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(Journal of Leukocyte Biology. 2002;72:598-607.)
© 2002 by Society for Leukocyte Biology

Human dendritic cells express functional formyl peptide receptor-like-2 (FPRL2) throughout maturation

De Yang*, Qian Chen*, Barry Gertz*, Rong He{dagger}, Michele Phulsuksombati*, Richard D. Ye{dagger} and Joost J. Oppenheim*

* Laboratory of Molecular Immunoregulation, Center for Cancer Research, National Cancer Institute at Frederick, National Institutes of Health, Maryland; and
{dagger} Department of Pharmacology, College of Medicine, University of Illinois, Chicago

Correspondence: Dr. Joost J. Oppenheim, LMI, CCR, NCI at Frederick, Building 560, Room 21-89, Frederick, MD 21702-1201. E-mail: oppenhei{at}mail.ncifcrf.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Immature and mature dendritic cells (iDC and mDC, respectively) migrate to different anatomical sites, e.g., sites of antigen (Ag) deposition and secondary lymphoid organs, respectively, to fulfill their roles in the induction of primary, Ag-specific immune responses. The trafficking pattern of iDC and mDC is based on their expression of functional chemotactic receptors and the in vivo sites expressing the corresponding ligands including chemokines and/or classical chemoattractants. In this study, we have evaluated the expression of the formyl peptide receptor like-2 (FPRL2) by human iDC and mDC. We show that iDC respond chemotactically and by Ca2+ mobilization to N-formyl-Met-Leu-Phe and a recently identified synthetic peptide Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), whereas mDC derived from the same donor only respond to WKYMVm. Furthermore, iDC and mDC express FPRL2 mRNA and protein. As mDC do not express any other members of the human FPR subfamily, FPRL2 expressed by DC must be functional and mediate the effect of WKYMVm on DC. Indeed, treatment of iDC and mDC with WKYMVm induces the internalization of FPRL2. Thus, human myeloid DC express functional FPRL2 and maintain its expression even after maturation, suggesting that the interaction of FPRL2 and its endogenous ligand(s) may be involved in regulating DC trafficking during Ag uptake and processing in the periphery as well as the T cell-stimulating phase of the immune responses.

Key Words: chemotaxis • Ca2+ mobilization • maturation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) are professional antigen (Ag)-presenting cells that have the unique capacity to stimulate naïve T cells in order to initiate primary Ag-specific immune responses [1 2 3 ]. Myeloid DC precursors are generated from hematopoietic stem cells in the bone marrow and differentiate into immature DC (iDC), which infiltrate most nonlymphoid tissues. After Ag uptake and processing in the peripheral tissues, DC mature (mDC) and acquire the capacity to migrate to secondary lymphoid organs and to activate Ag-specific naïve T cells. Thus, iDC and mDC are endowed with the capacity to traffic to different anatomical sites.

Regulation of DC trafficking in vivo like trafficking of other leukocytes is influenced by many chemotactic factors and adhesion molecules [2 , 4 ]. However, the capacity of iDC and mDC to migrate to different anatomical sites is primarily determined by their differential expression of G-protein-coupled seven-transmembrane domain receptors (GPCRs) specific for chemokines and classical chemoattractants [1 , 2 , 4 , 5 ]. Of the 20 chemokine receptors identified so far, in vitro studies have shown that iDC express CXC chemokine receptor (CXCR)1, 2, 4 and CCR1–6 and are able to migrate in response to their respective ligands [5 6 7 ]. In contrast, mDC express only CXCR4 and CCR7 and thus are able to migrate, respectively, in response to stromal cell-derived factor-1/CXCL12 and CCR7 ligands including 6Ckine/secondary lymphoid chemokine/exodus-2/CCL21 and macrophage inflammatory protein-3ß/EBI1 ligand chemokine/exodus-3/CCL19 [2 , 5 , 8 ]. The in vivo contribution of CCR6 to iDC migration and that of CCR7 to mDC trafficking has been clearly demonstrated [9 10 11 ], indicating that in vitro investigation of the expression of chemotactic receptors by DC provides a useful step for defining their involvement in DC trafficking.

The receptors for classical chemoattractants may also regulate the trafficking of DC precursors and DC. Classical chemoattractant receptors include C3a and C5a receptors, platelet-activating factor receptor (PAFR), and the receptors for formyl peptides (FPR). Human monocyte-derived myeloid iDC and mDC express functional PAFR [12 ]. Human DC derived from monocytes or CD34+ progenitors express functional C5a receptor even after maturation [13 ], indicating the interaction of C5a and its receptor may regulate trafficking of iDC and mDC. Indeed, it has been demonstrated that C5a is required for the induction of contact sensitivity in mice [14 15 16 ]. The human FPR subfamily has three members including FPR and two additional homologues termed FPR-like 1 and 2 (FPRL1 and FPRL2) [17 ]. We have previously shown that human iDC express functional FPR, and its expression is down-regulated on mDC [13 ]. We have recently demonstrated that FPRL1 is down-regulated even as DC precursors differentiate into iDC [18 ]. Whether human DC express FPRL2 has not been reported.

