Introduction

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Cystic fibrosis (CF) is the most common lethal inherited disease of Caucasians (1). It is characterized by decreased Cl permeability in epithelial tissues; however, the majority of patients (95%) die from chronic respiratory disease in their early adult life (2). The discovery of CF transmembrane conductance regulator (CFTR) and the recognition that the cellular defect can be complemented by replacement of a normal gene in CF airway cells has opened the door to gene transfer as a treatment for CF (3, 4). Recombinant adenoviral vectors (Ad) are currently being studied extensively as pulmonary gene transfer vectors for CF. Cellular and humoral immune responses to Ad have emerged as significant problems (5).

There is evidence that the cellular immune response is due in part to the expression of adenoviral proteins that are presented via major histocompatibility complex class I molecules and result in a cytotoxic T lymphocyte response in the transgene-expressing cells (6). In vitro and in vivo data suggest that psoralen-inactivated and ultraviolet light (UV)-inactivated recombinant adenovirus at high doses may be proinflammatory in the absence of gene expression. This may be due to early cellular responses to viral particle entry, to specific components of the viral capsid, or to the viral particle load (8, 9). In mice, inactivated Ad or incomplete Ad particles cause dose-dependent pulmonary inflammation similar to live recombinant Ad 6 d after intratracheal instillation (9). Inflammation caused by persistent bacterial infection (10) and the presence of bacterial endotoxins or bacterial CpG motifs (cytosine-guanine sequences) (11) in the airways of patients with CF may present a further impediment to the successful delivery, infection, and expression of CFTR. Of note, McElvaney and Crystal found increased interleukin (IL)-6 in bronchoalveolar lavage fluid (BALF) in patients with CF, compared with non-CF subjects after treatment with Ad (12).

Analysis of BALF from CF patients as young as 1 mo of age has demonstrated airway inflammation, documented by the presence of neutrophils, elastase activity, IL-1, and IL-8 in the CF BALF (13, 14). Nearly 70% of patients with CF are infected with Pseudomonas aeruginosa (15, 16). Endotoxins are lipopolysaccharide (LPS) components of the cell wall of gram-negative bacteria such as P. aeruginosa, and LPS is abundant in the lungs of CF patients chronically infected with these organisms. LPS triggers release of proinflammatory cytokines from alveolar macrophages and epithelial cells, leading to recruitment of neutrophils to the lung. Free LPS is toxic to lung cells in vitro at concentrations of 1,000 endotoxin units (EU)/ml (8) and there is evidence in vitro that the presence of LPS in the culture medium leads to an enhancement of the toxicity of entry-competent Ad (8). It is postulated that in the presence of Ad, entry of LPS into cells via endosomal pathways is enhanced. Thus, in CF patients with persistent gram-negative bacterial infections, the addition of Ad could lead to increased epithelial damage.

It has been known for more than a decade that C3H/ HeJ endotoxin-resistant (RES) mice are hyporesponsive to LPS (17). A closely related mouse strain, C3HeB/FeJ, exhibits a normal response in that these mice are sensitive (SEN) to LPS (18). The RES mice have an autosomal gene defect (the lps mutation) that results in an inability of these mice to produce significant quantities of tumor necrosis factor (TNF)- in response to LPS. Inhalation studies performed in our laboratory have established the dose-response curves and time course for the inflammatory response to inhaled LPS in the RES and SEN mice (18). Within 1 h of the end of a 4-h LPS inhalation exposure there is evidence of airway constriction and profound neutrophilia. BALF neutrophils, proinflammatory cytokines, and pulmonary function responses demonstrate a clear dose-response relationship over the range from 0.1 to 10 µg/m³ airborne concentrations (18). At concentrations of 3 to 50 µg/m³ there is a 100-fold increased responsiveness of the SEN mice to inhaled LPS-containing aerosols over the response of the RES mice (20). Dosimetry calculations suggest that 4 h exposure of the mice to 10 µg/m³ will yield a lung burden of about 200 EU or 10,000 EU/kg.

The overall goal of this research was to characterize early events associated with administration of recombinant Ad using an in vivo model. Such events may play a role in the subsequent immune response to vector-mediated gene transfer and influence the duration of gene expression. Experiments were performed in C3HeB/FeJ and C3H/HeJ mouse strains to compare Ad-induced inflammation in strains that have genetically based differential inflammatory responses. The pattern of response between these strains for Ad- and LPS-induced inflammation could reveal a common mechanism of early cytokine signaling and neutrophil recruitment. We sought to determine whether acute inflammation may be associated with the early events of gene transfer with Ad (i.e., viral entry) that precede the expression of any viral or therapeutic genes. Two hypotheses were tested using the two-strain C3H murine model. First, we hypothesized that in vivo Ad administration in the presence of LPS in the airways would induce greater toxicity than Ad without LPS pretreatment. Second, we hypothesized that Ad-induced early inflammatory responses could be blocked pharmacologically by pretreatment with a synthetic glucocorticoid immunosuppressant drug (dexamethasone [DEX]) or with methylxanthine derivatives, pentoxifylline (PTX) and lisofylline (LSF), that inhibit transcription of TNF- message (21, 22). These studies tested treatment regimens that could be used to improve the effectiveness of Ad-mediated gene transfer.

