REVIEWS OF INFECTIOUS DISEASES. VOL. 1, NO.6. NOVEMBER-DECEMBER 1979 © 1979 by The University of Chicago. 0162-0886/79/0106-0010$00.88
The Possible Role of Epstein-Barr Virus in Rheumatoid Arthritis Eng M. Tan
From the Division oj Rheumatic Diseases, University oj Colorado Medical Center, Denver, Colorado
For many years, our laboratory has been engaged in the study of autoantibodies to intracellular antigens. These autoantibodies are present in the sera of patients with systemic rheumatic diseases, including systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, scleroderma, and mixed connective tissue disease. In the course of these studies, we have demonstrated that antibodies to nuclear antigens are present in high frequency in patients with rheumatoid arthritis. These autoantibodies have specificity for a nuclear protein that is either a viral protein or a cellular protein induced by Epstein-Barr virus (EBV) in infected lymphoid cells. We will review the recent evidence supporting this hypothesis.
dation, La Jolla, Calif., and the University of Colorado Medical Center, Denver, Colo. In addition, sera from patients with Sjogren's syndrome were provided by Dr. Norman Talal, University of California, San Francisco. Dr. Gerald Rodnan, University of Pittsburgh, Pittsburgh, Pa. provided some sera from patients with scleroderma. The immunological methods used in these studies included Ouchterlony immunodiffusion, immunofluorescence, and hemagglutination. Human lymphoid cell lines and simian cell lines infected with EBV or simian herpesviruses were provided by Dr. Fred Jensen, Scripps Clinic and Research Foundation, and by Dr. Harvey Rabin, Frederick Cancer Research Center, Frederick, Md. Treatment of the tissue culture cells to obtain nuclear antigens for immunological studies has been described in detail previously [1-4]. In addition, preparation of tissue culture cells as substrates for immunofluorescence testing has also been described [5].
Materials and Methods
The materials and methods used in these studies have been described in detail previously [1-4]. Sera studied for their autoantibody content were obtained from patients with systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, scleroderma, and mixed connective tissue disease. These patients were seen in the clinics and hospitals of the Scripps Clinic and Research Foun-
Results
When a section of tissue, in this instance cryostat sections of mouse kidney, is used as substrate in the indirect immunofluorescence assay for detection of antibodies to nuclear antigens, in general, four patterns of nuclear staining are obtained, as illustrated in figure 1. Figure Ia shows a nuclear rim staining pattern, and figure 1b a speckled nuclear pattern. Figure Ic demonstrates homogeneous nuclear staining, and Figure 1d nucleolar staining. It has become clear that these staining
These studies were supported by grant no. AM 20705 from the National Institutes of Health and by a grant from the Kroc Foundation for the Advancement of Medical Science. The associates participating in these studies include Drs. M. Alspaugh, N. Talal, F. Jensen, and H. Rabin. Please address requests for reprints to Dr. Eng M. Tan, Division of Rheumatic Diseases, University of Colorado Medical Center, Denver, Colorado 80262.
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The sera of patients with rheumatoid arthritis form a precipitate with an antigen present in the sonicate of a human B (bone marrow-derived) lymphoid cell line (WiL 2) . The antibody is distinct from rheumatoid factor. The antigen can be demonstrated by an immunofluorescence technique to be distributed within the nucleus of the WiL 2 cell and shows a discrete speckled pattern of nuclear staining. The nuclear antigen is not detectable in organ extracts from many animal species or in human T (thymus-derived) lymphoid cell lines. The current evidence suggests that the nuclear antigen is detectable only in human B lymphoid cells infected with Epstein-Barr virus. It is not clear at present whether the nuclear antigen is a viral protein or a cell protein induced by the EpsteinBarr virus as a result of polyclonal B cell activation.
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patterns are the result of antinuclear antibodies of different specificities, reacting with nuclear antigens that are distributed within the nucleus in different areas. The speckled pattern of nuclear staining has recently become the object of intensive study, because autoantibodies of several different specificities, i.e., reactive with many different antigens, may produce this pattern of nuclear immunofluorescence. Antinuclear antibodies producing speckled patterns of nuclear staining are listed in table 1, and this is by no means a complete list. The nuclear antigens, which are protein in nature, may sometimes be complexed to nucleic acids such as the antibody reactive with nuclear ribonucleoprotein (RNP). All of these nuclear proteins are nonhistone proteins;
some of these proteins are "acidic," but others may be as "basic" as histones.
Antibodies reactive with different nonhistone proteins have been identified primarily by immunological methods, with the use of autoantibodies present in the sera of patients. One feature emerging from these studies has been the demonstration that some autoantibodies are highly specific serological markers. Antibody to the Sm antigen is a marker for systemic lupus erythematosus and is rarely seen in patients with other diseases [5]. If antibody to Sm is present in the context of other diseases, the patient may have overlapping syndromes consisting of systemic lupus erythematosus associated with another connective tissue disease such as rheumatoid arthritis or scleroderma. Another serological marker is antibody to the Sel-l antigen, which is seen in patients with scleroderma [6]. Antibody to nuclear RNP is present in high frequency in patients with
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Figure 1. Indirect immunofluorescence study using sections of mouse kidney as substrate to demonstrate the presence of antinuclear antibodies of different specificities: a, nuclear rim stain; b, speckled nuclear stain; c, homogenous nuclear stain, and d, nucleolar stain. These different patterns of nuclear staining are caused by antibodies reacting with different nuclear antigens. The speckled nuclear pattern is generally produced by antibodies to nonhistone nuclear proteins (x 4(0).
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EB V in Rheumatoid Arthritis
Table 1. Autoantibodies proteins. Antigenic specificity
to
nonhistone
nuclear
Disease association "Marker" antibody for SLE
2. Antibody to nuclear ribonucleoprotein
High titers in mixed connective tissue disease; also present in lower titers in other connective tissue diseases
3. Antibody to SS-B antigen
Present in 50010 of patients with Sjogreri's-sicca, 15% of patients with SLE
4. Antibody to SS-A antigen
Present in 70% of patients with Sjogren's-sicca, 30% of patients with SLE
5. Antibody to rheumatoid arthritis-associated nuclear antigen
Present in 66% of patients with rheumatoid arthritis, 62% of patients with SS-RA
6. Antibody to Sc1-1 antigen
"Marker" antibody for scleroderma; present in 10%-20%.
