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Clinical and epidemiological research
Extended report
Neutrophil-derived S100A12 as novel biomarker of inflammation in familial Mediterranean fever
  1. Tilmann Kallinich1,
  2. Helmut Wittkowski2,
  3. Rolf Keitzer1,
  4. Johannes Roth2,3,
  5. Dirk Foell2
  1. 1Pediatric Pneumology and Immunology, Charite University Hospital Berlin, Berlin, Germany
  2. 2Department of Pediatrics, University of Muenster, Muenster, Germany
  3. 3Institute of Immunology, University of Muenster, Muenster, Germany
  1. Correspondence to Tilmann Kallinich, Department of Pediatric Pneumology and Immunology, Charité–Universitaetsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany; tilmann.kallinich{at}charite.de

Abstract

Objective Familial Mediterranean fever (FMF) is characterised by recurrent periodic febrile attacks and persistent subclinical inflammation. The damage-associated molecular pattern (DAMP) protein S100A12 has proven to be a sensitive marker for disease activity and inflammation in numerous inflammatory disorders. The aim of this study was to analyse the role of S100A12 in the detection of inflammation in patients with FMF.

Methods 52 children and adolescents with a clinical and/or genetic diagnosis of FMF were prospectively followed-up over 18 months (in total 196 visits). During clinical visits, erythrocyte sedimentation rate, C reactive protein, serum amyloid A and S100A12 serum concentrations were determined. Patients were categorised into four groups according to the clinical activity of FMF.

Results Serum concentrations of S100A12 were excessively increased in patients with a mean increase of about 290-fold in active FMF above normal controls. S100A12 decreased significantly after introduction of colchicine therapy. Serum concentrations of S100A12 were significantly higher in patients treated with colchicine with persistent symptoms (mean±SEM, 6260±2120 ng/ml) than in those with clinically controlled disease (440±80 ng/ml, p<0.001). In contrast to classical markers of inflammation, S100A12 was significantly elevated in clinically unaffected homozygous MEFV gene mutation carriers, indicating subclinical inflammation.

Conclusions S100A12 is a valuable biomarker for monitoring disease activity, inflammation and response to colchicine treatment in patients with FMF. It might even be more sensitive in detecting subclinical inflammation than other available indicators.

https://doi.org/10.1136/ard.2009.114363

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    Introduction

    Familial Mediterranean fever (FMF) (OMIM database entry 249100) is the most common autoinflammatory disorder caused by mutations within the MEFV gene.1 2 This gene encodes for pyrin, a protein which mainly inhibits the production of the proinflammatory cytokine interleukin 1β by interfering with the so-called inflammasome reaction.3 4 This inhibitory control mechanism is lost through mutations within the MEFV gene, leading to a permanent activation of proinflammatory reactions.5 6

    The clinical course is characterised by unprovoked and self-limited febrile attacks occurring at irregular intervals and accompanied by signs of serositis. These local inflammatory reactions are mainly mediated by a massive infiltration of neutrophils.7 The most serious long-term complication is amyloid deposition, which occurs in at least one out of four untreated patients7 and is currently present in 11.4% of patients.8

    Continuous colchicine use is the well established prophylactic therapy to prevent the occurrence of febrile attacks as well as the development of amyloidosis.9,–,12 This drug interferes with neutrophilic functions, for example, the intracellular granula transport, the chemotaxis and the expression of adhesion molecules.13 However, the precise action of colchicine in FMF is still unknown. But despite sufficient colchicine treatment, a variable degree of subclinical inflammation persists.14,–,17 In light of the potential risk for the development of amyloid deposits and the impact of subclinical inflammation on the general wellbeing of the patient, it is therefore mandatory to establish inflammation markers with the highest possible sensitivity in order to thoroughly follow-up the patient and adjust the treatment.

