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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 322-329, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2001-0015OC

Gene Knockout or Pharmacological Inhibition of Poly(ADP-Ribose) Polymerase-1 Prevents Lung Inflammation in a Murine Model of Asthma

A. Hamid Boulares, Anna J. Zoltoski, Zaki A. Sherif, Puneet Jolly, Donald Massaro and Mark E. Smulson

Department of Biochemistry and Molecular Biology and Department of Medicine, Lung Laboratory, Georgetown University School of Medicine, Washington, District of Columbia

Address correspondence to: Hamid Boulares, Ph.D., Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, 1901 Perdido St., Room 5226, New Orleans, LA 70112. E-mail: hboulr{at}lsuhsc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Airway inflammation is a central feature of asthma and chronic obstructive pulmonary disease. Reactive oxygen species (ROS) contribute to inflammation by damaging DNA, which, in turn, results in the activation of poly(ADP-ribose) polymerase-1 (PARP-1) and depletion of its substrate, nicotinamide adenine dinucleotide. Here we show that prevention of PARP-1 activation protects against both ROS-induced airway epithelial cell injury in vitro and airway inflammation in vivo. H2O2 induced the generation of ROS, PARP-1 activation and concomitant nicotinamide adenine dinucleotide depletion, and release of lactate dehydrogenase in A549 human airway epithelial cells. These effects were blocked by the PARP-1 inhibitor 3-aminobenzamide (3-AB). Furthermore, 3-AB inhibited both activation of the proinflammatory transcription factor nuclear factor-{kappa}B and expression of the interleukin-8 gene induced by H2O2 in these cells. In a murine model of allergen-induced asthma, 3-AB prevented airway inflammation elicited by ovalbumin. Moreover, PARP-1 knockout mice were resistant to such ovalbumin-induced inflammation. These protective effects were associated with an inhibition of expression of the inducible nitric oxide synthase. These results implicate PARP-1 activation in airway inflammation, and suggest this enzyme as a potential target for the development of new therapeutic strategies in the treatment of asthma as well as other respiratory disorders such as chronic obstructive pulmonary disease.

Abbreviations: 3-aminobenzamide, 3-AB • bronchoalveolar lavage, BAL • chronic obstructive pulmonary disease, COPD • electrophoretic mobility shift assay, EMSA • dihydrodichlorofluorescein, H2DCF • interleukin, IL • inducible NOS, iNOS • lactate dehydrogenase, LDH • N-acetyl cysteine, NAC • nuclear factor-{kappa}B, NF-{kappa}B • nitric oxide synthase, NOS • ovalbumin, OVA • poly(ADP-ribose), PAR • PAR polymerase-1, PARP-1 • phosphate-buffered saline, PBS • reactive oxygen species, ROS • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Allergic asthma and chronic obstructive pulmonary disease (COPD) are complex respiratory disorders that affect millions of individuals worldwide. Oxidative injury plays an important role in the pathogenesis of both asthma and COPD (1–3). Both conditions are accompanied by the expression of multiple genes for inflammatory proteins, including cytokines, receptors, and adhesion molecules (4). Such gene expression is mediated, in part, by transcription factors, such as nuclear factor–{kappa}B (NF-{kappa}B), whose activation is induced by oxidative stress. In response to allergens, inflammatory cells in the airways and alveolar spaces release reactive oxygen species (ROS) (1, 2, 5). The released ROS are detrimental to the airway epithelium, with cell damage ultimately resulting in its shedding (1, 2). The ROS-induced increase in the expression of proinflammatory factors such as interleukin (IL)–8, tumor necrosis factor (TNF), RANTES, and eotaxin further promotes inflammatory cell infiltration (1, 2).

