98 1
CLINICAL AND SEROLOGIC ASSOCIATIONS OF THE
ANTIRIBOSOMAL P PROTEIN ANTIBODY
ELOISA BONFA and KEITH B. ELKON
Antibodies to the ribosomal P proteins (anti-P)
were detected, by Western blot analysis, in the sera of 20
of 114 patients with various autoimmune disorders.
Eighty-five percent of the patients with anti-P had
systemic lupus erythematosus (SLE). Of 93 randomly
selected patients, the frequency of anti-P was 7 of 59
SLE patients (12%) and 0 of 34 non-SLE patients.
Approximately one-third of the patients with anti-P
antibodies were male; approximately half were black. In
contrast to the findings of some previous studies which
used isolated ribosomes as antigen, an increased fre-
quency of renal disease was not observed. Although the
overall frequency of central nervous system lupus was
similar in SLE patients with and those without anti-P, 6
of 6 patients with psychosis had anti-P antibodies.
Western blotting was the most sensitive and specific
method for the detection of anti-P antibodies; counter-
immunoelectrophoresis and cytoplasmic indirect immu-
nofluorescence were positive in only 47% and 65% of
the anti-P-positive patients, respectively. Although 53%
of the SLE patients with anti-P had concomitant anti-Ro
antibodies, none had anti-La (as detected by counterim-
munoelectrophoresis). Anti-P antibodies, therefore, ap-
pear to be relatively specific serologic markers for SLE
and may be detected in the serum even when antibodies
to double-stranded DNA are not found.
Patients with systemic lupus erythematosus
SLE) and other autoimmune diseases manifest a di-
From the Hospital for Special Surgery and the Cornell
University Medical Center, New York, New York.
Supported by NIH grants AM-32845 and AM-14627 and the
New York SLE Foundation.
Eloisa Bonfa, MD; Keith B. Elkon, MD.
Address reprint requests to Dr. Eloisa Bonfa, Hospital for
Special Surgery, 535 East 70th Street, New York, NY 10021.
Submitted for publication November 18, 1985; accepted in
revised form February 10, 1986.
Arthritis and Rheumatism, Vol. 29, No. 8 (August 1986)
verse spectrum of antibodies to nuclear and cytoplas-
mic components of the cell (for review, see ref. 1).
Autoantibodies to isolated ribosomal preparations
were described more than 20 years ago (2,3), but the
prevalence, disease associations, and prognostic sig-
nificance of antiribosomal antibodies are all controver-
sial (2-9). The discordant data in the literature may be
partly explained by the different assays used to detect
antiribosomal antibodies; more importantly, however,
the differences are due to incomplete characterization
of the ribosomal antigens. Recently, the protein spec-
ificity of antiribosomal antibodies has been character-
ized and has been shown to react with cross-reactive
determinant(s) on 3 ribosomal phosphoproteins from
the large subunit PO, P1, and P2, having molecular
weights of 38,000, 19,000, and 17,000,respectively
(10). To define the clinical significance of these
autoantibodies, we studied the frequency of anti-P
antibodies in various autoimmune diseases, as well as
the clinical and serologic features of the patients who
have this antibody.
PATIENTS AND METHODS
Patients and controls. Serum samples from 114 pa-
tients with various autoimmune disorders were tested by
Western blot analysis for the presence of antiribosomal P
proteins (anti-P). These included 70 patients with SLE who
met the revised criteria of the American Rheumatism Asso-
ciation ( l l ) , 17 patients with rheumatoid arthritis, 5 patients
with idiopathic thrombocytopenic purpura, 6 patients with
polymyositis, 4 patients with scleroderma, and 12 patients
with miscellaneous autoimmune diseases (Sjogren’s syn-
drome, mixed connective tissue disease, seronegative arthri-
tis, and discoid lupus).
All but 21 of the patients were randomly selected.
These 21 patients (1 1 with SLE, 3 with polymyositis, 2 with
982 BONFA AND ELKON

