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Originally published online as doi:10.1189/jlb.0804435 on February 15, 2005

Published online before print February 15, 2005
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(Journal of Leukocyte Biology. 2005;77:739-747.)
© 2005 by Society for Leukocyte Biology

Novel monoclonal antibodies detect elevated levels of the chemokine CCL18/DC-CK1 in serum and body fluids in pathological conditions

Robbert van der Voort*, Matthijs Kramer*, Ernst Lindhout*,1, Ruurd Torensma*, Dagmar Eleveld*, Antoine W. T. van Lieshout{dagger}, Maaike Looman*, Theo Ruers{ddagger}, Timothy R. D. J. Radstake{dagger}, Carl G. Figdor* and Gosse J. Adema*,2

* Department of Tumor Immunology, Nijmegen Center for Molecular Life Sciences, and
{dagger} Rheumatology and
{ddagger} Surgical Oncology, University Medical Center St Radboud, Nijmegen, The Netherlands

2 Correspondence: Department of Tumor Immunology, NCMLS, University Medical Center St Radboud, Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: g.adema{at}ncmls.kun.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CC chemokine ligand 18/dendritic cell-chemokine 1 (CCL18/DC-CK1) is a CC chemokine, preferentially expressed by DC, which acts as a chemoattractant for naive T cells and mantle zone B cells. Applying a newly developed CCL18/DC-CK1 sandwich enzyme-linked immunosorbent assay, we demonstrate that DC secrete high amounts of CCL18/DC-CK1 and that this expression can be increased by interleukin-10. High levels of CCL18/DC-CK1 were also detected in human serum (average of 88 ng/ml). Moreover, elevated CCL18/DC-CK1 levels were detected in synovial fluid from rheumatoid arthritis patients and in drain fluid (average of 254 ng/ml and 122 ng/ml, respectively). Immunoprecipitation experiment using anti-CCL18/DC-CK1 monoclonal antibodies revealed a protein of 6–7 kDa in serum and drain fluid that was indistinguishable from recombinant CCL18/DC-CK1 on Western blot and in re-aggregation assays. The concentration of CCL18/DC-CK1 found in human serum is in the same order of magnitude as was previously reported to completely inhibit CCL11/eotaxin-induced CC chemokine receptor 3 (CCR3) activation and consequent migration of eosinophils. CCL18/DC-CK1 may therefore function as an agonist (for naive T and B cells) and as an antagonist for CCR3-expressing leukocytes such as eosinophils.

Key Words: human • dendritic cells • cell trafficking • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemokines play a central role in the migration of hematopoietic cells during immune surveillance, inflammation, and immune responses. In addition, chemokines have immune-regulatory and developmental functions in multiple cell types, including stem cells, neurons, microglia, and endothelial cells [1 2 3 4 5 ]. Chemokines are classified into four subfamilies based on the distribution of their N-terminal cystein residues: CC, CXC, CX3C, and C chemokines [6 ]. An alternative classification has been proposed on the basis of their function during homeostatic or inflammatory conditions [6 , 7 ]. The biological effects of chemokines are mediated by their interaction with members of the G-protein-coupled 7-transmembrane-spanning receptor family. To date, over 40 chemokines and 17 chemokine receptors have been identified in humans [6 7 8 ]. The involvement of chemokines and their corresponding receptors in inflammation, allergic reactions, and viral diseases makes them promising targets for clinical intervention [9 10 11 12 13 ]. We have reported the identification of a chemokine, CC chemokine ligand 18/dendritic cell-chemokine 1 [CCL18/DC-CK1; alternatively called pulmonary and activation-regulated chemokine (PARC), alternative macrophage activation-associated CC-CK1, and macrophage inflammatory protein 4 (MIP-4)], which is abundantly expressed by DC and preferentially attracts naive T cells and mantle zone B cells [14 15 16 17 18 ]. However, the receptor for DC-CK1 on T and B cells responsible for the observed migration has not yet been identified. Recently, Met-chemokine ß7 (Met-Ckß7), a modified form of CCL18/DC-CK1, was shown to bind to CC chemokine receptor 3 (CCR3) and exhibit potent antagonistic effects on CCL11/eotaxin and CCL13/monocyte chemoattractant protein 4 (MCP-4)-induced eosinophil migration [18 ]. As the native recombinant (r)CCL18/DC-CK1 protein was also shown to act antagonistically, these data suggest that CCL18/DC-CK1 may have dual functions.