ROS are potent inducers of DNA strand breakage both in vitro and in vivo. The resulting DNA strand breaks trigger the activation of poly(ADP-ribose) polymerase-1 (PARP-1), the activity of which is dependent on binding of the enzyme to the ends of broken DNA molecules (6). PARP-1 catalyzes the covalent attachment of long branched chains of poly(ADP-ribose) (PAR), with nicotinamide adenine dinucleotide (NAD) as its substrate, to a variety of nuclear DNA-binding proteins, including PARP-1 itself. Such poly(ADP-ribosyl)ation contributes to various physiologic and pathophysiologic events that are associated with DNA strand breakage, including DNA replication, repair of DNA damage, gene expression, and apoptosis (7–9). In several pathologic situations that involve massive DNA damage, excessive activation of PARP-1 depletes cellular stores of both NAD and its precursor ATP, leading to irreversible cytotoxicity and cell death (10). Excessive PARP-1 activation induced by ROS or nitric oxide (NO) has been associated with various pathologic conditions, including energetic failure and vascular collapse in shock (11, 12), streptozotocin-induced diabetes (13), cerebral ischemia (14), glutamate neurotoxicity (15), and parkinsonism (16). Consistent with a role for PARP-1 in these conditions, PARP-1 knockout mice are resistant to their onset.

Pharmacologic inhibition of PARP-1 offers a potential approach to the treatment of such conditions. 3-Aminobenzamide (3-AB), a competitive inhibitor of PARP-1, was recently shown to reduce markedly the extent of brain damage induced by focal ischemia (17) as well as to provide long-term protection against both myocardial reperfusion injury (11, 18) and chronic colitis (19) in rodents.

We have now investigated whether pharmacologic inhibition of PARP-1 by 3-AB protects against oxidant-induced injury of airway epithelial cells in an in vitro model of lung inflammation and determined the effect of PARP-1 activation on signal transduction through NF-{kappa}B and one of its target genes, that for IL-8, in these cells. We further examined the potential protective effect of this drug as well as of PARP-1 gene disruption in an ovalbumin (OVA)-triggered mouse model of allergic asthma. Our results suggest that PARP-1 is an appropriate target for new therapeutic approaches to modulation of asthma-associated inflammation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture and Treatment
Human A549 cells (American Type Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin. The cells were incubated in the absence or presence of 3 mM 3-AB for 1 h, after which they were treated (in the continued absence or presence of 3-AB) with 500 µM H2O2. PARP-1-/- and PARP-1+/+ fibroblasts were maintained as described for A549 cells; they were treated with 200 µM H2O2 in culture medium.

Animals and Protocol for Sensitization and Challenge
Wild-type and PARP-1-/- mice (SV129 x C57BL/6) were bred in a pathogen-free facility. At 5–6 wk of age, mice (six animals per group) were sensitized with an intraperitoneal injection of 100 µg of chicken OVA (Sigma, St. Louis, MO) mixed with aluminum hydroxide (Sigma). After 10 d, the animals were subjected to intranasal challenge with 1% OVA or saline (control). Wild-type mice received an intraperitoneal injection of 3-AB (20 mg/kg) 30 min before OVA challenge. The animals were killed by CO2 asphyxiation 48 h after challenge, and their lungs were either subjected to BAL or removed and fixed with formalin. The mice used in each experiment were of the same litter or the same family.

Measurement of LDH Release
The release of LDH from cells grown in 24-well plates was assessed with a nonradioactive cytotoxicity assay kit (CytoTox96; Promega, Madison, WI). Results were expressed as a percentage of the positive control (untreated cells lysed with lysis buffer supplied with the kit).

Immunofluorescence Microscopy
Cells were fixed, permeabilized, and stained with a mouse monoclonal antibody to PAR essentially as described (7). They were then examined with a Nikon fluorescence microscope.

