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Departments of
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Immunology/Microbiology,
General Surgery, and
Internal Medicine, Section of Rheumatology, Rush-Presbyterian-St. Luke’s Medical Center, Chicago, IL 60612; and
§
Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University of Chicago, IL 60153
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanism by which leflunomide inhibits B cell Ab production has not been fully elucidated. In a previous report we showed that one mechanism responsible for leflunomide’s inhibition of B cell Ab production was by its ability to suppress B cell proliferation (8). In agreement with other results (9, 10, 11, 12), the biochemical effect of leflunomide was to reduce pyrimidine triphosphate levels by inhibiting the enzyme dihydroorotate dehydrogenase (DHODH) in the pyrimidine synthesis biosynthetic pathway. This mechanism was responsible for the anti-proliferative effect of leflunomide since addition of uridine restored both pyrimidine nucleotide levels and proliferation in B cells (8).
We previously reported a second biochemical activity of leflunomide, which is to inhibit tyrosine kinase activity. In two separate systems, EGF3 and TCR activation, leflunomide blocks EGF receptor activity and TCR signaling by acting as a tyrosine kinase inhibitor (13, 14). Furthermore, in LSTRA cells treated with leflunomide, uridine restores proliferation without restoring tyrosine kinase activity (15). Based on these data, we hypothesized that leflunomide also prevents Ig production through inhibition of tyrosine kinase activity.
To test this hypothesis, we utilized a well-defined model of isotype
switch to examine the mechanism responsible for inhibition of Ig
secretion by leflunomide. IL-4 induces isotype switch to IgG1 and IgE
in the presence of LPS (16, 17, 18). IL-4R consists of two chains; an
-chain, which is the ligand binding chain, and a common 
chain
shared with IL-2, -7, -9, and -15R (19, 20, 21, 22, 23, 24, 25). IL-4 binding to its
receptor induces tyrosine phosphorylation of IL-4R (26, 27), 4PS
(IL-4-induced phosphotyrosine substrate) (28, 29), JAK1, JAK3, and
STAT6 (30). Activation of JAK1 and JAK3 induces rapid tyrosine
phosphorylation of a constitutive STAT6 protein (31, 32). Tyrosine
phosphorylation of STAT6 permits the STAT6 protein to form homodimers
that then translocate to the nucleus where they bind to IL-4 responsive
elements (33).
We report here that leflunomide appears to act as a pyrimidine synthesis inhibitor to suppress B cell proliferation and IgM secretion. However, leflunomide blocked IgG1 secretion and IL-4-induced tyrosine kinase activity independent of its effects on B cell proliferation. Specifically, leflunomide suppressed IL-4-induced tyrosine phosphorylation of JAK3 and STAT6 and prevented STAT6 binding to the STAT6 DNA binding site found in the IgG1 promoter. These data collectively suggest that leflunomide blocks IgG1 production due to its ability to prevent tyrosine phosphorylation of intracellular proteins required for IgG1 production. Thus, leflunomide could serve as a useful tool in dissecting elements important in isotype switch.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male B10.A mice 6 to 8 wk old were obtained from the Frederick Cancer Research Center (Frederick, MD.).
FACS Analysis
Single cell suspensions were prepared by mashing spleens between the frosted ends of two glass slides in RPMI 1640 complete media containing 7% FBS (Life Technologies, Grand Island, NY.), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 50 µM 2-ME, 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. Splenocytes (2 x 106 cells/ml) were stimulated with LPS (25 µg/ml) (Difco, Detroit, MI.) for 48 h and then either left untreated or were treated with leflunomide (50 µM) (Hoechst, AG, Weisbaden, Germany) for a 24-h period. IgM and IgG3 cell surface expression was then analyzed by flow cytometry using an anti-IgM (phycoerythrin) Ab (Fisher, Pittsburgh, PA.), an anti-IgG3 (FITC) (PharMingen, San Diego,CA.) and as control, rat isotype control (Becton Dickinson, Franklin Lakes, NJ.). FcRs were blocked using anti-FcR Ab 24G2. Analysis was performed on an EPICS Prolfile Cytometer (Epics Division of Coulter Corp., Hialeah, FL.).
