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15
Iron Metabolism, Microbial Virulence, and Host Defenses

Gerald T. Keusch1

Introduction

The relationship between iron nutritional status and susceptibility to infection, mediated by the effects of iron on the host and the pathogen, has become a topic of controversy in recent years. This controversy has followed the clear demonstration that iron is removed from the circulation into metabolically inaccessible forms during acute infection and the understanding that iron is required by both host and pathogen for survival and cellular replication. This documented ''iron withholding'' by the host has been seen by proponents of the so-called nutritional immunity hypothesis as evidence to suggest that it is of utility to host defense, providing a means to control infectious agents by limiting pathogen multiplication. The extension of this hypothesis has been that giving iron may be clinically harmful, either during an acute infection or as a supplement to control nutritional anemia. There is another camp, however, which believes that iron deficiency impairs immune function and increases susceptibility to infection.

1  

Gerald T. Keusch, Division of Geographic Medicine and Infectious Diseases, Tupper Research Institute, New England Medical Center, Boston, MA 02111



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--> 15 Iron Metabolism, Microbial Virulence, and Host Defenses Gerald T. Keusch1 Introduction The relationship between iron nutritional status and susceptibility to infection, mediated by the effects of iron on the host and the pathogen, has become a topic of controversy in recent years. This controversy has followed the clear demonstration that iron is removed from the circulation into metabolically inaccessible forms during acute infection and the understanding that iron is required by both host and pathogen for survival and cellular replication. This documented ''iron withholding'' by the host has been seen by proponents of the so-called nutritional immunity hypothesis as evidence to suggest that it is of utility to host defense, providing a means to control infectious agents by limiting pathogen multiplication. The extension of this hypothesis has been that giving iron may be clinically harmful, either during an acute infection or as a supplement to control nutritional anemia. There is another camp, however, which believes that iron deficiency impairs immune function and increases susceptibility to infection. 1   Gerald T. Keusch, Division of Geographic Medicine and Infectious Diseases, Tupper Research Institute, New England Medical Center, Boston, MA 02111

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--> This chapter addresses the physiology of iron metabolism by host and pathogen, discusses the immunological data, reviews the clinical studies in humans, and draws conclusions about the benefits and risks for military personnel, particularly premenopausal women, of giving iron supplements. Iron Metabolism Iron is a highly reactive metal able to generate oxygen free radicals that are toxic to cells. However, the aerobic nature of the earth creates a problem, because oxygen is also needed for life. Because iron can shift between the ferrous and ferric states and can readily and reversibly bind with oxygen, while containing it in a nonreactive state, iron-containing proteins have been selected as the predominant carrier of oxygen. In the environment, the problem of reactive free oxygen has been solved by converting inorganic iron to oxyhydroxide polymers, which reduces the amount of uncomplexed ferric (Fe+++) in solution at biological pH to tolerable levels (< 1018 M). In the mammalian host, iron is present predominantly as protein complexes, including transport, storage, enzyme, and oxygen transporter systems, primarily as heme, iron-sulfur proteins, and ferritin intracellularly, or bound to extracellular transport glycoproteins such as transferrin in serum or lactoferrin on mucosal surfaces. Mammals have overcome the environmental restrictions and biological imperatives of bound, insoluble iron by developing an effective iron acquisition system able to compete with hydroxyl ion for ferric iron. Iron absorption, transport to and into cells, and storage are closely regulated by systems that sense the amount of available free iron and rapidly respond to maintain iron homeostasis. This mechanism has been essential, because iron is the most abundant transition metal in humans, amounting to approximately 4 g in adult males, with about 1 g in storage forms. In young adult women, the amount of storage iron is considerably reduced to around 300 mg because of continuing iron losses during menstruation. Iron balance is tightly controlled, amounting to only 1 to 2 mg lost per day in men, but more in menstruating females, which is why iron-deficiency anemia is common among adult women. In addition to oxygen transport, iron is essential for many physiological processes, as iron metalloproteins are able to accept electrons from various donors, to shift oxidation state, and to participate in redox reactions and hence serve in the electron transport chain in the form of cytochromes and mitochondrial iron-sulphur proteins (Griffiths, 1987). Many iron metalloproteins are enzymes, including enzymes involved in oxygen metabolism itself (catalase, peroxidase, superoxide dismutase), as well as flavoproteins such as xanthine oxidase and dehydrogenase; NADH/NADPH dehydrogenases; amino acid hydroxylases; and a key enzyme for DNA synthesis, ribonucleotide reductase.

