This information is current as
of June 17, 2017.
Thalidomide Suppresses NF-κB Activation
Induced by TNF and H 2O2, But Not That
Activated by Ceramide, Lipopolysaccharides,
or Phorbol Ester
Sekhar Majumdar, Betty Lamothe and Bharat B. Aggarwal
J Immunol 2002; 168:2644-2651; ;
doi: 10.4049/jimmunol.168.6.2644
http://www.jimmunol.org/content/168/6/2644
Subscription
Permissions
Email Alerts
This article cites 70 articles, 35 of which you can access for free at:
http://www.jimmunol.org/content/168/6/2644.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2002 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
References
Thalidomide Suppresses NF-␬B Activation Induced by TNF
and H2O2, But Not That Activated by Ceramide,
Lipopolysaccharides, or Phorbol Ester1
Sekhar Majumdar, Betty Lamothe, and Bharat B. Aggarwal2
halidomide ([⫹]-␣-phthalimidoglutarimide), first synthesized almost 50 years ago as an antihistamine drug, was
soon found to have sedative effects in animal studies. Due
to lack of toxicity in animals even at 10 g/kg, it was approved in
1957 as an over-the-counter sedative during pregnancy in over 46
countries, and annual sales in Germany alone reached 14.58 tons
(1). However, this success did not last very long, because thalidomide was found to induce birth defects in humans; it was withdrawn from the market in 1965 (reviewed in Ref. 2). The demonstration in 1991 by Kaplan and colleagues (3) that thalidomide can
selectively inhibit TNF produced by stimulated human monocytes
and the central role of TNF in a wide variety of diseases placed this
novel derivative of glutamic acid on a comeback trail (4 – 6). In
1998, thalidomide was approved in the United States for the treatment
of erythema nodosum leprosum, a complication associated with
leprosy (7).
Thalidomide can modulate the role of TNF in replication of HIV
and in AIDS-associated wasting. It was found to block HIV replication (8, 9), suppress AIDS-associated wasting (10, 11), reduce
oral aphthous ulcers (12), and enhance weight gain in patients with
concomitant HIV-1 and Mycobacterium tuberculosis infection
(13). Thalidomide has shown significant promise in the treatment
of various immunological disorders including microsporidial diar-
T
Cytokine Research Laboratory, Department of Bioimmunotherapy, University of
Texas M. D. Anderson Cancer Center, Houston, TX 77030
Received for publication October 19, 2001. Accepted for publication January 2, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This research was supported by The Clayton Foundation for Research.
2
Address correspondence and reprint requests to Dr. Bharat B. Aggarwal, Cytokine
Research Laboratory, Department of Bioimmunotherapy, University of Texas M. D.
Anderson Cancer Center, 1515 Holcombe Boulevard, Box 143, Houston, TX 770304095. E-mail address: [email protected]
Copyright © 2002 by The American Association of Immunologists
rhea (14), bacterial meningitis (15, 16), chronic graft-vs-host disease (17), Crohn’s disease (18), and septic shock (19). Additionally, thalidomide was found to suppress angiogenesis (20), possibly through the inhibition of endothelial cell proliferation (21).
Because angiogenesis is critical for growth of most solid tumors,
thalidomide was tested against recurrent high-grade gliomas,
where it showed significant activity (22). Thalidomide is also active against multiple myeloma (23, 24), another cancer highly dependent on TNF (25).
The effect of thalidomide on various immunological disorders as
outlined above (1, 4, 6) is most likely mediated through its effect
on the modulation of cytokine production. Besides TNF, thalidomide has also been shown to decrease the production of chemokines, IL-6, and IL-12 (26 –30). Thalidomide increased the LPSinduced IL-10 production from PBMC but had no affect on antiCD3-induced IL-10 levels (28). This drug was also found to
suppress NO synthesis (31).
Suppression of the nuclear transcription factor NF-␬B activation
may explain several of the effects of thalidomide. A multisubunit
factor known to play a role in inflammation, immune modulation,
and cell proliferation (32), NF-␬B is primarily composed of proteins with molecular mass of 50 (p50) and 65 kDa (p65), and is
retained in the cytoplasm by I␬B␣. In its unstimulated form,
NF-␬B is activated by a wide variety of inflammatory stimuli,
including TNF, IL-1, okadaic acid, phorbol ester, H2O2, ceramide,
endotoxin, and gamma irradiation. Most of these agents induce the
phosphorylation-dependent degradation of I␬B␣ proteins, allowing active NF-␬B to translocate to the nucleus, where it regulates
gene expression. Constitutive activation of NF-␬B has been detected in various chronic inflammatory diseases treated by thalidomide, including Crohn’s disease, gastric ulcers, graft-vs-host disease, and AIDS (33–36). Angiogenesis also requires NF-␬B
activation (37). As a result, we postulated that thalidomide may
mediate its effects through suppression of NF-␬B. Therefore, in the
0022-1767/02/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Thalidomide ([ⴙ]-␣-phthalimidoglutarimide), a psychoactive drug that readily crosses the blood-brain barrier, has been shown
to exhibit anti-inflammatory, antiangiogenic, and immunosuppressive properties through a mechanism that is not fully established. Due to the central role of NF-␬B in these responses, we postulated that thalidomide mediates its effects through suppression
of NF-␬B activation. We investigated the effects of thalidomide on NF-␬B activation induced by various inflammatory agents in
Jurkat cells. The treatment of these cells with thalidomide suppressed TNF-induced NF-␬B activation, with optimum effect
occurring at 50 ␮g/ml thalidomide. These effects were not restricted to T cells, as other hematopoietic and epithelial cell types were
also inhibited. Thalidomide suppressed H2O2-induced NF-␬B activation but had no effect on NF-␬B activation induced by PMA,
LPS, okadaic acid, or ceramide, suggesting selectivity in suppression of NF-␬B. The suppression of TNF-induced NF-␬B activation
by thalidomide correlated with partial inhibition of TNF-induced degradation of an inhibitory subunit of NF-␬B (I␬B␣), abrogation of I␬B␣ kinase activation, and inhibition of NF-␬B-dependent reporter gene expression. Thalidomide abolished the NF␬B-dependent reporter gene expression activated by overexpression of TNFR1, TNFR-associated factor-2, and NF-␬B-inducing
kinase, but not that activated by the p65 subunit of NF-␬B. Overall, our results clearly demonstrate that thalidomide suppresses
NF-␬B activation specifically induced by TNF and H2O2 and that this may contribute to its role in suppression of proliferation,
inflammation, angiogenesis, and the immune system. The Journal of Immunology, 2002, 168: 2644 –2651.
The Journal of Immunology
present report we investigated whether thalidomide suppresses the
NF-␬B activation induced by various inflammatory stimuli and
whether it does so in different cell types. What pathway thalidomide employs to suppress NF-␬B activation was also investigated.
