Department of Biochemistry and Biophysics Karolinska Institutet, Stockholm, Sweden

STUDIES ON LEUKOTRIENE B4 AND ALARMINS IN INFLAMMATORY RESPONSES

Min Wan

万 敏

Stockholm 2010 All previously published papers were reproduced with permission from the publisher.

Published by Karolinska Institutet.

© Min Wan, 2010 ISBN 978-91-7409-767-2

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2010

Gårdsvägen 4, 169 70 Solna

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-- Albert Einstein The most exciting phrase to hear in science, the one that heralds the most discoveries, is not "Eureka!", but "That's funny..."

-- Isaac Asimov

ABSTRACT

Leukotriene B4 (LTB4) is a potent proinflammatory lipid mediator that is involved in host defense and inflammatory diseases, such as atherosclerosis. LL-37 and heparin binding protein (HBP) are cationic antimicrobial polypeptides, which belong to the alarmin family known to promote innate and adaptive immune reactions in response to tissue infection or injury. In the present thesis, the aims were to investigate the expression profile of enzymes and receptors for LT biosynthesis in atherosclerotic lesions and study the mechanisms of LTB4/LL-37 and LTB4/HBP interactions, the functions of these interactions and how anti-inflammatory lipids may interfere with the LTB4/LL-37 circuit. We found that mRNA levels of 5-lipoxygenase (5-LO), 5- lipoxygenase-activating protein (FLAP) and leukotriene A4 hydrolase (LTA4H), are significantly increased in human atherosclerotic plaques. Immunostaining confirmed abundant expression of these enzymes, colocalized in macrophages of intimal lesions. Furthermore, we have shown that in lesions of human tissues arachidonic acid may be converted into LTB4, which is blocked by a selective LTA4H inhibitor. In addition, expression of 5-LO and LTA4H, but not FLAP, is increased in patients with recent or ongoing symptoms of plaque instability. In search for the mechanisms by which LTB4 could exert a proinflammatory action within the vascular wall, we have found that LTB4 strongly promotes the release of LL- 37 and HBP from human polymorphonuclear neutrophils (PMNs) in a time- and dose- dependent manner. The induced release of LL-37 and HBP by LTB4 stimulation is mediated by the BLT1 receptor. Furthermore, protein phosphatase-1 (PP-1) inhibits the release by suppressing the BLT1-mediated exocytosis of PMN granules. LL-37 does not only exhibit potent antimicrobial activities, but the stimulation of 2+ PMNs with LL-37 also induces intracellular calcium ([Ca ]i) mobilization in a dose- dependent manner resulting in cPLA2 phosphorylation and translocation of 5-LO from the cytosol to the perinuclear membrane. Thus, LL-37 promotes the synthesis and release of LTB4 in intact or primed PMNs. This response is mediated by formyl peptide receptor like-1 (FPRL-1). Apparently, in human PMNs, positive feedback circuits exist between LTB4 and LL-37. Furthermore, this LTB4/LL-37 feedback loop is extended to functional responses, such as phagocytosis. We have also found that the two anti- inflammatory lipids, resolvin E1 (RvE1) and lipoxin A4 (LXA4) inhibit the release of LL-37 by LTB4 stimulation and the production of LTB4 by LL-37 induction, respectively. These compounds may serve as negative “brake signals” for the positive LTB4/LL-37 feedback circuit. Moreover, we have shown that postsecretory supernatants from LTB4-stimulated 2+ PMNs induce [Ca ]i mobilization in endothelial cells in vitro and enhance vascular permeability in vivo by employing a mouse model of pleurisy. Selective removal of HBP from the supernatants significantly reduces these activities, attributing a key role to HBP in LTB4-induced increase in vascular permeability. Taken together, we have provided indirect evidence that LTB4 plays a role in plaque instability and this mediator may act in synergy with LL-37 and HBP to promote vascular inflammation. These lipid-petide interactions may be regulated by endogenous anti-inflammatory lipids and offer novel opportunities for pharmacological intervention in inflammation.

LIST OF PUBLICATIONS

This thesis is based on the following articles, which are referred to in the text by their Roman numerals.

I. Qiu H, Gabrielsen A, Agardh HE, Wan M, Wetterholm A, Wong CH, Hedin U, Swedenborg J, Hansson GK, Samuelsson B, Paulsson-Berne G, Haeggström JZ. Expression of 5-lipoxygenase and leukotriene A4 hydrolase in human atherosclerotic lesions correlates with symptoms of plaque instability. Proc Natl Acad Sci U S A. 2006,103(21), 8161-8166.

II. Wan M, Sabirsh A, Wetterholm A, Agerberth B, Haeggström JZ. Leukotriene B4 triggers release of the cathelicidin LL-37 from human neutrophils: novel lipid-peptide interactions in innate immune responses. FASEB J. 2007, 21(11), 2897-2905.

III. Wan M, Godson C, Agerberth B, Haeggström JZ. Leukotriene B4/LL-37 proinflammatory circuits are mediated by BLT1 and FPRL-1, and are counter-regulated by lipoxin A4 and resolvin E1. Manuscript

IV. Di Gennaro A, Kenne E*, Wan M*, Soehnlein O, Lindbom L, Haeggström JZ. Leukotriene B4-induced changes in vascular permeability are mediated by neutrophil release of heparin-binding protein (HBP/CAP37/azurocidin). FASEB J. 2009, 23(6), 1750-1757. * contributed equally

Related articles not included in the thesis

1. Qiu H, Johansson AS, Sjöström M, Wan M, Schröder O, Palmblad J, Haeggström JZ. Differential induction of BLT receptor expression on human endothelial cells by lipopolysaccharide, cytokines, and leukotriene B4. Proc Natl Acad Sci U S A. 2006, 103(18), 6913-6918.

2. Qiu H, Strååt K, Rahbar A, Wan M, Söderberg-Nauclér C, Haeggström JZ. Human CMV infection induces 5-lipoxygenase expression and leukotriene B4 production in vascular smooth muscle cells. J Exp Med. 2008, 205(1), 19-24.

3. Sveinbjörnsson B, Rasmuson A, Baryawno N, Wan M, Pettersen I, Ponthan F, Orrego A, Haeggström JZ, Johnsen JI, Kogner P. Expression of enzymes and receptors of the leukotriene pathway in human neuroblastoma promotes tumor survival and provides a target for therapy. FASEB J. 2009, 23(6), 1750-1757.

4. Johansson AS, Wan M, Qiu H, Sjöström M, Haeggström JZ, Palmblad J. Effects of ethanol and ethyl pyruvate on expression of leukotriene B4 (BLT) receptors on human endothelial cells. Manuscript

5. Hua X, Wan M, Su J, Cederholm A, Haeggström JZ, Frostegård J. Oxidized cardiolipin has pro-inflammatory effects which are inhibited by Annexin A5: implications for cardiovascular disease and chronic inflammation. Manuscript

CONTENTS

CHAPTER 1. INTRODUCTION……………………………………………....……..1 1.1 Polymorphonuclear neutrophils (PMNs)………….….………………….1 1.1.1 Granules of PMNs……………………………………………...... 2 1.1.2 Recruitment of PMNs to inflammatory sites……………………..4 1.1.3 Role of PMN in inflammation…………………………………....5 1.2 Lipid mediators derived from arachidonic acid…...... 6 1.2.1 Arachidonic acid …………………………….…………………...6 1.2.2 Eicosanoids…………………………….…………………...... 7 1.2.3 Leukotrienes…….………………………………………………..7 1.2.4 Lipoxins………….……………………………………………...18 1.3 Resolvin E1 (RvE1)…………………………………….……………...21 1.3.1 Biosynthesis of RvE1..………….…………………………...... 22 1.3.2 RvE1 receptors...... …...23 1.3.3 Anti-inflammatory and pro-resolving properties………….…...23 1.3.4 Impact in disease models…...... 24 1.4 Alarmins………………………………………………….………….....24 1.4.1 LL-37/hCAP18……………………………………………...... 25 1.4.2 Defensins…………………………………………………….....28 1.4.3 Heparin-binding protein………………………………...... 29 CHAPTER 2. AIMS……………….…………………………...... 31 CHAPTER 3. METHODOLOGY……………………………….…...... 32 CHAPTER 4. RESULTS AND DISCUSSION…………………….…...... 33 CHARPET 5. CONCLUSIONS………………………………………….………....43 CHAPTER 6. ACKNOWLEDGEMENTS..……………………………….……...... 45 CHAPTER 7. REFERENCES…………………………………………….………....48

LIST OF ABBREVIATIONS

12-HHT 12(S)-hydroxy-heptadeca-5Z, 8E, 10E-trienoic acid 12-LO 12-lipoxygenase 15-LO 15-lipoxygenase 5-HPETE 5-hydroperoxy-eicosatetraenoic acid 5-LO 5-lipoxygenase AA Arachidonic acid AMPs Antimicrobial peptides ATL Aspirin-triggered lipoxins

BLT1 Leukotriene B4 receptor 1

BLT2 Leukotriene B4 receptor 2 CMV Cytomegalovirus COPD Chronic obstructive pulmonary disease COX Cyclooxygenase cPLA2 Cytosolic phospholipase A2 CXCR2 Chemokine receptor 2 CysLT1 Cysteinyl leukotriene receptor 1 CysLT2 Cysteinyl leukotriene receptor 2 cys-LTs Cysteinyl leukotrienes DC Dendritic cell DHA Docosahexaenoic acid EC Endothelial cell EPA Eicosapentaenoic acid FLAP 5-lipoxygenase-activating protein fMLP Formyl-Met-Leu-Phe FPRL-1 Formyl peptide receptor like-1 GPCR G protein-coupled receptor HBP Heparin-binding protein hCAP18 Human cationic protein 18 HIV Human immunodeficiency virus HUVEC Human umbilical vein endothelial cell ICAM-1 Intercellular adhesion molecule-1 IL Interleukin LPS Lipopolysaccharide LT Leukotriene

LTA4 Leukotriene A4

LTA4H Leukotriene A4 hydrolase

LTB4 Leukotriene B4

LX Lipoxin

LXA4 Lipoxin A4 MAPEG Membrane-associated proteins in eicosanoid and glutathione metabolism MAPK Mitogen activated protein kinases MMP Matrix metalloprotease PAF Platelet activating factor PG Prostaglandin PMN Polymorphonuclear neutrophil PPARα Peroxisome proliferator activated receptor α PSGL-1 P-selectin glycoprotein ligand-1 PUFA Polyunsaturated fatty acid ROS Reactive oxygen species SRS-A Slow reacting substance of anaphylaxis TLR4 Toll-like receptor 4 TNF-α Tumor necrosis factor-α

TXA2 Thromboxane A2 VCAM-1 Vascular cell adhesion molecule-1

Studies on Leukotriene B4 and Alarmins in Inflammatory Responses

CHAPTER 1 INTRODUCTION

Inflammation was defined around AD40 as rubor, calor, dolor, tumour (redness, heat, pain, and swelling). Nowadays it is known that inflammation occurs in response to an injurious stimulus, is beneficial to the host, and leads to removal of offending factors and restoration of tissue structure and functions. Thus, the hallmark of a successful inflammatory response is not only the clearance of injurious stimuli, but also restoration of normal physiology. Excessive or inappropriate inflammation contributes to a range of acute and chronic human diseases. Polymorphonuclear neutrophils (PMNs) are the most abundant leukocytes in the human blood and the first line of innate defense. PMNs recruitment is an integral part of the immune response to infections as well as of inflammatory disorders. A number of chemotactic factors are responsible for attracting PMNs, such as leukotriene B4

(LTB4), PAF, IL-8, fMLP and C5a. As a powerful weapon of the immune system,

PMNs can produce a wide range of products, such as lipids (LTB4, PAF, TXA2), antimicrobial polypeptides (defensins, cathelicidins, lactoferrins, lysozyme), cytokines (IL-1β, IL-6, IL-8, TNF-α), proteases (elastase, collagenase, MMP-9, proteinase 3), - reactive oxygen intermediates (ROS) (superoxide, H2O2, OH ) and nitric oxide. These products participate in various aspects of innate immunity in addition to activate the adaptive immune system. In this thesis, we highlight several products of PMNs: the proinflammatory mediators LTB4, LL-37 and HBP as well as the anti-inflammatory lipids LXA4 and RvE1. We have focused on how these proinflammatory and anti-inflammatory mediators are involved in different inflammatory responses both in vitro and in vivo.

