Localization of hCAP-18 on the surface of chemoattractant-stimulated human granulocytes: analysis using two novel hCAP-18-specific monoclonal antibodies.
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.
Stie
Jamal
J
Montana State University, Department of Microbiology, 109 Lewis Hall, Bozeman, MT 59715, USA.
Jesaitis
Andrew V
AV
Lord
Connie I
CI
Gripentrog
Jeannie M
JM
Taylor
Ross M
RM
Burritt
James B
JB
Jesaitis
Algirdas J
AJ
D000469:Alkaline Phosphatase; D000911:Antibodies, Monoclonal; D023181:Antimicrobial Cationic Peptides / Q000032:analysis* / Q000378:metabolism; D002630:Chemotactic Factors / Q000494:pharmacology; D018604:Epitope Mapping; D000939:Epitopes; D006098:Granulocytes / Q000737:chemistry* / Q000187:drug effects; D006801:Humans; D007781:Lactoferrin; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils; D011485:Protein Binding; D021381:Protein Transport; D054804:Cathelicidins
D000911:Antibodies, Monoclonal; D023181:Antimicrobial Cationic Peptides; D002630:Chemotactic Factors; D000939:Epitopes; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000469:Alkaline Phosphatase; D007781:Lactoferrin; D054804:Cathelicidins