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Synovial lymphocyte response to chlamydial stimulation associated with intrasynovial chlamydial antigen in a patient with Вrheumatoid arthritis.

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A 48-year-oldman with “rheumatoid arthritis” of
3 years duration was found to have synovial fluid lymphocytes that were maximally stimulated in vitro by chlamydial antigen, on 5 of 6 tests over 18 months. Immunocytochemical staining of a synovectomy specimen, using the
peroxidase-antiperoxidase technique, subsequently revealed chlamydial antigen in the synovium. The possibility
that Chlamydia in the synovium may produce features of
rheumatoid arthritis is discussed.
The 3H-thymidine uptake response of synovial
lymphocytes to microbial antigen stimulation appears
to suggest the origin of the arthritis in enteric and
sexually transmitted Reiter’s syndrome (1-4). The
finding that the synovial lymphocytes of Lyme disease
patients react to Borreliu burgdorferi antigens to a
greater extent than do their peripheral blood lymphocytes ( 5 ) lends evidence to support the validity of this
Studies in patients with rheumatoid arthritis
(RA) have raised the possibility that the response of
synovial lymphocytes to microbial antigen stimulation
might also be helpful for research into that disease.
Over 70 patients with RA have been studied and the
From the Department of Medicine, University of British
Columbia, Vancouver, British Columbia, Canada, and the Department of Medicine, University of Pennsylvania, Philadelphia.
Denys K. Ford, MD: Professor of Medicine, University of
British Columbia: Graham D. Reid, MB: Clinical Assistant Professor of Medicine, University of British Columbia; Sathish Magge,
MD: Research Fellow, University of Pennsylvania; H. Ralph Schumacher, MD: Professor of Medicine, University of Pennsylvania.
Address reprint requests to Denys K. Ford, MD, The
Arthritis Centre, 895 West 10th Avenue, Vancouver, British Columbia, V5Z 1L7, Canada.
Submitted for publication September 29, 1987: accepted in
revised form January 18, 1988.
Arthritis and Rheumatism, Vol. 31, No. 7 (July 1988)
results reported, either as observations on “controls”
for the findings in patients with Reiter’s syndrome
(2,3) or as observations on RA patients whose synovial
lymphocytes reacted maximally to a particular antigen
(6-10). We report the association of a repeated synovial lymphocyte response to chlamydial stimulation
with the presence of chlamydial antigen in a surgically
removed synovial specimen from a patient diagnosed
as having RA.
Case report. The patient injured his right knee
while playing soccer in 1953, when he was 16 years old.
Surgery was performed, and he had 3 episodes of
“water on the knee” over the next 10 years. In 1982, at
the age of 45,he had an episode of pain and swelling at
the base of his right thumb. About 2 months later, he
noted generalized stiffness, fatigue, and weight loss of
approximately 7 kg. He developed pain in the fingers,
wrists, shoulders, neck, and knees, and his right knee
became swollen.
The patient was referred to one of us (GDR) 5
months after the onset of his polyarthritis. At that time
he had pain and swelling of several finger joints, both
wrists, both shoulders, and both knees. There was a
large effusion in the right knee. All of the metatarsophalangeal joints were tender, and both fifth toes were
swollen and red; in addition, there was tenderness
beneath the left heel. Based on the clinical assessment,
it was concluded that the patient had an asymmetric
polyarthritis. It was noted in his chart that, “in view of
the swelling of the little toes and the heel pain, one
might suspect that this will be a seronegative type.”
The patient denied having diarrhea or genitourinary
Over the next 2 years, the polyarthritis persisted; the right knee was always the most involved
joint. Rheumatoid factor was found (titer 1:640 by
latex fixation). Radiographs showed erosions in the
hands and feet, and normal sacroiliac joints. Gold
treatment for rheumatoid arthritis was started in
March 1984 and continued at varying dosages up to the
present. Various nonsteroidal antiinflammatory drugs
were also given.
