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Lymphocyte activation in rheumatoid synovial effusion.

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I48 I
LYMPHOCYTE ACTIVATION IN
RHEUMATOID SYNOVIAL EFFUSION
J. SEBOK, A. TALERMAN, and H . W. WOUTERS
A rapid method based upon increase in intranuclear birefringence occurring in early stages of lymphocyte activation was used to examine whether there is
any difference in lymphocyte activation between lymphocytes from synovial fluid and those from peripheral
blood in patients with rheumatoid arthritis and other joint
diseases. Increased activation of synovial fluid lymphocytes compared with peripheral blood lymphocytes was
seen only in synovial effusions obtained from patients with
rheumatoid arthritis.
It has been reported recently that cell-free rheumatoid synovial effusion in appropriate dilution induces
in vitro blastic transformation of autologous lymphocytes (1,2).
I t has been suggested by Kinsella ( 1 ) that if a
similar reaction occurs in vivo in patients with rheumatoid arthritis it could induce intraarticular lymphocyte
transformation in synovial effusion. It has also been
From the Departments of Pathology and Orthopaedic Surgery. Dr. Daniel den Hoed Kliniek, Postbus 5201. Rotterdam, Holland.
J . Sebok. M.D.: Temporary Research Fellow in Rheumatology. Dr. Daniel den Hoed Kliniek, Rotterdam, Holland: A . Talerman. M.D.. M.R.C. Path., Head of the Department of Pathology.
Dr. Daniel den Hoed Kliniek. Rotterdam. Holland: H. W. Wouters.
M.D.: Head of the Department oforthpaedic Surgery, Dr. Daniel den
Hoed Kliniek. Rotterdam. Holland.
Address reprint requests to Dr. A . Talerman. Department
of Pathology. Dr. Daniel den Hoed Kliniek. Postbus 5201. Rotterdam. Holland.
Suhrnitted for publication August 31, 1976: accepted June 10.
1977.
Arthritis and Rheumatism, Vol. 20, No. 8 (November-December 1977)
reported recently that lymphocytes present in the synovial membrane of patients with rheumatoid arthritis
show appearances of intermediate stages of blastic
transformation or of well developed blastic cells (3,4). I n
view of this we have decided to study whether lymphocytes present in synovial effusion from patients with
rheumatoid arthritis show similar changes.
I t has been reported that an increase in intranuclear birefringence is a feature of activated lymphocytes (5). Therefore retardation of polarized light
was measured in isolated blood and synovial fluid lymphocytes obtained from patients with rheumatoid arthritis and other joint diseases. This measure was taken
to find whether there is any difference between the average retardation of lymphocytes taken from peripheral
blood and those from synovial fluid. We have also studied whether cell-free synovial fluid can induce activation
in isolated autologous blood lymphocytes.
MATERIAL AND METHODS
T h e synovial fluid was obtained by puncture o f knee
joints. A t t h e same time 15 ml of blood were taken using a
syringe containing 1,000 IU of heparin. The patients under
study were known t o be suffering from rheumatoid arthritis
with definite or classical diagnosis according to t h e A R A
criteria. Lymphocytes were isolated from both t h e blood a n d
the synovial effusion using gradient centrifugation a s described
by Boyum (6). O n e d r o p of t h e fluid containing isolated synovial lymphocytes a n d o n e d r o p containing those from t h e
blood were placed on t h e same glass slide.
SEBOK ET A L
I482
Table 1. The Clinical and Laboratory Findings and Average Retardation of Blood and Synovial Fluid
Lymphocytes in Patients Suflering from Seronegative Rheumatoid Arthritis
Age
(years)
Sex
Average
Retardation
of Blood
Lymphocytes
(nm)
Duration
of
Disease
(years)
~
*B
64
41
32
60
2
6
12
2
21.72
18.91
19.66
21.33
F
74
5
20.68
brufen. I = indomethacin;P
=
Site
of
ElTusion
~~~
F
M
F
F
=
Average
Retardation
of
Synovial
Fluid
Lymphocytes
(nm)
P
Treatment
in Last 3
Months*
~~~~
Knee
Knee
Knee
Left Knee
Right Knee
Left Knee
Right Knee
penicillamine. S
=
28.53
28.14
27.69
30.46
30.67
28.23
28.54
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
P
I
I. B
S
steroids.
microscopic analysis have been reported previously (5).
Synovial fluid from 16 patients, 8 of whom had bilateral effusions was examined. The retardation of 25 cells was
measured i n each sample using two rotatory compensators
with retardation of 31.2 nm and 51 nm.
The retardation of 50 random selected lymphocytes
was measured in the incubation experiments both from the
control and from the treated sample.
The results were evaluated using the t test.
