CONCISE
PUBLICATIONS
Antinuclear Antibody with Distinct Specificity
for Polymyositis
J. FREDERICK WOLFE, EDWARD ADELSTEIN, and GORDON C. SHARP
From the Department of Medicine, University of Missouri Medical Center, and the Department of
Pathology, Harry S. Truman Veterans Administration Hospital, Columbia, Missouri 65201
A B S T R A C T In the course of studying antinuclear
antibodies in the rheumatic diseases, a new precipitin
reaction (provisionally referred to as PM-1) was observed between calf thymus nuclear extract and polymyositis sera. Objectives of this study were to further
define the immunologic nature of this reaction and to
determine its specificity for polymyositis. Immunodiffusion studies using calf thymus nuclear extract revealed the PM-1 precipitin line in 17 of 28 patients
with polymyositis. This reaction was not produced by
sera of 460 patients with other diseases. Enzyme and
heat treatments of the nuclear extract showed that PM-I
was distinct from native DNA, ribonucleoprotein, and
Sm antigens. Fractionation of PM-i-positive serum
by 30% ammonium sulphate and Sephadex G-200
chromatography revealed that the factor producing the
PM-1 precipitin reaction was in a serum fraction which
showed only IgG by immunoelectrophoresis against
anti-whole human serum. Because of the apparent
strong specificity, the PM-1 system may represent a
marker antibody for polymyositis.
rheumatic diseases. The findings of circulating antibody to native DNA help to establish a diagnosis of
systemic lupus erythematosus (1), and antibody to nuclear ribonucleoprotein (RNP)' is helpful in the diagnosis of mixed connective tissue disease (2). In polymyositis, however, there has been no consistent
serological finding which is sufficiently sensitive and
specific to be useful as a diagnostic test.
Fluorescent antinuclear antibody tests have been
found to be positive to a variable degree, ranging
from 6 to 30% in different studies of polymyositis
(3, 4). However, the specific antigen involved in the
nuclear staining was not identified in these studies.
In another study, an IgG antibody which reacted
with capillary walls in mouse kidney was detected
in the sera of patients with a number of rheumatic
diseases including dermatopolymyositis (5). In the
course of studying the correlation of various antinuclear antibodies with clinical characteristics in the
rheumatic diseases, we have observed a new precipitin
reaction with calfthymus nuclear extract which appears
to be specific for polymyositis-dermatomyositis.
INTRODUCTION
Serological findings, sufficiently sensitive and specific,
have provided useful diagnostic tools in certain of the
This work was presented in part at the Meeting of the
American Federation for Clinical Research, Atlantic City,
N. J., 2 May 1976 (1976. Clin. Res. 24: 49A.) and at the
American Rheumatism Association Meeting in Chicago,
Ill., 11 June 1976.
Received for publication 12 August 1976 and in revised
form 7 October 1976.
176
METHODS
Sera of patients were collected during hospitalization or
clinic visits at this and other medical centers and frozen in
small aliquots before testing. A crude calf thymus nuclear
extract (CTNE) (6), DNA (7), and extractable nuclear
antigen (2) were prepared according to published methods.
Native DNA and extractable nuclear antigen were used in
passive hemagglutination (1).
' Abbreviations uised in this paper: CTNE, Calf
thymus nuclear extract; RNP, ribonucleoprotein.
The Journal of Clinical Investigation Volume 59 January 1977 176-178
Double diffusion was performed in 0.4%. Agarose (SeaKem Irish Moss Extractives, Springfield, N. J.) as previously
described (6). After plates had been incubated at 230C
and read at 24 h, they were placed at 40C and observed
again at 48, 72, and 96 h. Sera containing antibodies specific
for RNP, Sm, native, and denatured DNA were placed in
adjacent wells to
sera
being tested to characterize the
immune precipitates which resulted. Positive sera were also
tested in immunodiffusion with CTNE which had been
treated with deoxyribonuclease, ribonuclease, or trypsin, or
exposed to 37 or 560C according to published methods
(6). Immunoelectrophoresis using anti-whole human serum,
fractionation of gamma globulin by 30% ammonium sulfate
precipitation, and Sephadex G-200 (Pharmacia Fine Chemicals, Inc., Piscataway, N. J.) chromatography, and fluorescent
antinuclear antibody tests were performed by standard laboratory methods.
Diagnosis of polymyositis was made on the basis of: (a)
proximal muscle pain and (or) weakness involving pelvic
girdle, pectoral girdle, and (or) neck flexors; (b) elevation
of serum creatine phosphokinase and aldolase (c) electromyogram showing myopathic changes; and (d) muscle biopsy showing inflammatory infiltrates and (or) muscle necrosis. Patients were classified as dermatomyositis if they
had the above criteria and characteristic skin changes. Patients were classified as polymyositis-scleroderma overlap if
they had skin changes consistent with scleroderma in addition to the findings of polymyositis. Using these criteria,
28 patients were classified as having a polymyositis syndrome.
