Rheuni [ngy

Rheumatol Int (1990) 10:203-210

Clinical and Experimental investigations

© Springer-Verlag 1990

Stimulation of rheumatoid synovial and blood T cells and lines by synovial fluid and interleukin-2: characterization of clones and recognition of a co-stimulatory effect N. Hain, S. Alsalameh, W . M . Bertling, J.R. Kalden, and G . R . Burmester *

Institute of Clinical Immunology and Rheumatology and the Max-Planck Society, Clinical Research Unit for Rheumatology, Department of Medicine III, University of Erlangen-Niirnberg, Krankenhausstrasse 12, W-8520 Erlangen, Federal Republic of Germany Received May 29, 1990/AcceptedAugust 1, 1990 Summary. Rheumatoid arthritis (RA) is characterized by

the presence of interleukin-2 (I1-2) receptor-positive T ceils in the peripheral blood and synovial compartments. Utilizing the limiting dilution technique, the precursor frequencies of I1-2 responsive T cells were determined in peripheral blood and synovial sites from RA patients and in the blood of normal donors. The frequencies of I1-2 responsive T cells were significantly higher in RA patients (range from 1/180 to 1/7432) compared to normal donors (range from 1/400 to 1/8163). T-cell clones raised by the addition of I1-2 alone were predominantly of the CD4positive phenotype. Peripheral blood T cells, synovial Tcell clones and lines derived from RA patients were costimulated with I1-2 and synovial fluid or supernatants from cultured synovial lining cells. This co-stimulation induced a strikingly enhanced proliferative T-cell response while synovial fluid alone was without effect. This stimulatory activity was found in the high molecular weight range (approximately 150 kDa) and could not be attributed to the action of immunoglobulins or known cytokines such as I1-2 or interleukin-1 (II-1), suggesting the activity of a material that modulates the I1-2-dependent growth of T cells. The co-stimulatory capacity of synovial fluid with I1-2 may be relevant to the activated state, especially of synovial T cells. Key words: Rheumatoid arthritis - Synovial fluid - Inter-

leukin-2 - Synovial T cells - Co-stimulatory effect

Introduction

Rheumatoid arthritis (RA) is characterized by the presence of activated T cells in the peripheral blood and synovial sites of inflammation. The activation is associated * To whom offprint requests should be sent at: Institute of Clinical Immunology and Rheumatology, Department of Medicine III, University of Erlangen-Niirnberg, Krankenhausstrasse 12, W-8520 Erlangen, Federal Republic of Germany

with the expression of certain cell surface markers, particularly MHC class II (Ia) antigens and the receptor for interleukin-2 (I1-2). Increased levels of activated T cells in RA patients have especially been found in inflamed synovial compartments where T cells represent the predominant infiltrating lymphoid cells [1-3]. Thus, these T cells may play a vital role in the auto-immune process, having acquired the receptor for I1-2 in vivo with the ability to respond to exogenous I1-2, resulting in the proliferation of cells [4, 5]. This state of pre-activation is accompanied by a reciprocally reduced responsiveness to mitogens and allogeneic cells, as well as an increased modulation of the T-cell receptor/CD3-complex [6-9]. Furthermore, since pre-activated T cells have been found abundantly in the synovial fluid, this may be of interest for studying activating substances possibly present in this environment. Investigations on the influence of synovial fluid on T-cell function and proliferation have shown high levels of II-I [10, 11], whereas II-2 was not demonstrable by most investigators [12-16]. Moreover, rather than factors enhancing T-cell activation, inhibitory properties for T-cell functions have recently been described and attributed to constituents of synovial fluid [17-20]. In addition, synovial fluid contains high levels of hyaluronic acid, which may exert various effects on the functions of T cells [21, 22]. Since it is possible to clone T cells and to maintain them in long-term culture by the addition of growth factors, functional properties and reactivity patterns are accessible for studies. For this purpose, the limiting dilution technique provides the opportunity of investigating T-cell functions at a clonal level. The most interesting cells appear to be those T cells which can be raised and selected by their ability to respond to II-2 without other stimuli [23]. In the present investigation, T cells from the peripheral blood of RA patients and normal blood donors were investigated using a limiting dilution system to determine the frequencies of I1-2 responsive T cells, as indicated by subsequent proliferation. T-cell clones, T-cell lines and fresh mononuclear cells, both derived from synovial sites and peripheral blood, were co-cultured with cell free

204 synovial fluids. These e x p e r i m e n t s d e m o n s t r a t e d a stimulatory capacity o f synovial fluids o n the p r o l i f e r a t i o n o f T cells, which was entirely d e p e n d e n t o n the co-stimulat i o n with I1-2.

