Gestaltung des Titelblattes

Immunphänotypische Charakterisierung CD11c-positiver Zellen des Gehirns im
direkten Vergleich zu CD11c-positiven Zellen von Lunge, Leber und Milz
Dissertation
zur Erlangung des akademischen Grades
Dr. rer. med.
an der Medizinischen Fakultät
der Universität Leipzig
eingereicht von:
Kerstin Immig
geb. am 11.08.1983 in Leipzig
angefertigt an der
Medizinischen Fakultät der Universität Leipzig
am Institut für Anatomie
Betreuer: Herr Prof. Dr. med. Ingo Bechmann
Beschluss über die Verleihung des Doktorgrades vom: 23.03.2016
2
Bibliografische Beschreibung:
Immig, Kerstin
Immunphänotypische Charakterisierung CD11c-positiver Zellen des Gehirns im direkten
Vergleich zu CD11c-positiven Zellen von Lunge, Leber und Milz
Universität Leipzig, Dissertation
71 S.1, 101 Lit.2, 3 Abb., 2 Publikationen
Referat
Bei der vorliegenden Arbeit handelt es sich um eine experimentell durchgeführte
Charakterisierung von CD11c-positiven Zellen des Gehirns im direkten Vergleich zu CD11cpositiven Zellen aus Lunge, Leber und Milz. Mittels Konfokal- und Fluoreszenzmikroskopie
wurde die Existenz von intraparenchymalen Zellen nachgewiesen, welche den Zellmarker für
Dendritische Zellen CD11c exprimieren. Durch die Etablierung einer einheitlichen
Isolierungsmethode von CD11c-positiven mononukleären Zellen aus dem Gehirn, Milz,
Lunge und Leber, war es uns möglich, diese mittels Durchflusszytometrie, auf die Expression
wichtiger Marker für mononukleäre Zellen zu untersuchen und phänotypisch miteinander zu
vergleichen. Durch diese Zellanalysen zeigten wir, dass CD11c-positive Zellen des Gehirns
sowohl aufgrund ihrer spezifischen CD45-Expression, als auch durch die Expression von
CD11b einen Mikrogliaphänotyp aufwiesen. Dabei konnten wir beobachten, dass CD11cpositive Zellen aus dem Gehirn einzigartig in der Eigenschaft ihrer geringen Major
Histocompatibility Complex (MHC)-II-Expression sind. Mit Hilfe einer transgenen Mauslinie,
welche unter dem Promotor von MHC-II das grün-fluoreszierende Protein (GFP) exprimiert,
konnten wir nachweisen, dass Mikroglia selbst in der Umgebung von MHC-II-positiven
Zellen, kultiviert auf Schnittkulturen der Milz, ihre MHC-II-Negativität behalten. Im Vergleich
dazu adaptierten sich MHC-II-positive Splenozyten, kultiviert auf Schnittkulturen vom
Hippocampus, an die neue Umgebung und verringerten die Expression von MHC-II.
Unsere Daten lassen also die Schlussfolgerung zu, dass sich CD11c-positive Mikroglia
hinsichtlich ihrer Expression von MHC-II intrinsisch von CD11c-positiven Zellen anderer
Organe unterscheiden. Ebenso scheinen auch lokale Faktoren im Gehirn dazu beizutragen,
die
Expression
von
MHC-II
unter
physiologischen
unterdrücken.
________________
1 Seitenzahl
2 Zahl
insgesamt
der im Literaturverzeichnis der Einleitung ausgewiesenen Literaturangaben
Bedingungen
wirkungsvoll
zu
3
Inhaltsverzeichnis
1 Einleitung
4
1.1 Das Gehirn als immunprivilegiertes Organ
4
1.1.1 Das Immunprivileg und der fehlende lymphatische Abfluss
4
1.1.2 Das Immunprivileg und die Kompartimentierung im Gehirn
5
1.1.3 Das Immunprivileg und der afferente und efferente Arm des
Immunsystems
6
1.1.4 Das Immunprivileg und die Bluthirnschranke
6
1.1.5 Das Immunprivileg und das Fehlen von Dendritischen Zellen im Gehirn
9
1.2 Neuroinflammation
9
1.2.1 Multiple Sklerose
11
1.3 Myeloide Zellen des ZNS
13
1.3.1 Dendritische Zellen
14
1.3.2 Makrophagen
15
1.3.3 Mikroglia
15
1.4 CD11c als Marker für Dendritische Zellen
17
2 Fragestellung
19
3 Literaturverzeichnis der Einleitung
20
4 Publikationen
30
4.1 CD11c-expressing cells reside in the juxtavascular parenchyma and extend
processes into the glia limitans of the mouse nervous system
30
4.2 CD11c-positive cells from brain, spleen, lung and liver exhibit site-specific
immune phenotypes and plastically adapt to new environments
45
5 Zusammenfassung
61
6 Summary
65
7 Danksagung
68
8 Erklärung über die eigenständige Abfassung der Arbeit
69
9 Curriculum vitae
70
4
1 Einleitung
1.1 Das Gehirn als immunprivilegiertes Organ
Die verzögerte oder unterdrückte Reaktion des angeborenen und adaptiven Immunsystems,
zum Beispiel auf Fremdantigene eines Virus oder allogenetische Transplantate, bezeichnet
man als Immunprivileg und stellt somit eine besondere immunologische Situation dar.
Immunprivilegierte Orte im Körper sind zum Beispiel die vordere Augenkammer, der Hoden
oder die Plazenta, aber auch das Gehirn zählt zu den immunprivilegierten Organen.
Der
Begriff
„Immunprivileg“
wurde
erstmals
aufgrund
von
frühen
Transplantationsexperimenten von Billingham und Boswell verwendet. Deren Definition des
Immunprivilegs: „immunologically privileged, i.e., grafts transplanted to them are in some way
partially or fully exempted from the normal rigors imposed by their histoincompatible status.”,
beschreibt die Toleranz gegenüber Transplantaten (Billingham und Boswell, 1953). Schon in
den zwanziger Jahren des letzen Jahrhunderts wurde von Shirai eine immunologische
Barriere zwischen dem Gehirn und dem peripheren Immunsystem vermutet. Er beobachtete
ein starkes Tumorwachstum, nachdem er Tumorzellen aus Ratten in das Hirnparenchym von
Mäusen transplantiert hatte. Bei einer Transplantation von Tumorzellen aus Ratten
außerhalb des Gehirns, in Muskelgewebe oder in die Haut, beobachtete er kein Wachstum
(Shirai, 1921). Ebenfalls kam es zu keinem Tumorwachstum bei einer simultanen
Transplantation des Tumorgewebes in das Hirnparenchym und in die Milz (Murphy und
Sturm, 1923). Auch Medawar zeigte im Kaninchen, dass es zu keiner Abstoßungsreaktion
von Hauttransplantaten im Hirnparenchym und in der vorderen Augenkammer kam. Erst als
er die Haut zusätzlich in die Bauchhaut transplantierte, konnte er eine immunologische
Reaktion gegen die Transplantate beobachten. Daraus schlussfolgerte er, dass Antigene im
Gehirn und in der vorderen Augenkammer einer Immunantwort unterliegen können, aber sie
nicht selbst auslösen (Medawar, 1948).
1.1.1 Das Immunprivileg und der fehlende lymphatische Abfluss
Nachdem man das Phänomen des Immunprivilegs im zentralen Nervensystem (ZNS)
beobachtet hatte, wurden verschiedene Theorien entwickelt, dies zu erklären. Aufgrund
seiner oben beschriebenen Beobachtungen schlussfolgerte Medawar das Fehlen eines
lymphatischen Abfluss aus dem Hirnparenchym und in der vorderen Augenkammer
(Medawar, 1948).
Später zeigte man, dass eine Verbindung zwischen dem Gehirn und dem lymphatischen
System existiert. So besteht eine Verbindung über die Zerebrospinalflüssigkeit, die
arachnoidalen Villi, das durale Sinusblut und den Subarachnoidalraum, entlang der
olfaktorischen Nerven durch die Lamina cribrosa zur nasalen Submukosa (Andres et al.,
5
1987; Cserr und Knopf, 1992; Ichimura et al., 1991; Kida et al., 1993). Von der nasalen
Submukosa besteht ein Zugang zu den terminalen Lymphgefäßen, über welche Substanzen
aus dem Gehirn über einen aktiven oder passiven Transport zu den Lymphknoten gelangen
können (Cserr und Knopf, 1992).
1.1.2 Das Immunprivileg und die Kompartimentierung im Gehirn
Das Gehirn unterteilt sich in das Parenchym, den Hirnhäuten und den ventrikulären Räumen
mit dem Plexus choroideus und der Zerebrospinalflüssigkeit (Abbildung 1) (Galea et al.,
2007). Das Immunprivileg ist jedoch nicht in allen diesen Kompartimenten nachweisbar. So
beobachtete man eine Abstoßung von Maustumorgewebe, wenn dies in die Hirnventrikel von
Ratten transplantiert wurde. Injizierte man das Tumorgewebe direkt in das Hirnparenchym,
konnte keine Immunantwort ausgelöst werden (Murphy und Sturm, 1923). Auch
allogenetische Transplantate riefen eine Abstoßungsreaktion hervor, wenn diese in die
Ventrikel injiziert wurden (Mason et al., 1986). Ähnliche Beobachtungen wurden nach
Injektionen von bakteriellem und viralem Material in die Ventrikel gemacht. So konnte nur
eine Immunantwort bei einer Injektion in die Ventrikel ausgelöst werden, jedoch nicht bei
einer Transplantation in das Hirnparenchym (Matyszak und Perry, 1998; Stevenson et al.,
1997).
Abbildung
1:
Überblick
(Galea et al., 2007)
über
die
Kompartimente
des
Gehirns
Die verminderte Immunreaktion auf Fremdantigene im Hirnparenchym wurde auch für die
angeborene Immunantwort gezeigt. So führte eine intraventrikuläre Injektion von
Lipopolysacchariden zu einer Rekrutierung von neutrophilen Granulozyten und Monozyten
innerhalb von 2 h, ähnlich wie sie bei einer subkutanen Injektion beobachtet werden konnte.
Wurde jedoch die gleiche Dosis in das Hirnparenchym injiziert, konnte keine Rekrutierung
dieser Zellen beobachtet werden (Andersson et al., 1992).
6
1.1.3 Das Immunprivileg und der afferente und efferente Arm des Immunsystems
Der afferente Arm einer Immunantwort umfasst den Transport von Antigenen zu den
Lymphknoten und die Antigenpräsentation für naive T-Zellen sowie T-Gedächtniszellen.
Daraufhin erfolgt eine Aktivierung und Proliferation der antigenspezifischen T-Zellen (Galea
et al., 2007). Die für die Aktivierung notwendigen Antigene können aktiv mit Hilfe von Zellen,
den antigenpräsentierenden Zellen, zu den Lymphknoten transportiert werden. Ein anderer
Weg ergibt sich aus den sogenannten „löslichen“ Antigenen, welche vom perivaskulären
Raum in die Subarachnoidalräume und, wie bereits oben beschrieben, entlang des Nervus
olfaktorius durch die Lamina cribrosa gelangen (Galea et al., 2007). Von dort erreichen die
Antigene über die nasale Submukosa die drainierenden Lymphbahnen (Ichimura et al.,
1991).
Der efferente Arm einer Immunantwort des ZNS beschreibt den Zufluss von peripheren
Immunzellen, wie zum Beispiel Monozyten, neutrophile Granulozyten und T-Zellen zum
Gehirn (Cserr und Knopf, 1992; Galea et al., 2007). Unter gesunden Bedingungen ist diese
Migration in das Hirnparenchym stark reguliert. So wurde gezeigt, dass infiltrierende T-Zellen
zur Apoptose gezwungen werden können, welche über den Todesliganden FasL initiiert wird.
Die Expression von FasL konnte auf Neuronen, Mikroglia der weißen Substanz und
Astrozyten, welche in der Nähe von Blutgefäßen lokalisiert sind, nachgewiesen werden
(Bechmann et
al., 1999). Weiterhin scheint
die geringe Expression von
Major
Histocompatibility Complex (MHC)-II ein limitierender Faktor für die MHC-II-abhängige
Erkennung des spezifischen Antigens im Gehirn zu sein (Perry, 1998). Auch die Expression
der co-stimulatorischen Moleküle CD80 und CD86 hat einen wichtigen Einfluss auf die MHCII-abhängige Antigenpräsentation. So wurde beschrieben, dass CD80 die Proliferation
inflammatorischer T-Helfer-Zellen fördert, wohingegen CD86 die Proliferation schützender THelfer-Zellen unterstützt und eine Toleranz im Gehirn gegenüber dem Antigen entwickelt
werden konnte (Kuchroo et al., 1995). Am Beispiel der Experimente von Medawar als auch
von Matyszak und Perry wurde gezeigt, dass eine Immunantwort im Gehirn ausgelöst
werden kann, wenn die Transplantate in das Gehirn als auch in die Haut verpflanzt wurden
(Matyszak und Perry, 1996; Medawar, 1948). Daraus schlussfolgernd gibt es einen Zufluss
von peripheren Immunzellen in das Gehirn, welcher jedoch streng reguliert wird.
1.1.4 Das Immunprivileg und die Bluthirnschranke
Die Bluthirnschranke (BHS) beschreibt den fehlenden Übergang bestimmter Stoffe vom Blut
in das ZNS. Erste Beobachtungen wurden von Paul Ehrlich im Rahmen von Studien über
das Sauerstoffbedürfnis verschiedener Organe im Jahre 1885 beschrieben, als er bei der
Injektion von hydrophilen Farbstoffen beobachtete, dass sich das Gehirn und das
Rückenmark nicht anfärben ließen (Ehrlich, 1885). In einer späteren Arbeit wurden die
7
Kapillarwände als eine der Barrieren identifiziert, welche den Übertritt bestimmter Stoffe in
das ZNS verhinderten (Lewandowski, 1900). Später prägte Goldmann den Begriff der
physiologischen Grenzmembran. So beschrieb er, dass sich der hinter der Plazenta liegende
Fetus sowie fett- und glykogenspeichernde Zellen nicht von bestimmten Farbstoffen
anfärben ließen. Daraus schlussfolgerte er, dass physiologische Grenzmembranen und
deren Fähigkeit zur Phagozytose diese Gewebe von anderen abschirmen. In weiteren
Untersuchungen stellte er jedoch fest, dass das Gehirn nicht komplett von der Umgebung
abgeschirmt ist und sich Zellen des Plexus choroideus, der weichen Hirnhäute und entlang
der perivaskulären Räume mit Farbstoffen anfärben ließen. Die perivaskulären Räume
interpretierte er als Lymphräume. Da die Farbstoffe in Zellen eingeschlossen waren,
mutmaßte er, dass diese Zellen zur Phagozytose und Migration fähig sind. Diese
perivaskulären Phagozyten wurden seither mehrfach beschrieben (Angelov et al., 1998;
Fabriek et al., 2005). Weiterhin vermutet man, dass einige der perivaskulären Zellen zur
Antigenpräsentation fähig sind und daher eine entscheidende Funktion für den Ausbruch
autoimmuner Erkrankungen haben (Greter et al., 2005; Hickey und Kimura, 1988). Mit Hilfe
von elektronenmikroskopischen Studien konnte in einer weiteren Arbeit gezeigt werden, dass
das
morphologische
Korrelat
der
BHS
bestimmte
Verschlusskontakte
zwischen
benachbarten Endothelzellen in Form von Tight junctions (TJ) sind. Die Autoren
schlussfolgerten, dass diese den Durchtritt bestimmter Stoffe aus der Blutbahn in das Gehirn
zwischen benachbarten Endothelzellen verhindern (Reese und Karnovsky, 1967).
Abbildung 2: Topografie der neurovaskulären Einheit (Krueger und Bechmann, 2010)
Weitere Arbeiten zeigten zudem, dass nicht nur das Endothel, sondern auch benachbarte
Zellpopulationen gleichermaßen an der Aufrechterhaltung der Funktion der BHS beteiligt
sind und prägten daher den Begriff der neurovaskulären Einheit (Abbildung 2). Diese besteht
8
aus drei Kompartimenten: der Gefäßwand, dem perivaskulären Raum und dem
juxtavaskulären Neuropil. Alle drei Kompartimente sind durch Basalmembranen voneinander
getrennt (Bechmann et al., 2001a). Die Gefäßwand kann aus Endothelzellen, Perizyten und,
außerhalb des Kapillarbettes, glatten Muskelzellen bestehen. Der perivaskuläre Raum, auch
Virchow-Robin-Raum genannt, enthält perivaskuläre Flüssigkeit und perivaskuläre Zellen
(Zellen der weichen Hirnhäute und perivaskuläre Makrophagen). Das dritte Kompartiment
besteht aus dem Neuropil bzw. dem Hirnparenchym. Dieses wird durch die Glia limitans
begrenzt, welche sich hauptsächlich aus den Endfüßen der Astrozyten und wenigen
juxtavaskulären Mikrogliazellen zusammensetzt (Bechmann et al., 2007; Krueger und
Bechmann, 2010). Alle Komponenten der neurovaskulären Einheit haben einen Einfluss auf
die Funktion der BHS und die Aufrechterhaltung der Homöostase im ZNS (Weiss et al.,
2009).
Da der Begriff der BHS parallel mit den verstärkten Untersuchungen des Immunprivilegs des
Gehirns aufkam, wurde versucht das Immunprivileg mit dem Phänomen der BHS zu erklären
(Barker und Billingham, 1977). Unter physiologischen Bedingungen kommt es zu einer
kontrollierten Migration von Leukozyten in das Gehirn (Hickey, 2001), welche sich unter
inflammatorischen Bedingungen stark erhöht (Ajami et al., 2011; Priller et al., 2001; Wekerle,
1993). Ebenso konnte bei einer Transplantation von neuronalem Mausgewebe in Rattenhirn
eine Infiltration von T-Zellen und Makrophagen aus der Peripherie in das Transplantat 35
Tage nach der Transplantation beobachtet werden (Finsen et al., 1991). Das Immunprivileg
lässt sich jedoch nicht vollständig mit den Beobachtungen der BHS erklären. So wurde
gezeigt, dass Leukozyten hauptsächlich über postkapilläre Venulen in das Gehirnparenchym
migrieren und nicht über die Kapillaren, an welchen die TJ besonders ausgeprägt sind
(Raine et al., 1990). Darüber hinaus konnte eine transzelluläre Migration von Leukozyten in
das Gehirn beschrieben werden (Engelhardt und Ransohoff, 2005; Wolburg et al., 2005).
Weiterhin wurde belegt, dass die Infiltration von Leukozyten nicht mit dem Ort der größten
Bluthirnschrankenpermeabilität korreliert (Muller et al., 2005). Im Modell einer anterograden,
axonalen Degeneration konnte gezeigt werden, dass es zu keinem Einstrom der
Permeabilitätsmarker Meerrettichperoxidase oder Evans Blau in das Neuropil kam (Jensen
et al., 1997). Beide Substanzen sind häufig benutzte Marker um die Durchlässigkeit der BHS
bzw. von Gefäßen zu kontrollieren. Zusätzlich konnte im gleichen Tiermodell gezeigt werden,
dass es unter diesen inflammatorischen Bedingungen jedoch zu einer Infiltration von TZellen kommt (Bechmann et al., 2001b). Ebenfalls konnte gezeigt werden, dass zirkulierende
Monozyten
unter
dem
Einfluss
einer
anterograden,
axonalen
Degeneration
im
Hirnparenchym eine mikrogliaähnliche Form annehmen können (Bechmann et al., 2005).
Desweiteren muss man bei den Beschreibungen über die Leukozytenrekrutierung in das
Gehirn
beachten,
dass
postkapilläre
Venulen
vom
Neuropil
durch
ein
weiteres
9
Kompartiment, den perivaskulären Raum, getrennt sein können. Auch unter gesunden
Bedingungen konnte mit der intraventrikulären Injektion von verschiedenen fluoreszierenden
Farbstoffen, welche über Phagozytose aufgenommen werden, periphere Makrophagen im
perivaskulären Raum in Ratten beobachtet werden, allerdings nicht im Parenchym. Auch bei
der Reinjektion von markierten Makrophagen in die Schwanzvene konnte eine Migration von
Makrophagen aus der Peripherie in den perivaskulären Raum gezeigt werden. (Bechmann et
al., 2001a). Von daher ist der Begriff der BHS, welche den Übertritt bestimmter Stoffe vom
Blut in das ZNS verhindert, nicht mit dem Mechanismus der Migration von Leukozyten aus
der Peripherie in das ZNS gleichzusetzen.
