Journal of Autoimmunity (1992) $333-350

Rheumatoid Arthritis Synovial Fluid Enhances T Cell Effector Functions

Anna Ridderstad,* Manuchehr Abedi-Valugerdi,* and Erna Miiller*$

Hikan Striimt

*Department of Immunology, Arrhenius Laboratories for Natural Sciences, University of Stockholm, SDepartment of Clinical Immunology, Karolinska Institute at Huddinge Hospital and TDivision of Rheumatology, Department of Internal Medicine, Karolinska Institute at Danderyd Hospital, Stockholm, Sweden (Received 4 March 1991 and accepted 20 February 1992) Rheumatoid arthritis is a chronic autoimmune joint disease of unknown etiology. T cells are believed to be important in the pathogenesis of rheumatoid arthritis since they infiltrate the joints and express several activation markers, such as MHC class II and IL-2R. In this study we have elucidated the effect on freshly isolated T cells of rheumatoid arthritis synovial fluid @A-SF), which contains in vivo produced cytokines and enzymes. The mouse mixed lymphocyte culture (MLC) has been used as a model and specific cytotoxicity was evaluated against 51Cr-labelled sensitive target cells. Studies have shown that RA-SF contains a B cell dlfferentiation activity that can cross-react between the human and murine species. Here we have shown that the addition of RA-SF strongly potentiates cytotoxic activity as well as lymphokine production by allogeneic activated effector T cells. The enhanced cytotoxicity induced by RA-SF was found to be due to a combined effect of increased cytotoxic T lymphocyte (CTL) precursor frequency, measured by limiting dilution analysis, and a more efficient hilling on a per cell basis. Kinetic studies show that RA-SF must be added within 48 h after initiation of the MLC, otherwise the effect is lost. The target cell specificity of RA-SF was studied, using enriched CD4+ or CDS+ responder cells in the MLC. It was found that RA-SF could act directly on the CDS+ cells and potentiate their development to cytotoxic effector cells: this activity was not found when CD4+ responder cells were used instead. RA-SF could, on the other hand, greatly enhance IL-2 production by CD4+ responder cells. We suggest that B and T cell activity in RA-SF is important in the propagation of chronic inflammation in the joints of patients with rheumatoid arthritis.

Correspondence to: Anna Ridderstad, Department of Immunology, Arrhenius Laboratories for Natural Sciences, University of Stockholm, S-10691 Stockholm, Sweden. Fax: 46 8 154163. Phone: 46 8 157882.

0896-841

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0 1992 Academic Press Limited

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Introduction Rheumatoid arthritis (RA) is a chronic autoimmune disease of the joints. Joints with rheumatoid arthritis are sites of chronic inflammation involving T cells, B cells, macrophages and dendritic cells. The disease is also characterized by immune complex formation (involving rheumatoid factors, immunoglobulins and antigens) causing vasculitis in many organs. The pathology of the disease has been studied for a long time, but several questions remain to be answered. However, some conclusions can be drawn from our current knowledge. More than half of the B cells in the inflamed joint are active antibody producing plasma cells [ 11. In 1980, we proposed that the high autoantibody production found in patients with rheumatoid arthritis is due to polyclonal activation of the B cells, either caused by a viral or bacterial substance, or indirectly via cytokines produced by activated specific T cell clones [2]. There is high MHC-class II expression among the infiltrating macrophages and T cells, suggesting that these cells are activated [3]. The T cells have been implicated to have an active role in the disease process, although this has been currrently debated [4]. When inflammatory cells interact, cytokines are likely to be produced and indeed have been found. Cytokines, such as IL-6, IL-l and tumour necrosis factor-u (TNF-a) are found in very high concentrations in the synovial fluid of FU patients @A-SF). These cytokines, although they can be produced by T cells, are produced locally in the joint by macrophages (TNF-a and IL-l) and fibroblasts (IL-6), recently demonstrated using in situ hybridization techniques combined with phenotypic characterization [5]. Granulocyte macrophage-colony stimulating factor (GM-CSF), transforming growth factor-8 (TGF-8) and IL-8 are also produced locally in the joint, whereas T cell produced cytokines, such as IL-2, IL-3, IL-4 and interferon-y (EN-y) are conspicuously absent (for review see 6). There are several explanations for this: T cell derived cytokines are produced only during short time periods; they are consumed directly or act at very short distances. Alternatively, as yet undefined T cell produced cytokines exist in the SF. When examining the B cell stimulatory properties of RA-SF, we found that it could act as a T cell replacing factor for human peripheral blood B cells [ 11, as well as a B cell differentiation factor for murine spleen B cells [7]. RA-SF induces selectively IgG2b antibody production in these cells in the presence of LPS [7,8]. This isotype selection has not been found for any of the other presently defined cytokines [9, lo]. The IgG2b inducing factor in F&4-SF recently has been partially purified on the basis of its ability to bind Cibacron blue sepharose and Heparin sepharose. It is a protein, sensitive to trypsin and protease, to heat above 60°C and extreme pH values. The molecular weight is between 50 and 70 kDa (manuscript submitted). In this study we examined RA-SF, which is a physiological mixture of in vim produced cytokines and enzymes, for its properties to stimulate T cells in vitro. The murine mixed lymphocyte reaction (MLC) has been used as a model system. We chose the murine system since it has earlier been shown that the B cell differentiating activity of RA-SF could cross-react between the human and mouse species. C57B1/6 spleen T cells were chosen as responder cells and BALB/c spleen cells as stimulator cells because this combination gives a high rate of cytotoxic effector T cells. Here we show that RA-SF strongly enhances the cytotoxic activity of the developing effector cells in mouse MLC. This effect is caused by increased cytotoxic T lymphocyte (CTL)

