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LL-37, a human host defense peptide with immunomodulatory properties Bowdish, Dawn Marie Edith 2005

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L L - 3 7 , A H U M A N H O S T D E F E N C E P E P T I D E W I T H I M M U N O M O D U L A T O R Y P R O P E R T I E S by DAWN MARIE EDITH BOWDISH B.Sc. (Honours Microbiology), University of Guelph, 2000 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (MICROBIOLOGY) THE UNIVERSITY OF BRITISH COLUMBIA JULY 2005 © D a w n M . E . B o w d i s h , 2005 Abstract Human cationic antimicrobial protein-18 (hCAP-18) is the sole human cathelicidin. It is found at high concentrations in the specific granules o f neutrophils, is modestly expressed by epithelial and other cells and can be induced during the course of infection and/or inflammation. The mature, extracellular form o f h C A P - 1 8 is termed L L - 3 7 , which is a positively-charged, 37-amino acid peptide. Neutrophil-derived host defence peptides were initially discovered as components o f the non-oxidative killing mechanisms o f neutrophils; however, it is unclear whether L L - 3 7 kills bacteria directly at mucosal surfaces where it is found at lower concentrations. I demonstrated that although L L - 3 7 is antimicrobial in vitro under low salt conditions, it has little or no antimicrobial activity in media containing physiologically relevant cation concentrations. Thus I hypothesised that direct antimicrobial activity was probably not its primary function in vivo at mucosal surfaces. The immunomodulatory properties o f L L - 3 7 were investigated in tissue culture media which contains physiological concentrations of cations. When monocytes were treated with L L - 3 7 , lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines was blocked but T N F - a - induced cytokine production was unaffected and IL- ip- induced cytokine production was enhanced. Consistent with this observation, L L - 3 7 was able to block LPS- induced translocation of the p65 subunit o f the pro-inflammatory transcription factor, N F - K B . Other early signalling events mediated by L L - 3 7 included activation of two mitogen-activated protein kinases, p38 and extracellular-regulated kinase. The activation of these kinases was required for IL-8 transcription and release as well as transcription of the chemokines M I P - l a , M I P - i p , and M C P - 1 . The activation o f these kinases and subsequent production and release of IL-8 could be synergistically increased by the presence of granulocyte macrophage-colony stimulating factor, but not related cytokines, indicating that the composition of the inflammatory milieu may affect monocyte responses to L L - 3 7 . The data presented here demonstrate that L L - 3 7 is a multi-functional immunomodulator with both pro- and anti-inflammatory properties which are maintained at physiological cation concentrations. The observation that L L - 3 7 induced chemokines production suggests that it may recruit leukocytes to sites of infection or inflammation thus providing a possible explanation for the observed antimicrobial properties o f this peptide in vivo. ii T a b l e of Contents LL-37, A H U M A N HOST D E F E N C E PEPTIDE W I T H I M M U N O M O D U L A T O R Y PROPERTIES I A B S T R A C T I T A B L E OF CONTENTS I l l LIST OF T A B L E S V LIST OF FIGURES VI LIST OF A B B R E V I A T I O N S VIII A C K N O W L E D G E M E N T S X S T A T E M E N T OF A U T H O R S H I P XI 1.0 INTRODUCTION 1 1.1 HOST DEFENCE PEPTIDES OF MAMMALS 2 1.2 ANTIMICROBIAL OR HOST DEFENCE PEPTIDES? 5 1.3 EVIDENCE FOR ANTIMICROBIAL ACTIVITY IN VITRO 5 1.4 ANTIMICROBIAL ACTIVITY IN VIVO 7 1.5 IMMUNOMODULATORY PROPERTIES OF CATHELICIDINS 10 1.6 MODEL FOR THE PHYSIOLOGICAL ROLE OF L L - 3 7 13 1.7 HYPOTHESIS AND EXPERIMENTAL GOALS 15 1.8 BIBLIOGRAPHY 16 2.0 I M M U N O M O D U L A T O R Y ACTIVITIES OF S M A L L HOST D E F E N C E PEPTIDES 24 2.1 INTRODUCTION 25 2.2 MATERIALS & METHODS 26 2.3 RESULTS 28 2.4 DISCUSSION 33 2.5 BIBLIOGRAPHY 35 3.0 T H E H U M A N CATIONIC PEPTIDE LL-37 INDUCES A C T I V A T I O N OF T H E E X T R A C E L L U L A R SIGNAL R E G U L A T E D K I N A S E A N D P38 K I N A S E P A T H W A Y S IN P R I M A R Y H U M A N M O N O C Y T E S 39 3.1 INTRODUCTION 40 3.2 MATERIALS & METHODS 41 3.3 RESULTS 45 3.4 DISCUSSION 53 3.5 BIBLIOGRAPHY 57 4.0 LL-37 IS A POTENT ANTI-ENDOTOXIC A G E N T W I T H M U L T I P L E M E C H A N I S M S OF A C T I O N 61 4.1 INTRODUCTION 62 4.2 MATERIALS & METHODS 63 4.3 RESULTS 66 4.4 DISCUSSION 71 4.5 BIBLIOGRAPHY 74 5.0 LL-37 INDUCES RAPID & E A R L Y C H E M O K I N E P R O D U C T I O N V I A A C T I V A T I O N OF NF-K B A N D T H E M A P K 78 5.1 INTRODUCTION 79 5.2 MATERIALS & METHODS 80 5.3 RESULTS 83 5.4 DISCUSSION 89 5.5 BIBLIOGRAPHY 91 iii 6.0 DISCUSSION - THE PRIMARY ROLE OF LL-37 IN VIVO MAY BE IMMUNOMODULATORY 95 6.1 E A R L Y EVIDENCE THAT HOST DEFENCE PEPTIDES M I G H T H A V E IMMUNOMODULATORY FUNCTIONS 96 6.2 T H E ANTIBACTERIAL A N D IMMUNOMODULATORY PROPERTIES OF L L - 3 7 ARE PHYSIOLOGICALLY DISTINCT 99 6.3 INTERACTIONS BETWEEN L L - 3 7 A N D EUKARYOTIC C E L L S 100 6.4 L L - 3 7 INTERACTIONS WITH THE EFFECTOR C E L L S OF THE I M M U N E RESPONSE A R E SPECIFIC B U T N O T NECESSARILY RECEPTOR MEDIATED 101 6.5 CHEMOTAXIS , L E U K O C Y T E INFILTRATION A N D L L - 3 7 105 6.6 SYNERGISTIC INTERACTIONS BETWEEN L L - 3 7 A N D COMPONENTS OF THE INFLAMMATORY M I L E U 107 6.7 FUTURE DIRECTIONS - T H E R O L E OF L L - 3 7 IN H E A L T H & DISEASE A N D ITS POTENTIAL AS A N O V E L THERAPEUTIC A G E N T 109 6.8 B IBLIOGRAPHY 110 iv List of Tables 1.0 INTRODUCTION 1 T A B L E 1.1. T H E ANTIMICROBIAL PROPERTIES OF HOST DEFENCE PEPTIDES ARE REDUCED OR ELIMINATED IN THE PRESENCE OF SALTS 6 T A B L E 1.2. CONCENTRATIONS OF HOST DEFENCE PEPTIDES FOUND IN THE L U N G 7 2.0 I M M U N O M O D U L A T O R Y ACTIVITIES O F S M A L L HOST D E F E N C E PEPTIDES 24 T A B L E 2.1. SEQUENCES OF THE PEPTIDES USED IN THESE STUDIES 26 T A B L E 2.2. COMPARISON OF THE ACTIVITIES OF INDOLICIDIN, B A C 2 A A N D LL-37 (UG/ML) 33 3.0 T H E H U M A N CATIONIC PEPTIDE LL-37 INDUCES A C T I V A T I O N O F T H E E X T R A C E L L U L A R SIGNAL R E G U L A T E D KINASE AND P38 KINASE P A T H W A Y S IN PRIMARY H U M A N M O N O C Y T E S 39 T A B L E 3.1. PRIMER SEQUENCES OF CYTOKINE GENES 45 4.0 LL-37 IS A P O T E N T ANTI-ENDOTOXIC A G E N T W I T H M U L T I P L E M E C H A N I S M S O F A C T I O N 61 5.0 LL-37 INDUCES RAPID & E A R L Y C H E M O K I N E P R O D U C T I O N VIA A C T I V A T I O N O F NF-K B & T H E M A P K 78 T A B L E 5.1. PRIMER SEQUENCES USED IN THIS STUDY 82 6.0 DISCUSSION - T H E P R I M A R Y R O L E O F LL-37 IN VIVO M A Y B E I M M U N O M O D U L A T O R Y 95 T A B L E 6.1. CONCENTRATIONS OF H C A P - 1 8/LL-37 IN HEALTH A N D DISEASE 98 v List of Figures 1.0 INTRODUCTION 1 FIGURE 1.1. THE STRUCTURE OF THE CATHELICIDINS 4 FIGURE 1.2. INFLUENCE OF TISSUE CULTURE MEDIUM ON THE ANTIMICROBIAL ACTIVITY OF HOST DEFENCE PEPTIDES U FIGURE 1.3. EXPERIMENTAL MODEL 15 2.0 I M M U N O M O D U L A T O R Y ACTIVITIES O F S M A L L HOST D E F E N C E PEPTIDES 24 FIGURE 2.1. CYTOTOXICITY OF LL-37, BAC2A AND INDOLICIDIN 29 FIGURE 2.2. ANTI-ENDOTOXIN PROPERTIES OF INDOLICIDIN, BAC2A AND LL-37 30 FIGURE 2.3. ADDITION OF INDOLICIDIN UP TO 60 MINUTES AFTER THE ADDITION OF LPS RESULTS IN INHIBITION OF LPS INDUCED TNF-A FROM PMA TREATED THP-1 CELLS 30 FIGURE 2.4. THE ADDITION OF INDOLICIDIN AND LL-37 IN COMBINATION PRODUCES A GREATER THAN ADDITIVE INHIBITION OF LPS-INDUCED TNF-OC 31 FIGURE 2.5. BAC2A IS A CHEMOATTRACTANT FOR UNDIFFERENTIATED THP-1 CELLS 32 FIGURE 2.6. INDOLICIDIN INDUCES IL-8 PRODUCTION IN A DOSE DEPENDENT MANNER IN THE HUMAN BRONCHIAL EPITHELIAL CELL LINE, 16HBE40- 32 3.0 T H E H U M A N CATIONIC PEPTIDE LL-37 INDUCES A C T I V A T I O N O F T H E E X T R A C E L L U L A R SIGNAL R E G U L A T E D KINASE AND P38 KINASE P A T H W A Y S IN P R I M A R Y H U M A N M O N O C Y T E S 39 FIGURE 3.1. EXPOSURE TO LL-37 INDUCES PHOSPHORYLATION OF ERK1/2 AND P38 46 FIGURE 3.2. LL-37 is UNABLE TO INDUCE MAPK ACTIVATION IN HUMAN MONOCYTES UNDER SERUM FREE CONDITIONS 47 FIGURE 3.3. ACTIVATION OF ERK.1/2 AND P38 PHOSPHORYLATION BY LL-37 is INDUCED IN CELLS OF THE INNATE IMMUNE SYSTEM 48 FIGURE 3.4. LL-37 INDUCED PHOSPHORYLATION OF ERK1/2 AND P38 KINASES IS NOT MEDIATED BY G-PROTEIN COUPLED RECEPTORS 49 FIGURE 3.5. ERK1/2 ACTIVATION IS AMPLIFIED AND OCCURS AT LOWER CONCENTRATIONS OF LL-37 WHEN IN THE PRESENCE OF GM-CSF 50 FIGURE 3.6. LL-37 INDUCES IL-8 PRODUCTION IN HUMAN BLOOD DERIVED MONOCYTES IN THE ABSENCE OF CYTOTOXICITY 51 FIGURE 3.7. LL-37 INDUCES IL-8 SECRETION AND CHEMOKINE TRANSCRIPTION IN A P38 AND ERK1/2 KINASE DEPENDENT MANNER 53 4.0 LL-37 IS A P O T E N T ANTI-ENDOTOXIC A G E N T W I T H M U L T I P L E M E C H A N I S M S OF A C T I O N 61 FIGURE 4.1. LL-37 REDUCES LPS INDUCED CYTOKINE PRODUCTION IN PERIPHERAL BLOOD DERIVED MONOCYTES AND A MONOCYTE-LIKE CELL LINE 66 FIGURE 4.2. LL-37 CAN BE ADDED AFTER LPS AND REDUCE PRO-INFLAMMATORY CYTOKINE PRODUCTION 68 FIGURE 4.3. PRE-TREATMENT WITH LL-37 REDUCES PRO-INFLAMMATORY CYTOKINE PRODUCTION 69 FIGURE 4.4. LL-37 DOES NOT REDUCE PRO-INFLAMMATORY CYTOKINE PRODUCTION INDUCED BY IL-1 B OR T N F - A 70 FIGURE 4.5. LL-37 BLOCKS LPS INDUCED TRANSLOCATION OF P65 BUT INDUCES TRANSLOCATION OF P50 71 5.0 LL-37 INDUCES RAPID & E A R L Y C H E M O K I N E P R O D U C T I O N VIA A C T I V A T I O N O F NF-KB & T H E M A P K 78 FIGURE 5.1. LL-37 INDUCTION OF CHEMOKINE AND CYTOKINE TRANSCRIPTION IN PRIMARY MONOCYTES 84 FIGURE 5.2. INCREASES IN CHEMOKINE TRANSCRIPTION AS A RESULT OF DE NOVO TRANSCRIPTIONAL EVENTS 85 FIGURE 5.3. EFFECTS OF INHIBITORS OF THE P38 AND ERK1/2 KINASES ON LL-37 INDUCED TRANSCRIPTION 86 FIGURE 5.4. EFFECT OF CYCLOHEXIMIDE TREATMENT ON IL-8 RELEASE FROM MONOCYTES IN RESPONSE TO LL-37 STIMULATION 87 FIGURE 5.5. REQUIREMENT OF BOTH P38 AND ERK1/2 IN THE EARLY RELEASE OF IL-8 IN RESPONSE TO LL-37. ...88 FIGURE 5.6. LL-37 INDUCED TRANSLOCATION O F NF - K B SUBUNITS 88 FIGURE 5.7. Two MODELS OF SIGNALLING PATHWAY ACTIVATION IN LL-37 TREATED MONOCYTES 90 vi 6.0 DISCUSSION - T H E P R I M A R Y R O L E OF LL-37 IN VIVO M A Y B E I M M U N O M O D U L A T O R Y 95 FIGURE 6.1. THE EFFECT OF HUMAN SERUM ON IL -8 PRODUCTION FROM T H P - 1 CELLS 103 FIGURE 6.2. THREE MODELS ILLUSTRATING THE ROLE OF L L - 3 7 IN LEUKOCYTE INFILTRATION 106 FIGURE 6.3. THE INFLUENCE OF THE CYTOKINE MILIEU ON L L - 3 7 RESPONSES 109 vii List of Abbreviations A S L airway surface liquid A T C C American Type Culture Collection B A L bronchial alveolar lavage B P I bactericidal / permeability increasing protein C C L chemokine ( C - C motif) ligand C F cystic fibrosis D C dendritic cell E C L enhanced chemiluminescence 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 C S foetal calf serum F P R L - 1 formyl peptide like receptor-1 G - C S F granulocyte colony stimulating factor G M - C S F granulocyte macrophage- colony stimulating factor H B D human beta defensin H B E human bronchial epithelial cells H D L high-density lipoproteins H N P human neutrophil peptide hr hour H S human serum I L - interleukin L B P lipopolysaccharide binding protein L D H lactate dehydrogenase L D L low-density lipoproteins L A R C liver and activation regulated chemokine L P S lipopolysaccharide L T A lipoteichoic acid M A P K mitogen activated protein kinase M C P - 1 macrophage chemotattractant protein 1 M C P - 3 monocyte chemotactic protein 3 M - C S F macrophage colony stimulating factor M H C major histocompatibility complex class min minute MIP-lcx macrophage inflammatory protein alpha M I P - l p macrophage inflammatory protein beta P B D M peripheral blood derived monocytes P B M C peripheral blood mononuclear cells P B S phosphate buffered saline P M S F phenyl methyl sulfonyl fluoride Ptx pertussis toxin q R T - P C R quantitative reverse transcriptase polymerase chain reaction R T - P C R reverse transcriptase polymerase chain reaction S D S sodium dodecyl sulphate S D S - P A G E S D S polyacrylamide gel electrophoresis T B S tris buffered saline T B S T tris buffered saline + 0.1% Tween 20 T L R toll-like receptor TNF-oc tumor necrosis factor alpha Acknowledgements I would like to start by thanking my supervisor Dr. B o b Hancock for his support and guidance. I have benefited enormously under his tutelage and I truly appreciate all the opportunities he has provided me. I know that I have grown as a scientist because of Bob's mentoring style: he gives his students freedom to make mistakes (and discoveries) but reels us in when we might be going astray. I would like to thank my committee members, Dr . Brett Finlay, Dr . Vincent Duronio, Dr . Michael G o l d and Dr . Francois Jean for a very productive and helpful relationship over the years. I would also like to acknowledge Dr. Dav id Speert who provided me with advice and support and was like an honorary committee member. The Hancock lab has been a very inspiring place to do research and I have been very fortunate to work with some extremely hardworking, committed, bright and enthusiastic people. There are many more people deserving of thanks than I can list here but I would like to acknowledge my co-workers and fellow graduate students for the wonderful impact they have had on me. T w o people deserve special mention though, Elaine L a u and Donald Davidson. I would like to thank you, Elaine, for all those hours and hours o f discussion on our work and for listening to my half-baked ideas ("Yes Dawn, but do you actually have any proof o f that?"). Those conversations were extremely productive and inspired me to work towards getting that proof. I would also like to express my deep and sincere gratitude to you, Donald, for mentoring me over the course of my degree. I credit so much of my academic success to your friendship, encouragement, gentle and not so gentle pushes to get things finished, to your amazing insight and careful, thorough and critical comments on my work. There are so many people who were behind the scenes during the course o f my degree and who I would like to thank for their emotional support. Those people include my grandparents Jay and V i o l a Bowdish, my parents Wayne Bowdish and Donna and Rick Bryson, my brother Ryan Bowdish and sister Jennifer deHaan, and who could forget little Josh!, my dear friend Cor in Forrester, and the Vanderwal family for lending me their son. f was always encouraged in my academic endeavours by my grandmother, Barbara Pargeter, and I wil l always be inspired by her memory. There is one person who stands out most o f all. I would like to express my most heartfelt appreciation for my husband, Aaron Vanderwal. Thank you for all the late night pickups at the lab, for understanding that I may never be on time for dinner, for your careful editing of figures, for fine-tuning my presentations, and for your most impressive feat yet - formatting this thesis. Thank you for all your encouraging words, for holding up the home front when I needed to focus on work, and for making many personal sacrifices to support my school and career choices. Thank you for the many conversations which contributed to my successes over the course of my degree and its timely completion. I can't even imagine how different my experience with grad school would have been without you. Thank you. x Statement of A u t h o r s h i p Large sections of the introduction have been published in: Bowdish, D . M . E . , D . J . Davidson, and R. E . W . Hancock. 2005. A re-evaluation of the role o f host defence peptides in mammalian immunity. Curr Protein Pept Sci 6:35. Bowdish, D . M . E . , D . J . Davidson, Y . E . Lau , K . Lee, M . G . Scott, and R. E . W . Hancock. 2005. Impact o f L L - 3 7 on anti-infective immunity. J Leukoc Biol 77:451-9. and have been submitted for publication in: Bowdish, D . M . E . , D . J . Davidson, and R . E . W . Hancock. 2005. The role o f cathelicidins and defensins in host defence, in "Antimicrobial Peptides and H u m a n Disease" ed. by Dr. Wi l l i am Shafer. Chapter 2 was originally published as: Bowdish, D . M . E . , D . J . Davidson, M . G . Scott, and R. E . W . Hancock. 2005. Immunomodulatory activities o f small host defense peptides. Antimicrob Agents Chemother 49:1727-32. Chapter 3 was originally published as; Bowdish, D . M . E . , Davidson, D . J . , Speert, D .P . , Hancock, R . E . W . 2004. The Human Cationic Peptide L L - 3 7 Induces Activation ofthe Extracellular Signal-Regulated Kinase and p38 Kinase Pathways in Primary Human Monocytes. Journal of Immunology 772:3758-65 Sections of chapter 4 wi l l be published as; D . M . E . Bowdish, and R . E . W . Hancock. 2005 Antiendotoxin properties o f cationic host defence peptides and proteins. Journal of Endotoxin Research. In press. Chapter 5 is being prepared for publication as: Bowdish, D . M . E . , Davidson, D . J . , and Hancock, R . E . W . 2005. L L - 3 7 induces rapid & early chemokine production via activation o f N F - K B and the M A P K . The majority of Chapter 6 has not been published, however, Figure 6.1 is supplementary data for: L a u , Y . E . , Bowdish, D . M . E . , Hancock, R . E . W . , Davidson, D J . 2005. Apoptosis o f airway epithelial cells: human serum sensitive induction by the cathelicidin L L - 3 7 . Submitted. In all instances where previously published materials, or materials submitted for publication, are reproduced in this thesis, they represent the original research and writing of the author. A footnote at the beginning o f each chapter clearly indicates the contributions o f each author. Prof. R . E . W . Hancock has co-authored all of the publications listed here and his reading of the thesis will serve to verify this statement of authorship. xi 1.0 Introduction 1.0 INTRODUCTION 1 1.1 HOST DEFENCE PEPTIDES OF M A M M A L S 2 1.2 ANTIMICROBIAL OR HOST DEFENCE PEPTIDES? 5 1.3 EVIDENCE FOR ANTIMICROBIAL ACTIVITY IN VITRO 5 1.4 ANTIMICROBIAL ACTIVITY IN VIVO 7 1.5 IMMUNOMODULATORY PROPERTIES OF CATHELICIDINS 10 1.6 M O D E L FOR THE PHYSIOLOGICAL ROLE OF L L - 3 7 13 1.7 HYPOTHESIS A N D EXPERIMENTAL GOALS 15 1.8 BIBLIOGRAPHY 16 * Components o f this chapter have been published in Bowdish, D . M . E . , D . J . Davidson, Y . E . Lau , K . Lee, M . G . Scott, and R . E . W . Hancock. 2005. J Leukoc Biol. Apr;77(4):451-9., Bowdish, D . M . E . , D . J . Davidson, and R . E . W . Hancock. 2005. A re-evaluation of the role o f host defence peptides in mammalian immunity. Curr Protein Pept Sci 6:35. and are in review for publication in "Antimicrobial Peptides and Human Disease" ed. by Dr . Wi l l i am Shafer. 1 1.1 Host Defence Peptides of M a m m a l s Small cationic peptides with antimicrobial activity have been isolated from virtually every class o f l iving organsims (reviewed in (1)). Early on, these peptides were recognised to be a major component of the immune response in invertebrates (2, 3). In mammals, they were shown to be a major constituent o f neutrophils and macrophages, consisting of as much as 50% and 1.5% o f the total protein content o f these cells respectively (4, 5). Consequently these peptides were demonstrated to be an important component of the non-oxidative killing mechanisms of mammalian leukocytes (6). It has since been discovered that these peptides are expressed by a variety of different cell types and are found at low concentrations throughout the body. The classical description of cationic antimicrobial peptides includes molecules that are between 12 and 50 amino acids long, although there is a continuum of sizes o f such molecules up to large proteins, with 2 or more positively charged residues provided by arginine, lysine or, in acidic environments, histidine, and a large proportion (generally >50%) of hydrophobic residues (reviewed in (7-9)). The secondary structures o f these molecules follow four themes, including i) a-helical, ii) |3-stranded due to the presence of two or more disulphide bonds, iii) P-hairpin or loop due to the presence o f a single disulphide bond and/or cyclization of the peptide chain, and iv) extended. M a n y o f these peptides are in fact unstructured in free solution, and fold into their final configuration upon partitioning into biological membranes. In mammals the best-characterized host defence peptides are the defensins and the cathelicidins. Defensins The defensins are between 29-30 amino acids long (with a molecular mass of approximately 3.5 kDa) and contain three conserved disulphide bridges and are thus p-stranded (10). They are further subdivided to include the a- and P- defensins, a distinction based on the organization o f the 3 characteristic cysteine disulphide bonds. These peptides are rich in cysteine, arginine, and aromatic residues (11). The a-defensins were initially isolated from neutrophils and are thus called human neutrophil peptides ( H N P ) - l to -3 (12). Because of the high sequence similarity and difficulties in purifying the individual peptides, as well as the high degree of functional similarity, H N P 1-3 are often studied as a group, although certain studies have demonstrated that there are differences in their antimicrobial (13) and immunomodulatory activities (14). H N P - 4 was identified as an H N P due to its structural homology to H N P 1-3 (15). T w o other a-defensins, H D 5 and H D 6 , are found solely in the intestinal tract. H D 5 and H D 6 were found to be expressed at the transcriptional level solely in the small intestine and in situ 2 hybridization demonstrated that this expression occurs in the Paneth cells (16, 17). Southern blot analysis using a nucleotide probe for the conserved signal sequence o f the defensins indicated that a number of genes with high homology to H N P s exist within the human genome (16, 17). The (5-defensins are expressed in a variety of tissue types including epithelial cells from the trachea and lung, in the salivary and mammary glands, in the plasma and skin [(18-20), reviewed in (21)]. The expression of certain p-defensins is inducible upon stimulation with bacterial components or pro-inflammatory cytokines and thus these peptides are presumed to be an important component o f host defences against infection or inflammation. The best characterised members of the (3-defensin family are H B D 1 - 3 , however the antimicrobial properties o f H B D - 4 have been recently published (22) and over 20 potential p-defensin homologs have been identified in the human genome based on sequence similarity to H B D - 1 - 4 (23). Cathelicidins The cathelicidins are an evolutionarily conserved family of peptides which have been found in cows, sheep, guinea pigs, rabbits, mice, goats, horses and primates [reviewed in (24)]. They are characterised by having an evolutionarily conserved N-terminal domain with greater than 70% sequence homology to a protein of uncertain function called cathelin. Consequently the evolutionarily conserved component of these peptides is called the cathelin domain. These peptides have a signal sequence that is believed to target their delivery to the secondary granules of neutrophils. Despite the conserved nature of the cathelin domain, its function remains unclear, although it has been proposed to block the antimicrobial activity o f the cleaved product, presumably as a mechanism which allows storage o f the peptide in its inactive form (25), and there is some evidence that it has anti-protease activity (25). The C terminal domain, which has very little homology across species, is released by cleavage by proteases, and can consist o f a number of different structural classes including a-helical, extended or cysteine bridged [reviewed in (26)]. This mature form of the peptide has both antimicrobial and immunomodulatory properties. The structure of the cathelicidins is illustrated in Figure 1.1. Cathelicidins are synthesised at the myelocyte and metamyelocyte stage o f neutrophil maturation (27-29). Interesting differences in expression occur between species. For example, there have been at least eleven cathelicidin genes identified in cows, eight in sheep and many more in pigs (30-33). In contrast humans and mice appear to have only one cathelicidin. In artiodactyls (even toed hoofed mammals) expression of the cathelicidins appears to be restricted to cells o f myeloid or 3 lymphoid origin whereas in mice and humans cathelicidins are expressed by a variety of cell types including epithelial cells (34). Mature Peptide I Conserved Pre-Pro Region 1 Antimicrobial & Immunomodulatory 29-30 Amino Acids 98-114 Amino Acids 12-100 Amino Acids PRE PRO Signal Peptide [ PEPTIDE t Cleavage Site Figure 1.1. The Structure of the Cathelicidins. The cathelicidins shared a conserved pre-proregion with homology to the cathelin domain. The N-terminal domain contains a signal sequence while the C terminal contains both the antimicrobial and immunomodulatory properties. The sole human cathelicidin hCAP-18 /LL-37 is found at high concentrations (-630 pg per 109 cells) in its unprocessed form in the granules of neutrophils (35, 36). The hCAP-18 precursor protein is also found at more modest concentrations in lymphocytes, macrophages and a range of epithelial cells (37, 38). Upon encountering invading microorganisms, neutrophils release the contents of azurophilic and specific granules into the resultant phagolysosome (by phagosome-lysosome fusion), or to the cell exterior. Consequently LL-37 is found at sites of neutrophil degranulation. It is also produced by epithelial cells and is found in a number of tissues and bodily fluids including gastric juices, saliva, semen, sweat, plasma, airway surface liquid and breast milk (38-41). Generally, epithelial cells produce the hCAP-18 form. Although hCAP-18 is cleaved by the neutrophil protease, protease 3, when released from neutrophils, it is not entirely clear how or when hCAP-18 is cleaved when it is produced by epithelial cells. There are a variety of processed forms of hCAP-18 that result from as-yet uncharacterized cleavage processes. For example, a 6 kDa form is found in gastric juice (41), hCAP-18 from semen is cleaved to a 38 amino acid antimicrobial peptide ALL-38 in the vagina, (42), while numerous cleavage products are found in the sweat and saliva (39, 43). There appears to be some overlapping, complementary and possibly even enhanced antimicrobial activity of these isoforms (44) . To date there is no information about the immunomodulatory properties of naturally occurring variants of LL-37 although it has been demonstrated that synthetic variants of LL-37 which are between 12-31 amino acids long have varying capacities to induce IL-8 production (45) . 4 1.2 Antimicrobial or Host Defence Peptides? Small cationic peptides with antimicrobial activity are expressed in all classes of life and in lower vertebrates they appear to constitute the entire immune system. In mammals small cationic peptides have been clearly demonstrated to be a component o f the non-oxidative killing mechanisms of leukocytes (12) and thus they have traditionally been called "cationic antimicrobial peptides". However, it has since been discovered that these peptides are expressed by a variety of different cell types and are found at low concentrations throughout the body. It is not entirely clear whether- these peptides have significant antimicrobial activity at lower concentrations and this is compounded by the fact that the optimal antimicrobial activity o f these peptides occurs under low salt conditions which, in general, are not physiologically relevant in mammals. It has been proposed that the term "antimicrobial peptide" is misleading in some cases, relating more to a bias for in vitro antimicrobial testing at the point o f discovery rather than their likely in vivo function. Peptides initially isolated as and termed "antimicrobial peptides" have been shown to have more significant alternative functions in vivo (e.g. hepcidin is primarily involved in iron absorption but is also antimicrobial (46)), while conversely a variety of other molecules with previously-established functions (e.g. anti-proteases like serum leukoprotease inhibitor (47) and elafin (48), and certain chemokines (49)) have been shown to have antimicrobial activity in vitro under low salt conditions. In contrast, the immunomodulatory properties o f these peptides are generally studied in standard tissue culture media (50-54) which contains physiological concentrations of ions and it has been proposed that the term "host defence peptide" might be a more accurate label (55). Thus whether these peptides have meaningful microbicidal or immunoregulatory activities in vivo must be examined by considering two fundamental issues; i) the environment in which these activities are assessed in vitro compared to in vivo conditions, and ii) the concentrations at which such peptides are found in vivo. 1.3 Evidence for Antimicrobial Activity In Vitro The antimicrobial activity o f all host defence peptides is highest in media of low ionic strength, and the activity o f most peptides is inhibited by physiological concentrations of ions such as N a + , M g 2 + and C a 2 + . The antimicrobial properties o f the |3 defensins, for example, demonstrate profound salt sensitivity and in some cases their antimicrobial activity is completely lost at concentrations of 100 m M NaCI (22, 56, 57). Most biological fluids, including sputum (58), airway surface liquid (59) and serum/plasma (60), contain M g 2 + and C a 2 + at free concentrations between 1 and 2 m M and the presence o f these ions is generally more detrimental 5 to antimicrobial activity than N a + alone. The a-defensins are susceptible to concentrations of C a 2 + and M g 2 + as low as 0.5 m M (13). Table 1.1 summarises the effects o f physiological concentrations o f sodium ions on the antimicrobial activity o f human host defence peptides. Table 1.1. The antimicrobial properties of host defence peptides are reduced or eliminated in the presence of salts. Peptide Class o f Bacteria Inhibitory concentration 1 in low salt (Ug/ml) Inhibitory concentration in presence o f N a + 2 (ug/ml) References H N P 1-3 G r a m +ve 7 ->100 * (61-63) G r a m - v e 2.0 - 40 >250 (64) H B D - 1 G r a m +ve 1 .0-10 > 1 0 - N D 3 (65, 66) G r a m - v e >50 N D 4 (66) H B D - 2 G r a m +ve 0.01-0.1 0.5 - N D (65, 66) Gram - v e 10->100 N D (66, 67) H B D - 3 G r a m +ve 5 .0 -25 >50 (66, 68-70) G r a m - v e 10 - >25 1 0 - > 2 5 4 , N D i (68-70) H B D - 4 Gram +ve 4 .0 - 100 * (22) G r a m - v e 4.5 ->100 > 7 2 (22) L L - 3 7 G r a m +ve 15 - 30 125 (38) G r a m - v e 1.5 - 13 2.2 - >79 (64) * Experiment not performed 1 Concentration required to induce killing of at least two log orders in low salt (< 10 m M NaCI) 2 100 m M NaCI unless otherwise stated. 