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Mutations in cryopyrinBypassing roadblocks in the caspase 1 inflammasome for interleukin-1 secretion and disease activity.

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Vol. 56, No. 9, September 2007, pp 2817–2822
DOI 10.1002/art.22841
© 2007, American College of Rheumatology
Mutations in Cryopyrin: Bypassing Roadblocks in the Caspase 1 Inflammasome
for Interleukin-1␤ Secretion and Disease Activity
Charles A. Dinarello
Unlike the release of tumor necrosis factor ␣
(TNF␣), there are several roadblocks to the release of
interleukin-1␤ (IL-1␤), beginning with the transcription
of the IL1B gene and ending with the exit of the active
cytokine from the cell. There is also the additional
roadblock of the high level of synthesis and secretion of
the naturally occurring IL-1 receptor antagonist (IL1Ra), with its tight binding to the IL-1 receptor type I
and an affinity higher than that of either IL-1␤ or IL-1␣.
If these roadblocks are not enough to limit the activity of
IL-1␤, there is nature’s decoy mechanism, which consists
of the preferential binding of the cell surface IL-1
receptor type II to IL-1␤, preventing it from triggering
the signal-transducing type I receptor. The soluble form
of the type II receptor also prevents active IL-1␤ from
engaging the signaling receptor by forming an inactive
complex with the soluble IL-1 receptor accessory protein, which is constitutively produced in the liver. The
facilitation of IL-1␤ processing by the caspase 1 inflammasome through ATP activation of the P2X7 receptor
can also be viewed as a potential roadblock to the
activity of IL-1␤.
It seems clear that in the evolution of cytokines,
some cytokines, such as IL-6 and IL-1Ra, are quickly
driven from the cell by the Golgi machinery, whereas
others encounter a series of roadblocks that limit their
activity. The most revealing justifications for nature’s
wisdom in placing several roadblocks to IL-1␤ activity
are the consequences of the effects of IL-1␤ in the loss
of insulin-producing pancreatic beta cells (1) and the
manifestations of autoinflammatory syndromes, diseases
that carry a portfolio of destructive inflammation (2–4).
Familial Mediterranean fever (5) and hyper-IgD syndrome (6) are also classified as autoinflammatory diseases because of the poor control of the processing and
secretion of IL-1␤.
In this issue of Arthritis and Rheumatism (7), the
study reported by Gattorno and coworkers offers important new lessons about these roadblocks. The investigators studied the release of IL-1␤ from primary blood
monocytes obtained from patients with chronic infantile
neurologic, cutaneous, articular (CINCA) syndrome
(known in North America as neonatal-onset multisystem
inflammatory disease [NOMID]) and from a patient
with Muckle-Wells syndrome (MWS). These 2 syndromes, as well as familial cold-induced autoinflammatory syndrome (FCAS), are the primary examples of
dysregulated processing and secretion of IL-1␤. The
investigators also studied the effects of in vivo treatment
with anakinra, a recombinant human IL-1Ra. Specifically, the study examined the secretion of IL-1␤ from
peripheral blood monocytes obtained from the patients
before and during treatment with anakinra. The study
focused on the function of the IL-1␤ “inflammasome”
(more appropriately termed the caspase 1 inflammasome) and the effect of mutations in cryopyrin, the
protein of the NALP3 gene (8). The lessons learned
from these studies provide new insights into the tight
control of the processing and secretion of IL-1␤. While
the syndromes studied are rare, the mechanisms revealed by these studies have broad implications for how
evolution favored the restriction of biologic responses to
IL-1␤ by placing several roadblocks to its egress from
the cell.
The clinical manifestations of these syndromes
are characterized by recurrent fevers, neutrophilic leukocytosis, rashes, deforming arthritis, and high serum
levels of acute-phase reactants. Progressive deafness,
Supported by NIH grant AI-15614.
Charles A. Dinarello, MD: University of Colorado Health
Sciences Center, Denver.
Dr. Dinarello has received consulting fees (less than $10,000)
from Amgen and has given expert testimony for which he received a
fee (less than $10,000) from Chiron.
