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B Super-Repressor in Human Intestinal Epithelial Cells1
*
Departments of Medicine, Microbiology, Immunology and
the Center for Gastrointestinal Biology and Disease, University of North Carolina, Chapel Hill, NC 27599;
Division of Clinical Immunology, Mount Sinai Medical Center, New York, NY 10029; and
§
Department of Pharmacology, University of California, San Diego, CA 92093
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B plays a major role in the transcriptional regulation of
many proinflammatory genes in multiple cell lineages, including
intestinal epithelial cells (IEC). Activation of NF-B requires both
phosphorylation and degradation of its natural cytoplasmic inhibitor,
IB. We tested whether a super-repressor of NF-B activity, which
is a mutated nondegradable IB
resistant to phosphorylation and
degradation, could be delivered into IEC using an adenoviral vector
(Ad5IB) and determined the anti-inflammatory potential of this
inhibitor following different stimuli. We showed for the first time
that recombinant adenovirus efficiently infected (>80%) transformed
as well as primary IEC. Cytoplasmic levels of the NF-B
super-repressor protein were more than 50-fold higher than those of
endogenous IB, and this mutated IB was resistant to
IL-1ß-induced degradation. Immunofluorescent RelA nuclear staining
was strongly inhibited in Ad5IB-infected IEC compared with control
Ad5LacZ, and NF-B, but not AP-1 binding activity, was reduced by
more than 70% as measured by electrophoretic mobility shift assay
(EMSA). Induction of inducible nitric-oxide synthase (iNOS), IL-1ß,
and IL-8 genes by IL-1ß, TNF-, or PMA was blocked in
Ad5IB-infected cells but not in Ad5LacZ controls as assayed by
RT-PCR and ELISA. In addition, IL-1ß-induced IL-8 secretion was
totally inhibited by Ad5IB in primary colonic IEC. We conclude that
an adenoviral vector efficiently transfers a nondegradable IB in
both transformed and native IEC. The strong inhibition of NF-B
activity and the resulting down-regulation of multiple proinflammatory
molecules by Ad5IB suggests an exciting approach for in vivo
intestinal gene therapy and illustrates the key role of NF-B in
transcriptional regulation of the inflammatory phenotype of IEC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many cellular genes involved in the early process of immune, acute
phase and inflammatory responses are regulated at the level of
transcription by NF-B (10, 11). NF-B is an inducible dimeric
transcription factor that belongs to the Rel family of transcription
factors (12), whose prototype in many cells is composed of the RelA
(p65) and NF-B1 (p50) heterodimer subunits. This heterodimer is the
most potent gene trans-activator among the NF-B family
(13, 14) and is also the major NF-B protein found in the nucleus of
cytokine-stimulated IEC (15). A wide variety of agents (e.g. phorbol
esters, IL-1, TNF-, dsRNA, cAMP, viral trans-activators,
and reactive oxygen metabolites) are able to activate NF-B (10).
NF-B activation is tightly regulated by its endogenous inhibitor,
IB, which complexes NF-B in the cytoplasm. Phosphorylation and
proteolytic degradation of IB allows the release and nuclear
transmigration of NF-B (16, 17). Inducible IB degradation is
linked to phosphorylation of serine residues 32 and 36 located in the
N-terminal part of the polypeptide (18, 19, 20, 21, 22). Mutation of these two
amino acids has been shown to effectively prevent IB degradation and
NF-B activation (18, 19, 22, 23).
Viral vectors are potent vehicles for in vivo/in vitro gene delivery
into various cells (24, 25, 26). Among the viral vectors, adenoviruses are
particularly attractive for gene therapy since they can easily be
rendered replication deficient, are efficient gene delivery vehicles,
transduce dividing and nondividing cells from different organs and
tissues, and from the practical stand point can be produced at high
titers (24, 25). We constructed an adenoviral vector bearing a mutant
form of IB where serine 32 and 36 were replaced by alanine residues
(S32A/S36A), therefore preventing the inducible IB phosphorylation.
