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Leflunomide inhibits transendothelial migration of peripheral blood mononuclear cells
  1. J Grisar1,
  2. M Aringer1,
  3. M D Köller1,
  4. G H Stummvoll1,
  5. D Eselböck1,
  6. B Zwölfer1,
  7. C W Steiner1,
  8. B Zierhut2,
  9. L Wagner2,
  10. P Pietschmann3,
  11. J S Smolen1
  1. 1Division of Rheumatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria
  2. 2Division of Endocrinology, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria
  3. 3Department of Pathophysiology, Medical University of Vienna, Vienna, Austria
  1. Correspondence to:
    Dr J Grisar
    Division of Rheumatology, Department of Internal Medicine III, Medical University of Vienna, Waehringer Guertel 18–20, A-1090 Vienna, Austria; johannes.grisarmedun-wien.ac.at

Abstract

Objectives: To test whether the active metabolite of leflunomide (LEF-M), in addition to blocking the proliferation of activated lymphocytes by inhibiting dihydro-orotate dehydrogenase (DHODH), influences the transendothelial migration (TEM) of peripheral blood mononuclear cells (PBMC).

Methods: In an in vitro model of PBMC transmigration through an endothelial cell (EC) barrier, PBMC were re-collected in three groups: cells not adherent to the EC, cells bound to, and cells which had migrated through, the EC layer. Experiments in which cells were pretreated with LEF-M (in the absence or in the presence of uridine) were compared with parallel experiments in the presence of medium alone.

Results: Preincubation of EC with LEF-M led to a 36 (SEM 16)% reduction in PBMC TEM (p<0.05). Likewise, preincubation of PBMC induced a reduction in their TEM of 39 (9)% (p<0.005). Incubation of both PBMC and EC with LEF-M had an additive effect (mean reduction of 48 (6)%, p<0.005). Incubation of PBMC with LEF-M also decreased monocytic CD44 expression (p<0.005) and PBMC-hyaluronan binding (p<0.05). Incubation of cells with LEF-M and uridine in addition to LEF-M reversed the inhibition of migration, suggesting that the observed effects were due to DHODH inhibition. Fluorocytometric analysis of PBMC subsets within the migrated population showed a decrease of monocytes, but not of B or T cells, after LEF-M treatment.

Conclusions: LEF-M reduces monocytic adhesion molecule expression and TEM and may thus interfere with monocyte and EC activities in RA. Thus, the clinical effects of leflunomide may, at least in part, be due to blocking cell traffic into the inflamed synovia.

  • BND, bound
  • Dex, dexamethasone
  • DHODH, dihydro-orotate dehydrogenase
  • DMARD, disease modifying antirheumatic drug
  • EC, endothelial cell(s)
  • LEF-M, metabolite of leflunomide, A771 726
  • MCP, monocyte chemotactic protein
  • MFI, mean fluorescence intensity
  • MIG, migrated
  • MIP-1α, macrophage inhibitory protein-1α
  • MTX, methotrexate
  • NAD, non-adherent
  • NF-κB, nuclear factor κB
  • PBMC, peripheral blood mononuclear cell(s)
  • PBS, phosphate buffered saline
  • RA, rheumatoid arthritis
  • TEM, transendothelial migration
  • leflunomide
  • A77 1726
  • transendothelial migration
  • endothelium
  • peripheral blood mononuclear cells

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Although rheumatoid arthritis (RA) is a systemic disease, most of its pathology of takes place within the joints.1,2 One of the hallmarks of RA is the accumulation of inflammatory cells and subsequent hypercellularity in the synovial membrane, and the replenishment of these cells is an important process in the continuing inflammation of RA.1–3 To a great extent such recruitment to inflammatory sites is mediated by endothelial events1 and, in fact, blocking the transendothelial migration (TEM) by monoclonal antibodies to adhesion molecules constitutes an interesting therapeutic approach in RA.4 Both endothelial cells (EC) and leucocytes participate in the process of migration, which can be mimicked, at least in part, in vitro.5

