Epstein-Barr Virus Antibody Levels in Systemic Lupus Erythematosus

Transcription

1 Epstein-Barr Virus Antibody Levels in Systemic Lupus Erythematosus Paul E. Phillips and Yashar Hirshaut Mean Epstein-Barr virus antibody was not significantly elevated in sera from 50 patients with systemic lupus erythematosus compared to 50 ageand sex-matched blood donor controls. As shown previously for measles and parainfluenza type 1 antibodies, there was a significant direct correlation between Epstein-Barr virus antibody and IgG levels. The variable elevations of virus antibodies found so far in systemic lupus erythematosus probably result from hyperactivity of the humoral immune system. There has been recent interest in the possibility that viruses are involved in the pathogenesis of human connective tissue diseases, in particular, of systemic lupus erythematosus (SLE). This has been stimulated by the role of a murine leukemia-like virus in the autoimmune disease of New Zealand mice (l), by the discovery of multitubular ( virus-like ) inclusions in tissues from patients with SLE (2) and by the increased levels of virus antibodies found in the sera of patients with SLE (3-6). Evans et al recently reported that Epstein-Barr virus (EBV) antibody was more elevated in SLE than were several other virus antibodies and suggested From the Department of Rheumatic Diseases and the Research Department of the Hospital for Special Surgery, affiliated with the New York Hospital-Cornell University Medical College, the Clinical Immunology Service, Department of Medicine, Sloan-Kettering Institute for Cancer Research and the Department of Medicine, Cornell University Medical College, New York, NY. Supported by grants from The John A. Hartford Foundation, The New York Cancer Research Institute and the US Public Health Service (1-R01-AM14627 and CA ). Presented in part at the Annual Meeting of the AMERICAN RHEUMATISM ASSOCIATION, Dallas, Texas, June 8-9,1972. Research Department Publication No PAUL E. PHILLIPS, MD and YASHAR HIRSHAUT, MD: Assistant Professors of Medicine, Cornell University Medical College, New York, NY. Reprint requests should be addressed to: Dr. Paul E. Phillips, The Hospital for Special Surgery, 535 East 70th Street, New York, NY Submitted for publication June 28, 1972; accepted August 30,1972. that EBV might be involved in the pathogenesis of SLE (6). We report here studies of EBV antibody in our population of patients with SLE, who have been shown previously to have significantly increased levels of measles and parainfluenza type 1 antibodies (3). MATERIALS AND METHODS Sera were obtained from patients with SLE diagnosed by the presence of typical multisystem disease with positive lupus erythematosus cell and/or antinuclear antibody tests. Control sera were obtained from blood donors, age- and sex-matched with patients with SLE (& 2 yr). The matched groups each contained 47 females and 4 males; the mean age of the patients with SLE was 31.3 years (range: 8 to 70) and controls, 31.5 years (range: 8 to 69). Sera were stored at -2W C and coded prior to testing. Epstein-Barr virus antibody tests were done by indirect immunofluorescence as previously described (7), except that the EB-3, as well as the Jijoye, EBV-infected cell lines were used, and the sera were diluted in twofold steps beyond the endpoint of specific fluorescence. All sera were tested on a single batch of Jijoye cells, and then retested on a single batch of EB-3 cells. Saline controls were included on each slide. Epstein-Barr virus antibody titers were expressed as the log, of the reciprocal of the highest serum dilution showing specific fluorescence. For statistical analysis, subjects without antibody at the lowest dilution tested, 1:lO (3.3 log,), were arbitrarily considered to have antibody present at the next lower dilution (2.3 log,). No material differences resulted if these subjects were assigned log, titers of 0. The EBV antibody titer of SLE sera was rarely obscured by fluorescence of a nonspecific pattern (affecting all cells, rather than the minority in which the EBV genome is ex- Arthritis and Rheumatism, Vol. 16, No. 1 (January-February 1973) 97

