MODULATORS OF THE IMMUNE RESPONSE

Thesis for doctoral degree (Ph.D.)2008

Oliver Söhnlein, M.D.

Thesis for doctoral degree (Ph.D.) 2008

Oliver Söhnlein, M

.D.

NEUTROPHIL GRANULE PROTEINS:

MODULATORS OF THE IMMUNE RESPONSE

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From the Department of Physiology and Pharmacology

Karolinska Institutet, Stockholm, Sweden

NEUTROPHIL GRANULE PROTEINS:

MODULATORS OF THE IMMUNE RESPONSE

Oliver Söhnlein

M.D.

Stockholm 2008

2008

Gårdsvägen 4, 169 70 Solna

Printed by

All previously published papers were reproduced with kind permission from the

publishers.

Published by Karolinska Institutet.

© Oliver Söhnlein, 2008

ISBN 978-91-7357-560-7

”There is no need to be a doctor or scientist

to wonder why the human body

is capable of resisting so many harmful agents

in the course of everyday life.”

Elie Metchnikoff, 1908.

”…. it is likely that the leukocyte

granulations are in fact secretory

products, which the cell dissolves

and spreads to the environment

as needed.”

Paul Ehrlich, 1900.

To Joana To Joana

ABSTRACT Polymorphonuclear leukocytes (PMN) are multifunctional cells of the innate

immune system which represent the advance guard in inflammatory reactions. Via the release of granule proteins the PMN communicates with the endothelium, monocytes, dendritic cells, and lymphocytes and is thereby critically involved in the fine tuning of the host defense system. This thesis aimed at elucidating mechanisms by which the PMN and its granule proteins recruit, activate, and stimulate monocytes and macrophages. Moreover, PMN secretory products may contribute to tissue damage, which is another focus of this project.

Extravasation of PMN to the site of injury or infection precedes the recruitment of monocytes. In the first part of the project we addressed causal connections between these two events. There we demonstrate that PMN deposit heparin-binding protein (HBP) from rapidly mobilized secretory vesicles on the endothelial cell surface. In this location HBP interacts with monocytes rolling along the endothelium. Consequently, monocytes become activated and adhere to the vessel wall in increased numbers. In a second study we applied two in vivo models to elucidate the importance of the primary PMN extravasation for the subsequent recruitment of monocytes. We show that PMN extravasation is crucial for the early recruitment of inflammatory monocytes, a subset of monoytes that has recently been shown to be of high significance in inflammatory reactions. As principal mediators of this effect we identify the PMN granule components LL-37 and HBP which employ monocytic formyl peptide receptors to stimulate recruitment of inflammatory monocytes.

Macrophages are of crucial importance in bacterial clearance. In the second part of the project we investigated the impact of PMN secretion products on the macrophage’s capacity to phagocytose IgG-opsonized bacteria. PMN secretion was found to strongly enhance the phagocytic capacity of macrophages and thorough investigation revealed HBP and human neutrophil peptides 1-3 ( -defensins) as principal mediators of this effect. Mechanistically, these two proteins induce the release of TNF and IFN from macrophages. These activate the macrophage in an autocrine fashion resulting in upregulation of the Fc receptors CD64 and CD32.

Infection with Streptococcus pyogenes of the M1 serotype may lead to acute lung injury and in the third part of the project we addressed the involvement of PMN secretion products in this response. We demonstrate that depletion of PMN, but not of monocytes, completely abolishes the M1 protein-induced lung damage. The lung damage was restored by injection of PMN secretion into neutropenic mice indicating that secretion products of PMN are principle inducers of lung damage following infection with S. pyogens.

LIST OF PUBLICATIONS

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

Roman numerals:

I. O. SOEHNLEIN, X. Xie, H. Ulbrich, E. Kenne, P. Rotzius, H. Flodgaard, E.E.

Eriksson, and L. Lindbom.

Neutrophil-derived heparin-binding protein (HBP/CAP37) deposited on

endothelium enhances monocyte arrest under flow conditions. J. Immunol.,

2005,174:6399-405

II. O. SOEHNLEIN, A. Zernecke, E.E. Eriksson, A.G. Rothfuchs, C.T. Pham, K.

Bidzhekov, H. Herwald, M.E. Rottenberg, C. Weber, and L. Lindbom.

Neutrophil secretion products pave the way for inflammatory monocytes.

submitted. III. O. SOEHNLEIN, E. Kenne, P. Rotzius, E.E. Eriksson, and L. Lindbom.

Neutrophil secretion products regulate anti-bacterial activity in monocytes and

macrophages. Clin. Exp. Immunol., 2008, 151:139-45. IV. O. SOEHNLEIN, Y. Kai-Larsen, R. Frithiof, O.E. Sorensen, A.F. Gombart,

H.P. Koeffler, E. Kenne, E.E. Eriksson, H. Herwald, B. Agerberth, and L.

Lindbom.

Neutrophil-derived HBP and HNP1-3 boost bacterial phagocytosis by

macrophages. submitted.

V. L.I. Påhlman, M. Mörgelin, J. Eckert, L. Johansson, W. Russell, K. Riesbeck, O.

SOEHNLEIN, L. Lindbom, A. Norrby-Teglund, R.R. Schumann, L. Björck,

and H. Herwald.

Streptococcal M protein: a multipotent and powerful inducer of inflammation. J.

Immunol., 2006, 177:1221-8. VI. O. SOEHNLEIN, S. Oehmcke, X. Ma, A.G. Rothfuchs, R. Frithiof, M.

Mörgelin, H. Herwald, and L. Lindbom.

Neutrophil degranulation mediates severe lung damage triggered by

streptococcal M1 protein. Eur. Resp. J., in press.

Publications by the author, which are not included in the thesis

O. SOEHNLEIN, S. Eskafi, A. Schmeisser, H. Kloos, W.G. Daniel, and C.D.

Garlichs.

Atorvastatin induces tissue transglutaminase in human endothelial cells. Biochem.

Biophys. Res. Commun., 2004, 322:105-9.

A. Schmeisser*, O. SOEHNLEIN*, T. Illmer, H.M. Lorenz, S. Eskafi, O. Roerick, C.

Gabler, R. Strasser, W.G. Daniel, and C.D. Garlichs.

ACE inhibition lowers angiotensin II-induced chemokine expression by reduction of

NF-kappaB activity and AT1 receptor expression. Biochem. Biophys. Res. Commun.,

2004, 325:532-40. * contributed equally

H. Ulbrich, O. SOEHNLEIN, X. Xie, E.E. Eriksson, L. Lindbom, W. Albrecht, S.

Laufer, and G. Dannhardt.

Licofelone, a novel 5-LOX/COX-inhibitor, attenuates leukocyte rolling and adhesion

on endothelium under flow. Biochem. Pharmacol. 2005, 70:30-6.

O. SOEHNLEIN, A. Schmeisser, I. Cicha, C. Reiss, H. Ulbrich, L. Lindbom, W.G.

Daniel, and C.D. Garlichs.

ACE inhibition lowers angiotensin-II-induced monocyte adhesion to HUVEC by

reduction of p65 translocation and AT 1 expression. J. Vasc. Res., 2005, 42:399-407.

H.K. Ulbrich, A. Luxenburger, P. Prech, E.E. Eriksson, O. SOEHNLEIN, P. Rotzius,

L. Lindbom, and G. Dannhardt.

A novel class of potent nonglycosidic and nonpeptidic pan-selectin inhibitors. J. Med.

Chem., 2006, 49:5988-99.

A. Zernecke, I. Bot, Y.D. Talab, E. Shagdarsuren, K. Bidzhekov, S. Meiler, R. Krohn,

A. Schober, M. Sperandio, O. SOEHNLEIN, J. Bornemann, F. Tacke, E.A. Biessen,

and C. Weber.

Protective Role of CXC Receptor 4/CXC Ligand 12 Unveils the Importance of

Neutrophils in Atherosclerosis. Circ. Res., 2008, 102:209-17.

O. SOEHNLEIN.

Changing views in neutrophil biology: from legionnaires to paratroopers. Front.

Biosci., in press.

P. Rotzius, O. SOEHNLEIN, E. Kenne, L. Lindbom, K. Nystrom, S. Thams, and E.E.

Eriksson.

Demonstration of ApoE/Lysozyme M eGFP/eGFP mice as a versatile model to study

monocyte and neutrophil trafficking in atherosclerosis. submitted.

M. Scholz, O. SOEHNLEIN, L. Lindbom, A. Mattern, G. Dannhardt, and H.K.

Ulbrich.

Diaryl-dithiolanes and isothiazoles: COX-1/COX-2 and 5-LOX-inhibitory, OH

scavenging and anti-adhesive activities. submitted.

R.R. Koenen, J. Pruessmeyer, O. SOEHNLEIN, L. Lindbom, A. Zernecke, L.

Fraemohs, N. Schwarz, K. Reiss, C. Weber, and A. Ludwig.

Regulated release and functional modulation of junctional adhesion molecule A by

disintegrin-like metalloproteinases. submitted.

LIST OF CONTENTS

INTRODUCTION .............................................................................................1

Innate Immunity.....................................................................................................................................2

PMN.........................................................................................................................................................2 PMN granule subsets ...........................................................................................................................3

Secretory vesicles............................................................................................................................3 Secondary and tertiary granules ......................................................................................................4 Primary granules .............................................................................................................................4 Focus on LL-37, HNP1-3, and HBP ...............................................................................................5

LL-37 .........................................................................................................................................5 HNP1-3 ......................................................................................................................................6 HBP............................................................................................................................................6

Control of degranulation......................................................................................................................7 Role of PMN in inflammation .............................................................................................................8

Monocytes..............................................................................................................................................10 Monocyte subsets ..............................................................................................................................10 Recruitment of monocytes to sites of inflammation ..........................................................................11 Formyl peptide receptors ...................................................................................................................12

Macrophages.........................................................................................................................................13 Phagocytosis ......................................................................................................................................14 Antigen presentation..........................................................................................................................15 Cytokine production ..........................................................................................................................15 Activation patterns.............................................................................................................................16

PMN granule proteins as regulators of innate and adaptive immunity...........................................17 Endothelial cells ................................................................................................................................17 Monocytes and macrophages.............................................................................................................18 Dendritic cells....................................................................................................................................19 T cells ................................................................................................................................................19

AIMS..............................................................................................................20

RESULTS AND DISCUSSION ......................................................................21

Establishment and validation of methodology ...................................................................................21 PMN depletion...................................................................................................................................21 Activation of PMN by antibody cross-linking of CD18 ....................................................................21 Subcellular fractionation....................................................................................................................22

PMN secretion products command extravasation of monocytes......................................................23 Paper I................................................................................................................................................23 Paper II ..............................................................................................................................................25

PMN secretion products boost antimicrobial activity in macrophages ...........................................27 Paper III .............................................................................................................................................28 Paper IV.............................................................................................................................................29

PMN secretion products induce phenotypic changes in monocytes and macrophages and induce cytokine release.....................................................................................................................................31

Paper IV.............................................................................................................................................32 Paper V ..............................................................................................................................................34

The pernicious effect of PMN secretion..............................................................................................34

PERSPECTIVES ...........................................................................................37

Blocking PMN functions ......................................................................................................................37 Block adhesion ..................................................................................................................................37 Block degranulation...........................................................................................................................38 Antagonize PMN granule proteins ....................................................................................................38

Enhance PMN functions ......................................................................................................................39 Tumor biology ...................................................................................................................................39 PMN-derived proteins as antibiotics..................................................................................................39 Anti-endotoxic activity of cationic host defence peptides .................................................................40 Modulation of protective innate immunity by peptides .....................................................................40

Conclusion.............................................................................................................................................42

METHODOLOGY ..........................................................................................43

Cell culture............................................................................................................................................43 Isolation and activation of PMN........................................................................................................43 Isolation and culture of monocytes and macrophages .......................................................................43 Isolation and culture of BAEC and HUVEC .....................................................................................43 Culture of cell lines THP-1, Mono Mac 6, WEHI-3B, and RAW264.7 ............................................44

Physiological assays..............................................................................................................................44 Flow chamber ....................................................................................................................................44 Ca2+ mobilization...............................................................................................................................45 Phagocytosis assay.............................................................................................................................46 ROS measurement .............................................................................................................................46

Animal experiments..............................................................................................................................47 Animals..............................................................................................................................................47 Air pouch ...........................................................................................................................................47 Intravital microscopy .........................................................................................................................47 Peritonitis...........................................................................................................................................48 Bronchioalveolar lavage (BAL) and vascular permeability assay .....................................................48

Molecular Biology.................................................................................................................................49 HPLC.................................................................................................................................................49 Subcellular fractionation....................................................................................................................49 Immunoabsorption.............................................................................................................................50 ELISA................................................................................................................................................50 Immunocytochemistry .......................................................................................................................50 PCR....................................................................................................................................................51 Histology and electron microscopy ...................................................................................................51 Western Blot analysis ........................................................................................................................51

Statistics.................................................................................................................................................52

LIST OF ABBREVIATIONS ..........................................................................53

REFERENCES ..............................................................................................55

ACKNOWLEDGEMENTS .............................................................................72

Neutrophil granule proteins: Modulators of the immune response

1

Introduction The function of the immune system is to prevent the takeover of the body by

genomes other than that encoded in the germline (Nathan, 2002). Central to this function is the ability to kill, which is one of the core competences of the polymorphonuclear leukocyte (PMN), one of the body’s main cellular components for the destruction of microorganisms. The discovery of PMN dates back to the German Nobel laureate Paul Ehrlich, who already at that time realized the functional potential of the PMN granule contents in the inflammatory process: “… it is likely that the leukocyte granulations are in fact secretory products, which the cell dissolves and spreads to the environment as needed” (Ehrlich, 1908). However, it was also realized, that the emigration of the PMN is associated with cell and tissue damage to the host (Weiss, 1989). In fact, PMN-mediated tissue damage at sites of infection is so common that the host takes stock of it when judging whether to mobilize an immune response. Indeed, tissue injury is an important source of information that launches inflammation. However, the PMN’s role in triggering an immune response has long been underestimated. At its best, the PMN was acknowledged as rudimentary for its most-studied behaviors - crawling, eating, and disgorging prepacked enzymes and reactive oxygen species (ROS) (Nathan, 2002; Nathan, 2006).

Over the last years the view of the PMN has changed dramatically. It was realized that the PMN not only exerts functiones privatae such as bacterial killing by phagocytosis, release of reactive oxygen species (ROS) or antimicrobial peptides, but the PMN carries out plenty of functiones publicae. The PMN is a decision-shaper in launching the immune response (Nathan et al., 2006). Much of this perception is derived from diseases caused by dysfunctions of PMN, which are not only associated with recurrent infections, but also secondary dysfunctions of monocytes, macrophages, dendritic cells (DC), and lymphocytes (Lekstrom-Himes and Gallin, 2000). These studies have strongly contributed to two theories explaining the ignition of the immune response. The danger theory holds that injured host cells release alarm signals that activate antigen-presenting cells (APC) thereby initiating an immune response (Matzinger, 2002). The pattern-recognition theory postulates that 'microbial non-self' induces an innate immune response, which in turn triggers an adaptive immune response (Medzhitov and Janeway, 2002). No matter which theory turns out to be more important, the PMN holds a central position in both and is therefore an important checkpoint in controlling the immune response.

Introduction

2

Innate Immunity All multicellular organisms, including plants, intervertebrates, and vertebrates,

possess intrinsic mechanisms for defending themselves against microbial infections. Because these defense mechanisms are always present, ready to recognize and eliminate microbes, they are said to constitute innate immunity. The shared characteristic of the mechanisms of innate immunity is that they recognize and respond to microbes but do not react against nonmicrobial substances. An innate immune response may also be triggered by host cells that are damaged by microbes. Innate immunity contrasts to adaptive immunity, which must be stimulated by and adapts to encounters with microbes before it can be effective. Furthermore, adaptive immune responses may be directed against microbial as well as nonmicrobial antigens. The innate immune system consists of epithelia, which provide barriers to infection, cells in the circulation and tissues, the complement system and cytokines of the innate immune system (Abbas and Lichtman, 2004; Janeway et al., 2005).