Leflunomide is a new disease modifying antirheumatic drug (DMARD).6,7 Its active metabolite, A771 726, blocks the enzyme dihydro-orotate dehydrogenase (DHODH),8 thereby inhibiting the de novo synthesis of pyrimidines. Because pyrimidine synthesis is of critical importance to allow cell division, leflunomide appears to target proliferating cells such as activated T cells.9 Leflunomide also inhibits nuclear factor κB (NF-κB), a transcription factor activated by proinflammatory cytokines.10

Recent studies of synovial biopsies and magnetic resonance imaging in patients with RA undergoing DMARD treatment have shown that leflunomide, when compared with baseline and even with methotrexate treatment, significantly reduced synovial cellularity.11,12 Although this finding could be explained by the effect of leflunomide on proliferating cells or by effects on cytokine production or other downstream events, we wondered if leflunomide might also affect peripheral blood mononuclear cell (PBMC) migration.

In this study we show that leflunomide significantly reduces TEM and that the mode of action of this DMARD is due to effects on both EC and PBMC.

MATERIALS AND METHODS

Cell cultures

EC were isolated from human umbilical cord veins by collagenase digestion, as previously described,13 and then cultured in MCDB-M 104 medium (Gibco, Paisley, United Kingdom) supplemented with 20% fetal calf serum, 24 μg/ml EC growth supplement (TC Laevosan, Vienna, Austria), 50 U/ml heparin, l-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 μg/ml; all from Gibco). For all experiments EC in the third to fifth passage were used. To form a confluent monolayer on the collagen gels (see below), 5×105 EC per well were incubated for 24 hours.

PBMC were obtained from heparinised venous blood of healthy volunteers and of patients with RA fulfilling the 1987 American College of Rheumatology criteria, by density gradient centrifugation (Ficoll-Paque plus; Amersham Pharmacia Biotech AB, Uppsala, Sweden). For additional experiments, highly purified (to >95%) monocyte and/or T cell populations were prepared from the PBMC using negative selection by magnetic cell sorting (monocyte isolation kit and pan T cell isolation kit, MACS; Miltenyi, Bergisch Gladbach, Germany). PBMC or T cells or monocytes were then cultured for 24 hours in 33 mm macrowell plates (Nunc A/S, Roskilde, Denmark).

In some experiments, PBMC, T cells, monocytes, or EC were incubated with the active metabolite of leflunomide, A771 726 (LEF-M, courtesy of Aventis, Strasbourg, France), at different concentrations. Unless mentioned otherwise, 100 μM LEF-M was used. Among tested concentrations in the range of therapeutic serum levels, this concentration was found to give maximal effects without toxicity on PBMC (see below).

For comparative purposes, PBMC and EC were preincubated with methotrexate (MTX; Alexis Corporation, Lausen, Switzerland) or dexamethasone (Dex; Sigma-Aldrich, St Louis, MI, USA) in concentrations from 0.1 to 10 µmol/l14 for MTX and 1-100 nmol/l15 for Dex, respectively.

Transendothelial migration and adhesion capacity

Cell migration was studied on hydrated bovine collagen gels in 16 mm macrowell tissue culture plates (Nunc A/S), as previously described.5,13 To investigate the degree of TEM, PBMC (3×106) were layered on top of collagen gels with EC and incubated at 37°C for 1 hour.13 We have previously shown that the EC layer is a decisive element in this assay and clearly enhances transmigration.13 Cells that were non-adherent (NAD) to EC were harvested by washing twice with culture medium. Cells bound (BND) to the EC surface were removed by washing twice with warm (37°C) Puck’s EDTA, twice with warm (37°C) EGTA (0.5x10−3 M EGTA in phosphate buffered saline (PBS)), and once with cold (4°C) Puck’s EDTA.5 Finally, for the recovery of those cells that had migrated (MIG) into the collagen gels, 0.7 ml of a collagenase solution (0.1% collagenase, Sigma) was added to each well. Then the collagen gels were gently minced with a pipette and incubated for 60 minutes at 37°C. After incubation, MIG PBMC were recovered by washing the wells twice with PBS. Each population (NAD, BND, and MIG) was washed, resuspended in culture medium, and subjected to cell counting and analyses of cell surface marker expression by flow cytometry (see below). The percentage decrease of MIG PBMC was calculated by the formula:

