Lupus Erythematosus and Infectious Mononucleosis to Epstein-Barr ... Infection of human B-lymphocytes by Epstein-Barr, irus (EBV) results in the induction of a.
J. gen. Virol. (1986), 67, 2253-2258. Printed in Great Britain
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Key words: EBV/polypeptides/protein immunoblot
Reactions of Sera from Patients with Rh mmatoid Arthritis, Systemic Lupus Erythematosus and Infectious Mononucleosis to Epstein-Barr Virus-induced Poly t eptides By D. G. S C U L L E Y , * T. B. S C U L L E Y AND J. H. P O P E Queensland Institute of Medical Research, Bramston Te'race, Herston, Brisbane, Australia 4006 (Accepted 1 July 1586)
SUMMARY
P3HR-1 and Ramos cells induced with sodium butyrate and 12-O-tetradecanoylphorbol 13-acetate were used in the protein immure,blot technique to identify EpsteinBarr virus (EBV)-specific antibodies present in ser~ from clinically normal individuals and patients with systemic lupus erythematosus (SI,E), rheumatoid arthritis (RA) and infectious mononucleosis (IM). Sixteen EBV-sp,'cific polypeptides were detected ranging in tool. wt. from 22000 (22K) to 140K. Many of the sera contained antibodies to different subsets of these antigens, and a high pJ oportion expressed autoantibodies which reacted with cellular components from an EB V genome-negative cell line. About 50% of the sera from each category reacted with the 44K to 48K and 36K and 38K early antigen (EA) components. A high proportion of 1he SLE sera (64~) were found to contain anti-EA antibodies, suggesting an associati on between EBV and SLE. Almost all of the EBV-seropositive sera examined contai ned antibodies against a 22K late antigen, but none of the sera from IM patients r~ acted with this polypeptide. Infection of human B-lymphocytes by Epstein-Barr, irus (EBV) results in the induction of a large number of EBV-associated antigen complexes. In non-permissive infections, a number of EBV-induced antigens have been described, including EBNA1 and EBNA2 (Reedman & Klein, 1973; Hennessy & Kieff, 1983; ScuUey et al., 1~.'84). Virus-producing cell lines express additional antigen complexes which have been termed membrane antigens (MA), viral capsid antigens (VCA) and early antigens (EA) (Pearson, 1980~. Healthy EBV-seropositive individuals usually have antibodies against EBNA1, MA and VC~ ~. In addition, antibodies against EBV EA often can be found in the sera of patients with vario~is diseases, such as Burkitt's lymphoma (BL), nasopharyngeal carcinoma (NPC), systemic lu ~us erythematosus (SLE), rheumatoid arthritis (RA) and infectious mononucleosis (IM). Because of the nature of these diseases, a major problem in the detection of antibodies to tlte EBV antigens is the presence of autoantibodies in the sera of many patients (Gilliland & idannik, 1980; Hardin & Mimori, 1985 ; Sutton et at., 1974). However, this problem can be ov ",rcome by the use of immunoblotting, employing extracts of both EBV-positive and EBV-ne ,ative cell lines. P3HR-1 and Ramos cell lines were grown in RPMI 1640 medium supplemented with 10% foetal calf serum at 37 °C under 5% CO2. Cells at a der sity of 106 per ml were induced with 5 mM-sodium butyrate and 20 ng/ml 12-O-tetradecan~ytphorbol 13-acetate (TPA) (Baker Chemical, Philipsburg, N.J., U.S.A. and Sigma, respectively) for 3 days. SDS extracts were prepared from these two lines and layered across the tot of 10% polyacrylamide-SDS slab gels. Proteins were separated by electrophoresis (Laemmli, 1970), then electrophoretically transferred to nitrocellulose papers (Burnette, 1981), which were cut into strips and incubated with individual samples of serum. When sera collected Irom clinically normal individuals were screened, each of the 16 EBV-seropositive sera reacted with two polypeptides, the 72000 mol. wt. (72K) EBNA1 antigen (Sculley et al., 1984) and th,: 22K late viral antigen (Sculley et al., 1985) in P3HR-1 cells, but not in Ramos cells (Table 2) i 50% of these sera also detected E B V 0000-7183©1986 SGM
