This web page was produced as an assignment for an undergraduate course at Davidson College.
Click here to see an enlarged chime of the Fas death domain and the figure legend.
Fas is my favorite immunology protein because it initiates one of the most important pathways to programmed cell death, or apoptosis, in the body. Apoptosis is important in cell-mediated immune response, autoimmune tolerance, and cancer control. Fas is a surface receptor that is expressed throughout the body. The Fas ligand is expressed mostly on activated T cells, natural killer cells, and microglia, but is sometimes found on other cells of the immune system. When Fas binds to its ligand, a caspase cascade is initiated within the cell that eventually leads to its death (Maher et al. 2002). The first half of my website will give more details about the structure and function of the Fas protein in the immune system. The second half will explore the clinical aspects of Fas.
Fas (also referred to as CD95 or APO-1) is a 48-kDa (319 amino acids) member of the TNF/nerve growth factor receptor family. It has three regions: a N-terminal extracellular domain, a single transmembrane domain, and a cytoplasmic domain. The extracellular domain has three repeats of a cysteine-rich subdomain common to all members of the TNF receptor family. The extracellular domain also contains two glycosylation sites where sugars probably bind. The transmembrane domain is 17 amino acids long (Itoh et al. 1991). Within the cytoplasm lies an abundantly charged region known as the death domain. Figures 1 and 2 (click to see enlarged images and figure legend) show models of the Fas death domain. The structure consists of six antiparallel a helices capable of self-association. This capability is extremely important to internal cell signaling following Fas binding, as you will see below.
When a cell expressing Fas encounters another cell expressing Fas ligand, the receptor molecules trimerize in order to bind the ligand. Trimerization of the death domains in the cytoplasmic regions of each molecule is the signal for activation. After activation, the death domains are capable of interacting with each other, which leads to the recruitment of FADD, or Fas-associated death domain protein. In addition to the death domain, FADD has a death-effector domain (DED) capable of recruiting and splicing procaspase-8 into its active form, caspase-8 (Krammer 2000). The concentration of caspase-8 determines which of two paths are taken towards apoptosis.
If caspase-8 levels are high, it directly cleaves caspase-3 (Maher et al. 2002). Caspase-3 inactivates the inhibitor of CAD (caspase-activatable DNAse). CAD is then free to enter the nucleus and cleave DNA into lengths of approximately 180 base pairs (Ju, Matsui, and Ozdemirli 1999).
If caspase-8 concentration is low, it truncates Bid into tBID, a form capable of releasing cytochrome c from the mitochondria. Cytochrome c interacts with proteins such as Apaf-1, dATP, and procaspase-9 to produce active caspase-9. Caspase-9 then cleaves caspase-3, which activates CAD in the same way as it does in the first pathway. Apoptosis inhibitors Bcl-2 and Bcl-XL block apoptosis through the BID-mediated pathway but not the direct (caspase cascade) pathway (Maher et al. 2002).
Another group of inhibitors, known as FLIPs (FLICE-inhibitory proteins-- caspase-8 used to be called FLICE), contain two DED domains capable of binding to the Fas--FADD complex. In this way they prevent the recruitment of caspase-8 and inhibit both pathways to apoptosis. FLIPs were first identified in a class of herpes virus, but two human homologues have recently been identified (Krammer 2000).
Figure 3 (at left) summarizes the two Fas-mediated apoptotic pathways. Click on the image to see a larger version and the figure legend.
Role in Immune Response
Cytotoxic T cells and natural killer cells are responsible for destroying cells infected with intracellular cytoplasmic pathogens and cancer cells. It is important that these cells die by apoptosis so that the pathogen does not survive. Necrosis, which occurs when a cell dies due to infection or other types of damage, allows the pathogen to safely exit the cell and infect others. During apoptosis, CAD cleaves DNA and toxic proteases are released into the cytoplasm. The toxic environment sacrifices the cell but usually kills the pathogen with it. This pathway is initiated through Fas in conjunction with the perforin/granzyme pathway.
Both methods of inducing apoptosis are activated when the T cell receptor binds its specific antigen, at which point the cell proliferates and FasL, perforin, and granzyme are expressed. When the active T cell encounters its antigen again, FasL stimulates the target cell to began a caspase cleavage cascade as described above (Nagata and Golstein 1995). Perforin attacks the cell by creating a pore in the nuclear membrane. Granzymes then diffuse through the pore into the cell cytoplasm, where they are capable of activating caspases in a similar way to the Fas pathway (Darmon, Nicholson, and Bleackley 1995). Whether there is a preference for one method or another depending on the infected cell type or pathogen has not been determined. A similar mechanism occurs during Fas-induced apoptosis by natural killer cells, although the details are less clear ( Nagata 1997).