Materials and Methods
Materials
Cell lines
The cell lines T-Jurkat (T cells), HeLa (human epithelial cells), 293 (human embryonic kidney), and U937 (human histiocytic lymphoma) were
obtained from American Type Culture Collection (Manassas, VA). HeLa
and A293 cells were maintained in MEM, and the other cell lines were
cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml
penicillin, and 100 ␮g/ml streptomycin. For most studies, Jurkat cells were
used because these cells express both types of TNFR, and TNF-induced
responses in this cell type are well characterized in our laboratory.
NF-␬B activation
To determine NF-␬B activation, EMSA was conducted essentially as described (38). Briefly, nuclear extracts prepared from TNF-treated cells (2 ⫻
106/ml) were incubated with 32P-end-labeled 45-mer double-stranded
NF-␬B oligonucleotide (4 ␮g of protein with 16 fmoles DNA) from the
HIV long terminal repeat, 5⬘-TTGTTACAAGGGACTTTCCGCTGGG
GACTTTCCAGGGAGGCGTGG-3⬘ (underlining indicates NF-␬B binding sites) for 15 min at 37°C, and the DNA-protein complex formed was
separated from free oligonucleotide on 6.6% native polyacrylamide gels. A
double-stranded mutated oligonucleotide, 5⬘-TTGTTACAACTCACTTTC
CGCTGCTCACTTTCCAGGGAGGCGTGG-3⬘, was used to examine the
specificity of binding of NF-␬B to the DNA. The specificity of binding was
also examined by competition with the unlabeled oligonucleotide. For supershift assays, nuclear extracts prepared from TNF-treated cells were incubated with the Abs against either p50 or p65 of NF-␬B for 30 min at
room temperature before the complex was analyzed by EMSA. Abs against
c-Rel B and cyclin D1 and preimmune serum were included as negative
controls. The dried gels were visualized, and radioactive bands were quantitated by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using
ImageQuant software.
Degradation of I␬B␣
To determine the levels of I␬B␣, postnuclear (cytoplasmic) extracts were
prepared (39) from TNF-treated cells and resolved on 10% SDS-polyacrylamide gels. After electrophoresis, the proteins were electrotransferred to
nitrocellulose filters, probed with rabbit polyclonal Abs against I␬B␣, and
detected by ECL (Amersham Pharmacia Biotech, Arlington Heights, IL).
The bands obtained were quantitated using Personal Densitometer Scan
v1.30 using ImageQuant software version 3.3 (Molecular Dynamics).
IKK assay
The IKK assay was performed by a method described previously (40).
Briefly, IKK complex from cytoplasm was precipitated with Ab to IKK-␣,
followed by treatment with 20 ␮l of protein A/G-Sepharose (Pierce, RockAbbreviations used in this paper: IKK, I␬B␣ kinase; SEAP, secretory alkaline phosphatase; TRAF2, TNFR-associated factor-2; NIK, NF-␬B-inducing kinase; VEGF,
vascular endothelial growth factor; TPCK, N-tosyl-L-phenylalanyl chloromethyl
ketone.
3
ford, IL). After 2 h, the beads were washed with lysis buffer and then
assayed in kinase assay mixture containing 50 mM HEPES (pH 7.4), 20
mM MgCl2, 2 mM DTT, 20 ␮Ci [␥-32P]ATP, 10 ␮M unlabeled ATP, and
2 ␮g of substrate GST-I␬B␣. After incubation at 30°C for 30 min, the
reaction was terminated by boiling with 5 ␮l of 5⫻ SDS sample buffer for
5 min. Finally, the protein was resolved on 10% polyacrylamide gel under
reducing conditions, the gel was dried, and the radioactive bands were
visualized by PhosphorImager. To determine the total amounts of IKK-␣
and IKK-␤ in each sample, 60 ␮g of the cytoplasmic protein was resolved
on a 7.5% acrylamide gel and then electrotransferred to a nitrocellulose
membrane; the membrane was blocked with 5% nonfat milk protein for 1 h
and then incubated with either anti-IKK-␣ or anti-IKK-␤ (1/500 dilution)
for 1 h. The membrane was then washed and treated with HRP-conjugated
secondary anti-mouse IgG Ab and finally detected by chemiluminescence
(Amersham Pharmacia Biotech).
NF-␬B-dependent reporter gene transcription
The effect of thalidomide on TNF, TNFRI, TNFR-associated factor-2
(TRAF2), NF-␬B-inducing kinase (NIK), and p65 (transactivation subunit
of NF-␬B)-induced NF-␬B-dependent reporter gene transcription was measured as previously described (41). Briefly, human embryonic 293 cells
(0.5 million cells/well) were plated in six-well plates and transiently transfected the next day by the calcium phosphate method with pNF-␬B-secretory alkaline phosphatase (SEAP) (0.5 ␮g) and 0.5 ␮g of the expression
plasmids (TNFRI, TRAF2, NIK, and p65). The total final amount of DNA
was maintained at 2.5 ␮g by the addition of the control plasmid pCMVFLAG1 DNA. The cells were transfected for 18 h and after a medium
change treated with 10 ␮g/ml thalidomide for 24 h. To examine TNFinduced reporter gene expression, we transfected the cells with 0.5 ␮g of
the SEAP expression plasmid and 2 ␮g of the control plasmid pCMVFLAG1 DNA for 18 h and then pretreated for 2 h with 10 ␮g/ml thalidomide before treating them with 1 nM TNF. The cell culture medium was
harvested after 24 h of treatment and analyzed for SEAP activity according
to the protocol essentially as described by the manufacturer (Clontech Laboratories, Palo Alto, CA) using a 96-well fluorescence plate reader (Fluoroscan II; Labsystems, Chicago, IL) with excitation set at 360 nm and
emission at 460 nm.
Results
In this report we examined the effect of thalidomide on NF-␬B
activation induced by TNF and various other inflammatory stimuli
in different cell types. In the preliminary experiments, the concentration of thalidomide and the duration of exposure used had no
effect on cell viability (data not shown).
Thalidomide inhibits TNF-dependent NF-␬B activation
Jurkat cells were preincubated for 2 h with different concentrations
of thalidomide and treated with TNF (0.1 nM) for 30 min at 37°C,
and then nuclear extracts were prepared and assayed for NF-␬B
activation by EMSA. As shown in Fig. 1A, thalidomide inhibited
TNF-mediated NF-␬B activation in a dose-dependent manner,
with maximum inhibition occurring at 50 ␮g/ml. Thalidomide by
itself did not activate NF-␬B (data not shown).
Because NF-␬B is a family of proteins, various combinations of
Rel/NF-␬B protein can constitute an active NF-␬B heterodimer
that binds to a specific sequence in DNA (32). To show that the
retarded band visualized by EMSA in TNF-treated cells was indeed NF-␬B, we incubated nuclear extracts from TNF-activated
cells with Ab to either the p50 (NF-␬B1) or the p65 (RelA) subunit
of NF-␬B. Both shifted the band to a higher molecular mass (Fig.
