Host inflammatory and immune responses limit viral gene expression after administration of replication-deficient adenoviruses to the lung. The current study asks whether inducible nitric oxide synthase (iNOS) expression and peroxynitrite generation accompanied the inflammatory response following intratracheal administration of adenovirus. Pulmonary iNOS mRNA and protein were increased 2, 7, and 14 days following administration of 2 × 109plaque-forming units of recombinant adenovirus (Av1Luc1) to BALB/c mice. Adenovirus infection was associated with a marked increase in nitrotyrosine staining. Intense nitrotyrosine staining was observed in alveolar macrophages, respiratory epithelial cells, conducting airways, and alveolar spaces 2 days postinfection. Two weeks after exposure to adenovirus, nitrotyrosine staining was detected within alveolar macrophages, suggesting adenovirus enhanced the nitration of proteins that were subsequently taken up by alveolar macrophages. Western blot analysis using anti-nitrotyrosine antibody did not demonstrate accumulation of nitrated surfactant protein A (SP-A), although a small fraction of aggregated SP-A comigrated with a nitrotyrosine-positive protein. iNOS expression, peroxynitrite, and nitrotyrosine generation accompany and may contribute to inflammatory responses to adenovirus in the lung.
- lung inflammation
- inducible nitric oxide synthase
recombinant adenoviruses have been used to transfer DNAs to the respiratory epithelium (6, 8, 28, 29, 38-41, 44, 46). However, host inflammatory responses, limit the extent and duration of adenovirus-mediated gene transfer in the lung (10, 32, 42, 43,51). Administration of recombinant adenovirus to the lung causes acute inflammation and both Th1 and Th2 cell responses, leading to clearance of the infected cells and production of neutralizing antibody to the virus (42, 43). Administration of recombinant adenovirus has been shown to injure bronchiolar and alveolar epithelial cells in association with an accumulation of neutrophils and alveolar macrophages (25, 51). Ginsberg and colleagues (11,12) demonstrated that adenovirus induces the expression of several cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL)-1, and IL-6, accompanying the infiltration of the lung by alveolar macrophages and neutrophils. Our laboratory has shown that recombinant adenoviral vectors induce the expression of TNF-α, IL-1, monocyte chemoattractant protein-1, macrophage inflammatory protein-1α, IL-6, and granulocyte/macrophage colony-stimulating factor (17, 25). During the acute inflammatory phase of infection, immunostaining of surfactant proteins A and B (SP-A and SP-B, respectively) increases markedly in the airway lumen (50).
Proinflammatory cytokines TNF-α, IL-1, and interferon-γ (IFN-γ) have been reported to stimulate expression of the inducible nitric oxide synthase (iNOS) (24, 35). iNOS is stimulated during pulmonary viral infection in vivo (1, 2). The simultaneous production of nitric oxide (NO) and superoxide favors the generation of peroxynitrite, a cytotoxic oxidant (4). Peroxynitrite accompanies various inflammatory responses, and has been proposed to play an important role in mediating NO-related cellular injury (30, 33, 48). In vitro studies support the concept that peroxynitrite may damage various components of the respiratory epithelium and pulmonary surfactant (14, 15). In vitro nitration of SP-A by peroxynitrite (14) or tetranitromethane (TNM) (16) decreased its ability to aggregate and enhance the surface properties of phospholipids. Whether surfactant proteins are modified or inactivated by reactive nitrogen species following adenovirus inflammation in vivo is currently unknown.
The aim of the current study was to investigate whether pulmonary administration of adenovirus altered expression of iNOS and generated peroxynitrite. Because peroxynitrite reacts with SP-A in vitro (14), we assessed whether nitration of surfactant proteins occurred following intratracheal administration of adenovirus.
MATERIALS AND METHODS
Inbred strains of BalbCAnNCrlBR (BALB/c) mice were obtained from Charles River Laboratories (Wilmington, MA). Male and female mice of ∼20–25 g (48–60 days old) were utilized for the present experiments.
Intratracheal instillation of recombinant adenovirus.
