Correlation with Receptor Fmlp and C5a Upon Dendritic Cell Differential

Home , C5a receptor, Chemokine receptor

Differential Regulation of Responsiveness to fMLP and C5a Upon Dendritic Cell Maturation: Correlation with Receptor Expression1,2

De Yang,3* Qian Chen,3* Sabine Stoll,‡ Xin Chen,* O. M. Zack Howard,† and Joost J. Oppenheim4*

The trafficking of immature and mature dendritic cells (DCs) to different anatomical sites in vivo is critical for fulfilling their roles in the induction of Ag-specific immune responses. Although this process is complex and regulated by many mediators, the capacity of DCs to migrate is predominantly dependent on the expression of particular chemotactic receptors on the surface of DCs that enable them to move along chemotactic gradients formed by the corresponding chemokines and/or classical chemoattractants. ,Here we show that immature DCs (iDCs) respond to both fMLP and C5a as determined by chemotaxis and Ca2؉ mobilization whereas mature DCs (mDCs) respond to C5a, but not fMLP. Additionally, iDCs express the receptors for both fMLP and C5a at mRNA and protein levels. Upon maturation of DCs, fMLP receptor expression is almost completely absent, whereas C5a receptor mRNA and protein expression is maintained. Concomitantly, mDCs migrate chemotactically and mobilize intracellular Ca2؉ in response to C5a, but not fMLP. Thus the interaction between C5a and its receptor is likely involved in the regulation of trafficking of both iDCs and mDCs, whereas fMLP mobilizes only iDCs. The differential responsiveness to fMLP and C5a of iDCs and mDCs suggests that they play different roles in the initiation of immune responses. The Journal of Immunology, 2000, 165: 2694–2702.

yeloid dendritic cells (DCs)5 are highly specialized fulfill their respective roles in Ag uptake, processing, and APCs that initiate immune responses (1–3). DCs orig- presentation. M inate from bone marrow and migrate through the The trafficking pattern of DCs, like that of other leukocytes, is blood stream to populate nonlymphoid tissues in an immature state presumably controlled by many mediators. However, the direction (1). Upon the introduction of Ags into a host such as by an infec- of DC migration is primarily determined by chemotactic gradients tion, immature DCs (iDCs) migrate to the site of Ag deposition, formed by a variety of chemotactic factors, including classical che- take up and process Ags, and simultaneously undergo a process of moattractants and chemokines (3, 5, 10–16). Classical chemoat- phenotypic and functional maturation (2–6). Subsequently, the re- tractants include formyl peptides (e.g., cleavage products of bac- sultant mature DCs (mDCs) traffic via the afferent lymphatic to the terial and mitochondrial proteins such as fMLP) (5, 10), products T cell area of secondary lymphoid organs where they activate Ag- of host complement activation (e.g., C5a) (10), and lipid metabo- specific lymphocytes (6–9). Therefore, iDCs and mDCs traffic to lites (e.g., platelet activating factor) (15), whereas chemokines different anatomical sites (site of Ag deposition vs T cell area) and consist of a superfamily of Ͼ30 structurally related proteins clas- sified into the CXC, CC, CX3C, and C chemokine subfamilies (16–18). The effects of classical chemoattractants and chemokines *Laboratory of Molecular Immunoregulation, Division of Basic Sciences, and †In- tramural Research Support Program, Science Applications International Corp.-Fred- are mediated by members of the G protein-coupled seven-trans- erick, National Cancer Institute-Frederick Cancer Research and Development Center, membrane domain receptor superfamily (18–20). The reason that ‡ National Institutes of Health, Frederick, MD 21702-1201; and Laboratory of Immu- iDCs and mDCs are able to migrate toward different chemotactic nology, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20892 factors is due to the fact that they express different sets of chemo- Received for publication February 4, 2000. Accepted for publication June 12, 2000. tactic receptors. So far, human iDCs have been shown to express The costs of publication of this article were defrayed in part by the payment of page CXCR1 (12, 21), CXCR2 (12), CXCR4 (12, 21–24), CCR1 (12, 21, charges. This article must therefore be hereby marked advertisement in accordance 22, 24), CCR2 (12, 21, 22), CCR3 (22, 24), CCR4 (21, 22), CCR5 with 18 U.S.C. Section 1734 solely to indicate this fact. (12, 21–24), CCR6 (14, 25–29), CCR8,6 and probably CCR9 (30– 1 This work was supported in part by a fellowship from the Office of the International 32), whereas mDCs express only CXCR4 (21, 23) and CCR7 (14, 21, Affairs, National Cancer Institute, National Institutes of Health (to D.Y.) and by the National Cancer Institute, National Institutes of Health Contract N01-CO-56000 (to 33–35). O.M.Z.H.). Studies of DC responses to chemoattractants have shown that 2 The content of this publication does not necessarily reflect the views or policies of human iDCs derived from either peripheral blood monocytes or the Department of Health and Human Services, nor does mention of trade names, cord blood CD34ϩ cells respond chemotactically to and express commercial products, or organizations imply endorsement by the U.S. Government. The publisher or recipient acknowledges right of the U.S. Government to retain a the receptor for platelet activating factor (15). DCs isolated from nonexclusive, royalty-free license in and to any copyright covering the article. rat respiratory tract tissue or generated in vitro from human pe- 3 D.Y. and Q.C. contributed equally to this study. ripheral blood monocytes can be chemoattracted by fMLP and C5a 4 Address correspondence and reprint requests to Dr. Joost J. Oppenheim, Laboratory (5, 10). Human skin-derived Langerhans’ cells, a prototype of of Molecular Immunoregulation, Division of Basic Sciences, National Cancer Insti- tute-Frederick Cancer Research and Development Center, Building 560, Room 21-89, Frederick, MD 21702-1201. E-mail address: [email protected] 5 Abbreviations used in this paper: DCs, dendritic cells; mDC, mature DC; iDC, 6 O. M. Z. Howard, H. F. Dong, J. Subleski, S. Strobl, A.-K. Shirakawa, J. J. Oppenheim, immature DC; C5aR, C5a receptor; FPR, formyl peptide receptor; HPC, hemopoietic and E. L. Nelson. TARC and I-309 utilize CCR8 to induce chemotaxis of a CD83Ϫ subset progenitor cells; rh, recombinant human; SDF-1␣, stromal cell-derived factor 1␣. of human monocyte-derived dendritic cells. Submitted for publication.

FIGURE 1. Migration of monocyte-derived DCs in response to fMLP and C5a. The migration of DCs induced by different concentrations of fMLP and C5a was studied by chemotaxis assay with the use of SDF-1␣ as a positive control. CM, Chemotactic medium, indicating spontaneous DC migration. The results are shown as the average (mean Ϯ SD) of triplicated wells. A and C, Monocyte-derived iDCs; B and D, monocyte-derived mDCs; A and B, cell migration induced by fMLP; C and D, cell migration induced by C5a. Similar results were obtained from more than ﬁve separate experiments.