7. Antibody to proliferating cell nuclear antigen
Present in 10010 of patients with SLE
NOTE. SLE = systemic lupus erythematosus; Sjogren's sicca = Sjogren's syndrome with sicca complex only; SS-RA = Sjogren's syndrome with rheumatoid arthritis.
mixed connective tissue disease but is seen in lower frequency in patients with other rheumatic diseases. Antibody to the SS-B antigen is present in high frequency in those with Sjogren's sicca syndrome but is seen in lower frequency in those with systemic lupus erythematosus. These studies have led to the concept of the existence of distinct profiles of antinuclear antibodies in the systemic diseases. Data on antibody to rheumatoid arthritisassociated nuclear antigen (RANA) are listed in table 1. Antibody to RANA is present in high frequency in patients with rheumatoid arthritis and the type of Sjogren's syndrome seen in association with rheumatoid arthritis [2, 3]. In 1965, Bloch et al. published a comprehensive study on Sjogren's syndrome in which they divided Sjogren's syndrome into several subclasses [7]. Two major subclasses were comprised of patients with Sjogren's syndrome who only had the sicca complex and patients with Sjogren's syndrome who had definite or classical rheumatoid arthritis. These two subgroups were defined on the basis of clinical features. In our studies on these two subclasses of patients with Sjogren's syn-
Figure 2. Immunodiffusion study showing the lack of identity between precipitins in sera from patients with rheumatoid arthritis (RA) and Sjogren's syndrome and rheumatoid arthritis (SS-RA) on the one hand, and precipitins in sera from patients with Sjogren's syndrome with sicca complex only (SS-sicca) on the other. An extract of Wil., lymphoid cells is in the center well. The SS-sicca precipitin lines cross over the lines formed by SS-RA and RA sera, a result demonstrating immunological nonidentity. However, the SS-RA and RA precipitin lines demonstrate immunological identity.
drome, we noticed that patients who had Sjogren's syndrome with only the sicca complex (Sjogren's-sicca) had autoantibodies that we termed the SS-A and SS-B systems [1]. In contrast, patients with Sjogren's syndrome associated with rheumatoid arthritis (SS-RA) had precipitating antibodies of a different specificity [1, 2]. This distinction could be clearly demonstrated by immunodiffusion analysis, as shown in figure 2. The serum from a patient with Sjogren's-sicca reacted with a lymphoid cell extract (WiL 2 ) to produce a precipitin line that, in this illustration, represented the SS-B system. Serum from the patient with SSRA reacted with WiL 2 extract to produce one precipitin line, which completely crossed over the line from the reaction between Sjogren's-sicca serum and WiL 2 extract, a finding demonstrating immunological nonidentity. Another patient with
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1. Antibody to Sm antigen
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Table 2. Prevalence of antibody to rheumatoid arthritis-associated nuclear antigen (RANA) in connective tissue diseases. Disease
59/90 (67) 34/55 (62) 10/77 (13) 3/51 (6) 0/12 0122 4/46 (9) 6/71 (8)
rheumatoid arthritis who did not have Sjogren's syndrome also had a precipitating antibody that showed complete immunological identity with the SS-RA line. When we analyzed sera from patients with other diseases, as shown in table 2, it became
Figure 3. Immunofluorescence of WiL 2 lymphoid cells fixed at 37 C for 30 min. Serum from a patient with rheumatoid arthritis that contained precipitating antibody to WiL 2 cell extract was used to demonstrate that the reactive antigens in WiL 2 cells were distributed as distinct speckles in the nucleus (approximately x 1,000).
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Rheumatoid arthritis (RA) Sjogren's syndrome associated with RA Sjogren's syndrome with only the sicca complex Systemic lupus erythematosus Mixed connective tissue disease Discoid lupus erythematosus Progressive systemic sclerosis Normal human serum
No. positive/no. tested (OJo)
clear that the SS-A and SS-B autoantibodies were segregated in patients with Sjogren's-sicca, and the other precipitating antibody was segregated in patients with SS-RA and in patients with rheumatoid arthritis who did not have Sjogren's syndrome. Thus, the latter precipitating antibody was subsequently called rheumatoid arthritis precipitin (RAP). It is seen that the RAP is present in twothirds or more of patients with either rheumatoid arthritis or SS-RA. It is present in 1'\J8OJo of normal human sera. Although the antibody in sera of patients with rheumatoid arthritis could be demonstrated by immunodiffusion, it was desirable to determine whether the antibody could be detected by a different immunological assay. Figure 3 shows that it was possible to demonstrate this antigen by immunofluorescence. The human B lymphoid cell line WiL 2 was used as the substrate, and sera of
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EB Vin RheumatoidArthritis
Table 3. Presence of rheumatoid arthritis-associated nuclear antigen (RANA) in extracts of various organs or cells as demonstrated by immunodiffusion and immunofluorescence. Animal of origin, extracts of organs or cultured cells Mouse Stomach Spleen Kidney Liver Cultured lymphocytes (SCR-60) Monkey Cultured kidney cells (CV -1) Calf Thymus Rabbit Thymus Human Thyroid Kidney Normal white blood cells B lymphoid cells WiL 2 Raji Daudi T lymphoid cells Molt 4
Fluorescent Precipitin staining in immuneof tissues" diffusion"
NO NO NO
NO NO NO NO NO
+ + +
+ + NO
1301
• (-) = negative; (+) = positive; NO = not done.
