"This web page was produced as an assignment for an undergraduate course at Davidson College."
My Favorite Immunology Proteins: Fas and FasL
Fas is also named CD95 and Apo-1
The Role of Fas protein and Fas ligand in the Immune System:
Structure: The Fas protein and Fas ligand (FasL) are two proteins that interact to activate one of the best defined apoptotic (programmed cell death) pathways (Janeway et al., 2001). Fas and FasL are both members of the TNF (Tumor Necrosis Factor) family -- Fas is part of the transmembrane receptor family and FasL is part of the membrane-associated cytokine family. Both proteins contain cysteine repeats in their extracellular regions and death domains in their cytosolic tails to which adaptor proteins bind (Krammer, 2000).
Figure 1: Structure of TNF family receptors. TNF family molecules and receptors occur as trimers as seen in c. One subunit of a TNF receptor is seen binding to a monomeric TNF molecule in d (Janeway et al., 2001). Figure 8.33 from Immunobiology text.
Figure 2: Chime image of Fas Death Domain. Molecule 1DDF. (PDB, 2003; http://www.rcsb.org/pdb/cgi/explore.cgi?pid=219131048238273&page=0&pdbId=1DDF).
Pathway: When the homotrimer of FasL binds to Fas, it causes Fas to trimerize and brings together the death domains on the cytoplasmic tails of the protein (Janeway et al., 2001). The adaptor protein, FADD (Fas-associating protein with death domain), binds to these activated death domains and themselves bind to pro-caspase 8 through a set of death effector domains (DED). When pro-caspase 8's are brought together, they transactivate and cleave themselves to release caspase 8, a protease that cleaves protein chains after aspartic acid residues. Caspase 8 then cleaves and activates other caspases which eventually leads to activation of caspase 3. Caspase 3 cleaves I-CAD, the inhibitor of CAD (caspase activated DNAse), which frees CAD to enter the nucleus and cleave DNA. Small 200 basepair fragments of DNA in the nucleus are one of the characteristics of apoptosis and lead to death of the cell (Janeway et al., 2001).
Figure 3. The binding of Fas with FasL activates an apoptotic pathway that leads to cleaved DNA. The trimeric FasL induces trimerization of Fas which initiates this apoptotic cascade (Janeway et al., 2001). Figure 6.23 from Immunobiology text.
Other apoptotic pathways also lead to the activation of caspases and CAD. Granzymes secreted by CD8 Cytotoxic T cells activate CAD and other proteins activate other parts of the caspase cascade (Krammer, 2000). Although the Fas-FasL pathway is not unique in its activation of the caspase cascade, there is no pathway redudant with the Fas apoptotic pathway because when FADD is deleted, Fas induced apoptosis is blocked (Zhang et al., 1998).
Function in Apoptosis: Passive apoptosis occurs when cells are in the absence of survival factors, have irreparable damage, or are receiving conflicting signals (http://www.celldeath.de/encyclo/misc/deathrec.htm#PrtFour). Active apoptosis is important for the immune system and is involved in the negative selection of auto-reactive T cells, clonal deletion of self-antigen producing B-cells, and the death of lymphocytes after they have removed a pathogen (Osborne,1996). Death receptors of the TNF receptor family control active apoptosis and the binding of Fas and FasL is activated in a number of circumstances by the immune system
CD8 T cells and CD4 Th1 and Th2 cells produce different effector molecules and thus kill and/or effect different types of cells (Janeway et al., 2001). CD8 T cells (Cytotoxic T cells) express FasL upon recognition of a target cell and are thus able to kill target cells expressing Fas (Nagata & Golstein, 1995). Expression of FasL can be increased by interferon-gamma, which is synthesized in cells after viral infection and one of the main functions of CD8 T cells is to kill virus infected cells. Although it is believed that CD8 T cells most often kill their targets by releasing granule contents that contain perforin and granzymes, the activation of the Fas-FasL apoptotic pathway by CD8 T cells is also very important in peripheral immune responses to kill infected cells and to kill activated lymphocytes that have removed their pathogen and are no longer needed (Nagata & Golstein, 1995).
