| BackgroundSystemic lupus erythematosus(SLE) is an autoimmune disease, characterized by multiple systems and organs damage, complicated clinical manifestations, and has been recognized to be an important public issue. Although the pathogenesis of SLE has not been fully elucidated, available evidence suggests that genetic and environmental factors are associated with the pathogenesis of SLE, resulting in abnormal immunity, especially aberrant expression and(or) function of immune cells. For instance, imbalance of tolerance to self-antigens, dysregulated autoreactive T, B lymphocytes activation, uncontrolled roles of cytokines played in disease, and overexpression of autoantibodies against ds DNA, nucleosome, nucleoprotein. All these abnormality can lead to accumulative autoantibodies, and therefore result in complement activation, immune-complex deposition, and finally occurred tissues and(or) organs damage.Dendritic cells(DCs) are widely expressed in human body that functions in immunosurveillance, antigen-presentation and tolerance. DCs are specialized sentinel cells that bridge the innate and adaptive immune systems. They uniquely express a plethora of innate pattern-recognition receptors(PRRs), including Toll-like receptors(TLRs), C-type lectin receptors(CLRs), RIG-I-like receptors and NOD-like receptors. The general categories of DCs are the conventional dendritic cells(c DC) and the plasmacytoid dendritic cells(p DC). The PRRs can capture antigens(Ags) through binding to the pathogen-associated molecular pattern molecules(PAMPs) of microbes or damage-associated molecular pattern molecules(DAMPs) of endogenous tissues. Upon capturing activating Ags, DCs undergo a cascade of coordinated maturationevents involving(i) up-regulating surface expression of MHC class II molecules and co-stimulatory molecules,(ii) priming and modulating T, B lymphocytes function,(iii) generating some cytokines that polarize and modulate the function of other effector cells. DCs could influence SLE in several ways: presentation of self-antigen to autoreactive T cells, secretion of proinflammatory cytokines. SLE shows a strong association with MHC haplotypes, which suggests that T cell responses are essential for the development of this disease. Indeed, even an SLE-like disease that is driven by TLR7 overexpression in mice, and that presumably involves an innate initiating stimulus, is completely MHC haplotype dependent. This circumstantial evidence suggests that DCs are possibly involved in the presentation of chromatin and RNA-associated proteins to self-reactive T cells. Activated c DCs secrete IL-12p70 which elicits the production of IFNγ by T cells and promotes the differentiation of na?ve T helper(Th) cells into Th1 effectors. Furthermore, type I IFNs produced by p DCs lead to c DC maturation and lower the activation threshold for TLR agonists. Therefore, DCs could boost disease by secreting inflammatory cytokines.Reactive oxygen species(ROS) represent a large number of molecular entities that are either free radicals containing unpaired electrons, such as superoxide, NO, and hydroxyl radical, or nonradical oxidizing agents, such as H2O2 and the NO derivative peroxynitrite. NADPH oxidase within cells in response to stimuli can generate ROS, and cellular elements such as mitochondria and peroxisomes generate ROS from biological reactions during oxygen metabolism. Evidence suggests that ROS have emerged as crucial regulators in cellular motility, differentiation, and apoptosis through a direct influence on signal transducing proteins. ROS induce DC maturation and affect the production of proinflammatory cytokines. DCs have all the components of a functional NADPH oxidase within their plasma membranes. After interacting with cognate T cells or microbial components, DCs generate ROS. Therefore, ROS could serve as endocrine regulators of DC function.The extracellular signal-regulated kinase(ERK) signaling pathway is one of the highly conserved signaling pathways in mitogen-activated protein kinase(MAPK). It can regulate a variety of functions of immune cells. Although there is abnormal expression of(phosphorylation) ERK, ROS in SLE, and correlates with disease activity, current studies have not reported the expression of p ERK, ROS in monocytes-induced dendritic cells(mo DCs), the generation of inflammatory cytokines from mo DCs, and whether the expression of p ERK is related to ROS and the generation of inflammatory cytokines is realted to ROS. Furthermore, the present study will discuss the antigen presentation ability of mo DCs based on Th17 cell differentiation, and discuss whether the expression of Th17 cell correlates with ROS. Consequently, these findings will add the knowledge of the expression and(or) function of ROS in mo DCs.Part 1 Expression of ROS in monocytes-induced dendritic cells from systemic lupus erythematosus patientsObjective We isolate the monocytes from peripheral blood mononuclear cells(PBMCs) and induce dendritic cells(DCs). After mo DCs induction, we compare the expression of ROS in mo DCs from SLE patients(newly diagnosed patients, patients in steady state) with healthy controls so as to discuss the correlation between ROS and SLE.Methods All subjects