Download Functional Characterization of the CD300e Leukocyte Receptor Tamara Brckalo

Functional
Characterization
of the CD300e Leukocyte
Receptor
Tamara Brckalo
TESI DOCTORAL UPF / 2010
DEPARTAMENT DE CIENCIES EXPERIMENTALS I DE
LA SALUT
DIRECTOR DE LA TESI
Dr Miguel López-Botet
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To Filip and Raša
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ACKNOWLEDGEMENTS
My deepest gratitude is to my thesis advisor Prof. Dr. Miguel LópezBotet for his continuous support, guidance and encouragement he
provided in all stages of this thesis.
I would also like to thank my colleagues at the UPF: Andrea Saez,
Aura Muntasell, Gemma Heredia, Diogo Baia, Neus Romo, Giuliana
Magri and Medya Shikhagaie for their help and support during all
these years.
I owe deep respect and gratitude to Prof. Dr. Marco Cassatella for his
time, expertise and everything I had the opportunity to learn under his
guidance. I am very grateful to Federica Calzetti for being an excellent
teacher, exceptional colleague and a great friend.
I am eternally grateful to my parents Rajko and Slobodanka and my
brother Miloš, for their unconditional love and infinite
encouragement.
But above all, I would like to thank my husband Raša Karapandža that
has been there for me more than any other person. Thank you for
your love, your patience and for believing in me. At the end, I would
like to thank my son Filip for giving my life a new meaning and for
doing everything possible to make me write this thesis slowly but
surely.
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SUMMARY
The focus of this work was to functionally characterize the CD300e
receptor expressed in human monocytes and myeloid dendritic cells
and investigate the implications that receptor engagement has on their
biology. We provide evidence formally supporting that CD300e
functions as an activating receptor capable of regulating the innate
immune response by triggering various pro-inflammatory functions
including intracellular calcium mobilization, superoxide anion
production, pro-inflammatory cytokine release and up-regulation of
co-stimulatory molecules in myeloid cells. We also report that ligation
of CD300e on the surface of monocytes results in their differentiation
to functional MΦ2-like macrophages by an autocrine mechanism that
involves M-CSF and its receptor (CD115).
RESUM
L’objectiu d’aquest treball ha estat caracteritzar funcionalment el
receptor CD300e expressat en monòcits i cèl·lules dendrítiques
mieloides humanes, així com investigar les implicacions que l’activació
d’aquest receptor pot tenir en la seva biologia. Demostrem
formalment que el receptor CD300e funciona com un receptor
activador capaç de regular la resposta immune innata activant diverses
funcions proinflamatòries, incloent la mobilització de calci
intracel·lular, la producció d’anió superòxid, la secreció de citocines
proinflamatòries i la inducció de molècules coestimuladores en
cèl·lules mieloides. També descrivim que l’activació del receptor
CD300e a la superfície dels monòcits provoca la seva diferenciació cap
a macròfags funcionals del tipus MΦ2 gràcies a un mecanisme autocrí
que funciona a través del M-CSF i el seu receptor (CD115).
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PREFACE
The CD300 glycoproteins represent a family of recently identified cell
surface molecules with inhibitory and activating functions expressed
by cells of the myeloid lineage. Since there are only few characterized
reagents available, there is no information about their natural ligands,
and because most studies have used transfectants rather than primary
cells, our understanding of the biology of this receptor family is still
rather limited. On that basis, we have investigated in detail the
functional role of the CD300e receptor in human monocytes and
myeloid dendritic cells.
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ABBREVIATIONS
BCR
BMMC
CD
CR
DAP10
DAP12
DC
FcR
fMLP
G-CSF
GM-CSF
HCMV
HIV
IFN
Ig
IgSF
IL
ILT
IREM
ITAM
ITIM
ITSM
KIR
LPS
mAb
M-CSF
mDC
MHC
moDC
MΦ
NFAT
NF-κB
NK
PBMC
PI3K
PMN
RBL
ROS
SCF
B cell Receptor
Bone Marrow-derived Mast Cells
Cluster of Differentiation
Complement Receptor
DNAX Adaptor protein of 10kDa
DNAX Adaptor protein of 12kDa
Dendritic Cell
Receptor for the Constant Region of Immunoglobulins
Formyl-Methionyl-Leucyl-Phenylalanine
Granulocyte Colony Stimulating Factor
Granulocyte-Macrophage Colony Stimulating Factor
Human Cytomegalovirus
Human Immunodeficiency Virus
Interferon
Immunoglobulin
Immunoglobulin Super Family
Interleukin
Immunoglobulin-Like Transcript
Immune Receptor Expressed by Myeloid Cells
Immune Receptor Tyrosine-based Activating Motif
Immune Receptor Tyrosine-based Inhibitory Motif
Immune Receptor Tyrosine-based Switch Motif
Killer Immunoglobulin-Like Receptor
Lipopolysaccharide
Monoclonal Antibody
Macrophage Colony Stimulating Factor
Myeloid Dendritic Cell
Major Histocompatibility Complex
Monocyte-derived Dendritic Cell
Macrophage
Nuclear Factor of Activated T cells
Nuclear Factor - κB
Natural Killer Cells
Peripheral Blood Mononuclear Cells
Phosphatydil Inositol-3 Kinase
Polymorphonuclear Neutrophils
Rat Basophilic Leukemia
Reactive Oxygen Species
Stem Cell Factor
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SH-2
SHIP
SHP
SIGLEC
SP-A/D
TCR
TLR
TNF
TREM
ZAP-70
Src Homology-2 domain
SH2 domain containing Inositol Phosphatase
SH2 domain containing Protein Tyrosine Phosphatase
Sialic Acid Binding Ig-like Lectins
Surfactant protein A/D
T cell Receptor
Toll-like Receptor
Tumor Necrosis Factor
Triggering Receptor Expressed by Myeloid Cells
ξ – chain Associated Protein 70
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SUMMARY............................................................................... VII PREFACE ..................................................................................IX ABBREVIATIONS ....................................................................XI 1. INTRODUCTION................................................................... 3 1.1 Development of myeloid cells........................................... 3 1.2 Myeloid cells and their effector functions ........................ 6 1.2.1 Monocytes....................................................................................... 6 1.2.2 Macrophages.................................................................................. 8 1.2.3 Myeloid (Conventional) Dendritic cells...............................12 1.2.4 Granulocytes ................................................................................15 1.3 Immune receptor families in myeloid cells .....................19 1.3.1 CD85...............................................................................................19 1.3.2 TREM............................................................................................21 1.3.3 CD172 .............................................................................................23 1.3.4 CD200R .........................................................................................23 1.3.5 SIGLEC .........................................................................................24 1.3.6 CD300.............................................................................................26 1.3.6.1 Human CD300 receptors .....................................................29 1.3.6.2 Mouse CD300 receptors .......................................................33 2. AIMS ........................................................................................41 3. RESULTS ............................................................................... 45 Article 1: ...................................................................................... 47 Functional analysis of the CD300e receptor in human monocytes and myeloid
dendritic cells ...................................................................................................49 Article 2: ......................................................................................................77 Engagement of CD300e induces the differentiation of human monocytes to
macrophages involving an autocrine M-CSF-dependent pathway........................79 Appendix ...................................................................................107 Functional Characterization of CD300f Inhibitory Receptor in Human
Granulocytes...................................................................................107 4. DISCUSSION........................................................................126 5. CONCLUSIONS................................................................... 141 BIBLIOGRAPHY .....................................................................144 xiii
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Introduction
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INTRODUCTION
1. INTRODUCTION
Monocytes, granulocytes, macrophages and dendritic cells, collectively
called myeloid cells, are differentiated from common progenitors
derived from hematopoietic stem cells in the bone marrow.
Commitment to either lineage of myeloid cells is controlled by distinct
transcription factors, followed by differentiation in response to
specific colony-stimulating factors and release into the circulation.
Upon pathogen invasion, myeloid cells are rapidly recruited via
chemokine receptors into tissues, where they become activated,
developing different effector mechanisms that contribute to pathogen
elimination and promoting further recruitment of other leukocytes at
the site of the infection. Therefore, by serving as a first line of defense
myeloid cells play a major role in innate immunity.
1.1
Development of myeloid cells
All immune cells develop from hematopoietic stem cells (HSCs) that
can be subdivided in long-term HSCs (LT-HSCs), short-term
repopulating HSCs (ST-HSCs), and multipotent progenitor (MPP), as
defined by combinations of cell surface markers. Progenitors with a
more restricted differentiation potential identified in the bone marrow
include the common progenitor for lymphoid lineages (CLP) and
common myeloid progenitor (CMP) that generates granulocyticmacrophage (GM) and megakaryocytic-erythroid (ME) lineages.
Alternatively, MPPs may develop to recently described lymphoidprimed multipotent progenitors (LMPPs) that have lost
megakaryocytic-erythroid potential but retain myeloid and lymphoid
developmental options [1]. CLPs give rise to pro-B and pro-T cells,
uncommitted lymphoid progenitors that will differentiate further into
mature B and T cells, and cells of the NK lineage. CMPs in turn
generate two more restricted progenitors: granulocyte-macrophage
progenitors (GMPs) and megakaryocyte-erythrocyte progenitors
(MEPs). The offspring of GMPs includes monocytes, neutrophils,
eosinophils, and basophils/mast cells [2].
It is believed that CMPs can also give rise to macrophage-DC
progenitors (MDPs) that will give rise to monocytes, macrophages,
classical or myeloid DCs (cDCs or mDCs), and plasmacytoid DCs
(pDCs). MDPs-derived monocytes can further differentiate into
inflammatory DCs. MDPs lie upstream of the common DC
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INTRODUCTION
progenitors (CDPs), which are DC-restricted, giving rise to pDCs and,
via pre-DCs, to cDCs (Figure 1)1.
Figure 1. Hematopoietic tree for the development of myeloid cells [1].
Macrophage-dendritic progenitors (MDPs) and common DCsprogenitors (CDPs) commitment is dependent on cytokines. MDPs
and CDPs express the receptors for Fms-like tyrosine kinase 3 ligand
(Flt3L), GM-CSF and M-CSF. MDPs have the potential to
differentiate into macrophages, monocytes, and inflammatory DCs,
and via CDPs to cDCs and pDCs. Different microenvironments with
variations in the combination and concentration of the three cytokines
influence the lineage commitment and differentiation of MDPs and
CDPs to mature cells (Figure 2).
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Solid arrows show demonstrated pathways; dotted arrows show suggested
pathways that have not been formally proven.
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INTRODUCTION
Figure 2. Cytokine-induced differentiation of macrophage dendritic
progenitors (MDP) and common DCs-progenitors CDP progenitors [1].
The development of blood monocytes is dependent on the M-CSF
(also known as Csf-1). M-CSF receptor (CD115, Csf-1R) is expressed
on monocytes, macrophages, DCs and their precursors [3, 4]. The two
known ligands for CD115, M-CSF [5] and the more recently described
IL-34 [6], are both important for the development of myeloid lineage.
Other cytokines, such as GM-CSF, Flt3, and lymphotoxin α1β2 [7-9],
control the development and homeostasis of the macrophage and DC
networks but appear to be unnecessary for monocyte development
(Figure 2).
Myeloid cells develop from hematopoietic stem cells in the bone
marrow via several commitment steps and intermediate progenitor
stages that pass through the common myeloid progenitor (CMP), the
granulocyte/macrophage progenitor (GMP), and the macrophage/DC
progenitor (MDP) stages. Each of these differentiation steps involves
cell fate decisions that successively restrict developmental potential
and many are controlled by the Ets family transcription factor PU.1
[10]. Besides being able to induce myeloid commitment in immature
multipotent progenitor cells [11], PU.1 is required for the generation
of CMP in early myelopoiesis [12, 13]. It also controls several cell fate
decisions along the myelomonocytic pathway by engaging in
antagonistic interactions with different transcription factors. Initially,
inhibitory interactions with GATA-1 shut down the
megakaryocytic/erythroid pathway, and repression of GATA-2 blocks
mast cell development [14]. At the later bipotent GMP stage, PU.1 is
critical for driving monocytic differentiation, at the expense of
granulocytic differentiation [12], by antagonizing C/EBPα [15], a
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INTRODUCTION
transcription factor required for granulocytic development [16]. Two
additional transcription factors that can selectively drive monocyte fate
in myeloid progenitors are MafB and c-Maf [17, 18].
1.2
Myeloid cells and their effector functions
1.2.1 Monocytes
Monocytes represent 5-10% of leukocytes in human blood, and
contribute together with polymorphonuclear (PMN) and natural killer
(NK) cells to constitute the innate arm of the immune system.
Monocytes originate in the bone marrow from a common myeloid
progenitor shared with neutrophils, and are then released into
peripheral blood where they circulate for several days before entering
tissues. They constitute a systemic reservoir of myeloid precursors that
give rise to a variety of tissue resident macrophages, and also to
specialized cells such as dendritic cells (DCs) and osteoclasts [19-22].
Thus, circulating monocytes represent accessory cells that are able to
link inflammation and the innate defense against pathogens to
adaptive immune responses [10].
Blood monocytes also represent a large pool of scavenger and
potential effector cells inside blood vessels, in homeostasis and also
during inflammatory processes [23]. They are armed with a large array
of scavenger receptors that recognize microorganisms but also lipids
and dying cells. Upon stimulation, monocytes become activated and
produce different effector molecules involved in the defense against
pathogens. Stimulated monocytes can produce ROS, complement
factors, prostaglandins, nitric oxide (NO) (in mice), cytokines (i.e.
TNF-α, IL-1β, CXCL8, IL-6, and IL-10), vascular endothelial growth
factor and proteolytic enzymes [10]. Even though antigen presentation
has been described as a functional feature of monocytes, they have
been found to be far less efficient than DCs [24].
Monocytes have some typical morphological features such as irregular
cell shape, oval- or kidney-shaped nucleus, cytoplasmic vesicles, and
high cytoplasm-to-nucleus ratio. However, they are still very
heterogeneous in size and shape and are difficult to distinguish by
morphology from blood DCs, activated lymphocytes, and NK cells
[10]. Human blood monocytes can be defined by the expression of the
CSF-1 receptor (MCSF-R or CD115) and the chemokine receptor
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INTRODUCTION
CX3CR1. They are distinct from PMNs, NK cells, and lymphoid T
and B cells and do not express NKp46, CD3, CD19 or CD15 [10].
Table I summarizes cell surface receptors expressed by monocytes .
Antigen
CD14+CD16- CD14+CD16+
Antigen
CD14+CD16- CD14+CD16+
Chemokine receptors
Other receptors
CCR1
+
CD4
+
+
CCR2
+
CD11a
ND
ND
CCR4
+
CD11b
++
++
CCR5
+
CD11c
++
+++
CCR7
+
CD14
+++
+
CXCR1
+
CD31
+++
+++
CXCR2
+
CD32
+++
+
CXCR4
+
++
CD33
+++
+
CX3CR1
+
++
CD43
ND
ND
CD49b
ND
ND
CD62
++
CD80
ND
ND
CD86
+
++
CD115
++
++
CD116
++
++
CD200R
ND
ND
MHC class II
+
++
Table 1. Phenotype of the two best-characterized monocyte subsets in
humans [25].
In recent years, investigators have identified three distinct populations
of blood monocytes defined by the expression of CD14 and CD16
(CD14+CD16−, CD14+CD16+, and CD14dimCD16+) [26-29]. The
CD14+CD16− monocytes which represent the majority of human
blood monocytes (80%-90%), express high levels of the chemokine
receptor CCR2 and low levels of CX3CR1, and upon LPS stimulation
produce IL-10 rather than TNF and IL-1 [21, 30-32]. In contrast to
this subset, human CD16+ monocytes express high levels of CX3CR1
and low levels of CCR2 [21, 30, 32] and are responsible for the
production of TNF-α in response to LPS. They are found in larger
numbers in the blood of patients with acute inflammation [33] and
infectious diseases [34, 35]. The CD16+ monocytes, also called proinflammatory monocytes are composed of at least two populations
with strikingly distinct functions [27]. Monocytes that co-express
CD16 and CD14 (CD14+CD16+) also express other Fc receptors (i.e.
CD64 and CD32), have phagocytic activity, and are mainly responsible
for the production of TNF-α and IL-1 in response to LPS [36]. In
contrast, monocytes that express CD16 but very low levels of CD14
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INTRODUCTION
(CD14dimCD16+) lack the expression of other Fc receptors, are poorly
phagocytic and do not produce neither TNF-α nor IL-1 in response to
LPS [37]. Even though they may be expanded in the blood of septic
patients [34] the function of CD14dimCD16+ monocytes remains thus
far elusive.
1.2.2 Macrophages
Tissue macrophages are generally considered to be derived from
circulating monocytes and show a high degree of heterogeneity [25].
They have a broad role in the maintenance of tissue homeostasis,
through the clearance of senescent cells and the re-modeling and
repair of tissues after inflammation [38]. The heterogeneity of tissue
macrophages reflects the specialization of function that they have
adopted in different locations, such as the ability of osteoclasts to
remodel bone, or the high expression of pattern recognition receptors
and scavenger receptors by alveolar macrophages that are involved in
clearance of air-borne microorganisms, and environmental particles in
the lungs [25].
Osteoclasts. Osteoclast precursors are found in the
granulocyte/macrophage precursor (GMPs) and can be derived from
unfractionated, mature monocytes from peripheral blood [39, 40].