Av1Luc1 is a replication-deficient adenoviral vector expressing a luciferase reporter transgene derived from human serotype 5 adenovirus whose detailed structure has been previously reported (45). Recombinant adenoviral vector was propagated, purified, and titered using 293 cells as previously described (23). Vector preparations were checked for the presence of replication-competent adenovirus contamination by PCR directed at E1a sequences (36), and contained <10 plaque-forming units (pfu) of E1a-containing virus per 108 pfu of vector. Adenovirus vector was administered to the respiratory tract of BALB/c mice (n = 16 each) as previously described. Briefly, mice were anesthetized by isoflurane inhalation, and the trachea was exposed by a midline skin incision. Av1Luc1 (1 × 109pfu/mouse) was administered in 80–100 μl of indicator-free Hanks' balanced salt solution (HBSS) by transtracheal injection using a 1-ml syringe and 30-gauge needle. Control mice received sham injections of equivalent amounts of virus dilution buffer (10 mM Tris, pH 7.4, 1 mM MgCl2, and 10% glycerol) in HBSS. This method results in delivery of adenovirus throughout the respiratory tract (49).
Quantitation of iNOS mRNA by Northern blot analysis.
Two, seven, and fourteen days after Av1Luc1 administration, lungs were removed. Total RNA was isolated from the mouse lung using a modification of the method of Chomzynski and Sacchi (7). Briefly, lungs were homogenized in guanidine thiocyanate buffer and extracted with acid-phenol using the Phase Lock Gel II System (5 Prime → 3 Prime, Boulder, CO) as previously described (26). RNA samples (10 μg) were electrophoresed in a 1% denaturing agarose gel and blotted onto Hybond-N filter. The blotted filters were then hybridized with a [32P]dCTP-labeled murine macrophage iNOS cDNA, which was kindly provided by Dr. Charles Lowenstein. The cDNA probe was synthesized by random-primer labeling using the Decamer labeling kit (Ambion, Austin, TX). The filters were washed at a final stringency of 0.1× sodium chloride-sodium citrate-0.1% SDS at 55°C and exposed to KODAK X-OMAT film at −70°C for 1–2 days. After autoradiography, the filters were stripped and hybridized with a [32P]dCTP-labeled mrpL32, an mRNA for one of the large ribosomal subunit proteins, as an internal control. Relative mRNA levels were quantified by scanning densitometry using Alpha Imager 2000 Analysis Software (AlphaInnotech, San Leandro, CA). Specific iNOS mRNA levels were calculated as the ratio of iNOS to mrpL32 mRNA expression.
Measurement of iNOS activity.
Lungs were excised and frozen at −80°C for later measurement of iNOS enzymatic activity. The lungs were first homogenized in HEPES buffer (pH 7.5). Conversion of l-[3H]arginine tol-[3H]citrulline was measured in the homogenates as previously described (34). Briefly, 50 μl of tissue homogenate (∼100 μg protein/sample and equal for all groups) were incubated in the presence ofl-arginine/[3H]arginine (10 μM, 5,000 Bq/tube), NADPH (1 mM), calmodulin (30 nM), tetrahydrobiopterin (5 μM) and calcium (2 mM) for 20 min in HEPES buffer. Reactions were stopped by dilution with 1 ml of ice-cold HEPES buffer, pH 5.5, 2 mM EGTA, and 2 mM EDTA. Reaction mixtures were applied to Dowex 50W (Na+ form) columns;l-[3H]citrulline was eluted and measured by scintillation counting. To measure the calcium-independent (i.e., induced) NOS activity, incubations were performed with NADPH and EGTA (5 mM) and without calcium. l-[3H]arginine was obtained from DuPont NEN. All other chemicals were purchased from Sigma (St. Louis, MO). Protein concentration was measured spectrophotometrically in 96-well plates using a Bio-Rad protein assay (Bio-Rad, Hercules, CA).
Animals were killed 2, 7, and 14 days after Av1Luc1 administration. Lungs were inflation-fixed with 4% paraformaldehyde in PBS and then immersed in the same fixative at 4°C overnight. The next day, the lungs were washed in PBS, dehydrated in ethanol, and embedded in paraffin. Tissue sections were cut at 5 μm and stained with hematoxylin and eosin for morphological analysis.
Tyrosine nitration was measured in pulmonary sections by immunohistochemistry as previously described (9). Lungs were fixed in 4% paraformaldehyde in PBS, and 5-μm sections were prepared from paraffin-embedded tissues. Endogenous peroxidase was quenched with 0.3% H2O2 in 60% methanol for 15 min. The sections were incubated overnight with 1:1,000–1:2,000 dilution of primary anti-nitrotyrosine antibody (Upstate Biotechnology, Lake Placid, NY). In control measurements, tissues were incubated with the primary antibody in the presence of 10 mM nitrotyrosine. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin-peroxidase complex. Immunostaining for SP-A and SP-B was performed as previously described (50). In each experimental group, sections were evaluated by an investigator blinded to the treatment protocol. The photomicrographs shown are representative sections (n = 5–6) for each experimental group. All panels represent 690-fold magnification.
