Restoring effect of oral supplementation of zinc and arginine on thymic endocrine activity and peripheral immune functions in aged mice

Arch. Gerontol. Geriatr. suppl. 3 (1992)267-276 9 1992 Elsevier Science Publishers B.V. All rights reserved. 0167-4932/92/$05.00

267

RESTORING EFFECT OF ORAL SUPPLEMENTATION OF ZINC AND ARGININE ON THYMIC ENDOCRINE ACTIVITY AND PERIPHERAL IMMUNE FUNCTIONS IN AGED MICE

E~ MOCCHEGIANI, M. MUZZlOLI, L. SANTARELLI and N. FABRIS a Immunological Center, Gerontological Research Department, Institute of National Research Centers on Aging (INRCA), Via Birarelli, 8, 1-60121 Ancona, Italy, and Chair of Immunology, University of Pavia, Piazza Botta, 10, 1-27100 Pavia, Italy SUMMARY Previous work from this laboratory has demonstrated that both a trace element, zinc, and an amino acid, arginine, are capable, when orally administered, to recover some age-related immune dysfunctions. In the present paper the effectiveness of a zinc-arginine combination versus the single nutrients in restoring age-related immunological alterations in old Balb/c mice has been investigated. The zinc-arginine combination is more effective than the treatments of single nutrients alone, in particular on the reactivation of thymic endocrine activity, as measured by the circulating level of one of the best known thymic factors, i.e. thymulin (Zn-FTS) and the natural killer (NK) cell activ i t y , particularly under boosting condition by interleukin-2 (IL-2) or interferon (IFN). On the other parameters tested the zinc-arginine combination either was not more effective than the single nutrients or a prevalence of one of them was detectable, such as of arginine on mitogen response or of zinc on basal NK act i v t i y . The findings clearly suggest that nutritional interventions and particularly combination of nutrients may represent an interesting and side-effect deprived approach for immunorestoration in elderly people. Keywords: Zinc, arginine, aging, thymulin immune function

INTRODUCTION The frequent association between malnutrition,

infective diseases and aging

has provoked a good body of experimental and clinical studies, aimed to analyze either the effect of single nutrients on the immunological in immunological

aging (Chandra,

efficiency or their role

1985, 1989). While the number of single nut-

rients relevant of immune efficiency is somewhat consistent, ranging from traceelements

(Underwood,

(Daly et al., immunological

1977) to vitamins

(Bendich,

1988) and to amino acids

1990), the studies directed to investigate their possible role in aging have remained untill now fragmentary.

Nevertheless, it has been demonstrated that a trace element, zinc and an amino acid, arginine, may exert a restorative effect of some age-related immunological alterations.

In particular,

zinc has been found capable to restore cuta-

neous hypersensitivity reactions and responses to tetanus texoid vaccination in healthy elderly (Duchateau et al., in elderly patients suffering

1981), to recover thymic endocrine function

from uremia" or hyperprolactinemia

(Travaglini et

al., 1989) and to correct both thymic function and peripheral immune efficiency

268 in subjects suffering from Down's syndrome (Franceschi et al., 1988), which is considered an early aging syndrome.

These human findings

have got f u r t h e r

support by experimental studies showing that zinc supplementation

in old mice

can recover thymic structure and function and increase mitogen responsiveness of spleen cells (Fabris et al., 1990). One of the mechanisms of zinc action has been identified in the capacity of this trace element to confer biological activity to one of the best known thymic factor,

i.e.

thymulin [ Z n - F T S ) ,

the zinc-unbound

(FTS) form of which is not

only biologically inactive but competes with the active form for the binding to the specific target (Dardenne et al., 1982; Fabris et al., 1984). The relevance of arginine for the efficiency of the immune system has been hypothesized by the observation that supplementary dietary arginine in youngadult injured rats can accelerate wound healing and prevent post-traumatic atrophy of the thymus (Barbul et al., 1983). A possible role for arginine

in

immunological

aging

has been suggested

because arginine has a secretagogue action on growth hormone (GH, al.,

Penny et

1970) and that GH is capable to modulating thymic endocrine activity and

various immune functions during development and aging (Fabris et a l . ,

1988).

It has been,

in old

in fact,

demonstrated

that oral arginine supplementation

mice causes a complete recovery of the reduced thymic endocrine activity with an improvement of peripheral immune efficiency, such as T-cell subsets, mitogen responses and NK activity [Fabris et al.,

1986b). Arginine supplementation has

been found effective in increasing thymic endocrine activity and percentage of peripheral giani,

blood T-helper

(CD4) cells in healthy elderly

1991) and in neoplastic patients

[Fabris and Mocche-

[Mocchegiani et al.,

and arginine seem to act on similar targets,

1990).

