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The myeloid-related proteins 8 and 14 complex a novel ligand of toll-like receptor 4 and interleukin-1 form a positive feedback mechanism in systemic-onset juvenile idiopathic arthritis.

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ARTHRITIS & RHEUMATISM
Vol. 60, No. 3, March 2009, pp 883–891
DOI 10.1002/art.24349
© 2009, American College of Rheumatology
The Myeloid-Related Proteins 8 and 14 Complex,
a Novel Ligand of Toll-Like Receptor 4, and Interleukin-1␤
Form a Positive Feedback Mechanism in
Systemic-Onset Juvenile Idiopathic Arthritis
Michael Frosch,1 Martina Ahlmann,1 Thomas Vogl,1 Helmut Wittkowski,1 Nico Wulffraat,2
Dirk Foell,1 and Johannes Roth1
and patients with NOMID (2,830 ⴞ 580 ng/ml). In
contrast to C-reactive protein levels, MRP-8/MRP-14
concentrations distinguished systemic-onset JIA from
infections, with a specificity of 95%. MRP-14 in serum of
patients with systemic-onset JIA was a strong inducer of
IL-1␤ expression in phagocytes.
Conclusion. The analysis of MRP-8/MRP-14 in
serum is an excellent tool for the diagnosis of systemiconset JIA, allowing early differentiation between patients with systemic-onset JIA and those with other
inflammatory diseases. MRP-8/MRP-14 and IL-1␤ represent a novel positive feedback mechanism activating
phagocytes via 2 major signaling pathways of innate
immunity during the pathogenesis of systemic-onset JIA.
Objective. Fever of unknown origin is a diagnostic
challenge in children, especially for differentiation of
systemic-onset juvenile idiopathic arthritis (systemiconset JIA) and infectious diseases. We undertook this
study to analyze the relevance of myeloid-related proteins (MRPs) 8 and 14, endogenous activators of Tolllike receptor 4, in diagnosis and pathogenesis of
systemic-onset JIA.
Methods. Serum concentrations of MRP-8/
MRP-14 were analyzed in 60 patients with systemiconset JIA, 85 patients with systemic infections, 40
patients with acute lymphoblastic leukemia, 5 patients
with acute myeloblastic leukemia, 18 patients with
neonatal-onset multisystem inflammatory disease
(NOMID), and 50 healthy controls. In addition, we
investigated the link between interleukin-1␤ (IL-1␤)
and MRP-8/MRP-14 in systemic-onset JIA.
Results. Serum MRP-8/MRP-14 concentrations
were significantly (P < 0.001) elevated in patients with
active systemic-onset JIA (mean ⴞ 95% confidence
interval 14,920 ⴞ 4,030 ng/ml) compared with those in
healthy controls (340 ⴞ 70 ng/ml), patients with systemic infections (2,640 ⴞ 720 ng/ml), patients with acute
lymphoblastic leukemia (650 ⴞ 280 ng/ml), patients
with acute myeloblastic leukemia (840 ⴞ 940 ng/ml),
The differential diagnosis of fever of unknown
origin (FUO) is one of the major challenges in pediatrics. The main causes of FUO are infectious diseases,
but up to 200 conditions have to be ruled out. An
important differential diagnosis of FUO in children is
systemic-onset juvenile idiopathic arthritis (systemiconset JIA, or Still’s disease), an aggressive autoinflammatory disease presenting with fever and activation of
the innate immune system mimicking clinical signs of
severe infection (1). Specific immunologic features
found in other systemic autoimmune diseases are absent
in systemic-onset JIA, and characteristic signs of active
arthritis often develop in the later course of the disease.
Due to the nonspecific inflammatory pattern at initial
presentation, systemic-onset JIA cannot be distinguished from systemic infections by clinical and common
laboratory parameters. However, identification of
systemic-onset JIA as the basis of FUO is of particular
relevance for the early initiation of appropriate antiinflammatory therapy (1).
1
Michael Frosch, MD, Martina Ahlmann, MD, Thomas Vogl,
PhD, Helmut Wittkowski, MD, Dirk Foell, MD, Johannes Roth, MD:
University of Muenster, Muenster, Germany; 2Nico Wulffraat, MD:
Wilhelmina Children’s Hospital, and University Medical Centre,
Utrecht, The Netherlands.
Drs. Frosch and Ahlmann contributed equally to this work.