1B), thus suggesting that the TNF-activated complex consisted of
p50 and p65 subunits. Neither preimmune serum nor such irrelevant Abs as anti-cyclin D1 had any effect. Excess unlabeled
NF-␬B (100-fold) caused complete disappearance of the band but
mutant oligonucleotide did not (data not shown), indicating the
specificity of NF-␬B.
Thalidomide does not block NF-␬B activation induced by
phorbol ester, okadaic acid, LPS, or ceramide
Besides TNF, NF-␬B is also activated by a wide variety of other
agents, including phorbol ester, okadaic acid, LPS, and ceramide.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Thalidomide was obtained from Tocris Cookson (St. Louis, MO). It was
dissolved in DMSO to give a stock solution of 20 mg/ml. Bacterial-derived
human rTNF with a specific activity of 5 ⫻ 107 U/mg was kindly provided
by Genentech (South San Francisco, CA). Penicillin, streptomycin, RPMI
1640 medium, and FBS were obtained from Life Technologies (Grand
Island, NY). Tris, glycine, NaCl, SDS, PMA, and BSA were obtained from
Sigma-Aldrich (St. Louis, MO). The polyclonal Abs used were as follows:
anti-p65, against the epitope corresponding to amino acids mapping within
the amino-terminal domain of human NF-␬B p65; anti-p50, against a peptide 15-aa long mapping to the nuclear localization region of NF-␬B p50;
anti-I␬B␣, against amino acids 297–317 mapping to the carboxyl terminus
of I␬B␣; anti-c-Rel; and anti-cyclin D1 against amino acids 1–295, which
represents full-length cyclin D1 of human origin. All these Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-I␬B␣
(Ser32) Ab was purchased from New England Biolabs (Beverly, MA). Anti-I␬B␣ kinase (IKK)3-␣ and anti-IKK-␤ Abs were kindly provided by
Imgenex (San Diego, CA).
2645
2646
THALIDOMIDE BLOCKS TNF-MEDIATED NF-␬B ACTIVATION
mechanism of NF-␬B activation by TNF and H2O2. The initial
potentiation of NF-␬B activation may have been due to oxidative
free radicals generated by thalidomide (2), which could contribute
to the H2O2-induced NF-␬B activation.
Inhibition of NF-␬B activation by thalidomide is not cell type
specific
That distinct signal transduction pathways could mediate NF-␬B
induction in epithelial and lymphoid cells has been demonstrated
(48). All of the effects of thalidomide described until now were
observed in human Jurkat T cells. Therefore, we also studied
whether thalidomide could block TNF-induced NF-␬B activation
in myeloid (U937), epithelial (HeLa), and embryonic kidney (293)
cells. These cells were pretreated with different concentrations of
thalidomide for 2 h and NF-␬B activated by treatment with TNF
for 30 min. Thalidomide inhibited most TNF-induced NF-␬B activation in all cell types (Fig. 3), thus suggesting that the suppression is not cell type specific.
FIGURE 1. Thalidomide blocks TNF-induced NF-␬B activation. A,
The effect of different concentrations of thalidomide on TNF-dependent
NF-␬B activation. Jurkat cells (2 ⫻ 106/ml) were preincubated at 37°C for
2 h with different concentrations (0 –50 ␮g/ml) of thalidomide and then
treated with 0.1 nM TNF for 30 min. After these treatments nuclear extracts were prepared and then assayed for NF-␬B as described in Materials
and Methods. B, The binding of NF-␬B to the DNA is specific and consists
of p50 and p65 subunits. Nuclear extracts were prepared from untreated or
TNF (0.1 nM)-treated Jurkat cells (2 ⫻ 106/ml), incubated for 30 min with
different Abs or cold NF-␬B oligo probes, and then assayed for NF-␬B.
However, the signal transduction pathway induced by these agents
may differ (42– 46). Therefore, we examined the effect of thalidomide on the activation of NF-␬B by these agents. As shown in
Fig. 2, A and B, thalidomide had no effect on the activation of
NF-␬B induced by PMA, LPS, okadaic acid, and ceramide. This
suggests that the mechanism by which these agents activate NF-␬B
is different from that of TNF.
Thalidomide blocks NF-␬B activation induced by H2O2
It has recently been reported that NF-␬B activation by H2O2 occurs through a mechanism distinct from that by TNF (47). To
examine the effect of thalidomide on NF-␬B activation induced by
H2O2, we pretreated cells with 50 ␮g/ml thalidomide for 2 h and
treated them with 250 ␮M H2O2 for different times. As shown in
Fig. 2C, thalidomide potentiated the H2O2-induced NF-␬B activation at shorter incubation time (30 min) but suppressed it at longer
incubations (120 min). The suppression suggests a similarity in the
It has been shown that N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) (serine protease inhibitor), herbimycin A (protein
tyrosine kinase inhibitor), and caffeic acid phenylethyl ester downregulate NF-␬B activation by chemical modification of the NF-␬B
subunits, thus preventing its binding to DNA (49 –51). To determine whether thalidomide also modifies NF-␬B proteins, we incubated nuclear extracts prepared from TNF-activated cells with
different concentrations of thalidomide in vitro for either 1 or 2 h
and then performed EMSA (Fig. 4). The results in Fig. 4A indicate
that incubation of NF-␬B with 50 ␮g/ml thalidomide for 1 h completely suppressed its ability to bind to the DNA. Two hours of
treatment with thalidomide had no additional effect on the suppression of NF-␬B activity (Fig. 4B). These results suggest that
thalidomide may suppress NF-␬B activation through a mechanism
similar to that of TPCK, herbimycin A, and caffeic acid phenylethyl ester.
Thalidomide inhibits TNF-dependent degradation of I␬B␣
The activation of NF-␬B by TNF requires the proteolytic degradation of I␬B␣ (32). To determine whether inhibition of TNFinduced NF-␬B activation was due to inhibition of I␬B␣ degradation, we pretreated cells with 50 ␮g/ml thalidomide for 2 h,
exposed them to 0.1 nM TNF for different times, and then examined them for NF-␬B in the nucleus by EMSA and for I␬B␣ in the
cytoplasm by Western blot. As shown in Fig. 5A, TNF activated
NF-␬B in the control cells in a time-dependent manner but had no
effect in thalidomide-pretreated cells. TNF induced I␬B␣ degradation in control cells as early as 5 min, but in thalidomide-pretreated cells TNF-induced I␬B␣ degradation was suppressed (Fig.
5B), although not completely. In TNF-treated cells, a complete
resynthesis of I␬B␣ occurred at 60 min, when NF-␬B is still active. The resynthesis of I␬B␣ appeared to occur faster in cells
pretreated with thalidomide.
