The Activating Effects And Potential Mechanisms Of Immune Complexes On Human Endothelial Cells And Monocytes

Part ⅠThe activating effects and potential mechanisms of immune complexes on human endothelial cellsBackgrounds:Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by formation of immune complexes (ICs), which contain autoantigens nucleic acids, nucleic acids-associated protein and corresponding antibodies. The apoptotic cells which are impared cleared by monocytes/macrophages result in secondary necrosis, autoantigens nucleic acids and nucleic acids-associated protein are released by the nuclei of these secondary apoptotic cells in patients with SLE. The corresponding autoantibodies against these nuclear autoantigens, such as the anti-double-stranded DNA (dsDNA) antibodies are generated and lead to the formation of ICs. Deposition of ICs on target organs, such as skin and kidney, results in end organs damage.Vasculopathy and vasculitis, which are typical complications of SLE, are reported in10%to40%of SLE patients and are usually seen in cutaneous vessels, renal glomeruli, coronary and brain vessels. Vasculopathy and vasculitis are reported to be associated with the deposition of ICs on endothelium, endothelial cells activation and inflammatory cells infiltration into the inflammatory sites. However, the effects of ICs on the endothelial cells and the potential mechanisms remain unclear. HMGB1, also known as amphotericin, which belongs to the nucleic acids-associated protein, is an essential component of ICs. HMGB1is expressed in almost all eukaryotic cell types and is located mainly in the cell nucleus, where it binds to nucleotides to stabilize the structure of nucleosomes and to induce DNA binding to regulate transcription. In SLE patients, HMGB1-nucleic acids complexes are released from the secondary necrotic cells, form ICs with the corresponding antibodies and are found to be significantly elevated in the sera. Depositon of HMGB1-containing ICs are also found in target tissues, such as the skin and kidney, contributing to the chronic inflammation and tissue injury. The proinflammatory activity of HMGB1is mostly attributed to its ligation with RAGE. RAGE, a member of the immunoglobulin superfamily of cell surface molecules, is a central signal transduction receptor for HMGB1-modified adducts. It is expressed on cells such as endothelial cells, monocytes and macrophages and can signal to transcription factor NF-κB family member p65through the classical activation pathway. Furthermore, RAGE signaling to the NF-κB p65regulates the expression of RAGE itself.Knowing that HMGB1is one important component of ICs and the proinflammatory activity of HMGB1is mostly attributed to its ligation with RAGE. The cell signaling HMGB1-RAGE axis may play important roles in the effects of ICs on the endothelial cells. However, the roles of cell signaling HMGB1-RAGE axis in the effects of ICs on the endothelial cells has not been evaluated so far. It would be attracted and worthy to evaluate the involvement of the cell signaling HMGB1-RAGE axis in the effects of ICs on endothelial cells to examine whether it is one potential mechanism of ICs’effects on human endothelial cells.The effects of HMGB1can be specially blocked by HMGB1A-box, RAGE can be blocked by sRAGE. HMGB1A-box, sRAGE, Bay117082and combination of these blockers were used to block the effects of the HMGB1component in ICs, RAGE, NF-κB p65and combine effects of them in human endothelial cells, respectively to detect whether the effects of ICs on human endothelial cells occur involving in the cell signaling HMGB1-RAGE axis. p38 mitogen-activated protein kinases (MAPK) and extracellular signal-related kinases1and2(ERK1/2) pathways are reported to be involved in the signaling pathway of RAGE-mediated NF-κB activation. Specific inhibitors of p38MAPK (SB203580) and ERK1/2(PD98059) were also used to assess the contribution of p38MAPK and ERK1/2pathways to ICs-stimulated cytokine responses in human endothelial cells.Objectives:To explored the effects of ICs on the endothelial cells and evaluate the involvement of the cell signaling HMGB1-RAGE axis in these effects to examine one potential