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Department of Biochemistry and Molecular Biology and Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48823
3To whom correspondence should be addressed. E-mail: fraker{at}msu.edu.
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ABSTRACT |
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 TOP ABSTRACT LITERATURE CITED |
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KEY WORDS: • apoptosis • autophagy • embryos • lymphopoiesis • zinc deficiency
For a number of decades, developmental biologists knew that apoptosis or programmed cell death (PCD)4 played a key role in the normal development of embryos. Removal of rudimentary tissues in the embryo via apoptosis was programmed by specific hormonal cues (1,2). The recognition that apoptosis also played vital roles in immune function began with the pioneering work of Wyllie (1), who showed that glucocorticoids (Gcs) were potent inducers of apoptosis in thymocytes in the 1980s. This discovery spawned over a decade of intense interest in apoptosis, with Gc-induced cell death of thymocytes becoming a classical system for investigation (1,2). Eventually, UV light, radiation, certain chemotherapeutic drugs, deficits in cytokines, and serum deprivation were also shown to be effective death cues, especially among cells of the immune system (2). More recently, the killing of malignant, aberrant, and virally infected cells by natural killer and cytolytic T-cells has been shown to rely on the initiation of apoptosis in the target cell (2).
As time progresses, additional roles for apoptosis have been identified. With regard to the nutritional sciences, it was recently shown that zinc deficiency (ZD) greatly accelerates apoptosis among pre T- and B-cells (3). ZD has also been shown to initiate apoptosis during development, altering embryogenesis (4). Although these studies were done in mice, they raise concern about the impact of cell death in humans who are deficient in zinc due to suboptimal diet or chronic disease (5). These studies also suggest that the role of apoptosis and other forms of cell death in nutritional imbalances is probably extensive. ZD is therefore a paradigm that begins to elucidate the key roles that apoptosis plays in nutritional imbalances and it is the prime focus of this review.
Zinc Deficiency Can Induce Apoptosis in Immature Cells of the Immune System. The fact that serum and cytokine deprivation induced PCD in certain cell lines and thymocytes led to our hypothesis that nutritional deficiencies might also induce apoptosis in vulnerable cells. Moreover, deficiencies in zinc and protein calorie malnutrition (PCM) cause elevated production of Gcs that initiate apoptosis in thymocytes (5–7). Because thymic atrophy and lymphopenia accompanied these deficiencies, it seemed probable that apoptosis might well play a key role in the disruption of lymphopoiesis in these two common forms of malnutrition. To that end a series of studies was begun to determine whether ZD could initiate apoptosis in immature T- and B-cells, thereby causing the reduction in peripheral lymphocytes that contributed to decreased host defense (5).
The study of lymphopoiesis, which is the study of the maturation, proliferation, and development of lymphocytes in the marrow and thymus, can be difficult. This is partially because of the heterogeneity of the bone marrow and difficulties faced when harvesting marrow from the bones of mice, which are small, or the marrow of humans, which can be invasive. Plus, there were several other unknowns regarding the impact of ZD on lymphopoiesis. It was not yet known whether precursor B-cells that develop entirely in the marrow of young adult mammals were as susceptible to apoptotic cues as precursor T-cells or thymocytes. Moreover, it was not known whether the moderate concentrations of endogenously produced Gc generated during stress and some forms of malnutrition were sufficient to induce apoptosis among cells of the immune system (5–7). Most of the pioneering studies were done with synthetic steroids, such as dexamethasone, which were used at pharmacological concentrations (1,2).
The above obstacles were overcome by the utilization of multicolor flow cytometry, which not only provided quantitative analysis of multiple populations of cells within the marrow and thymus, but also determined the degree of apoptosis-survival among specific subsets of cells at the same time (3,8–11). It was quickly found that a 30-d period of ZD among young adult mice substantially reduced the B-cell compartment of the marrow by 30 to 70%, depending on the degree of deficiency (9). Not surprisingly, the heaviest losses were among the pre B-cells, with substantial losses also noted in the immature B-cells. As we expected, there was reasonable survival of the more mature B-cells. However, the greater survival of some of the earliest B-cells, or so-called pro B-cells, was a surprise (9). An analogous pattern of death-survival was noted in the thymus (3). Large losses in both the proportion and the absolute numbers of pre T-cells were noted, but there was reasonable survival of pro T-cells. Both pro T- and pro B-cells are the earliest precursors of the lineages, so it seemed odd that they should survive.