genome, and there is evidence that transformation of human B lymphoid cells into continuous cell lines is much facilitated by or may require infection with EBV. We thus studied many different lymphoid cell lines, some of human origin and others of simian origin (table 4). These cell lines were assayed for the presence of RANA as well as Epstein-Barr nuclear antigen (EBNA). RANA was assayed by indirect immunofluorescence with use of sera from patients with rheumatoid arthritis that had been determined previously to contain RAP and that were positive in immunofluorescence assays using WiL 2 cells as substrate. EBNA was determined by the complement amplification technique described by Reedman and Klein [8]. With these techniques, we determined that human lymphoid cell lines infected with EB virus contained RANA, but those infected with a herpesrelated virus of simian origin did not contain RANA. In addition, several different simian lymphoid cell lines, when infected with EBV, did contain RANA, but when infected with a simian herpesvirus, did not contain RANA. In most instances, EBNA and RANA were present in the same cell line, but this was not always the case since cell line CP-81 infected with Herpesvirus pongo contained EBNA but not RANA. Further studies were performed to determine the relationship between EBV and RANA. Normal human peripheral blood lymphocytes were isolated from several volunteers and divided for paired studies. One aliquot was maintained in culture as control, and the other aliquot was intentionally infected with EBV. Figure 4a shows WiL 2 lymphoid cells with speckled nuclear staining (RANA) produced by reaction with serum from a patient with rheumatoid arthritis. This was our standard positive. Figure 4b shows a lymphocyte culture infected with EB virus after culture for three to four weeks. Speckled nuclear staining has begun to appear in these cells incubated with sera from rheumatoid arthritis patients. As the cultures became transformed into continuous cell lines, the speckled nuclear staining became more intense and denser. Figures 4c and 4d are controls showing an uninfected culture incubated with serum from a patient with rheumatoid arthritis and an EBV-infected cell culture incubated with normal serum. respectively [4]. At present, we do not have further information concerning the molecular and chemical characteri-
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patients with rheumatoid arthritis showed a very finely discrete speckled nuclear stain. It was extremely difficult to develop this assay because the speckled nuclear antigen was highly soluble and many different methods of fixation caused the nuclear antigen to bleach out. Finally, it was determined that gentle heating at 37 C for 30 min in a dry oven preserved the labile nuclear antigen in the cell smear [3]. The availability of two different methods for the detection of autoantibody and conversely for the detection of nuclear antigen made it possible to perform the following series of studies. It was demonstrated (table 3) that RANA could not be detected by immunodiffusion or by immunofluorescence in tissues from species other than humans (i.e., mouse, monkey, calf, or rabbit). RANA was present in human B lymphoid cell lines but not in T cell lines and was not present in a mouse lymphoid cell line or in monkey kidney cells. It has been well documented that most human B lymphoid cell lines contain the Epstein-Barr virus
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Table 4. Epstein-Barr nuclear antigen (EBNA) and rheumatoid arthritis-associated nuclear antigen (RANA) in human and simian cell lines.
Cell line
Origin
Transforming virus
Immunofluorescent staining for
Lymphocyte cell type
EBNA
RANA
WiL 2
Human spleen
EBV
B
+++
+++
HCL-3/B95-8
Human umbilicus
EBV
B
+++
++
Human umbilicus
Herpesvirus papio
B
Owl monkey with in vitro EBVinduced lymphoreticular hyperplasia
EBV
B
+++
+
B95-8
Cotton-topped marmoset with in vitro EBV-transformed lymphocytes
EBV
B
+++
+
CP-81
Orangutan with monomyelogenous leukemia
Herpesvirus pongo
Undifferentiated
+
594-5
Baboon with lymphoreticular hyperplasia
H. papio
B
MLC-l
Cotton-topped marmoset with Herpesvirus saimiri-induced lymphoma
H. saimiri
T
NOTE.
EBV = Epstein-Barr virus.
zation of RANA. EBNA, however, has been purified by both Klein and Strominger with their associates [9, 10]. Further studies, which we have published, demonstrate that RANA appears to be different from EBNA in several characteristics. Data in table 5 show that most patients with Burkitt's lymphoma are positive for antibody to EBNA but are negative for antibody to RANA. Thus, there is a clear dissociation between autoimmune responses to EBNA and RANA. In addition, certain patients with rheumatoid arthritis may have antibody to RANA without antibody to EBNA. Differences in the immunofluorescent characteristics of RANA and EBNA are illustrated in figure 5. Figure Sa shows RANA nuclear staining demonstrated by indirect immunofluorescence. In figure Sc, the complement amplification method was used for the same serum. It is apparent that there was no amplification with the complement staining method of Reedman and Klein [8]. Figure Sb shows that a serum positive for antibody to EBNA did not stain nuclei when the indirect immunofluorescence technique was used. However, when the complement amplification technique was used (figure 5d), the nuclear staining was intense.
Discussion
These studies have demonstrated an association between EBV and rheumatoid arthritis. This association is related to the presence of an antibody in serum directed against a nuclear antigen present in human B lymphoid cells infected with EBV. This autoantibody is present in high frequency (66%) in patients with rheumatoid arthritis but in much lower frequency in patients with other connective tissue diseases or in normal populations. The nature of the nuclear antigen that induces the immune response in patients with rheumatoid arthritis is not known. The antigen that is present in nuclei of lymphoid B cells may be a viral protein, but it also could be a host cell protein induced by the EBV. These studies do not yet tell us if there is an etiological relationship between EBV infection and rheumatoid arthritis. If a relationship is present, specific host and other factors must play important roles since EBV has been implicated in such diverse diseases as Burkitt's lymphoma, nasopharyngeal carcinoma, and infectious mononucleosis. Studies supporting a relationship between EBV and rheumatoid arthritis have been reported
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HCL-3/KMPG-l 531-H
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ERVin Rheumatoid Arthritis
Table 5. Relationship between antibodies to rheumatoid arthritis-associated nuclear antigen (RANA) and EpsteinBarr nuclear antigen (EBNA). Immunofluorescent staining for Serum Patients with rheumatoid arthritis 1
2 3 Patients with Burkitt's lymphoma 1 2 3 4
RANA
EBNA
+ (32)* + (16) + (16)
+ (256) + (512) + + + +
Precipitating antibody to RANA
+ + +
(16) (4) (4)
(5,120) (2,560) (80) (1,280)
5
* Numbers in parentheses represent
the reciprocal of the highest dilution of serum positive for staining or precipitation.
