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Multiple Sclerosis Research Center, Department of Neurology, Vanderbilt University School of Medicine, Nashville, TN 37212
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production. We have further tested the in vivo
effects of tyrphostin B42 in experimental allergic encephalomyelitis, a
Th1 cell-mediated autoimmune disease. In vivo treatment with tyrphostin
B42 decreased the proliferation and IFN- production of neural
Ag-specific T cells. Treatment of mice with tyrphostin B42 also reduced
the incidence and severity of active and passive EAE. These results
suggest that tyrphostin B42 prevents EAE by inhibiting IL-12 signaling
and IL-12-mediated Th1 differentiation in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, heat shock proteins,
and CD40 ligand (1, 2, 3, 4). The biologically active IL-12 is a 70-kDa
heterodimeric protein, composed of covalently linked p35 and p40
subunits, encoded by two separate genes (5). This cytokine is capable
of regulating both innate and adaptive immune responses such as
proliferation, cytokine production, and cytotoxic activity of NK cells
and T cells (1). The importance of IL-12 in the development of the
Th1-type immune response in tuberculosis, leprosy, leishmaniasis, HIV,
and other infectious diseases has been well documented (6, 7, 8, 9, 10, 11).
Although the induction of IL-12 is necessary for protective Th1-type
host immune responses to infection, overexpression of IL-12 and other
Th1 cytokines contributes to the development of chronic inflammatory
and autoimmune diseases (12). Experimental allergic encephalomyelitis (EAE)3 is an autoimmune inflammatory demyelinating disease of the CNS that shows many pathological and clinical similarities with human multiple sclerosis (MS), and serves as a model system to test potential therapeutic agents for MS (12, 13, 14). EAE is induced in susceptible strains of mice, rats, and guinea pigs either by immunization with neural Ags or by adoptive transfer of neural Ag-specific T cells. Ags well known to induce EAE include myelin basic protein (MBP), proteolipoprotein, and myelin oligodendrocyte protein. EAE is an archetypal CD4+ Th1 cell-mediated autoimmune disease, and proinflammatory cytokines play an important role in the pathogenesis of the disease. Previous analyses showed a typical Th1-type cytokine profile in the CNS of animals with EAE (12, 13, 14). The detection of IL-12 p40 mRNA in MS plaque lesions suggested the involvement of IL-12 in the pathogenesis of demyelinating disease of the CNS in humans (15). Expression of IL-12 p40 in the target and lymphoid organs of mice with EAE and exacerbation or abrogation of EAE by systemic administration of rIL-12 or anti-IL-12 Ab, respectively, suggested the critical role of IL-12 in the pathogenesis of Th1 cell-mediated CNS demyelination (16, 17).
Signaling through its receptor, IL-12, induces the immune and inflammatory responses by tyrosine phosphorylation and activation of Janus kinases, Jak-2 and Tyk-2, and of Stat3 and Stat4 (18, 19, 20, 21). Activation of the Jak-Stat pathway leads to transcription of many IL-12 response genes associated with proliferation, cytokine production, and cytotoxic activity of T and NK cells. Modulation of signal transduction pathways by targeting protein tyrosine kinases has been considered a novel strategy for the treatment of infectious diseases, cancer, and autoimmune diseases (22, 23, 24, 25). Tyrphostin, a group of compounds derived from benzylidenemalononitrile nucleus, are potent inhibitors of protein tyrosine kinases (26). Tyrphostins inhibit the tyrosine kinase activities of epidermal growth factor receptor, platelet-derived growth factor receptor, insulin receptor, HER2-Neu, Trk, and Cdk2 (26, 27). Tyrphostins also block the replication of Moloney murine leukemia virus and HSV by inhibiting the phosphorylation of viral proteins (28, 29). The prevention of LPS-induced septic shock by tyrphostins suggested their use in the management of inflammatory diseases (24). The recent reports on the selective inhibition of constitutively active Jak-2 kinase in acute lymphoblastic leukemia cells and Stat3 in the myosis fungoides tumor cell line by AG490 (B42) further suggested the use of tyrphostins in examining the regulation of signals mediated through the Jak-Stat pathway (30, 31).
