Supplementation with selenium and human immune cell functions

�9 1994 by Humana Press Inc. All rights of any nature, whatsoever, reserved, 0163-4984/94/4101-2-0103 $03,40

Supplementation with Selenium and Human Immune Cell Functions

I. Effect on L y m p h o c y t e Prol i ferat ion and Interleukin 2 R e c e p t o r E x p r e s s i o n

MARTIN ROY, *,1 LIDIA KIREMIDJIAN-SCHUMACHER, 2 HARVEY I. WISHE, ~ MARTIN W. COHEN, 2

AND GLINTHER STOTZKY 3

New York University Dental Center, ~Department of Histology and Cell Biology; 2Department of Oral Medicine

and Pathology, New York; and 3New York University, Biology Department, New York

Received August 10, 1993; Accepted September 26, 1993

ABSTRACT

Selenium (Se) is an essential nutritional factor that was shown by us to alter the expression of the high affinity interleukin 2 receptor (II2-R) and its subunits, cell proliferation, and clonal expansion of cytotoxic Tqymphocytes in mice. This study shows that dietary supplementation of Se-replete humans with 200 Bg/d of sodium selenite for 8 wk, or in vitro supplementation with 1 x 10-7 M Se (as sodium selenite), result in a significant augmentation of the ability of peripheral blood lymphocytes to respond to stimulation with 1 btg/mL of phytohemagglutinin or alloantigen (mixed lymphocyte reaction) and to express high affinity II2-R on their surface. There was a clear correlation between supplementation with Se and enhanced 3H-thymidine incorporation into nuclear DNA, preceded by enhanced expression of high affinity I12-R. Supplementation with Se can appar- ently modulate T-lymphocyte mediated immune responses in humans that depend on signals generated by the interaction of interleukin 2 with II2-R.

Index Entries: Selenium; human; immunoenhancement; II2-R (interleukin 2 receptor); lymphocytes; proliferation.

*Author to whom all correspondence and reprint requests should be addressed.

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104 Roy et al.

INTRODUCTION

The interaction of mature peripheral blood T-lymphocytes with specific antigen or mitogen in the presence of accessory cells and in the con- text of major histocompatibility complex (MHC) molecules results in the initiation of signal transduction cascades that lead to T-cell proliferation and effector function. This finely tuned chain of biochemical events requires the participation of a number of cytokines, among which interleukin 2 (I12) is critical for T-cell proliferation (1). To exert its effect, II2 must interact with high affinity II2 receptors (II2-R) on the surface of the cells, which results in a signal transduction that culminates with the entry of the cell into the S phase of the cell cycle (2). The resulting proliferation and clonal expansion of lymphocytes represents the basis for the expression of all major immunologic functions.

Our previous studies utilizing the mouse as a model system have shown that selenium (Se) can modify the expression of high affinity II2- R (3,4) and, therefore, can exert a significant effect on T-cell proliferation and differentiation (3-7). Cells from animals maintained on Se-supplemented diets had an augmented capacity to proliferate in response to stimulation with antigen or mitogen and to differentiate into cytotoxic effector cells (5,6,8). The purpose of the present study was to determine whether dietary supplementatiop with Se would affect the expression of immune functions in humans by enhancing the expression of high affinity II2-R and, consequently, the ability of peripheral blood lymphocytes to proliferate in response to stimulation with mitogen or specific antigen.

MATERIALS AND METHODS

S u b j e c t s and S u p p l e m e n t a t i o n

Twenty-two healthy volunteers from the third- and fourth-year student population at New York University Dental Center were random- ized for sex, race, age, body weight, height, dietary habits, and history of vitamin intake and tobacco and alcohol consumption in their assignment to the placebo or experimental groups. There were 6 males and 5 females in each group; the average age was 27.4 yr, range 24-36 yr; average body weight was 67.81 kg, range 50.7-108.8 kg; average height was 169.2 cm, range 152.4-187.9 cm. The participants were instructed to take one tablet/d, for 8 wk, of the assigned supplement. The tablets contained either 200 gg of sodium selenite or the inactive ingredients of the active tablets (prepared by Garden State Nutritionals, Fairfield, NJ). Peripheral blood samples from the subjects were used for the high affinity II2-R expression assays; blood samples from 5 subjects selected from the placebo group were used for the mitogen-stimulated cell proliferation studies where Se was supplemented in vitro.

