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Review article
The sixth sense: hematopoietic stem cells detect danger through purinergic
signaling
Lara Rossi,1 Valentina Salvestrini,1 Davide Ferrari,2 Francesco Di Virgilio,2 and Roberto M. Lemoli1
1Department of Hematology and Oncological Sciences, L. and A. Seràgnoli, Institute of Hematology, University of Bologna and S. Orsola-Malpighi Hospital,
Bologna, Italy; and 2Department of Experimental and Diagnostic Medicine, Section of General Pathology and Interdisciplinary Center for the Study of
Inflammation, University of Ferrara, Ferrara, Italy
Over the past decade, extracellular nucleotides (such as ATP and UTP) have
emerged as key immunomodulators. This
family of molecules, already known for its
key metabolic functions, has been the
focus of intense investigation that has
unambiguously shown its crucial role as
mediators of cell-to-cell communication.
More recently, in addition to its involvement in inflammation and immunity, purinergic signaling has also been shown to
modulate BM-derived stem cells. Extracel-
lular nucleotides promote proliferation,
CXCL12-driven migration, and BM engraftment of hematopoietic progenitor and
stem cells. In addition, purinergic signaling acts indirectly on hematopoietic progenitor and stem cells by regulating differentiation and release of proinflammatory
cytokines in BM-derived human mesenchymal stromal cells, which are part of
the hematopoietic stem cell (HSC) niche.
HSC research has recently blended into
the field of immunology, as new findings
highlighted the role played by immunologic signals (such as IFN-␣, IFN-␥, or
TNF-␣) in the regulation of the HSC compartment. In this review, we summarize
recent reports unveiling a previously unsuspected ability of HSCs to integrate
inflammatory signals released by immune and stromal cells, with particular
emphasis on the dual role of extracellular
nucleotides as mediators of both immunologic responses and BM stem cell functions. (Blood. 2012;120(12):2365-2375)
Introduction
Living organisms are constantly exposed to foreign, and sometimes
harmful, agents. To protect tissues from damage and preserve
homeostasis, multicellular organisms have developed an array of
defense responses of which inflammation is a major manifestation.
As part of this defense mechanism, inflammation is instrumental
for mounting an effective innate and adaptive immune response.
Terminally differentiated cells (granulocytes, monocytes/macrophages, dendritic cells, B- and T-lymphocytes) have classically
been considered the principal players in inflammation and immunity. Conversely, hematopoietic stem cells (HSCs), from which all
immune and inflammatory cells derive, are usually thought not to
be part of the immune system. Localized in the nurturing environment of the BM niche, HSCs were thought to reside within an
“immunologic sanctuary,” protected from the insults affecting
peripheral tissues. However, recent findings suggest that HSCs are
not confined in a “splendid isolation”: BM-HSCs and circulating
HSCs can sense the presence of danger or stress signals in the
surrounding microenvironment and switch to an activated state or
reach injured tissues in need of repair.1,2 Therefore, HSCs respond
to early mediators of inflammation and distress that were so far
thought to be active on immune cells only, such as TNF-␣, IFNs,
Toll-like receptor (TLR) ligands and, most interestingly, extracellular nucleotides (eNTPs).
From the evolutionary standpoint, nucleotides are among the
most ancient biologic molecules; thus, it is not surprising that they
have been used by living organisms for multiple purposes: storage
and transmission of genetic information, energy metabolism, and
extracellular communication.3 eNTPs compose both extracellular
purines (ATP and its derivatives, ADP and adenosine) and extracellular pyrimidines (uridine-5⬘-phosphate [UTP] and [UDP]), which
intervene in a variety of biologic processes by binding NTPspecific cell-membrane receptors, collectively named purinergic
receptors. The presence of purinergic signaling in taxa as diverse as
mammals, plants, yeasts and bacteria suggests that nucleotides are
indeed an archaic, ubiquitous, communication system.4,5 However,
their messenger role has been realized comparatively late, and only
thanks to the pioneering studies of Geoffrey Burnstock in the
central and peripheral nervous system.6 It is now well established
that nucleotides mediate intercellular communication in virtually
all tissues and typify one of the most important indicators of cell
stress in the pericellular environment.7 In the hematopoietic and
immune systems, eNTPs act as potent immunomodulators of
neutrophil, monocyte, macrophage, dendritic cell, and T-lymphocyte
responses.8 In addition, eNTPs drive blood cell proliferation and
differentiation at different levels, including the compartment of
hematopoietic progenitors and stem cells (HPSCs). Here we
discuss how HSCs react to signals of cell injury and inflammation
to mount an integrated response to pathogens and tissue damage. In
our view, purinergic signaling activated by eNTPs emerges as a key
element bridging inflammation to HSC activation.
Submitted April 5, 2012; accepted July 2, 2012. Prepublished online as Blood
First Edition paper, July 11, 2012; DOI 10.1182/blood-2012-04-422378.
© 2012 by The American Society of Hematology
BLOOD, 20 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 12
How immunology blends into stem cell
biology: the effect of immunomodulators on
HSCs
Lying at the roots of the hematopoietic system, HSCs are a
reservoir of rare, multipotent stem cells that provide a continuous
supply of cells circulating in the peripheral blood (PB), such as
erythrocytes, platelets, lymphoid cells, and myeloid cells. Under
2365
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2366
ROSSI et al
steady-state conditions, HSCs are highly quiescent cells that divide
infrequently and are found mainly in the G0 phase of the cell cycle.9
Despite dormancy being the privileged status for HSCs, these cells
retain a considerable resilience that enables them to adjust their
cell-cycle dynamics and undergo proliferation when needed. Hemorrhagic stress is among the strongest triggers of HSC proliferation,
aiming at replenishing the population of circulating erythrocytes
lost through bleeding.10 Similarly, chemotherapic drugs (such as
5-fluorouracil or hydroxyurea), which kill proliferating hematopoietic progenitors, recruit HSCs out of quiescence to reconstitute the
whole downstream subset of differentiated cells.11
Interestingly, increased HSC proliferation has also been observed in response to immunologic stress, such as infections. Under
these conditions, HSCs skew their differentiation toward lymphoid
and/or myeloid cells to maintain an efficient army of pathogenfighting cells, until infectious agents are cleared. In analogy to
stimulation of erythropoiesis by massive bleeding, pathogeninduced HSC proliferation has traditionally been interpreted as a
physiologic reaction to infections, aimed at counterbalancing the
loss of immune cells from PB. However, it is now clear that HSCs
do not only react to the depletion of differentiated PB cells; rather,
the HSC compartment reacts directly to danger signals and
inflammatory cytokines released by pathogens, tissues, and resident immune cells. Several recent studies highlighted the role of
IFNs as major regulators of HSC cell cycle.12-14 Both IFN-␣ and
IFN-␥ stimulate HSC proliferation in mice,14,15 and HSCs from
IFN-␥-deficient mice are more quiescent and engraft better in
transplantation assays than those from wild-type animals.12 Conversely, overactivation of IFN pathway impairs HSC functions and
decreases their engraftment, suggesting a link between chronic
infections and HSC exhaustion.16 The impact of infections on
HSCs is also shown by the dual role of TNF-␣ on their proliferation
and differentiation. Depending on the context, TNF-␣ plays
inhibitory or stimulatory effects on HSCs.17,18 For instance, murine
Tnfrsf1a⫺/⫺ HSCs perform poorly in competitive repopulation
assays because of self-renewal defects. On the other hand, excessive activation of TNF-␣ signaling is associated with impaired
hematopoiesis,16 suggesting that any drastic imbalance in TNF-␣
signaling may be detrimental for HSC function.
HSCs sensing danger: the role of PRRs
Modulation of HSC functions by inflammatory cytokines is of
great interest, but not novel and surprising by itself. More
interesting and unexpected is the ability of HPSCs to respond
directly to factors released by pathogens or by injured tissues, in
other words to sense danger directly, unveiling a new skill for these
cells and a potentially novel function in the activation of first-line
immunity.1
Traditionally, immunology has focused on the self/non–selfdiscrimination. However, it is now gaining support the view that
immune responses would be more efficient if discrimination was
based not only on “foreignness” but also on “dangerousness,” as
proposed by Matzinger’s danger model.19 Multicellular organisms
have evolved a wide array of extracellular receptors, named pattern
recognition receptors (PRRs), specialized in recognizing pathogens
or molecules released from injured cells (danger signals).20 PRRs
bind viral or microbial pathogen-associated molecular patterns
(PAMPs, such as lipopolysaccharide, peptidogycans, single-strand
RNA, or unmethylated CpG motifs), and may also detect damageassociated molecular patterns. Damage-associated molecular pat-
BLOOD, 20 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 12
terns are endogenous molecules released by damaged or distressed
tissues, may be of intracellular or extracellular origin, and include
proteins (such as calgranulins) and nonprotein molecules (uric acid,
reactive oxygen species, heparin sulfate, biglycans, tenascin C, and
eNTPs).21 These molecules are collectively known as alarmins, a
term recently coined to indicate endogenous molecules that alert
immunity on tissue injury.22 Vertebrates possess a vast array of
conserved PRRs (TLRs, RIG-I–like receptors, NOD-like receptors,
AIM2-like receptors, and purinergic receptors), mostly expressed
by monocytes, macrophages, neutrophils, and dendritic cells.
