PERFORIN
 

    Perforin is a 60-kDa cytotoxic protein (Spaner et al. 1999), which is stored in lytic granules on the surface of cytotoxic T cells. When a cytotoxic T-cell receptor recognizes antigen on the surface of a target (i.e infected) cell, perforin as well as other cytotoxic effector proteins, are released by local exocytosis and induce the target cell to undergo  apoptosis (Spaner et al. 1999; Alberts et al. 1994).

APOPTOSIS

    Cells can die in either of two ways. First, they can die due to physical or chemical injury or to membrane damage, which leads to necrosis or cell disintegration. The dead tissue is then taken up and degraded by phagocytic cells. Second, cells may undergo programmed cell death, also referred to as apoptosis, in which the cellular DNA is broken down into 200 base pair fragments and the cell is destroyed from within (Janeway et al., 1999). Neighboring phagocytic cells ingest the cell rapidly and prevent the release of cytosolic contents into the extracellular space. Thus, no inflammatory response is elicited and the DNA of the engulfed cell can be reused. For this reason, apoptosis is often referred to as the “quiet death” (Alberts et al. 1994).
    Cytotoxic T cells kill infected cells by inducing them to undergo apoptosis. The two main pathways utilized by cytotoxic T cells are the perforin/granzyme pathway and the Fas/Fas-L pathway (Figures a and b). There are, however, slower pathways such as those mediated by lymphotoxin and tumor necrosis factor (TNF), which are also involved in cytotoxic T-cell induced apoptosis (Spielman et al., 1998).
 

THE PERFORIN/GRANZYME PATHWAY

    When a cytotoxic T cell recognizes antigen on the surface of a target cell, it releases the contents of its lytic granules through a calcium-dependent process. These granules contain two major classes of cytotoxic effector proteins- perforin and proteases known as granzymes (Janeway et al., 1999). Perforin is released through exocytosis at the point of contact (Alberts et al., 1994) and polymerizes within the membrane of the target cell (Spaner et al., 1999), producing a cylindrical structure in the lipid bilayer that is lipophilic on the outside and hydrophilic down the length of its hollow center. Water and salts are then able to enter the cell through these pores, destroying the integrity of the target cell membrane (Janeway et al., 1999).
In addition to water and salts, granzymes (specifically granzyme A and B), which have also been released from the lytic granules, can now enter the target cell. In vitro experiments indicate that the perforin-dependent entry of granzymes precedes the DNA fragmentation and breakdown of the nuclear envelope associated with apoptosis.(Blink et al., 1999). More specifically, granzyme A and B are introduced into the target cell and activate the caspase family of proteases (Hashimoto et al., 2000). The caspase cascade eventually leads to the activation of caspase-activatable DNase (CAD) which can then enter the nucleus of the target cell and cleave the DNA into 200 base pair fragments. Prevention of granzyme translocation through treatment of target cells with caspase inhibitors, blocks these apoptotic events (Blink et al., 1999).

    Received permission to use this figure from Scientific American. Please visit there website at www.sciam.com
(a) apoptosis through nonsecretory Fas-Fas ligand interaction     (b) apoptosis through secretory mechanisms
 

Gene Knock-Out

    Mice that are perforin/Fas-L double-deficient suffer from severe autoimmune disease, indicating an interesting role for these two cytotoxic pathways in the regulation of cell-mediated tissue destruction. It appears that each pathway alone is capable of effectively regulating this type of tissue destruction, since neither perforin nor Fas-L single-deficiency produced a similar syndrome. The maintenance of homeostasis in the immune system is a novel function for perforin and one which seems to occur only in the absence of Fas-L. Spielman et al. hypothesize that cytotoxic T cells control their own down-regulation through a negative-feedback loop, in which they lyse the APCs containing the original antigenic stimulus. Continued survival of the APCs in the presence of activated T cells may result in constant restimulation and proliferation, eventually leading to severe tissue destruction by mechanisms independent of the perforin- and Fas-L-pathways. Thus, the negative-feedback loop is eliminated in perforin/Fas-L double-deficient mice and autoimmunity ensues (Spielman et al., 1998).
    These findings are supported by other recent data concerning the induction of vascular leak syndrome (VLS). Cancer patients treated with high doses of IL-2 suffer significant damage to their endothelial cells which eventually leads to toxicity characterized by dypsnea, ascites, weight gain and pulmonary edema. This toxicity is caused by VLS which involves increased capillary leak. Rafi et al. found that IL-2 upregulates the activity of perforin and Fas-L. In perforin knockout mice, there was no significant damage to endothelial cells and VLS was notably reduced. Fas-L does not appear to play a significant role in tissue damage within the lungs as Fas-L deficient mice exhibited no decrease in VLS in this area. However, perforin knockout mice exhibited a marked decrease in VLS in the lungs. Thus, though IL-2 has proven effective in the treatment of certain types of cancer, there must also be therapeutic intervention during treatment to prevent endothelial cell damage (Rafi et al., 1998).
    Mice who have had the perforin gene knocked-out are severely impaired in their ability to mount effective cytotoxic T cell responses to many, but not all, viruses. Alternatively, mice that are defective in the gene for granzyme B suffer a less profound deficit. This is most likely due to the fact that there are several genes encoding for ganzymes (Janeway et al., 1999).
    The action of cytotoxic T cells mediated by perforin- or Fas ligand dependent mechanisms are essential for donor T cells to prevent allogeneic bone marrow rejection. A recent study found that the ability to prevent graft rejection in mice was severely impaired by the absence of perforin and was completely eliminated in double mutants that had perforin-deficient and Fas-ligand-defective CD8 cells (Clarke, 1998).
 

