Ann Rheum Dis 2011;70:747–754. doi:10.1136/ard.2010.134254747
Accepted 11 November 2010
Published Online First
20 December 2010
Objectives To assess the effi cacy of the interleukin 1
receptor antagonist anakinra in systemic-onset juvenile
idiopathic arthritis (SJIA).
Methods A multicentre, randomised, double-blind,
placebo-controlled trial was conducted. The primary
objective was to compare the effi cacy of a 1-month
treatment with anakinra (2 mg/kg subcutaneous daily,
maximum 100 mg) with a placebo between two groups
each with 12 patients with SJIA. Response was defi ned
by a 30% improvement of the paediatric American
College of Rheumatology criteria for JIA, resolution of
systemic symptoms and a decrease of at least 50% of
both C-reactive protein and erythrocyte sedimentation
rate compared with baseline. After month 1 (M1),
patients taking placebo were switched to anakinra.
Secondary objectives included tolerance and effi cacy
assessment for 12 months, and analyses of treatment
effect on blood gene expression profi ling.
Results At M1, 8/12 responders were receiving anakinra
and 1 responder receiving placebo (p=0.003). Ten
patients from the placebo group switched to anakinra;
nine were responders at M2. Between M1 and M12, six
patients stopped treatment owing to an adverse event
(n=2), lack of effi cacy (n=2) or a disease fl are (n=2).
Blood gene expression profi ling at enrolment and at 6
months’ follow-up showed one set of dysregulated genes
that reverted to normal values in the clinical responders
and a different set, including interferon (IFN)-inducible
genes, that was induced by anakinra.
Conclusions Anakinra treatment is effective in SJIA, at
least in the short term. It is associated with normalisation
of blood gene expression profi les in clinical responders
and induces a de novo IFN signature.
Trial Registration Number: NCT00339157.
Systemic-onset juvenile idiopathic arthritis (SJIA)
is a disease of unknown aetiology characterised
by arthritis and systemic symptoms starting before
the age of 16. 1 The most characteristic feature at
onset is spiking fever, which is often associated
with an evanescent skin rash, hepatosplenomegaly
and pericarditis. Non-steroidal anti-infl ammatory
drugs, corticosteroids, methotrexate and antitu-
mour necrosis factor (anti-TNF) agents are often
only partially effective. 2 3 The anti-interleukin
6 receptor antagonist tocilizumab was effective in
one randomised withdrawal trial. 4 Patients with a
persistently systematically active course 6 months
after disease onset, while receiving corticosteroid
treatment, are at high risk of longlasting, non-
remitting or polycyclic disease. 5
Interleukin 1 (IL-1) has an important role in SJIA.
In particular, serum samples from patients with SJIA
induce IL-1β transcription on healthy peripheral
blood mononuclear cells (PBMCs), and treatment
with the IL-1 receptor antagonist (IL-1Ra) anakinra
leads to the normalisation of a disease-specifi c gene
expression profi le. 6 7 Non-controlled pilot studies
provide evidence of dramatic, quick responses to
anakinra in a subgroup of patients with SJIA. 6 8 – 10
However, some patients may have a self-remitting
course, and no placebo-controlled studies have been
published to evaluate the effects of IL-1 blockade.
We therefore designed a trial aiming at assess-
ing the effi cacy of anakinra treatment, and its effect
on blood gene expression profi ling, in patients with
SJIA displaying active systemic disease despite cor-
PATIENTS AND METHODS
This was a multicentre trial including six centres.
Inclusion criteria were age 2–20 years, a diagno-
sis of SJIA, 1 more than 6 months’ disease duration,
active systemic disease (disease-related fever and/
or C-reactive protein (CRP) >20 mg/l and/or fi rst
hour erythrocyte sedimentation rate (ESR) >20)
and signifi cant overall disease activity at day 1 (D1)
(at least three of the following criteria: (1) physi-
cian global assessment of disease activity ≥20/100;
(2) parent/patient assessment of disease effect on
overall wellbeing ≥20/100; (3) Childhood Health
Assessment Questionnaire score ≥0.375/3; (4) ≥2
joints with active arthritis; (5) ≥2 joints with non-
irreversible limited range of motion and (6) ESR ≥30)
despite oral prednisone or prednisolone ≥0.3 mg/kg
or 10 mg/day (whichever was lower). Female sub-
jects entering the study were prepubescent, sexually
inactive or required to use effective contraception.
