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Anti-inflammatory effect of adenovirus-mediated IB overexpression in respiratory epithelial cells

¹ Dept of Internal Medicine, Seoul National University College of Medicine, Chongno-Gu, Seoul, Korea. ² Clinical Research Institute, Seoul National University Hospital, Chongno-Gu, Seoul, Korea. ³ Lung Institute, Seoul National University Medical Research Center, Chongno-Gu, Seoul, Korea

CORRESPONDENCE: C.G. Yoo, Dept of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul, 110-799, Korea. Fax: 82 27629662

Keywords: adenovirus, interleukin-;8, IB super-repressor, nuclear factor-B, respiratory epithelial cells, tumour necrosis factor-;

Received: November 28, 2000
This work was supported by the 1999 National Research and Development Programme (Ministry of Science and Technology).

	Abstract

TOP Abstract Materials and methods Results Discussion Acknowledgements References

 
Many studies into basic biological characteristics of inflammation and tissue injury have implicated pro-inflammatory cytokine-mediated tissue injury in the pathogenesis of inflammatory lung diseases. Because transcription of most pro-inflammatory cytokines is dependent on the activation of nuclear factor (NF)-B, NF-B could be a good potential target to suppress the cytokine cascade. Cytokine-induced activation of NF-B requires phosphorylation and subsequent degradation of IBa. Therefore, the blocking NF-B activation by IB could inhibit the pro-inflammatory cytokine-induced tissue injury.

To evaluate whether blocking of NF-B activation shows an anti-inflammatory effect, this study investigated the effect of adenovirus-mediated overexpression of IB super-repressor (IB-SR) on the pro-inflammatory cytokine expression in respiratory epithelial cells.

The transduction efficiency of adenovirus was >90% in both A549 and NCI-H157 cells. Ad5IB-SR-transduced cells expressed high levels of IB-SR, which was resistant to tumour necrosis factor (TNF)-;-;induced degradation. Adenovirus-mediated overexpression of IB-SR blocked cytokine-induced nuclear translocation of p65 and NF-B deoxyribonucleic acid binding activity without affecting total cellular expression level of NF-B. Ad5IB-SR transduction suppressed cytokine-induced interleukin-;8 and TNF-; expressions at both ribonucleic acid and protein levels.

These results suggest that blocking the nuclear factor-B pathway by adenovirus-mediated overexpression of IB-super-repressor shows an effective anti-inflammatory effect in respiratory epithelial cells.

Nuclear factor (NF)-B is an ubiquitous inducible transcription factor involved in immune, inflammatory, stress and developmental processes. It is sequestered in the cytoplasm in an inactive state by association with the inhibitory molecule IB. NF-B is rapidly activated in response to various stimuli, including viral infection, lipopolysaccharide (LPS), ultraviolet irradiation, and pro-inflammatory cytokines such as tumour necrosis factor (TNF)-; and interleukin (IL)-1ß 1–3. TNF-; leads to the sequential activation of the downstream NF-B-inducing kinase (NIK) and the recently isolated TNF-;-;inducible IB kinase complex (IKK) 4–8. When activated, IKK directly phosphorylates Ser³² and Ser³⁶ of IB, triggering ubiquitination at Lys²¹ and Lys²², and rapid degradation of IB in 26S proteasome 1–3. This process liberates NF-B, allowing it to translocate to the nucleus. In the nucleus, NF-B binds to its cognate B site and transactivates the downstream genes.

In inflammatory lung diseases, such as acute lung injury (ALI), pro-inflammatory cytokines, especially TNF-; and IL-1ß, are known to be involved in tissue damage 9. Pro-inflammatory cytokines function in redundant and overlapping ways through the cytokine "cascade" or "network". As most genes for inflammatory mediators (such as TNF-;, IL-;2, IL-;6, IL-;8, lymphotoxin, granulocyte macrophage-colony stimulating factor (GM-CSF), ß-;interferon, and adhesion molecules) have B site in the 5' flanking region 1–3, the transcription of most pro-inflammatory cytokine genes are regulated by NF-B activation. Therefore, NF-B could be a good potential target to suppress the cytokine cascade.

NF-B activation can be inhibited by blocking various points in the NF-B/IB pathway. One method of NF-B inhibition is overexpression of IB. The overexpression of wild-type IB partially blocked NF-B responsive gene expression in vitro 10, 11. Since wild type IB is degraded in response to inflammatory mediators which are present at the site of inflammation, overexpression of wild type IB would not be effective in blocking NF-B activation. In order to block the activation of NF-B effectively, IB should be resistant to degradation. Since inducible phosphorylation of IB at Ser³² and Ser³⁶ is a critical step in the degradation of IB, IB mutants in which Ser³² and Ser³⁶ are substituted with nonphosphorylatable alanine (IB super-repressor, IB-SR) are resistant to degradation.

