Introduction
Dendritic cells (DCs) are the most potent antigen-presenting cells in the immune system [
1,
2]. They represent a heterogeneous population of bone-marrow-derived cells located in lymphoid as well as in nonlymphoid organs. In peripheral tissues these antigen-presenting cells are immature and are functionally equipped to capture and process antigens. DCs are activated by pathogen-associated microbial patterns such as lipopolysaccharide (LPS) or by proinflammatory cytokines such as tumour necrosis factor (TNF)-α, and via the interaction of CD40 with its ligand (CD154), which is expressed on activated T cells [
3]. Mature DCs possess optimal immunostimulatory properties because of maximal expression of their antigen-presenting and co-stimulatory molecules (i.e. CD40, CD80 and CD86) and their increased production of proinflammatory cytokines, including IL-12 and TNF-α. In contrast to the central role played by mature DCs in the initiation of primary immune responses, immature DCs stimulate T-cell responses only weakly or they may even induce tolerance to potential autoantigens [
4].
Pharmacological modulation of DC activation has been demonstrated to prevent disease progression in several T-cell-mediated diseases [
5], and it may therefore represent a promising approach to specific treatment of immunological disorders [
6,
7]. Notably, corticosteroids and another well known antirheumatic drug, namely gold thiomalate, significantly inhibit DC function, which may contribute to their clinical effectiveness [
8,
9].
Leflunomide is a novel disease-modifying antirheumatic drug that exerts its effects after metabolic opening of the isoxazole ring via its active metabolite A77 1726 (LEF-M). Its major target is supposed to be dihydro-orotate-dehydrogenase (DHODH) [
10], which is a key enzyme in
de novo pyrimidine synthesis. Leflunomide reversibly inhibits DHODH activity with subsequent depletion of nucleotides, leading to cell cycle arrest in proliferating lymphocytes [
11]. This effect can be reversed to a certain degree by supplying the product of DHODH activity (i.e. uridine) to target cells. Other targets of LEF-M are tyrosine kinases such as Lck or JAK3 in activated T and B cells [
12]. Immunosuppressive effects of leflunomide have been described including, inhibition of T cells and antibody production [
13]. Furthermore, it was demonstrated that leflunomide blocks activation of nuclear factor-κB (NF-κB), which is a central proinflammatory transcription factor in several cell lines [
14], and impairs transendothelial migration of peripheral blood mononuclear cells [
15]. Apart from its well established beneficial effects in the treatment of rheumatoid arthritis (RA) [
16,
17], leflunomide is also effective in treatment against chronic allograft rejection [
18,
19].
DCs were postulated to play an important role in RA pathogenesis because they may perpetuate the disease by presenting self-antigen(s) [
20,
21]. Thus, DCs could represent an interesting target for dampening the disease process in RA. Moreover, DCs also play a fundamental role in allograft rejection [
22].
Because the effect of leflunomide on DC function has not yet been investigated, we analyzed the influence of leflunomide on the complete DC life cycle in vitro. We found that LEF-M potently altered the phenotype and function of DCs, independent of its well known antimetabolite activity, revealing a novel immunomodulatory activity of this agent with potential clinical implications for the treatment of RA and other immune cell mediated disorders.
Materials and methods
RPMI 1640 (GIBCO BRL, Grand Island, NY, USA) supplemented with 2 mmol/l L-glutamine, 100 μg/ml streptomycin, 100 U/ml penicillin and 10% foetal calf serum (FCS; Hyclone, Logan, UT, USA) was used as culture medium. LPS (
Escherichia coli 0111:B4) and uridine were purchased from Sigma Chemie GmbH Co. (Deisenhofen, Germany). Recombinant human (rh) granulocyte–macrophage colony-stimulating factor (GM-CSF) was obtained from Schering-Plough (Kenilworth, NJ, USA) and rh-IL-4 was from Strathmann Biotech GmbH (Hannover, Germany). Plasma concentrations in RA patients of A77 1726 (the active metabolite of leflunomide) achieved with a leflunomide maintenance dose of 20 mg/day are 46 ± 31 μg/ml (approximately 150 ± 100 μmol/l [
23]). Therefore, we chose concentrations from 75 to 150 μmol/l of A77 1726 (kindly provided by Aventis, Strasbourg, France) for DC treatment. A77 1726 is referred to as 'LEF-M' throughout the report. In some experiments uridine was added to test the reversibility of the observed effects of LEF-M.