3 N D = N o detectable decrease in antibacterial activity at less than 200 m M NaCI 4 50 m M NaCI 5 Antimicrobial activity abrogated in 150 m M NaCI In some cases, for example in the granules o f neutrophils, the concentration of host defence peptides is estimated to be as great as 10 mg/ml and there is no doubt that upon ingestion of bacteria these concentrations are sufficient to cause direct antimicrobial activity despite the presence of divalent cations or other inhibitory substances (71). However it is questionable whether these concentrations are reached at mucosal surfaces. For example, in patients suffering from inflammatory lung disease or infection the concentration of FfNPs in the bronchoalveolar lavage has been estimated to be 0.7 - 1.2 or 10 ug/ml respectively (72, 73). This contrasts with the concentration required for antimicrobial activity (> 10 ug/ml) or antiviral activity ( 8 - 5 0 ug/ml) under optimised in vitro conditions (72, 74). Concentrations o f host defence peptides found in the lung during health and disease are summarised in Table 1.2. 6 Table 1.2. Concentrations of host defence peptides found in the lung. 1 Peptide Cellular Sources Concentration i ug/ml) Healthy ( B A L ) C F ( B A L ) Infection ( B A L ) Inflammatory L u n g Disease ( B A L ) References H B D - 1 Neutrophils, epithelia 0 -0 .1 0 - 0.002 0 - 0.00007 0 - 0 . 2 (65, 75-77) H B D - 2 Neutrophils, epithelia 0 - 0.0004 0.0001 -0.01 0.0002 0.01-0.1 (78) L L - 3 7 Neutrophils, epithelia, submucosal glands 2.5 - 20* 0 - 16 2.5 - 30* - (79, 80) HNP-1-3 Neutrophils 0.2 300 ->1600J 0.2- 1.2 ~ 10 (72, 73) 1 A l l values were determined for adult volunteers, except those indicated by an asterisk (*) which were determined from infants. 0 implies not detected in a particular assay or patient; "-" indicates no information available, t These include concentrations found in sputum. Interestingly the antimicrobial activity o f host defence peptides may also be inhibited by components o f serum. For example, H N P - 1 has antiviral activity towards enveloped viruses, but this activity is abrogated by the presence o f serum or albumin (81). It is believed, but not conclusively shown that the high concentrations of a-defensins in neutrophils would overcome any localized serum effects (81). The antibacterial activity o f L L - 3 7 is abrogated by the presence of apolipoprotein A - l (82). Thus it is unlikely that under conditions of high ionic strength and in which serum proteins are present that the primary role o f host defence peptides is direct antimicrobial activity. 1.4 Antimicrobial Activity In Vivo There has been some debate about whether host defence peptides might be directly antimicrobial in vivo. Certainly, neutrophil granules and the crypts o f the lumen contain sufficiently high concentrations of peptides to ensure substantial antimicrobial activity. However, it is less clear whether antimicrobial activity occurs at lower concentrations such as those found at mucosal surfaces, and it is worth noting that such sites are often heavily colonized by a rich and diverse collection o f commensal bacteria. The evidence for antimicrobial activity in certain body sites is also inconclusive. O n one hand, certain bodily fluids such as sinus fluid (77) and gastric fluids (41) can directly kil l certain microrganisms, and this antimicrobial activity is ablated or reduced by removal o f proteins or immunodepletion with host defence peptide specific antibodies. However, in certain animal models in which peptides and bacteria are instilled 7 simultaneously, bacterial counts are often not significantly different from mice treated with bacteria alone, despite improved outcome or reduced pro-inflammatory responses (83, 84). The difficulties in assessing the role o f host defence peptides in vivo are profound as it is almost impossible to account for synergistic interactions between peptides and other factors, to assess the actual concentrations at the sites o f infection and to discriminate the direct antimicrobial activity o f peptides from other less direct effects such as enhancement o f inflammatory mechanisms (chemotaxis and recruitment of effector cells, enhancement of non-opsonic phagocytosis, etc). Nonetheless creative experiments and animal models have begun to elucidate the roles o f these peptides in vivo. In transgenic mouse model studies in which the expression o f certain host defence peptides is ablated, these mice are more susceptible to infection and carry increased bacterial loads when challenged (85, 86). Although this has been interpreted as being due to direct antimicrobial activity, other components o f host defences must be considered. For example, in a mouse model o f peritoneal Klebsiella pneumoniae infection, small doses of H N P - 1 (4 ng - 4 pg) caused an increase in leukocyte accumulation (87). In this model it was the accumulation of leukocytes that was linked to H N P - 1 induced antimicrobial activity as the reduction in bacterial counts was ablated in leukocytopenic mice. Similar results were observed in Staphylococcus aureus thigh infections (87). Gain of function studies have found that introducing or increasing the expression of a host defence peptide can reduce bacterial loads in certain animal models o f infection. For example adenovirus-mediated transfer of the L L - 3 7 / h C A P - 1 8 gene into the lungs of mice that were subsequently challenged with Pseudomonas aeruginosa led to a reduction in both the bacterial load and in the production of the pro-inflammatory cytokine, T N F - a (88). In a mouse model o f adenovirus mediated gene therapy in which L L - 3 7 expression was increased systemically, mice demonstrated a decreased susceptibility to lipopolysaccharide (LPS) induced septic shock in the complete absence o f bacterial infection (88). In other models the simultaneous instillation into the mouse lung of P. aeruginosa and either of H B D 2 or an L L - 3 7 derivative led to reduced lung damage and pro-inflammatory cytokine production, but did not affect bacterial counts (83). Perhaps the most convincing evidence that host defence peptides reduce bacterial counts through indirect antimicrobial activity is that in experimental models o f infection in mice administration of host defence peptides results in both decreases in bacterial numbers and increased infiltration of leukocytes. When these same studies are performed in leukocytopenic mice there is no decrease in bacterial numbers indicating that the perceived 8 antibacterial activity is due to the infiltration and subsequent activity o f leukocytes (87). In order to dissociate direct antibacterial activity from the immunomodulatory properties o f host defence peptides, synthetic peptides based on natural sequences o f peptides have been designed. These peptides were tested and found not to have antimicrobial activity in vitro, however they were protective in experimental models o f infection in vivo indicating that in vitro antimicrobial activity is not a necessary requirement for reduction of bacterial counts during the course of infection (89). T o date there are no identified individuals who are completely defective in host defence peptide expression. Perhaps this is a testament to the importance o f these peptides in the immune response. However, the neutrophils o f individuals with specific granule deficiency, a disease characterized by frequent and severe infections, have a reduction in the size of the peroxidase positive, defensin-containing granules (90), and are deficient in defensins (6). Although the reduction in defensin expression is obviously a contributor to the reduction in bacterial kill ing by the patient's neutrophils, it is difficult to assess the extent to which these infections are due to the lack of defensins, as these patients are also deficient in other neutrophil components. Patients, who suffer with morbus Kostman, a severe congenital neutropenia, and are treated with G - C S F to restore neutrophils level, do not express L L - 3 7 and have reduced expression o f a-defensins in these cells. One o f the manifestations of this disease is frequent and severe infections and periodontal disease (91). It is believed that the constitutive production and deposition of neutrophils is o f crucial importance to maintaining the immunological balance of the mouth. It has been proposed that the absence of L L - 3 7 may give a selective advantage to bacteria that at low levels are commensals but at higher levels are responsible for periodontal disease. It is unclear, however, whether L L - 3 7 is directly microbicidal towards common pathogens of the mouth or whether it has other immunomodulatory properties which might be involved in maintaining homeostasis in the mouth. Although a number of oral bacteria are susceptible to L L -37 (<10 ug/ml) at 10 m M NaCI in vitro, far fewer bacteria are susceptible in physiologically more relevant isotonic environments (92). Although L L - 3 7 has been detected in saliva, the actual concentration has not been determined (93). This indirect evidence implies that host defence peptides are an important component of the innate immune response in humans. Other indirect evidence for in vivo antimicrobial activity o f host defence peptides is that a decreased level o f expression often correlates with frequency or severity o f disease. For example, H B D - 2 and L L - 3 7 expression is depressed in patients with atopic dermatitis who often present with cases of acute or chronic colonization by Staphylococcus aureus (94). In contrast to atopic 9 dermatitis, H B D - 2 expression is increased in psoriatic skin, a disease in which patients are fairly resistant to bacterial infections (68, 95). It has recently been noted that the neutrophils o f patients with acute myeloid leukemia have markedly decreased expression of L L - 3 7 and it has been proposed that this might contribute to the enhanced susceptibility to infection experienced by these patients (96). Lastly, decreased expression of H B D - 2 in the bronchoalveolar lavage of cystic fibrosis patients who are generally heavily colonised with either S. aureus or P. aeruginosa, is correlated with increased severity of disease (80). Thus is does appear that host defence peptides are involved in the antimicrobial defences in humans, although it is not entirely clear i f this is because they are ki l l bacteria directly. Whether host defence peptides are directly or indirectly antimicrobial, it is apparent that it is o f advantage for bacterial pathogens to subvert their expression or activity. For example Streptococcus pyogenes binds to ci2-microglobin and secretes a small proteinase which inhibits L L - 3 7 from interacting with the bacteria and thus prevents L L - 3 7 mediated kill ing (97). L L - 3 7 expression has been shown to be decreased in Shigella infection, consistent with a proposed mechanism of evasion by this bacterium (98). However, it is not clear whether this is a direct down-regulation o f expression, or a consequence of denuding the epithelium, with reduced expression in the replacement cells. In summary, there is a substantial amount o f indirect evidence in humans and direct evidence in animal models indicating that host defence peptides play a pivotal role in the innate immune response. 1.5 I m m u n o m o d u l a t o r y Properties of Cathel ic idins It is becoming increasingly apparent that host defence peptides have a wide range of immunomodulatory properties that are either complementary to, or independent of, antimicrobial activity. Interest in the immunomodulatory functions of these has increased tremendously in the last few years. In contrast to the antimicrobial activity o f these peptides, their immunomodulatory properties are generally studied in tissue culture medium, which contains physiologically relevant concentrations o f ions and serum proteins. For example, even at a concentration as high as 100 pg/ml, L L - 3 7 is not antimicrobial in standard tissue culture medium (Figure 1.2). However, all o f its immunomodulatory properties are maintained under these conditions. Because these peptides lose their antimicrobial properties under these physiological conditions, but maintain their immunomodulatory properties, I and others have proposed that for certain peptides such as L L - 3 7 , antimicrobial activity may be secondary to immunomodulatory activity in vivo (89). 10 Figure 1.2. Influence of tissue culture medium on the antimicrobial activity of host defence peptides. Overnight cultures o f Salmonella Typhimurium serovar enterica or Staphylococcus aureus A T C C 25923 were diluted 50-fold in Lur ia Broth and allowed to grow to exponential phase (optical density at 600nm of 0.5) at 3 7 ° C . The cultures were spun down and resuspended in either l O m M phosphate buffer, p H 7.0 or in tissue culture medium, Dulbecco's Modif ied Eagle medium ( D M E M ) , with or without 10% foetal bovine serum. L L - 3 7 was added at a concentration of 100 ug/ml. Samples (100 ul) were removed at 0 min and after 15, 30 and 60 min viability was assessed by appropriate dilution and plating for viable bacteria on L agar ( L broth containing 1% agar). A ) Salmonella Typhimurium serovar enterica or B) Staphylococcus aureus A T C C 25923 was re-suspended in either l O m M phosphate buffer, p H 7.0 ( • ) or in Dulbecco's Modif ied Eagle medium ( D M E M ) , with (x) or without (A)10% foetal bovine serum. L L - 3 7 was antimicrobial at 15 minutes in phosphate buffer; however it lost all antimicrobial activity for both strains in tissue culture media. From Bowdish et al., J Leukoc Bio l . 2005 Apr;77(4):451-9. It is possible that many o f the antimicrobial effects observed in animal models may in fact be due to these immunomodulatory properties. This perspective is quite controversial, and will remain so since it is extremely difficult to discriminate between direct and indirect (i.e. through stimulation of innate immunity) mechanisms of kill ing. However, an increasing body of evidence suggests that L L - 3 7 and similar peptides might be involved in the clearance of infection by recruiting leukocytes to the site o f infection, by inducing cytokine and chemokine production, and altering leukocyte responses at the transcriptional and functional level. The a-defensins and cathelicidins have also been proposed to be involved in the promotion of wound healing. Thus their role may extend beyond just the resolution of infection to the maintenance and repair o f damaged tissues. A t very low concentrations many host defence peptides have the ability to neutralise L P S and other bacterial components, thereby attenuating the potential for an inflammatory response. Thus these peptides might also be involved in homeostasis at mucosal surfaces by preventing small amounts o f bacterial components from triggering an inflammatory response. The immunomodulatory properties o f L L - 3 7 and the related cathelicidins are summarised below. T ime (min) 11 One of the earliest discovered immunomodulatory properties o f the cathelicidins was the ability o f these peptides to bind and neutralise L P S (99, 100). Early experiments determined that cathelicidins from a variety o f different species could bind to many different chemotypes of L P S and reduce LPS- induced release of pro-inflammatory cytokines (e.g. TNF-cx, IL-1 , IL-6) and nitric oxide by monocytes or macrophages (101-104), although these two observations are not obligately linked (105). L L - 3 7 has been demonstrated to reduce pro-inflammatory cytokine production induced by other bacterial components such as S. aureus lipotechoic acid ( L T A ) and Mycobacterium A r a L A M (53). It has been clearly demonstrated that various host defence peptides protect mice from LPS-induced lethality (53, 88, 106-109). A s the ability o f L L - 3 7 to neutralise LPS- induced cytokine occurs at very low levels o f L L - 3 7 (1 pg/ml or less) (53, 110) I and others have suggested that one of the functions of these peptides at mucosal surfaces is to minimize inflammation due to low levels o f bacterial components during health or aid in the resolution of inflammation during an immune response induced by infection. L L - 3 7 may also be involved in the clearance of infection by recruiting leukocytes to sites o f high concentrations o f the peptide. A common property o f many host defence peptides in general and the cathelicidins in particular is the ability to induce chemotaxis. PR-39 is chemotactic for neutrophils (111). Similarily, the pro-forms o f a bovine bactenecin are chemotactic for neutrophils (112). L L - 3 7 has been demonstrated to be chemotactic for neutrophils (113), mouse mononuclear cells and P M N s (14), as well as human neutrophils, monocytes and T cells (114). Thus it is believed that elevated concentrations of host defence peptides could contribute to the recruitment of the effector cells o f the innate and adaptive immune response. However, the concentrations required to induce chemotaxis by host defence peptides is 10-100 fold higher than that o f conventional chemokines and thus it is expected that this would only be a function of these peptides at the high concentrations found in the acute inflammatory response. In addition to a role for cathelicidins in recruitment of immune cells, L L -37 has also been demonstrated to interact with epithelial cells and to promote chemokine production (53, 54, 115, 116). Host defence peptides may also be involved in the resolution o f inflammatory responses. Early experiments indicated that these peptides might be involved in wound healing. Consistent with this hypothesis, both human and mouse cathelicidins are up-regulated at sites of incision or wounding, even i f the wound is sterile. The appearance of cathelicidins in the skin has been ascribed to both synthesis within epidermal keratinocytes, and deposition from granulocyctes that migrate to the site o f injury (117). Upon incision, h C A P - 1 8 (the precursor to L L - 3 7 ) has 12 been shown to be up-regulated in the epidermis bordering the wound. This increase in expression at both the R N A and protein levels was clearly evident at the migrating front o f the wound during re-epithelialization. Levels o f h C A P - 1 8 decreased following wound closure and eventually returned to baseline levels when the wound was intact and re-epithelization was complete. h C A P - 1 8 was found to be an active component in the process o f re-epithelization since antibodies specific for the peptide decreased the rate o f re-epithelization in a concentration dependent manner (50). Consistent with this observation, low levels o f L L - 3 7 (as low as 50 ng/ml) have been demonstrated to increase proliferation in an endothelial cell line (51). The importance of this peptide in re-epithelization has been further inferred from its presence in wounds which are healing normally, but its absence in chronic ulcers (50). The porcine cathelicidin, PR-39, has been demonstrated to induce the production o f heparan sulfate proteoglycans, called syndecans, at sites of wound repair. The expression of syndecans increased during wound repair, causing the cells to become more responsive to growth factors and regulate cell proliferation and migration in response to these effectors. The ability o f PR-39 to induce syndecan expression indicated that this peptide, and possibly other cathelicidins, might function as signalling molecules in wound repair. Since I began these studies the range of immunomodulatory functions ascribed to L L - 3 7 have been expanded to include the promotion of angiogenesis (118), the modulation of the differentiation of immature dendritic cells from precursor cells, with consequent impact on T h cell polarization (119) and the enhancement of anti-tumour responses (120). In conclusion, the immunomodulatory properties o f host defence peptides in general and L L - 3 7 in particular are not as well characterised as their antimicrobial properties. Despite the fact that studies on the immunmodulatory properties o f the peptide are in their infancy it is apparent that this peptide and others like it have a variety of role in the immune response, the nature of which is just beginning to be understood. 1.6 M o d e l for the Physiological Role of LL-37 L L - 3 7 , the 37-residue C-terminal peptide of h C A P - 1 8 , is cleaved by proteinase-3 after exocytosis from neutrophils (36). Thus, during infection and inflammation high concentrations of L L - 3 7 will be released at sites of neutrophil accumulation. This occurs in gingival tissues and in the saliva where it has been proposed that L L - 3 7 is not produced by these tissues, but instead is deposited by the homeostatic degranulation of neutrophils (91, 121). The precursor h C A P - 1 8 is also produced by a variety of epithelial cell types. Its expression is moderately inducible upon stimulation with pro-inflammatory cytokines such as I L - l a (122) or bacterial components (41). 13 Although it is not yet clear how h C A P - 1 8 is released from these cell types and processed, the processed peptide L L - 3 7 is found at increased concentrations at cell surfaces in a number of inflammatory conditions, including psoriasis (123) and inflammatory lung conditions (124). Thus in the context o f infection and inflammation, L L - 3 7 can be derived via release by neutrophils recruited to the site o f infection, and via secretion by epithelial cells. The first cells that are recruited to sites of infection and inflammation are neutrophils. If the infection is not resolved in minutes to hours, a second wave o f cells including monocytes wil l be conscripted to the site o f infection (125). These monocytes wil l arrive at sites of elevated local concentrations of L L - 3 7 . U p o n exposure to L L - 3 7 , epithelial cells produce IL-8 and peripheral-blood-derived monocytes produce chemokines such as IL-8, M C P - 1 and M C P - 3 (53, 54, 115). These chemokines would attract neutrophils, monocytes and macrophages respectively. L L - 3 7 has also been demonstrated to be directly chemotactic for neutrophils, T cells, and monocytes (114). Thus, when the first line response is insufficient, levels o f L L - 3 7 are expected to be high enough to induce chemotaxis o f these cells types directly and LL-37-activated monocytes and epithelial cells may produce chemokines which wil l lead to the recruitment o f important immune response effector cells to assist in the resolution of infection. Figure 1.3 summarises this experimental model. Less is known about the expression of cathelicidins in other species. For example, it is not known i f they are produced by epithelial cells or i f they are found at equivalent concentrations to L L - 3 7 . However it has been demonstrated that they are found at high concentrations in the granules o f neutrophils and are chemotactic for a variety of different cell types thus elements of this model may also be applicable. 14 LUMEN Inflammatory st imulus M o n o c y t e Figure 1.3. Experimental model. Upon detection of infection or stimulation with inflammatory mediators, resident or recruited neutrophils release granule contents including high concentrations of antimicrobial peptides such as LL-37. Epithelial cells respond to inflammatory stimuli produce LL-37. A concentration gradient of LL-37 would occur at the site of infection. This concentration gradient could lead directly to chemotaxis of neutrophils, monocytes and other cell types. In addition, LL-37 can induce epithelial cells to produce IL-8 and other chemokines. Increased concentrations of IL-8 would lead to increased infiltration of neutrophils and monocytes. These cells would be arriving at sites with relatively high concentrations of LL-37 which would promote a variety of immunomodulatory effects. From Bowdish et al., J Leukoc Biol. 2005.77(4):451-9 1.7 Hypothesis and Experimental Goals It has become increasingly apparent that LL-37 is an immunomodulator, however at the time that I began this thesis it was not apparent how this peptide interacts with the effector cells of the innate immune response. It has been proposed that LL-37 may mediate its effects through either specific receptor mediated interactions or through more non-specific interactions (i.e. generalised membrane disruption or cytotoxicity). At the start of this research I hypothesised that LL-37 interacted directly with the cells of the innate immune response and that this interaction would not occur with cells of the adaptive immune response. These interactions were measured by activation of certain signalling pathways. I theorised that this cell specificity would not occur if LL-37 interacted with eukaryotic cells through a non-specific interaction. I also hypothesised 15 that L L - 3 7 induced signalling would be linked to some or all o f the immunomodulatory properties o f this peptide. One o f the goals o f these studies was to characterise early interactions of L L - 3 7 and the effector cells o f the innate immune response, with particular emphasis on monocytes. Once it was determined that L L - 3 7 interacted directly with the effector cells o f the innate immune response I hypothesised that activation of these signalling pathways was a requirement for the immunomodulatory properties o f this peptide such as chemokine production and the anti-endotoxin response. 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Y a n g , 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 ( J 6 - 1 ) D N A vaccine. LeukRes 29:535. 121. Dale, B . A . , J. R. Kimba l l , S. Krisanaprakornkit, F . Roberts, M . Robinovitch, R. O'Neal, E . V . Valore, T . Ganz, G . M . Anderson, and A . Weinberg. 2001. Local ized antimicrobial peptide expression in human gingiva. J Periodontal Res 36:285. 122. Erdag, G , and J. R. Morgan. 2002. Interleukin-1 alpha and interleukin-6 enhance the antibacterial properties o f cultured composite keratinocyte grafts. Ann Surg 235:113. 123. Frohm, M . , B . Agerberth, G . Ahangari, M . Stahle-Backdahl, S. L iden , H . Wigzel l , and G . H . Gudmundsson. 1997. The expression o f the gene coding for the antibacterial peptide L L - 3 7 is induced in human keratinocytes during inflammatory disorders. J Biol Chem 272:15258. 124. Agerberth, B . , J. Grunewald, E . Castanos-Velez, B . Olsson, H . Jornvall, H . Wigzel l , A . Eklund, and G . H . Gudmundsson. 1999. Antibacterial components in bronchoalveolar lavage fluid from healthy individuals and sarcoidosis patients. Am J Respir Crit Care Med 160:283. 125. Witte, M . B . , and A . Barbul . 1997. General principles o f wound healing. Surg Clin North Am 77:509. 23 2.0 Immunomodulatory Activities of Small Host Defence Peptides 2.0 I M M U N O M O D U L A T O R Y ACTIVITIES OF S M A L L HOST D E F E N C E PEPTIDES 24 2.1 INTRODUCTION 25 2.2 MATERIALS & METHODS 26 2.3 RESULTS 28 2.4 DISCUSSION 33 2.5 B IBLIOGRAPHY 35 T The majority of the work in this chapter has been published in Bowdish, D . M . E . , D . J . Davidson, M . G . Scott and R . E . W . Hancock. 2005. Immunomodulatory properties o f small host defense peptides. Antimicrob Agents & Chemother. 2005 May;49(5): 1727-32. Additional data, not included in the original publication, are presented in Table 2.1, Figure 2.1, Figure 2.2B, and Figure 2.3. The text has been modified accordingly. A l l data were collected and analysed by D . Bowdish. 