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Immunobiology

REGULATION OF B CELL FUNCTION BY THE IMMUNOSUPPRESSIVE AGENT LEFLUNOMIDE1

Siemasko, Karyn F.; Chong, Anita S.F.; Williams, James W.; Bremer, Eric G.; Finnegan, Alison2

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Abstract

Earlier studies have identified leflunomide, an isoxazol derivative, as a unique immunosuppressive drug capable of alleviating the severity of autoimmune disease and preventing allograft and xenograft rejection. Leflunomide is chemically and structurally unrelated to other immunosuppressive drugs currently under investigation. In a number of autoimmune diseases, autoantibodies and immune complexes are responsible for the immunopathology of the disease. Leflunomide is effective in controlling disease progression in a number of animal models of autoimmune disease, including systemic lupus erythematosis (SLE),* adjuvant arthritis, and experimental allergic encephalomyelitis(1-4). In an animal model of SLE, the MRL/lpr mouse, leflunomide is capable of inhibiting the formation of antibodies to double-stranded DNA (3-5).

Leflunomide is effective in prolonging the survival of solid organ allografts and skin allografts in animal models (6, 7). The production of alloantibodies that accompany rejection is also suppressed by leflunomide (7). The knowledge that leflunomide inhibits antibody production led Xiao et al. to investigate its effectiveness in controlling xenoantibodies that mediate xenograft rejection(8). Leflunomide, but not cyclosporine, prevented the increase in xenospecific IgM and IgG. Chronic rejection, which is thought to be mediated, in part, by alloantibodies and immune complexes, is also controlled by leflunomide (9, 10), unpublished data, Xiao F, personal communication). These data suggest that leflunomide directly downregulates the humoral immune response in vivo.

The mechanisms by which leflunomide inhibits B cell antibody production are presently unknown. Potential direct mechanisms by which leflunomide may inhibit B cell antibody production include inhibition of B cell proliferation, B cell differentiation, or B cell antibody secretion. Leflunomide may also act indirectly through suppression of T cell helper activity. In this study, we show that leflunomide has the capacity to inhibit B cell antibody production stimulated by both T cell-dependent and T cell-independent antigens in vivo, indicating that leflunomide directly inhibits B cell responses. We find that much of the antibody production in response to LPS in vitro is suppressed by blocking clonal expansion of B cells capable of secreting antibody. We demonstrate that leflunomide can inhibit the proliferation of murine B cells stimulated by a variety of stimuli that induce unique signal transduction pathways. This inhibition of proliferation is associated with the ability of leflunomide to block cell cycle transition from G1 to S phase and from entering G2/M phase. Accompanying the block in progression through the cell cycle, leflunomide was capable of inhibiting the upregulation of Cdk2 seen 48 hr after LPS stimulation, which correlates with the block in the S phase. These data indicate that leflunomide inhibits the majority of B cell antibody production by blocking the expansion of antibody-secreting cells. The reversibility of inhibition by the addition of exogenous uridine shows that in B cells leflunomide acts as a pyrimidine synthesis inhibitor in its ability to suppress proliferation and cell cycle progression.

MATERIALS AND METHODS

Animals. B10.A male mice 6-8 weeks old were obtained from the Frederick Cancer Research Center (Frederick, MD).

Antigens. Keyhole limpet hemocyanin (KLH) (Calbiochem, San Diego, CA.) and ovalbumin (OVA) (Sigma, St. Louis, MO) were conjugated with 2,4,6-trinitrobenzenesulfate (Pierce, Rockford, IL) as described(11). TNP-conjugated lipopolysaccharide (LPS) was purchased from Sigma.

Immunization. For primary antibody responses, mice were immunized with 50 μg of TNP-KLH in complete Freund's adjuvant (CFA) (Difco Laboratories, Detroit, MI) intraperitoneally (i.p.) or with 50 μg of TNP-LPS in saline, and serum was collected after one week. For secondary antibody responses, mice were boosted at 4 weeks with 50 μg of TNP-KLH in Freund's incomplete adjuvant and sera was collected one week later. For preexisting anti-TNP antibody responses mice were immunized with 50 μg of TNP-KLH in CFA 4 months before treatment with leflunomide and sera collected one week after treatment was begun.

