between fliC-DCs and LPS-DCs is that LPS stimulationresults in TLR4-mediated early IFN-� secretion through an IRF3-mediated signaling pathway, leading to the expression of MIG,IP-10, and I-TAC. TLR5-mediated signals do not result in thisearly IFN-� production and consequently do not mediate the ex-pression of MIG, IP-10, and I-TAC (Figs. 5, A and C, and 8). To

FIGURE 6. Exogenous human IFN-� restores the inducibility of expression of IP-10, MIG, and I-TAC in flagellin-treated DCs. A–D, Human monocyte-derived DCs were treated with flagellin (20 nM) or LPS (100 ng/ml) for 8 h in the absence or presence of exogenous rIFN-� (5–50 U/ml). IP-10, MIG,I-TAC, and IL-8 mRNA were detected by QPCR. E, The concentration of IP-10 in the above cultured supernatant was measured by ELISA.

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test this hypothesis, we stimulated DCs with flagellin in combina-tion with exogenous rIFN-�. Interestingly, stimulation with bothflagellin and IFN-� resulted in IP-10, MIG, I-TAC expression,whereas flagellin or IFN-� treatment alone failed to induce thesechemokines (Fig. 6). These data suggest that IFN-� productionafter LPS stimulation provides an additional signal required forIP-10, MIG, and I-TAC expression. The biological consequence ofthis disparity between the LPS and flagellin responses is notclear; however, in an actual infection, a number of differentTLRs are likely to be engaged simultaneously, perhaps makingthe specific signaling pathways used by any one TLR biologi-cally less relevant (2, 4).

Flagellin and LPS induced the expression of a number of otherchemokines. The neutrophil-active chemokines GRO-�, GRO-�,GRO-�, and IL-8 were expressed in both LPS- and fliC-treatedDCs, as were the monocyte/macrophage/NK-active chemokinesMCP-1, MIP-1�, MIP-1�, and RANTES. These findings suggestthat DCs not only play a role in the initiation of an acquired im-mune response, but that they may also play a role in amplifying theearly immune response by participating in the trafficking of innateimmune cells to sites of infection. The early expression of these

chemokines (1–3 h) is consistent with the notion that iDCs, uponencounter with pathogens, express these chemokines before theexpression of the lymph node homing chemokine receptor CCR7,which is maximally expressed at 24 h after stimulation (Fig. 2B).

Another intriguing finding was that murine splenic DCs do notmature to purified bacterial flagellin. Besides providing a controlfor the purity of our fliC preparation, this seems at odds with therecent findings by McSorley et al. (47) demonstrating the ability ofbacterial flagellin to effectively elicit a robust CD4 T cell responsein vivo. Though our in vitro findings predict flagellin will notdirectly stimulate DC maturation in vivo, it is still possible thatother cells, such as skin epithelia and gut epithelia, which expressTLR5 (Fig. 7D), are able to produce cytokines (such as TNF andIFNs), which may then trigger DC maturation (34, 51). This isconsistent with our previous data demonstrating IL-6 production inthe serum of flagellin-injected wild-type mice, but not of MyD88knockout mice (20).

A recent report by Renshaw et al. (52) demonstrated murineTLR5 expression in thioglycollate-elicited peritoneal macrophagesfrom 2- to 3-mo-old C57BL/6 mice. Moreover, this group found

FIGURE 7. TLR5-mediated flagellin stimulation fails to mature murine DCs. A, Murine DCs were purified from spleens using anti-CD11c microbeads(Miltenyi Biotec). Murine splenic DCs were stained with a FITC-conjugated mAb specific for CD80 or CD86 (open histograms) or isotype control (filledhistograms) and were examined by flow cytometry. Histograms depict the level of surface expression, and the value indicated on each histogram is the MFIof the marker-specific Ab. Murine CCR7 was detected using MIP-3�/CCL19-Fc, followed by addition of biotin-conjugated goat anti-human Fc, followedby PE-conjugated streptavidin. B and C, Portions of cells (3 � 105 cells each) used in A were harvested for RNA. Murine CD83, CD80, CD86, CCR7,and TLR1–9 mRNA expression was determined by QPCR. D, TLR5 mRNA expression in the RAW 264.7 murine macrophage cell line, murine bonemarrow-derived DCs, peritoneal macrophages, and skin epithelia was determined by QPCR and is depicted as the number of copies of the gene of interestper copy of the housekeeping gene GAPDH.