The present study investigated the effect of blocking the activation of NF-B by adenovirus-mediated overexpression of IB-SR on the pro-inflammatory cytokine expression in respiratory epithelial cells to evaluate the therapeutic potential of Ad5IB-SR in inflammatory lung diseases.

	Materials and methods

TOP Abstract Materials and methods Results Discussion Acknowledgements References

 
Cells
A549 and NCI-H157 human respiratory epithelial cell lines were maintained in a monolayer in Roswell Park Memorial Institute (RPMI) 1640 containing 10% foetal bovine serum (FBS), penicillin (60 µg·mL^–1), and streptomycin (100 µg·mL^–1) at 37°C under 5% carbon dioxide (CO₂).

Study design
Adenovirus vectors expressing IB-SR (Ad5IB-SR) and ß-;galactosidase (Ad5LacZ) were constructed. The transduction efficiency of adenovirus vector, its effect on the cell viability, and the stability of IB-SR were evaluated. The effect of adenovirus-mediated overexpression of IB-SR on the NF-B activity and the pro-inflammatory cytokine expressions in respiratory epithelial cells were evaluated.

Methods
Transduction of adenoviruses
An adenovirus vector expressing IB-SR was constructed as previously described 12. A recombinant adenovirus expressing ß-;galactosidase gene was used as a control virus. Cells were transduced at multiplicities of infection (moi) of 20 by adenovirus vector in serum free RPMI for 1 h with gentle shaking and then washed with phosphate buffered saline (PBS) and incubated with growth medium at 37°C, 5% CO₂ until use.

Analysis of cell viability
Cell viability was measured by a 3-;(4,5-dimethyl thiazol-;2-;yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. MTT solution was added to cells in 96-well plates to the final concentration of 0.5 mg·mL^–1, and cells were incubated at 37°C for 4 h. After removing culture media, 50 µL of dimethyl sulphoxide (DMSO) was added, and the optical density of each well was read at 590 nm.

Electrophoretic mobility shift assays
NF-B deoxyribonucleic acid (DNA) binding activity was assessed as described previously 13. Briefly, nuclear extracts were incubated for 20 min at room temperature with radiolabelled NF-B consensus sequence in the light chain enhancer in B-;cells (5'-AGT TGA GGG GAC TTT CCC AGG C-3'). In competition experiments, 50-fold molar excess of unlabeled oligonucleotide was added to the binding reaction. In supershift experiments, anti-p65 or anti-p50 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added and allowed to react for 45 min at room temperature. DNA-protein complexes were resolved on 4% nondenaturing polyacrylamide gel. Gels were dried and autoradiographed at –70°C.

Western blot analysis
Cytoplasmic, nuclear, and whole cell extracts were prepared as described previously 13. Twenty micrograms of protein was resolved on 10% sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose. The membranes were blocked with 5% skim milk-PBS/0.1% Tween 20 for 1 h prior to overnight incubation at room temperature with rabbit polyclonal anti-p65 or anti-IB antibodies from Santa Cruz Biotechnology, diluted 1:1000 in 5% skim milk-PBS/0.1% Tween 20. Membranes were incubated with goat anti-rabbit horseradish peroxidase (HRP)-conjugated antibody (Promega, Madison, WI, USA), diluted 1:2000 in 5% skim milk-PBS/0.1% Tween 20 for 1 h. Following successive washes, membranes were developed with an enhanced chemoluminescence (ECL) kit (Amersham Pharmacia Biotech, Uppsala, Sweden).

Immunofluorescent staining for nuclear factor-B
Cells grown in 2-;well chamber slides were fixed and permeabilized as described previously 13. Cells were incubated with rabbit polyclonal anti-p65 antibody, diluted 1:100 in 1% bovine serum albumin (BSA), for 30 min, and then incubated with rhodamine isothiocyanate-conjugated goat anti-rabbit immunoglobulin-;G antibody (Jackson ImmunoResearch, West Grove, PA, USA), diluted 1:100 in 1% BSA, for 30 min. Slides were analysed using a fluorescent light microscope.