Exclusion criteria included previous treatment with
an IL-1 inhibitor or any condition contraindicat-
ing immunosuppressive treatment. Intravenous or
intra-articular steroids, immunosuppressive drugs
▶ Additional data
(supplementary tables and
fi gures) are published online
only. To view these fi les please
visit the journal online at ( http://
1 Université Paris-Descartes
and Hôpital Necker-Enfants
Malades, Assistance Publique
Hôpitaux de Paris, Paris, France
2 Baylor Institute for Immunology
Research, Dallas, Texas, USA
3 Hôpital Femme-Mère-Enfant,
4 Hôpital Pellegrin Enfants,
5 Hôpital Hôtel Dieu, Assistance
Publique Hôpitaux de Paris,
6 University of Leuven, Leuven,
7 Hôpital Xavier Bichat,
Assistance Publique Hôpitaux
de Paris, Paris, France
8 Université Lyon Sud, Lyon,
9 INSERM Unit 899, Dallas,
10 Mount Sinai School of
Medicine, New York, New York,
Dr Pierre Quartier, Unite
Malades, 149 rue de Sevres 75
015 Paris, France;
PL and VP contributed equally to
A multicentre, randomised, double-blind,
placebo-controlled trial with the interleukin-1 receptor
antagonist anakinra in patients with systemic-onset
juvenile idiopathic arthritis (ANAJIS trial)
Pierre Quartier, 1 Florence Allantaz, 2 Rolando Cimaz, 3 Pascal Pillet, 4 Claude Messiaen, 1
Christophe Bardin, 5 Xavier Bossuyt, 6 Anne Boutten, 7 Jacques Bienvenu, 8
Agnes Duquesne, 3 Olivier Richer, 4 Damien Chaussabel, 2 Agnes Mogenet, 1
Jacques Banchereau, 2,9,10 Jean-Marc Treluyer, 1 Paul Landais, 1 Virginia Pascual 2
Ann Rheum Dis 2011;70:747–754. doi:10.1136/ard.2010.134254748
Gene expression profi ling and cytokine measurements
Blood samples were collected in Tempus tubes (ABI, Foster City,
California, USA) from the patients at D1, M1 and M6 and from
age and gender-matched healthy controls.
Transcriptional module-based analysis
This mining strategy has been described in detail elsewhere. 14 In
this study, RefSeq IDs were used to match probes between the
Affymetrix U133 platform, which was used to generate the orig-
inal set of modules using PBMC expression data, 14 and Illumina
Hu6 platform, which was used to hybridise whole blood sam-
ples from patients enrolled in this study and healthy controls.
Unambiguous matches in Illumina were found for 2109 out of
the 5348 Affymetrix probe sets. Illumina modules containing
<10 genes were not included in the analysis.
Cytokine multiplex analysis
Serum samples were analysed using the cytokine assay kit (Bio-
Rad, Hercules, California, USA) according to the manufacturer’s
Assessment, outcome and safety measures
The primary objective was to compare the effi cacy after
1 month’s treatment with anakinra (2 mg/kg subcutaneously daily,
maximum 100 mg) or placebo in the two groups of patients. To
be responders to a modifi ed American College of Rheumatology
Pediatric (ACRpedi) 30 score built for the purpose of the trial,
patients had to fulfi l the three following conditions: (1) ACRpedi
30 response 15 ; (2) absence of disease-related fever (body tempera-
ture <38°C over the past 8 days) and (3) 50% decrease compared
with D1 or normalisation of both CRP and ESR values.
Modifi ed ACRpedi 30, 50, 70 and 100 responses, assessed
throughout the study, included an improvement of 30%, 50%,
70% or more and 100% respectively, in at least three of the six
core criteria for juvenile rheumatoid arthritis and a worsening of
30 or more in no more than one of the criteria. The criteria were
the physician’s global assessment of disease activity and the
patient’s or the parents’ global assessment of overall wellbeing,
the number of joints with active arthritis, the number of joints
with limited range of motion, the Childhood Health Assessment
Questionnaire and ESR. 15 A disease fl are was defi ned as either
(1) a reoccurrence of disease-related fever or any systemic symp-
tom, or (2) an increase by twofold of either the ESR or CRP value,
or (3) a worsening of ≥30% in at least three of the six ACRpedi
core criteria and an improvement of ≥30% in no more than one
of the criteria. If the number of joints with active arthritis was
used as a criterion of fl are and the patient initially had no active
joints or only one active joint, an increase in the number of joints
with active arthritis to at least two was required.
We also aimed to assess the number of patients who reached
M6 with inactive disease, as defi ned by Wallace et al 16 under a
daily dose of predniso(lo)ne <0.3 mg/kg or 10 mg, whichever
Adverse events (AEs) were recorded throughout the study.
Specifi c procedures were set up for serious AEs (SAEs). This
trial was registered at http://www.clinicaltrials.gov (registration
We expected at least 60% difference in the percentage of
patients obtaining improvement in the anakinra-treated group
(group 1) compared with the control group (group 2), with no
and disease- modifying antirheumatic drugs (DMARDs) had to be
stopped at least 1 month before study onset or for longer periods
of time depending on their half-life. All patients entering the study,
and their parents for patients aged <18, gave written informed
The study was approved by the local independent ethics com-
mittee and consisted of two parts ( fi gure 1 ). Part 1 was a ran-
domised, double-blind, placebo-controlled phase. At D1, eligible
patients were randomised to receive either anakinra or placebo
(1:1) from D1 to month 1 (M1) using a computer-generated ran-
dom list. Patients were stratifi ed by centres and randomisation
was balanced across treatments and centres. The randomisation
information included the randomisation number, the centre, the
assigned treatment and the date of randomisation. Investigators,
other caregivers, the patients and their parents remained blinded
to the assigned treatment. The primary objective was to demon-
strate a higher proportion of responders in group 1 than group 2.
No immunosuppressive drugs or DMARDs were allowed dur-
ing the trial. Non-steroidal anti-infl ammatory drugs and corti-
costeroids had to be taken at stable dosage for 1 month before
D1 and until M1.
