Materials and Methods

Materials Blood was obtained from normal human volunteers and mixed with one-tenth vol of 3.8% sodium citrate. Plateletpoor plasma was prepared by centrifuging citrated blood at 2,000 x g for 20 min at 4 C. Rabbit brain thromboplastin (tissue factor), human thrombin and fibrinogen, specific coagulation deficient plasmas, phorbol myristate acetate (PMA), and bacterial lipopolysaccharide (LPS) were obtained from Sigma Chemical Co.(St. Louis, MO). A fulllength murine tissue factor cDNA in pGEM2 was purchased from American Type Culture Collection (Rockville, MD)(21).(32P] dCTP was obtained from Amersham (Arlington Heights, IL).

Cell Isolation All cells were obtained from specific pathogen-free, male Sprague-Dawley rats weighing 200 to 300 g. AEC were isolated as previously described (18, 19, 22). This procedure involves the intratracheal instillation of elastase to release epithelial cells from the alveolar basement membrane. Cells were purified by panning over IgG-coated plastic tissue culture plates to remove contaminating cells bearing Fc receptors. Freshly isolated epithelial cells were plated in 6-, 16-, or 60-mm tissue culture dishes (Costar, Cambridge, MA) at approximately 2.5 x 105 cells/em'in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 100 U/ml penicillin, 100 pg/mlstreptomycin, and 0.25 pg/mlamphotericin B (GIBCO, Grand Island, NY). All cells were incubated at 37 C in humidified 5% CO2/95% air. Epithelial cells formed confluent monolayers by 48 h in culture. At that time, there were> 90% AEC, which were> 95% viable as assessed by trypan blue dye exclusion, as previously reported (18, 19, 22). To determine cell number on the day of harvest, the monolayers were lysed with a solution containing 0.1% crystal violet, 0.1 M citric acid, and 0.1% Nonidet P-40 (Sigma) and the stained nuclei counted with a hemocytometer.