A small number of CD4 Th1 cells also express FasL and can kill cells expressing Fas. These CD4 Th1 cells also play an important role in killing activated lymphocytes (such as activated effector T cells) that are expressing Fas and thus augment this function that is performed by CD8 T cells (Krammer, 2000). The redundancy of this mechanism points to the importance of lymphocyte homeostatis, which will be again discussed with diseases caused by Fas mutants. Activation of mature effector B and T cells induces these cells to express Fas and thus they are killed by interaction with a cell expressing FasL or DAXX. Also, since activated T cells express FasL, they can kill activated B cells that are self-reactive by Fas-FasL apoptosis. CD4 Th1 cells also perform another role utilizing the Fas-FasL pathway. Macrophages activate CD4 Th1 cells, which express FasL and can kill macrophages expressing Fas (Janeway et al., 2001). Therefore, the CD4 Th1 cells kill macrophages that are chronically infected with intracellular bacteria, releasing the bacteria to be destroyed by other macrophages. The Fas-FasL apoptotic pathway is also utilized by CD4 T cells to kill self-reactive B cells that have become anergic (Janeway et al., 2001). After encountering soluble self antigen, a B cell becomes anergic. If this B cell encounters a CD4 T cell that is specific for the self antigen and expresses FasL, the two cells will bind via the FasL-Fas interaction and the B cell will be induced to undergo apoptosis (Janeway et al., 2001).
Therefore, the four main roles of Fas binding to FasL appear to be for CD4 T cells to maintain lymphocyte homeostasis (by killing activated effector T cells more so than B cells), kill macrophages infected with bacteria intracellularly, and kill anergic B cells and for CD8 T cells to kill some virally infected target cells (Janeway et al., 2001).
In addition, after T cells have survived positive and negative selection in the thymus, they are considered mature T cells and move to peripheral lymphoid organs in an attempt to encounter their antigen. But they still undergo selection in the periphery. T cells that bind to self antigens in the peripheral lymphoid tissues die through Fas-FasL mediated apoptosis (http://www.celldeath.de/encyclo/misc/deathrec.htm#PrtFour).
FasL has also been implicated in maintaining immune privileged sites such as the eye, testis, brain, joints, and pregnant uterus by inducing apoptosis in activated infiltrating leukocytes and lymphocytes that express Fas (Hammer et al., 2002). The immune system does not function in these certain areas of the body, immune-privileged sites, because immune responses cause inflammation which could cause irreparable damage to these tissues (Griffith et al., 1995). These immune-privileged sites express FasL and react to induce apoptosis immediately when activated immune cells expressing Fas enter the tissues (Griffith et al., 1995).
Although much has been learned about Fas and its interaction with FasL since the identification of Fas in 1989, there are still many aspects of its induction, expression, and function that remain debated and unknown. Some investigators initially thought that since Fas is expressed in the thymus and activated T cells express FasL, then the Fas-FasL pathway was probably the cause of apoptosis in negative selection of T cells in the thymus (Watanabe-Fukunaga et al., 1992). But then after studying Fas and FasL mutants in which negative selection of T cells in the thymus is not altered, some investigators changed their minds and decided that the Fas-FasL pathway is probably not involved in negative selection of T cells in the thymus (Nagata & Golstein, 1995). The current opinion is that the Fas-FasL pathway is not involved in negative selection, but Krammer et al. states that "negative selection might involve the Fas-FasL system when T cells encounter high antigen concentrations," so more research must be done in this area (Krammer et al., 2000). In a recent paper, Wood et al. states that natural killer (NK) cells express FasL and are involved in Fas-FasL apoptosis, which I found interesting, but he didn't elaborate anymore (Wood et al., 2003). There have also been many papers recently published that focus on the ability of various molecules to induce expression of Fas and FasL. The molecules range from interleukin 2 (IL-2) (Akashi & Kuwano., 2002) and interferon regulatory factor 4 (IRF-4) (Fanzo et al., 2003) to estrogen (Mor et al., 2003) and vitamin C (Perez-Cruz et al., 2003) and more information can be found on medline.
The Effects of Mutated or Deleted Fas
Mutations in the genes encoding Fas or FasL, which are both on human chromosome 10, lead to an excessive accumulation of lymphocytes, often T cells, that lack co-receptors and other proteins (http://www.ncbi.nlm.nih.gov/omim/). The T cells have presumably been activated, but failed to die. These mutations result in a condition called Autoimmune Lymphoproliferative Syndrome (ALPS) that is usually recessive, but the mutant phenotype has been seen in heterozygous humans. Some patients have mutations in the intracellular domain of the Fas gene which have a dominant-negative effect (Janeway et al., 2001). The symptoms and signs of ALPS are massively enlarged lymph nodes (lymphadenopathy) and increased numbers of double negative T cells. Other signs and symptoms are enlargement of the spleen (splenomegaly), decreased numbers of blood platelets (thrombocytopenia), anemia, skin rashes, and kidney dysfunction (http://www.ncbi.nlm.nih.gov/omim/). The mutations in Fas lead to the symptoms of ALPS by disrupting the death-domain interactions of Fas with the adaptor proteins FADD or DAXX and disrupts the ability of Fas-FasL interactions to cause apoptosis. A change in one Fas or FasL protein inhibits the trimer from forming and thus inhibits the apoptotic pathway (Janeway et al, 2001). Therefore, lymphocytes that are self-reactive and/or activated effector cells and should be triggered to undergo apoptosis remain alive.
