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Modulation of immune function and cytokine production by various levels of vitamin E supplementation during murine AIDS
Immunopharmacology ELSEVIER
Immunopharmacology 29 (1995) 225-233
Modulation of immune function and cytokine production by various levels of vitamin E supplementation during murine AIDS Yuejian Wang a'b, Dennis S. Huang a'c, Steve
W o o d a'b,
Ronald R. Watson a'*
aDepartment of Family and Community Medicine, University of Arizona, Tucson, A Z 85724, USA bNutritional Sciences Program University of Arizona Tucson, A Z 85724, USA CDepartment of Microbiology and Immunology, University of Arizona, Tucson, A Z 85724, USA Received 3 August 1994; accepted 18 October 1994
Abstract
Female C57BL/6 mice were infected with LP-BM5 retrovirus, causing murine AIDS which is functionally similar to human AIDS. Dietary supplementation, with a 15-, 150~ and 450-fold increase of vitamin E in a liquid diet, significantly restored levels of interleukin-2 (IL) and interferon-7 produced by splenocytes, which were suppressed by retrovirus infection. Retrovirus infection elevated levels of IL-6 and IL-10 produced by splenocytes, which were significantly normalized by all levels of vitamin E supplementation, respectively. Increased levels of IL-6 and tumor necrosis factor-a, produced by splenocytes during progression to routine AIDS, were also significantly normalized by all levels of vitamin E supplementation. Vitamin E supplementation restored retrovirus-suppressed splenocyte proliferation and natural killer cell cytotoxicity. Vitamin E supplementation also alleviated the AIDS symptoms: splenomegaly and hypergammaglobulinemia. These data indicate that dietary vitamin E supplementation at extremely high levels was not immunotoxic, and can modulate cytokine release and normalize immune dysfunctions during progression to murine AIDS. It should favorably affect host resistance and thereby retard the development of AIDS.
Keywords: Vitamin E; Cytokine; Murine AIDS; Immune response
1. Introduction
Acquired immune deficiency syndrome (AIDS) is a clinical disorder caused by human immunodeficiency virus, representing the end point in a progressive sequence of immunosuppressive changes. Murine LP-BM5 retrovirus induces murine AIDS with many functional similarities to human AIDS, even though the etiology of the two diseases is different. * Corresponding author. Tel: ( + 1 602) 626-6001. Fax ( + 1 602) 626-2030. 0162-3109/95/$9.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 2 - 3 1 0 9 ( 9 5 ) 0 0 0 6 1- l
It is characterized by lymphadenopathy, splenomegaly, hypergammaglobulinemia, deficient B-cell response to mitogen, T-cell functional deficiency, loss of disease resistance and cytokine dysregulation (Watson, 1989). Thus, LP-BM5 retrovirus infection in mice is a useful functional model to screen some therapies for AIDS treatment. Vitamin E functions as an antioxidant and an immunoenhancer. High doses of vitamin E decrease CD8 + T cells, and increase CD4 +/CD8 + ratio and total lymphocyte count, and stimulate activity of cytotoxic cells, natural killer (NK) cell activity, phago-
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cytosis of macrophages and mitogen responsiveness (Odeleye and Watson, 1991). The role of vitamin E in the clinical manifestation of HIV has recently been noticed (Coodley and Girard, 1991). Some patients with varying stages of HIV infection have low plasma levels of vitamin E (Bogden et al., 1990). The data from murine AIDS study further confirmed that the deficiencies of vitamin E occurs in the liver as well as spleen and thymus (Wang et al., 1994c). In rats, depressed antibody-dependent cell mediated cytotoxicity, decreased lymphocytes blastogenesis in response to mitogens, and depressed NK cell mediated cytotoxicity have been reported in vitamin deficient states (Odeleye and Watson, 1991). Thus, a vicious cycle may be set up in which the underlying immunological defects related to HIV infection are exacerbated by retrovirus-induced vitamin E deficiency. In LP-BM5 murine retrovirus infected mice tissue levels of vitamin E are greatly reduced (Wang et al,, 1994c), which could affect immune functions. We recently showed that supplementation with vitamin E partially restored tissue vitamin E and immune functions (Wang et al., 1994b). Consequently, even higher levels of vitamin E supplementation might completely normalize vitamin E deficiency, favorably modulating the immune response in retrovirus infection. Since the combination of existing medical therapy with vitamin E supplementation may provide a more successful and novel therapeutic approach for treatment of HIV infected individuals (Wang et al., 1993, 1994b), the strategy in this study was developed to determine whether supplementation of vitamin E at extremely high levels (150- and 450-fold) would more extensively modulate cytokine production and restore some immune functions altered by retrovirus infection, or whether it would be immunotoxic. 2. Materials and methods 2. I. A nimals
Female C57BL",,6 mice, 5 weeks old, were obtained from the Charles River Laboratories Inc. (Wilmington, DE). The animals were virus-free according to the vendor, and some were tested for virus by University Animal Care. The mice were housed in transparent plastic cages with stainless
steel wire lids with 4 mice per cage. The housing facility was maintained at 20 to 22 ° C and 60 to 80 °o relative humidity. Animals were exposed to a 12:12 h light-dark cycle. Water and mouse chow diet (4 °~o mouse diet, ~7001, Texlad, Madison, WI) were provided ad libitum. Animals were cared for as required by the University of Arizona Committee on Animal Research. After 2 week of adaptation in the animal facility in the Arizona Health Sciences Center, mice were then randomly assigned to one of the following eight treatments: control diets including two groups: uninfected and infected mice, vitamin E supplemented diets (15-fold increase) including two groups: uninfected and infected mice, vitamin E supplemented diets ( 150-fold increase) including two groups: uninfected and infected mice, vitamin E supplemented diets (450-fold increase) including two groups: uninfected and infected mice. All diets were provided ad libitum. The mice consumed 12-15 ml liquid diet per mice/day, No significant differences in diet consumption between groups were observed (data not shown). 2.2. Diet and treatment
All mice were given the National Research Council liquid diet (Watzl et al., 1993). Dietary ingredients were obtained from Dyets (~710279, Bethlehem, PA). The vitamin E supplemented diet had 150 (15-fold increase), 1,500 (150-fold increase) and 4,500 (450-fold increase) IU/L of vitamin E as D-Stocopherol acetate (Sigma, St. Louis, MO) added to liquid diet in addition to the 10.5 IU/L D-z~tocopherol acetate contained in the basal liquid diet. Vitamin E supplementation was started the day after retrovirus infection. The length of dietary treatment and retrovirus infection was 10 weeks. 2.3. L P - B M 5 murine leukemia retrovirus infection
The LP-BM5 retrovirus at 0.1 ml/mouse was injected intraperitoneally to mice. The virus titer had an ecotropic (XC) of 4.5 log10 PFU/ml. It induces disease with a time course comparable to that previously published (Watson, 1989). Infection of adult female C57BL/6 mice with LP-BM5 MuLV leads to the rapid induction of clinical symptoms with virtually no latent phase.
