Journal of Leukocyte Biology
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Originally published online as doi:10.1189/jlb.0906586 on March 30, 2007

Published online before print March 30, 2007
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(Journal of Leukocyte Biology. 2007;82:161-172.)
© 2007 by Society for Leukocyte Biology

Localization of hCAP-18 on the surface of chemoattractant-stimulated human granulocytes: analysis using two novel hCAP-18-specific monoclonal antibodies

Jamal Stie, Andrew V. Jesaitis, Connie I. Lord, Jeannie M. Gripentrog, Ross M. Taylor, James B. Burritt and Algirdas J. Jesaitis1

Montana State University, Department of Microbiology, Bozeman, Montana, USA

1 Correspondence: Montana State University, Department of Microbiology, 109 Lewis Hall, Bozeman, MT 59715, USA. E-mail: umbaj{at}montana.edu

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 ~15% of the total cellular hCAP-18 recovered from culture media, and ~10% and ~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.

Key Words: inflammation • leukocyte • exocytosis • cathelicidin






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