Part 2 was an open-label treatment period: all patients received
anakinra after M1. Tapering the dose of corticosteroids was
allowed after the M1 visit (reduction of 0.4–0.5 mg/kg monthly for
daily doses of ≥1.5 mg/kg, 0.3–0.4 mg/kg for doses between 1 and
1.5 mg/kg, 0.2–0.3 mg/kg between 0.6 and 1 mg/kg, 0.1–0.2 mg/kg
between 0.3 and 0.6 mg/kg, ≤0.10 mg/kg for doses <0.3 mg/kg).
Measurement of serum amyloid A (SAA) and ferritin levels
and the percentage of glycosylated ferritin were performed at
D1, M1 and M6. Total ferritin concentration was measured on
a Dimension RXL HM according to the guidelines of the manu-
facturer (Dade Behring, Paris, France). Glycosylated ferritin was
determined according to the method of Worwood et al , 11 with
minor modifi cations as previously described. 12 Pharmacokinetic
(PK) analyses were performed on blood taken at M2 and M6.
Concentrations of anakinra in plasma samples were determined
using the antibody (Ab) ELISA purchased from R&D Systems
(Minneapolis, Minnesota, USA).
Patients who were naive from anti-pneumococcal immunisa-
tion received Pneumo23 immunisation at D1 in order to assess
at M1 and M12 the effect of anakinra treatment on anti-pneu-
mococcal Ab response to fi ve capsular polysaccharides. 13
Figure 1 Study design. *Measurement of serum amyloid A and
ferritin levels, assessment of the percentage of glycosylated ferritin,
gene expression profi ling analysis and cytokine measurements.
†Measurement of the concentration of anakinra in plasma
(pharmacokinetic analyses). ‡Measurement of serum anti-
pneumococcal antibodies. D, day; M, month.
Ann Rheum Dis 2011;70:747–754. doi:10.1136/ard.2010.134254 749
Randomised, double-blind, placebo-controlled phase
Response to the primary objective and assessment of individual
Eight of 12 patients (67%) in group 1 and only 1 of 12 (8%) in
group 2 were responders at M1 (p=0.003), therefore the primary
objective of the trial was met.
Two patients from the control group stopped treatment after
5 and 11 days, respectively, owing to pain from injections and
were withdrawn from the trial after the M1 visit; one of them, a
child who presented a marked disease fl are at D1, was the only
responder at M1 in the control group.
There was a signifi cant difference in favour of group 1 in the
number of joints with active disease, physician general assess-
ment of disease activity, CRP, ESR and SAA values at M1 com-
pared with D1 ( table 2 ).
A univariate analysis to explore whether any covariable might
be associated with the response at M1 according to the treatment
more than 10% patients improving in group 2. Given a 5% type
I error, a 20% type II error and a two-sided Fisher exact test, 12
patients per group were required. An intention-to-treat analysis
To explore whether each variable from the ACRpedi score,
CRP, SAA and/or parent/patient assessment of pain were
associated with response to treatment, the ratio (value at
inclusion − value at M1)/value at inclusion, was compared in
both groups. Qualitative and quantitative data were compared
using Wilcoxon test and Fisher exact test, respectively. The
R statistical software was used for statistical analysis.
Demographic and baseline characteristics
Twenty-seven patients were screened for entry into the study;
24 were eligible for randomisation ( fi gure 2A ). Demographic
and disease characteristics are summarised in table 1 .
Figure 2 Patients’ disposition. (A) Randomised placebo-controlled, double-blind trial (until M1). (B) Open-labelled phase (from M1 to M12). Arthritis
activity leading to treatment withdrawal (= two patients withdrawn for a disease fl are-up, at M2 and M3, respectively, and two patients withdrawn
for a lack of response, at M4 and M5, respectively). *Two patients from the control group stopped treatment after 5 and 11 days, respectively, owing
to pain from injections and were withdrawn from the trial after the M1 visit. †Cutaneous and digestive symptoms leading to the diagnosis of Crohn’s
disease shortly after M2. ‡Increase of serum transaminases over fi ve times the upper limit of normal at M6. AE, adverse event; JIA, juvenile idiopathic
arthritis; M, month; SAE, serious adverse event.
Ann Rheum Dis 2011;70:747–754. doi:10.1136/ard.2010.134254750
CD163 and several members of the S100 family). Downregulated
transcripts encode proteins involved in antigen presentation,
lymphocyte lineage and signalling, as well as RNA processing/
splicing and DNA modifi cation (supplementary fi gure S1B and
table S2). Cytokine transcripts encoding IL1-β and IL-6 were not
differentially expressed, but oncostatin (OSM) and the nega-
tive regulator of cytokine signalling 3 (SOCS3) transcripts were
Changes in blood transcriptional signatures after 1 month
At M1, 522 transcripts were signifi cantly changed in patients
treated with anakinra but not in the placebo-treated group (sup-
plementary table S3). We found a signifi cant upregulation of the
purinergic receptor P2RX7, an ATP-gated ion channel involved
in IL-1β processing and secretion, 17 HLA-class II genes, the pro-
teasome activator PSME2 and transcripts involved in interferon
(IFN) signalling (SOCS1, STAT1 and STAT2). IL-1B was among
the most signifi cantly downregulated transcripts.
