Negative Regulation Of Immune Response By Apoptotic Cells And The Underlying Mechanisms

The liver is generally regarded as a special organ prone to immune tolerance,and it is also regarded as the major grave of aging erythrocytes and neutrophils.In addition, the liver is a site of activated CD8~+ T trapped and destroyed at the end of immune response.These phenomena suggest that liver may be the main scavenger of apoptotic leukocytes in blood.Considering that Kupffer cells,the resident professional phagocytes in liver,have been shown to actively mediate immune tolerance in liver, thus,we wonder whether Kuppfer cells are the critical phagocytes for clearance of apoptotic cells in liver,and what's the effect of apoptotic cell clearance on the immune response and inflammation.The presence of apoptotic cells is generally accompanied by immunosuppression. Although contact with apoptotic cells during the inflammation or in remodeling tissue may educate not only macrophages to adopt an immunoregulatory property,dendritic cells(De)and monocytes have been also showed to produce more anti-inflammatory cytokines,such as transforming growth factor(TGF)-βand interleukin(IL)-10,and less proinflammatory cytokines.The process appears to require identification of specific molecules on apoptotic cells,such as anionic lipid phosphatidylserine(PS). Interestingly,PS-carrying vesicles may simulate the anti-inflammatory response of apoptotic cells.However,the mechanisms for the control of immune response and inflammation by apoptotic cell clearance remain to be fully clarified up to now.IL-10 is an important immunoregulatory cytokine,which can be produced by Th2 cells,B cells,mast cells,and macrophages.Numerous studies demonstrate that IL-10 plays a central role in control of inflammation,for examples,collagen-induced arthritis,pancreatitis,uveitis,keratitis,hepatitis,peritonitis,lung injury,and neural injury.It has been shown that the increasing of IL-10 production was along with the decrease of proinflammatory cytokines from phagocytes being treated with LPS and apoptotic cells.And IL-10 secretion from macrophages interacting with apoptotic cells was dependent on the activation of p38MAPK and receptor CD36,and cell-cell contact,but not phagocytosis.The primary aim of this study is to investigate whether apoptotic cells could negatively regulate immune response and inflammation both in vitro and in vivo,and if so,what is the underlying mechanism.Therefore,in this study,we performed in vivo experiments by using apoptotic cells stained by 5,6-carboxyfluorescein diacetate succimidyl ester(CFSE).Our results showed that the in vivo transferred apoptotic cells were quickly captured(within 4 h)by host macrophages in vivo.The apoptotic cells derived from the activated splenocytes or blood leukocytes were mainly captured by hepatic Kupffer cells when infused through intravenous injection via tail or portal vein,however,intravenous infusion(via tail vein)with apoptotic cells derived from the unactivated splenocytes would lead to the accumulation of the apoptotic cells in the spleen and then activation of splenocytes.The above results suggested that the site where apoptotic cells were phagocytosed was determined by not only infusion pathway,but also cell activation state before apoptosis.Then we inhibited the activation of Kupffer cells in vivo with in vivo administration of Gadolinium Chloride (GdCl3)or cytochalasin,or deleted CD11c~+ DC in vivo with diphtheria toxin in vivo of the CD11c-DTR model mice to test which kind of cells responsible for the clearance of the infused apoptotic cells.Our results showed that macrophages in spleen and Kupffer cells in liver are the major phagocytes during clearing of apoptotic cells in vivo.,We compared the capacity of splenic macrophages,peritoneal thioglycollate-elicited macrophages and Kupffer cells to phagocytose apoptotic cells in vitro,and found that Kupffer cells displayed more profoundly capacity to engulf apoptotic cells. We observed that mice with LPS-induced fulminant hepatitis had a high mortality within 12 h,whereas infusion with apoptotic cells prior to the