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B Nuclear Localization Is Efficacious in Models of Inflammatory Disease
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B regulates many genes involved in
proinflammatory and immune responses. The transport of NF-B into the
nucleus is essential for its biologic activity. We describe a novel,
potent, and selective NF-B inhibitor composed of a cell-permeable
peptide carrying two nuclear localization sequences (NLS). This peptide
blocks NF-B nuclear localization, resulting in inhibition of cell
surface protein expression, cytokine production, and T cell
proliferation. The peptide is efficacious in vivo in a mouse septic
shock model as well as a mouse model of inflammatory bowel disease,
demonstrating that NF-B nuclear import plays a role in these acute
inflammatory disease models. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B is an inducible transcription
factor comprised of homo- and heterodimers of p50, p65, p52, relB, and
c-rel subunits whose effects on cells of the immune system have been
widely studied (1, 2, 3, 4, 5, 6, 7). NF-B is expressed as a cytosolic
protein that translocates to the nucleus following cell activation,
where it regulates the expression of a number of genes whose products
are involved in inflammation, lymphocyte activation, and cell growth
and differentiation. Therefore, inhibitors of NF-B are important for
the control of proteins involved in the amplification and perpetuation
of chronic inflammatory disease (1). A number of NF-B
inhibitors have been described to date, including glucocorticoids
(8), aspirin (9), gliotoxin
(10), antioxidants such as pyrrolidine dithiocarbamate
(PDTC) and acetylcysteine (11), and the cytokine IL-10
(12, 13). However, glucocorticoids have endocrine and
metabolic side effects when given systemically at high doses and for
long periods of time. Antioxidants are relatively weak, short-lived,
and affect kinase and phosphatase activity (14).
We report here a promising new approach to NF-B inhibition using
intracellularly targeted peptide inhibitors of nuclear translocation.
They use short amino acid sequences termed nuclear localization
sequences (NLSs)2
(15, 16, 17, 18, 19, 20), which mediate nuclear protein import. There are
at least two categories of NLS: "simple," containing five to seven
residues, or "bipartite," consisting of two sets of positively
charged residues separated by 10–11 aa (15). While there
is no universal consensus sequence among the known NLSs, they generally
contain at least two basic amino acids which constitute the core of the
NLS (15). The amino acid sequence of each NLS is specific
and essential for its function; point mutations of critical basic
residues destroy nuclear targeting activity (21, 22).
Facilitated nuclear import is a receptor-mediated process involving
NLS-binding proteins, called karyopherins, which reside in the
cytoplasm and at the nuclear pore (19). Several other
cytoplasmic factors have also been shown to play a role in this
process, including p10, Ran, and Hsc70 (23, 24, 25). Previous studies using
the immunosuppressive drug deoxyspergualin, which has some structural
similarities to the core NLS, have demonstrated that this agent
inhibits nuclear transport of the transcription factor NF-B perhaps
by interfering with Hsc70-mediated nuclear import (26, 27). We have
shown previously that distinct NLSs have varying affinities for the
different forms of karyopherin 
and that the different affinities
play a major role in driving nuclear transport (28).
Collectively, these studies suggested that 1) transcription factor
nuclear transport plays a critical role in influencing cellular immune
processes (29) and 2) inhibitors of nuclear translocation
may be selective for NF-B. We proposed that NLS peptides could be
designed that would competitively inhibit the interaction between
NF-B and karyopherins and lead to an inhibition of the
activation-induced transport of this transcription factor to the
nucleus.
Other groups have shown that microinjection or treatment of cells with
NLS peptides could inhibit nuclear transport in vitro (30, 31). The peptide we designed uses NLS sequences that alter the
interaction of NF-B with karyopherin and block its transport
into the nucleus. The NLS peptides are delivered into the cell by an
amino acid sequence derived from fibroblast growth factor, which
confers cell membrane permeability (32, 33, 34). Other
examples of peptides that translocate through membranes include
sequences from Antennapedia homeodomain, HIV Tat, and the
HSV structural protein VP22 (33, 35).
Our peptide approach is different from the studies reported by Lin et
al. (32) for several reasons. First, we used two NLSs, one
on either end of the fibroblast growth factor translocation sequence.
