Background
Rheumatoid arthritis (RA) is a chronic autoimmune disease that is mainly characterized by inflammation of the synovial tissue (ST), leading to cartilage and bone destruction. Influx of different inflammatory cells into the ST and enhanced production of cytokines and chemokines are all well known features of RA. Chemokines are small proteins that act as key players in the chemo-attraction of different leucocytes and perform their chemo-attractive task through interaction with their receptor on the target cell. Several chemokines have been shown to be abundantly present in RA ST at highly strategic sites [
1‐
3], which suggests a role for these chemokines in the pathogenesis of RA. In this respect, chemokines could be regarded as promising therapeutic targets in RA. This concept has already been translated to the clinic, since the blockade of C Chemokine Receptor 1 (CCR1) has recently been shown to be clinically effective in the treatment of RA [
4].
Antigen presenting cells (APC), such as dendritic cells (DC) and macrophages (MΦ), are generally accepted as critical mediators in the complex pathogenesis of RA [
5‐
7]. APC produce a multitude of chemokines that attract specific T cell subsets. Such chemokines are likely to play a critical role in the regulation of immune responses, since they orchestrate the spatial and temporal interaction between APC and T cells, which determines the fate and nature of the immune response. Evidence for this conceptual framework came recently from the observation that blocking APC-T cell interactions using CTLA4-Ig led to a significant reduction of disease activity in RA [
8]. Several chemokines orchestrate the attraction of T cells toward DC. It is tempting to speculate that interfering with these chemokines would lead to similar effects on disease activity as the direct blockade of T-cell DC interaction. Of this group of T- cell attracting chemokines, CCL18 and CXCL16 recently came out as potentially interesting targets in RA from previous research by our group and others [
9‐
13].
CC chemokine ligand 18 (CCL18, also DC-CK-1, PARC, AMAC-1) was first identified as a naïve T cell attracting chemokine [
14‐
16]. Next to chemo-attraction, CCL18 plays a role in stimulation of collagen production by fibroblasts [
17]. Despite numerous attempts to identify its receptor, CCL18 is still an orphan chemokine.
In vivo, CCL18 expression was first found in high quantities in the lung, which is caused by the abundant expression by alveolar macrophages [
15].
In vitro, DC and MΦ have been identified as CCL18 producers [
14‐
16,
18,
19]. To date, a substantial amount of data points toward the enrichment of DC and MΦ in the synovial tissue which likely to be responsible for the increased levels of CCL18 in RA synovial tissue and synovial fluid (SF) compared with that from healthy individuals [
18,
20]. In this line, CCL18 has been identified as a clinical marker in Gaucher's disease, a condition in which MΦ activation is likely to play a role in the pathogenesis [
21,
22]. In addition, a role for CCL18 has been suggested in a large variety of diseases, such as systemic sclerosis and acute lymphoblastic leukaemia [
23,
24]. In RA, we recently found that circulating CCL18 levels are elevated compared with controls and correlated with disease activity (van Lieshout
et al. manuscript submitted). Moreover, CCL18 mRNA expression by DC from RA patients was shown to be higher than by DC from healthy controls, which could be influenced by blockade of TNF-α [
10,
13]. The exact regulation of CCL18 protein secretion however is complicated and the studies published thus far have led to controversial results [
18,
19,
25‐
27], as elegantly reviewed by Schutyser
et al [
28].
In order to clarify the mechanism of CCL18 expression and secretion in RA, we investigated the role of a large panel of inflammatory mediators known to play a role in the disease process on CCL18 secretion. Here we show that CCL18 secretion by monocytes and DC is regulated by synergistic effects between IL-4/IL-13, IL-10 and RA SF, whereas pro-inflammatory cytokines and Toll-like receptor (TLR) ligands did not have any influence on CCL18 secretion. These data add novel information to the puzzle of increased CCL18 expression in RA.
