Shift toward T lymphocytes with a T helper 1 cytokine-secretion profile in the joints of patients with rheumatoid arthritis.
код для вставкиСкачатьARTHRITIS & RHEUMATISM Vol. 39, No. 12, December 1996, pp 1961-1969 0 1996, American College of Rheumatology 1961 SHIFT TOWARD T LYMPHOCYTES WITH A T HELPER 1 CYTOKINE-SECRETION PROFILE IN THE JOINTS OF PATIENTS WITH RHEUMATOID ARTHRITIS RADBOUD J. E. M. DOLHAIN, ANNETTE N. VAN DER HEIDEN, NATALJA T. TER HAAR, FERDINAND C. BREEDVELD, and ANDRE M. M. MILTENBURG Objective. To investigate whether T cells in the inflamed joints of patients with rheumatoid arthritis (RA) preferentially produce the T helper 1 (Thl) cytokines, interferon-y (IFNy) and interleukin-2 (IL-2), or the Th2 cytokine, IL-4, when compared with corresponding peripheral blood-derived T cells. Methods. Synovial fluid mononuclear cells (SFMC) and corresponding peripheral blood mononuclear cells (PBMC) from 10 patients with RA were analyzed, either directly or after in vitro stimulation, for the intracellular presence of T h l and Th2 cytokines. The amount of secreted cytokine in the cell culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA). Results. IFNy-containing cells were detected in the unstirnulated SFMC, but not in the PBMC, of 3 patients with RA. Cells positive for IL-2 or IL-4 were not detected in the unstimulated samples. Following stimulation, the mean percentage of cells containing T h l cytokines was significantly increased in the SFMC compared with the PBMC; no differences were found in the mean percentage of IL-4-containing cells. A comparable shift toward T h l cytokines was observed when the amount of secreted cytokine was determined by ELISA. Conclusion. A shift toward T cells with a T h l cytokine profile was observed in the joints of patients with RA. Since an imbalance between T h l and Th2 cells is thought to be of pathogenic significance, this finding Dr. Dolhain’s work was supported by a grant from “Het Nationaal Reumafonds.” Radboud J. E. M. Dolhain, MD, Annette N. van der Heiden, Natalja T. ter Haar, Ferdinand C. Breedveld, MD, PhD, Andre M. M. Miltenburg, PhD: Leiden University Hospital, Leiden, The Netherlands. Address reprint requests to Radboud J. E. M. Dolhain. MD, Leiden University Hospital, Department of Rheumatology, C4-R, PO Box 9600, 2300 RC Leiden, The Netherlands. Submitted for publication March 19, 1996; accepted in revised form July 1, 1996. might have implications for the development of new therapies for RA. T cell-mediated autoimmune responses are considered to play a role in the pathogenesis of rheumatoid arthritis (RA) (1,2). At least 2 functional subsets of T cells can be distinguished according to their cytokinesecretion profiles: T helper 1 (Thl) cells, which produce interleukin-2 (IL-2), interferon-y (IFNy), and lymphotoxin, and Th2 cells, which secrete IL-4, IL-5, and IL-10 (3,4). At present, an accumulation of evidence supports the concept that a T cell response with either a T h l or a Th2 character is associated with different manifestations of disease (5). In animal models of T cell-mediated autoimmune disease, T h l cells were responsible for a pathogenic response, whereas a protective immunosuppressive response resided within the Th2 population (6-9). In experimental Lyme disease, C3H/HeJ mice developed severe arthritis based upon antigen recognition by T h l cells, whereas BALB/c mice that developed a Th2 response have only shown mild disease (7). Further evidence indicates that therapies that induce a shift of the immune response from T h l to Th2, or that directly influence the balance between T h l and Th2 cells (e.g., the infusion of autoreactive T cell clones with a Th2 character into animals with established disease), both result in disease amelioration (8,9). Moreover, in human T cell-mediated autoimmune diseases, a T h l response is believed to be pathogenic. In multiple sclerosis, for example, proteolipid protein reactive clones isolated from peripheral blood during an acute attack exhibit a T h l character, whereas during remission, such clones can exhibit either Tho, T h l , or Th2 characteristics (10). Although in most studies the T cell