Interleukin-17A- or tumor necrosis factor α-mediated increase in proliferation of T cells cocultured with synovium-derived mesenchymal stem cells in rheumatoid arthritis

The study was approved by the Ethics Committee at Sun Yat-sen Memorial Hospital, and informed consent was obtained from all study subjects. Synovial tissue biopsies from the suprapatella pouch were obtained from 22 RAp and eight HD (For practical reasons, we chose patients with meniscus injury who were undergoing arthroscopy, and without any systemic immune disease or connective tissue disease, as the healthy donors) by using 3.5-mm grasping biopsy forceps under direct vision with arthroscopy. The RAp fulfilled the American College of Rheumatology criteria for RA [25]. The degree of macroscopic joint inflammation was evaluated by using the Visual Analogue Score (VAS). Scores (scaled between 0 and 100) were based on visual image of vasculature (redness and vessels due to hyperemia) in arthroscopy [26]. Synovial tissues were finely minced and digested with 0.4% collagenase (Gibco BRL Co.Ltd.,Gaithersburg, MD, USA) in high-glucose Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 U/ml streptomycin. After overnight incubation at 37°C, cells were collected by centrifugation, washed twice, resuspended in high-glucose DMEM supplemented with 10% FBS, plated in a T25 culture flask, and allowed to attach for 7 days. Nonadherent cells were removed by changing the medium. Cells were passaged when reaching near confluence according to previous report [7].

PBMCs were from HD and RAp by Ficoll-Hypaque density gradient (density, 1.077 g/L; Sigma). The clinical status of two groups is described in Table 1.

Synovial T cells were expanded from synovial tissues of RAp cultured for 14 days in RPMI-1640 medium supplemented with 10% FBS, in the presence of recombinant IL-2 (20 IU/ml; R&D Systems, Minneapolis, MN, USA). Cultures were fed every 3 days. T cells were then collected and analyzed with a cell sorter (FACS Vantage SE cell sorter; Becton Dickinson). More than 95% of the cells expressed CD3.

SMSCs (P4) were vaccinated into 96-well plates at a concentration of 1 × 10⁴/ml, in a final volume of 100 μl fresh medium (10% FBS + high-glucose DMEM). For cell counting, three wells of each sample were digested by using 0.25% trypsin-ethylenediamine tetraacetic acid per day up to 12 days. With these data for cell-population doubling, we acquired the SMSC growth curves. With methyl thiazolyl tetrazolium (MTT, 5 mg/ml; Sigma), dimethylsulfoxide (DMSO; Sigma), and an EL800 microplate reader (BioTek Instruments, Winooski, VT, USA) that was to detect absorbance at 490 nm, we made the SMSCs viability curves in the same way, according to the day and the absorbance. Fresh medium was used as a negative control.

For the in vitro differentiation assays, three procedures (adipogenic differentiation, osteogenic differentiation, and chondrogenic differentiation) were used, as previous described [7]. The intracellular lipid accumulation as an indicator was visualized on day 21 with Oil Red O staining. The alkaline phosphatase (ALP) of SMSCs was assayed by using Cell ALP Staining assay (Nanjing Jiancheng, China), according to the recommendations of the manufacturers on day7 and alizarin red staining (AR-S, 1%, pH 7.2; Sigma) on day 28, respectively. The chondrogenic differentiations were confirmed with alcian blue staining. Meanwhile, the experimental controls were established by culture of SMSCs (P4) in fresh medium, and only fresh medium was used as a negative control. All measurements were tested in triplicate. An inverted phase-contrast microscope (Nikon Eclipse Ti-S; Nikon Corporation, Japan) visualized the images.

For quantitative assay, three procedures (adipogenic differentiation value, calcium deposits, alcian blue intensity) were used as previous represented [27, 28].

After treatment with 0.25% trypsin-ethylenediamine tetraacetic acid, SMSCs (P4) were then resuspended in PBS containing 0.5% BSA and 0.1% sodium azide. Cell aliquots (1 × 10⁶ cells/ml) were incubated on ice with conjugated mAbs against CD105, CD166, CD44, CD90, CD14, CD34, CD45, and HLA-DR (Table 2) or conjugated isotypic controls. Flow cytometry was performed on a FACScan laser flow-cytometry system (Becton Dickinson, San Jose, CA, USA), and data were analyzed with the CellQuest software (BD Bioscience, San Jose, CA, USA).

The suppressive effects of SMSCs (P4) on mixed PBMCs reaction (MLR) and PBMCs proliferation stimulated by phytohemagglutinin (PHA) (4 μg/ml; Roche, Mannheim, Germany) were measured by using the MTT assay [29] and the ³H-TdR assay [30], as described previously. SMSCs were seeded in 96-well culture plates for 6 hours for adherence, and then irradiated (30 Gy) with Co⁶⁰.

For the MLR, allogeneic PBMCs (15 × 10⁴ cells/cm²) from an HD were mixed with PBMCs from another unrelated HD in identical mounts. The mixed PBMCs were then mixed with different ratios (3 × 10⁵ cells/cm² = 1:1 SMSC: PBMCs ratio, 15 × 10⁴ cells/cm² =1:2, 6 × 10⁴ cells/cm² =1:5, 3 × 10⁴ cells/cm² = 1:10, 15 × 10³ cells/cm² = 1:20) of SMSCs (experiment wells) or without SMSCs (blank wells) in 96-well culture plates to ensure efficient cell-cell contact for 5 days in 0.2 ml modified RPMI-1640 medium (Gibco) supplemented with 10% FBS.

