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[22] Vitamin E status and immune function
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VITAMIN E STATUS AND IMMUNE FUNCTION
[22] V i t a m i n E S t a t u s
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Function
ALISON BEHARKA, SUSAN REDICAN, LYNETrE LEKA,
and SIMIN NIKBIN MEYDANI
Introduction Vitamin E is the generic name used to describe eight naturally occurring, fat-soluble compounds known as tocopherols and tocotrienols. Vitamin E has many functions in the body, including protecting cell membranes from oxidative damage. I Although vitamin E is a constituent of all cellular membranes, it is found in high concentrations in the membranes of immune cells because they are at especially high risk for oxidative damage. 2-4 In fact, vitamin E is essential for normal function of the immune system. 5,6 A deficiency in vitamin E diminishes the ability of the immune system to respond to infectious microorganisms, to produce a delayed-type hypersensitivity (DTH) reaction, or to mount an antibody response to antigen. 7-9 Under some conditions, a pharmacological level of vitamin E is needed to achieve an optimal immune response. 8,1°In several studies, data have shown that elderly humans, as well as laboratory and farm animals, consuming diets that contain more than five times the recommended dietary allowance (RDA) of vitamin E for their species had significantly increased humoral and cell-mediated immune responses and increased resistance to infectious diseases compared to nonsupplemented controls. 8,1°-15These results suggest 1 L. Packer and V. E. Kagan, in "Vitamin E in Health and Disease" (L. Packer and J. Fuchs, eds.), p. 179. Marcel Dekker, New York, 1993. 2 A. Coquette, B. Vray, and J. Vanderpas, Arch. Int. Physiol. Biochem. 94, 529 (1986). 3 L. J. Hatman and H. J. Kayden, J. Lipid Res. 20, 639 (1979). 4 L. J. Machlin, in "Handbook of Vitamins: Nutritional, Biochemical, and Clinical Aspects" (2nd ed.), p. 99. Marcel Dekker, New York, 1991. 5 S. N. Meydani and R. P. Tengerdy, in "Vitamin E: Biochemical and Clinical Applications" (L. Packer and J. Fuchs, eds.), p. 549. Marcel Dekker. New York, 1991. S. N. Meydani and M. Hayek, in "Proceedings of the International Congress on Nutrition and Immunity," p. 105. ARTS Biomedical Publishers, St. Johns, Newfoundland, 1992. 7 M. L. Scott, Fed. Proc. 39, 2726 (1980). 8 R. P. Tengerdy, Avian Dis. J. 34, 848 (1990). 9 K. N. Jeejeebhoy, in "Modern Nutrition in Health and Disease" (M. E. Shils, J. A. Olson, and M. Shike, eds.), p. 805. Lea and Febiger, Philadelphia, 1994. 10A. Bendich, G. E. Gabriel, and L. J. Machlin, J, Nutr. 116, 675 (1986). 11 S. Moriguchi, N. Kobayashi, and Y. Kishino, J. Nutr. 120, 1096 (1990). 12 R. P. Tengerdy, in "Antioxidant Nutrients and the Immune Response" (A. Bendich, M. Phillips, and R. Tengerdy, eds.), p. 103. Plenum Press, New York, 1989.
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that under some conditions the R D A for vitamin E might not be adequate for immunological vigor and health. 16 The vitamin E requirement for ideal i m m u n e function depends on its interactions with other antioxidant and pro-oxidant nutrients, especially with polyunsaturated fatty acids, and on other factors that modulate the i m m u n e response, such as age and stress. 5,17 Elderly individuals respond to supplementation levels of vitamin E above the R D A with increased immunological vigor including enhanced D T H responsivenessJ 8 Epidemiological studies have shown correlations between high plasma vitamin E levels and decreased incidence of infections in the e l d e d y J 9,2° Recent studies also suggest that vitamin E supplementation above the R D A m a y improve the i m m u n e responsiveness of mice infected with the murine acquired immunodeficiency syndrome ( A I D S ) virus. 21 Therefore, conventional methods for determining the R D A , while adequate for arriving at the level of vitamin E required to prevent clinical deficiency symptoms, m a y not adequately predict the optimal level of vitamin E needed to maintain immunological health. Because vitamin E is involved in the maintenance of i m m u n e function, which appears to be especially sensitive to changes in vitamin E status, it m a y be possible to utilize selected immune p a r a m e t e r s as indicators of vitamin E status. This chapter provides protocols to study selected cell-mediated immune responses that are influenced by vitamin E status. In addition, quality control measures for h u m a n studies involving i m m u n e p a r a m e t e r s are discussed. G e n e r a l Q u a l i t y Control M e a s u r e s for H u m a n S t u d i e s M a n y m e a s u r e m e n t s of i m m u n e function are subject to interpretation. Therefore, reliable test results can only be achieved by assay standardization. This includes, but is not limited to, the following criteria:
I3 A. Bendich, Ann. N.Y. Acad. Sci. 587, 168 (1990). z4S. N. Meydani and J. B. Blumberg, in "Micronutrients in Health and in Disease Prevention" (A. Bendich and C. E. Butterworth, Jr., eds.), p. 289. Marcel Dekker, New York, 1991. 15M. Meydani, S. N. Meydani, L. Leka, J. Gong, and J. B. Blumberg, F A S E B J. 7, A415 (1993). 16L. M. Corwin and R. K. Gordon, Ann. N.Y. Acad. Sci. 393, 437 (1982). 17M. L. Eskew, W. J. Scheuchenzuker, R. W. Scholz, C. C. Reddy, and A. Zarkower, Environ. Res. 40, 274 (1986). 18S. N. Meydani and J. B. Blumberg, in "Nutritional Modulation of the Immune Response" (S. Cunningham-Rundles, ed.), p. 223. Marcel Dekker, New York, 1992. 19M. Chavance, G. Brubacher, B. Herbeth, in "Lymphoid Cell Functions in Aging" (A. L. Dewick, ed.), p. 231. Eurage, Interlaken, 1984. 2oM. Chavance, G. Brubacher, B. Herbeth, G. Vernhers, T. Mistacki, F. Dete, C. Fournier, and C. Janot, in "Nutritional Immunity and Illness in the Elderly" (R. K. Chandra, ed.), p. 137. Pergamon Press, New York, 1985. 21y. Wang, D. S. Hwang, B. Licing, and R. R. Watson, J. Nutr. 124, 2024 (1994).
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1. Potential subjects should be carefully screened before acceptance into a study. Exclusion criteria include most chronic medical conditions such as diabetes, cardiovascular or neoplastic diseases, calcium channel blocker therapy, steroidal or nonsteroidal antiinflammatory drug use, smoking or excessive alcohol use, nutrient supplementation. 2. At each visit, subjects should be asked a standardized set of questions regarding recent illness, medication, vaccinations, and stress. Protocols should be developed before the study begins specifying the conditions under which a subject may be excluded from the study. For example, a subject must not receive a flu vaccine less than 1 month before an immune test. In addition, the recorded answers to standardized questions may allow for interpretation of unusual results when the data are analyzed. 3. Theoretically, performance and evaluation of all tests should be done by the same technician; however, this may not be possible for large studies. During a longitudinal study, the subjects may need to be divided among more than one technician. Within a cross-sectional study, alternate technicians may be utilized as long as they have been identically trained and demonstrate low variability in replicating each other's observations. 4. Any test kits, reagents (especially mitogens), and media should be purchased from the same lot from the same company. 5. Appropriate controls, both positive and negative, must be included every time a test is performed. 6. A reliable baseline value for the parameter being tested must be determined for each subject. This will require multiple sampling. For example, three baseline blood draws within 2 weeks of each other would be utilized for tests involving blood mononuclear cells. 7. Common sense should be used at all times. For example, D T H skin test antigens should not be applied at sites that are difficult to assess due to pigmentation, inflammation, or extreme hairiness.
Cell-Mediated Immune Responses General
Cell-mediated immunity is an immune response specific to antigens and mediated by lymphocytes and macrophages with minor participation by other cell types. Cellular immunity is responsible for D T H reactions, foreign graft rejection, resistance to many pathogenic microorganisms, and tumor immunosurveillance.22 22 M. S. Meltzer and C. A. Nacy, in "Fundamental Immunology" (W. E. Paul, ed.), 2nd ed., p. 765. Raven Press, New York, 1989.
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D e l a y e d - T y p e Hypersensitivity Reaction
The D T H reaction is used as an in vivo assay to determine cell-mediated immune function. D T H is an antigen-specific, T-cell-dependent, recall response manifested as an inflammatory reaction that reaches peak intensity approximately 24-48 hr after antigenic challenge in primed animals. It is the kinetics of the inflammatory process following deposition of antigen in the skin that gives this response its name. Whereas allergic reactions occur within minutes and immune complex reactions occur within a day, D T H reactions peak at 2 days. 22,23 The D T H test involves introducing a relatively small amount of soluble antigen into the epidermis and superficial dermal tissue by needle puncture. Circulating T cells sensitized to the antigen from prior contact react with the antigen in the skin and induce a specific immune response, which includes mitosis (blastogenesis) and the release of soluble mediators (cytokines). Concurrently, antigen is encountered and processed by macrophages. The macrophages are activated to produce and release cytokines such as interleukin 1 (IL-1) and tumor necrosis factor (TNF). The processed antigen is presented by macrophages to T lymphocytes, which then produce cytokines such as IL-2 and interferon 3' (IFN). The cytokines produced by the activated macrophages and T cells are involved in the inflammation associated with a D T H reaction, The intensity of the overall dermal inflammation reaches its peak 24-48 hr after antigen application and is resolved within days or weeks. 22 Measuring the intensity of D T H involves quantitating some aspect of the local inflammatory response. In humans and guinea pigs, this is readily done by measuring the redness and induration of an area of shaven skin. This approach has failed in mice; however, alternative assays that involve measuring changes in tissue thickness of the footpad with a caliper or determining cellular infiltration have proven reliable. 22-24 D T H as a tool for assessing immunocompetence in vivo has been widely used for many years. 25 In humans, D T H was traditionally evaluated by the intradermal reactions observed following Mantoux-like techniques. However, this method has the potentially serious side effect of boosting D T H reactivity after repeated testingY '26 An alternative method that results in 23y. Luo and M. E. Doff, in "Current Protocols in Immunology" (J. E. Coligan, A. M. Kruisbeek, D. H. Margulies,E. M. Shevach,and W. Strober, eds.), p. 4.5.1. John Wiley& Sons, New York, 1995. 24M. A. Vadas, J. F. A. P. Miller, J. Gamble, and A. Whitelaw, Int. Arch. Allergy Appl. ImmunoL 49, 670 (1975). 2sB. M. Lesourd, A. Wang, and R. Moulias,Ann. Allergy 55, 729 (1985). 26E. C. Keystone,P. Demerieux, and D. Gladman, Clin. Exp. Immunol. 40, 202 (1980).
