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Oxidative Stress, Carotenoids and Immune Response
Posted on: March 16, 2004

Immune System and Reactive Oxygen Species
The immune system is comprised of innate (natural) and acquired (adaptive) immunity. Acquired immunity is composed of lymphocytes; these are highly active cells that constantly generate reactive oxidative products (ROS) as a part of their normal cellular activity. Oxidizing pollutants and many viruses also can induce ROS production by normal cells. The ROS are highly reactive and can destroy cellular membranes, cellular proteins and nucleic acids. One mechanism by which the innate branch of the immune system protects the animal is by phagocytizing and subsequently killing antigens through an oxidative bactericidal mechanism termed respiratory burst. Phagocytosis of a foreign particle by a macrophage or neutrophil activates NADP oxidase, resulting in the production of a large amount of superoxide anion (O2) from molecular oxygen. The O2 is then rapidly converted to hydrogen peroxide (H2O2) by superoxide dismutase. Neutrophils contain myeloperoxidase that converts H2O2 to the highly potent bactericidal component, hypochloride ions (OCl). Macrophages do not possess myeloperoxidase and, instead, depend on a myeloperoxidase-independent mechanism to generate other biological oxygen-derived free radicals. The latter include the generation of the hydroxyl radical (OH·). Even though the ROS are produced as part of the killing mechanism, nevertheless excessive phagocytic activity can lead to ROS-induced tissue damage.
As a defense mechanism, the body produces a number of endogenous antioxidants capable of scavenging these harmful ROS to maintain an optimal oxidant:antioxidant balance, thereby maintaining normal cellular function and health. However, under conditions of high oxidative stress, the ability of these antioxidants to eliminate ROS is often exceeded and, therefore, dietary sources of antioxidants or drugs are required. The most widely used dietary antioxidants include vitamin E, vitamin C, carotenoids, flavanoids, zinc and selenium. The harmful affects of ROS are not unique to immune cells but affect all cell types. However, immune cells are particularly sensitive to oxidative stress.
Studies on the role of carotenoids on immune response have generally used several key immune function assays. These include:

  1. Immunoglobulin (Ig) production. Production of Ig has traditionally been used to assess B cell function in a humoral immune response. B cells produce Ig that circulates freely to protect the body against foreign materials. The Ig serve to neutralize toxins, immobilize certain microorganisms, neutralize viral activity, agglutinate microorganisms or antigen particles and precipitate soluble antigens. B cell function requires the help of helper T (Th) cells.
  2. Lymphoblastogenesis. Antigen-stimulated lymphocyte proliferation normally occurs in lymphoid tissues. However, the ability of isolated lymphoid cells to proliferate when cultured in the presence of certain mitogens has given researchers an important tool to assess both T and B cell function in vitro. Commonly used mitogens include concanavalin A that stimulates T cells, lipopolysaccharide that stimulates B cells and pokeweed mitogen that stimulates T and B cells.
  3. Lymphocyte cytotoxic activity. Natural killer (NK) cells are a critical component of innate resistance against viruses, bacteria, fungi and parasites. They regulate the adaptive immune system and hematopoiesis, and serve as an immuno-surveillance system against tumors.
  4. Cytokine production. Cytokines are soluble molecules that mediate cell-to-cell interactions. Cytokines commonly measured include IL-2, TNF-α and IFN-γ produced by the CD4+Th1 cell subset, and IL-4, IL-5, IL-6 and IL-10 produced by the Th2 subset. The Th1 cells mediate cytotoxic and local inflammatory reactions, and therefore play important roles in combating intracellular pathogens including viruses, bacteria and parasites. The Th2 cells are more effective in humoral immunity, i.e., they stimulate B cells to proliferate and produce antibodies against free-living microorganisms. Therefore, a normal immune response will require a balance between the Th1 and Th2 subsets.
  5. Delayed type hypersensitivity (DTH). This is a cellular reaction involving T cells and macrophages without involving an antibody component. Antigen-presenting cells (e.g., dendritic cells) present the antigen or allergen to T cells that become activated and release lymphokines. These lymphokines activate macrophages and cause them to become voracious killers of the foreign invaders. The DTH response is simple to conduct. More importantly, it is a good indicator of in vivo cell-mediated immune response and is a predictor of morbidity and mortality in elderly human.
  6. Modern advances in flow cytometry techniques have enabled researchers to phenotype blood lymphocyte subsets by identifying cell surface molecules. Identifying the population shifts for a given cell subset will provide additional supporting evidence for the immune responses assessed using the functional assays described earlier. Also, flow cytometry can be used to identify cell activation through the induction of certain surface markers. Availability of reagents has further extended the application of flow cytometry to include apoptosis, cell cycle progression and cell signaling.
  7. Last, but not least, molecular bioscience techniques have provided exciting new research tools for studying mechanism of action of carotenoids in regulating intracellular events.

