Clin. exp. Immunol. (1990) 82, 499-503

Cytolytic activity in T cell clones derived from human synovial rheumatoid membrane: inhibition by synovial fluid A. M. M. MILTENBURG, J. M. VAN LAAR, P. DE KUIPER, M. R. DAHA* & F. C. BREEDVELD Departments of Rheumatology and *Nephrology, University Hosptial, Leiden, The Netherlands

(Acceptedfor publication 14 June 1990)

SUMMARY A panel of T cell clones was derived from the synovial membrane of a patient with rheumatoid arthritis (RA). We investigated whether T cell clones with cytolytic properties were present and whether T cell cytotoxicity was influenced by the presence of synovial fluid. These issues were studied using anti-CD3 and lectin-induced cytotoxicity assays. The majority of the T cell clones derived from the synovial membrane showed cytotoxic properties although non-cytotoxic clones were also found. Three clones (Nil N6 and N15) showed strong cytotoxicity (more than 40% lysis at an effector-totarget cell ratio of 10: 1) whereas three clones (N 16, N4 and N 14) were non-cytotoxic (less than 20% lysis at an effector-to-target cell ratio of 10: 1). The induction of cytotoxicity in the anti-CD3-driven system was shown to be dependent on the dose of anti-CD3 present. When synovial fluid was added to these assays a strong inhibition of cytotoxicity was found. This inhibition of cytotoxicity was found with synovial fluid samples of RA patients, as well as with non-RA synovial fluids. Both anti-CD3 and lectin-dependent cytotoxicity assays were strongly inhibited. In conclusion, T cell clones with cytotoxic activity can be isolated from rheumatoid synovial membrane. In the presence of synovial fluid these cytotoxic cells are inhibited to exert their cytotoxic function. ,

Keywords rheumatoid arthritis T cell clones cytotoxicity synovial fluid

INTRODUCTION

1978; Keystone et al., 1988; Hovdenes et al., 1989b) have been found. However, no data are available concerning functional properties of clonal populations of T cells derived from the synovial membrane of RA patients. We therefore generated a panel of T cell clones derived from the synovial membrane and investigated whether T cell clones with cytolytic properties may be present at the site of inflammation. Since we do not know the target antigen of the T cell clones that were generated, we started to investigate their cytotoxic potential by means of non-specific cytotoxicity induction, using anti-CD3 and lectin-dependent cellular cytotoxicity (Leeuwenberg et al., 1985; Spits et al., 1985; Miltenburg et al., 1987a). Because T cells with similar function may also be found in the synovial fluid, we investigated whether the cytotoxic function of these T cell clones was influenced by micro-environmental factors present in synovial fluid samples.

The inflammatory cells that are predominantly present in the synovial membrane of patients with rheumatoid arthritis (RA) have been demonstrated to be of T cell origin (Abrahamsen et al., 1975; van Boxel & Paget, 1975; Meijer et al., 1982). A large proportion of these tissue-infiltrating T cells may be activated since they react with monoclonal antibodies (MoAbs) directed against cell surface molecules that are absent on resting T cells, such as HLA-DR or the Tac antigen (CD25) (Cush & Lipsky, 1988; Hovdenes et al., 1989a). These activated T cell populations may be present at the site of inflammation as the result of an active ongoing immune response directed against putative autoantigens. When cytotoxic T cells are present, lysis of the autoantigen-presenting cells may occur, possibly resulting in tissue damage (Koga et al., 1989). Studies on T cell function in RA have been performed using whole T cell populations or mononuclear cell suspensions derived from the synovial fluid, the synovial membrane or from the peripheral blood. Alterations in responsiveness to mitogens, anti-CD3 MoAb and interleukin-2 (IL-2) (Burmester et al.,

Patient

Correspondence: Dr A. M. M. Miltenburg, University Hospital Leiden, Department of Rheumatology, Building 1, C2-Q, P.O. Box 9600, 2300 RC Leiden, The Netherlands.

Synovial membrane was obtained during knee replacement. The patient (a 58-year-old woman) was diagnosed as having a classical, seropositive RA in 1974. She was treated with antimalarial drugs at the time of the operation.

