| Rheumatoid arthritis (RA) is a common autoimmune disease in which a chronic inflammatory response develops, particularly in synovial joints. The joints are characteristic of inflammatory cells infiltration, synovium proliferation and pannus formation, resulting to the injury of cartilage and bones. The incidence in China is 0.26～0.5%。It can occur at any age and the incidence increases with the age. The female is more liable to RA and the peak age is between 45 to 50 years old. Nearly all the RA patients have the variable loss of joint functions which bring about severe social problems and economic burden.The pathogenesis of RA is complex. It has been generally accepted cell immunity plays an important role in the pathogenesis of RA. Invasive antigens are phagocytized and processed by synovial cells and T cells are activated, releasing many soluble factors such as interlukin-1(IL-1) and tumor necrosis factor (TNF) and promoting synovium proliferation and inflammation and final destruction of joint. Based on the important role of adaptive immunity in the pathogenesis of RA, there are many biologies tageted at adaptive immune system including immune cells, signal molecules and pivotal inflammatory cytokines. Among these biologies, TNF-αantagonist is used widely. But many randomized controlled trials have shown that 40-50% RA patients can not acquire ACR 50 improvements after treatment by one biologies. This suggests theories above-mentioned can not explain the pathogenesis of RA completely. Recent studies have demonstrated innate immunity also plays a crucial role in the pathogenesis of RA, but the mechanisms of innate immunity are poorly understood.In recent years, innate immune cells have been found to participate in immune regulation including nature killer cells (NK cells), dentritic cells and so on. Among them, with the advances in NK cell phetotypes and functions, the role of NK cells in immune regulation has raised much interest. NK cells are an important component of the innate immune system which account for 10-15% of peripheral blood lymphocytes (PBLs). NK cells have a number of effector functions, including recognition and lysis of virus-infected or tumor cells and production of immunoregulatory cytokines. Their activity is regulated by integration of both activatory and inhibitory signals from a wide range of cell surface receptors. Better understanding of the biology of NK receptors has led to a greater knowledge of their physiological role, and consequently their role in disease can now be investigated. The receptors involved in the regulation of NK cell activity fall into three categories: (1) Natural cytotoxicity receptors (NCR), including NKp30, NKp44, and NKp46, bind mostly undefined ligands on potential target cells. The resulting activating signal is essential for NK cell-mediated cytotoxicity. (2) C-type lectin-like receptors such as NKR-P1, NKG2D and CD94/NKG2A. (3) immunoglobulin (Ig) superfamily member receptors including CD158a,CD 158b. These receptor families contain both activating and inhibitory forms, and recognize HLA class I molecules and structurally related proteins as their ligands. In general terms, NK-cell activity is regulated by integrated signals provided by an array of activating and inhibitory cell surface receptors. More recent reports have also shown NK cells have the ability of releasing cytokines and can influence the initiation and development of the subsequent adaptive immune response. NK cells can secret many cytokines following activation which can bring about the proliferation, activation and cytokine production of T and B cells. More and more evidence has been obtained that NK cells play positive or negative immune regulatory effect by secreting various cytokines or cell-to-cell contact and maintain immune homeostasis. The malfunction or loss of NK cells is involved in the pathogenesis of many diseases (e.g., tumor, autoimmune diseases). It is promising to study the role of NK cells in the pathogenesis of rheumatoid arthritis.In RA, several studies have investigated the potential involvement of NK cells. Since the early 1980s, many studies have documented decreased NK cell numbers and impairment of NK cell function in the peripheral blood of patients with rheumatoid arthritis. Many studies have shown that NK cells play a role in the pathogenesis of RA through the following mechanisims:1,NK cells are capable of secreting many cytokines which are important for the development of RA. The CD56bright NK subset that is predominant in the RA synovium is capable of secreting large amounts of proinflammatory cytokines including TNF-alpha, IFN-γ, IL-10, TGF-βand so on. CD56bright NK cells can interact in a reciprocal fashion with T cells and dendritic cells thereby promoting the development of adaptive immune response. NK cells are also