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Figure 4. Indirect immunofluorescence demonstrating the induction of rheumatoid arthritis-associated nuclear antigen (RANA) after infection of lymphocytes with Epstein-Barr virus (EBV): a, discretely distributed speckled nuclear staining on WiL 2 cells as a result of reaction with a standard reference serum from a patient with rheumatoid arthritis (RA); b, EBV-infected peripheral blood leukocytes on day 20 of culture (a few discretely distributed fine nuclear speckles were seen at this time and became more numerous as the cells became transformed into continuous lines); c, the same EBV-infected cells (day 20) incubated with normal human serum; and d, an uninfected control cell culture on day 20, incubated with the same RA serum. Neither c nor d shows nuclear staining (x 400).
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recently by two other groups of investigators. Slaughter et al. showed that peripheral blood lymphocytes of patients with rheumatoid arthritis produce small amounts of rheumatoid factor in cell culture [11]. When these cells are infected with EBV, the production of rheumatoid factor is greatly increased. These and other studies show that the EBV may be a highly selective polyclonal B cell activator for patients with rheumatoid arthritis. Depper et al. recently reported that the peripheral blood lymphocytes of patients with rheumatoid arthritis are activated more rapidly by EBV than are normal lymphocytes [12]. They measured the "outgrowth" time of rheumatoid cells and normal cells after infection with EBV and showed a significantly shortened outgrowth
time of rheumatoid cells as compared with that of normal cells.
References
1. Alspaugh, M. A., Tan, E. M. Antibodies to cellular antigens in Sjogren's syndrome. J. Clin. Invest. 55:10671073, 1975. 2. Alspaugh, M. A., Talal, N., Tan, E. M. Differentiation and characterization of autoantibodies and their antigens in Sjogren's syndrome. Arth. Rheum. 19:216-222, 1976. 3. Alspaugh, M. A., Tan, E. M. Serum antibody in rheumatoid arthritis reactive with a cell-associated antigen. Demonstration by precipitation and immunofluorescence. Arth, Rheum. 19:711-719, 1976. 4. Alspaugh, M. A., Jensen, F. c., Rabin, H., Tan, E. M. Lymphocytes transformed by Epstein-Barr virus. Induction of nuclear antigen reactive with antibody in rheumatoid arthritis. J. Exp. Med. 147:1018-1027, 1978. 5. Notman, D. D., Kurata, N., Tan, E. M. Profiles of antinuclear antibodies in systemic rheumatic diseases. Ann. Intern. Med. 83:464, 1975. 6. Tan, E. M., Rodnan, G. P. Profile of antinuclear anti-
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Figure 5. Immunofluorescence study to demonstrate the difference between rheumatoid arthritis-associated nuclear antigen (RANA) and Epstein-Barr nuclear antigen (EBNA). When WiL 2 lymphoid cells were incubated with serum from a patient with rheumatoid arthritis (RA) and stained by the indirect immunofluorescence technique, the presence of speckled nuclear staining was demonstrated (0). When the complement amplification technique was used, the nuclear staining was not intensified (c). In contrast, WiL 2 lymphoid cells tested with a serum containing antibody to EBNA but negative for antibody to RANA showed no nuclear staining by the indirect fluorescence technique (b), but when the complement amplification technique was used, intense nuclear staining was observed (d). (x 400).
EBV in Rheumatoid Arthritis
to. Baron, D., Strominger, J. L. Partial purification and properties of the Epstein-Barr virus-associated nuclear antigen. J. BioI. Chern. 253:2875-2881, 1978. 11. Slaughter, L., Carson, D. A., Jensen, F. C., Holbrook, T. L., Vaughan, J. H. In vitro effects of Epstein-Barr virus on peripheral blood mononuclear cells from patients with rheumatoid arthritis and normal subjects. J. Exp. Med. 148:1429-1434, 1978. 12. Depper, J. M., Bardwick, P. A., Bluestein, H. G., Zvaifler, N. J., Seegmiller, J. E. Abnormal regulation of Epstein-Barr virus transformation of rheumatoid lymphoid cells [abstract]. Arth. Rheum. 22:605, 1979.
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bodies in progressive systemic sclerosis [abstract]. Arth. Rheum. 18:430, 1975. 7. Bloch, K. J., Buchanan, W. W., Wohl, M. J., Bunirn, J. J. Sjogren's syndrome: a clinical, pathological and serological study of sixty-two cases. Medicine (Baltimore) 44:187-231, 1965. 8. Reedman, B. M., Klein, G. Cellular localization of an Epstein-Barr virus (EBV)-associated complement fixing antigen in producer and non-producer lymphoblastoid cell lines. Int. J. Cancer 11:499-520, 1973. 9. Ohno, S., Luka, J., Lindahl, T., Klein, G. Identification of a purified complement-fixing antigen as the EpsteinBarr virus-determined nuclear antigen (EBNA) by its binding to metaphase chromosomes. Proc. Natl. Acad, Sci. USA 74:1605-1609, 1977.
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Discussion I didn't mention in my presentation was that CMV [cytomegalovirus] seems to induce endogenous viruses. Do you have electron microscopic or other evidence that the effect of EB virus is not the induction of identifiable, unculturable viral products? DR. TAN. This is one of the points that I referred to in noting that other factors might be involved, and the mechanism you described should be considered as a possibility. One suggestion that has come from studies by other investigators who are extending this work is that the EB virus may be a polyclonal cell activator that induces a de-differentiation type of nuclear antigen that is expressed in cells infected with the virus. This is a very intriguing possibility. There have been many studies showing that the EB virus is a polyclonal B-cell activator. The question is whether, in rheumatoid arthritis, it is acting as a more specific kind of B-cell activator that induces a specific nuclear protein.