Thalidomide inhibits TNF-induced IKK-␤ activation
The translocation of NF-␬B to the nucleus is preceded by the phosphorylation, ubiquitination, and proteolytic degradation of I␬B␣
(32). Because TNF-induced phosphorylation of I␬B␣ is mediated
through IKK-␤, these results suggest that thalidomide must inhibit
IKK-␤ activation. Indeed, as shown in Fig. 5C, in immune complex kinase assays, TNF activated IKK-␤ in a time-dependent
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Thalidomide suppresses the DNA-binding ability of NF-␬B
proteins
The Journal of Immunology
2647
manner and thalidomide treatment suppressed the activation. Under these conditions, IFN-␣ had no effect on the IKK-␣ and IKK-␤
protein levels (data not shown).
Thalidomide represses TNF-induced NF-␬B-dependent reporter
gene expression
Although we have shown by EMSA that thalidomide blocks
NF-␬B activation and phosphorylation and degradation of I␬B␣,
DNA binding alone does not always correlate with NF-␬B-dependent gene transcription, suggesting the role of additional regulatory
steps (52). To determine the effect of thalidomide on TNF-induced
NF-␬B-dependent reporter gene expression, we transiently transfected thalidomide-pretreated or untreated cells with the NF-␬Bregulated SEAP reporter construct and then stimulated the cells
with TNF. An almost 10-fold increase in SEAP activity over the
vector control was noted upon stimulation with TNF (Fig. 6).
TNF-induced SEAP activity was almost completely abolished by
dominant-negative I␬B␣, indicating the specificity. When the cells
were pretreated with thalidomide, TNF-induced NF-␬B-dependent
SEAP expression was inhibited in a dose-dependent manner.
These results demonstrate that thalidomide also represses NF-␬Bdependent reporter gene expression induced by TNF.
Thalidomide inhibits NF-␬B-dependent reporter gene expression
induced by TNFR1, TRAF2, and NIK
TNF-induced NF-␬B activation is mediated through sequential interaction of the TNFR with TNFR-associated death domain,
TRAF2, NIK, and IKK-␤, resulting in phosphorylation of I␬B␣.
To delineate the site of action of thalidomide in the TNF signaling
pathway leading to NF-␬B activation, cells were transfected with
TNFR1, TRAF2, NIK, and p65 plasmids, and then NF-␬B-dependent SEAP expression was monitored in untreated and thalidomide-treated cells. As shown in Fig. 7, TNFR1, TRAF2, NIK, and
p65 plasmids induced gene expression and thalidomide suppressed
TNFR1-, TRAF2-, and NIK-induced but not p65-induced NF-␬B
reporter expression. Thus thalidomide must act at a step downstream from NIK. Because NIK is known to activate IKK-␤, which
in turn phosphorylates I␬B␣, it appears that thalidomide must
block the activity of IKK-␤. Unlike the in vitro modification of
NF-␬B activity, thalidomide had no effect on the in vivo transcriptional activity of p65 subunit of NF-␬B.
Discussion
Because several in vitro and in vivo anti-inflammatory and immunomodulatory effects assigned to thalidomide require suppression
of NF-␬B activation, we tested the hypothesis that thalidomide
directly blocks NF-␬B activation. We found that thalidomide was
indeed a potent inhibitor of NF-␬B activation induced by TNF and
H2O2 but had no effect on NF-␬B activated by PMA, LPS, and
ceramide. Thalidomide suppressed inducible but not constitutive
NF-␬B activation. The suppression of NF-␬B by thalidomide accompanied inhibition of NF-␬B binding to the DNA, and suppression of I␬B␣ degradation and of I␬B␣ kinase. NF-␬B-dependent
reporter gene transcription induced by TNF, TNFR1, TRAF2, and
NIK was also suppressed by thalidomide.
Treatment of Jurkat cells with thalidomide completely suppressed NF-␬B activation induced by TNF and H2O2 but not that
activated by PMA, LPS, or ceramide. This differential effect is in
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 2. A, Thalidomide blocks H2O2-induced but not LPS-, ceramide-, okadaic acid-, and
PMA-induced NF-␬B activation. A, Jurkat cells
(2 ⫻ 106/ml) were preincubated for 120 min at 37°C
with thalidomide (50 ␮g/ml), treated with TNF (0.1
nM), PMA (25 ng/ml), or LPS (10 ␮g/ml) for 30
min, and then tested for NF-␬B activation as described in Materials and Methods. B, Jurkat cells
(2 ⫻ 106/ml) were preincubated for 120 min at 37°C
with thalidomide (50 ␮g/ml), treated with H2O2
(250 ␮M), okadaic acid (500 nM), or ceramide (10
␮M) for 30 min, and then tested for NF-␬B activation as described in Materials and Methods. C, Effect of thalidomide on the kinetics for NF-␬B activation by H2O2. Jurkat cells (2 ⫻ 106/ml) were
pretreated for 2 h with thalidomide (50 ␮g/ml) at
37°C, activated by H2O2 at different time points, and
tested for NF-␬B activation as described in Materials and Methods.
2648
THALIDOMIDE BLOCKS TNF-MEDIATED NF-␬B ACTIVATION
FIGURE 3. Thalidomide suppresses TNF-induced NF-␬B in different
cell types. U937 (A), 293 (B), and HeLa (C) cells (2 ⫻ 106/ml) were
preincubated at 37°C for 2 h with different concentrations of thalidomide
and then treated with 0.1 nM TNF for 30 min. Nuclear extracts were prepared and tested for NF-␬B activation as described in Materials and Methods. C, Medium control.
agreement with Rowland et al. (53), who examined the effect of
thalidomide on NF-␬B activation in PBMCs activated by PMA
and ionophore. Our results are also in agreement with a recent
report from Keifer et al. (54), who found that thalidomide blocks
TNF- and IL-1-induced NF-␬B activation. Why thalidomide
blocks TNF-induced NF-␬B activation but not that activated by
LPS, ceramide, or phorbol ester suggests a difference in the mechanism of NF-␬B activation by different agents. For instance, we
have shown that p56lck protein tyrosine kinase is required for ceramide-induced NF-␬B activation but not for TNF (43). Similarly,
ribosomal protein S6 kinase (pp90rsk) has been shown to be involved in phorbol ester-induced NF-␬B activation but not in TNFinduced activation (55, 56).
We also found that thalidomide blocks H2O2-induced NF-␬B
activation. Recent reports indicate that H2O2 suppresses NF-␬B
activation through a very different mechanism from that of TNF
(47). For example, TNF-induced NF-␬B activation requires I␬B␣
serine phosphorylation and then degradation, whereas that by
H2O2 leads to tyrosine phosphorylation but not degradation. Despite these differences, our results suggest that thalidomide must
act at a step that is common to both TNF- and H2O2-induced
activation.
Our results also indicate that thalidomide prevents NF-␬B from
binding to the DNA, but how is not clear. It has been previously
shown that herbimycin A, caffeic acid phenethyl ester, selenite,
and TPCK also prevent the DNA binding of NF-␬B, most likely
through modification of the p50 subunit of NF-␬B (49 –51, 57).