a major source of IFN-gamma which may contribute to inflammation in RA by promoting Bcell activation and classs witching and promoting dendritic cell maturation.2,NK cells may also contribute to the pathogenesis of RA through direct cell-cell interaction with other lymphocytes present in the inflamed synovium. NK cells can provide co-stimulation for T and B lymphocytes via expression of co-stimulatory molecules such as CD40L, OX40, CD70 and CD86. Activated NK cells can kill immature DC via NKp30, NKp46 and DNAM-1mediated recognition. A recent study has suggested that NK cells isolated from synovial fluid of RA patients are capable of inducing osteoclast differentiation from CD14+ monocytes when co-cultured. This process was dependent on RANKL and MCSF, both expressed by synovial fluid NKcells, suggesting NKcells may play a role in the progressive bone destruction observed in RA.Rather than consisting of a homogeneous population, NK-cell subsets with distinct functional characteristics have been defined in both mice and humans. Two subsets of NK cells in peripheral blood have been recognized. The majority (CD56dim NK cells) express moderate levels of CD56 and high levels of CD16. The minor subset of NK cells (CD56bright NK cells) accounts for only 10% of circulating NK cells. The two subsets of NK cells show important functional differences; the CD56dim subset has superior cytotoxic capacity, whereas the CD56bright subset has greater ability to produce proinflammatory cytokines including TNF-α. NKp44 receptor is a member of natural cytotoxicity receptors (NCR) which is an activation marker of NK cells. So many scholars have been interested in the role of NKp44+NK cells in the pathogenesis of many diseases.NKp44+NK cell is a subset of human NK cells and it phenotype is CD56+NKp44+NK cell. The structure of NKp44 ligand is not clear. It has been shown that NKp44 receptor can bind to viral hemagglutinins resulting in the activation of NK cells. Previous studies on NKp44+NK cells in RA patients have documented that NKp44+NK cells are higher in RA synovial fluid than those in RA peripheral blood and in healthy controls. But the role of NKp44+NK cells in the pathogenesis of RA is not clear. Recently, professor Marina Cella and his colleagues in Washington Medical University has found NKp44+NK cells in patients with inflammatory bowel disease are different from traditional NK cells showing decreased cytotoxicity due to lower intracellular IFN and granzymes and increased cytokines production. Activated NKp44+NK cells can induce aggregation of themselves and other immune cells and adhesion of lymphocytes. They also found a new human NK cell subset-NK-22 cell in patients with inflammatory bowel disease. NK-22 cell is a subset of NKp44+NK cell and its phenotype is NKp44+CCR6+NK cell. NK-22 cells can secret many cytokines including IL-22, IL-26 and leukemia inhibitory factor. They can stimulate epithelial cells to secrete IL-10, proliferate and express a variety of mitogenic and anti-apoptotic molecules. The study has suggested that NK-22 cells provide an innate source of IL-22 that may help constrain inflammation and protect mucosal sites. Like inflammatory bowel disease, RA is also an autoimmune disease. There is similar immunopathogenesis in the devolpment of RA and inflammatory bowel disease. It is unclear whether NK-22 cells exist in RA patients and may play a role in the pathogenesis of RA. IL-22 is the most important cytokine produced by NK-22 cells. It is a member of IL-10 family and can be induced by special immune cells including NK-22 cells, NKT cells, T cells and so on. IL-22 can induce the production of acute-phase proteins, MMPs and chemokines. It can also enhance the role of inflammatory cytokines such as IL-1, TNF-a, IL-17. IL-22 also produces an effect in the pathogenesis of RA. It has been proved that high levels of IL-22 were expressed in sublining and lining of RA synovial tissues. IL-22 can induce the proliferation and chemokine production of synovial fibroblasts. In animal models, consistent high level of IL-22 was detected in C57BL/6 CIA mice. IL-22-/-mice were less susceptible to CIA than were wild-type mice, as evidenced by their decreased incidence of arthritis and decreased pannus formation and low expression of IL-1β, IL-6, TNF-a and MMP9. IL-22 has been found to promote clastogenesis suggesting it plays an important role in the inflammation and bone destruction of RA.Our study has shown that there was NKp44+NK cell subset in PB and SF of RA patients whereas NKp44+NK cell subset was nearly absent in PB of healthy controls. NK-22 cells play an important role in the pathogenesis of patients with inflammatory bowl disease. Like inflammatory bowl disease, RA is also an autoimmune disease. It is unknown whether NK-22 cells may play an important role in the