In this study, we describe the generation of CCL18/DC-CK1-specific monoclonal antibodies (mAb). Using these mAb, we demonstrated expression of CCL18/DC-CK1 in monocyte-derived DC (MO-DC) culture supernatants after incubation with inflammatory and anti-inflammatory stimuli. It is surprising that applying this enzyme-linked immunosorbent assay (ELISA), we also detected large amounts of a 6- to 7-kDa protein, which is indistinguishable from rCCL18/DC-CK1, in human serum and other body fluids. The average concentration in serum was in the same order of magnitude as the concentration reported to completely inhibit CCR3-mediated eosinophil chemotaxis. Possible implications of these findings are discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and human samples
The cell lines HL-60, THP-1, and U937 and human embryonic kidney (HEK)293 cells transfected with CCL22/macrophage-derived chemokine-green fluorescent protein (MDC-GFP) or CCL18/DC-CK1-GFP were cultured in RPMI 1640 supplemented with L-glutamine, antibiotics plus antimycotics (all Invitrogen, Breda, The Netherlands), and 10% fetal calf serum (FCS; Greiner Bio-One, Alphen a/d Rijn). Mouse bone marrow-derived DC were cultured as described previously [19 ]. Dr. Pilar Barrera (UMC Nijmegen, The Netherlands) kindly provided synovial fluid from patients with rheumatoid arthritis (RA). Human serum from healthy individuals and synovial fluid from RA patients were isolated according to standard procedures [20 ]. Drain fluid was collected during lymph node resection of patients with mammacarcinoma. The Ethics Committee of UMC Nijmegen approved this study.

Generation of MO-DC
MO-DC were essentially generated as described previously [21 ]. In brief, peripheral blood mononuclear cells from healthy individuals were separated by Ficoll-density centrifugation. Monocytes were purified by adhesion and resuspended in RPMI 1640 supplemented with L-glutamine, antibiotics plus antimycotics (all Invitrogen), 10% FCS (Greiner Bio-One), 500 U/ml recombinant human interleukin (rhIL)-4 (Schering-Plough, Amstelveen, The Netherlands), and 800 U/ml rh granulocyte macrophage-colony stimulating factor (GM-CSF; Schering-Plough). Monocytes were cultured in 75 cm2 culture flasks (Costar, Badhoevedorp, The Netherlands) for a total of 6 days. At day 3, cells were isolated and resuspended in fresh culture medium containing IL-4 and GM-CSF. At day 6, immature DC were matured at a density of 0.8 x 106/ml in fresh, cytokine-containing culture medium in the absence or presence of 100 ng/ml lipopolysaccharide (LPS; Sigma Chemical Co., St. Louis, MO), 20 ng/ml rhIL-10 (PeproTech, London, UK), or a mixture of 50 ng/ml tumor necrosis factor {alpha} (TNF-{alpha}; PeproTech), 20 ng/ml IL-1ß (CellGenix, Freiburg, Germany), 20 ng/ml IL-6 (CellGenix), and 10 µg/ml prostaglandin E2 (PGE2; Pharmacia and Upjohn, Puurs, Belgium). DC were cultured in six-well tissue-culture plates (Costar) for up to 2 additional days. At days 3, 6, 7, and 8, culture supernatant was analyzed by ELISA, and DC were characterized by flow cytometry.

Direct chemokine ELISA
mAb were raised in mice as described previously [22 ]. In brief, BALB/c mice were immunized with 100 µg rhCCL18/DC-CK1 coupled to keyhole limpet hemocyanin weekly (Imject Carrier Proteins, Pierce, Rockford, IL). After 5 weeks, splenocytes were isolated and used to obtain hybridomas, which were cloned and screened by ELISA for reactivity against rhCCL18/DC-CK1 coupled to bovine serum albumin (BSA; Imject Carrier Proteins, Pierce). Prot A-purified anti-CCL18/DC-CK1 mAb (clones AZN-CK18, AZN-CK18B, and AZN-CK18C) were all of the immunoglobulin G1 (IgG1) isotype. To test the specificity of the generated mouse anti-CCL18/DC-CK1 mAb, direct ELISA was performed. Maxisorb ELISA plates (Nunc, Roskilde, Denmark) were incubated overnight at 4°C with 1–5 µg/ml rCCL18/DC-CK1, CCL2/MCP-1, CCL3/MIP-1{alpha}, CCL4/MIP-1ß, CCL5/regulated on activation, normal T expressed and secreted (RANTES), CCL7/MCP-3, CCL14/hemofiltrate CC chemokine 1 (HCC-1), CCL17/thymus and activation-regulated chemokine (TARC), CCL19/MIP-3ß, CCL22/MDC, CXC chemokine ligand 8 (CXCL8)/IL-8, or the nonchemokine hepatocyte growth factor (HGF) in phosphate-buffered saline (PBS). Next, plates were washed 3x with PBS and blocked with 100 µl/well 1% human serum albumin (HSA; CLB, Amsterdam, The Netherlands) in PBS for 30 min at 37°C. After washing 3x with PBS-Tween, the plates were incubated with 5 µg/ml mouse anti-CCL18/DC-CK1 mAb (AZN-CK18, AZN-CK18B, or AZN-CK18C) for 1 h at room temperature (RT) followed by 3x washing with PBS-Tween and incubation with 50 µl/well horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (250 ng/ml, Zymed Laboratories, Inc., San Francisco, CA). After washing, the ELISA was developed using 3,3',5,5'-tetramethylbenzidine (TMB) substrate, and absorption was measured using a Titertek ELISA plate reader at 450 nm.