Osteoclast precursors express the receptor for macrophage colonystimulating factor (M-CSF R or CD115) and depend on it for their
development. Since culture of peripheral-blood monocytes with MCSF and RANKL is sufficient to induce their differentiation into
osteoclasts it has been assumed that osteoclast precursors are
monocytes, although this has not been formally proven in vivo [25].
Alveolar macrophages. Alveolar macrophages have been reported to
be derived both from peripheral blood precursors and from their local
proliferation. GM-CSF has a crucial role in the maintenance,
maturation and activity of alveolar-macrophage populations. Although
it is assumed that a monocyte subset might be the precursor of
alveolar macrophages, this has not been directly proven[25].
Macrophages in the central nervous system. The central nervous
system (CNS) contains various macrophage subsets, including
microglia, perivascular macrophages, meningeal macrophages and
choroid-plexus macrophages [25]. Meningeal macrophages are thought
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INTRODUCTION
to be rapidly replaced by cells of bone-marrow origin, whereas the
turnover of of perivascular and choroid-plexus macrophages is slower;
the basis for the different turnover rate is unknown [41]. It is assumed
that monocytes can enter the CNS and differentiate into microglia
[25].
Splenic macrophages. Macrophages in the white pulp include
tingible-body macrophages. Marginal zone macrophages are found
next to the marginal sinus and they express pattern-recognition
receptors and scavenger receptors, which aid in the clearance of
blood-borne pathogens [42]. Metallophilic macrophages, found nearby
the white pulp and marginal sinus, can sample the circulation and are
thought to play an important role during infections [25]. Regarding the
origin of splenic macrophages, it seems that local proliferation does
occur under steady-state conditions in the case of white-pulp
macrophages and metallophilic macrophages, but circulating
precursors also contribute, even though their nature is uncertain.
Kupffer cells. Kupffer cells are an important component of the
mononuclear-phagocyte system that is present in the liver. The origin
of Kupffer cells has been speculated to involve two mechanisms:
replenishment by local proliferation and recruitment of circulating
precursors. Both mechanisms are presumably affected by
inflammation and other factors [25].
Inflammatory-monocyte-derived macrophages. It has been known
for a long while that monocytes are recruited to the inflammatory
lesion where they differentiate into macrophages [43]. At the site of
the infection macrophages undergo activation depending on local
environmental signals, among them microbial products and cytokines
[44]. Bacterial products (e.g., LPS) and immune stimuli such as
interferon-γ (IFN-γ), promote classic (or M1) macrophage activation
which produce abundant reactive nitrogen (RNI) and oxygen
intermediates (ROI) and IL-12 [45, 46]. M1-activated macrophages
participate in the Th1 responses mediating resistance against
intracellular parasites and tumors, associated to tissue disruptive
reactions [44]. Alternative (or M2) macrophage activation was
originally discovered as a response to IL-4 [45, 46]. M2-activated
macrophages may present different forms depending on the
stimulating signals, which include IL-4 and IL-13, immune complexes
plus signals mediated through receptors that involve downstream
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INTRODUCTION
signaling through MyD88 (i.e., IL-1 or LPS), glucocorticoid hormones,
and IL-10. M-CSF–cultured monocytes have a transcriptional profile
close to IL-4–activated cells, suggesting that this is a default pathway
of differentiation [44]. In general, M2-activated cells share high
expression of scavenger and mannose receptors, and have an IL-12low,
IL-10high, IL-1decoyRhigh, IL-1rahigh phenotype. They also have a
distinct chemokine expression pattern (i.e. CCL17 and CCL22) as
shown in Figure 3. Macrophage polarization is also characterized by
changes in various metabolic pathways [47] such as iron metabolism.
MΦ1 cells are characterized by an increased iron uptake and
intracellular retention of the metal whereas MΦ2 cells release iron to
the extracellular milieu.
The various forms of M2 activation are oriented to the promotion of
tissue remodeling and angiogenesis, parasite encapsulation, regulation
of immune responses, and have been associated to tumor growth
stimulation [44]. Recent results have highlighted the integration of M2polarized macrophages with immunoregulatory pathways since MΦ2
cells were shown to induce the differentiation of regulatory T cells
(Treg) [48], which have been reported to reciprocally contribute to the
alternative activation of human mononuclear phagocytes [49].
Macrophages that infiltrate tumor tissues have been shown to acquire
a polarized M2 phenotype [45], playing a key role in subversion of
adaptive immunity and in inflammatory circuits that promote tumor
growth and progression. Human peritoneal macrophages (pMΦ)
represent another paradigm of MΦ2 cells [50].
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INTRODUCTION
Figure 3. M1 and M2 macrophages, the extremes of a continuum [45].
Despite these classifications, the degree of macrophage plasticity is still
incompletely defined since it is unclear whether macrophage fate is
irreversibly determined or remains flexible [25].
Macrophages express a number of receptors that mediate their diverse
functions. Since these receptors are located on the surface but also in
intracellular compartments, they mediate recognition of both
extracellular and intracellular pathogens [51] (Figure 4). Complement
and Fc receptors function in phagocytosis and endocytosis of
opsonized particles [52, 53]. In addition, Fc receptors via NF-κB
regulate the production of pro-inflammatory mediators in
macrophages. Another group of phagocytic/endocytic surface
receptors are the non-Toll-like receptors (NTLR), which include the
family of scavenger receptors and C-type lectins [54]. Non-opsonic
surface receptors that do not mediate phagocytosis/endocytosis but
are important sensors of bacteria, fungi and viruses are the Toll-like
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INTRODUCTION
receptors (TLR) [55]. Scavenger receptors including CD36, SREC and
LOX-1 have been shown to collaborate with TLR to induce NF-κB
and may also directly mediate its induction upon interaction with their
ligands. Some TLRs are located within vacuoles and play a role in
recognition of intracellular pathogens. Cytosolic viruses and bacterial
products in macrophages are recognized by the NOD-like receptors
(NLR) and RIG-like helicases (RLH). NLR induce NF-kB either
directly or in collaboration with TLR.
Figure 4. Pattern-recognition receptor system in phagocytes [56]
1.2.3 Myeloid (Conventional) Dendritic cells
Dendritic cells (DCs) represent the migratory group of bone-marrowderived leukocytes that are specialized for the uptake, transport,
processing and presentation of antigens to T cells [57]. They are found
in all tissues including blood and lymphoid organs. In peripheral
tissues and in their 'immature' stage of development, DCs act as
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INTRODUCTION
sentinels continuously sampling the antigenic environment [57]. Any
encounter with microbial products or tissue damage initiates the
migration of the DCs to lymph nodes and their maturation process.
Antigens are processed and presented on the DC surface as peptides
bound to major histocompatibility complex (MHC) molecules.
Concomitantly, DCs up-regulate co-stimulatory molecules that are
required for effective interaction with T cells. In lymph nodes, mature
DCs efficiently trigger the response of T cells bearing receptors
specific for the foreign-peptide–MHC complexes on the DC surface.
Besides them, DC can also present glycolipids and glycopeptides to T
cells and NKT cells. In the steady state, DCs also migrate at a low rate
without undergoing activation and, by presenting self-antigens to
lymphocytes in the absence of co-stimulation they contribute to
tolerance [58].
There are two main pathways of DC ontogeny from hematopoietic
progenitor cells (HPCs), one pathway generates myeloid DCs (mDCs);
while another generates plasmacytoid DCs (pDCs). Myeloid (or
conventional) DCs are found in three compartments: in peripheral
tissue, secondary lymphoid organs and blood. Peripheral blood
myeloid dendritic cells (mDCs) differ from the other DC subsets by
expression of CD11c and CD123dim, higher levels of MHC class II
molecules and lower levels of CD62L. They are also characterized by
the presence of CD2 and Fc receptors CD32, CD64 and FcεRI. Some
myeloid DCs are also positive for CD14 and CD11b [59]. Peripheral
blood myeloid DCs express TLR1, 2, 3, 4, 5, 6, 7, 8 and 10, but not
TLR9 [60].
In the skin, two distinct types of mDCs are found in two distinct
layers. Langerhans cells (LCs), which express CD1a and Langerin
reside in the epidermis, while interstitial DCs (intDCs), which express
DC-SIGN and CD14 reside in the dermis [58]. Epidermal Langerhans
cells isolated from skin lack the expression of TLR4 and TLR5, while
dermal interstitial DCs express many TLRs including TLR2, 4 and 5
[61].
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INTRODUCTION
Figure 5. The current view of the ways in which the activation states of DCs
can determine the nature of T-cell responses [57].
Numerous agents can activate DCs including microbes, dead cells, as
well as innate and adaptive immune system components. Pathogenassociated molecular patterns (PAMPs) signal DCs and other cell types
through a variety of pattern-recognition receptors (PRR) including
Toll-like receptors (TLRs), cell surface C-type lectins receptors (CLRs)
and intracytoplasmic NOD-like receptors (NLRs) [58]. Cells
undergoing necrosis induce the maturation of DCs, and some
components involved may enhance antigen presentation by DCs
leading to T cell immunity. These endogenous activating molecules are
collectively called damage-associated molecular pattern molecules
(DAMPs) [62].
DCs can secrete a diversified panel of chemokines that attract
different cell types at different times of the immune response [63].
They also express a unique set of co-stimulatory molecules among
them early activation markers CD40, CD80 and CD86 [64] which
permit the activation of naïve T cells promoting primary immune
responses. Through the cytokines they secrete (e.g. IL-8/CXCL8, IL12, IL-23 or IL-10) as well as the surface molecules they express (e.g.
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INTRODUCTION
OX40-L or ICOS-l) DCs can contribute to polarize naïve T cells into
Th1, Th2, Treg or Th17 [58].
1.2.4 Granulocytes
Polymorphonuclear neutrophils (PMNs or neutrophils) are an
essential component of the innate immune system. Mature PMNs are
incapable of cell division, and their sustained generation by the bone
marrow at impressive numbers (1011 cells per day in a normal adult) is
the result of a highly controlled process of myelopoiesis. Common
myeloid progenitor (CMPs) divide and differentiate from the
pluripotent haematopoietic stem cell and generate granulocyticmacrophage (GM) lineage progenitors. Their differentiation proceeds
to committed stem cells, which provide granulocyte-restricted
progenies.
Once activated, neutrophils undergo a complex series of functional
responses culminating in the destruction of invading microbes. These
functions include chemotaxis to sites of infection, transmigration
across capillary endothelium, phagocytosis of opsonized microbes, and
killing of the pathogens. During maturation, neutrophils produce
multiple enzymes critical to these functional responses, each packaged
into primary (azurophil), secondary (specific) or tertiary (gelatinase)
granules, lysosomes and secretory vesicles [65]. Azurophil (or primary)
granules largely contain proteins and peptides directed toward
microbial killing and digestion, whereas specific granules replenish
membrane components and help to limit free radical reactions.
Azurophil granules are the first to be produced and they contain
MPO; three predominant neutral proteinases (i.e. cathepsin G,
elastase, and proteinase 3); bactericidal/permeability-increasing protein
(BPI); defensins and an abundant matrix composed of strongly
negatively charged sulphated proteoglycans [65] that binds almost all
the peptides and proteins other than lysozyme, which are strongly
cationic. Specific granules contain unsaturated lactoferrin that binds
and sequesters iron and copper, transcobalamin II that binds
cyanocobalamin, about two thirds of the lysozyme, neutrophil
gelatinase-associated lipocalin and a number of membrane proteins
also present in the plasma membrane, including flavocytochrome b558
of the NADPH oxidase. Gelatinase or tertiary granules contain
gelatinase in the absence of lactoferrin and may represent one end of
the spectrum of a single type of granule with the same contents but in
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INTRODUCTION
differing proportions [65]. Acid hydrolases are found it the lysosomes.
Endocytic vesicles contain serum albumin and provide a valuable
reservoir of membrane components. Their re-association with the
plasma membrane replenishes the membrane parts that were
consumed during phagocytosis, as well its component proteins like
complement receptor and flavocytochrome b558 [65].
In order to effectively eliminate pathogens neutrophils also produce
superoxide anions (O2–) that are generated by the reduced
nicotinamide adenine dinucleotide phosphate (NADPH) oxidase
complex. Once generated they are converted into potent microbicidal
reactive oxygen species (ROS), such as hydroxyl radical, hydrogen
peroxide (H2O2), and hyperchlorous acid [66].
Figure 6. Neutrophils deliver multiple anti-microbial molecules [67].
A variety of surface molecules regulates neutrophil functions[56]. FcR
and CR are expressed on both neutrophils and monocytes playing a
critical role in phagocytosis through pathways regulating cytoskeletal
reorganization [68]. Neutrophils and monocytes express cell-adhesion
molecules (selectins and integrins) that regulate their transendothelial
migration into tissues [69]. Neutrophils also express different
receptors for chemotactic factors that promote their migration. Among
others, receptors specific for complement (i.e.C5aR), bacterial N16
INTRODUCTION
formyl peptides (fMLP), platelet activating factor (PAF) and
leukotriene B-4 (LTB-4) [70].
Besides serving as a first line of defense, they contribute to the
recruitment, activation and programming of antigen presenting cells
(Figure 7). Neutrophils generate chemotactic signals that attract
monocytes and dendritic cells (DCs), and influence whether
macrophages differentiate to a predominantly pro- or antiinflammatory type [67]. By proteolytically activating prochemerin to
generate chemerin, neutrophils attract both immature DCs and
plasmacytoid DCs [71] and by producing tumor-necrosis factor (TNF)
and other cytokines they induce DC and macrophage differentiation
and activation [72]. Moreover, neutrophils secrete TNF-related ligand
B-lymphocyte stimulator (BLyS) [73] that induce B cell proliferation
and maturation, and interferon-γ that promotes macrophage
activation. Nevertheless, neutrophils can also function as powerful
suppressors of T-cell activation by impairing the T-cell receptor (TCR)
ζ-chain expression and cytokine production [74]. The ability of
neutrophils to control lymphocyte activation is counteracted by the
adaptive immune system regulation of the rate of neutrophil
production via granulocyte colony-stimulating factor (G-CSF)
production (Figure 6). Stromal-cell-derived G-CSF triggers bonemarrow neutrophils to release matrix metallo-proteinase 9 (MMP9)
that helps to mobilize progenitor cells. In the same time, G-CSF also
acts directly on the progenitors to increase their proliferation and
suppresses stromal-cell expression of CXC-chemokine ligand 12
(CXCL12) which helps to retain neutrophils in the bone marrow. In
turn, G-CSF production is regulated by interleukin-17 (IL-17), that is
produced by a T cell subset. T-cell production of IL-17 is governed by
IL-23, which is released from macrophages and is suppressed when
macrophages ingest apoptotic neutrophils [67].
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INTRODUCTION
Figure 7. Neutrophils interact with monocytes, dendritic cells, T cells and B
cells in a bidirectional, multi-compartmental manner [67].
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INTRODUCTION
1.3
Immune receptor families in myeloid cells
The innate immune response is regulated by an array of activating and
inhibitory surface receptors [75]. In myeloid cells, recognition of a
wide range of endogenous and exogenous ligands regulates cell
differentiation, growth and survival, adhesion, migration,
phagocytosis, cytotoxicity and cytokine secretion [76]. Biology and
function of activating and inhibitory receptors involved in regulating
these processes have been extensively explored in the last decade.
However, the nature of ligand/s for a great number of them still
remains unknown and for that reason the role that they have in the
immune response is only partially understood.
Immune receptors expressed by myeloid cells are often members of
multigenic families that include both inhibitory and activating
molecules. It is believed that both receptor types contribute to
establish an activation threshold that determines the initiation,
amplitude and the duration of the immune response triggered by
pathogenic stimuli. Inhibitory and activating members of the same
receptor family have similar extracellular regions but different
transmembrane and cytoplasmic domains that confer them with
distinct signaling properties; most of them can be classified as a C-type
lectins or members of the immunoglobulin superfamily (IgSF). Our
focus will be on superfamily members that are expressed by myeloid
cells.
1.3.1 CD85
Members of the CD85/ILT/LIR/MIR family are characterized by
either 2 or 4 homologous extracellular C-2 type Ig-like domains and
subclassified by the structure of the transmembrane and cytoplasmic
regions. They are related to other IgSF receptors whose genes are
clustered in human chromosome 19, including human FcαR, LAIR,
NKp46 and KIRs. Moreover, they are also homologous to murine
gp49B1/B2 antigens and PIR-A and PIR-B receptors. Activating
members expressed by myeloid cells CD85h, CD85h-like protein and
CD85g have a short cytoplasmic domain that lacks recognizable
docking motifs for signaling mediators. In addition, they are
characterized by the presence of a single basic arginine residue within
the hydrophobic transmembrane region [76, 77]. Activating CD85s
associate with the gamma chain of Fc receptors (FcRγ) that contains a
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INTRODUCTION
negatively charged residue in the transmembrane region and which
transduces the stimulatory signals by recruiting protein tyrosine
kinases through its cytoplasmic ITAMs. CD85h (ILT1) is selectively
expressed by myeloid cells and is able to induce intracellular Ca2+
mobilization in monocytes [77]. Human pDCs preferentially express
another triggering member, CD85g (ILT7), that can inhibit Toll-like
receptor-induced interferon production upon its association with the
Fc epsilon RI gamma [78]. CD85g directly binds to and can be
activated by bone marrow stromal cell antigen 2 (BST2; CD317)
protein. The interaction between CD85g and CD317 functions to
assure an appropriate TLR response by pDCs during viral infection
and likely participates in pDC-tumor crosstalk [79].
