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BκMaturation: Possible Role of CCL2 and NF-CCR2 Plays a Critical Role in Dendritic Cell

Ibarra, Peter C. Melby and Seema S. AhujaCarlos A. Estrada, Kassandra Clark, Edgar Garavito, Jessica Fabio Jimenez, Marlon P. Quinones, Hernan G. Martinez,

http://www.jimmunol.org/content/184/10/5571doi: 10.4049/jimmunol.0803494April 2010;

2010; 184:5571-5581; Prepublished online 19J Immunol 

Referenceshttp://www.jimmunol.org/content/184/10/5571.full#ref-list-1

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The Journal of Immunology

CCR2 Plays a Critical Role in Dendritic Cell Maturation:Possible Role of CCL2 and NF-kB

Fabio Jimenez,*,†,1 Marlon P. Quinones,*,†,1 Hernan G. Martinez,*,† Carlos A. Estrada,†

Kassandra Clark,*,† Edgar Garavito,† Jessica Ibarra,† Peter C. Melby,*,† and

Seema S. Ahuja*,†

We postulated that CCR2-driven activation of the transcription factor NF-kB plays a critical role in dendritic cell (DC) maturation

(e.g., migration, costimulation, and IL-12p70 production), necessary for the generation of protective immune responses against the

intracellular pathogen Leishmania major. Supporting this notion, we found that CCR2, its ligand CCL2, and NF-kB were required

for CCL19 production and adequate Langerhans cell (LC) migration both ex vivo and in vivo. Furthermore, a role for CCR2 in

upregulating costimulatory molecules was indicated by the reduced expression of CD80, CD86, and CD40 in Ccr22/2 bone

marrow-derived dendritic cells (BMDCs) compared with wild-type (WT) BMDCs. Four lines of evidence suggested that CCR2

plays a critical role in the induction of protective immunity against L. major by regulating IL-12p70 production and migration of

DC populations such as LCs. First, compared with WT, Ccr22/2 lymph node cells, splenocytes, BMDCs, and LCs produced lower

levels of IL-12p70 following stimulation with LPS/IFN-g or L. major. Second, a reduced number of LCs carried L. major from the

skin to the draining lymph nodes in Ccr22/2 mice compared with WT mice. Third, early treatment with exogenous IL-12 reversed

the susceptibility to L. major infection in Ccr22/2 mice. Finally, disruption of IL-12p70 in radioresistant cells, such as LCs, but not

in BMDCs resulted in the inability to mount a fully protective immune response in bone marrow chimeric mice. Collectively, our

data point to an important role for CCR2-driven activation of NF-kB in the regulation of DC/LC maturation processes that

regulate protective immunity against intracellular pathogens. The Journal of Immunology, 2010, 184: 5571–5581.

Dendritic cell (DC)maturation is a highly regulated cellularevent that includes at least three processes: migration,upregulation of costimulatory surface molecules (along

with Ag presentation), and the release of polarizing cytokines, suchas IL-12 (1–4), by Th cells. Models describing events occurring ineach of the three processes described above have emerged (5, 6), yetthe full array of factors coordinating DC maturation remains un-known. To date, these models build on disjointed evidence sug-gesting that DC maturation processes are driven by gene programstriggered by transcription factors, such as NF-kB (7–10).The study of the biology of DCs residing in the skin, such as

Langerhans cells (LCs) and dermal DCs, has provided significantinsight into the mechanisms underlying DC maturation (11–13),particularly as it relates to migration (5, 6). For instance, LC mi-gration is dependent on selective changes in the levels of chemo-attractant molecules, known as chemokines, and to changes in LCresponsiveness to these chemoattractants, a consequence in theupregulation of chemokine receptors such as CCR7 (6, 14, 15).

CCL19, a major ligand for CCR7, is produced by maturing DCsand by high endothelial venules, creating a gradient from the siteof its release to the lymphatics (5, 6, 16, 17). Cognate interactionsbetween this gradient and CCR7, present on the DC’s surface,drives the cells into the draining lymph nodes (DLNs) (6, 17, 18).Moreover, it is likely that induction of CCL19 production and theparallel maturation process are initially triggered by chemokinesproduced early on, which have the ability to provide initial signalsleading to NF-kB–dependent gene transactivation.Several models have been widely used in the literature to study

LCs. In this study, we took advantage of an ex vivo model of earskin explants and an in vivo model of contact hypersensitivity. (19–21). In the former model, skin from mouse ears is separated intotwo layers and placed in a petri dish containing culture media. Thephysical separation of the two skin layers induces LCs migrationfrom the epidermis into the dermal lymphatics. LCs are collectedas they fall off from the lymphatic channels directly into the petridish (19). The in vitro model involves painting the skin with FITCplus an irritant to induce the migration of LC carrying FITC to theDLNs (20, 21). In a previous study, our laboratory successfullytested the hypothesis that, in addition to CCR7, CCR2 plays a keyrole in LC trafficking during inflammation using these models.We showed that mice with genetic inactivation of CCR2

exhibited impaired LC migration and an inability to control in-fection with the intracellular pathogen Leishmania major (21).Notably, our group (and others) has previously shown that CCR2-dependent signals result in the induction of NF-kB translocation inastrocytes, but it remains unknown whether CCR2-dependentactivation of NF-kB is also linked to CCR2-dependent modulationof DC function. This seems plausible considering that DC matu-ration has been shown associated with changes in the expressionof CCR2 (22) and that maturation is thought to be modulated byNF-kB, as described above.

*Audie L. Murphy Division, Veterans Administration Center for Research on AIDSand HIV-1 Infection, South Texas Veterans Health Care System; and †Department ofMedicine, University of Texas Health Science Center, San Antonio, TX 78229

1F.J. and M.P.Q. contributed equally to this work.

Received for publication October 21, 2008. Accepted for publication March 8, 2010.

This work was supported by National Institutes of Health Grants RO1-AR052755 andRO1-AI48644 and a Veterans Affairs merit grant.

Address correspondence and reprint requests to Dr. Seema S. Ahuja, Department ofMedicine (MC 7870), University of Texas Health Science Center at San Antonio, 7703Floyd Curl Drive, San Antonio, TX 78229-3900. E-mail address: ahuja@uthscsa.edu

Abbreviations used in this paper: BM, bone marrow; BMDC, bone marrow-deriveddendritic cell; DC, dendritic cell; DLN, draining lymph node; Flt3L, Flt3 ligand;KLH, keyhole limpet hemocyanin; KO, knockout; LC, Langerhans cell; LTC4, leu-kotriene C4; PB, parasite burden; PDTC, pyrrolidine dithiocarbamate; rm, recombi-nant murine; RPA, RNase protection assay; RR, radioresistant; WT, wild-type.

