Myeloid related protein induces muscle derived inflammatory mediators in juvenile dermatomyositis
- Kiran Nistala†1,
- Hemlata Varsani†1,
- Helmut Wittkowski3,
- Thomas Vogl2,
- Petra Krol4,
- Vanita Shah1,
- Kamel Mamchaoui5,
- Paul A Brogan1,
- Johannes Roth2 and
- Lucy R Wedderburn1Email author
© Nistala et al.; licensee BioMed Central Ltd. 2013
Received: 10 February 2013
Accepted: 2 September 2013
Published: 23 September 2013
The aetiopathogenesis of juvenile dermatomyositis (JDM) remains poorly understood. In particular the contribution of monocytes or macrophages, which are frequently observed to be an infiltrate within muscle tissue very early in the disease process, is unknown. We hypothesised that these cells secrete the pro-inflammatory myeloid related protein (MRP) 8/14 which may then contribute to muscle pathology in JDM.
In this study of 56 JDM patients, serum MRP8/14 levels were compared with clinical measures of disease activity. Muscle biopsies taken early in disease were assessed by immunohistochemistry to determine the frequency and identity of MRP-expressing cells. The effects of MRP stimulation and endoplasmic reticulum (ER) stress on muscle were tested in vitro. Serum or supernatant levels of cytokines were analyzed by multiplex immunoassay.
Serum MRP8/14 correlated with physician’s global assessment of disease activity in JDM (R = 0.65, p = 0.0003) and muscle strength/endurance, childhood myositis assessment score (CMAS, R = −0.55, p = 0.004). MRP8/14 was widely expressed by CD68+ macrophages in JDM muscle tissue. When cultured with human myoblasts, MRP8 led to the secretion of MCP-1 and IL-6, which was enhanced by ER stress. Both inflammatory mediators were detected in significantly higher levels in the serum of JDM patients compared to healthy controls.
This study is the first to identify serum MRP8/14 as a potential biomarker for disease activity in JDM. We propose that tissue infiltrating macrophages secreting MRP8/14 may contribute to myositis, by driving the local production of cytokines directly from muscle.
Juvenile dermatomyositis (JDM) is a rare inflammatory disease of childhood affecting skin and muscle, frequently resulting in calcinosis, and sometimes with potentially life threatening complications including gut vasculitis and interstitial lung disease . Research into the disease pathogenesis has identified abnormalities of the immune system including circulating autoantibodies targeting nuclear antigens , dysregulation of T helper cell subsets  and a prominent interferon alpha (IFNα) gene signature in the peripheral blood of JDM patients . In JDM muscle, one of the earliest detectable changes is increased expression of Class I major histocompatibility complex (MHC) on skeletal muscle . Class I MHC can be upregulated non-specifically in response to muscle injury , but in JDM is itself thought to perpetuate the disease process . In animal models, forced over-expression of Class I protein leads to muscle fibre damage and a marked myeloid infiltrate into the muscle . One of the mechanisms of cellular injury, known as endoplasmic reticulum (ER) stress, results from accumulation of Class I protein within the ER which then activates multiple pathways, including the unfolded protein response (UPR) culminating in muscle cell death. Gene expression profiling from patients with adult myositis, as well as animal models, confirm that the UPR is up-regulated in myositis [7, 9]. Despite clear evidence that ER stress contributes to muscle cell death, it remains unclear if myocytes directly promote the inflammatory process and in particular the myeloid cell infiltrate frequently seen early in myositis, or whether this is merely a secondary response to tissue necrosis.
Once recruited, myeloid cells may potentiate the loss of muscle tissue by secreting a potent inflammatory heterodimeric protein, myeloid related protein (MRP) 8/14 (S100A8/A9)  which signals in a Toll-like receptor (TLR) 4 dependent manner  to induce apoptosis of skeletal muscle. MRP plays a pathogenic role in other childhood rheumatic diseases, including arthritis and vasculitis, and in these conditions it is a valuable biomarker of disease severity [12, 13]. In JDM, the clinical and serological markers of skin and muscle disease currently available lack sensitivity . In this study we examined the role of MRP8/14 as a biomarker of myositis and the mechanisms by which MRP8/14 may contribute to muscle disease. Our results show a close correlation between MRP8/14 levels in JDM serum and disease activity scores. MRP8/14 was expressed by CD68+ macrophages within JDM muscle and led to release of inflammatory mediators from muscle, an effect that was accentuated by ER stress. Our study identifies a novel pathway by which macrophage-muscle crosstalk can perpetuate inflammatory myositis.
