Lymphocytes were highly purified from synovial fluid and peripheral blood of 10 rheumatoid arthritis patients and assessed for responsiveness to PHA-P and Con-A. In all cases, both synovial and blood lymphocytes showed a marked reduction in response to these mitogens compared with normal blood lymphocytes. The factors responsible for this low T cell responsiveness are discussed.</p

Ezawa, Hidemitsu

Katsuyama, Shunsaku

Matsueda, Hideki

Miyake, Susumu

Ofuji, Tadashi

Yamana, Seizo

Okayama University Digital Information Repository

Low responsiveness of synovial and peripheral blood lymphocytes stimulated by either PHA-P or CON-A in patients with chronic rheumatoid arthritis

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 32, Issue 6 1978 Article 8DECEMBER 1978Low responsiveness of synovial and peripheralblood lymphocytes stimulated by eitherPHA-P or CON-A in patients with chronicrheumatoid arthritisSeizo Yamana∗ Hideki Matsueda† Hidemitsu Ezawa‡Susumu Miyake∗∗ Shunsaku Katsuyama†† Tadashi Ofuji‡‡∗Okayama University,†Okayama University,‡Okayama University,∗∗Okayama University,††Okayama University,‡‡Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Low responsiveness of synovial and peripheralblood lymphocytes stimulated by eitherPHA-P or CON-A in patients with chronicrheumatoid arthritis∗Seizo Yamana, Hideki Matsueda, Hidemitsu Ezawa, Susumu Miyake, ShunsakuKatsuyama, and Tadashi OfujiAbstractLymphocytes were highly purified from synovial fluid and peripheral blood of 10 rheumatoidarthritis patients and assessed for responsiveness to PHA-P and Con-A. In all cases, both syn-ovial and blood lymphocytes showed a marked reduction in response to these mitogens comparedwith normal blood lymphocytes. The factors responsible for this low T cell responsiveness arediscussed.KEYWORDS: rheumatoid arthritis, blastogenesis of lymphocytes, synovial lymphocytes∗Copyright (C) OKAYAMA UNIVERSITY MEDICAL SCHOOL PMID: 154829Acta Merl. Okayama 32, (6),441-447 (1978)LOW RESPONSIVENESS OF SYNOVIAL AND PERIP~ERALBLOOD LYMPHOCYTES STIMULATED BY EITHERPHA-P OR CON-A IN PATIENTS WITH CHRONICRHEUMATOID A,RTHRITISSeizo YAMANA, Hideki MATsuEDA, Hidemitsu EZAWA,Susumu MIYAKE, Shunsaku KATSUYAMAand Tadashi OFUJIClinical Immunology Division, 3rd Department of Internal Medicine,Okayama University Medical School, Okayama 700, JapanReceived August 11, 1978Abstract. Lymphocytes were highly purified from synovial fluidand peripheral blood of 10 rheumatoid arthritis patients and assessed forresponsiveness to PHA-P and Con-A. In all cases, both synovial andblood lymphocytes showed a marked reduction in response to thesemitogens compared with normal blood lymphocytes. The factors re-sponsible for this low T cell responsiveness are discussed.Key words: rheumatoid arthritis, blastogenesis of lymphocytes,synovial lymphocytesClinical and laboratory findings suggested a relationship between cell-mediated immunity and joint lesions in chronic rheumatoid arthritis (RA) (1-3).To investigate the role of cell-mediated tissue damage in RA, the responsivenessof blood lymphocytes to nonspecific mitogens such as PHA and Con-A or topossible specific antigens such as denatured IgG and synovial tissue extracts hasbeen studied by a large number of workers. Howevers, no consistent resultshave been obtained: e.g. the response was reported normal by Kacaki et at. (4),Waxman et at. (5), and Menard et at. (6) ; increased by Ivanyi et at. (7) ; anddecreased by Griswold and McIntosh (8), Reynolds and Abdou (9), Lance andKnight (10), and Rawson et at. (11). The findings remain controversial.In contrast, there have only been a few reports of work using lymphocytestaken directly from the synovial cavity of damaged sites. Ivanyi et at. (7),Reynolds