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DR. MELVIN KAPLAN. Could you tell us more about the nuclear constituent RANA [rheumatoid arthritis nuclear antigen]? Is this a product only of infected cells? Do you find any evidence of the antigen in uninfected cells by using more sensitive techniques? DR. ENG TAN. We know something about the nucleoprotein antigen that reacts with antibodies in sera from patients with rheumatoid arthritis, but our information is not complete. It is a protein antigen that is highly soluble in saline, and heretofore we have identified it in the nuclei of human B lymphoid cell lines carrying the EB [Epstein-Barr] virus genome. The antigen is not detectable by immunodiffusion or immunofluorescence in cell lines that do not carry the EB virus genome or in organs of many animal species. Whether it is an endogenous protein of the EB virus or a host cell protein induced by viral infection is not known, but we are more inclined to accept the latter interpretation. DR. JOHN HAMILTON. One of the findings that
The Possible Role of Epstein-Barr Virus in Rheumatoid Arthritis Eng M. Tan
From the Division oj Rheumatic Diseases, University oj Colorado Medical Center, Denver, Colorado
For many years, our laboratory has been engaged in the study of autoantibodies to intracellular antigens. These autoantibodies are present in the sera of patients with systemic rheumatic diseases, including systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, scleroderma, and mixed connective tissue disease. In the course of these studies, we have demonstrated that antibodies to nuclear antigens are present in high frequency in patients with rheumatoid arthritis. These autoantibodies have specificity for a nuclear protein that is either a viral protein or a cellular protein induced by Epstein-Barr virus (EBV) in infected lymphoid cells. We will review the recent evidence supporting this hypothesis.
dation, La Jolla, Calif., and the University of Colorado Medical Center, Denver, Colo. In addition, sera from patients with Sjogren's syndrome were provided by Dr. Norman Talal, University of California, San Francisco. Dr. Gerald Rodnan, University of Pittsburgh, Pittsburgh, Pa. provided some sera from patients with scleroderma. The immunological methods used in these studies included Ouchterlony immunodiffusion, immunofluorescence, and hemagglutination. Human lymphoid cell lines and simian cell lines infected with EBV or simian herpesviruses were provided by Dr. Fred Jensen, Scripps Clinic and Research Foundation, and by Dr. Harvey Rabin, Frederick Cancer Research Center, Frederick, Md. Treatment of the tissue culture cells to obtain nuclear antigens for immunological studies has been described in detail previously [1-4]. In addition, preparation of tissue culture cells as substrates for immunofluorescence testing has also been described [5].
Materials and Methods
The materials and methods used in these studies have been described in detail previously [1-4]. Sera studied for their autoantibody content were obtained from patients with systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, scleroderma, and mixed connective tissue disease. These patients were seen in the clinics and hospitals of the Scripps Clinic and Research Foun-
Results
When a section of tissue, in this instance cryostat sections of mouse kidney, is used as substrate in the indirect immunofluorescence assay for detection of antibodies to nuclear antigens, in general, four patterns of nuclear staining are obtained, as illustrated in figure 1. Figure Ia shows a nuclear rim staining pattern, and figure 1b a speckled nuclear pattern. Figure Ic demonstrates homogeneous nuclear staining, and Figure 1d nucleolar staining. It has become clear that these staining
These studies were supported by grant no. AM 20705 from the National Institutes of Health and by a grant from the Kroc Foundation for the Advancement of Medical Science. The associates participating in these studies include Drs. M. Alspaugh, N. Talal, F. Jensen, and H. Rabin. Please address requests for reprints to Dr. Eng M. Tan, Division of Rheumatic Diseases, University of Colorado Medical Center, Denver, Colorado 80262.
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The sera of patients with rheumatoid arthritis form a precipitate with an antigen present in the sonicate of a human B (bone marrow-derived) lymphoid cell line (WiL 2) . The antibody is distinct from rheumatoid factor. The antigen can be demonstrated by an immunofluorescence technique to be distributed within the nucleus of the WiL 2 cell and shows a discrete speckled pattern of nuclear staining. The nuclear antigen is not detectable in organ extracts from many animal species or in human T (thymus-derived) lymphoid cell lines. The current evidence suggests that the nuclear antigen is detectable only in human B lymphoid cells infected with Epstein-Barr virus. It is not clear at present whether the nuclear antigen is a viral protein or a cell protein induced by the EpsteinBarr virus as a result of polyclonal B cell activation.
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patterns are the result of antinuclear antibodies of different specificities, reacting with nuclear antigens that are distributed within the nucleus in different areas. The speckled pattern of nuclear staining has recently become the object of intensive study, because autoantibodies of several different specificities, i.e., reactive with many different antigens, may produce this pattern of nuclear immunofluorescence. Antinuclear antibodies producing speckled patterns of nuclear staining are listed in table 1, and this is by no means a complete list. The nuclear antigens, which are protein in nature, may sometimes be complexed to nucleic acids such as the antibody reactive with nuclear ribonucleoprotein (RNP). All of these nuclear proteins are nonhistone proteins;
some of these proteins are "acidic," but others may be as "basic" as histones.
Antibodies reactive with different nonhistone proteins have been identified primarily by immunological methods, with the use of autoantibodies present in the sera of patients. One feature emerging from these studies has been the demonstration that some autoantibodies are highly specific serological markers. Antibody to the Sm antigen is a marker for systemic lupus erythematosus and is rarely seen in patients with other diseases [5]. If antibody to Sm is present in the context of other diseases, the patient may have overlapping syndromes consisting of systemic lupus erythematosus associated with another connective tissue disease such as rheumatoid arthritis or scleroderma. Another serological marker is antibody to the Sel-l antigen, which is seen in patients with scleroderma [6]. Antibody to nuclear RNP is present in high frequency in patients with
Downloaded from http://cid.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 24, 2015
Figure 1. Indirect immunofluorescence study using sections of mouse kidney as substrate to demonstrate the presence of antinuclear antibodies of different specificities: a, nuclear rim stain; b, speckled nuclear stain; c, homogenous nuclear stain, and d, nucleolar stain. These different patterns of nuclear staining are caused by antibodies reacting with different nuclear antigens. The speckled nuclear pattern is generally produced by antibodies to nonhistone nuclear proteins (x 4(0).
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EB V in Rheumatoid Arthritis
Table 1. Autoantibodies proteins. Antigenic specificity
to
nonhistone
nuclear
Disease association "Marker" antibody for SLE
2. Antibody to nuclear ribonucleoprotein
High titers in mixed connective tissue disease; also present in lower titers in other connective tissue diseases
3. Antibody to SS-B antigen
Present in 50010 of patients with Sjogreri's-sicca, 15% of patients with SLE
4. Antibody to SS-A antigen
Present in 70% of patients with Sjogren's-sicca, 30% of patients with SLE
5. Antibody to rheumatoid arthritis-associated nuclear antigen
Present in 66% of patients with rheumatoid arthritis, 62% of patients with SS-RA
6. Antibody to Sc1-1 antigen
"Marker" antibody for scleroderma; present in 10%-20%.
