Functional Regulation Of Dendritic Cells By The Apoptotic Signal And Immune Microenvironment

Homeostasis is one of the main characteristics of the immune system, which guarantees that the immune responses to foreign pathogens or antigens are self-limited and wane as the antigens are eliminated, returning the immune system to its basal resting state. So there must be some negative feedback mechanisms to regulate and terminate the immune responses, keeping the balance of the organisms.Antigen-presenting cells (APC) play a key role in immune responses by acting as a bridge between antigens and effector cells and triggering the immune response. Dendritic cells (DC) are the most potent APC, possessing the unique capacity of activating naive T cells. They live in the peripheral non-lymphoid organ, where they capture the invaded microbial antigens and migrate to the peripheral lymphoid organs. During this process, DC have gained powerful capability of presenting antigen, with their ability of taking up antigens decreased. In the lymphoid organs, DC present the antigen to the antigen specific helper T cells and trigger the immune response. According to the principle of the homeostasis, DC can not keep activating T cells in the immune response. Then, what is the fate of DC after interacting with T cells and completing their antigen-presenting function? Researchers have found that while DC present antigens to T cells, DC receive signals from T cells which lead to apoptosis of DC to downregulate the immune response. But most researchers had ignored the effect of the microenvironment in their interaction. Since the immune response occurs in the in vivo immune microenvironment, where there is an abundance of cytokines and protein molecules, it makes sense for it to be regulated by the microenvironment. By studying the fate of immune cells after the immune response without considering the effect of the microenvironment, the conclusion will not reflect what really happened in vivo. Although there are many reports demonstrating that the microenvironment plays an important role in the cells differentiation and development and the tumor's arise and progress, there is no report about the regulation of the fate of DC by the microenvironment. Therefore, we speculate the microenvironment will play important roles in regulating the apoptotic DC to accommodate the characteristics of DC that DC are quite rare in vivo but DC have important biological functions in the immune system.To observe the effect of the microenvironment on the fate of DC, we used the endothelial splenic stromal cells (ESSC) to mimic the microenvironment of secondary immune organ where DC present antigens to T cella and initiate immune response. We aslo set up a model in which DC are induced to apoptosis. Then, the apoptotic DC were sorted by FACSDiva onto the culture plate seeded with ESSC and we observed the biological changes of apoptotic DC under the influence of ESSC. Our results showed that DC will undergo apoptosis if they are treated with high dose LPS plus CHX, or cocultured with activated T cells, or IFNy plus antibody against MHC class II molecule. And apoptotic DC could be devided into three kinds of apoptotic DC at different stages by staining with Annexin V/Jc-1/7AAD (or Annexin V/R123/7AAD) , ie early, intermediate or late apoptotic DC. The early phase of apoptotic DC is Annexin V^RnS^^AAD", intermediate apoptotic DC is Annexin ^KU^IAAD' while late apoptotic DC is Annexin V+R123low7AAD+. We sorted apoptotic DC in different phases by FACSdiva onto the monolayer ESSC cells or the culture medium containing GM-CSF and IL-4, and found that early apoptotic DC can revive in the culture medium containing GM-CSF and IL-4, the early and intermediate apoptotic DC can be revived after sorted onto ESSC and go into proliferation while late apoptotic DC can not be revived. The phenotype of the reversed and proliferating cells obviously changed, the expression of MHC class II, CD40,CD86 and CD lie decreased while high CDllb remained unchanged. Founctionally, the ability of reversed DC to stimulate T cell proliferation decreased and reversed DC could inhibit mature DC-induced T cell proliferation, suggesting that such DC have negative immune regulatory functions. These data indicate that some specific microenvironment could strongly regulate the biological functions of many immune cells. It can recue the apoptotic DC and induced these cells into regulatory cells, therefore efficiently regulating the immune responses.These findings have three main biological significances. The first is that our experiments for the first time proved that apoptosis which has proceeded to some phase could be rescued. The current studies about the regulation of apoptosis mainly focus on the intracellular genetic regulation, including caspaseSx Bcl-2 family % p53< c-Myc and IAPs, which can promote or inhibit the process of apoptosis. However, up to now, no experiments could directly prove that apoptosis which has proceed to some phase could be rescued. Our experiments have addressed this question with direct evidence. Therefore, when the organism receives some transient injury, some cells will go into apoptosis. After the injury withdraws, some apoptotic cells will go on while some cells will be rescued and then exhibit their functions again. For example, if the heart or brainexperiences a transient ischemic event, it will recover and has normal functions. Perhaps rescue of some apoptotic cells contributes to this phenomenon.Secondly, the microenvironment