Northern blot analysis
Total cellular ribonucleic acid (RNA) was isolated using TRIZOL Reagent (Gibco BRL, Gaithersburg, MD, USA). Equal amounts of total RNA (20 µg·lane^–1) from each sample were loaded into each lane of 1.0% agarose/2% formaldehyde gel, and capillary transferred to a nylon membrane. The human complementary DNA (cDNA) for TNF-; and IL-;8 (ATCC, Rockville, MD, USA) were radiolabelled with [-³²P]deoxycytidine triphosphate (dCTP) using a random priming kit (Stratagene, La Jolla, CA, USA). After prehybridizing the membranes for 2 h at 45°C in hybridization buffer, radiolabelled cDNA probe (1x10⁶ cpm·mL^–1 final concentration) was added and incubated overnight at 45°C. The membranes were exposed to X-;ray film (Eastman Kodak Co., Rochester, NY, USA) with intensifying screen for up to 5 days at –70°C.

Interleukin-;8 enzyme-linked immunosorbent assay
Cells (1x10⁴) were grown in 96-well culture plates in equal numbers. The supernatants were collected and stored at –70°C until being analysed. IL-;8 concentrations were quantitated using enzyme-linked immunosorbent assay (ELISA) kit (R & D System, Minneapolis, MN, USA) according to the manufacturer's specifications.

Statistical analysis
All experiments were carried more than three times. The cell viability data were shown as mean±SD percentage of control of three different experiments. The ELISA data were reported as means±SD of three different experiments. Paired t-;tests were used for comparisons and a value of p<0.05 was considered as significant.

	Results

TOP Abstract Materials and methods Results Discussion Acknowledgements References

 
Adenovirus efficiently transduces IB super-repressor gene into respiratory epithelial cells
The transduction efficiency of adenovirus vector in A549 cells was evaluated using a different adenovirus vector encoding ß-;galactosidase (Ad5LacZ) moi and observing the subsequent ß-;galactosidase staining. More than 90% of cells expressed ß-;galactosidase at the virus dose of 20 moi (fig. 1). To assess the adenovirus-mediated expression of IB-SR, Western blot analysis was performed with extracts of cells 48 h after infection with Ad5IB-SR. The exogenous IB-SR was heavily expressed compared to cells infected with Ad5LacZ (fig. 2a). To see the intracellular distribution of IB-SR, Western blot analysis was performed with cytoplasmic and nuclear extracts from Ad5LacZ or Ad5IB-SR transduced cells. While the endogenous IB was located mainly in the cytoplasm, some of the overexpressed IB-SR translocated to the nucleus (fig. 2b). In accordance with this observation, most cells expressed IB-SR in varying amounts both in the cytoplasm and nucleus in immunofluorescent staining (fig. 2c).

View larger version (82K): [in this window] [in a new window]   Fig. 1.— Transduction efficiency of adenovirus vector. A549 cells were infected with various doses of Ad5LacZ and stained for ß-;galactosidase 48 h after infection. a) Control; b) 5 multiples of infection (moi) Ad5LacZ; c) 10 moi; d) 20 moi; e) 50 moi; f) 100 moi.

View larger version (96K): [in this window] [in a new window]   Fig. 2.— Overexpression and subcellular localization of exogenous IB super-repressor (IB-SR) using adenoviral vectors in respiratory epithelial cells. a) Adenovirus-mediated overexpression of IB-SR. A549 and NCI-H157 cells were infected with Ad5LacZ or Ad5IB-SR at 20 multiples of infection (moi). Forty-eight hours after infection, whole cell extracts were separated by 10% sodium dodecylsulphate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, and IB was detected by rabbit polyclonal IB antibody. b) Subcellular localization of exogenous IB-SR. A549 cells were infected with Ad5LacZ or Ad5IB-SR. Cytoplasmic (C) and nuclear (N) extracts were assayed for IB by Western blot analysis 48 h after infection. c) and d) Cytoplasmic and nuclear localization of exogenous IB–SR. A549 cells were infected with c) Ad5LacZ or d) Ad5IB-SR. Immunofluorescent staining for IB was performed using IB antibody followed by rhodamine-conjugated detection antibody.

IB super-repressor is resistant to tumour necrosis factor-;-;induced degradation
Since various pro-inflammatory cytokines are produced during an inflammatory process, exogenous IB should be resistant to cytokine-induced degradation in order to block NF-B activation effectively at the site of inflammation. To investigate the stability of IB-SR, time dependent degradation of IB in response to TNF-; stimulation was determined by Western blot analysis after transduction with Ad5LacZ or Ad5IB-SR. In Ad5LacZ infected cells, endogenous IB was completely degraded in 30 min, followed by resynthesis in 60 min in both A549 and NCI-H157 cells (fig. 3a). In contrast, stimulation of Ad5IB-SR infected cells with TNF-; did not detectably decrease IB- SR protein levels (fig. 3b).

