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Introduction
Atopic asthma, characteristic of eosinophilic airway inflammation, is now regarded as a T-helper 2 (Th2) cell-mediated
disorder, which is under the control of dendritic cells
(DC)[1]. Studies in rodents and humans have revealed the presence of
an extensive network of DC in
airways[2,3]. Upon maturation in
vivo or in vitro, DC become less phagocytic and more potent
antigen-presenting cells (APC),
expressing high levels of major histocompatibility complex (MHC) class II and costimulatory molecules as well as
proin-flammatory cytokines. As the most potent professional APC identified, DC play a central role in inducing the primary
immune response, priming the naive T cells, initiating the immune tolerance, and regulating the types of T cell
responses[4].
Nuclear factor kB (NF-κB), which is typical of p50/p65 heterodimers, is sequestered as inactive trimers in the cytoplasm
of quiescent cells through interaction with IκBα, the most important member of the inhibitors of
NF-κB (IkB) family. Multiple signals converge on the common signaling pathway for
NF-κB activation upon sequential phosphorylation of
IκBα at the Ser 32/36 residues of the N-terminal domain, and subsequent proteolytic
degradation[5]. Recent data suggest that
NF-κB overactivation may be the basis for increased expression of myriad inflammatory genes and for
the perpetuation of chronic airway inflammation in
asthma[6,7]. More recently, we have cloned the 801 bp Chinese
IκBα mutant (IκBαM) gene (203-1003 bp) encoding 267 amino acids from human placenta, a novel nondegradable super-repressor of
NF-κB, by site-directed deletion of the N-terminal phosphorylation sites of Ser 32/36, and constructed a replication-deficient recombinant
adenovirus vector AdIκBαM[8]. These mutations prevent
IκBαM phosphoryla-tion, retaining NF-κB in its inactivated
cytosolic location complexed with IκBαM. DC maturation and cytokine production are
NF-κB-dependent, thus, we investigated the effects of adenoviral gene transfer of
IκBαM on apoptosis, phenotype and function of DC derived from human
monocytes to explore a possible approach for future DC-based immunotherapy of asthma.
Materials and methods
Reagents Recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin 4 (IL-4), and
tumor necrosis factor a (TNF-α) were generously provided by Dr Guang-yong PENG (Baylor College of Medicine, Dallas,
USA). Lipopolysaccharide (LPS) was purchased from Sigma Chemical Co (St Louis, MO, USA). Fluorescein isothio-
cyanate (FITC)-conjugated mouse anti-human CD14, HLA-DR, CD83 and CD80 were bought from Serotec (UK), and
phycoerythrin (PE)-conjugated mouse anti-human CD86 were bought from Diaclone (USA). Taq DNA polymerase and T4 DNA
ligase were obtained from Gibco BRL (USA).
[γ-32P] adenosine triphosphate and
[3H] thymidine (TdR) were provided by Beijing Furui Biotechnology (China).
Vectors The serotype 5-, E1-, and E3-defective
adeno-viruses expressing Escherichia coli β-galactosidase
(AdLacZ) and human IκBαM (AdIκBαM) were constructed as describ-ed
previously[8]. Remarkably, the molecular weight of novel
IκBαM was deduced to be 30 kDa. The generated titers of AdLacZ and
AdIκBαM were approximately
1.0×1010 and
4.0×109 pfu/mL, respectively.
Cell culture Blood buffy coat from healthy donors (Jiangsu Institute of Hematology) was centrifuged on Ficoll-Paque
(Pharmacia, Sweden) to acquire human peripheral blood mononuclear cells (PBMC). Monocytes were derived from PBMC
depleted of NK, B-, and T cells with anti-CD16, anti-CD19 and anti-CD3, as well as goat anti-mouse Ig-conjugated magnetic
beads (Miltenyi Biotec, USA) as previously
described[9]. These were plated
(1×107 cells/3 mL per well) into 6-well plates in
RPMI-1640 medium supplemented with 10% fetal calf serum (FCS; Gibco BRL, USA). Cells were cultured at
37 °C in 5% CO2 in medium supplemented with GM-CSF (900 ng/mL) and IL-4 (300 ng/mL). Cultures were fed every 2 d with half of the culture
volume of full doses of cytokines. On d 5 cells were stimulated with LPS (100
ng/mL) to induce maturation, and the suspended cells were harvested on d 7.