By contrast, CD85a (ILT5), CD85j (ILT2), CD85k (ILT3) and CD85d
(ILT4) contain cytoplasmic ITIMs [80-85]. When these receptors are
engaged in close proximity to activating receptors, their ITIMs
become phosphorylated by protein tyrosine kinases of the Src family.
Phosphorylation of these residues creates the docking site for SH-2
domain containing phosphatases such as SHP-1 and SHIP, which
deliver inhibitory signals by dephosphorylating ITAM-containing
signal transduction molecules. It has been reported that upon ITIM
tyrosine phosphorylation CD85j, CD85k and CD84d bind SHP-1
protein tyrosine phosphatase [81, 83, 84]. The mechanism of
inhibition so far has been studied for CD85j in T cells, B cells and
monocytes. In the CD85j-transfected Jurkat T cell line, receptor was
tyrosine phosphorylated when engaged with the TCR by p56Lck
kinase. In the same cells, crosslinking of CD85j and TCR reduced the
CD3ξ phosphorylation. Similarly, CD85j was tyrosine phosphorylated
in B cells when co-ligated with BCR [86]. In monocytes, Ca2+
mobilization and tyrosine phosphorylation triggered via either HLADR, the Fc gamma receptor II (FcγRII or CD32) or FcγRI (CD64)
were inhibited when triggering receptors were co-engaged with CD85j
or CD85d [83-85].
Peripheral blood monocytes express most CD85 members, while
granulocytes express only CD85h and CD85a. Peripheral blood DCs
express CD85h and/or CD85k and can be divided into two subsets
based on differential expression of these two receptors [87].
The homology with KIRs originally suggested that CD85s might
constitute receptors for MHC class I molecules. However, only CD85j
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INTRODUCTION
and CD85d were shown to interact with a broad range of cellular class
I molecules, including HLA-A, -B, and -G [83]. In addition, CD85j
was shown to bind the human cytomegalovirus (HCMV)-encoded
class I molecule UL18. CD85g binds bone marrow stromal cell
antigen 2 (BST2 or CD317). Other members of CD85 family are still
ligand-orphan.
1.3.2 TREM
Triggering receptors expressed by myeloid cells (TREMs) belong to
the family that includes both activating and inhibitory isoforms and
are encoded by a gene cluster on a human chromosome 6p21 that is
linked to the MHC. TREMs have limited homology with other
members of the immunoglobulin gene superfamily since their closest
relative is NKp44, an activating NK-cell receptor [88]. More distant
relatives of TREMs include the CD300 family members [89, 90] and a
receptor for a polymeric immunoglobulin (PigR) [91].
The human TREM family contains at least one inhibitory (TREM-like
transcript-1, TLT-1) and two activating receptors (TREM-1 and
TREM-2) that both signal via DAP12. The activating isoforms are
unique in that one receptor, TREM1, controls inflammation, whereas
another, TREM-2, regulates the development and function of
dendritic cells (DCs), as well as microglia and osteoclasts [91].
Human TREM-1 is expressed by peripheral blood neutrophils and a
subset of monocytes/macrophages [92]. In normal tissues, TREM-1 is
selectively expressed by alveolar macrophages [93]. Furthermore,
TREM-1 is expressed at high levels by neutrophilic infiltrates and
epithelial cells in human skin and lymph nodes after sepsis [94]. Coengagement of TREM-1 has been shown to stimulate the production
of pro-inflammatory chemokines and cytokines such as IL-8/CXCL8,
monocyte chemoattractant protein 1 (MCP1, CCL2), MCP3 (CCL7)
and macrophage inflammatory protein 1 (MIP1, CCL3). Triggering of
TREM-1 also induces granulocytes to release myeloperoxidase, but
does not induce phagocytosis [92, 95]. Crosslinking of TREM-1 on
LPS primed human monocytes induces the release of TNF and IL-1α.
These findings indicate that TREM-1 acts as an amplifier of
inflammatory responses initiated by TLRs [94]. Since LPS and other
TLR ligands up-regulate the expression of TREM-1, it is reasonable to
21
INTRODUCTION
believe that TREM-1 and TLRs cooperate in order to produce an
inflammatory response (Figure 8).
Figure 8. Schematic presentation of the role of TREM1 in inflammatory
responses [91].
While the main role of TREM-1 is the responses of granulocyte and
monocyte/macrophage, TREM-2 mainly controls the function of
other myeloid cells, including DCs, osteoclasts and microglia. Ligation
of TREM-2 immature monocyte-derived DCs induces their
incomplete maturation [96]. Some data indicate that the TREM2–
DAP12 pathway directs the differentiation of myeloid precursors
towards the formation of mature DCs, osteoclasts, microglia and
possibly oligodendrocytes but it is unclear how [91].
Even though TLT-1 displays two ITIMs and is able to recruit SHP-1
[97], some reports have shown that its cross-linking with FcεR induces
calcium influx [98] and therefore promotes activation instead of
inhibition.
Despite the fact that some studies indicate the presence of TREM-1
ligand on human platelets [99] and that TREM-2 ligands could be
22
INTRODUCTION
polyanionic bacterial products [100], TREM receptors are still
considered to be ligand-orphans.
1.3.3 CD172
Signal regulatory proteins (SIRPs or CD172) comprise a family of
transmembrane glycoproteins expressed in myeloid cells, including
macrophages, monocytes, granulocytes, and DCs [86]. Structurally,
CD172 receptors are characterized by three homologous extracellular
Ig-SF domains. CD172a (SIRPα) displays cytoplasmic ITIMs, recruits
SHP-2 and SHP-1, and inhibits receptor tyrosine kinase-coupled
signaling pathways [101-103]. Interaction with SCAP2 (Src-family
Associated Phosphoprotein 2), FYB (Fyn-binding protein) and Grb-2
have also been reported [101]. In the same time CD172a is able to
induce early events in integrin signaling and to positively regulate the
MAP-kinase signaling cascade in response to insulin when
overexpressed in transfected cells [86] which suggest that in some
circumstances CD172a may mediate stimulation rather than inhibition.
CD172b (SIRPβ) contains a short cytoplasmic domain that lack
cytoplasmic sequence motifs and associates with the transmembrane
ITAM bearing adapter protein DAP12 [104]. DAP12 is required for
cell surface expression of CD172b and it mediates activation in
CD172b-transfected cells by promoting Syk and MAPK
phosphorylation. CD172b signaling induces neutrophil migration
[105] and triggers phagocytosis in macrophages [106].
The ligands for CD172a are shown to be CD47, an integrin-associated
protein with multiple functions in immunological and neuronal
processes [86] and surfactant proteins A and D [107]. Ligand/s for
CD172b are still unknown.
1.3.4 CD200R
The CD200 gene cluster is located on human chromosome 3q12-13
and contains CD200R and its ligand CD200 [108, 109]. Expression of
the CD200R is restricted to the cells of myeloid lineages including
monocytes, macrophages and DC. On the other hand, CD200 has
been detected on the surface of thymocytes, B cells, activated T cells,
neurons, endothelial cells and macrophages [110]. Both CD200 and its
23
INTRODUCTION
receptor have two extracellular Ig domains, essential for their ligandreceptor interaction that was shown to modulate macrophage fusion
and differentiation of osteoclasts [111, 112].
That CD200R is an inhibitory receptor that in contrast to other welldescribed myeloid-inhibitory immune receptors does not contain any
immunotyrosine-based inhibitory motif (ITIM) and appears to deliver
inhibitory signals via a novel myeloid cell inhibitory pathway. The
crucial phosphorylation of CD200R is mediated through its tyrosine
residue within the NPxY motif. Upon receptor clustering and tyrosine
phosphorylation by Src, Dok1 and Dok2 adaptor proteins are
recruited and subsequently bound RasGAP and SHIP allowing the
downstream inhibition of the Ras MAPK pathways [113].
There is evidence supporting an immunoregulatory role for CD200
[110]. In particular, interaction with CD200R that is constitutively
expressed by monocytic myeloid cells such as macrophages and
dendritic cells represses their pro-inflammatory activation in vivo
[114-118], reduces activation of MAPKs in mononuclear cells, and
represses degranulation of human mast cells and basophils [119-121].
It is also probable that CD200 expressed by endothelium has a role in
controlling circulating neutrophil degranulation, but this has not been
directly tested. CD200R is the only definitively characterized receptor
for CD200, and functional ligands for the activating isoforms of the
receptor have yet to be found in humans or mice [110].
1.3.5 SIGLEC
The SIGLECs (sialic-acid-binding immunoglobulin-like lectins) are the
best characterized immunoglobulin-type lectins belonging to IgSF
[122]. They represent the type 1 membrane proteins with aminoterminal V-set immunoglobulin domain that binds sialic acid and
variable numbers of C2-set immunoglobulin domains. Human
monocytes express a great numbers of inhibitory SIGLEC molecules
including Siglec-3 (CD33), Siglec-5 (CD170), Siglec-7 (CD328), Siglec9 (CD329) and Siglec-10 that are all sub-classified as CD33-releated
SIGLECs. Siglec-11 is expressed exclusively by macrophages. Human
neutrophils and myeloid (conventional) dendritic cells express Siglec-9
(CD329).
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INTRODUCTION
Figure 9. Siglec-family proteins in humans and rodents [122]. B, B cells; Ba,
basophils; cDCs, conventional dendritic cells; Eo, eosinophils; GRB2, growthfactor-receptor-bound protein 2; ITIM, immunoreceptor tyrosine-based inhibitory
motif; Mac, macrophages; Mo, monocytes; MyP, myeloid progenitors; N,
neutrophils; ND, not determined; NK, natural killer cells; OligoD, oligodendrocytes;
pDCs, plasmacytoid dendritic cells; Schw, Schwann cells; Troph, trophoblasts.
Sialoadhesin (Siglec-1 or CD169), one of the largest memebrs of IgSF
with 17 Ig domains in the extracellular part, was discovered as a sialicacid-dependent macrophage adhesion molecule [123]. Contrasting to
most SIGLECs, sialoadhesin lacks tyrosine-based signaling motifs and
its cytoplasmic tail is poorly conserved, which suggests a primary role
as a binding partner in cell–cell interactions, rather than in cell
signaling [122]. Sialoadhesin is constitutively expressed on
subpopulations of tissue-resident macrophages [124] and is rapidly upregulated on inflammatory macrophages which indicate its
contribution in their pro-inflammatory functions.
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INTRODUCTION
The CD33-related SIGLECs are mainly expressed by mature cells of
the innate immune system, such as neutrophils, eosinophils,
monocytes, macrophages, NK cells, DCs and mast cells (Figure 8).
CD33 itself is a marker of myeloid progenitor cells, indicating a
potential role for CD33 in the regulation of cellular proliferation
and/or differentiation. There are great number of studies showing the
important roles of CD33-related SIGLECs in modulating leukocyte
behavior, including inhibition of cellular proliferation, induction of
apoptosis, inhibition of cellular activation and induction of proinflammatory cytokine secretion [122]. The SIGLECs signaling
pathways are poorly understood but in most cases they are assumed to
involve ITIM and ITIM-like motifs and recruitment of tyrosine
phosphatases [122].
1.3.6 CD300
CD300 represents a multigenic family of leukocyte surface receptors
belonging to the Ig superfamily, originally termed CMRF-35 and
Immune Receptor Expressed by Myeloid cells (IREM), that is
conserved in different mammalian species [125].
The human CD300 gene cluster is located in chromosome 17
(17q25.1). The molecules are encoded by genes with intron-exon
structures that are conserved between human and rodents ,[126]
(Figure 10). The locus contains six genes encoding for two inhibitory
receptors (CD300a/IRp60 and CD300f/IREM-1), two activating
receptors (CD300e/IREM-2 and CD300b/IREM-3), and two
receptors with still undefined function (CD300c/CMRF-35 and
CD300d/IREM-4). CD300g/Nepmucin is the most distantly related
CD300 molecule encoded by a gene that is mapped upstream from the
main complex (17q21). It shares similar IgV-like domain but is
exclusively expressed by endothelial cells unlike the other family
members [127]. In the close proximity to the CD300c/CMRF-35
gene is located a pseudogene (φ) that has a high degree of sequence
similarity with CD300c [128].
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INTRODUCTION
Figure 10. Shematic diagram showing the organization of CD300 gene complexes
in (a) human and (b) mouse. Gene orthologues are shaded similarly. [126].
Murine orthologues of CD300 molecules are members of CD300L
(CD300-Like) family with a gene custer located in mouse chromosome
11 that is syntenic to human chromosome 17 (25). It contains nine
genes encoding for six triggering (CLM-2, -3, -4, -5, -6 and -7) and two
inhibitory receptors (CLM-1 and CLM-8). CLM-9 is a counterpart of
human CD300g and is also located upstream of the CD300L locus. It
is of noteworthy that sequence homology in most cases is not
correlated with functional similarity between human and mouse
molecules. Only CD300g, CD300a and CD300f have clear orthologs
within the murine locus.
The CD300 molecules are type I transmembrane glycoproteins (Figure
11) with a single IgV-like extracellular domain and an extended
membrane proximal region that is rich in prolines, serines and
threonines, which links the Ig and transmembrane domains. The
CD300 IgV domain contains a conserved amino acid motif YWCR
and two disulfide bonds instead of one that makes them a distant
relatives of Fc receptors for polymeric A and M, TREMs and CD336
(NKp46) [126]. The transmembrane domains of CD300b, CD300c,
CD300d, and CD300e contain a charged amino acid residue, which
enables association with other transmembrane adaptor molecules such
as DAP12, whereas the cytoplasmic domains of CD300a and CD300f
contain tyrosine-based signalling motifs, particularly immunoreceptor
tyrosine-based inhibitory motifs (ITIM).
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INTRODUCTION
Figure 11. Schema of the individual CD300 molecules in (a) human and (b)
mouse [126].
Distribution of CD300 molecules has been determined by a
combination of mRNA and protein expression analyses when mAbs
are available. CD300d and CD300f transcripts are present in high
levels in the lung and CD300c in spleen and thymus. Expression of
CD300 in peripheral blood (PB) leukocytes follows four patterns.
First, CD300a and CD300c have a broad expression on leukocytes
including CD34+ hematopoietic stem cells but not B lymphocytes nor
a subpopulation of T lymphocytes [90][129-132]. Their surface
expression on PB CD4+ T lymphocytes subdivides those cells into two
naïve and two memory populations that have distinct functional and
survival capabilities [133]. Second, transcripts for all CD300 molecules,
except CD300g, are expressed by peripheral blood myeloid and DC
lineages, although in vitro-generated monocyte-derived DC (moDC)
down-regulate CD300a, CD300c and CD300f. There is still limited
information on the cellular distribution of CD300b and CD300d due
to the lack of available specific mAbs. Third, CD300e shows a
restricted expression, being present only in some mature myeloid
populations, monocytes and myeloid dendritic cells, and is also downregulated in in vitro-derived moDC [134, 135]. Finally, CD300g and its
28
INTRODUCTION
mouse ortholog CLM-9 mRNA are not expressed by leukocytes but
are expressed at high levels in the heart and in placenta [127].
Ligands for the CD300 molecules remain elusive. The data showing
that a fusion protein expressing the CLM-1 IgV domain was able to
bind to T cells, indicates that endogenous CD300 ligands probably
regulate interactions between cells [136]. The high degree of amino
acid identity between the CD300a and CD300c Ig domain sequences
implies that they might bind similar ligands, eventually with different
affinities [126]. CD300a was considered to be a potential NK cell
inhibitory molecule, but it does not bind to HLA-class I molecules
[129].
Despite the considerable sequence identity between the CD300 Ig
domains and the Ig binding domains of the Poly Ig receptor, only
CD300g has been shown to bind human Ig [89, 90, 127, 137, 138].
but its physiological relevance is unclear. Some CD300g isoforms can
bind L-Selectin that is expressed in lymphocytes and is known to
enhance lymphocyte adhesion [139].
1.3.6.1 Human CD300 receptors
CD300a
The first characterized inhibitory member of CD300 family, CD300a
(also termed Inhibitory Receptor protein 60, IRp60) is a type-I
transmembrane glycoprotein that contains an extracellular V-type Ig
domain, highly homologous to CD300c. Its cytoplasmatic region
displays four tyrosine residues out of which three are in the context of
ITIMs. Upon tyrosine phosphorylation these ITIMs are able to recruit
SHP-1, SHP-2 and SHIP phosphatases [129]. The extracellular region
of CD300a is highly N- and O-glycosilated in the membrane proximal
region.
CD300a is expressed by human NK cells, monocytes, neutrophils and
a subset of CD3+ T cells [129, 130]. CD300a might be expressed on all
NK clones, but not all NK clones could deliver an inhibitory signal,
probably due to the presence of CD300c with whom CD300a shares a
high degree of homology [140]. It is also expressed by eosinophils and
mast cells [131, 132]. In human neutrophils CD300a is rapidly upregulated upon stimulation with LPS and GM-CSF [130] due to the
translocation to the plasma membrane of the pre-synthesized receptor.
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INTRODUCTION
Treatment of cord-blood mast cells with human eosinophil-derived
neurotoxin and major basic protein down-regulated the expression of
CD300a [131].