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--> Thus, to limit iron-mediated catalysis of oxygen to produce extremely reactive oxygen radicals such as the hydroxyl radical, OH; which is highly toxic to cell membranes and can totally degrade DNA, virtually all iron in mammalian hosts is tied up with proteins, and the "free iron" pool is maintained at exceedingly low levels. The plasma-iron binding protein, transferrin, and a related protein, lactoferrin, released from the secondary granules of polymorphonuclear neutrophils (PMNs) (and also present in milk), keep the circulating free iron concentration close to zero. A major function of iron-transferrin is to provide a continuous source of iron for the rapid enzyme turnover, such as demonstrated by DNA synthesis, because the rate limiting enzyme for the production of nucleotides, ribonucleotide reductase is required for DNA synthesis. Uptake of iron from transferrin to the cell follows the binding of iron-transferrin to the transferrin receptor (TfR), a transmembrane protein for receptor-mediated endocytosis of iron-transferrin (Pollack, 1992). Transport of the TfR-iron complex into an acidified endosome results in the release of iron, with recirculation of the apotransferrin-TfR complex to the cell surface where the apotransferrin is released (Harford et al., 1990). If iron is taken up in excess, it is rapidly complexed within the cell by ferritin, a large, hollow, spherical protein that can accumulate large amounts of iron (Theil, 1987) and modulate iron-mediated reactions under conditions of excess intracellular iron (Joshi and Zimmerman, 1988). Ferritin is present in small amounts in the circulation and mirrors iron nutrition, with low levels found in people with iron deficiency and high levels in people with normal amounts of iron. Thus, serum ferritin serves in the diagnosis of iron-deficiency anemia (Worwood, 1986), and every μg per liter is equivalent to 8 to 10 mg of storage iron (Bothwell, 1995). Transport and storage of iron are finely tuned, as both transferrin-receptor and ferritin synthesis are coordinately and reciprocally regulated by iron concentration (Johnson et al., 1983; Klausner et al., 1993). When iron availability is low, TfR synthesis increases and ferritin synthesis decreases, with the reverse happening when iron availability is high. The mechanism by which this occurs is posttranscriptional and involves regions of the messenger RNA (mRNA) for ferritin and TfR able to bind a stem-loop region known as the iron-responsive element (IRE) located in the 5' and 3' regions, respectively. Specific iron-binding proteins that recognize these IREs, known as IRE-binding proteins (IRE-BP), are the effectors. The ferritin IRE-BP serves as a translational repressor of the ferritin gene (Leibold and Munro, 1988). That is, binding of the IRE-BP to the ferritin IRE blocks the translation of ferritin mRNA. The TfR IRE-BP operates by controlling mRNA half-life (Owen and Kuhn, 1987) via a more complex mechanism, involving the IREs and additional iron-independent "instability elements" affecting rapid degradation of the mRNA transcript (Harford and Klausner, 1990). When IRE-BP binds to the IRE, the instability element is inhibited, mRNA half-life increases, and TfR synthesis increases.

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--> Iron Acquisition by Microbes Because of the tightly regulated availability of iron in the human, microorganisms must compete with the host for iron, with a few exceptions (for example, Lactobacillus spp., which use manganese and cobalt instead) (Archibald, 1983). In fact, in most instances, microbes use systems of acquisition and transport of iron that are analogous to those of humans, because they too both require iron and a detoxification mechanism to prevent iron-mediated damage. These goals can be realized in several ways: Find another, less-toxic biocatalyst (e.g., Lactobacilli), which uses manganese and cobalt instead of iron. Make Fe+++ more soluble and easily transported by reduction to Fe++, a strategy adopted by several organisms (Bifidobacterium bifidum, Legionella pneumophila, Streptococcus mutans, and Streptomyces cerevisiae ). Produce chelators to bind ferric iron and transport proteins to safely obtain and use the metal, a solution adopted by most microbes. Produce receptors for iron protein that is bound by host in the form of transferrin, lactoferrin, or heme (e.g., pathogenic Neisseria, Hemophilus influenzae, Helicobacter pylori, Vibrios, and Yersinia). Develop multiple mechanisms to obtain and transport iron safely. These may be turned on in sequence as the conditions of iron restriction become more severe, thus allowing survival under different conditions of environmental stress conditions and protecting against mutational loss of the survival advantage. A few organisms (for example, Legionella pneumophila, Streptococcus mutans, or the yeast Saccharomyces cervisiae) reduce ferric iron and transport the more soluble, less toxic ferrous form of the metal. Most, however, simply make ferric iron-specific, high-affinity chelators (siderophores) (Guerinot, 1994) that effectively remove iron from host iron-binding proteins, even from ferritin (Zahringer et al., 1976), which means that intracellular storage is not a safe strategy to withhold iron from intracellular pathogens. Moreover, in at least a few known cases, certain microorganisms obtain iron directly from host sources, for example Neisseria meningiditis or N. gonorrhoea and Hemophilus influenzae, which make human-like transferrin receptors to transport iron-transferrin just as the host cells do (Martinez et al., 1990). The paradigm for iron acquisition and best-studied example is Escherichia coli. Under conditions of restricted iron availability, when a pathogen enters a human, the microbe rapidly and coordinately derepresses a set of genes responsible for the synthesis of iron acquisition and transport proteins that constitutes a system of iron chelators, outer membrane iron-siderophore receptors, and periplasmic and inner membrane transport proteins. This system is complex, involves multiple structural and regulatory genes and proteins, uses iron concentration as the signal, and is highly efficient (Neilands, 1995). It