pathogenesis of RA through the production of IL-22 or other cytokines. This study was focused on the role of NKp44+NK cell subset in the pathogenesis of RA and included three parts:1. Clinical significance of NKp44+NK cells was discussed by detecting the expressions of NKp44+NK cells in PB and SF of RA patients and analyzing the correlation between NKp44+NK cells and disease activity.2. The role of NK-22 cells in the pathogenesis of RA was discussed by studying the function of NK-22 cells and the significance of NK-22 cells for the judgement of disease activity and severity.3. Further discuss the role of NK-22 cells in the pathogenesis of RA and its significance for the judgement of disease activity and severity by studying the change in NK-22 cells after treatment with Sinomenine preparation combined with MTX and analyzing the correlation between NK-22 cells and DAS28 after treatment. In a word, it is of importance to interpret the function of NKp44+NK cell subset in RA and clinical significance for further understanding of RA pathogenesis and future treatment.In Traditional Chinese medieine (TCM), the condition that is congruent with arthritis is called "Bi syndrome." Bi syndrome manifests as pain, soreness, or numbness of muscles, tendons and joints which is the result of the body being "invaded" by the external environmental factors such as Wind, Cold, Heat, and/or Dampness. The individual symptoms depend on which external pathogenic factor is strongest. Long-term Bi syndrome can lead to the formation of Phlegn and the deformation of bones and joints. In TCM pain and reduced range of motion indicates the presence of blockage. Conditions such as RA, therefore are always treated by means of removing Wind-Dampness and smoothing meridian. Sinomenine is an alkaloid extracted from the Chinese medicinal plant, Sinomenine acutum, which has been utilized to treat RA in China for over 2000 years. Sinomenine has been shown to mediate a wide range of pharmacological actions which include anti-inflammatory and anti-rheumatic effects. Many sinomenien preparations have been used in clinic and have acquired good effects. Qingfengtong capsules (Sinomenine preparation) has an effect of removing wind and dispersing dampness, clearing meridians and collaterals, and expelling pathogenic cold from channel. Previous Pharmacological and clinical study on acute and chronic inflammation has shown a good anti-inflammatory and analgesic effect of Qingfengtong capsules.Methotrexate is one of disease-modifying antirheumatic drugs which competitively inhibites dihydrofolate reductase (DHFR), an enzyme that participates in the tetrahydrofolate synthesis. Dihydrofolate reductase catalyses the conversion of dihydrofolate to the active tetrahydrofolate. Folic acid is needed for the de novo synthesis of the nucleoside thymidine, required for DNA synthesis. Also, folate is needed for purine base synthesis, so all purine synthesis will be inhibited. Methotrexate, therefore, inhibits the proliferation and replication of cells. MTX is cytotoxic during the S-phase of the cell cycle and can inhibit humoral immunity and cellular immunity. It is the first choice for the treatment of rheumatoid arthritis.Methotrexate and Sinomenine preparation (Qingfengtong capsules) are both effective for RA. It is still unknown whether MTX plus Sinomenine preparation can make an effect through the inhibition of NK-22 cells.Quantitative detection of the expression of genes at the same time can not be achieved by traditional methods. QGP(QuantiGene Plex) technique combines b-DNA (Branched DNA) signal amplification and the Luminex xMAP liquid-chip platform. The advantage is available directly from whole blood samples or cultured cells to detect mRNA expression, no RNA purification, no reverse transcription, no PCR amplification, and it can also high-throughput quantitatively detect 3 to 36 genes from less samples. QGP technique is an advanced method for quantitative detection of multiple gene expression.ObjectivesThe general objectives of the study are to discuss the role of NKp44+NK cell subset in the pathogenesis of RA and clinical significance for the judgement of disease activity, severity and therapeutic effect.1. To study the expressions of NKp44+NK cells in RA patients and discuss the relationship between NKp44+NK cells and disease activity.2. To study the function of NK-22 cells and investigate the role of NK-22 cells in the pathogenesis of RA and significance for the judgement of disease activity and severity.3. To study the effect of Sinomenine preparation combined with MTX on NK-22 cells and discuss the significance of NK-22 cells for the judgement of disease activity and therapeutic effect.Methods1. Research Subjects35 RA patients were obtained from the department of rheumatology in Nan-fang hospital from October 2009 to March 