24 2.1 Introduction Cationic antimicrobial peptides are conserved across virtually all forms of life as a primitive component o f the innate immune response. They can be expressed either constitutively or in response to pathogen-associated molecular pattern molecules, such as bacterial lipopolysaccharide (LPS) , or inflammatory mediators such as IL-6 and TNF-cc (1, 2). Although they can be potent antimicrobial agents, a key element of their therapeutic potential may involve the myriad o f other activities attributed to them (3). Indeed, some peptides such as the human cathelicidin L L - 3 7 , have been proposed to have far more potent immunomodulatory activities than antimicrobial functions (4, 5). When considering the use o f peptides like L L - 3 7 in immunotherapy one must take into account the large size o f this peptide and the corresponding issues this raises including cost o f goods, protease lability and pharmacokinetics. In this study, we investigated the immunomodulatory properties o f two o f the smallest known active peptides, both derived from bovine cathelicidins, and contrasted those activities to L L - 3 7 , a known immunomodulator, with the goal o f developing novel immunomodulatory therapies. Naturally occurring cationic peptides can vary in size from 12 to 50 amino acids and have the property of folding into amphipathic structures (often after contact with membranes) that have a positively-charged, hydrophilic face and a hydrophobic face. In humans the major linear peptide is the sole cathelicidin characterized to date, L L - 3 7 . L L - 3 7 is the proteolytically-processed, extracellular form of h C A P - 1 8 , a cathelicidin peptide which is constitutively produced in the secondary granules o f neutrophils and by a variety o f other cells. Although found at mucosal surfaces at concentrations o f around 2 ug/ml, its expression is induced upon exposure to pro-inflammatory mediators or during the course o f infection or inflammation in a variety of tissues (5-8). Although cathelicidins are not well conserved between species, the evolutionary relationship between these peptides can be inferred from the highly conserved pro-region called the cathelin domain that is cleaved to release the active peptide. Cathelicidins have been found in cows ( B M A P - 2 7 , indolicidin, bactenecin), pigs (protegrins), mice ( C R A M P ) , rabbits ( C A P 18) and humans ( h C A P - 1 8 / L L - 3 7 ) and this evolutionary conservation suggests an important role in innate immunity (reviewed in (9)). T o date studies o f the influence o f peptides as effectors o f innate immunity have tended to utilize larger peptides o f 26 amino acids or more in size (10). In this study, two o f the shortest known peptides, indolicidin and B a c 2 A , a derivative of bactenecin, were investigated for their ability to affect a variety of innate immune responses such as cytokine production, anti-endotoxin activity and chemotaxis. Indolicidin, a 13 amino acid, proline and 25 tryptophan rich cathelicidin, folds into a characteristic boat shaped structure when associated with membranes (11). Its moderate antimicrobial activity ( M I C values o f between 16-64 | ig/ml for common Gram-negative bacteria and between 4-8 u.g/ml for Gram-positive bacteria) and ability to interact with microbial membranes have been well characterized (12). Bactenecin is a 12 amino acid cathelicidin that is also found in the large granules o f bovine neutrophils and is also moderately active against many common Gram-negative pathogens ( M I C values o f approximately 8 u.g/ml) and Gram-positive bacteria ( M I C values o f 64 u,g/ml and greater). The functional structure o f bactenecin in vivo has not been well characterized, and there is some evidence for a linear structure (13). B a c 2 A is a linearized derivative o f bactenecin in which two cysteine residues have been replaced with two alanine residues. It has modest broad-spectrum activity with M I C values o f between 2-32 p.g/ml for Gram-negative bacteria and between 0.25-16 p.g/ml for Gram-positive bacteria that is consistent with the activity o f the disulphide bonded peptide (14). This modification prevents the possibility o f concatemer formation thus making B a c 2 A a better target for drug development. Sequence information for the peptides used in this study is presented in Table 2.1. Table 2.1. Sequences of the peptides used in these studies. Name Sequence Amino acids Charge B a c 2 A R L A R I V V I R V A R 12 +4 Indolicidin I L P W K W P W W P W R R 13 +3 L L - 3 7 L L G D F F R K S K E K I G K E F r C R I V Q R I K D F L R N L V P R T E S 37 +6 Previous research has shown that L L - 3 7 is a potent immunomodulator. L L - 3 7 has been demonstrated to be a chemoattractant for human monocytes, T cells (15) and mast cells (16), is a potent anti-endotoxic agent (17) and induces chemokine production (4). In light o f these observations we compared the immunomodulatory activities o f the two bovine cathelicidins, indolicidin and B a c 2 A , to those of the better characterized L L - 3 7 with respect to anti-endotoxin properties, chemotaxis and chemokine production. W e found that indolicidin and B a c 2 A have different immunomodulatory properties, which make them candidates for development as novel therapies. 2.2 Materials & Methods Cell lines and culture conditions The human monocyte-like cell line, T H P - 1 (18), was obtained from the A T C C (No. T I B -202, Rockvil le , M D ) , and grown in supplemented R P M I 1640 media containing 10% foetal calf serum and 1% L-glutamine (Gibco B R L , Burlington, O N ) . T H P - 1 cells were differentiated into 26 adherent macrophage-like cells by addition of l O O m M phorbyl myristate acetate and incubation at 3 7 ° C , 5% C 0 2 for three days as described previously (19). Undifferentiated T H P - 1 monocytes were used for the chemotaxis experiments. Differentiated macrophage-like cells were used for the anti-endotoxin assays. The human bronchial epithelial cell line 16HBE14o- ( H B E ) was a gift from Dr . D . Gruenert (Cardiovascular Research Institute, University o f California, U C S F ) (20) and was maintained in minimal essential media ( M E M ) with Earle's salts (Gibco B R L , Burlington, O N ) . H B E cells were maintained in flasks and seeded into wells that had been treated with mouse type-4 collagen and human fibronectin solution (8.5 ml M E M with Earle's salts, 1% fibronectin, 1% collagen and 1 mg B S A ; Gibco B R L , Burlington, O N ) . Peptide synthesis. A l l peptides were synthesized by N - (9-fluorenyl) methoxycarbonyl (frnoc) chemistry at the Nucleic Acid/Protein Service unit at the University o f British Columbia , as previously described (21). Peptides were purified by reverse-phase high-performance liquid chromatography and were at least 98% pure. The concentration o f the peptides in solution was determined by amino acid analysis. Cytotoxicity assay A n M T T assay ( A T C C ) was used to assess the cytotoxicity o f the peptides and was performed as per the manufacturer's instructions. The assay is based on the cleavage of the yellow tetrazolium salt M T T into purple formazan by metabolically active cells. Cells were cultured as described above and peptides were added for 24 h. A decrease in the absorbance at 550 nm indicated a decrease in cell viability. Cytokine production IL-8 or T N F - a found in the supernatants o f the treated cells was measured using commercially prepared E L I S A plates in accordance to the manufacturer's suggestion ( R & D systems, Minneapolis , N M ) . Cells were seeded in 24 well tissue culture plates at a concentration of 2 x 10 5 cells/ml (THP-1) or 5 x 10 5 cells/ml ( H B E ) and incubated for either three days ( T H P -1) or 48 hours ( H B E ) . Cells were then incubated in at least triplicate, for six hours (to measure T N F - a production) or eight hours (to measure IL-8 production) in the presence of either media alone, or with Escherichia coli L P S 0111:B4 (Sigma Chemical C o . , St Louis , Mo. ) , cationic peptides or a combination of L P S and peptide (concentrations stated in text) in media. Supernatants were collected and stored at - 2 0 ° C until use. 27 Chemotaxis assay Chemotaxis o f the monocyte-like cell line T H P - 1 was assayed in a 96-well disposable chemotaxis system with 5 p m polycarbonate filters (Chemotx, Neuroprobe, Gaithersburg, M D ) . Chemotactic factors were diluted in R P M I media supplemented with 1% bovine serum albumin (chemotaxis medium) and added to the bottom chamber o f the chemotaxis plate. A 1 x 10 6 cell/ml suspension of T H P - 1 cells was made in chemotaxis media and 50 pl was added to the top of the chamber. The plate was incubated at 3 7 ° C , 5% CO2 for three hours. In order to quantify the number of cells that migrated through the filter, a standard curve of cell number represented by colour change was created using a colorimetric assay o f cell viability based on cleavage o f tetrazolium salt, W S T - 1 (Roche Diagnostics). W S T - 1 (10% v/v) was added and the plate was incubated at 3 7 ° C in 5% CO2 until colour development was complete. The plate was then read at 570 nm in a PowerWave 340 E L I S A plate reader. The number o f cells that had migrated through the membrane was expressed as a percentage of the total number o f cells added (chemotaxis index). Statistical analysis Student's t test was performed to determine statistical significance. Values are expressed as mean +/- standard error. Significance was determined as p value <0.05. 2.3 Results Indolicidin, Bad A and LL-37 are not cytotoxic at concentrations used in this study. Cytotoxicity was assessed using the M T T assay. None of the peptides caused significant cytotoxicity in either the H B E or T H P - 1 cell lines at concentrations o f < 100 pg/ml (Figure 2.1). In contrast, the cytotoxic peptide CP29 caused significant cell death at a concentration of 50 pg/ml. 28 Figure 2.1. Cytotoxicity of LL-37, Bac2A and Indolicidin. MTT assays were performed and it was found that LL-37, Bac2A and indolicidin are not cytotoxic to THP-1 cells (A) or HBE cells (B) at concentrations < 100 ug/ml. At 300 ug/ml, significant cytotoxicity was observed in indolicidin treated H B E cells and in indolicidin and LL-37 treated THP-1 cells. Mean values of three independent experiments are represented ± standard error of the mean. A Student's t test was performed and asterisks denote significance at a p<0.005 level compared to cells treated with a vehicle control only. Peptide concentration (ug/ml) Indolicidin, but not Bac2A, displays potent anti-endotoxic properties. Certain cationic peptides have the ability to block the production of cytokines produced in response to LPS by either directly up-regulating inhibitory pathways in cells (22) or interfering with the ability of LPS to bind LBP (17). THP-1 cells were stimulated with E. coli LPS and the amount of TNF-a in the supernatant was assayed by ELISA. The cells produced between 2,600-3,900 ng/ml of TNF-a. The ability of the short peptides indolicidin and Bac2A to inhibit LPS-induced TNF-a production in the THP-1 cell line was investigated at low concentrations of host defence peptides such as would be found at mucosal surfaces. The addition of indolicidin at the same time as LPS suppressed the LPS-induced production of TNF-a in a dose dependent manner (Figure 2.2A) in the differentiated macrophage-like THP-1 cell line and was statistically significant at > 5 ug/ml (p<0.001). This inhibition was not as potent at low concentrations as was observed for LL-37 (Figure 2.2A), a human cathelicidin previously shown to have anti-endotoxin ability (17), however, at concentrations of 50 ug/ml over 80% inhibition was observed (Figure 2.2B). In contrast, Bac2A caused no inhibition over the concentration range of 1-10 p.g/ml and demonstrated only moderate anti-endotoxin activity (approximately 30%) at 20 ug/ml. Interestingly, as shown previously with LL-37 and the insect-derived peptide C E M A (4, 21), the inhibition of cytokine production was observed even when indolicidin was added to the THP-1 cells up to one hour after LPS (Figure 2.3). This indicates that the anti-endotoxin properties of indolicidin may be due to an interaction of the peptide with cells in addition to direct neutralization of LPS. 29 100 .2 80 t3 CD 20 O ' Bac2A I „ I a l l 1 5 10 20 Peptide concentration (ug/ml) Indolicidin concentration (ug/ml) Figure 2.2. Anti-endotoxin properties of indolicidin, Bac2A and LL-37. A) Indolicidin and LL-37 significantly reduced the amount of TNF-a produced by differentiated THP-1 cells upon stimulation with 100 ng/ml ofE. coli 0111:B4 LPS. Bac2A did not reduce the amount of TNF-a at the concentrations tested (N/D, no detectable decrease in TNF-a). B) Higher doses of indolicidin are required to inhibit LPS induced TNF-a production. Mean values of one representative experiment of at least three are represented ± standard error of the mean. A Student's t test was performed and * denotes significant at p< 0.05, ** denotes significant at a p<0.001, *** denotes p<0.005 level compared to LPS-only treated controls. •M 60 Figure 2.3. Addition of indolicidin up to 60 minutes after the addition of LPS results in inhibition of LPS induced TNF-a from PMA treated THP-1 cells. THP-1 cells were stimulated with E. coli 0111:B4 LPS (lOOng/ml) and 20 pg/ml of indolicidin which was added either simultaneously, or 20, 40 or 60 minutes after the addition of LPS. There was no statistically significant change in inhibition of LPS induced TNF-a production when indolicidin was added up to 60 minutes after the LPS. Mean values of the average of three independent experiments ± the standard error of the mean shown. The anti-endotoxin activity of indolicidin and LL-37 in combination is greater than additive. It has been well established that certain cationic peptides used in combination can demonstrate synergistic antimicrobial activity (23). To determine whether this phenomenon was applicable to the non-antimicrobial activities of cationic peptides, we examined the ability of L L -37 to work synergistically with indolicidin to inhibit LPS-induced TNF-a production. The anti-20 40 Minutes post LPS addition 60 30 endotoxin activity o f indolicidin and L L - 3 7 in combination was significantly greater than either peptide alone and greater than the additive effect o f these two peptides (Figure 2.4; p<0.05) Figure 2.4. The addition of indolicidin and LL-37 in combination produces a greater than additive inhibition of LPS-induced TNF-a. Indolicidin and L L - 3 7 significantly reduced the amount of T N F - a produced by differentiated T H P - 1 cells upon stimulation with 100 ng/ml of E. coli 0111:B4 L P S . The combination o f very small amounts o f indolicidin (1 | ig/ml) or L L - 3 7 (1 ug/ml or 100 ng/ml) resulted in a greater reduction o f LPS- induced T N F -a than would be predicted to occur i f this effect was strictly additive. Mean values o f the average of three independent experiments ± the standard error o f the mean shown. A one-tailed Student's t test was performed and the results significant at a p<0.05 level are marked with an asterisk. s? 10 Bac2A is a more potent chemotactic agent than indolicidin. It has been shown previously that L L - 3 7 is chemotactic for various cells types, including monocytes, mast cells and T cells (15, 24). Little is known about the chemoattractant properties of other cathelicidins, although a relative of bactenecin, Bac7, has been shown to chemoattract monocytes (25). In this experiment the abilities o f indolicidin, B a c 2 A and L L - 3 7 to induce chemotaxis o f undifferentiated T H P - 1 cells were investigated. The addition o f B a c 2 A to the bottom chamber o f the Neuroprobe Chemotx filter, induced the migration o f T H P - 1 cells in a statistically significant (p<0.05 at 10 (ig/ml Bac2A, p<0.001 at >50 U-g/ml B a c 2 A ) and dose-dependant manner (Figure 2.5). Indolicidin did not induce a statistically significant increase in chemotaxis, except at a concentration of 300 |J.g/ml, at which concentration it demonstrated some cytotoxicity. Interestingly L L - 3 7 , which has previously been shown to be chemotactic for blood-derived monocytes (15), was not chemotactic for T H P - 1 cells. 31 o o in = x 9 3 — o CO E o <u t ; sz in " 1 o TJ CO s I 80 70 60 50 40 30 20 10 » No peptide •Bac2A a Indolicidin • LL-37 10 50 Peptide (ug/ml) 100 Figure 2.5. B a c 2 A is a chemoattractant for undifferentiated T H P - 1 cells. Indolicidin, B a c 2 A , L L - 3 7 , or water (vehicle control) were added to the bottom well o f a chemotaxis chamber at the concentrations shown. Undifferentiated T H P - 1 cells were added to the top well and after three hours chemotaxis was assessed. Chemotaxis was measured by calculating the total number of cells that migrated as a percentage o f the total number of cells added. M e a n values o f the average of three independent experiments ± the standard error of the mean shown. A Students's two-tailed t test was performed and the results significant at a p<0.05 level are marked with an asterisk and those significant at p<0.001 are marked by a double asterisk. Indolicidin, but not Bac2A, can induce IL-8 expression in 16HBE4o- cells. It has been shown previously that L L - 3 7 induces the release o f the chemokines IL-8 and/or M C P - 3 in both the murine lung and in various cell lines (4). Therefore we investigated the possibility that indolicidin and/or B a c 2 A might have similar properties. In differentiated T H P - 1 cells, indolicidin inhibited the L P S induced production o f the cytokine T N F - a , but did not itself induce T N F - a or IL-8 (data not shown). However, in the human bronchial cell line, 16HBE4o- , indolicidin concentrations > 10 pg/ml induced significant production o f the chemokine IL-8 (p < 0.05) in a dose dependant manner (Figure 2.6), but did not induce T N F - a (data not shown). B a c 2 A did not induce any cytokine or chemokine production, even at 50 pg/ml (data not shown). 3000 " I Q. 2000" a §o o P -a p 1000 -a oo ' 1 0 0 0.1 1 10 50 Indolicidin (ug/ml) 100 Figure 2.6. Indol ic id in induces I L - 8 product ion in a dose dependent m a n n e r in the h u m a n bronchia l epithelial cell l ine, 1 6 H B E 4 o - . Cells were grown to confluency and subsequently incubated with indolicidin for eight hours. The presence of IL-8 in the supernatant was detected by E L I S A . M e a n values o f one representative experiment o f at least three are represented ± standard error o f the mean, n=4 replicates for each condition. A two tailed T test was performed and results significant at a p<0.05 are marked with an asterisk. 32 2.4 Discussion In this study the bovine neutrophil-derived peptides indolicidin and B a c 2 A were demonstrated to have diverse and complementary immunomodulatory functions, as summarized in Table 2.2, in addition to their established antimicrobial activities. Indolicidin inhibited the LPS-induced pro-inflammatory cytokine responses in a macrophage-like cell line and induced chemokine production in a dose dependant manner in a bronchial epithelial cell line. Conversely, B a c 2 A had very weak anti-endotoxic or chemokine inducing properties but acted directly to induce chemotaxis o f macrophage-like T H P - 1 cells. Table 2.2. Comparison of t le activities of Indolicidin, Bac2A and L L - 3 7 (Ug/ml). Property M i n i m a l concentration demonstrating the given property (pg/ml) Reference Indolicidin B a c 2 A L L - 3 7 M I C G r a m Positive (e.g. S. aureus) 8 4 64 (26, 27) G r a m Negative (e.g. P. aeruginosa) 64 8 64 (23, 26) Anti-endotoxin activity 5 20 1 Figure 2.2 IL-8 production 10 N D ( > 100)1 10 Figure 2.6(28, 29) Chemotaxis N D ( > 100) 2 10 N D (> 300) 3 Figure 2.5 1 N D = not detectable at concentrations < 100 pg/ml. 2 N D = chemotaxis was observed at 300 pg/ml (data not shown) 3 N D = not detectable at concentrations < 300 pg/ml. Cationic peptides have traditionally been studied as antimicrobial agents. In bovine neutrophils, multiple classes o f peptides have been found, including the P- sheet defensins (30), the a-helical peptide B M A P , and the short (12-13 amino acids) cationic peptides bactenecin (31) and indolicidin (32). It seems reasonable to assume that these peptides have evolved to work in combination as opposed to independently, and that each peptide has distinct (but possibly overlapping) functions. Indeed, p-defensins have different antimicrobial activity spectra (33) while certain combinations of peptides have been demonstrated to be synergistic in their antimicrobial activities (23). However direct kill ing of microbes may not always be the primary role that these agents perform in the innate immune response as the physiological concentrations of peptides and o f antagonistic mono- and di-valent cations are often difficult to rationalize with a primary antimicrobial role in vivo (5, 34) Alternatively, their possible primary action is modulation of immune mechanisms, as these functions occur at physiological salt and peptide concentrations (4, 5). However, the degree to which these functions contrast and/or complement each other in different host defence peptides is largely unknown. 33 Individual peptides have a distinct spectrum of immune defense functions. T o test the possibility that different peptides may have diverse immunomodulatory functions on human cells, and thus have alternatively directed potential as immune boosting drugs, we chose to evaluate the properties o f two of the smallest related cationic peptides. It was interesting to observe that B a c 2 A , despite having a stronger binding affinity than indolicidin for L P S (17), was unable to block LPS- induced T N F - a production at equivalent concentrations. This was consistent with our previous results indicating that the ability o f peptides to block LPS-induced responses does not rely entirely on their ability to bind to L P S (35). Indeed, gene array studies on the R A W 264.7 mouse macrophage cell line demonstrated that peptides can block the expression of only a subset o f the genes induced by L P S , while themselves inducing the expression of a unique subset o f genes (22). This is consistent with the interpretation that the addition of peptides does not merely prevent L P S from binding to the macrophages, but alters LPS-induced gene expression in a more directed fashion. Indeed, we showed that indolicidin can block cytokine production when added up to an hour after the addition o f L P S to cells as previously demonstrated for L L - 3 7 and the insect peptide C E M A . In this time period, we assume that L P S would already bind to L P S receptors on macrophages, and initiate signalling (36). The ability o f peptides to selectively block pro-inflammatory cytokine gene expression implies that although their ability to bind to L P S and disrupt L P S binding to LPS-b ind ing protein (17) may be a component o f their anti-endotoxin ability, it is not the sole method by which they block pro-inflammatory responses. Indeed amongst the genes that are up regulated directly by peptides are anti-inflammatory cytokines such as IL-10 (4). The ability o f peptides to block the excess cytokine production that is a hallmark of sepsis has lead to their consideration as anti-sepsis agents (37-39). However toxicity due to the relatively high concentrations o f peptide required is a major concern. In this study we demonstrate that low concentrations of various peptides may be more effective in blocking L P S induced cytokine responses than higher concentrations o f a single peptide and that exogenous addition of peptides might lead to synergy with the hosts own naturally occurring peptides. It has been postulated that antimicrobial peptides have evolved from the deletion products of C X C chemokines (40) and, consistent with this, certain peptides are chemotactic for distinct subsets o f leukocytes. For example, L L - 3 7 is chemotactic for human blood-derived monocytes and T cells (15) through formyl peptide receptor-like 1 ( F P R L 1 ) at concentrations of approximately 50 ug/ml, whereas it is chemotactic towards mast cells through two different receptors between 5-10 ug/ml (16). Thus chemotaxis occurs at concentrations that are generally 34 higher than those required for anti-endotoxin activity. Although there is a paucity of data indicating the exact concentrations o f host defence peptides in vivo it is known that the cathelicidins are found at extremely high concentrations in the granules o f neutrophils and it seems reasonable that chemotaxis in vivo might occur in response to somewhat higher concentrations o f peptides, such as would occur at sites of neutrophil degranulation. Interestingly in our study, L L - 3 7 was not chemotactic for the T H P - 1 monocyte-like cell line (Figure 2.5) even though this cell line has been shown to express F P R L - 1 (41) and we found that these cells express F P R L - 1 at the R T - P C R level. Conversely, B a c 2 A , but not indolicidin, was shown to induce chemotaxis o f the cells at concentrations of > 10 ng/ml (Figure 2.5). Future experiments should aim to elucidate the chemotactic receptor for B a c 2 A . These data indicate that short peptides such as indolicidin and B a c 2 A possess contrasting immunomodulatory properties, which appear to be comparable to certain activities o f the known immunomodulator, L L - 3 7 . Interestingly, these bovine derived peptides function on human cell lines, indicating that host defence peptides may demonstrate conserved function across species. The differences in their immunomodulatory properties may reflect physiologically complementary functions in vivo, and/or be a consequence of the modifications made to the naturally occurring bactenecin to produce the derivative B a c 2 A . Regardless o f this, these contrasting activities make these peptides valuable in the design of alternatively directed therapeutic agents, and as tools in dissecting the variations in the mechanisms that underpin these diverse activities. In addition their smaller size compared to L L - 3 7 makes them potentially exciting prototypes for development as novel immunomodulatory drugs, especially since their collective ability to enhance chemokine production, induce chemotaxis and block endotoxin responses resembles the properties o f L L - 3 7 . Future work wil l involve optimizing these peptides and better characterising their immunomodulatory properties to further illuminate their potential as novel therapeutic agents. 2.5 B i b l i o g r a p h y 1. Diamond, G . , J . P. Russell, and C . L . Bevins. 1996. Inducible expression o f an antibiotic peptide gene in lipopolysaccharide-challenged tracheal epithelial cells. Proc Natl Acad Sci US A 93:5156. 2. Zhang, G . , H . W u , C . R. Ross, J . E . Minton, and F . Blecha. 2000. Cloning of porcine N R A M P 1 and its induction by lipopolysaccharide, tumor necrosis factor alpha, and interleukin-lbeta: role o f C D 14 and mitogen-activated protein kinases. Infect Immun 68:1086. 3. Scott, M . G . , and R. E . Hancock. 2000. Cationic antimicrobial peptides and their multifunctional role in the immune system. Crit Rev Immunol 20:407. 35 4. Scott, M . G . , D . J. Davidson, M . R. G o l d , D . Bowdish, and R. E . Hancock. 2002. The human antimicrobial peptide L L - 3 7 is a multifunctional modulator of innate immune responses. J Immunol 169:3883. 5. Bowdish, D . M . E . , Davidson, D . J . , Hancock, R . E . W . 2004. A re-evaluation o f the role o f host defence peptides in mammalian immunity. Combinatorial Chemistry & High Throughput Screening In press. 6. K i m , S. T . , H . E . Cha , D . Y . K i m , G . C . Han, Y . S. Chung, Y . J . Lee, Y . J . Hwang, and H . M . Lee. 2003. Antimicrobial peptide L L - 3 7 is upregulated in chronic nasal inflammatory disease. Acta Otolaryngol 123:81. 7. Woo , J. S., J. Y . Jeong, Y . J. Hwang, S. W . Chae, S. J . Hwang, and H . M . Lee. 2003. Expression of cathelicidin in human salivary glands. Arch Otolaryngol Head Neck Surg 129:211. 8. Ong, P. Y . , T . Ohtake, C . Brandt, I. Strickland, M . Boguniewicz, T . Ganz, R. L . Gallo, and D . Y . Leung. 2002. Endogenous antimicrobial peptides and skin infections in atopic dermatitis. NEngl J Med 347:1151. 9. Zanetti, M . 2004. Cathelicidins, multifunctional peptides o f the innate immunity. J Leukoc Biol 75:39. 10. Gennaro, R. , M . Zanetti, M . Benincasa, E . Podda, and M . M i a n i . 2002. Pro-rich antimicrobial peptides from animals: structure, biological functions and mechanism of action. Curr Pharm Des 8:763. 11. Rozek, A . , C . L . Friedrich, and R. E . Hancock. 2000. Structure of the bovine antimicrobial peptide indolicidin bound to dodecylphosphocholine and sodium dodecyl sulfate micelles. Biochemistry 39:15765. 12. Falla, T . J . , D . N . Karunaratne, and R. E . Hancock. 1996. M o d e of action of the antimicrobial peptide indolicidin. J Biol Chem 271:19298. 13. W u , M . , E . Maier , R . Benz, and R. E . Hancock. 1999. Mechanism of interaction of different classes o f cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli. Biochemistry 38:7235. 14. Romeo, D . , B . Skerlavaj, M . Bolognesi, and R. Gennaro. 1988. Structure and bactericidal activity o f an antibiotic dodecapeptide purified from bovine neutrophils. J Biol Chem 263:9573. 15. De, Y . , Q . Chen, A . P. Schmidt, G . M . Anderson, J. M . Wang, J . Wooters, J. J. Oppenheim, and O . Chertov. 2000. L L - 3 7 , the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 ( F P R L 1 ) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells. J Exp Med 192:1069. 16. Niyonsaba, F . , K . Iwabuchi, A . Someya, M . Hirata, H . Matsuda, H . Ogawa, and I. Nagaoka. 2002. A cathelicidin family of human antibacterial peptide L L - 3 7 induces mast cell chemotaxis. Immunology 106:20. 17. Scott, M . G . , A . C . Vreugdenhil , W . A . Buurman, R. E . Hancock, and M . R. Go ld . 2000. Cutting edge: cationic antimicrobial peptides block the binding o f lipopolysaccharide (LPS) to L P S binding protein. J Immunol 164:549. 18. Tsuchiya, S., M . Yamabe, Y . Yamaguchi , Y . Kobayashi , T . K o n n o , and K . Tada. 1980. Establishment and characterization of a human acute monocytic leukemia cell line ( T H P -1). Int J Cancer 26:171. 19. Stokes, R. W . , and D . Doxsee. 1999. The receptor-mediated uptake, survival, replication, and drug sensitivity of Mycobacterium tuberculosis within the macrophage-like cell line T H P - 1 : a comparison with human monocyte-derived macrophages. Cell Immunol 197:1. 36 20. Cozens, A . L . , M . J. Y e z z i , K . Kunzelmann, T . Ohrui , L . C h i n , K . E n g , W . E . Finkbeiner, J. H . Widdicombe, and D . C . Gruenert. 1994. C F T R expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am JRespir Cell Mol Biol 10:38. 21. Gough, M . , R. E . Hancock, and N . M . Kel ly . 1996. Antiendotoxin activity o f cationic peptide antimicrobial agents. Infect Immun 64:4922. 22. Scott, M . G , C . M . Rosenberger, M . R. G o l d , B . B . Finlay, and R. E . Hancock. 2000. A n alpha-helical cationic antimicrobial peptide selectively modulates macrophage responses to lipopolysaccharide and directly alters macrophage gene expression. J Immunol 165:3358. 23. Y a n , H . , and R. E . Hancock. 2001. Synergistic Interactions between Mammalian Antimicrobial Defense Peptides. Antimicrob Agents Chemother 45:1558. 24. Niyonsaba, F . , A . Someya, M . Hirata, H . Ogawa, and I. Nagaoka. 2001. Evaluation ofthe effects o f peptide antibiotics human beta-defensins-1/-2 and L L - 3 7 on histamine release and prostaglandin D(2) production from mast cells. Eur J Immunol 31:1066. 25. Verbanac, D . , M . Zanetti, and D . Romeo. 1993. Chemotactic and protease-inhibiting activities o f antibiotic peptide precursors. FEBS Lett 317:255. 