iDCs, express the receptor for C5a (C5aR) and respond chemo- ence of IL-4) was purchased from PharMingen. Anti-CD83 was purchased tactically to C5a (36). However, the regulation of DC responsive- from Coulter-Immunotech (Marseille, France). The other Abs used for flow 3 ness to fMLP and C5a and expression of formyl peptide receptor cytometry were purchased from PharMingen. [ H]fMLP with a specific radioactivity of 2000 Ci/mmol and [3H]TdR with a specific radioactivity of (FPR), the high-affinity receptor for fMLP, and C5aR upon DC 2 Ci/mmol were purchased from NEN (Boston, MA). PCR primers for maturation, have not been fully elucidated. Here, we investigated C5aR were purchased from Stratagene (La Jolla, CA). this issue by using human and murine DCs generated in vitro from either hemopoietic progenitor cells (HPC) or CD14ϩ peripheral blood monocytes. The results show that iDCs of both species ex- Cell isolation and purification press FPR and C5aR and are thus able to respond to fMLP and Human PBMC were isolated by routine Ficoll-Hypaque density gradient C5a. In contrast, mDCs differentially down-regulate FPR, but not centrifugation. Monocytes were purified (Ͼ95%) from human PBMC with C5aR, expression at mRNA and protein levels, resulting in a se- the use of a MACS CD14 monocyte isolation kit (Miltenyi Biotech, Au- lective retention of responsiveness to C5a as compared with fMLP. burn, CA) according to the manufacturer’s recommendation. Cord blood CD34ϩ HPC (Ͼ95%) were purchased from Poietics (Gaithersburg, MD). The DC precursors were amplified from CD34ϩ HPC exactly as described Materials and Methods (37) by culturing the cells at 5 ϫ 104 cells/ml in IMDM supplemented with Media and reagents 20% FBS, 10Ϫ5 M DTT, 25 ng/ml rhFlt3-ligand, 10 ng/ml rh thrombopoi- IMDM was purchased from Life Technologies (Rockville, MD). RPMI etin, and 20 ng/ml rh stem cell factor for 4 wk. The amplified DC precur- 1640 was purchased from BioWhittaker (Walkersville, MD). Recombinant sors were cryopreserved in IMDM containing 20% FBS and 10% DMSO human (rh) stromal cell-derived factor-1␣ (SDF-1␣), TNF-␣, GM-CSF, until later usage. Murine HPC were prepared from the bone marrow of IL-4, Flt3-ligand, stem cell factor, and thrombopoietin were purchased C57BL/6 mice (female, 7 wk) as described (38). Briefly, bone marrow cells from PeproTech (Rocky Hill, NJ). Recombinant murine (rm) GM-CSF, flushed from femur and tibia were depleted of RBC by ammonium chloride IL-4, and TNF-␣ were purchased from Biosource International (Camarillo, treatment. For depletion of lymphocytes and Ia-positive cells, the remain- CA), R&D Systems (Minneapolis, MN), and PharMingen (San Diego, ing cells were incubated with a mixture of mAb for1hat4°Cfollowed by CA), respectively. fMLP, rhC5a, and chemicals unless otherwise specified depletion with immunomagnetic beads coated with anti-rat IgG (Dynal, were purchased from Sigma (St. Louis, MO). FBS was purchased from Great Neck, NY). The mAbs used were anti-B220/CD45R (PharMingen), HyClone (Logan, UT). FITC-conjugated mouse anti-human C5aR (CD88) anti-MHC class II (M5/114.15.2 anti-I-Ab, d, q and I-Ed, k; American Type was purchased from Serotec (Oxford, U.K.). Mouse anti-human CD40 ag- Culture Collection, Manassas, VA), and anti-CD90 (Thy1; PharMingen). onistic Ab (IgG1, ␬, capable of stimulating B cell proliferation in the pres- The resulting cells (ϳ90% Sca-1ϩ/LinϪ) were used as murine HPC.

FIGURE 2. Ca2ϩ mobilization of monocyte-derived DCs. Arrows indicate the time points where stimulants were added at the final concentrations (M) as specified. A, fMLP-induced Ca2ϩ flux by iDCs (upper panel) and mDCs (lower panel). B, C5a-induced Ca2ϩ flux by iDCs (upper panel) and mDCs (lower panel). Three separate experiments showed almost identical results. 2696 REGULATION OF FPR AND C5aR EXPRESSION UPON DC MATURATION

Table I. Surface marker expression of monocyte-derived DCsa

iDCs mDCs

Marker % Positive MFIb % Positive MFI

CD1a 67 Ϯ 632Ϯ 16 75 Ϯ 269Ϯ 19 CD11a 91 Ϯ 129Ϯ 292Ϯ 0.4 32 Ϯ 0.7 CD14 NDc N/A ND N/A CD40 93 Ϯ 943Ϯ 10 98 Ϯ 278Ϯ 18 CD83 6 Ϯ 62Ϯ 0.4 54 Ϯ 9 9.4 Ϯ 1 CD86 57 Ϯ 21 22 Ϯ 1.4 85 Ϯ 17 41 Ϯ 3 HLA-DR 99 Ϯ 0.1 235 Ϯ 70 100 Ϯ 0.1 303 Ϯ 80

a iDCs and mDCs were generated from peripheral blood monocytes and analyzed by FACScan as described in Materials and Methods. Approximately 10,000 events were collected for each sample. Shown is the average (mean Ϯ SD) of three separate experiments. b MFI, Median ﬂuorescence intensity. c ND and N/A, Not detectable and not applicable, respectively.

DC preparation Beaconsfield, U.K). Ca2ϩ mobilization of the cells was measured by re- cording the ratio of fluorescence emitted at 510 nm after sequential exci- To generate human monocyte-derived iDCs, purified monocytes were in- tation at 340 and 380 nm in response to chemotactic factors at various ϫ 6 cubated at 1 10 /ml in RPMI 1640 medium (RPMI 1640 plus 10% FBS, concentrations. 2 mM glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 ␮g/ml strep- tomycin) in the presence of rhGM-CSF (50 ng/ml) and rhIL-4 (10ϳ50 FACS ng/ml) at 37°C in a CO2 (5%) incubator for 7 days. For the generation of human CD34ϩ-derived iDCs, DC precursors amplified from CD34ϩ HPC DCs were first washed three times with FACS buffer (PBS, 1% FBS, 5 were incubated at 5 ϫ 10 /ml in RPMI 1640 medium in the presence of 0.02% NaN3, pH 7.4) and then stained with FITC-conjugated anti-CD88 or control Ab at room temperature for 30 min as recommended by the man- rhGM-CSF (50 ng/ml) and rhIL-4 (50 ng/ml) at 37°C in a CO2 (5%) incubator for 2 days (37). Murine iDCs were generated by the incubation of ufacturer. After washing three times with PBS, the stained DCs were fixed murine HPC at 1 ϫ 106/ml in RPMI 1640 medium in the presence of with 1% paraformaldehyde in PBS, stored at 4°C overnight, and analyzed the next day with a flow cytometer (Coulter Epics, Miami, FL). rmGM-CSF (50 U/ml) and rmIL-4 (10 ng/ml) at 37°C in a CO2 (5%) incubator for 5 days (38). All of the cultures were fed with the same cytokine-containing medium every 2–3 days. To induce DC maturation, MLR iDCs were cultured in the same cytokine mixtures with added TNF-␣ (50 Allogeneic MLR was performed as described (11). Briefly, purified allo- ␮ 5 ng/ml) or anti-CD40 Ab (100 g/ml) for 48 h at 37°C in a CO2 (5%) geneic T cells (10 /well) were cultured with different numbers of iDCs or incubator (21, 23, 27, 28). mDCs in a 96-well flat-bottom plate for 7 days at 37°C in humidified air

with 5% CO2. The proliferative response of T cells was examined by puls- Chemotaxis assay ing the culture with [3H]TdR (0.5 ␮Ci/well) for another 18 h before har- 3 DC migration was assessed using a 48-well microchemotaxis chamber vesting. [ H]TdR incorporation was measured with a microbeta counter technique as previously described (27, 39). Briefly, different concentrations (Wallac, Gaithersburg, MD). of chemotactic factors were placed in the wells of the lower compartment Binding assay of the chamber (Neuroprobe, Cabin John, MD), and DCs (106 cells/ml) were added to the wells of the upper compartment. The lower and upper Equilibrium binding was performed in triplicate by adding a constant compartments were separated by a 5-␮m polycarbonate filter (Osmonics, amount of [3H]fMLP and increasing amounts of unlabeled fMLP to indi- 6 Livermore, CA). After incubation at 37°C in humidified air with 5% CO2 vidual 1.5-ml microfuge tubes, each containing 2 ϫ 10 DCs suspended in for 1.5 h, the filters were removed and stained, and the cells migrating RPMI 1640 containing 1% BSA, 2.5 mM HEPES, 0.05% NaN3. After across the filter were counted with the use of a Bioquant semiautomatic incubation at 24°C with constant mixing for 20 min, the cells were exten- counting system. The results are presented as number of cells per high sively washed with cold PBS, and the cell-associated radioactivity was power field. measured with a microbeta counter (Wallac). Calcium mobilization RNA isolation, RT-PCR, and Northern blot DCs (107 cells/ml in RPMI 1640 containing 10% FBS) were loaded with Total RNA from DCs was isolated by the use of TRIzol Reagent (Life dye by incubating with 5 ␮M fura-2 (Molecular Probes, Eugene, OR) at Technologies). The RNAs were cleaned by treatment with RNase-free 24°C for 30 min in the dark. Subsequently, the loaded cells were washed DNase I (Stratagene). RT-PCR was performed by the use of GeneAmp and resuspended (106 cells/ml) in saline buffer (138 mM NaCl, 6 mM KCl, RNA PCR Kit (Roche Molecular Systems, Branchburg, NJ). Briefly, 100

