Correlation of antisignal recognition particle autoantibody levels with creatine kinase activity in patients with necrotizing myopathy.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 63, No. 7, July 2011, pp 1961–1971 DOI 10.1002/art.30344 © 2011, American College of Rheumatology Correlation of Anti–Signal Recognition Particle Autoantibody Levels With Creatine Kinase Activity in Patients With Necrotizing Myopathy Olivier Benveniste,1 Laurent Drouot,2 Fabienne Jouen,3 Jean-Luc Charuel,4 Coralie Bloch-Queyrat,1 Anthony Behin,5 Zahir Amoura,4 Isabelle Marie,3 Marguerite Guiguet,6 Bruno Eymard,5 Danièle Gilbert,2 François Tron,3 Serge Herson,1 Lucile Musset,4 and Olivier Boyer3 autoimmune conditions or polyclonal hypergammaglobulinemia. Among the 31 anti-SRP–positive patients, serum samples from 8 patients were monitored over time for levels of anti-SRP autoantibodies and levels of CK (determined at least 3 times, consecutively, over a mean followup period of 783 days). The relationship between levels of anti-SRP autoantibodies and levels of CK was tested using a linear mixed model. Results. The assay yielded positive results for anti-SRP in all anti-SRP immunodot–positive serum samples tested, while all control sera tested negative. The 8 anti-SRP–positive patients who were followed up longitudinally were found to have normalized CK levels and improved muscle strength. There was a striking correlation between the degree of myolysis, as measured by CK levels, in patients receiving therapy and the anti-SRP54 autoantibody levels in these same patients (P ⴝ 0.002). Conclusion. Anti-SRP–positive myositis appears to be one of the few autoimmune diseases in which specific autoantibody levels are correlated with surrogate disease activity markers. These results reveal the usefulness of monitoring anti-SRP autoantibody levels in patients receiving therapy, and may also suggest a possible pathogenic role for anti-SRP autoantibodies in the necrotizing myopathies. Objective. Anti–signal recognition particle (antiSRP) autoantibodies are associated with severe acquired necrotizing myopathies. The role of these autoantibodies remains elusive, and the evolution of antiSRP levels over time is unknown. In this study, we developed an addressable laser bead immunoassay (ALBIA) technique to investigate a correlation between anti-SRP levels, serum creatine kinase (CK) levels, and muscle strength in patients with necrotizing myopathy. Methods. The diagnostic value of the ALBIA assay was determined by comparing serum levels of anti-SRP autoantibodies in 31 anti-SRP immunodot– positive patients to those in 190 healthy blood donors and 199 control patients with different inflammatory/ Supported in part by the Association Française Contre les Myopathies. 1 Olivier Benveniste, MD, PhD, Coralie Bloch-Queyrat, MD, PhD, Serge Herson, MD: Université Pierre et Marie Curie, Hôpital Pitié-Salpêtrière, Assistance Publique–Hôpitaux de Paris, Paris, France; 2Laurent Drouot, MSc, Danièle Gilbert, PhD: INSERM, U905, Université de Rouen, Rouen, France; 3Fabienne Jouen, MD, Isabelle Marie, MD, PhD, François Tron, MD, PhD, Olivier Boyer, MD, PhD: INSERM, U905, Université de Rouen, CHU de Rouen, Rouen, France; 4Jean-Luc Charuel, PharmD, Zahir Amoura, MD, PhD, Lucile Musset, PharmD: Hôpital Pitié-Salpêtrière, Assistance Publique–Hôpitaux de Paris, Paris, France; 5Anthony Behin, MD, Bruno Eymard, MD, PhD: Institut de Myologie, Paris, France; 6 Marguerite Guiguet, PhD: INSERM, U943, Université Pierre et Marie Curie, Paris, France. Dr. Benveniste and Mr. Drouot contributed equally to this work. Drs. Benveniste, Jouen, and Boyer and Mr. Drouot have filed a European patent application for a diagnostic method for assaying anti-SRP antibodies. Address correspondence to Olivier Boyer, MD, PhD, INSERM, U905, Université de Rouen, Faculté de Médecine et Pharmacie, 22 Boulevard Gambetta, F-76000 Rouen, France. E-mail: email@example.com. Submitted for publication August 18, 2010; accepted in revised form March 3, 2011. Autoantibodies directed against signal recognition particles (anti-SRP) are present in a minority (4–6%) of patients with acquired inflammatory and/or necrotizing myopathies (1–5). Anti-SRP autoantibodies are generally associated with severe clinical forms of the disease, particularly those with heart and lung involvement (6,7) and resistance to steroid therapy (8–10). 1961 1962 BENVENISTE ET AL Slowly progressive myopathies associated with anti-SRP autoantibodies and mimicking limb-girdle muscular dystrophies have also been described (11,12). Growing pathologic evidence further suggests that anti-SRP– positive myopathy is a distinct form of myositis, characterized by substantial muscle necrosis contrasting with little or no inflammatory infiltrates, expression of HLA class I, and particular patterns of complement C5b-9 deposition. The SRP complex is composed of 6 SRP family proteins associated with a small RNA. Its physiologic role is to guide the translocation of growing polypeptides into the endoplasmic reticulum during protein synthesis. Although recognition of each of the 6 SRP subunits or the 7S RNA has been detected in anti-SRP–positive sera, the signal peptide–binding 54-kd subunit of SRP (SRP54) (13) remains the main target, and reactivity to SRP54 is always present (14–16). Since the expression of SRP is ubiquitous, the role of anti-SRP autoantibodies in myositis remains elusive. Anti-SRP autoantibodies purified from patients’ sera can inhibit the in vitro translocation of secretory proteins into the endoplasmic reticulum (16). Therefore, one can hypothesize that anti-SRP autoantibodies may play a role in disease pathogenesis. Given that the level of serum creatine kinase (CK) reflects muscle necrosis in a given patient, studying the correlation between anti-SRP autoantibody levels and CK activity would be helpful to better understand