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Poor Expression of T Cell-Derived Cytokines and Activation and Proliferation Markers in Early Rheumatoid Synovial Tissue
CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY
Vol. 88, No. 1, July, pp. 84–90, 1998 Article No. II984525
Poor Expression of T Cell-Derived Cytokines and Activation and Proliferation Markers in Early Rheumatoid Synovial Tissue Tom J. M. Smeets, Radboud J. E. M. Dolhain, Andre´ M. M. Miltenburg, Ronella de Kuiper, Ferdinand C. Breedveld, and Paul P. Tak1 Department of Rheumatology, Leiden University Medical Center, The Netherlands
layed-type hypersensitivity (DTH) reactions in the skin and the cellular infiltrate in rheumatoid ST characterized by a large number of CD4/ T cells, which express activation markers (5–7), and the beneficial effect of some interventions directed at T cells (8, 9) form additional arguments. The T cell primacy in chronic RA has been questioned (10) on the basis of the hyporesponsiveness of T cells, reflected by a reduced response to mitogenic stimulation and decreased Ca2/ influxes (11, 12), impaired T cell receptor (TCR) z-chain mediated signaling (13), a lowered T cell proliferation rate (14), and a reduced expression of T-cell-derived cytokines like interferon-g (IFN-g) and interleukin 2 (IL-2) (15–17). The difficulty to demonstrate oligoclonal synovial T cell populations (18, 19) and the high number of synovial macrophages, which is correlated with disease activity (20) as well as with the progression of radiographic signs of joint destruction (21) as well as of fibroblast-like synoviocytes and other inflammatory cells in the synovial membrane could also suggest a restricted role for T cells in RA. The two paradigms in which either T cells or macrophages have a central role in the pathogenesis of RA are not mutually exclusive. It is conceivable that after a first phase initiated by an inciting stimulus operating through T-cell-dependent pathways, macrophage- and fibroblast-dependent processes emerge that promote synovial inflammation and joint destruction (22). In line with this hypothesis, it has been shown that during a T-cell-mediated disease the percentage of IFN-g producing T cells declines with time (23). Arguments in favor of the contention that T cells are involved in early disease processes are found in animal models of arthritis by showing T cell activation, which precedes a proinflammatory cytokine release by macrophages in collagen 2 induced arthritis (CIA) (24). In addition, a decline in IFN-g production in later stages CIA has been reported (25). Several clinical studies provided evidence to support a prominent role for T cells during early stages of RA, such as responses to possible auto-antigens which were particularly found in early disease phases (26, 27), the use of a more re-
We compared the state of activation and proliferation of T cells in synovial tissue (ST) from rheumatoid arthritis (RA) patients in early and late stages of the disease to find out whether T-cell-driven immune responses vary during the course of the disease. ST was obtained from 12 patients with early RA (õ 1 year) and 12 patients with longstanding RA (ú 5 years). T cells and interferon-g (IFN-g)-positive cells were detected in ST using immunohistologic methods. To determine the percentage of T cells expressing the interleukin-2 receptor, IFN-g, or the proliferation associated antigen Ki-67, immunofluorescence double-staining techniques were used. The scores for the number of T cells and for the expression of IFN-g as well as the percentages of T cells expressing CD25, IFN-g, or Ki-67 in rheumatoid synovium were not dependent on disease duration. These results do not support the assumption that the responsiveness of T cells in ST of RA patients differs between early and late stages of the disease. The data indicate that at present no arguments exist that the effect of T-cell-directed interventions on synovial inflammation might vary in different stages of the disease. q 1998 Academic Press Key Words: T cells; cytokines; early rheumatoid arthritis; immunohistochemistry.
INTRODUCTION
The role of T cells in the pathogenesis of rheumatoid arthritis (RA) is still not completely understood. The strongest argument in favor of the assumption that T cells play a critical role in RA is the association of disease susceptibility and outcome with HLA-DR4 antigens (1), and in particular with the ‘‘shared epitope’’ (2). Other evidence is derived from experimental animal models of arthritis where a single T cell clone can transfer the disease (3, 4). The similarities between the de1 To whom correspondence and reprint requests should be addressed at Leiden University Hospital, Department of Rheumatology, C4, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Fax: /3171 526 6752. E-mail: [email protected].