Immunohistochemistry
Paraffin-embedded tissue sections were subjected to removal of paraffin with xylene, dehydrated in ethanol, and then incubated in phosphate-buffered saline (PBS). Endogenous peroxidase activity was neutralized by incubation of the sections for 20 min in PBS containing 1% H2O2. After several washes with PBS, the sections were incubated for 30–60 min with 10% normal mouse serum and then for 2 h with the mouse monoclonal antibody to PAR. The sections were washed again with PBS, after which they were exposed consecutively to biotinylated horse antibodies to mouse immunoglobulin G, avidin-conjugated horseradish peroxidase, and peroxidase substrate (ABC kit; Vector Laboratories, Burlingame, CA). The sections were then counterstained with hematoxylin for 20 s, exposed to a graded series of ethanol solutions and Histoclear, covered with coverslips, and examined by light microscopy.

Assay of NAD and H2DCF Oxidation Assay for ROS
Cells incubated in six-well dishes were scraped into the culture medium, washed with PBS, and then subjected to extraction as described previously (9). The amount of NAD in the extract was determined with an enzymatic cycling assay as described (9). The assay for ROS was performed as described (20).

Preparation of Nuclear Extracts, Electrophoretic Mobility Shift Assay, Transient Transfection, and Luciferase Assay
Nuclear extracts were prepared and electrophoretic mobility shift assay (EMSA) analysis of DNA-binding activity was performed as described (20). A reporter plasmid containing the luciferase gene under the control of a promoter containing eight repeats of the DNA consensus sequence for NF-{kappa}B was introduced into A549 cells by transient transfection with the use of Transit I (Panvera, Madison, WI). Luciferase activity was determined essentially as described (9).

Northern Blot Analysis
Total RNA was extracted with the use of RNeasy reagent (Qiagen, Valencia, CA), and Northern blot analysis, as well as the preparation of the human IL-8 cDNA probe, were performed essentially as described (21).

Immunoblot Analysis
After lung removal, lung homogenates were prepared sonication in lysis buffer, after which a portion (50 µg of protein) of each homogenate was fractionated by SDS-polyacrylamide gel electrophoresis on a 4–20% gradient gel, and the separated proteins were transferred to a nitrocellulose filter as previously described (7). The filter was probed with antibodies to iNOS (BD Biosciences, Boston, MA) or ß-actin (Santa Cruz Biotech, Santa Cruz, CA). Immune complexes were detected with appropriate secondary antibodies and chemiluminescence reagents (Pierce, Rockford, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Protective Effect of 3-AB Against H2O2-Induced Injury in Airway Epithelial Cells
To determine whether inhibition of PARP-1 protects airway epithelial cells against oxidant-mediated injury, we examined the effect of 3-AB on H2O2-induced cytotoxicity in the human airway epithelial cell line A549. We assessed cell injury by measuring the release into the medium of lactate dehydrogenase (LDH), a marker of cell injury that also reflects the level of inflammation. Exposure of A549 cells to H2O2 resulted in a time-dependent increase in LDH release, and this effect was markedly inhibited (by > 70% at 12 h) by 3-AB (Figure 1A). The PARP-1 inhibitor 3-AB thus protected the airway epithelial cells against H2O2-induced injury.



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  		Figure 1. Protection by 3-AB against H2O2-induced LDH release and NAD depletion in A549 cells. (A) Cells were incubated in the absence or presence of 3-AB and then treated with H2O2 for 6 or 12 h. The activity of LDH in the medium was then measured. Data are means ± SD of values from three wells (each well assayed in duplicate) from a representative experiment. *Difference from untreated cells, P < 0.05; +Difference from cells treated with H2O2 for 6 h, P < 0.05; #Difference from cells treated with H2O2 for 12 h, P < 0.05 (Student's t test). (B) Cells were treated for 30 min with H2O2 in the absence (middle panels) or presence (right panels) of 3-AB and were then subjected to immunofluorescence staining with antibodies to PAR (top panels) and staining with Hoechst 33342 (bottom panels). Control cells were incubated with 3-AB alone (left panels) (magnification: x400). (C) Cells were incubated in the absence (open circles) or presence (closed circles) of 3-AB and then treated with H2O2 for the indicated times. Cell extracts were then assayed for NAD. Data are expressed as a percentage of the control value (time zero value for cells not exposed to 3-AB) and are means ± SD of triplicates from a representative experiment. *Difference from untreated cells, P < 0.05 (Student's t test).

