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. 2004 Dec;63(12):1632–1637. doi: 10.1136/ard.2003.018440

Leflunomide inhibits transendothelial migration of peripheral blood mononuclear cells

J Grisar 1, M Aringer 1, M Koller 1, G Stummvoll 1, D Eselbock 1, B Zwolfer 1, C Steiner 1, B Zierhut 1, L Wagner 1, P Pietschmann 1, J Smolen 1
PMCID: PMC1754829  PMID: 15547088

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.

Full Text

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Figure 1.

Figure 1

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 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).

Figure 2.

Figure 2

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 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).

Figure 3.

Figure 3

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 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).

Figure 4.

Figure 4

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 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.

Figure 5.

Figure 5

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 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.

Figure 6.

Figure 6

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 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.

Figure 7.

Figure 7

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 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.

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