©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Protein Tyrosine Phosphorylation in T Cells by a Novel Immunosuppressive Agent, Leflunomide (*)

Xiulong Xu (1)(§), James W. Williams (1), Eric G. Bremer (2) (4), Alison Finnegan (2) (3), Anita S.-F. Chong (1) (2)

From the (1) Department of General Surgery, (2) Immunology/Microbiology, (3) Internal Medicine, Section of Rheumatology, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612 and the (4) Chicago Institute for Neurosurgery and Neuroresearch, Chicago, Illinois 60614

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Leflunomide, a novel immunosuppressive drug, is able to prevent and reverse allograft and xenograft rejection in rodents, dogs, and monkeys. It is also effective in the treatment of several rodent models of arthritis and autoimmune disease. In vitro studies indicate that leflunomide is capable of inhibiting anti-CD3- and interleukin-2 (IL-2)-stimulated T cell proliferation. However, the biochemical mechanism for the inhibitory activity of leflunomide has not been elucidated. In this study, we characterized the inhibitory effects of leflunomide on Src family (p56 and p59 )-mediated protein tyrosine phosphorylation. Leflunomide was able to inhibit p59 and p56 activity in in vitro tyrosine kinase assays. The IC values for p59 (immunoprecipitated from either Jurkat or CTLL-4 cell lysate) autophosphorylation and phosphorylation of the exogenous substrate, histone 2B, were 125-175 and 22-40 µM respectively, while the IC values for p56 (immunoprecipitated from Jurkat cell lysates) autophosphorylation and phosphorylation of histone 2B were 160 and 65 µM respectively. We also demonstrated the ability of leflunomide to inhibit protein tyrosine phosphorylation induced by anti-CD3 monoclonal antibody in Jurkat cells. The IC values for total intracellular tyrosine phosphorylation ranged from 5 to 45 µM, with the IC values for the chain and phospholipase C isoform 1 being 35 and 44 µM respectively. Leflunomide also inhibited Ca mobilization in Jurkat cells stimulated by anti-CD3 antibody but not in those stimulated by ionomycin. Distal events of anti-CD3 monoclonal antibody stimulation, namely, IL-2 production and IL-2 receptor expression on human T lymphocytes, were also inhibited by leflunomide. Finally, tyrosine phosphorylation in CTLL-4 cells stimulated by IL-2 was also inhibited by leflunomide. These data collectively demonstrate the ability of leflunomide to inhibit tyrosine kinase activity in vitro, and suggest that inhibition of tyrosine phosphorylation events may be the mechanism by which leflunomide functions as an immunosuppressive agent.


INTRODUCTION

Protein-tyrosine kinases are thought to play essential roles in signal transduction by the T cell antigen receptor (TCR)() complex and cytokine receptors. The TCR complex comprises multiple components, including heterodimer / or /, which is linked to CD3 subunits (, , ), and the - homodimer or - heterodimer (1) . The occupancy of TCR by specific antigen/major histocompatibility complex or antibodies immediately activates Src-related tyrosine kinases, p59 and/or p56, which then induce tyrosine phosphorylation of several intracellular substrates (2, 3, 4, 5) . Tyrosine phosphorylation of one identified intracellular substrate, the chain, occurs very rapidly (10 s) upon TCRCD3 ligation, presumably by constitutively associated p59. The phosphorylated chain is then able to bind and activate a non-Src family tyrosine kinase ZAP-70 (6, 7) . Another substrate, phospholipase C isozyme 1 (PLC-1), is either directly or indirectly phosphorylated by p59 and/or p56 tyrosine kinase (8) . Tyrosine phosphorylation activates PLC-1, which then hydrolyzes phosphatidylinositol 4, 5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol. Inositol 1,4,5-trisphosphate mobilizes intracellular Ca, while diacylglycerol in combination with Ca then activates protein kinase C (9) . Ca binding to calmodulin leads to activation of calcineurin, a phosphatase that dephosphorylates the transcription factor NF-AT. Dephosphorylated NF-AT then translocates to nucleus where it stimulates the transcription of IL-2 gene (9) .