Patient and control study groups
All patients were recruited to the Juvenile Dermatomyositis National Cohort Biomarker Study and Repository for Idiopathic Inflammatory Myopathies (hereafter called the JDM Cohort study), a UK-wide multicentre cohort study and biobank for JDM through the UK JDM Research Group (JDRG) . The JDM cohort study had ethical approval from the North Yorkshire Multi-Centre Research Ethics Committee and was also approved by the Steering Committee of the UK JDM Cohort Study. A total of 56 patients who fulfilled the Bohan and Peter criteria for definite or probable JDM  were included in this study. Eighteen children (6 boys, 12 girls, mean age 5.3 years), who had a muscle biopsy taken as part of the investigation of diverse symptoms, such as clumsiness and falls, were used as controls. As previously reported , none of the controls received a diagnosis of myositis or myopathy and the biopsies were all reviewed by two expert histopathologists and confirmed to show normal muscle histology for age and, therefore, considered to represent suitable control samples. Ethical approval was obtained from the National Hospital for Neurology and Neurosurgery and the Institute Of Neurology Joint research ethics committee to use control muscle biopsies and serum from healthy controls. All patients (or their carers) provided consent to participate in the study.
Laboratory and clinical measurements
Serum levels of the muscle enzymes lactate dehydrogenase (LDH) and creatine kinase (CK) were analysed by the Great Ormond Street Hospital clinical biochemistry department. Erythrocyte sedimentation rate (ESR) was measured by Great Ormond Street Hospital haematology department. Standardised outcome variables for the assessment of JDM, physician determined disease activity visual analogue score (VAS), childhood health assessment questionnaire (CHAQ), childhood myositis assessment score (CMAS) and parental global disease activity VAS [18, 19] were collected prospectively as part of the UK JDM Cohort Study, as described .
Measurement of MRP8/14, MCP-1 and IL-6 concentrations
Serum was extracted within three hours of collection of peripheral blood and stored at −80°C. Serum concentrations of MRP8/14 were determined by sandwich enzyme-linked immunosorbent assay (ELISA) as previously described . Monocyte chemoattractant protein-1 (MCP-1) levels were measured in serum using multiplex immunoassay (Meso Scale Discovery MSD technology, Meso Scale Diagnostics, USA). MCP-1 was measured in cell culture supernatants by ELISA (eBioscience, Hatfield, UK). IL-6, and in some experiments IL-1β, IL-10, IL-17, TNF-α and IFN-γ, measurements were performed using multiplex immunoassay .
Immunohistochemistry and immunofluorescence staining
Immunohistochemistry and immunofluorescence staining were performed on biopsies from quadriceps (vastus lateralis) which were snap frozen within one hour and stored at −80°C. Staining was performed on acetone fixed 7 μm cryostat sections. Immunohistochemistry was performed as described  using anti-human monoclonal antibodies to CD68 (KP1), CD3 (UCHT1), MHC class l heavy chain (W6/32; Novacastra, Milton Keynes, UK) and MRP8/14 (27E10 ). Degree of CD3 and CD68 infiltration was scored as 0, 1 or 2 as described : briefly <4 cells in ×20 field scored 0, >4 cells in ×20 and/or 1 cluster scored 1, >20 cells in ×20 and/or >2 clusters in whole biopsy scored 2. For double immunofluorescence staining the following anti-human monoclonal antibodies were used: CD68 (KP1; Novocastra,), CD14 (TUK4), CD15 (C3D-1; DakoCytomation, Cambridgeshire, UK), CD163 (RM3/1 ) and anti-human polyclonal MRP14 . Sections were incubated with primary antibodies at 4°C overnight followed by 30 minutes at room temperature with secondary antibodies sheep anti-mouse fluorescein isothiocyanate (FITC) (Sigma, Dorset, UK) and goat anti-rabbit alexa 568 (Life Technologies, Paisley, UK). Sections were mounted with Vectashield mounting medium with diamidino-2-phenylindole (DAPI) (Vector, Peterborough, UK) and analysed by fluorescent microscopy. For each patient, cells positive for lineage markers and MRP14 were counted in ten 10× fields. The mean percentage MRP14 cells expressing specific lineage markers were determined.