K Dang et al. / Immunopharmacology 29 (1995j 225-233
2.4. Hepatic and serum vitamin E Hepatic concentration of total tocopherol was determined by the fluorometric method as described previously (Dugan et al., 1964). Briefly, about 0.1 g of liver was homogenized in 5 ml water and 5 ml ethanol, and vitamin E extracted with 5.0 ml n-hexane by shaking for 1 min and centrifugation at 3000 rpm for 5 min. Then, 0.5 ml of 60°.o sulfuric acid was added, the solution mixed thoroughly and the fluorescence intensity for vitamin E determined in the hexane at an emission of 340 nm and an excitation maxima of 295 nm using a fluorescence spectrophotometer (Hitachi F-2000, Hitachi, Tokyo, Japan). D-c~-Tocopherol was used as a standard. Serum vitamin E was measured by mixing 0.2 ml of serum with 2 ml of ethanol, extracting with 4.0 ml n-hexane by shaking for 5 min and centrifugation at 3000 rpm for 5 min. Vitamin E level in the n-hexane layers was measured as indicated above.
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chloride Tris buffer, pH 7.2) at 37°C for 2 min. Then the cells were washed twice with CM. Cells were counted and concentrations were adjusted to 1 x 107 cells/ml. Splenocyte viability was more than 95 ~o as determined by trypan blue exclusion. Splenocytes (1 x 107/ml, 0.1 ml/well) from each mouse were cultured in triplicate on 96-well flat-bottom culture plates (Falcon, Lincoln Park, NJ) with CM. Splenocytes were then stimulated with concanavalin A (ConA, 10 #g/ml, 0.1 ml/well, Sigma) for induction of IL-2, IL-6 and IL-10 with 24 h incubation, IFN-7 with 72 h incubation at 37°C, 5°; CO2 incubator. Splenocytes were also stimulated with lipopolysaccharide (LPS, 5 ~,g/ml, Gibco, Grand Island, NY) for 24 h induction for IL-6 and TNF-c~ production, and 72 h for induction of IgG. After incubation indicated above, the plates were centrifuged for 10 rain at 800 g. Supernatant was collected and stored at - 7 0 ° C until analysis. Cytokines and IgG were determined by sandwich ELISA as described previously (Wang et al., 1994a).
2.5. Standard cytokines and their antibodies 2.7. Mitogenesis of splenocvtes Rat anti-murine IFN-7, monoclonal antibody, standard recombinant IFN-7, hamster anti-TNFmonoclonal antibody, standard recombinant TNF-:~, rabbit anti-murine TNF-c~ serum, rat antimurine IL-6 monoclonal antibody and recombinant murine IL-6 were obtained from Genzyme (Boston, MA). Rat anti-routine IL-10 antibody, biotin-rat anti-murine IL-10 monoclonal antibody, and recombinant murine IL-10 were obtained from PharMingen (San Diego, CA). Goat anti-routine IL-6 polyclonal antibody was obtained from RandD System (Minneapolis, MN). Rabbit anti-murine IFN- 7 antiserum was prepared in our lab. 2.6. Cytokine and immunoglobulin G (IgG) production and their measurements IL-2, IFN- 7, IL-6 and IL-10 were produced by splenocytes as described previously (Wang et al., 1994a). Briefly, spleens were gently teased with forceps in culture medium (RPMI 1640 containing 10 ..Jo °j FCS, 2 mM glutamine, 100 units/ml penicillin and streptomycin, CM), producing a single cell suspension of splenocytes. Red blood cells were lysed by the addition of a lysing buffer (0.16 M ammonia
Splenic T and B cell proliferation response to mitogens was determined by [3H]thymidine incorporation as described previously (Wang et al., 1994a). Briefly, splenocytes in 0.1 ml of CM (1 x 107/ml) were cultured in 96-well flat-bottom cultured plates (Falcon) with ConA or LPS (5/~g/ml) in CM. They were incubated at 37°C, 5Yo CO2 incubator for 20 h for ConA-induced T Cell proliferation and 44 h for LPS-induced B cell proliferation, and then pulsed with [3H]thymidine (1 #Ci/well, New England Nuclear, Boston, MA). After 4 h, they were harvested by a cell sample harvester (Cambridge Technology, Cambridge, MA). Radioactivity was determined using a liquid scintillation counter (TriCarb, 2200CA, Packand, Lagunahills, CA). Data are presented as counts per minute (cpm). 2.8. Natural killer (NK) cell O,tOtoxicity N K cell cytotoxicity was measured by a fluorescent concentration release assay modified from the method of Wierda et al. (1989). Briefly, this method measures the fluorescent dye-2,7'-bis(carboxyethyl)-5,6'-carboxyfluorescein (BCECF) (Molecular
Y. Wang et al. / lmmunopharmaeology 29 (1995) 225-233
228
Probes, Eugene, O R ) remaining in the target cells using the Pandex Fluorescence Concentration and Analyzer (FCA) ( I D E X , Portland, ME). The N K sensitive cell line YAC-1 was propagated as a suspension culture in CM. Target cells were washed once with PBS and labeled with the carboxyfluorescein derivative. Effector to target (E:T) ratios were adjusted to 50:1 and plated in U - b o t t o m microtiter plates (Falcon, Lincoln Park, NJ) containing 4 x 10 4 target cells/0.1 ml/well. The cells were then incubated at 3 7 ° C in a humidified atmosphere of 5.0~o carbon dioxide in air for 3 h. After incubation, 20 #1 of 1~o inert fluoricon polystyrene assay particles were added to each well of plate (Pandex Harvesting Plate, I D E X , Portland, Maine), and 80 #1 aliquot from each well of irradiation plate were transferred to a Pantex plate. Epifluorescence of each well in the harvest plate was automatically read at 485/533 nm excitation/emission wavelengths for B C E C F using the Pandex FCA. Specific cytotoxicity (~/o) was calculated as follows:
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3.1. Hepatic and serum levels of vitamin E The concentrations o f hepatic and serum vitamin E were significantly reduced by the retrovirus infection (Fig. 1). Vitamin E supplementation at all levels significantly ( p < 0 . 0 5 ) increased hepatic level of vitamin E in both uninfected and infected mice. However, 450-fold vitamin E supplementation did not further increase hepatic vitamin E in infected mice c o m p a r e d to that increased by 150-fold vitamin E supplementation (Fig. 1A). While the 15-fold vitamin E significantly increased the serum levels of
Fig. 1. Effects of vitamin E supplementation on hepatic (A) and serum (B) vitamin E concentrations during murine AIDS. The values are mean + SD for 4 or 8 mice in each group. Mouse number of groups for 1 × and 15 × was 4 mice per group. Mouse number of groups for 150 × and 450 × was 8 mice per group. (a), p < 0.05 compared to I x vitamin E-fed uninfected or infected mice; (b), p<0.05 compared to 1 × vitamin E-fed uninfected mice; (c), p< 0.05 compared to 15 × vitamin E-fed uninfected or infected mice; (d), p<0.05 compared to 150× vitamin E-fed uninfected or infected mice. vitamin E in both non-infected mice and retrovirusinfected mice (Fig. 1B), 150- and 450-fold vitamin E supplementation did not further increase the levels o f serum vitamin E compared to that increased by 15-fold vitamin E supplementation. There was no effect of vitamin E concentration o f body weight gain (Table 1) or diet consumed (Table 2).