1.1 Polymorphonuclear neutrophils (PMNs) PMNs are the most abundant blood-borne leukocytes in healthy human and account for around 40-70% of leukocytes under normal conditions. PMNs are generated in the bone marrow, and the average life span is around 12 hours. Although these cells have a short circulating half-life, PMNs play a crucial role in the first-line defense of in the innate immune system. The various subsets of granules of PMNs constitute an important reservoir not only of antimicrobial proteins, proteases, and components of the respiratory burst oxidase,

1 Min Wan, 2010 but also of a wide range of membrane-bound receptors for endothelial adhesion molecules, extracellular matrix proteins and soluble mediators of inflammation (1).

1.1.1 Granules of PMNs The granules of the human PMNs have long been recognized for their content of proteolytic and bactericidal proteins (2). The formation of the granules starts at the stage of neutrophil maturation marked by transition from myeloblast to promyelocyte. The synthesis of granule proteins continues up to the stage of segmented cells (Figure 1), and the targeting of proteins into distinct granule subtypes is determined simply by the time point at which they are biosynthesized during the development of PMNs (3).

Figure 1. The formation of PMN granules. MB: myeloblast; PM: promyelocyte; MC: myelocyte; MM: metamyelocyte; BC: band cell.

According to their size, morphology, electron density or reference to a given protein, the granules are classified into secretary vesicles, tertiary (gelatinase) granules, secondary (specific) granules and primary (azurophil) granules. The rank of mobilizable availability is secretary vesicles > gelatinase granules > secondary granules > primary granules (4, 5). During their journey from the blood stream to the inflammatory site, PMNs release their granule contents in a hierarchical order: Secretory vesicles are characterized by their immediate release when contact is established between PMNs and the endothelium. Gelatinase granules are mobilized when PMNs transmigrate through the endothelium, and secondary and primary granules are liberated at the site of inflammation (1, 2). A number of proteins have been identified in these granules, and Table 1 shows how these proteins are distributed among the different granules (2). In many studies

2 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses

Table 1. Content of Human Neutrophil Granules. Adopted from (1).

several proteins or peptides have been selected as markers of the different granules, for instance, MPO is the marker for the primary granule and lactoferrin for the secondary granule. Degranulation is defined as the secretion by receptor-mediated exocytosis of granule-derived substances (6). All of the granules are retained in the cell cytoplasm and their contents are released when receptors in the plasma membrane or phagosomal membrane signal to activate the movement of the granules to cell membrane with subsequent exocytosis. Upon exocytosis of granules, neutrophils release a diverse array of antimicrobial proteins and enzymes, many of which also possess tissue-damaging properties. In many inflammatory disorders, excessive

3 Min Wan, 2010 neutrophil degranulation is a common feature. Therefore, exocytosis of neutrophils is a fine-tuned and tightly controlled process. Actin remodeling, intracellular calcium mobilization, phospholipid signaling and Src family kinases have been reported to be involved in PMNs degranulation (6).

1.1.2 Recruitment of PMNs to inflammatory sites The process of neutrophil extravasation comprises a complex multistep cascade that is orchestrated by a tightly coordinated sequence of adhesive interactions with vessel wall endothelial cells (ECs). Adhesion receptors as well as signaling molecules in both neutrophils and ECs regulate the recruitment of neutrophils into the site of inflammation or infection (7).

1.1.2.1 Rolling In most tissues, PMNs start rolling along the walls of postcapillary venules even after a slight perturbation. Rolling interactions last for seconds and are reversible. In addition, these interactions are mostly mediated by P-selectin, a highly conserved carbohydrate-binding molecule expressed on the endothelium and on activated platelets (8), while P-selectin glycoprotein ligand-1 (PSGL-1) is constitutively expressed on all neutrophils, eosinophils, monocytes, and lymphocytes (9). The P- selectin / PSGL-1 interaction is predominantly involved in the initial tethering of PMNs (10).

1.1.2.2 Activation PMNs do not rely on a single input for activation. As part of the innate immune system, PMNs have evolved to respond to many stimuli from bacteria (formylated peptides, lipopolysaccharides (LPS), noxious agents and immunologic signals (11). The concept of multiple inputs seems to be a much better description of neutrophil function. Although the signals generated by ligation of different receptors may be low and far from triggering complete neutrophil activation, the signaling pathways overlap and can be additive or even synergistic (11). This concept was introduced earlier as “priming” of neutrophils by one agent facilitating activation by another (12). Priming is important for modulating inflammatory responses, since a direct activation of PMNs at inappropriate times or distances from the inflammatory site, would be potentially deleterious to the host.

4 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses

1.1.2.3 Adhesion

During the activation step, chemotactic agents including IL-8, C5a, LTB4, PAF or fMLP induce rapid PMN adhesion, by converting the low-affinity, selectin-mediated interaction into the high-affinity, integrin-mediated firm adhesion (7). The adhesion of neutrophils to ECs requires regulated expression of molecules on the two cell- types. Firm adhesion requires activation of CD18 integrins on the surface of PMNs and the intercellular adhesion molecule-1 (ICAM-1) on the endothelium. For several adhesive functions of neutrophils, LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) are the dominant members of this family (13). ICAM-1 exhibits a low expression on ECs but is greatly increased at inflammatory sites and by stimulation with lipopolysaccharides (14).

1.1.2.4 Locomotion and transendothelial migration After firm adhesion, PMNs crawl over the EC surface to the nearest junction using their integrins Mac-1 and LFA-1, a process called locomotion or crawling (15). Afterwards, PMNs continue the transendothelial migration. Endothelial junctions represent the major barrier for the transmigrating PMNs (7). Activation of VCAM-1 and ICAM-1 on ECs stimulates an increase in cytosolic free calcium ions, leading to actin–myosin fiber contraction, which helps ECs to separate (16).

1.1.3 Role of PMN in inflammation PMNs are capacitated as they progress from a resting state in the bloodstream through the states of rolling and adherent cells to the fully activated cells at a site of injury. Neutrophils recognize and engulf microorganisms by a process known as phagocytosis. Subsequently, PMNs kill and degrade microorganisms via the production and fine tuned release of ROS. Furthermore, antimicrobial peptides and proteolytic granule proteins are liberated, which are delivered to phagosomes and to the extracellular environment. In addition to the immediate killing and degradation of microorganisms, activated PMNs synthesize chemotactic factors and cytokines, which recruit and regulate the inflammatory response of other effector cells including macrophages, mast cells and T-cells. Finally, activated PMNs initiate an apoptotic programme, facilitating resolution of inflammation and preventing tissue damage (17). As the major effector cells of innate immunity, PMNs act as a double-edged sword. If PMNs are absent, as in congenital neutropenia or the more common cyclic

5 Min Wan, 2010 neutropenia, opportunistic infections occur, resulting from overgrowth of normally resident skin and gut bacteria as well as fungi at sites of injury, or exposed mucosal tissues. In contrast, accumulation and overactivation of neutrophils can be fatal in disorders such as septic shock or acute respiratory distress (6). On the other hand, PMNs are not only producing proinflammatory mediators but also, in cooperation with other resident cells, produce several anti-inflammatory mediators, such as lipoxins and resolvins, acting as “stop signals” in inflammation.

1.2 Lipid mediators derived from arachidonic acid 1.2.1 Arachidonic acid (AA) AA, also known as 5, 8, 11, 14-eicosatetraenoic acid, is a polyunsaturated fatty acid (PUFA) with twenty carbons and four cis double bonds, the ultimate at the omega-6 position (20:4 ω-6). These double bonds are the physical explanation to its flexibility which contributes to the membrane fluidity of mammalian cells at physiological temperatures. The double bonds are also the key to the propensity of AA to react with molecular oxygen through the actions of three types of oxygenases: cyclooxygenase (COX), lipoxygenase (LO) and cytochrome P450 (Figure 2).

Figure 2. The pathways of arachidonic acid metabolism

6 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses

1.2.2 Eicosanoids The compounds generated from C20:4 ω-6 PUFA along the COX, LO and cytochrome P450 pthways comprise the major part of a family of structurally related lipid mediators that are collectively referred to as eicosanoids (eicosa, Greek for twenty). Major groups of eicosanoids include prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs) and lipoxins (LXs). Each compound also has a single letter code to indicate certain chemical properties or position within a metabolic pathway, and a number, which refers to the number of double bonds present in the molecule.

For example, leukotriene A4 (LTA4), is the first compound in the leukotriene pathway and contains 4 double bonds (18). In this thesis, we will focus on the lipoxygenase pathway.

1.2.3 Leukotrienes (LTs) 1.2.3.1 Brief history The LTs are biologically active fatty acids, originally isolated from leukocytes, which contain a conjugated triene moiety (18). The history of LTs can be traced back to 1930’s when Harkavy reported that sputum from patients suffering from asthma contained a compound that led to contraction of intestinal smooth muscle cells. In 1938 Kellaway and Feldberg discovered the presence of a compound in perfusates of guinea pig lung injected with cobra venom (19). This compound had the capacity to induce a slow and long lasting contraction of guinea-pig intestinal smooth muscle cells, which led to the creation of the name, slow-reacting substance (SRS). Later work by Brocklehurst confirmed the release of SRS-like activity following anaphylactic challenge of sensitized tissue (20), and he named this biological activity slow reacting substance of anaphylaxis (SRS-A). Studies of the properties of SRS-A revealed it was an AA derivative with three conjugated double bonds in the structure (21, 22). In 1979, Samuelsson’s group at Karolinska Institutet identified one product of the 5-lipoxygenase (5-LO) pathway in the AA metabolism as 5S, 12R-dihydroxy-6,

14-cis-8, 10-trans-eicosatetraenoic acid, which was named leukotriene B4 (23, 24). An extension of these studies led to the discovery of the pivotal epoxide intermediate,

LTA4 (25). During the same period, the SRS-A was identified as a cysteine- containing derivative of 5-hydroxy-7,9,11,14-eicosatetraenoic acid, structurally related to LTB4 and derived from the same intermediate as LTB4. Thus, SRS-A was first named leukotriene C4 (26). Later it was revealed that SRS-A is a mixture of the

7 Min Wan, 2010 cysteine-containing LTs: the parent compound LTC4 and its metabolites LTD4 and

LTE4 (27, 28).

1.2.3.2 Biosynthesis of LTs The biosynthesis of LTs requires free AA cleaved from membrane phospholipids. Phospholipases are the enzymes to hydrolyze phospholipids, and the largest class of phospholipases is the phospholipase A2 (PLA2), which primarily catalyze hydrolysis at the sn-2 position of glycerol phospholipids to generate a lysophospholipid and a free fatty acid (Figure 3). Among the PLA2 family, cytosolic phospholipase A2

(cPLA2) specifically liberates AA at sn-2 position from glycerol phospholipids (29, 30).

Figure 3. The hydrolytic site of PLA2 on glycerol phospholipids

As Figure 4 illustrates, leukotriene biosynthesis from AA is catalyzed by a series of enzymes, starting with 5-LO. After cell activation and in response to increased intracellular Ca2+, 5-LO translocates to the nuclear envelope, where it acts in concert with 5-lipoxygenase-activating protein (FLAP), a protein that is required for 5-LO to

8 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses function enzymatically in intact cells (31). 5-LO oxygenates AA into 5-hydroperoxy- eicosatetraenoic acid (5-HPETE), which is further converted into the unstable epoxide LTA4, the key intermediate in leukotriene biosynthesis. LTA4 is further metabolized by either LTA4 hydrolase, producing LTB4, or conjugated with reduced glutathione by LTC4 synthase, yielding LTC4. LTC4 is further metabolized by γ- glutamyl transpeptidase and dipeptidase into LTD4 and LTE4 (32).