 
To confirm that H2O2-induced cell injury was accompanied by activation of PARP-1, we examined PAR synthesis in treated cells by immunofluorescence staining with antibodies to PAR. Marked immunoreactivity, indicative of PARP-1 activity, was apparent in the nuclei of H2O2-treated cells (Figure 1B). Furthermore, this H2O2-induced increase in PAR synthesis was reduced to undetectable levels in the presence of 3-AB.

The excessive activation of PARP-1 that occurs in response to DNA strand breakage induced by oxidants (such as H2O2) results in the depletion of intracellular NAD, which is thought to be primarily responsible for the cell death that follows such lesions (22). Exposure of A549 cells to H2O2 resulted in a pronounced time-dependent decrease in the intracellular abundance of NAD that was apparent as early as 15 min (Figure 1C). However, the NAD concentration of H2O2-treated cells also incubated in the presence of 3-AB remained essentially unchanged. These results suggest that 3-AB protects A549 cells against oxidative injury at least in part by preventing PARP-1 activation and consequent NAD depletion.

Inhibition by 3-AB of ROS Generation in H2O2-Treated A549 Cells
The mechanism of the protective effect of 3-AB against oxidant-mediated injury in A549 cells was further investigated by examining whether this drug affected the generation of ROS in H2O2-treated cells. The production of ROS was monitored with the probe dihydrodichlorofluorescein (H2DCF). Treatment of A549 cells with H2O2 resulted in marked oxidation of H2DCF to the fluorescent DCF, indicating the generation of large amounts of ROS (Figure 2). Furthermore, this effect of H2O2 was inhibited by 3-AB. As a positive control, we showed that the antioxidant N-acetylcysteine (NAC) completely blocked the H2O2-induced oxidation of H2DCF. These results thus suggested that 3-AB exerts an antioxidant effect by inhibiting poly(ADP-ribosyl)-ation and depletion of NAD and ATP.



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  		Figure 2. Effect of 3-AB on ROS generation in H2O2-treated A549 cells. Cells were incubated in the absence or presence of 3-AB, loaded with H2DCF, washed, and then incubated for 1 h with or without H2O2. The oxidation of H2DCF was assessed with a fluorometer. As a positive control for antioxidant activity, cells were incubated with 3 mM NAC before and during H2O2 exposure. Data are expressed in arbitrary units (fluorescence) and are means ± SD of quadruplicate values from a representative experiment. *Difference from untreated cells, P < 0.05; #difference from cells treated with H2O2, P < 0.05 (Student's t test).

 
Inhibition by 3-AB of H2O2-Induced Activation of NF-{kappa}B and IL-8 Gene Expression in A549 Cells
NF-{kappa}B regulates the expression of many genes whose products contribute to inflammation (23). H2O2 has been suggested to function as a second messenger in NF-{kappa}B signaling (24). Treatment of A549 cells with H2O2 resulted in a marked increase in the DNA-binding activity of NF-{kappa}B as assessed by EMSA, and this effect was markedly inhibited (by > 75% as determined by densitometry) by 3-AB (Figure 3A). The specificity of NF-{kappa}B DNA binding activity was confirmed by supershifting analysis using antibodies to the p65 subunit of NF-{kappa}B (data not shown). The extent of inhibition of this effect of H2O2 by 3-AB was similar to that observed with NAC, suggesting that 3-AB modulates NF-{kappa}B activation, at least in part, by interfering with the oxidant signal that activates this transcription factor. Furthermore, in A549 cells transiently transfected with a plasmid that contains a luciferase reporter gene under the control of an NF-{kappa}B–dependent promoter, treatment with H2O2 induced a pronounced increase in luciferase activity (Figure 3B). This effect of H2O2 was markedly inhibited (by > 50%) by 3-AB, consistent with the level of inhibition by 3-AB of the H2O2-induced increase in the DNA-binding activity of NF-{kappa}B (Figure 3A). We confirmed the role of PARP-1 in the effect of H2O2 on NF-{kappa}B–mediated gene expression by subjecting fibroblasts derived from PARP-1 knockout (PARP-1-/-) and wild-type (PARP-1+/+) mice to similar analysis. The H2O2-induced increase in luciferase activity was greatly reduced in PARP-1-/- cells compared with that apparent in PARP-1+/+ cells (Figure 3C).



