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The pattern of response to antiinterleukin-1 treatment distinguishes two subsets of patients with systemic-onset juvenile idiopathic arthritis.

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ARTHRITIS & RHEUMATISM
Vol. 58, No. 5, May 2008, pp 1505–1515
DOI 10.1002/art.23437
© 2008, American College of Rheumatology
The Pattern of Response to Anti–Interleukin-1 Treatment
Distinguishes Two Subsets of Patients With
Systemic-Onset Juvenile Idiopathic Arthritis
Marco Gattorno,1 Alessandra Piccini,2 Denise Lasigliè,1 Sara Tassi,2 Giacomo Brisca,1
Sonia Carta,2 Laura Delfino,2 Francesca Ferlito,1 Maria Antonietta Pelagatti,1
Francesco Caroli,3 Antonella Buoncompagni,1 Stefania Viola,1 Anna Loy,1 Marina Sironi,4
Annunciata Vecchi,4 Angelo Ravelli,1 Alberto Martini,1 and Anna Rubartelli2
Objective. To assess the clinical response to
interleukin-1 (IL-1) blockade and in vitro IL-1␤ and
IL-18 secretion in patients with systemic-onset juvenile
idiopathic arthritis (JIA).
Methods. Twenty-two patients with systemiconset JIA were treated with the IL-1 receptor antagonist
(IL-1Ra) anakinra. Monocytes from 18 patients and 20
healthy donors were activated by different Toll-like
receptor ligands. Intracellular and secreted IL-1␤ and
IL-18 were analyzed by Western blotting and enzymelinked immunosorbent assay.
Results. Ten patients with systemic-onset JIA
exhibited a dramatic response to anakinra and were
classified as complete responders. Eleven patients had
an incomplete response or no response, and 1 patient
could not be classified in terms of response. Compared
with patients who had an incomplete response or no
response, complete responders had a lower number of
active joints (P ⴝ 0.02) and an increased absolute
neutrophil count (P ⴝ 0.02). In vitro IL-1␤ and IL-18
secretion in response to various stimuli was not increased and was independent of treatment efficacy.
Likewise, secretion of IL-1Ra by monocytes from patients with systemic-onset JIA was not impaired. An
overall low level of IL-1␤ secretion upon exposure to
exogenous ATP was observed, unrelated to treatment
responsiveness or disease activity.
Conclusion. Two subsets of systemic-onset JIA
can be identified according to patient response to IL-1
blockade. The 2 subsets appear to be characterized by
some distinct clinical features. In vitro secretion of
IL-1␤ and IL-18 by monocytes from patients with
systemic-onset JIA is not increased and is independent
of both treatment outcome and disease activity.
Treatment with anakinra, a recombinant
interleukin-1 receptor antagonist (IL-1Ra), is the most
effective therapy for hereditary autoinflammatory syndromes related to mutations in the inflammasome component cryopyrin/NALP3 (1–4), which result in partial
loss of control of IL-1␤ processing and in secretion of
large amounts of IL-1␤ (4–7). Anakinra has also shown
promising results in the treatment of systemic-onset
juvenile idiopathic arthritis (JIA) (8,9) and adult-onset
Still’s disease (10), 2 severe inflammatory diseases characterized by arthritis along with systemic features such as
high spiking fever, rash, hepatosplenomegaly, and serositis (11). The clinical benefit observed after treatment
of systemic-onset JIA with IL-1Ra and the finding of
enhanced in vitro production of IL-1␤ by patient monocytes has suggested that this disease could also be related
to dysregulated production and secretion of IL-1␤ (9).
IL-1␤ is a powerful proinflammatory cytokine
and plays a key role in innate and adaptive immunity
(12). Secretion of IL-1␤ occurs through a non-classical
Supported in part by grants from the Ministero Salute,
Istituto Superiore Sanità, Telethon Italy, and the Associazione Italiana
per la Ricerca sul Cancro. Drs. Tassi and Carta’s work was supported
by Italfarmaco, Milan, Italy.
1
Marco Gattorno, MD, Denise Lasigliè, Giacomo Brisca,
MD, Francesca Ferlito, PhD, Maria Antonietta Pelagatti, MD, Antonella Buoncompagni, MD, Stefania Viola, MD, Anna Loy, MD,
Angelo Ravelli, MD, Alberto Martini, MD: G. Gaslini Institute, and
University of Genoa, Genoa, Italy; 2Alessandra Piccini, PhD, Sara
Tassi, PhD, Sonia Carta, PhD, Laura Delfino, Anna Rubartelli, MD:
Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy; 3Francesco
Caroli, BS: G. Gaslini Institute, Genoa, Italy; 4Marina Sironi, PhD,
Annunciata Vecchi, PhD: Istituto Humanitas, Rozzano, Milan, Italy.
Address correspondence and reprint requests to Marco Gattorno, 2nd Division of Pediatrics, G. Gaslini Institute, Largo G. Gaslini
5, 16147 Genoa, Italy. E-mail: marcogattorno@ospedale-gaslini.ge.it.
Submitted for publication August 27, 2007; accepted in
revised form January 18, 2008.