Materials and methods Patients and controls. We studied 36 patients with RA, diagnosed according to the 1987 revised American College of Rheumatology Criteria, [24]. The median age was 51 years, with a range of 21 to 78. Of these, 14 were male and 22 were female. All patients had active disease, as determined by elevated erythrocyte sedimentation rate (> 50 mm/h), C-reactive protein (CRP) values (> 40 mg/1) and high joint scores. The majority received non-steroidal drugs and remission-inducing agents, primarily gold salts. Patients receiving more than 6 mg of prednisone or cytotoxic drugs were excluded from the study. Healthy blood donors served as controls.

Synovialfluid samples. Synovial fluid samples were usually aspirated from the knee joint of patients with RA. Cells were removed by centrifugation and the samples were stored at -20°C prior to the assays. Further samples were obtained from a traumatic joint injury, a reactive arthritis induced by Yersinia enterocolitica and from apparently normal joints undergoing surgery for bone tumours.

Preparation ofsynovial lining cells. Synovial tissues from seven patients were obtained after synovectomy. Tissue specimens were finely minced and enzymatically digested for 1-2 h (collagenase 1 mg/ ml, Seromed, Munich, FRG, DNAse 0.15 mg/ml, Paesel, Frankfurt, FRG). The resulting cell suspensions were filtered (88-m pores) and the cells were obtained from a Ficoll-Hypaque density gradient. The cells were washed twice and seeded in a culture flask (5 x 105/ml). Generally, synovial cells were divided once a week and maintained for several passages in continuous culture. Supernatants of cultures were collected after keeping them in serum-free medium for 24 h.

Isolation of peripheral blood and synovial fluid/tissue lymphocytes. Lymphoeytes were isolated by Ficoll-Hypaque density gradient centrifugation. They were washed twice and resuspended in RPMI containing 10% FCS, penicillin and streptomycin. Synovial tissue T cells were isolated from enzymaticallydigested tissue by E-rosetting and Percoll gradients as described previously [1].

Generation ofT-cell clones and T-cell lines. T-cell lines from synovial fluid/tissue and peripheral blood were generated by the addition of recombinant I1-2 (10 U/ml, Biotest, Offenbach, FRG) to the culture medium. We seeded 1 x l0 s ceils in 96 well plates (Nunc). Outgrowing cultures were fed with fresh I1-2containing medium once a week. T-cell clones were raised by limiting dilution technique according to a modified cloning protocol [25] as described below. Around day 14, growing clones were transferred to 48-well plates in fresh medium supplemented with I1-2 and suboptimal concentration of mitogen (PHA 0.1 ~tg/ml). Clones were fed with I1-2 every 3rd to 7th day according to their growth properties. Stimulation oJ T cells with synovial fluids and supernatants. We incubated 50 000 freshly isolated mononuclear cells from different compartments with cell free synovial fluids (10%) in the presence or absence of exogenous II-2 (10 U/ml). From each T-cell clone or line, 20 000 cells were incubated with synovial fluid (10%) and supernatants obtained from rheumatoid non-lymphoid synovial cells (10%). Cultures were grown for 3 and 5 days, respectively, and labelled with ~H-thymidin (1 p.Ci/well) for 18 h. Cells were harvested on glass fibre filters. The filters were dried and counted in a liquid scintillation counter.

