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Thesis for doctoral degree (Ph.D.)
2008
MODULATORS OF THE IMMUNE RESPONSE
Thesis for doctoral degree (Ph.D.) 2008
NEUTROPHIL GRANULE PROTEINS:
NEUTROPHIL GRANULE PROTEINS:
MODULATORS OF THE IMMUNE RESPONSE
Oliver Söhnlein, M.D.
Oliver Söhnlein, M.D.
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
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
Printed by
2008
Gårdsvägen 4, 169 70 Solna
”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 (Į-defensins) as
principal mediators of this effect. Mechanistically, these two proteins induce the
release of TNFĮ and IFNȖ from macrophages. These activate the macrophage in an
autocrine fashion resulting in upregulation of the FcȖ 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
streptococcal M1 protein. Eur. Resp. J., in press.
triggered by
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
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.
1
Introduction
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 myeloidspecific 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).
2
Neutrophil granule proteins: Modulators of the immune response
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,
E2-integrins
tertiary
granules:
MMP-9,
lactoferrin
secondary and
primary granules:
LL-37, HNP1-3
HBP, 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 E2-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
3
Introduction
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 peroxidasenegative 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, Į-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
4
Neutrophil granule proteins: Modulators of the immune response
G (Salvesen et al., 1993), elastase (Sinha et al., 1987), and HBP, the latter being
proteolytically inactive.
Table 3: PMN granule composition
Membrane
Matrix
Primary granule
CD63, CD68
HBP, Cathepsin G,
HNP1-3, Elastase,
MPO
Secondary granule
Laminin-R,
Vitronektin-R, TNFR, fpr
Lactoferrin, hCAP18
(LL-37), NGAL
Tertiary granule
CD11b, fpr
MMP-9,
Lysozyme
Secretory vesicles
CD11, CD14,
CD16, fpr, CD10,
CD13
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 Pmol or 630 Pg/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 twodomain 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, LL37 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).
5
Introduction
HNP1-3
Į-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 Į-defensins in human PMN, HNP1-4
(Ganz et al., 1985; Wilde et al., 1989), and two well-described human Paneth cell Įdefensins, 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 Į-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-1E 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-1E in that the
transcription of TNF, GM-CSF, MCP-1, TGF-E1 and VEGF, and the release of GMCSF, IL-1E and TGF-E1 remain at levels comparable to the untreated control cells
(Sakamoto et al., 2005). It was shown some time ago that rabbit Į-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
6
Neutrophil granule proteins: Modulators of the immune response
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 Gramnegative 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
7
Introduction
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 membranebound 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;
8
Neutrophil granule proteins: Modulators of the immune response
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 premade 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
Differentiation
chemokines
IFN-ĮR1/2, IFNGM-CSFR, GȖR1/2,
CSFR, IL-6R
IL(1,4,6,10)R,
TNFR-1/2, TGFER2, CCR-1, -2, -3,
IL-8RĮ/E, CXCR-4
Fc
IgG, IgA, IgE
Cytokines and
chemokines
Recognition
IL-1Į/E, IL-8,
VEGF, TNF, MIPTLR-1,-2,-4,-6,-8, 1Į, MIP-2Į, MIP-2E,
CD14, MyD88,
MIP-3Į, MCP-1
MD-2, fpr,
TREM1
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 IFNȖ 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.