Nonetheless, recent evidence indicates that also nonimmune cells,
such as endothelial cells, and, more recently, HSCs express PRRs.
These findings suggest that the network created by PAMPs,
damage-associated molecular patterns, and PRRs transversally
affects the whole hematopoietic system, from the compartment of
immature HSCs up to the terminally differentiated immune cells.23,24
Through this signaling network, HSCs can directly sense a
proinflammatory microenvironment even before inflammatory cytokines are released, driving a swift response to pathogens and
accelerating the replenishment of circulating immune cells. As a
direct consequence, repeated or chronic infections could negatively
affect HSC long-term survival. As recently demonstrated in a
model of chronic low-grade infection,25 the prolonged systemic
exposure to TLR ligands is detrimental to long-term HSCs, thus
highlighting a possible link between HSCs defects and different
disease states, including infection, inflammation, autoimmune
diseases, and aging.26-28
Purinergic danger signals and HSCs
Extracellular nucleotides as alarmins
Endogenous danger signals, collectively named alarmins, originate
from cells or from the extracellular matrix. Those of cellular origin
share a few fundamental features: (1) are rapidly released on
necrotic cell death; (2) can be released either passively or through
specialized efflux pathways; (3) recruit, by chemotaxis, target cells
and activate innate and adaptive immune responses; (4) promote
recovery from tissue injuries and restore homeostasis; and (5) their
activity is counterbalanced by inhibitory pathways that avoid
inflammatory loops.22 Purinergic signaling fulfils all these requirements, as nucleotides (1) are released into the extracellular environment on tissue injuries and cells death (passively or via specialized
pathways)29; (2) stimulate immune cell chemotaxis and recruitment
at inflammation sites by binding nucleotide-selective receptors,
also known as purinergic receptors or P2 receptors (P2Rs)30-32;
(3) activate innate and adaptive immune responses by stimulating
antigen-presenting cells, neutrophils, monocytes- and macrophages8; (4) drive tissue recovery by stimulating cell proliferation33; and (5) promote resolution of the immune response by
activating anti-inflammatory pathways dependent on ATP degradation products (eg, adenosine).34,35
Similarly to other inflammatory cascades, purinergic signaling
progresses through 3 phases: initiation, amplification, and resolution (Figure 1).
Initiation: release of eNTPs. Several mechanisms responsible
for nucleotide release into the extracellular fluids have been
described so far. Extensive tissue injury, traumatic shock, or
inflammation produces massive, nonspecific leakage of nucleotides
because of cell lysis.8 However, nonlytic mechanisms are concurrently activated after stimulation with PAMPs. ATP, ADP, and other
nucleotides are released via constitutive or stimulated exocytosis,
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BLOOD, 20 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 12
PURINERGIC SIGNALING AND INFLAMMATION IN HSCs
2367
Figure 1. The role of extracellular ATP and purinergic
signaling in inflammation. On insults inducing cell
damage and stress, cells can release into the extracellular environment nucleotides, such as ATP. Once released, ATP can take part in all the different steps of
inflammatory responses. 1. Initiation: after being secreted into the pericellular environment, ATP can act as a
danger signal and alert the immune system, thus helping
initiate the inflammatory response. 2. Progression: ATP
can bind to P2 receptors exposed on the cell membrane
of target cells and induce granulocyte and macrophage
chemotactic attraction toward the inflammatory focus, as
well as activation of antigen-presenting cells. These
responses induce the activation of both the innate and
the adaptive branches of the immune system, leading to
the amplification of the inflammatory response. 3. Resolution: under physiologic conditions, inflammatory responses need to be restrained from self-amplifying without control. To protect tissues from extended damages,
anti-inflammatory responses are activated. With specific
regard to purinergic signaling, CD39 and CD73 endonucleosidases concur to down-regulate inflammation by
hydrolyzing ATP in adenosine, with a double effect:
prevent ATP from further activate P2R signaling and
increase adenosine concentration, which activates antiinflammatory responses by binding to P1 receptors.
as well as through poorly selective plasma membrane channels,
such as connexins and pannexins.29 P2Rs themselves may participate in ATP release, as there is convincing evidence that the P2X7R
subtype allows nucleotide efflux.36,37 It is now clear that virtually
all cell types (hematopoietic cells included) constitutively release
ATP to maintain a steady nucleotide concentration in the nanomolar range in the pericellular environment. However, different cell
types release ATP in response to different stimuli (eg, erythrocytes
in response to deformation, endothelial cells to shear stress,
macrophage to endotoxin, osteoblasts, to mechanical loading).
Thus, it is conceivable that eNTP release is a general signal of
cellular stress, subsequently decoded by other cells to ignite
inflammation.8,38
It is probable that a steady ATP level causes a tonic stimulation
of P2Rs that is responsible for chronic, low-level, cellular activation. This is clearly shown by the in activity of ATP-hydrolyzing
enzymes (eg, apyrase), which cause a decrease in cytoplasmic Ca2⫹
concentration or mitochondrial potential. Conversely, transient
increases in eNTP concentration above steady-state levels are
induced by several mechanical (shear stress, osmotic swelling, cell
shrinking), chemical (reactive oxygen species, xenobiotics) or
biologic agents (lipopolysaccharide)39,40 (Figure 1).
Although initial evidence dates back to several decades ago, it is
only during the last 10 years that the role of ATP as extracellular
messenger has been generally accepted despite some initial skepticism.41 One of the main arguments against the involvement of ATP
in extracellular communication is the lack of an unequivocal in
vivo demonstration that ATP is present in the extracellular space
and that its levels are modulated during pathophysiologic responses. The very high cellular ATP content and its instability make
ATP measurement very liable to sampling artifacts secondary to
tissue manipulation. These experimental limitations have been
finally overcome by developing ATP-sensing probes capable of
detecting ATP in the pericellular space, where most of the biologic
activity of eNTPs is localized.42,43 This innovative probe is a
modified firefly luciferase engineered with a leader sequence and a
glycosylphosphatidylinositol anchor, which enables routing and
localization on the outer surface of the plasma membrane. Thanks
to this bioluminescent ATP probe, pmeLUC (plasma membrane
luciferase), we have been able to perform noninvasive sampling of
eATP in vivo and to demonstrate conclusively that eATP is present
at sites of inflammation and in tumor microenvironments.43 Similar
detection assays for extracellular uridine nucleotides, based on the
highly selective UDP-glucose pyrophosphorylase-coupled reaction, have been developed by other groups.44 This technical
breakthrough paved the way for a systematic in vivo investigation
of eATP involvement in inflammation and immunity and should
also allow measurements in the BM. Average eATP concentration
ranges under steady-state conditions in the 10⫺9-10⫺6M range. In
proximity of the plasma membrane, it is expected to be higher,
especially in protected pouches, which do not easily equilibrate
with the pericellular space, but as of now it is very difficult to give
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ROSSI et al
BLOOD, 20 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 12
Figure 2. The family of purinergic receptors: classification, structure, and signaling pathway. The family of
purinergic receptors comprises 2 major groups of membrane receptors: adenosine-activated P1 receptors
(P1Rs) and nucleotide-activated P2 receptors (P2Rs).
Based on pharmacologic profile and molecular structure,
2 subfamilies have been identified within P2Rs, namely,
P2XRs and P2YRs. P2XRs (composing 7 subtypes,
P2X1R through P2X7R) are trimeric ATP-gated plasma
membrane channels, whereas P2YRs are classic G
protein-coupled receptors. Based on their ligand selectivity, P2YRs are further subdivided in subtypes preferentially responding to ATP (human and rodent P2Y1R, and
human P2Y11R), to ADP (P2Y12R and P2Y13R), uridine5⬘-phosphate (UTP) and UDP (human P2Y4R and
P2Y6R), and ATP and UTP (human and rodent P2Y2R
and rodent P2Y4R), whereas human P2Y14R binds
sugar-linked nucleotides, such as UDP-glucose and
UDP-galactose. Two different signaling cascades are
associated with P2YRs: some subtypes mainly couple to
Gq proteins (P2Y1R, P2Y2R, P2Y4R, P2Y6R, and
P2Y11R), whereas others preferentially couple to Gi
proteins (P2Y11R, P2Y12R, P2Y13R, and P2Y14R), with
P2Y11R activating both molecular pathways.
accurate estimates. On cell stimulation, extracellular ATP levels
increase to reach 10⫺4M40 (Figure 1). Although robust evidence
shows that terminally differentiated erythroid, myeloid, and lymphoid cells release ATP, the precise pattern of eATP release in the
BM microenvironment remains largely unknown: future studies
measuring in situ eNTP concentrations in the BM are highly
warranted.