	Materials and Methods

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Mice

Male C3H/HeJ and C3HeB/FeJ mice were purchased from Jackson Laboratories (Bar Harbor, ME) at 5 wk of age. They were housed in the American Association for the Accreditation of Laboratory Animal Care-accredited vivarium of the University of Iowa Inhalation Toxicology Facility in polypropylene fiber-covered cages in high-efficiency particulate air (HEPA)-filtered Thoren caging units and quarantined 8 d after receipt and before use. Two mice from each shipment were necropsied as sentinels and tested for infection before enrolling the rest of the mice in the study. They were supplied with food (sterile Teklad 5% stock diet; Harlan, Madison, WI) and water ad libitum, and maintained on a 12-h light-dark cycle. All protocols were approved by the Institutional Animal Care and Use Committee and procedures conformed to the NIH Guide for the Care and Use of Laboratory Animals. Upon receipt, each mouse was instrumented with an identifying microchip injected subcutaneously. Identification was verified at each step of the protocol (Mini Tracker; Avid, Inc., Norco, CA).

Sham Exposure

Control mice were handled identically to treated animals except that the inhalation exposure was to nebulized sterile, pyrogen-free (pf) saline (0.9% NaCl; USP Baxter Healthcare Corp., Deerfield, IL) matched to the generation rate of LPS solutions. The instillation at time 0 h was 100 µl sterile, pf buffered saline (University of Iowa Tissue Culture/Hybridoma Facility, Iowa City, IA). Totally naive mice housed under the same conditions as experimental animals served as sentinel controls.

Experimental Design

The study was designed in three phases: the Ad time-course study, the Ad and LPS additivity studies, and the therapeutic intervention studies. The Ad time-course study was performed in one experimental session such that all Ad instillations and controls occurred together. The Ad and LPS additivity studies were conducted with the LPS and Ad + LPS exposures to SEN and RES mice performed together for each LPS-exposure concentration. Intervention experiments were conducted simultaneously on groups matched for therapeutic drug and LPS exposure level. The exposure protocol for these studies is illustrated in Figure 1. The time at which the Ad instillation occurred was designated as 0 h. Mice were placed into the exposure chamber at 3 h to acclimate. One hour later, the 4-h LPS inhalation was begun. Halfway through that period, mice were briefly removed from the chamber for Ad instillation. After completion of the 4-h LPS exposure, mice were returned to cages, where food and water were available, to await bronchoalveolar lavage (BAL) and necropsy. In this protocol, mice treated with LPS only were sham-instilled with buffered saline. Mice treated with Ad alone were sham-exposed to saline aerosols. Sham-treated animals were both sham-instilled and sham-aerosol exposed.

View larger version (10K): [in this window] [in a new window]   Figure 1.   Exposure protocol for experiments investigating inflammation of Ad and LPS. Mice received 4 h inhalation exposure to LPS with instillation of 1010 Ad particles 2 h into the exposure (time = 0 h). Mice were necropsied 3 h after Ad dosing (1 h after the end of LPS exposure). In the intervention studies shown below the timeline, DEX was administered at 24 and 3 h whereas PTX and LSF were given at 3 and 0 h.

Adenoviral Vectors

Ad were obtained from the University of Iowa Gene Transfer Vector Core and included Ad2.CMV.Gal that was used for the Ad time-course study and Ad5.RSV.lacZ used for all subsequent experiments. The viruses were grown, purified in phosphate-buffered saline (PBS), and titered as described previously (23). The preparations were next inactivated by treatment with 8-methoxypsoralen and intense UV irradiation as reported by Cotten and associates (24). It has been demonstrated previously that this method of inactivation effectively blocks production of messenger RNA and protein by replication-defective adenovirus (25). In our control experiments, the psoralen-inactivated particles were verified as incapable of directing -galactosidase production in cultured cells by reverse transcriptase-polymerase chain reaction, X-Gal histochemistry, and galactolight assays. It is common for viral preparations to be contaminated with endotoxin concentrations as high as 30 EU/ml (P. S. Thorne, unpublished data). Therefore, the endotoxin content of the Ad preparations was tested as described below, and preparations selected for use in these studies had contamination less than 0.05 EU/ml.