Chemokine sandwich ELISA and immunohistochemistry
For the detection of chemokines in supernatants, sandwich ELISAs were set up for detection of CCL18/DC-CK1. ELISA plates were coated overnight with 50 µl 5 µg/ml AZN-CK18B (mouse anti-CCL18/DC-CK1 mAb) or 1 µg/ml 25D8 (rat anti-CCL17/TARC mAb, DNAX Research Institute, Palo Alto, CA) in PBS at 4°C. Next, the plates were washed 3x with PBS and blocked with 100 µl 1% BSA in PBS for 30 min at 37°C. After washing 3x with PBS + 0.05% Tween-20, the plates were incubated with 50 µl sample for 60 min at RT. MO-DC supernatants were diluted in culture medium. Other samples were diluted in 1% BSA/PBS. Dilution in 5% BSA/PBS or 10–50% FCS/PBS gave similar results (data not shown). Again, after washing 3x with PBS-Tween, the plates were incubated with 50 µl 1 µg/ml goat anti-CCL18/DC-CK1 (R&D Systems, Abingdon, UK) in PBS-Tween for 30 min at RT. Next, the plates were washed 3x with PBS-Tween and incubated with 50 µl HRP-conjugated donkey anti-goat IgG (80 ng/ml, Jackson ImmunoResearch Laboratories, West Grove, PA) in PBS-Tween for 30 min at RT. After washing 3x with PBS-Tween, HRP presence was detected using TMB substrate and a Titertek ELISA plate reader at 450 nm. Detection limit of this ELISA is ~750 pg/ml.

Antibodies, immunohistochemistry, and flow cytometry
Immunohistochemistry was performed as described previously [15 ]. Flow cytometric analyses were performed on a FACSCalibur using CellQuest software (BD Biosciences PharMingen, Alphen aan den Rijn, The Netherlands). Next to the isotype controls mIgG1, mIgG2a, and mIgG2b, the following mouse mAb were used (clone name given in parentheses): anti-human leukocyte antigen (HLA)-DR/DP (Q5/13), anti-CD80 (L307.4), anti-CD86 (2331; all BD Biosciences PharMingen), anti-CD14 (RM052), anti-CD83 (HB15A; both Beckman Coulter, Mijdrecht, The Netherlands), and anti-DC-specific intercellular adhesion molecule-grabbing nonintegrin (SIGN; AZN-D1) [22 ]. As a secondary antibody, we used phycoerythrin-conjugated goat anti-mouse (BD Biosciences PharMingen).

Antibody coupling and immunoprecipitation
Packed Prot A sepharose CL-4B beads (500 µl, Pharmacia Biotechnologies, Uppsala, Sweden) were washed with 1 ml 0.2 M triethanolamine (TEA; Sigma Chemical Co.), pH 8.2, followed by washing with 1 ml 0.1 M Na2B4O7, pH 8.2. Next, the beads were resuspended in 500 µl 0.1 M Na2B4O7 and incubated with 1.5–3 mg AZN-CK18 mAb (1 h, 4°C). Next, the beads were washed 2x with 0.1 M Na2B4O7 and 1x with TEA. Subsequently, the beads were incubated with 50 mM dimethylpimelidate hydrochloride (Sigma Chemical Co.) in TEA for 45 min at RT. After spinning down, the beads were resuspended in 1 ml 50 mM ethanolamine, pH 8.2, and incubated 15 min at RT followed by washing 3x with 0.1 M Na2B4O7. Finally, the beads were resuspended in ~10 vol 0.1 M Na2B4O7 containing 0.02% NaN3 and stored at 4°C until use. Immunoprecipitation was performed using human serum or human drain fluid. First, the sample was centrifuged for 15 min at 14,000 rpm in an Eppendorf centrifuge. The sample was transferred to Eppendorf tubes, and ~50 µl packed Prot A sepharose CL-4B beads were added to the sample tubes and incubated at 4°C overnight while tumbling to preclear the samples. Next, the tubes were centrifuged, and the precleared samples were transferred to new tubes. To each tube, 15–25 µl AZN-CK18-coupled beads were added and incubated for 24 h at 4°C while tumbling. Subsequently, the beads were spun down and washed twice with PBS, pooled, transferred to a new Eppendorf tube, and washed again with PBS. Finally, the beads were resuspended in sodium dodecyl sulfate (SDS) sample buffer and stored at –80°C until SDS-polyacrylamide gel electrophoresis (PAGE).

SDS-PAGE and Western blotting
Precipitation samples were run on polyacrylamide gels for Western blotting. The samples were run under denaturing conditions on a 15% polyacrylamide gel with a 4% polyacrylamide stacking gel. Samples were thawed and incubated 5 min at 95°C before loading. First, the sample was stacked at 50 V followed by running at 100 V using the Bio-Rad Mini-Protean II gel system (Bio-Rad Laboratories, Hercules, CA). The gels were processed for silver staining or Western blotting. Silver staining was performed using the Bio-Rad Silverstain Plus kit according to the manual.

For enhanced resolution, after the first run, the gel was fixated in a solution of 10% MeOH, 7% acetic acid, for 30 min at RT and stained with Sypro Ruby gel stain (Molecular Probes, Leiden, The Netherlands) for 3 h at RT. After washing in 10% MeOH, 7% acetic acid, proteins were visualized using a 300 nm UV transilluminator. Proteins of interest were cut out and incubated in stacking buffer (125 mM Tris-HCl, pH 6.8, 0.1% SDS) for 1 h, and next, the gel slices were minced and run on a 16.5% Tris-Tricine ready gel (Bio-Rad Laboratories).