Human FPR was cloned in 1990 [19 ] and uses the prototype formyl peptide N-formyl-Met-Leu-Phe (fMLP) as a high affinity agonist [17 , 20 21 22 ]. Human FPRL1 (also known as FPR2 or FPRH1) was cloned in 1992 by several independent groups [23 24 25 ]. Although sharing 69% amino acid identity to FPR, FPRL1 only has very low affinity for fMLP [17 , 24 ]. Subsequently, FPRL1 was reported to be a functional, high affinity receptor for lipoxin A4 and was given another name, LXA4R [26 ]. In recent years, a number of diverse peptides/proteins including MMK-1, a peptide isolated from a random peptide library [27 ], have been reported to act as FPRL1-selective agonistic ligands [28 29 30 31 ]. The hexapeptide Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), originally identified from a combinatorial peptide library by its capability to stimulate phosphoinositide hydrolysis in lymphocyte cell lines [32 ], is a potent leukocyte activator [33 34 35 ] and has been shown to act on human FPR and FPRL1 [36 , 37 ]. Human FPRL2, also cloned in 1992, shares 56% and 83% amino acid identity with FPR and FPRL1, respectively [23 , 25 ]. FPRL2 mRNA is expressed by monocytes, but not by neutrophils [38 ]. Cells transfected to overexpress FPRL2 have recently been shown to respond to WKYMVm [39 ]. However, it is not clear whether functional FPRL2 is expressed by primary cells.

In the course of investigating the regulation of FPR and FPRL1 during human myeloid DC differentiation and maturation [13 , 18 ], we observed that mDC, although unable to respond to the fMLP, could nevertheless migrate in response to WKYMVm. As mDC exhibited no functional FPR and FPRL1 expression [13 , 18 ], we speculated that mDC must express another receptor that enabled them to respond to WKYMVm. Searching for this receptor, we found that human myeloid DC express FPRL2 at mRNA and protein levels and maintain FPRL2 expression even after maturation. We also demonstrated that FPRL2 expressed by DC was functional and mediated the effect of WKYMVm on mDC. Thus, we propose that the interaction of FPRL2 with its unknown endogenous or exogenous ligand(s) may be involved in regulating trafficking of iDC to sites of Ag deposition and of mDC from peripheral tissues to secondary lymphoid organs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
RPMI 1640 was purchased from BioWhittaker (Walkersville, MD). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT). Recombinant human tumor necrosis factor {alpha} (rhTNF-{alpha}; sp. Act.=2x107 U/mg), rh granulocyte macrophage-colony stimulating factor (GM-CSF; sp. Act.=107 U/mg), and rh interleukin (IL)-4 (sp. Act.=2x106 U/mg) were purchased from PeproTech (Rocky Hill, NJ). Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody, synthetic fMLP, Escherichia coli lipopolysaccharide (LPS; serotype 026:B5), and pertussis toxin (PTX) were purchased from Sigma Chemical Co. (St. Louis, MO). The other antibodies used for flow cytometry were purchased from BD PharMingen (San Diego, CA). Polyclonal anti-FPRL1 and 2 antiserum was raised in a rabbit against a synthetic peptide AANSASPPAETELQAM, which corresponds to the carboxyl terminal 16 amino acid residues of FPRL1 [23 , 24 ]. As a result of the high homology of the carboxyl terminus of FPRL1 with that of FPRL2 (12 out of the last 16 and 9 out of the last 10 amino acids are identical) [17 , 23 24 25 ], the antiserum generated was found to cross-react with the corresponding carboxyl terminus of FPRL2 by flow cytometry analysis (R. He and R. D. Ye, unpublished results). MMK-1, an FPRL1-specific ligand of 13 amino acid (LESIFRSLLFRVM) [27 ], and the hexapeptide WKYMVm (m represents a D-Met residue) [32 ] were synthesized and purified by the Department of Biochemistry, Colorado State University (Fort Collins). The purity for both peptides was more than 90%, and the amino acid composition was verified by mass spectrometry.

Isolation of human peripheral blood monocytes and DC culture
Human peripheral blood mononuclear cells (PBMC) were isolated from leukopacks (Courtesy of the Transfusion Medicine Department, NIH Clinic Center, Bethesda, MD) by routine Ficoll-Hypaque density gradient centrifugation. Monocytes were purified from human PBMC with the use of the magnetic cell sorter CD14 monocyte isolation kit (Miltenyi Biotech Inc., Auburn, CA), according to the manufacturer’s recommendation. The purity of monocytes was checked by FACScan analysis. Monocyte preparation with purity less than 95% was not used. The generation of DC from monocytes was carried out as described previously [13 , 18 ]. Briefly, monocytes were differentiated to iDC by incubating at 1 x 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 (50 ng/ml) at 37°C in a humidified CO2 (5%) incubator for 7 days. To induce DC maturation, iDC were cultured in the same cytokine cocktails with added rhTNF-{alpha} (50 ng/ml) or LPS (100 ng/ml) for 48 h at 37°C in a humidified CO2 (5%) incubator. As confirmed by flow cytometry analysis, iDC were CD1a+, CD14-, CD40low, CD83-, CD86low, human leukocyte Ag (HLA)-DRmedium, whereas mDC were CD1a+, CD14-, CD40high, CD83+, CD86high, and HLA-DRhigh (data not shown; and refs. [13 , 18 ]).