(1−(MIG LEF−M/MIG control))×100

Analyses of the influence of leflunomide on cell migration required PBMC preincubated with LEF-M and untreated EC, untreated PBMC, and pretreated EC, as well as coculture of both populations, either pretreated or untreated. In addition, we performed experiments, pretreating PBMC and EC with both LEF-M and uridine (at a concentration of 100 μmol/l) to test if uridine could rescue PBMC from the observed effects of LEF-M on TEM, which would suggest dependence on the enzyme DHODH.16 Addition of LEF-M to PBMC or EC immediately before testing for migration induced no significant change in cell migration (data not shown). Therefore, to imitate in vivo conditions, in those experiments where PBMC and/or EC were preincubated with LEF-M before the migration assay, cells were resuspended in medium containing LEF-M after washing them with PBS and before incubation on the monolayer.

To further test if LEF-M influenced the adhesion to EC, the following experiments were performed: EC with or without LEF-M were incubated overnight on gelatin covered 16 mm macrowell tissue culture plates (5×105 cells/well) without the presence of a subendothelial collagen gel. Subsequently PBMC, untreated or LEF-M pretreated, were added to the EC covered wells at 3×106 PBMC/well and incubated for 1 hour. Non-adherent cells were recovered by gentle washing, adherent cells by gentle mincing and washing; cells were then counted and analysed by flow cytometry.

To test for potential toxicity of LEF-M we analysed cell mortality by trypan blue exclusion after re-collecting cells from the monolayer. In addition, we incubated PBMC for 24 hours with and without 100 μM LEF-M and used a Coulter counter to determine cell loss (Coulter Electronics Ltd, Luton, UK).

Flow cytometry of PBMC

To characterise potential preferential adhesion or migration of PBMC subpopulations, PBMC and PBMC subsets re-collected in the respective cell populations were analysed by flow cytometry. Cells were stained with fluorescein isothiocyanate or phycoerythrin conjugated monoclonal antibodies against CD3 and CD14 (Becton & Dickinson, Sparks, MD, USA), and in some experiments also against CD4, CD8 (Becton & Dickinson), and CD19 (Immunotech, Westbrook, ME, USA), according to standard procedures.13 Analysis was performed on a Becton & Dickinson “FACScan” flow cytometer. Results are expressed as the percentage of positive cells, or when indicated, as the mean fluorescence intensity (MFI).

Analysis of the influence of LEF-M on PBMC and EC adhesion molecule expression and hyaluronan binding

PBMC or EC were incubated for 24 hours with LEF-M at a concentration of 100 μmol/l. Expression of the following cell surface molecules, which are known to have important roles in TEM, was measured as described above, using isotype matched appropriate non-specific antibodies (Becton & Dickinson) as negative controls: CD2, CD25, CD44 (Becton & Dickinson), CD11a, CD29, CD49d (Immunotech), CD18, CD69 (Serotec, Oxfordshire, United Kingdom), and CD62L (Pharmingen, San Diego, CA, USA) were analysed on PBMC. Anti-CD29 and anti-CD44 and antibodies to CD50 (ICAM-3), CD54 (ICAM-1), CD62E (E-selectin), CD62P (P-selectin), CD102 (ICAM-2), CD106 (from Serotec), and HLA-DR (Beckmann Coulter, Fullerton, CA, USA) were used to analyse EC.

To test the influence of LEF-M on the function of CD44, we determined the binding of fluorescein conjugated hyaluronic acid (Molecular Probes Inc, Eugene, OR, USA) to untreated or LEF-M pretreated PBMC by fluorescence activated cell sorter analysis.

Results are expressed as the MFI.

Chemotaxis

To determine, if LEF-M would also influence basal chemotaxis, chemotaxis assays were performed: 2×104 PBMC or 1×104 negatively selected monocytes from healthy donors, untreated or pretreated with 100 μM LEF-M for 24 hours, washed and resuspended in RPMI 1640 containing 1% bovine serum albumin, were added to the top of a chemotaxis chamber (Neuro Probe Inc, Gaithersburg, MD, USA). 30 μl of 10-fold serially diluted (0.1–1000 ng/ml) monocyte chemotactic protein-3 (MCP-3; R & D Systems, Wiesbaden, Germany) or, for PBMC only, MCP-1 plus macrophage inhibitory protein-1α (MIP-1α; both Sigma-Aldrich) was placed in the bottom wells of the chamber, separated from the cell suspension by a polycarbonate filter (5 μm pore size). The chamber was incubated at 37°C and 5% CO2 for 90 minutes. Cells which migrated were then counted in the bottom well in duplicate samples.