Progression: activation of purinergic signaling. eNTPs bind
to nucleotide-selective receptors, collectively named P2Rs. Based
on pharmacologic profile and on molecular structure, 2 subfamilies
have been identified, P2XRs and P2YRs.45 P2XRs (composing
7 subtypes, P2X1R through P2X7R) are trimeric ATP-gated plasma
membrane channels.46 P2YRs are classic G protein-coupled receptors that, based on their ligand selectivity, are further subdivided in
subtypes preferentially responding to ATP, ADP, UTP, UDP, or
sugar-linked nucleotides, such as UDP-glucose and UDPgalactose30 (Figure 2).
Depending on tissue, engagement of P2XRs and P2YRs
triggers a vast array of responses, ranging from cell proliferation or
differentiation to necrotic cell death or apoptosis, from secretory
exocytosis to chemotaxis, from reactive oxygen species generation
to cytokine release. P2YRs respond to low nucleotide concentrations; thus, they are particularly suited to “sense” minute changes
in nucleotide levels within the extracellular milieu and start
chemotaxis. P2Y2R, P2Y6R, P2Y12R, and possibly P2Y13R
appear to be the main subtypes involved in chemotaxis or
inflammatory cell recruitment. Conversely, P2X7R seems to deliver a “stop signal” once cells have reached the core of inflammation.47 This is consistent with the different signals delivered by
increasing ATP gradients: first, ATP activates high-affinity P2YRs
and then the low-affinity P2X7R subtype, where ATP release and
inflammation are higher. P2X7R plays an undisputed role in
cytokine release. This receptor has emerged as a potent activator of
the inflammasome and thus of IL-1␤ processing and release.48
Molecular details of this activity are however as yet obscure. The
ability of eATP and P2X7R to promote inflammation has been
established through several lines of in vivo evidence: of particular
interest is their participation in GVHD. Like severe infections or
traumas, GVHD leads to systemic inflammatory responses and
causes tissue destruction and massive release of danger signals. In
both humans and mice, ATP concentrations are increased during the
development of GVHD, contributing to ignite a self-sustaining
proinflammatory cascade involving IL-1␣, IFN-␥ production,
donor T-cell expansion, and regulatory T-cell decrease.49
Resolution: down-regulation of purinergic signaling. In the
extracellular environment, nucleotides are also a substrate for
nucleotide- and nucleoside-converting ectoenzymes, which rapidly
hydrolyze ATP to ADP, adenosine-monophosphate (AMP), and
eventually adenosine. The activity of ectoenzymes makes eNTPs
very labile in the blood (and in general in the extracellular
environment): consequently, eNTPs are mainly viewed as “shortrange” mediators, although it is unlikely that they can act in an
“hormone-like” fashion affecting responses of remote tissues and
organs.
Enzymes known to hydrolyze nucleotides include the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family,
the ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP)
family, the ecto-5⬘-nucleotidase, and alkaline phosphatases.40 NTPDase1/CD39 is one of the major nucleotide-hydrolyzing enzymes;
and accordingly, CD39-null mice show clinical signs of exacerbated inflammatory responses, as seen in a model of skin inflammation secondary to chemical insults.50,51 The ecto-5⬘-nucleotidase
CD73, which converts AMP into adenosine, is also critical in
inflammation because of its anti-inflammatory/immunosuppressive
activity. The anti-inflammatory role of CD73 is supported by in
vivo experiments showing that CD73⫺/⫺ mice develop enhanced
inflammation and an accelerated graft-versus-host reaction.52 Antiinflammatory effects of adenosine are mediated via a specific
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BLOOD, 20 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 12
subfamily of purinergic receptors, named P1 receptors (ie, the A1,
A2A, A2B, and A3 receptors.35 Interestingly, ATP is also a key
modulator of immunosuppressive activity of T-regulatory lymphocytes. For a detailed description of ATP and adenosine function in
immune cells, we refer readers to previously published reviews.53,54
Purinergic signaling in HPSCs
Purinergic signaling in hematopoiesis has been mostly investigated
in terminally differentiated cells55 and found to participate in
several cell functions, including platelet aggregation,56 chemotaxis,31,50 cell death, and proinflammatory activity,57 as summarized in Table 1. Historically, the investigation of purinergic
signaling in hematopoietic cells has focused on ATP, UTP, and
derivatives. On the contrary, little information is currently available
on other purines or pyrimidines, reflecting the limited activity that
nucleotides, such as CTP, GTP, ITP, and TTP, have generally
displayed.30 Despite the large body of data on purinergic signaling
in immune effector cells, investigation of eNTP-mediated responses on HPSCs started only a few years ago. Here, we
summarize the effects of eNTPs on HPSC proliferation, differentiation, and migration.
Proliferation. In 2004, we found that P2Rs are expressed on
CD34⫹ hematopoietic progenitors and that their engagement
produced fast changes in the intracellular calcium homeostasis.
ATP and UTP enhanced the stimulatory activity of several cytokines on clonogenic CD34⫹ cells. Similar effects were also
observed in lineage-negative CD34⫺ progenitors, as well as in
CD34⫹-derived long-term culture-initiating cells.58 Interestingly,
stimulation with UTP increased the number of human BMrepopulating CD34⫹ cells after transplantation into immunodeficient mice.58 ATP-induced increase in the intracellular Ca2⫹
concentration also stimulated murine HSC proliferation and differentiation.59 More recently, eATP has been shown to reduce the
number of HSCs as well as that of common myeloid progenitors
and granulocyte myeloid progenitors, whereas mature BM myeloid
cells were expanded. Interestingly, ATP-induced proliferation of
HSCs resulted in a reduction in Notch expression, a marker of HSC
quiescence.60
More recently, the link between ATP autocrine loops and HSC
proliferation was investigated using the nucleotide-binding fluorescent probe quinacrine. ATP was found to actively accumulate
within cytoplasmic vescicles in murine HPSCs.61 On stimulation of
Ca2⫹-sensitive pathways, HPSCs can release these vesicles, igniting a positive autocrine loop that leads to P2Rs stimulation and
further ATP release.61 Similarly to what previously observed,58 ATP
positively affected cell-cycle dynamics in Lineage⫺c-Kit⫹Sca-1⫹
HPSCs in a cell-autonomous manner, with P2X1R and P2X4R
subtypes most likely involved in the process.61 Finally, the role of
endogenous ATP on HSCs becomes more prominent under inflammatory conditions. As assessed in 2 mouse models of T-cellmediated chronic inflammation, ATP positively contributed to the
expansion of both Lineage⫺c-Kit⫹Sca-1⫹ cells and phenotypically
defined HSCs via the activation of P2XRs.61 However, inflammatory conditions have been shown to modify the phenotype of HSCs,
potentially leading to imprecise conclusions. In the future, it will be
important to address how purinergic signaling affects HSC ability
to repopulate the BM in the context of inflammation.
The findings collected over the past decade suggest that
purinergic signaling may promote the expansion of hematopoietic
progenitors at the expense of more immature HSC subsets.60
Quiescence is fundamental for the long-term maintenance of
hematopoiesis: a continuous stimulation of primitive HSCs with
PURINERGIC SIGNALING AND INFLAMMATION IN HSCs
2369
high concentrations of danger signals, such as eNTPs, may drive
HSCs out of quiescence and lead to a premature exhaustion of
hematopoiesis. In the future, knockout mice lacking P2 receptors or
endonucleotidases are expected to provide new clues on the effects
of eNTPs on HSC quiescence and long-term hematopoiesis. If
confirmed, such a scenario may help understand the detrimental
effect that chronic inflammation might have on long-term hematopoiesis (Figure 3).
Differentiation. Historically, investigations of purinergic signaling in hematopoiesis focused on lineage differentiation. Several
reports contributed over the years to demonstrate the role of ATP in
the differentiation of leukemia cell lines (HL-60 and NB4).
