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.

Myeloid dendritic cells (DCs)5 are highly specialized APCs that initiate immune responses (1, 2, 3). DCs originate from bone marrow and migrate through the blood stream to populate nonlymphoid tissues in an immature state (1). Upon the introduction of Ags into a host such as by an infection, immature DCs (iDCs) migrate to the site of Ag deposition, take up and process Ags, and simultaneously undergo a process of phenotypic and functional maturation (2, 3, 4, 5, 6). Subsequently, the resultant mature DCs (mDCs) traffic via the afferent lymphatic to the T cell area of secondary lymphoid organs where they activate Ag-specific lymphocytes (6, 7, 8, 9). Therefore, iDCs and mDCs traffic to different anatomical sites (site of Ag deposition vs T cell area) and fulfill their respective roles in Ag uptake, processing, and presentation.

The trafficking pattern of DCs, like that of other leukocytes, is presumably controlled by many mediators. However, the direction of DC migration is primarily determined by chemotactic gradients formed by a variety of chemotactic factors, including classical chemoattractants and chemokines (3, 5, 10, 11, 12, 13, 14, 15, 16). Classical chemoattractants include formyl peptides (e.g., cleavage products of bacterial and mitochondrial proteins such as fMLP) (5, 10), products of host complement activation (e.g., C5a) (10), and lipid metabolites (e.g., platelet activating factor) (15), whereas chemokines consist of a superfamily of >30 structurally related proteins classified into the CXC, CC, CX3C, and C chemokine subfamilies (16, 17, 18). The effects of classical chemoattractants and chemokines are mediated by members of the G protein-coupled seven-transmembrane domain receptor superfamily (18, 19, 20). The reason that iDCs and mDCs are able to migrate toward different chemotactic factors is due to the fact that they express different sets of chemotactic receptors. So far, human iDCs have been shown to express CXCR1 (12, 21), CXCR2 (12), CXCR4 (12, 21, 22, 23, 24), CCR1 (12, 21, 22, 24), CCR2 (12, 21, 22), CCR3 (22, 24), CCR4 (21, 22), CCR5 (12, 21, 22, 23, 24), CCR6 (14, 25, 26, 27, 28, 29), CCR8,6 and probably CCR9 (30, 31, 32), whereas mDCs express only CXCR4 (21, 23) and CCR7 (14, 21, 33, 34, 35).

Studies of DC responses to chemoattractants have shown that human iDCs derived from either peripheral blood monocytes or cord blood CD34+ cells respond chemotactically to and express the receptor for platelet activating factor (15). DCs isolated from rat respiratory tract tissue or generated in vitro from human peripheral blood monocytes can be chemoattracted by fMLP and C5a (5, 10). Human skin-derived Langerhans’ cells, a prototype of iDCs, express the receptor for C5a (C5aR) and respond chemotactically to C5a (36). However, the regulation of DC responsiveness to fMLP and C5a and expression of formyl peptide receptor (FPR), the high-affinity receptor for fMLP, and C5aR upon DC maturation, have not been fully elucidated. Here, we investigated this issue by using human and murine DCs generated in vitro from either hemopoietic progenitor cells (HPC) or CD14+ peripheral blood monocytes. The results show that iDCs of both species express FPR and C5aR and are thus able to respond to fMLP and C5a. In contrast, mDCs differentially down-regulate FPR, but not C5aR, expression at mRNA and protein levels, resulting in a selective retention of responsiveness to C5a as compared with fMLP.

IMDM was purchased from Life Technologies (Rockville, MD). RPMI 1640 was purchased from BioWhittaker (Walkersville, MD). Recombinant human (rh) stromal cell-derived factor-1α (SDF-1α), TNF-α, GM-CSF, IL-4, Flt3-ligand, stem cell factor, and thrombopoietin were purchased from PeproTech (Rocky Hill, NJ). Recombinant murine (rm) GM-CSF, IL-4, and TNF-α were purchased from Biosource International (Camarillo, CA), R&D Systems (Minneapolis, MN), and PharMingen (San Diego, CA), respectively. fMLP, rhC5a, and chemicals unless otherwise specified were purchased from Sigma (St. Louis, MO). FBS was purchased from HyClone (Logan, UT). FITC-conjugated mouse anti-human C5aR (CD88) was purchased from Serotec (Oxford, U.K.). Mouse anti-human CD40 agonistic Ab (IgG1, κ, capable of stimulating B cell proliferation in the presence of IL-4) was purchased from PharMingen. Anti-CD83 was purchased from Coulter-Immunotech (Marseille, France). The other Abs used for flow cytometry were purchased from PharMingen. [3H]fMLP with a specific radioactivity of 2000 Ci/mmol and [3H]TdR with a specific radioactivity of 2 Ci/mmol were purchased from NEN (Boston, MA). PCR primers for C5aR were purchased from Stratagene (La Jolla, CA).