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0090-1229/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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stricted repertoire of T cell receptor b genes in early disease phases (28, 29), and immunohistopathologic studies of the synovial membrane showing different amounts and distribution of inflammatory cells in various stages of synovitis (30). Based on these observations, it was suggested that therapeutic strategies directed particularly at autoimmune responses should be investigated for use in early stage RA (31). Insight into the activation state of T cells in different stages of disease may be important to understand pathogenetic mechanisms underlying RA and could be a lead for the design of future therapeutic strategies. The aim of the present study was to investigate the activation state and proliferation rate of ST T cells in patients with a wide range of disease duration. Tonsils were incorporated for comparison with a chronic T-cell-mediated immunological reaction. PATIENTS AND METHODS
Twelve patients with RA of õ1 year’s duration as measured from the first clinical signs of arthritis and 12 patients with RA of ú5 years’ duration were investigated. All patients had active arthritis and fulfilled the American College of Rheumatology (formerly, the American Rheumatism Association) criteria for RA (32). None of the patients were treated with corticosteroids at the time the biopsies were taken. All patients gave informed consent, and the study protocol was approved by the Medical Ethics Committee of the Leiden University Medical Center. Tissue Specimens An average of 20 biopsies of ST were randomly taken from the knee joint of the RA patients with a Parker– Pearson needle (33). Furthermore, human palatine tonsils were obtained from patients with recurrent tonsillitis undergoing tonsillectomy. The tissue samples were placed together and snap frozen in Tissue-Tek OCT (Miles, Elkhart, IN) by immersion in methylbutane (0707C) and stored in liquid nitrogen. Cryostat sections (5 mm) were placed on adhesive glass slides (Starfrost, Knittelgla¨ser, Germany), air-dried overnight, and stored at 0807C until immunohistochemical analysis could be performed. Immunohistochemistry Serial sections of RA ST were stained with the following mouse monoclonal antibodies (Mab): anti-IFN-g (MD2, Dr. P van der Meide, BPRC, Rijswijk, The Netherlands) (16, 34) and anti-CD3 (OKT3, Cilag, Herenthals, Belgium). Staining was performed as described previously (35). Before use, the slides were allowed to
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warm to room temperature and air-dried. Fixation was performed in acetone for 10 min and slides were again air-dried. The sections were washed between all steps with phosphate-buffered saline (PBS). Endogenous peroxidase activity was inhibited using 0.1% sodium azide and 0.3% hydrogen peroxide in PBS. All primary and secondary antibodies were diluted in PBS containing 10% normal human AB serum (NHS). The primary antibodies were incubated overnight at 47C. HRP-conjugated goat anti-mouse (DAKO, Glostrup, Denmark) was added for 30 min at room temperature, followed by incubation with HRP-conjugated swine anti-goat antibody (Tago, Burlingame, CA) for another 30 min at room temperature. HRP activity was detected using 3-amino-9-ethylcarbazole (Sigma, St. Louis, MO). Slides were counterstained with Mayer’s hemalum solution (Merck, Darmstadt, Germany) and, after washing with distilled water, mounted in Kaiser’s glycerol gelatin (Merck). Immunofluorescence Double-Staining Procedures
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To investigate the expression of IL-2R, IFN-g, and the proliferation associated antigen Ki-67 by T cells in ST and tonsils, double-staining procedures were performed. The Mab against IL-2R (CD25) and Ki-67 were obtained from DAKO. Slides were allowed to warm to room temperature, air-dried, fixed with acetone for 10 min at room temperature, and air-dried. Slides were incubated with mouse anti-Ki-67 or anti-CD25 Mab in PBS with 1% bovine serum albumin (BSA) or MD2 in PBS with 10% NHS on sequential sections overnight at 47C, followed by incubation with biotinylated horse anti-mouse Ig (Vector Laboratories, Burlingame, CA) for 45 min at room temperature, followed by an additional incubation period with streptavidin-HRP (Zymed, San Francisco, CA) for 45 min at room temperature. The free binding sites of the horse anti-mouse polyclonal antibody were blocked using 20% normal mouse serum (NMS) in PBS, for 20 min at room temperature. Subsequently, slides were incubated for 1 h at room temperature with a mixture of fluorescein isothiocyanate (FITC)-conjugated mouse anti-CD2 (DAKO) and anti-CD3 (Becton–Dickinson, Mountain View, CA), diluted in PBS with 5% NMS and 5% NHS. Afterward, slides were fixed with 1% paraformaldehyde in PBS for 15 min at room temperature. Histochemical revelation of HRP was performed using tetramethylrhodamine-conjugated tyramine, diluted in 0.2 M Tris-HCl, 10 mM imidazole, 0.01% hydrogen peroxide 30% at pH 8.8 for 30 min at room temperature. Subsequently, slides were counterstained using bisbenzimide. Between all incubation periods, slides were washed with PBS. Slides were mounted with PBS/glycerol 1:9 (v/v) containing a 0.01 M solution of p-phenylene diamine (Sigma) and sealed and stored at 0207C until microscopical examination was performed.
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TABLE 1 Demographic and Clinical Data of the 12 Patients with Early Rheumatoid Arthritis (RA) and the 12 Patients with Longstanding RA, Who Were Studied for the Expression of Activation Markers on Synovial T Cells
Patient
Age (years)
Sex (M/F)
Disease duration (months)
Disease modifying antirheumatic drugs
CRP (Mg/L)
RF (//0)
Erosions (//0)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
80 67 73 72 52 56 74 72 63 79 55 79 75 68 39 75 49 60 54 32 67 56 65 42
M F F M F F F M M F F M M F M F F M F F M F F M
3 10 4 3 7 5 7 2 4 4 8 6 576 60 72 156 156 204 312 72 288 60 216 156
— — — — — — — — — — — Plaquenil D-Penicillamine — — Hydroxychloroquine Sulfasalazine — — — — Methotrexate — —
118 11 80 16 74 20 70 23 78 107 108 80 124 36 25 32 33 57 51 33 96 92 33 66
/ / / / / / / / / / / 0 / / / / 0 / / / / / / 0
0 / 0 0 0 0 0 0 0 0 0 0 / / / / / / / / / / / /
Microscopic Analysis Immunohistochemical staining for IFN-g and CD3 was scored randomly by two observers, who were unaware of the clinical data, on a five-point scale (0–4) as described previously (20, 35–37, 48). Individual readings were identical or differed only one point. Minor differences between the observers were resolved by mutual agreement. This scoring system is calibrated for each marker and has been developed previously by examining many rheumatoid arthritis synovial tissues. Because some activation and proliferation markers or cytokines are more abundant than others, the evaluation of each requires a different sensitivity level. The scoring system of 0–4 therefore refers to different numbers of positively staining for each marker. In immunofluorescence double-staining experiments coexpression of CD3/CD2 with Ki-67, CD25, or MD2 was determined by counting at least 300 CD3//CD2/ cells.
the RA group and comparing the scores for ST from RA patients with those for tonsil). RESULTS
Clinical Features Demographic and clinical data of the RA patients included in the study are presented in Table 1. Both the early and the longstanding RA group consisted of seven females and five males each. The mean age was in the early RA group 69 { 10 (mean { standard deviation, SD) years and in the longstanding RA group 57 { 14 years. Patients with early RA had a disease duration of 5 { 2 months and patients with longstanding RA had a disease duration of 194 { 147 months. All patients were treated with nonsteroidal anti-inflammatory drugs (NSAIDs).