cytokine profile is determined following antigen-specific stimulation, differences in the T cell cytokine-secretion DOLHAIN ET AL 1962 Table 1. Characteristics of the study patients; Patient 1 2 3 4 5 6 7 8 9 10 Medication Sulfasalazine NSAID NSAID None Cyclosporine, hydroxychloroquine NSAID, acetaminophen Prednisone, NSAID Hydroxychloroquine, NSAID NSAID NSAID Disease duration (years) Ritchie Articular Index score ESR CRP (mmihour) (rnglliter) Leukocytes in synovial fluid ( X 103/mrn3) 4 2 24 20 22 14 71 103 47 36 91 78 60 NA 81 23 NA 13.4 5.2 4.6 17.0 8 38 111 134 10.1 4 13 22 15 74 44 21 32 8.7 8.0 3 15 7 5 NA 15 78 9.1 5.5 15 5 43 21 * ESR = erythrocyte sedimentation rate; CRP nonsteroidal antiinflammatory drug. = profile between patients and controls have also been observed following mitogenic stimulation (11-13). The available data concerning the presence of T lymphocytes with a Thl or Th2 character in the rheumatic joint are limited. T cell clones isolated from RA synovial tissue were proven to be primarily of the Thl type (14,15). However, only limited numbers of clones have been analyzed, and the possibility cannot be excluded that the procedure for cloning T cells favored the outgrowth of T cells with a specific Th type. Another study demonstrated the dominant presence of IFNy messenger RNA (mRNA), relative to IL-4, in the rheumatoid synovial membrane (16). Although this approach reflects the situation in vivo, no conclusion can be drawn regarding the full potential of T cells derived from the inflamed joint to produce cytokines following appropriate stimulation. To further investigate the balance (relative presence) of Thl and Th2 cells at the site of rheumatoid inflammation, paired synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) were obtained from RA patients in order to determine the percentage of cells producing the Thl cytokines, IFNy and IL-2, and the Th2 cytokine, IL-4. Staining for the different cytokines was performed either directly, after activation with phorbol myristate acetate (PMA) and a calcium ionophore, or after activation with immobilized anti-CD3 antibodies. Cytokine-producing cells were identified using cytokine-specific monoclonal antibodies (MAb) and immunohistochemical detection procedures. The amount of secreted cytokine in the cell culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA). To determine whether differences in the T cell subsets present in the PBMC 36 C-reactive protein; NA = not available; NSAID = and SFMC could be responsible for a shift toward a cytokine-secretion pattern with either a Thl or a Th2 character, the percentage of T cells and their activation and maturation state in these 2 compartments was evaluated by fluorescence-activated cell sorter (FACS) analysis. Finally, triple-staining techniques were used to examine the phenotype of cytokine-producing cells. PATIENTS AND METHODS Patients. Paired PBMC and SFMC were obtained from 10 patients (Table 1) with active RA (mean 2 SD number of swollen joints 6.2 ? 3.6; mean -C SD Ritchie Articular Index [17] 16.9 -C 10.7; mean -C SD erythrocyte sedimentation rate 64.4 2 31.0 mm/hour). All but 1 patient (patient 7) were positive for rheumatoid factor, and all patients had periarticular joint erosions detected on radiographs of the hands and feet. RA was defined according to the 1987 revised criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) (18). Synovial fluid was obtained from knee joints (mean ? SD number of leukocytes per mm3 of synovial fluid 9.1 2 4.1 X 10’; mean 2 SD visual analog scale for knee pain 5.9 -C 1.7, range 0-10). PBMC were also obtained from 5 healthy individuals who were matched for age and sex in order to compare cytokineproduction profiles. Lymphocytes were isolated from blood or synovial fluid by Ficoll-amidotrizoate gradient centrifugation and stored in tissue culture medium (TCM) supplemented with 10% dimethyl sulfoxide (Merck, Darmstadt, Germany) in liquid nitrogen. TCM consisted of Iscove’s modified DulbecCO’S medium (Whittaker, Baltimore, MD) supplemented with the antibiotics penicillin and streptomycin (100 IU/ml and 100 pg/ml, respectively; Boehringer