The cloning of karyopherin and a recently published crystal
structure shows that at least two NLSs can bind to a single molecule of
karyopherin (36). Therefore, we rationalized that
delivery of multiple NLS peptides on a single molecule would have a
much higher affinity for karyopherin than a single NLS. Secondly,
we synthesized the peptide in the D-amino acid form because
it is known that D-form peptides are metabolized more
slowly and are more resistant to proteolysis than their
L-form counterparts. Finally, in contrast to the studies by
Lin et al. (32), our studies focused on biological events
downstream of NF-B nuclear localization in cells of the immune
system. This study demonstrates the efficacy of cell-permeable NLS
peptide-induced inhibition of NF-B activation in modulating immune
responses in vivo and in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptides were synthesized on a Gilson multiple peptide synthesizer using F-moc (37) amino acids and purified by C18 reverse-phase HPLC. All peptides were analyzed by mass spectrometry and yielded the correct m.w.
Cells and reagents
The murine pre-B cell line 70Z/3 (American Type Culture
Collection, Manassas, VA) was grown in RPMI 1640 supplemented with 10%
heat-inactivated FBS and 5 x
10-5 M 2-ME. The murine
monocyte/macrophage cell line RAW 264.7 (TIB71; American Type Culture
Collection) was grown in DMEM containing 10% FBS. LPS was obtained
from Salmonella typhosa 0901 (Difco, Detroit, MI). Anti-CD3
and anti-CD28 Abs were kind gifts of Hung Theh and Jim Allison,
respectively (both of University of California, Berkeley, CA). Rabbit
anti-hamster Ab was obtained from Jackson ImmunoResearch (West
Grove, PA), and anti- Ig was obtained from Southern
Biotechnology Associates (Birmingham, AL). Cytokine levels were
measured using the appropriate Genzyme (Framingham, MA) ELISA kits.
Human peripheral blood T, B, and monocytic cells were isolated by
E-rosetting as described previously (38). For the IB
Western blot, we used Ab C-21 from Santa Cruz Biotechnology (Santa
Cruz, CA). Dextran sodium sulfate (DSS) (m.w. = 40,000) was obtained
from ICN Biomedical (Costa Mesa, CA).
EMSA
A total of 2 x 107 cells were washed
once in PBS followed by preparation of nuclear extracts as described
(14). The cells were lysed in buffer A (10 mM HEPES, 1.5
mM MgCl2, 10 mM NaCl, 0.25% Nonidet P-40, pH
7.5) for 5 min at 4°C, followed by centrifugation at 4000 rpm for 5
min. The supernatant (cytosol) was removed, and the nuclei were
extracted with buffer C (20 mM HEPES, 25% glycerol, 0.42 M NaCl, 0.2
mM EDTA, 1.5 mM MgCl2, 0.25% Nonidet P-40, pH
7.5). The nuclei were vortexed vigorously several times over 20 min,
followed by centrifugation at 14,000 rpm for 5 min. The supernatant
(nuclear extract) was removed and diluted 1:2 with buffer D (20 mM
HEPES, 50 mM KCl, 0.2 mM EDTA, 20% glycerol, pH7.5) and then frozen at
-80°C until use. The 32P-labeled probes used
contained the sequence: NF-B, 5'-AGTTGAGGGGACTTTCCCAGG-3'; AP-1,
5'-CGCTTGATGAGTCAGCCGGAA-3'; NF-AT, 5'-GGAGGAAAAACTGTTTCATACAGAAGG-3';
and Oct, 5'-TGTCGAATGCAAATCACTAGA-3'. A 6% polyacrylamide gel was
used with 0.5x TBE as running buffer to resolve the complexes.
Measurement of cytokine secretion
RAW 264.7 cells (1 x 105/well) were
preincubated for 2 h with BMS 205820 peptide before stimulation
with LPS (0.5 ng/ml) and additional incubation for either 6 or 24
h. The culture supernatants were then analyzed for IL-10, IL-2, IL-6,
or TNF- levels by ELISA (Genzyme).