and Abdou (9), and Sheldon et at. (12) reported a decreased response toPHA. Our experience with synovial fluid of RA patients has revealed thatthe cytological picture differs widely among cases. In addition, the Hypaque-Ficoll separation technique used in previous studies is not sufficient to purifythe lymphocytes.In this paper, therefore, we used highly purified lymphocytes from synovial4411Yamana et al.: Low responsiveness of synovial and peripheral blood lymphocytes sProduced by The Berkeley Electronic Press, 1978442 S. YAMANA et ai.fluid and tested the responsiveness of a constant number of lymphocytes to PHAand Con-A. Low responsiveness was confirmed for both synovial fluid andblood lymphocytes. In a following paper, the screening of environmental factorswhich might cause this decreased response to nonspecific mitogens will be re-ported.J,lATIENTS AND METHODSPatients. Seventeen patients with definite and classical RA were studied.Only 10 cases yielded sufficient numbers of purified synovial lymphocytes (morethan 5 x 105 cells) for testing blastogenesis. All patients were under medication,mainly oral administration of small amounts of prednisolone (less than 10 mg aday) and non-steroid hormones. Some patients had been receiving intra-articularadministration of steroid hormones which was stopped at least 2 weeks beforecollection of synovial fluid.Purification Procedures. Synovial fluid was collected in the presence of hyalu-ronidase, 5 units/ml and sodium. heparin, 250 units/ml. This was then diluted3-fold in Hanks' BSS (balanced salt solution), and Hypaque-Ficoll centrifugationwas performed in the usual manner to remove the majority of the highly con-taminating polymorphonuclear cells. Cells at the interface were suspended inTissue Culture Medium-199 containing 10% (v/v) foetal calf serum (FCS) and in-cubated in a glass Petri-dish for 40 min at 37°C twice to remove adherent cells.After incubation, loosely adherent lymphocytes were recovered by agitation withpipetting. The differential cell count for the resultant lymphocyte-rich cell sus-pension was checked on cytocentrifuge preparations stained with May-Giemsa orPeroxidase. Peripheral blood lymphocytes were separated on Hypaque-Ficoll.All procedures were performed under sterile conditions.Blastogenesis. Lymphoid cell suspensions were adjusted to exactly 5 X 105cells/ml in TC-199 containing 10% FCS. Aliquots of 200 ttl were added to thewells of a Microplate II tissue culture plate. PHA-P (Bacto-phytohemagglutininP, Difco), 300 fI or Con-A (Sigma Co.), 2.0 fig was then added. The concentrationof mitogens used was determined from a dose-response curve which included 0.3,3.0 and 30 fig for PHA-P and 002, 2.0 and 20 t-tg for Con-A. Experiments wereperformed in triplicate for each preparation. Incubation was continued for 72 hin a CO2 incubator and 4 h before cell harvesting, I pCi of 3H-thymidine wasadded in each well. A Mini-MASH automatic cell harvester was used to collectand wash the cells and radioactivity as counts per minute (cop.m) was counted ina Liquid Scintillator. Nor~al peripheral blood lymphocytes were used as con-trols under exactly the same conditions.T and B cell counts. T cells were counted by spontaneous sheep red blood cell(SRBC) rosette formation, and B cells by the presence of surface immunoglobulins(S-Ig). For detecting S-Ig, e;oat anti-hUman heavy chain (i, (x, fI-chain) mono-specific antibody labelled with FITC (Hyland) was used. Briefly, after the puri-fication procedure, approximately I x 105 cells were incubated with I drop ofconjugate (centrifuged at IO,OOOg before use) at room temperature for 30 min,I2Acta Medica Okayama, Vol. 32 [1978], Iss. 6, Art. 8http://escholarship.lib.okayama-u.ac.jp/amo/vol32/iss6/8Reduced