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We surmised that migration defects in CCR2-deficient micemight be a component of the broader disturbances affecting DCmaturation. Hence, using mice with genetic inactivation of CCR2,we sought to investigate the impact of CCR2 in DC migration andmaturation, aiming to test the hypothesis that CCR2-driven acti-vation of NF-kB could play a critical role in DC maturationprocesses, which in turn might be necessary for the generation ofprotective immune responses against the intracellular pathogenL. major. In this paper, we show that CCR2, potentially throughthe induction of NF-kB translocation, plays a role in DC matu-ration involving CCL19 production, LC migration; Ag pre-sentation with upregulation of costimulatory molecules in DCs;and the release of the Th1-inducing cytokine IL-12. Furthermore,we also provide evidence suggesting that IL-12p70, production byradioresistant (RR) cells, such as LCs, contributes to the genera-tion of protection against L. major infection. Thus, the role ofCCR2 in the elicitation of protective immunity is 2-fold; on onehand, CCR2 modulates the amount of IL-12 produced by DCs,and on the other hand, CCR2 regulates the migrating number ofIL-12p70 producing LCs.

Materials and MethodsMaterials

RPMI 1640 medium, medium 199, antibiotics, FBS, HEPES, PBS, andtrypsin were obtained from Invitrogen (Carlsbad, CA). Leukotriene C4

(LTC4) was from Cayman Chemical (Ann Arbor, MI), and recombinantproteins CCL19 (ED50, 3–15 ng/ml; endotoxin , 0.1 ng/mg) and CXCL1(ED50, 3–15 ng/ml; endotoxin , 1.0 EU/mg cytokine) were from R&DSystems (Minneapolis, MN). All other chemicals used were from Sigma-Aldrich (St. Louis, MO), and Abs were from BD Biosciences (SanDiego, CA), unless stated otherwise. Intracellular staining for Langerin(CD207) was conducted using the clone 929F3.01 from Dendritics(Lyon, France).

Mice, CCL19 blockade, and bone marrow transplantation

The Institutional Animal Care and Use Committee approved all protocols.The genetic inactivation of CCR2 mice and genotyping has been describedpreviously (23). Wild-type (WT) and gene knockout (KO) mice were onthe C57BL/6J background (n . 10 generations). In some experiments, weused WT and CCR2 KO mice of C57BL/6 3 129 background, and weobtained similar results to those in the C57BL/6J. Mice were born and bredunder specific pathogen-free conditions at the University of Texas HealthScience Center (San Antonio, TX). SCID mice were purchased from Ta-conic Farms (Germantown, NY). C57BL/6J IL-12p352/2 mice were pur-chased from The Jackson Laboratory (Bar Harbor, ME).

CCL19 Ab neutralization was performed in WT C57BL/6J, as describedpreviously (16). Briefly, 50 mg purified goat IgG or neutralizing polyclonalAbs against CCL19 (R&D Systems) was administered 1 d prior to L. majorinfection and weekly thereafter. Ear thickness was recorded on a weeklybasis, mice were sacrificed 4 wk postinfection, and parasite burdens weredetermined in DLNs.

For bone marrow (BM) transplantation, recipient mice received 1000cGy (lethal dose) irradiation from a 137Cs source 6–8 h before the celltransfer. Donor mice were euthanized, and BM was harvested, as describedbelow. BM cell suspensions were depleted of RBCs using RBC lysisbuffer, resuspended in PBS, and transferred into recipient mice (10 3 106

in 200 ml PBS) via tail vein injection. Recipient mice were infected 8 wkafter BM transfer.

Parasites, infection, and tracking of infected LCs

L. major clone VI (MHOM/IL/80/Friedlin) was a gift from Dr. D.L. Sacks(Laboratory of Parasitic Diseases, National Institutes of Health, Bethesda,MD). The virulence of the L. major strain was maintained by propagating itin mice (21). Protocols similar to those used to prepare soluble LeishmaniadonovaniAgs were used to make soluble L. majorAg (21). The intradermalroute was used to infect both ears with 23 106 or 13 107 stationary-phaseL. major promastigotes resuspended in 20 ml PBS (21). Higher doses ofparasites were used with similar results in short-term experiments. A dial-thickness gauge caliper (Fred V. Fowler, Newton, MA) was used to measureear thicknesses at weekly intervals for 5 wk (21). The parasites were grownin medium 199 containing 20% FBS and supplemented with hemin, ade-

nine, and antibiotics. The limiting dilution culture method was used toquantify parasite burden (21). In brief, relevant organs were weighed andhomogenized between two frosted slides. The homogenate was placed in96-well plates in triplicate, and 5-fold serial dilutions were performed incomplete medium 199. Parasites were cultured for 2 wk at 27˚C. The plateswere scored for the presence of motile parasites in individual wells using aninverted microscope at3200 magnification. The parasite scores are the logvalues of the reciprocal from the last positive dilution.

PKH26 (Sigma-Aldrich) labeling of L. major was performed as de-scribed by Quinones et al. (24). A minimum of 2.5 3 107 PKH-labeledparasites were used to infect mice. Injection with a lower amount ofparasites made the identification of cells carrying the parasite unreliable.This adjustment in the dose was performed to account for the loss ofparasite viability associated with PKH labeling and to increase our abilityto detect fluorescent-labeled parasites in vivo. Parasite viability was pre-served following PKH labeling and in vivo inoculation. Confirming thepathogenicity and viability of labeled parasites in vivo, we found that in-fection with PKH-labeled parasites did in fact result in chronic infection asdocumented by parasite recovery from the DLNs of infected animals.Three days following intradermal injection of PKH-labeled L. major, micewere euthanized, and FACS was used to determine the proportion ofCD11c+ cells carrying PKH-labeled L. major in the DLNs. Cell sorting andsubsequent culture of PKH+CD11c+ cells under conditions favoring par-asite growth gave rise to L. major, indicating that PKH labeling of L. majordid not significantly affect viability and pathogenicity (data not shown).We inferred that LCs constituted a large proportion of the population ofPKH+CD11c+-labeled cells considering that the majority of these cells alsoexpressed CD11b+ and DEC205+ and were CD8a2/low (data not shown).Furthermore, in vitro, PKH+CD11c+ cells promoted Ag-specific pro-liferation of purified T cells derived from the L. major-infected mice.

LC migration to lymph nodes and NF-kB in vivo inhibition

The protocols described by Sato et al. (21) were used to track the migrationof FITC+ LCs, from the ear skin to the DLNs in uninfected animals. In brief,freshly prepared 5% FITC in 1:1 acetone/dibutylphthalate was painted onboth sides of the ears (10 ml each) and on the abdomen (400 ml) (to quantifythe number of FITC-positive DLNs following Flt3 ligand [Flt3L] treat-ment). The DLNs were harvested 48 h later and analyzed by FACS or frozenin OCT compound (Sakura Finetek, Torrance, CA), and 5-mm cryostatsections were prepared and examined by fluorescence microscopy (BX60Olympus microscope; Olympus, Melville, NY). Animals were given a 0.1-ml i.p. and 10-ml intradermal (ear) injection of the NF-kB inhibitors pyr-rolidine dithiocarbamate (PDTC; 6 mg/ml) and MG-132 (5 mM) 1 h beforeFITC painting or ear explant experiments.