Electron microscopy On d 7 cells were washed once in phosphate-buffered saline (PBS), resuspended, and fixed
overnight in 2% paraformaldehyde and 2.5% glutaraldehyde, followed by fixation for 1 h in 1% cacodylate-buffered
osmium tetraoxide. After dehydration in a series of ethanol and propylene oxide solutions, the cells were embedded in epoxy
resin, ultrathinly sectioned, stained with uranyl
acetate and lead citrate, and examined under a scanning electron microscope (SEM; SX-40; Akashi, Japan). Additionally,
the prepared cells were routinely stained and examined under a transmission electron microscope (TEM; JEM-1010; Jeol,
Japan).
Gene transfer Adenoviral transfections were performed in 60 mm plates with mature DC at
5×105 cells/plate on d 7. For adenoviral transfections, medium was removed from each plate and replaced with 2 mL of medium containing either
AdIκBαM at multiplicity of infection (MOI) of 25, 50, or 100 pfu/cell, or AdLacZ at an MOI of 50 pfu/cell for 4 h, followed by incubation
in fresh RPMI-1640 medium supplemented with 10% FCS at
37 °C and in 5% CO2 for an additional 24 h and
48 h, respectively. The untransfected mature DC were used as normal controls.
PCR and RT-PCR DNA and RNA were extracted from mature DC, either untransfected or transfected with
AdIκBαM and AdLacZ, using Wizard plasmid isolation and Trizol kits (Promega, USA), according to the manufacturer¡¯s instruc-tions,
and the RNA was reverse-transcribed into cDNA for templates. Primers were designed using Primer Express software and
were as follows: forward primer 5¡¯-CCTCTAGAA-TGAAAGACGAGGAGTACGAG-3¡¯
and reverse primer 5¡¯-TGGTACCTCACAGCTCGTCCTCTGTGAACTCCGTG-3¡¯. The reaction started with an initial denaturation at
94 °C for 5 min, followed by 35 cycles of denaturation at
94 °C for
1 min, annealing at 54 °C for 1 min and extension at
72 °C for 1 min and additional extension of 10 min, after which 5
mL of each product was quantified by 1% agarose gel electro-phoresis.
Western blot analysis After 24 or 48 h of overexpression of the genes of
interest, cytosolic proteins were prepared as
described elsewhere[10] and quantitated by using the Brad-ford assay. Equivalent amounts of denatured protein
(100 µg) were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel, and electrophoretically transferred to
nitrocellulose membranes. Nonspecific binding to the membrane was blocked with 5% nonfat dry milk in PBS-Tween overnight at
4 °C. Blots were washed in PBS-Tween, and then probed with
anti-human IκBα antibody (1:1000 dilution) and horseradish
peroxidase-conjugated anti-goat IgG (1:5000 dilution, Santa Cruz Biotechnology, USA)
for 2 h at room temperature. Immunoblots were visualized with an enhanced chemiluminescence detection kit (Amersham Life Sciences, USA).
Electrophoretic mobility shift assay After 48 h over-expression of target genes,
DC were harvested upon stimulation with 10 ng/mL
TNF-α for 30 min. Preparation of nuclear extracts and the electrophoretic mobility shift assay (EMSA) were
carried out as described previously[11]. The
NF-κB oligonucleotide (5¡¯-AGTTGAGGGGACTTTCCCA-GGC-3¡¯) contained a
consensus NF-κB motif (underlined), and the mutant
NF-κB oligonucleotide
(5¡¯-AGTTGAGGGT-CCTTTCC-CAGGC-3¡¯; Promega, USA) was used as a mutant competitor. Five micrograms of nuclear extracts were incubated with 30 fmol of
[32P]-labeled oligonucleotide by T4 polynucleotide kinase for 30 min at room temperature. DNA-protein complexes were
electrophoretically resolved on 5% nondenatured polyacrylamide gel, which was visualized and quantitated by PhosphorImager
(Molecular Dynamics) using ImageQuant software (Amersham Life Sciences, USA). For competition
assays, a 100-fold excess of unlabeled NF-κB or mutant
NF-κB oligonucleotide was added to the nuclear extracts from
TNF-α-stimulated cells 10 min before exposure to the
[32P]-labeled probe.