ELISA
Single cell suspensions were prepared as described above. Splenocytes (4 x 105 cells/ml) were either unstimulated or stimulated with LPS (25 µg/ml) in a 96-well microtiter plate (Corning Costar Corp., Cambridge, MA.) for 48 h followed by the addition of IL-4 (4 ng/ml) (PharMingen) in the presence or absence of titrated concentrations of leflunomide. Cells were incubated for a total of 7 days at 37°C in a 5% CO2 incubator, and then supernatants were harvested for IgG1 analysis by ELISA. Briefly, goat anti-mouse IgG1 Ab was coated (5 µg/ml) in buffer (0.1 M NaHCO3, pH 8.2) onto an Immunolon microtiter plate (Dynatech, Inc., Chantilly, VA.) at 4°C overnight. IgG1 Abs and IgG1 standard were purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Plates were washed three times with PBS/Tween 20, blocked with PBS/3% BSA for 2 h at room temperature, and then washed three times. Supernatants (100 µl) were incubated on coated wells to detect IgG1 at room temperature for 1 h and then washed three times in PBS-Tween 20. Goat anti-mouse IgG1 HRP Ab (2 µg/ml) in PBS/1% BSA was added for 45 min at room temperature and then washed five times in PBS/Tween. The substrate, 2,2'-azino-di-3-ethylbenzthiazoline sulfonic acid (ABTS) and 0.012% H2O2 were added and the plates were read on an ELISA reader (Coulter, Hialeah, FL) at 405 nm. The data reported represent the percent inhibition of IgG1 production as compared with cells stimulated with LPS and IL-4.
Metabolic labeling, immunoprecipitation, and SDS-PAGE
Cells (1 x 107 cells/ml) were stimulated
with LPS for 84 h in the absence or presence of varying
concentrations of uridine as indicated in the text. Leflunomide was
added at either the beginning of the assay or 48 h after LPS
stimulation. In each experiment, the same number of viable cells
(2 x 106) was washed twice in PBS and then incubated
for 1 h in labeling media (RPMI 1640 without methionine) (ICN
Biomedicals Inc., Cleveland, OH), 4 mM glutamine, 100 U/ml penicillin,
100 µg/ml streptomycin, 1 mM sodium pyruvate, 50 µM 2-ME, and 10%
dialyzed FCS). For leflunomide and uridine experiments, labeling was
performed in the continuous presence of leflunomide or leflunomide and
uridine. After 1 h, 50 µCi of Tran 35S-label (ICN
Biomedicals, Inc.) was added for 3 h at 37°C. After incubation,
the cells were centrifuged and the supernatants were collected for
immunoprecipitation. Cellular extracts from the same number of cells
(2 x 106) were prepared by lysing the cellular
pellets in 250 µl lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM
EDTA, 0.1% NaN3, and 0.5% Triton), vortexed, and kept on
ice for 20 min. The lysates were then centrifuged (12,000 x
g) for 15 min at 4°C, and the supernatants were
collected for immunoprecipitation. Before immunoprecipitation,
formalin-fixed Staphylococcus aureus cells (10% w/v) (Life
Technologies) were washed three times in washing buffer (1 mM
methionine, 5 mM Na EDTA, pH 8.0, 50 mM Tris, pH 8.0, 0.02%
NaN3, 0.05% Triton-X-100, 0.5% NaDOC, 0.1% SDS, 500 mM
NaCl, and 1 mg/ml chicken albumin) and kept on ice. To
immunoprecipitate murine IgG, fixed S. aureus
cells (0.12 ml) were added to the supernatants (0.75 ml) for 1 h
on ice, centrifuged, and the pellet washed three times in washing
buffer, and one time in low salt buffer (50 mM Tris-HCl/pH 6.8). To
immunoprecipitate IgM, 2 µg rabbit IgG anti-mouse IgM/
Abs
were added to each group of supernatants and incubated overnight at
4°C. For immunoprecipitation, we used protein A-purified rabbit
anti-mouse serum from a rabbit that had been immunized with
purified IgM/ Ig. Then 120 µl of fixed S.aureus cells was added to each tube (0.75) for 1 h, spun
down, and the pellets were washed three times in wash buffer, and one
time in low salt buffer. One hundred microliters of 1x SDS sample
buffer was added to the pellet and boiled for 3 min. The
immunoprecipitated materials were run on an SDS-PAGE gel (34). The gels
were incubated for 1 h in 5% methanol, 5% acetic acid, and then
incubated for 1 h in Entensify (NEN Research Products, Boston,
MA). The gels were then dried and exposed to an x-ray film between two
intensifying screens. Band intensity was determined by scanning the
film with a Molecular Dynamics densitometer (Sunnyvale, CA).
