Previous Article | Next Article 
Infection and Immunity, December 1998, p. 5948-5954, Vol. 66, No. 12
Respiratory and Neurologic Disease Research
Unit, National Animal Disease Center, Agricultural Research Service,
U.S. Department of Agriculture, Ames, Iowa
500101;
Department of Veterinary
Pathology, Iowa State University, Ames, Iowa
500112; and
Joint Program in
Neonatology, Department of Medicine, Boston Children's Hospital,
Boston, Massachusetts 021153
Received 6 May 1998/Returned for modification 13 July 1998/Accepted 4 September 1998
Three small antimicrobial anionic peptides (AP) were originally
isolated from an ovine pulmonary surfactant. However, their presence in
bronchoalveolar lavage (BAL) fluid and tissues of the respiratory tract
is unknown. In this study, we made affinity-purified rabbit polyclonal
and mouse monoclonal antibodies to synthetic H-DDDDDDD-OH. Antibody
specificity was assessed by a competitive enzyme-linked immunosorbent
assay (ELISA), and the exact epitope binding sites were determined with
analog peptides synthesized on derivatized cellulose. These antibodies
were used to detect AP in BAL fluid by ELISA and in respiratory tissues
by Western blot analysis and immunocytochemistry. BAL fluid from 25 sheep contained 0.83 ± 0.33 mM AP (mean ± standard
deviation; range, 0.10 to 1.59 mM) and was antimicrobial. The presence
of AP in BAL fluid was confirmed by reverse-phase high-pressure liquid chromatography fractionation followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry on those fractions which were positive by competitive ELISA and demonstrated antimicrobial activity. In Western blots, polyclonal antibody PAB96-1 and monoclonal antibody 1G9-1C2 (5.0 µg/ml) detected four bands in solubilized turbinate and tracheal epithelial cells (53.7, 31.2, 28.0, and 25.7 kDa) and five bands in lung homogenates (53.5, 37.1, 31.2, 28.0, and
25.7 kDa). Only a single band was seen in solubilized liver and
small-intestine homogenates, and no bands were seen in blots containing
BAL fluid, albumin, or kidney or spleen homogenates. In
pulmonary-tissue sections, both antibodies PAB96-1 and 1G9-1C2 identified accumulated protein in the apical cytoplasm of the bronchial
and bronchiolar epithelia, in the cytoplasm of pulmonary endothelial
cells, and in an occasional alveolar macrophage. As a first step in
identifying a candidate AP precursor gene(s), degenerate
oligonucleotides representing all possible coding combinations for
H-GADDDDD-OH and H-DDDDDDD-OH were synthesized and used to probe
Southern blots of sheep genomic DNA. Following low-stringency washes
and a 2-day exposure, strongly hybridizing bands could be identified.
One degenerate oligonucleotide, SH87, was used as a hybridization probe
to screen a sheep phage genomic library. Two independent phage
contained the H-GADDDDD-OH coding sequence as part of a larger
predicted protein. AP may originate as part of an intracellular
precursor protein, with multistep processing leading to the release of
the heptapeptide into mucosal secretions. There it may interact with
other innate pulmonary defenses to prevent microbial infection.
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Detection of Anionic Antimicrobial Peptides in
Ovine Bronchoalveolar Lavage Fluid and Respiratory
Epithelium
*
Corresponding author. Mailing address: Respiratory and
Neurologic Disease Research Unit, National Animal Disease Center,
Agricultural Research Service, U.S. Department of Agriculture, P.O. Box
70, Ames, IA 50010. Phone: (515) 239-8593. Fax: (515) 239-8458. E-mail: kbrogden{at}nadc.ars.usda.gov.
Infection and Immunity, December 1998, p. 5948-5954, Vol. 66, No. 12
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Grubor, B., Meyerholz, D. K., Ackermann, M. R.
(2006). Collectins and cationic antimicrobial peptides of the respiratory epithelia.. Vet Pathol
43: 595-612
[Abstract] [Full Text] -
Rao, A., Chopra, S., Ram, G., Gupta, A., Ranganathan, A.
(2005). Application of the "Codon-shuffling" Method: SYNTHESIS AND SELECTION OF DE NOVO PROTEINS AS ANTIBACTERIALS. J. Biol. Chem.
280: 23605-23614
[Abstract] [Full Text] -
Levy, O.
(2004). Antimicrobial proteins and peptides: anti-infective molecules of mammalian leukocytes. J. Leukoc. Biol.
76: 909-925
[Abstract] [Full Text] -
Cole, A. M., Liao, H.-I, Stuchlik, O., Tilan, J., Pohl, J., Ganz, T.
(2002). Cationic Polypeptides Are Required for Antibacterial Activity of Human Airway Fluid. J. Immunol.
169: 6985-6991
[Abstract] [Full Text] -
Fales-Williams, A. J., Brogden, K. A., Huffman, E., Gallup, J. M., Ackermann, M. R.
(2002). Cellular Distribution of Anionic Antimicrobial Peptide in Normal Lung and during Acute Pulmonary Inflammation. Vet Pathol
39: 706-711
[Abstract] [Full Text] -
Fales-Williams, A. J., Gallup, J. M., Ramirez-Romero, R., Brogden, K. A., Ackermann, M. R.
(2002). Increased Anionic Peptide Distribution and Intensity during Progression and Resolution of Bacterial Pneumonia. CVI
9: 28-32
[Abstract] [Full Text] -
Augusto, L., Le Blay, K., Auger, G., Blanot, D., Chaby, R.
(2001). Interaction of bacterial lipopolysaccharide with mouse surfactant protein C inserted into lipid vesicles. Am. J. Physiol. Lung Cell. Mol. Physiol.
281: L776-L785
[Abstract] [Full Text] -
Brogden, K. A., Ackermann, M. R., McCray, P. B. Jr., Huttner, K. M.
(1999). Differences in the Concentrations of Small, Anionic, Antimicrobial Peptides in Bronchoalveolar Lavage Fluid and in Respiratory Epithelia of Patients with and without Cystic Fibrosis. Infect. Immun.
67: 4256-4259
[Abstract] [Full Text] -
Cole, A. M., Dewan, P., Ganz, T.
(1999). Innate Antimicrobial Activity of Nasal Secretions. Infect. Immun.
67: 3267-3275
[Abstract] [Full Text]
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»