Purification of the antigenic components of pigeon dropping extract, the responsible agent for cellular immunity in pigeon breeder’s disease☆,☆☆,★

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Abstract

Background: Pigeon breeder’s disease (PBD) is a lung disease caused by inhalation of antigens derived from pigeons. Objective: This study was undertaken to characterize the responsible component of pigeon dropping extract (PDE) for PBD. Methods: First, crude PDE was applied to SDS-PAGE followed by immunoblotting by using antibodies in bronchoalveolar lavage (BAL) fluid. Second, 9 bands of PDE were separated by SDS-PAGE and used for antigen-induced PBMCs. Finally, amino-terminal sequencing was conducted on an isolated 21-kd protein by 2-dimensional electrophoresis. Results: Immunoblots with BAL fluid from patients with PBD identified 9 bands. Similar patterns were observed by using BAL fluid from 10 control patients (9 with summer-type hypersensitivity pneumonitis or idiopathic pulmonary fibrosis and 1 asymptomatic breeder), except for the 21-kd protein, which was detected in 10 patients with PBD and 1 asymptomatic breeder. The stimulation indices of PBMCs determined by using proteins electroeluted from the 9 bands were higher in patients with PBD than in the 10 control patients. The 21-kd protein was separated into 5 spots by 2-dimensional electrophoresis; these spots were all reactive with BAL fluid from patients with PBD as determined by immunoblotting. The sequence of the 21-kd protein had 57% identity to a Saccharomyces cerevisiae chromosome X reading frame. A synthetic peptide, derived from the amino acid sequence of the N-terminal of the native protein, induced significant proliferation of PBMCs obtained from 5 patients with PBD, but not with PBMCs obtained from control patients. Conclusion: The 21-kd protein is the only protein that identified individuals exposed to pigeons by immunoblotting. Only PBMCs from patients with PBD showed significant proliferation to the 21-kd protein and to the synthetic peptide on the basis of the N-terminal sequence of the native peptide. The 21-kd protein will be an important antigen for studies on the epidemiology, diagnosis, and pathogenesis of PBD. (J Allergy Clin Immunol 1999;103:1158-65.)

Section snippets

Preparation of PDE

Fresh pigeon droppings were collected and stirred with 20 volumes of PBS (0.15 mol/L NaH2 PO4 /K2 HPO4 /0.15 mol/L NaCl, pH 7.4) for 24 hours. The extract was centrifuged 3 times at 12,000 rpm for 30 minutes. The supernatant was collected and dialyzed against distilled water (DW) for 7 days and freeze-dried. This crude PDE was stored at –20°C until used.

Collection of patients’ bronchoalveolar lavage fluids and PBMCs

The diagnosis of PBD was confirmed by history, clinical symptoms, white blood cell count, C-reactive protein, arterial blood gas analysis,

SDS-PAGE

For SDS-PAGE, 2-dimensional electrophoresis, and Western blotting, 80 mg/mL crude PDE was dissolved in PBS and filtered through MINISART (Sartorius AG, Gottingen, Germany). Crude PDE was separated by SDS-PAGE with 10 mm high stacking gels composed of 4.95% acrylamide and bis acrylamide (T), with a cross-linking of 2.67% (C) (C, percentage ratio of bis-acrylamide to T), and 80 mm high running gels (T = 30% and C = 2.67 %) in the buffer system according to the Laemmli method.17 PDE was mixed with

Identification of major allergens in PDE

SDS-PAGE analysis of crude PDE revealed multiple protein bands (Fig 2).

. SDS-PAGE (15% polyacrylamide gel) patterns of PDE. Lane 1, Molecular weight standard marker; lane 2, CBB-R350 stain; lane 3, silver stain.

The spectrum of protein-staining bands ranged from about 21 to 199 kd. To determine which proteins in PDE were responsible for specific IgG binding, Western blotting was performed with crude PDE and BAL fluid from patients with PBD and control patients. Analysis of the IgG immunoblots with

DISCUSSION

HP is an immunologic lung disease that results from repeated, intense exposure to a wide variety of inhaled antigens. The list of inhaled antigens for HP continues to enlarge.21 The responsible antigenic components have been identified as mannose and mannaoproteins of Trichosporon asahii and Trichosporon mucoides for SHP and as the carbohydrate moieties (glucose, mannose, and galactose units) of the glycoproteins from Saccharopolyspora rectivirgula (formerly Micropolyspora faeni ) for farmer’s

Acknowledgements

We thank Dr Vernon L. Moore, Merck Research Laboratories (retired) for his critical review of the manuscript.

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    Supported by the Research Committee of the Japanese Ministry of Health and Welfare on Diffuse Pulmonary Diseases, Grant-in-Aid for Scientific Research 9670604 from the Japanese Ministry of Education, and the Charitable Trust of Okamoto Satoshi Fund for Fibrotic Lung Disorders.

    ☆☆

    Reprint requests: Yasuyuki Yoshizawa, MD, PhD, The Pulmonary Medicine, Tokyo Medical and Dental University, 5-45, Yushima 1-chome, Bunkyo-ku, Tokyo 113-8519, Japan.

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