Expression of TLiSA1 on T cells from patients with rheumatoid arthritis and systemic lupus erythematosus

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

52, 366375 (1989)

Expression of TLiSAl on T Cells from Patients with Rheumatoid Arthritis and Systemic Lupus Erythematosus HIROTSUGU TABATA, MASAKO HARA, ATSUSHI KENICHI NORIOKA, MASAYOSHI HARIGAI, MAKOTO KAWAKAMI, MITSUHIRO KAWAGOE,

KITANI, TATSUO HIROSE, KIMIHIRO SUZUKI, AND HARUO NAKAMURA

1st Department of Internal Medicine, National Defense Medical College. Saitama, Japan The expression of a new activation antigen, T cell lineage specific activation antigen (TLiSAl) on peripheral blood T cells from 16 rheumatoid arthritis (RA) and 8 systemic lupus erythematosus (SLE) patients and synovial fluid T cells from RA patients was determined in the context of T cell activation. The percentages of TLiSAl positive T cells from inactive (4.6 2 5.2, means +- SE) or active RA (19.3 t 8.6) or inactive (1.7 t 2.1) or active SLE (8.7 2 2.7) were signitkantly increased compared with that of normal controls (0.7 rfr 0.4) (P < 0.01). All patients with vasculitis showed relatively high positive percentages. The mean fluorocytometric intensity of TLiSAl positive T cells from RA and SLE patients was significantly higher than that from normals. Percentages of TLiSAl positive T cells from synovial fluids (21.8 ? 4.9%) were significantly increased compared with those from peripheral blood of the same patients, indicating the local activation of T cells in patients with RA. An increase in the expression of TLiSAl with no increase in the expression of the very late activating antigen 1 (VLA-I) was found in peripheral blood from RA, suggesting a difference in the stage of T cell activation in RA. In RA, there was a clinical correlation with levels of TLiSAl expression on peripheral T cells. After stimulation with PHA, TLiSAl positive percentages were increased on Day 2 and continued to increase through 5 days of culture. The maximum expression was obtained on Day 5. An increased number of TLiSAl positive T cells belonged to OKT8. These results suggest that there is the systemic and the local activation of T cells in RA, following antigen stimulation, or a generalized nonspecific activation of immune system that could provide a means to monitor the abnormal immunologic activity in RA. Q 1989 Academic

Press. Inc.

INTRODUCTION

Several immunopathological conditions have been associated with rheumatoid arthritis (RA). Despite the immunoregulatory defects observed in T cells from patients with RA, evidence for in viva activation of RA peripheral blood T cells is the main issue to be resolved. Activated proliferating lymphocytes are a feature of RA. They are present in the synovial membrane, synovial fluid (SF), and peripheral blood (PB), probably as a result of activation by several immune substances, including interleukin (IL) 1 (1, 2), IL-2 (3, 4), and interferon (5, 6) and BCDF activity which we have previously reported (7). Several activation antigens have been measured on T cells from patients with RA in order to understand the role of activated T cells in disease. Increased numbers of the class II (Ia) antigen positive T cells have been found in peripheral blood (8), synovial fluid (9), and synovium (IO). Interleukin 2 receptor (I I), OKTlO (12), and very late activating antigen 1 (VLA-1) (13) have also been found 366 OWO-1229189 $1.50 Copyright 0 1989 by Academic Press. Inc. All rights of reproduction in any form reserved.

TLiSAl

ON

T CELLS

FROM

RA AND

367

SLE

on synovial T cells in RA. These results imply that injury in RA involves activated T cells. Recently, Bums et al. (14) reported a new monoclonal antibody to TLiSAl, a T lineage specific activation antigen, which were expressed on activated T cells. They indicated that the presence of antibodies to TLiSAl inhibits the differentiation to cytotoxic T lymphocyte (CTL) during the stimulation of PHA or mixed lymphocyte culture (MLC), while having no effect on cell proliferation or on effector cell function and that this antigen may function as a receptor for the T cell differentiation factor. Therefore, TLiSAl may be useful in the study of major disease associated with the presence of CTL. In autoimmune disorders such as RA and multiple sclerosis (MS), CTL may be involved in the tissue destruction which is characteristic of these diseases. This marker will be especially useful for analyzing the characteristics of T cells involved in collagen diseases. We determined the extent of TLiSAl expression on peripheral blood T cells from RA and systemic lupus erythematosus (SLE) and synovial fluid T cells from RA patients in the context of T cell activation. MATERIALS

