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Although Staphylococcus aureus is a significant cause of nosocomial pneumonia (19), little is known about the role of specific virulence factors for the induction of lung injury. Alpha-toxin is an important virulence factor in experimental models of mastitis (4), peritonitis (14), and corneal keratitis (13). Alpha-toxin monomers bind cell membranes and then associate in a heptameric complex to form a pore (17). The effects of alpha-toxin are both concentration and cell-type dependent and include cell lysis (1, 2), release of proinflammatory mediators and cytokines (6), and induction of apoptosis (7).

Purified alpha-toxin, administered through the vasculature, causes injury to the air-blood barrier in isolated perfused lungs (16). Specifically, alpha-toxin increases the permeability of the lung to fluid and causes necrosis of capillary endothelial cells (16). In this study, we determined the role of alpha-toxin in vivo by developing a rat model of S. aureus-induced pneumonia.

Alveolar epithelial type I cells cover 95% of the lungs' surface and are part of a tight epithelial barrier that is vital for maintaining a dry alveolus (8). Until recently, it was difficult to assess the extent of injury to type I cells other than by quantitative morphologic analysis at the electron microscopic level. However, we have recently demonstrated that the content of a type I cell-specific protein, rTI₄₀ (5, 15), in bronchoalveolar lavage (BAL) fluid is associated with morphologic injury to type I cells (9-11). In this study, we used the rTI₄₀ assay to determine the extent of damage to alveolar epithelial type I cells in S. aureus-induced pneumonia.

Bacterial strains 8325-4 and DU1090 were used to establish pneumonia. 8325-4 is an hla-positive strain derived from NCTC 8325. DU1090 is an alpha-toxin-defective mutant of 8325-4 constructed by allelic replacement (14). For inoculations, cultures of S. aureus were grown for 18 h with aeration in Todd-Hewitt broth (Oxoid). S. aureus was washed twice in sterile phosphate-buffered saline (PBS) before finally being resuspended in PBS. The number of viable bacterial cells was measured by colony counts. Production of alpha-toxin by S. aureus isolated from BAL fluid samples was confirmed by culture on 5% calf blood agar plates (4).

Rat model. Rats (Sprague-Dawley, male, 300 to 350 g; University College, Dublin, Ireland) were anesthetized with 4% halothane. Rats were then intubated through a tracheotomy with a blunt 16-gauge needle and ventilated with 100% oxygen at a rate of 70 breaths per min, a tidal volume of 3.5 ml, and positive end expiratory pressure of 1.5 cm of H₂O. Anesthesia was maintained with halothane (0.5 to 1.0%). Mean arterial pressure was measured with a carotid arterial catheter. The neuromuscular response was blocked with pancuronium (0.3 mg per kg of body weight).

BAL fluid analysis. The left bronchus was intubated, and the lung was lavaged with a total of 7 ml of PBS (4 ml and then 3 ml). The returned BAL fluid was pooled, and the total number of leukocytes was counted. The amount of rTI₄₀ in BAL fluid was determined with an enzyme-linked immunosorbent assay (ELISA)-based assay, as previously described (9-11). Data are presented in relative densitometry units (RDU). The molecular weight of rTI₄₀ was determined in BAL fluid by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting by standard methods (9, 11). The protein concentration of BAL fluid was determined by using the Bradford assay. The hemoglobin content was determined by measuring the oxidation of 3,3'5,5'-tetramethylbenzidine (Sigma diagnostic kit). Alpha-toxin protein was detected by Western and ELISA-based dot blot analyses.

Histology. Lungs were stained with hematoxylin and eosin to determine the overall extent of leukocyte recruitment to the airspaces or Gram stain to confirm the location of S. aureus. For detection of rTI₄₀, lungs were fixed with 4% paraformaldehyde and frozen; thin sections were then cut (5 µm). Lung sections were incubated with anti-rTI₄₀ hybridoma supernatant (5), followed by fluorescein isothiocyanate-linked antimouse immunoglobulin (Organon Teknika, West Chester, Pa.) sections were viewed in a Zeiss fluorescent microscope.

Alveolar epithelial cell isolation. Alveolar type II cells were isolated from rat lungs by using elastase (5). Type II cells were plated into 12-well plates and grown at 37°C in 5% CO₂, in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Alveolar epithelial cells acquire a type I cell-like phenotype after 7 days in culture, including the expression of rTI₄₀ (3, 5). Day 7 cells were exposed to purified alpha-toxin (0.01 to 10 µg/ml) for 4 h in serum-free medium at 37°C. Cell necrosis was determined by measuring lactate dehydrogenase activity and rTI₄₀ content in cells and culture medium.

View larger version (63K): [in this window] [in a new window]   FIG. 1.   Representative photographs of hematoxylin-and-eosin-stained lung 4 h after distal airway instillation of PBS (A) or S. aureus 8325-4 (B). Strain 8325-4 caused an influx of inflammatory cells into the airspaces (arrows) in comparison with the section from control lung. Original magnification, ×200.

View larger version (63K): [in this window] [in a new window]   FIG. 2.   Immunofluorescence detection of rTI40 4 h after distal airway instillation of either PBS (A) or S. aureus 8325-4 (B). White arrows show binding of anti-rTI40 monoclonal antibody to alveolar type I cells. Stars show binding of secondary antibody to S. aureus in the airspaces (B and C) (probably due to antibody binding to protein A in the cell wall of S. aureus). DU1090-infected lungs were not different from control lungs (data not shown). Original magnification, ×500.