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  		Figure 3. Effects of PARP-1 inhibition on H2O2-induced NF-{kappa}B activation and IL-8 gene expression. (A) A549 cells were incubated in the absence or presence of 3-AB and then treated with H2O2 for 90 min. Nuclear extracts were prepared and assayed for the DNA-binding activity of NF-{kappa}B by EMSA. As a positive control for antioxidant activity, cells were incubated with 3 mM NAC before and during H2O2 exposure. The positions of NF-{kappa}B–probe complexes are indicated. (B) A549 cells were transiently transfected with a luciferase reporter plasmid for NF-{kappa}B activity. The cells were treated with H2O2 for 12 h in the absence or presence of 3-AB, after which cell extracts were assayed for luciferase activity. (C) PARP-1-/- and PARP-1+/+ fibroblasts were transiently transfected with the luciferase reporter plasmid for NF-{kappa}B activity. They were then incubated in the absence or presence of H2O2 for 12 h, after which cell extracts were assayed for luciferase activity. Data in B and C are expressed in arbitrary units and are means ± SD of triplicate values from representative experiments; *difference from untreated cells, P < 0.05; #difference from cells treated with H2O2, P < 0.05 (Student's t test). (D) A549 cells were incubated in the absence or presence of 3-AB and then treated with H2O2 for 6 h. Total RNA was then isolated from the cells and subjected to Northern blot analysis with a 32P-labeled human IL-8 cDNA probe (top panel). The gel was also stained with ethidium bromide to confirm equal loading of samples (bottom panel).

 
Expression of the IL-8 gene is regulated by NF-{kappa}B. IL-8 plays an important role in asthma as well as in COPD-associated lung inflammation, at least in part, by inducing the chemotaxis of polymorphonuclear leukocytes (25). Northern blot analysis revealed that treatment of A549 cells with H2O2 induced a pronounced increase in the abundance of IL-8 mRNA, and this effect was almost completely inhibited by 3-AB (Figure 3D). The inhibition of PARP-1 activity thus not only protects against H2O2-mediated cell injury, but also may prevent exacerbation of inflammation by blocking the production of IL-8 through inhibition of NF-{kappa}B activation.