Flow cytometry DC that were either untransfected or transfected with
AdIκBαM or AdLacZ were incubated at a density of
1×105 cells/100 µL with 10 µL FITC- or PE-conjugated monoclonal antibodies for 30 min at room temperature. The cells
were washed once in PBS or fixed with 1% para-formaldehyde, then the surface markers of mature DC were assessed by
FACScan (Becton Dickinson, USA). In addition, apoptosis of mature DC was immediately analyzed by FACScan after
staining with 5 µL annexin V (AV)-FITC and 5 µL propidium iodide (PI) for 20 min at 4 °C. The total cell population consisted
of DC that were AV single positive (apoptotic), PI single positive (necrotic), AV and PI double positive (apo-necrotic), or AV
and PI double negative (live).
Enzyme-linked immunosorbent assay Supernatants from mature DC were harvested 48 h after transfection with
AdIκBαM and AdLacZ. Levels of IL-12 (p70) were determined using a sandwich enzyme-linked immunosorbent
assay (ELISA) kit (PharMingen, USA) in compliance with the manufacturer¡¯s instructions.
Mixed leukocyte reaction Allogeneic T cells were
obtained from nylon wool nonadherent PBMC by negative selection, with a purity of more than
95%[12]. T cells were cultured in triplicate in 96-well flat-bottomed plates at
2×105 cells/well, to which nonautologous mature DC, either
untrans-fected or transfected with AdIκBαM and AdLacZ, were added at
2×103, 4×103,
10×103, or 20×103 cells/well. Plates were
incubated at 37 °C and 5% CO2 for 96 h, and pulsed with 0.5
µCi/well of [3H]thymidine for the final 16 h before harvesting on filters for scintillation counting. Counts per minute (cpm)
of triplicate wells were deduced by subtracting the background cpm of the medium alone.
Statistical analysis Data were expressed as mean±SD, and assessed by one-way analysis of variance (ANOVA) and
paired Student¡¯s t-tests with SPSS 11.0 software. Statistical difference was assumed at
P<0.05.
Results
Morphology of mature DC In culture with GM-CSF and IL-4 for 5 d, monocytes differentiated into mature DC after
stimulation with LPS for 2 d. The mature DC showed prominent dendritic and veiled projections (>10 µm) on d 7 under the
SEM (Figure 1A), and displayed plentiful surface dendrites and cytosolic vesicles mostly of endocytic type, but a relatively
immature Golgi zone under the TEM (Figure 1B).
Detection of IκBαM gene in mature DC Electrophoretic analysis of both PCR and RT-PCR products revealed the unique
801 bp IκBαM cDNA in AdIκBαM-transfected, but not in AdLacZ-transfected or untransfected, mature DC,
indicating that the IκBαM gene is integrated into the
genome and transcribed in infected mature DC (Figure 2A). As determined by Western blot analysis, the tagged transgene
product can be visualized as being slightly larger than the native
IκBα. A dose- and time-dependent increase in IκBαM
expression can be seen in AdIκBαM-transfected mature DC, which peaked at an MOI of 100 pfu/cell and at 48 h (Figure 2B).
