Nutritional Status and Immune Responses in Mice

Nutrient Requirements

and Interactions

Nutritional Status and Immune Responses in Mice with Murine AIDS Are Normalized by Vitamin E Supplementation1-2'3 YUEJIAN WANG,*' DENNIS S. HUANG,*T' RONALD R. WATSON*4

BAILIN LIANG*'

AND

*Department of Family and Community Medicine, 'Nutritional Sciences Program, "Department of Microbiology and Immunology and 'Department of Nutritional Sciences, University of Arizona, Tucson, AZ 85724

'Part of this work was presented as a poster at Experimental Biology 93, March 28-April 1, 1993, New Orleans, LA, and pub lished in abstract form [Wang, Y., Huang, D. S., Wood, S., Lopez, M. C, Giger, P. T., Murphy, L. & Watson, R. R. (1993) Vitamin E supplementation assists in the restoration of imbalanced cytokine production induced by murine LP-BM5 retrovirus causing murine AIDS. FASEB J. 7: A285 (abs.)]. Supported by NIH AA 08037. 3The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734

INDEXING KEY WORDS:

â€¢vitamin E â€¢cytokine â€¢mice â€¢murine AIDS â€¢immune response

Acquired immune deficiency syndrome (AIDS)5 is a

solely to indicate this fact. 4To whom correspondence should be addressed. Abbreviations used: AIDS, acquired immune deficiency syn drome; Con A, concanavalin A; HIV, human immunodeficiency virus; IFN, interferon; Ig, immunoglobulin,- IL, interleukin; LPS, lipopolysaccharide; NF, nuclear factor; NK, natural killer; Th, T helper; TNF, tumor necrosis factor.

clinical disorder in humans caused by human im munodeficiency virus (HIV) infection. It represents the end point in a progressive sequence of immunosuppressive changes that render the body highly susceptible to tumors and opportunistic infections. 0022-3166/94 $3.00 Â©1994 American Institute of Nutrition. Manuscript received 24 September 1993. Initial review completed

26 November 2024

1993. Revision accepted

18 April 1994.

Downloaded from jn.nutrition.org by guest on July 15, 2011

AIDS has been identified as a major public health priority in the United States, with heavy social and economic impact. Immune and other physiological defects induced by HIV infection seem to be progressive and irreversible, with a mortality rate that approaches 100% (Berkelman et al. 1989). Therefore, there is a pressing need for valid and effective ther apeutic or preventive interventions in individuals with AIDS. The LP-BM5 leukemia retrovirus-infected murine model provides an exciting model for studying traditional AIDS therapies (Basham et al. 1991). Murine LP-BM5 retrovirus induces murine AIDS with many functional similarities to human AIDS, even though the etiologies of the two diseases are different. Both diseases are characterized by lymphadenopathy, splenomegaly, hypergammaglobulinemia, deficient B cell response to mitogen, T cell functional deficiency, loss of disease resistance and cytokine dysregulation (Wang et al. 1993, Watson

ABSTRACT Female C57BL/6 mice were infected with LP-BM5 retrovirus, causing murine AIDS, which is func tionally similar to human AIDS. Vitamin E effects on immune functions, cytokine production and nutritional concentrations in retrovirus-infected mice were deter mined. Retrovirus infection inhibited release of interleukin-2 (IL) and Interferon--) (IFN) and some immune functions, whereas it stimulated secretion of 1L-4. IL-5. IL-6 and tumor necrosis factor-Â« (TNF) and immunoglobulin (Ig) production. Furthermore, retrovirus in fection induced some nutritional deficiencies in the tissues. A 15-fold increase in dietary vitamin E largely restored concentrations of some micronutrients (vitamins A and E, zinc and copper) in the liver, in testine, serum and thymus. It also partially restored production of IL-2 and IFN-? by splenocytes. Retrovirusinduced elevated production of IL-4. IL-5 and IL-6 by splenocytes in vitro was normalized by vitamin E. Elevated release of IL-6, TNF-a, IgA and IgG produced by splenocytes in vitro during murine AIDS were also completely or partially normalized by vitamin E. Vitamin E also prevented retrovirus-induced suppression of splenocyte proliferation and natural killer cell activity. These data indicate that vitamin E supplementation during murine AIDS can help to ameliorate the disorders during murine AIDS, suggesting vitamin E usefulness in treatment of AIDS in humans. J. Nutr. 124: 2024-2032, 1994.

