Helmut Wittkowski

S100 proteins expressed in phagocytes: a novel group of damage-associated molecular pattern molecules

Damage‐associated molecular pattern (DAMP) molecules have been introduced as important proinflammatory factors of innate immunity. One example known for many years to be expressed in cells of myeloid origin are phagocytic S100 proteins, which mediate inflammatory responses and recruit inflammatory cells to sites of tissue damage. An emerging concept of pattern recognition involves the multiligand receptor foradvanced glycation end products (RAGE) and Toll‐like receptors (TLRs) in sensing not only pathogen‐associated molecular patterns (PAMPs) but also endogenous DAMPs, including S100 proteins. S100A8, S100A9, and S100A12 are found at high concentrations in inflamed tissue, where neutrophils and monocytes belong to the most abundant cell types. They exhibit proinflammatory effects in vitro at concentrations found at sites of inflammation in vivo. Although S100A12 binds to RAGE, at least part of the proinflammatory effects of the S100A8/S100A9 complex depend upon interaction with other receptors. Because of the divergent expression patterns, the absence of S100A12 in rodents, the different interaction partners described, and the specific intracellular and extracellular effects reported for these proteins, it is important to differentiate between distinct S100 proteins rather than subsuming them with the term “S100/calgranulins.” Analyzing the molecular basis of the specific effects exhibited by these proteins in greater detail bears the potential to elucidate important mechanisms of innate immunity, to establish valid biomarkers of phagocytic inflammation, and eventually to reveal novel targets for innovative anti‐inflammatory therapies.

In vivo regulation of interleukin 1β in patients with cryopyrin-associated periodic syndromes

The investigation of interleukin 1β (IL-1β) in human inflammatory diseases is hampered by the fact that it is virtually undetectable in human plasma. We demonstrate that by administering the anti–human IL-1β antibody canakinumab (ACZ885) to humans, the resulting formation of IL-1β–antibody complexes allowed the detection of in vivo–produced IL-1β. A two-compartment mathematical model was generated that predicted a constitutive production rate of 6 ng/d IL-1β in healthy subjects. In contrast, patients with cryopyrin-associated periodic syndromes (CAPS), a rare monogenetic disease driven by uncontrolled caspase-1 activity and IL-1 production, produced a mean of 31 ng/d. Treatment with canakinumab not only induced long-lasting complete clinical response but also reduced the production rate of IL-1β to normal levels within 8 wk of treatment, suggesting that IL-1β production in these patients was mainly IL-1β driven. The model further indicated that IL-1β is the only cytokine driving disease severity and duration of response to canakinumab. A correction for natural IL-1 antagonists was not required to fit the data. Together, the study allowed new insights into the production and regulation of IL-1β in man. It also indicated that CAPS is entirely mediated by IL-1β and that canakinumab treatment restores physiological IL-1β production.

Methotrexate withdrawal at 6 vs 12 months in juvenile idiopathic arthritis in remission: a randomized clinical trial.

CONTEXT Novel therapies have improved the remission rate in chronic inflammatory disorders including juvenile idiopathic arthritis (JIA). Therefore, strategies of tapering therapy and reliable parameters for detecting subclinical inflammation have now become challenging questions. OBJECTIVES To analyze whether longer methotrexate treatment during remission of JIA prevents flares after withdrawal of medication and whether specific biomarkers identify patients at risk for flares. DESIGN, SETTING, AND PATIENTS Prospective, open, multicenter, medication-withdrawal randomized clinical trial including 364 patients (median age, 11.0 years) with JIA recruited in 61 centers from 29 countries between February 2005 and June 2006. Patients were included at first confirmation of clinical remission while continuing medication. At the time of therapy withdrawal, levels of the phagocyte activation marker myeloid-related proteins 8 and 14 heterocomplex (MRP8/14) were determined. INTERVENTION Patients were randomly assigned to continue with methotrexate therapy for either 6 months (group 1 [n = 183]) or 12 months (group 2 [n = 181]) after induction of disease remission. MAIN OUTCOME MEASURES Primary outcome was relapse rate in the 2 treatment groups; secondary outcome was time to relapse. In a prespecified cohort analysis, the prognostic accuracy of MRP8/14 concentrations for the risk of flares was assessed. RESULTS Intention-to-treat analysis of the primary outcome revealed relapse within 24 months after the inclusion into the study in 98 of 183 patients (relapse rate, 56.7%) in group 1 and 94 of 181 (55.6%) in group 2. The odds ratio for group 1 vs group 2 was 1.02 (95% CI, 0.82-1.27; P = .86). The median relapse-free interval after inclusion was 21.0 months in group 1 and 23.0 months in group 2. The hazard ratio for group 1 vs group 2 was 1.07 (95% CI, 0.82-1.41; P = .61). Median follow-up duration after inclusion was 34.2 and 34.3 months in groups 1 and 2, respectively. Levels of MRP8/14 during remission were significantly higher in patients who subsequently developed flares (median, 715 [IQR, 320-1 110] ng/mL) compared with patients maintaining stable remission (400 [IQR, 220-800] ng/mL; P = .003). Low MRP8/14 levels indicated a low risk of flares within the next 3 months following the biomarker test (area under the receiver operating characteristic curve, 0.76; 95% CI, 0.62-0.90). CONCLUSIONS In patients with JIA in remission, a 12-month vs 6-month withdrawal of methotrexate did not reduce the relapse rate. Higher MRP8/14 concentrations were associated with risk of relapse after discontinuing methotrexate. TRIAL REGISTRATION isrctn.org Identifier: ISRCTN18186313.

