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BRIEF REPORT

PERSISTENT SYNOVIAL LYMPHOCYTE RESPONSES TO CYTOMEGALOVIRUS ANTIGEN IN SOME PATIENTS WITH RHEUMATOID ARTHRITIS

DENYS K. FORD, DOREEN M. DA ROZA, MICHAEL SCHULZER, GRAHAM D. REID, and JORGE F. DENEGRI

Synovial lymphocytes from 6 of 40 patients with rheumatoid arthritis responded to cytomegalovirus an- tigen stimulation. 3H-thymidine uptakes were more than 3 times greater than were those of the responses to 13 other microbial antigens. Similar results were obtained in 1 patient on 7 occasions over 17 months, and in the 5 other patients on each of 2 occasions. In 3 of the 6 patients, synovial lymphocyte responses to cytomega- lovirus antigen were markedly different from simulta- neous peripheral blood lymphocyte responses.

Previous studies have indicated that synovial fluid lymphocyte responses to microbial antigen stim- ulation may indicate the cause of enteric and sexually transmitted reactive arthritis, although peripheral blood lymphocytes have not shown this capability (1-3). Subsequent similar studies suggested that ap- proximately 50% of chronic or recurrent inflammatory arthritis confined to knee joints was a reactive arthri- tis, but was distinct from enteric and sexually trans- mitted Reiter’s syndrome (4). Investigation of cases of rheumatoid arthritis, using the same methods, has provided some support for the viewpoint that common viruses may be involved in the pathogenesis of rheu- -____

From the Departments of Medicine and Pathology, Univer- sity of British Columbia, Vancouver, British Columbia, Canada.

Supported by the Arthritis Society of Canada. Denys K . Ford, MD: Professor of Medicine; Doreen M. da

Roza, I~SC, RT: Research Technologist; Michael Schulzer, MD, PhD: Associate Professor of Medicine and Mathematics; Graham D. Reid, MB, ChB: Clinical Assistant Professor of Medicine; Jorge F. Denegri, MD: Clinical Assistant Professor of Pathology.

Address reprint requests to Dr. Denys K. Ford, The Arthritis Centre, 895 West 10th Avenue, Vancouver, British Colum- bia, Canada V5Z 1L7.

Submitted for publication July 3 1, 1986; accepted in revised form November 11. 1986.

matoid arthritis. Initially, we described a patient with rheumatoid arthritis and a patient with recurrent knee arthritis (9, in whom a maximum response of synovial fluid lymphocytes to rubella antigen was associated with the isolation of rubella virus from both patients, although neither had had any recent illness suggestive of the presence of rubella. Later, we described a third patient (6), in whom rheumatoid arthritis and a history of thyroiditis and retroperitoneal fibrosis were associ- ated with both a persistent maximum synovial lympho- cyte response to rubella and the presence of rubella virus in synovial fluid. We have also found that the synovial lymphocytes of other patients with rheuma- toid arthritis responded to stimulation with a variety of other viral antigens (7), and this responsiveness has been demonstrated to be consistent (8).

In the present report, we describe 6 patients with rheumatoid arthritis, in whom the synovial lym- phocytes responded maximally and consistently to cytomegalovirus (CMV) antigen.

PATIENTS AND METHODS

Patients. Forty patients with definite rheuma- toid arthritis, according to the American Rheumatism Association criteria (9), were selected for study. These patients had synovial knee effusions that were associ- ated with warmth of the involved knee, which indi- cated the presence of some degree of active inflamma- tion. In 6 patients, the synovial fluid lymphocytes responded maximally to CMV antigen; the other 34 patients, who were similarly tested, did not have a maximum synovial lymphocyte response to CMV antigen.

Arthritis and Rheumatism, Vol. 30, No. 6 (June 1987)

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I I I I I I Ureaplasma

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Figure 1. Synovial lymphocyte stimulation ipdices for the antigens used in successive tests (on the dates noted) in patient RG. Resp. syn. = respiratory syncytial virus; CMV = cytomegalovirus; PHA = phytohemagglutinin.

Four of the 6 patients whose lymphocytes re- sponded to CMV antigen were women. The ages of these 6 patients ranged from 42 to 71. The duration of arthritis ranged from 1 year to 15 years (mean 7 years). One of the patients had a persistently high titer of rheumatoid factor, and 1 had a persistently low titer; the others were rheumatoid factor-negative.

Laboratory methods. The methods were the same as those used in previous studies (1-8). Synovial and peripheral blood mononuclear cells were obtained by Ficoll-Hypaque separation from heparinized sam- ples. Lymphocytes, at a concentration of 10,000 per Terasaki plate well in 10% human AB plasma medium, were cultured as hanging-drops, at 37°C in 5% COz, for 7 days in the presence of 3 threefold dilutions of antigens. Triplicate wells were used for each determi- nation. The antigens were all crude preparations from the same sources described previously (1-8).