2025

VITAMIN E AND AIDS THERAPY

MATERIALS

AND METHODS

Animals. Five-week-old female C57BL/6 mice were obtained from Charles River Laboratories (Wil mington, DE) and housed in transparent plastic cages with stainless steel wire lids (four mice per cage) in the animal facility of the Arizona Health Sciences Center. The housing facility was maintained at 20 to 22Â°C and 60 to 80% relative humidity, with a 12-h light:dark cycle. Water and semipurified diet (4%

mouse diet, #7001, Teklad, Madison, WI) were freely available. After 2 wk, mice were randomly assigned to one of the following four treatments: control (nonsupplemented and uninfected), vitamin E-supplemented diet (supplemented and uninfected), LP-BM5 infection (nonsupplemented and infected), and LP-BM5 in fection plus vitamin E-supplemented diet (sup plemented and infected). All diets were freely available. Mice consumed 12-15 mL of the liquid diets per day. No significant differences in diet con sumption or body weight were observed among the four groups (data not shown). Animal care was ap proved by the University of Arizona Committee on Animal Research. Diet and treatment. All mice were fed the Na tional Research Council liquid diet (NRC 1978), which meets the nutritional requirement of mice (see Table 1 for diet composition). Dietary ingredients were obtained from Dyets (#710279, Bethlehem, PA). The vitamin E-supplemented diet had 0.110 g/L of vitamin E as flÃŸÃŸ-a-tocopherol acetate (Sigma Chemical, St. Louis, MO) added to the liquid diet in addition to the 0.0077 g/L ÃŸÃŸÃŸ-a-tocopherol acetate contained in the basal liquid diet. Vitamin E dietary supplementation was initiated the day of retrovirus

TABLE 1 Composition of the control liquid diet1 Ingredient

Concentration

g/L 29.9 0.2 7.5 7.5 18.0 139.6 10.0 3.0 0.3 2.5 6.0 'Mice were fed the National Research Council (1978) liquid diet. The fatty acid pattern was 31.4% saturated, 35.9% monounsaturated and 32.7% polyunsaturated fatty acid. 2Vitamin mix provided the following (mg/kg diet, except as noted): thiamin-HCl, 0.75; riboflavin, 1.05; pyridoxine-HCl, 0.26; nicotinic acid, 2.63 ngÂ¡calcium pantothenate, 2.1; folie acid, 0.13; biotin, 0.05; vitamin B-12, 2.63 jtg; retinyl palmitate, 39.6 ng, cholecalciferol, 1.0 Â¿Â¿g; RKK-a-tocopherol acetate, 10.5; menadione sodium, 0.79. â€¢^Mineral mix provided the following (mg/kg mix): calcium phosphate dibasic, 595.64; potassium phosphate monobasic, 175.10; sodium chloride, 36.27; sodium bicarbonate, 28.25; potassium sulfate, 83.22; magnesium oxide, 36.27; manganous sulfate-HjO, 1.35; ferrous sulfate-7H2O, 5.46; zinc carbonate, 2.52; cupric car bonate, 0.34; potassium iodate, 0.02; sodium selenite, 0.01; chromium potassium sulfate-12H2O, 0.85. Casein (vitamin free) DL-Methionine Corn oil Safflower oil Lard Maltose-dextrin Cellulose Xanthan gum Choline bitartrate Vitamin mix2 Mineral mix3


1989). Thus, LP-BM5 retrovirus infection in mice is a useful functional model to screen some therapies for AIDS treatment. Gastrointestinal dysfunction, including diarrhea and malabsorption, is a common manifestation of AIDS (Ullrich et al. 1989). HIV infection may play a pathogenic role in gastrointestinal cells, deleteriously affecting their nutritional absorption capacities. In situ hybridization studies have localized HIV in fection in various types of epithelial cells of the bowel mucosa (Barnett et al. 1991, Levy et al. 1989). The roles of vitamins and minerals in the clinical manifestation of HIV infection have recently been noticed (Coodley and Girad 1991). Patients at various stages of HIV infection had low serum vitamin A and E concentrations (Bogden et al. 1990). Serum zinc and copper deficiencies in HIV patients were also reported (Falutz et al. 1988, Fordyce-Baum et al. 1989). Undernutrition has been associated with immunological dysfunction and development of infectious processes. Thus, a vicious cycle may be set up in which the underlying immunological defects related to HIV in fection are exacerbated by the immune dysfunction associated with undernutrition, initiated by retrovirus infection. High intakes of vitamin E have enhanced immune response (Odeleye and Watson 1991) and reduced the size and numbers of carcinogen-induced tumors, which had been increased in mice with murine AIDS (Odeleye et al. 1992). Although vitamin E supplemen tation in animals or humans has been shown to in crease immune responses, little is known about vitamin E modulation of cytokine production (Wang and Watson 1993). The balance of cellular and hu moral immune response to antigens is controlled by communication between immunocompetent cells. They are regulated to a great extent by soluble medi ators, cytokines, produced mostly by T helper (Th) cells and macrophages. Cytokines play extremely im portant roles in the communication network that links inducer and effector immune and inflammatory cells (Arai et al. 1990). Thus, we investigated whether dietary vitamin E supplementation would help ame liorate the undernutrition, cytokine dysregulation and immune dysfunctions associated with retrovirus in fection in mice with murine AIDS.