Bronchoalveolar lavage, measurement of SP-A and nitrotyrosine by Western blotting.
Human SP-A (hSP-A; 100 μg/ml) purified from alveolar proteinosis material was incubated (25°C) with 0.5 mM TNM for 1 h in 50 mM Tris · HCl, pH 8.0, buffer. Nitrated hSP-A was separated from unreacted TNM using a PD-10 desalting column (Pharmacia Biotech, Piscataway, NJ) which was equilibrated in the same Tris buffer. Aliquots containing 100 ng of nitrated hSP-A were subjected to SDS-PAGE for Western blot analysis. To detect hSP-A, we used a rabbit anti-human SP-A antibody.
Lung surfactant was isolated from the bronchoalveolar lavage fluid of Av1Luc1-treated and control mice. The lungs were lavaged with three 1-ml aliquots of PBS. The recovered fluid (90% of the infusate) was first centrifuged at 1,000 rpm for 2 min to remove cellular debris, then at 20,000 g for 30 min at 4°C to sediment the surfactant components that were resuspended in 500 μl of 50 mM potassium phosphate buffer. Protein concentration was determined by the Lowry assay (22). SDS-PAGE of SP-A was carried out in a Schleicher & Schuell minigel apparatus using the discontinuous buffer system of Laemmli (21). All protein samples were boiled for 5 min, then centrifuged and cooled to room temperature before use. Five percent 2-mercaptoethanol was included as a reducing agent in SP-A samples. SP-A was separated on 8–16% polyacrylamide gels (Novex), and the apparent molecular masses were determined by comparison with marker proteins of known size. SP-A gels were transferred to nitrocellulose. Nonspecific protein binding sites on the nitrocellulose were blocked by incubating with 5% bovine serum albumin in Tris-buffered saline containing 0.1% (vol/vol) Tween 20 (TBS-T) for 15 min. To detect SP-A, the nitrocellulose-bound antigen was incubated overnight with (1:25,000 dilution) purified guinea pig anti-rat SP-A antibody at 4°C. To detect nitrotyrosine, nitrocellulose-bound antigen was incubated with (1:10,000 dilution) polyclonal rabbit anti-nitrotyrosine antibody overnight at 4°C. Rabbit anti-rat SP-D and rabbit anti-bovine SP-B (50) primary antisera were also utilized as previously described. After three TBS-T buffer washes (5 min each), the nitrocellulose was placed in a solution of rabbit anti-guinea pig or goat anti-rabbit immunoglobulin G (IgG), conjugated to horseradish peroxidase at 1:10,000 dilution for an additional hour. After three washes in TBS-T buffer (5 min each), the membrane was developed by enhanced chemiluminescence (Amersham).
Data are expressed as means ± SD. Statistical analysis of the data was performed by ANOVA, and P < 0.05 was considered statistically significant.
iNOS expression after Av1Luc1 administration.
Mice were killed 2, 7, and 14 days after Av1Luc1 administration. After intratracheal instillation of recombinant adenovirus, iNOS mRNA levels increased (Fig. 1). Elevated iNOS mRNA persisted 2 wk after the administration of the adenovirus. iNOS enzymatic activity in the lung, as detected by measurement of calcium-independent conversion of l-arginine tol-citrulline, was increased from 6 ± 2 fmol · mg−1 · min−1 (control) to 14 ± 2 and 21 ± 3 fmol · mg−1 · min−1 at 2 days and 7 days after Av1Luc1 administration (n = 6–8,P < 0.001; Fig. 2).
Histopathology and surfactant protein immunostaining.
