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The human cationic host defense peptide LL-37 modulates neutrophil apoptosis and chemokine responses Li, Yue Xin 2007

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THE HUMAN CATIONIC HOST DEFENSE PEPTIDE LL-37 MODULATES NEUTROPHIL APOPTOSIS AND CHEMOKINE RESPONSES by Yue X i n L i B.Med, Tianjin Medical University, 1998 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F Master of Science in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Microbioloy & Immunology) T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A January 2007 ® Yue Xin L i , 2007 Abstract L L - 3 7 is a human cationic peptide expressed primarily by neutrophils and epithelial cells. It is a 37 amino acid peptide that belongs to the cathelicidin family of the cationic host defense peptides. Accumulating evidence has demonstrated that L L - 3 7 has multiple immunomodulatory properties. The modulatory effects o f L L - 3 7 on neutrophils were investigated here, and L L - 3 7 was shown to be a potent inhibitor of spontaneous apoptosis in human neutrophils, signalling through P2X7 receptors and G-protein-coupled receptors other than the formyl peptide receptor-like-1 molecule. Inhibition of neutrophil apoptosis involved modulation of Mcl-1 expression, inhibition of B I D and procaspase-3 cleavage, and the activation of phosphatidylinositol-3 kinase and protein kinase C but not the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2) pathway. In addition, LL-37 modified neutrophil cytokine/chemokine responses to pro-inflammatory stimuli in a stimulus-specific manner. Specifically, L L - 3 7 abrogated LPS-induced T N F - a cytokine production while enhancing I L - i p elicited release of T N F - a as well as a number of chemokines including IL-8, Gro-a, C C L - 2 2 and M i p - l a . The increased release of chemokines induced synergistically by L L - 3 7 and I L - i p resulted from de novo protein synthesis and was found to be associated with the signalling through the ERK1/2 and p38 M A P kinases and nuclear factor KB pathways. These novel immunomodulatory properties of L L - 3 7 may contribute to peptide-mediated enhancement of innate host defenses against acute infection and are of considerable significance in the development of such peptides and their synthetic analogs as potential therapeutics for use against multiple antibiotic-resistant infectious diseases. i i Table of Contents A B S T R A C T ii T A B L E O F C O N T E N T S iii L I S T O F T A B L E S iv LIST O F F I G U R E S v LIST O F A B B R E V I A T I O N S vi A C K N O W L E D G E M E N T S viii C O - A U T H O R S H I P S T A T E M E N T ix 1.0 I N T R O D U C T I O N .....1 1.1 CATIONIC HOST DEFENSE PEPTIDES , 1 1.2 CATHELICIDIN FAMILY PEPTIDES AND THE HUMAN HOST DEFENSE PEPTIDE LL-37 2 1.3 NEUTROPHILS ARE IMPORTANT PLAYERS IN IMMUNE RESPONSE 8 1.3.1 THE APOPTOSIS PATHWAYS 9 1.3.2 R O L E OF B C L - 2 FAMILY PROTEINS IN MITOCHONDRIAL APOPTOSIS PATHWAY 10 1.3.3 NEUTROPHILS ARE IMMUNOMODULATORY .1. 11 1.4 EXPERIMENTAL M O D E L 12 1.5 HYPOTHESIS & EXPERIMENTAL GOALS 14 1.6 BIBLIOGRAPHY 15 2.0 T H E H U M A N C A T I O N I C H O S T D E F E N S E P E P T I D E L L - 3 7 IS A N T I - A P O P T O T I C F O R 19 N E U T R O P H H J S 2.1 INTRODUCTION '. 19 2.2 MATERIALS AND METHODS 21 2.3 RESULTS 26 2.4 DISCUSSION ) 36 2.5 BIBLIOGRAPHY ; 41 3.0 T H E H U M A N H O S T D E F E N S E P E P T I D E L L - 3 7 D I F F E R E N C I A L L Y M O D U L A T E S NEUTR. . .45 N E U T R O P H I L C Y T O K I N E / C H E M O K I N E R E S P O N S E S T O I N F L A M M A T O R Y S T I M U L I 3.1 INTRODUCTION 45 3.2 MATERIALS & METHODS 47 3.3 RESULTS 50 3.4 DISCUSSION 61 3.5 BIBLIOGRAPHY 65 4.0 DISCUSSION 68 4.1 PROLONGED NEUTROPHIL SURVIVAL IS NECESSARY TO CONTROL ACUTE INFECTIONS BUT DETRIMENTAL68 WHEN DYSREGULATED 4.2 THE PL3K AND P K C PATHWAYS ARE IMPORTANT IN LL-37-MEDIATED NEUTROPHIL SURVIVAL 69 4.3 LL-37 MEDIATES INHIBITION OF NEUTROPHIL APOPTOSIS THROUGH EFFECTS ON BOTH INTRINSIC AND ....74 EXTRINSIC APOPTOTIC PATHWAYS 4.4 LL-37 AMPLIFIES BENEFICIAL INFLAMMATORY SIGNALS BY ENHANCING NEUTROPHIL CHEMOK.INE 77 RESPONSES INDUCED BY IL-1B 4.5 CONCLUSIONS 80 4.6 BIBLIOGRAPHY 81 i i i List of Tables TABLE 3.1. PRIMER SEQUENCES USED IN THIS STUDY List of Figures FIGURE 1 . 1 . T H E STRUCTURE OF THE CATHELICIDINS 3 FIGURE 1 . 2 . EXPERIMENTAL M O D E L 1 3 FIGURE 2 . 1 . INHIBITION OF NEUTROPHIL APOPTOSIS BY L L - 3 7 2 6 FIGURE 2 . 2 . MODULATION OF NEUTROPHIL M C L - 1 EXPRESSION AND CLEAVAGE OF B I D IN RESPONSE TO L L - 3 7 . 3 0 FIGURE 2 . 3 . MODULATION OF PRO-CASPASE-3 CLEAVAGE IN L L - 3 7 - T R E A T E D NEUTROPHILS 3 1 FIGURE 2 . 4 . L L - 3 7 - I N D U C E D INHIBITION OF NEUTROPHIL APOPTOSIS IS MEDIATED BY P 2 X 7 AND A G-PROTEIN . . . 3 3 COUPLED RECEPTOR FIGURE 2 . 5 . L L - 3 7 - I N D U C E D NEUTROPHIL SURVIVAL INVOLVES MULTIPLE SIGNALLING PATHWAYS 3 5 FIGURE 3 . 1 . MODULATION OF NEUTROPHIL T N F - A PRODUCTION BY L L - 3 7 5 1 FIGURE 3 . 2 . L L - 3 7 ENHANCES IL-1B-INDUCED CHEMOKINE SECRETION BY NEUTROPHILS 5 4 FIGURE 3 . 3 . EFFECT OF L L - 3 7 AND I L - 1 B ON CHEMOKINE GENE EXPRESSION IN NEUTROPHILS 5 6 FIGURE 3 . 4 . INCREASED CHEMOKINE RELEASE AS A RESULT OF DENOVO PROTEIN SYNTHESIS 5 8 FIGURE 3 . 5 . SIGNALLING REGULATION OF CHEMOKINE RESPONSE INDUCED BY L L - 3 7 AND I L - 1 B 6 0 FIGURE 4 . 1 . M C L - 1 A N D A I GENE EXPRESSION IN NEUTROPHILS 7 2 FIGURE 4 . 2 . T H E P 3 8 SIGNALLING PATHWAY IS INVOLVED IN L L - 3 7 - I N D U C E D NEUTROPHIL SURVIVAL 7 4 FIGURE 4 . 3 . BID GENE EXPRESSION IN NEUTROPHILS 7 6 FIGURE 4 . 4 . EFFECT OF L L - 3 7 AND G M - C S F CO-STIMULATION ON CHEMOKINE RELEASE IN NEUTROPHILS 7 8 v List of Abbreviations A k t v-akt murine thymoma viral oncogene homolog 3 A p a f l apoptotic protease-activating factor 1 B A K BCL2-antagonist/killer 1 B A L bronchial alveolar lavage B A X BCL2-associated X protein Bcl-2 B-cel l leukemia/lymphoma 2 B H 3 Bcl-2 homologous region 3 B I D B H 3 interacting death agonist B I K BCL2-interacting killer (apoptosis-inducing) C F cystic fibrosis C G D chronic granulomatous disease C H D P cationic host defense peptide C R E B cAMP-responsive element-binding protein E G F R epidermal growth factor receptor E L I S A enzyme-linked immunosorbent assay E R K 1 / 2 extracellular regulated protein kinase F B S foetal bovine serum F P R L - 1 formyl peptide like receptor-1 G A P D H glyseraldehyde-3-phosphate dehydrogenase G - C S F granulocyte-colony stimulating factor G M - C S F granulocyte macrophage- colony stimulating factor Gro-a growth-regulated oncogene alpha H B E human bronchial epithelial cells H B - E G F heparin-binding EGF- l ike growth factor H E K human embryonic kidney H R P horseradish peroxidase IAP inhibitor of apoptosis protein IL- interleukin IKB Inhibitory KB IP-10 interferon-inducible protein 10 J N K Jun N-terminal kinase L B P lipopolysaccharide binding protein L P S lipopolysaccharide M A P K mitogen activated protein kinase Mcl-1 myeloid cell leukemia 1 M C P - 1 macrophage chemotattractant protein 1 M C P - 3 monocyte chemdtactic protein 3 M I G monokine induced by interferon-y M l P - l a macrophage inflammatory protein alpha M I P - i p macrophage inflammatory protein beta NF-KB nuclear factor KB N K natural killer P B M C peripheral blood mononuclear cells P B S phosphate buffered saline P E phycoerythrin PI3K phosphatidylinositol-3 -kinase P K C protein kinase C P L C phospholipase C P T X pertussis toxin R A N T E S regulated on activation, normal T-cell expressed and secreted ROI Reactive oxygen intermediates R T - P C R reverse transcriptase polymerase chain reaction SDS sodium dodecyl sulphate S D S - P A G E SDS polyacrylamide gel electrophoresis S T A T signal transducer and activator of transcription T B S tris buffered saline T B S T tris buffered saline + 0.1% Tween 20 T L R toll-like receptor T N F - a tumor necrosis factor alpha Acknowledgements I sincerely thank my supervisor, Dr. Bob Hancock, for his support and mentoring at every step of the way. The trust he always placed in me has helped me become an independent thinker and above all a responsible professional. Bob, I cannot thank you enough. I would also like to thank my committee members Dr.Brett Finlay and Dr. Linda Matssuchi who always provided me with excellent suggestions and comments in our meetings. I also thank the professors at the B . C . Childrens' hospital including Dr. David Speert, Dr. Stuart Turvey and Dr.Tobias Kollmann, for their invaluable advice and support whenever I was in need for it. David, I w i l l always remember your delicious deep-fried turkey at your Christmas parties as well as the wacky gift exchange! In addition, my very special thanks to Dr. Donald Davidson: Donald, you mentored me, all the way from Scotland: the distance didn't matter, I emailed you all kinds of questions from statistics to graph making. I could not have published my paper without you. I cannot express my gratitude enough, to all the wonderful people at the Hancock lab. In particular, I would like to thank Jelena, Jessie and Linda for the brainstorming sessions we engaged in over our projects. The most important thing though, was the mental support we provided for each other. I could not have made it through this program without you. Finally huge thanks to Susan, Barbara and Bernadette for helping me with various things: Susan, I always borrowed your swipe card for L S C and o f course, it rarely ever made it back on time to its rightful owner. Barb, without your help with all the paper work and the hectic scheduling, I wouldn't have made it to the finish line in time. Finally, I would like to thank my parents, sister and of course Ra l f who have always been there for me. M y friends, Richa Anand and Kate Tanner, have helped me with everything, especially with putting my misplaced confidence and commas back in place, at the times when I seemed to have lost both. I hope I w i l l do justice to all the trust and effort you all have placed in me, in all aspects o f my life. v i i i Co-authorship Statement Chapter 2 was originally published as; L i , Y . , Barlow P .G. , Wilkinson, T. S., Bowdish, D . M . E . , Lau, Y . E . , Cosseau, C , Haslet, C , John Simpson, A . , Hancock, R. E . W . and Donald J. Davidson. 2006. The human cationic host defense peptide L L - 3 7 mediates contrasting effects on apoptotic pathways in different primary cells o f the innate immune system. J Leukoc Biol Sep;80(3):509-520. Chapter 3 is being prepared for publication as; L i , Y . , Pistolic, J. and Robert E . W . Hancock. 2006. The role of human cationic host defense peptide L L - 3 7 in regulating chemokine responses in neutrophil and airway epithelial cell and their interactions. In all instances where previously published materials are reproduced in this thesis, they represent the original research and writing of the author. A footnote at the beginning of each chapter clearly indicates the contributions of each author. Professor R . E . W . Hancock has co-authored all of the publications listed here and his reading of the thesis w i l l serve to verify this statement of authorship. ix 1.0 Introduction 1.1 Cationic host defense peptides Cationic host defense peptides are widely distributed in nature. Initially, these peptides were viewed as "nature's antibiotics" owing to their broad spectrum of antimicrobial activity. More recent advances in the field of immunology suggest that in addition to their ability to k i l l microorganisms directly, many of these peptides have varied immunomodulatory effects (1). For example, the porcine peptide PR-39 has been shown to have the ability to stimulate wound healing via the induction of proteoglycans termed syndecans (2). Following this, the same peptide has been demonstrated to protect against acute myocardial infarction and acute pancreatitis in rodent models (2). It has become clear that cationic host defense peptides, an ancient component of the innate immunity, are more than simply "nature's antibiotics" but rather play a complex role in attenuating inflammation and resolving infections. To date, more than 700 eukaryotic cationic host defense peptides have been described (http://www.bbcm.univ.trieste.it/~tossi/antimic.htmlV with all deriving from larger precursors which undergo post-translational modifications to generate mature peptides (3). Mature cationic host defense peptides show an extraordinary molecular diversity, with most ranging in size from 12 to 50 amino acids, containing two or more positively charged residues (due to the presence of basic residues such as arginine and lysine) and a large proportion (generally >50%) o f hydrophobic residues [reviewed in (4)]. Although varying greatly in size, these peptides assume secondary structures which fall into four classes, including: i) a-helical, ii) P-stranded due to the presence of two or more disulphide bonds, i i i) P-hairpin or loop due to the presence of a single disulphide bond and/or cyclization of the peptide chain, and iv) extended (Figure 1.1). It should 1 be noted that many of these peptides are unstructured in free solution, and only fold into their final configurations upon partitioning into biological membranes (4-6). In mammals, one o f the best characterized families of peptides is the cathelicidins, which have been isolated from all mammalian species studied, including humans, monkeys, rats, mice, cattle, pigs, rabbits [reviewed in (7)]. Whi le humans and mice each encode one cathelicidin peptide, namely L L - 3 7 and C R A M P , some mammalian species contain multiple genes encoding peptides that belong to this family (7). 1.2 Cathelicidin family peptides and the human host defense peptide LL-37 The first well studied peptide in the cathelicidin family was the bovine neutrophil peptide Bac5. In the process of cloning Bac5 more than ten years ago, Zanetti's group detected additional c D N A s which displayed a high degree of identity to Bac5, yet predicted different peptide sequences (8). These c D N A sequences also showed a significant sequence homology in the pro-region to an earlier identified porcine leukocyte peptide named cathelin (9). Consequently, this group o f peptides were termed cathelicidin after the porcine peptide (now known as PR-39) and thus, initiated the discovery of other members within this peptide family (7). Like any other cationic host defense peptide, cathelicidins are also derived from larger precursors. The precursor molecules consists of an N-terminal pre-region called the cathelin domain and a C-terminal domain encoding for the mature peptides, as illustrated in Figure 1.1. There is significant conservation of amino-acid sequence (greater than 70% sequence identity) in the cathelin domain of the precursor molecules across this class of peptides (10). The C-terminal peptide domain shares little homology, and is released in its active form via proteolytic processing. The first event occurring is the cleavage by the signal peptidase to remove the signal 2 sequences, which serves to direct the cellular trafficking of the peptide during biosynthesis. For most of the bovine and porcine cathelicidins, the release of the mature peptide from the cathelin domain is accomplished by elastase derived from neutrophils (11). Whi le the mature peptides have been shown to have diverse biological activities, our current understanding of the roles of the cathelin domain is limited. Conserved Pre-pro region 29-30 amino acids Signal Peptide LL-37 BMAP-27 - CAP18 Protegrin-1 PR39 Indo licit) in Bactenecin f t 1 cc-helical 98-114 amino acids PRO Mature Peptide H Antimicrobial & Immunomodulatory 12-100 amino acids Cleavage site LLG DFFR KS KEKIGKEFKRIVQ RIKDFLRN LVP RTES • RFKRFR K K F K K L F K K L S P VIP LLH LG G LR KR LR KFRN KIKEKLKKIG QKIQ G LLP K I A P RTDY R G G R L C Y C R R R F C V C V G R G RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPG ILPWKWPWWPWRRG RLCRIWIEVCR p-sheet extended loop Figure 1.1. The Structure of the Cathelicidins. The cathelicidins shared a conseved pre-proregion with homology to the cathelin doman. The N-terminal domain contains a signal sequence while the C terminal contains both the antimicrobial and immunomodulatory properties. The four classes o f secondary structure are represented by various peptides with their sequences shown [this figure is modified from (12, 13) and http://www.bbcm.univ.trieste.it/~tossi/antimic.html1 3 The sole human cathelicidin h C A P 1 8 / L L - 3 7 was discovered in 1995 independently by three groups (14-16). High concentrations o f the unprocessed pro-peptide were first found in the specific granules of neutrophils (14). Following this, it became clear that when hCAP18 is released extracellularly as a result o f neutrophil degranulation, the pro-peptide is processed by proteinase 3 (17), liberating the mature peptide L L - 