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MATERIALS AND METHODS

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T cell clones and lines used in this study Synovial-tissue-derived T cell clones were generated using an anti-CD3-driven cloning and propagation system. Synovial tissue fragments were placed in 24-well tissue culture plates (Costar 3524, Cambridge, MA) in Iscove's modified Dulbecco's medium (IMDM) supplemented with antibiotics (penicillin 100 U/mI, streptomycin 100 pg/ml; Boehringer Mannheim, Mannheim, FRG), fetal calf serum (FCS, 10%) and 5%o v/v T cell growth factor (TCGF) (Miltenburg et al., 1987b). This TCGF concentration (5% v/v) corresponds to approximately 102 U/ml interleukin-2 (IL-2). Growing cells were separated from the tissue fragments and cloned by limiting dilution. Cells were cloned at 0-5 cell/well in a feeder mixture consisting of 30-Gy-irradiated third-party peripheral blood mononuclear cells (PBMC) (106/ml), OKT3 ascites (10-5 dilution), and 500 v/v TCGF (modified according to Londei et al., 1988). Cloned T cells were expanded using the above-described feeder cell mixture at a cell concentration of 2 x 105 responder cells/well in 24-well plates. Restimulation was performed every 10-14 days and in between those periods T cell clones were maintained in culture in the presence of 5%. v/v TCGF. The T cell clones studied here in detail were clone N6 (CD3 +CD4- CD8 +yTTcRVI5- V8- ), clone N15 (CD3+CD4 -CD8+y6TcR -V15 -V8+) and clone N I (CD3+CD4 -CD8+ocfTcR - VJ15 V#8 ). T cell lines

initiated from the peripheral blood of healthy volunteers by stimulation with OKT3 ascites (10-5 dilution) and were expanded using 5% TCGF, essentially as described for T cell clones. were

Cytotoxicity assays The natural killer (NK) sensitive target cell line K562 was cultured in IMDM (GIBCO, Grand Island, NY) in T75 tissue culture flasks (Greiner, Alphen aan den Rijn, The Netherlands). These cells were used in a standard 4-h chromium release assay (Miltenburg et al., 1987a). Briefly, cells were labelled with 100 pCi 51Cr-sodium chromate (New England Nuclear, Boston, MA), washed, and plated at 5 x 103 target cells/well. Effector cells were added at 50: 1, 10: 1, 2: 1 and 1: 1 effector-to-target cell ratios. Test plates were incubated for 4 h at 37 C 500 CO2. In all assays a positive (1% Triton X-100) and negative (medium) control was included, and all samples were tested in triplicate. The percentage of specific release was determined as follows: ct/min test sample- ct/min control ct/min Triton X-100-ct/min control

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T cell clones were tested for their ability to lyse K562 cells in the presence or absence of inducing agents. The concentrations of

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Fig. 1. Dose-dependent induction of cytotoxicity by anti-CD3 antibody. The synovial-membrane-derived T cell clones N6 (5) and N 15 (-) were tested for induction of cytolytic activity in the presence of various dilutions of anti-CD3 antibody. No natural killer cell activity was found (see medium control: less than 10° specific lysis). The effector-to-target cell ratio was 10: 1.

Fig. 2. Inhibition of anti-CD3-induced cytotoxicity with a series of rheumatoid arthritis synovial fluid samples from patients 79 (U), 80 (E3), 81 (0), 82 (DJ) and 83 (U). The synovial membrane derived T cell clones N6 (a, *) and N15 (b, *) were tested against chromium-labelled K562 cells in the presence of OKT3 ascites (1/400). Various doses of synovial fluid samples from five patients with rheumatoid arthritis were tested for inhibition of the induced cytotoxic properties of clone N6 and clone N 15. Effector-to-target cell ratio 10: 1.

Synovial fluid inhibits T cell cytotoxicity anti-CD3 or concanavalin A (ConA) used to induce cytotoxicity were as depicted in the legends to the figures.

