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Pattern of interleukin-1 secretion in response to lipopolysaccharide and ATP before and after interleukin-1 blockade in patients with CIAS1 mutations.

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ARTHRITIS & RHEUMATISM
Vol. 56, No. 9, September 2007, pp 3138–3148
DOI 10.1002/art.22842
© 2007, American College of Rheumatology
Pattern of Interleukin-1␤ Secretion in Response to
Lipopolysaccharide and ATP Before and After Interleukin-1
Blockade in Patients With CIAS1 Mutations
Marco Gattorno,1 Sara Tassi,2 Sonia Carta,2 Laura Delfino,2 Francesca Ferlito,1
Maria Antonietta Pelagatti,1 Andrea D’Osualdo,3 Antonella Buoncompagni,1
Maria Giannina Alpigiani,4 Maria Alessio,5 Alberto Martini,1 and Anna Rubartelli2
Results. LPS-induced IL-1␤ secretion was markedly increased in monocytes from patients with CIAS1
mutations. However, unlike in healthy subjects, secretion of IL-1␤ was not induced by exogenous ATP.
Treatment with IL-1Ra resulted in a dramatic clinical
improvement, which was paralleled by an early and
strong down-regulation of LPS-induced IL-1␤ secretion
by the patients’ cells in vitro.
Conclusion. Our results showed that the requirements of ATP stimulation for IL-1␤ release observed in
healthy individuals are bypassed in patients bearing
CIAS1 mutations. This indicates that cryopyrin is the
direct target of ATP and that the mutations release the
protein from the requirement of ATP for activation. In
addition, the dramatic amelioration induced by IL-1Ra
treatment is at least partly due to the strong decrease in
IL-1␤ secretion that follows the first injections of the
antagonist. These findings may have implications for
other chronic inflammatory conditions characterized by
increased IL-1␤.
Objective. To examine the synthesis, processing,
and secretion of interleukin-1␤ (IL-1␤), as well as the
clinical and biologic effects of IL-1 blockade, in patients
with chronic infantile neurologic, cutaneous, articular
(CINCA) syndrome and Muckle-Wells syndrome
(MWS), in an effort to understand the molecular mechanisms linking mutations of the CIAS1 gene and IL-1␤
hypersecretion, and the underlying response to IL-1
receptor antagonist (IL-1Ra).
Methods. Six patients with CINCA syndrome or
MWS were treated with IL-1Ra and followed up longitudinally. Monocytes obtained from the patients and
from 24 healthy donors were activated with lipopolysaccharide (LPS) for 3 hours, and intracellular and secreted IL-1␤ levels were determined by Western blotting
and enzyme-linked immunosorbent assay before and
after exposure to exogenous ATP.
Supported in part by grants from the Associazione Italiana
per la Ricerca sul Cancro, the Ministero della Salute (Ricerca Corrente e Finalizzata), and the Comitato Interministeriale per la Programmazione Economica (grant 02/07/2004, CBA project).
1
Marco Gattorno, MD, Francesca Ferlito, PhD, Maria Antonietta Pelagatti, MD, Antonella Buoncompagni, MD, Alberto Martini, MD: Second Division of Pediatrics, IRCCS, Istituto G. Gaslini,
and University of Genoa, Genoa, Italy; 2Sara Tassi, PhD, Sonia Carta,
PhD, Laura Delfino, Technician, Anna Rubartelli, MD: Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy; 3Andrea D’Osualdo,
PhD: Laboratory of Molecular Gentics, IRCCS, Istituto G. Gaslini,
Genoa, Italy; 4Maria Giannina Alpigiani, MD: University of Genoa,
Genoa, Italy; 5Maria Alessio, MD: Federico II University, Naples,
Italy.
Drs. Gattorno and Tassi contributed equally to this work.
Address correspondence and reprint requests to Marco Gattorno, MD, IRCCS, Istituto G. Gaslini, Pediatria II, Largo G. Gaslini
5, 16147 Genoa, Italy. E-mail: marcogattorno@ospedale-gaslini.ge.it,
or to Anna Rubartelli, MD, Istituto Nazionale per la Ricerca sul
Cancro, Largo Rosanna Benzi 10, 16132 Genoa, Italy. E-mail:
anna.rubartelli@istge.it.
Submitted for publication September 15, 2006; accepted in
revised form May 18, 2007.
Interleukin-1␤ (IL-1␤) plays a pivotal role in the
pathogenesis of autoinflammatory diseases and represents a potential target of therapeutic intervention in
both monogenic and multifactorial inflammatory diseases (1). Unlike most cytokines, IL-1␤ lacks a secretory
signal peptide and is externalized by monocytic cells
through a nonclassic pathway, arranged in 2 steps (2,3).