3 7 which contains 37 amino acids ( L L G D F F R K S K E K I G K E F K R I V Q R I K D F L R N L V P R T E S ) (18). In neutrophils, proteinase 3 is stored in a different compartment, referred to as the azurophil granule, thereby preventing the cleavage o f h C A P I 8 while it is still inside of specific granules (17). In addition to neutrophils, h C A P 1 8 / L L - 3 7 has been found in a variety of blood cell populations including N K cells, y8T cells, B cells, monocytes and mast cells, although at relatively small concentrations. The hCAP18 precursor is also expressed in various epithelial tissues and keratinocytes in response to stimulation by inflammation signals [reviewed in (19)], suggesting its important role at the mucosal surfaces where initial host-pathogen encounters occur. How these epithelium-derived hCAP18 molecules are processed to generate mature L L - 3 7 in general remains unclear. One recent report demonstrated that seminal hCAP-18 is processed in acidic vaginal fluid (pH 4) by a prostate-derived protease gastricsin, resulting in a 38 amino acid peptide, A L L - 3 8 (20). However, the biological functions o f the peptide variants have not been extensively studied. The earliest information on the biological functions of L L - 3 7 (other than antimicrobial activity) defined its ability to bind L P S , and to confer protection against sepsis in mice and inhibit the LPS-induced release of nitric oxide from macrophages (14). In the past five years, there has been a marked increase in the information on novel immunomodulatory activities of L L - 3 7 , gained from in vivo and in vitro studies. For example, recent microarray experiments in our laboratory have shown that L L - 3 7 selectively modulates the transcription of specific L P S -induced inflammatory genes in human monocytic cells (21). Moreover, L L - 3 7 is directly 4 chemotactic for a variety of cells in the innate and adaptive immune response, including human peripheral blood derived neutrophils, mast cells, eosinophils and CD4+ T cells (22-24). L L - 3 7 also induces mast cell degranulation, which results in the release o f inflammatory mediators such as chemoattractants and histamine (25). These substances increase vascular permeability thereby aiding neutrophils to extravasate to inflamed tissues. In addition, L L - 3 7 has been shown to induce chemokine (such as M C P - 1 , M C P - 3 , M l P - l a and M I P - i p ) gene expression in human peripheral blood derived monocytes, and Gro-a and R A N T E S production from airway epithelial cells (Bowdish, Pistolic and Hancock, unpublished results). Further, the chemokine IL-8 has been well documented to be induced by L L - 3 7 in various cells types (20, 26, 27). Collectively, these activities enhance the recruitment of effector cells o f the immune response to the site o f infection to eliminate invading pathogens. Interestingly, L L - 3 7 does not induce release o f pro-inflammatory cytokine T N F - a from monocytes or epithelial cells [(21), and Pistolic and Hancock, unpublished results]. Indeed, L L - 3 7 has been shown to inhibit the production of T N F -a induced by L P S in human monocytic cells (21), thereby protecting the host from excessive induction of immune responses which often result in sepsis. LL-37 further facilitates hosts to resolve infection by stimulating wound neovascularization and re-epithelialization of healing skin, thereby promoting wound healing, as demonstrated ex vivo and in an animal model (28, 29). Interestingly, high concentrations of L L - 3 7 (> 50 |ig/ml) have been recently shown to induce apoptosis in cultured lung and airway epithelial cell lines, and in vivo in the murine airway (30). In that report, it was shown that LL-37-induced apoptosis in vitro involved the activation of caspase pathways and was substantially inhibited by an inhibitor o f caspase 3. It was not investigated, however, which signalling pathways were implicated and whether any o f the proposed L L - 3 7 receptors were involved. Indeed, the signalling mechanism through which L L - 3 7 exerts its immunomodulatory effects has been o f particular interest and continues to be a very active area of research. 5 To date, three receptors have been shown to be associated with LL-37-mediated immonomodulatory activities, including P2X7 (31), F P R L - 1 (formyl peptide receptor-like 1) (22), and E G F R (epidermal growth factor receptor) (20). The purinergic receptor P2X7 has been shown to mediate LL-37-induced I L - i p release from LPS-primed human primary monocytes, as inhibitors of P 2 X 7 partially blocked this effect. F P R L - 1 is a G-protein coupled receptor and is found to be associated with a number of LL-37-mediated effects. For example, LL-37-induced angiogenesis via a direct effect on endothelial cells was suggested to be mediated by FPRL-1 (32). F P R L - 1 is also utilized by L L - 3 7 to induce chemotaxis of a number o f human blood cell types (22). In addition, FPRLl-transfected H E K 2 9 3 cells migrated in response to L L - 3 7 whereas the parental cells did not. It has been proposed that L L - 3 7 activates this receptor via specific, yet low-affinity binding (22). Indeed, L L - 3 7 has been demonstrated to bind two receptors in lung epithelial cells, a high affinity and a low affinity receptor, and the low-affinity receptor appears to be FPRL-1 (33). It is also thought that a G-protein coupled receptor other than F P R L - 1 may be involved in L L - 3 7 mediated effects. For example, LL-37-induced IL-8 production in keratinocytes is pertussis toxin sensitive, but a specific agonist for F P R L - 1 , W K Y M V M - N H 2 , does not induce these effects even at high concentrations (29). Unlike P2X7 and F P R L - 1 , E G F R has been proposed to be trans-activated by L L - 3 7 . In airway epithelial cells, LL-37 induces E G F R activation and subsequent IL-8 production, and this effect requires metalloproteinase activity because broad spectrum metalloproteinase inhibitors block IL-8 release (20). It has therefore been proposed that L L - 3 7 , by as-yet undetermined means, activates a metalloproteinase which subsequently cleaves membrane-anchored E G F R ligands, resulting in E G F R activation. It is important to note that there are indications that not all of the LL-37-induced effects require a receptor. Probably the strongest evidence is that IL-8 can be induced by the D amino acid analog o f L L - 3 7 in keratinocytes, and the induction is even more 6 potent than caused by the naturally occurring L-form peptide, possibly due to the increased resistance of the D peptide to proteolytic degradation (29). Although there are exceptions, in general most receptors do not recognize D-amino acids formed ligands owing to stereospecificity. Wi th regards to signalling pathways, it appears that M A P kinases play an important role in LL-37 mediated effects. L L - 3 7 has been shown to induce E R K 1 / 2 , p38 and I N K activation in the airway epithelial cell line NCI-H292 (20). E R K 1 / 2 and p38 are also phosphorylated in response to L L - 3 7 in human keratinocytes, mast cells, 16HBE4o" and monocytes (24, 26, 27). In addition to M A P kinases, L L - 3 7 phosphorylates S T A T 3 via HB-EGF-mediated E G F R transactivation in keratinocytes (34). Y u , Bowdish and Hancock found that in human P B M C s , L L - 3 7 induces transient degradation of phosphorylated IKB and subsequent nuclear translocation of NFKB subunit p50 ( Y u et al, manuscript submitted), indicating that L L - 3 7 may interact with the T L R pathways at undetermined levels. Y u also demonstrated, through conventional inhibitor studies, that the PI3 kinase and downstream Akt pathways are also involved in LL-37-mediated IL-8 release in human P B M C s . Although the knowledge of how L L - 3 7 modulates the immune response has increased markedly in recent years, in-depth studies are still needed to fully understand the complex roles of this peptide in innate immunity. To date, much of the information on the immunomodulatory properties of L L - 3 7 has been derived from studies on monocytes, airway epithelial and keratinocytes. However, at the infection foci, the most abundant leukocytes are neutrophils, and as discussed earlier, the concentration of L L - 3 7 at these sites can be high owing to neutrophil degranulation, yet very little is known how L L - 3 7 interacts with neutrophils. Neutrophils are major effector cells in innate immunity and are able to regulate immune responses via secretion 7 of cytokines, it is therefore logical and meaningful to examine how L L - 3 7 affects the functions of neutrophils and modulates their immune responses in physiological settings. 1.3 Neutrophils are important players of immune response Human neutrophils represent the most abundant granulocyte subtype (50% to 60% of the total leukocytes) in circulation (35). They play a key role in the early stages of the acute inflammatory responses to microbial infections. After pathogen invasion, neutrophils are quickly recruited from circulation to the site of infection where they, along with tissue macrophages, eliminate pathogens via phagocytosis (36). Activated neutophils also release toxic granular contents such as reactive oxygen species and granular enzymes like proteases to k i l l pathogens; however, these toxic compounds also cause collateral host tissue damage (36). In addition to their ability to directly k i l l pathogens, neutrophils can, when exposed to inflammatory mediators such as L P S , produce a variety of cytokines and chemokines which subsequently modulate the inflammatory response (37). In this sense, neutrophils are also considered immunomudulators in addition to their long recognized role in phagocytosing invading microbes. Neutrophils are developed in bone marrow from myeloid stem cells and are then released into the circulation where they survive for approximately 24 to 36 hours (38). The turnover of neutrophils is rapid and is important for the maintenance of the homeostasis of the immune system. Efficient and safe elimination of aging neutrophils is achieved by constitutive apoptosis, a process permitting the removal o f dead cells without the massive release o f cytotoxic contents into the surrounding tissues (36). Aged, apoptotic neutrophils migrate to tissues and are removed by macrophages or other cells (e.g., fibroblasts) by phagocytosis (39). While neutrophils have a very short life-span in the circulation, their survival time can increase significantly once they extravasate into the inflamed tissues where they become exposed to pro-inflammatory mediators such as G M - C S F and L P S , or to certain environmental conditions such as hypoxia. On the 8 contrary, a number of so-called death factors [for example, reactive oxygen intermediates (ROI) or IL-10] can accelerate spontaneous apoptosis of the neutrophils (40). Neutrophils from patients with chronic granulomatous disease have prolonged life spans as these cells are deficient in generating ROI (41). While extended survival of neutrophils is essential to clear pathogens during acute infection, dysregulation of the apoptosis processes may be responsible for the persistence of immune cells at inflammatory sites and the development o f chronic inflammatory disease (40). For example, cystic fibrosis (CF) is a disease characterized by neutrophil dominated lung inflammation. Even in the absence of detectable infection, substantially elevated amounts of pro-inflammatory cytokines and large numbers of neutrophils are found in the airways of C F infants. It has been proposed that enhanced neutrophil survival may be attributed to the effects of increased expression of epithelial derived G M - C S F and G - C S F (42). 1.3.1 The apoptosis pathways In humans cells, apoptosis can be initiated through two distinct but ultimately converging signalling pathways, namely the extrinsic and the intrinsic pathways [reviewed in (43)] . The extrinsic pathway can be induced by the activation o f death receptors such as T N F R family and Fas with their cognate ligands. (44). A s an example, the ligation of FasL triggers the trimerization of the Fas receptors and recruits initiator caspases (predominantly procaspase 8 or procaspase 10) through specialized adaptor proteins (45, 46). This promotes the aggregation of procaspase 8 and triggers autoproteolysis to generate active caspase 8 (47). Activated caspase-8 elicits apoptosis by means of two parallel cascades: it directly cleaves and activates caspase-3 (48) and it cleaves B i d , a pro-apoptotic Bcl-2 family protein (see below). Truncated B I D (tBID) translocates to the outer membrane of mitochondria and induces cytochrome c release which ultimately leads to the activation o f caspase 3 (38). 9 The intrinsic pathway can be induced by developmental cues such as growth factor withdrawal, and intrinsic factors such as D N A damage and unfolded proteins [reviewed in (38)]. These factors promote the depolarization o f the mitochondrial membrane and subsequent release of cytochrome c. Upon release from mitochondria, cytochrome c binds A p a f l (apoptotic protease-activating factor 1) and forms an activation complex with caspase-9, leading to the activation o f the latter which subsequently cleaves and activates caspase 3 (49). 1.3.2 Role of Bcl-2 family proteins in mitochondrial apoptosis pathway A s mentioned earlier, B I D belongs to the Bcl-2 family which can be functionally categorized into two groups by ability to inhibit or activate cell death: a) the pro-apoptotic proteins ( B A X , B A D and B I K , to name a few) and b) the anti-apoptotic proteins (such as Bcl -2 , Bc l -x , Mcl-1 and A l ) [reviewed in (50)]. These proteins form a complex network of checkpoints and balances that regulate the mitochondrial permeability and the release of cytochrome c. Although it is not currently known how exactly these proteins interact to control cell death, it has been proposed that following death signalling, pro-apoptotic B A X and B A K disrupt the outer mitochondrial membrane by forming channels or by modifying membrane pores (51). Such disruptions w i l l cause the release of cytochrome c and other apoptotic proteins, irreversibly executing the death program. In contrast, anti-apoptotic proteins have been shown to form heterodimers w i t h , B A X and B A K , suggesting their role as a "sink" to reduce the level of free pro-apoptotic Bcl-2 proteins (52). A subgroup o f pro-apoptotic proteins (including BID) does not directly disrupt mitochondrial membrane; however, they "sensitize" cells to death stimuli by a) binding to anti-apoptotic proteins thereby displacing and relieving inhibition of the bound B A K / B A K , or b) directly triggering the activity o f B A X / B A K (52). 10 Neutrophils constitutively express a variety of pro-apoptotic Bcl-2 proteins including B A X , B A D and B A K ; however, their cellular levels change very little when neutrophils are exposed to agents that either delay or accelerate apoptosis (53). Among the anti-apoptotic Bcl-2 proteins, M c l - 1 , A l and BC1-XL have been reported to be expressed in neutrophils, yet immunoblotting fails to detect significant levels of BC1-XL protein using a variety of commercial antibodies in all studies (54) despite the detection of BC1-XL m R N A in RNase protection assays. To date, Mcl-1 is the best characterized anti-apoptotic Bcl-2 family member in human neutrophils apoptosis studies. Mcl-1 is a unique member of the Bcl-2 family in that it has a very short half life, estimated between 1 hour and a few hours before degradation by proteinases (53). The expression of this protein can be up-regulated at the transcriptional level in conditions like hypoxia (55) or in response to certain cytokines such as IL-3 and G M - C S F (54). G M - C S F can also increase Mcl-1 protein stability thereby protecting it from degradation (56). Conversely, deprivation of G M - C S F has been shown to result in a significant decrease of Mcl-1 protein in the human myeloid progenitor cell line TF-1 (57). The high turnover rate of Mcl-1 leads to rapid loss of this anti-apoptotic Bcl-2 protein in neutrophils undergoing apoptosis. Indeed the loss of Mcl-1 has been postulated to be the major inducer of spontaneous apoptosis in neutrophils (53). It is also conceivable that the apoptotic program is initiated in neutrophils as soon as they are released from bone marrow into the circulation, in which survival factors such as G M - C S F are no longer present. 