Faecal S100A12 as a non-invasive marker distinguishing inflammatory bowel disease from irritable bowel syndrome

Objective: S100A12 is a pro-inflammatory protein that is secreted by granulocytes. S100A12 serum levels increase during inflammatory bowel disease (IBD). We performed the first study analysing faecal S100A12 in adults with signs of intestinal inflammation. Methods: Faecal S100A12 was determined by ELISA in faecal specimens of 171 consecutive patients and 24 healthy controls. Patients either suffered from infectious gastroenteritis confirmed by stool analysis (65 bacterial, 23 viral) or underwent endoscopic and histological investigation (32 with Crohn’s disease, 27 with ulcerative colitis, and 24 with irritable bowel syndrome; IBS). Intestinal S100A12 expression was analysed in biopsies obtained from all patients. Faecal calprotectin was used as an additional non-invasive surrogate marker. Results: Faecal S100A12 was significantly higher in patients with active IBD (2.45 ± 1.15 mg/kg) compared with healthy controls (0.006 ± 0.03 mg/kg; p<0.001) or patients with IBS (0.05 ± 0.11 mg/kg; p<0.001). Faecal S100A12 distinguished active IBD from healthy controls with a sensitivity of 86% and a specificity of 100%. We also found excellent sensitivity of 86% and specificity of 96% for distinguishing IBD from IBS. Faecal S100A12 was also elevated in bacterial enteritis but not in viral gastroenteritis. Faecal S100A12 correlated better with intestinal inflammation than faecal calprotectin or other biomarkers. Conclusions: Faecal S100A12 is a novel non-invasive marker distinguishing IBD from IBS or healthy individuals with a high sensitivity and specificity. Furthermore, S100A12 reflects inflammatory activity of chronic IBD. As a marker for neutrophil activation, faecal S100A12 may significantly improve our arsenal of non-invasive biomarkers of intestinal inflammation.

Monitoring disease activity by stool analyses: from occult blood to molecular markers of intestinal inflammation and damage

It is a common experience that gastrointestinal symptoms urge us to differentiate inflammatory bowel disease (IBD) from functional disorders. Furthermore, in patients with proven IBD the disease activity has to be accurately monitored. Faecal markers of neutrophil influx into the mucosa are promising indicators of intestinal inflammation. Some neutrophil-derived proteins may be linked to the pathogenesis of IBD due to their functions as damage-associated molecular pattern molecules (DAMPs). Phagocyte-specific DAMPs of the S100 family are released from neutrophils or monocytes, followed by pro-inflammatory activation of pattern recognition receptors. The complex of S100A8/S100A9 was termed “calprotectin” and has been in use as a faecal marker for 10 years. More recently, faecal S100A12 has been reported to be an even more accurate faecal marker of inflammation. We review the biology of this novel group of molecules which can be used as surrogate markers directly linked to the molecular mechanisms of gut inflammation.