mechanism of the effects of ICs on human endothelial cells in the pathogenesis SLE vasculitis.Methods:1. For ICs stimulation and blocking assays, human CRL-1730cells were seeded into6cm-dishes at80%confluent monolayers and pretreated with HMGB1A-box (10μg/ml), sRAGE (20μg/ml) SB203580(10μM), PD98059(25μM), Bay117082(1μM) or combination of them for1hour before the addition of ICs. After incubation for2hours, immunocytochemistry (ICC), immunofluorescence (IF) and Western blot analyses of NF-κB family member p65levels in the nucleus were performed. After incubation for6hours, RAGE, ICAM-1, VCAM-1, IL-8, MCP-1, IL-6, TNF-α and IL-1β mRNA levels were detected by Quantitative real-time-PCR. After incubation for16hours, cell surface RAGE, ICAM-1, VCAM-1proteins expression levels were analyzed by ICC, IF, flow cytometry (FCM) or cellular enzyme-linked immunosorbent assay (cellular ELISA) analysis. After incubation for24hours, the concentration of cytokines IL-8, MCP-1, IL-6, TNF-a and IL-1β in the cell-free supernatants were detected by ELISA.2. For monocytes transendothelial migration experiments, human CRL-1730cells were seeded on gelatin-coated permeable Transwell filters to form a monolayer and pretreated by blockers and ICs as described above for24hours before monocytes migration. After incubation for20hours, the cells that had transmigrated to the lower chamber were harvested and counted using a TC10Automated Cell Counter. And the difference of monocytes transendothelial migration between different endothelial cells groups are analysed.3. For cell viability assay, human CRL-1730cells were seeded onto24-well plates at105cells/well and allowed to attach and grow overnight to confluence, and then they were incubated with control medium alone, HMGB1A-box (10μg/ml), sRAGE (20μg/ml), SB203580(10μM), PD98059(25μM), Bay117082(1μM) or combination of them. After treatment for24hours, cells were harvested and overall cell counts were measured and the number of viable cells was determined by hemocytometer counts of Trypan Blue-impermeable cells. In another test, cell viability was determined using the CCK-8kit.4. Statistical analyses were performed using SPSS statistics software version17.0. Unless otherwise indicated, data are presented as mean±SE. Differences of group comparisons were analyzed using one-way ANOVA and subsequent appropriate post-hoc analysis by unpaired Student’s t-tests. A P value<0.05was considered statistically significant.Results:1. ICs up-regulate RAGE expression involving in the cell signaling HMGB1-RAGE axis in human endothelial cells2. ICs up-rcgulate the cell surface adhesion molecules ICAM-1and VCAM-1expression involving in the cell signaling HMGB1-RAGE axis in human endothelial cells3. ICs increase the secretion of chemokines IL-8, MCP-1and proinflammatory cytokines IL-6, TNF-a involving in the cell signaling HMGB1-RAGE axis in human endothelial cells4. Activation of p38MAPK and ERK1/2pathways in the cell siganling HMGB1-RAGE axis induced by ICs in human endothelial cells5. ICs lead to activation of NF-κB p65involving in the cell signaling HMGB1-RAGE axis in human endothelial cells 6. Monocytes transendothelial migration is enhanced by ICs pretreated human endothelial cells which occurs involving in the cell signaling HMGB1-RAGE axis of human endothelial cells7. Blockers HMGB1-A-box, sRAGE, Bay117082, SB203580, PD98059or combination of them used in the experiments were not toxic to human endothelial cells.Conclusions:This study demonstrates that ICs elicit proinflammatory responses in human endothelial cells and contribute to alterations in human endothelial cells function involving in the cell signaling HMGB1-RAGE axis, it provides a demonstration for the effects and a potential mechanism of the effects of ICs on human endothelial cells, which is important in understanding the pathogenesis of lupus vasculitis and may provide important evidence for the therapy of SLE vasculitis in the future. Part IIThe activating effects and potential mechanisms of immune complexes on human monocytes and its possible therapeutic methodsBackgrounds:Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by formation of immune complexes (ICs), which