Subsequently, new lines of evidence emerged supporting the
widespread expression of P2Rs in hematopoietic cells. However,
the pharmacologic profile of P2Rs sensibly differs among lineages
and changes over time depending on the differentiation step. The
differentiation of myeloid, erythroid, mekacaryocitic, and lymphoid progenitors has been shown to be influenced by eNTPs,
although different receptors appear to be involved, depending on
the specific lineage. For a detailed discussion on purinergic
signaling in hematopoietic differentiation, we refer the reader to
more comprehensive reviews on this topic.55,62
More recently, eNTPs have been found to have a tuning role on
more immature myeloid progenitors. Barbosa et al showed that the
in vivo administration of ATP depletes the number of granulocytemacrophage progenitors in mice while increasing the fraction of
mature (Gr1⫹, Mac1⫹) myeloid cells.60 Prospectively, this activity
could be a direct response of the hematopoietic system to generic
alarm signals, such as those released during infections. Besides
accelerating the production of a larger army of immune cells from
primitive progenitors, eNTPs can also provide a molecular bridge
between cell damage and immune responses: ATP and UTP can act
as “find-me” signals when released by apoptotic cells, helping
recruit phagocytes and allowing clearance of dying cells from
tissues.63
Migration. Several reports suggested that molecules belonging to the circuit of innate immunity and regulating chemotaxis in
immune cells may also play a role in enforcing HSC migration
from the BM to peripheral tissues. Under physiologic conditions,
HSCs are thought to primarily dwell in the BM, within the
nurturing environment of the stem cell niche. The niche shields
HSCs from external injury and helps maintain their survival,
quiescence, and self-renewal. Nevertheless, HSCs have been found
to circulate in the PB and other tissues as well. Although HSC
peripheral traffic has been extensively characterized in clinical
settings, their ability to egress the BM, circulate in peripheral
tissues, and eventually home back to the BM under physiologic
conditions is yet to be fully characterized. At any given time,
between 100 and 400 HSCs are found circulating in the mouse
PB64: some of these reenter the BM, in a continuous exchange
between the 2 compartments.65 It has been suggested that circulating HSCs are actually patrolling peripheral tissues, looking for
potential injuries and infections.66 Lipopolysaccharide and other
bacterial products have also been shown to easily reach the BM
environment, where they stimulate TLR-expressing HSCs to
produce more immune effectors. Nonetheless, migrating HSCs
have an advantage over BM-resident HSCs: they may act as scouts
in the peripheral tissues and sense the presence of pathogens
directly at infection sites, possibly promoting a rapid and localized
production of immune cells. For instance, complement cleavagefragments released during immune responses have been shown to
take part in HSC migration: C5 cleavage-fragments drive HSC
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2370
BLOOD, 20 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 12
ROSSI et al
Table 1. Expression and function of P1 and P2 receptor subtypes in normal human hematopoietic cells
Cell type
CD34⫹
cells
Receptor subtype
P2X1, P2X2, P2X3, P2X4, P2X5, P2X6, P2X7
Responses/functions
References
In vivo and in vitro proliferation, chemotaxis, cell adhesion*
58,83
IL-12 inhibition
84
P2Y1, P2Y2, P2Y11, P2Y12, P2Y13
DCs
A1
A2A
84,85
A3
86,87
Chemotaxis*
87
Chemotaxis
88
P2Y4
CXCL8
88
P2Y6
IL-8, maturation of moncytes derived DCs, chemotaxis
88,89
IL-1␤ secretion
88
Chemotaxis
92
P2Y1
P2Y2
31,88
P2Y11
P2X1, P2X4
88,90
P2X7
88,91
Plasmacytoid DCs
A1
A2A
Down-regulation IL-6, IL-12, IFN-␣
92
Eosinophils
A3
Apoptosis
93
P2X1, P2X4, P2X7
Chemotaxis
94
P2Y1
94
P2Y2
31,94
P2Y4, P2Y6, P2Y11
94
CD11 up-regulation, ROIs, IL-8 and ECP secretion*
94,95
P2X7
Shedding of L-selectin and CD23
83,96
TCD4⫹ lymphocytes
A2A
Inhibition of proliferation
97
TCD8⫹ lymphocytes
A2A
Inhibition of cytotoxicity
97
T lymphocytes
A1
Anergy, induction of Treg
99
B lymphocytes
P2X1, P2X2, P2X4
A2A
83,96
98
A2B
100
A3
87
P2X1, P2X4, P2X7
83
P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13
Macrophages
83
A1
Enhancement of Fc␥ receptor-mediated phagocytosis
101
A2A
IL-4-induced TIMP-1 secretory, inhibitory IL-12, NO, MIP-1␣, TNF-␣
102
decrease of Fc␥ receptor-mediated phagocytosis
A2B
IL-4-induced TIMP-1 secretory, inhibitory IL-12, NO, MIP-1␣, TNF-␣
102
Polycarion formation, IL-1␤ secretion
103
P2Y2
Enhancement of ROI production
104,105
A1
Activation of mast cell degranulation
106
A2A
Potentiation of Fc⑀RI-induced degranulation
107
A2B
Inhibition of mast cell degranulation
106
A3
P2X1, P2X4, P2X7
102
P2Y1, P2Y4, P2Y6
Mast cells
104
P2X1, P2X4
108
P2Y1, P2Y2, P2Y11, P2Y12, P2Y13
Monocytes
108
A2A
Inhibition of TNF-␣, IL-6, IL-8, IL-12
102
Stimulation of IL-10 secretion
102
A3
Inhibition ROIs production
109
P2X1, P2X4, P2X7
83,110
P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13
Neutrophils
A1
83,104
Increase of chemotaxis
Increase of Fc␥R-mediated phagocytosis and ROI production
101
Inhibition of Fc␥R-mediated phagocytosis and ROI production
101
Inhibition of cell adhesion
102
A3
Degranulation and ROI production inhibition, chemotaxis
111
P2X1
Chemotaxis
112
Chemotaxis
111
Inhibition of cytotoxicity
97
A2A
A2B
100
P2X7
P2Y2
110
P2Y4, P2Y6, P2Y11
NK cells
102
A2A
103,111
P2X1, P2X4, P2X5, P2X6, P2X7
110,113
P2Y1, P2Y2, P2Y4, P2Y6, , P2Y12, P2Y13, P2Y14
P2Y11
113
Inhibition of CX3CL1-elicited NK-mediated killing
ROI indicates reactive oxygen intermediate; ECP, eosinophil cationic protein; and TIMP-1, tissue inhibitor of matrix metalloproteinase-1.
*Responses/functions that have been documented in a cell type but not linked to a specific receptor subtype.
113
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PURINERGIC SIGNALING AND INFLAMMATION IN HSCs
2371
Table 1. Expression and function of P1 and P2 receptor subtypes in normal human hematopoietic cells (continued)
Cell type
Platelets
Receptor subtype
Responses/functions
References
A2A
Antiaggregation
114
P2Y1
Aggregation and shape changes
114
P2Y12
Aggregation, phosphatidylserine exposure
115
P2X1
Transient shape changes
109
Phosphatidylserine exposure
116
P2X7
Red blood cells
P2X2
P2X7
P2Y1
116
ROI indicates reactive oxygen intermediate; ECP, eosinophil cationic protein; and TIMP-1, tissue inhibitor of matrix metalloproteinase-1.
*Responses/functions that have been documented in a cell type but not linked to a specific receptor subtype.
mobilization, whereas C3 cleavage-products promote HSC retention in the BM. This finding has also been confirmed in animal
models, with C5-deficient mice acting as poor mobilizers and
C3-deficient mice mobilizing HSCs more efficiently.67 Such a
scenario help envision HSC mobilization as an integral part of
innate immunity.68
Recently, eNTPs have been shown to modulate HSC migration
in the presence of CXC-chemokine 12 (CXCL12), considered the
most important chemotactic factor for HSCs and responsible of
stem cell retention in the BM niche. eUTP improves human HSC
migration toward CXCL12 gradients and inhibits the downregulation of membrane CXCR4 (CXCL12 receptor) in migrating
CD34⫹ cells.69 Similarly, pretreatment with eUTP significantly
increases BM homing of CD34⫹ HSCs when transplanted into
immunodeficient mice.69 Of note, purinergic signaling can affect
HPSCs also indirectly, by acting on the HSC niche. BM-derived
mesenchymal stromal cells (BM-MSCs) represent a key component of the hematopoietic niche and secrete several HSC regulatory
molecules, such as CXCL12 and stem cell factor. Recent findings
Figure 3. Purinergic signaling in BM-derived HPSCs,
leukemic cells, and BM-derived MSCs. Recent findings suggest that purinergic signaling may affect HSCs at
2 different levels: by directly binding P2Rs on HPSCs or
by targeting cells that are part of the HSC niche (such as
MSCs). This figure summarizes how eNTPs directly
modulate proliferation, migration, and engraftment in
HPSCs and leukemic stem cells. BM-derived MSCs are
also a target for eNTPs: activation of purinergic signaling
in these cells has been shown to enhance their adipogenic/osteogenic differentiation, as well as the release of
proinflammatory cytokines.
showed that ATP treatment is associated with an increase in the
production of proinflammatory cytokines in BM-MSCs (such as
IL-2, IFN-␥, and IL-12p70), and the expression of purinergic
receptors is modulated during adipogenic and osteogenic
differentiation.70,71
Purinergic signaling in malignant
hematopoiesis
It is becoming increasingly evident that tumor onset and progression depend not only on properties intrinsic to cancer cells. Both
the biochemical composition of the tumor microenvironment and
the cancer-host interaction appear to be important in disease
evolution. eNTPs may now acquire a new biologic significance as
part of the complex molecular miscellany composing the tumor
environment.