Address correspondence and reprint requests to Johannes
Roth, MD, Institute of Immunology, University of Muenster, Roentgenstrasse 21, D-48149 Muenster, Germany. E-mail: rothj@unimuenster.de.
Submitted for publication August 12, 2008; accepted in
revised form November 25, 2008.
883
884
FROSCH ET AL
Table 1. Characteristics of the patients with active systemic-onset JIA and of those with infections, acute lymphoblastic leukemia, acute
myeloblastic leukemia, and NOMID*
Age, median (range)
years
No. of men/no. of women
MRP-8/MRP-14 level,
ng/ml
ESR, mm/hour
CRP level, mg/liter
Joints with active disease,
median (range)
Leukocytes/␮l
Active
systemic-onset
JIA
(n ⫽ 60)
Infections
(n ⫽ 85)
9.1 (1.8–18.1)
9.5 (1.2–33.2)
32/28
14,920 ⫾ 4,030
43/42
2,640 ⫾ 720
76 ⫾ 23
84 ⫾ 19
5 (3–7)
40 ⫾ 18
111 ⫾ 11
ND
16,120 ⫾ 2,220
13,300 ⫾ 1,210
P†
Acute
lymphoblastic
leukemia
(n ⫽ 40)
Acute
myeloblastic
leukemia
(n ⫽ 5)
NOMID
(n ⫽ 18)
–
6.2 (0.9–14.9)
11.0 (0.7–16.9)
11.0 (4.1–32.0)
18/22
650 ⫾ 280
3/2
840 ⫾ 940
10/8
2,830 ⫾ 580
0.948
0.297
–
75 ⫾ 33
33 ⫾ 16
ND
ND
17 ⫾ 49
ND
60 ⫾ 16
68 ⫾ 19
ND
0.851
38,290 ⫾ 36,400
33,120 ⫾ 71,800
17,200 ⫾ 3,600
–
⬍0.001
* Except where indicated otherwise, values are the mean ⫾ 95% confidence interval. NOMID ⫽ neonatal-onset multisystem inflammatory disease;
MRP-8 ⫽ myeloid-related protein 8; ESR ⫽ erythrocyte sedimentation rate; ND ⫽ not determined; CRP ⫽ C-reactive protein.
† Active systemic-onset juvenile idiopathic arthritis (JIA) versus infections.
The pathogenesis of systemic-onset JIA includes
overwhelming activation of innate immunity, particularly of the phagocyte system (2,3). Myeloid-related
proteins (MRPs) 8 (S100A8) and 14 (S100A9) are the
major calcium-binding proteins expressed in granulocytes, in monocytes, and in macrophages during early
differentiation stages. Complexes of MRP-8/MRP-14
are secreted after activation of phagocytes via a so-called
alternative pathway (4–6). MRP-8 and MRP-14 are
useful markers for followup of disease activity in several
autoimmune diseases (7). These proteins exert strong
proinflammatory effects on phagocytes and endothelial
cells in vitro (8,9). The recent identification of the
MRP-8/MRP-14 complex as a new, endogenous ligand
of Toll-like receptor 4 (TLR-4) has provided novel
molecular insights into the proinflammatory actions of
these proteins (10).
In the present study, we obtained evidence that
serum levels of MRP-8/MRP-14 are an excellent diagnostic tool allowing early differentiation between patients with systemic-onset JIA and those with systemic
infections. In addition, we describe a novel positive
feedback mechanism of innate immunity by which the
endogenous TLR-4 ligand MRP-8/MRP-14 and
interleukin-1␤ (IL-1␤) promote inflammation, which
may explain similarities in the inflammatory response
patterns of severe infections and systemic-onset JIA.
PATIENTS AND METHODS
Patients with systemic-onset JIA. Serum samples from
60 patients who fulfilled the International League of Associations for Rheumatology criteria for systemic-onset JIA (11)
were obtained at initial presentation and during the course of
disease. Clinical disease activity was determined on the basis of
the core set criteria for JIA (12,13). Collection of patient data
included medical history and physical examination, number of
joints with active disease, number of joints with limited range
of motion (ROM), physician’s global assessment of disease
activity, parent’s/patient’s assessment of overall well-being,
and functional ability (as measured by the Childhood Health
Assessment Questionnaire [14]). Leukocyte count, absolute
neutrophil count, red blood cell count, platelet count, erythrocyte sedimentation rate (ESR), and C-reactive protein
(CRP) level were determined as parameters of inflammation
(Table 1).