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minimal booster effect on DTH response is Multi-test CMI (Merieux Institute, Inc., Miami, FL), a multipuncture system that permits simultaneous application of seven antigens and a negative controlY
Multi-Test CMI for the Determination of DTH in Human Subjects 1. Select a test site. Preferred sites are the volar surfaces of the arms and the back. The site should be easily accessible for reading. Cleanse the site with ethanol and allow the area to dry. Identify the area by drawing lines above and below the test site. 2. A single-use disposable applicator is used to deliver several recall antigens and a control antigen into the epidermis and superficial dermal tissue by puncture. Commonly used recall antigens and respective concentrations include tetanus toxoid antigen (biologically equivalent to 5.5 x 105 Merieux tetanus units), diphtheria toxoid antigen (1.1 x 10 6 Merieux diphtheria units), Streptococcus antigen (2000 Merieux Streptococcus units), tuberculin antigen (3 × 105 U.S. tuberculin units), Candida antigen (2000 Merieux Candida units), Trichophyton antigen (150 Merieux Trichophyton units), and Proteus antigen (150 Merieux Proteus units). A negative control must also be applied and should contain the same solution that serves as a vehicle for the skin test antigens. In most cases, the vehicle will be a 70% (w/v) sterile glycerin solution. 3. Test sites are evaluated at both 24 and 48 hr. The larger reaction recorded from the two readings is used. The size of an indurated area is determined by inspection and palpation followed by measurements across two diameters at right angles. These measurements are averaged to determine mean diameter. An induration of 2 mm or greater at a test site is considered positive, providing there is no induration at the negative control site. 4. An antigen score is calculated as the total number of positive antigens, and the cumulative score is calculated as the total diameter of induration of all the positive reactions.
Interpretation of Delayed-Type Hypersensitivity Results. Delayed cellular hypersensitivity is a valuable measure of immune response because it involves a complex series of immunologic, cellular, mediator-associated, and vascular effects. Although numerous in vitro tests have been developed to assess T-cell activity, these in vitro assays do not necessarily monitor the same cells that mediate DTH nor do they consider the influences of regulatory events that may influence in vivo T-cell function. Therefore, local DTH 27 W. T. Kniker, C. T. Anderson, J. L. McBryde, M. Roumiantzeff, and B. Lesourd, Ann. Allergy 52, 75 (1984).
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responses represent an important source of information concerning in vivo T-cell function, z2,23 Clinically, D T H skin tests are of value in the overall assessment of immunocompetence. The inability of human adults to react to a battery of common skin antigens suggests hyporeactivity or anergy (nonresponsiveness to antigens). Cutaneous anergy may indicate functional impairment of or abnormalities in the cellular immune system. However, D T H is not an absolute determinant of immune system dysfunction, and any such interpretation must be avoided. D T H may be diminished or absent when there is in vitro evidence that T-cell function remains intact and when antibodyassociated immunity and phagocytic function appear normal. Reactivity to D T H test antigens may decrease or disappear temporarily as a result of febrile illness; measles and other viral infections; or live virus vaccination including measles, mumps, and rubella. It is also possible to observe loss of reactivity in patients undergoing treatment with drugs such as corticosteroids or procedures that suppress immunity. Moreover, recent infections and vaccinations influence the magnitude of the response. A lack of previous exposure to the administered antigen will result in a negative D T H response to that antigen. 22'23,25
Quality Control Measures for Multi-Test CM127 1. All applicator kits should be from the same lot. 2. The same technician should administer and read the test results (see the General Guidelines section). 3. Sterility of the test antigens is essential: opened or damaged applicators should be discarded; and the selected test site should be cleaned with ethanol prior to application; the applicator should never be reused. 4. Systemic reactions can occur in those persons sensitive to allergenic media components. Subjects who react severely to any of the test antigens should only be tested after the test heads containing the problem antigens have been removed. 5. Individuals may acquire skin testing sensitivity resulting from either immunization or infection.
Effect o f Vitamin E Status on Delayed-Type Hypersensitivity Response. The most widely accepted index of in vivo immune function is DTH. 28 D T H can be used as a predictor of morbidity and mortality in the elderly. 29 zs Report of an ILS/WHO Working Group, Clin. Exp. Immunol. 46~ 662 (1981). 29 S. J. Wayne, R. L. Rhyne, P. J. Garry, and J. S. Goodwin, J. Gerontol. Med. Sci. 45, M45 (1990).
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Christou e t al. 3° showed that in preoperative patients a significant correlation exists between D T H and mortality from sepsis. Improved D T H response in hospitalized patients has been associated with a decrease in sepsis and mortality. 31 Wayne et al. 29 showed that healthy elderly subjects who were anergic when tested for D T H had significantly higher mortality from all causes in subsequent years than those subjects with higher D T H responses. Supplementation with vitamin E above the R D A can increase DTH response in some situations. This is especially true for elderly persons. D T H responses are significantly diminished in the elderly, but supplementation with vitamin E improves D T H responsiveness in both older laboratory animals and humans. 5,32 Old mice supplemented with 500 ppm vitamin E for 30 days had significantly higher D T H in response to 2,4-dinitrofluorobenzene than mice fed the R D A . 32 Short-term supplementation of healthy elderly persons with 800 mg vitamin E for 30 days increased D T H scores. 33 In addition, long-term supplementation with 60, 200, or 800 IU of vitamin E a day increased D T H scores over time, 34 In the elderly, plasma vitamin E levels have been positively correlated with positive D T H responses to a variety of antigens, and epidemiologic studies indicate a lower incidence of infectious disease in elderly subjects with high plasma tocopherol levels. 2°'35Population groups maintaining high plasma tocopherol levels have also been noted to possess a lower incidence of cancer. 36-38 Lymphocyte Responsiveness: Proliferation and Interleukin 2 Production The cell-mediated immune responses involving lymphocyte functions are very sensitive to changes in vitamin E level. This section describes 30 N. V. Christou, J. Rodriguez-Tellado, L. Chartrand, B. Giannas, B. Kapadia, J. Meakins, and H. Rode, Ann. Surg. 210, 69 (1989). 31 j. D. Fletcher, G. A. Koch, and E. Endres, Aust. N.Z.J. Surg. 56, 17 (1986). 32 S. N. Meydani, M. Meydani, C. P. Verdon, A. C. Shapiro, J. B. Blumberg, and K. C. Hayes, Mech. Ageing Dev. 34, 191 (1986). 33 S. N. Meydani, M. P. Barklund, S. Liu, M. Meydani, R. A. Miller, J. G. Cannon, F. D. Morrow, R. Rocklin, and J. B. Blumberg, Am. J. Clin. Nutr. 52, 557 (1990). 34 S. N. Meydani, M. Meydani, J. B. Blumberg, L. S. Leka, G. Siber, R. Loszewski, C. Thompson, M. C. Pedrosa, R. D. Diamond, and B. D. Stollar, J A M A 277, 1380 (1997). 35 M. Chavance, G. Brubacher, and B. Herbeth, in "Lymphoid Cell Functions in Aging" (A. L. DeWick, ed.), p. 231. Eurage, Paris, 1984. 36 D. Trickier and G. Shktar, JNC 78, 1615 (1987). 37 M. S. Menkes, G. W. Comstock, J. P. Vuilleumier, K. J. Helsing, A. A. Rider, and R. Brookmezer, N. Engl. J. Med. 315, 1250 (1986). 38 R. M. Bostick, J. D. Potter, and D. R. McKenzie, Cancer Res. 53, 4230 (1993).
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procedures for assessing the basic immunologic function of lymphocytes: measurement of proliferative responses and cytokine production to a variety of stimuli.
Proliferation Assay General Measurement of proliferative responses by lymphocytes is a fundamental technique for the assessment of their biological responses to various stimuli. The assessment of cellular proliferation is perhaps the most often used technique in cellular immunology. Lymphocytes proliferate in response to mitogens, antigens, allogeneic or autologous cells, and many soluble factors. Quantitating the proliferative response involves measuring the number of cells present in a culture before and after the addition of a stimulating agent. Although cellular proliferation can be assessed by determining the increase in viable cell numbers through their actual enumeration using direct microscopy or automated cell counting, this approach can be time consuming. Therefore, the two most commonly used techniques are reduction of tetrazolium dyes (MqT) by active mitochondria or incorporation of tritiated thymidine into newly synthesized D N A . 39 Cell proliferation determined by estimating incorporation of [3H]thymidine into DNA is a process that is related to underlying changes in cell number. As cells enter the S phase of the cell cycle, chromosome replication takes place through the incorporation of soluble nucleotide precursors into newly synthesized DNA. In this assay, dividing cells are incubated (pulsed) with radioactive thymidine for several hours after which the amount of radioactivity incorporated into their DNA is determined. 39 Proliferative assays are applied in clinical studies as an assessment of overall immunologic competence of lymphocytes, as manifested by their ability to respond to proliferation signals. Defects in proliferation may be indicative of a fundamental cellular immunologic defect. Low proliferation is often found as a nonspecific secondary effect of chronic disease. 39 Procedure for Mitogen-Induced Proliferation of Peripheral Blood Mononuclear Cells Utilizing Incorporation of [3H]Thymidine into DNA 1. Peripheral blood mononuclear cells (PBMCs) are separated by density gradient from heparinized blood. Several procedures can be used to accomplish this. 4° 2. PBMCs are washed twice in RPMI 1640 medium supplemented with 100 mg/liter penicillin, 100 mg/liter streptomycin, 100 ml/liter heat39 R. Fernandez and V. Vetvivka, "Methods in Cellular Immunology." CRC Press, Boca Raton, Florida, 1995. 40 A. I. Boyum, Scand. J. Clin. Invest. 97 (Suppl. 21), 77 (1968).
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inactivated autologous plasma, L-glutamine (final concentration 2 mmol/ liter), and 25 mmol HEPES (final concentration 25 mmol/liter) (complete RPMI). Cells are resuspended in complete RPMI and counted under a light microscope. Cell viability is assessed by using the trypan blue exclusion method. Cell concentration is adjusted to 1 x 106 cell/ml with complete RPMI. 3. One hundred microliters of the cell suspension is pipetted into each well of a 96-well plate (1 x 105 cells/well). Triplicate wells are prepared for each experimental condition. Wells with no mitogen are included to measure background response. 4. Dilutions of mitogens between 1 and 100/xg/ml for P H A and concanavalin A (Con A) and 0.015% to 0.15% (w/v) for Staphylococcus aureus are prepared in complete RPMI. Optimal mitogen concentrations should be predetermined for each individual experimental condition. One hundred microliters of the diluted mitogen solution is added to each well. 5. Plates are incubated for 72 hr in a humidified 37°, 5% (v/v) CO2 incubator. 6. Four hours before termination of incubation, wells are pulsed with 20/zl of 25/zCi/ml [3H]thymidine. 7. Cells are harvested using an automated harvester, which aspirates the wells, lyses the cells, and retains DNA on glass filter paper, while allowing unincorporated [3H]thymidine to wash through. 8. Filters are air dried and placed into a liquid scintillation cocktail. 9. Counting is done using a liquid scintillation counter. 10. Results are reported as corrected counts per minute (cpm): the average cpm of mitogen-stimulated cultures minus the average cpm of cultures without mitogens. Alternatively, proliferation data can be expressed as the stimulation index, calculated by dividing the proliferation of the stimulated cells in cpm by the background proliferation.
