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Inflammasome 2014; 1: 76–80
Mini Review
Open Access
Takato Takenouchi*, Mitsutoshi Tsukimoto, Makoto Hashimoto, Hiroshi Kitani*
Inflammasome activation by danger signals:
extracellular ATP and pH
Abstract: Extracellular ATP has been recognized as a
danger signal that alerts the innate immune system.
High extracellular concentrations of ATP can trigger the
maturation and secretion of pro-inflammatory cytokines
(e.g., interleukin-1β and interleukin-18) through the
inflammasome-dependent activation of caspase-1. The
P2X7 receptor, an ATP-gated cation channel, plays a pivotal
role in ATP-induced NLRP3 inflammasome assembly.
Recently, intriguing evidence has emerged that acidic
extracellular pH acts as a danger signal that activates
inflammasomes. Extracellular acidification frequently
occurs at sites of inflammation, infection, or injury. In
addition, large amounts of ATP are readily released into the
extracellular space from damaged cells at such sites. Thus,
it is assumed that the ATP/P2X7 receptor pathway regulates
the inflammatory response under acidic extracellular
conditions. Here, we briefly discuss the mutual effects of
extracellular ATP and pH on inflammasome activation and
consider their roles in the regulation of inflammation.
Keywords:
inflammasome;
interleukin-1β;
extracellular acidosis; P2X7 receptor
ATP;
DOI 10.2478/infl-2014-0008
Received September 2, 2014; accepted September 22, 2014
1 Introduction
Inflammasomes are large intracellular multiprotein
complexes that are basically composed of a cytosolic
*Corresponding authors: Takato Takenouchi, Hiroshi Kitani: Animal
Immune and Cell Biology Research Unit, Division of Animal Sciences,
National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba,
Ibaraki 305-8634, Japan Tel&Fax: +81-29-838-6034; E-mail:
[email protected], [email protected]
Mitsutoshi Tsukimoto: Faculty of Pharmaceutical Sciences, Tokyo
University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
Makoto Hashimoto: Division of Sensory and Motor Systems, Tokyo
Metropolitan Institute of Medical Science, Tokyo, 156-0057, Japan
pattern recognition receptor (PRR) [e.g., nucleotidebinding domain and leucine-rich repeat containing
receptor (NLR) protein 1 (NLRP1), NLRP3, NLR family
CARD (caspase recruitment domain) domain-containing
protein 4 (NLRC4; also known as IPAF), or AIM2 (absent
in melanoma 2)] and an inactive procaspase [1]. Some
PRR (e.g., NLRP3 and AIM2) require the adaptor protein
ASC (apoptosis-associated speck-like protein containing
a CARD) in order to form inflammasomes [2]. Upon
PRR stimulation by “danger signals” that are released
or elaborated by pathogens or damaged/stressed
host cells [3], procaspase-1 is canonically recruited to
inflammasomes and converted into its catalytically active
form, caspase-1 [2]. Then, active caspase-1 proteolytically
cleaves the precursor forms of interleukin (IL)-1β (proIL-1β) and IL-18 (pro-IL-18) into their mature biologically
active forms, which are subsequently released into the
extracellular milieu through unconventional secretory
pathways [4]. Both of these cytokines are initially
synthesized as inactive precursor forms in response to
inflammatory stimuli, e.g., lipopolysaccharides (LPS),
and require post-translational processing by active
caspase-1 to exert their biological functions [5]. Since
the mature forms of IL-1β and IL-18 are potent proinflammatory cytokines of the innate immune system,
inflammasomes are considered to play an important
role in the homeostasis of the innate immune system.
Moreover, the uncontrolled production and release
of these cytokines is linked to various inflammatory
conditions [6], suggesting that inflammasomes are
potential therapeutic targets for the treatment of
inflammation-related disorders [7].