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WANG ET AL.

infection. The infection and treatment period was 8 wk. Mice were killed while under ether anesthesia. All tissues were then dissected, removed and kept at 4Â°C.The tissues for nutritional analysis were col lected and stored at -70Â°C until assayed. Blood col lection was from the axillary plexus of mice. Blood was kept at 4Â°Cfor 24 h and then centrifuged at 800 x g for 10 min. Sera were collected and stored at -70Â°C until assayed. LP-BM5 murine leukemia retrovirus infection. The retrovirus was administered intraperitoneally to mice in 0.1 mL with an ecotropic (XC) of 4.5 logio plaque forming units x 10~3/L, which induces disease with a

the sample was cooled, 5 mL of 11.1 mol/L nitric acid was added. After the sample was mixed and predigested in a 60Â°Cwater bath for 1 h, the water bath was heated to boiling. Five drops of 8.82 mol/L hydrogen peroxide were added, and the sample was heated for 3 h. Before the tubes were removed from the water bath, another drop of hydrogen peroxide was added. The solution was either measured directly or diluted to the appropriate concentrations for elemental analysis using a flame atomic absorption

were washed twice with culture medium. Cell con centration was determined and adjusted to IO10 cells/ L. Splenocyte viability was >95% as determined by trypan blue exclusion. Standard cytokines and their antibodies. Rat antimurine interferon-7 (IFN-7) monoclonal antibody, standard recombinant IFN-7, hamster anti-tumor necrosis factor-a (TNF-a) monoclonal antibody, standard recombinant TNF-a, rabbit anti-murine TNF-a serum, rat anti-murine interleukin (IL)-2, standard recombinant IL-2, rat anti-murine IL-6 monoclonal antibody and recombinant murine IL-6 were obtained from Genzyme (Boston, MA). Rat antimurine IL-4 antibody, biotin-rat anti-murine IL-4 monoclonal antibody, and recombinant murine IL-4 and IL-5 were obtained from PharMingen (San Diego, CA). Goat anti-murine IL-6 polyclonal antibody was obtained from R&D System (Minneapolis, MN). Rat anti-murine IL-5 (TRFK5) and biotin-rat anti-murineIL-5 (TRFK4) monoclonal antibodies were kind gifts from Robert L. Coffman of DNAX Institute (Palo Alto, CA). Rabbit anti-murine IL-2 antibody was ob tained from Collaborative Biomedicai (Bedford, MA). Rabbit anti-murine IFN-7 antiserum was prepared in our laboratory. Cytokine and immunoglobulin production. The spleens from the experimental mice were handled individually. Splenocytes (1010/L, 10~4 L/well) from one experimental mouse were cultured in triplicate on 96-well flat-bottom culture plates (Falcon, Lincoln Park, NJ) with culture medium. Splenocytes were then stimulated with concanavalin A (Con A, 10 mg/ L, 10~4 L/well; Sigma Chemical) for induction of IL-2, IL-4 and IL-6 with 24 h incubation and for induction of IL-5 and IFN-7 with 72 h incubation at 37Â°Cin a 5% CÃ›2 incubator. Splenocytes were also stimulated by lipopolysaccharide (LPS, 5 mg/L; Gibco, Grand Island, NY) for 24 h induction for IL-6 and TNF-a, and immunoglobulin (Ig)A and IgG with 72 h incubation. After incubation, the plates were centrifuged for 10 min at 800 x g. Supernatant was collected and stored at -70Â°C until assayed.


time course comparable to that previously studied (Watson 1989). Infection of adult female C57BL/6 mice with LP-BM5 retrovirus leads to the rapid in duction of symptoms with virtually no latent phase. Tissue vitamin A and vitamin E. Tissue concentra tions of total #jR.R-a-tocopherol and all-trans retinol were determined by the fluorometric method described previously (Dugan et al. 1964). Briefly, -0.1 g of liver was homogenized in 5 mL of water and 5 mL of ethanol, and vitamins A and E were extracted with 5 mL of n-hexane by shaking for 1 min and centrifugation at 800 x g for 5 min. The n-hexane layer was removed and measured at an emission of 430 nm and an excitation maximum of 365 nm for retinol measurement. Then, 0.5 mL of 6 mol/L sulfuric acid was added, the solution was mixed thoroughly, and the fluorescence intensity for tocopherol was determined in the hexane at an emission of 340 nm and an excitation maximum of 295 nm using a fluorescence spectrophotometer (F2000, Hitachi, Tokyo, Japan); a-tocopherol or all-trans retinol (Eastman Chemical, Kingsport, TN) was used as a standard. All concentrations of vitamins are reported as micromoles per gram of wet tissue. Serum vitamin A and vitamin E. Serum vitamin A and vitamin E were measured by mixing 0.5 mL of serum with 2 mL of ethanol, extracting with 4 mL of n-hexane by shaking for 5 min and centrifuging at 800 x g for 5 min. The vitamin A and E concentrations in n-hexane layers were measured as indicated above. Serum vitamin concentrations are reported as micromoles per liter of serum. Tissue zinc and copper. The concentrations of zinc and copper in the tissue were determined by flame atomic absorption spectrophotometry. Briefly, -0.1 g of tissue was dried at 56Â°Cin an oven overnight. After