Such a mutant, IB has been shown to act as an NF-B
super-repressor (27). The infectibility, as well as the
anti-inflammatory properties of this adenoviral vector (Ad5IB),
was tested in transformed and primary IEC. We report an efficient
transduction of exogenous super-repressor NF-B into both primary and
transformed IEC. This super-repressor blocked NF-B activation and
prevented inducible nitric oxide synthase (iNOS), IL-1ß, and IL-8
gene induction by IL-1ß, TNF-, or PMA. Our data show that Ad5IB
is a powerful anti-inflammatory tool and represents a promising
candidate for in vivo intestinal gene therapy. Moreover, complete
blockade of inducible IL-8 secretion by a nondegradable form of
IB illustrates the key regulatory role of this endogenous
inhibitor in IEC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transformed human HT-29 epithelial cells (ATCC HTB 38) were used between passages 10 and 25, and Caco-2 epithelial cells (ATCC HTB 37) were used between passages 29 and 40. HT-29 cells were grown in DMEM with high glucose (Life Technologies, Grand Island, NY) and Caco-2 cells in Eagle’s minimum essential medium (EMEM; Life Technologies), both supplemented with 10% heat-inactivated FBS (Life Technologies), 2 mM L-glutamine, antibiotics (Pen/Strep/Fungizone, 1X; Life Technologies), and 1% nonessential amino acids. Cells were cultured in a water-saturated atmosphere of 95% air and 5% CO2.
Isolation and stimulation of IEC
Colonic epithelial cells were isolated from resected specimens obtained from patients without inflammation by dispase treatment and percoll density gradient centrifugation, as previously described (28). Enriched epithelial cells were free of B cells and monocytes/macrophages with 2 to 4% contaminating T cells, as assayed by flow cytometric analysis using anti-CD14, anti-CD3, anti-CD20, and L-12 (anti-epithelial cell) mAbs (28). Viability was assessed by trypan blue and propidium iodide staining. Cells were used for experiments only when viability was >95%.
Ad5IB construction
The recombinant replicative-deficient adenovirus was constructed
by the method of Graham et al. (29). The IB S32A/S36A plasmid
used in this study was previously described (30). In brief, the cDNA
insert of plasmid pRc/CMV-IB S32A/S36A, which expresses an IB
super-repressor, was subcloned into the XbaI site of the
pACCMV.PLPASR (+) plasmid to construct the plasmid pACCMV/IB, in
which IB is driven by the CMV promoter/enhancer (see Fig. 1
). The plasmid DNA was prepared by the
alkaline lysis method and purified by Cesium chloride-ethidium bromide
density gradient centrifugation. Recombinant adenovirus IB
(Ad5IB) was constructed by cotransfection of 293 cells (embryonic
human kidney cells) with the pACCMV/IB plasmid plus the purified
fragment of ClaI-digested DNA from E1-deleted adenovirus
type 5.4 The presence of the
mutant IB sequence packaged into the recombinant Ad5 virus
(Ad5IB) was confirmed by PCR and by Western blotting as described
below. Ad5IB was grown in 293 cells and purified by banding twice on
cesium chloride gradients. Viral titers were determined by optical
densitometry (particles per ml) and by plaque assay; recombinant
viruses were then stored in 10% (v/v) glycerol at -20°C. The
Ad5IB gene contains an extra 27 base pair DNA nucleotides coding for
a peptide derived from hemagglutinin gene (YPYDVPDYA) to discriminate
between endogenous and exogenous IB. Ad5LacZ, which contains the
Escherichia coli ß-galactosidase gene, was grown and
purified as described above and used as a control virus throughout the
study.4
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IEC cell lines were cultured to post confluency, after which
they were infected with Ad5IB or Ad5LacZ in serum-free media
(Opti-MEM; Life Technologies) at different multiplicity of infection
(MOI; 0, 1:10, 1:50, and 1:100 IEC/viral particules) for 12 h. The
adenovirus was then washed off, fresh media-containing serum was added
to the transfection media, and cells were incubated for another 12
h. Cells were treated at various time points with IL-1ß (2 ng/ml),
TNF- (10ng/ml; both from Intergen, Purchase, NY), or PMA (100
ng/ml). Primary IEC were treated identically except that the infection
period was decreased to 4 h because of the limited period of
viability of freshly isolated IEC.
RNA extraction and amplification by RT-PCR
Infected Ad5IB or Ad5LacZ cells were stimulated with IL-1ß
(2 ng/ml), TNF- (10 ng/ml), or PMA (100 ng/ml) for 3 h. RNA was
isolated using the Trizol method (Life Technologies), and 1 µg of
total RNA was reversed transcribed and amplified (RT-PCR) using
specific primers for IL-1ß, IL-8, IB, and actin as described
previously (15). The iNOS oligonucleotide primers were: (5) 5'-AGG ATC
CAG TGG TCC AAC C-3' and (3) 5'-GCC CAC TTC CTC CAG GAT G-3.