Enhancement of T cell differentiationby RA-SF

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precursor frequency, as well as a more efficient cytotoxicity on a per cell basis. This study also shows that there is a RA-SF dependent increase in production of cytokines in the MLC. Materials and methods Purijkation of T cells by cell afinity chromatography

Antibodies against mouse IgG and IgM (Rabbit anti-mouse immunoglobulins, Dakopatts, Glostrup, Denmark, 2259) were covalently coupled to CNBr-activated sepharose 6 MB (Pharmacia, Uppsala, Sweden, code nr. 17-0820-01). Standard methods were used, according to the Pharmacia product information. Five ml of anti-mouse Ig-coupled Macro Beads suspended in Earles Balanced Salt Solution (E-BSS) were transferred to sterilized glass columns, to which 225 x IO6washed and RBC-depleted mouse spleen cells were added. After 30 min of incubation at room temperature, the cells were eluted with 15-20 ml BSS. The procedure resulted in 90-95 ?&Thy 1.2 positive T cells as determined by FACS analysis. Purification of Lyt2 (CD8)

or L3 T4 (CD4)

positive T lymphocytes

Purified murine T cells were mixed with an a-CD4 antibody (Ab) (GK 1.5). The T cell-Ab suspension was washed twice and passed through a CNBr-activated sepharose 6MB column to which antibodies against rat Ig (Rabbit anti-rat immunoglobulins, Dakopatts, 2147) were covalently coupled. This treatment depletes the CD4+ T cells and enriches the CD8+ cells resulting in about 75:/, CD8+ cells and 4-8Y, CD4+ cells as determined by immunofluorescence in the FACS analyser. Purification of CD4 positive cells was performed using the same protocol, with an aCD8 Ab (31M), resulting in about 80% CD4+ cells and 57” CD8+ cells. These enriched CD8+ or CD4+ cells were used as responder cells in some of the mixed lymphocyte cultures described below. Cell culture

MLC were established, with splenic T cells (as responders) and y-irradiated (2,OOOR from a 137Cssource) whole spleen stimulator cells, in Costar (Badhoevedorp, The Netherlands) 16-mm diameter culture wells containing Gibco (Gaithersburg, USA) RPMI-medium supplemented with 10% fetal calf serum (FCS), 1Y0glutamine, 1‘?A penicillin-streptomycin and 5 x 10F5 M 2-mercaptoethanol(2-ME). The cell concentrations used were 5 x lo5 responder cells and 5 x lo6 stimulator cells per well. Cultures were supplemented initially with RA-SF at a final dilution of l/l5 and incubated in sir/5% CO, at 37°C for 5 days. MLC were also performed in 96-well microcultures to measure proliferation. In these cultures lo5 responder cells and 10” stimulator cells per well were used. The proliferation assay was performed as described below for the lymphokine test on CTLL cells. Monoclonal antibodies

Supernatants from the llBl1 cell line producing rat anti-mouse IL-4 antibodies were added to some cultures at a final concentration of 10%. This concentration completely inhibits IL-4 dependent IgGl production by lipopolysaccharide (LPS)

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stimulated mouse spleen B cells. Purified mouse monoclonal antibody against human IL-6 (BSF2-166) was a kind gift from Dr T. Kishimoto (Osaka University, Japan). IL-6 was used at a final concentration of 1.75 ug/ml, which was the optimal concentration to inhibit the IL-6 dependent growth of the B45 cell line. Preparation

of RA-SF

samples

SF specimens were obtained from RA-patients fulfilling the criteria of the American Rheumatism Association [ 111. The patients were treated with non-steroidal antiinflammatory drugs at the time of joint puncture. Before use, the samples were treated with 10 ug hyaluronidase (Sigma Chemical Co., St Louis, MO, USA) per ml SF and incubated at 37°C for 45 min. Thereafter the SF samples were centrifuged to remove the cell debris (10 min at 2,000 rpm). Test for alloactivated

cytotoxic

T lymphocytes

After 5 days of culture, the cytotoxic activity of the effector cells was tested against 5’Cr-labelled target cells (48 h concanavalin A (Con-A)-activated spleen cells or P815 mastocytoma cells of DBA/2 origin). The harvested effector cells were washed in BSS and counted. The indicated effector:target cell ratio was distributed in Vshaped 96-well microtitre plates. The plates were centrifuged followed by 3 h of incubation at 37°C. One hundred and fifty ul of supernatant was then collected from each well and counted in a y-counter. Specific cytotoxic activity was calculated as 100x(

ex erimental cpm - spontaneous cpm) p (maximum cpm - spontaneous cpm)

Spontaneous release was determined by incubating target cells with culture medium only and the maximum release by incubating target cells with 5% Triton X-100. Spontaneous chromium release was between 8 and 18% of maximum in all experiments. Lymphokine

test on CTLL

cells

MLC supernatants and RA-SF as such were tested for the presence of IL-2 and IL-4 on a CTLL indicator cell line [12, 131. RPM1 medium containing 6 x lo3 actively dividing CTLL cells, 5% FCS, 2% glutamine, 2% sodium pyruvate, 1y0 penicillinstreptomycin and 5 x lop5 M 2-ME was added to each well in flat bottomed microculture plates. The supernatants to be tested were titrated and the proliferation was measured on day 1. For the last 6 h of incubation, the cultures were pulsed with 4 uCi/20 ul [3H]-thymidine and thereafter harvested on a Skatron 7035 filter, using a Skatron harvesting machine (Lier, Norway), and counted in a liquid scintillator (LKB, Pharmacia) using 0.3ml Insta Fluor (United Technologies Packard) scintillation fluid per sample. Immunojborescence

Immunofluorescence staining was performed with FITC- or PE-conjugated antibodies against mouse B and T cell markers, followed by FACS analysis. Anti-mouse

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L3T4 (CD4), Lyt-2 (CDS) and Thy-l were purchased from Becton Dickinson (Mountain View, CA, USA) and rabbit anti-mouse Ig (F(ab’), fragments) from Dakopatts. The cells were stained with the conjugated antibodies described above and then analysed on a Beckton Dickinson FACScan.