Mannheim, Mannheim, Germany) and 10% fetal calf serum (Gibco BRL, Breda, The Netherlands). Induction of cytokines. Paired PBMC and SFMC samples were thawed and washed with phosphate buffered saline (PBS). A portion of the cells was directly analyzed for the presence of cytokine-producing cells. These cells were 1963 Thl CYTOKINE-SECRETION PROFILE IN RA Table 2. Specificity - Characteristics of the monoclonal antibodies used in the present study" Antibody CD2 CD3 CD4 D9D10 MD1 17H12 25D2 MT910 Leu-4 Leu-3A CD8 Leu-2A CD16 CD20 CDE CD45RA CLB-CD16 LEU-16 Tac CD45RO Leu-45RO CD69 Leu-23 B8.11.2 - - - - LEU-18 Reactivity Source IFNy IFNy IL-2 IL-4 Pan T cell Pan T cell Helper/inducer T cells, subset of monocytes Cytotoxic/suppressor T cells, natural killer cells Natural killer cells B cells I L 2 receptor Naive T cells, B cells, monocytes Memory T cells, B cells, monocytes Activated T and B cells HLA-DR Laskay et a1 (see ref. 19) Van der Meide et al (see ref. 21) DNAX (Palo Alto, CA) (see ref. 22) DNAX (Palo Alto, CA) (see ref. 22) Dakopatts (Glostrup, Denmark) Becton Dickinson (Mountain View, CA) Becton Dickinson (Mountain View, CA) - Biotin - FITC PFITC FITC Becton Dickinson (Mountain View, CA) P CLB (Amsterdam, The Netherlands) Becton Dickinson (Mountain View, CA) ATCC (Rockville, MD) Becton Dickinson (Mountain View, CA) - - FITC Becton Dickinson (Mountain View, CA) P Becton Dickinson (Mountain View, CA) Rebai et a1 (see ref. 23) - * IFNy = interferon-y; IL-2 = interleukin-2; FITC = fluorescein isothiocyanate; P = R-phycoerythrin; CLB Netherlands Red Cross Blood Transfusion Service; ATCC = American Type Culture Collection. stained as described below. The remaining cells were stimulated in 96-well tissue culture plates (Greiner, Solingen, Germany) with a combination of PMA (1 ng/ml) (Sigma, St. Louis, MO) and the calcium ionophore A23187 (ionomycin, 500 ng/ml; Sigma) or with immobilized anti-CD3 (OKT3; Cilag, Herenthals, Belgium) in TCM. For the latter purpose, 96-well plates were coated overnight at room temperature with OKT3 (10 pg/ml, 75 pl/well) in PBS. Wells were aspirated and washed twice with PBS before use. The cells were cultured for 4 hours at 37°C and 5% CO, at a concentration of 2 X lo5 cells/well, after which cells and supernatants were harvested. Supernatants were stored at -20°C until further analysis. Staining procedure. Light microscopy. Both stimulated and unstimulated cells were stained for the presence of the Thl cytokines, IFNy and IL-2, and the Th2 cytokine, IL-4, according to the paraformaldehyde saponin procedure (19,20). The sources and specificities for all of the antibodies used are listed in Table 2 (21-23). Cells were allowed to adhere t o adhesion slides (Biorad, Munich, Germany), fixed with 4% paraformaldehyde (P6148; Sigma) for 1 hour, and endogenous peroxidase activity was blocked by incubating the cells with 0.3% H,O, (30%) and 0.1% NaN, in PBS for 10 minutes. Cells were permeabilized with 0.1% saponin (Riedel de Haen, Seelze, Germany) in PBS. Cytobine-producing cells were detected using cytokine-specific MAb (Table 2) followed by 2 steps of horseradish peroxidase-conjugated reagents (rabbit anti-mouse Ig (P161), rabbit anti-rat Ig (P450), and swine anti-rabbit Ig (P399) polyclonal antibodies; all from Dakopatts, Glostrup, Denmark). All first-step reagents were diluted in PBS containing 0.1% saponin and 1% bovine serum albumin (BSA Organon Teknika, Boxtel, The Netherlands). Second- and third-step reagents were diluted in PBS containing 0.1% saponin and 10% normal human serum. Between all incubation periods, slides were washed with PBS containing 0.1% saponin. Histochemical revelation of horseradish peroxidase was performed with diaminobenzidine tetrahydrochloride. Label = Central Laboratory of The Fluorescence microscopy (triple-stainingprocedures). In order to identify cellular subsets involved in the production of Thl cytokines, triple-staining techniques were developed. Both stimulated and unstimulated cells were allowed to adhere to reaction fields of adhesion slides. Cells were first incubated for 30 minutes at 4°C with unconjugated mouse anti-human MAb against the maturation markers CD45RA and