1 mM CaCl2, 10 mM HEPES, 5 mM glucose, and 1% BSA, pH 7.4). Each ng of RNAs was used in the RT-PCR. After reverse transcription, C5a and 2 ml of loaded DC suspension was then transferred into a quartz cuvette, GAPDH cDNA fragments were ampliﬁed by 30 cycles of PCR (denatured which was placed in a luminescence spectrometer LS50 B (Perkin-Elmer, at 95°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 1

Table II. Stimulation of allogeneic MLR by human monocyte-derived DCsa

[3H]TdR Incorporation (cpm/well)

DC DCs (no./well) alone DCs (no./well) ϩ T cells (105/well) Maturation Stage 100 1,000 10,000 100 1,000 10,000

iDCs 125 Ϯ 26 99 Ϯ 34 13 Ϯ 2 116 Ϯ 23 164 Ϯ 33 102 Ϯ 32 mDCs 46 Ϯ 19 71 Ϯ 22 83 Ϯ 16 91 Ϯ 29 4,751 Ϯ 203 36,231 Ϯ 1,537

a Human monocyte-derived DCs at various numbers per well as indicated were cultured alone or with allogeneic peripheral blood T cells (105/well) in RPMI 1640 containing 10% FBS in wells of 96-well tissue culture plate for 7 days. [3H]TdR was added to each well (0.5 ␮Ci/10 ␮l/well) and cultured for another 18 h before cell harvest. The radioactivity (cpm) incorporated is shown as the average (mean Ϯ SD) of six wells. The [3H]TdR incorporation for T cells cultured alone and in the presence of 5 ␮g/ml of phytohemagglutinin was 47 Ϯ 18 and 105,945 Ϯ 2,233 cpm, respectively. The Journal of Immunology 2697

FIGURE 5. Regulation of responsiveness to, and expression of receptors for, fMLP and C5a upon DC maturation induced by CD40 ligation. FIGURE 3. Expression of FPR and C5aR by monocyte-derived DCs. A, Immature DCs were generated from purified monocyte by incubation at 3 F E Binding of [ H]fMLP by iDCs ( ) and mDCs ( ) in the presence of 37°C for 7 days in the presence of GM-CSF and IL-4. The resulting iDCs increasing concentrations of unlabeled fMLP. The results shown are the were divided into two parts. One part was used for chemotaxis and RNA Ϯ average (mean SD) of four separate experiments. B, FACS analysis of extraction and one part was incubated with anti-CD40 Ab at a concentra- C5aR expression by iDCs (solid line) and mDCs (asterisk line). Dotted line tion of 100 ␮g/ml for 48 h to induce DC maturation. mDCs were also used indicates fluorescence of cells stained with control mouse IgG. One rep- for chemotaxis and RNA extraction. A, Migration of iDCs (open symbols) resentative experiment of three is shown. and anti-CD40-induced mDCs (closed symbols) in response to fMLP (circles) and C5a (triangles). B, FPR mRNA expression by iDCs (lane 1) and anti-CD40-induced mDCs (lane 2) as detected by Northern blot. The nitrocellulose filter was first probed with 32P-labeled FPR cDNA fragment, min) with the last extension being performed at 72°C for 10 min. The 32 primers for human C5aR were 5Ј-CCCAAGCTTGGGGCGGGGAAT stripped, and reprobed with P-labeled actin cDNA fragment. C, RT-PCR CAATGAATTTCAGCGA-3Ј and 5Ј-CCGCTCGAGCGGCTATCACAT products of C5aR (upper panel) and GAPDH (lower panel) displayed by AGTGAAGGAGGACGCA-3Ј. The primers for human GAPDH were 5Ј- agarose gel electrophoresis. The anticipated sizes for C5aR and GAPDH GATGACATCAAGAAGGTGGTGAA-3Јand5Ј-GTCTTACTCCTTGGA are 500 and 246 bp, respectively. M, Marker that is a 1-kb DNA ladder. GGCCATGT-3Ј. PCR products were identified on 1–2% agarose gel after Lane 1, iDCs; lane 2, anti-CD40-induced mDCs. ethidium bromide staining and photodocumented. Northern blot was performed as described elsewhere (40) with minor modification. Briefly, total RNA (20 ␮g/lane) was fractionated on 1% agarose-formaldehyde gel and Results transferred to a nitrocellulose filter. The specific mRNA on the filter was Maturation of monocyte-derived DCs down-regulates their detected by hybridization with a 32P-labeled cDNA probe at 42°C overnight in buffer comprising 50% formamide, 5ϫ SSPE (1ϫ SSPE is 0.15 M responsiveness to fMLP, but not to C5a ϫ NaCl, 10 mM NaH2PO4, 10 mM EDTA, pH 7.4), 5 Denhardt’s solution, ␮ To address the effect of maturation of DCs on their response to 1% SDS, and 100 g/ml denatured salmon sperm DNA. Two cDNA 2ϩ probes were used: a 1000-bp HindIII-EcoRI fragment of human FPR fMLP and C5a, we compared the chemotaxis and Ca mobiliza- cDNA and a 400-bp ␤-actin cDNA fragment (41). The probes were labeled tion of iDCs and mDCs derived from human monocytes in re- by a RadPrime DNA labeling kit (Life Technologies). After hybridization, sponse to fMLP and C5a. iDCs migrated to both fMLP and C5a the filters were washed with 2ϫ SSC (1ϫ SSC is 0.15 M NaCl, 15 mM with bell-shaped dose-response curves with optimal concentrations ϫ sodium citrate, pH 7.0) containing 0.2% SDS and 0.1 SSC containing at 10Ϫ8 M and 10Ϫ9 M, respectively (Fig. 1, A and C), confirming 0.1% SSC until a reasonably low background was obtained. The filter was dried and autoradiographed overnight at Ϫ80°C using a Kodak x-ray film the previous reports (5, 10). Both fMLP and C5a also induced ϩ (Rochester, NY). intracellular Ca2 mobilization by iDCs in a dose-dependent manner (Fig. 2, A and B, upper panels). After treatment of iDCs with rhTNF-␣ for 48 h to induce maturation, mDCs lost their responsiveness to fMLP as measured by chemotaxis (Fig. 1B) and Ca2ϩ mobilization (Fig. 2A, lower panel), but maintained responsiveness to C5a in terms of both chemotaxis (Fig. 1D) and Ca2ϩ mobilization (Fig. 2B, lower panel). Furthermore, C5a was equally potent and efficacious for iDCs and mDCs because 1) it induced the migration of similar numbers of iDCs and mDCs at identical optimal concentration (10Ϫ9 M) under similar experimental conditions FIGURE 4. Expression of FPR and C5aR at mRNA level by monocyte- (Fig. 1, C and D), and 2) it mobilized intracellular Ca2ϩ to a derived DCs. A, FPR mRNA expression by iDCs (lane 1) and mDCs (lane Ϫ Ϫ similar extent in concentrations ranging from 10 11 to 10 7 M 2) as detected by Northern blot. The nitrocellulose filter was first probed ␣ with 32P-labeled FPR cDNA fragment, stripped, and reprobed with 32P- (Fig. 2B). The result that SDF-1 was chemotactic for both iDCs labeled actin cDNA fragment. B, RP-PCR products of C5aR (upper panel) and mDCs (Fig. 1) is in accordance with previous reports (21, 27). and GAPDH (lower panel) displayed by agarose gel electrophoresis. The To ensure that monocyte-derived iDCs and mDCs used in this anticipated sizes for C5aR and GAPDH are 500 and 246 bp, respectively. study show phenotypic characteristics of iDCs and mDCs, we ex- M, Marker that is a 1-kb DNA ladder. Lane 1, iDCs; lane 2, mDCs. amined their surface marker expression and capacity to stimulate 2698 REGULATION OF FPR AND C5aR EXPRESSION UPON DC MATURATION

