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Sensitivity and specificity of anti-Jo-1 antibodies in autoimmune diseases with myositis.

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ARTHRITIS & RHEUMATISM
Vol. 39, No. 2, February 1996, pp 292-296
0 1996, American College of Rheumatology
292
SENSITIVITY AND SPECIFICITY OF ANTI-Jo-1 ANTIBODIES IN
AUTOIMMUNE DISEASES WITH MYOSITIS
DOLORES VAZQUEZ-ABAD and NAOMI F. ROTHFIELD
Objective. To determine the sensitivity and specificity of anti-Jo-1 in systemic sclerosis (SSc) patients
with and without myositis.
Methods. Immunoblots on HeLa nuclei were used
to screen sera from 554 consecutive connective tissue
disease patients. Those who had 45-55-kd bands, all
patients with polymyositis/derrnatomyositis (PM/DM),
and a random selection of SSc, Raynaud’s disease,
systemic lupus erythematosus, and rheumatoid arthritis
patients were also studied by anti-Jo-1 enzyme-linked
immunosorbent assay and by immunoblots on rabbit
pooled aminoacyl-transfer RNA synthetase.
Results. Anti-Jo-1 was present only in 8 of the 40
PM/DM patients.
Conclusion. Anti-Jo-1 is specific for PM/DM.
Anti-Jo- 1 antibodies are found in polymyositis/
dermatomyositis (PM/DM) patients, especially in
those with lung involvement (1). Anti-Jo-1 is directed
at histidyl-transfer RNA (tRNA) synthetase (Jo-1
autoantigen), which is a dimer of 50-kd subunits found
in the cytoplasm (1,2). Anti-Jo-1 may be detected
by double immunodiffusion, immunoblotting, and
enzyme-linked immunosorbent assay (ELISA) (3) using an antigen containing Jo-1 and standard positive
and negative control sera.
The presence of anti-Jo-1 in systemic sclerosis
(SSc) patients has not been examined in large-scale
studies. SSc patients frequently present with lung
involvement, and, in some cases, with myositis.
Therefore, in the present study, we decided to screen
our large series of consecutive SSc patients for the
Dr. Rothfield’s work was supported by NIH grants AR31986 and AR-20621.
Dolores VAzquez-Abad, MD, Naomi F. Rothfield, MD:
University of Connecticut School of Medicine, Farmington.
Address reprint requests to Dolores Vhzquez-Abad, MD,
Division of Rheumatic Diseases, University of Connecticut School
of Medicine, 263 Farmington Ave, Farmington, CT 06030-13 10.
Submitted for publication July 25, 1995; accepted in revised
form September 29, 1995.
presence of anti-Jo-1 and compare them with other
connective tissue disease patients with and without
myositis.
Our results show that anti-Jo-1 is present in
20.0% of PM/DM patients (8 of 40), and is absent from
SSc patients’ sera, including those with PM/DM overlap and those with elevated creatine phosphokinase
(CPK) levels and muscle pain, but who do not fulfill
criteria for PM/DM, with or without interstitial lung
fibrosis.
PATIENTS AND METHODS
Patients and controls. All patients and controls were
seen in the Rheumatology Clinics at the University of
Connecticut School of Medicine, Farmington. A total of 429
patients with SSc or Raynaud’s disease with or without
overlap syndromes (excluding patients with PM/DM overlap), 31 patients with rheumatoid arthritis (RA), 54 patients
with systemic lupus erythematosus (SLE) who did not have
Raynaud’s disease or SSc, and 40 patients with PMIDM (21
PM and 19 DM), including 6 DM and 4 PM patients with
Raynaud’s overlap and 6 PM and 4 DM patients with SSc
overlap, were studied (Table 1).
All patients met the American College of Rheumatology criteria for classification (4-6), and the polymyositis
group met the criteria of Bohan and Peter (7). Sera were
collected from all patients and kept at -70°C. An anti-Jo-1
positive control from the Centers for Disease Control and
Prevention (CDC anti-Jo-I) was used for all experiments. A
pool of normal human serum devoid of autoantibodies
(NHS) was used as a negative control (8).
HeLa immunoblots. All sera were immunoblotted on
HeLa cell nuclei, as previously described (8,9).