    Numerous studies in patients with inflammatory disorders showed an excellent correlation between disease activity and serum concentrations of S100A12, thus demonstrating its potential as a sensitive marker of inflammation.18 In a recently published study we demonstrated that patients with active FMF exhibit vastly elevated serum levels of S100A12. Similar concentrations were only found in systemic juvenile idiopathic arthritis (JIA) but not in other inflammatory disorders.19 This protein is a member of the damage-associated molecular pattern (DAMP) family of molecules, which represent a group of danger signals sharing characteristics of cytokines. After being released by activated or necrotic cells these proteins mediate inflammatory responses and recruit immune cells to the damaged tissue. S100A12 is exclusively expressed by activated granulocytes20 and binds to the multiligand receptor for advanced glycation end products on endothelium, mononuclear phagocytes and lymphocytes21 leading to induction of adhesion molecules and nuclear factor (NF)κB-mediated upregulation of proinflammatory cytokines.21 22

    Since (1) S100A12 was shown to be associated with active FMF, (2) S100A12 is exclusively expressed and released by activated neutrophils, the cell type most abundantly found in the affected sites in FMF, (3) the most effective drug for the treatment of FMF, colchicine, interacts with neutrophil function and (4) S100A12 was shown to be a sensitive marker of inflammation in other conditions driven by the action of phagocytic cells, we performed a prospective study to further delineate the potential of S100A12 to be used as a sensitive marker of inflammation in children and adolescents with FMF. Therefore, the serum levels of S100A12 were compared to conventional biomarkers of inflammation and correlated to the clinical phenotype.

    Methods

    Determination of erythrocyte sedimentation rate, C reactive protein and serum amyloid A protein

    Erythrocyte sedimentation rate (ESR) was determined directly in the outpatient department by means of the Westerngren method. The Sediplus kit (Sarstedt, Nuernbrecht, Germany) was applied. C reactive protein (CRP) was analysed in the hospital laboratory of the Charité, Berlin. According to the manufacturer normal range is below 5 mg/litre. Samples for the analysis of serum amyloid A protein (SAA) were sent to an external reference laboratory (Limbach, Heidelberg, Germany). Concentrations were determined by a latex-enhanced nephelometric immunoassay (N Latex SAA; Dade Boehring Diagnostic, Schwalbach, Germany). The normal range is <10 mg/litre. All patients gave informed consent.

    Determination of S100A12

    Serum samples were centrifuged within 2 h after acquisition and were frozen at −20°C until measurement. Concentrations of S100A12 were determined in patient sera by a double sandwich ELISA system established in our laboratory, as described previously.23 The interassay and intra-assay coefficients of variation were 12.1 and 4.8%, respectively.24 The readers of the laboratory assay were blinded for the diagnosis. For comparison with earlier studies internal control sera were included in all ELISA studies.

    Statistical analysis

    SPSS V.13.0 (SPSS, Chicago, Illinois, USA) for Windows was used for statistical analyses. Rank differences were analysed using the Mann–Whitney U test. Data are expressed as mean±SEM except where stated otherwise. There were no missing test results, and no indeterminate or outliers were excluded.

    Results

    Patients

    Between April 2006 and October 2007 196 visits and samples from 52 children and adolescents, who attended the outpatient clinic for paediatric rheumatology at the Children's Hospital of the Charité, Berlin, were analysed. Patients, with highly suspected or definite FMF as well as healthy MEFV carriers, were consecutively recruited and followed-up during the study period. This study was approved by the institutional review board.

    Five children had two disease-associated mutations within the MEFV gene, but never showed any symptoms of FMF. A total of 47 patients fulfilled the clinical criteria for FMF.25 As soon as the diagnosis was established, these patients had been treated with daily colchicine doses.26 The median age of the patients at the start of the study was 10.3 years (range 3.2–20.4 years). A total of 18 girls and 34 boys were included into the study. Detailed patients characteristics are summarised in table 1.

    View this table:
    Table 1

    Patient characteristics

    During all visits the history was taken and a clinical examination was performed by a paediatrician experienced in the treatment of FMF (TK). Blood was drawn for routine examination and one additional serum sample was taken for the S100A12 analysis.

