title: A neutrophil-derived proteolytic inactive elastase homologue (hHBP) mediates reversible contraction of fibroblasts and endothelial cell monolayers and stimulates monocyte survival and thrombospondin secretion.
abstract: Human heparin-binding protein (hHBP) is a recently discovered proteolytically inactive neutrophil elastase homologue with sequence identity to azurocidin and CAP37. The protein has antibacterial properties and chemotactic activity toward monocytes. In the present work, we show that monocytes, cultured under serum-free conditions, developed morphological changes and formed multicellular aggregates 4 h after the addition of hHBP at a concentration of 10 micrograms/ml. However, after prolonged incubation (11 days) with unchanged medium, the cells spread again. The hHBP-treated cells had a two- to threefold increase in survival compared to control cells, measured using trypan blue as an indicator of living cells. Differentiation of the alive cells to macrophages was detected by changes in morphology, a threefold increase in protein content, and a three- to fourfold increase in acid phosphatase activity. When monocytes in parallel experiments were labelled with [35S]methionine de novo synthesis and secretion of thrombospondin in a dose-dependent manner was observed after 16 h, with half-maximal secretion at 2 micrograms hHBP/ml and a maximal 12-fold increase in secretion with respect to controls at 16 micrograms/ml. Supplementary labeling with [35S]sulfate revealed that the same monocytes down-regulated the secretion of a large proteoglycan (300-400 kd), apparently also with a half-maximal decrease rate at 2 micrograms/ml hHBP. Exposure of confluent fibroblast and endothelial cell monolayers to hHBP (10 micrograms/ml) in the absence of fetal calf serum resulted in cell contraction leaving gaps between cells, the phenomenon being recognizable within 4 h after addition of hHBP. Addition of fetal calf serum to a concentration of 10% completely restored the monolayers. A unique role of hHBP in host defense involving recruitment of monocytes and a key function of hHBP in neutrophil extravasation in response to inflammatory chemotactic signals such as leukotriene B4, complement peptide C5a, and N-formyl-methionyl-leucyl-phenylalanine are suggested.
authors.affiliation: Novo Nordisk A/S, Gentofte, Denmark.
authors.forename: E
authors.initials: E
authors.lastname: Ostergaard
authors.forename: H
authors.initials: H
authors.lastname: Flodgaard
mesh_terms: D000595:Amino Acid Sequence; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002634:Chemotaxis, Leukocyte; D016207:Cytokines / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology* / Q000378:metabolism; D009504:Neutrophils / Q000502:physiology*; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011480:Protease Inhibitors / Q000737:chemistry; D012636:Secretory Rate / Q000187:drug effects; D019699:Thrombospondins; D013997:Time Factors
chemical_list: D016207:Cytokines; D010980:Platelet Membrane Glycoproteins; D011480:Protease Inhibitors; D019699:Thrombospondins; D010196:Pancreatic Elastase