Address correspondence and reprint requests to Charles A.
Dinarello, MD, University of Colorado Health Sciences Center, 4200
East Ninth Avenue B168, Denver, CO 80262. E-mail:
Submitted for publication May 13, 2007; accepted in revised
form May 18, 2007.
leptomeningitis, and amyloidosis may be present in some
patients. These syndromes are part of a larger group of
diseases that are called “autoinflammatory” to distinguish them from those considered to be “autoimmune,”
although there is clearly overlap between the 2 groups.
Both the adult-onset and the juvenile-onset forms of
Still’s disease (systemic-onset juvenile idiopathic arthritis) fall into the autoinflammatory group. The best
criterion for identifying an autoinflammatory disease is
that the clinical, biochemical, and hematologic manifestations are rapidly and impressively reversed upon initiation of treatment with IL-1Ra, with the IL-1 trap, or
with monoclonal anti–IL-1␤ antibodies. Hence, the culprit in these diseases is IL-1␤ and not IL-1␣; essentially,
autoinflammatory diseases are diseases that occur as a
result of poor control of the release of active IL-1␤.
Compared with monocytes from healthy subjects,
freshly isolated peripheral blood monocytes from patients with autoinflammatory diseases secrete more
IL-1␤ when incubated in vitro. For example, peripheral
blood mononuclear cells (PBMCs) from patients with
NOMID secreted ⬎1 ng/ml more IL-1␤ during a 24hour incubation in the absence of any exogenous stimulus, as compared with ⬍50 pg/ml secreted by cells from
healthy controls (2). When stimulated with endotoxin,
PBMCs from NOMID patients secreted 3–4 times more
IL-1␤ than PBMCs from controls. In the Gattorno study
(7), monocytes from patients with CINCA syndrome
secreted even greater amounts of IL-1␤ when stimulated
with lipopolysaccharide as compared with monocytes
from controls. Although patients with FCAS exhibit
dramatic reversal of disease manifestations upon treatment with anakinra, elevated levels of IL-1␤ in the
circulation following cold exposure is not observed (4).
Rather, the circulating cytokine is IL-6, which is under
the control of IL-1.
These observations reinforce an important clinical lesson in cytokine-mediated diseases: causation is
established with specific receptor blockade or specific
cytokine neutralization, and not with elevated circulating levels. For example, many diseases are associated
with elevated serum levels of IL-6 because this cytokine
is readily secreted, but few are due to a pathologic role
of IL-6. Although increased secretion of IL-1␤ by stimulated blood monocytes in vitro can be helpful in the
diagnosis of an IL-1␤–mediated disease, a few days of
treatment with anakinra is safe and will unambiguously
reveal the role of IL-1 in the disease in question. Despite
this rather “easy IL-1 test of causation,” in the report by
Gattorno, there is a lesson about the control of active
IL-1␤ secretion and this lesson may apply to other
cytokines, such as IL-18 and IL-33. Although IL-18 likely
contributes to several autoinflammatory diseases, macrophage activation syndrome appears to be a disease
that is attributable to IL-18 (9). IL-33 may mediate mast
cell–related and other T helper type 2 diseases. Both
cytokines are members of the IL-1 family and are
caspase 1 dependent for secretion of the active cytokine.
The first lesson from the Gattorno study is that
monocytes obtained from their patients secreted IL-1␤
without a second signal. What is meant by the expression
“second signal”? There are 2 ways to study the secretion
of IL-1␤ from fresh human blood monocytes. Most
studies stimulate the cells with endotoxin (usually at
concentrations of 1 ng/ml or lower) and, after 24 hours
of incubation, measure the “mature” cytokine released
into the supernatant, using a specific enzyme-linked
immunosorbent assay. In the absence of cell leakage or
cell death, starting after 4 hours and over the course of
24 hours, IL-1␤ is steadily released into the supernatant
medium. The IL-1␤ that is released is called “mature”
because it represents the “active” cytokine following
cleavage of the inactive IL-1␤ precursor by caspase 1. As
described below, activation of the intracellular cysteine
protease caspase 1 is the primary function of the inflammasome. In general, most, if not all, Toll-like receptor
(TLR) ligands, as well as microorganisms themselves,
stimulate the secretion of IL-1 ␤ via a caspase
1–dependent processing of the IL-1␤ precursor. Like
endotoxins from gram-negative bacteria (via TLR-4),
microbial products from gram-positive bacteria (via
TLR-2) will result in the progressive release of IL-1␤
from monocytes starting at 4 hours and continuing for
the next 20–36 hours. The vast majority of studies on the
secretion of IL-1␤ use a 24-hour stimulation period.