Limiting dilution analysis Limiting dilution experiments were performed in 96-well round bottomed microculture plates. Affinity purified splenic T cells from C57B1/6 mice were used as responder cells and irradiated BALB/c spleen cells as stimulator cells. Cells were cultured for 9 days in RPM1 supplemented with 10% FCS, 10% rat Con-A supernatant, 1% glutamine, 1% penicillin-streptomycin and 5 x lop5 M 2mercaptoethanol. Limiting numbers of responder cells and 2 x lo5 stimulator cells were added to each culture, with 24 wells per cell concentration. On day 9, 100 ul culture supernatant was removed from each well and 1,000 *lCr-labelled P815 target cells in 100 ul was added. Plates were centrifuged and incubated in 37°C for 3 h. One hundred ul of supematant was collected from each well and counted in a y-counter. Wells were considered as positive when experiment cpm exceeded the mean of spontaneous cpm + 3 standard deviations (SD). The results were analysed as previously described [ 141.

Results Eflect of RA-SF

on MLC and CTL development

RA-SF, by itself, does not stimulate resting T cells to proliferate (data not shown), but a clear potentiating effect of RA-SF on the generation of cytotoxic effector cells in a mouse MLC is observed (Figure 1). Here highly purified C57BL/6 (H-2b) T cells (90yo Thy- I+) were used as responder cells, and irradiated BALB/c (H-2d) spleen cells as stimulator cells. Cytotoxicity was evaluated against 51Cr-labelled sensitive target cells (P815) on day 5, which was found to be optimal. The data demonstrate that cultures supplemented with RA-SF throughout the culture period showed much greater cytolytic activity than control cultures. Consistent data were obtained in repeat experiments. Upon cell harvest from the MLC, the number of blast cells in the cultures supplemented with RA-SF was found to be increased. This observation was made both when the number of surviving blast cells were counted under the light microscope and when the forward scatter parameter using the FACScan was studied (Figure 2, top). In addition to increased blast cell number, increased granularity among the cells from RA-SF treated MLC was also observed (Figure 2, bottom). The proliferative capacity of the T cells in MLC was tested in the presence or absence of RA-SF on days 3,4,5 and 6. Figure 3b shows that &A-SF clearly induces higher proliferation, particularly at the peak on day 4, but it does not alter the kinetics of the proliferative response. Figure 3a shows that the IL-2 content in the MLC supernatant (measured on the IL-2 sensitive CTLL cells) peaks on day 3, but in the cultures stimulated with RA-SF, the concentration of IL-2 remains high for a more prolonged period.

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0 Effector:

Figure 1. MLC with C57BL/6 spleen T cells as stimulator cells. Cultures were supplemented concentration of l/30. The specific cytotoxic 5’Cr-labelled P815 tumor cells. -OMLC+

Target

cell ratio

(90% Thy-l +) as responder cells and BALB/c spleen cells at initiation with synovial fluid from one patient at a final activity of the effector cells was tested on day 5 against SF; -0MLC control.

The enhanced cytotoxic activity of the developing effector cells (Figure 4) could be induced by addition of U-SF to MLC on days 0,l or 2. However, when added on day 3 or later, RA-SF did not affect the cytotoxic activity. Enriched CD8+ or CD4’

T cells as responder cells in MLC

To determine the target cell specificity of RA-SF, we used enriched CD8+ or CD4+ C57BL/6 spleen T cells as responder cells in MLC, with BALB/c spleen cells as stimulator cells, in two different experiments. Cultures of CD8+ responder cells, supplemented with F2A-SF, typically showed increased cytotoxic activity (Figure 5), whereas F&I-SF could not induce higher cytotoxic activity in cultures containing CD4+ T cells as responder cells (Figure 6). This indicates that RA-SF could act directly on CD8+ cytotoxic T cell precursors to potentiate cytotoxicity. Detection of lymphokine production in MLC T cell activation leads to proliferation, lymphokine secretion and cytotoxic activity. Since we found that RA-SF selectively potentiated CTL development and enhanced the proliferation in MLC, we also tested lymphokine production. MLC supematants were collected when effector cells were harvested and tested on CTLL cells for the

Enhancemeht of T cell differentiation by FM-SF

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Figure 2. The size and granularity distribution of harvested effector cells, from untreated MLC (dotted line) and from MLC stimulated with RA-SF at initiation of the culture period (solid line), was measured. The cells were analysed in the FACScan, with forward scatter (FSC) and side scatter (SSC) parameters.

presence of mainly IL-2. The results, shown in Figure 7, demonstrate that the supernatants from RA-SF-stimulated MLC induced proliferation in the CTLL cell line, whereas supernatants from control cultures had no effect. RA-SF as such, does not stimulate CTLL cell proliferation, which shows that the lymphokines that exist in the MLC + RA-SF supernatants are produced by the R4-SF stimulated T cells. Supernatants from RA-SF-stimulated MLC with CD8 or CD4 positive responder cells were also tested for growth stimulating activity on CTLL cells. Cultures with CD8+ responder cells contained no detectable CTLL stimulating activity, after addition of RA-SF. On the other hand, cultures with CD4’ responder cells, when stimulated with RA-SF, contained high amounts of CTLL stimulating activity (Figure 7).