Protein-tyrosine kinase inhibitors have been useful in defining the role of these enzymes in signal transduction events. Herbimycin A, a benzoquinonoid ansamycin antibiotic, increases the turnover of p56 and p59, which in turn decreases tyrosine kinase activity in human T lymphocytes, and thus impairs signal transduction by the TCR complex. Tyrosine phosphorylation, PLC-1 activity, phosphatidylinositol 4, 5-bisphosphate hydrolysis, and [Ca] mobilization, as well as the expression of distal markers of T cell activation, such as interleukin-2 (IL-2) and IL-2 receptor (IL-2R), are all inhibited by herbimycin A (2) . Genistein, a natural isoflavone, also inhibits tyrosine phosphorylation of PLC-1 and other substrates, although it is less effective than herbimycin A and has only marginal effects on the generation of inositol 1,4,5-trisphosphate and Ca mobilization (10, 11) . However, genistein still efficiently blocks IL-2 production and IL-2 receptor expression (12) . Both herbimycin A and genistein are capable of blocking proliferation of T cells stimulated by phytohemagglutinin or by anti-TCR antibody, but neither has yet been tested in any in vivo animal models for immunosuppression.

Leflunomide is an immunosuppressant that may act by inhibiting tyrosine phosphorylation. This isoxazol derivative, which shares no apparent chemical relationship to the other known immunosuppressive drugs, is able to prevent autoimmune diseases and rejection of transplant allografts and xenografts (13, 14, 15) . In vitro cell culture experiments revealed that the active metabolite of leflunomide, A771726, inhibited proliferation of peripheral blood mononuclear cells in a one-way mixed lymphocyte reaction. In addition, leflunomide inhibited T cell proliferation stimulated by anti-CD3 antibody plus PMA, anti-CD28 antibody plus PMA, as well as by IL-2 (16) .

Earlier studies showed that leflunomide inhibited the tyrosine kinase activity of the epidermal growth receptor (17) . We have extended these studies and here report that leflunomide inhibits the activity of p59 and p56, two Src family protein-tyrosine kinases implicated in signal transduction of T cells. We also examined the effects of leflunomide on the TCR signal transduction cascade and have observed that leflunomide blocks tyrosine phosphorylation of the PLC-1 and chain, and consequently inhibits Ca mobilization, IL-2 secretion, and IL-2R expression. In addition, leflunomide is also able to inhibit IL-2-stimulated tyrosine phosphorylation in CTLL-4 cells. These data collectively suggest that immunosuppression mediated by leflunomide is due to its ability to inhibit tyrosine phosphorylation of intracellular proteins required for T cell activation and clonal expansion.


MATERIALS AND METHODS

Cells

Human leukemia cell line Jurkat clone E6-1 was a generous gift from Dr. Greg Spear (Department of Immunol/Microbiology, Rush Presbyterian St. Luke's Medical Center, Chicago, IL). Human T lymphocytes were isolated; the 95% purity was reached as examined by immunofluroscence staining following the procedure described earlier (16). CTLL-4, a subclone of murine T cell clone, CTLL-2, was maintained as suspension cultures in 50 Cetus units/ml IL-2 in RMPI 1640 supplemented with 5% fetal bovine serum, 2 mML-glutamine, and 5 10M -mercaptoethanol.