In this study we have examined the effects of tyrphostin B42 (AG490) on IL-12-induced tyrosine phosphorylation and activation of Jak-2 kinase and the associated Tyk-2, Stat3, and Stat4 in normal T cells. We have also examined the inhibitory effects of tyrphostin B42 on the development of neural Ag-specific Th1 responses in vivo and the pathogenesis of EAE.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SJL/J mice (4–6 wk old) were purchased from Clarence Reader (National Institutes of Health, Bethesda, MD) and were maintained in the animal care facility at Vanderbilt University Medical Center (Nashville, TN).
Cytokines and Abs
Recombinant murine IL-12 was a gift from Genetics Institute
(Cambridge, MA). Tyrphostin A1
(C11H8N2O), A10
(C10H5N3O3), and B42
(C17H14N2O3) were
obtained from LC Laboratories (Woburn, MA). Anti-Jak-2 Ab and
anti-phosphotyrosine mAb 4G10 were purchased from Upstate
Biotechnology (Lake Placid, NY). Anti-Tyk-2 Ab and anti-Stat3 Ab
were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti-Stat4 Ab was purchased from Transduction Laboratories (Lexington,
KY). Recombinant murine IFN- was a gift from R. Budd (Vermont
University, Burlington, VT). The anti-IFN- mAb, R4-6A2 was
purified from ascitic fluid collected from nude mice following
transplantation of R4-6A2 hybridoma cells (HB 170, American Type
Culture Collection, Manassas, VA). The anti-IFN- mAb MM700 was
obtained from Endogen (Woburn, MA) and was conjugated with biotin
according to a standard protocol. Anti-IL-12 mAb C17.8 (anti-p40)
was prepared from hybridoma cells provided by Dr. G. Trinchieri (Wistar
Institute, Philadelphia, PA), and anti-IL-12p35 mAb was obtained
from PharMingen (San Diego, CA).
T cells and T cell clones
Con A-activated T cells were prepared by stimulation of mouse spleen cells (2 x 106/ml) with 5 µg/ml of Con A (Pharmacia Biotech, Uppsala, Sweden) in RPMI 1640 medium supplemented with 10% FBS (HyClone, Logan, UT) at 37°C in 5% CO2. After 3 days of culture, cells were harvested and recultured in medium containing 0.5% FBS for an additional 18 h to synchronize to G1 phase of the cell cycle. Dead cells were removed by centrifugation over Histopaque-1077 (Sigma, St. Louis, MO) at 1200 x g for 15 min, and the T cell blasts were used for experiments. This population of cells normally contains >98% T cell blasts as measured by flow cytometry (32).
HS-17 is an MBP/p91–103 peptide-specific CD4+ Th1 cell clone, developed by limiting dilution of spleen cells from the peptide-immunized SJL/J mice. The phenotype and maintenance of HS-17 cells have been described previously (33). Briefly, HS-17 T cells (1 x 106) were stimulated with 100 µg/ml of MBP/p91–103 peptide and irradiated spleen cells (1 x 107) for 2 days. The cells were harvested, washed, and expanded in RPMI 1640 medium supplemented with 10% FBS (HyClone) in the presence of 20 U/ml of IL-2.
Immunoprecipitation and Western blotting
The immunoprecipitation and Western blot analyses of Jak and Stat proteins were performed as described previously (32). Briefly, activated T cells (2.5 x 107/lane) were treated with 10 µM tyrphostins for 30 min, and then stimulated with 1 ng/ml of IL-12 at 37°C for 15 min. Cell lysates were prepared, and Jak-2, Tyk-2, Stat3, and Stat4 proteins were immunoprecipitated. The phosphoproteins in the immune complexes were analyzed by SDS-PAGE and Western blotting and visualized by the ECL method. The blots were stripped and reprobed with specific Ab to ensure equal protein loading.