2 PHILLIPS & HIRSHAUT Table 1. Comparison of Epstein-Barr Virus Antibody Levels in SLE and Matched Controls, Using Jijoye Cell Line Table 2. Comparison of Epstein-Barr Virus Antibody Levels in SLE and Matched Controls, Using 6-3 Cell Line Antibody titer Subjects (log 2) Significance Grouo No. Mean+SD t P Antibody titer Subjects (log2) Significance Group No. MeankSD t P SLE * 2.04 Controls f ns* SLE f 1.68 Controls ns* *Not significant, >0.05, <0.10 *Not significant, >0.05. <0.10 pressed, 30% in the Jijoye line, 10% in the EB-3). Such sera (and their matched controls) were excluded from analysis. Serum I& levels were measured by radial immunodiffusion as described previously (8). All sera were tested in duplicate. Two controls could not be tested because no serum was left. Standard statistical methods were used for the 1 test and correlation coefficient (9). RESULTS Using the Jijoye EBV-infected cell line, the geometric mean antibody titer of the group with SLE was not significantly greater than the controls (Table 1). Since Evans et a1 used the EB-3 cell line (6), we retested our groups using this cell line, but again the mean antibody titer of the group with SLE was not significantly greater than the controls (Table 2). While the mean titer of both the SLE and control groups was somewhat higher using the EB-3 cell line, there was excellent correlation between titers obtained on the two cell lines (T , P < 0.001). Age and sex had no significant influence on EBV antibody titers in either the SLE or control groups. No difference was found in the mean antibody levels of the patients with SLE classified by disease activity or dose of corticosteroid taken. The mean level of IS; was significantly higher in the group with SLE, mg/ml compared to 7.74 mg/ml in the 49 matched controls (P < 0.001). There was a significant direct correlation between EBV antibody (measured on either cell line or using the mean of cate determinations, and EBV rn &A A * * A 3._ AA A antibody levels of titers on the L - > L Jijoye and EB-3 cell lines. The m 2 4- A regression line (y = c a ~, r , t = ) rn. B CL for this direct correlation in i 2- the combined group is signifi- 5 cant (P<O.OOl), as is that (not plotted) for the group I I, I I I 98 Arthritis and Rheumatism, Vol. 16, No. 1 (January-February 1973)

3 EPSTEIN-BARR ANTIBODY LEVELS IN SLE Table 3. Correlation Between Epstein-Barr Virus Antibody and IgG Levels in SLE and Control Subjects EBV antibody Subjects Mean titer Mean IgG Group No. Type (log,) (mdrnl) SLE* 57 Jijoye EB Meant Controls 48 Mean TOTAL 103 Mean r Correlation P < < < ns% <0.001 *Includes patients with SLE who had no matched controls tmean of Jijoye and EB-3 determinations %Not significant, >0.05, <0.10 both determinations) and IgG levels in patients with SLE (Table 3). This correlation was not significant for the controls alone, but their range of IgG levels was narrow compared to the group with SLE (Figure 1). However, when the SLE and control groups were combined, the direct correlation between EBV antibody and IgG levels was again significant (Table 3, Figure 1). These correlations were not significantly affected by excluding the subjects who had either no EBV antibody or the highest IgG level. Since measles antibody levels also correlated directly with IgG levels in this group with SLE (8), it was not surprising to find a significant correlation between measles and EBV antibody levels in these patients (r , P < 0.025). Sequential studies of EBV antibody and IgG levels were done on 2 to 12 sera (mean: 3.4) from 23 patients with SLE followed for periods of 2 weeks to 75 months (mean: 17.3). There were large changes in both EBV antibody and IgG levels in some of these patients (Figure 2). The 45% frequency of corresponding changes in the two parameters (both rising or falling or unchanged together) was similar to the 50% frequency expected on a random basis. No relationship was found between changes in EBV antibody level and changes in disease activity in these sequentially studied patients. c m i O b I 1 ' I 2 ' I 3 ' ' Years from initial serum specimen Fig 2. Sequential IgG (0-0) and EBV antibody (0-0) levels of a 21-year-old woman with SLE. measuredover a 4-year period. IgG levels are the mean of duplicate determinations, and EBV antibody levels of titers on the Jijoye and EB-3 cell lines. Significant correspondence between changes in the two parameters is not apparent. DISCUSSION Using the same cell line, we were unable to confirm the significant increase of EBV antibody level in SLE found by Evans et al(6). The mean titer for their group of 34 matched patients with SLE (1:216.8 converted to log, = 7.76) was almost identical with that of our 50 patients. The major discrepancy between our results lies in the titers of the matched control groups. They used patients with tuberculosis Arthritis and Rheumatism, Vol. 16, No. 1 (January-February 1973) 99