First, Toll-like receptor ligands, such as lipopolysaccharide (LPS), induce gene expression and synthesis of the
inactive IL-1␤ precursor (proIL-1␤). Monocytes stimulated with LPS alone release only ⬃20% of the IL-1␤
over 24–48 hours (4). A second stimulus, such as
exogenous ATP, strongly enhances the proteolytic maturation and secretion of IL-1␤ (5,6). ATP-triggered
3138
IL-1␤ SECRETION IN CINCA SYNDROME AND MWS PATIENTS
3139
Table 1. Clinical characteristics of the patients with CINCA syndrome or MWS*
Patient/age
(years)/sex
C1/8.5/M
C2/5.9/F
C3/18.5/M
C4/33.8/M
C5/3.1/M
MWS1/12.8/F
Age
Treatment
at onset of
Hearing
Uveitis/
Facies/bone Developmental Chronic
CIAS1 at the time
CINCA/MWS Fever Rash Arthritis
loss
papilledema dysplasia
delay
meningitis mutation of study
1 week
Birth
2 weeks
3 weeks
6 months
16 months
⫹⫹ ⫹⫹⫹
⫹
⫹⫹
⫹⫹ ⫹⫹⫹
⫹⫹ ⫹⫹⫹
⫹
⫹
⫹⫹ ⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹⫹
–
⫹⫹
⫹⫹
–
⫹
⫹
–
–
⫹⫹
–
⫹
⫹
⫹
–
⫹⫹
⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹
–
⫹⫹
–
⫹
⫹
⫹
–
⫹⫹
–
⫹
–
–
–
N477K
D303N
T348M
Negative
M406I
E525K
NSAID
NSAID
NSAID
NSAID
NSAID
NSAID
* The intensity of the clinical manifestations was scored as follows: – ⫽ absent, ⫹ ⫽ present, ⫹⫹ ⫽ severe, and ⫹⫹⫹ ⫽ very severe. Patients C1–C5
have been described in part elsewhere (28). CINCA syndrome ⫽ chronic infantile neurologic, cutaneous, articular syndrome; MWS ⫽ Muckle-Wells
syndrome; NSAID ⫽ nonsteroidal antiinflammatory drug.
IL-1␤ secretion is mediated by P2X7 receptors expressed
on the surface of monocytes (7) and involves a series of
events that have only been partly clarified (8,9).
A crucial role in IL-1␤ processing is played by
the inflammasome, a multiprotein complex responsible
for the activation of caspase 1, which, in turn, converts
proIL-1␤ to the mature, active 17-kd form (10). A key
protein of the inflammasome is cryopyrin (also known
as NALP3 or cold-induced autoinflammatory syndrome
1 [CIAS-1]) (11,12). Experimental mouse models have
recently revealed that monocytes from knockout mice
deficient in cryopyrin cannot activate caspase 1 upon
stimulation with LPS and ATP, resulting in a lack of
IL-1␤ secretion (13–15). In contrast, mutations in the
CIAS1 gene in humans are associated with diseases
characterized by excessive production of IL-1␤ (11,
16–20). However, the molecular mechanisms connecting mutations in CIAS1 to IL-1␤ hypersecretion are
unclear.
Cryopyrin-associated autoinflammatory diseases
include familial cold autoinflammatory syndrome
(FCAS; MIM no. #120100), Muckle-Wells syndrome
(MWS; MIM no. #191900), and chronic infantile neurologic, cutaneous, articular (CINCA) syndrome (MIM
no. #607115), which is also known as neonatal-onset
multisystem inflammatory disease (NOMID) (21–23).
These conditions represent different phenotypes, from
the milder to the most severe, in the context of a clinical
continuum (24). FCAS is characterized by episodes of
rash, fever, and arthralgia after exposure to cold (18,21).
MWS consists of recurrent episodes of urticarial rash,
fever, and abdominal pain. Sensorineural deafness and
amyloidosis may represent late complications (22).
CINCA syndrome is characterized by a neonatal onset,
with urticarial-like skin lesions, fever, arthritis, persistent
elevation of acute-phase reactants, and early involvement of the central nervous system (chronic meningitis,
central deafness), eyes (uveitis, papilledema), and bones
(frontal bossing, epiphyseal and metaphyseal anomalies)
(19,23,25).
In each of these diseases, blocking the IL-1
receptors by daily injections of the recombinant human
IL-1 receptor antagonist (IL-1Ra) anakinra returns the
patients to normalcy, thus indicating the strong IL-1
dependency of the pathogenesis of these syndromes
(18,25–27). Despite these clear clinical results, the in
vivo target of anakinra remains to be elucidated.
The present study was aimed at investigating the
mode of IL-1 synthesis, processing, and secretion in
patients with CIAS1 mutations. Our findings indicate
excessive IL-1 secretion by monocytes from these patients. Strikingly, treatment of patients with IL-1Ra
resulted in normalization of this in vitro hypersecretion.
Both findings have value in elucidating the pathogenesis
of CINCA syndrome and MWS and help in understanding the therapeutic effects of treatment with IL-1Ra.
PATIENTS AND METHODS
CINCA syndrome and MWS patients. Six patients, 5
with CINCA syndrome and 1 with MWS (2 females and 4
males; mean age 13.8 years [range 3.1–33.8 years]), were
enrolled in the study. The clinical characteristics of the study
patients are shown in Table 1. The CINCA syndrome patients
have been described in part elsewhere (28). Disease onset
occurred within the first month of life in most of the study
patients. The disease course in the CINCA syndrome patients
was characterized by daily urticarial rashes, persistent elevation of levels of acute-phase reactants, and neurosensory,
ocular, and bone involvement. In the MWS patient, symptoms
began at the age of 16 months, with recurrent episodes of
fever, urticarial rash, and arthritis/arthralgia, with persistent
mild elevations of acute-phase reactant levels, consistent with
a clinical diagnosis of MWS. Five of the 6 study patients had a
mutation in exon 3 of the CIAS1 gene (Table 1).