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--> includes several types of chemically distinct siderophores (catechol enterobactins, hydroxamates such as aerobactin, and diverse molecules such as ferrichrome), all of which are characterized by an extremely high affinity for iron and a general similarity in the transport mechanisms. For example, the association constant of enterobactin for iron at neutral pH is, astoundingly, 1052, which explains why it can strip iron from transferrin, for which the association constant is "only" 1036. Certain Enterobacteriaceae can use siderophores made by other organisms, for example the fungal products ferrioxamine B and ferrichrome. In fact, the methane sulfonate salt of deferrated ferrioxamine B is used clinically as an iron chelator drug, Desferal, to treat iron overload states. Finally, when the iron-siderophore complex reaches the cytoplasm, iron is released from ferric enterochelin by an esterase or from aerobactin or citrate siderophores by reduction mediated through flavin reductases (ferri-siderophore reductase). Some organisms produce and use several different siderophores. In some instances, there is a stepwise synthesis of distinct chelators of increasing affinity for iron as the iron concentration is reduced, which suggests a carefully evolved strategy of iron acquisition that may confer competitive advantages in vivo (Sevinc and Page, 1992). In contrast, in an anaerobic environment, a specialized system to solubilize iron may be unnecessary because reduced ferrous iron is both available and soluble. A ferrous iron transport gene, feoAB, has recently been cloned from E. coli (Kammler et al., 1993), suggesting that organisms with an anaerobic ecological niche may have specialized systems for taking up reduced iron. This assumption is consistent with the finding that feo mutants are worse colonizers of the mouse gut than the parental wild type E. coli (Stojiljkovic et al., 1993). Some organisms, for example pathogenic Neisseria, do not make siderophores, albeit they can use exogenously supplied chelators (West and Sparling, 1985; Yancey and Finkelstein, 1981). N. gonorrhoeae and N. meningitidis obtain iron from transferrin, whereas nonpathogenic strains are inhibited in the presence of transferrin (Michelsen and Sparling, 1981; Michelsen et al., 1982). These pathogens produce a transferrin receptor that specifically binds human transferrin and directly obtains iron in this manner (Schryners and Morris, 1988). Many iron-regulated bacterial proteins are regulated at the transcriptional level by a common gene, fur, which encodes the iron-binding Fur protein. When iron is readily available, Fur binds the metal, which allows it to recognize a sequence with dyad symmetry in the promoter region of iron-regulated genes known as the "iron" or "Fur box." The iron-Fur complex acts as a repressor of the gene and stops the synthesis of the gene product. Under conditions of iron deprivation, Fur does not bind and the iron-regulated proteins are made. In this manner, the multiple genes involved in iron acquisition can be turned on together by this single mechanism. Fur proteins that recognize the consensus iron-box sequence have been sequenced from Shigella spp., Yersinia pestis, Vibrio cholerae and V. vulnificus, Pseudomonas aeruginosa , and Neisseria spp.,