PARP-1 Inhibition by 3-AB or Gene Knockout Reduces Inflammatory Cell Infiltration and PAR Synthesis in Airways of OVA-Challenged Mice
The OVA-triggered mouse model of allergic asthma exhibits many of the features of human asthma, including airway hyperresponsiveness and inflammation as well as increased expression of proinflammatory cytokines (26). This model also manifests inflammation in alveoli and bronchi (see Figure 4C) similar to that apparent in COPD. To determine whether the protective effect of inhibition of poly(ADP-ribosyl)ation by 3-AB observed with airway epithelial cells in vitro was also manifest in this animal model, we examined the effects of 3-AB treatment on OVA-induced inflammation. Mice that had been sensitized to OVA were challenged intranasally either with OVA or with saline as a control. Forty-eight hours after challenge, the mice were killed and their lungs were either subjected to bronchoalveolar lavage (BAL) or removed and fixed with formalin. OVA challenge induced a marked increase in the number of inflammatory cells in BAL fluid, and this effect was greatly reduced by intraperitoneal injection of 3-AB (20 mg per kilogram of body mass) 30 min before challenge (Figure 4A). Histologic examination of lung sections also revealed pronounced infiltration of inflammatory cells into the bronchi and alveoli of OVA-challenged mice, and, again, this infiltration was substantially reduced by 3-AB (Figure 4B-D). We also subjected PARP-1-/- mice to the same protocol of OVA sensitization and intranasal challenge. The number of inflammatory cells present in BAL fluid after OVA challenge was greatly reduced in PARP-1-/- mice compared with wild-type controls (Figure 4A). Histologic examination of lung sections also revealed a pronounced reduction in the extent of infiltration of inflammatory cells into the airways of OVA-challenged PARP-1–/– mice (Figures 4E and 4F). These results confirm that the protective effect of 3-AB in vivo is attributable to its inhibition of PARP-1, and they are consistent with the ability of 3-AB to inhibit indirectly the activation of NF-{kappa}B and the expression of IL-8 in A549 cells. PARP-1 inhibition by gene deletion or by 3-AB treatment exhibited no effect on anti-OVA IgG levels in sera from the different mice as assessed by ELISA; thus, the protective effect of PARP-1 inhibition against OVA-induced lung inflammation, apparently, was not the result of a decrease in the immune response to OVA challenge (data not shown).



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  		Figure 4. Effect of PARP-1 inhibition on inflammatory cell infiltration in a murine model of asthma. (A) Wild-type or PARP-1–/– mice were sensitized to OVA and then subjected to an intranasal challenge with OVA or saline vehicle (control). Wild-type mice received an intraperitoneal injection of 3-AB or vehicle before OVA challenge. Cells in BAL fluid were subsequently stained with trypan blue and counted with a hemocytometer. Data are means ± SD of values from six mice per group. *Difference from unchallenged mice, P < 0.05; #difference from wild-type mice challenged with OVA, P < 0.05 (Student's t test). (B–F) Fixed lungs from wild-type mice challenged with saline (B), challenged with OVA (C), or treated with 3-AB and then challenged with OVA (D) as well as those from PARP-1-/- mice challenged with saline (E) or with OVA (F) were sectioned, stained with hematoxylin and eosin, and observed by light microscopy (magnification: x100). Arrows indicate sites of inflammatory cell infiltration. The results are representative of at least three experiments performed at different times.

 
We next examined whether PARP-1 activation was apparent in the airways of OVA-challenged mice by subjecting lung sections to immunohistochemical analysis with antibodies to PAR. Indeed, PARP-1 was highly activated in the airways of OVA-challenged mice (Figure 5B) compared with the level of activity apparent in the airways of saline-challenged animals (Figure 5A). This increased level of PARP-1 activity is indicative of a substantial extent of DNA strand breakage. Such DNA strand breaks might be the consequence of the generation of large amounts of oxidants by both inflammatory cells and epithelial cells. PARP-1 inhibition by prior treatment of mice with 3-AB or by gene disruption markedly reduced the extent of OVA-induced PAR staining (Figures 5C–5E), indicating a relation between PARP-1 activation and cell infiltration. Thus, overall, the results obtained with the animal model of asthma are consistent with those observed in the cell culture system.



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  		Figure 5. PAR synthesis induced in the lungs of OVA-challenged mice and its inhibition by 3-AB or PARP-1 gene disruption. OVA-sensitized wild-type mice were challenged with saline (A) or OVA (B) or were treated with 3-AB before OVA challenge (C). OVA-sensitized PARP-1-/- mice were challenged with saline (D) or OVA (E). Lung sections were then prepared and subjected to immunohistochemical staining (brown reaction product) with antibodies to PAR (magnification: x100). The results are representative of at least three experiments performed at different times.