Northern blot analysis
Total RNA (10 µg) was electrophoresed on 1.5% denaturing gels as described (31). The RNA was blotted onto Hybond-N paper (Amersham, Arlington Heights, IL) overnight, followed by UV fixation. Integrity of RNA was checked by methylene blue staining as described (31). Membranes were hybridized to [32P]dCTP-labeled cDNA probe encoding human IL-8, washed, and exposed as described previously (32).
Nuclear extracts and EMSA
Infected Ad5IB and Ad5LacZ cells were stimulated 30 min with
IL-1ß (2 ng/ml) or TNF- (10 ng/ml), lysed, and nuclear protein
extracts were prepared as described previously (15). Nuclear extracts
(5 µg) were incubated with double-stranded class I MHC B sites
(GGCTGGGGATTCCCCAT CT), separated by electrophoresis, and analyzed
by autoradiography as described previously (15). For AP-1 binding
activity, HT-29 cells were starved in 0.5% serum for 24 h.
Nuclear proteins were prepared as described above with the inclusion of
a phosphatase inhibitor mixture as described previously (33). The AP-1
probe used in the shift assay was derived from the consensus AP-1 site
found in the human collagenase gene (5'-TAAAGCATGAGTCAGGACACCTC-3').
The specificity of the probe was evaluated by incubating the nuclear
extract with an excess (100X) of unlabeled AP-1 oligonucleotide. In
addition, Jun Ab (1 µl) was used to ascertain the identity of the
shifted complexes.
Western blot analysis
Uninfected or Ad5IB-infected cells were lysed in
Laemmli buffer and 20 µg of proteins were electrophoresed
on 10% SDS-polyacrylamide gels. Immunoreactive IB was detected
using the enhanced chemiluminescence (ECL) light-detecting kit
(Amersham) as described previously (15).
Immunofluorescence study and ß-galactosidase staining
Uninfected, Ad5IB- or Ad5LacZ-infected cells were stimulated
30 min with IL-1ß (2 ng/ml). Cells were fixed with 100% ice cold
methanol. Blocking was performed using 25% nonimmune goat serum (NGS;
Sigma, St. Louis, MO) for 30 min. After blocking, rabbit anti-RelA
Ab (diluted 1:200 in 25% NGS) or rabbit anti-IB Ab (1
µg/ml; C-21, Santa Cruz Biotechnology, Santa Cruz, CA) was added for
30 min after which rhodamine isothiocyanate-conjugated goat
anti-rabbit IgG Ab (Jackson ImmunoResearch, West Grove, PA) diluted
1:100 in 25% NGS was added for 30 min. RelA and IB expression
was visualized with a fluorescent light microscope. For
ß-galactosidase staining, Ad5LacZ-infected cells were fixed 24 h
postinfection in a solution of 1% glutaraldehyde for 15 min.
ß-galactosidase staining was detected using a solution of 10 mM
K4Fe(CN)6, 10 mM K4Fe3(CN)6, 2 mM MgCl2 (Sigma), and 400 µg/ml of
5-bromo-4-chloro-3-indolyl ß-D-galactoside
(Boehringer Mannheim, Indianapolis, IN).
IL-8 ELISA
An IL-8 ELISA of cell culture supernatants from noninfected,
Ad5LacZ-infected, and Ad5IB-infected IEC was performed in duplicate
according to the manufacturer’s specifications (R&D Systems,
Minneapolis, MN).
Statistical analysis
Data are expressed as means ± SEM. Statistical significance was performed by the two-tailed Student t test for paired data and considered significant if p values were <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
The IB S32A/S36A mutant (Fig. 1
) was cloned into the
adenoviral transfer vector pACCMV.PLPASR, and recombinant adenoviruses
were generated by cotransfection of the transfer vector with adenoviral
DNA restriction endonuclease fragment. Among the different adenoviral
clones (6 of 20) expressing high level of mutant
IB4, one clone was further characterized (clone
18). IEC infectibility was evaluated by using different multiplicity of
infection (MOI) of either Ad5IB or Ad5LacZ (control virus). Large
numbers of positively transfected HT-29 cells (>80%) could be
detected with increased MOI by either ß-galactosidase staining (Fig. 2A) or IB
immunofluorescence staining (Fig. 2B). Western blot
analysis performed from both Ad5IB-infected Caco-2 and HT-29 cell
protein extracts revealed an increased expression of exogenous mutant
IB with increased MOI (Fig. 2C). The level of
exogenous IB was approximately 50-fold higher than levels of
endogenous IB at an MOI of 50. Based on data provided by the
ß-galactosidase staining, IB immunofluorescence and Western blots,
subsequent analysis of Ad5IB function was then performed using an
MOI of 50. These data show that Ad5IB efficiently transduced the
IB super-repressor into IEC.