Patients were categorized as having active disease
(presence of any joint with active disease or signs of systemic
disease) or were considered to have disease in remission based
on proposed criteria for at least 3 consecutive months, including the absence of any systemic symptoms, no active arthritis,
and normal CRP level and ESR, regardless of medication (15).
Relapse was defined according to the preliminary definition of
disease flare in JIA (16). Serum samples from 3 patients (2
boys ages 14 and 17 years and 1 girl age 5 years) with
systemic-onset JIA were analyzed in a prospective manner
prior to and after treatment with IL-1 receptor antagonist
(IL-1Ra). The study was approved by the institutional ethics
committee, and informed consent was obtained from patients
or parents.
Patients with systemic infections, malignancies, and
neonatal-onset multisystem inflammatory disease (NOMID)
and healthy controls. We included 85 patients with severe
systemic infections (median age 9 years [range 1–33 years],
CRP level ⬎50 mg/liter, fever ⬎38.5°C). A total of 66 patients
had proven bacterial infections (40 with pneumonia, 8 with
urinary tract infections, 3 with gastrointestinal [GI] tract
infections, 2 with osteomyelitis, 2 with soft tissue infections, 6
with sepsis, 3 with peritonitis, and 2 with appendicitis). All
serum samples were obtained prior to the start of antibiotic
treatment. Nineteen patients presented with typical viral infec-
MRP-8 AND MRP-14 IN SYSTEMIC-ONSET JUVENILE IDIOPATHIC ARTHRITIS
tions (11 with respiratory tract infections, 7 with GI tract
infections, and 1 with Epstein-Barr virus infection).
Furthermore, we included 40 patients with acute lymphoblastic leukemia, 5 patients with acute myeloblastic leukemia, and 18 patients with NOMID. Of the 18 patients with
NOMID, 12 had proven mutations in exon 3 of the coldinduced autoinflammatory syndrome 1 (CIAS1) gene. Patients
presented with active disease showing at least 2 of the following clinical manifestations: urticarial rash, central nervous
system involvement (e.g., papilledema, pleocytosis in the cerebrospinal fluid, and sensorineural hearing loss), or epiphyseal
or patellar overgrowth seen on radiography. At the time of
sampling, all patients had active inflammatory disease despite
antiinflammatory treatment, but none of the patients was
treated with recombinant IL-1Ra.
Serum samples were obtained at initial presentation
before institution of therapy. Normal MRP-8/MRP-14 levels
were determined in 50 healthy controls (median age 16 years
[range 1–34 years]). As previously described, there were no
significant differences in serum MRP-8/MRP-14 concentrations with regard to age or sex distribution (4).
Determination of concentrations of MRP-8/MRP-14
by sandwich enzyme-linked immunosorbent assay (ELISA).
Concentrations of MRP-8/MRP-14 in sera and culture supernatants were determined by ELISA as described previously
(4). For comparison with earlier studies, internal control sera
were used as a reference in all ELISA studies.
Stimulation of monocytes. Monocytes were isolated
from human buffy coats and cultured as described previously
(4). Monocytes were incubated for 24 hours with lipopolysaccharide (LPS) (10 ng/ml; Sigma, Deisenhofen, Germany) or
MRP-14 (5 ␮g/ml) (10), and IL-1␤ concentrations in supernatants were determined by ELISA (Becton Dickinson, Heidelberg, Germany). The interassay variability in baseline IL-1␤
production between different monocyte preparations is the
reason for presentation of data as a percent of control. The
maximal LPS contamination in our MRP-14 preparation was
⬍3 endotoxin units/mg protein, as described (10). Polymyxin B
(25 ␮g/ml; Sigma) was added to MRP-14 in control experiments to exclude stimulatory effects due to LPS contamination. In parallel sets of experiments, monocytes were primed
for 16 hours with interferon-␥ (IFN␥) (500 IU/ml; Bender
MedSystems, Vienna, Austria) prior to stimulation with LPS
or MRP-14. In order to block the effects of MRP-8/MRP-14
complexes, 500 ␮l of serum obtained during active disease
from 7 patients with systemic-onset JIA was incubated with
anti–MRP-14 antibodies (1 ␮g/␮l) or nonspecific isotypematched rabbit antibodies (1 ␮g/ml). Immune complexes were
precipitated with protein G–Sepharose (Amersham Biosciences, Freiburg, Germany). MRP-8/MRP-14 concentrations
were analyzed before and after immunoprecipitation. Monocytes (1 ⫻ 106/ml) were cultured in the presence of 20% serum
from patients with systemic-onset JIA, 20% precipitated serum
from patients with systemic-onset JIA, or 20% control serum
for 6 hours, and IL-1␤ concentrations in supernatants were
determined by ELISA.