At baseline, the patients were experiencing daily urticarial rashes (5 of 6 patients), arthritis (4 of 6 patients), and
3140
GATTORNO ET AL
Figure 1. Production, processing, and secretion of interleukin-1␤ (IL-1␤) by monocytes from
healthy adult donors (HD). a, Normal monocytes were cultured for 3 hours in the absence (top)
and presence (bottom) of 1 ␮g/ml of lipopolysaccharide (LPS). Aliquots from cell lysates were
separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by Western
blotting with anti–IL-1␤. Migration of the 35-kd and 29-kd proIL-1␤ molecular forms is indicated.
The lower IL-1␤ bands were not blocked by caspase 1 inhibitors (results not shown) and are
probably nonspecific endoproteases that were activated during the preparation of the samples (3).
b, Supernatants from normal monocytes cultured for 3 hours with LPS (top) or for 3 hours with LPS
followed by 15 minutes with ATP (bottom) were concentrated and analyzed as in a. Shown are 6
representative controls displaying different levels of basal secretion and different responses to ATP:
high (donors D and B), low (donors I and O), and intermediate (donors E and F). Migration of the
17-kd mature IL-1␤ is indicated. Detection of secreted IL-1␤ was not associated with the release
of lactate dehydrogenase (results not shown), which rules out a role of cell lysis in the
externalization of the cytokine. c, IL-1␤ secreted by monocytes from 24 healthy donors during 3
hours of incubation with LPS or during 15 minutes of exposure to ATP following LPS stimulation
was quantified by enzyme-linked immunosorbent assay. Results are expressed as ng/ml of IL-1␤
secreted per 106 cells. Values are the mean of at least 3 different experiments.
headaches (2 of 6 patients). Elevation of acute-phase reactants
and severe leukocytosis were observed in all patients. Hypochromic anemia was present in 4 of the 6 patients. Patients
were treated with IL-1Ra (anakinra) at a starting dosage of 1–2
mg/kg/day, delivered subcutaneously (maximum daily dose 100
mg).
Informed consent was obtained from all patients and
healthy donors. The study was approved by the Board of Ethics
of G. Gaslini Institute.
Cell cultures. Monocytes from buffy coats obtained
from the blood of 24 healthy adult donors matched for sex and
ethnicity or from heparinized blood obtained from the study
patients were enriched by adherence in RPMI 1640 medium
containing 10% fetal bovine serum, and then activated with 1
IL-1␤ SECRETION IN CINCA SYNDROME AND MWS PATIENTS
␮g/ml of LPS (all from Sigma-Aldrich, Milan, Italy) for 3 hours
at 37°C in RPMI 1640 medium supplemented with 1%
Nutridoma-HU (Roche Applied Science, Monza, Italy) as
described elsewhere (3,8,9). Supernatants were collected and
replaced with RPMI 1640/1% Nutridoma-HU in the presence
or absence of 1 mM ATP (Sigma-Aldrich) for 15 minutes.
After the addition of ATP, supernatants were collected and
cells were lysed in 1% Triton X-100 lysis buffer.
Determination of lactate dehydrogenase (LDH) release. The release of LDH into supernatants as a marker of cell
lysis during cocultures of dendritic cells and natural killer cells
was determined by standard methods, using an LDH colorimetric assay (Sigma-Aldrich) (3).
Enzyme-linked immunosorbent assay (ELISA). IL-1␤
content in supernatants was determined by ELISA (R&D
Systems, Minneapolis, MN).
Western blot analysis. Cell lysates and trichloroacetic
acid–concentrated supernatants were prepared as described
previously (8), resolved by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis on 12% gels, and then
electrotransferred (4,8). Filters were probed with 3ZD anti–
IL-1␤ monoclonal antibody (IgG1; Biological Resources
Branch, National Cancer Institute, Frederick, MD) or with
rabbit anti–caspase 1 antiserum R105 (kind gift of Dr. D. K.
Miller, Merck Research Laboratories, Rahway, NJ) followed
by the relevant horseradish peroxidase–conjugated secondary
antibody (Dako, Glostrup, Denmark) and developed by enhanced chemiluminescence using ECL Plus (Amersham Pharmacia Biotech, Milan, Italy).
Mutational screening of the CIAS1 gene and detection
of P2RX7 polymorphisms. DNA was extracted from peripheral
blood samples by standard methods. All coding regions and
intronic flanking sequences of the CIAS1 gene were amplified
by polymerase chain reaction (PCR) using specific primers
(available upon request from the authors) designed with
Primer Express 2.0 software (Applied Biosystems, Foster City,
CA). Four primer pairs were used to selectively amplify exons
5, 9, 10, 11, and 13 of the P2RX7 gene, as previously described (29).