Ad Instillation Exposure

Mice were lightly anesthetized under methoxyflurane vapor (Metofane; Pitman-Moore, Mundelein, IL), and 100 µl of inactivated Ad at 10¹¹ particles/ml (viral load 10¹⁰ particles/mouse) were instilled intratracheally using a 25-gauge cannula (Critkon, Tampa, FL) and a tuberculin syringe under fiberoptic illumination. The volume delivered was measured using an Eppendorf micropipetter before being loaded into the syringe. Mice were allowed to recover from the anesthetic before being reintroduced to the exposure chamber.

LPS Inhalation Exposure

All inhalation exposures were carried out in the Inhalation Toxicology Facility. Mice were placed in a 40-liter glass whole-body exposure chamber and exposed, by inhalation, to LPS at concentrations ranging from 0.076 to 6.1 µg/m³. Aerosols of LPS were generated from solutions of purified Escherichia coli 0111:B4 LPS in sterile, pf saline using a glass Pitt #1 nebulizer operated at 101.5 kPa-gauge pressure and supplied via a precision syringe pump (Harvard Apparatus, Cambridge, MA) (26). The generation system was supplied with temperature- and humidity-controlled HEPA-filtered air.

Exposure Quantitation

The airborne concentrations of the LPS aerosols were monitored through sampling and analysis of LPS, real-time aerosol monitoring, and aerosol size analysis. Air samples were collected using 47-mm closed-face in-line cassettes with glass fiber filters rated for 99.99% collection of 0.3-µm particles (Gelman Sciences, Ann Arbor, MI). Sampling flow rate was monitored using rotometers calibrated with a Gillibrator flow meter (Gillian Instrument Corp., Wayne, NJ). A minimum of four integrated 15-min samples and at least one laboratory blank were used for each inhalation exposure to determine the endotoxin concentration. Real-time monitoring of the aerosol (DataRAM; MIE, Inc., Billerica, MA) was performed in some experiments to insure stability of the aerosol. Aerosol size analysis was performed using an Aerodynamic Particle Sizer (APS-33B; TSI, Inc., St. Paul, MN) operated at a total flow of 5 liters/min, with sheath flow at 4 liters/min and with a 1:1,000 aerosol dilution (Aerosol Diluter; TSI, Inc.). A seven-stage Mercer cascade impactor (In-Tox, Inc., Albuquerque, NM) operated at 2.0 liters/min was used for independent determination of the aerosol size distribution. Mass median aerodynamic diameter of 1.2 µm and a geometric standard deviation of 1.8 were determined based on sampling with the cascade impactor. The count median aerodynamic diameter determined using the aerodynamic particle sizer was less than 1.0 µm.

Necropsy and BAL

At 3 or 24 h after instillation, mice were killed by cervical dislocation under methoxyflurane anesthesia and BALF was collected by washing the lungs three times, 1 ml/lavage with sterile, pf saline under a pressure head of 25 cm. The BALF was centrifuged and the pellet was resuspended in Cellogro (Fischer Scientific, Inc., Itaska, IL) and used to assess total cells per milliliter by hemocytometer and differential cell counts by light microscopy after cytospinning and Diff-Quik staining (Baxter Scientific Products, McGaw Park, IL). The lavage supernatants were split into 150-µl aliquots and frozen at 80°C for later cytokine analysis. The lungs of some mice from each group were not lavaged but rather were perfused with Karnofsky's fixative (1% formaldehyde, 2.5% glutaraldehyde, pH 7.2, in NaH₂PO₄-Na₂HPO₄ buffer) and processed for histopathologic study.

Endotoxin Assay

Sputum samples were weighed and aliquots were disbursed in 1.0 ml of pf water by aggressive vortexing. Suspensions were then diluted 1:1,000, 1:10,000, and 1:100,000 in pf water and analyzed as described later. Air-sampling filters for endotoxin quantitation were extracted and analyzed using the endpoint chromogenic Limulus amebocyte lysate (LAL) assay (QCL-1000; BioWhittaker, Walkersville, MD). Sampling filters were extracted by placing the filters in sterile, pf, 50-ml polypropylene tubes (Corning, Inc., Corning, NY) with 30 ml of sterile, pf water and incubating at 24°C for 60 min with agitation on a rocker table (27). Lyophilized standard endotoxin from E. coli 0111:B4, chromogenic substrate, and LAL preparations were reconstituted with sterile, pf water from our Nanopure system. Duplicate serial dilutions of filter extract, endotoxin standards, and Nanopure water samples were prepared using sterile, pf water in borosilicate glass tubes that had been heated for 4 h at 200°C to remove endotoxin activity. Aliquots (50 µl) of the serial dilutions of endotoxin standard and filter extracts were pipetted into a pf polystyrene microplate (Corning), and equal volumes (50 µl) of the lysate were added to each well. Solutions in the microplate wells were mixed and incubated at 37°C for 10 min. Chromogenic substrate solution (100 µl) was added to the wells, followed by 6 min incubation at 37°C in a heat block. The reaction was quenched by addition of 100 µl of 25% acetic acid (Fisher Scientific, Pittsburgh, PA). The microplate was mixed once again and the absorbance measured at 405 nm (BT2000 MicroKinetics Reader; Bio-Tek Instruments, Palo Alto, CA). Change in absorbance relative to the mean of four assay reagent blank wells was calculated and a standard curve of absorbance versus endotoxin concentration was generated. The standard curve ranged from 0.1 to 1.0 EU of National Institute of Standards and Technology traceable EC-5 standard endotoxin (10 EU = 1 ng endotoxin). Only those assays in which standard curves had correlation coefficients greater than 0.995 were accepted.