Carotenoids, ROS and Immunity
The traditional concept of ROS function is that they indiscriminately destroy cell components. However, exciting research has more recently elucidated the role of these reactive species in signal transduction, gene regulation, and disease etiology. This has infused new excitement and challenges into research on the possible role of carotenoids as antioxidants in disease prevention. Early studies demonstrated that dietary β-carotene prevented bladder, kidney, ear and gut infection in vitamin A-deficient rats and reduced ear infection in young children. Because of the provitamin A activity of β-carotene, these studies raised the possibility that the action of the carotenoid is due to its prior conversion to vitamin A.
Numerous studies using nonprovitamin A carotenoids and, more recently, using cats as the animal model have demonstrated the immuno-modulatory action of dietary carotenoids. Many earlier studies focused on β-carotene. β-carotene possesses a marked stimulatory action on the growth of the thymus gland and a large increase in the number of thymic small lymphocytes. The stimulatory activity of β-carotene on lymphocyte blastogenesis has similarly been demonstrated in rats, pigs, and cattle. Increased numbers of Th and T inducer lymphocytes have been reported in human adults given oral β-carotene supplementation. The number of lymphoid cells with surface markers for NK cells and for IL-2 and transferrin receptors also was increased substantially in peripheral blood mononuclear cells (PBMC) from individuals supplemented with β-carotene. Enhanced NK cell cytotoxicity was observed in human subjects given oral β-carotene. Similarly, long-term β-carotene supplementation to elderly but not middle-age men increased NK cell activity. In vitro, β-carotene induced hamster macrophages to produce TNF-α. Activation of TNF-α by ROS increases the dissociation of IκB from NFκB, and the subsequent translocation of this transcription factor to the nucleus, resulting in the production of cytokines, chemokines, cell adhesion molecules, and acute phase proteins; this activation also produces an anti-apoptotic effect. Alternatively, intracellular ROS may directly increase NFκB. Therefore, ROS are important in primary immune response; conversely, antioxidants can produce the opposite effect. In fact, the antioxidant molecule N-acetyl-L-cysteine can inhibit NFκB, and consequently down-regulate the production of cytokines (IL-6, IL-8, IL-12, and TNF-α), as well as down-regulate the expression of surface molecules (HLA-DR, B7-2 and CD40) in human dendritic cells. Therefore, an antioxidant may impair the generation of primary immune responses through its inhibitory action on dendritic cells. While this scenario occurs in a normal cell, under conditions of high oxidative stress, excess ROS may be produced, resulting in the inhibition of NFκB. Excess ROS is known to cause abnormal cell proliferation and to decrease apoptosis; both are undesirable responses in tumor cells. Therefore, antioxidants are desirable under conditions of high oxidative stress. Analogous to this situation, high concentrations of intracellular nitric oxide induced oxidative killing of isolated rat hepatocytes while low nitric oxide concentrations was protective.
Besides cell-mediated and humoral immune responses, β-carotene has been shown to regulate nonspecific cellular host defense. Blood neutrophils isolated from cattle fed β-carotene had higher killing ability during the peripartum period. The increased bacterial killing could be accounted for partly by increased myeloperoxidase activity in the neutrophils. Dietary β-carotene stimulates phagocytic and bacterial killing ability of neutrophils from dairy cows during the stressful drying off period. In contrast, retinol and retinoic acid generally decreased phagocytosis and had no effect on killing activity.

