From the
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
Protein-tyrosine kinases are thought to play essential roles in
signal transduction by the T cell antigen receptor (TCR)
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
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
Tyrosine phosphorylation of immunoprecipitated
PLC-
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
The cross-linking of the
TCR
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
The mechanism by which
leflunmide inhibits protein-tyrosine kinases is not known. CTLL-4 cells
stimulated with IL-2 express dramatically elevated p59
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 (
We thank Dr. Christopher E. Rudd for generously
providing us with anti-fyn serum, Dr. Craig Hall for providing
us with anti-
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)
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 TCR
CD3 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) .
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.
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.
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 10
M
-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 Ca
5 Influx
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.
Ability of Leflunomide to Inhibit p59
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 and
p56
Activity in in Vitro Tyrosine Kinase Assays
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
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
TCR Chain, and
PLC-
1 in Anti-CD3-stimulated Jurkat Cells
CD3 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.
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 Ca
To test the effect of leflunomide on
anti-CD3-stimulated [CaMobilization
]
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.
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 TCR
CD3
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 TCR
CD3-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).
CD3 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.
= 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.
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.
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.
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.
mAb, Dr. Robert Bartlett (Hoechst AG, Wiesbald,
Germany) for leflunomide (A771726), and Cetus Corp. (Emeryville, CA)
for human recombinant IL-2.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.