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that flagellin treatment could induce IL-6 and TNF productionfrom thioglycollate-elicited peritoneal macrophages isolated fromyoung mice, but not old mice. In addition, splenic macrophageswere found to express lower levels of TLR5 and were nonrespon-sive to flagellin treatment. In Fig. 7, we demonstrated that residentperitoneal macrophages did not express TLR5 mRNA. We alsoassessed TLR5 mRNA expression in resident and thioglycollate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice.Unlike Renshaw et al. (52), we were only able to detect low levelsof TLR5 mRNA expression in these peritoneal macrophages (Fig.7; data not shown). Furthermore, these cells did not produce TNFor IL-8 in response to flagellin stimulation (data not shown). Thesedifferences may be due to the way the cells were handled or cul-tured. Moreover, Renshaw et al. (52) did not provide any infor-mation about the purity of their flagellin preparation. We havefound that it is very difficult to eliminate TLR2/TLR4 agonistsfrom flagellin preparations. Our flagellin preparations are purifiedusing extensive dialysis and size fractionation and are rigorouslytested on Chinese hamster ovary cells stably transfected withTLR2, TLR4, or TLR5. Only flagellin preparations/fractions thatsolely activated TLR5-expressing cells were used in our studies.Alternatively, Renshaw et al.’s flagellin preparation (52) may bemodified, enabling it to stimulate through murine TLR5 or in com-bination with another unidentified receptor. Indeed, we have foundthat monomeric flagellin (induced by sonicating) stimulates DCsmore potently than does aggregated flagellin. However, both ofthese forms failed to activate murine macrophages or murine DCs

in our experiments. Interestingly, Renshaw et al. (52) reported thatsplenic macrophages expressed only low levels of TLR5 and didnot respond to flagellin, whereas thioglycollate-elicited peritonealmacrophages expressed higher levels of TLR5 and responded toflagellin. This might indicate that TLR5 varies based on the acti-vation state (thioglycollate elicited) and anatomical location(spleen vs peritoneum). However, we have not been able to repro-duce these findings.

The biological significance of murine DC unresponsiveness toflagellin is unclear. Again, a number of different TLRs are likelystimulated during exposure to microbes and the stimulation ofTLR5 by flagellin is just one of these possible interactions. There-fore, it is unlikely that the lack of recognition of bacterial flagellinby murine DCs will result in mice displaying enhanced suscepti-bility to flagellated bacteria compared with humans; however, itmay lead to qualitative differences in the immune response to flag-ellated bacteria between mice and humans (53, 54).

Recently, Rehli (reviewed in Ref. 54) clarified the species-spe-cific variations of TLR expression in mice and humans. Recentdata from several groups suggest that constitutive and inducibleTLR expression in different cell types and in different species arecontrolled by transcriptional regulation (55–57). Moreover, generegulatory elements found in the proximal promoters of TLR genescontrol cell-type specificity and inducible TLR expression in miceand humans. For example, the human TLR2 gene is constitutivelyexpressed in monocytes and is not inducible by microbial patho-gens. In contrast, the mouse TLR2 gene, which has NF-�B and

FIGURE 8. Model of TLR5- vs TLR4-induced chemokine production in human DCs. Activation of TLR5 and TLR4 by flagellin and LPS,respectively, induces a differential set of chemokines. Both TLR5 and TLR4 agonists induced the expression of chemokines active on neutrophils(IL-8/CXCL8, GRO-�,�,�/CXCL1–3), monocytes (MCP-1/CCL2), NK cells (RANTES/CCL5), and iDCs (MIP-1�/CCL3, MIP-1�/CCL4), but onlythe TLR4 agonist LPS induced chemokines active on effector T cells (IP-10/CXCL10, MIG/CXCL9, I-TAC/CXCL11). TLR4 and TLR3 agonistshave been shown to induce nuclear translocation of IRF3, which leads to the expression of IFN-�. IFN-� stimulates the IFN-�/� receptor throughan autocrine loop on DCs, which leads to the phosphorylation of STAT1�/�. STAT1�/� then induces expression of the T cell attracting chemokinesIP-10, MIG, and I-TAC. Stimulation with flagellin in combination with exogenous rIFN-� induces IP-10, MIG, and I-TAC, whereas stimulation withIFN-� or flagellin alone fails to induce these chemokines. This suggests that IFN-� production after LPS stimulation provides an additional signalrequired for IP-10, MIG, and I-TAC expression.

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STAT5 transcription factor binding sites in its proximal promoter,is rapidly induced by microbial stimuli (57). The human TLR3gene is constitutively and selectively expressed in myeloid DCs,whereas the mouse TLR3 gene is inducible and expressed in mac-rophages. Likewise, TLR9 and TLR5 appear to be regulated in acell type- and species-specific manner. Human DCs express TLR5,but not TLR9 (Fig. 1C), whereas mouse DCs express TLR9, butnot TLR5 (Fig. 7C). The biological consequence of this species-specific difference remains unclear.

Our findings and other studies suggest that the stimulation ofany TLR expressed on DCs is sufficient for maturation, as mea-sured by surface expression of coreceptors, T cell stimulatory ac-tivity, and endocytic activity (9, 38). Finally, we demonstrate aqualitative difference in gene expression of IFN-�/� and the T cellchemokines IP-10, I-TAC, and MIG between fliC-DCs andLPS-DCs.

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