PCR fragments were purified with enzymatic mix Sap
I–Exo III by incubation for 40 minutes at 37°C and for 15
minutes at 80°C, and then analyzed for mutations by direct
DNA sequencing using the BigDye Terminator cycle sequencing kit v3.1 and an ABI 3100 automated DNA sequencer (both
from Applied Biosystems). DNA analysis of each exon was also
performed in 50 adult individuals attending our blood transfusion center.
Statistical analysis. Comparisons among clinical and
laboratory parameters before and after treatment with anakinra were performed using Wilcoxon’s matched pairs test for
continuous variables and McNemar’s chi-square test for categorical variables. All statistical tests were 2-tailed with an alpha
level of 5%.
RESULTS
IL-1␤ production and secretion by monocytes
from CINCA syndrome and MWS patients with CIAS1
gene mutations. The production and secretion of IL-1␤
by monocytes from 4 patients with CINCA syndrome
3141
(patients C1–C4, Table 1), 1 patient with MWS (patient
MWS1, Table 1), and 24 healthy individuals (Figure 1)
were compared under resting conditions and after activation with LPS alone or with LPS followed by a brief
exposure to exogenous ATP.
In the absence of LPS, adherent control monocytes synthesized little or no proIL-1␤ (Figure 1a, top,
showing 6 representative subjects). Similarly, monocytes
from the CINCA syndrome and MWS patients spontaneously produced small or moderate amounts of
proIL-1␤ (Figure 2a, lanes 1–4 and Figure 2c, lane 1).
Neither control monocytes (results not shown) nor patient monocytes (Figure 2b, lanes 1, 4, 7, and 10 and
Figure 2c, lane 3) secreted detectable IL-1␤ under these
conditions. LPS stimulation drove the synthesis and
intracellular accumulation of proIL-1␤ to a similar extent in monocytes from normal donors (Figure 1a,
bottom) and patients (Figure 2a, lanes 5–8 and Figure
2C, lane 2). Notably, the lower IL-1␤ bands shown in
Figure 1a were not blocked by treatment with caspase 1
inhibitors (results not shown) and are probably due to
nonspecific endoproteases being activated during the
sample preparation (3).
Control monocytes activated with LPS for 3
hours secreted variable amounts of IL-1␤, which was
detected as a faint 17-kd band (Figure 1b, top). As
previously reported (6–9), brief exposure (15 minutes)
to extracellular ATP strongly induces the secretion of
mature IL-1␤ (Figure 1b, bottom). Quantification by
ELISA (Figure 1c) confirmed the variable secretion of
IL-1␤ during 3 hours of LPS stimulation and the different, but consistent, increases after exposure to ATP in
the 24 normal donors. The presence of variable amounts
of proIL-1␤ in supernatants was erratic and was not
paralleled by a release of the cytosolic enzyme LDH
(results not shown), ruling out a role of cell lysis in the
externalization of the cytokine (2,3).
In contrast, the LPS-induced secretion of mature
17-kd IL-1␤ by monocytes from the 3 CINCA syndrome
patients bearing a CIAS1 mutation who were tested
(patients C1–C3, Table 1) was dramatically high (Figure
2b, lanes 2, 5, and 8). Remarkably, the addition of
exogenous ATP failed to stimulate further IL-1␤ secretion in these patients (Figure 2b, lanes 3, 6, and 9).
Conversely, monocytes from the single CINCA syndrome patient who lacked CIAS1 mutations (patient C4,
Table 1) displayed a pattern of IL-1␤ secretion similar to
that of healthy donors, with low secretion of IL-1␤
during the 3 hours of stimulation with LPS, which
increased following induction with ATP (Figure 2b,
lanes 11 and 12). Monocytes from the MWS patient
3142
GATTORNO ET AL
Figure 2. Production, processing, and secretion of interleukin-1␤ (IL-1␤) by monocytes from 4 patients with chronic infantile neurologic, cutaneous,
articular (CINCA) syndrome and 1 patient with Muckle-Wells syndrome (MWS). Peripheral blood monocytes were separated by sodium dodecyl
sulfate–polyacrylamide gel electrophoresis and analyzed by Western blotting with anti–IL-1␤. Migration of the 35-kd and 29-kd proIL-1␤ molecular
forms in cell lysates and the 17-kd mature IL-1␤ form in supernatants is indicated. a, Aliquots of cell lysates from untreated or lipopolysaccharide
(LPS)–treated monocytes from CINCA syndrome patients were analyzed by Western blotting. b, Supernatants from CINCA syndrome patient
monocytes were incubated for 3 hours without LPS (lanes 1, 4, 7, and 10), with LPS (lanes 2, 5, 8, and 11), or with LPS followed by 15 minutes with
ATP (lanes 3, 6, 9, and 12) and analyzed by Western blotting. Detection of secreted IL-1␤ was not associated with the release of lactate
dehydrogenase (results not shown). c, Aliquots of cell lysates from untreated (lane 1) or LPS-treated (lane 2) monocytes from an MWS patient were
analyzed by Western blotting. Supernatants from the MWS patient monocytes were incubated for 3 hours without LPS (lane 3), with LPS (lane 4),
or with LPS followed by 15 minutes with ATP (lane 5) and analyzed by Western blotting. d, IL-1␤ secreted by patient monocytes during 3 hours
of incubation with LPS or during 15 minutes of exposure to ATP following LPS stimulation was quantified by enzyme-linked immunosorbent assay.