The above mystery was resolved upon the discovery that not unlike more mature T- and B-cells, pro T- and pro B-cells expressed modest amounts of the so-called anti-apoptotic members of the Bcl-2 family that appear to prevent the release of cytochrome c from the mitochondria (12,13). The latter event initiates the caspase cascade, which is a hallmark of apoptosis that begins the digestion of the cell cytoskeleton, DNA, and other vital structures (12,13). Thus, these early lineage cells were afforded some protection against ZD and accompanying Gc. Conversely, pre T- and pre B-cells, which may generate anti-self and nonsense clones, do not have such protection, because they express little Bcl-2-like protein. The ability to readily induce apoptosis in the latter cells, which can generate anti-self or aberrant clones, is key to our survival (2,12). So, in fact, the heavy losses among pre T- and pre B-cells with greater retention of the earliest precursors during ZD correlated with their degree of protection or lack thereof to apoptosis.
Experiments were then designed to demonstrate that the huge loss of precursor lymphocytes discussed above was indeed due to apoptosis. In a separate series of studies, cells of the thymus and marrow were exposed both in vivo and in vitro to concentrations of Gc analogous to that found in ZD and PCM mice (8,10). Substantial amounts of apoptosis were induced in both pre T- and pre B-cells with better survival noted for the earliest members or pro T- and B-cells (5,8). The pattern of losses matched that observed in ZD mice. Moreover, pre B-cells of the marrow of human subjects were equally sensitive to Gc-induced PCD (10,14). These studies served to reaffirm the key role Gc plays in changing the distribution of cells of the immune system during ZD in both mice and humans and showed that the concentrations of endogenously produced Gc were sufficient to induce apoptosis in precursor cells (5). When we used the thymus as our indicator organ because it contains over 80% pre T-cells, we were subsequently able to demonstrate that ZD accelerated apoptosis in this population by 50 to 300% (3). This, of course, is a remarkable increase in apoptosis, which over time would clearly be a major contributor to the thymic atrophy and the reduced production of lymphocytes needed to replenish the peripheral immune system (Fig. 1).
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Morphological Changes Created by ZD-Induced Apoptosis. The vulnerability of immature and embryonic cells to ZD-induced apoptosis is affirmed by an important study from the laboratory of Keen and colleagues (4) (Fig. 1). Examination of rat embryos from ZD dams revealed substantial morphological changes in the optic and head regions and among neural crest cells. Within these tissues there was greatly increased activity of caspase 3, which is indicative of apoptosis (13). Thus, not unlike the cells of the immune system, certain cells and tissues of the ZD embryos were more inclined to undergo apoptosis than others. Therefore, the distribution and degree of susceptibility of apoptosis can vary among cells and tissues. Nevertheless, the morphological changes noted in these ZD embryos were substantial, raising renewed concerns about the impact of even modest deficiencies in zinc on the development of the human fetus.
Diversity of Tissues and Cells That Undergo Apoptosis When Zinc Is Low. A recent study of ZD in young rats revealed that the atrophy of the thymus and testes was accompanied by increases in apoptosis in these tissues (15). As ZD progressed, apoptosis was also noted in the liver and kidney. Collectively, these mouse model studies continue to raise concern about the impact of ZD-induced apoptosis on the integrity of human tissues and organs. The latter is of concern not only in the case of limited dietary zinc, but also for the numerous chronic diseases where suboptimal zinc and PCM are components of the etiology of the disease (5). For example, renal disease is accompanied by low plasma zinc. A recent study of peripheral blood mononuclear cells in a group of patients with renal failure noted a 300% increase in apoptosis in these cells (16). This study reaffirms the sensitivity of the lymphocytic cells of the immune system to ZD-induced apoptosis and demonstrates the value of mouse models in predicting mechanistic outcomes.