CD45RO or the activation marker HLA-DR; these antibodies were diluted in PBS with 1% BSA. Visualization of these antibodies was performed by a second incubation period (30 minutes at 4°C) of the reaction fields with a sheep anti-mouse Ig polyclonal antibody conjugated to aminomethylcoumarin acetic acid (1533983; Boehringer Mannheim) diluted in PBS with 1% BSA. Subsequently, free binding sites of the sheep anti-mouse Ig polyclonal antibody were blocked by incubating the reaction fields with 20% normal mouse serum (NMS) in PBS for 20 minutes at 4°C. To detect T cells, reaction fields were then incubated with a mouse anti-human CD2 antibody conjugated to fluorescein isothiocyanate in PBS with 5% NMS for 30 minutes at 4°C. Between all incubation periods, slides were washed with cold (4°C) PBS. When staining for surface markers was completed, cells were fixed using 4% paraformaldehyde for 30 minutes at 4°C and permeabilized with saponin 0.1% in PBS. To detect cytokine-producing cells, reaction fields were incubated for 30 minutes at 37°C with either a biotinylated mouse anti-human IFNy antibody or a rat anti-human IL-2 antibody, both diluted in PBS with 5% NMS and 0.1% saponin. Incubation with the biotinylated anti-IFNy antibody was directly followed by a final incubation period of 30 minutes at room temperature with streptavidin conjugated to tetramethyl rhodamine isothiocyanate (A7169; Sigma) diluted in PBS with 0.1% saponin and 2% BSA. For the detection of IL-2-producing cells, the final incubation period (30 minutes at room temperature) was preceded by an additional incubation with a biotinylated goat anti-rat Ig polyclonal antibody (BA9400; Vector Laboratories, DOLHAIN ET AL 1964 A B Figure 1. A, Morphologic aspects of the intracellular presence of a cytokine product, using interleukin-2 (IL-2) as an example. Positive cells (in this case, activated synovial fluid mononuclear cells that stain positive for IL-2) are characterized by a distinct perinuclear dot, indicative of Golgi localization (by immunoperoxidase staining). B, Negative control. (Original magnification X 250.) Burlingame, CA) diluted in PBS with 0.1% saponin and 5% NMS. During the procedure of intracellular staining for cytokines, slides were washed with PBS with 0.1% saponin between all incubation periods. Primary, secondary, and tertiary reagents were titrated to obtain optimal results. Specificity controls. The specificity of all antibodies used has been described in detail (Table 2). As controls for nonspecific interactions, isotype-matched irrelevant antibodies were included and cytokine-specific antibodies were omitted from the procedure. As a positive control for the accuracy of the staining procedure, activated cells from a CD4+ Tho clone were included in all experiments. Examination of slides. Cytokine-producing cells were identified by a clear perinuclear dot indicative of Golgi localization. To determine the total number of cytokine-producing cells, at least 2,000 cells were scored in case the cells were not stimulated in vitro or following stimulation with immobilized OKT3. Following activation with PMA and ionomycin, at least 1,000 cells were analyzed. For double- and triple-staining techniques, -200 cells expressing a specific marker were scored on a Leitz Diaplane fluorescence microscope (Leica Mikroskopie und Systeme, Wetzlar, Germany). FACS analysis. Phenotyping of PBMC and SFMC was performed by flow cytometry on nonstimulated cells using a FACS scan (Becton Dickinson, Mountain View, CA). The percentage of cells expressing CD14 (monocytes), CD16 (natural killer cells), and CD20 (B cells) was assessed using monolabel fluorescence techniques. To determine the percentage of T cells and to analyze subpopulations of cells within the T cell subset, double-label immunofluorescence techniques were used. All antibodies used for FACS analysis are listed in Table 2. ELISA. ELISA was used to detect the amount of cytokine secreted in the cell culture supernatant. The ELISA for IFNy was performed as described previously (21). To detect IL-2, the mouse anti-human IL-2 MAb 419 A7A3 (58.1 19.08; Medgenix Diagnostics SA, Fleurus, Belgium) was used as a