For Western blotting, the proteins were transferred to nitrocellulose [Hi-bond enhanced chemiluminescence (ECL), Amersham Biosciences, Little Chalfont, UK] by blotting the gel for 1 h at 400 mA. The nitrocellulose blot was blocked with 1.5% horse serum in 20 mM Tris-HCl, pH 7.4, + 0.1% Tween [Tris-buffered saline/Tween 20 (TBS-T)] for 1 h. The blot was washed 5 min with TBS-T followed by incubation with 500 ng/ml biotinylated goat anti-PARC (R&D Systems) in TBS-T + 1% horse serum for 1 h at RT. After washing 6x with TBS-T, the blot was incubated with HRP-conjugated streptavidin (1:5000 diluted in TBS-T, Pierce) for 1 h at RT. Subsequently, the blot was washed and incubated for 1 min with ECL substrate (Amersham). Finally, the blot was imaged on a scientific imaging film (Kodak, Rochester, NY).

Statistical analysis
For statistical analyses, we first logarithmically transformed the values from groups with a skewed distribution. Next, differences between groups were calculated by using the two-sample t-test. P values of less than 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of CCL18/DC-CK1-specific antibodies
We recently identified a novel chemokine, CCL18/DC-CK1, which is preferentially expressed by DC and attracts naive T cells and mantle zone B cells. To further explore the expression of this chemokine, we raised mAb against CCL18/DC-CK1. Three hybridomas, AZN-CK18, AZN-CK18B, and AZN-CK18C, were isolated and further characterized. As shown in Figure 1A , all three anti-CCL18/DC-CK1 antibodies are specific for CCL18/DC-CK1 in a direct ELISA. The mAb do not cross-react with any of 12 other chemokines tested, including the CC chemokines CCL3/MIP-1{alpha} (63% homology), CCL4/MIP-1ß (42% homology), and CCL14/HCC-1 (40% homology), to which CCL18/DC-CK1 is most homologues, nor with the heparan-sulfate-binding protein HGF.



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  		Figure 1. Direct and sandwich ELISA specifically detects CCL18/DC-CK1. (A) The anti-CCL18/DC-CK1 mAb AZN-CK18 (open bars), AZN-CK18B (solid bars), and AZN-CK18C (dotted bars) were tested in a direct ELISA against a panel of related chemokines indicated in the figure. All three antibodies are specific for CCL18/DC-CK1 and do not react with the most homologues CC chemokines, CCL3/MIP-1{alpha}, CCL4/MIP-1ß, and CCL14/HCC-1, nor with any of the other tested chemokines. (B) Specificity of the CCL18/DC-CK1 sandwich ELISA using AZN-CK18, AZN-CK18B, or AZN-CK18C as catching antibody and polyclonal anti-CCL18/DC-CK1 as detection antibody. Different concentrations of rCCL18/DC-CK1 (12.5 or 25 ng/ml), rCCL3/MIP-1{alpha} (100 ng/ml), or rCCL5/RANTES (100 ng/ml) were tested for recognition in the CCL18/DC-CK1 sandwich ELISA. Especially AZN-CK18B, in combination with polyclonal anti-CCL18/DC-CK1, detects CCL18/DC-CK1 in a high, specific absorption and does not detect CCL3/MIP-1{alpha} or CCL5/RANTES. Mean data of three wells are presented as specific absorption minus background absorption (~170 units). ND, Not detectable. (C) Titration curve for the CCL18/DC-CK1 sandwich ELISA indicating that amounts as low as 780 pg/ml can be detected. (D) Analysis of the culture supernatants of the monocytic cell lines HL-60, THP-1, U937, murine and human DC, and CCL22/MDC-GFP or CCL18/DC-CK1-GFP expressing transfectants further demonstrates the specificity of the CCL18/DC-CK1 ELISA. The results represent the means of three measurements and are representative of at least two independent experiments.

 
To develop a CCL18/DC-CK1-specific sandwich ELISA, the CCL18/DC-CK1 mAb were tested as catching antibody and a commercially available goat anti-CCL18/DC-CK1 polyclonal antibody as detecting antibody. Initial experiments showed that the application of AZN-CK18B as the catching gave the best results (Fig. 1B , and data not shown). This ELISA is highly specific for CCL18/DC-CK1, as neither CCL3/MIP-1{alpha} nor CCL5/RANTES was detected at concentrations of up to 100 ng/ml (Fig. 1B) , whereas amounts as low as 780 pg/ml CCL18/DC-CK1 could be measured (Fig. 1C) . Using this sandwich ELISA, we were able to detect high levels of CCL18/DC-CK1 in the culture supernatant of mature MO-DC but not in those from the human monocytic cell lines THP-1, HL-60, U937, and murine bone marrow-derived DC (Fig. 1D) . These results are in line with the absence of CCL18/DC-CK1 mRNA in these cells [14 ]. Moreover, we detected more than 1 µg/ml CCL18/DC-CK1 in the supernatant of CCL18/DC-CK1-GFP-transfected HEK293 cells, whereas the supernatant of CCL22/MDC-GFP-transfected HEK293 was negative (Fig. 1D) .