Chemotaxis assay
Migration of monocytes and DC in response to chemotactic factors was assessed using a 48-well microchemotaxis chamber technique as previously described [6 , 13 ]. Briefly, different concentrations of chemotactic factors were placed in triplicates in the wells of the lower compartment of the chamber (Neuro Probe, Cabin John, MD), and cells (106 cells/ml) were added to wells of the upper compartment. In some experiments, various concentrations of chemotactic factor were also included in the wells of the upper compartment. The lower compartment was separated from the upper compartment 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 chemotactic index (C.I.), which is defined as the "fold" increase in the number of migrating cells in the presence of test factors over the spontaneous cell migration (in the absence of test factors). The statistical significance of the increase in cell migration was determined by an unpaired t-test.

Measurement of calcium flux
Monocytes and DC (107 cells/ml in RPMI 1640 containing 10% FBS) were loaded by incubating with 5 µM Fura-2 (Molecular Probes, Eugene, OR) at 24°C for 30 min in the dark. Subsequently, the cells were washed with 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% bovine serum albumin (BSA), pH 7.4]. Each 2 ml cell suspension was then transferred into a quartz cuvette, which was placed in a luminescence spectrometer LS50 B (Perkin-Elmer Limited, Beaconsfield, UK). 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. In some experiments, loaded cells were treated with PTX (final concentration of 200 ng/ml) at 37°C for 30 min and were washed twice with saline buffer before measuring the calcium flux of the cells in response to chemotactic factors.

Flow cytometry
DC (106/sample) were washed three times with phosphate-buffered saline (PBS) and fixed for 10 min at room temperature in 3.7% paraformaldehyde (W/V in PBS). Subsequently, the cells were permeabilized/blocked by treatment with permeabilizing/blocking (P/B) buffer (PBS containing 0.1% saponin, 1% BSA, and 10 µg/ml human IgG). Thereafter, the cells were stained with rabbit preimmune serum or rabbit anti-FPRL1 and 2 immune serum (both at 1:400 dilution with P/B buffer) at room temperature for 1 h. After washing three times with P/B buffer, the cells were further stained with FITC-conjugated goat anti-rabbit IgG (1:40 dilution with P/B buffer) for 30 min at room temperature. The stained cells were washed twice with P/B buffer, twice with PBS, fixed with 1% paraformaldehyde in PBS at 4°C overnight, and analyzed the next day with a flow cytometer (Coulter Epics®, Miami, FL).

RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA from monocytes, DC, and macrophages was isolated by the use of TRIzol® Reagent (Life Technologies, Grand Island, NY). The RNAs were cleaned by treatment with RNase-free DNase I (Stratagene, La Jolla, CA). RT-PCR was performed by the use of the ProSTARTM HF single-tube RT-PCR system (Stratagene). Briefly, 5–50 ng total RNA was used in the RT-PCR reaction. After reverse transcription at 37°C for 15 min and inactivation of Moloney murine leukemia virus reverse transcriptase at 95°C for 1 min, FPR, FPRL2, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA fragments were amplified by 40 cycles of PCR (denature at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 68°C for 2 min) reaction, and the last extension was performed at 68°C for 10 min. The sense and antisense primers for FPR were 5'-CTCCAGTTGGACTAGCCACA-3' (nucleotides 1639~1658 in the coding region of exon 2) and 5'-CCATCACCCAGGGCCCAATG-3' (nucleotides 5341~5359 in the coding region of exon 3), respectively, which resulted in the amplification of a 500-bp FPR-specific cDNA fragment as previously reported [40 ]. The sense and antisense primers for FPRL2 were 5'-GCCAAGGTCTTTCTGATCC-3' (nucleotides 592~610 as counted from the starting position of the open reading frame) and 5'-GGTCTGGGCTGAGTCAGGGA-3' (nucleotides 977~996), which enabled the amplification of a 404-bp FPRL2-specific cDNA fragment as verified by sequencing. The primers for FPRL1 were 5'-CTGCTGGTGCTGCTGGCAAG-3' and 5'-AATATCCCTGACCCCATCCTCA-3', which, as previously shown, enabled the amplification of a 1.1-kb FPRL1-specific cDNA fragment [18 ]. The primers for human GAPDH were 5'-GATGACATCAAGAAGGTGGTGAA-3' and 5'-GTCTTACTCCTTGGAGGCCATGT-3', which resulted in the amplification of a fragment of 246 bp as previously described [13 , 18 ]. PCR products were identified on 2% agarose gel, stained with ethidium bromide, and photodocumented after washing off excessive dye with water.