For many years it was believed that innate immunity is non-specific, weak, and ineffective in combating most infections. We now know that innate immunity specifically targets microbes and launches a powerful early defense mechanism capable of controlling and even eradicating infections before adaptive immunity becomes active. In addition to providing an early defense against infections, innate immunity also orchestrates the response of the adaptive immune system to respond to different microbes in ways that are effective at combating these microbes. Conversely, the adaptive immune response often uses mechanisms of innate immunity to eradicate infections. Therefore, elements of the innate immune system are of central importance in the host defense and some of the cellular components shall be highlighted here.

PMN PMN form the major type of leukocytes in human peripheral blood, with

counts ranging from 40 to 70 % of leukocytes under normal conditions. These cells mature in the bone marrow in about two weeks, a process in which the myeloid-specific growth factors G-CSF and GM-CSF play an important role. In the first half of this 2-week period, the PMN precursors undergo five divisions and differentiate from myeloblasts through promyelocytes to neutrophilic myelocytes. In the promyelocyte stage, the primary granules are formed, whereas the secondary granules are formed in the myelocytic stage. Later stages of PMN differentiation comprise metamyelocytes, band forms, and segmented cells. At the metamyelocyte stage tertiary granules are produced, while secretory vesicles are likely to be formed by endocytosis when the PMN is circulating in the blood (Borregaard et al., 1987).


3

PMN granule subsets Granule proteins of PMN are key effectors in the immune response initiated by PMN (Chertov et al., 2000). The subsets of PMN can be classified as primary, secondary, and tertiary granules, as well as secretory vesicles (Borregaard and Cowland, 1997; Faurschou and Borregaard, 2003). Distinctions between granule classes can be made upon analysis of marker proteins. About 300 different proteins are stored in PMN granules (Lominadze et al., 2005), which will be released into the surrounding, incorporated into the cell membrane or remain attached to the membrane upon granule mobilization. During its journey from the blood stream to the inflammatory site, the PMN releases its granules in a hierarchical order (Figure 1) (Borregaard and Cowland, 1997; Faurschou and Borregaard, 2003): Secretory vesicles are characterized by their immediate release when contact is established between the PMN and the endothelium. Tertiary granules are mobilized when the PMN transmigrates, and secondary and primary granules are liberated at the site of inflammation. Proteins inside the granules are thereby set free when needed for the PMN to extravasate and kill microorganisms. Furthermore, these proteins are deposited in the extravascular space and functionally affect inflammatory cells nearby.

secretory vesicles:HBP, fpr, CD14,

2-integrins

tertiarygranules:MMP-9, lactoferrin

secondary and primary granules:LL-37, HNP1-3HBP, cathepsin G,Elastase, MPO

secretory vesicles:HBP, fpr, CD14,

2-integrins

tertiarygranules:MMP-9, lactoferrin

secondary and primary granules:LL-37, HNP1-3HBP, cathepsin G,Elastase, MPO

Figure 1: Hierarchical organization of PMN granule release.

Secretory vesicles Secretory vesicles are of endocytotic origin (Borregaard et al., 1987) and represent a pool of membrane-associated receptors, which will be incorporated into the plasma membrane after release of the vesicles (Sengelov et al., 1993). Abundant receptors within the secretory vesicle membrane are 2-integrins, CR1, formyl peptide receptors (fpr), CD14, CD16 and the metalloproteinase leukolysin (Table 1). Mobilization of secretory vesicles and incorporation of these receptors into the cell

Introduction

4

membrane changes the phenotype of the PMN completely. Thereby the relatively inactive, on the endothelium rolling PMN is transformed into a cell that is capable of interacting with the endothelium, monocytes and dendritic cells and to receive inflammatory signals from the environment. Besides plasma proteins like albumin only one additional protein is described to be stored in secretory vesicles: heparin binding protein (HBP, also known as CAP37 and azurocidin) (Tapper et al., 2002). The release of HBP at the initial stage of PMN extravasation is thought to be of essential importance in the PMN-induced increase in vascular permeability (Gautam et al., 2001) and the adhesion of monocytes to endothelial cells (Lee et al., 2003, Soehnlein et al., 2005).

Secondary and tertiary granules Peroxidase-negative granules can be divided into secondary granules and

tertiary granules (Kjeldsen et al., 1992). This separation is arbitrary, since peroxidase-negative granules are formed as a continuum in myelocytes and metamyelocytes. However, from a physiological point of view, the conceptual separation of peroxidase-negative granules into two subsets is meaningful, since secondary granules and tertiary granules differ significantly from each other with respect to protein content and secretory properties. Thus, whereas the larger secondary granules are rich in antibiotic substances, the smaller tertiary granules are not (Table 1). Conversely, tertiary granules are more easily exocytosed than secondary granules. These characteristics reflect that tertiary granules are important primarily as a reservoir of matrix-degrading enzymes and membrane receptors needed during PMN extravasation and diapedesis (Joiner et al., 1989; Jesaitis et al., 1990; Mollinedo et al., 1997). In contrast, secondary granules participate mainly in the antimicrobial activities of the PMN by mobilization of their arsenal of antimicrobial substances (e.g. lactoferrin, NGAL, lysozyme, and hCAP18, the proform of LL-37) either to the phagosome or the exterior of the cell.

Primary granules Primary granules are packed with acid hydrolases and antimicrobial proteins, while the membrane is devoid of receptors. In addition to the marker protein myeloperoxidase (MPO) a magnitude of antimicrobial substances can be found in this granule subsets, among them human neutrophil peptides 1-3 (HNP1-3, -defensins) (Rich et al., 1987), and the members of the serprocidin family (Table 1), serine proteases with antimicrobial activity: proteinase-3 (Campenelli et al., 1990), cathepsin


5

G (Salvesen et al., 1993), elastase (Sinha et al., 1987), and HBP, the latter being proteolytically inactive.

Table 3: PMN granule composition Primary granule Secondary granule Tertiary granule Secretory vesicles Membrane CD63, CD68 Laminin-R,

Vitronektin-R, TNF-R, fpr

CD11b, fpr CD11, CD14, CD16, fpr, CD10, CD13

Matrix HBP, Cathepsin G, HNP1-3, Elastase, MPO

Lactoferrin, hCAP18 (LL-37), NGAL

MMP-9, Lysozyme

HBP, Albumin

Focus on LL-37, HNP1-3, and HBP Several constituents of PMN granules were found to be of essential

importance during the course of the experimental progress of this thesis. Therefore, the following summary shall highlight the basic facts known about these proteins.

LL-37 LL-37 is the only cathelicidin peptide identified in humans. It is found

predominantly in PMN and various epithelial linings (Gudmundsson et al., 1996; Frohm Nilsson et al., 1999), but it is also present in monocytes, NK-cells, lymphocytes and mast cells (Agerberth et al., 2000; Di Nardo et al., 2003). The name LL-37 derives from the first two leucine (L) residues at the N-terminus and the number of amino acids (37) constituting the peptide. hCAP18 is the name of the inactive proform of LL-37 and has an approximate mass of 18 kDa. This inactive proprotein is stored in secondary granules of PMN at a molar concentration as high as lactoferrin (40 mol or 630 g/109 cells), constituting a substantial part of the proteins in PMN secondary granules (Sorensen et al., 1997a; Sorensen et al., 1997b). Three proteases in PMN (elastase, cathepsin G and proteinase 3) were shown to cleave hCAP18 in vitro. However, only proteinase 3 has been found to cleave the two-domain protein into the mature peptide and the cathelin part in exocytosed material from PMN (Sorensen et al., 2001). Besides its broad antimicrobial activity against both Gram-negative and Gram-positive bacteria, LL-37 has gained attention for its immunomodulating properties (Kai-Larsen and Agerberth, 2008). In this context, LL-37 was shown to exert chemotactic activity towards monocytes, PMN, and T cells, which was found to be mediated via Formyl Peptide Receptor Like-1 (FPRL1) (De Yang et al., 2000; Yang et al., 2001). By the induction of angiogenesis, LL-37 was shown to be beneficial in wound healing (Kuczulla et al., 2003).

Introduction

6

HNP1-3 -defensins are constitutively produced and comprise 5-7 % of the PMN

content. They represent the major protein species within the primary granules making up 50-70 % of their content. There are four -defensins in human PMN, HNP1-4 (Ganz et al., 1985; Wilde et al., 1989), and two well-described human Paneth cell -defensins, HD-5 and HD-6 (Mallow et al., 1996). HNP1-3, differ from each other by one amino acid at the N-terminal end of the mature peptide (Lourenzoni et al., 2007), and whilst HNP4 significantly differs in primary sequence, it maintains the conserved cysteine residues and the characteristic array of disulphide bonds (Wilde et al., 1989). Initially, PMN-derived HNPs were identified as members of the cationic antimicrobial peptides that exert antimicrobial activity towards bacteria, viruses, and parasites. A growing body of data indicates that -defensins have a very broad range of immunomodulatory properties. HNP-1 chemoattracts monocytes, naive T cells and immature dendritic cells (DC) (Territo et al., 1989; Chertov et al., 1996). Conversely, HNP-1 inhibits the migration of PMN in response to fMLP and leukotriene B4

(Grutkoski et al., 2003). This inhibition appears to be specific, as HNP-1 was not able to inhibit the recruitment of PMN in response to IL-8. Additionally, whereas HNP-2 chemoattracts naive T cells, immature DCs, and monocytes, HNP-3 does not have any of these activities (Territo et al., 1989; Chertov et al., 1996). Like LL-37 (Lau et al., 2005), HNP-1 induces IL-8 and MCP-1 production (Van Wetering et al., 1997a; Van Wetering et al., 1997b; Liu et al., 2007). Transcription of IL-8 and IL-1 is induced in primary human bronchial epithelial cells after treatment with HNP-1 (Sakamoto et al., 2005). This effect of HNP-1 appears to be selective for IL-8 and IL-1 in that the transcription of TNF, GM-CSF, MCP-1, TGF- 1 and VEGF, and the release of GM-CSF, IL-1 and TGF- 1 remain at levels comparable to the untreated control cells (Sakamoto et al., 2005). It was shown some time ago that rabbit -defensins increased non-opsonic phagocytosis of P. aeruginosa by macrophages by more than 2-fold, thereby contributing to bacterial clearance (Sawyer et al., 1988)

HBP HBP was first identified and isolated by Shafer et al. in 1984. Because of its

potent antimicrobial activity, its cationicity, and hydrophobicity, it was considered a component of the oxygen-independent host defense. Due to its charge and its size, it gained the name cationic antimicrobial protein 37 (CAP37) (Shafer et al., 1984). Gabay et al. identified azurocidin, a PMN-derived bactericidal protein from the azurophilic granules of human PMN (Gabay et al., 1989). In parallel, Flodgaard et al. isolated a protein from human and porcine PMN that displayed strong binding


7

capability for heparin, earning it the name heparin-binding protein (Flodgaard et al., 1991). Complete sequencing has shown that CAP37, azurocidin, and HBP are the same proteins.

HBP is stored in secretory vesicles as well as primary granules (Tapper et al., 2002). Due to this distribution, HBP from rapidly mobilized secretory vesicles may target the endothelium and blood cells, while HBP from primary granules may affect cells at the site of inflammation. The interaction between HBP and the endothelium results in activation of endothelial cells (Olofsson et al., 1999) with subsequent intercellular gap formation and plasma leakage (Gautam et al., 2001). In the long run, activation of endothelial cells also leads to enhanced adhesion molecule expression increasing leukocyte adhesion (Lee et al., 2003). At the site of inflammation, intracellular stores of HBP are released which activate monocytes and macrophages to release cytokines and chemokines (Rasmussen et al., 1996; Heinzelmann et al., 1998a). Moreover, HBP was shown to exert a direct chemotactic effect on PMN, monocytes, and T cells (Pereira et al., 1990; Chertov et al., 1996; Chertov et al., 1997). Besides its antimicrobial activity, which is mainly directed against Gram-negative bacteria, HBP opsonizes bacteria, thereby facilitating bacterial phagocytosis by macrophages (Heinzelmann et al., 1998b).

Control of degranulation Receptor stimulation of PMN by a secretagogue triggers granule translocation to the phagosomal or plasma membrane, where they dock and fuse, releasing their contents. The release of granule-derived mediators from PMN occurs by a tightly controlled receptor-coupled mechanism termed “regulated exocytosis”. Exocytosis is postulated to take place in four discrete steps (Toonen and Verhage, 2003). The first step of exocytosis is granule recruitment from the cytoplasm and translocation to the target membrane, which is dependent on actin cytoskeleton remodeling and microtubule assembly (Burgoyne et al., 2003). This is followed by the second step of granule tethering and docking, leading to contact of the outer surface of the granule lipid bilayer membrane with the inner surface of the target membrane. Granule priming then follows as the third step to make granules fusion-competent and ensures that they fuse rapidly, in which a reversible fusion pore structure develops between the granule and the target membrane. Tethering and docking steps are dependent on the sequential action of SNARE proteins (Stow et al., 2006). The fourth and final step, granule fusion, occurs by the formation and rapid expansion of the fusion pore, leading to complete fusion of the granule membrane with the target membrane and expulsion of granular contents to the outside of the cell. This increases the total

Introduction

8

surface area of the cell, and exposes the interior surface of the granule membrane to the exterior.

Translocation and exocytosis of granules in PMN require, as a minimum, increases in intracellular Ca2+, GTP as well as hydrolysis of ATP (Nusse and Lindau, 1988; Theander et al., 2002). Many other proteins have shown to be involved in granule exocytosis. The final step of exocytosis, however, has been postulated to involve the mutual recognition of secretory granules and target membranes, associated with the existence of intracellular receptors that guide the docking and fusion of granules. This led to the formation of the SNARE paradigm, which states that secretory granules possess membrane-bound receptor molecules that bind another set of membrane-bound receptors on target membranes (Söllner et al., 1993). The functional importance of components of the SNARE complex has been shown in studies using permeabilized PMN. There antibodies to VAMP-2 and VAMP-7, as well as syntaxin 4 and 6 block granule release (Martin-Martin et al., 2000; Mollinedo et al., 2003; Mollinedo et al., 2006).

Role of PMN in inflammation The mobilization and extravasation of circulating leukocytes is a key event in

the immune response after initiation of an inflammatory condition. Extravasation of PMN normally precedes a second wave of emigrating monocytes (Witko-Sarsat et al., 2000). PMN were identified a century ago by the German Nobel laureate Paul Ehrlich, who already at that time realized the functional impact of this cell type (Ehrlich, 1900). Nevertheless, PMN are traditionally looked upon as storage for antimicrobial peptides and proteolytic enzymes. The function of the PMN in bacterial clearance is regarded rudimentary and potentially harmful if proteolytic granule components are released uncontrolled.

In recent years, however, this view underwent substantial changes. It is now established that PMN granules are not just emptied like bags, but rather released upon specific signals that the PMN senses in the local environment (Lacy and Eitzen, 2008). In addition, granule exocytosis changes the phenotype of the PMN since membrane-bound granule components will be incorporated into the plasma membrane thereby allowing a promiscuous interaction with neighboring cells. Individual proteins of the PMN granules were primarily recognized for their antimicrobial and proteolytic function. However, many of these proteins have potent immunomodulatory functions, many of which are effective at lower concentrations which can be reached in vivo. These proteins are at the PMN’s immediate command and therefore represent the first powerful line of defense in injury and infection (Borregaard and Cowland, 1997;


9

Chertov et al., 2000; Faurschou and Borregaard, 2003). Interestingly, granule proteins are synthesized at defined stages of PMN maturation in the bone marrow. These are transported by the PMN in the blood. No further de novo expression of proteins occurs once the PMN has entered the bloodstream. In the past peripheral and emigrated PMN were looked upon as being incapable of producing new proteins thereby accounting for the short life span of these cells. However, evidence is accumulating that emigrated PMN launch an extensive gene expression programme (Table 2) (Theilgaard-Mönch et al., 2006). Hence, the PMN contains not only pre-made granule proteins for the immediate immune response at hand, but also a wide range of cytokines, chemokines as well as receptors for differentiation, recognition, cytokines and chemokines. This permits a tight interaction of PMN with monocytes, macrophages, dendritic cells and T cells.