Immunofluoreseence staining. For fluorescence staining of T-cell surface antigens, the following monoclonal antibodies were used: 91d6

[26] (anti-CD4, helper/inducer T cells), 89bl [26] (anti-CD3 "pan" mature T cells), OKT8 [27] (suppressor/cytotoxic T cells, Ortho, Heidelberg, FRG), Tac [28] (anti-CD 25), and OkIal (HLA-DR constant region, Ortho). The monoelonal antibody anti-Tac detecting the 55 kDA chain of the I1-2 receptor was kindly provided by Dr. T. Waldman (NIH, Bethesda, Md., USA). The evaluation of lymphocyte subsets was performed by cell-sorter analysis (EPICS V, Coulter) as previously described [1].

Precursor frequency analysis. Precursor frequency analysis were performed by limiting dilution technique, carried out in 96-well round bottom microtiter-plates (Nunc). Each well contained 100 000 autologous mononuclear blood cells as feeder cells irradiated with 60 Gy in a final volume of 0.2 ml. Lymphoid cells used for dilution were incubated in plastic dishes to deplete adherent monocytes. These cells were stained for Tac antigen expression. Each single dilution step was comprised of 16 wells containing serial twofold dilutions of cells, starting with 16 384 down to 16 cells per well. The culture medium, RPMI 1640 was supplemented either with autologous serum or AB serum and exogenous I1-2 (Biotest, 10 U/ml). After 10 days of incubation (6.5% CO/37 °C), cultures were microscopically scored for growth (Leitz, Wetzlar). Gel filtration of synovial fluids. We applied 0.5 ml synovial fluid to a gelfiltration column (Superose 12, Pharmacia) of a FPLC system. Fractions of 1 ml were collected, dialysed against medium and tested in the proliferation assay in a dilution of 1/4. For the prepartion of human IgG a protein A column (Pharmacia) was used. Five milliliters of synovial fluid containing I1-2 enhancing activity was passed through the column (Tris-HCl buffer) and IgG was eluted by different pH values ranging from 8 to 4.5 (citrate buffer). These samples were dialysed against culture medium and adjusted to the original concentration. In the biological test system, samples were diluted to a final of 1/4. Statistical evaluation. The results of the limiting dilution experiments were analysed according to Poisson distribution and maximum likelihood was used as a statistical estimator [29]. The probabilities for the computed chi-square, which is a value of the goodness of fit to Poisson distribution, were determined for all frequencies. Values of less than 0.05 were consistent with the Poisson model at the 95% level. The Mann-Whitney rank-order test was used for the evaluation of statistical significance of corresponding samples. The relationship between precursor frequencies towards Tac + T cells was calculated using stepwise multiple regression analysis.

Results Increased frequencies o f II-2 responsive T cells in patients with R A . T h e limiting d i l u t i o n cell system used allows the possibility o f e v a l u a t i n g the p r e c u r s o r frequency o f a n y cell t h a t is able to r e s p o n d to I1-2 b y establishing a cell culture. The results are s h o w n in Table 1. The precursor frequencies o f T cells giving rise to a single cell culture were significantly higher in the peripheral b l o o d o f R A patients ( P < 0 . 0 5 ) c o m p a r e d to n o r m a l donors. T h e y r a n g e d f r o m 1/184 to 1/7432 (median: 393) c o m p a r e d to 1/400 to 1/8163 (median: 1113) in n o r m a l b l o o d d o n o r s . These results were c o m p a r e d to the p r o p o r t i o n o f I1-2 receptor-positive T cells in the original cell samples as j u d g e d by the anti-Tac a n t i b o d y . T h e p r o p o r t i o n o f Tac positive T cells was significantly elevated in the p a t i e n t g r o u p (range 4 to 2 4 % , P < 0 . 0 5 ) . However, the high n u m b e r s of I1-2-receptor-positive cells did n o t correlate with the n u m b e r s o f precursors (r----0.25 P = 0.5). T h u s , in parallel to the d e t e r m i n a t i o n of p r e c u r s o r frequencies,

205 Table 1. Precursor frequencies of in vivo activated T cells as compared to the expression of the I1-2 receptor (CD 25) and the I1-2 response of T lymphocytes. PB = Peripheral blood; ST = synovial tissue; Tn/Ts/c= T helper/T suppressor/cytotoxic cells

•~

Controls

"~

30

,~

25

Frequency

CD25+T cells (%)