LPS/D-GalN challenge could dramatically ameliorate the pathological damage of liver.When the apoptotic cells infused through portal vein,the more profound preventive effect was observed. In addition,we also observed that portal veinous infusion of 1×10~7 to 3×10~7 apoptotic cells for each mouse during 3 to 7 days before challenge of LPS/D-GalN to induce fulminant hepatitis could ameliorate hepatic injury more efficiently.To determine whether Kupffer cells are critical for LPS/D-GalN-induced hepatitis and apoptotic cell-induced protection against hepatitis,we used GdCl3 to specifically inhibit Kupffer cell phagocytosis and function.Once the activation of Kupffer cells was inhibited by GdCl3,LPS/D-GalN could not induce fulminant hepatitis.Moreover, hepatits would happen when Kupffer cells were transferred into the mouse model via portal veinous injection.However,fulminant hepatits would not happen when apoptotic cell-stimulated Kupffer cells were transfered.With the in vivo deletion of DCs or CD25+ regulatory T cells,our further experiments proved that DCs or CD25+ regulatory T cells were not responsible for the apoptotic cell-induced protection against hepatitis.Thaken together,these results demonstrate that Kupffer cells are critical mediator responsible for the prevention of inflammation induced by apoptotic cell infusion..In order to confirm the changes of Kupffer cells after apoptotic cell phagocytosis, we isolated and cultured Kupffer cells from normal mice and mice infused with or without apoptotic cells.We first analyzed the morphology of Kupffer cells 3 days after cocultured with apoptotic cells.Striking morphologic changes occurred after phagocytosis of apoptotic cells.Kupffer cells were stained with Wright-Gimmsa dyes and observed under phase contrast microscope.Unactivated Kupffer cells appeared to be rounded and relatively non-adherent to the tissue culture plate.Three days after culture with apoptotic cells,Kupffer cells became firmly adherent and long elliptoid on the tissue culture plate.Additionally,after overnight culture with 10 ng/mL LPS, Kupffer cells became more tightly adherent and spread out on the tissue culture plastic.Then we wondered if the interaction with apoptotic cells could modify phenotype of Kupffer cells.Next,we tested the expression of costimulatory molecules (CD40,CD80,CD86),CD14 and B7-H1 of Kupffer cells by FACS.However,no distinct changes were observed.Interestingly,inflammatory cytokines(TNF-α,IL-1β, IFN-γ,and IL-6)production by Kupffer cells pretreated with apoptotic cells was significantly decreased as compared to that of un-pretreated Kupffer cells under stimulation of LPS.On the other hand,production of anti-inflammatory cytokines such as IL-10,TGF-β,IL-4 by apoptotic cell-engulfed Kupffer cells was enhanced more significantly in response to LPS.However,no dramatically alteration of cytokines secretion by Kupffer cells was observed if not stimulated by LPS.The previous studies demonstrate that Kupffer cells are the mediator of the endotoxin-induced hepatitis.We used GdCl3 to block the function of Kupffer cells and then infused Kupffer cells via portal vein,these Kupffer cells were purified from wild type mice or IL-10 Knock-out mice,pretreated with or without apoptotic cells. Wild type Kupffer cells cultured with apoptotic cells could prevent hepatitis in vivo, however,when IL-10-deficient Kupffer cells were used to repeat the above experiments,the preventive effect on hepatitis was disappeared.Similarly,when we pretreated IL-10 knock-out mice with apoptotic cells via portal vein,there was no preventive effect on hepatitis.Taking together,these data demonstrated that apoptotic cell-stimulated Kupffer cells could dampen inflammation during LPS-induced hepatitis.To determine the time of IL-10 secretion during hepatitis,we assayed the serum IL-10 level in the mice after LPS/D-GalN injection.Data showed that IL-10 in serum significantly increased at 4 h after stimulation;however,once GdCl3 was injected before apoptotic cell infusion,there was substantially less IL-10 in serum.We further determined which factor was responsible for the