Rabbit tRNA synthetase-enriched supernatant immunoblots (RSI). Rabbit kidney cells (LCC-RKI) were kindly
provided by Drs. Deutschter and Filonenko. The LCC-RK1
were grown, harvested, washed with PBS and suspended in
20 mM HEPES-KOH buffer (pH 7.4) containing 1 mM
dithiothreitol, 0.2 mM EDTA, 1 mM MgCI,, 0.4 mM phenylmethylsulfonyl fluoride, 5 mg/ml leupeptin, and 5 mg/ml
cu,-macroglobulin. After 10 minutes of incubation at 4”C,
lysis was achieved by 10 strokes of a Dounce homogenizer.
The homogenate was supplemented with 50% volume/
volume of the same buffer containing 21% glycerol and
293
ANTI-Jo- 1 IN MY OSITIS PATIENTS
Table 1. Characteristics of the patients and sera studied*
Diagnosis (n)
SScICREST (213)
RD (216)
RA (31)
SLE (54)
PMIDM (40)
Pnmary (16)
Overlap with
RD (10)
SScICREST (10)
Other CTD (4)
Myositis only
(n = 50)
Pulmonary fibrosis
only (n = 113)
Myositislpulmonary
fibrosis (n = 49)
45-55 kd + HeLa
blots (n = 554)t
ELISA and RSI
(n = 161)t
25
0
1 I3
32
0
0165
0
16
4
2412 13
141216
113
9/54
8/40
4/16
4
5
2
2/10
0110
214
24
12
0
0
0
NA
NA
6
5
2
NA
NA
NA
1
0
1
0125
013
0129
8/40
4/16
2/10
0110
214
* ELISA = enzyme-linked immunosorbent assay; RSI = rabbit transfer RNA synthetase-enriched supernatant immunoblot; SSc = systemic
sclerosis; CREST = calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias; RD = Raynaud’s disease; RA
= rheumatoid arthritis; SLE = systemic lupus erythematosus: PMIDM = polymyositisldermatomyositis;CTD = connective tissue disease.
t Values are the number positiveltotal number tested.
0.495M potassium acetate, and centrifuged at 10,OOOg for 15
minutes. The supernatant (RS) was withdrawn and stored at
-20°C.
RS, which are enriched for aminoacyl-tRNA synthetases, were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation and for
immunoblots, as previously published (10). Briefly, RS were
separated on 10% SDS-PAGE under reducing conditions,
and transferred to nitrocellulose paper (BioRad, Hercules,
CA). After 1 hour at room temperature in PBS containing 4%
bovine serum albumin, the strips were incubated with the
different sera at a 1 :1,OOO dilution in the same buffer. After 1
hour of incubation, the strips were thoroughly washed and
incubated with rabbit anti-human 7, a, p, K , and A chains
coupled to horseradish peroxidase (HRP; Dakopatts, Copenhagen, Denmark) for 1 hour and after thorough washes, they
were developed with the ECL system (Amersham, Arlington
Heights, IL), or with the KPI system (Kirkegaard & Perry,
Gaithersburg, MD). Besides the human sera, a mouse monoclonal anti-rabbit arginyl-tRNA transferase (kindly provided
by Drs. Filonenko and Deutscher) was used in the RSI blots
to verify the quality of the RS blots. This strip was incubated
with goat anti-mouse IgG coupled to HRP (Dakopatts)
before development.
All 40 sera from PM/DM patients, random samples of
sera from the other groups of patients (65 of 213 SScl
CREST, 25 of 216 Raynaud’s disease, 2 of 31 RA, and 29 of
54 SLE patients) that includes all sera with a band at 45-55
kd on HeLa immunoblots were further tested by RSI (Table
1). Tested sera included 13 of 25 SSc/CREST patients and 1
of 1 RA patient with myositis without pulmonary fibrosis, 18
of 32 SSc/CREST patients and 1 of 1 RA patient with
myositis and pulmonary fibrosis, and 15 of 113 SSc/CREST
patients with pulmonary fibrosis without myositis. Not all
sera were so tested because of the limited supply of the
purified antigen.
Anti-Jo-1 ELISA. An ELISA kit with affinity-purified
Jo-1 from calf thymus (Diamedix, Miami, FL) was used to
screen the same sera that was tested on RSI (see above).