2010, including 7 males and 28 females. The avrage age was (46.8±10.3) years. There were 20 active RA cases consisted of 4 males and 16 females and 15 remissive RA cases consisted of 3 males and 12 females. The average ages of active patients and remissive patients were (48.1±10.8) years and (46.6±8.7) years respectively.20 healthy controls include 3 males and 17 females and the average age was (47±11.6) years. None of cases with other autoimmune diseases or severe heart, kidney and liver diseases.2. Research Methods2.1 The expression and clinical significance of NKp44+NK cells in RA patientsUsing flow cytometry, the proportions of NKp44+NK cells in PB and SF of RA patients and intracellar content of perforin, granzyme B and IFN-γin NKp44+NK cells were detected. The relationship between NKp44+NK cells and disease activity including DAS28, anti-CCP antibody and RF was analyzed.2.2 The Study of the function of NK-22 cells in RA patients2.2.1 Detection of NK-22 cells in PB, SF and synovium of RA patientsUsing flow cytometry, the proportions of NK-22 cells in PB and SF of RA patients were detected. NK-22 cells in RA synovium were detected by immunoflueresence staining.2.2.2 Detection of cytotoxicity of NK-22 cellsNK-22 cells were sorted using flow cytometry and cultured in vitro. The purity of NK-22 cells and intracellar content of perforin, granzyme B and IFN-γwere detected using flow cytometry.2.2.3 The levels of IL-22 and TNF-a and gene expressions in NK-22 cells and correlation between NK-22 proportions and the level of IL-22 and TNF-a in PB and SF of RA patientsThe levels of IL-22 and TNF-a in the supernatant of NK-22 culture solution were measured by ELISA and gene expressions of IL-22 and TNF-a of NK-22 cells were measured by QGP assay. The levels of IL-22 and TNF-a in PB and SF of active patients were also detected by ELISA. Correlations between NK-22 cells and the levels of IL-22 and TNF-a in PB and SF were analyzed.2.2.4 The effect of NK-22 culture supernatant on the proliferation of fibroblast-like synoviocytesThe effect of NK-22 culture supernatant on the proliferation of fibroblast-like synoviocytes was detected by MTT. Meanwhile the effect of IL-22 antibody and TNF-a antibody on the proliferation of fibroblast-like synoviocytes induced by NK-22 supernatants were observed.2.2.5 The effect of rhIL-22 on the production of MCP-1 from RA fibroblast-like synoviocytesThe effect of rhIL-22 on the production of MCP-1 from RA fibroblast-like synoviocytes was determined by ELISA.2.2.6 Correlation between NK-22 cells and disease activity of RACorrelation between NK-22 cells and disease activity including DAS28, anti-CCP antibody and RF was analyzed.2.3 The effect of Sinomenine preparation (Qingfengtong capsules) combined with MTX on NK-22 cells in RA PB14 RA patients were treated with Sinomenine preparation (Qingfengtong capsules) plus MTX for 12 weeks. The proportions of NK-22 cells in RA patients after treatment were detected using flow cytometry. Correlation between NK-22 cells and DAS28 after treatment was analyzed.3 StatisticsAll data were analyzed using statistical software SPSS 13.0. Measurement data were expressed as means±SD or median±INQ. Comparison between measurement data of normal distribution was done by one-Way ANOVA followed by bonferroni test. Comparison between measurement data of abnormal distribution was done by multiple nonparametric tests or two sample nonparametric tests. Spearman correlation was used for correlation analysis. A two-sided P-value less than 0.05 was considered statistically significant.Results1 The expression and clinical significance of NKp44+NK cells in RA patientsThe proportions of NKp44+NK cells in PB of active and remissive patients and healthy individuals are 1.480%,0.540%,0.000% respectively. The proportion of NKp44+NK cells in PB of active patients are significantly higer than remissive patients and healthy individuals (χ2=46.708,P=0.000). The proportion of NKp44+NK cells in SF of ten active RA patients is significantly higher than matched PB (17.624% vs 3.473%, z=-3.780, P=0.000). NKp44+NK cells produce a small amount of perforin (0.08%), granzyme B (0.34%) and IFN-y (0.12%). There was positive correlation between NKp44+NK cells proportion in PB of active patients and DAS 28 and anti-CCP antibody level (r=0.777, P=0.000; r=0.967, P=0.000). There was no correlation between NKp44+NK cells proportion in PB and RF (r=-0.343, P=0.138). There was positive correlation between NKp44+NK cells proportion in SF of active patients and DAS 28 and anti-CCP antibody level (r=0.930, P=0.000; r=0.867, P=0.001).There was no correlation between NKp44+NK cells proportion in SF and RF (r=0.564, P=0.09).2 The study of function of NK-22 cells2.1 Detection of NK-22 cells in PB, SF and synovium of RA patientsThe proportions of NK-22 cells in PB of RA