Nucleotide triphosphate analysis
B cells (2 x 105 cells/ml) were stimulated with LPS incubated with leflunomide plus or minus uridine for 24 h. Samples were prepared as described (35). Briefly, preparation of samples required extraction in 0.4 M trichloroacetic acid on ice for 20 min followed by extraction with tri-N-octylamine and 1,1,2-trichloro-trifluoro-ethane. For HPLC analysis, a Waters HPLC system with a 616 pump, a 600 S gradient controller, a 717 plus autosampler, and 996 PDA detector (Milford, MA) using a strong anion exchange column of Partisil-10 SAX which was eluted with a gradient of 10 to 500 mM potassium phosphate (pH 4.5), was used. Quantitation of the four nucleotide peaks was done by integration and comparison to standardization samples.
Immunoprecipitation and Western blot analysis
Murine spleen cells were isolated as described above. Cells (5 x 105 cells/ml) were stimulated with LPS (25 µg/ml) in the presence or absence of leflunomide plus or minus uridine for varying amounts of time. For the immunoprecipitation experiments, cells were stimulated with LPS overnight in the presence or absence of leflunomide plus or minus uridine and then stimulated for 5 min with IL-4 at 37°C before lysis. 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 x g for 15 min at 4°C. Protein measurements of the lysates were performed using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). For the immunoprecipitation experiments, the lysates were precleared with 100 µl of Pansorbin (CalBiochem, San Diego, CA) for 30 min. After preclearing, anti-JAK3 antiserum (UBI) at a dilution of 1:500, anti-STAT6 affinity purified Ab (1 µg) (Santa Cruz, CA), or normal rabbit serum were added to the lysates and incubated overnight at 4°C. Protein G/A beads (25 µl) (CalBiochem) were added for 4 h at 4°C to immunoprecipitate the proteins and then washed three times with lysis buffer. The immunoprecipitates were resuspended in 2x sample buffer and boiled for 5 min. All samples were run on a 10% 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 three, 5 min washes in TBST. The nitrocellulose was then incubated for 18 h in anti-phosphotyrosine 4G10 (0.33 µg/ml in 5%-milk/TBST), followed by three washes with TBST, and then incubated in goat anti-mouse IgG HRP-labeled Ab (UBI) at a 1:2500 dilution followed by 10 washes with TBST. The membrane was then 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. To reprobe the blot, nitrocellulose membranes were placed in a solution of 62.5 mM Tris, pH 6.7, 2% SDS, and 100 mM 2-ME at room temperature for 30 min to strip the membrane of Abs. The nitrocellulose was then probed as described above with either anti-JAK3 antiserum (UBI) or anti-STAT6 affinity purified Ab (Santa Cruz, CA) (both Abs were used at 1 µg/ml in 5%-milk/TBST), and the secondary was goat anti-rabbit IgG HRP-labeled Ab at a 1:5000 dilution. Band intensity was determined by scanning the film with a Molecular Dynamics densitometer (Sunnyvale, CA).