Table 2: Gene expression of the emigrated PMN

Receptors for Cytokines and chemokines

Cytokines and chemokines

Differentiation Fc Recognition

IFN- R1/2, IFN-R1/2,

IL(1,4,6,10)R, TNFR-1/2, TGF-

R2, CCR-1, -2, -3, IL-8R / , CXCR-4

GM-CSFR, G-CSFR, IL-6R

IgG, IgA, IgE TLR-1,-2,-4,-6,-8, CD14, MyD88, MD-2, fpr, TREM1

IL-1 / , IL-8, VEGF, TNF, MIP-1 , MIP-2 , MIP-2 , MIP-3 , MCP-1

The biology of the PMN gains further complexity by the discovery that several PMN subtypes possess distinct functional differences (Tsuda et al., 2004), and that PMN may preferentially differentiate towards cells that phenotypically and functionally resemble macrophages and dendritic cells (Galligan and Yoshimura, 2003). The differentiation depends on the local milieu the PMN is surrounded by: GM-CSF, which prolongs the life span of the PMN after its extravasation, induces expression of MHC II allowing for antigen presentation towards T cells. Additional presence of TNF and IFN induces the expression of MHC II, CD40, CD83, CCR6 and MCP-1, driving the PMN differentiation towards a dendritic cell phenotype (Fanger et al., 1997).

Taken together, the picture of the PMN has changed in recent years from a short-lived, unspecific, tissue-damaging cell to a specific and readily mobilizable weapon, which is crucially involved in activation of monocytes, macrophages, dendritic cells and T cells. Mediators of this powerful panoply of actions are the preformed PMN granules, de novo synthesized proteins, as well as direct interactions with cells of the innate and adaptive immune system.

Introduction

10

Monocytes Monocytes represent about 5–10 % of peripheral blood leukocytes in both

humans and mice. They originate from a myeloid precursor in the bone marrow (Fogg et al., 2006). Monocytes are released in the circulation and then enter tissues. The half-life of monocytes in blood is believed to be relatively short, about one day in mice (Van Furth and Cohn, 1968) and three days in humans (Whitelaw, 1972). This short half-life in blood has fostered the concept that blood monocytes may continuously repopulate macrophage or DC populations to maintain homeostasis and, during inflammation, fulfill critical roles in innate and adaptive immunity (Ziegler-Heitbrock, 2000).

Monocyte subsets Heterogeneity among human monocytes was recognized in the late 1980s.

Differential expression of CD14 and CD16 was used to define the two major monocyte subsets in peripheral blood: (i) “classical” CD14+CD16- monocytes, typically representing up to 95 % of peripheral monocytes in a healthy individual and (ii) “non-classical” CD14loCD16+ monocytes (Ziegler-Heitbrock et al., 1988; Ziegler-Heitbrock, 2000; Strauss-Ayali et al., 2007). These subsets differ phenotypically in their adhesion molecule and chemokine receptor expression. Functionally, classical monocytes produce higher amounts of cytokines and contribute more effectively to bacterial clearance by phagocytosis as compared to non-classical monocytes (Grage-Griebenow et al., 1993; Grage-Griebenow et al., 2000). In contrast, non-classical monocytes are more potent in presenting antigens (Table 3) (Grage-Griebenow et al., 2001; Strauss-Ayali et al., 2007).

Mouse blood monocytes are identified primarily by their expression of CD115, CD11b, and the F4/80 antigen. Only recently have murine monocyte subpopulations been identified (Geissmann et al., 2003). In the mouse, one population of monocytes was found to express intermediate levels of CX3CR1 and high levels of granulocyte

antigen 1 (Gr1) (Table 3). These cells also express CCR2. This population of murine monocytes (CX3CR1+CCR2+Gr1hi) shares morphological characteristics and chemokine receptor expression patterns with the classical human CD14+CD16–

monocytes. However, because of their rapid recruitment to areas of damage or inflamed tissues, this population of murine monocytes was called "inflammatory"

monocytes. Thus, although these two populations share morphological characteristics, current studies suggest that they may have different functions. This difference has led to some confusion in extrapolating information from the murine system into the


11

human system and vice versa. The other main murine monocyte subpopulation was found to express high levels of CX3CR1 and low levels of Gr1 and CCR2. These monocytes appear to be orthologs of the human CD14loCD16+ counterparts described above. Murine CX3CR1hiCCR2–Gr1low monocytes and human CD14loCD16+ (CX3CR1hiCCR2–) monocytes were found to be smaller in size and less granular. In the mouse, the CX3CR1hiCCR2–Gr1low cells tended to persist longer in blood and normal tissues. For this reason, these monocyte subpopulations have also been referred to as resident monocytes.

Table 3: Murine monocyte subpopulations Inflammatory

monocyte Intermediate

monocyte Resident monocyte

Gr-1 expression Gr-1hi Gr-1int Gr-1lo Surface markers CD115+, F4/80+, CD11b+

CD62L+ CD62L- Chemokine receptor expression

CCR2+, CX3CR1+ CCR2+, CX3CR1+, CCR7+, CCR8+

CCR2-, CX3CR1++, CCR5+

Functional properties of the respective human ortholog

Phagocytosis , T cell activation (MLR, APC) , Cytokine release (IL-1, IL-6, TNF)

Phagocytosis , T cell activation (MLR, APC) , Cytokine release (IL-1, IL-6, TNF)

Recruitment of monocytes to sites of inflammation The extravasation of monocytes from the vascular lumen to the tissues

involves a series of sequential molecular interactions between monocytes and endothelial cells. Selectins are cell-surface proteins on the monocyte that interact with glycoprotein ligands on the endothelial cells, allowing the monocyte to bind weakly and initiate the adhesion cascade. The rolling monocyte is then stimulated – by chemokines or other chemotactic compounds, such as PAF or LTB4 – to engage its surface integrins with counter-receptors expressed by endothelial cells. This results in firm adhesion of monocytes to the endothelium before they emigrate through the vessel wall. Monocytic 1- and 2-integrins are activated by signals that are elicited by chemokines. This leads to the tight adhesion of monocytes to the vascular endothelium and the induction of active, cytoskeleton-driven migration (Kim et al., 2003). Chemokines are small secreted proteins that are produced by endothelial cells, leukocytes or stromal cells. Several chemokines can bind to transmembrane heparin sulfate proteoglycans on the luminal surface of vascular endothelial cells and be presented to leukocytes (Tanaka et al., 1993; Spillmann et al., 1998; Roscic-Mrkic et al., 2003). When chemokines bind to these proteoglycans, the binding site for chemokine receptors remains exposed. Thus, when a rolling monocyte encounters a chemokine bound to the endothelium, the chemokine interacts with a G protein-

Introduction

12

coupled receptor expressed on the monocyte and elicits a rapid integrin-activation signal.

Once the monocyte has adhered to the endothelium it may start to migrate through the vessel wall. For inflammatory and resident monocytes different mechanisms have been proposed. Because Gr1hi monocytes express CCR2, a molecule well known to be involved in inflammatory monocyte recruitment (Imhof and Aurrand-Lions, 2004), it was proposed that Gr1hi monocytes are rapidly recruited to sites of inflammation. In typical models of acute inflammation, Gr1hi monocyte recruitment is critically dependent on CCR2 (Boring et al., 1997; Merad et al., 2002), but not on CX3CR1 (Jung et al., 2000). In addition to CCR2, CCR6/CCL20 is involved in the recruitment of Gr1hi monocytes (Merad et al., 2004; Le Borgne et al., 2006).

Less is known about the trafficking and fate of Gr1lo monocytes. In contrast to Gr1hi monocytes, they migrate scarcely or not at all to inflamed tissue in mice, including the acutely inflamed peritoneum (Geissmann et al., 2003; Sunderkotter et al., 2004), or to skin after intracutaneous injection of latex beads (Qu et al., 2004), administration of vaccine formulations (Le Borgne et al., 2006), or epicutaneous UV exposure (Ginhoux et al., 2006). It has therefore been hypothesized that Gr1lo monocytes may fulfill critical roles in replacing resident macrophages or DCs in the steady state (Geissmann et al., 2003). It has further been suggested that the Gr1lo monocytes utilize CX3CR1 to migrate into non-inflamed tissue for replacing resident macrophages or DCs, given their higher surface expression of CX3CR1 and the CX3CL1-dependent transendothelial migration of human CD16+ monocytes in vitro (Ancuta et al., 2003; Geissmann et al., 2003). However, the experimental evidence for the trafficking pattern and the potential role of Gr1lo monocytes as precursors for resident tissue cells is rather limited.

Formyl peptide receptors The family of formyl peptide receptors (fpr) is a promiscuous subfamily of the G protein-coupled receptors and they are involved in the control of immune responses (Le et al., 2002; Migeotte et al., 2006). Fprs were first described in phagocytic leukocytes, monocytes and PMN. Later, these receptors were also found in immature dendritic cells, microglial cells, platelets, spleen and bone marrow. They are also described in non-hematopoitic cell populations and tissues, such as hepatocytes, fibroblasts, astrocytes, neurons of the autonomous nervous system, lung cells, thyroid, heart, kidney, stomach, and colon (Migeotte et al., 2006). In humans the fpr family consists of FPR1, FPRL1, and FPRL2. The murine ortholog of FPR1 is fpr1, the


13

orthologs of FPRL1 are fpr2 and LXA4R. In addition to these, there are at least four more receptors in the mouse making the murine fpr family much more diverse.

The classical evidence supporting fprs as an anti-microbial receptor is that bacteria are the major biological source of chemotactic formyl peptides. fMLP binds to fpr and activates chemotactic and antimicrobial responses in PMN. Besides this classical ligation of fprs several ligands for fprs have recently been identified, most of which are involved in initiating an inflammatory response. In this context, HP2-20 from Helicobacter pylori (Betten et al., 2001), various peptides derived from HIV (Deng et al., 1999; Le et al., 2000), the annexin I holoprotein or its N-terminal peptides (Hayhoe et al., 2006), as well as the lipid mediator LXA4 (Fiore et al., 1994) are worth mentioning. Ligation of fprs results in various functional consequences including Ca2+ mobilization, migration, cytokine production, mediator release, and desensitization of other chemokine receptors. The importance of these functions has been demonstrated in a fpr1-/- mouse exhibiting a higher susceptibility for Listeria monocytogenes (Gao et al., 1999).

Macrophages Macrophages are thought to be evolutionary ancient cells, because related cell

types are already found in the hemolymph of primitive multicellular organisms. In 1882, the Russian biologist Elie Metchnikoff recognized the presence of highly mobile mononuclear phagocytes in invertebrates (Metchnikoff, 1882). Based on his observation that these cells take up and digest bacteria and organic matter he postulated that in complex higher organisms phagocytes patrol the various organs and epithelial surfaces in order to remove ‘nonself’ material, ranging from aged or malignant cells to microbial invaders. This revolutionary theory emerged as true. In the bone marrow of mammalian organisms, macrophages are derived from myeloid progenitor cells, which ultimately develop into monocytes that enter the peripheral bloodstream. Circulating monocytes give rise to a variety of tissue-resident macrophages throughout the body, as well as to specialized cells such as DCs (Burke and Lewis, 2002). Even as early as 1939, Ebert and Florey reported the observation that monocytes emigrated from blood vessels and developed into macrophages in the tissue. Pro-inflammatory, metabolic and immune stimuli all elicit increased recruitment of monocytes to peripheral sites, where differentiation into macrophages and DCs occurs, contributing to host defense, tissue remodeling and repair. (Gordon, 1999; Muller and Randolph, 1999)

Introduction

14

Phagocytosis A crucial task of macrophages is to recognize and endocytose a large number

of genetically diverse infectious pathogens and to discriminate these from noninfectious, innocuous, intact self-structures. In addition, macrophages are also important for the clearance of senescent or apoptotic cells in the host organism (Gregory and Devitt, 2004). Macrophages are equipped with a set of “nonopsonic receptors” that are thought to recognize certain pathogen-associated molecules, motifs or patterns and to mediate subsequent phagocytosis (Aderem and Underhill, 1999). For example the mannose receptor (MR) and the DEC205 receptor on the macrophage recognize mannan, a component of the cell wall of yeasts. Also, the CD14 molecule binds lipopolysaccharide (LPS) and lipoteichoic acid (LTA) found on the surface of Gram-negative and -positive bacteria, respectively. In addition, the scavenger receptor A family binds polyanionic ligands, bacterial lipid A, LTA and oxLDL. Moreover, a group of evolutionarily highly conserved receptors is named after the toll protein originally described in Drosophila (Medzhitov and Janeway, 2000). Toll is a transmembrane protein that is linked to signal transduction pathways that lead to the activation of NF- B and the production of certain peptides with antimicrobial activity. Drosophila flies, expressing a loss-of-function mutant of the toll gene are highly susceptible to fungal infections. To date, the genes for 11 different toll-like receptors (TLRs) have been discovered in mammals (Akira and Takeda, 2004). Not surprisingly, macrophages, PMNs and DCs express most of these TLRs. Crosslinking of TLRs by microbial products usually elicits a strong immune response with the release of pro-inflammatory cytokines and antimicrobial effector molecules, which results from the activation of NF- B and IRF-3 (Akira and Takeda, 2004). Finally, NOD1 and NOD2, two members of the group of nucleotide-binding oligomerization domain (NOD) proteins (Inohara and Nunez, 2003) also recognize specific components of Gram-negative or -positive bacteria. NOD1 and NOD2 sense different derivatives of bacterial cell wall peptidoglycan. Unlike other macrophage receptors, they are located in the cytosol, which enables them to detect intracellular bacteria or bacterial products.

The uptake of microbial pathogens by macrophages can be enhanced by opsonization with humoral products of the host organisms, which then mediate binding to the so-called opsonic phagocytic receptors (Aderem and Underhill, 1999). Recognition after opsonization occurs by either complement receptors or by Fc receptors. Among the Fc receptors are those recognizing IgG: Fc RI (CD64) with high affinity for monomeric IgG, Fc RII (CD32) and Fc RIII (CD16), which have low affinity for monomeric IgG but can effectively bind immune complexes by multiple receptor-ligand interactions. Complement receptors on the macrophage are involved in the binding and ingestion of opsonized particles. CR1 (CD35) recognizes


15

C3b whereas CR3, the Mac-1 molecule (CD11b/CD18), recognizes iC3b. The ligand for CR4 (CD11c/CD18) is still unclear but it may also be iC3b.

Antigen presentation Macrophages fulfill all requirements for an APC. They efficiently endocytose

and phagocytose soluble and particulate antigens, they are equipped with a proteolytic machinery consisting of nonlysosomal proteasomes and lysosomal proteases, and they can be activated for the surface expression of MHC class II antigens and costimulatory molecules. Many of the known elements of APC–T cell-interaction (MHC class II restriction, cytokine production, receptor–ligand pairs of costimulatory molecules and adhesins such as sialoadhesin) were first demonstrated with macrophages (Unanue and Allen, 1987). A particularly important example is the production of interleukin (IL)-12 by antigen-presenting macrophages, which turned out to be a prerequisite for the induction of IFN -producing, CD4+ and MHC class II-restricted type 1 T helper cells in vitro and in vivo (Desmedt et al., 1998; Hsieh et al., 1993). Macrophages are also capable of presenting antigens derived from intracellular pathogens (e.g. Mycobacterium tuberculosis, Leishmania major) in a MHC class I-restricted manner and thereby elicit a CD8+ T cell response. The T cell-stimulatory capacity of macrophages is generally inferior to that of mature DCs. In fact, inflammatory macrophages frequently downregulate T cell activation and proliferation. This “suppressor phenotype” can be caused by the release of T cell-inhibitory compounds (e.g. ROS, NO, prostaglandins) or a reduced production of bioactive IL-12 by macrophages after crosslinking of inhibitory receptors (Bogdan and Nathan, 1993; Mosser and Karp, 1999; Ravetch and Lanier, 2000; Bogdan et al., 2004).

Cytokine production Macrophages are an important source of cytokines. Under functional aspects,

the plethora of products can be placed into four major groups (Burke and Lewis, 2002; Bogdan, 2006): (i) cytokines that mediate a pro-inflammatory response, i.e. help to recruit further inflammatory cells (e.g. IL-1, TNF, CC and CXC chemokines such as IL-8 and monocyte-chemotactic protein-1); (ii) cytokines that mediate T cell and NK cell activation (e.g. IL-1, IL-12, IL-18, IL-23); (iii) cytokines that exert an activating feedback effect on the macrophage itself (e.g. IL-1, TNF, IL-12, IL-18, IFN / ); (iv) cytokines that downregulate the macrophage and/or help to terminate the inflammation (e.g. IL-10, transforming growth factor (TGF)- ). The production of cytokines by macrophages can be triggered by microbial products such as LPS, by

Introduction

16

cell–cell contact-dependent interaction with type 1 T helper cells, or by soluble factors including prostaglandins, leukotrienes, and – most importantly – other cytokines (e.g. IFN ).