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--> and a similar protein in Corynebacterium diphtheriae, DtxR, plays a Fur-like role in this organism. It should be noted that the Fur system controls a number of proteins other than those involved in iron uptake, including carbon utilization pathway proteins, superoxide dismutase, acid adaptation responses, and others. Iron regulation of gene transcription and translation is not just an in vitro phenomena; iron-regulated proteins are also made by bacteria growing in vivo in mammalian hosts, including clinical isolates obtained from human urinary and respiratory tract infections as well as from experimental animal infections using human pathogens (Camilli et al., 1994; Chart et al., 1988; Shand et al., 1985). Moreover, a number of virulence properties of microbes are iron regulated in vitro and are specifically turned on by low iron availability (Table 15-1). This mechanism is consistent with the generally held view that virulence genes are preferentially turned on in vivo in order to ensure biosynthetic economy of the organism. That is, to enhance the survival of pathogenic microbes outside of the host, the organism does not waste energy and substrate making things it does not need. The clear evidence that pathogens adapt to low iron availability, that they have developed intricate iron acquisition mechanisms, and that they use iron levels as the signal for transcriptional regulation of these systems raises doubt about the validity of the iron-withholding hypothesis of nutritional immunity (Kontoghiorghies and Weinberg, 1995). Given the existence of these iron-regulated systems for iron acquisition, it may be reasonable to believe that iron deficiency or acute phase shifts of iron from the circulation to intracellular storage sites during infection will restrict microbial acquisition of iron in vivo. In the context of biologic regulation and the postulated role of iron in the TABLE 15-1 Iron Regulated Bacterial Virulence Factors Organism Virulence Factor Escherichia coli Aerobactin, Shiga toxin-1, α-hemolysin Shigella dysenteriae type Shiga toxin Serratia marcescens Hemolysin Vibrio cholerae Iron-regulated gene A (IrgA) Vibrio anguillarum Anguibactin Yersinia spp. Iron-regulated outer membrane proteins Neisseria gonorrhoeae Transferrin-binding protein 1 and 2 Pseudomonas aeruginosa Exotoxin A, elastase, protease Corynebacterium diphtheriae Diphtheria toxin

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--> nutritional immunity hypothesis, iron is required by the host (as well as the microbe) and its uptake and distribution is so finely regulated by such a complex mechanism, that nature would chose a simple single solution (for example, iron withholding) as a major host defense against most infections. Effects of Iron Deficiency on Immune Function Because iron is as essential for mammalian ribonucleotide reductase as it is for the microbial enzyme, it is not surprising that transferrin-iron is needed for clonal expansion of lymphocytes in immune responses (Kay and Benzie, 1986; Philips and Azari, 1975). In fact, iron uptake must precede DNA synthesis (Kronke et al., 1985), and therefore, transferrin receptors (CD71) must be expressed as T-lymphocytes become activated in response to interleukin (IL)-2/IL-2 receptor interactions (Brock and Rankin, 1981; Neckers and Cossman, 1983). Dependency on the uptake of transferrin iron has been reported for B-cell proliferation and the mixed lymphocyte reaction (Futran et al., 1989; Kemp et al., 1987). However, B-cells may have larger iron storage pools than T-cells and may be more resistant to iron-limiting conditions, for example in the presence of antitransferrin receptor antibody plus the iron chelator desferrioxamine (Kemp et al., 1987). Among T-cells, T-helper (Th)1 cells are much more sensitive than Th2 clones to the intracellular iron depletion produced by the combination of antitransferrin receptor antibody plus desferrioxamine (Thorson et al., 1991). Human peripheral blood mononuclear cells still respond to phytohemagglutinin (PHA) in iron-depleted medium (Taylor et al., 1988), and it is necessary to chelate intracellular iron with desferrioxamine to block this (Golding and Young, 1995), providing further evidence of iron stores and efficient uptake systems in activated lymphocytes. Chronic iron deficiency would deplete these stores, because a source of diferric-transferrin to replenish the intracellular iron pool is lacking. In humans with iron-deficiency anemia, a decrease in total CD3-positive and CD4-positive lymphocytes, B-lymphocytes, and K-cell activity has been reported, with a significant recovery of all but the K-cell activity with repletion of iron (Santos and Falcao, 1990). Similar findings are reported in mice that were made iron deficient, with reduced density of T-lymphocytes in the thymus and spleen, but no changes in lymphocyte subpopulation ratios (Kuvibidila et al., 1990). This study demonstrated no alteration in the serum or thymic concentration of the thymic hormone, thymulin, which is involved in T-lymphocyte differentiation. Sufficient data exist to document that iron deficiency is associated with diminished, delayed-type skin test reactivity to recall antigens (Joynson et al., 1972; Krantman et al., 1982; Macdougall et al., 1975). In vitro mitogen-stimulated lymphocyte proliferation is also impaired (Fletcher et al., 1975; Sawitsky et al., 1976). Although some investigators have found no effects (Grosch-Warner et al., 1984; Gupta et al., 1982), negative in vitro studies could be confounded by rapid repletion of cellular iron from the culture medium. In