340 AnnaRidderstadetal.

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Day Figure 3. MLC were performed in 96-well flat bottomed microculture plates for proliferation assay and 24-well plates for cytokine detection assay, with purified C57BL/6 spleen T cells (92% Thy l+) as responder cells and BALB/c spleen cells as stimulator cells, in the presence or absence of RA-SF (final dilution l/15). (a) Supematants were harvested on days 3, 4, 5 and 6 and the IL-2 contents in these supematants (finally diluted l/2) were measured on the CTLL cell-line. (b) The proliferation of MLC was measured on days 3,4,5 and 6 with [)H]-thymidine incorporation according to Materials and methods. A MLC+RA-SF; A MLC control.

200

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0.2

ratio

Figure 4. RA-SF was added to MLC (described in Figure 1) at days 0, 1,2,3 and 4 at a final dilution of 1/15 and the cytotoxic activity of harvested effector cells was measured on day 5 as described above. C = control.

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Figure 5. Purified CDS+ T cells from C57BL/6 spleen were used as responder cells and BALB/c spleen cells were used as stimulator cells. MLC were performed after the same culture conditions as in Figure 1. The cytotoxicity was measured at day 5 as described above. -OMLC+ SF; 0 MLC control.

Do antibodies against IL-4 or IL-6 inhibit the increased cytotoxicity by RA-SF?

MLC were performed using purified C57BL/6 T cells as responder cells and BALB/ c spleen cells as stimulator cells. RA-SF was added at initiation and in some cultures together with u-mouse IL-4 antibodies (11Bll). These antibodies, which could inhibit the IgGl inducing effect of IL-4 (not shown), did not decrease the RA-SF induced increased cytotoxicity of harvested effector cells in the MLC (Figure 8). The results indicate that IL-4, known to stimulate the development of CTL, is neither produced nor responsible for the enhanced cytotoxic activity observed. Since RA-SF does not contain IL-4 [30] we have not tested antibodies against human IL-4. In similar experiments, purified recombinant u-human IL-6 monoclonal antibodies were used to inhibit the augmented cytotoxicity induced by RA-SF. The reason why antibodies against human IL-6 were used was that our RA-SF samples do contain IL-6 and it has also been shown that IL-6 can cross-react between mouse and human. However, antibodies against IL-6 could not decrease the cytotoxicity induced by RA-SF (Figure 9), indicating that IL-6 in our RA-SF preparations is not responsible for the increased cytotoxicity. ESfect of RA synovialfluid

on the CTL precursor frequency

To establish whether the increased cytotoxic activity, induced by RA-SF,

on an increased CTL

depended precursor frequency or on more effective cytotoxic T cells,

342 Anna Ridderstad et al.

20

Effector:

Target

cell ratio

Figure 6. MLC were performed as described in Figure 1 with purified T cells or CD4+ cells from C57BL/6 spleen as responder cells and BALB/c spleen cells as stimulator cells. Cytotoxicity was measured on day 5. -0MLC ‘T’ control; -OMLC ‘T’+ SF; -OMLC ‘CD4’ control; -mMLC ‘CD4’+ SF.

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Figure 7. Detection of lymphokine activity in MLC supematants, tested on a CTLL indicator cell line. The proliferation of CTLL cells stimulated with different concentrations of MLC supematants or PA-SF alone was measured by [3H]-thymidine incorporation according to the Materials and methods. Proliferation of CTLL cells after stimulation with rat Con-A supematant (IL-2) was measured as a control and gave about lO,OOO-50,000 cpm (data not shown). (a) -APA-SF alone; -•MLC+RA-SF CD4/ CD8 responders; -0MLC control CD4/CDS responders. (b) -OMLC+RA-SF CD4/CD8 responders; -0MLC control CD4/CD8 responders; - W- MLC + PA-SF CD8 responders; -OMLC control CD8 responders. (c) -AMLC+RA-SF CD4 responders; -AMLC control CD4 responders.

Enhancement of T cell differentiationby RA-SF

12.5 Effector:

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3.1 0.8 Target cell ratio

Figure 8. MLC were performed as before in the presence or absence of 10% culture supernatant from 11 Bl 1cells, which produce rat a-mouse IL-4 antibodies. Cytotoxicity was measured on day 5 as described before.-@-MLCcontrol;-O-MLC+SF;--O-MLC+llB11;-~-MLC+SF+llB11.

limiting dilution studies were performed. In two independent experiments (Figure lo), we measured the precursor frequency of CTL in MLC with C57BL/6 responder cells and BALB/c stimulator cells in the presence or absence of RA-SF, using the limiting dilution technique (see Materials and methods). The CTL precursor frequency in MLC stimulated with RA-SF is about twice that of untreated MLC. It increased from about l/550 to l/275. Thus, the increased cytotoxic potential, induced by RA-SF, is a combined effect of increased precursor frequency and more efficient killing by individual CTL. Discussion Here we have shown that RA-SF, a physiological fluid produced in excess in RApatients, that contains in viwoproduced cytokines and enzymes, has profound effects on T cell differentiation including development into cytotoxic and lymphokineproducing effector cells. Instead of mixing recombinant cytokines we have examined the effect of an in vim mixture of cytokines. The joints of patients with rheumatoid arthritis are infiltrated by large numbers of mononuclear cells and the predominant cell type seems to be the activated T lymphocyte [ 151. These cells express increased levels of IL-2 receptors, MHC class II, as well as other activation markers [ 16,171, It is likely that T cells play an important role in the pathogenesis of rheumatoid arthritis at least early in the disease, before the acute inflammation becomes chronic. RA is an HLA-associated autoimmune disease

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Figure 9. MLC cultures, described in Figure bodies against human IL-6 at a final concentration MLCcontrol;-•-MLCfRA-SF;-0-MLC+RA-SF+ahrIL-6.