Module-level analysis of the SJIA transcriptional signature to follow
response to anakinra treatment
A recently described analysis algorithm 14 that permits com-
parison of the expression of 28 groups of transcriptionally co-
regulated genes (modules) in patients and controls was applied
to the blood expression profi les ( fi gure 3 ). At D1, patients from
both groups displayed fairly homogeneous modular expres-
sion patterns with signifi cant upregulation of innate immunity
and underexpression of adaptive immunity modules. Modules
group failed to fi nd any association. A multivariate analysis was
not performed given the restricted number of patients.
Safety and tolerability
Fourteen AEs were recorded in group 1 and 13 in group 2
( table 3 ). There were no SAEs.
Anti-pneumococcal antibody response
At M1, the levels of postvaccination Abs against fi ve pneumo-
coccal capsular polysaccharide serotypes were not signifi cantly
different between the two groups. In group 1, 11 patients
adequately responded to all serotypes and the 12th patient
had a normal response to three of the fi ve serotypes (online
supplementary table S1).
Gene expression profi ling analyses
Samples from 21 patients were included in these analyses, as
blood from two patients was not properly collected and one
patient was eventually diagnosed with Crohn’s disease.
Baseline SJIA blood gene expression profi ling
Analyses of blood from 21 patients with SJIA and 21 controls
at D1 showed the differential expression of 986 transcripts
(supplementary fi gure S1A and table S2). Among those, some of
the most highly upregulated encode proteins involved in innate
immunity activation, including (1) signalling components of the
IL-1, IL-18 and Toll-like receptors (IL-1R2, IL-18RAP, IRAK3,
TLR5, PGLYRP1), (2) infl ammasome (CARD12/IPAF) and (3)
anti-bacterial defence/infl ammation (DEFA1, BPI, MPO ELA2,
Table 1 Patients characteristics at study treatment onset
Female, n (%)
Age, mean value, years (SD)
Disease mean duration, years (SD)
Fever (>38°C), no. of patients (%)
CRP, mg/l (n≤6), mean value (SD)
ESR fi rst hour (n≤10), mean value (SD)
SAA, mg/l (n≤6.4), mean value (SD)
High serum ferritin*, no. of patients
Active joints, mean no. (SD)
Joints with LOM, mean no. (SD)
Physician’s VAS, mean value (SD)
Parent’s global VAS, mean value (SD)
Parent’s pain VAS, mean value (SD)
CHAQ, mean value (SD)
Treatment with steroids (predniso(lo)ne)
Duration, mean, years (SD)
Daily dose, mean, mg/kg (SD)
Previous treatments with DMARDs, biological agents
DMARD and/or biological agent, no. of patients
DMARD, no biological agent, no. of patients
DMARD and biological agent, no. of patients
Methotrexate, no. of patients
Etanercept, no. of patients
Others, no. of patients (no. of DMARDs)
Anakinra (n = 12) Placebo (n = 12) All patients (n = 24)
*Ferritin level was highly variable and it was elevated (>100 µg/l in patients <13 years, >200 in female patients >13 years and
>300 in male patients >13 years) in only fi ve patients (range 347–3135 µg/l), with low glycosylated ferritin (<40%) in these fi ve
patients (range 14–30%).
†Thalidomide (n=2), tocilizumab (n=2, one single infusion, phase II trial), azathioprine (n=1), ciclosporin (n=1), lefl unomide (n=1).
‡Azathioprine (n=2), thalidomide (n=1), tocilizumab (n=1, one single infusion, phase II trial), ciclosporin (n=1), intravenous
CHAQ, Childhood Health Assessment Questionnaire (0–3); CRP, C-reactive protein; DMARDs, disease-modifying antirheumatic drugs;
ESR, erythrocyte sedimentation rate; LOM, joints with limitation of passive motion; SAA, serum amyloid A; VAS, visual assessment
(0–100 mm scale) of disease activity by the physician, disease effect on overall wellbeing and pain by the parents.
Ann Rheum Dis 2011;70:747–754. doi:10.1136/ard.2010.134254 751
responded to treatment ( fi gure 3 ). Of the four non-responders,
one patient was later diagnosed with Crohn’s disease and blood
from another patient was not properly collected; the two other
patients did not show dysregulation of the modules at baseline
and remained the same at M1. The modular alterations corrected
in responders include transcripts within the platelet (M1.2), cyto-
toxic (M2.1) and erythropoiesis (M2.3) modules. None of these
modules changed in the placebo group, therefore supporting the
including myeloid linage (M1.5 and M2.6), neutrophil (M2.2)
and infl ammation (M3.2 and 3.3) related transcripts were
upregulated. Modules associated with erythrocytes (M2.3)
and platelets (M1.2) were also upregulated. Conversely, mod-
ules containing transcripts related to lymphoid/T-cell lineage
(M2.8 and M3.8), B cells (M1.3), T cells (M1.4) and cytotoxic
cells (M2.1) were downregulated. At M1, several modular
alterations were corrected in the anakinra-treated patients who
Table 3 Adverse events (AEs)
Number of patients (patient-years*)
Any AE, no. (/patient-year)†
Serious AE, no. (/patient-year)
Pain to injection, no. (/patient-year)
Post-injection erythema, no.
Infections, no. (/patient-year)
ENT infections and laryngitis, no.
Skin infections, no.
