Certified pathogen-free, Sprague Dawley male rats (body weight, 280–380 g) were purchased from Charles River Laboratories (Wilmington, MA). The rats were housed in cages with filter tops in specific pathogen-free conditions. Sterile food and water were provided ad lib. All experiments were done in compliance with Animal Care Committee rules of the University of California at San Francisco, U.S.A., and all protocols were approved prior to the start of the experiments.

P. aeruginosa PA103 was used in this study. Bacteria from frozen stocks, stored at -70°C in 10% sterile skim milk solutions, were streaked onto trypticase soy agar plates. Five milliliters of a deferrated dialysate of trypticase soy broth supplemented with 10 mM nitrilotriacetic acid (Sigma Chemical, St. Louis, MO), 1% glycerol, and 100 mM monosodium glutamate was inoculated with a loop of bacteria and grown at 33°C for 13 h under shaking conditions. Cultures were centrifuged at 8,500 × g for 5 min and the media discarded. The bacterial pellet was washed twice in lactated Ringer's (L/R) solution and diluted to the appropriate concentration of CFU/ml in L/R solution, as determined by spectrophotometry. Plating out the known dilutions on sheep blood agar plates confirmed the bacterial concentrations.

The rat model for P. aeruginosa pneumonia was reported previously 2223. Briefly, rats were anesthetized with 100 mg/kg of pentobarbital sodium administered intraperitoneally. An endotracheal tube (PE-240, Clay Adams, Parsippany, NJ) was inserted into the trachea via an open tracheostomy. The rats were ventilated with a constant-volume respirator (Harvard Apparatus, South Natick, MA) with an inspired O₂ fraction of 1.0, peak airway pressures of 8–12 cmH₂O and a 2 cm positive end expiratory pressure (PEEP). The respiratory rate was adjusted to maintain PaCO₂ between 35 and 45 mmHg. The rats remained anesthetized, intubated and ventilated throughout the entire experiment. The right carotid artery was canulated with a polyethylene tube (PE-50, Clay Adams) to monitor systemic arterial pressure, administrate drugs and obtain blood samples.

The instillate consisted of 5% bovine serum albumin (BSA), 2 mg of Evans blue dye, and 1 μCi of ¹³¹I-labeled albumin, and P. aeruginosa, at a final concentration of 5 × 10⁷ CFU/ml in L/R solution to a total volume of 1 milliliter; Colloid osmotic pressure of the instillate was adjusted by adding 5% BSA as an established method to quantify liquid clearance of lung epithelial barriers as an index of lung edema 24. The bacteria were added just before airspace instillation if the experiment was to include bacteria. A sample of the instillate was saved for radioactivity measurement (counts/min/g) in a γ-ray counter (Auto-Gamma, model 5550, Packard, Downers Grove, IL) and quantitative bacterial cultures on sheep blood agar plates to assure accurate inoculations. The instillates were delivered slowly, over a 30 min period using a polyethylene tube (PE-10, Clay Adams) into the left lungs.

Mab166 IgG (IgG2bκ) or its Fab fragments were previously prepared in PBS and stored at -70°C 21. Experimental groups are listed in Table 1. One additional group of rats was used as the sham control group; rats received L/R solution not containing IgG. In three groups of rats, we co-instilled P. aeruginosa PA103 (5 × 10⁷ CFU) with 4 mg/kg of either mouse monoclonal isotype-matched control IgG (IgG2b, clone #20116.11, R&D System, Minneapolis, MN), rabbit anti-PcrV polyclonal IgG, or murine monoclonal Mab166 IgG intratracheally. In another three groups, rats received either PBS or 4 mg/kg of either anti-PcrV polyclonal IgG, Mab166 IgG, or Mab166 Fab fragments one hour after the airspace instillation of P. aeruginosa PA103 (5 × 10⁷ CFU).

After surgical preparation, blood pressure and gas exchanges were allowed to stabilize. Systemic arterial pressure and airway pressure were continuously monitored using an on-line data logging system (Powerlab, ADInstruments, Mountain View, CA). Blood samples were collected every hour for gas exchange measurement, ¹³¹I-albumin radioactivity count and bacterial culture. The rats were kept anesthetized and paralyzed throughout the experiment. Four hours after bacterial instillation, rats were deeply anesthetized and exsanguinated. Pleural fluids were obtained for radioactivity counts. The lungs were removed through a sternotomy; the left and right lobes were weighed and homogenized separately for water to dry weight ratio measurement and radioactivity counts.

Lung injury was quantified in two different ways, as previously described 2223. The first method evaluates the integrity of the lung epithelial barrier by quantifying the efflux of ¹³¹I-albumin from the alveolar to the bloodstream. Total ¹³¹I-albumin instilled into the lung was determined by measuring duplicate samples of the instillate for total radioactivity (cpm/g) and multiplying this amount by the total volume instilled into the lung. Circulating plasma ¹³¹I-albumin was measured from blood samples obtained every hour and at the end of the experiment. The plasma fraction was calculated by multiplying the counts per gram times the plasma volume [body weight × 0.07 (1-hematocrit)]. The second method, the water to dry weight ratio, is a well-accepted index of lung edema. Lung homogenates were placed in pre-weighed aluminum pans and dried to a constant weight in an oven at 80°C for 3 days. The excess water in the experimental lung was calculated with an equation described previously 2223.