Reagents

The active metabolite of leflunomide, A771726, used in all in vitro experiments, was a gift from Hoechst AG Werk Albert (Wiesbaden, Germany). Leflunomide was solubilized in distilled water and used at the indicated concentrations. PMA and ionomycin were purchased from Sigma, were made up at 1 mg/ml in 200-proof alcohol, and were stored at -20 °C for use. Anti-CD3 mAb or anti-CD25 mAb was purified from the culture supernatants of OKT-3 or 2A3A1H hybridoma (ATCC, Bethesda, MD), respectively, using a protein G plus/protein A-agarose column (Oncogene Science, Manhasset, NY). The concentration of mAbs was measured by absorption at 280 nm in a spectrophotometer. Rabbit anti-p59 antiserum was kindly provided by Dr. Christopher Rudd (Division of Tumor Immunology, Dana-Farber Cancer Institute). Anti-phosphotyrosine mAb, 4G10, anti-p56 antiserum, and the pooled anti-PLC-1 mAbs were purchased from UBI (Placid Lake, NY). Anti- mAb, 1D4.1, was a kind gift from Dr. Craig Hall (Division of Immunology, Beth Israel Hospital, Harvard Medical School, Boston). Histone 2B was purchased from Calbiochem (San Diego, CA).

In Vitro Tyrosine Kinase Assay

P59 was immunoprecipitated from 5 10 Jurkat cells or from 5 10 IL-2-stimulated CTLL-4 cells with anti-p59 antiserum as described previously (5) . P56 was immunoprecipitated from 5 10 Jurkat cells with anti-p56 antiserum. Tyrosine kinase assays were performed as described previously (5) . Autophosphorylation of p59 and phosphorylation of exogenous substrate were analyzed by electrophoresis on a 12.5% SDS-polyacrylamide gel followed by autoradiography.

Detection of Protein Tyrosine Phosphorylation

CTLL-4 cells and Jurkat cells were stimulated by IL-2 and anti-CD3 mAb, respectively. Cell lystates were prepared, and protein tyrosine phosphorylation was examined by Western blotting using the anti-phosphotyrosine mAb, 4G10, and enhanced chemiluminescence (ECL) (Amersham Corp.) following manufacturer's instruction. Tyrosine phosphorylation of the chain was similarly examined and identity of chain was confirmed by striping nitrocellulose membrane and reprobing with anti- chain mAb. To detect tyrosine phosphorylation of PLC-1, PLC-1 was first immunoprecipitated from Jurkat cell lysates with pooled anti-PLC-1 mAbs and then probed by Western blotting with anti-phosphotyrosine mAb (4G10) and detected with ECL. After stripping the membrane, PLC-1 protein was reprobed with anti-PLC-1 mAbs as described previously (18) .

Measurement of CaInflux

5 10 Jurkat cells were preincubated with various concentrations of leflunomide in serum-free RPMI 1640 medium at 37 °C for 10 min. Cells were then loaded with fura-2/AM as described previously (19) . Leflunomide was present throughout the entire process. Cells were stimulated with 4 µM anti-CD3 mAb or 2 µg of ionomycin. Fura-2/AM fluorescence was measured in a SLM Aminco SPF-500C spectrophotometer. The concentration of intracellular [Ca] was calculated using an equation derived by Grynkiewicz et al.(20) .

IL-2 Assay

5 10 cells/ml of enriched human T cells were preincubated with various concentrations of leflunomide in serum-free medium at 37 °C for 10 min and then stimulated with plastic-immobilized anti-CD3 mAb. Cells were cultured in complete RPMI 1640 supplemented with 5% fetal bovine serum for 24 h in the presence of 10 µg/ml of anti-human CD25 mAb to prevent IL-2 autocrine consumption. The supernatants were then harvested and centrifuged, and the IL-2 in the supernatant was measured in a bioassay using the murine IL-2-dependent cell line, CTLL-4, as described previously (11). To avoid the influence of residual leflunomide in the supernatants on proliferation of CTLL-4 cells, leflunomide was removed by extensive dialysis against phosphate-buffered saline (0.1 M, pH 7.4). A control supernatant harvested from anti-CD3-stimulated T cells and spiked with 200 µM leflunomide was included to ascertain that all leflunomide was removed by the dialysis procedure. This control supernatant stimulated CTLL-4 proliferation as well as did the positive control supernatant without added leflunomide.

