Materials and Methods

Cytokines, Antibodies, and Other Reagents Recombinant human interleukin-lji (IL-1) was purchased from Cistron (Pine Brook, NJ); recombinant human tumor necrosis factor-a (TNF-a) was purchased from Genzyme (Cambridge, MA). N-formyl-methionyl-leucyl-phenylalanine (fMLP) and diaminobenzidine (DAB) were purchased from Sigma Chemical Co.(St. Louis, MO). Murine monoclonal antibodies (mAbs) to CDlla (TSlI22) and CD18 (TSl/18) were provided by Dr. Timothy Springer (Boston, MA); mAb to CDllb (anti-Mol, clone 44) was provided by Dr. Robert Todd, Jr.(Ann Arbor, MI); mAb to the type 1 complement receptor (anti-CRl, clone 3D9) was provided by Dr. Melvin Berger (Cleveland, OH); mAb to CDw32 (anti-FcR IT, clone N. 3) was provided by Drs. Michael Fanger and Paul Guyre (Hanover, NH); mAb to ICAM-l was prepared from clone R6. 5-D6, hereafter abbreviated R6. 5 (16); mAb W6/32 to a common HLA determinant was purchased from Seralab (Sussex, UK). Purified collagen (Vitrogen-100@) was purchased from Collagen Corp.(Palo Alto, CA). Fibronectin was purchased from Telios Pharmaceuticals (San Diego, CA).

Tracheal Epithelial Cell (TEC) Cultures Human tracheal segments obtained at autopsy were treated as previously described with trypsin and EDTA to isolate the mixed population of separate epithelial cells from the underlying connective tissue (17). The epithelial cells were cultured as previously described on either 25-mm-diameter round glass coverslips or 25-cm2culture flasks that had been coated with a mixture that contained fibronectin (1 p. gl dl), bovine serum albumin (1mg/dl), and collagen (3 mg/dl). When the cells reached confluence, the monolayers on the coverslips were employed in the PMN adherence assay described below, and the monolayers in the flasks were harvested in unicellular suspension for measurement ofICAM-l surface expression by immunofluorescence flow cytometry. For some experiments, confluent epithelial monolayers were treated with TNF-a (250 U/m!) or IL-l (5 U/ml) for 24 or 48 h before subsequent assays. An immortalized cell line, designated 9HTEo-, derived from human TEC by transformation with an origin-defective simian virus-40 (18), was similarly cultured on coverslips and in 25-cm2flasks, and these cells were studied in the adherence and ICAM-l expression assays.