7. Antibody to proliferating cell nuclear antigen
Present in 10010 of patients with SLE
NOTE. SLE = systemic lupus erythematosus; Sjogren's sicca = Sjogren's syndrome with sicca complex only; SS-RA = Sjogren's syndrome with rheumatoid arthritis.
mixed connective tissue disease but is seen in lower frequency in patients with other rheumatic diseases. Antibody to the SS-B antigen is present in high frequency in those with Sjogren's sicca syndrome but is seen in lower frequency in those with systemic lupus erythematosus. These studies have led to the concept of the existence of distinct profiles of antinuclear antibodies in the systemic diseases. Data on antibody to rheumatoid arthritisassociated nuclear antigen (RANA) are listed in table 1. Antibody to RANA is present in high frequency in patients with rheumatoid arthritis and the type of Sjogren's syndrome seen in association with rheumatoid arthritis [2, 3]. In 1965, Bloch et al. published a comprehensive study on Sjogren's syndrome in which they divided Sjogren's syndrome into several subclasses [7]. Two major subclasses were comprised of patients with Sjogren's syndrome who only had the sicca complex and patients with Sjogren's syndrome who had definite or classical rheumatoid arthritis. These two subgroups were defined on the basis of clinical features. In our studies on these two subclasses of patients with Sjogren's syn-
Figure 2. Immunodiffusion study showing the lack of identity between precipitins in sera from patients with rheumatoid arthritis (RA) and Sjogren's syndrome and rheumatoid arthritis (SS-RA) on the one hand, and precipitins in sera from patients with Sjogren's syndrome with sicca complex only (SS-sicca) on the other. An extract of Wil., lymphoid cells is in the center well. The SS-sicca precipitin lines cross over the lines formed by SS-RA and RA sera, a result demonstrating immunological nonidentity. However, the SS-RA and RA precipitin lines demonstrate immunological identity.
drome, we noticed that patients who had Sjogren's syndrome with only the sicca complex (Sjogren's-sicca) had autoantibodies that we termed the SS-A and SS-B systems [1]. In contrast, patients with Sjogren's syndrome associated with rheumatoid arthritis (SS-RA) had precipitating antibodies of a different specificity [1, 2]. This distinction could be clearly demonstrated by immunodiffusion analysis, as shown in figure 2. The serum from a patient with Sjogren's-sicca reacted with a lymphoid cell extract (WiL 2 ) to produce a precipitin line that, in this illustration, represented the SS-B system. Serum from the patient with SSRA reacted with WiL 2 extract to produce one precipitin line, which completely crossed over the line from the reaction between Sjogren's-sicca serum and WiL 2 extract, a finding demonstrating immunological nonidentity. Another patient with
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1. Antibody to Sm antigen
1000
Tan
Table 2. Prevalence of antibody to rheumatoid arthritis-associated nuclear antigen (RANA) in connective tissue diseases. Disease
59/90 (67) 34/55 (62) 10/77 (13) 3/51 (6) 0/12 0122 4/46 (9) 6/71 (8)
rheumatoid arthritis who did not have Sjogren's syndrome also had a precipitating antibody that showed complete immunological identity with the SS-RA line. When we analyzed sera from patients with other diseases, as shown in table 2, it became
Figure 3. Immunofluorescence of WiL 2 lymphoid cells fixed at 37 C for 30 min. Serum from a patient with rheumatoid arthritis that contained precipitating antibody to WiL 2 cell extract was used to demonstrate that the reactive antigens in WiL 2 cells were distributed as distinct speckles in the nucleus (approximately x 1,000).
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Rheumatoid arthritis (RA) Sjogren's syndrome associated with RA Sjogren's syndrome with only the sicca complex Systemic lupus erythematosus Mixed connective tissue disease Discoid lupus erythematosus Progressive systemic sclerosis Normal human serum
No. positive/no. tested (OJo)
clear that the SS-A and SS-B autoantibodies were segregated in patients with Sjogren's-sicca, and the other precipitating antibody was segregated in patients with SS-RA and in patients with rheumatoid arthritis who did not have Sjogren's syndrome. Thus, the latter precipitating antibody was subsequently called rheumatoid arthritis precipitin (RAP). It is seen that the RAP is present in twothirds or more of patients with either rheumatoid arthritis or SS-RA. It is present in 1'\J8OJo of normal human sera. Although the antibody in sera of patients with rheumatoid arthritis could be demonstrated by immunodiffusion, it was desirable to determine whether the antibody could be detected by a different immunological assay. Figure 3 shows that it was possible to demonstrate this antigen by immunofluorescence. The human B lymphoid cell line WiL 2 was used as the substrate, and sera of
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EB Vin RheumatoidArthritis
Table 3. Presence of rheumatoid arthritis-associated nuclear antigen (RANA) in extracts of various organs or cells as demonstrated by immunodiffusion and immunofluorescence. Animal of origin, extracts of organs or cultured cells Mouse Stomach Spleen Kidney Liver Cultured lymphocytes (SCR-60) Monkey Cultured kidney cells (CV -1) Calf Thymus Rabbit Thymus Human Thyroid Kidney Normal white blood cells B lymphoid cells WiL 2 Raji Daudi T lymphoid cells Molt 4
Fluorescent Precipitin staining in immuneof tissues" diffusion"
NO NO NO
NO NO NO NO NO
+ + +
+ + NO
1301
• (-) = negative; (+) = positive; NO = not done.