Statistical Analysis
Immunohistologic Features
The following nonparametric tests were used: the Kruskal–Wallis test for several group means (comparing the scores for the three groups: ST from early RA, ST from longstanding RA, and tonsil) and the Mann– Whitney U test (comparing the scores for ST within
CD3 and IFN-g (Fig. 1) could be detected in ST samples from all RA patients. Staining for IFN-g could be detected in association with various structures, especially with blood vessels and the synovial lining layer. The mean scores for CD3 and IFN-g in patients with
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FIG. 1. IFN-g / (A) cells and negative control (B) in sequential slides of rheumatoid synovial tissue. Single stain peroxidase technique; Mayer’s Hemalum counterstained; original magnification 1 250.
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FIG. 2. Double immunofluorescence staining showing CD3/CD2 / (A) Ki-67 / (B) cells in a perivascular lymphocytic aggregate in rheumatoid synovial tissue. Original magnification 1 250/1630.
early RA were not different from those in patients with longstanding RA (P ú 0.1) (Table 2). Double Staining Procedures No difference was found for the mean percentages of T cells expressing Ki-67 (Fig. 2), CD25, and IFN-g TABLE 2 Semiquantitative Scoresa (0–4) for the Expression of CD3 and IFN-g in Synovial Tissue from Patients with Early (õ1 Year) and Longstanding (ú5 Years) Rheumatoid Arthritis
between early and late RA patients (all P ú 0.1) (Table 3). The mean percentages of Ki-67, CD25, and IFN-g positive T cells in patients with RA were lower compared with those in tonsils (Kruskal–Wallis test and Mann–Whitney test: P õ 0.05) (Table 3). Some individual patients exhibited similar numbers of T cells expressing activation markers compared with those observed in tonsils after recurrent tonsillitis. These patients did not differ from the other patients regarding disease activity and medication.
Disease duration
CD3 IFN-g a
DISCUSSION
õ1 year (n Å 12)
ú5 years (n Å 12)
2.5 { 1.0 (1–4) 2.9 { 1.0 (1–4)
2.3 { 1.0 (1–4) 3.2 { 0.7 (2–4)
Values are the mean { SD (range) (all P values ú 0.1).
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The results presented here demonstrate a reduced activation and proliferation state of rheumatoid ST T cells compared with T cells from tonsils. The results are in line with previous findings (11–17) suggesting that T cells in rheumatoid ST are functionally deficient.
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TABLE 3 a
Percentages of T Cells Coexpressing CD25, IFN-g, and Ki-67 in Synovial Tissue (ST) from Patients with Early Rheumatoid Arthritis (RA) (õ1 Year), Longstanding RA (ú5 Years), and in Tonsil
% CD25 of CD3/CD2 subset % IFN-g of CD3/CD2 subset % KI-67 of CD3/CD2 subset
ST RA õ1 year (n Å 11)
ST RA ú5 years (n Å 12)
Tonsil (n Å 5)
1.9 { 1.7 (0.0–3.7) 3.2 { 2.8 (0.0–8.3) 2.2 { 1.2 (0.0–4.3)
1.9 { 2.2 (0.0–7.7) 3.7 { 2.7 (0.7–10.0) 2.3 { 2.2 (0.0–6.3)
10.5* { 2.3 (7.0–12.7) 22.9* { 5.0 (16.0–28.7) 7.1** { 2.3 (4.3–10.0)
a Values are the mean { SD (range). * P õ 0.005, Kruskal–Wallis test (comparing scores for the three groups) and Mann–Whitney test (RA versus tonsil). ** P õ 0.05, Kruskal–Wallis test, and P õ 0.005, Mann–Whitney test.
The hyporesponsive state of ST T cells could also be observed in the so-called ‘‘early’’ RA patients group. In contrast to previous studies, the application of doublestaining techniques in the present study clearly shows that the reduced expression of IFN-g can be attributed to T cells. Double staining is important to interpret the expression of IFN-g, IL-2R, and Ki-67, because they can be expressed by multiple cells. The reduced expression of IFN-g in ST from patients with RA described here is in concordance with previous studies (16, 38) as is the finding concerning the low IL-2R and Ki-67 expression in ST (39–41). In the present study we demonstrate a hyporesponsive state of T cells from RA patients compared with T cells from patients with recurrent tonsillitis. To reduce the change of bias as a result of differences in technique or patient selection, all biopsy specimens were obtained with the same biopsy technique and patients treated with corticosteroids were excluded. It is unlikely that the findings described in the present study are influenced by sampling error due to the use of blind needle biopsies in the light of results from a recent study, which shows that most microscopic measures of inflammation in biopsy specimens obtained from the suprapatellar pouch by blind needle biopsy are similar to those in samples selected at arthroscopy (42). Furthermore, sections from a large number of biopsy specimens were used to minimize sampling error. Inhibitory cytokines present in ST, such as TGF-b (43) and IL-10 (44), may play a role in the reduced function of T cells in active RA. Furthermore, observations in a transgenic mice model showed attenuating of TCR signaling by chronic exposure of TNF-a (45) and it has been suggested that chronic exposure of TNF-a may also be responsible for reduced T cell functions in RA (46). Recently, a direct correlation was found between hyporesponsiveness of ST T cells of RA patients and TCR-mediated signaling transduction by severely diminished phosphorylation of TCR z-chain and p38 (13). High levels of thioredoxine, released under conditions of oxidative stress, and decreased intracellular levels of the antioxidant gluthatione (GSH) were
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found in RA synovial fluid (47), which may also be relevant for the reduced T cell functions. The similar findings of hyporesponsiveness of T cells for early and late RA, as reported here, do not support the paradigm that the inflammatory mechanisms in ST of RA patients differ between early and late stages of the clinically manifest disease. These findings are in concordance with recent studies, reporting that the immunohistologic features of rheumatoid synovium were not dependent on disease duration (20, 48). It has been suggested that asymptomatic synovitis may precede the clinical manifestations of RA and that so-called early RA is already a chronic disease (20, 49). Thus, synovitis of clinically early RA patients should already be regarded as a chronic form of synovial inflammation. Therefore, it may be questioned whether effects of Tcell-directed therapies might vary in different stages of RA. On average, the percentages of T cells in rheumatoid ST expressing activation markers were found to be variable. In some RA patients with either early or longstanding disease, the expression of activation markers in the ST was similar to the findings in tonsils. It is conceivable that T cell activity waxes and wanes during the whole course of the disease, or that T cell activity plays a dominant role in subsets of the patient population. Whether this may be clinically relevant remains to be determined. In conclusion, ST T cells of early RA patients reveal already a hyporesponsive state, which is indistinguishable from what is found in longstanding disease. This implies that there is no rationale for studying T-cellmediated therapies preferably in early RA based on the hypothesis that downregulation T cell function should be more effective during earlier stages of the disease. REFERENCES 1. Van Zeben, D., Hazes, J. M., Zwinderman, A. H., Cats, A., Schreuder, G. M., D’Amaro, J., and Breedveld, F. C., Association of HLA-DR4 with a more progressive disease course in patients with rheumatoid arthritis: results of a follow-up study. Arthritis Rheum. 34, 822–830, 1991.