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Short communication T a b l e 1. Serology of test sera Antibody titre to EBV antigen Status of donors SLE
¢
A
IgM~ Serum EBNA* EA-D + R t VCA~ SLE1 ANA§ SLE2 ANA SLE3 10 40 SLE4 ANA SLE5 ANA SLE6 40 SLE7 ANA SLE8 ANA SLE9 40 SLE10 ANA SLEll ANA SLE12 ANA SLE13 ANA SLE14 ANA RA 320 RA1 320 40 >640 RA2 160 40 160 RA3 160 40 40 RA4 640 40 640 RA5 160 160 40960 RA6 40 160 640 RA7 10 40 160 RA8 40 40 2560 RA9 160 160 160 RA10 < I0 10 160 80 RAIl 160 40 160 80 RA12 160 40 640 160 RA13 < 10 160 40 40 RA14 40 ANA 40 40 RA15 ANA ANA 160 160 RA16 ANA 320 ANA 160 RA17 10 10 640 320 IM IMI 160 40 640 IM2 40 160 160 IM3 160 640 40 IM4 40 640 40 IM5 40 160 40 IM6 10 160 40 1M7 640 640 IM8 < 10 40 IM9 40 < 10 IM10 40 < 10 160 IMI1 40 < 10 160 IMI2 40 640 640 IMI3 40 40 IMI4 10 40 40 IMI5 10 40 160 * Titres measured according to Reedman & Klein (1973). "t"Titres measured according to Klein & Dombos (1973). :~ Titres measured according to Henle & Henle (1966)~ § ANA denotes the presence of autoantibodies, which make assessment of antibody titres impossible.
specific p o l y p e p t i d e s at 5 0 K a n d 4 2 K ( T a b l e 2). N o n e o f t h e s e a n t i g e n s w a s d e t e c t e d w i t h four E B V - s e r o n e g a t i v e sera. T h e r e was little o r n o r e a c t i o n w i t h t h e R a m o s cell l i n e i n c o n t r a s t t o t h e results o b t a i n e d w i t h S L E sera. M o s t o f t h e sera f r o m S L E p a t i e n t s w e r e f o u n d to c o n t a i n a u t o a n t i b o d i e s b y i m m u n o f l u o r e s c e n c e ( T a b l e 1) a n d t h i s w a s also e v i d e n t i n t h e i m m u n o b l o t s o n R a m o s cells (Fig. 1 b).
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Short communication Table 2. EBV-induced polypeptides detected by each of the different groups of sera
Mol. wt. of polypeptides detected ( x 10 -3) 140 130 120 90 82 72 67 60 52 50 44-48 42 38 36 30 22 Total number of sera studied
Percentage of sera in each group that detect the polypeptide • A Normal SLE 29 . . . 100 93 7 21 50 50 50 50 64 64 21 100 86 16
14
RA 18 18 35 -
IM 20 20 7 13
100 12 12 12 100 24 88 40 69 12 94
13 20 33 40 47 33 13 26 -
17
15
.
20
Incubation of the sera with extracts of the P3HR-1 cell line also detected these cellular components, as well as a n u m b e r of additional EBV-specific polypeptides (Fig. 1 a). The most p r o m i n e n t E A polypeptides detected by immunoblotting are the 44K to 48K E A - D complex and the 36K and 38K antigens (Sculley et al., 1985). Lanes 17 and 18 represent an R A and an N P C serum respectively which were anti-EA-positive by immunofluorescence and were known to react with the 44K to 48K and/or the 36K and 38K polypeptides in immunoblots (Sculley et al., 1985) and were included as reference sera. The results demonstrated that nine of the 14 sera from SLE patients ( 6 4 ~ ) reacted with the 36K and 38K proteins, and 5 0 ~ detected the 44K to 48K E A - D antigen. The high percentage of SLE sera containing a n t i - E A antibodies suggests an association between the disease and EBV. A preliminary study, where samples of sera were taken from the same patients at different stages of the disease, indicated that a n t i - E A antibodies m a y persist for an extended length of time (lanes 3 and 19 represent serum taken from patient SLE1, with an 8 month interval; lanes 5 and 20 were from patient SLE3, with a 22 m o n t h interval). W h e t h e r this is brought about by treatment of SLE patients (i.e. activation of the EBV genome by various drugs) or by the disease itself requires further study. Unlike the SLE sera, which were selected randomly, the sera from R A patients were tested by immunoblotting only if they were anti-EA-positive by immunofluorescence, with three autoantibody-positive sera being included. All of the R A patients included in this study were diagnosed through