Treatment. Leflunomide is insoluble in water, therefore carboxymethylcellulose (CMC) (Serva, Heidelberg, Germany) of medium viscosity was used as a carrier. Leflunomide is rapidly converted to its active isoxazol open ring form in vivo designated as A77 1726 (Fig. 1).(We will use the term leflunomide to refer to the closed ring form in the in vivo experiments and the open ring form in the in vitro experiments.) Mice were treated with leflunomide (Hoechst, AG, Weisbaden, FRG.) 35 mg/kg/day (prepared fresh every day using sonication to emulsify the drug in solution) in 1% CMC or 1% alone by gavage for a total of 7 weeks beginning 7 days before the first immunization. For the preexisting antibody response leflunomide in 1% CMC or 1% CMC alone was started 4 months after immunization with TNP-KLH. Groups consisted of 5 mice per group.

ELISA. Serum anti-TNP antibodies and LPS-induced production of anti-IgM antibodies were detected by ELISA. TNP-OVA (10 μg/ml) (Sigma) or 10 μg/ml of goat antimouse IgM, unlabeled (Southern Biotechnology Associates Inc., Birmingham, AL) diluted in PBS was used to coat the bottom of an Immunolon microtiter plate (Dynatech, Inc., Chantilly, VA) at 4°C overnight. Following incubation, plates were washed twice with PBS-TWEEN 20(0.05%). Plates were blocked with blocking buffer (2% BSA in PBS) for 1 hr, washed, and 100 μl of serially diluted serum in blocking buffer or neat supernatants was added to each of the coated wells and incubated at room temperature for 1 hr and then washed twice in PBS-TWEEN 20. Goat antimouse Ig antibody or goat antimouse IgM antibody conjugated with horseradish peroxidase diluted 1:2000 in blocking buffer (0.01 ml) was added for 1 hr at room temperature and then washed extensively in PBS-TWEEN 20. The substrate, 2,2′-azino-di-3-ethylbenzthiazoline sulfonic acid (ABTS) and 0.012% H2O2 was added to all wells. The plates were read on an ELISA reader (Coulter, Hialeah, FL) at 405 nm. The data reported represent the mean and standard deviation of 5 individual sera from groups of mice treated in vivo or triplicate wells from B cells activated in vitro.

B cell isolation. Single-cell suspensions were prepared by mashing spleens between the frosted ends of two glass slides in RPMI complete medium containing 7% fetal bovine serum (Gibco), 100 U/ml penicillin, 100μg/ml streptomycin, 2 mm L-glutamine, 50 μM 2-mercaptoethanol, 1 mm sodium pyruvate, 0.01 mm nonessential amino acids, and 10 mm HEPES. Red blood cells were lysed by treatment with Tris-ammonium chloride. To prepare B cells plus macrophages, spleen cells were depleted of T cells by treatment with a T cell-specific cytotoxic rabbit anti-mouse brain serum and guinea pig complement as previously described (12). B cell population purity was analyzed by flow cytometry using an anti-IgM (FITC) antibody (Fisher, Pittsburgh, PA.) or as a control, an IgG2a (FITC) antibody(Southern Biotechnology Associates) and found to be >90% B cells(Fig. 2). Analysis was performed on an EPICS Profile Flow Cytometer (Epics Division of Coulter Corp., Hialeah, FL). Fluorescence intensity is represented by a 3-decade logarithmic scale.

B cell proliferation assay. Murine splenic B cells were seeded in 96-well microtiter plates at 4×105 cells/well in triplicate. The B cells were stimulated with F(ab′)2 anti-IgM antibody (10μg/ml), whole goat antimouse IgM antibody (2 μg/ml), PMA (5 ng/ml) plus calcium ionophore (2 μg/ml) (Sigma, St. Louis, MO) or LPS (25 μg/ml)(Difco, Detroit, MI) in the presence or absence of leflunomide, added at varying concentrations and at different times to each well, for a final volume of 200 μl. After a 48- or 72-hr incubation (37°C, 5% CO2), cells were pulsed with 1 μCi per well of [3H]-TdR (ICN Biomedicals, Inc., Costa Mesa, CA.). After 16 hr (unless otherwise indicated), cells were harvested onto glass fiber filters and the [3H]-TdR incorporation was measured via scintillation counting. The purine compounds and uridine were purchased from Sigma. The controls for the proliferative experiments, cells alone and cells plus leflunomide, gave proliferative responses of less than 1×104 cpm. The mean and standard deviation of triplicate wells were reported. When cells were visually counted, viability was determined by staining with trypan Blue.