Over the past decade, several in vitro studies described ATP’s
capability of suppressing cell growth in several cancer cell lines,
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2372
ROSSI et al
including human HL-60 acute promyelocytic leukemia cells.72 In
line with these findings, our group recently investigated the role of
purinergic signaling in the tumor microenvironment associated
with acute myeloid leukemia (AML). We found that AML cells
express several functional P2XRs and P2YRs. Very interestingly,
and conversely to HSCs from healthy donors,58 ATP inhibits cell
proliferation and colony formation in AML cells, by restraining
AML cells in G0, and reduces AML cell engraftment in immunodeficient mice.73
Among P2Rs, P2X7R appears to be a major mediator of
ATP-dependent inhibition of cell growth in AML cells. As previously reported in chronic lymphocytic leukemia,74 also in AML
P2X7 expression is higher, with highest levels in patients with a
decreased remission rate. Remarkably, the highest P2X7R levels
were detected in patients undergoing relapse, suggesting a role for
this receptor in AML progression.75 P2X7R is also overexpressed
in adult AML cells where its tonic stimulation promotes cell
proliferation. Other P2XRs, such as P2X1R, P2X4R, and P2X5R
are abnormally expressed in AML cells, suggesting a wide
dysregulation and a possible pathogenic role of purinergic signaling in hematologic malignancies.76 On the contrary, ATP exerts an
antiproliferative activity on leukemic blasts by down-modulating
P2X7R expression.73 Based on this findings, eNTPs emerge as key
components of the leukemic microenvironment where they appear
to modulate proliferation and differentiation (Figure 3). In addition,
eNTPs affect the motility of cells derived from AML patients.73 In
migration assays, ATP inhibited the spontaneous migration of AML
cells in vitro, whereas UTP also reduced CXCL12-driven migration. P2Y2R and P2Y4R receptors appeared to be the subtypes
mainly involved in the process. Interestingly, AML BM homing
and long-term engraftment are also decreased on exposure to
eNTPs. Remarkably, ATP and UTP, as well as P2YR-agonists,
significantly inhibited the long-term engraftment of highly purified
CD38⫺CD34⫹ leukemic stem cells,73 suggesting that purinergic
signaling can also affect rare leukemic stem cells, which usually
escape most therapeutic approaches, making AML difficult to
eradicate (Figure 3).
Translational implications and future
perspectives
Translational studies on nucleotides are a rapidly expanding field
that has prompted experimental therapeutic strategies against
chronic inflammation and autoimmune diseases. In hematology,
blocking purinergic signaling is now regarded as a promising
pharmacologic approach for reducing the detrimental effects of
GVHD without compromising graft-versus-leukemia responses.77
Purinergic signaling could be targeted at different levels: (1) by
counteracting ATP release in response to chemical or infective
insults; (2) by preventing the activation of P2Rs signaling cascades,
such as the P2X7R-NALP3-inflammasome pathway; and (3) by
enhancing ATP hydrolysis by ecto-enzymes (CD39 or CD73),
promoting a quicker resolution of inflammation.
On the contrary, therapies enhancing purinergic signaling in the
tumor microenvironment may prove useful in firing immunity
against cancer cells.78 In addition, regulation of cell proliferation
by nucleotides presents sensible differences between leukemic
stem cells and HSCs: inhibition of AML proliferation and engraftment highlights novel routes of investigations for treating hematopoietic tumors while sparing healthy HSCs.
BLOOD, 20 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 12
Deciphering what role purinergic signaling plays in vivo will
also be critical for the mechanistic integration of eNTP effects on
HSCs and the environment in which they reside. Because of the
widespread expression of P2Rs, the biologic outcome of their
stimulation will probably reflect the concerted activity on different
cell subsets, including hematopoietic and nonhematopoietic cells.
New insights are also expected to arise from the role of
purinergic signaling in bone physiology. Osteoblast cells play a
pivotal role in the BM niche and osteoblast defects have previously
been associated with impaired maintenance of HSCs. Interestingly,
osteoblasts and osteoclasts are important sources of extracellular
ATP, potentially affecting HPSC responses in the niche. In
addition, several investigators reported bone deficiency in knockout mice lacking specific P2Rs. P2Y1R⫺/⫺, P2Y2R⫺/⫺, P2Y6R⫺/⫺,
and P2Y13R⫺/⫺ mice present bone abnormalities, and P2X7R⫺/⫺
mice display a striking reduction in bone formation and remodeling,79 suggesting a role in balancing osteoclasts and osteoblast
activity.80 Recently, studies in A2B knockout mice also highlighted
a reduction in osteoblast differentiation, hinting at a role for
adenosine in bone formation and resorption. Similar conclusions
were drawn from the analysis of mice lacking CD73, suggesting
that the disruption of adenosine signaling is highly detrimental to
osteoblasts. Interestingly, CD73 blocking has been shown to
significantly reduce tumor growth and metastasis.81 Of note, both
BM-MSCs (from which osteoblasts are derived) and HPSCs
(which give rise, among other lineages, to osteoclasts) express both
CD39 and CD73,60 suggesting that new insights could arise from
the pharmacologic targeting of endonucleosidases. If confirmed,
purinergic signaling may emerge as a regulator of HSC-stromal cell
interactions within the osteoblastic stem cell niche, and future
studies may help elucidate the role of P2Rs in both steady-state and
stressed hematopoiesis.
In conclusion, over the past 2 decades, a new picture of defense
mechanisms in vertebrates has emerged. Not just specialized
immune cells, but nonimmune cells as well may be involved,
adding versatility and resilience to the whole system. Strikingly,
some of these cells are primitive progenitors and stem cells,
suggesting that immune effectors only represent the visible tip of a
complex defense mechanism that deepens its roots into the stem
cell compartment. In this view, the hematopoietic system no longer
appears as a hierarchical model where the different compartments
are hermetically organized. Instead, hematopoiesis is a dynamic
system that reacts as a whole, where cells, either terminally
differentiated or HSCs, speak a common language and react
synergistically to protect the organism from danger and reestablish
homeostasis (Figure 4). Several insults can subvert homeostatic
conditions. Ionizing radiations, infections, chemical insults, and
chemotherapeutic drugs all contribute to the release of nucleotides
into the extracellular milieu. In the hematopoietic system, eNTPs
can act at different levels: (1) by stimulating HPSC proliferation
and differentiation, eNTPs and other alarmins contribute to immune cell production, invigorating defense mechanisms; and
(2) circulating HSCs may sense danger signals coming from
injured or inflamed tissues and migrate where immune effectors are
needed the most, providing an in loco supply of granulocytes or
antigen-presenting cells. Future insights may arise from other cell
types participating to the healing of injured areas by responding to
purinergic signaling, such as endothelial progenitor cells: in these
cells, ATP has been shown to negatively affect proliferation and
inhibit TLR4 expression, decreasing the release of proinflammatory cytokines.82
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BLOOD, 20 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 12
PURINERGIC SIGNALING AND INFLAMMATION IN HSCs
2373
Figure 4. Extracellular nucleotides: pleiotropic players in the maintenance of homeostasis. A prospective
overview of the role played by eNTPs in modulating
responses to danger and how purinergic pathway integrates with immunomodulatory responses. Several insults can contribute to subvert the homeostatic condition
in the hematopoietic system. Infective agents, ionizing
radiations, chemical insults, and mechanical stress contribute to the release of nucleotides into the extracellular
environment and the engagement of P2Rs on target cells
(HSCs, regulatory T cells, MSCs, and endothelial progenitor cells), where eNTPs can cooperate in containing
damage at different levels: (1) stimulate HSC proliferation and the subsequent production of immune effectors
that will invigorate defense mechanisms; (2) attract HSCs,
in synergy with other chemokines (eg, CXCL12), toward
sites of inflammation and infection to activate tissue
repair; and (3) contain detrimental effects of prolonged
inflammation and immune responses acting on mature
immune cells (eg, regulatory T cells). The net result of
such a widespread effect on the hematopoietic system is
aimed at damage containment and return to homeostatic
conditions. EPC indicates endothelial progenitor cells.