Quantitative reverse transcriptase–polymerase chain
reaction (RT-PCR). Expression of selected genes was confirmed by real-time RT-PCR as described previously (8).
Briefly, complementary DNA was synthesized from 5 ␮g of
total RNA using Reverd Aid H minus transcriptase (Fermen-
885
tas, Hanover, MD). Primers were designed using the Primer
Express software package (Applied Biosystems, Foster City,
CA) and obtained from Qiagen (Chatsworth, CA). Real-time
RT-PCR was performed using the Quanti-Tect SYBR Green
PCR kit (Qiagen), and data were acquired with the ABI
PRISM 7900 HT instrument (Applied Biosystems). Each
measurement was set up in duplicate, and 3 independent
experiments were performed. After normalization to the endogenous housekeeping control gene GAPDH, the relative
expression was calculated. The primers used for PCR analysis
for detection of IL-1␤ RNA were 5⬘-GCGGCCAGGATATAACTGACTTC-3⬘ (forward) and 5⬘-TCCACATTCAGCACAGGACTCTC-3⬘ (reverse).
Statistical analysis. Analysis of variance was used to
analyze differences between subgroups of patients. Confirmed
differences were tested for statistical significance by using
Dunnett’s post-test with Bonferroni adjustment. Rank differences were analyzed using the Mann-Whitney U test. Correlations were calculated using Spearman’s rho. Receiver
operating characteristic (ROC) curves were plotted to determine the accuracy of inflammation marker measurements as a
diagnostic test. SPSS 12.0 for Windows (SPSS, Chicago, IL)
was used for statistical analyses. Unless stated otherwise, data
are expressed as the mean ⫾ 95% confidence interval
(95% CI).
RESULTS
Serum MRP-8/MRP-14 concentrations differ significantly in systemic-onset JIA and systemic infections.
Serum levels of MRP-8/MRP-14 during active disease in
patients with systemic-onset JIA were ⬃44-fold higher
than those in healthy controls (mean ⫾ 95% CI 14,920 ⫾
4,030 ng/ml versus 340 ⫾ 70 ng/ml) (P ⬍ 0.001). Mean
concentrations during active disease in patients with
systemic-onset JIA were also significantly higher than
those in patients with systemic infections (2,640 ⫾ 720
ng/ml), acute lymphoblastic leukemia (650 ⫾ 280 ng/ml),
acute myeloblastic leukemia (840 ⫾ 940 ng/ml), and
NOMID (2,830 ⫾ 580 ng/ml) (P ⬍ 0.001 for all) (Table 1
and Figure 1A). Patients with systemic-onset JIA in
clinical remission had almost normal MRP-8/MRP-14
levels (530 ⫾ 440 ng/ml). CRP concentrations did not
differ significantly between patients with active systemiconset JIA (84 ⫾ 19 mg/liter) and patients with systemic
infections (111 ⫾ 11 mg/liter) (P ⫽ 0.297) (Table 1 and
Figure 1B).
ROC analyses confirmed the specificity of MRP8/MRP-14 levels for systemic-onset JIA. The area under
the curve was 0.747 ⫾ 0.097 for MRP-8/MRP-14 level
and 0.257 ⫾ 0.101 for CRP level (Figure 1C), confirming
that CRP values were not reliable markers for the
diagnosis of systemic-onset JIA. An MRP-8/MRP-14
cutoff concentration of 9,200 ng/ml had a specificity of
886
Figure 1. A and B, Serum concentrations of myeloid-related proteins
(MRPs) 8 and 14 (A) and C-reactive protein (CRP) (B) in patients
with active systemic-onset juvenile idiopathic arthritis (systemic-onset
JIA [SJIA]), systemic infections, inactive systemic-onset JIA, neonatalonset multisystem inflammatory disease (NOMID), acute lymphoblastic leukemia (ALL), and acute myeloblastic leukemia (AML). Also
shown is the serum concentration of MRP-8/MRP-14 in a group of
healthy controls (A). Box plots show the median (thin horizontal line),
the mean (thick horizontal line), and the 25th and 75th percentiles.