Statistical analyses

Where applicable, data are presented as mean (SEM). A paired Student’s t test or, when distributions were not normal, a Kruskal-Wallis test was used for the statistical analyses.

RESULTS

Leflunomide reduces transendothelial migration of PBMC

To test if leflunomide affected the TEM of PBMC, both PBMC and EC were preincubated for 24 hours with 100 μM LEF-M (established as the optimal dose in dose-response analyses) before the migration assay. In healthy volunteers, a significant decrease in the percentage of migrated PBMC from a mean (SEM) of 17 (3)% (range 6–47%) without LEF-M to a mean of 7 (1)% (range 3–26%) with LEF-M pretreatment (p<0.005) was seen (fig 1). Incubation of PBMC of patients with RA with LEF-M likewise led to a significant reduction in TEM (16 (2)% without LEF-M v 12 (2)% with LEF-M of migrated PBMC, ranges: 7–30% without v 5–28% with LEF-M, p<0.005, paired t test).

Figure 1

 LEF-M decreases the transendothelial migration of PBMC both in healthy subjects and patients with RA. In 14 experiments, EC and PBMC of healthy volunteers were preincubated with 100 μM LEF-M for 24 hours. A significant decrease in the percentage of migrated PBMC was found as compared with control experiments (17 (3)% v 7 (1)%, p<0.005).

As expected, the proportion of the NAD PBMC increased accordingly (57 (5)% without v 69 (3)% with LEF-M, p<0.01); the BND population was not significantly influenced.

The observed decrease in spontaneous TEM was not due to toxic effects of LEF-M, because no significant difference in the percentage of trypan blue positive cells was seen (9 (3)% without v 10 (2)% with LEF-M, p = NS). Also, when counting pretreated and untreated PBMC after 24 hours’ incubation no significant difference between the two groups was detected (PBMC incubated in medium: 100%, PBMC incubated with LEF-M: 107%, mean of six experiments).

MTX does not inhibit TEM

Preincubation of PBMC and EC with MTX showed that this DMARD did not significantly reduce TEM (fig 2) compared with control, suggesting that the pronounced inhibition in TEM is not common to all DMARDs. In contrast, Dex was highly effective in inhibiting TEM (fig 2).

Figure 2

 MTX does not significantly affect TEM (MTX at all doses v controls p = NS), contrasting with the effect of LEF-M (p<0.02 v control). Dex was highly effective in TEM inhibition (p<0.02 at doses of 1 and 10 nmol/l Dex v controls, p<0.05 at a dose of 100 nmol/l Dex v control).

PBMC and EC are affected by leflunomide

To elucidate if LEF-M interfered with PBMC, EC, or both, a series of experiments preincubating the populations separately was performed. When PBMC, but not EC, were preincubated with LEF-M for 24 hours, the frequency of migrated cells decreased by a mean (SEM) of 39 (9)% (p<0.005) compared with untreated PBMC (fig 3). Likewise, when only EC were preincubated with LEF-M, migration also tended to decrease by 36 (16)% (p<0.05) compared with migration on untreated EC (fig 3). Thus, LEF-M inhibited cell migration through events both in PBMC and EC. However, each of these effects was somewhat less pronounced than the simultaneous incubation of both cell populations with LEF-M (mean percentage inhibition of migration 48 (6)%, p<0.005), suggesting additive effects.

Figure 3

 Treatment of either PBMC or EC with LEF-M decreases the transendothelial migration of PBMC. Preincubation for 24 hours of PBMC or EC or PBMC and EC with 100 μM LEF-M induced a significant decrease in the percentage of migrated cells (PBMC 39 (9)% v 48 (6)%, p<0.005; EC: 36 (16)% v 48 (6)%, p<0.05).