IL-2 Receptor Assay

The expression of IL-2R chain was determined using fluorescein isothiocyanate-conjugated anti-CD25 mAb (Becton Dickinson, Mountain View, CA). The percent of cells expressing CD25 was determined from 2500 cells using an EPICS C flow cytometer (Coulter, Hialeah, FL).

Quantitation of Tyrosine Phosphorylation

The exposed X-Omat films from the in vitro tyrosine kinase assays or the phosphotyrosine proteins detected on Western blots were scanned in a LKB densitometer (2202 Ultrascan laser densitometer). The peaks corresponding to the bands of interest were integrated to determine the relative amounts of phosphorylation.


RESULTS

Ability of Leflunomide to Inhibit p59 and p56 Activity in in Vitro Tyrosine Kinase Assays

It has been well documented that Src-related tyrosine kinases are involved in signal transduction of hematopoetic cells. Mattar et al. (17) reported that leflunomide inhibited protein-tyrosine kinase activity of the EGF receptor. Therefore, we first tested whether leflunomide was able to inhibit p59 activity in immune complex tyrosine kinase assays. The enzymatic activity of protein-tyrosine kinases, p59 and p56, immunoprecipitated from Jurkat cells or from IL-2-stimulated CTLL-4 cells, was inhibited by leflunomide in a dose-dependent manner (Fig. 1). The half-maximal dose (IC) required for inhibiting p59 -mediated phosphorylation of the exogenous substrate histone 2B was 22-40 µM, whereas the IC value for inhibiting the autophosphorylation of p59 was 125-175 µM (Fig. 1, A and B). Leflunomide also inhibited p56 autophosphorylation in a dose-dependent manner with an IC of 160 µM and phosphorylation of histone 2B with an IC of 65 µM (Fig. 1C).


Figure 1: Ability of leflunomide to inhibit p59 and p56 activity in in vitro tyrosine kinase assays. P59 from 1 10 Jurkat cells (A) or from 5 10 IL-2-stimulated CTLL-4 cells (B) was immunoprecipitated with 5 µl of anti-p59 antiserum, and a protein-tyrosine kinase assay was performed as described under ``Materials and Methods.'' C, inhibition of p56 activity by leflunomide in in vitro tyrosine kinase assay. P56 was immunoprecipitated from 5 10 Jurkat cells, and a protein-tyrosine kinase assay was performed as described under ``Materials and Methods.'' The concentrations of leflunomide are indicated on the top of figure. This experiment was repeated three times, and one representative experiment is presented.



Ability of Leflunomide to Inhibit Tyrosine Phosphorylation of Total Intracellular Proteins, the Chain, and PLC-1 in Anti-CD3-stimulated Jurkat Cells

The ability of leflunomide to inhibit protein tyrosine phosphorylation was further investigated in Jurkat cells stimulated with anti-CD3 mAb. Consistent with previous reports (2, 3) , aggregation of TCRCD3 complex resulted in the appearance of a number of new phosphotyrosine-containing proteins (Fig. 2A). The molecular masses of these proteins were approximately 135, 100, 97, 80, 72, 56, and 42 kDa. Tyrosine phosphorylation of these substrates was differentially inhibited in Jurkat cells preincubated with leflunomide for 2 h with IC values ranging from 5 µM to 45 µM for the various substrates.