26. W u , M . , and R. E . Hancock. 1999. Improved derivatives o f bactenecin, a cyclic dodecameric antimicrobial cationic peptide. Antimicrob Agents Chemother 43:1274. 21. Turner, J . , Y . C h o , N . N . Dinh , A . J. Waring, and R. I. Lehrer. 1998. Activities o f L L - 3 7 , a cathelin-associated antimicrobial peptide of human neutrophils. Antimicrob Agents Chemother 42:2206. 28. L a u , Y . E . , A . Rozek, M . G . Scott, D . L . Goosney, D . J . Davidson, and R. E . W . Hancock. 2005. Interaction and cellular localization of the human host defense peptide L L - 3 7 with lung epithelial cells. Infect Immun 73:583. 29. Bowdish, D . M . E . , Davidson, D . J . , Speert, D .P . , Hancock, R . E . W . 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. Journal of Immunology 172:3758 30. Selsted, M . E . , Y . Q . Tang, W . L . Morris , P. A . M c G u i r e , M . J . Novotny, W . Smith, A . H . Henschen, and J. S. Cullor. 1993. Purification, primary structures, and antibacterial activities o f beta-defensins, a new family of antimicrobial peptides from bovine neutrophils. J Biol Chem 268:6641. 31. Gennaro, R. , L . Dolzani , and D . Romeo. 1983. Potency o f bactericidal proteins purified from the large granules o f bovine neutrophils. Infect Immun 40:684. 32. Selsted, M . E . , M . J. Novotny, W . L . Morris , Y . Q . Tang, W . Smith, and J. S. Cullor. 1992. Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils. J Biol Chem 267:4292. 33. Singh, P. K . , H . P. Jia, K . Wiles, J. Hesselberth, L . L i u , B . A . Conway, E . P. Greenberg, E . V . Valore, M . J . Welsh, T . Ganz, B . F . Tack, and P. B . M c C r a y , Jr. 1998. Production o f beta-defensins by human airway epithelia. Proc Natl Acad Sci U SA 95:14961. 34. Hancock, R. E . W . , Devine, D . 2004. "Antimicrobial" or "host defence" peptides? In Mammalian host defence peptides. R. E . W . Hancock, Devine, D . , ed. Cambridge University Press, N e w York . 35. Scott, M . G , H . Y a n , and R. E . Hancock. 1999. Biological properties o f structurally related alpha-helical cationic antimicrobial peptides. Infect Immun 67:2005. 36. Hsu , Y . W . , K . H . C h i , W . C . Huang, and W . W . L i n . 2001. Ceramide inhibits lipopolysaccharide-mediated nitric oxide synthase and cyclooxygenase-2 induction in macrophages: effects on protein kinases and transcription factors. J Immunol 166:5388. 37 37. Ciornei , C . D . , A . Egesten, M . Engstrom, K . Tornebrandt, and M . Bodelsson. 2002. Bactericidal/permeability-increasing protein inhibits endotoxin-induced vascular nitric oxide synthesis. Acta Anaesthesiol Scand 46:1111. 38. Jiang, J . , P. Z h u , Z . Wang, Y . He, D . L i u , K . Tian, and Y . Diao. 1998. Protective effect o f bactericidal/permeability-increasing protein in mice with E . coli sepsis. Chin J Traumatol 1:21. 39. Lev in , M . , P. A . Quint, B . Goldstein, P. Barton, J. S. Bradley, S. D . Shemie, T . Y e h , S. S. K i m , D . P. Cafaro, P. J. Scannon, and B . P. Giroir . 2000. Recombinant bactericidal/permeability-increasing protein (rBPI21) as adjunctive treatment for children with severe meningococcal sepsis: a randomised trial. rBPI21 Meningococcal Sepsis Study Group. Lancet 356:961. 40. Krijgsveld, J . , S. A . Zaat, J. Meeldijk, P. A . van Veelen, G . Fang, B . Poolman, E . Brandt, J. E . Ehlert, A . J . Kuijpers, G . H . Engbers, J. Feijen, and J. Dankert. 2000. Thrombocidins, microbicidal proteins from human blood platelets, are C-terminal deletion products o f C X C chemokines. J Biol Chem 275:20374. 41. Resnati, M . , I. Pallavicini, J . M . Wang, J . Oppenheim, C . N . Serhan, M . Romano, and F . Blasi . 2002. The fibrinolytic receptor for urokinase activates the G protein-coupled chemotactic receptor F P R L 1 / L X A 4 R . Proc Natl Acad Sci USA 99:1359. 38 3.0 The Human Cationic Peptide LL-37 Induces Activation of the Extracellular Signal Regulated Kinase and p38 Kinase Pathways in Primary Human Monocytes* 3.0 T H E H U M A N CATIONIC PEPTIDE LL-37 INDUCES A C T I V A T I O N OF T H E E X T R A C E L L U L A R SIGNAL R E G U L A T E D K I N A S E A N D P38 K I N A S E P A T H W A Y S IN P R I M A R Y H U M A N M O N O C Y T E S 39 3.1 INTRODUCTION 40 3.2 MATERIALS & METHODS 41 3.3 RESULTS 45 3.4 DISCUSSION 53 3.5 B IBLIOGRAPHY 57 1 The majority o f this work was originally published in the Bowdish, D . M . E . , D . J . Davidson, D . P. Speert and R . E . W . Hancock. The human cationic peptide L L - 3 7 induces activation of the extracellular signal regulated kinase and p38 kinase pathways in primary human monocytes. Journal of Immunol. 2004 M a r 15;172(6):3758-65. A l l data was collected and analysed by D . Bowdish except Figure 3 .6A which was produced by D.J.Davidson. Figure 3.5B represents new data. The text has been updated to reflect advances in the literature. 39 3.1 Introduction Cationic host defense peptides are a primitive and conserved component of the innate immune response. These peptides can be expressed either constitutively or induced in response to pathogen associated molecular patterns, such as bacterial lipopolysaccharide (LPS) , or inflammatory mediators such as interleukin-6 and tumor necrosis factor alpha ( T N F - a ) (1, 2). While some o f these peptides have the ability to ki l l microorganisms (3, 4), leading to their categorization as antimicrobial peptides, they also appear to have functions in modulating immune responses (5-7). The role o f cationic peptides has been considered to be of primary significance in the innate immune response. However, it is becoming increasingly apparent that they provide a key link between the innate and adaptive responses (8, 9). The cathelicidins are an evolutionarily conserved group of peptides o f which h C A P 1 8 / L L - 3 7 is the only human member. In neutrophils, h C A P 1 8 is found in its unprocessed form, however upon release from cells the C terminal domain, which is called L L - 3 7 , is cleaved from the N terminal cathelin domain (10). L L - 3 7 is a potent modulator of the immune response and to date a wide variety o f interactions with the effector cells o f the immune response have been described (11). Monocytes, the precursors o f both macrophages and certain lineages of dendritic cells, have been shown to demonstrate chemotaxis towards L L - 3 7 (12), which is also able to stimulate the production of chemokines and chemokine receptors (13). A variety o f cationic peptides can also reduce pro-inflammatory cytokine production by macrophages to T o l l -like receptor agonists, such as L P S (14). Consistent with this function, L L - 3 7 can suppress endotoxin-mediated lethality in animals (15). Dendritic cells are also affected by L L - 3 7 treatment. Recent studies have shown that differentiation, function and surface expression of a variety of cell surface markers of human monocyte-derived dendritic cells are altered by exposure to L L - 3 7 , which generates cells with enhanced antigen uptake and presentation capacity (16). L L - 3 7 also has a variety of other functions including promotion of histamine release from mast cells, inhibition of tissue proteases, stimulation of wound healing, and angiogenesis (17-20). Despite burgeoning interest in the immunomodulatory properties o f cationic peptides, little is known about their mechanism of stimulation of the effector cells o f the innate immune response. A t the time this work was originally published there was only one proposed receptor for L L - 3 7 , F P R L - 1 , a pertussis-toxin sensitive, G-protein coupled receptor that has been proposed to be the chemotactic receptor on monocytes, neutrophils and subsets o f T cells (12). The recently-demonstrated ability o f L L - 3 7 to induce mast cell chemotaxis is also pertussis toxin 40 sensitive and thus mediated by a G-protein coupled receptor, however the F P R L - 1 receptor was not found to be involved (21). These studies indicated that there may be two types of receptors for L L - 3 7 on mast cells, a high affinity receptor that is responsible for L L - 3 7 induced chemotaxis and a low affinity receptor with an undefined function (21). The downstream signalling and consequences of L L - 3 7 binding to these receptors has not been characterized. Since this work was published it has been proposed that the epidermal growth factor receptor ( E G F R ) is the receptor linked to mitogen activated protein kinases ( M A P K ) activation and IL-8 production in epithelial cells and that P2X7, an purinergic receptor is linked to L L - 3 7 induced I L -i p processing in L P S primed monocytes (22, 23). Although there is ample evidence that cationic host defence peptides interact directly with the effector cells o f the innate and adaptive immune response, very little is known about eukaryotic cell signalling induced by peptide interaction. In this paper we demonstrate that L L -37 can signal via the induction o f phosphorylation of the M A P K , E R K 1 / 2 and p38 in peripheral blood derived monocytes and in a human bronchial epithelial cell line. W e demonstrate that L L -37 promotes the activation o f E R K 1 / 2 and p38, at concentrations equivalent to those likely to be found at the site o f acute inflammation (25-50 pg/ml). In addition, in the presence o f the cytokine G M - C S F , activation o f these kinases occurs at concentrations of L L - 3 7 as low as 5-10 pg/ml, which would be predicted to be present at the onset o f inflammation. Thus we hypothesize that low levels o f peptide are homeostatic and do not possess a major immunomodulatory effect unless they are expressed in the presence of other agonists such as cytokines. Lastly, we demonstrate that at least two of the immunomodulatory properties o f L L - 3 7 , increases in secretion of IL-8 and the transcription of the chemokines, M C P - 1 , M C P - 3 and IL-8, are dependent upon the activation o f the p38 and E R K 1 / 2 kinases. 3.2 Mater ia l s & M e t h o d s Cell purification and culture Blood monocytes were prepared using standard techniques (24). Briefly, 100 ml of fresh human venous blood was collected in sodium heparin-containing Vacutainer collection tubes (Becton Dickinson, Mississauga, O N , Canada) from volunteers according to U B C Clinical Research Ethics Board protocol C02-0091. The blood was mixed, at a 1:1 ratio, with RIO media ( R P M I 1640 medium supplemented with 10% v/v fetal calf serum (FBS) , 1% L-glutamine, 1 n M sodium pyruvate) in an E-toxa-clean (Sigma-Aldrich, Oakvil le , O N , Canada) washed, endotoxin-free bottle. P B M C were separated using Ficoll-Paque Plus (Amersham Pharmacia Biotech, Baie D 'Urfe , P Q , Canada) at room temperature and washed with phosphate buffered saline (PBS). 41 Monocytes were enriched by removal o f T-cells by resetting with fresh sheep red blood cells ( U B C animal care unit) pre-treated with Vibrio cholerae neuraminidase (Calbiochem Biosciences Inc., L a Jolla, C A , U S A ) and repeat separation by F ico l l Paque Plus (25). The enriched monocytes were washed with P B S , then cultured (approximately 2-3 x 106 per well) in RIO media for 1 hour at 3 7 ° C followed by the removal o f non-adherent cells; monocytes were >95% pure as determined by flow cytometry (data not shown). B lymphocytes were isolated by removing non-adherent cells and adding them to a new plate for one hour at 3 7 ° C . This was repeated a total o f three times. A n y remaining monocytes adhered to the plates, and residual non-adherent cells were primarily B cells. Cells were cultured in Falcon tissue culture 6-well plates (Becton Dickinson, Mississauga, O N , Canada). The adherent monocytes were cultured in 1 ml RIO media at 3 7 ° C in which L L - 3 7 and/or cytokines dissolved in endotoxin-free water (Sigma-Aldr ich , Oakville, O N , Canada) were added. Endotoxin-free water was added as a vehicle control. For studies using pertussis toxin the media was replaced with 1 ml of RIO media containing 100 ng/ml o f toxin and incubated for 60 min at 3 7 ° C . L L - 3 7 and cytokines were added directly to the media containing pertussis toxin. For the isolation of T lymphocytes, the rosetted T cells and sheep red blood cells were resuspended in 20 ml P B S and 10 ml of distilled water was added to lyse the latter. The cells were then centrifuged at 1000 rpm for 5 min after which the supernatant was removed. The pelleted T cells were promptly washed in P B S and increasing amounts o f water were added until all sheep red blood cells had lysed. The remaining T cells were washed once in P B S , and viability was confirmed using a 0.4% Trypan blue solution. Primary human blood monocytes and T cells were cultured in R P M I 1640 supplemented with 10%> v/v heat-inactivated F B S , 1% v/v L - glutamine, 1 n M sodium pyruvate ( G I B C O Invitrogen Corporation, Burlington, O N , Canada). For each experiment between two and eight donors were used. The simian virus 40-transformed, immortalized 16HBE14o- bronchial epithelial cell line was a generous gift o f Dr . D . Gruenert (University o f California, San Francisco, C A ) (26). Cells were routinely cultured to confluence in 100% humidity and 5%> C 0 2 at 3 7 ° C . They were grown in Min imal Essential media with Earles' salts ( G I B C O Invitrogen Corporation, Burlington, O N , Canada) containing 10%> F B S (Hyclone), 2 m M L-glutamine. For experiments, cells were seeded at 1x10 s cells per ml o f media in 24 well plates and cultured at 3 7 ° C and 5%> CO2 for two days. The cells were used between passages 8 and 20. 42 Media and reagents L L - 3 7 (sequence L L G D F F R K S K E K I G K E F P C R I V Q R I K D F L R N L V P R T E S ) and CP29 (sequence K W K S F I K K L T T A V K K V L T T G L P A L I S ) , were synthesized by Fmoc [(N-(9-fluorenyl) methoxycarbonyl)] chemistry at the Nucleic Acid/Protein Synthesis ( N A P S ) Unit at U B C . Human recombinant granulocyte-macrophage colony-stimulating factor ( G M - C S F ) , interleukin-4 (IL-4) and macrophage colony-stimulating factor ( M - C S F ) were purchased from Research Diagnostics Inc. (Flanders, N J , U S A ) . Pertussis toxin was supplied by List Biological Laboratories Inc. (Campbell , C A , U S A ) . S B 203580, a specific inhibitor o f p38 kinase was purchased from Sigma-Aldrich (Oakville, O N , Canada) and PD98059, a specific inhibitor of E R K 1 / 2 kinase was purchased from Cel l Signaling Technology (Beverly, M A ) . The peptide W K Y M V M was a kind gift from Dr. Claes Dahlgren, Phagocyte Research Laboratory, Dept. o f Rheumatology & Inflammation Research, University o f Goteborg, Sweden. Analysis of cytotoxicity and cell viability Viabil i ty after cell isolation was determined by resuspending cells in a 0.4% Trypan blue solution (Gibco). Peptide cytotoxicity was assessed by collecting culture supernatants after stimulation with L L - 3 7 or vehicle control. The concentration o f L D H - 1 in the supernatants was quantified using a Cytotoxicity Detection kit (Roche Diagnostics, Laval , P Q , Canada) according to the manufacturer's instructions. Analysis of endotoxin contaimination. The media, peptide and chemokine stocks, and vehicle controls were tested for endotoxin contamination using a L A L Chromagenic Endpoint Assay (HyCul t Biotechnology). Endotoxin levels were less than 0.3 E U / m l . Immunob lotting After stimulation, cells were washed with ice cold P B S containing 1 m M vanadate (Sigma). Next 125 p l o f R I P A buffer (50 m M T r i s - H C l , p H 7.4, 1% NP-40 , 0.25% sodium deoxycholate, 150 m M NaCI , 1 m M E D T A , 1 m M P M S F , 1 pg/ml each of aprotinin, leupeptin, pepstatin, 1 m M sodium orthovanadate, 1 m M NaF) was added and the cells were incubated on ice until they were completely lysed as assessed by visual inspection. The lysates were quantitated using a B C A assay (Pierce). 30 pg o f lysate was loaded onto 1.5 m m thick gels, which were run at 100 volts for approximately 2 hours. Proteins were transferred to nitrocellulose filters for 75 min at 70 V . The filters were blocked for 2 hours at room temperature with 5% skim milk in T B S T (10 m M T r i s - H C l p H 8, 150 m M N a C I , 0.1% Tween-20). The 43 filters were then incubated overnight at 4 ° C with the a n t i - E R K l / 2 - P or anti-p38-P (Cell Signalling Technology, M a ) polyclonal antibodies. Immunoreactive bands were detected using horseradish peroxidase-conjugated sheep anti-mouse IgG antibodies (Amersham Pharmacia, N e w Jersey) and chemiluminescence detection (Sigma, M o ) . T o quantify bands, the films were scanned and then quantified by densitometry using the software program, ImageJ. The blots were reprobed with a P-actin antibody ( I C N Biomedical Incorporated, Ohio) and densitometry was performed to allow correction for protein loading. Kinase Assay A n E R K 1 / 2 activity assay was performed using a non-radioactive kit (Cell Signalling Technology). Briefly, cells were treated for 15 min and lysed in lysis buffer. Equal amounts o f proteins were immunoprecipitated with an immobilized p h o s p h o - E R K l / 2 antibody that reacts only with the phosphorylated (i.e. active) form of E R K 1 / 2 . The immobilized precipitated enzymes were then used for the kinase assay using Elk-1 followed by immunoblot analysis with antibodies that allow detection and quantitation of phosphorylated substrates. Quantification of IL-8 Human IL-8 from supernatants o f 16HBE140- cells and blood-derived monocytes was measured by using the commercially available enzyme-linked immunosorbent assay kit (Biosource, Montreal , Q C ) according to the manufacturer's instructions. Semiquantitative RT-PCR Total R N A was isolated from donor blood derived monocytes using an RNaqueous Micro-kit (Ambion) as described by the manufacturer. The samples were DNase treated, and then c D N A synthesis was accomplished by using a first-strand c D N A synthesis kit (Gibco). The resultant c D N A s were used as a template in P C R s for various cytokine genes the primer sequences for which are listed in Table 3.1. Each R T - P C R reaction was performed in at least duplicate. Results were analyzed in the linear phase o f amplification and normalized to the housekeeping control, glyceraldehyde-3-phosphate dehydrogenase. The absence o f contaiminating genomic D N A was verified by including controls generated without reverse transcriptase. 44 Table 3.1. Primer sequences of cytokine genes. Primer Forward sequence (5'-3') Reverse Sequence (5'-3') M C P - 1 T C A T A G C A G C C A C C T T C A T T C T A G C G C A G A T T C T T G G G T T G IL-8 G T G C A G A G G G T T G T G G A G A A G T T C T C C C G T G C A A T A T C T A G G M C P - 3 T G T C C T T T C T C A G A G T G G T T C T T G C T T C C A T A G G G A C A T C A T A 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 IL-P G G A T A T G G A G C A A C A A G T G G A T G T A C C A G T T G G G G A A C T 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 3.3 Results LL-3 7 induces ERK1/2 andp38 phosphorylation in peripheral blood derived monocytes. T o determine i f L L - 3 7 induced the activation of the M A P kinases, E R K 1 / 2 and/or p38, peripheral blood derived monocytes were treated with 50 pg/ml L L - 3 7 or water (as a vehicle control) for 15 min. T o visualize the activated (phosphorylated) form of the kinases, immunoblots were performed with antibodies specific for the dually phosphorylated form of the kinases (phosphorylation on Thr202+Tyr204 and T h r l 8 0 + T y r l 8 2 for E R K 1 / 2 and p38 respectively). The gels were re-probed with an antibody for P-actin to normalize for loading differences. In all cases, an increase in phosphorylation o f E R K 1 / 2 (n=8) and p38 (n=4) was observed in response to L L - 3 7 treatment (Figure 3.1). 45 F i g u r e 3.1. E x p o s u r e to L L - 3 7 induces phosphorylat ion of E R K 1 / 2 a n d p38. Human peripheral blood derived monocytes were exposed to 50 ug/ml o f L L - 3 7 (+), or endotoxin free water (-) as a vehicle control, for 15 minutes. Antibodies specific for the phosphorylated forms of E R K 1 / 2 and p38 were used to detect activation o f E R K 1 / 2 and p38 in cell lysates. B-actin was quantified to allow correction for protein loading. A l l donors tested showed increased phosphorylation of E R K 1 / 2 and p38 in response to L L - 3 7 treatment. A ) One representative donor is shown. B) The phosphorylation o f E R K 1 / 2 (n=9 donors) and p38 (n=4 donors) was quantified by densitometry. A student's one-tailed t-test was performed. Double asterisk indicates p < 0.01, asterisk indicates p<0.05. M e a n values ± S E M are shown. A serum component is required for LL-37-induced activation It has been proposed that L L - 3 7 binds to at least one serum protein, and that this binding modifies its activities (27). Thus, we tested the ability o f L L - 3 7 to activate E R K 1 / 2 and p38 in the presence and absence o f serum. N o cytotoxicity was observed in response to a 15 minute L L -37 exposure in the presence or absence of serum, as assessed by trypan blue exclusion or the L D H - 1 assay (data not shown). In the absence of serum, no detectable activation of E R K 1 / 2 or p38 was observed using antibodies specific for the phosphorylated forms o f the kinases, in contrast to the activation observed in the presence of F C S (n=3, Figure 3.2). In addition, a functional assay for E R K 1 / 2 activation was performed. In this assay, activation of an E R K 1 / 2 controlled transcription factor, E lk -1 , was detected in media containing serum but not in serum free media (n=2, Figure 3.2). This indicates that L L - 3 7 requires the addition of at least one serum component to induce activation o f the M A P kinases, E R K 1 / 2 and p38. 46 Figure 3.2. L L - 3 7 is unable to induce M A P K activation in h u m a n monocytes under serum free conditions. Cells were exposed to 50 pg/ml of L L -37 (+), or endotoxin free water (-) as a vehicle control, for 15 minutes. A ) After exposure to L L - 3 7 in media containing 10% F C S , phosphorylated E R K 1 / 2 was detectable, however, no phosphorylation o f E R K 1 / 2 was detected in the absence o f serum (n=3). B ) E l k - 1 , a transcription factor downstream o f E R K 1 / 2 , was activated (phosphorylated) upon exposure to 50 pg/ml of L L -37 in media containing 10% F C S , but not in the absence of serum (n=2). LL-3 7 activation of MAPK pathways is specific for monocytes and epithelial cells. T o assess whether L L - 3 7 induced activation o f the E R K 1 / 2 and p38 kinases was specific to effector cells o f the innate immune response, phosphorylation of p38 and E R K 1 / 2 was studied in freshly isolated primary monocytes, B cells, T cells and the human epithelial cell line 16HBE14o- (Figure 3.3). N o cytotoxicity was detected in any cell type under the assay conditions, as assessed by trypan blue exclusion or the L D H - 1 assay (data not shown). Immunoblot analysis demonstrated that both the epithelial cell line (n=2 experiments) and primary monocytes (n=8 donors) showed substantial increases in E R K 1 / 2 and p38 phosphorylation upon exposure to L L - 3 7 for 15 min (as assessed after normalization for loading differences, by re-probing with an antibody for P-actin). However, no phosphorylation of either E R K 1 / 2 or p38 kinases was evident in human T cells or B cells (n=4 donors and n=2 donors respectively). This cellular specificity indicates that activation o f the M A P kinases is specific to certain cell types and therefore may relate to similar receptors in these types o f cells or to similarities in membrane organization. 47 PBDM T cells B cells HBE LL-37 + - + Figure 3.3. Act ivat ion of E R K 1 / 2 and p38 phosphorylat ion by L L - 3 7 is induced in cells of the innate i m m u n e system. Freshly isolated human peripheral blood derived monocytes ( P B D M ) , T cells, and B cells, and the human bronchial epithelial cell line ( H B E ) were stimulated for 15 minutes with 50 | lg /ml of L L - 3 7 (+), or endotoxin free water as a vehicle control (-), in media containing 10% F C S . Antibodies specific for the phosphorylated forms of E R K 1 / 2 and p38 were used to detect their activation in cell lysates. Activation o f E R K 1 / 2 and p38 was evident in monocytes and the bronchial epithelial cell line, but not T or B cells. For monocytes, T cells, and B cells, one representative donor is shown from n=8, 4 and 2 respectively. For the 16HBE14o- cell line, one representative experiment o f two is shown, fi-actin was quantified to allow correction for protein loading. G-coupled proteins are not involved in LL-3 7 induced activation of MAPK. F P R L - 1 , a pertussis toxin sensitive G-coupled protein has been proposed to be a receptor for L L - 3 7 on monocytes, certain subsets o f T cells, and neutrophils (12). T o test whether F P R L - 1 or other G-protein coupled receptors were involved in LL-37- induced signalling, sensitivity to inhibition with pertussis toxin was examined. N o significant reduction o f E R K 1 / 2 phosphorylation was observed as a result of incubation with pertussis toxin. E R K 1 / 2 phosphorylation upon stimulation with 50 ug/ml L L - 3 7 was increased an average o f 8.3 fold in the absence (n=9) and 7.6 fold in the presence (n=3) o f pertussis toxin. (Figure 3.4). A synthetic peptide activator/agonist ( W K Y M V M ) of F P R L - 1 and F P R L - 2 was used as a positive control. The F P R L - 1 agonist induced massive E R K 1 / 2 phosphorylation, which was partially inhibited by the incubation with pertussis toxin (approximately 6 and 9 fold inhibition as determined by densitometry was seen in two donors; Figure 3.4), indicating that the incubation with pertussis toxin under the conditions studied did indeed inhibit G-protein coupled receptor signalling. Since L L - 3 7 induced signalling was not affected by pertussis toxin treatment these data suggest that activation o f E R K 1 / 2 was not linked to a pertussis toxin sensitive receptor, and in particular was not linked to the F P R L - 1 receptor. 48 Figure 3.4. L L - 3 7 induced phosphorylat ion of ERK1/2 a n d p38 kinases is not mediated by G-prote in coupled receptors. Antibodies were used to detect the phosphorylated forms of E R K 1 / 2 and p38 in cell lysates from human peripheral blood derived monocytes. 13-actin was quantified to allow correction for protein loading. Cells were exposed to 50 pg /ml o f L L - 3 7 (+), or endotoxin free water (-) as a vehicle control, for 15 minutes in media containing 10% F C S . Phosphorylation induced by L L - 3 7 was not inhibited by lOOng/ml pertussis toxin (+) added thirty minutes before exposure to L L - 3 7 and present during L L - 3 7 induced stimulation. A s a positive control cells were exposed to 100 ng/ml of W K Y M V M , a synthetic agonist o f F P R L - 1 (+). Phosphorylation o f E R K induced by F P R L - 1 was reduced approximately nine-fold by the addition of pertussis toxin (+). One representative experiment o f two shown. LL-37 induced activation ofERKl/2 andp38 is dose dependent and demonstrates synergy with GM-CSF. The precursor h C A P 1 8 is found at low levels in blood and serum while L L - 3 7 , the processed form o f the peptide, is found at much higher concentrations in the context o f infection (28-30). W e hypothesized that L L - 3 7 induced activation o f the M A P kinases may be more pronounced in the presence o f cytokines which are up-regulated during the course of infection. T o test this hypothesis we added G M - C S F , IL-4 or M - C S F (each at lOOng/ml) concurrently with L L - 3 7 and measured phosphorylation of E R K 1/2 in freshly isolated human blood monocytes (Figure 3.5A). E R K 1 / 2 phosphorylation was evident when cells were treated with 50 pg/ml of L L - 3 7 (8.3 fold increase over untreated, n=9) but not at lower concentrations (n=2). In the presence of 100 ng/ml G M - C S F , LL-37- induced E R K 1/2 phosphorylation increased markedly (58 fold greater than untreated, n=5). This synergistic activation did not occur in the presence o f 100 ng/ml M - C S F or IL-4. Furthermore, in the presence o f G M - C S F , activation o f E R K 1 / 2 occurred in response to concentrations of 5 and 10 pg/ml o f L L - 3 7 , respectively, in the two donors tested (Figure 3.5B). This demonstrates that L L - 3 7 induced activation of E R K 1 / 2 occurred at a lower threshold in the presence of G M - C S F , a cytokine found locally at sites of infection. The presence o f G M - C S F also enhanced L L - 3 7 induced phosphorylation of p38 (data not shown). T o test whether increased phosphorylation of the M A P K in the presence of G M - C S F 49 had a biologically significant outcome, IL-8 production was measured. In the presence of GM-CSF, IL-8 production was increased in 3 of 4 donors (Figure 3.5C). A 45000 .1 40000 | 35000 g 30000 £ 25000 j£ 20000 LU o 15000 2> £ 10000 s - 5000 0 * • 0 fig/mt I I SO nn/ml 1 1 Ju LIU/Mil : • IL-4 B LL-37 (ng/ml) 0 FCS M-CSF No Cytokines GM-CSF 10 25 50 FCS + GM-CSF 6000 MMB flfefll <MM • 0 ng/ml GMCSF • 10 ng/ml GM-CSF • 25 ng/ml GM-CSF • 50 ng/ml GM-CSF • 100 ng/ml GM-CSF 0 25 Concentration of LL-37 (ug/ml) Figure 3.5. ERK1/2 activation is amplified and occurs at lower concentrations of LL-37 when in the presence of GM-CSF. A) Freshly isolated monocytes were stimulated with LL-37 (50 pg/ml), or endotoxin free water (0 pg/ml LL-37) as a vehicle control, in media containing 10% FCS and either no cytokines, GM-CSF (100 ng/ml), M-CSF (100 ng/ml) or IL-4 (100 ng/ml). A minimum of three donors was used for each experiment. LL-37 induced phosphorylation of ERK 1/2 was significantly enhanced in the presence of GM-CSF, but not IL-4 or M-CSF. Mean ± SE. Asterisk indicates p=0.004. B) The concentration of LL-37 required to induce phosphorylation of ERK 1/2 was decreased from 50 pg/ml to 5 pg/ml. One representative donor of two is shown. C) LL-37 and GM-CSF synergy results in increased IL-8 production. LL-37 induced IL-8 production is increased in the presence of GM-CSF in three of four donors tested. One representative donor is shown. Mean of three wells ± SE, p<0.002. LL-37 induced MAPK activation is not related to cytotoxicity in human blood derived monocytes. Some cationic peptides induce lysis of eukaryotic cells. To test whether LL-37 was cytotoxic in our model system over longer exposure periods, human blood derived monocytes from two donors were incubated with LL-37 (10-50 pg/ml), the cytotoxic peptide CP29 (50 pg/ml) or a vehicle control for 0.5 to 4 hours (Figure 3.6A). The concentration of LDH in the supernatants was quantified as a measure of cytotoxicity. The cytotoxic peptide, CP29, induced 50 significant cell lysis beginning at 0.5 hours. In contrast, cells exposed to up to 50 p.g/ml L L - 3 7 demonstrated no more lysis than the controls (Figure 3.6A). These data demonstrate that M A P K phosphorylation induced by 15 minute exposure to L L - 3 7 in human blood derived monocytes does not correlate with later cytotoxicity at the concentrations used in this study. Figure 3.6. LL -37 induces IL-8 production in human blood derived monocytes in the absence of cytotoxicity. A ) H u m a n peripheral blood derived monocytes from two separate donors were exposed to concentrations o f between 1 0 - 5 0 ug/ml o f L L - 3 7 or endotoxin free water as a vehicle control, for up to four hours. The concentration of L D H - 1 in the supernatants induces a colourmetric change which provides a measure o f cytotoxicity. The average release of L D H by the two donors is shown. Error bars represent the range of results for the two donors. B ) After assaying for cytotoxicity the supernatants collected at 4 hrs were assayed for IL-8 by E L I S A . The average of 2 donors is shown. Error bars represent the range o f results for the two donors. LL-3 7 exposure induces IL-8 secretion in human blood-derived monocytes W e have recently demonstrated that L L - 3 7 induced the secretion o f chemokines in the human A549 epithelial cell line (IL-8), and whole human blood ( M C P - 1 and IL-8) (13). In addition to testing for L D H levels, the supernatants from human blood derived monocytes treated with 10-200 ug/ml, the cytotoxic peptide CP29 (50 ug/ml) or a vehicle control for up to four hours were assayed for IL-8 secretion by E L I S A . In both donors tested, the addition of L L - 3 7 led to an increase in IL-8 secretion; however, the cytotoxic peptide C P 2 9 did not induce IL-8 secretion (Figure 3.6B). These data confirm that the immunomodulatory properties o f L L - 3 7 and cytotoxicity are independent. 