Blastogenesis of Lymphocytes in RA 443followed by 3 washes in PBS containing 0.02% (w/v) NaN3• Stained cells weremounted on a glass slide in phosphate-buffered glycerol and observed by con-ventional fluorescent microscopy.RESULTSFigure 1 shows the cell type distribution after purification. Larger closedcircles indicate the cases used in this experiment. Smaller circles are the caseswhich could not supply sufficient cells for testing blastogenesis. Six out of tencases yielded more than 80 % mononuclear cells; other cases still contained largenumbers of neutrophils and macrophages even after purification. These resultswere supported by peroxidase staining (lower column).10 20 30 40 50 60 70 80 90 100 (%)Lymphoid• • .........mononuclear cellMacrophage :I-:::NeutrophilUnidentified cell ....Perioxidase R (-) • .. • :.."'._IFig. 1. Proportion of lymphoid mononuclear cells from synovial fluid of 17 RA patientspurified by Hypaque-Ficoll sedimentation and double absorption to a glass Petri-dish, largerclosed circles indicate cases used in this experiment.30.01.5 3.0~P-i 3(l).~"1ji 2..,±'"4-<o~ 1....,:5'+~';::; 4x15.0PHA-P (jIg/well)Fig. 2. Dose-respnse curve for PHA-P. Peripheral blood lymphocytes, I X 105 cells ineach well were incubated with I pCi 3H-thymidine.3Yamana et al.: Low responsiveness of synovial and peripheral blood lymphocytes sProduced by The Berkeley Electronic Press, 1978444 S. YAMANA et ai.0.1 0.5 1.0 2.0 3.0 4.0 5.0Con-A lPg/well)Fig. 3. Dose-response curve for Con-A. Peripheral blood lymphocytes, I X 105 cells ineach well were incubated with I pCi 3H-thymidine.06 0000050000<t'-"8'0.--< 4x~~ 0~ 000Q) 0 0~ 3 0 0.,..,'"Cl 0 b.,..,]-....,.i4-< 00 0Q) 0~ttl....,:5" • 001 • 0• 000• ~o.:. !e • ••J:. .:. ••••••N B S N B S44943 12410 1148 19703 4071 1150t10963 ±8642 ±1039 t7630 ±2613 ±1268PHA-P Con-AFig. 4. Blastogenesis of synovial fluid and peripheral blood lymphocytes to PHA-P andCon-A. N, normal peripheral blood lymphocytes; B, blood lymphocytes of RA; S, synovialfluid lymphocytes of RA; open circle (0), before adhension to glass dish; closed circle (el ;after adhension to glass dish.4Acta Medica Okayama, Vol. 32 [1978], Iss. 6, Art. 8http://escholarship.lib.okayama-u.ac.jp/amo/vol32/iss6/8Reduced Blastogenesis of Lymphocytes in RA 445In Figs. 2 and 3, dose-response curves for PHA-P and Con-A are given.PHA-P showed maximal response at a concentration of 3.0pg/1 X 105 cells andCon-A at 20 ftg/l X 105 cells.Fig. 4 summarizes the results. Blood and synovial lymphocytes showedmarkedly low responsiveness to both PHA-P and Con-A; approximately ten-foldfor synovial lymphocytes compared with that of control blood lymphocytes.Table I shows the data for each case. The proportion of T cells in synovialfluid correlated well with response to mitogens except for case No.3. B cellproportions varied from 4.3% to 63.7% and did not correlate with mitogenresponsiveness.TABLE 1. BLASTOGENESIS, CELL DIFFERENTIATION AND T, B CELL SUBPOPULATIONSIN SYNOVIAL FLUID OF RA PATIENTSBlastgenesisa Mononue1ear Perioxidase T cell B cellPatients Class Stage PHA Con-A lymphoid cells reaction ( +) (%) (0/0)(3.0) (2.0) (%) (0/0)1 Y.M. ]I[ rV 1086 1655 92.0 7.9 35.0 17.82 S. T. ][ N 1498 90.8 14.6 40.6 11. 63 M.K. I N 228 37 50.0 44.6 41. 0 37.44 Y. U. ][ I 157 141 71. 9 28. 15 T. C. ][ III 682 95. 7 3.66 M.O. ][ N 2854 16627 T. Y. II I 1939 897 56.4 36.2 61. 8 26.28 H. S. I ][ 2634 3543 89.9 9.0 53.0 4.39 Y. S. I N 250 112 21. 0 85.1 14.8 67. 