3.2. Production of cytokines IL-2 and I F N - ? are secreted by T h l cells, and are responsible for regulation of cell-mediated immunity
Y. Wang et al. / Immunopharmacology 29 {I995) 225-233
against virus infections and tumors. As the retrovirus infection progressed to murine AIDS, production of IL-2 and IFN- 7 was suppressed (Fig. 2). IL-2 and IFN- 7 production was significantly restored by vitamin E at 15-, 150- and 450-fold vitamin E supplementation, while 150- and 450-fold vitamin E supplementation further normalized the levels of IL-2, but not IFN-7 compared to that increased by 15-fold vitamin E supplementation (Fig. 2). The in-
crease in IL-2 production correlated with the increase of vitamin E supplementation (coefficient of correlation r = 0.981, p<0.019). IL-6 and IL-10 are produced by Th2 cells, and are responsible for regulation of humoral immune responses. The retrovirus infection significantly increased IL-6 and IL-10 production. Elevated IL-6 and IL-10 secretion during murine AID S was significantly decreased by vitamin E supplementation at all levels (Fig. 3). However
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Y. Wang et al. /' lmmunopharmacology 29 (1995) 225-233
230
150- and 450-fold vitamin E supplementation did not further reduce the levels of IL-6 and IL-10 compared to the reduction caused by 15-fold vitamin E supplementation. Production of IL-6 and TNF-zt by LPS-induced splenocytes could be released by macrophages or B cells. Increased secretion of IL-6 and TNF-:~ during murine AIDS could be significantly reduced by all levels of vitamin E supplementation, whereas 150- and 450-fold vitamin E supplementation further reduced the levels of TNF-:~ and
IL-6 compared to that lessened by 15-fold vitamin E supplementation (Fig. 4). The reduction of IL-6 and TNF-c~ release correlated with the increase of vitamin E supplementation (IL-6: r = - 0 . 9 6 , p<0.0397; TNF-~: r = - 0.9396, p<0.049). 3.3. Immune responses
T and B cell proliferation was significantly inhibited by' retrovirus infection (Fig. 5). Higher levels of
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Groups Fig. 5. Effects of vitamin E supplementation on splenic T (A) and B cell (B) proliferation during murine AIDS. Every sample was determined in triplicate. The values are mean + SD for 4 or 8 mice in each group. Mouse number of groups for 1 x and 15 x was 4 mice per group. Mouse number of groups for 150 x and 450 x was 8 mice per group. ( a ) , p < 0 . 0 5 compared to 1 x vitamin E-fed uninfected or infected mice; (b), p < 0 . 0 5 compared to 1 × vitamin E-fed uninfected mice; (c), p < 0 . 0 5 compared to 15 x vitamin E-fed uninfected or infected mice; (d), p < 0.05 compared to 150 x vitamin E-fed uninfected or infected mice.
Y. Wang et al. /lmmunopharmacology 29 (1995) 225-233
dietary vitamin E significantly restored T-cell and B-cell mitogenesis, while 150- or 450-fold increase of vitamin E supplementation further enhanced splenic T and B cell proliferation compared to that increased by 15-fold vitamin E supplementation (Fig. 5). The increase o f T and B cell proliferation was correlated with the increase of vitamin E supplementation (T cells: r= 0.89, p < 0.043; B cells: r= 0.94, p < 0.032). The suppressed NK cell activity by retrovirus infections significantly restored by all levels of vitamin E supplementation during murine AIDS, while 150and 450-fold vitamin E supplementation further increased splenic NK cell activity compared to that increased by that 15-fold vitamin E supplementation. The increase of NK cell activity was correlated with the increase of vitamin E supplementation (r= 0.956, p < 0.0445).
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Splenomegaly and hypergammaglobulinemia are hallmarks of murine AIDS. Level of IgG produced by LPS-stimulated splenocytes significantly increased by retrovirus infection (Fig. 6B). All levels of vitamin E supplementation in the murine AIDS significantly reduced the IgG production, whereas 150- and 450-fold vitamin E supplementation did not further decrease production of IgG compared to that reduced by 15-fold vitamin E supplementation (Fig. 6B). The body weight of mice given vitamin E supplementation during progression to murine AIDS was not significantly different (data not shown). Vitamin E supplementation at all levels significantly reduced spleen weight, but 150- and 450-fold increase of vitamin E supplementation did not further reduce the spleen weight compared to that lessened by 15-fold increase of vitamin E (data not shown). 4. Discussion
The present study investigated the effects of different supplemental vitamin E levels during murine AIDS on cytokine secretion and immune functions. The findings presented in this study confirm and expand our previous findings that vitamin E supplementation could be effective treatment, favorably affecting immune response during murine AIDS (Wang et al., 1994b). Vitamin E supplementation at
20
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M u r i n e AIDS
Groups Fig. 6. Effects of vitamin E supplementation on splenic NK (A) cell cytotoxicity and lgG production by LPS-stimulated splenocytes (B) during murine AIDS. Every sample was determined in triplicate. The values are mean ± SD for 4 or 8 mice in each group. Mouse number of groups for 1 x and 15 x was 4 mice per group. Mouse number of groups for 150 x and 450 × was 8 mice per group. (a),p<0.05 compared to 1 × vitamin E-fed uninfected or infected mice; (b), p < 0.05 compared to 1 x vitamin E-fed uninfected mice; (c), p<0.05 compared to 15 x vitamin E-fed uninfected or infected mice; (d), p < 0.05 compared to I50 x vitamin E-fed uninfected or infected mice.
