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Molecular and functional characterization of murine macrophage subtypes [Elektronische Ressource] / vorgelegt von Daniela Dreymüller

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135 pages
“Molecular and functional characterization of murinemacrophage subtypes”Von der Fakultät für Mathematik, Informatik undNaturwissenschaften der RWTH Aachen University zurErlangung des akademischen Grades einer Doktorin derNaturwissenschaften genehmigte Dissertationvorgelegt vonDiplom-Biologin Daniela Dreymülleraus AachenBerichter:Universitätsprofessor Dr. Wilhelm Jahnen-DechentUniversitätsprofessor Dr. Lothar EllingTag der mündlichen Prüfung: 16.06.2009Diese Dissertation ist auf den Internetseiten der Hochschulbibliothekonline verfügbar.Content ISummary................................................................................................................................11. Introduction ............................................................................................................31.1. Macrophage biology ...............................................................................................31.1.1. Murine macrophage subtypes..............................................................................31.1.1.1. Murine macrophage subtypes from embryo to newborn...................................31.1.1.2. Macrophage subtypes in adult mice.................................................................41.1.1.3. Markers for embryonic/fetal and adult macrophages........................................51.1.2. The macrophage microenvironment ....................................................................61.1.2.1.
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“Molecular and functional characterization of murine
macrophage subtypes”
Von der Fakultät für Mathematik, Informatik und
Naturwissenschaften der RWTH Aachen University zur
Erlangung des akademischen Grades einer Doktorin der
Naturwissenschaften genehmigte Dissertation
vorgelegt von
Diplom-Biologin Daniela Dreymüller
aus Aachen
Berichter:
Universitätsprofessor Dr. Wilhelm Jahnen-Dechent
Universitätsprofessor Dr. Lothar Elling
Tag der mündlichen Prüfung: 16.06.2009
Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek
online verfügbar.Content I
Summary................................................................................................................................1
1. Introduction ............................................................................................................3
1.1. Macrophage biology ...............................................................................................3
1.1.1. Murine macrophage subtypes..............................................................................3
1.1.1.1. Murine macrophage subtypes from embryo to newborn...................................3
1.1.1.2. Macrophage subtypes in adult mice.................................................................4
1.1.1.3. Markers for embryonic/fetal and adult macrophages........................................5
1.1.2. The macrophage microenvironment ....................................................................6
1.1.2.1. The extracellular matrix – macrophage interplay .............................................6
1.1.2.2. Macrophage activation states...........................................................................7
1.1.3. Macrophage function ..........................................................................................8
1.1.3.1. Endocytosis.....................................................................................................9
1.1.3.2. Lysosomal degradation..................................................................................11
1.1.3.3. Antigen presentation .....................................................................................12
1.1.3.4. Migration ......................................................................................................13
1.2. Wound healing......................................................................................................14
1.2.1. Wound healing in the embryo/fetus...................................................................16
1.2.2. Wound healing and macrophages – inflammation and scaring...........................16
1.2.3. Models for wound healing.................................................................................18
1.3. Macrophage model ...............................................................................................18
1.4. Experimental aims ................................................................................................20
2. Material and Methods ...........................................................................................21
2.1. Cell culture ...........................................................................................................21
2.1.1. Murine feeder cell culture .................................................................................21
2.1.2. R1 cell culture...................................................................................................21
2.1.3. ES macrophage differentiation and culture ........................................................21
2.1.4. BM macrophage differentiation and culture.......................................................22
2.1.5. Saphenous vein endothelial cell (SVEC) culture................................................22
2.1.6. Rat hepatoma cell line FTO2B culture...............................................................22
2.2. Determination of cell size .....................................................................................22
2.3. Activation profiling...............................................................................................23
2.3.1. Stimulation by LPS ...........................................................................................23
2.3.2. Oxidative burst measurement ............................................................................23
2.3.3. Classical and alternative activation....................................................................23II Content
2.3.3.1. Expression of marker genes CD163, CD206 and CCL3.................................23
2.3.3.2. Cytokine expression profile...........................................................................23
2.3.4. Leukocyte acidic phosphatase activity...............................................................24
2.3.5. Lysozyme activity.............................................................................................24
2.3.6. Osteoclast differentiation...................................................................................24
2.4. Surface marker profiling .......................................................................................24
2.5. Analysis of macrophage function ..........................................................................25
2.5.1. Endocytosis.......................................................................................................25
2.5.1.1. Receptor-mediated endocytosis .....................................................................25
2.5.1.2. Pinocytosis....................................................................................................26
2.5.1.3. Pinocytosis and phagocytosis of latex beads..................................................26