Interpretation of Proliferation Assay Results. The relative ease of acquiring cells and measuring lymphocyte proliferation has made this assay a popular measure of immune function. In many cases, the proliferation response appears to be correlated with the vitamin E content of the diet. However, results from this assay must be critically analyzed before a recommendation about the vitamin E status of an individual is made. Cell proliferation in response to external stimuli is a very complex process often involving delivery of a signal or set of signals to cell membranes, activation of intracellular enzymatic pathways which are not well understood, activation and transcription of multiple genes, D N A and protein synthesis, and, finally, cell division. The mechanism of how vitamin E status affects the proliferative response is not clear.
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Experimental conditions must be thoroughly worked out for the proliferation assay to be of value. Cell populations used in this protocol must have high viability and contain an appropriate cell population for the response being tested. The composition of the culture medium, and especially the serum source, can be critical. 41 The selection of mitogen is important. There can be considerable variability in individual responses to particular activating agents; for example, a small portion of the normal human population has a low or absent response to pokeweed mitogen. In addition, the level of mitogen utilized becomes very important in the interpretation of proliferative responses. Effect o f Vitamin E on Mitogen-Induced Lymphocyte Proliferation. The level of in vitro proliferation by lymphocytes in response to mitogens is influenced by vitamin E level. Lymphocytes derived from animals maintained on vitamin E-deficient diets show depressed mitogenic response to T-cell mitogens, a defect that is reversible following vitamin E supplementation. 42 Eskew et al. 43 found that either vitamin E or selenium deficiency suppressed rat lymphocyte proliferation. Low levels of dietary vitamin E, which were sufficient to protect against many of the adverse effects of vitamin E deficiency, were insufficient to enhance lymphocyte proliferative responses even though the diet contained all other nutrients at recommended levels. Addition of vitamin E above the recommended dietary levels enhances mitogenesis in mixed populations of lymphocytes from humans and a variety of animal species compared to controls. 5 Cells from healthy elderly humans consuming a diet supplemented with 800 IU/day of vitamin E demonstrated increased mitogenic responses to optimal doses of Con A compared to cells from elderly subjects consuming the R D A . 33 Moriguchi et al. 11 reported that as dietary intakes of vitamin E increased from 100 to 2500 mg/kg of diet, there was a corresponding increase in rat splenic lymphocyte responses to Con A. Lymphocytes from mice supplemented with varying levels of vitamin E only showed enhanced Con A proliferation when dietary levels were in excess of the vitamin content of normal chow diet. 44 Interleukin-2 Production General. The T-cell growth factor, IL-2, is a lymphokine produced by T-helper type 1 cells following induction by mitogens such as lectin or by 41 S. P. James, in "Current Protocols in Immunology" (R. Coico, ed.), p. 7.10.1. John Wiley & Sons, New York, 1995. 42 A. Bendich, in "Antioxidant Nutrients and Immune Functions" (A. Bendich, M. Phillips, and R. P. Tengerdy, eds.), p. 36. Plenum Press, New York, 1990. 43 M. L. Eskew, R. W. Scholz, C. C. Reddy, D. A. Todhunter, and A. Zarkower, Immunology 54, 173 (1985). 44 M. Corwin and J. Shloss, J. Nutr. 110, 916 (1980).
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a specific antigen.45 IL-2 induces proliferation and differentiation of both T and B cells and thus, to a major degree, it regulates the magnitude and duration of the immune response. In addition to promoting the proliferation of cells, IL-2 induces the release of cytokines such as interferon gamma from activated T lymphocytes.46 Vitamin E level in the diet has been shown to influence IL-2 production. It has been hypothesized that vitamin E may exert some of its immunostimulatory effects by indirectly increasing IL-2 production. 32 Expression, synthesis, and secretion of IL-2 can be investigated by a variety of techniques. The following assay can be used to determine biologically active, secreted IL-2 in culture supernatants or biological fluids. This assay utilizes clones of the murine CTLL cell line first described by Gillis et al. 47 Other cell lines that can be used include HT-2, FDC-2, or MT-I. CTLL cells proliferate in the presence of IL-2, and to a lesser extent, in the presence of murine IL-448 and IL-15. 49 Proliferation can be measured by using [3H]thymidine or MTT. Procedure for Production of Interleukin 2 in Vitro. Cells are cultured in the presence or absence of phorbol myristic acetate (PMA), PHA, or Con A in 12-well plates. Plates are incubated in a 5% CO2 incubator at 37 °. Following 48 hr of incubation, supernatants are collected and stored at - 2 0 ° until analyzed.
Procedure for Detection of Biologically Active Human Interleukin
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1. The murine IL-2-dependent T-cell line, CTLL-2, is used to monitor IL-2 production. Prior to conducting the assay, logarithmically growing cells are washed three times with media to remove any residual IL-2. 2. Washed CTLL-2 cells are cultured at a concentration of 5 × 103 cells/well (100-/zl well) in 96-well microtiter plates in the presence of serially diluted test or control supernatants (100-/zl well). 3. The recombinant IL-2 standard and samples are diluted in complete RPMI. Culture supernatants are diluted 1:1-1:4. The IL-2 standard is titrated such that the least dilution allows the cells to achieve maximum DNA synthesis (positive control), usually at 20-40 U/ml (this is dependent 45 K. A. Smith, Science 240, 1169 (1988). 46 E. S. Kimball and P. M. Grob, in "The Lymphocyte: Structure and Function" (J. J. Marchalonis, ed.), p. 71. Marcel Dekker, New York, 1988. 47 S. Gillis, M. M. Ferm, W. Ou, and K. A. Smith, J. ImrnunoL 120, 2027 (1978). 48 R. Fernandez-Botran, P. H. Krammer, T. Diamanstein, J. W. Uhr, and E. S, Vitetta, J. Exp. Med. 164, 580 (1986). 49 K. H. Grabstein, J. Eisenman, K. Shanebeck, C. Rauch, S. Srinivasan, V. Fung, C. Beers, J. Richardson, M. Schoenborn, M. Ahdieh, L. Johnson, M. R. Alderson, J. D. Watson, D. M. Anderson, and J. G. Girl, Science 264, 965 (1994). 50 L. S. Davis, P. E. Lipsky, and K. Bottomly, in "Current Protocols in Immunology" (R. Coico, ed.), p. 6.3.1. John Wiley & Sons, New York, 1995.
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on the brand of recombinant IL-2 used). A typical standard curve ranges from 20 U/ml down to 0.3 U/ml recombinant IL-2. 4. CTLL-2 cells cultured in the absence of IL-2 serve as background proliferation (negative control). 5. After an 18-hr incubation at 37° in 5% (v/v) CO2 incubator, wells are pulsed with 0.5/~Ci [3H]thymidine. After 6 hr, the cells are harvested onto glass fiber filters with an automatic cell harvester and are then dried. 6. Isotope uptake (cpm) is determined using a liquid scintillation counter.
Interpretation of Interleukin-2 Bioassay Results. We have described a biological assay for IL-2. This bioassay has the advantage of being more sensitive than many of the other available techniques, and it can differentiate biologically active forms from inert forms of the cytokine. However, care must be taken to rule out the involvement of contaminating cytokines. Almost all the cloned murine cell lines used to measure IL-2 activity are also capable of responding to murine IL-4 and IL-15. 48'49'51 Other compounds present in serum can potentially influence the outcome of an IL-2 bioassay. 39'51 CTLL cells are susceptible to inhibitors (e.g., cytokine antagonists, soluble cytokine receptors) present in biological fluids. In addition, stimulants that may be in the samples (e.g., mitogens) may have unforeseen effects on the CTLL responses. Therefore, appropriate controls must always be included. Controls should include negative samples such as medium alone, and medium plus any stimulants. Antibodies against IL-4 and IL-15 should be included in samples containing these cytokines in addition to IL-2. Appropriate controls must be included in every assay because CTLL cells are subject to assay-to-assay variability in their responsiveness to IL-2. Effect of Vitamin E Status on Interleukin 2 Production. IL-2 production can be influenced by the level of available vitamin E. Elderly individuals produce less IL-2 than their young counterparts. This age-induced reduction in IL-2 production can be partially reversed by vitamin E supplementation above the RDA. For example, Meydani et al. 32 reported that older mice fed diets containing 500 ppm vitamin E had enhanced IL-2 production compared to mice fed a diet containing 30 ppmP 2 In addition, elderly healthy humans supplemented with 800 IU/day dl-a-tocopherol for 30 days had increased Con A-induced IL-2 production versus nonsupplemented subjects. 33
51 j. L. Rossio and A. J. H. Gearing, in "Clinical Applications of Cytokines: Role in Pathogenesis, Diagnosis and Therapy" (J. J. Oppenheim, J. Rossio, and A. Gearing, eds.), p. 16. Oxford University Press, New York, 1993.
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Microbial disease can reduce IL-2 production. Human immunodeficiency virus (HIV)-positive persons and mice infected with murine acquired immunodeficiency (MAIDS) produce less IL-2 than uninfected controls. Vitamin E supplementation over the murine requirement increased IL-2 production by splenocytes from mice infected with the retrovirus that causes MAIDS. 52 Mononuclear Phagocytic Cell Functions Affected by Vitamin E Status General. Mononuclear phagocytic cells are often called accessory cells and include monocytes and macrophages. An accessory cell helps lymphocytes, enabling them to carry out their function of recognition and elimination of antigens. Accessory cells that can present antigens to lymphocytes are called antigen-presenting cells and include mononuclear phagocytes. These cells play a crucial role in the immune system through their functions as antigen-presenting cells and as regulatory cells, which synthesize and release soluble immune-enhancing and -suppressing cytokines. Because of the diversity of their functions, mononuclear phagocytes are key cellular elements in the immunological control of a number of diseases. The mononuclear phagocyte is a highly secretory cell. The variety of secreted molecules is large and includes cytokines that are involved in the acute inflammatory response as well as lymphocyte response. Macrophage secretion of several cytokines appears to be affected by vitamin E level. However, the literature has been inconsistent in characterizing this effect. Two cytokines that consistently change concentration in response to vitamin E are TNF-a and IL-6. Cytokine Production of Interleukin-6 and Tumor Necrosis Factor-a. IL-6 is produced by many cell types in response to a variety of immunological or pathological stimuli. The most potent sources include endotoxin-stimulated monocytes and macrophages and IL-l-stimulated fibroblasts. There are many assays available to detect IL-6, including commercially available enzyme-linked immunosorbent assay (ELISA) kits. Biologically active IL-6 can be detected using IL-6-dependent murine hybridomas. The most commonly used hybridoma lines are B-9 and 7TD1. 53,54 The general bioassay procedure was described previously in more detail for IL-2. Briefly, the hybridomas are maintained in recombinant IL-6, washed prior to assay,
52y. Wang,D. S. Huang,S. Wood,and R. R. Watson,Immunopharmacology 29, 225 (1995). 53j. Van Snick,S. Cayphas,A. Vink, C. Uyttenhove,P. Coulie,and R. Simpson,Proc. Natl. Acad. Sci. U.S.A. 83, 9679 (1986). 54L. A. Aarden, E. R. De Groot, O. L. Schaap, and P. M. Lansdorp, Eur. J. ImmunoL 17, 1411 (1987).
