ScaaJ. J. Immunol., Vol. 6, 1977.
Antibody-Dependent Cytotoxicity Mediated by Cells Eluted from Synovial Tissues of Patients with Rheumatoid Arthritis and Juvenile Rheumatoid Arthritis T. G. ABRAHAMSEN, S. S. FR0LAND, J. B. NATVIG & J. PAHLE Institute of Immunology' and Rheumatology, Rikshospitalet University Hospital, und Oslo Saaitet5forening Rheumatism Hospital, Oslo, Norway
Abrahamsen, T. G., Fraland, S. S.. Natvig, J, B. & Pahle, J. Antibody-Dependent Cytotoxicity Mediated by Cells Eluted from Synovial Tissues of Patients with Rheumatoid Arthritis and Juvenile Rheumatoid Arthritis. Scand. J. Immunol. 6, 1251-1261, 1977. Cell suspensions containing an average of 7 8 % lymphocytes were obtained from synovial tissues of 26 patients with rheumatoid arthritis and 10 patients with juvenile rheumatoid arthritis. These cells were shown to mediate cytotoxicity against ^'Cr-labeled chicken erythrocytes sensitized with a rabbit anti-cliicken erythrocj'te antiserum. Nylon column filtration of the cells increased tlio proportion of lymphocytes and usually also the cytotoxicitj', which suggested that at least some of the effector cells were lymphocytes. The cj'totoxic activity uf the cells obtained from rheumatoid synovial tissue wa.s always lower than that obtained with the patients' peripheral blood lymphocytes. No significant change in cytotoxicity of normal peripheral blood lymphocytes was observed after these cells had been treated in the same manner as the rheumatoid synovial tissues. r . G. Abrahamsen, Institute of immunology Oslo 1, Norway
Anti-target cell antibodies can induce lymphoqte-mediated q'totoxicitj' in various in vitro systems (10). Lysis is dependent on effector cell interaction with the Fc part of the antibody molecule attached to the target cell (20). There is evidence that the lymphoid effector cells in peripheral blood are found among a lymphocyte population that forms rosettes with antibody-sensitized er}'throcytes (EA-RFC) but largely lacks the usual T- and B-lymphocyte markers (9, 30). In peripheral blood of rheumatoid arthritis patients the proportion of the Fc-receptorbearing cells, determined as EA-RFC, is within the normal range (15), and the antibodydependent cytotoxic activity of unfractionated
and Rheumatology,
F. Qvamsgt. 1,
lymphocyte suspensions is similar to that found in suspensions from nonrheumatoid arthritis patients and normal controls (31)- The latter finding has been confirmed in several studies (11, 13, 25). The data on synovial fluid cells are less conclusive; in comparison with the patients' peripheral blood lymphocytes, both ei^ual (25) and lower ( U ) cytotoxicity have been obser\'ed. Rheumatoid synovial tissue is usually infiltrated by mononuclear cells (32). It has recently become possible to elute lymphocytes from these tissues, and membrane marker studies have revealed a predominance of T lymphocytes compared with B lymphocytes in the resulting cell suspensions both in rheu-
1252 T. G. Ahrahamsett, S. S. Freland, J. B. Natvig & J. Pahle
matoid arthritis Oi 28) and juvenile rheumatoid arthritis patients (3). The average proportion of Fc-receptor-bearing lymhocytes (EARFC) is similar to that obtained for B lymphocytes (1, 3). The present study vi-as undertaken to determine the cytotoxic activity of the cells eiuted from synovial tissue.s from patients with rheumatoid arthritis and juvenile rheumatoid arthritis in a ^^Cr-release assay u.sing as target cells chicken er)'throcytes sensitized with a rabbit anti-chicken etythrocyte antiserum.