Cell migration from ear skin explants

As previously described (19, 21), ears were dissected, rinsed in 70%ethanol with vigorous shaking, and allowed to dry for 15 min. The ventraland dorsal sheets of ear skin were separated with a pair of fine forceps. Thetwo leaflets were transferred dermal side down for 24 or 72 h in a 10-cmpetri dish containing culture media. The loosely adherent populations ofcells that spontaneously emigrated out of the dermal layers were recoveredby incubating the dermal layers in PBS containing 2 mg/ml glucose for20 min at 37˚C. The collected cells were pooled together, filtered througha 70-mm nylon cell strainer, washed with PBS, and then stained. In theseexperiments, LCs were identified as I-Ab bright and DEC-205+. In selectedexperiments, different compounds were added to the culture media of theear explants.

Generation BM-derived DC and in vitro infections

BM-derived DCs (BMDCs) were generated as described previously (25).Briefly, BM was collected from 6- to 8-wk-old mice and cultured in thepresence of: recombinant murine (rm)GM-CSF (50 ng/ml) and rmIL-4(1 ng/ml). BMDC cultures have been shown to contain a large proportionof mature DCs (25). For infection studies, BMDCs were cultured with liveL. major parasites in a 1:10 ratio.

In vitro Ag presentation activity

The Ag-specific syngeneic proliferation assays adapted for this experimenthavebeendescribedpreviously (21).WTmicewere immunizedwith keyholelimpet hemocyanin (KLH) and sacrificed 10–15 d postimmunization, and theDLNs were collected as a source of responding cells. KLH-respondingT cells (isolated using murine T cells, enrichment columns [R&D Systems])were cocultured (1:4 ratio) with KLH-pulsed (200mg/ml) BMDCs fromWTor Ccr22/2 mice. After 48 h, culture supernatants were harvested, and cy-tokine levels were measured by ELISA.

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Flt3L treatment

In a set of experiments, 10 d prior to infection, WT and Ccr22/2 micereceived a daily i.p. dose of 10 mg recombinant human Flt3L, as previouslydescribed (26, 27), and followed during a period of 7 wk. Groups of un-infected animals were used for corroboration of the Flt3L effect on DCs byFACS and analysis of DC migration using ear explants and FITC painting.

Administration of rmIL-12 or LPS and measurement ofendogenous heterodimeric IL-12p70 production

Mice were injected i.p. with 0.5 mg rmIL-12 (ED50 , 0.1 ng/ml, endotoxin, 0.1 ng/mg; PeproTech, Rocky Hill, NJ) or 100 mg LPS at the time ofinfection and repeated at 24 and 48 h postinfection. The IL-12 adminis-tration was a modification from a protocol described by Heinzel et al. (28).The administration period of IL-12 was shortened from 7 to 2 d given thatwe wanted to capture the early release of this cytokine. A total of 1 mg/mlLPS and 10 ng/ml IFN-g (ED50 0.1 ng/ml, endotoxin , 0.1 ng/mg; Pe-proTech) were used to induce IL-12p70 production in DLNs using pro-tocols described previously (24). At different time points (6, 12, 24, and48 h poststimulation with LPS plus IFN-g), cultured supernatants werecollected. ELISA was used to determine IL-12p70 levels in the super-natants, as described previously (24). Unless indicated otherwise, back-ground values obtained in the absence of stimulation are subtracted fromthe data presented.

NF-kB translocation in ear explants

Ears from WT and Ccr22/2 mice were split and immediately frozen (0 h)or left in culture for 2 h and then frozen. Subsequently, ears were cut intosmall pieces and homogenized, and the nuclear proteins were extractedusing the nuclear protein extraction kit from Panomics (Fremont, CA)following the manufacturer’s protocols. NF-kB was quantified in the nu-clear extracts using a previously validated beads-based system fromPanomics. The coefficient of variation for the assay was under 5% basedon the company’s data. The amount of NF-kB minus background signalwas normalized based on 1) the levels of the housekeeping gene tran-scription factor IID and 2) by the total amount of nuclear extracts origi-nally recovered from the ear explants.

RNase protection assay

RNase protection assay (RPA) was performed as described previously (23,29). Total RNA was isolated from homogenized ear explants and used toperform the RPA using RiboQuant Multiprobe RNase Protection Assay Kit(BD Biosciences). The data are presented as the ratio of the densitometricsignals of a housekeeping gene L32 to the mRNA for the gene of interest.

CCL19 ELISA

CCL19 protein levels from homogenized ear explants at 6, 12, 24, and 72 hwere analyzed using a custom ELISA. Protein tissue lysates were prepared,as described previously (24). CCL19 capture and detection Ab, strepta-vidin-conjugated HRP, as well as rmCCL19 were purchased from R&DSystems and used at recommended dilutions for the ELISA. To calculatethe CCL19 protein ratio, total protein concentrations were measured withBradford reagent (Sigma-Aldrich), using the microplate assay followingthe manufacturer’s instructions.

Ag-specific cytokine production in infected mice

Single-cell suspensions (5 3 106/ml) were cultured with or without 50 mg/ml soluble L. major Ag in 24-well plates, as described previously (21).After 48 h of culture, supernatants were harvested, and ELISAwas used todetermine the IL-4, IL-5, and IFN-g protein levels. The range of detectionby ELISAs for IL-4 was 2.0–125 and 15.6–1000 pg/ml for IL-5 and IFN-g.

Statistical analysis and data modeling

Data represent themean6 SD.Groupswere analyzedwith Stata (StataCorp,College Station, TX) or SPSS (Chicago, IL) statistical software. Accordingto the number of groups and the distribution (normally distributed or not),nonpaired t test, one-way ANOVA, Kruskal-Wallis, Mann-Whitney U, orFisher’s exact test were performed. Statistical significance was accepted atp , 0.05. Pathways Analysis Software (Ingenuity Systems, RedwoodCity, CA) was used to visualize potential pathways suggested from our dataand emerging from published literature by looking at interactions amongCCR2, CCL2, CCL19, NF-kB, and IL-12.

