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Via Serine Proteases Pneumoniae Streptococcus Human Neutrophils Kill Human Neutrophils Kill Streptococcus pneumoniae via Serine Proteases Alistair J. Standish and Jeffrey N. Weiser This information is current as J Immunol 2009; 183:2602-2609; Prepublished online 20 of October 2, 2021. July 2009; doi: 10.4049/jimmunol.0900688 http://www.jimmunol.org/content/183/4/2602 Downloaded from References This article cites 48 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/183/4/2602.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 2, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Human Neutrophils Kill Streptococcus pneumoniae via Serine Proteases1 Alistair J. Standish and Jeffrey N. Weiser2 Neutrophils, or polymorphonuclear leukocytes, comprise a crucial component of innate immunity, controlling bacterial and fungal infection through a combination of both oxidative and nonoxidative mechanisms. Indeed, neutrophils are believed to play an important role in controlling infection caused by the major human pathogen Streptococcus pneumoniae. However, the method by which neutrophils kill the pneumococcus as well as other Gram-positive bacteria, is not fully understood. We investigated human neutrophil killing of the pneumococcus in a complement-dependent opsonophagocytic assay. In contrast to other Gram-positive organisms, inhibition of the NADPH oxidase did not affect killing of S. pneumoniae. Supernatant from degranulated neutrophils killed the pneumococcus, suggesting a role for granular products. When neutrophil granule proteases were inhibited with either a protease mixture, or specific serine protease inhibitors 4-(2-Aminoethyl)benzenesulfonylfluoride and diisopropylfluorophos- phate, killing by neutrophils was inhibited in a manner that correlated with increased intracellular survival. All three compounds Downloaded from inhibited intracellular activity of the three major neutrophil serine proteases: elastase, cathepsin G, and proteinase 3. Additionally, purified elastase and cathepsin G were sufficient to kill S. pneumoniae in a serine protease dependent-manner in in vitro assays. Inhibition studies using specific inhibitors of these serine proteases suggested that while each serine protease is sufficient to kill the pneumococcus, none is essential. Our findings show that Gram-positive pathogens are killed by human neutrophils via different mechanisms involving serine proteases. The Journal of Immunology, 2009, 183: 2602–2609. http://www.jimmunol.org/ olymorphonuclear leukocytes (neutrophils) are a major host defense against this Gram-positive diplococcus. Neutrophils component of innate immunity, providing a crucial first are among the first cells recruited during many stages of Spn P barrier against both bacterial and fungal infection. Indeed, pathogenesis, including asymptomatic nasopharyngeal coloniza- neutropenic individuals are highly susceptible to a multitude of life tion, as well as invasive infection of the lungs (2, 3). Recently, we threatening infections as a direct result of the loss of these immune reported that a normally asymptomatic colonizing strain became cells. In healthy individuals, neutrophils circulating in the blood- invasive when mice were depleted of neutrophils (4). Additionally, stream are the first cells recruited to the site of infection, where neutrophil killing early in infection contributed to the subsequent they engulf pathogens into the phagolysosome thus exposing them development of the adaptive immune response. These observa- by guest on October 2, 2021 to a multitude of both oxidative and nonoxidative mechanisms of tions, therefore, confirm that neutrophils play an important role in killing. It has also been suggested that neutrophils produce neu- controlling Spn infection. However, the mechanism by which neu- trophil extracellular traps (NETs),3, which first trap pathogens trophils kill Gram-positive pathogens such as Spn is still largely within chromatin structures, and subsequently kill with various unknown. antimicrobial proteins (1). Neutrophil antimicrobial mechanisms are generally divided into Streptococcus pneumoniae (Spn), commonly known as the two distinct groups, the oxidative and nonoxidative. The oxidative pneumococcus, is a major human pathogen responsible for much burst, which generates reactive oxidative species (ROS) through an morbidity and mortality