In vitro kinase assay
The in vitro kinase activity of Jak proteins was determined as
described previously (32). Briefly, Con A-activated T cells (2 x
107/lane) were treated with 10 µM tyrphostin at 37°C
for 30 min and stimulated with 1 ng/ml of IL-12 at 37°C for 15 min.
The Jak-2 and Tyk-2 proteins were immunoprecipitated using specific Ab.
The binding of [-32P]ATP to Jak proteins in the immune
complexes was analyzed by SDS-PAGE followed by autoradiography.
Proliferation assay
Con A-activated T cells and HS-17 T cells were cultured in RPMI 1640 medium with different concentrations of IL-12 in the absence or the presence of different concentrations of tyrphostins for 48 h. Spleen cells from mice treated with tyrphostin B42 following immunization with MBP/CFA were cultured in the presence of different concentrations of Ag for 96 h. [3H]Thymidine (0.5 µCi/ml) was added during the last 18 h, and the uptake of radiolabel was measured as an index of proliferation (16, 32, 34).
Cell cycle analysis
The cell cycle progression of activated T cells was analyzed as described previously (32, 34). Con A-activated T cells were cultured with 1 ng/ml IL-12 in the absence or the presence of 10 µM tyrphostin A1 or B42 for 24 h. The cells were stained with propidium iodide and were analyzed based on DNA content by flow cytometry.
TdT assay for apoptosis
The apoptosis in activated T cells was determined by TdT assay as described previously (32, 34). Con A-activated T cells were cultured with 1 ng/ml of IL-12 in the absence or the presence of 10 µM tyrphostin A1 or B42 for 48 h. The cells were fixed, stained, and analyzed for DNA double-strand break by flow cytometry.
ELISA for IL-12, IFN-, and IL-4
HS-17 Th1 cells were stimulated with 1 ng/ml IL-12 in the
presence or the absence of 10 µM tyrphostins A1 or B42, and the
culture supernatants were collected at 48 h. Spleen cells from
mice immunized with MBP/CFA and treated with tyrphostin B42 were
cultured in the presence of 100 µg/ml of MBP, and the culture
supernatants were collected at 24, 48, 72, and 96 h. The levels of
IFN- in the culture supernatants were measured by a sandwich ELISA
using anti-IFN- mAb R4-6A2 as capture Ab and biotin-conjugated
anti-IFN- mAb MM700 as detection Ab. The levels of biologically
active IL-12 in the culture supernatants were measured by ELISA using
C17.8 mAb (anti-p40) as capture Ab and biotin-conjugated mAb
(anti-p35, PharMingen) as detection Ab. The levels of IL-4 in the
culture supernatants were measured using an ELISA kit obtained from
Endogen (16, 34).
Induction and treatment of EAE
To induce active EAE, 4- to 6-wk-old female SJL/J mice were immunized with 800 µg of mouse spinal cord homogenate (MSCH) in 150 µl of an emulsion of IFA containing 50 µg/ml of H37Ra in the lower dorsum on days 0, 7, and 14 (35). Mice in the test group were treated s.c. with tyrphostin B42 (200 µg/50 µl of DMSO) on days 0, 3, 7, 10, 14, 17, 20, and 24. Mice in the control group received 50 µl of DMSO. To induce passive EAE, 4- to 6-wk-old female SJL/J mice (donor) were immunized with 400 µg of MBP in CFA on days 0 and 7. On day 14, the lymph node and spleen cells were harvested and cultured in RPMI 1640 medium (5 x 106 or 2.5 x 106 cells/ml, respectively) containing 100 µg/ml MBP. After 4 days, the cells were harvested, T cell blasts were isolated by Ficoll-Hypaque gradient centrifugation, and 1 x 107 cells were injected i.p. into naive recipient female SJL/J mice (16). The recipient mice were treated s.c. with 200 µg of tyrphostin B42 in 50 µl of DMSO or with 50 µl of DMSO alone on alternative days from days 0–15. The disease in active and passive EAE was graded as follows; 0, normal; 0.5, stiff tail; 1, limp tail; 1.5, limp tail with inability to right; 2, paralysis of one limb; 2.5, paralysis of one limb and weakness of one other limb; 3, complete paralysis of both hind limbs; 4, moribund; and 5, death (16, 35).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-12 signal transduction involves tyrosine phosphorylation and
activation of Jak-2 and Tyk-2. Tyrphostin B42 was shown to be a
specific inhibitor of constitutively active Jak-2 in B cell lymphoma
(30). We examined whether tyrphostin B42 decreases the IL-12-induced
tyrosine phosphorylation of Jak-2 in normal T cells. Western blot
analysis of immunoprecipitates from Con A-activated T cells showed an
increase in the tyrosine phosphorylation of Jak-2 (Fig. 1
A) and Tyk-2 (Fig. 1B) following stimulation with 1 ng/ml of IL-12 for 15 min.