Values are the mean of at least 3 different experiments. The mean and SD values in the 24 healthy donors (HD) shown in Figure 1d are provided
for comparison. Results are expressed as ng/ml of IL-1␤ secreted per 106 cells.
carrying the E525K mutation of the CIAS1 gene (patient
MWS1, Table 1) secreted in response to LPS an IL-1␤
band that was weaker than that observed in the CINCA
syndrome patients (Figure 2c, lane 2; compare with
Figure 1b, top). However, similar to patients with CIAS1
mutations, ATP was unable to further induce IL-1␤
secretion (Figure 2c, lane 5).
The results of the Western blot analyses were
confirmed and quantified by ELISA (Figure 2d).
Induction of caspase 1 secretion by LPSstimulated monocytes from CINCA syndrome patients.
ATP-induced IL-1␤ secretion by monocytes from
healthy individuals was accompanied by the conversion
of procaspase 1 to active caspase 1, followed by secretion
of the enzyme (8,30,31). Comparison of caspase 1 pro-
duction and secretion in healthy individuals and CINCA
syndrome patients revealed that both normal and mutated monocytes constitutively produced procaspase 1
(p46), with only a little, if any, increase being detected
after LPS stimulation (Figure 3a, lanes 1 and 2). However, while control monocytes did not secrete mature
20-kd caspase 1 unless they were exposed to LPS plus
ATP treatment (Figure 3a, top blot, lanes 3–5), in
monocytes from patients C1 and C3 (Figure 3a, middle
and bottom blots) caspase 1 activation and secretion of
abundant p20 active caspase 1 occurred following activation with LPS alone (Figure 3a, lane 4). ATP was
unable to drive caspase 1 secretion in cells from CINCA
syndrome patients (Figure 3a, lane 5), a finding similar
to that for the secretion of IL-1␤.
IL-1␤ SECRETION IN CINCA SYNDROME AND MWS PATIENTS
Figure 3. Production, processing, and secretion of caspase 1 by monocytes from a healthy donor (donor A) and from 2 chronic infantile
neurologic, cutaneous, articular (CINCA) syndrome patients with a
CIAS1 mutation (patients C1 and C3). Monocytes were obtained a,
before or b, 2 days after initiation of treatment with anakinra. Cells
were cultured as described in Figures 1 and 2, and cell lysates and
supernatants were analyzed for the presence of procaspase 1 (procasp-1 p46) or active caspase 1 (casp-1 p20) by Western blotting with
anti–caspase 1 antibody. Shown are cell lysates obtained from untreated (lane 1) or lipopolysaccharide (LPS)–treated (lane 2) monocytes and supernatants obtained from monocytes incubated for 3 hours
without LPS (lane 3), with LPS (lane 4), or with LPS followed by 15
minutes with ATP (lane 5).
Lack of relationship between resistance to ATP
stimulation in CINCA syndrome or MWS patients and
mutations of the gene for the P2X7 receptor. P2RX7, the
gene for the purinergic receptor responsible for the
Table 2.
3143
ATP-dependent release of IL-1␤ (7), is highly polymorphic, and a number of loss-of-function mutations have
been characterized (29). The presence of functionally
relevant P2RX7 polymorphisms in CINCA syndrome
and MWS patients was investigated (Table 2). Patients
C1, C2, and C3, whose monocytes secreted little IL-1␤
following ATP triggering, expressed the wild-type
P2RX7, ruling out a role of this receptor in the lack of
response to ATP. In contrast, patient C4, who lacked
CIAS1 mutations and whose monocytes secreted IL-1␤
in response to ATP at levels similar to those in healthy
individuals, was heterozygous for the 1513 A⬎C nucleotide mutation; this mutation is associated with a complete loss of function only when 2 copies are present
(32,33). The P2RX7 gene in patient MWS1 carried on
both alleles the nucleotide substitution 489 C⬎T. This
variant is present in ⬃50% of the population, and
although it was proposed to increase the function of
P2X7 (34), its functional relevance with respect to IL-1␤
secretion is uncertain (29).
Clinical and biologic effects of anakinra treatment in patients with CINCA syndrome or MWS. The
clinical response to anakinra treatment in the study
patients is summarized in Figures 4a and b. After the
first dose of anakinra was administered, all patients
displayed dramatic improvement, with complete resolution of urticarial rash, arthritis, and fever within 1 week
from the beginning of the treatment (Figure 4a). A rapid
decrease in the levels of acute-phase reactants was also
observed in the first weeks of treatment, with complete
normalization in the majority of the patients (Figure 4b).
