Effect of L-arginine on thymic function. Possible role of L-arginine: Nitric oxide (no) pathway

Arch. Gerontol. Geriatr. suppl. 4 (1994) 1 6 3 - 1 7 0 9 1994 Elsevier Science Ireland Ltd. All rights reserved. 0167-4943/94/$07o00

163

EFFECT OF L-ARGININE ON THYMIC FUNCTION. POSSIBLE ROLE OF L - A R G I N I N E : N I T R I C OXIDE (NO) PATHWAY

E. MOCCHEGIANI a, G. NISTICO b, L. SANTARELLI a and N. FABRIS a'c a

Immunologlcal

Birarelli, logy,

Center

of

8, 60121 Ancona,

University

of

Rome,

Gerontological Italy; Tot

Research

Department,

bChair of Pharmacology,

Vergata,

Roma,

Italy,

]NRCA,

Via

Department of Bio-

CChair of

Immunology,

School of Medicine, U n i v e r s i t y of Pavia, Pavia, italy

SUMMARY The thymus and in particular its epithelial component produces hormonal peptides which are required for differentiation of stem cells into mature T-cells. With advancing age, there occurs a progressive reduction of the plasma level of one of the best known thymic p.e~tides, i.e. thymulin. In old mice, oral supplementation with arginine (9 x 10- g r / d a y / m o u s e ) for 1 month is able to induce a regrowth of the thymus and recovery of the reduced thymulin plasma level to the values observed in young animals. The direct immunological target of a r g i r.ine seems to be the thymus gland. In fact, the transplantation of thymus from old arginine treated mice into young thymectomized recipients is able to restore thymulin plasma level in thymectomized recipients to nearly the same level as do thymuses from young mice. Furthermore, arginine supplementation young t h y m ectomized recipients is unable to induce the reappearance of thymulin a c t i v i t y in the blood. With regard to the mechanism of action of arginine, two pathways may be suggested. The f i r s t one may be indirect and mediated by the secretagogue action of arginine on growth hormone. The second one, may rely on a direct action t h r o u g h the L-arginine:NO pathway. Lymphocyte-depleted thymic explants from young mice, when incubated in v i t r o with the NO-synthetase inhibitor L-NAME (6 mM), are, in fact, incapable of producing and realising t h y mulin in the supernatant. The in v i t r o addition of L-arg C60 raM) is able to recover such a production to the values observed in supernatants of control thymic cultures. The present findings offer the f i r s t evidence that also the thymic endocrine a c t i v i t y is modulated by L - a r g i n i n e : N O pathway. Keywords: arginine, t h y m u l i n ,

n i t r i c oxide, thymus

INTRODUCTION A good body of experimental and clinical evidences have been accumulated in

the

last years

on the

effect

of arginine

on the

immune

system

(Barbul,

1990). The f i r s t evidence comes from the observation of the role of arginine in wound healing in experimentally injured rats (Seifter et a l . , 1978). A number of experimental studies have f u r t h e r demonstrated that arginine supplementation is able to prevent post-traumatic atrophy of the thymus (Barbul et a l . , 1980), and to increase thymus weight and cellularity

in otherwise normal rats (Daly et a l . ,

1990). In addition,

arginine treatment in old mice induces thymus regrowth with a

recovery of the thymic endocrine a c t i v i t y to the values observed in young mice

164

(Mocchegiani et a l . , gical

functions

1992). At peripheral

occurring

supplementation

during

aging,

(Mocchegiani et a l . ,

of an immunomodulatory

level, the derangements of immunoloare consistently

restored

1992). All these findings

role of arginine,

by arginine

s u p p o r t the idea

but they do not give precise informa-

tions on its mechanism of action. One mechanism proposed is based on the well known secretagogue action of arginine

on p i t u i t a r y

growth

hormone

(GH)

(Isidori

recent evidence that GH is capable of modulating

et a l . ,

1981) and on the

thymic endocrine

activity

as

well as peripheral

immune functions both d u r i n g development and aging (Fabris

and Mocchegiani,

1994). Experimental evidences in young rats have shown that

the derangement of immunological functions, injury

is restored

1980;

1983).