Table III. Surface marker expression of human HPC-derived DCsa

iDCs mDCs

Marker % positive MFIb % positive MFI*

CD1a 30 Ϯ 43Ϯ 0.3 80 Ϯ 10 12 Ϯ 5 CD83 2 Ϯ 1 1.5 Ϯ 0.1 69 Ϯ 146Ϯ 3 CD86 26 Ϯ 49Ϯ 1.5 77 Ϯ 246Ϯ 3 HLA-DR 94 Ϯ 0.5 48 Ϯ 497Ϯ 2 317 Ϯ 10

a iDCs and mDCs were generated from human HPC and analyzed by FACScan as described in Materials and Methods. At least 5000 or more events were collected for each sample. Shown is the average (mean Ϯ SD) of two separate experiments. b MFI, Median fluorescence intensity. allogeneic MLR. As outlined in Table I, iDCs were CD1aϩ, amounts of RNAs were loaded in both lanes 1 and 2 (Fig. 4A). As ϩ Ϫ low Ϫ low me CD11a , CD14 , CD40 , CD83 , CD86 , and HLA-DR - expected, both iDCs and mDCs expressed a similar level of C5aR ϩ ϩ Ϫ high dium, whereas mDCs were CD1a , CD11a , CD14 , CD40 , mRNA as measured by RT-PCR using C5aR-specific primers (Fig. CD83ϩ, CD86high, and HLA-DRhigh. In addition, iDCs were un- 4B, lanes 1 and 2). These results indicate that DCs, upon matura- able to stimulate allogeneic MLR whereas mDCs stimulated tion induced by TNF-␣, down-regulate their FPR expression while marked proliferation of allogeneic T cells, especially at DC:T ra- maintaining their C5aR expression. tios equal to 1:100 or 1:10, as detected by [3H]TdR incorporation (Table II). These data confirmed that monocyte-derived iDCs and mDCs used in this study had the characteristics of iDCs DC maturation induced by CD40 ligation also differentially and mDCs. regulates their responsiveness to and receptor expression for fMLP and C5a The responsiveness of iDCs and mDCs to fMLP and C5a DC maturation can be induced in vitro by treatment of iDCs with correlates with FPR and C5aR expression a variety of agents including bacterial products (LPS, etc.) (14, 21, In vivo, down-regulation of the response of DCs to fMLP may 23, 33), proinflammatory cytokines (TNF-␣, IL-1, etc.) (14, 21, 23, result from either down-regulation of FPR expression or homolo- 27, 28, 33), synthetic nucleic acids (CpG-oligodeoxynucleotides, gous desensitization due to the presence of an excessive amount of poly(I:C), etc.) (44, 45), and macrophage-conditioned medium (34, formyl peptides in the environment, which can derive from either 45), by cross-linking of membrane CD43 (46) or by CD40 ligation microbes or disrupted mitochondria (19, 42, 43). Although homol- with CD40 ligand or anti-CD40 Ab (14, 21, 33). However, DC ogous desensitization is less likely to occur when DCs are induced maturation induced by TNF-␣ and many other agents is considered to mature in an in vitro culture system, we examined the expres- reversible and only that induced by poly(I:C), macrophage-condi- sion of FPR and C5aR by iDCs and mDCs to rule out this possi- tioned medium, or CD40 ligation is stable (21, 45, 47, 48). To bility. To this end, iDCs and mDCs were tested for binding to determine whether stable maturation of DCs affects the regulation [3H]fMLP. iDCs specifically bound [3H]fMLP, and this was in- of FPR and C5aR expression, we prepared human mDCs by treat- hibited in a dose-dependent manner by unlabeled fMLP, whereas ment of monocyte-derived iDCs with anti-CD40 agonistic Ab. mDCs did not bind [3H]fMLP at all, suggesting that mDCs greatly mDCs generated in this manner showed phenotypic (CD40high, decrease their surface FPR expression (Fig. 3A). In contrast, both CD83ϩ, CD86high, and HLA-DRhigh) and functional (capable of iDCs and mDCs expressed comparable amounts of C5aR on their stimulating allogeneic MLR) characteristics similar to those of surfaces as determined by FACS analysis after staining of the cells TNF-␣-induced mDCs (data not shown). As shown by Fig. 5A, with FITC-conjugated anti-CD88 (Fig. 3B). To determine whether mDCs induced by anti-CD40 Ab did not migrate to fMLP (F), yet TNF-␣ induces differential regulation of FPR and C5aR expression they still migrated in response to C5a (Œ). In agreement with the at the transcriptional or posttranscriptional level, the expression of chemotaxis data (Fig. 5A), iDCs expressed both FPR (Fig. 5B, lane FPR and C5aR mRNAs by iDCs and mDCs was further investi- 1) and C5aR (Fig. 5C, lane 1) mRNAs. Upon anti-CD40 Ab-in- gated. By Northern blot analysis, iDCs were shown to express FPR duced maturation, mDCs down-regulated FPR mRNA (Fig. 5B, mRNA (Fig. 4A, lane 1), whereas FPR mRNA expression by lane 2) while maintaining a comparable level of C5aR mRNA mDCs was undetectable (Fig. 4A, lane 2). Reprobing of the same expression (Fig. 5C, lane 2). Therefore, DC maturation induced by filter with a 32P-labeled actin cDNA fragment after stripping either TNF-␣ or CD40 ligation results in selective down-regulation yielded bands of nearly identical intensity, confirming that equal of FPR, but not C5aR, expression.

Table IV. Stimulation of allogeneic MLR by human HPC-derived DCsa

[3H]TdR Incorporation (cpm/well)

DC DCs (no./well) alone DCs (no./well) ϩ T cells (105/well) Maturation Stage 100 1,000 10,000 100 1,000 10,000

iDCs 179 Ϯ 27 113 Ϯ 14 108 Ϯ 10 62 Ϯ 21 92 Ϯ 15 206 Ϯ 9 mDCs 100 Ϯ 18 58 Ϯ 15 201 Ϯ 883Ϯ 29 3,129 Ϯ 565 22,265 Ϯ 915

a Human HPC-derived DCs at various numbers per well as indicated were cultured alone or with allogeneic peripheral blood T cells (105/well) in RPMI 1640 containing 10% FBS in wells of 96-well tissue culture plate for 7 days. [3H]TdR was added to each well (0.5 ␮Ci/10 ␮l/well) and cultured for another 18 h before cell harvest. The radioactivity (cpm) incorporated is shown as the average (mean Ϯ SD) of six wells. The [3H]TdR incorporation for T cells cultured alone and in the presence of 5 ␮g/ml of phytohemagglutinin was 56 Ϯ 21 and 97,291 Ϯ 2,784 cpm, respectively. The Journal of Immunology 2699