Bars indicate the 10th and 90th percentiles. There was a significant
difference in MRP-8/MRP-14 concentrations between patients with
active systemic-onset JIA and patients with systemic infections (P ⬍
0.001) or healthy controls (P ⬍ 0.001) (note the break in the y-axis in
A). CRP levels did not differ significantly between patients with active
systemic-onset JIA and patients with systemic infections. C, Receiver
operating characteristic curve analysis of MRP-8/MRP-14 and CRP
concentrations displayed as sensitivity against 1 – specificity for the
differentiation between systemic-onset JIA and systemic infections.
95% for the diagnosis of systemic-onset JIA, which
resulted in a positive likelihood ratio of 8.0.
FROSCH ET AL
MRP-8/MRP-14 concentrations during response
to IL-1Ra treatment. We analyzed serum MRP-8/
MRP-14 concentrations in 3 patients with systemic-onset
JIA after treatment with IL-1Ra (anakinra; 2 mg/kg/day
administered subcutaneously). These patients’ disease
had been refractory to any other antiinflammatory therapy including tumor necrosis factor (TNF) blockade. We
found an impressive decrease in MRP-8/MRP-14 concentrations after initiation of IL-1Ra therapy, and this
decrease was stable for at least 3 months (Figure 2A).
This response was paralleled by a significant decrease in
disease activity (number of joints with limited ROM
4–10 before treatment and 0 after treatment, ESR 33–67
mm/hour before treatment and 7–8 mm/hour after treatment, CRP level 81–141 mg/liter before treatment and
⬍5 mg/liter after treatment).
MRP-14 induces IL-1 secretion in human monocytes. Stimulation of human monocytes in vitro revealed
that MRP-14 was capable of inducing IL-1␤ secretion in
a dose-dependent manner, with levels comparable with
those observed using the inflammatory stimulus LPS
(Figures 2B and D). Addition of the LPS inhibitor
Polymyxin B resulted in no significant inhibition of
MRP-14, whereas activity of 10 ng/ml LPS was completely blocked. Priming of monocytes with IFN␥ prior
to stimulation amplified the effects of both MRP-14 and
LPS (Figure 2B). MRP-14 increased the expression of
IL-1␤ messenger RNA ⬎200-fold compared with controls (Figure 2C). In control experiments, we excluded
the possibility of LPS contamination of our MRP-14
preparations, as described previously (10).
These data indicate that MRP-14 might be the
recently described missing serum factor responsible for
IL-1␤ release in systemic-onset JIA (17). We therefore tested whether this effect could be blocked by
anti–MRP-14 antibodies. IL-1␤ secretion induced by
serum from patients with systemic-onset JIA was almost completely blocked by immunoabsorption with
anti–MRP-14 (Figure 2E), but not by irrelevant rabbit
control antibodies. The efficiency and specificity of
immunoabsorption were evaluated by determining the
concentrations of MRP-8/MRP-14 complexes in sera
before and after immunoprecipitation (Figure 2F). Our
results confirm the specificity of MRP-14 effects and
exclude the possibility of any effect due to LPS contamination in our in vitro experiments, as previously shown
in detail (10).
DISCUSSION
Although diagnosing FUO in children is a common clinical task, the evaluation of underlying causes is
MRP-8 AND MRP-14 IN SYSTEMIC-ONSET JUVENILE IDIOPATHIC ARTHRITIS
887
Figure 2. A, Three patients (Pat) with systemic-onset JIA were treated daily with 2 mg/kg recombinant interleukin-1 receptor antagonist (IL-1Ra).