Human PBMC were isolated by routine Ficoll-Hypaque density gradient centrifugation. Monocytes were purified (>95%) from human PBMC with the use of a MACS CD14 monocyte isolation kit (Miltenyi Biotech, Auburn, CA) according to the manufacturer’s recommendation. Cord blood CD34+ HPC (>95%) were purchased from Poietics (Gaithersburg, MD). The DC precursors were amplified from CD34+ HPC exactly as described (37) by culturing the cells at 5 × 104 cells/ml in IMDM supplemented with 20% FBS, 10−5 M DTT, 25 ng/ml rhFlt3-ligand, 10 ng/ml rh thrombopoietin, and 20 ng/ml rh stem cell factor for 4 wk. The amplified DC precursors were cryopreserved in IMDM containing 20% FBS and 10% DMSO until later usage. Murine HPC were prepared from the bone marrow of C57BL/6 mice (female, 7 wk) as described (38). Briefly, bone marrow cells flushed from femur and tibia were depleted of RBC by ammonium chloride treatment. For depletion of lymphocytes and Ia-positive cells, the remaining cells were incubated with a mixture of mAb for 1 h at 4°C followed by depletion with immunomagnetic beads coated with anti-rat IgG (Dynal, Great Neck, NY). The mAbs used were anti-B220/CD45R (PharMingen), anti-MHC class II (M5/114.15.2 anti-I-Ab, d, q and I-Ed, k; American Type Culture Collection, Manassas, VA), and anti-CD90 (Thy1; PharMingen). The resulting cells (∼90% Sca-1+/Lin) were used as murine HPC.

To generate human monocyte-derived iDCs, purified monocytes were incubated at 1 × 106/ml in RPMI 1640 medium (RPMI 1640 plus 10% FBS, 2 mM glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin) in the presence of rhGM-CSF (50 ng/ml) and rhIL-4 (10∼50 ng/ml) at 37°C in a CO2 (5%) incubator for 7 days. For the generation of human CD34+-derived iDCs, DC precursors amplified from CD34+ HPC were incubated at 5 × 105/ml in RPMI 1640 medium in the presence of 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 murine HPC at 1 × 106/ml in RPMI 1640 medium in the presence of 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, iDCs were cultured in the same cytokine mixtures with added TNF-α (50 ng/ml) or anti-CD40 Ab (100 μg/ml) for 48 h at 37°C in a CO2 (5%) incubator (21, 23, 27, 28).

DC migration was assessed using a 48-well microchemotaxis chamber technique as previously described (27, 39). Briefly, different concentrations of chemotactic factors were placed in the wells of the lower compartment 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 compartments were separated by a 5-μm polycarbonate filter (Osmonics, Livermore, CA). After incubation at 37°C in humidified air with 5% CO2 for 1.5 h, the filters were removed and stained, and the cells migrating across the filter were counted with the use of a Bioquant semiautomatic counting system. The results are presented as number of cells per high power field.

DCs (107 cells/ml in RPMI 1640 containing 10% FBS) were loaded with dye by incubating with 5 μM fura-2 (Molecular Probes, Eugene, OR) at 24°C for 30 min in the dark. Subsequently, the loaded cells were washed and resuspended (106 cells/ml) in saline buffer (138 mM NaCl, 6 mM KCl, 1 mM CaCl2, 10 mM HEPES, 5 mM glucose, and 1% BSA, pH 7.4). Each 2 ml of loaded DC suspension was then transferred into a quartz cuvette, which was placed in a luminescence spectrometer LS50 B (Perkin-Elmer, Beaconsfield, U.K). Ca2+ mobilization of the cells was measured by recording the ratio of fluorescence emitted at 510 nm after sequential excitation at 340 and 380 nm in response to chemotactic factors at various concentrations.

DCs were first washed three times with FACS buffer (PBS, 1% FBS, 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 manufacturer. After washing three times with PBS, the stained DCs were fixed with 1% paraformaldehyde in PBS, stored at 4°C overnight, and analyzed the next day with a flow cytometer (Coulter Epics, Miami, FL).

Allogeneic MLR was performed as described (11). Briefly, purified allogeneic T cells (105/well) were cultured with different numbers of iDCs or 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 pulsing the culture with [3H]TdR (0.5 μCi/well) for another 18 h before harvesting. [3H]TdR incorporation was measured with a microbeta counter (Wallac, Gaithersburg, MD).

Equilibrium binding was performed in triplicate by adding a constant amount of [3H]fMLP and increasing amounts of unlabeled fMLP to individual 1.5-ml microfuge tubes, each containing 2 × 106 DCs suspended in RPMI 1640 containing 1% BSA, 2.5 mM HEPES, 0.05% NaN3. After incubation at 24°C with constant mixing for 20 min, the cells were extensively washed with cold PBS, and the cell-associated radioactivity was measured with a microbeta counter (Wallac).

