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.