Cytokine Assays

The concentrations of murine cytokines in the BALF were determined using commercial enzyme-linked immunosorbent assay kits (TNF-: Genzyme Immunologicals, Cambridge, MA; IL-6: Endogen, Inc., Cambridge, MA). The lower limit of detection for these cytokines in mouse BALF is 17.5 pg/ml for TNF- and 15 pg/ml for IL-6. Neither of these assay kits shows cross-reactivity with other murine cytokines.

Intervention Therapeutics

Three drugs were tested for their efficacy to block Ad- and LPS-induced inflammation. Doses were selected to be at the high end of values reported in the literature for studies in rodents. Times of administration were based on the pharmacokinetics of the drugs in rodents. DEX sodium phosphate USP (Elkins-Sinn, Inc., Cherry Hill, NJ) was administered by intraperitoneal (i.p.) injection of 0.25 ml at a dose of 40 mg/kg at two time points, 24 h before exposure and at 3 h (see Figure 1). PTX (Hoechst-Roussel Pharmaceuticals, Inc., Sommerville, PA) was administered intraperitoneally at a dose of 50 mg/kg in 0.25 ml at 3 h and at time 0 h. LSF (Cell Therapeutics, Inc., Seattle, WA) was injected intraperitoneally at 3 h and at time 0 h at the same dose as PTX. An additional LSF efficacy study tested 4 i.p. injections at 50 mg/kg each at 2, 1, 0, and 1 h. For the sham-injected control animals, 0.25 ml of sterile, pf saline (Baxter Healthcare) was injected at 3 and at 0 h.

Statistical Analyses

The size of the experimental groups was determined from power calculations based on preliminary data. For 1- of 0.90 and  of 0.05, five animals per group were indicated and six were actually used. Differences between RES and SEN groups used Wilcoxon rank tests with z approximation for large n. Within groups, differences were tested using Kruskal-Wallis rank tests. If significant differences in means were observed, pair-wise comparisons were performed using Wilcoxon ranked sum tests with t-test approximation for small n. The chi-square P value with 1 degree of freedom was used to determine significance at  = 0.05. All analyses were run in SAS Version 6.04 (SAS Institute, Cary, NC, 1987).

	Results

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Endotoxin in Sputum

To establish that CF patients have an airway epithelial burden of endotoxin, we collected sputum samples from five patients harboring Pseudomonas and ranging from moderately ill (forced expiratory volume in 1 s [FEV₁] = 70% of predicted) to severely diseased (FEV₁ = 32% of predicted). These data are presented in Table 1 and illustrate moderate to extremely high concentrations of endotoxin ranging from 5,070 to 337,000 EU/ml of sputum. In a previous study (19) we reported inflammation in five healthy, nonsmoking human volunteers exposed acutely to a comparable level (280,000 EU) of inhaled endotoxin in aqueous grain-dust extract. That endotoxin exposure produced a mean 35% drop in FEV₁ within 20 min of the end of exposure and an increase from < 0.1 × 10⁵ to 15 × 10⁵ neutrophils/ml lavage fluid with corresponding increases in TNF-, IL-6, and IL-8.