150- and 450-fold was not imnmnotoxic, even though in some cases this extremely high vitamin E supplementation appears to only marginally further normalize immune dysfunction and cytokine dysregulation compared to that normalized by 15-fold increase of vitamin E. Our results also indicate that very high levels of vitamin E supplementation appear more effective than 15 fold supplementation (Wang et al., 1994b) to maintain some immune func-
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tions from declining and producing murine AIDS. These results suggest a possible role for high vitamin E dosages as a potential therapeutic nutrient to help normalize immune dysfunction caused by HIV infection, thereby retarding the development of AID S. It is intriguing that during murine AIDS vitamin E supplementation stimulates production of Thl cytokines and cell-mediated immunity, whereas it inhibits secretion of Th2 cytokines and hypergammaglobulinemia. Thl and Th2 regulation by glucocorticoid during vitamin E supplementation could lead to the stimulation of Thl cytokines and suppression of Th2 cytokines. Glucocorticoid inhibited cell-mediated immunity as assayed by human and murine primary mixed lymphocyte cultures (Bertoglio and Leroux, 1988), which correlates directly with the amount IL-2 produced (Bertoglio and Leroux (1988; Gills etal., 1979). Daynes etal. (Daynes and Araneo, 1989) demonstrated a differential regulation of the production of Th 1 and Th2 cytokines by bulk T cell populations. In vivo treatment of ovalbumin immunized mice with glucocorticoid led to increased production of IL-4 with decreased secretion of IL-2. Similarly, in vitro exposure of splenocytes to glucocorticoid also shifted the balance of Thl and Th2 cytokines from IL-2 to IL-4 dominance (Daynes and Araneo, 1989). Additional glucocorticoid exposure to ovalbumin specific T cell clones and a T cell hybridoma, that produce both IL-2 and IL-4, also resulted in an inhibition of the IL-2 and IL-4 production and an increase in the IL-4 production (Daynes and Araneo, 1989; Daynes et al., 1990). Thus, we speculate that reduced exposure of T cells to glucocorticoid by vitamin E may shift the differentiation of Th cells to Thl cells. The hypothesis is consistent with the observations that vitamin E supplementation in mice caused a sustained reduction of circulating glucocorticoid levels (Lira et al., 1981), and increased level of circulating glucocorticoid during human AIDS (Coodley et al., 1994), which are correlated with the suppression of immune response (Coodley et al., 1994). Thus, the restoration of imbalance of Th 1/Th2 cytokine secretion by vitamin E explains that vitamin E supplementation alleviated hypergammaglobulinemia and restored cell-mediated immune response during murine AIDS. Another underlying mechanism by which vitamin E supplementation restores the imbalance of
Th 1 and Th2 cytokine secretion is vitamin E-induced reduction of prostaglandin E 2 (PG), a potent inhibitor of T cells and cytokine production. PGE 2 can raise intracellular cyclic AMP (cAMP), which inhibited IL-2 and IFN-7 production by isolated T cells (Chouaib et al., 1985; Feldman et al., 1987), whereas IL-4 and IL-5 production by a Th2 clone was not affected (Betz and Fox, 1991; Krause and Deutsch, 1991). This notion is supported by the findings that PGE 2 produced by splenocytes is drastically increased during murine AIDS (Fernandes etal., 1992). Consequently, the reduction of PGE 2 by vitamin E (Meydani et al., 1986) would be expected to normalize the imbalance of Thl/Th2 cytokine production, explaining vitamin E as a therapeutic agent to stimulate cell-mediated immune response. Since conversion of T h l to Th2 cytokine release may be a critical factor during development of AIDS (Clerici and Shearer, 1993), immunomodulatory nature of vitamin E provides a basis for vitamin E nutritional therapy in AIDS. TNF-e levels are elevated in HIV patients and murine AIDS (Wang et al., 1993; Boue et al., 1991). The pathological effects of TNF-c~ in the body include generation of oxidants (Klebanoff et al., 1986), a potent inducer of HIV replication in monocytes (Toledano and Leonard, 1991, Spriggs et al., 1990; Roederer et al., 1990; Paeck et al., 1991), and stimulating synthesis of PGE 2 (Akama et al., 1990), an immunosuppressive substance produced by macrophages/ monocytes and lymphocytes in humans. These pathological effects of TNF-c~ could also be directly lessened by vitamin E, because vitamin E reduced TNF-:~ level in murine AIDS. Thus, vitamin E supplementation during murine AIDS would alleviate the AIDS symptoms and retard the development of AIDS through reducing TNF-:~, which had been drastically increased by retrovirus infection. Vitamin E was effective in mice that lost tissue vitamin E (Wang et al., 1994a,b). In addition these changes occurred without evidence of reduced food intake or body weight loss (Chen and Watson, 1991). In conclusion, vitamin E supplementation in murine AIDS acts on various immune components to modify immune defects induced by retrovirus infection without any immunotoxicity at extremely high oral supplementation. It tends to normalize immune dysfunction and cytokine dysregulation during mu-
}~ Wang et al. / Immunopharmacology 29 (1995) 225-233
rine AIDS. Thus, the vitamin E nutritional supplementation may provide an additional therapeutic approach for treatment of HIV infected patients without additional immunotoxicity. References Akama H, Ichikawa Y, Matsushita Y, Shinozawa T, Homma M. Mononuclear cells enhance prostaglandin E2 production of polymorphonuclear leukocytes via tumor necrosis factor-:t. Biochem Biophys Res Commun 1990; 168: 857. Bogden JD, Baker H, Frank, O. Micronutrient status and human immuno- deficiency virus (HIV) infection. Ann NY Acad Sci 1990; 587: 189. Betz M, Fox BS. Prostagiandin E2 inhibits production of Thl lymphocytes but no ofTh2 lymphokines. J Immunol 1991; 146: 103. Boue F, Wallon C, Goujard C, Barresinouss F, Galand P. and Defraissy, JF. HIV induced IL-6 production by human B lymphocytes: Role of IL-4. J Immunol 1991; 148: 3761. Bertoglio JH, Leroux E. Differential effects of glucocorticoid on the proliferation of a murine helper and a cytolytic T cell clone in response to IL-2 and IL-4. J Immunol 1988; 141: 1191. Chen, GJ, Watson, RR. Modulation of tumor necrosis factor and gamma interferon production by cocaine and morphine in aging mice infected with LP-BM5, a murine retrovirus. J. Leuk. Biol. 1991; 50: 349-355. Chouaib S, Welte K, Mertelsmann R, Dupont T. PGE2 acts at two distinct pathways ofT lymphocyte activation: inhibition of IL-2 production and downregulation of transferrin expression. J Immunol 1985; 135: 1172. Clerici M, Shearer GM. A THI-~TH2 switch is a critical step in the etiology of HIV infection. Immunol Today 1993; 14: 107. Coodley G, Girard DE. Vitamins and Minerals in HIV infection J Gen Int Med 1991; 6: 472. Coodley GO, Loveless MO, Heidi D, Nelson HD, Coodley MK. Endocrine function in the HIV wasting syndrome. J AIDS 1994; 7: 46. Daynes RA, Araneo BA. Contrasting effects ofglucocorticoid on the capacity ofT cells to produce the growth factors interleukin 2 and interleukin 4. Eur J Immunol 1989; 19: 2319. Daynes RA, Dudley D J, Araneo BA. Regulation of murine lymphokine production in vivo. II. Dehydroepiandrosterone is a natural enhancer of IL-2 synthesis in helper T cells. Eur J Immunol 1990; 20: 793. Dugan RE, Frigero NA, Siebert JM. Colorimetric determination of vitamin A and its derivative with trifluoroacetic acid. Anal Chem 1964; 36: 114. Fauci AS, Dale DC. The effect of in vivo hydrocortisone on subpopulations of human lymphocytes. J Clin Invest 1974; 53: 240. Feldman RD, Hunninghake GW, McArdle WL. fl-Adrenergic receptor-mediated suppression of IL-2 receptor in human lymphocytes. J Immunol 1987; 139: 3355. Fernandes G, Tomar V, Mohan N, Venkatraman JA. Potential of diet therapy on murine AIDS. J Nutr 1992; 122: 716.
