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Persistent synovial lymphocyte responses to cytomegalovirus antigen in some patients with rheumatoid arthritis.

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700
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 antigen stimulation. 3H-thymidineuptakes 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 cytomegalovirus antigen were markedly different from simultaneous peripheral blood lymphocyte responses.
Previous studies have indicated that synovial
fluid lymphocyte responses to microbial antigen stimulation 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 approximately 50% of chronic or recurrent inflammatory
arthritis confined to knee joints was a reactive arthritis, but was distinct from enteric and sexually transmitted 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, University 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 ~ S CRT:
,
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 Columbia, Canada V5Z 1L7.
Submitted for publication July 3 1, 1986; accepted in revised
form November 11. 1986.
Arthritis and Rheumatism, Vol. 30, No. 6 (June 1987)
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 associated with both a persistent maximum synovial lymphocyte response to rubella and the presence of rubella
virus in synovial fluid. We have also found that the
synovial lymphocytes of other patients with rheumatoid 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 lymphocytes responded maximally and consistently to
cytomegalovirus (CMV) antigen.
PATIENTS AND METHODS
Patients. Forty patients with definite rheumatoid arthritis, according to the American Rheumatism
Association criteria (9), were selected for study. These
patients had synovial knee effusions that were associated with warmth of the involved knee, which indicated the presence of some degree of active inflammation. 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.
701
BRIEF REPORTS
SYNOVIAL
STIMULATION
10
I
Ureaplasma
Chlamydia
Salmonella
Candida
Rubella
I
I
I
I
I
I
I
I
I
50
70
10
I1
I
I
I
I
I
I
Adenovirus
Parainfluenza
I
Resp syn
Reovirus
CMV
Coxsackie
Vartcella
Measles
CMV control
PHA
30
INDICES
I
I
I
Date
Aug 15
Aug 29
Novl3
Feb 20
Apr 10 Sept 3
Dec 9
R.G.
1984
1985
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 responded 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 samples. 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 determination. 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 isothiocyanateconjugated 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 3Hthymidine 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
BRIEF REPORTS
702
BLOOD
SY NOVlAL
T8
T4 DRt
-
T8
0
0
-
T8 DRt
Pre
L
Pre
Post
Post
CMV Antigen Stimulation
Figure 2. Classification of synovial and peripheral blood lymphocytes 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 lymphocyte 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. Rheumatoid factor test results were strongly, and repeatedly, 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
1
1
1
1
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 lymphocytes 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+ lymphocytes, 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
I
1
I
I
I
1
1
I
I
UreapIasrna
Chlamydia
Salmonella
I
I
I
I
Candido
Rubella
I
Mumps
Adenovirus
I
Parainfluenza
Resp syn
=
Reovirus
CMV
C M V control
E e
I
I
1
1
I
?
m I
I
Coxsackie
Varicella
Measles
I
I
I
I
703
BRIEF REPORTS
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 demonstrable response in these patients’ peripheral blood lymphocytes (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, Canada), to determine if CMV nucleic acid was present.
Using a dot hybridization technique, nucleotide sequences 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 demonstrate CMV antigen in synovial mononuclear cells by
indirect fluorescence staining with monoclonal antiCMV 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 specimen demonstrated immunoperoxidase-positive staining 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 stimulus. 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 demonstrate consistent responses of synovial lymphocytes, 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 immunodiagnostic techniques are not yet available for use in cases
of consistent synovial responses to other viral antigens. 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 consistently 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 laboratory 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 rubella arthritis and with Reiter’s syndrome, this research program has not included serologic investigations 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
704
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 stimulation 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
BRIEF REPORTS
8.
9.
10.
11.
arthritis and Reiter’s syndrome. J Rheumatol 10543646, 1983
Ford DK, d a Roza DM: Further observatiotts on the
responses of synovial lymphocytes to viral antigens in
rheumatoid arthritis. J Rheumatol 13: 113-1 17, 1986
Ropes MW, Bennett GA, Cobb S, Jacox R, Jessar RA:
1958 revision of diagnostic criteria for rheumatoid arthritis. gull Rheum Dis 9:175-176, 1958
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 Association. New Orleans, June 3-7, 1986
Sigal LH, Steere AC, Freeman DH, Dwyer JM: Proliferative 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
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