Confocal microscopy
iDC and mDC were seeded into gelatin-coated, glass-bottom, 35-mm dishes (MatTek Corp., Ahland, MA; Cat. No. P35GC-0-14-C) and were incubated in appropriate medium at 37°C for 24 h prior to the experiments. Cycloheximide was added to a final concentration of 100 µg/ml during the last 3 h of incubation to stop protein synthesis. Twenty minutes before the end of the incubation period, WKYMVm was added to some dishes to a final concentration of 10 µM to induce receptor internalization. The cells were then rinsed three times with ice-cold PBS to remove ligand and FCS and were fixed in 3.7% paraformaldehyde at room temperature for 10 min. After three rinses with PBS, the cells were permeabilized with PBS containing 0.1% saponin and 0.02% Nonidet P-40 at room temperature for 30 min. The permeabilized cells were sequentially stained with the primary polyclonal antibody (rabbit preimmune serum or anti-FPRL1 and 2; both were diluted by 1:500 in PBS containing 0.1% saponin and 10 µg/ml human IgG) and the secondary FITC-labeled goat anti-rabbit IgG (at 1:100 dilution). The stained cells were extensively washed, air-dried, and covered with several drops of Gel/MountTM (Biomeda Corp., Foster City, CA). A confocal laser-activated scanning microscope (Carl Zeiss 410) was used for the microscopic analysis of stained cells. FITC-stained cells were excited at 488 nm, and the fluorescence emitted between 510 nm and 565 nm was collected.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
WKYMVm, but not fMLP or MMK-1, induces chemotaxis of mDC
MMK-1 and fMLP are selective high affinity ligands for FPRL1 and FPR, respectively [24 , 27 ], whereas WKYMVm has been reported to act on FPR and FPRL1 [36 , 37 ]. When the migration of monocytes (DC precursors), iDC, and mDC toward all of these three ligands was examined, an interesting pattern appeared (Fig. 1 ). As expected, monocytes migrated in response to these three ligands (Fig. 1 , upper left panel). iDC migrated in response to fMLP and WKYMVm, but not to MMK-1 (Fig. 1 , lower left panel), a result compatible with our previous report that iDC express no functional FPRL1 [18 ]. To investigate whether maturation of DC affects their responsiveness to fMLP, MMK-1, and WKYMVm, DC were matured in the presence of TNF-{alpha} and examined. Interestingly, mDC, although unable to migrate to fMLP or MMK-1, nevertheless migrated in response to WKYMVm (Fig. 1 , upper right panel). Almost identical results were obtained when mDC generated in the presence of LPS instead of TNF-{alpha} were used for the chemotaxis (Fig. 1 , lower right panel). Thus, mDC generated in the presence of TNF-{alpha} were used in subsequent experiments.



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  		Figure 1. Migration of monocytes and monocyte-derived DC in response to fMLP, MMK-1, and WKYMVm. Human peripheral blood monocytes were isolated from a single donor and incubated in the presence of rhGM-CSF and rhIL-4 for 7 days in humidified air containing 5% CO2 to generate iDC. mDC were generated by culturing iDC in the presence of rhGM-CSF, rhIL-4, and rhTNF-{alpha} or LPS for an additional 2 days. The migration of monocytes, iDC, and mDC in response to various concentrations of fMLP, MMK-1, and WKYMVm was tested by the use of 48-well chemotaxis chambers, and the results are shown as the average C. I. (mean±SD) of triplicated wells. The spontaneous migration (in the absence of chemotactic factors) for monocytes and DC was 30~50 cells and 50~70 cells per high-powered field, respectively. Similar results were obtained using cells of more than five individual donors.

 
To address whether WKYMVm-induced migration of mDC resulted from chemotaxis or chemokinesis, checkerboard analysis was performed, and the results are summarized in Figure 2 . WKYMVm dose-dependently induced the migration of mDC when added in the lower wells of the chemotaxis chamber. However, increasing concentration of WKYMVm added simultaneously with the cells to the upper wells of the chamber was not only unable to induce directional mDC migration, but also dose-dependently abrogated mDC migration induced by WKYMVm in the lower wells. Thus WKYMVm-induced migration of mDC was based on chemotaxis rather than chemokinesis.



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  		Figure 2. Checkerboard analysis of WKYMVm-induced mDC migration. Various concentrations of WKYMVm were added to the lower wells, and mDC in the absence or presence of various concentrations of WKYMVm, were added to the upper wells of a chamber. After a 90-min incubation at 37°C, the membrane was removed, scraped, stained, and dried. The migration of mDC to the lower surface of the membrane was determined and shown as the average C. I. (mean±SD) of triplicated wells. Two separate experiments showed almost identical results.

