Micronutrients and Innate Immunity

Abstract

Micronutrients such as zinc, selenium, iron, copper, β-carotene, vitamins A, C, and E, and folic acid can influence several components of innate immunity. Select micronutrients play an important role in alteration of oxidant-mediated tissue injury, and phagocytic cells produce reactive oxidants as part of the defense against infectious agents. Thus, adequate micronutrients are required to prevent damage of cells participating in innate immunity. Deficiencies in zinc and vitamins A and D may reduce natural killer cell function, whereas supplemental zinc or vitamin C may enhance their activity. The specific effects of micronutrients on neutrophil functions are not clear. Select micronutrients may play a role in innate immunity associated with some disease processes. Future studies should focus on issues such as age-related micronutrient status and innate immunity, alterations of micronutrients in disease states and their effect on innate immunity, and the mechanisms by which micronutrients alter innate immunity.

Nonspecific, innate, or natural immunity is present from birth and comprises components that serve as defense barriers. The innate immune system can play an important role as the first-line defense or barrier against foreign organisms and substances. It also plays a role in acute and chronic inflammation and in select disease states. One type of barrier that functions to prevent entry of pathogens is structural. Examples include the skin and mucous membranes; these are effective in preventing the entry of most pathogens. Because of low pH caused by various fatty acids and enzymes, the skin can limit the growth of most bacteria. In addition, secretory products (e.g., saliva, tears, and mucus) contain proteins that can destroy pathogens.

Another component of innate immunity consists of physiologic barriers and includes parameters such as temperature, pH, and oxygen levels. An example is the low pH of the stomach, which provides an innate barrier to infection, as few ingested microorganisms can survive that environment. In addition, various soluble factors may function in innate immunity. For example, the hydrolytic enzyme found in mucous membrane secretions, lysozyme, can cleave the peptidoglycans found in the bacterial cell wall.

Phagocytosis and pinocytosis constitute important defense mechanisms, which may initiate the innate immune response and play a role in the adaptive immune response. During phagocytosis, the appropriate immune cell internalizes bacteria and macromolecules such as foreign antigens. The internalization is initiated by the interaction of specific receptors on the surface of the phagocytic cell with ligands associated with the material to be internalized [1]. That leads to the polymerization of actin at a specific site, which is followed by the internalization of the material by actin-based mechanisms. Internalization occurs by invagination of small regions of the plasma membrane forming small endocytic vesicles. After internalization, actin is shed from the phagocytic vesicle, and those vesicles fuse with each other and with components of the endocytic pathway leading to the formation of the mature phagolysosome. The trafficking of the endosome-lysosome occurs in association with microtubules. Thus, the maturation of phagosomes requires the interaction of actin and tubulin of the cytoskeleton. Ultimately, engulfed particles are degraded in mature phagosomes by hydrolytic enzymes.

A major component or barrier created in nonspecific immunity is the inflammatory response. This is a complex process with many events caused by endogenous and exogenous factors. The principal signs of inflammation include increased blood flow, increased capillary permeability, and influx of phagocytic cells. Those cells consist of polymorphonuclear leukocytes (PMNL), macrophages, and lymphocytes. Events in the response are initiated by interactions among several mediators. Some come from the invading microorganisms, some are released from the damaged tissue, some are from leukocytes participating in the inflammatory response, and others are generated by enzyme systems in the blood. Those in the serum constitute the acute-phase proteins. The inflammatory response may be augmented by some components of acquired immunity, which includes both antibodies and T cells. Those lymphocytes can release cytokines, which have a profound effect on activation of macrophages to destroy the foreign organism. Micronutrients may potentially influence some of these processes of nonspecific immunity by modulating inflammatory cell function. Here we review select published studies on micronutrients and innate immunity and studies that focus on micronutrients and macrophages, natural killer (NK) cells, and PMNL. Where relevant, the relationship of micronutrients to the prevention and modulation of select diseases through alterations in the activities of those cells will also be discussed. Finally, we discuss some possible future directions for research with respect to micronutrient status and innate immunity.

Micronutrients and Macrophage Function

Most macrophage functions are performed after that cell acquires various capacities (e.g., the ability to produce nitric oxide). Those capacities are often attained through interaction with the microenvironment. Signals from a variety of sources, including the extracellular matrix, foreign macromolecules, and other cells, can determine the activational state and thus the role macrophages will play in homeostasis or in pathologic processes. Those functions, in vivo and in vitro, are exquisitely sensitive to alterations by several dietary components. While a major focus of dietary studies has been on macrophage function and macronutrients (e.g., dietary fat), several recent studies focused on the effects of micronutrients (e.g., vitamins and trace elements). Those studies confirmed that micronutrients can significantly alter macrophage function and thus alter their role in innate immunity, inflammation, and several disease processes.