the pathogenesis of this form of acquired myopathy and may define a surrogate marker of disease activity. Nevertheless, this has been hampered to date by the lack of a quantitative anti-SRP assay. Currently, the detection of anti-SRP autoantibodies is based on an indirect immunofluorescence test on HEp-2 cells, which typically shows a cytoplasmic pattern (1). Since this profile is not specific, the diagnosis has to be confirmed by an immunodot assay or by protein immunoprecipitation. Yet, these latter assays are not quantitative and do not allow for reproducible titration of anti-SRP autoantibodies. The objective of this study was to develop a quantitative test of anti-SRP autoantibodies and to determine whether there is a correlation between anti-SRP autoantibody levels, serum CK activity, and muscle strength. PATIENTS AND METHODS Patients. This study assessing the development of a new technique for the diagnosis and monitoring of anti-SRP antibodies was approved by the ethics review committee of Cochin Hospital in Paris. Thirty-one patients from 2 centers in France (Rouen University Hospital and Paris Pitié-Salpêtrière University Hospital) whose sera were tested for anti-SRP autoantibodies by immunodot were included in the study. Among these patients, 9 fulfilled the Bohan and Peter criteria for polymyositis (PM) (17) and 22 fulfilled the Hoogendijk criteria for acquired necrotizing myopathy (5). Among the 31 anti-SRP–positive patients, serum samples from 8 patients were monitored over time for anti-SRP autoantibody levels and serum CK activity (determined at least 3 times, consecutively). The date of the first sample assessment was referred to as day 0, and this date also corresponded to the initiation of treatment (for those patients who had never received therapy) or the restart of a modified treatment regimen (for patients who had experienced a relapse). The clinical characteristics, treatments, and outcomes in these 8 patients are summarized in Table 1. Controls. Control sera were collected from 190 healthy blood donors and from 172 patients with different inflammatory/ autoimmune diseases, diagnosed according to the established classification criteria for each disease: the American College of Rheumatology (ACR) revised criteria for systemic lupus erythematosus (SLE) (18) with anti–double-stranded DNA (antidsDNA) autoantibodies (n ⫽ 20), the ACR criteria for rheumatoid arthritis (RA) (19) with anti–cyclic citrullinated peptide antibodies and/or rheumatoid factor (n ⫽ 40), the revised European criteria for primary Sjögren’s syndrome (20) with anti-SSA and/or anti-SSB autoantibodies (n ⫽ 20), the Bohan and Peter criteria (17) for dermatomyositis (DM) (n ⫽ 22) or PM (n ⫽ 10), the Troyanov criteria for overlap myositis (2) with anti–transfer RNA (tRNA) synthetase antibodies (anti–Jo-1 [n ⫽ 27], anti–PL-7 [n ⫽ 1], anti–PL-12 [n ⫽ 2]), and the Griggs criteria for inclusion body myositis (21) (n ⫽ 30). Another disease control consisted of sera from patients with polyclonal hypergammaglobulinemia (mean ⫾ SD serum IgG level 23.1 ⫾ 6.9 gm/liter) (n ⫽ 27). Serum samples were stored at ⫺80°C until used. Detection of autoantibodies. Indirect immunofluorescence was performed on HEp-2000 cells (Immunoconcepts). Myositis-associated autoantibodies were detected by dot-blot immunoassay using BlueDOT Polymyositis/Scleroderma dot (PMS8D-24; D-Tek SA). Western blot analysis of recombinant SRP54 protein. The purity of the recombinant SRP54 protein was determined using 4–10% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions, followed by Coomassie blue staining. Western blot analysis was then performed by transfer of proteins, separated by nonreducing SDS-PAGE, to a nitrocellulose membrane, followed by incubation with antihistidine antibodies, antiSRP54 antibodies, or an anti-SRP54–positive human serum. Development of the addressable laser bead immunoassay (ALBIA) for quantification of SRP54-specific antibodies. Full-length human recombinant SRP54 protein fused to a hexa-histidine tag was obtained from Diarect. LiquiChip nickel–nitrilotriacetic acid (Ni-NTA) beads and streptavidinR–phycoerythrin were from Qiagen. The concentration of SRP54 protein coupled to Ni-NTA microspheres was 10 g/ml. When indicated, hexa-histidine–tagged human recombinant Jo-1 and intrinsic factor proteins (Diarect) were used in place of SRP54. Beads were incubated with serum from patients or EVOLUTION OF ANTI-SRP LEVELS IN NECROTIZING MYOPATHY 1963 Table 1. Clinical characteristics and followup of 8 patients with anti–signal recognition particle autoantibodies* Patient/sex/ age/origin† Disease duration/ relapse‡ 1/M/62/C 48 months/yes 2/F/76/C 36 months/no 3/F/29/A 72 months/yes 4/M/52/A 3 months/no 5/F/57/C 50 months/yes 6/F/45/C 30 months/no 7/M/38/C 120 months (first diagnosed as LGMD)/no 9 months/no 8/F/20/A Clinical signs on day 0/duration of AOF/ MRC deltoids/MRC psoas§ Proximal weakness, wheelchair/60/3/2 Mild proximal weakness/ 150/4/4 Proximal weakness, impossible to get out of bed, ICU/5/3/2 Proximal weakness, wheelchair/0/2/2 Proximal weakness, wheelchair/0/2/2 Proximal weakness, walk with walker/20/3/2 Treatments received before day 0 Treatments started on day 0 Outcomes (day of followup)/duration of AOF/MRC deltoids/MRC psoas§ Pred., MTX, IVIG Pred., AZA, RTX Mild proximal weakness, walk without support (day 917)/250/5/4 Normal strength (day 1,161)/250/5/5 Pred., AZA Pred., AZA, MTX, CYC, MMF, IVIG, PE Pred. Proximal weakness, walk with walker/100/4/2 Proximal weakness, walk with cane/70/4/4 Pred., RTX Mild proximal weakness, walk with cane (day 1,078)/75/4/4 Pred., AZA, IVIG Normal strength (day 165)/250/5/5 Pred., MMF, IVIG, RTX Pred., MTX, PE, IVIG; RTX, AZA (on day 329) Pred., AZA, IVIG Mild proximal weakness, walk with cane (day 1,318)/100/4/4 Slight improvement, walk with cane (day 559), change of treatment (day 