Activation patterns The prototypic soluble activator of macrophage function is IFN . This

cytokine is produced by CD4+ type 1 T-helper cells, CD8+ T cells, NK cells, NKT-cells, and macrophages themselves (Schleicher et al., 2005). IFN is best known for the induction of antimicrobial effector pathways, the upregulation of macrophage cytokine production (e.g. IL-1, IL-6, IL4-12, TNF), and the enhanced expression of class II MHC antigens and immune receptors (e.g. Fc R type I, II and III) on the cell surface (Table 4). Apart from IFN , several other soluble mediators (e.g. IFN / , TNF, IL-1, IL-2, IL-7, MIF) have been shown to promote certain macrophage functions when added to macrophages in combination or in the presence of a microbial stimulus (e.g. LPS).

Some of these processes can be antagonized by anti-inflammatory cytokines such as IL-4, IL-10, IL-13 and TGF . To date, TGF is still the only cytokine that is able to truly deactivate macrophages (Bogdan and Nathan, 1993). IL-4 and IL-13 are not solely inhibitory cytokines, but also exert certain stimulatory effects: they increase the production of anti-inflammatory cytokines (IL-10 and IL-1 receptor antagonist), upregulate the expression of certain phagocytic receptors (MR, Fc R type II, scavenger receptor) and enhance fluid-phase pinocytosis and MR-mediated uptake by activation of phosphatidylinositol-3-kinase (Table 4). Macrophages exposed to IL-4 and IL-13 can therefore also be viewed as alternatively activated macrophages (Gordon, 2003). Importantly, by virtue of their cytokine and receptor profile these macrophages help to limit inflammatory reactions and to heal tissue injuries. The clearance of dead tissue cells by these macrophages is likely to further promote their anti-inflammatory and immunosuppressive functions (Freire-de-Lima et al., 2000; Gregory and Devitt, 2004) (Table 4). In addition to soluble factors the activity of macrophages is also regulated by cell surface receptors. Macrophage activation, i.e. increased release of pro-inflammatory cytokines and/or NO, is observed after ligation of CD16, which mediates phagocytosis of IgG-coated particles, and after binding of microbial products to CD14/TLR. The CD40, CD80 and CD86 molecules on the surface of macrophage function as costimulatory macrophage receptors during the MHC class II/T cell receptor-based interaction with T helper lymphocytes. On the other hand, there are a number of receptor molecules on macrophages that after crosslinking transmit inhibitory signals. These include CD64 and CD32, the class A scavenger receptors and CR3 all of which cause suppression of IL-12 production


17

(and/or an increase of IL-10 release). Inhibitory or restrictive signals to macrophages may also be delivered by CD200R (Nathan and Muller, 2001) and by the SIRP R family (Oldenborg et al., 2000). Considering the fact that some of these ‘inhibitory’ receptors are utilized by microbes, the silencing of the pro-inflammatory function of macrophages (which will delay the subsequent immune response), might represent a survival strategy for both extra- and intracellular pathogens.

Table 4: Macrophage activation patterns Innate activation Classical

activation Alternative activation

Deactivation

Signal Ligation of TLR, CD14, TREM, -Glucan-R

IFN , TNF IL-4, IL-13 Ligation of Scavenger-R, CD47, CD200R, Uptake of apoptotic cells, TGF-

Secretion product ROS, NO, IFN / TNF; IL-12, IL-1, IL-6, NO, ROS, IP-10, MIP-1 , MCP-1

IL-1RA, IL-10, AMAC-1

PGE2, TGF- , IL-10

Marker MHCII MHCII , CD86 , CD40 , Mannose-R

Mannose-R , Scavenger-R , CD23 , CD14

MHCII

Functional features

Microbicidal, tissue destruction, cellular immunity, delayed hypersensitivity

Humoral immunity, allergic and anti-parasitic effects, tissue repair

Immunosuppression

PMN granule proteins as regulators of innate and adaptive immunity PMN granules are released during the journey of the PMN to the site of injury or infection and modulate the function of endothelial cells, monocytes, macrophages, dendritic cells and T cells in various ways.

Endothelial cells PMN mobilize secretory vesicles after the initial contact with the vessel wall has been established via interaction of 2-integrins with their endothelial ligands. Consequently, both PMN and endothelial cells become activated. Mobilization of secretory vesicles results in discharge of HBP which diversely modulates the endothelial cell function. In this respect, Gautam et al. ascribed an exclusive role for HBP in PMN-mediated permeability changes (Gautam et al., 2001). In addition, HBP

Introduction

18

enhances the expression of VCAM-1 and ICAM-1 priming the adhesion of subsequent leukocyte subsets (Lee et al., 2003). After the initial contact between HBP and the endothelial cells, the protein will be internalized and targeted to the mitochondria thereby reducing apoptosis in endothelial cells (Olofsson et al., 1999).

Monocytes and macrophages Monocytes emigrate to the site of inflammation hours and days after the initial invasion of PMN. Studies on patients with specific granule deficiency (SGD) lacking some of their PMN granule proteins reveal reduced monocyte recruitment into a skin blister (Gallin et al., 1982; Gallin, 1985). This may point at a potential causal connection between primary PMN recruitment, release of granule proteins and extravasation of monocytes. Following these studies several granule proteins with monocyte chemoattracting activity were identified: cathepsin G, HBP, and HNPs (all of which are stored in the primary granules) and LL-37 (from secondary granules). The chemotactic effect of these proteins was shown to be Gi-protein dependent and members of the fpr family were identified as potential docking stations (De Yang et al., 2000; Sun et al., 2004). Release of these proteins may facilitate the early recruitment of monocytes, while other mechanisms may be important at a later time point. PMN are capable of producing CCL2, CCL3, and CCL4 directly and cytokines which may activate neighboring cells to produce chemokines, both of which may contribute to the recruitment of inflammatory cells (Scapini et al., 2000; Theilgaard et al., 2006).

The inflammatory cytokine network is subjected to intensive modifications by PMN granule proteins. In this respect, HBP and HNPs are able to activate monocytes and macrophages to release TNF, while proteinase-3 proteolytically cleaves the pro-forms of TNF, IL-1 and IL-18 transforming them into their active form. In contrast, proteinase-3 inactivates IL-32 thereby contributing to the fine-tuning of the cytokine network (Meyer-Hoffert, 2008).

The antimicrobial activity of macrophages is enhanced by PMN granule proteins in several ways. Bacteria may be opsonized by HNPs and HBP allowing macrophages to more efficiently phagocytose bacteria (Fleischmann et al., 1985; Heinzelmann et al., 1998b). Moreover, HNPs, cathepsin G, and LL-37 induce the production and release of reactive oxygen species in macrophages (Zughaier et al., 2005).


19

Dendritic cells Like monocytes, DCs are piloted to the inflammatory focus by PMN-derived HNPs. Furthermore, the PMN is able to synthesize vast amounts of CCL19 and CCL20 (Scapini et al., 2000) which allow a recruitment of immature DCs but not mature DCs. In contrast to monocytes, the activation of DCs by PMN is mainly dependent on direct cell-cell interaction resulting in upregulation of MHC II, CD86, and CD40 (Boudaly, 2008). The contact is established via 2-integrins on PMN and DC-SIGN on DCs. This phenotypic change allows for a more potent T cell activation by DCs (Boudaly, 2008).

Figure 2: Interface between PMN and innate and adaptive immunity. During their journey from the blood flow to the inflammatory site, PMN secrete their granule contents, which interact with endothelial cells, monocytes, macrophages, dendritic cells, and T cells.

T cells Depletion of PMN in mice revealed that PMN contribute to the induction of a T cell-specific immune response in an unexpected manner. PMN are involved in the production of the T cell-specific chemokines IP-10 and Mig thereby contributing to the recruitment of CD4+ T cells (Molesworth-Kenyon et al., 2005; Shiratsuchi et al., 2007). Moreover, PMN are able to carry out functions of APCs in process termed transdifferentiation. In this, PMN express MHC II, CD80, CD83, and CD86 and thereby present antigens to T cells (Tvinnereim et al., 2004; Beauvillain et al., 2007). Recently it was demonstrated, that PMN are able to cross-present, a process which was formerly described for DCs. As a consequence naive CD8+ T cells differentiate to cytotoxic T cells.

PMN Monocyte/Macrophage

Cytokine releasePhagocytosis Recruitment ROS release

Dendritic Cell

T-cell RecruitmentActivation (MHCII, CD40, CD86)

Recruitment Activation Differentiation

Secretory Vesicles Tertiary Granules Secondary Granules Primary Granules

Adhesion molecule expressionPresentation of granule proteins Permeability Apoptosis

Endothelial cell

Aims

20

Aims

The presented work started when the Lindbom group realized the importance of PMN-derived HBP in endothelial cell function. From there I initially explored the potential interaction between HBP and monocytes. Soon it became apparent that a deductive approach is more physiological and I used crude PMN secretion in my experiments to try to elucidate the active component in several biological settings. Therefore the specific aims were:

To uncover how PMN secretion products instruct the extravasation of monocytes (papers I and II).

To determine whether PMN secretion products possess antimicrobial functions except for directly killing bacteria. Specifically the activation of macrophages to phagocytose was investigated (papers III and IV).

To discover by which mechanism PMN secretion products activate monocytes and macrophages, induce cytokine release, and alter their phenotype (papers IV and V).

To understand to what extent PMN secretion products contribute to acute lung damage induced by Streptococcus pyogenes (papers V and VI).


21

Results and Discussion

Establishment and validation of methodology

PMN depletion Depletion of circulating PMN by antibody injection is a common and frequently applied technique to elucidate the involvement of the PMN in various pathophysiological processes (Tateda et al., 2001; Bliss et al., 2001; Melnikov et al., 2002; Zhou et al., 2003; Mednick et al., 2003). The clones RB6-8C5 and 1A8 are commonly used for PMN depletion. While the latter binds to Ly6G which is expressed on PMN only, the first one also binds to Ly6C expressed on PMN and inflammatory monocytes (Fleming et al., 1993). Therefore, injection of 1A8 has been suggested to specifically deplete PMN, while RB6-8C5 may deplete PMN and inflammatory monocytes.

We established our protocol for the depletion of PMN in CX3CR1eGFP/+ mice, having fluorescent monocytes. Injection of RB6-8C5 (100 µg, i.p.) and subsequent quantification of PMN (Gr1hi, eGFP-), resident monocytes (Gr1lo, eGFPhi), and inflammatory monocytes (Gr1hi, eGFPlo) by FACS indicated that the number of circulating PMN dropped rapidly after antibody injection, while the number of circulating resident or inflammatory monocytes was not affected (Figure 3A). However, at higher doses of RB6-8C5 (500 µg and more) inflammatory monocytes will also be depleted (not shown). Similarly, we found that injection of 1A8 causes a selective depletion of PMN. In addition, we assessed the activation of monocytes after antibody injection. The expression of neither CD11b nor CD62L is affected on the surface of inflammatory monocytes after injection of RB6-8C5 (Figure 3B). Serum concentrations of TNF and IFN were also not affected in the presence of the antibody (not shown). Functionally, we investigated adhesion and transmigration of monocytes treated with RB6-8C5 in vitro. Compared to treatment with an isotype control antibody, neither adhesion to rmICAM-1 nor migration across a filter in response to MCP-1 were affected (not shown). In conclusion, we establish that given the right dose of RB6-8C5 a specific depletion of PMN can be reached.

Activation of PMN by antibody cross-linking of CD18 To obtain the content of PMN granules, secretion was induced through antibody cross-linking of CD18 in freshly isolated human PMN using the primary


22

antibody IB4 and a secondary goat anti-mouse F(ab’)2-fragment (Figure 3C) (Walzog et al., 1994). Treatment in such way resulted in release of secretory vesicles, tertiary, secondary, and primary granules as shown by western blot analysis for marker proteins (Figure 3C). Furthermore, we identified substantial amounts of HBP, elastase, cathepsin G and HNP1-3 in the secretion. However, the secretion was found to be devoid of the cytokines TNF and IFN as well as the chemokines IL-8 and MCP-1. In contrast to activation of PMN by chemical stimuli such as fMLP or PMA, treatment in the way described allows to harvest PMN secretion that is clear of the activating agent and is contaminated with a secondary antibody only. This will minimize unspecific side effects.

A

IB4+

Sek.

Ab

MMP-9

LL-37

Elastase

Albumin

IB4

0

0.2

0.4

0.6

0.8

1

1.2

1.4pr

imar

y

seco

ndar

y

tert

iary

secr

etor

y

Lactoferrin

CD35MMP-9

5 10 15 20 25 300

MPOactivity

[U/ml]

C

B

D

A

IB4+

Sek.

Ab

MMP-9

LL-37

Elastase

Albumin

IB4

0

0.2

0.4

0.6

0.8

1

1.2

1.4pr

imar

y

seco

ndar

y

tert

iary

secr

etor

y

Lactoferrin

CD35MMP-9

5 10 15 20 25 300

MPOactivity

[U/ml]

0

0.2

0.4

0.6

0.8

1

1.2

1.4pr

imar

y

seco

ndar

y

tert

iary

secr

etor

y

Lactoferrin

CD35MMP-9

5 10 15 20 25 300

MPOactivity

[U/ml]

C

B

D

Figure 3: Validation of methodology. (A/B) PMN depletion by injection of RB6-8C5. Intraperitoneal application of the antibody induces selective depletion of PMN (A). Surface expression of CD11b and CD62L on inflammatory monocytes is not affected by injection of the antibody (B). (C) Cross-linking of CD18 on human PMN induces release of secretory vesicles (albumin), tertiary (MMP-9), secondary (LL-37), and primary (elastase) granules. (D) Subsets of PMN granules can be separated by subcellular fractionation (left). Individual granule subclasses are identified by marker analysis using spectophotometry and Western blot analysis.

Subcellular fractionation Subcellular fractionation was essentially performed as described by Kjeldsen et al. (Kjeldsen et al., 1999). Postnuclear supernatants were separated by density gradient centrifugation after PMN were disrupted via nitrogen cavitation. Thereby, the


23

PMN granule subsets are separated. Individual fractions are tested thereafter for the presence of marker proteins specific for one granule subsets. Fractions in which only one granule type is present were then used for further experimentation.

PMN secretion products command extravasation of monocytes PMN dominate the initial leukocyte influx to sites of acute infection and inflammation (Witko-Sarsat et al., 2000). This first wave of PMN extravasation precedes a second wave of monocyte extravasation. Recruited PMN are thought to trigger this cellular switch by releasing soluble factors that initiate monocyte recruitment (Doherty et al., 1988; Fillion et al., 2001; Janardhan et al., 2006). Monocytes are of central importance in bacterial clearance and cytokine production, and subsequently contribute to the resolution of inflammation and repair of damaged tissue. Since the early phase of monocyte recruitment sets in just a few hours after PMN recruitment, ready-made PMN granule proteins deposited at the site of inflammation may be of critical importance in this response (Antony et al., 1985; Zhou et al., 2003). Indeed, supernatants of activated PMN from patients with SGD lacking proteins in their primary, secondary, and tertiary granules show a reduced capacity to attract monocytes despite normal monocyte chemotaxis in vitro to other stimuli (Gallin et al., 1982; Gallin et al., 1985). Following this initial observation, several PMN-derived granule proteins with monocyte-chemotactic activity have been identified, among them LL-37, cathepsin G, human neutrophil peptide 1-3 (HNP1-3, alpha-defensins), and heparin-binding protein (HBP, also known as CAP37 and azurocidin) (Territo et al., 1989; Pereira et al., 1990; Chertov et al., 1996; Chertov et al., 1997; De Yang et al., 2000). Their action was found to be Pertussis toxin (PTx)-sensitive and several receptors have been suggested to mediate the chemotactic effect (Chertov et al., 1997; De Yang et al., 2000).

Paper I In a first approach we aimed at investigating the involvement of PMN-derived HBP in adhesion of monocytes to the endothelial lining. Due to its localization in secretory vesicles as well as primary granules (Tapper et al., 2002), HBP is of special significance among the PMN granule proteins. While HBP from primary granules is likely to be released when the PMN has reached the extravascular space where it exerts its antimicrobial activity and modulates the function of neighboring cells, HBP from secretory vesicles is rapidly mobilized allowing a prompt interaction with endothelial cells.