Treatment of cells with 10 µM tyrphostin B42 decreased the
IL-12-induced tyrosine phosphorylation of Jak-2 (Fig. 1A)
without affecting Tyk-2 (Fig. 1B). In contrast, treatment of
T cells with tyrphostin A1 inhibited the IL-12-induced tyrosine
phosphorylation of Tyk-2 (Fig. 1B), but not that of Jak-2
(Fig. 1A). Tyrphostin A10 had little or no effect on the
IL-12-induced tyrosine phosphorylation of Jak-2 and Tyk-2. Reprobing
the blots with anti-Jak-2 (Fig. 1A) or anti-Tyk-2
(Fig. 1B) Ab showed no difference in the amount of proteins
present in tyrphostin-treated vs untreated cells.
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To determine whether the inhibition of IL-12-induced tyrosine
phosphorylation of Jak kinases by tyrphostins leads to a decrease in
the catalytic activity, Jak-2 and Tyk-2 immunoprecipitates were tested
for their in vitro kinase activity. Stimulation of Con A-activated T
cells with 1 ng/ml of IL-12 increased the in vitro kinase activity of
Jak-2 and Tyk-2 kinases. Treatment of cells with 10 µM tyrphostin B42
specifically decreased the IL-12-induced activation of Jak-2 without
affecting Tyk-2 (Fig. 2). In contrast,
treatment of cells with A1 decreased the IL-12-induced activation of
Tyk-2, but not Jak-2, kinase (Fig. 2). Tyrphostin A10 had little or no
effect on the IL-12-induced activation of Jak-2 and Tyk-2 kinases.
These results suggest that tyrphostin B42 and A1 have mutually
exclusive inhibitory actions on the IL-12-induced tyrosine
phosphorylation and activation of Jak-2 and Tyk-2 kinases,
respectively, in T cells.
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IL-12-induced activation of Jak kinases leads to the tyrosine
phosphorylation and activation of transcription factors Stat3 and
Stat4. We examined the relationship between the activation of Jak
kinases and Stat proteins in IL-12 signaling. Stimulation of Con
A-activated T cells with 1 ng/ml of IL-12 resulted in the tyrosine
phosphorylation of Stat3 and Stat4 in 15 min (Fig. 3). Treatment of T cells with either
tyrphostin A1 or B42 inhibited the IL-12-induced tyrosine
phosphorylation of Stat3 (Fig. 3A), whereas treatment of
cells with A1 or B42 had little or no effect on the tyrosine
phosphorylation of Stat4 (Fig. 3B). These results suggest
that the inhibition of either Jak-2 or Tyk-2 leads to a decrease in the
tyrosine phosphorylation of Stat3, but not that of Stat4, protein in T
cells.
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To study the functional consequences following inhibition of IL-12
signaling, we examined the effect of tyrphostin B42 on IL-12-induced
proliferation of T cells. Stimulation of Con A-activated T cells with
IL-12 resulted in a dose-dependent increase in proliferation, with a
maximal response at 1 ng/ml (34). [3H]Thymidine uptake
increased from 1,745 ± 328 cpm in the absence of IL-12 to
129,963 ± 189 cpm following the addition of 1 ng/ml of IL-12 in
48 h. Addition of tyrphostin B42 to the cultures resulted a
dose-dependent decrease in the IL-12-induced proliferation of T cells
(Fig. 4A).