51 Activation of ERK1/2 and p38 is necessary for transcription of Elk-1 controlled genes and secretion of IL-8. IL-8 release is known to be governed, at least in part, by activation o f the E R K 1 / 2 and p38 kinases (31). In order to determine i f activation of the M A P kinases was required for IL-8 release, cells were incubated with a p38 kinase-specific inhibitor, S B 203580, and/or an E R K 1 / 2 -specific inhibitor, PD98059, for one hour prior to a four hour incubation with 50 pg/ml L L - 3 7 . IL-8 in the culture media was assayed by E L I S A . In both donors tested, L L - 3 7 induced IL-8 secretion was reduced by the presence of either 10 u M E R K 1 / 2 or p38 kinase inhibitors and was abrogated in the presence o f the combination of both inhibitors (Figure 3.7A). When the human bronchial epithelial cells were stimulated with 50 ug/ml of L L - 3 7 for four hours, a significant increase in the amount of IL-8 released into the media was detected (Figure 3.7B). This L L - 3 7 induced secretion of IL-8 was reduced in the presence of either the p38 inhibitor or the E R K 1 / 2 inhibitor. In the presence of both inhibitors in combination IL-8 secretion was not significantly different from the baseline level o f IL-8 secretion (Figure 3.7B). These data indicate that both p38 and E R K 1 / 2 kinases are involved in L L - 3 7 mediated IL-8 secretion in blood derived monocytes and epithelial cells. Activation of ERK1/2 and p38 is necessary for LL-37 induced transcription of certain chemokines. T o determine the downstream transcriptional effects o f LL-37- induced M A P kinase activation, the expression o f genes known to be regulated by E R K 1 / 2 or p38 was assessed. Semi-quantitative R T - P C R was performed on R N A collected from monocytes isolated from two donors and pre-treated with the E R K 1 / 2 and p38 inhibitors or vehicle control prior to a four hour exposure with 50 ug/ml o f L L - 3 7 (n=2). M C P - 1 and IL-8 have been demonstrated to be under the transcriptional control o f both E R K 1 / 2 and p38 (32, 33). Consistent with these observations the expression o f these genes was up-regulated following exposure to L L - 3 7 and this up-regulation was abolished in cells pretreated with either E R K 1 / 2 or p38 inhibitors (Figure 3.7C). Transcription o f the gene encoding M C P - 3 was also increased in the presence of L L - 3 7 and was dependent on the activation o f both p38 and E R K 1 / 2 kinase. Conversely, there were no increases in the transcription of the genes encoding the pro-inflammatory cytokines T N F - a , I L -1(3 or IL-6 in either donor (data not shown). These data are consistent with the hypothesis that the activation of the E R K 1 / 2 and p38 signalling pathways has functional effects on transcription of cytokine genes with immunomodulatory functions. 52 J » • Donor 1 1 • Donor 2 1 1 ^ 1 LL-37 +10 PD LL-37 LL-37+ +10 SB 10 PD&10 SB MCP-3 .V \V MCP-1 3.4 Discussion Figure 3.7. LL-37 induces IL-8 secretion and chemokine transcription in a p38 and ERK1/2 kinase dependent manner. A ) Human peripheral blood derived monocytes from 2 donors were incubated with 10 u M p38 kinase specific inhibitor SB 203580 (10 SB) , and/or 10 u M E R K 1 / 2 specific inhibitor PD98059 (10 P D ) for one hour, after which the cells were exposed for four hours to 50 ug/ml L L -37 or endotoxin free water as a vehicle control. IL-8 was assayed in the culture media by E L I S A . B ) Approximately l x l 0 5 cells o f the human bronchial epithelial cell line, 16HBE14o- , were seeded per well of a 24 well plate and grown for two days. The cells were then incubated with 50 u M p38 kinase specific inhibitor S B 203580 (50 SB) , and/or 50 u M E R K 1 / 2 specific inhibitor PD98059 (50 P D ) for one hour, after which the cells were exposed for four hours to 50 ug/ml o f L L - 3 7 or endotoxin free water as a vehicle control. IL-8 was assayed in the culture media by E L I S A . M e a n ± S E M o f three independent experiments shown. The asterisk indicates significantly difference at p<0.05. C ) Human peripheral blood derived monocytes were incubated with kinase inhibitors and L L - 3 7 as above. After four hours R N A was collected and R T - P C R was performed. The m R N A expression of the chemokine genes was corrected for G A P D H expression. M C P - 1 , M C P - 3 and IL-8 were up-regulated by L L - 3 7 treatment and this up-regulation was abrogated by treatment with inhibitors o f either E R K 1 / 2 and p38. Representative data for one o f two donors tested is shown. There is a paucity o f information on how antimicrobial peptides exert their effects on eukaryotic cells. Despite the fact that there have been hundreds, i f not thousands of antimicrobial peptides characterized to date, many of which have immuno-modulatory properties, little or nothing is known about how they initiate signalling within eukaryotic cells. In this paper we demonstrate for the first time that the human cathelicidin, L L - 3 7 , induces activation of both the p38 and E R K 1 / 2 kinases in monocytes and epithelial cells. Activation o f these kinases has been demonstrated to have pleiotropic influences on the effector cells o f the immune response including cytokine production, cellular activation, proliferation and differentiation (34). Since L L - 3 7 has been demonstrated to modulate all o f these processes, it is tempting to speculate that 53 its immunomodulatory abilities may be regulated in part by its ability to signal through these pathways. In a study by Y a n g et al. L L - 3 7 was demonstrated to be a chemoattractant for monocytes, neutrophils and T cells (12). This activity o f L L - 3 7 was proposed to be receptor-mediated, functioning via the G-protein coupled receptor F P R L - 1 and inhibited by an peptide agonist o f F P R L - 1 (12). Due to the insensitivity o f L L - 3 7 induced M A P K activation to both pertussis toxin (Figure 3.4) and cholera toxin (data not shown), we believe that activation of E R K 1 / 2 and p38 by L L - 3 7 is not linked to a G-protein coupled receptor, such as F P R L - 1 . Indeed, although F P R L - 1 is expressed on both monocytes and T cells, L L - 3 7 induced activation of E R K 1 / 2 and p38 was observed in monocytes and epithelial cells, but not on T or B cells, indicating that this receptor is not the mechanism by which the M A P kinases are activated. Niyonsaba et al. demonstrated that F P R L - 1 was not the receptor on mast cells, which undergo both chemotaxis and degranulation in response to L L - 3 7 . In fact, the authors suggest that L L - 3 7 has two receptors in this cell type, one high affinity pertussis toxin sensitive receptor which is linked to L L - 3 7 induced chemotaxis and one low affinity receptor with as o f yet undefined function (21). Our study indicates that L L - 3 7 induces activation o f p38 and E R K 1/2 kinases, directly or indirectly, through a pertussis toxin insensitive mechanism. L L - 3 7 induced signalling was not observed in the absence o f serum. The properties o f L L - 3 7 have been demonstrated to be altered in the presence o f serum (35). In fact, in the absence o f serum, moderate concentrations (30-100 pg/ml) o f L L - 3 7 and related peptides were cytotoxic towards both prokaryotic and eukaryotic cells (27, 36, 37). However in the presence of serum, cytotoxicity towards both cell types was substantially reduced (36, 38). In the current study it was demonstrated that in media containing serum, L L - 3 7 does not induce substantial cytotoxicity in human blood derived monocytes. In other studies we have shown that in the absence of serum, 50 pg/ml of L L - 3 7 does result in increased cell lysis in as little as two hours (39). L L - 3 7 induced activation of the M A P K is not a result o f cytotoxicity as it does not occur in the absence o f serum. When L L - 3 7 is found in the blood or tissues during the course of infection it may exists coupled to one or several proteins. The presence o f apolipoprotein 1 has been proposed to reduce L L - 3 7 induced cytotoxicity while also inhibiting its antimicrobial functions (27). It wil l be of interest to determine i f the presence of apolipoprotein can restore the ability o f L L - 3 7 to induce signalling in serum free medium. However, it is not unreasonable to assume that there are multiple components in serum that may interact with the positively charged L L - 3 7 . 54 These data illustrate the importance of studying the immunomodulatory effects o f L L - 3 7 , and perhaps other cationic peptides, in the presence of serum. L L - 3 7 induces chemokine secretion in airway epithelial cell lines, human whole blood and mouse models (13, 40, 41). In this study we demonstrated that the activation of E R K 1 / 2 and p38 kinases is required for L L - 3 7 induced transcription of the chemokines IL-8 , M C P - 1 and M C P - 3 in human blood derived monocytes. W e also demonstrated that this transcription is specific for chemokine genes, and not pro-inflammatory cytokines such as TNF-c t , IL-1 (3 and I L -6. Transcription of IL-8 and other chemokine genes was completely abrogated in the presence of inhibitors o f either the p38 or E R K 1 / 2 kinases. In addition, secretion o f IL-8 in both the epithelial cell line and the monocytes was only partially inhibited by the presence of either inhibitor and only completely inhibited in the presence of both inhibitors, indicating that the kinases may effect chemokine production at both the transcriptional and post-transcriptional levels. Monocytes circulate in the blood and enter the tissues via the process o f extravasation upon detection o f chemokines such as IL-8 or potentially L L - 3 7 . During this migration from the bloodstream to the tissues the processed of differentiation and activation begin (42). Upon arrival at sites of high concentrations of L L - 3 7 we believe that activation o f the M A P K would occur and that this activation would have important physiological outcomes. Interestingly, L L - 3 7 induced activation o f E R K 1/2 and p38 signalling is enhanced in the presence of G M - C S F , but not M - C S F or IL-4. Although initial characterization of G M - C S F focused on its ability to generate granulocytes and macrophage colonies from common precursor cells, it has more recently been proposed to have many roles in the inflammatory response. These include generating increased numbers of circulating neutrophils and peritoneal macrophages (43), promotion of peripheral blood monocyte survival (30), and potentiation of LPS- induced cytokine release and subsequent lethality in vivo (44). G M - C S F is not generally found in the serum o f healthy adults (45), however it is present in the serum after stimulation with L P S (46) and is produced by a variety o f tissues, including upper airway epithelial cells, fibroblasts, endothelial cells and monocytes themselves (47). This pattern of expression mimics that o f L L - 3 7 during the course o f infection or stimulation with inflammatory mediators (29, 48-50). Hamilton et al. propose that hematopoietic cells respond to infection by stimulating surrounding tissues to produce G M - C S F . This localised increase in the concentration of G M - C S F encourages survival and differentiation and alters the activation state of local monocytes and polymorphonuclear neutrophils, thus promoting clearance o f infectious organisms (51). The production and action o f G M - C S F occurs 55 locally at the site o f inflammation indicating that this activation and proliferation of hematopoietic cells would be a localized, rather than systemic, orchestration o f the immune response. L L - 3 7 alone activates cell signalling pathways only at high concentrations, which in vivo would be found only at sites o f acute inflammation. The presence o f G M - C S F lowers the threshold for L L - 3 7 induced activation of the M A P kinases to between 5-10 ug/ml, concentrations that would be found in vivo at the onset o f infection and which are modestly higher than the normal concentration o f circulating h C A P - 1 8 (28, 29, 52). This increase in phosphorylation results in an increase in IL-8 production. W e hypothesize that low concentrations o f L L - 3 7 (< 5 ug/ml) are homeostatic and do not activate the effector cells o f the innate immune response unless they exist in the presence of pro-inflammatory signals such as the presence of pro-inflammatory cytokines. The presence of both L L - 3 7 and G M - C S F may thus lead to novel functional phenotypes of effector cells o f the innate immune response. Although cationic peptides often have antimicrobial activity, the key to their therapeutic potential may lie amongst the myriad of other activities attributed to them, such as their ability to alter the inflammatory response (53). For example, under conditions analogous to those found in vivo, L L - 3 7 is a weak antimicrobial agent [Chapter 1, Figure 1.2]. Conversely, it is one of the most potent anti-endotoxic agents among cationic host defense peptides, is directly chemotactic, and induces dramatic changes in the phenotype of monocyte derived dendritic cells, indicating a substantial role in the innate immune response (13). Thus it is becoming increasingly clear that peptides are more than simply "nature's antibiotics" but rather play a complex role in resolving infection, attenuating inflammation and, when this attempt at resolution is not sufficient, in alerting the adaptive immune response. Characterization o f the mechanism o f action of these peptides is shedding light on the properties that make them potential therapeutic agents. W e propose that LL-37- induced activation of the E R K 1 / 2 and p38 kinases is a pivotal function of this peptide, acting via an as yet undetermined G-protein coupled receptor-independent, but serum-dependent mechanism. 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Effects o f human cathelicidin antimicrobial peptide L L - 3 7 on lipopolysaccharide-induced nitric oxide release from rat aorta in vitro. Acta Anaesthesiol Scand 47:213. 38. Wang, Y . , B . Agerberth, and J. Johansson. 1998. Structure and activity o f cathelicidin antibacterial proteins. J Protein Chem 17:522. 39. Bowdish, D . M . E . , D . J. Davidson, and R. E . W . Hancock. 2005. A re-evaluation of the role o f host defence peptides in mammalian immunity. Curr Protein Pept Sci 6:35. 40. V a n Wetering, S., S. P. Mannesse-Lazeroms, J. H . Dijkman, and P. S. Hiemstra. 1997. Effect o f neutrophil serine proteinases and defensins on lung epithelial cells: modulation of cytotoxicity and IL-8 production. JLeukoc Biol 62:217. 41. V a n Wetering, S., S. P. Mannesse-Lazeroms, M . A . V a n Sterkenburg, M . R. Daha, J . H . Dijkman, and P. S. Hiemstra. 1997. Effect o f defensins on interleukin-8 synthesis in airway epithelial cells. Am J Physiol 272:L888. 42. Muller , W . A . , and G . J. 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Colony stimulating factors, cytokines and monocyte-macrophages--some controversies. Immunol Today 14:18. 52. Murakami , M . , T . Ohtake, R. A . Dorschner, B . Schittek, C . Garbe, and R. L . Gallo. 2002. Cathelicidin anti-microbial peptide expression in sweat, an innate defense system for the skin. J Invest Dermatol 119:1090. 53. Scott, M . G . , and R. E . Hancock. 2000. Cationic antimicrobial peptides and their multifunctional role in the immune system. Crit Rev Immunol 20:407. 60 4.0 LL-37 is a Potent Anti-endotoxic Agent With Multiple Mechanisms of Action 4.0 LL-37 IS A POTENT ANTI-ENDOTOXIC AGENT WITH MULTIPLE MECHANISMS OF ACTION 61 4.1 INTRODUCTION 62 4.2 MATERIALS & METHODS 63 4.3 RESULTS 66 4.4 DISCUSSION 71 4.5 B IBLIOGRAPHY 74 61 4.1 Introduction Host defence peptides are small, positively charged peptides which are an evolutionarily conserved component of the innate immune response. Individual peptides are found in high concentrations in the granules o f neutrophils and some can be produced by epithelial and other cell types upon stimulation with bacterial components or inflammatory mediators (1, 2). Originally these peptides were believed to function simply as natural antibiotic compounds in the antibacterial defences o f neutrophils; however, it has become apparent that their antimicrobial activity extends to viruses and eukaryotic microbes, and they appear to enhance host defences by interacting with neutrophils (3), monocytes (4), macrophages (5), dendritic cells (6), T cells (3), and epithelial cells (7, 8). Thus they are being developed as novel antimicrobial and immunostimulatory agents. Although the mechanisms by which they ki l l bacteria are not fully elucidated, many mammalian antimicrobial peptides ki l l bacterial cells in a non-lytic manner (9). This is an especially valuable asset considering that the use of antibiotics which lyse bacteria, and consequently release immunostimulatory bacterial components has been linked to the development or enhancement of septic shock (10, 11). Early experiments also determined that a number of host defence peptides from various sources bound to diverse strains o f L P S and reduced LPS- induced release of pro-inflammatory cytokines (e.g. T N F - a , IL-1 , IL-6) and nitric oxide from monocytes or macrophages (12-15), although these two observations are not obligately linked (16). Nevertheless it has been clearly demonstrated that various host defence peptides protect mice from LPS- induced lethality (17-21). A s the excessive production of pro-inflammatory cytokines precedes the development o f full-blown septic shock, the ability o f these peptides to reduce pro-inflammatory cytokine production induced by a variety o f pathogen molecules, including L T A , C p G and L P S , indicates that they might function as broad-spectrum anti-sepsis agents (22, 23). The cathelicidins are host defence peptides found at high concentrations in the granules o f neutrophils. The ability o f cathelicidins to bind to L P S and block L P S induced pro-inflammatory cytokine production seems to be conserved in all identified homologs to date, including those found in sheep (24), cows (25, 26), and humans (13). These peptides are characterised by their evolutionarily-conserved N-terminal cathelin domain/pro-piece which is cleaved from the antimicrobial/immunomodulatory C-terminus upon degranulation from neutrophils. The human cathelicidin, named h C A P - 1 8 in its unprocessed form, can be processed to several peptides including the 37 amino acid peptide called L L - 3 7 (27). Initial studies on the anti-endotoxin properties o f this peptide focussed on h C A P - 1 8 (17), however, it was later found 62 that the L P S binding properties o f the peptide were contained within the processed peptide, L L -37 (28). It has been proposed that the anti-endotoxic properties o f this peptides are due in part to an inhibition o f the binding o f L P S the cell surface molecule/receptor component C D 14 (29) and lipopolysaccharide binding protein ( L B P ) (16), although the binding affinity o f L L - 3 7 for L P S is less that o f either o f these components. L L - 3 7 has been shown to block a number of L P S -induced inflammatory responses including contractility and N O release in aortic rings (30), infiltration of leukocytes and chemokine production (19), and pro-inflammatory cytokine production in a macrophage cell line or in whole human blood (18). It is possible that at physiological concentrations L L - 3 7 may provide homeostatic buffering o f the effects o f low concentrations o f L P S or other pathogen components. It is not entirely clear whether L L - 3 7 and other host defence peptides are able to mediate these effects exclusively through direct binding and neutralisation o f L P S and other negatively charged bacterial components or whether this is due to a more subtle interaction with host cells. Evidence for both possibilities exists. In this study I investigated the ability o f L L - 3 7 to block L P S induced pro-inflammatory cytokine production from human peripheral blood derived monocytes and a monocyte/macrophage-like cell line and demonstrate that although L L - 3 7 appears to block L P S induced cytokine production in vitro by binding and neutralising L P S directly, evidence exists for a possible secondary mechanism o f action. 4.2 Materials & Methods Isolation of Peripheral Blood Derived Monocytes. Blood monocytes were prepared using standard techniques (31). Briefly, 100 ml of fresh human venous blood was collected in sodium heparin Vacutainer collection tubes (Becton Dickinson, Mississauga, O N , Canada) from volunteers according to U B C Clinical Research Ethics Board protocol C02-0091. The blood was mixed, at a 1:1 ratio, with R P M I 1640 media [supplemented with 10% v/v foetal calf serum ( F C S ) , 1% L-glutamine, 1 n M sodium pyruvate] in an E-toxa-clean (Sigma-Aldrich, Oakville, O N , Canada) washed, endotoxin-free bottle. Peripheral blood mononuclear cells ( P B M C ) were separated using Ficoll-Paque Plus (Amersham Pharmacia Biotech, Baie D ' U r f e , P Q , Canada) at room temperature and washed with phosphate buffered saline (PBS) . Monocytes were enriched by the removal o f T-cells by resetting with fresh sheep red blood cells ( U B C animal care unit) pre-treated with Vibrio cholerae neuraminidase (Calbiochem Biosciences Inc., L a Jolla, C A , U S A ) and repeated separation by using Ficol l Paque Plus (32). The enriched monocytes were washed with P B S , then cultured in tissue culture plates for 1 hour at 3 7 ° C followed by the removal o f non-adherent cells. 63 Monocytes isolated in this way were >95% pure as determined by flow cytometry (data not shown). Cells were cultured in Falcon tissue culture 48-well plates (Becton Dickinson, Mississauga, O N , Canada). The adherent monocytes were cultured in 0.25 ml media at 3 7 ° C in which L P S , L L - 3 7 and/or cytokines dissolved in endotoxin-free water (Sigma-Aldrich, Oakville, O N , Canada) were added. Endotoxin-free water was added as a vehicle control. Cell Culture The human monocyte-like cell line, T H P - 1 (33), was obtained from the A T C C (No. T I B -202, Rockvil le , M D ) , and grown in supplemented R P M I 1640 media containing 10% foetal calf serum , 1% sodium pyruvate and 1%> L-glutamine (Gibco B R L , Burlington, O N ) . T H P - 1 cells were differentiated into adherent macrophage-like cells by the addition o f 100 m M phorbyl myristate acetate ( P M A ) and incubation at 3 7 ° C , 5% CO2 for three days as described previously (34) . Peptide Synthesis. L L - 3 7 was synthesized by N-(9-fluorenyl) methoxycarbonyl (Fmoc) chemistry at the Nucleic Acid/Protein Service unit at the University o f British Columbia , as previously described (35) . 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, St. Louis , M O ) and the concentration of the peptides in solution was determined by amino acid analysis. Cytokine Production The concentrations o f either T N F - a or IL-6 found in the supernatants o f the treated cells were measured using commercially prepared E L I S A plates in accordance to the manufacturer's suggestion ( R & D systems, Minneapolis, M N ) . For T H P - 1 cells, cells were treated with P M A and 2 x 10 5 cells were seeded in 1 ml of media containing 10%> F C S in 24 well tissue culture plates and allowed to differentiate for three days. For primary peripheral blood derived monocytes between 0.5-1 x 10 5 cells were seeded in 250 ul in 48 well tissue culture plates. Cells were then incubated in at least triplicate, for four hours in the presence of either media alone, or with Pseudomonas aeruginosa HI03 L P S , cationic peptides or a combination of L P S and peptide (concentrations stated in text) in media. Supernatants were collected and stored at - 2 0 ° C until use. Immunoblotting T H P - 1 cells were cultured as above. O n the day of the assay the cells were gently detached using Ce l l Dissociation Solution (Sigma, St. Louis , M O ) . Cells were washed, re-64 suspended in media and added to 5 ml polystyrene tubes (Becton Dickinson) and allowed to rest for 2 hours. Approximately 1 x 10 6 T H P - 1 cells were used per condition. Cells were then stimulated by adding L P S , L L - 3 7 or water as a vehicle control. After stimulation the cells were centrifuged, washed one time with ice-cold P B S with I m M vanadate and nuclear extracts were isolated using N E - P E R Nuclear and Cytoplasmic Extraction Reagents K i t (Pierce, Fisher Canada, O N ) as per the manufacturer's directions. The lysates were assayed for protein concentration using a B C A assay (Pierce, Fisher Canada, O N ) . Lysate (7.5 pg) was loaded per lane onto 1.0 m m thick gels, which were run at 100 V for approximately 2-3 hr. Proteins were transferred to P V D F filters for 80 min at 80 V . The filters were blocked for 1 hour at room temperature with 5% skim milk in T B S T (10 m M T r i s - H C l p H 8, 150 m M NaCI , 0.1% Tween-20). The filters were then incubated overnight at 4 ° C with the anti-p50 or anti-p65 (Santa Cruz, C A ) monoclonal antibodies. Immunoreactive bands were detected using horseradish peroxidase-conjugated sheep anti-mouse or donkey anti-rabbit IgG antibodies (Amersham Pharmacia, Piscataway, NJ) and chemiluminescence detection (Sigma, St. Louis , M O ) . Gel Shift Assays G e l shift assays using a biotinylated N F - K B consensus sequence were performed with the above mentioned nuclear extracts using a LightShift Chemiluminescent E M S A K i t (Pierce, Fisher Canada, O N ) as per the manufacturer's directions. Briefly, a N F - K B probe consensus sequence ( 5 ' - A G T T G A G G G G T C T T C C C A G G C - 3 ' , Panomics, Redwood City, Ca) labelled with biotin was mixed with approximately 7.5 pg of nuclear lysate and binding buffer supplemented with glyercol (8%), M g C l 2 (5 m M ) , K C I (100 m M ) , E D T A (1 m M ) , D T T (1 m M ) and poly IC:dC (1.5 pg). T o determine the specificity o f the reaction an unlabelled biotin probe was used to block the shift. Extracts were incubated for 20 min and then run on a 7% acrylamide gel for 1 -2 hours. The gels were transferred to nitrocellulose membranes at a 380 m A for 30 minutes and U . V . cross-linked before detection as per the manufacturer's directions. Statistical Analysis Student's t test was performed to determine statistical significance. Values are expressed as mean ± standard error o f the mean. 65 4.3 Results 0 10 LL-37 (ug/ml) 50 LL-37 blocks LPS induced pro-inflammatory cytokine production from primary human monocytes and a monocyte-like cell line L L - 3 7 has been demonstrated to block L P S induced T N F - a production by a mouse macrophage cell line (18) and from a monocyte cell line (26) but it's ability to block the production of other pro-inflammatory cytokines by primary human cells has not been evaluated. In this experiment peripheral blood derived monocytes were treated with 10 ng/ml L P S with or without L L - 3 7 (10 or 50 pg/ml) for four hours and supernatants were assayed for either T N F - a (n=5), or IL-6 (n=2)(one representative donor shown, Figure 4.1 A & B) . Figure 4.1. LL-37 reduces LPS induced cytokine production in peripheral blood derived monocytes and a monocyte-like cell line. A ) Peripheral blood derived monocytes from 5 donors were treated with 10 ng/ml o f L P S for 4 hr. L L -37 (10 or 50 ug/ml) reduced L P S induced T N F -a production to baseline levels. The average of three separate wells o f one representative donor +/-standard deviation is shown. B ) L L - 3 7 reduces L P S induced production o f IL-6. The average of three separate wells o f one representative donor +/-standard deviation is shown. C ) T H P - 1 cells were treated with L P S or L P S & L L - 3 7 for 4 hrs. The presence o f 10 ug/ml L L - 3 7 reduced the amount o f T N F - a produced by approximately 90%. The average of three independent experiments ± 0 10 standard deviation is LL-37 (pg/ml) shown. 