24

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A pre-requisite for the direct induction of monocyte adhesion to endothelial cells by HBP is that HBP and monocytes encounter each other. Therefore we investigated the deposition of HBP on the endothelial cell surface after superfusion with freshly isolated PMN. Our results clearly indicate that adherent, but not rolling PMN release HBP which then anchors on endothelial cells (Figure 4A). In further experiments we identify endothelial proteoglycan side chains as primary binding sites for HBP. In the next step isolated leukocyte subpopulations were superfused over endothelial cells on which recombinant HBP was deposited. We found a significant enhancement of adherent monocytes and Mono Mac 6 cells, but not of lymphocytes or PMN (Figure 4B). This fits well with the preferential interaction of HBP with monocytes (Heinzelmann et al., 1998a). Since enhanced monocyte adhesion in response to immobilized HBP may be due to activation of monocytes or endothelial cells, we excluded endothelial cells from the system. Instead, we perfused isolated monocytes over recombinant ICAM-1, VCAM-1, P-selectin, or E-selectin in the presence or absence of HBP. We found a significant enhancement of monocyte


25

adhesion when HBP is present indicating that HBP activates monocytes directly (Figure 4C). Support for this direct activation comes from experiments in which we labeled monocytes with the Ca2+-sensitive fluorochrome fluo4. There, we show an enhanced Ca2+-mobilization in the presence of HBP compared to recombinant adhesion molecules only (Figure 4D).

In conclusion, we show that adherent PMN set HBP free, which is then deposited on endothelial cells. HBP in this location activates monocytes resulting in their firm adhesion. An interesting feature with regard to the HBP-evoked effect on monocyte arrest is that the activating agent is not derived from the inflammatory lesion itself, but rather seeded on the endothelium by a third party, the emigrating PMN. A similar mechanism has previously been observed in relation to leukocyte recruitment during atherogenesis, where activated platelets are postulated to spread the proinflammatory chemokine RANTES (von Hundelshausen et al., 2001) on the endothelial surface, which in turn triggers increased arrest of monocytes.

Paper II In a second approach we aimed at investigating the monocyte-recruiting capacity of PMN granule components in vivo. The extravasation of monocytes was analyzed in a subcutaneous air pouch model and by intravital microscopy of the cremaster muscle. The latter offers the advantage of analyzing adhesion and transmigration, while the first allows to further differentiate extravasated cells. In the cremaster model monocytes were identified by using CX3CR1eGFP/+ mice which have fluorescent monocytes (Jung et al., 2000; Geissmann et al., 2003) or by labeling monocyte subsets with fluorescent latex beads (Tacke et al., 2006; Tacke et al., 2007). In both models we confirm that PMN precede the extravasation of monocytes by several hours raising the question of a causal contribution of PMN and their secretion products.

To address this question we investigated the extravasation of monocytes in neutropenic mice. Depletion of PMN significantly reduced the number of adherent and extravasated monocytes (Figure 5A). The extravasation of monocytes could be restored by injecting PMN secretion which was obtained as described above (Figure 5A). Peripheral monocytes in humans and mice consist of at least two different subsets, which have distinct functional characteristics. To differentiate between inflammatory and resident monocytes in the mouse the expression level of CX3CR1 and Gr1 may be analyzed. Inflammatory, classical monocytes are Gr1hi and CX3CR1lo, while resident, non-classical monocytes are Gr1lo und CX3CR1hi. In both recruitment models, we found that depletion of PMN and local injection of PMN secretion


26

selectively affected the number of recruited inflammatory monocytes (Figure 5B). Causative for this selective action may be the activation of inflammatory monocytes by PMN secretion as implied by intracellular Ca2+ mobilization (Figure 5C). Further support comes from experiments where the migration speed of inflammatory and resident monocytes was measured in the cremaster muscle in the presence or absence of PMN secretion. In the presence of PMN secretion the migration speed of inflammatory monocytes is significantly higher than the migration speed of resident monocytes (Figure 5C).

Figure 5: PMN granule contents induce recruitment of inflammatory monocytes. (A) Depletion of monocytes reduces the number of monocytes recruited to the cremaster muscle (left) and to the air pouch (right). Local instillation of PMN secretion restores the recruitment of monocytes. (B) PMN granule contents specifically contribute to recruitment of inflammatory monocytes to the cremaster muscle (left) or air pouch (right). (C) PMN secretion enhances the migration speed of inflammatory monocytes significantly more than of resident monocytes (left). PMN secretion induces Ca2+

mobilization in inflammatory monocytes, but not in resident monocytes. (D) Constituents of primary and secondary granules induce recruitment of inflammatory monocytes to the air pouch, while tertiary granule and secretory vesicle contents are without effect. To narrow the number of candidates involved in the monocyte recruitment, we performed subcellular fractionation of PMN and used the individual granule subsets instead of PMN secretion in the air pouch model. While contents of secretory vesicles and tertiary granules were without effect, constituents of primary and secondary granules enhanced the recruitment of inflammatory monocytes significantly (Figure 5D). Immunodepletion of HBP, HNP1-3, LL-37, and cathepsin G, all of which were previously shown to attract monocytes, renders the PMN secretion non-functional. In

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the next step, recombinant or isolated forms of these four proteins were injected into the air pouch and the subsequent recruitment of inflammatory monocytes was investigated. While, cathepsin G failed to enhance extravasation of inflammatory monocytes, HBP, LL-37, and HNP1-3 all induced a significant recruitment of inflammatory monocytes. To elucidate the contribution of these three proteins to the activity of the PMN secretion, we immunodepleted the PMN secretion of HBP, LL-37, or HNP1-3. However, only depletion of LL-37 and HBP reduced the potency of the PMN secretion to attract inflammatory monocytes. Depletion of HNP1-3 was without effect, indicating that LL-37 and HBP are the primary mediators of recruitment of inflammatory monocytes. LL-37 has previously been shown to mediate its chemotactic effect via fprs. To investigate the potential involvement of this receptor family in the recruitment of inflammatory monocytes in response to LL-37 and HBP, we injected the fprs antagonist BOC-PLPLP intravenously before the experiment. We found a clear reduction of monocyte recruitment in the presence of the antagonist, indicating that this receptor family is of crucial importance in the extravasation of inflammatory monocytes in response to PMN secretion products.

Our study suggests an essential role of the primary PMN extravasation for the subsequent recruitment of inflammatory monocytes. At later time points, additional mechanisms, such as chemokine release from PMN (Scapini et al., 2000) or other immune cells (Weber and Koenen, 2006), likely gain increasing importance for attracting monocytes to the inflamed tissue. We demonstrate that the early recruitment of inflammatory monocytes by PMN is linked to the rapid discharge and deposition of PMN granule proteins LL-37 and HBP. These granule proteins employ monocytic formyl peptide receptors thereby initiating recruitment of these cells to the inflamed locus.

PMN secretion products boost antimicrobial activity in macrophages

Monocytes and macrophages possess a rich panoply of antimicrobial devices, such as phagocytic uptake of microorganisms and intracellular killing of bacteria using oxygen-dependent mechanisms (Aderem and Underhill, 1999). The efficiency of bacterial phagocytosis crucially depends on the opsonisation of the pathogen as well as the state of activation of the macrophage (Bogdan, 2006). Clinical evidence for the importance of PMN granule proteins in the communication with monocytes and macrophages comes from humans lacking PMN or suffering from disorders in the composition of PMN granules. The lack of granule proteins in SGD is associated with impaired macrophage functions, like macrophage maturation, cytokine production, and phagocytosis in humans and mice (Tavor et al., 2002; Shiohara et al., 2004).


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Therefore, we addressed the influence of PMN secretion products in the regulation of antimicrobial activity in macrophages in papers III and IV.

Paper III In a first approach we investigated the uptake of IgG-opsonized Staphylococcus aureus bacteria by human monocytic THP-1, human macrophages derived from monocytes, murine monocytic WEHI-3B, and murine RAW 264.7 macrophages. These cell types were incubated with PMN secretion for 24 hours before addition of bacteria. For all four types of monocytes and macrophages we found a significant enhancement of bacterial phagocytosis compared to treatment with medium alone (Figure 6A), indicating that PMN secretion may activate macrophages directly. This was supported in experiments where we measured the Ca2+ mobilization in monocytes and macrophages in response to PMN secretion.

Bacterial clearance is not only dependent on effective phagocytosis but also on intracellular killing. Bacterial killing by macrophages critically relies on oxygen-dependent microbicidal mechanisms (Burke and Lewis, 2002). Similarly to our experiments above, we treated human macrophages with PMN secretion for 24 hours and analyzed the ROS formation after addition of IgG-opsonized S. aureus. Compared to control, S. aureus significantly enhanced the intracellular ROS formation, but pre-treatment with PMN secretion did not further enhance this response. However, when we added PMN secretion acutely, we found a strong increase of intracellular ROS formation (Figure 6B). Interestingly, the formation of ROS induced by S. aureus was several fold enhanced by addition of PMN secretion (Figure 6B), indicating a significant role of PMN-derived secretion products in stimulating ROS formation in macrophages.

Neutropenia and neutrophil disorders are regularly associated with bacterial infections. Besides the primary lack or insufficiency of PMN a secondary antimicrobial dysfunction of macrophages contributes to the onset of such clinical events. PMN granules contain a wide diversity of proteins, many of which have direct antimicrobial activity (Levy, 2004). Besides this direct microbicidal function, several granule proteins were shown to opsonize bacteria, allowing a more efficient phagocytosis (Fleischmann et al., 1985; Heinzelmann et al., 1998b). A third antimicrobial mechanism of PMN granule proteins was recently identified by Tan et al., who showed that the transfer of PMN granule proteins from apoptotic PMN to macrophages enhances the intracellular killing of M. tuberculosis (Tan et al., 2006). In paper III we report yet another antimicrobial mechanism of PMN granule constituents, showing that PMN secretion products activate macrophages and


29

stimulate bacterial phagocytosis and intracellular killing in monocytes and macrophages. This study points at the importance of the PMN and its secretion products in regulating antimicrobial activities of monocytes and macrophages.

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Figure 6: PMN secretion products enhance phagocytosis and ROS production in monocytes and macrophages. A: THP-1 cells, WEHI-3B cells, RAW264.7 cells, and human macrophages were treated with PMN secretion for 24 hours. The uptake of IgG-opsonized S. aureus was analyzed thereafter. Phagocytosis is expressed as bacteria per cell. B: Human macrophages were grown in a 96 well plate, labeled with H2DCFDA and the fluorescence was measured over 80 min after stimulation with medium (ctrl) PMN secretion, S. aureus or PMN secretion plus S. aureus.

Paper IV In this paper we aimed at identifying the mechanisms by which PMN secretion products induce phagocytosis of bacteria and the active components within the PMN secretion responsible for this effect. In initial experiments we found that PMN secretion mediates enhanced phagocytosis only when bacteria were opsonized with IgG (Figure 7A). Neither uptake of complement-opsonized bacteria nor uptake of non-opsonized bacteria was affected by pre-treatment of macrophages with PMN secretion (Figure 7A), indicative of an altered Fc R expression in the presence of PMN secretion. Immunocytochemical analysis of macrophages treated with PMN secretion indeed revealed enhanced expression of the high affinity receptor CD64 and CD32 (Figure 7B), but not the low affinity receptor CD16. In addition, expression of complement receptors CD11b, CD11c, and CD35 was not affected.

Two approaches were chosen to identify the active component(s) in the PMN secretion. First, we performed subcellular fractionation of human PMN and incubated macrophages with individual granule subsets. Interestingly, only the primary granule fraction induced enhanced uptake of IgG-opsonized S. aureus (Figure 7C). This was


30

not different from the effect exerted by crude PMN secretion. Several proteins localized in PMN primary granules were shown to activate macrophages. Thus, we used both recombinant and isolated forms of these proteins in subsequent experiments. There, only HBP and HNP1-3 enhanced phagocytosis of IgG-opsonized S. aureus (Figure 7D), indicating that these two proteins are critically involved in the PMN-mediated bacterial phagocytosis by macrophages. In a second approach, PMN secretion was separated by HPLC and the phagocytosis-enhancing capacity of three neighboring pooled fractions was analyzed. By screening the phagocytic activity in this way, we found strong activity in fractions 60-62 and 45-47 (Figure 7E). Results from the previous approach led us to screen all fractions for the presence of HBP and HNP1-3 by dot blot. Interestingly, we found immunoreactivity for HBP in fractions 60-62 and for HNP1-3 in fractions 46 and 47 (Figure 7E). The presence of HBP in fractions 60-62 was further supported by Western blot analysis and N-terminal sequencing. Similarly, we used mass spectrometry and N-terminal sequencing to confirm the presence of HNP1-3 in fractions 46 and 47. Thereby, both approaches provide strong evidence for the importance of HBP and HNP1-3 in PMN-derived increase of bacterial phagocytosis by macrophages. In a more deductive approach we depleted PMN secretion of HBP or HNP1-3 individually or in combination and analyzed the remaining activity in the phagocytosis assay. Depletion of HBP reduced the activity of the PMN secretion grossly, while depletion of HNP1-3 lowered the activity to a minor extent (Figure 7F). Depletion of both proteins abolished the activity of the PMN secretion almost completely, indicating that these two proteins are the principal mediators of the enhanced phagocytosis. Interestingly, we could also establish the same relationship in an in vivo model. We used a peritonitis model to investigate the contribution of PMN to the phagocytic capacity of macrophages. Depletion of PMN from the murine circulation significantly reduced the ability to phagocytose IgG-opsonized bacteria by peritoneal macrophages. Intraperitoneal application of HBP and HNP1-3 restored most of the defect to phagocytose (Figure 7F), further supporting the importance of these two proteins.

In an attempt to elucidate the mechanism for enhanced phagocytosis we investigated the levels of TNF and IFN in the cell culture medium after stimulation with PMN secretion, HBP, or HNP1-3. We found a clear increase of these two cytokines (Figure 8B), both of which are prototypic activators of macrophages. Using neutralizing antibodies we can dissect the importance of the individual cytokines in activating macrophages and the subsequent phagocytosis. We could clearly show that neutralizing TNF efficiently reduces the enhanced expression of CD64 and CD32 as well as the enhanced phagocytosis. Blocking TNF and IFN together almost completely abolished the effect of HBP and HNP1-3.


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In conclusion, we identify a novel mechanism of bacterial clearance involving HBP and HNP1-3. These two PMN-derived granule components directly activate the macrophage and strongly enhance its phagocytic activity. This is mediated by an endogenous activation via release of the cytokines TNF and IFN which enhance the expression of CD64 and CD32.

Figure 7: PMN-derived HBP and HNP1-3 induce the expression of CD64 and CD32 thereby enhancing the phagocytosis of IgG-opsonized bacteria. (A) PMN secretion enhances the uptake of IgG-opsonized S. aureus bacteria in human macrophages. (B) Stimulation of human macrophages with PMN secretion enhances the expression of CD64 and CD32. (C) Treatment of human macrophages with isolated granule subpopulations allows identification of primary granule contents as phagocytosis-enhancing compartment. (D) Recombinant HBP and purified HNP1-3 enhance phagocytosis by human macrophages. (E) Testing of PMN secretion fractionated by HPLC allows identification of two active fractions (top). Employing dot blot and western blot HBP is detected in fractions 60-62 (lower left). HNP1-3 is detected in fractions 44-46 by mass spectometry and dot blot (lower right). (F) Immunodepletion of HBP and HNP1-3 from the PMN secretion illustrates the importance of these two polypeptides fort he phagocytosis-enhancing capacity of the PMN secretion of macrophages (left). In a murine model we show that peritoneal macrophages of neutropenic mice have a reduced phagocytic capacity compared to mice with intact white blood count. The majority of this defect can be rescued by local application of HBP and HNP1-3.

PMN secretion products induce phenotypic changes in monocytes and macrophages and induce cytokine release Until about ten years ago the macrophage was viewed as a cell that releases inflammatory mediators, phagocytoses bacteria and presents antigens. However,

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much of this is true only for the classically activated macrophage. This view has changed by discoveries by Gordon et al. that describe alternative ways of macrophage activation. We now know that macrophage activation can occur classically (IFN and TNF), alternatively (IL-4, IL-13), and by innate signals (e.g. ligation of TLRs). Macrophages can be deactivated e.g. by ligation of scavenger receptors or by uptake of apoptotic cells. Each of these activation forms is characterized by a change in phenotype and functional capacities.