    Patients were grouped into the following categories:

    1. Stable FMF: patients continuously taking colchicine and not showing any symptoms characteristic for FMF during the study period (n=28).

    2. Unstable FMF: patients continuously taking colchicine and showing symptoms characteristic for FMF at any time during the study period (n=19).

    3. MEFV mutation carriers: individuals with two mutations within the MEFV gene (detailed characteristics depicted in table 1), who never had any symptoms characteristic for FMF and thus never received colchicine (n=5).

    4. Untreated FMF: patients newly diagnosed as having FMF during the study period (n=7). After introduction of colchicine these patients switched into group 1 or 2.

    Serum concentrations of S100A12 in different activity stages of FMF

    Serum samples from patients with newly diagnosed FMF (‘untreated FMF’) revealed a mean (±SEM) S100A12 concentration of 33 500±22 200 ng/ml (figure 1A). This concentration is significantly increased compared to the other groups. In patients treated with colchicine and showing symptoms characteristic for FMF at any time during the study period (‘unstable FMF’) the mean serum concentration was 6260±2120 ng/ml, and thus being significantly increased compared to the group of patients with well controlled disease (‘stable FMF’, p<0.001), who had the lowest serum concentration (440±80 ng/ml). However, even in this group the levels were above the normative age-matched paediatric cut-off value (120 ng/ml).27

    Figure 1
    Figure 1

    Inflammatory markers in familial Mediterranean fever (FMF). Serum concentration of S100A12 (A), C reactive protein (CRP) (B), erythrocyte sedimentation rate (ESR) (C) and serum amyloid A protein (SAA) (D) were determined at different activity stages of FMF. Patients were grouped into the following categories: ‘stable FMF’ treated with colchicine and not showing any symptoms; ‘unstable FMF’ treated with colchicine and showing any symptoms during study period; ‘MEFV mutation carriers’ exhibiting two mutations within the MEFV gene, never having shown any symptoms characteristic for FMF and thus not being treated with colchicine, and newly diagnosed ‘untreated FMF’. Scatter plot with y axis in logarithmic scale. Horizontal grey line indicates mean. Grey dashed lines represent upper limits of the normal range for each parameter.

    Interestingly, in participants exhibiting two mutations within the MEFV gene but never showing symptoms characteristic for FMF (‘MEFV mutation carriers’) the mean serum concentration was 1710±1160 ng/ml. This concentration is significantly higher than the concentration in the serum samples from the stable patients with FMF (p=0.02), but did not show any statistical significant difference when compared to unstable patients with FMF (p=0.1). As in patients with overt FMF, the most significant elevation above normal was also seen in one apparently healthy individual with a homozygous M694V mutation (6260 ng/ml).

    Other markers of inflammation in the different groups

    The concentrations for CRP (99±39 mg/litre) and SAA (469±84 mg/litre) as well as the ESR (61±8 mm/h) were significantly higher in patients with newly diagnosed as having FMF (‘untreated FMF’) compared to all other activity stages of FMF (figure 1B–D). The serum levels of CRP (34±6 mg/litre) and SAA (185±71 mg/litre) were also clearly increased in the group of unstably treated patients with FMF compared to stably treated patients showing no symptoms (CRP: p<0.001; SAA: p=0.04) and participants with the genetic diagnosis of FMF (CRP: p=0.007; SAA: p=0.006). In contrast, the ESR did not discriminate between unstably treated patients with FMF and clinically unaffected individuals with genetically FMF (p=0.09).

    Comparison of inflammation markers in active FMF

    In a group of seven patients with newly diagnosed FMF the increase of the different inflammation markers were compared to normal values of healthy participants. The fold increase is shown as the ratio between individual values of the different biomarkers and the regular cut-offs (S100A12 120 ng/ml, CRP 5 mg/litre, ESR 15 mm/h, SAA 10 mg/litre; figure 2). Comparing the different inflammation markers demonstrates the unique increase of S100A12 in active FMF, with a mean increase of 290-fold increase above the regular cut-off. The other inflammation markers were increased to a much lower extent (mean fold increase for CRP 20, for ESR 4 and for SAA 40).