Discussion
In this study, we add new pieces to the complicated puzzle of CCL18 regulation in RA. Firstly, we demonstrate that CCL18 production can be induced by IL-4, IL-13 and IL-10 in monocyte derived cells. Secondly, we show that a large panel of pro-inflammatory stimuli and TLR mediated signals leading to DC maturation are of no influence on CCL18 production. Thirdly, IL-10 only induces a minor CCL18 secretion, but acts in synergy with both IL-4 and IL-13 on monocytes and monocyte derived cells. Finally, we provide evidence that RA SF is able to induce CCL18 secretion in strong synergy with IL-4, IL-13 and IL-10, which could not be inhibited by a blockade of IL-10 and IL-13.
CCL18 can be produced by MoDC as well as by certain types of MΦ. Often these cell types are considered to be totally different cells. However, the differences between MoDC and AaMΦ are not that large, since monocyte derived macrophages are cultured in the presence of GM-CSF by some groups [
36] and both cells require the presence of IL-4 or IL-13. Penna and co workers demonstrated that several
in vivo DC subtypes were not able to produce CCL18 [
37], which is in contrast with previous findings, where CCL18 mRNA expression was found on CD11c+ myeloid blood DC [
27]. Moreover,
in vitro cultured MoDC have been identified as potent CCL18 producers [
18,
19]. These data suggest that a CD14+ monocyte origin in combination with a stimulation by IL-4/IL-13 is critical for CCL18 secretion. This hypothesis is strengthened our data, demonstrating that non-adherent monocytes/macrophages were able to produce CCL18 under the influence of IL-4. In addition, the synergistic effects of IL-4/IL-13 and IL-10 on CCL18 secretion by freshly isolated monocytes were already clearly visible after 24 hours. This indicates that a full differentiation into DC or MΦ is not essential for CCL18 production as has been suggested previously for CCL18 mRNA expression [
16]. Thus monocytes rapidly secrete CCL18 upon triggering with the right stimuli.
In the literature there is still some controversy regarding the effect of DC maturation on CCL18 production. Vulcano and co workers suggested that DC down regulate their CCL18 secretion upon maturation [
19]. This is in contrast with results from other studies, where maturation caused an increased mRNA expression [
10,
26,
27]. A similar contrast between protein and mRNA was found on blood DC [
27,
37]. The reason for these differences between mRNA expression and protein secretion patterns still needs to be investigated in detail. Recently, we already provided evidence that DC maturation does not influence CCL18 protein secretion [
18], which is further strengthened by the data from the present study, in which different TLR stimulatory pathways did not induce CCL18 production, whereas full DC maturation was achieved [
31]. Also TNF-α and CD40L, both well appreciated inducers of DC maturation [
38,
39], did not enhance CCL18 production. Perhaps the discrepancy between the different reports is hidden in subtle differences in culture conditions, which are difficult to trace in the published data. Intriguingly, stimulation with IL-10 alone only lead to a marginal induction of CCL18 secretion by monocytes/macrophages, but did act in a strong synergy with IL-4 or IL-13. The latter is not caused by an up regulation of the receptors IL-10Rα, IL-4Rα or IL-13Rα2 (data not shown). Probably intracellular pathways direct the synergy between these cytokines, which is an interesting topic that warrants further investigation.
We showed that RA SF induces CCL18 production and strongly synergizes with IL-4, IL-13 and IL-10. Blocking studies revealed that neither IL-10 nor IL-13 in SF were responsible for this effect. This suggests the presence of another, yet unidentified CCL18 inducing factor in RA SF. Another explanation for this fact might be the presence of inhibiting factors in SF that counter-regulate the effects of IL-10 and IL-13. The identification of the factor in SF that drives the effects on CCL18 secretion may provide important new insights to the pro-vs. anti inflammatory balance in RA. In order to find this factor in a complex fluid like SF, more knowledge on the pathways of CCL18 regulation is critical. Another intriguing observation from our study is the finding that pre-incubation with SF lead to a sustained synergistic CCL18 secretion upon stimulation with IL-4, IL-13 and IL-10. This could be regarded as an "imprinting effect", meaning that the cell's previous environment determines the nature of response to stimuli, even when the cell is no longer in such an environment. Results from previous studies, in which we showed that moDC from RA patients differ in phenotype and cytokine response from control DC after 6 days in culture might also be explained by such a phenomenon [
40,
41].