: Target

1, were supplemented with purified monoclonal antiof 1.75 ug/ml. Cytotoxicity was assayed at day 5. -fl-

Number

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of cells/culture 0

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Figure 10. Limiting dilution analyses were performed with limiting numbers of responder cells (C57BL/6 spleen T cells) and a constant number of stimulator cells (BALB/c spleen cells) with 24 wells of each cell concentration. Two independent experiments were carried out. -OMLC control; -OMLC+RA-SF.

for which DR4 positive individuals have a higher risk of developing the disease. It has been suggested recently [ 181 that a disease-associated MHC phenotype might allow the generation of potentially pathogenic T cell clones during thymic selection.

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Several examples of T cell-mediated autoimmune diseases exist, such as experimental allergic encephalomyelitis, which is an induced autoimmune disease mediated by CD4’ T lymphocytes [ 19,201. It has been shown that CD4 and CD8 positive T cells can transfer autoimmune diabetes from diabetic NOD mice to healthy neonates [2 l] and islet cell specific T cell clones can be isolated from the same mice [22]. Infiltrating T cells from the thyroid glands of two patients with Graves’ disease were cloned and some clones were found to recognize autologous thyroid cells [23] also indicating an important role of specific T cell clones. T lymphocytes were the predominant cells isolated from synovial tissue of RA patients [ 151. Clonal analysis of T cell infiltrates in synovial tissues of patients with rheumatoid arthritis revealed that CD4+ clones were more frequent [24] and ‘self reactive’ T cell clones have been established, recognizing as yet undefined antigen(s) in RA-SF in a DR4Dw4 restricted manner [25]. Several T cell clones, isolated from RA-SF, were reactive with different mycobacterial proteins [26], which have been implicated as being important for the pathology of the disease. Although T cells are thought to be important for the pathogenesis of rheumatoid arthritis, no T cell derived cytokines were found (see ref. 6), which led Firestein and Zwaifler to question the importance of T cells [4]. The explanation for the absence of T cell-produced cytokines could be that they are produced for only short time periods, they are consumed directly or act at very short distances, but it does not necessarily mean that T cells are unimportant. The time when synovial fluids are aspirated or joints are operated on is most probably late in the course of disease, when the T cells might already have played their role, which could be another explanation for the lack of T cell derived cytokines. Alternatively, a yet unidentified T cell product may exist in the RA-SF. When the inflammatory cells in the arthritic joint interact, cytokines are likely to be produced. Synovial fluid and synovial tissue from patients with rheumatoid arthritis and other forms of arthritis, contain high levels of IL-6, produced by fibroblasts, and IL-l and TNF-a, produced by macrophages [5]. IL-8 [27], GM-CSF [28] and TGF-P [29] are also abundant, but not IL-2, IL-3, IL-4 or IFN-)I [5,30]. We have shown previously that RA-SF contains a B cell active factor that permits the differentiation of murine B lymphocytes into IgG2b producing plasma cells in the presence of the polyclonal B cell activator LPS, these results were obtained both in vitro and in vivo [7,8]. Here we demonstrate that RA-SF, in addition to its effects on B cells also contains soluble substances which can act on murine spleen T cells. Increased cytotoxic activity of activated effector cells, formed as a result of allogeneic stimulation, was induced by RA-SF. These substances could not, however, activate resting T cells (not shown). The enhanced cytotoxicity, induced by RA-SF, was found to be the result of a combined effect of a higher CTL precursor frequency, as well as more efficient killing by individual CTL. We found increased granularity among the effector cells from RA-SF treated MLC. This also indicates more efficient killing on a per cell basis, since the number of enzyme containing granules within the CTL increases before delivery ofthe ‘lethal hit’ to the target cell [31]. Kinetic studies show that RA-SF must be added within 48 h after initiation of the MLC, otherwise the effect is lost i.e., the cells are not responsive to RA-SF after this time, which might be the result of downregulation of a specific receptor. This, however, requires further elucidation.

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In order to determine the target cell specificity of RA-SF, purified CD4+ or CD8+ spleen T cells were used as responder cells. We found that enriched CD4+ T lymphocytes produce a CTLL stimulating activity, mainly IL-2, when activated by allogeneic stimulator cells in the presence of RA-SF. As such, RA-SF does not contain any measurable amounts of IL-2, tested on the same cell line. When CD8 positive T cells were used as responder cells, no CTLL stimulatory activity was induced by KA-SF, whereas the cytotoxic activity was greatly enhanced. Based on these results, we conclude that RA-SF contains two activities; it induces differentiation of CD8+ T cells and it induces lymphokine production by CD4+ T cells. Another possibility is that RA-SF acts via CD4+ cells, which produce soluble factors, different from IL-4 or IL-6, that in turn stimulate the CD8+ cells to increased cytotoxic activity. Several of the known cytokines have been shown to affect T cells in different ways [12,32-391. IL-l and IL-6 synergize in some situations to induce effector functions of both T, and CTL cells [40-42]. Several studies have suggested that factors, such as killer helper factor (KHF) or cytotoxic T cell differentiation factor (CDF), are involved in the generation of CTL, but little is known about the molecular nature of these factors [43,44]. IL-6 can act as a CDF [45] as well as a late acting KHF in CTL generation, but only together with IL-2 [46,47]. IL-4 can also act as a helper factor for the generation of cytotoxic T lymphocytes in primary MLC [48] and is a more potent helper factor for unprimed precursors than is IL-2. High doses of IL-2, however, could induce non-specific syngeneic killing, an activity not ascribed to IL-4. IL-5 permits the development of cytotoxic T lymphocytes from thymocytes activated by (TNP modified syngeneic) stimulator cells in the presence of IL-2 [49]. In addition, we were not able to mimic the effect of RA-SF by adding recombinant IL-2 to the MLC (not shown). Seemingly in contrast to our findings of a T cell stimulating activity in RA-SF, it has been reported that addition of RA-SF strongly inhibited the cytotoxicity of T cell clones isolated from rheumatoid synovial membrane or normal human T cells from peripheral blood [50]. The reason for this discrepancy could be that the synovial fluids were treated differently from those of our study and the cytotoxicity was measured by different assays, using human peripheral blood lymphocytes instead of spleen cells. The effect of PA-SF on human T cells should continue to be studied. It is unlikely that IL-4 is responsible for the effect mediated by RA-SF, since firstly RA-SF does not contain IL-4 [30] and secondly, IL-4 is not believed to crossreact between human and mouse [51]. It does not seem that KA-SF induces IL-4 production, which in turn mediates the increased cytotoxicity of effector cells, since antibodies against murine IL-4 do not inhibit the activity. The addition of recombinant anti-human IL-6 antibodies did not decrease the effect of RA-SF, thus indicating that IL-6 in the PA-SF is not mediating the effect, although it is present in high amounts [ 52-561. To summarize, we have found that RA-SF can induce differentiation of IgG2b producing murine B cells in the presence of the polyclonal B cell activator LPS. In addition, RA-SF potentiates the development of effector T cells formed by allogeneic stimulation. By using biochemically purified IgG2b-inducing factor, which was separated according to its ability to bind Blue Sepharose, in the T cell system, we found the activity in the fraction that does not bind to Blue Sepharose. Therefore,

