Dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (PC), dipalmitoylphosphatidylglycerol (DPPG) and cholesterol were purchased from Avanti Polar Lipids (Birmingham, AL). L-α-dipalmitoyl [2-palmitoyl-9,10-³H(N)]PC was obtained from DuPont New England Nuclear (Boston, MA). Elastase for type II cell isolations was purchased from Worthington Biochemicals (Freehold, NJ). Dulbecco's PBS, DMEM, and fetal bovine serum (FBS) were obtained from Life Technologies (Gaithersburg, MD). Low-endotoxin BSA, O26:B6 Escherichia coli LPS, Rat IgG and all other chemicals were purchased from Sigma (St. Louis, MO). Chloroform and methanol were obtained from EM Science (Gibbstown, NJ).

Small unilamellar liposomes were prepared with a lipid composition similar to pulmonary surfactant: 52% DPPC, 26% egg PC, 15% DPPG and 7% cholesterol by weight with trace amounts of ³H-DPPC (12 μCi/mg phospholipid). The lipids were dried under nitrogen, reconstituted in 0.15 M saline and extruded from a French Press cell under 900 psi. This resulted in small unilamellar liposomes at a concentration of approximately 1 mg lipid/ml.

Male pathogen-free Sprague Dawley rats (150–200 g; Taconic Farms, Germantown, NY) were used for the current study. For LPS animals, endotoxin (0.1 mg/kg of O26:B6 E. coli LPS) was suspended in 300 μl of sterile 0.15 M saline. Control animals received an equal volume (300 μl) of sterile 0.15 M saline. For instillation, animals were anesthetized with halothane such that they remained unconscious throughout the entire instillation procedure and had no cough reflex upon intubation. Animals were placed on a board at a 45° angle, intubated with an 18-gauge blunt ended catheter and either sterile saline or LPS suspension was instilled followed by five 1 ml boluses of air to facilitate the distribution of the instilled fluid. Two hours prior to killing, liposomes (100 μg lipid) were intratracheally instilled in all groups following the identical instillation procedure. Separate groups of both control and LPS animals were sacrificed at 18 or 72 hours after saline or LPS instillation.

One control animal and one LPS animal were investigated simultaneously, thus all procedures were done in parallel. At 18 or 72 hours after saline or LPS instillation, animals were anesthetized with 0.3 mg of sodium pentobarbital and 700 U of heparin. After loss of toe pinch reflex, the trachea was cannulated and the rat was exsanguinated via transection of the descending aorta. The chest cavity was subsequently opened and the lungs perfused through the pulmonary artery with 40–50 ml of a calcium buffer (140 mM NaCl, 5 mM KCl, 2.5 mM Na₂HPO₄, 10 mM HEPES, 2.0 mM CaCl₂, and 1.3 mM MgSO₄ at 37°C). The lung from the first animal was carefully removed from the thoracic cavity and placed between saline soaked gauze pads while the second lung was perfused and removed in identical fashion. The time for lung perfusion and removal was approximately 10 minutes. After lungs were isolated from both a control and LPS animal, they were lavaged simultaneously with eight 10-ml volumes of EGTA buffer (140 mM NaCl, 5 mM KCl, 2.5 mM Na₂HPO₄, 10 mM HEPES, and 0.2 mM EGTA at 37°C). For each animal the individual lavages were collected and combined to make up the bronchoalveolar lavage fluid (BALF), which was immediately stored on ice. After the lavage procedure, individual lobes were dissected away from the major airways. The lung tissue was then cut into 5 mm pieces in 5 ml calcium buffer and subsequently homogenized with a Polytron PT-MR-2100. After complete homogenization, total lung tissue volume was diluted to 40 ml with calcium buffer and stored on ice. A small aliquot (2 ml) of the total BALF was removed and the remainder was centrifuged at 250 g for 10 minutes at 4°C to generate a pellet that was primarily BALF cells. The cell pellet was subsequently suspended in 10 ml of PBS. Lavage cell numbers were determined by a hemocytometer, viability determined by trypan blue exclusion and cell differential determined by Hemacolor staining of cytospins that were prepared using a Shandon Cytospin 2 centrifuge. Liposome-association in total BALF, cell-free BALF, BALF cells and homogenized lung tissue was determined by scintillation counting.

Alveolar phospholipid levels were measured in the cell-free BALF by phospholipid-phosphorous measurement. Lipids were extracted using the method of Bligh & Dyer 19 and phospholipid levels were determined using a modification of the Duck-Chong phosphorous assay 20. Briefly, 100 μl of 10% magnesium nitrate in methanol was added to the extracted lipids. After drying, the samples were ashed in a fume hood on an electric rack for approximately 1 min. After 1 ml of 1 M HCl was added, the samples were reheated on a heating block while covered for 15 min at 95°C. After cooling, a 66 μl aliquot of each sample was added to individual wells of a 96 well plate along with 134 μl of a dye consisting of 4.2% ammonium molybdate in 4.5 M HCl with 0.3% malachite green (1:3 vol/vol). The absorbency of each sample was read at 650 nm using a Biorad 550 microplate reader and compared to reference standards on the same plate.