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SMEETS ET AL. tochemical detection of co-localizing cytokine and antibody producing cells in the extrafollicular area of human palatine tonsils. Clin. Exp. Immunol. 93, 223–228, 1993. Tak, P. P., van der Lubbe, P. A., Cauli, A., Daha, M. R., Smeets, T. J. M., Kluin, P. M., Meinders, A. E., Yanni, G., Panayi, G. S., and Breedveld, F. C., Reduction of synovial inflammation after anti-CD4 monoclonal antibody treatment in early rheumatoid arthritis. Arthritis Rheum. 38, 1457–1465, 1995. Tak, P. P., Kummer, J. A., Hack, C. E., Daha, M. R., Smeets, T. J. M., Erkelens, G. W., Meinders, A. E., Kluin, P. M., and Breedveld, F. C., Granzyme-positive cytotoxic cells are specifically increased in early rheumatoid synovial tissue. Arthritis Rheum. 37, 1735–1743, 1994. Thurkow, E. W., Van der Heijden, I. M., Breedveld, F. C., Smeets, T. J. M., Daha, M. R., Kluin, P. M., Meinders, A. E., and Tak, P. P., Increased expression of IL-15 in the synovium of patients with rheumatoid arthritis compared with patients with yersinia induced arthritis and osteoarthritis. J. Pathol. 181, 444–450, 1997. Husby, G., and Williams, R. C., Immunohistochemical studies of interleukin-2 and gamma-interferon in rheumatoid arthritis. Arthritis Rheum. 28, 174–181, 1985. Haraoui, B., Pelletier, J. P., Cloutier, J. M., Faure, M. P., and Martel-Pelletier, J., Synovial membrane histology and immunopathology in rheumatoid arthritis and osteoarthritis. In vivo effects of antirheumatic drugs. Arthritis Rheum. 34, 153–163, 1991. El-Gabalawy, H. S., and Keillor, J., Immunohistologic study of T cell receptor d-chain expression in rheumatoid synovial membranes. Sem. Arthr. Rheum. 21, 239–245, 1992. Lalor, P. A., Mapp, P. I., Hall, P. A., and Revell, P. A., Proliferative activity of cells in the synovium as demonstrated by a monoclonal antibody, Ki-67. Rheumatol. Int. 7, 183–186, 1987. Youssef, P. P., Kraan, M., Bresnihan, B., Cassidy, N., Cunnane,
43.
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G., Emery, P., FitzGerald, O., Kane, D., Breedveld, F. C., Lindblad, S., Reece, R., Veale, D., and Tak, P. P., Quantitative microscopic analysis of inflammation in rheumatoid synovial membrane samples selected at arthroscopy compared with samples taken blindly by needle biopsy. Arthritis Rheum. 41, 663–669, 1998. Fava, R., Olsen, N., Keski-Oja, J., Moses, H., and Pincus, T., Active and latent forms of transforming growth factor b activity in synovial effusions. J. Exp. Med. 169, 291–296, 1989. Katsikis, P., Chu, C. Q., Brennan, F. M., Maini, R. N., and Feldmann, M., Immunoregulatory role of interleukin-10 (IL10) in rheumatoid arthritis. J. Exp. Med. 179, 1517–1527, 1994. Cope, A. P., Liblau, R. S., Yang, X. D., Congia, M., Laudanna, C., Schreiber, R. D., Probert, L., Kollias, G., and McDevitt, H. O., Chronic tumor necrosis factor alters T cell responses by attenuating T cell receptor signalling. J. Exp. Med. 185, 1573–1584, 1997. Cope, A. P., Londei, M., Chu, N. R., Cohen, S. B. A., Elliot, M. J., Brennan, F. M., Maini, R. N., and Feldmann, M., Chronic exposure to tumor necrosis factor (TNF) in vitro impairs the activation of T cells through the T cell receptor/CD3 complex: reversal in vivo by anti-TNF antibodies in patients with rheumatoid arthritis. J. Clin. Invest. 94, 749–760, 1994. Maurice, M. M., Nakamura, H., Van der Voort, E. A. M., Van Vliet, A. I., Staal, F. J. T., Tak, P. P., Breedveld, F. C., and Verweij, C. L., Evidence for the role of an altered redox state in hyporesponsiveness of synovial T cells in rheumatoid arthritis. J. Immunol. 158, 1458–1465, 1997. Tak, P. P., Thurkow, E. W., Daha, M. R., Kluin, P. M., Smeets, T. J. M., Meinders, A. E., and Breedveld, F. C., Expression of adhesion molecules in early rheumatoid synovial tissue. Clin. Immunol. Immunopathol. 77, 236–242, 1995. Tak, P. P., Smeets, T. J. M., Daha, M. R., Meijers, K. A. E., Brand, R., Meiders, A. E., and Breedveld, F. C., Is one year early, or too late? Reply. Arthritis Rheum. 40, 1913–1914, 1997.