In sham-treated mice, no pulmonary inflammation was observed (Fig.3). In contrast, 2 days after Av1Luc1 administration, acute inflammatory changes that were characterized by epithelial cell injury, sloughing of epithelial cells, and infiltration with macrophages and neutrophils were observed in the lungs of BALB/c mice (Fig. 4) (see also Ref.51). Rabbit polyclonal SP-A and SP-B antibodies immunostained alveolar type II, nonciliated bronchial epithelial cells and macrophages in the sham-treated mouse lung (Fig. 3). At 2 days, the acute inflammatory response was associated with marked accumulation of SP-A and SP-B in the airways and decreased staining in type II cells and bronchiolar epithelial cells (Fig. 4) (see also Ref.50). There was increased nitrotyrosine staining in extracellular material, lung interstitium, epithelium, and subendothelial tissue (Fig. 4) compared with sham-treated mouse lung in which nitrotyrosine was only detected at low levels in alveolar macrophages (Fig. 3). By 7 days, the inflammatory response in BALB/c mice consisted primarily of perivascular and peribronchial lymphocytic infiltrates (Fig. 5). Decreased intracellular staining for SP-A and SP-B was still a prominent finding, and SP-B-stained material was associated with enlarged alveolar macrophages (Fig. 5). Nitrotyrosine staining was associated with alveolar macrophages and pulmonary epithelial cells.
Two weeks after adenovirus administration, focal areas of lymphocytic infiltrates persisted in the lung parenchyma (Fig.6). At this time, SP-A was again apparent in type II cells and bronchiolar epithelial cells in a pattern typical of uninfected mice. However, SP-A and SP-B proteins remained undetectable in focal regions of chronic inflammation. At this time, most of the nitrotyrosine staining was present in macrophages (Fig. 6).
Western blot analysis of bronchoalveolar lavage surfactant of Av1Luc1-treated mice.
To determine whether extracellular surfactant was nitrated, Western blot analysis was performed on surfactant pellets from mice that were infected with high doses (4 × 109 pfu) of adenovirus (Fig. 7 A). In surfactant from Av1Luc1-infected mice, intact isoforms of SP-A (36-kDa monomer) were observed. The nitrotyrosine antibody did not bind to SP-A (36 kDa) in lavage samples from Av1Luc1-treated mice (Fig.7 A). However, some larger bands (120,000 Da) reacted with both SP-A and nitrotyrosine antibody and may represent nitrated and aggregated SP-A induced in Av1Luc1-treated lungs. These nitrated forms represent <10% of the total SP-A. In parallel Western blot experiments, neither SP-B nor SP-D stained for nitrotyrosine after Av1Luc1 exposure. Total protein analysis and Coomassie blue gel staining of bronchoalveolar lavage samples were consistent with serum protein leakage into adenoviral-infected airways. In addition, no specific nitrated protein bands could be identified in postsurfactant supernatants of infected mice (data not shown).
In control experiments (Fig. 7 B), hSP-A was nitrated with TNM, demonstrating some aggregation of hSP-A and nitration of both SP-A monomers and oligomers. The position of monomeric and dimeric SP-A was identified, and no immunoreactivity was seen with anti-nitrotyrosine antibody in the lane containing unmodified human SP-A.
Intratracheal exposure to a ΔE1-E3 replication-deficient adenoviral vector enhanced iNOS activity and iNOS mRNA in the lung. Nitrotyrosine immunostaining, a marker of peroxynitrite production, was observed in the extracellular material, respiratory epithelium, and subendothelial tissue. However, nitration of the major forms of SP-A, SP-B, and SP-D was not observed in bronchoalveolar lavage fluid from mice exposed to adenovirus.
Human alveolar and bronchial epithelial cells exhibit a low baseline level of iNOS expression (13). In pathophysiological conditions associated with the generation of proinflammatory cytokines, increased iNOS activity has been observed (5, 27, 31). Two and seven days after Av1Luc1 treatment, we have observed damage to the alveolar and bronchiolar epithelium, including sloughing of epithelial cells and infiltration with neutrophils and macrophages, in association with release of cytokines (50, 51). Increased production of various proinflammatory cytokines has been detected in adenoviral vector-related early inflammatory processes (17, 25, 49). Increased IFN-γ, IL-1β, and TNF-α were detected in early inflammatory processes, associated with increased NO production by alveolar macrophages and respiratory epithelial cells. In the present study, we detected modestly increased iNOS mRNA 2, 7, and 14 days following recombinant adenovirus infection, consistent with persistent inflammatory and immune responses to the virus. iNOS activity in the lung was also detected by measurement of calcium-independent conversion of l-arginine to l-citrulline in lung samples. Based on previous studies wherein inhibitors of NO synthase improved pulmonary function and survival in models of viral pulmonary inflammation (1, 2), it has been proposed that iNOS-derived NO or its reaction products exacerbate rather than attenuate the inflammatory process.