Endotoxin Dose-Response

Our laboratory has previously investigated the pulmonary effects of several different inhaled endotoxin-containing compounds comparing biomarkers from C3H/HeJ endotoxin-hyporesponsive mice to those from normoresponsive C3HeB/FeJ mice and LPS-exposed humans (18). The same two-strain mouse model for investigating lung inflammation was used here to test the inflammatory potency of Ad and to determine whether endotoxin in the lungs of CF patients with gram-negative bacterial infections would enhance Ad-induced inflammation. Figure 2 illustrates the endotoxin dose-response relationship for BAL neutrophilia between the SEN and RES mice. Each data point represents the mean of five to eight mice exposed for 4 h and lavaged 1 h later. Exposure concentrations up to 0.03 µg/m³ produced insignificant differences between the two strains. Exposures above 0.08 µg/m³ were significantly different and produced as much as 3.5 orders of magnitude difference between strains. It is noteworthy that at high exposures (above 10 µg/m³), the RES mice demonstrated a mild neutrophilic response. These dose- response curves were used to select LPS exposure concentrations for subsequent studies (Table 2).

View larger version (14K): [in this window] [in a new window]   Figure 2.   Endotoxin dose-response curve illustrating the impaired neutrophilic response in the C3H/HeJ LPS-resistant mice (RES, filled squares) in comparison with the C3HeB/FeJ sensitive mice (SEN, filled circles). Data show the concentration of neutrophils in the BALF (PMN in Lavage) taken 1 h after the termination of a 4-h inhalation exposure. Some of these data were drawn from previous studies using this animal model (18, 20).

View larger version (30K): [in this window] [in a new window]   Figure 4.   Inflammatory responses of SEN and RES mice to Ad instillation alone (Ad5.RSV.LacZ), LPS inhalation alone, or Ad and LPS together. Sham-exposed control mice received saline inhalation and PBS instillation to control for both treatments. (a) illustrates the percentage of neutrophils in the BALF; (b) and (c) show the concentrations of TNF- and IL-6, respectively, in the BALF from the same groups of animals. Error bars indicate standard error; ns, not significant. *Significant difference between groups. Low and high LPS concentrations are as listed in Table 2.

View larger version (26K): [in this window] [in a new window]   Figure 5.   Tests of the effectiveness of DEX and PTX therapeutic interventions are illustrated using percentage of neutrophils (a) and the concentration of TNF- (b) in the BALF as biomarkers and Ad (Ad5.RSV.LacZ), LPS, or Ad + LPS as the inflammatory stimuli. Control animals in each exposure regimen were injected with saline rather than treated with the drug. The sham- exposed group received saline injection, saline inhalation, and PBS instillation to control for all three treatments. Both the neutrophilic response (a) and the TNF- concentration (b) revealed no significant reduction in inflammation with the PTX but a substantial therapeutic effect of the DEX. Low and medium LPS concentrations are as listed in Table 2.

Time Course of Ad Inflammation

Our first goal was to determine whether pulmonary administration of endotoxin-free Ad at doses relevant for gene transfer could induce early inflammation independent of viral protein expression. Ginsberg and Prince reported that 10⁸ plaque-forming units (pfu) of infectious type 5 adenovirus produced mild to moderate early histopathologic responses in cotton rats (28). Assuming a 100:1 particle-to-pfu ratio, this corresponds to 10¹⁰ Ad particles. Our initial dose-response study with intratracheal instillation of 10⁹, 10¹⁰, or 10¹¹ Ad particles demonstrated that 10¹⁰ psoralen- and UV-inactivated Ad particles in a 100-µl volume yielded a tenfold increase in neutrophils in the lung lavage fluid compared with controls at 3 h after exposure. This dose was chosen for subsequent studies because this level of response would allow detection of additive or inhibitory effects. We next established the time course for Ad-induced inflammation. In this study, inactivated Ad were instilled at time 0 and the concentration of neutrophils in the BALF as well as the TNF- concentrations were measured at 3, 6, 12, 24, and 48 h. As shown in Figure 3, SEN and RES control mice did not differ in neutrophil concentrations (Figure 3a) and both had TNF- concentrations (Figure 3b) below detection (< 15 pg/ml). The neutrophil concentration was above the concentration typically observed in naive mice (0.2 × 10³ cells/ml), likely because of the tracheal cannulation required for instillation of the sham challenge. Among the Ad-exposed groups, both the SEN and RES mice showed time-dependent increases in neutrophilia that differed by about 1 log unit up to 24 h after instillation. The SEN mice demonstrated a tenfold increase in neutrophil concentration over controls at 3 h, 30-fold at 6 h, and 60-fold at 12 h. No neutrophil response was observed in sham-instilled mice 3 or 24 h after instillation. Assay of cell-free lavage fluid for TNF- concentration revealed an increase from nondetectable levels in control mice to a mean 750 pg/ml concentration at 3 h in SEN mice and 100 pg/ml in RES mice. By 48 h after instillation, the TNF- concentration had returned to baseline levels. The two strains of mice followed essentially the same time course of response to Ad dosing, but with a significant blunting of inflammation in the RES mice.