MATERIALS AND METHODS Sources of materials. Synovial tissues were collected from 26 patients with rheumatoid arthritis (RA) diagnosed in accordance with the criteria of the American Rheumatism Association and 10 patients with juvenile rheumatoid arthritis (JRA) diagnosed in accordance with the criteria of Ansell & Bywaters (5). Two of the patients with RA and one witli JRA were studied twice, using specimens removed during operation of different joints. Four of the RA patients were seronegative (Waaler-Rose test), whereas the others showed various degrees of seropositivit)', with titers ranging from 64 to more than 1,024. The patients with JRA were seronegative. An indirect immunofluorescence test for antinuclear factors in serum was negative for the RA patients, but two JRA patients had titers of 128 and 512, respectively. Four of the patients with RA received no medication before operation, whereas the others and the patients with JRA were treated with various anti-inflammatory drugs. Nine of the RA patients and two of the jRA patients received corticosteroids in addition. The specimens were obtained during synovectomy of various joints and tendon sheaths, usually the wrist ot the knee joint. From ten patients, eight with RA and two with JRA, venous blood samples (in 10 IU heparin per ml blood) were taken within 13 days after tlie operation. Two of these RA patients were also studied before the operation. Control peripheral blood lymphocytes were oh-
tained from healthy donors. The blood lyniphocytes were separated by the Isopaque-i'icoll gradient centrifugation method (7). Elation and characterization nf cells from the rheumatoid tissue. The dissociation of tissue and separation of lymphocytes were performed as described in detail previously (I). Briefly, after the tissue had been minced with iri.s scissors, it was treated with crude collagenase and deoxyribonuclease. Debris was removed by filtration through one layer of nylon mesh. The amount of peripheral blood in the tissue was determined by counting the numbers of erj'throcytes and total cells in stained (May-Grunwald/ Giemsa) cytocentrifuge smears of the resulting cell suspension. An average ratio of 1:20 (that is, erv'throcytes to total cells) (range, 'totoxic aaivity oi cells eluted from synovial tissues of patients with rheumatoid arthritis (RA) and juvenile rht-uniatoid arthritis (JRA) cumparc-d with the patients' peripht-ral blood lymphocytes studied within 15 days after the operation Source of effector cells Synovial tissue Patient
RF*
Antiserumt (Corr. Cl)
Peripheral blood
Medium'* (Cl)
Antiserum (Corr. Cl)
Medium (Cl)
38.5 51.2 30.0 34.2 24.8 31.2 18.4 15.7
10.4
40.1 32.9 36.3 (20.2-60.5)
7,0 5.0
RA
R.K. G.L. A.M.N. L.R. E.K. O.L. E.S. T.G.
64 128 128 128 128 128 ^52
Antibody-Dependent Cytotoxicity Mediated by Cells Eluted from Synovial Tissues of Patients with Rheumatoid Arthritis and Juvenile Rheumatoid Arthritis T. G. ABRAHAMSEN, S. S. FR0LAND, J. B. NATVIG & J. PAHLE Institute of Immunology' and Rheumatology, Rikshospitalet University Hospital, und Oslo Saaitet5forening Rheumatism Hospital, Oslo, Norway
Abrahamsen, T. G., Fraland, S. S.. Natvig, J, B. & Pahle, J. Antibody-Dependent Cytotoxicity Mediated by Cells Eluted from Synovial Tissues of Patients with Rheumatoid Arthritis and Juvenile Rheumatoid Arthritis. Scand. J. Immunol. 6, 1251-1261, 1977. Cell suspensions containing an average of 7 8 % lymphocytes were obtained from synovial tissues of 26 patients with rheumatoid arthritis and 10 patients with juvenile rheumatoid arthritis. These cells were shown to mediate cytotoxicity against ^'Cr-labeled chicken erythrocytes sensitized with a rabbit anti-cliicken erythrocj'te antiserum. Nylon column filtration of the cells increased tlio proportion of lymphocytes and usually also the cytotoxicitj', which suggested that at least some of the effector cells were lymphocytes. The cj'totoxic activity uf the cells obtained from rheumatoid synovial tissue wa.s always lower than that obtained with the patients' peripheral blood lymphocytes. No significant change in cytotoxicity of normal peripheral blood lymphocytes was observed after these cells had been treated in the same manner as the rheumatoid synovial tissues. r . G. Abrahamsen, Institute of immunology Oslo 1, Norway