AND METHODS

1. Materials

Sixteen patients with RA, including 5 patients with vasculitis characterized by leg ulcer, cutaneous vasculitis, peripheral neuropathy, and gangrane, fulfilled the American Rheumatism Associations (ARA) criteria of the diagnosis of RA (15), and 8 patients with SLE fulfilled the ARA revised criteria of SLE (16) and 10 normal controls, age and sex matched, were examined. Activity was determined by Lansbury’s index (17) for RA and Urowitz’s activity criteria count (18) for SLE. The clinical activity of RA with vasculitis was evaluated basically by Lansbury’s activity index and additionally by the progression of vasculitis according to the report by Abe et al. (19). 2. Cell Separation

Mononuclear cells (MNCs) were separated from heparinized peripheral blood or synovial fluid by Ficoll
T cells (1

x

lO’?ml) were cultured in RPM1 1640 (GIBCO)

medium

containing

TABATA

368

ET AL.

10% FCS, penicillin (100 U/ml), and streptomycin (Difco, Detroit, MI) at 37°C in 5% CO, humidified 4. Fluorocytometric

(100 pg/ml) with 1% PHA-M atmosphere for several days.

Analysis

After 1 x lo6 T cells were treated with mouse irrelevant y-globulin at 0°C for 30 min for blocking the Fc receptor, 5 pl of FITC-conjugated TLiSAl antibody (T Cell Sciences Inc., Cambridge, MA) or FITC-conjugated VLA-I antibody (T Cell Sciences Inc.) was added and incubated at 4°C for 30 min. Cells were then washed three times with PBS and analyzed using a fluorescence-activated cell sorter, Orth Spectrum III (Ortho Diagnostic Systems Inc., Raritan, NY). Control cells were stained FITC-labeled mouse IgG (control IgG; Ortho Diagnostic Systems Inc). In some cases, T cells were analyzed by dual staining with FITC-TLiSAl and PEOKT3, PE-OKT4, or PE-OKT8 (Ortho Diagnostic Systems Inc.). A two-color analysis by PE-OKT3 and FITC-TLiSAl revealed that TLiSAI positive cells were always within T cell subset. 5. Statistical Statistical

Analysis analysis was done by means of Wilcoxon

and paired

t

test.

RESULTS

1. Fluorocytometric

Profile

of T Cells Stained with TLiSAl

(Fig. I)

T cells were isolated from peripheral blood of a normal control and RA and from synovial fluid of the same RA patient. The cells were stained with FITCconjugated TLiSAl. Figure 1 shows a representative fluorocytometric profile.

A-l (PBL)

control

0 100200

B-l (PBL)

0

GR-FL A-P(PBL)

100

control

C-l (SFL)

200

0

B-2(PBL)

C-P(SFL)

TLiSA-1

7.3%

n

TLiSA-1

15.9%

GR-FL (Normal)

200

GR-FL

GR-FL TLISA-1

100

control

GR-FL
FIG. 1. Fluorocytometric profile of T cells stained with TLiSA 1. T cells were isolated from peripheral blood of a normal control (A) and RA (B) and synovial fluid (C) of the same patient. T cells were stained with FITC-conjugated TLiSAl .

TLiSAl

ON T CELLS FROM RA AND SLE

369

Although normal peripheral T cells had almost negative TLiSAl staining, peripheral T cells from RA showed 7.3% positive TLiSAl and the mean fluorointensity (MFI) was increased. Moreover, T cells from synovial fluid of the same patients showed increased positive percentages of TLiSAl (15.9%) compared with T cells from peripheral blood. 2. Expression of TLiSAl

on Peripheral T Cells (Fig. 2)

T cells from peripheral blood of 10 normals and 10 inactive and 6 active RA patients, including 5 patients with vasculitis, and 5 inactive and 4 active SLE were stained with FITC-conjugated TLiSAl and analyzed by flow cytometry. The percentages of TLiSAl positive T cells from active FU (19.3 + 8.6 means 2 SE) were significantly increased compared with those of normal controls (0.7 f 0.4) (P < 0.01). There was also a significant difference between active and inactive RA (4.6 k 5.2) (P < 0.01). Active SLE (8.7 f 2.7) showed the tendency of increased percentages of TLiSAl positive T cells, but there was no significant difference between active and inactive SLE (1.7 + 2.1). All patients with vasculitis (0) showed the tendency of high positive percentages.

Q

. .

-I& normal N

; 0 .I . . . Am

inactive

active

RA

. .. I. I. .

active

inactive

SLE

FIG. 2. Expression of TLiSAl on peripheral T cells. Peripheral T cells from 10 normal, 16 RA, and 8 SLE were stained with TLiSAl and analyzed by Ortho Spectrum III. Double circle (8) means RA patients with vasculitis. Horizontal lines expressed mean ? SE. NS, not significant.