Electrophoretic mobility shift and supershift analysis
Cells (5 x 105) were stimulated with LPS in the presence or absence of varying concentrations of leflunomide or uridine in media with 2% FCS overnight. Viable cells (1 x 107 cells/ml) were then stimulated for 5 min with IL-4 (4 ng/ml). Nuclear extracts were prepared by treating the cell pellet with cold buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF), which causes the cells to swell, and then 25 µl of 10% NP40 was added for 15 min, vortexed vigorously, and the nuclei pelleted. The nuclear pellet was resuspended in buffer (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF) for 15 min at 4°C. Debris was pelleted and supernatants were frozen at -70°C. The DNA binding reaction was performed in a 25-µl volume with 0.5 ng 32P-end-labeled double stranded synthetic oligonucleotide probe for a STAT6 consensus binding site (5'-TGCCTTAGTCAACTTCCCAAGAACAGA-3') (Integrated DNA Technologies, Inc., Coralville, IA) (36), 5 µg protein, 12% glycerol, 12 mM HEPES-NaOH (pH 7.9), 60 mM KCl, 5 mM MgCl2, 4 mM Tris-HCl (pH7.9), 0.6 mM EDTA (pH 7.9), 0.6 mM EDTA (pH 7.9), 0.6 mM DTT, and 1 µg/ml poly dI/dC for 30 min on ice. For supershift analysis, nuclear extracts (3–5 µg) were incubated with 2 µg anti-STAT6 Ab for 30 min before incubation with the labeled probe. Protein-DNA complexes were resolved in 5% native polyacrylamide gels pre-electrophoresed for 30 min at room temperature in 0.25x TBE (22.5 mM Tris-Borate and 0.5 mM EDTA, pH 8.3). The gels were dried and exposed overnight to x-ray film (Eastman Kodak, Co., Rochester, NY). Band intensity was determined by scanning the film with a Molecular Dynamics densitometer.
Statistical analysis
Data are represented as mean and SD. Comparison between groups was performed using an unpaired, two-tailed t test and p values < 0.05 were regarded as significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously showed that leflunomide inhibits B cell proliferation and Ig secretion from LPS-stimulated B cells in a dose-dependent manner (8). Since B cell proliferation was inhibited, the inability to detect Ig was directly due to a reduction in the number of B cells. These experiments, however, did not exclude the possibility that leflunomide also prevented the differentiation of B cells into Ab-secreting plasma cells. The inhibition of B cell Ig secretion may be solely dependent on the ability of leflunomide to inhibit B cell proliferation. Alternatively, leflunomide may also have an additional inhibitory effect on the signals required for stimulating B cells to differentiate into IgM- and IgG-secreting cells.
If leflunomide inhibits B cell differentiation, then the frequency of
Ig-secreting cells in the LPS culture will be reduced in the presence
of the drug. The level of Ig secreted from the same number of B cells
stimulated with LPS in the presence and absence of leflunomide was
therefore examined. Cells were stimulated with LPS in the presence of
varying concentrations of leflunomide for 84 h. At the end of the
84-h culture period, viable cells were counted. In agreement with our
previous data (8), leflunomide inhibited proliferation in a
dose-dependent manner (Fig. 1
B). The cells in each
treatment group were then normalized for cell number and metabolically
labeled with Tran 35S label for 3 h before
immunoprecipitation of IgM and IgG from the supernatants. Leflunomide
inhibited IgM and IgG secretion in a dose-dependent manner although the
IgG response was more sensitive to inhibition than the IgM response. At
the highest concentration of leflunomide (50 µM), IgM secretion was
inhibited by 61% while IgG secretion was inhibited by 96%. At 12.5
µM leflunomide, IgM secretion was inhibited by 26% and IgG was
inhibited by 80% (Fig. 1, A, C, and
D). These results suggest that leflunomide may block
the differentiation of B cells into Ig-secreting cells by a mechanism
that is independent of inhibition of B cell proliferation.
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E, upper right panel) while in the
leflunomide group only 12% were IgM and IgG3 positive (Fig. 1E, lower right panel). From these data,
we conclude that leflunomide has an effect on both LPS-induced B cell
switching to IgG3, and, for cells that are capable of switching in the
presence of the drug, leflunomide inhibits the differentiation of these
IgG3+ cells into secreting cells. Leflunomide blocks the IgG Ab response even when proliferation is no longer inhibited.