Total RNA from DCs was isolated by the use of TRIzol Reagent (Life Technologies). The RNAs were cleaned by treatment with RNase-free DNase I (Stratagene). RT-PCR was performed by the use of GeneAmp RNA PCR Kit (Roche Molecular Systems, Branchburg, NJ). Briefly, 100 ng of RNAs was used in the RT-PCR. After reverse transcription, C5a and GAPDH cDNA fragments were amplified by 30 cycles of PCR (denatured at 95°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 1 min) with the last extension being performed at 72°C for 10 min. The primers for human C5aR were 5′-CCCAAGCTTGGGGCGGGGAATCAATGAATTTCAGCGA-3′ and 5′-CCGCTCGAGCGGCTATCACATAGTGAAGGAGGACGCA-3′. The primers for human GAPDH were 5′-GATGACATCAAGAAGGTGGTGAA-3′ and 5′-GTCTTACTCCTTGGAGGCCATGT-3′. PCR products were identified on 1–2% agarose gel after ethidium bromide staining and photodocumented. Northern blot was performed as described elsewhere (40) with minor modification. Briefly, total RNA (20 μg/lane) was fractionated on 1% agarose-formaldehyde gel and transferred to a nitrocellulose filter. The specific mRNA on the filter was detected by hybridization with a 32P-labeled cDNA probe at 42°C overnight in buffer comprising 50% formamide, 5× SSPE (1× SSPE is 0.15 M NaCl, 10 mM NaH2PO4, 10 mM EDTA, pH 7.4), 5× Denhardt’s solution, 1% SDS, and 100 μg/ml denatured salmon sperm DNA. Two cDNA probes were used: a 1000-bp HindIII-EcoRI fragment of human FPR cDNA and a 400-bp β-actin cDNA fragment (41). The probes were labeled by a RadPrime DNA labeling kit (Life Technologies). After hybridization, the filters were washed with 2× SSC (1× SSC is 0.15 M NaCl, 15 mM sodium citrate, pH 7.0) containing 0.2% SDS and 0.1× SSC containing 0.1% SSC until a reasonably low background was obtained. The filter was dried and autoradiographed overnight at −80°C using a Kodak x-ray film (Rochester, NY).

To address the effect of maturation of DCs on their response to fMLP and C5a, we compared the chemotaxis and Ca2+ mobilization of iDCs and mDCs derived from human monocytes in response to fMLP and C5a. iDCs migrated to both fMLP and C5a with bell-shaped dose-response curves with optimal concentrations at 10−8 M and 10−9 M, respectively (Fig. 1, A and C), confirming the previous reports (5, 10). Both fMLP and C5a also induced intracellular Ca2+ mobilization by iDCs in a dose-dependent manner (Fig. 2, A and B, upper panels). After treatment of iDCs with rhTNF-α for 48 h to induce maturation, mDCs lost their responsiveness to fMLP as measured by chemotaxis (Fig. 1,B) and Ca2+ mobilization (Fig. 2,A, lower panel), but maintained responsiveness to C5a in terms of both chemotaxis (Fig. 1,D) and Ca2+ mobilization (Fig. 2,B, 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 (10−9 M) under similar experimental conditions (Fig. 1, C and D), and 2) it mobilized intracellular Ca2+ to a similar extent in concentrations ranging from 10−11 to 10−7 M (Fig. 2,B). The result that SDF-1α was chemotactic for both iDCs and mDCs (Fig. 1) is in accordance with previous reports (21, 27).

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-1α as a positive control. CM, Chemotactic medium, indicating spontaneous DC migration. The results are shown as the average (mean ± 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.

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-1α as a positive control. CM, Chemotactic medium, indicating spontaneous DC migration. The results are shown as the average (mean ± 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.

Close modal
FIGURE 2.

Ca2+ mobilization of monocyte-derived DCs. Arrows indicate the time points where stimulants were added at the final concentrations (M) as specified. A, fMLP-induced Ca2+ flux by iDCs (upper panel) and mDCs (lower panel). B, C5a-induced Ca2+ flux by iDCs (upper panel) and mDCs (lower panel). Three separate experiments showed almost identical results.

FIGURE 2.

Ca2+ mobilization of monocyte-derived DCs. Arrows indicate the time points where stimulants were added at the final concentrations (M) as specified. A, fMLP-induced Ca2+ flux by iDCs (upper panel) and mDCs (lower panel). B, C5a-induced Ca2+ flux by iDCs (upper panel) and mDCs (lower panel). Three separate experiments showed almost identical results.

Close modal

To ensure that monocyte-derived iDCs and mDCs used in this study show phenotypic characteristics of iDCs and mDCs, we examined their surface marker expression and capacity to stimulate allogeneic MLR. As outlined in Table I, iDCs were CD1a+, CD11a+, CD14, CD40low, CD83, CD86low, and HLA-DRmedium, whereas mDCs were CD1a+, CD11a+, CD14, CD40high, CD83+, CD86high, and HLA-DRhigh. In addition, iDCs were unable to stimulate allogeneic MLR whereas mDCs stimulated marked proliferation of allogeneic T cells, especially at DC:T ratios 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 and mDCs.

Table I.