Other infections, no.
Vomiting, abdominal pain, no.
Other AE¶, no. (/patient-year)
Part A (double-blind)
Part 2 (M1–12) (open label)
*Patient-years = 12 patients in each group followed up for 1 month during the double-blind phase, 22 patients exposed to study treatment for a total of 182 months during the
open-label phase (eight patients were withdrawn from the trial between M1 and M6).
†Disease activity/fl ares was not systematically recorded as an AE.
‡Infections in four patients, vertebral collapse in one patient (these fi ve patients continued the trial), skin and digestive symptoms leading to the diagnosis of Crohn’s disease in one
§Varicella (n=3), vulvar candidiasis (n=2), isolated fever (n=2), atypical pneumonitis, urinary tract infection. Favourable outcome in all cases, no patient withdrawn from the trial.
¶Skin lesions (n=5), haematuria (n=2), back pain (n=2), dental fracture, asthenia, vertigo.
Table 2 Responses at month 1
Primary objective (modifi ed ACRpedi 30)†
Systemic symptoms responders†
Primary objectives used in other trials
ACRpedi 30 responders
ACRpedi 30 and no fever‡
ACRpedi 30, no fever and CRP <15 mg/l§
Modifi ed ACRpedi 50, 70 and 100 response†
Modifi ed ACRpedi 50 responders
Modifi ed ACRpedi 70 responders
Modifi ed ACRpedi 100 responders
Response to individual variables
Number of active joints
Number of joints with LOM
Physician’s disease activity assessment**
Parent/patient’s global assessment**
Parent/patient’s assessment of pain**
Anakinra (n = 12)
Number of responders (%)
Placebo (n = 12)
Mean variation from D1 to M1 (%)
*χ 2 Test.
†Body temperature <38°C for more than 7 days, CRP and ESR normalised or decreased by at least 50% (=systemic symptoms
responders) and also, in responders to the trial primary objective, ACRpedi 30, 50, 70 or 100 (whichever level is indicated) response
compared with D1.
‡Body temperature <38°C for more than 7 days and ACRpedi 30 response compared with D1.
§Body temperature <38°C for more than 7 days, CRP <15 mg/l and ACRpedi 30 response compared with D1 as in a recent trial with
the anti-interleukin 6 receptor antibody tocilizumab.
**Using a visual analogue scale (0–100 mm).
ACRpedi 30, American College of Rheumatology Pediatric 30 response; CHAQ, Childhood Health Assessment Questionnaire; CRP,
C-reactive protein; ESR, erythrocyte sedimentation rate; LOM, joints with limitation of passive motion; SAA, serum amyloid A.
Ann Rheum Dis 2011;70:747–754. doi:10.1136/ard.2010.134254752
The dose of predniso(lo)ne had been reduced after the M1
visit in accordance with the protocol recommendations in all
the responders who continued the trial (eight patients, all from
group 1) and in seven other patients (four patients from group 1
and three patients from group 2) who had shown some improve-
ment (investigator’s decision). Three responders from group 1 in
whom the dose of corticosteroids had been reduced were not
responders at M2 anymore.
The dose of corticosteroids was then either maintained stable
or reduced in accordance with the protocol recommendations at
each visit (supplementary table S4).
Seventeen patients continued the trial until M6. Their mean
daily dose of predniso(lo)ne was 0.18 mg/kg (median 0.16,
validity of the data at D1. We also observed the coordinated
upregulation of type I IFN-inducible transcripts (M3.1) in the
anakinra- but not in the placebo-treated group.
Twenty-two patients entered the second phase of the study
( fi gure 2B ). Nine out of 10 patients from group 2 who switched
to anakinra at M1 responded at M2. PK analyses revealed a trend
towards lower anakinra concentrations at M2 and M6 in patients
with lower weight and those who had failed to respond to anak-
inra after 1 month (residual anakinra concentration of 45.5 ± 51 ng/
ml (range 20–122) vs 136.5 ± 106 ng/ml (range 20–353) in respond-
ers), but overall this difference was not signifi cant.
Figure 3 Modular analysis. (A) Annotated module map of patients with systemic-onset juvenile idiopathic arthritis (SJIA) at D1, M1 and M6.
Expression levels in patients were compared with those of healthy controls. Spots indicate the proportion of genes that were signifi cantly changed
for each module (Mann–Whitney rank test, p<0.05). The black outline indicates the core signature common in the two groups of patients at D1. (B)
Forty-nine representative genes from the module 3.1 were rearranged by hierarchical clustering in order to reveal differential expression. Expression
values are normalised for each gene to the healthy group. Transformed expression levels are indicated by colour scale, with red representing relatively
high expression and blue indicating relatively low expression. (C) Levels of inducible proteins IP10 and TRAIL were measured in the serum of
patients with SJIA at D1 and M6. All results were analysed using a paired t-test. (D) SOCS3 expression was measured in whole blood of patients by
microarray at D1 and M6. Results were analysed using a paired t-test. D, day; M, month.