[3H]Thymidine uptake in T cells stimulated with 1 ng/ml
of IL-12 decreased to 13,866 ± 745 (90% inhibition;
p < 0.001) following the addition of 10 µM
tyrphostin B42. Addition of tyrphostin A1 or A10 showed no decrease in
the IL-12-induced proliferation of T cells.
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B). [3H]Thymidine uptake increased from
26,230 ± 3,138 cpm in the absence of IL-12 to 91,413 ±
17,724 cpm (3.5-fold increase) following the addition of 2 ng/ml of
IL-12 in 48 h. Addition of tyrphostin B42 to the cultures resulted
in a dose-dependent decrease in the IL-12-induced proliferation of
HS-17 T cells (Fig. 4C). [3H]Thymidine uptake
in HS-17 T cells stimulated with 1 ng/ml of IL-12 decreased to
18,084 ± 2,772 (80% inhibition; p < 0.001)
following the addition of 10 µM tyrphostin B42. These results suggest
that the inhibition of Jak-2 by tyrphostin B42 leads to a decrease in
the IL-12-induced proliferation of T cells. Tyrphostin B42 inhibits IL-12-induced T cell cycle progression
To examine whether the inhibition of IL-12-induced T cell
proliferation by tyrphostin B42 was due to cell cycle arrest, Con
A-activated T cells were stimulated with IL-12 in the presence or the
absence of tyrphostin for 24 h, and the cells in different cell
cycle stages were analyzed based on DNA content by flow cytometry (Fig. 5). T cells stimulated with 1 ng/ml of
IL-12 in the presence of 10 µM tyrphostin B42 showed growth arrest at
G1 phase of the cell cycle (G0-G1,
71%; S-G2-M, 29%). Cells that were stimulated with IL-12
in the absence of tyrphostin showed normal cell cycle progression
through S phase (G0-G1, 39%;
S-G2-M, 61%). Cells stimulated with IL-12 in the presence
of 10 µM tyrphostin A1 also showed normal cell cycle progression
through S phase (G0-G1, 41%;
S-G2-M, 59%). These results suggest that the inhibition of
Jak-2 by B42 leads to a decrease in DNA synthesis and T cell
proliferation.
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We next studied whether the inhibition of IL-12-induced T cell
proliferation by B42 leads to apoptotic cell death. Flow cytometric
analysis of TdT-stained T cells cultured for 48 h with 1 ng/ml of
IL-12 in the absence of tyrphostin showed apoptosis in 4% of cells,
whereas those cultured with IL-12 in the presence of 10 µM tyrphostin
B42 showed apoptosis in 48% of cells (p <
0.005). T cells cultured in medium alone in the absence of IL-12 and
tyrphostin showed 23% apoptosis and those cultured with IL-12 in the
presence of tyrphostin A1 showed only 7% apoptotic cells (Fig. 6). These results suggest that IL-12
prevents T cell apoptosis, and tyrphostin B42 blocks IL-12-mediated
rescue of T cells from death.
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in Th1
cells
To further study the effects of tyrphostin B42 on IL-12-induced
production of IFN-, HS-17 T cells were stimulated with IL-12 in the
presence or the absence of different concentrations of tyrphostins, and
the levels of IFN- in 48-h culture supernatants were measured by
ELISA. HS-17 T cells stimulated with 1 ng/ml of IL-12 produced
6.97 ± 0.58 ng/ml of IFN- in 48 h. Treatment of cells
with tyrphostin B42 resulted in a dose-dependent decrease in the
IL-12-induced production of IFN- (Fig. 7). The level of IFN- decreased to
1.06 ± 0.04 ng/ml (85% inhibition; p < 0.001)
following the addition of 10 µM tyrphostin B42 to HS-17 T cell
cultures. Addition of 10 µM tyrphostin A1 also decreased the level of
IFN- production to 3.21 ± 0.33 ng/ml (54% inhibition;
p < 0.005). These results suggest that the inhibition
of IL-12 signaling through Jak-2 or Tyk-2 by tyrphostin B42 or A1,
respectively, leads to a decrease in the IL-12-induced production of
IFN- in T cells.