Since B cell differentiation may be dependent on B cell
proliferation, we next tested whether leflunomide inhibited
differentiation of B cells independent of its effects on cell
proliferation. B cells were allowed to proliferate before the addition
of leflunomide. As we previously showed, the addition of leflunomide to
LPS-activated B cells at 48 h no longer blocks B cell
proliferation (8). B cells were stimulated with LPS for 48 h and
then incubated for the remaining 36 h in the presence of
leflunomide. At the end of the 84-h incubation period, B cells were
metabolically labeled with Tran 35S label for 3
h, and IgM and IgG were immunoprecipitated from the supernatants and
analyzed by SDS-PAGE. When leflunomide was added at the initiation of
LPS-stimulated cultures, B cell proliferation was blocked by 74%, IgM
secretion was inhibited by 65%, and IgG secretion was inhibited by
95% (Fig. 2). However, when leflunomide
was added at 48 h, there was only a 7% inhibition of
proliferation and only a 16% inhibition in IgM secretion (Fig. 2, A-C). Under these conditions, leflunomide
was still able to block IgG secretion by 70% (Fig. 2, A and
D). In these experiments, the inhibition of the IgM
response correlated with the ability of leflunomide to suppress B cell
proliferation. The IgG response, however, was blocked by leflunomide
even when proliferation was restored. These results suggest that
leflunomide inhibition of IgG production is by a mechanism other than
blocking proliferation.
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The fact that uridine reverses the inhibitory effect of
leflunomide on B cell proliferation is strong evidence that leflunomide
functions as a pyrimidine synthesis inhibitor (8). Thus, the addition
of uridine in the presence of leflunomide provides another approach to
distinguish between the effects of leflunomide on B cell proliferation
and differentiation. B cells were stimulated with LPS in the presence
of leflunomide (50 µM) and a concentration of uridine (125 µM) that
has been shown to block the ability of leflunomide to inhibit
proliferation (8). Proliferation of B cells in the presence of
leflunomide was inhibited by 62% while the addition of 125 µM
uridine restored proliferation (Fig. 3B). The levels of IgM
and IgG secreted in the supernatants were blocked by 69% and 93%
respectively when leflunomide was present for the entire 84-h
incubation period and by 22% and 73% respectively when cells were
stimulated with LPS in the presence of leflunomide and uridine (Fig. 3, A, C, and D). Thus, while
uridine could reverse the inhibitory effects of leflunomide on B cell
proliferation and IgM production, the IgG response was not fully
reversed by the addition of 125 µM uridine. An alternative
explanation for these results is that Ab synthesis is more dependent on
uridine 5' triphosphate nucleotide pools than is proliferation. To
exclude this possibility, we examined the level of cellular uridine 5'
triphosphate in the presence of leflunomide and 125 µM uridine (Table I). The cellular nucleotides were
analyzed by HPLC in the presence of leflunomide with and without 125
µM uridine. The addition of exogenous uridine fully restored the
uridine nucleotide pools. These data demonstrate that leflunomide
inhibited IgG secretion independently of its pyrimidine synthesis
inhibitory activity.
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Since the signal transduction pathways for LPS-induced IgG
secretion are not known, we chose to study the well-defined model of Ig
secretion, LPS plus IL-4-induced IgG1 secretion. Supernatants from B
cells stimulated with LPS for 48 h to allow B cells to
proliferate, followed by the addition of IL-4 in the presence or
absence of leflunomide, were collected 7 days after the initiation of
LPS stimulation, and the IgG1 levels were measured by ELISA. The
leflunomide-treated cells had a decreased level of IgG1
(IC50 of approximately 12 µM) as compared with
the supernatants from cells stimulated with LPS plus IL-4 (Fig. 4). Thus, leflunomide inhibits
IL-4-induced IgG1 production in LPS-activated B cells. Since
leflunomide has been described to function as a tyrosine kinase
inhibitor (13, 14, 15), the ability of leflunomide to prevent IgG1
production by inhibition of tyrosine kinase activity was examined.
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IL-4 stimulation of B cells induces rapid tyrosine phosphorylation
of JAK3 (37). To determine whether leflunomide blocked JAK3 tyrosine
phosphorylation induced by IL-4, cells were stimulated overnight with
LPS in the absence or presence of varying concentrations of
leflunomide. Experiments were performed in the presence and absence of
uridine (125 µM). After incubation, the same number of cells were
stimulated for 5 min with IL-4, lysed, and immunoprecipitated with
anti-JAK3 antiserum. Precipitates were resolved on SDS-PAGE,
transferred to nitrocellulose, and probed with an
anti-phosphotyrosine Ab (4G10). As shown in Figure 5A, leflunomide inhibited the
IL-4-induced JAK3 phosphorylation in a dose-dependent manner with an
IC50 of approximately 100 µM. In the presence of
exogenous uridine (125 µM), JAK3 tyrosine phosphorylation was still
inhibited in a dose-dependent manner by leflunomide with an
IC50 of approximately 75 µM (Fig. 5B).