the Rheumatology Clinic at the Princess A l e x a n d r a Hospital, Brisbane and sera were obtained from patients with either classical or definite R A ( A m e r i c a n R h e u m a t i s m Association criteria). Even though these sera were anti-EA-positive by immunofluorescence, only four of the 17 sera reacted with the 44K to 48K E A - D antigen, and a further eight sera reacted with the 36K and 38K antigens (predominantly the 36K component) (results not shown, but summarized in Table 2). Two samples o f sera which did not react with either the 44K to 48K or 36K to 38K antigens were autoantibody-positive by immunofluorescence and were therefore presumed to be anti-EA-negative. The failure of the remaining three anti-EA-positive sera to react with known E A components in the immunoblots indicates that not all the E A polypeptides, as defined by immunofluorescence, were detected using immunoblotfing. All of the sera reacted with E B N A 1 despite the fact that three of the sera had titres o f < 10 in antiE B N A immunofluorescence tests (Table 1), and all but one of the sera detected the 22K component. Once again, the immunoblot with R a m o s cells indicated the presence of
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2256 (a)
t
2
3 4
7 8 9 10 11 12 13 14 15 16 17 18 19 20
5 6
92-67--
4 EBNA 1
43--
30--
,,t 22K
2015--
(b)
1 2 3 4 5 6
7
8
9 10 11 12 13 14 15 16 17 18 19 20
Fig. 1. Detection of EBV-specific antigens by protein immunoblot using sera from patients with systemic lupus erythematosus. SDS extracts were prepared from P3HR-1 (a) or Ramos (b) cells which had been incubated with sodium butyrate and TPA for 3 days. These extracts were each applied across the top of a polyacrylamide-SDS slab gel. Proteins were separated by electrophoresis then transferred to nitrocellulose paper. The paper was cut into strips and each strip incubated with an individual serum. The strips were realigned prior to exposure to X-ray film. Serum samples: lane 1, NEG2; lane 2, POS 1; lanes 3 and 19, SLE1 ; lane 4, SLE2; lanes 5 and 20, SLE3; lane 6, SLE4; lane 7, SLE5; lane 8, SLE6; lane 9, SLE7; lane 10, SLE8; lane 11, SLE9; lane 12, SLE10; lane 13, SLE11 ; lane 14, SLE12; lane 15, SLE13; lane 16, SLE14; lane 17, RA1 ; lane 18, NPC3. The positions of molecular weight standards are indicated on the left.
autoantibodies, w i t h reaction mostly occurring with a polypeptide a r o u n d 32K (results n o t shown). A l m o s t 6 0 ~ of the R A sera detected this 32K protein, while n o n e of the other groups studied reacted with the antigen. T h i s a p p a r e n t l y RA-specific a u t o a n t i b o d y m a y have some i m p o r t a n c e as a diagnostic tool a n d bears further study. Sera from suspected I M p a t i e n t s were received from the Q u e e n s l a n d U n i v e r s i t y H e a l t h Services a n d a positive diagnosis confirmed by i m m u n o f l u o r e s c e n c e tests for I g M a n t i b o d i e s to EBV. W i t h these sera, a variety of non-specific proteins were detected in the i m m u n o b l o t using R a m o s cell extracts (Fig. 2b). Only 10 of the I M sera tested ( 6 6 ~ ) were found to have positive
Short communication (a)
S
1 2 3 4
5
6
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7 8 9 10 11 12 13 14 15 16 17 18 19120
92-67 .
.
.
-q EBNA1
.
43~
i 30 -- ~
~
"q 22K
(b)
S
1 2
3 4 5
6 7 8 9 10 11 12 13 14 15 16 17 18 19:20
92-67-43--
30--
20-15--
Fig. 2. Detection of EBV-specific antigens by immunoblot using sera from patients with infectious mononucleosis. SDS extracts were prepared from P3HR-1 (a) or Ramos (b) cells as described in the legend to Fig. 1. Serum samples: lane 1, NEG2; lane 2, POS1; lane 3, POS2; lane 4, POS3; lane 5, POS4; lane 6, IM1 ; lane 7, IM2; lane 8, IM3; lane 9, IM4; lane 10, IM5; lane 11, IM6; lane 12, IM7; lane 13, IM8 ; lane 14, IM9; lane 15, IM10; lane 16, IM11 ; lane 17, IM12; lane 18, IM13; lane 19, IM14; lane 20, IM15. Lane S contains molecular weight standards.