370

TABATA

ET AL.

3. Mean Fluorocytometric Intensity of Peripheral T Cells Stained with FITC-TLiSAl (Table 1) The MFI of TLiSAl positive T cells from 16 RA and 8 SLE patients was significantly higher than that from 10 normal controls (P < 0.005). 4. The Comparison of the Expression of TLiSAl and VLA-I on T Cells (Table 2) The expression of TLiSAl was compared with VLA-1 in peripheral T cells soon after drown from 8 cases of RA, 5 cases of SLE, and 5 normal without stimulation. VLA-1 usually appears slowly and is not highly expressed until 2-3 weeks after stimulation. The expression of VLA-1 on T cells from RA did not significantly increase, in contrast with the increased percentages of TLiSAl positive T cells. These results imply a certain activation stage of RA T cells. 5. Comparison of Percentage of TLiSAl Positive T Cells from Peripheral Blood and Synovial Fluid of Patients with RA (Fig. 3) The expression of TLiSAl on T cells from peripheral blood and synovial fluid was examined simultaneously in 10 RA patients. Percentages of TLiSAl positive T cells from synovial fluid (21.8 + 4.9%) were significantly increased compared with those from peripheral blood (11.7 2 3.6%, P < O.Ol), indicating the local activation of T cells in patients with RA. 6. Relationship between Percentages of TLiSAl Positive T Cells and Clinical Features in RA (Fig. 4) These are cases whose percentages of TLiSAl positive T cells were serially examined. Case 1 showed low percentages of TLiSAl during the inactive stage but the percentages were gradually increased with exacerbation. Case 2 shows that the high percentages of TLiSAl positive T cells decreased following the reduction of disease activity. Two additional cases showed the same course as case 2. 7. Kinetics of the Expression of TLiSAl on Peripheral T Cells after Stimulation with PHA (Fig. 5) Peripheral T cells were stimulated with 1% PHA-M and monitored for the expression of TLiSAl at various intervals. Six representative experiments are TABLE MEAN

FLUOROCYTOMETRIC

-~ Normal RA SLE

INTENSITY

1

OF PERIPHERAL

TLiSAl

‘I

26.3 2 8.5 51.7 k 19.3 * 51.8 -t 27.7

Note. Results were expressed as mean k SD, * P < 0.005.

T CELLS

STAINED

* ____--

WITH

FITC-TLiSAI

Control __--~ 23.6 + 9.7 25.0 k 9.5 22.9 -+ 7.6

TLiSAl

TABLE THE COMPARISON

371

ON T CELLS FROM RA AND SLE

OF THE EXPRESION

2 TLiSAl

AND

VLA-1 ON T CELLS

TLiSAl

VLA-1

Normal RA

11.0 + 7.8**

0.7 k 0.8%

1.0 f 1.1

SLE

9.8 f 8.2*

0.5 2 0.1

0.5 + 0.3%

Now. Values are means 2 SE. * P < 0.01; **p < 0.005.

illustrated in Fig. 5. An increase of TLiSAl positive percentages was seen on Day 2. The number of TLiSAl positive T cells continued to increase through 5 days of culture. The maximum expression of TLiSAl was obtained on Day 5 in all cases. Percentages of TLiSAl positive T cells in KA (0) and R4 with vasculitis (0) were higher than that of normal T cells (0) after Day 3. 8. The Distribution of TLiSAl Positive T Cells Determined with OKT4, OKT8, and TLiSAl (Fig. 6)

by Dual Staining

Peripheral T cells from normals were stimulated with 1% PHA-M for 2 days and stained with FITC-TLiSAl and PE-OKT4 or PE-OKT8. Figure 6. shows three representative cases. The percentages of T8+TLiSAlf cells were increased compared with T4+TLiSAl+ cells.

50 -

gj 40=VI 8 I.: c

30-

H 6 t z

x-

: e $ lo-

0 Peripheral blood

Syy$

FIG. 3. Comparison of percentages of TLiSAl positive T cells from peripheral blood and synovial fluid of patients with RA. The expression of TLiSAl on T cells from peripheral blood and synovial fluid was expressed simultaneously in 10 RA patients. Statistical analysis was done by means of paired f test (P < 0.01).

TABATA

372

1 > Case

Arthritis

e

bier

w

ESR(mm/l') RAHA CH50(u/m@ cf?P("g/d~) GP(mmHg) Jont Count.