Herbimycin A and selenite have been shown to cause a covalent
modification of a key thiol at cysteine 62 of the p50 subunit of
NF-␬B, thereby inhibiting its binding to the DNA. Conversely,
thioredoxin was found to stimulate the DNA binding of NF-␬B by
reduction of the disulfide bond involving cysteine 62 (58).
Whether the same thiol is modified by thalidomide is not clear.
That thalidomide can induce DNA damage through generation of
oxidative free radical has been recently reported (2). The same
mechanism could apply to suppression of NF-␬B activation. It is
unlikely, however, that the oxidative damage is the only mechanism for suppression of NF-␬B by thalidomide, because this drug
had no effect on NF-␬B activation by PMA, ceramide, and LPS. In
this respect, thalidomide appears to be specific. The inability of
thalidomide to block NF-␬B activation by other agents suggests a
complex mechanism of action.
Thalidomide also blocked TNF-induced I␬B␣ degradation and
activation of IKK needed for NF-␬B activation. These results are
in agreement with those of Keifer et al. (54). Like thalidomide,
TPCK-suppressed NF-␬B activation also correlated with inhibition
of in vitro DNA binding of NF-␬B and with inhibition of the
phosphorylation and degradation of I␬B␣ (49). TNF-induced
NF-␬B activation is mediated through sequential interaction of the
TNFR with TNFR-associated death domain, TRAF2, NIK, and
IKK-␤, resulting in phosphorylation of I␬B␣. We found that thalidomide blocked the NF-␬B activation induced by the TNFR,
TRAF2, and NIK but had no effect on p65-mediated gene transcription. These results also suggest the site of NF-␬B activation
lies between NIK and p65, thus pointing to IKK.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 4. Thalidomide inhibits in vitro DNA binding of NF-␬B protein. Nuclear extracts (NE) were prepared from Jurkat cells treated with 0.1
nM TNF for 30 min; 5 ␮g/sample NE protein was treated with indicated
concentrations of thalidomide for either 1 (A) or 2 h (B) and then assayed
for DNA binding by EMSA.
The Journal of Immunology
2649
We found that thalidomide blocks NF-␬B activation in a wide variety of cells including T cells, myeloid cells, and epithelial cells.
Others have reported that thalidomide blocks TNF-induced NF-␬B
activation in endothelial cells (54). Thus suppression of NF-␬B activation by thalidomide does not appear to be cell type specific.
Our results demonstrate that thalidomide blocks not only NF-␬B
activation as monitored by DNA binding but also NF-␬B-dependent reporter gene transcription. These results are consistent with
reports that showed the inhibition of expression by thalidomide of
several genes regulated by NF-␬B, including inflammatory cyto-
FIGURE 6. Thalidomide inhibits TNF-induced NF-␬B-dependent
SEAP reporter gene expression. 293 cells treated with different concentrations of thalidomide were transiently transfected with NF-␬B containing
plasmid linked to the SEAP gene. After 24 h in culture with TNF, cell
supernatants were collected and assayed for SEAP activity as described in
Materials and Methods. Results are expressed as fold activity over the
nontransfected control. The specificity of the assay was examined by suppression of TNF-induced NF-␬B reporter activity by I␬B␣ dominant-negative mutant plasmid.
FIGURE 7. Thalidomide inhibits NF-␬B-dependent reporter gene expression induced by TNFRI, TRAF2, and NIK. Human embryonic 293 cells were
transiently transfected with indicated plasmids along with NF-␬B containing
plasmid linked to the SEAP gene for 18 h. After a medium change, the cells
were treated with 10 ␮g/ml thalidomide for 24 h. Where indicated, cells were
exposed to 1 nM TNF for 24 h after 2 h of preincubation with 10 ␮g/ml
thalidomide. Supernatants were collected and assayed for SEAP activity as
described in Materials and Methods. Results are expressed as fold activity over
the nontransfected control. Horizontal bars, SD.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 5. A and B, Effect of thalidomide on
TNF-induced degradation of I␬B␣. Jurkat cells were
incubated at 37°C with thalidomide (50 ␮g/ml) for 2 h
and then treated with 0.1 nM TNF at 37°C for different
times as indicated and then tested for NF-␬B activation by EMSA (A) and for I␬B␣ in cytosolic fractions
by Western blot analysis (B). ␤-Actin shows equal
protein loading. C, Thalidomide inhibits TNF-induced
IKK activity. Cells (2 ⫻ 106/ml) were incubated first
with thalidomide (50 ␮g/ml) for 2 h and then with
TNF (1 nM) for different times and then analyzed for
IKK by the immune complex kinase assay as described in Materials and Methods.
2650
THALIDOMIDE BLOCKS TNF-MEDIATED NF-␬B ACTIVATION
Acknowledgments
We thank Walter Pagel for a careful review of the manuscript.
References
1. Zwingenberger, K., and S. Wnendt. 1995. Immunomodulation by thalidomide:
systematic review of the literature and of unpublished observations. J. Inflamm.
46:177.
2. Parman, T., M. J. Wiley, and P. G. Wells. 1999. Free radical-mediated oxidative
DNA damage in the mechanism of thalidomide teratogenicity. Nat. Med. 5:582.
3. Sampaio, E. P., E. N. Sarno, R. Galilly, Z. A. Cohn, and G. Kaplan. 1991.
Thalidomide selectively inhibits tumor necrosis factor ␣ production by stimulated
human monocytes. J. Exp. Med. 173:699.
4. Klausner, J. D., V. H. Freedman, and G. Kaplan. 1996. Thalidomide as an antiTNF-␣ inhibitor: implications for clinical use. Clin. Immunol. Immunopathol.
81:219.
5. Hales, B. F. 1999. Thalidomide on the comeback trail. Nat. Med. 5:489.
6. Schuler, U., and G. Ehninger. 1995. Thalidomide: rationale for renewed use in
immunological disorders. Drug Safety 12:364.
7. Sampaio, E. P., G. Kaplan, A. Miranda, J. A. Nery, C. P. Miguel, S. M. Viana,
and E. N. Sarno. 1993. The influence of thalidomide on the clinical and immunologic manifestation of erythema nodosum leprosum. J. Infect. Dis. 168:408.
8. Makonkawkeyoon, S., R. N. Limson-Pobre, A. L. Moreira, V. Schauf, and
G. Kaplan. 1993. Thalidomide inhibits the replication of human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 90:5974.
9. Moreira, A. L., L. G. Corral, W. Ye, B. Johnson, D. Stirling, G. W. Muller,
V. H. Freedman, and G. Kaplan. 1997. Thalidomide and thalidomide analogs
reduce HIV type 1 replication in human macrophages in vitro. AIDS Res. Hum.
Retroviruses 13:857.