1.3.3 Neutrophils are immunomodulatory Neutrophils are terminally differentiated cells, and they have been conventionally considered to lack the ability to synthesize new proteins in circulation. However in recent years, they have been shown to express and secret a variety o f pro- and anti-inflammatory proteins 11 including cytokines, chemokines, growth factors, and interferons in characteristic patterns in response to stimulatory agents (37). Among these, chemokines represent a group of structurally related cytokines with the ability to specifically recruit distinct leukocyte subpopulations (58). In addition to their role in coordinating the immune responses, these proteins have also been shown to be involved in B and T-cell development and in the modulation of angiogenesis (59). Neutrophils have been demonstrated to express and secrete a number o f chemokines including IL-8, Gro-a, M i p - l a , M i p - i p , IP-10, and M I G (60). It is interesting to note that, while neutrophils are a major source o f IL-8 production, these cells are also the primary targets of IL-8. Specifically, IL-8 is chemotactic for neutrophils and induces neutrophil respiratory burst activity, enhanced expression o f surface adhesion molecules, and increased adherence to endothelial cells (60). These reciprocal interactions would amplify inflammatory responses, allowing the host to rapidly resolve acute infections. Given the essential roles of cytokines and chemokines in the host immune defense, it is reasonable to consider neutrophils as important regulators of the immune responses. 1.4 Experimental Model A t the site of infection, the expression o f hCAP-18 is induced in local epithelia upon detection of infection (61, 62). Elevated levels o f L L - 3 7 , together with other inflammatory mediators such as IL-8, would create a gradient permitting chemotaxis of neutrophils to the infection focus. Recruited neutrophils would release granule contents including high concentrations of antimicrobial peptides such as hCAP18 . This would further increase the local concentration of L L - 3 7 as the precursor hCAP-18 is processed by proteinase 3. Raised levels of L L - 3 7 would then, according to this thesis, enhance the survival o f neutrophils, and modulate neutrophil chemokine responses to endogenous inflammatory signals. Thus collectively, L L - 3 7 12 would augment the innate host defenses mediated through neutrophils to counter acute infectious challenge through multiple mechanisms. The experimental model proposed and pursued in this thesis is summarized in Figure 1.2. ACUTE INFECTION D. J. Davidson Figure 1.2. Experimental Model. Upon infection, local epithelia release h C A P l 8 / L L - 3 7 . L L - 3 7 , together w i t h other i n f l a m m a t o r y media tors , chemoattract neutrophils to the site o f infection. Recruited neutrophils release granule contents, and this further increases the local concentration of L L - 3 7 . Thus, raised levels o f LL-37 in an acute inflammatory scenario could contribute to innate host defenses by mediating recruitment and enhanced survival o f neutrophils and the modulation of innate immune cell responses to endogenous inflammatory signals, to enhance the resolution o f an acute infectious insult (Art by Donald Davidson). 13 1.5 Hypothesis & Experimental Goals The primary goal of this study was to define the immunomodulatory effects of L L - 3 7 on human neutrophil responses, with particular focus on their apoptotic processes. I hypothesized that L L - 3 7 enhances innate immunity at sites of infection / inflammation by affecting apoptosis and other functions of neutrophils. A t the beginning of this study, I examined the ability of L L -37 to inhibit neutrophil apoptosis in an efficient and timely manner, as well as the mechanisms through which L L - 3 7 exerts its effects. In particular, because Bcl-2 family proteins play an important role in regulating apoptosis, one of the goals was to evaluate the cellular levels of important members of this family such as Mcl-1 and B I D . M y aim was also to elucidate signalling pathways and putative receptors through which L L - 3 7 mediates its inhibitory effects on neutrophil apoptosis. Another important aspect of this study was to evaluate how L L - 3 7 affects the chemokine responses of neutrophils, in the presence or absence of other inflammatory stimuli including I L - i p and L P S . 14 1.6 Bibliography 1. Finlay, B . B . , and R. E . Hancock. 2004. Can innate immunity be enhanced to treat microbial infections? Nat Rev Microbiol 2:497-504. 2. L i , J. , M . Post, R. Vo lk , Y . Gao, M . L i , C . Metais, K . Sato, J. Tsai, W . Ai rd , R. D . Rosenberg, T. G . Hampton, F. Sellke, P. Carmeliet, and M . Simons. 2000. PR39, a peptide regulator o f angiogenesis. Nat Med 6:49-55. 3. Gao, Y . , S. Lecker, M . J. 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The neutrophil as a cellular source of chemokines. Immunol Rev 177:195-203. 63. Erdag, G . , and J. R. Morgan. 2002. Interleukin-1 alpha and interleukin-6 enhance the antibacterial properties of cultured composite keratinocyte grafts. Ann Surg 235:113-124. 64. Hase, K . , M . Murakami, M . Iimura, S. P. Cole, Y . Horibe, T. Ohtake, M . Obonyo, R. L . Gallo, L . Eckmann, and M . F. Kagnoff. 2003. Expression of LL-37 by human gastric epithelial cells as a potential host defense mechanism against Helicobacter pylori. Gastroenterology 125:1613-1625. 18 2.0 The Human Cationic Host Defense Peptide LL-37 is Anti-apoptotic For Neutrophils" 2.1 Introduction Cationic host defense peptides ( C H D P ; also known as antimicrobial peptides) are key, evolutionarily-conserved components o f host defenses (1). The importance of C H D P to human immunity is indicated by the increased susceptibility to infection o f individuals with specific granule deficiency (2) and of morbus Kostmann patients (3) (representing conditions in which neutrophil deficiency in defensins and cathelicidin, respectively, constitute key components of the disease) and by the up-regulation of peptide expression in the lung (4), skin (5), or other sites in response to inflammation. Furthermore, studies using knockout mouse models, transgenics, and gene therapy augmentation clearly demonstrate the significance of C H D P to host defense against infections (6-11). Recently, various C H D P have been shown to have immunomodulatory properties (11), with a growing number of these demonstrated in vivo, including chemokine production, angiogenesis, anti-endotoxin activity, and chemotaxis (12-15). In addition, multiple studies of C H D P have demonstrated broad-spectrum antimicrobial activities in vitro. Consequently, the development of C H D P and their synthetic derivatives is likely to yield future antimicrobial therapeutics relatively unaffected by common bacterial resistance mechanisms and potentially combining microbicidal activities with immunomodulatory properties (16). * The majority of this work was originally published in the Journal o f Luecocyte Biology 2006 Sep; 80(3):509-520. A l l data was generated by Y . L i except Figure 2.1 A and 2.4 that was produced by Barlow, Wilson and Davidson. Figure 2.1 B - D and 2.5 represent updated data. The text has been updated to reflect advances in the literature. 19 The C H D P demonstrating the most significant immunomodulatory potential to date is L L - 3 7 , the predominant, mature peptide fragment of the sole human cathelicidin, human cationic antimicrobial protein (hCAP)-18. This peptide is found in the specific granules of neutrophils, and is also produced by a range of epithelial cells and to a lesser extent, by lymphocytes and macrophages (17). L L - 3 7 expression has been detected at ~5 ng /ml in bronchoalveolar lavage ( B A L ) from healthy infants and is up-regulated by inflammation, being detected in B A L from infants with pulmonary infections and in individuals with cystic fibrosis lung disease at up to -30 pg/ml and -15 pg/ml, respectively (4, 18), and at levels of -1.5 mg/ml in psoriatic skin lesions (5). The full extent of the immunomodulatory functions of L L - 3 7 and the underlying mechanisms involved remain undetermined and are of considerable significance in the development of therapeutic, synthetic analogs. W e hypothesized that the potential for L L - 3 7 to modulate the function and survival of key innate-immune effector cells could be critical to enhancing the clearance o f infection and resolution of inflammation. In particular, neutrophils, a cell type for which L L - 3 7 is both directly and indirectly chemotactic (12, 19-22), represent a key component of innate defenses against infection. These cells have a short half-life, "programmed" to die by apoptosis, and exert a defensive role by intracellular k i l l ing and the release of defensins and cathelicidins, lytic enzymes, proteases, and inflammatory mediators (23, 24). In this study, we demonstrate that L L - 3 7 acted as a potent inhibitor o f human neutrophil apoptosis, signalling through P2X7 receptors and G-protein-coupled receptors, other than formyl peptide receptor-like-1 molecule (FPRL-1) . L L - 3 7 modulated Mcl-1 expression and inhibited cleavage of B I D and procaspase-3, acting through activation o f phosphatidylinositol-3 kinase (PI3K) and protein kinase C (PKC) , but not the extracellular signal-regulated kinase ( E R K ) 1/2 mitogen-activated protein kinase ( M A P K ) pathway. 20 2.2 Materials & Methods Reagents H-Trp-Arg-Trp-Trp-Trp-Trp - C O N H 2 (WRW4) and H-Trp-Lys-Tyr-Met-Val-D-Met-C O N H 2 ( W K Y M V m ; both reconstituted in dimethyl sulfoxide, according to the manufacturer's instructions), and pertussis toxin (PTX) were purchased from Calbiochem, Merk Biosciences (Nottingham, U K ) . Oxidized adenosine 5'-triphosphate (ATP) , SB203580 and rottlerin were purchased from Sigma-Aldrich (Poole, U K , or Oakville, Ontario, Canada). Inhibitors LY294002 and PD98059 were purchased from Cel l Signalling Technology, Inc. (Mississauga, O N , Canada). Rabbit polyclonal antibodies against M c l - 1 , B I D , and cleaved caspase-3 were purchased from Cel l Signalling Technology (Mississauga, Ontario, Canada). Mouse polyclonal antibody against caspase-3 was purchased from Alexis Biochemicals (Axxora, San Diego, C A ) . Horseradish peroxidase (HRP)-conjugated goat antirabbit and antimouse immunoglobulin G antibodies were purchased from Cel l Signalling Technology (Beverly, M A ) and Amersham Biosciences (Piscataway, NJ) , respectively. Lipopolysaccharide (LPS) from Pseudomonas aeruginosa Strain HI03 was highly purified free o f proteins and lipids using the Darveau-Hancock method as 1 described previously (25). Briefly, P. aeruginosa was grown overnight in Luria-Bertani broth at 37°C. Cells were collected and washed, and the isolated L P S pellets were extracted with a 2:1 chloroform:methanol solution to remove contaminating lipids. Purified L P S samples were quantitated using an assay for the specific sugar 2-keto-3-deoxyoctosonic acid assay and then resuspended in endotoxin-free water (Sigma-Aldrich). LL-37 was synthesized by N-(9-fluorenyl) methoxycarbonyl chemistry at the Nucleic Acid/Protein Service unit at the University of British Columbia ( U B C , Vancouver, Canada), as described previously (26). Peptides were purified by reverse-phase high-performance liquid chromatography and were at least 98% pure. L L - 3 7 was dissolved in endotoxin-free water (Sigma-Aldrich) and stored at - 2 0 ° C until further use. The 21 concentration of the peptides in solution was determined by amino acid analysis. A l l reagents were tested to ensure that they were free of endotoxin and reconstituted in endotoxin-free water. Isolation of human blood neutrophils Fresh human venous blood was collected from volunteers, according to U B C Clinical Research Ethics Board protocol C02-0091 or University of Edinburgh (Scotland) Research Ethics Committee approval #1702/95/4/72, using Vacutainer® collection tubes containing sodium heparin (BD Biosciences, Mississauga, Ontario, Canada) or sodium citrate solution (Phoenix Pharma Ltd., Gloucester, U K ) as an anticoagulant. Neutrophils were purified by Ficoll-Paque gradient centrifugation as described previously (27). Briefly, blood was mixed, at 1:1 ratio, with 2 % Dextran T-500 (Amersham Pharmacia Biotech, Buckingham, U K ) in 0.9% saline and sedimented for 30 min at room temperature. The leukocyte-rich upper layer was then centrifuged at 200 g for 7 min, and remaining erythrocytes were lysed hypotonically with ice-cold, distilled water for 30 s, followed by restoration of tonicity with 2.5% saline. Neutrophils were then separated by centrifugation over a Ficoll-Paque Plus (Amersham Pharmacia Biotech) density gradient at 400 gfor 25 min at 4°C. The cells were washed with Krebs-Ringerphosphate buffer (pH 7.3), containing glucose (10 m M ) and M g 2 + ( 1 . 5 m M ) , and resuspended in RPMI-1640 media (Invitrogen), supplemented with 10% (v/v) heat-inactivated F B S , 1% (v/v) L-glutamine, and 1 n M sodium pyruvate. Alternatively, blood was centrifuged at 300 g for 20 min at room temperature, platelet-rich plasma was removed, and cells were suspended gently in 1% Dextran T-500 (Amersham Pharmacia Biotech, Buckingham, U K ) in 0.9% saline and sedimented for 30 min at room temperature. The leukocyte-rich upper layer was then fractionated by using three-step discontinuous, isotonic Percoll gradients as described previously (28). Briefly, cells were centrifuged at 300 g for 6 min, resuspended in 55% isotonic 22 Percoll (Amersham Pharmacia Biotech), layered on top of 68% and 81% isotonic Percoll layers, and centrifuged at 700 g for 20 min at room temperature. Neutrophils were collected, washed in phosphate-buffered saline (PBS) without calcium or magnesium, and resuspended in Iscoves's Dulbecco's modified Eagle's medium ( I D M E M ; Invitrogen, Paisley, U K , or Burlington, Ontario, Canada) with 10% (v/v) heat-inactivated fetal bovine serum (FBS). Polymorphonuclear leukocytes isolated were 95-4>8% neutrophils using morphological criteria, and viability was assessed by trypan blue exclusion. Dose-dependent LL-37-mediated inhibition of neutrophil apoptosis was observed by fluorescent-activated cell sorter ( F A C S ) , irrespective of the anticoagulant or methodology used to isolate cells. The former method was used in analyses by Western immunoblot and other inhibitor studies, and the latter method in morphological studies and receptor inhibitor studies. Neutrophil apoptosis Freshly isolated human blood neutrophils were incubated at 37°C, 5% C O 2 , for 0, 1,4, or 20 h, at 1 x 10 6 cells in 1 ml R P M I with 10% (v/v) heat-inactivated F B S in 24 well tissue culture plates in the presence of L L - 3 7 , G M - C S F , or W K Y M V m at the stated concentrations or a vehicle control, at least in duplicate. Where inhibitors were used, these (or vehicle control) were added 30 min before the addition of L L - 3 7 . Apoptosis was assessed by flow cytometry using Phycoerythrin (PE)-labeled Annexin V (Biolegend, San Diego, C A ) and nucleic acid dye 7 A A D ( B D Biosciences, Mississauga, O N , Canada) diluted 1:100 with binding buffer provided by Biolegend (San Diego, C A ) at room temperature. The samples were analyzed using a FACSCal ibu r system ( B D Biosciences), counting a minimum of 10,000 cells. In addition to F A C S analyses, apoptosis was assessed using standard morphological analysis of distinctive apoptotic morphology. Samples (100 pi) o f cells were cytocentrifiiged, fixed in methanol, stained with Reastain Quick-Diff (Reagena, Garridor, U K ) , and examined using oi l immersion 23 microscopy. Total cell counts were performed in duplicate by haemocytometer and light microscopy (using standard methodology) for each well upon removal o f gently resuspended cells for F A C S analysis and cytospin. Western immunoblotting Fresh human blood neutrophils (7.5xl0 6 ) , per condition, in 5 ml RPMI-1640 media [supplemented with 10% (v/v) heat-inactivated F B S , 1% (v/v) L-glutamine, and 1 n M sodium pyruvate] were exposed to LL-37 at the concentrations detailed or endotoxin-free water as a vehicle control and incubated at 37°C, 5% C O 2 , for 4 h. Cells were washed with ice-cold P B S containing 1 raM sodium orthovanadate (Sigma-Aldrich) and lysed with 150 jxl 1% Triton X-100, 10 m M Tris, p H 7.5, 150 m M N a C l , 2 m M E D T A , 1 m M phenylmethylsulfonyl fluoride buffer containing Sigma protease inhibitor cocktail [104 m M 4-(2-aminoethyl) benzenesulfonyl fluoride, 0.08 m M aprotinin, 2 m M leupeptin, 4 m M bestatin, 1.5 m M pepstatin A , 1.4 m M E-64] and phosphatase inhibitor cocktail 2 (sodium orthovanadate, sodium molybdate, sodium tartrate, imidazole). The protein concentrations o f lysates were determined using a bicinchoninic acid assay (Pierce, Rockford, IL, or Cramlington, U K ) . Equivalent lysate (15-40 pg) was resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( S D S - P A G E ) and transferred to nitrocellulose membranes (Bio-Rad, Hercules, C A ) , which were blocked for 1 h with 20 m M Tris, p H 7.4, 150 m M N a C l , and 0.1% Tween 20 (TBST) containing 5% skimmed milk powder (TBST/milk) . Subsequently, the nitrocellulose membranes were incubated with anti-Mcl-1, anti-B I D , anti-caspase-3, or anti-cleaved caspase-3 antibodies at 1/1000 dilution in TBST/mi lk for 1 h at room temperature. Membranes were washed for 30 min in T B S T and then incubated with a 1/5000 dilution of HRP-conjugated goat anti-mouse or anti-rabbit antibody (in TBST/mi lk ) for 1 h. The membranes were washed for 30 min in T B S T and developed with chemiluminescence peroxidase substrate (Sigma-Aldrich), according to the manufacturer's instructions. To quantify 24 bands, the luminescence was detected with Versadoc (Bio-Rad). The blots were stripped using Restore™ Western blot stripping buffer (Pierce), according to the manufacturer's instructions, and reprobed with an anti-glyceraldehyde 3-phosphate dehydrogenase ( G A P D H ) antibody, and densitometry was performed to allow correction for protein loading. Chemotaxis Assay Transwell® polyester permeable supports (pore size 3.0 urn, diameter 6.5 mm; Corning Life Sciences, U K ) were utilised for chemotaxis studies. Briefly, each chemoattractant sample (600 Lil) was loaded into the lower well and 100 p:l of P B S without C a 2 + and M g 2 + containing 1 x 10 5 fresh human blood neutrophils added to the apical compartment of each transwell. The plates were incubated for 60 min at 37°C in 5% C O 2 . Chemoattractants tested were sterile P B S without Ca and M g (negative control) and W K Y M V m . For inhibition studies, neutrophils were pre-treated with 10 f i M W R W 4 for 30 min at room temperature prior to use in the chemotaxis assay. Following incubation, the supernatants from both the upper and lower compartments were removed and a cytospin was performed. The upper surface o f the polyester membranes were wiped with cotton buds to remove non-migrated cells and the cells on the underside of the membranes were fixed with methanol and stained with a Reastain Quick-Dif f (Reagena). The polyester membranes with migrated cells were removed with a curved serrated scalpel, and mounted 'cel l side up' on a microscope slide. The migrated cells were then counted using a Zeiss light microscope at lOOOx magnification. For each membrane, five fields of view were counted and results expressed as a chemotactic index correct for migration observed in the negative control. Statistical analysis Student's Mests were performed to determine statistical significance, and P < 0.05 was considered significant. Values shown are expressed as mean ± S E M . 