Document Detail

Localization of hCAP-18 on the surface of chemoattractant-stimulated human granulocytes: analysis using two novel hCAP-18-specific monoclonal antibodies.
MedLine Citation:
PMID:  17400609     Owner:  NLM     Status:  MEDLINE    
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.
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:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2007-03-30
Journal Detail:
Title:  Journal of leukocyte biology     Volume:  82     ISSN:  0741-5400     ISO Abbreviation:  J. Leukoc. Biol.     Publication Date:  2007 Jul 
Date Detail:
Created Date:  2007-07-02     Completed Date:  2007-09-05     Revised Date:  2007-12-03    
Medline Journal Info:
Nlm Unique ID:  8405628     Medline TA:  J Leukoc Biol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  161-72     Citation Subset:  IM    
Montana State University, Department of Microbiology, 109 Lewis Hall, Bozeman, MT 59715, USA.
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MeSH Terms
Alkaline Phosphatase
Antibodies, Monoclonal / diagnostic use
Antimicrobial Cationic Peptides / analysis*,  metabolism
Chemotactic Factors / pharmacology
Epitope Mapping
Granulocytes / chemistry*,  drug effects
N-Formylmethionine Leucyl-Phenylalanine / pharmacology*
Protein Binding
Protein Transport
Grant Support
Reg. No./Substance:
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 Phosphatase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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