2.5.1.4. Phagocytosis of apoptotic cells......................................................................26
2.5.1.5. Phagocytosis of rat hepatoma cells (F2OTB cells)........................................26
2.5.1.6. Phagocytosis of bioparticles ..........................................................................26
2.5.1.7. Endocytosis of bovine fetuin-A monomer and calciprotein particles (CPPs) ..27
2.5.2. Adhesion assays................................................................................................27
2.5.2.1. Coated glass slides ........................................................................................27
TM2.5.2.2. MSA cell adhesion assay ...........................................................................27
2.5.3. Matrix metalloproteinase activity ......................................................................27
2.5.4. T cell activation assays......................................................................................28
2.5.4.1. OT-II proliferation assay ...............................................................................28
2.5.4.2. OT-I proliferation assay ................................................................................28
2.5.5. Transmigration assay ........................................................................................29
2.6. Generation of transgenic E. coli strains .................................................................29
2.6.1. Generation of calcium competent bacteria.........................................................29
2.6.2. Cloning of PCR fragments ................................................................................29
2.6.2.1. Vector-insert-ligation ....................................................................................29
2.6.2.2. Transformation of bacteria ............................................................................29
2.6.2.3. Plasmid preparation.......................................................................................30
2.7. Analysis of gene expression ..................................................................................30
2.7.1. Semiquantitative RT-PCR.................................................................................30
2.7.2. RealTime-PCR..................................................................................................30
2.7.3. Primer systems..................................................................................................31
2.8. In vivo-tail wound model.......................................................................................31Content III
2.8.1. Wounding and cell application ..........................................................................31
2.8.2. Histology and immunhistochemical staining procedure .....................................31
2.8.3. Statistical analysis.............................................................................................33
3. Results..................................................................................................................34
3.1. Determination of cell size......................................................................................34
3.2. Surface marker profiling .......................................................................................34
3.3. Developmental classification.................................................................................35
3.4. Activation profiling...............................................................................................38
3.4.1. Activation by LPS treatment .............................................................................38
3.4.2. Oxidative burst measurement ............................................................................39
3.4.3. Expression of marker genes CD163, CD206, and CCL3....................................40
3.4.4. Cytokine expression profile...............................................................................41
3.4.5. Acid phosphatase and tartrate resistant acid phosphatase acitivity .....................45
3.4.6. Differentiation to osteoclasts .............................................................................46
3.5. Gene expression profiling .....................................................................................47
3.5.1. Analysis of DNA chip array data.......................................................................47
3.5.1.1. Confirmation of most differentially expressed genes by RT-PCR methods ....48
TM3.5.1.2. Gene Ontology and Pathway Architect anaylsis .........................................50
3.5.2. Expression of matrix metalloproteinases ...........................................................52
3.5.3. Expression of antigen processing proteins and coagulation factors ....................52
3.5.4. Scavenger receptor profile.................................................................................54
3.5.5. Extracellular matrix proteins .............................................................................55
3.5.6. Integrin expression profile.................................................................................56
3.6. Macrophage Function ...........................................................................................57
3.6.1. Adhesion profiling ............................................................................................57
3.6.2. Endocytosis.......................................................................................................61
3.6.3. Phagocytosis of apoptotic mouse fibroblasts......................................................63
3.6.4. Phagocytosis of rat hepatoma cells (F2OTB).....................................................65
3.6.5. Antigen presentation .........................................................................................66
3.6.6. Extracellular matrix remodeling ........................................................................70
3.6.7. Transmigration assay ........................................................................................70
3.7. Wound healing......................................................................................................73
4. Discussion ............................................................................................................83
5. Conclusion..........................................................................................................104IV Content
6. Appendix ............................................................................................................105
6.1. Abbreviations......................................................................................................105
6.2. Figures................................................................................................................109
6.3. Tables .................................................................................................................111
6.4. References ..........................................................................................................111
Acknowledgement..............................................................................................................129Summary 1
Summary
Macrophages are professional phagocytes involved in pathogen defense, antigen presentation,
particle and debris clearing, as well as in removal of apoptotic cells and tissue remodeling.