25 2.3 Results LL-37 inhibits neutrophil apoptosis To evaluate the effect of L L - 3 7 exposure on spontaneous neutrophil apoptosis, fresh human peripheral blood neutrophils were isolated and incubated in culture over a range of L L - 3 7 concentrations. The level o f apoptosis was assessed by F A C S quantification o f PE-labeled annexin V and 7 A A D staining. Confirmation of apoptosis was obtained by morphological evaluation of cytospins, revealing the classical appearance of cell shrinkage and nuclear condensation in apoptotic cells (Figure 2.1 A ) . In untreated cells, spontaneous apoptosis was observed in 65.3% ± 3.2% (n=5) of cells at 20 h. Exposure to L L - 3 7 was demonstrated to inhibit neutrophil apoptosis significantly at 20 h (Figure 2.IB) . This inhibition was dose-dependent, with significant inhibition (P=0.03) at L L - 3 7 concentrations o f 250 ng/ml or greater, and profound inhibition of apoptosis observed at 10 |ag/ml (diminished by 49% of control levels; P=0.004)or above. 0 Lig/ml LL-37 1 ng/ml LL-37 Figure 2.1A. Inhibition of neutrophil apoptosis by LL-37. 26 B) 0 0.1 0.25 0.5 1 10 25 LL-37 (ug/ml) 0 0.1 0.25 0.5 1 10 25 LL-37 (Mg/ml) Figure 2.1 (B-D). Inhibition of neutrophil apoptosis by LL-37. Figure 2.1. Inhibition of neutrophil apoptosis by L L - 3 7 . Neutrophils were incubated for 20 h over a range of L L - 3 7 concentrations. Modulation of spontaneous apoptosis was examined by F A C S analysis and morphology. (A) Representative fields from cytospins. White arrows indicate examples of apoptotic neutrophils, 320x original magnification. ( B - D ) F A C S analysis was used to determine the percentage of neutrophils, which were (B) apoptotic (PE-annexin V-positive, 7AAD-negative), (C) viable (PE-annexin V -negative, 7AAD-negative), and (D) necrotic (PE-annexin V-positive, 7AAD-posit ive). Figures represent the percentage of cells in LL-37-treated samples as a proportion o f that observed in the vehicle alone-treated controls to correct for donor variation and indicate mean values ± S E M , for three replicates for each condition, using five different donors. Significance was assessed using relative values in LL-37-treated samples compared with vehicle alone-treated controls by paired sample Student's Mest analyses. *, P <0.05; **, P<0.01. The consequence of LL-37-mediated inhibition of apoptosis in neutrophils at 20 h was evaluated by F A C S analysis, assessing the number o f annexin V-negative/7AAD-negative (viable) and annexin V-positive/7AAD-positive (necrotic) cells. In the absence of L L - 3 7 , 27.3% ± 2.4% (n=5) of cells were viable, and 6.32% ± 1.3% (n=5) of cells were necrotic at 20 h. Exposure to L L - 3 7 resulted in dose-dependent increases in the proportions of viable (Figure 2.1C) and necrotic (Figure 2. ID) cells. The proportion of viable cells increased significantly after exposure to 250 ng/ml L L - 3 7 (P=0.02) or greater and by up to 93.8% of control levels at 10 Hg/ml. In contrast, the proportion of necrotic cells did not increase significantly at concentrations of L L - 3 7 below 1 |ag/ml but was greatly increased by exposure to higher levels of L L - 3 7 , by approximately 2.5 fold o f control levels at 10 jxg/ml. Neutrophils were also examined over a time course using F A C S analyses and total cell counts. In contrast to inhibition o f apoptosis by L L - 3 7 at 20 hours, negligible levels o f apoptosis and necrosis were observed by F A C S at 0 hours, 1 hour and 4 hours (data not shown). 28 LL-37 exposure modulates expression of Mcl-1 and inhibits cleavage of BID and procaspase-3 in neutrophils To study the mechanisms by which L L - 3 7 can modulate the apoptosis of neutrophils, the expression of critical apoptosis-regulating Bcl-2 family proteins and the cleavage o f procaspase-3 were examined by Western immunoblot. Fresh human peripheral blood neutrophils were examined at 4 h after exposure to L L - 3 7 in the presence of 10% F B S , a time-point chosen to precede any substantial apoptosis or necrosis with 94-96% cell viability observed, irrespective of exposure to LL-37 . Expression of the anti-apoptotic protein Mcl-1 is of particular importance in neutrophils (29) and was found to be significantly higher in neutrophils after 4 h exposure to L L -37 (Figure 2.2A) with dose-dependent regulation. Cleavage o f the "BH3 domain only" Bcl-2 protein B I D by caspase-8 generates a pro-apoptotic fragment (30, 31) and was demonstrated to be inhibited significantly in neutrophils exposed to L L - 3 7 (Figure 2.2B). In addition, significantly higher levels of the inactive procaspase-3 protein, with a corresponding, significant decrease in levels o f active-cleaved caspase-3, were observed after exposure to L L - 3 7 (Figure 2.3A and 2.3B), indicating inhibition of the activation pathways for the effector caspase-3 and correlating with LL-37-mediated inhibition of neutrophil apoptosis. 29 Mcl-1 GAPDH c 6 o </> Q-•40 kDa BID -36kDa GAPDH 1 0 0.25 1 10 50 GM-CSF , i LL-37 (ug/ml) 5 .9 4 in (J) o a J 5 2 Q 5 1 •22 kDa •36 kDa 1 0 0.25 1 10 50 GM-CSF i LL-37 (ug/ml) Figure 2.2. Modulation of neutrophil Mcl-1 expression and cleavage of B I D in response to L L - 3 7 . Neutrophils were exposed to a concentration range o f 0.25-50 pg/ml L L - 3 7 , 30 ng/ml G M - C S F as a positive control, or endotoxin-free water as a vehicle control for 4 h. Whole cell protein lysates were prepared and analyzed by S D S - P A G E and Western immunoblotting. Immunoblots for expression o f (A) Mcl-1 and (B) uncleaved B I D are shown, each representative o f three different donors, and expression of the housekeeping protein G A P D H was assessed as a loading control. Quantitative densitometry was performed, corrected for protein loading, expressed as a proportion o f the vehicle alone-treated control sample, and displayed as mean values ± S E M for three different donors. Student's f-test analyses were used to compare Mcl-1 or B I D expression in L L - 3 7 - or GM-CSF-exposed samples with vehicle alone-treated controls. *, P <0.05. 30 A) 3 . o ro E T3> i n co E ro Pro-caspase-3 Cleaved caspase-3 GAPDH CO c n o i n co o I O cn c: o CO 32 kDa 17 kDa 19 kDa 36 kDa B) CO c CO O II 2 co 8 6 4 2 0 o o co <n _ CL O CO 1 0.75 <-> co 0.5 1 $ 0.25 CD Q> o 0 * i 0 0.25 1 10 50 GM-CSF LL-37 (ng/ml) ** 0 0.25 1 10 50 GM-CSF LL-37 (ng/ml) Figure 2.3. Modulation of pro-caspase-3 cleavage in LL-37-treated neutrophils. Neutrophils were exposed to a concentration range o f 0.25 - 50 (ig/ml o f L L - 3 7 , 30 ng/ml G M - C S F as a positive control, or endotoxin free water as a vehicle control, for 4 hours. Whole cell protein lysates were prepared and analysed by S D S - P A G E and Western Immunoblotting. A ) Immunoblots for expression o f inactive pro-caspase-3 and active cleaved caspase-3 are shown, representative of five different donors, with expression o f the house-keeping protein G A P D H assessed as a loading control. B) Quantitative densitometry was performed, corrected for protein loading, expressed as a proportion o f the vehicle-alone treated control sample, and displayed as mean values ± S E M , for five different donors. Student's t-test analyses were used to compare pro-caspase-3 and cleaved caspase-3 expression in L L - 3 7 or G M - C S F exposed samples with vehicle-alone treated controls, *, p<0.05, **,p<0.01. P2X7 and a G-protein involved receptor is involved in LL-37-induced inhibition of neutrophil apoptosis The immunomodulatory effects o f LL-37 have been proposed to be dependent on signalling through a number of receptors, potentially relevant in this system, including the G -31 protein-coupled receptor FPRL-1 (19) and the purinergic receptor P 2 X 7 (32). Additional, as-yet-unidentified G-protein-coupled receptors and undetermined high- and low-affinity receptors have also been proposed (33-35). To assess the possible significance of these receptors, human peripheral blood neutrophils were pre-incubated with P T X (to inhibit G-protein-coupled receptors), W R W 4 , or oxidized A T P (specific inhibitors of FPRL-1 (36) and P 2 X 7 (32) receptors, respectively) before exposure to a concentration range o f L L - 3 7 . The levels o f spontaneous apoptosis were subsequently determined after 20 h incubation (Figure 2.4A). N o significant LL-37-mediated inhibition of neutrophil apoptosis occurred in the presence of oxidized A T P or P T X at concentrations of L L - 3 7 up to 1 pg/ml, demonstrating effective blockade of L L - 3 7 activity. In contrast, W R W 4 , at the optimal concentration for near-complete inhibition of the FPRL-1-specific a g o n i s t W K Y M V m (36), did not impair the activity of L L - 3 7 , and significant LL-37-mediated inhibition of neutrophil apoptosis was observed at 250 ng/ml (P=0.009) and 1 ug/ml LL-37 (/M).01) in the presence of W R W 4 . To further evaluate the role of F P R L - 1 in this system, human peripheral blood neutrophils were incubated with the F P R L - 1 -specific agonist W K Y M V m at 0.2 p M and 10 u M (approximately equimolar with 1 pg/ml and 50 pg/ml L L - 3 7 , respectively). N o significant effects of this agonist were observed on the level of spontaneous apoptosis after 20 h incubation (Figure 2.4B). To confirm the biological activity o f our W K Y M V m and W R W 4 peptides, chemotaxis of fresh human neutrophils was studied. A s described previously (37), significant chemotaxis of neutrophils was observed in response to 10 u M W K Y M V m (P=0.001). This chemotaxis was inhibited significantly by preincubation with 10 p M W R W 4 (P=0.02; Figure 2.4C). These data suggest that P 2 X 7 receptors and an undetermined G-protein-coupled receptor other than F P R L - 1 are required for LL-37-mediated inhibition of neutrophil apoptosis. 32 C) x 1 0 I 8 .y 6 -«—• o 3 4 I 2 O 0 • OHM WRW4 • 10nMWRW4 ** • 0 10 10 WKYMVm (\xM) Figure 2.4. LL-37-induced inhibition of neutrophil apoptosis is mediated by P2X7 and a G-protein coupled receptor. Neutrophil apoptosis over 20 h incubation was examined in duplicate by F A C S analysis for PE-annexin V-positive, 7AAD-negative cells after (A) incubation with 0.25 pg/ml or 1 |!g/ml LL-37 or endotoxin-free water as a vehicle control, added 30 min after 100 uM-oxidized A T P , 10 u M W R W 4 , 200 ng/ml P T X , or a vehicle-alone control in the presence of 10% F B S . Results represent the percentage of apoptotic cells as mean ± S E M for four or more replicates per condition from five different donors. Paired sample Student's Mest analyses were used to compare LL-37-treated samples with controls under the same inhibitor controls under the same inhibitor conditions. **, P <0.01, or (B) incubation with 1 ug/ml L L - 3 7 , 0.2 u M or 10 \iM W K Y M V m , or a vehicle-alone control. Results represent the percentage of apoptotic cells as mean ± S E M for three replicates from three different donors. Paired sample Student Mest analyses were used to compare treated samples with controls. **, P <0.01. (C) Neutrophil chemotaxis was assessed in triplicate in response to 10 u M W K Y M V m or vehicle-alone control after preincubation with 10 u M W R W 4 or vehicle-alone control, and the chemotactic index was displayed as mean ± S E M for 3 repeats from three different donors. Paired sample Student's M e s t analyses were used to compare WKYMVm-t r ea t ed samples with controls and WRW4-pretreated samples with W K Y M V m alone. *, P <0.05; **, P £0 .01 . LL-3 7-induced inhibition of neutrophil apoptosis involves multiple signalling pathways LL-37 has been demonstrated to induce M A P K activation in a cell type-specific manner, through a PTX-insensitive pathway (22, 27), and to enhance the GM-CSF-dependent activation of these pathways in primary monocytes (38). G M - C S F is a potent inhibitor of neutrophil apoptosis, with this effect mediated through the activation of E R K 1 / 2 M A P K and PI3K pathways (39). In addition, P K C signalling has also been implicated in LL-37-induced angiogenesis by a direct effect on endothelial cells v ia P L C - y / P K C / N F - K B . Thus, to determine the significance of these pathways in this system, human peripheral blood neutrophils were. pre-incubated with PD098059 (inhibitor of the E R K 1 / 2 M A P K pathway via M A P K kinase), LY294002 (PI3K inhibitor), or rottlerin (inhibitor of P K C ) before exposure to 1 tig/ml L L - 3 7 . The levels of spontaneous apoptosis were subsequently determined after 20 h incubation (Figure 2.5). The inhibition of the E R K 1 / 2 M A P K pathway alone resulted in a degree of inhibition of neutrophil apoptosis but did not impair the effects of L L - 3 7 , and significant LL-37-mediated inhibition o f neutrophil apoptosis was observed at 1 ng/ml L L - 3 7 (P=0.03) in the presence of PD098059. In contrast, in the presence of LY294002 or rottlerin, 1 ng/ml o f LL-37 was unable to significantly 34 inhibit neutrophil apoptosis. These data suggest that L L - 3 7 may use the P I3K and P K C but not the E R K 1 / 2 M A P K pathway in the inhibition of neutrophil apoptosis. • 0 no/ml LL-37 • 1 pg/ml LL-37 Figure 2.5. LL-37-induced neutrophil survival involves multiple signalling pathways. Neutrophil apoptosis over 20 h incubation was examined by F A C S analysis for PE-annexin V-positive, 7AAD-negative cells after incubation with 1 pg/ml L L - 3 7 or endotoxin-free water as a vehicle control, added 30 min after 10 u M LY294002, 10 u M rottlerin, 10 u M PD98059, or a vehicle-alone control. Results represent the percentage of apoptotic cells as mean ± S E M for three replicates per condition from four different donors. Paired sample Student's Mest analyses were used to compare LL-37-treated samples with controls under the same inhibitor conditions, or to compare LL-37-treated samples under inhibitor conditions to samples without inhibitor treatments. *, P <0.05. 