[http://www.cc.nih.gov/ccc/aboutcc/backgrounder/BALPS8_1.html : Interesting story about discovery of ALPS disease]
Mutations in Fas (lpr-lymphoproliferation mutations) and FasL (gld-generalized lymphoproliferative disease mutations) genes were first observed and studied in mice (Krammer, 2000). In lpr mice, there is a splicing defect, which results in decreased expressed of Fas, while in gld mice, there is a point mutation in the C terminus that inhibits FasL to interact with Fas (Wood et al., 2003). Recessive mouse mutations lpr and gld, result in the accumulation of peripheral lymphoid cells and the formation of autoreactive antibodies that lead to lymphadenopathy, spenomegaly, nephritis, and arthritis, which are similar signs and symptoms to those observed in the human disease Systemic Lupus Erthematosus (SLE) (Rathmell et al, 1995).
Lupus (SLE) is is chronic inflammatory disease with periods of remission and relapse that affects multiple systems of connective tissue of the skin, joints, kidneys, lungs, heart, nervous system, blood vessels (http://www.ncbi.nlm.nih.gov/omim/). SLE is thought to be a failure of the regulatory mechanisms of the autoimmune system, since patients have increased levels of monocyte apoptosis and apoptosis eliminates lymphocytes that attack self tissues and produce autoantibodies. The cause of SLE is thought to be viral or mutations in a combination of various immune proteins and the Fas and FasL genes are thought to possibly play a role in the disease, through increased expression, although currently other genes such as those encoding immunoglobulin G Fc receptor II or IL-2 are thought to play more of a role (http://www.ncbi.nlm.nih.gov/omim/).
Accumulation of monocytes also leads to vasculititis, arteriosclerosis, and rheumatoid arthritis and thus defects in the Fas-FasL apoptotic pathway are also implicated and being studied in these diseases (Mor et al., 2003). Many other diseases including Hashimoto's thyroiditis (HT), toxic epidermal necrolysis (TEN), and Acute Lymphoblastic Leukemia (ALL) are thought to result from mutations in Fas or FasL (http://www.celldeath.de/encyclo/misc/deathrec.htm#PrtFour). ALL is believed to result from alternately spliced forms of Fas that lack intact transmembrane domains and therefore encode soluble forms of the protein that are not functional (Wood et al.,2003 ).
Diabetic vascular permeability results from Fas-FasL apoptosis of the retinal vasculature and results in diabetic retinopathy. Targeting of the Fas-FasL pathway may provide a treatment for this complication of diabetes (Joussen et al, 2003).
Excessive activity of the Fas system has been linked to the sensitivity of HIV and AIDS patients to Fas-mediated apoptosis and increased expression of Fas on their peripheral blood lymphocytes. Finally, tumors that express FasL are thought to suppress the immune response by killing tumor reactive immune cells (http://www.celldeath.de/encyclo/misc/deathrec.htm#PrtFour). In conclusion, mutations and deletions of the Fas and FasL gene lead directly to APLS, but are also thought to contribute to many other diseases.
Drugs that Bind to Fas
Since mutations in Fas and FasL lead to autoimmune disease, targeting these genes and receptors by inducing or blocking apoptosis may be an important therapy in treating autoimmune diseases (Zhou et al., 2002). Pharmaceutical companies are also targeting FADD and DAXX (Fas death domain associated protein), two adaptor proteins that bind to the death domains of Fas and themselves have death effector domains that activate the caspase cascade. Daxx is being advertised already as a drug target to treat conditions resulting from both insufficient apoptosis and excess apoptosis (container.pharmalicensing.com). Daxx activates the JNK kinase MAP3K5 and thus the MAP kinase pathway (http://www.ncbi.nlm.nih.gov/omim/). There are quite a few molecules that stimulate or inhibit the Fas-Induced apoptosis pathway, but none of these molecules interact directly with Fas besides FADD and DAXX. All of the other molecules bind to FADD, DAXX, or caspases.
Molecules that Inhibit the Fas-Induced Pathway
The Fas protein induces apoptosis by activation of pro-caspase 8 and downstream events include an increase in reactive oxygen species (ROS) and the release of pro-apoptotic factors from the mitochondria which leads to caspase 3 activation. Bcl-2 can block the activation of caspase-8 and caspase 3 and thus inhibit the Fas-Induced apoptotic pathway, but it does not affect Fas directly (Janeway, 2001).