Figure 4. Leukotriene biosynthesis

9 Min Wan, 2010

LTB4 is degraded along two pathways: microsomal ω-oxidation and peroxisomal β-oxidation. It has been shown that ω-oxidation is the major pathway for the catabolism of LTB4 in human PMNs (33), while β-oxidation occurs in the kidney, lung and liver (34, 35). It has been demonstrated that LTB4 binds to peroxisome proliferator-activated receptor α (PPARα) to induce transcription of enzymes involved in ω- and β-oxidation (36), contributing to the control of the duration of LTB4 actions.

1.2.3.3 Key enzymes in LT biosynthesis

1.2.3.3.1 Cytosolic phospholipase A2 (cPLA2)

PLA2 catalyzes the release of fatty acid from the sn-2 position of phospholipids.

There are 15 genes encoding PLA2 in mammals comprising three main types: secreted PLA2 (sPLA2) with low molecular weight, calcium-independent PLA2

(iPLA2) and cytosolic PLA2 (cPLA2) with high molecular weight (37). Among these, cPLA2 is the only enzyme that preferentially hydrolyzes sn-2 AA, and is believed to play a pivotal role in providing free AA for eicosanoid biosynthesis (38).

cPLA2 is widely distributed at a relatively constant level in almost all human tissues (39). An important step in the regulation of cPLA2 involves its translocation from the cytosol to the nuclear membrane. The translocation is induced by increased 2+ intracellular concentrations of calcium ([Ca ]i), which bind the N-terminal C2 domain of cPLA2 (40). Calcium-induced docking of cPLA2 to the membrane allows interaction of the catalytic domain of the enzyme with phospholipid substrates in order to release arachidonic acid. The crystal structure of human cPLA2 also confirmed that the enzyme consists of two distinct, independently folded domains: the N-terminal C2 domain and the C-terminal catalytic domain (41). The active site in the catalytic domain is located at the bottom of a deep, narrow cleft, which is partially covered by a “lid”. Upon membrane binding, conformational changes in the enzyme might take place to move the lid and allow substrate access to the active site (41).

Maximal cPLA2 activation requires both mobilization of intracellular calcium and sustained phosphorylation (42, 43). The catalytic domain also contains several functionally important phosphorylation sites including Ser505, Ser727 and Ser515 which are phosphorylated by mitogen activated protein kinases (MAPK) (42, 44), MAPK-regulated kinase MNK1 (45), and calcium-calmodulin kinase II, respectively (46).

10 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses

1.2.3.3.2 5-lipoxygenase (5-LO) In leukotriene biosynthesis, 5-LO catalyzes oxygenation of AA to 5-HPETE, and its further dehydration to the allylic epoxide LTA4 (32). 5-LO expression is mainly restricted to various leukocytes, i.e. PMNs, eosinophils, monocytes/macrophages, mast cells, B-lymphocytes and dendritic cells (DCs) (47). In addition to the normal expression in various leukocytes, 5-LO is also found in many tumor cells (48). Mammalian 5-LO is a soluble enzyme consisting of 672 or 673 amino acid residues (49). Based on the crystal structure of rabbit reticulocyte 15-LO (50), 5-LO structure can be modeled as a monomeric enzyme with two domains: the catalytic C- terminal domain, which is mainly helical in structure and contains iron; the smaller N-terminal domain, which is a C2-like β sandwich with typical ligand-binding loops, which have been shown to bind Ca2+ and cellular membranes (49). An important determinant of 5-LO action is its activation by a transient rise in 2+ [Ca ]i that causes intracellular trafficking of 5-LO. In resting cells, 5-LO resides either in the cytosol (e.g., in PMNs, eosinophils and peritoneal macrophages) or in a nuclear soluble compartment associated with chromatin (e.g., in alveolar macrophages, Langerhans cells or rat basophilic leukemia cells). Responding to various stimuli, 5-LO tranlocates to the nuclear membrane. Both nuclear import and export sequences have been identified in the primary structure of 5-LO (51). For intact cells, 5-LO phosphorylation modulates nuclear import and export, and contributes to the regulation of 5-LO activity. Kinase-mediated phosphorylation can also affect 5-LO activity. 5-LO can be phosphorylated in vitro on three residues: Ser- 271, by MAPK activated protein kinase 2 (52); Ser-663 by extracellular signal- regulated kinase 2 (ERK2) (52); and Ser-523 by PKA catalytic subunit (53). There has been great interest in identifying inhibitors of 5-LO activity, because of the importance of 5-LO derived LTs in the pathogenesis of asthma and other inflammatory diseases. Several competitive inhibitors of AA metabolism, including zileuton, AA-861, ABT-761, ZD-2138 and L-739010, are very specific for 5-LO (54). Zileuton was the first 5-LO inhibitor demonstrating efficacy in the treatment of asthma patients (55).

1.2.3.3.3 5-lipoxygenase-activating protein (FLAP) In the late 1980s, it was found that MK-886 inhibited both in vitro and in vivo leukotriene synthesis without affecting 5-LO, phospholipases or other non-selective

11 Min Wan, 2010 mechanisms of inhibiting leukotriene production (56). Subsequently, a novel 18 kDa integral membrane protein was identified that specifically binds the inhibitor MK-886 (57). Because this protein was required for the cellular activity of 5-LO, it was named FLAP. It has been suggested that the role of FLAP is to present AA to 5-LO (54). FLAP was shown to be an AA binding protein and this binding could compete with compounds such as MK-886 (58). FLAP also stimulates the utilization of AA by 5-

LO and increases the efficiency with which 5-LO converts 5-HPETE into LTA4 (59). FLAP is recognized as a member of a protein superfamily designated as membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG). In 2007, a crystal structure of FLAP in complex with inhibitors of leukotriene biosynthesis were reported, revealing a homotrimeric structure with four transmembrane helices in each monomer (60). Recently the FLAP gene has been linked to a risk of myocardial infarction and stroke (61-64). Beneficial effects of FLAP inhibitors have been seen in both acute and chronic cardiovascular diseases (CVD) in animal models (65-67).

1.2.3.3.4 Leukotriene A4 hydrolase (LTA4H)

LTA4H has been purified and cloned from several mammalian sources, such as human, mouse, rat and guinea pig (reviewed in (68)). This protein contains 610 amino acid residues, and the calculated molecular mass of the human enzyme is 69 kDa (69). The amino acid sequences of LTA4H are highly conserved and exhibit > 90% identity among these species.

It has been known for long time that LTA4H catalyzes the hydrolysis of the unstable epoxide intermediate LTA4 into LTB4, which is a highly substrate specific process.

Besides the epoxide hydrolase activity, LTA4H was found to possess a zinc- dependent peptidase activity (70). Although it has never been experimentally verified, it is generally assumed that the aminopeptidase activity is involved in the processing of peptides related to inflammation and host defense (69).

LTA4H is a monomeric soluble protein containing one zinc atom which is essential for the activities of the enzyme (71). The high resolution crystal structure of LTA4H in complex with the competitive inhibitor bestatin reveals that this protein is folded into three domains (N-terminal, C-terminal and catalytic domains) that together create a deep cleft haboring the catalytic Zn2+ site (72).

12 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses

LTA4H is widely distributed and resides in the cytosol, although nuclear localization has also been reported (73). The 5-LO expression is predominantly found in leukocytes, while LTA4H is present in many cell types that lack significant 5-LO activity. This uneven distribution of two intimately coupled enzymes has been explained in terms of transcellular biosynthesis. Indeed transfer of LTA4 from activated leukocytes to a variety of other cell types has been demonstrated in vitro (74) and in vivo (75, 76), a phenomenon that is promoted by tight cell–cell interactions.

Variants in the LTA4H gene have been linked to susceptibility to asthma (77) and myocardial infarction (78). The study of LTA4H inhibitors revealed that LTA4H is a potential target for the treatment of inflammatory bowel diseases (79, 80). The LTA4H inhibitor JNJ-26993135 selectively inhibits LTB4 production, without affecting cys- LTs production, while maintaining or increasing the production of the anti- inflammatory mediator, LXA4 (81).

1.2.3.3.5 Leukotriene C4 Synthase (LTC4S)

LTC4S conjugates LTA4 with glutathione to form LTC4, the parent compound of cys-LTs. This enzyme is expressed in a limited number of cell types such as eosinophils, mast cells, basophils and monocytes/macrophages. It is also present in platelets that lack 5-LO (82).

LTC4S is an 18 kDa integral membrane protein that initially was found to function as a homodimer (83). Studies on the crystal structure revealed that LTC4S is a homotrimer, where each monomer is comprised of four transmembrane segments (84,

85). Similarly to FLAP, human LTC4S belongs to the MAPEG protein family (86).

The cDNA of LTC4S encodes a protein with 150 amino acid residues (87), and the deduced amino acid sequence reveals 31% overall identity to FLAP. The subcellular localization of LTC4S has been determined to be in the outer nuclear membrane (88).

Furthermore, it has been demonstrated that LTC4S and FLAP interact with each other, and mixed complexes of FLAP and LTC4S have been detected (89).

Phenotypic characterization of LTC4S-deficient mice have revealed a prominent role of the cys-LTs in the augmentation of mast cell-dependent vascular permeability (90) and the regulation of Th2 cell-dependent pulmonary inflammation (91).

Promoter polymorphisms in the LTC4S gene have been associated with a risk of ischemic cerebrovascular disease (92) and transient ischemic stroke (93).

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1.2.3.4 G protein-coupled receptors (GPCRs) for leukotrienes

As depicted in Figure 2, so far, LTB4 has been found to bind to two GPCRs, BLT1 and BLT2 with high and low affinity, respectively. CysLT1, CysLT2 and a putative receptor for LTE4 (CysLT3) have been reported for cys-LTs.

1.2.3.4.1 BLT1

The human high-affinity LTB4 receptor, i.e. BLT1, was cloned by Yokomizo et al. in 1997 (94). Amino acid sequence analysis of BLT1 in human (95, 96) and mouse (97) shows the presence of seven hydrophobic transmembrane domains common for GPCRs. So far the gene of BLT1 in human (94), guinea pig (98, 99), mouse (97) and rat (100) have been cloned. BLT1 shares 70% amino acid identity among the four species, and exhibits low similarities to other known GPCR families. Human BLT1 is highly expressed on peripheral blood leukocytes, and lower expression has been detected in spleen, thymus, bone marrow, lymph nodes, heart, skeletal muscle, brain and liver (Table 2). The gene encoding human BLT1 is located on chromosome 14q11.2-q12 and spans about 5.5 kbp (102). The BLT1 gene consists of three exons, and the ORF is in exon 3. Human BLT1 protein consists of 352 amino acid residues with an approximate

Table 2. Comparison between BLT1 and BLT2. Modified from (101). BLT1 BLT2 Human (352: D89078) Human (358: AB029892) Structure (amino acids: Mouse (351: AF044030) Mouse (360: AB029893) Accession number) Rat (351: AB025230) Rat (359: AB052660) Guinea pig (348: AB0050490)

Affinity for LTB4 Human PMNs, Kd = 0.39-1.5 nM hBLT2 transfected HEK293

Cells, Kd = 22.7 nM

Ligand LTB4 > 20-OH-LTB4 > 12(R)- 12-HHT > LTB4 > 12(S)- HETE > 5-HETE HETE > 15(S)-HETE Expression Leukocytes >> Thymus, spleen Spleen > Ovary, liver, leukocytes G-protein Gi, G16 Gq Chromosome (human) 14q11.2-q12 14q11.2-q12

14 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses molecular weight of 43 kDa. BLT1 expression on leukocytes is upregulated in inflammation, and the BLT1 gene appears to be tightly regulated at the transcriptional level and is induced by various stimuli. Rat BLT1 transcription is elevated in activated peritoneal macrophages (100) and human BLT1 transcription is induced in PMNs by dexamethasone (103). In human monocytes, proinflammatory mediators, such as interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) and LPS, down- regulated the mRNA expression of BLT1, whereas the anti-inflammatory cytokine, IL- 10 and dexamethasone up-regulated the mRNA expression of BLT1 (104). Moreover, the treatment with LPS or LTB4 induces the expression of mRNA and protein levels of BLT1 in HUVEC (105). Two lines of BLT1-deficient mice (106, 107) and one line of transgenic mice overexpressing BLT1 (108) have been generated. Functional studies of these mice demonstrate pivotal roles of the LTB4-BLT1 pathway in leukocyte functions, such as chemotaxis, adhesion and migration to inflamed tissues.