In neutrophils, cross-linking of CD300a inhibits ITAM-initiated
calcium influx and production of ROS [130]. Moreover, engagement
of the receptor is able to inhibit NK cell mediated cytotoxicity upon
recruitment of SHP-1 and SHP-2 [129]. In mast cells, CD300a inhibits
the release of β-hexosaminidase and IL-4, as well as stem cell factormediated survival and differentiation [131, 132, 141]. In eosinophils,
CD300a associates with SHP-1 and is able to inhibit eotaxin-induced
transmigration, and secretion of TNF-α, IL-β1, IL-4 and IFN-γ [132];
it also suppresses the anti-apoptotic effects of IL-5 and GM-CSF.
CD300b
CD300b (also known as IREM-3) is an activating member of the
CD300 family. The extracellular region of CD300b displays a single Vtype domain, followed by membrane proximal region, transmembrane
domain containing positively charged lysine residue and short
cytoplasmatic tail with tyrosine-based motif [142]. In transfected cells,
CD300b was able to interact with DAP12 and deliver activating
signals through this ITAM-bearing adaptor protein. In addition, a
tyrosine-based motif in the cytoplasmatic tail of CD300b is able to
recruit growth-factor receptor-bound protein 2 (Grb-2) in a
phosphorylation dependent manner [142].
In the absence of specific monoclonal antibodies, information about
CD300b expression was obtained using RT-PCR. CD300b transcripts
were found abundantly in human monocytes and myelomonocytic cell
lines [142], as well as in a wide set of human tissues, including colon,
lung, placenta, bone marrow and fetal liver.
Cross-linking of CD300b on the surface of a RBL/CD300b
transfectant resulted in NFAT/AP-1-dependent transcriptional
activity and hexosaminidase release both in the presence and absence
of DAP12, suggesting the association with an unknown adaptor in
RBL cells that would account for DAP12-independent signaling [142].
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INTRODUCTION
CD300c
Despite being the first cloned member of the CD300 family back in
1992 as a CMRF-35A, precise information about its function and
expression pattern is still unclear. None of the recently generated antiCD300c mAbs could discriminate between CD300a and CD300c,
since these two molecules show 80% identity at the amino acid level in
their Ig domains [140]. RT-PCR analyses indicate the presence of
CD300c transcripts in all peripheral blood populations at a different
level. A higher expression was found in monocytes, CD34+ cord
blood and NK cells. Both CD4+ and CD8+ T cells, together with
plasmacytoid and CD11c+ blood DC have lower expression of
CD300c [126].
The CD300c transcripts encode for a type-I transmembrane protein
with single extracellular V-type Ig domain followed by a membrane
proximal region, a transmembrane domain bearing a positively
charged glutamic acid and a short cytoplasmatic domain [143]. Very
recently it has been shown that CD300c receptor is able to deliver
activating signals in transfected RBL-2H3 mast cells and that its
signaling was partially mediated by association with FcεRγ [144].
CD300d
CD300d is a recently cloned gene that, according to RT-PCR
expression analysis, is present at high levels in the lung, monocytes
and CD11c+ blood DC [126]. Sequence analysis predicts that CD300d
is structurally similar to other triggering CD300 members, having a
short cytoplasmatic tail with no signaling motifs and a positively
charged lysine residue within its transmembrane domain (Figure 11).
At the moment no information about its function and cellular
distribution is available.
CD300e
CD300e was originally termed IREM-2 and according to the sequence
analysis and some data obtained with transfected cells, corresponds to
a triggering receptor. Like the other CD300 molecules, it consists of a
single V-type Ig domain, a transmembrane domain bearing a lysine
residue and a short cytoplasmatic tail [134].
CD300e expression is restricted to cells of myeloid origin, and in
peripheral blood CD300e can be detected on the surface of
monocytes and myeloid DC [134, 135]. On the other hand, monocyte31
INTRODUCTION
derived dendritic cells generated in vitro and myeloid cell lines do not
express CD300e [134]. CD300e is displayed at a relatively late stage of
monocyte differentiation, when precursors have already acquired high
levels of CD14 and CD64 [134]. CD300e transcripts were detected in
bone marrow, thymus, lungs and spleen [126].
In a CD300e/RBL transfectant engagement of the receptor was able
to induce transcriptional activity and it interacted with DAP12 in
transiently transfected COS-7 cells [134]. Even though it was shown
that CD300e engagement can induce TNF-α secretion in human
monocytes information regarding its functional role remained rather
limited.
CD300f
CD300f also known as IREM-1 is another inhibitory member of the
family. It is composed of a single V-type Ig domain, a membrane
proximal region followed by a transmembrane region and a long
cytoplasmatic tail with five tyrosine residues [145]. Two of them are in
the context of ITIMs and two provide docking sites for the p85α
regulatory subunit of PI3K.
CD300f transcripts are present in the spleen and lungs and are found
at high levels in monocytes, neutrophils, mast cells, eosinophils, and
CD11c+ DC [126]. CD300f has been detected on the surface of
monocytes and neutrophils by using specific mAbs [145].
CD300f was able to recruit SHP-1 in a phosphorylation dependent
manner and one of the ITIMs was shown to be essential for this
interaction [145]. The inhibitory role of CD300f was studied in
CD300f transfected RBL cells, where it was shown that its
engagement prevents activation induced by signals delivered through
the FcεRI [145]. In the same experimental system, CD300f was able to
recruit the p85α subunit of PI3K and deliver activating signals when
ITIMs and the distal tyrosine-based motif were disabled. Moreover, in
myeloid cell lines CD300f can bind both SHP-1 phosphatase and p85α
simultaneously consistent with a putative functional duality of the
receptor [146].
CD300g
The CD300g or nepmucin is a distant member of the CD300 family.
The gene encoding for CD300g is mapped at some distance from the
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INTRODUCTION
main gene cluster. CD300g contains a single V-type immunoglobulin
(Ig) domain and a mucin-like domain, a transmembrane region and a
long cytoplasmatic tail lacking the structural features suggestive of
stimulatory or inhibitory potential [127].
Unlike other CD300 members, CD300g is not expressed by
leukocytes. Instead its expression is restricted to endothelial cells [139].
CD300g transcripts were identified abundantly in heart and placenta
[127].
CD300g was shown to mediate L-selectin-dependent lymphocyte
rolling and promotes lymphocyte adhesion [139]. Moreover, it is the
only CD300 member shown to bind Ig, inlcuding human IgA2 and
IgM but not IgG [127, 138].
1.3.6.2
Mouse CD300 receptors
CLM-1
CLM-1 encoded by CD300LF gene, was the first identified murine
CD300 molecule and thus far the best characterized. It the literature it
can also be found under the following names: DIgR2, MAIR-Va/b
and LMIR32 [136, 147-149]. The CLM-1/LMIR3/MAIRVa and
DIgR2/MAIRVb are two alternative slicing variants of the molecules
that differ in seven amino acids.
CLM-1 is functional and the orthologue of human CD300f. It
contains within the cytoplasmatic tail two ITIMs, a potential PI3K
docking site and an additional tyrosine residue in the context of a
putative SAP (SLAM-associated protein) motif. Through ITIMs,
CLM-1 is able to recruit SHP-1 and SHP-2 upon tyrosine
phosphorylation but not SHIP [136, 150, 151]. Phosphorylated CLM1 also associates with PI3K Grb-2 and, unexpectedly, with FcRγ in
bone-marrow derived mast cells (BMMCs) [151].
The receptor is expressed in myeloid cells such as granulocytes,
dendritic and mast cells [126]. CLM-1 expression in granulocytes is
up-regulated by TLR4 ligand and G-CSF [149].
2 CLM - CMRF-35 like molecule; DIgR – Dendritic cell-derived Ig-like Receptor ;
MAIR – Myeloid-Associated Ig-like Receptor; LMIR – Leukocyte Mono-Ig-like
Receptor; IgSF – Immunglobulin superfamily
33
INTRODUCTION
The inhibitory role of the CLM-1 was studied in the myeloid
differentiation process and regulation of DC priming. It was shown
that receptor engagement inhibits differentiation of myeloid cells to
osteoclasts in response to RANKL and TGFβ [150] and prevents
dendritic cell-initiated antigen-specific Th1 responses [136]. It was
recently reported that CLM-1 cross-linking enhanced cytokine
production of BMMCs stimulated with LPS, while suppressing the
production stimulated by other TLR agonists and stem cell factor
[151]. This data support that CLM-1 has a dual function in mouse
BMMCs as proposed for the human CD300f receptor [146].
Inhibitory signals were mediated by SHP-1 and SHP-2 phosphatases,
whereas the activating signal was dependent on its association with
FcRγ. An additional inhibitory role of CLM-1 was recently identified
in the experimental autoimmune encephalomyelitis, a preclinical
model of multiple sclerosis, where it was shown that the receptor acts
as a negative regulator of myeloid effector cells in demyelination [152].
In several transfected cell types, CLM-1 engagement induced
apoptosis through the caspase- and endoplasmic reticulumindependent mechanisms [148]. CLM-5 was identified as an activating
counterpart of CLM-1 [137, 138, 149].
Other CLM molecules
The CLM-2 (also termed MAIR-VIII and IgSF18) is encoded by
CD300E gene and represents a mouse orthologue of human CD300e
activating receptor. Thus far, there is no available information
regarding its expression or functional role. CLM3 or MAIR-VI is
another member of the mouse CD300 family that has not been
characterized so far.
CLM-4, also known as MAIR-II, DIgR1 and IgSF7 is encoded by
CD300LD gene. Even though it is the orthologue of CD300d,
structurally it is more similar to CD300b. It is expressed by peritoneal,
spleen and bone marrow-derived macrophages and a small subset of B
cells in the spleen that upon LPS stimulation up-regulate its expression
[153]. CLM-4 transcripts were found in antigen-presenting cells,
including DC, monocytes/macrophages and are highly abundant in
spleen, granulocytes and NK cells [126, 154]. CLM-4 is able to bind
DAP12 in resting and LPS-stimulated B cells isolated from spleen and
peritoneum [153, 155]. In peritoneal and bone marrow derived
macrophages CLM-4 interacts with another ITAM-bearing adaptor
34
INTRODUCTION
FcRγ [155]. CLM-4 engagement in peritoneal and spleen macrophages
results in the release of TNF-α, IL-6 and MCP-1[153, 155] .
CLM-5 (also termed as MAIR-IV and LMIR-4) is preferentially
expressed by peripheral blood neutrophils, bone marrow and spleen.
It is also expressed by bone marrow and spleen macrophages and
CD11c+ DC [156]. Stimulation of granulocytes with LPS resulted in
complete down-regulation of CLM-5 [149]. Upon cross-linking in
mast cells, it induces IL-6, TNF-α, histamine secretion and cell
survival [149]. CLM-5 was reported to be the activating counterpart of
the inhibitory CLM-1 [137, 149]. In mast cells and granulocytes CLM5 synergizes with TLR4 [149] and additionally in mast cells also with
FcεR [149]. In transfected cell and peritoneal macrophages CLM-5
was shown to associated with FcRγ [156].
CLM-6 corresponds to the orthologue of human CD300c and is also
termed MAIR-III and LMIR2. It is a type I transmembrane protein,
with a single extracellular variable Ig domain that shares about 90%
amino acid identity with CLM-8. This indicates that CLM-8 and CLM6 represent a pair of molecules that regulate mast cell-mediated
inflammatory responses. In COS-1 transfectants CLM-6 is associating
with DAP10, DAP12, and FcRγ adaptor proteins [138]. The
expression pattern of CLM-6 is still poorly defined.
CLM-7 (also termed LMIR5, MAIR-VII, IgSF17, mIREM-3 and
CD300Lb) is the orthologue of CD300b. The molecule was detected
in the spleen and peritoneal macrophages, granulocytes and spleen
DCs. Mast cells and in vitro derived bone-marrow populations also
express CLM-7 [157]. Cross-linking of transduced CLM-7 in bone
marrow-derived mast cells (BMMCs) triggered activation, promoting
cytokine and chemokine production (TNF-α, IL-6 and MCP-1), cell
survival, degranulation, and adhesion to the extracellular matrix. In the
same cells CLM-7 was shown to associate with both DAP12 and
DAP10 [157]. Very recently T cell Ig mucin 1 (TIM1) was identified as
a ligand for CLM-7 [158].
CLM-8 is also known as MAIR-I, LMIR1 and CD300La. It has the
typical structure of inhibitory receptors with five tyrosine residues in
cytoplasmatic tail in the context of ITIMs. Through those residues
CLM-8 is able to bind SHP-1, SHP-2 and SHIP phosphatases in
transfected cells [138, 159] but only SHIP in BMMCs [138, 153].
35
INTRODUCTION
CLM-8 has a wide expressional pattern. It is present in myeloid cells,
including monocytes, neutrophils, eosinophils and basophils,
peritoneal and spleen macrophages, spleen DCs, a small subset of B
cells isolated from spleen and in bone-marrow-granulocytes and mast
cells. It can also be induced on the surface of NK cells in response to
IL-12 [153, 160]. The functional role of CLM-8 was broadly studied in
mouse models of allergy since it is highly expressed by mast cells and
eosinophils. Cross-linking of CLM-8 in the mouse model of asthma
resulted in decreased secretion of Th2 cytokines and mast cells
mediators including IL-4, IL-5, IL-13, tryptase and eotaxin-2 [141,
160]. Engagement of CLM-8 impaired mast cell degranulation and
anaphylactic reactions in the passive cutaneous anaphylaxis model
[141, 161]. CLM-8 was also shown to inhibit both IgE-dependent
serotonin and hexosaminidase release [153, 159].
Nepmucin or CLM-9 is the Ig domain-containing sialomucin and
orthologue to human CD300g receptor. Unlike the other CLM
members it is expressed in vascular endothelial cells of various tissues
including those of high endothelial venules in lymph nodes [139].
There nepmucin supports L-selectin-dependent lymphocyte rolling
through its mucin-like domain and mediates lymphocyte binding
through its Ig domain. Nepmucin-expressing endothelial cells also
showed enhanced lymphocyte transendothelial migration and it is
suggested that endothelial nepmucin promotes this by using multiple
adhesion pathways [162].
36
INTRODUCTION
37
INTRODUCTION
38
Aims
39
40
2. AIMS
The present work was originally developed in the context of the
project focused on the characterization of CD300e/IREM-2 and
CD300f/IREM-1 myeloid cell receptors. The main aims were:
1. To functionally characterize CD300e in human monocytes and
myeloid dendritic cells and investigate the implications that its
engagement has on cells that naturally express the receptor.
2. To examine the functional role of the inhibitory CD300f
receptor in human granulocytes.
41
42
Results
43
44
3. RESULTS
3.1 Article 1:
Functional analysis of the CD300e receptor in human
monocytes and myeloid dendritic cells
Eur J Immunol. 2010 Mar; 40(3):722-32.
3.2 Article 2:
Engagement of CD300e induces the differentiation of
human monocytes to macrophages involving an
autocrine M-CSF-dependent pathway
Submitted manuscript
3.3 Appendix I
Functional characterization of CD300f inhibitory
receptor in human granulocytes
Preliminary results
45
46
Article 1: Functional analysis of the CD300e receptor in
human monocytes and myeloid dendritic cells
Eur J Immunol. 2010 Mar; 40(3):722-32
47
Brckalo T, Calzetti F, Pérez-Cabezas B, Borràs FE, Cassatella MA, López-Botet M.
Functional analysis of the CD300e receptor in human monocytes and myeloid dendrictic
cells. Eur J Immunol. 2010; 40(3): 722-32.
48
RESULTS: ARTICLE 1
74
RESULTS: ARTICLE 1
75
RESULTS: ARTICLE 1
76
Article 2:
Engagement
of
CD300e
induces
the
differentiation of human monocytes to
macrophages involving an autocrine M-CSFdependent pathway
Submitted manuscript
77
78
RESULTS: ARTICLE 2
Engagement of CD300e induces the differentiation of
human monocytes to macrophages involving an
autocrine M-CSF-dependent pathway
Tamara Brckalo*, Diogo Baia*, and Miguel Lopez-Bótet*(†)
(*)
Immunology Unit, Department of Experimental and Health
Sciences, University Pompeu Fabra, Barcelona, Spain; (†)IMIMHospital del Mar, Barcelona, Spain
Key words: human,
differentiation, M-CSF
monocytes,
macrophages,
CD300,
cell
Correspondence: Miguel López-Botet, Immunology Unit,
Department of Experimental and Health Sciences, University Pompeu
Fabra, Dr Aiguader 88, 08003 Barcelona, Spain; fax. +34933160410, email: [email protected]
Abbreviations: IREM, Immune receptor expressed by myeloid cells;
DAP, DNAX-activating protein; DC, dendritic cells; FADD, Fasassociated death domain ; FLIP-L, FLICE Inhibitory protein L ; OxLDL, oxidized low-density lipoprotein; MΦ, macrophages; CD300e
MΦ, macrophages obtained by stimulating monocytes with antiCD300e mAb; MΦ1, classically activated macrophages; MΦ2,
alternatively activated macrophages; moDC, monocyte derived
dendritic cell; PAK2, p21-activated protein kinase 2; ROS, reactive
oxygen species; RIP1, Receptor-Interacting Protein 1; pMΦ, human
peritoneal macrophages.