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Se and Immunity 105

For the antigen-stimulated cell proliferation studies (mixed lymphocyte reaction; MLR), blood samples were collected from 3 of the investi- gators in this study; the average age was 43.0 y, range 36--47 y; average body weight 66.6 kg, range 54.4-97~5 kg; average height was 165.9 cm, range 157.4-177.8 cm. Se was supplemented in vitro.

Determination of Se Levels

Peripheral blood samples were drawn before, and 4 and 8 wk after initiation of treatment. The Se content of the plasma was determined flu- orometrically, according to the method of Spallholz et al. (9), using stan- dard solutions of Se (25-200 ng Se/mL, as selenomethionine) to calibrate the assay. The sensitivity of the assay was 10 ng of Se.

Preparation of Peripheral Blood Mononuclear Cells (PBMC)

The peripheral blood samples were centrifuged, the plasma removed for determination of Se levels, and the buffy coat was layered over lymphocyte separation medium (Organon Teknica Corp., Durham, NC) to separate the PBMC. The PBMC were washed twice in RPMI-1640 supplemented with 25 mM HEPES, 100 U / m L of penicillin, 100 ~tg/mL of streptomycin, 0.I mM nonessential amino acids, and 2 m M glutamine (Gibco, Grand Island, NY; basic medium), counted, and suspended in basic m e d i u m with addit ional supplementa t ion as specified for each experiment.

MLR

2.5 x 106 PBMC were cocultured with 0.833 x 106 Raji cells that had been treated with mitomycin C (100 p.g/mL, 60 min at 37~ Sigma Chemical Co., St. Louis, MO) in 1.5 mL of basic medium supplemented with 5 x 10-5M 2-mercaptoethanol (2ME), to 10% with Se-defined fetal bovine serum (FBS; Se content I gg/DL; Hyclone, Logan, UT; equal to 1.2 x 10-8M Se in the final culture medium) and 1 x 10-7M Se (as sodium selenite). The cells were plated in 24-well culture plates and incubated for 4 d. Control cultures consisted of PBMC and Raji cells incubated in the absence of Se and of PBMC cultured in the presence and absence of Se. At the end of the incubation period, the ceils were collected, centrifuged, and plated at 2 x 105 cells/well in 96-well flat bottom plates in a total of 200 gL of the respective culture supernatants . The cultures (8-10 replicates/sample) received 1 gCi /wel l of [methyl-3H] thymidine (sp. act. 6.7 C i /mmol ; New England Nuclear, Boston, MA) and were incubated for an additional 24 h. The ceils were harvested with an auto- matic cell harvester (Skatron, Sterling, VA), lysed in distilled water, and the nuclei collected on filters. The amounts of radioisotope incorporated, in cpm, were determined by liquid scintillation counting.

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Mitogen Stimulation Assay PBMC were plated in 96-well fiat bot tom plates at 2 x 105 cel ls /wel l

in 200 gL basic m e d i u m supp lemen ted with 5 x 10-5M 2ME, to 10% with Se-defined FBS, 1 x 10-7M Se (as sod ium selenite), and with 1 g g / m L of phy tohemagglu t in in -P (PHA; Sigma). Control cultures were incubated in the absence of Se a n d / o r PHA, i.e., -Se +PHA; +Se -PHA; -Se -PHA. The cultures (6 repl icates/sample) were incubated for 48 or 72 h. Four hours before the end of the cul ture per iod , the cells received 1 ~tCi/well of [methyl-3H] thymidine , and the a m o u n t of rad io iso tope incorpora t ion de te rmined as described above.

125I-I12 B i n d i n g Assay PBMC from blood samples d r awn after 8 wk of t reatment were cul-