A variety of endogenous and exogenous danger
signals have been identified as inducers of inflammasome
assembly [8]. Among them, extracellular ATP has been
widely recognized as an endogenous danger signal that
activates inflammasomes. The P2X7 receptor (P2X7R),
an extracellular ATP-gated cation channel, plays a
key role in ATP-induced inflammasome activation [9].
Inflammasomes can also sense cellular stress caused
by microenvironmental changes such as a reduction in
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Inflammasome activation by danger signals: extracellular ATP and pH
extracellular osmolarity [10]. In this regard, emerging
evidence suggests that extracellular acidosis represents
a danger signal that alerts the innate immune system [11].
It is conceivable that extracellular acidosis modulates
the innate immune system because its development
is a hallmark of inflammatory processes [12]. In this
commentary, we summarize the recent advances in our
knowledge regarding the involvement of extracellular
ATP and pH in the activation of inflammasomes and
consider their putative roles in the regulation of
inflammation.
2 Extracellular ATP activates
inflammasomes via the P2X7R
The biological actions of extracellular ATP are generally
mediated by cell surface P2R. To date, two types of P2R,
ligand-gated P2X ion channels and G protein-coupled P2Y
receptors, have been identified [13]. The P2X7R is a member
of the P2X subfamily, and its activation by ATP opens
cation channels that are permeable to several cations
such as K+, Na+, Ca2+, and Mg2+ [14]. P2X7R are abundantly
expressed by cells involved in the inflammatory and
immune systems, such as monocytes, macrophages,
dendritic cells, and T cells. In contrast to other P2R, higher
concentrations of extracellular ATP (in the mM range) are
required to elicit P2X7R-dependent cellular responses
in vitro [15].
Accumulating evidence suggests that the P2X7R
plays a critical role in ATP-induced inflammasome
activation [9]. NLRP3 inflammasomes are known to sense
P2X7R-mediated cytoplasmic signals in monocytes and
macrophages [4,16]. It has also been reported that the P2X7R
mediates the activation of novel NLRP2 inflammasomes
by ATP in human astrocytes [17]. Similar to other poreforming toxins, such as maitotoxin and nigericin, the
depletion of intracellular K+ following K+ efflux through
P2X7R channels is a key event in the ATP-induced
activation of NLRP3 inflammasomes (Fig. 1, left side) [14].
Based on the close association between reactive oxygen
species (ROS) and NLRP3 inflammasome activation, it is
also suggested that P2X7R-mediated ROS production leads
to the activation of NLRP3 inflammasomes [18].
2.1 Acidic extracellular pH acts as a danger
signal via inflammasome activation
Local extracellular acidosis is often observed at sites of
ischemia and inflammation as well in injured or malignant
tissues, probably due to the stimulation of anaerobic
77
glycolysis [19]. Thus, it is plausible that extracellular
acidosis plays a role in the regulation of inflammatory
processes by modulating the functions of innate immune
cells [12]. In this context, acidic extracellular pH (6.5)
selectively stimulated the secretion of mature IL-1β from
human monocytes without affecting the production
of other pro-inflammatory cytokines, such as tumor
necrosis factor (TNF)-α and IL-6 [20,21]. The finding that
mature IL-1β secretion by monocytes that were cultured
at pH 6.5 was dependent on caspase-1 activity would
suggest that inflammasome activation is involved in the
abovementioned process [20]. It is worth noting that the
latter researchers detected increased pro-IL-1β mRNA
expression in the monocytes cultured at pH 6.5, suggesting
that the acidic extracellular pH-induced rise in mature
IL-1β secretion was partly due to the enhanced synthesis
of pro-IL-1β [20].
Recent reports have described the role of acidic
extracellular pH in the activation of inflammasomes.