1.1.5 Das Immunprivileg und das Fehlen von Dendritischen Zellen im Gehirn
Eine weitere Theorie zur Erklärung des Immunprivilegs im Gehirn bezieht sich auf das
Fehlen
von
Dendritischen
Zellen
(DCs)
im
ZNS
(Sedgwick,
1995).
DCs
sind
antigenpräsentierende Zellen, welche Antigene am Ort der Entzündung aufnehmen, zu den
assoziierten Lymphknoten migrieren, um dort T-Zellen zu aktivieren. Auch diese Sichtweise
wurde jedoch in Frage gestellt nachdem im gesunden Rattenhirn DCs sowohl in den
Meningen als auch im Plexus choroideus gefunden wurden (Matyszak und Perry, 1996;
McMenamin, 1999; McMenamin et al., 2003). Man nimmt jedoch an, dass die Anzahl von
DCs im Gehirn unter gesunden Bedingungen sehr gering ist. Ein vielfach verwendeter
Marker für die Erkennung von DCs ist das Integrin CD11c. Da immunhistochemische
Untersuchungen und derzeit verfügbare Antikörper kaum positive Färbungen im Gehirn
zeigten, war es schwer DCs unter gesunden Bedingungen im ZNS und speziell im
Hirnparenchym und im perivaskulären Raum nachzuweisen. Mit der Entwicklung von
transgenen Mausmodellen, welche unter dem Promotor von CD11c (itgax) das grünfluoreszierende Protein (GFP) (Jung et al., 2002) oder das gelb-fluoreszierende Protein
(YFP) (Lindquist et al., 2004) exprimieren, war es nun möglich, diese Zellen unter
physiologischen Bedingungen zu untersuchen und die Verteilung im Gehirn zu analysieren
(Bulloch et al., 2008; Prodinger et al.,2011; Wieghöfer et al., 2014). Benutzt man also CD11c
als alleinigen DC-Marker, so lässt sich das Immunprivileg des ZNS nicht mit dem Fehlen von
DCs erklären, da man CD11c-exprimierende Zellen im perivaskulären Raum als auch im
Parenchym beobachten konnte.
1.2 Neuroinflammation
Unter inflammatorischen Bedingungen kommt es zu einem erhöhten Aufkommen von
Leukozyten wie DCs, neutrophilen Granulozyten, T-Zellen und B-Zellen im Gehirn (Matyszak
und Perry, 1996). Durch den perivaskulären Raum zwischen Gefäßwand und dem Neuropil
ist die Leukozyteninfiltration aus der Peripherie in den postkapillären Gefäßen in zwei
10
Schritte unterteilt. Zuerst muss die innere und äußere Basalmembran der Gefäßwand
durchquert werden, um in den perivaskulären Raum zu gelangen. Viele Leukozyten
verbleiben auch unter inflammatorischen Bedingungen in diesem Kompartiment. Weiterhin
ist der CC-Chemokin-Ligand (CCL2), welcher an den Rezeptor CCR2 bindet, wichtig für die
Migration durch die Gefäßwand in den perivaskulären Raum. Neben der Aufgabe als
chemischer Lockstoff für periphere Leukozyten, kann
die Bindung von CCL2 an den
Rezeptor eine Umverteilung der TJ-Proteine und Änderungen des Aktinzytoskelettes von
Endothelzellen bewirken (Babcock et al., 2003; Semple et al., 2010; Toft-Hansen et al.,
2006). Mäuse welche kein CCR2 oder CCL2 exprimierten, zeigten eine verringerte Migration
von infiltrierenden Leukozyten als auch keine oder abgeschwächte Symptome bei der
experimentellen Autoimmunenzephalomyelitis (EAE) (Huang et al., 2002; Semple et al.,
2010).
In einem zweiten Schritt muss die Glia limitans passiert werden, welche sich hauptsächlich
aus den astrozytären Endfüßen, einigen juxtavaskulären Mikroglia und der angrenzenden
Basalmembran zusammensetzt. Die Basalmembranen der Gefäßwand unterscheiden sich in
ihrem molekularen Aufbau von der Basalmembran der Glia limitans. So enthält nur die
Basalmembran der Glia limitans die Isoformen Laminin 1 und 2 (Owens et al., 2008; Sixt et
al., 2001). Diese Beobachtung könnte erklären, warum infiltrierende Leukozyten die
Gefäßwand durchqueren können, aber nicht die Glia limitans und im perivaskulärem Raum
verbleiben.
In
weiteren
Studien
konnte
gezeigt
werden,
dass
auch
die
Matrixmetalloproteinasen (MMP) 2 und 9 benötigt werden, um eine Neuroinflammation
auszulösen (Agrawal et al., 2006). MMP 2 und 9 besitzen eine hohe Affinität zu
Dystroglykan, welches Laminin 1 und 2 verankert und so die astrozytären Endfüße mit der
Basalmembran der Glia limitans verbindet. Transgene Mäuse, welche kein MMP 2 und 9
exprimierten, entwickelten keine EAE, da die Bindung von Dystroglykan durch MMP 2 und 9
verhindert wurde und es so zu keiner Leukozyteninfiltration in das Neuropil kam (Agrawal et
al., 2006). In weiteren Arbeiten in der Maus konnte gezeigt werden, dass auch perivaskuläre
Makrophagen eine wichtige Funktion bei der Entwicklung einer Neuroinflammation haben.
Durch die Depletion der perivaskulären Makrophagen kam es nach einer Induktion einer
EAE zwar zu einer Ansammlung von Leukozyten im perivaskulären Raum, aber nicht zu
einer Infiltration in das Neuropil. Zudem waren keine klinischen Symptome einer EAE
sichtbar (Tran et al., 1998). Die gleiche Arbeitsgruppe benutzte Pertussistoxin, um eine
Entzündung in Mäusen auszulösen, welche CCL2 überexprimieren. In diesem Mausmodell
kommt
es
durch
die
Injektion
von
Pertussistoxin
zu
einer
Erhöhung
von
proinflammatorischen Zytokinen, welche dann zu einer Enzephalopathie führen. Dabei
beobachteten sie eine Infiltration der Leukozyten in das Hirnparenchym. Bei einer
Behandlung
dieser
Mäuse
mit
einem
Matrixmetalloproteinaseninhibitor,
nach
der
11
Behandlung mit Pertussistoxin, konnte nur eine Leukozyteninfiltration in den perivaskulären
Raum
beobachtet
werden,
aber
nicht
in
das
Neuropil.
Ebenso
waren
keine
inflammatorischen Symptome in diesen Mäusen erkennbar (Toft-Hansen et al., 2006). Diese
Arbeiten zeigen, dass MMPs, welche wahrscheinlich von perivaskulären Makrophagen oder
T-Zellen gebildet werden (Agrawal et al., 2006), eine wichtige Rolle bei der Durchführung
des zweiten Schrittes der Neuroinflammation und der eigentlichen Entstehung von klinischen
Symptomen spielen.
Des Weiteren ist in diesem Kontext zu erwähnen, dass auch andere Regulationssysteme die
Leukozyteninfiltration aus dem perivaskulären Raum in das Hirnparenchym beeinflussen
können. So wurde für den Todesliganden FasL gezeigt, dass dieser einen Einfluss auf die
Infiltration in das Hirnparenchym hat (Bechmann et al., 1999; Sabelko-Downes et al., 1999).
Wie oben beschrieben, sind Mikroglia ein wichtiger Bestandteil des Neuropils und werden
auch als Makrophagen des Gehirns bezeichnet. Unter stabilen Bedingungen zeigen sie eher
einen
antiinflammatorischen
Phänotyp.
Jedoch
konnte
auch
bei
ihnen
unter
inflammatorischen Konditionen ein Anstieg von Zellmarkern beschrieben werden, die eher
als proinflammatorisch angesehen werden (Carson et al., 1998; Matyszak und Perry, 1998).
1.2.1 Multiple Sklerose
Multiple Sklerose (MS) ist mit rund 2,5 Millionen Betroffenen eine der häufigsten
Autoimmunerkrankungen weltweit, welche mit einer Demyelinisierung im ZNS einhergeht
(Goldmann und Prinz, 2013; Ortiz et al., 2014; Ransohoff, 2012). Die Krankheit kann das
Gehirn, das Rückenmark und den Nervus opticus betreffen. Im Falle von MS ist die
Autoreaktivität meistens gegen das basische Myelinprotein (MBP), das Proteolipid-Protein
(PLP) oder das Myelin-Oligodendrozyten-Glykoprotein (MOG) gerichtet. Die klinischen
Symptome dieser Erkrankung sind sehr heterogen und zeigen sich unter anderem in
Sehstörungen,
Störungen
der
Motorik,
Störungen
der
Koordination,
Blasen-
und
Darminkontinenz, Spastiken, Störungen der Sensorik, Störungen der Propriozeption und der
Sprache (Ortiz et al., 2014; Steinman, 1996).
Durch die Infiltration von autoreaktiven, myelinspezifischen T-Zellen in das ZNS kommt es zu
einer T-Zell-vermittelten Immunantwort, welche mit einer weiteren Rekrutierung von
peripheren Makrophagen und einer Aktivierung der Mikroglia einhergeht (Lassmann et al.,
2012). Die Interaktion der T-Zellen mit antigenpräsentierenden Zellen im ZNS führt zu einer
Expression von weiteren löslichen Faktoren wie Zytokinen, Chemokinen und Proteasen,
welche eine weitere Infiltration von inflammatorischen autoreaktiven Leukozyten und die
Umstrukturierung der BHS zur Folge hat (Babcock et al., 2003; Ransohoff, 2012; Semple et
al., 2010; Toft-Hansen et al., 2006). Ebenso wurde in mehreren Publikationen gezeigt, dass
Myelinantigene in Lymphknoten von MS-Patienten und in Tiermodellen nachweisbar waren
12
(Locatelli et al., 2012; Mutlu et al., 2007; van Zwam et al., 2009). Daraus ergab sich die
Fragestellung, auf welchem Weg die Antigene die assoziierten Lymphknoten erreichen
können. Hierfür werden zwei Möglichkeiten diskutiert. Zum einem ist dies der passive
Transport über die Zerebrospinalflüssigkeit und zum anderen der aktive Transport über DCs.
In ersten Arbeiten wurden bereits 12 h nach der Injektion von T-Zellen und Monozyten in das
Areal einer entorhinalen Kortexläsion erste injizierte Leukozyten in den zervikalen
Lymphknoten beobachtet (Goldmann et al., 2006; Kaminski et al., 2012). Dies unterstützt
wiederum die Annahme, dass ein zellulärer Transport ZNS-spezifischer Antigene möglich
sein könnte, um drainierende Lymphknoten zu erreichen.
Die Überwindung der BHS ist ein wichtiger Prozess bei der Entwicklung einer
Neuroinflammation. Wie bereits oben beschrieben sind MMP 2 und 9 wichtig für die Passage
der Glia limitans und deren Basalmembran (Agrawal et al., 2006; Toft-Hansen et al., 2006).
Diese können von T-Zellen, Makrophagen und Mikroglia gebildet werden (Agrawal et al.,
2006; Correale und Villa, 2007; Ortiz et al., 2014). Auch Chemokine fördern das Passieren
der BHS von T-Zellen. So spielt CCL2, wie bereits unter 1.2 beschrieben, beim Passieren
des Gefäßendothels und deren Basalmembranen eine Rolle (Babcock et al., 2003; ToftHansen et al., 2006). So wurde gezeigt, dass das Chemokin CCL2 von Astrozyten und
Mikroglia gebildet werden kann (Babcock et al., 2003). Der dazu gehörige Chemokinrezeptor
konnte auf infiltrierenden Leukozyten, Mikroglia, Makrophagen und Endothelzellen
nachgewiesen werden (Correale und Villa, 2007; Semple et al., 2010). Weiterhin werden
inflammatorische Zytokine von T-Zellen, Makrophagen, Astrozyten und Mikroglia gebildet,
wie zum Beispiel Interferon-ϒ, Tumornekrosefaktor-α, Interleukin-23 und Interleukin-17.
Diese können neurotoxisch wirken und haben eine weitere Rekrutierung inflammatorischer
Leukozyten aus der Peripherie zur Folge (Ortiz et al., 2014; Ransohoff, 2012).
Grundsätzlich wird die MS in zwei Subtypen unterteilt. Zum einen gibt es die langsamere
Verlaufsform, bei welcher eine primäre intermittierende Form einer sekundären progressiven
Verlaufsform voran geht. Bei der zweiten Form der MS gibt es nur einen progressiven
Verlauf (Prinz et al., 2011; Ransohoff, 2012). Therapiemaßnahmen bestehen hauptsächlich
aus der Verabreichung von antiinflammatorischen Substanzen wie Interferon-ß, welche zu
einem langsameren Krankheitsverlauf führen. Trotz aller Bemühungen, ist die Krankheit bis
heute nicht heilbar.
Die EAE ist ein Tiermodell der MS und simuliert die progressive Verlaufsform dieser
Erkrankung. Die verschiedenen EAE-Formen werden häufig in Nagetieren wie Ratten oder
Mäusen ausgelöst (Constantinescu et al., 2011; Ransohoff, 2012), um Störungen der
Motorik, inflammatorische Demyelinisierung und Gewichtsverlust zu untersuchen. Es gibt
zwei Arten eine EAE zu induzieren. Zum einen besteht die Möglichkeit, gegen die Antigene
MBP, PLP oder MOG voraktivierte T-Zellen intravenös zu injizieren oder direkt Peptide von
13
MBP, PLP oder MOG in Kombination mit dem vollständigen Freudschen Adjuvans (CFA),
einer Emulsion welche das abgetötete Mycobacterium tuberculosis enthält. Durch die
Bindung des CFA von den Toll-Like-Rezeptoren kommt es zu einer Immunreaktion gegen
das gespritzte, körpereigene Antigen. Ungefähr ab dem 10. Tag zeigen sich die ersten
klinischen Symptome, welche sich in einer aszendierenden Lähmung äußern.
1.3 Myeloide Zellen des ZNS
Eine wesentliche Komponente des Immunsystems im ZNS sind myeloide Zellen, welche eine
besondere Rolle für das Immunprivileg im Gehirn einnehmen, aber auch in der Pathogenese
verschiedenster neuroimmunologischer Erkrankungen (Prinz et al., 2011). Alle myeloiden
Zellen
werden
im
mononukleären
phagozytierenden
System
(Abbildung
3)
zusammengefasst.
Abbildung 3: Überblick über
(Ransohoff und Cardona, 2010)
das
mononukleäre
phagozytierende
System
Eine der wichtigsten myeloiden Zellpopulationen des Gehirns ist die Mikroglia, deren
Vorläuferzellen zum größten Teil während der Embryogenese vom Dottersack in das
Gehirnparenchym migrieren (Ginhoux et al., 2010). Weitere myeloide Zellen im Gehirn sind
perivaskuläre, meningeale sowie aus dem Plexus choroideus stammende Makrophagen und
DCs (Bulloch et al. 2008, Greter et al. 2005, Prodinger et al. 2011, Ransohoff and Cardona
2010). Diese Zellen stammen von einer gemeinsamen hämatopoetischen Stammzelle (HSC)
14
aus dem Knochenmark ab. Daraus entwickelt sich ein gemeinsamer myeloider Vorläufer, der
Common myeloid progenitor (CMP). Aus dem CMP entsteht ein granulozytärer und
monozytärer Vorläufer, der Granulocyte monocyte progenitor (GMP), um sich anschließend
über den Monocyte dendritic cell progenitor (MDP) in Makrophagen, Monozyten und DCs zu
differenzieren (Bulloch et al. 2008, Greter et al. 2005, Prodinger et al. 2011, Ransohoff and
Cardona 2010). Unter bestimmten inflammatorischen Bedingungen können Monozyten aus
der Peripherie in das Hirnparenchym migrieren, sich der Morphologie der Mikroglia anpassen
und deren Aufgaben übernehmen (Bechmann et al., 2005; Varvel et al. 2012).
1.3.1 Dendritische Zellen
DCs wurden erstmals im Jahr 1973 beschrieben (Steinman und Cohn, 1973) und erfüllen
eine Schlüsselfunktion in der Aktivierung zwischen dem angeborenen und adaptiven
Immunsystem. DCs sind spezialisierte antigenprozessierende und -präsentierende Zellen
(Satpathy et al., 2012). Generell unterscheidet man zwischen reifen und unreifen DCs.
Unter inflammatorischen Bedingungen kommt es durch verschiedene Stimuli wie zum
Beispiel durch inflammatorische Zytokine zur Reifung von DCs. Parallel kommt es zur
Aufnahme von Antigenen und Migration zu den Lymphknoten bzw. lymphatischen Organen.
Durch die Erhöhung der Expression der antigenpräsentierenden Moleküle MHC-I und II
sowie der T-Zell-bindenden und co-stimulatorischen Moleküle CD80 und CD86 auf der
Zelloberfläche, kommt es zu einer Veränderung des Zellphänotyps. Dabei bezeichnet man
die Präsentation der Antigene über die MHC-Moleküle als „Signal 1“ und die Stimulation der
T-Zellen über CD80 und CD86 als „Signal 2“. Im lymphatischen Gewebe kommt es dann zur
Präsentation der aufgenommenen Antigene, um dort naive T-Zellen zu aktivieren (Steinman
et al., 2003a; Steinman et al., 2003b). Auch unter nichtinflammatorischen Bedingungen
kommt den „unreifen“ DCs eine wichtige Funktion zu. So sind sie bedeutend für die
Vermittlung von Toleranz, indem sie T-Zellen depletieren und für die Proliferation von
regulatorischen T-Zellen verantwortlich sind (Mohammad et al., 2012; Steinman et al.,
2003b). Da durch sterbende körpereigene Zellen ständig körpereigene Antigene präsentiert
werden, ist diese Aufgabe essentiell, um einen Ausbruch von Autoimmunerkrankungen zu
vermeiden. Eine wichtige Rolle spielt dabei die periphere Toleranz, welche in der Peripherie
außerhalb des lymphatischen Gewebes vermittelt wird, da nicht alle reaktiven T-Zellen durch
zentrale Toleranz inaktiviert werden können. Auch unreife DCs können Antigene über die
MHC-Moleküle präsentieren. Allerdings kommt es dabei zu einer Veränderung des zweiten
Signals, welche eine Unterdrückung einer T-Zell-Antwort, eine T-Zell-Anergie, zur Folge hat
(Greenfield et al., 1998).
Zusätzlich unterscheidet man zwei Arten von DCs. Die klassischen DCs (cDCs) können
überall in Geweben vorkommen (Wu et al., 2009). Weiterhin sind spezielle cDCs in der Haut,
15
Leber und Lunge beschrieben (Mesnil et al. ,2012; Nakamoto et al. 2012; van Rijt et al.,
2005). Sie sind überwiegend kurzlebig und werden durch Vorläufer aus dem Blut gebildet.
Plasmazytoide DCs (pDCs) besitzen den gleichen Vorläufer wie cDCs, sind aber langlebiger
als cDCs und hauptsächlich im Knochenmark und in allen peripheren Organen präsent. Sie
sind darauf spezialisiert auf virale Infektionen zu reagieren, indem sie eine massive
Interferon-I-Antwort auslösen. Ebenso können sie wie cDCs Antigene präsentieren und eine
T-Zellantwort auslösen (Geissmann et al., 2010b). Als Marker für DCs werden in der Maus
hauptsächlich CD11c und MHC-II benutzt (Gottfried-Blackmore et al. 2009; Greter et al.
2005; Jung et al. 2002; McMahon et al. 2005; van Rijt et al., 2005). Mit Hilfe einer MHC-IIFärbung konnten DCs in den weichen Hirnhäuten und im Plexus choroideus im Rattenhirn
nachgewiesen werden und somit die Theorie über das Fehlen von DCs im ZNS (Sedgwick,
1995) widerlegen (Matyszak und Perry, 1996; McMenamin, 1999). Mit der Entwicklung von
transgenen Mauslinien, welche unter dem Promotor von CD11c (Itgax) GFP (Jung et al.,
2002) bzw. YFP (Lindquist et al., 2004) exprimieren, konnte auch im gesunden Gehirn die
Existenz von parenchymalen CD11c-positiven Zellen nachgewiesen werden (Bulloch et al.,
2008; Prodinger et al., 2011).
