Male Sprague-Dawley rats (300 to 350 g, pathogen free) were purchased from Sasco Korea. Not otherwise mentioned, all experimental reagents were purchased from Sigma-Aldrich Company. (St. Louis, MO, USA). Recombinant human interleukin-1α (IL-1, endotoxin level 1< EU/mg) was purchased from R&D systems (Mineapolis, MN, USA), frozen in aliquots, and thawed daily before use. ¹²⁵I-labeled bovine serum albumin (¹²⁵I-BSA) was obtained from ICN Radiochemicals (Irvine, CA, USA). Goat anti-human cPLA2 polyclonal antibody was purchased from Santa Cruz Biochem (Santa Cruz, CA, USA).

Animals were fed ad libitum till 24 hours before experiments. ALI was induced as described previously13. Fifty nanogram of IL-1 was dissolved in 1.0 mL of normal saline and this solution was insufflated into the trachea. The NADPH oxidase inhibitor apocynin (8 mg/kg)4 was administered through the femoral vein just after instillation of IL-1. To sham treated rats, 1.0 mL of normal saline was instilled into the trachea.

Lung lavage was performed by cannulating the trachea and instilling 8.0 mL of normal saline with a syringe. Approximately 6.0 mL of lavage fluid was recovered from each rat. After the recovered lung lavage was centrifuged (1,000 g; 10 minutes), the supernatant was collected and stored at -20℃ for measurement of protein. The sedimented cellular pellet was resuspended in 1.0 mL of distilled water and 1.0 mL of Hank's balanced salt solution (HBSS) for 2 seconds to lyse erythrocytes, then centrifuged again. The supernatant was discarded and the pellet was resuspended in 1.0 mL of normal saline. Using 200µL of suspended solution, cytospin slides were prepared and Wright stained for differential cell count. Total leukocyte counts were performed with a hemocytometer and the fraction of neutrophils was calculated by determining the percentage of neutrophils.

BAL protein was determined as described previously by Brown et al14. using supernatant of BAL. Bicinchoninic acid was used as chromogenic substrate.

Four and half hours after IL-1 instillation, ¹²⁵I-BSA (1.0µCi) was injected through jugular vein. One mL of blood was withdrawn 30 minutes later from the right atrium and then assayed for radioactivity. Subsequently, lungs were perfused blood free with normal saline and removed. Lung leak index was calculated as the ratio of radioactivity in the right lung to radioactivity in 1.0 mL of blood.

To assess the accumulation of neutrophils in the lung, lung MPO was determined as previously described by Goldblum et al15. with modification. MPO activity was measured using o-dianisidine as the chromogenic substrate and H₂O₂ to initiate the reaction. Light absorbance at 460 nM was proportional to MPO activity.

Cytochrome-C reduction assay; Neutrophils were isolated as described previously according to Botha et al16. Briefly, the mixture of 15 mL of pentaspan and 0.1 mL of heparin (1,000 units) were mixed with 30 mL of fresh human blood before standing for 40 minutes at room temperature. Then the plasma layered on 74%, 55% isotonic percoll gradient after separation from erythrocytes, centrifuged (1,500 rpm; 20 minutes) and neutrophils were collected from the interface of 74, 55% of percoll gradients. With isolated neutrophils, NADPH oxidase activity was determined by the method of cytochrome-c reduction assay described previously17. Collected suspension of neutrophils was hypotonic lysed for removal of erythrocytes and washed twice with PBS. For cytochrome-c assay, except control samples, phorbol myristate acetate (PMA, 2.5µg/mL) and apocynin (100µM) were added to the neutrophil suspension (2×10⁷ cells) before incubating for 15 minutes at 37℃. Light absorbance at 550 nM was proportional to the reduction of cytochrome-c by free radicals.

As an index of oxidative stress in lung tissues, GGT activity was measured as previously described by Meister et al.18 Briefly, 0.2 mL of L-γ-p-nitroanilide (1 mM), 0.2 mL of glycylglycine (20 mM) and 0.4 mL of Tris-Hcl buffer, and 0.2 mL of lung homogenate were added in the tube and incubated at 37℃ for 30 minutes in shaking water bath. The reaction was stopped by adding 0.1 mL of 50 mM of serine borate solution. The release of p-nitroaniline was recorded as the change of absorbance at 410 nM. The specific activity was expressed as µ moles of p-nitraniline released per minute per mg of protein.

Small blocks (1 mm³) of lung tissue were rapidly fixed in the cold 2.5% glutaraldehyde and the small tissue blocks were degassed by the applying of the pressurized air (20 cm H₂O) into the sealed vial. The prefixed tissues were rinsed with PBS (pH 7.4) and post fixed 1% OsO₄. After dehydrated with graded ethanol-propylene, the tissues were embedded in epoxy-resin. The embedded tissues were polymerized at 37℃ for 12 hours and at 60℃ for 24 hours before ultra thin section. The sections were stained with uranyl acetate and lead citrate before electron microscopy (H600, Hitachi, Japan).

Cytospinned cells were subjected to staining for fluorescence for cPLA2 in BAL cells. Goat-anti-human-cPLA2 polyclonal antibody (C-20, Santa Cruz, CA, USA) was used as first antibody. The antibody complex was visualized by staining with propidium iodide.

Data were analyzed using a one-way analysis of variance with a Student-Newman-Keuls multiple comparisons test. A p-value of <0.05 was considered statistically significant.

As was shown in Table 1, numbers of neutrophils in BAL were higher in IL-1 treated rats than those of sham treated rats (p<0.001). Apocynin did not decrease the numbers of neutrophils in BAL of IL-1 treated rats. Apocynin did not affect the accumulation of neutrophils by IL-1 either. The MPO activity of the lung was much higher (p<0.001) in IL-1 treated rats compared with that of sham treated rats. But it was not decreased by apocynin.

Assay of BAL protein and measurement of lung leak index (LLI) revealed the increased protein leak in IL-1 treated rats (p<0.001) compared with those of sham treated rats. Apocynin was not effective to suppress the lung leak in IL-1 treated rats (Table 2).

In phorbol myristate acetate (PMA)-stimulated neutrophils, the amount of reduced cytochrome-c was very much more than that of non-stimulated neutrophils (p<0.001). Apocynin had decreased the generation of superoxide anion from PMA-stimulated neutrophils (p<0.001) (Table 3).

As an index of oxidative stress, GGT activity was higher in lung tissues of rats given IL-1 than that of sham treated rats (p<0.001). Apocynin was not effective to suppress the increased GGT activity by IL-1 (Table 4).

In normal alveolar type II cell, lamellar bodies are well preserved (Figure 1A). By contrast, IL-1 elicited typical morphological changes of oxidative stress in alveolar type II cell such as vacuolization, enlargement of lamellar bodies and disruption of basement membrane (Figure 1B). These pathological changes were not altered by apocynin (Figure 1C).

The BAL cells from the rat given IL-1 showed enhanced expression of cPLA2 in neutrophilic cytoplasms compared with the expression in the cellular cytoplasms of sham treated rats. Apocynin did not alter the increased expression of cPLA2 by IL-1 (Figure 2).