79
RESULTS: ARTICLE 2
ABSTRACT
Human monocytes may alternatively differentiate to dendritic cells or
macrophages depending on the nature of the environmental signals. In
vitro monocytes stimulated with M-CSF or GM-CSF yield the socalled alternatively (MΦ2) or classically activated macrophages (MΦ1),
respectively. Cross-linking of the CD300e receptor restricted to the
monocytic lineage was reported to promote cell activation and
survival. We provide evidence supporting that CD300e engagement
led to monocyte differentiation to macrophages displaying a
CD14brightCD16+CD163low/negCD1a-CD11bbrightCD209phenotype.
Upon stimulation with LPS these cells exhibited a low respiratory
burst but high phagocytic activity, and mainly produced IL-10, thus
resembling type 2 macrophages (MΦ2). Monocyte differentiation
induced by CD300e was inhibited by anti-M-CSF and anti-CD115
(CSF-1R or M-CSF R) mAbs. CD300e engagement did not upregulate CD115 surface expression but rather enhanced its
internalization associated to M-CSF production. Detection of caspase3 active fragments in monocytes activated via CD300e further
indirectly supported M-CSF/CD115 involvement in the
differentiation process. Overall, our data indicate that CD300e
regulates monocyte differentiation to functional MΦ2 macrophages by
a mechanism that entails an autocrine production and consumption of
M-CSF.
80
RESULTS: ARTICLE 2
INTRODUCTION
Differentiation of monocytes to macrophages (MΦ) or dendritic cells
(DC) depends on environmental factors encountered during migration
to peripheral tissues [172, 184-186]. A number of in vitro conditions
have been reported to induce different patterns of monocyte
differentiation. Monocytes differentiate to immature dendritic cells in
the presence of serum from systemic lupus erythematosus patients
that contains IFN-α [187], in response to IL-4 plus GM-CSF
produced by mast cells [162-164] and also upon transendothelial
trafficking [188]. Culturing monocytes in the presence of GM-CSF
together with either LPS, TNF-α or calcium ionophore leads to their
differentiation to mature dendritic-like cells [189, 190]. DCs that have
features of Langerhans cells can be obtained in culture with TGF-β or
GM-CSF plus IL-15 [191]. On the other hand, monocyte
differentiation to macrophages is efficiently induced in the presence of
either macrophage colony-stimulating factor (M-CSF or CSF-1) [192,
193] or granulocyte/macrophage colony-stimulating factor (GM-CSF
or CSF-2) [194] . In addition, , interleukin 32 (IL-32) [195], human
cytomegalovirus (HCMV) infection [196] and oxidized low-density
lipoprotein (Ox-LDL) [197] can also induce differentiation of
monocyte to macrophage-like cells. Some cytokines such as IL-10
[198], IL-6 [199] and IFN-γ [200] are able to switch monocyte
differentiation from dendritic cells to macrophages either inducing MCSF secretion or up-regulating surface expression of the M-CSF
receptor (CD115 or CSF-1R). Recently, Toll-like receptor (TLR)
activation of human monocytes has been reported to induce
differentiation to macrophages and dendritic cells, by up-regulating
IL-15 and IL-15-R and GM-CSF and CD114 (GM-CSF-R),
respectively [201].
M-CSF is the main regulator of survival, proliferation and
differentiation of mononuclear phagocytes, which include monocytes,
macrophages, osteoclasts, and their precursors [202, 203]. M-CSF is
detected in the circulation under steady-state conditions and may be
produced in vitro by several cell types including fibroblasts,
endothelial cells, stromal cells, macrophages, smooth muscle cells and
osteoblasts [204]. The M-CSF receptor, encoded by the protooncogene Csf1r [205] is a tyrosine kinase exclusively expressed on
mononuclear phagocytes. Binding of M-CSF to CD115 induces
receptor dimerization, autophosporylation of cytoplasmatic tyrosine
residues and phosphorylation of signaling proteins (e.g. SHP-1, Src
81
RESULTS: ARTICLE 2
kinases, PLC-γ, PI(3)K, Akt and Erk) [206]. The M-CSF/CD115
complex undergoes internalization and further degradation by
lysosomes. CD115 signaling was shown to be essential for cell survival
[207] and their entry into the S phase of cell cycle [208].
CD300e is an activating receptor selectively expressed by mononuclear
cells (i.e.) that has been shown to trigger various inflammatory
responses in peripheral blood monocytes and mDCs, including
intracellular calcium mobilization, respiratory burst activity, upregulation of surface markers and cytokine production [209]. CD300e
is expressed at a relatively late stage of the monocyte maturation
process, when their precursors have already acquired high levels of
CD14 [134], and has been used as a specific monocyte marker [135].
We recently reported that cross-linking of CD300e promoted
monocyte survival by a still unknown mechanism [209]. In the present
study, we addressed whether prolonged survival of monocytes was
eventually associated to their differentiation. Our results indicate that
CD300e ligation leads to differentiation of monocytes to macrophages
that show an anti-inflammatory or alternatively activated (MΦ2)
profile, according to the expression of specific surface markers,
cytokine secretion and phagocytic activity. Moreover, CD300einduced monocyte differentiation appeared mediated by an autocrine
effect of M-CSF associated with caspase-3 activation. Our results
support that activation through a monocyte-specific receptor results in
their differentiation into functional macrophages.
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RESULTS: ARTICLE 2
MATERIALS AND METHODS
Isolation and differentiation of monocytes
Human peripheral blood samples were obtained from healthy donors
according to guidelines approved by the Clinical Research Ethical
Committee (CEIC-IMAS). Peripheral blood mononuclear cells
(PBMCs) were isolated under the endotoxin-free conditions from
fresh blood by Ficoll-Paque PLUS centrifugation (GE Healthcare BioSciences AB, Uppsala, Sweden) and extensively washed with PBS for
platelet removal. Monocytes were isolated from total PBMC by
negative selection using the EasySep™ Human Monocytes
Enrichment Kit without CD16 Depletion (StemCell Technologies,
Seattle, WA, USA) following the manufacturer’s guidelines. Purity of
cell preparation was assessed by FACS using the CD14 as a monocyte
marker. 80-95% cells were CD14+ and viability was greater than 98%
according to Trypan blue exclusion staining (Sigma Aldrich, Sent Lois,
MO, USA). Monocytes were cultured at 1 x 106 cells/ml in RPMI1640/Glutamax source medium (Invitrogen Life Technologies,
Paisley, UK) supplemented with 10% (v/v) heat-inactivated fetal calf
serum with low endotoxin level (Greiner Bio-One GmbH,
Frickenhausen, Germany) at 37OC in 5% CO2 atmosphere and
endotoxin-free conditions.
Differentiation of monocytes into alternatively activated macrophages
(MΦ2) or classically activated macrophages (MΦ1) was performed in
the presence of M-CSF (100 ng/ml, ImmunoTools GmbH,
Friesoythe, Germany) or GM-CSF (50 ng/ml, R&D Systems,
Minneapolis, MN, USA), respectively. In order to differentiate
monocyte to dendritic cells GM-CSF plus IL-4 (bouth from R&D
Systems, Minneapolis, MN, USA) were used at 50 ng/ml and 25
ng/ml, respectively.
In vitro stimulation of monocytes with anti-CD300e mAb
For cell activation through the CD300e receptor, an agonistic antiCD300e mAb (clone UP-H2, IgG1) was used [134]. Flat-bottom 24
well plates (Greiner Bio-One, GmbH, Frickenhausen, Germany) were
coated with 10 μg/ml of anti-CD300e or isotype-matched control
mAb MOPC-21 (mouse IgG1, from Sigma Aldrich, Sent Luis, MO,
USA) for 3-4 hours at 37oC. Freshly isolated monocytes were added to
the wells at a concentration of 1 x 106/ml and cultured for 6 days. MCSF and IL-6 neutralizing antibodies (both from R&D Systems,
Minneapolis, MN, USA) were used as previously described [199].
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RESULTS: ARTICLE 2
Briefly, anti-M-CSF mAb was added to the cultures at 0.5 µg/ml at
days 0 and 3; neutralizing anti-IL-6 mAb was used at daily doses of 2.5
µg/ml. Neutralizing anti-CD115 mAb was also used at 0.5 µg/ml at
days 0 and 3 (Santa Cruz Biotechnology, San Diego, CA, USA).
Flow cytometry analysis
Cells were incubated on ice in 15% human serum to block Fc
receptors in a round bottom 96-well culture plate (Corning
Incorporated, Corning, NY, USA). The following murine monoclonal
antibodies were used: PE-conjugated anti-CD1a, anti-CD14, antiCD16 (BD Biosciences, San Jose, CA, USA), anti-CD163 (BioLegend,
San Diego, CA, USA). For indirect staining, cells were incubated with
either anti-CD11b (mouse IgG1, clone BEAR-1), anti-CD209 (clone
MAB161, IgG2b), anti-cleaved caspase-3 mAb (clone 5A1E, Cell
Signaling Technology, Danvers, MA, USA) or anti-CD115 (clone 24A5, Santa Cruz Biotechnology, San Diego, CA, USA) followed by
either PE-conjugated rabbit anti-mouse IgG (DakoCytomation
Denmark A/S, Glostrup, Denmark), FITC-conjugated goat anti-rat
IgG1/2 (BD Pharmingen, San Diego, CA, USA) or PE-conjugated goat
anti-rabbit IgG (Sigma Aldrich, Sent Lois, MO, USA). For each
staining the appropriate PE-, or FITC- isotype controls were included
(ImmunoTools GmbH, Friesoythe, Germany) and cells were analyzed
by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA,
USA) . For each staining at least 10 000 events were collected and data
analysis was performed using the FlowJo software (Three Star,
Ashland, OR, USA).
To compare the staining intensity of different samples we calculated
the ratios between the geometric mean fluorescence intensity (geo
MFI) of samples and isotype-matched controls (MFI=geo
MFIsample/geo MFIisotype control). The number of cells (Y axis) was
normalized for the different overlaid samples and represented as “%
of Max” using the FlowJo software.
Intracellular staining
In order to detect the active fragment of caspase-3 we performed the
intracellular staining with anti-cleaved caspase-3 mAb (clone 5A1E,
Cell Signaling Technology, Danvers, MA, USA) according to
manufacturer’s instructions. Briefly, monocytes incubated for 72h with
either anti-CD300e, M-CSF or GM-CSF plus IL-4 were collected and
fixed with 4% formaldehyde. Cells were then permeabilized with 90%
methanol and incubated on ice for 30 minutes. After blocking with
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RESULTS: ARTICLE 2
0.5% BSA in PBS, cells were incubated with anti-cleaved caspase-3
mAb followed by conjugated secondary antibody and analyzed by flow
cytometry.
Production of reactive oxygen intermediates
Production of superoxide anion (O2_) was assayed by detection of
reduced Cytochrome c as previously described [209]. PMA at the
concentration of 4 µg/ml was used to trigger the respiratory burst in
macrophages.
Cytokine detection
Cytokines were detected in cell free culture supernatants using
commercial ELISA kits for IL-6, IL-10, TNF-α (all from
ImmunoTools GmbH, Friesoythe, Germany), IL-12p70 (Bioscience,
San Diego, CA, USA) and M-CSF (R&D Systems, Minneapolis, MN,
USA).
Detection of phagocytosis
Phagocytic activity was evaluated by the internalization of PE-labeled
polystyrene microspheres (FluoresbriteR Polychromatic red
microspheres 1μm, Polysciences, Warrington, PA, USA) as described
elsewhere [210]. At the end of the culture cells were harvested,
incubated with PE-labeled polystyrene microspheres (E:T ratio 1:25)
for 60 and 120 minutes at 37OC and then transferred to ice. Samples
were washed twice with ice cold PBS, fixed with 1%
paraformaldehyde in PBS and analyzed by FACS. Phagocytic activity
was quantified as the percentage of PE-positive events detected by
flow cytometry (FACScan, Becton Dickinson, San Jose, CA, USA).
Data analysis was carried out using the FlowJo software (Three Star,
Ashland, OR, USA).
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RESULTS: ARTICLE 2
RESULTS
Engagement of CD300e in monocytes promotes their
differentiation to M2-like macrophages
We have recently reported that activation via CD300e triggers the
prolonged survival of monocytes in culture [209]. Monocytes cultured
for 6 days in anti-CD300e mAb-coated plates displayed morphological
changes suggestive of an ongoing differentiation process and were
firmly attached to the wells (Figure 1A, magnification x200). By
contrast, a marked loss of viability (<1%) was observed in monocytes
cultured over the control IgG1 mAb (MOPC-21), thus indicating that
survival and differentiation could not be attributed to FcR engagement
(data not shown).
Flow cytometry analysis of cells recovered after CD300e engagement
revealed that they displayed a CD14brightCD11bbright CD16+ phenotype;
moreover, they were negative for CD1a and CD209 (DC-SIGN), but
expressed CD163, an MΦ2 specific marker (Fig 1B and data not
shown). As compared to MΦ1 and MΦ2 differentiated in parallel with
GM-CSF and M-CSF, respectively, CD300e MΦ clearly resembled
anti-inflammatory macrophages (MΦ2).
These cells are characterized by a high IL-10 production, associated to
low secretion of pro-inflammatory cytokines such as IL-6, IL-12 and
TNF-α [194, 211]. The pattern of cytokine production by CD300e
MΦ was assessed measuring IL-10, IL-6, IL-12p70 and TNF-α
secretion upon stimulation with LPS. Though cytokine production
levels were lower than those detected in MΦ2 generated with M-CSF,
CD300e MΦ produced higher amounts of IL-10 as compared to IL-6
or TNF-α, whereas no IL-12p70 was detected (Fig 2A and data not
shown). Thus, according to their cellular phenotype and cytokine
secretion pattern, CD300e derived macrophages resembled antiinflammatory macrophages.
Phagocytosis and respiratory burst activity of CD300e M
An important functional characteristic of MΦ2, as compared to MΦ1
and moDC, is their higher capacity for antigen uptake, in particular
that of early apoptotic cells [211]. We assessed the capacity of CD300e
derived MΦ for uptaking polystyrene-PE labeled microspheres (1μm)
by FACS, and quantified phagocytic activity as the percentage of PEpositive events (Fig 2B). Similarly to MΦ2, CD300e MΦ exhibited a
higher uptake of microspheres as compared to MΦ1 at 1h (CD300e
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RESULTS: ARTICLE 2
MΦ 56.9 ± 2.2 %; MΦ2 43.9 ± 1.1%; MΦ1 23.4 ± 1.1% ) and 2h of
incubation (CD300e MΦ 82.2 ± 1.1%; MΦ2 81.3 ± 1.3%; MΦ1 46.4
± 2.3%) suggesting that CD300e MΦ share phagocytic properties with
MΦ2.
The production of ROS by activated macrophages is known to play a
key microbicidal role contributing also to inflammation [212, 213].
Thus, we tested the ability of CD300e MΦ to generate ROS in
response to PMA, as compared to MΦ1 and MΦ2. As shown in Fig
2C, superoxide anion production was detected in all three types of
macrophages, being considerably higher in both MΦ1 (332.8 ± 80.2
nmol/106 cells) and MΦ2 (366.6 ± 92.4 nmol/106 cells) as compared
to CD300e MΦ (162.3 ± 3.2 nmol/106 cells). It is of note that basal
respiratory burst was virtually undetectable in CD300e MΦ (1.5 ± 2.2
nmol/106 cells) whereas it was relatively high in MΦ2 and MΦ1 (46.9
± 2.1 and 84.1 ± 6.6 nmol/106 cells, respectively) which might be a
consequence of M-CSF and GM-CSF priming of differentiated
macrophages.
Expression of co-stimulatory molecules
An effective antigen presentation by activated macrophages to T cells
involves the CD80 and CD86 co-stimulatory molecules [214]. Thus,
we examined their expression upon stimulation of the the three
different MΦ populations with LPS. It is of note that basal expression
levels of CD80 were different, being higher in M1Φ and lower in
CD300e MΦ; by contrast CD86 expression was comparable (Fig 3).
Beyond the differences in their basal levels of CD80, both CD300e
MΦ and M2Φ increased its expression in response to LPS, downregulating CD86, as reported for M2Φ [50]. By contrast, activation of
M1Φ with LPS resulted in a marked up-regulation of both costimulatory molecules. These results support that CD300e MΦ may
display a reduced T cell-stimulatory capacity upon LPS exposure,
further supporting their functional resemblance with M2Φ.
Autocrine M-CSF and CD115 play a central role in monocyte-tomacrophage differentiation induced by CD300e engagement
M2Φ are generated in vitro in the presence of exogenous M-CSF, the
most potent macrophage differentiation factor described [172]. Taking
into account the resemblance of M2Φ to CD300e MΦ [172], we
addressed the putative autocrine contribution of M-CSF and its
receptor (CD115). We considered the possibility that CD300e
stimulation might induce M-CSF secretion and/or CD115 expression.
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RESULTS: ARTICLE 2
As shown in Fig 4, a neutralizing anti M-CSF mAb markedly
decreased the number of differentiated macrophages recovered (antiCD300e: 360 ± 19 x103 cells; anti-CD300e plus anti-M-CSF 166 ± 6
x103 cells). Similar results were obtained when CD300e stimulation
was carried out in the presence of an antagonistc anti-CD115 (M-CSF
R) mAb (anti-CD300e: 431 ± 44 x103 cells; anti-CD300e plus antiCD115 281 ± 6 x103 cells) (Fig 4D).