Upon the encounter of an antigen, DC normally mature and migrate to lymphoid tissues in order to perform their task of antigen presentation to T cells. Immature DC or MΦ can also encounter naïve T cells in the periphery, which subsequently might result in tolerance [
42]. This peripheral tolerance is a critical mechanism to prevent auto-immunity. A role for CCL18 in this part might explain the high expression of CCL18 by alveolar MΦ [
15,
16], which are located at a site where the maintenance of tolerance to non-pathogenic antigens, that are constantly present, is crucial. Also the synergistic effect on CCL18 secretion that we found with IL-10, a cytokine that is well appreciated as a pivotal regulator of the immune system, fits in this picture. The synovial lining in the joints has similarities with the alveolar lining in the lung. They both consist of MΦ-like cells and both form a barrier to a site in which self- and non-pathogenic antigens are constantly present. The disease process in RA is considered to be driven by pro inflammatory cytokines such as IL-1β, TNF-α, IL-17 and IL-18 [
43‐
48], whereas CCL18 is regulated by IL-10, IL-4 and IL-13. It is therefore tempting to speculate that the high CCL18 expression in RA is designed to uphold peripheral tolerance, which however seems to fail. This failure might be explained in two ways. The first explanation might be that the skewing in the balance towards Th1 is still present despite the upregulation of anti inflammatory mediators. Secondly, mature DC are present in the synovial tissue in perivascular regions and secondary lymphoid organs [
3,
49], which is in sharp contrast with healthy synovial tissue. Therefore an explanation for the ongoing immune process might be that these mature DC direct naïve T cells towards a phenotype that drives the pro-inflammatory immune response in the synovial tissue.
Conclusion
In summary, we provide evidence that monocyte derived cells produce CCL18 under the influence of IL-4 and IL-13. IL-10 acts in strong synergy with IL-4 and IL-13 as a key regulator of CCL18 production by monocytes, which indicates that CCL18 secretion is not confined to fully developed DC and MΦ. In addition, the effects of IL-4, IL-13 and IL-10 are strongly enhanced by RASF, which is due to yet unidentified factors. Both the in vivo expression pattern and the contributing factors to its regulation in vitro are suggestive for a role for CCL18 in the regulation of the immune system, both in health and auto-immune diseases such as RA.
Methods
Patients and samples
For cell culture experiments, 50 ml peripheral blood was taken from healthy volunteers and RA patients after receiving informed consent in 10 ml lithium heparine (Vacutainer, USA) tubes. Synovial biopsies from RA patients were taken with small needle arthroscopy (Storz, Tutlingen, Germany). Synovial fluid from RA patients was obtained during arthroscopy. For our experiments in which monocytes were stimulated with SF, a pool of SF from 10 different RA patients was used. Synovial samples from healthy controls were taken during scheduled arthroscopic procedures by orthopedic surgeons in patients with traumatic knee injuries. The Nijmegen medical ethics committee (MEC) approved these studies.
Recombinant proteins and antibodies
For stimulation of iDC, we used 20 ng/ml recombinant (rh) IL-1β, rhTNF-α, rhIL-10, rhIL-13, rhIL-15, rhIL-17, rhIL18, 10 ng/ml IFN-γ (all R&D systems, Minneapolis, USA), or 20 ng/ml RANKL and CD40L (Pepro Tech, Rocky Hill, USA). DCs were cultured with 500 U/ml IL-4 and 800 U/ml GM-CSF. The same IL-4 concentration was used for monocyte stimulations. For Toll-like receptor stimulation, 10 μg/ml pam
3cys (TLR2), 25 μg/ml poly (i:c) (TLR3), 2 μg/ml lipopolysacharide (LPS) (TLR4), or 1 μg/ml R848 (TLR7/8) was used [
31]. Blockade of IL-10 (Ebioscience, San Diego, USA) and blockade of IL-13 (Diaclone, Becanson, France) was achieved with a 1000× excess of a neutralizing antibody. For FACS analysis, we used mouse-anti human antibodies against CD14, (Dako, Glostrup, Denmark), CD83 (Beckman Coulter, Mijdrecht, The Netherlands), IL-4Rα (Santa Cruz, California USA), IL-13Rα II (R&D systems, Minneapolis, USA) and IL-10Rα (R&D systems, Minneapolis, USA) or mouse-isotype control (goat IgG for IL-13RαII). For ELISA, mouse anti-human and biotynilated goat anti-human CCL18 were used as capture and detection antibody (R&D systems, Minneapolis, USA). A standard curve was made with rhCCL18 (R&D systems, Minneapolis, USA). Immuno histochemistry for CCL18 was performed with AZN-CK18B [
18] as a primary antibody.