Cell preparation and culture
Peripheral blood mononuclear cells were obtained from buffy coats of healthy blood donors (courtesy of the Austrian Red Cross) by density gradient centrifugation over Ficoll-Paque PLUS (Amersham Biosciences, Uppsala, Sweden). For isolation of monocytes, peripheral blood mononuclear cells were depleted of T cells by sheep erythrocyte-rosetting overnight.
Monocytes (>85% CD14+) were cultured in six-well plates (Costar, Cambridge, MA, USA) at a cell density of 5 × 105 cells/ml in RPMI 1640/10% FCS medium in a humidified atmosphere containing 5% carbon dioxide. For induction of DC differentiation, the culture medium was supplemented for 5 days with 50 ng/ml rh-GM-CSF and 10 ng/ml rh-IL-4. For initation of maturation, LPS (100 ng/ml) was added for an additional 48 hours. For the DC differentiation and maturation experiments, different concentrations of LEF-M, or medium as control, were added either at the beginning of the culture or 6 hours before the addition of LPS. Cell viability was assessed by staining with propidium iodide (PI; Sigma, Saint Louis, MO, USA) and subsequent flow cytometric analysis of the cells.
Surface marker expression
For evaluation of surface marker expression, cells (50 μl at 5 × 106 cells/ml) were incubated with fluorescein isothiocyanate (FITC)-conjugated or phycoerythrin (PE)-conjugated mAbs for 45 min at 4°C. For control purposes, nonbinding isotype-matched FITC-conjugated and PE-conjugated mouse IgG (An der Grub, Kaumberg, Austria) were employed. After extensive washing cells were analyzed on a COULTER EPICS XL-MLC flowcytometer (Beckman Coulter, Fullerton, CA, USA) using EXPO32 software. All measurements were done using a three-colour setup, which was established using standard compensation procedures. FITC-labelled mAbs to CD1a (IgG1; clone HI149), CD14 (IgG2b; clone MΦP9), CD83 (IgG1; clone HB15e) and HLA-DR (IgG2a; L243), and R-PE-labelled mAbs to CD80 (IgG1; L307.4), CD86 (IgG2b; clone IT2.2) and CD206 (mannose receptor; IgG1; clone 19.2) were obtained from Becton Dickinson (San Diego, CA, USA). FITC-conjugated anti-CD40 (IgG1; clone LOB7/6) was purchased from Serotec (Oxford, UK). R-PE-labelled anti-major histocompatibility complex (MHC) class I antibody (IgG2a; clone 3F10) was obtained from Ancell (Bayport, MN, USA).
Morphological cell analysis
Microscopy was performed in parallel to all other analyses to assess cell morphology by using a light optical microscope (Olympus Corporation, Tokyo, Japan).
Assessment of T-cell stimulatory capability
Stimulator cells were irradiated (3000 rad, 137Cs source) and added at increasing cell numbers to 1 × 105 allogeneic T cells in 96-well culture plates in RPMI 1640 medium supplemented with 10% FCS (total volume 200 μl/well). After 4–5 days, cells were pulsed with 1 μCi [3H]thymidine (ICN Pharmaceuticals, Irvine, CA, USA). After another 18 hours the cells were harvested on glass-fibre filters (Packard, Meriden, CT, USA) and DNA-associated radioactivity was determined using a microplate scintillation counter (Packard, Meriden, CT, USA). DNA synthesis was expressed as mean counts/min of triplicate cultures.
Measurement of cytokine production
DCs were differentiated and subsequently activated (100 ng/ml LPS) in the presence or absence of different concentrations of LEF-M. Cell-free supernatants were harvested 48 hours after cell activation. Cytokines were measured by sandwich enzyme-linked immunosorbent assays using matched pair antibodies. Capture as well as detection antibodies to human IL-12p40 were obtained from R&D Systems (Minneapolis, MN, USA). Antibodies to human TNF-α were from PharMingen (San Diego, CA, USA). Standards consisted of human recombinant material from R&D Systems. Assays were set up in duplicate and were performed in accordance with recommendations from the manufacturers. The lower limit of detection was 20 pg/ml for all cytokines.
Analysis of nuclear factor-κB activation
NF-κB activation was assessed using an electrophoretic mobility shift assay (EMSA).