Statistical analysis. Fisher’s exact test was used to
compare the prevalence of anti-Jo-1 in the different groups
of patients. Odds ratios were calculated to determine the
relative risk of interstitial lung fibrosis in the presence or
absence of anti-Jo-1 in the PM/DM patients. Bayes’ theorem
was used to calculate the sensitivity and specificity of the
anti-Jo-1 result by ELISA and RSI.
RESULTS
The CDC anti-Jo-1 serum showed a distinct
50-kd band when tested by immunoblotting on extracts
of HeLa cell chromosomes separated on SDS-PAGE
(+ in Figure 1A). The CDC anti-Jo-1 serum reacted
with only a single band at 50 kd on the RSI blot (+ in
Figure lB), and was positive by ELISA. The NHS was
negative when tested for the presence of anti-Jo-1 by
all 3 methods (- in Figures 1A and B; ELISA results
not shown).
Fifty-five sera found to react with a band in the
range of 45-55 kd and 106 random sera with no bands
between 45 and 55 kd were further tested for the
presence of anti-Jo-1 by ELISA and RSI blots. Eight
of the 40 PM/DM patients had a 50-kd band on HeLa
blots, and were positive for anti-Jo-1 using ELISA and
RSI blots (Table 1). Figure 1A shows the HeLa blots
from 4 of these 8 anti-Jo-1 positive patients with the
distinct 50-kd band. Figure 1B shows the results of the
RSI blots using a mouse monoclonal antibody to
anti-rabbit arginyl-tRNA transferase that reacts with
60-kd, 66-kd, and 120-kd bands, and 4 of the 8 antiJo-1 positive sera reacting with the 50-kd band. All
other sera, whether or not they reacted with a 4555-kd band, were negative for anti-Jo-1 by ELISA and
RSI blots. The results of the ELISA and RSI blots
correlated completely. All 8 anti-Jo-1 positive sera by
ELISA and RSI, had a 50-kd band by HeLa chromo-
294
VAZQUEZ-ABAD AND ROTHFIELD
B
A
2
3
4
(-)
(+) MoAb
1
2
3
4
92
66
50
H
50
Figure 1. Immunoblots of normal human serum negative control (-), Centers for Disease Control and Prevention anti-Jo-I
and a mouse monoclonal anti-rabbit arginyl-transfer RNA
positive control (+), 4 anti-Jo-1 positive patients (lanes M),
transferase (MoAb), using A, extracts of HeLa nuclei and B, extracts of rabbit kidney cell supernatants. Numbers to the left are
molecular weight markers (in kd); arrows show the Jo-1 band; H = histones.
some blots; thus, there were no false-negative results
by HeLa blots.
Three of the 8 anti-Jo-1 positive patients had
primary PM, 1 had primary DM, 2 had Raynaud’s
disease and DM, 1 fulfilled criteria for RA and DM,
and 1 fulfilled criteria for RA and PM. Using the
Bayes’ theorem, the specificity and sensitivity of antiJo-1 for PM/DM are 100% and 20%, respectively. Six
of the 8 anti-Jo-1 positive patients had interstitial lung
disease, compared with 10 of the 32 anti-Jo-1 negative
PM/DM patients (odds ratio 6.6, confidence interval
between 0.88 and 47.37; P < 0.04).
DISCUSSION
Anti-Jo-1 is the most frequent autoantibody in
PM/DM and is specific for this disease, identifying a
clinical subset of patients different from those with
other PM/DM-related autoantibodies (1-3). Among
these other antibodies, a low percentage of patients
shares an antibody with 4% of SSc patients, i.e., the
PM/Scl autoantibodies that detect an 80-kd and a
100-kd band on HeLa cell nuclear blots (2). On the
other hand, in SSc the 3 most frequently seen autoantibodies are anticentromere, anti-topoisomerase I, and
anti-RNA polymerase 1 and I11 autoantibodies (1 1).
These autoantibodies are specific for the SSc spectrum
of diseases and identify different clinical subsets
(2,ll). None of these clinical subsets is characterized
by the presence of rnyositis with or without the interstitial lung fibrosis frequently found in SSc patients.
In this study, we have shown that none of the
213 SSc patients had anti-Jo-1. Among the 213 SSc
patients studied, 57 had elevated CPK levels and
muscle pain, but did not fulfill criteria for PM/DM.
Their electromyograms and muscle biopsy samples
were negative or were deemed unnecessary (and were
not considered overlap syndrome). Of these 57 SSc
patients with rnyositis, 32 had interstitial lung fibrosis.