Monocyte/macrophage and MoDC isolation and culture
MoDC were cultured using essentially the same protocol as described previously [
13,
40]. In brief, peripheral blood mononuclear cells were isolated from venous blood by density gradient centrifugation over Ficoll-Hypaque (Amersham Biosciences, Roosendaal, The Netherlands). The interphase was collected and washed with citrated phosphate buffered saline, and the cells were allowed to adhere for 1 hour at 37°C in RPMI-1640 (Life Technologies, Breda, The Netherlands) supplemented with 2% human serum in culture plates (Costar, Badhoeverdorp, The Netherlands). Adherent cells were cultured in RPMI-1640 Dutch modification supplemented with 10% fetal calf serum L-glutamine (Life Technologies, Breda, The Netherlands) and antibiotic-antimycotic agents (Life Technologies, Breda, The Netherlands) (culture medium) in the presence of IL-4 (500 U/ml; Strathmann Biotech, Hamburg Germany) and granulocyte monocyte-colony stimulating factor (GM-CSF) (800 U/ml; R&D systems, Minneapolis USA) for 6 days. Fresh culture medium with the same supplements was added at day 3, and then iDC were harvested at day 6. To generate mature DC, immature DC were re-suspended in a concentration of 0,5 × 10
6/ml in fresh IL-4 and GM-CSF containing culture medium. Immature DC were then stimulated with cytokines or maturation stimuli in the presence of IL-4 and GM-CSF. DC were harvested after another 48 hours of culture. For CCL18 measurements in supernatant of cells stimulated with TLR ligands, aliquoted culture supernatant from previous experiments was used [
31].
For the culture of monocytes/macrophages, CD14+ cells were isolated with magnetic cell sorting and separation (MACS). In brief, mononuclear cells were labelled with anti CD14 microbeads (Miltenyi Biotec, Amsterdam, the Netherlands) and incubated for 30 minutes at 4°C. CD14 positive cells were then separated from the other cells using a MACS column (Miltenyi Biotec, Amsterdam, the Netherlands) according to the manufacturers instructions. CD14+ cells were cultured in a concentration of 0,5 × 10
6 cells/ml in culture medium for up to 6 days. Where appropriate, fresh culture medium was added on day 3. After 6 days, supernatant was collected for ELISA and cells were prepared for FACS analysis. In some additional experiments, monocytes/macrophages were cultured for three days in the same concentration and in the same media in teflon bags [
34] or in rotation discs (Cellon, Luxembourg) [
35] to prevent adherence of the cells. In experiments in which monocytes/macrophages were stimulated with RA SF, the cells were cultured for three days in the presence of 100 μl RA SF. Cells were then harvested and re-suspended in fresh culture medium without SF, but with the cytokines that were present in the first three days. Anti-IL-10 or anti-IL-13 neutralizing antibodies were only present during the first three days.