4 PHILLIPS & HIRSHAUT who had a more than fourfold lower EBV antibody titer (1:35.3 = 5.14 log,) than our blood donor controls. The distribution of antibody titers of our controls was similar to that of other groups (including patients with tuberculosis) previously tested in our laboratory and having normal ranges of antibody titers. We have previously compared our results with those of another laboratory and found generally excellent agreement, except for a moderately greater sensitivity of our test (7). Thus, we can find no evidence that our control group was atypical in any respect. The EBV antibody titer elevation found in our group with SLE, although not significant, was similar to the elevations of many other virus antibodies found in SLE by others (4,5). In our group with SLE it was similar to the elevation found in adenovirus antibody (1 0) but was less than that of measles and parainfluenza type 1 antibodies (3, 11). We have shown previously that levels of the latter two antibodies have a direct relationship to gammaglobulin or IgG levels (8, 11). The same relationship was found here between EBV antibody and IgG levels in the group with SLE. We have also found a direct correlation between measles and parainfluenza type 1 antibody levels in SLE (11) and, in this study, between measles and EBV antibody levels. No other significant correlations between virus antibody levels and clinical or laboratory parameters were found in this or our earlier studies (3, 10, 11). Thus, measles, parainfluenza type 1 and EBV antibody levels all show a significant tendency to be higher in patients with high levels of gammaglobulin or IgG. In addition, elevations of these three virus antibodies tend significantly to occur in the same patients with SLE. Although the sequential studies of changes in EBV antibody and IgG levels in patients with SLE did not reveal a significant level of correspondence, as was found for changes in measles antibody and IgG (8, lo), this may be related to the fact that EBV infection is often chronic even in normal individuals, while measles is usually an acute infec- tion. Therefore the immunologic response may differ in the two infections. We have concluded previously that the elevated measles and parainfluenza type 1 antibody levels in SLE are a result of hyperactivity of the humoral immune system in this disease (8, 1 l), a conclusion shared by Hollinger et af (4). The similar relationship found for EBV antibody in this study strengthens this argument and makes it more likely that all the moderate virus antibody elevations found to date in SLE are also a result of this immunologic hyperactivity. While the hypothesis that chronic virus infection is involved in the pathogenesis of SLE merits further investigation, studies of virus antibodies in this disease have not yet provided any supportive evidence. Note added in proof: While this report was in press. DA Stevens, MB Stevens, GR Newell et af (Arch Intern Med 130:23-28, 1972) reported no significant difference between Epstein-Barr virus antibody levels of patients either with SLE or other connective tissue disease compared to normal controls, using indirect immunofluorescence on the HRIK Epstein-Barr virus-infected cell line. ACKNOWLEDGMENTS We thank Rita Hargrave and Bozena Dvorak for excellent technical assistance, and Dr. Charles Christian for his helpful advice. REFERENCES 1. Mellors RC, Shirai T, Aoki T, et al: Wild-type Gross leukemia virus and the pathogenesis of the glomerulonephritis of New Zealand mice. J Exp Med 133: , Gyorkey F, Min K-W, Sinkovics JG, et al: Systemic lupus erythematosus and myxovirus. N Engl J Med 280:333,1969 (letter) 3. Phillips PE, Christian CL: Myxovirus antibody increases in human connective tissue disease. Science 168: , Hollinger FB, Sharp JT, Lidsky MD, et al: Antibodies to viral antigens in systemic lupus erythematosus. Arthritis Rheum 14:l-11, Hurd ER, Dowdle W, Casey H, et al: Virus an- 100 Arthritis and Rheumatism, Vol. 16. No. 1 (January-February 1973)

5 EPSTEIN-BARR ANTIBODY LEVELS IN SLE tibody levels in systemic lupus erythematosus. Arthritis Rheum 15: , Evans AS, Rothfield NF, Niederman JC: Raised antibody titers to Epstein-Barr virus in systemic lupus erythematosus. Lancet 1: , Hirshaut Y, Glade P, Vieira LO, et al. Sarcoidosis, another disease associated with evidence for herpes-like virus infection. N Engl J Med 283: , Phillips PE, Christian CL: The influence of se- rum immunoglobulin concentration on measles antibody level. Proc SOC Exp Biol Med 140: , Croxton FE: Elementary Statistics with Applications in Medicine and the Biological Sciences. New York, Dover, 1969, pp , Phillips PE, Christian CL: Unpublished data 11. Phillips PE, Christian CL: Virus antibody studies in the connective tissue diseases. Arthritis Rheum 14: ,1971 (abstr) CALENDAR OF FUTURE MEETINGS OF THE AMERICAN RHEUMATISM ASSOCIATION.June 6-9,1973 ARA Annual Meeting Century Plaza Hotel Los Angeles, Calif September 30- XI11 International Congress Kyoto, Japan October 6, 1973 of Rheumatology (For information write to: Secretariat, the Organizing Committee, XIIIth International Congress of Rheumatology, Japan Convention Services, Inc, 3-23, 7-chome, Roppongi, Minato-ku, Tokyo 106, Japan. Cable address: JACONSERVINC TOKYO.) June 16-21,1974 VI Pan American Congress Sheraton Four Seasons on Rheumatic Diseases Hotel and Toronto, Canada ARA Annual Meeting Winter ARA Annual Meeting Undetermined Arthritis and Rheumatism, Vol. 16, No. 1 (January-February 1973) 101