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B expression and resistance to
IL-1ß-induced proteolysis following Ad5LacZ or Ad5IB infection of
IEC. Constitutive IB mRNA was detected in resting (nonstimulated)
Ad5IB-infected IEC but not in noninfected or Ad5LacZ-infected cells
(Fig. 3A) as measured
by RT-PCR. When protein was extracted from Ad5IB infected or
noninfected IEC after IL-1ß stimulation and analyzed by Western blot,
no degradation of the mutated exogenous IB was observed (Fig. 3B). More importantly, levels of exogenous IB
remained much higher than endogenous IB, consistent with data
present in Figure 2. Note that endogenous IB from IL-1ß-stimulated
Caco-2 cells was totally degraded as opposed to incomplete degradation
in HT-29 cells. This altered HT-29 IB degradation pathway is typical
of most transformed IEC lines and primary IEC, as previously reported
by our group (15). These data show that following Ad5IB infection,
the IB super-repressor is efficiently transcribed and translated
into a protein that is resistant to IL-1ß-mediated proteolysis.
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B activity by Ad5IB
The high level of Ad5IB expression combined with its
resistance to degradation suggested a potential inhibitory effect of
this reagent on NF-B activity. Effects of Ad5IB on NF-B
activation was investigated by studying RelA nuclear translocation and
NF-B binding activity. Noninfected, Ad5IB-infected, and
Ad5LacZ-infected IEC were stimulated with IL-1ß after which RelA
nuclear localization was visualized by immunofluorescence. A strong
nuclear staining was observed in IL-1ß-stimulated Ad5LacZ-infected
Caco-2 cells but only modest staining in HT-29 cells compared with
control (Fig. 4; compare panels
2 and 5 with 1 and 4,
respectively). This pattern of staining is consistent with our previous
findings (15). In Ad5IB-infected IEC, a clear cytoplasmic pattern of
RelA staining with no appreciable nuclear immunofluorescence was
observed (Fig. 4; compare panel 3 with 2 and
6 with 5). We next sought to demonstrate
NF-B DNA binding activity using a consensus B-probe. Nuclear
extracts derived from uninfected, Ad5IB-infected, or
Ad5LacZ-infected IEC were analyzed 30 min after media or IL-1ß
stimulation. IL-1ß strongly induced NF-B binding activity in both
Ad5LacZ-infected Caco-2 or HT-29 cells (Fig. 5A). A significant
reduction of NF-B binding activity was observed in Ad5IB-infected
IEC when compared with stimulated control virus. The specificity of
Ad5IB-mediated NF-B inhibition was tested by measuring AP-1
binding activity. Figure 5B shows that AP-1 binding activity
was not affected in Ad5IB-infected HT-29 cells nor in Caco-2 cells
(data not shown). In contrast to NF-B, AP-1 is constitutively
present in the nucleus of IEC lines and is not significantly activated
by IL-1ß (1.3-fold over nonstimulated).
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B
The strong inhibition of NF-B activity by Ad5IB in IEC lines
suggested that cytokine-mediated B-dependent gene induction could be
down-regulated by this reagent. As shown in Figure 6A, IL-1ß induction of iNOS,
IL-1ß, and IL-8 mRNA expression is inhibited in Ad5IB-infected IEC
when compared with control Ad5LacZ-infected cells. Inhibition of IL-8
mRNA accumulation by Ad5IkB was also measured by Northern analysis.
Figure 6B shows that Ad5IkB strongly inhibits
IL-1ß-induced IL-8 mRNA accumulation in Caco-2 cells. Interestingly,
Ad5LacZ but not Ad5IB leads to an up-regulation of IL-8 mRNA in
HT-29 cells in the absence of exogenous stimuli (Fig. 6C). A two- to threefold increased in IL-8 secretion
was also found in Ad5LacZ-infected but not in Ad5IB-infected HT-29
cells (Fig. 6D). Nevertheless, IL-1ß-induced IL-8
mRNA accumulation was totally inhibited in Ad5IB-infected cells but
not in Ad5LacZ cells as shown in Figure 6, A to
C. These data suggest that adenoviral infection stimulates
IL-8 secretion in HT-29 but not in Caco-2 cells. This effect is blocked
when the gene delivered is an NF-B super-repressor. This inhibitory
effect was verified at the protein level by measuring IL-8 secretion
from infected or noninfected IEC lines stimulated with IL-1ß. IL-8
secretion induced by IL-1ß was blocked in Ad5IB-infected but not
in Ad5LacZ-infected cells after 12 h of IL-1ß stimulation (Fig. 6D).