ResultsImpaired LC migration in CCR2 KO mice is associated withreduced CCL19 production

We have shown that compared with WT mice, Ccr22/2 miceexhibit decreased numbers of LCs migrating out of ear skin ex-plants (21). This reduced migration is seen despite comparablenumbers of resident LCs in the epidermis of WT and Ccr22/2

mice (21). A similar outcome has been described in mice lackingCCR7 or its ligand CCL19, suggesting that the CCR7–CCL19 axisis essential in coordinating LC migration (6, 16, 17, 30). Thus, wehypothesized that there could be a connection between signalsgenerated by CCR2 and the activation of the CCR7–CCL19 axisthat occurs during DC maturation. To test this notion, we firstmeasured the levels of expression of CCR7 and CCL19 mRNA in24- and 72-h ear skin explants from WT and Ccr22/2 mice.Compared with the WT mice, ear skin explants derived from

Ccr22/2 mice significantly expressed lower levels of CCL19 butnot CCR7 or CCL2, a control chemokine (Fig. 1A). Furthermore,this reduction in mRNA translated into significantly lower CCL19protein concentrations in the ear skin explants of Ccr22/2 com-pared with WT mice (Fig. 1B). Because DCs are a major source ofCCL19 in the ear explants (16), it seemed plausible that Ccr2-nullDCs had a reduced ability to produce this chemokine.Next, we surmised that if LC migration in Ccr22/2 mice was

defective, because of lower levels of CCL19 in the explants, thenreconstitution of CCL19 to normal levels should ameliorate theLC migration defect. To determine whether this was indeed thecase, we added CCL19 into the culture medium of the ear skinexplants of WT and Ccr22/2 mice and analyzed LC migration. Wefound that the addition of CCL19, but not the unrelated chemokineCXCL1, into the culture medium significantly increased thenumber of LCs that migrated out from ear explants of WT miceas well as those of Ccr22/2 mice (Fig. 1C). However, the mag-nitude of the increase induced by CCL19 was comparativelyhigher in ear explants derived from Ccr22/2 mice (Fig. 1C). Next,we asked whether this increase in LC migration by CCL19 wasdependent on LTC4. The rationale for this line of inquiry wasprovided by two studies that showed that LTC4 is required to in-duce chemotaxis toward CCL19 in LCs (16) and that CCR2 ac-tivation induces the release of LTC4 in a model of airwayhyperreactivity (31). We found that addition of LTC4 did not en-hance the migration of LC from ear skin explants derived fromCcr22/2 mice (Fig. 1C). Taken together, these results suggest thatthe LC migration defect seen in Ccr22/2 mice may be due toreduced local upregulation of CCL19.

Possible role of CCL2, the principal ligand of CCR2, and theinduction of NF-kB–dependent gene transactivation of DCmigration

We then focused on determining the possible upstream and down-stream mechanistic pathways that could account for the defect inCCL19 production and the related LC migration defect in Ccr22/2

mice.We envisioned a scenario in which CCL2 released locally actson its receptor CCR2 to induce the translocation of the transcriptionfactor NF-kB into the nucleus. This transcription factor promotesDC migration manifested by the release of CCL19 and LC migra-tion from the epidermis toward the DLNs. The following con-verging lines of evidence supported this line of reasoning.First, we found that, similar to the findings in Ccr22/2 mice,

genetic inactivation of the CCR2 ligand CCL2 was also associatedwith abnormal LC migration (Fig. 1D). As expected, decreased LCmigration was accompanied by a reduction in CCL19 mRNA (datanot shown). Furthermore, providing a more direct link between the

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CCL2–CCR2 axis and CCL19, we found that the addition of CCL2to murine splenocytes induced CCL19 production (Fig. 1E), whichwas localized in DCs (data not shown). Second, 2 h after ear peelingthere was a significant increase in nuclear NF-kB translocation inWTexplants (1.85-fold increase from time 0 to 2 h; p = 0.009; n = 12explants for each time point). However, inCcr22/2 explants,NF-kBtranslocation was blunted, failing to reach statistical significance

(1.47-fold increase from time 0 to 2 h; p= NS; n = 12 explants foreach time point). Third, in vivo systemic treatment of micewith NF-kB inhibitors disrupted LC migration. We used two different com-pounds routinely used for in vitro and in vivo studies (32–39). BothPDTC (32, 33) and MG-132 (40), administered to mice prior toepidermal FITC painting, inhibited LC migration from ear skin toDLNs (Fig. 1F). A similar effect was seenwhen inhibition ofNF-kB

1 2 3 40

CCL19Ccr2 LTC4

++

+

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A B

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E F G

FIGURE 1. CCL19 and LC migration in Ccr22/2 mice. A, RPA assay for CCL19, CCR7, and CCL2 mRNA expression at 24 h in ear explants. Data

presented as the ratio between the levels of CCL19, CCR7, and CCL2 mRNA to L-32 from five independent experiments with ear explants from two to

three mice per experiment. B, CCL19 protein levels in ear explants. Levels of CCL19 are expressed in micrograms per milligram total protein. Samples

were composed of at least two WT or Ccr22/2 ears per time point. C, Effects of exogenous administration of CCL19 on LCs migration out from ear

explants. Recombinant mouse CCL19, CXCL1, or LTC4 was added to the culture medium. After 72 h, cells recovered from the medium were stained for I-

Ab and DEC-205. The data represent the mean of three independent experiments, each consisting of at least three WT orCcr22/2 mice per treatment.

Migration is shown as normalized values for each group regarding their unstimulated counterpart (numbers of migrated DCs from representative ex-

periments are as follows: WT 32600, WT plus CCL19 67100, Ccr22/2 12418, and Ccr22/2 plus CCL19 68513). Dotted lines represent fold increase or

decrease over media alone in WT or Ccr2-null mice (N). D, Migration of LCs (DEC-205+/I-Ab+) out of ear explants from WT and Ccl22/2 mice. A

representative experiment of the three performed is shown. Each independent experiment performed included ears from at least three WT or Ccl22/2 mice.

E, Dose-response induction of CCL19 production by CCL2. WT splenocytes were treated with increasing concentrations of CCL2, and after 24 h, the levels

of CCL19 were measured in culture supernatants. A representative experiment of the three performed is shown. F, WT mice were pretreated with NF-kB

chemical antagonists as described in Materials and Methods. Subsequently, their ears were painted with FITC/acetone. Migration of LCs from the skin to

DLNs was quantified by assessing the percentage of FITC+ cells in LN cell suspensions. The majority of these cells also expressed I-Ab. A representative

experiment from four performed for MG-132 and five for PDTC is shown. Vehicle (2) or active antagonists (+). Each experiment included a minimum of

two mice. G, Ear explants of WT mice were treated with or without the NF-kB antagonist MG-132, and mRNA levels for CCL19 using RPA were

quantified. pp , 0.05; ppp , 0.01.