329)/60/3/3 Pred., MTX, IVIG Normal strength (day 484)/250/5/5 Slight improvement, walk with cane (day 578)/150/4/3 * Pred. ⫽ prednisone; MTX ⫽ methotrexate; IVIG ⫽ intravenous immunoglobulin; AZA ⫽ azathioprine; RTX ⫽ rituximab; ICU ⫽ intensive care unit; CYC ⫽ cyclosporine; MMF ⫽ mycophenolate mofetil; PE ⫽ plasma exchange; LGMD ⫽ limb-girdle muscular dystrophy. † Patients’ age was the age (in years) on day 0. Patients’ origin was determined as either Caucasian (C) or African (A). ‡ Disease duration was determined as the number of months since appearance of the first signs of disease or number of months since day 0 of the study. § The arms outstretched forward (AOF) test measures the duration of time (number of seconds) during which the arms are upheld in this position (normal ⬎150 seconds) (42). Muscle strength in the deltoid and psoas muscles is determined as a score of severity (0 ⫽ absence of any movement to 5 ⫽ normal strength) using the 6-point British Medical Research Council (MRC) manual muscle strength test. controls for 2 hours at room temperature. Blanks (no serum, secondary antibody only), negative controls (anti-SRP autoantibody–negative serum), and positive controls (highly anti-SRP autoantibody–positive human serum or goat antiSRP autoantibodies with appropriate secondary antibody) were included in every assay. Biotinylated mouse anti-human IgG antibodies (or isotype-specific antibodies) were then added to the incubations for 1 hour. After washing, beads were incubated with 50 l of streptavidin-R–phycoerythrin (Qiagen) at 1:1,000 dilution for 15 minutes. For each assay, the mean fluorescence intensity (MFI) of the reaction was determined on a Bio-Plex apparatus (Bio-Rad). Fifty beads were counted for each analysis. An internal quality control yielding a fluorescence value close to 50% of the plateau was added in each experiment. The specificity of the ALBIA method was determined using human sera that were highly positive for anti-SRP54, anti–Jo-1, or anti–intrinsic factor antibodies, added at 1:1,000 dilution. Specific inhibition in the ALBIA assay for SRP54 was performed using growing concentrations of recombinant SRP54 protein. The percent inhibition was calculated as (1 ⫺ [MFIpreadsorbed serum/MFIserum]) ⫻ 100. Homologous and heterologous inhibition in the ALBIA assays was further performed by preabsorption of 4 anti-SRP–positive serum samples, 4 anti–Jo-1–positive serum samples, or 4 anti–intrinsic factor antibody–positive serum samples with 100 g/ml of recombinant SRP54, Jo-1, or intrinsic factor protein. Serum samples from all of the patients were initially assayed at a 1:500 dilution. The anti-SRP autoantibody levels were determined at a dilution of 1:D, calculated using the following formula: ([MFIserum/MFIcalibrator] ⫻ level of calibrator) ⫻ D/500. The calibrator was a human serum that was highly positive for anti-SRP autoantibodies (the same as used throughout the study), whose level in the assay was arbitrarily set to 100 arbitrary units (AU)/ml. When the MFI of a given serum sample at 1:500 dilution was higher than 80% of the calibrator MFI, further dilutions were performed, and the first dilution yielding an MFI inferior to 80% of the calibrator MFI was retained for calculation of autoantibody levels. Statistical analysis. The normality of data distribution was analyzed using the D’Agostino and Pearson test. The relationship between anti-SRP autoantibody levels and CK levels was tested using a linear mixed model. RESULTS Findings on ALBIA quantitative assay for antiSRP54 autoantibodies. To allow the quantitative analysis of anti-SRP54 autoantibodies in patients and to allow followup of these autoantibody levels over time, we developed the ALBIA technique (22). For this assay, we used a recombinant human SRP54 protein harboring 1964 Figure 1. Development of a quantitative assay for the detection of anti–signal recognition particle 54-kd subunit (anti-SRP54) antibodies. A, Structure of the recombinant human SRP54 protein, containing a hexa-histidine tag in C-terminal position (arrowhead). N, G, and M represent the N-terminal, GTP-binding, and methionine-rich domains, respectively. The epitopes recognized by the goat and chicken antiSRP antibodies (raised against SRP peptides) are depicted by arrows. B, Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of the recombinant SRP54 protein after Coomassie blue staining. C, Western blot analysis of the recombinant SRP54 protein, using an antibody directed to the C-terminal histidine tag and different antiSRP54 antibodies. D, Validation of the results using an addressable laser bead immunoassay with serial dilutions of goat and chicken anti-SRP54 antibodies. The MFI values are the mean ⫾ SD of triplicate determinations. Inset, Higher magnification of the curve for the lower anti-SRP54 antibody concentrations (horizontal broken line depicts the mean ⫹ 2 SD in negative controls [n ⫽ 18]). BENVENISTE ET AL Figure 2. Specificity of the signal recognition particle 54-kd subunit (SRP54)–coated beads. A, SRP54-, Jo-1–, and intrinsic factor–specific beads were prepared using 10 g/ml of the corresponding hexahistidine–tagged protein and analyzed by addressable laser bead immunoassay using a human anti-SRP54, anti–Jo-1, or anti–intrinsic factor–positive serum. B, Free human recombinant SRP54 protein inhibited human anti-SRP antibody binding to SRP54-coated beads, in a dose-dependent manner. The percent inhibition is given relative to the MFI value in the absence of free SRP54. C, Specificity of inhibition by the proteins in the assay was assessed as the percent inhibition of 4 different anti-SRP54–positive human serum samples by 100 g/ml of free homologous (SRP54) or heterologous (Jo-1 and intrinsic factor) proteins. EVOLUTION OF ANTI-SRP LEVELS IN NECROTIZING MYOPATHY a hexa-histidine tag in its C-terminal, methionine-rich domain (Figure 