worldwide. Protection against disease and NADPH oxidase system, was long thought of as the neutrophil’s colonization is mainly mediated by opsonin-dependent phagocy- primary mode of killing pathogens. The essential nature of this tosis. Neutrophils have long been considered key effector cells in defense is seen by the fact that individuals with chronic granulo- tomas disease (CGD), which results in reduced production of ROS, have recurrent bacterial and fungal infections. However, Spn and Department of Microbiology, University of Pennsylvania School of Medicine, Phil- other catalase-negative organisms are not frequently associated adelphia, Pennsylvania 19104 with infection in CGD patients. Indeed, neutrophils isolated from Received for publication March 4, 2009. Accepted for publication June 18, 2009. CGD patients killed Spn just as efficiently as the control in ex vivo The costs of publication of this article were defrayed in part by the payment of page experiments (5). Additionally, mice deficient in enzymes associ- charges. This article must therefore be hereby marked advertisement in accordance ated with phagocyte production of ROS, have shown that ROS do with 18 U.S.C. Section 1734 solely to indicate this fact. not play a role in controlling Spn infection in either the lungs or 1 This work was supported by grants from the U.S. Public Health Service to J.N.W. (AI44231 and AI38446). brain (6, 7). Thus, this suggests that other neutrophil killing mech- 2 Address correspondence and reprint requests to Dr. Jeffrey N. Weiser, 402A John- anisms are necessary to control Spn. son Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104. E-mail address: Other than ROS, neutrophils also produce a multitude of anti- [email protected] microbial molecules in their granules. The two major granule sub- 3 Abbreviations used in this paper: NET, neutrophil extracellular trap; Spn, Strepto- sets are the azurophilic and specific granules. The azurophilic coccus pneumoniae; ROS, reactive oxygen species; CGD, chronic granulotomas dis- ease; BPI, bactericidal permeability-increasing protein; TS, tryptic soy broth; DPI, granules, are the first to fuse with the phagolysosome and contain diphenyleneiodonium; PI, protease inhibitor cocktail; AEBSF, 4-(2-Aminoethyl)ben- defensins, bactericidal permeability-increasing protein (BPI) as zenesulfonylfluoride, Hcl; DFP, diisopropylfluorophosphate; wt, wild type; OPH, op- well as serine proteases such as elastase, cathepsin G and protein- sonophagocytic; CytB, cytochalasin B; HNP, human neutrophil peptides. ase 3. Although BPI and defensins function by disrupting anionic Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 bacterial surfaces, the serine proteases generally degrade bacterial www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900688 The Journal of Immunology 2603 proteins, including virulence factors. The invading pathogen is CD18)-deficient mice (Jackson ImmunoResearch Laboratories) have a tar- then exposed to the specific granules that contain additional mi- geted mutation in the gene for integrin ␣ M or CR3 (19). crobicides such as lactoferrin, which has been implicated in de- Opsonophagocytic killing assays fense against Spn (8). The importance of these nonoxidative mechanisms is exempli- Opsonophagocytic (OPH) killing assays were conducted essentially as de- scribed by Davis et al. (2008). Bacterial strains were grown to mid-log fied by Chedaki-Higashi syndrome. This syndrome results in re- phase, PBS-washed, and resuspended in ϩϩϩ solution. In brief, 103 bac- duced levels of microbicidals as well as lower mobilization of its terial cells (in 10 ␮l) were preopsonized with infant rabbit serum shown to granules (9). Patients with this syndrome exhibit recurrent infec- be free of Spn Abs (40 ␮l) (Pel-Freez) for 30 min at 37°C. When Abs were tions and lower life expectancy. Interestingly, neutrophils isolated required, a 1/100 dilution of type 6 specific capsular anitbodies (Statens serum Institut) was added to the preopsonisaton reaction. Neutrophils were from a Chedaki-Higashi syndrome patient had a diminished ca- then added to reactions (105 cells per reaction in 40 ␮l) with ϩϩϩ solution pacity to kill Spn, indicating that nonoxidative mechanisms play an (110 ␮l) and incubated for 45 min at 37°C with rotation. Reactions were important role in neutrophil-mediated killing (10).
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