Low zinc cultures substantially increased apoptosis among Jurkat T-cells, 3T3 fibroblasts, and neuroblastoma cells (17). Although the use of zinc chelators to produce suboptimal zinc status in vitro can be problematic, glioma cells and human fibroblasts exhibited DNA strand breaks that could be a precursor of mutations and/or apoptosis in cultures containing low amounts of free zinc (18). A human monocytic cell line, THP-1, when maintained in zinc chelated cultures, exhibited reduced viability followed by apoptosis (19). Increased mRNA for the ZIP2 transporter was noted in these cells, suggesting that the cells were attempting to maintain zinc homeostasis. In sum, suboptimal zinc can induce apoptosis in a variety of types of cells. Identification of cells resistant to ZD-induced apoptosis could be very important to our understanding of how some cells maintain zinc homeostasis and survive the stress suboptimal zinc.
New Ways to Die. There are a variety of means whereby cell death can be brought about (Fig. 2). Of particular interest are the recent studies of autophagy, which appears to be a highly regulated process for the degradation of intracellular proteins and organelles. Autophagy is initiated by starvation or hormonal cues (20,21). Hepatocytes cultured for several hours in the absence of amino acids begin a process of partial self digestion that appears to be a means of providing the cell with needed amino acids to maintain gluconeogenesis under stressful conditions. Incubation of a variety of cells with glucagons also initiated autophagy. Amazingly, a membranous structure is generated from the endoplasmic reticulum that surrounds portions of the organelles in the cytoplasm. This membrane, which is called an autophagosome, eventually fuses with lysosomes to begin the digestion of intracellular proteins (Fig. 1).
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Additional attention is being given to necrosis, a form of so-called accidental death, which is presumed to be unregulated (21). Significant changes in osmolarity, pH temperature, oxygen tension, etc., can initiate necrosis. Unlike PCD where cells condense, preventing the release of viruses, defective genes, inflammatory factors, etc., into the surrounding environment, the necrotic cell swells and often ruptures, thereby releasing its contents. It can thereby exacerbate an already problematic situation. Moreover, it is not uncommon to note some necrosis among populations of cells dying apoptotically (21). Various cell lines made deficient in caspases eventually undergo necrosis in response to cues that normally initiated PCD (21). Whether necrosis proves to be more regulated than originally thought awaits further experimentation.
Conclusions and Perspectives. New forms of cell death will likely continue to emerge as we examine more carefully the impact of various stresses on cell survival. The studies discussed herein and the literature show that the type of stress in combination with its intensity and duration can affect the death path chosen (Fig. 2). The type of death initiated in a cell or tissue by a specific nutrient may have to be defined on a case-by-case basis. It may also prove overly simplistic to presume that only one type of death is initiated, especially in tissues that are substantially compromised by deficiencies in a variety of nutrients (Fig. 2) (21). It is also important to remember that as our understanding of the biochemistry of PCD advances, specific drugs are being developed that can modulate the different forms of apoptosis. This offers the possibility of reducing the tissue damage caused by PCD (21). Thus, the correct identification of the death pathways initiated by nutritional imbalances may become very important in the treatment and management of affected subjects. Clearly, the study of the interrelationships between nutritional status and cell death is in its infancy, with much remaining to be learned.
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FOOTNOTES |
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2 Manuscript received 16 September 2004.
4 Abbreviations used: Gc, glucocorticoid; PCD, programmed cell death; PCM, protein calorie malnutrition; ZD, zinc deficiency.
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LITERATURE CITED |
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TOPABSTRACTLITERATURE CITED |
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1. Wyllie, A. H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284:555-556.[Medline]
2. Cohen, J. (1991) Programmed cell death in the immune system. Adv. Immunol. 50:55-85.[Medline]
3. King, L., Osati-Ashtiani, F. & Fraker, P. (2002) Apoptosis plays a distinct role in the loss of precursor lymphocytes during zinc deficiency in mice. J. Nutr. 132:974-979.