cyte-derived DCs, we investigated the response to fMLP and C5a as well as FPR and C5aR expression of HPC-derived DCs before and after maturation. Human HPC-derived iDCs were CD1alow, CD83Ϫ, CD86low, and HLA-DRmedium (Table III) and lacked the capacity to stimulate allogeneic MLR (Table IV), whereas HPC- derived mDCs were CD1ahigh, CD83ϩ, CD86high, and HLA- DRhigh (Table III) with potent capacity to stimulate allogeneic MLR (Table IV), indicating that they exhibited immature and mature phenotypes, respectively. Interestingly, human HPC-derived iDCs migrated, similar to monocyte-derived iDCs, toward both fMLP (Fig. 6A, E) and C5a (Fig. 6A, ‚). After TNF-␣-induced maturation, CD34ϩ HPC-derived mDCs migrated toward C5a (Fig. 6A, Œ), but not to fMLP (Fig. 6A, F). Furthermore, CD34ϩ HPC-derived iDCs mobilized intracellular Ca2ϩ in response to both fMLP and C5a, whereas mDCs did so only in response to C5a, but not to fMLP (Fig. 6B). Concomitantly, HPC-derived iDCs expressed both FPR (Fig. 6C, left, lane 1) and C5aR (Fig. 6C, right, lane 1) mRNAs, whereas mDCs maintained comparable levels of C5aR mRNA (Fig. 6C, right, lane 2), but down-regulated FPR mRNA to an undetectable level (Fig. 6C, left, lane 2). Thus, upon maturation, human HPC-derived DCs also differentially regulate their responsiveness to and receptor expression for fMLP and C5a.

C5a-induced migration of mDCs is based on chemotaxis FIGURE 6. Regulation of responsiveness to, and expression of recep- It has been reported that induction of iDC migration by fMLP and ϩ tors for, fMLP and C5a upon maturation of CD34 progenitor-derived C5a is based on chemotaxis (5, 10, 36). To assure whether C5a- DCs. iDCs and mDCs were generated from DC precursors amplified induced mDC migration is due to chemotaxis or chemokinesis, CD34ϩ progenitors as described in Materials and Methods. A, Migration of ϩ checkerboard analysis was performed. As shown by Table V, ad- CD34 -derived iDCs (open symbols) and mDCs (closed symbols) induced dition of C5a into the upper wells alone did not cause mDCs to by fMLP (circles) and C5a (triangles). The results are shown as the aver- migrate across the membrane (row 1), indicating that C5a did not age (mean Ϯ SD) of triplicated wells. Error bars were omitted for clarity. Similar results were obtained from three separate experiments. B,Ca2ϩ increase chemokinesis of mDCs. Addition of C5a into the lower mobilization of CD34ϩ-derived DCs. Shown are the peak heights induced wells alone resulted in dose-dependent mDC migration (column 1). by fMLP or C5a at the final concentrations (M) as specified on the right Moreover, when C5a was added into both the upper and lower side. Two separate experiments yielded almost identical results. C, Expres- wells, mDC migration was inhibited to various degrees, depending sion of FPR and C5aR at mRNA level by CD34ϩ-derived DCs. Left, FPR on C5a concentrations in the lower and upper wells (desensitiza- mRNA expression by iDCs (lane 1) and mDCs (lane 2) as detected by tion). Taken together, checkerboard analysis indicates that C5a- Northern blot. The nitrocellulose filter was first probed with 32P-labeled induced mDC migration is also based on chemotaxis. FPR cDNA fragment, stripped, and reprobed with 32P-labeled actin cDNA fragment. Right, RT-PCR products of C5aR (upper panel) and GAPDH Chemotaxis of murine HPC-derived DCs to fMLP and C5a (lower panel) displayed by agarose gel electrophoresis. The anticipated before and after maturation sizes for C5aR and GAPDH are 500 and 246 bp, respectively. M, Marker that is a 1-kb DNA ladder. Lane 1, iDCs; lane 2, mDCs. Murine iDCs express CXCR4 (49), CCR1 (49, 50), CCR2 (49), and CCR5 (34, 49) whereas murine mDCs are CXCR4-positive (49) and CCR7-positive (49–52). Unlike human iDCs, murine Differential regulation of the responsiveness to, and receptor iDCs do not express CCR4 (49) and CCR6 (50), highlighting sev- expression for, fMLP and C5a upon maturation of human ϩ eral differences between human and mouse DCs. This led us to CD34 HPC-derived DCs investigate whether murine DCs respond similarly to maturation Human DCs can be generated in vitro in a large number from signals as do human DCs with particular regard to the regulation of either monocytes or CD34ϩ HPC (11–15, 21, 22, 24–28, 33, 34). responsiveness to fMLP and C5a. As demonstrated by Fig. 7, C5a To address whether HPC-derived DCs behave similarly to mono- induced the migration of both murine iDCs (‚) and mDCs (Œ).

Table V. Checkerboard analysis of C5a-induced mDC migrationa

C5a in Upper Wells (nM) C5a in Lower Wells (nM) 0 0.1 1 10

08Ϯ 19Ϯ 27Ϯ 17Ϯ 1 0.1 25 Ϯ 39Ϯ 27Ϯ 16Ϯ 2 178Ϯ 924Ϯ 511Ϯ 27Ϯ 1 10 30 Ϯ 312Ϯ 48Ϯ 27Ϯ 2

a Human monocyte-derived mDCs were used at 5 ϫ 105/ml. C5a at specified concentrations was added to the lower wells of the chemotaxis chamber, and mDCs in the absence or presence of specified concentrations of C5a was added to the upper wells of the chemotaxis chamber. The results are shown as the average (mean Ϯ SD) of migrated mDCs of triplicated wells (per high-power field). Two additional experiments yielded similar results. 2700 REGULATION OF FPR AND C5aR EXPRESSION UPON DC MATURATION