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B would be able to prevent IL-8
secretion resulting from other stimuli. Ad5LacZ- or Ad5IB-infected
HT-29 cells were stimulated with either TNF- (10 ng/ml) or PMA (100
ng/ml) for 3 h after which IL-8 expression was analyzed by RT-PCR.
TNF- and PMA-mediated IL-8 mRNA induction was also inhibited in
Ad5IB-infected cells (Fig. 7A; compare lanes 2
and 3 with 4 and 5, respectively). In
addition, IL-8 secretion induced by TNF- was suppressed in
Ad5IB-infected HT-29 cells after 12 h stimulation (Fig. 7B); similar results were obtained following the PMA
induction (data not shown).
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B was then
investigated in cultured primary IEC. Cells freshly isolated from
resected normal colon specimens were infected with Ad5IB or Ad5LacZ
for 4 h and then stimulated with IL-1ß (100 ng/ml) for 12
h. Figure 8A demonstrates that
protein extract from Ad5IB-infected but not Ad5LacZ-infected or
noninfected primary IEC shows the presence of an immunoreactive mutant
IB protein as assayed by Western blot. The IB mutant delivered
into primary IEC by the adenovirus was functional as seen in Figure 8B where Ad5IB-infected primary IEC showed a total
inhibition of IL-8 secretion when compared with media controls.
Therefore, Ad5IB was able to infect both transformed and primary IEC
and blocked proinflammatory gene expression. These data proved that
Ad5IB is a potent inhibitor of NF-B activation in IEC and
therefore could potentially be used in vivo as a tool to down-regulate
inflammation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B super-repressor
represents a novel means to block NF-B activity in many types of
mammalian cells. Since NF-B is a key transcription factor in a
number of mucosal immune responses and represents a master switch in
many chronic inflammatory diseases (11), blocking its activity is
conceptually superior to independently blocking each different
inflammatory mediator. Our group has shown that cytokine and adhesion
molecule expression could be modulated in IEC by targeting the
IB/NF-B system with pharmacologic products (15, 34, 35). For
example, blockade of IB degradation by proteasome inhibitors
almost completly attenuated cytokine-induced IL-8 and ICAM-1 gene
expression (15, 35). In this study, we report the generation of an
adenovirus bearing an NF-B super-repressor (Ad5IB) and explore
the possibility of using this virus to suppress the induction of
several inflammatory mediators. We show for the first time that a
recombinant adenovirus can efficiently infect (>80%) transformed IEC
as well as primary colonic epithelial cells. IEC expressed high levels
of the mutant IB, which resists IL-1ß-induced proteolysis due to
its inability to be phosphorylated at serines 32 and 36. High Ad5IB
expression levels combined with proteolysis resistance leads to almost
total inhibition both of NF-B activity and of iNOS, IL-1ß, and
IL-8 gene expression in IEC following stimulation with several
different inducing agents. Identical inhibitory responses in two
transformed IEC lines with different degrees of IB degradation
(Caco-2 and HT-29; (15)) and primary colonic epithelial cells
demonstrate universality of this approach to IEC in various stages of
differentiation. These results suggest that Ad5IB is a potential
candidate for in vivo intestinal gene therapy and illustrate the
central role of IB as a regulator of IEC proinflammatory gene
expression.
The balance between inactive and active NF-B following cell
stimulation relies mostly on IB. Overexpression or degradation of
IB can disrupt this balance and shift NF-B toward an inactive or
active state, respectively. Most of the different approaches to inhibit
NF-B activity have focused on endothelial or mononuclear hemopoietic
cells. Most of these strategies have targeted IB, such as
proteasome blockade, phosphorylation inhibition, and protein
overexpression (36, 37, 38, 39, 40) although antisense oligonucleotides to RelA
(p65) inhibit experimental colitis (41). In this study, we demonstrated
the feasibility of delivering an NF-B super-repressor into IEC. The
IB protein delivered into IEC was a S32A/S36A mutant form of IB
that mutated the inducible amino acid phosphoreceptor and therefore
abolished the degradation process. This approach is conceptually
superior to delivering the wild-type protein that is still degradable
and therefore solely relies on overexpression of the exogenous gene to
achieve NF-B inhibition.