Mouse T cell purification and proliferation
To isolate splenic T cells, BALB/c spleens were teased apart and pressed through a wire mesh screen. The spleen cells were washed two times, and RBCs and other materials were removed using Lympholyte M (Cedarlane Laboratories, Westbury, NY). The spleen cells were enriched for T cells using a mouse T cell enrichment column (R&D Systems, Minneapolis, MN). The cells were then incubated at 37°C for 3 h with media (RPMI 1640 plus 10% FCS) plus or minus BMS 214572 at 0.5 or 2.0 µM. The cells were then centrifuged, resuspended in either media alone or containing the appropriate concentration of peptide, plated into 96-well plates, and stimulated as described in the figure legend. After 72 h, the cells were pulsed with [3H]thymidine for 6 h and counts were determined.
Animal studies
For the sepsis model, BALB/c mice were administered a bolus injection of 200 µg LPS in PBS i.v., and mortality was measured after day 7. For the DSS study, Swiss-Webster mice were administered 5% DSS in drinking water from day 0 to day 6 and peptide on days 1–9. On day 10, the mice were sacrificed, and colons were removed and analyzed for disease. Sections of the entire colon were graded as to the severity of crypt injury and degree of inflammation. The crypt injury was scored as follows: grade 0, intact crypt; grade 1, loss of the basilar one-third of the crypt; grade 2, loss of the basilar two-thirds of the crypt; grade 3, loss of the entire crypt with the surface epithelium remaining intact; grade 4, loss of both the entire crypt and surface epithelium resulting in an erosion. The scoring severity of inflammation was as follows: grade 0, unremarkable; grade 1, minimal; grade 2, mild; grade 3, moderate; grade 4, severe. Both scores also included a measure of the extent (percentage) of involvement as follows: grade 1, 0–25%; grade 2, 26–50%; grade 3, 51–75%; grade 4, 76–100%. The final score is the product of either the inflammation or injury grade by the grade for extent of involvement.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
We synthesized a D-amino acid peptide (referred to as
BMS 214572) containing a cell membrane-translocating sequence flanked
by two nuclear localization sequences derived from c-myc
(16, 39) (Table I
). We show
elsewhere using confocal microscopy that BMS 214572 is readily taken up
by cells within 30 min and accumulates in the cytoplasm (Nadler et al.,
manuscript in preparation). In all of the experiments described below,
each peptide was used at concentrations that were nontoxic, in the time
frame of the experiment, based on trypan blue exclusion (data not
shown). In addition, we have also synthesized another peptide, BMS
205820, that contains the NLS from SV40 large T Ag (16, 40). To evaluate the effect of the peptides on NF-B nuclear
translocation, we performed EMSA using nuclear extracts from 70Z/3
cells. NF-B activation in these cells has been well studied, making
this cell line an ideal candidate for these studies. As shown in Fig. 1A, both BMS 205820 and BMS
214572 potently inhibited LPS-induced NF-B translocation in the high
nanomolar range. The two peptides also inhibited PMA-induced NF-B
translocation; however, the concentration of peptides required for
inhibition was several-fold higher. The inhibition was selective for
NF-B, as Oct-1 DNA binding from the same extracts was not affected.
The step immediately preceding nuclear import is the degradation of
IB (subsequent to phosphorylation) to expose the nuclear
localization sequence on NF-B (1). As seen in Fig. 1B, BMS 205820 at a concentration that was shown to inhibit
NF-B translocation did not affect the degradation of IB,
indicating that the peptide likely inhibits the nuclear import of
NF-B and not an upstream signaling pathway.
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B in normal human PBL as
well. As shown in Fig. 2, NF-B nuclear
localization in T cells was inhibited only by 2 µM peptide, whereas
the B plus monocyte cell population was sensitive to inhibition by both
concentrations of the peptide. There was no significant effect on the
nuclear levels of octamer binding protein. Together with the data
showing differential effects on PMA vs LPS nuclear transport, these
data suggest there may be different nuclear transport pathways in
different cell types. The different concentrations of peptide required
for inhibition of NF-B in different cell types and methods of
stimulation may also be due to different levels of karyopherins. In
fact, we have shown that there is a differential expression of
karyopherins in different cell types and that stimulation of cells with
PMA causes a rapid increase in the levels of karyopherins compared with
LPS stimulation (28).
