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.код для вставкиСкачать
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: email@example.com. 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 ). 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. REFERENCES 1. Woo P. Systemic juvenile idiopathic arthritis: diagnosis, management, and outcome. Nat Clin Pract Rheumatol 2006;2:28–34. 2. Jarvis JN. Pathogenesis and mechanisms of inflammation in the childhood rheumatic diseases. Curr Opin Rheumatol 1998;10: 459–67. 3. Niki Y, Yamada H, Seki S, Kikuchi T, Takaishi H, Toyama Y, et al. Macrophage- and neutrophil-dominant arthritis in human IL-1␣ transgenic mice. J Clin Invest 2001;107:1127–35. 4. 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