Cell culture and stimulation
LHCNM2, a human skeletal myoblast cell line, was derived from the pectoralis major muscle of a 41-year-old male Caucasian heart-transplant donor, in accordance with local ethical legislation . Myoblasts were grown in (Dulbecco’s) modified Eagle’s medium ((D)MEM) containing 4.5 mg/ml glucose + MEM 199 (at a ratio of 4:1), supplemented with 20% foetal calf serum (FCS), 100 IU penicillin, 100 μg streptomycin (GIBCO/Invitrogen, Paisley, UK). Myoblasts were cultured at 5 × 104/ml in six-well plates at 37°C in 5% C02 and the medium was refreshed when cells reached 60% confluence. To test the effects of TLR4 agonists, myoblasts were stimulated with lipopolysaccharide (LPS) or recombinant human MRP8, MRP14 or MRP8/14  or left in culture medium alone. In some experiments myoblasts were pre-incubated for four hours with thapsigargin, an ER calcium-ATPase inhibitor which induces ER stress . Thapsigargin was used at 0.05 μM, LPS at 100 ng/ml and MRP proteins at 5 μg/ml. Supernatants and cells were harvested as a time course over a 24-hour period. Supernatants were stored at −80°C for cytokine and chemokine measurements and cells were lysed in TRizol reagent (Invitrogen) for RNA extraction.
RNA isolation, cDNA synthesis and PCR
RNA from both myoblast cells and muscle tissue was isolated with TRIzol according to the manufacturer’s instructions. cDNA was synthesised using superscript II RT and random hexamers (Invitrogen). TLR4 expression on muscle tissue was analysed using Quantitect TLR4 primers (Qiagen, Crawley, UK). As a read out of ER stress, expression of x-box binding protein (XBP)-1 splice variants were analysed by PCR using the following primers: forward CGGAAGCCAAGGGGAATGAA, reverse CCCAACAGGATATCAGACTCTGA, at an annealing temperature of 62°C for 35 cycles. Primers to detect HPRT mRNA (Qiagen) were used as a housekeeping control for all PCR experiments. PCR products were visualised by 1.5% agarose gel electrophoresis. MCP-1 mRNA levels were quantified using real time PCR,  and HPRT gene expression was used for normalisation. cDNA was amplified using SYBR green (Bio-Rad Hertfordshire, UK) and thermo cycler rotor-gene 6000 Corbert (Qiagen). Data were analysed using Rotor-gene 6000 software (Qiagen).
Serum MRP levels, muscle enzymes and ESR were compared with clinical measures of disease activity using Spearman’s Rank. Where parametrically distributed, data were compared using 1 way or 2 way analysis of variance (ANOVA) and if appropriate paired t-tests. For non-parametric results, unpaired data were compared using the Mann–Whitney test. Differences in CD3/CD68 frequency were analysed by the Chi-Square test for trend. Results of P <0.05 were considered statistically significant. Analyses were carried out in SPSS version 18 (IBM, New York, USA).
Summary of patient demographics
Median (inter-quartile range)
Age at disease onset
6.0 (3.8 to 9.9) years
Number of male: female patients
0.5 years (0.25 to 1.8)
1.3 (1.0 to 2.2)
33 (14 to 48)
Physician’s Global Assessment
4.2 (2.4 to 7.4)
1018 (781 to 1759)
223 (56 to 1165)
19 (13 to 37)
Patients receiving methotrexate
14/48 (29%, 8 missing data)
Patients receiving cortico-steroids
24/51 (47%, 5 missing data)
Disease activity measures in JDM correlate with serum levels of MRP8/14
Identification of MRP8/14 expressing cells in inflamed muscle
Examining muscle biopsies for myeloid sub-populations, we frequently observed a heavy infiltration of macrophages, often without a T cell aggregate (Figure 2C, top panels). In contrast biopsies with large numbers of T cells invariably had a significant macrophage infiltrate (Figure 2C, bottom panels). This observation raised the possibility that T cell recruitment could depend on the early macrophage infiltrate, which establishes the appropriate chemokine milieu to attract lymphocytes from the circulation. To quantify the relative abundance of macrophages and T cells in JDM muscle, we used our biopsy score tool  which includes a semi quantitative assessment of cell infiltration (macrophage infiltration and T cell infiltration, each scored as 0, 1, or 2). Analysis of these biopsies (n = 28) showed that muscle sections scored significantly higher for macrophage infiltration, than for T cells (P <0.001, Chi square test for trend, Figure 2D); indeed, 25% of these early biopsies scored 0 for T cell infiltrate. Interestingly, none of the biopsies assessed had T cell infiltration in the absence of a macrophage population, which suggests that myeloid cells may be more important in the early tissue inflammation of JDM than previously recognised.