JAK3 tyrosine phosphorylation was not detectable in cells stimulated
with LPS alone (Fig. 5A, lane 1). To
control for the specificity of anti-JAK3 Abs, cell lysates from LPS
plus IL-4-activated cells were immunoprecipitated with normal rabbit
serum, and JAK3 phosphorylation was not detectable (Fig. 5A,
lane 2). To determine relevant amounts of JAK3, the
membrane was stripped and reprobed with anti-JAK3 (data not shown),
and the percent decrease in JAK3 phosphorylation was quantified based
on the relative amount of JAK3 protein as determined by laser
densitometry (Fig. 5C). These data demonstrate that
leflunomide reduced IL-4-induced JAK3 tyrosine phosphorylation. Based
on the decrease in JAK3 tyrosine phosphorylation, and presumably JAK3
tyrosine kinase activation, we hypothesized that leflunomide should
also diminish STAT6 tyrosine phosphorylation.
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Activation of JAK3 correlates with tyrosine phosphorylation of
STAT6 (38). To determine whether reduced tyrosine phosphorylation of
JAK3 by leflunomide was associated with reduced STAT6 phosphorylation,
STAT6 was immunoprecipitated from cells stimulated with LPS for 24
h in the presence of leflunomide or leflunomide plus uridine. The same
number of cells were treated with IL-4 and then immunoprecipitated with
anti-STAT6 Ab. The immune complexes were resolved on SDS-PAGE gel
and blotted with an antiphosphotyrosine Ab (4G10). As shown in Figure 6 (lanes 1 and
2), LPS alone did not induce STAT6 phosphorylation
but required induction by IL-4. STAT6 phosphorylation induced in
LPS-stimulated B cells by IL-4 was inhibited by leflunomide in the
absence or presence of uridine in a dose-dependent manner with an
IC50 of approximately 100 µM (Fig. 6, A and
B). These data support our hypothesis that, by
inhibiting the tyrosine phosphorylation of JAK3, the tyrosine
phosphorylation of STAT6 is reduced in the presence of leflunomide, as
well as in the presence of leflunomide plus uridine.
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IL-4 induces transcription of IgG1 through STAT6 binding to a site
in the promoter of the C1 gene (39). Since leflunomide inhibited
STAT6 tyrosine phosphorylation, we hypothesized that leflunomide would
suppress STAT6 binding to STAT6-specific DNA binding elements. We
synthesized an oligonucleotide probe corresponding to the consensus
STAT6 binding site (36). B cells were stimulated with LPS for 24 h
in the presence of leflunomide or leflunomide plus uridine. The same
number of cells were stimulated with IL-4. After incubation, nuclear
extracts were prepared and incubated with the
32P-labeled STAT6 binding oligonucleotide. DNA binding
was assessed by electrophoretic mobility shift assay (EMSA). As shown
in Figure 7, IL-4 activation induced
formation of a DNA binding complex (lane 2)
that could not be activated with LPS alone (lane
1). This complex was specific for the STAT6 binding element
since 100 molar excess unlabeled probe competed for binding of the
labeled probe while no competition was observed for an unrelated
NF-
B binding site (Fig. 7, lane 4). Leflunomide
inhibited STAT6 binding in a dose-dependent manner with an
IC50 of 100 µM. In cells stimulated in the presence of
leflunomide plus uridine, STAT6 binding was also prevented in a
dose-dependent manner (IC50 of 100 µM). These data
demonstrate that leflunomide is acting by a mechanism independent of
pyrimidine synthesis inhibition to block binding of DNA binding
proteins to the promoter regions of the IgG1 gene. These results are
consistent with the inhibition of the JAK3 and STAT6 tyrosine
phosphorylation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In B cells, Ig production is closely coupled to proliferation. When B cell proliferation was restored by the addition of exogenous uridine in the presence of leflunomide, B cells secreted IgM. These data support the hypothesis that signals causing the differentiation into IgM-producing cells are coupled to B cell proliferation (43). In contrast, restoring proliferation in the presence of leflunomide did not restore IgG secretion. LPS-induced B cell proliferation, therefore, is not sufficient for inducing IgG secretion, suggesting that a second signal is required for the differentiation of B cells into IgG secreting cells.