1.3.2 Makrophagen
Makrophagen sind residente und phagozytierende Zellen, die nicht nur im lymphatischen
Gewebe, sondern auch in allen anderen Organen und Geweben vorkommen (Geissmann et
al., 2010b). Unter nichtinflammatorischen Bedingungen spielen sie eine wichtige Rolle bei
der Aufrechterhaltung der Homöostase, indem sie apoptotische Zellen beseitigen und
Wachstumsfaktoren sezernieren (Geissmann et al., 2010b; Satpathy et al., 2012). Darüber
hinaus besitzen Makrophagen eine Vielfalt an Rezeptoren, um Pathogene schnell zu
erkennen, diese zu phagozytieren und mit Hilfe der Produktion von bestimmten
inflammatorischen Zytokinen und Chemokinen eine gezielte Immunantwort auszulösen. Als
typische Erkennungsmarker für Makrophagen in der Maus werden häufig F4/80 und CD11b
verwendet (McMahon et al., 2005). Wie bereits erwähnt, existieren auch im Gehirn
Makrophagen. Hier unterscheidet man zwischen den aus der Peripherie abstammenden und
hauptsächlich in den Meningen, dem Plexus choroideus oder perivaskulär lokalisierten
Makrophagen (Ransohoff und Cardona, 2010) sowie den residenten, parenchymal
lokalisierten Makrophagen, der Mikroglia (Ransohoff und Cardona, 2010).
1.3.3 Mikroglia
Mikroglia sind die residenten Gewebsmakrophagen des Gehirns. CX3CR1 ist ein typischer
Marker für residente Makrophagen. Dieser Marker wird auch genutzt, um Mikroglia zu
identifizieren und zu charakterisieren. Weitere häufig verwendete Marker für Mikroglia sind
16
F4/80, CD11b und IBA-1 (Carson et al., 1998; Sedgwick et al., 1991). Auch durch ihre
spezifische Expression von CD45 kann man Mikroglia von anderen peripheren Makrophagen
und mononukleären Zellen unterscheiden. So exprimieren Mikroglia CD45 auf mittlerem
(CD45int) Niveau, während andere Leukozyten CD45 auf hohem (CD45high) Niveau
exprimieren (Sedgwick et al., 1991).
Unter der Verwendung des Modells der entorhinalen Kortexläsion, welches zu einer
anterograden, axonalen Degeneration führt, konnte gezeigt werden, dass Vorläuferzellen der
Mikroglia von zirkulierenden, knochenmarksabstammenden Monozyten des Blutes in das
adulte Gehirn migrieren können (Bechmann et al., 2005). Später konnte gezeigt werden,
dass
die
von
Blutmonozyten
abstammenden
Mikrogliavorläufer
unter
bestimmten
Voraussetzungen, wie zum Beispiel Bestrahlung, in das adulte Gehirn migrieren (Mildner et
al., 2007). Ein Großteil der residenten Mikroglia stammt von einem Mirkogliavorläufer ab,
welcher bereits während der Embryogenese aus dem extraembryonalen Dottersack ins
Gehirn wandert (Ginhoux et al., 2010; Kierdorf et al., 2013). Damit nehmen residente
Mikroglia eine besondere Stellung im mononukleären phagozytierenden System ein.
Im gesunden Gehirn und unter nichtinflammatorischen Bedingungen besitzen Mikroglia
einen kleinen Zellkörper und lange, ramifizierte Fortsätze. Mit diesen Fortsätzen können sie
ihre Umgebung überwachen (Nimmerjahn, 2005). Nach Gewebeschaden bzw. einer
Entzündung wechseln Mikroglia ihre Morphologie in einen aktiven amöboiden Zustand. Je
nach Art der Aktivierung können Mikroglia akut immunologisch auf die Entzündung
reagieren, in dem sie aktiv an einer T-Zellantwort beteiligt sind oder eine schnelle Heilung
und Reparatur initiieren, um das Gewebe vor einem größeren Schaden durch eine akute
Immunantwort zu schützen (Murphy et al., 2010; Prinz et al., 2011).
Mikroglia sind ebenfalls in der Umgestaltung und Beseitigung von synaptischen Strukturen
der Neurone involviert. Zudem unterstützen sie den Turnover von Myelin im ZNS (Fitzner et
al., 2011; Goldmann und Prinz, 2013; Tremblay, 2011). Unter inflammatorischen
Bedingungen, zum Beispiel im Falle einer MS, kann regelmäßig eine Aktivierung und
Proliferation von Mikroglia beobachtet werden. Mikroglia können proinflammatorische
Proteine sezernieren, welche zu einer Aktivierung weiterer Mikroglia bzw. eingewanderter
Leukozyten führt (Babcock et al., 2003; Goldmann und Prinz, 2013; Semple et al., 2010). So
wurde in einem Modell der entorhinalen Kortexläsion in Mäusen gezeigt, dass Mikroglia im
Gehirn, neben Astrozyten, der Hauptproduzent von CCL2 sind (Babcock et al., 2003). Wie
oben beschrieben, ist CCL2 wichtig für die Migration durch das Gefäßendothel in den
perivaskulären Raum postkapillärer Venulen. Dadurch bewirkt es die Rekrutierung sowie
Aktivierung peripherer Leukozyten und Mikroglia (Babcock et al., 2003; Mahad und
Ransohoff, 2003; Owens et al., 2008; Semple et al., 2010).
17
Bei einer EAE wurde gezeigt, dass auch Mikroglia als antigenpräsentierene Zellen agieren,
in dem sie die dafür wichtigen Oberflächenmoleküle wie MHC-II, CD80 und CD86
exprimieren. Durch die Aufnahme und Prozessierung von Myelin zu kleinen Peptiden,
welche dann über MHC-II den myelinspezifischen, autoreaktiven T-Helfer-Zellen präsentiert
werden, kann es zum „Epitope spreading“ kommen (Goldmann und Prinz, 2013; Mack et al.,
2003; McMahon et al., 2005; Satoh et al., 1995; Tran et al., 1998). Dabei werden diese
autoreaktiven
T-Zellen
aktiviert,
welche
dann
wiederum
über
die
Expression
proinflammatorischer Zytokine zu einer weiteren Infiltration peripherer Leukozyten bzw. zur
Aktivierung weiterer peripherer Makrophagen und Mikroglia führt (Constantinescu et al.,
2011; Goldmann und Prinz, 2013; Mack et al., 2003; McMahon et al., 2005; Ortiz et al.,
2014). Andererseits konnte beobachtet werden, dass Mikroglia auch Toleranz induzieren
können, indem sie das 2. Signal der Antigenpräsentation so veränderten, dass es zu einer
Anergie der infiltrierten antigenspezifischen T-Zellen kam (Bechmann et al., 2001b; Kuchroo
et al., 1995).
1.4 CD11c als Marker für Dendritische Zellen
Einer der am häufigsten verwendeten Marker für verschiedene Subpopulationen der DCs ist
CD11c. Da jedoch gezeigt werden konnte, dass dieser Marker nicht nur allein auf DCs
exprimiert wird, sondern auch auf anderen Zelltypen, wie den Makrophagen, wird die
Existenz von DCs als eigenständige Zellpopulation in Frage gestellt (Geissmann et al.,
2010a; Hume, 2008; Randolph und Merad, 2013). Dabei scheint, dass die Berücksichtigung
der Herkunft dieser Zellen eine wichtige Rolle spielt. So sollte beachtet werden, ob man
Zellen aus einem lymphatischen Gewebe oder nichtlymphatischen Gewebe beschreibt.
Typische Charakterisierungsmarker von Zellen des mononukleären Systems wie MHC-II,
F4/80, CD11b und CD11c, sind nicht unbedingt vergleichbar zwischen den einzelnen
Populationen und Geweben (Geissmann et al., 2010a; Merad et al., 2013). Prinzipiell
unterscheidet man DCs durch ihre Expression von CD11c und MHC-II von Makrophagen.
Makrophagen zeichnen sich eher durch ihre Expression von F4/80 und CD11b aus (Bulloch
et al., 2008; Ji et al., 2013; Jung et al., 2002; McMahon et al., 2005). Jedoch gibt es Arbeiten,
welche zeigten, dass auch Lungen- und Milzmakrophagen fähig sind, CD11c zu exprimieren
(Probst et al., 2005; van Rijt et al., 2005). Darüber hinaus wurde beschrieben, dass auch
DCs typische Makrophagenmarker aufweisen können (Bogunovic et al., 2009; Denning et
al., 2007; Merad et al., 2013). Zum Beispiel wurden F4/80-positive Zellen der Lamina propria
aufgrund der Expression von CD11c als DCs beschrieben (Bogunovic et al., 2009). In einer
anderen Arbeit wurden die gleichen Zellen als Makrophagenpopulation bezeichnet. Obwohl
die Autoren diese Zellen hier als Makrophagen bezeichnen, wurde gezeigt, dass diese
Population regulatorische T-Zellen induzieren kann und wichtig für die Vermittlung der
18
Toleranz in der Lamina propria des Darms ist, eine Eigenschaft die vornehmlich DCs
zugesprochen
wird
(Denning
et
al.,
2007).
Weiterhin
konnte
im
Gehirn
unter
inflammatorischen Bedingungen gezeigt werden, dass auch eingewanderte Makrophagen
den DC-Marker CD11c exprimieren (McMahon et al., 2005). Ein weiteres funktionelles
Merkmal für die Charakterisierung von DCs ist die Antigenpräsentation gegenüber naiven TZellen. Auch hier konnte in verschiedenen Tiermodellen beobachtet werden, dass
Makrophagen fähig sind, Antigene naiven T-Zellen zu präsentieren (Mesnil et al., 2012;
Nakamoto et al., 2012).
Demzufolge ergibt sich daraus das Problem, dass keine uniforme Definition von DCs bzw.
Makrophagen in den untersuchten Geweben, als auch unter den verschiedenen
Voraussetzungen,
unter
welchen
diese
Charakterisierungen
stattfanden,
existiert
(Geissmann et al., 2010a; Hume, 2008; Hume et al., 2013; Randolph und Merad, 2013).
Durch die Verwendung des transgenen Tiermodells CD11c-DTR/GFP (Jung et al., 2002)
zeigten wir erstmals intraparenchymal lokalisierte CD11c-positive Zellen unter gesunden
Bedingungen im Gehirn. Wir konnten zeigen, dass diese Zellen vorwiegend positiv für die
Mikroglia/Makrophagenmarker Iba-1 und CD11b sind. Bis jetzt existiert keine einheitliche
Definition
dieser
intraparenchymal
lokalisierten
Zellen
unter
nichtinflammatorischen
Voraussetzungen.
Aufgrund der oben beschriebenen Heterogenität in der Charakterisierung von Makrophagen
und DCs sowie mit dem Aufkommen der Diskussion, ob DCs als unabhängige und klar
definierte Zelllinie überhaupt existieren (Geissmann et al., 2010a; Hume, 2008; Hume et al.,
2013; Randolph und Merad, 2013), stellte sich uns die Frage, wie sich intraparenchymale
CD11c-positive Zellen des Gehirns von CD11c-positiven Zellen aus anderen Organen
unterscheiden. Dafür verglichen wir diese auf die Expression spezifischer monozytärer und
inflammatorischer Zellmarker.
19
2 Fragestellung
Durch die Heterogenität in der Charakterisierung von DCs ist die Existenz derselben als
eigenständige Zelllinie umstritten, da bislang keine klare Definition existiert. Zudem konnte
gezeigt werden, dass einer der Hauptmarker für DCs, CD11c, auch auf anderen Zelltypen
wie Makrophagen exprimiert werden kann. Ziel dieser Arbeit war daher, die von uns
identifizierten CD11c-positiven Zellen mit CD11c-positiven Zellen aus Lunge, Leber und Milz
immunphänotypisch zu vergleichen. Dadurch wollten wir Klarheit erlangen inwiefern
Ähnlichkeiten zwischen den untersuchten Zellen den Begriff Dendritische Zelle reflektieren.
Dabei untersuchten wir die folgenden Fragestellungen:
1. Wie unterscheiden sich CD11c-positive Zellen im Gehirn von CD11c-positiven
Zellen aus anderen Organen?
2. Existieren CD11c-positive Mikroglia als eigenständige Population?
3. Besitzen intraparenchymale CD11c-positive Zellen intrinsische Eigenschaften
oder können diese abhängig vom Umgebungsmillieu beeinflusst werden?
Im Rahmen dieser angefertigten Arbeit konnten wir zeigen, dass die intraparenchymale
CD11c-positive Population im Gehirn immunphänotypisch den Mikroglia ähnelt und sich vor
allem in ihrer MHC-II-Expression von den anderen untersuchten CD11c-positiven
Populationen unterscheidet. Auch die MHC-II-Expression unterscheidet sich zwischen den
CD11c-positiven Mikroglia und den CD11c-negativen Mikroglia nicht. Bei der Untersuchung,
ob sich die MHC-II-Expression der Mikroglia in der Umgebung eines Immunorgans ändern
kann, beobachteten wir in unseren Experimenten keine Veränderung der MHC-II-Expression
und schlussfolgern daher, dass Mikroglia einen intrinsischen Phänotyp besitzen. Dieser
einzigartige Phänotyp der CD11c-positiven Mikroglia könnte einen Einfluss auf das
Immunprivileg des Gehirns vermuten lassen. Zudem erlaubt die Charakterisierung der
CD11c-positiven Zellen im Gehirn und deren immunphänotypischer Vergleich mit peripheren
CD11c-positiven Populationen die Untersuchung von Veränderungen im Rahmen der
Neuroinflammation und könnte so zu einem besseren Verständnis der Pathophysiologie
zahlreicher Erkrankungen wie der MS beitragen.
20
3 Literaturverzeichnis der Einleitung
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4 Publikationen
4.1
CD11c-expressing cells reside in the juxtavascular parenchyma and extend
processes into the glia limitans of the mouse nervous system
Carolin Prodinger, Jörg Bunse, Martin Krüger, Fridtjof Schiefenhövel, Christine Brandt, Jon
D. Laman, Melanie Greter, Kerstin Immig, Frank Heppner, Burkhard Becher, Ingo Bechmann
Carolin Prodinger, Jörg Bunse have contributed equally.
C. Prodinger , M. Krüger, F. Schiefenhövel, I. Bechmann
Institute of Clinical Neuroanatomy, Johann Wolfgang Goethe-University, 60590
Frankfurt/Main, Germany
e-mail: [email protected]
J. Bunse, M. Krüger, F. Schiefenhövel, K. Immig, I. Bechmann
Institute of Anatomy, University of Leipzig, 04103 Leipzig, Germany
J. Bunse, C. Brandt
Institute of Cell and Neurobiology, Charité,10098 Berlin, Germany
J. D. Laman
Department of Immunology, MS Centre ErasMS, Erasmus MC, 3000 DR Rotterdam,
The Netherlands
M. Greter, B. Becher
Neuroimmunology Unit, Institute of Experimental Immunology, University Hospital of
Zurich, Zurich, Switzerland
F. Heppner
Institute for Neuropathology, Charité, 10098 Berlin, Germany
31
Acta Neuropathol (2011) 121:445–458
DOI 10.1007/s00401-010-0774-y
ORIGINAL PAPER
CD11c-expressing cells reside in the juxtavascular parenchyma
and extend processes into the glia limitans of the mouse nervous
system
Carolin Prodinger • Jörg Bunse • Martin Krüger • Fridtjof Schiefenhövel
Christine Brandt • Jon D. Laman • Melanie Greter • Kerstin Immig •
Frank Heppner • Burkhard Becher • Ingo Bechmann
•
Received: 9 August 2010 / Revised: 27 October 2010 / Accepted: 31 October 2010 / Published online: 13 November 2010
Ó Springer-Verlag 2010
Abstract Recent studies demonstrated that primary
immune responses can be induced within the brain
depending on vessel-associated cells expressing markers of
dendritic cells (DC). Using mice transcribing the green
fluorescent protein (GFP) under the promoter of the DC
marker CD11c, we determined the distribution, phenotype,
and source of CD11c? cells in non-diseased brains. Predilection areas of multiple sclerosis (MS) lesions
(periventricular area, adjacent fibre tracts, and optical
nerve) were preferentially populated by CD11c? cells.
Most CD11c? cells were located within the juxtavascular
parenchyma rather than the perivascular spaces. Virtually
Carolin Prodinger, Jörg Bunse have contributed equally.
C. Prodinger M. Krüger F. Schiefenhövel I. Bechmann (&)
Institute of Clinical Neuroanatomy, Johann Wolfgang
Goethe-University, 60590 Frankfurt/Main, Germany
e-mail: [email protected]
J. Bunse M. Krüger F. Schiefenhövel K. Immig I. Bechmann
Institute of Anatomy, University of Leipzig,
04103 Leipzig, Germany
J. Bunse C. Brandt
Institute of Cell and Neurobiology, Charité,
10098 Berlin, Germany
all CD11c? cells co-expressed ionized calcium-binding
adaptor molecule 1 (IBA-1), CD11b, while detectable
levels of major histocompatibility complex II (MHC-II) in
non-diseased mice was restricted to CD11c? cells of the
choroid plexus. Cellular processes project into the glia
limitans which may allow transport and/or presentation of
intraparenchymal antigens to extravasated T cells in perivascular spaces. In chimeric mice bearing CD11c-GFP
bone marrow, fluorescent cells appeared in the CNS
between 8 and 12 weeks after transplantation. In organotypic slice cultures from CD11c-GFP mice, the number of
fluorescent cells strongly increased within 72 h. Strikingly,
using anti-CD209, an established marker for human DC, a
similar population was detected in human brains. Thus, we
show for the first time that CD11c? cells can not only be
recruited from the blood into the parenchyma, but also
develop from an intraneural precursor in situ. Dysbalance
in their recruitment/development may be an initial step in
the pathogenesis of chronic (autoimmune) neuroinflammatory diseases such as MS.
Keywords Neurodegeneration Alzheimer Innate immunity Multiple sclerosis (MS) Microglia Immune privilege
Introduction
J. D. Laman
Department of Immunology, MS Centre ErasMS,
Erasmus MC, 3000 DR Rotterdam, The Netherlands
M. Greter B. Becher
Neuroimmunology Unit, Institute of Experimental Immunology,
University Hospital of Zurich, Zurich, Switzerland
F. Heppner
Institute for Neuropathology, Charité, Berlin, Germany
Based on the observation that many foreign antigens, such
as allografts or heat-killed Bacillus Calmette-Guérin
(BCG), inoculated into the parenchyma are tolerated [31,
35, 43] the brain has been addressed as an ‘‘immunologically privileged’’ site [4, 8, 14]. The concept of immune
privilege has often been misapprehended as describing a
state of ignorance, although Medawar had already shown
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32
that the observed tolerance to ‘‘foreign’’ antigens within the
brain parenchyma can readily be broken by peripherally
exposing the same antigen [3, 35]. Therefore, he concluded
‘‘that skin homografts transplanted to the brain submit to,
but cannot elicit an immune state’’ and that ‘‘a lymphatic
drainage system is necessary for immunity to be called into
being’’ [35]. Such drainage is now established for solutes
involving pathways such as perivascular spaces, the cribroid plate, and the perineural sheath [10, 61]. Recently,
myelin-associated as well as axonal antigens have been
detected in cervical lymph nodes during experimental
autoimmune encephalitis (EAE) [11], multiple sclerosis
(MS) [12] and after axotomy [37, 59], but it is unclear
whether they are drained passively within the cerebrospinal
fluid (CSF) or transported actively out of the brain by a sort
of dendritic (DC) or monocytic cell. Such transportation
would conflict with the predominant current view that
antigen-presenting cells (APC) do not leave the parenchyma of the brain. However, cells expressing DC/APC
markers have been detected in the meninges, the choroid
plexus [30, 33, 34], and the cerebrospinal fluid [41, 42] and
they appear in the parenchyma under inflammatory conditions [13, 16, 39, 46, 47, 51, 52, 55].
Recently, it has been shown that the induction of neuroinflammation depends on antigen-presenting dendritic
cells associated with brain vessels [16, 32], but their
topographic localization and distribution remained
unknown. This was at least in part due to the problem that
currently available antibodies raised against DC markers
such as CD11c and CD205 provided no or questionable
signals within the normal mouse brain.
Here, we used mice expressing green fluorescent protein
(GFP) under the control of the CD11c promoter itgax for
an in-depth analysis of the CD11c? population within the
central nervous system (CNS) [19]. In line with previous
reports, we found numerous ramified CD11c? cells within
the meninges and the choroid plexus. However, we report
that they also reside within the brain, the spinal cord, and
the optic nerve, where they are mostly located within the
parenchyma from where they extend processes to the glia
limitans. Since re-stimulation of T cells by their cognate
antigen in perivascular spaces is indispensable for their
progression across the glia limitans [4, 58], it may be the
presence of CD11c? cells at the glia limitans rendering
them crucial for the onset of neuroinflammation [16].
