Leflunomide is a synthetic, low molecular weight isoxazole derivative that is clinically applied for the treatment of rheumatoid arthritis (RA) due to its potent antiinflammatory and immunosuppressive properties (1–3). Furthermore, it is currently being evaluated for the treatment of inflammatory bowel disease (4) and as a novel immunosuppressive agent for the amelioration of chronic allograft rejection (5). Leflunomide is a prodrug that is rapidly converted into its active metabolite A77 1726, called “teriflunomide,” which constitutes >95% of the drug in the circulation. Two major mechanisms have been reported to underlie the inhibitory effects of leflunomide on T cell activation. First, teriflunomide blocks de novo pyrimidine synthesis by inhibiting dihydroorotate dehydrogenase (DHODH), which leads to diminished DNA synthesis and thus impaired proliferative capacity (6). Second, teriflunomide is an inhibitor of the Src family tyrosine kinases LCK and Fyn and, as such, brought about a diminished CD3-induced calcium response in Jurkat cells (7) and diminished interleukin-mediated signaling by inhibition of JAK-3 phosphorylation, as demonstrated in a murine cell line (8). However, LCK was shown to be dispensable for antigen-presenting cell (APC)–induced T cell activation and even to be a negative regulator of T cell receptor (TCR)–induced signals (9). Moreover, it is unknown whether teriflunomide exerts any effects on untransformed T cells, independent of de novo pyrimidine synthesis.
Successful T cell activation depends on prolonged stimulation by professional APCs, which is enabled by the formation of a so-called immunologic synapse (IS) (10). The IS is a supramolecular structure initiated by congregating of specific adhesion and signaling molecules at the contact site between the T cell and the APC, which facilitates activation and further differentiation of T cells (11). Stabilized T cell/APC conjugates are characterized by clustering of CD3 within the IS (12). Relocalization of T cell molecules to the T cell/APC interface is an active and highly regulated process, which is induced by the integration of signals derived from the TCR/CD3 complex and costimulatory receptors and requires cytoskeletal rearrangements (10, 13, 14). The β2 integrin lymphocyte function–associated antigen 1 (LFA-1) plays a dual and crucial role in the formation of T cell/APC conjugates. First, due to TCR-mediated signals, LFA-1 alters its adhesive state mainly by enhanced avidity, which is controlled by the actin cytoskeleton (15–17). This process of “inside-out signaling” leads to enhanced binding of LFA-1 to its ligand intercellular adhesion molecule 1 (ICAM-1) and thus adhesion to APCs (18,19). Second, ligand binding of LFA-1 itself transduces critical signals (“outside-in signaling”), which augment TCR/CD3-induced T cell adhesion, surface molecule expression, and interleukin production, and then lead to Th1 differentiation (20–22).
In the present study, we demonstrated a novel mechanism of action of the disease-modifying antirheumatic drug (DMARD) leflunomide, which is independent of its activity as an antimetabolite. Despite only moderate inhibition of proximal T cell signaling, the active leflunomide metabolite teriflunomide suppressed integrin avidity after TCR/CD3 engagement. Furthermore, strong abrogation of ICAM-1–mediated costimulation of T cells by teriflunomide was observed. Consistent with these alterations, the formation of the mature IS was profoundly disrupted by teriflunomide, culminating in the abrogation of antigen-specific conjugate formation occurring between APCs and T cells. Since undisturbed interaction of T cells with APCs is a prerequisite for eliciting an effective immune response, these results could explain the potency of leflunomide in the treatment of a variety of disorders involving activated T cells, independent of its antimetabolic activity.
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- MATERIALS AND METHODS
In this study, a novel aspect of the immunosuppressive action of leflunomide distinct from its proposed action as an antimetabolite was revealed, namely, the profound interference of the active leflunomide metabolite teriflunomide with T cell/APC interactions. By mimicking in vivo stimulation of T cells by APC, we showed that teriflunomide disrupts not only maturation of the IS by relocalization of molecules to the contact site, but also antigen-specific T cell/APC conjugate formation. In human T cells, teriflunomide moderately inhibited antibody-mediated T cell activation via CD3 and CD3/CD28, but strongly interfered with physiologically relevant T cell activation via APCs. As a molecular basis of this phenomenon, the activation-driven increase in integrin avidity and costimulation via integrins were strongly impaired by teriflunomide.
These findings may have a substantial impact on our understanding of the influence of teriflunomide as a DMARD, since the completion of an immune response critically depends on the successful interaction of T cells with APCs (32). Furthermore, infiltration of the synovial membrane with leukocytes is a pivotal event in RA, and recent data have demonstrated decreased synovial cellularity in patients treated with leflunomide (33). Also, inhibited activation of monocytic cells by A77 1726–treated T cells (34) may be related to altered integrin avidity. Our data showing a complex influence of teriflunomide on integrin function are consistent with recent data that show that this drug also interferes with leukocyte/endothelial interactions (35). This peculiar property, which is not shared by most other currently used immunosuppressive drugs, such as calcineurin inhibitors, might also be involved in the beneficial effects of malononitrilamides on chronic allograft rejection (5, 36).