To further characterize the mAb, they were used to stain MO-DC, which are known to express CCL18/DC-CK1 mRNA. Therefore, freshly isolated monocytes were cultured in the presence of GM-CSF and IL-4, and cytospin preparations were made on days 6 and 8, the latter after 48 h in the presence of LPS. As shown in Figure 2 , some CCL18/DC-CK1 immunoreactivity was observed on day 6 immature DC (Fig. 2B) , but over 90% of the day 8, mature DC (Fig. 2D) was strongly positive for CCL18/DC-CK1. It is remarkable that a significant percentage of this CCL18/DC-CK1 was localized close to the cell membrane. No staining was observed with an isotype-matched, control mAb (Fig. 2C) . As expected, fresh monocytes do not express CCL18/DC-CK1 (data not shown). It is striking that at day 8, the intensity of the staining was increased, and a larger number of LPS-matured DC expressed CCL18/DC-CK1 (Fig. 2B and 2D) . The increase in CCL18/DC-CK1 protein nicely paralleled the previously observed increase in CCL18/DC-CK1 mRNA levels in time [14 ], further emphasizing the specificity of our novel anti-CCL18/DC-CK1 antibodies. We note, however, that DC maturation by LPS is not essential for an increased secretion of CCL18/DC-CK1. MO-DC cultured for 8 days in the absence of LPS also secrete increased amounts of CCL18/DC-CK1 as compared with day 6 MO-DC (see Fig. 3 ).



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  		Figure 2. CCL18/DC-CK1 mAb strongly stain mature DC. Cytospin preparations of day 6, immature DC (A and B) and day 8, mature DC (C and D) were stained with the anti-CCL18/DC-CK1 mAb AZN-CK18 (B and D) or an isotype-matched, control mAb (A and C). Original magnification, 200x.

 


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  		Figure 3. IL-10 strongly induces the expression of CCL18/DC-CK1 by DC. (A) Expression of MHC II (HLA-DR/DP), CD80, and CD83 by day 6 DC and by DC cultured for 2 additional days in the presence or absence of LPS, maturation mix (TNF-{alpha}, IL-1ß, IL-6 plus PGE2), or IL-10. Cells were analyzed by flow cytometry. The numbers indicate the mean fluoresence intensity for each marker. (B) DC produce large amounts of CCL18/DC-CK1. DC medium was refreshed at day 6, and the production of CCL18/DC-CK1 by the DC was measured after 24 and 48 h. Results represent the means of duplicate measurements and are representative of more than three sandwich ELISAs performed. (C) Expression of CCL18/DC-CK1 by DC is strongly induced by IL-10 but not by maturation stimuli. DC from multiple donors were cultured for 8 days, of which 2 days were in the absence (eight donors) or presence of LPS (five donors with conventional LPS plus three with ultrapure LPS), maturation mix (two donors), or IL-10 (three donors). (A) Representative phenotypes of these DC are shown. Duplicate culture supernatants were analyzed by sandwich ELISA (three independent experiments). ***, P < 0.001, for the group indicated compared with the control group.

 
IL-10 strongly induces the expression of CCL18/DC-CK1 by DC
To study the expression of CCL18/DC-CK1 by DC in more detail, we cultured MO-DC in the presence of GM-CSF and IL-4 for 6 days. These DC had an immature phenotype with high expression of the DC marker DC-SIGN and low-to-intermediate expression levels for major histocompatibility complex class II (MHC II) and the costimulatory molecules CD80 and CD86 (Fig. 3A , and data not shown). In addition, these DC were negative for the monocyte marker CD14 and the maturation marker CD83 (Fig. 3A , and data not shown). Then, from day 6 on, DC were harvested and further cultured in the absence or presence of LPS, IL-10, or a maturation mix consisting of TNF-{alpha}, IL-1ß, IL-6, and PGE2. DC matured with LPS or the maturation mix showed a mature phenotype with strong expression of MHC II, CD80, CD86, and CD83 (Fig. 3A , and data not shown). In contrast, IL-10-treated DC demonstrated a phenotype with immature features: lack of CD83 and low-to-intermediate expression of MHC II, CD80, and CD86 (Fig. 3A , and data not shown).

Next, using our novel sandwich ELISA, we analyzed the expression of CCL18/DC-CK1 by the DC described above. Whereas little CCL18/DC-CK1 was produced by DC cultured for up to 6 days (data not shown), a large amount of CCL18/DC-CK1 was detected in the culture supernatant of DC cultured for 7 or 8 days in the absence of additional stimuli (Fig. 3B) . Furthermore, DC cultured in the presence of IL-10 for 2 days showed strongly induced CCL18/DC-CK1 at day 8 (Fig. 3C) . In contrast, LPS, conventional or ultrapure, or maturation mix did not induce a significant, further increase in the expression of CCL18/DC-CK1 by fully matured DC (Fig. 3A) from a total of eight donors.

Detection of CCL18/DC-CK1 in serum and drain fluid by a specific sandwich ELISA
As the presence of chemokines in human serum has been described [23 ], we tested human serum for the presence of CCL18/DC-CK1. It is striking that as shown in Table 1 , human serum contains on average of 88 ng/ml CCL18/DC-CK1. In serum of horse, donkey, goat, fetal calf, rabbit, rat, and mouse, no specific signal could be detected (data not shown).