Effects of leflunomide are inhibited by addition of uridine

Next, we tested if the impairment of TEM could be prevented by incubation in the presence of uridine as an external pyrimidine source. When EC and PBMC were incubated with both LEF-M and uridine, the decrease in TEM, compared with simultaneous control experiments with LEF-M only, was almost completely inhibited (fig 4).

Figure 4

 The effect of transendothelial migration is reversed by uridine. When PBMC and EC were incubated for 24 hours with both LEF-M and uridine, transendothelial migration significantly increased as compared with parallel control experiments where cells were incubated solely with LEF-M (decreased by 48 (8)% with LEF-M v 8 (1)8% with LEF-M and uridine, p<0.025). However, no significant decrease between untreated PBMC and PBMC preincubated with LEF-M and uridine was seen.

Effects of leflunomide on monocyte and lymphocyte migration and adhesion

We then analysed which PBMC subsets were preferentially influenced within their migration behaviour by LEF-M. After incubation with LEF-M, the percentage of migrated CD14 positive cells (that is, monocytes) was significantly decreased by 15 (4)% (p = 0.006) compared with migration in the absence of LEF-M, while B and T cell subsets were similarly distributed among the migrated populations preincubated with or without LEF-M (fig 5).

Figure 5

 LEF-M decreases the migration of CD14 positive cells. In the presence of LEF-M the percentage of CD14 positive cells (that is, monocytes) in the migrated population significantly decreased (by 15 (4)%, p = 0.006), whereas T and B lymphocytes showed no altered cell counts in the migrated fraction.

This observation was further supported by data obtained in analyses of adhesion experiments: In these experiments the percentage of PBMC adherent to an EC monolayer decreased from 51 (8)% without LEF-M to 40 (6)% after pretreatment with LEF-M (p<0.05). When analysing the subsets, CD14 positive cells among the adherent PBMC decreased from 9 (2)% without, to 5 (1)% with, LEF-M treatment (p<0.05). These data were confirmed by using highly purified monocytes whose potential to transmigrate was significantly inhibited by increasing dosages of LEF-M to a maximal degree of 55% at a concentration of 100 μmol/l, results similar to those obtained by staining for CD14+ cells among transmigrated PBMC (fig 6).

Figure 6

 Dose-response of LEF-M. Dose-response experiments with highly purified monocytes indicated that a dose of 100 μmol/l LEF-M clearly inhibits TEM. This concentration was used for all other experiments.

Many ligands and receptors are known to have important roles in the different steps of extravasation (adhesion, rolling, binding, shearing) (tables 1 and 2). Several adhesion molecules on PBMC tended to decrease with LEF-M pretreatment, but most of these differences were not significant. However, CD44 expression on PBMC, and CD102 and HLA-DR expression on EC decreased significantly (tables 1 and 2).

Table 1

 Surface adhesion molecules on PBMC incubated with 100 μM LEF-M for 24 hours v control experiments

Table 2

 Surface adhesion molecule expression on EC incubated with 100 μM LEF-M for 24 hours v control experiments

Effects on adhesion and hyaluronan binding

As we observed a mild but significant decrease in the expression of CD44 after incubation with LEF-M (MFI 351 (15) without LEF-M v 325 (18) with LEF-M, p<0.05; table 1), further experiments were performed, which showed that this alteration in CD44 expression was limited to monocytes (MFI of CD44 on CD14 positive cells 949 (66) without v 885 (66) with LEF-M, p<0.005). This decrease was paralleled also by a significant decrease in receptor functions—namely, binding of hyaluronic acid (MFI 23 (6) without LEF-M v 17 (4) with LEF-M, p<0.05), as observed in hyaluronan binding experiments. Thus, the reduction in CD44 expression had a functional equivalent in reduced hyaluronan binding.

Chemotaxis is not significantly influenced by LEF-M

We also investigated the potential influence of LEF-M on the chemotaxis of both PBMC and monocytes. To this end, increasing doses of MCP-3 (fig 7) or a combination of MCP-1 and MIP-1α (not shown) were used. Neither PBMC nor purified monocytes were affected in their chemotactic behaviour by LEF-M (fig 7). These data supported the notion that the altered migratory behaviour, of PBMC in general and of monocytes in particular, was not due to effects on chemotaxis but rather to other mechanisms such as the changes in adhesion described above.