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Ig and other
cell-surface Ags
To determine the effect of BMS 205820 on NF-B-regulated immune
responses in vitro, we used 70Z/3 cells as a model of B cell
differentiation (41). These cells can be activated to
express surface IgM, indicative of conversion to a more mature B cell
phenotype. This increase in surface IgM expression is due to induction
of light chain transcription, which is regulated in large part by
NF-B. We stimulated 70Z/3 cells overnight with 500 ng/ml LPS, under
which conditions 
95% of cells become light chain positive (data
not shown). Fig. 3A shows that
both the L-amino acid form and the
D-amino acid form of BMS 205820 are equally
effective at inhibiting LPS-induced expression. As predicted based
on its ability to inhibit NF-B, BMS 214572 was also very effective
at inhibiting surface expression. In contrast, the single NLS
L-amino acid JBC peptide was significantly less
inhibitory, as were the SV40 and translocation peptide controls.
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to express surface Ig.
IFN- is known to activate the Jak/STAT transcription factor pathway,
which is NF-B independent (42). Interestingly, we found
that IFN--induced expression was not affected by BMS 205820
(Fig. 3
B), suggesting that the peptide inhibition is indeed
selective for the NF-B-mediated signaling pathway.
Besides Ig, other genes with putative NF-B binding sites include
cell-surface molecules involved in immune function such as CD40, class
I and class II MHC, and cell adhesion molecule CD54 (1, 2). We examined the effect of the peptide on surface levels of
these molecules and found that BMS 214572 was effective at inhibiting
LPS-induced but not IFN--induced surface expression of CD40, class I
MHC, and CD54, whereas class II MHC was not affected (data not
shown).
To determine whether the peptide could inhibit an ongoing immune
response, we treated 70Z/3 cells with the peptide at various times
after LPS activation. BMS 205820 was able to maximally inhibit
expression only when added before or at the same time as the LPS
stimulus (Fig. 3C); the inhibitory effect of the peptide
decreased when added after NF-B activation. These data are
consistent with the hypothesis that the peptide is inhibiting
translocation of NF-B to the nucleus rather than interfering with
later events such as DNA binding or cellular processes in general. In
fact, we have shown that there was no effect on NF-B binding to DNA
at concentrations of peptide up to 1 µM (data not shown).
BMS 205820 and BMS 214572 inhibit proinflammatory cytokine production
Production of proinflammatory cytokines by monocytes is known to
be regulated by transcription factors such as NF-B. TNF-, in
particular, appears to be involved in disease states such as rheumatoid
arthritis, inflammatory bowel disease (IBD), and septic shock (1, 43). Therefore, we examined the effect of BMS 205820 on TNF-
and IL-6 cytokine production induced by LPS activation of RAW 264.7
cells. As shown in Fig. 4, BMS 205820 at
5 µM nearly completely inhibited both TNF- and IL-6 production in
response to LPS. BMS 214572 had a similar ability to suppress TNF-
production. In contrast, neither the c-myc nor SV40 single
NLS peptide had much effect in these assays.
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To assess the peptide effect on other cells of the immune system,
mouse splenic T cells were examined in a proliferation assay. As shown
in Fig. 5A, when T cells were
stimulated with anti-CD3 plus anti-CD28, pretreatment with
increasing amounts of peptide resulted in an augmentation of
proliferation. However, when cells were stimulated instead with PMA
plus anti-CD28, the peptide effect was reversed and T cell
proliferation was inhibited. Both effects were most dramatic when low
concentrations of the stimuli were used (data not shown). There was no
toxicity observed in the time frame of the experiment. One explanation
for these divergent effects is that the CD3 plus CD28 activation
pathway is calcium dependent and partially cyclosporin A sensitive and
therefore signals predominantly through a different transcription
factor such as NF-AT (44, 45). BMS 214572, by inhibiting
NF-B signaling, may act to indirectly enhance NF-AT-mediated T cell
activation. In contrast, PMA plus anti-CD28-induced proliferation,
which is cyclosporin A resistant, may be more NF-B dependent.
Therefore, peptide inhibition of T cell activation would be more
evident under these conditions.
