| IntroductionRheumatoid arthritis (RA) is a chronic, systemic, autoimmune disorder characterized by symmetric and polyarthric joint disease. RA affects approximately 0.5 to 1.0% of the adult population across the globe. The main features of RA pathogenesis include massive and persistent synovial proliferation in multiple joints followed by destruction of cartilages and bones. With the progression of the disease, almost the majority of RA patients have the different degrees of joint deformity, functional disability, and systemic involvement, which lead to severe long-term economic consequences and social issues.Currently, the exact etiology and pathogenesis of RA is still unclear. It is considered to be the result of a complex interplay among multiple factors including genetic predisposition, environmental triggers, immune status, and stochastic factors. Environmental exposure such as smoking leads to the loss of tolerance to self-proteins by environment-gene interactions. Thereafter, antigens processed by dendritic cells (DCs) drive the activation of CD4+ T and B lymphocytes in synovial germinal centres or lymph nodes. Further, the production of pathogenic antibodies, abnormal activation of cytokine networks, and other molecular products of damage lead to chronic synovitis and progressive bone destruction in joints as well as systemic disorders through migration and local positive feedback mechanisms. With the progress of RA pathogenesis and the development of targeted drugs, several new anti-rheumatic drugs have been widely used in the clinical. But a systematic review study based on a number of randomized, double-blind, controlled clinical trial showed that after the use of anti-TNFa drugs for at least six months,40～50% patients failed to achieve the American college of rheumatology (ACR) 50% response. And at least 70% of patients failed to achieve the remission of disease activity score 28 joint counts (DAS28). Other drug such as tocilizumab or tofacitinib also has the same therapeutic effect, when compared with anti-TNFa drugs. Therefore, it is important for basic research and clinical treatment to further explore the pathogenesis of RA.As an important component of innate immune system, natural killer (NK) cells play an important biological effect in the resistance of pathogen infection and tumor and have also been implicated in the regulation of autoimmune disease through limit or amplification of the immune response. According to the expression of cell surface molecules in NK cells, it is divided into a CD56bright NK cells subset and a CD56dim NK cells subset. The previous subset primarily plays a role in immune regulation by secreting the cytokine and chemokines. The later subset mainly plays the role of natural killer and cell cytotoxicity by antibody-dependent and cell-mediated. Although the exact mechanism of NK cells in the pathogenesis of RA is not fully clarified, previous studies have suggested that the possible mechanisms include the following aspects at least. Firstly, CD56bright NK cells are enriched in RA synovial tissue and involved in the inflammatory response of arthritis by recruiting a large number of proinflammatory cytokines. Secondly, NK cells are able to promote the differentiation and activation of T and B lymphocytes as well as the maturation of DCs, while the DCs conversely activate NK cells by the production of interleukin (IL)-12. And also the NK cells are able to kill the immature DCs by the cytotoxicity mediated by NKp30 and NKp46, which result in the lack of tolerance in DCs. Thirdly, NK cells express the costimulatory molecules, such as CD40L, OX40, CD70 and CD86, which promote the maturation and activation of T and B lymphocytes by direct contact to provide costimulation. Finally, NK cells isolated from synovial fluid of RA patients not only promote monocytes secrete the tumor necrosis factor a (TNFa), but also accelerate itself secrete interferon-gamma (IFN-γ) when co-cultured both of them. By another way, NK cells could induce the CD 14+ monocytes differentiate into osteoclasts which dependent on the receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF).NKp44+NK cells marked with CD3-CD56+NKp44+is a CD56bright NK cells subset. NKp44 belonging to natural cytotoxicity receptor (NCR) family members selectively expresses in NK cells activated by IL-2 and is one of the important surface markers of NK cells. The study in patients with inflammatory bowel disease (IBD) revealed that NKp44+NKp46-NK cells with IL-22 secretion decreased and NKp44-NKp46+NK cells with IFN-y secretion increased in inflammation sites of intestinal mucosa. The imbalance of those two cell subsets plays a pathogenic mechanism during the proliferation of intestinal mucosa epithelial cells. In addition, High proportion of NKp44+NK cells have been detected in patients with ankylosing spondylitis, and primary Sjogren’s syndrome and these cells play a