Western blot analysis. Murine splenic B cells were isolated as described above. Cells were stimulated with LPS (25 μg/ml) in the presence or absence of leflunomide for varying amounts of time. Cells were lysed in lysis buffer (50 mM Tris-HCl, 2 mM EDTA, 0.15 M NaCl, 1% NP40, 200 μM Na3VO4, 1 mM PMSF, 1 μg/ml aprotinin, and 1 μg/ml leupeptin) for 15 min. Particulate debris was removed by centrifugation at 12,000×g for 15 min at 4°C. Protein measurements of the lysates were performed using the Bio Rad Protein Assay (Bio Rad, Hercules, CA.). The lysates were boiled with equal amounts of 2X Laemmli's sample buffer for 5 min. These samples were run on a 15% SDS-PAGE gel and were transferred to Hybond-ECL nitrocellulose paper (Amersham Corporation, Arlington Heights, IL). The nitrocellulose was blocked in 5% nonfat dry milk in TBST (20 mM Tris-base, 137 mM NaCl, and 0.1% TWEEN) for 90 min followed by 3 5-min washes in TBST. The nitrocellulose was then incubated for 18 hr in 0.2 μg/ml of anti-Cdk2 antibody diluted in 5%-milk/TBST, followed by 10 washes with TBST(13). The membrane was then incubated in donkey anti-rabbit IgG HRP-labeled antibody at 1:2500 (Amersham). The membrane was incubated for 1 min in equal volumes of Enhanced Chemiluminescence detection reagents 1 and 2 (Amersham). The excess reagent was poured off and the membrane was exposed to film. The film was scanned with a Molecular Dynamics densitometer.

Cell cycle analysis. Cells were stimulated for 48 hr with 25μg/ml LPS in the presence or absence of leflunomide (6.25 μM to 100μM), actinomycin D (0.05 μg/ml) (Sigma), or hydroxyurea (2 mM) (Sigma). After 48 hr, the cell population was adjusted to 5×106 cells/ml in complete medium. Cells (100 μl) from each group were incubated with 500μl of DNA assay solution (New Concept Scientific LTD., Ontario, Canada) containing propidium iodide, detergent, and RNase on ice for 30 min. Cell cycle analysis was performed on an EPICS Profile Flow Cytometer (Epics Division of Coulter Corp.). DNA histograms were analyzed using the Multicycle computer program (Phoenix Flow, San Diego, CA).

RESULTS

Leflunomide inhibits T cell-dependent and T cell-independent B cell antibody production. To determine whether leflunomide was inhibiting B cell antibody responses by blocking T cell helper function and/or by directly inhibiting B cell function, we examined the role of leflunomide in suppressing B cell antibody production in vivo to the T cell-dependent antigen, TNP-KLH, and T cell-independent antigen, TNP-LPS. Mice were treated with leflunomide(35 mg/kg/day) for 7 weeks beginning one week before the primary immunization. One week following primary immunization, mice were bled and serial dilutions of serum were tested for anti-TNP antibody levels by a TNP-specific ELISA. Leflunomide inhibited the primary anti-TNP antibody response to both TNP-KLH and TNP-LPS antigens (Fig. 3, A and C) in comparison with carrier-treated controls. Mice boosted with TNP-KLH in the presence of leflunomide and bled one week later for an anti-TNP antibody response had a reduced antibody response. This is most likely due to the inhibition of priming by leflunomide (Fig. 3B). These results indicate that leflunomide is effective in inhibiting the B cell antibody responses to both T cell-dependent and T cell-independent antigens in vivo and suggests that leflunomide directly effects the function of B cells. Leflunomide may inhibit B cell antibody responses by blocking expansion of antibody secreting B cells or it may inhibit B cell differentiation or secretion.

Leflunomide inhibits a preexisting T cell-dependent antibody response. We next examined the ability of leflunomide to inhibit an ongoing antibody response in mice which were immunized with TNP-KLH (50 μg/ml) in CFA. These TNP-KLH-primed mice expressed detectable levels of circulating anti-TNP antibodies 4 months after immunization. Treatment with leflunomide for one week significantly reduced the total immunoglobulin levels of anti-TNP antibodies in the serum (Fig. 3D). The leflunomide inhibition of a preexisting antibody response was reversible because 8 weeks after cessation of leflunomide treatment, the antibody levels were partially restored (data not shown). These data indicate that leflunomide can inhibit the production of antibodies by activated B cells.