View larger version (45K): [in this window] [in a new window]   Fig. 3.— Exogenous IB super-repressor (IB-SR) is resistant to tumour necrosis factor (TNF)-;-;induced degradation. A549 and NCI-H157 cells were infected with a) Ad5LacZ or b) Ad5IB-SR at 20 multiples of infection (moi) for 48 h, and then stimulated with TNF-; (5 ng·mL–1) for 5, 30, 60, and 90 min. Whole cell extracts were assayed for IB by Western blot analysis.

Adenovirus-mediated gene transfer of IB super-repressor blocks tumour necrosis factor-;-;induced nuclear factor-B activation

View larger version (57K): [in this window] [in a new window]   Fig. 4.— Adenovirus-mediated overexpression of IB super-repressor (IB-SR) blocks tumour necrosis factor (TNF)-;-;induced activation of nuclear factor (NF)-B. a) Effect of overexpression of IB-SR on NF-B deoxyribonucleic acid (DNA) binding activity. A549 and NCI-H157 cells were infected with Ad5LacZ or Ad5IB-SR for 48 h, and then incubated for 30 min in the presence or absence of TNF-; (5 ng·mL–1). Nuclear extracts were subjected to electrophoretic mobility shift assay with B site DNA probe. b) Effect of overexpression of IB-SR on blocking of TNF-;-;induced nuclear translocation of NF-B subunit p65. Ad5LacZ or Ad5IB-SR-infected A549 cells were treated with media alone or TNF-; (5 ng·mL–1) for 30 min and then cytoplasmic and nuclear extract were prepared. Cytoplasmic (C) and nuclear (N) extracts were analysed for the presence of p65 by Western blot analysis. c) Effect of overexpression of IB-SR on subcellular localization of p65. Ad5LacZ- or Ad5IB-SR-infected A549 cells were treated with media alone (control) or TNF-; (5 ng·mL–1) for 30 min and then fixed and permeabilized for 5 min. Immunofluorescent staining for p65 was performed using anti-p65 antibody followed by a rhodamine-conjugated antibody.

Adenovirus-mediated gene transfer of IB super-repressor blocks pro-inflammatory cytokine expression

View larger version (54K): [in this window] [in a new window]   Fig. 5.— Adenovirus-mediated overexpression of IB super-repressor (IB-SR) blocks interleukin (IL)-1ß- and tumour necrosis factor (TNF)-induced pro-inflammatory cytokine expression. a) and b) Effect of exogenous IB-SR on pro-inflammatory cytokine messenger ribonucleic acid (mRNA) expression. A549 and NCI-H157 cells were infected with Ad5LacZ or Ad5IB-SR, then stimulated with a) TNF-; (5 ng·mL–1) and b) IL-1ß (5 ng·mL–1) for 4 h. IL-1ß-induced TNF-; mRNA, and TNF-;-;induced IL-;8 mRNA expressions were assayed by Northern blot analysis. c) and d) Effect of exogenous IB-SR on the production of IL-;8. Ad5LacZ- or Ad5IB-SR-infected cells were stimulated with TNF- for 18 h. Concentrations of IL-;8 in supernatant fluid were quantitated in c) A549 cells and d) NCI-H157 cells, by enzyme-linked immunosorbent assay (ELISA). : nonstimulated cells; : cells stimulated with TNF-; (5 ng·mL–1 for 18 h). Data are presented as mean±sd of three different experiments. *: p<0.05 versus Ad5LacZ.

	Discussion

TOP Abstract Materials and methods Results Discussion Acknowledgements References

The present study constructed an adenovirus vector-expressing nondegradable IB to block NF-B activation. Adenovirus vectors have advantages over other viral vector systems. Because an adenovirus does not integrate into chromosomal DNA, it poses a reduced risk for cellular transformation caused by insertional mutagenesis or activation of endogenous retroviruses. An adenovirus also has the capacity for expression of relatively large DNA inserts (up to 7 kb), the ability to be propagated easily and purified to high titres, an extremely broad host range, and the availability of a large number of vectors containing different promoters 14. In addition, in contrast to retroviral vectors, which can express transduced genes only in proliferating cells with relatively low efficiency, adenovirus vectors can express their gene products with much higher efficiency in dividing, nondividing, or slowly proliferating cells, without prolonged in vitro culture or selection 15, 16. To assay the inhibitory effect of IB on the expression of endogenous genes, and to allow biochemical analysis, it is necessary to utilize a highly efficient transfection system. Adenovirus-mediated gene transfer meets this criterion 17–20. In this study, the transduction efficiency was >90% and IB-SR was highly expressed in both A549 and NCI-H157 cells. Adenoviral gene therapy is particularly applicable to the respiratory system because of its easy accessibility by bronchoscopy or inhalation.