View larger version (20K): [in this window] [in a new window]   Figure 3.   Time course for inflammation after instillation of 1010 replication-deficient Ad particles (Ad2.CMV.Gal). (a) Ad- induced neutrophil recruitment (PMN in Lavage) demonstrating maximal neutrophilic BAL response at 12 to 24 h after instillation. The endotoxin-sensitive mice (SEN) demonstrated an order of magnitude greater response than did the resistant mice (RES). (b) Maximal TNF- concentration in BALF after Ad instillation occurred at the first time point, 3 h after instillation.

Effects of Ad and LPS Exposure

We next developed experiments to address our hypothesis that administration of replication-defective Ad in the presence of LPS in the extracellular milieu would produce additive or even supra-additive (synergistic) effects. These experiments followed the protocol outlined in Figure 1 and are summarized in Figure 4. To ensure adequate deposition of LPS aerosol in the murine airways, the aerosol was carefully sized and optimized (see MATERIALS AND METHODS). The presence of large numbers of neutrophils in peribronchiolar tissues in fixed and stained lung slides validated aerosol delivery to the bronchiolar region.

Figure 4 includes data for SEN mice (left six bars) and RES mice (right six bars). Sham-exposed mice in both groups received saline inhalation exposure and saline instillation, and demonstrated no neutrophilia, TNF- concentrations below the limit of detection, and minimal IL-6 in the lung lavage fluid. For Ad and LPS treatments, SEN mice demonstrated highly significantly greater percent neutrophils (polymorphonuclear leukocytes [PMN]), TNF-, and IL-6 responses than did RES mice (P < 0.0001).

Figure 4 shows that exposure to Ad, low LPS, and Ad + low LPS produced the same degree of neutrophilic infiltration into the lavage fluid, as represented by differential counting. This illustrates no additivity of the Ad- and LPS-induced inflammation. When lavage samples from these mice were assayed for TNF- and IL-6, findings were consistent; there were no significant differences between SEN mice exposed to Ad, LPS, or Ad + LPS with LPS aerosol at 0.097 µg/m³.

To establish a higher degree of inflammation, the protocol was carried out with an LPS aerosol exposure concentration of 6.1 µg/m³ (high). At this concentration, the LPS produced a significantly greater (P < 0.05) degree of inflammation than did Ad without LPS (96% versus 44.5% PMN, 7,250 versus 769 pg/ml TNF-, and 1,306 versus 114 pg/ml IL-6). When mice were exposed to both high LPS and Ad, there was no significant increase in inflammatory responses over exposure to the high LPS alone. This experiment clearly demonstrates three points. First, both Ad and LPS induce lung inflammation within hours of exposure in SEN mice. Second, mice resistant to LPS are also hyporesponsive to Ad-induced inflammation. Third, there is no additivity or synergism in the inflammatory stimuli of Ad and LPS.

Effects of Therapeutic Interventions

The protocol used to test our hypothesis regarding Ad and LPS additivity was also suitable for testing our second hypothesis, that Ad-induced early inflammation in the presence or absence of LPS could be blocked with an immunosuppressant drug or with TNF- inhibitors. To accomplish this, the protocol was amended to include drug administration as shown below the timeline in Figure 1. LPS exposure concentrations used in these experiments are listed in Table 2. Figure 5 illustrates the results of efficacy trials using i.p. injections of DEX, PTX, or saline (control). Percentages of PMN and TNF- concentrations in the BALF served as biomarkers of inflammation. The treatment groups shown on the abscissa are the same as those in Figure 4 except that the low LPS concentration was 0.076 µg/m³ and a medium LPS concentration (0.60 µg/m³) was substituted for the high concentration used previously. Figure 5 shows that treatment with DEX reduced the percent of PMN and TNF- concentrations in the lavage fluid in most of the treatment groups. Significant reductions (P < 0.05) were observed for Ad, low LPS, Ad + low LPS (TNF- only), medium LPS, and Ad + medium LPS (TNF- only). Although DEX reduced inflammation when compared with saline, there was still measurable inflammation in these animals. PTX did not prove efficacious in any of the treatment groups for either response biomarker. Subsequently, studies were performed using a second methylxanthine derivative, LSF, and this same protocol. In those experiments, no significant reductions in inflammatory response biomarkers were observed. The LSF trial was repeated, this time using four doses (2, 1, 0, and 1 h) of the drug (50 mg/kg each) and an LPS inhalation concentration of 5.0 µg/m³ (high). Again, no significant reductions in percent of PMN, TNF-, or IL-6 were observed (data not shown). Thus, DEX did blunt the Ad- and LPS-induced inflammation, whereas neither PTX nor LSF proved effective at blocking the inflammation, even at high doses.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Recombinant Ad have shown promise for pulmonary gene transfer for treating CF (23). However, several problems have arisen that have limited this approach. First, patients with CF experience chronic bacterial pulmonary infections, and LPS shed from the outer cell wall of gram-negative organisms in the lungs of these patients (see Table 1) may induce severe inflammation. Second, there is evidence that the adenoviral capsid itself may induce airway inflammation (9). Thus, attempts at gene transfer using Ad in patients who already have LPS-induced airways inflammation could be problematic.