Paper IV In papers III and IV we describe the enhancement of bacterial phagocytosis in response to PMN secretion products. In the course of these studies we identified the granule proteins HBP and HNP1-3 to be the principal mediators of this response. As central mechanism we demonstrate an upregulation of Fc receptors CD32 and CD64 on the macrophage surface, which may indicate a classical activation pattern. This is normally mediated by IFN or TNF (Bogdan, 2006). We therefore hypothesized, that macrophages release IFN and TNF in response to HBP and HNP1-3. Release of these cytokines may stimulate macrophages in an autocrine fashion, thereby serving as the underlying mechanism to enhanced phagocytosis in response to HBP and HNP1-3.

To investigate the activation pattern of macrophages in more detail, we treated human macrophages with PMN secretion, HBP, or HNP1-3. Either treatment resulted in upregulation of HLAII, CD40, and CD86 (Figure 8A and 8C) indicating a classical activation pattern. In further experiments we analyzed the IFN and TNF concentration in the poststimulatory supernatant of macrophages treated with PMN secretion, HBP, or HNP1-3. With no stimulation, concentrations of these two cytokine were low. In the PMN secretion none of the two cytokines were detected. Treatment of macrophages with PMN secretion, HBP or HNP1-3, on the other hand, significantly increased the IFN and TNF concentration (Figure 8B). HBP mediated its effect via activation of 2-integrins as indicated by the use of an antibody to CD18, which blocked the cytokine release in response to HBP. Several inhibitors were used to find out via which receptor HNP1-3 mediate their action. However, blockage of CD18 (with IB4), P2Y6 receptor (with suramin), Gi protein (with PTx), or tyrosine kinases (with genistein) did not reduce the release of either IFN or TNF. In further experiments we aimed to elucidate the importance of this cytokine release for the activation pattern observed in our studies. Blocking antibodies to TNF greatly reduced the HBP and HNP1-3 mediated upregulation of HLAII, CD40, and CD86 (Figure 8C). Smaller effects were found using blocking antibodies to IFN .


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Figure 8: PMN secretion induces classical macrophage activation via release of TNF and IFN . (A) Incubation of human macrophages with PMN secretion enhances the surface expression of CD86, HLA II, CD40, and CD25 indicating a classical activation pattern. (B) Incubation of human macrophages with HBP (left) or HNP1-3 (right) induces release of TNF and IFN . (C) Neutralizing antibodies to TNF and IFN significantly reduce the HBP- and HNP1-3-mediated upregulation of HLA II, CD40, and CD86.

Taken together our data indicate that PMN-derived HBP and HNP1-3 induce the release of TNF and IFN from macrophages. This activates macrophages in an autocrine fashion leading to phenotypical changes observed in classically activated macrophages. Functionally this relates to a enhanced phagocytic capacity of macrophages.

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Paper V In this manuscript we investigate the activation of peripheral blood monocytes by PMN-derived HBP and complexes formed between fibrinogen and M protein shed from Streptococcus pyogenes. Previous work has shown that these complexes bind to

2-integrins on PMN inducing the release of HBP (Herwald et al., 2004), which was previously shown to be a potent mediator in vascular leak formation (Gautam et al., 2001).

Incubation of whole blood or peripheral blood mononuclear cells (PBMC) with M1 protein induced a strong increase in the release of IL-6, IL-1 , and TNF. Additional stimulation of PBMCs with HBP further increased release of these three cytokines. Experiments where TLR2-transfected cells were used indicate that M1 protein acts via TLR2. Further support for this comes from electron microscopy images that clearly show the colocalization of M1 protein and TLR2 on monocytes. On the other hand, HBP released from PMN potentiates the cytokine release from PBMCs. In paper I we show that HBP induces Ca2+-mobilization in monocytes. Ligation of 2-integrins has previously been shown to induce Ca2+ mobilization and cytokine release (Berton and Lowell, 1999). Moreover, it has been suggested that HBP binds to 2-integrins on PMN (Cai and Wright, 1996) and we were therefore wondering if HBP activates monocytes via 2-integrins. In Ca2+ mobilization assays we found that a blocking antibody to 2 integrins completely abolishes the HBP-induced Ca2+ response, while a control antibody was without effect. In line with these observations we also found in paper IV that the release of IFN and TNF from human macrophages was blocked in the presence of a 2-integrin antibody.

In conclusion, this study provides insight into the crosstalk of the multifunctional PMN-derived HBP and M protein shed from S. pyogenes. For HBP,

2-integrins were identified as receptors, while TLR2 acts as receptor for M proteins. In a concerted effort they boost the cytokine release from peripheral blood monocytes.

The pernicious effect of PMN secretion Constituents of the PMN granules are not just immunomodulating or –activating, they may also possess harmful effects. This becomes obvious in various clinical conditions such as reperfusion injury (Buras and Reenstra, 2007) or in the case of septic complications (Joyce et al., 2004).


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Figure 9: Intravascular activation and degranulation of PMN after injection of M1 protein results in acute lung damage (A) M1 protein forms complexes with fibrinogen, which bind to PMN via 2-integrins. (B) M1/fibrinogen complexes induce PMN degranulation, as indicated by release of MPO (top), MMP-9 (middle), and the upregulation of CD14 (bottom). (C) Light microscopy and electron microscopy of the murine lung. Images represent examples of control mice (top), mice injected with M1 protein (2nd panel), neutropenic mice injected with M1 protein (3rd panel), and neutropenic mice receiving M1 protein and PMN secretion (bottom). (D-F) The BAL fluid of mice treated as described in C is analyzed for the number of cells (D) and the protein concentration (F). Wet weight/dry weight ratio of the lung (E) is analyzed as a measure of the pulmonary permeability.

The streptococcal toxic shock syndrome (STSS) which is normally triggered by the M1 serotype of S. pyogenes, is characterized by a strong activation of PMN, edema formation and acute lung injury (Cunningham, 2000). We have previously identified a new virulence mechanism of M1 protein. Therein, we show that M1 protein is shed form the bacterial surface by the action of PMN-derived proteases. M1 protein will form complexes with fibrinogen which will bind to PMN via 2-integrins (Figure 9A) (Herwald et al., 2004).

In paper VI we studied the degranulation of PMN in response to activation with M1/fibrinogen and addressed the pathophysiological consequences in an in vivo mouse model. Incubation of whole blood with M1 protein induces released of MPO and MMP-9, which are marker proteins for primary, secondary, and tertiary granules(Figure 9B). In addition, we show a rapid upregulation of CD16, CD11b, CD66b, and CD63 further supporting the mobilization of all four PMN granule subsets. In a mouse model intravenous injection of M1 protein induced acute lung

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damage, characterized by intrapulmonary hemorrhage, deposition of fibrinogen aggregates and swelling of the alveolar membrane. Inhibition of attachment of the M1/fibrinogen complexes to PMN by the fibrinogen peptide Gly-Pro-Arg-Pro or the CD18 antibody IB4 inhibited PMN degranulation as well destruction of the lung tissue (Figure 9C). To investigate a causal link between PMN degranulation and lung damage we rendered mice neutropenic. Injection of M1 protein into neutropenic mice completely prevented the lung damage, putting the PMN in a central position. In a next step we injected human PMN secretion into neutropenic mice and could thereby restore the damage induced after M1 protein application. A similar response was obtained in mice treated with murine PMN secretion.

Taken together, this study demonstrates that complexes formed by M1 protein shed from S. pyogenes and fibrinogen stimulate circulating PMN to degranulate. This response induces the whole picture of acute lung damage. Similar pathophysiological connections may exist where intravascular activation of PMN is associated with acute lung damage, such as disseminated intravascular coagulation. These data point to the powerful pernicious effect of PMN granule proteins in the early stages of acute lung injury and provide not only mechanistic insight but also stimulate therapeutic approaches that target PMN activation and degranulation rather than individual granule components.


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Perspectives

Scientists interested in anti-inflammatory therapeutics are discouraged from targeting the PMN because it seems almost impossible to suppress PMN-dependent tissue damage without the serious side effect of increasing the host's risk for infection (Nathan, 2002). On the other hand, stimulating PMN-dependent inflammatory actions to battle infection may also induce tissue damage. However, approaches are accumulating that target the PMN, its granule proteins, PMN extravasation or degranulation to enhance or suppress PMN function.

Blocking PMN functions

Block adhesion The most common goal of PMN-targeted pharmacology is to suppress

inflammation, for example in rheumatoid arthritis or chronic obstructive pulmonary disease. Blocking CAMs that mediate the accumulation of PMN in inflammation is thought to be an effective treatment strategy in clinical inflammatory disorders. However, despite promising preclinical results, the outcome of clinical trials has been inconsistent. With the exception of some beneficial effects in treating psoriasis and asthma (Aydt and Wolff, 2002), preventing activity of either the selectins or CD18 has had limited effects (Ulbrich et al., 2003). Given the redundancy and overlapping functions of molecules that are involved in PMN extravasation (Ley, 2001) alternative pathways might govern PMN adhesion and emigration in these disorders. Consequently, selective inhibition of single CAMs might not be sufficient to prevent PMN recruitment whereas combined targeting of more than one CAM might be effective. Of note, targeting specific CAMs important for recruitment also interferes with other leukocyte activities, and not only extravasation. For example, blocking LFA-1 inhibits activation of lymphocytes by antigen-presenting cells in secondary lymphoid organs, which will influence the progression of autoimmune diseases (Sims and Dustin, 2002). In addition, blocking selectin and CD18 adhesion pathways mimics the clinical syndromes of leukocyte-adhesion deficiency (LAD I and II) (Bunting et al., 2002) and could compromise innate immune responses more severely.

Although most clinical trials of anti-adhesion therapies have used humanized mAbs, several groups are designing and testing low-molecular-weight compounds that will eventually replace these antibodies. Small-molecule antagonists may have several advantages over antibodies and are less likely to trigger adverse immune reactions. In addition to their involvement in cell adhesion, ligand binding by CAMs

Perspectives

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initiates intracellular signaling events that are crucial for numerous immune cell activities, such as motile responses, exocytosis, cytokine production and the respiratory burst (Berton and Lowell, 1999). Hence, it is conceivable that impaired intracellular signaling contributes to the beneficial effect of CAM inhibition.

Block degranulation Blocking degranulation appears an elegant way of interfering with harmful effects of PMN granule constituents. Many substances have been developed to reduce PMN degranulation, among them inhibitors of Rac and other small molecules (Lacy and Eitzen, 2008). Most of these inhibitors are, however, looked upon as a study tool rather than a potential drug. Recently a peptide against the N-terminus of myristoylated alanine-rich C kinase (MARCKs) has been developed (Singer et al., 2004). This peptide was proven to be extremely effective in blocking mucin secretion in a mouse model of asthma, thereby improving the clinical situation (Singer et al., 2004). It was demonstrated in vitro that this peptide blocks degranulation of PMN (Takashi et al., 2006). We used this peptide within the experiments of paper II and found that it was effective when injected into the air pouch. Since these inhibitors act far upstream of the degranulation cascade they may also affect other functions, such as respiratory burst and phagocytosis. The further downstream an inhibitor interferes with degranulation the more specific the inhibition may be and the less side effects will occur. The last step in PMN degranulation is the docking and fusion of the granule with the cell membrane, a process that is tightly regulated by SNAREs (Mollinedo et al., 2006; Stow et al., 2006; Lacy and Eitzen, 2008). Several groups are currently working to target this process. To date neither preclinical nor clinical data are available. However, the dilemma of targeting PMN degranulation is the fact that this will also reduce accumulation of PMN at the site of inflammation, since release of proteinases may be needed to dissect the tissue.

Antagonize PMN granule proteins An alternative therapeutic approach has been to block individual PMN

enzymes. This might afford some benefit, since the blockade would occur after the granules are released, allowing a normal accumulation of PMN. This approach, however, is confounded by the redundancy of the inflammatory pathways involved. In particular, inhibition of neutrophil elastase (Luisetti et al., 1996) might be relatively ineffective if not at the same time inhibiting also cathepsin G, proteinase-3 and MMPs (de Garavilla et al., 2005).


39

Enhance PMN functions

Tumor biology The tissue-damaging power of PMN might be appalling to the rheumatologist,

but it should be appealing to the oncologist. Whereas inflammation can contribute to tumor formation and progression (Coussens and Werb, 2002), PMN can have an important role in tumor destruction in mice (Rothstein and Schreiber, 1988; Tepper et al., 1989; Stoppacciaro et al., 1993) and, under certain circumstances, also in humans (Soiffer et al., 1998). Once a malignant tumor has become metastatic, it is a fair try to induce a neutrophil-driven inflammatory response that is robust enough to destroy the tumor directly (Soiffer et al., 1998) or to damage its vasculature (Rothstein and Schreiber, 1988). In a clinical study, a complement-fixing, PMN-recruiting tumor-specific monoclonal antibody was administered to eight patients with end-stage metastatic melanoma (Minasian et al., 1994). In one patient, hemorrhagic necrosis ensued in all the tumor deposits throughout the body, leading to the first instance of tumor-lysis syndrome reported in a patient with a solid tumor. Therefore, PMN might be especially valuable as anti-tumor effector cells when one considers their enormous numerical preponderance over tumor-specific cytotoxic T cells.

PMN-derived proteins as antibiotics PMN-derived antimicrobial peptides and proteins are important in protection

against microorganisms (Levy, 2004). In the human, cathelicidins (LL-37) and defensins are the dominant class of innate antimicrobial peptides (Yang et al., 2001). Under laboratory conditions, LL-37 has a broad range of activity against both gram-negative and gram-positive bacteria. Among the microbial strains that have been analysed Escherichia coli, Salmonella typhimurium and Neisseria gonorrhoeae have been found to be most sensitive (Turner et al., 1998; Bergman et al., 2005). Furthermore, LL-37 exhibits potent activity against Pseudomonas aeriginosa (Turner et al., 1998) and group A Streptococcus (Dorschner et al., 2001), whereas low activity has been detected against the yeast Candida albicans (Turner et al., 1998). LL-37 has also been shown to possess some antiviral activity against herpes simplex virus (Yasin et al., 2000) and vaccinia virus (Howell et al., 2004), making it a promising broad spectrum antimicrobial agent for therapeutic use. In vivo, LL-37 acts in concert with other antimicrobial components, i.e. defensins (Ong et al., 2002), lysozyme and lactoferrin (Bals et al., pnas, 1998), to exert optimal killing capacity. Defensins also possess potent antimicrobial, antiviral, and antiparasitic functions (Rehaume and Hancock, 2008). In addition, -defensins neutralize the toxins produced by Bacillus

Perspectives

40

anthracis, Pseudomonas aeruginosa and Corynebacterium diphtheriae (Kim et al., 2005; Kim et al., 2006).

Companies have started to use the antimicrobial activity of PMN-derived granule proteins as a template for developing drugs with similar activities. Based on the structure of lactoferrin, LL-37, and the defensins, several drugs have been developed, some of which are in clinical studies (Zhang and Falla, 2004; Zhang and Falla, 2006). The spectrum these drugs aim at is broad and ranges from diabetic foot ulcers to sepsis, and candidiasis. However, the current success is limited by the proteolytic degradation of these drugs and their toxicity due to charge and size.

Anti-endotoxic activity of cationic host defence peptides It has been known for some time that polymyxin B can bind to endotoxin and

has anti-endotoxin activity, as evaluated by the ability to suppress the production of pro-inflammatory cytokines (including TNF, IL-1 and IL-6) and protect against endotoxin-mediated lethality in animal models. Small cationic peptides derived from polymyxin show similar anti-endotoxic activity (Rustici et al., 1993), even in the absence of antimicrobial activity. It was subsequently shown that PMN-derived BPI was able to neutralize the ability of E. coli 055:B5 endotoxin to cause gelation of a Limulus amoebocyte lysate (Gray et al., 1994). Moreover, LL-37 had anti-endotoxic activity, including an ability to protect mice against LPS lethality (Scott et al., 2002; Hirata et al., 1994), as did cationic peptides from Limulus anti-lipopolysaccharide factor and lipopolysaccharide-binding protein. Gough et al. demonstrated similar activities in the synthetic cationic antimicrobial peptides CEMA and CEME (Gough et al., 1996). The in vivo protection provided by these peptides correlated with an ability to neutralize the LPS-induced hyperexpression of TNF in murine macrophages. As an alternative mechanism it was found that cationic peptides such as CEMA act directly on macrophages to regulate signaling pathways, and that this differentially affects the ability of LPS to upregulate the expression of selected genes (Scott et al., 2000). It was subsequently shown that the human host defense peptide LL-37 had a similar ability to upregulate some genes and downregulate others (Scott et al., 2002). Therefore, endogenous or synthetic cationic antimicrobial peptides may prove useful as tools in combating sepsis.