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--> contrast, B-cell number and function, measured by the antibody response to a strong protein antigen, tetanus toxoid, are normal (Chandra and Saraya, 1975). However, the kinetics of the response have not been studied. The critical role of iron metalloenzymes for DNA synthesis and cell proliferation makes it likely that iron deficiency can impair the speed of both T- and B-lymphocyte proliferative responses during activation. In fact, limited data in animals are consistent with the concept that antibody production is impaired in iron deficiency states (Kochanowski and Sherman, 1985). Because the rapidity with which host responses are mobilized in vivo plays an important role in determining whether or not exposure to an infectious agent results in illness, these effects of iron deficiency can have clinical significance. Neutrophil myeloperoxidase, an iron metalloenzyme that generates reactive bactericidal halides during PMN phagocytosis, is reduced in iron deficiency states (Prasad, 1979; Turgeon-O'Brien et al., 1985; Yetgin et al., 1979). Myeloperoxidase-mediated killing is a redundant system; however, iron is also required for the production by phagocytic cells of reactive microbicidal oxygen species via the Fenton reaction (Fridovich, 1978). Impairment of this pathway in iron-deficient individuals is evidenced by the diminished ability of their PMNs to reduce the dye, nitroblue tetrazolium (NBT)2 (Celada et al., 1979; Chandra, 1975). A modest diminution in in vitro intracellular bactericidal activity is also reported in some studies (Chandra, 1973; Moore and Humbert, 1984; Walter et al., 1986), although contradictory data also exist (Kulapongs et al., 1974; Van Heerden et al., 1981). Bactericidal assays are, however, not very sensitive to small changes in functional capacity and are most useful to detect profound defects. Other neutrophil functions, such as chemotaxis, phagocytosis, and degranulation, appear to be normal in iron deficiency, which suggests that the effects of iron deficiency are specific and related to the biological functions of the metal. Macrophage functions, such as antigen presentation, have not been studied in iron-deficient subjects. Animal experiments have shown that clearance of polyvinylpyrrolidone and the generation of reactive oxygen species is impaired in iron-deficient mice (Kuvibidila and Wade, 1987; Thompson and Brock, 1986), and that IL-1 production is reduced in iron-deficient rat leukocytes (Heylar and Sherman, 1987). Although it is difficult to extrapolate from in vitro studies of immune function to altered host susceptibility to infection, it can be concluded that iron deficiency is not going to enhance host immune responses. Whether or not delayed or diminished responses affect the course of the host-pathogen interaction at the clinical level remains to be conclusively shown. This, no doubt, differs from subject to subject and will relate to the severity of the iron 2   The NBT test is used to detect the oxidative ability of PMN leukocytes by measuring the concomitant reduction of NBT, reflected in a measurable color change.

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--> deficiency, the presence of concomitant nutritional defects, and the nature of the infecting organism. Effects of Iron Excess on Immune Function In developing the nutritional immunity concept, the evidence that iron promotes microbial growth while conditions characterized by iron excess impair immune function and increase susceptibility to infection has been used to support the argument that iron withholding protects the host. That is, if withholding iron protects, then excess iron will enhance infection (Weinberg, 1984). In fact, many examples exist in which the provision of exogenous iron or the addition of unsaturated iron-binding proteins to reduce iron availability correlates respectively with increased or diminished growth of organisms in vitro (Bullen et al., 1978; Chart and Griffiths, 1985). Furthermore, when microbial growth in complete medium is inhibited by the addition of apolactoferrin, this can be reversed by addition of excess iron. Iron overload states, such as β-thalassemia or sickle-cell anemia with multiple transfusions, idiopathic hemochromatosis, or "Bantu" hemosiderosis due to grossly excessive oral iron loads are characterized by iron-saturated transferrin with the excess iron present as loose complexes of iron and albumin readily available to microorganisms (Hershko and Peto, 1987). In some of these disease states, the incidence of infection and fatal outcomes is increased (Barret-Connor, 1971; Buchanan, 1971). This association is supported by the increased mortality of infectious challenge of experimental animal hemochromatosis models compared with euferric states (Fletcher and Goldstein, 1970; Robins-Browne and Pripic, 1985). However, when the clinical parameters are reexamined to separate the effect of increased free iron from the effects due to damage of the liver and spleen and secondary diabetes, it becomes difficult to attribute increased susceptibility to iron itself rather than to damage to the reticuloendothelial system and cellular function as a consequence of iron-mediated oxidation/peroxidation effects (Hershko et al., 1988). Thus, while approximately 20 percent of deaths in β-thalassemia are attributed to infection, nearly all of these deaths occur in splenectomized patients. Similarly, the enhanced susceptibility to infection in sickle-cell patients occurs primarily in the young, before significant iron overload occurs. Clinical analysis thus suggests that it is not increased free iron availability in iron overload that is associated with increased incidence or severity of infection. Because free iron is known to damage cells, iron excess is likely to impair immune function. PMNs from β-thalassemia or transfusion hemosiderosis patients produce less superoxide and hydrogen peroxide than controls (Flament et al., 1986; Martino et al., 1984; Waterlot et al., 1985), and NBT dye reduction is impaired as well (Cantinieaux et al., 1987; Tavo et al., 1977). Cells from thalassemia patients also show diminished chemotactic responses and reduced random migration (Khan et al., 1983). Peripheral blood monocytes from