tured in 24-well plates at 2 x 106 cel ls /well in 2 mL basic m e d i u m sup- p l e m e n t e d wi th 1 ~ g / m L PHA and to 10% wi th Se-def ined FBS. The lymphob la s t s were collected 48 or 72 h later, separa ted f rom cellular debris by cent r i fugat ion over l y m p h o c y t e separa t ion m e d i u m , and washed in 5 mL of binding buffer (RPMI-1640 with 25 m M HEPES and 10 m g / m L of fraction V of bovine se rum albumin, Sigma). The cells were r e s u s p e n d e d in 5 mL of b ind ing buffer and incuba ted in a Dubnof f shaker at 37~ for two 1-h periods, with extensive washes be tween incu- bations to remove bound I12, and washed cells were used for the assay. The assay was per formed essentially as descr ibed by Robb et al. (10). Serial 2 /3 dilutions (2 nM-6 pM) of 125I-labeled h u m a n recombinant I12 (New England Nuclear), wi th and wi thou t 500 t imes molar excess of cold I12 (mur ine recombinant I12; a gift from Gerard Zurawski , DNAX Research Inst i tu te of Molecular and Cellular Biology, Palo Alto, CA), were incubated at 37~ with 5 x 105 lymphoblas ts in 150 ~tL of b inding buffer in E p p e n d o r f tubes unde r l a in wi th 150 gL of a 3:1 mix tu re of d ibu ty lph tha l a t e (Aldrich Chemica l Co., Mi lwaukee , WI) to d inony l phthalate (ICN Biochemicals, Cleveland, OH) (11). After 15 min, 350 gL of ice-cold binding buffer was added to each tube, the tubes were cen- t r i fuged at 9000 rpm for 60 s in a microcentr ifuge, and the a m o u n t of free radiolabeled ligand in the aqueous phase was determined. The oil mixture was discarded, and the a m o u n t of cel l -bound rad io iso tope in the cell pellet was determined. Specific b inding was calculated by sub- tracting nonspecific binding (in the presence of cold I12) from total binding. Computer-ass is ted Scatchard analyses were used to de te rmine the number of high affinity binding sites and dissociation constants (Kd).

Statistical Analysis The results are presented as the ari thmetic means +_ s tandard error

of the means (SEM) for each control and experimental group. The Kd are p re sen ted as the geometr ic mean _+ SEM. Differences be tween g roup


Se and Immunity 107

Table 1 Mean Plasma Selenium Levels Determined Before and After 4 and 8 wk

of Supplementation with 200 ~tg/d of Sodium Selenite or a Placebo

Plasma Selenium Levels, btg/DL

Treatment Baseline 4 w 8 w

Selenite Mean _+ SEM 13.10 +_ 0.73 13.86 +_ 0.65 15.27 +_ 0.68

Range 9.6-15.5 10.8-15.5 12.56-17.9 Placebo

Mean _+ SEM 12.86 _+ 0.55 12.78 _+ 0.54 14.34 +_ 0.74 Range 10.9-16.1 10.7-17.1 9.1-17.4

means were determined using the Student 's two-tailed t-test and p-values <_ 0.05 were considered to be significantly different.

RESULTS

Plasma Se Levels

All individuals in the dietary Se supplementat ion s tudy had a Se- replete status (12,13), as indicated by the mean and range of baseline plasma Se levels (Table 1). There were no significant changes in the plasma Se levels after 4 and 8 w of daily supplementation with 200 gg of sodium selenite as compared with the baseline values or the values for subjects taking the placebo tablets. These results are in agreement with other data that indicate that supplementa t ion with 200 ~g of sod ium selenite produces only a small increase in Se in the blood of replete individuals (14,15). The mean plasma Se level in blood samples used for the in vitro studies was 14.65 + 1.22 g g / D L that also indicted a Se-replete status in the participants.

MLR

Stimulation of human peripheral blood lymphocytes with alloantigen in the presence of 1 x 10-7M Se (as selenite) resulted in a significant increase in [methyl-3H] thymidine incorporation (Fig. 1). The mean incorporation in the nuclei of these cells was 41.5% higher (p<0.035) than in control cells in the absence of Se. The intrasample variation within replicates was less than 10%.

Mitogen Stimulation

Stimulation with PHA of peripheral blood lymphocytes from Se- replete individuals in the presence of 1 x 10-7M Se resulted in a signifi-


108 Roy et al.

12

Z o E-~

0

o ~D o

E~

Z

I

11

10

CONTROL l x 1 0 -TM Se Fig. 1. Effect of supplementation with Se in vitro (1 x 10-7M,

as sodium selenite) on the ability of human peripheral blood lymphocytes to respond to stimulation with alloantigen (Raji cells) for 5 d in a mixed lymphocyte reaction.

cant increase in the ability of the cells to undergo blastogenesis (Fig~ 2). The effect was apparent after 72 h of incubation when cultures stimulated in the presence of Se showed a 45.1% increase in [methyl-3H] thymidine incorporat ion (p<0.004) as compared with control cultures. There were no significant differences in radioisotope incorporation after 48 h of incubation. Cells cultured in the presence of Se without mitogen showed background radioisotope incorporat ion values that indicated that Se alone does not stimulate lymphocyte activation or proliferation.