1505
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GATTORNO ET AL
Table 1. Baseline characteristics of the patients with systemic-onset juvenile idiopathic arthritis*
Patient
Age,
years/
sex
Disease
duration,
years
No. of
active
joints
CRP,
mg/dl
WBCs (neutrophils),
⫻ 103/mm3
Treatment
Prednisone
dosage,
mg/kg/day
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12
S13
S14
S15
S16
S17
S18
S19
S20
S21
S22
7.8/M
8.5/M
14.3/M
12.0/F
9.1/F
11.7/F
12.5/M
10.4/F
13.2/F
7.5/F
16.8/M
12.6/F
6.0/M
12.4/M
17.0/M
18.7/M
3.58/M
4.3/F
3.3/M
0.9/F
12.8/F
11.7/F
3.9
1.5
3.2
2.01
2.4
2.2
2.6
0.6
10.9
0.5
3.3
0.5
3.0
0.9
10.8
4.8
2.12
0.7
1.6
0.3
10.5
5.5
6
2
4
8
3
7
3
2
10
1
4
3
4
55
9
45
7
6
20
16
4
10
15.3
23.4
5.9
4.1
11.5
10.3
8.7
14.8
14.9
5.27
8.9
1.6
11.9
13.9
14.7
1.3
7.1
12.7
12.9
16.5
15.0
2.8
25.8 (23.8)
23.4 (22.3)
29.4 (27.9)
14.9 (9.7)
8.9 (6.1)
13.4 (9.3)
18.0 (16.3)
26.3 (23.7)
35.8 (30.9)
14.8 (9.5)
17.3 (14.6)
22.0 (19.7)
16.2 (8.1)
11.9 (8.1)
18.0 (15.7)
11.4 (8.8)
17.1 (8.9)
13.6 (9.1)
14.3 (10.1)
20.3 (7.3)
13.0 (8.2)
18.2 (15.7)
Steroid, MTX, AZA, etan.
NSAID, steroid, MTX, CSA
NSAID, steroid, MTX, etan.
Steroid, MTX, CSA
NSAID, steroid, MTX, CSA
NSAID, steroid, MTX, inflix.
Steroid, MTX
Steroid, CSA
NSAID, steroid
Steroid, MTX
NSAID, steroid
Steroid, MTX
NSAID, steroid, MTX, etan.
Steroid, MTX, inflix.
Steroid, CSA, AZA
NSAID, steroid
NSAID, steroid, etan.
Steroid, CSA
Steroid, thal., MTX, etan.
NSAID, steroid, thal.
NSAID, steroid, etan.
Steroid, etan.
2
3
0.5
0.5
0.8
0.4
0.2
0.5
1.1
0.4
0.1
1.5
0.3
0.15
0.15
0.2
0.5
0.78
1
2.5
0.5
0.2
* CRP ⫽ C-reactive protein; WBCs ⫽ white blood cells; MTX ⫽ methotrexate; AZA ⫽ azathioprine; etan. ⫽ etanercept; NSAID ⫽ nonsteroidal
antiinflammatory drug; CSA ⫽ cyclosporin A; inflix. ⫽ infliximab; thal. ⫽ thalidomide.
pathway (13,14) and requires 2 signals (13,14). First,
Toll-like receptor (TLR) signaling induces gene expression and synthesis of the inactive IL-1␤ precursor
(proIL-1␤). Then, a second signal drives processing and
secretion of the cytokine. A highly effective second
signal is exogenous ATP, which binds P2X7 receptors
expressed on the surface of monocytes (15–17). Processing requires the assembly of the inflammasome, a multiprotein intracellular complex responsible for the activation of caspase 1. Active caspase 1 in turn converts proIL1␤ into the biologically active cytokine (7,18,19). Generation of mature IL-1␤ is associated with its secretion.
In this study, we evaluated the clinical response
to IL-1 blockade in a cohort of 22 patients with systemiconset JIA and studied the levels of IL-1␤ and IL-18
secreted in vitro by patient monocytes. We found that
⬃40% of patients with systemic-onset JIA respond very
satisfactorily to IL-1 blockade, with a pattern strongly
reminiscent of that observed in autoinflammatory diseases related to cryopyrin mutation. In vitro IL-1␤ and
IL-18 secretion in response to various stimuli was not
increased and was independent of treatment efficacy. A
lower number of involved joints and a higher absolute
neutrophil count were associated with a greater likelihood of response to anakinra.
PATIENTS AND METHODS
Patients. The study was approved by the Ethics Board
of G. Gaslini Institute. Twenty-two patients with systemiconset JIA (20) (11 girls and 11 boys) were selected for
treatment with anakinra (Table 1). All patients had received
long-term corticosteroid therapy, mostly in association with 1
or more second-line agents. Patients previously treated with
etanercept or infliximab underwent a washout period of at
least 2 weeks or 8 weeks, respectively.
Anakinra treatment was started after informed consent
was provided by parents and, when applicable, by patients. The
starting dosage of anakinra was 1 mg/kg/day, subcutaneously
(maximum 100 mg). Response to treatment was evaluated
based on findings in a number of clinical and laboratory
parameters (fever, rash, number of active joints, erythrocyte
sedimentation rate [ESR], C-reactive protein [CRP] level,
white blood cell count, hemoglobin level) during followup.
Six additional patients with active systemic-onset JIA
who had not received steroid treatment were also analyzed.
Their mean age was 8.1 years (range 3.5–13.3 years), and
their mean disease duration was 3.2 years (range 0.4–10.8
years).