Serum concentrations of MRP-8/MRP-14 were analyzed at different time points after starting IL-1Ra treatment as indicated. B, Peripheral blood
monocytes (1 ⫻ 106/ml) were incubated for 24 hours with either 10 ng/ml lipopolysaccharide (LPS) or 5 ␮g/ml MRP-14 or left untreated as controls
(medium). IL-1␤ concentrations in supernatants were determined by enzyme-linked immunosorbent assay (ELISA). In control experiments 25
␮g/ml Polymyxin B (PM) was added to MRP-14 to exclude stimulatory effects due to LPS contamination. In another set of experiments monocytes
were primed for 16 hours with interferon-␥ (IFN) and subsequently stimulated with LPS or MRP-14. Shown are data from 3 independent
experiments. Values are the mean and SEM. Results were normalized to those in untreated controls (set at 100%). ⴱ ⫽ P ⱕ 0.05 versus controls;
§ ⫽ P ⱕ 0.05 versus LPS stimulation without concomitant application of Polymyxin B, by Wilcoxon test. C, Human monocytes were treated with
5 ␮g/ml MRP-14 or 10 ng/ml LPS for 4 hours, and IL-1␤ mRNA was detected by polymerase chain reaction (PCR). The PCR data were normalized
to GAPDH expression, and the mean and SEM n-fold regulation in comparison with phosphate buffered saline–treated controls (Con) was
determined in 3 independent experiments. IL-1␤ mRNA expression in control cells was set at 1. D, Human monocytes were treated with different
concentrations of MRP-14 (0.5–50 ␮g/ml) for 24 hours, and IL-1␤ secretion into supernatants was analyzed by ELISA. Values are the mean. E,
Monocytes (1 ⫻ 106/ml) were cultured for 6 hours in the presence of 20% serum (volume/volume) from 5 patients with active systemic-onset JIA
and 2 healthy controls. In addition, MRP-8/MRP-14 complexes were removed from serum of patients with systemic-onset JIA by immunoprecipitation (IP) with anti–MRP-14 antibodies prior to stimulation. Secretion of IL-1␤ into supernatants was analyzed by ELISA. F, Serum concentrations
of MRP-8/MRP-14 were determined before and after immunoprecipitation with anti–MRP-14 antibodies and with nonspecific rabbit control
antibodies (1 ␮g/␮l). Shown are data from 5 independent experiments. Values are the mean and SEM. See Figure 1 for other definitions.
888
FROSCH ET AL
Table 2. Serum concentrations of MRP-8/MRP-14 in healthy controls and in patients with systemiconset JIA and other inflammatory diseases*
MRP-8/MRP-14,
ng/ml
No. of
subjects
340 ⫾ 70
50
†
14,920 ⫾ 4,030
2,380 ⫾ 530
640 ⫾ 110
1,010 ⫾ 150
910 ⫾ 250
60
89
40
40
28
†
4, 37, 38
39
39
40
3,630 ⫾ 480
810 ⫾ 90
570 ⫾ 245
450 ⫾ 80
21
42
6
6
8
41
42
42
2,830 ⫾ 580
18
†
3,720 ⫾ 870
1,960 ⫾ 620
2,530 ⫾ 670
66
19
16
†
†
43
650 ⫾ 280
840 ⫾ 940
40
5
†
†
Healthy controls
Arthritis
Systemic-onset JIA
JIA
RA
SpA
PsA
Vasculitis and autoimmune diseases
Kawasaki disease
Giant cell arteritis
SLE
DM
Autoinflammatory diseases
NOMID
Infections
Proven bacterial infections
Pneumonia
Leprosy (type 2 reaction)
Malignancies
Acute lymphoblastic leukemia
Acute myeloblastic leukemia
Ref.
* Values are the mean ⫾ 95% confidence interval. All serum concentrations were determined in our
laboratory by the same calibrated MRP-8/MRP-14 enzyme-linked immunosorbent assay, including
internal control sera for direct comparison of different studies. Serum concentrations in patients with
systemic-onset JIA were significantly higher (P ⱕ 0.01) compared with those in healthy controls and
compared with those in patients with all other inflammatory diseases presented in this table. RA ⫽
rheumatoid arthritis; SpA ⫽ spondylarthritis; PsA ⫽ psoriatic arthritis; SLE ⫽ systemic lupus erythematosus; DM ⫽ dermatomyositis (see Table 1 for other definitions).
† Data presented for the first time in the present study.
still a challenging problem in clinical practice. By far,
infections account for the largest group of children
presenting with FUO; however, malignancies and rheumatic diseases have to be identified or excluded early to
start appropriate therapies. As a prototype of systemic
rheumatic diseases in childhood, systemic-onset JIA
represents a special challenge in diagnosis (1). Clinicians
are not able to ascertain diagnosis using common laboratory tests, and in a number of patients specific clinical
signs (i.e., arthritis) develop later in the course of
systemic-onset JIA. The clinical presentation of
systemic-onset JIA in many patients resembles systemic
that of infections.