At present, the origin of the DC observed in (autoimmune) neuroinflammation is unclear. They may either
derive from microglia stimulated with granulocyte macrophage colony-stimulating factor (GM-CSF) [49] or
infiltrate the parenchyma from the blood along with other
leukocytes [16, 39]. Using slice cultures derived from p3
and adult CD11c-GFP mice and chimeras grafted with
CD11c-GFP bone marrow, we demonstrate for the first
123
time that CD11c? cells can differentiate from an intraparenchymal precursor in situ and—in principle—can be
recruited from the systemic circulation. Due to the irradiation used to create bone marrow chimeras, the latter
finding, however, must be interpreted with care since
Mildner et al. [36] demonstrated that shielding of brain
areas during irradiation specifically blocks infiltration into
the respective regions. Finally, we identified a population
reflecting the intraparenchymal location of CD11c? cells
in human brains using the DC-marker CD209.
Materials and methods
Mouse strains and housing
CD11c-diphtheria toxin receptor (DTR)-GFP transgenic
(tg) mice [19], i.e., mice expressing GFP under the control
of the CD11c promoter itgax, on a C57BL/6 background
were purchased from Charles River (Charles River Laboratories, Inc., Wilmington, MA) and housed under standard
conditions with free access to food and water. Care was
been taken to minimize any pain and discomfort to animals. All experiments were performed after approval by
the Animal Ethical Committee according to German legislation on animal experiments.
Perfusion and fixation
Animals (n = 30; 7–21 weeks of age) were killed and
transcardially perfused with 100 ml 0.9% NaCl (AppliChem. Darmstadt, Germany) followed by 100 ml of a
fixative containing 4% paraformaldehyde (PFA) (Merck,
Germany) in 0.1 M phosphate buffered saline (PBS; pH
7.4). Brains were removed and post-fixed overnight in the
same fixative and cut on a vibratome (30–50 lm sections)
(Microm, HM 650V) or incubated in a solution of 30%
sucrose (Fluka Biochemika, Germany) in 0.1 M phosphate
buffer (PB; pH 7) for 24 h. These sections were embedded
in Neg-50 medium (Richard-Allan Scientific, Kalamazoo,
MI), snap-frozen in -80°C cold methylbutane (Roth,
Karlsruhe, Germany), and cut further into 10–20 lm
cryostat sections. For semithin sections and electron
microscopy, the fixative contained 4% sucrose and 0.2%
glutaraldehyde (AgarScientific, United Kingdom) in carbonate buffered PFA. The brains were removed and postfixed over night in the same solution.
Immunostaining for (confocal) fluorescence
microscopy
For the immunocytochemistry, slices were fixed at 1, 24,
48 and 72 h in 4% paraformaldehyde overnight at 4°C.
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Acta Neuropathol (2011) 121:445–458
Subsequently, the slices were stored in 0.8 M saccharose-solution over 2 weeks at 4°C. Afterwards, the slices
were cut into 10 lm sections with a cryostat.
Unspecific binding was blocked using 10% normal
goat serum (NGS) (Chemicon, Temecula, CA) and 0.5%
Triton X-100 (VWR, Prolabo, Darmstadt, Germany) in
PB. After incubation in this solution for 30 min, sections
were incubated at ?4°C overnight in PB containing
1% NGS and 0,5% Triton X-100 and the respective
primary antibodies: polyclonal anti-ionized calciumbinding adaptor molecule 1 (anti-IBA-1) (1:1,000; rabbit
anti mouse; WAKO Chemicals, Neuss, Germany); antiCD11b (1:100; rat anti mouse; Leinco, St. Louis, MO);
anti-major histocompatibility complex II (anti-MHC-II)
(1:100; rat anti mouse; BD, Heidelberg, Germany); antipan-laminin (1:200; rabbit anti mouse; DakoCytomation,
Denmark); anti-glial fibrillary acidic protein (anti-GFAP)
(1:1,000; mouse anti mouse; Chemicon, Temecula, CA).
After several washes in PB, sections were incubated for
90 min at room temperature in PB containing 1% NGS
and 0.5% Triton X-100, and the corresponding secondary
antibody (Alexa 350, Alexa 488, Alexa 568, or Alexa
688; all 1:250; Invitrogen, Karlsruhe, Germany). For
nuclear staining the Hoechst reagent (Sigma, Steinheim,
Germany) was used according to the manufacturer’s
protocol. Sections were embedded in fluorescence
mounting medium (DakoCytomation, Denmark).
Diaminobenzidine (DAB)-staining
For DAB staining, slices were incubated in 1% Naborohydride (Sigma, St. Louis, MO) to reduce background fluorescence. Lipids were unhinged by short
incubation in an ascending and descending alcohol series
(20–30–40–30–20%). Unspecific binding was blocked
using 5% bovine serum albumin (BSA) (Biomol, Hamburg, Germany) in PB for 60 min. Sections were
subsequently incubated with the primary antibody
(polyclonal goat anti-GFP 1:1,000; Acris, Hiddenhausen,
Germany) and 1% BSA dissolved in PB at 4°C overnight. Section were then washed several times in PB and
incubated in a solution containing 1% BSA and the
biotin-coupled secondary antibody (1:200; Vector-Laboratories, Wertheim-Bettingen, Germany) in PB for
90 min at room temperature. Binding of the primary
antibody was visualized using an avidin–biotin (ABC) kit
(Vector-Laboratories, Wertheim-Bettingen, Germany)
with DAB as chromogen according to the manufacturer’s
instructions. Slices were dehydrated and embedded in
Durcupan (ACM Fluka, Sigma-Aldrich, Gillingham,
UK). Semithin and ultrathin sections were cut using a
Leica Ultracut (Ultracut UCT, Leica).
Organotypic slice cultures
Organotypic slice cultures were prepared from either 3-dayold or adult CD11c-DTR-GFPtg mice (Jackson Laboratories, Boston, MA, USA) as previously described [24]. In
brief, mice were perfused with saline to eliminate blood
leukocytes from the circulation. After decapitation, the
brain was rapidly removed under sterile conditions and
placed in ice-cold preparation medium consisting of 50%
minimum essential medium (MEM) (Gibco, Karlsruhe,
Germany), 49 ml aqua pro inject, with 2 mM L-glutamine
(Gibco, Karlsruhe, Germany) at pH 7.35. The hippocampi
were taken from the brain and cut into 350 lm thick vertical
slices on a tissue chopper (Technical Products International,
St. Louis, MO, USA). Subsequently, the slices were cultured on Millipore cell culture inserts (pore size 0.4 lm;
Millipore, USA) in six-well plates containing cultivation
medium. The sterile medium contained 25% MEM, 25%
basal medium eagle (BME) (Gibco, Karlsruhe, Germany),
25% heat-inactivated normal horse serum (Gibco), 20.9 ml
aqua pro inject, 2 mM L-glutamine, 0.65% glucose-20
(Braun, Melsungen, Germany), at pH 7.2. The organotypic
slice cultures were incubated at 37°C in a humidified
atmosphere with 5% CO2 for 1, 24, 48 and 72 h. The culture
medium was changed every 48 h (n = 10). To exclude that
exogenous effects induce the differentiation of dendritic
cell in the slice culture, we performed the same experiments
with serum-free medium using p3 and adult animals
(n = 10). The serum-free medium contained (for 100 ml)
37.5 ml MEM, 20.9 ml aqua pro inject, 37.5 ml BME with
2 mM glutamine and 0.65% glucose-20 (Gibco, Karlsruhe,
Germany) at pH 7.2 (n = 3).
Irradiation of bone marrow chimeric mice
CD11c-GFP mice were killed by cervical dislocation. Bone
marrow (BM) cells were isolated by flushing femur and
tibia bones with Dulbecco’s Modified Eagle Medium
(DMEM) containing 1% penicillin/streptomycin and 1%
fetal bovine serum (all from GIBCO, Invitrogen, Karlsruhe, Germany) filtered through a 40 lm cell strainer
(BD, Heidelberg, Germany). C57BL/6J wild type (wt)
recipient mice were lethally irradiated with 11 Gy (split
dose 2 9 5.5 Gy) and intravenously injected with
4–6 9 106 BM cells. On each of days 5, 15, 28, 46, 75, 105
and 161 (15 and 23 weeks; n = 4) after transplantation
mice were killed for histological analysis.
Entorhinal cortex lesion (ECL)
Stereotaxic lesioning of the left entorhinal cortex was
performed as described in detail elsewhere [6, 25]. In brief,
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34
mice were anaesthetized with a mixture of ketamine (20%)
(Cura Med, Germany) and Rompun (8%) (Bayer Vital,
Germany) (10 ll/g body weight). They were then fixed in
an stereotactic apparatus (Kopf Instruments, Tujunga, CA)
and the eyes were protected with a humid soft tissue. For
ECL, the left entorhinal cortex was lesioned using a 2 mm
broad knife. After exposing the skull the following coordinates measured from k (where the longitudinal and the k
suture meet) were used: anteroposterior ?0.4 mm, lateral
?1 mm and dorsoventral, down to the base of the skull.
The wound was carefully sutured and the animal was
placed back in its cage. Three days after entorhinal lesion,
animals were killed and perfused as described above.
Microscopy
To analyze the colocalization of GFP? cells with the
expression of common microglia markers and to determine
the distribution of the GFP? cells in the slice, a Leica TCS
SL laser scanning microscope with was used. 3D-reconstruction was performed with Volocity 3 software
(Improvision, Tübingen, Germany) (n = 3). Fluorescent
stainings and semithin-sections were studied using an
Olympus BX51 fluorescence and light- microscope or a
Zeiss Axiovert 100 M LSM510 confocal microscope. For
analysis of the 3D-extension of CD11c-GFP cells within
slices and the stainings of BM-chimeras, an Olympus
Fluoview FV1000 was used. The 3D-reconstruction was
performed with Imaris software. Electron-microscopy was
performed using a Zeiss EM109.
Human tissues
Formalin-fixed and paraffin-embedded human autopsy
material with or without neuropathological alteration was
used. All cases underwent detailed neuropathologic examination. Informed consent for autopsy and subsequent use of
tissue for research purposes was given. To mark human DC
a primary antibody to human DC-SIGN (CD209; R&D
Systems; 1:10 dilution) was used upon ethylenediaminetetraacetic acid (EDTA)-driven antigen retrieval. The
immunohistochemical staining was carried out on an automated Benchmark staining apparatus (Ventana Medical
Systems/Roche) following the manufacturer’s guidelines.
Results
Distribution of CD11c-GFP cells throughout the CNS
(Fig. 1)
Serial sections of brain, spinal cord, and optic nerve
revealed a consistent rather than random distribution
123
pattern of CD11c-GFP cells (Fig. 1a). In the brain, they
were preferentially situated within the tela of the choroid
plexus, beneath the ependyma of the lateral ventricles,
along the adjacent corpus callosum and the fornix (Fig. 1b–
d). Regularly, but at lower numbers, CD11c-GFP cells
were also present in the white matter of the olfactory bulbs,
the brain stem and the cerebellum. Such preferential
location within the white matter was also evident in the
spinal cord (Fig. 1e), where the largest numbers were
found along the spinobulbar tract and the lateral fibre
tracts, while the spinal nerves were not populated by
CD11c-GFP cells (Fig. 1f–h). Importantly, the optic nerve
was found to be densely populated by CD11c-GFP cells
(Fig. 1i, k), while fluorescent cells were extremely rare
within other cranial nerves, e.g. the trigeminal nerve
(Fig. 1l) and the trigeminal ganglion.
Parenchymal, but not choroid plexus CD11c-GFP cells
express microglial markers but lack MHC-II (Fig. 2)
At higher magnification CD11c-GFP cells exhibited ramified morphologies with more plump processes in the
ependyma of the lateral ventricles and tiny ramifications
within fibre tracts. Counterstaining with IBA-1 and CD11b
revealed that almost all CD11c-GFP cells expressed these
microglial markers. The ratio of CD11c-GFP cells among
all microglia stained with IBA-1 or CD11b ranged between
none or occasional cells (e.g., cortex) and a maximum of
up to two thirds (optic nerve). MHC-II was expressed at
detectable levels by CD11c-GFP cells only in the choroid
plexus. In contrast to all other regions studied, CD11c-GFP
cells of the plexus did not regularly express IBA-1 and
CD11b (Fig. 2g–i). Meningeal and perivascular macrophages expressed MHC-II in all brain areas (Fig. 2c–m).
CD11c-GFP cells are preferentially localized
in the juxtavascular neuropil (Fig. 3)
As anticipated from fluorescence microscopy and a previous
report [16], CD11c-GFP cells were regularly found in close
vicinity to blood vessels (Fig. 3a) raising the question of
whether they are part of the vascular wall, reside in perivascular spaces (i.e., the compartment between the vascular
basement membrane and the glia limitans) or within the
juxtavascular parenchyma (i.e., the neuropil beyond the glia
limitans) [4, 40] (Fig. 3b). To address this issue, the borders
between these three compartments (vascular wall, perivascular space, and juxtavascular parenchyma) were
demarcated using GFAP and/or laminin antibodies. As the
vascular and the glial basement membranes differ in their
laminin contents [53], an anti-pan-laminin serum was used
allowing simultaneous detection of both membranes
(Fig. 3d, e). To unequivocally distinguish the perivascular
Acta Neuropathol (2011) 121:445–458
35
Fig. 1 Distribution of CD11cGFP cells throughout the CNS.
a–d Distribution of CD11c-GFP
cells in the forebrain. a Four
horizontal brain hematoxylin
and eosin-stained sections from
different levels schematically
show the typical distribution of
CD11c-GFP cells. Sites of
evident accumulation included
the periventricular/
subependymal area, the alveus,
fimbria, the choroid plexus, and
the corpus callosum. b–
d CD11c-GFP cells are shown
at sites of typical accumulation
in 16 lm cryosections as
indicated by frames in
a (sections 2 and 3).
b Subventricular zone of the
frontal pole of the lateral
ventricle; c fornix; d choroid
plexus/tela choroidea of the
third ventricle. e–h Distribution
of CD11c-GFP cells in the
spinal cord. The frames depicted
in e are shown under high
magnification in f–h. f The
spinothalamic tract is densely
populated by CD11c-GFP cells.
The white line indicates the
surface of the spinal cord.
g Many CD11c-GFP cells are
regularly found in the
spinobulbar tract. h No cells
were found in the spinal nerve
and its roots, visualized with
Hoechst staining. i, k, l While
the optic nerve (i, k) is densely
populated by CD11c-GFP cells,
the trigeminal nerve (l) and
other cranial nerves contain
only very few fluorescent cells
(arrow in l)
space from the parenchyma, the localization of identified
CD11c-GFP cells was further analysed in semi- and ultrathin
sections (1.0 and 0.055 lm, respectively). In laminin-stained
samples, most CD11c-GFP cells appeared to reside within
the juxtavascular parenchyma (Fig. 3a). This finding was
confirmed in semithin sections (Figs. 3c, 4c). Only occasional cells were located within perivascular spaces. In
contrast to the parenchymal population, these cells lacked
tiny ramifications (Fig. 3d–e).
CD11c-GFP cells participate in the glia limitans
organization (Fig. 4)
Confocal analysis of sections stained for laminin or GFAP
suggested that processes of CD11c-GFP cells participate in
the formation of the glia limitans (Fig. 4a, b). The same
was observed in semithin sections, where GFP was stained
with a polyclonal antiserum (Fig. 4c). In fact, electron
microscopical analysis revealed that CD11c-GFP cells
directly attach to the basement membrane of the glia limitans (Fig. 4d) thus representing an integral element of this
glial barrier.
Origin of CD11c-GFP cells (I): appearance of CD11c?
cells in organotypic brain slices (Figs. 5, 6, 7)
The origin of CD11c? dendritic cells in inflamed neural
tissue is currently unclear. They may either be recruited
from blood or derive from intrinsic (microglial) precursors.
To test the latter possibility, we prepared organotypic slice
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36
Fig. 2 Parenchymal, but not choroid plexus CD11c-GFP cells
express microglial markers but lack MHC-II. Brain (a–c), spinal
cord (d–f), choroid plexus (g–l) and optic nerve (k–m) of CD11c-GFP
mice were counterstained with antibodies for the microglial markers
IBA-1 (a, d, g, k) and CD11b (b, e, h, l) and for MHC-II (c, f, i, m).
CD11c-GFP is shown in green, IBA-1, CD11b, and MHC-II in red,
nuclei stained with Hoechst are shown in blue. The inserts represent
one characteristic cell for each staining. Note that the antibody against
MHC-II strongly labels meningeal macrophages, but not intraparenchymal GFP-expressing cells. Green arrows in g and h point to CD11cGFP cells, which are negative for either IBA-1 (d) or CD11c (e)
cultures obtained from CD11c-GFP mice and searched for
GFP? cells in living cultures using an epifluorescence
microscope. Despite the relatively low resolution of this
mode of observation, it was already evident that the
number of GFP-expressing cells strongly increased over
time. For a more detailed analysis of the respective cells,
slices were subsequently fixed at various time points and
cryosections were prepared for immunocytochemistry.
As shown in Fig. 5a–c, few or no fluorescent cells were
visible in slices from p3 animals at 1 h after incubation. At
24 h, numerous GFP? cells could be detected in all cultures (Fig. 5d–f). These cells were located throughout the
whole width of the slices which was evident in cross
sections cut perpendicularly to the surface of the slice
(Fig. 5f). Cryosections of these slices revealed that the
fluorescent cells exhibited ramified morphologies and
stained positive for the microglial markers MAC-1 and
IBA-1 (Fig. 6). No additional increase of GFP? cells was
apparent at 48 and 72 h.
An important question was whether the induction of
CD11c on cells within slice cultures was restricted to the
early postnatal phase. Therefore, we prepared acute brain
slices from adult (8–12 weeks old) mice and studied the
appearance of fluorescent cells in these tissues. Again,
fluorescent cells were rare or absent directly after incubation (Fig. 5g–i), but numerous cells were found after 24 h
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Acta Neuropathol (2011) 121:445–458
37
Fig. 3 CD11c-GFP cells are preferentially localized in the juxtavascular neuropil. a Confocal 3D-reconstruction of CD11c-GFP cells and
their relation to a brain vessel (stained for pan-laminin in red) in a
50 lm vibratome section. As described previously [16], all cells are
to be vessel-associated raising the question of whether they are in the
juxtavascular parenchyma or in the perivascular space. Both
compartments seem to be populated (arrowhead juxtavascular, arrow
perivascular), but it is impossible to unequivocally distinguish
between the two in these sections. b Schematic illustration of the
different compartments of the neurovascular interface. The vascular
wall represents the first compartment and consists of endothelial cells,
pericytes and (if present) smooth muscle cells of the tunica media.
The second is the perivascular (Virchow/Robin) space which harbors
perivascular macrophages and leptomeningeal mesothelial cells. It is
on the one side bordered by the basement membrane of the vascular
wall, and by the basement membrane of the glia limitans on the other.
The third compartment is the juxtavascular parenchyma starting
beyond the astrocytic endfeet forming the glia limitans. c The
existence of juxtavascular, and thus intraparenchymal CD11c-GFP
cells was confirmed in semi-thin sections (1.0 lm) in which GFP was
visualized with an antibody and DAB staining. The neuropil is
counterstained with toluidine-blue. The vast majority of CD11c-GFP
cells turned out to be located within the juxtavascular parenchyma. d,
e In addition, scattered individual cells cells were also found in
perivascular spaces depicted either by laminin staining (red) to show
the outer vascular and parenchymal basement membranes (arrows)
and thus the perivascular space in d or by combined laminin (red) and
GFAP (blue) staining. The insert in d shows the same cell after
cutting for semithin analysis which clearly confirms its location
within the perivascular space (arrows point to basement membranes)
in vitro (Fig. 5j–l). Thus, just like in the early postnatal
phase, adult brain tissue apparently contains a cell type
with the potential to express CD11c.
As shown in Fig. 7, 3D-reconstruction of the CD11cGFP cells revealed tiny extensions of up to 35 lm with
rare or invisible secondary ramifications. Due to the
counterstaining of nuclei with Hoechst, it also became
apparent that the cells were clearly located within the
parenchyma rather than being part of (peri-)vascular
remnants (Fig. 7).