TH/Ts/c Responseto ratio I1-2 of PBMC

16 0 5 10 6 3 0 9 0 3

2

1.4 1.9 1.7 1.3 1.1 2.2 2.7 1.3 1.8

27 231 22 580 31 428 3 482 1 740 6 636 12010 997 33 752

9 4 24 14 10 9 12 6 7 21 12

3.5 1.4 6.1 6.6 4.6 1.4 4.6 1.8 -

10 243 22 087 23 901 3 887 6 869 1 925 14 332 14913 1 829 21 794 3 627

45 40

M

~

I

sera

RA

I synovial fluids

EeA TR

PB 400 a 2 PB 713 ~ 3 PB 748 4 PB 789 c 5 PB 943 c 6 PB 1282c 7 PB 2494 8 PB 2696 ¢ 9 PB 2788 10 PB 8163 RA patients 1 PB 184 2 PB 307 ¢ 3 PB 358 4 PB 360 5 PB 393 c 6 PB 749 7 PB 818 c 8 PB 884 ~ 9 PB 5915 10 ST 360 11 ST 7432 1

6 721 b

a Reciprocal value b Adpm Experiment done with autologous serum bulk cultures of peripheral blood mononuclear cells (PBMC) were generated by the addition of I1-2, and the proliferative responses were determined. These experiments were indicative of a similarity between precursor frequencies and the I1-2 induced proliferation of mononuclear cells in bulk cultures. In three patients, however (nos. 7, 8, 10), and two normal blood donors (nos. 8, 10), there was a divergence with donors showing low-precursor frequencies, but demonstrating a marked proliferative response in the bulk culture system.

T-cell clones generated by a primary cloning procedure. Utilizing the limiting dilution technique, T-cell clones from R A patients were raised from peripheral blood and synovial compartments solely by the addition of exogenous I1-2. Wells containing positive cultures fitting the linear curve expected from the Poisson distribution were considered to be o f monoclonal origin. These clones were expanded by I1-2 and suboptimal concentration of P H A (0.1 gg/ml). They were generally expanded up to an average of 3 million cells before being tested for restimulation. Fluorescence staining showed that 92% of clones (n = 52) displayed the CD4 antigen irrespective of their origin. Interestingly, three clones in the patient group were double-positive for the CD4 and CD8 antigen, whereas no clone with these characteristics emerged in normal blood donors.

Stimulation of T cells by synovial fluids: synovial fluid enhances T-cell proliferation in conjunction with Il-2. In

20

.C

10

o

Fig. 1. Stimulation of PBMC of one patient by various sera and synovial fluids in the presence of IL-2. We cultured 50 000 PBMC in FCS (5%) and I1-2(10 U/ml) containing medium that was additionally supplemented with 10% of different synovial fluid and serum samples. TR=traumatic fluid; ReA=Yersmia reactive arthritis; MA=monarthritis. Similar results were obtained with PBMC from another two RA patients (not illustrated) search for an antigen reactivity of activated rheumatoid T cells, initial experiments were designed to investigate whether the intra-articular environment, as examplified by synovial fluid, contains material stimulatory for T cells. Thus, peripheral blood and synovial mononuclear cells from patients with R A were incubated with cell free synovial fluid and autologous or allogeneic sera of R A and normal donors in the presence of I1-2. Out of the 15 R A synovial fluids used, 11 were tested in an autologous cell system (donors 1 to 11) and 9 were examined on 17 allogeneic cell preparations (donors 12 to 28). The data shown in Table 2 demonstrate a striking enhancement of T-cell reactivity induced by R A synovial fluid, while control sera either of R A or normal origin generally were not effective. In the vast majority of experiments, incubation with R A synovial fluid enhanced the proliferative response of T cells to I1-2. Thus, synovial fluid appeared to contain material co-stimulatory with I1-2. The augmentation of T-cell proliferation, however, was only evident upon co-stimulation with I1-2, while synovial fluid usually had no significant stimulatory capacity except for two experiments (nos. 5, 15). N o co-stimulation was detectable in experiments with apparently normal joint fluid (nos. 29, 30). Similar enhancement of the T-cell proliferation was not observed by the addition of exogenous II-1. Figure 1 illustrates a typical experiment where synovial fluid and sera o f various sources were examined showing the strong activating capacity of rheumatoid synovial fluid. The stimulating property was not only demonstrable with synovial fluid from RA, but was also evident in joint effusions from a reactive arthritis and a traumatic arthritis, but not with serum from patients with R A or normal donors. Moreover, the synergistic effect with I1-2 was apparent in mitogen and semi-antigendriven T-cell systems, as seen with P H A and the antiCD3 antibody 89bl (Fig. 2). When PBMCs were pre-incubated both in sera and synovial fluid for periods of 4, 12, and 18 h, respectively, and then washed, there was no enhancement of the subsequent I1-2 induced proliferation