In a previous study we have shown that systemiconset JIA is associated with high concentrations of the
most abundant calcium-binding proteins in phagocytes,
MRP-8 and MRP-14 (18). Both proteins are released
after inflammatory activation of phagocytes. Serum concentrations correlate with phagocyte activity in different
inflammatory diseases (4,7). The primary goal of our
present study was to investigate the predictive value of
MRP-8/MRP-14 in the differential diagnosis of systemic-
onset JIA versus severe infections and hematologic
malignancies, since systemic-onset JIA is associated with
an extraordinary activation of the phagocyte system.
Classic parameters such as ESR, CRP level, and leukocyte count did not distinguish between active systemiconset JIA and severe infectious diseases. In contrast,
MRP-8/MRP-14 concentrations were significantly
higher in systemic-onset JIA than in infectious diseases
independent of the underlying infectious disease. MRP8/MRP-14 concentrations ⱖ9,200 ng/ml exhibited sensitivity of 95% for systemic-onset JIA and a resulting high
likelihood ratio of 8.0, which, according to the American
College of Rheumatology Ad Hoc Committee on Immunologic Testing Guidelines, is considered “very useful,” the highest category for a diagnostic test (19). Such
a test constitutes significant progress in the differential
diagnosis of FUO, especially since patients with
systemic-onset JIA represent only ⬃10% of patients
with prolonged FUO (20,21).
Concentrations of MRP-8/MRP-14 in systemiconset JIA also differ significantly from those in other
inflammatory disorders, such as other forms of arthritis,
MRP-8 AND MRP-14 IN SYSTEMIC-ONSET JUVENILE IDIOPATHIC ARTHRITIS
systemic lupus erythematosus, dermatomyositis, and systemic vasculitis, e.g., Kawasaki disease (Table 2). Measurement of serum concentrations of IL-1, IL-6, IL-8,
IL-12, or TNF is not helpful in resolving the clinical
dilemma regarding differential diagnosis of systemiconset JIA versus other inflammatory diseases (22,23). It
is well known that high concentrations of IL-1␤ are not
found in systemic-onset JIA (17,23). This is probably due
to a short half-life and a low stability of this protein in
serum. However, the biologic relevance of IL-1␤ is
confirmed by the efficiency of anti–IL-1␤ treatment in
systemic-onset JIA. In contrast, IL-18 is found in high
concentrations in serum of patients with systemic-onset
JIA (23). Ferritin levels are frequently elevated in
systemic-onset JIA, but (at least in children) they are
normal in a significant proportion of patients and are
therefore not a reliable marker for diagnosis of systemiconset JIA (24–26). In addition, MRP-8/MRP-14 levels in
patients with acute lymphoblastic leukemia, acute myeloblastic leukemia, and NOMID were significantly
lower than in patients with active systemic-onset JIA,
thus helping to rule out another important differential
diagnosis of FUO. Hence, MRP-8/MRP-14 concentrations are so far the only individual parameter distinguishing between systemic-onset JIA and other systemic
inflammatory causes of FUO.
In addition to this diagnostic implication, our
data underline the key role of innate immune processes
in the pathogenesis of systemic-onset JIA. Recently, the
MRP-8/MRP-14 complex has come into focus as an
endogenous ligand of TLR-4 promoting expression of
proinflammatory proteins such as cytokines, chemokines, NADPH oxidase, or signal transduction molecules
(10). In parallel, MRP-8 and MRP-14 activate the
integrin receptor CD11b/CD18 on phagocytes and modulate transendothelial migration of leukocytes (9). In
endothelial cells, MRP-8 and MRP-14 induce a proinflammatory and prothrombotic response (8). A recently
identified inflammatory disorder, the hallmark of which
is an extraordinary abundance of MRP-8 and MRP-14
(27), provides strong evidence of a direct pathogenetic
role of these 2 molecules in chronic inflammation in
vivo, especially in arthritis and systemic inflammation.
The high abundance of an internal TLR-4 activator in
systemic-onset JIA may be the molecular basis for
similarities in the inflammatory response patterns of
severe infections and systemic-onset JIA.
In contrast to other forms of rheumatoid arthritis, systemic-onset JIA shows an unsatisfactory response
to TNF blockade (28,29). However, response to treatment with IL-1Ra demonstrated that IL-1␤ is a key
889
cytokine in systemic-onset JIA that is released excessively by blood mononuclear cells (17,30,31). Pascual et
al described a serum factor that is responsible for IL-1␤
release in systemic-onset JIA (17). We now demonstrate
that stimulation of human monocytes by MRP-14 at
concentrations found in sera from patients with
systemic-onset JIA can indeed induce IL-1␤ secretion.