1A). The purity of the SRP54 protein was confirmed by Coomassie blue staining after SDS-PAGE, which revealed a unique band of 60 kd (Figure 1B), specifically recognized by chicken or goat anti-SRP54 autoantibodies and by human anti-SRP autoantibody–positive serum (Figure 1C). The tagged recombinant SRP54 protein was coupled to fluorescent beads through interaction of histidines with the nickel moiety of the beads (23), and was further used to measure the levels of anti-SRP autoantibodies. This assay was used for quantification of SRP54-specific antibodies, and therefore it is hereafter referred to as the ALBIA-SRP54. A series of parameters were optimized, including incubation times and buffers, washing steps, secondary antibodies, and the concentration of recombinant SRP54 protein for bead coating, which was set to 10 g/ml (details available from the corresponding author upon request). The ALBIASRP54 could detect protein concentrations as low as 2 ng/ml in assays using chicken anti-SRP54–specific antibodies or 8 ng/ml in those using goat anti-SRP54– specific antibodies, indicating that the assay had good analytic sensitivity (Figure 1D). Only anti-SRP54–positive serum, but not irrelevant anti–Jo-1–positive or anti–intrinsic factor antibody– positive serum, reacted with SRP54 beads (Figure 2A). Similarly, beads coated with Jo-1 or intrinsic factor recombinant proteins that were produced and purified using the same process as that used for SRP54 revealed positivity only for the corresponding autoantibodies in the serum. Further experiments showed that the free SRP54 protein could inhibit the binding of anti-SRP54 autoantibodies in a dose-dependent manner (Figure 2B), whereas no inhibition was observed using a heterologous protein (Figure 2C). The method used for calculating the level of anti-SRP54 autoantibodies is illustrated in Figure 3. The patient’s serum used in this example displayed a characteristic cytoplasmic pattern on immunofluorescence (Figure 3A) and was positive for anti-SRP54 on dot-blot immunoassay (Figure 3B). Using the ALBIA-SRP54, this serum showed a saturating signal at the 1:500 screening dilution, and a further 1:5,000 dilution was retained to perform the calculations in reference to the calibrator used throughout the study, which, as mentioned above, had a level arbitrarily fixed to 100 AU/ml (Figure 3C). The reproducibility of the test was assessed by determining the level of intra- and interassay variations among sera with high, medium, and low anti-SRP54 levels. The intraassay coefficients of variation were 1965 Figure 3. Determination of the level of anti–signal recognition particle 54-kd subunit (anti-SRP54) antibodies in human serum. The same anti-SRP54–positive human serum was used in each experiment. A, Classic indirect immunofluorescence (IFI) assay on HEp-2000 cells in the serum showed a characteristic cytoplasmic staining pattern after incubation of a 1:80 dilution of the serum, revealed with an anti-human IgG conjugate. The titer of the serum was ⬎1,200. B, Dot-blot analysis of 1:150-diluted serum showed specific reactivity against SRP54, but not other antigens. C, Addressable laser bead immunoassay (ALBIA) was used to calculate the anti-SRP54 antibody level. Anti-SRP54 levels were determined in reference to the MFI value of a calibrator (human serum highly positive for anti-SRP54) in the same assay. The level of the calibrator (cal) is arbitrarily set to 100 arbitrary units (AU)/ml. The assay is first performed using a 1:500 screening dilution of the serum. In those cases in which the MFI of the sample at 1:500 dilution is higher than 80% of the MFI of the calibrator, further dilutions are performed, and the first dilution yielding an MFI inferior to 80% of the calibrator MFI is retained for calculation. An example is given for a serum with an anti-SRP54 level of 408 AU/ml. 1966 lower than 5% (details available from the corresponding author upon request). Similarly, assay-to-assay and batch-to-batch variations ranged from 0.4% to 3.3%. Taken together, these data indicate the excellent sensitivity, specificity, and reproducibility of the ALBIASRP54 technique. Diagnostic value of the ALBIA-SRP54. The diagnostic value of the assay was determined by comparing the levels of anti-SRP in the serum of anti-SRP immunodot–positive patients to those in the serum of healthy blood donors. The values obtained with control sera did not follow a normal distribution (P ⬍ 0.0001; n ⫽ 190); the median level of anti-SRP in controls was 2 AU/ml and the 99th percentile of the distribution was 35 AU/ml. At the threshold of 35 AU/ml, all sera from anti-SRP–positive patients scored positive for anti-SRP, with levels ranging from 66 to 22,080 AU/ml, whereas 189 (99.5%) of 190 control sera were negative for anti-SRP (Figure 4). Using a cutoff of 60 AU/ml, all sera from the patients scored positive for anti-SRP and all healthy control sera were negative. Sera from patients with different inflammatory/autoimmune conditions, including RA, primary Sjögren’s syndrome, SLE, PM, DM, anti–tRNA synthetase antibody–positive myositis, or inclusion body myositis, as well as sera from patients with polyclonal hypergammaglobulinemia were all negative for anti-SRP (Figure 4). IgG isotypes of anti-SRP autoantibodies. The majority of patients (16 of 21, or 76%) had only a single subclass of anti-SRP54 IgG, whereas the presence of ⱖ2 IgG isotypes was found in the remaining 24% of patients (5 of 21). The most frequent isotype of antiSRP autoantibodies was IgG1, which was present in 81% (17 of 21) of the sera analyzed (details available from the corresponding author upon request). Anti-SRP IgG1 antibodies were either present alone (12 of 17, or 71%) or associated with 1 or 2 other isotypes. The second more frequent isotype was IgG4, found in 29% of sera (6 of 21). IgG2 was never detected as a single isotype, but