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--> β-thalassemic patients are impaired in their microbicidal activity (Ballart et al., 1986; Van Asbeck et al., 1984a, b). Oxidative damage most likely impairs the functional responses of lymphocytes exposed to iron excess in vitro. For example, addition of increasing amounts of iron to normal T-lymphocytes diminishes cloning efficiency and reduces proliferative responses to mitogens (Good et al., 1988; Munn et al., 1981). Several studies report a reduction in the number and function of CD4 cells (Dwyer et al., 1987; Grady et al., 1985; Pardalos et al., 1987) and decreased natural killer (NK) cell activity (Akbar et al., 1986; Neri et al., 1984) in iron overload patients. Pokeweed mitogen-induced generation of immunoglobulin-producing cells is diminished in thalassemia major patients (Nualart et al., 1987). Co-culture with normal cells suggests that the defect resides at the Th-cell level. Some of these defects are corrected by iron chelation therapy, which suggests that they are due to the toxic effects of free iron. Clinical Data in Iron Deficiency and Iron Excess Published clinical studies to date that examined the effect of iron deficiency on susceptibility to infection are typically flawed in design, fail to include sufficient numbers of subjects to interpret the results, or do not account for confounders commonly encountered in field studies (Keusch and Farthing, 1986). Some studies have simply compared the hematological status of infants admitted to the hospital because of infection with that of another hospital control group. However, because inflammation results in shifts of iron and iron status markers, these studies cannot distinguish cause and effect. The most cited clinical studies of the past 70 years have involved a comparison of the morbidity among infants fed an iron-fortified formula and the same food without added iron (Andelman and Sered, 1966; Heresi et al., 1985; Hussein et al., 1985; MacKay, 1928). In general, the iron-fortified group was reported to experience fewer respiratory and intestinal infections. Unfortunately, the data are suspect because the controls were inadequate, and morbidity data were collected via recall interviews of the mothers, rather than active surveillance of illness by trained personnel. However, others reported no differences between unsupplemented and supplemented groups (Darsdaran et al., 1979; James and Combes, 1960), while some studies found an increased incidence of infections in the iron-treated subjects (Oppenheimer and Hendrickse, 1983). Although one study in adults suggested increased morbidity among iron-deficient adults compared with iron-supplemented adults (Basta et al., 1979), the clinical consequences of iron deficiency remain uncertain (Dhur et al., 1989; Strauss, 1978). Nonetheless, there are a few examples where the association of iron overload and susceptibility to infection is related to the increased free iron available for the growth of pathogens that do not compete well for protein-bound iron in vivo. For example, low virulence Yersinia enterocolitica, which