The biochemical mechanism responsible for isotype switch has not been
fully elucidated. It has been well documented that LPS induces
preferential switch to IgG3; as we have shown here, inhibition by
leflunomide is partly due to its suppression of LPS-induced switch to
IgG3. Since the LPS signal for the induction of this switch is not
known (43), we decided to examine the effects of leflunomide on the
IL-4-signaling pathway. The biochemical signals for IL-4-induced
isotype switch to IgG1 and IgE are more clearly defined. In this model
system, the signal LPS plus IL-4 induces JAK1, JAK3, and STAT6 tyrosine
phosphorylation (30). In both IL-4- and STAT6-deficient mice, generated
by gene targeting in embryonic stem cells, B cells do not produce IgE.
In our studies, leflunomide at an IC50 of 100 µM, blocked
IL-4-induced tyrosine phosphorylation of JAK3 and STAT6. Once STAT6 is
tyrosine phosphorylated it forms a homodimer that translocates to the
nucleus, where it binds to a STAT6 binding site located in the promoter
region of 1 and 
genes (33). Leflunomide, at an IC50
of 100 µM, inhibited IL-4-induced STAT6 tyrosine phosphorylation.
This concentration is similar to the concentration of leflunomide that
reduces binding of STAT6 to the STAT6 binding element. Therefore, it is
likely that this inhibition of binding is due to a blockade in STAT6
phosphorylation. Our results further demonstrate that leflunomide
inhibits IgG1 secretion; however, the IC50 of 12 µM is
much lower than the IC50 for inhibition of tyrosine
phosphorylation. Since the conditions for detecting IgG1 secretion
require culturing for 7 days, while those for tyrosine phosphorylation
of JAK and STAT proteins are several minutes, it is conceivable that
inhibition of other signals acting at a later timepoint might compound
the inhibitor activity of leflunomide.
These studies indicate that inhibition of both pyrimidine biosynthesis and tyrosine phosphorylation may lead to effects on B cell proliferation and Ab production in vitro. The important question is how leflunomide inhibits B cell responses in vivo. The effect of leflunomide on B cells in vivo may be dependent on the differential activation requirements of virgin and memory B cells. It has been clearly shown that differentiation of virgin B cells into IgM-secreting B cells requires proliferation (43, 44). Therefore, leflunomide may inhibit B cell proliferation and ultimately IgM secretion either by blocking pyrimidine synthesis or by blocking tyrosine kinases. However, memory B cells have less stringent requirements for differentiation into IgG secreting cells. Memory B cells do not require a signal to proliferate, and, in fact, induction of proliferation reduces Ab production (45). Therefore, Ig production from memory B cells in vivo may be inhibited by blocking differentiation signals such as IL-4-induced IgG1 and IgE. We postulate that, for this subset of B cells, leflunomide blocks differentiation.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alison Finnegan, Section of Rheumatology, Rush-Presbyterian-St. Luke’s Medical Center, 1653 W. Congress Parkway, Chicago, Il 60612.
3 Abbreviations used in this paper: EGF, epidermal growth factor; HRP, horseradish peroxidase; TBST, 20 mM Tris-base, 137 mM NaCl, 0.1% Tween.