1.2.3.4.2 BLT2

BLT2, an additional LTB4 receptor, was identified by Yokomizo et al. in 2000 (109). Human BLT1 and BLT2 are structurally similar with 45% amino acid identity. However, the tissue distributions of BLT1 and BLT2 are quite different (Table 2). Human and mouse BLT1 are expressed primarily in leukoctyes, while BLT2 is expressed more ubiquitously in most human tissues, with high expression in spleen, ovary, liver and leukocytes (109). The human BLT2 gene is located approximately 3 kb upstream of the human BLT1 gene, and the ORF of human BLT2 overlaps the BLT1 gene promoter, suggesting a complex regulatory system for the gene transcription (102).

LTB4 binds to BLT2 with significantly lower specificity than to BLT1. Membrane fractions of HEK 293 cells transfected with the cloned human BLT2 sequence demonstrated specific and saturable LTB4 binding with a Kd of 22.7 nM, which is approximately 20-fold higher than human BLT1 transfectants (109). Recently it was reported that 12(S)-hydroxy-heptadeca-5Z, 8E, 10E-trienoic acid (12-HHT) is a 3 natural ligand for BLT2 (110). Displacement analysis using [ H] LTB4 showed that

12-HHT binds to BLT2 with a higher affinity (IC50=2.8 nM) than LTB4 (IC50=25 nM), and may in fact represent the primary ligand for BLT2.

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Multiple LTB4 receptor antagonists have been developed. BLT1-specific antagonists include CP105696 (111) and U-75302 (112), while LY255283 competes with LTB4 binding to human BLT2 but not to human BLT1 (113). ZK158252 and

ONO4057 (114) and CP195543 (115) compete with LTB4 binding to both receptors.

1.2.3.4.3 Receptors of cys-LTs Early pharmacological studies provided evidence that cys-LTs exert their action through specific cellular receptors (reviewed in (116)). Additional experimental results strongly suggested that these are GPCRs (117, 118). Furthermore, a number of data rapidly accumulated suggesting the existence of at least two receptor subtypes (119, 120) that were later named CysLT1 and CysLT2. Subsequently, human CysLT1 was cloned in 1999 (121, 122), and CysLT2 in 2000 (123-125). The human CysLT1 consists of 337 amino acid residues with a calculated molecular weight 38 kDa, while human CysLT2 protein contains 346 amino acids with 38% identity to human CysLT1. Human CysLT1 recptor is highly expressed in spleen and peripheral blood leukocytes, while the expression in lung, small intestine, pancreas and placenta is not so pronounced (121, 122). CysLT2, on the other hand, was found to be abundantly expressed in human heart, adrenal gland, peripheral blood leukocytes, spleen and lymph nodes (123-125). The preferred ligands for

CysLT1 are LTD4 followed by LTC4 and LTE4 (122), while the rank order of the affinity to CysLT2 is: LTC4 = LTD4 >> LTE4 (123). By targeted gene disruption, CysLT1 and CysLT2 deficient mice have been generated (126, 127). Phenotypic characterization for these mice unexpectedly revealed that CysLT2, rather than CysLT1, mediates the signal for pulmonary inflammation and fibrosis. Thus, it has been suggested that CysLT2 is involved in chronic inflammation, whereas CysLT1 mediates bronchoconstriction and microvascular leakage in acute inflammation.

LTE4 exerts potent biological functions in vitro (128) and in vivo (129-131); however, neither CysLT1 nor CysLT2 has signifcant affinity for LTE4. Thus, the existence of an additional cys-LT receptor with a preference for LTE4 (CysLT3) has long been suggested. Recently, it has been reported that LTE4-mediated production of

PGD2 by human mast cells was unaffected by knockdown of either CysLT1 or CysLT2 (132). Moreover, mice lacking both CysLT1 and CysLT2 (Cysltr1/Cysltr2−/−) exhibit enhanced skin swelling in response to intracutaneous LTE4 relative to wild type

16 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses controls (133). These results confirmed the presence of an unidentified CysLT3.

Consequent studies revealed that LTE4–induced pulmonary infammation is mediated by the P2Y12 receptor (134). However, the facts that P2Y12 receptors do not directly −/− bind LTE4 (134) and that the enhanced ear edema to LTE4 in the Cysltr1/Cysltr2 mice was not affected by pretreatment with a P2Y12 inhibitor (133), indicate that P2Y12 receptors are not the putative CysLT3. It has been suggested that perharps P2Y12 are components of a complex with the putative CysLT3 (134). During the process of seeking CysLT3, GPR17 had been found as a candidate (135). However, further work unexpectedly revealed that GPR17 is a ligand- independent, constitutive negative regulator for CysLT1, suppressing CysLT1- mediated functions at the cell membrane (136).

1.2.3.5 Biological effects of LTs

1.2.3.5.1 Biological effects of LTB4

After LTB4 was discovered and chemically synthesized, its ability to activate

PMNs was demonstrated (137). LTB4 is now known to be a major product of activated leukocytes, in particular neutrophils and macrophages, with the ability to recruit and activate a range of immune effector cells including PMNs, macrophages and eosinophils. Recent studies also demonstrated that LTB4 mediates migration of mast cells (138), DCs (139), T-cells (140-142), NK cells (143) and smooth muscle cells (144, 145). It has also been reported that LTB4 can activate ECs to express adhesion molecules (146, 147) as well as BLT1 (105). A key role has therefore been proposed for LTB4 in the pathogenesis of a variety of inflammatory disorders, including inflammatory arthritis (148, 149), asthma (150-152), chronic obstructive pulmonary disease (COPD) (150, 153), atopic dermatitis (154), psoriasis (155) and inflammatory bowel disease (156). Synthesis of LTB4 in human atherosclerotic plaques were reported 20 years ago (157), and recent research indicates a contribution of LTB4 to the pathology of various cardiovascular diseases, such as myocardial infarction (61, 78), atherosclerosis (158), and aortic aneurysms (159).

Although LTB4 is recognized as a potent chemotactic factor, it is also known to exert other roles in innate immune responses (160). It has been shown that LTB4 augments neutrophil and macrophage phagocytosis (161, 162), and enhances the killing of a variety of microorganisms, including bacteria (163, 164), fungi (165) and parasites (166). It has also been found that LTB4 has the potential to protect against

17 Min Wan, 2010 different viruses, such as HIV (167), CMV (168-170), and herpes simplex virus type 1

(171). These antimicrobial activities are supposed to be mediated by various LTB4- induced antimicrobial peptides/proteins, nitric oxide and ROS (160).

1.2.3.5.2 Biological effects of Cys-LTs

Cys-LTs, namely LTC4, LTD4, and LTE4, are potent smooth muscle contracting agents, particularly in the respiratory tract and microcirculation (172). They also increase microvascular permeability, allowing extravasation of inflammatory cells and leakage of plasma components, which leads to the development of edema (173). In addition, cys-LTs enhance mucous secretion, eosinophil infiltration in human airway, as well as bronchial smooth muscle cell proliferation (130, 174). Cys-LTs may also modulate the activities of several components of the immune system. It was reported that cys-LTs promote the maturation, migration and activation of DCs (175, 176), prolong eosinophil survival (177) and enhance cytokine production by human monocytes, T-cells (178) and mast cells (179). Furthermore, cys-LTs are known to play important roles in the pathogenesis of different inflammatory diseases, such as asthma (reviewed in (180)), cardiovascular diseases (181, 182), rheumatoid arthritis (183) and atopic dermatitis (184).

1.2.4 Lipoxins (LXs)

In 1984, Serhan and colleagues discovered the lipoxins (lipoxin A4 and B4) (185, 186). Interestingly, aspirin-acetylated COX-2 can trigger the formation of a 15- epimeric form of LXs (187). Further research has shown that LXs and aspirin- triggered LXs (ATL) evoke protective actions in a range of physiologic and pathophysiologic processes. Hence, they function as “stop signals” in inflammation and actively participate in dampening host responses (188).

1.2.4.1 Biosynthesis of LXA4 and 15-epi-LXA4

So far three transcellular pathways have been identified for the biosynthesis of LXA4 and 15-epi-LXA4 (Figure 5). The two major routes of LXA4 biosynthesis in human cell types are depicted in Figure 3(a) and 3(b). One pathway involves peripheral blood leukocyte-platelet interactions. The enzyme 5-LO in leukocytes converts AA to LTA4, which is released from leukocytes and further transformed by adherent platelets to

LXA4 via 12-LO (189, 190). The second biosynthetic route is initiated at mucosal

18 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses

Figure 5. The biosynthetic pathways of LXA4 and 15-epi-LXA4 surfaces by 15-LO that transforms AA to 15S-hydroxy-eicosatetraenoic acid (15S- HETE) (191). This latter metabolite is rapidly taken up by PMNs and is subsequently converted via 5-LO to LXA4. In addition to these two main routes, it has been discovered that aspirin-acetylated COX-2 transforms AA to 15R-HETE, which is taken up by leukocytes and is converted via 5-LO to 15-epi-LXA4, a product that is also called ATL. Notably, biosynthesis of LXA4 not only leads to LX formation but also reduces LT production.

LXA4 was subsequently found to undergo rapid metabolic inactivation via ω- hydroxylation as well as via prostaglandin dehydrogenase (PGDH)-mediated reduction of the ∆C13 double bond and oxidation at C15 (192). Therefore, different analogs of LXA4 have been synthesized, which are resistant to rapid metabolism, but maintain the biological actions of native LXA4 (193-195).

1.2.4.2 LXA4 receptors Early studies indicated the presence of a high affinity and relatively selective receptor for LXA4 and additional cross-talk between LXA4 and LTD4 mediated by a shared receptor (196, 197). The cDNA of the LXA4 receptor with high affinity was identified as a homolog (~70%) to fMLP receptor (FPR), named as formyl peptide receptor like-1 (FPRL-1) (198), which also belongs to the GPCR family.

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Actually, multiple additional ligands for human FPRL-1 have now been identified, including serum amyloid A (SAA) (199), LL-37 (200), synthetic peptides derived from HIV glycoprotein 120 (201), MHC binding peptide (202) and peptides derived from annexin 1 (203) (Table 3). Most of them appear to have much lower affinity than LXs. These various ligands of FPRL-1 have been shown to possess different proinflammatory and anti-inflammatory properties. However, interactions between FPRL-1 and various ligands occur with different affinities and these lipid or peptide ligands may bind to various sites of the receptor. The activation of the receptor may also evoke separate intracellular signaling pathways that depends on the cell type and system, and may reflect, at the genomic level, the economy of using one receptor structure for multiple recognitions and functions of the immune system (204). So far knockout mice of the cloned murine FPRL-1 are not available. However, transgenic mice have been generated in which myeloid-specific expression of the human FPRL-1 receptor (hALX-R-Tg mice) is driven by the human CD11b promoter (205). The results from these transgenic mice demonstrated that FPRL-1 is a key receptor and sensor in formation of acute exudates and their resolution.

1.2.4.3 Anti-inflammatory and pro-resolving properties

The biological actions of LXA4 and its synthetic analogs have been characterized in many cell and tissue types, both in vitro and in vivo. It is well-known that LXA4 provides counterregulatory effects on inflammatory processes in order to promote resolution of inflammation (206). In acute inflammatory responses, LXA4 inhibits human PMNs chemotaxis in response to LTB4 in vitro, decreases PMNs transmigration across both human microvessel endothelial and epithelial cells (207). Furthermore,

LXA4 blocks P-selectin expression on HUVEC (208), while it upregulates the chemotaxis and adhesion of monocytes without increasing cytotoxicity (209). In addition, LXA4 stimulates macrophages to ingest apoptotic neutrophils (210), and inhibits multiple steps of vascular endothelial growth factor (VEGF)-induced angiogenesis (211). LXA4 also has the potential of immune-modulatory functions, by inhibiting TNF-α secretion from activated T-cells (212) and block cytotoxicity of NK cells (213). Moreover, LXA4 counterregulates the proinflammatory responses during microbial infection by reducing IL-12 production from DCs (214). However, Bafica et al. addressed the other side of the coin (215), by showing that mice become more resistant to M. tuberculosis infection in the absence of endogenously generated LXA4.