206 Table 2. Stimulation of peripheral blood, synovial fluid and synovial tissue T lymphocytes by addition of I1-2 and synovial fluid (SF) Donor

1 SF ST 2 PB SF 3 ST 4 PB 5 PB 6 SF

Autologous sera and autologous SF

(1) a (1) [11] (2) (2) (3) (4) (5) (6)

SF (10%)

Sera + IL-2 (10%/10 U/ml)

SF + IL-2 (10%/10 U/ml)

IL-2 + IL-1 (10 U + 10 U/ml)

1.089 b 2.245 0.681 0.963 1.198 2.923 30.185 4.052

4.359 4.922 8.952 1.924 8.062 4.676 18.622 17.125

32.302 17.756 18.981 3.370 7.987 16.522 51.797 15.669

13.840 7.433 -

52.332 82.184 16.051 154.508 12.885 12.239 13.284

5.133

18.831 9.660 18.336 9.738 5.457 -

Allogeneic sera and autologous SF 7 PB SF 8 PB 9 PB 10 PB SF 11 SF

(7) (7) (8) (9) c (10) (10) (11)

1.170 0.903 5.667 2.522 0.710 1.545 2.004

0.000 0.607 16.312 15.049 7.769 4.945 9.970

Allogeneic sera and allogeneic SF 12 ST 13 ST 14 ST 15 PB 16 PB 17 PB 18 PB 19 PB 20 PB 21 PB 22 PB 23 PB 24 PB 25 SF 26 ST ST 27 PB 28 PB

(G) a (G) (F) (7) [1] (7) [5] (9) [3] (7) (7) (7) [4] (8) [9] (8) (S) (2) (7) (F) (T) (1) [6] (S)

29 PB (nSF) e 30 PB (nSF)

1.454 4.199 2.320 10.069 1.763 2.311 0.791 1.113 1.492 0.774 0.960 1.271 1.335 0.777 0.986 1.039 1.080 1.859

17.003 12.642 21.349 10.616 7.241 33.185 0.922 16.278 7.150 2.098 0.814 14.487 17.262 129.074 28.519 28.519 3.819 5.546

38.289 22.812 28.753 19.007 23.683 32.919 21.867 44.624 44.811 4.884 2.159 12.461 17.307 60.514 34.703 36.353 6.107 28.708

5.067 14.310

13.427 54.955

14.058 41.525

-

a Synovial fluid sample of corresponding or allogeneic donors respectively b A dpm, mean values of triplicate cultures Value measured on day 10 d Initial of donor e nSF = normal SF. Numbers in [] correspond to patients in Table 1. Medium values for all experiments < 1,500 dpm (data n o t shown). To f u r t h e r characterize the effect o f synovial fluid o n T cells, synovial fluids were diluted several times a n d tested i n a similar fashion. These experim e n t s d e m o n s t r a t e d that even in very low c o n c e n t r a t i o n s ( d o w n to 0 . 3 % ) s y n o v i a l fluid r e t a i n e d its s t i m u l a t o r y activity whereas high c o n c e n t r a t i o n s o f synovial fluid (20 to 100% o f culture m e d i u m ) d i m i n i s h e d the proliferative s t r e n g t h o f the cells (data n o t shown).