RESULTS

Cytolytic potential of T cell clones isolatedfrom synovial tissue A series of T cell clones derived from the synovial membrane of a patient with RA was studied with regard to their cytolytic potential. The results of an initial screening of this panel using K562 target cells in the presence of OKT3 ascites (1/400)

Inhibition of anti-CD3-induced cytotoxicity by synovialfluid In the next set of experiments, a synovial fluid sample was added in various doses to the cytotoxicity assay and a strong inhibition of target cell lysis as exerted by clone N6 and N15 was found. This inhibition was dependent on the concentration of synovial fluid that was added to the cytotoxicity assay. Using a series of RA synovial fluid samples, anti-CD3-induced cytotoxicity of

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Fig. 3. Inhibition of anti-CD3-induced cytotoxicity with a series of nonO). The synovial RA, traumatic synovial fluid samples (O, E3, membrane T cell clone N 1 (U) was incubated with chromium-labelled K562 target cells in the presence of OKT3 ascites (dilution 1/400). The effects of non-RA traumatic synovial fluid samples were tested by adding various doses of these fluids to the assay. The effector-to-target cell ratio was 20:1. 5,

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demonstrated that clones with a high cytotoxic potential (more than 40% lysis at an effector-to-target cell ratio of 10: 1; clones N N6, N1 5) as well as non-cytotoxic clones (less than 20% lysis at an effector-to-target cell ratio of 10: 1; clones N16, N4, N14) were found. Most of the clones demonstrated intermediate cytotoxic activity (20-40% specific lysis at an effector-to-target cell ratio of 10: 1). Clones N6, N 15 and N I have been used further in this study and their activation requirements in terms of cytotoxicity have been studied in detail. The induction of cytotoxicity in clones N6 and N 15 was shown to be dependent on the dose of OKT3 antibody (or the dilution of ascites) that was added to the mixture of effector cells and K562 target cells (Fig. 1). Neither of the T cell clones N6 nor N1 5 showed NK activity, since in the absence of OKT3 ascites no target cell lysis occurred (Fig. 1). 11,

Synovialfluid samples Heparinized synovial fluid samples were centrifuged for 10 min at 2000 g, aliquoted, and frozen at - 20'C. The fluids were derived from the knees of patients with ankylosing spondylitis (patient 79), RA (patients 80-82) and a patient with an unclassified arthritis (patient 83). Their age range was 28-77 years. Two patients (81 and 82) received non-steroidal antiinflammatory drugs. Patient 80 received a second-line antirheumatic drug. The other synovial fluid samples were derived from patients who were not receiving a treatment at the time of fluid aspiration.

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Fig. 4. Lack of inhibition of natural killer (NK) activity by synovial fluid. Normal peripheral blood mononuclear cells (U) were tested against K562 target cells and NK activity was found (80% lysis at an effector-to-target cell ratio of 50:1). Various doses of rheumatoid arthritis (RA) synovial fluid samples from patients 79 (E), 80 (E3), 81 (a), 82 (0) and 83 (U) were added to the NK cell assay system.

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)00 1:4000 1: 8000 1: Dilution of OKT3 ascites Fig. 5. Effects of synovial fluid on anti-CD3-induced cytotoxicity exerted by normal human T cells. Normal donor T cells were incubated with K562 target cells and anti-CD3 monoclonal antibody, in the presence (U) or absence (l) of 4°/% synovial fluid (from patient 81). The effector-to-target cell ratio was 10: 1.

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the T cell clones N6 and N1 5 was inhibited and again the effects were dependent on the dose of synovial fluid added (Fig. 2). A series of non-RA, traumatic synovial fluid samples also inhibited anti-CD3 induced cytotoxicity now tested using clone N 1 (Fig. 3). The RA synovial fluid samples were also titrated into a NK cell assay, in which fresh PBMC were used as effector cells and K562 cells as target cells, in order to study the specificity of the inhibitory properties. No inhibition of NK cell activity was detected (Fig. 4) indicating that the inhibitory effect was not a non-specific effect of cell-cell interactions due to the viscosity of the synovial fluid samples. Furthermore, the effects of RA synovial fluid samples on T cell-mediated cytotoxicity exerted by a T cell line obtained from a healthy individual was examined. The synovial fluid was demonstrated to inhibit the cytotoxic function of normal T cells (Fig. 5).