The patient demonstrated 3 types of skin lesions during the 3-year observation period. He was
referred to a dermatologist who noted scattered scaling plaques typical of psoriasis, tinea versicolor of the
trunk, and stasis eczema of both lower legs. Significant
lymphadenopathy of the hilar, axillary, and epitrochlear nodes was also noted, but only reactive hyperplasia was shown on biopsy. HLA typing revealed
Synovial fluid was obtained from the patient’s
right knee on 6 occasions between August 1983 and
January 1985, as shown in Figure 1. In 5 of the 6
studies, the synovial lymphocytes were stimulated
maximally by chlamydial antigen, while once (August
1984) there was no significant stimulation from any
antigen. On the first test (August 1983), reactivity with
phytohemagglutinin was also studied; it, too, elicited
less of a stimulation response than the chlamydial
antigen. In August 1985, similar testing was performed
with the patient’s peripheral blood lymphocytes.
These lymphocytes also responded to a chlamydial
antigen more than to other antigens; the stimulation
index was twice that obtained from the antigen with
the next highest stimulation index. These lymphocyte
studies were performed using techniques that have
been standard for the last 8 years and have been
extensively described in earlier reports (1-4,641).
In May 1985, a total right knee replacement was
performed, and the synovial tissue removed was dissected to select hypertrophied villous synovium, both
for electron microscopy and for the freezing of aliquots. The routine pathology report stated, “the synovium is heavily inflamed with chronic inflammatory
cells including many plasma cells-consistent with
rheumatoid arthritis.” A frozen aliquot was forwarded
to Dr. Wanda Wenman (Division of Pediatrics, Infectious Diseases, University of Alberta, Edmonton, Al-
20 40
2040 60
2040 60 a0100120
Resp. Syn.
M a y Aug.
Figure 1. ’H-thymidine uptake response of the patient’s synovial fluid lymphocytes to various antigens, on 6
occasions between August 1983 and January 1985. Resp. Syn. = respiratory syncytial virus; CMV =
cytomegalovirus; PHA = phytohemagglutinin.
berta, Canada), who had developed a DNA hybridization probe for chlamydial nucleic acid. She was unable
to demonstrate any chlamydial nucleic acid in this
frozen specimen. Another sample of synovium was
fixed in 1% glutaraldehyde and forwarded to one of the
authors (HRS) in Philadelphia for ultrastructural and
immunocytochemical studies.
Routine transmission electron microscopy
showed proliferation of types A and B synovial lining
cells, scattered lymphocytes and plasma cells, and
vessels with endothelial proliferation and multilaminated basement membrane. There were many macrophage-like cells with phagocytic vacuoles containing
finely granular and particulate material, but no suggestion of any identifiable microorganisms. Some of the
vacuoles contained typical aurosomes (12); energy
dispersive elemental analysis (I 3) confirmed that these
contained gold. Some dense inclusions contained both
gold and small, round, darker particles with indistinct
Portions of tissue were processed for immunocytochemical studies to search for chlamydial antigen.
Endogenous peroxidase was blocked by incubation in
0.03% H,O, for 30 minutes. The specimen was next
incubated in normal goat globulin and then in 0.01%
saponin to permeabilize membranes. This was followed by incubation in rabbit antichlamydial antibody
(Abbott, Chicago, IL) diluted 1: 100 (previously determined to be the optimum dilution) for 1 hour at room
temperature. After washing, goat anti-rabbit IgG (1 :20)
was added in excess, so that binding sites were available for the peroxidase-antiperoxidase complex,
which was added in 1 :40 concentration after a further
wash. The tissue was finally incubated in Tris buffer,
H,O,, and diaminobenzidine, which would produce a
dark reaction if chlamydial antigen was present. This
procedure revealed marked dark staining of the particles in many vacuoles of the macrophage-like cells. No
reaction product was seen in any other sites.