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  		Table 1. CCL18/DC-CK1 Levels in Human Serum and Body Fluids

 
To further investigate the presence of CCL18/DC-CK1 in pathological conditions, we measured the amount of CCL18/DC-CK1 in drain fluids obtained from patients after surgery. In drain fluids from different patients, the amount of CCL18/DC-CK1 varied between 21 and 315 ng/ml (Table 1) , with an average of 122 ng/ml. Furthermore, analysis of synovial fluid samples from RA patients also revealed the presence of high concentrations of CCL18/DC-CK1, with an average of 254 ng/ml (Table 1) . These data suggest that the level of CCL18/DC-CK1 in body fluids increases during inflammatory or anti-inflammatory conditions.

Anti-CCL18/DC-CK1 mAb detect a 6- to 7-kDa protein indistinguishable from CCL18/DC-CK1
To further investigate the presence of CCL18/DC-CK1 in human body fluids, we performed immunoprecipitations on drain fluid samples using mAb AZN-CK18C. Analysis of the immunoprecipitate by PAGE and silver staining indicated that a protein of the expected size (6–7 kDa) was specifically immunoprecipitated by the AZN-CK18C mAb (Fig. 4A ). The amount of this precipitated protein was estimated to be 250 ng by comparison with known amounts of rCCL18/DC-CK1, which is in line with the quantities detected in ELISA (Table 1) .



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  		Figure 4. CCL18/DC-CK1 in drain fluid is indistinguishable from rCCL18/DC-CK1. (A) Silver staining of a 15% Tris-glycine gel containing different concentration of rCCL18/DC-CK1 and the AZN-CK18C immunoprecipitate (IP) from drain fluid. The size of the dominant product in the IP sample migrates at the same position as rCCL18/DC-CK1. M, marker. (B) Western blot analysis using the anti-CCL18/DC-CK1 polyclonal antibody on different concentration of rCCL18/DC-CK1 and the AZN-CK18C immunoprecipitate (IP) from drain fluid. (C) A mixture of the chemokines, CCL3/MIP-1{alpha}, CCL4/MIP-1ß, and CCL5/RANTES (5 µg/ml each in RPMI+1% HSA; CK-mix), human serum (HuS), FCS, and human serum supplemented with 1 µg/ml CCL18/DC-CK1 (HuS+) were subjected to immunoprecipitation using AZN-18C, and the immunoprecipitated proteins were analyzed by Western blotting using the anti-CCL18/DC-CK1 polyclonal antibodies. rCCL18/DC-CK1 was used as a control. PAGE, Western blotting, and re-aggregation experiments were performed on drain fluid samples after immunoprecipitation by AZN-CK18C and on rCCL18/DC-CK1. Sypro Ruby staining (D) and Western blot analysis (E) of 15% Tris-glycine gels run in parallel demonstrate the presence of a clear protein band at 6.5 kDa. The 250 ng rCCL18/DC-CK1 band and the corresponding band (boxed area) of the IP sample were excised from the Sypro Ruby-stained gel. Both samples were soaked in Tris-Tricine stacking buffer and without further denaturation, applied to a new 16.5% Tris-glycine gel together with new rCCL18/DC-CK1 (F). Western blotting of this second gel, using goat anti-CCL18/DC-CK1, clearly reveals that both excised samples have identical re-aggregation patterns (arrows) and migrate at the same position as the new CCL18/DC-CK1 sample. Mw, Molecular weight.

 
Western blot analysis of the precipitated sample further showed that the precipitated protein of 6–7 kDa was specifically detected by the anti-CCL18/DC-CK1 polyclonal antibody and migrated at the same position as rCCL18/DC-CK1 (Fig. 4B) . In addition, in a further specificity test, we precipitated a protein of the correct size from human serum but not from FCS or from a mix containing CCL3/MIP-1{alpha}, CCL4/MIP-1ß, and CCL5/RANTES (Fig. 4C) . More of this protein was precipitated when human serum was supplemented with rCCL18/DC-CK1 (Fig. 4C) .