To assess M-CSF production by CD300e activated monocytes, cells
were cultured in anti-CD300e mAb coated plates and supernatants
collected at different time points were assayed by ELISA. As shown in
Fig 4C, samples from all tested donors secreted M-CSF upon CD300e
engagement. At day 3, the M-CSF concentrations reached maximal
levels decreasing by the end of the culture period (day 6). Altogether,
these results support an autocrine contribution of M-CSF to the
observed effects upon CD300e stimulation. IL-6 is known to upregulate the surface expression of M-CSF receptor (CD115) in human
monocytes [199]. In order to test whether IL-6 production triggered
by CD300e ligation may up-regulate CD115, cultures were carried out
in the presence of a neutralizing anti-IL-6 mAb. Under these
experimental settings no changes in the total number of differentiated
macrophages recovered were noticed (anti-CD300e: 431 ± 44 x103
cells; anti-CD300e plus anti-IL-6 506 ± 27 x103 cells) (Fig 4D),
indirectly ruling out an involvement of IL-6 in the differentiation
process promoted by CD300e activation.
Taken together these results indicate that monocyte-to-macrophage
differentiation initiated by CD300e ligation is dependent on M-CSF
and that the M-CSF receptor (CD115) plays an essential role in the
process.
Surface expression of CD115 is down-regulated in CD300e M
Since consumption of M-CSF is associated with internalization of MCSF receptor [215] we evaluated whether CD300e cross-linking
modulated CD115 expression. Freshly isolated human monocytes
were cultured for various days in the presence of plate-bound antiCD300e mAb and subsequently assessed for CD115 expression. As
monocytes cultured in GM-CSF plus IL-4 (moDC) express CD115
stably during the differentiation process [199, 216] we used them as a
control. As shown in Fig 5, CD300e derived M had a reduced
surface expression of CD115 at all time points as compared moDC.
Analysis of permeabilized cells showed similar levels of CD115 in
both cell types (data not shown) thereby suggesting an internalization
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RESULTS: ARTICLE 2
of CD115 by CD300e M as indicated elsewhere [200]. These results
suggest that the up-regulation of M-CSF production by monocytes
triggered by CD300e engagement leads to an autocrine M-CSF
consumption.
CD300e induced differentiation of monocytes to macrophages is
associated with caspase-3 activation
The differentiation of human peripheral blood monocytes to
macrophages in response to M-CSF is associated with activation of
caspases, in particular, caspase-3 and caspase-9 [217, 218]. Since the
neutralizing anti-M-CSF antibody reduced monocyte differentiation
upon CD300e cross-linking, indicating the involvement of the
cytokine in the differentiation process, we tested activation of caspase3 in these cells. Cleaved caspase-3 fragments were detectable in
monocytes undergoing differentiation to M2 as early as 24h,
reaching a peak at 72-96h [217]. By contrast, differentiation of
monocytes to moDC (in the presence of GM-CSF plus IL-4) did not
involve caspase-3 activation. In line with these observations, active
caspase-3 fragments were detected by intracellular staining at 72h in
both CD300e M and M2 (Fig 5), being undetectable in moDC
generated in parallel from the same donor. These results  further
support the participation of M-CSF/CD115 signaling cascade in the
CD300e induced differentiation process.
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DISCUSSION
The CD300e surface molecule, originally termed Immune Receptor
Expressed by Myeloid cells-2 (IREM-2), is an activating receptor
restricted to mature monocytes and myeloid dendritic cells. We have
recently reported that CD300e ligation besides triggering a proinflammatory response in both cell types, also leads to their prolonged
survival in culture. Here we report that survival of monocytes induced
by CD300e ligation involves M-CSF release and its autocrine
consumption by CD115, being associated to their differentiation to
MΦ2-like macrophages.
M-CSF is the primary regulator of survival, proliferation,
differentiation and function of monocytes and macrophages [202,
203]. Besides having a significant homeostatic contribution to
macrophage-lineage development and maintenance of macrophage
numbers [181, [219], M-CSF enhances cytotoxicity, superoxide
production, phagocytosis, chemotaxis and cytokine production in
monocytes and macrophages [220, 221]. It also enhances in
monocytes the expression of cell surface molecules such as CD11b,
CD14, CD16, CD23, HLA-I and HLA-II [222]. On the other hand,
stimulation of monocytes and macrophages with M-CSF downregulates the expression of different TLRs, including TLR1, TLR2,
TLR6 and TLR9 [223]. By contrast, the P2X7 extracellular ATP
receptor that regulates DCs and macrophage inflammatory functions
is up-regulated on human monocytes in response to M-CSF [224].
M-CSF exists in three isoforms including a membrane-spanning cellsurface glycoprotein as well as a secreted glycoprotein and a secreted
proteoglycan that are released to the circulation under steady state
conditions. The circulating isoforms are synthesized by endothelial
cells and have an endocrine action, whereas the cell-surface isoform
mediates cell-contact dependent regulatory effects [225]. M-CSF levels
increase at sites of inflammation, forming a part of a cytokine network
involved in communication between myeloid cells and neighboring
cells during the inflammatory reaction [226]. Different stimuli have
been shown to induce M-CSF secretion by human monocytes in vitro
including GM-CSF [227, 228], interleukin-2 (IL-2) [229], HIV
infection [230-232], tumor necrosis factor alpha (TNF-α) [233] and
stimulation via CD44, CD45 and LFA-3 [234]. PMA, LPS,
cycloheximide and IL-3 are all able to induce M-CSF transcription but
not protein secretion [209].
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We recently showed that CD300e ligation activates human monocytes
promoting their survival in culture. Our results indicate that
monocytesurvival and their differentiation to macrophages depends
on an autocrine M-CSF production upon CD300e cross-linking, since
the effect was partially inhibited by neutralizing M-CSF and blocking
CD115. The mechanism by which CD300e signaling triggers M-CSF
secretion in vitro thus far remains elusive. Since activation of
monocytes and their adherence to plastic are not sufficient to induce
M-CSF production in culture [235] we hypothesized that this might
occur as a result of the initial cytokine secretion triggered upon
CD300e cross-linking. Indeed, TNF-α [209] was identified as a
powerful inducer of M-CSF secretion by human monocytes [233]. It is
noteworthy that other surface molecules CD44, CD45 and LFA-3
reported to induce M-CSF upon cross-linking also trigger TNF-α
production in monocytes [236].
M-CSF binding stabilizes the noncovalent dimerization of CD115,
inducing its kinase activation and tyrosine autophosphorylation. This
is followed by a covalent dimerization of the receptor leading to
subsequent events that include a second wave of tyrosine
phosphorylation, serine phosphorylation, receptor ubiquitination and
internalization of the receptor-ligand complex for lysosomal
degradation [225]. The second wave of tyrosine phosphorylation
triggers CD115 interaction with down-stream signaling molecules such
as PI3K, Grb2, Cbl, Lyn, Mona, PLCγ2, Socs1 and Src. The Src/Pyk2
and PI3K signaling pathways were identified as important regulators
of cell adhesion, polarization, spreading, motility and monocyte
differentiation [225]. Even though signaling pathways that connect
M-CSF receptor with direct effectors of these processes have not been
precisely dissected, it has recently been reported that signaling through
CD115 induces the stabilization and nuclear translocation of βcatenin, which among other functions anchors the actin cytoskeleton
[206]. An important step forward was the discovery of caspase
involvement in M-SCF induced monocyte–to-macrophage
differentiation. Caspase-8 is activated in response to M-CSF and it
induces the formation of a multimolecular platform comprised of
FADD, FLIP-L and RIP1 that contributes with its cleaved fragment to
the down-regulation of NF-kB [237]. Caspase-3 that is activated
downstream of caspase-8, cleaves other target proteins such as PAK2
and nucleophosmin [217, 238], that contribute to cytoskeleton
rearrangements required for macrophage motility [218]. We have
shown that CD300e cross-linking in monocytes results in CD115
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RESULTS: ARTICLE 2
internalization and activation of caspase-3, processes that are
associated to their differentiation to macrophages.
Cells belonging to the monocytic lineage are quite heterogeneous. In
the case of macrophages such heterogeneity probably reflects their
plasticity and versatility in the response to microenvironmental signals
(i.e. cytokines and microbial products) [45]. In order to define
macrophage phenotypes and activation states, macrophage
polarization to the so-called classically activated (MΦ1) or alternatively
activated (MΦ2) macrophage states have been proposed, based in part
on their cytokine profile [45]. The adherent cell populations that result
by culturing monocytes with GM-CSF or M-CSF have been related to
MΦ1- and MΦ2- polarized macrophages, respectively [193, 194]
which differ in terms of receptor expression and effector functions
[45]. Culture with GM-CSF leads to cells that produce proinflammatory cytokines, (i.e. TNF, IL-6, IL-12p70 and IL-23)
following LPS stimulation, whereas after culture with M-CSF cells
tend to produce predominantly IL-10 and CC-chemokine ligand 2
(CCL2 or MCP1) [226]. MΦ2 express Scavenger receptor-A, -B,
CD163 and mannose receptor which are absent in MΦ1 [45].
Classically activated M1 macrophages are potent effector cells capable
of killing microorganisms and tumor cells and produce high levels of
pro-inflammatory cytokines. In contrast, M2 cells promote
angiogenesis, tissue remodeling and repair [45].
Activation of monocytes via CD300e results in their differentiation
toward MΦ2 macrophages. Yet, though their phenotypic and
functional properties correspond to alternatively activated
macrophages they appear less efficient than cells differentiated in
parallel in response to exogenous M-CSF. Recently macrophages with
distinct functional properties have been termed as classically activated
(microbicidal activity), wound-healing (tissue repair) and regulatory
(anti-inflammatory activity) MΦ [239]. The latter, correspond to
human MΦ2 and are characterized by high production of IL-10 and
low secretion of inflammatory cytokines, but they require two stimuli
to fully induce their anti-inflammatory activity. A first signal (i.e.
immune complexes, prostaglandins, adenosine or apoptotic cells),
generally has little or no stimulatory function on its own [239].
However when combined with a second stimulus, such as a TLR
ligand, these two signals re-program macrophages to produce IL-10
[214] and down-regulate IL-12 production [240]. Thus, the low IL-10
production of CD300e MΦ in response to LPS, may simply reflect the
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RESULTS: ARTICLE 2
absence of an additional signal to fully induce their anti-inflammatory
potential.
According to the ‘M-CSF resistance’ model, monocyte-derived
macrophages tend to initially acquire the properties of a polarized
MΦ2 population [204]. Depending on the exposure to proinflammatory cytokines they may convert to a MΦ1 state when proinflammatory stimuli, such as GM-CSF, IFN-γ and LPS, overcome MCSF-driven polarization interfering with M-CSF signaling. CD300einduced monocyte differentiation in the absence of pro-inflammatory
signals is consistent with this model. Moreover, monocyte
differentiation triggered by other cell surface receptors in the absence
of pro-inflammatory stimuli is expected to follow a similar pattern. In
this line with this prediction, we have observed that signaling via
hOSCAR, another triggering receptor capable of inducing monocyte
survival [29] also led to differentiation of monocytes to MΦ2-like cells
(data not shown).
It has been shown that human peritoneal macrophages (pMΦ) freshly
isolated from patients on peritoneal dialysis have phenotypical features
of MΦ2 and are likely involved in maintenance of anti-inflammatory
conditions in peritoneal cavity [50]. It is of note that the M-CSF level
in human peritoneal fluid is 2.5-fold higher as compared to plasma,
and it has been shown also that M-CSF levels correlate to the numbers
of pMΦ [241]. The available information indicate that macrophages
that infiltrate tumors (tumor-asociated macrophages, TAMs) also
acquire the properties of a polarized MΦ2 population [45].
A model is proposed (Figure 7) to interpret how activation via
CD300e activation may induce monocyte differentiation towards
inflammatory MΦ2-like cells. Upon interaction with its still unknown
ligand, CD300e triggers cellular activation and the secretion of proinflammatory cytokines including TNF-α. Subsequently, TNF-αinduced M-CSF binds to CD115 promoting its dimerization,
activation and initiation of the signaling cascade that via caspase-3
activates downstream proteins leading to differentiation. After
receiving appropriate signals in the tissue, macrophages induce their
anti-inflammatory activity and take part in either immune responses or
homeostasis.
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ACKNOWLEDGEMENTS
This work was supported by a grant from Plan Nacional de I+D
(SAF2007-61814) and Red Heracles, Ministerio de Ciencia e
Innovación (MICINN). TB and DB are supported by fellowships
from MICINN.
We thank Dr. Oscar Fornas (University Pompeu Fabra, Barcelona,
Spain) for advice in flow cytometry analysis and blood donors for their
contribution.
CONFLICT OF INTEREST
The authors declare no financial or commercial conflict of interest.
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FIGURES
Figure 1. CD300e activation induces monocyte differentiation
into macrophages. (A) Monocytes were cultured for six days over
the plate coated anti-CD300e mAb with no added cytokines
(magnification x 200). (B) At the end of the culture cells were
harvested and analyzed for surface expression of CD14, CD11b,
CD16 (FcγRIII) and CD163 molecules. MΦ1 and MΦ2 were
generated in parallel from the same donor. Open histograms represent
matched isotype controls. Data are representative of at least five
independent experiments performed with different donors.
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RESULTS: ARTICLE 2
Figure 2. Functional analysis of CD300e derived MΦ. (A)
Cytokine production by CD300e MΦ, M2Φ and M2Φ following the
LPS stimulation. Cell that were generated by culturing monocytes for
six days over the anti-CD300e mAb or in the presence of M-CSF
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RESULTS: ARTICLE 2
(M2Φ) and GM-CSF (M1Φ), were stimulated with LPS for 24h. After
the incubation, supernatants were harvested and analyzed for IL-10
and IL-6 release by ELISA. (B) Phagocytic activity of CD300eMΦ,
MΦ1 and MΦ2 was assessed by the uptake of fluorescent polystyrene
microspheres at 37OC at different time points and analyzed by FACS.
(C) All three types of macrophages (CD300eMΦ, MΦ1 and MΦ2)
were stimulated or not with PMA and production of superoxide anion
(O2-) was measured by detection of reduced Cytochrome c at 6h. (BC) Data (mean ± SD of triplicate samples) correspond to a
representative experiment out of three performed with different
donors. CD300eMΦ, MΦ1 and MΦ2 were generated in parallel from
the same donor.
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RESULTS: ARTICLE 2
Figure 3. CD300e MΦ up-regulate expression of CD80 upon
activation with LPS. CD300e MΦ, MΦ2 and MΦ1, all generated
from a same donor, were stimulated or not with LPS and analyzed by
FACS for the expression of co-stimulatory molecules after 24h. Data
are representative of four independent experiments performed with
different donors.
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RESULTS: ARTICLE 2
Figure 4. CD300e mediated monocyte differentiation to
macrophages is M-CSF and CD115 dependent. Monocytes were
cultured for six days over the plate-coated anti-CD300e mAb with or
without neutralizing anti-M-CSF mAb (A-B), anti-CD115 (anti-MCSF R) or anti-IL-6 mAbs (D). At the end of the culture, cells were
photographed (magnification x 200) and viable cells were counted.
Data (mean ± SD of duplicate samples) correspond to a representative
experiment out of three performed with different donors. (C) M-SCF
production by CD300e MΦ at day 3 and day 6. Data represent mean
± SD of duplicate samples for four independent donors.
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Figure 5. Surface expression of CD115 is down-regulated in
CD300e macrophages. Monocytes were cultured over the platecoated anti-CD300e mAb or in the presence of GM-CSF plus IL-4
(moDC) for various days and analyzed by flow cytometry for the
surface expression of CD115. Open histograms correspond to binding
of an isotype-matched control mAb and numbers in the corners
correspond to the MFI. One representative experiment is shown out
of two performed with different donors.
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RESULTS: ARTICLE 2
Figure 6. Differentiation of monocytes to macrophages induced
by CD300e is associated with capsase-3 activation. Monocytes
were cultured for 72h over the plate-coated anti-CD300e mAb, in the
presence of M-CSF (100ng/ml) or GM-CSF (100ng/ml) and IL-4
(25ng/ml) when they were harvested and analyzed by flow cytometry
for the presence of caspase-3 active fragments. Open histograms
represent matched isotype control. One representative experiment out
of three performed with different donors.
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Figure 7. CD300e is able to trigger differentiation of monocytes
to macrophages by inducing the M-CSF release and its
consumption. Cross-linking of CD300e on the surface of monocytes
triggers the M-CSF release. CD115 (M-CSF R) binds and consumes
autocrine M-CSF, inducing the cascade of events that lead to
differentiation of monocytes to macrophages.
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RESULT: APPENDIX
Appendix Functional
Characterization
of
CD300f
Inhibitory Receptor in Human Granulocytes
Preliminary results
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RESULT: APPENDIX
INTRODUCTION
The Immune receptor expressed by myeloid cell 1 (CD300f or IREM1) is a monomeric type I transmembrane protein that displays five
cytoplasmatic tyrosine residues (Y205, Y236, Y249, Y263 and Y284),
among which Y205 and Y249 are located in ITIM, whereas Y236 and
Y263 constitute putative binding sites for PI3 kinase [145, 146].
Biochemical analysis revealed that CD300f is capable of recruiting Src
homology region 2 domain-containing phosphatase-1 (SHP-1) in a
phosphorylation dependent manner and that the main docking site for
this interaction is Y205. In addition crosslinking of the receptor in
transfected RBL cells inhibited FcRε-induced activation [145] and
degranulation [144]. A loss of inhibitory function and the ability to
interact with SHP-1, was observed when Y205, Y249 and Y284 were
mutated to phenylalanine [144]. Remarkably, engagement of the Y205,
249,284F CD300f mutant was able to induce RBL cell degranulation.