10 50 LL-37 (pg/ml) 66 L L - 3 7 reduced L P S induced pro-inflammatory cytokine production, with almost complete inhibition occurring at 10 pg/ml of L L - 3 7 . L L - 3 7 did not itself induce T N F - a production although a small increase in IL-6 was observed in one donor (data not shown). L L - 3 7 effectively blocks pro-inflammatory cytokine production from L P S stimulated monocytes. T H P - 1 cells were seeded at a higher concentration than the primary cells (2 x 10 5 cells per well versus approximately 5 x 10 4 for the primary cells) and thus the concentrations of T N F -a detected in response to L P S were greater than the primary cells (between 4000-7000 ng/ml versus between 1000-6000 ng/ml depending on the donor). L L - 3 7 also induces a dose dependent decrease in T N F - a in T H P - 1 cells (Chapter 2, Figure 2.2) with an approximate 90% reduction occurring at 10 pg/ml (Figure 4.1C). Consistent with the published data these T H P - 1 cells did not produce IL-6 (36). Thus L L - 3 7 is as effective at blocking L P S induce pro-inflammatory cytokine production from primary cells as it is from cell lines. LL-3 7 can be added after LPS and reduce TNF-a production W e have previously shown that L L - 3 7 can be added up to an hour after L P S and reduce pro-inflammatory cytokine production and it has been proposed that this reduction is due to a direct interaction between L L - 3 7 and host cells (18). T o date it has not been tested whether the resulting decrease in T N F - a after delayed addition o f L L - 3 7 is because L P S must be continually present to maximally induce pro-inflammatory signalling as has been demonstrated for other peptides in other model systems (37). T o test this, T H P - 1 cells were stimulated with 10 ng/ml L P S for four hours and T N F - a production was assessed. L L - 3 7 (10 pg/ml) was added either simultaneously or 30, 60 or 90 minutes after the L P S . Alternatively, the cells were washed three times with 1 ml o f media at these same timepoints. Fresh media without L P S was added and the cells were incubated for a total o f 4 hr at which point the supernatants were collected and T N F - a production was assessed. The total amount of T N F - a produced in the L P S stimulated cells was calculated to be 100%) and the amount of T N F - a in the supernatants o f the cells treated with L L -37 or washed was expressed as a percentage of the total amount o f T N F - a . The addition of L L -37 or the removal o f L P S at 30 or 60 minutes resulted in an equivalent reduction in cytokine production (Figure 4.2). 67 t = 0 T LPS added • LL-37 • Wash o •g 80 supernatants collected and TNF-a production measured t = 4hr 30 60 Time (minutes) 90 Figure 4.2. LL -37 can be added after LPS and reduce pro-inflammatory cytokine production. THP-1 cells were treated with 10 ng/ml LPS for four hours. At 30, 60 or 90 min either LL-3 7(10 pg/ml) was added or the supernatant was removed and fresh media without LPS was added. At 4 hr the supernatants were removed and TNF-a production was assessed. There was no statistically significant difference between LL-37 treatment and removal of the LPS. Average of three independent experiments +/- standard deviation from the mean shown. Pre-treatment with LL-37 results in a reduction of LPS induced TNF-a production In order to test whether LL-37 is required to be present to result in a decrease in pro-inflammatory cytokine production or if there might be an additional interaction between the peptide and the cells which results in a subsequent decrease in cytokine production, THP-1 cells were pre-treated with either endotoxin-free water as a vehicle control or LL-37 (50 pg/ml) for 30 minutes. The cells were washed three times with 1 ml of media and then fresh media (including 10% FCS) containing LPS (10 ng/ml) was added. TNF-a production was assessed after four hours. Although the cells pre-treated with LL-37 produced less TNF-a the reduction did not reach statistical significance (Figure 4.3). 68 t = -30 min T L L " 3 7 O R V E N I C L E control added t = 0 min Cells washed and L P S added 1600-E 1200 Q. U_ z Supernatants t = 4 hr J , collected and T T N F - a production measured 0 30 Minutes of LL-37 pre-treatment Figure 4 .3. Pre-treatment with LL-37 reduces pro-inflammatory cytokine production. T H P - 1 cells were treated with L L - 3 7 (50 ng/ml) for 30 min prior to the addition of L P S (10 ng/ml) and then washed three times. Average of three independent experiments +/- standard deviation from the mean shown. The anti-inflammatory properties of LL-37 are specific for LPS In order to test whether the ability o f L L - 3 7 to reduce pro-inflammatory cytokine production was specific for L P S induced responses or was applicable to other pro-inflammatory stimuli, monocytes were stimulated with either I L - l p (100 ng/ml) or T N F - a (10 ng/ml) in the presence or absence o f L L - 3 7 (10 & 50 ug/ml) for 4 hr. Stimulation with I L - i p resulted in an increase in T N F - a found in the supernatants (between 150 - >2000 pg/ml, donor dependent). The presence o f L L - 3 7 enhanced IL-1B induced T N F - a production in a dose dependent manner and this increase in T N F - a was statistically significant in four o f five donors (p values range from p<0.06 to p<0.0006, donor dependent, Figure 4.4A). Although I L - i p also induced the production of IL-6, L L - 3 7 did not significantly alter the amount o f IL-6 produced in response to I L - i p (n=2, data not shown). Stimulation with T N F - a (10 ng/ml) also resulted in autocrine production o f T N F - a (approximately 60 ng/ml, n=2) and IL-6 production in one o f two donors (data not shown). The presence o f L L - 3 7 at high concentrations (50 pg/ml) resulted in a slight decrease in T N F - a production in both donors tested but this was not statistically significant (Figure 4.4B). In summary, the presence of L L - 3 7 enhances IL-1 P induced pro-inflammatory cytokine production and does not appear to alter T N F - a induced pro-inflammatory cytokine production. 69 Figure 4.4. LL-37 does not reduce pro-inflammatory cytokine production induced by IL-ip or TNF-a. A ) Primary monocytes were treated with lOOng/ml o f IL-1 p for four hr. Supernatants were collected and assayed for T N F - a production. A l l five donors showed an increase in T N F - a production in response to IL-1 p. In all donors tested there was an increase in I L - i p induced cytokine production in the presence of L L - 3 7 . This increase was statistically significant in four out o f five donors. One representative donor of five shown. Average of three wells +/- standard deviation is shown. B ) L L - 3 7 does not reduce T N F - a induced T N F - a production. T N F - a (10 ng/ml) stimulation of primary monocytes stimulated production of T N F - a and there was no statistically significant decrease in the presence o f L L - 3 7 . One representative donor of two shown. Average of three wells +/- standard deviation is shown. LL-3 7 inhibits p65 translocation in response to LPS It is not entirely clear i f L L - 3 7 blocks L P S induced pro-inflammatory cytokine production by binding to L P S and thus preventing N F - K B activation and pro-inflammatory responses or i f there may be another more subtle interaction with host cells. In order to determine i f L L - 3 7 was able to alter L P S induced activation and translocation o f N F - K B , gel shift assays were performed. T H P - 1 cells were treated with L P S (10 ng/ml), L L - 3 7 (10 ug/ml) or both L P S and L L - 3 7 for 20 minutes. N F - K B is found primarily in the cytoplasm in unstimulated cells. U p o n exposure to pro-inflammatory stimuli activation and translocation to the nucleus occurs. Nuclear extracts were collected and gel shift assays were performed. A shift in the gel occurs when N F - K B is present to bind to the biotinylated probe. The specificity o f the reaction was determined by blocking the reaction with excess unbiotinylated probe ("cold" probe). L P S induces translocation o f N F - K B as previously described. L L - 3 7 did not prevent L P S induced translocation o f N F - K B and in fact L L - 3 7 stimulation resulted in nuclear translocation of N F - K B (Figure 4.5A). A s the N F - K B transcription factor family consists o f five different proteins which form homo- and heterodimers that have different specificities for D N A binding. The best characterised heterodimer consists o f the p65:p50 subunits. Binding of this heterodimer is required for L P S induced transcription o f pro-inflammatory cytokine genes such as T N F - a . In order to determine i f L L - 3 7 had any effect on L P S induced p65:p50 translocation, Western blots o f the nuclear extracts were performed. L P S induced translocation o f both p65 and p50 (Figure 4.5B). L L - 3 7 70 did not induce translocation o f p65 but did induce translocation of p50. L L - 3 7 could block L P S induced p65 translocation when it was added either simultaneously or after 10 min o f L P S stimulation (Figure 4.5B). F igure 4.5. L L - 3 7 blocks L P S induced translocation of p65 but induces translocation o f p50. T H P - 1 cells were stimulated with either L P S (10 ng/ml), L L - 3 7 (10 ug/ml) or a combination o f L P S or L L - 3 7 for 20 min. Nuclear extracts were isolated and gel shift assays and Western blots were performed. A ) L P S and L L - 3 7 induced translocation of N F - K B . L L - 3 7 did not block L P S induced N F - K B translocation. One representative gel o f two independent experiments is shown. B ) L L - 3 7 blocked L P S induced translocation of p65 but not o f p50. L P S induced translocation o f p65 could be blocked when L L - 3 7 was added either simultaneously or when added after 10 min of L P S stimulation. L L - 3 7 induced translocation o f p50 and did not reduce L P S induced translocation of p50. One representative experiment of three shown. 4.4 Discussion Host defence peptides and synthetic derivatives bind to the anionic sugars, phosphate groups and lipid A core of L P S and L P S binding has been demonstrated to be an important requirement for bacterial ki l l ing (38). Indeed, it has been proposed that a conserved L P S binding motif exists which consists o f both basic and hydrophobic amino acids and accounts for the ability o f natural and synthetic peptides to bind and neutralise L P S (39, 40). Binding of host defence peptides to L P S has been proposed to mask its biological effects (41) since high affinity binding induces changes in the endotoxic L P S aggregate structure and a neutralisation of the negative charges o f L P S . L L - 3 7 binds directly to L P S and this binding is o f sufficiently high affinity to displace L B P . There is some in vitro evidence that the inhibition o f pro-inflammatory cytokine production induced by bacterial components correlates to some extent with binding and neutralisation o f these components by host defence peptides (16). Nevertheless doubt exists as to whether this is the only mechanism of action as most peptides have L P S binding affinities that are orders o f magnitude lower than well established L P S neutralising molecules such as bacterial permeability increasing protein (BPI) and yet are as effective, or in some cases more effective, at preventing mortality in animal models o f septic shock. Similarly, although L P S binding affinity correlates with an ability to block T N F - a production from a macrophage cell line, there are certain peptides, such as a derivative of bovine bactenecin, that has a relatively high binding 71 affinity for L P S but is virtually unable to block L P S induced cytokine production (16, 26). There is emerging evidence suggesting that these peptides might have other, subtler mechanisms of altering the pro-inflammatory response which may be applicable to in vivo experiments or human clinical trials. L L - 3 7 , for example, has been demonstrated to increase the expression of anti-inflammatory genes such as IL-10 in a macrophage cell line (18). Although low concentrations o f B P I reduce pro-inflammatory cytokine production induced by L P S , much higher concentrations o f B P I are required to neutralize the anti-inflammatory cytokine I L - 1 R A (42). This selective modulation of L P S induced responses also occurs with C E M A , an insect derived peptide with anti-inflammatory properties in vitro and in vivo, which blocks some but not all, o f the transcriptional responses induced by L P S in a macrophage-like cell line and induces expression of genes believed to be involved in host responses to infection (43). Thus there is a subtle growing body o f evidence suggesting that host defence peptides may be able to modulate host responses to L P S at the transcriptional and post-transcriptional level. L L - 3 7 and other host defence peptides show potential as anti-sepsis agents. The ability o f L L - 3 7 and other host defence peptides to block L P S induced pro-inflammatory cytokine production is believed to provide an advantage over treatment with conventional antibiotics. In many animal models in which sepsis, or septic shock, is induced by injection o f high numbers of bacteria, host defence peptides do not reduce bacterial counts. Thus, at first blush it might appear that they are no more effective than currently established therapies. However, in all cases in which they were measured, the serum levels o f cytokines and L P S were reduced and in many studies mortality is decreased (17). In human disease it is clear that treatment with antibiotics is not sufficient to prevent mortality, as decreases in viable bacteria do not correlate with survival. It is thus believed that the fatal aspect o f septic shock is mediated due to overwhelming production of pro-inflammatory cytokines. In studies where the efficacy o f host defence peptides is compared with that o f conventional antibiotics, it is clear that only the peptides have an ability to reduce pro-inflammatory cytokine production. In this study we confirm that L L - 3 7 can reduce L P S induced pro-inflammatory cytokine production but demonstrate that in vitro this is likely due to binding and sequestering the L P S since this does not occur in response to I L - i p or T N F - a . This is consistent with previous studies demonstrating that B P I can reduce L P S , but not I L - i p -induced, N O synthesis (44). This data is somewhat contradictory to one study in an animal model in which the cathelicidin peptide B M A P - 2 8 can often be added up to six hours after administration o f bacterial components, by which point a cascade o f pro-inflammatory cytokines has been produced and clearance of L P S has begun, and still reduce pro-inflammatory cytokine 72 production and mortality (20). This indicates that in vitro models o f L P S induced cytokine responses may not necessarily reflect in vivo conditions. It also appears as though L L - 3 7 may enhance pro-inflammatory cytokine production by other stimuli such as I L - l p \ Although L L - 3 7 treatment has been demonstrated to increase IL-1B release in L P S primed monocytes (4) this is the first indication that synergistic increases in cytokine production may occur in unprimed cells as well. This observation may have clinical relevance as these peptides are developed into immunomodulatory therapies. L L - 3 7 has been demonstrated to be protective in animal models o f sepsis i f administered at low concentrations but to increase mortality at higher concentrations (45). A s has previously been demonstrated for the insect derived peptide C E M E , and the bovine cathelicidin indolicidin, L L - 3 7 can be added to a monocyte cell line up to an hour after L P S and still reduce pro-inflammatory cytokine production (18, 35). Initially this was attributed to the ability o f the peptide to interact directly with the host cell and block LPS- induced cytokine production through an unspecified mechanism o f action, however, these data are also consistent with the hypothesis that L L - 3 7 and other host defence peptides bind to and neutralise the existing L P S . This has also been demonstrated for a BPI derivative which reduces LPS-induced E -selectin expression and activation of the key pro-inflammatory transcription factor N F - K B when added concurrently with L P S or as much as 6 hours afterwards (37). This implies that L P S must be continually present to maximally induce pro-inflammatory signalling and L P S induced responses (37). Consistent with this, washing away the L P S results in similar reductions in T N F -rx production. It is clear that further experiments wil l be required to determine i f interactions o f L L - 3 7 and other host defence peptides with monocytes are involved in suppressing pro-inflammatory responses. This study also demonstrates that L L - 3 7 might counteract the LPS- induced translocation of the p65 subunit o f N F - K B in a manner consistent with binding and neutralising L P S interactions with monocytes. However L L - 3 7 induced p50 translocation and did not appear to alter p50 translocation in response to L P S . This is an especially intriguing observation as the accumulation of p50 homodimers in the nucleus has been demonstrated to block transcription o f T N F - a (46) and to be an important component in inducing L P S tolerance (47). Future studies should focus on elucidating the role of alternative subunit formation in L L - 3 7 treated cells and to determine i f accumulation of these subunits occurs in cells pre-treated with L L - 3 7 thus elucidating a possible mechanism of action by which pre-treatment prevents T N F - a production. 73 In animal models cathelicidins have been demonstrated to reduce LPS- induced T N F - a production(18, 19), to reduce circulating levels o f endotoxin (24), to suppress leukocyte infiltration in a model o f endotoxin-induced uveitis (19) and to prevent lethality in animal models o f sepsis in which the animals are injected with high concentrations o f bacteria or L P S (18, 45). Although current in vitro models o f host defence peptide interactions study host defence peptide and L P S interactions over short time points and indicate that the suppressive effects o f these peptides are due primarily to their L P S binding and neutralising functions, there may be additional more subtle mechanisms by which L L - 3 7 and other peptides also alter pro-inflammatory responses which may ultimately be more useful in the development of these peptides into drugs with immunomodulatory properties. 4.5 Bibliography 1. Diamond, G . , J . P. Russell, and C . L . Bevins. 1996. Inducible expression of an antibiotic peptide gene in lipopolysaccharide-challenged tracheal epithelial cells. Proc Natl Acad Sci USA 93:5156. 2. Ne l l , M . J . , G . Sandra Tjabringa, M . J. Vonk , P. S. Hiemstra, and J. J. Grote. 2004. Bacterial products increase expression of the human cathelicidin h C A P - 1 8 / L L - 3 7 in cultured human sinus epithelial cells. 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Johnson. 1998. Tumor necrosis factor alpha transcription in macrophages is attenuated by an autocrine factor that preferentially induces NF-kappaB p50. Mol Cell Biol 18:5678. 47. Bohuslav, J . , V . V . Kravchenko, G . C . Parry, J. H . Erl ich , S. Gerondakis, N . Mackman, and R. J. Ulevitch. 1998. Regulation o f an essential innate immune response by the p50 subunit o f N F - k a p p a B . J Clin Invest 102:1645. 11 5.0 LL-37 Induces Rapid & Early Chemokine Production Via Activation of N F - K B and the MAPK 5.0 LL-37 INDUCES RAPID & EARLY CHEMOKINE PRODUCTION VIA ACTIVATION OF NF-KB & THE MAPK 78 5.1 INTRODUCTION 79 5.2 MATERIALS & METHODS 80 5.3 RESULTS 83 5.4 DISCUSSION 89 5.5 B IBLIOGRAPHY 91 78 5.1 Introduction Leukocyte infiltration is a hallmark of inflammation and is an essential component of the early immune response. Leukocytes accumulate at infection foci early in the course o f infection and dissipate as the infection is managed. In general, this is a tightly regulated process which results in the resolution o f infection without damaging the host; however, dysregulation has been linked to conditions such as asthma (1), rheumatoid arthritis (2), neurodegenerative disease (3) and tumour formation (4). In certain diseases such as arthritis and cystic fibrosis, the infiltration of excessive numbers o f leukocytes is believed to be a major contributing factor to disease pathology (5-7). Thus it is essential to characterise and understand the involvement of novel chemoattractants and chemokine-inducing agents. Host defence peptides are essential components o f the innate immune response which are found at elevated concentrations during the course o f infection and inflammation (8-10). These are small, positively charged peptides that are found in high concentrations in the granules o f neutrophils and at lower concentrations in other cell types such as monocytes, T cells and N K cells (11). They are also expressed by epithelial cells and keratinocytes upon stimulation with bacterial components or pro-inflammatory cytokines (12, 13). They have been shown to have diverse functions in the innate immune response including altering the differentiation of dendritic cells (14), promoting angiogenesis and wound healing (15-17), and inducing chemotaxis and chemokine production (18-20). There are three major classes o f host defence peptides in humans, the alpha defensins, beta defensins and the sole cathelicidin, h C A P - 1 8 / L L - 3 7 . Members of each of these classes have been demonstrated to function as chemoattractants for dendritic cells, neutrophils, monocytes, and/or T cells although the concentrations required for chemotaxis tend to be much higher than conventional chemokines (21, 22). These peptides may also be involved in the recruitment of leukocytes by inducing the production o f chemokines. L L - 3 7 , for example, has been demonstrated to induce IL-8 production from epithelial cells and epithelial cell lines, from keratinocytes and from peripheral blood derived monocytes (23-26). Interestingly, increased pulmonary concentrations o f L L - 3 7 correlate with granulocyte infiltrations as well as the presence o f neutrophil chemokines and neutrophil markers in cystic fibrosis (27). Other cell types are also found at sites o f high concentrations of L L - 3 7 including mononuclear cells. Although L L - 3 7 may function as a chemoattractant for these cells, the high concentrations required for chemotactic activity in vitro imply that this might not be the primary role o f this peptide in leukocyte recruitment. 79 I have demonstrated in Chapter 3 that L L - 3 7 induces activation o f the mitogen activated protein kinases ( M A P K ) , extracellular regulated kinase ( E R K 1 / 2 ) and p38, in monocytes and this activation is linked to transcription of the chemokines IL-8 , M C P - 1 and M C P - 3 . I also demonstrated that L L - 3 7 induces the production o f substantial amounts o f IL-8 from peripheral blood derived monocytes. I propose that the ability o f this peptide to induce chemokine production may be involved in the recruitment o f leukocytes to sites o f elevated concentrations of L L - 3 7 which occur at site o f infection and inflammation. In this chapter the ability o f L L - 3 7 to induce transcription of other C C chemokines was investigated. L L - 3 7 stimulation of peripheral blood monocytes resulted in release of IL-8 which can be detected in supernatants after only 15 min o f stimulation. Because o f the quick kinetics o f this response, the possibility that L L - 3 7 was inducing a transcript stabilisation event or was stimulating the release of pre-formed stores o f IL-8 was investigated. This study demonstrates that L L - 3 7 mediated activation of the M A P K results in de novo transcription of various C C chemokines and is required for in de novo IL-8 production which is detectable in as short a time as 15 minutes after stimulation. L L -37 is thus a potent immunostimulatory agent which is likely involved in the early recruitment o f leukocytes to sites o f infection or inflammation. 5.2 Mater ia l s & M e t h o d s Isolation of Peripheral Blood Derived Monocytes. Blood monocytes were prepared using standard techniques (28). Briefly, 100ml of fresh human venous blood was collected in sodium heparin-containing Vacutainer collection tubes (Becton Dickinson, Mississauga, O N , Canada) from volunteers according to U B C Clinical Research Ethics Board protocol C02-0091. The blood was mixed, at a 1:1 ratio, with R P M I 1640 media [supplemented with 10% v/v foetal calf serum (FBS) , 1% L-glutamine, 1 m M sodium pyruvate] in an E-toxa-clean (Sigma-Aldrich, Oakvil le , O N , Canada) washed, endotoxin-free bottle. Peripheral blood mononuclear cells ( P B M C ) were separated using Ficoll-Paque Plus (Amersham Pharmacia Biotech, Baie D 'Urfe , P Q , Canada) at room temperature and washed with phosphate buffered saline (PBS). Monocytes were enriched with the removal o f T-cells by resetting with fresh sheep red blood cells ( U B C animal care unit) pre-treated with Vibrio cholerae neuraminidase (Calbiochem Biosciences Inc., L a Jolla, C A ) and repeat separation by Ficol l Paque Plus (29). The enriched monocytes were washed with P B S , then cultured (approximately 0.5-1 x 10 5 per well) for 1 hour at 3 7 ° C followed by the removal o f non-adherent cells; monocytes were >95% pure as determined by flow cytometry. Cells were cultured in Falcon tissue culture 48-well plates (Becton Dickinson, Mississauga, O N , Canada). The adherent 80 monocytes were cultured in 0.25 ml media at 3 7 ° C in which L L - 3 7 dissolved in endotoxin-free water (Sigma-Aldrich, Oakvil le , O N , Canada) were added. Endotoxin-free water was added as a vehicle control. A total o f six donors were used in these experiments. Cell Culture The human monocyte-like cell line, T H P - 1 (30), was obtained from the A T C C (No. T I B -202, Rockvil le , M D ) , and grown in supplemented R P M I 1640 medium containing 10% foetal calf serum , 1%> sodium pyruvate and 1% L-glutamine (Gibco B R L , Burlington, O N ) . T H P - 1 cells were differentiated into adherent macrophage-like cells by addition of 100 m M phorbyl myristate acetate and incubation at 3 7 ° C , 5%> CO2 for three days as described previously (31). Inhibitor Studies SB 203580, a specific inhibitor of p38 kinase was purchased from Sigma-Aldrich (Oakville, O N , Canada) and PD98059, a specific inhibitor o f E R f G / 2 kinase was purchased from Cel l Signaling Technology. Cells were incubated with the kinase inhibitors (5 or 10 u M ) for 1 hr prior to stimulation with L L - 3 7 . The transcriptional inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide were incubated at a concentration o f 5 ug/ml and 1 ug/ml respectively for 30 min prior to stimulation with L L - 3 7 (Calbiochem Biosciences Inc., L a Jolla, C A ) . A l l inhibitors were present over the course of L L - 3 7 stimulation. Peptide Synthesis. L L - 3 7 was synthesized by N-(9-fluorenyl) methoxycarbonyl (Fmoc) chemistry at the Nucleic Acid/Protein Service unit at the University o f British Columbia , as previously described (32). 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, St. Louis , M O ) and the concentration of the peptides in solution was determined by amino acid analysis. Semi-Quantitiative and Quantitative Reverse-Transcriptase-PCR (RT-PCR) Total R N A was isolated from donor blood derived monocytes using an RNaqueous Micro-kit (Ambion) as described by the manufacturer. The samples were DNase treated, and then c D N A synthesis was accomplished by using a first-strand c D N A synthesis kit (Gibco). The resultant c D N A s were used as a template in P C R s for various cytokine genes (Table 5.1). A l l quantitative R T - P C R experiments were performed in duplicate on an A B I 7700. Sequence Detection System. Results were analyzed in the linear phase o f amplification and normalized to the R T - P C R results for the housekeeping control, glyceraldehyde-3-phosphate dehydrogenase ( G A P D H ) . T o quantify bands o f the semi-quantitative R T - P C R the gels were quantified by 81 densitometry using the software program ImageJ and normalised to the housekeeping gene, G A P D H . Reactions were verified by including controls lacking reverse transcriptase. T a b l e 5.1. P r i m e r sequences used in this study. Primer Forward sequence (5'-3') Reverse Sequence (5'-3') M C P - 1 / C C L 4 T C A T A G C A G C C A C C T T C A T T C T A G C G C A G A T T C T T G G G T T G 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 MIP-1(3/CCL2 C T T T T C T T A C A C C G C G A G G A A G C A G A G G C T G C T G G T C T C A T I L - 8 / C X C L 8 G T G C A G A G G G T T G T G G A G A A G T T C T C C C G T G C A A T A T C T A G G IL-6 A C C T G A A C C T T C C A A A G A T G G G C G C A G A A T G A G A T G A G T T G 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 IL-p G G A T A T G G A G C A A C A A G T G G A T G T A C C A G T T G G G G A A C T 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 Cytokine Production The concentration o f IL-8 in the supernatants o f the treated cells was measured using commercially prepared E L I S A plates in accordance to the manufacturer's suggestion (Biosource, Montreal, Q C , Canada). Monocytes were seeded at approximately 0.5-1 x 10 5 cells per well in 48 well plates. Cells were then stimulated with either endotoxin free water as a vehicle control or L L - 3 7 in at least triplicate. Supernatants were collected and stored at - 2 0 ° C until use. Immunoblotting T H P - 1 cells were cultured as above. O n the day of the assay the cells were gently detached using Ce l l Dissociation Solution (Sigma). Cells were washed, re-suspended in media and added to 5 ml polystyrene tubes (Becton Dickinson) and allowed to rest for 2 hours. Approximately 1 x 10 6 T H P - 1 cells were used per condition. Cells were then stimulated by adding L P S , L L - 3 7 or water as a vehicle control. After stimulation the cells were centrifuged, washed one time with ice-cold P B S with I m M vanadate and nuclear extracts were isolated using N E - P E R Nuclear and Cytoplasmic Extraction Reagents K i t (Pierce, Fisher Canada, O N ) as per the manufacturer's directions. The protein concentrations o f the lysates were quantitated using a B C A assay (Pierce, Fisher Canada, O N ) . Lysate (7.5 pg) was loaded onto 1.0 m m thick gels, which were run at 100 V for approximately 2-3 hours. Proteins were transferred to P V D F filters for 80 min at 80 V . The filters were blocked for 1 hour at room temperature with 5% skimmed milk in T B S T (10 m M T r i s - H C l p H 8, 150 m M NaCI , 0.1% Tween-20). The filters were then incubated overnight at 4 ° C with the anti-p50 or anti-p65 (Cell Signalling Technology) monoclonal antibodies. Immunoreactive bands were detected using horseradish peroxidase-conjugated donkey anti-rabbit IgG antibodies (Amersham Pharmacia) and chemiluminescence detection (Sigma). 