10. Reyes-Teran, G., J. G. Sierra-Madero, V. Martinez del Cerro, H. Arroyo-Figueroa,
A. Pasquetti, J. J. Calva, and G. M. Ruiz-Palacios. 1996. Effects of thalidomide on
HIV-associated wasting syndrome: a randomized, double-blind, placebo-controlled
clinical trial. AIDS 10:1501.
11. Kaplan, G., S. Thomas, D. S. Fierer, K. Mulligan, P. A. Haslett, W. J. Fessel,
L. G. Smith, K. A. Kook, D. Stirling, and M. Schambelan. 2000. Thalidomide for
the treatment of AIDS-associated wasting. AIDS Res. Hum. Retroviruses 16:
1345.
12. Jacobson, J. M., J. S. Greenspan, J. Spritzler, N. Ketter, J. L. Fahey, J. B. Jackson,
L. Fox, M. Chernoff, A. W. Wu, L. A. MacPhail, et al. 1997. Thalidomide for the
treatment of oral aphthous ulcers in patients with human immunodeficiency virus
infection. N. Engl. J. Med. 336:1487.
13. Klausner, J. D., S. Makonkawkeyoon, P. Akarasewi, K. Nakata, W. Kasinrerk,
L. Corral, R. L. Dewar, H. C. Lane, V. H. Freedman, and G. Kaplan. 1996. The
effect of thalidomide on the pathogenesis of human immunodeficiency virus type
1 and M. tuberculosis infection. J. Acquired Immune Defic. Syndr. Hum. Retrovirol. 11:247.
14. Sharpstone, D., A. Rowbottom, M. Nelson, and B. Gazzard. 1995. The treatment
of microsporidial diarrhoea with thalidomide. AIDS 9:658.
15. Burroughs, M. H., L. Tsenova-Berkova, K. Sokol, J. Ossig, E. Tuomanen, and
G. Kaplan. 1995. Effect of thalidomide on the inflammatory response in cerebrospinal fluid in experimental bacterial meningitis. Microb. Pathog. 19:245.
16. Schoeman, J. F., P. Springer, A. Ravenscroft, P. R. Donald, L. G. Bekker,
A. J. van Rensburg, W. A. Hanekom, P. A. Haslett, and G. Kaplan. 2000. Adjunctive thalidomide therapy of childhood tuberculous meningitis: possible antiinflammatory role. J. Child Neurol. 15:497.
17. Vogelsang, G. B., E. R. Farmer, A. D. Hess, V. Altamonte, W. E. Beschorner,
D. A. Jabs, R. L. Corio, L. S. Levin, O. M. Colvin, J. R. Wingard, et al. 1992.
Thalidomide for the treatment of chronic graft-versus-host disease. N. Engl.
J. Med. 326:1055.
18. Ginsburg, P. M., I. Hanan, and E. D. Ehrenpreis. 2001. Treatment of severe
esophageal Crohn’s disease with thalidomide. Am. J. Gastroenterol. 96:1305.
19. Moreira, A. L., J. Wang, E. N. Sarno, and G. Kaplan. 1997. Thalidomide protects
mice against LPS-induced shock. Braz. J. Med. Biol. Res. 30:1199.
20. D’Amato, R. J., M. S. Loughnan, E. Flynn, and J. Folkman. 1994. Thalidomide
is an inhibitor of angiogenesis. Proc. Natl. Acad. Sci. USA 91:4082.
21. Moreira, A. L., D. R. Friedlander, B. Shif, G. Kaplan, and D. Zagzag. 1999.
Thalidomide and a thalidomide analogue inhibit endothelial cell proliferation in
vitro. J. Neurooncol. 43:109.
22. Fine, H. A., W. D. Figg, K. Jaeckle, P. Y. Wen, A. P. Kyritsis, J. S. Loeffler,
V. A. Levin, P. M. Black, R. Kaplan, J. M. Pluda, and W. K. Yung. 2000. Phase
II trial of the antiangiogenic agent thalidomide in patients with recurrent highgrade gliomas. J. Clin. Oncol. 18:708.
23. Davies, F. E., N. Raje, T. Hideshima, S. Lentzsch, G. Young, Y. T. Tai, B. Lin,
K. Podar, D. Gupta, D. Chauhan, et al. 2001. Thalidomide and immunomodulatory derivatives augment natural killer cell cytotoxicity in multiple myeloma.
Blood 98:210.
24. Hideshima, T., D. Chauhan, Y, Shima, N. Raje, F. E. Davies, Y. T. Tai,
S. P. Treon, B. Lin, R. L. Schlossman, P. Richardson, et al. 2000. Thalidomide
and its analogs overcome drug resistance of human multiple myeloma cells to
conventional therapy. Blood 96:2943.
25. Hideshima, T., D. Chauhan, R. Schlossman, P. Richardson, and K. C. Anderson.
2001. The role of tumor necrosis factor ␣ in the pathophysiology of human
multiple myeloma: therapeutic applications. Oncogene 20:4519.
26. Lokensgard, J. R., S. Hu, E. M. van Fenema, W. S. Sheng, and P. K. Peterson.
2000. Effect of thalidomide on chemokine production by human microglia. J. Infect. Dis. 182:983.
27. Moreira, A. L., L. Tsenova-Berkova, J. Wang, P. Laochumroonvorapong,
S. Freeman, V. H. Freedman, and G. Kaplan. 1997. Effect of cytokine modulation
by thalidomide on the granulomatous response in murine tuberculosis. Tuber.
Lung Dis. 78:47.
28. Corral, L. G., P. A. Haslett, G. W. Muller, R. Chen, L. M. Wong, C. J. Ocampo,
R. T. Patterson, D. I. Stirling, and G. Kaplan. 1999. Differential cytokine modulation and T cell activation by two distinct classes of thalidomide analogs that
are potent inhibitors of TNF-␣. J. Immunol. 163:380.
29. Haslett, P. A., J. D. Klausner, S. Makonkawkeyoon, A. Moreira, P. Metatratip,
B. Boyle, W. Kunachiwa, N. Maneekarn, P. Vongchan, L. G. Corral, et al. 1999.
Thalidomide stimulates T cell responses and interleukin 12 production in HIVinfected patients. AIDS Res. Hum. Retroviruses 15:1169.
30. Haslett, P. A., L. G. Corral, M. Albert, and G. Kaplan. 1998. Thalidomide costimulates primary human T lymphocytes, preferentially inducing proliferation,
cytokine production, and cytotoxic responses in the CD8⫹ subset. J. Exp. Med.
187:1885.
31. Lopez-Talavera, J. C., G. Cadelina, J. Olchowski, W. Merrill, and
R. J. Groszmann. 1996. Thalidomide inhibits tumor necrosis factor ␣, decreases
nitric oxide synthesis, and ameliorates the hyperdynamic circulatory syndrome in
portal-hypertensive rats. Hepatology 23:1616.
