SUMMARY Synovial effusions from 14 patients with rheumatoid arthritis contain factors capable of enhancing and suppressing the production of immunoglobulin when added to normal peripheral lymphocyte cultures in vitro. Utilizing a radioactive immune coprecipitation technique it could be demonstrated that enhancement was observed upon addition of relatively small amounts of synovial fluid, while addition of higher amounts resulted in suppression of immunoglobulin synthesis. Nine of ten non-rheumatoid joint effusions showed neither enhancing nor suppressing activities. The possible relationship of these events to lymphokine production in the joint is discusse

ROSENTHAL, M.

RERO DOC Digital Library

Rheumatology and Rehabilitation, 1976, 15, 71ORIGINAL PAPERSUPPRESSION AND STIMULATION OFIMMUNOGLOBULIN SYNTHESIS BY AGENTS PRESENTIN RHEUMATOID SYNOVIAL EFFUSIONSBY M. ROSENTHALRheumatologische UniversitStsklinik, Felix Platter-Spital, Burgfelderstrasse 101, CH-4055Basel, SwitzerlandSUMMARYSynovia! effusions from 14 patients with rheumatoid arthritis contain factors capable of enhan-cing and suppressing the production of immunoglobulin when added to normal peripherallymphocyte cultures in vitro. Utilizing a radioactive immune coprecipitation technique it could bedemonstrated that enhancement was observed upon addition of relatively small amounts ofsynovial fluid, while addition of higher amounts resulted in suppression of immunoglobulinsynthesis. Nine of ten non-rheumatoid joint effusions showed neither enhancing nor suppressingactivities. The possible relationship of these events to lymphokine production in the joint isdiscussed.THE participation of immune mechanisms in the pathogenesis of rheumatoid arthritis(RA) is well established (Pekin et al., 1964; Smiley et al, 1968; Winchester et al, 1969;Rodman et al., 1967). Recently, we described the presence in rheumatoid synovialeffusions of enhancing factors capable of stimulating immunoglobulin synthesis inperipheral-blood lymphocyte cultures in vitro (Rosenthal et al., 1976). The factorsdescribed are a low-molecular factor which elutes from an Agarose A 5m column withserum albumin, presumably a lymphokine, and a high-molecular factor, which elutesfrom such a column with an IgM marker, presumably an immune complex. The low-molecular factor is non-specific and stimulates immunoglobulin synthesis in bothrheumatoid and normal lymphocyte cultures. The high-molecular factor showedspecificity in its exclusive stimulation of rheumatoid cells. Recently, evidence has accumu-lated that in the collaboration between T-B cells and macrophages, T cells not only havean enhancing effect on antibody response, but also have a suppressive effect on antibodyformation (Baker et al., 1973; Hardin et al, 1973; Dutton, 1973; Herzenberg et al.,1973; SjSberg, 1972). The experiments presented here were designed to investigate thepossibility that not only a B-cell enhancing function, but also B-cell suppression functionis mediated by soluble factors. Such B-cell inhibitory factors may be produced in vivoby the inflamed synovium in rheumatoid arthritis and be present in the synovial effusion.MATERIALS AND METHODSKnee-joint effusions from 14 patients with classical or definite rheumatoid arthritis(RA) as defined by the A.R.A. criteria (Ropes et al., 1959) and from 10 non-rheumatoidpatients were obtained. The non-rheumatoid patients included six with osteoarthritis,two with ankylosing spondylitis, one with Reiter's syndrome and one with post-traumaticeffusion.The effusions were immediately centrifuged at 2500 rev./min for 20 minutes at roomtemperature to remove cellular elements and the cell-free effusions treated with 0.02mg of hyaluronidase/ml of joint fluid (bovine testes hyaluronidase, type I, Sigma Chem.Accepted for publication August 1975.7172 RHEUMATOLOGY AND REHABILITATION VOL. XV NO. 2Corp., St. Louis, Mo.) for 2 hours at 37°C. The treated synovial effusions were centrifugedagain and then either tested immediately or frozen at —70°C until tested.Lymphocyte cultures were prepared from heparinized peripheral blood obtainedfrom eight different normal donors utilizing the Ficoll-Hypaque gradient separationprocedure (Boyum, 1968). Five to 15 x 106 lymphocytes were cultured in 2 ml of RPMI1640 medium buffered with Hepes buffer (Gibco, Grand Island, N.Y.) and supplementedwith 