In this study, it was shown that instillation of an LPS-free solution of 10¹⁰ inactivated Ad particles into the lungs of mice produced a neutrophilic inflammation with concomitant increases in TNF- and IL-6 in the lung lavage fluid. Responses were maximal within 12 h. Inhaled LPS at 0.097 µg/m³ produced similar levels of early pulmonary neutrophilia and production of proinflammatory cytokines. Importantly, there was no evidence of an additive effect of these inflammatory stimuli at low, medium, or high LPS exposures. Assuming human responses are similar to those of mice, this suggests that patients with LPS-induced airway inflammation will not significantly upregulate proinflammatory cytokines soon after treatment with Ad for gene therapy. This is an encouraging finding for the use of Ad in gene transfer.

Cotten and associates reported that LPS commonly contaminates plasmid DNA preparations grown in E. coli, and hypothesized that LPS contaminating DNA preparations can be toxic to primary cells in the presence of adenovirus particles (8). They found that incubation of primary human melanoma cells in vitro with Salmonella minnesota LPS alone at 250 µg/ml did not produce cytotoxicity as measured by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction assay. Treatment of cell cultures with LPS-free, inactivated Ad at 10⁴ or 10⁵ particles per cell produced up to a 22% loss in viability. When 10⁵ Ad particles per cell and 2 µg/ml or higher LPS concentrations were introduced to the cultures, loss of viability increased. This effect was not observed at 10⁴ Ad particles per cell, where addition of up to 250 µg/ml of LPS produced no increase in cytotoxicity. It should be noted that this study did not use physiologically relevant doses of LPS and used very high doses of Ad. The dose of inhaled LPS in our 4-h low-concentration exposure (0.097 µg/m³; see Figure 4) that produced marked inflammation was approximately 0.42 ng LPS per mouse, whereas Cotten and coworkers used 6.25 ng per cell (25,000 ng per microplate well). Further, we delivered approximately 10³ Ad particles per mouse airway cell whereas Cotten and colleagues used 10⁵ Ad particles per cell. Thus, the severe inflammation observed in vivo occurred at exposures that were several orders of magnitude below those that produced loss of viability in vitro.

McCoy and associates investigated lung inflammation in CBA/J mice 6 d after intratracheal instillation of intact, inactivated, or incomplete adenoviral particles (9). As in our study, inactivated Ad was produced by treating the virus with psoralen and UV light. When compared with PBS-instilled controls, mice treated with 7 × 10¹⁰ particles of intact, inactivated, or incomplete Ad.RSV.IL-1ra produced 2- to 2.6-fold increased numbers of inflammatory cells per collagenase-digested mouse lung. McCoy and coworkers reported that treatment groups all had the following differential cell distribution in the lung digest: 71.3% lymphocytes, 22.1% monocytes/macrophages, and 6.6% neutrophils. Mouse lungs were lavaged before mincing and digestion but only IL-1ra assays were performed on the lavage solutions to test for reporter gene expression. These investigators concluded that high doses of instilled Ad caused inflammation independent of any gene expression. Because they focused on the responses 6 d after the instillation and studied only the cells from collagenase-digested lungs, key early events in the inflammatory process were not investigated.

Noah and coworkers investigated Ad-induced inflammation using an in vitro system. They measured IL-6 and IL-8 production by cultured human bronchial epithelial cells after treatment with replication-deficient Ad.CMV.lacZ, active wild-type Ad 5, TNF-, or Rous sarcoma virus (RSV) (29). Treatment with the Ad at 10¹ to 10⁴ pfu per cultured cell did not produce significant release of cytokines into the culture media. Conversely, both TNF- and RSV induced significant IL-6 and IL-8 production at 24 h after treatment in some of the cultures. These experiments were repeated using cultured human alveolar macrophages with similar results. Based on these studies, Noah and colleagues (29) concluded that neither adenovirus or adenoviral gene transfer vectors induced cultured airway epithelial cells or macrophages to produce inflammatory cytokines. This, however, is inconsistent with the work in vivo of McCoy and associates (9) and the experiments described in this manuscript, and may suggest that this cell-culture system lacked the necessary signal transduction pathways or metabolic machinery to serve as a predictive model for Ad- induced inflammation.