was always found in association with other isotypes, and IgG3 was found in only 1 serum sample. Effect of plasma exchange on anti-SRP autoantibody levels. In 1 patient (patient 6 in Table 1 and Figure 5), an evolutive form of anti-SRP–positive myopathy was observed, since, not long after the time of diagnosis, this patient required the use of a walker. On day 0, we started her on a treatment regimen of prednisone (1 mg/kg/day), methotrexate (0.3 mg/kg/week), and intravenous immunoglobulins (2 gm/kg/month). Because of the absence of any treatment effect on muscular strength, plasma exchanges were additionally performed BENVENISTE ET AL Figure 4. Diagnostic value of the addressable laser bead immunoassay for signal recognition particle 54-kd subunit (ALBIA-SRP54). Sera from anti-SRP–positive patients were compared to sera from healthy blood donors as control. The threshold for anti-SRP positivity corresponding to the 99th percentile of the control distribution (35 arbitrary units [AU]/ml) is depicted by a broken line, while the threshold of 60 AU/ml is indicated as a solid line. Using these thresholds, sera from patients with different inflammatory/autoimmune conditions, including rheumatoid arthritis (RA), primary Sjögren’s syndrome (SS), systemic lupus erythematosus (SLE), polymyositis (PM), dermatomyositis (DM), anti–transfer RNA (tRNA) synthetase–positive myositis, or inclusion body myositis (IBM), as well as patients with polyclonal hypergammaglobulinemia, were also assayed. Inset, Higher magnification of the values around the threshold. on days 49, 58, and 60. An aliquot was sampled from the machine tubing in order to determine plasma anti-SRP levels at the beginning and end of each procedure (Figure 5A). As expected, anti-SRP autoantibody levels always decreased following plasma exchange (autoantibody depletion ranging from 54% to 64%), with a rebound observed on subsequent days, as indicated by augmented levels, albeit still reduced compared to previous values, at the beginning of the next exchange. Thus, plasma exchange indeed diminished the levels of anti-SRP54 autoantibodies, and the ALBIA-SRP54 technique allowed us to quantify the level of autoantibody depletion in this patient. EVOLUTION OF ANTI-SRP LEVELS IN NECROTIZING MYOPATHY 1967 Figure 5. Monitoring of anti–signal recognition particle 54-kd subunit (anti-SRP54) levels and serum creatine kinase (CK) levels in patients with necrotizing myopathy. A, Evolution of plasma levels of anti-SRP54 in a patient before and after plasma exchanges performed on days 49, 58, and 60. B, Evolution of serum anti-SRP54 levels and CK levels in 8 patients receiving therapy (see Table 1 for treatment details in each patient). AU ⫽ arbitrary units. Evolution of anti-SRP autoantibody levels, CK levels, and muscle strength during therapy. Serum samples from 8 patients were obtained over 3 consecutive time points in order to monitor the anti-SRP autoantibody levels and CK levels over time. All of these patients received a new or modified treatment on day 0, and were followed up for a period ranging from 165 days to 1,318 days thereafter (mean 783 days). During treatment, 6 of the 8 patients showed a clear clinical improvement (Table 1), which was manifested by a noticeable regression of their muscular deficit (patients 1, 3, and 5) or even normalization of muscle strength (patients 2, 4, and 8), whereas in patients 6 and 7, only a modest improvement was observed (Table 1). Regarding the levels of CK during therapy, the general feature was a decrease in levels and subsequent normalization over the followup (Figure 5B). The CK level on day 0 greatly varied between patients and was not predictive of muscular weakness. For instance, patient 8, who could still walk (although with a cane), had a CK level of 8,000 units/liter, whereas patient 3, who was the more severely affected patient in this series (i.e., required hospitalization in an intensive care unit for respiratory assistance), had a 10 times lower CK level. Similarly, all 8 patients had very different anti-SRP levels at baseline, but in all of these patients, the autoantibody levels significantly decreased over time, and even reached levels below the cutoff for positivity (⬍60 AU/ml) in patients 6 and 8. Importantly, the evolution of anti-SRP autoantibody levels closely paralleled that of CK levels. Indeed, autoantibody levels dropped with a decrease in CK in all patients, and conversely, the anti-SRP level augmented with a rise in CK. Using a linear mixed model, in which the correlations among measurements in the same patient were taken into account, anti-SRP autoantibody levels appeared to be significantly associated with CK levels (P ⫽ 0.002) but not with disease duration (P ⫽ 0.26). Similarly, there was a parallel evolution between anti-SRP autoantibody levels and improvement in muscle strength in all 8 of the patients examined (Table 1). DISCUSSION This is the first report of a positive correlation between the levels of anti-SRP autoantibodies and CK 1968 levels in patients with this severe form of myositis, a finding that was further paralleled by observations of clinical improvement in muscle strength in every patient. These findings, observed in the largest series of anti-SRP autoantibody–positive patients reported so far (4,10), were obtained using a newly developed assay that allowed us to quantitatively assess the levels of anti-SRP IgG autoantibodies. The diagnostic value of this new assay was established by comparing anti-SRP IgG antibody levels between a large number of control subjects with or without inflammatory/autoimmune diseases, including myositis, and 31 anti-SRP autoantibody–positive patients whose sera exhibited a characteristic cytoplasmic pattern on immunofluorescence of Hep-2 cells and binding of SRP in a commercial immunodot assay. We used a histidine-tagged recombinant human SRP54 