Surface marker expression of monocyte-derived DCsa

MarkeriDCsmDCs
% PositiveMFIb% PositiveMFI
CD1a 67 ± 6 32 ± 16 75 ± 2 69 ± 19 
CD11a 91 ± 1 29 ± 2 92 ± 0.4 32 ± 0.7 
CD14 NDc N/A ND N/A 
CD40 93 ± 9 43 ± 10 98 ± 2 78 ± 18 
CD83 6 ± 6 2 ± 0.4 54 ± 9 9.4 ± 1 
CD86 57 ± 21 22 ± 1.4 85 ± 17 41 ± 3 
HLA-DR 99 ± 0.1 235 ± 70 100 ± 0.1 303 ± 80 
MarkeriDCsmDCs
% PositiveMFIb% PositiveMFI
CD1a 67 ± 6 32 ± 16 75 ± 2 69 ± 19 
CD11a 91 ± 1 29 ± 2 92 ± 0.4 32 ± 0.7 
CD14 NDc N/A ND N/A 
CD40 93 ± 9 43 ± 10 98 ± 2 78 ± 18 
CD83 6 ± 6 2 ± 0.4 54 ± 9 9.4 ± 1 
CD86 57 ± 21 22 ± 1.4 85 ± 17 41 ± 3 
HLA-DR 99 ± 0.1 235 ± 70 100 ± 0.1 303 ± 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 ± SD) of three separate experiments.

b

MFI, Median fluorescence intensity.

c

ND and N/A, Not detectable and not applicable, respectively.

Table II.

Stimulation of allogeneic MLR by human monocyte-derived DCsa

DC Maturation Stage[3H]TdR Incorporation (cpm/well)
DCs (no./well) aloneDCs (no./well) + T cells (105/well)
1001,00010,0001001,00010,000
iDCs 125 ± 26 99 ± 34 13 ± 2 116 ± 23 164 ± 33 102 ± 32 
mDCs 46 ± 19 71 ± 22 83 ± 16 91 ± 29 4,751 ± 203 36,231 ± 1,537 
DC Maturation Stage[3H]TdR Incorporation (cpm/well)
DCs (no./well) aloneDCs (no./well) + T cells (105/well)
1001,00010,0001001,00010,000
iDCs 125 ± 26 99 ± 34 13 ± 2 116 ± 23 164 ± 33 102 ± 32 
mDCs 46 ± 19 71 ± 22 83 ± 16 91 ± 29 4,751 ± 203 36,231 ± 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 μCi/10 μl/well) and cultured for another 18 h before cell harvest. The radioactivity (cpm) incorporated is shown as the average (mean ± SD) of six wells. The [3H]TdR incorporation for T cells cultured alone and in the presence of 5 μg/ml of phytohemagglutinin was 47 ± 18 and 105,945 ± 2,233 cpm, respectively.

In vivo, down-regulation of the response of DCs to fMLP may result from either down-regulation of FPR expression or homologous desensitization due to the presence of an excessive amount of formyl peptides in the environment, which can derive from either microbes or disrupted mitochondria (19, 42, 43). Although homologous desensitization is less likely to occur when DCs are induced to mature in an in vitro culture system, we examined the expression of FPR and C5aR by iDCs and mDCs to rule out this possibility. To this end, iDCs and mDCs were tested for binding to [3H]fMLP. iDCs specifically bound [3H]fMLP, and this was inhibited in a dose-dependent manner by unlabeled fMLP, whereas mDCs did not bind [3H]fMLP at all, suggesting that mDCs greatly decrease their surface FPR expression (Fig. 3,A). In contrast, both iDCs and mDCs expressed comparable amounts of C5aR on their surfaces as determined by FACS analysis after staining of the cells with FITC-conjugated anti-CD88 (Fig. 3,B). To determine whether TNF-α induces differential regulation of FPR and C5aR expression at the transcriptional or posttranscriptional level, the expression of FPR and C5aR mRNAs by iDCs and mDCs was further investigated. By Northern blot analysis, iDCs were shown to express FPR mRNA (Fig. 4,A, lane 1), whereas FPR mRNA expression by mDCs was undetectable (Fig. 4,A, lane 2). Reprobing of the same filter with a 32P-labeled actin cDNA fragment after stripping yielded bands of nearly identical intensity, confirming that equal amounts of RNAs were loaded in both lanes 1 and 2 (Fig. 4,A). As expected, both iDCs and mDCs expressed a similar level of C5aR mRNA as measured by RT-PCR using C5aR-specific primers (Fig. 4 B, lanes 1 and 2). These results indicate that DCs, upon maturation induced by TNF-α, down-regulate their FPR expression while maintaining their C5aR expression.

FIGURE 3.

Expression of FPR and C5aR by monocyte-derived DCs. A, Binding of [3H]fMLP by iDCs (•) and mDCs (○) in the presence of increasing concentrations of unlabeled fMLP. The results shown are the average (mean ± SD) of four separate experiments. B, FACS analysis of C5aR expression by iDCs (solid line) and mDCs (asterisk line). Dotted line indicates fluorescence of cells stained with control mouse IgG. One representative experiment of three is shown.

FIGURE 3.