    Figure 2
    Figure 2

    Fold increase of inflammation markers above regular cut-off in seven patients with untreated active familial Mediterranean fever (FMF) (new-onset FMF group). The fold increase of the S100A12 concentration above 120 ng/ml, the C reactive protein (CRP) concentration above 5 mg/litre, the erythrocyte sedimentation rate (ESR) above 15 mm in the first hour and the serum amyloid A protein (SAA) concentration above 120 ng/ml were calculated. Scatter plot with y axis in logarithmic scale. Horizontal grey line indicates mean.

    Response to colchicine treatment

    In seven patients with newly diagnosed FMF a continuous prophylactic colchicine therapy was introduced and S100A12 levels were determined before treatment and during three follow-up visits (figure 3). Colchicine treatment led to a dramatic decrease of serum S100A12 concentration to 0.3% to 3% of the initial concentrations. Two patients initially exhibited excessively increased S100A12 serum concentrations (grey dashed line, circles; black solid line, circles). In the first patient, age-adapted colchicine medication (0.5 mg/day) initially did not induce a significant reduction of S100A12 serum levels, whereas in the second age-adapted colchicine medication (1.5 mg/day) led to a rapid decrease of concentration from 163 300 to 740 ng/ml. In all other patients serum levels rapidly decreased to normal values (<120 ng/ml) after introduction of colchicine medication.

    Figure 3
    Figure 3

    Decrease of S100A12 serum concentration in response to therapy. S100A12 concentrations were analysed in seven patients with new-onset familial Mediterranean fever (FMF) at time of diagnosis (baseline) and at three follow-up visits after introduction of colchicine therapy. Depending on the therapy response and possible dose adaption the follow-up period lasted from 3.5 to 12 months. The highest initial values were from the patients with homozygous M694V mutations. The y axis is in logarithmic scale. Grey dashed lines represent upper limits of the normal range for each parameter.

    S100A12 concentrations in patients with different mutations

    Figure 4 shows the S100A12 concentrations according to the different mutations, which were found in nine patients of the cohort. S100A12 concentration was significantly higher (p<0.001) in the serum from patients with a homozygous carrier status for M694V (n=19) compared to patients harbouring M694x/V726A (8 × M694V/V726A and 2 × M694I/V726A) and M680I/M694V (n=9). CRP, ESR and SAA were not significantly different between these groups (data not shown). The 11 patients with unstable FMF and homozygous M964V mutations had significantly higher levels (p<0.001) than the 7 patients with these mutations but stable disease, indicating that the mutation does influence levels in patients with incomplete control of their disease activity. In patients who are clinically stable, the mutation does not influence S100A12 levels. The patients with homozygous M694V mutations were underrepresented in the group of stable patients with FMF, a fact underlying the greater likelihood of unstable disease in these patients.

    Figure 4
    Figure 4

    S100A12 serum concentrations according to the most frequently found mutations. Bars represent the serum concentration of S100A12 in patients harbouring the most frequently found mutations according to disease activity (***p<0.001). Scatter plot with y axis in logarithmic scale. Horizontal grey line indicates mean. FMF, familial Mediterranean fever.

    Discussion

    The primary goal of this study was to delineate the potential of S100A12 molecules as sensitive biomarker of inflammation in patients with FMF. We found S100A12 highly expressed in active forms of FMF with a roughly 290-fold rise in concentration, which exceeds by far the observed values for the other markers of inflammation, that is, ESR, CRP and SAA. These findings confirm that excessively elevated concentrations of S100A12 are exclusively found in the systemic form of JIA and active forms of FMF and can be used to differentiate these conditions from other forms of fever of unknown origin.19 Genetic analysis can verify the clinical diagnosis of FMF, but since FMF can also be present in patients harbouring only one or even no mutation within the MEFV gene28 and since the impact of many mutations on the disease expression is still a matter of debate, this method cannot be taken as gold standard. The measurement of S100A12 serum levels as an additional diagnostic tool can now help to rapidly establish the correct diagnosis of FMF and to differentiate it from other causes of recurrent fever.19