S100A12 is a novel molecular marker differentiating systemic‐onset juvenile idiopathic arthritis from other causes of fever of unknown origin

OBJECTIVE Fever of unknown origin (FUO) in children presents a diagnostic challenge. The differential diagnosis includes systemic-onset juvenile idiopathic arthritis (JIA), an autoinflammatory syndrome associated with activation of phagocytic cells that, at presentation, is difficult to differentiate from severe systemic infections. The aim of this study was to investigate whether serum concentrations of the phagocytic proinflammatory protein S100A12 may help in deciding whether to treat patients with FUO with antibiotics or immunosuppressive agents. METHODS Serum samples were obtained from 45 healthy control subjects and from 240 patients (60 with systemic-onset JIA, 17 with familial Mediterranean fever [FMF], 18 with neonatal-onset multisystem inflammatory disease [NOMID], 17 with Muckle-Wells syndrome [MWS], 40 with acute lymphoblastic leukemia [ALL], 5 with acute myeloblastic leukemia [AML], and 83 with systemic infections). All samples were collected at the time of presentation, before the initiation of any treatment, and concentrations of S100A12 were determined by enzyme-linked immunosorbent assay. RESULTS The mean +/- 95% confidence interval serum levels of S100A12 were as follows: in patients with JIA, 7,190 +/- 2,690 ng/ml; in patients with FMF, 6,720 +/- 4,960 ng/ml; in patients with NOMID, 720 +/- 450 ng/ml; in patients with MWS, 150 +/- 60 ng/ml; in patients with infections, 470 +/- 160 ng/ml; in patients with ALL, 130 +/- 80 ng/ml; in patients with AML, 45 +/- 60 ng/ml; in healthy control subjects, 50 +/- 10 ng/ml. The sensitivity and specificity of S100A12 to distinguish between systemic-onset JIA and infections were 66% and 94%, respectively. CONCLUSION S100A12, a marker of granulocyte activation, is highly overexpressed in patients with systemic-onset JIA or FMF, which may point to as-yet unknown common inflammatory mechanisms in these diseases. The measurement of S100A12 serum levels may provide a valuable diagnostic tool in the evaluation of FUO.

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

OBJECTIVE Fever of unknown origin is a diagnostic challenge in children, especially for differentiation of systemic-onset juvenile idiopathic arthritis (systemic-onset JIA) and infectious diseases. We undertook this study to analyze the relevance of myeloid-related proteins (MRPs) 8 and 14, endogenous activators of Toll-like receptor 4, in diagnosis and pathogenesis of systemic-onset JIA. METHODS Serum concentrations of MRP-8/MRP-14 were analyzed in 60 patients with systemic-onset 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-1beta (IL-1beta) 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 +/- 95% confidence interval 14,920 +/- 4,030 ng/ml) compared with those in healthy controls (340 +/- 70 ng/ml), patients with systemic infections (2,640 +/- 720 ng/ml), patients with acute lymphoblastic leukemia (650 +/- 280 ng/ml), patients with acute myeloblastic leukemia (840 +/- 940 ng/ml), and patients with NOMID (2,830 +/- 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-1beta expression in phagocytes. CONCLUSION The analysis of MRP-8/MRP-14 in serum is an excellent tool for the diagnosis of systemic-onset JIA, allowing early differentiation between patients with systemic-onset JIA and those with other inflammatory diseases. MRP-8/MRP-14 and IL-1beta represent a novel positive feedback mechanism activating phagocytes via 2 major signaling pathways of innate immunity during the pathogenesis of systemic-onset JIA.

Phagocyte‐specific S100 proteins are released from affected mucosa and promote immune responses during inflammatory bowel disease

Phagocyte‐derived S100 proteins are endogenous activators of innate immune responses. S100A12 binds to the receptor for advanced glycation end‐products, while complexes of S100A8/S100A9 (myeloid‐related proteins, MRP8/14; calprotectin) are ligands of toll‐like receptor 4. These S100 proteins can be detected in stool. In the present study we analyse the release of S100A12 and MRP8/14 from intestinal tissue. Specimens from patients with Crohns disease (CD; n = 30), ulcerative colitis (UC; n = 30), irritable bowel syndrome (IBS; n = 30) or without inflammation (n = 30) were obtained during endoscopy. After 24 h culture, S100A12 and MRP8/14 were analysed in supernatants. Endoscopic, histological, laboratory and clinical disease activity measures were documented. We found an increased spontaneous release of S100A12 from tissue in inflammatory bowel disease (IBD). The release of S100A12 into the supernatants was 28‐fold enhanced in inflamed tissue when compared to non‐inflamed tissue (mean 46.9 vs. 1.7 ng/ml, p < 0.0001). In active CD, release of S100A12 and MRP8/14 was strongly dependent on localization, with little release from sites of active ileal inflammation compared to colonic inflammation. This difference was more pronounced for S100A12 than for MRP8/14. S100A12 and MRP8/14 provoked up‐regulation of adhesion molecules and chemokines on human intestinal microvascular endothelial cells (HIMECs) isolated from normal colonic tissue. The direct release of phagocyte‐derived S100 proteins from inflamed tissues may reflect secretion from infiltrating neutrophils (S100A12) and also monocytes or epithelial cells (MRP8/14). Via activation of pattern recognition receptors, these proteins promote inflammation in intestinal tissue. The enhanced mucosal release can explain the correlation of fecal markers with disease activity in IBD. Copyright