gave written informed consent prior to study enrollment, and then, we collected 15 ml EDTA-anticoagulated blood, finished the questionaire. PBMCs were isolated by Ficoll-Hypaque centrifugation. After the isolation of PBMCs, monocytes were magnetically selected using anti-human CD14 micro Beads. Freshly isolated monocytes were cultured in complete medium containing RPMI-1640 supplemented with 10% heat-inactivated FBS, 2 m M L-glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin, 50 u M β-mercaptoethanol, and inducers 50 ng/ml rh GM-CSF and 50 ng/ml rh IL-4 for 7 days. After mo DCs induction, we examined the expression of ROS in mo DCs. In this study, we recruited 20 SLE patients, including 11 newly diagnosed SLE patients and 9 patients in steady state, and 20 healthy controls. mo DCs are either stimulated with LPS or not stimulated with LPS. Similarly, freshly isolated monocytes were cultured in complete medium containing RPMI-1640 supplemented with 25% newly diagnosed SLE patient’s serum, 2 m M L-glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin, 50 u M β-mercaptoethanol for 7 days. In this part, 10 SLE patients, including 5 newly diagnosed SLE patients and 5 patients in steady state, and 10 healthy controls were recruited. mo DCs are also either stimulated with LPS or not stimulated with LPS. Of the newly diagnosed patients, they had not used anti-rheumatic drugs and SLE Disease Activity Index(SLEDAI) was more than 10, while the patients in steady state had used anti-rheumatic drugs for more than 1 month and the SLEDAI was less than 10. The statistical significance of the differences between groups was determined by Student’s t-test, when the data were normally distributed and the equal variance testwas satisfied. Or, Mann-Whitney rank sum test was used, when the data were not normally distributed or ranked. All statistics were performed with SPSS 10.01. P value less than 0.05 was considered statistically significant.Results(1) With respect to the first method that mo DCs were cultured in complete medium and inducers, there are 20 female SLE patients, and 19 female, 1 male healthy controls. Age of SLE patients is 32.0±7.9 years, ranges from 20 to 44 year. Age of healthy controls is 34.1 ±12.8 years, ranges from 22 to 59 year. Expression of ROS in mo DCs between healthy controls and patients in steady state, newly diagnosed patients showed the P value of 0.402, 0.358 before mo DCs stimulated with LPS, respectively, and the P value was 0.566, 0.670 after mo DCs stimulated with LPS, respectively. In spite of the disease activity, we compared the expression of ROS in mo DCs between all healthy controls and all SLE patients, the P value was 0.194 before mo DCs stimulated with LPS, and the P value was 0.646 after mo DCs stimulated with LPS. With respect to the effect of LPS stimulation on SLE patients or healthy controls themselves, expression of ROS in mo DCs without LPS stimulation from healthy controls was compared with patients in steady state without LPS stimulation. Similarly, expression of ROS in mo DCs with LPS stimulation from healthy controls was compared with patients in steady state with LPS stimulation. Then, these two ratios were compared, and the P value was 0.441. We also compared these ratios in healthy controls and newly diagnosed patients, the P value was 0.075. In spite of the disease activity, we compared the expression of ROS in mo DCs from all healthy controls and all SLE patients before and after LPS stimulation. Expression of ROS in mo DCs without LPS stimulation from all healthy controls was compared with all patients without LPS stimulation. Similarly, expression of ROS in mo DCs with LPS stimulation from all healthy controls was compared with all patients with LPS stimulation. Then, these two ratios were compared, and the P value was 0.062.(2) With respect to the second method that mo DCs were cultured in complete mediumwith SLE patient’s serum, both SLE patients and healthy controls showed 9 female, 1 male. Age of SLE patients is 33.3±9.1 years, ranges from 20 to 48 year. Age of healthy controls is 32.7 ±8.9 years, ranges from 23 to 48 year. Expression of ROS in mo DCs between healthy controls and patients in steady state, newly diagnosed patients showed the P value of 0.064, 0.530 before mo DCs stimulated with LPS, respectively, and the P value was 0.834, 0.292 after mo DCs stimulated with LPS, respectively. In spite of the disease activity, we compared the expression of ROS in mo DCs between all healthy controls and all SLE patients, the P value was 0.791 before mo DCs stimulated with LPS, and the P value was 0.496 after mo DCs stimulated with LPS. With respect to the effect of LPS stimulation on SLE patients or healthy controls themselves, expression of ROS in mo DCs without LPS stimulation from healthy controls was compared with patients in steady state without LPS stimulation. Similarly, expression of ROS in mo DCs with LPS stimulation from healthy controls was compared with patients in steady state with LPS stimulation. Then, these two ratios were compared, and the P value was 0.447. We also compared these ratios in healthy controls and newly diagnosed patients, the P value was 0.293. In spite of the disease activity, we