Inhibition by AdIκBαM of TNF-α-induced NF-κB activation in mature DC
The function of AdIκBαM in blocking NF-κB binding was assessed by EMSA. In contrast to a basal
NF-κB activity in the control (untransfected and nonstimulated) DC, the
TNF-α-stimulated DC exhibited a dramatically promoted
NF-κB activity (P<0.01). Gene transfer of
IκBαM, but not LacZ, at an MOI of 100 pfu/cell and 48 h sharply inhibited the
NF-κB activity compared with the TNF-α-stimulated DC
(P<0.01), suggesting that AdIκBαM inhibits
TNF-α-induced NF-κB activation in mature DC (Figure 3A, 3B). In competition assays, addition of
100-fold excess of unlabeled NF-κB, but not mutant
NF-κB, oligonucleotide affected NF-κB binding, confirming the specificity of
NF-κB binding (Figure 3A).
Induction by AdIκBαM of apoptosis in mature DC
Flow cytometry revealed low levels of normal apoptosis in mature DC on
d 7, with a higher level of apoptosis 48 h later
(P<
0.01). Optimal gene transfer of IκBαM, but not LacZ, significantly augmented the apoptosis of mature DC
(P<0.01), demonstrating that AdIκBαM facilitates apoptosis in mature DC (Figure 4).
Effects of AdIκBαM on phenotype and function of
mature DC Flow cytometry revealed low levels of CD14, but high levels of HLA-DR, CD83, CD80, and CD86 in mature DC.
Except for similar levels of CD14 and HLA-DR
(P>0.05), optimal gene transfer of IκBαM, but not LacZ, greatly downregulated the
levels of CD83, CD80, and CD86 in mature DC, among which CD80 had a smaller decrease than did CD86 (all
P<0.01). Notably, the mature DC overexpressing
IκBαM still secreted high levels of CD83, which is a hallmark of maturation. These results imply
that downregulation of the B7 surface molecules by
AdIκBαM in mature DC may contribute to the immune shift of T cells (Figure
5, Table 1). ELISA confirmed that gene transfer of
IκBαM, but not LacZ, resulted in a lower secretion levels of IL-12 (p70) in
mature DC (P<0.01, Figure 6). The mixed leukocyte reaction revealed, in parallel with the ratio of DC:T cells, a similar potency for
stimulating the proliferation of allogeneic T cells between AdLacZ-transfected and untransfected mature DC. In contrast, the
AdIκBαM-transfected mature DC embodied a strikingly decreased mixed leukocyte reaction in comparison with the
AdLacZ-transfected mature DC (P<0.05 or 0.01;
Figure 7).
Discussion
T-cell stimulation and Th1/Th2-cell polarization require 3 DC-derived signals: signal 1 (recognizing signal), which is mediated
through T-cell receptor (TCR) triggering by MHC class II-associated peptides processed from pathogens; signal 2 (costimulatory
signal), which is mainly mediated by triggering of T cell CD28 by CD80 (B7-1) and CD86 (B7-2); and signal 3 (polarizing signal),
which is mediated by various soluble or membrane-bound
factors[13-15].
NF-κB plays a crucial role in antiapoptosis by regulation of the transcription of cytokine genes, induction of the antiapoptotic
genes, and promotion of the TNF receptor-associated factors and the inhibitor-of-apoptosis proteins to block the activation of
caspase-8[16,17]. IκBα, a 37 kDa inhibitory protein, is arranged in 3 domains: the N-terminal domain-containing specific
phosphorylation sites, the internal domain of 5 tandem ankyrin repeat sequences, and the C-terminal domain containing Pro-Glu-Ser-Thr
polypeptides for regulation of NF-κB activity, binding of
NF-κB, and rapid protein turnover,
respectively[18]. IκBα retains the p65-containing
NF-κB complex in the cytoplasm by masking its nuclear localization sequence (NLS). Upon appropriate stimulation,
for example by IL-1b, TNF-α, or LPS, IκBα
undergoes phosphorylation on two serine residues, Ser 32 and Ser 36, which renders
IκBα susceptible to proteolytic degradation via the ubiquitin-proteasome pathway, resulting in release and nuclear translocation of
NF-κB. Conse-quently, the active NF-κB, singlely or in combination with other nuclear factors, initiates transcription of target genes associated with immune and
inflammatory responses. As for the inflammatory cascade,
NF-κB repression in airways through suppression of
IκBα degradation or augmentation of IκBα synthesis can downregulate transcription of
an
array of the NF-κB-dependent genes, which is more effective than blockade of single downstream inflammatory or immune
genes[19].