molecule whose high apparent molecular weight of 60 kd is attributable to the increased level of glycosylation occurring in the insect SF9 cell line in which the recombinant molecule was produced. Mapping studies previously showed that anti-SRP54 autoantibodies from patients react with the amino-terminal N domain and central G domain, but do not react with the carboxyterminal M domain of the molecule, whose function is to bind endoplasmic reticulum signal sequences (13,16). The goat anti-SRP54 autoantibodies (raised against an amino-terminal peptide) and even the chicken antiSRP54 autoantibodies (raised against a peptide corresponding to residues 300–504 of the M domain) recognized the tagged recombinant SRP54 protein both on Western blotting (Figure 1C) and in the ALBIA-SRP54 assay (Figure 1D). Therefore, the carboxy-terminal– oriented coupling to beads through a small-sized hexahistidine tag, which does not interfere with protein structure, allows a good accessibility of SRP54 epitopes to specific antibodies. The ALBIA-SRP54 detected anti-SRP in all antiSRP–positive sera tested in this series of patients. None of the serum samples from patients with other inflammatory/autoimmune conditions or other forms of myositis, such as PM, DM, anti–tRNA synthetase antibody–positive myositis, or inclusion body myositis, scored positive for anti-SRP. The threshold for positivity that gave the best diagnostic value was 60 AU/ml. Nevertheless, values above the 99th percentile of the control distribution, 35 AU/ml, could also be considered positive, thus defining the 35–60 AU/ml range as a critical zone for this assay. The assay also allowed us to analyze anti-SRP IgG subclass distribution, which has, as yet, been unknown in this disease. Most patients had IgG1 and/or BENVENISTE ET AL IgG4, in line with the IgG1/IgG4 specificity that has been reported in patients with other autoimmune diseases, such as myasthenia gravis, membranous glomerulonephritis, or pemphigus, in whom both isotypes have been found to be pathogenic (24). In the present study, treatment of a patient with plasma exchange repeatedly reduced anti-SRP54 autoantibody levels by more than 2-fold (Figure 5A). Thus, the assay constitutes a new tool to monitor the efficacy of autoantibody-depleting strategies. Importantly, longitudinal followup of patients receiving therapy revealed a strong positive correlation between the intensity of myolysis, as measured by CK activity, and anti-SRP54 autoantibody levels (Figure 5B). It is well established that the serum CK activity level is an excellent indicator of the severity of myolysis in a given patient, whereas it is a poor marker of disease activity at the population level in patients with myositis, as noted in patients with DM, for instance (25). As expected, the CK levels at baseline varied between patients, and, individually, these levels were not indicative of disease severity. Although the initial anti-SRP level is not representative of disease severity in terms of muscular strength, the correlation between serum CK activity and the anti-SRP level over time in each given patient was found to be highly representative of an achievement of disease control (Table 1 and Figure 5B). Two patients (patients 6 and 7) appeared less responsive to therapy, as indicated by the scores of muscle strength at the end of the followup period, which may represent an incomplete control of the disease in patient 6 and a too-short period of observation for patient 7. The sera from patient 6 showed an increase (after normalization of the values) in both the anti-SRP level and CK activity (Figure 5B), which led us to reinforce the treatment with the addition of rituximab and azathioprine on day 329 (Table 1). Patient 7 had a long history (⬎10 years) of disease evolution since the initial diagnosis of myopathy as limb-girdle muscular dystrophy from unknown gene origin (as has also been the case for other patients already reported in the literature [11,12]), which preceded our assay for anti-SRP autoantibodies and the initiation of immunosuppressive treatments. On day 0, magnetic resonance imaging of the muscle of patient 7 showed an important fatty involvement of limb-girdle muscles that led us to anticipate that a rapid resolution of the muscle weakness would not be obtained. Nevertheless, the CK level normalized under treatment, a situation that had never been observed previously in this patient. The known examples of autoimmune diseases in EVOLUTION OF ANTI-SRP LEVELS IN NECROTIZING MYOPATHY which specific autoantibody levels are clearly correlated with surrogate disease activity markers or clinical signs are sparse. In non–organ-specific autoimmune disorders, the best-documented case is the association of SLE with anti-dsDNA autoantibodies (26,27). A reduction in anti-dsDNA levels is indeed associated with remission of SLE, whereas an increase in anti-dsDNA may prompt a disease flare. Monitoring of anti-dsDNA antibodies is thus recommended during followup of SLE and for clinical trials (28). A correlation between clinical remission and reduction or disappearance of pathogenic autoantibodies was also reported in antineutrophil cytoplasmic antibody–associated vasculitides (29,30), although the results have been inconsistent (31), and in anti–Jo-1–positive myositis (32). Among organ-specific autoimmune diseases, pemphigus constitutes another well-established example, as demonstrated by our previous analysis of antidesmoglein antibody levels in patients receiving anti-CD20 therapy (33,34). However, in most autoimmune diseases, autoantibodies are generally not indicative of disease activity. In RA, for instance, although rheumatoid factor and anti–citrullinated protein autoantibodies can sometimes disappear with treatment (35), their levels generally remain stable over time (36,37). The followup of these markers has therefore