| BackgroundAnti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (ANCA associatedvasculitis, AAV) represents a group of systemic autoimmune diseases, including Wegener ’sgranulomatosis (WG), microscopic polyangiitis (MPA), Churg-Strauss syndrome (CSS) andrenal-limited vasculitis(RLV). When idneys are involved, they usually presentpauci-immune focal segmental necrotising glomerulonephritis (PiFSGN), which leads torapid and irreversible renal failure. It is acknowledged as a local clinical manifestation ofanti-neutrophil cytoplasmic autoantibody-associated system vasculitis (AAV).ANCAs are a heterogeneous group of autoantibodies produced by stiumulatingneutrophil cytoplasmic antigens, which are sensitive serological markers for diagnosis ofANCA-associated vasculitis (AAV). Myeloperoxidase (MPO) and Proteinase3(PR3) aretwo major ANCA antigens. The present study has confirmed that ANCAs play a pathogenicrole in ANCA-associated vasculitis (AAV), and it is the core pathogenic mechanism thatANCAs bind with target antigens on the surface of neutrophils. Neutrophils can beactivated when ANCAs recognize the conformational epitopes of MPO and PR3. Understeady circumstances, neutrophils do not express target antigens for ANCA on cell surfaces,but only in the cytoplasm of neutrophils. These target antigens would be transferred fromthe cytoplasm to the cell surface and exposed the binding antigens only when neutrophilswere primed or stimulated by inflammatory cytokines. In this case, ANCAs can bind withtarget antigens on neutrophil surface through their Fab2segment, and then neutrophilswould be activated, resulting in local inflammation. Apoptotic neutrophils and their cell debris which ingested by nearby phagocytoticcells and timely removed are imperative conditions to eliminate the inflammatory response,maintaining homeostasis. However, the obstacled clearance of apoptotic neutrophils wouldresult in inflammatory diseases. In patients with ANCA associated pauci-immune focalsegmental necrotising glomerulonephritis (PiFSGN), numbers of activated neutrophils andlysosomal enzymes, which released from its nuclear debris, are accumulated aroundinjuried capillaries. These neutrophils infiltration and cellular components deposit areclosely associated with kidney damages. Research indicates that many diseases incidence(such as SIRS, ARDS and MODS etc.) are due to neutrophil delayed apoptosis or apoptosisobstructions. A large number of adhesion molecules expressed on cell surface whenneutrophil apoptosis disorders or even delay, which enhance the ability of adhesion tovascular endothelial cells, destroy the integrity of endothelial cells, and induce theinflammatory response.Neutrophil extracellular traps (NETs) is a newly recognized mode of neutrophilcell-death, which extrudes its structures of decondensed chromatin decorated withantimicrobial peptides, such as myeloperoxidase (MPO), elastase, proteinase3(PR3),cathepsin G, lactoferrin and others. Recent studies have demonstrated that the formation ofneutrophil extracellular traps (NETs) also participate in pathogenesis of AAV. It couldtransfer cytoplasmic neutrophil autoantigens to dendritic cells, which induce ANCAassociated autoimmunity (AAV) in mice. And ANCA can be further effect on neutrophils bypromoting more neutrophil extracellular nets (NETs) formation, which results in a viciouscycle of autoimmune reactions.DCs act as the most powerful potent antigen-presenting cells and the only antigenspresentation cells to CD4~+T cells in human immune system, which play a vital role inmediation of immune response and maintaining tolerance. Th17cells are recognized assubsets of CD4~+T cell relevanting to autoimmune diseasese. It is proved that highercontents of Th17cells in serum of ANCA associated vasculitis patients than that in ANCAnegative vasculitis and healthy people. Th17cells participate in the occurrence of ANCAassociated vasculitis and tissue damage. Research has been confirmed that ithere are a lot of immature dendritic cell infiltrationn renal pathological specimens of patients withANCA-associated vasculitis, and involved in activation of Th17cell proliferation anddifferentiation.Human anti-lysosome-associated membrane protein2(LAMP-2) antibody, a newsubtype of ANCA family, is universal prevalence in patients with active disease of PiFSGNand capable to lead to PiFSGN in WKY rats, as well as activating human neutrophils orprovoking endothelium apoptosis in vitro. The results have clearly demonstrated thatanti-LAMP-2antibody plays a pathogenic role in the development of AAV. However, howanti-LAMP-2antibody leads to AAV remains unknown.This project intends to adopt magnetic activated cell sorting, flow cytometry, ELISA,immunohistochemistry and confocal laser scanning techniques to observe the influence ofanti-LAMP-2antibody on neutrophil (neutrophil apoptosis process, neutrophil extracellularnets (NETs) formation), and the effects of neutrophil treated by anti-LAMP-2antibody onactivation of immature dendritic cells, and the secretion of inflammatory cytokinesmicroenvironment on initial CD4~+T cells into Th17differentiation. Based on these findings,we aim to test the feasibility that anti-LAMP-2antibody induce apoptosis disturbance orneutrophils extracelluar traps formation in AAV.Materials and MethodsPart11. CD14~+monocytes were separated and purityed by immunomagnetic beads, werecultured to become immature dendritic cells in vitro, and were detected by opticalmicroscopy and flow cytometry.2. Density gradient centrifugation method was used to isolate neutrophils fromperipheral bloods of healthy