In the present study, monocytes cocultured with GM-CSF and IL-4 differentiated into mature DC upon stimulation with
maturation inducer LPS. The IκBαM gene was successfully delivered into the monocyte-derived mature DC, and was expressed
time- and dose-dependently. Importantly, NF-κB-blocked mature DC by
AdIκBαM obviously inhibited the proliferation of T
cells, which is in agreement with the report that selective inhibition of
NF-κB in DC by the NF-κB essential modulator-binding
domain peptide blocks maturation and prevents T-cell
proliferation, accompanied by less Th1 and Th2
polarization[20]. Furthermore, TNF-α-induced
NF-κB activation of mature DC was significantly attenuated by
AdIκBαM, and the NF-κB-blocked mature DC were more susceptible to apoptosis than those transfected with AdLacZ. Nevertheless, blockade of
NF-κB in mature DC, other than little change in HLA-DR related to antigen-presentation, caused striking downregulation of the B7 costimulatory molecules.
It is deduced, therefore, that AdIκBαM might, in part, facilitate
the antigen-specific
immune tolerance of T cells via induction of DC apoptosis and downregulation of B7 molecules.
Asthma is, per se, a multigenetically susceptible clinical syndrome, in which sensitized individuals develop an aberrant
Th2-dominated immunity for the underlying basis of eosinophilic airway
inflammation[10,14]. B7-1 and B7-2 differentiate the Th0 cells
into a Th1 subset for cellular immunity and a Th2 subset for humoral immunity,
respectively[1,13,15]. Interestingly, downregulation
of B7-2 was more effective than that of B7-1 in the mature DC overexpressing
IκBαM, indicating that blockade of NF-κB in DC may, to some extent, contribute to rectifying the Th1/Th2 imbalance in asthmatics. It is noteworthy that Th2 development could
be the default pathway induced by DC when the production of IL-12 is quite
low[2]. As the major effective treatment for asthma,
corticosteroids, like all pharmacologic agents that have been shown to inhibit
NF-κB, have numerous other effects that could limit their therapeutic
usefulness[21]. Currently, there is much interest in identifying more specific and effective
NF-κB inhibitors, among which the IκBαM is a novel
NF-κB inhibitor. Although blocking the NF-κB pathway is unlikely to be a clinically beneficial
approach due to the broad range of genes involved, it is of importance to understand the role of
NF-κB during specific immune responses for identifying relevant molecules as potential therapeutic
targets[22].
In summary, our findings suggest that the
AdIκBαM is applicable to both effective gene transfer of
IκBαM and specific blockade of NF-κB in monocyte-derived mature DC. Moreover, the ability of mature DC overexpressing
IκBαM to induce DC apoptosis and downregulate B7 molecules highlights an important mechanism for T-cell immune suppres-sion. The successful
downregulation of T-cell responses following inhibition of
NF-κB in mature DC offers a potential strategy for future
immunotherapy of asthma[23,24]. Antigen-specific T-cell immune tolerance could be induced by
selective blockade of NF-κB with AdIκBαM in antigen-primed mature DC
(ie by pollen, ragweed, house dust mite, etc). Alternatively, the DC-mediated Th1-dominated
response is promising for amelioration of the undue Th2 by
AdIκBαM in combination with Th1-inducing agents, such as CpG
oligodeoxynucleotide, purified protein derivative and bacilli
Calmette-Guerin[25,26].
Acknowledgements
We would like to thank Prof Joel N KLINE (Division of Pulmonary Medicine, University of Iowa Hospitals and Clinics) and
Prof Gang HU (Department of Pharmacology and Neurobiology, Nanjing Medical University) for helpful comments and critical
review of the manuscript.
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