Relative quantities of alveolar SP-A from 18-hour control animals, 18-hour LPS animals and 72-hour LPS animals were determined by loading equal volumes of BALF on 15% SDS-PAGE gels under reducing conditions. Total BALF recovery was not different among the individual animals. Proteins were then transferred to nitrocellulose where SP-A was probed with a well characterized polyclonal rabbit anti-rat SP-A 21. The nitrocellulose was subsequently developed using the ECL system (Amersham Pharmacia Biotech, Piscataway, NJ).

A separate cohort of animals was required for type II cell isolation studies. As with the whole lung studies, type II cell isolation was completed for one control and one LPS rat in parallel for both 18- and 72-hour time points after instillation of saline or LPS. The type II cell isolation procedure was described previously with minor modifications of the original protocol 2223. The modifications were designed for isolation of type II cells from inflamed lungs, and included increasing the elastase to 3,000 orcein units/lung, and increasing the surface area for IgG panning by two-fold. Briefly the isolation procedure involved killing the rats, removing the lungs and lavaging the lungs eight times as stated in Whole Lung Studies. Subsequently the lungs were lavaged twice with calcium buffer (37°C) and once with elastase solution (3000 orcein/40 ml calcium buffer; 37°C). The lungs were then filled with the elastase solution and suspended in warmed saline (37°C) for 20 minutes. Lung tissue was removed from the major airways, cut into 5-mm pieces and chopped 200 times with sharp scissors in 5 ml of the calcium solution along with 2 mg of DNase. The tissue suspension was added to a flask with 4 ml of FBS and 35 ml of calcium buffer and shaken vigorously for 2 min in a 37°C water bath. The resultant tissue suspension was strained through gauze, and then decreasing sizes of nylon mesh (150-, 15-, and 8-μm). The cell suspension was centrifuged at 250 g for 10 min at 4°C, the resulting cellular pellet was suspended in 20 ml of warm DMEM (37°C) and incubated on two IgG coated Petri dishes (150 × 15 mm) for 30 minutes at 37°C. After the incubation period, nonadherent cells were removed from the plate, washed once with PBS, suspended in calcium buffer and used for determination of radiolabel recovery associated with Type II cells. The remaining adherent cells were gently scraped off the plate in 5 ml of DMEM and transferred into a polypropylene tube; this population of cells is referred to as "plate cells" and consisted primarily of macrophages and/or neutrophils as described in detail below. The cells were washed once and suspended in calcium buffer for determination of associated radioactivity. A small aliquot from the isolated type II cells and plate cells was saved for differential cell count and viability. Cell purity was determined by counting a minimum of 250 cells from random fields after staining by the Papanicolaou method 22.

All data reported are means ± standard error (SE). Repetitions (n) used to calculate means ± SE were from independent experiments, not from replicates within an experiment. An analysis of variance (ANOVA) was used to determine differences between all experimental groups at a specific time point, followed by a Tukey post-hoc test for multiple comparisons. Significance was accepted when p < 0.05.

Table 1 reveals total alveolar cell numbers and differentials from the BALF of control and LPS groups killed at the 18- and 72-hour time points. Eighteen hours after instillation of LPS there were significantly more cells recovered in the BALF from the LPS treated animals compared to the saline control group (p < 0.01). Cell differentials from the two 18-hour groups revealed that the LPS group had primarily neutrophils in the BALF, whereas the cells recovered from the control group were predominantly macrophages. Seventy-two hours after LPS instillation there were significantly greater numbers of cells in the BALF of the LPS group compared to the 72-hour control group (p < 0.01), but significantly fewer numbers of cells compared to the LPS 18-hour group (p < 0.01). Cell differentials from the 72-hour groups revealed that the cells were predominately macrophages in both the LPS and control groups. Of note, there were no differences in cell numbers or differentials between the two control groups at the different time points.

Table 1 also displays the total phospholipid levels measured in the BALF of the four experimental groups. There was an approximate 15% decrease in total phospholipid recovered by lung lavage from both LPS groups compared to their respective saline control groups, however this did not reach statistical significance. Of note, there was no difference in mean body weights among the four experimental groups (Con 18: 177 ± 7 g; LPS 18: 180 ± 10 g; Con 72: 186 ± 9 g; LPS 72: 192 ± 7 g). Figure 1 shows Western blot analysis of SP-A measured in the BALF recovered from animals killed 18 hours post saline instillation (control), and 18 hours and 72 hours after LPS instillation. There was relatively greater quantity of SP-A in the BALF of animals killed at both 18 and 72 hours after LPS instillation compared to the control animals, consistent with previous reports using similar models of intrapulmonary LPS administration 91011.