The isolated blood lymphocytes were incubated in T C
199/Difco/medium. The number of living mononuclear cells
was 0.9 - 1.5 X 108 in each milliter of the culture. The
cultures were divided into samples (each 2 ml in volume). One
sample was used as a control while the other was treated with
0.1 ml of cell-free supernatant of the synovial effusion. After
incubating for 30 minutes at 37" C, one drop of the control
and one drop of the treated sample were placed on the same
glass slide. The methods of staining and of the polarization
Table 2. Clinical and Laboratory Findings and Average Retardation of Blood and Synovial Fluid
Lymphocytes in Patients Suffering from Seropositive Rheumatoid A rthritis
Sex
Duration
Age of Disease
(years) (years)
F
69
M
M
6
5
Rose
Test
Average
Retardation
o f Blood
Lymphocytes
(nm)
1/32
23.62
45
I
1/64
4
5
1/32
M
56
3
1/128
F
70
7
1/32
F
55
2
1/64
M
F
F
M
M
45
34
65
65
33
3
4
20
8
4
1/32
1/64
1/32
1/32
1/32
* Au
=
gold salts, B
=
brufen, I
=
Site of
Synovial
Effusion
Left Knee
Right Knee
20.17 Left Knee
Right Knee
21.93 Left Knee
Right Knee
19.93 Left Knee
Right Knee
18.61 Left Knee
Right Knee
19.34 Left Knee
Right Knee
19.25 Knee
20.14 Knee
18.99 Knee
18.61 Knee
19.42 Knee
indomethacin, P
=
Average
Retardation
of Synovial
Lymphocytes
(nm)
30.97
30.44
27.70
28.21
28.43
28.87
28.02
27.37
28.47
27.56
28.89
30.82
27.70
26.95
27.66
27.64
27.63
P
Treatment
in Last 3
Months*
<0.0005
Au
<0.0005
<0.0005
<0.0005
P
<0.0005
I
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
penicillamine. N = naprosim.
B
Au
B
I
N
I
Au, I
LYMPHOCYTE ACTIVATION
1483
Table 3. Clinical and Laboratory Findings and Average Retardation of Blood and Synovial Fluid
Lymphocytes in Patients Suffering from Diseases Other than Rheumatoid Arthritis
Age
(years)
Sex
M
46
64
F
M
M
60
56
23
M
Treatment in
Last 3 Months
Average
Retardation
of Blood
Lymphocytes
Average
Retardation
of Synovial
Fluid
Lymphocytes
19.28
18.00
18.72
17.49
Ochronosis
Osteoarthrosis
17.48
17.41
20.12
16.95
16.50
19.25
Osteoarthrosis
Gout
Posttraumatic effusion
of the knee joint
Salicylates
Brufen
Phenyl butazone
lndomethacin
lndomethacin
None
RESULTS
The age, sex, duration of disease, and average
retardation of blood and synovial fluid lymphocytes in
patients suffering from seronegative rheumatoid arthritis are shown in Table 1 . The findings in patients with
seropositive rheumatoid arthritis are given in Table 2.
Table 3 presents data on patients suffering from diseases
other than rheumatoid arthritis. Table 4 shows the main
clinical and serologic findings in patients with rheumatoid arthritis from whom isolated peripheral blood lym-
Diagnosis
phocytes were incubated with autologous synovial effusion.
DISCUSSION
The results of the present study show that synovial fluid lymphocytes from patients with seropositive
and seronegative rheumatoid arthritis exhibit significantly increased retardation of polarized light, as compared to blood lymphocytes. This difference in retarda-
Table 4. Clinical and Laboratory Findings and Retardation of Blood and Synovial Fluid Lymphocytes and Retardation of Blood Lynrphocjres
Incubated with Cell-free Synovial Effusion in Patients Suffering from Rheumatoid A rthritis
Sex
Duration
of Disease
Age (years)
(years)
Rose Test
F
F
M
36
63
54
36
54
54
65
62
54
71
56
18
36
13
12
18
22
8
15
22
18
7
Neg
Neg
N eg
Neg
17.99
17.39
17.56
17.74
17.75
18.85
18.29
19.66
18.43
17.39
19.99
M
46
12
Neg
19.28
M
45
17
N eg
19.23
F
F
M
F
M
F
M
F
1/32
1/8
I /256
1/32
I /256
1 /a
1/32
Average
Retardation
of Blood
Lymphocytes
(nm)
Site of
Effusion
Knee
Knee
Knee
Knee
Knee
Knee
Knee
Knee
Left Knee
Knee
Left Knee
Right Knee
Left Knee
Right Knee
Left Knee
Right Knee
Average
Retardation of
Synovial Fluid
Lymphocytes
(nm)
27.56
27.88
29.68
29.68
29.13
29.39
29.31
29.31
29.38
29.68
29.21
28.42
26.38
29.45
25.35
27.61
P
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
Average
Retardation
of Blood
Lymphocytes
Incubated
with Synovial
Fluid (nm)
26.56
25.79
26.71
30.19
29.36
26.29
26.29
30.10
29.06
25.79
28.66
27.41
26.39
27.45
27.70
28.21
P
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
SEBOK ET A L
tion is not seen in patients suffering from diseases other
than rheumatoid arthritis. It has also been shown that
blood lymphocytes when incubated with cell-free synovial effusion from patients with rheumatoid arthritis
exhibit similar retardation t o autologous synovial fluid
lymphocytes. Their retardation is significantly increased
as compared t o nonincubated blood lymphocytes.