Nuclear extracts from DCs were prepared as described perviously [
24]. Oligonucleotides resembling the consensus binding site for NF-κB (5'-AGTTGAGGGGACTTTCCCAGGC-3') and activator protein-1 (5'-CGCTTGATGACTCAGCCGGAA-3') were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The double-stranded oligonucleotides used in all experiments were end-labelled using T4 polynucleotide kinase and [γ-
32P]-ATP. After labelling, 5 μg nuclear extract was incubated with 120,000 counts/min labelled probe in the presence of 3 μg poly(dI-DCs) at room temperature for 30 min. This mixture was separated on a 6% polyacrylamide gel in Tris/glycine/EDTA buffer at pH 8.5. Control experiments were performed as described previously [
25]. The specificity of NF-κB binding was proven using excess, unlabelled NF-κB probe that competed successfully for NF-κB binding, whereas an unrelated competitor (activator protein-1 oligonucleotide) did not (data not shown).
Statistical analysis
Comparisons were performed using two-tailed paired Student's t-tests. P < 0.05 was considered statistically significant.
Discussion
This study reveals a novel aspect of the immunomodulatory action of leflunomide, namely the profound interference of LEF-M (A77 1726) with DC function. Using human monocyte-derived DCs as a model system, we demonstrated that LEF-M disrupts differentiation of DCs from uncommitted monocytic precursor cells, resulting in maturation-insensitive DCs. Furthermore, we showed that the maturation process of uncommitted immature DCs was markedly impaired by LEF-M. The metabolite LEF-M differentially affected the expression of critical surface molecules, inhibited the production of proinflammatory cytokines and, at the functional level, profoundly impaired the T-cell stimulatory capacity of DCs. As a molecular basis for the ability of LEF-M to interfere with several aspects of DC function, the activation-driven nuclear transmigration of the essential transcription factor NF-κB was markedly impaired by LEF-M. These findings have substantial implications for our understanding of the effects of leflunomide as a disease-modifying antirheumatic drug, because the initiation of an immune response critically depends on proper DC function. Furthermore, interference with DC maturation and function could also be involved in the beneficial effects of leflunomide on chronic allograft rejection [
18], which is not shared by most other currently used immunosuppressive drugs such as calcineurin inhibitors.
The observation that DCs could play a pivotal role in the formation and maintenance of joint inflammation in RA [
31] was confirmed by the finding reported by Balanescu and coworkers [
32] of a correlation between co-stimulatory molecule expression of synovial DCs and disease activity in RA patients. Moreover, mature DCs might be central in the development of perivascular aggregates in synovial inflammation areas, the formation of organized lymphoid structures, and in the perpetuation of inflammatory and erosive activity [
20,
21]. Although there is sufficient evidence for an impact of leflunomide on synoviocytes, chondrocytes and osteoclasts [
33‐
36], our data suggest that the potent inhibition of DC function by LEF-M might contribute to the beneficial effects of leflunomide treatment in patients with RA.
Exposure of DCs to LEF-M led to an alteration in the surface marker profile. Our findings concerning the impact of LEF-M on critical co-stimulatory molecules might be especially important in RA because the expression level of co-stimulatory molecules on DCs correlates with disease activity in patients with RA [
32]. Another important finding in the present study was the observed disruption by LEF-M of the DC differentiation process. Interestingly, neo-expression of CD1a – the classic Langerhans cell-associated marker – was strongly inhibited in LEF-M-treated DCs. This finding is accordance with observations of significant efficacy of leflunomide in psoriasis [
37], in which CD1a is highly overexpressed in involved skin [
38]. Importantly, CD14 – a classic monocyte/macrophage marker – was downregulated, indicating that LEF-M does not subvert the DC differentiation programme toward macrophages as has been shown for IL-6, IL-10 and corticosteroids [
39,
40].
A central observation in our study was the functional alteration of DCs differentiated in the presence of LEF-M; these cells exhibited a marked reduction in their T-cell stimulatory capacity upon activation. These data indicate that LEF-M, by blocking the differentiation of monocytic precursors into mature DCs, potentially impairs proper DC function and might therefore modulate immune responsiveness against potential autoantigens and other antigens. Our finding of markedly decreased production of TNF-α and IL-12 by LEF-M-treated DCs, in conjunction with insufficient co-stimulatory molecule expression of DCs, may be of interest for further DC studies with LEF-M, because recent reports demonstrated this phenotype to be potentially tolerogenic [
41,
42].