not been recommended in clinical practice. During systemic sclerosis, autoantibodies are highly valuable as markers of diagnosis or prognosis (38), but not of disease activity, since they persist over time. In myasthenia gravis, a paradigm of autoantibodymediated disorders (39), the demonstration of the pathogenic role of anti–acetylcholine receptor (antiAchR) antibodies has been firmly established. However, even if anti-AChR antibodies can occasionally disappear with treatment, their levels generally remain unchanged over time, and no correlation has been found between autoantibody levels and disease flares (40). Therefore, the positive correlation between autoantibody levels and a surrogate marker of disease, as revealed herein, represents an original finding in the field of autoantibodyassociated diseases and allows us to assign to anti-SRP autoantibodies a new role in myositis from both a scientific and a practical point of view. Future clinical trials in anti-SRP–positive myopathies could benefit from the monitoring of autoantibody levels. Preliminary observations suggest that B cell– targeting therapies, such as anti-CD20, may be efficient in otherwise–treatment-resistant anti-SRP–positive patients (8,41). Since there are very few lymphocytic infiltrates in the muscle of these patients, B cell– targeting therapies presumably act on the autoreactive 1969 B cell response, which may include antigen presentation to T cells, cytokine production, and/or autoantibody secretion. We are currently investigating the effect of rituximab in anti-SRP–positive patients in an ongoing clinical trial (ClinicalTrials.gov Identifier: NCT00774462). Due to the ubiquitous distribution of SRP, there is still no clear understanding of the basis for an association between anti-SRP autoantibodies and muscle disease. Since the levels of anti-SRP54 autoantibodies are closely correlated with the level of myolysis, the present results suggest that anti-SRP might play a pathogenic role. Experimental studies, such as the evaluation of the effect of anti-SRP autoantibodies on cultures of myogenic cells or their passive transfer to animals, would be helpful to further elucidate this point. The quantity of circulating anti-SRP autoantibodies may also depend on the abundance of SRP autoantigens released by muscle necrosis that would trigger the autoimmune humoral response. Finally, the possibility cannot be excluded that the SRP54 protein may not be the main target of anti-SRP54 autoantibodies in vivo in the necrotizing myopathies, and that polyspecific anti-SRP54 autoantibodies may recognize other, as yet unknown, targets on muscle cells. ACKNOWLEDGMENTS We acknowledge Sabine Nebrel and Florence Aubrée for technical assistance, Sahil Adriouch and Serge Jacquot for helpful discussions, and Jean-François Ménard for his help in statistical analyses. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Boyer 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 conception and design. Benveniste, Boyer. Acquisition of data. Benveniste, Drouot, Jouen, Charuel, BlochQueyrat, Behin, Amoura, Marie, Eymard, Herson, Musset. Analysis and interpretation of data. Benveniste, Drouot, Jouen, Guiguet, Gilbert, Tron, Boyer. REFERENCES 1. Targoff IN, Johnson AE, Miller FW. Antibody to signal recognition particle in polymyositis. Arthritis Rheum 1990;33:1361–70. 2. Troyanov Y, Targoff IN, Tremblay JL, Goulet JR, Raymond Y, Senecal JL. Novel classification of idiopathic inflammatory myopathies based on overlap syndrome features and autoantibodies: analysis of 100 French Canadian patients. Medicine (Baltimore) 2005;84:231–49. 1970 3. Brouwer R, Hengstman GJ, Vree Egberts W, Ehrfeld H, Bozic B, Ghirardello A, et al. Autoantibody profiles in the sera of European patients with myositis. Ann Rheum Dis 2001;60: 116–23. 4. Hengstman GJ, ter Laak HJ, Vree Egberts WT, Lundberg IE, Moutsopoulos HM, Vencovsky J, et al. Anti-signal recognition particle autoantibodies: marker of a necrotising myopathy. Ann Rheum Dis 2006;65:1635–8. 5. Hoogendijk JE, Amato AA, Lecky BR, Choy EH, Lundberg IE, Rose MR, et al. 119th ENMC international workshop: trial design in adult idiopathic inflammatory myopathies, with the exception of inclusion body myositis, 10–12 October 2003, Naarden, The Netherlands. Neuromuscul Disord 2004;14:337–45. 6. Kao AH, Lacomis D, Lucas M, Fertig N, Oddis CV. Anti–signal recognition particle autoantibody in patients with and patients without idiopathic inflammatory myopathy. Arthritis Rheum 2004; 50:209–15. 7. Miller T, Al-Lozi MT, Lopate G, Pestronk A. Myopathy with antibodies to the signal recognition particle: clinical and pathological features. J Neurol Neurosurg Psychiatry 2002;73:420–8. 8. Arlet JB, Dimitri D, Pagnoux C, Boyer O, Maisonobe T, Authier FJ, et al. Marked efficacy of a therapeutic strategy associating prednisone and plasma exchange followed by rituximab in two patients with refractory myopathy associated with antibodies to the signal recognition particle (SRP). Neuromuscul Disord 2006;16: 334–6. 9. Love LA, Leff RL, Fraser DD, Targoff IN, Dalakas M, Plotz PH, et al. A new approach to the classification of idiopathic inflammatory myopathy: myositis-specific autoantibodies define useful homogeneous patient groups. Medicine (Baltimore) 1991;70:360–74. 10. Takada T, Hirakata M, Suwa A, Kaneko Y, Kuwana M, Ishihara T, et al. Clinical and histopathological features of myopathies in Japanese patients with anti-SRP autoantibodies. Mod Rheumatol 2009;19:156–64. 11. Dimitri D, Andre C, Roucoules J, Hosseini H, Humbel RL, Authier FJ. Myopathy associated with anti-signal recognition peptide antibodies: clinical heterogeneity contrasts with stereotyped histopathology. Muscle Nerve 2007;35:389–95. 12. Suzuki S, Satoh T, Sato S, Otomo M, Hirayama Y, Sato H, et al. Clinical utility of anti-signal recognition particle antibody in the differential diagnosis of myopathies. Rheumatology (Oxford) 2008;47:1539–42. 