35 2.4 Discussion Various cationic host-defense peptides [including L L - 3 7 and the murine homologue cathelicidin-related antimicrobial peptide ( m C R A M P ) ] have been demonstrated recently to have multiple immunomodulatory capabilities, potentially representing key mechanisms by which these peptides can enhance host clearance of infection in vivo. The full extent of these immuno-modulatory functions and the precise mechanisms by which these peptides contribute to innate immunity remain undetermined in vivo and are of clear significance in the development of such peptides and their synthetic analogs as novel, antimicrobial therapeutics for multiple antibiotic-resistant infections. The microbicidal activity of L L - 3 7 is acutely sensitive to serum proteins and largely inhibitable under physiologically relevant, ionic conditions (40), although other recently proposed cleavage forms o f hCAP-18 may have more effective microbicidal activities (41, 42). However, various immuno-modulatory properties have been demonstrated in physiological ionic environments, including leukocyte chemotaxis, stimulation o f epithelial cell IL-8 production, post-translational modification of I L - i p , modulation of macrophage transcription, angiogenesis, enhanced wound-healing, modulation of dendritic cell differentiation and function, and adjuvant properties (12, 14, 19-22, 32, 34, 35, 43-46). It remains challenging to demonstrate the relative contribution of direct microbicidal activity versus antimicrobial immuno-modulatory functions in vivo. Studies of m C R A M P indicate that despite high minimum inhibitory concentration values (even against a susceptible mutant bacteria), direct microbicidal activity may contribute to host defense in vivo in specific systems, perhaps at higher concentrations under favorable conditions in a protected niche or functioning synergistically (47, 48). However, our laboratory has recently described in vivo protection against infection in animal models using a synthetic C H D P 36 derivative with no direct in vitro antimicrobial activity (40). Thus, the multiple immuno-modulatory properties described for L L - 3 7 in vitro and in vivo seem likely to be fundamental to the importance of this peptide to the innate immune system. The precise mechanisms underlying the immunomodulatory effects of L L - 3 7 are in many cases unknown. A number of identified and undefined receptors have been described for L L - 3 7 , including F P R L - 1 and other G-protein-coupled receptors, epidermal growth factor receptor (EGFR) , responsive via metalloproteinase-mediated cleavage of membrane-anchored E G F R ligands, and P 2 X 7 receptors acting via caspase-1 (19, 22, 32-35, 38). However, the relative significance of these receptors remains unclear, and traditional, ligand-binding receptor mechanisms may not fully determine peptide activities (42). In addition, M A P K activation is involved in G-protein-independent L L - 3 7 stimulation o f monocytes (38), and endocytic peptide uptake in airway epithelial cells in vitro is required for LL-37-induced IL-8 expression (33, 40). It is demonstrated here that, in addition to the previously described immunomodulatory functions of L L - 3 7 , this C H D P is capable o f modulating apoptotic pathways in primary human neutrophils. The capacity of L L - 3 7 to act as a potent inhibitor o f spontaneous neutrophil apoptosis was demonstrated to involve signalling via P 2 X 7 receptors and G-protein-coupled receptors other than F P R L - 1 . These data confirm and complement some o f the observations made in a recent related paper (49). However, in contrast to our observations, Nagaoka et al. observed a role for FPRL-1 in LL-37-mediated inhibition of neutrophil apoptosis (49). The reasons for this difference remain to be determined but may relate to alternate sources o f FPRL-1 antagonistic and agonistic peptides (subjected to functional validation by reproducing wel l -characterized activities of these peptides in other systems in our study), different peptide solvents, or technical differences in neutrophil purification, which might result in differential background 37 levels of contaminant cell types with FPRL-1-mediated responses to L L - 3 7 , which could influence the response of neutrophils in these assays. Nevertheless, the observation that a combination of receptor types could be involved in LL-37 signalling was also the conclusion of a recent study examining the effects of L L - 3 7 on keratinocyte functions (42). That study, interestingly, also demonstrated similar responses when using D - L L - 3 7 (composed of amino acids in the synthetic D form), arguing against a highly structure-specific interaction between L L - 3 7 and cell surface receptors. Thus, we cannot exclude a complexity exceeding the use of P2X7 receptors and G-protein-coupled receptors in LL-37-mediated inhibition of neutrophil apoptosis. Regardless, our data suggest that this process involves subsequent downstream activation of PI3K and P K C but not the ERK1 /2 M A P K pathway. The latter observation is in keeping with the recent demonstration that in contrast to its effects in monocytes and epithelial cells (22, 38), L L - 3 7 does not stimulate ERK1 /2 phosphorylation in neutrophils (20). We also demonstrate that L L - 3 7 mediates anti-apoptotic effects in neutrophils via an inhibitory effect on the activation of the effector caspase-3 and by altering the balance of Bcl-2 family proteins. It is interesting to note that these effects, observed after 4 h of incubation with L L - 3 7 , preceded substantial membrane translocation of phosphatidylserine (PS), assessed by F A C S analyses of PE-annexin V/7AAD-sta ined cells. After this time-point, apoptosis determined by translocation of PS increased rapidly, and -15% of control neutrophils were PE-annexin V -positive/7AAD-negative at 6 h (data not shown), demonstrating the temporal sequence o f events in apoptosis of these cells. These data support previous studies indicating that the translocation of PS is a caspase-3-dependent event downstream o f caspase activation (50) and that L L - 3 7 -mediated inhibition of caspase-3 cleavage precedes effects on translocation of PS in neutrophils undergoing spontaneous apoptosis. 38 L L - 3 7 modulated expression of the anti-inflammatory protein M c l - 1 , preventing the loss of expression associated with spontaneous apoptosis. This Bcl-2 family protein has a short half-life, providing rapid response to environmental stimuli and playing a critical role in promoting neutrophil survival by inhibiting mitochondrial damage and cytochrome c release (29, 51). L L - 3 7 also inhibited cleavage of the B H 3 domain-only Bcl-2 protein B I D , thereby decreasing generation o f the pro-apoptotic p i 5 B I D cleavage fragment, believed to translocate to the mitochondria and trigger release o f cytochrome c during the induction of apoptosis (30, 31). Cleavage o f B I D by caspase-8 is critical for death receptor-induced apoptosis but also plays an important role in spontaneous neutrophil apoptosis, even in the absence of death receptor stimulation (52). In addition to the inhibition of cleavage of the key effector caspase-3, these observations suggest that L L - 3 7 exerts its anti-apoptotic effect on neutrophils by acting upstream of mitochondria on intrinsic and traditionally extrinsic pathways for the induction o f programmed cell death. The consequences for LL-37-mediated inhibition o f neutrophil apoptosis in vitro were demonstrated to be dependent on the concentration of L L - 3 7 . After 20 h exposure to lower concentrations of L L - 3 7 , significant increases were observed in the proportion o f viable cells at the expense of apoptotic cells. However, at higher concentrations of L L - 3 7 , despite further increases in the proportion of viable cells, the more dramatic decreases in apoptotic cells were accompanied by an increase in neutrophil necrosis. This switch in cellular fate, evident at approximately 10 pg/ml, could represent a transition to inflammatory concentrations o f L L - 3 7 in vivo (4). The neutrophil necrosis observed is likely a consequence of the inhibition of apoptotic pathways, rather than secondary necrosis o f apoptotic cells, given the potent inhibition o f apoptotic pathways observed after 4 h exposure to L L - 3 7 , before any substantial, detectable cell death, and bears similarities to the recently reported effects o f L P S in inhibiting apoptosis but 39 inducing necrosis in neutrophils (53). In contrast, the anti-apoptotic factor G M - C S F acts primarily as a potent neutrophil-survival factor (54). Thus, although LL-37-mediated inhibition of apoptosis led to a dose-dependent increase in neutrophil survival, higher concentrations o f peptide also promoted an additional switch from apoptotic to necrotic cell death. The in vivo consequences of LL-37-mediated inhibition of neutrophil apoptosis remain to be determined. Low-level , acute increases in L L - 3 7 may primarily enhance neutrophil survival, promoting the clearance of infection. However, although necrosis induced by higher concentrations of L L - 3 7 could be beneficial to the host by amplifying the acute inflammatory response [as proposed previously for LPS-induced necrosis (53)], the uncontrolled release of neutrophil contents in chronic inflammation would be expected to result in host damage and impair the resolution of inflammation. In addition to enhancing neutrophil survival, L L - 3 7 has previously been demonstrated to be chemotactic for neutrophils in vitro, directly (19) and indirectly, by inducing IL-8 production by epithelial cells (12), and the murine homologue C R A M P has been shown to induce neutrophil recruitment in vivo in a mouse model (21). Further, L L - 3 7 release by recruited neutrophils would therefore amplify these responses and the effects of L L - 3 7 on neutrophil apoptosis. Thus, raised levels of L L - 3 7 in an acute inflammatory scenario could contribute to innate host defenses by mediating recruitment and enhanced survival o f neutrophils to enhance the resolution o f an acute, infectious insult. A full knowledge of the in vivo significance o f these L L -37-mediated immunomodulatory activities and their applicability to other C H D P is essential in developing the potential of C H D P as future immunomodulatory, antimicrobial therapeutics. 40 2.5 Bibliography 1. Zasloff, M . 2002. 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Modulation of the TLR-mediated inflammatory response by the endogenous human host defense peptide L L - 3 7 . J Immunol 176:2455-2464. 44. Heilborn, J. D . , M . F. Nilsson, G . Kratz, G . Weber, O. Sorensen, N . Borregaard, and M . Stahle-Backdahl. 2003. The cathelicidin anti-microbial peptide L L - 3 7 is involved in re-epithelialization of human skin wounds and is lacking in chronic ulcer epithelium. J Invest Dermatol'120:379-389. 45. Shaykhiev, R., C. Beisswenger, K . Kandler, J. Senske, A . Puchner, T. Damm, J. Behr, and R. Bals. 2005. Human endogenous antibiotic L L - 3 7 stimulates airway epithelial cell proliferation and wound closure. Am J Physiol Lung Cell Mol Physiol 289:L842-848. 43 46. A n , L . L . , Y . H . Yang, X . T. M a , Y . M . L i n , G . L i , Y . H . Song, and K . F. W u . 2005. L L -37 enhances adaptive antitumor immune response in a murine model when genetically fused with M - C S F R (J6-1) D N A vaccine. LeukRes 29:535-543. 47. Braff, M . H . , M . 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Activities of L L - 3 7 , a cathelin-associated antimicrobial peptide of human neutrophils. Antimicrob Agents Chemother 42:2206-2214. 57. Scott, M . G . , A . C. Vreugdenhil, W . A . Buurman, R. E . Hancock, and M . R. Gold. 2000. Cutting edge: cationic antimicrobial peptides block the binding of lipopolysaccharide (LPS) to L P S binding protein. J Immunol 164:549-553. 44 3.0 The Human Host Defense Peptide LL-37 Differentially Modulates Neutrophil Cytokine /chemokine Responses To Inflammatory Stimuli 3.1 Introduction Cathelicidins are cationic host defense peptides found in mammals. These peptides are important components in host defense against pathogenic microbial challenge. Cathelicidins have been demonstrated to have a broad range of biological functions in modulating the immune response as well as direct antimicrobial activities that are observed for only certain peptides under physiological conditions. hCAP-18 is the sole human cathelicidin and is stored at high concentrations in the specific granules of neutrophils and upon release, is processed to its active form, L L - 3 7 , a 37 amino acid peptide (1). This peptide is also found at low concentrations in a variety of blood cell populations including N K cells, ybT cells, B cells, monocytes and mast cell (2). The precursor hCAP-18 is also expressed in a range of epithelial cells and keratinocytes upon stimulation with bacterial components or pro-inflammatory cytokines (3, 4), and the processed peptide LL-37 is found in various body fluids including sweat, gastric fluid, airway surface liquid, breast milk, and saliva (2). LL-37 has been shown to have diverse functions in the innate immune response exemplified by its ability to promote angiogenesis and wound healing (5-7), to prolong the life span of neutrophils as demonstrated in Chapter 2, and to protect rodents against endotoxemia/sepsis induced by L P S (8, 9). Consistent with this, LL-37 has also been shown to inhibit LPS-induced pro-inflammatory cytokine T N F - a production in human monocytic cells (10). In addition, LL-37 acts in synergy with cytokine G M - C S F in inducing increased IL-8 * The majority of this work is being prepared for publication: L i , Y . , Pistolic, J. and Robert E .W. Hancock. The human cationic host defense peptide L L - 3 7 in regulating chemokine responses of neutrophils and airway epithelial cells and their interactions. 45 production via enhanced activation of E R K 1 / 2 and p38 M A P kinases (11). One of the important roles of inflammation is to deliver additional effector molecules and cells to sites of infection to augment the elimination of invading pathogens. Leukocytes, in response to chemoattractic agents, accumulate in the inflamed tissues during the course o f early infection and dissipate as the infection is resolved (12). L L - 3 7 has been shown to act as a chemoattractant to directly induce chemotaxis of a range of blood cells including neutrophils, monocytes, eosinophils, mast cells and CD4+ T cells although the L L - 3 7 concentrations necessary for chemotaxis appear to be much higher than conventional chemokines (13, 14). L L -37 may also contribute to the recruitment of leukocytes by inducing the production of chemokines. For example, LL-37 has been well demonstrated to induce IL-8 production from airway epithelial cells, keratinocytes and human peripheral blood derived monocytes (6, 15-17). Although neutrophils are terminally differentiated cells, they are able to express and secrete inflammatory cytokines such as T N F - a and a number o f chemokines including IL-8, Gro-a, M I G , IP-10, M i p - l a and Mip-1(3 in response to inflammatory signals such as L P S (18). A s neutrophils are predominate leukocytes at the site of infection, it is essential to understand the effects of LL-37 on cytokine/chemokine production of these cells. In addition to L L - 3 7 , there are many other important inflammatory mediators, such as endogenous I L - i p or bacterial molecules like L P S , in the inflammatory milieu, and these various effector molecules interact to ensure efficient and balanced functioning of the innate immune system. To understand the role of LL-37 in regulating neutrophil cytokine/chemokine responses in the presence or absence of other key inflammatory signals, I examined the production of T N F - a cytokine and chemokines including Gro-a, M i p - l a , IL-8 and C C L 2 2 in neutrophils. I provide evidence in this Chapter that human host defense peptide L L - 3 7 has potent anti-endotoxin properties in neutrophils, acting at the transcriptional level. It is further 46 demonstrated here that LL-37 enhances IL-ip-induced release of chemokines including C C L 2 2 , IL-8, M i p - l a , and Gro-a, all o f which are important in recruiting leukocytes to the site of infection/inflammation. These secreted chemokines result from de novo protein synthesis but are not regulated at the transcriptional level. In addition, the NFKB and E R K 1 / 2 and p38 M A P K signalling pathways are involved in the enhanced release of most chemokines induced by co-stimulation with L L - 3 7 and IL-1 p. 3.2 Materials & Methods Reagents Recombinant human interleukin I L - i p was purchased from Research Diagnostics Inc. (Flanders, NJ) . Pharmacological inhibitor BAY11-7085 was purchased from Biomol International (Plymouth Meeting, PA. ) and PD98059 was from Cel l Signalling Technology, Inc. (Mississauga, O N , Canada). Cycloheximide and SB203580 were purchased from Sigma (Oakville, Ontario, Canada). Lipopolysaccharide (LPS) from Pseudomonas aeruginosa Strain HI03 was highly purified free of proteins and lipids using the Darveau-Hancock method as described previously (19). Briefly, P. aeruginosa was grown overnight in Luria-Bertani broth at 37°C. Cells were collected and washed, and the isolated L P S pellets were extracted with a 2:1 chloroform:methanol solution to remove contaminating lipids. Purified L P S samples were quantitated using an assay for the specific sugar 2-keto-3-deoxyoctosonic acid assay and then resuspended in endotoxin-free