 
WKYMVm, but not fMLP or MMK-1, can mobilize Ca2+ in mDC
The capacity of fMLP, MMK-1, or WKYMVm to induce Ca2+ mobilization in monocytes, iDC, and mDC was also examined and compared (Fig. 3 ). In agreement to chemotaxis data shown in Figure 1 , although MMK-1 induced Ca2+ flux in monocytes, and fMLP did so in monocytes and iDC, WKYMVm was able to induce Ca2+ flux in monocytes, iDC, and mDC. Thus, only WKYMVm could activate mDC. Furthermore, at a final concentration of 10-9 M, WKYMVm induced considerable Ca2+ flux in monocytes, an appreciable degree of Ca2+ flux in iDC, but no Ca2+ flux in mDC. At a concentration of 10-8 M, WKYMVm also induced a significant degree of Ca2+ flux in mDC. Therefore, monocytes, iDC, and mDC differ in sensitivity to WKYMVm-mobilized Ca2+ signal, and monocytes > iDC > mDC. As mDC express neither FPR nor FPRL1 [13 , 18 ], we hypothesized that WKYMVm must also act as an agonistic ligand on a receptor other than FPR or FPRL1.



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  		Figure 3. Ca2+ mobilization of monocytes and monocyte-derived DC in response to fMLP, MMK-1, and WKYMVm. Monocytes and monocyte-derived iDC and mDC were from the same donors. Stimulants (fMLP, MMK-1, and WKYMVm) were added 36 s after starting the recording at the final concentration (M) as specified. One representative experiment out of three is shown.

 
The effect of WKYMVm on mDC is mediated by a G-protein-coupled receptor
As WKYMVm can act on FPR and FPRL1, both of which are GPCRs, we further investigated whether the putative receptor that enabled mDC to respond to WKYMVm was also a G-protein-coupled receptor by determining whether the effect of WKYMVm on mDC could be inhibited by PTX, a toxin that specifically inhibits G-protein-coupled receptor signaling by adenosine 5'-diphosphate-ribosylating Gi protein [41 ]. As shown in Figure 4 A , incubation of mDC at 37°C for 30 min in the presence of PTX (200 ng/ml) prior to chemotaxis assay completely inhibited the migration of mDC in response to WKYMVm (hatched bar). The inhibition was not a result of the preincubation per se, as incubation of mDC similarly in the absence of PTX did not inhibit mDC migration in response to WKYMVm (Fig. 4A , dotted bar). Similarly, WKYMVm could not induce Ca2+ mobilization in PTX-treated mDC, although sham-treated mDC mobilized Ca2+ in response to WKYMVm (Fig. 4B) .



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  		Figure 4. Inhibition by PTX of the effect of WKYMVm on mDC. (A) Chemotaxis. mDC were treated in the absence (dotted bar) or presence (hatched bar) of PTX at 200 ng/ml at 37°C for 30 min before performing chemotaxis assay. *, P < 0.05; **, P < 0.01. (B) Ca2+ flux. Fura-2-loaded mDC were incubated with or without 200 ng/ml PTX (PTX+ and PTX-, respectively), washed once with saline buffer, and examined for their Ca2+ mobilization in response to WKYMVm. Arrows indicate the time points where WKYMVm were added at the final concentrations as specified. PTX treatment of DC did not decrease their viability (data not shown) or motility, as the random migration of treated mDC was similar to untreated cells [the first two bars (A)]. Two separate experiments showed almost identical results.

 
iDC and mDC express FPRL2 at mRNA and protein levels
We hypothesized that FPRL2 might be the receptor that mDC used to respond to WKYMVm. Several pieces of evidence supported this hypothesis including: 1) The orphan receptor FPRL2 is structually a seven-transmembrane domain GPCR and has high homology with FPR and FPRL1 at the amino acid level (56% and 83%, respectively) [17 , 23 , 24 ]; 2) WKYMVm acts on FPR and FPRL1 with a preference for FPRL1 [36 , 37 ]; 3) FPRL2 is expressed by monocytes [38 ], and its expression might be preserved during the differentiation and maturation of monocyte-derived DC; and 4) Christophe et al. [39 ] recently reported that WKYMVm can activate FPRL2-transfected cell lines. To test this hypothesis, we investigated whether FPRL2 mRNA was transcribed by DC. Total RNAs were isolated from iDC and mDC derived from the same donor, and an identical amount of total RNA was used to amplify FPRL2 and GAPDH by RT-PCR with the use of gene-specific primer pairs (Fig. 5 A ). Similar FPRL2 cDNA bands of expected size (404 bp) were amplified from iDC and mDC total RNAs, indicating that iDC and mDC expressed FPRL2 at mRNA level (Fig. 6 , middle panel). Amplification of identical GAPDH cDNA bands of 246 bp from 5 ng iDC and mDC total RNAs (lanes 2 and 4, respectively) confirmed that indeed equal amounts of RNAs were used (Fig. 5A , bottom panel). In full agreement with our previous Northern blot analysis [13 ] as well as data shown in Figures 1 and 3 , RT-PCR with FPR-specific primers revealed that FPR mRNA was only expressed by iDC, but not by mDC (Fig. 5A , top panel). The lack of FPRL1 expression by iDC and mDC [18 ] was confirmed by RT-PCR (Fig. 5B) .