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In view of the central role played by IL-12 in the pathogenesis of
EAE, a Th1 cell-mediated autoimmune disease, and the inhibitory effects
of tyrphostin B42 on IL-12 signal transduction in T cells, we set out
to examine the in vivo effects of tyrphostin B42 on the pathogenesis of
EAE. To test the effects on active EAE, SJL/J mice were treated with
tyrphostin B42 or DMSO following induction of EAE by immunization with
MSCH in CFA. Only 2 of 11 mice in the test group that received
tyrphostin B42 developed clinical paralysis, with a mean maximum
clinical score (MMCS) of 0.14. All 13 mice in the control group that
received DMSO developed paralysis with a MMCS of 1.92
(p < 0.001; Fig. 8A). The duration of disease
was also decreased following treatment with tyrphostin B42 (control,
12.2 days; B42, 6.5 days).
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To examine the effects of tyrphostin B42 on passive EAE, mice were
treated s.c. with 200 µg of tyrphostin B42 on alternate days from
0–15 following passive transfer of MBP-reactive T cells. None of the
six mice that received tyrphostin B42 developed EAE. In contrast,
five of six control mice developed clinical paralysis (MMCS, 0.95;
p < 0.001; Fig. 8B). These results suggest that the
tyrphostin B42 prevents the development of active and passive EAE.
Tyrphostin B42 prevents EAE by inhibiting the differentiation of neural Ag-specific Th1 cells in vivo
To confirm that tyrphostin B42 interfered with the generation of
encephalitogenic T cells, we examined the development of MBP-reactive
Th1 cells in vivo. Spleen cells were isolated from mice treated with
tyrphostin B42 or DMSO following immunization with MBP/CFA, and their
proliferative response to MBP was examined. Spleen cells from mice
treated with tyrphostin B42 showed a 36% decrease
(p < 0.005) in the proliferative response to
MBP compared with that in DMSO-treated cells (Fig. 9A). Spleen cells from
tyrphostin B42-treated mice also showed a decrease in the levels of
IL-12 and IFN- secretion in response to MBP in culture. The level of
IL-12p70 secretion in control mice was 1750 ± 240 pg/ml, and it
decreased to 600 ± 20 pg/ml (64% inhibition; p
< 0.005) following treatment with tyrphostin B42 (Fig. 9B).
IFN- production in control mice was 8 ± 0.56 ng/ml, and it
also decreased to 4.16 ± 0.29 ng/ml (48% inhibition;
p < 0.005) following treatment with tyrphostin B42
(Fig. 9C). No decrease in the levels of IL-4 production was
seen in the tyrphostin B42-treated mice compared with that in
DMSO-treated control mice (Fig. 9D). These results suggest
that treatment with tyrphostin B42 inhibits the generation of
MBP-specific Th1 cells in vivo and may account for its protective
effects on EAE.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and 4)
the pathogenesis of EAE. Inhibition of either Jak-2 or Tyk-2 leads to
impaired activation of Stat3 without altering the phosphorylation of
Stat4. Consequent to the inhibition of Jak-2 and Stat3, tyrphostin B42
decreased the IL-12-induced proliferation and survival of T cells.
