Secretion of heparin-binding protein from human neutrophils is determined by its localization in azurophilic granules and secretory vesicles

Abstract

Human neutrophils have an important role in host defense against microbial infection. At different stages of an infectious process, neutrophils progressively up-regulate receptors and release various effector molecules. These are stored in several distinct types of granules with varying propensity to be secreted. Heparin-binding protein (HBP), also known as CAP37 or azurocidin, is a multifunctional, inactive serine-protease homologue. The present work shows that HBP is released from neutrophils on stimulation with secretagogues that do not trigger the secretion of azurophilic granule content. Therefore, the subcellular localization of HBP was investigated in more detail. Immunofluorescence microscopy revealed that HBP was localized close to the plasma membrane. Further analysis by fractionation of postnuclear supernatants from cavitated neutrophils showed that HBP is stored in azurophilic granules and secretory vesicles but that it is also detected to a minor extent in the plasma membrane. IntroductionPolymorphonuclear leukocytes (PMNs) have an important role in early host defense against invading microorganisms (for reviews, see references 1 and 2). Recruitment of these cells from the bloodstream to a site of infection involves their recognition of inflammatory mediators, their binding to adhesion molecules of the vascular endothelium, and their migration across the endothelial barrier. 3 How efficiently neutrophils perform these tasks depends on a sophisticated mobilization mechanism that triggers the release of granule contents and the concomitant up-regulation of various receptors to the plasma membrane. 4 Secretory processes are also important for the extravascular migration of neutrophils through tissues. Once the cells have reached the focus of infection, they are fully activated and are able to fight the infection by secreting reactive oxygen intermediates, antimicrobial peptides, and degradative enzymes. 2 These substances can be preferentially targeted to phagosome compartments to achieve efficient killing and degradation of internalized microorganisms.Lately, much interest has been focused on the various granule types of neutrophils and their sequential mobilization during the inflammatory process (for review, see reference 5). Analysis of these granules by electron microscopy and subcellular fractionation has demonstrated that neutrophils have at least 4 different granule or vesicle types. [6][7][8][9] These are the primary or azurophilic granules that contain myeloperoxidase (MPO), bactericidal proteins, and proteinases; the secondary or specific granules that store lactoferrin and enzymes such as collagenase and gelatinase; the tertiary or gelatinase granules that, like specific granules, contain tissuedegrading enzymes; and the secretory vesicles, an easily mobilizable compartment, that contain alkaline phosphatase and plasma proteins such as human serum albumin. The 4 granule types are mobilized at different stages of the inflammatory process; secretory vesicles are more readily secreted than th...

Neutrophil-derived azurocidin alarms the immune system

“…Azurocidin is released almost completely after granule mobilization. In contrast to, e.g., HNPs, which are released mainly into the phagolysosome [21], 90% of the azurocidin are released upon degranulation [17,19]. These three distinct properties of azurocidin favor the promiscuous mode of action that this protein displays.…”

“…Azurocidin, however, possesses some features that make it unique among the PMN granule proteins: Azurocidin is the only PMN granule protein stored in two different compartments. As a result of its storage in secretory vesicles and primary granules, azurocidin is released at a very early stage of PMN extravasation as well as at a later stage when the PMN has reached the site of inflammation [17], thereby allowing it to target cells in the bloodstream, the endothelial lining, and the extravascular environment. The amino acid sequence and the three-dimensional structure of azurocidin have been unveiled and show that azurocidin is a member of the serine protease superfamily.…”

“…Another intriguing feature of azurocidin-mediated chemotaxis is the relatively low concentration required to carry out this action. Although micromolar concentrations are needed for its antimicrobial activity, nanomolar concentrations are sufficient for its chemotactic activity, a concentration range that is reachable in the tissue [17].…”

Attenuated, Oncolytic, but Not Wild-Type Measles Virus Infection Has Pleiotropic Effects on Human Neutrophil Function

“…To determine whether MV-Vac infection was directly affecting neutrophil granule release, we quantified granule exocytosis by flow cytometry, taking advantage of the fact that increases in cell surface expression of CD66b (specific granule marker), CD35 (secretory granule marker), and CD63 (azurophil granule marker) are consequent on the relevant granule exocytosis (25,26). Fig.…”

Neutrophil-Derived Hyperresistinemia in Severe Acute Streptococcal Infections

“…Subcellular fractionation of neutrophils was performed by density centrifugation on Percoll gradient as described by Kjedsel et al (19), with some minor modifications (20). Briefly, polymorphonuclear neutrophils were purified and adjusted to 5 ϫ 10 7 cells/ml, followed by their disruption by nitrogen cavitation.…”