tissue-protective or a proinflammatory role by overexpression of interleukin 22 (IL-22). In patients with RA, human chemokine receptor 6 (CCR6) positive NKp44+NK cells (NK-22 cells) are significantly expanded in RA peripheral blood and synovial fluid, which were correlated positively with RA disease activity. It suggests that NK-22 cells, as a cell subset of NKp44+NK, may play an important role in the pathogenesis of RA. However, the expression of NKp44+NK cells in RA different tissues, especially in RA synovial tissue, and their molecular mechanisms and signaling pathways in the development of arthritis are still unclear.ObjectivesThe purpose of this study was to investigate not only the proportion of NKp44+NK cells in the peripheral blood and synovial fluid of patients with RA, but also the expression of those cells in RA synovial tissue. To clarify the molecular mechanisms of the proliferation of fibroblast-like synoviocytes (FLS) promoted by NKp44+NK cells and aime to evaluate the IL-22-dependent signal pathway that could promote FLS proliferation.Methods1 Patients’samplesDuring the period of February 2012 and December 2013, peripheral blood samples from 37 patients with RA as well as 35 healthy volunteers and synovial fluid samples from the knee joint of 16 patients with knee osteoarthritis (KOA) were collected from the department of Traditional Chinese Medicine Rheumatology or Bone and Joint Surgery, Nanfang Hospital, Southern Medical University. Synovial fluid samples were obtained during the therapeutic arthrocentesis from patients with KOA and 21 patients with RA who donated their PB samples synchronously. Synovial tissues specimens from patients with RA and KOA (3 patients each) were obtained during the joint replacement surgery. The study was conducted according to the principles expressed in the Declaration of Helsinki. All samples, clinical data, and demographic data were obtained after patients had given their written consent which was approved by the Institutional Medical Ethics Review Board of Nanfang Hospital (NO. NFEC-20120201).2 Diagnostic classification criteria and disease activity assessment of RAPatients with RA were fulfilled the 1987 American College of Rheumatology (ACR) classification criteria or 2010 ACR/European League Against Rheumatism (EULAR) classification criteria for RA. Patients with KOA were fulfilled the 1995 American College of Rheumatology (ACR) classification criteria for KOA. The disease activity assessment of RA was conducted according to disease activity score in 28 joints (DAS28) and clinical disease activity index (CDIA).3 Experimental methods3.1 Flow cytometric analysis of NKp44+NK cells and the clinical data collectionThe proportion of NKp44+NK cells from the peripheral blood and synovial fluid were analyzed by flow cytometry. And the detection of NKp44+NK cells from synovial tissue was performed by dual-labeling immunofluorescence. The clinical data were collected. Basic clinical information from patients with KOA and healthy volunteers was also collected.3.2 The flow sorting, culture, and cytokine detection of NKp44+NK cells in vitroNK cells and NKp44+NK cells were sorted from the synovial fluid of patients with RA by flow cytometry. The separated NK and NKp44+NK cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) complete medium which contains 10% heat-inactivated fetal bovine serum (FBS),2 mM L-glutamine,100 U/ml penicillin, and 100 μg/ml streptomycin at 37℃ in 5% carbon dioxide (CO2). The cells were treated with cell stimulation cocktail (l×) containing phorbol 12-myristate 13-acetate and ionomycin for 5 hours before the supernatant was collected as previously described. IL-22, M-CSF and RANKL concentrations in NK and NKp44+NK cells culture supernatants were determined by using Enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s protocol.3.3 The isolation, culture, and identification of FLS in vitroFLS were isolated from knee joint synovial tissues by the method of tissue block and then cultured in DMEM complete medium which contains 10% heat-inactivated FBS,100 U/ml penicillin, and 100 μg/ml streptomycin at 37℃ in 5% CO2. The identification of RA FLS between passages 4 and 5 was performed by flow cytometry as well as immunohistochemical analysis.3.4 Drug treatment of the cells in vitroFirstly, to identify the FLS proliferation, the cells were stimulated with a final concentration of 50% NKp44+NK cells culture supernatant,50μg/ml IL-22 antagonist, combination of cells culture supernatant and IL-22 antagonist, different concentrations of recombinant human IL-22 (rhIL-22) (1,10,50, and 100 ng/ml), and negative controls for 24,48, or 72 hours separately.Secondly, to clarify the signaling pathways in IL-22 treated FLS, the cells were treated with 50 ng/ml rhIL-22 for 0,0.25,0.5,1,1.5,2,4, and 8 hours. And the relative expression of total protein and phosphor-protein for STAT3, ERK1/2 and P38 were detected by