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was achieved by adding the inhibitors to cultured ear explants (datanot shown).Finally,weobserved that chemical inhibitionofNF-kBresulted in

a reduction in CCL19 levels in the tissue (Fig. 1G) linking NF-kBand CCL19 and falling in linewith our findings that LCmigration inCcr22/2 and Ccl22/2 mice is disrupted. We used two differentinhibitors because in general chemical inhibitors are not 100%specific. Therefore, PDTC and MG-142 could have other effectsbeyond NF-kB inhibition that could also affect LC migration.In agreement with these data sets, it has been reported that the

promoter region of the CCL19 gene has several NF-kB bindingsites (8, 41).

Abnormal upregulation of costimulatory molecules in Ccr22/2

DCs

The results presented so far suggest that CCL2-CCR2–mediatedinduction of NF-kB gene transactivation could be required forCCL19-driven LC migration from the skin to the DLNs. Next, wetested the notion that absence of Ccr2 and the related impairmentof DC maturation could also be manifested by a failure to up-regulate costimulatory molecules and MHC class II. To test thisnotion, we studied DC differentiation and maturation occurringin vitro from BM progenitors induced by GM-CSF and IL-4 (24,25). In this well-characterized in vitro model system, we foundthat despite comparable numbers of CD11c+ DCs in cultures fromWT and Ccr22/2 mice (Fig. 2A), the percentages of DCs ex-pressing costimulatory molecules and MHC class II were signif-icantly lower in the KO mice (Fig. 2B–E). Overall, this differencewas also evident when looking at the mean fluorescence intensityvalues (mean fluorescence intensities in a representative experi-ment WT versus Ccr22/2 BMDCs included: CD80, 469 versus384; CD86, 264 versus 289; and I-Ab, 749 versus 443). Thisfinding is in line with the view that CCR2 participates in DCmaturation in a broader context.

Susceptibility of Ccr22/2 mice to L. major infection is relatedto abnormal maturation of DC populations including LCs

We postulated that genetic inactivation of CCR2 leads to suscep-tibility to L. major infection by disrupting LCs and other DCmaturation, such as migration and IL-12p70 production. To test this

notion, we first asked whether CCR2-dependent signals influencethe migration of LCs carrying L. major from the skin to the DLNs.To this end, we quantified the proportion of DCs carrying PKH-labeled L. major from the epidermis into the DLNs of WT andCcr22/2 3 d after intradermal injection. We found that comparedwith WT mice, Ccr22/2 DLNs contained a smaller proportion ofcells likely to constitute PKH-labeled LCs (Fig. 3A).Next, we determined whether genetic inactivation of CCR2 af-

fected the production of IL-12p70 by DCs. We first measured IL-12p70 production by DLN cells and splenocytes in vitro, becausethese lymphoid organs contain diverse populations of DCs, whichhave been shown to be an important source of this cytokine (42–44). This observation was confirmed using intracellular stainingfor IL-12, along with the surface staining for the DC markerCD11c (data not shown). In this in vitro system, we found thatLPS/IFN-g–induced production of IL-12p70 by splenocytes andlymph node (LN) cells from Ccr22/2 mice was significantly lowerthan in WT mice (Fig. 3B, 3C). Next, we sought to determinewhether CCR2 also regulates IL-12p70 production and other DCmaturation processes triggered by exposure L. major. Indeed, us-ing BMDCs as a model system, we found that L. major-inducedupregulation of class II expression was reduced in Ccr22/2 DCscompared with WT DCs (Fig. 3D). Likewise, Ccr22/2 BMDCsproduced lower levels of IL-12p70 than WT DCs in response toactivation with live L. major (Fig. 3E). The reduction in IL-12p70production seemed to be functionally relevant given that, com-pared with WT BMDCs, Ccr22/2 BMDCs had a reduced capacityto induce Ag-specific production of IFN-g by KLH-primed WTT cells (Fig. 3F). Furthermore, there was a correlation between thedefect in IL-12 production of DC Ccr22/2 identified in vitro andin vivo. This was suggested by the observation that there wasa reduced proportion of mature LCs producing IL-12 (CD205+I-Ab+IL-12+ cells) in the DLNs of L. major-infected Ccr22/2 micecompared with infected WT mice (Fig. 3G).We surmised that if the defective production of IL-12p70 in

Ccr22/2 DCs seen in vitro was an important determinant of sus-ceptibility to L. major infection, then early exogenous adminis-tration of this cytokine to Ccr22/2 mice, at the time of exposure toL. major, would switch the susceptible phenotype to resistance.Supporting this posit, administration of rIL-12 to Ccr22/2 miceduring the initial 3 d postinfection corrected their inability to

CD11c

WT

CC

R2-/

-

CD40 I-Ab CD80 CD86

8.8

21.0 *48.6 *

11.3

79.6 *

50.3

32.4 *

6.542.6

49.0

A B C D E

FIGURE 2. Defective maturation of Ccr22/2 BMDCs. WT and Ccr22/2 BM cells were cultured in the presence of GM-CSF and IL-4 for a total of 7 d to

generate BMDCs. Surface staining for the following DC maturation markers is shown. A, CD11c; B, CD40L; C, I-Ab; D, CD80; and E, CD86. A rep-

resentative experiment with percentages of positive cells is shown. pp , 0.05, comparing the average of the percentages for individual markers.

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mount a protective response to L. major infection (Fig. 4A, 4B).Compared with untreated Ccr22/2 mice, rIL-12–treated Ccr22/2

mice had reduced ear swelling and reduced spleen parasite bur-dens at 6 wk postinfection (Fig. 4A, 4B). Furthermore, Ag-specificimmune responses in IL-12–treated Ccr22/2 mice were charac-terized by significantly higher Ag-specific IFN-g and lower IL-4and IL-5 compared with untreated Ccr22/2 mice (Fig. 4C–E).An important issue to address was the specificity of exogenous

IL-12 administration for both the phenotype of Ccr22/2 mice andthe treatment itself. First, the observation that administration ofrIL-12p70 did not have any measurable effect in WTmice (Fig. 4A,4B) rules out a simple overall beneficial effect of IL-12p70 ad-ministration in resistant mice strains. Second, the effect of exoge-nous IL-12p70 administration in the phenotype of Ccr22/2 micewas not a consequence of nonspecific inflammatory effects assuggested by the lack of efficacy of treatment with LPS. In thisexperiment, mice received three doses of LPS or saline priorto infection with L. major. Doses of LPS in this range are known topromote DC migration and IL-12 production in vivo (45). Ccr22/2

mice were equally susceptible to infection regardless of receivingeither PBS or LPS (Fig. 4F). LPS administration was sufficient toinduce systemic effects in WT mice, including DC mobilizationfrom the marginal zone to T cell zone in the spleen (data notshown). This outcome was also in line with previous researchdemonstrating the inability of LPS administration to changea L. major-susceptible phenotype in mice in vivo (46).