compared the expression of ROS in mo DCs from all healthy controls and all SLE patients before and after LPS stimulation. Expression of ROS in mo DCs without LPS stimulation from all healthy controls was compared with all patients without LPS stimulation. Similarly, expression of ROS in mo DCs with LPS stimulation from all healthy controls was compared with all patients with LPS stimulation. Then, these two ratios were compared, and the P value was 0.821. Conclusion The expression of ROS was not significantly aberrant in mo DCs between SLE patients and healthy controls, and may not correlate with SLE.Part 2 Association study of ROS with the generation of inflammatory cytokines from mo DCsObjective To compare the expression of p ERK and Th1,Th2,Th17 realted inflammatory cytokines in mo DCs from SLE patients(newly diagnosed patients) with healthy controls so as to discuss the correlation between ROS and p ERK, inflammatory cytokines.Methods We collected the supernatant from mo DCs cultured in complete medium and inducers(11 patients and controls), and mo DCs cultured in complete medium with SLE patient’s serum(5 patients and controls). Supernatant from healthy controls and newly diagnosed patients was preserved in-80℃, and then used to examine the expression of inflammatory cytokines IL-2, IL-6, IL-10, IL-17 by ELISA kits according to the manufacture’s instructions. mo DCs were either stimulated with LPS or not stimulated with LPS. Furthermore, we collected the mo DCs cultured in complete medium and inducers from 11 newly diagnosed patients and 11 healthy controls. mo DCs were not stimulated with LPS and lysed, and western blot analysis examined the expression of p ERK. We also discuss the correlation between ROS and inflammatory cytokines, p ERK expression. In this part, the mo DCs used were from the first part. All statistics were performed with SPSS 10.01. P value less than 0.05 was considered statistically significant.Results(1) Expression of Th1,Th2,Th17 related inflammatory cytokines IL-2, IL-6, IL-10 and IL-17 from mo DCs was comparable between healthy controls and newly diagnosed patients either stimulated with LPS or not stimulated with LPS(P>0.05).(2) Expression of p ERK in SLE patients was significantly lower in comparison with healthy controls without stimulation with LPS(P=0.031).(3) There was no significant correlation between ROS expression, inflammatory cytokines and p ERK expression(P>0.05), and the correlation between p ERK expression and inflammatory cytokines expression was not significant(P>0.05).Conclusion There were no strong differences in inflammatory cytokines expression between healthy controls and newly diagnosed patients, but the p ERK expression was markedly lower in SLE patients compared with the controls. However, the generation of inflammatory cytokines(IL-2, IL-6, IL-10, IL-17) may not correalte with ROS.Part 3 Association study of ROS with the antigen presentation ability of mo DCs, take the differentiation of Th17 for instanceObjective To coculture the mo DCs from healthy controls and CD4+ T cells from healthy controls, or mo DCs from newly diagnosed patients and CD4+ T cells from healthy controls, respectively, so as to discuss the antigen presentation ability of mo DCs, take the differentiation of Th17 for instance. Furthermore, to discuss the correlation between ROS expression and differentiation of Th17, in order to infer whether the change of antigen presentation ability of mo DCs is correlated with ROS.Methods Monocytes from newly diagnosed patients(5 samples) and healthy controls(5 samples) were cultured in complete medium with SLE patient’s serum. Cells were cultured for 4 days and collected. After mo DCs induction, we made different concentrations of mo DCs and cocultured with T cells from healthy controls(one person) for 6 days. Then, supernatant was examined for inflammatory cytokines TGF-β, IL-6 between patients and controls by ELISA kits according to the manufacture’s instructions. Similarly, the differentiation of Th17 cell was examined by flow cytometry. We also discuss the relationship between expression of ROS and expression of the inflammatory cytokines, Th17 cell differentiation. In this part, the mo DCs used were from the first part. All statistics were performed with SPSS 10.01. P value less than 0.05 was considered statistically significant.Results(1) Different concentrations of mo DCs either from newly diagnosed patients or healthy controls cocultured with T cells from healthy controls, there were no significant differences of TGF-β, IL-6 expression between patients and controls(P>0.05).(2) Similarly, the expression of Th17 cell was comparable between patients and controls, though there were distinct concentrations of mo DCs(P>0.05).(3) We found no strong correlation between ROS expression and inflammatory cytokines, Th17 cell differentiation(P>0.05).Conclusion Take the differentiation of Th17 for instance, the antigen presentation ability of mo DCs between newly diagnosed patients and healthy controls was not significantly different, and the change of antigen presentation ability of mo DCs(to induce Th17 differentiation) may not correlate with ROS. |
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