32. Verma, I. M., J. K. Stevenson, E. M. Schwarz, D. Van Antwerp, and
S. Miyamoto. 1995. Rel/NF-␬B/I␬B family: intimate tales of association and
dissociation. Genes Dev. 9:2723.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
kines (such as TNF, IL-6, IL-12, and chemokines), antiapoptotic
molecules (TRAF1 and c-IAP2), and NO synthase (26 –32, 54).
The revival of thalidomide as a therapeutic agent is based on reports that this drug can suppress TNF production (3, 53, 59 – 61).
What role inhibition of NF-␬B activation by thalidomide plays in
suppression of TNF production is less clear. Many investigators
have examined the effect of thalidomide on TNF production in
macrophages induced by either LPS (3, 59 – 61) or phorbol ester
(53). We found that thalidomide had no effect on NF-␬B activation
induced by either of the agents. Although NF-␬B is needed for
TNF production, thalidomide may not mediate its effects on TNF
through NF-␬B suppression. Thalidomide has been shown to block
TNF production by either enhancing mRNA degradation (59) or
by binding the drug to ␣1-acid glycoprotein (60). This suggests
that thalidomide could modulate cytokine production by multiple
mechanisms. Thus our results indicate that the ability of thalidomide to modulate gene expression through suppression of NF-␬B
is determined by the pathway activated by the inducer.
Thalidomide has been shown to inhibit angiogenesis (20). Several reports suggest the critical role of TNF in angiogenesis and the
involvement of IL-8, vascular endothelial growth factor (VEGF),
and basic fibroblast growth factor in TNF-dependent angiogenesis
(62, 63). Because TNF regulates the expression of VEGF and IL-8
through activation of NF-␬B, our results suggest that thalidomide
may regulate TNF-induced angiogenesis through down-regulation
of the expression of VEGF and IL-8. The suppression of proliferation of endothelial cells by thalidomide also appears to correlate
with suppression of activation of NF-␬B and another transcription
factor, SP-1 (21). TNF is also known to stimulate the HIV enhancer by activation of NF-␬B (36). The ability of thalidomide to
suppress HIV replication (8) may also be mediated through inhibition of NF-␬B activation (9), as may the activity of thalidomide
against multiple myeloma (22, 23) because, first, NF-␬B is constitutively active in multiple myeloma cells (64) and, second, TNF
plays a critical role in the pathophysiology of human multiple myeloma (25). Similarly, activated NF-␬B has been found during
both arthritis and Crohn’s disease (65, 66), which may explain the
inhibitory effects of thalidomide against these diseases (18, 67).
The suppression of NF-␬B may also explain the effects of thalidomide observed against various cancers (22–25, 68) and other inflammatory diseases (69, 70). Overall our results indicate that thalidomide blocks NF-␬B activation and NF-␬B-dependent gene
expression in most cell types but only that induced by certain
agents, and that this suppression occurs through multiple
mechanisms.
The Journal of Immunology
52. Nasuhara, Y., I. M. Adcock, M. Catley, P. J. Barnes, and R. Newton. 1999.
Differential I␬B kinase activation and I␬B␣ degradation by interleukin-1␤ and
tumor necrosis factor-␣ in human U937 monocytic cells: evidence for additional
regulatory steps in ␬B-dependent transcription. J. Biol. Chem. 274:19965.
53. Rowland, T. L., S. M. McHugh, J. Deighton, P. W. Ewan, R. J. Dearman, and
I. Kimber. 2001. Differential effect of thalidomide and dexamethasone on the
transcription factor NF-␬B. Int. Immunopharmacol. 1:49.
54. Keifer, J. A., D. C. Guttridge, B. P. Ashburner, and A. S. Baldwin, Jr. 2001.
Inhibition of NF-␬B activity by thalidomide through suppression of I␬B kinase
activity. J. Biol. Chem. 276:22382.
55. Ghoda, L., X. Lin, and W. C. Greene. 1997. The 90-kDa ribosomal S6 kinase
(pp90rsk) phosphorylates the N-terminal regulatory domain of I␬B␣ and stimulates its degradation in vitro. J. Biol. Chem. 272:21281.
56. Schouten, G. J., A. C. Vertegaal, S. T. Whiteside, A. Israel, M. Toebes,
J. C. Dorsman, A. J. van der Eb, and A. Zantema. 1997. I␬B␣ is a target for the
mitogen-activated 90 kDa ribosomal S6 kinase. EMBO J. 16:3133.
57. Kim, I. Y., and T. Stadtman. 1997. Inhibition of NF-␬B DNA binding and nitric
oxide induction in human T cells and lung adenocarcinoma cells by selenite
treatment. Proc. Natl. Acad. Sci. USA 94:12904.
58. Matthews, J. R., N. Wakasugi, J. L. Virelizier, J. Yodoi, and R. T. Hay. 1992.
Thioredoxin regulates the DNA binding activity of NF-␬B by reduction of a
disulphide bond involving cysteine 62. Nucleic Acids Res. 20:3821.
59. Moreira, A. L., E. P. Sampaio, A. Zmuidzinas, P. Frindt, K. A. Smith, and
G. Kaplan. 1993. Thalidomide exerts its inhibitory action on tumor necrosis factor ␣ by enhancing mRNA degradation. J. Exp. Med. 177:1675.
60. Turk, B. E., H. Jiang, and J. O. Liu. 1996. Binding of thalidomide to ␣1-acid
glycoprotein may be involved in its inhibition of tumor necrosis factor ␣ production. Proc. Natl. Acad. Sci. USA 93:7552.
61. Tavares, J. L., A. Wangoo, P. Dilworth, B. Marshall, S. Kotecha, and R. J. Shaw.
1997. Thalidomide reduces tumour necrosis factor-␣ production by human alveolar macrophages. Respir. Med. 91:31.
62. Leibovich, S. J., P. J. Polverini, H. M. Shepard, D. M. Wiseman, V. Shively, and
N. Nuseir. 1987. Macrophage-induced angiogenesis is mediated by tumour necrosis factor-␣. Nature 329:630.
63. Yoshida, S., M. Ono, T. Shono, H. Izumi, T. Ishibashi, H. Suzuki, and
M. Kuwano. 1997. Involvement of interleukin-8, vascular endothelial growth
factor, and basic fibroblast growth factor in tumor necrosis factor ␣-dependent
angiogenesis. Mol. Cell. Biol. 17:4015.
64. Feinman, R., J. Koury, M. Thames, B. Barlogie, J. Epstein, and D. S. Siegel.
1999. Role of NF-␬B in the rescue of multiple myeloma cells from glucocorticoid-induced apoptosis by bcl-2. Blood 93:3044.
65. Marok, R., P. G. Winyard, A. Coumbe, M. L. Kus, K. Gaffney, S. Blades,
P. I. Mapp, C. J. Morris, D. R. Blake, C. Kaltschmidt, and P. A. Baeuerle. 1996.
Activation of the transcription factor nuclear factor-␬B in human inflamed synovial tissue. Arthritis Rheum. 39:583.