This effect was shown to be dependent on PI3K recruitment via both
Y236 and Y263 residues [144]. The putative involvement of PI3K
raised the question as to whether CD300f may be able to deliver
activating signals in cells that constitutively express it
Human monocytes and granulocytes (mainly polymorphonuclear
neutrophils, PMNs) are the only peripheral blood cells expressing
surface CD300f [144]. Because of their capacity to release proteases,
together with great amounts of reactive oxygen species and
bactericidal proteins, PMNs play a crucial role in host defense [242].
In addition to their classical pro-inflammatory functions, upon
appropriate stimulation they are able to produce different cytokines
and chemokines [243]. PMN express a variety of surface receptors that
regulate their functions. Among them, FcR -I, -II and –III are critical
for facilitating phagocytosis whereas C5aR, C3bR, chemotactic
receptors for PAF, LTB-4 and fMLP, Toll-like receptors and also
GM-CSF R and G-CSF R regulate their migration [56].
Human neutrophils express various ITIM-bearing inhibitory receptors
including: CD305, CLECSF6, CD31 (or PECAM-1), CD66a (or
CEACAM1 or BGPa), SIRPα, p75/AIRM1, CD33, CD300a
[129][129] and CD300f [143]. Recently it was shown that CD300a can
inhibit CD32a (FcγRIIa) mediated reactive oxygen species production
in human PMN by inhibiting Ca2+ flux, [129]. The receptor was rapidly
translocated from an intracellular pool to the cell surface in response
to pro-inflammatory stimuli, suggesting an important role of CD300a
in the modulation of PMN function [129].
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So far, the role of CD300f in cells that constitutively express the
receptor, including human neutrophils is uncertain. In this regard, we
investigated the functional effects of CD300f ligation in freshly
isolated human PMN by using an agonistic anti-CD300f monoclonal
antibody to mimic its still unknown ligand.
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MATERIALS AND METHODS
Cell purification and culture
Granulocytes (neutrophils >96.5%, eosinophils <3%) were isolated
under endotoxin-free conditions from buffy coats of healthy donors
[244] and cultured in RPMI-1640/Glutamax medium (Invitrogen Life
Technologies) supplemented with 10% low endotoxin FBS (Greiner
Bio-One GmbH) for various times at 37OC in 5% CO2 atmosphere.
After incubation, neutrophils were collected and centrifuged at 300 x g
for 5 min Supernatants wereharvested and stored at -80OC whereas
cells were either used for immunofluorescencet staining, mRNA
extraction or lysed for protein analysis.
Ex vivo stimulation of cells
To study cell activation through the CD300f receptor, an agonistic
anti-CD300f mAb (clone UP-D2, IgG1) was used [143]. The antiCD161 (clone HP-3G10, mouse IgG1), anti-TREM-1 (clone 21C7,
mouse IgG1, kindly provided by Dr. Marco Colonna, University of
Washington, Saint Louis, MO) and anti-ILT2 (clone HP-F1, mouse
IgG1) mAbs were used as isotype-matched controls. Ex vivo
stimulation of cells was performed as previously described [209].
Flow cytometry
Surface expression of CD300f, CD11b, CD62L and CD66b was tested
by immunofluorescence and flow cytometry analysis using the
FACScan or FACSCalibur (Becton Dickinson) as described previously
[209]. Cells were stained with UP-D2 mAb (anti-CD300f) or isotype
matched control mAb followed by secondary PE-conjugated goat
rabbit anti-mouse Ab (DakoCytomation Denmark A/S). For direct
immunofluorescence the following murine mAb were used: PEconjugated anti-CD11b, anti-CD66b, FITC-conjugated CD62L (all
from BD Pharmingen) in combination with their isotype-matched
controls (BioLegend).
Production of reactive oxygen intermediates
The production of superoxide anion (O2-) was assayed by detection of
reduced Cytochrome c by freshly isolated PMN as previously
described [209].
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Soluble mediator release assays
Concentrations of mediators in cell-free supernatants were measured
by specific human ELISA kits for IL-8/CXCL8 (ImmunoTools
GmbH), albumin and lactoferrin (Bethyl Laboratories, INC) according
to manufacturer’s instructions.
Real time RT-PCR
Real time RT-PCR has been performed as previously described [245,
246]. Data were calculated using the Q-Gene software
(BioTechniques) and expressed as mean normalized expression
(MNE) units after GAPDH normalization.
Survival assay
Estimation of apoptotic and necrotic neutrophils was determined
using the Annexin-V-FLUOS Staining Kit (Roche Applied Sciences)
following the manufacturer’s guidelines in combination with
propidium iodide labeling, followed by by flow cytometry analysis.
Immunoprecipitation and western blot analysis
After incubation with 1mM sodium pervanadate for 15min at 37OC
purified, PMN were biotinylated using EZ-Link Sulfo-NHS-Biotin
(Pierce) following manufacturer’s instructions, lysed and
immunoprecipitated as described [144]. Samples were resolved by
SDS-PAGE in a 10% acrilamide gel and transferred onto polyvinyl
difluoride membranes (Millipore). Membranes were blocked for 1h
with 3% BSA in TTBS and then probed with HRP-conjugated antiphosphotyrosine mAb mixture (PY-7E1, PY-1B2 and PY20) (Zymed
Laboratories). After stripping with Restore Western Blot Stripping
Buffer (Pierce) membranes were re-probed with HRP-conjugated
Streptavidine (Roche) and, after an additional stripping step, with antip85 (Upstate Biotechnology) followed by HRP-conjugated
secondary Ab.
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RESULT: APPENDIX
RESULTS AND DISCUSSION
Inflammatory stimuli up-regulate the expression of CD300f in
human neutrophils
We first investigated whether basal expression of CD300f in freshly
isolated neutrophils (Fig.1) varied in response to various proinflammatory agents [67]. As shown in Fig. 2, GM-CSF or a
combination of LPS and IFN-γ induced a significant increase in
CD300f surface expression levels; by contrast, neither G-CSF, IL-4,
LPS nor IFN-γ alone had any effect. The increase in the CD300f
expression occurred rapidly, becoming detectable one hour after
stimulation with GM-CSF or with LPS and IFN-γ (data not shown).
Further studies are required to assess whether this results from the
translocation of an existing intracellular receptor pool.
This expression pattern was similar to that reported for CD300a,
which was also rapidly up-regulated upon neutrophil stimulation with
GM-CSF and LPS [130]. Moreover, the mouse homolog of CD300f,
known as CD300Lf/CLM-1/LMIR3/DIgR2/MAIR-Va/b was also
shown to be up-regulated in murine neutrophils upon stimulation with
LPS and G-CSF both in vitro and in vivo [149]. Because the
expression of these receptors is up-regulated by inflammatory stimuli
in human neutrophils they may play an important role in controlling
inflammation.
Engagement of the CD300f receptor triggers a respiratory burst
in PMN
Experiments were designed to explore whether CD300f inhibited the
respiratory burst in neutrophils. Yet, unexpectedly, upon engagement
of CD300f in neutrophils, stimulation of superoxide anion (O-2)
production was detected. ROS production over control (medium or
isotype control mAb) was detectable within 10 minutes after
incubation with anti CD300f mAb and linearly increased for 140 min
(Fig. 3); anti-TREM-1 and fMLP were used in the assay as positive
controls. Further studies are needed in order to test wheter CD300f
induced respiratory burst in PMN is results from its association to
PI3K.
CD300f cross-linking on freshly isolated neutrophils leads to
phenotypic changes
To further investigate the functional role of CD300f, PMN were
stimulated for 18h with plate-coated mAb and analyzed for the
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RESULT: APPENDIX
expression of surface molecules known to be modulated upon
activation. Neutrophils treated with anti-CD300f mAb significantly
cleaved surface CD62L that mediates leukocyte rolling on activated
endothelium in inflamed tissues. On the other hand, CD300f
engagement had no effect on CD11b and CD66b surface expression
(data not shown) indirectly supporting that neutrophils do not
degranulate upon CD300f ligation.
Engagement of CD300f enhances proinflammatory responses
induced by TLR4 ligand in neutrophils
Since our results have shown that CD300f is able to activate the
respiratory burst, we further explored its putative role as an activating
receptor. Cross-linking of CD300f strongly enhanced IL-8/CXCL8
production in human neutrophils stimulated by LPS and fMLP (Fig.
5A). Real-time PCR confirmed the up-regulation of IL-8/CXCL8
expression when CD300f was ligated together with TLR4 (Fig. 5B). By
contrast, the synergistic effect of CD300f and LPS was not observed
for TNFα production. In the same line, CD300f engagement did not
enhance the degranulation of neutrophils triggered by fMLP (Fig. 5A),
measured by albumin and lactoferrin release, markers of secretory
vesicles and secondary granules, respectively.
A recent study in mice is in line with these observations, since crosslinking of the endogenous CD300f homolog (CD300Lf/CLM1/LMIR3) significantly enhanced IL-6 production in BMMCs
stimulated with LPS [149]. Interestingly, receptor engagement
impaired cytokine production of CLM-1 transduced BMMCs
stimulated with ligands for TLR2, TLR3 and TLR9 or SCF. These
observations suggest that CD300Lf has a dual function in BMMCs,
co-stimulating TLR4 signalling, but repressing the response to other
TLR agonists or SCF. Our results confirm that CD300f may induce
activating signals in human neutrophils but further studies are needed
to test whether it also exerts an inhibitory function.
CD300f ligation has no effect on neutrophil life span in culture
We tested next whether CD300f can modify the ex vivo life span of
neutrophils cultured in the absence of survival factors for 18h. As
shown in Fig. 5, neither control mAb nor anti-CD300f decreased the
proportion of apoptotic and necrotic neutrophils, whereas rhGM-CSF
promoted neutrophil survival. This is in contrast with the observation
that the murine CD300Lf/CLM-1/MAIR-V/DIgR1 induced cell
death in myeloid cells by caspase and endoplasmatic reticulum stress114
RESULT: APPENDIX
independent mechanism [146]. CD300Lf-mediated cell death was
dependent on the cytoplasmatic region, but it did not require ITIM
nor ITSM.
CD300f is not tyrosine phosphorylated in human neutrophils
Biochemical analysis performed in CD300f transfectants and the U937
cell line, that does constitutively express the receptor, revealed that
CD300f is displayed as a monomeric molecule. Its migration in SDSPAGE as a smear between 50 and 99 kDa suggests the existence of
different isoforms, together with different degrees of glycosylation.
Upon cellular treatment with sodium pervanadate, CD300f was
tyrosine phosphorylated [143, 144]. When similar studies were
performed in human neutrophils treated with sodium pervanadate and
surface-labeled with biotin, no evidence for CD300f phosphorylation
was obtained (Fig. 7A), despite that the receptor was efficiently
immunoprecipitated as confirmed by re-blotting the membrane with
Streptavidin (Fig. 7B). In the same experiments, we were unable to
detect any association of CD300f with the p85α subunit of PI3K. A
recent study performed with the mouse homolog CLM-1/LMIR3
showed that this inhibitory receptor associates with the ITAM bearing
adaptor protein FcRγ [149]. Whether the human receptor is also
capable of coupling to FcRγ in neutrophils remains to be determined.
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FIGURES
Figure 1. Expression of CD300f (IREM-1) receptor by peripheral
blood neutrophils. Freshly isolated PMN were stained with antiCD300f (UP-D2) mAb followed by anti-mouse IgG1-FITC and
analyzed by flow cytometry for the surface expression of CD300f. A
representative experiment out of 10 performed with samples from
different donors is shown.
Figure 2. CD300f is up-regulated on human neutrophils in
response to pro-inflammatory agents. Surface expression of
CD300f on human neutrophils after overnight treatment with rhGMCSF (10ng/ml), rhG-CSF (103U/ml), rhIL-4 (25ng/ml), rhIFN-γ
(100U/ml) or LPS (100ng/ml). Results are representative of five
independent experiments performed with different donors.
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Figure 3. Cross-linking of CD300f triggers the release of reactive
oxygen intermediates in PMN. (A) Freshly-isolated PMN were
stimulated during 2.5h with plate-coated mAb, as indicated, and the
production of superoxide anion (O2-) was analyzed by detection of
reduced Cytochrome c at ten minute intervals. (B) Superoxide anion
production after 140 minutes of stimulation with plate-coated mAbs.
Data (mean ± SD of triplicate samples) in (A-B) correspond to a
representative experiment out of five performed with samples from
different donors.
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Figure 4. Neutrophils down-regulate CD62L upon CD300f crosslinking. PMN were incubated overnight with plate-coated control
IgG1 (HP-3G10), anti-CD300f (UP-D2), medium or with 100ng/ml
LPS and analyzed by flow cytometry for the expression of CD62L.
One representative experiment out three performed with similar
results.
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Figure 5. CD300f ligation synergistically up-regulates IL8/CXCL8 release by neutrophils upon LPS and fMLP
stimulation. (A) PMN were incubated for 18h with plate-coated
control IgG1 (HP-3G10) or anti-CD300f (UP-D2) mAbs and in the
presence or absence of 100ng/ml LPS and 10nM fMLP. 10mM
Cytochalasin B was used as a positive control. Culture supernatants
were tested by ELISA for IL-8/CXCL8, lactoferrin and albumin
release. Data are mean ± SD of duplicate samples from one
representative experiment out of five performed with various donors.
(B) Neutrophils were cultured for the indicated times with antiCD300f, 100 ng/ml LPS or both. Total RNA was extracted and
analyzed for IL-8/CXCL8, TNF- and GAPDH mRNA expression
by real-time PCR. A representative experiment is shown. Gene
expression is depicted as MNE units after GAPDH mRNA
normalization of triplicate reactions for each sample.
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RESULT: APPENDIX
Figure 6. Engagement of CD300f has no effect on survival of
neutrophils. PMN were incubated with plate-coated mAb (control
IgG1, anti-ILT2, HP-F1; anti-CD300f, UP-D2), medium alone or 10
ng/ml rhGM-CSF, as indicated. After 18h, cells were harvested and
analyzed for Annexin V and propidium iodine binding (viable cells:
AnnexinV-PI-, apoptotic cells: AnnexinV+PI-, necrotic cells:
AnnexinV+PI+). Data show mean ± SD of three independent
experiments performed with different donors.
Figure 7. CD300f is not tyrosine phosphorylated in neutrophils.
PMN were surface labeled with biotin, treated with sodium
pervanadate, lysed and immunoprecipitated with anti-CD300f, antiCEACAM-1 (positive control) or isotype negative control mAbs.
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RESULT: APPENDIX
Western blots were conducted with anti-phosphotyrosine mAb
mixture (A) and Streptavidine (B).
121
RESULT: APPENDIX
122
RESULT: APPENDIX
123
Discussion
124
DISCUSSION
125
DISCUSSION
4. DISCUSSION The human CD300 family of immune receptors is expressed by
myeloid cells. The first member (CD300c or CMRF-35A) was
identified by expressional cloning back in 1992 [89] and its gene was
mapped to human chromosome 17q22-25. During the last decade, five
more genes with a related Ig domain were subsequently mapped to the
same region; encoding for paired triggering and inhibitory surface
glycoproteins of the immunoglobulin superfamily. Despite the time
elapsed our understanding of the biological role of CD300 receptors
is still quite limited mainly due to the lack of information regarding the
nature of their ligands and the unavailability of specific reagents for all
of them. Moreover, most data come from studies in transfectants
rather than in primary cells. Our goal was was to pursue the functional
characterization of CD300e and CD300f in peripheral blood cells
constitutively expressing them.
CD300e was originally identified as a result of an Ensembl genome
data base search for homologues of CD300c (CMRF-35) and CD300f
(IREM-1) and was termed Immune Receptor Expressed by Myeloid
cells-2 (IREM-2). The cloned nucleotide sequence contained an open
reading frame of 618 nucleotides encoding for a polypeptide of 205
amino acids with a predicted molecular mass of 23kDa [134].
According to the amino acid sequence analysis, CD300e is a type I
membrane protein with a 12 amino acid (aa) long signal peptide. The
extracellular region contains a single Ig variable domain and a 44-aa
linker with a potential N-glycosylation site. The transmembrane
domain displays a lysine residue followed by a short cytoplasmatic tail
of 10-aa. The extracellular Ig domain of CD300e has a high degree of
homology with other myeloid receptors including CD300a (71%), and
CD300c (73%) from a same family, but also with TREM-1 (49%) and
TREM-2 (58%) [134].
CD300e was shown to be selectively expressed by both subsets of
peripheral blood monocytes, CD14+CD16- and CD14+CD16+, and by
myeloid dendritic cells [134]. Analysis of CD300e in bone marrow
samples showed that all hematopoietic precursors tested (erythroid,
myeloid and B cells precursors) were negative and that the receptor
was detected only at relatively late stages of monocyte differentiation,
when their precursors have already acquired high levels of CD14 and
126
DISCUSSION
CD64 [134]. It is of note that none of the leukemic cell lines tested,
including those belonging to the myeloid lineage (THP-1, U937,
HL60, K562 nor MonoMac6) [134] expressed CD300e.