Expression of FPR and C5aR by monocyte-derived DCs. A, Binding of [3H]fMLP by iDCs (•) and mDCs (○) in the presence of increasing concentrations of unlabeled fMLP. The results shown are the average (mean ± SD) of four separate experiments. B, FACS analysis of C5aR expression by iDCs (solid line) and mDCs (asterisk line). Dotted line indicates fluorescence of cells stained with control mouse IgG. One representative experiment of three is shown.

Close modal
FIGURE 4.

Expression of FPR and C5aR at mRNA level by monocyte-derived DCs. A, FPR mRNA expression by iDCs (lane 1) and mDCs (lane 2) as detected by Northern blot. The nitrocellulose filter was first probed with 32P-labeled FPR cDNA fragment, stripped, and reprobed with 32P-labeled actin cDNA fragment. B, RP-PCR products of C5aR (upper panel) and GAPDH (lower panel) displayed by agarose gel electrophoresis. The anticipated 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.

FIGURE 4.

Expression of FPR and C5aR at mRNA level by monocyte-derived DCs. A, FPR mRNA expression by iDCs (lane 1) and mDCs (lane 2) as detected by Northern blot. The nitrocellulose filter was first probed with 32P-labeled FPR cDNA fragment, stripped, and reprobed with 32P-labeled actin cDNA fragment. B, RP-PCR products of C5aR (upper panel) and GAPDH (lower panel) displayed by agarose gel electrophoresis. The anticipated 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.

Close modal

DC maturation can be induced in vitro by treatment of iDCs with a variety of agents including bacterial products (LPS, etc.) (14, 21, 23, 33), proinflammatory cytokines (TNF-α, IL-1, etc.) (14, 21, 23, 27, 28, 33), synthetic nucleic acids (CpG-oligodeoxynucleotides, poly(I:C), etc.) (44, 45), and macrophage-conditioned medium (34, 45), by cross-linking of membrane CD43 (46) or by CD40 ligation with CD40 ligand or anti-CD40 Ab (14, 21, 33). However, DC maturation induced by TNF-α and many other agents is considered reversible and only that induced by poly(I:C), macrophage-conditioned medium, or CD40 ligation is stable (21, 45, 47, 48). To determine whether stable maturation of DCs affects the regulation of FPR and C5aR expression, we prepared human mDCs by treatment of monocyte-derived iDCs with anti-CD40 agonistic Ab. mDCs generated in this manner showed phenotypic (CD40high, CD83+, CD86high, and HLA-DRhigh) and functional (capable of stimulating allogeneic MLR) characteristics similar to those of TNF-α-induced mDCs (data not shown). As shown by Fig. 5,A, mDCs induced by anti-CD40 Ab did not migrate to fMLP (•), yet they still migrated in response to C5a (▴). In agreement with the chemotaxis data (Fig. 5,A), iDCs expressed both FPR (Fig. 5,B, lane 1) and C5aR (Fig. 5,C, lane 1) mRNAs. Upon anti-CD40 Ab-induced maturation, mDCs down-regulated FPR mRNA (Fig. 5,B, lane 2) while maintaining a comparable level of C5aR mRNA expression (Fig. 5 C, lane 2). Therefore, DC maturation induced by either TNF-α or CD40 ligation results in selective down-regulation of FPR, but not C5aR, expression.

FIGURE 5.

Regulation of responsiveness to, and expression of receptors for, fMLP and C5a upon DC maturation induced by CD40 ligation. Immature DCs were generated from purified monocyte by incubation at 37°C for 7 days in the presence of GM-CSF and IL-4. The resulting iDCs were divided into two parts. One part was used for chemotaxis and RNA extraction and one part was incubated with anti-CD40 Ab at a concentration of 100 μg/ml for 48 h to induce DC maturation. mDCs were also used for chemotaxis and RNA extraction. A, Migration of iDCs (open symbols) 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, stripped, and reprobed with 32P-labeled actin cDNA fragment. C, RT-PCR products of C5aR (upper panel) and GAPDH (lower panel) displayed by agarose gel electrophoresis. The anticipated 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, anti-CD40-induced mDCs.

FIGURE 5.

Regulation of responsiveness to, and expression of receptors for, fMLP and C5a upon DC maturation induced by CD40 ligation. Immature DCs were generated from purified monocyte by incubation at 37°C for 7 days in the presence of GM-CSF and IL-4. The resulting iDCs were divided into two parts. One part was used for chemotaxis and RNA extraction and one part was incubated with anti-CD40 Ab at a concentration of 100 μg/ml for 48 h to induce DC maturation. mDCs were also used for chemotaxis and RNA extraction. A, Migration of iDCs (open symbols) 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, stripped, and reprobed with 32P-labeled actin cDNA fragment. C, RT-PCR products of C5aR (upper panel) and GAPDH (lower panel) displayed by agarose gel electrophoresis. The anticipated 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, anti-CD40-induced mDCs.