Molecules that Stimulate the Fas-Induced Pathway
Corticotropin-releasing hormone (CRH) increases the expression of FasL on trophoblast and maternal cells at the fetal-maternal junction. CRH also effects the death of activated T lymphocytes through FasL which allows the fetus to implant and helps regulate the maternal immune toleration of the fetus. Regulation of the Fas-FasL interaction is thus crucial for pregnancy (Makrigiannakis et al., 2003). Also, the Fas pathway activates sphingomyelinase and ceramides which are known to stimulate apoptosis, but imipramine (sphingomyelin inhibitor) and caspase inhibitors (such as CrmA) prevent activation of the apoptotic pathway (http://www.celldeath.de/encyclo/misc/deathrec.htm#PrtFour).
Death Receptors: http://www.celldeath.de/encyclo/misc/deathrec.htm#PrtFour. Accessed on March 20, 2003.
OMIM: http://www.ncbi.nlm.nih.gov/omim/. Accessed on March 20, 2003.
Pharmalicensing: container.pharmalicensing.com. Accessed on March 20, 2003.
Akashi A, Kuwano K. (2002) "Fas-mediated lysis of target cell by IL-2 treated cytotoxic T lymphocytes. Kurume." Med J. 49:119-29.
Fanzo JC, Hu CM, Jang SY, Pernis AB. (2003). "Regulation of lymphocyte
apoptosis by interferon regulatory factor 4 (IRF-4)."
J Exp Med. 197:303-14.
Griffith TS, Brunner T, Fletcher SM, Green DR, Ferguson TA. (1995). "Fas ligand-induced apoptosis as a mechanism of immune privilege." Science. 270:1189-92.
Hammer A, Hartmann M, Sedlmayr P, Walcher W, Kohnen G, Dohr
G. (2002). "Expression of functional Fas ligand in choriocarcinoma."
Am J Reprod Immunol. 48:226-34.
Janeway, C., Travers, P. Walport, M., Shlomchik, M. Immunobiology: The Immune System in Health and Disease. New York, New York: Garland Publishing. 2001.
Joussen AM, Poulaki V, Mitsiades N, Cai WY, Suzuma I, Pak J, Ju ST, Rook SL, Esser P, Mitsiades CS, Kirchhof B, Adamis AP, Aiello LP. (2003) "Suppression of Fas-FasL-induced endothelial cell apoptosis prevents diabetic blood-retinal barrier breakdown in a model of streptozotocin-induced diabetes." FASEB J. 17:76-8.
Krammer, P. Dhein, J., Walczack, H., et al. (1994). "The Role of APO-1-Mediated Apoptosis in the Immune System." Immunological Reviews. 124:175-191.
Makrigiannakis A, Zoumakis E, Kalantaridou S, Mitsiades N, Margioris A, Chrousos GP, Gravanis A. (2003) "Corticotropin-releasing hormone (CRH) and immunotolerance of the fetus." Biochem Pharmacol. 65: 917-921.
Mor G, Sapi E, Abrahams VM, Rutherford T, Song J, Hao XY, Muzaffar S, Kohen F. (2003) "Interaction of the estrogen receptors with the Fas ligand promoter in human monocytes." J Immunol. 170:114-22.
Nagata, S., Goldstein, P. (1995) "The Fas Death Factor." Science. 267: 1449-1456.
Osborne, BA. (1996) "Apoptosis and the maintenance of homoeostasis in the immune system." Curr Opin Immunol. 245-54.
Perez-Cruz I, Carcamo JM, Golde DW. (2003) "Vitamin C inhibits FAS-induced apoptosis in monocytes and U937 cells." Blood.pre-published.
Rathmell JC, Cooke MP, Ho WY, Grein J, Townsend SE, Davis MM, Goodnow CC. (1995). "CD95 (Fas)-dependent elimination of self-reactive B cells upon interaction with CD4+ T cells." Nature 376:181-4.
Wood CM, Goodman PA, Vassilev AO, Uckun FM. (2003). "CD95 (APO-1/FAS) deficiency in infant acute lymphoblastic leukemia: detection of novel soluble Fas splice variants." Eur J Haematol 70:156-71.
Zhang J, Cado D, Chen A, Kabra NH, Winoto A. (1998). "Fas-mediated apoptosis and activation-induced T-cell proliferation are defective in mice lacking FADD/Mort1." Nature 392:296-300.
Zhou T, Mountz JD, Kimberly RP. (2002). "Immunobiology of tumor necrosis factor receptor superfamily." Immunol Res. 26:323-336.
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