Potential role of RR/LC-derived IL-12 in the generation ofprotective immunity against L. major infection

DCsubsetsvaryintheirabilitytoproduceIL-12p70,andconsequently,they differ in their capability to polarize T cell responses toward Th1.

However, considering all the redundancy in the DC network, it is

unclear whether the production of IL-12p70 by any DC subtype is

indispensable for induction of protective immune responses. To infer

that L. major susceptibility in Ccr22/2 mice is linked to a decreased

amount of IL-12 released by LCs, we first needed to demonstrate that

IL-12p70 production by LCs does play a nonredundant role in the

generation of protective immune responses against L. major. Aiming

-

-

FIGURE 3. IL-12 production, and response to L.major infection is defective inCcr22/2mice.A, Impairedmigration of parasite-bearing LCs to theDLNs in

Ccr22/2mice. Absolute numbers of PKH+CD11c+ per LN of a representative experiment is shown.B andC, LPS/IFN-g–induced IL-12p70 production by LNs

or splenocytes is decreased in CCR2-deficient mice. Results from one representative experiment out of two performed with five mice each are shown (mean6SD). D, Ccr22/2 BMDCs have diminished induction of class II molecules upon encounter of viable L. major promastigotes (1:5 ratio). E, Lower production

of IL-12p70 by Ccr22/2 BMDCs in response to live L. major promastigotes challenges (1:10 ratio). Similar results were obtained in two independent ex-

periments. F, KLH-pulsed BMDCT cells from Ccr22/2mice have a reduced ability to induce the release of IFN-g protein compared withWT. Similar results

were obtained in two independent experiments. G, Decreased production of IL-12p70 by Ccr22/2 by activated CD205 cells in the DLNs in 4-wk infected

animals. In vitro stimulation of cells with known inductors of IL-12 restore the ability of these cells to produce this cytokine. pp , 0.05; ppp, 0.01.

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to provide evidence for a role of LC-derived IL-12, we engineeredBM chimeric mice in which IL-12p70 production was deficient ineither the BM-derived cell populations, the RR cell compartment, orin both compartments or was normal.The rationale for the use of BM chimeras mice was the knowl-

edge that a large proportion of LCs is derived from RR/LC pro-genitors (47) and that therefore selective replacement of all BMDCpopulations but not LCs could be accomplished in a lethally irra-diated and syngeneically reconstituted host. Using WT and IL-12p352/2 mice as donor or recipients, the following four groups ofchimeric mice were created. The first group was lethally irradiatedIL-12p352/2 recipients of WT mice BM (WT→IL-12p352/2). Inthese mice, LCs should be unable to produce IL-12p70; however,all other populations of BMDCs would release normal IL-12p70levels. Group 2 consisted of lethally irradiated WT recipient miceof IL-12p352/2 BM (IL-12p352/2→WT). In these mice, normalrelease of IL-12p70 is expected in LCs but disrupted in all BMDCpopulations. The third group consisted of IL-12p352/2recipients of

IL-12p352/2BM, and the fourth group comprisedWT recipients ofWT BM. The final two groups served as controls because IL-12production was blunted in both LCs and BMDCs (IL-12p352/2

→IL-12p352/2), or it was normal in both cell compartments(WT→WT).After allowing sufficient time for their reconstitution, chimeric

mice were infected intradermally with L. major and followed over7 wk. Ear swelling and DLN parasite burdens were used as in-dicators of protective immunity. Importantly, we found that theproportion of LCs (CD11c+CD207+ or CD11c+DEC205+ cells)residing in the DLNs 7 wk postinfection was comparable acrossall four groups of chimeras, indicating that genetic inactivation ofIL-12p352/2 was not influencing DC migration (data not shown).As anticipated, the control groupsWT→WTand IL-12p352/2→IL-

12p352/2 were completely resistant or susceptible to L. major in-fection, respectively (Fig. 4G, Table I). Interestingly, WT→IL-12p352/2 chimeras had significantly more ear swelling and higherDLN parasite burdens thanWT→WTmice but less than IL-12p352/2

**

*

*

*

*

**

***

**** ** **

-

FIGURE 4. Defective production of IL-12 by

RR/LC may account for Ccr22/2 susceptibility to

L. major infection. A and B, Administration of IL-

12 reverses the L. major-susceptible phenotype of

Ccr22/2mice. Exogenous administration of rmIL-

12 inCCR2-deficientmice for 1d prior and2dafter

L. major infection corrected their susceptible phe-

notype as measured by ear swelling and parasite

burden. Results are themean6SD for fivemice. A

representative experiment of two independent ex-

periments is shown. Differences between Ccr22/2

mice treated with or without IL-12: pp, 0.05; ppp

, 0.01. C–E, Skewing from Th2 cytokine pro-

duction to a Th1 pattern in Ccr22/2 mice. Each

symbol represents the Ag-specific cytokine levels

produced from a single mouse. F, Treatment with

LPS prior to infection and every 24 h for 2 d post-

infectiondidnot improve theL.major susceptibility

in Ccr22/2mice.G, Lethal radiation of WTor IL-

12p352/2mice and BM reconstitution with WTor

IL-12p352/2 cells showed that IL-12 from the LC

or RR compartment is necessary but not sufficient

to control L. major infection. ppp , 0.01, com-

paring WT + WT versus IL-12p352/2 + WT and

WT + WT versus IL-12p352/2 + IL-12p352/2.

Results are the mean6 SD of four to five mice per

group. H and I, T and B cell-deficient mice recipi-

ents of Ccr22/2 (Scid + Ccr22/2) or WT (Scid +

Ccr22/2) T cells exhibit similarly enhanced pro-

tection against L. major infection compared with

Scidmice that did not receiveT cells (Scid). Results

are the mean6 SD of three to four mice per group.

pp, 0.05; ppp, 0.01.