In vitro production and secretion of IL-1␤, IL-18, and
IL-1Ra. Fresh monocytes from 9 of the patients with systemiconset JIA (patients 4, 5, 6, 8, 10, 12, 17, 19, and 22) were
analyzed for secretion of IL-1␤, IL-1Ra, and IL-18 before and
after anakinra treatment. Monocytes from the 6 additional,
non–steroid-treated patients with active systemic-onset JIA
were also analyzed. Monocytes from 20 age-matched healthy
JIA SUBSETS DISTINGUISHED BY RESPONSE TO ANTI–IL-1
individuals attending our clinic for routine examinations prior
to minor surgical procedures or from other young donors were
used as controls, after informed consent was obtained.
Cell preparation and in vitro treatment. Monocytes
isolated from patients with systemic-onset JIA and healthy
controls were enriched by adherence in RPMI 1640 medium
containing 10% fetal bovine serum (Sigma-Aldrich, Milan,
Italy). Purity was assessed by fluorescence-activated cell sorter
analysis. The proportion of CD14⫹ cells was consistently
ⱖ75%, and that of CD3⫹ cells was ⱕ5%. Peripheral blood
mononuclear cells (PBMCs) or enriched monocytes were
activated with different stimuli at 37°C in RPMI 1640 supplemented with 1% Nutridoma-HU (Roche, Milan, Italy), for 3
hours or 24 hours as previously described (14,21,22). The
stimuli used were as follows: lipopolysaccharide (LPS) (1
␮g/ml; Sigma-Aldrich), muramyldipeptide (3 ␮g/ml; Calbiochem, La Jolla, CA), Staphylococcus aureus (107 heat-killed S
aureus/ml; InvivoGen, San Diego, CA), the yeast cell wall
derivative zymosan (50 ␮g/ml; kindly provided by Dr. Guido
Frumento, Genoa, Italy), phorbol myristate acetate (PMA) (50
ng/ml; Sigma-Aldrich), and ionomycin (1 ␮g/ml; SigmaAldrich).
In some experiments, after 3 hours of stimulation with
LPS, supernatants were replaced with RPMI 1640–1%
Nutridoma-HU to which 1 mM ATP (Sigma-Aldrich) had been
added, with further incubation for 15 minutes. At the end of
each experiment, supernatants were collected and cells lysed in
1% Triton X-100 lysis buffer.
Enzyme-linked immunosorbent assay (ELISA). To determine the concentrations of IL-1␤, IL-1Ra (R&D Systems,
Minneapolis, MN), and IL-18 (MBL, Nagoya, Japan), ELISAs
were performed on supernatants of monocyte cultures.
Western blot analysis. Cell lysates and trichloroacetic
acid–concentrated supernatants, prepared as previously described (22), were boiled in reducing Laemmli sample buffer,
resolved by sodium dodecyl sulfate–12% polyacrylamide gel
electrophoresis, and electrotransferred as described (14,21,22).
Filters were probed with anti–IL-1␤ monoclonal antibody 3ZD
(IgG1; obtained from the National Cancer Institute Biological
Resources Branch, Frederick, MD) followed by horseradish
peroxidase–conjugated goat anti-mouse IgG (Dako, Glostrup,
Denmark) and developed with ECL-Plus (GE Healthcare,
Milan, Italy).
Multicytokine determination. Sera from 16 patients
with systemic-onset JIA were collected before and 1 week after
the beginning of treatment with anakinra and immediately
stored at ⫺80°C until analysis. Serum levels of 28 different
soluble molecules (IL-1␤, IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL-10, IL-12 [p70], IL-13, IL-15, IL-17, total IL-18,
basic fibroblast growth factor, eotaxin, granulocyte colonystimulating factor [G-CSF], granulocyte–macrophage colonystimulating factor [GM-CSF], interferon-␥ [IFN␥], interferoninducible protein 10 [IP-10], monocyte chemotactic protein 1,
macrophage inflammatory protein 1␣ [⌴⌱P-1␣], MIP-1␤,
platelet-derived growth factor, RANTES, tumor necrosis factor ␣ [TNF␣], vascular endothelial growth factor [VEGF])
were analyzed by Bio-Plex cytokine multiplexable bead assay
(Bio-Rad, Richmond, CA). Soluble IL-1 decoy receptor and
pentraxin 3 (PTX3) were also measured by ELISA, as previously described (23,24). Sera from 10 age-matched healthy
1507
subjects were used as controls, after informed consent was
obtained.
Genetic analysis of CIAS1 mutations and P2X7 receptor polymorphisms. DNA was extracted from peripheral blood
by standard methods. All coding regions and intronic flanking
sequences of CIAS1 were amplified by polymerase chain
reaction using specific primers designed with Primer Express
2.0 software (Applied Biosystems, Foster City, CA), as previously described (4).
Four primer pairs designed with Primer Express 2.0
were used to selectively amplify exons 5, 9, 10, 11, and 13 of the
P2X7 gene, as previously described (4). DNA analysis of each
exon was also performed in 50 Italian control subjects.
Statistical analysis. Comparisons of clinical and laboratory parameters before and after treatment were performed
using Wilcoxon’s matched pairs test and McNemar’s chi-square
test for continuous and categorical variables, respectively. Differences in serum cytokine levels and in vitro cytokine secretion
between patients with systemic-onset JIA and healthy controls or
between disease subgroups (responders versus nonresponders)
were analyzed by Mann-Whitney U test.