genome, and there is evidence that transformation of human B lymphoid cells into continuous cell lines is much facilitated by or may require infection with EBV. We thus studied many different lymphoid cell lines, some of human origin and others of simian origin (table 4). These cell lines were assayed for the presence of RANA as well as Epstein-Barr nuclear antigen (EBNA). RANA was assayed by indirect immunofluorescence with use of sera from patients with rheumatoid arthritis that had been determined previously to contain RAP and that were positive in immunofluorescence assays using WiL 2 cells as substrate. EBNA was determined by the complement amplification technique described by Reedman and Klein [8]. With these techniques, we determined that human lymphoid cell lines infected with EB virus contained RANA, but those infected with a herpesrelated virus of simian origin did not contain RANA. In addition, several different simian lymphoid cell lines, when infected with EBV, did contain RANA, but when infected with a simian herpesvirus, did not contain RANA. In most instances, EBNA and RANA were present in the same cell line, but this was not always the case since cell line CP-81 infected with Herpesvirus pongo contained EBNA but not RANA. Further studies were performed to determine the relationship between EBV and RANA. Normal human peripheral blood lymphocytes were isolated from several volunteers and divided for paired studies. One aliquot was maintained in culture as control, and the other aliquot was intentionally infected with EBV. Figure 4a shows WiL 2 lymphoid cells with speckled nuclear staining (RANA) produced by reaction with serum from a patient with rheumatoid arthritis. This was our standard positive. Figure 4b shows a lymphocyte culture infected with EB virus after culture for three to four weeks. Speckled nuclear staining has begun to appear in these cells incubated with sera from rheumatoid arthritis patients. As the cultures became transformed into continuous cell lines, the speckled nuclear staining became more intense and denser. Figures 4c and 4d are controls showing an uninfected culture incubated with serum from a patient with rheumatoid arthritis and an EBV-infected cell culture incubated with normal serum. respectively [4]. At present, we do not have further information concerning the molecular and chemical characteri-
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patients with rheumatoid arthritis showed a very finely discrete speckled nuclear stain. It was extremely difficult to develop this assay because the speckled nuclear antigen was highly soluble and many different methods of fixation caused the nuclear antigen to bleach out. Finally, it was determined that gentle heating at 37 C for 30 min in a dry oven preserved the labile nuclear antigen in the cell smear [3]. The availability of two different methods for the detection of autoantibody and conversely for the detection of nuclear antigen made it possible to perform the following series of studies. It was demonstrated (table 3) that RANA could not be detected by immunodiffusion or by immunofluorescence in tissues from species other than humans (i.e., mouse, monkey, calf, or rabbit). RANA was present in human B lymphoid cell lines but not in T cell lines and was not present in a mouse lymphoid cell line or in monkey kidney cells. It has been well documented that most human B lymphoid cell lines contain the Epstein-Barr virus
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Tan
Table 4. Epstein-Barr nuclear antigen (EBNA) and rheumatoid arthritis-associated nuclear antigen (RANA) in human and simian cell lines.
Cell line
Origin
Transforming virus
Immunofluorescent staining for
Lymphocyte cell type
EBNA
RANA
WiL 2
Human spleen
EBV
B
+++
+++
HCL-3/B95-8
Human umbilicus
EBV
B
+++
++
Human umbilicus
Herpesvirus papio
B
Owl monkey with in vitro EBVinduced lymphoreticular hyperplasia
EBV
B
+++
+
B95-8
Cotton-topped marmoset with in vitro EBV-transformed lymphocytes
EBV
B
+++
+
CP-81
Orangutan with monomyelogenous leukemia
Herpesvirus pongo
Undifferentiated
+
594-5
Baboon with lymphoreticular hyperplasia
H. papio
B
MLC-l
Cotton-topped marmoset with Herpesvirus saimiri-induced lymphoma
H. saimiri
T
NOTE.
EBV = Epstein-Barr virus.
zation of RANA. EBNA, however, has been purified by both Klein and Strominger with their associates [9, 10]. Further studies, which we have published, demonstrate that RANA appears to be different from EBNA in several characteristics. Data in table 5 show that most patients with Burkitt's lymphoma are positive for antibody to EBNA but are negative for antibody to RANA. Thus, there is a clear dissociation between autoimmune responses to EBNA and RANA. In addition, certain patients with rheumatoid arthritis may have antibody to RANA without antibody to EBNA. Differences in the immunofluorescent characteristics of RANA and EBNA are illustrated in figure 5. Figure Sa shows RANA nuclear staining demonstrated by indirect immunofluorescence. In figure Sc, the complement amplification method was used for the same serum. It is apparent that there was no amplification with the complement staining method of Reedman and Klein [8]. Figure Sb shows that a serum positive for antibody to EBNA did not stain nuclei when the indirect immunofluorescence technique was used. However, when the complement amplification technique was used (figure 5d), the nuclear staining was intense.
Discussion
These studies have demonstrated an association between EBV and rheumatoid arthritis. This association is related to the presence of an antibody in serum directed against a nuclear antigen present in human B lymphoid cells infected with EBV. This autoantibody is present in high frequency (66%) in patients with rheumatoid arthritis but in much lower frequency in patients with other connective tissue diseases or in normal populations. The nature of the nuclear antigen that induces the immune response in patients with rheumatoid arthritis is not known. The antigen that is present in nuclei of lymphoid B cells may be a viral protein, but it also could be a host cell protein induced by the EBV. These studies do not yet tell us if there is an etiological relationship between EBV infection and rheumatoid arthritis. If a relationship is present, specific host and other factors must play important roles since EBV has been implicated in such diverse diseases as Burkitt's lymphoma, nasopharyngeal carcinoma, and infectious mononucleosis. Studies supporting a relationship between EBV and rheumatoid arthritis have been reported
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HCL-3/KMPG-l 531-H
1003
ERVin Rheumatoid Arthritis
Table 5. Relationship between antibodies to rheumatoid arthritis-associated nuclear antigen (RANA) and EpsteinBarr nuclear antigen (EBNA). Immunofluorescent staining for Serum Patients with rheumatoid arthritis 1
2 3 Patients with Burkitt's lymphoma 1 2 3 4
RANA
EBNA
+ (32)* + (16) + (16)
+ (256) + (512) + + + +
Precipitating antibody to RANA
+ + +
(16) (4) (4)
(5,120) (2,560) (80) (1,280)
5
* Numbers in parentheses represent
the reciprocal of the highest dilution of serum positive for staining or precipitation.
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Figure 4. Indirect immunofluorescence demonstrating the induction of rheumatoid arthritis-associated nuclear antigen (RANA) after infection of lymphocytes with Epstein-Barr virus (EBV): a, discretely distributed speckled nuclear staining on WiL 2 cells as a result of reaction with a standard reference serum from a patient with rheumatoid arthritis (RA); b, EBV-infected peripheral blood leukocytes on day 20 of culture (a few discretely distributed fine nuclear speckles were seen at this time and became more numerous as the cells became transformed into continuous lines); c, the same EBV-infected cells (day 20) incubated with normal human serum; and d, an uninfected control cell culture on day 20, incubated with the same RA serum. Neither c nor d shows nuclear staining (x 400).