Leflunomide inhibition of B cell proliferation. It is clear from previous results that leflunomide has the capacity to inhibit proliferation of various cell types (5). The next set of experiments was designed to determine the ability of leflunomide to inhibit B cell proliferation stimulated by three different agents. B cells can be induced to proliferate by anti-IgM or PMA plus calcium ionophore, which trigger a rise in intracellular calcium (14, 15, or by LPS, which does not induce a rise in intracellular calcium (16). Leflunomide inhibited B cell proliferation to the three different agents at a similar IC50, approximately 10-20 μM (Fig. 4, A-C). The observation that similar concentrations of leflunomide are capable of inhibiting these 3 different activators of B cell proliferation suggests that leflunomide is targeting a common component necessary for B cell proliferation.

Time course analysis of the effect of leflunomide on LPS-stimulated B cell proliferation and antibody levels. Time course experiments were performed to determine at what point in the activation process leflunomide inhibits B cell proliferation. Leflunomide was added to cultures at 0, 24, 48, and 72 hr and the proliferation assay was assayed at 88 hr. Addition of leflunomide at either 0 or 24 hr suppressed B cell proliferation. Addition of leflunomide at 48 hr and 72 hr of stimulation had a minimal inhibitory effect on proliferation (Fig. 5A). The fact that leflunomide was inhibitory when added at the 24-hr time point suggests that leflunomide does not block the initial activation signals necessary for B cell proliferation.

To examine the effect of leflunomide on the immunoglobulin production, B cells were cultured with LPS (25 μg/ml) in the presence of leflunomide added at different times after activation for 7 days, and the amount of IgM antibody in the culture supernatants was determined by ELISA. Leflunomide inhibited LPS-induced antibody production in a dose-dependent manner when added at either the 0- or 24-hr time points (Fig. 5B). Leflunomide was not as inhibitory at the 48- or 72-hr time points. The IC50 for antibody production when leflunomide was added at the 0 time point was approximately 5 μM. Taken together these data suggest that leflunomide inhibits B cell antibody production by suppressing the expansion of antibody-secreting B cells.

Effects of leflunomide on the cell cycle. To investigate where in the cell cycle the leflunomide-sensitive point resided, resting B cells were analyzed for changes in DNA content following stimulation with LPS. Resting B cells were stimulated with LPS for 24 and 48 hr. Cells with a greater-than-2N complement of DNA (S and G2/M phases of the cell cycle) were observed at the 48 hr time point but not at the 24-hr time point (data not shown). Addition of leflunomide at the beginning of culture partially inhibited cells from entering the S phase in a dose-dependent manner (Table 1). Approximately 57% of the cells at 100 μM leflunomide and 44% at 50μM leflunomide were blocked from entering the S phase as compared with LPS-stimulated control cells. The percentage of cells blocked from entering S phase decreased in a dose-dependent manner.

Known cell cycle inhibitors were compared with leflunomide to determine where in the cell cycle leflunomide acted. At the initiation of LPS stimulation cultures were treated with actinomycin D (0.05 μg/ml), which blocks at the G0 to G1 transition, or leflunomide, while hydroxyurea (2 mM), which blocks the G1 to S transition, was added at 24 hr. DNA content was determined at 48 hr (Table 1). Leflunomide was comparable to hydroxyurea in its ability to partially block B cell entry into the S phase.

Addition of leflunomide at 16 hr after LPS stimulation inhibited 52% of the cells from entering S phase. Addition at 39 hr increased the percentage of cells in S phase in comparison with control cells, but these cells were inhibited from entering into G2/M as compared with the control group(Table 2). These data are in agreement with DNA synthesis data in which addition of leflunomide at 24 hr blocks proliferation, and suggests that leflunomide is blocking B cell proliferation at two different points in the cell cycle, G1 to S phase and G2 to M phase.

In our proliferation data we showed that the addition of leflunomide 72 hr after LPS stimulation no longer had an inhibitory effect on proliferation(Fig 5A). However, based on the above data, it became evident that there may be artifacts in measuring proliferation by thymidine incorporation. If cells enter the S phase before they are treated with leflunomide, proliferation as measured by 3H-TdR incorporation may be inaccurate. Hence, if cells are in the S phase but are blocked from entering the G2/M phase, they would still be able to incorporate thymidine. To circumvent this problem, cells were stimulated with LPS and then leflunomide was added 72 hr after stimulation. By counting viable cells at 96 hr, it was determined that there was an approximately 30% decrease in cell number in the leflunomide-treated group. These data indicate that some cells were blocked at the G2/M boundary, confirming the cell cycle data.