Stimulation of T-cell clones and lines with supernatants from synovial lining cells and rheumatoid synovial fluid. T-cell clones f r o m R A p a t i e n t s were generated as described above. These clones were tested for r e s t i m u l a t i o n a n d r e - a c t i v a t i o n b y m i t o g e n , s u p e r n a t a n t s f r o m syn-

ovial lining cells a n d cell-free synovial fluids f r o m R A patients. T-cell clones generated f r o m the synovial tissue were i n c u b a t e d with s u p e r n a t a n t s f r o m r h e u m a t o i d n o n l y m p h o i d s y n o v i a l - l i n i n g cells. T h e s u p e r n a t a n t s enh a n c e d the proliferative responses o f the T-cell clones u p to m o r e t h a n twice the c o n t r o l values (Fig. 3: clones 1 a n d 4). I n f u r t h e r experiments, T-cell clones from synovial fluid were tested for the proliferative response to 11-2 in the presence o f synovial fluid, the a n t i - C D 3 a n t i b o d y 8 9 b l , a n d a s u p e r n a t a n t o f T cells activated by P H A (Fig. 4). All T-cell clones were m o s t effectively s t i m u l a t e d by the s u p e r n a t a n t o f P H A s t i m u l a t e d T cells. The antib o d y 8 9 b l was able to stimulate the 3 H - t h y m i d i n u p t a k e even in the absence o f m a c r o p h a g e s w h i c h were n o longer

207 50 ~o

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AB s e r u m RA SF RA SF TR SF

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No.

anti-CD3

PHA

I 5

Fig. 2. Synovial fluid supports the anti-CD3 triggered activation of T cells. From one RA patient 50 000 PBMC were activated by PHA or the anti-CD3 antibody 89bl in the presence of synovial fluids and control serum. This result was reproduced with another two RA patients. TR = trauma; CTC = cultured T cells without stimulus

of

fraction

Fig. 5. Gel filtration of synovial fluids. RA synovial-fluid-derived fresh T cells were used as target cells in the proliferation assay. The main peak of stimulation is included in the high molecular weight fractions, as indicated by the molecular markers

7O

111-2

I )¢

6O

~

I1-2 + SLC-SN

50

~x 40

.E

30 20

I ~" clone

1

2

3

6

5

4

wiLhouL I1-2

Fig. 3. Stimulating effect of supernatants from synovial lining cells (SLC-SN) on synovial tissue derived T-cell clones. T-cell clones were raised under limiting dilution conditions and expanded with 11-2. After 4 weeks, 20 000 cells of a T-cell clones were restimulated by I1-2and serum-free supernatants from synovial lining cells of the third passage

2O

['-'-'7 IL-2 [ ~ IL-2 and SF

N "0

~L

15

lO

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n§ 2 T-cell

n

3

4

clones

from

5 synovial

6

7

fluid

Fig. 4. Synovial fluid supports the restimulation of synovial fluidderived T-cell clones; 20 000 cells of 28-day-old T-cell clones were incubated with I1-2 and SF plus I1-2

present in the clonal T-cell population after 20 days of culture (data not shown). Furthermore, the proliferation of six clones ( 1 - 6 ) in the presence of aUogeneic synovial fluid was two- to fourfold higher c o m p a r e d to control sera. In this assay, synovial fluid alone exerted only a marginal effect on the proliferation of the T-cell clones. Similarly, the enhancement of the proliferation was also evident, when T-cell lines derived from the peripheral blood from patients were used. In contrast, the co-stimulatory activity was not obtained with a T-cell line derived from a normal d o n o r and specific for tetanus toxoid (Table 3) and with the cytotoxic mouse T-cell line CTLL-2.

Gelfiltration ofsynovialfluids. Three R A synovial fluids were filtered on a molecular sieve column and the fractions obtained were tested for stimulatory activity. Three T-cell preparations derived f r o m the synovial fluid/tissue (two from synovial fluid, one f r o m synovial tissue) were used in the proliferation assay. These experiments revealed a co-stimulatory activity in the high molecular weight fractions with a peak at a b o u t 150 kDa. Figure 5 shows a representative example for the proliferation of synovial fluid T cells in the presence of I1-2 and the fractions, which were eluted f r o m the molecular sieve column. Similar data were obtained in two additional experiments (not illustrated). P B M C from three normal donors tested in parallel were not co-stimulated by the fractions of 150 kDA. In order to test whether the T-cell stimulation was due to the activity of immunoglobulins, synovial fluid with strong T-cell stimulatory activity was applied to a protein A column. The IgG-containing eluent was tested for stimulatory capacity on peripheral blood cells from two R A patients and a T-cell line derived from R A synovial tissue. Synovial I g G was not able to enhance the proliferation of T cells; however, no co-stimulation could be detected in the effluent of the column, suggesting a possible loss of biological activity of synovial fluid by application to the column (Table 4).