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and the cells are combined with the samples to be analyzed. Proliferation is measured 3 days later by [3H]thymidine incorporation. 39 TNF-a is produced by activated macrophages and other cells and has a broad spectrum of biological actions on many different target cells, both immune and nonimmune. TNF is considered a major inflammatory mediator. TNF can be measured by both ELISA and biological assay. There are many commercially available ELISA kits for detecting TNF. The most commonly used bioassay for TNF is a cytotoxicity assay based on murine fibroblasts such as L929 or L-M cells. These cells deteriorate and die over a 3-day period in the presence of TNF. Sensitivity levels vary with cell isolates, passage number, and temperature. 55 EFFECT OF VITAMIN E STATUS ON TUMOR NECROSIS FACTOR-Or AND INTERLEUKIN-6 SECRETION BY MONONUCLEAR PHAGOCYTES. Infection with
the retrovirus that causes AIDS in humans and MAIDS in mice results in elevated levels of IL-6 and TNF-a as the disease progresses. 52'56'57 Supplementation over the murine requirement with vitamin E appears to lower the retrovirus-induced production of IL-6 and TNF in mice. 21'57 Wang et a/. 2t'52 reported that mice infected with MAIDS and supplemented with vitamin E at 15-450 times the NRC recommendation showed normalization of IL-6 and TNF-a produced by splenocytes during progression to MAIDS at all levels of vitamin E supplementation. Moreover, aging affects IL-6 and TNF-ot production. Depending on the experimental conditions, aging decreased or increased production of IL-6 and TNF. 58'59Vitamin E supplementation of elderly human subjects inhibited IL-6 production and prevented the exercise-induced increase in TNF-a production by peripheral blood mononuclear cells. 6°'61 Dietary alcohol (ethanol) consumption decreases production of IL-6 and TNF-ot. When a 15-fold increase over the mouse NRC-recommended level of vitamin E was supplied to ethanol-fed mice for 10 weeks, production of IL-6 by Con A-stimulated splenocytes, and IL-6 and TNF-o~ by LPSstimulated splenocytes was restored. 62 55 M. M. Hogan and S. N. Vogel, in "Current Protocols in Immunology" (R. Coico, ed.), p. 6.10.1. John Wiley & Sons, New York, 1993. 56 F. Boue, C. Wallon, C. Goujard, F. Barresinouss, P. Galand, and J. F. Defraissy, J. Imrnunol. 148, 3761 (1992). 57 L. Y. Wang, D. S. Huang, P. T. Giger, and R. R. Watson, Adv. Biol. Sci. 86, 335 (1993). 58 R. B. Effros, R. L. Walford, R. Weindruch, and C. Mitcheltree, J. Gerontol. 46, B142 (1991). 59 j. G. Cannon, S. F. Orencole, R. A. Fielding, M. Meydani, S. N. Meydani, M. A. Fiatarone, J. B. Blumberg, and W. J. Evans, Am. J. Physiol. 259, R1214 (1990). 6o j. G. Cannon, S. N. Meydani, R. A. Fielding, M. A. Fitarone, M. Meydani, M. Farhangmehr, S. F. Orencole, J. B. Blumberg, and W. J. Evans, Am. J. Physiol. 260, R1235 (1991). 61 y. Wang, D. S. Huang, P. T. Giger, and R. R. Watson, Alcohol. Clin. Exp. Res. 18, 64 (1994). 62 S. M. Wahl, J. B. Allen, S. Gartner, J. M. Orenstein, M. Popovic, D. E. Chenoweth, L. O. Arthur, W. L. Farrar, and L. M. Wahl, J. lmmunol. 142, 3553 (1989).
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Phagocytosis and Chemotaxis Phagocytosis is the engulfment of cells, microorganisms, and particulate materials. Phagocytosis is very important in a variety of circumstances including the clearance of microbial infections and in the daily turnover of senescent tissues and cells. Chemotaxis is important because the phagocytes must be able to move to where they are needed. Factors that inhibit macrophage migration can have a deleterious effect on host protective function.63 Phagocytosis can be measured a variety of ways. The following protocol is used for the measurement of Fcy receptor-mediated binding and phagocytosis.64 PHAGOCYTOSIS ASSAY.
1. Sheep red blood cells (SRBC) are maintained in Alsever's solution (1:1, v/v) at 4°. 2. Five milliliters SRBC are placed into a 15-ml conical centrifuge tube, diluted to 15 ml with physiological saline, and centrifuged at 300g for 10 min at room temperature. Discard supernatant and repeat wash two times. Count using a hemacytometer. Resuspend SRBC at 1 × 108 cells/ml in saline. 3. Opsonization is accomplished by incubating 10 ml of SRBC suspension with 200/xl of rat anti-SRBC antiserum (heat inactivated: optimum concentration previously determined) for 60 min at 37°. 4. Radiolabeling of the opsonized SRBC is accomplished by incubation with 200/zCi of sodium [51Cr]chromate for 1 hr at 37°. The opsonized 51Crlabeled SRBC cultures are then washed three times with RPMI to remove excess S~Cr, and the final volume is adjusted to give a 0.6% suspension of SRBC. Prepare macrophage/monocyte samples. 1. Purified macrophages/monocytes are washed two times by adding 10 ml PBS, centrifuging at 1200 rpm for 10 min at 4°, and discarding supernatant. Cells are resuspended in complete RPMI to a final concentration of 2 × 10 6 cells/ml. 2. One hundred microliters of cell suspension is added to each well of a 96-well fiat bottom tissue culture plate. Prepare at least three replicate wells for each treatment and include positive and negative controls. Allow cells to adhere (4 hr in a 5% CO2, 37° incubator), then add treatment (interferon) if needed. 3. The opsonized [51Cr]SRBC cultures are added. After 2 hr at 37°, the 63 A. D. Politis and S. N. Vogel, in "Current Protocols in Immunology" (R. Coico, ed.), p. 14.8.1. John Wiley & Sons, New York, 1995. 64 R. E. Harris, L. A. Boxer, and R. L. Bahner, Blood 55, 338 (1980).
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cultures are rinsed once with distilled water to lyse nonphagocytosed SRBC and then washed twice with 0.1 mol/liter phosphate buffer, pH 7.2. All remaining cells are lysed with 0.1 mol/liter NaOH. 4. The radioactivity of the lysate is measured in a gamma counter. The stimulation index is calculated by assigning a value of 1 to the phagocytic activity of control macrophages incubated with medium only, then by comparing this value to the phagocytic activity of macrophages from each treatment. EFFECT OF VITAMIN E ON PHAGOCYTIC CELL RESPONSES. T h e e f f e c t s
of dietary vitamin E on phagocytic cell function have been well documented. Rats fed vitamin E-deficient diets exhibit impaired macrophage chemotaxis, reduced ability to ingest complement-coated beads, and decreased protection from autooxidative damage. 65 Dietary vitamin E supplementation has been shown to ameliorate endotoxin-induced inhibition of monocyte migration and phagocytosis in r a t s , 66 t o reduce immunosuppressive cytokine production by macrophages in mice, and to normalize monocyte chemotaxis in humans with diabetes. 67 The ability of alveolar macrophages to phagocytose opsonized sheep red blood cells has been reported to increase with increasing concentrations of vitamin E in diets and showed a fivefold increase in rats fed the diet with the highest vitamin E content compared to rats in the control group. 11 Yano and Ichikawa 67 reported that alveolar macrophages from vitamin E-supplemented rats demonstrated enhanced phagocytosis of opsonized particles.
Mechanism The mechanism underlying the immunostimulatory effect of vitamin E has not been completely delineated. 6 Although some of the effects of vitamin E on immune cell functions can be attributed to its antioxidant activities, other mechanisms appear to be involved. 6a,69It has been hypothesized that one of these alternative mechanisms is the reduction of immunoinhibitory molecules secreted by immune cells themselves, particularly by macrophages. Activated macrophages secrete molecules such as H2027°,vI and prostaglandin E2 (PGE2), which have been shown to depress lymphocyte 65 N. P. Rocha, Brazilian J. Med. Biol. Res. 22, 1401 (1989). 66 n . R. Hill, N. H, Augustine, M. L. Rallison, and J. I. Santos, J. Clin. Immunol. 3, 70 (1983). 67 T. Yano and T. Ichikawa, Nutr. Res. 14, 1387 (1994). 68 L. M. Corwin and J. Shloss, Z Nutr. 110, 2497 (1980). 69 n . Sies, Am. J. Med. 91, 31 (1991). 70 R. I. Fisher and F. Bostick-Bruton, J. Immunol. 129, 1770 (1982). 71 Z. Metzger, J. T. Hoffeld, and J. J. Oppenheim, J. lmmunol. 124, 938 (1980).
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proliferation, 7°'72,73 lymphokine production, TM and the generation of cytotoxic T cells.75 Vitamin E modulates the formation of these potentially immunosuppressive molecules, particularly PGE2. Peritoneal macrophages and splenocytes from mice supplemented with vitamin E produced less PGE2 after stimulation than unsupplemented control mice. 32'76 Additional research is needed to understand fully the mechanisms of the effect of vitamin E on immune responsiveness. Summary Evidence from animal and human studies indicates that vitamin E plays an important role in the maintenance of the immune system. Even a marginal vitamin E deficiency impairs the immune response, while supplementation with higher than recommended dietary levels of vitamin E enhances humoral and cell-mediated immunity. The current R D A level of vitamin E prevents clinical deficiency syndrome but in some situations, especially in older subjects or in a disease state, fails to maintain optimal host defense. The immunological parameters reviewed are all sensitive to changes in the availability of vitamin E and, therefore, may reflect the vitamin E status of a given individual more accurately than conventional methods. Acknowledgments This project has been funded at least in part with federal funds from the U.S. Department of Agriculture, Agricultural Research Service, under contract 53-K06-01. The contents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government. The authors thank Timothy S. McElreavy for preparation of this manuscript.
v2 M. Rola-Plezczunski, Immunol. Today 6, 302 (1985). 73 D. R. Webb, T. J. Rogers, and E. Nowoiejski, Proc. Natl. Acad. Sci. U.S.A. 332, 260 (1980). 74 R. P. Phipps, S. H. Stein, and R. L. Roper, lmrnunol. Today 12, 349 (1991). 75 M. Plaut, J. Immunol. 123, 692 (1979). 76 E. H. Romach, S. Kidao, B. G. Sanders, and K. Kline, Nutr. Cancer 20, 205 (1993).