patients and healthy individuals were 0.690% and 0.000% respectively. There was significant difference between two groups (z=-6.225, P=0.000). The proportion of NK-22 cells in SF of ten active RA patients was significantly higher than matched PB (4.165% vs 1.195%, z=-3.781, P=0.000). NK-22 cells were also detected in RA synovium and they were double positive cells (NKp44+CCR6+).2.2 Cytotoxicity of NK-22 cells2×104NK-22 cells were sorted using flow cytometry. After cultured in vitro for2 weeks, the purity was more than 90%. NK-22 cells produced low intracellular content of perforin (0.07%), granzyme B (0.26%) and IFN-y (0.11%).2.3 The levels of IL-22 and TNF-a and gene expressions in NK-22 cells and correlation between NK-22 proportions and the level of IL-22 and TNF-a in PB and SF of RA patientsThe levels of IL-22 and TNF-a in the the supernatant of NK-22 culture solution were (1293.971±345.048) pg/ml and (272.238±83.471) pg/ml respectively. Gene expressions of IL-22 and TNF-a of NK-22 cells are significantly higher than normal NK cells (P<0.01). The levels of TNF-αand IL-22 in PB of 20 active RA patients were (313.059±90.513) pg/ml and (5.34±1.614) ng/ml respectively, while the levels of TNF-αand IL-22 in SF of 10 RA patients were (681.245±65.996) pg/ml and (7.22±1.249) ng/ml. There was positive correlation between NK-22 cells in PB and the levels of IL-22 and TNF-a in PB (r=0.869, P=0.000; r=0.957, P=0.000). There was also positive correlation between NK-22 cells in SF and the levels of IL-22 and TNF-αin SF (r=0.930, P=0.000; r=0.967, P=0.000).2.4 The effect of NK-22 culture supernatant on the proliferation of fibroblast-like synoviocytesThe rapid proliferation of fibroblast-like synoviocytes was observed at 24h after the addition of culture supernatant of NK-22 cells. Moreover, the proliferation was time and dose dependent.At 48 hours, RA fibroblast-like synoviocytes proliferated obviously in culture supernatant group and 1:2 dilution group compared with blank group (P<0.05). At 72 hours and 96 hours, RA fibroblast-like synoviocytes proliferated obviously in three groups of different concentrations of culture supernatant compared with blank group (P<0.05). IL-22 antibody (50ug/ml) united with TNF-αantibody (1.2ug/ml) could completely inhibite the proliferation of RA fibroblast-like synoviocytes induced by NK-22 culture supernatant.2.5 The effect of rhIL-22 on the production of MCP-1 from RA fibroblast-like synoviocytesThe levels of MCP-1 after 72 hours' culture in blank group, lng/ml rhIL-22 group and 10ng/ml rhIL-22 group were 52.17pg/ml,81.18 pg/ml,129.208 pg/ml respectively. The levels of MCP-1 in lng/ml rhIL-22 group and lOng/ml rhIL-22 group were higher than those in blank group (P<0.05). The level of MCP-1 in 10ng/ml rhIL-22 group was higher than that in 1ng/ml rhIL-22 group (P<0.05).2.6 Correlation between NK-22 cells and the disease activityNK-22 proportions in active RA patients, remissive patients and healthy controls were 0.805%,0.510%,0.000% respectively. The proportions of NK-22 cells in active patients were higher than those in remissive patients and healthy controls (x2=40.193, P=0.000). There was positive correlation between NK-22 proportion in PB and DAS 28 (r=0.975, P=0.000) and the level of anti-CCP antibody (r=0.717, P=0.000), while NK-22 proportion in SF was positively related to DAS 28 (r=0.918, P=0.000), anti-CCP antibody (r=0.851, P=0.002) and RF (r=0.784, P=0.007).3 The effect of Sinomenine preparation (Qingfengtong capsules) combined with MTX on NK-22 cells in RA PBNK-22 cells proportions in RA PB were significantly decreased after Sinomenine preparation (Qingfengtong capsules) plus MTX treatment vs before treatment (P<0.05). The proportions of NK-22 cells after treatment in RA PB were highly correlated with DAS28 after treatment (r=0.836, P=0.000).Conclusions1. NKp44+NK cells with decreased cytotoxicity increase in PB and SF of RA patients.2. NKp44+NK cells are highly correlated with disease activity and severity.3. NK-22 cells increase in PB and SF of RA patients and the cytotoxicity of NK-22 cells has decreased. NK-22 cells also exist in RA synovium.4. IL-22 and TNF-a are main cytokines produced by NK-22 cells and NK-22 cells may play an important role in the pathogenesis of RA through the production of IL-22 and TNF-a. The pathogenic actions include (1) NK-22 cell supernatant can promote the proliferation of RA FLS. Moreover, the proliferation was time and dose dependent. (2) rhIL-22 can induce the production of MCP-1 from RA FLS.5. There is close correlation between NK-22 cells and disease activity and severity.6. Sinomenine preparation combined with MTX can decrease the number of NK-22 cells in RA patients suggesting the significance of NK-22 cells for the judgement of therapeutic effect.
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