Vitamins and macrophage function

Several studies show that vitamins can significantly alter macrophage phagocytosis and several of its constituent steps (table 1). Phagocytosis is a component of several macrophage functions (e.g., removal of effete red blood cells) and requires various steps. Steps, such as adherence, migration, ingestion, and superoxide anion production, can be evaluated separately. For example, the entire process of phagocytosis and of several steps were enhanced after in vitro treatment of peritoneal macrophages with antioxidant vitamins such as α-tocopherol, vitamin E, and ascorbic acid [7]. In that study, substrate adherence was increased by α-tocopherol and ascorbic acid. Vitamin E, α-tocopherol, and ascorbic acid increased random migration, chemotaxis, ingestion, and superoxide anion production. A similar finding was observed in the Kupffer cell. Both phagocytic activity and the production of the reactive oxygen species, superoxide anion, were enhanced in Kupffer cells of rats treated in vivo with all-trans-retinol [5]. Peripheral blood monocytes from the vitamin A—treated rats also had increased respiratory burst activity compared with controls. In contrast, the effects of vitamin D₃ on macrophage phagocytosis may be related to the ability of that vitamin to alter monocyte maturation. Thus, D₃ enhances immunoglobulin and complement-mediated phagocytosis by human monocytes through its stimulation of monocyte maturation to macrophages [2].

Table 1

Vitamin effects on macrophage function.

Another important macrophage function that has been studied with regard to micronutrient effects is the production of cytokines such as interleukin (IL)-1 and -6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ) and inflammatory mediators such as prostaglandin E₂ (PGE₂). These studies were prompted by the role that macrophages play in inflammation and specific chronic inflammatory diseases. One study showed that vitamin E inadequacy led to increased PGE₂ production in rat alveolar macrophages [9]. In another, the in vivo treatment of rats with all-trans-retinol significantly increased mononuclear cell production of both PGE₂ and TNF-α [5]. In addition to phagocytosis and cytokine production, macrophages can present antigen in conjunction with major histocompatibility complex (MHC) class II molecules on their surface. MHC II was down-regulated in human monocytes treated in vitro with vitamin D₃ [2]. Thus, the effect of some vitamins on some innate immune functions demonstrates the potential for enhancement. The optimal levels of those micronutrients remain to be determined.

Trace elements and macrophage function

The relationship between intake of select trace elements and macrophage functions appears to be as important as with vitamins. The most extensively studied of those trace elements are zinc and selenium (table 2). Low plasma levels of zinc lead to impaired immune function and several disease processes lead to zinc deficiency [13]. Surprisingly, only a few studies have assessed zinc and macrophage phagocytosis. In one, decreased human monocyte phagocytosis was observed as a result of zinc deficiency [14]. In contrast, more studies have focused on trace elements and cytokine production. Zinc supplementation directly induced cytokine production, predominantly IL-1, IL-6, and TNF-α, by mononuclear cells in vitro [15]. Addition of copper in vitro had no affect. In contrast, dietary selenium deficiency stimulated an increase in rat macrophage production of PGE₂ and transforming growth factor-β [9, 11]. Several of those cytokines can be important regulators for macrophage tumor cytotoxicity. Dietary supplementation with selenium resulted in a significant increase in the tumor cytotoxicity by mouse macrophages [10]. That effect, however, may not have been specific for macrophages, as selenium supplementation also increased the activity of cytotoxic lymphocytes and lymphokine-activated killer cells.

Table 2

Trace element effects on macrophage function.

Micronutrients may play a significant role in alteration of macrophage function and in inflammatory diseases such as rheumatoid arthritis. Excess of some micronutrients such as iron can aggravate arthritic inflammation, whereas pharmacologic doses of zinc may immobilize macrophages. Selenium may act as an oxygen radical scavenger. Thus, it has been hypothesized that antioxidants may reduce rheumatic inflammation. In some cases, the effect of the micronutrient can be direct (e.g., the effects of zinc on cytokine production). For the most part, however, there is no real understanding of the mechanism of action of these vitamins and trace elements on altered macrophage function.

Micronutrients and NK Cell Function

NK cells can recognize and kill select tumor cells and virus-infected cells. They are also responsible for antibody-dependent cytotoxicity. When activated, NK cells can also release IFN-γ and other cytokines, such as IL-1, and granulocyte macrophage colony-stimulating factor. Earlier dietary studies with NK cells focused on macronutrients such as protein and fat. More recent studies focused on the effects of micronutrients.