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→IL-12p352/2, suggesting that IL-12p70 derived fromRR cells, suchas LCs, was necessary to elicit some degree of protective immunityagainst L. major (Fig. 4G, Table I). Moreover, IL-12p352/2→WTandIL-12p352/2→IL-12p352/2 chimeras were equally susceptible to L.major infection, suggesting that IL-12p70 produced by RR/LCs wasinsufficient for the elicitation of adequate immune responses againstL. major (Fig. 4G, Table I), and therefore, BM-derived populationswere an indispensable source of IL-12p70. Taken together, the resultsfrom the BM chimeras suggest that IL-12p70 derived from RR cells,namely LCs, contributes but is not sufficient for the elicitation of fullprotection against intradermal L. major infection.On the basis of the results above, we surmised that if direct effects

of CCR2 on T cells could be ruled out, then at least two interrelatedAPC factors might underlie the susceptibility inCcr22/2mice to L.major infection. The first factor being a reduced amount of IL-12p70 produced by Ccr22/2 DC populations including LCs (Figs.3A, 3B, 3E, 3F, 4G, Table I). The second, a reduced number of LCsin the DLN, would factor the production of lower amounts of IL-12p70 (Fig. 3C, 3G) and reduced support adequate T cell activa-tion. To address these factors, the following sets of experimentswere conducted.To rule out the possibility that intrinsic defects in T cells were

responsible for abnormalities in Ccr22/2 mice, we conductedadoptive T cell transfer studies from WT or Ccr2-null mice intoSCID mice. As shown in Fig. 4H and 4I, T cells from WT orCcr22/2 mice were comparable in their ability to protect SCIDmice against the infectious challenge. This finding is in line withour previous report that WT and Ccr22/2 T cells are comparablein their ability to polarize into Th1/Th2 cells (48).Next, we attempted to gather evidence for a nonredundant role of

the reduced number of LCs arriving in the DLNs inCcr22/2mice’ssusceptibility to L. major infection. We successfully increasedonly the number of DCs migrating in Ccr22/2 mice withoutchanging the functional maturation defect (e.g., ability to produceIL-12p70; data not shown) using 10-d treatment with Flt3L (TableII). Administration of Ftl3L is a well characterized approach toexpand the multiple subsets of DCs, including LCs and dermalDCs (49). Flt3L treatment partially increased the detection ofFITC+ LCs in the DLNs of Ccr22/2 mice (Table II) and LC mi-

grating cells out of the ear explants (data not shown). However,this increase was not sufficient to change the susceptibly state ofCcr22/2 mice to L. major infection as PBS-treated and Flt3L-treated Ccr22/2 mice had a similar outcome following L. majorinfection (Table II, data not shown).Apossible interpretationof thisfinding is that although thenumerical

DC/LC defect may be partially corrected by administering Flt3L toCcr22/2 mice, increasing the availability of DCs seems ineffectivebecause their functional defects (abnormal IL-12p70 release and ex-pression of costimulatory molecules) remained unchanged.

DiscussionIn this study, we show that CCR2-dependent signals triggered bybindingofits ligandCCL2andsubsequentnucleartranslocationofNF-kB are required for DC maturation and LC migration from the epi-dermis to the DLNs. Interestingly, CCR2-dependent signals con-tribute to the production of CCL19, upregulation of costimulatorymolecules, and the production of IL-12 (a proposedmodel is shown inFig. 5). In particular, the later process was found to be critical for thegeneration of protective immune responses againstL.major infectionin resistant mice. Taken together, these data extend our previous re-ports that the susceptibility of Ccr22/2 mice to L. major infectioncould, in fact, be secondary to the inability of LCs to produce IL-12and a failure to mount a protective Th1 response.The role of the chemokine system inDCbiology is classically seen

as being critical for cell migration. However, increasing evidencesuggests that in addition to their role in chemotaxis, chemokines andtheir receptors also regulateDCmaturation (50).For instance, humanDCs treated with neutralizing Abs against the chemokine receptorsCCR1 or CCR3 reduced their ability to activate T cells in vitro (51).Inmice, CCL19was found to inducematuration of DCs, resulting inupregulation of costimulatory molecules, production of proinfla-mmatory cytokines, and enhancement of DCs’ ability to activateT cells (52). More recently, evidence consistent with our findingssuggests that CCR2-dependent signals are critical forDCmaturation(22). The authors showed that genetic inactivation of CCR2 in mu-rine DCs lead to reduced expression of costimulatory moleculesupon activation with LPS, reduced allostimulatory capacity, andabnormalmigration (22). Unfortunately, themechanismsunderlyingthese observations were not reported. In this paper, we extend thoseobservations by revealing the potential role of the CCL2, CCR2’smain ligand CCL2, and of NF-kB–mediated signals, as well as thecritical role ofCCR2 in regulation of IL-12 production bymaturatingDCs. We also present data supporting the in vivo relevance of theseobservations in the context of a specific DC subset, namely LCs, andtest the relevance of our findings in an in vivo infectious model.A role for the CCL2–CCR2 axis in LC migration/maturation is

also supported by three recent reports. First, human CCL2 trans-genic mice exhibit an acceleration of migration of LC from theepidermis into the DLN, following sensitization with haptens, aneffect that is accompanied with increased expression of class IIand costimulatory molecules (53). Second, CCL2 has been shown

Table I. Sources of IL-12 production and susceptibility to L. major in mice receiving adoptive transplants

Groups Donor→RecipientIL-12 fromRR/LCs

IL-12 fromBMDCs Log PB (LN) p , 0.05

A WT→WT Yes Yes 1.54 6 0.12 AvsCAvsD

B WT→IL-12p35 No Yes 2.22 6 0.53 BvsCBvsD

C IL-12p35→WT Yes No 3.40 6 0.81 NSD IL-12p35→IL-12p35 No No 4.64 6 0.63 NS

PB, parasite burden.

Table II. Increased numbers of DCs with Flt3L treatment does notchange susceptibility toward L. major in Ccr22/2 mice

Flt3L LN FITC+ (%) Log PB (Spleen)

WT 2 19/19 (100) 0.0 6 0.0WT + 15/19 (78) 0.0 6 0.0Ccr22/2 2 6/22 (28) 0.9 6 1.07*Ccr22/2 + 11/25 (44) 3.7 6 0.6**

LN FITC+ cells indicate the number of slides positive for FITC cells over the totalnumber of analyzed slides in the DLNs.

pp = 0.054, difference between WT and Ccr22/2; ppp = 0.001, comparing WTand Ccr22/2 mice receiving Flt3L. Results are the mean 6 SD for five mice.

PB, parasite burden.

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to have potent chemoattractant activity for murine LCs in vitro(54). And finally immature DCs respond to CCL2 (55).Another line of convergent evidence supporting a role for NF-kB

translocation by the CCR2 ligand CCL2 is highlighted in recentwork fromour laboratory (29). Using an EMSA-based approach, wefound CCL2-induced nuclear translocation of NF-kB in astrocytes.The effect of CCL2 on NF-kB translocation was 1) time dependent,peaking at 120 min after the addition of CCL2; 2) dose dependentwith maximal effects at 100 ng/ml for the dose-range tested; and 3)dependent predominantly on the p65 component of NF-kB (29).Wealso demonstrated that CCL2-induced NF-kB translocation wasmarkedly diminished in the absence of CCR2. (29).A logical extension of this work was to investigate the functional

implications of CCL2-CCR2 activation of NF-kB in DC biology.NF-kB is highly expressed by DCs, and it mediates key DC func-