Figure 2: Ability of leflunomide to inhibit tyrosine phosphorylation of total intracellular substrates, chain and PLC-1 in anti-CD3-stimulated Jurkat cells. A, inhibition of total protein tyrosine phosphorylation in Jurkat cells stimulated with anti-CD3 mAb. 5 10 Jurkat cells were preincubated with the indicated concentrations of leflunomide for 2 h and then unstimulated or stimulated with 2 µg anti-CD3 mAb for 2 min and cross-linked with 8 µg of goat anti-mouse IgG for a further 2 min. 50 µg of cell lysates from each sample were separated by a 10% SDS-polyacrylamide gel electrophoresis, and tyrosine phosphorylation was analyzed by Western blotting. Results are a representative of two separate experiments. B, 5 10 Jurkat cells were preincubated with indicated concentrations of leflunomide for 10 min and unstimulated or stimulated with 2 µg of anti-CD3 mAb for 2 min plus cross-linked with 8 µg of goat anti-mouse IgG for another 2 min. Cell lysates were prepared, 200 µg of postnuclear lysates were separated by a 14% SDS-polyacrylamide gel electrophoresis, and Western blotting was performed to detect phosphorylated chain using anti-phosphotyrosine mAb (4G10) and ECL. The membrane was reprobed with anti- mAb to confirm the identity of chain. Results represent one of three independent experiments. C, inhibition of PLC-1 tyrosine phosphorylation by leflunomide. 2 10 Jurkat cells were preincubated with various concentrations of leflunomide for 2 h, unstimulated or stimulated with 8 µg of anti-CD3 mAb for 2 min and then cross-linked with 32 µg of goat anti-mouse IgG for another 2 min. PLC-1 was immunoprecipitated with a pool of anti-PLC-1 monoclonal antibodies, and tyrosine phosphorylation was probed with anti-phosphotyrosine mAb (4G10) and ECL technique. After stripping the nitrocellulose membrane, PLC-1 protein was identified with anti-PLC-1 mAbs. Results represent one of three experiments.



Two substrates that are tyrosine-phosphorylated in Jurkat cells following the stimulation of TCRCD3 complex have been characterized; one is the chain of the CD3 complex and the other is PLC-1 (8, 21) . To examine the effect of leflunomide on tyrosine phosphorylation of the chain, Jurkat cells were preincubated with various concentrations of leflunomide for 10 min and then stimulated with anti-CD3 mAb. Cell lysates were resolved by a 14% SDS-polyacrylamide gel electrophoresis; the tyrosine phosphorylated chain was detected by anti-phosphotyrosine mAb (4G10) antibody and ECL on Western blot (Fig. 2B, upperpanel), and the identity of the chain was confirmed by reblotting with anti- chain mAb (Fig. 2B, bottompanel). Densitometric analyses revealed that leflunomide inhibited tyrosine phosphorylation of the chain with an IC of 35 µM.

Tyrosine phosphorylation of immunoprecipitated PLC-1 from untreated or leflunomide-treated Jurkat cells was monitored by Western blot and ECL. As shown in Fig. 2C (toppanel), stimulation of Jurkat cells with anti-CD3 mAb greatly induced tyrosine phosphorylation of PLC-1, while preincubation of Jurkat cells with the indicated doses of leflunomide for 2 h prior to stimulation significantly inhibited anti-CD3-stimulated tyrosine phosphorylation of PLC-1. The IC was calculated as 44 µM. The nitrocellulose membrane was subsequently stripped and reprobed with anti-PLC-1 antibody to confirm that approximately equal amounts of PLC-1 had been immunoprecipitated from each sample (Fig. 2C, bottompanel).

Ability of Leflunomide to Inhibit CaMobilization

To test the effect of leflunomide on anti-CD3-stimulated [Ca] mobilization, Jurkat cells were preincubated with leflunomide for 10 min and then loaded with fura-2/AM in the presence of leflunomide. As shown in Fig. 3A, 50 µM leflunomide partially (50%) inhibited [Ca] mobilization in Jurkat cells stimulated by anti-CD3 mAb, and 100 µM of leflunomide inhibited [Ca] mobilization by 80%. However, 100 µM leflunomide did not significantly inhibit [Ca] mobilization in response to stimulation of ionomycin, a calcium ionophore (Fig. 3B), suggesting that leflunomide most likely reduced Ca mobilization by inhibiting tyrosine phosphorylation and activation of PLC-1.