Inhibition of Tyk-2 and Stat3 phosphorylation by tyrphostin A1 had no
effect on T cell proliferation or survival, whereas treatment with
either tyrphostin A1 or B42 attenuated the IL-12-induced secretion of
IFN- in T cells. In vivo treatment with tyrphostin B42 inhibited the
development of neural Ag-specific Th1 cells and the pathogenesis of
EAE. IL-12R belongs to the type I cytokine receptor superfamily, which is characterized by the absence of a catalytic domain within the cytoplasmic region (18). IL-12Rß1 and IL-12Rß2 are the two IL-12R subunits known to be responsible for IL-12 signal transduction. IL-12 signaling involves the tyrosine phosphorylation and activation of two members of the Janus family nonreceptor tyrosine kinases, Jak-2 and Tyk-2 (19). Treatment with tyrphostin B42 blocks IL-12-induced tyrosine phosphorylation and activation of Jak-2 kinase in normal T cells. An earlier report showed the in vitro and in vivo effects of tyrphostin B42 on the inhibition of constitutively active Jak-2 kinase in B cell lymphoma (30). Our study showed that tyrphostin B42 is equally effective in preventing IL-12, a potent proinflammatory cytokine-induced tyrosine phosphorylation, and activation of Jak-2 kinase in nontransformed T cells.
Although tyrphostin A1 has no inhibitory effect on a number of tyrosine kinases (26) and hence was used as a negative control in prior studies, we observed that A1 is effective in inhibiting IL-12-induced tyrosine phosphorylation and activation of Tyk-2. At equimolar concentrations, the inhibitory effect of tyrphostin A1 on Tyk-2 was comparable to that of tyrphostin B42 on Jak-2. The inhibition of IL-12-induced activation of Tyk-2 without affecting Jak-2 suggests that tyrphostin A1 is a specific inhibitor of Tyk-2 kinase. Since B42 inhibited the activation of Jak-2, but not Tyk-2, it was possible to dissect out the role of individual Jak kinases in IL-12 signaling and IL-12-mediated cellular functions. Although the physical association of Jak kinases with IL-12Rß subunits has not been well established, the specific inhibition of Jak-2 and Tyk-2 by tyrphostin B42 and A1, respectively, suggests their specificity to Jak kinases. Whether the inhibition of B42 and A1 is exclusively restricted to these Jak kinases is not known. The epidermal growth factor receptor kinase inhibitor, tyrphostin A10, did not have any effect on Jak-2, but showed partial inhibition of Tyk-2 kinase.
Stat transcription factors are cytoplasmic proteins that are phosphorylated and translocated into the nucleus following activation of associated kinases (36, 37). Although the activation of Stat proteins proceeds with the activation of Jak kinases in cytokine signaling, the association between the activation of Jak-2 and Tyk-2 and phosphorylation of Stat3 or Stat4 in IL-12 signaling is not known. Our study showed that the inhibition of either Jak-2 or Tyk-2 leads to a decrease in IL-12-induced tyrosine phosphorylation and activation of Stat3. Little or no inhibition of Stat4 was observed following inhibition of Jak-2 or Tyk-2. We have shown earlier that the concurrent inhibition of Jak-2 and Tyk-2 by TGF-ß leads to a decrease in the IL-12-induced tyrosine phosphorylation of Stat4 in T cells (34). In this study we have not examined the IL-12-induced tyrosine phosphorylation of Stat4 after concurrent inhibition of Jak-2 and Tyk-2 by treatment with A1 and B42 together. A recent study also showed that the inhibition of Jak-2 kinase by dominant negative mutants did not block the IL-3-induced tyrosine phosphorylation of Stat5, suggesting that the tyrosine kinases other than Jak-2 may also mediate the phosphorylation of Stat proteins (38). Taken together, these findings suggest that the activation of either Jak-2 or Tyk-2 kinase is sufficient to mediate the IL-12-induced tyrosine phosphorylation of Stat4, and the coactivation of both Jak-2 and Tyk-2 kinases appears to be required for the phosphorylation of Stat3.