Efficacy and safety of anakinra therapy in pediatric and adult patients with the autoinflammatory Muckle-Wells syndrome

OBJECTIVE Muckle-Wells syndrome (MWS) is an inherited autoinflammatory disease caused by mutations in the NLRP3 gene that result in excessive interleukin-1 (IL-1) release. It is characterized by severe fevers, rashes, arthralgia, and conjunctivitis, leading to sensorineural deafness and amyloidosis. The recombinant IL-1 receptor antagonist anakinra blocks the biologic activity of IL-1. The aim of this study was to determine the short- and long-term efficacy and safety of anakinra therapy in children and adults with severe MWS. METHODS A single-center observational study was performed. Standardized assessments included clinical features, the Disease Activity Score (DAS) for MWS, classic and novel markers of inflammation, and patient-derived measures of health status. The primary outcome was a score of <10 on the DAS for MWS at 2 weeks and at the last followup visit. Measures of MWS disease activity were investigated using descriptive statistics and paired comparative analysis. RESULTS A total of 12 patients with severe MWS (5 children and 7 adults) received anakinra for a median of 11 months (range 5-14 months). The median followup was 11 months (range 5-14 months). Disease activity was significantly lower in all patients at 2 weeks (P = 0.0005). Organ manifestations of MWS improved, as did all patient-derived measures of health status, markers of inflammation, and hearing loss in 2 of the patients. Levels of the novel neutrophil activation biomarker S100A12 followed clinical disease activity. Treatment was well tolerated, and no serious adverse events were observed. CONCLUSION Anakinra was found to be a safe and effective treatment of severe MWS, leading to a significant improvement in disease activity at 2 weeks as well as long-term. Anakinra therapy should therefore be considered in children and adults with severe MWS disease requiring IL-1 blockade.

The toll-like receptor 4 agonist MRP8/14 protein complex is a sensitive indicator for disease activity and predicts relapses in systemic-onset juvenile idiopathic arthritis

Background Analysis of myeloid-related protein 8 and 14 complex (MRP8/14) serum concentrations is a potential new tool to support the diagnosis of systemic-onset juvenile idiopathic arthritis (SJIA) in the presence of fever of unknown origin. Objective To test the ability of MRP8/14 serum concentrations to monitor disease activity in patients with SJIA and stratify patients at risk of relapse. Methods Serum concentrations of MRP8/14 in 52 patients with SJIA were determined by a sandwich ELISA. The monitoring of therapeutic regimens targeting interleukin 1 and tumour necrosis factor α, and methotrexate treatment was analysed and diagnostic power to predict flares was tested. Results MRP8/14 levels were clearly raised in active disease and decreased significantly in response to successful treatments. Serum concentrations of MRP8/14 increased significantly (p<0.001) (mean±95% CI 12.030±3.090 ng/ml) during disease flares compared with patients with inactive disease (864±86 ng/ml). During clinical remission MRP8/14 serum levels of >740 ng/ml predicted disease flares accurately (sensitivity 92%, specificity 88%). MRP8/14 levels correlated well with clinical disease activity, as assessed by physicians global assessment of disease activity (r=0.62), Childhood Health Assessment Questionnaire (r=0.56), active joint count (r=0.46) and with C-reactive protein (r=0.71) and erythrocyte sedimentation rate (r=0.72) (for all p<0.001). Conclusion MRP8/14 serum concentrations correlate closely with response to drug treatment and disease activity and therefore might be an additional measurement for monitoring anti-inflammatory treatment of individual patients with SJIA. MRP8/14 serum concentrations are the first predictive biomarker indicating subclinical disease activity and stratifying patients at risk of relapse during times of clinically inactive disease.