The authors apologize to those authors whose work could not be
referenced or discussed because of space limitations.
Acknowledgments
The authors thank Margaret A. Goodell for her valuable comments
and suggestions.
This research was supported by the Italian Ministry for University Education and Research (PRIN 2008), Cassa di Risparmio di
Bologna (project on Leukemic Stem Cells), the Italian Association
for Cancer Research (IG 5354), Telethon of Italy (GGP11014), the
Regione Emilia Romagna (Research Programs “Innovative approaches to the diagnosis of inflammatory diseases” and “Moniter”), and the University of Ferrara (institutional funds). L.R. was
also supported by the Italian Leukemia and Lymphoma Association, section of Bologna (BolognaAIL).
Authorship
Contribution: L.R., V.S., D.F., F.D.V., and R.M.L. designed and
wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Lara Rossi, Department of Hematology and
Oncological Sciences, L. and A. Seràgnoli, Institute of Hematology, University of Bologna, Orsola-Malpighi Hospital, via Massarenti, 9, I-40138 Bologna, Italy; e-mail: [email protected]
References
1. Baldridge MT, King KY, Goodell MA. Inflammatory
signals regulate hematopoietic stem cells. Trends
Immunol. 2011;32(2):57-65.
2. Boiko JR, Borghesi L. Hematopoiesis sculpted by
pathogens: toll-like receptors and inflammatory
mediators directly activate stem cells. Cytokine.
2012;57(1):1-8.
3. Lazarowski ER, Boucher RC. UTP as an extracellular signaling molecule. News Physiol Sci. 2001;
16:1-5.
4. Tanaka K, Gilroy S, Jones AM, Stacey G. Extracellular ATP signaling in plants. Trends Cell Biol.
2010;20(10):601-608.
5. Esther CR Jr, Dohlman HG, Ault AD, et al. Similarities between UDP-glucose and adenine nucleotide release in yeast: involvement of the secretory pathway. Biochemistry. 2008;47(35):92699278.
6. Burnstock G. Purine and pyrimidine receptors.
Cell Mol Life Sci. 2007;64(12):1471-1483.
7. Burnstock G. Pathophysiology and therapeutic
potential of purinergic signaling. Pharmacol Rev.
2006;58(1):58-86.
8. Bours MJL, Swennen ELR, Di Virgilio F,
Cronstein BN, Dagnelie PC. Adenosine 5⬘triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation.
Pharmacol Ther. 2006;112(2):358-404.
9. Trumpp A, Essers M, Wilson A. Awakening dormant haematopoietic stem cells. Nat Rev Immunol. 2010;10(3):201-209.
10. Cheshier SH, Prohaska SS, Weissman IL. The
effect of bleeding on hematopoietic stem cell cycling and self-renewal. Stem Cells Dev. 2007;
16(5):707-717.
chronic infection. Nature. 2010;465(7299):793797.
13. Sato T, Onai N, Yoshihara H, et al. Interferon
regulatory factor-2 protects quiescent hematopoietic stem cells from type I interferon-dependent
exhaustion. Nat Med. 2009;15(6):696-700.
14. Essers MG, Offner S, Blanco-Bose WE, et al. IFNalpha activates dormant haematopoietic stem
cells in vivo. Nature. 2009;458(7240):904-908.
15. Zhao X, Ren G, Liang L, et al. Brief report:
interferon-gamma induces expansion of
Lin(⫺)Sca-1(⫹)c-Kit(⫹) cells. Stem Cells. 2010;
28(1):122-126.
11. Harrison D, Lerner C. Most primitive hematopoietic stem cells are stimulated to cycle rapidly after
treatment. Blood. 1991;78(5):1237-1240.
16. Kitagawa M, Saito I, Kuwata T, et al. Overexpression of tumor necrosis factor (TNF)-alpha and
interferon (IFN)-gamma by bone marrow cells
from patients with myelodysplastic syndromes.
Leukemia. 1997;11(12):2049-2054.
12. Baldridge MT, King KY, Boles NC, Weksberg DC,
Goodell MA. Quiescent haematopoietic stem
cells are activated by IFN-gamma in response to
17. Zhang Y, Harada A, Bluethmann H, et al. Tumor
necrosis factor (TNF) is a physiologic regulator of
hematopoietic progenitor cells: increase of early
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2374
ROSSI et al
hematopoietic progenitor cells in TNF receptor
p55-deficient mice in vivo and potent inhibition of
progenitor cell proliferation by TNF alpha in vitro.
Blood. 1995;86(8):2930-2937.
18. Rebel VI, Hartnett S, Hill GR, et al. Essential role
for the p55 tumor necrosis factor receptor in regulating hematopoiesis at a stem cell level. J Exp
Med. 1999;190(10):1493-1504.
19. Matzinger P. The danger model: a renewed sense
of self. Science. 2002;296(5566):301-305.
20. Hansen JD, Vojtech LN, Laing KJ. Sensing disease and danger: a survey of vertebrate PRRs
and their origins. Dev Comp Immunol. 2011;
35(9):886-897.
21. Bianchi ME. DAMPs, PAMPs and alarmins: all we
need to know about danger. J Leukoc Biol. 2007;
81(1):1-5.
22. Oppenheim JJ, Yang D. Alarmins: chemotactic
activators of immune responses. Curr Opin Immunol. 2005;17(4):359-365.
23. Nardini E, Morelli D, Aiello P, et al. CpGoligodeoxynucleotides induce mobilization of hematopoietic progenitor cells into peripheral blood
in association with mouse KC (IL-8) production.
J Cell Physiol. 2005;204(3):889-895.
24. Nagai Y, Garrett KP, Ohta S, et al. Toll-like receptors on hematopoietic progenitor cells stimulate
innate immune system replenishment. Immunity.
2006;24(6):801-812.
25. Esplin BL, Shimazu T, Welner RS, et al. Chronic
exposure to a TLR ligand injures hematopoietic
stem cells. J Immunol. 2011;186(9):5367-5375.
26. Chen C, Liu Y, Liu Y, Zheng P. Mammalian target
of rapamycin activation underlies HSC defects in
autoimmune disease and inflammation in mice.
Bone. 2010;120(11):4091-4101.
BLOOD, 20 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 12
38. Fredholm BB. Adenosine, an endogenous distress signal, modulates tissue damage and repair.
Cell Death Differ. 2007;14(7):1315-1323.
39. Lazarowski ER, Boucher RC, Harden TK. Constitutive release of ATP and evidence for major contribution of ecto-nucleotide pyrophosphatase and
nucleoside diphosphokinase to extracellular nucleotide concentrations. J Biol Chem. 2000;
275(40):31061-31068.
40. Yegutkin GG. Nucleotide- and nucleosideconverting ectoenzymes: important modulators of
purinergic signalling cascade. Biochim Biophys
Acta. 2008;1783(5):673-694.
41. Burnstock G. Purinergic signalling: its unpopular
beginning, its acceptance and its exciting future.
Bioessays. 2012;218-225.
42. Joseph SM, Buchakjian MR, Dubyak GR. Colocalization of ATP release sites and ecto-ATPase
activity at the extracellular surface of human astrocytes. J Biol Chem. 2003;278(26):2333123342.
59. Paredes-Gamero EJ, Leon CMMP, Borojevic R,
Oshiro MEM, Ferreira AT. Changes in intracellular
Ca2⫹ levels induced by cytokines and P2 agonists differentially modulate proliferation or commitment with macrophage differentiation in murine hematopoietic cells. J Biol Chem. 2008;
283(46):31909-31919.
60. Barbosa CMV, Leon CMMP, Nogueira-Pedro A, et
al. Differentiation of hematopoietic stem cell and
myeloid populations by ATP is modulated by cytokines. Cell Death Dis. 2011;2:e165.
61. Casati A, Frascoli M, Traggiai E, et al. Cellautonomous regulation of hematopoietic stem
cell cycling activity by ATP. Cell Death Differ.
2011;18(3):396-404.
62. Sak K, Boeynaems JM, Everaus H. Involvement
of P2Y receptors in the differentiation of haematopoietic cells. J Leukoc Biol. 2003;73:442-447.
43. Pellegatti P, Raffaghello L, Bianchi G, et al. Increased level of extracellular ATP at tumor sites:
in vivo imaging with plasma membrane luciferase. PloS One. 2008;3(7):e2599.
63. Elliott MR, Chekeni FB, Trampont PC, et al. Nucleotides released by apoptotic cells act as a findme signal to promote phagocytic clearance. Nature. 2009;461(7261):282-286.
44. Lazarowski ER, Homolya L, Boucher RC,
Harden TK. Direct demonstration of mechanically
induced release of cellular UTP and its implication for uridine nucleotide receptor activation.