208 Table 3. Synovial fluid does not affect a tetanus toxoid dependent T cell line and the mouse T cell line CTLL-2.50 000 T cells from the T-cell lines were incubated with various SF and AB serum as control. On day 30 the T-cell lines were tested for restimulation with I1-2 and supplements as described; 10 000 cells from the CTLL-2 line were tested for growth in the presence of SFs and Ab serum

AB serum IL-2 SF 1 b SF 29 SF 30

TT line

CTLL-2

ST-T-line 1

PB-line 2

PB-1 line 3

PB-line4

54 249 a 53 568 26 918 48 679 43 350

8 442 166 279 1 043 I 127 1 396

6 190 8 178 29 315 30 096 -

18 622 19 257 -46 177 33 177

6 494 -24 575

4 546 -8 889

Adpm b Number corresponds to those given in Table 2 Table 4. SF-immunoglobulin has no effect on the T-cell prolifera-

tion. RA peripheral blood and a synovial tissue derived T-cell lines were incubated with serum, SF, and the corresponding preparation of IgG, which was adjusted to the former IgG level of SF Target cell

Serum + IL-2

SF + IL-2

SF-IgG + IL-2

Effluent

PB (RA) PB (RA) RA-ST-T line

50 000 a 13 312 525

82 637 32 980 6 327

50 147 15 961 959

42 354 16 217 199

a Adpm

Discussion

The objective of the present study was to characterize T cells capable of responding spontaneously to 11-2 and to investigate synovial fluid for its effects on the proliferation of T cells. Two principle findings were obtained: firstly, the precursor frequencies of T cells responsive to exogenous I1-2 were significantly higher in individuals with R A as c o m p a r e d to normal blood donors. This fact reflected the elevated levels of 11-2 receptor positive T cells in the peripheral blood and joints of R A patients. Secondly, synovial fluid stimulated the proliferation of R A P B M C , T-cell lines and T-cell clones in a co-stimulatory fashion, together with exogenous 11-2. The evaluation of the precursor frequencies of I1-2-responsive T cells by limiting dilution is based on their capacity to f o r m a monoclonal cell culture u p o n addition of 11-2. Accordingly, the test system selects mainly I1-2-receptor-positive T cells which have presumably acquired 11-2 responsiveness in vivo by contact with unidentified antigen(s). A significantly increased outgrowth of peripheral blood T cells could be demonstrated in patients c o m p a r e d with control subjects. These results are in agreement with the investigations of other laboratories [30-32], which obtained similar frequencies in peripheral blood, but with less variations in the frequencies. H o w ever, additional data obtained in the present investigation demonstrated that it was not possible to correlate directly the precursor frequencies with the proportion of T cells expressing the 11-2 receptor, as determined with the anti-Tac antibody. This divergence is m o s t likely caused by the differential binding affinity for 11-2 by the 11-2 receptor, which is composed of two chains with low or high affinity for 11-2, indicating different stages of T-cell activation. Since the anti-Tac antibody detects the