 
PARP-1 Inhibition by 3-AB or Gene Knockout Reduces iNOS Expression in the Lungs of OVA-Challenged Mice
The inducible form of NO synthase (iNOS), its product (NO), and the by-product peroxynitrite (ONOO-) contribute to the pathogenesis of asthma and COPD (2). Mice deficient in iNOS are thus resistant to OVA-induced inflammation (27). Furthermore, ONOO- is a potent activator of PARP-1 and, through its activation of this enzyme, is thought to participate in cell and tissue injury associated with cerebral ischemia (14), glutamate neurotoxicity (15), and parkinsonism (16). Other studies have also implicated PARP-1 in signal transduction upstream of iNOS (19, 28, 29). We examined whether the protective effects of 3-AB and PARP-1 gene disruption against OVA-induced inflammation were associated with a decrease in iNOS expression. Immunoblot analysis of lung extracts revealed that the amount of iNOS was substantially increased by OVA challenge in wild-type mice (Figure 6). This effect was blocked by prior treatment of wild-type mice with 3-AB and was not apparent in PARP-1-/- mice, consistent with the fact that the iNOS gene is regulated by NF-{kappa}B.



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  		Figure 6. Effects of 3-AB or PARP-1 gene disruption on iNOS expression induced by OVA challenge. OVA-sensitized mice of the indicated PARP-1 genotypes were challenged with saline or OVA, with or without prior injection of 3-AB. Lung tissue was subsequently subjected to immunoblot analysis with antibodies to iNOS or to ß-actin (control). The results are representative of at least three experiments performed at different times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Oxidative injury plays an important role in the inflammation observed both in individuals with asthma or COPD and in animal models of these conditions (1–3). Oxidants such as H2O2 and NO are generated in large amounts during an asthma attack and serve as indicators of the severity of such attacks (30). They are also associated with inflammation in individuals with COPD (3).

PARP-1 and its activation by DNA strand breaks have been closely associated with numerous pathologic conditions in which oxidative stress has been implicated. These conditions include ischemia, parkinsonism, stroke, diabetes, glutamate neurotoxicity, and shock (11–13, 15, 16, 31). PARP-1-/- animals are protected from such conditions because, although they manifest a similar extent of DNA damage as do wild-type animals, their intracellular energy stores are not depleted as a result of excessive activation of PARP-1. We now provide evidence that PARP-1 plays an important role in asthma-associated inflammation and may therefore represent a new therapeutic target for modulating the course of inflammation in individuals with this disease.

We have used both a cell culture system and an animal model to investigate the role of PARP-1 in airway inflammation. The PARP-1 inhibitor 3-AB protected A549 human airway epithelial cells from oxidant-mediated cell damage. Inhibition of PARP-1 by 3-AB also prevented the H2O2-induced depletion of intracellular NAD in these cells. Depletion of NAD results in a cellular energy crisis that is closely associated with cell death (10). We also detected pronounced activation of PARP-1 in lung sections of OVA-challenged mice. This observation is indicative of substantial DNA damage in the lung cells, which may be the result of oxidative stress. The ability of 3-AB to reduce the level of PARP-1 activation in the lungs of OVA-challenged mice is consistent with the similar effect of this agent in H2O2-treated epithelial cells, as well as with the antioxidant action of 3-AB observed in other cell systems (31–33). We confirmed the role of PARP-1 in the pathogenesis of asthma by showing that PARP-1-/- mice are resistant to the induction of this condition. The protective effect of PARP-1 inhibition against OVA-induced lung inflammation, apparently, was not the result of a decrease in the immune response to OVA challenge (data not shown).

We and others have recently associated NAD depletion with perturbation of mitochondrial function and integrity (9, 34, 35). Mitochondria presumably attempt to compensate for the rapid loss of NAD caused by excessive poly(ADP-ribosyl)ation by increasing the rate of respiration (36), which, in turn, results in an increase in the generation of ROS, more DNA damage, and further activation of PARP-1. We have shown that this cross-talk between PARP-1 and mitochondria creates an amplification loop that contributes to cell death induced by the proinflammatory cytokine TNF (9). This cross-talk is interrupted when PARP-1 activation is inhibited by 3-AB. The ability of 3-AB to protect against TNF-induced cell death (9) might also be related to the protection afforded by this drug against proinflammatory events in asthma, given that TNF is thought to be a major mediator of inflammation in this disease.

