Recovery of an Epitope Recognized by a Novel Monoclonal Antibody from Airway Lavage during Experimental Induction of Chronic Bronchitis Takeshi Koshino, K. Ramakrishnan Bhaskar, Lynne M. Reid, Craig Gerard, Angeline Warner, Stephanie A. Shore, Kristin Anderson, Greg Butler, Hideya Iijima, and Jeffrey M. Drazen Respiratory Biology Program, Harvard School of Public Health, Departments of Pathology, Children's Hospital, and Departments of Medicine, Beth Israel Hospital, Brigham and Women's Hospital, Children's Hospital (Ina Sue Perlmutter Laboratory), and Harvard Medical School, Boston, Massachusetts

Prolonged exposure of dogs to high concentrations of S02 gas results in a syndrome with many of the characteristics of human chronic bronchitis, including cough and chronic mucous hypersecretion as well as airway obstruction. We developed and used a novel monoclonal antibody, GB-4B, raised against epithelial glycoprotein isolated from human hypersecretory mucus to probe airway lavage samples from dogs before and during prolonged exposure to S02 gas. There were relatively low mean titers of the epitope recognized by GB-4B in airway lavage fluid as evidenced by enzyme-linked immunosorbent assay before exposure to S02 gas. After 25 to 50 wk of S02 exposure, the dogs showed a significant increase in pulmonary resistance and there was a significant increase in the titer of the epitope in the airway lavage fluid. Using the same antibody immunohistochemical analysis of airway tissues from SOrexposed dogs revealed patchy staining of the mucous glands and airway secretory cells and dense staining along the airway surface; airway tissue from control dogs and one S02-exposed dog whose lavage fluid did not contain the epitope showed little or no staining. These data demonstrate that similar mucin epitopes appear in airway lavage fluid under hypersecretory conditions in both animals and humans. The epitope may have utility as a marker of chronic mucous hypersecretion.

The clinical syndrome of chronic bronchitis in humans is characterized by prolonged sputum production (1). The pathological counterpart ofthis syndrome is hypertrophy and hyperplasia of the airway mucous-producing cells and airway inflammation (2). Although chronic bronchitis usually takes decades to develop in humans, Chakrin and Saunders (3) described a canine model of chronic bronchitis induced by high-dose S02 exposure in which a syndrome reminiscent of the human disease develops in 4 to 6 mo. We have previously not only confirmed these aspects of the model (4, 5), we have also demonstrated that epithelial glycoprotein of typical buoyant density was not detectable in "normal" canine airway lavage fluid but appeared after prolonged exposure to S02 (5, 6). In the present study, we used a monoclonal antibody raised against human bronchial epithelial glycoprotein obtained from an individual with sterile muKey Words: sulfur dioxide, chronic bronchitis, mucous glycoprotein, immunohistochemistry, enzyme-linked immunosorbent assay (Received in original form August 15, 1989 and in revised form December 28, 1989) Address correspondence to: Jeffrey M. Drazen, M.D., Respiratory Biology Program, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115. Abbreviations: density gradient ultracentrifugation, DGU; periodic acid Schiff, PAS; pulmonary resistance, RL. Am. J. Respir. Cell Mol. BioI. Vol. 2. pp. 453-462, 1990

cous hypersecretion, monoclonal GB-4B, as a probe to study canine airway lavage fluid before and during the development of bronchitis induced by the exposure to S02 gas. We found that this antibody recognized an epitope in airway lavage fluid from some dogs before S02 exposure but at higher titer in all but one dog after chronic exposure to S02 gas. These data demonstrate that there are similar alterations in the processing of airway mucins after prolonged exposure to inflammatory stimuli in animals and in humans; such differences may have utility as markers of the bronchitic state.

Materials and Methods Materials and Statistics Guanidine HCI, BSA, horseradish peroxidase-labeled goat anti-mouse IgG, 2-mercaptoethanol, 3, 3'-diaminobenzidine (Sigma Chemical Co., St. Louis, MO), light green SF histochemical stain (Fisher Scientific, Boston, MA), polyvinyl microtiter plates (96-well EIA plate; Costar, Cambridge, MA), nonfat dry milk (Carnation Co., Los Angeles, CA), protein assay reagent (Bio-Rad No. 23200; Pierce Chemical Co., Rockford, IL), and Vector ABC staining kits (Vector Laboratories, Burlingame, CA) were obtained as noted. All other chemicals were reagent grade. Student's t test or analysis of variance (ANOVA)were used to determine the significance of the data obtained; a P value of 0.05 or less was considered significant.

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Monoclonal Antibody Immunogen preparation. Sterile bronchial mucus aspirated from an acute quadriplegic patient was used as the source for the purification of mucous glycoprotein, which served as the immunogen for the monoclonal antibody. The mucous glycoprotein was isolated from the bronchial secretions using density gradient ultracentrifugation (DGD) in cesium salts (6, 7) (Figure 1). After initial purification using a CsBr gradient, the glycoconjugate fraction was treated with DNAse I, suspended in CsCI, and subjected to a second DG U. Seven fractions of equal volume were collected by pipetting from the top, and aliquots of the fractions were assayed for hexose content. Densities of the fractions were determined for the aliquots. Fraction no. 4, with the highest hexose content, was used as the immunogen. To further establish macromolecular purity, an aliquot of fraction no. 4 was analyzed by analytical DGD (Figure 1). Monoclonal antibody development. One hundred microliters of the immunogen (5.0 mg/ml) (in complete Freund's adjuvant) was injected intraperitoneally into BALB/c mice. Each mouse was boosted at 3-wk intervals with subcutaneously administered immunogen in incomplete Freund's adjuvant (first booster) followed by intravenously administered

A.

immunogen in phosphate-buffered saline, pH 7.4 (second booster). One week after the second booster, immune spleen cells were fused with NSI myeloma cells to produce hybridoma cell lines using standard protocols (8); those that gave a positive reaction with the antigen were split and subcloned to assure monoclonality. Medium from one subclone, GB4B, an IgG 1 subtype, was used as noted below. Characterization ofimmunogen and antibody. The glycoprotein fraction used as the immunogen (Figure 1) was subjected to analytical ultracentrifugation (Model E; Beckman) in CsBr to establish purity (9, 10). Carbohydrate composition of the glycoprotein was determined by gas-liquid chromatography oftrimethylsilyl derivatives followingthe procedures of Clamp and colleagues (11). To establish that the antigen was associated with a macromolecule, the glycoprotein was subjected to gel filtration (Biogel A5M; 50 mM Tris, pH 7.0, 1.5 M KCI) and the eluate screened for immunoreactivity using GB-4B. The immunogen's ability to react with GB-4B in the solid phase ELISA was tested after treatment with alkaline borohydride (12), trifluoromethanesulfonic acid (13), or proteases. Reactivity of the antibody with bovine gall bladder mucus and pig gastric mucus (kindly provided by Dr. 1. Thomas Lamont) was also tested.

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