Received October 20, 1997; accepted with revision January 8, 1998
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Vol. 88, No. 1, July, pp. 84–90, 1998 Article No. II984525
Poor Expression of T Cell-Derived Cytokines and Activation and Proliferation Markers in Early Rheumatoid Synovial Tissue Tom J. M. Smeets, Radboud J. E. M. Dolhain, Andre´ M. M. Miltenburg, Ronella de Kuiper, Ferdinand C. Breedveld, and Paul P. Tak1 Department of Rheumatology, Leiden University Medical Center, The Netherlands
layed-type hypersensitivity (DTH) reactions in the skin and the cellular infiltrate in rheumatoid ST characterized by a large number of CD4/ T cells, which express activation markers (5–7), and the beneficial effect of some interventions directed at T cells (8, 9) form additional arguments. The T cell primacy in chronic RA has been questioned (10) on the basis of the hyporesponsiveness of T cells, reflected by a reduced response to mitogenic stimulation and decreased Ca2/ influxes (11, 12), impaired T cell receptor (TCR) z-chain mediated signaling (13), a lowered T cell proliferation rate (14), and a reduced expression of T-cell-derived cytokines like interferon-g (IFN-g) and interleukin 2 (IL-2) (15–17). The difficulty to demonstrate oligoclonal synovial T cell populations (18, 19) and the high number of synovial macrophages, which is correlated with disease activity (20) as well as with the progression of radiographic signs of joint destruction (21) as well as of fibroblast-like synoviocytes and other inflammatory cells in the synovial membrane could also suggest a restricted role for T cells in RA. The two paradigms in which either T cells or macrophages have a central role in the pathogenesis of RA are not mutually exclusive. It is conceivable that after a first phase initiated by an inciting stimulus operating through T-cell-dependent pathways, macrophage- and fibroblast-dependent processes emerge that promote synovial inflammation and joint destruction (22). In line with this hypothesis, it has been shown that during a T-cell-mediated disease the percentage of IFN-g producing T cells declines with time (23). Arguments in favor of the contention that T cells are involved in early disease processes are found in animal models of arthritis by showing T cell activation, which precedes a proinflammatory cytokine release by macrophages in collagen 2 induced arthritis (CIA) (24). In addition, a decline in IFN-g production in later stages CIA has been reported (25). Several clinical studies provided evidence to support a prominent role for T cells during early stages of RA, such as responses to possible auto-antigens which were particularly found in early disease phases (26, 27), the use of a more re-
We compared the state of activation and proliferation of T cells in synovial tissue (ST) from rheumatoid arthritis (RA) patients in early and late stages of the disease to find out whether T-cell-driven immune responses vary during the course of the disease. ST was obtained from 12 patients with early RA (õ 1 year) and 12 patients with longstanding RA (ú 5 years). T cells and interferon-g (IFN-g)-positive cells were detected in ST using immunohistologic methods. To determine the percentage of T cells expressing the interleukin-2 receptor, IFN-g, or the proliferation associated antigen Ki-67, immunofluorescence double-staining techniques were used. The scores for the number of T cells and for the expression of IFN-g as well as the percentages of T cells expressing CD25, IFN-g, or Ki-67 in rheumatoid synovium were not dependent on disease duration. These results do not support the assumption that the responsiveness of T cells in ST of RA patients differs between early and late stages of the disease. The data indicate that at present no arguments exist that the effect of T-cell-directed interventions on synovial inflammation might vary in different stages of the disease. q 1998 Academic Press Key Words: T cells; cytokines; early rheumatoid arthritis; immunohistochemistry.
INTRODUCTION
The role of T cells in the pathogenesis of rheumatoid arthritis (RA) is still not completely understood. The strongest argument in favor of the assumption that T cells play a critical role in RA is the association of disease susceptibility and outcome with HLA-DR4 antigens (1), and in particular with the ‘‘shared epitope’’ (2). Other evidence is derived from experimental animal models of arthritis where a single T cell clone can transfer the disease (3, 4). The similarities between the de1 To whom correspondence and reprint requests should be addressed at Leiden University Hospital, Department of Rheumatology, C4, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Fax: /3171 526 6752. E-mail: [email protected].