Inhibition of a cell cycle component by leflunomide. Recent data from Tanguay et al. (13) demonstrate that Cdk2 is upregulated in B cells at the beginning of the S phase. In primary B cells, Cdk2 is not detected before the G1 to S phase transition. Cells incubated with LPS in the presence of 50 μM leflunomide for 48 hr showed a 3-fold decrease in Cdk2 protein levels in comparison with the control group(Fig. 6, A and B). Our results suggest that inhibition of progression through the S phase by leflunomide correlates with a reduced level of the Cdk2 protein.

Reversal of proliferation and cell cycle inhibition by uridine. Recent studies have suggested that leflunomide may be acting as a pyrimidine synthesis inhibitor, similar to the immunosuppressive agent brequinar sodium(17, 18). The inhibitory effects of a pyrimidine synthesis inhibitor can be overcome by the addition of exogenous uridine(19). Uridine (25-100 μM) was added with titrated concentrations of leflunomide to LPS-activated B cells and proliferation and cell cycle progress were measured (Fig. 7 and Table 3). Uridine had a minimal effect on cell growth when leflunomide was not present. B cell proliferation induced by LPS in the presence of leflunomide was reduced as compared with the control cells. However, when uridine was added at the same time as leflunomide, cell proliferation and cell cycle were comparable to that in the control groups. However, at leflunomide concentrations of 100 μM, uridine could not reverse the inhibitory effects(Fig. 7). These data indicate that uridine was capable of reversing the inhibitory effects of leflunomide at lower concentrations. At high concentrations, leflunomide may inhibit B cell proliferation by another mechanism.

To examine the possibility that leflunomide may be acting as a purine synthesis inhibitor in B cells, various concentrations (1-100 μM) of adenosine, adenine, guanosine, or guanine were added to cells stimulated with LPS in the presence of leflunomide. At all concentrations of purines tested, there was no reversal of leflunomide's ability to inhibit B cell proliferation, while in the same experiment uridine blocked the inhibitory activity of leflunomide. The results are presented for purine compounds at 10μM concentration (Table 4). The addition of exogenous purines were unable to reverse the antiproliferative effects of leflunomide, suggesting that leflunomide is not acting as a purine synthesis inhibitor.

DISCUSSION

In this study, we define the inhibitory effects of leflunomide on B cell antibody production and proliferation. We first showed that leflunomide directly controls B cell function in vivo. B cell antibody responses in vivo to T cell-dependent (TNP-KLH) or T cell-independent (TNP-LPS) antigens were inhibited by leflunomide. Even a second immunization with TNP-KLH did not overcome the suppressive effects of leflunomide. For T cell-dependent antigens, the synergistic inhibition of both T cells and B cells may be responsible for the suppressed primary (Fig. 3A) and secondary (Fig. 3B) antibody responses to T cell-dependent antigens in vivo. Additionally, it has been shown that cytokine production by T cells is only partially sensitive to leflunomide(20, 21). Thus, it is possible that primed T cells may act as effective helper cells in secondary responses even in the presence of leflunomide. In addition, we observed that leflunomide inhibited a preexisting anti-TNP-KLH antibody response. Williams et al.(7, 8) have previously reported that leflunomide suppressed in vivo antibody responses to allogeneic organ grafts. The generation of allospecific antibodies was suppressed when treatment was initiated on the day of transplant or 4 days after transplant. More important, the preexisting natural antibodies against xenografts are suppressed by leflunomide (8). In both autoimmune diseases and in xenografts, antibodies play an important role in the immunopathology of disease and organ rejection. From our observations it can be concluded that leflunomide can inhibit preexisting antibodies and could therefore serve as an agent to inhibit the progression of these diseases.