water (Sigma-Aldrich). L L - 3 7 was synthesized by N-(9-fluorenyl) methoxycarbonyl chemistry at the Nucleic Acid/Protein Service unit at the University of British Columbia ( U B C , Vancouver, Canada), as described previously (20). Peptides were purified by reverse-phase high-performance liquid chromatography and were at least 98% pure. LL-37 was dissolved in endotoxin-free water (Sigma-Aldrich) and stored at - 2 0 ° C until further 47 use. The concentration of the peptides in solution was determined by amino acid analysis. A l l reagents were tested to ensure that they were free of endotoxin and reconstituted in endotoxin-free water. Isolation of human blood neutrophils Fresh human venous blood was collected from volunteers, according to University of British Columbia Clinical Research Ethics Board protocol C02-0091, using Vacutainer® collection tubes containing sodium heparin ( B D Biosciences, Mississauga, Ontario, Canada). Blood was mixed, at 1:1 ratio with 2 % Dextran T-500 (Amersham Pharmacia Biotech, Buckingham, U K ) in 0.9% saline and sedimented for 30 min at room temperature. The leukocyte-rich upper layer was then fractionated by Ficoll-Paque gradient centrifugation as described previously (21). Briefly, cells were centrifuged at 200 g for 7 min, remaining erythrocytes were lysed hypotonically with ice-cold, distilled water for 30 seconds, followed by restoration of tonicity with 2.5% saline, and neutrophils were separated by centrifugation over a Ficoll-Paque Plus (Amersham Pharmacia Biotech) density gradient at 400 g for 25 min at 4°C. The cells were washed with Krebs-Ringer phosphate buffer (pH 7.3), containing glucose (10 m M ) and M g 2 + (1.5 mM) , and resuspended in RPMI-1640 media (Invitrogen), supplemented with 10% (v/v) heat-inactivated F B S , 1% (v/v) L-glutamine, and 1 n M sodium pyruvate. Detection of cytokines Fresh human blood neutrophils were plated at 1 x 10 6 cells in 1 ml RPMI-1640 media (supplemented with 10% (v/v) heat-inactivated F B S , 1% (v/v) L-glutamine, 1 n M sodium pyruvate) in 24-well plates. Cells were then incubated in media for 20 h in the presence of P. aeruginosa HI03 L P S , L L - 3 7 , IL-1B (at the stated concentrations), or endotoxin-free water as a vehicle control, in at least triplicate. Supernatants were collected and stored at - 2 0 ° C until used. 48 The concentrations of cytokine or chemokines in the supernatants were measured using commercially prepared E L I S A kits in accordance with the manufacturer's instructions. Specifically, E L I S A kits were purchased for T N F - a (eBioscience, San Diego, C A ) , M i p - l a and IL-8 (BioSource International, Camarillo, C A ) , and Gro-a /CCL22 ( R & D Systems, Minneapolis, M N ) . RNA extraction and quantitative PCR Fresh human blood neutrophils were plated at 3 x 10 6 cells in 3 ml RPMI-1640 media (supplemented with 10% (v/v) heat-inactivated F B S , 1% (v/v) L-glutamine, 1 n M sodium pyruvate) in 6-well plates. Cells were then incubated for 2 h in the presence of P. aeruginosa H103 L P S , L L - 3 7 , I L - i p (at the stated concentrations), or endotoxin-free water as a vehicle control. R N A was isolated from neutrophils with RNeasy M i n i kit, digested with RNase-Free DNase (Qiagen, Mississauga, Ontario), and eluted in RNase-free water (Ambion, Austin, T X ) in accordance with the manufacturer's instructions. R N A concentration, integrity, and purity were assessed by Agilent 2100 Bioanalyzer using R N A 6000 Nano kits (Agilent Technologies, Santa Clara, C A ) . Reverse transcription and q P C R was performed using Superscript III Platinum Two-Step q R T - P C R kit with S Y B R Green (Invitrogen Life Technologies, Burlington, Ontario) according to the manufacturer's instructions, in the A B I Prism 7000 sequence detection system (Applied Biosystems). A melting curve was performed to ensure that any product detected was specific to the desired amplicon. Fold changes were calculated after normalization to endogenous G A P D H and using the comparative Ct method (22). The primers used for qRT-P C R are reported in Table 3.1. 49 Table 3.1. Primer sequences used in this study. Primer Forward sequence (5'-3') Reverse Sequence (5'-3') M I P - l a / C C L 3 G C A T C A C T T G C T G C T G A C A C C T G G A C C C A C T C C T C A C T G G G r o - a / C X C L l G C C A G T G C T T G C A G A C C C T G G C T A T G A C T T C G G T T T G G G I L - 8 / C X C L 8 * G A C C A C A C T G C G C C A A C A C C T T C T C C A C A A C C C T C T G C A C C C L 2 2 T G C C G T G A T T A C G T C C G T T A A A G G T T A G C A A C A C C A C G C C T N F - a A G G G A G C C T T T G G T T C T G G T C A G C A A T G A G T G A C A G T T G G G A P D H G A A A C T G T G G C G T G A T G G G T C G C T G T T G A A G T C A G A G G Statistical analysis Student's Mests were performed to determine statistical significance, and P <0.05 was considered significant. Values shown are expressed as mean ± S E M . 3.3 Results LL-37 differentially modulates neutrophil TNF-a cytokine response to inflammatory stimuli It is well known that L L - 3 7 neutralizes T N F - a induced by L P S in human monocytes and a monocytic cell line (THP-1) (10). To evaluate the effect of L L - 3 7 on T N F - a release from neutrophils induced by L P S or another inflammatory cytokine I L - i p , neutrophils were incubated for 20 h with L P S or I L - i p in the presence or absence of 10 pg/ml L L - 3 7 , with subsequent determination of the T N F - a release by E L I S A analysis of cell supernatants (Figure 3.1 A ) . A s expected, on the basis of the well-characterized, anti-endotoxic effects ascribed to this peptide in the responses of other cell types, L L - 3 7 inhibited the release o f T N F - a in response to L P S exposure. However, in contrast, L L - 3 7 exposure of neutrophils led to an significant but modest enhancement of the release o f T N F - a (PK).04) in the presence of I L - i p . These data demonstrate functional cytokine responses in LL-37-treated neutrophils and indicate that L L - 3 7 modulates the cytokine responses of neutrophils to inflammatory signals in a stimulus-specific manner. To examine whether the effect of L L - 3 7 on T N F - a release was regulated at the transcriptional level, peripheral blood neutrophils were exposed for 2 hours to L P S or I L - i p in 50 the presence or absence of L L - 3 7 . Total R N A was extracted, and the expression of the T N F - a gene was examined by quantitative R T - P C R analysis. A s shown in Figure 3 . IB, exposure to L L - 3 7 substantially inhibited LPS-induced T N F - a m R N A (by approximately 89%), which is consistent with the decreased T N F - a release at the protein level. In contrast, L L - 3 7 did not significantly alter IL-ip-mediated increased T N F - a transcription. In summary, L L - 3 7 inhibited LPS-induced pro-inflammatory cytokine T N F - a production at the transcriptional level, but through different mechanisms, it modestly enhanced IL-ip-induced T N F - a release from neutrophils. A ) 300 J= 200 o> 3 a' £ 100 h-0 O 0 (ig/ml LL-37 • 10 Ltg/ml LL-37 ** Control IL-13 LPS Figure 3.1 A. Modulation of neutrophil TNF-a production by LL-37. 51 B) 25 n • 0 HQ/ml LL-37 • 10 ng/ml LL-37 20 -U 2 10 o LL * 5 0 Control IL-1R LPS Figure 3.1. Modulation of neutrophil TNF-a production by LL-37. A ) The T N F - a release by neutrophils was assessed by E L I S A analysis o f culture supernatants following incubation of cells for 20 h in the presence of 100 ng/ml L P S , 10 ng/ml IL-1B or vehicle-alone control, in the presence of absence of 10 ng/ml L L - 3 7 . Data represent means ± S E M for three replicates per condition from four different donors. Paired sample Student's Mest analyses were used to compare LL-37-treated samples with controls under the same stimulatory conditions. *, P <0.05; **, P <0.01. B ) Neutrophils were incubated for 2 hours with 100 ng/ml L P S , 10 ng/ml IL-1 p, or vehicle-alone control, in the presence or absence of 10 ng/ml L L - 3 7 , and the T N F - a gene expression was examined by qPCR. Fold changes (y-axis) were normalized to G A P D H and are relative to the gene expression in unstimulated cells (normalized to 1) using the comparative Ct method (see Materials and Methods for details). q P C R data represent means ± S E M for duplicates per condition from four different donors. Paired sample Student's Mest analyses were used to compare LL-37-treated samples with controls under the same stimulatory conditions. *, P<0.05. 52 LL-37 synergizes with IL-l(5in regulating neutrophil chemokine responses It is well documented that LL-37 is able to induce chemokine IL-8 production in human leukocytes, airway epithelial cells and keratinocytes (6, 11, 23). In addition, L L - 3 7 induces the gene expression of chemokines including M C P - 1 , M l P - l a , M I P - i p in human monocytes (11). It is known that neutrophils express a number of chemokines in response to inflammatory signals (24). To examine the role o f L L - 3 7 in modulating neutrophil chemokine response to I L - i p , human peripheral blood derived neutrophils were stimulated for 20 h with L L - 3 7 and/or I L - i p , with subsequent determination o f the chemokine responses by E L I S A analysis o f cell supernatants. A s demonstrated by Figure 3.2, L L - 3 7 alone induced a small but significant amount of C C L - 2 2 , Gro-a, M i p - l a and IL-8 release from neutrophils. Neutrophil chemokine responses to I L - i p appear to be donor dependent, ranging from virtually unresponsive to moderately induction of chemokines. However, L L - 3 7 exposure of neutrophils significantly enhanced the release of all chemokines tested (P <0.05) in the presence of I L - i p . These results demonstrated that the host defense peptide L L - 3 7 acted in synergy with I L - i p to augment the inflammatory chemokine responses in neutrophils. 53 E "B) a c o "•S3 (0 c a> o c o o Control IL-1 B Mip-1a G r o - a • 0 pg/ml LL-37 • 10|jg/mlLL-37 400 300 200 100 0 Control IL-1 B IL-8 800 600 400 200 Control IL-1B Control IL-1 B Figure 3.2. LL-37 enhances IL-lp-induced chemokine secretion by neutrophils. The C C L 2 2 , Gro-a , M i p - l a , and IL-8 release by neutrophils was assessed by E L I S A analysis of culture supernatants following incubation of neutrophils for 20 hours with 10 ng/ml IL-1 B, or a vehicle-control alone, in the presence or absence o f 10 )J.g/ml L L -37. Data represent means ± S E M for three replicates per condition from four different donors. Paired sample Student's Mest analyses were used to compare LL-37-treated samples with controls under the same stimulatory conditions. * P <0.05. 54 Synergistic inductions of the chemokines by LL-37 and IL-1(5 are not regulated at the mRNA level To examine whether the synergistic effect between L L - 3 7 and I L - i p in inducing chemokine release is regulated at the transcriptional level, neutrophils were exposed for 2 hours to LL-37 in the presence or absence of I L - i p , and total R N A was extracted from the cells. Quantitative R T - P C R analysis was used to examine the gene expression of C C L 2 2 , Gro-a , M i p -l a , and IL-8. A s shown in Figure 3.3, the gene expression of these chemokines was not induced by L L - 3 7 alone but was enhanced in various degrees in response to IL-1 B stimulation. In contrast to augmented chemokine protein release, L L - 3 7 did not further increase gene expression o f these chemokines induced by I L - i p . These results suggest that the synergistic effect of L L - 3 7 and IL-1 p in chemokine production is not regulated at the transcriptional level in neutrophils. 55 CD O) c co .c o ; g o CCL22 Gro-a Control IL-1p Mip-1a 20. 16 12 8 4 0 • 0 (jg/ml LL-37 • lOpo/ml LL-37 Control IL-1P IL-8 1 4 1 i i 3 1 1 • Control IL-1P 1 Control IL-ip Figure 3.3. Effect of LL-37 and IL- ip on chemokine gene expression in neutrophils. Neutrophils were incubated for 2 hours with 10 ng/ml I L - i p or vehicle-alone control, with or without 10 ug/ml L L - 3 7 , and the gene expression of C C L 2 2 , Gro-a , M i p - l a , and IL-8 were examined by qPCR. Fold changes (y-axis) were normalized to G A P D H and are relative to the gene expression in unstimulated cells (normalized to 1) using the comparative Ct method (see Materials and Methods for details). The result of an experiment from one donor, representative of four, is shown. 56 De novo protein synthesis is required for chemokine release induced by co-stimulation of LL-37 andlL-lB L L - 3 7 has been shown to induce rapid release of IL-8 from monocytes, as early as 15 minutes after stimulation (Bowdish and Hancock, unpublished results). It has been suggested that this induction results from de novo synthesis o f IL-8, since it can be inhibited by cycloheximide, an inhibitor of protein synthesis (25). To examine whether the chemokines induced by co-stimulation of LL-37 and I L - i p were the result of de novo protein synthesis or release from internal stores, neutrophils were pre-treated with cycloheximide prior to stimulation with LL-37 and/or I L - i p for 20 hours (Figure 3.4). A s expected, the presence of cycloheximide substantially reduced the amount of all chemokines released in the supernatant, indicating these chemokines were primarily newly synthesized due to co-stimulation of L L - 3 7 and I L - i p . 57 E Q. C o (0 c o c o o CCL22 400 300 ^ 200 100 -0 -800 600 400 200 0 Mip-1a I I . A S Gro-a • (-) cycloheximide • (+) cycloheximide 400 300 I 2 0 0 100 IL-8 800 600 400 * 200 I , 0 1^  CO CO. o O co + CO. CO c o o X I CO + oa. Figure 3.4. Increased chemokines release as a result of de novo protein synthesis. Neutrophils were incubated for 30 minutes with 1 ng/ml cycloheximide before exposure to 10 ng/ml I L - i p or vehicle-alone control, in the presence or absence of 10 ng/ml L L - 3 7 . The IL-8, M i p l - a , C C L 2 2 , and Gro-a release was assessed by E L I S A analysis of culture supernatants following incubation o f cells for 20 hours. Data represent means ± S E M , for three replicates per condition from four donors. Paired sample Student's Mest analyses were used to compare LL-37-treated samples with controls under the same stimulatory conditions. *, p<0.05. 58 ERK1/2 and p38 signalling pathways are involved in the synergistic induction of chemokines by LL-37 and IL-ip L L - 3 7 has been shown to mediate its effects through the activation of a number of signalling pathways. For example, LL-37-induced IL-8 production is mediated via the p38 and E R K 1 / 2 M A P K signalling pathways in human monocytes and airway epithelial cells (6, 11). NFKB signalling has also been implicated in inducing the transcription of IL-8 (Bowdish and Hancock, unpublished results). To determine the significance o f these pathways in the enhanced chemokine production induced by co-stimulation of L L - 3 7 and I L - i p , neutrophils were pre-incubated with inhibitors including PD098059 (inhibitor of the E R K 1 / 2 M A P K pathway via M A P K kinase), SB203580 (inhibitor of p38 M A P K ) or BAY11-7058 (inhibitor of the NFKB pathway via IKB) prior to exposure to L L - 3 7 and/or I L - i p . Subsequent chemokine responses were determined by E L I S A analysis o f cell supernatants (Figure 3.5). The inhibition o f the E R K 1 / 2 , p38 M A P K or NFKB pathways significantly suppressed production of IL-8, M i p l - a , and C C L 2 2 ; however, among the four donors, only two donors showed a reduction on Gro -a levels in the presence of these inhibitors (data not shown). The other two donors had very low level of Gro-a release even in the presence of both L L - 3 7 and I L - i p . These results demonstrate that the M A P K and NFKB pathways are involved in regulating the release o f IL-8, M i p l - a , and C C L 2 2 induced synergistically by L L - 3 7 and I L - i p . 59 C o co v-+-» C (D O C o O CCL22 800 600 400 200 0 800 600 Mip-1a IL-8 800 600 400 200 0 co c o O • (-) Inhibitor • (+) PD98059 • (+) SB203580 • (+) BAY11-7085 t * % co + C O . 400 200 0 * ^ * * * * CO i -c o O CO + CO. Figure 3.5. Signalling regulation of chemokine responses induced by LL-37 and IL-1(3 in neutrophils. Neutrophils were pre-treated for 30 minutes with 12.5 u M SB203580, 10 u M PD098059 or 8 u M BAY11-7085 before they were exposed to 10 ng/ml I L - l p or vehicle-alone control, in the presence or absence o f 10 fig/ml L L - 3 7 . The IL-8, C C L 2 2 , and M i p - l a release by neutrophils was assessed by E L I S A analysis o f culture supernatants following incubation of cells for 20 hours. Data represent means ± S E M , for three replicates per condition from 4 different donors. Paired sample Student's Mest analyses were used to compare inhibitor-treated samples with controls under the same stimulatory conditions. * p<0.05. 