Inhibition of lectin-dependent cellular cytotoxicity by synovial fluid The inhibition of cytotoxic activity by synovial fluid in the antiCD3-induced cytotoxicity assays could be the result of a competition for Fc receptors on the K562 cells by human antibodies present in the synovial fluid. To circumvent this problem we used another type of assay to induce cytotoxic activity and again looked for the effects of the addition of a dose response of synovial fluid. To induce cytotoxic activity we now used a lectin-driven system. K562 cells were treated for 30 min with ConA (10 jig/ml), and incubated with the T cell clone N 1. A strong induction of cytotoxicity was observed (Fig. 6). This cytolytic activity could again be inhibited using synovial fluid samples and was dependent on the dose of synovial fluid added to the test system (Fig. 6).

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Fig. 6. Inhibition of lectin-dependent cellular cytotoxicity by synovial fluid. K562 cells pretreated with concanavalin A were incubated with clone Ni (-) (effector-to-target cell ratio 50: 1) and induction of cytotoxicity was found. The effects of rheumatoid arthritis synovial fluid samples from patients 79 (a), 80 (a), 81 (X), 82 (El) and 83 (H) were tested by adding these fluids in various doses to the cytotoxicity assay system.

DISCUSSION In this study the effects of synovial fluid on cytotoxicity exerted by T cell clones isolated from human rheumatoid synovial membrane were investigated. A panel of synovial tissue T cell clones was generated using an anti-CD3-driven proliferation system. The majority of these T cell clones demonstrated cytotoxic potential. Three T cell clones, all with strong cytolytic properties, were selected for this investigation. Two clones, N6 and N15, were studied in detail. The induction of cytotoxicity, when tested in an anti-CD3-triggered cytotoxicity assay system (Leeuwenberg et al., 1985; Spits et al., 1985), was dependent on the dose of anti-CD3 MoAb used. A series of synovial fluid samples was shown to inhibit strongly this induction of cytotoxicity, and this inhibitory effect was dependent on the dose of synovial fluid added. The synovial fluid samples did not prevent cell-cell interactions needed for cytotoxicity induction non-specifically, since the NK cell-mediated lysis of K562 target cells by normal PBMC was not influenced. The effect of synovial fluid therefore seemed T cell specific. This was further confirmed using T cell lines obtained from the peripheral blood of healthy

Synovialfluid inhibits T cell cytotoxicity volunteers. Since immunoglobulins present in synovial fluid may interfere in the anti-CD3-driven cytotoxicity system, we investigated whether synovial fluid samples were able to inhibit T cell-mediated cytotoxicity as induced by the lectin ConA. It was demonstrated that this lectin-dependent cellular cytotoxicity was also strongly inhibited by synovial fluid. The amount of inhibition was again dependent on the dose of synovial fluid that was added. The mechanism by which synovial fluid inhibits T cellmediated cytotoxicity is unknown. Since NK cell-mediated lysis of K562 cells is not influenced by synovial fluid, we assume that the inhibitory effect on T cell cytotoxicity is related either to the recognition events occurring via the T cell receptor for antigen or to the post-recognition triggering events involving the T cell receptor. Studies using antigen-specific cytotoxic T cell clones should be helpful to elucidate further the way in which synovial fluid samples influence T cell-mediated target cell lysis. T lymphocytes have been demonstrated to be predominantly present in the cellular infiltrate found in RA synovial membranes. Part of these lymphocytes may react with putative autoantigens. When autoantigen-reactive cytotoxic T cells encounter their specific antigen on the membrane of autologous macrophages or other cells, this recognition might result in the lysis of such targets (Koga et al., 1989). Since clones with strong cytotoxic activity were isolated from the synovial membrane, this type of interaction is likely to occur in vivo. The activation of cytotoxic T cells could take place within the environment of the synovial tissue. Furthermore, cells may migrate to the synovial fluid. Therefore we investigated whether cell-cell interactions leading to lysis of the target cells, which may take place in tissue, can be influenced by the presence of synovial fluid. Here we demonstrated that T cell cytotoxicity, induced by anti-CD3 MoAb or by the lectin ConA, was strongly inhibited by synovial fluid samples. This suggests that specific cell-cell interactions may be possible only within the synovial tissue, whereas cells leaking to the synovial fluid may be inhibited to exert their functional repertoire.