To further compare the protein detected in the body fluid, we made use of the property of CCL18/DC-CK1 to form aggregates under suboptimal-reducing conditions. Hereto, CCL18/DC-CK1 was precipitated from drain fluid and analyzed together with different concentrations of rCCL18/DC-CK1 on two separate 15% PAGE gels. One gel was used for Western blot analysis, and the other the gel was stained with Sypro Ruby stain. The Western blot stained with goat anti-CCL18/DC-CK1 showed a single band of 6.5 kDa in the immunoprecipitate, which migrated at exactly the same position as rCCL18/DC-CK1 (Fig. 4E) . Also, the Sypro Ruby-stained gel run in parallel revealed a band of 6.5 kDa in the rCCL18/DC-CK1 lanes and in the lane containing the immunoprecipitate (Fig. 4D) . From this gel, we excised the 250 ng rCCL18/DC-CK1 band and the corresponding band from the IP sample. Both samples were soaked in Tris-Tricine stacking buffer and loaded (without further denaturation) on a 16.5% Tris-Tricine gel that yields an optimal separation of small proteins. Subsequent Western blotting of this second gel clearly demonstrated that the protein isolated from the IP sample has an identical re-aggregation pattern as rCCL18/DC-CK1 (Fig. 4F) , indicating that the immunoprecipitated protein is indistinguishable from CCL18/DC-CK1.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here, we report on the isolation and characterization of mAb raised against CCL18/DC-CK1, a chemokine that is abundantly expressed by DC. The mAb obtained specifically recognize CCL18/DC-CK1 out of a panel of 12 chemokines in direct ELISA screening. Even the most homologues chemokines, CCL3/MIP-1{alpha}, CCL4/MIP-1ß, and CCL14/HCC-1 (homology ranging from 40% to 65% overall but over 90% homology in the C-terminal region), were not recognized by the anti-CCL18/DC-CK1 antibodies (Fig. 1A) . In addition, the sandwich ELISA developed to measure CCL18/DC-CK1 concentrations in culture supernatants and other samples also demonstrates this specificity for CCL18/DC-CK1. The commercial anti-CCL18/DC-CK1 polyclonal antibody, used as detection antibody, cross-reacts with CCL3/MIP-1{alpha} and CCL4/MIP-1ß to some extent (~10% and 5%, respectively; datasheet, R&D Systems). However, using the CCL18/DC-CK1 mAb as the catching antibody in the sandwich ELISA, high concentrations of CCL3/MIP-1{alpha} were not detected (Fig. 1B) . The detection limit of the CCL18/DC-CK1 sandwich ELISA was determined at 780 pg/ml (Fig. 1C) . As CCL18/DC-CK1 is expressed by DC, cytospin preparations and culture supernatant of GM-CSF- and IL-4-generated MO-DC were analyzed at different time-points. Staining of cytospin preparations of MO-DC with our CCL18/DC-CK1 mAb showed an increased expression of CCL18/DC-CK1 between days 6 and 8 (Fig. 2) . In the sandwich ELISA, CCL18/DC-CK1 was clearly detected from day 3 onward and increased to high amounts at day 8 (Fig. 3B , and data not shown). Also, for other chemokines such as CCL3/MIP-1{alpha} and CCL4/MIP-1ß, it was reported that DC are capable of producing large amounts in short periods of time in vitro [24 , 25 ]. Our data confirm conventional and quantitative, real-time polymerase chain reaction experiments performed by us and others [24 , 25 ]. Moreover, our data, demonstrating that maturation of DC has no significant additive effect on the expression of CCL18/DC-CK1, are in line with those recently reported by Pivarcsi et al. [26 ]. These authors demonstrated that MO-DC cultured for 7 days express CCL18/DC-CK1 constitutively and independent of maturation signals such as LPS, peptidoglycan, or CD40L. In contrast, Vulcano et al. [27 ] reported that maturation of MO-DC down-regulates the production of CCL18/DC-CK1. A possible explanation is that the effect of DC maturation on CCL18/DC-CK1 expression is strongly donor-dependent. We have detected increased and decreased concentrations of CCL18/DC-CK in the supernatants of fully matured DC (data not shown). However, the net effect of LPS or maturation mix on the expression of CCL18/DC-CK1 by DC from eight different donors was not significant (Fig. 3C) . An alternative explanation for the discrepancy could be that the DC described by Vulcano et al. [27 ] were cultured in the absence of cytokines between days 6 and 8. We and Pivarcsi and colleagues still provide GM-CSF plus IL-4 during that period. Lack of these cytokines could skew the cells toward a macrophage-like phenotype.

In contrast to the maturation stimuli described above, the anti-inflammatory cytokine IL-10 did strongly induce the expression of CCL18/DC-CK1 by MO-DC (Fig. 3C) . These data confirm experiments with MO-DC and macrophages reported by others [17 , 27 ] and suggest that CCL18/DC-CK1 plays an important role during tolerance or humoral immunity in which high levels of IL-10 are present [28 , 29 ]. It is interesting that IL-10 is also known to block DC maturation, and immature or semi-mature DC have recently been reported to induce immunosuppressive regulatory T cells [29 , 30 ]. Although the distinction between tolerogenic and immunogenic DC is still poorly defined, the observed expression profile of CCL18/DC-CK1 implies that immunogenic as well as tolerogenic DC might secrete this chemokine.

As several other chemokines in human serum have been detected, the presence of CCL18/DC-CK1 in serum is not unexpected. However, the concentration of CCL18/DC-CK1 found in serum (average 88 ng/ml) is surprisingly high (Table 1) . Recently, similar concentrations of CCL18/DC-CK1 in human serum were reported [31 , 32 ]. The absence of CCL18/DC-CK1 reactivity in mouse serum (data not shown) is in line with genetic data, implicating that the CCL18/DC-CK1 gene has only arisen relatively late in evolution [33 , 34 ].