60 3.4 Discussion Growing body of evidence shows that the human cathelicidin L L - 3 7 is a multi-functional immunomodulator, and the underlying mechanism of these effects has been investigated extensively in recent years. The functions of L L - 3 7 tend to be studied in an isolated manner; however, the immune system comprises numerous immune mediators which would also arise in the inflammatory milieu. The interplay of these molecules is important in defining the outcome of the immune responses. In this Chapter, I examined how L L - 3 7 modified the cytokine/chemokine responses induced by different inflammatory mediators, and I demonstrate that, while L L - 3 7 alone induced a small amount of chemokine release from neutrophils, it greatly enhanced IL-ip-induced release of chemokines including IL-8, Gro-a, C C L 2 2 , and M i p -l a . The increased production of chemokine was not regulated at the m R N A level, but resulted from de novo protein synthesis, presumably from existing pools of m R N A s . The E R K 1 / 2 and p38 M A P K and NFKB signalling pathways were involved in regulating the synergistic release o f IL-8, C C L 2 2 , and M i p - l a , but not Gro-a. In contrast to its effect on IL-ip-induced chemokine responses, L L - 3 7 abrogated LPS-induced T N F - a gene expression as well as protein release. These results demonstrated that the modulatory effects o f L L - 3 7 on neutrophil cytokine/chemokine responses to distinct inflammatory signals are stimulus-specific. Pro-inflammatory mediators are essential for an effective host defense to clear infections; however, uncontrolled and excessive production of T N F - a often results in sepsis and even septic shock (26-29). L P S is the most common agent to cause sepsis. The ability of L L - 3 7 to block L P S induced pro-inflammatory cytokine production is thought to confer a protective effect to the host against sepsis. Neutrophils account for the majority of infiltrated leukocytes at the infection foci and are a main source o f T N F - a induced by inflammatory stimuli including 61 L P S (24). I demonstrate here that LPS-induced T N F - a release from neutrophils can be substantially blocked by L L - 3 7 and that this blockade of T N F - a is regulated at the transcriptional level. This could be due in part to the direct binding o f L L - 3 7 to the l ipid A core of L P S , thereby impeding the binding o f L P S to L B P and subsequent activation of T L R signalling (30). However, more recent evidence strongly suggests that the anti-endotoxin activity o f L L - 3 7 involves additional mechanisms o f action (10). However, the potential binding between L L - 3 7 and L P S complicates analysis of the effects resulting from other regulatory systems, and further well-designed experiments are needed to gain full understanding. In contrast to its anti-endotoxin activity, L L - 3 7 was demonstrated here to synergize with the inflammatory cytokine IL-1 P in inducing chemokine production from neutrophils. IL-1B is an important cytokine induced via the activation o f T L R signalling by pathogen-derived stimuli at the sites o f infection (31). The concentration o f L L - 3 7 wi l l also rise at these sites due to neutrophil degranulation and increased expression in epithelia (32, 33). Thus it is not surprising that I L - i p and L L - 3 7 would co-exist in the inflammatory milieu at the infection focus. Neutrophils co-stimulated with LL-37 and I L - i p released significantly higher level of chemokines than those o f exposed to either stimulus alone. These released chemokines are essential components of the immune response as they recruit effector cells to infected tissues to eliminate pathogens. The synergistic effect between LL-37 and IL-1 B is of advantage particularly at the onset of infection when the concentration of either is too low to independently induce sufficient chemokines. The ability o f L L - 3 7 to amplify favourable inflammatory signals further demonstrates that L L - 3 7 functions as a modulator of immune responses. To gain insights into the mechanisms by which L L - 3 7 and IL-1B synergistically regulate neutrophil chemokine response, I examined the signalling pathways that have been implicated in 62 the LL-37-mediated effects. For example, low concentrations of L L - 3 7 (5-10 ug/ml) have previously been shown to induce activation of E R K 1 / 2 and p38 M A P kinases in synergy with G M - C S F , resulting in increased IL-8 production in monocytes (11). These M A P K s are clearly involved in the synergistic effect of L L - 3 7 and I L - i p induced release of IL-8 , C C L 2 2 , and M i p -l a from neutrophils. The p38 M A P K is known to regulate cytokine/chemokine production at the post-transcriptional level by controlling the stability of m R N A (34). A common feature of inflammatory m R N A s , including chemokines, is the presence of A U - r i c h sequences in the 3' untranslated regions (35). The sequences o f A U - r i c h elements ( A R E ) target m R N A for rapid degradation through mechanisms such as deadenylation (36) or by exosome which is recruited to m R N A via certain A R E binding proteins (34). The activated M A P kinase p38 has been shown to stabilize these m R N A s , although the precise mechanism is not entirely understood. The involvement of the p38 pathway suggests that the enhanced chemokine production induced by IL-1 B and L L - 3 7 is partly due to increased chemokine m R N A stability. In agreement with this, I showed here that the translation inhibitor cycloheximide abrogated the release of all chemokines, indicating that the chemokines were newly synthesised proteins rather than released from a pre-stored pool. In contrast to the aforementioned well-established role of p38, it is not entirely clear how E R K 1 / 2 regulates chemokine production. It has been proposed that the IL-8 expression regulated by E R K 1 / 2 is exerted through the activation of NFKB (35). NFKB signalling has also been implicated in LL-37-induced production o f IL-8 in human P B M C s , since an inhibitor o f IKB degradation suppressed IL-8 release by approximately 96% (Mookerjee and Hancock, unpublished results). In addition, Y u , Bowdish and Hancock have shown that L L - 3 7 induces transient IKB degradation and nuclear translocation of certain NFKB subunits in P B M C s , and in the presence of I L - i p , the magnitude of IKB degradation is increased ( Y u et al, manuscript 63 submitted). However, in my study, L L - 3 7 alone did not induce expression of any of the chemokines genes, nor did it enhance IL-ip-induced chemokine gene expression. These results indicate that the LL-37-mediated effects in this context may not involve the activation of NFKB, and the observed inhibition of chemokine release in the presence of the inhibitor of NFKB signalling is primarily due to its known inhibitory effect on IL-ip-induced activities. This further demonstrates that some, i f not all, o f the effects of L L - 3 7 on the immune response are cell type specific. Unlike other chemokines, the inhibitors for M A P K and NFKB pathways had inconsistent effects on the release of Gro-a. Among the four donors tested, two donors showed reduction of Gro-a release in response to the inhibitors while others did not. Associated with this, the two donors who did not respond to the inhibitors had very low Gro-a protein release in all conditions, which could be responsible for the insignificant inhibition due to relatively large errors. Further studies involving a large number of donors are needed to solve the issues related to donor variation and to ultimately draw clear conclusions with this chemokine. Elevated cytokine/chemokine production at the site of infection and leukocyte infiltration are general features of inflammation and essential to control infections. L L - 3 7 contributes to innate host defenses by modulating neutrophil cytokine/chemokine responses. Specifically, this peptide enhances the release of chemokines in the presence o f endogenous inflammatory mediators while minimizing the level of detrimental pro-inflammatory cytokine T N F - a resulted from L P S stimulation. These abilities to augment the innate immune response clearly represent novel and potentially powerful means to prevent or treat infections. 64 3.5 Bibliography 1. Sorensen, O. E . , P. Fol l in , A . H . Johnsen, J. Calafat, G . S. Tjabringa, P. S. Hiemstra, and N . Borregaard. 2001. Human cathelicidin, hCAP-18 , is processed to the antimicrobial peptide L L - 3 7 by extracellular cleavage with proteinase 3. Blood 97:3951-3959. 2. Zanetti, M . 2004. Cathelicidins, multifunctional peptides of the innate immunity. J Leukoc 5/0/75:39-48. 3. Frohm, M . , B . Agerberth, G . Ahangari, M . Stahle-Backdahl, S. Liden, H . Wigzel l , and G . H . Gudmundsson. 1997. 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Popowych, R. Falsafi, J. J. Y u , Y . L i , S. Veatch, F. M . Roche, K . L . Brown, F. S. Brinkman, K . Hokamp, A . Potter, L . A . Babiuk, P. J. Griebel, and R. E . Hancock. 2006. Bovine and human cathelicidin cationic host defense peptides similarly suppress transcriptional responses to bacterial lipopolysaccharide. J Leukoc Biol. 67 4.0 Discussion 4.1 Prolonged neutrophil survival is necessary to control acute infections but detrimental when dysregulated Neutrophils generally have a critical role in eliminating pathogenic bacteria and fungi. This important role can be demonstrated in those conditions in which neutrophils do not function properly. For example, neutrophils with C D 1 8 / L F A deficiency are not able to transmigrate from circulation to the site of infection; and the patients with these defective neutrophils suffer from recurrent infections (1). To function properly, neutrophils need to be present in adequate numbers and to have an appropriately functioning machinery to phagocytose and k i l l infecting microorganisms. However their disadvantageous short life span may pose a problem in resolving acute infections as rapidly aging neutrophils lose the ability to k i l l pathogens. Fortunately in such conditions, there are ample micro-environmental inflammatory signals which would activate neutrophils, not only enhancing their microbicidal functions, but also prolonging their life span. A s demonstrated in Chapter 2, L L - 3 7 serves as a survival agent, potently extending the half life of neutrophils even at low concentrations (Figure 2.1). This essentially enables rapid removal o f invading pathogens to avoid unnecessary activation of adaptive immunity and/or chronic infectious conditions. However, because activated neutrophils release cytotoxic cellular components to the surrounding tissues while eliminating pathogens, a common view is that this can cause detrimental host tissue damage (2). Conversely, early in infection, small-scale neutrophil-mediated tissue destruction would serve to disassemble local collagen fibrils that could potentially obstruct neutrophil-pathogen contact (3). Obviously, prolonged neutrophil survival should be minimized in situations like chronic infections or pathological conditions such as chronic granulomatous disease (CGD) . C G D is a rare condition in which one of several components of the N A D P H oxidase system is dysfunctional, rendering all cells incapable of 68 generating toxic reactive oxygen species (ROS) [reviewed in ref (4)]. R O S , a so-called death factor for neutrophils, is largely responsible for causing cells to become apoptotic and susceptible to non-inflammatory removal (5). A s the neutrophils in C G D patients lose the capacity to make R O S , they have extended life spans and have been shown to have enhanced production of pro-inflammatory cytokines (1). A s a result, these patients are highly susceptible to infections with a narrow range of pathogens and are plagued by many different and profound inflammatory conditions, including inflammatory bowel disease, periodontal inflammation, granulomatous obstruction of the urinary and gastrointestinal tract and "sterile" inflammation of the lungs and other organs (1). The aforementioned clearly demonstrates that dysregulation of neutrophil apoptosis is detrimental to the host, and in conditions such as C G D , the prolonged survival of neutrophils caused by L L - 3 7 and other survival factors might be unfavorable and even in normal individuals must be managed. 4.2 The PI3K and P K C pathways are important in LL-37-mediated neutrophil survival. The phosphatidylinositol 3-kinases (PI3Ks) are members of a conserved family of intracellular l ipid kinases that phosphorylate phosphatidylinositol and phosphoinositides [reviewed in (6)]. These phosphorylated lipids subsequently contribute to the activation of various intracellular signalling pathways that regulate diverse functions such as cell survival and proliferation, differentiation, trafficking and metabolism. PI3 kinases can be activated by a number of receptors including growth factor receptor tyrosine kinases (RTKs) , G protein-coupled receptors (GPCRs) , cytokine receptors and integrins (7). In addition, the purinergic receptor P 2 X 7 has been demonstrated to activate the PI3K/Akt pathway in astrocytes (8). In Chapter 2, ?2X-j receptor and an unidentified G protein-coupled receptor were shown to be 69 associated with LL-37-induced inhibition of apoptosis (Figure 2.4). However, it is not clear whether this effect was linked to PI3K activation, which was also shown to mediate L L - 3 7 -induced neutrophil survival (Figure 2.5). To test this, the above-mentioned two receptors could be blocked using specific inhibitors and the effect on the subsequent phosphorylation status of PI3K could be examined. This would help to link these isolated events in a coherent paradigm and to gain a complete understanding of the precise mechanism by which L L - 3 7 regulates neutrophil apoptosis. The most characterized downstream effector molecule of P I3K is A k t (also called protein kinase B , P K B ) . Once activated, Ak t can phosphorylate a host of other proteins that affect diverse cellular responses including cell growth, cell cycle entry, and cell survival (6). One of the target proteins o f A k t is the apoptosis-inducing protein B A D . Phosphorylation o f B A D by Ak t prevents B A D from binding to anti-apoptotic Bcl-2 family members Bcl -2 and B c l - x L , thereby promoting cell survival (9). A s an example, G M - C S F has been shown to inhibit neutrophil spontaneous apoptosis through activating PI3K/Akt and subsequent phosphorylation of B A D (10). Since it was recently shown that L L - 3 7 induces phosphorylation o f A k t in human P B M C s and that this event is linked to chemokine regulation in these cells ( Y u and Hancock, manuscript submitted), it is possible that LL-37 exerts its anti-apoptotic effect through the activation of Akt . Thus in future studies the phosphorylation state of A k t and B A D as well as the mitochondrial translocation o f B A D should be examined, and this information w i l l be valuable to gain insights into the mechanism through which L L - 3 7 regulates apoptosis in neutrophils. In addition to PI3K, P K C signalling also appears to be involved in LL-37-mediated neutrophil survival, since rottlerin, an inhibitor for P K C 8 (which also inhibits other P K C isoforms to a lesser extent), suppressed LL-37-induced neutrophil survival (Chapter 2, Figure 70 2.5). P K C 8 has been implicated in regulating cell cycle and apoptosis in many cell types, and one key downstream pathway that P K C 8 can activate is the NFKB signalling (11). A P K C - 8 to NFKB pathway has been shown to be involved in enhanced neutrophil adhesion in endothelial cells and in the induction of LAP (inhibitor of apoptosis protein, which functions as an endogenous caspase inhibitors) (12, 13). P K C signalling has also been implicated in L L - 3 7 -induced angiogenesis by a direct effect on endothelial cells v ia the P L C - y / P K C / N F - K B signalling pathway (14). In neutrophils, inhibition o f P K C - 8 attenuates TNF-a-mediated activation of NFKB, resulting in diminished antiapoptotic signalling (15). These facts demonstrate that the NFKB pathway is an important downstream signalling pathway involved in various effects mediated by certain P K C isoforms including P K C 8 . The NFKB pathway regulates cell apoptotic processes through its effects on the transcription of survival proteins including M c l - 1 , A l and IAP (16, 17). However, I have found that L L - 3 7 failed to induce Mcl-1 and A l gene expression in neutrophils (Figure 4.1), indicating that mechanisms other than transcriptional regulation of anti-apoptotic Bcl-2 proteins are involved in LL-37-mediated neutrophil survival. The gene expression of Mcl-1 and A l in these experiments was examined at 2 hours post-stimulation by L L - 3 7 , which might not be an appropriate time point. Thus, a time course experiment would clarify whether L L - 3 7 regulates the expression of these proteins at the transcriptional level. In addition, although L L - 3 7 has been shown to induce transient activation o f NFKB in human P B M C s ( Y u and Hancock, manuscript submitted), this effect has not been investigated in neutrophils. Thus nuclear translocation o f NFKB subunits needs to be examined in neutrophils treated with L L - 3 7 to draw any further conclusions. 