The initial activation of B cells leads to a dramatic expansion in the number of antigen-specific B cells that can produce antibodies. Thus, blocking this expansion would result in a decrease of antigen-specific antibody titers in vivo. In vitro experiments demonstrated that leflunomide dramatically reduced the proliferation of B cells, indicating that this may be the primary mode by which leflunomide controls B cell activation in vivo. We used a variety of different stimuli, those that do and do not induce intracellular calcium, in an attempt to dissect the signal transduction pathway that was being inhibited by leflunomide. Activation by cross-linking with anti-IgM induces phosphorylation of a number of tyrosine kinases and induces intracellular calcium (22, 14). PMA bypasses the early tyrosine kinases and directly activates protein kinase C(15). The activation signals delivered by LPS are less clear but have been shown not to involve the early tyrosine kinases activated by anti-IgM and do not induce a rise in intracellular calcium(16). It was significant that even though different stimuli activate B cell proliferation by different signal cascades their proliferation was inhibited at a similar concentration (IC50 = 10-20μM). These data suggest that leflunomide is blocking an essential component, or components, necessary for B cell proliferation and common to each of the stimuli.

To identify the leflunomide-sensitive block in B cell proliferation, we analyzed the progression of B cells through the cell cycle. Leflunomide blocked cell cycle progression at two different points. Similar to hydroxyurea, leflunomide partially blocked progression at the G1/S boundary. However, when cells were allowed to enter S phase before the addition of leflunomide cells were blocked from entering G2/M. Bartlett et al. have observed that leflunomide inhibits IL-2 receptor expression in resting human lymphocytes when stimulated with anti-CD3, suggesting an arrest in the G0/G1 phase of the cell cycle (23). When B lymphoma cells(already cycling) were treated with leflunomide, they were held in the early S phase of the cell cycle (24). Evidence from these two separate systems agrees with our data. Tanguay et al.(13) have recently identified a cyclin-dependent kinase that is involved in cell cycle progression in primary cultures of murine B cells. Cdk2 expression was observed to parallel DNA replication and entry into the S phase. In the presence of leflunomide, Cdk2 levels were decreased, consistent with a block in entry into S phase.

Recent reports have shown that the inhibitory effects of leflunomide on cell proliferation are reversible by the addition of uridine(17, 18). We report here that inhibition of B cell proliferation is also reversible by uridine. BQR is a 4-substituted quinoline carboxylic acid that inhibits the fourth enzyme dehydroorotate dehydrogenase (DHO-DH) of the de novo pyrimidine nucleotide biosynthesis pathway (25). Blocking DHO-DH activity leads to depletion of the intracellular nucleotide pools, thus resulting in the inhibition of DNA and RNA synthesis. Depletion of the nucleotide pools prevents cells from proliferating, leading to a build-up of cells in the S phase (19). A number of groups have evidence that leflunomide is inhibiting the DHO-DH enzyme, similar to brequinar sodium(unpublished data and Ransom J, Williamson R, personal communication).

Leflunomide and BQR both function as pyrimidine synthesis inhibitors in vitro. However, in vivo the differences in toxic side effects suggest that they may function by different mechanisms. BQR treatment in vivo is associated with lymphocyte depletion, hypoplasia of the bone marrow, and gastrointestinal symptoms (26). However, leflunomide is well tolerated in human studies. There were few side effects in 500 rheumatoid arthritis patients treated with leflunomide in a Phase II clinical study(27). Hence, there are indications that leflunomide may be acting by a different mechanism in vivo. Another mechanism by which leflunomide inhibits activation has recently been described. Activation signals through either the EGF receptor or the IL-2 receptor are inhibited by leflunomide's ability to function as a tyrosine kinase inhibitor(21, 28, and Nickevich D, Bremer E, personal communication). We are currently investigating the role of leflunomide as a tyrosine kinase inhibitor in B cell proliferation. Therefore, in addition to leflunomide acting as a pyrimidine synthesis inhibitor, it may also function as a tyrosine kinase inhibitor in vivo.

Our observations that leflunomide directly inhibits B cell function in vivo suggest that leflunomide is a highly effective drug for controlling humoral immune responses. Leflunomide may be a very exciting drug for treatment of chronic and xenograft rejection, where alloantibodies and naturally occurring xenoantibodies participate in graft rejection. Moreover, the lack of toxicity in humans makes leflunomide's potential as single drug therapy, or in combination with other immunosuppressive agents, highly efficacious.

Acknowledgments. We thank Dr. Robert Bartlett for kindly supplying leflunomide and Thomas Mattar for his technical assistance.