Among the 40 PM/DM patients, 10 also fulfilled criteria for SSc (PM/DM overlap with SSc), and were all
negative anti-Jo-l . Hence, the absence of anti-Jo-1
antibodies in SSc patients is not explained by selection
bias and exclusion of patients with rnyositis and/or
interstitial lung fibrosis in that disease group. To
confirm that these negative results were not due to a
detection error in our technique, we screened our
population of PM/DM patients and found 8 of 40
patients (20%) with PMDM to be positive for antiJo-1. This prevalence is similar to that previously
reported (1). Furthermore, the association between
interstitial lung disease in PMDM and anti-Jo-1 positivity was similar to that previously published (1,2).
Two previous studies described investigations
of large series of sera from SSc patients by immunoprecipitation (12,13). In the figures published with
those studies, a few sera can be seen to precipitate a
295
ANTI-Jo-1 IN MYOSITIS PATIENTS
protein at approximately 50 kd. Anti-Jo-1 was not
specifically investigated in either study (12,13). In the
report by Bernstein et al, a survey of autoantibodies in
1,11 I sera was carried out (14). Anti-Jo- I was found in
9 of 36 patients with myositis and in 1 of 161 patients
with SSc by counterimmunoelectrophoresis; however,
no definitions or criteria for the diagnosis of SSc or
myositis were provided. An evaluation of an ELISA
using purified Jo-1 included 21 SSc sera along with a
large series of DM/PM patients and patients with other
connective tissue diseases (15). Some of the sera were
gifts to the investigators from 2 other institutions.
None of the 21 SSc sera had anti-Jo-1. Of the 19
patients with PM/DM overlap, 3 had anti-Jo-1 (2
overlap with SSc, 1 with RA). We did not find any
anti-Jo-1 positive sera among 10 patients with PM/DM
and SSc overlap. Perhaps the difference between our
findings and those of Biswas et al results from studying
only 10 patients with PM/DM and SSc overlap in our
series. However, the exact number of patients with
PM/DM and SSc overlap in their study was not stated
(15). On the other hand, the discrepancy may be due to
differences in criteria used for the diagnosis of PM/DM
and SSc overlap.
There is a retrospective collaborative study in
which chart reviews of patients whose sera were
positive for anti-Jo-1 in 2 hospital laboratories over an
8-year period were conducted (16). Sera testing positive by counterimmunoelectrophoresis were further
tested for anti-Jo-1 by immunoprecipitation and enzyme inhibition assays. Sixteen of the 19 anti-Jo-1
positive patients met criteria for myositis. An unusual
finding was that of the 19 anti-Jo-1 positive patients,
17 had Raynaud’s phenomenon, 15 sclerodactyly, 6
dysphagia, 6 telangiectasias, and 4 had soft tissue
calcification. Thus, there was a high prevalence of
anti-Jo-1 in patients with the CREST features in that
retrospective study. We did not find anti-Jo-1 in any of
our CREST patients. The criteria used to diagnose SSc
were not stated, and the clinical manifestations were
obtained from a chart review. There is the possibility
that the clinical manifestations cannot be compared
within the patient groups in that study, nor between
that patient group and ours. Another explanation for
these remarkably contrasting results may be that the
study populations, and thus, the humoral responses,
are different. This is possible, since antinuclear antibodies are associated with dissimilar clinical manifestations in SSc patients of different ethnic backgrounds
(17). In myositis patients from different ethnic back-
grounds, the prevalence of anti-Jo-1 is different (Uthman I et al: unpublished data).
From the present study, we conclude that antiJo-1 antibodies are disease-specific autoantibodies
similar to anti-topoisomerase I in SSc (11). SSc and
PMDM have highly specific and exclusive autoantibodies, suggesting that in both diseases, the patients’
humoral responses undergo similar selection and proliferation processes that determine the presence of
anti-Jo-I only in PMDM and of anti-topoisomerase I
only in SSc. These results support the concept of a
selected and restricted B cell dysregulation associated
with the establishment of PM/DM and SSc (18).
ACKNOWLEDGMENTS
We appreciate t h e helpful discussions and technical
s u g g e s t i o n s o f Murray D e u t s c h e r , MD and V a l e r y
Filonenko, P h D , and t h e excellent technical s u p p o r t of
Lesley S. Daniels and Sean M. Cusick.
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