Other Recent Studies on Perforin

    A recent study demonstrated the effect of perforin-mediated cytotoxic T cell function in neurological disease. Mice that were deficient for perforin were injected with Theiler's Murine Encephalomyelitis Virus (TMEW) (model for Multiple Sclerosis). These mice showed viral persistence in the central nervous system, demyelination in the white matter of the spinal cord, and chronic brain pathology. However, these mice revealed only minimal neurological defects as a result of demyelination. Mice with functional CD8+ T-cells and perforin molecules suffered comparable demyelination, but had severe clinical disease. Thus, this study indicates that perforin release by CD8+ T-cells may contribute to the induction of neurological disease following demyelination (Murray et al., 1998).
    Perforin has also been implicated in the pathogenesis of acute lung injury. Quantitative polymerase chain reaction (PCR) analysis revealed that the mRNAs for perforin were highly upregulated in the acute phase of acute respiratory distress syndrome (ARDS) following sepsis. This finding combined with other results suggest that the dual apoptosis pathway (perforin/granzyme) (Fas-ligand/Fas) is a likely contributor to the accumulation and activation of inflammatory cells in the lungs (Hashimoto et al., 2000).
    It appears that perforin/Fas-ligand double deficiency may also contribute to the expansion of macrophages, pancreatitis and, in females, infertility and hysterosalpingitis. Double deficient CD4 or CD8 cytotoxic T-cells are unable to lyse cognate-activated macrophages. Thus, they cannot mediate negative feedback regulation by lysis of antigen presenting cells and T cells continue to be activated. These findings provide insight into a possible homeostatic role for perforin in the immune system (Spielman, 1998).

                                                       WORKS CITED
 

Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., Watson, J. Molecular Biology of the Cell, 3rd ed. New York:
    Garland Publishing, Inc., 1994.

Blink, E., Trapani, J., & Jans, D. 1999. Perforin-dependent nuclear targeting of granzymes: A central role in the nuclear events
    of granule-exocytosis-mediated apoptosis? Immunological Cell Biology 77(3): 206-215.

Clarke, C. 1999. Absence of Perforin Affects Ability to Prevent Rejection. Blood Weekly: 1.

Hashimoto, S., Kobayashi, A., Kooguchi, K., Kitamura, Y., Onodera, H., Nakajima, H. 2000. Upregulation of two death
    pathways of Perforin/Granzyme and FasL/Fas in septic acute respiratory distress syndrome. American Journal of
    Respiratory Critical Care Medicine 161(1): 237-243.

Janeway, Charles A. Jr., Paul Travers, Mark Walport, and J. Donald Capra. ImmunoBiology: The immune system in health
    and disease, 4th ed. London: Elsevier Science, 1999.

Murray, P., McGavern, D., Lin, X., Njenga, M., Leibowitz, J., Pease, L., Rodriguez, M. 1998. Perforin-Dependent
    Neurologic Injury In A Viral Model of Multiple Sclerosis. Journal of Neuroscience 18: 7306-7314.

Rafi, A., Zeytun, A., Bradley, M., Sponenberg, D., Grayson, R., Nagarkatti, M., & Nagarkatti, P. 1998. Evidence for the
    Involvement of Fas Ligand and Perforin in the Induction of Vascular Leak Syndrome. The Journal of Immunology
    161(6):3077-86.

Spaner, D., Raju, K., Rabinovich, B., & Miller, R. 1999. A Role for Perforin in Activation-Induced T Cell Death in Vivo:
    Increased Expansion of Allogeneic Perforin-Deficient T Cells in SCID mice. The Journal of Immunology 162(2):1192-9.

Spielman, J., Lee, R., & Podack, E. 1998. Perforin/Fas-Ligand Double Deficiency is Associated with Macrophage Expansion
    and Severe Pancreatitis. The Journal of Immunology 161: 7063-7070.

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