Interestingly, we found the effects of LEF-M on DCs to be mediated independent of its inhibition of DHODH. As shown for several other leflunomide-mediated effects on other cell types, such as osteoclasts in the RA joint [
43], memory T-cell lines in an autoimmune encephalomyelitis model [
44] and in articular chondrocytes [
34], or on functional effects such as repression of viral replication [
45,
46], the inhibitory effects of LEF-M in the present study are clearly independent of pyrimidine synthesis.
The transcription factor NF-κB plays a decisive role in proper DC function. NF-κB translocation is essential to the ability of mature DC to present antigen to naïve T cells [
28,
29]. Recently reported data demonstrate that LEF-M inhibits TNF-α-induced NF-κB activation in several cell lines [
14,
47]. Interestingly, we found a profound suppression of NF-κB transactivation in activated DCs by LEF-M. These results are in accordance with our findings showing impaired expression of maturation markers and reduced allo-stimulatory capacity of leflunomide-treated DCs, because selective inhibition of NF-κB activity has been shown to impair maturation of DCs [
48]. Our findings concerning cytokine production are also consistent with NF-κB inhibition, because the human IL-12 promoter contains crucial NF-κB binding sites and TNF-α production is also NF-κB dependent [
49]. Although the mechanisms underlying this profound NF-κB inhibitory activity of LEF-M on DCs are currently unknown, it is tempting to speculate that leflunomide may interfere with phosphorylation/dephosphorylation events in the LPS-triggered signalling program. Apart from the possibility that LEF-M might directly induce the transcription of distinct IκB family members, LEF-M could also induce particular phosphatases to inhibit the IκB-inactivating kinase IKK. Furthermore, recent studies have shown that leflunomide acts at the level of IκB-α phosphorylation via interference with IKK-α activation, ultimately leading to defective IκB-α phosphorylaton. Although further studies are required to unravel the detailed molecular mechanisms of suppressed NF-κB transactivation in LEF-M-treated DCs, our findings indicate that NF-κB inhibition is a central feature of the molecular actions of LEF-M on DCs.
Importantly, the results from the present study were obtained with monocyte-derived DCs generated from healthy volunteers. Hence, further studies will be necessary to clarify the effects of LEF-M on peripheral and synovial DCs in experimental models of arthritis and on DCs obtained from RA patients. Nevertheless, our finding of DC inhibition induced by LEF-M reveals a novel view of the disease-modifying effects of this drug, which appear to act on both T cells and DCs. In fact, the involvement of DC–T cell interactions in the pathways leading to and perpetuating RA and the effects of inhibiting this process are supported by recent findings on the significant clinical effects of interference with CD80/86–CD28 co-stimulation [
50].
Conclusion
The present study shows that monocyte-derived DCs are sensitive targets of LEF-M, possibly by inhibitory effects on NF-κB. DCs are affected by LEF-M at all major stages in their life cycle, ultimately leading to an impairment in DC function. In addition to a direct inhibitory action on specific T-cell responses, modulation of the immune system may therefore also be explained through the effects of leflunomide on DCs rendering these cells less able to support immunoinflammatory responses. Thus, the versatile role played by leflunomide as an immunomodulatory agent in vitro and in vivo is further supported by its effect on DCs. These findings reveal a novel mode of action of the active leflunomide metabolite during induction of cellular immune responses, which may contribute to the clinical effectiveness of leflunomide in diseases that involve exaggerated immune responsiveness.
Acknowledgements
We thank Bianca Weissenhorn and Margarethe Merio for expert technical assistance.
This study was supported in part by grants of the Austrian Jubilee Fund (ÖNB 10282; to MDS), the Austrian Science Fund (P16788-B13; to TMS) and the Center of Molecular Medicine, a basic research institute within the companies of the Austrian Academy of Sciences (to TMS and JSS).
Authors' contributions
BK performed all flow cytometric and proliferation experiments, wrote the draft version of the manuscript and compiled the figures. MZ performed the uridine experiments. KS performed the electrophoretic mobility shift assays. JG analyzed the statistical data. JSS provided substantial input into the study design and helped in writing the manuscript. BW helped with statistical analysis and with finalizing the manuscript. WHH provided substantial input into the study design and helped with finalizing the manuscript. TMS was involved in all phases of the experimental process. GJZ performed the cytokine measurements. MDS designed the experiments, controlled all experimental steps and finalized the manuscript. All authors read and approved the final manuscipt.