T. K.

10 160X 41.5 0.4 60 4

58 320 46.3 1.2 56 6

68 320 35.4 1.9 54 8

9.6

19.9

6.1

2 ) Case PSL

1

10 160 33.1 ‘:0.3 58 2

I. K. 12/14

2124 1

Arthritis1 ESR(mm/l') RAHA CH50(u/mP) CRP(mglW WmmHg) Joint Count

ET AL.

100 1280X 49.8 3.7 80 8

4

1

lOmg/dw 50 640 42.0 1.3 75 7

28 640 42.0 1.3 108 3

25 320 38.1 c'0.3 112 4

TLtSAl

17.1

24.7

FIG. 4. Relationship between percentages of TLiSAl positive T cells and clinical features in RA. Case T.K. was RA with vasculitis. Case I.K. had severe arthritis and was treated with corticosteroid.

DISCUSSION

TLiSAl is a monoclonal antibody that binds to the T lineage specific activation antigen which appears on the surface of activated T cells. This molecule is a polypeptide of about 70 kDa. The maximal expression of TLiSAl on PHAstimulated T cells occurs 3-6 days after activation, 1 or 2 days after maximal IL-2 receptor expression, and remains highly expressed for several weeks. TLiSAl may be important in studying differentiation of activated T cells into functional cytotoxic T lymphocytes. The antibody to TLiSAl may interface with the cell uptake of a differentiation factor or the function of such a factor (14). In this study, we disclosed that percentages of TLiSAl positive T cells were

TLiSAl

ON T CELLS FROM RA AND SLE

Days

373

in culture

FIG. 5. Kinetics of expression of TLiSAl on PHA-stimulated peripheral T cells. Peripheral T cells from normal (O), RA (a), and RA with vasculitis (0) were stimulated with PHA-M (1%) and stained with FITC-TLiSAl at the indicated days.

increased in both peripheral blood and synovial fluid from RA patients. A markedly increased number of TLiSAl positive T cells were observed in SF from RA compared with autologous PB. Several activation antigens known to appear in vitro have been measured in vivo on T cells from patients with RA. T cells bearing elevated levels of the class II (Ia) activation antigens have been found in peripheral blood (8), synovial fluid (9), and synovium (10) of patients with this disease. Other activation markers, including OKTlO and interleukin 2 receptor (IL-2R), have also been found on synovial T cells in RA (11). Our results extend those observations. Additionally, Hemler et al. (13) reported the elevated expression of the very late activating antigen 1, which is maximally expressed following approximately 2 to 4 weeks of in vitro culture in RASF. We compared the appearance of the VLAl with TLiSAl in a

E s akl

0

Zdays 0 0 Ldays 0 tdays M. H. T.Y. Case S. M. FIG. 6. The distribution of TLiSAl positive T cells determined by dual staining with OKT4,OKT8, and TLiSAl. Peripheral T cells from normals were stimulated with PHA-M (1%) for 2 days and double stained with FITC-TLiSAl and PE-T4 or PE-T8. Values are means the percentages of double positive T cells.

374

TABATA

ET AL.

group of patients with RA, SLE, and normal controls. An increase in the expression of TLiSAl with no increase in the expression of VLAI was found in peripheral blood from RA. This may suggest differences in stages of T cell activation in RA. The presence of activated T cells in the peripheral blood of RA suggests that there is systemic immune activation in RA. Moreover, the markedly increase TLiSAl expression on T cells from RASF indicates the local activation of T cells in the intlammatory region that could provide a means to monitor abnormal immunologic activity in RA when these cells are functionally characterized. There was a clinical correlation with levels of TLiSAl expression in RA. This may assess immunological activity which might correlate with the disease process and might be useful as a clinical monitor for RA. Given the accumulation of unusual T cells at the site of the joint, certain populations of which displayed cytolytic activity and were likely to represent cells activated in vivo, one may speculate that these cells may be involved in the injury process. TLiSAl is associated with the differentiation of cytotoxic T cells from precurser cells. By double stain analysis, after PHA stimulation, mainly T8 cells obtained TLiSAl antigen in our study. In fact, Fox et al. (20) and Goto et al. (21) found that patients with RA had an increased proportion of OKT8 positive cells in SF when compared with the proportion found in autologous PB T cells. Clark et al. (22) generated long term T cell lines from RA synovial fluid with IL-2 and noted the high proportion of phenotypic suppressor/cytotoxic T cells among those lines. Theoretically, only activated T cells are responsive and can be propagated with IL-2. The generation of T8 cell lines by IL-2 therefore suggests that some of the SFT cells are activated in viva, consistent with our findings. Although these findings are not unique to RA, the high percentage of TLiSAl positive T cells in RA suggests that these cells probably represent secondarily activated cells following antigenic stimulation or are the consequence of a generalized nonspecific activation of the immune system. These results do not directly represent the ability of CTL in RA but suggest the state of activation and differentiation processes to CTL. The biological significance of TLiSAl positive T cells in a RA patient is as yet unknown. We are presently evaluating TLiSAl positive T cells in RASF for their ability and their role in disease process. REFERENCES 1. Nouri, A. M. E., Panagi, G. S., and Goodman, S. M.. Cytokines and the chronic inflammation of rheumatoid diseases. I. The presence of interleukin 1 in synovial fluids. C/in. Exp. Immunol. 55, 295-302, 1984. 2. Fontane, A., Hengartner, H., Weber, E., er al., Interleukin I activity in the synovial fluid of patients with rheumatoid arthritis. Rheumatol. Inc. 2, 49-53, 1982. 3. Wilkins, J. A., Warrington, R. J., Sigurdson, S. L., and Rutherford, S. J., The demonstration of an interleukin 2-like activity in the synovial fluids of rheumatoid arthritis patients. J. Rheumatol. 10, 109-113, 1983. 4. Nouri, A. M. E., Panayi, G. S., and Goodman, S. M., Cytokines and the chronic inflammation of rheumatic disease. I. The presence of interleukin 2 in synovial fluids. Clin. Erp. immunol. 58, 402-409, 1984. 5. Casario, T. C., Androws, B. S., Martin, D. A., et al., Interferon in synovial fluid and serum of patients with rheumatic disease. J. Rheumatol. 10, 647-650. 1983.