Figure 3: Ability of leflunomide to inhibit Ca mobilization. 5 10 cells were preincubated in the absence or presence of various concentrations of leflunomide for 10 min and loaded with fura-2/AM for 30 min as described in text. Cells were stimulated with 4 µg of anti-CD3 mAb (A) or 2 µg of ionomycin (B) for the indicated times. The concentration of [Ca] was calculated and plotted against time.



Ability of Leflunomide to Inhibit IL-2 Production and IL-2 Receptor Expression

The ability of leflunomide to inhibit the early anti-CD3-stimulated signal transduction events suggests that leflunomide should also inhibit IL-2 production and IL-2R expression induced by anti-CD3 mAb. This supposition is supported in part by a previous report (25) that leflunomide inhibits anti-CD3-stimulated IL-2R expression. To further test this hypothesis, enriched human T cells were stimulated by plastic-immobilized anti-CD3 mAb alone or by plastic-immobilized anti-CD3 mAb plus PMA either in the absence or presence of the indicated concentrations of leflunomide. Cell culture supernatants were harvested at 24 h, and IL-2 in the supernatant was quantitated. The results in Fig. 4A show that leflunomide partially inhibited IL-2 production in human T cells following stimulation with anti-CD3 mAb alone (IC = 40 µM) or with anti-CD3 mAb plus PMA (IC = 50-100 µM). In contrast, leflunomide inhibited IL-2R expression in human T cells stimulated by CD3 alone with an IC of approximately 50 µM, but did not inhibit IL-2R expression when T cells were stimulated with PMA alone or anti-CD3 mAb plus PMA (Fig. 4B).


Figure 4: Inhibitory effect of leflunomide on IL-2 and IL-2 receptor expression in PBL cells. A, inhibition of IL-2 production by leflunomide in T cells stimulated by immobilized anti-CD3 mAb or anti-CD3 plus PMA; B, inhibition of IL-2 receptor expression on T cells stimulated by anti-CD3 antibody but not on those stimulated by PMA alone or anti-CD3 plus PMA.



Ability of Leflunomide to Inhibit Protein Tyrosine Phosphorylation in IL-2-stimulated CTLL-4 Cells

The inhibitory effect of leflunomide on tyrosine phosphorylation was further investigated by testing whether leflunomide was able to inhibit protein tyrosine phosphorylation in IL-2-stimulated CTLL-4 cells. The data presented in Fig. 5show that stimulation of CTLL-4 cells with IL-2 induced tyrosine phosphorylation of several proteins with molecular masses of approximately 42, 60, 78-80, 85, 90, 110, and 130 kDa. Tyrosine phosphorylation of an approximately 60 kDa protein, whose identity is not known, was strongly induced by IL-2, but this induction was not inhibited by leflunomide. The IC values of leflunomide required for inhibiting tyrosine phosphorylation of 42-, 78-80-, 85-, 90-, 110-, and 130-kDa proteins were calculated as 120, 48, 100, 36, 88, and 170 µM, respectively.


Figure 5: Leflunomide inhibited protein tyrosine phosphorylation in IL-2-stimulated CTLL-4 cells. 5 10 of CTLL-4 cells were starved of IL-2 for 4 h and then preincubated with the indicated concentrations of leflunomide for 10 min and then unstimulated or stimulated with a total of 500 units of IL-2 for 30 min. Postnuclear cell lysates were resolved by a 10% SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membrane. Protein tyrosine phosphorylation was detected by using anti-phosphotyrosine mAb (4G10) and ECL. This figure represents one of three representative experiments.