The vehicle used for delivering the exogenous gene is also a critical
parameter in determining response of the transfected cell population to
stimulation. For example, less than 10% of HT-29 cells are positively
transfected using the lipofectamine method (15). Therefore, endogenous
gene expression will likely not be inhibited at such a low level of
transfected cells. In contrast, in vitro and in vivo adenovirus
infection achieves a high percentage of cells bearing the exogenous
gene. Although adenoviruses efficiently infect in vivo a number of
different organs including hepatocytes and respiratory epithelial cells
(25), there is still no data available on cells derived from the
intestine (25). The intestine constitutes an attractive site for viral
gene therapy because of its accessibility by endoscopy or site-specific
oral delivery systems. Our data demonstrate for the first time that
adenoviruses have the potential to effectively deliver in vitro an
NF-B super-repressor into both transformed and primary IEC. However,
the ability of Ad5IB to infect IEC is more efficient in transformed
than in primary IEC. This could be due to the heterogeneity of the cell
populations isolated from our resected tissue, which contain a
distribution of crypt to villous cells. Cells at various stage of
differentiation express various levels of adenoviral receptor that
potentially influence the infection susceptibility. The biggest
remaining challenges for in vivo intestinal gene therapy are to
overcome the natural antiviral defenses provided by the mucus barrier
and the rapid turnover of intestinal epithelial cells.
Primary IEC spontaneously release high levels of IL-8 into the cell
supernatant without exogenous stimulation, as previously shown by Yang
et al. (42). This IL-8 production might originate from cell activation
triggered during the isolation procedure. The inability of Ad5IB to
prevent this release could be explained by the fact that the
super-repressor inhibits only new gene transcription. Therefore, any
previous IL-8 mRNA transcripts present in the isolated cells before
Ad5IB infection will continue to be translated and released into the
cell supernatant. The incomplete inhibition of IL-8 production in
Ad5IB-infected transformed IEC may be explained by a partial
suppression of NF-B activity and/or by release of IL-8 by the
uninfected cells, which comprise 
20% of the total population.
In addition, it is possible that transcription factors other than
NF-B participate in the induction of IL-8 in IEC. Nevertheless, we
demonstrated that Ad5IB significantly suppresses new IL-8 protein
released by IL-1ß-stimulated IEC.
Another theoretical barrier to in vivo gene therapy with viral vectors
is a local inflammatory response, as suggested by induction of cytokine
expression (Fig. 6, C and D) following
Ad5LacZ infection of IEC. An inflammatory response has been previously
documented in the literature (36, 43). In accordance with these
findings, we documented that Ad5LacZ infection leads to an
up-regulation of the IL-8 gene in IEC. This effect was not seen when
Ad5IB was used to infect IEC. This suggests that any potential in
vivo inflammation caused by recombinant adenovirus infection will be
prevented or suppressed by the Ad5IB encoding gene. The development
of a new generation of adenoviral vector with less immunogenic
properties combined with oral tolerance should eventually decrease this
inflammation response.
It should be emphasized that overexpression of the nondegradable form
of IB will not only affect the phenotype of infected IEC but also
would prevent the release of proinflammatory molecules that affect
adjacent epithelial cells and lamina propria immune cells. Eckmann and
Kagnoff have postulated that IEC are the sentinels that detect
bacterial and parasite invasion and initiate the mucosal immune
response (6, 7). We have reported that inhibition of NF-B activation
with different proteasome inhibitors totally suppresses
TNF--mediated ICAM-1 gene expression in IEC (35). It is reasonable
to speculate that inhibition of IL-8 secretion and ICAM-1 protein
synthesis by epithelial cells could prevent neutrophil recruitment and
transepithelial inflammatory cell migration (7, 44, 45). In addition,
our data show that iNOS gene expression, which catalyzes the production
of NO, is inhibited by AD5IB. This supports a previous finding that
emphasizes the critical role of NF-B in iNOS gene expression
(46).
NO concentration is increased in the mucosa of patients with
inflammatory bowel diseases and may be implicated in the
pathophysiology of these disorders (47). Therefore, inhibition of a
wide variety of proinflammatory molecules by transfection of a
nondegradable IB molecule illustrates the key role of NF-B
transcriptional regulation of the inflammatory response in IEC. In
addition, IEC could theoretically be induced to secrete
immunosuppressive molecules delivered by gene therapy. It remains to be
seen if utilizing in vivo IEC as a factory for production of secreted
immunosuppressive proteins (e.g. IL-4, IL-10, IL-RA, TGF-ß) or
whether nondegradable IB expression will down-regulate
experimental inflammation.