spectrophotometer (model 180-70, Hitachi, Tokyo, Japan) at 213.8 nm for zinc and at 324.8 nm for copper. The same nitric acid containing 8.82 mol/L hydrogen peroxide was used for the standard and blank solutions in the mineral analyses. All concen trations of minerals are reported as micromoles per gram of dry tissue. Preparation of splenocytes. Spleens from mice receiving vitamin E treatment or from controls were gently teased with forceps in culture medium (RPMI 1640 medium containing 10% fetal bovine serum, 2 mmol/L glutamine, 100 mg/L penicillin-strep tomycin), producing a single cell suspension of splenocytes. Red blood cells were lysed by the ad dition of a lysis buffer (0.16 mol/L ammonia chloride Tris buffer, pH 7.2) at 37Â°Cfor 2 min. Then the cells


ELISA for cytokines. All splenocytes were determined described previously (Wang wells of 96-well microtiter natech, Chantilly, VA) were with* 50 fiL of anti-cytokine

cytokines produced by by sandwich ELISA as et al. 1994). Briefly, the plates (Immulon II, Dycoated overnight at 4Â°C capture rat or hamster

monoclonal antibody specific for the measured cytokines, diluted to 1-4 mg/L in 0.05 mol/L bicar bonate buffer (pH 9.6). Plates were washed once with PBS (0.01 mol/L, pH 7.2-7.4) containing 0.05% (v/v) Tween 20 (PBS-Tween). To each well 100 Â¡tLof 10 g/L bovine serum albumin in PBS was added, and the plates were incubated (37Â°C,5% CO2, 95% air) for 1

zolinesulfonic acid) (in citrate buffer, 0.1 mol/L, pH 4.2, containing 8.82 mmol/L hydrogen peroxide; Sigma Chemical) was added to each well, and the plates were allowed to develop for 20-30 min at room temperature. Optical density was determined at 405 nm by Titertek Multiscan (Flow Lab, Mclean, VA). Sensitivity of the ELISA assays for IL-2, IL-4, IL-5, IL6, TNF-a and IFN-7 were determined to be 156, 156, 320, 300, 156 and 78 ng/L, respectively. No crossreactions between these measured cytokines in the ELISA kits were observed (data not shown). Cytotoxicity for natural killer cells. Activity of natural killer (NK) cells was measured using 51Cr release as described previously (Wang et al. 1994). Natural killer cell-sensitive YAC-1 cells were used as target cells. Briefly, YAC-1 target cells were labeled with 3.7 MBq of 51Cr (Du Pont NEN Products, Boston, MA) at 37Â°Cfor 2 h, washed, and incubated in PBS for 30 min. After incubation, target cells were washed and adjusted to IO9 cells/L. Splenocytes for NK cell activity and target cells in triplicate were suspended in round-bottom microtiter plate wells producing effector:target ratios of 100:1 and 50:1. The plates were then centrifuged (200 x g, 2 min), in cubated for 4 h at 37Â°Cand again centrifuged (200 x g, 10 min). Supernatant

liquid (100 Â¿iL)was harvested

and radioactivity (release in becquerels, ER) measured by a gamma counter. Spontaneous release (SR) is bec querels was obtained by incubating labeled target cells in medium without splenocytes. Maximal re lease (MR) in becquerels was induced by the presence of 100 tiL of 16.6 mmol/L Tergitol (type NP-40, Sigma Chemical). The amount of lysis was then calculated in terms of the percentage of chromium release: % Specific chromium release = (ER - SR)/ (MR - SR) x 100. Mitogenesis of splenocytes. The proliferation of splenocytes was determined by incorporation of thymidine as described previously (Wang et al. 1994). Briefly, each mouse was separately handled as one sample. Splenocytes from each experimental mouse were cultured in triplicate in 100 /Â¿Lof culture medium (1010/L) on 96-well flat-bottom cultured plates (Falcon) with Con A and LPS (5 mg/L) with culture medium. They were incubated at 37Â°Cin a 5% COj incubator for 20 h for Con A-induced T cell proliferation and 44 h for LPS-induced B cell prolifer ation, and then pulsed with [3H]thymidine (37 kBq/ well, Du Pont NEN Products). After 4 h, they were harvested by a cell sample harvester (Cambridge Technology, Cambridge, MA). Radioactivity was de termined by a liquid scintillation counter (Tri-Carb, 2200CA, Packard, Laguna Hills, CA). Data units are reported as becquerels. ELISA for IgA and IgG. The IgA and IgG concen trations produced by splenocytes were determined by sandwich ELISA as described previously (Wang et al. 1994). Briefly, the wells of 96-well microtiter plates (Immulon II, Dynatech) were coated overnight at 4Â°C with 50 /Â¿Lof goat anti-murine IgA and IgG 7-chain (Sigma Chemical), diluted to 2 mg/L in 0.05 mol/L bicarbonate buffer (pH 9.6). Plates were washed one time with PBS-Tween. Plates were washed twice with PBS, and 50 /Â¿Lof samples or standard IgA and IgG (Sigma Chemical) diluted in culture medium was added to plates. Plates were incubated as above for 1 h, then washed three times as above; 5 x 10~5 L of diluted biotinylated goat anti-murine IgA and IgG (2 mg/L) in PBS was added, followed by incubation at 37Â°C for another hour. Then 50 Â¿iLof diluted strepavidin-horseradish peroxidase (1:5000; Jackson) was added for another hour of incubation at 37Â°C. Finally, the development of substrate color and meas urement of optical density were as described above. Sensitivities of ELISA assays for IgA and IgG were determined to be 20 and 40 ng/L, respectively. No cross-reaction between ELISA kits or with murine IgM were observed (data not shown). Statistics. Group means were compared by the Kruskal-Wallace test (Ott 1988). Significantly different group means (P < 0.05) were determined by Duncan's multiple range test (Ott 1988).