recruitment because fMLP and C5a were found to be most potent and efficacious for DCs isolated from airway tissues (5). Upon maturation induced by TNF-␣ or CD40 ligation, mDCs down-regulated their responsiveness to and receptor expression for fMLP while maintaining their responsiveness to and receptor expression for C5a. These results are compatible with the finding that human skin-derived DCs up-regulate their responsiveness to C5a after treatment with TNF-␣ for 24 h (36). Moreover, the differential regulation of mDCs responsiveness to fMLP and C5a paral- leled the expression of FPR and C5aR. This differential regulation seems to be DC maturation-dependent rather than TNF-␣-depen- FIGURE 7. Chemotaxis of murine HPC-derived DCs in response to dent because 1) DCs matured by CD40 ligation also exhibited the fMLP and C5a. The migration of DCs induced by different concentrations same pattern, and 2) TNF-␣ has been shown to promote, rather of fMLP and C5a was studied by chemotaxis assay. The results are shown than suppress, FPR expression in human neutrophils (59). Because Ϯ as the average (mean SD) of triplicated wells. Spontaneous DC migra- the expression of FPR mRNA was also down-regulated, it can be ϭ ϳ tion (chemotactic index 1) was 40 50 cells per high-power field. One speculated that a reduction in either FPR mRNA stability and/or experiment representative of four is shown. FPR gene transcription is responsible. FPR and C5aR genes are known to cluster at the same narrow region of a chromosome (19 in human and 17 in mouse) (60–62), however, the structure of However, fMLP was only chemotactic for murine iDCs (E), but their promoter regions is not well understood. Furthermore, how not for murine mDCs (F), suggesting that maturation of murine the expression of FPR and C5aR genes is controlled remains un- DCs also differentially regulates their responsiveness to fMLP and known. Therefore, how and why DC maturation results in differ- C5a. The optimal chemotactic concentration of fMLP for murine ential regulation of FPR and C5aR expression needs further Ϫ iDCs was 10 6 M, which was 100-fold higher than that for human investigation. Ϫ iDCs (10 8 M). This difference presumably reflects the different DCs generated from CD34ϩ HPC are heterogeneous. At least sensitivities of human and murine FPR to fMLP rather than the two subsets of DCs, CD1aϩ and CD1aϪ, can be derived from difference between human and murine iDCs because 1) to induce CD34ϩ HPC-derived DCs (63). Although our results show that, ϩ comparable level of intracellular Ca2 mobilization in murine neu- similar to monocyte-derived DCs, human HPC-derived DCs also trophils, 100-fold or more fMLP is needed than with human neu- differentially down-regulate FPR, but not C5aR, expression upon trophils (53); 2) about 100-fold more fMLP is required to induce maturation, whether different subsets of HPC-derived DCs behave ϩ comparable level of intracellular Ca2 mobilization in murine as similarly or differently in the course of maturation in terms of FPR compared with human FPR-expressing Xenopus oocytes (53); and and C5aR expression awaits further investigation. 3) human embryonic kidney 293 cells expressing murine FPR mi- Interaction of two chemokine receptors, CXCR4 and CCR7 that Ϫ grate in response to fMLP with an optimal concentration of 10 6 are known to be expressed by mDCs (14, 21, 23, 33–35), with their M (54), whereas those expressing human FPR migrate in response ligands, e.g., SDF-1␣, secondary lymphoid chemokine (also Ϫ to fMLP with an optimal concentration of 10 8 M (55). known as 6Ckine, Exodus-2, or TCA4), and EBI1 ligand chemokine (also known as macrophage inflammatory protein-3␤), is in- Discussion volved in the recruitment of mDCs to lymphoid tissues (35, 51, To execute their roles in initiating and modulating immune re- 64). This leads us to propose that the interaction of C5a and C5aR sponses, iDCs need to migrate to sites of pathogen entry in the may also participate in the recruitment of mDCs to lymphoid tis- peripheral tissues to take up Ags, whereas mDCs have to migrate sues, specifically in guiding and/or sorting mDCs to B cell folli- to lymphoid organs to transfer native Ag to naive B cells and to cles, where naive B cells acquire native Ags delivered by mDCs present processed Ag to naive T cells (1–9, 56, 57). Studies of the (57, 65). Besides macrophages, B cells have recently been found to in vitro effect of chemotactic factors on DC (10–15, 23, 26, 27, be a source of C5a in secondary lymphoid tissue (66). Follicular 30–34, 49, 50, 52) have increased our understanding of their in DCs, a particular type of DC localizing in B cell follicles, retain vivo roles in guiding DC trafficking (35, 51). Although human Ag-Ab complexes on their surfaces (67). Therefore, C5a may be monocyte-derived DCs or DCs isolated from human skin and rat generated via the classical pathway of complement activation trig- respiratory tract tissue have previously been shown to migrate in gered by follicular DC-bound immune complexes, thereby forming response to fMLP and/or C5a (5, 10, 36), the relationship between a C5a gradient. C5a is also a chemoattractant for B cells (66, 68). maturation of DC and their responsiveness to fMLP and C5a is not Thus, locally generated C5a gradient may attract both mDCs and known. Our results showing that both human and murine iDCs naive B cells to B cell follicles to facilitate Ag transfer (56, 57, 65). generated from either monocytes or HPC migrated to fMLP and Collectively, our results suggest that the interaction of FPR with C5a suggest that formyl peptides and C5a may participate in the its ligands is possibly involved in the recruitment of iDCs whereas recruitment of iDCs to sites of infection. Although this needs to be that of C5a and C5aR may participate in the recruitment of both confirmed by animal model studies, the following evidence sup- iDCs and mDCs in vivo to distinct anatomical sites. The observa- ports this possibility. Formyl peptides can be released by invading tion that murine DC responded to fMLP and C5a similarly to hu- microorganisms (42) or disrupted mitochondria due to microor- man DCs indicates that mouse models can be used to decipher the ganism-induced host cell damage (43). C5a can be produced as a in vivo roles of fMLP and C5a in DC trafficking, particularly by the result of complement activation through classical or alternative use of recently established FPR and C5aR knockout mice (69, 70). pathway at local or systemic inflammatory sites (58). Thus, gradients of formyl peptides and C5a are presumably formed at the Acknowledgments sites of infection. Furthermore, pathogen inhalation induces a very We thank N. Dunlop for technical assistance and Dr. Ronald N. Germain, rapid recruitment of DCs to the rat airway epithelium (4, 5). Chief of the Laboratory of Immunology, National Institute of Allergy and Formyl peptides and C5a may be at least in part involved in this Infectious Diseases, National Institutes of Health, for helpful discussion The Journal of Immunology 2701 and critical review of the manuscript. The support of the laboratory manager 29. Carramolino, L., L. Kremer, I. Goya, R. Varona, J. M. Buesa, J. Gutierrez, A. Zaballos, ␤ C. Fogle and secretarial assistance of C. Nolan is gratefully appreciated. C. Martinez-A., and G. Marquez. 1999. Down-regulation of the -chemokine receptor CCR6 in dendritic cells mediated by TNF-␣ and IL-4. J. Leukocyte Biol. 66:837. References 30. Vicari, A. P., D. J. Figueroa, J. A. Hedrick, J. S. Foster, K. P. Singh, S. Menon, N. G. Copeland, D. J. Gilbert, N. A. Jenkins, K. B. Bacon, and A. Zlotnik. 1997. 1. Cella, M., F. Sallusto, and A. Lanzavecchia. 1997. Origin, maturation and antigen TECK: a novel CC chemokine specifically expressed by thymic dendritic cells presenting function of dendritic cells. Curr. Opin. Immunol. 9:10. and potentially involved in T cell development. Immunity 7:291. 2. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of 31. Zabel, B. A., W. W. Agace, J. J. Campbell, H. M. Heath, D. Parent, A. I. Roberts, immunity. Nature 392:245. E. C. Ebert, N. Kassam, S. Qin, M. Zovko, et al. 1999. Human G protein-coupled 3. Sallusto, F., and A. Lanzavecchia. 1999. Mobilizing dendritic cells for tolerance, receptor GPR-9–6/CC chemokine receptor 9 is selectively expressed on intestinal priming, and chronic inflammation. J. Exp. Med. 189:611. homing T lymphocytes, mucosal lymphocytes, and thymocytes and is required for 4. McWilliam, A. S., D. J. Nelson, J. A. Thomas, and P. G. Holt. 1994. Rapid thymus-expressed chemokine-mediated chemotaxis. J. Exp. Med. 190:1241. dendritic cell recruitment is a hallmark of the acute inflammatory response at mucosal surfaces. J. Exp. Med. 179:1331. 32. Zaballos, A., J. Gutierrez, R. Varona, C. Ardavin, and G. Marquez. 1999. Cutting edge: identification of the orphan chemokine receptor GPR-9–6 as CCR9, the 5. McWilliam, A. S., S. Napoli, A. M. Marsh, F. L. Pemper, D. J. Nelson, receptor for chemokine TECK. J. Immunol. 162:5671. C. L. Pimm, P. A. Stumbles, T. N. C. Wells, and P. G. Holt. 1996. Dendritic cells are recruited into the airway epithelium during the inflammatory response to a 33. Sozzani, S., P. Allavena, G. D’Amico, W. Luini, G. Bianchi, M. Kataura, T. Imai, broad spectrum of stimuli. J. Exp. Med. 184:2429. O. Yoshie, R. Bonecchi, and A. Mantovani. 1998. Differential regulation of che- 6. Matsuno, K., T. Ezaki, S. Kudo, and Y. Uehara. 1996. A life stage of particle- mokine receptors during dendritic cell maturation: a model for their trafficking laden rat dendritic cells in vivo: their terminal division, active phagocytosis, and properties. J. Immunol. 