Monitoring of the patients during anakinra treatment
Functionally relevant polymorphisms of P2RX7 in patients with CINCA syndrome or MWS*
P2RX7 genotype
Patient
nt 489 C⬎T
(His155Tyr)†
nt 946 G⬎A
(Arg307Gln)‡
nt 1096 C⬎G
(Thr357Ser)§
nt 1513 A⬎C
(Glu496Ala)§
nt 1729 T⬎A
(Ile568Asn)‡
C1
C2
C3
C4
MWS1
C/C
C/C
C/C
C/C
T/T¶
G/G
G/G
G/G
G/G
G/G
C/C
C/C
C/C
C/C
C/C
A/A
A/A
A/A
A/C†
A/A
T/T
T/T
T/T
T/T
T/T
* CINCA syndrome ⫽ chronic infantile neurologic, cutaneous, articular syndrome; MWS ⫽ Muckle-Wells
syndrome; nt ⫽ nucleotide.
† The effect of His155Tyr substitution is a subject of controversy. Shemon and coworkers (29) reported
this mutation as nonfunctional, whereas Cabrini and coworkers (34) proposed that it increases the activity
of P2RX7.
‡ Arg307Gln and Ile568Asn substitutions have also been reported to confer complete loss of function
when 2 copies were present (29).
§ The most common substitutions at amino acid residues 357 and 496 of P2RX7 were demonstrated to
induce a complete loss of receptor function when 2 copies were present or in combination with another
loss-of-function polymorphism (32).
¶ Mutation.
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GATTORNO ET AL
Figure 4. Clinical response to anakinra treatment in 5 patients with chronic infantile neurologic,
cutaneous, articular (CINCA) syndrome (patients C1–C5) and 1 patient with Muckle-Wells
syndrome (MWS) (patient MW1). Assessments were made at baseline and at 1 week, 4 weeks, and
the last followup visit (mean 12.6 months [range 5–15 months]) after treatment with anakinra. a,
Number of patients with active arthritis, fever, and cutaneous manifestations before and after
treatment with anakinra. ⴱ ⫽ P ⫽ 0.04 versus baseline, by nonparametric McNemar’s chi-square
test. b, C-reactive protein (CRP) and hemoglobin (Hb) values before and after treatment with
anakinra. (See also Table 1.) P values are versus baseline, as determined by nonparametric
Wilcoxon’s matched pairs test. NS ⫽ not significant.
(mean 12.6 months [range 5–15 months]) revealed that
all of them continued to experience complete control of
their symptoms of inflammation, with nearly complete
amelioration of their general conditions (Figures 4a and
b). However, no significant improvement in the visual
and/or acoustic impairments has so far been observed in
these patients.
Monocytes were obtained from the patients at
different time intervals following the initiation of anakinra therapy and analyzed for IL-1␤ production and
secretion. Whereas LPS-induced IL-1␤ synthesis remained unchanged in individual patients as compared
with the findings before anakinra treatment (results not
shown), the amount of IL-1␤ secreted in response to
LPS activation by monocytes from the 3 CINCA syndrome patients with CIAS1 mutations (patients C1–C3)
was dramatically decreased (Figure 5a, lanes 2, 5, and 8).
Interestingly, the decrease in IL-1␤ secretion induced by
LPS was not accompanied by reestablishment of the
responsiveness to ATP, which again failed to induce
IL-1␤ secretion (Figure 5a, lanes 3, 6, and 9). Notably, in
patients C1 and C3, secretion of caspase 1 was also
strongly reduced after anakinra treatment (Figure 3b),
indicating that the secretion of the 2 proteins is tightly
associated.
In contrast, in the CINCA syndrome patient who
lacked a CIAS1 mutation (patient C4) and who displayed a pattern of IL-1␤ secretion comparable to that
in normal individuals before treatment (see Figure 2b),
the amount of IL-1␤ secreted following either LPS
activation or ATP stimulation did not change significantly (Figure 5a, lanes 10–12), despite a very good
clinical response to anakinra. In addition, cells from
patient MWS1, who similarly displayed a good clinical
response to anakinra, did not present significant variations in the amount of IL-1␤ secreted over 3 hours of
LPS stimulation and remained insensitive to ATP stimulation (Figure 5b).
Figure 5c shows the quantification of IL-1␤
present in cell supernatants, as determined by ELISA.
Close monitoring of IL-1␤ secretion by monocytes from
CINCA syndrome patients during anakinra treatment
IL-1␤ SECRETION IN CINCA SYNDROME AND MWS PATIENTS
3145
Figure 5. In vitro secretion of interleukin-1␤ (IL-1␤) by cells from 4 patients with chronic infantile
neurologic, cutaneous, articular (CINCA) syndrome (patients C1–C4) and 1 patient with MuckleWells syndrome (MWS) (patient MWS1) treated with anakinra. Peripheral blood monocytes were
obtained at 2 days (C1 and C3), 1 month (C4 and MWS1), and 4 months (C2) after initiation of
anakinra, cultured as described in Figure 2, and analyzed by Western blotting or enzyme-linked
immunosorbent assay (ELISA). a, Supernatants from CINCA syndrome patient monocytes were
incubated for 3 hours without lipopolysaccharide (LPS) (lanes 1, 4, 7, and 10), with LPS (lanes 2,
5, 8, and 11), or with LPS followed by 15 minutes with ATP (lanes 3, 6, 9, and 12) and analyzed by
Western blotting. b, Supernatants from MWS patient monocytes were incubated for 3 hours
without LPS (lanes 1), with LPS (lanes 2), or with LPS followed by 15 minutes with ATP (lanes 3)
and analyzed by Western blotting. c, IL-1␤ secreted by patient monocytes during 3 hours of
incubation with LPS or during 15 minutes of exposure to ATP following LPS stimulation was
quantified by ELISA. Values are the mean of at least 3 different experiments. The mean and SD
values in the 24 healthy donors (HD) shown in Figure 1c are provided for comparison. Results are
expressed as ng/ml of IL-1␤ secreted per 106 cells.
showed that, although some fluctuations occurred during the year of therapy, IL-1␤ secretion was still low after
12 months (data not shown).