Modulation of protective innate immunity by peptides There is excellent evidence that PMN-derived proteins and peptides have

activities that correspond to many aspects of innate immunity usually associated with acute inflammation and host defense (Gudmundsson and Agerberth, 1999; Hancock


41

and Diamond, 2000; Otte et al., 2003). PMN-derived proteins not only possess chemotactic effects for PMN, monocytes, mast cells and T cells, they also stimulate the release of chemokines from host cells, which result in recruitment of cells of the innate immune system to the infection site. They can also stimulate mast-cell degranulation, which leads to histamine release and a consequent increase in permeability, or they directly induce permeability changes by induction of endothelial cell contraction. PMN-derived proteins can also increase production of integrins that are involved in chemotactic responses and promote phagocytosis. They inhibit the lysis of fibrin clots by tissue plasminogen activator, thereby reducing the spread of bacteria. Moreover, they induce expression of inducible nitric oxide synthase to increase the rates of bacterial killing. In addition, they can stimulate tissue and wound repair through promotion of fibroblast chemotaxis and growth. Finally, they can promote angiogenesis in endothelial cells and wound healing. Although these responses would be considered pro-inflammatory, with certain exceptions they are quite distinct in representing only a modest subset of typical pro-inflammatory responses to infectious agents and their signaling molecules like LPS and lipoteichoic acid (LTA), and cationic host defense peptides actually suppress the stimulated production of pro-inflammatory cytokines like TNF and IL-6.

The mechanisms by which these multiple events are mediated by cationic host defense peptides are becoming more clear. As mentioned above, peptides like CEMA and LL-37 stimulate the expression of many genes in macrophages, including some innate-response genes (Scott et al., 2000; Scott et al., 2002). Consistent with these gene-array experiments, LL-37 directly upregulates certain chemokines, including MCP-1 and IL-8, and chemokine receptors, such as CXCR4, CCR2 and IL-8 receptor-B, without stimulating the pro-inflammatory cytokine TNF (Gough et al., 1996). Other upregulated genes include certain growth factors and growth-factor receptors, as well as oncogenes. One possibility is that LL-37 causes differentiation of cells of innate immunity to allow them to more effectively perform anti-infective functions in innate immunity. In this regard, LL-37 is a potent modulator of DC differentiation (Biragyn et al., 2002; Davidson et al., 2004). Although it is difficult to determine the exact contribution of each of the many activities of cationic host defense peptides, the emerging picture is that these compounds generally enhance host defenses at the site of infection, while dampening harmful inflammatory effects mediated by TNF and related TLR-induced cytokines.

This knowledge has recently led to the development of a compound that selectively modulates innate immune responses thereby being anti-infective (Scott et al., 2007). Scott et al. developed innate defense-regulator peptide (IDR-1), which in its activity is reminiscent of the activities of defensins and LL-37. IDR-1 was effective in in vivo settings of Gram positive and Gram negative infections by local or

Perspectives

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systemic application. However, the peptide lacks direct antimicrobial effects and acts through gene and protein expression in monocytes and macrophages.

Conclusion The ability to enhance or augment the innate immune response clearly

represents a potentially powerful way to prevent or treat infections and inflammatory disease. As the innate response is not pathogen-specific, such stimulatory agents could have a broad spectrum of activity, and biotechnology companies have begun to look at using cationic peptides and other PMN-derived proteins as potential enhancers of the innate response to treat infectious diseases. Central to the design of therapeutic agents that modulate innate immune responses will be greater knowledge of the various host-defense reactions innate responses to microorganisms, and the classification of these responses as beneficial or harmful. This PhD thesis hopefully contributes to the elucidation of the multiple cross-talks in innate immunity ultimately allowing to target the involved proteins.


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Methodology

Cell culture

Isolation and activation of PMN Human PMN were isolated from whole blood by single-step density

centrifugation over Polymorphprep (Nycomed Pharma AS), washed twice and resuspended in RPMI containing 1% FBS. PMN activation and degranulation was induced through Ab cross-linking of integrin 2-chain CD18. Isolated PMN were incubated with mAb IB4 against CD18 (3 µg of IB4 per 106 PMN), washed and subjected to CD18 receptor cross-linking through addition of goat anti-mouse F(ab')2 (diluted 1/20; Jackson ImmunoResearch Laboratories). PMN were sedimented by centrifugation and the cell-free supernatant containing PMN secretion was collected, filtered, and stored at –70 °C until use.

Isolation and culture of monocytes and macrophages Human mononuclear leukocytes were isolated from buffy coat by density

gradient centrifugation. Buffy coats were diluted 3-fold with PBS and layered on Ficoll-Paque PLUS (Amersham Pharmacia Biotech AB), centrifuged at 400 g for 40 minutes at room temperature and mononuclear cells were collected. The cells were then washed twice with PBS and seeded in culture flasks. After one hour incubation, lymphocytes in the supernatant were centrifuged and resuspended in RPMI supplemented with 1 % FBS and subsequently used in flow chamber experiments. Cells adherent to the flask’s bottom after four washes were considered as representing the monocyte fraction. These cells were detached with 5 mM EDTA, washed four times and resuspended in RPMI 1640 containing 1 % FBS and then used. Monocyte purity assessed by FACS analysis always exceeded 80 %. Differentiation of monocytes to macrophages was achieved by plating monocytes on cell culture dishes and subsequent culture for eight days in M199 supplemented with 10 % FBS.

Isolation and culture of BAEC and HUVEC BAEC were isolated as described previously (Booyse et al., 1975). Harvested

cells were cultured in medium containing RPMI 1640 and M199 (1:1), penicillin (100 U/ml) and streptomycin (100 µg/ml) in 20 % FBS. Primary cultures of HUVEC were obtained from fresh umbilical cords. The umbilical vein was incubated with 0.1 %

Methodology

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dispase for 30 minutes. After washing with PBS, HUVEC were cultured in M199 containing 20 % human serum.

Culture of cell lines THP-1, Mono Mac 6, WEHI-3B, and RAW264.7 Human monocytic THP-1 cells were cultured in RPMI 1640 containing 1 mM

sodium pyruvate, 0.05 mM 2-mercapotoethanol, and 10 % FBS. Human monocytic Mono Mac 6 cells (gift from H.W.L. Ziegler-Heitbrock, University of Munich, Germany), were grown in RPMI 1640 medium supplemented with 10 % FBS, 1× nonessential amino acids, sodium pyruvate (1 mM), oxalacetate (1 mM), and insulin (10 µg/ml). Mouse monocytic WEHI-3B cells (gift from D. Vestweber, Max Planck Institute, Münster, Germany) were cultured in RPMI1640 containing 10 % FBS and mouse RAW 264.7 macrophages (gift from M. Mäurer, University of Mainz, Germany) were cultured in D-MEM containing 10 % FBS. All cell culture media were supplemented with streptomycin (100 µg/ml) and penicillin (100 U/ml).

Physiological assays

Flow chamber Laminar flow chamber assays (Glycotech) were performed on confluent

BAEC or HUVEC monolayers or on plates coated with recombinant CAM. Confluent EC were activated with LPS at a concentration of 1 g/ml for 4 hours. Pre-perfusion of EC with PMN was obtained by perfusion of freshly isolated PMN over LPS-activated BAEC for 1 hour, followed by perfusion with Mono Mac 6 medium for 5 minutes. Thereafter Mono Mac 6 cells were injected for 4 minutes. The number of adherent Mono Mac 6 cells was quantified after Türck staining which allows to distinguish between PMN and Mono Mac 6. Preincubation of EC with HBP was achieved by adding 10 µg/ml recombinant HBP to the wells 15 minutes prior to the experiment. PMN secretion was added to the EC 15 minutes prior to the experiment when indicated. Plates coated with CAMs (P-selectin, E-selectin, VCAM-1, ICAM-1) and recombinant HBP were produced by adding 50 µl of recombinant human CAM (1

g/ml, R&D systems) to the centre of a well in a 6-well plate. Recombinant HBP (1 g/ml) was added together with CAMs when indicated. Plates were then incubated

overnight at 4 °C. The coated plates and the plates with EC were washed with PBS prior to the experiment to remove excessive reagents. Flow chamber experiments were performed with Mono Mac 6 cells and with isolated human monocytes, lymphocytes and PMN (106 cells/ml) resuspended in medium containing 1 % FBS


45

and infused with a syringe pump at 1 dyne/cm2 for 4 minutes. The experiments were televised and recorded on video for off-line analysis. Leukocyte adhesion was quantified by counting the number of arrested cells in four separate randomly chosen fields in each well.

Ca2+ mobilization Mono Mac 6 cells were incubated (37 °C, 30 minutes) with the Ca2+ sensitive

fluorophore fluo-4/AM (Molecular Probes) according to manufacturer’s instructions and washed twice with PBS before use. 500 l Mono Mac 6 cells (106 cells/ml) were subjected to stimulation with rHBP 500 ng/ml. Changes in intracellular free Ca2+ were analyzed by flow cytometry (FACSort, Becton Dickinson) immediately and every 30 seconds after stimulation with HBP for up to 3 minutes. Additional experiments were performed by fluorescence plate readings. 100 µl Mono Mac 6 cells (106 cells/ml) loaded with fluo4/AM were added to wells of a 96 well plate, which had been coated with CAMs (1 µg/ml) and rHBP. Fluorescence intensity was measured after cells were added to the well. Fluorescence intensity of Mono Mac 6 cells added to BSA-coated wells was considered as background fluorescence and subtracted from fluorescence intensity values of CAM-coated wells. Similar experiments were performed on 24 well plates coated as described above. Fluorescence of fluo4/AM labeled Mono Mac 6 was measured by fluorescence microscopy for individual cells 30, 60 and 90 seconds after cell injection.

In paper II Ca2+ mobilization is measured in murine monocytes. Whole blood from CCR2–/– and C57BL/6 mice was harvested by cardiac puncture. After lysis of red blood cells the leukocytes were resuspended in 0.5 % BSA in HBSS. Anti-Gr1 and anti-F4/80 were used to allow identification of the leukocyte subsets. In addition, leukocytes were incubated with the Ca2+-sensitive fluorochrome X-rhod-1 (Invitrogen). Mobilization of intracellular Ca2+ was analyzed by FACS in inflammatory monocytes (Gr1+, F4/80+) and resident monocytes (Gr1-, F4/80+) following stimulation with medium (neg. ctrl), ionomycin (1 µM, pos. ctrl) or PMN secretion. In separate experiments, LL-37, cathepsin G and HBP were used to induce Ca2+ mobilization. Fluorescence was measured 60 seconds before and every 30 seconds after stimulation up to 150 seconds.

In paper III Ca2+ mobilization was analyzed in human macrophages. Human macrophages were grown in a 96 well plate and incubated (37 °C, 30 minutes) with the Ca2+ sensitive fluorophore fluo-4/AM (Molecular Probes) according to the manufacturer’s instructions and washed twice with PBS before use. Cells were then

Methodology

46

subjected to PMN secretion or sham treatment. The fluorescence intensity was recorded in a plate reader before and at 30, 90, and 150 seconds after injection.

Phagocytosis assay Fluorescent S. aureus and E. coli as well as opsonizing reagent (Molecular

Probes) were reconstituted as indicated by the manufacturer. IgG-opsonization was achieved according to the manufacturer’s instructions. Complement opsonization was attained by incubation of bacteria with fresh human serum at 37 °C for 1 hour. Opsonized particles were washed three times. Bacteria were then seeded onto macrophages grown in a 96 well plate at a ratio of 10 bacteria per cell for 1 hour at 37 °C. Thereafter, trypane blue was added and cells were fixed. The amount of fluorescent particles per cell was quantified by a blinded observer using fluorescence microscopy (Nikon TE300) counting two random microscopic fields per well.

Anti-CD16 (MEM-154, BioVendor), anti-CD32 (AT10, GeneTex), and anti-CD64 (10.1, Immunotools) were added to the macrophages at 10 µg/ml 30 minutes before addition of IgG-opsonized bacteria. Anti-TNF (5 µg/ml, Mab1, BioLegend) and anti-IFN (10 µg/ml, NIB42, BioLegend) were added 30 minutes before stimulation and were present throughout the experiment.

The following proteins were used at the indicated concentrations to stimulate human macrophages for 24 hours before accomplishment of the phagocytosis assay: elastase (Costa Mesa), proteinase 3 (Wieslab), HNP1-3 (purified from human PMN; Faurschou et al., 2002), lysozyme, cathepsin G (both Sigma), MPO (Green Corporation), HBP (produced as described by Rasmussen et al., 1996; gift from Hans Flodgaard).

ROS measurement Human macrophages cultured in 96 well plates were labeled with H2DCFDA

(Molecular Probes) in PBS at a final concentration of 10 µM. Cells were incubated for 30 minutes at 37 °C and the background fluorescence was read before activation. Thereafter, the cells were stimulated with PMN secretion or S. aureus and the fluorescence was measured in a fluorescence plate reader for 80 minutes.


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Animal experiments

Animals Wild-type C57BL/6 mice and Balb/c mice as well as gene-targeted CCR2–/–,

CX3CR1eGFP/+, CX3CR1eGFP/eGFP and DPPI-/- mice were used for in vivo experiments. All genetically modified animals were on C57BL/6 background. All animal experiments were approved by the local ethical committee for animal experimentation.

Air pouch A subcutaneous air pouch was performed as previously described (Werr et al.,

2000). In brief, mice of either sex were injected at day 0 and 4 with 5 ml sterile air subcutaneously in the back. At day 7 1 ml sterile PBS containing PAF (10-6 M) was injected into the pouch. Alternatively TNF (50 µg/ml) or L. monocytogenes (106) were used as stimuli. Twelve hours later the pouch was lavaged with 5 mM EDTA in PBS. Mice receiving PMN secretion were injected with PAF in 500 µl PBS + 500 µl PMN secretion.

Intravital microscopy Monocyte adhesion and extravasation in the M. cremaster was observed by intravital microscopy in CX3CR1eGFP/+ mice and in C57BL/6 mice in which inflammatory monocytes were tracked after taking up fluorescently labeled latex beads (Tacke et al., 2006; Tacke et al., 2007). Mice were injected intrascrotally with PAF (10-6 M, 300 µl, Sigma) and the M. cremaster was exteriorized for microscopic observation 12 hours later. For time course experiments, the M. cremaster was exposed and the number of adherent and extravasated cells was quantified in six random fields of the tissue. One hour later the muscle was superfused with PAF and the subsequent leukocyte extravasation was quantified for periods up to four hours. Intravital microscopy of leukocyte adhesion and extravasation in the M. cremaster was performed as described previously (Thorlacius et al., 1997).

Fluorescence intensity of monocytes extravasated into the cremaster muscle was quantified by Image J software (http://rsb.info.nih.gov/ij) by subtracting the background fluorescence from the monocyte fluorescence. Arbitrary fluorescence units were plotted vs. the cell count revealing two populations with a clear cut-off at 70.

Methodology

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For analysis of monocyte migration, the muscle of neutropenic CX3CR1eGFP/+ mice was exposed 12 hours after local injection of PAF. The migration of fluorescent cells was tracked for 30 minutes. Thereafter, the cremaster muscle was superfused with PMN secretion and the same cells were tracked for another 30 minutes. Migration speed was assessed by off-line analysis of video tapes, and the distinction between the monocyte subsets was made based on their fluorescence intensity as described above.