On the seventh day of incubation, 1 pCi of 'H-thymidine was added. After 4-6 hours, the cells were harvested, and the 3H-thymidine uptake was determined by scintillation counting. The degree of stimulation was expressed as the stimulation index (SI), which was the (triplicate) average counts per minute of the antigen dilution that gave the maximum response divided by the average cpm of 9 or more wells of unstimulated cells. The accuracy of the

method was assessed by examining the coefficients of variation of the triplicate cpm readings of the CMV antigen response. The median coefticient of variation for the sets of cpm readings for this response was 21%.

Lymphocyte subpopulations were analyzed with 2-color immunafluorescence, using commercial monoclonal antibodies and a fluorescence-activated cell sorter (FACS) 420 (Becton Dickinson, Sunnyvale, CA). Phycoerythrin and fluorescein isothiocyanate- conjugated monoclonal antibodies were used in a direct immunofluorescence technique.

RESULTS Because the 6 patients described here were

selected based on a numerically maximum response to repeated CMV antigen stimulation, it was necessary to demonstrate that this CMV response was, in fact, significantly greater than the response to the antigen that gave the next highest stimulation. The 3H- thymidine uptake data in the 6 CMV responders gave a median SI of 58 to stimulation with CMV; the median SI for the antigen that gave the next highest response was 17.

Statistical analysis of these data, based on a weighted paired t-test, confirmed that the response to the CMV antigen was significantly greater than the second highest response (P < 0.0001). The median of

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Figure 2. Classification of synovial and peripheral blood lympho- cytes before, and 7 days after, stimulation with cytornegalovirus (CMV) antigen in patient RG.

the ratio of the CMV response to that of the second highest antigen was 3.6. The 95% confidence interval for this ratio was between 2.7 and 6.8.

Figure 1 shows the results of synovial lympho- cyte stimulation, as tested on 7 occasions over a period of 17 months, in patient RG. This patient had definite rheumatoid arthritis of 6 years duration. Rheu- matoid factor test results were strongly, and repeat- edly, positive. Although the degree of stimulation varied from test to test, the greatest antigenic response was always to the CMV antigen. Tests performed in November 1984 showed that the response of synovial

lymphocytes to CMV antigen was greater than the response to phytohemagglutinin (PHA).

In comparisons of the responses of synovial fluid lymphocytes with those of peripheral blood lym- phocytes in this patient, significant differences were found. The response of peripheral blood lymphocytes was tested on 3 occasions, and at each testing, there was a stimulation by CMV antigen. On 2 of the 3 occasions, the CMV response was the maximum antigenic response. There was, however, a difference in the responses when comparisons were made with PHA stimulation. On November 13, 1984, the synovial CMV SI was 19, versus a PHA SI of 9; in contrast, the peripheral blood lymphocyte SI for CMV stimulation was 8, while the SI for PHA was 75. An additional difference was observed when FACS analysis of synovial and peripheral blood mononuclear cells was performed before and after CMV antigen stimulation. Figure 2 shows that prior to stimulation, 11% of the synovial mononuclear cells were T4 DR+ lympho- cytes, and on the seventh day of exposure to CMV antigen, this value increased to 25%. In contrast, no T4 DR+ peripheral blood lymphocytes were present before or after CMV antigen stimulation. Moreover, following stimulation with CMV antigen, 90% of the synovial lymphocytes, compared with only 5% of the peripheral blood lymphocytes, had undergone blast transformation.

Synovial lymphocytes responded maximally to CMV antigen in 5 other patients, as shown in Figure 3. Each of these patients was tested on 2 occasions, and

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the results were uniformly confirmatory on the second testing. In patients IC and FK, very marked responses were observed in synovial lymphocytes, with SI of 40 and 264, respectively, while there was no demonstra- ble response in these patients’ peripheral blood lym- phocytes (SI of 1 in both patients). In 2 other patients, MJ and RN, there were responses from peripheral blood lymphocytes, although these were markedly less responsive than were their synovial lymphocytes.

In view of our experience with the 3 patients who had a maximum response to rubella antigen, in whom rubella virus was identified (5,6), we attempted to demonstrate evidence of CMV infection in the patients described in this report. In 3 of the patients, synovial fluid was added to human foreskin fibroblast cell cultures, but no cytopathogenic changes were observed. In patient RG, synovial fluid and blood mononuclear cell deposits were treated with sodium dodecyl sulfate (SDS) and delivered to Drs. Sharon Cassol and David Hoar of the Department of Medical Biochemistry (University of Calgary, Alberta, Can- ada), to determine if CMV nucleic acid was present. Using a dot hybridization technique, nucleotide se- quences characteristic of CMV were demonstrable in these SDS-treated cell deposits. The significance of this was not clear, however, because most healthy donors, whose blood had been studied with the same technique, had similar CMV nucleotide sequences in SDS-treated peripheral blood cells.