DISCUSSION
In this study, we analyzed the synthesis, processing, and secretion of IL-1␤ in patients with CINCA
syndrome and MWS, both before and after treatment
with anakinra, and compared the in vitro data with the
clinical responses. Our study has generated 2 major new
findings. First, monocytes from CINCA syndrome patients following LPS stimulation secrete impressively
greater amounts of mature IL-1␤ despite a cytoplasmic
accumulation of the precursor, comparable to that of
healthy individuals, but fail to increase IL-1␤ secretion
in response to ATP. These peculiarities are likely to be
dependent on the mutated cryopyrin, since they were
absent in the single CINCA syndrome patient who
lacked known mutations. Interestingly, the MWS patient, who had a CIAS1 mutation and displayed milder
clinical manifestations, failed to respond to ATP despite
having levels of LPS-induced IL-1␤ secretion that were
comparable to those secreted by normal individuals. The
analysis of the sequence coding for the ATP receptor
P2X7 revealed that none of the patients expressed
nonfunctional P2RX7 variants, which ruled out the pos-
3146
sibility that the failure of ATP to induce IL-1␤ secretion
was due to a loss-of-function mutation of the highly
polymorphic P2RX7 gene (7,29).
The second main finding was that the treatment
with anakinra in CINCA syndrome or MWS patients,
which was associated with a dramatic clinical response,
was paralleled by a sharp decrease in LPS-induced IL-1␤
secretion, but did not restore the ability of the patients’
monocytes to secrete IL-1␤ in response to ATP.
We found that 2 signals are required for inflammasome activation. The dramatic secretion of IL-1␤
following LPS stimulation of CINCA syndrome monocytes could be dependent upon the induction of
proIL-1␤ synthesis in a cell that, bearing a CIAS1
mutation, displays a constitutively activated inflammasome (11,12). However, if this were the case, caspase 1,
which, unlike IL-1␤, is also synthesized by resting monocytes (8), would be processed and secreted even in the
absence of LPS stimulation. Our study ruled out this
possibility by showing that caspase 1, although normally
produced by resting CINCA syndrome monocytes, was
processed and secreted only after exposure to LPS.
The different behavior of normal and CIAS1mutated monocytes (showing low versus high IL-1␤ and
caspase 1 secretion in response to LPS and different
responsiveness to ATP) suggests that in normal monocytes, 2 stimuli (LPS and ATP) are required to achieve
a rapid activation of the inflammasome, whereas in
patients carrying a CIAS1 mutation, LPS alone is sufficient to overactivate procaspase 1 and proIL-1␤ processing and secretion. In principle, the dramatic increase in
IL-1␤ secretion following LPS stimulation of monocytes
from CINCA syndrome patients could account for a
“secretory exhaustion” of a cell that is no longer responsive to ATP triggering. However, “secretory exhaustion”
cannot account for the lack of response to ATP of MWS
monocytes that secreted IL-1␤ in physiologic amounts
following LPS stimulation. Furthermore, the lack of
response to ATP persisted in monocytes from CINCA
syndrome patients after treatment with anakinra, which
dramatically decreased the LPS-induced IL-1␤ secretion.
Recent data obtained from studies of cryopyrindeficient mice indicate that cryopyrin is essential for
assembly of the inflammasome in response to components of the signaling pathway mediated by Toll-like
receptor and ATP (13,14). Our present findings in
humans support the hypothesis that cryopyrin could be
the direct target of ATP (most probably mediated by the
low intracellular levels of K⫹ induced by P2X7 receptor
triggering [5]), and when mutated, cryopyrin is freed
GATTORNO ET AL
from the requirement of ATP for its activation. LPS
would act upstream, possibly inducing the expression
and/or activation of a different (known or unknown)
inflammasome component.
In the present study, unstimulated monocytes
from CINCA syndrome or MWS patients secreted little,
if any, IL-1␤. This result differs partially from data
obtained by other groups of investigators, which showed
an increased synthesis (19) and secretion (11,20,25) of
IL-1␤ by purified monocytes or mononuclear cells from
CINCA syndrome or MWS patients, even in the absence
of LPS stimulation. This discrepancy is likely explained
by the methods used. The cell adherence time we used in
this study before testing proIL-1␤ content and IL-1␤
secretion is much shorter than that used by the other
groups. In our experience, a longer adherence time also
results in the activation of normal monocytes in the
absence of LPS, although to different extents in different
individuals.