volunteers and identification the neutrophils throughimmunofluorescence and flow cytometry.3. Neutrophils were in presense of Anti-LAMP-2antibody for24h, and then apoptosisrate changes of neutrophils were detected by flow cytometry.4. Apoptotic neutrophils labeled by FITC-Annexin V were cultured with immaturedendritic cells (DCs), and then the state of dendritic cells phagocyting apoptotic neutrophils were assayed by confocal detection.5. Neutrophils were stimulated by anti-LAMP-2antibody for24h, and their influenceson MFI of immature dendritic cells (DCs) activation markers (CD80, CD83, CD86andHLA-DR) were detected by flow cytometry.6. Neutrophils were stimulated by anti-LAMP-2antibody for24h, and their influenceson the secretion of (IL-1β, IL-6, IL-23) from dendritic cells were detected by ELISA.7. CD4~+T cells were separated and purifyed by immunomagnetic beads and then wereidentificated them through flow cytometry.8. CD4~+T lymphocytes were added into the coculture system of neutrophils anddendritic cell.5days later, we use of flow cytometry to determine whether CD4~+T cells toTh17cells or not.Part21. Neutrophils were stimulated by anti-LAMP-2antibody for3h, and then neutrophilextracellular traps (NETs) formation, as well as the exposure of antigen (LAMP-2, MPO,PR3) was detected by immunofluorescence.2. Immature dendritic cells labeled with PKH-26were cultured with anti-LAMP-2antibody-induced neutrophilextracellular traps (NETs) stained with SYTOX green for4h,then the interaction of NETs with dendritic cells were observe by confocal detection.3. Immature dendritic cells labeled with PKH-26were cultured with anti-LAMP-2antibody-induced neutrophilextracellular traps(NETs) stained with FITC-PR3, MPO andLAMP-2monoclonal antibody for18h, then the stages of extracellular autoantigens(LAMP-2, MPO, PR3) transferred to dendritic cells were observe by confocal detection.4. Neutrophils were stimulated by anti-LAMP-2antibody for3h, and their influenceson MFI of immature dendritic cells (DCs) activation markers (CD80, CD83, CD86andHLA-DR) were detected by flow cytometry.5. Neutrophils were stimulated by anti-LAMP-2antibody for3h, and their influenceson the secretion of (IL-1β, IL-6, IL-23) from dendritic cells were detected by ELISA.6. CD4~+T lymphocytes were added into the coculture system of neutrophils anddendritic cell.5days later, we use of flow cytometry to determine whether CD4~+T cells toTh17cells or not. ResultsPart11. Neutrophil apoptosis rates were significantly decreased when they were in thepresence of anti-hLAMP-2antibody for24h.2. Apoptotic neutrophils labeled with FITC-Annexin V appeared in the cytoplasm ofimmature dendritic cells.3. Anti-LAMP-2antibody distrubed the process of neutrophil apoptosis, whichincreased the the surface activation markers (CD80, CD83, CD86and HLA-DR) ofdendritic cell; however, no significant changes of the activated phenotype (CD80, CD83,CD86and HLA-DR) were detected between anti-LAMP-2antibody treated neutrophilsgroup and routinely apoptotic group.4. Anti-LAMP-2antibody-induced abnormal neutrophil apoptosis promoted dendriticcells to secretion of IL-1β, IL-6, but no IL-23has been detected.5. CD4~+T lymphocytes were added into the coculture system of neutrophils anddendritic cell.5days later, we found that part of CD4~+T cells differenticate into Treg cellsPart21. Anti-LAMP-2antibody induced neutrophil extracellular traps (NETs) formation,exposing LAMP-2, MPO and PR3along with NETs.2. Neutrophils were aged for3h in the presence of anti-LAMP-2antibody, and thenNETs stings were found to interact with imDCs after4hours coculture. On18hourscoculture, imDC to uptook of NETs autoantigens stained along with a mAb to PR3, MPOand LAMP-2, including LAMP-2, MPO and PR3directly conjugated with FITC dye.3. Anti-LAMP-2antibody-induced NETs significantly increased the markers indicativeof maturation on dendritic cells (CD80, CD83, CD86and HLA-DR).4. Anti-LAMP-2antibody-induced NETs significantly increased the secretion of IL-1β,IL-6,IL-23by dendritic cells.5. CD4~+T lymphocytes were added into neutrophils and dendritic cell coculturesystem.5days later, Th17cells and the secretion of IL-17a were detected.6. It is believed that NETs are adequately digested by DNase I. When DNase I addedinto Anti-LAMP-2antibody induced NETs, all chromatin fibers exposed along with NETs were all vanished. The increases of markers indicative of maturation on dendritic cells byanti-LAMP-2antibody–induced NETs were disappeared. And the differentiation of Th17cells and the secretion of IL-17a were prevented by using DNase I.Conclusions:1. Anti-LAMP-2antibody descreases the apoptotic rates of neutrophils.2.Dendritic cells engulfs LAMP-2antibody-induced apoptotic neutrophils. Thephagocytosis fluorescent tracer system of dendritic cells-apoptotic neutrophil wasestablished.3. Anti-LAMP-2antibody distrubs the process of neutrophil apoptosis, which partlyincreases the MFI of dendritic cell surface activation markers, and promotes the secretionof IL-1β,IL-6.In this case, no na ve CD4~+T cells differentiate into Th17cells are founded.4. Anti-LAMP-2antibody induces neutrophil extracellular traps (NETs) formation andexposes intracellular autoantigens (MPO, PR3, and LAMP-2).5. Dendritic cells engulfs autoantigens released from LAMP-2antibody-induced NETs.The phagocytosis fluorescent tracer system of dendritic cells-autoantigens was established.6. PBMC-derived DCs are actived by anti-hLAMP-2antibody induced-NETs.Moreover, coculture supernatants set up the microenviroment leading CD4~+T cellsdifferentiate into Th17cells. |
PDF Full Text Request