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--> lack the Yersinia virulence plasmid encoding high-efficiency iron acquisition systems can use host free iron under conditions of iron overload, or even more efficiently, desferrioxamine chelated iron (Carniel et al., 1987, 1989). Life-threatening infections due to such strains in patients on chelation therapy (Blei and Puder, 1993; Green, 1992; Kelly et al., 1987; Rabson et al., 1975) or following massive oral ingestion of iron (Fakir et al., 1992; Melby et al., 1982; Mofenson et al., 1988) are described. In some instances, presentation with Yersinia sepsis has led to the discovery of an iron overload disease (Vadillo et al., 1994). Vibrio vulnificus also uses heme-iron or desferrioxamine-chelated iron (Helms et al., 1984; Wright et al., 1981), and infections are documented in iron overload patients (Blake et al., 1979). Other, less common associations between chelation therapy and sepsis include Listeria (Mossey and Sondheimer, 1985), and certain fungi, including pathogenic Mucor and Rhizopus species (Abe et al., 1990; Daly et al., 1989; Goodili and Abuelo, 1987). The effect of iron chelators may be dual, including serving as a source of iron and depressing host phagocytic and lymphocyte-mediated defenses (Autenrieth et al., 1994, 1995; Ewald et al., 1994). Transient increases in free iron can result from parenteral administration of iron-polysaccharide complexes. Following the introduction of one such agent, iron-dextran, for rapid correction of neonatal iron deficiency in New Zealand, a striking increase in E. coli septicemia was detected (Barry and Reeve, 1973). The infections occurred shortly after the iron loading, which suggests that they were related to increased available iron. When this therapy was abandoned, the sepsis rate soon dropped by 10-fold (Barry and Reeve, 1977; Farmer and Becroft, 1976). Iron-sorbital-citrate, another loosely bound, low-molecular-weight iron complex for parenteral administration, rapidly saturates serum transferrin and is excreted in the urine. Use of this drug has been associated with increased pyuria in patients with chronic pyelonephritis, which suggests local exacerbation of infection (Briggs et al., 1963). This hypothesis is consistent with a report that this drug stimulates the growth of E. coli already present in the kidney in a model experimental murine pyelonephritis (Buchanan, 1971). Several studies have shown an increase in malaria parasitemia in individuals given parenteral or oral iron (Masawe et al., 1974; Murray et al., 1978a). Although these reports are not entirely convincing because they lack appropriate placebo control groups, fail to control for the presence of protein energy malnutrition and associated reduced transferrin levels, and lack proper blinding, a recent, well-designed, double-blind study in Papua New Guinea confirms the observation (Oppenheimer et al., 1986a). Parenteral iron administration resulted in a 64 percent increase in parasitemia rate at both 6 and 12 months, along with a 20 to 40 percent increase in spleen rates3 and significantly increased admissions to the hospital for malaria. In addition, 3   In malaria endemic regions, repeated malarial infections and resulting hemolysis lead to splenomegaly in young children. Thus, the prevalence of splenomegaly (''spleen rate'') reflects the frequency of clinical malaria.

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--> admissions for measles, otitis media, and pneumonia were increased in the iron group as well (Oppenheimer et al., 1986b). The detrimental effects of the iron-dextran were most apparent in infants with the highest birth hemoglobin level and, therefore, the highest level of iron stores. Another careful study of total-dose, iron-dextran infusion in pregnant women in Papua New Guinea corroborated the increased risk of malaria parasitemia associated with the drug (Oppenheimer et al., 1986c). There are a number of explanations for this. Iron administration will increase erythropoiesis, and Plasmodia preferably invade young cells. Iron also increases hemoglobin synthesis, which is essential for parasite metabolism (Hershko and Peto, 1988), and provides the metal for growth rate-limiting iron-metalloenzymes (Scheibel, 1988; Scheibel and Sherman, 1988) (dihyroorotate dehydrogenase, phosphoenol pyruvate carboxykinase, cytochrome oxidase, and ribonucleotide reductase) necessary for parasite replication. Hemolysis of any etiology is a known risk factor for Salmonella infection (Black et al., 1960; Jones et al., 1977), presumably related to the uptake of iron-hemoglobin by macrophages and secondary macrophage defects in phagocytosis and/or killing of the organism. In experimental murine models, hemolysis induced by Plasmodium berghei infection or phenylhydrazine dramatically increases the virulence of Salmonella typhimurium, whereas iron deficiency induced by bleeding did not (Kaye et al., 1967). These data demonstrate that excess free iron may have a direct enhancing effect on a limited number of pathogens and that chronic iron overload results in oxidative and peroxidative damage to the immune system and impairs its function. The effects of iron overload cannot therefore be considered to be a continuum ranging from protection of the host due to iron withholding at one end to increased susceptibility due to iron excess at the other, as the nutritional immunity hypothesis presumes. Indeed, iron deficiency and iron excess both exert an adverse effect on host defenses but by entirely different mechanisms. Implications of Iron Deficiency for the Military The evidence reviewed suggests that iron deficiency offers little nonspecific protection against infections and that iron overload stimulates the growth of a very limited number of pathogens, but results in real damage of the immune system with more general effects on host defenses. At the same time, it is the parenteral administration of iron or the use of iron chelation therapy in iron overload diseases that have been associated with enhanced susceptibility to infections. However, no convincing data show that oral iron supplements for repletion of stores and treatment of iron-deficiency anemia have any adverse impact on infectious diseases. Diminished iron stores among military personnel are most likely to be in women of child-bearing age. Screening for iron-deficiency anemia among recruits, and at regular examinations thereafter, represents an opportunity to