Ad-induced inflammation was compared in C3HeB/FeJ and C3H/HeJ mice to determine the importance of this genetically based differential response to the induction of early inflammation by inactivated Ad particles. It has been shown previously that C3H/HeJ mice compared with other mouse strains (C3H/HeN, C3H/HeOUJ, C3HeB/ FeJ, and C57/Bl6) are hyporesponsive to purified endotoxin (17, 30, 31), grain-dust extract (18, 19), microbially contaminated metal-working fluids (20), Staphylococcal enterotoxin B (32), instilled dextran beads (33), acetylcholine (34), hyperoxia (35), ozone (36), and nitrogen dioxide (37). It was reported in 1986 that C3H/HeJ mice have reduced TNF- gene expression after endotoxin treatment when compared with C3H/HeN mice (17). Subsequent studies demonstrated reduced expression and production of IL-1, which stimulates TNF- production (38, 39), and reduced expression of the immunosuppressive cytokine IL-10 (40). Most of these studies used injection or intratracheal instillation of endotoxin as the stimulus for inflammation. Our studies clearly demonstrate that C3H/HeJ mice are hyporesponsive to inflammation induced by inhalation of LPS (Figures 2 and 4). Data in Figures 3 and 4 also demonstrate a reduced response to the inflammatory stimulus of the Ad treatment in the C3H/ HeJ mice. Although the C3H/HeJ mice do produce TNF- and IL-6 with the same time course as the C3HeB/FeJ mice, these cytokines appear in significantly lower concentrations, resulting in diminished neutrophil infiltration. Ad instillation without LPS inhalation stimulated production of TNF- and IL-6 in the C3H/HeJ mice that was 10-fold and 3-fold lower (respectively) than the levels produced in C3HeB/FeJ mice. This differential response for Ad- and LPS-induced inflammation is evidence for a common mechanism of early cytokine signaling and neutrophil recruitment. However, the fact that there was no additivity of response even at doses selected to produce submaximal responses is perplexing. Together, these findings demonstrate a strain difference and a lack of additivity of effect but fail to reveal a difference in the nature of the proinflammatory stimuli.

Our finding of early IL-6 production by Ad instillation has been reported in human studies (12). Treatment of CF patients with an adenovirus vector expressing the CFTR complementary DNA led to increased serum IL-6 within 4 h of Ad instillation in one patient. Further study revealed increased IL-6 in BALF from patients with CF but not in non-CF control subjects. These investigators postulated that the patients with CF had neutrophilic and mononuclear infiltrates into the epithelium and that Ad triggered the release of IL-6. Our Ad preparation was demonstrated to be incapable of RNA or protein production. In addition, it is unlikely that there would be substantial Ad gene expression within our 3-h time frame, even if there were some active Ad particles. If the capsid proteins are important to the induction of inflammation, deletion of viral genes alone will not effectively reduce the inflammatory potency. This may point to the need for therapeutic interventions to limit inflammation before gene transfer using Ad. Alternatively, improvements in the therapeutic index of Ad may reduce early dose-dependent inflammatory events. Incorporation of adenovirus in calcium-phosphate precipitates enhances gene transfer to airway epithelia in vitro and in vivo at a lower multiplicity of infection (41).

In our attempts at blocking Ad-induced lung inflammation we tested DEX, a glucocorticoid immunosuppressant, and two methylxanthine derivatives we postulated would blunt the inflammation by impairing the production of TNF-. Studies by Rice and colleagues (42) demonstrated that mice injected with 100 mg/kg of either PTX or LSF had lower plasma TNF- levels than did controls after intravenous administration of 10,000 µg/kg LPS. Our results were disappointing for the methylxanthine derivatives because they showed no significant effects, even with four hourly doses at 50 mg/kg. DEX did significantly reduce both the neutrophilic and the cytokine responses, particularly for the Ad-induced inflammation. Thus, DEX may be a useful element of a gene transfer protocol for CF. Zsengeller and coworkers (43) investigated cotton rats treated with DEX 1 d before and 3 d after intratracheal instillation of Ad and found reduced cellularity on histopathology. Our studies suggest that CF patients who already harbor gram-negative bacterial infections may experience little additional early inflammation after gene therapy with Ad. If gene therapy with Ad is extended to patients with less advanced disease or to children diagnosed with CF, treatment with immunosuppressants including DEX may help in reducing Ad-induced inflammation and may facilitate gene transfer.

In summary, in these studies we showed that replication-defective Ad induced profound inflammatory responses within 3 h. RES mice were hyporesponsive to Ad-induced inflammation as well as to LPS. Co-administration of a high dose of Ad with LPS was not significantly more inflammatory than LPS alone. DEX blunted both the Ad- and LPS-induced inflammation, but the two methylxanthines tested did not significantly inhibit inflammation.