161:1083. translocation from the liver to the draining lymph. J. Exp. Med. 183:1865. 34. Yanagihara, S., E. Komura, J. Nagafune, H. Watarai, and Y. Yamaguchi. 1998. 7. MacPherson, G. G., C. D. Jenkins, M. J. Stein, and C. Edwards. 1995. Endotoxin- EBI1/CCR7 is a new member of dendritic cell chemokine receptor that is up- mediated dendritic cell release from intestine. J. Immunol. 154:1317. regulated upon maturation. J. Immunol. 161:3096. 8. Roake, J. A., A. S. Rao, P. J. Morris, C. P. Larsen, D. F. Hankins, and 35. Foster, R., A. Schubel, D. Breitfeld, E. Kremmer, I. Renner-Muller, E. Wolf, and J. M. Austyn. 1995. Dendritic cell loss from nonlymphoid tissues after systemic M. Lipp. 1999. CCR7 coordinates the primary immune response by establishing administration of lipopolysaccharide, tumor necrosis factor, and interleukin 1. functional microenvironments in secondary lymphoid organs. Cell 99:23. J. Exp. Med. 181:2237. 36. Morelli, A., A. Larregina, E. Chuluyan, E. Kolkowski, and L. Fainboim. 1996. 9. Smedt, T. D., B. Pajak, E. Muraille, L. Lespagnard, E. Heinen, P. D. Baetselier, Expression and modulation of C5a receptor (CD88) on skin dendritic cells: che- J. Urbain, O. Leo, and M. Moser. 1996. Regulation of dendritic cell numbers and motactic effect of C5a on skin migratory dendritic cells. Immunology 89:126. maturation by lipopolysaccharide in vivo. J. Exp. Med. 184:1413. 37. Arrighi, J.-F., C. Hauser, B. Chapuis, R. H. Zubler, and V. Kindler. 1999. Long- ϩ 10. Sozzani, S., F. Sallusto, W. Luini, D. Zhou, L. Piemonti, P. Allavena, term culture of human CD34 progenitors with Flt3-ligand, thrombopoietin, and Ϫ Ϫ J. V. Damme, S. Valitutti, A. Lanzavecchia, and A. Mantovani. 1995. Migration stem cell factor induces extensive amplification of a CD34 CD14 and a Ϫ ϩ of dendritic cells in response to formyl peptides, C5a, and a distinct set of che- CD34 CD14 dendritic cell precursor. Blood 93:2244. mokine. J. Immunol. 155:3292. 38. Inaba, K., M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu, 11. Xu, L. L., K. M. Warren, W. L. Rose, W. Gong, and J. M. Wang. 1996. Human and R. M. Steinman. 1992. Generation of large numbers of dendritic cells from recombinant monocyte chemotactic protein and other C-C chemokines bind and mouse bone marrow cultures supplemented with granulocyte/macrophage colo- induce directional migration of dendritic cells in vitro. J. Leukocyte Biol. 60:365. ny-stimulating factor. J. Exp. Med. 176:1693. 12. Sozzani, S., W. Luini, A. Borsatti, N. Polentarutti, D. Zhou, L. Piemonti, G. 39. Falk, W., R. H. Goodwin, and E. J. Leonard. 1980. A 48-well micro chemotaxis assembly D’Amico, C. A. Power, T. N. C. Wells, M. Gobbi, P. Allavena, and for rapid and accurate measurement of leukocyte migration. J. Immunol. Methods 33:239. A. Mantovani. 1997. Receptor expression and responsiveness of human dendritic 40. Yang, D., H. Hayashi, T. Takii, Y. Mizutani, Y. Inukai, and K. Onozaki. 1997. cells to a defined set of CC and CXC chemokines. J. Immunol. 159:1993. Interleukin-1-induced growth inhibition of human melanoma cells: interleukin- 13. D’Amico, G., G. Bianchi, S. Bernasconi, L. Bersani, L. Piemonti, S. Sozzani, 1-induced antizyme expression is responsible for ornithine decarboxylase activity A. Mantovani, and P. Allavena. 1998. Adhesion, transendothelial migration, and down-regulation. J. Biol. Chem. 272:3376. reverse transmigration of in vitro cultured dendritic cells. Blood 92:207. 41. Howard, O. M. Z., M. Dean, H. Young, M. Ramsburg, J. A. Turpin, 14. Dieu, M.-C., B. Vanbervliet, A. Vicari, F. Briere, A. Zlotnik, S. Lebecgue, and D. F. Michiel, D. J. Kelvin, L. Lee, and W. L. Parrar. 1992. Characterization of C. Caux. 1998. Selective recruitment of immature and mature dendritic cells by a class 3 tyrosine kinase. Oncogene 7:895. distinct chemokine expressed in different anatomic sites. J. Exp. Med. 188:373. 42. Schiffmann, E., B. A. Corcoran, and S. M. Wahl. 1975. N-Formyl-methionyl 15. Sozzani, S., D. Longoni, R. Bonecchi, W. Luini, L. Bersani, G. D’Amico, peptides as chemoattractants for leukocytes. Proc. Natl. Acad. Sci. USA 72:1059. A. Borsatti, F. Bussolino, P. Allavena, and A. Mantovani. 1997. Human mono- ϩ 43. Carp, H. 1982. Mitochondrial N-formylmethionyl proteins as chemoattractants cyte-derived and CD34 cell-derived dendritic cells express functional receptors for neutrophils. J. Exp. Med. 155:264. for platelet activating factor. FEBS Lett. 418:98. 44. Hartmann, G., G. J. Weiner, and A. M. Krieg. 1999. CpG DNA: a potent signal 16. Baggiolini, M. 1998. Chemokines and leukocyte traffic. Nature 392:565. for growth, activation, and maturation of human dendritic cells. Proc. Natl. Acad. 17. Rollins, B. J. 1997. Chemokines. Blood 90:909. Sci. USA 96:9305. 18. Zlotnick, A., J. Morales, and J. A. Hedrick. 1999. Recent advances in chemokines 45. Verdijk, R. M., T. Mutis, B. Esendam, J. Kamp, C. J. M. Melief, A. Brand, and and chemokine receptors. Crit. Rev. Immunol. 19:1. E. Goulmy. 1999. Polyriboinosinic polyribocytidylic acid (Poly(I:C)) induces stable 19. Murphy, P. M. 1994. The molecular biology of leukocyte chemoattractant recep- maturation of functionally active human dendritic cells. J. Immunol. 163:57. tors. Annu. Rev. Immunol. 12:593. 46. Corinti, S., E. Fanales-Belasio, C. Albanesi, A. Cavani, P. Angelisova, and 20. Murphy, P. M. 1996. Chemokine receptors: structure, function and role in mi- G. Girolomoni. 1999. Cross-linking of membrane CD43 mediates dendritic cell crobial pathogenesis. Cytokine Growth Factor Rev. 7:47. maturation. J. Immunol. 162:6331. 21. Sallusto, F., P. Schaerli, P. Loetscher, C. Schaniel, D. Lenig, C. R. Mackay, S. Qin, and A. Lanzavecchia. 1998. Rapid and coordinated switch in chemokine 47. Romani, N., D. Reider, M. Heuer, S. Ebner, E. Kampgen, B. Eibl, receptor expression during dendritic cell maturation. Eur. J. Immunol. 28:2760. D. Niederwieser, and G. Schuler. 1996. Generation of mature dendritic cells from 22. Ayehunie, S., E. A. Garcia-Zepeda, J. A. Hoxie, R. Horuk, T. S. Kupper, A. D. Luster, human blood: an improved method with special regard to clinical applicability. and R. M. Ruprecht. 1997. Human immunodeficiency virus-1 entry into purified blood J. Immunol. Methods 196:137. dendritic cells through CC and CXC chemokine coreceptors. Blood 90:1379. 48. Nelson, E. L., S. Strobl, J. Subleski, D. Prieto, W. C. Koop, and P. J. Nelson. ␣ 23. Lin, C.-L., R. M. Suri, R. A. Rahdon, J. M. Austyn, and J. A. Roake. 1998. 1999. Cycling of human dendritic cell effector phenotypes in response to TNF : Dendritic cell chemotaxis and transendothelial migration are induced by distinct modification of the current “maturation” paradigm and implication of in vivo chemokines and are regulated on maturation. Eur. J. Immunol. 28:4114. immunoregulation. FASEB J. 13:2021. 24. Sato, K., H. Kawasaki, H. Nagayama, R. Serizawa, J. Ikeda, C. Morimoto, K. Yasunaga, 49. Vecchi, A., L. Massimiliano, S. Ramponi, W. Luini, S. Bernasconi, R. Nonecchi, N. Yamaji, K. Tadokoro, T. Juji, and T. A. Takahashi. 1999. CC chemokine receptors, P. Allavena, M. Parmentier, A. Mantovani, and S. Sozzani. 1999. Differential CCR1 and CCR3, are potentially involved in antigen-presenting cell function of human responsiveness to constitutive vs. inducible chemokines of immature and mature peripheral blood monocyte-derived dendritic cells. Blood 93:34. mouse dendritic cells. J. Leukocyte Biol. 66:489. 25. Power, C. A., D. J. Church, A. Meyer, S. Alouani, A. E. I. Proudfoot, 50. Ogata, M., Y. Zhang, Y. Wang, M. Itakura, Y.-Y. Zhang, A. Harada, S. Hashimoto, I. Clark-Lewis, S. Sozzani, A. Mantovani, and T. N. C. Wells. 1997. Cloning and and K. Matsushima. 1999. Chemotactic response toward chemokines and its regu- characterization of a specific receptor for the novel CC chemokine MIP-3␣ from lation by transforming growth factor-␤1 of murine bone marrow hematopoietic pro- lung dendritic cells. J. Exp. Med. 186:825. genitor cell-derived different subset of dendritic cells. Blood 93:3225. 26. Greaves, D. R., W. Wang, D. J. Dairaghi, M. C. Dieu, B. Saint-Vis, K. Franz-Bacon, 51. Saeki, H., A. M. Moore, M. J. Brown, and S. T. Hwang. 1999. Cutting edge: D. Rossi, C. Caux, T. McClanahan, S. Gordon, A. Zlotnik, and T. J. Schall. 1997. CCR6, secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 a CC chemokine receptor that interacts with macrophage inflammatory protein 3␣ and is (CCR7) participate in the emigration pathway of mature dendritic cells from the highly expressed in human dendritic cells. J. Exp. Med. 186:837. skin to regional lymph nodes. J. Immunol. 162:2472. 27. Yang, D., O. M. Z. Howard, Q. Chen, and J. J. Oppenheim. 1999. Cutting edge: 52. Kellermann, S.-A., S. Hudak, E. R. Oldham, Y.-J. Liu, and L. M. McEvoy. 1999. immature dendritic cells generated from monocytes in the presence of TGF-␤1 The CC chemokine receptor-7 ligands 6Ckine and macrophage inflammatory express functional C-C chemokine receptor 6. J. Immunol. 163:1737. protein-3␤ are potent chemoattractants for in vitro- and in vivo-derived dendritic 28. Yang, D., O. Chertov, S. N. Bykovskaia, Q. Chen, M. J. Buffo, J. Shogan, cells. J. Immunol. 162:3859. M. Anderson, J. M. Schroder, J. M. Wang, O. M. Z. Howard, and 53. Gao, J.-L., and P. M. Murphy. 1993. Species and subtype variants of the N-formyl J. J. Oppenheim. 1999. ␤-Defensins: linking innate and adaptive immunity peptide chemotactic receptor reveal multiple important functional domains. through dendritic and T cell CCR6. Science 286:525. J. Biol. Chem. 268:25395. 2702 REGULATION OF FPR AND C5aR EXPRESSION UPON DC MATURATION