RESULTS
Heterogeneity of clinical response to anti–IL-1
treatment among patients with systemic-onset JIA. At
baseline, all 22 patients with systemic-onset JIA exhibited active arthritis and elevated levels of acute-phase
reactants. High fever was present in 17, and rash in 12.
Soon after the initiation of anakinra treatment, a general
amelioration of the disease was observed, with normalization of the systemic features (fever and rash) and
decrease in the levels of acute-phase reactants in the vast
majority of the patients (details on clinical and laboratory findings at baseline and over time available from
the authors upon request).
However, as early as the first week of treatment,
it was clear that there were 2 distinct patterns of
response to anakinra among the patients with systemiconset JRA (Figure 1). One group of 10 patients (patients
S1–S10) showed prompt control of systemic and articular manifestations, with normalization of acute-phase
reactant levels and persistence of complete control of
the disease after a mean followup of 1.36 years (range
0.3–2.59 years). These patients were designated complete responders (Figure 1A). Four months after the
start of therapy, they were able to discontinue all
medications except anakinra. Two of these patients have
now been receiving anakinra for ⬎2.5 years, and 5 others
have received anakinra for ⬎1 year.
A second group of 11 patients (patients S11–S20
and S22 [incomplete responders or nonresponders])
exhibited a variable response to anakinra treatment,
with general improvement soon after the initiation of the
therapy, but with a tendency toward recurrence of the
1508
GATTORNO ET AL
Figure 1. Response to anakinra in patients with systemic-onset juvenile idiopathic arthritis who were designated
good responders (A) and those designated incomplete responders or nonresponders (B). At the time of statistical
analysis, the duration of followup did not differ between the 2 groups. Number of active joints and C-reactive
protein (CRP) level are shown as representative of clinical and laboratory parameters, respectively. The course of
findings for other acute-phase reactants (white blood cell count and erythrocyte sedimentation rate) and
hemoglobin levels was highly consistent with that for CRP. Numbers preceded by the letter S are individual patient
numbers. P values (versus baseline) were determined by Wilcoxon’s nonparametric matched pairs test. NS ⫽ not
significant.
disease manifestations, especially arthritis and elevation
of acute-phase reactant levels, during followup, despite
increases in the daily dosage to 3 or 4 mg/kg (Figure 1B).
Conversely, the systemic features in these patients, such
as fever and rash, were generally well controlled over
time. Seven patients in this group (patients S11–S17
[incomplete responders]) are still receiving anakinra
treatment with a mean followup of 1.34 years (range
0.3–2.1). However, they are also being treated with
second-line agents (5 of 7 patients) and/or oral steroids
(6 of 7 patients). Due to the persistence of active disease
even at higher doses of anakinra and poor compliance with
the daily injection protocol, patients S18, S19, S20, and S22
discontinued the treatment after 6 months, 3 months, 6
months, and 1 month, respectively (nonresponders).
Apart from variable skin reactions at the injection site, no major adverse events were observed. How-
ever, on day 13 of treatment, 2 patients (patients S11 and
S21) presented with laboratory features consistent with
macrophage activation syndrome (MAS), i.e., marked
elevation of liver enzyme, ferritin, and triglyceride levels,
and decreased platelet count. Both patients discontinued anakinra and were treated with steroids and cyclosporin A, with rapid control of the MAS. Six months
later, patient S11 was re-treated with anakinra for a
relapse of his underlying disease, without any further
sign of MAS. The parents of patient S21 did not allow
reinstitution of the treatment with anakinra after complete control of MAS was achieved. This patient therefore was not classified as a responder or a nonresponder.
Lack of increase in IL-1␤ secretion by monocytes
from both responders and nonresponders after stimulation with LPS and ATP. To investigate whether the
different responses to anakinra among patients with
JIA SUBSETS DISTINGUISHED BY RESPONSE TO ANTI–IL-1
1509
Figure 2. Interleukin-1␤ (IL-1␤) production, processing, and secretion by monocytes from healthy controls and
patients with systemic-onset juvenile idiopathic arthritis (SoJIA). A and B, Monocytes from healthy controls (A)
or from representative patients with systemic-onset JIA (B) were left unstimulated or were cultured with
lipopolysaccharide (LPS) for 3 hours or with LPS for 3 hours and ATP for 15 minutes, and the presence of IL-1␤
in cell lysates and supernatants was analyzed by Western blotting. Numbers preceded by the letter S are individual
patient numbers. SF ⫽ steroid free. C, Supernatants of monocytes from 20 healthy controls, 9 patients with
systemic-onset JIA before and after anakinra treatment, and 6 patients with active JIA who had not received
steroids were treated with LPS or with LPS and ATP as described above, and IL-1␤ levels were measured. Data
are presented as box blots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes
represent the median, and the lines outside the boxes represent the highest and lowest values. ⴱ ⫽ P ⬍ 0.05 versus
controls, by Mann-Whitney U test.
systemic-onset JIA were related to different levels of
IL-1␤ secretion, spontaneous and LPS-induced production and secretion of IL-1␤ were studied in monocytes
from 9 anakinra-treated patients with active systemiconset JIA and 20 healthy controls. IL-1␤ is an inducible
cytokine that is not expressed by resting monocytes (12).