The protective effect of inhibition of poly(ADP-ribosyl)ation by 3-AB or by PARP-1 gene disruption is likely also related to the observed inhibition of NF-{kappa}B activation. ROS play a pivotal role in mediating the activation of NF-{kappa}B (24), and NF-{kappa}B is implicated in the pathogenesis of asthma and lung epithelial cell injury (23). The activity of NF-{kappa}B is thus increased in the airways of patients with asthma (37). A role for NF-{kappa}B in promoting airway hyperresponsiveness and allergic pulmonary inflammation has also been demonstrated by the observation that NF-{kappa}B (c-Rel)–deficient mice are resistant to OVA-triggered asthma (38). These various observations have led to the selection of NF-{kappa}B as a target for the development of new anti-inflammatory drugs for asthma.

The iNOS enzyme is thought to contribute to the pathogenesis of asthma by mediating the associated marked increase in NO synthesis (39). Both 3-AB and PARP-1 gene disruption each completely blocked the OVA-induced accumulation of iNOS, an effect that is likely important in the observed protection against lung inflammation. Given that iNOS gene expression is mediated, in part, by NF-{kappa}B, the inhibition of iNOS accumulation observed in vivo is consistent with the inhibitory effects of 3-AB and PARP-1 gene disruption on NF-{kappa}B–mediated gene expression in airway epithelial cells in vitro. Inhibition of PARP-1 by auto-poly(ADP-ribosyl)ation was recently shown to result in an increase in the DNA-binding activity of the p50 subunit of NF-{kappa}B in HeLa cells (40).

IL-8 is a potent chemoattractant for neutrophils and plays an important role in the recruitment of these cells to sites of inflammation both in asthma and in COPD. The expression of this cytokine is increased by oxidative stress (23). The inhibition by 3-AB or by PARP-1 gene disruption of inflammatory cell infiltration into the airways of OVA-challenged mice is thus consistent with their inhibitory effects on H2O2-induced NF-{kappa}B activation and IL-8 gene expression in cultured airway epithelial cells. Inhibition of PARP-1 may therefore prevent cell injury in vivo both by preserving NAD concentrations and by blocking the infiltration of harmful inflammatory cells into the lungs. This notion might explain the long-lasting protective effect of 3-AB (48 h) after OVA challenge.

Several recent animal studies have shown that 3-AB protects against the deleterious morphologic and functional consequences of myocardial ischemia (18), prevents intestinal mucosal barrier dysfunction after mesenteric ischemia (41), attenuates cerebral vasospasm after subarachnoid hemorrhage (42), and reduces inflammation in a model of chronic colitis (19). It is possible that 3-AB possesses additional properties that contribute to its protective effects. Benzamides, from which 3-AB is derived, have recently been suggested to harbor antioxidant and anti-inflammatory properties (43). Inhibitors of PARP-1, however, may prove effective, either alone or in combination with glucocorticoids or other asthma or COPD drugs, in the treatment of these common debilitating conditions.


   Acknowledgments
 
The authors thank Z.-Q. Wang for providing the PARP-1-/- mice, C. Giardina for the NF-{kappa}B–luciferase plasmid, and S. Hassan for help in establishing the murine asthma model. This work was supported in part by grants from the National Cancer Institute (PO1CA-74175 and CA25344), the U.S. Air Force Office of Scientific Research (AF-F49620-98-1-042), the U.S. Army Medical Research and Development Command (DAMD17-95-C-5001) (to M.E.S.), and the National Institutes of Health (HL-20366) (to D.M.).

Received in original form December 27, 2001

Received in final form October 8, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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