contain autoantigens nucleic acids, nucleic acids-associated protein and corresponding antibodies. In SLE patients, the apoptotic cells which are impaired cleared by monocytes/macrophages result in secondary necrosis, autoantigens nucleic acids and nucleic acids-associated protein are released by the nuclei of these secondary apoptotic cells. The corresponding autoantibodies against these nuclear autoantigens, such as the anti-double-stranded DNA (dsDNA) antibodies are generated and lead to the formation of ICs. Deposition of ICs on target organs, such as the kidney, results in end organs damage.Monocytes that are aberrantly activated in the patients of SLE initiate and maintain autoimmune responses via production of several cytokines and functional alteration. IL-6, TNF-a and MCP-1are cytokines secreted by monocytes, which are aberrantly up-regulated in the monocytes of SLE patients and play very important roles in the pathogenesis of SLE. Monocytes are the primary source of IL-6in the peripheral blood, IL-6promotes the production of autoantibodies and is required for the differentiation of B cells into antibodies secreting plasma cells. TNF-a is a pro-inflammatory cytokine and its over-expression leads to perpetuation of inflammation. MCP-1, which is known for its ability as a potent chemoattractant of monocytes, facilitates the recruitment of monocytes into the inflammatory sites. Recent studies on lupus nephritis demonstrated that activated monocytes/macrophages vigorously participate in and amplify renal inflammation and injury, which influence the outcome of lupus nephritis.ICs which contain nucleic acids, nucleic acids-associated protein and corresponding antibodies are pathogenic. It has been reported that the nucleic acids component in ICs isolated from SLE patients are of similar size as the cleaved chromatin fragments released from apoptotic cells, these fragments are rich in oligonucleotide class A CpG (CpG-A) and can be specifically recognized by TLR9, which is a pattern recognition receptor expressed on monocytes, pDCs, B cells and so on. TLR9activation can be specifically blocked by its antagonist inhibitory oligodeoxynucleotide ODN2088. High Mobility Group Box1(HMGB1; also known as amphotericin) which belongs to the nucleic acids-associated protein in ICs, is another essential component of ICs. It is expressed in eukaryotic cells and is located mainly in the cell nucleus, where it binds to nucleotides to stabilize the structure of nucleosomes and to induce DNA binding to regulate transcription. In SLE patients, HMGB1released from the secondary necrotic cells are found to be significantly elevated in the sera. Once released into the extracellular milieu, it functions as a cytokine that activates inflammatory cells to the further production of cytokines. The pro-inflammatory cytokine activity of HMGB1is mostly attributed to its association with RAGE. RAGE is the first described receptor for HMGB1and is expressed on the surface of monocytes, vascular smooth muscle cells, neurons, endothelial cells and so on. RAGE can be blocked by soluble RAGE (sRAGE). It has been reported that ICs can stimulate pDCs and autoreactive B cells to induce IFN-a and other pro-inflammatory cytokines production and disease development through RAGE and TLR9due to the nucleic acids-associated protein HMGB1and nucleic acids component in the ICs. However, the effects of ICs on monocytes and the potential mechanisms remain unclear.Both cell surface receptor RAGE and cytosolic receptor TLR9can signal to transcription factor NF-κB family member p65through the classical activation pathway, which can regulate the expression of IL-6, TNF-α and MCP-1. Furthermore, RAGE signal to NF-κB p65can regulate the expression of RAGE itself. In this paper, ICs were used to detect its effects on monocytes, sRAGE, ODN2088, and Bay117082were used to block RAGE, TLR9and NF-κB p65, respectively, to detect the possible mechanisms of ICs’effects on monocytes.Despite the recent advances in anti-inflammatory therapy, current treatment options for SLE are diverse and poorly defined. It has been reported that PPAR-y agonist is a very useful therapeutic strategy in mouse models of