| Localization of hCAP-18 on the surface of chemoattractant-stimulated human granulocytes: analysis using two novel hCAP-18-specific monoclonal antibodies. | |
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MedLine Citation:
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PMID: 17400609 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The well-described antimicrobial and immunoregulatory properties of human cathelicidin antimicrobial protein 18 (hCAP-18) derive in part from the ability of its proteolytic fragment, LL-37 (a.k.a. CAP-37), to associate with activated immune and epithelial cells during inflammation. We now show a stable association between hCAP-18 and the cell surface of formyl-Met-Leu-Phe (fMLF)-stimulated neutrophils using two novel hCAP-18-specific mAb, H7 and N9, which recognize a single 16-kDa band, identified by N-terminal sequencing and mass spectrometry as hCAP-18. Phage display analysis of epitope-binding sites showed that both mAb probably recognize a similar five amino acid sequence near the C terminus of the prodomain. Immunoblot analysis of degranulated neutrophil supernatants resulted in mAb recognition of the 14-kDa prodomain of hCAP-18. Subcellular fractionation of unstimulated neutrophils on density gradients showed expected cosedimentation of hCAP-18 with specific granule lactoferrin (LF). fMLF stimulation resulted in an average 25% release of specific granule hCAP-18, with approximately 15% of the total cellular hCAP-18 recovered from culture media, and approximately 10% and approximately 75%, respectively, codistributing with plasma membrane alkaline phosphatase and specific granule LF. Surface association of hCAP-18 on fMLF-stimulated neutrophils was confirmed by immunofluorescence microscopy and flow cytometry analysis, which also suggested a significant up-regulation of surface hCAP-18 on cytochalasin B-pretreated, fully degranulated neutrophils. hCAP-18 surface association was labile to 10 mM NaOH treatment but resistant to 1 M NaCl and also partitioned into the detergent phase following Triton X-114 solubilization, possibly suggesting a stable association with one or more integral membrane proteins. We conclude that fMLF stimulation promotes redistribution of hCAP-18 to the surface of human neutrophils. |
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Authors:
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Jamal Stie; Andrew V Jesaitis; Connie I Lord; Jeannie M Gripentrog; Ross M Taylor; James B Burritt; Algirdas J Jesaitis |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't Date: 2007-03-30 |
Journal Detail:
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Title: Journal of leukocyte biology Volume: 82 ISSN: 0741-5400 ISO Abbreviation: J. Leukoc. Biol. Publication Date: 2007 Jul |
Date Detail:
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Created Date: 2007-07-02 Completed Date: 2007-09-05 Revised Date: 2007-12-03 |
Medline Journal Info:
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Nlm Unique ID: 8405628 Medline TA: J Leukoc Biol Country: United States |
Other Details:
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Languages: eng Pagination: 161-72 Citation Subset: IM |
Affiliation:
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Montana State University, Department of Microbiology, 109 Lewis Hall, Bozeman, MT 59715, USA. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Alkaline Phosphatase Antibodies, Monoclonal / diagnostic use Antimicrobial Cationic Peptides / analysis*, metabolism Chemotactic Factors / pharmacology Epitope Mapping Epitopes Granulocytes / chemistry*, drug effects Humans Lactoferrin N-Formylmethionine Leucyl-Phenylalanine / pharmacology* Neutrophils Protein Binding Protein Transport |
| Grant Support | |
ID/Acronym/Agency:
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2R01-AI22735/AI/NIAID NIH HHS; 2R01-AI26711/AI/NIAID NIH HHS; T32 AI 07465/AI/NIAID NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Antibodies, Monoclonal; 0/Antimicrobial Cationic Peptides; 0/Chemotactic Factors; 0/Epitopes; 0/Lactoferrin; 143108-26-3/CAP18 lipopolysaccharide-binding protein; 59880-97-6/N-Formylmethionine Leucyl-Phenylalanine; EC 3.1.3.1/Alkaline Phosphatase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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