western blot analysis. To inhibit the STAT3 signaling pathways,100 μM AG490, a tyrphostin, was added 2 hours before stimulation of 50 ng/ml rhIL-22; and the FLS were continually incubated for 0.25,0.5,1,1.5, and 2 hours.At last, the FLS were treated with 100 p. M AG490 combined with 50% NKp44+NK cells culture supernatant or 50 ng/ml rhIL-22 for 24,48, or 72 hours to identify the blocking effect of FLS proliferation.3.5 Cell proliferation assay and western blot analysisCell proliferation was examined by MTT assay. A total of 3×103 FLS/well between passages 4 and 5 was incubated in a 96-well plate for adherent growth at 37℃ in 5% CO2. After the intervention, MTT was then added to a final concentration of 0.5mg/ml, and the FLS were incubated again for 4 hours. At last,150 μg DMSO was added to the empty wells which were shaken for 10 minutes to fully melt the crystals. Optical density at 490 nm was determined by a Model EL309 microplate reader.The signaling pathways were detected by western blot analysis. A total of 1×105 FLS/well between passages 4 and 5 was incubated in a 6-well plate for 24 hours. After the intervention, the cells were washed with ice-cold PBS, homogenized on ice in the Radioimmunoprecipitation assay (RIPA) lysis buffer I with protease inhibitors, and centrifuged at 10,000 rpm for 10 minutes at 4℃. The total protein concentration was determined using the BCA protein assay kit. Equal amounts of total proteins (30μg) were separated on a 12% sodium dodecyl sulfate-polyacrylamide gelelectrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane. After being blocked for 1 hour with Tris buffered saline containing 0.5% Tween 20 and 5% skim milk at room temperature, the membranes were incubated overnight at 4℃ with primary antibodies. Membranes were then incubated with peroxidase AffiniPure goat anti-mouse or rabbit IgG for 1 hour at room temperature. The western blot was visualized by the ECL Plus kit. Human glyceraldehyde phosphate dehydrogenase (GAPDH) proteins expression was measured as an internal control.4 Statistical analysisAll statistics were calculated with SPSS 20.0. Measurements data were presented as mean±standard deviation (mean±SD), while count data were presented as numbers (n) or constituent ratio (%). Shapiro-Wilk test was used for normality and Levene test was used for homogeneity of variance test in small sample (3≤n≤50). A p<0.1 was considered statistically significant. Student’s t test or one-way analysis of variance followed by Bonferroni or Tambane’s T2 test was used to evaluate the significance of the differences. The non-normal distribution measurement data were tested with the Wilcoxon or Kruskal-Wallis rank-sum test. Pearson Chi-square test were used for the comparison of count data and Fisher exact test were used when theoretical frequency was less than 5 or the total observation frequency was less than 30. Spearman Correlation analysis was performed to evaluate the association between variables. Ap<0.05 was considered statistically significant (* p<0.05,** p<0.01,*** p O.001). All p values were two tailed.Results1 NKp44+NK cells expand in the different tissues of patients with RA and were correlated with RA disease activity1.1 NKp44+NK cells expand in the different tissues of patients with RACompared with healthy volunteer controls, the proportion of NKp44+NK cells in the peripheral blood NK cells increased significantly in patients with RA (3.05 ± 2.40% vs 0.53±0.73%; p<.0.001). The substantial increase of these cells was also detected in the synovial fluid of these patients, when compared with in the synovial fluid of KOA controls (6.58±4.31% vs 0.90±.13%; p<0.001) as well as compared with in the peripheral blood of matched patients with RA (6.58±4.31% vs 3.05± 2.40%; p<0.001). Additionally, the expressions of NKp44+NK cells with CD56+ and NKp44+ examined by dual-labeling immunofluorescence were observed in RA synovial tissues, whereas those cells were hardly expressed in KOA synovial tissues.1.2 Expanded NKp44+NK cells were correlated with RA disease activityAccording to the scores of DAS28, the patients with RA were divided into active RA (≥ 2.6) and remission RA (< 2.6). And the active RA patients have higher proportion of NKp44+NK cells in the peripheral blood and synovial fluid, when compared with the remission RA patients (=33.286,p<0.001). The average scores for disease activity score in 28 joints (DAS28) and clinical disease activity index (CDIA) were 4.0±1.4 and 16.4±13.2 in RA peripheral blood group and 4.4±1.5 and 19.8± 14.2 in RA synovial fluid group, respectively. The frequency of NKp44+NK cells in the peripheral blood of patients with RA positively correlated with the levels of DAS28 (r=0.886,p<0.001) or CDIA (r=0.895,p<0.001). Similar in the synovial fluid of patients with RA, NKp44+NK cells were also positively correlated with the levels of DAS28 (r=0.930,p<0.001) or CDIA (r=0.904,p<0.001).2 NKp44+NK cells