Expres s ion o f II2-R Af ter Die tary S u p p l e m e n t a t i o n wi th Se

Supplementat ion with 200 ~ g / d of sodium selenite for 8 wk exerted a significant effect on the number of II2-R/cell expressed on st imulated peripheral blood lymphocytes. Scatchard plots of the 125I-I12 binding data (0.2 nM to 6.0 pM) on stimulated ]ymphocytes indicated that Se exerted its effect on the expression of high affinity II2 binding sites, since the mean Kds ranged from 4.7 to 7.9 x 10-11M (Fig. 3). After 48 h of stimulation with PHA, lymphoblasts from individuals receiving the Se supplement showed a significant increase (43.8%; p < 0.001) in the number of

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S e a n d I m m u n i t y 109

Z o [..,

o

o

Z

Z c~

1

3 0

2 5

2O

o

z

:I 10

-PHA 8

z 8

4

~ 2

u --- 0

4 8 72

TIME IN CULTURE (HOURS)

Fig. 2. Effect of supplementation with Se in vitro (1 x 10-7M, as sodium selenite) on the ability of human peripheral blood lymphocytes to respond to stimulation with PHA (1 ~tg/mL).

high affinity II2-R/cell as compared with cells from individuals receiving the placebo tablets (Fig. 4). After 72 h of stimulation, cells from Se-supplemented individuals showed a 19.1% decrease in the number of high affinity It2-R/cell, whereas cells from individuals in the placebo group showed no significant changes in the number of high affinity II2-R.

DISCUSSION

The interaction of an t igen/MHC molecules with the T-lymphocyte receptor complex triggers the expression of I12 and I12-R. The induction of I12 and II:-R are independent events, and the induction of II2-R. To

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1 10 Roy et al.

~n

u

o m

Z D o

18

16

14

12

10

8

8

4

2

0

0

Kd=84 pM

2 4 8 8 10 12

SELENIUM

�9 �9

,, ,,,, ,,,, ,,,, ,,,, ,,,, ,

2 4 5 8 10 12

SITES/CELL

(MOLECULES IN HUNDREDS)

Fig. 3. Scatchard plots of the 125I-II2 binding data on lymphocytes from Se-supplemented (200 ~tg/d of Se as sodium selenite for 8 wk) and nonsupplemented individuals. Data rep- resent the binding patterns from a typical experiment after 48 h of stimulation Of the cells with 1 ~tg/mL PHA.

exert its proliferative effect on T-cells, I12 must interact with the high affinity requires fewer and weaker signals than does the induction of I12 (16). I12-R (Ka -- 10-11M), which results in the formation of a stable complex that consists of I12 and three, noncovalently associated subunits: 0~ (p55), ~ (p70/75), and 7 (p64) (17,18). Binding of the ligand is followed by rapid endocytosis of the complex and the transduction of signals that initiate cell division (2,17). II2-R ct, inducible after stimulation, is non- functional with respect to t12 internalization and II2-signaling (19,20). Biologic responses and I12 internalization are mediated through the II2-R ~/7 component of the high affinity receptor (18), which is l inked to at least two intracellular signaling pathways that mediate the induction of nuclear protooncogenes (21).

Mitogen stimulation of T-cells from Se supplemented individuals resulted in the expression of a significantly greater number of high affinity II2-R/cell after 48 h of stimulation as compared with cells from nonsupplemented individuals. This was followed by a decline in the number of receptors after 72 h, which probably reflected the internalization of

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Se and Immuni ty 111

1 4 0 0

1 3 0 0

o 1 2 0 0

m l iO0

1000

0 900

8 0 0

7OO

6 0 0

48 HOURS

PLACEBO SELENITE

72 HOURS

Fig. 4. Effect of dietary supplementation with Se (200 btg/d) as sodium selenite for 8 wk on the ability of human peripheral blood lymphocytes to express high affinity II2-R after stimulation with PHA (1 ~g/mL) for 48 and 72 h.