There is evidence that IB, which is resynthesized after degradation, can enter the nucleus and remove NF-B from DNA. The inactive NF-B-IB complex is then transported back into the cytoplasm, thereby completing a cycle of activation and inactivation of NF-B 21–23. In the present study, some of the over-expressed IB-SR translocated to the nucleus whereas the endogenous IB remained in the cytoplasm. This observation suggests that over-expressed IB-SR suppressed the function of activated NF-B in addition to sequestering NF-B in the cytoplasm.

Excessive adenovirus infection itself is cytotoxic. In addition, overexpression of IB-SR alone resulted in increased cytotoxicity in lung cancer cells 24. To evaluate whether the decreased pro-inflammatory cytokine production after transduction with Ad5IB-SR may be due to a cytotoxic effect of adenovirus infection, cell viability was assessed by MTT assay at 24 and 48 h after adenovirus infection. Neither Ad5LacZ nor Ad5IB-SR transduction decreased cell viabilities of A549 or NCI-H157 cells, up to 48 h, at a dose of 20 moi, minimizing the possibility of a cytotoxic effect of Ad5IB-SR in these experiments. This is not consistent with a previous study by Batra et al. 24, which showed a marked decrease in cell viability by Ad5IB-SR transduction. One factor that may contribute to this discrepancy is the dose of adenovirus. In contrast to 20 moi used in the present study, cells were transduced at 100 moi in the Batra et al. 24 study.

Respiratory epithelial cells are the main target of injury in various inflammatory lung diseases. In addition, respiratory epithelial cells are actively involved in initiating and maintaining inflammation by producing pro-inflammatory mediators. This inflammatory process delays the normal epithelial cell repair following lung injury, which is critical for restoration of lung function. For these reasons, suppression of the inflammatory response in respiratory epithelial cells seems to be a prerequisite for accelerating the repair process in inflammatory lung diseases. In this study, both TNF-;-;induced IL-;8 and IL-;1ß-induced TNF-; expressions were inhibited at the mRNA and protein levels in Ad5IB-SR-infected respiratory epithelial cells compared to Ad5LacZ-infected cells. Because TNF-; and IL-1ß are both chemotactic and mitogenic for lung fibroblasts and stimulate collagen synthesis by these cells 25, the inhibition of NF-B activation with Ad5IB-SR may be an effective gene therapy for various inflammatory lung diseases, by suppressing both inflammation and fibrosis. In animal studies, intravenous somatic gene transfer with IB reduced endotoxin-induced activation of inflammatory mediators and increased survival 26. However, there are many problems that need to be solved before gene therapy can be widely used in clinical settings. First, an efficient method of delivering the adenoviral vector into the respiratory epithelial cells should be developed. In addition, host immunological responses against viral vector proteins need to be modulated to allow long-term exogene expression 27, 28.

In diverse cell types, NF-B has been shown to regulate the apoptotic program, either as essential for the induction of apoptosis or, perhaps more commonly, as blockers of apoptosis. This depends on the specific cell type and the type of inducer. For example, in respiratory epithelial cells, the inhibition of NF-B activation by Ad5IB-SR made NCI-H157 cells more susceptible to TNF-;-;induced apoptosis 12, but not in A549 cells (data not shown). Therefore, TNF-;, which is released in large amounts from inflammatory cells, may induce apoptosis in cells with adenovirus-mediated overexpression of IB-SR at the regional inflammation site. This may decrease the usefulness of Ad5IB-SR as a therapeutic tool to manage the excessive inflammation. The development of specific and safe NF-B inhibitors, which does not impair NF-B dependent normal cell functions, would be necessary for the effective suppression of inflammation.

In conclusion, the present data suggest the feasibility of adenovirus-mediated overexpression of IB super-repressor to suppress the inflammatory response in respiratory epithelial cells. Because there is no evidence that adenovirus-mediated overexpression of IB super-repressor would be effective in vivo, additional studies are needed to determine the effectiveness of this approach in lung inflammation in vivo.

	Acknowledgements

TOP Abstract Materials and methods Results Discussion Acknowledgements References

 
The authors thank A.S. Baldwin (University of North Carolina, Chapel Hill, NC, USA) for providing IB-SR cDNA, and R.D. Gerard (University of Texas Southwestern Medical Center, Dallas, TX) for providing pAC-CMVpLpA and pJM17. The authors also thank S. Kim for proofreading this manuscript.

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