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Gills S, Crabtree GR, Smith KA. Glucocorticoid-induced inhibition of T cell growth factor production. I. The effect on mitogen-induced lymphocyte proliferation. J Immunol 1979; 123: 1624. Krause DS, Deutsch C. Cyclic AMP directly inhibits IL-2 receptor expression in human T cells: expression of both p55 and p75 subunits is affected. J Immunol (1991) 146: 2285. Klebanoff SJ, Vadas MA, Harlan JM. Stimulation of neutrophils by tumor necrosis factor. J Immunol 1986; 136: 4220. Lim TS, Putt N, Satranski D, Chung C, Watson RR. Effect of vitamin E on cell-mediated immune responses and serum corticosterone in young and maturing mice. Immunol 1981; 44: 289. Meydani SN, Meydani M, Verdon CP, Shapiro AA, Blumberg JB, Hayes KC. Vitamin E supplementation suppresses PGE2 synthesis and enhances the immune response of aged mice. Mech Ageing Dev 1986; 12: 191. Odeleye OE, Watson RR. The potential role of vitamin E in the treatment of immunological abnormalities during acquired immune deficiency syndrome. Prog Food Nutr Sci 1991; 15: 1. Paeck R, Rubin P, Bauerle PA. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kB transcription factor and HIV. EMBO J 1991; 10: 2247. Roederer M, Stall FJ, Raju PA. ELA SW, Herzenberg LA. Cytokine-stimulated human immunodeficiency virus replication is inhibited by N-acetyl-L-cysteine. Proc Natl Acad Sci USA 1990; 87: 4884. Spriggs DR, Sherman ML, Imamura K. Phospholipase A2 activation and autoinduction of tumor necrosis factor gene expression by tumor necrosis factor. Cancer Res 1990; 50: 7101. Toledano M, Leonard W. Modulation of transcription factor NF-kB by oxidation-reduction in vitro. Proc Natl Acad Sci USA 1991; 88: 4328. Wang Y, Huang D S, Giger PT, Watson RR. Kinetics of cytokine production by T cells and macrophages during murine AIDS. Adv Biol Sci 1993; 86: 335. Wang Y, Huang DS, Giger PT, Watson RR. Influence of chronic dietary ethanol on cytokine production by murine splenocytes and thymocytes. Alcohol Clin Exp Res 1994a; 18: 64. Wang Y, Huang DS, Liang B, Watson, RR, Normalization of restoration of nutritional status and immune functions by vitamin E supplementation in routine AIDS. J Nutr 1994b; in press. Wang Y, Liang B, Watson RR. Suppression of tissue levels of vitamin A, E, zinc and copper in murine AIDS. Nutr Res 1994b 14: 1031. Wang Y, Watson RR. Is Vitamin E supplementation a useful agent in AIDS therapy? Prog Food Nutr Sci 1993; 17: 351. Watson RR. Murine models for acquired deficiency syndrome. Life Sci 1989; 44: i. Watzl B, Lopez M, Shabhazian M, Chen G-J, Colombo LL, Huang DS, Witte, M, Watson, RR. Diet and ethanol modulate immune responses in young C57BL/6 mice. Alcohol Clin Exp Res 1993; 17: 623. Wierda WG, Mehr DS, Kim YB. Comparison of fluorochromelabeled and 5tCr-labeled targets for natural killer cytotoxicity assay. J Immunol Methods 1989; 122: 15.
Immunopharmacology 29 (1995) 225-233
Modulation of immune function and cytokine production by various levels of vitamin E supplementation during murine AIDS Yuejian Wang a'b, Dennis S. Huang a'c, Steve
W o o d a'b,
Ronald R. Watson a'*
aDepartment of Family and Community Medicine, University of Arizona, Tucson, A Z 85724, USA bNutritional Sciences Program University of Arizona Tucson, A Z 85724, USA CDepartment of Microbiology and Immunology, University of Arizona, Tucson, A Z 85724, USA Received 3 August 1994; accepted 18 October 1994
Abstract
Female C57BL/6 mice were infected with LP-BM5 retrovirus, causing murine AIDS which is functionally similar to human AIDS. Dietary supplementation, with a 15-, 150~ and 450-fold increase of vitamin E in a liquid diet, significantly restored levels of interleukin-2 (IL) and interferon-7 produced by splenocytes, which were suppressed by retrovirus infection. Retrovirus infection elevated levels of IL-6 and IL-10 produced by splenocytes, which were significantly normalized by all levels of vitamin E supplementation, respectively. Increased levels of IL-6 and tumor necrosis factor-a, produced by splenocytes during progression to routine AIDS, were also significantly normalized by all levels of vitamin E supplementation. Vitamin E supplementation restored retrovirus-suppressed splenocyte proliferation and natural killer cell cytotoxicity. Vitamin E supplementation also alleviated the AIDS symptoms: splenomegaly and hypergammaglobulinemia. These data indicate that dietary vitamin E supplementation at extremely high levels was not immunotoxic, and can modulate cytokine release and normalize immune dysfunctions during progression to murine AIDS. It should favorably affect host resistance and thereby retard the development of AIDS.