They represent a highly diverse cell type, varying between the embryo and the adult, different
tissues and even within one tissue. Macrophages express a specific repertoire of surface
markers, enabling phagocytosis, and cell interaction. This involves scavenger receptors, Toll-
like-receptors, major histocompatibility complex (MHC) I and II molecules, and integrins.
Furthermore, macrophages secrete mediators regulating the inflammatory response, and pro-
teolytic enzymes involved in migration processes and tissue remodeling. Macrophages can be
differently activated, depending on the macrophage microenvironment. The macrophage
subtype and the distinct activation state are key determinants for the outcome of wound heal-
ing and tissue regeneration. Macrophages can be differentiated ex vivo from bone marrow
hematopoietic stem cells, so-called bone marrow derived macrophages (BM macrophages),
but also from embryonic stem cells resembling the embryonic/fetal development of macro-
phages, so-called ES macrophages. I hypothesized, that ES macrophages may represent a
source of naïve macrophages resembling the embryonic macrophage subtype, which should
be non-inflammatory, highly proficient in apoptotic cell clearing and tissue remodeling, and
should facilitate scar-free wound healing. In contrast, BM macrophages are thought to repre-
sent a macrophage subtype predominantly mediating inflammation related immune responses
and antigen presentation. ES macrophages were to be molecularly and functionally charac-
terized in vitro in comparison to BM macrophages. To this end, I employed genome wide
expression profiling, activation profiling, surface marker profiling, and functional assays
testing endocytosis, adhesion, antigen presentation, and cell migration. Surface marker pro-
filing and gene expression analysis revealed similar expression of the macrophage specific
markers CD14, CD115, F4/80, and CD68 in BM and ES macrophages. In contrast, the two
macrophage subtypes differed in the expression of the receptor repertoire involved in endo-
cytosis of foreign body particles, calciprotein particles and apoptotic cells. This involved Toll-
like receptors 4 and 6 and macrophage scavenger receptor 2, and resulted in higher clearing
capacity in BM macrophages in comparison to ES macrophages. Developmental profiling
revealed that ES resembled a liver hematopoietic embryonic or an adult macro-
phage subtype. Both macrophage subtypes were classified as resident macrophages that could
be differentiated into prefusion osteoclasts, and showed differential activation properties con-
cerning the classical and alternative activation pathway. BM macrophages were more sensi-2 Summary
tive to pro-inflammatory stimuli (classical activation), whereas ES macrophages showed
stronger reaction to alternative activation and deactivation stimuli. Gene expression analysis
revealed IL-1 and IL-3-dependent differential expression of coagulation factors and MHC I
and II molecules in BM and ES macrophages, but an inductor-independent up-regulation of
matrix metalloproteinase expression in the latter macrophage subtype. Functional analysis
revealed that BM macrophages had a higher and more prolonged cross-presentation capacity
in comparison to ES macrophages, whereas ES macrophages showed higher proteolytic ac-
tivity in cell culture supernatant. The differential expression of integrins was associated with a
higher adherence capacity of ES macrophages, e.g. on laminin and collagen types I and III.
The transmigration properties of the two macrophage subtypes showed the complex network
of the interplay between MMP secretion and integrin adhesion and was influenced by the in-
teraction potential of the two macrophage subtypes with the endothelial monolayer. Macro-
phages are key players during all phases of wound healing, strongly associated with the se-
verity of inflammation and the level of scar formation. Investigation of the influence of the
two macrophage subtypes on wound healing in an in vivo-tail wound model showed dimin-
ished wound contraction, delayed eschar shedding and delayed wound closure in both macro-
phage-treated wound types compared to cell-free treated control wounds. Furthermore,
macrophage-treatment of wounds resulted in prolonged neutrophil influx and prolonged in-
flammatory response. ES macrophage-treatment resulted in a higher cellularity of the wound
after complete wound closure, mainly caused by fibroblast accumulation. These findings con-
firmed the hypothesis that BM macrophages resemble a macrophage subtype predominantly
mediating inflammation related immune responses and tissue homeostatis, thus classified as
M1 macrophage according to Mantovani and colleagues (Mantovani et al. 2004). My original
hypothesis held that ES macrophages should be non-inflammatory and predominantly in-
volved in apoptotic cell clearing and tissue remodeling, resembling an embryonic/fetal
macrophage subtypes, thus probably mediating scar-free healing. Contrary to this hypothesis,
the present study showed that ES macrophages efficiently mediated inflammation related
processes, showed less clearing capacity and were associated with fibroblast accumulation in
tail wounds after complete re-epithelialization, eventually resulting in fibrosis. Consistent
with the activation profiles, ES macrophages thus do not constitute an embryonic/fetal, but
rather an alternatively activated macrophage subtype of the M2 type according to current clas-
sification criteria (Mantovani et al. 2004).Introduction 3
1. Introduction
1.1. Macrophage biology
Macrophages (Greek: big eater, from makros "large" and phagein "eat") are the major differ-
entiated cells of the mononuclear-phagocyte system, and act in both innate and cell-mediated
immunity. They play an essential role in body homeostasis by engulfment of senescent cells
and pathogens, the regulation of inflammation (secretion of mediators and complement), the
control of the inflammatory response (antigen presentation and T cell activation), and are key
players in tissue remodeling and wound healing (Shepard et al. 2000).