Findings in monocytes from healthy donors confirmed
that in most cases (70%), no intracellular proIL-1␤
could be detected (Figure 2A). In contrast, monocytes
from 6 of 9 patients with systemic-onset JIA (67%)
exhibited intracellular proIL-1␤ in the absence of stimulation, indicating a certain degree of in vivo preactivation. Very little, if any, IL-1␤ was spontaneously secreted by monocytes from either controls or JIA patients
during 3 hours of incubation in the absence of stimuli
(Figures 2A and B).
Treatment with LPS induced a strong increase in
levels of intracellular proIL-1␤ in all controls (Figure
2A), whereas only variable induction was observed in
patient monocytes (Figure 2B), with no response to LPS
in some cases. IL-1␤ secretion detected by ELISA after
3 hours of LPS stimulation was variable but overall was
low in all patients with systemic-onset JIA compared
with healthy controls, in supernatants both from purified
monocytes (P ⫽ 0.004 by Mann-Whitney U test) (Figure
2C) and from PBMCs (data not shown). No differences
in IL-1␤ secretion were found between anakinra responders and incomplete responders or nonresponders.
Secretion of IL-1␤ was strongly enhanced by extracellular ATP, which triggers the purinergic receptor P2X7
expressed on the surface of monocytes (Figure 2).
Interestingly, patients with active systemic-onset
JIA exhibited defective responses to ATP (Figure 2B),
with levels of ATP-induced IL-1␤ secretion significantly
lower than those observed in healthy donors (P ⫽ 0.004)
(Figure 2C). Again, no differences were found between
1510
GATTORNO ET AL
Table 2. Functionally relevant P2X7 receptor polymorphisms in 9 patients with systemic-onset juvenile
idiopathic arthritis
P2X7 receptor genotype
Patient
Nucleotide
489 C⬎T
(His155Tyr)*
Nucleotide
946 G⬎A
(Arg307Gln)†
Nucleotide
1096 C⬎G
(Thr357Ser)‡
Nucleotide
1513 A⬎C
(Glu496Ala)‡
Nucleotide
1729 T⬎A
(Ile568Asn)†
S4
S5
S6
S8
S10
S12
S17
S19
S22
T/T
T/T
C/C
C/T
C/T
C/T
C/C
C/T
C/C
G/G
G/G
G/G
G/G
G/G
G/G
G/G
G/G
G/G
C/C
C/G
C/C
C/G
C/C
C/C
C/C
C/C
C/C
A/A
A/A
A/C
A/C
A/A
A/C
A/A
A/A
A/A
T/T
T/T
T/T
T/T
T/T
T/A
T/T
T/T
T/T
* The effect of the His155Tyr substitution is controversial: Shemon et al reported this mutation as
nonfunctional (27), whereas Cabrini et al proposed that it increases P2X7 receptor activity (41).
† Arg307Gln and Ile568Asn substitutions have been reported to confer complete loss of function also in
heterozygous dosage (27).
‡ The most common substitutions, at amino acid residues 357 and 496 of P2X7, have been demonstrated
to induce complete loss of receptor function when present in homozygous dosage or in combination with
another loss-of-function polymorphism (27).
responders and incomplete responders or nonresponders. ATP stimulation of monocytes from healthy
donors results in caspase 1 activation and secretion
(21,25). In accordance with the low IL-1␤ secretion
induced by ATP in monocytes from patients with
systemic-onset JIA, caspase 1 activation or secretion was
undetectable (results not shown). We have recently
observed a severe impairment of the response to ATP in
patients carrying CIAS1 mutations (4). However, none
of our patients with systemic-onset JIA had mutations in
the CIAS1 gene (results not shown).
Reduced secretion of IL-1␤ in response to ATP
could be due to expression of nonfunctional variants of
the highly polymorphic P2X7 receptor (26). The P2X7
gene was analyzed in the 9 patients with systemic-onset
JIA represented in Figure 2C. Patient S8 expressed only
the 2 variants T357S and E496A, a combination found in
3% of healthy subjects and considered the most common
cause of near-absent P2X7 function in PBMCs (27)
(Table 2). Patient S12 was heterozygous for E496A (26)
and I568N (28), 2 mutations that, when associated, are
indicative of nonfunctional or low-functional P2X7 (27).
The other patients did not express variants known to
result in loss of function (27).
Effects of ongoing steroid treatment and disease
activity on IL-1␤ secretion after LPS and ATP stimulation. All 22 patients with systemic-onset JIA in the
responder analysis were treated with steroids, which are
known to down-regulate IL-1␤ (29,30), raising the possibility of interference of the therapy with IL-1␤ produc-
tion and secretion. Monocytes from these patients were
therefore compared with monocytes from 6 additional
patients with active systemic-onset JIA who had not
received steroids. Although there was variation in the
amount of proIL-1␤ constitutively produced by resting
monocytes from non–steroid-treated patients, LPS induced an increase in proIL-1␤ synthesis (Figure 2B) and
levels of IL-1␤ secretion (Figure 2C) to levels comparable with those observed in healthy individuals. In contrast, levels of ATP-induced IL-1␤ secretion were significantly lower than in healthy controls (Figure 2C),
indicating that, although steroid treatment may be involved in the low IL-1␤ production and secretion detected in patients with active systemic-onset JIA, it does
not explain the defective response to ATP stimulation.