In vitro differentiation of monocytes to immature dendritic cells (iDC)
or classically activated macrophages (MΦ1) resulted in the complete
loss of CD300e expression [134]. In our hands, (Figure 12) monocytes
down-regulated CD300e as early as 24 hours upon in vitro culture
and similar results were obtained in the presence of IL-4. By contrast,
GM-CSF maintained partially CD300e expression, which was fully
preserved by M-CSF stimulated cells at levels comparable to those of
fresh monocytes. Neither LPS, TNF-α nor IL-10 were able to
significantly alter cell surface expression of CD300e at 24h post
culture (data not shown).
Figure 12. CD300e expression is modulated upon monocyte culture and
treatment with cytokines. Purified monocytes were cultured for 24h with 10ng/ml
M-CSF, 50ng/ml GM-CSF, 25ng/ml IL-4 or medium alone when the viable cells
were analyzed by flow cytometry for CD300e expression. Data are expressed as
mean fluorescence intensity (MFI) ± SD of three experiments performed with
different donors. Grey bar: freshly isolated monocytes; black bars: monocytes
cultured for 24h.
Anti-inflammatory macrophages (MΦ2) derived from monocytes in
the presence of M-CSF expressed CD300e that was lost in proinflammatory MΦ1 and DC (data not shown). Identification of the
CD300e ligand should provide clues to understand the biological
implications for its selective expression along monocyte
differentiation.
127
DISCUSSION
The structure of CD300e indicated a potential triggering role in
myeloid cells and its initial characterization in transfected cells
confirmed this hypothesis [134]. In the first part of this work, we
investigated the functional role of CD300e in peripheral blood
monocytes and myeloid dendritic cells (mDC) using an agonistic mAb
to mimic its ligand. Our data formally support that CD300e functions
as an activating receptor that is capable of regulating the innate
immune response in myelomonocytic cells. Upon engagement by an
agonistic mAb, CD300e triggered an intracellular calcium mobilization
and superoxide anion production in monocytes. Activation via
CD300e provided survival signals that prevented monocyte and mDC
apoptosis, triggered the production of pro-inflammatory cytokines
(including IL-8/CXCL8, TNF- α, IL-6) and up-regulated the
expression of cell surface co-stimulatory molecules CD83 and CD86
in both cell types. In addition, CD300e activation of myeloid dendritic
cells enhanced the alloreactive response of naïve T cells.
Beyond their function as a reservoir of precursors that give rise to
tissue resident macrophages, dendritic cells (DCs) and osteoclasts,
circulating monocytes represent accessory cells that link inflammation
and innate defense to adaptive immune responses, and may also
function as scavenger and effector cells in homeostasis [23]. These
cells are armed with scavenger receptors that recognize
microorganisms but also lipids and dying cells. Upon pathogen
recognition, monocytes become activated and produce a wide array of
effector molecules that are involved in the defense including among
others: ROS, complement factors, eicosanoids, cytokines (i.e. TNF-α,
IL-1β, IL-8/CXCL8, IL-6, and IL-10), vascular endothelial growth
factor, and proteolytic enzymes [10]. To some extent, monocytes are
able to present antigens and provide co-stimulatory signals to T cells.
Myeloid DCs are professional antigen presenting cells that are
specialized for the uptake, transport, processing and presentation of
antigens to naïve T cells [57]. Upon the encounter with microbial
products or tissue damage, mDCs become activated and besides
migrating to lymph nodes, they may contribute to the elimination of
pathogens. The production of chemokines and pro-inflammatory
cytokines (IL-8/CXCL8, IL-12, IL-23 or IL-10) together with upregulation of co-stimulatory (e.g. CD40, CD80, CD86, OX40-L and
ICOS-l) contribute to the activation of naïve T cells.
128
DISCUSSION
Engagement of CD300e resulted in activation of effector functions in
both monocytes and mDC including the production of molecules
involved in the defense against pathogens (i.e. reactive oxygen species,
cytokines, and chemokines) and in the activation of the adaptive
immune response. Yet, unlike other well-characterized triggering
receptors that belong to the same subset (e.g. TREM-1), the signaling
pathways initiated by CD300e remain elusive. The presence of a lysine
residue in the transmembrane domain suggested that CD300e might
associate with an ITAM-containing adaptor molecule bearing a
negatively charged amino acid residue in its trasmembrane region and,
in fact, CD300e was shown to associate with DAP12 in COS-7
transfected cells [134]. Yet, we failed to prove that this association is
taking place in human monocytes. In fact, we were unable to coimmunoprecipitate any phosphorylated molecule together with
CD300e (data not shown). Beyond putative technical reasons limiting
the sensitivity of these assays, it is conceivable that CD300e may not
be constitutively associated with its adaptor on the surface of
monocytes. The possibility that the receptor might couple to an
adaptor different from DAP12, is uncertain since tyrosinephosphorylated molecules were not co-immunoprecipitated.
Additional experiments are required in order to define the molecular
basis for CD300e signalling in monocytes.
We recently reported that CD300e ligation besides triggering various
pro-inflammatory responses in myeloid cells, also leads to their
prolonged survival in culture by a mechanism independent of caspase3 [187]. In the present study, we observed that CD300e ligation led to
differentiation of monocytes to alternatively activated macrophages
(MΦ2) displaying an anti-inflammatory functional profile according to
their surface phenotype, cytokine secretion and phagocytic activity.
Cells obtained as a result of CD300e activation of monocytes
displayed phenotypic and functional characteristics closer to MΦ2
alternatively activated macrophages. MΦ2-like cells have been found
in human peritoneal cavity [50] and infiltrated in tumors [45], for that
reason they have been designated human peritoneal macrophages
(pMΦ) and tumor-associated macrophages (TAMs), respectively.
Alternatively activated macrophages preferentially produce IL-10 and
CCL2 in response to LPS, contrary to classically activated MΦ that
under same conditions favor the secretion of pro-inflammatory
cytokines such as TNF, IL-6, IL-12p70 and IL-23. Hamilton, J. has
129
DISCUSSION
recently formulated a so-called ‘M-SCF resistance’ model for
macrophage-dependent inflammation (Figure 15) [226]. He proposed
that macrophage populations in various tissues are normally exposed
to sufficient levels of tissue-derived M-CSF to maintain them in an
'MΦ2-like' polarized state [45, 193], with a relatively compromised
ability to produce pro-inflammatory mediators. During inflammation,
autoimmunity and/or infection, exposure of macrophages to
increasing concentrations of pro-inflammatory stimuli, such as GMCSF, IFN-γ and LPS, overcomes this M-CSF-driven polarization by
interfering with M-CSF signaling leading to a pro-inflammatory
('MΦ1-like') state [45, 193]. This polarization would be dependent on
the degree of exposure to M-CSF. In other words, at least at the level
of the macrophage lineage, inflammation can be viewed as a state of
'M-CSF resistance' or compromised M-CSF-receptor signaling. This
hypothesis also proposes that when the levels of pro-inflammatory
stimuli diminish, M-CSF at the reaction site will assist in the resolution
of the lesion by favoring the MΦ2-like macrophage phenotype.
Figure 15. Macrophage-driven inflammation: a state of ‘M-CSF resistance’ or
compromised M-CSF receptor signaling [204].
According to the ‘M-CSF resistance’ model all monocyte-derived
macrophages acquire at the beginning the properties of a polarized
MΦ2 population. Depending on the exposure to pro-inflammatory
cytokines during the processes of inflammation, autoimmunity and/or
infection, they may later convert to a pro-inflammatory ‘MΦ1’ state.
The CD300e induced differentiation pattern in the absence of proinflamatory signals is consistent with this model. In the same line,
signaling via hOSCAR, another triggering receptor capable of inducing
130
DISCUSSION
monocyte survival [175] also led to differentiation of monocytes to
MΦ2-like cells (data not shown).
Our results indicate that prolonged survival of monocytes and their
differentiation to macrophages depends on an autocrine effect of MCSF that is released upon CD300e cross-linking. By neutralizing MCSF and blocking its receptor, CD115, the effect was only partially
inhibited probably due to the continuous secretion of M-CSF by
CD300e activated monocytes that was detected in all donors tested.
On the other hand, the mechanism by which CD300e signaling
triggers M-CSF secretion in vitro remains elusive. Activation of
monocytes and their adherence to plastic are not sufficient to induce
M-CSF release [235] and we have postulated that this may occur as a
consequence of initial cytokine secretion induced by CD300e
engagement. Various stimuli are shown to induce M-CSF in vitro:
GM-CSF [227, 228], IL-2 [229], HIV infection [230-232], TNF-α [233]
and stimulation via surface molecules CD44, CD45 and LFA-3 [234].
Among the mentioned cytokines, TNF-α was a clear candidate as it
was reported to be a powerful inducer of M-CSF secretion by human
monocytes [233].Further studies are needed in order to confirm this
hypothesis. It is noteworthy that surface molecules CD44, CD45 and
LFA-3 which are reported to induce M-CSF are also known to trigger
TNF-α release by human monocytes [236].
Besides identifying M-CSF as a key player in the CD300e derived
monocyte-to-macrophage differentiation, we provided evidence
supporting an involvement of the M-CSF signaling pathway in the
process. M-CSF binding to CD115 results in dimerization of the
receptor, autophosporylation of cytoplasmatic tyrosine residues and
phosphorylation of various substrates including SHP-1, Src kinases,
PLC-γ, PI(3)K, Akt and Erk. The second wave of phosphorylation
activates numerous down-stream signaling molecules including Grb2,
Cbl, Lyn, Mona, PLCγ2, Socs1 and Src [206]. Once formed, the MCSF/CD115 complex gets internalized and later degraded by
lysosomes. The Src/Pyk2 and PI3K signaling pathways were identified
as important regulators of cell adhesion, polarization, spreading,
motility and monocyte differentiation [225]. The CD115 signaling
pathway is connected to the ‘caspase network’ shown to be essential
for macrophage differentiation. Caspase-8 is activated in response to
M-CSF and induces the formation of a multimolecular platform
comprised of FADD, FLIP-L and RIP1 that contributes with its
131
DISCUSSION
cleaved fragment to the down-regulation of NF-κB [237]. Caspase-3
which is activated downstream of caspase-8 cleaves other target
proteins such as PAK2 and nucleophosmin [217, 238], that play role in
cytoskeleton rearrangements required for the macrophage to acquire
motility [218]. We were able to show that CD300e cross-linking on the
surface of monocytes results in both CD115 internalization and
activation of caspase-3, two processes that are associated to
monocyte-to-macrophage differentiation.
A key issue for understanding the biological role of CD300e is the
identification of its putative ligand/s. Even though the function of
myeloid activating receptors has been extensively studied in the past,
their ligand specificity remains often ill defined. Some of these
molecules recognize exogenous pathogen-related ligands like
polyanionic bacterial products recognized by TREM-2 [99]. Others
bind endogenous ligands – CD85g binds CD317 [78], CD172a both
CD47 [85] and soluble pattern recognition receptors SP-A and SP-D
[106]. On that basis, CD300e may function as a pathogen-associated
molecular pattern receptor (PRR) or, alternatively, could contribute to
sensing self stress-inducible molecules. Further studies are required to
identify the CD300e ligand.
Part of our work was focused on CD300f, another ligand-orphan
inhibitory receptor. CD300f receptor that was originally termed
IREM-1, is composed of a single V-type extracelluar Ig domain, a
membrane proximal region that is followed by transmembrane domain
and a long cytoplasmatic tail with five tyrosine residues [145]. Two of
these tyrosines are in the context of ITIMs and other two constitute
binding sites for the PI3K regulatory subunit p85α. CD300f has been
detected on the surface of human monocytes and neutrophils. As
expected for an ITIM bearing surface protein, CD300f was able to
recruit SHP-1 phosphatase in a phosphorylation dependent manner
[145]. Its inhibitory role was studied in transfected RBL cells, where it
was shown that CD300f engagement prevents gene transcription
induced by signals delivered through the activating FcεRI [145]. In the
same experimental system, CD300f was able to recruit the p85α
subunit of PI3K and deliver activating signals when ITIMs and the
distal tyrosine-based motif were disabled. In the U937 myeloid
leukemia cell line CD300f binds SHP-1 phosphatase and p85α
simultaneously, thus suggesting its putative functional duality [146].
132
DISCUSSION
Preliminary data on the functional role of CD300f in human
granulocytes, indicated that its engagement triggered the production of
reactive oxygen species, resulted in the decreased expression of
CD62L that is a characteristic of activated neutrophils and showed a
synergistic effect with TLR4. We were however unable to confirm that
these functional effects result of preferential signaling via PI3K in
human granulocytes, even though we observed that CD300f was not
recruiting SHP-1 in these cells, presumably due to the lack of tyrosine
phosphorylation essential for this interaction. Warning on the
possibility that granulocyte activation by anti-CD300f mAb might be
due to its potential cross-reactivity with another triggering molecule of
the same family (CD300d) should be also considered. Yet,
interestingly similar results have been obtained recently on the
functional characterization of the murine orthologue receptor CLM1/LMIR3. In fact CLM-1 cross-linking enhanced cytokine production
of BMMCs in response to TLR4 ligand, while suppressing the
production stimulated by other TLR agonist and stem cell factor [151].
Inhibition involved SHP-1 and SHP-2, whereas the activating signal
appeared mainly mediated by its association to FcRγ. Further studies
are required to understand the physiological role of CD300f and, in
particular, the identification of its endogenous ligand/s becomes an
essential step.
In that regard we have tested a panel of human cells for the binding of
CD300f/IgG2a fusion protein and were able to conclude that it
reproducibly stained the Jurkat T cell leukemia and HeLa cell lines
(Figure 16). Since Jurkat cells are CD4+ T lymphocytes, we analyzed
the expression of CD300f putative ligand/s in CD3+ T lymphocytes
and the CD4+ subset, using both fresh and PHA-activated T cells. The
CD300f/IgG2a supernatant marginally stained CD3+ lymphocytes,
particularly the CD4+ subset among PHA-activated T cells, whereas
no binding was seen with resting T cells. No staining of CD8+ T, NK,
B cells and monocytes was detected.
133
DISCUSSION
Figure 16. Staining of Jurkat and HeLa cells with CD300f/IgG2a fusion
protein. Anti-myc was used as a isotype control. The staining pattern was
reproduced in three experiments.
As an additional strategy to seek for CD300f ligand/s we designed a
reporter assays in murine T cell line BW36 that were stably transfected
with CD300f/CD3ξ chimeric receptor. The CD3ξ cytoplasmic domain
of the chimeric CD300f receptor contains three immunoreceptor
tyrosine-based activation motifs (ITAM) which upon receptor
engagement should recruit the ZAP-70 and Syk tyrosine kinases,
resulting in the activation of a signaling cascade that includes the
activation of NFAT transcription factors and, ultimately, the
production of interleukin 2 (IL-2) by the BW T cell line.
To confirm the results of binding studies with CD300f/IgG2a, the BW
CD300f/CD3ξ reporter cells were co-cultured with Jurkat and HeLa
for 24h and the cell supernatant was tested for IL-2 production. Even
though the chimeric receptor was functional in BW cell upon crosslinking with agonistic anti-CD300f (UP-D2) antibody, no response
was obtained upon interaction with the stimulatory cells, despite the
fact that, as mentioned above, both cell types interacted with the
soluble fusion protein (Figure 17). Similar results were obtained using
the RBL CD300f/CD3ξ as a reporter system indicating that the
sensitivity of the system is presumably insufficient to detect weak
receptor-ligand interactions. It is also conceivable that some accessory
molecules required to optimize signaling may be missing in the
heterologous BW-Jurkat interaction.
As stressed in the introduction, a substantial number of leukocyte
receptors in different families still remain “ligand-orphan”, despite the
long time elapsed since they were cloned, presumably reflecting
common technological limitations. As a main open issue for
understanding the biological role of CD300e and CD300f is the
134
DISCUSSION
identification of their ligands, our laboratory has currently established
a collaboration (Dr. Claudia Cantoni and Carola Prato, University of
Genova) in a joint effort to design novel strategies allowing to
overcome a number of technical difficulties to progress thus far
encountered
135
DISSCUSION
136
137
138
Conclusions
139
140
CONCLUSIONS
5. CONCLUSIONS
1. Upon engagement by an agonistic mAb, CD300e triggers
intracellular calcium mobilization and superoxide anion production in
human monocytes.
2. Activation via CD300e provides survival signals that prevent
monocyte and mDC apoptosis by a mechanism independent of TNF-mediated inhibition of caspase-3.
3. CD300e cross-linking on the surface of monocytes and myeloid
dendritic cells induces the production of pro-inflammatory cytokines
(TNF-α and IL-8/CXCL8) and up-regulates the expression of CD83
and CD86 .
4. In myeloid dendritic cells, stimulation via CD300e enhanced the
alloreactive response of naïve T cells.
5. CD300e engagement promotes the differentiation of monocytes to
macrophages that display a CD14bright CD16+ CD163low/neg CD1aCD11bbright CD209- phenotype, exhibit a low respiratory burst but high
phagocytic activity and mainly produce IL-10 upon stimulation with
LPS, thus resembling anti-inflammatory type 2 macrophages (MΦ2).
6. Monocyte-to-macrophage differentiation induced by CD300e
appears dependent on the production of M-CSF and autocrine
stimulation via its receptor (CD115).
7. Overall, our data support that CD300e functions as an activating
receptor capable of regulating the innate immune response of cells of
the monocytic lineage and promotes their differentiation to MΦ2-like
macrophages .
141
CONCLUSIONS
142
CONCLUSIONS
143
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