Although IL-12 is an important growth factor for Th1 cells (39), the role of Jak-2 and Tyk-2 kinases in mediating IL-12-induced growth and survival signals is not well understood. Our study showed that the inhibition of IL-12 signaling through Jak-2 kinase by tyrphostin B42 results in decreased proliferation and increased apoptosis of T cells. Earlier studies have shown the antimitogenic effects of tyrphostins on normal and transformed cell lines (26). These findings are consistent with other reports showing the inhibition of T cell proliferation and induction of apoptosis following blockade of IL-2 and IL-12 signaling by TGF-ß in T cells (32, 40). The inactivation of Tyk-2 and Stat3 by A1 had no effect on proliferation or apoptosis, suggesting that Tyk-2 and Stat3 are not involved in regulating these T cell responses. Since T cell apoptosis was induced following inhibition of Jak-2 and Stat3 without affecting Stat4, B42 appears to block Jak-2 substrates other than Stat3. This would suggest the activation of other pathways that require Jak-2 kinase, but not Stat, proteins in IL-12 signaling. IL-3-induced activation of the Ras pathway is mediated by Jak-2 kinase that, in turn, prevents apoptosis in myeloid cells (41). However, activation of the Ras pathway by IL-12, and its regulation by Jak-2 kinase in T cells is not known.
The induction of IFN- is a key event in the IL-12-mediated
differentiation of Th1 cells. In this study we observed that the
inhibition of either Jak-2 or Tyk-2 resulted in a decrease in the
IL-12-induced secretion of IFN-, suggesting the involvement of both
kinases in the induction of IFN-. This study also showed that the
activation of Stat3 is essential for the induction of IFN- by IL-12.
A recent study using T cells from Stat4 knockout mice showed that Stat4
is required for T cell proliferation and IFN- production in response
to IL-12 (42). Taken together, these findings suggest that the
concurrent activation and heterodimerization of Stat3 and Stat4 may be
necessary for the induction of IFN- in IL-12 signaling, and the
inhibition of any one of these may lead to a blockade of IFN- gene
expression, whereas IL-12-induced T cell proliferation is mediated by
Stat4 and Jak-2 substrates other than Stat3, and the inhibition of any
one of these would lead to the blockade of T cell proliferation.
Consistent with proliferation, the inhibition of Jak-2 by tyrphostin
B42 leads to T cell apoptosis, whereas the apoptotic response in T
cells from Stat4 knockout mice is not known.
EAE is a Th1 cell-mediated autoimmune disease of the CNS that serves as
a prototypic animal model for MS (12). Although the presence of a
particular cytokine and its causal role for the development of a
disease have been difficult to predict, the central role played by
IL-12 in the development of neural Ag-specific Th1 responses and the
protective effects of neutralizing anti-IL-12 Ab make a strong
argument for its central role in EAE (6, 16, 17). In this study we have
successfully tested our hypothesis that the inhibition of IL-12
signaling be a novel approach to block the development of the Th1
cell-mediated autoimmune disease EAE. The inhibition of Ag-induced T
cell proliferation and IFN- production in MBP-sensitized spleen
cells following in vivo treatment with tyrphostin B42 suggests that
tyrphostin B42 inhibits IL-12 signaling and IL-12-mediated Th1
differentiation in vivo. The prevention of active and passive EAE by
treatment with tyrphostin B42 suggests that the in vivo inhibition of
IL-12 signaling impairs the development of neural Ag-specific Th1 cells
and the pathogenesis of CNS demyelination. No evidence for a switch
from Th1- to Th2-type immune response was seen in EAE mice following
treatment with tyrphostin B42. Recent studies also showed the
inhibition of EAE by treatment with tyrphostins, including B42 (AG490)
(43, 44). One of these studies noted an increase in IFN production
following treatment with B42 (AG490), which is not consistent with our
observation in this study. Further investigations on the effects of
tyrphostin B42 on Jak-2 kinase associated with other cytokine signaling
pathways will substantiate its role in the pathogenesis of EAE and
other autoimmune diseases.
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2 Address correspondence and reprint requests to Dr. Subramaniam Sriram, Multiple Sclerosis Research Center, Vanderbilt University School of Medicine, 1222 VSRH, 2201 Capers Ave., Nashville, TN 37212. E-mail address:
3 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental allergic encephalomyelitis; CNS, central nervous system; MBP/p91–103, myelin basic protein peptide/p91–103; Jak, Janus kinase; MSCH, mouse spinal cord homogenate; MMCS, mean maximum clinical score.
Received for publication April 30, 1998. Accepted for publication February 23, 1999.
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