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0090-1229/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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stricted repertoire of T cell receptor b genes in early disease phases (28, 29), and immunohistopathologic studies of the synovial membrane showing different amounts and distribution of inflammatory cells in various stages of synovitis (30). Based on these observations, it was suggested that therapeutic strategies directed particularly at autoimmune responses should be investigated for use in early stage RA (31). Insight into the activation state of T cells in different stages of disease may be important to understand pathogenetic mechanisms underlying RA and could be a lead for the design of future therapeutic strategies. The aim of the present study was to investigate the activation state and proliferation rate of ST T cells in patients with a wide range of disease duration. Tonsils were incorporated for comparison with a chronic T-cell-mediated immunological reaction. PATIENTS AND METHODS
Twelve patients with RA of õ1 year’s duration as measured from the first clinical signs of arthritis and 12 patients with RA of ú5 years’ duration were investigated. All patients had active arthritis and fulfilled the American College of Rheumatology (formerly, the American Rheumatism Association) criteria for RA (32). None of the patients were treated with corticosteroids at the time the biopsies were taken. All patients gave informed consent, and the study protocol was approved by the Medical Ethics Committee of the Leiden University Medical Center. Tissue Specimens An average of 20 biopsies of ST were randomly taken from the knee joint of the RA patients with a Parker– Pearson needle (33). Furthermore, human palatine tonsils were obtained from patients with recurrent tonsillitis undergoing tonsillectomy. The tissue samples were placed together and snap frozen in Tissue-Tek OCT (Miles, Elkhart, IN) by immersion in methylbutane (0707C) and stored in liquid nitrogen. Cryostat sections (5 mm) were placed on adhesive glass slides (Starfrost, Knittelgla¨ser, Germany), air-dried overnight, and stored at 0807C until immunohistochemical analysis could be performed. Immunohistochemistry Serial sections of RA ST were stained with the following mouse monoclonal antibodies (Mab): anti-IFN-g (MD2, Dr. P van der Meide, BPRC, Rijswijk, The Netherlands) (16, 34) and anti-CD3 (OKT3, Cilag, Herenthals, Belgium). Staining was performed as described previously (35). Before use, the slides were allowed to
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warm to room temperature and air-dried. Fixation was performed in acetone for 10 min and slides were again air-dried. The sections were washed between all steps with phosphate-buffered saline (PBS). Endogenous peroxidase activity was inhibited using 0.1% sodium azide and 0.3% hydrogen peroxide in PBS. All primary and secondary antibodies were diluted in PBS containing 10% normal human AB serum (NHS). The primary antibodies were incubated overnight at 47C. HRP-conjugated goat anti-mouse (DAKO, Glostrup, Denmark) was added for 30 min at room temperature, followed by incubation with HRP-conjugated swine anti-goat antibody (Tago, Burlingame, CA) for another 30 min at room temperature. HRP activity was detected using 3-amino-9-ethylcarbazole (Sigma, St. Louis, MO). Slides were counterstained with Mayer’s hemalum solution (Merck, Darmstadt, Germany) and, after washing with distilled water, mounted in Kaiser’s glycerol gelatin (Merck). Immunofluorescence Double-Staining Procedures
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To investigate the expression of IL-2R, IFN-g, and the proliferation associated antigen Ki-67 by T cells in ST and tonsils, double-staining procedures were performed. The Mab against IL-2R (CD25) and Ki-67 were obtained from DAKO. Slides were allowed to warm to room temperature, air-dried, fixed with acetone for 10 min at room temperature, and air-dried. Slides were incubated with mouse anti-Ki-67 or anti-CD25 Mab in PBS with 1% bovine serum albumin (BSA) or MD2 in PBS with 10% NHS on sequential sections overnight at 47C, followed by incubation with biotinylated horse anti-mouse Ig (Vector Laboratories, Burlingame, CA) for 45 min at room temperature, followed by an additional incubation period with streptavidin-HRP (Zymed, San Francisco, CA) for 45 min at room temperature. The free binding sites of the horse anti-mouse polyclonal antibody were blocked using 20% normal mouse serum (NMS) in PBS, for 20 min at room temperature. Subsequently, slides were incubated for 1 h at room temperature with a mixture of fluorescein isothiocyanate (FITC)-conjugated mouse anti-CD2 (DAKO) and anti-CD3 (Becton–Dickinson, Mountain View, CA), diluted in PBS with 5% NMS and 5% NHS. Afterward, slides were fixed with 1% paraformaldehyde in PBS for 15 min at room temperature. Histochemical revelation of HRP was performed using tetramethylrhodamine-conjugated tyramine, diluted in 0.2 M Tris-HCl, 10 mM imidazole, 0.01% hydrogen peroxide 30% at pH 8.8 for 30 min at room temperature. Subsequently, slides were counterstained using bisbenzimide. Between all incubation periods, slides were washed with PBS. Slides were mounted with PBS/glycerol 1:9 (v/v) containing a 0.01 M solution of p-phenylene diamine (Sigma) and sealed and stored at 0207C until microscopical examination was performed.
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TABLE 1 Demographic and Clinical Data of the 12 Patients with Early Rheumatoid Arthritis (RA) and the 12 Patients with Longstanding RA, Who Were Studied for the Expression of Activation Markers on Synovial T Cells
Patient
Age (years)
Sex (M/F)
Disease duration (months)
Disease modifying antirheumatic drugs
CRP (Mg/L)
RF (//0)
Erosions (//0)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
80 67 73 72 52 56 74 72 63 79 55 79 75 68 39 75 49 60 54 32 67 56 65 42
M F F M F F F M M F F M M F M F F M F F M F F M
3 10 4 3 7 5 7 2 4 4 8 6 576 60 72 156 156 204 312 72 288 60 216 156
— — — — — — — — — — — Plaquenil D-Penicillamine — — Hydroxychloroquine Sulfasalazine — — — — Methotrexate — —
118 11 80 16 74 20 70 23 78 107 108 80 124 36 25 32 33 57 51 33 96 92 33 66
/ / / / / / / / / / / 0 / / / / 0 / / / / / / 0
0 / 0 0 0 0 0 0 0 0 0 0 / / / / / / / / / / / /
Microscopic Analysis Immunohistochemical staining for IFN-g and CD3 was scored randomly by two observers, who were unaware of the clinical data, on a five-point scale (0–4) as described previously (20, 35–37, 48). Individual readings were identical or differed only one point. Minor differences between the observers were resolved by mutual agreement. This scoring system is calibrated for each marker and has been developed previously by examining many rheumatoid arthritis synovial tissues. Because some activation and proliferation markers or cytokines are more abundant than others, the evaluation of each requires a different sensitivity level. The scoring system of 0–4 therefore refers to different numbers of positively staining for each marker. In immunofluorescence double-staining experiments coexpression of CD3/CD2 with Ki-67, CD25, or MD2 was determined by counting at least 300 CD3//CD2/ cells.
the RA group and comparing the scores for ST from RA patients with those for tonsil). RESULTS
Clinical Features Demographic and clinical data of the RA patients included in the study are presented in Table 1. Both the early and the longstanding RA group consisted of seven females and five males each. The mean age was in the early RA group 69 { 10 (mean { standard deviation, SD) years and in the longstanding RA group 57 { 14 years. Patients with early RA had a disease duration of 5 { 2 months and patients with longstanding RA had a disease duration of 194 { 147 months. All patients were treated with nonsteroidal anti-inflammatory drugs (NSAIDs).