Keywords: Vitamin E; Cytokine; Murine AIDS; Immune response
1. Introduction
Acquired immune deficiency syndrome (AIDS) is a clinical disorder caused by human immunodeficiency virus, representing the end point in a progressive sequence of immunosuppressive changes. Murine LP-BM5 retrovirus induces murine AIDS with many functional similarities to human AIDS, even though the etiology of the two diseases is different. * Corresponding author. Tel: ( + 1 602) 626-6001. Fax ( + 1 602) 626-2030. 0162-3109/95/$9.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 2 - 3 1 0 9 ( 9 5 ) 0 0 0 6 1- l
It is characterized by lymphadenopathy, splenomegaly, hypergammaglobulinemia, deficient B-cell response to mitogen, T-cell functional deficiency, loss of disease resistance and cytokine dysregulation (Watson, 1989). Thus, LP-BM5 retrovirus infection in mice is a useful functional model to screen some therapies for AIDS treatment. Vitamin E functions as an antioxidant and an immunoenhancer. High doses of vitamin E decrease CD8 + T cells, and increase CD4 +/CD8 + ratio and total lymphocyte count, and stimulate activity of cytotoxic cells, natural killer (NK) cell activity, phago-
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K Wang et al. / lmmunopharmacology 29 (1995) 225-233
cytosis of macrophages and mitogen responsiveness (Odeleye and Watson, 1991). The role of vitamin E in the clinical manifestation of HIV has recently been noticed (Coodley and Girard, 1991). Some patients with varying stages of HIV infection have low plasma levels of vitamin E (Bogden et al., 1990). The data from murine AIDS study further confirmed that the deficiencies of vitamin E occurs in the liver as well as spleen and thymus (Wang et al., 1994c). In rats, depressed antibody-dependent cell mediated cytotoxicity, decreased lymphocytes blastogenesis in response to mitogens, and depressed NK cell mediated cytotoxicity have been reported in vitamin deficient states (Odeleye and Watson, 1991). Thus, a vicious cycle may be set up in which the underlying immunological defects related to HIV infection are exacerbated by retrovirus-induced vitamin E deficiency. In LP-BM5 murine retrovirus infected mice tissue levels of vitamin E are greatly reduced (Wang et al,, 1994c), which could affect immune functions. We recently showed that supplementation with vitamin E partially restored tissue vitamin E and immune functions (Wang et al., 1994b). Consequently, even higher levels of vitamin E supplementation might completely normalize vitamin E deficiency, favorably modulating the immune response in retrovirus infection. Since the combination of existing medical therapy with vitamin E supplementation may provide a more successful and novel therapeutic approach for treatment of HIV infected individuals (Wang et al., 1993, 1994b), the strategy in this study was developed to determine whether supplementation of vitamin E at extremely high levels (150- and 450-fold) would more extensively modulate cytokine production and restore some immune functions altered by retrovirus infection, or whether it would be immunotoxic. 2. Materials and methods 2. I. A nimals
Female C57BL",,6 mice, 5 weeks old, were obtained from the Charles River Laboratories Inc. (Wilmington, DE). The animals were virus-free according to the vendor, and some were tested for virus by University Animal Care. The mice were housed in transparent plastic cages with stainless
steel wire lids with 4 mice per cage. The housing facility was maintained at 20 to 22 ° C and 60 to 80 °o relative humidity. Animals were exposed to a 12:12 h light-dark cycle. Water and mouse chow diet (4 °~o mouse diet, ~7001, Texlad, Madison, WI) were provided ad libitum. Animals were cared for as required by the University of Arizona Committee on Animal Research. After 2 week of adaptation in the animal facility in the Arizona Health Sciences Center, mice were then randomly assigned to one of the following eight treatments: control diets including two groups: uninfected and infected mice, vitamin E supplemented diets (15-fold increase) including two groups: uninfected and infected mice, vitamin E supplemented diets ( 150-fold increase) including two groups: uninfected and infected mice, vitamin E supplemented diets (450-fold increase) including two groups: uninfected and infected mice. All diets were provided ad libitum. The mice consumed 12-15 ml liquid diet per mice/day, No significant differences in diet consumption between groups were observed (data not shown). 2.2. Diet and treatment
All mice were given the National Research Council liquid diet (Watzl et al., 1993). Dietary ingredients were obtained from Dyets (~710279, Bethlehem, PA). The vitamin E supplemented diet had 150 (15-fold increase), 1,500 (150-fold increase) and 4,500 (450-fold increase) IU/L of vitamin E as D-Stocopherol acetate (Sigma, St. Louis, MO) added to liquid diet in addition to the 10.5 IU/L D-z~tocopherol acetate contained in the basal liquid diet. Vitamin E supplementation was started the day after retrovirus infection. The length of dietary treatment and retrovirus infection was 10 weeks. 2.3. L P - B M 5 murine leukemia retrovirus infection
The LP-BM5 retrovirus at 0.1 ml/mouse was injected intraperitoneally to mice. The virus titer had an ecotropic (XC) of 4.5 log10 PFU/ml. It induces disease with a time course comparable to that previously published (Watson, 1989). Infection of adult female C57BL/6 mice with LP-BM5 MuLV leads to the rapid induction of clinical symptoms with virtually no latent phase.
K Dang et al. / Immunopharmacology 29 (1995j 225-233
2.4. Hepatic and serum vitamin E Hepatic concentration of total tocopherol was determined by the fluorometric method as described previously (Dugan et al., 1964). Briefly, about 0.1 g of liver was homogenized in 5 ml water and 5 ml ethanol, and vitamin E extracted with 5.0 ml n-hexane by shaking for 1 min and centrifugation at 3000 rpm for 5 min. Then, 0.5 ml of 60°.o sulfuric acid was added, the solution mixed thoroughly and the fluorescence intensity for vitamin E determined in the hexane at an emission of 340 nm and an excitation maxima of 295 nm using a fluorescence spectrophotometer (Hitachi F-2000, Hitachi, Tokyo, Japan). D-c~-Tocopherol was used as a standard. Serum vitamin E was measured by mixing 0.2 ml of serum with 2 ml of ethanol, extracting with 4.0 ml n-hexane by shaking for 5 min and centrifugation at 3000 rpm for 5 min. Vitamin E level in the n-hexane layers was measured as indicated above.
227
chloride Tris buffer, pH 7.2) at 37°C for 2 min. Then the cells were washed twice with CM. Cells were counted and concentrations were adjusted to 1 x 107 cells/ml. Splenocyte viability was more than 95 ~o as determined by trypan blue exclusion. Splenocytes (1 x 107/ml, 0.1 ml/well) from each mouse were cultured in triplicate on 96-well flat-bottom culture plates (Falcon, Lincoln Park, NJ) with CM. Splenocytes were then stimulated with concanavalin A (ConA, 10 #g/ml, 0.1 ml/well, Sigma) for induction of IL-2, IL-6 and IL-10 with 24 h incubation, IFN-7 with 72 h incubation at 37°C, 5°; CO2 incubator. Splenocytes were also stimulated with lipopolysaccharide (LPS, 5 ~,g/ml, Gibco, Grand Island, NY) for 24 h induction for IL-6 and TNF-c~ production, and 72 h for induction of IgG. After incubation indicated above, the plates were centrifuged for 10 rain at 800 g. Supernatant was collected and stored at - 7 0 ° C until analysis. Cytokines and IgG were determined by sandwich ELISA as described previously (Wang et al., 1994a).