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these are probably two separate activities (this issue will be discussed further elsewhere). The presence of a B cell differentiation factor and a T cell stimulating factor in RASF has several implications for the pathology of the disease. Increased antibody synthesis, particularly autoantibodies, leads to immune complex formation, which in turn activates the complement cascade that eventually results in tissue damage. RASF contains a substance that could enhance the differentiation of newly activated T cells and induce the production of lymphokines that in turn could stimulate proliferation of other T cells (exemplified by CTLL). These findings might explain the chronic presence of activated T cells in the synovial joint. We suggest that the B and T cell activity in RA-SF is important in the propagation of the chronic inflammation in the joints of patients with rheumatoid arthritis. Acknowledgements This investigation was supported by grants from the Swedish Medical Research Council (no B16X-02334), King Gustav V 80-year Foundation, the Swedish Association of Rheumatism and the Nanna Schwartz Foundation. The excellent technical assistance of MS Lotta Aveberger is gratefully acknowledged. References 1. Al-Balaghi, S., H. Strom, and E. Moller. 1984. Demonstration of a helper factor(s) with T cell replacing activity in synovial fluid. Sand. J. Immunol. 20: 493-501 2. Moller, E., H. Strom, and S. Al-Balaghi. 1980. Role of polyclonal activation in specific immune response. Relevance for findings of antibody activity in various diseases. Sand. J. Immunol. 12: 177-182 3. Klareskog, L., U. Forsum, A. Wigren, and H. Wigzell. 1982. Relationships between HLA-DR expressing cells and T lymphocytes of different synovial tissue. Stand. J. Immunol. 15: 501-507 4. Firestein, G. S. andN. J. Zvaifler. 1990. How important are T cells in chronic rheumatoid synovitis? Arthritis Rheum. 33: 768-773 G. S., J. M. Alvaro-Garcia, and R. Maki. 1990. Quantitative analysis of 5. Firestein, cytokine gene expression in rheumatoid arthritis. J. Immunol. 144: 3347-3353 A., M. Abedi-Valugerdi, and E. Moller. 1991. Cytokines in rheumatoid 6. Ridderstad, arthritis. Ann. Med. 23: 219-223 7. Al-Balaghi, S., H. Strom, and E. Moller. 1984. B cell differentiation factor in synovial fluid of rheumatoid arthritis patients. Immunol. Rev. 78: 7-23 M., A. Ridderstad, H. Strom, and E. MBller. 1989. Synovial fluid from 8. Abedi-Valugerdi, rheumatoid arthritis patients induces polyclonal antibody formation in vivo. Stand. J. Immunol. 30: 587-596 9. Moller, E. and H. Strom. 1988. Biological characterization of T cell replacing factor in the synovial fluid of rheumatoid arthritis patients. Scund. 3. Immunol. 27: 717-724 10. Abedi-Valugerdi, M., A. Ridderstad, H. Strom, and E. Moller. 1991. Relationship between IgG2b-inducing activity in rheumatoid arthritis synovial fluid and other well known cytokines and inflammatory mediators. Arthritis Rheum. 34: 1461-1465 11. Arnett, F. C., S. M. Edworth, D. A. Bloch, D. J. McShane, J. F. Fries, N. S. Cooper, L. A. Healey, S. R. Kaplan, M. H. Liang, H. S. Luthra, T. A. Medsger, D. M. Mitchell Jr, D. H. Neustadt, R. S. Pinals, J. G. Schaller, J. T. Sharp, R. L. Wilder, and G. G. Hunder. 1988. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 31: 315-324 12. Gillis, S., M. M. Ferm, W. Ou, and K. A. Smith. 1978. T cell growth factor: parameters of production and a quantitative microassay for activity. J. Zmmunol. 120: 2027-2032

348

Anna Ridderstad et al.