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--> prescribe iron for those in need who will benefit. This has two advantages to the military: first, improvement of physical fitness by correcting anemia and second, repletion of iron stores and optimization of immune function. There are no disadvantages and no risk of promoting infectious diseases. The major issue in compliance with supplemental iron is the intestinal side effects associated with oral iron, but this can be overcome by experimenting with the preparation used in a given patient and/or by lowering the daily dose. References Abe, F., H. Inaba, T. Katoh, and M. Hotachi. 1990. Effects of iron and desferrioxamine on Rhizopus infection. Mycopathologia 110:87-91. Akbar, A.N., P.A. Fitzgerald-Bocarsly, M. deSousa, P.J. Giardina, M.W. Hilgartner, and R.W. Grady. 1986. Decreased natural killer activity in thalassemia major: A possible consequence of iron overload. J. Immunol. 136:1635-1640. Andelman, M.B., and B.R. Sered. 1966. Utilization of dietary iron by term infants: A study of 1,048 infants from a low socioeconomic population. Am. J. Dis. Child. 111:45-55. Archibald, F. 1983. Lactobacillus plantarum, an organism not requiring iron. FEMS Microbiol. Lett. 19:29-32. Autenrieth, I.B., E. Bohn, J.H. Ewald, and J. Heesemann. 1995. Deferoxamine B but not deferoxamine G1 inhibits cytokine production in murine bone marrow macrophages. J. Infect. Dis. 172:490-496. Autenrieth, I.B., R. Reissbrodt, E. Saken, R. Berner, U. Vogel, W. Rabsch , and J. Heesemann. 1994. Desferrioxamine-promoted virulence of Yersinia enterocolitica in mice depends on both desferrioxamine type and mouse strain. J. Infect. Dis. 169:562-567. Ballart, I.J., M.E. Estevez, L. Sen, R.A. Diez, J. Giuntoli, S.A. de Miani, and J. Penalver. 1986. Progressive dysfunction of monocytes associated with iron overload and age in patients with thalassemia major. Blood 67:105-109. Barret-Connor, E.. 1971. Bacterial infection and sickle-cell anaemia. An analysis of 250 infections in 166 patients and a review of the literature. Medicine 50:97-112. Barry, D.M.J., and A.W. Reeve. 1973. Iron injections and serious Gram negative infection in Polynesian newborn. N. Zeal. J. Med. 78:376. Barry, D.M.J., and A.W. Reeve. 1977. Increased incidence of gram-negative neonatal sepsis with intramuscular iron administration. Pediatrics 60:908-912. Basta, S.S., Soekirman, A. Karyadi, and N.S. Scrimshaw. 1979. Iron-deficiency anaemia and the productivity of adult males in Indonesia . Am. J. Clin. Nutr. 32:916-925. Black, P.H., L.J. Kunz, and M.N. Swartz. 1960. Salmonellosis--a review of some usual aspects. N. Engl. J. Med. 262:811-817, 921-927. Blake, P.A., M.H. Merson, R.E. Weaver, D.G. Hollis, and P.C. Heublein. 1979. Disease caused by a marine vibrio: Clinical characteristics and epidemiology. N. Engl. J. Med. 300:1-5. Blei, F., and D.R. Puder. 1993. Yersinia enterocolitica bacteremia in a chronically transfused patient with sickle-cell anemia. Case report and review of the literature. Am. J. Pediatr. Hematol. Oncol. 15:430-434. Bothwell, T.H., 1995. Overview and mechanisms of iron regulation. Nutr. Rev. 53:237-245. Briggs, J.D., C.A. Kennedy, and A. Goldberg. 1963. Urinary white cell excretion after iron-sorbitol-citric acid. Br. Med. J. ii:352-354.

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--> Discussion PAUL ROATH: It seemed most of your talk centered on gram negatives. Is there any expectation of gram positives to have an iron uptake system because the membranes would conserve it differently? GERALD KEUSCH: There are parallels in the gram positives. Most of the information has really come from the paradigm organism, E. coli. But Corynebacterium diphtheriae is a gram positive organism in which toxic production is closely regulated by iron. There is a good source of information on the gram positive bacteria and iron in the monograph Iron and Infection, edited by Bullen and Griffiths (1987).