Statistical Analysis
Immunohistologic Features
The following nonparametric tests were used: the Kruskal–Wallis test for several group means (comparing the scores for the three groups: ST from early RA, ST from longstanding RA, and tonsil) and the Mann– Whitney U test (comparing the scores for ST within
CD3 and IFN-g (Fig. 1) could be detected in ST samples from all RA patients. Staining for IFN-g could be detected in association with various structures, especially with blood vessels and the synovial lining layer. The mean scores for CD3 and IFN-g in patients with
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FIG. 1. IFN-g / (A) cells and negative control (B) in sequential slides of rheumatoid synovial tissue. Single stain peroxidase technique; Mayer’s Hemalum counterstained; original magnification 1 250.
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FIG. 2. Double immunofluorescence staining showing CD3/CD2 / (A) Ki-67 / (B) cells in a perivascular lymphocytic aggregate in rheumatoid synovial tissue. Original magnification 1 250/1630.
early RA were not different from those in patients with longstanding RA (P ú 0.1) (Table 2). Double Staining Procedures No difference was found for the mean percentages of T cells expressing Ki-67 (Fig. 2), CD25, and IFN-g TABLE 2 Semiquantitative Scoresa (0–4) for the Expression of CD3 and IFN-g in Synovial Tissue from Patients with Early (õ1 Year) and Longstanding (ú5 Years) Rheumatoid Arthritis
between early and late RA patients (all P ú 0.1) (Table 3). The mean percentages of Ki-67, CD25, and IFN-g positive T cells in patients with RA were lower compared with those in tonsils (Kruskal–Wallis test and Mann–Whitney test: P õ 0.05) (Table 3). Some individual patients exhibited similar numbers of T cells expressing activation markers compared with those observed in tonsils after recurrent tonsillitis. These patients did not differ from the other patients regarding disease activity and medication.
Disease duration
CD3 IFN-g a
DISCUSSION
õ1 year (n Å 12)
ú5 years (n Å 12)
2.5 { 1.0 (1–4) 2.9 { 1.0 (1–4)
2.3 { 1.0 (1–4) 3.2 { 0.7 (2–4)
Values are the mean { SD (range) (all P values ú 0.1).
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The results presented here demonstrate a reduced activation and proliferation state of rheumatoid ST T cells compared with T cells from tonsils. The results are in line with previous findings (11–17) suggesting that T cells in rheumatoid ST are functionally deficient.
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TABLE 3 a
Percentages of T Cells Coexpressing CD25, IFN-g, and Ki-67 in Synovial Tissue (ST) from Patients with Early Rheumatoid Arthritis (RA) (õ1 Year), Longstanding RA (ú5 Years), and in Tonsil
% CD25 of CD3/CD2 subset % IFN-g of CD3/CD2 subset % KI-67 of CD3/CD2 subset
ST RA õ1 year (n Å 11)
ST RA ú5 years (n Å 12)
Tonsil (n Å 5)
1.9 { 1.7 (0.0–3.7) 3.2 { 2.8 (0.0–8.3) 2.2 { 1.2 (0.0–4.3)
1.9 { 2.2 (0.0–7.7) 3.7 { 2.7 (0.7–10.0) 2.3 { 2.2 (0.0–6.3)
10.5* { 2.3 (7.0–12.7) 22.9* { 5.0 (16.0–28.7) 7.1** { 2.3 (4.3–10.0)
a Values are the mean { SD (range). * P õ 0.005, Kruskal–Wallis test (comparing scores for the three groups) and Mann–Whitney test (RA versus tonsil). ** P õ 0.05, Kruskal–Wallis test, and P õ 0.005, Mann–Whitney test.
The hyporesponsive state of ST T cells could also be observed in the so-called ‘‘early’’ RA patients group. In contrast to previous studies, the application of doublestaining techniques in the present study clearly shows that the reduced expression of IFN-g can be attributed to T cells. Double staining is important to interpret the expression of IFN-g, IL-2R, and Ki-67, because they can be expressed by multiple cells. The reduced expression of IFN-g in ST from patients with RA described here is in concordance with previous studies (16, 38) as is the finding concerning the low IL-2R and Ki-67 expression in ST (39–41). In the present study we demonstrate a hyporesponsive state of T cells from RA patients compared with T cells from patients with recurrent tonsillitis. To reduce the change of bias as a result of differences in technique or patient selection, all biopsy specimens were obtained with the same biopsy technique and patients treated with corticosteroids were excluded. It is unlikely that the findings described in the present study are influenced by sampling error due to the use of blind needle biopsies in the light of results from a recent study, which shows that most microscopic measures of inflammation in biopsy specimens obtained from the suprapatellar pouch by blind needle biopsy are similar to those in samples selected at arthroscopy (42). Furthermore, sections from a large number of biopsy specimens were used to minimize sampling error. Inhibitory cytokines present in ST, such as TGF-b (43) and IL-10 (44), may play a role in the reduced function of T cells in active RA. Furthermore, observations in a transgenic mice model showed attenuating of TCR signaling by chronic exposure of TNF-a (45) and it has been suggested that chronic exposure of TNF-a may also be responsible for reduced T cell functions in RA (46). Recently, a direct correlation was found between hyporesponsiveness of ST T cells of RA patients and TCR-mediated signaling transduction by severely diminished phosphorylation of TCR z-chain and p38 (13). High levels of thioredoxine, released under conditions of oxidative stress, and decreased intracellular levels of the antioxidant gluthatione (GSH) were
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found in RA synovial fluid (47), which may also be relevant for the reduced T cell functions. The similar findings of hyporesponsiveness of T cells for early and late RA, as reported here, do not support the paradigm that the inflammatory mechanisms in ST of RA patients differ between early and late stages of the clinically manifest disease. These findings are in concordance with recent studies, reporting that the immunohistologic features of rheumatoid synovium were not dependent on disease duration (20, 48). It has been suggested that asymptomatic synovitis may precede the clinical manifestations of RA and that so-called early RA is already a chronic disease (20, 49). Thus, synovitis of clinically early RA patients should already be regarded as a chronic form of synovial inflammation. Therefore, it may be questioned whether effects of Tcell-directed therapies might vary in different stages of RA. On average, the percentages of T cells in rheumatoid ST expressing activation markers were found to be variable. In some RA patients with either early or longstanding disease, the expression of activation markers in the ST was similar to the findings in tonsils. It is conceivable that T cell activity waxes and wanes during the whole course of the disease, or that T cell activity plays a dominant role in subsets of the patient population. Whether this may be clinically relevant remains to be determined. In conclusion, ST T cells of early RA patients reveal already a hyporesponsive state, which is indistinguishable from what is found in longstanding disease. This implies that there is no rationale for studying T-cellmediated therapies preferably in early RA based on the hypothesis that downregulation T cell function should be more effective during earlier stages of the disease. REFERENCES 1. Van Zeben, D., Hazes, J. M., Zwinderman, A. H., Cats, A., Schreuder, G. M., D’Amaro, J., and Breedveld, F. C., Association of HLA-DR4 with a more progressive disease course in patients with rheumatoid arthritis: results of a follow-up study. Arthritis Rheum. 34, 822–830, 1991.
