Regulation Of Different Ways Of Cell Death On Macrophage Secretion Of Cytokines

BackgroundCell death is a vital biological process in multicellular organisms that plays important roles in their development,defense and homeostasis.Special markers (such as phosphatidylserine,PS) exposed on dead cell surface enable that dead cells are quickly engulfed and processed by phagocytes,which may cause different immune consequences.Two ways of cell death,apoptosis and necrosis,are important in vivo.Both apoptotic cells and necrotic cells are rapidly recognized by phagocytes, mainly macrophages.Apoptosis is an active,energy consuming,and programmatic cellular process,which is not accompanied by tissue injury or inflammation.On the contrary,necrosis is a passive,non energy consuming and non programmatic cellular process that usually causes inflammatory and tissue damage.Researchers during the past 10 years revealed that both forms of dead cells play critical roles in autoimmunity,tumor immunity,infection immunity and transplantation immunity.To address how dead cells regulate immune responses,Sun proposed the "Cell death recognition model for the immune system",which presumes that apoptotic cells induce immune tolerance,while necrotic cells initiate contrary aggressive immune responses.Our previous studies have shown that apoptotic cells could inhibit T cell activation,prolong graft survival and induce immune tolerance.But how dead cells take their effect on phagocytes remains unclear.It has been known that cells and tissues infected by Gram-negative bacteria release a large amount of LPS(Lipopolysaccharide,LPS) into surrounding tissues. LPS then activate macrophages to release tumor necrosis factor-α(TNF-α), interlukin-1(IL-1),interlukine-6(IL-6),interlukine-8(IL-8) and other inflammatory mediators that induce local or systemic inflammatory responses.Meanwhile, inflammation is always accompanied with an extensive cell necrosis.When bacterial infection is present and germ derived metabolic product released,whether necrotic cells or apoptotic cells which are frequently found along with bacterial infection could actively regulate the immune system remains obscure.To address these questions,we investigated the effect of apoptotic cells and necrotic cells on macrophage secretion of inflammatory cytokines,especially TNF-αand IL-6,in the absence or presence of LPS.PurposeInvestigate the effects of different ways of cell death on the secretion of pro-inflammatory cytokines by macrophages and provide experimental evidence for "cell death immune recognition model",which may not only help us to understand the recognition and responses of the immune system,but also be conducive to novel protocols for inducing immune tolerance or initiating more appropriate immune responses.Methods1.Culture of mouse macrophage cell lines,RAW264.7The mouse macrophage ceil lines,RAW264.7,were a generous gift from Professor.Jiang,Department of path physiology of South Medical University.Cells were maintained in RPMI-1640 culture medium supplemented with 5%fetal bovine serum,100U/ml penicillin,and 100g/ml streptomycin in a humidified incubator with 5%CO₂ at 37℃.2.Isolation and culture of mouse peritoneal maerophageSix-week-old mice were intraperitoneally injected with 1ml sterile liquid paraffin.Three to four days later,the mice were bled and sacrificed,and their abdominal cavity repeatedly flushed with sterile PBS.Exudates were collected and washed with RPMI-1640 medium 3 times.Cells were re-suspended and cultured in a humidified incubator with 5%CO₂ at 37℃. 3.Preparation of apoptotic/necrotic thymocytesThymocytes were isolated from Balb/c mouse and cell concentration adjusted to 1×10^6/ml with complete medium RPMI-1640.For apoptosis induction,the cells were incubated with dexamethasone(5×10^-6M) for 6hrs in RPMI-1640 containing 10%FBS.For necrosis induction,the cells were heated at 56℃in a water bath for 60min.The cells were collected and apoptosis/necrosis determined by Annexin V-FITC Apoptosis Detection Kit according to the manufacturer's instructions.4.Co-culture of dead cells with macrophagesRAW264.7 cells or freshly isolated peritoneal macrophages were aliquoted in 96-well plates in 3 groups:control,apoptotic cell group and necrotic cell group.In the control group,macrophages were co-cultured with complete