Since zinc

the question arose, whether the

combination of zinc and arginine supplementation might be more effective than the administration of the single nutrient alone. With these premises, zinc-sulphate

(Zn) and L-arginine

(Arg)

orally administered to aged mice either alone or in combination

have been

(Zn-Arg)

and

their effect on immune system evaluated in terms of thymic endocrine efficiency, peripheral T-mitogen response and NK activity. MATERIALS AND METHODS Animals. Young male (2 months) and old male (24 months) Balblc mice from our own colony were used. In both age groups mice were treated for 30 consecutive days either with arginine alone (added to the drinking water in a concentration assuring a total daily intake of 9 x 10-4 g/day/mouse, if we assume a water consumption of 5 mlldaylmouse), or with zinc sulphate alone (at the dose of 5 ]Jgldaylmouse), or with zinc-arginine combination at the same doses.

269 Thymulin T-

and

(Zn-FTS) determination.

B-lymphocytes

carry

Spleen cells from young mice including

receptors

for

sheep erythrocytes

(SRBC)

and,

therefore, form rosettes (RFC) when mixed with SRBC. T - and B-rosettes can be discriminated

by

adding

10 ug/ml

azathioprine,

which

selectively

inhibits

T-rosette formation. The azathioprine sensitivity of T-cells is strictly dependent on the thymic endocrine function: plete

disappearance

spleen

cells

(Zn-FTS).

from

of

the

in biological

fluids

azathioprine-sensitivity

thymectomized

This phenomenon

removal of the thymus in mice induces commice

are

while

incubated

it

with

reappears purified

when

thymulin

represents the basis for the bioassay of thymulin

(Bach et al.,

1975).

The bioassay is still

required

since

questions have been raised over the specificity of the radioimmunoassay developed till

now.

The maximum dilution

of plasma samples inducing

azathioprine

sensitivity in 50 % of RCFs from thymectomized mice was taken as the thymulin titer.

The percentage of RCFs that may become azathioprine sensitive in the

presence of excess thymulin concentration ranges between 50 ~ and 65 ~. This technique, as extensively described elsewhere (Bach et al., 1975; Fabris et al., 1984), is specific for thymulin,

since the assay is unaffected by other thymic

hormones, and the rosette-inducing

activity

plasma samples through an antithymulin

is completely removed by passing

immunoadsorbent

(Bach et al.,

1975).

The sensitivity of the bioassay allows detection of I pglml synthetic thymulin (from Serva,

Heidelberg,

Germany).

The assay is reliable,

since in two con-

secutive blind assays, no difference of more than I (expressed as 111og2) was found in various samples (Bach et a l . , 1975). Determination of inactive thymic hormone {FTS). The presence of inactive thymulin was detected by the method of Fabris et al. (1984, 1986a). The method is based on the capacity of inactive molecules to inhibit the biological activity of synthetic thymulin

in the rosette-inhibition

assay.

Sixty

~I of plasma diluted

1:50 with Hank's solution was mixed with 60 ul of different concentrations of synthetic pg/ml).

thymulin A I

(final

concentrations

= 0.1,

0.5,

I,

5,

10,

50 and

100

: 50 dilution of plasma was used to avoid interference by endo-

genous thymulin, while the lowest final concentration of synthetic thymulin used was 0.1 pglml as, in our experimental conditions, this was the lowest concentration

can inducing azathioprine sensitivity in the spleen cells from thymec-

tomized mice. The mixture was immediately filtered through a CF50 Amicon membrane

(cone size 50,000

daltons)

for 30 min at 4~

Filtrate

was tested

for

thymulin activity in the rosette assay. Inactive thymulin concentration was expressed as the highest concentration of synthetic thymulin

(pg/ml inhibited by

the plasma sample). Mitogen response. Spleen cells, obtained" by teasing the spleen through a 60-mesh

sieve, were washed twice in Hank's solution,

counted and resuspen-

270 ded,

to a final

RPMI-1640

concentration

(Gibco)

supplemented

activated male human serum. (Cook,

of 3 x

106 t r y p a n

with

glutamine,

blue excluding antibiotics

cells/ml,

in

and 5 ~ fresh

in-

Aliquots of 0.1 ml were d i s t r i b u t e d

in microwells

USA). Mitogen was then added in the amount of 10 ul/well.