Importantly, in animals or humans injected with endotoxins, the earliest detectable level of IL-1␤ in the
circulation is at 3 hours, and peak levels are present
between 4 and 8 hours.
The second way to study the secretion of mature
IL-1␤ is to accelerate the process by incubating the
monocytes with endotoxin for a short period of time (3
hours) and then triggering the rapid release of mature
IL-1␤ within 15 minutes. This experimental approach is
called the “two-signal” method. As shown in Figure 1A,
3 hours of exposure to endotoxin provides the first signal
and results in transcription and translation of the IL-1␤
precursor. A second signal results in the rapid release of
mature IL-1␤. This so-called second signal is triggered
by activation of the purinergic ion channel called P2X7
Figure 1. Steps in the activation of the caspase 1 inflammasome and the secretion of interleukin-1␤ (IL-1␤). A, Monocytes from unaffected, healthy
controls. Activation of Toll-like receptor (TLR) (step 1) results in transcription (step 2) and translation of the IL-1␤ precursor (step 3). The IL-1␤
precursor remains diffusely in the cytosol (14). Upon activation of the P2X7 receptor (P2X7 R) (step 4), there is a rapid efflux of potassium from
the cell (step 5a), resulting in a fall in intracellular potassium levels (step 5b). The fall in intracellular potassium levels triggers the assembly of the
components of the caspase 1 inflammasome (step 6) and its association with procaspase 1. The caspase 1 inflammasone is composed of cryopyrin
plus ASC (apoptosis-associated speck-like protein containing a caspase 1 recruitment domain [CARD]) and Cardinal (CARD inhibitor of
NF-␬B–activating ligands). Cryoprin is a large protein with 4 domains: PYR (a pyrin domain), NACHT (a domain found in NAIP [neuronal
apoptosis inhibitor protein], CIITA [class II major histocompatibility complex transcription activator], HET-E [incompatibility locus protein from
Podospora anserina, a bacterial mucleotide triphosphatase protein], and TP-1 [telomerase-associated protein 1]), NAD (NALP-associated domain),
and LRR (leucine-rich repeats). The processing of procaspase 1 results in the formation of the active caspase 1 heterodimer and the cleavage of the
IL-1␤ precursor. The enzymatic processing of the IL-1␤ precursor by caspase 1 may take place in the cytosol (step 7a) or in the secretory lysosome
(step 7b) or both. An influx of calcium into the cell (step 8), with an increase in intracellular calcium levels, provides a mechanism by which mature
IL-1␤ is released from the cell (10,15) (step 9). Calcium influx activates 3 phospholipases; phosphatidylcholine-specific phospholipase C and
calcium-dependent phospholipase A2 are required for secretion of IL-1␤, with exocytosis of the lysosomal contents (10). B, Monocytes from subjects
with mutations in cryopyrin. TLR activation and the synthesis of the IL-1␤ precursor take place (steps 1, 2, and 3) as in A. TLR triggering may also
result in the assembly of the inflammasome (step 4). However, there is constitutive activation of caspase 1 in monocytes from subjects with mutations
in cryopyrin, and activation of the inflammasome by a fall in intracellular potassium is not required. Similar to monocytes from healthy controls, the
processing of the IL-1␤ precursor by caspase 1 may take place in the cytosol (step 5a) or in the secretory lysosome (step 5b) or both. Also similar
to monocytes from healthy controls, an influx of calcium into the cell (step 6), with an increase in intracellular calcium levels, provides a mechanism
by which mature IL-1␤ is released from the cell (10,15) (step 7). TIR ⫽ Toll/interleukin-1 receptor.