Anti-target cell antibodies can induce lymphoqte-mediated q'totoxicitj' in various in vitro systems (10). Lysis is dependent on effector cell interaction with the Fc part of the antibody molecule attached to the target cell (20). There is evidence that the lymphoid effector cells in peripheral blood are found among a lymphocyte population that forms rosettes with antibody-sensitized er}'throcytes (EA-RFC) but largely lacks the usual T- and B-lymphocyte markers (9, 30). In peripheral blood of rheumatoid arthritis patients the proportion of the Fc-receptorbearing cells, determined as EA-RFC, is within the normal range (15), and the antibodydependent cytotoxic activity of unfractionated
and Rheumatology,
F. Qvamsgt. 1,
lymphocyte suspensions is similar to that found in suspensions from nonrheumatoid arthritis patients and normal controls (31)- The latter finding has been confirmed in several studies (11, 13, 25). The data on synovial fluid cells are less conclusive; in comparison with the patients' peripheral blood lymphocytes, both ei^ual (25) and lower ( U ) cytotoxicity have been obser\'ed. Rheumatoid synovial tissue is usually infiltrated by mononuclear cells (32). It has recently become possible to elute lymphocytes from these tissues, and membrane marker studies have revealed a predominance of T lymphocytes compared with B lymphocytes in the resulting cell suspensions both in rheu-
1252 T. G. Ahrahamsett, S. S. Freland, J. B. Natvig & J. Pahle
matoid arthritis Oi 28) and juvenile rheumatoid arthritis patients (3). The average proportion of Fc-receptor-bearing lymhocytes (EARFC) is similar to that obtained for B lymphocytes (1, 3). The present study vi-as undertaken to determine the cytotoxic activity of the cells eiuted from synovial tissue.s from patients with rheumatoid arthritis and juvenile rheumatoid arthritis in a ^^Cr-release assay u.sing as target cells chicken er)'throcytes sensitized with a rabbit anti-chicken etythrocyte antiserum.
MATERIALS AND METHODS Sources of materials. Synovial tissues were collected from 26 patients with rheumatoid arthritis (RA) diagnosed in accordance with the criteria of the American Rheumatism Association and 10 patients with juvenile rheumatoid arthritis (JRA) diagnosed in accordance with the criteria of Ansell & Bywaters (5). Two of the patients with RA and one witli JRA were studied twice, using specimens removed during operation of different joints. Four of the RA patients were seronegative (Waaler-Rose test), whereas the others showed various degrees of seropositivit)', with titers ranging from 64 to more than 1,024. The patients with JRA were seronegative. An indirect immunofluorescence test for antinuclear factors in serum was negative for the RA patients, but two JRA patients had titers of 128 and 512, respectively. Four of the patients with RA received no medication before operation, whereas the others and the patients with JRA were treated with various anti-inflammatory drugs. Nine of the RA patients and two of the jRA patients received corticosteroids in addition. The specimens were obtained during synovectomy of various joints and tendon sheaths, usually the wrist ot the knee joint. From ten patients, eight with RA and two with JRA, venous blood samples (in 10 IU heparin per ml blood) were taken within 13 days after tlie operation. Two of these RA patients were also studied before the operation. Control peripheral blood lymphocytes were oh-
tained from healthy donors. The blood lyniphocytes were separated by the Isopaque-i'icoll gradient centrifugation method (7). Elation and characterization nf cells from the rheumatoid tissue. The dissociation of tissue and separation of lymphocytes were performed as described in detail previously (I). Briefly, after the tissue had been minced with iri.s scissors, it was treated with crude collagenase and deoxyribonuclease. Debris was removed by filtration through one layer of nylon mesh. The amount of peripheral blood in the tissue was determined by counting the numbers of erj'throcytes and total cells in stained (May-Grunwald/ Giemsa) cytocentrifuge smears of the resulting cell suspension. An average ratio of 1:20 (that is, erv'throcytes to total cells) (range, 'totoxic aaivity oi cells eluted from synovial tissues of patients with rheumatoid arthritis (RA) and juvenile rht-uniatoid arthritis (JRA) cumparc-d with the patients' peripht-ral blood lymphocytes studied within 15 days after the operation Source of effector cells Synovial tissue Patient
RF*
Antiserumt (Corr. Cl)
Peripheral blood
Medium'* (Cl)
Antiserum (Corr. Cl)
Medium (Cl)
38.5 51.2 30.0 34.2 24.8 31.2 18.4 15.7
10.4
40.1 32.9 36.3 (20.2-60.5)
7,0 5.0
RA
R.K. G.L. A.M.N. L.R. E.K. O.L. E.S. T.G.
64 128 128 128 128 128 ^52