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B
activation correlated with inhibition of proliferation. We observed
that stimulation of T cells with PMA plus anti-CD28 resulted in
significant NF-B activation, and to a greater extent than
anti-CD3 plus anti-CD28 activation, as shown in Fig. 5B. When cells were pretreated with BMS 214572 under these
conditions, NF-B activation was inhibited. Although stimulation of T
cells with anti-CD3 plus anti-CD28 resulted in a much lower
level of NF-B activation compared with PMA plus anti-CD28,
pretreatment with peptide to a limited extent inhibited activation by
this stimulus as well. Both methods of stimulation resulted in similar
levels of NF-AT activation. Interestingly, we found that there was an
increase in binding of nuclear extract proteins to an NF-AT DNA probe
when cells were treated with peptide.
These data are consistent with the hypothesis that PMA plus CD28
stimulation signals primarily through the NF-B pathway, unlike CD3
plus CD28 stimulation, which signals mainly through a non-NF-B
pathway. In addition, we examined Oct-1 activation and found little
effect of the peptide on this transcription factor, further
demonstrating the specificity of the peptide for NF-B (data not
shown). Possible explanations for the differential effects on
proliferation induced by the two treatments are discussed below.
BMS 205820 inhibits in vivo inflammatory responses
Because of the intriguing in vitro results reported above, we were
interested in the effect of the peptide on in vivo models of disease.
As shown in Table II, BMS 205820 was able
to significantly protect mice from death in a murine model of septic
shock (46). Because the biologic consequences of sepsis
are widely thought to involve proinflammatory cytokines such as
TNF-, we also treated mice with a nonlethal dose of LPS and measured
cytokine levels. TNF- levels in these mice were shown to be
suppressed by peptide treatment. IL-6 levels were only slightly
affected, and IL-10 levels were significantly enhanced (Fig. 6). Similar to the peptide-induced
increase on anti-CD3 plus anti-CD28-mediated T cell
proliferation, it is possible that NF-B acts in a negative fashion
to limit IL-10 production. The efficacy of the peptide inhibitor in the
sepsis model suggests it may play a role in treatment of this
disease.
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(top), there
were dramatic changes in the histopathology of the colons of
DSS-treated mice before and after per os or i.v. treatment with
peptide. These changes are quantified in Fig. 7 (bottom),
which illustrates a peptide-mediated decrease in both the inflammation
and injury scores of the diseased colon. These data demonstrate that
BMS 205820 is efficacious in this mouse model of IBD.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B. Most of the previously described inhibitors of
NF-B have been shown to act on the early stages of NF-B
activation (8, 9, 10, 11, 12, 13). The peptide we designed acts at the
more downstream event of nuclear translocation; hence, this peptide
inhibitor represents a novel means of modulating transcription factor
activity.
The mechanism of action of the peptides is established; we have shown
that BMS 214572 and BMS 205820 block the nuclear localization of
NF-B. There was also a dose response relationship between inhibition
of NF-B nuclear translocation and the downstream effect on light
chain production (Fig. 1, left vs Fig. 3B). This
inhibition was selective for NF-B, as translocation of other nuclear
transcription factors such as Oct-1 and NF-AT was not significantly
inhibited. In addition, there was no effect of the peptides on
IFN-/STAT-mediated processes. While our data suggest that these
peptides are specific for inhibition of NF-B, we cannot rule out the
possibility that the localization of other proteins is affected as
well. We predicted the effect would be most specific for NF-B due to
its proposed lower affinity interaction (compared with other
transcription factors) for karyopherin . Torgerson et al.
(34) recently reported that an NLS peptide based on the
NF-B p50 sequence attached to the fibroblast growth factor
translocation sequence inhibited the nuclear translocation of several
transcription factors. Those results could be attributed to the
different peptide sequences used in their studies and suggest that
there are multiple pathways and various ways to modulate the import
process. Indeed, we found that the SN50 peptide reported by Torgerson
et al. (34), which has the identical sequence to the JBC
peptide reported herein, was 35-fold less effective at
inhibiting Ig expression compared with BMS 205820. These
differences in potency may contribute to the differences in selectivity
seen with these two peptides. In addition, SN50 may bind to different
armadillo motifs on karyopherin relative to BMS 205820, resulting in
differences in selectivity as well.
The inhibition by BMS peptides does not appear to be due to a toxic
effect, because cell viability and global protein levels, both
cytoplasmic and nuclear, were unaffected (data not shown). Not all
NF-B-regulated biological functions were affected by the peptides,
and some were affected to varying degrees, possibly because redundancy
within the cell allows other transcription factors to compensate for
the loss or inhibition of NF-B. One example is the lack of
inhibition of LPS-induced class II expression that is reported to be
regulated by NF-B. In addition, the peptide-mediated inhibition was
overcome when higher doses of LPS were used to stimulate cells,
suggesting that NF-B-mediated immune responses may require a
threshold level of NF-B to activate transcription (data not shown).