tions (6). Indeed, in line with our findings, previous reports havehighlighted that NF-kB strongly enhances DC longevity (56); well-known DC-activating mediators such CD40L promote maturation viasustained activationofNF-kB(57); and inhibition ofNF-kBactivationblocks maturation of DCs, in terms of upregulation of MHC andcostimulatory molecules (9, 58).The contribution of NF-kB to DCmigration has been less studied

ex vivo and in vivo. We were able to document blunting in Ccr22/2

skin explants of the rapid upregulation of NF-kB that occurredfollowing ear peeling in WT explants. Highlighting further the im-portance of this transcription factor, we found that administration ofthe NF-kB chemical antagonists PDTC and MG-132 to mice sig-nificantly reduced LC migration in the FITC-painted model. Cer-tainly, a major concern with the use of the chemical antagonists isthe lack of specificity. However, our finding of reduced LC migra-tion in mice receiving PDTC or MG-132 is further supported byreports indicating that the proteasome inhibitor-based (N-benzy-loxycarbonyl-Ile-Glu[O-tert-butyl]-Ala-leucinal [PSI]) or adeno-viral- mediated antagonism of NF-kBblocks DCmaturation events,such as upregulation of costimulatory molecules and production ofcytokines (59).Moreover,verysimilar towhatweshowinthecurrentreport,NF-kB

blockade using decoy oligodeoxynucleotides in vivo was shown toreduce LC migration and impair T cell responses (60). Nonetheless,our findings extend these observations by revealing that blockade of

NF-kB is linked to reduced production of CCL19, a critical chemo-kine forLCmigration (16), and further highlight the critical role of theCCL2–CCR2 axis in the induction of NF-kB. Moreover, the dataprovide a mechanistic backdrop to the observation that chemicalantagonism of CCR2 in vivo reduces the magnitude of delayed-typehypersensitivity responses (61), an immunological process that ishighly dependent on DCmigration from the skin to the DLNs (2, 60).Thus, collectively our data suggest that CCL2-dependent signalsacting via CCR2 lead to NF-kB translocation to the nucleus andtransactivation of the CCL19 gene (Fig. 5). This possibility is furthersupported by work documenting that both human and mouse CCL19promoters have several functional NF-kB binding sites (8, 41).Our data also indicate that many of the phenotypic consequences

of CCR2 inactivation may be due to deficits in NF-kB activation.Indeed, in IL-12p70 production in DCs evidently occurs in re-sponse to signals that require NF-kB activation (7, 62, 63).The observation that inactivation of CCR2 is associated with

abnormal production of IL-12p70 by DCs in vitro supports previousreports documenting abnormal IL-12p70 production in Ccr22/2

mice (64), as well as the fact that chemokines are increasinglyrecognized as regulators of the production of this cytokine by DCs(65). By contrast, in this study, we show the relevance of reducedproduction of IL-12p70 in Ccr22/2 mice, in vivo, as highlightedby the demonstration that administration of IL-12p70 prior toinfection corrects the L. major-susceptible phenotype but does notaffect the response of WT mice.The observation that treatment with LPS prior to infection,

a stimulus known to promote DCmaturation and mobilization (66),was not able to induce resistance in Ccr22/2 mice suggests that inthe absence of CCR2, DCs are incapable of overcoming using thenonspecific stimuli. Thus, effective signals via CCR2 might beindispensable. Aside from CCL2, other CCR2 ligands couldprovide these signals as suggested by our observation that CCL2-null mice are resistant to L. major infection (67) and that CCL2signals are not critical for IL-12p70 production in vivo (64).Our experiments using lethal radiation and BM transplantation

suggests that IL-12p70 released by a cell type with RR progenitors,such as LCs contributes, but is not sufficient, to induce protectiveimmunity against L. major. This finding allows us to infer that thesusceptible phenotype in Ccr22/2 mice could be related, in part, toabnormalities in IL-12p70 production by LCs and other DC pop-ulations. Importantly, the contribution of CCR2 to the generation ofprotective immune responses goes beyond its role in LC migrationand involves the modulation of maturation processes. Indeed,solely increasing the numbers of DCs available in Ccr22/2 prior toL. major infection using Flt3L failed to change their susceptibility.Our work confirms and further extends the role of DCs (68), spe-cifically LCs, in the generation of immune responses against L.major, including the observation that LCs produce IL-12p70 whenexposed to L. major parasites (43, 69) and that IL-12p70 released inthe early stages of infection is indispensable for the induction ofprotective immune responses against L. major (70). It has also beenshown that production of IL-12p70 by mature DCs is required togenerate protective immune responses against L. major, and workby Wiethe et al. (71) showed that immature DCs induce Th2 po-larization and are susceptibility to L. major infection as is likely tobe the case in Ccr22/2 mice.Although the relevance of LCs for the generation of protective

immune responses against L. major seems to be well established,some reports challenge this notion (72). For instance, skin-derivedDCs, including LCs and dermal DCs, were found to migratepoorly to LNs after L. major infection and have been shown toplay a minor role in early T cell activation (73). Moreover, resi-dent DCs in LNs (rather than LCs) were responsible for the

ERK 1/2

ERK

Akt

NF-kB (complex)

CCR7

IRF1

IL12B

CCL19

CCR2

CCL2

FIGURE 5. Potential molecular underpinnings of CCR2’s contribution

to DC maturation: pathway analysis and visualization. A model was

generated using Ingenuity Pathways Analysis software from published

literature and screening of direct (e.g., CCL2→CCR2) or indirect (e.g.,

CCL2→NF-kB) interactions emerging by the data presented in this paper,

involving CCR2, CCL2, CCL19, NF-kB, and IL-12. Our model suggests

that the interaction between CCR2 and its cognate ligand CCL2 induces

the expression of genes upregulated concurrently as part of DC maturation,

such as CCR7, CCL19, and IL-12B (IL-12p40), possibly via induction of

NF-kB transactivation. It also suggests other potential mediators are in-

volved in the pathway.

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induction of protective immune responses (74). Of note, in thesereports, the infectious challenge was administered via the s.c.route. By contrast, our study used the intradermal route. Clearly,the use of the intradermal route more likely engaged LCs and ledto differential immune responses (67, 75). Furthermore, the routeof administration is critical given that LCs promote T cell re-sponses to skin Ag, but only under defined conditions (76) andalso through our work indicating that the effects of the CCL2–CCR2 axis are modulated by the route of infectious challenge(67). The doses of parasite used also can be a critical factor. Thedoses of L. major used in this study were significantly higher thanexpected during human infection and did not use a live vector(sandfly) to inoculate the parasites; therefore, the underlying cel-lular and molecular physiological process observed may not rep-resent normal responses against infection in a natural setting.

AcknowledgmentsWe thank N. Sato, Jason Schaeffer, Jeniffer Perez, and George Chenaux for

performing some of the initial key experiments.

DisclosuresThe authors have no financial conflicts of interest.

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