h. Plates were washed twice as above, and 50 nL of standard and sample cytokines diluted in culture medium were added. Plates were incubated as above for 2 h and washed three times as above. Then, 50 Â¿iL of diluted rabbit anti-IL-2, TNF-a, and IFN-7 polyclonal, goat anti-IL-6-polyclonal, and biotinylated rat anti-IL-4 and IL-5 monoclonal detecting antibodies in PBS (1-4 mg/L) were added into each plate. Plates were incubated as above for 1.5 h and washed four times with PBS-Tween. Diluted strepavidin-horseradish peroxidase (50 /Â¿L, 1:5000; Jackson, West Grove, PA) for IL-4, IL-5 and goat anti-rabbit IgGhorseradish peroxidase (1:5000; American Qualex, La Mirada, CA) conjugates for IL-2, TNF-a and IFN-7 in PBS, and donkey anti-goat IgG-horseradish peroxidase (1:5000; Jackson) for IL-6 were added to each well. Plates were then incubated as above for l h and washed five times with PBS-Tween. Finally, 100 Â¿iL of substrate buffer of 2,2'-azinobis(3-ethylbenzthia-

2027

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WANG ET AL.

RESULTS

TABLE 2 Effect of vitamin E on hepatic, intestinal

and splenic vitamin and mineral concentrations

during murine AIDS1

Treatment Vitamin E

Retrovirus

Retinol\im

Ã¶l3.20 0.39+

1.1a+

0.36a+ + 0.019+ 0.016a+

0.062+ +

0.012+ 0.016a+

0.016+ +

wet tissue0.16

tissue0.30

Â± 4.35 Â± 2.47 Â± 0.49ab0.24 5.27 Â±

0.0190.21 Â± 0.018a0.06 Â± 0.017a0.18 Â± 0.016b0.13 Â±

0.081.35 Â± 0.091.51 Â± 0.261.97 Â± 0.20abIntestine0.84 Â±

0.0330.27 Â± 0.0420.25 Â± 0.036a0.35 Â± 0.042b0.09 Â±

Â± 0.22 Â± 0.18 Â± 0.18 Â± 0.0560.086

0.0130.12 Â± 0.0140.10 Â± 0.012a0.14 Â± 0.009b0.95 Â±

0.140.97 + Â±0.170.86 0.141.14 Â± 0.14abSpleen0.21 Â±

0.0190.14 Â± 0.0270.12 Â± 0.0380.15 Â± 0.022a0.12 Â±

0.0210.22 Â± 0.0200.23 Â± Â± 0.091.36 Â± 0.061a0.18 Â± 0.20a0.20 Â± 0.0180.11 Â± 0.089 Â± 0.018a0.16 Â± 0.046a0.19 Â± 0.027 Â± 0.08a0.39 Â± 0.038 Â±0.004abTocopherol'g Â±0.1 labZinc\imol/gLiver1.29 Â±0.021abCopperdry Â±0.025a

'Values are means Â±SD,n - 8 (-/- and +/-), n - 6 (-/+| and n - 7 (+/+). Letters indicate significant differences at P the untreated and uninfected group (-/-); b, compared with the untreated and infected group (-/+).

0.05: a, compared with


Tissue nutrient concentrations. Zinc concen tration in the liver was not significantly affected by retrovirus infection, whereas concentrations of vitamins A and E and of copper were significantly lower in the nonsupplemented, infected mice com pared with the non-supplemented, uninfected group (Table 2). Vitamin E supplementation significantly improved the hepatic concentrations of vitamin A, vitamin E, zinc and copper in the retrovirus-infected mice compared with the nonsupplemented, infected mice (Table 2). It had no effect on the hepatic concen trations of zinc and copper in the uninfected mice compared with the nonsupplemented, uninfected group, while significantly increasing their hepatic concentrations of vitamin A and vitamin E (Table 2). Intestinal concentrations of vitamins A and E were significantly reduced by the retrovirus infection com pared with concentrations in the nonsupplemented, infected group, whereas zinc and copper concentra tions were not affected (Table 2). Vitamin E sup plementation significantly increased the intestinal concentration of vitamin E in the retrovirus-infected mice compared with the untreated and infected group, but it had no effect on intestinal concentra tions of vitamin A, zinc and copper in the infected mice (Table 2). The concentrations of vitamin A, vitamin E and zinc in the spleen were significantly reduced by retrovirus infection compared with con centrations in the nonsupplemented, uninfected mice, whereas the copper concentration was signifi