9
Introduction
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 (ZieglerHeitbrock, 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; ZieglerHeitbrock, 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 (GrageGriebenow 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
10
Neutrophil granule proteins: Modulators of the immune response
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
Gr-1 expression
Surface markers
Chemokine receptor
expression
Functional properties of the
respective human ortholog
Inflammatory
monocyte
Gr-1hi
Intermediate
Resident monocyte
monocyte
Gr-1int
Gr-1lo
+
+
+
CD115 , F4/80 , CD11b
CD62L+
CD62L+
+
+
+
CCR2 , CX3CR1++,
CCR2 , CX3CR1
CCR2 , CX3CR1 ,
CCR7+, CCR8+
CCR5+
Phagocytosis n, T
Phagocytosis p, T
cell activation (MLR,
cell activation (MLR,
APC) p, Cytokine
APC) n, Cytokine
release (IL-1, IL-6,
release (IL-1, IL-6,
TNF) n
TNF) p
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 E1- and E2-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-
11
Introduction
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
12
Neutrophil granule proteins: Modulators of the immune response
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)
13
Introduction
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-țB 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 proinflammatory cytokines and antimicrobial effector molecules, which results from the
activation of NF-țB 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 Gramnegative 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: FcJRI (CD64) with
high affinity for monomeric IgG, FcJRII (CD32) and FcJRIII (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
14
Neutrophil granule proteins: Modulators of the immune response
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 IFNȖ-producing, CD4+ and MHC class IIrestricted 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 Irestricted 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 cellinhibitory 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, IFNĮ/E); (iv)
cytokines that downregulate the macrophage and/or help to terminate the
inflammation (e.g. IL-10, transforming growth factor (TGF)- E). The production of
cytokines by macrophages can be triggered by microbial products such as LPS, by
15
Introduction
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. IFNȖ).
Activation patterns
The prototypic soluble activator of macrophage function is IFNȖ. This
cytokine is produced by CD4+ type 1 T-helper cells, CD8+ T cells, NK cells, NKTcells, and macrophages themselves (Schleicher et al., 2005). IFNȖ 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. FcȖR type I, II and III) on the cell
surface (Table 4). Apart from IFNȖ, several other soluble mediators (e.g. IFN Į/E,
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 TGFE. To date, TGFE 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, FcİR 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
16
Neutrophil granule proteins: Modulators of the immune response
(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 SIRPĮR
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
IFNȖ, TNF
Signal
Ligation of TLR,
CD14, TREM, EGlucan-R
Secretion product
ROS, NO, IFNĮ/E TNF; IL-12, IL-1,
IL-6, NO, ROS,
IP-10, MIP-1Į,
MCP-1
MHCII n
MHCII n, CD86 n,
CD40 n, MannoseRp
Microbicidal,
tissue destruction,
cellular immunity,
delayed
hypersensitivity
Marker
Functional
features
Alternative
activation
IL-4, IL-13
IL-1RA, IL-10,
AMAC-1
Deactivation
Ligation of
Scavenger-R,
CD47, CD200R,
Uptake of apoptotic
cells, TGF-E
PGE2, TGF-E, IL10
Mannose-R n,
MHCII p
Scavenger-R n,
CD23 n, CD14 p
Humoral
Immunosuppression
immunity, allergic
and anti-parasitic
effects, tissue
repair
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 E2-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
17
Introduction
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 proforms of TNF, IL-1E 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).
18
Neutrophil granule proteins: Modulators of the immune response
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 E2-integrins on PMN and
DC-SIGN on DCs. This phenotypic change allows for a more potent T cell activation
by DCs (Boudaly, 2008).
Adhesion molecule expression
Presentation of granule proteins
Permeability
Apoptosis
Endothelial cell
PMN
Monocyte/Macrophage
Secretory Vesicles
Dendritic Cell
Tertiary Granules
Secondary Granules
T-cell
Primary Granules
Recruitment
Activation
Differentiation
Cytokine release
Phagocytosis
Recruitment
ROS release
Recruitment
Activation
(MHCII, CD40, CD86)
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.
19
Aims
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:
20
x
To uncover how PMN secretion products instruct the extravasation of
monocytes (papers I and II).
x
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).
x
To discover by which mechanism PMN secretion products activate monocytes
and macrophages, induce cytokine release, and alter their phenotype (papers
IV and V).
x
To understand to what extent PMN secretion products contribute to acute lung
damage induced by Streptococcus pyogenes (papers V and VI).
Neutrophil granule proteins: Modulators of the immune response
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 IFNȖ 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
21
Results and Discussion
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 IFNȖ 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.
B
D
1.4
1.2
Elastase
0.6
0.4
0.2
0
Lactoferrin
30
20
25
secretory
tertiary
CD35
15
MMP-9
10
IB4
IB4+
Sek. Ab
Albumin
0.8
secondary
MMP-9
1
MPO
activity
[U/ml]
5
LL-37
0
C
primary
A
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
22
Neutrophil granule proteins: Modulators of the immune response
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.