plays a great role in the rescue of the apoptotic DC. It has been reported that the immune microenvironment plays a great role in the development and differentiation of DC and help to prevent the apoptosis of different type of cells. Our pre\ious study also showed that the microenvironment could drive mature DC to proliferate and fuether diefferentiate into regulatory DC. All the results prove that the microenvironment has the basis to influence the apoptosis of DC. According to our results, if part of the apoptotic DC could be rescued in vivo, it will regulate the immune response and accommodate with the characteristics of DC that they are rare in vivo but their functions are very important.Thirdly, the functions of the rescued DC exhibit negatively regulation of T cell proliferation. After rescued by ESSC, the proliferating DC obtained the regulatory functions, which was consistent with our previous study about the effect of ESSC on mature DC to drive them into regulatory DC, however, the rescued DC have different phenotype with the regulatory DC derived from the mature DC under ESSC. All the results suggest that the immune microenvironment might regulate DC at different states including maturation and apoptosisof DC into DC with regulatory functions so that they could downregulate the immune response and control it to the normal condition after DC complete their antigen-presenting functions and prime the immune response to some extent, thus avoiding the possible injury caused by the overactivated immune system.During our investigation of a suitable method to induce DC apoptosis, we initially administered the classical apoptosis-inducers FasL or agonistic anti-Fas antibody to induce DC apoptosis. But the results showed that FasL or anti-Fas antibody induced secretion of cytokines like IL-ip, resulting in DC maturation instead of DC apoptosis, Fas signaling induces DC maturation via the autocrined IL-1J3 dependent manner. Moreover, to investigate the mechanisms by which Fas signal acitvates DC, we detected the activation of ERK, NF-kB and caspase 1 in Fas-ligated DC. The results showed that ERK signaling pathway was involved in Fas-induced production of cytokines and DC maturation. Activation of NF-kB is one of the mechanisms responsible for the resistance of DC to Fas-induced apoptosis (Guo Z et al, Blood, 2004, 102, 4441-4447). Considering that chemokines, just like DC, are important link between innate and adaptive immunity, we investigated the non-apoptotic function of Fas signaling in DC to induce DC to secrete chemokines which could chemoattract T cells and neuotrophils both in vivo and invitro. They also could enhan functions of the attracted T cells and neutrophils, for examples, to promote the proliferation of antigen specific T cells and the phagocytosis of neutrophils. These results showed that Fas signaling links innate and adaptive immunity by promoting DC secretion of CC and CXC chemokines.The observations that Fas signaling induces DC maturation and secretion of chemokines have important biological significance. FasL is expressed on activated T cells, some tumors, some nonlymphoid cells of liver, small intestine and in sites of immune privilege such as testis, eye and placenta, whereas Fas is broadly distributed on many types of cells, including DC. Under different physiological or pathological conditions, these FasL-expressing cells will encounter DC. As potent antigen-presenting cells, DC, activated by innate stimuli and loaded with processed foreign antigen, migrate to regional lymph nodes and prime T cells. Activated T cells subsequently find their way back to the site of inflammation and exert effector functions to eliminate pathogens. During this process, FasL-expressing activated T cells will inevitably interact with Fas on DC. Our results show that as a result of this interaction Fas-ligated DC could secret more chemokines and recruit more naive T cells and promote their activation and proliferation, thus enhancing adaptive immunity. Furthermore, at the site of infection, as our results demonstrated, Fas-FasL interaction will recruite more activated T cells and phagocytes and enhance the endocytosis of neutrophils, strengthening the innate immunity.Furthermore, FasL expressed on stromal cells in immune privileged sites and tumor cells was once considered to be the main mechanism for an immune privileged environment and tumor immune escape. However, in recent years, the role of FasL in immune privilege and tumor escape has been re-evaluated. Increasing data support that FasL expression induces potent inflammatory responses in many tissues. Reports regarding FasL-transfected cells-induced inflammation focused mainly on the infiltration of neutrophils but researchers find now that the early nonspecific inflammatory responses induced by FasL-transfected cells promote tumor-specific T cells response, which is ultimately responsible for protective immunity For this to occur, T cell subsets, including naive T cells, must be recruited to the site of tumor inoculation following neutrophils infiltration by some chemokines. Which cells produce this panel of chemokines? Most former researches ignored these Fas-expressing DC cells. Our experiments indicate that the interaction of widely-distributed Fas-expressing DC with FasL-transfected tumor cells and subsequent secretion of chemokines by DC may play an important role in this process. Given the established role of neutrophils in T cells responses, our data clearly suggest that Fas-ligation on DC promotes innate...