Vitamins and NK cell function

Like other cells of the immune system, it appears that deficiencies in certain vitamins can have adverse effects on NK cell function (table 3). These deficiencies can be caused by poor nutrition, disease, or aging. For example, a nutritional deficiency in vitamin A in both children [20] and rats [16] led to fewer NK cells with less activity. Certain diseases can play a significant role in nutritional status, which in turn can affect NK cell activity. That nutritional deficiency can be altered to normalize immune function was shown when mice with murine AIDS were supplemented with tocopherol, which not only restored vitamin E, but also vitamin A and zinc and copper to control levels in various tissues [19]. In addition, that treatment completely restored NK cell number and activity. Vitamin E deficiency also impairs NK cell activity due to a decreased number of CD16⁺ cells in patients with Shwachman's syndrome [18].

Table 3

Vitamin effects on natural killer (NK) cells.

Trace elements and NK cell function

The most commonly assessed function of NK cells is the determination of cytolysis of standardized tumor cell lines. The lines used are typically very sensitive to NK cell activity. Accordingly, a number of studies have reported the phenomenon in which a trace element deficiency decreased but supplementation increased NK cell cytolysis (e.g., in elderly patients, zinc supplementation transiently enhanced NK cell activity) [21]. Although zinc supplementation enhanced that activity, there appeared to be a threshold concentration of the supplement beyond which the NK cell activity was reduced [22]. Addition of zinc in vitro to NK cells also appeared to enhance their activity [23]. Similarly, dietary selenium increased NK cell activity and IL-2 receptor protein on the surface of mouse NK cells [10]. In contrast, iron decreased mouse NK cell cytolytic activity [24].

Some diseases may be caused or aggravated by overactive cells participating in the innate immune response and altered nonspecific immune function may be due to changes in micro-nutrient availability. For example, inflammatory bowel disease can cause disturbances in zinc metabolism and increased NK cell activity [25]. Zinc administration in vivo decreased peripheral blood NK cell activity in vitro in patients with that inflammatory disease.

Micronutrients and PMNL

Neutrophils are usually the first cells to arrive at sites of tissue damage or infection. Chemotactic factors that include certain components of the complement and fibrinolytic systems, leukotrienes and cytokines, mediate a transient leukocytosis during infection as well as the adherence to and extravasation of neutrophils through the vascular endothelium to accumulate at inflammatory sites. Neutrophils then utilize a variety of weapons to destroy microorganisms. Following phagocytosis, phagosomes fuse with intracellular granules; the largest granules contain myeloperoxidase, lysozyme, defensins, and hydrolytic enzymes. Neutrophils consume large amounts of oxygen to generate H²O²; this oxidative or respiratory burst is required for the destruction of ingested pathogens [26–28]. The synthesis or activity of several of the enzymes and reactions that are essential to neutrophil function can be altered by the deficiency or supplementation of micronutrients. For example, iron deficiency can lead to a reduction of the iron-dependent enzyme myeloperoxidase and impaired killing of ingested bacteria [29]. Micronutrients known for their antioxidant effects could also affect the oxidative reactions that neutrophils utilize to generate reactive oxygen species. However, that effect could be advantageous if the antioxidants scavenge those molecules before they damage surrounding tissues.

Vitamins and PMNL function

It is difficult to assess the direct effects of particular micronutrient deficiencies on human neutrophils since that condition occurs primarily in malnutrition. Moreover, both the depletion of macronutrients and micronutrients in malnutrition contribute to immune suppression (table 4). This is found in elderly persons: Deficits of zinc, selenium, and vitamin B₆ lower immune response, which in turn can predispose to infection [35]. However, knowledge regarding the importance of particular micronutrients on neutrophil function can be gleaned from work with animals and supplementation studies in humans. Bacterial killing by neutrophils is slightly decreased in vitamin B₁₂ but not folic acid deficiency; this impairment was reversed after supplementation [31]. Vitamin A deficiency in rodents reduces the adhesion and phagocytosis of Pseudomonas aeruginosa and the production of reactive oxidative molecules. When the animals were fed vitamin A, neutrophil function was restored [30].

Table 4

Effects of vitamins on neutrophil function.