DISCUSSION

The results described here support the hypothesis that leflunomide acts as an immunosuppressive agent by inhibiting the activity of protein-tyrosine kinases. Leflunomide inhibited p56 and p59 activity in in vitro kinase assays and also intracellular protein tyrosine phosphorylation in anti-CD3-stimulated Jurkat cell lines and IL-2-stimulated CTLL-4. Protein tyrosine phosphorylation is important for the initiation of cellular responses triggered by the TCRCD3 complex, despite the lack of intrinsic protein-tyrosine kinase activity of either the TCR or the CD3 molecular complex. It has been suggested by Weiss and Littman (22) that specific consensus sequences, antigen recognition activation motifs, on CD3 molecules become phosphorylated by constitutively associated protein-tyrosine kinases such as p59 and/or p56 (22, 23). These phosphorylated motifs then serve as substrates for additional cytoplasmic protein-tyrosine kinases, thereby allowing the recruitment of additional effector molecules to the aggregated receptors via SH2 domain-phosphotyrosine interactions. Tyrosine phosphorylation of the chain of the CD3 complex is critical for successful TCRCD3-mediated signaling. We here report that leflunomide inhibited CD3-stimulated tyrosine phosphorylation of total intracellular proteins (IC values of 5-45 µM) and specifically of the chain (IC of 35 µM).

The cross-linking of the TCRCD3 complex also induces PLC-1 activity. Biochemical and genetic data indicate that the activation of PLC-1 occurs by the tyrosine phosphorylation of PLC-1 (21) . The mechanism by which phosphorylation of PLC-1 occurs is not clear, but it appears to require, either directly or indirectly, p56 kinase function (22) . We report that the CD3-stimulated phosphorylation of PLC-1 was also inhibited by leflunomide at an IC of 44 µM. Since the activation of PLC-1 is thought to contribute directly to the rapid and sustained increase in [Ca], we predicted that leflunomide would also inhibit [Ca] mobilization in Jurkat cells stimulated with anti-CD3 mAb. Leflunomide was indeed able to inhibit [Ca] mobilization with an IC of approximately 50 µM in Jurkat cells stimulated by anti-CD3 mAb but not by ionomycin. These findings further corroborate the conclusion that leflunomide inhibits T cell activation by selectively inhibiting protein-tyrosine kinase activity.

The inhibitory effects of leflunomide on distal events of anti-CD3-mediated activation, namely, IL-2 production and IL-2R expression, were examined in purified human T lymphocytes since the production of IL-2 in Jurkat cells stimulated by anti-CD3 mAbs is undetectable and IL-2R expression is also marginally detectable or undetectable (24) . However, the production of IL-2 in T lymphocytes stimulated by PMA alone is also undetectable (11) . Consistent with our previous observations (16) , leflunomide partially inhibited IL-2 production when T lymphocytes were stimulated with anti-CD3 mAb or co-stimulated with anti-CD3 antibody and PMA. In agreement with a previous report (25) , leflunomide was able to inhibit IL-2R expression in human T cells stimulated with anti-CD3 mAb, but not IL-2R expression when the cells were stimulated with PMA alone or PMA plus anti-CD3 mAb. Since PMA can directly activate protein kinase C, thus bypassing the early activation of tyrosine kinase, our observations further support the notion that leflunomide inhibits early protein-tyrosine kinase activity, thereby blocking T cell activation. Differences in ability of leflunomide to inhibit IL-2 production versus IL-2R expression could reflect the differences in signals regulating IL-2 production and IL-2R expression. However, we have not excluded the possibility that leflunomide may inhibit T cell activation independent of inhibition of protein-tyrosine kinases.

A number of studies have demonstrated that activation of T cells by protein kinase C stimulates tyrosine phosphorylation of the serine/threonine kinase, microtubule-associated protein (MAP) 2-kinase (26, 27). Activation of MAP 2-kinase requires phosphorylation on both serine/threonine and tyrosine residues (28) . Phosphorylation of MAP 2-kinase is mediated by the serine/threonine/tyrosine kinase, MAP 2-kinase kinase (29, 30) . Our observation that leflunomide cannot inhibit PMA-stimulated IL-2R expression lead us to speculate that leflunomide cannot inhibit MAP 2-kinase kinase activity. Experiments demonstrating that genistein, a tyrosine kinase inhibitor, could not inhibit PMA-stimulated IL-2R expression (11) support this hypothesis.