Figure 2 shows distribution of the recovered radioactivity associated with the cell free BALF, the isolated BALF cells, and whole lung tissue for the two groups killed 18 hours after instillation of saline or LPS. There was no difference in total lung radioactivity recovered compared to the total radiolabel instilled between the Control 18 and LPS 18 groups (46.0 ± 4.6% and 48.4 ± 4.0%; respectively). There was significantly less radiolabel recovered in the BALF (p < 0.05) and significantly more radiolabel associated with the BALF cells (p < 0.01) in the LPS group compared to the control group. There was no difference in radiolabel association with lung tissue between the two groups.

Figure 3 displays total recovered radioactivity associated with the cell free BALF, the isolated BALF cells, and whole lung tissue for the two groups killed 72 hours after instillation of saline or LPS. There was no difference in the total lung radiolabel recovered compared to the total radiolabel instilled between the Control 72 and LPS 72 groups (47.3 ± 4.0% and 44.8 ± 3.8%; respectively). There was significantly less radiolabel recovered in the BALF (p < 0.05) and significantly more radiolabel liposomes associated with the BALF cells (p < 0.01) in the LPS group compared to the control group. There was no difference in radiolabel associated with lung tissue between the two groups.

Additional controls were performed in which animals (n = 2) received radiolabel liposomes followed by whole lung lavage and lung tissue homogenization 5 minutes after the instillation procedure. Mean total lung radiolabel recovery for this control group was 77% suggesting that ~25% of the radiolabel liposomes were lost in the instillation procedure (i.e., syringe, catheter), or possibly were adhered to the airway epithelium. The distribution of the recovered liposomal radioactivity within the 5-minute control lungs was 91% associated with the BALF, 2% associated with BALF cells, and 7% associated with lung tissue.

Table 2 demonstrates the cell recovery and cell differential after type II cell isolation for the two experimental groups at the 18-hour time point. Type II cell recovery for the control group ranged from 10.4 × 10⁶ to 21.6 × 10⁶ cells with a mean of 17.1 × 10⁶. The purity of this cell fraction averaged greater than 80% type II cells and viability was greater than 95%. Plate cell recovery from the control group ranged from 1.8 × 10⁶ to 4.7 × 10⁶ cells with a mean value of 3.2 × 10⁶. This cell fraction was predominately macrophages and the viability was greater than 90%. For the LPS group at the 18-hour time point, type II cell recovery ranged from 10.6 × 10⁶ to 20.5 × 10⁶ cells and a mean of 16.3 × 10⁶ cells, with a purity greater than 85% type II cells and viability greater than 95%. Plate cells from the LPS 18 group ranged from 2.7 × 10⁶ to 13 × 10⁶ cells with a mean of 6.6 × 10⁶ cells. This cell population consisted primarily of neutrophils and macrophages and viability was greater than 90%. There were no significant differences in cell numbers obtained from control and LPS animals 18 hours after saline or LPS instillation.

Figure 4 reveals the radioactivity per million tissue cells for the isolated type II cells and the other tissue associated cells (plate cells) for the control and LPS 18-hour groups. There were no significant differences in the radioactivity per type II cell or plate cell between the control group and the LPS group at the 18-hour time point after instillation.

Table 3 shows the cell recovery and cell differential after type II cell isolation for the two experimental groups at the 72-hour time point. Type II cell recovery for the control group ranged from 21.2 × 10⁶ to 40 × 10⁶ cells with a mean of 28.4 × 10⁶. The purity of this cell fraction averaged 75% type II cells and viability was greater than 95%. The number of cells obtained from the plate group ranged from 1.8 × 10⁶to 15.9 × 10⁶ cells with a mean value of 6.2 × 10⁶ with the primary cell type being macrophages with a small percentage of neutrophils and type II cells. Viability for the plate cells were greater than 90%. For the LPS group at the 72-hour time point, type II cell recovery ranged from 19.2 × 10⁶ to 64.6 × 10⁶ cells and a mean of 33.2 × 10⁶ cells, purity greater than 80% type II cells and viability greater than 95%. Plate cells from the LPS 18-hour group ranged from 3.3 × 10⁶ to 16.2 × 10⁶ cells with a mean of 7.8 × 10⁶ cells. This cell population consisted primarily of macrophages and viability was greater than 90%. There were no significant differences in cell recoveries between the control and LPS groups at the 72-hour time point. Of note, there were a greater number of cells recovered from both 72-hour groups compared to both 18-hour groups.

Figure 5 reveals the radioactivity per million tissue cells for the isolated type II cells and the other tissue-associated cells (plate cells) for the control and LPS 72-hour groups. There were no significant differences in the radioactivity per type II cell or plate cell between the LPS group and the control group 72 hours after the appropriate instillation. Of note there was no significant difference in radioactivity per cell for both the type II cells and plate cells between the two 72-hour groups and the two 18-hour groups.