Role in Immunoregulation
The immune system must be tightly controlled in order to prevent excessive tissue damage and cell accumulation. Developing T cells in the thymus must be destroyed if they do not produce a functional T cell receptor or are unable to bind with the right affinity for self MHC class I molecules. After infection, lymphocyte levels must return to normal, meaning that many of the newly formed effector cells will die. Some areas of the body, such as the ovaries, testis, and retina, cannot survive the damage they would sustain as a result of an inflammatory immune reaction. These areas are not subject to immune responses, and for that reason are known as immune privileged.
The Fas pathway has been implicated in all of these areas of immune regulation. A Fas knockout mouse strain, known as lpr (lymphoproliferation) has been developed that shows significant defects in these areas. Another strain expresses the receptor, but a point mutation produces a misfolded form incapable of transducing signals. The mutant strain is referred to as lprcg. Both the knockout and the mutation produce similar effects. These include: excessive numbers of CD4-/CD8- T cells, enlarged spleen and lymph nodes, and low red blood cell and platelets due to autoimmune reactions (Kimura and Matsuzawa 1994).
Similar effects are observed in patients with autoimmune lymphoproliferative syndrome (ALPS). Most patients are heterozygous for a mutant allele that disrupts the a3-helix of Fas. The mutation results in decreased recruitment of FADD and caspase-8. It is not fully understood why the mutant allele would inhibit the functional allele (loss of function is >50%), but it has been suggested that the Fas signaling complex may require that some components be preassembled, allowing the unfolded protein to interfere with FADD binding (Martin et al. 1999).
CD4-CD8- T cells are immature cells that have not produced a functional b-chain receptor. Normally they die when they don't receive a survival signal during positive selection. Their presence in the Fas knockout indicates that Fas must play a role in killing cells that don't pass positive selection (Krammer 2000). After positive selection, thymocytes must undergo negative selection, at which point the cells that are extremely autoreactive are killed. At first, scientists did not think that Fas played a major role in negative selection because T cell receptors in lpr mice are not excessively autoreactive (Kimura and Matsuzawa 1994). But in fact the process is Fas independent only at low antigen concentrations. lpr mice were capable of deleting highly reactive cells in the presence of small amounts of antigen but the cells survived in the presence of high antigen concentrations. Normal mice deleted reactive T cells at all antigen concentrations (Kishimoto, Surh, and Sprent 1998).
The enlarged spleen and lymph nodes associated with the Fas mutation occurs as a result of the accumulation and survival of lymphocytes after an immune response. The ways that Fas causes T cell apoptosis are better understood than the method of B cell apoptosis. Normally, activated T cells proliferate in response to IL-2, which is required for clonal expansion. But IL-2 also sensitizes T cells to Fas, making it easier for them to be targeted after the pathogen has been removed. There is also evidence that activation stimulates T cells to secrete FasL, which acts on the secreting cell and nearby sensitive cells expressing Fas. In this way, activation regulates itself. Without Fas, the signal to proliferate eventually turns off but the signal to die is never received. Surviving effector cells have a high propensity towards becoming self-reactive and may be the source of autoimmune symptoms (Krammer 2000).
There is a lot of controversy in the scientific community about the role of Fas in immune privilege. Some evidence has shown that Fas-expressing immune cells that enter the testis and retina are killed in a FasL-dependent process (Griffith et al. 1995), but other data suggest that FasL expression promotes inflammation and tissue rejection. It is not known whether lpr mice lack the ability to kill off T cells in the testis. Testis expressing FasL survived indefinitely after transplantation into normal mice (Bellgrau et al. 1995), but testis from gld mice (which do not produce FasL) were quickly rejected. Other research shows that FasL activates a granulocyte response that speeds the rejection process (Allison et al. 1997). Research in this area is extremely applicable to specific immune suppression during organ transplantation and cancer research. Many cancers express FasL, which prevents active T cells from attacking the tumor. If we can discover what factors influence whether FasL induces rejection or immune privilege, it may develop into a powerful mechanism for targeting cancer and protecting transplants (Maher et al. 2002).
Click here for the Autoimmune lymphoproliferative syndrome (ALPS) homepage.
With the exception of Fas-specific antibodies and the FasL, few drugs have been created that bind specifically to the Fas molecule. There are several drugs and substances that interact with the Fas signaling pathway. Some of them, including FLICE, Bcl-2, and Bcl-XL have been described above. Non-steroidal anti-inflammatory (NSAID) drugs have also been shown to initiate apoptosis through the Fas signaling mechanism, and may be useful in leukemia treatment. NSAIDs also kill colon epithelial cancer cells through an unrelated pathway (Han et al. 2001). There is much anticipation about application of apoptosis research to cancer therapy, transplant rejection, and even AIDS.
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Davidson College Immunology
taught by Dr. Malcolm Campbell