Both alveolar macrophage and respiratory epithelial cells release both NO and superoxide, which react at a near diffusion-limited rate (6.7 × 109M−1 · s−1) to form peroxynitrite, a potent oxidant capable of damaging the alveolar epithelium and surfactant system (3). The major product of the reaction of peroxynitrite with proteins is the addition of a nitro group in the ortho position of tyrosine to form nitrotyrosine (19). The abundance of nitrotyrosine staining observed in the adenoviral vector-treated lungs suggests that the generation of peroxynitrite occurs during adenovirus-mediated pulmonary inflammation. Indeed, nitrotyrosine has been reported in lungs of acute respiratory distress syndrome patients (15) and in acute lung injury (20). Interestingly, in the present study, nitrotyrosine was also detected in the respiratory epithelium, alveolar macrophages, and endothelium after adenovirus infection. Nitrotyrosine staining was also detected in the extracellular spaces of the lung.
Previous in vitro studies demonstrated the reaction of peroxynitrite with surfactant proteins (14); however, the physiological relevance of these findings remains unclear. Tyrosine nitration is reported to have biological effects on the structure and function of surfactant proteins in vitro. For example, nitration of SP-A inhibited its ability to enhance binding of Pneumocystis carinii to alveolar macrophages (47). Nitration of SP-A by peroxynitrite also decreased its ability to aggregate lipid (16). Peroxynitrite-induced fragmentation of purified SP-A was also observed (14). In our previous study (50) and in this study, no depletion of the SP-A pool has been observed. Adenovirus-induced SP-A and SP-B secretion is accompanied by elevated alveolar surfactant lipid pools (18), which we postulate may preserve surfactant function and host defense during the acute infection period.
In our experiments, immunostaining of SP-A colocalized with nitrotyrosine staining. However, by Western blotting analysis, only a trace amount of nitrated larger molecular mass protein was detected in surfactant from the Av1Luc1-infected mice. The identity of this protein was not precisely determined, but it may represent a small fraction of aggregated SP-A. While in the acute phase of inflammation, most of the tyrosine nitration occurred extracellularly. At 14 days, nitrotyrosine staining was noted primarily within alveolar macrophages. This finding is consistent with the hypothesis that extracellular nitrated proteins are taken up by pulmonary macrophages. SP-A enhances antibacterial and antiviral functions of alveolar macrophages (37). Therefore, potential inactivation of SP-A by reactive oxygen species might block its effect on host defense. However, the marked increase in SP-A content observed by immunostaining and the relatively low level of SP-A nitration following the adenovirus does not support the concept that the activity of SP-A is impaired.
Taken together, our results support the hypothesis that iNOS expression, peroxynitrite production, and tyrosine nitration may contribute to the pathophysiological response to adenoviral infection and adenovirus-mediated gene transfer. Currently, we have not determined the effect of inhibition or ablation of iNOS activity on the response to adenovirus. Despite enhanced iNOS expression and extensive protein nitration following adenoviral exposure, nitrated pulmonary SP-A or SP-B did not accumulate. Indeed, immunostaining of extracellular SP-A and SP-B increased considerably following adenoviral administration (Ref. 50 and present findings). Nitrated proteins accumulated in alveolar macrophages following adenoviral infection, indicating clearance of nitrated proteins. The precise identity and role of the nitrated proteins accompanying adenoviral infection of the lung remain to be discerned.
We thank Becky Gelfand and Jennifer Melzer for technical assistance, Sherri Profitt for advice on immunohistochemistry, and Ann Maher for help in preparing the manuscript.
This work was supported by the Cystic Fibrosis Foundation, Research and Development Program Center for CF Research (J. A. Whitsett), The Center for Gene Therapy of Cystic Fibrosis and Other Lung Diseases, and National Heart, Lung, and Blood Institute Grant HL-51832 (J. A. Whitsett).
Present addresses: Z. K. Zsengellér Perlmutter Laboratory Children's Hospital Medical Center, Boston, MA 02115; C. Szabó, INOTEK Corp., Beverly, MA.
Address for reprint requests and other correspondence: J. A. Whitsett, Children's Hospital Medical Center, Divisions of Neonatology and Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail:).
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- Copyright © 2001 the American Physiological Society