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ON T CELLS FROM RA AND SLE

375

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11. Burmester, G. R., Jahn, B., Gramatzki, M., Zacher, J., and Kalden, J. R., Activated T cells in vivo and in vitro: Divergence in expression of Tat and Ia antigens in the nonblastoid small T cells of inflammation and normal T cells activated in vitro. J. Zmmunol. 133, 1230-1234, 1984. 12. Fox, R. I., Fong, S., Sabharwal, N., Carstens, S. A., Kung, P. C., and Vaughan, J. H., Synovial fluid lymphocytes differ from peripheral blood lymphocytes in patients with rheumatoid arthritis. J. Zmmunol. 128, 351-354, 1982. 13. Hemler, M. E., Glass, D., Coblyn, J. S., and Jacobson, J. G., Very late activation antigen on rheumatoid synovial fluid T lymphocytes association with stages of T cell activation. 1. Clin. Invest. 78, 696-702, 1986. 14. Bums, G. F., Triglia, T., Werkmeister, J. A., Begley, C. G., and Boyd, A. W., TLiSAl, a human T lineage-specific activation antigen involved in the differentiation of cytotoxic T lymphocytes and anomalous killer cells from their precursors. J. Exp. Med. 161, 1063-1078, 1985. 15. Amett, F. C., Edworthy, S., Bloch, D. A., et al., The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 31, 315-332, 1988. 16. Tan, E. M., Cohen, A. S., Fries, J. F., Masi, A. T., McShane, D. J., Rothtield, N. F., Schaller, J. G., Talal, N., and Winchester, R. J., The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 258, 1271-1277, 1982. 17. Lansbery, J., Quantitation of activity of rheumatoid arthritis: Method for summation of systemic indices of rheumatoid activity. Amer. J. Med. Sci. 232, 300-317, 1956. 18. Urowitz, M. B., Gladman, D. D., Tozman, E. C., and Goldsmith, C. H., The lupus activity criteria count (LACC). J. Rheumotol. 11, 783-787, 1984. 19. Abe, T., Takeuchi, T., Koike, J., Hosono, O., Homma, M., Morimoto, C., and Yokohari, R., Suppressor T cell function in patients with rheumatoid arthritis complicated by vasculitis. Arthritis Rheum. 27, 752-759, 1984. 20. Fox, R. I., Fong, S., Sabhanval, N., Corstens, S. A., Kung, P. C., and Vaughan, J. H., Synovial fluid lymphocytes differ from peripheral blood lymphocytes in patients with rheumatoid arthritis. J. Zmmunol. 128, 351-354, 1982. 21. Goto, M., Miyamoto, T., Nishioka, K., and Uchida, S., T cytotoxic and helper cells are markedly increased, and T suppressor and inducer cells are decreased in rheumatoid synovial fluids. Arthritis Rheum. 30, 737-743, 1987. 22. Clark, R. B., Muirhead, S. P., and Kathryn Pollard, M., Generation of long-term T cell lines from synovial fluid. Clin. Zmmunol. Zmmunopathol. 33, 287-292, 1984. Received November 7, 1989; accepted with revision April 6, 1989