capture reagent. The biotinylated mouse anti-human IL-2 MAb 297 C 16GZ (58.124.03; Medgenix Diagnostics SA) was used as a secondary reagent. The IL-2 ELISA and IL-4 ELISA (M1914; CLB, Amsterdam, The Netherlands) were performed according to the specifications of the manufacturer. Statistical analysis. The Wilcoxon signed rank test was used to analyze matched pairs. The Mann-Whitney U test was used to compare group means. Correlation coefficients were determined by Spearman’s rank correlation test. RESULTS Enhanced presence of Thl cytokine-producing cells in SFMC of RA patients. PBMC and S F M C from 10 RA patients were stained either directly or after in vitro activation for t h e presence of intracellular cytokines. Cells that stained positive for cytokines could b e identified by a distinct perinuclear dot, indicative of Golgi localization (Figure 1A). These dots were absent when the first antibody was omitted from the procedure or when it was replaced by an irrelevant isotype-matched control antibody (Figure 1B). The occurrence of cells that were found to spontaneously contain a cytokine product in peripheral blood was below 1 in 1,000 cells. T h l CYTOKINE-SECRETION PROFILE IN RA 3.01 N.S, 2.0 0.30 0.20 N.S F T 1.o 0.0 SFMC PBMC SFMC PBMC SFMC PBMC Figure 2. Enhanced presence of T helper I cytokines in synovial fluid mononuclear cells (SFMC) of patients with rheumatoid arthritis (RA). SFMC and peripheral blood mononuclear cells (PBMC) of 10 patients with RA were stimulated for 4 hours with phorbol myristate acetate and ionomycin. The percentages of cells that stained positive for A, interferon-y (IFNy) (W), B, interleukin-2 (IL-2) ( were determined using immunohistochemical detection methods. The concentrations of these cytokines in the supernatants (D,IFNy. E, 1L-2, and F, IL-4) were detected by enzyme-linked immunosorbent assay. Bars show the mean and SD. P values represent levels of significance between paired SFMC and PBMC. N.S. = not significant. However, the T h l cytokine, IFNy, could be detected in 3 (2%, 2%, and lo%, respectively) of the unstimulated SFMC samples. No IL-2- or IL-4-containing cells could be detected in unstimulated samples. SFMC and corresponding PBMC samples were stimulated with a combination of PMA and ionomycin to investigate the full potential of cells from these 2 compartments to produce cytokines following stimulation. In SFMC samples, more cells were found to produce IFNy (P < 0.02) and IL-2 ( P < 0.05), compared with paired PBMC (Figures 2A and B). IL-4-producing cells could be detected in only 5 of 10 SFMC samples and 6 of 10 PBMC samples. The mean number of cells expressing this cytokine in all samples tested was low and similar in both populations (Figure 2C). PBMC of 5 age- and sex-matched healthy volunteers were included in this experiment to determine whether the difference in frequencies of IFNy- and IL-2-producing cells could be 1965 attributed to the PBMC or SFMC fraction. Similar frequencies of IFNy-, IL-2-, and IL-4-producing cells were detected in the PBMC of healthy volunteers when compared with PBMC of RA patients. To investigate the potential for cytokine production after treatment with a more physiologic stimulus, cells were activated using immobilized OKT3 antibodies. With this stimulus, IFNy- and IL-Zexpressing cells were detected in all PBMC and SFMC samples, although lower numbers of cells that stained positive were present when compared with PMA- and ionomycin-activated cellular preparations. The mean ? SD percentages of cells staining positive for IFNy were 2.9 ? 2.1 in SFMC and 1.8 2 1.7 in PBMC samples (P = 0.06). The mean ? SD percentages of IL-2-containing cells were 0.5 2 0.4 in SFMC and 0.3 ? 