In 1 patient, an attempt was made to demon- strate CMV antigen in synovial mononuclear cells by indirect fluorescence staining with monoclonal anti- CMV antibody and by FACS analysis; however, no antigen was found. It has not yet been possible to seek CMV antigen in synovial biopsy material from these patients. Such an approach was productive in a patient with a repeatedly maximum response to chlamydial antigen, studied by Schumacher et al; subsequently, a synovectomy was performed, and the synovium spec- imen demonstrated immunoperoxidase-positive stain- ing for Chlamydia (10).

DISCUSSION

The causes of the clinical syndrome called rheumatoid arthritis are unknown. There is an obvious immunopathologic process, and immunoregulatory mechanisms are probably aberrant. The fact remains that an immune response requires an antigenic stimu- lus. In the clinical syndrome of reactive arthritis, multiple microbial agents are causative, and it appears

that the synovial lymphocytes can identify the agent that is responsible for a particular patient’s reactive arthritis (1-3).

The present studies, performed over 6 years, have defined cases of rheumatoid arthritis which dem- onstrate consistent responses of synovial lympho- cytes, but (usually) not blood lymphocytes, to specific viral agents. Rubella virus was isolated from 3 patients who had a synovial response to rubella antigen (5,6). Equivalently effective viral isolation and immunodiag- nostic techniques are not yet available for use in cases of consistent synovial responses to other viral anti- gens. Thus, the evidence for a possible relationship of CMV to the rheumatoid arthritis in the 6 patients described here is incomplete. In these patients, the synovial lymphocytes responded maximally and con- sistently to CMV antigen, but this observation is not currently associated with other supportive findings, and its significance is therefore unproven.

The current work of investigators in this labo- ratory is based on the hypothesis that rheumatoid arthritis is an immunopathologic and clinical response to a variety of microbial agents, and that the particular agent that is responsible for a particular patient’s rheumatoid arthritis can probably be designated by the response of synovial lymphocytes to the antigens of that agent. Because of previous experience with ru- bella arthritis and with Reiter’s syndrome, this re- search program has not included serologic investiga- tions of levels of antibody against the microbiologic agents under study, including CMV. We believe that the validity of this approach is confirmed by the finding of greater synovial (than peripheral blood) lymphocyte responses to the causative antigen in patients with Lyme arthritis (1 1).

Acknowledgments. Drs. J. Hudson and C. Sherlock (Division of Medical Microbiology, University of British Columbia, Vancouver) performed the synovial fluid testing for cytomegalovirus; the Canadian Red Cross provided the human AB plasma; and Dr. W. R. Bowie (Division of Infectious Diseases, University of British Columbia, Vancouver) provided the chlamydial antigen. We thank Drs. A. Chalmers and B. E. Koehler for allowing us to study their patients.

REFERENCES

Ford DK, da Roza DM, Schulzer M: The specificity of synovial mononuclear cell responses to microbiological antigens in Reiter’s syndrome. J Rheumatol 9561-569, 1982 Ford DK: Infectious agents in Reiter’s syndrome. Clin Exp Rheumatol 1:273-277, 1983

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3. Ford DK, da Roza DM, Schulzer M: Lymphocytes from the site of disease but not blood lymphocytes indicate the cause of arthritis. Ann Rheum Dis 44:701-710, 1985

4. Ford DK, da Roza DM, Ward RH: Arthritis confided to knee joints: synovial lymphocyte responses to microbial antigens correlate with distribution of HLA. Arthritis Rheum 27: 1157-1 164, 1984

5. Ford DK, da Roza DM, Reid GD, Chantler JK, Tingle AJ: Synovial mononuclear cell responses to rubella antigen in rheumatoid arthritis and unexplained knee arthritis. J Rheumatol 9:42W23, 1982

6. Chantler JK, da Roza DM, Bottnie ME, Reid GD, Ford DK: Sequential studies on synovial lymphacyte stimu- lation by rubella antigen, and rubella virus isolation in an adult with persistent arthritis. Ann Rheum Dis 44: 564-568, 1985

7. Ford DK, da Roza DM: Observation on the responses of synovial lymphocytes to viral antigens in rheumatoid

arthritis and Reiter’s syndrome. J Rheumatol 10543- 646, 1983

8. Ford DK, da Roza DM: Further observatiotts on the responses of synovial lymphocytes to viral antigens in rheumatoid arthritis. J Rheumatol 13: 113-1 17, 1986

9. Ropes MW, Bennett GA, Cobb S, Jacox R, Jessar RA: 1958 revision of diagnostic criteria for rheumatoid arthri- tis. gull Rheum Dis 9:175-176, 1958

10. Schumacher HR Jr, Cherian PV, Sieck M, Clayburne G: Ultrastructural identification of chlamydial antigens in synovial membrane in acute Reiter’s syndrome, 50th Annual Meeting of the American Rheumatism Associa- tion. New Orleans, June 3-7, 1986

1 1 . Sigal LH, Steere AC, Freeman DH, Dwyer JM: Prolif- erative responses of mononuclear cells in Lyme disease: reactivity to Borrelia burgdorferi antigens is greater in joint fluid than in blood. Arthritis Rheum 29:761-769, 1986