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recently by two other groups of investigators. Slaughter et al. showed that peripheral blood lymphocytes of patients with rheumatoid arthritis produce small amounts of rheumatoid factor in cell culture [11]. When these cells are infected with EBV, the production of rheumatoid factor is greatly increased. These and other studies show that the EBV may be a highly selective polyclonal B cell activator for patients with rheumatoid arthritis. Depper et al. recently reported that the peripheral blood lymphocytes of patients with rheumatoid arthritis are activated more rapidly by EBV than are normal lymphocytes [12]. They measured the "outgrowth" time of rheumatoid cells and normal cells after infection with EBV and showed a significantly shortened outgrowth
time of rheumatoid cells as compared with that of normal cells.
References
1. Alspaugh, M. A., Tan, E. M. Antibodies to cellular antigens in Sjogren's syndrome. J. Clin. Invest. 55:10671073, 1975. 2. Alspaugh, M. A., Talal, N., Tan, E. M. Differentiation and characterization of autoantibodies and their antigens in Sjogren's syndrome. Arth. Rheum. 19:216-222, 1976. 3. Alspaugh, M. A., Tan, E. M. Serum antibody in rheumatoid arthritis reactive with a cell-associated antigen. Demonstration by precipitation and immunofluorescence. Arth, Rheum. 19:711-719, 1976. 4. Alspaugh, M. A., Jensen, F. c., Rabin, H., Tan, E. M. Lymphocytes transformed by Epstein-Barr virus. Induction of nuclear antigen reactive with antibody in rheumatoid arthritis. J. Exp. Med. 147:1018-1027, 1978. 5. Notman, D. D., Kurata, N., Tan, E. M. Profiles of antinuclear antibodies in systemic rheumatic diseases. Ann. Intern. Med. 83:464, 1975. 6. Tan, E. M., Rodnan, G. P. Profile of antinuclear anti-
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Figure 5. Immunofluorescence study to demonstrate the difference between rheumatoid arthritis-associated nuclear antigen (RANA) and Epstein-Barr nuclear antigen (EBNA). When WiL 2 lymphoid cells were incubated with serum from a patient with rheumatoid arthritis (RA) and stained by the indirect immunofluorescence technique, the presence of speckled nuclear staining was demonstrated (0). When the complement amplification technique was used, the nuclear staining was not intensified (c). In contrast, WiL 2 lymphoid cells tested with a serum containing antibody to EBNA but negative for antibody to RANA showed no nuclear staining by the indirect fluorescence technique (b), but when the complement amplification technique was used, intense nuclear staining was observed (d). (x 400).
EBV in Rheumatoid Arthritis
to. Baron, D., Strominger, J. L. Partial purification and properties of the Epstein-Barr virus-associated nuclear antigen. J. BioI. Chern. 253:2875-2881, 1978. 11. Slaughter, L., Carson, D. A., Jensen, F. C., Holbrook, T. L., Vaughan, J. H. In vitro effects of Epstein-Barr virus on peripheral blood mononuclear cells from patients with rheumatoid arthritis and normal subjects. J. Exp. Med. 148:1429-1434, 1978. 12. Depper, J. M., Bardwick, P. A., Bluestein, H. G., Zvaifler, N. J., Seegmiller, J. E. Abnormal regulation of Epstein-Barr virus transformation of rheumatoid lymphoid cells [abstract]. Arth. Rheum. 22:605, 1979.
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bodies in progressive systemic sclerosis [abstract]. Arth. Rheum. 18:430, 1975. 7. Bloch, K. J., Buchanan, W. W., Wohl, M. J., Bunirn, J. J. Sjogren's syndrome: a clinical, pathological and serological study of sixty-two cases. Medicine (Baltimore) 44:187-231, 1965. 8. Reedman, B. M., Klein, G. Cellular localization of an Epstein-Barr virus (EBV)-associated complement fixing antigen in producer and non-producer lymphoblastoid cell lines. Int. J. Cancer 11:499-520, 1973. 9. Ohno, S., Luka, J., Lindahl, T., Klein, G. Identification of a purified complement-fixing antigen as the EpsteinBarr virus-determined nuclear antigen (EBNA) by its binding to metaphase chromosomes. Proc. Natl. Acad, Sci. USA 74:1605-1609, 1977.
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Discussion I didn't mention in my presentation was that CMV [cytomegalovirus] seems to induce endogenous viruses. Do you have electron microscopic or other evidence that the effect of EB virus is not the induction of identifiable, unculturable viral products? DR. TAN. This is one of the points that I referred to in noting that other factors might be involved, and the mechanism you described should be considered as a possibility. One suggestion that has come from studies by other investigators who are extending this work is that the EB virus may be a polyclonal cell activator that induces a de-differentiation type of nuclear antigen that is expressed in cells infected with the virus. This is a very intriguing possibility. There have been many studies showing that the EB virus is a polyclonal B-cell activator. The question is whether, in rheumatoid arthritis, it is acting as a more specific kind of B-cell activator that induces a specific nuclear protein.
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DR. MELVIN KAPLAN. Could you tell us more about the nuclear constituent RANA [rheumatoid arthritis nuclear antigen]? Is this a product only of infected cells? Do you find any evidence of the antigen in uninfected cells by using more sensitive techniques? DR. ENG TAN. We know something about the nucleoprotein antigen that reacts with antibodies in sera from patients with rheumatoid arthritis, but our information is not complete. It is a protein antigen that is highly soluble in saline, and heretofore we have identified it in the nuclei of human B lymphoid cell lines carrying the EB [Epstein-Barr] virus genome. The antigen is not detectable by immunodiffusion or immunofluorescence in cell lines that do not carry the EB virus genome or in organs of many animal species. Whether it is an endogenous protein of the EB virus or a host cell protein induced by viral infection is not known, but we are more inclined to accept the latter interpretation. DR. JOHN HAMILTON. One of the findings that