Alternatively, higher concentrations of LPS may trigger other signaling
pathways that are able to mediate the same biological events. These
possibilities are being examined.
The peptide inhibitor of NF-B that we describe in this report has
yielded some surprising results with respect to the modulation of
immune responses, compared with other inhibitors of NF-B. We have
begun to determine a molecular basis for the differential effects of
the peptides on T cell proliferation. The effects do not appear to be
due to changes in IL-2 or IL-4 because the levels of these cytokines
were not significantly altered by peptide treatment (data not shown);
effects on the synthesis of other cytokines are presently being
investigated. Although IL-2 has been reported to be regulated by
NF-B, we have found that peptide inhibition of NF-B did not
affect IL-2 levels in mouse cells. This suggests that other
transcription factors such as NF-AT may be sufficient for driving
transcription of the IL-2 gene. With respect to the stimulation of
proliferation by the peptide upon CD3 plus CD28 activation, it is
possible that NF-B may be acting as a negative regulator of
transcription. Inhibition of NF-B by the peptide may actually
enhance transcription of certain genes. In fact, Casolaro et al.
(48) have shown that NF-B inhibits NF-AT-driven
transcription in particular T cell subsets. We also found that the
peptide enhanced NF-AT DNA binding, although at this point we do not
have an explanation for these results. Additional studies on the
effects of the peptides on T cells should further our understanding of
the role of NF-B on T cell responses.
The efficacy exhibited by the NLS peptides in two important established
in vivo models of inflammatory disease is encouraging. Not only were
animals protected from septic shock death, but TNF-, which is one of
the players implicated in septic shock, was significantly decreased
whereas IL-10 levels were dramatically enhanced. Because IL-10 is
antiinflammatory, its presence serves to limit the production of
proinflammatory cytokines. Interestingly, compounds such as SR31747A,
glucocorticoids, and cAMP enhancing agents (49) also cause
inhibition of TNF- and concomitant increases in IL-10 in response to
LPS (50). Some of these compounds have also been shown to
inhibit NF-B. The effects on IL-10 may either be transcriptional or
a secondary effect of decreasing TNF- levels. Reports describing
high levels of activated NF-B in the inflamed bowels of Crohn’s
disease patients as well as enhanced expression of NF-B-regulated
proinflammatory cytokines such as IL-1 and TNF- implicate the
transcription factor as a key player in IBD (51, 52).
Because of the fundamental roles of IL-1 and TNF- in the
inflammatory response, inhibition of these cytokines would be expected
to provide effective treatment of IBD. In fact, local administration of
antisense oligonucleotides targeted against p65 NF-B abrogated
established intestinal inflammation in mice, providing direct evidence
for the importance of NF-B in IBD (53). Remarkably, the
BMS 205820 peptide was efficacious when administered i.v. as well as
when administered orally. Because of the resistance of the
D-amino acid peptide to proteolysis, we believe that the
peptide may be acting topically at the site of inflammation in the
colon. The DSS model is very stringent, and, in fact, standard
therapies such as steroids are not effective in this model although
they are potent antiinflammatory agents in humans. That BMS 205820 is
efficacious in this model suggests it may be quite potent and indicates
its potential utility in treating inflammatory disease patients. In
order for this peptide to be used clinically it must first be shown to
be safe in animals. A 5-day i.v. dosing study in rats at doses up to 10
mg/kg showed no toxicities or histopathologic changes (data not shown).
In addition, a 2-wk oral dosing study in mice up to 20 mg/kg showed no
overt signs of toxicity or histopathologic changes, indicating that
this compound is safe in animals. In both of these toxicology studies,
a detailed histopathologic analysis of all organs and leukocytes was
performed.
Our work using cell-permeable nuclear-targeted D-amino acid
NLS peptides presents a useful tool for studying gene transcription and
downstream intracellular processes specific to a single transcription
factor. We have shown that these peptides inhibit a variety of
NF-B-dependent immune responses in several different cell types,
both in vitro and in vivo. Based on these results, we propose that
these peptides have potential as novel immunomodulatory therapeutic
agents. Future studies will determine whether modulating gene
expression at the level of nuclear transport is applicable to
transcription factors other than NF-B (54).