It has been previously reported that leflunomide is able to block proliferation of human T cells stimulated by IL-2 (16) and also CTLL-4 cell proliferation in response to IL-2 stimulation (IC = 40 µM).() We here demonstrate that IL-2-stimulated protein tyrosine phosphorylation in CTLL-4 cells was inhibited by leflunomide; the IC doses for the inhibition of tyrosine phosphorylation of 78-80- and 90-kDa proteins were 48 and 36 µM, respectively. These doses are close to the IC values for CTLL-4 cell proliferation. Therefore, the inhibition of protein tyrosine phosphorylation in IL-2-stimulated CTLL-4 cells may account for the inhibition of cell proliferation.

The mechanism by which leflunmide inhibits protein-tyrosine kinases is not known. CTLL-4 cells stimulated with IL-2 express dramatically elevated p59 activity. When CTLL-4 cells were stimulated with IL-2 in the presence of leflunomide and p59 activity was analyzed by in vitro kinase assay, in the absence of leflunomide, p59 activity was comparable with that from cells not treated with leflunomide (data not shown). This result therefore suggests that leflunomide does not inhibit the activation of p59 but inhibits the ability p56 to phosphorylate.

The potent immunosuppressive activity of leflunomide was revealed by in vivo studies of autoimmune disease, arthritis, and in transplantation models. In addition, preliminary clinical studies with rheumatoid arthritis patients revealed that leflunomide mediated clinical and immunogical improvements with minimal toxicity (13) . Despite these exciting results, little is known of the mechanism of action of leflunomide. We here report that leflunomide has the ability to inhibit Src family kinase activity and speculate that this may be one mechanism by which leflunomide exerts its immunosuppressive activity. The concentrations of leflunomide (A771726) necessary for inhibition of tyrosine kinase activity in T cells (50 µM) are easily attainable as blood levels in humans in the absence of significant toxicity.() The serum concentration of leflunomide (A771726) in Lewis rat that effectively prevents cardiac allograft rejection ranges from 10-100 µM.() These observations suggest that leflunomide may represent a new class of immunomodulatory agents with tyrosine kinase inhibitory activity, and it may be useful as a pharmacological tool to further dissect the role of tyrosine kinases in antigen- or cytokine-driven T cell activation in vivo.


FOOTNOTES

*
This project was supported in part by the National Institutes of Health Grant AI34061 and the Section of Transplantation, Rush Presbyterian St. Luke's Medical Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: TCR, T cell antigen receptor; PLC-1, phospholipase C isozyme 1; IL-2, interleukin-2; IL-2R, IL-2 receptor; PMA, phorbol 12-myristate 13-acetate; mAb, monoclonal antibody; MAP, microtubule-associated protein.

X. Xu, unpublished results.

R. R. Bartlett, personal communication.

J. W. Williams, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Christopher E. Rudd for generously providing us with anti-fyn serum, Dr. Craig Hall for providing us with anti- mAb, Dr. Robert Bartlett (Hoechst AG, Wiesbald, Germany) for leflunomide (A771726), and Cetus Corp. (Emeryville, CA) for human recombinant IL-2.

Addendum-After submission of this manuscript, a second activity of leflunomide, inhibition of pyrimidine synthesis, was reported (31, 32) . We have confirmed those studies and observed that the anti-proliferative activities of leflunomide can be reversed by the addition of uridine, and in some cases, cytidine. However, we also observed that inhibition of tyrosine kinase activity could not be reversed by uridine. Thus it appears that leflunomide has two independent activities on T cells and that the relative contribution of both activities to the immunosuppressive effect of leflunomide in vivo requires further definition.


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