SLE, it can reduce autoantibodies production, it can also reduce renal inflammation and injury. It is reported that the PPAR-y agonists which contains thiazolidinediones (TZDs), prostanoids and non-steroidal anti-inflammatory drugs (NSAIDs) activate the nuclear hormone receptor PPAR-y, which is expressed in monocytes, keratinocytes and so on, and regulate the expression of pro-inflammatory proteins by interacting with transcription factors NF-κB, it is also reported that PPAR-y agonists can inhibit the nuclear translocation of NF-κB p65and promote PPAR-y to form complexes with p65in the nucleus, which reduces the binding of p65to its target sequences. Here we chose pioglitazone, which belongs to TZDs and pharmacological agonist of PPAR-y as a representative of the PPAR-y agonists to examine the effects of PPAR-y agonists on monocytes aberrant activation and their mechanisms in SLE in vitro to further detect its therapeutic effects in SLE in addition to its effective therapeutic effects in mouse models of SLE reported before.Objectives:In this study, we try to explore the effects of ICs on monocytes aberrant activation in SLE and the potential mechanisms, and to propose a potential therapeutic strategy to the monocytes aberrant activation in SLE.Methods:1. Human U937cells were seeded onto6-well plates at106cells/well and pretreated with sRAGE (20μg/ml), ODN2088(12μg/ml), Bay117082(1μM) or PPAR-y agonist pioglitazone (10μM) for one hour before treating with ICs. After incubation for two hours, the level of NF-κB family member p65in the nucleus was analyzed by western blot and immunoprecipitation (IP) assays were performed to detect the formation of complexes PPAR-y-p65. After incubation for six hours, RAGE, IL-6, TNF-a and MCP-1mRNA expression levels were determined using real-time RT-PCT. After incubation for16hours, RAGE protein expression level was determined by flow cytometry and western blot analysis. After incubation for24hours, the concentrations of cytokines IL-6, TNF-a and MCP-1in the cell-free supernatants were examined and transendothelial migration assays were performed to detect the changes of monocytes transendothelial migration.2. For the cell viability assay, human U937cells were seeded onto12-well plates at5×105cells/well and incubated with control medium alone or sRAGE (20μg/ml), ODN2088(12μg/ml), Bay117082(1μM) or PPAR-y agonist pioglitazone (10μM) for24hours. Cell viability was determined by measuring overall cell counts, hemocytometer counts of Trypan Blue-impermeable cells and CCK-8.3. Statistical analyses were performed using SPSS Statistics software (version17.0). Unless otherwise indicated, the data were presented as mean±SE. Differences of group comparisons were analyzed using one-way ANOVA and subsequent appropriate post hoc analysis by unpaired Student’s t test. A P value<0.05was considered statistically significant.Results:1. Cell surface receptor RAGE expression are up-regulated by ICs through TLR9mediated NF-κB activation and can be inhibited by PPAR-y agonist in human monocytes2. The secretion of monocytic cytokines IL-6, MCP-1and TNF-a is increased by ICs through RAGE/TLR9mediated NF-κB activation and can be inhibited by PPAR-y agonist in human monocytes3. Increased nuclear import of NF-κB family member p65is induced by ICs through RAGE/TLR9and can be inhibited by PPAR-y agonist in human monocytes4. Monocytes transendothelial migration is increased by ICs stimulation through RAGE/TLR-9mediated NF-κB activation and can be inhibited by PPAR-y agonist5. The PPAR-y agonist pioglitazone inhibits ICs-induced NF-κB p65activation via formation of PPAR-y-p65complexes in a dose dependent manner in human monocytes6. sRAGE, ODN2088, Bay117982and pioglitazone used in the experiements are no cytotoxic to human monocytes.Conclusions:This study provides a demonstration of ICs’activating effects on the aberrant activation of monocytes and provides one possible mechanism of how ICs activate monocytes in SLE, which plays important roles in the pathogenesis of SLE. In addition, this study suggests a possible way to inhibit the monocytic activation in SLE, which has the potential to become one of the therapeutic options for SLE.