The PBMCs proliferation assay only uses one autologous or allogeneic PBMCs reaction (30 × 10⁴ cells/cm²) from a healthy donor or patient with RA stimulated with PHA. Inhibitory or proliferative effects were measured on day 5 by using the MTT assay or the ³H-TdR assay. All measurements were performed in triplicate. Results were expressed as the mean (% inhibition or % proliferation) ± SD.

Continuous variables were expressed as the mean ± SD, and categoric variables were presented as frequencies and percentages. The significance of the results was determined by using the unpaired Student t test, χ² test, and repeated-measure tests with Bonferroni correction. Data analysis was performed with statistical software (SPSS, version 15.0 for Windows; SPSS Inc., Armonk, NY, USA). P < 0.05 was considered statistically significant.

The RA-SMSCs growth curves (data not shown) have the same tendency as those for HD-SMSCs. The SMSCs population-doubling data of these two groups at each day (1 to 12 days) were tested by repeated-measures tests with Bonferroni correction, and the statistical result indicates that no statistically significant difference was present in SMSCs population doubling between RAp and HD (P = 0.157). For SMSCs viability at each point of time from 1 to 12 days, the difference of optical density (OD) at 490 nm between RAp and HD also was not statistically significant, as determined by cellular viability assays (P = 0.161).

In both groups, obvious differentiated adipocytes and osteocytes were detected as early as the day 14 after being induced for adipogenic and osteogenic differentiation, and obvious differentiated chondrocytes were seen at about 21 days since induction. For adipogenesis, the cells displayed the accumulation of lipid vacuoles, which stained with oil red O (Figure 1A1; B1). We analyzed the adipogenic differentiation value results by using IPP 6.0; no significant difference was found between RA-SMSCs and HD-SMSCs (P = 0.193). For osteogenesis, SMSCs from both populations treated with osteogenic medium underwent a change in their morphology from spindle shaped to cuboidal, and formed large nodules that stained for ALP and alizarin red (Figure 1A2, 3; B2, 3). For quantitative assay of mineral deposits, absorption of the dye at 562 nm revealed no significant difference in alizarin red-positive mineralized matrices (P = 0.088). For chondrogenesis, the cells displayed cartilage-specific metachromasia with alcian blue in vitro (Figure 1A4; B4). The alcian blue staining intensity of pellets in both groups had no significant difference (P = 0.075).

By FACS analysis, for SMSCs from both tissues, the expression of CD14, CD34, CD45, and HLA-DR was negative, and the expression of CD44, CD90, CD105, and CD166 was positive (Figure 2). The expression intensity of each marker was not statistically different between the two populations (P > 0.05). Indeed, the cell populations isolated from two different tissues displayed a similar phenotype, just as the typical MSCs did [12].

As shown in Figure 3, no statistically significant reduction in suppressive potential (percentage inhibition) of RA-SMSCs was found on MLR at all five ratios, compared with the percentage inhibition of HD-SMSCs (Figure 3A2, A3; P > 0.05). Similarly, RA-SMSCs produced no statistically significant decreased inhibitory effect on autologous or allogeneic PBMCs proliferation (Figure 3B2, B3; P > 0.05), ranging from a SMSC-to-PBMC ratio of 1:20 to 1:1. Furthermore, in either of them, the ³H-TdR assay data also suggested a significant relation between dose and inhibition of SMSCs from both populations was established.

To understand why SMSCs do not display any immunosuppressive effects in RAp, we tested the potential role of the inflammatory environment on SMSCs behavior. We therefore investigated the role of various cytokines (IL-17A, TNF-α, IFN-γ) on the interaction of synovial T cells from RAp with autologous SMSCs. Because the best ratio of SMSCs to PBMCs was 1:1, we used this cell ratio for subsequent experiments. Irradiated SMSCs and synovial T cells were cultured alone or were cocultured at a 1:1 ratio for 24 hours in the presence or absence of PHA (4 μg/ml), and exogenous IL-17A (50 ng/ml), TNF-α (50 ng/ml), or IFN-γ (50 ng/ml) was added either alone or in combination. Each experiment was performed in triplicate and repeated at least 3 times. The coculture of HD-SMSCs and synovial T cells from RAp were used as the experimental control.

As shown in Figure 4A1, RA-SMSCs totally inhibited the autologous response of synovial T-cells (P < 0.05). In contrast, the proliferation of synovial T cells was increased when cultured with RA-SMSCs in the presence of IL-17A or TNF-α (P < 0.05; Figure 4A2). IL-17A and TNF-α had no effect on RA-SMSC or synovial T cells cultured alone (P > 0.05; Figure 4A3). No effect was observed when IFN-γ was added to the reactions (P > 0.05). Moreover, HD-SMSCs were unable to suppress the proliferative response in the presence of TNF-α and IL-17A, either alone or in combination (Figure 4B; P < 0.05).