20 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses

Table 3. Ligands for FPRL-1. Adopted from (204).

1.2.4.4 Effects on inflammatory disorders

LXA4 exhibits protective functions in several inflammatory disorders, such as atherosclerosis (216), acute renal failure (217), cystic fibrosis (218) and periodontitis

(219). The role of LXA4 in asthma is more complex, since increased LXA4 production has been detected in mild asthma (220), while reduced levels are observed in severe asthma (221).

1.3 Resolvin E1 (RvE1) Numerous reports suggest that supplementation of dietary ω-3 PUFAs, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have beneficial effects

21 Min Wan, 2010 on a wide range of human diseases in which unresolved inflammation is suspected as a key component in disease pathogenesis (reviewed in(222)). Several possible mechanisms for the beneficial effects of ω-3 PUFAs have been suggested. One hypothesis is that ω-3 PUFAs can substitute AA and prevent its conversion into proinflammatory eicosanoids such as 4-series LTs and 2-series PGs. Consequently, ω-3 PUFAs are converted into less potent 5-series LTs and 3-series PGs (reviewed in (223)). Recently, Dr. Charles N. Serhan’s group has developed a systematic methodology of mediator informatics and lipidomics by employing LC- MS-MS (224). Inflammatory exudates from dorsal skin pouches of mice are analysed, which are known to resolve spontaneously. Based on these techniques, they discovered a series of novel ω-3 PUFA-derived lipid mediators which appeared in inflammatory exudates during the course of acute inflammatory responses. These include E-series resolvins such as RvE1 from EPA and D-series resolvins such as resolvin D1 (RvD1), protectin D1 and maresins from DHA (225-228). As lipoxins, these newly identified chemical mediators appear to exert potent anti-inflammatory and pro-resolving actions both in vitro and in vivo. In this thesis, we have studied one member of the resolvin family, i.e. RvE1.

1.3.1 Biosynthesis of RvE1 In 2000, Dr. Charles N. Serhan and his colleagues isolated the first bioactive compound from inflammatory exudates formed in murine air pouches via intrapouch injections of TNF-α together with ω-3 PUFA and aspirin. Structural studies revealed that this potent bioactive product is generated from EPA and proved to be 5,12,18- trihydroxy-6,8,10,14,16-eicosapentaenoic acid (225). Later on a series of bioactive compounds were discovered based on this strategy. The term resolvins (resolution- phase interaction products) was first introduced to signify that these new structures were endogenous mediators and they are biosynthesized in the resolution phase of inflammatory exudates, possessing very potent anti-inflammatory and immunoregulatory actions (226). Since the first discovered resolvin was generated from EPA, it was named Resolvin E1. The biosynthetic pathway of RvE1 was investigated in vitro (225). It was proposed that aspirin treatment at local sites of inflammation can convert EPA via acetylated COX-2 in vascular ECs to 18R-HEPE. This is followed by conversion of 18R-HEPE

22 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses

Figure 6. Biosynthetic pathways of RvE1 into RvE1 by 5-LO in activated human PMNs. This pathway involves transcellular biosynthesis via cell–cell interactions at local sites of inflammation. In addition to the aspirin–COX-2 pathway, RvE1 formation can be initiated via routes that do not require aspirin, involving cytochrome P450 monooxygenase present in microbes (229). These two pathways are illustrated in Figure 6.

1.3.2 RvE1 receptors To identify the receptor(s) mediating the activities of RvE1, Arita et al. screened 10 GPCRs closely related to FPRL-1 (230). This screening resulted in the identification of ChemR23 that is specifically activated by RvE1. In another study, it was found that RvE1 is a partial agonist to BLT1 serving as a local damper of LTB4-BLT1 signals on PMNs (231).

1.3.3 Anti-inflammatory and pro-resolving properties When RvE1 was discovered, this compound was found both to reduce inflammation in vivo and to target isolated human neutrophil transendothelial

23 Min Wan, 2010 migration in vitro (225). Further studies have demonstrated that RvE1 is a potent regulator of PMNs. In several inflammatory mouse models, RvE1 dramatically reduces PMN infiltration into inflammatory sites (230, 232, 233), and in vitro RvE1 regulates PMN transmigration (225, 226, 234). Further studies provided evidence that RvE1 induces L-selectin shedding together with reduction of CD18 surface expression on human PMNs and monocytes (235). It has also been found that RvE1 inhibits IL-8-stimulated PMN chemotaxis and enhances phagocytosis, ROS generation and pathogen killing by PMNs (236). In addition to regulating the functions of PMNs, RvE1 also displays potent activities on other cell types. RvE1 activates non-phlogistic phagocytosis of apoptotic PMNs by macrophages (233). Furthermore, RvE1 potently inhibits TNF-α, IL-6 and IL-23 release from LPS-stimulated bone marrow-derived DCs (237).

1.3.4 Impact in disease models RvE1 displayed protective effects against severe inammatory bowel disease (232). In a rabbit model of periodontal disease, topical application of RvE1 reduces periodontal inammation and exhibits protection from inammation-induced osteoclast activation and bone loss (238, 239). Evidence has been presented that RvE1 serves as a pivotal counter-regulatory signal in allergic inflammation and offers a novel therapeutic approach for human asthma (reviewed in (240)).

1.4 Alarmins Recent studies have identified several structurally diverse endogenous polypeptides as mediators of innate immunity. This subset of mediators alerts host defense to tissue injury and/or infection by augmenting innate and adaptive immune responses, therefore, they are called “alarmins” (241). So far, alarmins include high- mobility group box protein 1, eosinophil-derived neurotoxin, as well as PMN-derived defensins, cathelicidins and heparin-binding protein (HBP). Although these molecules are structurally diverse, they possess certain similar features: firstly, they are rapidly released in response to infection or tissue injury; secondly, they possess both chemotactic and activating effects on antigen-presenting cells (APCs); and thirdly, they exhibit potent in vivo immunomodulating activities. In our project, we highlight two polypetides which belong to alarmins: the cathelicidin LL-37/hCAP18 and HBP. Since defensins are one major member of alarmins in mammals, they will

24 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses also be introduced in this section, although they have not been investigated in our projects.

1.4.1 LL-37/hCAP18 Neutrophil granules contain several antimicrobial peptides (AMPs) that are important effector molecules of innate immunity. In mammals, the main families of these peptides are cathelicidins and defensins. LL-37 is the only cathelicidin peptide identified in human and has also been classified as an alarmin.

1.4.1.1 Structure and processing LL-37 is an amphipathic α helical peptide. The name LL-37 derives from the first two N-terminal leucine (L) residues and the number of amino acids. hCAP18 is the name of the inactive proform of LL-37. As illustrated in Figure 7, the gene encoding LL- 37/hCAP18 contains four exons. The first three exons code for the signal sequence and the cathelin proregion. Exon 4 encodes the mature and active LL-37 peptide (242). The primary translation product is a pre-pro-peptide. The signal sequence is cleaved off once it has fulfilled its purpose of targeting hCAP18 to the storage granules, and the amino acid sequence of the cathelin domain is highly conserved in the whole cathelicidin family.

Figure 7. Structure of the gene and peptide of LL-37/hCAP18

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LL-37/hCAP18 is mainly expressed in PMNs (243) and different epithelial cells (244), but has also been detected in monocytes, T-cells, NK cells and mast cells (245- 247). The inactive proprotein hCAP18 is mainly stored in specific granules of PMNs at a molar concentration, constituting a substantial part of the proteins in specific granules of neutrophils (243, 248). Proteinase 3 has been reported to be the only neutrophil-derived protease that can process hCAP18 into active LL-37. However, data suggest that also kallikreins (249, 250) and gastricsin (251) can process hCAP18 into active peptides, but with different sizes compared to LL-37. Very recently, it was demonstrated that LL-37 can be degraded by mast cell tryptase, and no typical activity comparing to intact LL-37 was detected from the resulting LL-37 fragments (252). In addition to proteases of human cells, it has also been reported that LL-37 is degraded by Staphylococcus aureus-derived proteases, such as aureolysin and V8 protease (253), contributing to the resistance of this pathogen to the innate immune system.

1.4.1.2 Receptors So far several receptors have been shown to mediate various functions of LL-37. LL- 37 utilizes FPRL-1 as a receptor to chemoattract human PMNs, monocytes, T-cells and eosinophils (200, 254). In addition, FPRL-1 mediates the angiogenic activity (255). LL- 37 promotes IL-1β processing and release from monocytes via direct activation of the

P2X7 receptor (256). Furthermore, LL-37 can promote IL-8 release from the airway epithelial cells and induce keratinocyte migration via epidermal growth factor receptor (EGFR) transactivation (257, 258). Recent studies have provided evidences that LL-37 may act as a functional ligand for CXCR2 on human PMNs (259). Moreover, intracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was reported to work as a functional direct binding partner of LL-37 in monocytes and macrophages (260).

1.4.1.3 Antimicrobial activity In vitro studies on antimicrobial properties of LL-37 have demonstrated that it possesses potent capability to kill a broad range of bacteria, viruses and parasites, making it a promising antimicrobial agent for therapeutic use (261). Studies on the mouse cathelicidin (mCRAMP) and mice deficient in the mCRAMP gene demonstrated that this peptide affords a pivotal protection against necrotic skin

26 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses infection (262) and bacterial clearance of the urinary tract (263). Recent studies have shown that vitamin D upregulates human cathelicidin gene (CAMP) and the corresponding peptide in different bronchial and skin epithelial cells as well as in myeloid cells (264-266). Furthermore, this induction of LL-37 contributes to the mechanism of vitamin D protection against tuberculosis (267, 268). In a rabbit model, oral butyrate treatment induces the expression of the endogenous cathelicidin CAP18 (the rabbit homologue to LL-37) in colonic epithelial cells and promotes elimination of Shigella bacteria (269). Schauber et al. also reported that butyrate and other short chain fatty acids modulate LL-37 expression in human colonic epithelial cells (270).

1.4.1.4 Additional activities LL-37 is a multifunctional peptide, and it displays a variety of additional functions on various cell types in host defense.

1.4.1.4.1 Effects on PMNs LL-37 utilizes FPRL-1 as a receptor to chemoattract human PMNs (200), and induces the generation of ROS and release of α-defensins from PMNs (271). In addition, LL-37 suppresses PMN apoptosis resulting in the prolongation of PMN life span (272, 273).

1.4.1.4.2 Effects on epithelial cells In contrast to the inhibition of neutrophil apoptosis, LL-37 induced apoptosis in primary airway epithelial cells (273, 274). However, it has also been reported that LL- 37 had anti-apoptotic effects on keratinocytes that is mediated by a COX-2-dependent mechanism (275). Moreover, LL-37 induces IL-18 secretion in keratinocytes (276).

1.4.1.4.3 Effects on mast cells Nardo et al. demonstrated that the presence of cathelicidins is vital for the ability of mammalian mast cells to participate in antimicrobial defense (277). LL-37 also induces mast cell chemotaxis (278), upregulates the level of TLR4 mRNA and protein in addition to induce cytokine release from mast cells (279). LL-37 increases vascular permeability in the skin via mast cell activation (280), and induces histamine release as well as PGD2 production from mast cells (281).

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1.4.1.4.4 Effects on dendritic cells (DCs) LL-37 modulates DC differentiation (282) and inhibits the activation of DCs by TLR ligands (283, 284). Interestingly, LL-37 binds human DNA in a complex that drives autoimmunity by the production of α-interferon in plasmacytoid DC in psoriasis (285).

1.4.1.4.5 Effects on other cell types LL-37 is a chemoattractant to human monocytes, T-cells and eosinophils (200, 245, 254), and it enhances IL-8 release from human airway smooth muscle cells (286). LL- 37 induces angiogenesis by a direct action on ECs via the specific receptor FPRL-1 (255).