Received for publication June 12, 1996. Accepted for publication October 23, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  P. Baumann, S. Mandl-Weber, A. Volkl, C. Adam, I. Bumeder, F. Oduncu, and R. Schmidmaier Dihydroorotate dehydrogenase inhibitor A771726 (leflunomide) induces apoptosis and diminishes proliferation of multiple myeloma cells Mol. Cancer Ther., February 1, 2009; 8(2): 366 - 375. [Abstract] [Full Text] [PDF]
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 N. Leca, K. A. Muczynski, J. A. Jefferson, I. H. de Boer, J. Kowalewska, E. A. Kendrick, R. Pichler, and C. L. Davis Higher Levels of Leflunomide Are Associated with Hemolysis and Are not Superior to Lower Levels for BK Virus Clearance in Renal Transplant Patients Clin. J. Am. Soc. Nephrol., May 1, 2008; 3(3): 829 - 835. [Abstract] [Full Text] [PDF]
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 E. Kreijveld, H. J. P. M. Koenen, L. B. Hilbrands, H. J. P. van Hooff, and I. Joosten The immunosuppressive drug FK778 induces regulatory activity in stimulated human CD4+CD25- T cells Blood, January 1, 2007; 109(1): 244 - 252. [Abstract] [Full Text] [PDF]
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 K. M. Stuhlmeier Effects of Leflunomide on Hyaluronan Synthases (HAS): NF-{kappa}B-Independent Suppression of IL-1-Induced HAS1 Transcription by Leflunomide J. Immunol., June 1, 2005; 174(11): 7376 - 7382. [Abstract] [Full Text] [PDF]
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 H. Akiho, P. Lovato, Y. Deng, P. J. M. Ceponis, P. Blennerhassett, and S. M. Collins Interleukin-4- and -13-induced hypercontractility of human intestinal muscle cells-implication for motility changes in Crohn's disease Am J Physiol Gastrointest Liver Physiol, April 1, 2005; 288(4): G609 - G615. [Abstract] [Full Text] [PDF]
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 T. Korn, T. Magnus, K. Toyka, and S. Jung Modulation of effector cell functions in experimental autoimmune encephalomyelitis by leflunomide-- mechanisms independent of pyrimidine depletion J. Leukoc. Biol., November 1, 2004; 76(5): 950 - 960. [Abstract] [Full Text] [PDF]
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 N. J. Olsen and C. M. Stein New Drugs for Rheumatoid Arthritis N. Engl. J. Med., May 20, 2004; 350(21): 2167 - 2179. [Full Text] [PDF]
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P. Dimitrova, A. Skapenko, M. L. Herrmann, R. Schleyerbach, J. R. Kalden, and H. Schulze-Koops Restriction of De Novo Pyrimidine Biosynthesis Inhibits Th1 Cell Activation and Promotes Th2 Cell Differentiation J. Immunol., September 15, 2002; 169(6): 3392 - 3399. [Abstract] [Full Text] [PDF]
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 T. Korn, K. Toyka, H.-P. Hartung, and S. Jung Suppression of experimental autoimmune neuritis by leflunomide Brain, September 1, 2001; 124(9): 1791 - 1802. [Abstract] [Full Text] [PDF]
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S. K. Manna, A. Mukhopadhyay, and B. B. Aggarwal Leflunomide Suppresses TNF-Induced Cellular Responses: Effects on NF-{kappa}B, Activator Protein-1, c-Jun N-Terminal Protein Kinase, and Apoptosis J. Immunol., November 15, 2000; 165(10): 5962 - 5969. [Abstract] [Full Text] [PDF]
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 F C Breedveld and J-M Dayer Leflunomide: mode of action in the treatment of rheumatoid arthritis Ann Rheum Dis, November 1, 2000; 59(11): 841 - 849. [Abstract] [Full Text]
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J. J O'Shea, R. Visconti, T. P Cheng, and M. Gadina Jaks and Stats as therapeutic targets Ann Rheum Dis, November 1, 2000; 59(90001): i115 - 118. [Abstract] [Full Text] [PDF]
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S. K. Manna and B. B. Aggarwal Immunosuppressive Leflunomide Metabolite (A77 1726) Blocks TNF-Dependent Nuclear Factor-{kappa}B Activation and Gene Expression J. Immunol., February 15, 1999; 162(4): 2095 - 2102. [Abstract] [Full Text] [PDF]
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L. M. Stamm, A. Raisanen-Sokolowski, M. Okano, M. E. Russell, J. R. David, and A. R. Satoskar Mice with STAT6-Targeted Gene Disruption Develop a Th1 Response and Control Cutaneous Leishmaniasis J. Immunol., December 1, 1998; 161(11): 6180 - 6188. [Abstract] [Full Text] [PDF]
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 P. J. M. Ceponis, F. Botelho, C. D. Richards, and D. M. McKay Interleukins 4 and 13 Increase Intestinal Epithelial Permeability by a Phosphatidylinositol 3-Kinase Pathway. LACK OF EVIDENCE FOR STAT 6 INVOLVEMENT J. Biol. Chem., September 8, 2000; 275(37): 29132 - 29137. [Abstract] [Full Text] [PDF]
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