J Biol Chem. 1997;272(39):24348-24354.
64. Morrison SJ, Wright DE, Weissman IL. Cyclophosphamide/granulocyte colony-stimulating factor induces hematopoietic stem cells to proliferate
prior to mobilization. Proc Natl Acad Sci U S A.
1997;94(5):1908-1913.
45. Ralevic V, Burnstock G. Receptors for purines
and pyrimidines. Pharmacol Rev. 1998;50(3):
413-492.
65. Wright DE, Wagers AJ, Gulati AP, Johnson FL,
Weissman IL. Physiological migration of hematopoietic stem and progenitor cells. Science. 2001;
294(5548):1933-1936.
46. Khakh BS, North RA. P2X receptors as cellsurface ATP sensors in health and disease. Nature. 2006;442(7102):527-532.
27. Chen C, Liu Y, Liu Y, Zheng P. mTOR regulation
and therapeutic rejuvenation of aging hematopoietic stem cells. Sci Signal. 2009;2(98):ra75.
47. Wiley JS, Sluyter R, Gu BJ, Stokes L, Fuller SJ.
The human P2X7 receptor and its role in innate
immunity. Tissue Antigens. 2011;78(5):321-332.
28. Takizawa H, Regoes RR, Boddupalli CS,
Bonhoeffer S, Manz MG. Dynamic variation in
cycling of hematopoietic stem cells in steady
state and inflammation. J Exp Med. 2011;208(2):
273-284.
48. Piccini A, Carta S, Tassi S, et al. ATP is released
by monocytes stimulated with pathogen-sensing
receptor ligands and induces IL-1beta and IL-18
secretion in an autocrine way. Proc Natl Acad Sci
U S A. 2008;105(23):8067-8072.
29. Lazarowski ER. Vesicular and conductive mechanisms of nucleotide release. Purinergic Signal.
2012;359-373.
49. Wilhelm K, Ganesan J, Müller T, et al. Graftversus-host disease is enhanced by extracellular
ATP activating P2X7R. Nat Med. 2010;16(12):
1434-1438.
30. Abbracchio MP, Burnstock G, Boeynaems JM, et
al. International union of pharmacology LVIII: update on the P2Y G protein-coupled nucleotide
receptors: from molecular mechanisms and
pathophysiology. Pharmacol Rev. 2006;58(3):
281-341.
nucleotides are potent stimulators of human hematopoietic stem cells in vitro and in vivo. Blood.
2004;104(6):1662-1670.
50. Hyman MC, Petrovic-Djergovic D, Visovatti SH,
et al. Self-regulation of inflammatory cell trafficking in mice by the leukocyte surface apyrase
CD39. J Clin Invest. 2009;119(5):1136.
66. Massberg S, Schaerli P, Knezevic-Maramica I, et
al. Immunosurveillance by hematopoietic progenitor cells trafficking through blood, lymph, and
peripheral tissues. Cell. 2007;131(5):994-1008.
67. Reca R, Cramer D, Yan J, et al. A novel role of
complement in mobilization: immunodeficient
mice are poor granulocyte-colony stimulating factor mobilizers because they lack complementactivating immunoglobulins. Stem Cells. 2007;
25(12):3093-3100.
68. Lee H, Ratajczak MZ. Innate immunity: a key
player in the mobilization of hematopoietic stem/
progenitor cells. Arch Immunol Ther Exp. 2009;
57(4):269-278.
69. Rossi L, Manfredini R, Bertolini F, et al. The extracellular nucleotide UTP is a potent inducer of hematopoietic stem cell migration. Blood. 2007;
109(2):533-542.
31. Müller T, Robaye B, Vieira R, et al. The purinergic
receptor P2Y2 receptor mediates chemotaxis of
dendritic cells and eosinophils in allergic lung inflammation. Allergy. 2010;65(12):1545-1553.
51. Mizumoto N, Kumamoto T, Robson SC, et al.
CD39 is the dominant Langerhans cellassociated ecto-NTPDase: modulatory roles in
inflammation and immune responsiveness. Nat
Med. 2002;8(4):358-365.
70. Ferrari D, Gulinelli S, Salvestrini V, et al. Purinergic stimulation of human mesenchymal stem cells
potentiates their chemotactic response to
CXCL12 and increases the homing capacity and
production of proinflammatory cytokines. Exp Hematol. 2011;39(3):360-374.
32. Lee B, Cheng T, Adams GB, et al. P2Y-like receptor, GPR105 (P2Y14) identifies and mediates
chemotaxis of bone-marrow hematopoietic stem
cells. Genes Dev. 2003;105:1592-1604.
52. Zernecke A, Bidzhekov K, Ozüyaman B, et al.
CD73/ecto-5⬘-nucleotidase protects against vascular inflammation and neointima formation. Circulation. 2006;113(17):2120-2127.
71. Zippel N, Limbach CA, Ratajski N, et al. Purinergic receptors influence the differentiation of human mesenchymal stem cells. Stem Cells Dev.
2012;21(6):884-900.
33. Burnstock G, Verkhratsky A. Long-term (trophic)
purinergic signalling: purinoceptors control cell
proliferation, differentiation and death. Cell Death
Dis. 2010;1(1):e9.
53. Bours MJL, Dagnelie PC, Giuliani AL, Wesselius A,
Di Virgilio F. P2 receptors and extracellular ATP: a
novel homeostatic pathway in inflammation. Front
Biosci (Schol Ed). 2011;3:1443-1456.
34. Di Virgilio F, Boeynaems J-M, Robson SC. Extracellular nucleotides as negative modulators of
immunity. Curr Opinion Pharmacol. 2009;9(4):
507-513.
54. Trautmann A. Extracellular ATP in the immune
system: more than just a “danger signal.” Sci Signal. 2009;2(56):19193605.
72. Conigrave AD, van der Weyden L, Holt L, et al.
Extracellular ATP-dependent suppression of proliferation and induction of differentiation of human
HL-60 leukemia cells by distinct mechanisms.
Biochem Pharmacol. 2000;60(11):1585-1591.
35. Blackburn M, Vance C, Morschl E, Wilson C.
Adenosine receptors and inflammation. Handb
Exp Pharmacol. 2009;193:215-269.
36. Suadicani SO, Brosnan CF, Scemes E. P2X7 receptors mediate ATP release and amplification of
astrocytic intercellular Ca2⫹ signaling. J Neurosci. 2006;26(5):1378-1385.
37. Brandao-Burch A, Key ML, Patel JJ, Arnett TR,
Orriss IR. The P2X7 receptor is an important
regulator of extracellular ATP levels. Front Endocrinol. 2012;3:41.
55. Di Virgilio F. Nucleotide receptors: an emerging
family of regulatory molecules in blood cells.
Blood. 2001;97(3):587-600.
56. Cattaneo M, Lecchi A, Ohno M, et al. Antiaggregatory activity in human platelets of potent antagonists of the P2Y 1 receptor. Biochem Pharmacol. 2004;68(10):1995-2002.
57. Pizzirani C, Ferrari D, Chiozzi P, et al. Stimulation
of P2 receptors causes release of IL-1betaloaded microvesicles from human dendritic cells.
Blood. 2007;109(9):3856-3864.
58. Lemoli RM, Ferrari D, Fogli M, et al. Extracellular
73. Salvestrini V, Zini R, Rossi L, et al. Purinergic signaling inhibits human acute myeloblastic leukemia cell proliferation, migration and engraftment
in immunodeficient mice. Blood. 2012;119(1):
217-226.
74. Adinolfi E. P2X7 receptor expression in evolutive
and indolent forms of chronic B lymphocytic leukemia. Blood. 2002;99(2):706-708.
75. Zhang X-J, Zheng G-G, Ma X-T, et al. Expression
of P2X7 in human hematopoietic cell lines and
leukemia patients. Leuk Res. 2004;28(12):13131322.
76. Chong J-H, Zheng G-G, Zhu X-F, et al. Abnormal
expression of P2X family receptors in Chinese
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 20 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 12
pediatric acute leukemias. Biochem Biophys Res
Commun. 2010;391(1):498-504.
77. Zeiser R, Penack O, Holler E, Idzko M. Danger
signals activating innate immunity in graft-versushost disease. J Mol Med. 2011;89(9):833-845.
PURINERGIC SIGNALING AND INFLAMMATION IN HSCs
91. Ferrari D, La Sala A, Chiozzi P, et al. The P2 purinergic receptors of human dendritic cells: identification and coupling to cytokine release. FASEB J.
2000;14(15):2466-2476.
2375
Virgilio F. P2X(7) receptor and polykarion formation. Mol Biol Cell. 2000;11(9):3169-3176.