23
Results and Discussion
A
Light
microscopy
Fluorescence
microscopy
ctrl
+IB4
PMN perfusion
*
* *
*
0
LPS +HBP
90s
BSA
E-selectin
40
*
E-selectin
+ HBP
20
0
IC
Ctrl
*
60s
-1
VC
A
M
-1
Pse
le
ct
in
Ese
le
ct
in
50
LPS
SA
100
*
*
60
30s
M
150
*
D
C
A
B
Lymphocytes
Neutrophils
Monocytes
MM6
200
80
A
B
A
250
Adherent monocytes/mm2
Adherent cells in % of LPS
TNF (50ng/ml, 24h)
Figure 4: HBP enhances monocyte adhesion. (A) Isolated PMN are perfused for 30 minutes over
TNF-activated endothelial cells. Thereafter, we stain surface-bound HBP (left) and quantify the
resulting fluorescence (right). Blockade of PMN adhesion by IB4 also inhibits deposition of HBP. (B)
Isolated leukocyte populations are superfused over LPS-activated HUVEC and the number of adherent
cells is quantified (dashed line). Deposition of HBP enhances adhesion of monocytes and monocytic
Mono Mac 6 cells only. (C) Monocytes are superfused over plates coated with recombinant adhesion
molecules and HBP. Thereafter, the number of adherent monocytes is quantified. (D) Monocytes are
labeled with the Ca2+-sensitive dye fluo4. Interaction with E-selectin + HBP induces significantly
higher Ca2+ mobilization as compared to interaction with E-selectin only.
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
24
Neutrophil granule proteins: Modulators of the immune response
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
25
Results and Discussion
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).
A
B
C
D
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
26
Neutrophil granule proteins: Modulators of the immune response
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).
27
Results and Discussion
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 oxygendependent 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
28
Neutrophil granule proteins: Modulators of the immune response
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.
A
B
ctrl
PMN secretion
S. aureus + PMN secretion
S. aureus
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 FcȖR 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
29
Results and Discussion
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 PMNmediated 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 IFNȖ 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 IFNȖ together almost
completely abolished the effect of HBP and HNP1-3.
30
Neutrophil granule proteins: Modulators of the immune response
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 IFNȖ which enhance the
expression of CD64 and CD32.
B
ctrl
PMN sec
E
40
p h a g o c y to s e d
S . a u r e u s /c e ll
5
CD32
4
CD64
30
3
20
2
10
1
0
0
0
C
A b s2 14 n m x 1 0 0
A
10 20 30 40 50 60 70
Fractions
Fraction 44-46
Fraction 60-62
100
% Intensity
0
(M+1)
3443.29
2.1E+4
(M+1)
3372.11
(M+1)
3487.41
0
3200 3300 3400 3500 3600 3700
Mass
D
F
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 phagocytosisenhancing 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,
31
Results and Discussion
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 (IFNȖ 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 FcȖ receptors CD32 and CD64
on the macrophage surface, which may indicate a classical activation pattern. This is
normally mediated by IFNȖ 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 IFNȖ 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 IFNȖ and TNF concentration (Figure 8B). HBP mediated
its effect via activation of E2-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 IFNȖ 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 IFNȖ.
32
Neutrophil granule proteins: Modulators of the immune response
A
B
H
H
C
Figure 8: PMN secretion induces classical macrophage activation via release of TNF and IFNȖ.
(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 IFNȖ. (C) Neutralizing
antibodies to TNF and IFNȖ 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 IFNȖ 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.
33
Results and Discussion
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
E2-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-1E, 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 E2-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 E2-integrins on PMN (Cai and Wright, 1996) and we were therefore
wondering if HBP activates monocytes via E2-integrins. In Ca2+ mobilization assays
we found that a blocking antibody to E2 integrins completely abolishes the HBPinduced Ca2+ response, while a control antibody was without effect. In line with these
observations we also found in paper IV that the release of IFNȖ and TNF from human
macrophages was blocked in the presence of a E2-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,
E2-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).
34
Neutrophil granule proteins: Modulators of the immune response
A
count
ctrl
CD14
E
M1
M1 +
Gly-Pro-Arg-Pro
M1 +
Gly-Pro-Arg-Pro
ctrl
M1
D
C
B
ctrl
M1
M1 +
Gly-Pro-Arg-Pro
F
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 E2-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 E2-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
35
Results and Discussion
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.