54. Hartt, J. K., G. Barish, P. M. Purphy, and J.-L. Gao. 1999. N-Formylpeptides 62. Gao, J.-L., H. Chen, J. D. Filie, C. A. Kozak, and P. M. Murphy. 1998. Differ- induce two distinct concentration optima for mouse neutrophil chemotaxis by dif- ential expansion of the N-formylpeptide receptor gene cluster in human and ferential interaction with two N-formylpeptide receptor (FPR) subtypes: molecular mouse. Genomics 51:270. characterization of FPR2, a second mouse neutrophil FPR. J. Exp. Med. 190:741. 63. Caux, C., B. Vanbervliet, C. Massacrier, C. Dezutter-Dambuyant, B. Saint-Vis, 55. Su, S. B., J.-L. Gao, W.-H. Gong, N. M. Dunlop, P. M. Murphy, J. J. Oppenheim, C. Jacquet, K. Yoneda, S. Imamura, D. Schmitt, and J. Banchereau. 1996. CD34ϩ and J. M. Wang. 1999. T21/DP107, a synthetic leucine zipper-like domain of the hematopoietic progenitors from human cord blood differentiate along two indepen- HIV-1 envelope gp41, attracts and activates human phagocytes by using G-pro- dent dendritic cell pathways in response to GM-CSFϩTNF␣. J. Exp. Med. 184:695. tein-coupled formyl peptide receptors. J. Immunol. 162:5924. 64. Saeki, H., and S. T. Hwang. 2000. Dermal expression of stromal cell-derived 56. Kushnir, N., L. Liu, and G. G. MacPherson. 1998. Dendritic cells and resting B factor (SDF)-1 contribute to the recruitment of mature dendritic cells from skin cells form clusters in vitro and in vivo: T cell independence, partial LFA-1 depen- to regional lymph nodes. J. Invest. Dermatol. 114:210. dence, and regulation by cross-linking surface molecules. J. Immunol. 160:1774. 65. Berney, C., S. Herren, C. A. Power, S. Gordon, L. Martinez-Pomares, and 57. Wykes, M., A. Pombo, C. Jenkins, and G. G. MacPherson. 1998. Dendritic cells M. H. Kosco-Vilbois. 1999. A member of the dendritic cell family that enters B interact directly with naive B lymphocytes to transfer antigen and initiate class cells follicles and stimulates primary antibody responses identified by a mannose switching in a primary T-dependent response. J. Immunol. 161:1313. receptor fusion protein. J. Exp. Med. 190:851. 58. Gerard, C., and N. P. Gerard. 1994. C5a anaphylatoxin and its seven transmem- 66. Ottonello, L., A. Corcione, G. Tortolina, I. Airoldi, E. Albesiano, A. Favre, R. brane-segment receptor. Annu. Rev. Immunol. 12:775. D’Agostino, F. Malavasi, V. Pistoia, and F. Dallegri. 1999. rC5a directs the in 59. O’Flaherty, J. T., A. G. Rossi, J. F. Redman, and D. P. Jacobson. 1991. Tumor vitro migration of human memory and naive tossillar B lymphocytes: implica- necrosis factor-␣ regulates expression of receptors for formyl-methionyl-leucyl- tions for B cell trafficking in secondary lymphoid tissues. J. Immunol. 162:6510. phenylalanine, leukotriene B4, and platelet-activating factor: dissociation from 67. MacLennan, I. C. 1994. Germinal centers. Annu. Rev. Immunol. 12:117. priming in human polymorphonuclear neutrophils. J. Immunol. 147:3842. 68. Kupp, L. I., M. H. Kosco, H. A. Schenkein, and J. G. Tew. 1991. Chemotaxis of 60. Gerard, N. P., L. Bao, H. Xiao-Ping, R. L. Eddy, Jr., T. B. Shows, and C. Gerard. germinal center B cells in response to C5a. Eur. J. Immunol. 21:2697. 1993. Human chemotaxis receptor genes cluster at 19q13.3–13.4: characteriza- 69. Hopken, U. E., B. Lu, N. P. Gerard, and C. Gerard. 1996. The C5a chemoattrac- tion of the human C5a receptor gene. Biochemistry 32:1243. tant receptor mediates mucosal defense to infection. Nature 383:86. 61. Alvarez, V., E. Coto, F. Setien, and C. Lopez-Larrea. 1994. A physical map of two 70. Gao, J.-L., E. J. Lee, and P. M. Murphy. 1999. Impaired antibacterial host defense clusters containing the genes for six proinflammatory receptors. Immunogenetics 40:100. in mice lacking the N-formylpeptide receptor. J. Exp. Med. 189:657.