0.3 in PBMC samples ( P = 0.21). Following stimulation with OKT3, only 1 SFMC sample was found to contain IL-4-positive cells. Enhanced presence of Thl cytokines in culture supernatants of activated SFMC. In all supernatants of cell cultures stimulated with PMA and ionomycin, IFNy and IL-4 could be detected. The concentrations of IFNy in the supernatants of stimulated SFMC were significantly higher when compared with those in supernatants of activated paired PBMC (P C 0.01) (Figure 2D). Similar concentrations of IL-4 could be detected in the supernatants of PBMC and SFMC cultures (Figure 2F). In 8 of 10 SFMC and 5 of 10 PBMC cell culture supernatants, IL-2 concentrations above the detection limit of the ELSA (625 pg/ml) were measured. Although the Supernatants of stimulated SFMC contained more IL-2 when compared with those of activated paired PBMC, the difference did not reach statistical significance (P = 0.07) (Figure 2E). T o obtain more insight into the balance between the production of T h l and Th2 cytokines in SFMC and PBMC, ratios of the levels of IFNy to IL-4 were calculated by dividing the concentration of IFNy in the cell culture supernatants by the concentration of IL-4. In 9 of 10 patients, this ratio was higher in the SFMC culture supernatants compared with paired PBMC culture supernatants (P < 0.01) (Figure 3). No differences were observed in the concentrations of IFNy, IL-2, and IL-4 in supernatants of stimulated PBMC of RA patients compared with PBMC of 5 healthy controls. To investigate whether the amount of a certain cytokine in the cell culture supernatant was the result of the number of cells producing that specific cytokine and to evaluate the accuracy of the technique for intracellular staining, the percentage of cells that stained positive for a certain cytokine was correlated with the concentration of that specific cytokine in the cell culture DOLHAIN ET AL 1966 P 500 ' 1 2 3 4 5 6 7 PATIENT 8 9 1 0 Figure 3. Ratio of the production of interferon-y (IFNy) to interleukin-4 (IL-4) in synovial fluid mononuclear cells (SFMC) (H) compared with peripheral blood mononuclear cells (PBMC) (H). To obtain insight into the balance between T helper 1 and T helper 2 cytokines, the ratio of IFNy to IL-4 was calculated by dividing the concentration of IFNy by that of IL-4 in the cell culture supernatant of phorbol myristate acetate- and ionomycin-stimulated SFMC and PBMC. P < 0.01. SFMC vs. PBMC. supernatant. For all cytokines evaluated, statistically significant correlations were found (IFNy p = 0.71, P < 0.0001; IL-2 p = 0.83, P < 0.0001; IL-4 p = 0.76, P < 0.000 1). Phenotypic characterization of the different PBMC and SFMC samples. Phenotyping of the different PBMC and SFMC samples revealed a significant increase in the number of CD3-positive cells in the SFMC samples compared with the PBMC samples, accompanied by a lower percentage of cells expressing the monocyte marker CD14, the marker for natural killer cells CD16, and the B cell marker CD20 (Table 3). The increase in the percentage of CD3-positive cells in the SFMC samples when compared with the PBMC samples cannot solely account for the difference in the number of cytokine-producing cells following activation, since, in patients who showed comparable frequencies of T cells in their SFMC and PBMC, a higher number of cytokine-producing cells could be demonstrated in the SFMC (patient 9 SFMC CD3 38%, IFNy 10.6%, PBMC CD3 48%, IFNy 6.2%; patient 10 SFMC CD3 52%, IFNy 14.9%, PBMC CD3 42%, IFNy 3.5%). To obtain more insight into the differences between T cell populations in SFMC and PBMC samples, double-staining procedures were performed. No differences were observed regarding the percentage of T cells expressing CD4 or CD8. However, the SFMC-derived fraction contained significantly more memory T cells (CD45RO) and fewer naive T cells (CD45RA) when compared with the PBMC-derived fraction. In the SFMC fraction, more T cells were found to express the activation markers HLA-DR and CD69; the expression of the activation marker CD25 on T cells was low, and T cells expressing this marker could not be detected in all samples (PBMC CD25 4 of 9 samples positive, SFMC CD25 7 of 10 samples positive). Nevertheless, the expression of CD25 was significantly increased on SFMCderived T cells compared with PBMC-derived T cells (Table 3). Phenotype of the Thl cytokine-producing cells. Triple stainings were performed to identify the phenotype of the T h l cytokine-producing cells (Figure 4). In all 3 patients tested, the vast majority of the cytokineproducing cells coexpressed the T cell marker CD2 (IFNy PBMC mean 94% CD2+, SFMC mean 