Close modal

Human DCs can be generated in vitro in a large number from either monocytes or CD34+ HPC (11, 12, 13, 14, 15, 21, 22, 24, 25, 26, 27, 28, 33, 34). To address whether HPC-derived DCs behave similarly to monocyte-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, CD83, CD86low, and HLA-DRmedium (Table III) and lacked the capacity to stimulate allogeneic MLR (Table IV), whereas HPC-derived mDCs were CD1ahigh, CD83+, 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. 6,A, ○) and C5a (Fig. 6,A, ▵). After TNF-α-induced maturation, CD34+ HPC-derived mDCs migrated toward C5a (Fig. 6,A, ▴), but not to fMLP (Fig. 6,A, •). Furthermore, CD34+ HPC-derived iDCs mobilized intracellular Ca2+ in response to both fMLP and C5a, whereas mDCs did so only in response to C5a, but not to fMLP (Fig. 6,B). Concomitantly, HPC-derived iDCs expressed both FPR (Fig. 6,C, left, lane 1) and C5aR (Fig. 6,C, right, lane 1) mRNAs, whereas mDCs maintained comparable levels of C5aR mRNA (Fig. 6,C, right, lane 2), but down-regulated FPR mRNA to an undetectable level (Fig. 6 C, left, lane 2). Thus, upon maturation, human HPC-derived DCs also differentially regulate their responsiveness to and receptor expression for fMLP and C5a.

Table III.

Surface marker expression of human HPC-derived DCsa

MarkeriDCsmDCs
% positiveMFIb% positiveMFI*
CD1a 30 ± 4 3 ± 0.3 80 ± 10 12 ± 5 
CD83 2 ± 1 1.5 ± 0.1 69 ± 1 46 ± 3 
CD86 26 ± 4 9 ± 1.5 77 ± 2 46 ± 3 
HLA-DR 94 ± 0.5 48 ± 4 97 ± 2 317 ± 10 
MarkeriDCsmDCs
% positiveMFIb% positiveMFI*
CD1a 30 ± 4 3 ± 0.3 80 ± 10 12 ± 5 
CD83 2 ± 1 1.5 ± 0.1 69 ± 1 46 ± 3 
CD86 26 ± 4 9 ± 1.5 77 ± 2 46 ± 3 
HLA-DR 94 ± 0.5 48 ± 4 97 ± 2 317 ± 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 ± SD) of two separate experiments.

b

MFI, Median fluorescence intensity.

Table IV.

Stimulation of allogeneic MLR by human HPC-derived DCsa

DC Maturation Stage[3H]TdR Incorporation (cpm/well)
DCs (no./well) aloneDCs (no./well) + T cells (105/well)
1001,00010,0001001,00010,000
iDCs 179 ± 27 113 ± 14 108 ± 10 62 ± 21 92 ± 15 206 ± 9 
mDCs 100 ± 18 58 ± 15 201 ± 8 83 ± 29 3,129 ± 565 22,265 ± 915 
DC Maturation Stage[3H]TdR Incorporation (cpm/well)
DCs (no./well) aloneDCs (no./well) + T cells (105/well)
1001,00010,0001001,00010,000
iDCs 179 ± 27 113 ± 14 108 ± 10 62 ± 21 92 ± 15 206 ± 9 
mDCs 100 ± 18 58 ± 15 201 ± 8 83 ± 29 3,129 ± 565 22,265 ± 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 μCi/10 μl/well) and cultured for another 18 h before cell harvest. The radioactivity (cpm) incorporated is shown as the average (mean ± SD) of six wells. The [3H]TdR incorporation for T cells cultured alone and in the presence of 5 μg/ml of phytohemagglutinin was 56 ± 21 and 97,291 ± 2,784 cpm, respectively.

FIGURE 6.

Regulation of responsiveness to, and expression of receptors for, fMLP and C5a upon maturation of CD34+ progenitor-derived DCs. iDCs and mDCs were generated from DC precursors amplified CD34+ progenitors as described in Materials and Methods. A, Migration of CD34+-derived iDCs (open symbols) and mDCs (closed symbols) induced by fMLP (circles) and C5a (triangles). The results are shown as the average (mean ± SD) of triplicated wells. Error bars were omitted for clarity. Similar results were obtained from three separate experiments. B, Ca2+ mobilization of CD34+-derived DCs. Shown are the peak heights induced by fMLP or C5a at the final concentrations (M) as specified on the right side. Two separate experiments yielded almost identical results. C, Expression of FPR and C5aR at mRNA level by CD34+-derived DCs. Left, FPR mRNA expression by iDCs (lane 1) and mDCs (lane 2) as detected by Northern blot. The nitrocellulose filter was first probed with 32P-labeled FPR cDNA fragment, stripped, and reprobed with 32P-labeled actin cDNA fragment. Right, RT-PCR products of C5aR (upper panel) and GAPDH (lower panel) displayed by agarose gel electrophoresis. The anticipated 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.

FIGURE 6.