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Fig. 4 CD11c-GFP cells
participate in the glia limitans
organization. As shown in
Fig. 3a and c, some cells
seemed to participate in the
organization of the glia limitans.
a This was confirmed by triple
fluorescence analysis depicting
GFAP (blue), laminin (red), and
GFP (green). Note that the
processes of CD11c-GFP cells
intermingle with the astrocytic
endfeet of the glia limitans.
b This was also evident in
sections cutting vessels
perpendicularly to their
longitudinal axis, where CD11cGFP processes were found to
embrace the parenchymal
basement membrane (stained
with laminin in red, nuclei
blue). The insert shows a
confocal picture in which
CD11c-GFP processes appeared
to be directly attached to the
parenchymal basement
membrane. c–d This was
confirmed in semithin sections
(c) and ultrastructural analysis
(original magnification 94,400)
(d) of GFP-DAB-stained
sections, where the direct
contact (green arrows) between
GFP-filled cells and the
basement membrane (dotted red
line) could be proven
Fig. 5 Overviews of CD11c-GFP cells in slice cultures. Organotypic
slice cultures were prepared from either p3 (a–f) or adult
(g–l) CD11c-DTR-GFPtg mice and kept in culture for either 1 h
(a–c, g–i) or 24 h (d–f, j–l). CD11c-GFP-expressing cells were rare or
absent at 24 h in p3 (a–c) and adult (g–h) slices. At 24 h, however,
fluorescent cells were abundant in all slices irrespective of whether
123
they derived from p3 (d–e) or adult (j–l) mice (n = 10). As a proof of
principle, these data demonstrate that neural tissue harbors a cell type
which can be induced to express CD11c. Thus, recruitment across the
blood–brain barrier is not an absolute prerequisite for such cells to
appear in the parenchyma
39
Acta Neuropathol (2011) 121:445–458
Fig. 6 Expression of microglial
markers on CD11c-GFP cells.
Immunocytochemistry was
performed in cryosections from
slice cultures of all groups,
constantly revealing that
CD11c-GFP cells were
immune-positive for MAC-1
(a–c) and IBA-1 (d–f). Pictures
shown here derive from an
untreated, p3-slice kept in
culture for 24 h. Note that
MAC-1 (b) is a membrane
protein (CD11b), while IBA-1
(e) and GFP (a, d) are located
within the cytoplasm. The same
observations were made in
slices incubated for 48 and 72 h
(n = 8)
Fig. 7 3D-reconstruction of
parenchymal CD11c-GFP cells.
Various CD11c-GFP cells in a
slice kept in serum-free medium
for 24 h. Nuclei are
counterstained with Hoechst.
The frames in the upper panel
are shown at higher
magnification and in 3Dreconstruction in a–c. a A cell
showing cardinal branches, but
no secondary ramifications,
reflecting a typical
morphological feature of the de
novo appearing CD11c-GFP
population. b Arrowheads point
to long and tiny extensions.
c Arrowheads point to putative
remnants of small vessels. The
fluorescent cells are clearly
located within the adjacent
parenchyma rather than in the
vascular wall or the perivascular
space
Origin of CD11c-GFP cells (II): CD11c-GFP cells can
be recruited from BM (Fig. 8)
In order to test whether the intraparenchymal population of
CD11c-GFP cells can also derive from a precursor of the
BM, C57BL/6J wt mice were lethally irradiated and
transplanted with BM from CD11c-GFP mice. Mice were
killed on each of days 5, 15, 28, 46, 75, 105 and 161 (15
and 23 weeks; n = 4) after transplantation. First intraparenchymal CD11c-GFP cells were detected within the
periaqueductal white matter at 46 days after transplantation. Subsequently, fornices, brainstem, spinal cord, and
optic nerve were populated. Confocal analyses confirmed
the intraparenchymal localization using laminin to distinguish perivascular spaces from the neuropil and
demonstrated the coexpression of microglia/macrophage
markers IBA-1 and CD11b (nearly all cells) and MHC-II
(occasional).
The number of CD11c-GFP cells is not increased
in zones of anterograde axonal degeneration (Fig. 9)
We have previously demonstrated that axonal injury
induced by entorhinal cortex lesion (ECL) causes induction
123
40
Fig. 8 CD11c-GFP cells can be recruited from BM. Within a time
period of 48 days to 23 weeks after transplantation of transgenic
CD11c-GFP BM into lethally irradiated wt mice, an increasing
number of ramified CD11c-GFP cells was found in characteristic
locations within the neuropil surrounding the aqueduct (a), brain
stem, fornix (b), fibre tracts surrounding the striatum, white matter of
the spinal cord (c) and the optic nerve (d). Counterstaining of the
basal laminae with laminin (e) demonstrates the intraparenchymal
localization of the CD11c-GFP cells. Further confocal analysis
reveals their regular expression of CD11b (f) and IBA-1 (g) (orange
arrows) and their occasional expression of MHC-II (h)
Fig. 9 The number of CD11c-GFP cells is not increased in zones of
anterograde axonal degeneration. We have previously shown that
myelin-phagocytosing cells in zones of axonal degeneration induced
by entorhinal cortex lesion (ECL) express MHC-II and CD86 [7], and
that mononuclear cells are recruited which subsequently transform
into microglia [5]. a–b To test whether these cells also express
CD11c, we performed ECL in adult mice. The distribution of
microglia in the normal hippocampus is shown in b. A massive
accumulation of IBA-1 (red) cells is visible in the zones of axonal
degeneration in the hippocampus, the molecular layer (ML). However, these cells did not express CD11c. The frame depicts a CD11cGFP cell located at a vessel in the entorhinal cortex which is shown at
higher magnification in the insert
of CD86 and MHC-II on microglia-like elements [7] as
well as the recruitment of hematogenous cells and their
subsequent transformation into microglia [5]. Similar
findings have been reported by others [2, 26, 62, 63].
Therefore, we speculated that CD11c expression may also
be induced on local microglia or CD11c? hematogeneous
cells may be recruited after ECL. However, an increase in
neither number nor distribution of CD11c-GFP cells was
evident in this setting (Fig. 9a, b). Thus, although axonal
degeneration provides cues for the recruitment of mononuclear cells from the blood and cells expressing the stem
cell marker CD34 [26], CD11c is not found on these
populations.
123
Anti-CD209 demarks a population of cells in human
brains the distribution of which reflects
intraparenchymal mouse CD11c cells (Fig. 10)
As shown above, CD11c-GFP cells do not only populate
the meninges, the choroid plexus and the perivascular
space, but also reside within the brain parenchyma (Figs. 1,
2, 3, 4). A similar population was identified in human
Acta Neuropathol (2011) 121:445–458
41
Fig. 10 In the human brain
parenchymal DC are present in
the periventricular area. To
analyze the distribution of DC
in human paraffin slices of
different human brains were
stained with CD209, an
established human DC-marker.
In addition to many perivascular
cells, that were described
earlier, a population of
juxtavascular DC reflecting the
intraparenchymal location of the
CD11c? cells of the CD11cGFPtg-mice (Figs. 1, 2, 3, 4)
could be detected in the
periventricular regions of the
human brains. a–
b Periventricular white matter.
c Optical nerve (c derives from
a patient with elective necrosis
of the cortex and hippocampus,
a and d from an individual
without brain pathology)
brains using the DC-marker CD209. These cells were also
located adjacent to blood vessels clearly beyond the glia
limitans within the parenchyma (Fig. 10).
Discussion
In continuation of the work by Hickey and Kimura [18],
two recent papers demonstrate that a population of brainbased antigen-presenting, CD11c? cells are required and
sufficient to stimulate autoimmune neuroinflammation
within the CNS [16, 32]. Both reports demonstrated that
the population of DC-like elements with the functional
capacity to support T cell activation derives from the systemic myeloid compartment and not from the CNSparenchyma, but it was unknown whether they reside in
perivascular spaces or within the parenchyma. Here, we
explored the distribution and localization of such CD11c?
cells within the normal mouse brain, spinal cord and optic
nerve. To this end, we took advantage of a transgenic
mouse strain expressing GFP under the itgax promoter,
since all available antibodies to mouse CD11c reveal weak,
questionable or unspecific staining in cryo-, vibratom-, or
paraffin sections of the brain. This may be explained by
low expression levels of the respective markers under
normal conditions, which may be driven by factors
exclusively present in the normal parenchyma, because DC
were found in the leptomeningeal compartment and during
neuroinflammation using the same antibodies [13, 16, 21,
30, 33, 51] (for review see [42]).
We found that (a) CD11c-GFP cells reside in virtually
all regions of the brain and the spinal cord, (b) they are not
randomly scattered, but their distribution shows a typical
pattern with preferential localization in the periventricular
areas and the adjacent fibre tracts as well as in the optic
nerve and the white matter of the spinal cord, (c) only few
cells are located in perivascular spaces, while the vast
majority resides in the juxtavascular parenchyma from
where they extend processes to the basement membrane of
the glia limitans, (d) CD11c can be induced in cells already
present in the brain, but CD11c cells are also recruited
from the blood, (e) CD209 labels an intraparenchymal
population of cells in human brains with features similar to
the CD11c-GFP cells described herein.
We regard it as the main finding that CD11c-GFP cells
are located within the parenchyma, but extend processes to
the glia limitans. We have previously proposed that neuroinflammation involves two differentially regulated steps:
(1) passage of the vascular wall into perivascular spaces,
and (2) progression across the glia limitans into the
123
42
neuropil [4, 40]. While the first step has been investigated
intensively (for review: [23, 27], much less is known about
the second. The group of Owens was the first to experimentally dissect steps one and two: they found that
elimination of perivascular cells and subsequent induction
of EAE does not interfere with perivascular accumulation
of leukocytes, but completely blocks their progression
across the glia limitans [58]. The same group demonstrated
that the chemokine CCL2 is a major player in driving
recruitment of leukocytes into the parenchyma [2]. In mice
overexpressing CCL2 in the brain, i.p. application of pertussis toxin initiates cellular infiltration across the glia
limitans causing weight loss and encephalopathy. Strikingly, treatment of these mice with the broad spectrum
metalloproteinase (MMP) inhibitor BB-94/Batimastat did
not interfere with perivascular infiltration either, but
instead abolished the second step along with clinical
symptoms [57]. Others showed that MMP-2/MMP-9 double knock-out mice are resistant to EAE, because they
cannot cleave dystroglycan, which anchors astrocytic
endfeet to the basement membrane of the glia limitans [1].
These studies strongly suggest that expression of MMPs by
either the T cells or macrophages in the perivascular space
is a prerequisite for passage of the glia limitans and the
induction of symptomatic neuroinflammation in EAE.
However, if animals are immunized with horseradish peroxidase (HRP) and the same antigen is subsequently
injected into the ventricle, T cells home to HRP-phagocytosing perivascular macrophages upon peripheral antigen
boosting, but they do not pass the glia limitans. Again, this
T cell extravasation over the first barrier provided by the
endothelium does not cause clinical symptoms [60].
All these studies are integrated by the view that antigenpresentation/recognition in the perivascular space may be
necessary, but per se are not sufficient for the induction of
intraparenchymal neuroinflammation [60]. The second step
of neuroinflammation may require presentation of an intraparenchymal antigen at the glia limitans itself. In fact,
non-astrocytic processes immune positive for microglial
markers have been demonstrated at the ultrastructural level
by Lassmann [28]. It has often been questioned how T cells
can be re-stimulated within the brain upon adoptive
transfer to cause passive EAE, since their cognate antigen
should not normally be present within perivascular macrophages. Given that the decisive antigen-presenting cell
type expresses CD11c [16, 32] and, as shown here, is
located at the glia limitans, one can picture a scenario in
which, e.g., physiological degradation of myelin and
uptake by local CD-11c? cells causes permanent presentation of its epitopes at the interface between parenchyma
and perivascular space. The arrival of encephalitogenic
Th1/Th17 cells and the accompanying secretion of proinflammatory cytokines could then induce maturation of
123
CD11c? antigen-presenting cells eventually leading to
MMP expression and permeabilization of the glia limitans.
Thus, the localization of CD11? cells at the glia limitans
provides a possible explanation for their recently described
crucial function in autoimmune neuroinflammation [16]
and raises the possibility that alterations in their recruitment, development, or activation state represents an early
step in the pathogenesis of autoimmune diseases such as
MS.
Our data clearly demonstrate the presence of ramified
cells expressing CD11c and several markers shared with,
but not specific for microglia. The question remains whether these cells should be regarded as DC. CD11c is a
membrane glycoprotein which belongs to the CD18 family
of integrins, including CD11a, CD11b, and CD11d. Functionally, CD11c is responsible for binding a diverse array
of ligands such as endothelial cell adhesion molecules,
bacterial cell wall components, complement factors such as
iC3b, and matrix proteins [20, 29, 38, 48, 50, 56]. In vivo,
CD11c expression is also induced during early stages of
myeloid cell differentiation [20] and is increased considerably during differentiation of monocytes into tissue
macrophages [50]. CD11c expression is mostly restricted
to cells of the myeloid lineage: macrophages, monocytes,
granulocytes, and at characteristically much higher levels—most DC express CD11c, but a small population of
CD11c? B and T lymphocytes (activated B and cytotoxic
T cells) has also been described [22, 23, 27, 44, 54]. Thus,
the cell type demonstrated herein can either be viewed as a
subpopulation of microglia, which belongs to the monocytic lineage, or might represent a form of immature,
quiescent, or tolerogenic DC which lack the expression of
MHC-II. Beyond pure terminology, the latter would imply
the capability of these cells to carry antigens from the CNS
parenchyma into the draining cervical and lumbar lymph
nodes and other lymphoid organs. Such migration has only
been observed upon injection of DC [9, 17, 21] and seems
to involve the cribriform plate as major exit route from
brain to nasal mucosa [15]. However, at present, direct
evidence for a cell type carrying antigens from the brain to
lymphoid organs is lacking.
Our data unequivocally demonstrate the presence of
CD11c? cells with ramified morphology in the parenchyma of normal adult mouse brain, spinal cord and optic
nerve and of a similar population in man. Their extensions
into the glia limitans provide a possible explanation of how
intraparenchymal myelin can be presented to adoptively
transferred T cells. Of note, we found MHC-II expressing
CD11? cells in the choroid plexus, which may be involved
in the previously reported CCR6-dependent recruitment of
Th17 cells into the brain [45]. This route would also be in
line with our observation that CD11c? cells reside in the
ependyma through which T cells could be guided from the
43
Acta Neuropathol (2011) 121:445–458
ventricles. Studying changes in the CD11? population
under neuroinflammatory conditions and subsequently
relating them to MS lesions could provide very useful
information on the development, chronicity, and progression of MS.
Acknowledgments This study was supported by the DFG (FoGr
‘‘Fate of barin macrophages’’ to I.B.), the Dutch MS Research
Foundation (JDL) and the COST consortium NEURINFNET
(BM0603: J.D.L., B.B., I.B.). The Olympus Fluoview microscope was
financed by the Kassel-Stiftung, the Messer-Stiftung, and the Dr.
Senckenberg-Stiftung. We gratefully acknowledge the help of these
Frankfurt foundations. I.B. is a fellow of the Frankfurt Institute of
Advanced Studies (FIAS).
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45
4.2 CD11c-positive cells from brain, spleen, lung, and liver exhibit site-specific
immune phenotypes and plastically adapt to new environments
Kerstin Immig, Martin Gericke, Franziska Menzel, Felizitas Merz, Martin Krueger, Fridtjof
Schiefenhövel, Andreas Lösche, Kathrin Jäger, Uwe-Karsten Hanisch, Knut Biber K, Ingo
Bechmann
K. Immig, M. Gericke, F. Menzel, F. Merz, M. Krueger, F. Schiefenhövel, K. Bechmann
Institute of Anatomy, University of Leipzig, 04103 Leipzig, Germany
A. Lösche, K. Jäger
IZKF-FACS-Core Unit, Leipzig University, 04103 Leipzig, Germany
Uwe-Karsten Hanisch
Institute of Neuropathology, University of Göttingen, 37075 Göttingen, Germany
Knut Biber
Department of Psychiatry and Psychotherapy, Section of Molecular Psychiatry, University of
Freiburg, 79104 Freiburg, Germany
46
RESEARCH ARTICLE
CD11c-Positive Cells from Brain, Spleen,
Lung, and Liver Exhibit Site-Specific Immune
Phenotypes and Plastically Adapt to New
Environments
Kerstin Immig,1 Martin Gericke,1 Franziska Menzel,1 Felicitas Merz,1 Martin Krueger,1
Fridtjof Schiefenh€
ovel,1 Andreas L€
osche,2 Kathrin J€
ager,2 Uwe-Karsten Hanisch,3
Knut Biber,4 and Ingo Bechmann1
The brain’s immune privilege has been also attributed to the lack of dendritic cells (DC) within its parenchyma and the adjacent meninges, an assumption, which implies maintenance of antigens rather than their presentation in lymphoid organs.
Using mice transcribing the green fluorescent protein under the promoter of the DC marker CD11c (itgax), we identified a
juxtavascular population of cells expressing this DC marker and demonstrated their origin from bone marrow and local microglia. We now phenotypically compared this population with CD11c/CD45 double-positive cells from lung, liver, and spleen in
healthy mice using seven-color flow cytometry. We identified unique, site-specific expression patterns of F4/80, CD80, CD86,
CX3CR1, CCR2, FLT3, CD103, and MHC-II. Furthermore, we observed the two known CD45-positive populations (CD45high
and CD45int) in the brain, whereas liver, lung, and spleen exhibited a homogeneous CD45high population. CD11c-positive
microglia lacked MHC-II expression and CD45high/CD11c-positive cells from the brain have a lower percentage of MHC-IIpositive cells. To test whether phenotypical differences are fixed by origin or specifically develop due to environmental factors, we transplanted brain and spleen mononuclear cells on organotypic slice cultures from brain (OHSC) and spleen (OSSC).
We demonstrate that adaption and ramification of MHC-II-positive splenocytes is paralleled by down-regulation of MHC-II,
whereas brain-derived mononuclear cells neither ramified nor up-regulated MHC-II in OSSCs. Thus, brain-derived mononuclear cells maintain their MHC-II-negative phenotype within the environment of an immune organ. Intraparenchymal CD11cpositive cells share immunophenotypical characteristics of DCs from other organs but remain unique for their low MHC-II
expression.
GLIA 2015;63:611–625
Key words: Key words CD11c-positive cells, immune privilege, microglia
Introduction
D
endritic cells (DC) were first described in 1973 (Steinman and Cohn, 2007) and represent key players in the
initiation of the innate and adaptive immune responses. They
are specialized antigen-processing and antigen-presenting cells
(APCs), which are capable of migration to T-cell zones where
they present antigens and activate na€ıve T-cells during infection (Steinman et al., 2003b). Under steady state conditions,
immature DCs are important for peripheral tolerance, deletion of T-cells (Mohammad et al., 2012; Steinman et al.,
2003a) or expansion of regulatory T-cells (Proietto et al.,
2008). Different subsets of DCs were described in lymphoid
tissues (lymph nodes and spleen) and nonlymphoid organs,
such as liver and lung (Mesnil et al., 2012; Nakamoto et al.,
2012; van Rijt, 2005). However, besides their function,
murine DCs are mainly distinguished by their expression of
View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22771
Published online December 3, 2014 in Wiley Online Library (wileyonlinelibrary.com). Received Sep 3, 2014, Accepted for publication Nov 6, 2014.
Address correspondence to Ingo Bechmann, Institute of Anatomy, Medical Faculty, Leipzig University, Liebigstraße 13, 04103, Leipzig, Germany. E-mail: [email protected]
From the 1Institute of Anatomy, Leipzig University, Leipzig, Germany; 2IZKF-FACS-Core Unit, Leipzig University, Leipzig, Germany; 3Institute of Neuropathology,
University of G€
ottingen, G€
ottingen, Germany; 4Department of Psychiatry and Psychotherapy, Section of Molecular Psychiatry, University of Freiburg, Freiburg,
Germany
Additional Supporting Information may be found in the online version of this article.
C
V
47
CD11c and major histocompatibility complex (MHC) II
(Bulloch et al., 2008; Gottfried-Blackmore et al., 2009;
Greter et al., 2005; Jung et al., 2002; Lv et al., 2011; McMahon et al., 2005; van Rijt, 2005).
The brain‘s immune privilege was originally related to
the lack of DCs and a complete isolation of the brain from
the peripheral immune system. However, this assumption was
challenged by the discovery of DCs in the meninges and choroid plexus in healthy rat and mouse brain (Matyszak and
Perry, 1996; McMenamin, 1999; McMenamin et al., 2003).