10% heat-inactivated fetal calf serum (FCS) and antibiotics (Penicillin 100 u/ml,Streptomycin 100^g/ml). Upon addition of prepared synovial fluid, lymphocyte cultureswere incubated at 37°C in 95 % air-5 % CO2. After 48 hours, cultures were centrifugedat 800 rev./min for 10 minutes and the medium containing all added materials removedand replaced by 2 ml of special minimal essential medium (MEM) (Gibco, Grand Island,N.Y.) lacking four essential amino acids and containing 5% pooled, heat-inactivatedhuman AB serum and antibiotics. To each culture, 5 fid of 14C-labelled amino acidmixture (Schwarz-Mann, Orangeburg, N.Y.) was added containing the four aminoacids L-arginine (specific activity 312 mCi/mmol), L-leucine (s.a. 312 mCi/mmol), L-lysine(s.a. 310 mCi/mmol) and L-valine (s.a. 258 mCi/mmol) missing in the special MEM, eachat a concentration of 250 nCi/ml. The cultures were then incubated under the sameconditions for another 24-hour period. On termination of culture, all cellular elementswere removed by ultracentrifugation at 35 000 rev./min for 1 hour and the supernatantsthen dialysed against four changes of 0.01 M-phosphate-buffered saline (PBS), pH 7.2.The determination of the incorporation of uC-amino acids into new synthesizedimmunoglobulin was performed according to the method of Jasin and Ziff (1969) asmodified by Jasin and Ziff (1975). In short, aliquots of 0.1 ml of the supernatants weretaken for the determinations of total radioactivity, the TCA-precipitable radioactivity,the radioactivity present in the immune precipitate formed in slight antibody excess withpolyvalent goat anti-human immunoglobulin antiserum (Meloy Labs., Springfield, Va.)and in a nonspecific precipitate formed in equivalence by egg albumin-anti-egg albumin.The counts obtained for the nonspecific precipitation represented about 2-5 % of thetotal counts and were subtracted from the counts obtained for the immune precipitate.The precipitates were counted upon addition of 1 ml of 0. lM-NaOH, 2 ml of saline and10 ml of Aquasol (New England Nuclear, Boston, Mass.) in a Beckman fluid scintillationcounter. Cell viability counts performed at the beginning and in some experiments at theend of the cultures were done utilizing the trypan blue dye exclusion technique.RESULTSThe effect of synovial effusions on cell viability counts and protein synthesis of normallymphocyte culturesDifferent peripheral lymphocyte cultures from different normal donors were incuba-ted in the presence of three different concentrations of six rheumatoid and four non-rheumatoid joint effusions. After 72 hours of incubation, cell viability counts using thetrypan blue exclusion technique were performed and are given in Fig. 1. It can be seenthat three different amounts of either rheumatoid or non-rheumatoid synovial effusionspresent in the lymphocyte culture did not change the cell viability counts significantly.Similar findings are observed in the estimation of protein synthesis as measured bythe counts of U C precipitated with 5 % TCA in 0.1 ml of culture supernatant followingan extensive dialysis (Fig. 2). The estimated protein synthesis shows no significant changefor the different concentration of effusions added, suggesting that not only is viabilitynot jeopardized, but also no toxic effect is exerted upon the lymphocyte cultures. TheROSENTHAL: IMMUNOGLOBULIN SYNTHESIS 73apparent trend toward a decreased incorporation of the uC-label in the TCA precipitatewas not present upon examination of the individual lymphocyte cultures.I.90m>80i -§£70RA NonRA RA NonRA RA NonRA0.01ml 0.05ml 0.25 mlSYNOVIAL EFFUSIONFio. 1.—Percentage viability at termination of cultures as determined by the trypan blue dyeexclusion technique. All determinations were performed hi duplicate. There is no significantstatistical difference between the values obtained for RA and non-RA synovial effusions con-taining cultures.oooo252015••••: •—•o o• 1—t•e ••••t 1 I t•*••••oo1 IRA NonRA RA NonRA RA NonRA0.01ml 0.05 ml 0.25mlSYNOVIAL EFFUSIONFIG. 2.