One report provided in vivo and in vitro evidence that a single dose of vitamin C could improve primary abnormalities in neutrophil motility and antimicrobial activity in humans with chronic granulomatous disease; vitamin C also improved neutrophil motility in some persons with bronchial asthma [36]. Supplementation with the antioxidant vitamins C and E in healthy and aged women suffering from depression and coronary heart disease resulted in a significant increase in neutrophil adherence, chemotaxis, and phagocytic capacity. However, those vitamins decreased superoxide production by neutrophils [32]. Another study also demonstrated that supplementation with vitamins C and E in healthy humans suppressed neutrophil production of oxygen free radicals [33]. Several investigators have suggested that such an antioxidant effect might be beneficial if it reduced oxidant-mediated damage in certain diseases.

Whether this reduction in superoxide production by neutrophils adversely affects microbicidal capacity has not been explored. Moreover, vitamin C inhibits the activation of the oxidant-sensitive transcription factor NF-κB, which mediates the production of proinflammatory cytokines such as IL-1 and TNF-α [37]. It appears that cellular signaling in and function of neutrophils can be influenced by vitamins C and E. Indeed, neutrophils in vitro were unable to phagocytose Candida albicans when high concentrations of vitamin C were added [34]; vitamin E supplementation did not adversely affect this capacity [38]. Since activated neutrophils accumulate vitamin C at levels 10-fold greater than normal neutrophils [39], it is not certain how this high concentration may alter neutrophil activity in the face of chronic vitamin C supplementation. while vitamins C and E supplementation might be advantageous for minimizing neutrophil-induced tissue damage in persons with ischemia and chronic inflammatory disease, perhaps it would impair the ability of neutrophils to destroy pathogens in healthy persons or in those vulnerable to infection. Given the current popularity of supplementation with vitamins C and E, the possibility of altered intracellular signaling and oxidant production by neutrophils needs to be addressed.

Trace elements and PMNL function

Fewer published studies have reported the effects of trace elements on neutrophil function (table 5). Copper deficiency decreased the number of circulating neutrophils and impaired their function in humans [40] and rodents [45]. Iron deficiency compromises the ability of neutrophils to kill bacteria [43, 46]. While numerous studies demonstrate the effects of deficiencies of copper and iron, fewer have focused on the effects of supplementation of these two micronutrients in healthy persons. Large amounts of oral zinc significantly impaired PMNL function [41] in one study. Alternatively, in vitro, zinc potentiated the neutrophil response against Staphylococcus aureus [42]. Zinc, copper, and nickel induced neutrophil chemotaxis in vitro. From that finding the investigators hypothesized that the effect was relevant to instances when those metals might be solubilized from prosthetic devices or utilized as therapeutic agents [47]. Selenium in vitro enhanced the phagocytic and bactericidal functions of human neutrophils [44]. Based on those findings, investigators suggested that supplementation might augment those functions in humans. However, a recent trial showed that sodium selenite supplementation was not an efficient stimulating agent of phagocytosis in humans [48]. Thus, while supplementation with trace elements to enhance neutrophil function in states of malnutrition can be effective, it is not clear whether supplementation in healthy persons would enhance or impair their microbicidal activity. Few studies have explored the mechanisms by which trace elements modulate neutrophil function.

Table 5

Trace element effects on neutrophil function.

Possible Future Directions

Studies to date with micronutrients indicate they have as much potential to alter innate immunity as macronutrients. Nevertheless, the study of individual micronutrients on individual components in innate immunity are extremely difficult to design and interpret. For example, in supplementation studies, micronutrients can have varied effects on inflammatory cells that may be either direct or indirect, making it difficult to link in vitro observations with those studies. While micronutrient deficiency, in general, can have a widespread effect on nearly all components of the innate immune response, that effect can be reversed by supplementation. However, it is not clear whether excesses of micronutrients will enhance or suppress overall innate immunity. Although preliminary work is promising, an extensive number of well-controlled studies need to be done to clarify which micronutrients and what concentrations are necessary to influence innate immunity. This would involve issues of micronutrient deficiency or excess. It will also be important to select appropriate methods for accurate assessment. Specific diseases, which invoke select components of innate immunity, should be included as relevant models. Thus, an important area of focus should be on innate immunity, micronutrient status, and disease. The primary question is whether it is possible to modulate the disease process by altering micronutrient intake.

Several studies have focused on elderly persons and micronutrient supplementation. It appears that age-related micronutrient status and innate immunity will be important issues for future investigation. Although we are just starting to understand which components of innate immunity can be altered by micronutrients, it will be important to determine the mechanisms that induce alteration. Those studies may reveal novel treatment modalities utilizing micronutrients.

Grant support: California Breast Cancer Research Program (4CB-0157).