Monocytes from the anakinra-treated patients
with systemic-onset JIA were also analyzed after 1 week
of treatment. Despite a significant improvement in
disease activity, spontaneous and induced IL-1␤ production and secretion did not change significantly (Figure
2C), with no evident differences between responders and
nonresponders (data not shown).
IL-1␤ secretion induced by different inflammatory stimuli in monocytes from patients with systemiconset JIA. The kinetics of IL-1␤ secretion and the
response to different pathogen-associated molecular
patterns were investigated in 7 patients with active
systemic-onset JIA and 12 healthy controls. As shown in
Figure 3A, 24 hours after plating in the presence of the
TLR-4 ligand LPS, the levels of IL-1␤ secreted by
JIA SUBSETS DISTINGUISHED BY RESPONSE TO ANTI–IL-1
Figure 3. Comparison of the secretion of IL-1␤, IL-18, and IL-1
receptor antagonist (IL-1Ra) by monocytes from healthy controls
(open bars) and patients with systemic-onset JIA (shaded bars). A,
IL-1␤ detected in supernatants from monocytes cultured for 3 hours or
24 hours in the presence of zymosan (Zym), muramyldipeptide
(MDP), Staphylococcus aureus (Staph), phorbol myristate acetate and
ionomycin (PMA/Iono), or LPS. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01 versus
controls, by Mann-Whitney U test. B, IL-18 detected in supernatants
from monocytes cultured for 24 hours in the absence (medium) or
presence of 1 ␮g/ml LPS. C, IL-1Ra detected by enzyme-linked
immunosorbent assay in supernatants from monocytes cultured for 24
hours in the absence or presence of 1 ␮g/ml LPS. No significant
differences in levels of IL-18 or IL-1Ra between patients and controls
were observed. Data are presented as box blots, where the boxes
represent the 25th to 75th percentiles, the lines within the boxes
represent the median, and the lines outside the boxes represent the
highest and lowest values. See Figure 2 for other definitions.
monocytes from patients with systemic-onset JIA before
anakinra treatment were still lower than those detected
in normal individuals, confirming the observations made
at 3 hours. Similarly, the levels of IL-1␤ secretion
induced on monocytes from the same JIA patients by
muramyldipeptide (a ligand for NOD-2), S aureus, and
zymosan (both more active on TLR-2) (31,32) were
consistently comparable with or lower than those in
healthy controls, after either 3 hours or 24 hours of
exposure. The effect of PMA plus ionomycin, which was
previously shown to trigger IL-1␤ secretion by PBMCs
from patients with systemic-onset JIA (9), was also
1511
investigated. In the present investigation, no increased
secretion of IL-1␤ by either monocytes (Figure 3A) or
PBMCs (data not shown) from the 7 JIA patients
studied was observed after PMA/ionomycin treatment.
Secretion of IL-18 and IL-1Ra by monocytes from
patients with systemic-onset JIA in levels comparable
with those in healthy controls. Unlike IL-1␤, IL-18 is
constitutively produced by monocytes. However, like
IL-1␤, IL-18 is processed by caspase 1, and its secretion
requires LPS priming (33). The secretion of IL-18 by
monocytes from patients with systemic-onset JIA, without stimulation or activated with LPS, was investigated.
IL-18 was barely detectable after 3 hours of exposure to
LPS, in monocytes from both healthy controls and
patients (data not shown). Detectable amounts of the
secreted mature cytokine were found after longer exposure to LPS (24 hours) and were comparable with or
even lower than those in healthy subjects (Figure 3B).
Activated human monocytes simultaneously synthesize both IL-1␤ and its specific inhibitor IL-1Ra (34).
Therefore, we wished to investigate whether the good
clinical response to anakinra among patients in the
responder group could be due to defective production of
IL-1Ra (rather than to increased release of IL-1␤) by
monocytes from these patients. However, the levels of
IL-1Ra secreted by monocytes from the JIA patients
before and after stimulation with LPS were comparable
with those observed in healthy controls (Figure 3C).
Analysis of clinical and laboratory parameters to
characterize the complete responder subset of systemiconset JIA. To try to identify parameters that could
distinguish complete responders from incomplete
responders/nonresponders to anakinra, several baseline
clinical and biologic features were investigated. Among
the clinical variables, complete responders had a significantly lower number of actively involved joints (median
3.5 [range 1–10]) compared with incomplete responders
and nonresponders (median 7 [range 3–55]) (P ⫽ 0.02).
Conversely, no significant differences in systemic features, i.e., presence of fever, rash, hepatosplenomegaly,
and serositis, were observed.
Among laboratory parameters measured at baseline, no differences were observed in levels of acutephase reactants (CRP, ESR, fibrinogen, ferritin) or
hemoglobin between the 2 groups. Conversely, complete
responders had a significantly higher number of circulating neutrophils (median 19.3 ⫻ 103/mm3 [range 6.1–
30.9]) compared with incomplete responders and nonresponders (9.1 ⫻ 103/mm3 [range 7.3–19.7]) (P ⫽ 0.02).