The altered IB degradation in IEC and the striking difference in
RelA nuclear staining pattern between IL-1ß-stimulated Caco-2 and
HT-29 cells have been previously reported by us (15). In the present
study, we demonstrate again strong nuclear RelA staining with a
concomitant decrease in cytoplasmic immunoreactivity in
IL-1ß-stimulated Caco-2 cells. In contrast, HT-29 cells shows only a
modest RelA nuclear staining with no attenuation of cytoplasmic
staining following IL-1ß stimulation. Despite the small amount of
nuclear RelA, HT-29 cells secrete at least as much IL-8 as Caco-2 cells
following IL-1ß stimulation, as demonstrated in Figure 6 and our
previous report (15). This suggests that there is no direct correlation
between RelA nuclear translocation and amount of IL-8 production in
HT-29 cells. The effect of Ad5IB is then best appreciated in
Caco-2-infected cells where more than 90% of the cells show a strong
inhibition of RelA nuclear staining. Nevertheless, both cell lines show
a strong inhibition of NF-B binding activity and IL-8 gene
expression after Ad5IB infection, validating the biologic effects of
the NF-B super-repressor in different IEC with variable patterns of
IB degradation, possibly representing different stages of epithelial
cell differentiation.
The constitutive presence of nuclear AP-1 binding activity and lack of
significant enhanced AP-1 activity following IEC cytokine stimulation
contrasts with the inducible feature of this pathway reported in many
cell types (48). The oligonucleotide probe used to measure AP-1 DNA
binding in our study is derived from the human collagenase promoter, a
known inducible promoter (48). Wu et al. have reported that the IL-8
promoter region of Caco-2 cells constitutively exhibit AP-1 binding
activity that is not up-regulated following IL-1ß stimulation (49).
In addition, porcine aortic endothelial cells show constitutive AP-1
binding activity that is not up-regulated following LPS stimulation
(36). It appears therefore that modulation of AP-1 binding activity is
cell type specific and that IEC exhibit constitutive AP-1 binding
activity, in contrast to the inducible nature of NF-B DNA binding.
The key role of NF-B and the lack of a contribution of AP-1 in
transcriptional regulation of IL-8 gene expression is documented by
almost complete blockade of IL-8 secretion following highly selective
trapping of NF-B by Ad5IB.
In summary, our results clearly demonstrate the anti-inflammatory
properties of Ad5IB in IEC and the pivotal role of NF-B
transcriptional regulation of a number of proinflammatory molecules
following cytokine stimulation. These findings suggest a potential new
therapeutic tool for intestinal gene therapy if an efficient in vivo
administration protocol can be developed for the intestine.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. R. Balfour Sartor, Division of Digestive Diseases and Nutrition, CB No. 7080, Burnett-Womack Bldg, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7080.
3 Abbreviations used in this paper: IEC, intestinal epithelial cells; IB, inhibitor B; Ad5, adenovirus type 5; LacZ, ß-galactosidase; EMSA, electrophoretic mobility shift assay; iNOS, inducible nitric oxide synthase; NO, nitric oxide; NGS, nonimmune goat serum; MOI, multiplicity of infection; ECL, enhanced chemiluminescence.
4 Y. Iimuro, T. Nishiura, C. Hellerbrand, K. Behrns, R. Shoonhoven, J. Grisham, and D. Brenner. NF-B prevents apoptosis in liver disfuncton during liver regeneration. Submitted for publication.
Received for publication June 18, 1997. Accepted for publication September 15, 1997.
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References |
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B in the immune system. Annu. Rev. Immunol. 12:141.[Medline]
B, a pivotal transcription factor in chronic inflammatory diseases. N. Engl. J. Med 336:1066.B: regulation by distinct protein subunits. Biochim. Biophys. Acta 1072:63.[Medline]
B p65 transcriptional activator and an alternatively spliced derivative. Mol. Cell. Biol. 12:444.B functions as a potent transcriptional activator and a target for v-Rel-mediated repression. Proc. Natl. Acad. Sci. USA 89:1875.B degradation in human colonic epithelial cells. J. Immunol. 158:226.[Abstract]
B: a lesson in family values. Cell 80:529.[Medline]
B regulation: the emerging role of phosphorylation and proteolysis. Immunity 3:263.[Medline]
B- proteolysis by site-specific, signal-induced phosphorylation. Science 267:1485.B to the ubiquitin-proteasome pathway. Genes Dev. 9:1586.B by novel ubiquitination-dependent protein kinase activity. Cell 84:853.[Medline]
B requires site-specific ubiquitination. Proc. Natl. Acad. Sci. USA 92:11259.B and stabilizes a newly phosphorylated form of IB- that is still bound to NF-B. EMBO J 13:5433.[Medline]
B to multiple pathway for NF-B activation. Mol. Cell. Biol. 15:2809.[Abstract]
B. Science 274:784.B phosphorylation sites that signal its ubiquitination and degradation. Mol. Cell. Biol. 16:1295.[Abstract]
stimulates AP-1 activity through prolonged activation of the c-Jun kinase. J. Biol. Chem. 269:26396. gene expression through an IB/NF-B pathway in intestinal IEC-6 cells. Gastroenterology 110:A333.