2.5. Standard cytokines and their antibodies 2.7. Mitogenesis of splenocvtes Rat anti-murine IFN-7, monoclonal antibody, standard recombinant IFN-7, hamster anti-TNFmonoclonal antibody, standard recombinant TNF-:~, rabbit anti-murine TNF-c~ serum, rat antimurine IL-6 monoclonal antibody and recombinant murine IL-6 were obtained from Genzyme (Boston, MA). Rat anti-routine IL-10 antibody, biotin-rat anti-murine IL-10 monoclonal antibody, and recombinant murine IL-10 were obtained from PharMingen (San Diego, CA). Goat anti-routine IL-6 polyclonal antibody was obtained from RandD System (Minneapolis, MN). Rabbit anti-murine IFN- 7 antiserum was prepared in our lab. 2.6. Cytokine and immunoglobulin G (IgG) production and their measurements IL-2, IFN- 7, IL-6 and IL-10 were produced by splenocytes as described previously (Wang et al., 1994a). Briefly, spleens were gently teased with forceps in culture medium (RPMI 1640 containing 10 ..Jo °j FCS, 2 mM glutamine, 100 units/ml penicillin and streptomycin, CM), producing a single cell suspension of splenocytes. Red blood cells were lysed by the addition of a lysing buffer (0.16 M ammonia
Splenic T and B cell proliferation response to mitogens was determined by [3H]thymidine incorporation as described previously (Wang et al., 1994a). Briefly, splenocytes in 0.1 ml of CM (1 x 107/ml) were cultured in 96-well flat-bottom cultured plates (Falcon) with ConA or LPS (5/~g/ml) in CM. They were incubated at 37°C, 5Yo CO2 incubator for 20 h for ConA-induced T Cell proliferation and 44 h for LPS-induced B cell proliferation, and then pulsed with [3H]thymidine (1 #Ci/well, New England Nuclear, Boston, MA). After 4 h, they were harvested by a cell sample harvester (Cambridge Technology, Cambridge, MA). Radioactivity was determined using a liquid scintillation counter (TriCarb, 2200CA, Packand, Lagunahills, CA). Data are presented as counts per minute (cpm). 2.8. Natural killer (NK) cell O,tOtoxicity N K cell cytotoxicity was measured by a fluorescent concentration release assay modified from the method of Wierda et al. (1989). Briefly, this method measures the fluorescent dye-2,7'-bis(carboxyethyl)-5,6'-carboxyfluorescein (BCECF) (Molecular
Y. Wang et al. / lmmunopharmaeology 29 (1995) 225-233
228
Probes, Eugene, O R ) remaining in the target cells using the Pandex Fluorescence Concentration and Analyzer (FCA) ( I D E X , Portland, ME). The N K sensitive cell line YAC-1 was propagated as a suspension culture in CM. Target cells were washed once with PBS and labeled with the carboxyfluorescein derivative. Effector to target (E:T) ratios were adjusted to 50:1 and plated in U - b o t t o m microtiter plates (Falcon, Lincoln Park, NJ) containing 4 x 10 4 target cells/0.1 ml/well. The cells were then incubated at 3 7 ° C in a humidified atmosphere of 5.0~o carbon dioxide in air for 3 h. After incubation, 20 #1 of 1~o inert fluoricon polystyrene assay particles were added to each well of plate (Pandex Harvesting Plate, I D E X , Portland, Maine), and 80 #1 aliquot from each well of irradiation plate were transferred to a Pantex plate. Epifluorescence of each well in the harvest plate was automatically read at 485/533 nm excitation/emission wavelengths for B C E C F using the Pandex FCA. Specific cytotoxicity (~/o) was calculated as follows:
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3.1. Hepatic and serum levels of vitamin E The concentrations o f hepatic and serum vitamin E were significantly reduced by the retrovirus infection (Fig. 1). Vitamin E supplementation at all levels significantly ( p < 0 . 0 5 ) increased hepatic level of vitamin E in both uninfected and infected mice. However, 450-fold vitamin E supplementation did not further increase hepatic vitamin E in infected mice c o m p a r e d to that increased by 150-fold vitamin E supplementation (Fig. 1A). While the 15-fold vitamin E significantly increased the serum levels of
Fig. 1. Effects of vitamin E supplementation on hepatic (A) and serum (B) vitamin E concentrations during murine AIDS. The values are mean + SD for 4 or 8 mice in each group. Mouse number of groups for 1 × and 15 × was 4 mice per group. Mouse number of groups for 150 × and 450 × was 8 mice per group. (a), p < 0.05 compared to I x vitamin E-fed uninfected or infected mice; (b), p<0.05 compared to 1 × vitamin E-fed uninfected mice; (c), p< 0.05 compared to 15 × vitamin E-fed uninfected or infected mice; (d), p<0.05 compared to 150× vitamin E-fed uninfected or infected mice. vitamin E in both non-infected mice and retrovirusinfected mice (Fig. 1B), 150- and 450-fold vitamin E supplementation did not further increase the levels o f serum vitamin E compared to that increased by 15-fold vitamin E supplementation. There was no effect of vitamin E concentration o f body weight gain (Table 1) or diet consumed (Table 2).
3.2. Production of cytokines IL-2 and I F N - ? are secreted by T h l cells, and are responsible for regulation of cell-mediated immunity
Y. Wang et al. / Immunopharmacology 29 {I995) 225-233
against virus infections and tumors. As the retrovirus infection progressed to murine AIDS, production of IL-2 and IFN- 7 was suppressed (Fig. 2). IL-2 and IFN- 7 production was significantly restored by vitamin E at 15-, 150- and 450-fold vitamin E supplementation, while 150- and 450-fold vitamin E supplementation further normalized the levels of IL-2, but not IFN-7 compared to that increased by 15-fold vitamin E supplementation (Fig. 2). The in-
crease in IL-2 production correlated with the increase of vitamin E supplementation (coefficient of correlation r = 0.981, p<0.019). IL-6 and IL-10 are produced by Th2 cells, and are responsible for regulation of humoral immune responses. The retrovirus infection significantly increased IL-6 and IL-10 production. Elevated IL-6 and IL-10 secretion during murine AID S was significantly decreased by vitamin E supplementation at all levels (Fig. 3). However
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Y. Wang et al. /' lmmunopharmacology 29 (1995) 225-233
230
150- and 450-fold vitamin E supplementation did not further reduce the levels of IL-6 and IL-10 compared to the reduction caused by 15-fold vitamin E supplementation. Production of IL-6 and TNF-zt by LPS-induced splenocytes could be released by macrophages or B cells. Increased secretion of IL-6 and TNF-:~ during murine AIDS could be significantly reduced by all levels of vitamin E supplementation, whereas 150- and 450-fold vitamin E supplementation further reduced the levels of TNF-:~ and
IL-6 compared to that lessened by 15-fold vitamin E supplementation (Fig. 4). The reduction of IL-6 and TNF-c~ release correlated with the increase of vitamin E supplementation (IL-6: r = - 0 . 9 6 , p<0.0397; TNF-~: r = - 0.9396, p<0.049). 3.3. Immune responses
T and B cell proliferation was significantly inhibited by' retrovirus infection (Fig. 5). Higher levels of
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Groups Fig. 5. Effects of vitamin E supplementation on splenic T (A) and B cell (B) proliferation during murine AIDS. Every sample was determined in triplicate. The values are mean + SD for 4 or 8 mice in each group. Mouse number of groups for 1 x and 15 x was 4 mice per group. Mouse number of groups for 150 x and 450 x was 8 mice per group. ( a ) , p < 0 . 0 5 compared to 1 x vitamin E-fed uninfected or infected mice; (b), p < 0 . 0 5 compared to 1 × vitamin E-fed uninfected mice; (c), p < 0 . 0 5 compared to 15 x vitamin E-fed uninfected or infected mice; (d), p < 0.05 compared to 150 x vitamin E-fed uninfected or infected mice.
Y. Wang et al. /lmmunopharmacology 29 (1995) 225-233
dietary vitamin E significantly restored T-cell and B-cell mitogenesis, while 150- or 450-fold increase of vitamin E supplementation further enhanced splenic T and B cell proliferation compared to that increased by 15-fold vitamin E supplementation (Fig. 5). The increase o f T and B cell proliferation was correlated with the increase of vitamin E supplementation (T cells: r= 0.89, p < 0.043; B cells: r= 0.94, p < 0.032). The suppressed NK cell activity by retrovirus infections significantly restored by all levels of vitamin E supplementation during murine AIDS, while 150and 450-fold vitamin E supplementation further increased splenic NK cell activity compared to that increased by that 15-fold vitamin E supplementation. The increase of NK cell activity was correlated with the increase of vitamin E supplementation (r= 0.956, p < 0.0445).