Immuno histochemistry
For immuno histochemistry, frozen ST was cut into 7 μm sections and mounted on slides, air-dried, and stored at -80°C. Before staining, the cryosections were air-dried, fixed in acetone for 10 min and air-dried again. The sections were then stained with 5 μg/ml mouse anti human CCL18 or isotype control at 37°C for 1 hour at room temperature (RT) and washed in PBS. Endogenous peroxidase was blocked with 0,3% H2O2/methanol. After another wash-step, the sections were incubated with a biotin-conjugated horse anti-mouse antibody at RT for 30 min. Next, the samples were washed and incubated with avidin-biotin-HRP complex (Vector, Burlingham, UK) at RT for 20 min. Next, the section were stained with diaminodenzidine (DAB) (Sigma, Zwijndrecht, the Netherlands). Finally, sections were then counterstained with hematoxylin, rehydrated and mounted in to allow microscopic evaluation of the samples.
Fluorescence-Activated Cell Sorter (FACS) analysis
The phenotype of cells was characterized by using flow cytometry techniques (FACS). For this aim, cells were harvested and collected by means of centrifugation and further processed on melting ice. Cells were diluted in buffer solution (PBS with 1% bovine albumine, pH 7,4) in a concentration of 1.106 cells/ml and plated in v-shaped 96 wells plates (1.105 cells per plate). Cells were labeled with monoclonal mouse- or goat anti human antibodies or mouse-isotype control (goat IgG for IL-13RαII) and incubated at a temperature of 4°C for 45 minutes. Cells were then washed and labeled with goat-anti-mouse (or rabbit anti-goat when appropriate) FITC (Zymed Laboratories, South San Francisco, USA) as a secondary antibody. After another 30 minute incubation at 4°C, cells were again washed, diluted in buffer solution and transferred into FACS tubes. Cells were gated according to their forward and side scatters and fluorescence was measured with a FACSCalibur® (Becton-Dickinson, San Jose, USA) and Cellquest® software.
Enzyme Linked Immuno Sorbent Assay (ELISA)
For the detection of CCL18 protein levels of CCL18 in supernatant, a sandwich ELISA was performed as described previously [
18,
50]. In brief, maxisorb ELISA plates (Nunc, Roskilde, Denmark) were coated overnight with 50 μl/well 1 μg/ml capture antibody in PBS. Next, the plates were washed 3 times with PBS and blocked with 300 μl 1% Bovine Albumin (Sigma, Zwijndrecht, the Netherlands) in PBS for a minimum of 1 h at RT. After washing 3 times with ELISA wash buffer (PBS containing 0.05% Tween-20), the plates were incubated with 50 μl/well of serial dilutions of the sample for 2 hrs at RT. Serial dilutions of rhCCL18 were used to obtain a standard curve. After washing 3 times with ELISA wash buffer, the plates were incubated with 50 μl/well of 0.05 μg/ml secondary antibody at RT for 1 hr. Thereafter, the plates were washed 3 times with ELISA wash buffer, and incubated with 50 μl/well of streptavidin conjugated to Poly-Horse Radish Peroxidase (CLB, Amsterdam) for 20 minutes at RT. After washing 3 times with ELISA wash buffer, the presence of HRP was detected using 50 μl/well 3,3',5,5-tetramethylbenzidine (TMB) (Biomerieux, Marcy l'Etoile, France) diluted in peroxide buffer (UP) (Biomerieux, Marcy l'Etoile, France). The reaction was stopped with 50 μl/well 2,5M H
2SO
4. Absorbance was measured at 450 nm using a Magellan Tracker V4.XX (Tecan Austria GMBH). As an internal control for inter-assay variability, a sample of pooled normal human serum (n = 300) was taken along in all assays. The maximal accepted inter-assay variability is 10%. The detection limit of the ELISA is 100 pg/ml.
Statistical analysis
CCL18 production levels by monocyte derived cells upon different stimulations were compared with a Wilcoxon Signed Rank test. P-values < 0,05 were considered significant.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
AvL performed the experiments, provided the RA synovial biopsies, designed the study and wrote the manuscript. RvdV co-designed the study and edited the manuscript. LlB performed and co-designed experiments. MR performed and designed all TLR experiments. BS provided synovial tissue from controls and edited the manuscript. PvR supervised the study and edited the manuscript. GA co-designed the study and edited the manuscript. TR co-designed the study and edited the manuscript. All authors read and approved the manuscript.