MRP8 drives the production of MCP-1 and IL-6 from inflamed muscle
XBP-1 and MRP8 interplay in muscle inflammation
The assessment of disease activity in JDM, and the distinction of disease flare from deconditioning or muscle atrophy, is still largely dependent on clinical evaluation. In this study, MRP8/14 has been identified as a novel biomarker of disease activity in JDM, and found to be superior to existing serological correlates of disease. By exploring the effects of MRP8 and MRP14 on skeletal muscle in vitro and correlating results with ex vivo JDM samples, we have identified a pro-inflammatory role of MRP8 for myositic muscle that we propose contributes to the enrichment of key inflammatory mediators MCP-1 and IL-6, seen in JDM muscle and serum, which then contribute to further recruitment of inflammatory cells to muscle. MRP8 has been shown to be the active component stimulating TLR4 in murine models of inflammation whereas MRP14 seems to have a regulatory function in the MRP8/14 complex. The exact mechanisms activating the MRP8/14 heterodimer in vivo are currently not clear but co-stimulation may be required .
MRP8/14 has been shown to be a highly sensitive marker of disease activity in a range of rheumatic disorders, including arthritis, vasculitis and autoinflammatory disease [12, 13]. As a biomarker, MRP8/14 has many characteristics that make it suitable for both clinical and research use; it is easily detected in serum even at low levels, is already in clinical use to detect gut inflammation  and is stable in clinical serum samples even when transported at room temperature. Most recently, we have successfully used MRP8/14 to identify patients with juvenile arthritis who are likely to remain in remission following withdrawal of immunosuppression by methotrexate . This proof of concept study confirms a role for MRP8/14 in the detection of sub-clinical disease activity in rheumatic disorders and could potentially apply to JDM to assist with the withdrawal of immunosuppression.
Other biomarkers have been identified in JDM, including the IFNα gene signature and IFN induced chemokines [4, 38–40]. IFNα is produced by plasmacytoid dendritic cells (pDC), and induces transcriptional activators which bind downstream response elements in promoter sequences (IFN-stimulated response elements, ISRE), enhancing the transcription of many immune related genes including the chemokine MCP-1 . Our results suggest that MCP-1 can also be produced by a MRP-dependent pathway, by muscle fibres themselves in JDM. It is, therefore, of interest, that in a recent report MCP-1, an IFNα associated chemokine correlated better with disease activity than the IFNα gene signature itself . This may suggest that IFNα-independent production of MCP-1, including muscle derived MCP-1, may play a role in juvenile myositis.
It is striking that JDM biopsies that were taken relatively early in disease (median disease duration six months), already show a major infiltration of monocytes/macrophages, often in the absence of cells from the adaptive immune system. Previous studies have not clearly defined the role of such infiltrating myeloid cells in muscle cell damage and inflammation during myositis. Our results clearly show that MRP8 and MRP14 are secreted by CD68+ infiltrating cells and that these are largely CD163- suggesting that they are indeed pro-inflammatory in phenotype. We propose that the local secretion of MRP proteins by these cells has several downstream effects, including the stimulation of muscle to produce MCP-1 and IL-6. MCP-1 secreted by skeletal muscle, in response to MRP, may then play an important role in propagating the inflammatory infiltrate. Cells recruited into DM muscle, including monocytes and memory T cells, express high levels of CCR2, the sole receptor for MCP-1 [42, 43]. MRP8 and MRP14 may be important in linking an initial innate immune response with a later adaptive one by recruiting CCR2+ memory T cells and supporting the differentiation of local B cells into plasma cells as this is dependent on signalling through CCR2 . As further evidence of the importance of MCP-1 in myositis, animal models have implicated this chemokine in a transgenic model of selective over expression of self MHC Class l in skeletal muscle, MHC over expression induced MCP-1 production  and, in a model of viral induced myositis, blockade of MCP-1 significantly attenuated muscle inflammation .
Our results demonstrating that MRP8 induced IL-6 and MCP-1 secretion by myoblasts add to a growing body of evidence that suggests that muscle itself contributes to the inflammatory process [8, 46]. To test how muscle derived cytokine secretion would be altered by non-immune insults to the muscle, known to occur in DM [7–9], we adopted a thapsigargin induced model of muscle ER stress. Using this system we have now identified ER stress as a mechanism for priming myoblasts to secrete IL-6 in response to a second signal, such as macrophage derived MRP8 binding TLR4. This is pertinent to myositis as IL-6 is known to correlate with disease activity in idiopathic inflammatory myositis (IIM) and IL-6 blockade attenuates muscle inflammation in some mouse models . Given that IL-6 is produced by a range of immune cells, including macrophages, B and T cells, it is difficult to discern the exact contribution made by skeletal muscle towards the enrichment of this cytokine in JDM serum. Nevertheless, MCP-1 and IL-6 appear to be tightly co-regulated in JDM serum  and, given that both are expressed by inflamed muscle, it is possible that the close correlation between serum levels and muscle disease activity [4, 40] is explained by the muscle itself being a key source of these cytokines in JDM.
