J. John Cohen

Programmed cell death in the immune system

Publisher Summary This chapter discusses the mechanisms of programmed cell death (PCD). In many different examples of PCD, regardless of the cell type involved and regardless of the stimulus, the cells undergo similar biochemical and morphological events, and PCD often follows a final common pathway. Evidence for the existence of programmed cell death and cell death program is discussed. Programmed cell death is an old phenomenon that has awakened new interest. It can be demonstrated in various systems.. They all have in common nuclear disintegration, the morphology of apoptosis, DNA damage, and early recognition by phagocytic cells. It is likely that there is a death program that all cells follow when they embark on this pathway, even if the specific (or nonspecific) triggers are very different. There are various stages in a B cells life when it risks undergoing a programmed death.

Apoptosis in leukocytes

All cells of the hematopoietic system have finite life spans, shorter by far than that of the host. They end their lives by committing a form of cellular suicide or programmed cell death. The morphology of this process is considerably different from that of necrosis and is called apoptosis. Apoptotic cells undergo a stereotyped sequence of changes, including shrinkage and nuclear collapse. The cell is quickly recognized and eaten by a phagocyte, without the elicitation of an inflammatory response. Although most cells have specific triggers of apoptosis, the killer T cell seems able to induce apoptosis in any cell it recognizes. The process of apoptosis is regulated by cytokines, and may be modulated both in vitro and in vivo. J. Leukoc. Biol. 57: 2–10; 1995.

Hyperthermia Induces Apoptosis in Thymocytes

Mild hyperthermia (43 degrees C for 1 h) induces extensive double-stranded DNA fragmentation and, at a later time, cell death in murine thymocytes. The cleavage of DNA into oligonucleosome-sized fragments resembles that observed in examples of apoptosis including radiation-induced death of thymocytes. Following hyperthermia, incubation at 37 degrees C is necessary to detect DNA fragmentation, although protein and RNA synthesis do not seem to be required. Two protein synthesis inhibitors, cycloheximide and emetine, and two RNA synthesis inhibitors, actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, do not inhibit DNA fragmentation or cell death in heated thymocytes at concentrations which significantly block these effects in irradiated thymocytes. We have used this difference in sensitivity to show that the DNA fragmentation induced in thymocytes which are irradiated and then heated seems to be caused only by the heating and not by the irradiation.

Apoptosis: mechanisms of life and death in the immune system.

When normal cells die, it is almost always by the process of apoptosis, which is a morphologic description. Some cells die readily and are rapidly replaced; others live as long as we do. Because apoptotic morphology is similar from cell to cell, researchers have embarked on an intense search for a common underlying biochemical process. There is an apoptotic mechanism or cell death program, which is expressed in embryos and throughout life. Most tissues, and especially the skin, gut, and immune system, depend on well-ordered apoptosis and cell replacement. Apoptosis can fail to occur, as in the newly described autoimmune lymphoproliferative syndrome, or be turned on pathologically, as in degenerative diseases. We would like to induce apoptosis in dangerous cells or block it to preserve valuable cells. We need to know more about how apoptosis is regulated and what we can do to influence the process.

DNA Fragmentation in Targets of CTL: An Example of Programmed Cell Death in the Immune System

Our studies of T cell-mediated cytotoxicity grew out of an interest in the mechanism of programmed cell death. The death of cells in the metazoan body can be categorized functionally, morphologically and biochemically. On the functional level, cell death is often acceptable or desirable for the system; it is part of the design. Examples of this programmed cell death include morphogenetic death, which occurs during early development and helps shape organs and limbs; death in systems with a normal cell turnover, for example epithelia or polymorphonuclear neutrophils; and the involution of hormone-dependent tissues. Opposed to this functionally acceptable cell death is accidental death, such as might follow physical, chemical or anoxic injury, and certain bacterial and viral infections.

Growth factors rescue embryonic dopamine neurons from programmed cell death.

Poor survival of embryonic dopamine neurons is a primary problem limiting the value of neurotransplantation for Parkinsons disease. Several neurotrophic factors have been shown to promote dopamine neuron survival when used individually in culture. We have found that two peptides, insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF), have additive effects on cell survival when used in combination. These growth factors reduced the number of dopamine cells undergoing apoptotic cell death. The neurotrophic factors induced proliferation of astrocytes but not dopamine neurons. When cell proliferation was blocked by cytosine arabinoside, the beneficial effects of IGF-I and bFGF were abolished, suggesting that effects of the growth factors were mediated, at least in part, by factors associated with glia. These results indicate that growth factors in combination may prove useful for enhancing dopamine neuron survival for neurotransplantation.

Cell-mediated cytotoxic mechanisms

There are two competing, but probably really complementary, models for the mechanism of cell-mediated cytotoxicity. One depends upon contact-mediated transmembrane signaling, and the other on the exocytosis of toxic materials by the killer cell. There is exciting news on both fronts. Transmembrane signaling has been shown to involve the surface molecule Fas/APO-1 on targets and its ligand on cytotoxic T cells. The Fas ligand has been cloned, and is a member of the tumor necrosis factor family. The major cytolytic molecule in the exocytosis pathway is perforin; perforin knock-out mice have been produced, and they display many intriguing abnormalities. It has been a bumper year for cytotoxicologists.

Standard quantitative assays for apoptosis

The term apoptosis refers to a peculiar morphology of cell death. It is of special interest because it can be triggered physiologically (and pathologically), and it is regulated by the actions of specific gene products. Therefore, it can in principle be activated and suppressed by medical intervention. It thus is often important to determine whether cells are dying by apoptosis (or its less regulated counterpart, necrosis) and also to quantify the effect in a population of cells. Here the classic methods of apoptosis quantitation are described; they will be of particular use to those whose laboratories are set up for standard microscopical and biochemical techniques, who do apoptosis assays infrequently but wish them to be widely accepted and reproducible. A simple microscopic observation, using blue light illumination and a pair of fluorescent dyes, is recommended for most applications.

Identification of genes involved in programmed cell death

Three modes of activation of apoptosis are described: induction, in which new gene expression occurs after the stimulus is applied; transduction, in which gene expression is unnecessary at the time of stimulation; and release, in which apoptosis is activated by the inhibition of gene expression. Genes activated in the induction mechanism were identified by a process of subtractive hybridization, whereby newly transcribed messenger RNAs could be isolated. Progress in characterizing some of these genes is described. There are many difficulties and conceptual problems associated with such a gene cloning approach, but the results will be worth the effort.

Apoptosis and Its Regulation

There is only one popular way to be conceived, but a myriad of ways to die. We die from some external force, such as an accident or foul play, or else from “natural causes”. It is interesting how good the parallel is with the cells out of which we are made. All our somatic cells arise by the process of mitosis, regardless of their location in the body or their ultimate destiny. And these cells die by either of two processes, roughly equivalent to accidents and natural causes. Just as a pathologist will determine cause of death by examining the body, we distinguish the two forms of cell death by morphology1.