66. Thiele, K., A. Bierhaus, F. Autschbach, M. Hofmann, W. Stremmel, H. Thiele,
R. Ziegler, and P. P. Nawroth. 1999. Cell specific effects of glucocorticoid treatment on the NF-␬Bp65/I␬B␣ system in patients with Crohn’s disease. Gut 45:
693.
67. Oliver, S. J., S. L. Freeman, L. G. Corral, C. J. Ocampo, and G. Kaplan. 1999.
Thalidomide analogue CC1069 inhibits development of rat adjuvant arthritis.
Clin. Exp. Immunol. 118:315.
68. Eisen, T., C. Boshoff, I. Mak, F. Sapunar, M. M. Vaughan, L. Pyle,
S. R. Johnston, R. Ahern, I. E. Smith, and M. E. Gore. 2000. Continuous low dose
thalidomide: a phase II study in advanced melanoma, renal cell, ovarian and
breast cancer. Br. J. Cancer 82:812.
69. Calabrese, L., and A. B. Fleischer. 2000. Thalidomide: current and potential
clinical applications. Am. J. Med. 108:487.
70. Marriott, J. B., G. Muller, and A. G. Dalgleish. 1999. Thalidomide as an emerging immunotherapeutic agent. Immunol. Today 20:538.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
33. Marok, R., P. G. Winyard, A. Coumbe, M. L. Kus, K. Gaffney, S. Blades,
P. I. Mapp, C. J. Morris, D. R. Blake, C. Kaltschmidt, and P. A. Baeuerle. 1996.
Activation of the transcription factor nuclear factor-␬B in human inflamed synovial tissue. Arthritis Rheum. 39:583.
34. Thiele, K., A. Bierhaus, F. Autschbach, M. Hofmann, W. Stremmel, H. Thiele,
R. Ziegler, and P. P. Nawroth. 1999. Cell specific effects of glucocorticoid treatment on the NF-␬Bp65/I␬B␣ system in patients with Crohn’s disease. Gut 45:
693.
35. Takahashi, S., T. Fujita, and A. Yamamoto. 2001. Role of nuclear factor-␬B in
gastric ulcer healing in rats. Am. J. Physiol. 280:G1296.
36. Osborn, L., S. Kunkel, and G. J. Nabel. 1989. Tumor necrosis factor ␣ and
interleukin 1 stimulate the human immunodeficiency virus enhancer by activation
of the nuclear factor ␬B. Proc. Natl. Acad. Sci. USA 86:2336.
37. Shono, T., M. Ono, H. Izumi, S. I. Jimi, K. Matsushima, T. Okamoto, K. Kohno,
and M. Kuwano. 1996. Involvement of the transcription factor NF-␬B in tubular
morphogenesis of human microvascular endothelial cells by oxidative stress.
Mol. Cell. Biol. 16:4231.
38. Chaturvedi, M. M., A. Mukhopadhyay, and B. B. Aggarwal. 2000. Assay for
redox-sensitive transcription factor. Methods Enzymol. 319:585.
39. Manna, S. K., A. Mukhopadhyay, and B. B. Aggarwal. 2000. Human chorionic
gonadotropin suppresses activation of nuclear transcription factor-␬B and activator protein-1 induced by tumor necrosis factor. J. Biol. Chem. 275:13307.
40. Manna, S. K., A. Mukhopadhyay, and B. B. Aggarwal. 2000. Leflunomide suppresses TNF-induced cellular responses: effects on NF-␬B, activator protein-1,
c-Jun N-terminal protein kinase, and apoptosis. J. Immunol. 165:5962.
41. Darnay, B., J. Ni, P. A. Moore, and B. B. Aggarwal. 1999. Activation of NF-␬B
by RANK requires tumor necrosis factor receptor-associated factor (TRAF) 6 and
NF-␬B-inducing kinase: identification of a novel TRAF6 interaction motif.
J. Biol. Chem. 274:7724.
42. Canty, T. G., Jr., E. M. Boyle, Jr., A. Farr, E. N. Morgan, E. D. Verrier, and
T. H. Pohlman. 1999. Oxidative stress induces NF-␬B nuclear translocation without degradation of I␬B␣. Circulation 100(Suppl.):II361.
43. Manna, S. K., N. K. Sah, and B. B. Aggarwal. 2000. Protein tyrosine kinase
p56lck is required for ceramide-induced but not tumor necrosis factor-induced
activation of NF-␬B, AP-1, JNK, and apoptosis. J. Biol. Chem. 275:13297.
44. Raju, U., G. J. Gumin, F. Noel, and P. J. Tofilon. 1998. I␬B␣ degradation is not
a requirement for the X-ray-induced activation of nuclear factor ␬B in normal rat
astrocytes and human brain tumour cells. Int. J. Radiat. Biol. 74:617.
45. Zwacka, R. M., Y. Zhang, W. Zhou, J. Halldorson, and J. F. Engelhardt. 1998.
Ischemia/reperfusion injury in the liver of BALB/c mice activates AP-1 and
nuclear factor ␬B independently of I␬B degradation. Hepatology 28:1022.
46. Mukhopadhyay, A., S. K. Manna, and B. B. Aggarwal. 2000. Pervanadate-induced nuclear factor-␬B activation requires tyrosine phosphorylation and degradation of I␬B␣: comparison with tumor necrosis factor-␣. J. Biol. Chem. 275:
8549.
47. Livolsi, A., V. Busuttil, V. Imbert, R. T. Abraham, and J. F. Peyron. 2001.
Tyrosine phosphorylation-dependent activation of NF-␬B: requirement for p56
LCK and ZAP-70 protein tyrosine kinases. Eur. J. Biochem. 268:1508.
48. Bonizzi, G., J. Piette, M. P. Merville, and V. Bours. 1997. Distinct signal transduction pathways mediate NF-␬B induction by IL-1␤ in epithelial and lymphoid
cells. J. Immunol. 159:5264.
49. Finco, T. S., A. A. Beg, and A. S. Baldwin. 1994. Inducible phosphorylation of
I␬B␣ is not sufficient for its dissociation from NF-␬B and is inhibited by protease
inhibitors. Proc. Natl. Acad. Sci. USA 91:11884.
50. Mahon, T. M., and L. A. O’Neill. 1995. Studies into the effect of the tyrosine
kinase inhibitor herbimycin A on NF-␬B activation in T lymphocytes: evidence
for covalent modification of the p50 subunit. J. Biol. Chem. 270:28557.
51. Natarajan, K., S. Singh, T. R. Burke, Jr., D. Grunberger, and B. B. Aggarwal.
1996. Caffeic acid phenethyl ester is a potent and specific inhibitor of activation
of nuclear transcription factor NF-␬B. Proc. Natl. Acad. Sci. USA 93:9090.
2651