with millimolar concentrations of ATP. The activation
of the P2X7 receptor by ATP results in the nearsimultaneous efflux of potassium from the cell (see
Figure 1A). The fall in intracellular potassium levels
provides the signal for the cleavage of the IL-1␤ precursor by caspase 1 (10). The cleavage may take place in
cellular organelles called secretory lysosomes, but also
possibly in the cytosol. The cleavage likely takes place
near the cell membrane. Within 15 minutes of ATP
activation of the P2X7 receptor, potassium levels fall,
and mature IL-1␤ is released from the cell. Activation of
the P2X7 receptor and the fall in intracellular K⫹
concentrations, with the subsequent events leading to
IL-1␤ processing and secretion, likely also take place in
the “long incubation” method, but at lower levels,
resulting in lower secretion of IL-1␤. Naturally occurring
peptides that activate the P2X7 receptor have been
described (11).
The expected finding in the study by Gattorno
was that after 3 hours of exposure to endotoxin, monocytes from the CINCA syndrome or MWS patients
secreted significantly more IL-1␤ than did those from
the healthy controls (7). The unexpected finding in the
study was that monocytes from the CINCA syndrome
and MWS patients did not respond to ATP with a burst
of IL-1␤ release. The rapid release of IL-1␤ by monocytes from healthy donors is triggered by a short exposure to ATP. Unlike monocytes from healthy donors, the
release of larger amounts of IL-1␤ did not require the
second signal from ATP. Thus, the ATP activation step
(step 4 in Figure 1A) was not needed in monocytes from
patients with the cryopyrin mutations. One interpretation is that the caspase 1 inflammasome is constitutively
active in these cells during the 3-hour incubation period
and does not require the ATP-driven efflux of potassium
to cleave the precursor (Figure 1B). But, there was yet
another unexpected finding.
In monocytes from healthy controls, ATP activated the processing and the rapid secretion not only of
mature IL-1␤, but also of caspase 1. However, caspase 1
release did not take place following the addition of ATP
to cells from the patients. Hence, the 2 ATP-driven
effects are not needed in monocytes from these patients.
Procaspase 1 is present in resting monocytes, whereas
synthesis of the IL-1␤ precursor requires TLR activation. Therefore, one would expect to detect spontaneous
secretion of caspase 1 in monocytes from patients with a
constitutively activated caspase 1 inflammasome. The
observation that caspase 1 secretion by the patients’
monocytes occurred only after TLR triggering suggests
that the caspase 1 inflammasome is partially, but not
fully, active in cells from patients with autoinflammatory
syndromes. Therefore, one can interpret the effect of
mutations in cryopyrin as bypassing one of the roadblocks that control the exit of active IL-1␤ from the cell.
Although all patients with CINCA syndrome
exhibit the same spectrum of clinical, biochemical, and
hematologic abnormalities, not all patients carry mutations in the CIAS1 gene. For example, in a study of 18
patients with NOMID (CINCA syndrome), only 67%
carried the mutation (2). Nevertheless, monocytes from
all of the patients released significantly more IL-1␤,
whether resting or after stimulation with endotoxin for
24 hours, as compared with monocytes from healthy
controls. The study by Gattorno and colleagues (7) went
one step further and discovered that the failure to
require ATP for the 3-hour release of IL-1␤ was only
observed in the CINCA syndrome and MWS patients
who had CIAS1 mutations. Monocytes from the CINCA
syndrome patient without CIAS1 mutations responded
to ATP, as did monocytes from healthy donors. We can
assume that this differential response to ATP also existed
in the cohort of 18 NOMID patients whose cells were
studied only for the release of IL-1␤ over 24 hours (2).