710 K. S. ][ N 151 97.0 0-------_...a Blastogenesis was measured as described in methods.Data are expressed as c.p.m. of 3H-thymidine incorporated.DISCUSSIONChronic rheumatoid arthritis is characterized by the infiltration of lymphoidmononuclear cells in the synovial tissue which might cause joint destruction (I,3). Functional analysis of these cells has been carried out by many workers. Inthe present study, the biological activities of lymphocytes from synovial fluidwere assessed by the incorporation of 3H-thymidine after stimulation with non-specific T cell mitogens. For this, it was necessary to purify lymphocyte pre-parations from contaminating macrophages and neutrophils. In many previouslyreported studies, the Hypaque-Ficoll sedimentation technique alone was used.This technique, however, is not sufficient to remove the large contamination withmacrophages and even neutrophils found in synovial fluid. Further adhesion toglass Petri-dish was required to remove these cells. Even though this purifica-5Yamana et al.: Low responsiveness of synovial and peripheral blood lymphocytes sProduced by The Berkeley Electronic Press, 1978446 S. YAMANA et at.tion technique was applied to synovial fluid in this study, some cases e.g. caseNo.9 still showed a low proportion of lymphocytes. Therefore, in the presentexperiment, the cell concentration was adjusted to give a constant total numberof lymphocytes for the blastogenesis to mitogens. Early death of contaminatingmacrophages and neutrophils during the 72 h incubation did not influence theresults.Ivanyi et at. (7) reported an increased response of RA synoviallymphoeytesto B cell mitogens such as PWM and LPS, but a decreased response to T cellmitogens such as PHA. Sheldon et at. (12) also reported a decreased response toPHA, approximately one quarter of that of normal blood lymphocytes. Ourdata showed a response approximately one tenth of normal. The average responsefor triplicate experiments varied case by case. Lymphoid cell suspensions for casesNo.5 and 10 contained more than 95 % lymphocytes before incubation andshowed a markedly low responsiveness. It is not clear whether the response wasreally low or was reduced by the lack of macrophages in these particular prepara-tions (13). Blood lymphocytes also showed a reduced response to PHA and Con-A. Our data confirm the results of previous studies (9, 11, 12, 14).Why do lymphocytes in RA show a low response to mitogens? Several ex-plantations can be considered. Low complement levels and the presence ofrheumatoid factor in the synovial fluid have been reported. These findingssuggest the presence of immune complexes which might cause a lowered respon-siveness to mitogens. Large amounts of synoval tissue antigens and denaturedIgG are also present in the synovial fluid which could have already stimulatedthe lymphocytes in vivo, thus resulting in poor in vitro blastogenesis to nonspecificmitogens. In addition, therapeutic drugs might cause a lowered response tomitogens. In this experiment, an attempt was made to avoid o;;uch an effect byceasing the administration of drugs at least two weeks before collection of the testsamples. Reduced biological function of synovial lymphocytes does not denytheir role in synovial tissue destruction. In fact, Stastny et at. (2) reported lym-phokine activity in synovial fluid. The lymphocytes, therefore, might have losttheir biological activity in vivo after releasing lymphokines. Data on blood lym-phocytes in RA, however, might suggest the presence of humoral factors whichdirectly cause a lowered responsiveness.Reported T and B lymphocyte proportions in synovial fluid vary widely (15,16). Sheldon et at. (12) found increased T cell and decreased B cell proportions.In contrast, Venon-Robert et at. (17) reported decreased T cells and increased Bcells. This discrepancy may be explained by differences in the purity of testlymphocyte suspensions, the nature of the conjugated anti-immunoglobulin usedor by the presence of exogenous cytophilic immunoglobulin