This induction was effectively blocked by anti–MRP-14
antibodies, indicating that MRP-14 might be the recently described missing serum factor responsible for
IL-1␤ release in systemic-onset JIA (17). However, we
also found an impressive and rapid decrease in MRP-8/
MRP-14 concentrations after initiation of IL-1Ra therapy in systemic-onset JIA, which was paralleled by a
significant decrease in disease activity, thus further
indicating a strong linkage of IL-1␤ and MRP-8/MRP14. Recent studies have shown that some patients with
systemic-onset JIA do not respond to IL-1 blockade
(32,33). It would therefore be of interest to determine
whether high levels of MRP-8/MRP-14 have a predictive
value with regard to disease activity and treatment
response. Possible differences in MRP-8/MRP-14 or
IL-1␤ expression between responders to anti–
IL-1 treatment and nonresponders may help to identify
molecular mechanisms underlying heterogeneity of
systemic-onset JIA.
At present it cannot be determined whether
IL-1␤ or MRP-8/MRP-14 comes first in the cause-andeffect chain. The response of serum MRP-8/MRP-14
concentrations after IL-1Ra treatment indicates the first
possibility, while blocking by anti–MRP-14 of IL-1␤
secretion induced by serum from patients with systemiconset JIA indicates the second possibility. However,
these molecules represent a positive feedback mechanism in the inflammatory process of systemic-onset JIA,
since IL-1␤ has previously been shown to induce further
MRP-8/MRP-14 release (5).
Several inborn, multisystemic syndromes reveal a
fundamental role of IL-1␤ in systemic inflammation.
IL-1␤ is activated from a precursor molecule by proteolytic cleavage of caspase 1 (34). Subsequently, IL-1␤ is
released by an alternative secretory pathway independent of the classical route via the endoplasmic reticulum
or Golgi complex (31). Different mutations involving the
genes for NALP3 or pyrin, both controlling IL-1␤ processing, lead to inborn inflammatory syndromes characterized by overwhelming activation of the innate immune system via IL-1␤ release. It is therefore a logical
conclusion that uncontrolled processing or release of
IL-1␤ might be the underlying molecular mechanism of
systemic-onset JIA (30,31,34,35). In this context it is
890
FROSCH ET AL
interesting that MRP-8 and MRP-14 are also released
via a so-called alternative pathway (5). Our data thus
indicate that the inflammatory process of systemic-onset
JIA is closely linked to alternative secretion of different
proinflammatory molecules. High serum concentrations
of IL-18, another member of the IL-1 family, could also
be explained by this mechanism (23,29,36).
Taken together, our findings demonstrate that
monitoring of surrogate markers for this activation
pathway is highly useful for diagnosis of systemic-onset
JIA. Furthermore, MRP-8/MRP-14 and IL-1␤ represent
a novel positive feedback mechanism activating phagocytes via 2 major signaling pathways of innate immunity
during the pathogenesis of systemic-onset JIA. Targeted
modulation of this inflammatory mechanism might be a
novel specific strategy to suppress undesirable inflammation in systemic-onset JIA or other diseases associated with overwhelming activation of the innate immune
system.
5.
6.
7.
8.
9.
10.
11.
ACKNOWLEDGMENTS
The authors thank Raphaela Goldbach-Mansky, MD
(National Institute of Arthritis and Musculoskeletal and Skin
Diseases, National Institutes of Health, Bethesda, MD) for
samples from and data on patients with NOMID and Michael
C. Frühwald, MD (Department of Pediatric Hematology and
Oncology, University Hospital Muenster) for samples from
and data on patients with acute lymphoblastic leukemia and
acute myeloblastic leukemia.
AUTHOR CONTRIBUTIONS
Dr. Roth 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. Frosch, Ahlmann, Vogl, Wittkowski, Foell, Roth.
Acquisition of data. Frosch, Ahlmann, Vogl, Wittkowski, Wulffraat,
Foell, Roth.
Analysis and interpretation of data. Frosch, Ahlmann, Vogl, Wittkowski, Foell, Roth.
Manuscript preparation. Frosch, Ahlmann, Wittkowski, Wulffraat,
Roth.
Statistical analysis. Wittkowski.
12.
13.
14.
15.
16.
17.
18.
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