4. Jankowski-Hennig, M., Clegg, M., Daston, G., Rogers, J. & Keen, C. (2000) Zinc-deficient rat embryos have increased caspase 3-like activity and apoptosis. Biochem. Biophys. Res. Commun. 271:250-256.[Medline]
5. Fraker, P. & King, L. (2004) Reprogramming of the immune system during zinc deficiency. Ann. Rev. Nutr. 24:277-298.[Medline]
6. Kubibidila, S., Yu, L., Ode, D. & Warrier, R. P. (1993) The immune response in protein-energy malnutrition and single nutrient deficiencies. Klurfeld, D. M. eds. Human Nutrition: A Comprehensive Treatise 8:121-157 Plenum Press New York, NY. .
7. DePasquale-Jardieu, P. & Fraker, P. J. (1980) Further characterization of the role of corticosterone in the loss of humoral immunity in zinc-deficient A/J mice as determined by adrenalectomy. J. Immunol. 124:2650-2655.[Medline]
8. Garvy, B., King, L., Telford, W., Morford, L. & Fraker, P. J. (1993) Chronic levels of corticosterone reduces the number of cycling cells of the B-lineage in murine bone marrow and induces apoptosis. Immunology 80:587-592.[Medline]
9. King, L. E., Osati-Ashtiani, F. & Fraker, P. (1995) Depletion of cells of the B-lineage in the bone marrow of zinc deficient mouse. Immunology 85:69-73.[Medline]
10. Lill-Elghanian, D., Schwartz, K., King, L. & Fraker, P. (2002) Glucocorticoid-induced apoptosis in early B-cells from human bone marrow. Exp. Biol. Med. 227:753-762.
11. King, L. & Fraker, P. (2002) Zinc deficiency in mice alters myelopoiesis and hematopoiesis. J. Nutr. 132:3301-3307.
12. Merino, R., Ding, L., Veis, D., Korsmeyer, S. & Nunez, G. (1994) Development of regulation of the Bcl-2 protein and susceptibility to cell death in B-lymphocytes. EMBO J. 13:683-691.[Medline]
13. Green, D. & Kroemer, G. (2004) The pathophysiology of mitochondrial cell death. Science 305:626-629.
14. Fraker, P. & Lill-Elghanian, D. (2004) The many roles for apoptosis in immunity as modified by aging and nutritional status. J. Nutr. Health Aging 8:129-136.
15. Yanagisawa, N. & Wada, O. (2001) Increased apoptosis in a variety of tissues of zinc deficient rats. Life Sci. 69:1639-1649.[Medline]
16. Weissgarten, J., Berman, S., Modar, R., Rosenberg, M., Rapoport, M., Cohen, M. & Averbukh, Z. (2002) Zn+2 metabolism affects apoptosis rate and proliferative responsiveness of PBMC from patients on chronic hemodialysis. Metabolism 51:1392-1396.[Medline]
17. Verstraeten, S., Zago, M., MacKenzie, G., Keen, C. & Oteiza, P. (2004) Influence of zinc deficiency on cell membrane fluidity in Jurkat, 3T3 and IMR-32 cells. Biochem. J. 378:579-587.[Medline]
18. Ho, E., Courtemanche, C. & Ames, B. (2003) Zinc deficiency induces oxidative DNA damage and increases p53 expression in human lung fibroblasts. J. Nutr. 133:2543-2548.
19. Cao, J., Bobo, J., Liazzi, J. & Cousins, R. (2001) Effects of intracellular zinc depletion on metallothionein and ZIP2 transporter expression and apoptosis. J. Leukoc. Biol. 70:559-566.
20. Yoshimori, T. (2004) Autophagy: A regulated bulk degradation process inside cells. Biochem. Biophys. Res. Commun. 313:453-458.[Medline]
21. Guimaraes, C. & Linden, R. (2004) Programmed cell death, apoptosis and alternate death styles. Eur. J. Biochem. 271:1638-1650.[Medline]
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