99% CD2-t; IL-2 PBMC mean 80% CD2+, SFMC mean 98% CD2-t). I F N y and IL-2-producing cells could be detected in both the CD2+, HLA-DR- and the CD2+, HLA-DR+ subsets. No clear differences in frequencies of cytokine-producing cells could be demonstrated in the 2 subsets tested. IFNy-producing cells were detected in both the CD2+, CD45RA and CD2+, CD45RO subsets. In all patients the expression of IL-2 seemed to be confined to the CD2+, CD45RO subset only. Triple staining with CD69 as a marker was not performed, since, upon activation, a very rapid increase in CD69+ T cells was noted (data not shown). Unfortunately, using immunofluorescence-based techniques, no IL-4producing cells could be demonstrated. This is probably due to the fact that, as reported previously (20), immunofluorescence-based techniques are somewhat less sensitive in demonstrating cytokine-producing cells when compared with techniques used with the light microscope. Table 3. Differences in phenotype of mononuclear cells isolated from synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC)* Mononuclear cells, T cells, '36 % Marker PBMC SFMC Marker PBMC SFMC CD3 CD14 CD16 CD20 45 t 9 t 23 t 11$ 18 2 10 7 % 75 7 0 % 16 9 % I1 4 +5 2%2 CD4 CD8 CD45RA CD45RO HLA-DR CD69 CD25 55 i 15 4 2 ? 15 43 ? lo$ 6 2 t 121 11 t 5 t 4 -t 4 t 2t64 54 t 9 41 2 9 11 ? 9 93t4 53 t 12 57 z 11 6'10 * Values are the mean t SD. f P < 0.01 versus SFMC. $. P < 0.025 versus SFMC. 5 P < 0.05 versus SFMC. Thl CYTOKINE-SECRETION PROFILE IN RA 1967 DISCUSSION A B C Figure 4. Phenotypic analysis of cytokine-producing cells using triplestaining immunofluorescence technology. The arrow in each figure indicates a cell coexpressing A, interferon-y (using tetramethylrhodamine isothiocyanate), B, CD2 (using fluorescein isothiocyanate), or C, CD45RO (using aminomethylcoumarin acetic acid). (Original magnification x 400.) The present data have provided evidence to support a role for Thl cytokine-producing T cells in the pathogenesis of RA. Cells that spontaneously produced [FNy within the synovial fluid mononuclear cell population of RA patients were detected. Furthermore, a shift toward lymphocytes with a Thl cytokine profile was Dbserved in activated SFMC compared with paired PBMC samples. This shift was observed not only when the percentage of cells producing Thl or Th2 cytokines was taken into account, but also when the amount of Thl 3r Th2 cytokmes in the cell culture supernatants was determined. The increase in T cells expressing a Thl cytokine profile was specific for the SFMC, since the balance between the Thl and Th2 cells from the PBMC of RA patients was not different from that of healthy controls. It was also shown that the SFMC-derived T cells were more often of the memory type, and a higher percentage of these cells expressed activation markers when compared with PBMC-derived T cells. Triple staining revealed that differences in maturation state might have contributed to the increased production of IL-2, but, for F N y , this was not the case. In addition, it was concluded that differences in the expression of activation markers were not associated with the increased production of cytokines. In this study, highly significant correlation coefficients were found between the percentage of cytokineproducing cells and the amount of that specific cytokine n the cell culture supernatant. These findings are conistent with those of a previous study (24) that suggested I model of T cell activation in which, at a single-cell evel, all-or-none rather than graded responses of cytoLine genes were dominant. Our data extrapolate from he results obtained in murine models to human disease. n addition, our results extend beyond the mRNA to the )rotein level. The finding that the concentrations of ,ytokines, as detected by ELISA, correlated well with he percentage of cells producing a specific cytokine, as Jetected by intracellular cytokine staining, underlines the usefulness of the latter method in quantifying cytokine production, even when, similar to IL-4, only a few producer cells may be present. The percentages of cells producing IFNy, IL-2, and IL-4 were in the same range in the PBMC of RA