The Gattorno study draws attention to a unique
property of the mutations, since one can now assign a
molecular mechanism to the increase in IL-1␤ secretion
relevant to the ATP-driven activation of caspase 1 by the
complex of interacting proteins that comprise the
caspase 1 inflammasome. It is a unique example of how
a single mutation reveals a great deal of new information
about a complex molecular mechanism. The assembly of
proteins of the mutated inflammasome, although not
requiring the fall in intracellular potassium, nevertheless
still requires an initiating signal, and in the laboratory,
endotoxin is used. In the patient, IL-1␤ itself likely
functions as the initiator.
The function of the caspase 1 inflammasome is
primarily to convert inactive procaspase 1 into the active
enzyme. In the rapid release method, this activation is
triggered by the efflux of potassium and is rapidly
followed by the appearance of mature IL-1␤ in the
supernatant together with the processed caspase 1.
Gattorno provides evidence that the mutation in
cryopyrin results in activation of caspase 1 without
requiring a sudden fall in the level of intracellular
potassium in order to activate the inflammasome. The
authors propose that mutated cryopyrin allows for the
assembly of the complex of interacting proteins that
comprise the inflammasome in the presence of normal
intracellular levels of potassium. This explanation
would, in fact, provide a molecular mechanism for the
ATP trigger in the wild-type cryopyrin. In the wild-type
inflammasome, ATP activation of the P2X7 receptor
opens the potassium channel, and as potassium levels
fall, caspase 1 is simultaneously activated by the inflammasome. Thus, cryopyrin, in which any one of several
mutations may occur, is the molecular target and results
in the activation of caspase 1. The model provides an
explanation for the consistent observation that monocytes from patients with CINCA/NOMID or MWS
secrete more IL-1␤ than do those from healthy controls.
Although studied using an exogenous stimulant such as
endotoxin, monocytes from these patients more likely
experience background stimulation from endogenous
inflammation in vivo. In fact, as discussed below, the
most likely endogenous stimulant is IL-1␤ itself.
The model also supports the concept that the
rate-limiting step in the secretion of IL-1␤ is activation
of caspase 1. Any disease process that includes an
increase in the steady-state levels of caspase 1, components of the inflammasome, or the IL-1␤ precursor
carries the potential to be an “autoinflammatory” disease. Gattorno and colleagues report that in vivo therapy with anakinra in patients with CIAS1 mutations was
associated with an in vitro reduction in the secretion of
IL-1␤ from monocytes as compared with secretion before treatment. Monocytes from the patient without
mutations, although secreting more IL-1␤ than those
from controls, secreted the same amount of IL-1␤
before, as well as after, anakinra treatment. The reduction in the secretion of IL-1␤ following initiation of
anakinra treatment was also reported by GoldbachMansky et al (2).
What accounts for these observations, whether
assessed by the long (24-hour) release method (2) or the
short (3 hours plus 15 minutes) release method (7)?
There is no magic about these observations. IL-1␤
stimulates both its own gene expression as well as the
translation of the messenger RNA into the precursor.
The original observations were made 20 years ago
(12,13). The most likely explanation for a downregulation of secretion with IL-1 receptor blockade is
that IL-1␤ stimulates the synthesis of caspase 1 and
IL-1␤ precursor. In fact, in NOMID patients treated
with anakinra, there was a down-regulation of both
caspase 1 and the IL-1␤ precursor (2), suggesting that
blocking the activity of IL-1␤ brings about a reduction in
the further release of IL-1␤. Thus, the rate-limiting step
is IL-1␤ activity itself. In support of this concept is the
observation that a single dose of a neutralizing anti–
IL-1␤ monoclonal antibody in patients with MWS resulted in a rapid amelioration of disease that lasted 6
months, but within 24 hours after the infusion of the
antibody, steady-state levels of IL-1␤ messenger RNA
from PBMCs were decreased (Lachmann H: personal
communication). It would therefore be of considerable
interest to activate the caspase 1 inflammasome in
healthy donor monocytes stimulated with IL-1␣ for 3
hours and then activate the P2X7 receptor with ATP.
Such a study would address a model closer to the clinical
reality, since IL-1 itself, rather than endotoxin, is the
challenge encountered by patients who have autoinflammatory diseases.
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