In these studies, DCs and macrophages were identified by
expression patterns of MHC-II/OX62 and CD11b/OX42. In
addition, the detection of central nervous system (CNS)
derived antigens in cervical lymph nodes (CLN) in multiple
sclerosis (MS) patients (van Zwam et al., 2009) or in mice
after induction of primary oligodendrocyte death (Locatelli
et al., 2012) and the observation that brain leukocytes can
leave the skull to reach CLNs (Goldmann et al., 2006;
Kaminski et al., 2012) challenged the concept of a strict isolation of the CNS from the peripheral immune system. Later,
the surface antigen CD11c became a widely used tool to
identify murine DCs and to characterize their activity. However, based on the problem that immunolabeling via available
antibodies for DC markers like CD11c only provided questionable results under healthy conditions in lymphoid tissue
and mouse brain, CD11c-positive DCs were only investigated
under inflammatory conditions. This issue was addressed by
developing two transgenic mouse strains. First, a transgenic
mouse expressing a fusion protein of simian diphtheria toxin
receptor (DTR) and GFP under the promoter of CD11c,
itgax (Jung et al., 2002), was developed. Later a transgenic
Abbreviations
APC
bm
CLN
CNS
CSFR-1
d
DC
DTR
EAE
EYFP
gfp
IB4
IBA-1
jv
LN
MFI
MHC-II
MS
NIH
OHSC
OSSC
pv
wt
antigen presenting cell
bone marrow
cervical lymph node
central nervous system
colony stimulating factor 1 receptor
day
dendritic cell
diphtheria toxin
experimental autoimmune encephalomyelitis
enhanced yellow fluorescent protein
green fluorescent protein
isolectin B4
calcium-binding adaptor protein 1
juxtavascular
lymph node
mean fluorescence intensity
major histocompatibility complex II
multiple sclerosis
National Institute of Health
organotypic hippocampal slice cultures
organotypic spleen slice cultures
perivascular
wild type
mouse expressing the enhanced yellow fluorescent protein
(EYFP) under the promoter of CD11c was generated (Lindquist et al., 2004). For the first time, these models allowed
differential analyses to investigate the distribution of CD11cpositive cells in the brain under steady-state conditions
(Anandasabapathy et al., 2011; Bulloch et al., 2008; Prodinger et al., 2011). In this context, our group identified an
intraparenchymal, juxtavascular (jv) population of CD11cpositive cells by immunolabeling for laminin and electron
microscopy (Prodinger et al., 2011). Furthermore, we showed
the co-expression of these CD11c-positive cells with common
microglia markers, such as the calcium-binding adaptor protein 1 (IBA-1) and CD11b (Prodinger et al., 2011).
Studies on the nature of DC and macrophagesubpopulations in different nonlymphoid tissues (lung, liver,
gut) recently re-initiated the discussion whether DCs can be
clearly differentiated from macrophages and whether DCs
really represent a unique cell type or rather exist as “a heterogeneous subset of mononuclear cells” (Geissmann et al.,
2010; Hume, 2008; Hume et al., 2013; Randolph and
Merad, 2013).
Confusion may further arise from the fact that studies
differentiating DCs from macrophages often neglected the
important issue of whether or not these cells were isolated
from lymphoid or nonlymphoid tissues. In fact, the expression patterns of both cell types for crucial markers, such as
MHC-II, F4/80, CD11b, and CD11c, may not necessarily
be comparable between different tissues (Geissmann et al.,
2010). In various studies, DCs are distinguished from other
populations by their expression of CD11c and MHC-II. In
contrast, macrophages are frequently characterized by their
expression of CD11b and F4/80, although they are CD11c
negative (Bulloch et al., 2008; Greter et al., 2005; Ji et al.,
2013; Jung et al., 2002; McMahon et al., 2005). However,
CD11c-positive cells from lung, liver, and spleen, which are
positive for F4/80 and CD11b were regarded as DCs. Further, cells addressed as macrophages in the brain were shown
to express CD11c (McMahon et al., 2005). Outside of the
CNS, mononuclear phagocytes isolated from the gut lamina
propria express F4/80, colony stimulating factor 1 receptor
(CSFR-1), and CD11c, and were thus classified as DCs
(Bogunovic et al., 2009; Denning et al., 2007). Further,
application of DT in CD11c-DTR-GFP mice did not exclusively lead to a depletion of DCs, but also targeted macrophages in lung, spleen, and LN (Probst et al., 2005; van Rijt,
2005). Although CD11c-positive cells are known to induce
T-cell responses, this capacity was also demonstrated for macrophages in animal models for hepatitis and experimental
autoimmune encephalomyelitis (EAE) (Mesnil et al., 2012;
Nakamoto et al., 2012). Taken together, there is no consent
to a uniform definition for DCs, and criteria are often either
Volume 63, No. 4
48
Immig et al: Comparison of CD11c1 Microglia
based on presence and absence of surface markers or solely
relying on functional features, such as activation of na€ıve Tcells, migration to lymphoid organs, and antigen-presentation
(Geissmann et al., 2010; Hume, 2008; Hume et al., 2013;
McMahon et al., 2005; Randolph and Merad, 2013).
In the CNS, microglia are regarded as a population of
resident macrophages, which are characterized by their expression of CD45 intermediate (CD45int), IBA-1, and CD11b
(Carson et al., 1998; Sedgwick et al., 1991). In the past, these
cells were regarded as a bone marrow (bm) derived population (Bechmann et al., 2005). However, later it has been
demonstrated that this microglia engraftment during adulthood arises only under specific host endogenous factors
(Mildner et al., 2007). Subsequent studies demonstrated that
adult microglia rather represent an ontogenetically distinct
population in the mononuclear phagocyte system, which is
derived from progenitors of the yolk sac (Ginhoux et al.,
2010; Kierdorf et al., 2013). As mentioned above, the intraparenchymal CD11c-positive cells have also been shown to
express macrophage markers. In this context, CX3CR1 and
CCR2 represent important markers to distinguish between
the resident microglial population and inflammatory, bloodderived mononuclear phagocytes (Geissmann et al., 2003;
Harrison et al., 1998; Jung et al., 2000; Lewis et al., 2011;
Niess, 2005; Prinz and Priller, 2010).
Because it was shown that the DC marker CD11c is
also expressed on macrophages (McMahon et al., 2005;
Probst et al., 2005; van Rijt, 2005), and the discussion raised
if DCs can be clearly differentiated from macrophages (Geissmann et al., 2010; Hume, 2008; Hume et al., 2013; Randolph and Merad, 2013), we wanted to compare brainderived CD11c-positive cells to CD11c-positive cells derived
from other organs. To characterize the CD11c-positive populations of the brain, we phenotypically compared both
CD45int/CD11c-positive and CD45high/CD11c-positive cells
to CD11c/CD45-positive populations from spleen, liver, and
lung by seven-color flow cytometry and further analyzed the
influence of local factors on MHC-II expression in organotypic spleen (OSSCs) and hippocampal slice cultures
(OHSCs).
Materials and Methods
Mice
Mice strains were maintained in our animal facility at 12 h light and
dark cycle and with free access to food and water. For flow cytometry analyses, 6 weeks old male wild type (wt) C57BL/6J (n 5 3–4)
mice were used. For the preparation of the organotypic slice cultures,
5–8 days (d) old wt C57BL/6J mice and 5–8 d old CD11c-GFP/
DTR mice (n 5 3) (Jung et al., 2002) were used. Adult mice
expressing an enhanced green fluorescent protein under the promoter
of MHC-II (MHC-II-eGFP; kindly provided by Ana-Maria
April 2015
Lennon-Dumenil, Institut Curie in Paris, France; n 5 4–5) (Boes
et al., 2002) were used to co-cultivate isolated mononuclear cells
from spleen and brain on wt organotypic slice cultures. Mice were
housed in the local animal facility of the Leipzig University under
the guidelines of the Animal Experimental Committee. All experiments were performed in accordance with the National Institute of
Health (NIH) guidelines and approved by the local authorities.
Isolation of Mononuclear Cells
Animals were killed by CO2 and subsequently perfused with ice-cold
PBS (Gibco, Life technologies, Darmstadt, Germany) to clear the
intravascular compartment of the brain from blood cells. Brain, liver,
lung, and spleen were removed and transferred into ice-cold medium
(50 ml HBSS (Gibco) supplemented with 650 ml 45% D-Glucose
(Sigma-Aldrich, Taufkirchen, Germany) and 750 ml 1M HEPES
(Gibco). Organs were minced and homogenized using a glass potter
(Novodirect, Kehl, Germany) followed by trituration of fire-polished
pasteur pipettes with decreasing diameters. Cell suspension was filtered through a 70 mm cell strainer (BD Falcon, BD Biosciences,
Heidelberg, Germany) followed by a careful centrifugation (Labofuge
400R, Heraeus Instruments) by 900 rpm at 4 C for 10 min. Pellets
of liver, lung, and spleen cells were incubated in red-blood-cell lysis
buffer for 30 min on ice and subsequently washed in PBS. Brain
cells were re-suspended in 10 ml of 75% Percoll solution (GE
Healthcare, M€
unchen, Germany) covered with 10 ml of 25% Percoll
solution. Finally, 6 ml of PBS were carefully added on the top. The
cell gradient was centrifuged at 1,800 rpm at 4 C for 30 min. After
centrifugation the myelin-containing layer was carefully removed.
Mononuclear brain cells were collected from the thin layer between
the 25 and 75% Percoll layer. Mononuclear cells were discriminated
from lymphocytes by granularity and size and for specific cell
markers (see below flow cytometry staining).
Preparation of OHSC
Brains from 5–8 d old CD11c-DTR/GFP or wt mouse pubs were
carefully removed. Forebrains were stored in ice-cold HBSS, 0.45%
D-glucose, 1% PenStrep (Gibco), and 1% L-glutamine (Gibco). Further, forebrains were carefully cut in 350 mm thick slices using a
vibratome (Leica VT1200S). Subsequently, hippocampi were collected and incubated for 30 min with Alexa647-conjugated isolectin
B4 from Griffonia simplicifolia (IB4, 1:100; Molecular Probes, Life
Technologies), diluted in pre-warmed medium containing 47%
MEM (Gibco), 25% HBSS, 25% normal horse serum (Gibco), 1%
glucose (45%), 1% PenStrep, and 1% L-glutamine. Slices containing
hippocampus (OHSC) were washed and transferred to cell culture
inserts (Millipore, Schwalbach, Germany). OHSCs from 5 to 6 d
old CD11c-GFP/DTR mice were directly used for live imaging.
OHSCs from 5 to 8 d old wt mice were used for co-cultivation
with brain or spleen mononuclear cells and were cultivated for 6 d
(37 C, 5% CO2, 60% humidity).
Preparation of OSSC
Spleens were removed from 5 to 8 d old mice and stored in ice-cold
medium as described above. About 3% agarose gel was prepared
using low melting agarose (Roth, Karlsruhe, Germany). Embedded
49
spleens were carefully cut in 350 mm thin slices with the vibratome.
Subsequently, slices were collected in preparation medium and
removed from agarose residues.
Co-Cultivation of Brain or Spleen Mononuclear Cells
on OHSC and OSSC
Mononuclear cells from spleen and brain were isolated from the
MHC-II-eGFP mice as described above. Isolated mononuclear cells
were incubated with the fluorescent dye PKH26 (Sigma-Aldrich) dissolved in culture medium as described in the providers protocol. Of
note, isolated mononuclear cells, using Percoll gradient, contain at
least up to 90–97% of microglia verified by CD45int and CD11b
expression. About 3 ml of the cell suspension was applied on the slice
cultures and incubated for 2 h. Subsequently, live imaging was performed. In addition, slices were fixed 2, 24, and 72 h after starting
co-cultivation using 1% PFA. Slices were washed with PBS and
counterstained by HOECHST 33312 (Sigma). Further analyses were
performed with an epifluorescence microscope (Olympus BX40)
equipped with an XM10 camera. Images were processed with ImageJ
software.
Live Imaging
Live imaging was performed using confocal microscopy (Olympus
FV1000) equipped with a climate chamber at 37 C, 5% CO2, and
60% humidity. Images were acquired every 15 min for up to 24 h
starting either 2, 24, or 72 h after application of MHC-II-eGFP cells.
Flow Cytometry Staining
Mononuclear cells were isolated and prepared as described above.
Cells were pre-incubated with anti-CD16/32 antibody (1:100, eBioscience, San Diego, CA) for 10 min to minimize unspecific binding
of antibodies on Fc-receptors. Next, cells were incubated for 30 min
with primary labeled antibodies on ice; CD11b-Alexa Flour 700
(1:100, eBioscience), CD45-FITC (1:250, eBioscience), CD11ceFlour450 (1:80, eBioscience,), CD80-APC (1:100, eBioscience),
CD86-PE-Cy5 (1:100, eBioscience), F4/80-PE-Cy7 (1:100, eBioscience), I-A[b]-PE (1:100, BD Pharmingen, BD Biosciences), or
CD103-PE (1:100, BD Pharmingen). Nonprimary-labeled antibodies
[CCR2 (1:200, Abcam, Cambridge, MA), CX3CR1 (1:200, Abcam),
and Flt3 (1:200, Abcam) were incubated for 30 min. After washing
twice with PBS, cells were incubated with the secondary antibody
(goat anti-rabbit-Alexa Flour 633 (1:200, Invitrogen)]. Further, cells
were rinsed in PBS and centrifuged for 2 min at 1,200 rpm. LSRII
(BD) was used for cell staining measurements. For all flow cytometry
stainings gates were set according to blank and isotype controls used
in each experiment (Supp. Info. Fig. S1A–D).
Statistical Analyses
Flow cytometry stainings (percentages and MFIs) were analyzed
using FlowJo (FlowJo). ImageJ was used for cell counting. Data of
at least three independent experiments were evaluated by Dunnetttest or Newman-Keuls multiple comparison and presented as standard derivation 6 SD using GraphPad Prism. P values <0.05 were
considered to be statistically significant (* P < 0.05, ** P 0.01, and
*** P 0.001).
Results
Identification of Two Different CD11c/CD45Positive Cell Fractions From the Brain
To phenotypically characterize the previously described population of CD11c-positive cells of the brain in direct comparison to CD11c-positive cells of other organs, we first
established an isolation method valid for all investigated
organs. Isolated cells were stained for CD45 with subsequent
gating to CD11c and analyzed by flow cytometry (Fig. 1A).
All gates were set according to the isotype controls used for
all organs and stainings. The MFI of each staining was calculated by the MFI of the appropriate antibody staining subtracted by the respective isotype control. Thus, no significant
differences in the percentage of CD11c-positive cells among
the CD45-positive fraction were observed between brain,
liver, and lung, whereas the ratio of spleen CD11-positive
cells appeared to be significantly lower (Fig. 1B).
According to the literature, there are two different populations of CD45-expressing cells in the brain (Carson et al.,
1998; Sedgwick et al., 1991). Although CD45int cells are
addressed as microglia, the CD45high fraction is represented
by brain macrophages including perivascular (pv) macrophages and meningeal DCs (Fig. 1C). Of note, both brain
populations contained CD11c-positive cells. In contrast,
spleen, liver, and lung showed a homogenous population of
CD45high-expressing cells. Further, both brain-derived CD45positive populations exhibited significantly lower amounts of
CD11c-positive cells, as compared with the CD45-positive
population of the lung (Fig. 1D). Of note, CD11c-positive
microglia and CD11c-positive brain macrophages also showed
significant lower expression levels of CD11c compared with
other organs, as illustrated by the MFI (Fig. 1E).
Up-Regulation of CD11c in Jv Microglia in OHSC
We have previously shown that the number of CD11cpositive cells is increasing in OHSCs over time (Prodinger
et al., 2011). We now performed, live imaging of IB4-labeled
microglial (arrow) cells observing the up-regulation of CD11c
in OHSC from 5 to 8 d old CD11c-DTR/GFP mice. We
found that, migrating microglia were induced to express
CD11c upon reaching microvessels (which were also
demarked by IB-4 labeling, dashed line) (Fig. 2).
CD11c-Positive Microglia are Lacking MHC-IIExpression
MHC-II represents an important cell surface marker for DCs
and APCs, relating to the ability to present antigens to CD4positive T-cells. Therefore, we in this study analyzed both
CD11c-positive fractions of the brain for their expression of
MHC-II in direct comparison to CD11c-positive cells from
peripheral organs. In this context, both brain fractions
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Immig et al: Comparison of CD11c1 Microglia
FIGURE 1: Overview of the CD11c-positive cell fraction among CD45-positive cells in brain, spleen, liver, and lung in healthy mice. (A)
Cells were gated in the forward scatter (FSC) for CD45. CD45-positive cells were further gated for CD11c-positive cells and (B) compared for their percentage among CD45-positive cells from brain, spleen, liver, and lung. (C) Discrimination between CD45int- and
CD45high-expressing cells in the brain. (D) Comparison to the CD11c/CD45high-positive cells from spleen, liver, and lung for their percentage and MFI.
April 2015
51
FIGURE 2: Up-regulation of CD11c of parenchymal and IB4-positive cells in OHSCs. OHSCs were pre-incubated with IB4 for 30 min. At
regular intervals of 34 min, images of the OHSCs were acquired using confocal microscopy. The arrow marks the IB4-positive cell and
the dashed line the blood vessel.
showed significantly lower ratios of MHC-II-expressing cells
compared with those obtained from spleen and liver (Fig.
3A). Moreover, the percentage of MHC-II expressing cells
among the population of CD11c-positive microglia was dramatically lower compared with brain macrophages. In fact,
the ratio of 0.47% in combination with significantly lower
expression levels of MHC-II impressively indicates that
MHC-II expression is almost lacking in CD11c-positive
microglia (Fig. 3B). Thus, intraparenchymal CD11c-positive
microglia are unique for their absence of MHC-II expression.
Unique and Site-Specific Expression Patterns of F4/
80, CX3CR1, CCR2, CD80, CD86, Flt3, and CD103
Because the above described comparison showed a unique phenotype of brain-derived CD11c/CD45-positive cells, we
extended our analysis for the expression of important
FIGURE 3: Comparison of MHC-II-positive cells among CD11c/CD45-positive cells in brain, liver, lung, and spleen in healthy mice. Flow
cytometry analyses of the percentages (A) and MFIs (B) of MHC-II-positive cells among CD11c/CD45-positive brain cells compared with
CD11c/CD45-positive cells from spleen, liver, and lung are shown.
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Immig et al: Comparison of CD11c1 Microglia
mononuclear and inflammatory cell markers (F4/80, CX3CR1,
CCR2, CD80, CD86, Flt3, CD103) (Supp. Info. Fig. S1 and
Table S1) with respect to the percentage of positive cells and
intensity of protein expression measured by MFI.
The highest amount of F4/80 expression was found in the
CD11c-positive microglia fraction (Fig. 4A). Also, brain macrophages showed a significantly higher ratio of F4/80-positive cells
compared with spleen, liver, and lung, whereas the MFI of F4/
FIGURE 4: Comparison of F4/80, CX3CR1, CD80, CD86, CCR2, CD103, Flt3-positive cells among CD11c/CD45-positive cells in brain,
spleen, liver, and lung. Flow cytometry analyses of the percentages (A) and MFIs (B) of positive cells among CD11c/CD45-positive cell
fractions are shown for F4/80, CX3CR1, CD80, CD86, CCR2, CD103, and Flt3.
April 2015
53
FIGURE 4: (Continued)
80 in both CD11c-positive brain populations was found to be
significantly lower than in spleen and liver (Fig. 4B).
CD11c-positive microglia contained by far the highest
percentage of CX3CR1-positive cells (Fig. 4A) with comparable MFI to mononuclear populations from other organs,
despite liver macrophages (Fig. 4B). Interestingly, brain macrophages expressed only low amounts of CX3CR1 (Fig. 4A,B).
The expression of the co-stimulatory molecules CD80
and CD86 measured by either percentage or MFI was similar
for CD11c-positive microglia and CD11c-positive brain macrophages. However, higher amounts of CD80- and CD86positive cells were detected in the liver, whereas lung CD11cpositive cells expressed the highest MFI for CD86 (Fig. 4A).
Although no significant differences were observed between
the percentages of CCR2-positive cells within the CD11cpositive fractions of all isolated mononuclear cells (Fig. 4A),
the CCR2 expression levels (measured by MFI) of both brain
CD11c-positive fractions were significantly lower compared
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Immig et al: Comparison of CD11c1 Microglia
FIGURE 5: Signatures of investigating CD11c/CD45-positive cells for their expression of F4/80, CX3CR1, CD80, CD86, CCR2, CD103,
and Flt3. Signatures of the (A) percentages and (B) MFI among CD11c/CD45-positive cells in brain, spleen, liver, and lung are shown for
F4/80, CX3CR1, CD80, CD86, CCR2, CD103, and Flt3.
with spleen, liver, and lung (Fig. 4B) and number of CCR2positive brain mononuclear cells was low under these steadystate conditions. Moreover, the highest amount of CD103positive cells was found in the CD11c-positive cell fraction of
the liver. In brain macrophages, CD103 could not be
detected (Fig. 4A). No differences were measured for the
expression intensity of CD103-positive cells between all investigated CD11c-positive populations (Fig. 4B). Of note,
CD11c-positive microglia showed the highest percentage of
Flt3-positive cells (Fig. 4A). In contrast, no Flt3-positive cells
were observed in the CD11c-positive brain macrophages. The
highest expression intensity for Flt3 was observed in lungderived CD11c-positive cells (Fig. 4B).