bation at 37~

in a CO 2 atmosphere for 48 h r s ,

A f t e r incu-

1 ]~Ci of 3H-thymidine

(spec.

act. 2000 mCi/mM; Amersham) was added to each well. Cells were collected 20 h later by an automatic cell h a r v e s t e r determined were

using

prepared

a scintillation

for

(Skatron,

counter

each mitogen

Norway)

and

{Tricarb-Packard).

concentration.

radioactivity Triplicate

Phytohaemagglutinin

Difco) was used at final concentration of 0.0125 ~g/ml.

was

cultures M (PHA,

Concanavalin A ( C o n - A ,

Serva) was used at the final concentration of 7.5 ~ g / m l . NK

assay.

A

51Cr-cytotoxicity

assay

in

0.2

ml

Linbro

microtest

plates

(Flow Laboratories) was used. YAC-1 target cells (106/ml) were labelled for 1 h 51 with 100 lJCi (specific a c t i v i t y 386.05 mCi/mg) Cr (sodium chromate, New England Nuclear)

at 37~

washed (three times) and added to effector cell po-

pulations in a 4 hrs 51Cr-release assay. Plates were centrifuged at 800 rpm for 5 min and 100 ul of supernatant was collected and counted in a Beckman 5500 51 gamma counter. The spontaneous release was determined by c u l t u r i n g Cr-labelled target cells alone.

Total counts were determined

by counting

an aliquot

of labelled target cells after resuspension in the well. The following formula was used to calculate the percentage specific lysis. Test cpm - spontaneous release Specific lysis =

x 100 Total counts - spontaneous release

For cell

interferon

populations

were

(IFN)

and

interleukin-2

incubated

for

24 h r

(IL-2) with

boosting,

IFN

effector

(1000 U/ml)

or

spleen

IL-2

(25

U/ml) and then the c y t o t o x i c i t y a c t i v i t y was detected as described above. Zinc determination.

Plasma zinc was determined by atomic absorption spec-

trophotometry against reference standard (Evenson and Warren, 1975}. Statistical two-tailed

analysis.

Student's

The significance

t test.

Differences

between

the means was assessed by

were considered

significant

when p <

0.05.

RESULTS Effect of zinc and arginine on thymic endocrine function Table I shows that old mice have a reduced thymus weight,

a low plasma

concentration of active thymulin / Z n - F T S ) and an increased level of the inactive form ( F T S ) .

The serum zinc levels are also reduced in old mice when compared

with the values of young controls.

271 Table I EFFECT OF ZINC AND ARGININE ADMINISTRATION ON VARIOUS PARAMETERS IN OLD MICE (mean + S . D . )

Animals

Treatment

Thymus

Thymulin

Thymulin

Plasma Zn

weight

(ZnFTS)

i n h i b , act.

level

mg

I I log 2

pglml

ugldl

Young

none

47.5 + 2.3

4.8 + 0.2

0.3 + 0.02

115 + 13

2 months

Arg

48.3 + 2.0

5.1 + 0.3

0.3 + 0.03

117 + 17

Zn

45.3 + 1.7

5.1 + 0.3

0.2 + 0.03

120 + 15

A r g + Zn

46.7 + 2.1

5.2 + 0.2

0.2 + 0.03

115 + 15

65.0 + 11.7

85 + 17

Old

none

18.5 + 2.5

1.5 + 0.3

24 months

Arg

37.3 + 2 . 7 * *

3.0 + 0.2*

2.3 + 1.0 * *

90 + 15

Zn

36.3 + 2 . 5 * *

3.8 + 0.2*

0.9 + 0.04**

98 + 12"

A r g + Zn

36.8 + 2 . 7 * *

5.0 + 0 . 3 * *

0.5 + 0.03**

100 + 11"

Notes: One and 2 a s t e r i s k s indicate p < 0.05 or 0.001, the age-matched u n t r e a t e d c o n t r o l s .

r e s p e c t i v e l y , against

All the treatments with a r g i n i n e or zinc alone or with

z i n c - a r g i n i n e com-

bination for 30 consecutive days in old mice induced a r e g r o w t h of the thymus up to 80 ~ of the weight values recorded in young mice w i t h o u t s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s between v a r i o u s treatments. c o v e r y of the mice,

reduced

plasma t h y m u l i n

the z i n c - a r g i n i n e combination

A statistically significant

level is also observed

being more e f f e c t i v e than

in

re-

old treated

the single n u t -

r i e n t s alone (p < 0.01). In all treated old g r o u p s ,

a reduction of plasma concentration

of thymic

hormone inactive molecules is observed when compared to values of old u n t r e a t ed

mice,

group

with

complete

disappearance

in

the

zinc

while in the a r g i n i n e alone treated group

and

zinc-arginine

a small residual

treated

level of the

inactive thymic hormone form is s t i l l detectable. Treatments with zinc and zinca r g i n i n e combinations increase also mean zinc plasma v a l u e s ,