While murine lymphocytes are highly sensitive to DHODH inhibition by teriflunomide (1, 8), its 50% inhibition concentration (IC50) for DHODH is ∼150 μM in human cells, similar to the IC50 for LCK and Fyn in Jurkat cells (7). Hence, the concentrations used in this study are the optimal inhibition concentration for tyrosine kinase inhibition and within the range of plasma concentrations in leflunomide-treated patients (23). As shown for several other leflunomide-mediated effects, such as repression of viral replication, response to IL-2, and effector cell functions in an autoimmune encephalomyelitis model (8, 37, 38), the inhibitory effects of teriflunomide in our study are not reversible by the addition of exogenous uridine. Thus, the effects of a diminished pyrimidine pool due to DHODH inhibition, such as reduced production of glycoproteins that are involved in cell/cell contact and adhesion (1) as well as inhibited tumor necrosis factor–induced cellular responses and promotion of Th2 cell differentiation (39, 40), probably do not underlie the effects observed in this study.
Src family kinases play a pivotal role in early T cell signal transduction, as well as in cytoskeletal rearrangements, integrin activation, adhesion, and T cell/APC conjugate formation (41, 42). Hence, the lack of inhibition by the Src family kinase inhibitor teriflunomide on CD3/CD28-mediated signaling via MAPK activation and IκB degradation is astonishing at first glance. However, the roles of Src family kinases in TCR signaling are complex, and LCK even acts as a negative regulator of T cell activation (9, 43, 44). Moreover, inhibition of PMA plus ionomycin–induced activation events by teriflunomide indicates that interference with crucial activation events is not limited to most proximal CD3-induced signaling events, but involves steps downstream of protein kinase C/Ras (PMA) and calcium response (ionomycin).
In addition, this study provides several lines of evidence that teriflunomide strongly affects costimulatory outside-in signaling provided by LFA-1, which is crucial for T cell activation, including effective T cell adhesion and Th1 differentiation (20, 22, 45). Mg2+ plus EGTA induces a high-affinity state of LFA-1, which allows increased binding of LFA-1 to ICAM-1 (21). However, T cell adhesion to immobilized ICAM-1 induced by Mg2+ plus EGTA requires cytoskeletal remodeling and therefore depends on active signaling rather than being a passive process (20). Hence, together with the suppression of ICAM-1–induced augmentation of surface marker and IL-2 expression, impaired T cell adhesion induced by Mg2+ plus EGTA in the presence of unaltered LFA-1 cell surface expression indicates disturbed LFA-1–mediated signaling. Interestingly, our results are consistent with those of other studies on the effects of leflunomide on CD43-induced actin cytoskeleton remodeling (46) and homotypic cell aggregation (47), suggesting that leflunomide generally affects cytoskeletal-driven events. Recently, a molecule that is pivotal for cytoskeletal-driven events such as integrin activation and is also involved in integrin signaling, termed RAPL (regulator of adhesion and polarization enriched in lymphocytes), has been described (48). Hence, future investigations might reveal an interaction of teriflunomide with the activation of this regulator of T cell–adhesive events.
Another putative target of teriflunomide action in T cells is cyclooxygenase 2 (COX-2), which has been shown to be inhibited by A77 1726 in fibroblast-like synoviocytes (49). Importantly, because our experiments analyzed very early events of T cell activation and only already-activated T cells express COX-2, it is conceivable that modulation of COX-2 or its products is not causally involved in the observed effects of teriflunomide. However, inhibition of COX-2 activity at later stages of the effector T cell response might contribute to the complete mode of action of teriflunomide. Collectively, our data indicate that teriflunomide hampers T cell responsiveness beyond the inhibition of critical signaling steps such as LCK activity, which might be reflected by impaired calcium mobilization. Teriflunomide also critically affects integrin functions, which results in impaired adhesion and integrin-mediated costimulation.
Integrin functions are crucial for T cell activation by APCs through the formation of the mature IS (19). Notably, teriflunomide affects not only LFA-1 avidity and outside-in signaling, but also its relocalization to the IS. Inhibited clustering of CD3 within the IS indicates that only an immature type of IS is formed in the presence of teriflunomide, which is insufficient for the formation of stable T cell/APC conjugates (12). Since stable conjugates are indispensable for induction of immune responses (32), the effects of teriflunomide on IS formation could therefore underlie hampered T cell responsiveness in vivo.
In conclusion, we have shown that the active leflunomide metabolite teriflunomide impairs integrin avidity and integrin-mediated signals, which leads to the abrogation of successful T cell/APC interactions as expressed by the formation of the mature IS and antigen-specific conjugates. Furthermore, suppressed adhesion to extracellular matrix proteins may be crucial for the effects of teriflunomide in modifying exaggerated immune responses by interfering with immune cell invasion of inflamed tissue, such as the synovial membrane in RA. These data therefore reveal a novel mode of action of teriflunomide during the induction of cellular immune responses, which may contribute to its clinical effectiveness in diseases involving exaggerated immune responses.