promote the proliferation of FLS by secreting IL-222.1 The culture and identification of FLS in vitroThe pattern of FLS in vitro present fusiform and grow adherently and spirally. Vimentin-positive FLS cells were almost found in all in vitro culture of FLS between passages 4 and 5, while CD68-positive macrophages were barely detected. The purity of FLS marked with CD55 was nearly 99% according to the flow cytometric analysis.2.2 The flow sorting, culture, and cytokine detection of NKp44+NK cells in vitroThere is no difference between NK cells and NKp44+NK cells on the cell morphology at optical microscope. Both of them are suspended growth in the medium. After treatment with cell stimulation cocktail, the levels of IL-22 in NKp44+NK and NK cell culture supernatants were 5826.5±284.2 and 801.4±158.9 pg/ml (t=26.729, p<0.001). The concentration of M-CSF in NKp44+NK and NK cell culture supernatants were 116.1±3.5 and 19.4±5.1 pg/ml (p=27.180,p<0.001). While the concentration of RANKL in NKp44+NK and NK cell culture supernatants were 309.2 ±12.8 and 391.0±23.1 pg/ml (t=5.357, p=0.006).2.3 NKp44+NK cells culture supernatant promote FLS proliferation by IL-22FLS was cultured with NKp44+NK cell culture supernatants. Compared with untreated cells, treatment with 50% cell culture supernatants after 24,48, or 72 hours increased the proliferation of FLS (p=0.006, p=0.021, p<0.001). Further, the proliferation of FLS decreased significantly when treated with the combination of 50% NKp44+NK cell culture supernatants and 50 μg/ml IL-22 antagonist compared with individual cell culture supernatants after 24,48, or 72 hours (p=0.022, p=0.032, p<0.01). However, no significant differences of the FLS proliferation were found in response to the treatment with or without 50μg/ml IL-22 antagonist (p=0.491, p=1.000,p=1.000).2.4 rhIL-22 promote the proliferation of FLS in a dose-dependent mannerAdditionally, the stimulation of rhIL-22 (0,1,10,50,100 ng/ml) on the proliferation of FLS was examined. The FLS proliferation increased with rhIL-22 concentration in a dose-dependent manner, especially treated after 72 hours (p<0.05).3 The signal pathway of FLS proliferation promoted by IL-22 from NKp44+NK cells3.1 The expression of total protein and phosphor-protein for STAT3, ERK1/2 and P38 in FLS treated by rhIL-22The phosphorylation and total protein of STAT3, ERK1/2 and P38 were examined in rhIL-22 treated FLS. Western blot analysis showed that 50 ng/ml rhIL-22 promoted the tyrosine phosphorylation of STAT3 from 0 to 8 hours (p<0.001), while the total protein expression of STAT3 and GAPDH had no change(p>0.05). Additionally, the phosphorylation and total protein of STAT3, ERK1/2 and P38 also had no change (p>0.05).3.2 AG490 inhibited the STAT3 signal pathway in FLS activated by rhIL-22Further,100 uM AG490 was used to selectively inhibit SATA3 signaling. The protein was collected for western blot analysis after the treatment of FLS for 0.25 h, 0.5 h,1 h,1.5 h and 2 h. A significant decrease in 50 ng/ml rhIL-22 stimulated tyrosine phosphorylation of STAT3 was found after treatment with the combination of 100 μM AG490 from 0.25 to 2 hours when compared with single rhIL-22 stimulation for 2 hours (p<0.001). The total protein expression of STAT3 and GAPDH had no change after the stimulation (p>0.05).3.3 AG490 inhibited the FLS proliferation promoted by rhIL-22When treated the FLS with both of 50 ng/ml rhIL-22 and/or 100 μM AG490, MTT assay revealed that a significant decrease in 50 ng/ml rhIL-22 inducing the FLS proliferation was found after 24,48, or 72 hours’ intervention with 100 μM AG490 when compared with intervention without inhibitor (p<0.01). In addition, AG490 could also inhibit the FLS proliferation significantly after 24,48, or 72 hours when compared with untreated cells (p<0.01).3.4 AG490 inhibited the FLS proliferation promoted by NKp44+NK cells culture supernatantSimilarly, MTT assay revealed significantly less proliferation in 100 μM AG490-treated than no inhibitor-treated cells when 50% NKp44+NK cell culture supernatants were used together for 24,48, or 72 hours’ intervention (p<0.001). Compared with untreated cells,100 μM AG490 decreased 50% NKp44+NK cell culture supernatants induced the FLS proliferation after 24,48, or 72 hours’ intervention (p<0.001).ConclusionFirstly, the present study clarifies the expansion of NKp44+NK cells in the PB and SF of patients with RA, especially in the synovial tissues of RA for the first time. The expanded NKp44+NK cells were correlated with RA disease activity.Secondly, NKp44+NK cells cultured in vitro could secret IL-22, M-CSF and RANKL. The high concentration of IL-22 expressed by NKp44+NK cells could promote the proliferation of FLS.At last, STAT3 is an essential signaling pathway in mediating the effects of IL-22 secreted by NKp44+NK cells on the proliferation of FLS in patients with RA. |
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