This difference was not related to ongoing steroid
treatment since there were no significant differences in
1512
GATTORNO ET AL
Figure 4. Levels of serum cytokines and other soluble molecules in patients with systemic-onset juvenile idiopathic arthritis who were designated
good responders to anakinra (n ⫽ 7) (shaded bars) and those designated incomplete responders or nonresponders (n ⫽ 9) (open bars). Values were
determined at baseline (pre) and after 1 week of treatment (post). Results are expressed as the mean and SD pg/ml (logarithmic scale). ⴱ ⫽ P ⬍
0.05 versus pretreatment level, by Wilcoxon’s nonparametric matched pairs test. P values of 0.04 and 0.02 shown on the figure (determined by
Mann-Whitney U test) refer to the difference between the group of responders and the group of incomplete responders or nonresponders. IL-1Ra ⫽
interleukin-1 receptor antagonist; GM-CSF ⫽ granulocyte–macrophage colony-stimulating factor; G-CSF ⫽ granulocyte colony-stimulating factor;
IP-10 ⫽ interferon-inducible protein 10; IFN␥ ⫽ interferon-␥; VEGF ⫽ vascular endothelial growth factor; TNF␣ ⫽ tumor necrosis factor ␣.
the daily dosage of steroids between the group of
complete responders (median 0.5 mg/kg [range 0.2–3])
and the incomplete responders and nonresponders (median 0.5 mg/kg [range 0.1–2.5]) (Table 1).
As noted above, no significant differences in the
amounts of secreted IL-1␤ and IL-18 were observed
between responders (patients S4, S5, S6, S8, and S10)
and incomplete responders/nonresponders (patients
S12, S17, S19, and S22).
With the aim of identifying possible serum markers, 30 different soluble molecules in sera from 16
patients with systemic-onset JIA (7 responders and 9
incomplete responders or nonresponders), collected the
day before the beginning of treatment, were analyzed
and compared with those in 10 age-matched healthy
controls (details available from the authors upon request). Levels of a number of soluble mediators were
found to be increased in serum from patients with active
systemic-onset JIA. Most of the soluble mediators displaying the highest degree of difference in comparison
with healthy controls (P ⬍ 0.0001 or P ⬍ 0.001) were
IL-1–related cytokines (IL-18, IL-6, GM-CSF, G-CSF,
PTX3, and soluble IL-1Ra). High levels of IL-17 were
also detected (P ⬍ 0.001). Levels of IL-10, IL-7, IP-10,
IFN␥, VEGF, IL-4, TNF␣, IL-12, and IL-13 were modestly, but significantly, elevated (P ⬍ 0.01 or P ⬍ 0.05).
Serum IL-1␤ was detectable in only 4 patients with
systemic-onset JIA.
The multi-analysis of the soluble molecules at
baseline in responders and incomplete responders/
nonresponders did not lead to identification of a specific
serologic pattern distinguishing the 2 subgroups of patients. However, the group of patients with an incomplete response or nonresponse to anakinra exhibited
increased levels of G-CSF (P ⫽ 0.04) and undetectable
levels of IL-9, the latter of which were increased in
patients with complete response to anti–IL-1 treatment
(P ⫽ 0.02) (Figure 4).
Serum levels of the different soluble mediators
were also analyzed after 1 week of treatment. Among
the soluble molecules that were overexpressed before
anakinra treatment, a significant down-modulation after
1 week of treatment was observed for IL-6 only (mean ⫾
SD serum concentration after treatment 79.9 ⫾ 66.8
pg/ml; P ⫽ 0.009). When responders and nonresponders
were analyzed separately, significant decreases in the
levels of IL-6 (P ⫽ 0.02) and GM-CSF (P ⬍ 0.05) were
observed in the responder group only, with no significant
decreases in levels of the other soluble molecules.
Conversely, increased levels of soluble IL-1Ra (likely
due to the ongoing treatment) were observed in both
responders and nonresponders (P ⫽ 0.02) (Figure 4).
DISCUSSION
Results of the present study show that systemiconset JIA can be divided into 2 subsets according to the
JIA SUBSETS DISTINGUISHED BY RESPONSE TO ANTI–IL-1
type of response to anakinra. One subset, accounting for
⬃40% of patients, had a dramatic and persistent response to IL-1 blockade, which allowed the rapid discontinuation of all other treatments. In the other group
of patients, the treatment, although showing an effect on
systemic manifestations, did not control arthritis and
inflammation and was either withdrawn or continued in
combination with second-line agents or steroids. These
findings support the hypothesis that systemic-onset JIA
is a heterogeneous condition, as also indicated by
marked differences in outcome (11). Indeed, in approximately half of patients the disease is characterized by a
monocyclic or intermittent course, in which arthritis
tends to remit when systemic features are controlled. In
the other half, the disease follows an unremitting course,
and chronic arthritis often leads to joint damage.
The proportion of patients who were responders
to anakinra in the present study was lower than that
observed in a previous investigation (9). This discrepancy may be related to differences in patient selection.
In fact, in the present study we found that complete
responders had a higher absolute neutrophil count and a
lower number of active joints at baseline compared with
patients who were incomplete responders or nonresponders. In the study by Pascual et al (9), however, 8 of
9 patients with systemic-onset JIA who showed good
response to anakinra also had mild articular involvement, with a mean number of active joints and total
white blood cell count that were very similar to those
observed in our group of responder patients.