In

by arginine

treatment

immunodepressed

thymic functions included, following via growth

subjects,

hormone

such as cancer

(Barbul

et a l . ,

patients,

arginine

treatment is able to induce a recovery of thymulin plasma concentrations and an improvement clear-cut

of

peripheral

evidences

immune efficiency

have been gathered

(Mocchegiani

on a possible

et a l . ,

1990a).

involvement

No

of growth

hormone in this clinical design. Recent findings have shown that the biological effect of arginine on many organs oxide

and tissues, (NO),

derives

may be mediated

a relevant

from arginine,

can be either

regulator through

constitutive,

by

its

role in the production

of cell function

(Moncada et a l . ,

the action of enzymes,

and

therefore

present

of n i t r i c 1989).

NO-synthetases,

in basal conditions,

NO

which or in-

duceable under the stimulation of various neuro-hormones or cytokines (Moncada et a l . ,

1991; Marcinkiewicz

and Chain,

1993).

Such an L - a r g i n i n e : N O

has been demonstrated to be involved

in various physiological

ing the immune system (Albina et a l . ,

1989; Liew et a l . ,

ry,

pathway

systems,

includ-

1990; Albina and Hen-

1991 ). These findings

have rised the question on whether also the effect of a r -

ginine on thymic endocrine a c t i v i t y

might be mediated by L - A r g : N O

pathway.

Aim of the present work was to analyze the role of arginine on thymic functions and

in particular

on thymic endocrine

activity

and to v e r i f y

the possible

in-

volvement of L - A r g : N O pathway. with

In order

to test this

arginine

in the d r i n k i n g

been evaluated thymulin

hypothesis,

by measuring

water,

firstly, and their

ester

(L-NAME),

thymic

endocrine

the plasma level of thymulin

secreting cells in the thymus.

ginine-methyl

old animals have been treated

a potent

Secondly,

activity

has

and the number of

the effect of N G - n i t r o - L - a r -

antagonist

of arginine

1989), and the effect of the concomitant addition of L-arginine

(Rees et a l . , on the in v i t r o

production and secretion of thymulin were evaluated by thymic c u l t u r e s .

165

MATERIALS AND METHODS Animals. Young (2 months) and old (18 months) male Balblc mice from our own colony were used.

Old mice or young

for 30 consecutive days with arginine

thymectomized

animals were treated

(added to the d r i n k i n g

water in a con-

centration assuring a total daily intake of 9 x 10 -4 g / d a y / m o u s e ) .

Young t h y m -

ectomized mice ( T X ) were used also for thymic transplantation. Surgical

procedures.

Thymectomy was performed

in young

Balb/c mice by

suction under ether anaesthesia; at sacrifice animals showing thymic remnants at specific

inspection

were discarded.

Transplants

of thymuses

from young,

old

and old arginine treated mice in thymectomized recipients were performed under the kidney capsule. Blood

samples

Animals were sacrificed

were obtained

stored at -70~

by

cardiac

15 days after

puncture.

The

the transplantation. plasma

samples were

until use.

Organ c u l t u r e .

Whole thymuses (two t h y m u s e s / c u l t u r e )

deprived of thymo-

cytes through squeezing and extensive washing with MI0 medium, were put into plate wells,

containing

I ml of MI0 medium supplemented

with 5 % heat inacti-

vated bovine fetal serum and incubated in CO 2 atmosphere at 37~ experimental conditions,

de novo synthesis of thymulin does occur and the ki-

netics of such a production can be monitored by thymulin supernatant

Under these

(Mocchegiani et a l . ,

1990b).

determination

Supernatant samples (60 1~I) were ta-

ken at d i f f e r e n t culture times (0, 5, 10, 15, 30 rain and I h r ) , volume was reconstituted

in the

with fresh medium.