Peritonitis Female Balb/c mice (20-25 g) were injected with thioglycollate broth and

peritoneal macrophages were harvested and isolated after 4 days as described (Davies and Gordon 2005). PMN depletion was performed by daily i.p. injections of RB6-8C5 mAb starting one day before thioglycollate injection. Controls were injected with isotype-matching antibody. To monitor neutropenia (< 500 PMN/µl and < 20 % of basal value) peripheral blood cells were counted manually and differentiated using FACS. Where indicated, 500 µl of PMN secretion were injected i.p. daily. HNP1-3 (2 µg/mouse) or HBP (10 µg/mouse) were injected i.p. 48 hours and 24 hours before peritoneal macrophages were harvested. Peritoneal macrophages were seeded onto a 96 well plate (105 cells/well) and the phagocytosis assay was performed. The fluorescence intensity reflecting the number of phagocytosed bacteria was quantified in a plate reader (Fluoroskan Ascent, Labsystems).

Peritonitis experiments with male Sprague-Dawley rats (500-550 g) were performed as described for mice. PMN depletion was achieved by injection of anti-PMN serum (1 ml/kg, Cedarlane). Injections with HNP1-3 (20 µg/rat) and HBP (100 µg/rat) and subsequent analysis of the phagocytic capacity of peritoneal macrophages was conducted as described for mice. All animal experiments were approved by the local ethical committee for animal experimentation.

Bronchioalveolar lavage (BAL) and vascular permeability assay After exsanguination via V. cava inferior, the trachea was catheterized and the

left lung was lavaged three times with 500 µl PBS. Leukocytes in the BAL fluid were manually counted, and the protein concentration was assessed using a standard protein assay (BioRad). BAL protein concentration and the wet weight/dry weight ratio were used as indicators of plasma exudation. To obtain the wet weight/dry weigh ration, excised lungs were weight, dried over night at 60 °C, and weight again. In mice subjected to injection of preformed M1/fibrinogen precipitate, Evans Blue dye was used to assess vascular leakage (Green et al., 1988). Evans Blue (50 mg/kg) was


49

administered i.v. and dye extravasation used to assess change in vascular permeability. At the end of the experiment, the pulmonary circulation was flushed with PBS and Evans Blue was extracted from homogenized lung tissue by incubating in formamide for 24 hours at 60 °C. The optical density of the supernatant and of serum was measured at 620 nm and Evans Blue-albumin extravasation was expressed as µl serum equivalents per g lung tissue.

Molecular Biology

HPLC Proteins in the PMN secretion were enriched with an OASIS column (Waters).

After addition of trifluoroacetic acid (TFA) to the PMN secretion to a final concentration of 0.1 % TFA, the solution was centrifuged. The supernatant was then loaded onto an OASIS 1cc column (Waters), activated by acetonitrile (ACN) and equilibrated in 0.1% aqueous TFA. The column was washed with 0.1 % TFA and proteins were eluted with 80 % ACN in 0.1 % TFA followed by lyophilization. The lyophilized material was redissolved in 0.1 % TFA and protein content was quantified.

Proteins and peptides were separated by HPLC using an ÄKTA purifier system (Amersham Pharmacia Biotech). Reversed phase (RP) chromatography was performed on a Vydac C18 column (Separations Group) equilibrated in 0.1 % TFA at a flow rate of 1 ml/min. A gradient of 0-30 %, 30-60 % and 60-80 % in 37 minutes, 30 minutes and 12 minutes, respectively, was employed and the effluent was monitored at 214 nm. The collected fractions were lyophilized and used for further analyses.

Subcellular fractionation Subcellular fractionation of PMN was performed by density centrifugation on

Percoll gradients, as described by Kjeldsen et al. (Kjeldsen et al., 1999). PMN were disrupted by nitrogen cavitation (350 psi, 5 minutes), and the cavitate was collected by the drop into a solution of EGTA (pH 7.4) final concentration of 1.5 mM. Nuclei and unbroken cells were sedimented by centrifugation at 500 g for 10 minutes at 4 °C. The postnuclear supernatant was mixed with a heavy Percoll solution (7 ml, 1.12 g/ml). The mixture was layered under 14 ml light Percoll solution (1.04 g/ml). Five millilitres heavy Percoll solution (1.12 g/ml) were applied to the bottom of the tube. Relaxation buffer (5 ml) was applied on top of the gradient. The gradient was centrifuged at 37 000 g for 35 minutes at 4 °C using a fixed-angle Beckman JA-20

Methodology

50

rotor. After centrifugation, 1 ml fractions were collected by aspiration from the bottom of the tube using a peristaltic pump. To get rid of the Percoll, fractions were centrifuged for 90 minutes at 45000 rpm in an ultracentrifuge (L-60, Beckman instrument) at 4 °C. To determine which fraction corresponded to which granule compartment, each individual fraction was analyzed for the presence of specific markers of the respective granule by western blot.

Immunoabsorption Monoclonal HNP1-3 antibody or monoclonal HBP antibody were coupled to

Protein A Sepharose (CL-4B, Amersham Pharmacia Biotech). After PMN secretion was precleared with Protein A sepharose the antibody/Protein A Sepharose complexes were incubated with the PMN secretion under gentle rotation over night at 4 °C. The beads were spun down, and the efficacy of immunoadsorption was verified with Western blot for HBP and dot blot for HNP1-3. HBP-specific staining was carried out as described before (Gautam et al., 2001) and HNP1-3 staining with anti-HNP1-3 (1:2000) (PanATecs GmbH).

ELISA Concentrations of TNF, IFN , IL-8, and MCP-1 (eBioscience) were analyzed

in the poststimulatory cell-free supernatant using a commercial ELISA kit following the manufacturer’s instruction. HBP concentrations were determined by ELISA as described before (Tapper et al., 2002).

Immunocytochemistry Human macrophages were cultured in 96 well plates and stimulated with PMN

secretion for 24 hours. Thereafter, the cells were washed and incubated with 0.5 % BSA in PBS for 15 minutes. Cell surface staining was carried out applying fluorescently labeled mAbs for 30 minutes at room temperature directed against the following antigens: CD36, CD80, CD86, HLAII, CD40, CD25, CD14, CD11c, CD35, CD32, CD64 (all FITC-labeled, Immunotools), FITC CD62L, PE CD11b, and PE CD16 (Becton Dickinson). After staining, macrophages were washed and the fluorescence intensity was analyzed in a fluorescence plate reader (Fluoroskan Ascent, Labsystems).


51

PCR Whole blood and spleenocytes from C57BL/6 mice were subjected to red cell

lysis, resuspended in HEPES-buffered HBSS + 0.5 % BSA and 2 mM EDTA and reacted with Gr-1-PercP-Cy5.5, CD45-APC-Cy7 (BD Biosciences) and CD115-PE (eBiosciences) for 30 minutes on ice. High-pressure sorting with purity precision mode was performed in a FACSAria sorter (BD) to separate and collect CD115+Gr-1+CD45+ inflammatory monocytes. Total RNA was isolated from sorted, inflammatory monocytes and reverse-transcribed into cDNA by Mo-MLV RT (Invitrogen). PCRs

were performed with 20 ng cDNA using Taq DNA polymerase (Promega) for 38 cycles cycles (30 seconds at 95 °C, 40 seconds at 60 °C, and 1 minute at 72 °C) and specific primers:

aldolase: 5'-AGCTGTCTGACATCGCTCACCG-3' 5'-CACATACTGGCAGCGCTTCAAG-3' fpr2: 5'-GTAAGAAGGAGACCTCAGCTG-3' 5'-CCCTCTAGCATCTCTAACTGTA GTC-3' fpr1: 5'-CTGTAGATCTGTCCAGAGC TGTTG-3' 5'-CAATGTAAGAAGAAA TGCACAAATC-3').

Histology and electron microscopy After completion of the experiment, one part of the right lung was fixed in

formalin, embedded in paraffin and stained with Mayer’s hematoxylin and eosin for histological examination (Herwald et al., 2004). Another part of the lung was prepared for scanning electron microscopy as described (Herwald et al., 2004).

Western Blot analysis The composition of the PMN secretion was analyzed by Western blot. SDS-

PAGE and Western Blot were done as described before (Schmeisser et al., 2004). After blocking of non-specific binding overnight at 4 °C, the blot membranes were incubated with primary antibodies for one hour at room temperature: anti-MMP-9 (1:500, Sigma), anti-albumin (1:500, DAKO), anti-LL-37 (1:200, Innovagen AB, gift from B. Agerberth, Karolinska Institute, Stockholm, Sweden), anti-elastase (1:500, DAKO). After washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse antibody or goat anti-rabbit antibody (1:3000, Pierce). Staining was visualized by an enhanced chemiluminescence system (Pierce). Membranes were photographed using BioRad GelDoc and analysed with BioRad Quanty One 4.5.2.

Methodology

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Statistics All data are presented as mean SD. All experiments were performed at least three times. A p of < 0.05 is considered significant. For details regarding calculation of statistical significances can be found in the method’s section of each individual manuscript.


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List of abbreviations

ACN acetonitrile APC antigen presenting cell BAEC bovine aortic endothelial cell BAL bronchio-alveolar lavage CAM cell adhesion molecule CAP37 cationic antimicrobial protein with a mass of 37 kDa CD cluster of differentiation DC dendritic cell DPPI dipeptidyl peptidase I EC endothelial cell FACS fluorescent-activated cell sorting FBS fetal bovine serum FITC fluoresceinisothiocyanate fMLP formyl-Methionyl-Leucyl-Phenylalanine fpr formyl peptide receptor family FPRL1 formyl Peptide Receptor Like-1 FPR1 formyl Peptide Receptor 1 fpr1 murine formyl peptide receptor 1 fpr2 murine formyl peptide receptor 2 G-CSF granulocyte colony stimulating factor GM-CSF granulocyte-macrophage colony stimulating factor HBP heparin binding protein hCAP18 human cationic antimicrobial protein with a mass of 18 kDa HNP human neutrophil peptide HPLC high-performance liquid chromatography HUVEC human umbilical vein endothelial cell IFN interferon Ig immunoglobulin IL interleukin kDa kilodalton LPS lipopolysaccharide LTA lipoteichoic acid LTB4 leukotrien B4 LXA4R lipoxin A4 receptor mAb monoclonal antibody MCP monocyte chemotactic protein MHC major histocompatibility complex

List of abbreviations

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MIF macrophage migration inhibitory factor MIP macrophage inflammatory protein MLR mixed leukocyte reaction MPO myeloperoxidase MR mannose receptor NOD nucleotide-binding oligomerization domain PAF platelet activating factor PE R-phycoerythrine PMN polymorphonuclear leukocyte PTx pertussis toxin ROS reactive oxygen species SGD specific granule deficiency SNARE soluble NSF attachment receptor STSS streptococcal toxic shock syndrome TFA trifluoroacetic acid TNF tumour necrosis factor TLR toll-like receptor VEGF vascular endothelial growth factor


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Acknowledgements

Doing a PhD is somewhat similar to the Tour de France - apart from the fact that a PhD takes much longer. In the beginning there are the flat stages, as an individual rider you don’t really know where you stand in the peloton. You might win a sprint and get some points (equals points for courses within the PhD program) but it does not really mean that you will make it through the Tour. More important are the mountain stages. You work hard to climb up the mountain, the closer you get to the top the more your legs hurt, and the closer you are to giving up. Once you made it to the top you come to the descent, which is fast and steep – just like getting a paper rejected. And the next ramp won’t be far. Once you made it to through the mountains nothing can go wrong. You write up your thesis and you may even have a glass of champagne on the last stage. I could not have chosen a better place for my PhD than the Physiology at KI – this place has earned the nickname “bicycle repair shop” within KI.

Finishing the Tour at the Champs-Élysées is a great triumph for every rider. However, this success would not be possible without a whole team. Here I would like to point out some people, who were of great support during my Tour de PhD.

Lennart Lindbom, the sporting manager: Thanks for being a good coach for the last four years and for giving me the freedom I needed to develop some creative thoughts, but also for teaching me patience, caution, and modesty. I know that you like HBP a lot, but there is a lot more in the granules than just that one protein.

Einar E. Eriksson: Thanks for bringing in CX3CR1eGFP/+ mice, for scientific advice especially during the flat stages, your input on the manuscripts, and good travel companionship in the US.

Ellinor: Thanks for your infectious laugh, for being my mediator to the Swedish society, for trying to teach me some Swedish, for organizing great parties at Physiology, and for proof-reading all these papers of mine.

Roban: Thanks for the great time in the corridor, for wrestling, talking about life, sheep and statistics, for running around Brunnsviken, travelling to Iceland and Munich, and making a fool out of my- and yourself in front of my future boss.

Pierre: Thanks for nerve-racking backgammon duels (still get sweaty fingers when I think of it), for a never ending repertoire of places to go out for dinner or have a


73

drink, for fun times in Iceland, and for dragging me to the football stadium watching Hammarby.

Thanks to Mats for the invitation to Höga Kusten – watch out, one day I’ll be there.

Thanks to all PhD students at the Physiology, especially to Lydia, Lars, Johanna, Daniel, Kristoffer, and Åsa for fun times at physiology parties. Thanks to Renee for always taking care of small and big computer problems.

Thanks to Ylva for helping me out with all these things I could not do myself – HPLC, mass spec, dot blot N-terminal sequencing. What would the phagocytosis story be without your input. Thanks also to the Germans at the Swedish West coast for a outstanding collaboration: Heiko, Sonja, and Matthias. Thanks to Antonio for bringing some Italian lifestyle into our collaboration. Thanks to Christian and Alma for the good time in Aachen – a lot more to come. Thanks also to Toni for tons of RB6 mAb and to Martin for teaching me some microbiology.

Thanks to Stefano, Paulo, Rachel, Emma, Joe, Maria, and Ana for great parties on Södermalm, and watching the sunrise from the roof while having fresh pasta.

Thanks to the folks from Värtavägen, Sarah, David, The Bear, Livia, Emilie, and all the others for the relaxed atmosphere and listening to my ever-repeating stories when I was home.

Thanks to all players of my soccer team ”Magrizos” - and since that turned out to be not so successful we had to try with another name - ”Periquita”. Thanks for great fights early Saturday mornings and Sunday evenings against the Röda Ugglan, Re Orient, and all the others. Razorblade tackles, fervid discussions with the ref …. so many memories. But why did we never win this stupid league?

Thanks to my biking buddy Ulle for not just biking out to Stenhamra, but also for all your words of wisdom (Du machst das schon alles richtig, Hol Dir das Emmy Noether, ….) - ever considered a career as a life couch?

Lots of thanks to Caro and Jessie for good friendship, fun Portuguese classes, travelling to Tallinn and the Archipelago, a great time in Berlin, and playing Wizard.

Thanks to Linda for always bringing good German wine, beer, and food, great Schnitzel parties, and relaxing trips in your Golf to the Archipelago.

Thanks to Megan for teaching me to appreciate beer (and she is American, I am Bavarian!!!), funny spinning classes, organizing my surprise birthday party, great

Acknowledgements

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biking trips, all the treats, sharing thoughts, concerns, and semla …., thanks for the good time.

Ratan, Swedish class paid off, because we got to know each other – who cares about the right pronunciation of a Swedish ‘jaha’, if you get to know a friend who likes to hike, drink beer, wine, and whiskey, teaches you how to ski, and many other things, and who appreciates live for what it is! Keystone – here we come for the next 50 years!

Toffer, my best friend: Thanks for so many things, long phone calls, late phone calls, phone calls at unusual times…. Thanks for always being there and thinking for me if I forgot once again. Thanks for travelling around the globe with me and for having been my most frequent visitor in Stockholm. I am looking forward to the great time to come when I’ve moved to Aachen.

Herzlichen Dank gilt vor allem auch meinen Eltern für unzählige Telefonate, schwere Fress- und Geschenkpakete, die Ihr immer an Ostern, meinem Geburtstag und Weihnachten nach Stockholm geschickt habt (um die auch immer beneidet wurde), und die Unterstützung in den letzten Jahren, wenngleich auch nicht immer offensichtlich war wofür dieses oder jenes schon wieder gut sein sollte.

Obrigada minha amada Joana, pelo teu amor, suporte e compreensão. Obrigada por me fazeres sorrir e rir e por teres trazido o teu sol Português para Estocolmo

This work was financially supported by the Swedish Medial Research Council, the Swedish Heart and Lung Foundation, the Lars Hierta Memorial Foundation, and the AFA Health Fund. Oliver Söhnlein is a recipient of a post-doc grant from the Deutsche Forschungsgemeinschaft (SO 876/1-1).

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