1.4.1.5 Involvement in inflammatory diseases LL-37 is present in atherosclerotic lesions (287-289) and the expression of LL-37 is altered in inflammatory bowel disease (290). It has been found that LL-37 is highly upregulated in psoriatic lesions, while downregulated in skin specimens from patients with atopic dermatitis (291). Interestingly, LL-37 exerts diverging functions in various cancers. For example, LL-37 acts as a growth factor in breast cancer (292, 293) and lung cancer cells (294). In contrast, LL-37 exerts antitumor effects against ovarian cancer (295). In induced sputum, the level of hCAP18 is elevated in cystic fibrosis (CF) and COPD patients, while asthma patients exhibit reduced level of hCAP18 (296). Furthermore, patients with rosacea express abnormally high levels of cathelicidin in their facial skin (297).

1.4.2 Defensins Defensins are cysteine-rich peptides and include α- and β-families. Human neutrophil peptides (HNP) 1-4 are α-defensins in PMNs, where they are stored in primary granules at high concentrations. Thus, α-defensins constitute more than 5% of the total protein content of PMNs and participate in the non-oxidative killing of ingested microbes (298). HNP1-3 differ from each other by one amino acid at the N- terminus of the mature peptides (299), while HNP-4 has a significantly different primary structure (300). PMN-derived HNPs have a broad antimicrobial spectrum against bacteria, viruses and parasites. Like LL-37, α-defensins possess additional immunomodulatory properties (reviewed in (301)). In addition to HNP1-4, there are two well-described human Paneth cell α-defensins, HD-5 (302) and HD-6 (303).

28 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses

Human β-defensins (HBDs) are mainly expressed in different epithelial cells, where their expression is either constitutive or induced by cytokines or bacterial components (304). Although only four HBDs have been characterized at the peptide level, 28 additional HBD genes have been identified in the human genome (305). HBDs also display potent antimicrobial activities and immunomodulatory functions.

1.4.3 Heparin-binding protein (HBP) Shafer et al isolated a protein from PMNs in 1984 (306), and according to its charge and size, it was named cationic antimicrobial protein with a mass of 37 kDa (CAP37). Several years later, Gabay et al. identified azurocidin, a PMN-derived bactericidal protein from the azurophilic granules of human PMNs (307). Flodgaard et al. isolated a protein from PMNs that displayed strong binding capability for heparin, earning its name heparin-binding protein (HBP) (308). After complete sequencing it has been shown that CAP37, azurocidin and HBP are the same protein.

1.4.3.1 Structure of HBP HBP accounts for approximately 20% of the primary granule protein or 0.45% of the total protein content of PMNs (309). The complete amino acid sequence of HBP showed that HBP is a single-chain glycoprotein consisting of 222 amino acid residues with three N-glycosylation sites (310). Hence, partially glycosylated or unglycosylated species of HBP could exist, which was evidenced by the presence of several protein bands with various molecular weights after its electrophoretic separation (310). Sequence analysis also indicates that HBP bears very substantial similarities with serine proteases, and the highest similarity (45%) is to human neutrophil elastase (311). However, HBP lacks catalytic activity caused by critical subsitutions of several conserved amino acid residues important for the catalytic activity of serine proteases (312).

1.4.3.2 Antimicrobial activity HBP exhibits a very potent antimicrobial action. It was demonstrated that HBP contributed significantly to the ability of PMNs to kill gram-negative bacteria (313). Gabay et al. also provided evidence that HBP has potent activity against E.coli (307). One study demonstrated that although HBP on its own is ineffective against

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Capnocytophaga sputigena, it can synergize with elastase and cathepsin C in killing these oral bacteria (314).

1.4.3.3 Additional activities HBP is also known as a multi-functional protein released during inflammation with a high potential of regulating monocyte/macrophage functions. HBP is stored in secretory vesicles as well as primary granules of PMNs (315). Due to this distribution, HBP from rapidly mobilized secretory vesicles may target the endothelium and blood cells, while HBP from primary guanules may affect cells at the site of inflammation. The interaction between HBP and the endothelium results in activation of ECs (316) with subsequent intercellular gap formation and plasma leakage (317) together with enhanced expression of adhesion molecules (318, 319). It has been demonstrated that HBP is a potent and highly specific chemoattractant for monocytes (320), PMNs (321) and T-cells (322). HBP also activates monocytes and macrophages to release cytokines and chemokines (323, 324), and enhances the bacterial phagocytosis of macrophages (325).

30 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses

CHAPTER 2 AIMS

The work of the present thesis was founded on experimental evidence from our laboratory, indicating the involvement of leukotrienes, particularly LTB4, in atherosclerosis. These data inspired us to search for mechanisms by which LTB4 may promote vascular inflammation. Unexpectedly, we discovered novel interactions between LTB4 and alarmins, which form the core of this thesis.

The general aims of the present thesis are to study the interactions between proinflammatory lipids and polypeptides, the physiological functions of these interactions, and how anti-inflammatory lipids may interfere with these interactions. The specific objectives are:

2.1 To evaluate the expression of proteins in the 5-LO pathway for LTs biosynthesis in atherosclerotic lesions and the possible correlation with plaque instability (Paper I).

2.2 To study the interactions between LTB4 and LL-37, the mechanisms and functional effects of these interactions and the potential impacts of the anti-inflammatory lipids

LXA4 and RvE1 on LTB4/LL-37 circuit (Paper II and III).

2.3 To investigate the interactions between LTB4 and HBP, the mechanisms and the effects of these interactions on vascular permeability in vivo (Paper IV).

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CHAPTER 3 METHODOLOGY

The methods employed in this thesis are listed below. A detailed description of the method can be found in the papers where they appear as referenced.

1) Isolation of PMNs from buffy coat: Paper II, III, IV 2) Cell culturing (HUVEC): Paper IV 3) Peptide/protein extraction: Paper II, III 4) SDS-PAGE and Western blot analysis: Paper I, II, III 5) ELISA: Paper I, II, III, IV 6) Intracellular calcium mobilization: Paper III, IV 7) HPLC: Paper I, II 8) Immunocytochemistry: Paper I, II 9) Inhibition zone assay: Paper II 10) Phagocytosis assay: Paper II 11) Real-Time PCR: Paper I 12) Mice models: Paper I, IV

32 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses

CHAPTER 4 RESULTS AND DISCUSSION

At the onset of my PhD studies, several reports in the literature had implicated that leukotrienes, in particular LTB4, participates in the pathogenesis of arteriosclerosis, myocardial infarction and stroke. For instance, it had been demonstrated that 5-LO is abundantly expressed in human atherosclerotic lesions and correlates with disease stages (326). Furthermore, deletion of the 5-LO gene provides protection in atherosclerosis-prone mice (327), and a selective BLT1 antagonist could protect against atherogenesis in two mouse models of atherosclerosis (328). Interestingly, variants of the 5-LO and FLAP genes were identified as significant risk factors for the development of atherosclerosis and myocardial infarction in human (61, 158). However, data obtained with mouse models of LT deficiency and atherosclerosis were inconclusive and failed to establish a link between LTs and CVD (159, 327). Therefore, we went back to human samples and carried out mRNA profiling of enzymes and receptors of the LT cascade in a unique collection of plaque tissues.

4.1 Expression of proteins in the 5-LO pathway and biosynthesis of LTs in atherosclerotic lesions (Paper I) In this project, we exploited the large Biobank of Karolinska Endarterectomies (BiKE) collection of human atherosclerotic vascular tissues obtained from patients undergoing carotid endarterectomy. In addition, we investigated plaque tissues from two mouse models of atherosclerosis. These analyses generated a comprehensive expression profile of key enzymes and receptors of the LT cascade in humans and mice.

4.1.1 Expression of 5-LO, FLAP and LTA4H is increased and colocalized in human atherosclerotic plaques Our results demonstrated that in human carotid atherosclerotic lesions, the mRNA levels of 5-LO, FLAP and LTA4H were significantly increased as compared to healthy controls (normal iliac arteries). The increased level of 5-LO mRNA confirmed previous data on human atherosclerotic specimens (326), while the finding of enhanced mRNA levels of FLAP and LTA4H verifies the importance of these key

33 Min Wan, 2010 enzymes required for LTB4 biosynthesis in human atherosclerosis. These results also indicate a critical involvement of LTB4 in this chronic inflammatory disease. Monocytes and macrophages are important inflammatory cell types during the process of atherosclerosis, and these cells are probably the main source of LTB4 production in atheroslerotic lesions. It has been reported that LTB4 not only promotes the recruitment of moncytes to the lesion (329), but also modulates the functions of monocytes, such as cytokine gene expressions (330). Moreover, LTB4 is involved in the macrophage-foam cell transformation (331). From our immunostaining results, we showed that 5-LO, FLAP and LTA4H are colocalized in intimal lesions, particularly in macrophages, presumably facilitating efficient enzyme coupling and

LTB4 synthesis. Indeed, we found that human carotid plaque tissues incubated with

AA generated significant amounts of LTB4, a synthesis which is reduced by ~80% after incubation with a selective LTA4H inhibitor.

4.1.2 Expression of 5-LO and LTA4H mRNA correlates with recent or ongoing atherothrombotic events The plaque tissues could be divided into three categories depending on the time elapsed between the last recorded ischemic symptoms (transitory ischemic attacks, amaurosis fugax) and surgical removal of the lesion, < 1 month, 1-3 moths, and > 3 months. Presumably, these time intervals represent unstable plaques at different stages, i.e. shorter time interval means more unstable plaques. In our experiments, we detected higher mRNA levels of 5-LO and LTA4H in the lesions obtained at a shorter time interval from ischemic symptoms, indicating an association between expression of 5-LO and LTA4H and plaque instability. Together with genetic data identifying 5-

LO and LTA4H as risk factors for atherosclerosis (78, 158), our results strongly suggest that LTB4 may be involved in driving local acute inflammatory processes that precede an acute thrombotic event. Hence, our findings are potentially of clinical significance and suggest that LTA4H appears to be a promising target for development of drugs in the prevention and treatment of atherogenesis and acute atherothrombotic complications.

34 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses

4.1.3 Differences exist in the expression profiles of proteins in the LT pathway between human and murine lesions In our study, we have detected profound differences between human and mouse atherosclerotic lesions with respect to the expression patterns and localization of the proteins in LTs biosynthesis. Thus, none of the changes in mRNA levels of 5-LO,

LTA4H and FLAP could be reproduced in ApoE (-/-) or ApoE/LDLR (-/-) mice. In the latter mouse model, 5-LO and LTA4H immunoreactivities were segregated into different layers of the vascular wall. These differences could be explained by one or several of the following factors: firstly, the expression patterns of the proteins of the 5-LO pathway in LTs biosynthesis reflect species differencies with respect to susceptibility to LT-dependent atherogenesis; secondly, the histology of mouse arteries is different from human; thirdly, mouse models mostly reflect early phases of atherogenesis, whereas human surgical specimens are end-stage lesions causing clinical symptoms after years of progression. The spectrum of factors operating in the destabilization of such advanced lesions and subsequent thrombus formation is likely to differ significantly from that promoting initiation of the disease process; fourthly, mice do not develop atherosclerosis without genetic manipulation, therefore genetically modified mouse models, targeting two pivotal genes involved in lipoprotein metabolism, namely ApoE and LDLR, have been utilized largely for atherosclerosis research. However, these genetic manipulations may induce other complicated physiological changes that may explain some of the LT-related differences in atherosclerosis development in mice and human. In any event, the observed differences between human and mouse plaque tissues should be taken into account when evaluating effects of gene deletions or pharmacological interventions in these mouse models.

4.2 LTB4/LL-37 and LTB4/HBP interactions in human PMNs (Paper II -IV) The studies of human atherosclerotic plaque inspired us to look for novel mechanisms by which LTB4 could exert a proinflammatory action within the vascular wall. One lead was the previous observation that human atherosclerotic plaques express increased levels of the antimicrobial polypeptide LL-37 (287) and HBP (332).

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4.2.1 LTB4 is a potent stimulus of PMN exocytosis with concomitant release of LL-37 and HBP (Paper II and IV) Once inflammation or infection is initiated by various noxious agents or microbes, PMNs are the first cells to be recruited to the sites of injury, guided by chemoattractants such as LTB4, which is released by resident inflammatory cells. In Paper II, exposure of human PMNs to as little as 1 nM LTB4 led to an almost instantaneous (within 60 s) degranulation and release of the human antimicrobial peptide LL-37 detected by Western blot analysis and ELISA. The precursor of LL-37, hCAP18, is stored in PMN secondary granules; while the processing enzyme proteinase-3, which cleaves hCAP18 into LL-37, is mainly stored in the primary granules (333).