Table 1. Frequency of detection of antiribosomal P (anti-P)
antibodies in patients with and those without systemic lupus
erythematosus (SLE)
Randomly
selected Cytoplasmic IIF
Group (n = 93) (n = 21)* Total
SLE patients 59 11 70
Anti-P-positive 7 10 17
Non-SLE patients 34 10 44
Anti-P-positive 0 3 3
* These 21 patients were selected for study on the basis of positive
cytoplasmic staining of serum on indirect immunofluorescence (IIF)
testing.
scleroderma, 4 with seronegative arthritis, and 1 with discoid
lupus I were referred for study because their serum samples
showed cytoplasmic immunofluorescence. Where appropri-
ate, these sera were analyzed separately. Sera were also
obtained from 12 normal subjects; these served as controls.
Clinical evaluation. Twenty patients with anti-P anti-
bodies and 33 SLE patients without anti-P antibodies (deter-
mined1 by Western blotting) were evaluated by retrospective
clinical chart review. Renal disease was diagnosed on the
basis of histologic criteria (12), persistent proteinuria >0.5
&day, or cellular casts seen on analysis of urine. Central
nervous system (CNS) involvement was diagnosed when
seizures, coma, or changes in mental status occurred in the
absence of drugs or other known metabolic causes (13).
Hematologic disorders were defined by persistent (at least 2
months duration) hemolytic anemia with reticulocytosis, leu-
kopenia <4,000/mm3, lymphopenia < 1,500/mm3, or throm-
bocytopenia <100,000/mm3.
Dermatologic disease was diagnosed by the presence
of a nialar rash, discoid rash, mucosal ulcers, or photosen-
sitivity. Articular disease was defined by a nonerosive ar-
thritis that involved 2 or more peripheral joints. Cardiovas-
cular iind pulmonary diseases included serositis, restrictive
lung disease, and cardiac valvular defects in the absence of
other known causes. Muscle involvement was diagnosed
when weakness was accompanied by an elevation of muscle
enzymies or an abnormal electromyograph result.
Serum antibody assays. Antinuclear antibodies
(ANA) and anticytoplasmic antibodies were detected by
indirect immunofluorescence (IIF) using a human epithelial
of Fairhurst et al (19). To strip off loosely bound proteins,
the ribosomes were centrifuged through buffers containing
500 mM KCI, as decribed (10).
Statistical analyses. Sera from SLE patients who
were positive for anti-P were compared with those from SLE
patients who were negative for anti-P, by chi-square analysis
with Yates’ correction. The means were compared by Stu-
dent’s t-test.
RESULTS
Sera from 114 patients with various autoim-
mune diseases and from 12 normal individuals were
studied (Table 1). Anti-P antibody was detected in 20
patients (7 of 93 who were randomly selected, and 13
of 21 who were selected for positive cytoplasmic
fluorescence). Eighty-five percent of the patients with
anti-P antibodies (17 of 20) had SLE. Normal individ-
uals and randomly selected patients with various
autoimmune disorders did not have this antibody.
Three women who had been selected for positive
cytoplasmic immunofluorescence were positive for
anti-P antibody. Their clinical features included
seronegative nonerosive arthritis (10 years duration),
mild arthralgias (5 years duration), and discoid lupus (4
years duration).
A clinical analysis of the 17 patients with defi-
nite SLE who had anti-P antibody and 33 SLE patients
who did not have the anti-P antibody is shown in Table
2. Approximately one-third of anti-P-positive patients
were male; approximately one-half were black. The
mean disease duration and frequency of multisystem
organ involvement were similar in both groups. The
lower frequency of hematologic and renal disease and
the higher frequency of CNS disease in the anti-P-
Table 2. Clinical findings in systemic lupus erythematosus
patients with and those without antiribosomal P (anti-P) antibody
Anti-P-positive Anti-P-negative
Finding (n = 17) (n = 33)
~ ~