cantly elevated in the infected mice (Table 2). Vitamin E supplementation of infected mice signifi cantly but only partially restored splenic concentra tions of all nutrients except copper in the infected mice compared with the nonsupplemented, infected group (Table 2). The concentrations of vitamins A*and E in the thymus were significantly lessened by retrovirus infection compared with concentrations in the nonsupplemented, uninfected group (Table 3). Vitamin E supplementation of infected mice signifi cantly elevated and thereby restored thymic vitamin A and E concentrations compared with results in the nonsupplemented, infected group (Table 3). Serum vitamin A and E concentrations were significantly reduced by retrovirus infection compared with con centrations in the nonsupplemented, uninfected mice (Table 3). Vitamin E supplementation significantly increased the serum concentration of vitamin E in the retrovirus-infected mice compared with the nonsup plemented, infected group (Table 3), whereas serum vitamin A was not significantly affected by vitamin E. Production of cytokines. Interleukin-2 and IFN--y are secreted by Thl cells and modulate cell-mediated immunity against viral infection. Dietary vitamin E supplementation significantly increased release of IL2 and IFN-7 by Con A-stimulated splenocytes from uninfected mice compared with the nonsup plemented, uninfected group (Table 4). Production of IL-2 and IFN-7 by splenocytes, suppressed by retrovirus infection, was significantly increased by vitamin E supplementation compared with results in the nonsupplemented, infected group (Table 4). These

2029


TABLE 3 Effect of vitamin E on thymic and serum vitamin A and vitamin E concentrations

Serum

Thymus

Treatment

Tocopherol

Retinol

Retrovirus

Vitamin E

during murine AIDS1

Retinol

\anol/L serum

\imollg wet thymus 0.12 0.10 0.07 0.11

Â±0.026 Â±0.019 Â±0.010a Â±0.016b

0.20 0.24 0.14 0.20

Â±0.03 Â±0.02 Â±0.03a Â±0.02b

Tocopherol

7.3 6.9 4.7 5.0

Â±1.5 Â±1.1 Â±1.8a Â±2.2

2.9 7.9 1.7 5.0

Â±1.2 Â±2.2a Â±0.2a Â±1.4ab

Values are means Â±SD,n = 8 (-/- and +/-}, n = 6 (-/+) and n = 7 (+/+)â€¢ Letters indicate significant differences at P < 0.05: a, compared with the untreated and uninfected group (-/-); b, compared with the untreated and infected group (-/+).

stimulated splenocytes from retrovirus-infected mice (Table 4). Immune responses. Vitamin E supplementation had no effect on the splenic NK cell activity in unin fected mice compared with the nonsupplemented, uninfected group. However, vitamin E supplemen tation significantly restored the NK cell activity that was inhibited by retrovirus infection as compared with the nonsupplemented, infected group (Fig. 1). Splenic T and B cell proliferation, suppressed by retrovirus infection, was significantly elevated and thus restored to normal values by vitamin E sup plementation (Fig. 2). Production of IgA and IgG. The vitamin Esupplemented diet had no effect of IgA or IgG production by LPS-induced splenocytes from the uninfected mice compared with the nonsup plemented, uninfected group (Table 5). However, elevated IgA and IgG concentrations, induced by retrovirus infection, were significantly reduced by vitamin E supplementation although normal concen trations were not restored (Table 5).

TABLE 4 Effect of vitamin E on concentrations

splenocytesVitamin TreatmentCon splenocytesIL-6TNF-aW/L0.50+ E

Retrovirus

of cytokines produced

by splenocytes

in vitro during murine AIDS1

A-stimulated

IL-2IFN-7IL-4IL-5IL-6LPS-stimulated

0.03Â±

1.00+ 0.02+ +

1.929.0Â± Â±0.030 0.03Â± 0.05Â± 3.3Â± 0.92.73 Â± 0.06aÂ± 2.4a1.10 Â± Â±0.031 0.03Â± 0.08Â± 2.4Â± 0.34.51 Â± 0.001a 0.15a Â±0.1a 0.13a 4.3a Â±0.6a Â± Â±0.03ab15.018.0 Â±0.9ab0.020 0.002b0.580.592.40 Â±0.01ab2.11.83.42.3Â± Â±0.13b20.521.532.4 Â±1.5b2.52 3.21 Â±O.lb 0.35Â± 0.017 Â±0.0010.001a0.001a 0.40Â± 24.5Â±

sample from each mouse was determined in triplicate. Values are means Â±SD,n = 8 (-/- and +/-), n - 6 (-/+) and n = 7 (+/+). Letters indicate significant differences at P < 0.05: a, compared with the untreated and uninfected group (-/-); b, compared with the untreated and infected group (-/+). 2Abbreviations used: Con A, concanavalin A; IFN-y, interferon-7; IL, interleukin; LPS, lipopolysaccharide; TNF-a, tumor necrosis factor-a.