VITAMIN E STATUS AND IMMUNE FUNCTION
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and Immune
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Function
ALISON BEHARKA, SUSAN REDICAN, LYNETrE LEKA,
and SIMIN NIKBIN MEYDANI
Introduction Vitamin E is the generic name used to describe eight naturally occurring, fat-soluble compounds known as tocopherols and tocotrienols. Vitamin E has many functions in the body, including protecting cell membranes from oxidative damage. I Although vitamin E is a constituent of all cellular membranes, it is found in high concentrations in the membranes of immune cells because they are at especially high risk for oxidative damage. 2-4 In fact, vitamin E is essential for normal function of the immune system. 5,6 A deficiency in vitamin E diminishes the ability of the immune system to respond to infectious microorganisms, to produce a delayed-type hypersensitivity (DTH) reaction, or to mount an antibody response to antigen. 7-9 Under some conditions, a pharmacological level of vitamin E is needed to achieve an optimal immune response. 8,1°In several studies, data have shown that elderly humans, as well as laboratory and farm animals, consuming diets that contain more than five times the recommended dietary allowance (RDA) of vitamin E for their species had significantly increased humoral and cell-mediated immune responses and increased resistance to infectious diseases compared to nonsupplemented controls. 8,1°-15These results suggest 1 L. Packer and V. E. Kagan, in "Vitamin E in Health and Disease" (L. Packer and J. Fuchs, eds.), p. 179. Marcel Dekker, New York, 1993. 2 A. Coquette, B. Vray, and J. Vanderpas, Arch. Int. Physiol. Biochem. 94, 529 (1986). 3 L. J. Hatman and H. J. Kayden, J. Lipid Res. 20, 639 (1979). 4 L. J. Machlin, in "Handbook of Vitamins: Nutritional, Biochemical, and Clinical Aspects" (2nd ed.), p. 99. Marcel Dekker, New York, 1991. 5 S. N. Meydani and R. P. Tengerdy, in "Vitamin E: Biochemical and Clinical Applications" (L. Packer and J. Fuchs, eds.), p. 549. Marcel Dekker. New York, 1991. S. N. Meydani and M. Hayek, in "Proceedings of the International Congress on Nutrition and Immunity," p. 105. ARTS Biomedical Publishers, St. Johns, Newfoundland, 1992. 7 M. L. Scott, Fed. Proc. 39, 2726 (1980). 8 R. P. Tengerdy, Avian Dis. J. 34, 848 (1990). 9 K. N. Jeejeebhoy, in "Modern Nutrition in Health and Disease" (M. E. Shils, J. A. Olson, and M. Shike, eds.), p. 805. Lea and Febiger, Philadelphia, 1994. 10A. Bendich, G. E. Gabriel, and L. J. Machlin, J, Nutr. 116, 675 (1986). 11 S. Moriguchi, N. Kobayashi, and Y. Kishino, J. Nutr. 120, 1096 (1990). 12 R. P. Tengerdy, in "Antioxidant Nutrients and the Immune Response" (A. Bendich, M. Phillips, and R. Tengerdy, eds.), p. 103. Plenum Press, New York, 1989.
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that under some conditions the R D A for vitamin E might not be adequate for immunological vigor and health. 16 The vitamin E requirement for ideal i m m u n e function depends on its interactions with other antioxidant and pro-oxidant nutrients, especially with polyunsaturated fatty acids, and on other factors that modulate the i m m u n e response, such as age and stress. 5,17 Elderly individuals respond to supplementation levels of vitamin E above the R D A with increased immunological vigor including enhanced D T H responsivenessJ 8 Epidemiological studies have shown correlations between high plasma vitamin E levels and decreased incidence of infections in the e l d e d y J 9,2° Recent studies also suggest that vitamin E supplementation above the R D A m a y improve the i m m u n e responsiveness of mice infected with the murine acquired immunodeficiency syndrome ( A I D S ) virus. 21 Therefore, conventional methods for determining the R D A , while adequate for arriving at the level of vitamin E required to prevent clinical deficiency symptoms, m a y not adequately predict the optimal level of vitamin E needed to maintain immunological health. Because vitamin E is involved in the maintenance of i m m u n e function, which appears to be especially sensitive to changes in vitamin E status, it m a y be possible to utilize selected immune p a r a m e t e r s as indicators of vitamin E status. This chapter provides protocols to study selected cell-mediated immune responses that are influenced by vitamin E status. In addition, quality control measures for h u m a n studies involving i m m u n e p a r a m e t e r s are discussed. G e n e r a l Q u a l i t y Control M e a s u r e s for H u m a n S t u d i e s M a n y m e a s u r e m e n t s of i m m u n e function are subject to interpretation. Therefore, reliable test results can only be achieved by assay standardization. This includes, but is not limited to, the following criteria:
I3 A. Bendich, Ann. N.Y. Acad. Sci. 587, 168 (1990). z4S. N. Meydani and J. B. Blumberg, in "Micronutrients in Health and in Disease Prevention" (A. Bendich and C. E. Butterworth, Jr., eds.), p. 289. Marcel Dekker, New York, 1991. 15M. Meydani, S. N. Meydani, L. Leka, J. Gong, and J. B. Blumberg, F A S E B J. 7, A415 (1993). 16L. M. Corwin and R. K. Gordon, Ann. N.Y. Acad. Sci. 393, 437 (1982). 17M. L. Eskew, W. J. Scheuchenzuker, R. W. Scholz, C. C. Reddy, and A. Zarkower, Environ. Res. 40, 274 (1986). 18S. N. Meydani and J. B. Blumberg, in "Nutritional Modulation of the Immune Response" (S. Cunningham-Rundles, ed.), p. 223. Marcel Dekker, New York, 1992. 19M. Chavance, G. Brubacher, B. Herbeth, in "Lymphoid Cell Functions in Aging" (A. L. Dewick, ed.), p. 231. Eurage, Interlaken, 1984. 2oM. Chavance, G. Brubacher, B. Herbeth, G. Vernhers, T. Mistacki, F. Dete, C. Fournier, and C. Janot, in "Nutritional Immunity and Illness in the Elderly" (R. K. Chandra, ed.), p. 137. Pergamon Press, New York, 1985. 21y. Wang, D. S. Hwang, B. Licing, and R. R. Watson, J. Nutr. 124, 2024 (1994).
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1. Potential subjects should be carefully screened before acceptance into a study. Exclusion criteria include most chronic medical conditions such as diabetes, cardiovascular or neoplastic diseases, calcium channel blocker therapy, steroidal or nonsteroidal antiinflammatory drug use, smoking or excessive alcohol use, nutrient supplementation. 2. At each visit, subjects should be asked a standardized set of questions regarding recent illness, medication, vaccinations, and stress. Protocols should be developed before the study begins specifying the conditions under which a subject may be excluded from the study. For example, a subject must not receive a flu vaccine less than 1 month before an immune test. In addition, the recorded answers to standardized questions may allow for interpretation of unusual results when the data are analyzed. 3. Theoretically, performance and evaluation of all tests should be done by the same technician; however, this may not be possible for large studies. During a longitudinal study, the subjects may need to be divided among more than one technician. Within a cross-sectional study, alternate technicians may be utilized as long as they have been identically trained and demonstrate low variability in replicating each other's observations. 4. Any test kits, reagents (especially mitogens), and media should be purchased from the same lot from the same company. 5. Appropriate controls, both positive and negative, must be included every time a test is performed. 6. A reliable baseline value for the parameter being tested must be determined for each subject. This will require multiple sampling. For example, three baseline blood draws within 2 weeks of each other would be utilized for tests involving blood mononuclear cells. 7. Common sense should be used at all times. For example, D T H skin test antigens should not be applied at sites that are difficult to assess due to pigmentation, inflammation, or extreme hairiness.
Cell-Mediated Immune Responses General
Cell-mediated immunity is an immune response specific to antigens and mediated by lymphocytes and macrophages with minor participation by other cell types. Cellular immunity is responsible for D T H reactions, foreign graft rejection, resistance to many pathogenic microorganisms, and tumor immunosurveillance.22 22 M. S. Meltzer and C. A. Nacy, in "Fundamental Immunology" (W. E. Paul, ed.), 2nd ed., p. 765. Raven Press, New York, 1989.
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D e l a y e d - T y p e Hypersensitivity Reaction
The D T H reaction is used as an in vivo assay to determine cell-mediated immune function. D T H is an antigen-specific, T-cell-dependent, recall response manifested as an inflammatory reaction that reaches peak intensity approximately 24-48 hr after antigenic challenge in primed animals. It is the kinetics of the inflammatory process following deposition of antigen in the skin that gives this response its name. Whereas allergic reactions occur within minutes and immune complex reactions occur within a day, D T H reactions peak at 2 days. 22,23 The D T H test involves introducing a relatively small amount of soluble antigen into the epidermis and superficial dermal tissue by needle puncture. Circulating T cells sensitized to the antigen from prior contact react with the antigen in the skin and induce a specific immune response, which includes mitosis (blastogenesis) and the release of soluble mediators (cytokines). Concurrently, antigen is encountered and processed by macrophages. The macrophages are activated to produce and release cytokines such as interleukin 1 (IL-1) and tumor necrosis factor (TNF). The processed antigen is presented by macrophages to T lymphocytes, which then produce cytokines such as IL-2 and interferon 3' (IFN). The cytokines produced by the activated macrophages and T cells are involved in the inflammation associated with a D T H reaction, The intensity of the overall dermal inflammation reaches its peak 24-48 hr after antigen application and is resolved within days or weeks. 22 Measuring the intensity of D T H involves quantitating some aspect of the local inflammatory response. In humans and guinea pigs, this is readily done by measuring the redness and induration of an area of shaven skin. This approach has failed in mice; however, alternative assays that involve measuring changes in tissue thickness of the footpad with a caliper or determining cellular infiltration have proven reliable. 22-24 D T H as a tool for assessing immunocompetence in vivo has been widely used for many years. 25 In humans, D T H was traditionally evaluated by the intradermal reactions observed following Mantoux-like techniques. However, this method has the potentially serious side effect of boosting D T H reactivity after repeated testingY '26 An alternative method that results in 23y. Luo and M. E. Doff, in "Current Protocols in Immunology" (J. E. Coligan, A. M. Kruisbeek, D. H. Margulies,E. M. Shevach,and W. Strober, eds.), p. 4.5.1. John Wiley& Sons, New York, 1995. 24M. A. Vadas, J. F. A. P. Miller, J. Gamble, and A. Whitelaw, Int. Arch. Allergy Appl. ImmunoL 49, 670 (1975). 2sB. M. Lesourd, A. Wang, and R. Moulias,Ann. Allergy 55, 729 (1985). 26E. C. Keystone,P. Demerieux, and D. Gladman, Clin. Exp. Immunol. 40, 202 (1980).