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  		Figure 5. Expression of FPRL2 mRNA by iDC and mDC. Total RNA was isolated from iDC and mDC derived from the same donor. (A) Total iDC or mDC RNAs (50 or 5 ng) were used for the RT-PCR amplification. RT-PCR products of FPR (top), FPRL2 (middle), and GAPDH (bottom) were displayed by agarose gel (2%) electrophoresis. The marker used was a 50-bp DNA ladder. (B) Each 5 ng total RNA derived from monocytes (used as a positive control), iDC, and mDC was used for the amplification of FPRL1 and GAPDH by RT-PCR. The products of FPRL1 (top) and GAPDH (bottom) were displayed similarly as in Figure 1A . The marker used was a 1-kb DNA ladder. The anticipated size for FPR, FPRL1, FPRL2, and GAPDH was 500-, 1100-, 404-, and 246-bp, respectively. One representative experiment out of three is shown.

 


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  		Figure 6. FPRL2 protein expression by iDC and mDC. iDC and mDC were generated from monocytes isolated from the same donor. Aliquots of 106 iDC or mDC were permeabilized and stained with rabbit preimmune serum (open area) or anti-FPRL1 and 2 serum (solid area). After subsequent staining with FITC-conjugated goat anti-rabbit IgG, the cells were analyzed with a flow cytometer. Shown are the overlay histograms for iDC (left panel) and mDC (right panel). Two separate experiments showed almost identical results.

 
We further investigated whether DC expressed FPRL2 at the protein level by flow cytometry. A polyclonal rabbit anti-FPRL1 and 2 antiserum, which recognizes the carboxyl terminal of human FPRL1 and FPRL2, was used to stain permeabilized iDC and mDC. For the negative control, preimmune serum from the same rabbit was used. We reasoned that as iDC and mDC have no FPRL1 expression (Figs. 1 and 3 ; and ref. [18 ]), if DC were positively stained, it must be a result of FPRL2. After staining with FITC-conjugated goat anti-rabbit IgG as the secondary antibody, the samples were analyzed. As demonstrated in Figure 6 , iDC (left panel) and mDC (right panel) exhibited increased fluorescence when stained with anti-FPRL1,2 (solid area) than when stained with preimmune serum (open area), indicating iDC and mDC expressed FPRL2 protein.

FPRL2 expressed on DC is functional
To conclude that the FPRL2 expressed on mDC is the receptor that mediates WKYMVm-induced mDC activation, direct evidence that FPRL2 on mDC can transduce signal from WKYMVm is essential. We therefore explored whether treatment of mDC with WKYMVm could induce the internalization of FPRL2, one of the characteristic responses common to all Gi protein-coupled chemotactic receptors upon agonist-induced activation [42 ]. To do so, iDC and mDC were generated from the same population of precursors, treated at 37°C for 20 min in the absence (sham) or presence of 10 µM WKYMVm, and immunostained for confocal microscopic analysis. Although FPRL2-specific antibody has not been available, we used the anti-FPRL1 and 2 antiserum to detect FPRL2 in DC, based on the fact that iDC and mDC do not express FPRL1 (Figs. 1 3 and 5 ; and ref. [18 ]). In iDC and mDC treated in the absense of WKYMVm (sham), staining with anti-FPRL1 and 2 revealed that FPRL2 was predominantly distributed at or close to the cell surface, as illustrated by the bright fluorescent staining in the cell periphery (Fig. 7 , left panels). The FPRL2 was unevenly distributed since a punctated fluorescence at or underneath the cell membrane was observed. In about 10% of the cells (iDC and mDC), fluorescent staining was visualized within the cytoplasm (e.g., white arrow). Why FPRL2 is not evenly distributed at the cell surface in DC is not clear, but a similar pattern was also observed in FPRL2-transfected cells [39 ]. Importantly, treatment of iDC and mDC with WKYMVm resulted in the accumulation of bright fluorescent staining predominantly in the cytoplasm (red arrows), albeit the membrane of some cells was still stained (Fig. 7 , right panels). iDC and mDC stained with rabbit preimmune serum showed very faint green fluorescence (data not shown), indicating that the bright fluorescence observed in Figure 7 was specifically a result of anti-FPRL1 and 2 antibody rather than nonspecific binding of rabbit IgG in the antiserum used. Thus, WKYMVm was able to trigger considerable FPRL2 internalization in iDC and mDC, suggesting that FPRL2 expressed by DC is functional.