Mean age at beginning of study 21 23

cell h e , HEp-2 (Antibodies Inc., Davis, CA), as substrate. (years)
Sera were diluted 150 in phosphate buffered saline, pH 7.4. Mean duration of disease (years) 10 12
Antibodies to double-stranded DNA (dsDNA) were mea- Malelfemale 5/12 4/29
sured by the Farr assay (14). Counterimmunoelectrophoresis Race
(CIE) was performed in 1.5% agarose in O.05M barbital Black 9 11
buffer, pH 8.2 (15). Reference sera of known specificities White 8 14
were used to detect and characterize the precipitating anti- Other 0 8
bodies. Extracts of rabbit thymus and dog spleen were used Multisystem organ involvement
Cardiovascular 4 8
as sources of antigens, as described previously (16). Renal 8 24
Polyacrylamide gel electrophoresis was performed Hematologic 9 21
using the discontinuous buffer system described by Laemmli Muscular 4 6
(17), in1 the presence of 0.1% sodium dodecyl sulfate. West- Dermatologic 15 26
ern blotting (18) was performed as described elsewhere (16). Articular 15 27
Ribosomes were isolated from dog liver by homogenization, Pulmonary 6 14
differential centrifugation, and discontinuous sucrose den- Central nervous system 7 10
Psychosis 6 0
sity ultracentrifugation, essentially according to the method
ANTI-P ANTIBODY AS SLE MARKER 983

positive group did not reach statistical significance.

However, of the 6 SLE patients who had psychosis as
a major clinical manifestation, all had the anti-P
antibody.
The laboratory findings in the 17 SLE patients
with and 33 SLE patients without anti-P antibodies are
shown in Table 3. Cytoplasmic IIF was detected in
65% of the anti-P-positive patients and in 3% of the
anti-P-negative patients. Nucleolar IIF was present in
47% of the anti-P-positive patients. The majority of
patients in both groups also had nuclear immunofluo-
rescence, but 3 patients with anti-P were ANA nega-
tive (Figure 1). Of the 21 patients whose sera were
referred because of positive cytoplasmic immunoflu-
orescence, 13 had anti-P activity; 10 of these 13 pa-
tients had SLE (Table l).
Most anti-P-positive sera tested by CIE con-
tained autoantibodies with other specificities, and
although 53% had anti-Ro antibodies, none had con-
comitant anti-La. Precipitating antibodies against P
antigens were positive in fewer than half of the anti-P-
positive patients. Western blotting was positive in all
of the anti-P-positive patients, with a frequency of
12% (7 of 59) in the unselected SLE patient group.
Only 2 of the 17 patient sera reactive with the P
proteins showed reactivity with other ribosomal
Figure 1. Indirect immunofluorescence staining of serum from a
proteins.
patient with antiribosomal P antibodies, showing prominent cyto-
plasmic and nucleolar staining and no nuclear fluorescence. HEp-2
DISCUSSION cell was used as substrate (original magnification x 900).