results are in accordance with the enhancement of NK cell activity and Con A-induced T cell prolifer ation in the spleen (Fig. 1 and 2A). Irtterleukin-4, IL-5 and IL-6 are produced by Th2 cells and modulate humoral responses. Release of IL-4 by Con Astimulated splenocytes from vitamin Esupplemented, uninfected mice was significantly en hanced compared with release by splenocytes from the nonsupplemented, uninfected group (Table 4), whereas IL-5 and IL-6 secretion by Con A-stimulated splenocytes was not significantly affected by vitamin E (Table 4). Retrovirus infection elevated IL-4, IL-5 and IL-6 secretion, while vitamin E supplementation significantly reduced IL-4, IL-5 and IL-6 secretion compared with results for the nonsupplemented, in fected group (Table 4). Production of IL-6 and TNF-a by LPS-stimulated splenocytes could be by macro phages and B cells. Vitamin E supplementation did not have an effect on IL-6 or TNF-a production by LPS-stimulated splenocytes in the uninfected mice compared with the nonsupplemented, uninfected group, whereas it significantly lowered and thereby normalized the high concentrations produced by LPS-

2030

WANG ET AL.

Retrovirus Control

Vitarain E

Control

1500

Vitamin E

M

e

>>

U

M

100:1 50:1 Ratio of Effectors/Target Cells

Control Vitamin E Control

DISCUSSION

Groups

The present study investigated the effects of sup plemental vitamin E during murine AIDS on tissue nutrient concentrations, cytokine secretion by splenocytes, and some immune functions. Increased dietary vitamin E generally restored tissue concentra tions of vitamin A, vitamin E, zinc and copper, which had been suppressed by murine AIDS. Vitamin E supplementation partially restored secretion of IL-2 and IFN-7, mitogenesis of splenocytes, and NK cell

TABLE 5

Effect of vitamin E on concentrations of immunoglobulin and IgG produced by splenocytes in vitro during murine AIDS1

(Ig)A

ImmunoglobulinVitamin Treatment E

+ +

Retrovirus

+ +

IgAVS/L44.5 Â±20.3 Â±0.3 40.0 Â±15.1 3.6 Â±0.3 165.0 Â±43.7a 24.5 Â±9.9a 110.0 Â± 5.0abIgGmg/L3.6 13.5 Â±1.3ab

1Every sample from each mouse was determined in triplicate. Values are means Â±SD,n = 8 (-/- and +/-), n = 6 (-/+) and n = 7 (+/+). Letters indicate significant differences at P < 0.05: a, compared with the untreated and uninfected group (-/-); b, compared with the untreated and infected group (-/+).

Vitamin B

Retrovirus

FIGURE 2 Effect of vitamin E on concanavalin Ainduced (A) and lipopolysaccharide-induced (B) splenic mitogenesis during murine AIDS. Every sample from each mouse was determined in triplicate. Bars represent means + SD. See Table 2 for n for each group. Letters indicate sig nificant differences at P < 0.05: a, compared with the un treated and uninfected group; b, compared with the nonsup plemented, infected group.

activity, which were suppressed by retrovirus in fection. Vitamin E treatment of infected mice reduced levels of IL-4, IL-5, IL-6 and TNF-a, which were elevated by retrovirus infection in nonsupplemented mice. These results suggest a possible role for vitamin E as a potential therapeutic nutrient to help ame liorate undernutrition and immune dysfunction caused by retrovirus infection in AIDS patients. Vitamin A and E deficiencies have also been as sociated with suppression of immune responses and loss of disease resistance. Vitamin A deficiency depresses lymphocyte activation by mitogens in rats and leads to a depletion of mature T cells in the spleen. Repletion with retinoic acid results in a rapid increase in the peripheral lymphocyte count (Sherman and Hallquist 1990). In vitamin E-deficient rats, depressed antibody-dependent cell-mediated cytotoxicity, decreased lymphocytes blastogenesis, and depressed NK cell-mediated cytotoxicity have been reported. Zinc has an important role in immunocompetence and exerts a crucial regulatory


FIGURE 1 Effect of vitamin E on cytotoxicity of splenic natural killer (NK) cells during murine AIDS. Every sample from each mouse was determined in triplicate. Bars rep resent means + SD. See Table 2 for n for each group. Letters indicate significant differences at P < 0.05: a, compared with the untreated and uninfected group; b, compared with the nonsupplemented, infected group.