During lymphocyte transformation provoked by
different mitogenic agents similar morphologic, biochemical, a n d molecular events take place although differences have been noted in the duration of DNA synthesis (7). Within the nuclei of the stimulated cells the
loosely packed euchromatin is very active according t o
the autoradiographic a n d electron microscopic autoradiographic studies (7). It has been reported previously
that the activated D N A shows an increased retardation
when examined with polarized light microscope (5). In
view of this and our observation of t h e significantly
increased intranuclear retardation of the synovial fluid
lymphocytes compared with blood lymphocytes from
patients with rheumatoid arthritis, we suggest that the
retardation indicates that the synovial fluid lymphocytes
have been activated in vivo by the rheumatoid arthritis.
The synovial fluid lymphocytes from patients
with diseases other than rheumatoid arthritis did not
show increased retardation compared with blood lymphocytes.
De Compos Vidal (8) has reported that the euchromatin molecule isolated from lymphocytes shows
an average retardation of approximately 19.5 nm. We
obtained similar results in studying isolated blood lymphocytes, although in a few cases the retardation was
increased. This is in accordance with the findings of
Klein et a1 (9) who observed a n increased uptake of
radioactive thymidine in blood lymphocytes from patients suffering from rheumatoid arthritis. There are
reports in the literature which provide evidence indicating that lymphocytes are being activated in the
synovial membrane of patients with rheumatoid arthritis (3,4,10).
The increased retardation of the isolated lymphocytes as observed in the present study indicates that
they have already been activated in the rheumatoid synovial effusion, while in diseases such as gout, ochronosis, or osteroarthrosis there were n o differences between the retardation of blood and synovial fluid
lymphocytes. Loewi and Gumpel ( 1 1 ) have reported an
increased radioactive thymidine uptake in isolated synovial fluid lymphocytes, which could be interpreted in the
same way. O u r findings suggest that there a r e factors in
the rheumatoid synovial effusion which can activate
autologous lymphocytes in vitro as has been suggested
by Kinsella (1) a n d Crout et a1 (2). It is possible that the
same factors a r e responsible for the lymphocytes being
activated within the synovial effusion in vivo.
REFERENCES
Kinsella TD: Induction of autologous lymphocyte transformation by synovial fluids from patients with rheumatoid arthritis. Clin Exp Immunol 14:187-191, 1973
2. Crout JE, McDuffie FC, Ritts RF Jr: Induction of peripheral blood lymphocyte transformation by autologous synovial fluid. Arthritis Rheum 19:523-531, 1976
3. Kobayashi K , Ziff M: Electron microscopic studies of
lymphoid cells in rheumatoid synovial membrane. Arthritis Rheum 16:471-486, 1973
4. lshikawa H, Ziff M: Electron microscopic observations of
immuno-reactive cells in the rheumatoid synovial membrane. Arthritis Rheum l9:l-l4, 1976
5. Surjhn L Jr, Sebok J: Increase in intranuclear birefringence during chromatin activation reaction. Exptl Cell
Res 78:241-243, 1973
6. Boyum A: Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest
21:51-109 (SUPPI97) 1968
7 . Douglas SD: H u m a n lymphocyte growth i n vitro: morphologic, biochemical and immunologic significance. Int
Rev Exper pathol 10:42-114, 1971
8. De Compos Vidal B: Birefringence and linear dichroism of
euchromatin stained with toluidin blue: cotton effect like
phenomenon. Beitr Path 145:269-285, 1972
9. Klein G, Altmann H, Wottawa A, et al: Untersuchungen
uber die DNA Synthese peripherer Lymphocyten bei progredient chronischer Polyarthritis. Blut 28:187-190, 1974
10. Norval M , Ogilvie MM, Marmion B P DNA polymerase
activity in rheumatoid synovial membranes. A n n Rheum
Dis 34:205-212, 1975
1 1 . Loewi G, Gumpel M: Joint exudate and peripheral blood
mononuclear cells in inflammatory joint disease. Short
Term Culture and Cytotoxicity in Rheumatoid Arthritis:
Pathogenic Mechanisms and Consequences in Therapeutics. Edited by W Miiller, H Harwerth. New York, Academic Press, 1971, pp 475-488
1.
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