Our results imply that LTB4 induces exocytosis of both primary and secondary granules of PMNs. In Paper IV, we analyzed the presence of markers for primary and secondary/tertiary granules, MPO and MMP-9 respectively, in the supernatants from

LTB4-triggered PMNs. Immunoblotting of MMP-9 and MPO activity assays revealed that LTB4 induced the release of primary, secondary, and tertiary granules, thus confirming our hypothesis in Paper II.

In Paper IV, we found by Western blot analysis that LTB4 also induces a rapid release of HBP (within 30 s) that continued up to 5 min after challenge. Similar responses were detected with fMLP stimulation. The concentration-response curve revealed that LTB4 induces the release of HBP with a threshold concentration of 10 nM and a maximal response at 1 µM. Tyrosine kinases of the Src family have been implicated in PMN degranulation (334). We showed that the release of both LL-37 and HBP from human PMNs depends on these kinases since the secretion could be blocked by the potent Src tyrosine kinase inhibitor PP-1. Notably, these experiments of PMN degranulation were carried out with PMNs pre- treated with 10 µM cytochalasin B (cytoB) for 5 min before LTB4 stimulation. It is known that actins act as a physical barrier, preventing granule access to the plasma membrane during the process of PMN exocytosis or degranulation (335). Thus, actin cytoskeleton remodeling is an important step for PMN exocytosis or degranulation. CytoB is a well-known inhibitor of actin polymerization (336) and widely used to disrupt the actin structure, enhancing basal and stimulus-coupled exocytosis and degranulation of PMNs. In pilot experiments, we have shown that the presence of cytoB alone did not induce PMN degranulation.

36 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses

4.2.2 LTB4-induced exocytosis of PMNs occurs through activation of the BLT1 receptor (Paper II and IV)

It is known that LTB4 acts via two specific G protein-coupled receptors (GPCR) termed BLT1 (high affinity) and BLT2 (low affinity). In our expriments, LTB4- induced release of LL-37/hCAP18 and HBP from PMNs could be efficiently inhibited by the BLT1-specific antagonist CP105696, whereas the BLT2 antagonist LY255283 was ineffective. Furthermore, the unique BLT2-specific agonist, Compound A (337), failed to induce LL-37/hCAP18 and HBP from PMNs. In addition, the effects of other eicosanoids on LL-37 release were consistent with their affinity for BLT1 rather than BLT2. Taken together, our results demonstrate that exocytosis from human PMNs after LTB4 stimulation is primarily mediated by BLT1.

4.2.3 Functional studies of released polypeptides from LTB4-stimulated PMNs (Paper II-IV) In our studies, we have demonstrated that the alarmins LL-37 and HBP are promptly released from PMNs, in response to the proinflammatory mediator LTB4. In this section, we continue to discuss the functions of released LL-37 and HBP.

4.2.3.1 The functional studies of released HBP from LTB4-stimulated PMNs (Paper IV) Although HBP has been known to possess antimicrobial activities, we have focused on its functions as alarmin. HBP has been shown to activate monocytes (338), macrophages (325) and lymphocytes (322), thereby enhancing leukocyte recruitment and the release of specific cytokines. Activation of ECs by HBP has previously been suggested to be a key mechanism behind PMN-induced changes in vascular permeability (317). Since LTB4 is known to stimulate enhanced vascular permeability through PMN activation (339), we investigated the potential contribution of HBP in

LTB4-induced vascular permeability. Furthermore, we analyzed the effects of HBP in the activation of ECs that is tightly regulated during changes of vascular permeability.

4.2.3.1.1 HBP, derived from neutrophils challenged with LTB4, activates endothelial cells 2+ The supernatant from LTB4-treated PMNs triggers a rise in EC [Ca ]i that could be completely prevented by pretreatment of PMNs with the selective BLT1

37 Min Wan, 2010 antagonist CP105696. Previous data obtained in our laboratory revealed that 2+ quiescent HUVEC exposed to LTB4 did not induce [Ca ]i change (105). Moreover, 2+ the ability of the postsecretory supernatants to induce changes in EC [Ca ]i decreased by almost 80% after selective removal of HBP by specific immunoadsorption. This result is in agreement with the finding of reduced release of HBP after CP105696 treatment as determined by Western blot analysis. HBP- 2+ depleted supernatants still exhibited partial activity of inducing changes of EC [Ca ]i, which is likely due to the presence of additional bioactive molecules in the supernatant of LTB4-treated PMNs, such as elastase (340) and cathepsin G (341).

4.2.3.1.2 LTB4 increases vascular permeability via neutrophil-derived HBP Animal experiments were performed to test the functions of HBP in vivo utilizing a mouse model in pleurisy. In this mouse model, FITC-conjugated dextran was monitored as a plasma tracer. Clearance volume (CV) and the number of PMNs were measured to evaluate the vascular permeability. Higher values in these two parameters indicate that more plasma enter the pleural cavity from blood vessels, representing higher vascular permeability. The antibody RB6-8C5, which specifically recognizes PMNs, was employed to create neutropenic mice by depleting PMNs.

Our results demonstrated that the injection of LTB4 into pleural cavity of normal mice induces increased vascular permeability. However, LTB4-induced plasma extravasation is abolished in PMN-depleted mice. Furthermore, both plasma exudation and PMN recruitment are reduced markedly after blockade of BLT1. These results clearly demonstrated that the LTB4-induced increase in vascular permeability is related to activation of PMNs and illustrates the fact that LTB4 does not directly affect EC barrier function.

Moreover, treatment of neutropenic mice with the supernatant from LTB4-treated PMNs showed that the increase in vascular permeability caused by this supernatant could be ascribed to the presence of HBP in the postsecretory supernatant. These data 2+ are in agreement with the results obtained in the experiments with EC [Ca ]i mobilization, confirming that HBP released from LTB4-stimulated PMNs is not only biologically active but also contributes to plasma extravasation during leukotriene- dependent inflammation.

38 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses

4.2.3.2 Functional studies of released LL-37 from LTB4-stimulated PMNs (Paper II and III) 4.2.3.2.1 Antimicrobial activity

The antimicrobial activity of released LL-37 from LTB4-stimulated PMNs was evaluated by an inhibition zone assay. The Gram+ bacterium B. megaterium strain Bm11 is an LL-37 sensitive strain; therefore it was utilized as our experimental model to test the antibacterial activity of LL-37. Our results demonstrated that the secreted material from LTB4-treated PMNs exhibited high antibacterial activity against Bm11. The antibacterial activity decreased significantly after the secreted material was incubated with a specic monoclonal LL-37 antibody, compared to material incubated with a nonspecic monoclonal antibody. Hence, in our experimental conditions most of the antibacterial activity observed in the material secreted from

LTB4-stimulated PMNs originated from functionally active LL-37.

LTB4 has been found to enhance the killing of several different microbes (163, 164, 167, 168). Besides LL-37, it is known that HBP also exhibits potent antimicrobial activity, although we did not analyze this activity in our project. Flamand et al. also provided the evidence that LTB4 causes the release of potent antimicrobial agents from PMNs in vitro as well as in vivo (171). Therefore, we assume that the release of these mediators upon treatment of PMNs with LTB4 most likely contributes to the previously described antimicrobial activities of LTB4.

4.2.3.2.2 Proinflammatory functions It has been reported that LL-37 exerts several proinflammatory funtions on PMNs, therefore, we hypothesized that LL-37 can induce LT synthesis in PMNs. 2+ It is known that changes in [Ca ]i activate early key enzymes of LT biosynthesis, such as cPLA2 and 5-LO. Upon cellular activation, these two enzymes are phosphorylated and translocate to the nuclear envelope. In our experiments, we found 2+ that incubation of PMNs with LL-37 induces mobilization of [Ca ]i in a dose- dependent manner. Interestingly, we also found that LL-37 promotes cPLA2 phosphorylation and the translocation of 5-LO from the cytosol to the nuclear membrane of human PMNs. Consequently, we could observe LTB4 synthesis as assessed by an LTB4 enzyme immunoassay kit (EIA) and HPLC. Under optimized conditions, LTB4 production was detected by EIA after direct incubation of PMNs

39 Min Wan, 2010 with 15 µg/ml of LL-37 for 20 min. However, the level of released LTB4 was too low to be detected by HPLC analysis. The pretreatment or “priming” of circulating PMNs with proinammatory stimuli, such as LPS or certain cytokines such as GM-CSF, would enhance the responses of

PMNs to subsequent stimulation, resulting in an increased potential to generate LTB4 (342, 343). Considering the in vivo situation during tissue inflammation, extravasated PMNs are in contact with surrounding cytokines and LPS from bacteria. Accordingly, we primed PMNs with LPS or GM-CSF to possibly produce larger quantities of LTB4, enabling HPLC analysis. This priming process together with LL-37 stimulation did indeed result in greater and more prompt production of LTB4 (5 min to reach peak concentrations) with levels that were easily detected by HPLC analysis. However, in control experiments with PMNs primed with LPS or GM-CSF alone, essentially no

LTB4 was produced. Notably, inhibitors of p38 MAP kinase and Src tyrosine kinases almost totally block the LTB4 release from LL-37-treated neutrophils, displaying a p38 MAP kinase and Src tyrosine kinase-dependent reaction.

In addition to LTB4, we also analyzed the production of cys-LTs from LL-37-treated PMNs. However, no change in concentration of cys-LTs was detected according to our preliminary data. Considering previous reports that LL-37 exerts proinflammatory effects on different cell types, it would be very interesting to acquire a more complete picture of the produced lipids after treatment of various inflammatory cells with LL-37. Such a screening would better evaluate the functions of LL-37 as an alarmin. Our results demonstrate that the endogenous host defense peptide LL-37 can stimulate both intact and primed PMNs to release LTB4. Thus, a positive feedback loop appears to exist between LTB4 and LL-37. Subsequently, we analyzed the effects of the LTB4/LL-37 circuit on an important function of PMN, i.e. phagocytosis. We found that LL-37 induces phagocytosis of E. coli particles and that this function is signicantly enhanced by concurrent exposure to LTB4. When PMNs were primed with GM-CSF, we observed reciprocal additive effects of both LTB4 and LL-37 on phagocytosis evoked by the respective mediator. Thus, the effect of cross-talk between LL-37 and LTB4 is not limited to their formation and signaling, but is extended to functional responses such as phagocytosis. This activity is important for the efcacy and guidance of human innate immune responses.

40 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses

4.2.4 FPRL-1 mediates LL-37-induced LTB4 release from human PMNs (Paper III)

We also analyzed the LTB4 production of PMNs treated with sequence-scrambled LL-37 peptide (sLL-37). Our results show that sLL-37 is almost inactive in inducing the production of LTB4, indicating that the effects of LL-37 are specic and presumably receptor mediated. We have shown that pre-incubation with pertussis toxin, which is a potent inhibitor of GPCR, totally blocks LTB4 release from PMNs in response to LL-37 incubation, indicating that this reaction is mediated by GPCR(s). Previous research has shown that various biological functions of LL-37 are mediated by several receptors including FPRL-1 and the recently reported CXCR2, both of which belong to the GPCR family (200, 344). Furthermore, preincubation with the specific FPRL-1 antagonist, WRW4 peptide, almost totally depletes LTB4 release from PMNs. Moreover, high concentrations of LTB4 could be detected when PMNs were incubated with different concentrations of the FPRL-1 specific agonist, WKYMVm. Taken together, these data indicate that LL-37-induced LTB4 release from human PMNs is mediated by FPRL-1.