1.1.1. Murine macrophage subtypes
During development of a newborn individual many different subtypes and activation states of
macrophages occur that are distinct from the adult subtypes of murine monocytes and macro-
phages.
1.1.1.1. Murine macrophage subtypes from embryo to newborn
During mouse embryogenesis, the first hematopoietic precursor cells, yielding cells that have
a macrophage-like phenotype, occur shortly after the onset of gastrulation (7.5 days post coi-
tum (dpc)) in the splanchic mesoderm of the yolk sac (Cline et al. 1972; Lichanska et al.
2000; Bertrand et al. 2005). These cells migrate to the head mesenchyme and its circulation
(Herbomel et al. 1999). In the mouse, Takahashi et al. first described two different lineages of
yolk sac macrophages, called ‘primitive’ and ‘definitive’, with different developmental ori-
gins. Definitive macrophages were suggested to develop through a monocytic cell series like
in adult organisms, whereas primitive macrophages were directly derived from hematopoietic
precursors (Takahashi et al. 1989). Recent studies revealed that yolk-sac derived macrophages
can be distinguished into three distinct developmental waves: the maternally derived wave
+ -(CD45 c-kit , only detectable until 9.5 dpc), the yolk sac-derived macrophage restricted pre-
+ - -cursor wave (CD45 c-kit ), and the yolk sac-derived erythromyeloid precursor wave (CD45 c-
+kit ) (Bertrand et al. 2005). The erythromyeloid precursor cells and an additional progenitor
cell subtype derived from intraembryonic angiogenic clusters in the aorta, gonads, and
mesonephros region contribute to the onset of a mixed hematopoietic progenitor cell popula-
tion in the fetal liver at 12 dpc (Morrison et al. 1995; Palis et al. 1999). Fetus-derived macro-4 Introduction
phages resembling an adult phenotype are first observed after this onset of fetal liver hema-
topoiesis (Lichanska et al. 2000), becoming the most abundant cell type in the embryo, in
some organs up to 10 to 15% of the total cell number. The last change in site of hematopoiesis
occurs at 17.5 dpc with the detection of hematopoietic stem cells in the fetal bone marrow
(Christensen et al. 2004). Macrophages play essential roles in embryogenesis, especially in
organ development and tissue remodeling, e.g. in the dorsal midline and the branchial arches
(Hopkinson-Woolley et al. 1994). In addition, studies in zebrafish revealed that embryonic
macrophages also show immune abilities like clearing of gram-negative and gram-positive
bacteria (Herbomel et al. 1999).