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HYPORESPONSIVENESS OF T CELLS IN EARLY RA 2. Winchester, R., The molecular basis of susceptibility to rheumatoid arthritis. Adv. Immunol. 56, 389–466, 1994. 3. Klareskog, L., Holmdahl, R., Larsson, E., and Wigzell, H., Role of T lymphocytes in collagen 2 induced arthritis in rats, Clin. Exp. Immunol. 51, 117–125, 1983. 4. Taurog, J. D., Sandberg, G. P., and Mahowald, M. L., The cellular basis of adjuvant arthritis: characterization of the cells mediating passive transfer. Cell. Immunol. 80, 198–204, 1983. 5. Klareskog, L., Forsum, U., Scheynius, A., Kabelitz, D., and Wigzell, H., Evidence in support of a self-perpetuating HLA-DR dependent delayed type cell reaction in rheumatoid arthritis. Proc. Natl. Acad. Sci. USA 79, 3632–3636, 1982. 6. Pitzalis, C., Kingsley, G. H., Murphy, J., and Panayi, G. S., Abnormal distribution of the helper-inducer and suppressor-inducer T lymphocyte subsets in the rheumatoid joint. Clin. Immunol. Immunopathol. 45, 252–258, 1987. 7. Tak, P. P., Hintzen, R. Q., Teunissen, J. J. M., Smeets, T. J. M., Daha, M. R., van Lier, R. A. W., Kluin, P. M., Meinders, A. E., Swaak, A. J. G., and Breedveld, F. C., Expression of the activation antigen CD27 in rheumatoid arthritis. Clin. Immunol. Immunopathol. 80, 129–138, 1996. 8. Panayi, G. S., Choy, E. H. S., Connoly, D. J. A., Regan, T., Manna, V. K., Rapson, N., Kingsley, G. H., and Johnston, J. M., T cell hypothesis in rheumatoid arthritis (RA) tested by humanized non-depleting anti-CD4 monoclonal antibody (Mab) treatment I: suppression of disease activity and acute phase response. Arthritis Rheum. 9(Suppl.), S1300, 1996. 9. Levy, R., Weisman, M., Wiesenhutter, C., Yocum, D., Schnitzer, T., Goldman, A., Schiff, M., Breedveld, F., Solinger, A., MacDonald, B., and Lipani, J., Results of a placebo controlled, multicenter trial using a primatized non-depleting, anti-CD4 monoclonal antibody in the treatment of rheumatoid arthritis. Arthritis Rheum. 9(Suppl.), S122, 1996. 10. Firestein, G. S., and Zvaifler, N. J., How important are T cells in chronic rheumatoid synovitis? Arthritis Rheum. 33, 768–773, 1990. 11. Kingsley, G. H., Pinalis, C., and Panayi, G. S., Abnormal lymphocyte reactivity to self-major histocompatibility antigens in rheumatoid arthritis. J. Rheumatol. 14, 667–673, 1987. 12. Allen, M. E., Young, S. P., Michell, R. H., and Bacon, P. A., Altered T lymphocyte signalling in rheumatoid arthritis. Eur. J. Immunol. 25, 1547–1554, 1995. 13. Maurice, M. M., Lankester, A. C., Bezemer, A. C., Geertsma, M. F., Tak, P. P., Breedveld, F. C., van Lier, R. A. W., and Verweij, C. L., Defective TCR-mediated signaling in synovial T cells in rheumatoid arthritis. J. Immunol. 159, 2973–2978, 1997. 14. Cush, J. J., and Lipsky, P. E., Cellular basis for rheumatoid inflammation. Clin. Orthop. 265, 9–22, 1991. 15. Firestein, G. S., Xu, W. D., Townsend, K., Broide, D., AlvaroGarcia, J. M., Glasebrook, A., and Zvaifler, N. J., Cytokines in chronic inflammatory arthritis. I. Failure to detect T cell lymphokines (interleukin 2 and interleukin 3) and presence of macrophage colony-stimulating factor (CSF-1) and a novel mast cell growth factor in rheumatoid synovitis. J. Exp. Med. 168, 1573– 1586, 1988. 16. Dolhain, R. J. E. M., Ter Haar, N. T., Hoefakker, S., Tak, P. P., De Ley, M., Classen, E., Breedveld, F. C., and Miltenburg, A. M. M., Increased expression of interferon (IFN)-gamma together with IFN-gamma receptor in the rheumatoid synovial membrane compared with synovium of patients with osteoarthritis. Br. J. Rheumatol. 35, 24–32, 1996. 17. Brennan, F. M., Field, M., Chu, C. Q., Feldmann, M., and Maini, R. N., Cytokine expression in rheumatoid arthritis. Br. J. Rheumatol. 30(Suppl. 1), 76–80, 1991.
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Received October 20, 1997; accepted with revision January 8, 1998
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