104. Jin J, Dasari VR, Sistare FD, Kunapuli SP. Distribution of P2Y receptor subtypes on haematopoietic cells. Br J Pharmacol. 1998;123(5):789-794.
78. Stagg J, Smyth MJ. Extracellular adenosine
triphosphate and adenosine in cancer. Oncogene. 2010;29(39):5346-5358.
92. Schnurr M, Toy T, Shin A, et al. Role of adenosine
receptors in regulating chemotaxis and cytokine
production of plasmacytoid dendritic cells. Blood.
2004;103(4):1391-1397.
79. Ke HZ, Qi H, Weidema AF, et al. Deletion of the
P2X7 nucleotide receptor reveals its regulatory
roles in bone formation and resorption. Mol Endocrinol. 2003;17(7):1356-1367.
93. Kohno Y, Ji X, Mawhorter SD, Koshiba M,
Jacobson KA. Activation of A3 adenosine receptors on human eosinophils elevates intracellular
calcium. Blood. 1996;88(9):3569-3574.
105. Schmid-Antomarchi H, Schmid-Alliana A,
Romey G, et al. Extracellular ATP and UTP control the generation of reactive oxygen intermediates in human macrophages through the opening
of a charybdotoxin-sensitive Ca2⫹-dependent
K⫹ channel. J Immunol. 1997;159(12):62096215.
80. Orriss IR, Wang N, Burnstock G, et al. The
P2Y(6) receptor stimulates bone resorption by
osteoclasts. Endocrinology. 2011;152(10):37063716.
94. Ferrari D, Idzko M, Dichmann S, et al. P2 purinergic receptors of human eosinophils: characterization and coupling to oxygen radical production.
FEBS Lett. 2000;486(3):217-224.
106. Yip KH, Lau HYA, Wise H. Reciprocal modulation
of anti-IgE induced histamine release from human mast cells by A1 and A(2b) adenosine receptors. Br J Pharmacol. 2011;164(2b):807-819.
95. Dichmann S, Idzko M, Zimpfer U, et al. Adenosine triphosphate-induced oxygen radical production and CD11b up-regulation: Ca mobilization
and actin reorganization in human eosinophils
adenosine triphosphate-induced oxygen radical
production and CD11b up-regulation. Blood.
2000;95(3):973-978.
107. Gomez G, Zhao W, Schwartz LB. Disparity in
Fc⑀RI-induced degranulation of primary human
lung and skin mast cells exposed to adenosine.
J Clin Immunol. 2011;31(3):479-487.
81. Stagg J, Divisekera U, Duret H, et al. CD73deficient mice have increased antitumor immunity
and are resistant to experimental metastasis.
Cancer Res. 2011;71(8):2892-2900.
82. Xiao Z, Yang M, Li F, Lv Q, He Q. Extracellular
nucleotide inhibits cell proliferation and negatively
regulates toll-like receptor 4 signaling in human
progenitor endothelial cells. Cell Biol Int. 2012;
36(7):625-633.
83. Wang L, Jacobsen SEW, Bengtsson A, Erlinge D.
P2 receptor mRNA expression profiles in human
lymphocytes, monocytes and CD34⫹ stem and
progenitor cells. BMC Immunol. 2004;5:16.
84. Gessi S, Varani K, Merighi S, et al. Expression of
A3 adenosine receptors in human lymphocytes:
up-regulation in T cell activation. Mol Pharmacol.
2004;65(3):711-719.
85. Haskó G, Cronstein B. Adenosine: an endogenous regulator of innate immunity. Trends Immunol. 2004;25(1):33-39.
86. Panther E, Corinti S, Idzko M, et al. Adenosine
affects expression of membrane molecules, cytokine and chemokine release, and the T-cell stimulatory capacity of human dendritic cells. Blood.
2003;101(10):3985-3990.
87. Panther E, Idzko M, Herouy Y, et al. Expression
and function of adenosine receptors in human
dendritic cells. FASEB J. 2001;15(11):1963-1970.
96. Gu B, Bendall LJ, Wiley JS. Adenosine
triphosphate-induced shedding of CD23 and
L-selectin (CD62L) from lymphocytes is mediated
by the same receptor but different metalloproteases. Blood. 1998;92(3):946-951.
97. Häusler SFM, Montalbán del Barrio I,
Strohschein J, et al. Ectonucleotidases CD39 and
CD73 on OvCA cells are potent adenosinegenerating enzymes responsible for adenosine
receptor 2A-dependent suppression of T cell
function and NK cell cytotoxicity. Cancer Immunol
Immunother. 2011;60(10):1405-1418.
98. Himer L, Csóka B, Selmeczy Z, et al. Adenosine
A2A receptor activation protects CD4⫹ T lymphocytes against activation-induced cell death.
FASEB J. 2010;24(8):2631-2640.
99. Zarek PE, Huang C-T, Lutz ER, et al. A2A receptor signaling promotes peripheral tolerance by
inducing T-cell anergy and the generation of
adaptive regulatory T cells. Blood. 2008;111(1):
251-259.
88. Idzko M. Nucleotides induce chemotaxis and actin polymerization in immature but not mature human dendritic cells via activation of pertussis
toxin-sensitive P2Y receptors. Blood. 2002;
100(3):925-932.
100. Gessi S, Varani K, Merighi S, et al. Expression,
pharmacological profile, and functional coupling
of a 2B receptors in a recombinant system and in
peripheral blood cells using a novel selective antagonist radioligand. Mol Pharmacol. 2005;67(6):
2137-2147.
89. Idzko M, Panther E, Sorichter S, et al. Characterization of the biological activities of uridine
diphosphate in human dendritic cells: influence
on chemotaxis and CXCL8 release. J Cell
Physiol. 2004;201(2):286-293.
101. Salmon JE, Cronstein BN. Fc gamma receptormediated functions in neutrophils are modulated
by adenosine receptor occupancy: A1 receptors
are stimulatory and A2 receptors are inhibitory.
J Immunol. 1990;145(7):2235-2240.
90. Wilkin F, Duhant X, Bruyns C, et al. The P2Y11
receptor mediates the ATP-induced maturation of
human monocyte-derived dendritic cells. J Immunol. 2001;166(12):7172-7177.
102. Cronstein BN. Adenosine, an endogenous antiinflammatory agent. J Appl Physiol. 1994;76(1):513.
103. Falzoni S, Chiozzi P, Ferrari D, Buell G, Di
108. Bradding P, Okayama Y, Kambe N, Saito H. Ion
channel gene expression in human lung, skin,
and cord blood-derived mast cells. J Leukoc Biol.
2003;73:614-620.
109. Broussas M, Cornillet-Lefèbvre P, Potron G,
Nguyen P. Inhibition of fMLP-triggered respiratory
burst of human monocytes by adenosine: involvement of A3 adenosine receptor. J Leukoc Biol.
1999;66(3):495-501.
110. Gu BJ, Zhang WY, Bendall LJ, et al. Expression
of P2X 7 purinoceptors on human lymphocytes
and monocytes: evidence for nonfunctional P2X 7
receptors. Am J Physiol Cell Physiol. 2000;279:
C1189-C1197.
111. Chen Y, Shukla A, Namiki S, Insel PA, Junger WG.
A putative osmoreceptor system that controls
neutrophil function through the release of ATP, its
conversion to adenosine, and activation of A2
adenosine and P2 receptors. J Leukoc Biol.
2004;76:245-253.
112. Lecut C, Frederix K, Johnson DM, et al. P2X1 ion
channels promote neutrophil chemotaxis through
Rho kinase activation. J Immunol. 2009;183(4):
2801-2809.
113. Gorini S, Callegari G, Romagnoli G, et al. ATP
secreted by endothelial cells blocks CX3CL1elicited natural killer cell chemotaxis and cytotoxicity via P2Y11 receptor activation. Blood. 2010;
116(22):4492-4500.
114. Cattaneo M. ADP receptors: inhibitory strategies
for antiplatelet therapy. Drug News Perspect.
2006;19(5):253-259.
115. Mahaut-Smith MP, Jones S, Evans RJ. The P2X1
receptor and platelet function. Purinergic Signal.
2011;7(3):341-356.
116. Sluyter R, Shemon AN, Barden JA, Wiley JS. Extracellular ATP increases cation fluxes in human
erythrocytes by activation of the P2X7 receptor.
J Biol Chem. 2004;279(43):44749-44755.
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2012 120: 2365-2375
doi:10.1182/blood-2012-04-422378 originally published
online July 11, 2012
The sixth sense: hematopoietic stem cells detect danger through
purinergic signaling
Lara Rossi, Valentina Salvestrini, Davide Ferrari, Francesco Di Virgilio and Roberto M. Lemoli
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