Endocytosis of heparin-binding protein (CAP37) is essential for the enhancement of lipopolysaccharide-induced TNF-alpha production in human monocytes.
Heparin-binding protein (HBP), also known as CAP37, is a proteolytically inactive serine protease homologue that is released from activated granulocytes. However, HBP is not a biologically inactive molecule but rather a multifunctional protein with properties that include the enhancement of LPS-induced TNF-alpha production from monocytes. We have previously demonstrated that HBP is internalized in monocytes. In the current study, we hypothesize that HBP is internalized in monocytes via endocytosis, and this internalization is an important mechanism by which HBP enhances LPS-induced TNF-alpha release. Using whole blood from healthy donors and flow cytometry, we found that colchicine (0.1-10 mM), cytochalasin D (1000 microM), NH4Cl (10-50 mM), and bafilomycin A1 (0.1-3 microM) significantly reduced the affinity of FITC-HBP for CD14-positive monocytes. Using isolated human monocytes and ELISA, we found that colchicine (0.1 mM), cytochalasin D (30 and 300 microM), NH4Cl (30 mM), and bafilomycin A1 (1 microM) significantly reduced the effect of HBP (10 microg/ml) to enhance LPS (10 ng/ml)-induced TNF-alpha release after 24 h. These findings demonstrate that internalization of HBP in monocytes is essential for the enhancement of LPS-induced TNF-alpha release. Transport of HBP to an activating compartment depends on intact F-actin polymerization and endosomal acidification, an important mechanism for endosomal protein sorting and trafficking.
Heinzelmann
M
M
Department of Physiology and Biophysics, The Price Institute of Surgical Research, University of Louisville, School of Medicine, KY 40292, USA. m.heinzelmann@bluewin.ch
Platz
A
A
Flodgaard
H
H
Polk
H C
HC
Miller
F N
FN
D000276:Adjuvants, Immunologic / Q000502:physiology*; D000643:Ammonium Chloride / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins / Q000378:metabolism* / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism* / Q000502:physiology; D003078:Colchicine / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects / Q000276:immunology*; D006801:Humans; D008070:Lipopolysaccharides / Q000276:immunology* / Q000494:pharmacology; D018942:Macrolides; D009000:Monocytes / Q000276:immunology / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
C062991:AZU1 protein, human; D000276:Adjuvants, Immunologic; D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins; D002352:Carrier Proteins; D008070:Lipopolysaccharides; D018942:Macrolides; D014409:Tumor Necrosis Factor-alpha; D000643:Ammonium Chloride; D015638:Cytochalasin D; C040929:bafilomycin A1; D003078:Colchicine