231
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Splenomegaly and hypergammaglobulinemia are hallmarks of murine AIDS. Level of IgG produced by LPS-stimulated splenocytes significantly increased by retrovirus infection (Fig. 6B). All levels of vitamin E supplementation in the murine AIDS significantly reduced the IgG production, whereas 150- and 450-fold vitamin E supplementation did not further decrease production of IgG compared to that reduced by 15-fold vitamin E supplementation (Fig. 6B). The body weight of mice given vitamin E supplementation during progression to murine AIDS was not significantly different (data not shown). Vitamin E supplementation at all levels significantly reduced spleen weight, but 150- and 450-fold increase of vitamin E supplementation did not further reduce the spleen weight compared to that lessened by 15-fold increase of vitamin E (data not shown). 4. Discussion
The present study investigated the effects of different supplemental vitamin E levels during murine AIDS on cytokine secretion and immune functions. The findings presented in this study confirm and expand our previous findings that vitamin E supplementation could be effective treatment, favorably affecting immune response during murine AIDS (Wang et al., 1994b). Vitamin E supplementation at
20
0 Unifected
M u r i n e AIDS
Groups Fig. 6. Effects of vitamin E supplementation on splenic NK (A) cell cytotoxicity and lgG production by LPS-stimulated splenocytes (B) during murine AIDS. Every sample was determined in triplicate. The values are mean ± SD for 4 or 8 mice in each group. Mouse number of groups for 1 x and 15 x was 4 mice per group. Mouse number of groups for 150 x and 450 × was 8 mice per group. (a),p<0.05 compared to 1 × vitamin E-fed uninfected or infected mice; (b), p < 0.05 compared to 1 x vitamin E-fed uninfected mice; (c), p<0.05 compared to 15 x vitamin E-fed uninfected or infected mice; (d), p < 0.05 compared to I50 x vitamin E-fed uninfected or infected mice.
150- and 450-fold was not imnmnotoxic, even though in some cases this extremely high vitamin E supplementation appears to only marginally further normalize immune dysfunction and cytokine dysregulation compared to that normalized by 15-fold increase of vitamin E. Our results also indicate that very high levels of vitamin E supplementation appear more effective than 15 fold supplementation (Wang et al., 1994b) to maintain some immune func-
232
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tions from declining and producing murine AIDS. These results suggest a possible role for high vitamin E dosages as a potential therapeutic nutrient to help normalize immune dysfunction caused by HIV infection, thereby retarding the development of AID S. It is intriguing that during murine AIDS vitamin E supplementation stimulates production of Thl cytokines and cell-mediated immunity, whereas it inhibits secretion of Th2 cytokines and hypergammaglobulinemia. Thl and Th2 regulation by glucocorticoid during vitamin E supplementation could lead to the stimulation of Thl cytokines and suppression of Th2 cytokines. Glucocorticoid inhibited cell-mediated immunity as assayed by human and murine primary mixed lymphocyte cultures (Bertoglio and Leroux, 1988), which correlates directly with the amount IL-2 produced (Bertoglio and Leroux (1988; Gills etal., 1979). Daynes etal. (Daynes and Araneo, 1989) demonstrated a differential regulation of the production of Th 1 and Th2 cytokines by bulk T cell populations. In vivo treatment of ovalbumin immunized mice with glucocorticoid led to increased production of IL-4 with decreased secretion of IL-2. Similarly, in vitro exposure of splenocytes to glucocorticoid also shifted the balance of Thl and Th2 cytokines from IL-2 to IL-4 dominance (Daynes and Araneo, 1989). Additional glucocorticoid exposure to ovalbumin specific T cell clones and a T cell hybridoma, that produce both IL-2 and IL-4, also resulted in an inhibition of the IL-2 and IL-4 production and an increase in the IL-4 production (Daynes and Araneo, 1989; Daynes et al., 1990). Thus, we speculate that reduced exposure of T cells to glucocorticoid by vitamin E may shift the differentiation of Th cells to Thl cells. The hypothesis is consistent with the observations that vitamin E supplementation in mice caused a sustained reduction of circulating glucocorticoid levels (Lira et al., 1981), and increased level of circulating glucocorticoid during human AIDS (Coodley et al., 1994), which are correlated with the suppression of immune response (Coodley et al., 1994). Thus, the restoration of imbalance of Th 1/Th2 cytokine secretion by vitamin E explains that vitamin E supplementation alleviated hypergammaglobulinemia and restored cell-mediated immune response during murine AIDS. Another underlying mechanism by which vitamin E supplementation restores the imbalance of
Th 1 and Th2 cytokine secretion is vitamin E-induced reduction of prostaglandin E 2 (PG), a potent inhibitor of T cells and cytokine production. PGE 2 can raise intracellular cyclic AMP (cAMP), which inhibited IL-2 and IFN-7 production by isolated T cells (Chouaib et al., 1985; Feldman et al., 1987), whereas IL-4 and IL-5 production by a Th2 clone was not affected (Betz and Fox, 1991; Krause and Deutsch, 1991). This notion is supported by the findings that PGE 2 produced by splenocytes is drastically increased during murine AIDS (Fernandes etal., 1992). Consequently, the reduction of PGE 2 by vitamin E (Meydani et al., 1986) would be expected to normalize the imbalance of Thl/Th2 cytokine production, explaining vitamin E as a therapeutic agent to stimulate cell-mediated immune response. Since conversion of T h l to Th2 cytokine release may be a critical factor during development of AIDS (Clerici and Shearer, 1993), immunomodulatory nature of vitamin E provides a basis for vitamin E nutritional therapy in AIDS. TNF-e levels are elevated in HIV patients and murine AIDS (Wang et al., 1993; Boue et al., 1991). The pathological effects of TNF-c~ in the body include generation of oxidants (Klebanoff et al., 1986), a potent inducer of HIV replication in monocytes (Toledano and Leonard, 1991, Spriggs et al., 1990; Roederer et al., 1990; Paeck et al., 1991), and stimulating synthesis of PGE 2 (Akama et al., 1990), an immunosuppressive substance produced by macrophages/ monocytes and lymphocytes in humans. These pathological effects of TNF-c~ could also be directly lessened by vitamin E, because vitamin E reduced TNF-:~ level in murine AIDS. Thus, vitamin E supplementation during murine AIDS would alleviate the AIDS symptoms and retard the development of AIDS through reducing TNF-:~, which had been drastically increased by retrovirus infection. Vitamin E was effective in mice that lost tissue vitamin E (Wang et al., 1994a,b). In addition these changes occurred without evidence of reduced food intake or body weight loss (Chen and Watson, 1991). In conclusion, vitamin E supplementation in murine AIDS acts on various immune components to modify immune defects induced by retrovirus infection without any immunotoxicity at extremely high oral supplementation. It tends to normalize immune dysfunction and cytokine dysregulation during mu-
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