It is interesting that high levels of CCL18/DC-CK1, ranging from 21 up to 315 ng/ml, were found in drain fluid samples obtained from breast cancer patients that have had recent lymph node resection (Table 1) . The average concentration in these drain fluid samples (122 ng/ml) is even higher than the average serum level (Table 1) . Silver staining demonstrated that CCL18/DC-CK1 is indeed present in large quantities in serum and drain fluid. Characterization of the protein detected by the mAb in serum and drain fluid by immunoprecipitation, Western blot, and re-aggregation analysis showed that the protein is indistinguishable from rCCL18/DC-CK1 (Fig. 4) . The observed aggregation and re-aggregation are specific features of chemokines, such as CCL18/DC-CK1, containing Asp and Glu at ~15 amino acids downstream of the N-terminal cysteins [35 ]. It is intriguing that the potential to form aggregates could result in a relative resistance to proteases and a consequent, prolonged half-life of CCL18/DC-CK1 in serum. This might (partially) explain the high levels of CCL18/DC-CK1 detected.

Furthermore, we detected high amounts (a mean of 254 ng/ml) of CCL18/DC-CK1 in synovial fluid from RA patients (Table 1) . These findings are in line with data presented by Schutyser et al. [36 ]. Moreover, our own recent data indicate that sera from RA patients also contain enhanced levels of CCL18/DC-CK1 and that MO-DC from RA patients produce much more CCL18/DC-CK1 than their normal counterparts [20 ]. It is interesting that tumor-infiltrating myeloid cells expressing high levels of CCL18/DC-CK1 have also been detected in ascitic fluid from patients with ovarian carcinoma [37 ]. In addition, expression of CCL18/DC-CK1 was found to be significantly increased in lungs affected by hypersensitivity pneumonitis (HP) [38 ]. The secretion of CCL18/DC-CK1 was related directly to the number of infiltrating lymphocytes, an important characteristic of HP. It is tempting to speculate that the increased levels of CCL18/DC-CK1 found in patients with RA, ovarian cancer, or HP result from the (partial) activation of the immune system. Currently, we are investigating the possibility of whether the amount of CCL18/DC-CK1 in serum can be used as an indicator to predict inflammatory or anti-inflammatory conditions.

As it is difficult to test the chemotactic properties of chemokines in the complexity of human serum and drain fluid, we have not been able to determine whether CCL18/DC-CK1 in serum is functionally active. Irrespective of whether the high amount of CCL18/DC-CK1 in serum represents a functional pool of chemokines or reflects inflammatory or anti-inflammatory encounters of the past, the significance of these high concentrations of CCL18/DC-CK1 is difficult to interpret in light of the observed chemotactic activity of CCL18/DC-CK1 on naïve T cells and B cells. However, one possible explanation is given by data demonstrating that CCL18/DC-CK1 can act as an antagonist of CCL11/eotaxin and CCL13/MCP-4 for binding to CCR3 [18 ]. IL-4 is known to induce CCL11/eotaxin, among others, in epithelial cells and fibroblasts [39 , 40 ]. As IL-4 is also critical in inducing CCL18/DC-CK1 production by DC [14 ], eosinophil migration could be regulated directly through the induction of CCL11/eotaxin and CCL18/DC-CK1, where CCL18/DC-CK1 could have a potential role in dampening eosinophil and/or allergic responses in the lung, for instance. This hypothesis fits the observation by Goerdt and colleagues [17 ] that reduced levels of CCL18/DC-CK1 are expressed by alveolar macrophages from asthmatics as compared with healthy individuals. Possibly, CCL18/DC-CK1 acts as an antagonist to reduce excessive eosinophil influx and consequent damage in the lung. Interesting in this respect is the recent observation that eosinophils themselves are also able to produce significant amounts of CCL18/DC-CK1 [41 ]. An antagonistic function has also been reported for other chemokines. The CXC receptor 3 ligands, CXCL9/monokine induced by interferon (IFN)-{gamma}, CXCL10/IFN-inducible protein 10, and CXCL11/IFN-inducible T cell-{alpha} chemoattractant, also function as natural antagonists for CCR3 [42 ], and naturally occurring, N-terminally truncated CCL2/MCP-1 and CCL8/MCP-2 variants act as receptor antagonists for their active homologues [43 ]. However, for blocking CCR3, CCL18/DC-CK1 does not have to be truncated, although a modified form of CCL18/DC-CK1 (Met-Ckß7) is a more potent inhibitor [18 ].

Collectively, using a new CCL18/DC-CK1 ELISA, we demonstrate that DC produce high levels of CCL18/DC-CK1 upon stimulation with inflammatory and anti-inflammatory stimuli. Moreover, large quantities of CCL18/DC-CK1 are present in serum, synovial fluid, and drain fluid. Our results further suggest that CCL18/DC-CK1 might play a stimulatory as well as an inhibitory role in the immune system.


   ACKNOWLEDGEMENTS
 
This work was supported by the Dutch Cancer Society (Grant No. KUN99-1947) and the Dutch Foundation of Scientific Research (Grant No. 90308). We thank Drs. P. Barrera (UMC Nijmegen) for synovial fluid and Dr. G. Zurawski (DNAX Research Institute) for rDC-CK1. We thank Lars Guelen and Nynke van Berkum for help with the experiments.


   FOOTNOTES
 
1 Current address: Future Diagnostics, Nieuweweg 172, 6603 BT Wijchen, The Netherlands. Back

Received August 2, 2004; revised January 20, 2005; accepted January 24, 2005.


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