1.1.1.2. Macrophage subtypes in adult mice
Circulating monocytes can be recruited to tissues by many different ways, including pro-
inflammatory cytokines and metabolic end products. This process involves rolling and tight
adherence to the endothelium, transmigration through the endothelial layer, and subsequent
migration through subendothelial structures. In the tissue microenvironment, monocytes dif-
ferentiate into tissue-specific macrophages, for example Langerhans cells (skin), Kupffer cells
(liver), microglia (brain) and osteoclasts (bone). Heterogeneity of tissue macrophages is mir-
rored by different subclasses of peripheral-blood monocytes. In humans, inflammatory mono-
high - + +cytes (CD14 CD16 ) are distinguished from resident monocytes (CD14 CD16 ). In mice,
the equivalent counterparts are marked by the expression of CC-chemokine receptor 2 (CCR2,
receptor for monocyte chemotatic proteins (MCPs) and eotaxin), CX C-chemokine receptor 13
(CX CR1, receptor for fractalkine), CD11c and GR1 (Ly6). ‘Inflammatory’ monocytes, which3
+ low - +are released from bone marrow, are CCR2 CX CR1 CD11c GR1 . In the absence of in-3
flammation or any other stimulus, these precursor cells develop into resident monocytes
- high + -(CCR2 CX CR1 CD11c GR1 ) that enter the tissue and differentiate to tissue-resident3
macrophages (Gordon et al. 2005). Macrophage colony-stimulating factor (M-CSF) and
granulocyte-macrophage colony-stimulating factor (GM-CSF) are key factors stimulating the
proliferation and function of mononuclear phagocytes (Jones et al. 1989). Both factors are
produced by fibroblasts and endothelial cells in response to inflammatory stimuli, but also by
macrophages themselves after stimulation with endotoxin, and follow the release of interleu-
kin 1 (IL-1) and IL-3, which are general stimulators of hematopoiesis. Thus, the tissue macro-
phage pool is replenished by circulating monocytes as well as by local proliferation.Introduction 5
1.1.1.3. Markers for embryonic/fetal and adult macrophages
In the last 20 years, many researchers focused on markers to distinguish between the embry-
onic/fetal and the adult macrophage subtypes. A comprehensive set of molecular markers for
the developmental states of embryonic/fetal and adult macrophages (Hume et al. 1983; Faust
et al. 1997; Lichanska et al. 1999; Palis et al. 1999; Lichanska et al. 2000; Lieschke et al.
2001; Bertrand et al. 2005; Gordon et al. 2005; Hume 2006) complements established light-
microscopic and ultrastructural morphological properties of macrophages (Cline et al. 1972;
Takahashi et al. 1989; Morioka et al. 1994; El-Nefiawy et al. 2002). Table 1 summarizes the
main characteristics of the different macrophage subtypes. With proceeding developmental
state, nuclei and chromatin as well as all organelles become more highly structured. Plasma
membrane ruffling indicates increasing endocytic and secretory activity. With the onset of
liver hematopoiesis, macrophages express the ETS-transcription factor Pu.1, the S100 calcium
binding protein A8 (S100A8), endogenous lysozyme, and F4/80. Thus, they can be clearly
distinguished from yolk sac-derived macrophages that lack the expression of the mentioned
markers.
Table 1 Characteristics of murine macrophage subtypes
-: negative in staining or function; +: positive in staining or function; dpc: days post coitum; ER: en-
doplasmatic reticulum; rER: rough endoplasmatic reticulum
yolk-sac yolk-sac yolk-sac fetal liver
characteristic adult
(9 dpc) (10/11 dpc) (12 dpc) (14 dpc)
one or two, large
nucleoli/marginated eccentric/ chromatin in eccentric/ chromatin in
nucleus/chromatin large/fine chromatin round or oblong/ chromatin fine clumps fine clumps
euchromatic
rough; high rate of
cell surface smooth deep invaginations ruffled ruffled
vesicle formation
small amount of
large mitochondria; large mitochondria; well-
mitochondria and mitochondria well-developed Golgi developed Golgi
Golgi apparatus,
organelles scarce elongated or circular; apparatus in apparatus in
short flattened granular ER juxtanuclear position; juxtanuclear position;
saccules;scarce
scattered strands of rER scattered strands of rER
rER
vesicles; vacuoles; vesicles, vacuoles,
vesicles; primary primary and
pinocytic vesicles; pinocyic vesicles, vesicles/lysosomes - and secondary membrane-bound
membrane-bound membrane-bound
lysosomes inclusions of debris
lysosomes lysosomes
zymosan phagocytosis - - - + +
latex bead phagocytosis + + + + +
peroxidase activtiy - - - + -/+
proliferative capacity + + + + -/(+)
secretory activity - - + + +
F4/80 - -/+ -/+ + +
CD115 - + + + +
S100A8 - - - + +
CD24a + + + + +
CD24a inducibility low low low low high
BM8 - + + + +
IgG receptor - + + + +
Pu.1 - - - + +
lysozyme M - - - + +

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