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phagocytosis of macrophages and neutrophils, and in creasing cytotoxicity of NK cells (Arai et al. 1990). Thus, stimulation of IFN-7 production by vitamin E may explain vitamin E's ability to increase tumor resistance during murine AIDS (Odeleye et al. 1992). Enhancement of IFN-7 production by vitamin E may be due to the decreased production of immunosuppressive prostaglandin Ej by immunocompetent cells (Meydani et al. 1986). Prostaglandin Ej is a potent inhibitor of NK cells (Brunda et al. 1990, Meydani et al. 1990), which are a major source of IFN-7 production. Because NK cell activity is susceptible to oxidative injury by hydrogen peroxide (El-Hag et al. 1980) and vitamin E supplementation decreases hydrogen peroxide level (Bray and Brahmi 1986), in creased NK cell activity and IFN-7 production in mice fed vitamin E was expected. Interleukin-2 is a pivotal cytokine in the growth and differentiation of T and B cells and in the activation of NK cells and lymphokine-activated killer cells (Arai et al. 1990). Thus, the stimulation of IL-2 production we observed in vitamin E-supplemented, uninfected mice and vitamin E-supplemented, retrovirus-infected mice may be responsible for vitamin E's immunoenhancing activity and reduction of tumor growth (Odeleye et al. 1992). Our murine AIDS studies suggest that vitamin E supplementation acts on various immune compo nents to modify immune defects induced by retrovirus infection. Vitamin E supplementation also effectively alleviates nutrient deficiencies induced by retrovirus infection in the immune organs. Thus, the combination of existing medical therapy with nutri tional supplementation may provide successful and novel therapeutic approaches for the treatment of HIV-infected individuals. Although the results of our animal experiments may not totally be extrapolated to HIV-positive humans, information obtained from such studies using murine AIDS may serve as a basis for the study of immunoenhancing properties of vitamin E in HIV-infected humans.

ACKNOWLEDGMENTS We gratefully acknowledge the support of Cleamond D. Eskelson of the Department of Surgery and David K. Y. Lei of the Department of Nutritional Sciences for use of their facility and advice in the vitamin and mineral analysis.

LITERATURE CITED Arai, K., Lee, F., Miyajima, A., Miyatake, S., Arai, N. & Yokotu, T. (1990! Cytokines: coordinators of immune and inflammatory responses. Annu. Rev. Biochem. 59: 783-836. Barnett, S. W., Barboza, A., Wilcox, C. M., Forsmark, L. E. & Levy, I. A. (1991) Characterization of human immunodeficiency virus type 1 strains recovered from the bowel of infected individuals. Virology 182: 802-809.


effect on specific immune functions. Zinc deficiency results in adverse effects on the immune system, including a depression of cell-mediated immunity with decreased T cells and B cells in lymphoid tissue, decreased T helper cell number and activity, an in verted ratio of T helper to T suppressor cells, a depressed response of T cells to tumor cells, decreased lymphokine production, decreased production of cytotoxic T cells, reduced antibody response, and decreased NK cell activity (Keen and Gershwin 1990). In HIV-infected patients, a decrease in serum zinc concentration was correlated with the suppression of lymphoproliferative responses of mononuclear cells to mitogen stimulation, and zinc supplementation augmented this response to mitogen (Falutz et al. 1988). Therefore, zinc deficiency could further pre dispose HIV-infected patients to more frequent or severe infections. Collectively, such nutritional changes may partly contribute to the loss of host resistance to the tumor and to opportunistic infec tions observed in murine AIDS (Darban et al. 1991, Odeleye et al. 1992). Thus, vitamin E supplemen tation during AIDS infection may help restore immune responses and host defenses in part via reversing nutrient deficiencies. It is not clear, however, why the level of copper in the spleen was elevated by retrovirus infection. It may be due to an abnormal absorption or transportation of nutrients associated with retrovirus infection. Clearly, murine retrovirus infection caused tissue deficiencies of nutrients which play important roles in immune functions and antioxidant activities. Free radicals play an important role in AIDS (Baruchel and Wainberg 1990). Tumor necrosis factora generates free radicals from neutrophils at concen trations as low as 0.5 mg/L (Larick and Wright 1992). Tumor necrosis factor-a concentrations are elevated in the serum of HIV patients and mice with murine AIDS, and thus TNF-a may use free radicals as a second messenger. Free radicals can induce the ex pression of HIV in human T cell lines by activating transcription of nuclear factor (NF)-/cB (Paeck et al. 1991, Toledano and Leonard 1991). Thus vitamin E, as an antioxidant, may block activation of NF-icB via lowering free radicals and reducing TNF-a level, thereby inhibiting HIV replication and retarding the progression to AIDS. A unique feature of the retrovirus is its persistence in a quiescent state, prior to activation, without production of either viral mRNA or proteins. Vitamin E may potentially inhibit retrovirus replication and keep the retrovirus in a quiescent state, and retard the progression to AIDS. Indeed, our previous results indicated that vitamin E supplementation significantly reduced free radical concentrations and hepatic lipid peroxidation, which were enhanced by retrovirus infection in murine AIDS (Odeleye et al. 1992). Interferon-7 has multiple distinct biological ac tivities including anti-viral activity, activating

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