In summary, the comparison of crucial cell surface
markers between the investigated CD11c-positive fractions of
the brain parenchyma by flow cytometry revealed unique and
site-specific expression patterns compared with the other
extraneural CD11c-positive cells. For visualization, we combined the results to create signatures for each of the analyzed
CD11c-positive cells, reflecting either the amount of positive
cells (Fig. 5A) or the MFI (Fig. 5B).
CD11c-Positive and Negative Microglia Show the
Same Expression Patterns for MHC-II
To study differences between CD11c-positive and CD11cnegative microglia, we compared the above mentioned cell
markers for their percentage (Fig. 6A) and MFI (Fig. 6B). In
this study, we detected a higher amount of F4/80, CCR2,
April 2015
CD86, and Flt3-positive cells in the fraction of CD11cpositive microglia (Fig. 6A). Measuring the expression we
observed significant higher expression of CX3CR1, but less
CCR2 in CD11c-positive microglia compared with the
CD11c-negative microglia (Fig. 6B). Most importantly,
MHC-II expression did not differ between CD11c-positive
and CD11c-negative microglia (Fig. 6A,B). Taken together,
we observed that CD11c-positive microglia are not equal to
CD11c-negative microglia in their immune phenotype (F4/
80, CCR2, CD86, Flt3), but both CD11c-negative and
CD11c-positive microglia shared uniquely low MHC-II
levels.
Microglia Maintain Their MHC-II-Negative
Phenotype in the Environment of an Immune Organ
To test whether the unique MHC-II-negative phenotype of
microglia is driven by local cues or inherent to microglia, we
transplanted microglia on OSSCs to determine whether this
environment causes up-regulation of MHC-II. Vice versa,
splenocytes were transplanted on entorhino-OHSCs (Hailer
et al., 1998; Vinet et al., 2012) to test whether this causes
down-regulation of MHC-II. As shown above, the low
MHC-II expression is similar in CD11c-positive and negative
microglia. Thus, we focused in this experiment on the expression of MHC-II alone, a key-molecule for antigen presentation, to investigate if the unique MHC-II-phenotype of
microglia is driven by local cues or inherent. Cells were isolated from MHC-II-eGFP mice (Boes et al., 2002) and
55
FIGURE 6: Comparison of CD11c-positive and CD11c-negative microglia (CD45int). Comparison of the percentages (A) and MFIs (B)
among CD11c-positive and CD11c-negative microglia for MHC-II, F4/80, CX3CR1, CCR2, CD80, CD86, CD103, and Flt3.
stained using PKH26 before transplantation on slices. Using
flow cytometry, we confirmed that the vast majority of brainderived mononuclear cells were MHC-II-negative, whereas
splenocytes regularly expressed MHC-II (Fig. 7A). Analysis of
histological sections further confirmed the absence of MHCII-positive cells in the brain parenchyma properly. However,
individual MHC-II-positive cells were regularly detected in
the meninges, choroid plexus and in the perivascular compartment (Fig. 7B). Upon transplantation onto organotypic
slice cultures from wt mice, live imaging was performed at 2,
24, and 72 h for at least 2 h. Additional slices were fixed
with 1% PFA and analyzed by fluorescence microscopy.
Amoeboid PKH26-labeled splenocytes lost their eGFP
expression and ramified within OHSCs (Fig. 8A,B,D),
whereas brain-derived mononuclear cells remained MHC-IInegative on OSSCs (Fig. 8A,B,D). No significant changes
were observed for splenocytes on OSSCs or brain mononuclear cells on OHSCs and OSSCs (Fig. 8A,B). Ramification
of splenocytes on OHSCs correlated to fate of MHC-II (Fig.
8A–D). Taken together, brain mononuclear cells maintain
their MHC-II-negative phenotype in the environment of an
immune organ, whereas splenocytes down-regulate MHC-II
and ramify to a microglia-like morphology in OHSCs.
Discussion
In the current study, we identified two distinct CD11cpositive cell populations within the CD45int and CD45high
cell fraction of the brain under physiological conditions.
CD45high- expressing cells are commonly addressed as meningeal and pv DCs, or as macrophages of peripheral origin
(Carson et al., 1998; Sedgwick et al., 1991), whereas
CD45int- expressing cells are commonly considered as microglia (Carson et al., 1998; Sedgwick et al., 1991), which originate from yolk sac-derived macrophage progenitors and
populate the brain parenchyma during the prenatal
Volume 63, No. 4
56
Immig et al: Comparison of CD11c1 Microglia
FIGURE 7: Analyses of brain and spleen from MHC-II-eGFP mice. (A) Flow cytometry analyses of isolated spleen and brain mononuclear
cells from MHC-II-eGFP mice labeled with PKH26. (B) Histological sections of brain and spleen from MHC-II-eGFP mice.
development (Alliot et al., 1999; Ginhoux et al., 2010; Vinet
et al., 2012).
In previous studies Greter et al. demonstrated the existence of vessel-associated CD11c-positive cells, which
appeared to be located in pv spaces (Greter et al., 2005). Furthermore, using fluorescence and electron microscopy our
group identified an intraparenchymal population of CD11cpositive cells (Prodinger et al., 2011), which express the macrophage marker IBA-1 and CD11b. In continuation of our
previous work, the current study demonstrates that cells
expressing a microglial phenotype (CD45int, CD11b) contain
a distinct subpopulation expressing CD11c. This is in line
with previous studies confirming the existence of CD11cpositive cells in the embryonic, neonatal, and adult mouse
brain parenchyma (Bulloch et al., 2008). However, due to the
recent debate on the nature of DCs (Geissmann et al., 2010;
April 2015
Hume, 2008; Randolph and Merad, 2013), the question
arose whether this parenchymal population can be addressed
as DCs, implying the ability to stimulate na€ıve T-cells and to
transfer antigens into lymphoid organs, or whether the
described population should more appropriately be addressed
as a CD11c-positive fraction of microglia. In recent studies,
Wlodarczyk et al. showed that CD11c-positive microglia did
not express Th1- or Th17-promoting cytokines in EAE but
could induce proliferation of primed CD4-positive T-cells
(Wlodarczyk et al., 2014). As mentioned before, in previous
studies cells expressing CD11c were addressed as macrophages, and cells addressed as DCs were shown to possess
macrophage features (Bogunovic et al., 2009; Denning et al.,
2007; McMahon et al., 2005; Probst et al., 2005). To avoid
further confusion in the current study, the population of
CD45high cells from the meninges or choroid plexus are
57
FIGURE 8: Analyses of brain and spleen mononuclear cells from MHC-II-eGFP mice, co-cultivated on wt OHSCs and OSSCs, for their
MHC-eGFP expression and ramification. Comparison of MHC-II-eGFP expression (A), cell ramification (B), and the correlation (C) between
MHC-II-eGFP-positive cells and ramification of brain and spleen mononuclear cells isolated from the MHC-II-eGFP mice, labeled with
PKH26 and co-cultivated on wt hippocampus and spleen slice cultures. (D) Representative microscopic analyses of brain and spleen
mononuclear cells isolated from the MHC-II-eGFP mouse, labeled with PKH26, are shown for the co-cultivation for the time points 2, 24,
and 72 h after application on wt organotypic slice cultures from hippocampus (OHSC) and spleen (OSSC).
addressed as brain macrophages. In contrast, intraparenchymal CD11c-positive cells are in this study addressed as
CD11c-positive microglia, which reflects their expression of
IBA-1, CD11b, and CD45int.
To compare this population with CD11c-positive cells
from other organs, we adapted the microglia Percoll isolation
protocol (Haas et al., 2008) to isolate in the same way
CD11c-positive cells from brain, spleen, liver, and lung.
Volume 63, No. 4
58
Immig et al: Comparison of CD11c1 Microglia
Direct comparison of the isolated CD11c-positive populations
by flow cytometry showed that in contrast to CD11c-positive
cells from peripheral organs, CD11c-positive microglia almost
completely lacked MHC-II expression. Consistent with our
observations, previous studies reported that CD11c-positive
cells of the parenchyma are lacking MHC-II; its expression
was found to be restricted to the meninges and choroid
plexus under physiological conditions (Lv et al., 2011; Prodinger et al., 2011). In this study, we further demonstrate
that the percentage of MHC-II-expressing CD11c-positive
brain macrophages is also significantly lower compared with
spleen and liver, which is likely to reflect the influence of
local factors suppressing MHC-II expression. Thus, in contrast to the CD11c-positive cells obtained from spleen and
lung, CD11c-positive microglia do not seem to be equipped
for antigen-presentation to CD4-positive cells under steady
state conditions.
To investigate further differences in the expression for
common macrophage or DC markers, we compared both
CD11c-positive populations of the brain to CD11c-positive
populations of peripheral organs and found that CD11cpositive microglia contain a higher percentage of cells expressing CX3CR1 and F4/80, which are known as markers for resident macrophages and microglia (Cardona et al., 2006;
Carson et al., 1998; Harrison et al., 1998; Jung et al., 2000;
Lewis et al., 2011; Niess, 2005; Prinz et al., 2011; Wlodarczyk et al., 2014). Further, in direct comparison to liver-,
lung- and spleen-derived CD11c-positive cells, CD11cpositive microglia and brain macrophages showed significantly
lower expression levels of the mononuclear cell marker CCR2
(MFI), whereas the overall percentage of CCR2-positive cells
among the different CD11c-positive populations did not differ. This is in line with the view that microglia are resident,
whereas CCR2-positive inflammatory monocytes are known
to be highly mobile (Prinz et al., 2011).
CD103-positive DCs were first identified within the
lamina propria of the gut and have been described as important key players to induce immune tolerance (Scott et al.,
2011). Among CD11c-positive microglia, we also detected a
small amount (4.7%) of cells positive for CD103, whereas
CD11-positive brain macrophages did not express CD103. In
line with our data, previous studies described CD45int and
CD45high CD103/CD11c double-positive cells in the olfactory bulb in a vesicular stomatitis virus model (D’Agostino
et al., 2012). In this study, the CD45high expressing cells have
been shown to promote T-cell proliferation, although an
immunological role for the CD45int microglia-like CD103/
CD11c-positive population has not been demonstrated
(D’Agostino et al., 2012).
Flt3L has been shown to drive maturation to DCs in
meningeal CD11c-positive cells (Anandasabapathy et al.,
April 2015
2011). We did not detect Flt3 in brain macrophages, but the
percentage of Flt3-positive cells was highest in microglia compared with CD11c-positive cells from spleen, lung, and liver.
CD80 and CD86 represent co-stimulatory molecules,
which are important for antigen-presentation to T-cells. Interestingly, although MHC-II expression was not detectable in
CD11c-positive microglia, the expression pattern of both costimulatory molecules turned out to be comparable to the other
CD11c-positive fractions from brain, spleen, and lung under
steady-state conditions. Also the MFI for both molecules was
found to be similar except for CD11c-positive cells of the lung,
which exhibited the highest expression intensity of CD86. Similar results have been observed in recent publications for both
CD11c/CD45-positive brain populations compared with
CD11c-positive cells from spleen under inflammatory conditions (Wlodarczyk et al., 2014). Further, the presence of at least
one of these co-stimulatory molecules is required for T-cell activation and their expression has been described for a small subset
of adult microglia (Carson et al., 1998) where CD86 seems to
be related to protection and tolerance (Bechmann et al., 2001;
Kuchroo et al., 1995; Mutlu et al., 2007).
In the current study, we analyzed CD11c-positive cells
obtained from different organs for their expression patterns of
crucial DC/macrophage markers and thereby created specific
signatures of their expression profiles reflecting the percentages of positive cells among the CD11c-positive fractions
and the expression intensity of the investigated antigens.
These signatures were strikingly different between brain, lung,
liver, and spleen. Therefore, we can conclude that CD11cpositive cells represent a heterogenic population exhibiting
features, which strongly depend on their origin and location.
Thus, efforts for their classification or denotation in publications should be made more carefully and with respect to the
mentioned parameters.
Of note, comparison of CD11c-positive microglia with
CD11c-negative microglia showed higher amounts of F4/80,
CCR2, CD86, and Flt3-positive cells in the fraction of
CD11c-positive microglia. Furthermore, CCR2 is expressed
less, whereas CX3CR1 is expressed at a higher intensity in
CD11c-positive microglia compared with CD11c-negative
microglia. Remarkably, both microglia fractions are lacking
MHC-II expression. This phenotype remained stable upon
transfer onto OSSCs, whereas spleen-derived MHC-II-eGFP
cells lost their green fluorescence and ramified on OHSCs
supporting the view that local factors specifically adopt
mononuclear cells within the brain’s parenchyma (Abutbul
et al., 2012; Hailer et al., 1998) where this ramified,
“inactivated” phenotype exhibits neuroprotective effects
(Abutbul et al., 2012).
Our data provide an overview of the phenotype of different CD11c-positive cells derived from different organs and
59
specifically demonstrate the unique and “inactivated” phenotype of CD11c-positive microglia under physiological conditions. These findings are useful in studying changes of brain
CD11c-positive populations under neuroinflammatory conditions in comparison to changes of CD11c-positive cells
derived from other organs. This could give new insights for
the understanding of the progression and development of
chronic neuroinflammatory diseases such as MS and Alzheimer‘s disease.
Randolph GJ, Merad M. 2009. Origin of the lamina propria dendritic cell network. Immunity 31:513–525.
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EM, Kaunzner UW, Liu K, Lindquist R, Nussenzweig MC, Steinman RM,
McEwen BS. 2008. CD11c/EYFP transgene illuminates a discrete network of
dendritic cells within the embryonic, neonatal, adult, and injured mouse
brain. J Comp Neurol 508:687–710.
Cardona AE, Pioro EP, Sasse ME, Kostenko V, Cardona SM, Dijkstra IM,
Huang D, Kidd G, Dombrowski S, Dutta R, Lee JC, Cook DN, Jung S, Lira
SA, Littman DR, Ransohoff RM. 2006. Control of microglial neurotoxicity by
the fractalkine receptor. Nat Neurosci 9:917–924.
Carson MJ, Reilly CR, Sutcliffe JG, Lo D. 1998. Mature microglia resemble
immature antigen-presenting cells. Glia 22:72–85.
Acknowledgment
Grant sponsor: Deutsche Forschungsgemeinschaft; Grant
number: DFG-FOR1336 (B2 to IB, C1 to KB, and A1 to
UKH); Grant sponsor: Helmholtz Alliance ICEMED (Imaging and Curing Environmental Metabolic Diseases).
The authors thank Sonja Kallendrusch (Institute of Anatomy, Leipzig University) and Kristin Schubert (Department of
Experimental Rheumatology, University Hospital Leipzig) for
the help in establishing the co-cultivation experiment. Further
the authors thank to Lena Wendeburg (Department of Psychiatry and Psychotherapy, Section of Molecular Psychiatry, University of Freiburg, Freiburg) for the help in establishing the
isolation of mononuclear cells and Constanze Hobusch (Institute of Anatomy, Leipzig University) and Bianka Graf (Institute of Anatomy, Leipzig University) for technical assistance in
tissue preparation. Also the authors thank to Dr. Petra FinkSterba, Dr. Petra Hirrlinger, and Sigrid Weisheit (MedizinischExperimentelles Zentrum, Leipzig University) for animal care.
D’Agostino PM, Kwak C, Vecchiarelli HA, Toth JG, Miller JM, Masheeb Z,
McEwen BS, Bulloch K. 2012. Viral-induced encephalitis initiates distinct and
functional CD1031 CD11b1 brain dendritic cell populations within the olfactory bulb. Proc Natl Acad Sci USA 109:6175–6180.
Denning TL, Wang Y-C, Patel SR, Williams IR, Pulendran B. 2007. Lamina
propria macrophages and dendritic cells differentially induce regulatory and
interleukin 17-producing T cell responses. Nat Immunol 8:1086–1094.
Geissmann F, Gordon S, Hume DA, Mowat AM, Randolph GJ. 2010. Unravelling mononuclear phagocyte heterogeneity. Nat Rev Immunol 10:453–460.
Geissmann F, Jung S, Littman DR. 2003. Blood monocytes consist of two
principal subsets with distinct migratory properties. Immunity 19:71–82.
Ginhoux F, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, Mehler MF,
Conway SJ, Ng LG, Stanley ER, Samokhvalov IM, Merad M. 2010. Fate mapping analysis reveals that adult microglia derive from primitive macrophages.
Science 330:841–845.
Goldmann J, Kwidzinski E, Brandt C, Mahlo J, Richter D, Bechmann I. 2006.
T cells traffic from brain to cervical lymph nodes via the cribroid plate and
the nasal mucosa. J Leukocyte Biol 80:797–801.
Gottfried-Blackmore A, Kaunzner UW, Idoyaga J, Felger JC, McEwen BS,
Bulloch K. 2009. Acute in vivo exposure to interferon- enables resident brain
dendritic cells to become effective antigen presenting cells. Proc Natl Acad
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61
5 Zusammenfassung
Dissertation zur Erlangung des akademischen Grades Dr. rer. med.
Titel: Immunphänotypische Charakterisierung CD11c-positiver Zellen des Gehirns im
direkten Vergleich zu CD11c-positiven Zellen von Lunge, Leber und Milz
Eingereicht von: Kerstin Immig
Angefertigt am: Institut für Anatomie der Medizinischen Fakultät der Universität Leipzig
Betreuer: Herr Professor Dr. med. Ingo Bechmann
Eingereicht im: 21.07.2015
Das Fehlen von Dendritischen Zellen (DCs) im Gehirn wurde oft in kausalen Zusammenhang
zum Immunprivileg des Gehirns gebracht. Die Ansicht, dass es keine DCs im Gehirn gibt,
wurde jedoch mit der Beobachtung von CD11c- und Major Histocompatibility Complex
(MHC)-II-positiven Zellen in den Meningen und im Plexus choroideus sowohl in Ratten als
auch in Mäusen in Frage gestellt. CD11c und MHC-II sind zwei Marker, welche am
häufigsten für die Identifizierung von DCs verwendet wurden. Da aber fast alle zur Verfügung
stehenden Antikörper gegen DCs unter nichtinflammatorischen Bedingungen keine klaren
Ergebnisse im neuralen Gewebe erbrachten, gab es kaum Untersuchungen von DCs unter
diesen Bedingungen. Die Entwicklung des transgenen Mausmodells, welche unter dem
Promotor von CD11c das grün-fluoreszierende Protein (GFP) exprimiert, war ein
entscheidender Schritt, DCs und CD11c-positive Zellen unter nichtinflammatorischen
Bedingungen im Gehirn zu untersuchen. Unter Benutzung dieses Mausmodells untersuchten
wir CD11c-positive Zellen aus dem Gehirn unter gesunden Bedingungen und verglichen
diese mit CD11c-positiven Zellen aus anderen Organen.
Dabei zeigten wir zum ersten Mal, dass auch im gesunden Mausgehirn juxtavaskuläre,
intraparenchymal lokalisierte Zellen existieren, welche CD11c exprimieren. Darüber hinaus
waren die Zellen in der Tela des Plexus choroideus, unter dem Ependym der lateralen
Ventrikel, entlang des Corpus callosum und um den Fornix verteilt. Im Gegensatz zu den
meisten im Plexus choroideus lokalisierten CD11c-positiven Zellen, zeigten wir, dass die
intraparenchymal lokalisierten Zellen ebenfalls die Mikroglia-/Makrophagenmarker IBA-1 und
CD11b exprimierten. Auch vereinzelte Fortsätze dieser CD11c-positiven Zellen waren am
Aufbau
der
Glia
limitans
beteiligt.
Sowohl
mittels
konfokaler,
als
auch
mittels
62
Elektronenmikroskopie, beobachteten wir, dass diese Fortsätze an der Bildung der Glia
limitans beteiligt waren und bis an ihre Basalmembran grenzten. Weitergehend nutzten wir
das Modell der organotypischen Schnittkulturen des Hippocampus (OHSC) und zeigten,
dass CD11c-positive Zellen sich aus intraparenchymalen Vorläufern entwickeln können.
Ebenso wiesen wir nach, dass sich die CD11c-positiven Zellen auch aus Vorläuferzellen des
Knochenmarks
entwickeln
können.