36
Neutrophil granule proteins: Modulators of the immune response
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
37
Perspectives
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).
38
Neutrophil granule proteins: Modulators of the immune response
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 tumorspecific 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 gramnegative 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, Į-defensins neutralize the toxins produced by Bacillus
39
Perspectives
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
40
Neutrophil granule proteins: Modulators of the immune response
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
41
Perspectives
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.
42
Neutrophil granule proteins: Modulators of the immune response
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 E2-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 %
43
Methodology
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 Pg/ml for 4 hours. Pre-perfusion
of EC with PMN was obtained by perfusion of freshly isolated PMN over LPSactivated 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
Pg/ml, R&D systems) to the centre of a well in a 6-well plate. Recombinant HBP (1
Pg/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
44
Neutrophil granule proteins: Modulators of the immune response
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 Pl 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 BSAcoated 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
45
Methodology
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 antiCD64 (10.1, Immunotools) were added to the macrophages at 10 µg/ml 30 minutes
before addition of IgG-opsonized bacteria. Anti-TNFĮ (5 µg/ml, Mab1, BioLegend)
and anti-IFNJ (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.
46
Neutrophil granule proteins: Modulators of the immune response
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.
47
Methodology
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 antiPMN 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
48
Neutrophil granule proteins: Modulators of the immune response
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
49
Methodology
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, IFNJ, 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).
50
Neutrophil granule proteins: Modulators of the immune response
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. SDSPAGE 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 peroxidaseconjugated 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.
51
Methodology
Statistics
All data are presented as mean r 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.
52
Neutrophil granule proteins: Modulators of the immune response
List of abbreviations
ACN
APC
BAEC
BAL
CAM
CAP37
CD
DC
DPPI
EC
FACS
FBS
FITC
fMLP
fpr
FPRL1
FPR1
fpr1
fpr2
G-CSF
GM-CSF
HBP
hCAP18
HNP
HPLC
HUVEC
IFN
Ig
IL
kDa
LPS
LTA
LTB4
LXA4R
mAb
MCP
MHC
acetonitrile
antigen presenting cell
bovine aortic endothelial cell
bronchio-alveolar lavage
cell adhesion molecule
cationic antimicrobial protein with a mass of 37 kDa
cluster of differentiation
dendritic cell
dipeptidyl peptidase I
endothelial cell
fluorescent-activated cell sorting
fetal bovine serum
fluoresceinisothiocyanate
formyl-Methionyl-Leucyl-Phenylalanine
formyl peptide receptor family
formyl Peptide Receptor Like-1
formyl Peptide Receptor 1
murine formyl peptide receptor 1
murine formyl peptide receptor 2
granulocyte colony stimulating factor
granulocyte-macrophage colony stimulating factor
heparin binding protein
human cationic antimicrobial protein with a mass of 18 kDa
human neutrophil peptide
high-performance liquid chromatography
human umbilical vein endothelial cell
interferon
immunoglobulin
interleukin
kilodalton
lipopolysaccharide
lipoteichoic acid
leukotrien B4
lipoxin A4 receptor
monoclonal antibody
monocyte chemotactic protein
major histocompatibility complex
53
List of abbreviations
MIF
MIP
MLR
MPO
MR
NOD
PAF
PE
PMN
PTx
ROS
SGD
SNARE
STSS
TFA
TNF
TLR
macrophage migration inhibitory factor
macrophage inflammatory protein
mixed leukocyte reaction
myeloperoxidase
mannose receptor
nucleotide-binding oligomerization domain
platelet activating factor
R-phycoerythrine
polymorphonuclear leukocyte
pertussis toxin
reactive oxygen species
specific granule deficiency
soluble NSF attachment receptor
streptococcal toxic shock syndrome
trifluoroacetic acid
tumour necrosis factor
toll-like receptor
VEGF
vascular endothelial growth factor
54
Neutrophil granule proteins: Modulators of the immune response
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Acknowledgements
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
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Neutrophil granule proteins: Modulators of the immune response
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
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Acknowledgements
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).
74