82 5.3 Results LL-37 induces transcription of chemokines and cytokines T o determine whether L L - 3 7 induced transcription o f chemokines and cytokines at early time points, monocytes from three donors were treated with 50 pg/ml o f L L - 3 7 for 30, 60, or 90 minutes. R N A was collected and quantitative R T - P C R ( q R T - P C R ) was performed. Each q R T -P C R reaction was performed in duplicate. The increases in expression were calculated by dividing the expression level in the L L - 3 7 treated samples by the expression level in the vehicle control treated samples. Increases in chemokine transcripts for the C C family chemokines M C P -1, M l P - l a , M I P - i p , for IL-8 and the cytokine IL-6 (Figure 5.1) occurred as a result o f L L - 3 7 treatment in all donors tested. However, there was considerable variation in both the amplitude and the kinetics o f the response between donors. For example, transcription o f M C P - 1 peaked at 60 min for LL-37-treated monocytes from donors 1 and 2 and at 30 min for donor 3 (Figure 5.1). Peak transcription of M I P - l p occurred for donors 2 and 3 at 30 min, however there was no increase in transcription at 30 or 60 min for donor 1 and instead, transcription was greatly increased at 90 min. Semi-quantitative P C R was also performed for the pro-inflammatory cytokines T N F - a , IL-12p40 and I L - l p \ However, no detectable increase in transcription of these genes was found due to L L - 3 7 treatment at 38 rounds o f amplification (data not shown). 83 i i Donor 1 • Donor 2 • Donor 3 30 60 Time (min) 30 60 Time (min) 30 CD OJ an 20 O old 10 MIP-ip/ . CCL4 1 30 60 Time (min) 90 30 60 Time (min) 60 Time (min) Figure 5.1. LL-37 induction of chemokine and cytokine transcription in primary monocytes. Monocytes from three different donors were stimulated with LL-37 (50 ug/ml) or endotoxin free water as a vehicle control for 30, 60 & 90 min. R N A was collected and quantitative RT-PCR was performed. Bars are the average of duplicate qRT-PCR reactions. Changes in gene expression were measured by dividing the expression level of the LL-37 treated samples by the expression level of the vehicle control treated samples. LL-37 induces transcription of CC family chemokines (A), a neutrophil chemokine (B) and at least one cytokine (C). Early production of chemokines is a result of de novo transcription which is controlled in part by activation of both the p38 and ERK1/2 kinases Many genes which are induced in response to stress or detection of infection demonstrate increases in expression which are due to increased stabilisation of the mRNA transcript rather than increases in de novo transcription (33). One possible explanation for the very rapid LL-37-induced increases in chemokine transcripts was that an LL-37-induced transcript stabilisation event was occurring. To test whether transcript stabilisation was increased after stimulation with 84 L L - 3 7 , cells were pre-treated with actinomycin D , a known inhibitor o f m R N A synthesis (34). If increases in the m R N A transcript were due to a stabilisation event, there would be no apparent decrease in gene expression with actinomycin D treatment. Conversely i f de novo transcription was required then actinomycin D treatment should result in a decrease in the chemokine transcripts. Monocytes were pre-treated with actinomycin D (5 pg/ml) for 30 min and then stimulated with L L - 3 7 . Act inomycin D was present for the entire course o f the experiment. R N A was collected, c D N A was synthesised and q R T - P C R was performed. Each q R T - P C R reaction was performed in duplicate and bars represent the average o f the duplicate experiments. One representative donor o f two shown. Pre-treatment with actinomycin D reduced transcription of chemokine genes to values close to baseline indicating that increases in transcription were primarily due to de novo transcription (Figure 5.2). 41 0 CD 8 C CD sz O A TJ 4 O LL 0 IL-8/CXCL8 • 0 pg/ml LL-37 50 pg/ml LL-37 V C Actinomycin D & 8 I 6 o TJ O 4 : 2 0 MIP-1a/CCL3 V C MIP-1p7CCL4 i • V C Actinomycin D MCP -1 /CCL2 Actinomycin D Figure 5.2. Increases in chemokine transcription as a result o f de novo transcript ional events. Monocytes from 2 donors were pre-treated with actinomycin D (5 pg/ml) or a vehicle control ( V C ) for 30 min then stimulated with L L - 3 7 (50 pg/ml) for 30 min. Increases in the chemokine transcripts were detected by quantitative P C R . Treatment with actinomycin D prior to L L - 3 7 stimulation reduced the transcription o f chemokine genes to baseline, indicating that chemokine transcription in response to L L - 3 7 was primarily due to de novo transcriptional events. Changes in the transcript are expressed as expression in L L -37-treated samples/expression in untreated samples. Each q R T - P C R reaction was performed in duplicate and bars represent the average o f these duplicates. One representative donor o f two shown. L L - 3 7 induced transcription of certain chemokines at 4 hr has previously been demonstrated to be controlled by LL-37- induced activation o f both the p38 and E R K 1 / 2 kinases [Chapter 3, Figure 3.3 & 3.7; (24)] but it was not determined i f activation o f these kinases was a 85 requirement for early transcriptional events. Treatment with inhibitors o f either p38 or E R K 1 / 2 (10 u M ) individually resulted in slight reductions in transcription o f chemokine genes in monocytes from two donors, however, when both inhibitors were used in combination the transcription of the chemokine genes was completely abrogated suggesting that activation o f both kinases is required for maximal transcription (Figure 5.3). F igure 5.3. Effects of inhibitors of the p38 and E R K 1 / 2 kinases on L L - 3 7 induced transcr ipt ion . Monocytes from two donors were pre-treated with an inhibitor for either E R K 1 / 2 or p38 kinase (10 u M ) or both inhibitors for 1 hr. Cells were then stimulated with L L - 3 7 (50 ug/ml) for 30 or 60 m i n . R N A was collected, c D N A was created and semi-quantitative R T - P C R was performed for M C P - 1 (60' L L - 3 7 treatment, 35 cycles), IL-8 (30' L L - 3 7 treatment, 26 cycle), M l P - l a (30' L L - 3 7 treatment, 28 cycles) and M I P - l p (30 'LL-37 treatment, 30 cycles). P C R products were run on a gel and normalised to G A P D H (30 & 60', 31 cycles).Treatment with either M A P K inhibitor individually resulted in a slight reduction in transcription but treatment with both inhibitors resulted in a complete abrogation of chemokine transcription. A ) One representative donor o f two shown. B) Average quantified results o f two donors shown. Bars represent the average of both donors and the error bars represent the range of responses between both donors. De novo protein synthesis is required for IL-8 release Certain chemokines have been demonstrated to be released independently o f de novo protein production (35). L L - 3 7 induces IL-8 release into the supernatants which can be detected as early as 15 minutes after stimulation (Figure 5.4) T o test whether L L - 3 7 induced IL-8 release was a result o f de novo protein synthesis or release from internal stores, monocytes were pre-treated with cycloheximide prior to stimulation with L L - 3 7 . Cycloheximide is an inhibitor o f protein synthesis (36). Thus, i f IL-8 levels in the supernatant decreased upon cycloheximide treatment, this would imply that IL-8 release is most likely subsequent to new protein synthesis. LL-37 E R K inhibitor p38 inhibitor M C P - 1 B c o '</) cn CD cL X LU CD > ro CD cm G A P D H 6000 4000 2000 LL-37 LL-37 + ERK inhibitor LL-37 + LL-37 + p38 ERK &p38 inhibitor inhibitor Treatment 86 L L - 3 7 induced release o f IL-8 in a time dependent manner in both donor tested. The presence of cycloheximide (1 pg/ml) substantially reduced the amount o f IL-8 released at most time points indicating that IL-8 release is primarily due to de novo translation. , , 800, , Time (min) T i m e ( m i n ) Figure 5.4. Effect of cycloheximide treatment on IL-8 release f r o m monocytes in response to LL-37 s t imulat ion. Monocytes were pre-treated with cycloheximide (1 pg/ml) or a vehicle control for 30 min prior to L L - 3 7 (50 pg/ml) stimulation. IL-8 in the supernatants was detected by E L I S A . Bars represent the mean o f three wells minus background levels o f IL-8 ± standard error of the mean. Results from both donors shown. The p38 and ERK 1/2 kinases are involved in early IL-8 release I have previously demonstrated that L L - 3 7 activated both the p38 and E R K 1/2 kinases after 15 min of stimulation and inhibition of this response reduced IL-8 production at 4 hrs [Chapter 3, Figures 3.3 & 3.7, (24)]. T o determine what the involvement o f these kinases in early IL-8 release, monocytes were pre-incubated with an inhibitor for either kinase for 1 hr prior to stimulation with L L - 3 7 (50 pg/ml). Supernatants were collected after 15, 30 and 60 minutes o f L L - 3 7 treatment and the amount of IL-8 was assessed by E L I S A . Interestingly, pre-treatment with the p38 inhibitor resulted in a greater reduction in IL-8 production than did pre-treatment with an E R K 1/2 inhibitor (Figure 5.5), in contrast to inhibition of transcription o f the IL-8 gene which appeared to be equally reduced by both kinase inhibitors (Figure 5.3). 87 800 2 400 • LL-37 alone D + 5 nM ERK inhibitor • + 5 nM p38 inhibitor 15 30 Time (min) F i g u r e 5.5. R e q u i r e m e n t of both p38 and E R K 1 / 2 in the early release of I L - 8 in response to L L - 3 7 . Monocytes from two donors were pre-treated with inhibitors for either p38 (SB 203580) or E R K 1 / 2 (PD98059) kinases (5 u M ) for 60 min prior to L L - 3 7 (50 ug/ml) stimulation. Supernatants were collected and assayed for IL-8 production by E L I S A . Treatment with either kinase inhibitor reduced the amount o f IL-8 release. Bars are the average o f three wells minus the background levels o f IL-8 production ± standard error. Results from both donors are shown. LL-3 7 induces translocation of p50 and this may be involved in chemokine transcription & secretion IL-8 transcription has been demonstrated to be highly dependent upon activation of N F -KB therefore the ability o f L L - 3 7 to induce activation o f N F - K B was investigated. A monocyte cell line (THP-1) was treated with 10 ug/ml of L L - 3 7 , or vehicle control, for 20 min at which time nuclear protein extracts were collected. In general N F - K B subunits are maintained in the cytoplasm and upon stimulation with activating agents the subunits become phosphorylated and translocate to the nucleus. L P S is known to induce translocation of both the p65 and p50 subunits and thus was utilised as a positive control. T o determine i f activation o f N F - K B occurred in response to L L - 3 7 treatment, Western blots o f the nuclear extracts were probed for the presence of two o f the major subunits. L L - 3 7 stimulation caused the translocation o f the p50 but not the p65 subunit to occur (Figure 5.6). LL-37 -p65 p50 LPS Figure 5.6. L L - 3 7 induced translocat ion of N F - K B subunits. A monocyte-like cell line ( T H P - 1 ) was stimulated with L L - 3 7 (10 ug/ml) for 20 min and nuclear extracts were collected. Western blots for the p50 and p65 subunits o f N F - K B were performed. L P S (10 ng/ml) which is a known inducer o f both p50 and p65 translocation, was used as a positive control. One representative experiment of three is shown. 88 5.4 Discussion I have demonstrated that L L - 3 7 stimulation o f peripheral blood derived monocytes induced the transcription and release o f the major neutrophil chemokine, IL-8 and the transcription o f four C C family chemokines. The induction of these chemokines was rapid, with increases at the transcriptional level occurring within 30 minutes. Cytokine and chemokine genes often have A U - r i c h sequences in the 3' untranslated region o f their m R N A . The presence of A U -rich sequences leads to rapid degradation of the transcripts unless they are bound by stabilisation proteins (37). This provides a mechanism by which rapid translation from the m R N A transcript can occur in response to inflammatory stimuli. Because IL-8 and other chemokines are known to contain A U - r i c h regions, the possibility that transcript levels were increased due to a stabilisation event was examined. Despite the rapid nature of this response, the substantial inhibition of IL-8 gene expression by actinomycin D or cycloheximide indicated that the production of these chemokines was most likely due to de novo transcription and protein production. The involvement o f the M A P K in IL-8 transcription and release has been well documented in a variety o f cell types and model systems (38-40). It has been demonstrated that activation of the E R K 1/2 and p38 kinases are involved in both transcriptional and post-transcriptional regulation of IL-8 although there is substantial variability in the contribution of these kinases which depends in part on the type o f stimuli and the target cell [reviewed in (41)]. Because o f the well-documented involvement of N F - K B in IL-8 transcription, I investigated the ability o f L L - 3 7 to induce N F - K B translocation. The observation that L L - 3 7 induced the translocation of the p50 subunit o f N F - K B but not the p65 subunit was a departure from the consensus within the literature. In a general model o f IL-8 production, the binding of N F - K B (p50/p65) and AP-1 to the promoter sequence is required for transcription while p38 activation is required for transcript stabilisation and protein production (42). The observation that activation of the p65 subunit is not required for IL-8 transcription in L L - 3 7 - treated monocytes is not entirely consistent with published reports, which indicate that although many N F - K B homo- and hetero- dimer combinations bind to the IL-8 promotor, subunits containing the p65 subunit are the major contributor to transcription (43). However there are several reports indicating that IL-8 release may occur independently of N F - K B activation and it is possible that this is the case for L L - 3 7 stimulated cells (44, 45). Other chemokines including M C P - 1 are also regulated at the transcriptional and post-translational levels by p38 and N F - K B (p65/p50)(46). This generalisation may however not be applicable to all situations or chemokines however as, for example, transcription of the M I P - i p gene requires p50 and Re lB activation (47, 48). It is obvious that the 89 control of chemokine genes at the transcriptional level is quite complicated and although there may be common themes there are differences between cell types, the type of stimuli, mouse and human cells and primary cells versus cell lines. Future studies should focus on the composition of the N F - K B dimers stimulated by LL-37. This study suggests but does not demonstrate that N F - K B is involved in LL-37-induced transcription of IL-8 and other CC-family chemokines. It is not evident whether N F - K B activation occurs in parallel to or downstream of MAPK activation (Figure 5.7). The requirement of ERK1/2 and p38 upstream of N F - K B activation has been discovered in a number of different systems (49, 50) and thus future experiments using inhibitors of these kinases should be performed to determine if LL-37 induced MAPK activation is upstream of or parallel to N F - K B activation. N F - K B ERK1/2 -P P 3 8 - P B ERK1/2 -P p38-P Figure 5.7. Two models of signalling pathway activation in LL-37 treated monocytes. A) N F -K B activation could occur in parallel with ERK 1/2 and p38 activation. B ) N F - K B activation could occur downstream of activation of the ERK 1/2 and or p38 kinases. 90 Certain diseases such as cystic fibrosis and psoriasis are characterised by both high levels of L L - 3 7 and high levels o f neutrophil markers and neutrophil specific chemokines such as IL-8 (12, 27, 51, 52). Elevated levels o f L L - 3 7 at sites o f infection correlate with elevated numbers of leukocytes including granulocytes and mononuclear cells (27). However, it is not known i f the increased number o f leukocytes is due to the chemotactic properties o f the peptide itself (21, 22) or whether these cells arrive at sites of high concentrations of L L - 3 7 due to the chemokine-inducing properties o f the peptide. Although L L - 3 7 is chemotactic for a number of cell types including neutrophils and monocytes (21) it is much less potent (50 u M ) than conventional chemokines such as IL-8 (1 n M ) (53). A s a result o f the ability o f L L - 3 7 to induce the production of chemokines in a rapid fashion it may not be the elevated levels o f L L - 3 7 that lead to increases in leukocyte infiltration but rather the increased levels o f chemokines produced in response to L L - 3 7 exposure which may contribute to LL-37-associated leukocyte infiltration. In certain diseases it is the tissue damage due to excessive inflammatory cell infiltration which appears to be the major contributor to pathology. In these cases it wi l l be necessary to develop therapies which target not only the causative agent but also target the detrimental host response. Nove l contributors to leukocyte recruitment such as L L - 3 7 are potential targets for such therapies. 5.5 B i b l i o g r a p h y 1. Schuh, J . M . , K . 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Biochem Pharmacol 69:385. 94 6.0 Discussion - The Primary Role of LL-37 In Vivo May Be Immunomodulatory8 6.0 DISCUSSION - T H E P R I M A R Y R O L E OF LL-37 IN VIVO M A Y B E I M M U N O M O D U L A T O R Y 95 6.1 E A R L Y EVIDENCE THAT HOST DEFENCE PEPTIDES M I G H T H A V E IMMUNOMODULATORY FUNCTIONS 96 6.2 T H E ANTIBACTERIAL A N D IMMUNOMODULATORY PROPERTIES OF L L - 3 7 ARE PHYSIOLOGICALLY DISTINCT 99 6.3 INTERACTIONS B E T W E E N L L - 3 7 A N D EUKARYOTIC C E L L S 100 6.4 L L - 3 7 INTERACTIONS WITH THE EFFECTOR C E L L S OF THE I M M U N E RESPONSE A R E SPECIFIC B U T N O T NECESSARILY RECEPTOR MEDIATED 101 6.5 CHEMOTAXIS , L E U K O C Y T E INFILTRATION A N D L L - 3 7 105 6.6 SYNERGISTIC INTERACTIONS BETWEEN L L - 3 7 A N D COMPONENTS OF THE INFLAMMATORY M I L E U 107 6.7 FUTURE DIRECTIONS - T H E R O L E OF L L - 3 7 IN H E A L T H & DISEASE A N D ITS POTENTIAL AS A N O V E L THERAPEUTIC A G E N T 109 6.8 B IBLIOGRAPHY 110 5 Figure 6.1 is supplemental data submitted for publication in L a u , Y . E . , Bowdish, D . M . E . , Hancock, R . E . W . , Davidson, D . J . 2005. Apoptosis o f airway epithelial cells: human serum sensitive induction by the cathelicidin L L - 3 7 . Submitted. 95 6.1 Early Evidence that Host Defence Peptides Might Have Immunomodulatory Functions When I began my degree in September 2000, the number of published reports on the immunomodulatory properties o f L L - 3 7 was minimal. There was one report that L L - 3 7 was a chemoattractant for monocytes, neutrophils and T cells (1) and one report that increased expression o f L L - 3 7 could protect mice against bacterial or L P S induced shock (2). Preliminary unpublished data from our laboratory suggested that L L - 3 7 had anti-endotoxin properties, which might be not be completely explained by L L - 3 7 ' s ability to bind and neutralise L P S . Moreover, L L - 3 7 could induce chemokine production from the mouse macrophage cell line, R A W 264.7, [later published in (3)]. There were scattered reports that other host defence peptides had immunomodulatory properties such as the ability to activate complement (4), promote chemotaxis (5) and alter cell signalling (6) but the emphasis in the published literature was clearly on the antimicrobial properties o f these peptides. In fact in 2000 published reports on the antimicrobial functions of these peptides were approximately 10-fold more prevalent than papers on their immunomodulatory properties. The emphasis in the published literature changed slightly upon the discovery that the P-defensins bound to a receptor on immature dendritic cells and T cells and that this receptor was linked to chemotaxis (7). This receptor was identified as C C R 6 , whose known ligand was the chemokine L A R C / M I P - 3 a (8). Chemokines and host defence peptides are both positively charged, or have regions of high positive charge and have antimicrobial and chemotactic properties and thus it was suggested that certain defensins were evolutionarily related to chemokines (9-11). This contributed to the enticing hypothesis that the chemotactic properties o f these peptides could be explained in part by their evolutionary and structural relatedness to chemokines (12). It followed that host defence peptides might have a broader role in host defence, however it was difficult to determine i f this was physiologically relevant due to the high concentrations required for chemotaxis. The discovery o f a receptor for some o f these peptides also indicated that interactions between host defence peptides and eukaryotic cells might not simply be a result o f interactions with membranes. These peptides have been demonstrated to interact with both prokaryote and eukaryote model membranes [reviewed in (13)] and in 2000 the literature favoured the hypothesis that the high concentrations of L L - 3 7 and other host peptides used in immunomodulatory studies were sufficient to induce cytotoxicity and thus that the observed 96 immunomodulatory effects o f these peptides might be a secondary response to this cytotoxicity. I and others have demonstrated that at the concentrations used in these studies, L L - 3 7 was not cytotoxic [Chapter 2, Figure 2.1; Chapter 3, Figure 3.6]. Another common criticism of research on the immunomodulatory properties o f L L - 3 7 was that the concentrations o f peptides used in in vitro studies are higher than would be expected to be found in vivo. Although very little was known about the concentrations o f this peptide at the time this thesis work began, it is now known that the concentrations of L L - 3 7 found at sites o f infection and inflammation are within the range of concentrations used in this thesis (Table 6.1). 97 Table 6.1. Concentrations of hCAP-18/LL-37 in health and disease. Body Site F o r m Produced by Concentration in Health (pg/ml) Concentration in Disease (pg/ml) References Lungs h C A P - 1 8 / L L - 3 7 / alternatively processed forms 3 Neutrophils, epithelia, submucosal glands, alveolar macrophages 2.5 - 20 2 . 5 - 3 0 (14-16) Nasal fluid L L - 3 7 Epithelia, mucosal glands, leukocytes +/- +++4 (17) B lood / Plasma h C A P - 1 8 ? 1.2 * (18) Mouth h C A P - 1 8 / alternatively processed forms / L L - 3 7 Salivary glands, infiltration of leukocytes + ++6 (19-21) Semen h C A P - 1 8 1 epithelium of the epididymis 42 - 143 * (22) Skin L L - 3 7 Keratinocytes / infiltrating leukocytes +/- 1300 (23) Female reproductive tract h C A P - 1 8 / A L L - 3 8 2 Deposited in ejaculatory fluid 12-40 * (24) Gastro-intestinal tract h C A P - 1 8 / L L - 3 7 Epithelial cells or Infiltrating 5 leukocytes +++ +/- (25) Breastmilk L L - 3 7 Ductule cells, leukocytes? 140 * (26) * Assay not done. 1 A post-translationally modified form of h C A P - 1 8 was also observed but not characterised. 2 A L L - 3 8 is an alternatively process form only detectable after sexual intercourse. 3 Western blot shown detected both h C A P - 1 8 & L L - 3 7 as well as uncharacterised bands of intermediate size(16). 4 The transcript for h C A P - 1 8 and immunostaining for L L - 3 7 was found at low levels in some of the healthy controls and was found at high levels for most o f the patients with nasal inflammatory disease 5 In healthy tissue h C A P - 1 8 / L L - 3 7 is expressed primarily in epithelial cells, in diseased tissues it is found primarily in infiltrating leukocytes 6 Expression of h C A P - 1 8 was increased at the transcript level in patients with chronic sialadenitis (21). Since 2000, multiple mouse knockouts that are deficient in host defence peptide expression have demonstrated that reduced expression of these peptides results in a reduced ability to clear infection and increased bacterial counts (27, 28). Consistent with this, mice that overexpress host defence peptides are resistant to infection and carry lower bacterial numbers 98 (29). A t first blush this might appear to indicate that the expression o f host defence peptides can result in direct bacterial kil l ing. However, these experiments do not distinguish between direct and indirect bacterial kil l ing. I and others have hypothesised that the perceived reduction in microbial counts in in vivo experiments might be due, at least in part, to the immunomodulatory properties o f these peptides. This perspective is gaining support as an increasing body of evidence suggests that L L - 3 7 and other host defence peptides have a variety of immunomodulatory properties in vitro and in vivo. This change in perspective is particularly apparent when examining the shift in the focus of published reports on the antimicrobial properties o f host defence peptides. In 2005 almost an equal number o f reports addressed the antimicrobial activity versus the immunomodulatory properties o f host defence peptides. Below I discuss the accumulating evidence for my hypothesis that L L - 3 7 is primarily an immunomodulatory agent. 6.2 The Antibacterial and Immunomodulatory Properties of LL-37 are Physiologically Distinct Increasing evidence suggests that a primary role o f various cationic host defence peptides in vivo may be to modulate or prime the immune response. Whi le there is no doubt that under some circumstances these peptides have antimicrobial activity in vivo, for example at the mg/ml concentrations at which a-defensins are found in the lysosomes o f neutrophils, this may not be the case for other host defence peptides such as L L - 3 7 or at other body locations such as at mucosal surfaces. Neutrophils are major contributors to antibacterial defences and are expected to be major sources o f host defence peptides. The unprocessed form o f h C A P - 1 8 is found at extremely high concentrations in the granules o f neutrophils (-630 u.g per 10 9 cells) and the processed form, L L - 3 7 is expected to be found at high concentrations at sites of neutrophil degranulation since neutrophils release both h C A P - 1 8 and proteinase 3, the enzyme that cleaves the peptide to its active form (18, 30). Thus it is possible that antimicrobial activity at sites o f neutrophil degranulation might be attributable in part to the presence o f L L - 3 7 . It is not clear, however, whether antimicrobial activity is a general property o f this peptide at mucosal surfaces where it is produced by epithelial cells and is constitutively found at lower concentrations. Although there are certain body fluids in which innate antimicrobial activity is attributed, at least in part, to the presence of L L - 3 7 (e.g. bronchoalveolar lavage fluid from patients with sarcoidosis (16)) oftentimes the antimicrobial activity o f L L - 3 7 in vivo has been inferred from the correlation o f high concentrations of the peptide with the absence o f bacterial infection. In psoriasis, for example, h C A P - 1 8 / L L - 3 7 has been demonstrated to be present at extremely high 99 concentrations (23, 31). Patients with psoriasis rarely have skin infections whereas patients with atopic dermatitis, a disease in which h C A P - 1 8 / L L - 3 7 expression is generally not detectable, are frequently colonised with S. aureus (23). Although there may be a correlation between high concentrations of L L - 3 7 and reduced bacterial numbers this does not necessarily mean that L L -37 kills bacteria directly. The possibility has not been thoroughly investigated as to whether L L -37 and other host defence peptides might reduce bacterial numbers through modulation or enhancement of the immune response, for example by recruiting neutrophils or other effector cells o f the innate immune response. The assumption that high levels o f host defence peptides lead to reductions in bacterial numbers does not hold up in other biological situations, such as in the lungs of cystic fibrosis patients. These patients have elevated concentrations o f L L - 3 7 in the bronchoalveolar lavage when compared to healthy controls but are chronically colonised with pathogens such as P. aeruginosa (15). Care must also be taken to distinguish between the presence of the antimicrobially inactive unprocessed form o f the peptide, h C A P - 1 8 and the processed form, L L - 3 7 , which has the antimicrobial activity. In certain body fluids the presence of h C A P - 1 8 is high (up to 120 ug/ml) but no antibacterial activity is detected (22, 25). Although it is not clear i f h C A P - 1 8 functions as an immunomodulatory agent, it is presumed to be an important component o f host defence as it is constitutively expressed in lymphocytes and macrophages, is produced at mucosal surfaces by epithelial cells, is up-regulated in response to infection and inflammation, and is released upon degranulation o f neutrophils (32-37). In contrast to studies on the antimicrobial properties o f L L - 3 7 , the studies on the immunomodulatory properties o f the peptide are performed in standard tissue culture medium that has physiological concentrations of salts. A s previously stated, the antibacterial activity o f L L - 3 7 is ablated in physiological concentrations o f salt while the immunomodulatory properties remain intact. Although it is obviously quite difficult to separate the immunomodulatory functions o f this peptide from its antimicrobial functions in vivo, it is fairly easy to distinguish the differences in these properties in vitro. The culture conditions used in this thesis did not support the antibacterial activity o f this peptide [Chapter 1, Figure 1.2] but did mimic ionic conditions in vivo and thus are an acceptable model in which to study the non-antimicrobial functions o f L L - 3 7 and its interactions with host cells. 