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minimal booster effect on DTH response is Multi-test CMI (Merieux Institute, Inc., Miami, FL), a multipuncture system that permits simultaneous application of seven antigens and a negative controlY
Multi-Test CMI for the Determination of DTH in Human Subjects 1. Select a test site. Preferred sites are the volar surfaces of the arms and the back. The site should be easily accessible for reading. Cleanse the site with ethanol and allow the area to dry. Identify the area by drawing lines above and below the test site. 2. A single-use disposable applicator is used to deliver several recall antigens and a control antigen into the epidermis and superficial dermal tissue by puncture. Commonly used recall antigens and respective concentrations include tetanus toxoid antigen (biologically equivalent to 5.5 x 105 Merieux tetanus units), diphtheria toxoid antigen (1.1 x 10 6 Merieux diphtheria units), Streptococcus antigen (2000 Merieux Streptococcus units), tuberculin antigen (3 × 105 U.S. tuberculin units), Candida antigen (2000 Merieux Candida units), Trichophyton antigen (150 Merieux Trichophyton units), and Proteus antigen (150 Merieux Proteus units). A negative control must also be applied and should contain the same solution that serves as a vehicle for the skin test antigens. In most cases, the vehicle will be a 70% (w/v) sterile glycerin solution. 3. Test sites are evaluated at both 24 and 48 hr. The larger reaction recorded from the two readings is used. The size of an indurated area is determined by inspection and palpation followed by measurements across two diameters at right angles. These measurements are averaged to determine mean diameter. An induration of 2 mm or greater at a test site is considered positive, providing there is no induration at the negative control site. 4. An antigen score is calculated as the total number of positive antigens, and the cumulative score is calculated as the total diameter of induration of all the positive reactions.
Interpretation of Delayed-Type Hypersensitivity Results. Delayed cellular hypersensitivity is a valuable measure of immune response because it involves a complex series of immunologic, cellular, mediator-associated, and vascular effects. Although numerous in vitro tests have been developed to assess T-cell activity, these in vitro assays do not necessarily monitor the same cells that mediate DTH nor do they consider the influences of regulatory events that may influence in vivo T-cell function. Therefore, local DTH 27 W. T. Kniker, C. T. Anderson, J. L. McBryde, M. Roumiantzeff, and B. Lesourd, Ann. Allergy 52, 75 (1984).
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responses represent an important source of information concerning in vivo T-cell function, z2,23 Clinically, D T H skin tests are of value in the overall assessment of immunocompetence. The inability of human adults to react to a battery of common skin antigens suggests hyporeactivity or anergy (nonresponsiveness to antigens). Cutaneous anergy may indicate functional impairment of or abnormalities in the cellular immune system. However, D T H is not an absolute determinant of immune system dysfunction, and any such interpretation must be avoided. D T H may be diminished or absent when there is in vitro evidence that T-cell function remains intact and when antibodyassociated immunity and phagocytic function appear normal. Reactivity to D T H test antigens may decrease or disappear temporarily as a result of febrile illness; measles and other viral infections; or live virus vaccination including measles, mumps, and rubella. It is also possible to observe loss of reactivity in patients undergoing treatment with drugs such as corticosteroids or procedures that suppress immunity. Moreover, recent infections and vaccinations influence the magnitude of the response. A lack of previous exposure to the administered antigen will result in a negative D T H response to that antigen. 22'23,25
Quality Control Measures for Multi-Test CM127 1. All applicator kits should be from the same lot. 2. The same technician should administer and read the test results (see the General Guidelines section). 3. Sterility of the test antigens is essential: opened or damaged applicators should be discarded; and the selected test site should be cleaned with ethanol prior to application; the applicator should never be reused. 4. Systemic reactions can occur in those persons sensitive to allergenic media components. Subjects who react severely to any of the test antigens should only be tested after the test heads containing the problem antigens have been removed. 5. Individuals may acquire skin testing sensitivity resulting from either immunization or infection.
Effect o f Vitamin E Status on Delayed-Type Hypersensitivity Response. The most widely accepted index of in vivo immune function is DTH. 28 D T H can be used as a predictor of morbidity and mortality in the elderly. 29 zs Report of an ILS/WHO Working Group, Clin. Exp. Immunol. 46~ 662 (1981). 29 S. J. Wayne, R. L. Rhyne, P. J. Garry, and J. S. Goodwin, J. Gerontol. Med. Sci. 45, M45 (1990).
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Christou e t al. 3° showed that in preoperative patients a significant correlation exists between D T H and mortality from sepsis. Improved D T H response in hospitalized patients has been associated with a decrease in sepsis and mortality. 31 Wayne et al. 29 showed that healthy elderly subjects who were anergic when tested for D T H had significantly higher mortality from all causes in subsequent years than those subjects with higher D T H responses. Supplementation with vitamin E above the R D A can increase DTH response in some situations. This is especially true for elderly persons. D T H responses are significantly diminished in the elderly, but supplementation with vitamin E improves D T H responsiveness in both older laboratory animals and humans. 5,32 Old mice supplemented with 500 ppm vitamin E for 30 days had significantly higher D T H in response to 2,4-dinitrofluorobenzene than mice fed the R D A . 32 Short-term supplementation of healthy elderly persons with 800 mg vitamin E for 30 days increased D T H scores. 33 In addition, long-term supplementation with 60, 200, or 800 IU of vitamin E a day increased D T H scores over time, 34 In the elderly, plasma vitamin E levels have been positively correlated with positive D T H responses to a variety of antigens, and epidemiologic studies indicate a lower incidence of infectious disease in elderly subjects with high plasma tocopherol levels. 2°'35Population groups maintaining high plasma tocopherol levels have also been noted to possess a lower incidence of cancer. 36-38 Lymphocyte Responsiveness: Proliferation and Interleukin 2 Production The cell-mediated immune responses involving lymphocyte functions are very sensitive to changes in vitamin E level. This section describes 30 N. V. Christou, J. Rodriguez-Tellado, L. Chartrand, B. Giannas, B. Kapadia, J. Meakins, and H. Rode, Ann. Surg. 210, 69 (1989). 31 j. D. Fletcher, G. A. Koch, and E. Endres, Aust. N.Z.J. Surg. 56, 17 (1986). 32 S. N. Meydani, M. Meydani, C. P. Verdon, A. C. Shapiro, J. B. Blumberg, and K. C. Hayes, Mech. Ageing Dev. 34, 191 (1986). 33 S. N. Meydani, M. P. Barklund, S. Liu, M. Meydani, R. A. Miller, J. G. Cannon, F. D. Morrow, R. Rocklin, and J. B. Blumberg, Am. J. Clin. Nutr. 52, 557 (1990). 34 S. N. Meydani, M. Meydani, J. B. Blumberg, L. S. Leka, G. Siber, R. Loszewski, C. Thompson, M. C. Pedrosa, R. D. Diamond, and B. D. Stollar, J A M A 277, 1380 (1997). 35 M. Chavance, G. Brubacher, and B. Herbeth, in "Lymphoid Cell Functions in Aging" (A. L. DeWick, ed.), p. 231. Eurage, Paris, 1984. 36 D. Trickier and G. Shktar, JNC 78, 1615 (1987). 37 M. S. Menkes, G. W. Comstock, J. P. Vuilleumier, K. J. Helsing, A. A. Rider, and R. Brookmezer, N. Engl. J. Med. 315, 1250 (1986). 38 R. M. Bostick, J. D. Potter, and D. R. McKenzie, Cancer Res. 53, 4230 (1993).
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procedures for assessing the basic immunologic function of lymphocytes: measurement of proliferative responses and cytokine production to a variety of stimuli.
Proliferation Assay General Measurement of proliferative responses by lymphocytes is a fundamental technique for the assessment of their biological responses to various stimuli. The assessment of cellular proliferation is perhaps the most often used technique in cellular immunology. Lymphocytes proliferate in response to mitogens, antigens, allogeneic or autologous cells, and many soluble factors. Quantitating the proliferative response involves measuring the number of cells present in a culture before and after the addition of a stimulating agent. Although cellular proliferation can be assessed by determining the increase in viable cell numbers through their actual enumeration using direct microscopy or automated cell counting, this approach can be time consuming. Therefore, the two most commonly used techniques are reduction of tetrazolium dyes (MqT) by active mitochondria or incorporation of tritiated thymidine into newly synthesized D N A . 39 Cell proliferation determined by estimating incorporation of [3H]thymidine into DNA is a process that is related to underlying changes in cell number. As cells enter the S phase of the cell cycle, chromosome replication takes place through the incorporation of soluble nucleotide precursors into newly synthesized DNA. In this assay, dividing cells are incubated (pulsed) with radioactive thymidine for several hours after which the amount of radioactivity incorporated into their DNA is determined. 39 Proliferative assays are applied in clinical studies as an assessment of overall immunologic competence of lymphocytes, as manifested by their ability to respond to proliferation signals. Defects in proliferation may be indicative of a fundamental cellular immunologic defect. Low proliferation is often found as a nonspecific secondary effect of chronic disease. 39 Procedure for Mitogen-Induced Proliferation of Peripheral Blood Mononuclear Cells Utilizing Incorporation of [3H]Thymidine into DNA 1. Peripheral blood mononuclear cells (PBMCs) are separated by density gradient from heparinized blood. Several procedures can be used to accomplish this. 4° 2. PBMCs are washed twice in RPMI 1640 medium supplemented with 100 mg/liter penicillin, 100 mg/liter streptomycin, 100 ml/liter heat39 R. Fernandez and V. Vetvivka, "Methods in Cellular Immunology." CRC Press, Boca Raton, Florida, 1995. 40 A. I. Boyum, Scand. J. Clin. Invest. 97 (Suppl. 21), 77 (1968).
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inactivated autologous plasma, L-glutamine (final concentration 2 mmol/ liter), and 25 mmol HEPES (final concentration 25 mmol/liter) (complete RPMI). Cells are resuspended in complete RPMI and counted under a light microscope. Cell viability is assessed by using the trypan blue exclusion method. Cell concentration is adjusted to 1 x 106 cell/ml with complete RPMI. 3. One hundred microliters of the cell suspension is pipetted into each well of a 96-well plate (1 x 105 cells/well). Triplicate wells are prepared for each experimental condition. Wells with no mitogen are included to measure background response. 4. Dilutions of mitogens between 1 and 100/xg/ml for P H A and concanavalin A (Con A) and 0.015% to 0.15% (w/v) for Staphylococcus aureus are prepared in complete RPMI. Optimal mitogen concentrations should be predetermined for each individual experimental condition. One hundred microliters of the diluted mitogen solution is added to each well. 5. Plates are incubated for 72 hr in a humidified 37°, 5% (v/v) CO2 incubator. 6. Four hours before termination of incubation, wells are pulsed with 20/zl of 25/zCi/ml [3H]thymidine. 7. Cells are harvested using an automated harvester, which aspirates the wells, lyses the cells, and retains DNA on glass filter paper, while allowing unincorporated [3H]thymidine to wash through. 8. Filters are air dried and placed into a liquid scintillation cocktail. 9. Counting is done using a liquid scintillation counter. 10. Results are reported as corrected counts per minute (cpm): the average cpm of mitogen-stimulated cultures minus the average cpm of cultures without mitogens. Alternatively, proliferation data can be expressed as the stimulation index, calculated by dividing the proliferation of the stimulated cells in cpm by the background proliferation.