on the cell surface.Winchester et at. (19) recommended the use of conjugated anti-F (ab')2 to avoid6Acta Medica Okayama, Vol. 32 [1978], Iss. 6, Art. 8http://escholarship.lib.okayama-u.ac.jp/amo/vol32/iss6/8Reduced Blastogenesis of Lymphocytes in RA 447binding via the Fc receptor. This is, however, still controversial. To removethe majority of exogenous immunoglobulin in this study, lymphocyte suspensionswere incubated at 37°C for I h and washed before adding the conjugate.In a subsequent paper, humoral factors in the synovial fluid which mightaffect lymphocytes will be examined.Acknowledgment. We wish to thank Dr. J. M. Rolland, Department of Pathology and Im-munology, Alfred Hospital, Prahran, Victoria, Australia for scientific discussion and assistancewith the completion of this manuscript.REFERENCES1. Ziff, M.: Relation of cellular infiltration of rheumatic synovial membrane to its im-mune response. Arthritis Rheum. 17, 313, 1974.2. Stastny, P., Rosenthal, M., Andreis, M. and Ziff, M.: Lymphokines in the rheumatoidjoint. Arthritis Rheum. 18, 237, 1975.3. Bankhust, A. D., Husby, G., Williams, Jr. R. C.: Predominance of T cells in the lympho-cytic infiltrates of synovial tissues in rheumatoid arthritis. Arthritis Rheum. 19, 555, 1976.4. Kacaki, J. N., Bullock, W. E. and Vaughan, J. H.: Failure of lymphocyte transformationin response to gamma globulin in rheumatoid arthritis. Lancet I. 1298, 1969.5. Waxman, j., Lockshin, M. D. and Schnapp, j. j.: Cellular immunity in rheumatic diseases.Arthritis Rheum. 16, 499, 1973.6. Menard, H. A., Haim, T. and Peltire, A. P.: In vitro studies on peripheral blood leuko-cytes in rheumatoid arthritis. Ann. rheum. 33, 361, 1974.7. Ivanyi, L., Lehner, T. and Burry, H. c.: The response of synovial fluid lymphoeytes toT & B stimulants in vitro. Immunology 25, 90S, 1973.8. Griswold, W. R. and McIntosh, R. M.: Lymphocyte responses to phytohemagglutinin inrheumatoid arthritis and glomerulonephritis and the effects of immunosuppression.Experimentia 15, 606, 1973.9. Reynolds, M. D. and Abdou, N. I.: Comparative study of the in vitro proliferative re-sponce of blood and synovial fluid leukocytes of rheumatoid arthritis patients. ]. Clin.Invest. 52, 1627, 1973.10. Lance, E. M. and Knight, S. C.: Immunologic reactivity in rheumatoid arthritis. ArthritisRheum. 17, 513, 1974.11. Rawson, A. J. and Huang, T. C.: Lymphocyte populations in rheumatoid arthritis. Ar-thritis Rheum. 19, 720, 1976.12. Sheldon, P. j., Papamichail, M. and Holborrow, E. j.: Studies on syovial fluid lympho-cytes in rheumatoid arthritis. Ann. Rheum. Dis. 33, 509, 1974.13. Rosenstreich, D. j., Farrar, j. j. and Dougherty, S. : Absolute macrophage dependency ofT lymphocyte activation by mitogens. ]. Immunol. 116, 131, 1976.14. Silverman, H. A, Johnson, J. S., Vaughan, J. H. and McGlamory, J. C.: Altered lympho-cytes reactivity in rheumatoid arthritis. Arthritis Rheum. 19, 509, 1976.15. Utsinger, P. D.: Lymphocyte responsiveness in systemic lupus erythematosus. ArthritisRheum. 18, 595, 1975.16. Winchester, R. j., Bentwick, Z. H. and Kunkel, H. G.: Alteration in the proportion of Band T lymphocytes in rheumatoid arthritis joint fluids with low complement and in-creased complexes. Arthritis Rheum, (Abstr), 16, 138, 1973.17. Vernon-Roberts, B., Curney, H. L. F. and Perrin j.: T and B cells in the blood and syno-vial fluid of rheumatoid patients. Ann. Rheum. Dis. 33, 430, 1974.7Yamana et al.: Low responsiveness of synovial and peripheral blood lymphocytes sProduced by The Berkeley Electronic Press, 1978

Low responsiveness of synovial and peripheral blood lymphocytes stimulated by either PHA-P or CON-A in patients with chronic rheumatoid arthritis

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