Blood samples (10 mL) were collected between 6:30 and 7:00 am from each subject. Following centrifugation, 5 mL of serum w8ls frozen at — 20°C for later analysis. On the day of the test, a standard BAL was performed as described previously. Briefly, after local anesthesia of the upper airway with 4% lidocaine, a flexible fiberoptic bronchoscope (type p-20; Olympus BF; Olympus Co; Tokyo) was wedged into a subsegmental bronchus of the right middle lobe, or in patients with focal bronchiectasis, into areas demonstrating bronchiectatic changes. An aliquot of 50 mL of sterile saline solution at body temperature was instilled through the bronchoscope. The fluid was immediately retrieved by gentle suction using a sterile syringe and the procedure was repeated three times. BALF was passed through two sheets of gauze and centrifuged at 500Xg for 10 min at 4°C. After washing twice with phosphate-buffered saline solution (PBS) without calcium and magnesium (PBS; Gibco; West Sussex, UK), the cell pellets were suspended in PBS supplemented with 10% heat-inactivated fetal calf serum (FCS) and counted using a hemocy-tometer. An aliquot was diluted to a concentration of 2X10° cells per milliliter, and a differential cell count was performed on May-Giemsa-stained preparations (Cytospin 2; Shan don Instruments; Sewickley, Pa). The remaining fluid was centrifuged at 500Xg for 5 min at 4°C, and the supernatant was stored at — 80°C until examined.
The serum levels of soluble L-, E-, and P-selectin (sL-, sE-, and sP-selectin), ICAM-1 (sICAM-1), and VCAM-1 (sVCAM-1) w’ere measured using commercial assay kits (R&D Systems Europe; Abingdon, OX, UK). The serum samples were dilluted 1:20 for sE-, sP-selectin, and sICAM-1, 1:50 for sVCAM-1, and 1:100 for sL-selectin assays before the assay was performed. An enzyme-linked immunosorbent assay (ELISA) was used. It was standardized against a purified form of recombinant soluble molecules. The concentrations of interleukin (IL)-lp and IL-8 were also quantified using ELISA kits (IL-ip from Medgenix Diagnostics SA; Fleurus, Belgium; and IL-8 from Tore Fuji Bionix; Tokyo). Plates were read at 450 nm in an ELISA reader. Duplicate assays were performed for serial dilutions of each sample and the average value was recorded. The sensitivities of these assays w^ere 9.0 ng/mL, 0.4 ng/mL, 4.6 ng/mL, 2.8 ng/mL, 140 ng/mL, 6.3 pg/mL, and 3 pg/mL for sL-, E-, and P-selectin, sICAM-1, sVCAM-1, IL-lp, and IL-8, respectively.
In Vitro Effects of L-selectin Expression on Peripheral Blood Neutrophils
Venous blood was collected by heparinization from five healthy individuals selected at random; erythrocytes were separated with dextran, and neutrophils were isolated with Ficoll-Hypaque density gradient centrifugation. The cells were suspended with Hanks’ balanced salt solution (Gibco; Grand Island, NY) containing 0.25% bovine serum albumin at a cell density of 1X106 cells per milliliter. The viability of the cells obtained at that stage wras 95% or higher as confirmed with the trypan blue exclusion. Neutrophils w^ere stimulated with N-formyl-methionyl-leucyl-phenylalanine (FMLP; Sigma; St. Louis), recombinant human IL-8 (Pepro Tech Inc; Rocky Hill, NJ) or recombinat IL-lp (Boehringer Mannheim Biochemicals; Indianapolis, Ind) at 37°C for 15 min. The cells were then stained as described previously, using fluorescein isothioeyanate-conjugated anti-Leu-8 (Becton Dickinson; Mountain View, Calif), a monoclonal antibody to the L-selectin, and analysis was performed (using a FACSean; Becton Dickinson). In the next step, the neutrophils were treated with 1.0, 10, or 50 jxg/mL erythromycin at 37°C for 30 min and then stimulated with 50 or 500 ng/mL IL-8 at 37°C for 15 min followed by analysis.