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Correlation of antisignal recognition particle autoantibody levels with creatine kinase activity in patients with necrotizing myopathy.

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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:
olivier.boyer@chu-rouen.fr.
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.
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