patients when compared with the PBMC of healthy controls, and similar concentrations of cytokines were measured in the supernatants of these cell popu~ations. This implies that the shift toward a Thl response is restricted to the actual site of inflammation. Such com- 1968 partmentalization of functional subsets of T cells in the direction of a T h l or a Th2 response has been reported previously. In experimental Lyme disease, a Thl-based T cell response against the inciting antigen was associated with severe disease manifestations. This polarization in T cell function was present only at the site of inflammation, and not in distant lymphoid organs (7). Comparable observations have been made in patients with conjunctivitis (13). Triple-staining techniques were used to determine whether the differences in activation and maturation states of the SFMC-derived T cells could be responsible for the increased production of T h l cytokines in the SFMC fraction. Consistent with findings in the literature, FACS analysis demonstrated that synovial fluid-derived T cells were mainly of the CD45RO memory type and showed an increased expression of the activation markers HLA-DR and CD69 (25). Most (26-29), but not all (30),studies have demonstrated that CD45RO T cells produce more IFNy than CD45RA cells. In this study, the percentage of CD45RO T cells producing IFNy was not significantly higher than that of CD45RA cells. Furthermore, consistent with previous findings, IL-2 production was mostly confined to the CD45RO subset (29,31). No differences in production of IFNy and IL-2 could be observed with regard to T cells with or without HLA-DR expression. From these data it can be concluded that an increase in CD45RO T cells, but not differences in activation state, might have contributed to the increased expression of T h l cytokines in SFMC. The finding that the cytokine profile of SFMCderived T cells was significantly different from that of PBMC-derived T cells underscores the importance of T cells in maintaining RA. Although RA has been considered to be a T cell-mediated autoimmune disease (1,2), the dominant role of T cells in the process of rheumatoid synovitis has been questioned recently (32). Moreover, mechanisms that lead to joint destruction but are not under the regulation of T cells have been proposed (33). The observations made in this study, however, favor the idea of either a selective influx or a specific outgrowth of T cells of the T h l type occurring in the synovial membrane. This suggests a role for T h l cytokine-producing T cells in the pathogenic mechanisms underlying RA. As a result, an increase in IFNy over I L 4 production may be of pathogenic significance and may account for several of the histomorphologic changes in RA, such as activation of monocytes/macrophages and up-regulation of HLA class I1 antigens, both of which are prominent hallmarks of the rheumatoid joint (1,34,35). Since T h l and Th2 cytokines are believed to have DOLHAIN ET AL mutual regulatory interactions, the balance between T cells with a T h l or Th2 phenotype is considered to be an important regulatory mechanism of T cell function. This balance is most profoundly influenced by those factors, including cytokines, that influence the initial priming of T cells in the direction of T h l or Th2. During priming, the monokine IL-12 causes a shift toward a T h l response, whereas the T cell-derived cytokine IL-4 does so for a Th2 response. The Th2 and monocyte-derived cytokine IL-10 is able to down-regulate T h l function, but does not influence the initial priming toward a T h l response (36). This could be an explanation for the predominance of T h l over Th2 cytokine-producing cells, despite the presence of 1L-10 in the rheumatoid joint (37). Therefore, similar to findings in animal models of autoimmune disease, treatment strategies that result in reversal of the observed shift in T cell function could be of therapeutic benefit (8,9). In experimental arthritis, infusion of IL-4 alone or in combination with IL-10 has been shown to have beneficial effects on the severity of disease (38,39). 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