Ann Rheum Dis 2011;70:747–754. doi:10.1136/ard.2010.134254753
the study design that precluded the use of associated DMARDs
and allowed tapering of the corticosteroid dose at an early stage,
a strategy designed to minimise the risk of treatment-related
This study has limitations owing to the small number of
patients and the short duration of the double-blind phase. In
particular, we were unable to properly assess which patients’
characteristics might predict response to treatment. In recent
reports, the absence of small joint involvement, a smaller num-
ber of joints with active disease and an increased absolute
neutrophil count were reported to be predictive of response to
anakinra. 9 10
No severe infections occurred in this trial and anti-pneumo-
coccal Ab response was not affected by anakinra treatment.
However, severe infections have been reported under anakinra
in adults with rheumatoid arthritis. 19 One patient here was
diagnosed with infl ammatory bowel disease, a complication
reported in some patients initially diagnosed with JIA and treated
with anti-TNF agents. 3 20 21 An additional patient experienced a
sudden rise in serum transaminases. In order to draw defi nitive
conclusions about the safety of anakinra in SJIA, further studies
with larger numbers of patients and longer follow-up need to be
As expected, the ex vivo genomic analysis of patient blood at
D1 showed upregulation of transcripts related to innate immu-
nity and haematopoiesis (supplementary table S2). Although
several hereditary and sporadic IL-1β-mediated diseases involve
alterations in NLRP3 infl ammasome components, 22 no altered
expression of NLRP3 infl ammasome-related transcripts was
observed. However, two different fl agellin receptors (CARD12
and TLR5) were signifi cantly upregulated in the majority of
patients at D1 (supplementary table S2) and normalised in the
responder group at M6 (supplementary table S5). Whether
the triggering of fl agellin receptors is involved in the patho-
genesis of SJIA remains to be elucidated. Alterations in the
blood gene expression profi les of patients with SJIA at the time
of randomisation were highly reproducible in the treatment
and placebo groups. This was best visualised using a strategy
based on the analysis of modules composed of transcription-
ally co-regulated genes. 14 Longitudinal changes correlating
with disease resolution were found upon follow-up. Changes
probably induced by treatment, regardless of clinical response,
were also identifi ed. Strikingly, an upregulation of type I IFN-
regulated transcripts, which is not a feature of untreated SJIA, 7
23 – 25 was induced in the majority of anakinra-treated patients
at M1 and M6. Accordingly, two IFN-inducible proteins were
also signifi cantly upregulated in the serum samples of patients
at M6. The molecular and cellular basis for an IL-1 β/type I
IFN cross-regulation in SJIA remains unknown. Inhibition of
type I IFN signalling by IL-1β might occur through the induc-
tion of SOCS3, which is a negative regulator of IL-1-dependent
acute infl ammatory arthritis and osteoclast generation. 26 27
Conversely, type I IFN decreases IL-1β and induces IL-1Ra
production by PBMCs in vitro. 28 A similar cross-regulation
has been described for type I IFN and TNFα. Thus, patients
with SJIA treated with TNF antagonists develop a blood IFN
signature. 29 Furthermore, a signifi cant proportion of patients
with rheumatoid arthritis and Crohn’s disease treated with
TNF antagonists develop anti-nuclear Abs and some of them
display symptoms of systemic lupus erythematosus, a type I
IFN-mediated disease. 29 Whether the upregulation of type I
IFN-inducible genes in anakinra-treated patients could have
clinical consequences through the triggering of adaptive immu-
nity remains to be determined.
range 0–0.58); the six responders at M6 had a daily prednisone
dose of <10 mg or 0.3 mg/kg. These six patients, whose mean
predniso(lo)ne dose at enrolment was 0.51 mg/kg (SD 0.32),
were all responders to 1 month of anakinra treatment and had
already achieved ACRpedi 50 or 70 improvement at this stage.
Sixteen patients reached M12; among seven responders, six
had stopped corticosteroid treatment and fi ve of them had inac-
Safety and tolerability
Six patients developed a SAE. Four infections and one vertebral
collapse had a favourable outcome and these fi ve patients con-
tinued the trial; one patient was diagnosed with Crohn’s disease
shortly after M2 and withdrawn from the trial. This patient ini-
tially belong to group 1; he was a non-responder at M1; how-
ever, minor improvement had led the treating doctor to start
tapering the dose of prednisone at this stage.
Other AEs mainly consisted of non-severe injection-site reac-
tions and common infections ( table 3 ). One patient stopped anak-
inra owing to a sudden increase in serum transaminases at M6.
Anti-pneumococcal (Pneumo23) antibody response
At M12, 12 patients were tested and an adequate Ab response
was maintained in all cases (supplementary table S1).
Changes in gene expression profi ling at M6, modular analysis and
correlation with clinical response to anakinra
Expression of more than 500 transcripts changed signifi cantly in
the six patients who responded to anakinra at M6 (supplemen-
tary table S5).
The modular analysis of responders at M6 (n=6) showed
the normalisation of 11/13 original modular alterations, while
non-responders still showed altered expression of 12 of them
( fi gure 3A ). Interestingly, overexpression of type I IFN-inducible
genes (module 3.1) was observed in every group after initiation
of anakinra treatment (both at M1 and M6) regardless of the
clinical response ( fi gure 3B ). To support the validity of the tran-
scriptional data, we measured the protein levels of two IFN-
inducible proteins (IP10 and TRAIL) and found that they were
signifi cantly increased in the serum of anakinra-treated patients
at M6 ( fi gure 3C ). Accordingly, SOCS3 expression, which could
explain the cross-regulation of IL-1β and type I IFN, was down-
regulated in the majority of patients at M6 ( fi gure 3D ).