Only minor differences between the 2 response
groups were observed when serum levels of 30 different
cytokines and other soluble molecules were analyzed in
this study. Consistently, lower levels of G-CSF were
found in responders, in association with higher neutrophil counts. This apparent discrepancy may relate to a
negative feedback mechanism. Indeed, G-CSF receptor
expressed by neutrophils has been found to mediate
uptake and clearance of G-CSF (35), indicating that
plasma levels of the growth factor are related to the
number of neutrophils.
Anakinra is highly effective in the treatment of
the cryopyrinopathies (1–4,36), which are characterized
by dramatically increased secretion of IL-1␤ (4–7),
raising the possibility that patients with systemic-onset
JIA who are complete responders to anakinra have
similar alterations in IL-1␤ secretion despite the absence
of mutations in cryopyrin. This hypothesis was, however,
ruled out by our data showing that in vitro IL-1␤
secretion by monocytes from patients with systemiconset JIA was comparable with or even lower than that
1513
by monocytes from healthy controls. This low level of
secretion was independent of the clinical response to
anakinra as well as of the pathogen-associated molecular
patterns used to induce IL-1␤ secretion.
The low levels of in vitro IL-1␤ release detected
in patients with systemic-onset JIA could be due to
ongoing steroid treatment (29,30). Monocytes from a
number of patients with systemic-onset JIA who had not
received steroids secreted IL-1␤ in levels comparable
with those in controls. Interestingly, however, IL-1␤
secretion in response to exogenous ATP was defective in
patients with systemic-onset JIA regardless of whether
they had taken steroids, suggesting a potential alteration
of ATP signaling. Several nonfunctional variants of the
ATP membrane receptor P2X7 have been described
(27) and might account for the low degree of ATPinduced IL-1␤ secretion observed in patients with
systemic-onset JIA. However, variants of the gene that
are possibly associated with abnormal function (27) were
expressed by only 2 of 9 patients. This suggests that, even
if mutations of P2X7 may have a role in the decreased
responsiveness to ATP in some patients with systemiconset JIA, in most patients P2X7 polymorphism is not
responsible for the low IL-1␤ secretion observed in vitro
following exposure to exogenous ATP. Thus, the mechanism underlying this resistance to ATP is as yet unelucidated and warrants further investigation.
Our data differ from those reported by Pascual et
al, who found that blood cells from patients with
systemic-onset JIA secreted more IL-1␤ than those from
healthy individuals (9). The high variability of the disease, together with the different methods of cell activation, may account in part for this discrepancy. In our
group of patients, PMA/ionomycin-induced secretion
(by either PBMCs or enriched monocytes) was comparable with or lower than that observed in healthy controls. Similarly, IL-1␤ secretion was consistently not
increased in systemic-onset JIA patient monocytes compared with control monocytes after stimulation with
different pathogen-associated molecular patterns that
trigger different pathogen-sensing receptors (31,32).
This suggests that monocytes from patients with
systemic-onset JIA are resistant to induction of IL-1␤
secretion in vitro, independent of the stimulus used and
of the support of other cell types.
The finding that patient monocytes did not show
increased IL-1␤ secretion in vitro was unexpected in a
disease with many IL-1–related symptoms. A possible
explanation may be that in systemic-onset JIA, unlike in
diseases in which there is a mutated cryopyrin, the
increase in IL-1␤ secretion occurs at privileged sites,
1514
GATTORNO ET AL
possibly controlled by the local microenvironment. Similarly, IL-18, one of the inflammation-related cytokines
that is significantly increased in sera from patients with
systemic-onset JIA (37,38), was secreted in vitro by
monocytes from patients in both systemic-onset JIA
subgroups (responders and nonresponders) at levels
falling into the range observed in healthy controls. Thus,
there is also an apparent discrepancy between in vivo
and in vitro production of IL-18. Interestingly, IL-18
overexpression has been observed in the bone marrow of
a patient with systemic-onset JIA who had MAS, suggesting that bone marrow may be the source not only of
increased serum IL-18 but also of other proinflammatory cytokines (39). In any case, our data showing
efficacy of anakinra treatment in spite of low levels of
IL-1␤ reinforce the concept that in cytokine-mediated
diseases, causation may be established by observing the
results of specific receptor blockade or specific cytokine
neutralization, rather than by elevated levels of the
cytokine (40).
In conclusion, we have shown that a subset of
patients with systemic-onset JIA has a dramatic response
to anakinra. This finding suggests a major role of IL-1 in
this disease subset and provides for the first time a
parameter with which to investigate the heterogeneity of
systemic-onset JIA. The 2 response subsets are characterized by some distinct clinical features. In vitro secretion of IL-1␤ and IL-18 by monocytes from patients with
systemic-onset JIA is not increased and is independent
of both treatment outcome and disease activity.
AUTHOR CONTRIBUTIONS
Dr. Gattorno had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
data analysis.
Study design. Gattorno, Martini, Rubartelli.
Acquisition of data. Piccini, Lasigliè, Tassi, Brisca, Carta, Delfino,
Ferlito, Pelagatti, Caroli, Buoncompagni, Viola, Loy, Sironi, Vecchi,
Ravelli,
Analysis and interpretation of data. Gattorno, Piccini, Buoncompagni,
Viola, Loy, Vecchi, Ravelli,
Manuscript preparation. Gattorno, Martini, Rubartelli.
Statistical analysis. Gattorno.
4.
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18.
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