Preincubation

L-arginine,

and the culture

with

L-NAME, used

at a final concentration

of 6 mM and/or

at a final concentration

60 mM (Moncada et a l . ,

1991) was carried out for 30 min at 37~

of

The L-NAME

or L-NAME+L-Arg conditioned medium was then removed and, after three washings, thymus cultures were replaced into the CO 2 incubator. Thymulin

determination.

B-lymphocytes c a r r y

Spleen cells

receptors

from

young

for sheep e r y t h r o c y t e s

mice including (SRBC) and,

T-

and

therefore,

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

by

mation. thymic

adding

The

azathioprine

endocrine

disappearance

10 1~glml

azathioprine

sensitivity

function:

removal

selectively

of of

T-cells the

of the a z a t h i o p r i n e - s e n s i t i v i t y

is

thymus while

inhibiting strictly in

T-rosette

dependent

mice

induces

it reappears

on

forthe

complete

with

purified

thymulin.

This phenomenon represents the basis for the bioassay of thymulin in

biological

fluids

questions

have been raised over the specificity

(Bach

et

al.,

1975).

The

bioassay

is

still

required

since

of the radioimmunoassays

de-

veloped till now. The maximum dilution of plasma samples inducing azathioprine s e n s i t i v i t y in 50 % of RFCs from thymectomized mice was taken as the thymulin titer.

The percentage

of RFCs that may become azathioprine

presence of excess thymulin concentration

sensitive

in the

ranges between 50 96 and 65 %. This

166

technique, as extensively described elsewhere (Bach et a l . , 1984),

is specific for t h y m u l i n ,

hormones,

since the assay is unaffected

and the rosette-inducing

samples t h r o u g h an antithymulin

activity

is completely

by other thymic

removed by passing

immunoabsorbent (Bach et a l . , 1975). The sen-

sitivity

of the bioassay allows detection

Serva,

Heidelberg,

Germany).

1975; Fabris et a l . ,

of I

pglml

synthetic

The assay is reliable,

thymulin

[from

since in two consecutive

blind assays, no differences of more than I (expressed as 111og2) was found in various samples (Bach et a l . , 1975). Immunofluorescence

studies

on thymic

section.

Thymulin

containing

were identified on cryostate sections of thymus by immunofluorescence, k i n d l y supplied by Dr.

MoAb,

anti-thymulin

MoAb is an IgG2a antibody whose specificity for thymulin has p r e -

GAMIIgG2alFITC

diluted

to 1:20.

of the organ.

cells

in

100 microscope fields of 135,000 iJm2 from

replacement of the antibody

serum employed at the same d i l u t i o n ,

The

1982). This MoAb was revealed by a

two micrometer thick sections obtained at d i f f e r e n t

Controls included

of antithymulin

France.

The number of thymulin-containing

the thymus was assessed by counting three or four frozen,

Paris,

using an

anti-thymulin

viously been demonstrated (Savino et a l . ,

M. Dardenne,

cells

by the buffer

monoclonal with synthetic thymulin

levels

by normal mouse

itself and by preabsorption (500 ~glml for I hr at 37~

and o v e r n i g h t at 4~ Statistical tailed

Student's

analysis. test.

Differences

Differences

between

in thymulin

evaluated using ANOVA ( t w o - w a y s ) ,

means

were

evaluated

concentrations

by

(Figure

two-

3) were

taking p < 0.05 for all tests as being sta-

tistically significant.

RESULTS In vivo effect

of arginine

on thymic

endocrine

activity.

Figure

I

shows

that thymulin plasma levels (Zn FTS) are s t r o n g l y reduced in old mice. Also the number of t h y m u l i n - s e c r e t i n g

cells decreased in old mice when compared to the

values of young animals. An oral supplementation with arginine for 30 consecutive

days

in old mice is able to induce a statistically

significant

recovery

thymulin plasma levels (p < 0.01) and of the number of t h y m u l i n - s e c r e t i n g (p < 0.01).

of

cells

Undetectable thymulin plasma levels are present in young thymecto-

mized mice. Oral supplementation

with arginine in thymectomized mice does not

induce reappearance of thymulin

in their

blood (Figure

I).