medium RPMI-1640, while in apoptotic cell group and necrotic cell groups,macrophages were co-cultured with apoptotic or necrotic cells(1:5) respectively.One hour later,un-engulfed apoptotic or necrotic cells were washed away and fresh complete medium RPMI-1640 added.The cells were continuously cultured a 37℃,5%CO₂ for18hrs and supernatants collected and kept in-20℃.5.Co-culture of dead cells with macrophages and LPSRAW264.7 cells or freshly isolated peritoneal macrophages were aliquoted in 96-well plates in 3 groups:control group,apoptotic cell group and necrotic cell group. In the control group,macrophages were co-cultured with complete medium RPMI-1640,while in apoptotic cell group or necrotic cell groups,macrophages were co-cultured with apoptotic or necrotic cells(1:5) respectively.One hour later, un-engulfed apoptotic or necrotic cells were washed away and fresh complete medium RPMI-1640 containing LPS(100ng/ml) supplemented.The cells were continuously cultured at 37℃,5%CO₂ for18hr and supernatants collected and kept in-20℃.6.Cytokine detectionCytokines in the supernatant of different groups were determined by Bio-Plex suspension system using LINCO cytokine detection kits.7.Statistical analysis Data were analyzed with GraphPad Prism5.0 software and comparisons between experimental groups and control group were made with t-tests.Results1.One thymus from a 4 to 6-week mouse yielded 1-2×10⁸ thymocytes.First,we compared the effect of dexamethasone(Dex) and ultra-violet(UV) on the induction of thymocyte apoptosis,and found that Dex is the better inducer.Then,we tested different conditions for apoptosis induction by Dex,including Dex concentrations, cell concentrations,incubation time and found that treating the cells(5×10⁶/ml) with 5×10^-6M Dex for 6hrs is the best for thymocyte apoptosis induction.Flow cytometry found that the early apoptotic rate was more than 40%,while late apoptosis rate or necrotic rate less than 10%.For necrotic cells induced by incubation at 56℃for 1h,more than 95%mouse thymocytes were Annexin-V and propidine iodide double positive(necrotic cells) with double negative(living cells) less than 5%.2.Compared with the control group,peritoneal macrophages that had been treated with apoptotic cells for 1 hr significantly reduced the secretion of inflammatory cytokines,especially TNF-αand IL-6,in response to LPS stimulation.Therefore, apoptotic cells down regulate the pro-inflammatory response of macrophages to LPS stimulation.3.Compared with the control group,in-flammatory cytokine production by macrophages co-cultured with necrotic cells was significantly enhanced.In addition, compared with necrotic cell treatment or LPS treatment alone,macrophages that had been treated with necrotic cells for 1 hr followed by LPS stimulation further dramatically enhanced secretion of inflammatory cytokines,especially TNF-αand IL-6.Therefore,necrotic cells themselves not only up regulate the pro-inflammatory response of macrophages,but also have synergistic effect with LPS to further enhance pro-inflammatory responses of macrophages.4.Remember that macrophages were co-cultured with apoptotic cells or necrotic cells for only 1 hr,suggesting that either apoptotic cells or necrotic cells seem to have a "turn off and turn on" effect on macrophages during which a continuous presence of dead cells is not necessary. Conclusions1.The best conditions for mouse thymocyte apoptosis induction are:4ml RPMI-1640 containing 10%FCS,Petri dish of 6cm diameter,Dex concentration of 5×10^-6M,cell concentration of 5×10⁶/ml and incubation for 5h.2.Apoptotic cells can inhibit the production of inflammatory cytokines,especially TNF-αand IL-6,in macrophages,which may be the mechanisms by which apoptotic cells inhibit inflammatory responses and induce immune tolerance.3.Compared with the control group,either necrotic cells or LPS significantly increased production of TNF-αand IL-6 by macrophage cell line RAW264.7.The secretion of these two inflammatory cytokines further dramatically increased while macrophages were treated a combined treatment of both necrotic cells and LPS. These findings suggest that cell necrosis,usually found in bacterial infection,leads to strong and sustained inflammatory responses that are able to more effectively activate the immune system to generate anti-infective immunity.