This double-blind, placebo-controlled study demonstrated
the effi cacy of anakinra in treating corticosteroid-dependent
patients with SJIA, as a signifi cantly higher proportion of
responders was observed after 1 month of treatment compared
with placebo. However, a loss of response was observed in
most patients over time.
The effi cacy of anakinra treatment in SJIA, at least in the short
term, concorded with previous reports. 6 8 – 10 However, the only
other placebo-controlled trial testing anakinra in JIA showed
no signifi cant improvement versus placebo, 18 illustrating that
marked response to IL-1 inhibition is not characteristic of all
JIA subtypes. The lack of sustained response in several patients
may have been related to several factors. First, most patients
had diffuse polyarthritis at enrolment but no fever; as previously
reported, 9 anakinra seemed less effective on the arthritis than on
systemic features. Second, PK data suggested that low-weight
children might have benefi ted from a higher anakinra dosage.
Third, we included patients with active SJIA, steroid-depen-
dency and a minimal disease duration of 6 months, therefore
diffi cult to treat 5 ; loss of responses may have been favoured by
Ann Rheum Dis 2011;70:747–754. doi:10.1136/ard.2010.134254754
9. Gattorno M, Piccini A, Lasigliè D, et al. The pattern of response to anti-interleukin-1
treatment distinguishes two subsets of patients with systemic-onset juvenile
idiopathic arthritis. Arthritis Rheum 2008 ; 58 : 1505 – 15 .
Zeft A, Hollister R, LaFleur B, et al. Anakinra for systemic juvenile arthritis: the Rocky
Mountain experience. J Clin Rheumatol 2009 ; 15 : 161 – 4 .
Worwood M, Cragg SJ, Wagstaff M, et al. Binding of human serum ferritin to
concanavalin A. Clin Sci 1979 ; 56 : 83 – 7 .
Vignes S, Le Moël G, Fautrel B, et al. Percentage of glycosylated serum ferritin
remains low throughout the course of adult onset Still’s disease. Ann Rheum Dis
2000 ; 59 : 347 – 50 .
Jeurissen A, Moens L, Raes M, et al. Laboratory diagnosis of specifi c antibody
defi ciency to pneumococcal capsular polysaccharide antigens. Clin Chem
2007 ; 53 : 505 – 10 .
Chaussabel D, Quinn C, Shen J, et al. A modular analysis framework for blood
genomics studies: application to systemic lupus erythematosus. Immunity
2008 ; 29 : 150 – 64 .
Giannini EH, Ruperto N, Ravelli A, et al. Preliminary defi nition of improvement in
juvenile arthritis. Arthritis Rheum 1997 ; 40 : 1202 – 9 .
16. Wallace CA, Ruperto N, Giannini E . Preliminary criteria for clinical remission for
select categories of juvenile idiopathic arthritis. J Rheumatol 2004 ; 31 : 2290 – 4 .
17. Solle M, Labasi J, Perregaux DG, et al. Altered cytokine production in mice lacking
P2X(7) receptors. J Biol Chem 2001 ; 276 : 125 – 32 .
18. Ilowite N, Porras O, Reiff A, et al. Anakinra in the treatment of polyarticular-course
juvenile rheumatoid arthritis: safety and preliminary effi cacy results of a randomized
multicenter study. Clin Rheumatol 2009 ; 28 : 129 – 37 .
19. Salliot C, Dougados M, Gossec L. Risk of serious infections during rituximab,
abatacept and anakinra treatments for rheumatoid arthritis: meta-analyses of
randomised placebo-controlled trials. Ann Rheum Dis 2009 ; 68 : 25 – 32 .
20. Ruemmele FM, Prieur AM, Talbotec C, et al. Development of Crohn disease
during anti-TNF-alpha therapy in a child with juvenile idiopathic arthritis. J Pediatr
Gastroenterol Nutr 2004 ; 39 : 203 – 6 .
21. Dallocchio A, Canioni D, Ruemmele F, et al. Occurrence of infl ammatory bowel
disease during treatment of juvenile idiopathic arthritis with etanercept: a French
retrospective study. Rheumatology (Oxford) 2010 ; 49 : 1694 – 8 .
22. Martinon F, Mayor A, Tschopp J. The infl ammasomes: guardians of the body. Annu
Rev Immunol 2009 ; 27 : 229 – 65 .
23. Fall N, Barnes M, Thornton S, et al. Gene expression profi ling of peripheral blood
from patients with untreated new-onset systemic juvenile idiopathic arthritis reveals
molecular heterogeneity that may predict macrophage activation syndrome. Arthritis
Rheum 2007 ; 56 : 3793 – 804 .
24. Palucka AK, Blanck JP, Bennett L, et al. Cross-regulation of TNF and IFN-alpha in
autoimmune diseases. Proc Natl Acad Sci USA 2005 ; 102 : 3372 – 7 .
25. Ogilvie EM, Khan A, Hubank M, et al. Specifi c gene expression profi les in systemic
juvenile idiopathic arthritis. Arthritis Rheum 2007 ; 56 : 1954 – 65 .