13. Severinson, E., T. Naito, H. Tokumoto, D. Fukushima, A. Hirano, K. Hama, and T. Homo. 1987. Interleukin 4 (IgGl induction factor): a multifunctional lymphokine acting also on T cells. Eur. 3. Immunol. 17: 67-72 14. S. Fazekas de St. Groth. 1982. The evaluation of limiting dilution assays. 3. Zmmunol. Methods 49: RI l-R28 15. Abrahamsen, T. G., S. S. Friiland, J. B. Natvig, and J. Pahle. 1975. Elution and characterization of lymphocytes from rheumatoid inflammatory tissue. Stand. 3. Zmmunol. 4: 823-830 16. Laffon, A., F. Sanchez-Madrid, M. Ortiz de Landazuri, A. Jimenez Cuesta, A. Ariza, C. Ossorio, and P. Sabando. 1989. Very late activation antigen on synovial fluid T cells from patients with rheumatoid arthritis and other rheumatic diseases. Arthritis Rheum. 32: 386-392 17. Potocnik, A. J., R. Kinne, H. Menninger, J. Zacher, F. Emmrich, and R. A. Kroczek. 1990. Expression of activation antigens on T cells in rheumatoid arthritis patients. Stand. 3. Immunol. 31: 213-224 18. Moller, E., J. Bohme, M. Abedi-Valugerdi, A. Ridderstad, and 0. Olerup. 1990. Speculations on mechanisms of HLA associations with autoimmune diseases and the specificity of “autoreactive” T lymphocytes. Immunol. Rev. 118:5-19 19. Paterson, P. Y. 1960. Transfer of allergic encephalomyelitis in rats by means of lymph nodecel1s.J. Exp. Med. 111: 119-135 20. Pettinelli, C. B. and D. E. McFarlin. 1981. Adoptive transfer of experimental allergic encephalomyelitis in S JL/ J mice after in vitro activation of lymph node cells by myelin basic protein:requirement for Lytl+2lymphocytes. 3. Zmmunol. 127: 1420-1423 21. Bendelac, A., C. Carnaud, C. Boitard, and J. F. Bach. 1987. Syngeneic transfer of autoimmune diabetes from diabetic NOD mice to healthy neonates. Requirement for both L3T4+ and Lyt-2+ T cel1s.J. Exp. Med. 166: 823-832 22. Haskins, K., M. Portas, B. Bergman, K. Lafferty, and B. Bradley. 1989. Pancreatic isletspecific T-cell clones from nonobese diabetic mice. Proc. Natl. Acad. Sci. USA 66: 8000-8004 23. Londei, M., G. F. Bottazzo, and M. Feldman. 1985. Human T-cell clones from autoimmune thyroid glands: specific recognition of autologous thyroid cells. Science 228: 85-89 24. Hakoda, M., T. Ishimoto, K. Yamamoto, K. Inoue, N. Kamatani, N. Miyasaka, and K. Nishioka. 1990. Clonal analysis of T cell infiltrates in synovial tissue of patients with rheumatoid arthritis. Clin. Immunol. Immunopathol. 57: 387-398 25. Devereux, D., R. E. O’Hehir, J. McGujire, W. C. A. van Schooten, and J. R. Lamb. 1991. HLA-DR4Dw4-restricted T cell recognition of self antigen(s) in the rheumatoid synovial compartment. ht. Immunol. 3: 635-640 26. Holoshitz, J., F. Koning, J. E. Coligan, J. De Bruyn, and S. Strober. 1989. Isolation of CD4_CD8mycobacteria-reactive T lymphocyte clones from rheumatoid arthritis synovial fluid. Nature 339: 226-229 27. Brennan, F. M., C. 0. C. Zachariae, D. Chantry, C. G. Larsen, M. Turner, R. N. Maini, K. Matsushima, and M. Feldman. 1990. Detection of interleukin 8 biological activity in synovial fluids from patients with rheumatoid arthritis and production of interleukin 8 mRNA by isolated synovial cells. Eur. 3. Zmmunol. 20: 2141-2144 28. Xu, W.-D., G. S. Firestein, R. Taetle, K. Kaushansky, and N. J. Zwaifler. 1989. Cytokines in chronic inflammatory arthritis II. Granulocyte-Macrophage colonystimulating factor in rheumatoid synovial effusions. 3. Clin. Invest. 83: 876-882 29. Brennan, F. M., D. Chantry, M. Turner, B. Foxwell, R. Maini, and M. Feldmann. 1990. Detection of transforming growth factor-beta in rheumatoid arthritis synovial tissue: lack of effect on spontaneous cytokine production in joint cell cultures. Clin. Exp. Zmmunol. 81: 278-285 30. Miossec, P., M. Naviliat, A. D. D’angerac, J. Sany, and J. Banchereau. 1990. Low levels of interleukin-4 and high levels of transforming growth factor j3 in rheumatoid synovitis. Arthritis Rheum. 33: 1180-l 187