(p

< 0.05);

argi-

nine treatment induces only a small increment of zinc v a l u e s . No s i g n i f i c a n t

effects

have

been

induced

by

any

of

the

treatments

in

young mice (Table I ) . Effect of zinc and a r g i n i n e on p e r i p h e r a l immune e f f i c i e n c y Table II shows that a r g i n i n e administration as well as zinc-supplementation are able to cause a s t a t i s t i c a l l y s i g n i f i c a n t increment of both PHA and Con-A (p

272 T a b l e II EFFECT

OF

ZINC

AND

ARGININE

ADMINISTRATION

ON

TH E

MITOGEN

RESPONSE OF SPLEEN CELLS OF OLD MICE (mean + S . D . )

Animals

Treatment

PHA

ConA (cpmlcult)x103

Young

none

78.7 + 13

245.6 + 13

2 months

Arg

92.4 + 15

243.1 + 15

Zn

70.2 + 13

239.7 + 15

A r g + Zn

90.4 + 15

245.7 + 12

16.1 + 16

58.5 + 11

Old

none

m

24 m o n t h s

Arg

60.2 + 15"*

124.4 + 12"*

m

Zn

46.8 + 15"*

87.0 + 15"

A r g + Zn

65.2 + 13"*

120.2 + 12"*

Notes: One and 2 a s t e r i s k s i n d i c a t e p < 0.05 the age-matched u n t r e a t e d controls.

or

0.001,

respectively,

against

Table III EFFECT OF ZINC A N D A R G I N I N E A D M I N I S T R A T I O N ON NK A C T I V I T Y IN OLD MICE (mean 96 o f l y s i s + S . D . )

Treatment

NK ( b a s a l )

None

NK ( y - I F N )

NK ( I L - 2 )

none

17.70 + 0,56

3,15 + 0.42

12.50 + 0,66

12.70 + 1.56

Arg

20.50 + 0 . 2 9 " *

7,52 + 0 , 6 8 * *

14.40 + 1,31

16.80 + 2,25

Zn

24,20 + 0 , 2 9 *

5,51 + 0 , 4 7 "

13,25 + 1,04

11,63 + 2,32

A r g + Zn

23.10 + 0 , 4 1 " *

9.90 + 1,96 ~'*

15,28 + 0 , 9 6 *

13.82 + 2.55

7.53 + 3.09

Young

Old none

2.86 + 0.19

2.63 + 2.62

3.22 + 0.43

Arg

3.02 + 0.22

2.67 + 0.22

8.34 + 1 . 2 3 " *

12.10 + 3.96

Zn

5.37 + 0.79

3.91 + 0.45

6.23 + 1 . 2 3 "

10.10 + 1.22

A r g + Zn

5.45 + 0 . 5 5 * *

5.21 + 0.54

10.83 + 1 . 0 8 " *

Notes: One and 2 a s t e r i s k s i n d i c a t e p < 0.05 the age-matched untreated controls.

or

0.001,

17.34 + 3 . 1 5 " *

respectively,

against

273 < 0.01)

response in cells from old mice,

being more active than zinc.

Consequently,

observed when zinc-arginine

combination

tivity

the

low

endogenous

NK

the arginine

treatment,

as a t r e n d ,

no f u r t h e r

significant

increment is

is used.

cytotoxicity

With regard

of old

to NK cell ac-

mice when

compared

with

young animals (p < 0.01) is s l i g h t l y modified only by zinc-arginine combination, although the increment is practically activity

induced

combination,

by

IFN and

IL-2

the single n u t r i e n t s

city (Table I I I ) .