One limitation of our study is that we are unable to exclude the action of MRP8 on other receptors apart from TLR4, such as the for advanced glycation end products (RAGE) . However, data from the murine system would suggest that TLR4 is the dominant receptor for MRP8 .
This study contributes novel insights into the possible roles of macrophage derived MRP8 and MRP14 in driving production of chemokines and cytokines by muscle cells. These data emphasise the importance of skeletal muscle as an organ with the potential for immune functions and demonstrate how cross talk between muscle and the innate immune system can be instrumental in sustaining further adaptive responses as well as on-going inflammation in autoimmune muscle disease.
Our results highlight the relationship between serum MRP8/14 levels and disease activity in JDM, and identify a novel mechanism by which macrophage derived MRP8/14 may directly activate muscle cells and, thereby, perpetuate inflammatory myositis. Further prospective studies are required to test the role of MRP8/14 as a putative biomarker for disease activity in JDM.
Analysis of variance
Childhood health assessment questionnaire
Childhood myositis assessment score
Enzyme-linked immunosorbent assay
Erythrocyte sedimentation rate
Monocyte chemoattractant protein-1
Major histocompatibility complex
Myeloid related protein
Polymerase chain reaction
Unfolded protein response
Visual analogue score
x-box binding protein.
The Juvenile Dermatomyositis Research Group would like to thank all of the patients and their families who contributed to the Juvenile Dermatomyositis Cohort Study. We thank all local research coordinators and principal investigators who have made this research possible. The members who contributed are as follows: Dr Kate Armon and Mr Joe Ellis-Gage (Norfolk and Norwich University Hospitals), Dr Liza McCann, Mr Ian Roberts, Dr Eileen Baildam and Ms Louise Hanna (The Royal Liverpool Children’s Hospital, Alder Hey, Liverpool), Dr Phil Riley and Ms Ann McGovern (Royal Manchester Children’s Hospital, Manchester), Dr Clive Ryder and Mrs. Janis Scott (Birmingham Children’s Hospital, Birmingham), Dr Sue Wyatt, Mrs Gillian Jackson, Dr Tania Amin, Mark Wood and Vanessa Van Rooyen (Leeds General Infirmary, Leeds), Dr Joyce Davidson, Dr Janet Gardner-Medwin, Dr Neil Martin and Ms Sue Ferguson (The Royal Hospital for Sick Children, Yorkhill, Glasgow), Dr Mark Friswell, Professor Helen Foster, Mrs Alison Swift, Dr Sharmila Jandial, Ms Vicky Stevenson and Ms Debbie Wade (Great North Children’s Hospital, Newcastle), Dr Helen Venning, Mrs Elizabeth Stretton and Ms Mary Jordan (Queens Medical Centre, Nottingham), Professor Lucy Wedderburn, Dr Clarissa Pilkington, Dr N. Hasson, Mrs Sue Maillard, Ms Elizabeth Halkon, Ms Virginia Brown, Ms Audrey Juggins, Dr Sally Smith, Mrs Sian Lunt, Ms Elli Enayat, Mrs Hemlata Varsani, Miss Laura Beard and Miss Katie Arnold (Great Ormond Street Hospital, London), Dr Kevin Murray (Princess Margaret Hospital, Perth, Western Australia) Dr John Ioannou (University College London Hospital).
We thank Dr Vincent Mouly, Institut de Myologie, Paris for advice on muscle cell lines and Dr Jane Goodall, Cambridge University for providing XBP-1 primer sequences.
The JDM Cohort Study and this work have been supported by generous grants from the Wellcome Trust UK (085860), Action Medical Research UK, (SP4252), and The Henry Smith Charity. The JDM Cohort study has been adopted onto the Comprehensive Research Network through the Medicines for Children Research Network (http://www.mcrn.org.uk). LW is supported in part by the Great Ormond Street Hospital Children’s Charity. KN is a Wellcome Trust Intermediate Clinical Fellow (097259). TV and JR were supported by grants from the Interdisciplinary Centre for Clinical Research at the University of Muenster (Vo2/014/09 to T.V. as well as grant Ro2/004/10 to J.R) and the Deutsche Forschungsgemeinschaft (DFG project RO 1190/9-1).
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