6.3 Interactions Between L L - 3 7 and E u k a r y o t i c Cel ls Due to the amphipathic properties o f some host defence peptides, their affinity for membrane binding and their membrane disrupting effects at high concentrations for both prokaryote and eukaryote model membranes (38, 39) it was initially believed that many of the 100 observed immunomodulatory properties might be a result o f membrane stress and thus non-specific. There are not many examples in the literature o f host defence peptide-like immunomodulatory properties resulting from non-specific membrane disruption such as the induction o f cytokine production (40). The perceived cytotoxicity o f these peptides for eukaryotic cells may be an artifact o f performing these assays in low ionic strength buffer, or in the absence o f serum, as the presence of serum abrogates the cytotoxic effect in a number o f different cell types (41-43). The experiments in this thesis were performed in standard tissue culture media using 10 % foetal calf serum, conditions under which I demonstrated that L L - 3 7 was not cytotoxic for the cells used in these studies [Chapter 2, Figure 2.1, Chapter 3, Figure 3.6]. I also demonstrated that peptide-induced IL-8 production was not a consequence o f cytotoxicity, as the cytotoxic peptide CP29 did not induce IL-8 production [Chapter 3, Figure 3.6]. Because o f these and analogous observations, the current consensus in the literature is that these peptides are not merely non-specific membrane disrupting agents. This change occurred due to published reports indicating that the immunomodulatory properties o f host defence peptides are not non-specific, but rather result from specific interaction with certain subsets o f the cells o f the innate and adaptive immune response, as well as epithelial cells, keratinocytes and cells o f the reproductive tract (1, 22, 41, 44). I demonstrated here that L L - 3 7 induced activation o f the M A P K in monocytes and epithelial cells but not B or T cells [Chapter 3, Figure 3.3], and this observation implied that there was specificity in responses to exposure to L L - 3 7 . The directed nature o f these responses indicates that there may even be a receptor or receptors for L L - 3 7 that is linked to the immunomodulatory properties o f this peptide. 6.4 L L - 3 7 Interactions With the Effector Cells of the Immune Response Are Specific But Not Necessarily Receptor Mediated T o date there have been a number of receptors associated with L L - 3 7 induced immunomodulation including formyl peptide receptor like-1 ( F P R L - 1 ) , P 2 X 7 , epidermal growth factor receptor ( E G F R ) , and as yet unidentified high and low affinity receptors (1, 45, 46). T w o of these putative receptors, P 2 X 7 and E G F R are not proposed to be direct receptors for L L - 3 7 . For example, although L L - 3 7 mediated I L - i p processing can be blocked by inhibitors o f the P 2 X 7 receptor, other effects such as an increase in membrane permeability and size and shape changes cannot be inhibited. The authors of this study propose that L L - 3 7 induces an upstream activation event that results in activation of P 2 X 7 and appears to be independent of other identified receptors (47). This upstream event must occur very rapidly, as altered membrane 101 permeability is observed in as little as one minute after L L - 3 7 stimulation. Similarly it is not proposed that L L - 3 7 binds E G F R directly, but rather that it induces cleavage o f surface bound metalloproteases in an undefined fashion, the result o f which is transactivation of E G F R (46). Inhibitors o f E G F R associated tyrosine kinases block L L - 3 7 induced IL-8 production in airway epithelial cells and partially inhibit IL-8 production f r o m . keratinocytes (44, 46). Interestingly the positively charged L P S binding lipopeptide, polymyxin B , also interacts with P 2 X 7 (48) and enhances E G F R mediated endocytosis (49), indicating that these receptors may have a general affinity for small positively charged peptides. T o date the only receptor to which L L - 3 7 has been proposed to bind directly is the pertussis-toxin-sensitive, G-protein coupled receptor F P R L - 1 (1). This receptor is a G-protein coupled receptor which can be inhibited by treatment with pertussis toxin. L L - 3 7 mediated chemotaxis can be inhibited either by an agonist o f this receptor or by pertussis toxin. Further complicating this story is the observation that IL-8 production in keratinocytes and mast cell chemotaxis is reduced but not abolished upon treatment with pertussis toxin, indicating that these events are linked to a Gi-protein coupled receptor, although this receptor cannot be F P R L - 1 , as agonists for F P R L - 1 do not have the same effects (44-46). However, other L L - 3 7 mediated effects such as M A P kinase activation and IL-8 production in monocytes are not pertussis-toxin-sensitive, indicating that L L - 3 7 may mediate these events through other receptors or other mechanisms o f action (41). These contradictory and sometimes conflicting reports suggest that L L - 3 7 , and possibly other host defence peptides, may not actually work through a conventional ligand-receptor interaction. For example, host defence peptides have often been compared to chemokines; however, unlike the majority o f chemokines, L L - 3 7 has cross-species reactivity. Whereas most chemokines have greatly reduced or abolished activity across species (50), L L - 3 7 has been shown to exert its immunomodulatory effects on cells from a wide range o f species including rat (45, 51), mouse (3, 52), rabbit (53), and humans (54). Although this does not eliminate the possibility o f a non-specific receptor, it is an unusual phenomenon which requires more investigation. A recent observation that L L - 3 7 synthesised with D-amino acids ( D - L L - 3 7 ) is a more potent inducer of IL-8 production by keratinocytes than is the conventionally synthesised L- form raises some interesting questions concerning whether L L - 3 7 has a conventional receptor. Similarly the L - and D - forms induced membrane permeability in a dose dependant manner, although the D - L L - 3 7 induces maximal permeability at a lower concentration (44). Thus the 102 authors of this study propose that LL-37 and D-LL-37 may interact with membranes rather than with a conventional receptor. Although many receptors do not recognise ligands made from D-amino acids, it has been proposed that receptors that bind a wide range of proteins and peptides such as the signalling protein calmodulin and the major histocompatibilty complex (MHC), might also be able to bind to peptides composed of D-amino acids (55, 56). Consistent with this it has been found that these receptors recognise lower-order structural properties of their ligands, specifically regions of segmental or helical amphipathicity. LL-37 is an alpha-helical peptide with such regions of amphipathicity and thus might interact with a promiscuous binding protein. Interesting differences exist between LL-37 mediated signalling events in epithelial cells and monocytes, and these differences are substantial enough to indicate that LL-37 may have a different receptor or interaction with these two cell types. In monocytes LL-37 does not activate either the ERK1/2 or p38 kinases with the resultant production of IL-8 unless there is serum present (41). In experiments performed in epithelial cells the presence of serum is not a requirement for MAPK activation or IL-8 production (41, 46). The type of serum also has profound effects on LL-37 interactions with epithelial cells. In the presence of 10% human serum in the culture medium IL-8 production is abolished in an airway epithelial cell line (43) but this did not occur in a monocyte cell line (Figure 6.1). 1400 1200 — 1000 E L L - 3 7 (pg/ml) Figure 6.1. The effect of human serum on IL-8 production from THP-1 cells. THP-1 cells were cultured as previously described. Cells were stimulated with LL-37 for 4 hr in the presence of either 10% human serum (HS) or 10% foetal calf serum (FCS) in the culture medium. IL-8 was produced in a dose dependant manner in the presence of both HS and FCS. Bars are the average of three independent experiments ± standard deviation from the mean. A single asterisk indicates p<0.004, double asterisk indicates p<0.0008. The observation that airway epithelial cells do not appear to require a serum component to respond to LL-37 is biologically relevant. The lung is an opsonin poor environment in which serum components are not present unless there is a serious breach in the integrity of the epithelial barrier. Thus LL-37 induced signalling in pulmonary epithelial cells would be expected to occur independent of serum proteins. The high rate of LL-37 induced cytotoxicity and apoptosis which occurs in these cell types under serum free conditions is abrogated by the presence of HDLs (43). Consistent with this, HDLs are present in the lung and play an important role in modulating the 103 inflammatory response in response to infection (57-59). In a manuscript prepared in collaboration, although we did not formally demonstrate that exogenous H D L s were inhibitory to cytotoxicity and that the presence of H D L s in serum reduced IL-8 production, it was demonstrated that the presence of human serum, in which the concentration o f H D L s is high, abrogated IL-8 production (43). Thus, the presence o f other L L - 3 7 binding components such as lipoproteins may thus be crucial to the in vivo biological functions of this peptide. In other biological contexts the presence o f serum may be important for L L - 3 7 signalling. It has been demonstrated that h C A P - 1 8 is predominantly bound to low density lipoprotein and/or very low density lipoprotein particles in human serum (60) whereas L L - 3 7 binds predominantly to apolipoprotein A - l , a component of H D L in human serum (61). Although apolipoprotein-Al blocks the antimicrobial and cytotoxic properties o f the peptide, it is not known i f this binding alters its immunomodulatory properties such as activation of signalling pathways and the induction o f IL-8 production. L L - 3 7 may bind to and interact with epithelial cells with relatively high affinity; however, binding to monocytes requires the presence o f lipoproteins which would bind many L L - 3 7 molecules and thus result in a high avidity interaction between bound L L - 3 7 and other types o f receptors such as the scavenger receptors. A n y model that suggests that there is a specific receptor for L L - 3 7 must account for the transient membrane permeabilization observed after L L - 3 7 treatment o f some cell types. In a LPS-pr imed monocyte-like cell line, L L - 3 7 causes a transient increase in lactate dehydrogenase ( L D H ) release, a marker for cell permeability (47) and this release cannot be blocked by inhibitors o f the P 2 X 7 receptor, indicating that it is independent o f activation of this receptor. These increases in membrane permeability correlate with L L - 3 7 induced increases in I L - i p processing (47). In keratinocytes there is no observed increase in L D H release, however there is evidence that some increase in membrane permeability occurs as propidium iodine incorporation occurs in a dose dependent manner (44). This membrane permeability correlates with IL-8 release by these cells. Membrane permeabilization may be an essential component of the mechanism o f action of L L - 3 7 , as it has been demonstrated that L L - 3 7 must enter epithelial cells for IL-8 production to occur (62). The evidence linking the immunomodulatory properties o f L L -37 with transient increases in membrane permeability, as a potential mechanism of action, is currently weak. It is also difficult to reconcile this mode o f action with cell specificity, however, this observation has the potential to provide a unifying link between the observed activation of a number of different receptors and signalling pathways, and to explain the cross-species interaction of the peptides 104 6 . 5 Chemotaxis, Leukocyte Infiltration and L L - 3 7 One o f the conserved properties o f cathelicidins from a number o f species is the ability to induce chemotaxis and it is believed that the increased expression o f cathelicidins during the course of infection and inflammation may play a role in leukocyte recruitment (63-65). In some situations increased concentrations o f L L - 3 7 correlate with disruption o f the integrity o f the epithelial barrier and as such may contribute to the pathology o f disease (43, 66). In cystic fibrosis, the increased concentration of L L - 3 7 in the lung correlates with severity o f lung dysfunction (15). It is fairly well established that some o f the lung damage in cystic fibrosis and other lung diseases is due to the accumulation o f neutrophils and the subsequent release of proteolytic neutrophil components [reviewed in (67, 68)]. Since exogenous expression of L L - 3 7 in both a xenograft model o f the C F lung, and in tissue culture systems, has been demonstrated to increase bacterial kil l ing o f P. aeruginosa, it has been proposed that this peptide may be a useful therapeutic treatment for the chronic lung infections in C F (2, 69). However, these models did not ascertain whether neutrophil accumulation would occur in response to increased levels o f L L - 3 7 , and i f this were to occur whether lung dysfunction would be exacerbated. Elucidating the time at which L L - 3 7 is produced and its role in the recruitment o f leukocytes wi l l be important in order to understand the role o f this peptide in disease. Increased concentrations of the neutrophil-derived peptides L L - 3 7 and the a-defensins during the course o f infections or chronic inflammation correlate with increased levels o f infiltrating leukocytes including granulocytes and mononuclear cells (15, 70) and correlate especially well with concentrations of neutrophils, neutrophil markers and neutrophil specific chemokines (15, 17, 71-74). During the course o f infection or inflammation, epithelial cells produce h C A P - 1 8 / L L - 3 7 , the latter o f which has been demonstrated to be a chemoattractant for neutrophils and other cells. Although it is possible that this initial production o f L L - 3 7 by epithelial cells (75), and possibly by other resident cells, results in infiltration of leukocytes (Figure 6.2A), this model is problematic due to the fact that L L - 3 7 is a much weaker chemoattractant than conventional chemokines. The correlation o f L L - 3 7 expression with the influx of neutrophils implies that elevated concentrations o f L L - 3 7 might result from release by incoming neutrophils. In this scenario, neutrophils and other cells would arrive at the site o f infection or inflammation due to the elevated concentrations o f chemokines, and the resulting neutrophil degranulation would result in increased levels o f L L - 3 7 (Figure 6.2B). 105 A Infectious or Pro-inflammatory stimuli B Infectious or Pro-inflammatory stimuli C Infectious or Pro-inflammatory stimuli Epithelium © LL-37 Leukocyte Infiltration © ® © Circulating cells • <@ (J), Figure 6.2. Three models illustrating the role of LL-37 in leukocyte infiltration. A) Elevated levels of LL-37 due to inducible expression in response to pro-inflammatory or infectious agents lead to chemotaxis of leukocytes. B) Infectious or pro-inflammatory stimuli lead to production of chemokines which recruit leukocytes to the site of infection. LL-37 is deposited at sites of infection by degranulation of neutrophils. C) Leukocytes are recruited to the site of infection due to increased chemokine production. LL-37 is produced by epithelial cells and is released by incoming neutrophils. As monocytes and other precursor cells arrive at sites with elevated concentrations of LL-37 they produce chemokines resulting in increased leukocyte infiltration. By collating what is currently known about LL-37 expression in the early stages of infection and accounting for the observed chemokine inducing properties of this peptide described in this thesis, it is possible to expand on these models. At the initial stages of infection, elevated levels of LL-37 would occur as a result of stimulation of epithelial cells with either bacterial components or with pro-inflammatory stimuli (33, 75), or by release from cells such as incoming neutrophils or resident macrophages. LL-37 induces IL-8 production from epithelial cells, thus initiating the recruitment of neutrophils (46, 62). IL-8 induces neutrophils to release a-defensins and may also be involved in release of LL-37 (71). If an infection is not resolved in minutes to hours a second wave of cells including monocytes will be conscripted to the site of infection (76). In this scenario monocytes would be arriving at sites containing high concentrations of LL-37 and other cytokines. Upon exposure to LL-37, peripheral-blood-derived monocytes produce chemokines such as IL-8, MCP-1, MCP-3, MlP-la and MIP-ip in a matter of minutes in a MAP kinase dependent manner [Chapter 5, Figure 5.1, (41)]. Production of these chemokines would result in the further recruitment of neutrophils, monocytes and macrophages. Consistent with this hypothesis, mononuclear cells are found at sites of high LL-37 concentration in the lung (15). Thus when the first line response is insufficient, LL-37-activated monocytes are induced to produce chemokines that will lead to the recruitment of important immune response effector cells to assist in the resolution of infection (Figure 6.2C). Although there is evidence to 106 support this hypothesis in the lungs, it is important to note that in other experimental models the administration o f exogeneous L L - 3 7 does not appear to lead to the infdtration o f leukocytes (53). However, as the supporting experiments were done in rabbits, it is unclear whether this is because the chemotactic properties o f L L - 3 7 are specific to human cells or whether L L - 3 7 -induced leukocyte recruitment in vivo does not occur in all tissue types. The data presented within this thesis is consistent with the hypothesis that the L L - 3 7 induced production o f chemokines results in increased recruitment o f the effector cells o f the innate immune response. Although L L - 3 7 and other host defence peptides are generally thought of as "anti-inflammatory" since they inhibit the production o f T N F - a and other pro-inflammatory cytokines [Chapter 2, Figure 2.2, (77-79)], it is difficult to reconcile this characterization with the observed influx o f leukocytes which is one of the hallmarks o f the inflammatory response. Indeed, it has been demonstrated that increases in L L - 3 7 and the a-defensins during the course of lung infections occurs in parallel with increased production o f the peptides in the bone marrow and elevated concentrations in the plasma, indicating that localized infections can be mirrored by a systemic enhancement of innate immunity (71). I believe that the data from in vivo studies of the expression o f host defence peptides in human disease and in vitro studies o f the immunomodulatory properties o f these peptides indicate that they are more accurately described as initiators o f a localised recruitment of the effector cells o f the innate immune response and a constitutive component of immune surveillance. 6.6 Synergistic Interactions Between LL-37 and Components of the Inflammatory Mileu A major criticism of host defence peptide research is that many o f the antimicrobial and immunomodulatory effects observed are only induced by concentrations o f peptides that are higher than would be expected in vivo, or that would only be expected to occur at sites o f severe, chronic inflammation. Determining the exact concentrations o f cationic peptides in vivo is technically difficult (80). With this caveat, L L - 3 7 can be detected at concentrations of 1 \xM (~5 Ug/ml) in the bronchoalveolar lavage fluid of healthy infants (14), and its concentration is increased by 2- to 3- fold in the bronchoalveolar lavage fluid from infants with either systemic or pulmonary inflammation (14). However, accurately assessing the dilution factor for the airway surface liquid, which has an estimated depth o f only 1 ul per c m 2 o f tissue (81), and accounting for any secretory response stimulated during the lavage procedure means that bronchoalveolar lavage estimations probably have low accuracy. A l s o this method measures the average increase in peptide concentration across a wide surface area and any localized increases, which could be much greater than are currently estimated, would be missed. Thus it seems possible that under 107 some physiological conditions the concentrations o f host defence peptides, and specifically the concentrations o f L L - 3 7 , might be quite high at localized sites o f infection. During the course o f infection or inflammation, increased concentrations of L L - 3 7 would form in the lung as a result o f both release of the peptide by neutrophils and de novo production by epithelial cells. However, many other cytokines and inflammatory mediators would also be present and the involvement of these other inflammatory mediators in peptide-mediated immunomodulation has not been fully assessed. T o date, synergies between host defence peptides and components o f the inflammatory milieu, including larger proteins such as lactoferrin, lysozyme, elastase, and S L P I , have only been tested with regards to antimicrobial activity (37, 82, 83). However, synergies between host defence components which would naturally be present in the course o f infection have not been investigated for other immunomodulatory activities. I have demonstrated that L L - 3 7 induces activation o f the M A P kinases, E R K 1 / 2 and p38, in peripheral blood derived monocytes at high concentrations o f L L - 3 7 (50 pg/ml). Interestingly the presence of granulocyte macrophage colony stimulating factor ( G M - C S F ) both increased the magnitude o f this activation and decreased, to between 5-10 ug/ml, the threshold amount of L L -37 required to induce activation [Chapter 3, Figure 3.5, (41)]. This synergy was found to be specific to G M - C S F as the structurally related polypeptides IL-4 and M - C S F did not have the same ability to enhance L L - 3 7 induced signalling. In studies performed by Davidson et al (84) in collaboration with me, D C s were exposed to both G M - C S F and L L - 3 7 . Statistically significant changes in both the size and surface complexity o f the cells and o f C D 18, and C D 1 l b expression were observed at 5 ug/ml of L L - 3 7 and it seems likely that this was due in part to the synergistic interaction between L L - 3 7 and G M - C S F (84). G M - C S F is a cytokine that is produced by macrophages and T lymphocytes (85), and is also produced by lung epithelial cell lines in response to pro-inflammatory cytokines or exposure to bacteria (86, 87). G M - C S F promotes the survival, proliferation, differentiation, and activation of haematopoietic cells, predominantly in the macrophage and neutrophil lineages (85). G M - C S F has a number o f other immunomodulatory properties including the enhancement of antigen presentation, promotion o f phagocytosis and antibody-dependent kill ing, induction of chemotaxis, and induction of the release of reactive oxygen intermediates and histamines, (88-91). G M - C S F is known to work synergistically with other cytokines. For example, the addition of G M - C S F with IL-10 leads to increased expression the chemokine receptor C C R 1 (92). In bronchial epithelial cells, G M - C S F is produced upon stimulation with Tol l - l ike receptor 108 agonists and pro-inflammatory cytokines (86, 93, 94). Thus I propose (Figure 6.3) that stimulation by pro-inflammatory components would result in GM-CSF production at local infection sites, the presence of which would serve to magnify the local immunomodulatory effects of LL-37. Incoming monocytes and pre-dendritic cells would then be attracted to sites containing higher concentrations of LL-37 and GM-CSF and which would modulate their function and/or differentiation. LUMEN Pro-inflammatory Stimuli Figure 6.3. The influence of the cytokine milieu on LL-37 responses. Circulating effector cells of innate and adaptive immunity arrive at sites of high LL-37 concentration. LL-37, produced by epithelial cells and neutrophils at sites of infection and inflammation, modulates the differentiation of iDC from precursor cells. These LL-37-derived second-line DCs have altered phenotypes with increased antigen capture capacity and promote a more robust Thl response. Synergy between GM-CSF and LL-37 may be important in this process. Adapted with permission precursor f r o m artwork by D. J. Davidson. cells J I believe that the modest levels of LL-37 found at a variety of sites throughout the body (14, 18) may not be sufficient to initiate all of the immunomodulatory events ascribed to this molecule. However in the presence of a secondary signal such as GM-CSF, the threshold for immunomodulation would be decreased. There are many precedents in the adaptive immune response for the requirement of secondary signals (95, 96). These observations highlight the inadequacy of studying the properties of these peptides in isolation, and highlight the possibility that combinations of cytokines, chemokines or inflammatory mediators may enhance or alter the immunomodulatory properties of these peptides. 6.7 Future Directions - The Role of LL-37 in Health & Disease and its Potential as a Novel Therapeutic Agent The importance of host defence peptides in the immune response is only beginning to be understood. The conservation of these peptides as a component of the immune response across all classes of life is a testament to both their importance and effectiveness. In humans a number of polymorphisms in related proteins such as bacterial permeability increasing protein (97) and the defensins (98, 99) have been identified as risk factors in certain disease conditions. To date no polymorphisms or genetic defects in expression have been identified in the gene encoding hCAP-18; however, in the one syndrome in which neutrophil expression of LL-37 and possibly 109 other host defence peptides is defective, although not abrogated, patients suffer from untreatable chronic infections as well as acute and severe infectious episodes which are often life-threatening (20). This indirect evidence is consistent with the idea that these peptides are an essential component o f the innate immune response in humans. A s interest in the role o f host defence peptides in health and disease is increasing, reports o f changes in expression during the course of infection and disease are beginning to emerge. It is increasingly clear that inducible expression is an important component o f the body's response to infectious and inflammatory stimuli and that these peptides may play a homeostatic role in neutralizing low levels o f bacterial components, thus minimizing inflammatory responses. The data in this thesis are consistent with the hypothesis that L L - 3 7 is involved in the resolution o f inflammation both by blocking the production of pro-inflammatory cytokines induced by low levels o f bacterial components, and by recruiting cells o f the immune response which are required to remove invading micro-organisms. However, L L - 3 7 is clearly multi-functional and when the resolution o f infection does not occur, or i f the inflammatory stimulus is an endogenous cytokine such as IL-1 p, it might help to enhance, sustain or even amplify o f the immune response. Host defence peptides in general, and L L - 3 7 in particular, show potential as novel therapeutic agents. Exogenous addition of host defence peptides or their modified derivatives reduce bacterial counts in a number of disease models including those of septic shock and bacterial infection, and demonstrate potential as novel adjuvants and immunostimulatory agents. A s virtually every antibiotic in the physician's repertoire is modified or adapted from nature's design, it is likely that further studies o f the immunomodulatory properties o f L L - 3 7 , as well as other naturally occurring host defence peptides and their derivatives wi l l provide insight into the complexity of the immune response and the potential o f these peptides to become invaluable additions to existing anti-infective therapies. 6.8 B i b l i o g r a p h y 1. De , Y . , Q . Chen, A . P. Schmidt, G . M . Anderson, J . M . Wang, J . Wooters, J. J. Oppenheim, and O . Chertov. 2000. 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