a n t i - E A - D + R titres by i m m u n o f l u o r e s c e n c e . O f these sera, s e v e n reacted w i t h either, or both, the 4 4 K to 4 8 K and 36K and 38K p o l y p e p t i d e s in P 3 H R - 1 cells. U n l i k e the o t h e r diseases studied, only 13 ~ (two out o f 15 patients) r e a c t e d w i t h E B N A 1. S o m e o f the sera, h o w e v e r , did react w i t h a cellular c o m p o n e n t at 72K in b o t h the P 3 H R - 1 and R a m o s cell extracts. T h e m o s t
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Short communication
interesting feature of the sera from I M patients was their inability to detect the 22K antigen, despite the fact that all the sera were anti-VCA-positive by immunofluorescence. This 22K protein has previously been shown to be a late viral product (Sculley et al., 1985) and m a y be either a V C A or M A component. Because anti-EA-negative, anti-VCA-positive sera react with this antigen (Table 2), and because it is not present in virus non-producing cell lines (Sculley et al., 1984), it is likely to be a component of the viral particle. It is therefore surprising that sera from patients in the acute phase of I M fail to react with the antigen. The method employed in the immunoblots utilizes the binding of radioiodinated Protein A to a n t i b o d y - a n t i g e n complexes and would not detect IgM antibodies. The possibility that only I g M antibodies, in I M patients, are directed against the 22K antigen was investigated by both employing anti-human I g M serum raised in rabbits, as a bridge in the immunoblots, and by using peroxidase-labelled antihuman Ig, but still no reaction with the 22K antigen was detected (results not shown). The presence of anti-22K antibodies in normal sera indicates that some time after the acute phase of IM, individuals eventually develop these antibodies and then a p p e a r to m a i n t a i n them for life. Further study will be required to determine the length of time after onset of I M that anti-22K antibodies a p p e a r and whether they m a y serve some protective function. We thank Drs D. Moss and R. Hazelton for supplying the sera. This work was supported by grants from the National Health and Medical Research Council and the Queensland Cancer Fund. REFERENCES BURNE'I'rE,W. N. (1981). "Western Blotting': electrophoretic transfer of proteins from SDS-polyacrylamidegels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated Protein A. Analytical Biochemistry 112, 195-203. GILLILAND,B. C. & MANNIK,M. (1980). Rheumatoid arthritis. In Principlesof Internal Medicine, 9th edn., pp. 18721880. Edited by T. R. Harrison. New York: McGraw-Hill. HARDIN,J. A. & MIMORI,T. (1985). Antibodies to ribonucleoproteins. Clinics in Rheumatic Diseases 11, 485-505. HENLE, G. ~ HENLE, W. (1966). Immunofluorescence in cells derived from Burkitt's lymphoma. Journal of Bacteriology 91, 1248-1256. HENNESSY,K. & KIEFF,E. (1983). One of two Epstein-Barr virus nuclear antigens contains a glycine-alanine copolymer domain. Proceedings of the National Academy of Sciences, U.S.A. 80, 1096-1100. KLEIN, G. & DOMBOS,L. (1973). Relationship between the sensitivity of EBV-carrying lymphoblastoid lines to superinfection and inducibility of the resident viral genomes. International Journal of Cancer 11, 327-337. LAEMMLI,U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, 680-685. PEARSON,G. R. (1980). Epstein-Barr virus: immunology. In Viral Ontology, pp. 739-767. Edited by G. Klein. New York: Raven Press. REEDMAN,T. B. M. &KLEIN,G. (1973). Cellular localisation of an Epstein-Barr virus (EBV)-associated complementfixing antigen in producer and non-producer lymphoblastoid cell lines. International Journal of Cancer 2, 499520. SCULLEY, T. B., WALKER, P. J., MOSS, D. J. & POPE, J. H. (1984). I d e n t i f i c a t i o n o f m u l t i p l e E p s t e i n - B a r r v i r u s - i n d u c e d
nuclear antigens with sera from patients with rheumatoid arthritis. Journal of Virology 52, 88-93. SCULLEY, T. B., SCULLEY, D. G. & POPE, J. H. (1985). I d e n t i f i c a t i o n o f E p s t e i n - B a r r v i r u s - i n d u c e d p o l y p e p t i d e s in
P3HR-1 cells by protein immunoblot. Journal of General Virology 66, 1113-1122. (1974). The occurrence of autoantibodies in infectious mononucleosis. Clinical and Experimental Immunology 17, 427-436.
SUTTON, R. N. P., EMOND, R. T. D., THOMAS, D. B. & DONIACH, D.
(Received 7 April 1986)