ACKNOWLEDGMENTS This study was supported by the Dutch League against Rheumatism. We thank Mrs J. Ravensbergen for secretarial assistance.

REFERENCES ABRAHAMSEN, T.G., FR0LAND, S.S., NATVIG, J.B. & PAHLE, J. (1975) Elution and characterization of lymphocytes from rheumatoid inflammatory tissue. Scand. J. Immunol. 4, 823.

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BURMESTER, G.R., KALDEN, J.R., PETER, H.H., SCHEDEL, I., BECK, P. & WITTENBERG, A. (1978) Immunological and functional characteristics of peripheral blood and synovial fluid lymphocytes from patients with rheumatoid arthritis. Scand. J. Immunol. 7, 405. CUSH, J.J. & LIPSKY, P.E. (1988) Phenotypic analysis of synovial tissue and peripheral blood lymphocytes isolated from patients with rheumatoid arthritis. Arthritis Rheum. 31, 1230. HOVDENES, J., GAUDERNACK, G., KVIEN, T.K. & EGELAND, T. (1989a) Expression of activation markers on CD4+ and CD8+ cells from synovial fluid, synovial tissue and peripheral blood of patients with inflammatory arthritides. Scand. J. Immunol. 29, 631. HOVDENES, J., GUADERNACK, G., KVIEN, T.K., EGELAND, T. & MELLBYE, O.J. (1989b) A functional study of purified CD4+ and CD8+ cells isolated from synovial fluid of patients with rheumatoid arthritis and other arthritides. Scand. J. Immunol. 29, 641. KEYSTONE, E.C., POPLONSKI, L., MILLER, R.G., GORCZYNSKI, R., GLADMAN, D. & SNOW, K. (1988) Reactivity of T cells from patients with rheumatoid arthritis to anti-CD3 antibody. Clin. Immunol. Immunopathol. 48, 325. KOGA, T., WAND-WURTTENBERGER, A., DE BRUYN, J., MUNK, M.E., SCHOEL, B. & KAUFMANN, S.H.E. (1989) T cells against a bacterial heat shock protein recognize stressed macrophages. Science, 245, 1112. LEEUWENBERG, J.F.M., SPITS, H., TAX, W.J.M. & CAPEL, P.J.A. (1985) Induction of nonspecific cytotoxicity by monoclonal anti-T3 antibodies. J. Immunol. 134, 3770. LONDEI, M., GRUBECK-LOEBENSTEIN, B., DE BERARDINIS, P., GREENALL, C. & FELDMANN, M. (1988) Efficient propagation and cloning of human T cells in the absence of antigen by means of OKT3, interleukin-2, and antigen-presenting cells. Scand. J. Immunol. 27, 35. MEIJER, C.J.L.M., DE GRAAFF-REITSMA, C.B., LAFEBER, G.J.M. & CATS, A. (1982) In situ localization of lymphocyte subsets in synovial membranes of patients with rheumatoid athritis with monoclonal antibodies. J. Rheumatol. 9, 359. MILTENBURG, A.M.M., MEIJER-PAAPE, M.E., DAHA, M.R. & PAUL, L.C. (1987a) Inhibition of T cell cytolytic potential by concanavalin A: a result of activation? Scand. J. Immunol. 26, 555. MILTENBURG, A.M.M., MEIJER-PAAPE, M.E., DAHA, M.R. & PAUL, L.C. (1987b) Endothelial cell lysis induced by lymphokine activated human peripheral blood mononuclear cells. Eur. J. Immunol. 17, 1383. SPITS, H., YSSEL, H., LEEUWENBERG, J. & DE VRIES, J.E. (1985) Antigenspecific cytotoxic T-cell and antigen-specific proliferating T cell clones can be induced to cytolytic activity by monoclonal antibodies against T3. Eur. J. Immunol. 15, 88. VAN BOXEL, J.A. & PAGET, S.A. (1975) Predominantly T-cell infiltrate in rheumatoid synovial membranes. N. Engl. J. Med. 293, 517.

Cytolytic activity in T cell clones derived from human synovial rheumatoid membrane: inhibition by synovial fluid.

A panel of T cell clones was derived from the synovial membrane of a patient with rheumatoid arthritis (RA). We investigated whether T cell clones wit...
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