B mediates cytokine induced expression of ICAM-1 in IEC-6 cells. Gastroenterology 110:A329.
B, an inhibitor of transcription factor NF-B. J. Exp. Med. 183:1013.B- phosphorylation. J. Immunol. 155:3538.[Abstract]
B block Tat-tumor necrosis factor synergistic activation of human immunodeficiency virus type 1 gene expression and virus multiplication. J. Virol. 70:5777.[Abstract]
B abrogates established experimental colitis in mice. Nat. Med. 2:998.[Medline]
B. Proc. Natl. Acad. Sci. USA 93:3308.This article has been cited by other articles:
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D. A. O'Neil, E. M. Porter, D. Elewaut, G. M. Anderson, L. Eckmann, T. Ganz, and M. F. Kagnoff Expression and Regulation of the Human {beta}-Defensins hBD-1 and hBD-2 in Intestinal Epithelium J. Immunol., December 15, 1999; 163(12): 6718 - 6724. [Abstract] [Full Text] [PDF]
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C. Jobin, C. A. Bradham, M. P. Russo, B. Juma, A. S. Narula, D. A. Brenner, and R. B. Sartor Curcumin Blocks Cytokine-Mediated NF-{kappa}B Activation and Proinflammatory Gene Expression by Inhibiting Inhibitory Factor I-{kappa}B Kinase Activity J. Immunol., September 15, 1999; 163(6): 3474 - 3483. [Abstract] [Full Text] [PDF]
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D. Elewaut, J. A. DiDonato, J. Mogg Kim, F. Truong, L. Eckmann, and M. F. Kagnoff NF-{kappa}B Is a Central Regulator of the Intestinal Epithelial Cell Innate Immune Response Induced by Infection with Enteroinvasive Bacteria J. Immunol., August 1, 1999; 163(3): 1457 - 1466. [Abstract] [Full Text] [PDF]
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X. Sun, J. Lee, T. Navas, D. T. Baldwin, T. A. Stewart, and V. M. Dixit RIP3, a Novel Apoptosis-inducing Kinase J. Biol. Chem., June 11, 1999; 274(24): 16871 - 16875. [Abstract] [Full Text] [PDF]
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 S Wirtz, P R Galle, and M F Neurath Efficient gene delivery to the inflamed colon by local administration of recombinant adenoviruses with normal or modified fibre structure Gut, June 1, 1999; 44(6): 800 - 807. [Abstract] [Full Text] [PDF]
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C. Jobin, L. Holt, C. A. Bradham, K. Streetz, D. A. Brenner, and R. B. Sartor TNF Receptor-Associated Factor-2 Is Involved in Both IL-1{beta} and TNF-{alpha} Signaling Cascades Leading to NF-{kappa}B Activation and IL-8 Expression in Human Intestinal Epithelial Cells J. Immunol., April 15, 1999; 162(8): 4447 - 4454. [Abstract] [Full Text] [PDF]
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E. G. A. Cafferata, A. M. G. Guerrico, O. H. Pivetta, and T. A. Santa-Coloma NF-kappa B Activation Is Involved in Regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Interleukin-1beta J. Biol. Chem., April 27, 2001; 276(18): 15441 - 15444. [Abstract] [Full Text] [PDF]
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R. LaMonica, S. S. Kocer, J. Nazarova, W. Dowling, E. Geimonen, R. D. Shaw, and E. R. Mackow VP4 Differentially Regulates TRAF2 Signaling, Disengaging JNK Activation while Directing NF-kappa B to Effect Rotavirus-specific Cellular Responses J. Biol. Chem., June 1, 2001; 276(23): 19889 - 19896. [Abstract] [Full Text] [PDF]
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A. K. Cardozo, H. Heimberg, Y. Heremans, R. Leeman, B. Kutlu, M. Kruhoffer, T. Orntoft, and D. L. Eizirik A Comprehensive Analysis of Cytokine-induced and Nuclear Factor-kappa B-dependent Genes in Primary Rat Pancreatic beta -Cells J. Biol. Chem., December 21, 2001; 276(52): 48879 - 48886. [Abstract] [Full Text] [PDF]
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