—Counts per minute obtained for the TCA-precipitable material in 0.1 ml of culturesupernatant. All determinations were performed in duplicate. There is no significant statisticaldifference between the values obtained for RA and non-RA synovial effusions containingcultures.74 RHEUMATOLOGY AND REHABILITATION VOL. XV NO. 2The effect of different amounts ofsynovial effusions on immunoglobulin synthesis of normallymphocyte culturesUtilizing a radioactive immune coprecipitation assay, immunoglobulin determina-tions of normal lymphocyte cultures in the presence of different amounts of joint effusionswere performed (Table). Immunoglobulin synthesis in the presence of varying amountsTABLETHE EFFECT OF DIFFERENT AMOUNTS OF RHEUMATOID AND NON-RHEUMATOID SYNOVIAL EFFUSIONSON IMMUNOGLOBULIN SYNTHESIS IN NORMAL PERIPHERAL BLOOD LYMPHOCYTESAmount ofeffusionadded0.25 ml0.20 ml0.10ml0.05 ml0.01 ml0.005 mlMediumRheumatoid arthritis1ct./min±s.D.2597 ±97(P <0.01)1190±489(N.S.)1783±33O(P <0.01)2027±880(P <0.01)3125±946(P <0.01)1962±879(P <0.01)Non-rheumatoid arthritis1S.I.±S.D.8 ct./min ±S.D.4O.75±0.13(P = 0.05)1.14±0.30(N.S.)1.77±0.17(P <0.001)2.14±0.88(P <0.001)3.56±1.17(P <0.001)1.87±0.65(P <0.01)902 ±145820±118(N.S.)920±55(N.S.)827 ±147(N.S.)852±79(N.S.)1009±260(N.S.)83O±197(N.S.)S.I.±S.D.8O.88±O.ll1.06±0.170.93±0.150.99±0.131.20±0.420.95±0.241 Fourteen different hyaluronidase-treated synovial effusions from rheumatoid arthritispatients and 10 synovial effusions from non-rheumatoid patients were assayed on eight differentnormal lymphocyte donors. The non-rheumatoid fluids were obtained from two patients withankylosing spondylitis, six with osteoarthritis, one with Reiter's syndrome and one with post-traumatic effusion. All experiments were performed in duplicate.2 Counts per minute per 0.1 ml of culture supernatant. The significance is determined ascompared to medium control._ _, . , . T , ct./min experimental culture8 Stimulation Index = ct./min medium controlThe significance determined between rheumatoid and non-rheumatoid effusions containingcultures.of non-rheumatoid effusion shows no significant changes. However, rheumatoid synovialeffusions exert profound effects on the production of immunoglobulins. Low concentra-tions, such as 0.005 ml added to 2 ml lymphocyte cultures are already actively enhancing.Maximal stimulation is present in cultures to which 0.01 ml of joint effusions was added.Increasing amounts of rheumatoid synovial fluids added to the cultures reduced thestimulatory effect with, eventually, no immunoglobulin synthesis upon addition of 0.25ml of synovial fluid.ROSENTHAL: IMMUNOGLOBULIN SYNTHESIS 75DISCUSSIONThe rheumatoid synovium is a playground for multiple immunological and meta-bolic events resulting in a chronic perpetuative inflammation. Both humoral and cellularimmune mechanisms have been shown to be involved in this process (Pekin et al., 1964;Smiley et al, 1968; Winchester et al, 1969; Rodman et al, 1967; Bacon et al., 1973;Waxman et al., 1973; Rothenberger and Thiele, 1970; Clot et al, 1973). It is thereforeto be expected that synovial effusions will contain material released during such events.In earlier publications, we have demonstrated that mediators of delayed hypersensitivitycan be found in rheumatoid synovial effusions (Stastny et al, 1975) and intra-articularinjections of such mediator preparations evoke the development of a chronic synovitisresembling to some extent rheumatoid arthritis (Andreis et al, 1974). In a recent publica-tion we adduced evidence for the presence of factors enhancing the synthesis of immuno-globulins in rheumatoid joint effusions (Rosenthal et al, 1976).The immune response to certain antigens is controlled not only by a T cell populationwith enhancing capacity on immunoglobulin synthesis, but also by a subpopulation ofT cells with suppressive effect on antibody response. Recent work done by Katz, Pauland Benacerraf (1973) interpreted the observation of inhibition of antibody response asdue to release of soluble inhibitory mediators most likely by T lymphocytes. In thiscommunication, evidence was brought that the two antagonistic activities can bedemonstrated in synovial fluids produced in vivo. To whai extent suppressive or enhancingactivities are manifested depends upon the dilution of the synovial effusion while exposedto lymphocyte cultures. The presence of 12.5 % rheumatoid synovial fluids in the lympho-cyte cultures inhibits the immunoglobulin synthesis specifically without exerting anydetectable toxic effect on the viability or impairment of the protein synthesis in thelymphocytes. Subsequent dilution of the synovial fluid gives rise to extensive productionof immunoglobulin reaching an optimum