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: NLS, nuclear localization sequence; DSS, dextran sodium sulfate; IBD, inflammatory bowel disease.
Received for publication November 23, 1999. Accepted for publication May 8, 2000.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B—a pivotal transcription factor in chronic inflammatory diseases. N. Engl. J. Med. 336:1066.B and Rel: participants in a multiform transcriptional regulatory system. Int. Rev. Cytol. 143:1.[Medline]
B and cytokine-inducible enhancers. FASEB J. 9:899.[Abstract]
B, a mediator of lipopolysaccharide effects. Immunobiology 187:233.[Medline]
B: a lesson in family values. Cell 80:529.[Medline]
B regulation: the emerging role of phosphorylation and proteolysis. Immunity 3:263.[Medline]
B: ten years after. Cell 87:13.[Medline]
B in mediation of immunosuppression by glucocorticoids. Science 270:283.B activation. Science 274:1383.B. J. Exp. Med. 183:1829.B transcription factor and HIV-1. EMBO J. 10:2247.[Medline]
B) which are involved in IL-6 regulation. Leukemia 10:1308.[Medline]
B (NF-B) activation in human monocytes. J. Biol. Chem. 270:9558.B. J. Biol. Chem. 272:11541. light chain expression in 70Z/3 pre-B cells blocking lipopolysaccharide induced NF-B activation. J. Immunol. 155:2427.[Abstract]
with nuclear localization sequences. J. Biol. Chem. 272:4310.B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence. J. Biol. Chem. 270:14255.B, AP-1, NFAT and STAT1 nuclear import in T lymphocytes by noninvasive delivery of peptide carrying the nuclear localization sequence of NF-B. J. Immunol. 161:6084.. Cell 94:193.[Medline]
light chain synthesis in 70Z/3 B lymphoma cells by chemically defined lipid A precursors. J. Biol. Chem. 263:5098.B: implications for differential gene expression in T helper cell subsets. Proc. Natl. Acad. Sci. USA 92:11623.B in Crohn’s Disease. Inflamm. Res. 47:440.[Medline]
B abrogates established experimental colitis in mice. Nat. Med. 2:998.
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D. Strassheim, K. Asehnoune, J.-S. Park, J.-Y. Kim, Q. He, D. Richter, S. Mitra, J. Arcaroli, K. Kuhn, and E. Abraham Modulation of bone marrow-derived neutrophil signaling by H2O2: disparate effects on kinases, NF-{kappa}B, and cytokine expression Am J Physiol Cell Physiol, March 1, 2004; 286(3): C683 - C692. [Abstract] [Full Text]
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 D. C. Harnish, L. M. Albert, Y. Leathurby, A. M. Eckert, A. Ciarletta, M. Kasaian, and J. C. Keith Jr. Beneficial effects of estrogen treatment in the HLA-B27 transgenic rat model of inflammatory bowel disease Am J Physiol Gastrointest Liver Physiol, January 1, 2004; 286(1): G118 - G125. [Abstract] [Full Text] [PDF]
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 J. Macdonald, H. F. Galley, and N. R. Webster Oxidative stress and gene expression in sepsis Br. J. Anaesth., February 1, 2003; 90(2): 221 - 232. [Abstract] [Full Text] [PDF]
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 F. D'Acquisto, M. J. May, and S. Ghosh Inhibition of Nuclear Factor Kappa B (NF-B):: An Emerging Theme in Anti-Inflammatory Therapies Mol. Interv., February 1, 2002; 2(1): 22 - 35. [Abstract] [Full Text] [PDF]
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J. G. Kupfner, J. J. Arcaroli, H.-K. Yum, S. G. Nadler, K.-Y. Yang, and E. Abraham Role of NF-{kappa}B in Endotoxemia-Induced Alterations of Lung Neutrophil Apoptosis J. Immunol., December 15, 2001; 167(12): 7044 - 7051. [Abstract] [Full Text] [PDF]
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0.01, difference between control and
peptide-treated samples using a multiple ANOVA analysis.