Zusammenfassend
konnten
wir
zeigen,
dass
intraparenchymal lokalisierte Zellen Eigenschaften von DCs teilen, in dem sie CD11c
exprimieren, aber zeitgleich auch typische Expressionsmarker für Mikroglia bzw.
Makrophagen aufwiesen.
Etwa zur gleichen Zeit wurde in zahlreichen Publikationen gezeigt, dass der DC-Marker
CD11c auch auf anderen Zelltypen, etwa wie B-Zellen und Makrophagen, exprimiert werden
kann. Dies führte zu der Diskussion, ob sich DCs als eigenständige Zellpopulation darstellen
lassen und ob sie überhaupt von Makrophagen unterschieden werden können. Deshalb
stellte sich uns die Frage wie sich CD11c-exprimierende Zellen des Gehirns von CD11cpositiven Zellen aus anderen Organen unterscheiden.
Dafür isolierten wir mit Hilfe eines einheitlichen Isolierungsprotokolls mononukläre Zellen aus
dem Gehirn sowie aus der Lunge, der Leber und der Milz. Danach färbten wir diese Zellen
mit etablierten monozytären und inflammatorischen Zellmarkern und verglichen diese Zellen
miteinander mittels Durchflusszytometrie. Dabei nutzten wir den prozentualen Anteil der
positiven Zellen für den entsprechenden Marker als auch die Expressionsintensität (MFI),
welche eine Aussage erlaubt, wie stark der entsprechende Marker von der Zelle exprimiert
wird. Zunächst wurden die isolierten Zellen auf den Panleukozytenmarker CD45 untersucht.
In der Lunge, Leber und Milz haben wir eine homogene CD45 hoch-exprimierende (CD45high)
Population isolieren können, wohingegen im Gehirn hinsichtlich der Expression von CD45
zwei unterschiedliche Populationen nachweisbar waren. Wie bereits in der Literatur
beschrieben, ist die CD45intermediär (CD45int)-exprimierende Population, welche einen Anteil
von bis zu 99 % der mononukleären Zellen im Gehirn ausmacht, als Mikroglia beschrieben.
Bei den wenigen CD45high-exprimierenden Zellen handelt es sich wiederum um
Makrophagen und DCs aus den Meningen und dem Plexus choroideus. Danach haben wir
unsere isolierten Zellen auf die Expression von CD11c untersucht und konnten in allen
Populationen CD11c-positive Zellen isolieren. Der prozentuale Anteil CD11c-positiver Zellen
in der Mikrogliapopulation war geringer als der prozentuale Anteil in der Lunge und Leber.
Auch
der
prozentuale
Anteil
CD11c-positiver
Zellen
unter
den
perivaskulären
Makrophagen/DCs war geringer als jener in der Lunge. Weiterhin untersuchten wir die
isolierten Populationen auf wichtige inflammatorische und mononukleäre Zellmarker
(CX3CR1, F4/80, CD80, CD86, MHC-II, CCR2, CD103) und identifizierten einzigartige und
umgebungsspezifische Eigenschaften dieser Zellen. So exprimierten die CD11c-positiven
63
Mikroglia nicht nur den DC-Marker CD11c, auch der Anteil an typischen Mikrogliamarkern
wie CX3CR1 und F4/80 war am höchsten im Vergleich zu CD11c-positiven Zellen aus
Lunge,
Leber
und
Milz.
Im
Gegensatz
dazu
waren
beide
CD11c-positiven
Gehirnpopulationen nahezu negativ in der Expressionsintensität für den inflammatorischen
Monozytenmarker CCR2. Durch die Zusammenfassung aller Marker haben wir spezifische
Signaturen erstellt, welche die Heterogenität jeder untersuchten CD11c-positiven Population
in Gehirn, Milz, Lunge und Leber verdeutlichen. Beim Vergleich der CD11c-positiven
Populationen für MHC-II, einem wichtigen Protein für die Antigenpräsentation, stellten wir
fest, dass beide Gehirnpopulationen im Vergleich zur Milz und Leber einen geringeren Anteil
an MHC-II-positiven Zellen beinhalteten. Darüber hinaus war die Expressionsintensität von
MHC-II in den CD11c-positiven Mikroglia fast nicht messbar. Schlussfolgernd lässt sich
daraus ableiten, dass die Mikroglia einzigartig in ihrer Expression für MHC-II im Vergleich zu
den anderen untersuchten CD11c-positiven Populationen aus Lunge, Leber und Milz sind.
Nachdem wir zeigen konnten, dass die CD11c-positiven Mikroglia in ihrer MHC-II-Expression
einzigartig sind und sich zudem in dieser Eigenschaft nicht von den CD11c-negativen
Mikroglia unterschieden, wollten wir anhand dieses Beispiels untersuchen, ob diese
Eigenschaft zellspezifisch ist oder durch die Umgebung im Gehirn beeinflusst wird. Dafür
haben wir isolierte mononukleäre Zellen von einer Mauslinie, welche unter dem Promotor
von
MHC-II
das
verstärkte
grün-fluoreszierende
Protein
(eGFP)
exprimiert,
auf
Schnittkulturen von Hippocampus (OHSC) und Milz (OSSC) von Wildtypmäusen (Wt) cokultiviert. Durch die Benutzung eines konfokalen Mikroskops und eines Live-Imaging
Systems konnten wir die Schnittkulturen, co-kultiviert mit den Einzelzellen, über einen
Zeitraum von mindestens 72 h beobachten. Mit Hilfe des rot-fluoreszierenden Farbstoffes
PKH26 konnten wir die transplantierten MHC-II-negativen Zellen aus der MHC-II-eGFPMaus von den Wt-Zellen unterscheiden. Hauptsächlich waren die Mikroglia MHC-II-negativ
und Milzzellen MHC-II-positiv. Während unserer Beobachtungen sahen wir, dass sich
Milzzellen begannen zu ramifizieren, wenn sie auf OHSCs kultiviert wurden, wobei zeitgleich
die Expression von MHC-II deutlich geringer wurde. Wenn diese Zellen hingegen auf OSSCs
co-kultiviert wurden, blieben diese hauptsächlich MHC-II-positiv und behielten ihre amöboide
Form. Weiterhin blieben Mikroglia, co-kultiviert auf OSSCs, MHC-II-negativ und ebenfalls
amöboid. Mikroglia, kultiviert auf OHSCs, ramifizierten dagegen und blieben MHC-II-negativ.
Dies ließ uns schlussfolgern, dass immunologisch aktive Zellen aus der Peripherie durch
Umgebungsfaktoren im Gehirn beeinflusst werden und unter nichtinflammatorischen
Bedingungen einen eher antiinflammatorischen Phänotyp entwickeln. Darüber hinaus
behalten Mikroglia außerhalb ihrer Umgebung, in diesem Fall in der Umgebung eines
Immunorgans, unter nichtinflammatorischen Bedingungen, ihren inaktiven Phänotyp.
64
Diese Daten unterstützen die Ansicht, dass lokale Faktoren mononukleäre Zellen
beeinflussen und nichtinflammatorische Bedingungen schaffen, um im immunprivilegierten
Gehirnparenchym die neuroprotektiven Effekte aufrecht zu erhalten. Darüber hinaus konnten
wir in unserem Modell zeigen, dass Mikroglia, unter nichtinflammatorischen Bedingungen,
auch in der Umgebung eines Immunorgans ihren intrinsischen Phänotyp behalten können.
Diese Ergebnisse charakterisieren CD11c-positive Zellen des Gehirns als eigenständigen
Typus, den wir als Signatur dargestellt haben. Interessanterweise zeigen sich solche aber
auch für CD11c-positive Zellen aus anderen Organen, sodass von einer ubiquitär
stattfindenden lokalen Adaption ausgegangen werden kann. Die Festlegung der Signaturen
von CD11c-positiven und CD11c-negativen Mikroglia erlaubt uns die Untersuchung von
Veränderungen in verschiedenen Pathologien wie der Multiplen Sklerose und der
Alzheimerschen Erkrankung.
Aufbauend auf den vorhergehenden Ergebnissen möchten wir nun herausfinden, ob
Mikroglia eine wichtige Rolle in Pathologien der Adipositas spielen können. Dafür
untersuchen wir Mäuse, welche eine spezifische fettreiche oder kohlenhydratreiche
Futterdiät erhalten haben. Da der Hypothalamus ein wichtiges Kontrollzentrum für Hunger
ist, wollen wir Mikroglia aus dem Hypothalamus mit Mikroglia aus dem Cortex
immunphänotypisch miteinander vergleichen. Erste Ergebnisse deuten auf ein verändertes
Phagozytoseverhalten von Mikroglia hin.
.
65
6 Summary
One of the concepts for the immune privilege of the brain has been attributed to the lack of
Dendritic Cells (DCs). This view became challenged by the identification of CD11c and Major
Histocompatibility Complex (MHC)-II-positive cells in the meninges and choroid plexus in the
brain of healthy mice and rats. These two proteins are the most prominent markers used for
the identification of DCs. However, under non-inflammatory conditions available antibodies
only provided questionable results when applied on central nervous system (CNS) tissue.
The development of the transgenic mouse model in which the green fluorescent protein
(GFP) is expressed under the promoter of CD11c (itgax) allowed the investigation of CD11cpositive cells under non-inflammatory conditions in the brain. Using the advantage of this
mouse model and flow cytometry analyzes, we have investigated CD11c-positive cells in the
brain parenchyma and compared these cells to CD11c-positive cells derived from other
organs.
In our studies we have demonstrated the existence of intraparenchymal CD11c-positive cells
in the brain of healthy mice for the first time. Our observations showed that these cells are
distributed in the tela of the choroid plexus, below the ependyma of the lateral ventricle,
along the corpus callosum and within the fornix. Beside the DC-marker CD11c, the cells
located within the parenchyma also expressed typical macrophage and microglia markers
such as IBA-1 and CD11b. Additionally, using electron and confocal microscopy, we
identified that some processes of the intraparenchymal CD11c-positive cells take part in the
formation of the glia limitans. Moreover, using organotypic hippocampal slice cultures
(OHSCs) we observed that these CD11c-positive cells can be derived from intrinsic
precursors as well as from the bone-marrow using a chimeric mouse model. To summarize,
we identified and characterized intraparenchymal cells, expressing the DC marker CD11c as
well as the macrophage/microglia marker IBA-1 and CD11b.
The demonstration that the DC-Marker CD11c can also be expressed from macrophages or
B-cells, gave rise to a discussion about the existence of DCs as an independent cell line and
if DCs can in fact be clearly distinguished from macrophages. Therefore, we addressed the
question on how brain-derived CD11c-positive cells differ from CD11c-positive cells derived
from other organs.
Here, we used acutely isolated mononuclear cells from brain, spleen, liver and lung and
analyzed them for the expression of important mononuclear and inflammatory cell markers
using flow cytometry. We labeled the isolated cells for the pan leukocyte marker CD45 and
observed two different CD45-expressing cells in the brain, whereas the other organs
exhibited a more homogenous CD45 high (CD45high) expressing population. As it is
described in the literature the CD45 intermediate (CD45int) expressing population is known as
66
microglia and the CD45high expressing population is defined as perivascular or meningeal
macrophages and DCs. From all investigated CD45-positive populations we isolated CD11cpositive cells. Here, the percentage of CD11c-positive cells among the microglia fraction was
significantly lower compared to that of lung and liver. Further, the percentage of CD11cpositive cells among the perivascular macrophages and DCs was significantly lower
compared to the lung. Moreover, comparing cells for other important inflammatory and
mononuclear cell markers (CX3CR1, F4/80, CD80, CD86, MHC-II, CCR2, CD103), we found
unique and site-specific expression patterns for each organ. The fraction of CD11c-positive
microglia contained the highest amount of CX3CR1 and F4/80-positive cells compared to the
other investigated CD11c-positive cell populations. In contrast, both CD11c-positive brainderived populations almost completely lacked the inflammatory monocyte marker CCR2.
Additionally, to demonstrate the heterogeneity of CD11c-positive populations from brain,
spleen, liver and lung we summarized all investigated cell markers in specific signatures.
Comparing the expression of MHC-II, an important cell marker for antigen presentation to Tcells, we observed that the amount of MHC-II-positive cells among both CD11c-positive brain
populations was significantly lower compared to spleen and liver. Investigating the
expression intensity of MHC-II, we found that CD11c-positive microglia almost completely
lacked the MHC-II expression. These results indicate that CD11c-positive microglia are
unique for their low MHC-II expression in comparison to the other investigated CD11cpositive populations. Besides, we did not find any differences between the MHC-II
expression of CD11c-positive microglia and CD11c-negative microglia.
To test whether phenotypical differences of microglia are fixed by origin or developed due to
environmental factors, we established the co-cultivation of acutely isolated mononuclear cells
from spleen and brain from the MHC-II-enhanced green fluorescent protein (eGFP) mice on
organotypic slice cultures from hippocampus (OHSC) and spleen (OSSC) from wild type (wt)
mice. Using confocal microscopy, we established a live-imaging set-up to observe our cocultivated single cells on wt slices over at least 72 h. To differentiate MHC-II-negative cells
isolated from the MHC-II-eGFP mouse, from wt cells, we used the red fluorescent dye
PKH26. Here, we observed that spleen cells co-cultivated on OHSCs mainly reduced their
MHC-II-expression, which was paralleled by enhanced ramification. In contrast, mononuclear
cells obtained from spleen did not reduce their MHC-II-expression nor ramified on OSSCs.
Investigating brain mononuclear cells co-cultivated on OSSCs, we observed no up-regulation
of MHC-II or any ramification. Brain mononuclear cells co-cultivated on OHSCs remained
MHC-II-negative but expressed a ramified morphology. In summary, peripheral cells adapt to
the environment of the brain and transform to an immunological inactivated phenotype while
microglia keep their immunological inactivated phenotype in the environment of an immune
organ under non-inflammatory conditions.
67
These investigations support the view, that local factors influence mononuclear cells and
inactivate pro-inflammatory features under physiological conditions to maintain the
neuroprotective effects of the brain`s immune privilege. Moreover, microglia show an intrinsic
phenotype, which does not change in the environment of an immune organ under noninflammatory conditions.
These findings characterize the intraparenchymally located CD11c-positive brain population
as an independent cell type which we illustrated in specific signatures. Interestingly, such
specific signatures can be displayed for other DCs derived from other organs. Therefore we
suppose that beside a local adaption of cells in the brain, also a ubiquitary adaption to the
local environment occurs. The definition of specific signatures of CD11c-positive and
negative microglia may help to investigate changes in different pathologies such as multiple
sclerosis and Alzheimer`s disease.
In continuation to our previous work we would like to invest if microglia may have an
important role in different pathologies of obesity. Therefore, we now investigate mice under
high fat or high carbohydrate diet in comparison to mice under chow diet. Since the
hypothalamus is an important brain region to regulate hunger we want to isolate microglia
from the hypothalamus and from the cortex to compare the immunological phenotype. Preliminary results indicate a different behavior of phagocytosis of microglia in mice which
received a high caloric diet.
68
7 Danksagung
Die vorliegende Promotionsarbeit entstand am Institut für Anatomie der Universität Leipzig.
Hiermit möchte ich mich für die Überlassung des Themas herzlichst bei meinem Doktorvater
Prof. Dr. med. Ingo Bechmann bedanken. Durch seine stete Betreuung, seine Ermutigung
zum selbständigen wissenschaftlichen Denken und sein vorgelebter Enthusiasmus an der
Wissenschaft, erlaubte mir große Freude an dieser Arbeit zu entwickeln.
Ein großes Dankeschön möchte ich auch an alle Mitarbeiter des Instituts für Anatomie und
den Mitgliedern unserer Arbeitsgruppe, besonders an Dr. med. Martin Gericke, Dr. med.
Martin Krüger, Dr. rer. med. Felicitas Merz, Franziska Menzel, Julia Landmann und PD. Dr.
phil. nat. Marco Koch für die Geduld und Unterstützung während der Doktorarbeit
aussprechen. Weiterhin möchte ich mich bei den Mitarbeitern der IZKF-FACS Core Unit
bedanken. Zusätzlich geht ein großes Dankeschön an alle Mitglieder der Forschergruppe
DFG 1336 für die tolle Organisation und den jährlichen Retreats, welche das Thema, durch
kritische Diskussionen, in großen Schritten vorangetrieben haben.
Meinen größten Dank möchte ich an meine Familie richten, welche mich mein ganzes Leben
unterstützt hat und mir immer Mut und Zuversicht zugesprochen hat. Zum Schluss geht noch
ein großes Dankeschön an alle meine Freunde, welche mich auf meinem bisherigen Weg
begleitet haben und mich unterstützen.
Dankeschön!
69
8 Erklärung über die eigenständige Abfassung der Arbeit
Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig und ohne unzulässige Hilfe
oder Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Ich versichere,
dass Dritte von mir weder unmittelbar noch mittelbar geldwerte Leistungen für Arbeiten
erhalten haben, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen,
und dass die vorgelegte Arbeit weder im Inland noch im Ausland in gleicher oder ähnlicher
Form einer anderen Prüfungsbehörde zum Zweck einer Promotion oder eines anderen
Prüfungsverfahrens vorgelegt wurde. Alles aus anderen Quellen und von anderen Personen
übernommene Material, das in der Arbeit verwendet wurde oder auf das direkt Bezug
genommen wird, wurde als solches kenntlich gemacht. Insbesondere wurden alle Personen
genannt, die direkt an der Entstehung der vorliegenden Arbeit beteiligt waren.
.................................
Datum
....................................
Unterschrift
70
9 Curriculum vitae
Name:
Kerstin Immig
Anschrift:
Bernhard-Göring-Straße 91
04275 Leipzig
Geburtsort:
11.08.1983, Leipzig
Familienstand:
ledig
Schulbildung:
1990-1994 Heinrich-Pestalozzi-Schule (Grundschule)
1994-1999 Uhlandschule (Gymnasium)
1999-2002 Max-Klinger-Schule (Gymnasium)
2002: Abitur
Studium:
2002-2004 Studium der Biologie (Grundstudium), Universität
Leipzig
2003-2004 ERASMUS an der Neuen Bulgarischen Universität
Sofia
2004-2008 Studium der Biologie (Diplom), Universität Rostock
Promotion:
seit: 2010 an der Universität Leipzig
Thema: Immunphänotypische Charakterisierung CD11c-positiver
Zellen des Gehirns im direkten Vergleich zu CD11c-positiven Zellen
von Lunge, Leber und Milz
Preise:
2011- 10. Research Festival der Universität Leipzig, Posterpreis
2012- 11. Research Festival der Universität Leipzig, Posterpreis
Berufserfahrung: - Oktober 2008 bis Mai 2010 wissenschaftliche Mitarbeiterin am
Institut für allgemeine Pharmakologie der Johann-Wolfgang Goethe
Universität Frankfurt
- seit Mai 2010 wissenschaftliche Mitarbeiterin am Institut für
Anatomie der Universität Leipzig
71
Publikationen:
Prodinger C, Bunse J, Krüger M, Schiefenhövel F, Brandt C, Laman JD, Greter M, Immig K,
Heppner F, Becher B, Bechmann I
(2010) CD11c-expressing cells reside in the
juxtavascular parenchyma and extend processes into the glia limitans of the mouse nervous
system. Acta Neuropathol. 121:445-58
Fischer W, Urban N, Immig K, Franke H, Schaefer M (2013) Natural compounds with P2X7
receptor-modulating properties. Purinergic Signal. 10:313-26
Haase J, Weyer U, Immig K, Klöting N, Blüher M, Eilers J, Bechmann I, Gericke M (2013)
Local proliferation of macrophages in adipose tissue during obesity-induced inflammation.
Diabetologia. 57:562-71
Preissler J, Grosche A, Lede V, Le Duc D, Krügel K, Matyash V, Szulzewsky F, Kallendrusch
S, Immig K, Kettenmann H, Bechmann I, Schöneberg T, Schulz A (2014) Altered microglial
phagocytosis in GPR34-deficient mice. Glia 63(2):206-15
Krueger M, Bechmann I, Immig K, Reichenbach A, Härtig W, Michalski D (2015)
Blood-brain barrier breakdown involves four distinct stages of vascular damage in various
models of experimental focal cerebral ischemia. J Cereb Blood Flow Metab. 35(2):292-30.
Immig K, Gericke M, Menzel F, Merz F, Krueger M, Schiefenhövel F, Lösche A, Jäger K,
Hanisch UK, Biber K, Bechmann I (2015) CD11c-positive cells from brain, spleen, lung, and
liver exhibit site-specific immune phenotypes and plastically adapt to new environments.
Glia. 63(4):611-25.