How does anakinra render CIAS1-mutated
monocytes refractory to LPS-induced IL-1␤ secretion?
All 6 of our CINCA syndrome or MWS patients experienced complete clinical remission following treatment
with anakinra, including the patient who lacked CIAS1
mutations; this supports the results recently reported
(25) in a different cohort of patients. In addition, while
Goldbach-Mansky et al (25) observed a decrease in
IL1B gene expression after 3 months of treatment, we
found an early (after 48 hours) and marked downmodulation of LPS-induced IL-1␤ secretion in all of our
patients with CIAS1 mutations, independently of
proIL-1␤ synthesis, that remained substantially unchanged as compared with pretreatment levels in each
patient. This observation is consistent with the prompt
and dramatic clinical response observed in all CINCA
syndrome patients soon after the introduction of anakinra. In contrast, no significant decrease in IL-1␤
secretion was observed in the CINCA syndrome patient
lacking a CIAS1 mutation or in the MWS patient.
Whether this is due to a low level of LPS-induced
secretion by cells from these 2 patients, which could
make the decrease less evident, or whether it is due to
other mechanisms remains to be clarified.
The mechanisms related to the dramatic effects
of anakinra in patients with autoinflammatory syndromes are still largely unclear. Our study suggests that
anakinra could play roles other than a simple competition with free circulating IL-1␤ for IL-1 receptor type I.
In fact, the rapid clinical remission obtained in our
patients was associated with a prompt decrease in IL-1␤
secretion by LPS-stimulated peripheral blood mono-
IL-1␤ SECRETION IN CINCA SYNDROME AND MWS PATIENTS
cytes, which strongly suggests that the monocytes themselves represent a major target of anakinra. This observation is consistent with previous studies highlighting the
capacity of IL-1 to induce IL-1 itself (35–37). Anakinra
can block this positive feedback, as confirmed by the
observation that normal monocytes activated in vitro
with LPS in the presence of recombinant IL-1Ra decrease IL-1␤ secretion (data not shown).
However, the simple occupancy of monocyte IL-1
receptors by anakinra in vivo is unlikely to be responsible for the decreased IL-1␤ secretion observed in vitro in
monocytes from CINCA syndrome patients, since the
procedures used to purify and wash the monocytes
should greatly reduce the amount of bound recombinant
IL-1Ra. If a consistent proportion of LPS-induced IL-1␤
secretion occurs as a result of IL-1␤ itself, one could
speculate that in monocytes from CINCA syndrome
patients treated with anakinra, IL-1 receptor type I is
down-regulated, resulting in a reduced secretion of
IL-1␤. It remains to be elucidated whether interference
by anakinra in the vicious circle of IL-1 inducing IL-1 is
the major cause of the decreased secretion or whether
other mechanisms are involved.
In conclusion, our findings confirm the pivotal
role of IL-1␤ in the pathogenesis of CINCA syndrome
or MWS and shed new light on the underlying molecular
mechanisms. ATP as a secretory trigger is not operative
in monocytes from patients with CINCA syndrome or
with MWS who carry mutated cryopyrin genes. This
suggests that while in normal monocytes, 2 signals are
required to activate the inflammasome, thus tightly
controlling IL-1␤ secretion, in patients with a CIAS1
mutation, a single stimulus, even one that in low
amounts (such as danger signals released by injured cells
or bacterial products) would be unable to trigger IL-1␤
secretion in healthy individuals, is sufficient to drive a
dramatic inflammatory cascade.
The in vivo encounter with anakinra not only
blocks the effects of IL-1 on target cells, but also inhibits
the production of IL-1␤ by monocytes, resulting in an
impressive amelioration of the clinical manifestations.
Our findings may also have implications for other
chronic inflammatory conditions characterized by an
increased expression of IL-1␤ and responsive to IL-1
blockade, such as systemic-onset juvenile idiopathic arthritis and adult Still’s disease (38–41).
ACKNOWLEDGMENTS
We thank Prof. M. Ferrarini for critically reading the
manuscript. We also thank the Biological Resources Branch of
3147
the National Cancer Institute (Frederick, MD) for providing
the 3ZD anti–IL-1 monoclonal antibody, Dr. D. K. Miller
(Merck Research Laboratories, Rahway, NJ) for the kind gift
of the rabbit anti–caspase 1 antiserum R105, and the Blood
Centers of Ospedale S. Martino (Genoa, Italy) for providing
the buffy coats.
AUTHOR CONTRIBUTIONS
Drs. Gattorno and Rubartelli had full access to all of the data
in the study and take responsibility for the integrity of the data and the
accuracy of the data analysis.
Study design. Gattorno, Martini, Rubartelli.
Acquisition of data. Gattorno, Tassi, Carta, Delfino, Ferlito, Pelagatti,
D’Osualdo, Buoncompagni, Alpigiani, Alessio.
Analysis and interpretation of data. Gattorno, Tassi, Carta, Delfino,
Ferlito, Pelagatti, Buoncompagni, Rubartelli.
Manuscript preparation. Gattorno, Martini, Rubartelli.
Statistical analysis. Gattorno.
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