Figure 1
Figure 1:
The chemical structures of the two forms of leflunomide. The prodrug form of leflunomide was used for the in vivo studies (A). The prodrug is quickly metabolized to the active metabolite, A77 1726, which is used in the in vitro experiments (B).
Figure 2
Figure 2:
Flow cytometric analysis of purified B cells. Cells obtained from the spleens of B10.A mice were stained with anti-IgG2a (FITC) or anti-IgM(FITC) either before (A and B, respectively) or after T cell depletion (C and D, respectively). The percentage of B cells in the undepleted group (A) was 42%. The percentage of B cells in the T cell-depleted group was >90%.
Figure 3
Figure 3:
Leflunomide inhibition of T cell-dependent and T cell-independent antibody responses in mice. B10. A mice were injected with 50 μg/ml TNP-KLH in CFA i.p. (A). Mice were either treated daily with leflunomide (35 mg/kg/day) in 1% CMC or CMC alone by gavage starting 1 week before immunization. Sera was collected from leflunomide treated or control mice one week after immunization. The same mice were injected a second time with TNP-KLH in ICA i.p. on day 28 and bled 1 week later (B). Mice were immunized with TNP-LPS (50 μg/ml) in saline and treated with leflunomide as in A (C). B10.A mice were injected with 50 μg/ml TNP-KLH in CFA 4 months before treatment with leflunomide (35 mg/kg/day) in 1% CMC or CMC alone (D). Sera was collected one week after leflunomide treatment. Serial dilutions of sera were assayed by a TNP- specific ELISA for total Ig levels. Data represent the mean and standard deviation of sera from 5 individual mice from each group.
Figure 4
Figure 4:
Leflunomide inhibits B cell proliferation stimulated by anti-IgM antibody, PMA plus calcium ionophore, or LPS in a dose-dependent manner. B cells (4×105 cells/well) were stimulated with whole IgM antibody(2 μg/ml), F(ab′)2 anti-IgM antibody (10 μg/ml) (A), PMA (5 ng/ml) plus calcium ionophore (0.2 μg/ml) (B) or LPS (25 μg/ml) (C) in the presence or absence of varying concentrations of leflunomide. After 48 hr of incubation cells were pulsed with 3[H]-TdR and harvested 16 hr later. The controls for these experiments consisted of cells alone, cells plus stimuli, and cells plus leflunomide. These data represent the mean and standard deviation of triplicate wells and are representative of 3 experiments performed.
Figure 5
Figure 5:
Kinetics of inhibition of B cell proliferation and antibody secretion by leflunomide. B cells (4×105 cells/ml) were stimulated with LPS (25 μg/ml) in the presence or absence of varying concentrations of leflunomide added at 0, 24, 48, or 72 hr. After 72 hr of incubation cells were pulsed with [3H]-TdR and harvested 16 hr later(A). Supernatants from LPS-stimulated B cells were assayed for IgM antibody by ELISA after a 5-day culture. These results are representative of 3 separate experiments. Data represent the mean of triplicate wells. The standard deviations were <15%.
Figure 6
Figure 6:
Cdk2 levels are decreased by leflunomide after a 48-hr stimulation with LPS. B cells (1×107) were stimulated with LPS in the presence or absence of 50 μM leflunomide for either 31 or 48 hr. Lysates (20 μg of protein) were run on a 10% SDS-PAGE gel and analyzed by immunoblotting with an anti-Cdk2 antibody. (Lane 1) unstimulated cells, (lane 2) stimulated 31 hr in the presence of leflunomide (50 μM), (lane 3) stimulated 31 hr, (lane 4) stimulated 48 hr in the presence of leflunomide (50μM), (lane 5) stimulated 48 hr (A). Bar graph representing the relative absorbance values of the Cdk2 signal (B). These results are representative of 2 separate experiments.
Figure 7
Figure 7:
Exogenous uridine reverses the inhibition of proliferation seen with leflunomide. B cells were stimulated as presented inFigure 5 with LPS with varying concentrations of leflunomide, in the presence or absence of varying concentrations of uridine for 48 hr. After 48 hr cells were pulsed with [3H]-TdR and harvested 6 hr later. These data represent the mean and standard deviation of triplicate wells and are representative of 3 experiments performed.

*Abbreviations: ABTS, 2, 2′-azino-di-3-ethylbenzthiazoline sulfonic acid; CFA, complete Freund's adjuvant; CMC, carboxymethylcellulose; IC50, 50% inhibition; ICA, incomplete Freund's adjuvant, KLH, keyhole limpet hemocyanin; OVA, ovalbumin; PMA, phorbol myristate acetate.

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