Rajamaki and coll. demonstrated that acidic media
(pH 6.0-7.0) can trigger the pH-dependent secretion of
mature IL-1β via activation of NLRP3 inflammasomes
in LPS-primed human macrophages [11]. Since it was
demonstrated that alkaline extracellular pH (pH 8.08.5) inversely inhibit the IL-1β secretion induced by
several known NLRP3 activators, it has been suggested
that the extracellular pH has the bipartite regulatory
potential on the activation of NLRP3 inflammasomes
[11]. Rajamaki and coll. did not observe any significant
change in pro-IL-1β mRNA expression under acidic
extracellular conditions [11], supporting the notion that
acidic extracellular pH-induced secretion of mature IL-1β
results from inflammasome activation rather than the
enhanced synthesis of its precursor. Consequently, this
study proposes the intriguing concept that extracellular
acidosis acts as a novel endogenous danger signal
that alerts the innate immune system via NLRP3
inflammasomes [11].
Rapid intracellular acidification caused by
extracellular acidosis seems to be important for acidic
pH-dependent inflammasome activation (Fig. 1, right side)
[11,20]. As for the ATP/P2X7R pathway, it has also been
reported that ATP induced a reduction in intracellular
pH in rat pancreatic ducts via P2X7R activation [22]. We
further observed that the activation of the P2X7R by ATP
induced an increase in lysosomal pH in microglial cells
[23], which might have caused a reduction in cytosolic
pH by impairing the intracellular proton balance.
These studies imply that P2X7R-mediated intracellular
acidification is implicated in the ATP-induced activation
of inflammasomes (Fig. 1).
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78 T. Takenouchi et al.
Figure 1: Schematic representation of the potential effects of extracellular ATP and acidic pH on inflammasome activation, and IL-1β processing and secretion in monocyte/macrophage-lineage cells. ATP-induced P2X7R activation triggers the NLRP3 inflammasome-dependent
production of active caspase-1 (casp1) followed by the unconventional secretion of mature IL-1β (mIL-1β) (left side). Acidic extracellular
pH-induced reductions in intracellular pH also trigger the NLRP3 inflammasome-dependent production and secretion of mIL-1β (right side).
At lower extracellular pH, P2X7R activation by ATP preferentially produces and secretes 20-kDa IL-1β rather than mIL-1β (central part).
The lysosomal enzyme cathepsin D seems to be responsible for the production of 20-kDa IL-1β. If cytoplasmic pro-IL1b translocates to
secretory lysosomes, as was suggested by previous reports [32,33], 20-kDa IL-1β might be produced within lysosomes and secreted into
the extracellular space via Ca2+-dependent exocytosis (pathway 1). If cathepsin D is released from lysosomes into the cytoplasm due to
P2X7R-dependent lysosomal membrane damage, 20-kDa IL-1β might be produced within the cytoplasm and secreted via an unconventional
secretory pathway (pathway 2). We have proposed an alternative pathway in which pro-IL-1β leaks from cells after P2X7R stimulation and is
then converted to the 20-kDa form by exocytosed cathepsin D in the acidic extracellular space (pathway 3).
2.2 Acidic extracellular pH inhibits P2X7R
ion channel functions
Extracellular protons represent an important regulatory
factor for voltage and ligand-gated ion channels, and
the P2X7R is subject to proton modulation [24]. Indeed,
a previous study showed that extracellular protons
potently inhibited P2X7R-dependent ion currents and
the induction of membrane pore formation through
the allosteric modulation of P2X7R channels [25]. The
charges of several amino acid residues in the extracellular
domain of the rat P2X7R, including Lys, Asp, and His, are
likely to be involved in the functional inhibition of this
receptor by acidic pH [26,27]. In addition, a reduction
in the concentration of the genuine P2X7R agonist ATP4by the protonation of ATP was reported to contribute to
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Inflammasome activation by danger signals: extracellular ATP and pH
the inhibitory effect of acidic pH on the P2X7R [28]. In
any case, P2X7R ion channel functions are negatively
regulated under acidic extracellular conditions (Fig. 1).
2.3 Acidic extracellular pH modulates ATPinduced IL-1β processing
Despite the inhibitory effect of acidic pH on P2X7R
channel activity, the production and secretion of
significant amounts of mature IL-1β have been observed
after treatment with high concentrations of extracellular
ATP, even under acidic extracellular conditions. We found
that P2X7R activation by ATP triggered the caspase-1dependent production and secretion of mature IL-1β at
pH 6.2-6.7 in LPS-primed mouse microglial cells [29].