Carotenoids and Tumor Immunity
The immuno-regulatory action of carotenoids has also been demonstrated through their role in tumor immunity. Mice fed β-carotene had augmented tumor immunity against syngeneic fibrosarcoma cells. Further was demonstrated that the action of β-carotene was specific against the antigens. In vitro, β-carotene inhibited the growth of MCF-7 and Hs578T while lycopene inhibited MCF-7 and MDA-MB-231 human breast cancer cells; canthaxanthin (carotenoid with no provitamin A activity) had no inhibitory effect. The authors concluded that the presence of estrogen receptors is an important, even though not essential, factor in the action of carotenoids on tumor cell growth. Comparison of antitumor activity of various carotenoids against the growth of a transplantable mammary tumor in mice showed that astaxanthin, canthaxanthin, and β-carotene inhibited tumor growth, astaxanthin showed the highest anti-tumor activity.
More detailed study on the mechanism of action of dietary lutein (carotenoid with no provitamin A activity) against mammary tumor growth showed that dietary lutein consistently inhibited the growth of mammary tumors in mice, and even lowered the incidence of tumor development. Dietary lutein decreased apoptosis in blood leukocytes from tumor-bearing mice compared to unsupplemented mice, suggesting a heightened immune status. On the other hand, apoptosis in tumor cells was increased by dietary lutein, suggesting increased death of tumor cells. These results demonstrate a selective action of lutein by decreasing apoptosis in immune cells but increasing apoptosis in tumor cells.
The possible gene regulatory action of carotenoids in hematopoietic cells was confirmed by lutein: lutein but not β-carotene or astaxanthin up-regulated the pim-1 gene in mouse splenocytes. Pim-1 is expressed in normal lymphocytes and is involved in hematopoietic cell proliferation, differentiation and apoptosis.
In order to find out the possible involvement of lutein in apoptosis, the BALB/c mouse model was used to study the ability of lutein to regulate the expression of genes involved in apoptosis. Results showed that dietary lutein decreased mammary tumor growth, increased the mRNA expression of the proapoptotic genes p53 and BAX, decreased the expression of the anti-apoptotic gene Bcl-2, and increased the BAX:Bcl-2 ratio in tumors. This selective action of lutein was similarly observed in human mammary cells. The p53 tumor suppressive gene can induce cell cycle arrest to allow DNA repair or apoptosis. Its pathway is independent of the mitochondria, and therefore of ROS. On the other hand, Bcl-2 functions as a suppressor of apoptotic death and is negatively regulated by wild type p53. The predominance of BAX over Bcl-2 accelerates apoptosis. Bcl-2 resides in the outer mitochondria membrane and prevents cytochrome c release. BAX is inactive until it is translocated to the mitochondria where it binds to Bcl-2 to induce cytochrome c release. Once released, cytochrome c activates caspases to bring about apoptosis. Apoptosis or programmed cell death is important in normal development and health. Uncontrolled cell proliferation can lead to cancer and autoimmune diseases whereas excessive cell death can lead to neurodegenerative diseases and AIDS.
The functionality of a carotenoid is determined by its subcellular localization. Studies in cats and dogs have shown significant uptake of orally fed lutein by the mitochondria, nuclei and microsomes of circulating lymphocytes, with the mitochondria showing the highest uptake in the cat. Similar studies in cats, dogs, cattle and pigs reported uptake of β-carotene by all lymphocyte subcellular fractions. In the case of cats and cattle, the mitochondria again took up the greatest proportion of β-carotene. The mitochondria electron transport system utilizes ~85% of the oxygen consumed by the cell to generate ATP; therefore, they are the most important source of ROS. Cytochrome c located between the inner and outer mitochondria membranes plays a critical role in the apoptotic process. The release of cytochrome c is regulated by the pro-apoptotic proteins BAX, BID and BIM, and by the anti-apoptotic proteins Bcl-2, Bcl-XL and BFL-1. Therefore, the mitochondria is likely a key player in immunity and disease, and the localization of the carotenoids in the mitochondria is therefore of particular relevance. The presence of these carotenoids in subcellular organelles can protect the immune cells against oxidative injury, and ensure optimal cellular functions, including apoptosis, cell signaling and gene regulation.
Besides acting through the various mechanisms described earlier, carotenoids can also influence immune function through their ability to regulate membrane fluidity, and gap-junction communication. Of course, all these actions are most likely interrelated in their modulation of an immune response. Evidence has suggested that the action of carotenoids on immunity and diseases may be mediated, at least in part, by their ability to quench ROS. However, the action of ROS is multifaceted: on the one hand, they are toxic to cellular components, but on the other hand, intracellular and extracellular ROS are important signaling molecules involved in the regulation of gene expression, cell growth and cell death. Therefore, to simply conclude whether carotenoids are heroes or villains is to oversimplify their true role in the body. The action of carotenoids on immune response hangs in a delicate balance with the intra- and extracellular milieu, the outcome of which depends not only on the type and concentration of the carotenoid but also on the cell type and animal species involved. Even though studies to date have provided evidence for a specific action of carotenoids, much has yet to be done to truly understand their molecular action. The use of molecular bioscience techniques can provide the necessary research tool to probe into the complex interaction of carotenoids with cell systems.

Source: Boon P Chew, Jean Soon Park. Carotenoid Action on the Immune Response. The Journal of Nutrition. Jan 2004. Vol. 134, Iss. 1; pg.257-262.
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