13. Janda CY, Li J, Oubridge C, Hernandez H, Robinson CV, Nagai K. Recognition of a signal peptide by the signal recognition particle. Nature 2010;465:507–10. 14. Okada N, Mimori T, Mukai R, Kashiwagi H, Hardin JA. Characterization of human autoantibodies that selectively precipitate the 7SL RNA component of the signal recognition particle. J Immunol 1987;138:3219–23. 15. Reeves WH, Nigam SK, Blobel G. Human autoantibodies reactive with the signal-recognition particle. Proc Natl Acad Sci U S A 1986;83:9507–11. 16. Romisch K, Miller FW, Dobberstein B, High S. Human autoantibodies against the 54 kDa protein of the signal recognition particle block function at multiple stages. Arthritis Res Ther 2006;8:R39. 17. Bohan A, Peter JB. Polymyositis and dermatomyositis (first of two parts). N Engl J Med 1975;292:344–7. 18. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25:1271–7. 19. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24. 20. Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexan- BENVENISTE ET AL 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. der EL, Carsons SE, et al, and the European Study Group on Diagnostic Criteria for Sjögren’s Syndrome. Classification criteria for Sjögren’s syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis 2002;61:554–8. Griggs RC, Askanas V, DiMauro S, Engel A, Karpati G, Mendell JR, et al. Inclusion body myositis and myopathies. Ann Neurol 1995;38:705–13. Fritzler MJ, Behmanesh F, Fritzler ML. Analysis of human sera that are polyreactive in an addressable laser bead immunoassay. Clin Immunol 2006;120:349–56. Verkaik N, Brouwer E, Hooijkaas H, van Belkum A, van Wamel W. Comparison of carboxylated and Penta-His microspheres for semi-quantitative measurement of antibody responses to Histagged proteins. J Immunol Methods 2008;335:121–5. Sitaru C, Mihai S, Zillikens D. The relevance of the IgG subclass of autoantibodies for blister induction in autoimmune bullous skin diseases. Arch Dermatol Res 2007;299:1–8. Komiya T, Negoro N, Kondo K, Miura K, Hirota Y, Yoshikawa J. Clinical significance of von Willebrand factor in patients with adult dermatomyositis. Clin Rheumatol 2005;24:352–7. Isenberg DA, Dudeney C, Williams W, Todd-Pokropek A, Stollar BD. Disease activity in systemic lupus erythematosus related to a range of antibodies binding DNA and synthetic polynucleotides. Ann Rheum Dis 1988;47:717–24. Isenberg DA, Shoenfeld Y, Schwartz RS. Multiple serologic reactions and their relationship to clinical activity in systemic lupus erythematosus. Arthritis Rheum 1984;27:132–8. Bertsias GK, Ioannidis JP, Boletis J, Bombardieri S, Cervera R, Dostal C, et al. EULAR points to consider for conducting clinical trials in systemic lupus erythematosus: literature based evidence for the selection of endpoints. Ann Rheum Dis 2009;68:477–83. Kyndt X, Reumaux D, Bridoux F, Tribout B, Bataille P, Hachulla E, et al. Serial measurements of antineutrophil cytoplasmic autoantibodies in patients with systemic vasculitis. Am J Med 1999; 106:527–33. Terrier B, Saadoun D, Sene D, Ghillani P, Amoura Z, Deray G, et al. Antimyeloperoxidase antibodies are a useful marker of disease activity in antineutrophil cytoplasmic antibody-associated vasculitides. Ann Rheum Dis 2009;68:1564–71. Girard T, Mahr A, Noel LH, Cordier JF, Lesavre P, Andre MH, et al. Are antineutrophil cytoplasmic antibodies a marker predictive of relapse in Wegener’s granulomatosis? A prospective study. Rheumatology (Oxford) 2001;40:147–51. Stone KB, Oddis CV, Fertig N, Katsumata Y, Lucas M, Vogt M, et al. Anti–Jo-1 antibody levels correlate with disease activity in idiopathic inflammatory myopathy. Arthritis Rheum 2007;56: 3125–31. Joly P, Mouquet H, Roujeau JC, D’Incan M, Gilbert D, Jacquot S, et al. A single cycle of rituximab for the treatment of severe pemphigus. N Engl J Med 2007;357:545–52. Mouquet H, Musette P, Gougeon ML, Jacquot S, Lemercier B, Lim A, et al. B-cell depletion immunotherapy in pemphigus: effects on cellular and humoral immune responses. J Invest Dermatol 2008;128:2859–69. Alessandri C, Bombardieri M, Papa N, Cinquini M, Magrini L, Tincani A, et al. Decrease of anti-cyclic citrullinated peptide antibodies and rheumatoid factor following anti-TNF␣ therapy (infliximab) in rheumatoid arthritis is associated with clinical improvement. Ann Rheum Dis 2004;63:1218–21. De Rycke L, Peene I, Hoffman IE, Kruithof E, Union A, Meheus L, et al. Rheumatoid factor and anticitrullinated protein antibodies in rheumatoid arthritis: diagnostic value, associations with radiological progression rate, and extra-articular manifestations. Ann Rheum Dis 2004;63:1587–93. Caramaschi P, Biasi D, Tonolli E, Pieropan S, Martinelli N, EVOLUTION OF ANTI-SRP LEVELS IN NECROTIZING MYOPATHY Carletto A, et al. Antibodies against cyclic citrullinated peptides in patients affected by rheumatoid arthritis before and after infliximab treatment. Rheumatol Int 2005;26:58–62. 38. Koenig M, Dieude M, Senecal JL. Predictive value of antinuclear autoantibodies: the lessons of the systemic sclerosis autoantibodies. Autoimmun Rev 2008;7:588–93. 39. Vincent A, Palace J, Hilton-Jones D. Myasthenia gravis. Lancet 2001;357:2122–8. 40. Sanders DB, Hart IK, Mantegazza R, Shukla SS, Siddiqi ZA, De Baets MH, et al. An international, phase III, randomized trial of 1971 mycophenolate mofetil in myasthenia gravis. Neurology 2008;71: 400–6. 41. Valiyil R, Casciola-Rosen L, Hong G, Mammen A, ChristopherStine L. Rituximab therapy for myopathy associated with anti–signal recognition particle antibodies: a case series. Arthritis Care Res (Hoboken) 2010;62:1328–34. 42. Sharshar T, Chevret S, Mazighi M, Chillet P, Huberfeld G, Berreotta C, et al. Validity and reliability of two muscle strength scores commonly used as endpoints in assessing treatment of myasthenia gravis. J Neurol 2000;247:286–90.