Regulation of responsiveness to, and expression of receptors for, fMLP and C5a upon maturation of CD34+ progenitor-derived DCs. iDCs and mDCs were generated from DC precursors amplified CD34+ progenitors as described in Materials and Methods. A, Migration of CD34+-derived iDCs (open symbols) and mDCs (closed symbols) induced by fMLP (circles) and C5a (triangles). The results are shown as the average (mean ± SD) of triplicated wells. Error bars were omitted for clarity. Similar results were obtained from three separate experiments. B, Ca2+ mobilization of CD34+-derived DCs. Shown are the peak heights induced by fMLP or C5a at the final concentrations (M) as specified on the right side. Two separate experiments yielded almost identical results. C, Expression of FPR and C5aR at mRNA level by CD34+-derived DCs. Left, FPR mRNA expression by iDCs (lane 1) and mDCs (lane 2) as detected by Northern blot. The nitrocellulose filter was first probed with 32P-labeled FPR cDNA fragment, stripped, and reprobed with 32P-labeled actin cDNA fragment. Right, RT-PCR products of C5aR (upper panel) and GAPDH (lower panel) displayed by agarose gel electrophoresis. The anticipated 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.

Close modal

It has been reported that induction of iDC migration by fMLP and C5a is based on chemotaxis (5, 10, 36). To assure whether C5a-induced mDC migration is due to chemotaxis or chemokinesis, checkerboard analysis was performed. As shown by Table V, addition of C5a into the upper wells alone did not cause mDCs to migrate across the membrane (row 1), indicating that C5a did not increase chemokinesis of mDCs. Addition of C5a into the lower wells alone resulted in dose-dependent mDC migration (column 1). Moreover, when C5a was added into both the upper and lower wells, mDC migration was inhibited to various degrees, depending on C5a concentrations in the lower and upper wells (desensitization). Taken together, checkerboard analysis indicates that C5a-induced mDC migration is also based on chemotaxis.

Table V.

Checkerboard analysis of C5a-induced mDC migrationa

C5a in Lower Wells (nM)C5a in Upper Wells (nM)
00.1110
8 ± 1 9 ± 2 7 ± 1 7 ± 1 
0.1 25 ± 3 9 ± 2 7 ± 1 6 ± 2 
78 ± 9 24 ± 5 11 ± 2 7 ± 1 
10 30 ± 3 12 ± 4 8 ± 2 7 ± 2 
C5a in Lower Wells (nM)C5a in Upper Wells (nM)
00.1110
8 ± 1 9 ± 2 7 ± 1 7 ± 1 
0.1 25 ± 3 9 ± 2 7 ± 1 6 ± 2 
78 ± 9 24 ± 5 11 ± 2 7 ± 1 
10 30 ± 3 12 ± 4 8 ± 2 7 ± 2 
a

Human monocyte-derived mDCs were used at 5 × 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 ± SD) of migrated mDCs of triplicated wells (per high-power field). Two additional experiments yielded similar results.

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, 50, 51, 52). Unlike human iDCs, murine iDCs do not express CCR4 (49) and CCR6 (50), highlighting several differences between human and mouse DCs. This led us to investigate whether murine DCs respond similarly to maturation signals as do human DCs with particular regard to the regulation of responsiveness to fMLP and C5a. As demonstrated by Fig. 7, C5a induced the migration of both murine iDCs (▵) and mDCs (▴). However, fMLP was only chemotactic for murine iDCs (○), but not for murine mDCs (•), suggesting that maturation of murine DCs also differentially regulates their responsiveness to fMLP and C5a. The optimal chemotactic concentration of fMLP for murine iDCs was 10−6 M, which was 100-fold higher than that for human iDCs (10−8 M). This difference presumably reflects the different sensitivities of human and murine FPR to fMLP rather than the difference between human and murine iDCs because 1) to induce comparable level of intracellular Ca2+ mobilization in murine neutrophils, 100-fold or more fMLP is needed than with human neutrophils (53); 2) about 100-fold more fMLP is required to induce comparable level of intracellular Ca2+ mobilization in murine as compared with human FPR-expressing Xenopus oocytes (53); and 3) human embryonic kidney 293 cells expressing murine FPR migrate in response to fMLP with an optimal concentration of 10−6 M (54), whereas those expressing human FPR migrate in response to fMLP with an optimal concentration of 10−8 M (55).

FIGURE 7.

Chemotaxis of murine HPC-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. The results are shown as the average (mean ± SD) of triplicated wells. Spontaneous DC migration (chemotactic index = 1) was 40∼50 cells per high-power field. One experiment representative of four is shown.

FIGURE 7.

Chemotaxis of murine HPC-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. The results are shown as the average (mean ± SD) of triplicated wells. Spontaneous DC migration (chemotactic index = 1) was 40∼50 cells per high-power field. One experiment representative of four is shown.