RESULTS
Fig. I shows the line of precipitation, provisionally
referred to as PM-1, which formed between the CTNE
and the sera of patients with polymyositis. Because the
PM-I precipitin line is rather weak, it is difficult to appreciate in a reproduction but it has clearly been
observed that the PM-1 line crosses both the RNP
and the Sm lines, indicating that the PM-1 antigen is
distinct from these antigens. It was often necessary
to observe plates for 48, 72, or 96 h to see PM-1
precipitates form and establish their identity. The reactions of all PM-i-positive sera showed lines of iden-
TABLE I
Characterization of Nuclear Extract A utige us
Treatmnent of
CTNE
PMI-i
Sm
RNP
RNase
Dnase
37°C-6 h
56°C-1 h
Trypsin
Resistant
Resistant
Resistant
Sensitive
Sensitive
Resistant
Resistant
Resistant
Resistant
Partially
sensitive
Sensitive
Resistant
Sensitive
Sensitive
Sensitive
tity. Table I lists various enzymatic and physical
treatments performed on the CTNE and their effect
on the precipitin reactions, which further demonstrates
that PM-1, RNP, and Sm are different antigens.
The PM-1 antigen was sensitive to trypsin and heating
at 56°C for 1 h, but was resistant to DNase and RNase
digestion. Fractionation of PM-i-positive serum by
30% ammonium sulfate precipitation and Sephadex
G-200 chromatography revealed that the factor produicing the PM-1 precipitin reaction was in a serum
fraction which showed only IgG by immunoelectrophoresis against anti-whole human serum.
As shown in Table II, 17 of 28 patients with polymyositis, dermatomyositis, or polymyositis-scleroderma overlap (61%) were positive for the PM-1
reaction. Serial studies over a 5-yr period on 10 of
these polymyositis patients, 5 positive and 5 negative
for the PM-I reaction, showed that if PM-I was present,
it remained whether or not the disease was active or
in remission. Likewise, if PM-1 was not present at the
time the patient was first seen it remained absent
regardless of the clinical course. Other groups of patients studied including 169 with mixed connective
tissue disease, 100 with systemic lupus erythematosus,
30 with scleroderma, 30 with rheumatoid arthritis,
10 with osteoarthritis, 21 with other rheumatic
diseases, and 100 other hospitalized patients were uniformly negative for the PM-1 precipitin reaction.
Fluorescent antinuclear antibody tests were performed on all 28 patients with polymyositis. 8 of the 17
TABLE II
Incidence of PM-i ini Polyinyositis Syndromnes
Positive
Clinical svndrome
FIG.
1
The well-marked CTNE contains calf thymus nuclear
extract. Well 1 contains serum from a patient with antibodies
to RNP and Sm. Well 2 contains serum from a patient with
polymyositis which produces the PM-1 line that is distinct
from both the RNP and Sm lines.
Polymyositis
Dermatomyositis
Polymyositis-scleroderma
overlap
Total
total
9/14
1/6
64
17
7/8
87
17/28
61
Antinuiclear Atntibody in Polyiniyositis
177
sera positive for PM-1 were positive in fluorescent
antinuclear antibody tests with titers ranging from 1:20
to 1:80, usually showing a speckled pattern. These
eight positive sera were from four patients with polymyositis and four with polymyositis-scleroderma
overlap. Only 2 of 11 sera negative for the PM-1
reaction were positive, both with a titer of 1:20.
DISCUSSION
The present study and a recent report by Reichlin
and Mattioli (8) suggest that a particular antinuclear
antibody may be a highly specific serological marker
for polymyositis-dermatomyositis. Because they found
the precipitin reaction to be weak and sporadic,
Reichlin and Mattioli employed an indirect test in
which Fab fragments of sera being tested were studied
for their capacity to produce significant inhibition of
complement fixation by a reference serum reacting
with CTNE. In their study, 10 of 17 (59%) polymyositis sera and 3 of 84 sera from normals or
patients with other diseases produced significant inhibition. Although the PM-1 precipitin reaction is not
strong, we have found it to be consistent and reproducible. The immunodiffusion method has the advantage that we can demonstrate directly that PM-1
reactions of all positive sera show lines of identity.
Even though immunodiffusion is insensitive, we were
able to show the PM-1 reaction in 17 of 28 (61%)
patients with polymyositis or dermatomyositis but did
not find the precipitin line in any of 460 sera from
patients with other diseases.
It is possible, because of the similarity in physiochemical characteristics of the antigen, that the same
system is being described in these two independent
investigations. However, the studies differ with respect
to the incidence of the antibody in patients with dermatomyositis. We are presently pursuing this question
through an exchange of sera.
It is not clear at this time whether the PM-1positive and PM-i-negative patients represent different clinical subgroups of polymyositis. No definite
clinical distinctions are apparent to us at this time.
Thus, our finding that 40% of polymyositis patients
are negative for PM-1 may simply be due to the
insensitivity of the methods used in detection.
Because muscle-like elements have been described
178
J. F. Wolfe, E. Adelstein, and G. C. Sharp
in thymus (9), it is conceivable that the PM-1 antigen
could be a component of such structures and may
not necessarily be derived from thymocytes or other
immunologic cells in thymus. The PM-1 antigen is
definitely distinct from RNP, Sm, and DNA antigens.
Since it is sensitive to trypsin, it may be a nuclear
protein. Further studies are in progress to define
this antigen and to determine whether the immunologic reaction is thymus specific.
ACKNOWLEDGMENTS
We thank Mrs. Meridith Manning and Miss Cindy Evers for
their skilled technical assistance and Mrs. Linda O'Connor,
Miss Debbie Harman, and Mrs. Nanci Hubbard for their
secretarial assistance.
This research was supported in part by a grant from the
U. S. Public Health Service (AM 16141), a grant from The
Arthritis Foundation, and a Clinical Research Center grant
(RR-00-287-10).
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