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  		Figure 7. WKYMVm-induced internalization of FPRL2 in iDC and mDC. iDC and mDC generated from monocytes isolated from the same donor were treated first at 37°C with cycloheximide (100 µg/ml) for 3 h and subsequently at 37°C for 20 min in the absence or presence of 10 µM WKYMVm (sham- and WKYMVm-treated, respectively). Thereafter, the cells were fixed, permeabilized, and sequentially stained with primary antibody (rabbit preimmune serum or anti-FPRL1 and 2 antiserum) and FITC-conjugated secondary antibody for confocal microscopy. Shown are the images of the medial plane of cells sham-treated or treated with WKYMVm. The optical section thickness was calculated to be 0.69 µm. DC stained with preimmune serum, as the primary antibody showed extremely faint green fluorescence (data not shown). The white arrow in the left lower panel indicates FPRL2 fluorescence inside a mDC. The red arrows point to examples that FPRL2 fluorescence was accumulated inside iDC and mDC. One representative experiment out of three is shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
FPR, FPRL1, and FPRL2, three members of the human FPR subfamily, were cloned in the early 1990s [19 , 23 24 25 ]. FPR and FPRL1 have long been shown to be the high and low affinity receptors for the prototype chemoattactant peptide fMLP, respectively [20 21 22 , 24 ]. In recent years, a number of FPRL1-specific ligands including MKK-1 have been identified [27 28 29 30 31 ]. FPRL2, however, has long been considered an orphan monocyte-expressed GPCR since its mRNA is selectively expressed by monocytes, but not by neutrophils [38 ], and no agonistic ligand(s) has been identified until recently [39 ]. We have demonstrated in the present study that iDC and mDC generated from purified peripheral blood monocytes express FPRL2 mRNA and protein, and FPRL2 acts as a functional receptor. To the best of our knowledge, this is the first indication that WKYMVm is an agonistic ligand for FPRL2 expressed on primary human cells, iDC and mDC express functional FPRL2, and only FPRL2, but not other members of the FPR family, is expressed in mDC.

Peripheral blood monocytes are the myeloid DC precursors that can be differentiated into DC in vitro, either by treatment with appropriate cytokines [43 , 44 ] or by incubation with endothelial cells grown on a collagen matrix [45 ], and in vivo [46 ]. Although it has not been directly demonstrated as a result of the unavailability of FPRL2-specific agonistic ligand or FPRL2-specific antibody, several lines of evidence indicate that monocytes, the precursors of myeloid DC, presumably also express functional FPRL2. First, monocytes express FPRL2 at mRNA level [38 ]. Secondly, FPRL2 expressed by monocyte-derived DC is functional. Finally, comparison of the Ca2+ mobilization signals induced by WKYMVm in monocytes, iDC, and mDC (Fig. 3) indicates that monocytes are most sensitive. This is presumably a result of the expression by monocytes of all three WKYMVm-responsive receptors, including FPR, FPRL1, and FPRL2. Thus, functional FPRL2 is expressed by human DC precursors (monocytes) and iDC, and its expression is maintained even after DC maturation.

Based on the results of our previous [13 , 18 ] and present studies, the functional expression of the three members of the human FPR subfamily can be summarized as in Table 1 . As the interaction of chemotactic receptor(s) on a particular leukocyte with corresponding ligand(s) regulates the trafficking of that cell [2 , 4 , 8 , 17 ], it can thus be hypothesized that all of the three receptors are differentially involved in the homing of DC precursors (monocytes), iDC, and mDC. Indeed, several previous studies suggest that the migration of monocytes to sites of inflammation and of iDC to sites of Ag deposition is regulated by FPR agonists [17 , 47 48 49 ]. The lack of FPRL1 expression by DC indicates that the interaction of FPRL1 with its endogenous ligands predominantly contributes to the recruitment of neutrophils, monocytes, and macrophages [18 , 36 , 38 ]. In addition to FPRL2, several other G-protein-coupled chemotactic receptors are expressed by mDC, including CCR7, CXCR4, C5a receptor, and PAFR [5 , 12 , 13 , 50 ]. The contribution of CCR7 and CXCR4 in regulating mDC trafficking has been clearly demonstrated [10 , 11 , 51 ]. C5a receptor may also contribute to mDC homing to regional lymph nodes, as mice lacking C5a or C5a receptor exhibit impaired contact sensitivity reaction [14 15 16 ]. Although mDC express only FPRL2 but not FPR or FPRL1, whether the interaction of FPRL2 on mDC with its endogenous ligand(s) may also contribute to regulating trafficking of mDC from peripheral tissues to secondary lymphoid organs remains to be investigated. The mouse as well as the human FPR subfamily is relatively well-characterized. In contrast to humans, the mouse FPR subfamily has six members [52 53 54 ]. Mouse FPRs share 77% amino acid identity with human FPRs and also use fMLP as an agonistic ligand [52 , 53 ]. Mouse FPRL1 is one ortholog for human FPRL1 that shares 74% amino acid identity and like human FPRL1, interacts with fMLP with low affinity [24 , 55 ] but acts as a high affinity receptor for LXA4 [26 , 56 ]. The gene product of Fpr-rs2 [52 ], recently named as mouse FPR2 [53 ], shares high homology with mouse FPRL1 (81% amino acid identity) and uses serum amyloid A (SAA) as an agonist [57 ]. Therefore, the functions of mouse FPRL1 and FPR2 are similar to that of human FPRL1. However, the mouse ortholog(s) for human FPRL2 has not been characterized.