Interpretation of Proliferation Assay Results. The relative ease of acquiring cells and measuring lymphocyte proliferation has made this assay a popular measure of immune function. In many cases, the proliferation response appears to be correlated with the vitamin E content of the diet. However, results from this assay must be critically analyzed before a recommendation about the vitamin E status of an individual is made. Cell proliferation in response to external stimuli is a very complex process often involving delivery of a signal or set of signals to cell membranes, activation of intracellular enzymatic pathways which are not well understood, activation and transcription of multiple genes, D N A and protein synthesis, and, finally, cell division. The mechanism of how vitamin E status affects the proliferative response is not clear.
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Experimental conditions must be thoroughly worked out for the proliferation assay to be of value. Cell populations used in this protocol must have high viability and contain an appropriate cell population for the response being tested. The composition of the culture medium, and especially the serum source, can be critical. 41 The selection of mitogen is important. There can be considerable variability in individual responses to particular activating agents; for example, a small portion of the normal human population has a low or absent response to pokeweed mitogen. In addition, the level of mitogen utilized becomes very important in the interpretation of proliferative responses. Effect o f Vitamin E on Mitogen-Induced Lymphocyte Proliferation. The level of in vitro proliferation by lymphocytes in response to mitogens is influenced by vitamin E level. Lymphocytes derived from animals maintained on vitamin E-deficient diets show depressed mitogenic response to T-cell mitogens, a defect that is reversible following vitamin E supplementation. 42 Eskew et al. 43 found that either vitamin E or selenium deficiency suppressed rat lymphocyte proliferation. Low levels of dietary vitamin E, which were sufficient to protect against many of the adverse effects of vitamin E deficiency, were insufficient to enhance lymphocyte proliferative responses even though the diet contained all other nutrients at recommended levels. Addition of vitamin E above the recommended dietary levels enhances mitogenesis in mixed populations of lymphocytes from humans and a variety of animal species compared to controls. 5 Cells from healthy elderly humans consuming a diet supplemented with 800 IU/day of vitamin E demonstrated increased mitogenic responses to optimal doses of Con A compared to cells from elderly subjects consuming the R D A . 33 Moriguchi et al. 11 reported that as dietary intakes of vitamin E increased from 100 to 2500 mg/kg of diet, there was a corresponding increase in rat splenic lymphocyte responses to Con A. Lymphocytes from mice supplemented with varying levels of vitamin E only showed enhanced Con A proliferation when dietary levels were in excess of the vitamin content of normal chow diet. 44 Interleukin-2 Production General. The T-cell growth factor, IL-2, is a lymphokine produced by T-helper type 1 cells following induction by mitogens such as lectin or by 41 S. P. James, in "Current Protocols in Immunology" (R. Coico, ed.), p. 7.10.1. John Wiley & Sons, New York, 1995. 42 A. Bendich, in "Antioxidant Nutrients and Immune Functions" (A. Bendich, M. Phillips, and R. P. Tengerdy, eds.), p. 36. Plenum Press, New York, 1990. 43 M. L. Eskew, R. W. Scholz, C. C. Reddy, D. A. Todhunter, and A. Zarkower, Immunology 54, 173 (1985). 44 M. Corwin and J. Shloss, J. Nutr. 110, 916 (1980).
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a specific antigen.45 IL-2 induces proliferation and differentiation of both T and B cells and thus, to a major degree, it regulates the magnitude and duration of the immune response. In addition to promoting the proliferation of cells, IL-2 induces the release of cytokines such as interferon gamma from activated T lymphocytes.46 Vitamin E level in the diet has been shown to influence IL-2 production. It has been hypothesized that vitamin E may exert some of its immunostimulatory effects by indirectly increasing IL-2 production. 32 Expression, synthesis, and secretion of IL-2 can be investigated by a variety of techniques. The following assay can be used to determine biologically active, secreted IL-2 in culture supernatants or biological fluids. This assay utilizes clones of the murine CTLL cell line first described by Gillis et al. 47 Other cell lines that can be used include HT-2, FDC-2, or MT-I. CTLL cells proliferate in the presence of IL-2, and to a lesser extent, in the presence of murine IL-448 and IL-15. 49 Proliferation can be measured by using [3H]thymidine or MTT. Procedure for Production of Interleukin 2 in Vitro. Cells are cultured in the presence or absence of phorbol myristic acetate (PMA), PHA, or Con A in 12-well plates. Plates are incubated in a 5% CO2 incubator at 37 °. Following 48 hr of incubation, supernatants are collected and stored at - 2 0 ° until analyzed.
Procedure for Detection of Biologically Active Human Interleukin
23950
1. The murine IL-2-dependent T-cell line, CTLL-2, is used to monitor IL-2 production. Prior to conducting the assay, logarithmically growing cells are washed three times with media to remove any residual IL-2. 2. Washed CTLL-2 cells are cultured at a concentration of 5 × 103 cells/well (100-/zl well) in 96-well microtiter plates in the presence of serially diluted test or control supernatants (100-/zl well). 3. The recombinant IL-2 standard and samples are diluted in complete RPMI. Culture supernatants are diluted 1:1-1:4. The IL-2 standard is titrated such that the least dilution allows the cells to achieve maximum DNA synthesis (positive control), usually at 20-40 U/ml (this is dependent 45 K. A. Smith, Science 240, 1169 (1988). 46 E. S. Kimball and P. M. Grob, in "The Lymphocyte: Structure and Function" (J. J. Marchalonis, ed.), p. 71. Marcel Dekker, New York, 1988. 47 S. Gillis, M. M. Ferm, W. Ou, and K. A. Smith, J. ImrnunoL 120, 2027 (1978). 48 R. Fernandez-Botran, P. H. Krammer, T. Diamanstein, J. W. Uhr, and E. S, Vitetta, J. Exp. Med. 164, 580 (1986). 49 K. H. Grabstein, J. Eisenman, K. Shanebeck, C. Rauch, S. Srinivasan, V. Fung, C. Beers, J. Richardson, M. Schoenborn, M. Ahdieh, L. Johnson, M. R. Alderson, J. D. Watson, D. M. Anderson, and J. G. Girl, Science 264, 965 (1994). 50 L. S. Davis, P. E. Lipsky, and K. Bottomly, in "Current Protocols in Immunology" (R. Coico, ed.), p. 6.3.1. John Wiley & Sons, New York, 1995.
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on the brand of recombinant IL-2 used). A typical standard curve ranges from 20 U/ml down to 0.3 U/ml recombinant IL-2. 4. CTLL-2 cells cultured in the absence of IL-2 serve as background proliferation (negative control). 5. After an 18-hr incubation at 37° in 5% (v/v) CO2 incubator, wells are pulsed with 0.5/~Ci [3H]thymidine. After 6 hr, the cells are harvested onto glass fiber filters with an automatic cell harvester and are then dried. 6. Isotope uptake (cpm) is determined using a liquid scintillation counter.
Interpretation of Interleukin-2 Bioassay Results. We have described a biological assay for IL-2. This bioassay has the advantage of being more sensitive than many of the other available techniques, and it can differentiate biologically active forms from inert forms of the cytokine. However, care must be taken to rule out the involvement of contaminating cytokines. Almost all the cloned murine cell lines used to measure IL-2 activity are also capable of responding to murine IL-4 and IL-15. 48'49'51 Other compounds present in serum can potentially influence the outcome of an IL-2 bioassay. 39'51 CTLL cells are susceptible to inhibitors (e.g., cytokine antagonists, soluble cytokine receptors) present in biological fluids. In addition, stimulants that may be in the samples (e.g., mitogens) may have unforeseen effects on the CTLL responses. Therefore, appropriate controls must always be included. Controls should include negative samples such as medium alone, and medium plus any stimulants. Antibodies against IL-4 and IL-15 should be included in samples containing these cytokines in addition to IL-2. Appropriate controls must be included in every assay because CTLL cells are subject to assay-to-assay variability in their responsiveness to IL-2. Effect of Vitamin E Status on Interleukin 2 Production. IL-2 production can be influenced by the level of available vitamin E. Elderly individuals produce less IL-2 than their young counterparts. This age-induced reduction in IL-2 production can be partially reversed by vitamin E supplementation above the RDA. For example, Meydani et al. 32 reported that older mice fed diets containing 500 ppm vitamin E had enhanced IL-2 production compared to mice fed a diet containing 30 ppmP 2 In addition, elderly healthy humans supplemented with 800 IU/day dl-a-tocopherol for 30 days had increased Con A-induced IL-2 production versus nonsupplemented subjects. 33
51 j. L. Rossio and A. J. H. Gearing, in "Clinical Applications of Cytokines: Role in Pathogenesis, Diagnosis and Therapy" (J. J. Oppenheim, J. Rossio, and A. Gearing, eds.), p. 16. Oxford University Press, New York, 1993.
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Microbial disease can reduce IL-2 production. Human immunodeficiency virus (HIV)-positive persons and mice infected with murine acquired immunodeficiency (MAIDS) produce less IL-2 than uninfected controls. Vitamin E supplementation over the murine requirement increased IL-2 production by splenocytes from mice infected with the retrovirus that causes MAIDS. 52 Mononuclear Phagocytic Cell Functions Affected by Vitamin E Status General. Mononuclear phagocytic cells are often called accessory cells and include monocytes and macrophages. An accessory cell helps lymphocytes, enabling them to carry out their function of recognition and elimination of antigens. Accessory cells that can present antigens to lymphocytes are called antigen-presenting cells and include mononuclear phagocytes. These cells play a crucial role in the immune system through their functions as antigen-presenting cells and as regulatory cells, which synthesize and release soluble immune-enhancing and -suppressing cytokines. Because of the diversity of their functions, mononuclear phagocytes are key cellular elements in the immunological control of a number of diseases. The mononuclear phagocyte is a highly secretory cell. The variety of secreted molecules is large and includes cytokines that are involved in the acute inflammatory response as well as lymphocyte response. Macrophage secretion of several cytokines appears to be affected by vitamin E level. However, the literature has been inconsistent in characterizing this effect. Two cytokines that consistently change concentration in response to vitamin E are TNF-a and IL-6. Cytokine Production of Interleukin-6 and Tumor Necrosis Factor-a. IL-6 is produced by many cell types in response to a variety of immunological or pathological stimuli. The most potent sources include endotoxin-stimulated monocytes and macrophages and IL-l-stimulated fibroblasts. There are many assays available to detect IL-6, including commercially available enzyme-linked immunosorbent assay (ELISA) kits. Biologically active IL-6 can be detected using IL-6-dependent murine hybridomas. The most commonly used hybridoma lines are B-9 and 7TD1. 53,54 The general bioassay procedure was described previously in more detail for IL-2. Briefly, the hybridomas are maintained in recombinant IL-6, washed prior to assay,
52y. Wang,D. S. Huang,S. Wood,and R. R. Watson,Immunopharmacology 29, 225 (1995). 53j. Van Snick,S. Cayphas,A. Vink, C. Uyttenhove,P. Coulie,and R. Simpson,Proc. Natl. Acad. Sci. U.S.A. 83, 9679 (1986). 54L. A. Aarden, E. R. De Groot, O. L. Schaap, and P. M. Lansdorp, Eur. J. ImmunoL 17, 1411 (1987).