beta chain, Tac antigen expression does not necessarily correspond to the strength o f T-cell proliferation [33, 34]. However, it was generally observed that R A T cells were more active since establishing and maintaining I1-2-dependent T-cell clones was a consistent finding in this patient group while it was only occasionally possible to raise lines from normal blood donors. In rheumatoid inflammation, the synovial compartments are the main targets o f T-cell activity, as demonstrated by the presence of large amounts of activated T cells [2, 3]. Possibly, synovial tissue and synovial fluid represent a special environment that either induces or maintains T-cell activation. To look for possible activating factors present in these compartments, co-culture experiments were performed in which T cells were incubated with both synovial fluid and I1-2. This experimental approach was chosen since preliminary experiments carried out in our laboratory and known data from the literature [35] had not demonstrated a significant stimulatory effect of synovial fluid alone. The results obtained showed the strong capability of synovial fluids to enhance the I1-2-induced proliferation of cell preparations containing activated T cells. This co-stimulatory effect was not confined to R A synovial fluid, but was also evident in effusions f r o m arthritides induced by Yersinia or trauma. However, it was not demonstrable in presumably normal joint fluid from surgical material. These findings suggest the presence of a lymphotropic factor(s), which is active in several conditions leading to inflammation. The observations of a similarly enhancing effect of synovial lining cell supernatants strongly suggest that upon joint inflammation synovial lining cells release a factor(s) into the joint fluid, which is able to enhance I1-2-induced Tcell proliferation. Considerable attention was paid to the possible action of known T-cell activating factors such as 11-1 or 11-2. While significant amounts of I1-1 were found in synovial fluid samples, free 11-2 was not detectable (data not shown). Moreover, in our study the addition of large amounts of I1-1 or 11-2 to the T cells investigated did not show the stimulatory effects seen with synovial fluids. Thus, it is highly unlikely that the effect described can be attributed to the action of 11-1 or II-2 present in the synovial fluid. Additional experiments demonstrated that synovial I g G was not able to enhance T-cell proliferation. The loss of T-cell stimulating activity after passage through the protein-A column m a y be due to a nonspecific trapping of the 11-2 enhancing activity. Furthermore, additional experiments argue against immunglobu-

209 lins being responsible for the effects described. Thus, filtration o f several synovial fluids on an anion-exchange c o l u m n did n o t show a co-elution o f T-cell stimulatory fractions with I g G or I g M fractions (Hain et al., to be published). O n the target cell level, the I1-2 enhancing effects were evident in a variety o f R A cell preparations that all contained activated T cells. Thus, peripheral b l o o d cells, b l o o d and synovial T-cell lines a n d clones raised by the addition o f I1-2 were equally susceptible to the co-stimulatory effect o f synovial fluids and I1-2. O f special interest, n o t only n o r m a l peripheral b l o o d cells, b u t also the restimulation o f a h u m a n tetanus t o x o i d - d e p e n d e n t Tcell line or the m o u s e C T L L line were unaffected by synovial fluids. These data suggest that the target cell is o f prime i m p o r t a n c e in the co-stimulation assays with synovial fluid and I1-2, and the best targets a p p e a r to be R A T cells. These results m a y also explain the seemingly contradictory data by Miossec et al. [17] and E m e r y et al. [19] w h o d e m o n s t r a t e d inhibitory effects o f R A synovial fluids in different assay systems o f m o u s e or h u m a n origin. These data could be reconciled by the hypothesis that synovial fluid contains a factor(s) binding or m o d u l a t i n g the action o f I1-2, either resulting in the activation or inhibition o f T cells strongly dependent on the target T cell used. I n this respect, biochemical d a t a o f a molecular weight o f 150 k D a o f such a substance (Miossec et al. [17]) are in g o o d agreement with the 11-2 co-stimulatory activity o f a high molecular weight material o f synovial fluid origin f o u n d in experiments by o u r g r o u p [23]. A n alternative hypothesis m a y be the presence o f antigenic material contained in synovial fluid and possibly released by synovial lining cells or the affected cartilage [36, 37]. Thus, proteoglycans [38] and collagens [39] have been d e m o n s t r a t e d as possible targets o f an a u t o i m m u n e process. However, in the present investigation, synovial fluid alone was never able to induce an antigen like stimulation in the T-cell preparations examined. This was always strongly dependent on the addition o f exogenous I1-2. A d d i t i o n a l biochemical analysis o f inflamed synovial fluid, which is o n g o i n g in o u r l a b o r a t o r y , on this c o m p l e x material will be necessary to gain m o r e insight into the activities m o d u l a t i n g T-cell functions. In any event, the d a t a obtained so far strongly s u p p o r t the hypothesis that the synovial c o m p a r t m e n t is an especially permissive env i r o n m e n t for T-cell activation.

Acknowledgements. Supported by the Sanderstiftung,

Grant 87.025.1, and the Deutsche Forschungs-Gemeinschaft, Grant Bu 445/2-5.

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