Transplantation

one thymus from old-arginine

treated mice into young thymectomized

induces

the

the

reappearance

in

host

of significant

plasma

recipients

thymulin

whereas thymuses from old, untreated mice are ineffective (Table I ) .

of

levels,

167

700

600 .~ ~b 500 8 4oo r3

.1

3oo

2oo "~ loo

Old

Young

Old+Arg.

Tx

Tx+Arg.

Figure I . Effect of in v i v o oral supplementation with a r g i n i n e in old mice on the thymulin plasma level ([--]) and on the number of t h y m u l i n - s e c r e t i n g cells in the thymus ( I I ) . Control data on thymectomized animals are also r e p o r t e d (5 anim a l s / g r o u p ) . One a s t e r i s k indicates p < 0.01 when compared to age matched old controls. Table I T H Y M U L I N PLASMA LEVELS IN YOUNG THYMECTOMIZED MICE T R A N S P L A N T E D WITH THYMUS FROM YOUNG, OLD AND OLD, ARGININE T R E A T E D SYNGENIC ANIMALS Experimental g r o u p s

n

Thymectomized mice ( T x )

5

T x + old thymus

5

1.2+0.3

Tx + old-arg,

5

3.6+0.2*

T x + young Thymus

5

4.5+0.3

Young controls

5

5.0+0.2

Old controls

5

1.0+0.3

thymus

T h y m u l i n plasma levels (log_2) 0.7+0.2

m

* p < 0.01 when compared to values of T x or T x + old thymus Effect of in v i t r o production

addition

of L-NAME and L - a r g i n i n e ,

by thymic organ c u l t u r e s .

on thymic hormone

F i g u r e 2 shows that the p r e i n c u b a t i o n of

y o u n g thymus e x p l a n t s with L-NAME completely p r e v e n t s the appearance of t h y inulin-like

activity

L-arginine

is able to restore the thymulin

in

the

supernatant.

The

concomitant

in

vitro

addition

of

p r o d u c t i o n to the values observed in

the s u p e r n a t a n t of control y o u n g thymic c u l t u r e s .

168 i

5-

o

?

0

5

I0 15

30

60

lime of Culture (rain.)

Figure 2. Inhibition of in v i t r o thymulin production by thymic explants by L-NAME ( [ ] ) and restoration by L-arginine [ 9 For comparison thymic control cultures are reported ( O ) . One asterisk indicates p < 0.001 (ANOVA test) when compared to values of young controls ( O ) or L-NAME + L - A r g - t r e a t m e n t

(1). DISCUSSION I

The data reported

in the present paper confirm the requirement of a r g i -

nine for thymic function

and in particular

for

its endocrine

1990; Fabris and Mocchegiani,

1992; Mocchegiani et a l . ,

supplementation

in old mice is able to recover

with arginine

plasma level of thymulin ducing

cells

1992).

The mechanism

is far from being

chanisms should be taken

by

which

understood:

both

into account.

Indirect

(Barbul,

In fact, an oral both the reduced

and the decreased number of epithelial

in the thymus.

endocrine a c t i v i t y

activity

arginine

thymulin affects

prothymic

indirect and direct me-

mechanisms are based on the

knowledge that arginine has a secretagogue action on the growth hormone { I s i dori et a l . ,

1981) and by the findings

that a growth hormone treatment in old

mice is able to induce a regrowth of the thymus associated with an increment of thymic endocrine a c t i v i t y

(Fabris and Mocchegiani,

1994). Receptors for growth

hormone have been found on thymic epithelial cells (Ban et a l . , more the presence of specific documented ( A r r e m b r e c h t ,

receptors

for

GH on thymocytes

1991). F u r t h e r has also been

1974).