Control procedures, consisting of replacement
of the antichlamydial antibody with normal rabbit
globulin, elimination of the peroxidase-antiperoxidase
complex, and absorption of the antibody with Chlamydia yielded totally negative results or showed only
very rare and irregular suggestions of a reaction product. Positive control procedures were also performed,
using synovia from 2 patients with Reiter’s syndrome
and cultured chlamydial organisms (14). Similar findings of chlamydial reactivity in synovial tissue and
cells of Reiter’s syndrome patients have recently been
described by Keat et al, who used a fluoresceinlabeled monoclonal antibody to Chlamydia trachoma-
tis (15). Three other synovial membranes from patients
with RA were negative for chlamydial reactivity.
Despite the above-described findings, a sample
of the patient’s serum, which was forwarded to Dr.
Robert C. Brunham (Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba,
Canada), did not reveal either IgM or IgG antibody
against C trachomatis.
Discussion. The results of several studies (6-10)
suggest that rheumatoid arthritis may be a clinical
syndrome caused by an aberrant immune response to
a variety of common infectious agents, and that the
microbiologic cause of a particular patient’s RA may
be designated by the response of synovial lymphocytes to that particular antigen, as compared with the
response to antigens of other, nonrelevant agents. This
viewpoint has recently been expressed in greater detail
( 16,17).
The demonstration of a maximal synovial lymphocyte response to a particular antigen is not, in
itself, sufficient evidence that the organism plays an
etiologic role in the patient’s disease; further documentation of a relationship between that particular
agent and the arthritis is needed. Earlier studies in
Vancouver have shown that, in each of 3 cases, the
finding of a maximal synovial lymphocyte response to
rubella antigen was associated with the isolation of
rubella virus from the patient (8,9). In the present
case, chlamydial antigen was found in the synovium,
following the observation that the patient’s synovial
lymphocytes responded maximally to stimulation with
chlamydial antigen on successive occasions. Although
an attempt was made to demonstrate chlamydial nucleic acid in the removed synovial specimen, it was
unsuccessful. The sensitivity of the hybridization technique employed and the suitability of the frozen specimen shipped to Edmonton are uncertainties in trying
to relate this negative finding to the finding of synovial
chlamydial antigen by the immunoperoxidase method
employed in the Philadelphia laboratory.
This patient had “rheumatoid arthritis,” with
radiographically evident erosions and positive rheumatoid factor. H e also had scattered skin lesions,
which a dermatologist confidently diagnosed as psoriasis; moreover, he had “dactylitis” and some heel
pain. In the opinion of the 2 rheumatologists responsible for his medical care, his disease did not fulfill
criteria for psoriatic arthritis; both treated him for
“rheumatoid arthritis.” The hilar, axillary, and epitrochlear lymphadenopathy showed only reactive hyperplasia on lymph node biopsy. At no time was there
any indication that his arthritis was related to genito-
urinary or intestinal symptoms, a n d he denied having
any such symptoms.
Only this patient and one other patient with
“rheumatoid arthritis” have shown a significant synovial lymphocyte response to chlamydial antigen; the
study group now includes 70 patients with RA. Thus, if
synovial lymphocyte response can suggest t h e underlying causative agents in RA, Chlamydia is likely to b e
related t o t h e disease in only a small percentage of
cases. Previous evidence that Chlamydia might be
related to unexplained knee arthritis (18), an entity
which is clinically distinct from sexually acquired reactive arthritis, seems to have received recent confirmation (19), a n d there is thus no a priori reason why
Chlamydia could not be responsible for the clinical
syndrome of rheumatoid arthritis in some cases. T h e
demonstration of gold in t h e same inclusions as t h e
chlamydial antigen is of interest and may have implications for t h e mechanism of action of gold as a treatment.
T h e failure to demonstrate serum antibody against
chlamydial antigen in our patient is perhaps not unexpected, in view of similar findings in 13 patients with
failed rubella immunization, of whom 3 had arthritis
(20). Studies of the synovial lymphocyte responses in
additional patients with RA are needed to further elucidate the possible role of infectious agents in this
Acknowledgments. We thank Doreen da Roza, Susan
Rothfuss, and Marie Sieck for technical assistance, and Dr.
W. R. Bowie of the Division of Infectious Diseases, University of British Columbia, for providing the chlamydial antigen.
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