4.2.5 The positive circuit of LTB4/LL-37 is counter-regulated by anti- inflammatory lipids (Paper III)

We have demonstrated the existence of a positive feedback loop between LTB4 and LL-37 in vitro. In acute inflammation, uncontrolled proinflammatory responses are harmful to the host, which could lead to chronic inflammation. Therefore, in physiological situations it is important to balance and regulate this positive feedback circuit. In the resolution phase of inflammation, the lipid profile produced by PMNs shifts from pro-inflammatory to anti-inflammatory mediators including resolvins and lipoxins, which are pivotal for the resolution of inflammation. In this paper, we show that ω-3 PUFA-derived RvE1 and AA-derived LXA4 down-regulate the LTB4-induced

LL-37 release and LL-37-induced LTB4 production, respectively, from human PMNs. This indicates that these two anti-inflammatory mediators could serve as “negative signals” for the LTB4/LL-37 positive circuit. These anti-inflammatory mediators are generated via transcellular routes between PMNs and other cell types. After PMNs have migrated into the inflamed sites, they make contact with various cell types, including epithelial cells and ECs, which facilitate the production of resolvins and lipoxins in the human body.

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Interestingly, it has been reported that RvE1 regulates inflammation in part by interaction with BLT1 (231), which is the high affinity receptor for LTB4. In our experiments RvE1 could compete with LTB4 and bind to BLT1, resulting in inhibition of LTB4-induced exocytosis of PMNs. However, it is known that ChemR23, another receptor of RvE1, also mediates several anti-inflammatory functions of RvE1 (231). According to our experiments, we can not exclude the possibility that ChemR23 is also involved in this counter-regulation. Nonetheless, our results are in excellent agreement with earlier data showing that binding of RvE1 to BLT1 attenuates LTB4-dependent pro-inflammatory signals (231).

On the other hand, LXA4 is recognized as a potent agonist of FRPL-1, and possesses anti-inflammatory and pro-resolving functions. Thus, LL-37 and LXA4 are ligands to the same receptor. How can pro-inflammatory and anti-inflammatory mediators share the same receptor regulating opposite responses? It has been suggested that distinct conformational changes of the receptor induced by different ligands may cause changes in interactions between the receptor and the heterotrimeric G proteins, resulting in differential activation of effector molecules (345). From other studies and our data, we suggest that LL-37 and LXA4 bind to different sites of FRPL-1, inducing conformational changes of the receptor and G protein coupling patterns. The binding of

LXA4 to FRPL-1 might induce down-regulation of key signaling molecules in the pathway of LL-37-induced LTB4 production. However, more studies are needed to verify our hypothesis.

42 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses

CHAPTER 5 CONCLUSIONS

5.1 Regulation of inflammatory responses by the pro-inflammatory mediators

(LTB4 and LL-37) and the anti-inflammatory lipids (LXA4 and RvE1) The conclusions of Paper II and III are illustrated as a schematic model in Figure 8. (1) Once inflammation or infection is initiated by various microbes, PMNs migrate into the site of injury, guided by chemoattractants including LTB4, which is released by resident macrophages or mast cells. According to our results, (2) LTB4 binds to the high affinity receptor BLT1 on human PMNs, inducing the activation of Src family tyrosine kinases followed by LL-37 release from PMNs. (3) The released LL-37 either kills microbes directly or (4) binds to the receptor FPRL-1 that induces phosphorylation of cPLA2 via p38 MAPK pathway and 5-LO translocation, resulting in more production of LTB4 in PMNs. (5) This in turn results in a positive feedback loop between LTB4 and LL-37. (6) In the resolution phase of inflammation, LXA4 and RvE1 are generated, competing with LL-37 and LTB4 in the binding of FPRL-1 and BLT1, respectively, and serve as negative signals for the positive LTB4/LL-37 circuit.

Figure 8. A schematic model of the regulation of inflammatory responses by the pro-inflammatory mediators and the anti-inflammatory lipids

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5.2 LTB4-HBP interactions in control of vascular permeability

The conclusions of Paper IV are illustrated in Figure 9. (1) LTB4 released by resident macrophages in inamed tissues interacts with BLT1 present on the surface of PMNs.

(2) The binding of LTB4 to BLT1 leads to the activation of PMNs and the β2 integrin- mediated adhesion of PMNs, (3) inducing release of PMNs granule contents including HBP via the 1-phosphatidylinositol 3-kinase (PI3K) pathway. (4) HBP is released into the interface between the adherent PMNs and ECs and interacts with ECs, (5) 2+ triggering [Ca ]i mobilization and formation of interendothelial gaps and consequent plasma leakage.

Figure 9. A schematic model of LTB4-HBP interactions in control of vascular permeability

44 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses

CHAPTER 6 ACKNOWLEDGEMENTS

My passed five years as a PhD student at Department of Medical Biochemistry and Biophysics, Karolinska Institutet, has been a pleasant experience. Hereby, I would like to give my sincere gratitude to all the people who have given me help and support, and shared the good and bad time with me during these years, in particular to:

My main supervisor Prof. Jesper Z. Haeggström. Thank you very much for accepting me as your PhD student, for sharing your extensive knowledge, enthusiasm and wisdom with me, for the endless encouragement and support, for giving me enough freedom to explore the fun of research. I am indebted to you for your critical reading of the thesis and for never getting annoyed by my interruptions from time to time. I also feel truly grateful to you for being like a father to me and giving me honest advice. I have learned so much from you not only in research-related matter.

My co-supervisor Prof. Birgitta Agerberth. I feel I was so lucky to have you as my co-supervisor. Thanks a lot for being such an excellent mentor, sharing your enormous knowledge, keeping an eye on the progress of my work and critical reading of the manuscripts and thesis. I really appreciate your thoughtful concerns for my life as a mother and for all your support and encouragement during these years.

My co-supervisor Dr. Anders Wetterholm. You are always the best! Thank you so much for sharing your great expertise, always giving me incredibly useful advice, endless help with my experiments and my life, and for your careful reading of the thesis. I also appreciate the fresh asparagus from you and the unforgettable trip to Öland.

Special thanks to Prof. emeritus Bengt Samuelsson, for establishing a wonderful research field and for providing a creative international research environment.

Prof. Göran Hansson, Anders Gabrielsen, Gabrielle Paulsson-Berne from the Department of Molecular Medicine and Surgery, for the wonderful collaboration on the atherosclerosis project; Prof. Jan Palmblad and Anne-Sofie Johansson from the Department of Medicine, Huddinge University Hospital, for the fruitful collaborations on the HUVEC projects, and the help with the ethical permit; Baldur Sveinbjörnsson, Agnes Rasmuson from the Department of Woman and Child health, for a very nice collaboration on the neuroblastma project, interesting discussions and support in the Mexican conference; Prof. Cecilia Söderberg-Nauclér and Klas Strååt from the Department of Molecular Medicine and Surgery, for the excellent collaboration on the CMV project; Prof. Johan Frostegård, Xiang Hua and Jun Su from the Department of Medicine, Huddinge University Hospital, for the collaboration on the interesting

45 Min Wan, 2010 cardiolipin project; Prof. Lennart Lindbom, Oliver Soehnlein and Ellinor Kenne for inspiring discussions and nice collaborations on the HBP project.

The previous and present colleagues and friends at Kemi II in MBB: Prof. Mats Hamberg and Olle Rådmark, for your generous help whenever I needed it; Hong Qiu, for your friendship, for guiding me to start in the lab and for our fruitful collaborations; Antonio Di Gennaro, for excellent collaborations and enjoyable discussion, for being such a gorgeous neighbour in the office; Alan Sabirsh, for your contribution to the LL- 37 project, and for all inspiring discussion about research and culture; Craig Wheelock, for the technical support, for sharing your great expertise; Eva Ohlson, for technical assistance and other countless help; Dolores Salvado Duro, for being such a good roommate for MBB and Mexican conferences, interesting discussion on our projects and our lives, and all delicious food and nice gifts from your mom; Agnes Rinaldo- Matthis, Alicia Hidalgo, Julia Esser, Marija Rakonjac, Susanna Lundström, Fabio Dalexandri, Jia Sun, Sven Pawelzik, and previous colleagues Fredrik Tholander, Sipra Saha, Ulrike Haas, Hiromi Hanaka, Tove Hammarberg, Vildan Dincbas-Renqvist, Yilmaz Mahshid, Gudrun Tibbelin, Lina Adriana, for sharing your expert knowledge in the lab, for the fun during summer schools, and for all pleasant talks and discussions during lunch and cake time. Anneli Svarén and Galina Stahmer for great help with all administrative issues and sharing the experience on taking care of babies. Thank all of you for making Kemi II such a wonderful place to work in.

Monica Lindh, for teaching me all the techniques related to LL-37, for always organizing the lab in good order, for the countless help and all pleasant talks with you; Ylva Kai-Larsen, Andreas Cederlund, Protim Sarker, Peter Bergman, Essam Refai, for sharing your expert knowledge in the lab and for enjoyable talks and discussion. I was always laughing in your lab, so much fun with you guys; and all other people in Kemi I, for scientific discussions in regular seminars and making comfortable atmosphere in the division.

The people in previous Stockholms Biovetenskapliga Forskarskola: Prof. Eva Severinson, thank you for giving me the opportunity to start my studies at Karolinska Institutet, for organizing all the interesting courses and seminars; Camilla Ahlqvist, thank you so much for giving me numerous help when I just came to Stockholm; all classmates, for all nice group work together, and the great trip to Reykjavik.

All my friends outside the lab: Xiaoqun Zhang, Hongshi Qi, Yan Li, Bing Zhang, Qiaolin Deng, Zhe Jin, Xiaowei Zheng, Jiangning Yang, Xiaofeng Zheng, Xin Wang, Jiakun Cai, Ying Sheng, Junhang Zhang, Rui Liu, Rong Liu, Yu Sun, for all the fun we have shared with Badminton, nice dinners and trips! Special thanks to

46 Studies on Leukotriene B4 and Alarmins in Inflammatory Responses

Qiaolin and Zhe, for picking me up at the airport when I came to Stockholm for the first time and making me feel not far away from home; Xiaoqun and Hongshi, for your countless help and thoughtful concern; Yan and Bing, for all honest advice and enjoyable talks; Xiaowei Zheng and Jiangning Yang, for all the help and flavorful food from you; Ying Sun and Liqun He, for sharing the experience as parents and the help with moving; Jiakun Cai and Ying Sheng, for being always like a brother and a sister to me and for your support during my difficult time; Shaobo Jin and Jian Zhao, for the nice trip to Helsinki and all delicious food at your home; Tiiu Saarne, for all the good time we have shared and all the help from you; Jinfeng Shen, JiYeun Hur and Yu Li, for lots of fun with you, and the memorable trip to the Netherlands; Zhuochun Peng, Li Wang and Yaping Li, for being such good roommates and great companionship; Junhang Zhang, Xiaoda Wang, Ling Zheng, Shucheng Ying and Jing Chang, for all the fun with you in Jägargatan 20; Wennie Wei, 感谢你的热情善 良和真诚的友谊，以及对我所有的帮助！Yuan Xue, Yingbo Lin, Mingmei Shang, Wei Jiao, Yanjiao Zhang, Xiao Wang, Jianguang Ji, Tong Liu, Ying Zhu, Jun Ma, Lina Yu, Sergio Montano, Qing Cheng, Bin Zhao, thanks a lot for your friendship, the nice talks and the help from you!

My special appreciation to my Master’s supervisor Prof. Kangsen Mai. Thank you so much for introducing me into research, and for your constant encouragement and support through all these years. You have set up such a successful example for younger researchers. Ms. Wei Xu, Dr. Beiping Tan, Qinghui Ai, Wenbing Zhang and all other previous colleagues in the lab of Prof. Mai, thanks a lot for your friendship and care during these years, for organization and invitation for “Hai Zhi Yuan” scientific club, and for keeping me updated on the situation back home.

Last but not least, my beloved family: My dear parents, 养儿方知父母恩，感谢你们 从小对我的教育和培养，一直以来对我的理解和支持！没有你们的牺牲和帮助 就没有这本论文的顺利完成，真心地感激你们为我所付出的一切！ My younger brother, Xiang, for being so supportive and helpful, I could not ask for a better brother than you! Enjoy your time in US, and looking forward to your PhD thesis☺. All my uncles, aunties and cousins, for all the nice talks with you by phone, always making me feel not far away from home. My husband Ming, life has not been easy for us during these years, thank you so much for your love, for your understanding, support and encouragement, for always patiently listening to my complaint! My angel Yiwei, you are the best gift I have ever received! Thank you for all the happiness and courage you have brought to me. You let me know the real meaning of family and love. I love you forever!

47 Min Wan, 2010

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