This indirect mechanism of action is in agreement with the observation that the enhancing effect of arginine owl allograft viral by

tumors, previous

rejection,

on resistance of rats to

and on recovery of thymus weight after

i n j u r y are all abrogated

hypophysectomy

1983). This would clearly

and

restored

by

GH treatment

(Barbul

suggests that an intact p i t u i t a r y - t h y m u s

et a l . ,

axis is r e -

quired for arginine action. An alternative, be proposed.

or additional,

Recently,

mechanism for arginine action can, however,

it has been demonstrated

that arginine

is required

by

many cells as a precursor of NO, a molecule which has fundamental roles in cell activity

regulation

(Moncada et a l . ,

1989).

L-arginine:NO

pathway

is also in-

169

volved in many immunological functions (Albina et a l . ,

1989; Liew et a l . ,

1990;

Albina and Henry, 1991). Evidences have been accumulated on the role of reactive nitrogen intermediates on macrophage f u n c t i o n s ,

in particular on tumor c i t o t o x i c i t y and macro-

phage killing of bacteria and parasites (Liew et a l . ,

1990). Also lymphocytes

f u n c t i o n s , such as mitogen responsiveness seem to be dependent on L-arginine: NO pathway

(Albina and H e n r y ,

1991).

The data

presented here show that

L - a r g i n i n e : N O pathway is involved in thymic hormone production by thymic epithelial cells. The in v i t r o preincubation of young thymic explants with L-NAME, a potent antagonist of arginine (Rees et a l . , duction,

1989), i n h i b i t s the thymulin pro-

whereas L - a r g i n i n e addition completely restores it.

Although f u r t h e r

works are required in order to definite the exact mechanism of action of a r g i nine, the present findings offer the f i r s t evidence that also thymic endocrine function is modulated by the L - a r g i n i n e : N O pathway. ACKNOWLEDGMENTS This work was supported by INRCA and, in part, by Italian Health Minist r y (40 %). We are grateful to Mr. Marzio Marcellini and Mrs. Nazzarena Gasparini for their excellent technical assistance. REFERENCES Albina, J.E. and Henry, W . L . , j r . (1991): Suppression of lymphocyte proliferation t h r o u g h the n i t r i c - o x i d e s y n t h e t i s i z i n g pathway. J. Surg. Res., 110, 327-334. Albina, J . E . , Caldwell, M . D . , H e n r y , W . L . , j r . and Mills, C.D. (1989): Regulation of macrophage functions by L - a r g i n i n e . J. Exp. Med., 169, 10211029. A r r e m b r e c h t , S. (1974): Specific binding of growth hormone to thymocytes. Nature, 252, 255-257. 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 hormone is the mouse and the humane. Ann. N . Y . Acad. Sci. USA, 249, 186-210. Ban, E., Gagnerault, M . C . , Jammes, H . , Postel-Vinay, M . C . , Haour, F. and Dardenne, M. (1991): Specific binding sites for growth hormone in cultured mouse thymic epithelial cells. Life Sci., 48, 2141-2148. B a r b u l , A. (1990): A r g i n i n e and immune function. N u t r i t i o n , 6, $53-$58. Barbul, A . , Wasserkrug, H . L . , Seifter, E., Retture, G., Levenson, S.M. and Effort, G. (1980): Immunostimulatory effect of arginine in normal and injured rats. J. Surg. Res., 29, 228-235. B a r b u l , A . , Rettura, G., Levenson, S.M. and Seifter, E. (1983): Wound healing and thymotropic effect 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. Daly, J . M . , Reynolds, J . , Sigal, R . K . , Shou, J. and Liberman, M.D. (1990): Effect of dietary protein and amino-acids on immune f u n c t i o n . C r i t . Care Med., 18 (Suppl. 2), 86-93. Fabris, N. and Mocchegiani, E. (1992): A r g i n i n e - c o n t a i n i n g compounds and thymic endocrine a c t i v i t y . Thymus, 19, $21-$30. Fabris, N. and Mocchegiani, E. (1994): Immunomodulating role of growth hormone. In: Growth hormone II: Basic and Clinical Aspects. Editors: B.13. Bercu and R.R. Walker. S p r i n g e r - V e r l a g , New Y o r k , U . S . A . , (in press).

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