increased IL-10 production in Kupffer cells induced by apoptotic cells.Apoptotic cells themselves could release IL-10 and TGF-βafter UV-B irradiation.To confirm whether soluble molecules or cell-cell contact was involved in this process,we used transwell system (0.4μM pore holes)to separate Kupffer cells and apoptotic cells.IL-10 production reduced dramatically when transwell was used in the coculture system.As apoptotic cell-Kupffer cell contact may lead to apoptotic cell phagocytosis by Kupffer cells,we used cytochalasin B,a kind of inhibitor of actin polymerization and phagocytosis,to determine whether apoptotic cell phagocytosis was required.Cytochalasin B could not block the production of IL-10 in Kupffer cells,suggesting that cell-cell contact, but not phagocytosis,is critical for the IL-10 production in Kupffer cells after interacted with apoptotic cells.Although apoptotic cells could release TGF-βand IL-10,culture supernatant of apoptotic cells could not induce IL-10 production of Kupffer cells.And using neutralizing anti-IL-10 antibodies during the interaction of Kupffer cells and apoptotic cells had no effect on IL-10 production in Kupffer cells;in contrast,using neutralizing anti-TGF-βantibodies significantly blocked the IL-10 production in Kupffer cells. However,rmTGF-β1 was not able to increase IL-10 production,suggesting that membrane-bound TGF-βon apoptotic cells may be a critical factor in the promotion of IL-10 secretion by Kupffer cells.Furtherly,we proved that Kupffer cells isolated from Smad3-deficient mice secreted little IL-10 in respond to apoptotic cells, compared to wild type ones.To confirm the expression of membrane-bound TGF-βof apoptotic cells,we test it with FACS assay.Our results showed that up to 50-60% apoptotic cells expressed membrane-bound TGF-β4 h after UV-B irradiation.Subsequently,we proved that IL-10 secretion by Kupffer cells was regulated in transcription level through RT-PCR assay.Then we went on to determine which signal pathway is responsible for the increased IL-10 production.The above results have shown that Smad3 pathway was involved in this process;and previous reports suggested that ERK could cross-talk with Smads.We postulated that ERK may be an important mediator in the signaling of apoptotic cells-stimulated IL-10 secretion. Using PD98059,a specific inhibitor of ERK,we observed that ERK activation was important for IL-10 production in Kupffer cells after apoptotic cells feeding.And FACS analysis showed there was obvious phosphorylation of ERK 30 min after interaction of apoptotic cells with Kupffer cells.Moreover,ERK activation in Kupffer cells was down-regulated by neutralizing antibodies of TGF-β.Nevertheless,ERK in Kupffer cells could not be activated by rmTGF-β,which demonstrated it was membrane-bound TGF-βbeing responsible for IL-10 production in Kupffer cells through ERK- associated pathway.Therefore,we postulated that Kupffer cell-derived IL-10 was responsible for the suppression of inflammationary response during the LPS-induced fulminant hepatitis via inhibition of TNF-αproduction.Data showed that the cytolysis of hepatocytes by Kupffer cells was significantly suppressed when apoptotic cells were present,or anti-TNF-αantibody was used.In addition,SMT,a kind of specific iNOS inhibitor also dramatically reduced the hepatic injury.And anti-TNF-αantibody could cooperate with SMT to suppress the cytotoxicity.We further determined the ability of Kupffer cells to produce TNF-αand NO when IL-10 was blocked via specific monoclonal antibody.TNF-αand NO production were markedly inhibited during the interaction with apoptotic cells,but both of them restored when anti-IL-10 antibody was added.In summary,our results demonstrate that IL-10 secreted by Kupffer cells induced by apoptotic cells can inhibit Kupffer cell-mediated cytolysis of hepatocytes through inhibiting the production of TNF-αand NO,thus suggesting preferential phagocytosis of apoptotic cells by Kupffer cells attenuates endotoxin-induced hepatitis through IL-10.Our study provides new mechanistic explanation for the induction and maintenance of immune tolerance in liver.