SummaryEffectors of the innate immune system, the antibacterial peptides, have pivotal roles in preventing infection at epithelial surfaces. Here we show that proteinases of the significant human pathogens Pseudomonas aeruginosa , Enterococcus faecalis , Proteus mirabilis and Streptococcus pyogenes, degrade the antibacterial peptide LL-37. Analysis by mass spectrometry of fragments generated by P. aeruginosa elastase in vitro revealed that the initial cleavages occurred at Asn-Leu and Asp-Phe, followed by two breaks at Arg-Ile, thus inactivating the peptide. Proteinases of the other pathogens also degraded LL-37 as determined by SDS-PAGE. Ex vivo , P. aeruginosa elastase induced LL-37 degradation in human wound fluid, leading to enhanced bacterial survival. The degradation was blocked by the metalloproteinase inhibitors GM6001 and 1, 10-phenantroline (both of which inhibited P. aeruginosa elastase, P. mirabilis proteinase, and E. faecalis gelatinase), or the inhibitor E64 (which inhibited S. pyogenes cysteine proteinase). Additional experiments demonstrated that dermatan sulphate and disaccharides of the structure GalNAc(4,6S)], or sucroseoctasulphate, inhibited the degradation of LL-37. The results indicate that proteolytic degradation of LL-37 is a common virulence mechanism and that molecules which block this degradation could have therapeutic potential.

The neutrophil is a key player in immunity, and its activities are essential for the resolution of infections. Neutrophil-pathogen interactions usually trigger a large arsenal of antimicrobial measures that leads to the highly efficient killing of pathogens. In neutrophils, the phagocytic process, including the formation and maturation of the phagosome, is in many respects very different from that in other phagocytes. Although the complex mechanisms that coordinate the membrane traffic, oxidative burst, and release of granule contents required for the microbicidal activities of neutrophils are not completely understood, it is evident that they are unique and differ from those in macrophages. Neutrophils exhibit more rapid rates of phagocytosis and higher intensity of oxidative respiratory response than do macrophages. The phagosome maturation pathway in macrophages, which is linked to the endocytic pathway, is replaced in neutrophils by the rapid delivery of preformed granules to nonacidic phagosomes. This review describes the plasticity and dynamics of the phagocytic process with a special focus on neutrophil phagosome maturation.

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...

Proteinase 3 (PR3) is found in granules of all neutrophils but also on the plasma membrane of a subset of neutrophils (mPR3). CD177, another neutrophil protein, also displays a bimodal surface expression. In this study, we have investigated the coexpression of these two molecules, as well as the effect of cell activation on their surface expression. We can show that CD177 is expressed on the same subset of neutrophils as mPR3. Experiments show that the expression of mPR3 and CD177 on the plasma membrane is increased or decreased in parallel during cell stimulation or spontaneous apoptosis. Furthermore, we observed a rapid internalization and recirculation of mPR3 and plasma membrane CD177, where all mPR3 is replaced within 30 min. Our findings suggest that the PR3 found on the plasma membrane has its origin in the same intracellular storage as CD177, i.e., secondary granules and secretory vesicles and not primary granules. PR3- and CD177-expressing neutrophils constitute a subpopulation of neutrophils with an unknown role in the innate immune system, which may play an important role in diseases such as Wegener's granulomatosis and polycythemia vera.

SummaryStrains of the Gram-positive human pathogen Streptococcus pyogenes (group A streptococcus ) that express surface-associated M or M-like proteins survive and grow in non-immune fresh human blood. This is generally accepted to be caused by an antiphagocytic property of these proteins. However, in most previous studies, an inhibition of the internalization of the bacteria into host cells has not been studied or not directly demonstrated. Therefore, in the present paper, we used flow cytometry, fluorescence microscopy and electron microscopy to study phagocytosis by human neutrophils of wild-type S. pyogenes and strains deficient in expression of M protein and/or the M-like protein H. The results demonstrate that all strains of S. pyogenes tested, including the wild-type AP1 strain, induce actin polymerization and are efficiently phagocytosed by human neutrophils. In addition, using classical bactericidal assays, we show that the wild-type AP1 strain can survive inside neutrophils, whereas mutant strains are rapidly killed. We conclude that the ability of virulent S. pyogenes to survive and multiply in whole blood is most likely not possible to explain only by an antiphagocytic effect of bacterial surface components. Instead, our data suggest that bacterial evasion of host defences occurs intracellularly and that survival inside human neutrophils may contribute to the pathogenesis of S. pyogenes and the recurrence of S. pyogenes infections.