the II2-II2-R complex (Fig. 4). In contrast, cells stimulated under the same conditions and supplemented with 1 x 10-7M Se in vitro showed a significantly higher nuclear 3H-thymidine incorporation after 72 h of stimulation. The temporal order of these events demons t ra ted a clear correlation between enhanced nuclear DNA synthesis, which was preceded by enhanced expression of high affinity II2-R, and supplementation with Se. Significantly enhanced nuclear DNA synthesis, as indicated by enhanced nuclear 3H-thymidine incorporation, was also demonstrated after allogenic stimulation of human lymphocytes for 5 d in the presence of 1 x 10-7M Se (Fig. 1).

Our previous results with a mouse model system have shown that supplementat ion with Se enhances the expression of both the c, and [3 subunits of the high affinity II2-R and that the higher number of high affinity II2-R/cell results in the internalization of greater amounts of I12 (3,4), Because supplementation with Se has no effect on the endogenous levels of II2 (5) and the concentrations of I12 and II2-R determine the number of T-cells that enter the cell mitotic cycle (1,2), it can be postulated that, in the presence of Se and continuous immunologic stimulation, cells

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with greater numbers of functional high affinity II2-R may replicate and expand faster. This was confirmed in our earlier studies that showed that supplementation with Se results in enhanced proliferation and in the generation of a greater number of cytotoxic lymphocytes within a given cell population after stimulation with antigen (5,6). As reported in the accompanying paper, stimulation of human lymphocytes under the same conditions produced the same results (22). Because of the quantita- tive changes within these human cell populations, the enhanced nuclear 3H-thymidine incorporation reported in the present study indicates a greater number of cells entering the S phase of the cell mitotic cycle, which results in an enhanced clonal expansion of the precursor cells.

The mechanism(s) by which Se enhances the expression of high affinity II2-R on the surface of activated lymphocytes is unknown. Among the mechanisms used by T-cells to regulate the activation of early genes, e.g., I12, II2-R, following stimulation are changes in transcription rate, termination of transcription, and stabilization of mRNA (23). As Se alone, without mitogen/antigen stimulation, has no effect on nuclear 3H-thymidine incorporation (Fig. 2) or II2-R expression (3), it is unlikely that it affects gene activation/transcription directly. Since the expression of high affinity II2-R has been shown to be regulated by posttranscriptional mechanisms (24,25), and Se has been shown to regulate posttran- scriptionally the production of several biologic molecules at the level of translation (26,27) or stabilization of mRNA (28), it is likely that Se exerts its effect on II2-R expression through a posttranscriptional mechanism(s). For example, as with iron, where the stability of the transferrin receptor mRNA is regulated by an iron-dependent binding of a cytosolic protein to the mRNA (29), a Se-binding protein(s) may regulate the stability of the high affinity II2-R mRNAs. Cytosolic proteins that do not contain selenocysLeine but readily bind trace amounts of Se have been character- ized, and it has been proposed that the effects of Se on cell growth may be related to its ability to modulate the function of such Se binding proteins (30,31).

Although there is no direct evidence for the role of Se in the regula- tion of the expression of high affinity II2-R by the above mechanism, our results provide the basis for further investigation in this area. The interaction of I12 with II2-R delivers various signals to a wide range of cell types. For example, signals generated by this interaction affect the growth and differentiation of T- and B-lymphocytes, the generation of lymphokine activated killer cells, the activity of natural killer cells, the proliferation and maturation of oligodendroglial cells, and the program- ming of mature T-cells for apoptosis (21). The presence or absence of Se in the environment can, thus, exert a significant effect on these processes through modulation of the expression of high affinity II2-R. The elucida- tion of the mechanism(s) involved in the modulatory effect of Se on the expression of high affinity II2-R may provide the rationale for the uti- lization of Se as a biologic response modifier.

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S e a n d I m m u n i t y 1 1 3

ACKNOWLEDGMENTS

This work was supported by Grant 91A01 from the American Insti- tute for Cancer Research. The authors thank R. J. Robb and Y. Yefremov for technical assistance and the members of the New York Universi ty College of Dentistry student body who participated in this study.

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Supplementation with selenium and human immune cell functions

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