Close modal

To execute their roles in initiating and modulating immune responses, iDCs need to migrate to sites of pathogen entry in the peripheral tissues to take up Ags, whereas mDCs have to migrate to lymphoid organs to transfer native Ag to naive B cells and to present processed Ag to naive T cells (1, 2, 3, 4, 5, 6, 7, 8, 9, 56, 57). Studies of the in vitro effect of chemotactic factors on DC (10, 11, 12, 13, 14, 15, 23, 26, 27, 30, 31, 32, 33, 34, 49, 50, 52) have increased our understanding of their in vivo roles in guiding DC trafficking (35, 51). Although human monocyte-derived DCs or DCs isolated from human skin and rat respiratory tract tissue have previously been shown to migrate in response to fMLP and/or C5a (5, 10, 36), the relationship between maturation of DC and their responsiveness to fMLP and C5a is not known. Our results showing that both human and murine iDCs generated from either monocytes or HPC migrated to fMLP and C5a suggest that formyl peptides and C5a may participate in the recruitment of iDCs to sites of infection. Although this needs to be confirmed by animal model studies, the following evidence supports this possibility. Formyl peptides can be released by invading microorganisms (42) or disrupted mitochondria due to microorganism-induced host cell damage (43). C5a can be produced as a result of complement activation through classical or alternative pathway at local or systemic inflammatory sites (58). Thus, gradients of formyl peptides and C5a are presumably formed at the sites of infection. Furthermore, pathogen inhalation induces a very rapid recruitment of DCs to the rat airway epithelium (4, 5). Formyl peptides and C5a may be at least in part involved in this 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-α 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-α for 24 h (36). Moreover, the differential regulation of mDCs responsiveness to fMLP and C5a paralleled the expression of FPR and C5aR. This differential regulation seems to be DC maturation-dependent rather than TNF-α-dependent because 1) DCs matured by CD40 ligation also exhibited the same pattern, and 2) TNF-α has been shown to promote, rather than suppress, FPR expression in human neutrophils (59). Because the expression of FPR mRNA was also down-regulated, it can be speculated that a reduction in either FPR mRNA stability and/or 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, 61, 62), however, the structure of their promoter regions is not well understood. Furthermore, how the expression of FPR and C5aR genes is controlled remains unknown. Therefore, how and why DC maturation results in differential regulation of FPR and C5aR expression needs further investigation.

DCs generated from CD34+ HPC are heterogeneous. At least two subsets of DCs, CD1a+ and CD1a, can be derived from CD34+ HPC-derived DCs (63). Although our results show that, similar to monocyte-derived DCs, human HPC-derived DCs also differentially down-regulate FPR, but not C5aR, expression upon maturation, whether different subsets of HPC-derived DCs behave similarly or differently in the course of maturation in terms of FPR and C5aR expression awaits further investigation.

Interaction of two chemokine receptors, CXCR4 and CCR7 that are known to be expressed by mDCs (14, 21, 23, 33, 34, 35), with their ligands, e.g., SDF-1α, secondary lymphoid chemokine (also known as 6Ckine, Exodus-2, or TCA4), and EBI1 ligand chemokine (also known as macrophage inflammatory protein-3β), is involved in the recruitment of mDCs to lymphoid tissues (35, 51, 64). This leads us to propose that the interaction of C5a and C5aR may also participate in the recruitment of mDCs to lymphoid tissues, specifically in guiding and/or sorting mDCs to B cell follicles, where naive B cells acquire native Ags delivered by mDCs (57, 65). Besides macrophages, B cells have recently been found to be a source of C5a in secondary lymphoid tissue (66). Follicular DCs, a particular type of DC localizing in B cell follicles, retain Ag-Ab complexes on their surfaces (67). Therefore, C5a may be generated via the classical pathway of complement activation triggered by follicular DC-bound immune complexes, thereby forming a C5a gradient. C5a is also a chemoattractant for B cells (66, 68). Thus, locally generated C5a gradient may attract both mDCs and naive B cells to B cell follicles to facilitate Ag transfer (56, 57, 65).

Collectively, our results suggest that the interaction of FPR with its ligands is possibly involved in the recruitment of iDCs whereas that of C5a and C5aR may participate in the recruitment of both iDCs and mDCs in vivo to distinct anatomical sites. The observation that murine DC responded to fMLP and C5a similarly to human DCs indicates that mouse models can be used to decipher the in vivo roles of fMLP and C5a in DC trafficking, particularly by the use of recently established FPR and C5aR knockout mice (69, 70).

We thank N. Dunlop for technical assistance and Dr. Ronald N. Germain, Chief of the Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, for helpful discussion and critical review of the manuscript. The support of the laboratory manager C. Fogle and secretarial assistance of C. Nolan is gratefully appreciated.

1

This work was supported in part by a fellowship from the Office of the International 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 O.M.Z.H.).

2

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, 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 nonexclusive, royalty-free license in and to any copyright covering the article.

5

Abbreviations used in this paper: DCs, dendritic cells; mDC, mature DC; iDC, immature DC; C5aR, C5a receptor; FPR, formyl peptide receptor; HPC, hemopoietic progenitor cells; rh, recombinant human; SDF-1α, stromal cell-derived factor 1α.

6

O. M. Z. Howard, H. F. Dong, J. Subleski, S. Strobl, A.-K. Shirakawa, J. J. Oppenheim, and E. L. Nelson. TARC and I-309 utilize CCR8 to induce chemotaxis of a CD83 subset of human monocyte-derived dendritic cells. Submitted for publication.

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