Many investigators have explored associations

between clinical autoimmune disorders and specific nephritis, and serum levels of the antibody have been
antibodies. The presence of anti-dsDNA has been correlated with disease activity (20,21). Other antibod-
strongly associated with the pathogenesis of lupus ies have also been considered to be markers for
different rheumatic diseases: anti-Sm for SLE, anti-
Table 3. Laboratory findings in systemic lupus erythematosus centromere for CREST syndrome (calcinosis, Ray-
patients with and those without antiribosomal P (anti-P) naud’s phenomenon, esophageal dysmotility, sclero-
antibodies*
dactyly, telangiectasias), and anti-U 1 RNP for mixed
Anti-P-positive Anti-P-negative connective tissue disease (22-24). The newer methods
Finding (n = 17) (n = 33) for precise characterization of antibody specificity,
Antinuclear antibodies including immunoprecipitation (25), Western blotting
Nuclear 14 29 (18), and 2-dimensional gel electrophoresis (26), allow
Nucleolar 8 2
Cytoplasmic 11 1 clinical associations to be reliably made.
Anti-double-stranded DNA 11 24 In the present study, the anti-P antibody was
Counterimmunoelectrophoresis predominantly found in patients with established SLE
Anti-Ro 9 12
Anti-La 0 7 and was not detected in a randomly selected control
Anti-Sm 6 4 population. The 3 patients who had the anti-P antibody
Anti-RNP 7 6 without full-blown lupus could have had a forme fruste
Antiribosomal 8 0
Western blot of SLE, since features of other autoimmune disorders
Anti-P 17 0 were not present in these patients. However, much
* Antinuclear antibodies were detected by indirect immunofluores- larger numbers of patients with other diseases should
cence; antidouble-stranded DNA was detected by Farr assay. be studied to determine the diagnostic value of anti-P.
984
Despite some reports of an increased frequency of
renal disease and adverse clinical course in patients
witlh antiribosomal antibodies (8,27), patients with
antii-P had a lower frequency of renal disease and a
similar mean duration of disease. These discrepancies
may be due to the detection of autoantibodies to
ribosomal RNA (rRNA), an RNA-protein complex, or
to loosely bound cytoplasmic proteins in the previous
studies. They cannot be explained by autoantibodies
to other ribosomal proteins, since these were rarely
obslerved in Western blots (this study) or in immuno-
precipitation of 35S-methionine-labeled HeLa cells
(unpublished observations). We and others (10,28)
have noted evidence for possible anti-rRNA antibod-
ies in some of the patients with anti-P protein antibod-
ies. Whether these antibodies are independently asso-
ciated with different clinical manifestations remains to
be determined.
Because racial origin (29,30) and sex (31) influ-
ence the expression of SLE in humans, the high
frequencies of anti-P antibodies in blacks and males
are d interest. However, studies of larger numbers of
patients will be needed to see whether a clear associ-
ation with anti-P can be established. Similarly, a
higher frequency of CNS disease was observed in
patients with the anti-P antibody, but this was not
statistically significant. Of 6 patients with psychosis as
a major clinical manifestation, all had the anti-P anti-
bod![. In at least 1 of these patients, selective enrich-
ment of the anti-P antibody was demonstrated in the
cerebrospinal fluid (32).
The anti-P antibody may serve as an additional
serologic marker for SLE, since anti-dsDNA antibod-
ies were not detected in 35% of the anti-P-positive
patients. However, most of these patients also had
antibodies directed against other saline-soluble cellu-
lar antigens, of which anti-Ro was the most frequently
observed (53% of anti-P-positive lupus patients). The
absence of anti-La precipitins in these patients’ sera is
surprising, considering the known association of anti-
Ro and anti-La in individual sera (33,34). Whether this
discordance reflects a dominant immune response
against cytoplasmic particles in the anti-P- positive
patients or a selective genetic dysregulation of anti-
body production is uncertain. La is located in the
nucleus, whereas the cytoplasmic location of Ro re-
mains controversial (35,36). More sensitive Western
blot studies will be required to determine whether low
conclentrations of anti-La antibodies are present in
these sera.
Immunofluorescence is a useful screening test
for anti-P: 10 of 11 SLE sera referred because of
BONFAANDELKON
known cytoplasmic immunofluroescence on HEp-2
cells showed anti-P activity on Western blots. How-
ever, about one-third of SLE patients with anti-P did
not show cytoplasmic immunofluorescence, and many
sera from patients with other autoimmune diseases
had cytoplasmic immunofluorescence and no anti-P
activify. Since the ribosome is assembled in the
nucleolus and the P proteins have been detected in
nuclear extracts (lo), nucleolar immunofluorescence is
expected. It is likely that in about half of the anti-
P-positive sera studied, nucleolar immunofluores-
cence was obscured by the presence of other nuclear
autoantibodies. CIE using dog spleen extract was the
least sensitive test for anti-P, and commercially ob-
tained thymus extracts showed an even lower fre-
quency of positive results (unpublished observations).
Isolated whole ribosomes produce nonspecific precip-
itation in CIE (lo), whereas immunodiffusion is likely
to be more specific but even less sensitive. Western
blotting and immunoprecipitation are, therefore, the
only 2 techniques currently available for sensitive and
specific detection of anti-P antibodies. Development of
radioimmunoassays for the purified P proteins should
allow routine detection of these antibodies and enable
investigators to determine correlations with clinical
disease activity.
ACKNOWLEDGMENTS
We thank Drs. C. L. Christian, M. D. Lockshin, J.
Markenson, S. Paget, L. Kagen, and S. J. Golombek for
allowing us to study patients under their care, and H. Bonfa
for helping with computer analysis.
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