Wong PK, Egan PJ, Croker BA, et al. SOCS-3 negatively regulates innate and
adaptive immune mechanisms in acute IL-1-dependent infl ammatory arthritis. J Clin
Invest 2006 ; 116 : 1571 – 81 .
27. Pauli EK, Schmolke M, Wolff T, et al. Infl uenza A virus inhibits type I IFN
signaling via NF-kappaB-dependent induction of SOCS-3 expression. PLoS Pathog
2008 ; 4 : e1000196 .
28. Martinelli S, Urosevic M, Daryadel A, et al. Induction of genes mediating interferon-
dependent extracellular trap formation during neutrophil differentiation. J Biol Chem
2004 ; 279 : 44123 – 32 .
29. Pascual V, Farkas L, Banchereau J. Systemic lupus erythematosus: all roads lead to
type I interferons. Curr Opin Immunol 2006 ; 18 : 676 – 82 .
30. Dinarello CA. Interleukin-1beta and the autoinfl ammatory diseases. N Engl J Med
2009 ; 360 : 2467 – 70 .
In conclusion, a signifi cant proportion of patients with SJIA
responded to IL-1 blockade as dramatically as patients with
autoinfl ammatory syndromes, confi rming a key role for IL-1 in a
wide spectrum of diseases. 30 Furthermore, clinical response and
cytokine cascades induced upon IL1 blockade can be followed
using blood gene expression profi ling.
Acknowledgements Professor Kone-Paut, Drs Lemelle, Fouillet-Desjonqueres,
Florkin and Neven were co-investigators. The Centres d’Investigation Clinique of
Necker-Enfants Malades, Paris (Pr Bresson), Lyon (Dr Kassai, Ms Masson), Bordeaux
(Ms Vautrat and Nacka), and Ms Marie, clinical research nurse from Bicetre took an
active part in the trial. Drs Broissand, Pivot, Ghezzoul, Prevot, Taburet and Sterlingot
were involved as pharmacists, Dr Urien in PK analyses, Ms Menoni and Sotou-Bere in
data management and Karolina Palucka in rewriting important sections of the paper.
Funding The promoter was the Institut Francais pour la Recherche Scientifi que et
Medicale (INSERM). Financial support was obtained from AMGEN (which had no
role in analysis and reporting phase), INSERM, the French Society for Infl ammatory
Diseases in Pediatric Rheumatology (SOFREMIP), the Association for the Development
of Pediatric Rheumatology (ADRI) and the French association for patients with
Juvenile Arthritis KOURIR.
Competing interests None.
Contributors All authors were involved in drafting of the article or revising it critically
for important intellectual content, and all authors approved the fi nal version to be
published. PQ had full access to all the data in the study and takes responsibility for
the integrity of the data and the accuracy of the data analysis. Study conception
and design: PQ and PL; acquisition of data: PQ, FA, RC, PP, AD and OR; analysis and
interpretation of data: PQ, FA, CM, CB, XB, AB, JB, DC, J-MT, JB, PL and VP.
Ethics approval This study was conducted with the approval of the Necker-Enfants
Malades Hospital, Paris, France, Commite pour la protection des personnes.
Provenance and peer review Not commissioned; externally peer reviewed.
1. Petty RE, Southwood TR, Manners P, et al. International League of Associations
for Rheumatology classifi cation of juvenile idiopathic arthritis: second revision,
Edmonton, 2001. J Rheumatol 2004 ; 31 : 390 – 2 .
2. Woo P, Southwood TR, Prieur AM, et al. Randomized, placebo-controlled, crossover
trial of low-dose oral methotrexate in children with extended oligoarticular or
systemic arthritis. Arthritis Rheum 2000 ; 43 : 1849 – 57 .
3. Quartier P, Taupin P, Bourdeaut F, et al. Effi cacy of etanercept for the treatment of juve-
nile idiopathic arthritis according to the onset type. Arthritis Rheum 2003 ; 48 : 1093 – 101 .
4. Yokota S, Imagawa T, Mori M, et al. Effi cacy and safety of tocilizumab in patients
with systemic-onset juvenile idiopathic arthritis: a randomised, double-blind,
placebo-controlled, withdrawal phase III trial. Lancet 2008 ; 371 : 998 – 1006 .
5. Spiegel LR, Schneider R, Lang BA, et al. Early predictors of poor functional outcome
in systemic-onset juvenile rheumatoid arthritis: a multicenter cohort study. Arthritis
Rheum 2000 ; 43 : 2402 – 9 .
6. Pascual V, Allantaz F, Arce E, et al. Role of interleukin-1 (IL-1) in the pathogenesis of
systemic onset juvenile idiopathic arthritis and clinical response to IL-1 blockade.
J Exp Med 2005 ; 201 : 1479 – 86 .
7. Allantaz F, Chaussabel D, Stichweh D, et al. Blood leukocyte microarrays to diagnose
systemic onset juvenile idiopathic arthritis and follow the response to IL-1 blockade.
J Exp Med 2007 ; 204 : 2131 – 44 .
8. Lequerré T, Quartier P, Rosellini D, et al. Interleukin-1 receptor antagonist (anakinra)
treatment in patients with systemic-onset juvenile idiopathic arthritis or adult onset
Still disease: preliminary experience in France. Ann Rheum Dis 2008 ; 67 : 302 – 8 .