Figure 7

 Leflunomide does not influence the chemotaxis of PBMC or monocytes. When chemotaxis induced by increasing concentrations of MCP-3 was assayed for PBMC (A) or monocytes (B), no difference could be seen between untreated cells or cells pretreated with 100 μM LEF-M.

DISCUSSION

The data obtained show a novel mode of action of leflunomide—namely, interference with TEM. This effect is seen when PBMC, whether derived from healthy subjects or RA donors, are preincubated with LEF-M. LEF-M also affects EC, although to a slightly smaller degree. However, the effect is particularly prominent when both EC and PBMC are exposed to LEF-M, as is the case in vivo. Under such circumstances PBMC migration is reduced by nearly 50%. Others have found similar effects on isolated neutrophil cell populations.17 MTX did not affect TEM, indicating differences in the cellular effects of the two drugs, which might help to explain the benefit of the combination of both drugs in patients with RA.18

The accumulation of lymphocytes and monocytes/macrophages in the synovial membrane is a pivotal event in RA, because these cells fuel the inflammatory process and secrete various cytokines. Among PBMC, the relative effects of LEF-M on migration are particularly pronounced on monocytes. These cells are of major pathogenic importance because of their large accumulation in the RA synovial membrane, their secretion of proinflammatory cytokines,19 and because they are precursor cells for osteoclast differentiation.20 These effects of LEF-M were confirmed when purified monocyte populations were used. Thus, the overall reduction in cell migration and the additional particular effects on the proportion of migrating monocytes/macrophages help to explain the clinical efficacy of leflunomide on joint inflammation and destruction.6,7,21 Studies of synovial biopsies have recently shown a decrease in synovial cellularity in patients treated with leflunomide.11 The concentration of LEF-M used in this study was the optimal inhibitory dose and within the therapeutic range.22

The effects observed seem to be mediated by the primary mode of action of LEF-M—namely, inhibition of DHODH, because uridine, which counteracts the DHODH mediated reduction in de novo pyrimidine synthesis,16 rescues cells from the inhibitory activity of LEF-M. Because there is evidence that DHODH inhibition changes the composition of the cell membrane,23 LEF-M may also alter cell membrane plasticity in addition to cell surface molecule expression. Thus, conceivably, the reduction in CD44 expression on monocytes has an important role. Because CD44 is ultimately involved in TEM,24–26 a decrease of its expression may have major effects on cell accumulation at inflammatory sites. Moreover, the down regulation of CD44 is also accompanied by decreased hyaluronan binding of PBMC, indicating that the LEF-M associated alteration of CD44 expression is associated with functional changes. This is supported by observations in experimental arthritis, where administration of anti-CD44 antibodies reduces arthritic activity.27–29 In addition, CD102 (intercellular adhesion molecule-2 (ICAM-2)), an adhesion molecule that has an important role in transmigration and costimulatory signalling,30 was down regulated on EC. In accordance with the data presented, LEF-M also appears to affect cell adhesion.31,32 Each of these mechanisms alone may be of limited influence; however, taken together these actions appear to exert a significant overall effect, which is manifested in a significant reduction in transendothelial migration and, consequently, in anti-inflammatory action. Some of the effects of LEF-M in these events may be due to its inhibitory activity on the glycosylation of adhesion molecules.33 Likewise, NF-κB, a transcription factor induced by proinflammatory cytokines, which is involved in adhesion molecule expression,34 is also inhibited by LEF-M.10,35 Importantly, however, chemotaxis, another important event that triggers TEM, was not affected by LEF-M.

In conclusion, LEF-M inhibits TEM of inflammatory cells in a DHODH dependent manner, at least in part by reducing adhesion and transmigratory capacity owing to its effects on adhesion molecules. In conjunction with its inhibitory activity on proliferation, tumour necrosis factor production, and NF-κB, these findings add yet another dimension to the molecular and cellular basis underlying the clinical effects of this DMARD.

Acknowledgments

We thank Aventis for kindly providing us with LEF-M.

REFERENCES

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