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  		Table 1. Functional Expression of FPR, FPRL1, and FPRL2 During the Differentiation and Maturation of Human Myeloid DC

 
It is interesting that FPR, FPRL1, and FPRL2, the three closely related receptors of the same subfamily [17 ], are differentially regulated during the differentiation and maturation of human myeloid DC (Table 1) . How this differential regulation occurs is unknown, partly because the regulatory elements in the promoter regions of FPR, FPRL1, and FPRL2 genes have not been well studied; the significance of the differential regulation is not clear at this time either. However, analyzing the natural and/or endogenous ligands for FPR, FPRL1, and FPRL2 may provide some clues, as the initiation of cell migration also depends on the availability of corresponding agonistic ligands. The natural ligands for FPR are probably fMLP and N-formulated peptides derived from bacteria [17 , 24 , 58 , 59 ], albeit some synthetic peptides can also activate this receptor [36 ]. The N-terminal domain of annexin has been reported to interact with human FPR and was proposed as an endogenous ligand for FPR [60 ]. The expression of annexin I (presumably also the generation of its N-terminal domain peptides) increases in response to glucocorticoids [61 , 62 ], which are available during (especially in the late phase of) inflammation.

Thus, FPR may have evolved to sense prokaryotic microorganism-derived peptides (such as fMLP) to recruit FPR-expressing cells including iDC to sites of bacterial entry in the initial phase of infection and to receive annexin I signal to suppress the accumulation of FPR-expressing cells in the late phase of inflammation. Of about one dozen of the agonistic ligands identified so far for FPRL1 [18 , 24 , 26 27 28 29 30 31 , 36 , 37 , 63 64 65 66 ], six ligands are natural/endogenous, including LXA4 [26 , 63 ], SAA [28 ], LL-37, the cleaved carboxyl terminal domain of human antimicrobial protein cathelicidin/hCAP18 [31 ], the mitochondrial necrotactic peptide (MYFINILTL) derived from reduced nicotinamide adenine dinucleotide dehydrogenase subunit 1 [30 ], amyloid ß1-42 [65 ], and a cellular prion peptide fragment PrP106–126 [66 ]. These endogenous ligands are generated predominantly in association with inflammation and/or tissue injury [30 , 56 , 63 , 67 68 69 70 71 ]. The lack of FPRL1 expression by DC is noteworthy and may indicate that DC activation through FPRL1 in circumstances where endogenous FPRL1 ligands are generated could be potentially inimical to the host (e.g., by initiating unwanted autoimmune responses). Furthermore, the interaction of FPRL1 with its endogenous ligands may have a negative feedback-inhibitory effect on FPRL1-positive cells, especially when produced systemically in large amounts such as during severe infection. For example, LXA4 has been reported to act as a potent inhibitor of acute inflammation by suppressing CD11/18 expression and chemokine production of FPRL1-positive cells [56 , 63 , 72 ]. In addition, serum amyloid is able to inhibit the oxidative burst response of neutrophils as well as cell adhesion [73 , 74 ]. Scrapie prion protein has also been reported to inhibit neutrophil functions [75 ]. Thus, it would be counterproductive for DC to sense these ligands, as DC should not be inhibited even during severe systemic inflammation to perform Ag uptake, processing, and presentation. Although no endogenous and/or natural ligand(s) for FPRL2 has been identified as yet, they are presumably to be generated along the trafficking routes of DC precursors and DC.

In brief, we have demonstrated that FPRL2 is expressed by primary human iDC and mDC and suggest that the interaction of FPRL2 with its natural endogenous ligand(s) may participate in regulating DC trafficking. Direct investigation of the function of FPRL2, in particular its involvement in regulating DC trafficking, will become more feasible when the FPRL2-specific neutralizing antibody becomes available, the natural and/or endogenous agonist(s) for FPRL2 is identified, and the mouse ortholog(s) for human FPRL2 has been characterized.


   ACKNOWLEDGEMENTS
 
We thank N. Dunlop for assistance in isolating peripheral blood PBMC and Dr. E. Cho for help in confocal microscopy. The support of the laboratory manager Mrs. C. Fogle-Lamb and secretarial assistance of Ms. C. Nolan are gratefully appreciated. We acknowledge Dr. O. M. Zack Howard for her critical reading of this manuscript. 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 the right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.

Received January 7, 2002; revised March 22, 2002; accepted March 27, 2002.


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 DISCUSSION
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