concentration of 0.5 % of synovial effusionpresent in the lymphocyte cultures. The effect observed here does not seem to be mediatedby a specific factor because it can be demonstrated in normal, unsensitized lymphocytes.The question of whether the suppression or stimulation of immunoglobulin synthesis ismediated by the same factor in different concentrations or by two different agents is thesubject of further studies.REFERENCESANDREIS, M., STASTNY, P. and ZIFF, M. (1974) "Experimental Arthritis Produced by Injection ofMediators of Delayed Hypersensitivity". Arthritis Rheum., 17,537.BACON, P. A., BLUESTONE, R., CRACCIOLO, A. and GOLDBERG, L. S. (1973) "Cell-mediatedImmunity to Synovial Antigens in Rheumatoid Arthritis". Lancet ii, 699.BAKER, P. J., REED, N. D., STASHAK, P. W., AMSBAUGH, DIANA, F. and PRESCOTT, B. (1973)"Regulation of the Antibody Response to Type HI Pneumococcal Polysaccharide: I. Natureof Regulatory Cells". / . Exp. Med., 137,1431.BOYUM, A. (1968) "Isolation of Mononuclear Cells and Granulocytes from Human Blood".Scand. J. Clin. Lab. Invest., 21,97.CLOT, J., SANY, J. and SERRE, H. (1973) "Cellular Immunity in Rheumatoid Arthritis: L Study ofthe Response to Undenatured IgG". Pathologie-Biologie, France, 21,465.DUTTON, R. W. (1973) "Inhibitory and Stimulatory Effects of Concanavalin A on the Responseof Mouse Spleen Cell Suspensions to Antigen: n . Evidence for Separate Stimulatory andInhibitory Cells". / . Exp. Med., 138,1496.HARDIN, J. A., CHUSED, T. M. and STEINBERG, A. D. (1973) "Suppressor Cells in the Graft v.Host Reaction". / . Immunol, 111, 650.HERZENBERG, LEONORE, A., CHAN, EVA, L., RAVITCH, MYRNICE, M., RIBLET, R. J. and HERZEN-BERG, LEONARD, A. (1973) "Active Suppression of Immunoglobulin Allotype Synthesis:HI. Identification of T Cells as Responsible for Suppression by Cells from Spleen, Thymus,Lymph Node and Bone Marrow". / . Exp. Med., 137,1311.76 RHEUMATOLOGY AND REHABILITATION VOL. XV NO. 2JASIN, H. E. and ZIFF, M. (1969) "Immunoglobulin and Specific Antibody Synthesis in ChronicInflammatory Focus: Antigen Induced Synoyitis". / . Immunol., 102,355.and (1975) "Immunoglobulin Synthesis by Peripheral Blood Cells in Systemic LupusErythematosus". Arthritis Rheum., 18,219.KATZ, D. H., PAUL, W. E. and BENACERRAF, B. (1973) "Carrier Function in Anti-hapten Anti-body Responses: VI. Establishment of Experimental Conditions for either Inhibitory orEnhancing Influences of Carrier-specific Cells on Antibody Production". / . Immunol., 110,107.PEKOJ, T. J. and ZVAIFLER, N. J. (1964) "Hemolytic Complement in Synovial Fluid". / . Clin.Invest., 43, 1372.RODMAN, W. S., WILLIAMS, R. C , BILKA, P. J. et al. (1967) "Immunofluorescent Localization ofthe Third and Fourth Component of Complement in Synovial Tissue from Patients withRheumatoid Arthritis". / . Lab. Clin. Med., 69, 141.ROPES, M., BENNETT, G. A., COBB, S., JACOX, R. and JESSAR, R. (1959) "Diagnostic Criteria forRheumatoid Arthritis". Bull. Rheum. Dis., 9,175.ROSENTHAL, M., STASTNY, P. and ZIFF, M. (1976) "Enhancement of Immunoglobulin and Anti-immunoglobulin Synthesis by Fractions of Rheumatoid Synovial Fluid and SynovialTissue Culture Supematants". In the press.RoTHENBERGER, W. and THIELE, H. G. (1970) "In vitro Studien zur Pathogenese der primar-chronischen Polyarthritis mittels des Migrations-Inhibitionstests". Klin. Wochenschr. 1308.SJOBERO, O. (1972) "Effect of Allogeneic Cell Interaction on the Primary Immune Responsein vitro. Cell Types Involved in Suppression and Stimulation of Antibody Synthesis".Clin. Exp. Immunol., 12, 365.SMILEY, J. D., SACKS, C. and ZIFF, M. (1968) "In vitro Synthesis of Immunoglobulin by Rheuma-toid Synovial Membrane". J. Clin. Invest., 47,664.STASTNY, P., ROSENTHAL, M., ANDREIS, M. and ZIFF, M. (1975) "Lymphokines in the Rheuma-toid Joint". Arthritis Rheum., 18, 237.WAXMAN, J., LOCKSHIN, M. D., SCHNAPP, J. J. and DONESON, IRA N. (1973) "Cellular Immunityin Rheumatic Disease: I. Rheumatoid Arthritis". Arthritis Rheum., 16,499.WINCHESTER, R. J., AONELLO, V. and KUNKEL, H. (1969) "The Joint Fluid Gamma-globulinComplexes and their Relationship to Intra-articular Complement Diminution". Ann. N. Y.Acad. Sci., 168, 195.

SUPPRESSION AND STIMULATION OF IMMUNOGLOBULIN SYNTHESIS BY AGENTS PRESENT IN RHEUMATOID SYNOVIAL EFFUSIONS

Abstract

Similar works

Full text

Available Versions

RERO DOC Digital Library