Enhancement of T cell differentiation by RA-SF

349

31. Peters, P. P., H. J. Geuze, H. A. van der Donk, and J. Borst. 1990. A new model for the lethal hit delivery by cytotoxic T lymphocytes. Zmmunol. Today 11: 28-32 32. Morgan, D. A., F. W. Roscetti, and R. Gallo. 1976. Selective in vitro growth of T lymphocytes from normal human bone marrow. Science 193: 1007-1008 33. Gillis, S. and K. A. Smith. 1977. Long term culture of tumor specific cytotoxic T cells. Nature 268: 154-156 34. Mosmann, T. R., M. W. Bond, R. L. Coffman, J. Ohara, and W. E. Paul. 1986. T cell and mast cell lines respond to B cell stimulatory factor-l. Proc. Nurl. Acad. Sci. USA 83: 5654-5658 M. Sanders, K. G. Oliver,Y. Chen,P. H. Krammer, J. W. Uhr, 35. Fernandez-Botran,R.,V. and E. S. Vitetta. 1986. Interleukin 4 mediates autocrine growth of helper T cells after antigenic stimulation. Proc. Natl. Acad. Sci. USA 83: 9689-9693 36. Lichtman, A. H., E. A. Kurt- Jones, and A. K. Abbas. 1987. B cell stimulatory factor 1 and not interleukin 2 is the autokrine growth factor for some helper T lymphocytes. Proc. Natl. Acad. Sci. USA 84: 824-827 37. Grabstein, K. H., L. S. Park, P. J. Morrissey, H. Sassenfeld, V. Price, D. Urdal, and M. B. Widmer. 1987. Regulation of murine T cell proliferation by B cell stimulatory factor-l. J Zmmunol. 139: 1148-l 153 38. Hu-Ii, J., E. M. Shevach, J, Mizuguchi, J. Ohara, T. Mosmann, and W. E Paul. 1987. B cell stimulatory factor 1 (interleukin 4) is a potent costimulant for normal and resting T lymphocytes.3. Exp. Med. 165: 157-172 39. Lee, F., T. Yokota, T. Otsuka, P. Meyerson, D. Villaret, R. Coffman, T. Mosmann, D. Rennick, N. Roehm, C. Smith, A. Zlotnik, and K. Arai. 1986. Isolation and characterization of a mouse cDNA clone that expresses B cell stimulatory factor 1 activities and T cell and mast cell stimulating activities. Proc. Natl. Acad. Sci. USA 83: 2061-2065 40. Renauld, J., A. Vink, and J. van Snick. 1989. Accessory signals in murine cytolytic T cell responses. Dual requirement for IL-l and IL-6.3. Zmmunol. 6: 1894-1898 41. Holsti, M. A. and D. H. Raulet. 1989. IL-6 and IL-l synergize to stimulate IL-2 production and proliferation of peripheral T cells. 3. Zmmunol. 8: 2514-25 19 42. Houssiau, F. A., P. G. Coulie, and J. van Snick. 1989. Distinct roles of IL-l and IL-6 in human T cell activation. 3. Zmmunol. 8: 2520-2524 43. Raulet, D. H. and M. J. Bevan. 1982. A differentiation factor required for the expression of cytotoxic T-cell function. Nature 296: 754-757 44. Yang, S. S., T. R. Malek, M. E. Hargrove, and C. Ting. 1985. Lymphokine induced cytotoxicity: Requirement of two cytokines for the induction of optimal cytotoxic responses. 3. Zmmunol. 134: 3912-3919 45. Ming, J. E., C. Cernetti, R. M. Steinman, and A. Granelli-Piperno. 1989. Interleukin 6 is the principal cytolytic T lymphocyte differentiation factor for thymocytes in human leukocyte conditioned medium. 3. Mol. Cell Zmmunol. 4: 203-212 46. Okada, M., M. Kitahara, S. Kishimoto, T. Matsuda, T. Hirano, and T. Kishimoto. 1988. IL-6/BSF-2 functions as a killer helper factor in the in vitro induction of cytotoxic T cells. 3. Zmmunol. 141: 1543-1549 47. Takai, Y., G. G. Wong, S. C. Clark, S. J. Burakoff, and S. H. Herrmann. 1988. B cell stimulatory factor-2 is involved in the differentiation of cytotoxic T lymphocytes. 3. Zmmunol. 140: 508-512 48. Widmer, M. B. and K. H. Grabstein. 1987. Regulation of cytotoxic T-lymphocyte generation by B cell stimulatory factor. Nature 326: 795-798 49. Takatsu, K., Y. Kikuchi, T. Takahashi, T. Homo, M. Matsumoto, N. Harada, N. Yamaguchi, and A. Tominaga. 1987. Interleukin 5, a T-cell-derived B-cell differentiation factor also induces cytotoxic T lymphocytes. Proc. Natl. Acad. Sci. USA 84: 4234-4238 50. Miltenburg, A. M. M., J. M. van Laar, P. de Kuiper, M. R. Daha, and F. C. Breedveld. 1990. Cytolytic activity in T cell clones derived from human synovial rheumatoid membrane: inhibition by synovial fluid. Clin. Exp. Zmmunol. 82: 499-503 51. Mosmann, T. R., T. Yokota, R. Kastelein, S. M. Zurawski, N. Arai, and Y. Takebe. 1987. Species specificity of T cell stimulating activities of IL 2 and BSF-1 (IL 4): comparison of

350

52.

53.

54.

55.

56.

Anna Ridderstad et al. normal and recombinant, mouse and human IL 2 and BSF-1 (IL 4). J. Zmmunol. 138: 1813-1816 Houssiau, F. A., J. Devogelaer, J. Van Darnme, C. N. de Deuxchaisnes, and J. Van Snick. 1988. Interleukin-6 in synovial fluid and serum of patients with rheumatoid arthritis and other inflammatory arthritides. Arthritis Rheum. 31: 784-788 Hirano, T., T. Matsuda, M. Turner, N. Miyasaka, G. Buchan, B. Tang, K. Sato, M. Shimitsu, R. Maini, M. Feldmann, and T. Kishimoto. 1988. Excessive production of interleukin 6/B cell stimulatory factor-2 in rheumatoid arthritis. Eur. J. Zmmunol. 18: 1797-1801 Guerne, P.-A., B. L. Zuraw, J. H. Vaughan, D. A. Carson, and M. Lotz. 1989. Synovium as a source of interleukin 6 in vitro. Contribution to local and systemic manifestations of arthritis. J. Clin. Invest. 83: 585-592 Nawata, Y., E. M. Eugui, S. W. Lee, and A. C. Allison. 1989. IL-6 is the principal factor produced by synovia of patients with rheumatoid arthritis that induces B lymphocytes to secrete immunoglobulins. Ann. NY. Acad. Sci. 557: 230-239 Bhardwaj, N., U. Santhanam, L. L. Lau, S. B. Tatter, J. Ghrayeb, M. Rivelis, R. M. Steinman, P. B. Sehgal, and L. T. May. 1989. ILd/IFN-82 in synovial effusions of patients with rheumatoid arthritis and other arthritides. Identification of several isoforms and studies of cellular s0urces.J. Zmmunol. 143: 2153-2159