71 Mcl-1 A1 0) c CO £ U o X X O) c (0 u o X * _r_ o O r--co UL co O co + LL CO o o c o o r--co CO O I o co + LL CO o I o Figure 4.1. Mcl-1 and A l gene expression in neutrophils. Neutrophils were exposed to 10 pg/ml LL-37 and/or 30 ng/ml G M - C S F , or vehicle-alone control for 2 hours, and the gene expression of Mcl-1 and A l was examined by qPCR. Fold changes (y-axis) were normalized to G A P D H and are relative to the gene expression in un-stimulated cells (normalized to 1) using the comparative Ct method (see Materials and Methods in Chapter 2 for details). Data represent means ± S E M for duplicates per condition from three different donors. Paired sample Student's r-test analyses were used to compare treated samples with controls. *, P <0.05. In addition to PI3K, I have also examined the role of E R K 1 / 2 and p38 M A P K in the regulation of LL-37-induced neutrophil survival and apoptosis. Although E R K 1 / 2 has been implicated in the anti-apoptotic effect of many survival factors, it appears to be irrelevant in L L -37-induced inhibition of neutrophil apoptosis (Figure 2.5). In contrast to the well-accepted role of E R K 1 / 2 in suppressing neutrophil spontaneous apoptosis, the function o f p38 M A P K seems to be complex. It appears that the involvement of p38 in stimuli-induced survival is stimulus dependent. For instance, G M - C S F stimulation does not activate p38 (18), whereas hypoxia-72 mediated inhibition of apoptosis requires p38 M A P K activity (19), and further, p38 activation promotes apoptosis in LPS-stimulated neutrophils (20). Indeed, it has also been shown that p38 is activated by cellular stress, a so-called death factor, and results in subsequent apoptosis (21). In resting neutrophils, Aoshiba et al demonstrated that p38 was constitutively phosphorylated and activated, and inhibition of p38 by a specific inhibitor and an antisense oligonucleotide delayed apoptosis (18). However, conflicting reports exist, such that the role of p38 in constitutive spontaneous apoptosis is not entirely clear (21). These contradicting data suggest that p38 M A P K activation can generate downstream signals that either promote cell death or survival, depending on the specific stimulus and the interactions between p38 and other signalling pathways (17). Nonetheless, in my experiments, inhibition of the p38 pathway by specific inhibitor SB203580 suppressed spontaneous apoptosis in resting neutrophils but also modestly impaired LL-37-induced neutrophil survival (Figure 4.2). These results support the concept that p38 activation promotes apoptosis in resting cells, and they also indicate that, like hypoxia, LL-37-mediated inhibition of neutrophil apoptosis involves p38 activation. 73 100 CO w 80 o a. 160 o 4 0 0> 20 0 III • 0 pg/ml LL-37 • 1 pg/ml LL-37 c o O o CO in CO o CM m co • CM O O CM >• X I in o oo o> Q CL Figure 4.2. The p38 signalling pathway is involved in LL-37-induced neutrophil survival. Neutrophil apoptosis over 20 h incubation was examined by F A C S analysis for PE-annexinV-positive, 7AAD-negative cells after incubation with 1 pg/ml L L - 3 7 or endotoxin-free water as a vehicle control, added 30 min after 12.5 p M SB203580, 10 u M LY294002, 10 p M PD98059, or a vehicle-alone control. Results represent the percentage of apoptotic cells as mean ± S E M for three replicates per condition from four different donors. Paired sample Student's Mest analyses were used to compare inhibitors-treated samples with samples that were not exposed to inhibitors under the same stimulation conditions (LL-37-treated or vehicle-control treated samples). *, P <0.05. 4.3 LL-37 mediates inhibition of neutrophil apoptosis through effects on both intrinsic and extrinsic apoptotic pathways. A s described in detail in Chapter 1, two pathways, the intrinsic mitochondrial pathway and extrinsic death receptor pathway, are known to initiate the apoptosis cascades in neutrophils. 74 M y results indicate that L L - 3 7 has effects on both pathways. Firstly, in Chapter 2,1 showed that LL-37-treated neutrophils have a significantly higher level of the anti-apoptotic Mcl-1 protein as opposed to unstimulated cells (Chapter 2, Figure 2.2). Mcl-1 is a key regulator o f the mitochondrial pathway, and it is known that the gene expression of Mcl-1 can be induced by certain cytokines including G M - C S F (22). In addition, the cellular level of this protein can also be regulated at the post-translational level since it has a P E N T motif which would target it for proteolysis via the proteasome (17). Interestingly, a recent report demonstrated that G M - C S F , in addition to its modest effect on Mcl-1 transcription, also increases the stability of Mcl-1 protein, rendering it more resistant to proteasome degradation (17). It was not investigated in this thesis whether L L - 3 7 has the similar Mcl-1 stabilization effect, but it is clear that L L - 3 7 does not regulate Mcl-1 expression at the transcriptional level, as demonstrated by Figure 4.1. However, this peptide may conceivably sustain Mcl-1 gene expression by stabilizing transcripts of Mcl-1 through activation of p38 M A P K . In addition to the mitochondrial intrinsic pathway, death receptor apoptotic pathway also appears to be regulated by L L - 3 7 , as I showed in Chapter 2 that higher levels of full length B I D were found in L L - 3 7 treated neutrophils than in unstimulated cells (Figure 2.2). In apoptotic cells, B I D is cleaved by activated caspase 8 via the activation o f death receptors. Desagher et al have demonstrated that when full length B I D is phosphorylated, it becomes insensitive to cleavage by caspase 8, thereby rendering H e L a cells resistant to death-receptor induced apoptosis (23). This group has also identified the candidate kinases that phosphorylate B I D , including both casein kinases ( C K ) I and II. Both kinases appear to be ubiquitously expressed and constitutively active. It is not entirely clear how casein kinases are regulated, but it has been proposed that casein kinases work only in conjunction with other protein kinases to phosphorylate certain substrates (23). For example, P K C is one such kinase which is implicated in PMA-induced B I D 75 phosphorylation via C K in Jurkat cells (24). Interestingly, P K C appears to play a role in L L - 3 7 mediated anti-apoptotic effect as demonstrated in Chapter 2 (Figure 2.5). Thus it is possible that L L - 3 7 activates P K C , which in turn phosphorylates B I D in conjunction with C K s . To test this hypothesis, the phosphorylation state of B I D could be examined under conditions in which P K C is blocked. Naturally, B I D can also be regulated at various levels through other mechanisms such as by controlling the expression or activation state of caspase 8, the up-stream molecule o f B I D . However, it seems that gene expression o f B I D is not affected by L L - 3 7 , as demonstrated by Figure 4.3. 0) o> c o o c o O r» LL _j o £- CO 0 0 Q_ « 1 CO o Figure 4.3. B I D gene expression in neutrophils. Neutrophils were exposed to 10 | ig/ml L L - 3 7 and/or 30 ng/ml G M - C S F , 100 ng/ml L P S or vehicle-alone control for 2 hours, and the gene expression of B I D was examined by qPCR. Fold changes (y-axis) were normalized to G A P D H and are relative to the gene expression in unstimulated cells (normalized to 1) using the comparative Ct method (see Materials and Methods in Chapter 2 for details). Data represent means ± S E M for duplicates per condition from three different donors. Student's /-test analyses were used to compare treated samples with controls. * P<0.05. 76 4.4 L L - 3 7 amplifies beneficial inflammatory signals by enhancing chemokine production induced by IL-lp\ Neutrophils are released from the bone marrow into circulation at a rate o f l x l O 1 2 cells / day and constitute 60 % of blood cells. Monocytes, on the other hand, represent a mere 10 % o f the blood cell population (25). Neutrophils are terminally differentiated cells and perhaps produce low level of cytokines on a per cell basis as compared to macrophages; but early on at the infection foci, they outnumber macrophages by a considerable extent (26). Thus as a whole, neutrophils are one of the major sources of cytokine/chemokine, should infection occur, and as a result, they are increasingly appreciated as an important immunomodulator (26). Neutrophils have been shown to produce a number o f chemokines in response to pro-inflammatory stimuli such as L P S (26). These chemokines recruit other effector cells to the infection foci to eliminate invading microbes, and therefore are an essential component o f the immune response. Among the chemokines tested, I have shown that L L - 3 7 is able to induce Gro-a, M i p - l a , IL-8, and C C L 2 2 release from human neutrophils (Chapter 3, Figure 3.2). Unlike monocytes and airway epithelial cells, neutrophils only release small amounts of these chemokines in response to L L - 3 7 , although they are capable of producing much larger quantities of chemokines in conditions such as co-stimulation with L L - 3 7 and IL-1B (Figure 3.2) and stimulation by L P S (Figure 3.1). These results demonstrate that some of the LL-37-mediated effects are specific to distinct cell types. This view is further supported by the fact that the synergistic effect between L L - 3 7 and cytokines is not a general mechanism that applies to all cell types. For example, in human peripheral derived monocytes, G M - C S F has also been to act in synergy with low concentrations of L L - 3 7 (< 10 ng/ml) in inducing augmented activation of E R K 1 / 2 and p38, an effect that would require at least 50 ng/ml L L - 3 7 used alone (27). However, according to my results, co-stimulation of G M - C S F and L L - 3 7 did not induce considerably higher levels of 77 chemokine release from neutrophils (Figure 4.4). Nor did these two molecules act in synergy in inducing an enhanced inhibition of apoptosis in neutrophils (data not shown), although they are both potent survival factors. 1200 Mip-1a 1000 -f a . 800 -£ a . 600 -n 400 -a . i 200 -0 -on • Q 2 CO o - 1 O LL CO o co CO O CO Q-1400 1200 ^1000 1> 800 a op 600 ~ 400 200 0 IL-8 . a • o £ c ± o - 1 O * CO o * co O L ! c o o * X I c o CL Figure 4.4. Effect of LL-37 and GM-CSF co-stimulation on chemokine release in neutrophils. The Mip-loc and IL-8 production by neutrophils was assessed by E L I S A analysis of culture supernatants following incubation of neutrophils for 20 hours with 10 ug/ml L L - 3 7 , 30 ng/ml G M - C S F with or without L L - 3 7 , 100 ng/ml L P S , or vehicle-alone control. Data represent means ± S E M for three replicates per condition. The result of an experiment from one donor, representative of three, is shown. Paired sample Student's /-test analyses were used to compare treated samples with controls. *, P <0.05. The synergistic effect between L L - 3 7 and I L - i p is particularly relevant as I L - i p is a major proinflammatory stimulus, and by activating the I L - 1 R / T L R signalling pathway, it induces the production of numerous inflammatory mediators. During the course of infection, neutrophils and 78 monocytes rapidly migrate to the site o f infection, where L L - 3 7 accumulates as a result o f neutrophil degranulation and up-regulated expression in epithelial cells. The elevated levels of L L - 3 7 also contribute in part to the induction of IL-1 (3 from human monocytes (28). Interestingly, IL-1 (3 on its own is not a potent inducer of chemokine release from neutrophils (Figure 3.2); however, the presence o f both stimuli significantly enhances neutrophil chemokine responses even with low concentrations of L L - 3 7 (10 jxg/ml). L L - 3 7 expression has been detected at ~5 pg/ml in bronchoalveolar lavage ( B A L ) from healthy infants and is up-regulated by inflammation, being detected in B A L from infants with pulmonary infections and in individuals with cystic fibrosis at up to -30 ng/ml and -15 ng/mh respectively (29, 30). Many effects o f L L -37 observed from in vitro studies cannot be induced by this peptide alone with these abovementioned concentrations (5-30 iig/m\); however, L L - 3 7 or any other inflammatory mediator would not exist in isolation because the innate immune system has numerous components with complex interactions to control the outcome of an infection. Thus studies on the synergistic effects between L L - 3 7 and IL-1 (3 would provide information towards understanding o f immune response under physiological conditions. 4.5 Conclusions M y results presented in this thesis add yet another aspect to our ever expanding knowledge o f the complex modulatory role o f L L - 3 7 in innate immunity. It is intriguing that this small peptide with a simple structure displays incredibly diverse and often times specific effects on distinct cell types. Because L L - 3 7 and other cationic host defense peptides generally enhance host defenses at the infection foci while diminishing harmful inflammatory effects such as T N F -a induction by certain T L R agonists, these peptides show great promise as a therapeutic adjuvant to prevent or treat infections (31). However, at high concentrations L L - 3 7 demonstrates 79 cytotoxicity due to cell membrane damage, which limits its potential for therapeutic use. Understanding the immunomodulatory functions and the underlying mechanisms would provide invaluable information in developing synthetic peptides. The successful synthetic peptide should have high potency o f antimicrobial activity yet low cytotoxicity to host cells, and the ability to boost beneficial immune responses and dampen detrimental effects. Results of this study wi l l assist further understanding o f how L L - 3 7 regulates infectious and inflammatory processes in vitro and ultimately in vivo, and point to new directions for controlling infectious diseases. 80 4.6 Bibliography 1. Lakshman, R., and A . Finn. 2001. Neutrophil disorders and their management. J Clin Pathol 54:7-19. 2. Simon, H . U . 2003. Neutrophil apoptosis pathways and their modifications in inflammation. Immunol Rev 193:101-110. 3. Nathan, C . 2006. Neutrophils and immunity: challenges and opportunities. Nat Rev Immunol 6:173-182. 4. Heyworth, P. G . , A . R. Cross, and J. T. Curnutte. 2003. Chronic granulomatous disease. Curr Opin Immunol 15:578-584. 5. Hofman, P. 2004. Molecular regulation of neutrophil apoptosis and potential targets for therapeutic strategy against the inflammatory process. Curr Drug Targets Inflamm Allergy 3:1-9. 6. Katso, R., K . Okkenhaug, K . Ahmadi, S. White, J. Timms, and M . D . Waterfield. 2001. Cellular function of phosphoinositide 3-kinases: implications for development, homeostasis, and cancer. Annu Rev Cell Dev Biol 17:615-675. 7. Engelman, J. A . , J. Luo, and L . C . Cantley. 2006. The evolution of phosphatidylinositol 3-kinases as regulators o f growth and metabolism. Nat Rev Genet 7:606-619. 8. Jacques-Silva, M . C , R. Rodnight, G . Lenz, Z . Liao, Q. Kong, M . Tran, Y . Kang, F. A . Gonzalez, G . A . Weisman, and J. T. Neary. 2004. P2X7 receptors stimulate A K T phosphorylation in astrocytes. Br J Pharmacol 141:1106-1117. 9. Zha, J., H . Harada, E . Yang, J. Jockel, and S. J. Korsmeyer. 1996. 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Wong, R. Craddock, L . K . Ass i , M . Salmon, and J . M . Lord. 2004. Early events in spontaneous neutrophil apoptosis. Biochem Soc Trans 32:461-464. 26. Cassatella, M . A . 1999. Neutrophil-derived proteins: selling cytokines by the pound. Adv Immunol 73:369-509. 27. Bowdish, D . M . , D . J. Davidson, D . P. Speert, and R. E . Hancock. 2004. The human cationic peptide L L - 3 7 induces activation of the extracellular signal-regulated kinase and p38 kinase pathways in primary human monocytes. J Immunol 172:3758-3765. 28. Elssner, A . , M . Duncan, M . Gavril in, and M . D . Wewers. 2004. A novel P 2 X 7 receptor activator, the human cathelicidin-derived peptide L L 3 7 , induces IL-1 beta processing and release. J Immunol 172:4987-4994. 29. Schaller-Bals, S., A . Schulze, and R. Bals. 2002. Increased levels o f antimicrobial peptides in tracheal aspirates o f newborn infants during infection. Am J Respir Crit Care Med 165:992-995. 30. Chen, C . I., S. Schaller-Bals, K . P. Paul, U . Wahn, and R. Bals. 2004. Beta-defensins and L L - 3 7 in bronchoalveolar lavage fluid o f patients with cystic fibrosis. J Cyst Fibros 3:45-50. 31. Finlay, B . B . , and R. E . Hancock. 2004. Can innate immunity be enhanced to treat microbial infections? Nat Rev Microbiol 2:497-504. 82 

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