This finding is supported by another study in which it
was found that extracellular ATP triggered mature IL-1β
secretion at pH 6.2 in LPS-primed mouse mixed glia [30].
In these experimental systems, acidic pH alone failed
to induce the production and secretion of mature IL-1β
[29,30]. Considering that acidic extracellular pH perturbs
the functions of P2X7R channels, acidic pH-induced
reductions in intracellular pH might be involved in the
facilitation of P2X7R-mediated inflammasome assembly.
It is worth noting that acidic extracellular pH promoted
the production and secretion of an unconventional 20-kDa
form of IL-1β instead of the mature 17-kDa form upon
stimulation with ATP in LPS-primed mouse microglial
cells in a low pH-dependent manner (Fig. 1, central part)
[29,30]. Interestingly, lactic acid, which causes a reduction
in tissue pH in vivo, induced the preferential production
of 20-kDa IL-1β in LPS-primed mouse mixed glia [30].
The production and secretion of 20-kDa IL-1β was also
observed in LPS-primed human THP-1 macrophages after
6 h of incubation at lower pH (6.0), even without ATP
stimulation [11]. The lysosomal protease cathepsin D was
shown to be responsible for the production of 20-kDa
IL-1β (Fig. 1, central part), whereas active caspase-1 is not
involved in this process [29,30]. Considering that proIL-1β is used as a substrate for the production of both
the 20-kDa and mature forms of IL-1β, it is likely that the
enhanced production of 20-kDa IL-1β induced by lower pH
results in a relative reduction in the amount of bioactive
mature IL-1β during acidosis, which might be involved in
the physiological regulation of inflammatory responses.
3 Conclusion and perspectives
Recent studies have demonstrated that NLRP3
inflammasomes are able to sense a diverse range of danger
79
signals and then initiate the innate immune response
[18]. Given the local accumulation of these danger signals
in affected tissues, their cooperative effects need to be
considered when attempting to understand the regulation
of inflammatory process in vivo. In particular, since
extracellular acidosis is a common feature of inflammatory
loci it is likely that some danger signals exert their
functions under acidic extracellular conditions. From this
point of view, we discussed the possible mutual effects
of extracellular ATP and acidic pH on inflammasome
activation and IL-1β-dependent innate immunity.
Extracellular ATP or acidic pH alone can stimulate
the NLRP3 inflammasome-dependent production and
secretion of mature IL-1β in monocyte/macrophagelineage cells (Fig. 1, left and right sides). However,
when they co-exist pro-IL-1β seems to be preferentially
converted into the unconventional 20-kDa form of IL-1β
rather than the 17-kDa mature form (Fig. 1, central part)
[29,30]. Since 20-kDa IL-1β is suggested to be ~5-fold less
active at the IL-1 receptor than the mature form [31], it
is speculated that extracellular acidosis suppresses ATPinduced IL-1β-dependent innate immune responses. This
system might contribute to the physiological control of
inflammation by preventing excessive inflammatory
responses. If so, it is possible that the dysregulation
of 20-kDa IL-1β production exacerbates IL-1β-driven
inflammation. Although further experiments are required
to clarify the physiological or pathophysiological roles
of 20-kDa IL-1β in vivo, this pathway is a potential target
for the development of therapeutic strategies against
inflammatory diseases.
Acknowledgements: This study was supported by a
Grant-in-Aid for Scientific Research (Category C: Grant#
25450521) from the Japan Society for the Promotion of
Science (JSPS); the NIAS Strategic Research Fund from
National Institute of Agrobiological Sciences; and a grant
from the Ministry of Agriculture, Forestry and Fisheries
of Japan (Genomic-based Technology for Agricultural
Improvement, AGB-1004).
The authors declare no other conflict of interest.
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