    Numerous studies performed in patients with different autoimmunological disorders demonstrated an excellent correlation of S100A12 serum concentrations with the degree of inflammation, favouring this proinflammatory molecule as diagnostic biomarker for monitoring disease activity.18 This holds also true for patients experiencing FMF: after introduction of a sufficient prophylactic colchicine therapy the serum levels of S100A12 dramatically dropped to 0.3% to 3% of the initial values. Moreover, in patients who have been treated S100A12 can differentiate between those patients with well controlled disease and those still showing symptoms of FMF. Interestingly, and in contrast to the other markers of inflammation, in the group of patients harbouring two mutations within the MEFV gene but never showing any symptoms characteristic for FMF (‘MEFV mutation carriers’) it is significantly elevated compared to the group of patients with well controlled disease. Thus, S100A12 molecules might even be more sensitive in the detection of inflammation then the other markers.

    It is striking that S100A12 levels in patients with active FMF clearly differ from those in other disorders including other autoinflammatory syndromes with genetic background. The only exception from this rule is active systemic-onset JIA. This is interesting since in both diseases a disturbed activation of alternative secretory pathways especially relevant in neutrophils has been proposed to be involved in the molecular processes.29

    The ideal tool to monitor inflammation should be directly involved in the pathophysiological processes of the disease, rather than being a sole surrogate marker. For S100A12 there is strong evidence, that this molecule indeed measures inflammation in FMF: (1) S100A12 is a DAMP which mediates inflammatory responses and recruit immune cells to the damaged tissue;30 (2) at concentrations measured in the serum of patients with active FMF, S100A12 induces clear proinflammatory effects in vitro (20 000 ng/ml), for example, upregulation of adhesion molecules, chemokines and cytokines, chemotaxis and loss of endothelial integrity;22 (3) S100A12 is expressed and released by activated granulocytes20 and overexpressed at sites of local inflammation.31 These cells also massively infiltrate affected sites in FMF7 and express the MEFV gene;32 (4) the effective prophylactic colchicine therapy disrupts neutrophilic function13 and leads to a dramatic decrease in the serum concentrations of the neutrophil-derived S100A12 (figure 3). Taken together, S100A12 molecules reflect the activity of the cell type mainly responsible for the pathopyhsiological processes in FMF and targeted by the standard therapy. Unfortunately, the period of follow-up of our study was not long enough to allow us to analyse, whether S100A12 can be used to predict disease flare and a necessary colchicine dose adjustment. However, for JIA it is known, that S100A12 serum concentrations can forecast relapses.33 This clinical observation underlines the suitability of S100A12 as a marker for inflammation.

    In summary, this study illustrates the accuracy of the S100A12 molecules as sensitive indicator for inflammation in FMF. Further studies are warranted to establish their role as an early indicator of relapses and/or amyloidosis and their potential to fine tune colchicine therapy more precisely.

    Acknowledgments

    The authors thank Melanie Saers and Dorothee Lagemann for excellent technical assistance and Annette Schmidt, Andrea Rothmeier and Hannelore Bollmann for helping with sample asservation.

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    Footnotes

    • TK and HW contributed equally to this work.

    • Funding The study was supported by grants of the Interdisciplinary Centre for Clinical Research, University of Muenster, Germany (Project Foe2/005/06), the German Research Foundation (DFG Project FO 354/2–2) and of the German Ministry of Education and Research (BMBF, project ‘AID-NET’). The funding sources had no involvement in the study design; in the collection, analysis and interpretation of the data; in the writing of the report; and in the decision to submit the paper for publication.

    • Competing interests JR has a pending patent application for S100A12 ELISA (US 20030175713). All other authors declare that they have no financial or personal competing interest related to this study.

    • Ethics approval This study was conducted with the approval of the University Muenster.

    • Provenance and peer review Not commissioned; externally peer reviewed.