irrelevant in respect of young values. is significantly

increased

by

NK

zinc-arginine

being effective only on IFN-induced c y t o t o x i -

A slight increase p a r t i c u l a r l y of basal NK a c t i v i t y is also p r e -

sent in young treated mice, regardless the kind of treatment applied. DISCUSSION Clinical

and

experimental

zinc (Fabris et a l . , al.,

evidences

1990) and L-arginine

have demonstrated (Fabris et a l . ,

the

relevance

of

1986b; Mocchegiani et

1990) for immunocompetence and the capacity of oral supplementation either

with zinc or with arginine to restore at least to some extent thymic endocrine a c t i v i t y and peripheral immune functions in old mice and humans (Fabris et a l . , 1990; Fabris and Mocchegiani, 1991). The findings reported in the present paper while confirming the effectiveness of zinc and arginine treatment in recovering age-related demonstrate that the zinc-arginine

immune functions,

combination is more effective that the t r e a t -

ments of single n u t r i e n t s alone in particular on the reactivation of thymic endocrine a c t i v i t y ,

as measured by the circulating

thymic factors,

i.e.,

level of one of the best known

thymulin ( Z n - F T S ) and the NK a c t i v i t y ,

p a r t i c u l a r l y under

boosting condition by IL-2 or IFN. On the other parameters tested,

the zinc-arginine

combination

either was

not more effective than the single n u t r i e n t s or a prevalence of one of them was detectable such as of arginine on mitogen response, or of zinc on basal NK activity. In all cases the effects

do not seem to be additive,

since the effect

of

zinc-arginine combination never represented the sum of the effect of the t r e a t ments with single n u t r i e n t s . Another consideration to be made is that the percentual restoration in respect of the values observed in young animals is d i f f e r e n t in dependence on the parameter

tested.

The percentual

increment

around 50-70 % for mitogen responses,

is marginal

in basal NK a c t i v i t y ,

nearly 100 % for IFN and IL-2 boosting

NK c y t o t o x i c i t y , and for thymic endocrine a c t i v i t y . These findings can suggest some considerations.

F i r s t of all,

it is out of

doubt that both zinc and arginine are effective in restoring immune efficiency in old mice, although the preferential

functional target for each of these n u t r i e n t s

274 may be different

and d i f f e r e n t also the effectiveness of the combination.

cases, any of the age-related intrinsic

and i r r e v e r s i b l e ,

In all

immune defects studied should not be considered

since one or the other treatment can restore it,

at

least to a significant extent. Secondly,

the degree of restoration

is not total for all parameters.

would suggest either that other factors,

in addition

to zinc and arginine,

This are

required for completely recovering some immunological defects in old age or that the length of the treatment was not enough to affect all parameters. second hypothesis should obviously

be tested,

cluded since various microenvironmental

While this

the f i r s t one might not be e x -

factors act on the immune system and

all of them may be required to f u l l y restore it during aging. Finally, that,

the lack of an additive

to a certain e x t e n t ,

effect of zinc and arginine

may suggest

the mechanism involved belong to a common physio-

logical cascade. This interpretation may find support in the knowledge that zinc may influence the t u r n o v e r of T 3 - T 4 (Fabris et a l . , and PRL ( T r a v a g l i n i et a l . ,

1991), GH (Prasad,

1985)

1989) that are hormones promoting immune efficien-

cy, and that arginine has a secretagogue action on the same hormones. On the other hand high blood levels of GH are associated with increased plasma levels of zinc

(Travaglini

still unknown.

et a l . ,

1990),

though

the reasons for this association are

These considerations make acceptable the work hypothesis that at

least p a r t of the physiological cascade activated by zinc or by arginine may be common and therefore

an additive

effect

of the two compounds

might

not be

observable. While this hypothesis

will

be matter of f u t u r e

sented clearly suggest that nutritional

works,

the findings

pre-

interventions and p a r t i c u l a r l y combination

of n u t r i e n t s may represent an interesting and side-effect deprived approach for immunorestoration in elderly people. ACKNOWLEDGEMENTS This

work

was supported

by

CNR and

Italian

Health

Ministry.

We are

grateful to Mr, M, Marcellini and Mrs. N. Gasparini for their excellent technical assistance, We thank Mrs, C. Chesi for t y p i n g the manuscript. REFERENCES Bach, J . F . , Dardenne, M., Pleau, J.M. and Bach, M.A. (1975): Isolation, biochemical characteristics and biological a c t i v i t y of a circulating thymic h o r mone in the mouse and the human. Ann. N . Y . Acad. Sci. USA, 249, 186-210. Barbul, A . , Rettura, G . , Levenson, S.M. and Seifter, E. (1983): Wound healing and thymotropic effects of arginine: a p i t u i t a r y mechanism of action. Am. J. Clin. N u t r . , 37, 786-794. Bendich, A. (1988): A n t i o x i d a n t vitamins and immune response. In: Nutrition and Immunology, pp.125-148. Editor: R . K . Chandra. Alan Liss, New Y o r k .

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