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and the cells are combined with the samples to be analyzed. Proliferation is measured 3 days later by [3H]thymidine incorporation. 39 TNF-a is produced by activated macrophages and other cells and has a broad spectrum of biological actions on many different target cells, both immune and nonimmune. TNF is considered a major inflammatory mediator. TNF can be measured by both ELISA and biological assay. There are many commercially available ELISA kits for detecting TNF. The most commonly used bioassay for TNF is a cytotoxicity assay based on murine fibroblasts such as L929 or L-M cells. These cells deteriorate and die over a 3-day period in the presence of TNF. Sensitivity levels vary with cell isolates, passage number, and temperature. 55 EFFECT OF VITAMIN E STATUS ON TUMOR NECROSIS FACTOR-Or AND INTERLEUKIN-6 SECRETION BY MONONUCLEAR PHAGOCYTES. Infection with
the retrovirus that causes AIDS in humans and MAIDS in mice results in elevated levels of IL-6 and TNF-a as the disease progresses. 52'56'57 Supplementation over the murine requirement with vitamin E appears to lower the retrovirus-induced production of IL-6 and TNF in mice. 21'57 Wang et a/. 2t'52 reported that mice infected with MAIDS and supplemented with vitamin E at 15-450 times the NRC recommendation showed normalization of IL-6 and TNF-a produced by splenocytes during progression to MAIDS at all levels of vitamin E supplementation. Moreover, aging affects IL-6 and TNF-ot production. Depending on the experimental conditions, aging decreased or increased production of IL-6 and TNF. 58'59Vitamin E supplementation of elderly human subjects inhibited IL-6 production and prevented the exercise-induced increase in TNF-a production by peripheral blood mononuclear cells. 6°'61 Dietary alcohol (ethanol) consumption decreases production of IL-6 and TNF-ot. When a 15-fold increase over the mouse NRC-recommended level of vitamin E was supplied to ethanol-fed mice for 10 weeks, production of IL-6 by Con A-stimulated splenocytes, and IL-6 and TNF-o~ by LPSstimulated splenocytes was restored. 62 55 M. M. Hogan and S. N. Vogel, in "Current Protocols in Immunology" (R. Coico, ed.), p. 6.10.1. John Wiley & Sons, New York, 1993. 56 F. Boue, C. Wallon, C. Goujard, F. Barresinouss, P. Galand, and J. F. Defraissy, J. Imrnunol. 148, 3761 (1992). 57 L. Y. Wang, D. S. Huang, P. T. Giger, and R. R. Watson, Adv. Biol. Sci. 86, 335 (1993). 58 R. B. Effros, R. L. Walford, R. Weindruch, and C. Mitcheltree, J. Gerontol. 46, B142 (1991). 59 j. G. Cannon, S. F. Orencole, R. A. Fielding, M. Meydani, S. N. Meydani, M. A. Fiatarone, J. B. Blumberg, and W. J. Evans, Am. J. Physiol. 259, R1214 (1990). 6o j. G. Cannon, S. N. Meydani, R. A. Fielding, M. A. Fitarone, M. Meydani, M. Farhangmehr, S. F. Orencole, J. B. Blumberg, and W. J. Evans, Am. J. Physiol. 260, R1235 (1991). 61 y. Wang, D. S. Huang, P. T. Giger, and R. R. Watson, Alcohol. Clin. Exp. Res. 18, 64 (1994). 62 S. M. Wahl, J. B. Allen, S. Gartner, J. M. Orenstein, M. Popovic, D. E. Chenoweth, L. O. Arthur, W. L. Farrar, and L. M. Wahl, J. lmmunol. 142, 3553 (1989).
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Phagocytosis and Chemotaxis Phagocytosis is the engulfment of cells, microorganisms, and particulate materials. Phagocytosis is very important in a variety of circumstances including the clearance of microbial infections and in the daily turnover of senescent tissues and cells. Chemotaxis is important because the phagocytes must be able to move to where they are needed. Factors that inhibit macrophage migration can have a deleterious effect on host protective function.63 Phagocytosis can be measured a variety of ways. The following protocol is used for the measurement of Fcy receptor-mediated binding and phagocytosis.64 PHAGOCYTOSIS ASSAY.
1. Sheep red blood cells (SRBC) are maintained in Alsever's solution (1:1, v/v) at 4°. 2. Five milliliters SRBC are placed into a 15-ml conical centrifuge tube, diluted to 15 ml with physiological saline, and centrifuged at 300g for 10 min at room temperature. Discard supernatant and repeat wash two times. Count using a hemacytometer. Resuspend SRBC at 1 × 108 cells/ml in saline. 3. Opsonization is accomplished by incubating 10 ml of SRBC suspension with 200/xl of rat anti-SRBC antiserum (heat inactivated: optimum concentration previously determined) for 60 min at 37°. 4. Radiolabeling of the opsonized SRBC is accomplished by incubation with 200/zCi of sodium [51Cr]chromate for 1 hr at 37°. The opsonized 51Crlabeled SRBC cultures are then washed three times with RPMI to remove excess S~Cr, and the final volume is adjusted to give a 0.6% suspension of SRBC. Prepare macrophage/monocyte samples. 1. Purified macrophages/monocytes are washed two times by adding 10 ml PBS, centrifuging at 1200 rpm for 10 min at 4°, and discarding supernatant. Cells are resuspended in complete RPMI to a final concentration of 2 × 10 6 cells/ml. 2. One hundred microliters of cell suspension is added to each well of a 96-well fiat bottom tissue culture plate. Prepare at least three replicate wells for each treatment and include positive and negative controls. Allow cells to adhere (4 hr in a 5% CO2, 37° incubator), then add treatment (interferon) if needed. 3. The opsonized [51Cr]SRBC cultures are added. After 2 hr at 37°, the 63 A. D. Politis and S. N. Vogel, in "Current Protocols in Immunology" (R. Coico, ed.), p. 14.8.1. John Wiley & Sons, New York, 1995. 64 R. E. Harris, L. A. Boxer, and R. L. Bahner, Blood 55, 338 (1980).
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cultures are rinsed once with distilled water to lyse nonphagocytosed SRBC and then washed twice with 0.1 mol/liter phosphate buffer, pH 7.2. All remaining cells are lysed with 0.1 mol/liter NaOH. 4. The radioactivity of the lysate is measured in a gamma counter. The stimulation index is calculated by assigning a value of 1 to the phagocytic activity of control macrophages incubated with medium only, then by comparing this value to the phagocytic activity of macrophages from each treatment. EFFECT OF VITAMIN E ON PHAGOCYTIC CELL RESPONSES. T h e e f f e c t s
of dietary vitamin E on phagocytic cell function have been well documented. Rats fed vitamin E-deficient diets exhibit impaired macrophage chemotaxis, reduced ability to ingest complement-coated beads, and decreased protection from autooxidative damage. 65 Dietary vitamin E supplementation has been shown to ameliorate endotoxin-induced inhibition of monocyte migration and phagocytosis in r a t s , 66 t o reduce immunosuppressive cytokine production by macrophages in mice, and to normalize monocyte chemotaxis in humans with diabetes. 67 The ability of alveolar macrophages to phagocytose opsonized sheep red blood cells has been reported to increase with increasing concentrations of vitamin E in diets and showed a fivefold increase in rats fed the diet with the highest vitamin E content compared to rats in the control group. 11 Yano and Ichikawa 67 reported that alveolar macrophages from vitamin E-supplemented rats demonstrated enhanced phagocytosis of opsonized particles.
Mechanism The mechanism underlying the immunostimulatory effect of vitamin E has not been completely delineated. 6 Although some of the effects of vitamin E on immune cell functions can be attributed to its antioxidant activities, other mechanisms appear to be involved. 6a,69It has been hypothesized that one of these alternative mechanisms is the reduction of immunoinhibitory molecules secreted by immune cells themselves, particularly by macrophages. Activated macrophages secrete molecules such as H2027°,vI and prostaglandin E2 (PGE2), which have been shown to depress lymphocyte 65 N. P. Rocha, Brazilian J. Med. Biol. Res. 22, 1401 (1989). 66 n . R. Hill, N. H, Augustine, M. L. Rallison, and J. I. Santos, J. Clin. Immunol. 3, 70 (1983). 67 T. Yano and T. Ichikawa, Nutr. Res. 14, 1387 (1994). 68 L. M. Corwin and J. Shloss, Z Nutr. 110, 2497 (1980). 69 n . Sies, Am. J. Med. 91, 31 (1991). 70 R. I. Fisher and F. Bostick-Bruton, J. Immunol. 129, 1770 (1982). 71 Z. Metzger, J. T. Hoffeld, and J. J. Oppenheim, J. lmmunol. 124, 938 (1980).
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proliferation, 7°'72,73 lymphokine production, TM and the generation of cytotoxic T cells.75 Vitamin E modulates the formation of these potentially immunosuppressive molecules, particularly PGE2. Peritoneal macrophages and splenocytes from mice supplemented with vitamin E produced less PGE2 after stimulation than unsupplemented control mice. 32'76 Additional research is needed to understand fully the mechanisms of the effect of vitamin E on immune responsiveness. Summary Evidence from animal and human studies indicates that vitamin E plays an important role in the maintenance of the immune system. Even a marginal vitamin E deficiency impairs the immune response, while supplementation with higher than recommended dietary levels of vitamin E enhances humoral and cell-mediated immunity. The current R D A level of vitamin E prevents clinical deficiency syndrome but in some situations, especially in older subjects or in a disease state, fails to maintain optimal host defense. The immunological parameters reviewed are all sensitive to changes in the availability of vitamin E and, therefore, may reflect the vitamin E status of a given individual more accurately than conventional methods. Acknowledgments This project has been funded at least in part with federal funds from the U.S. Department of Agriculture, Agricultural Research Service, under contract 53-K06-01. The contents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government. The authors thank Timothy S. McElreavy for preparation of this manuscript.
v2 M. Rola-Plezczunski, Immunol. Today 6, 302 (1985). 73 D. R. Webb, T. J. Rogers, and E. Nowoiejski, Proc. Natl. Acad. Sci. U.S.A. 332, 260 (1980). 74 R. P. Phipps, S. H. Stein, and R. L. Roper, lmrnunol. Today 12, 349 (1991). 75 M. Plaut, J. Immunol. 123, 692 (1979). 76 E. H. Romach, S. Kidao, B. G. Sanders, and K. Kline, Nutr. Cancer 20, 205 (1993).