Lungs were perfused with 10% buffered formalin phosphate for fixation and were embedded in paraffin. Mounted sections were stained with hematoxylin-eosin and observed under light microscopy.

Results are presented as mean ± standard errors. The difference between the control IgG-treated group and the Mab166 IgG or Mab166 Fab fragments-treated group was analyzed. Two-way analysis of variance (ANOVA), repeated measure, followed by the Newman-Keuls t-test or unpaired Student's t-test was used for comparisons of data. Significance was accepted at P value of < 0.05.

First, to evaluate the maximal blocking effects of anti-PcrV IgGs on P. aeruginosa-induced acute lung injury, we coinstilled either rabbit-derived polyclonal anti-PcrV IgG, murine monoclonal anti-PcrV IgG Mab166 (IgG2b), or irrelevant monoclonal control IgG (IgG2b) (4 mg/kg, respectively) with P. aeruginosa PA103 (5 × 10⁷ CFU) into the lungs of the anesthetized ventilated rats under artificially controlled ventilation. The antibodies were premixed with P. aeruginosa three min before the instillation. The acute alveolar lung injury was quantified as the efflux of the coinstilled radioactive alveolar protein tracers (¹³¹I-albumin) into the circulation every one-hour during the 4-h experimental period.

The control rats that received the lactated Ringer's solution supplemented with 5% bovine serum albumin but without bacteria did not show any lung epithelial injury (Fig. 1). Wet to dry weight ratios of the lungs increased to approximately 6 in the control rats (Fig. 2). Note a wet to dry weight ratio of the lung of a normal rat is between 3.5–4.0 (data not shown). The rats that received P. aeruginosa mixed with control irrelevant monoclonal IgG intratracheally developed significant acute epithelial injury in 4 h (Fig. 1). Severe lung edema was observed in this group of rats 4 h after bacterial instillation (Fig. 2). The arterial blood pressure decreased below 80 mmHg after 4 h time point (Fig. 3). Arterial blood oxygenation severely decreased to approx. 100 mmHg immediately after bacterial instillation, never normalized (Fig. 4). Metabolic acidosis gradually developed over the 4 h in this group of rats (Fig. 5).

The rats that received P. aeruginosa premixed with rabbit polyclonal anti-PcrV IgG intratracheally developed significantly lower levels of lung injury. Alveolar epithelial injury was significantly lower than that of rats that had received control IgG (Fig. 1), and lung edema was less, although not significantly (Fig. 2). Blood pressure was normal for the 4 h (Fig. 3), arterial blood oxygenation recovered by the 4 h time point (Fig. 4), and acidosis did not developed (Fig. 5). Finally, in the rats which received P. aeruginosa premixed with murine monoclonal anti-PcrV IgG Mab166, the lung epithelial injury and lung edema were significantly less than in the other groups (Fig. 1 and 2). Arterial blood pressures and acid-base status of these rats were normal for 4 h (Fig. 3 and 5), and the arterial blood oxygenation was the best among the three groups (Fig. 4). Thus, co-instillation of Mab166 with P. aeruginosa was the most protective.

Next, we evaluated the therapeutic administration of anti-PcrV IgG in our rat model. In this series of the experiments, we administered either rabbit polyclonal anti-PcrV IgG, murine monoclonal anti-PcrV IgG Mab166, Fab fragments of Mab166 (4 mg/kg, respectively), or PBS alone without IgG 1 h after the instillation of P. aeruginosa (5 × 10⁷ CFU) into the lungs of the anesthetized ventilated rats. The rats that received PBS alone 1 h after bacterial instillation showed a significant increase in lung epithelial injury and lung edema after 4 h. The arterial blood pressure gradually decreased to 80 mmHg over the experimental periods. The arterial blood oxygenation remained significantly decreased (Fig. 4). Severe metabolic acidosis developed over the 4 h (Fig. 5).

The rats that had received either rabbit polyclonal anti-PcrV IgG, murine monoclonal anti-PcrV IgG Mab166, or Fab fragments of Mab166 (4 mg/kg) intratracheally showed significant improvement of alveolar epithelial injury and lung edema 4 h after bacterial instillation (Fig. 1). The protective effect of Mab166 Fab fragments on lung epithelial injury was the most significant among the three antibodies, while rabbit polyclonal and murine monoclonal anti-PcrV IgGs were better in improving lung edema than Mab166 Fab fragments (Fig. 2). Hypotension did not develop in the three groups of rats that received any anti-PcrV antibodies (Fig. 3). The arterial oxygenation in the three treated groups of rats was significantly improved compared to the untreated rats (Fig. 4). Although mild metabolic acidosis did develop in the rats that had received either rabbit polyclonal anti-PcrV IgG or Mab166 Fab, the rats that had received Mab166 did not become acidotic (Fig. 5).

We compared the lung histology between the rats treated with Mab166 and the rats treated with control IgG (Fig. 6). While the rats that received control IgG one hour after bacterial instillation showed severe neutrophil recruitment and destruction of alveolar structures (Fig. 6A), the rats that received Mab166 had almost no neutrophils in their airspaces and had preservation of normal alveolar structures (Fig. 6B). As a result, therapeutic administration of Mab166 showed comparable effects to rabbit polyclonal anti-PcrV IgG in preventing acute lung injury and subsequently occurring systemic distress. The therapeutic administration of Mab166 Fab fragments also had the same or better effects than the administration of Mab166 IgG.