Spontaneous expression of interferon genes in murine peritoneal macrophages: Modulation during the in vitro aging

Arch. Oerontol. Geriatr. suppl. 3 (1992) 123-128 9 1992 Elsevier Science Publishers B.V. All rights reserved. 0167-4932/92/$05.00

123

SPONTANEOUS EXPRESSION OF INTERFERON GENES IN MURINE PERITONEAL MACROPHAGES:

L. CONTI,

MODULATION DURING THE IN VITRO AGING

P. DI MARZIO, S. GESSANI, P. BORGHI, B. VARANO,

C. DIEFFENBACH a and F. BELARDELLI Istituto Superiore di SanitY, Department of Virology, Viale Regina Elena 299, Rome, Italy; and aUSUHS, Department of Pathology, Bethesda, MA, USA

SUMMARY The expression of interferon (IFN)-B gene and its modulation during in v i t r o "aging" was studied in unstirnulated peritoneal macrophages (PM) explanted fc~Om lipopolysaccharide (LPS)-responsive (Lps N) and LPS-hyporesponsive ( L p s ) mice. A strong direct correlation between the LPS response of PM and their capacity to express low levels of IFN-13 was found. Moreover, the decay of antiviral state during in v i t r o cultivation was correlated with the turnover of IFN-8 mRNA. In the light of the multiple biologic effect of IFN, the constitutive expression of IFN-f3 gene in PM can play a crucial role not only in the restriction of viral replication, but also in the modulation of cell differentiation and immune response. Keywords: peritoneal macrophages, interferon, in vitro aging INTRODUCTION Several animal viruses do not multiply in mouse PM when these cells are freshly explanted, suggesting that this "intrinsic" antiviral activ.ity may play a role in the host defense against viral

infections

(reviewed by Morahan et al.,

1985). The in vivo resistance of PM to viral infection can be abolished by injection of mice with antibodies to IFN-~/13 (Belardelli et al.,

1984). Although IFN

was not detected in the nutrient medium of freshly isolated PM, we have previously demonstrated that these cells were capable of transferring their antiviral state to target mouse monolayers, permissive to virus replication,

in co-culture

experiments (Proietti et al., 1986). Moreover, this transfer was completely abolished when antibodies to I FN-ct/B were added to the culture medium. The capacity of PM of expressing endogenous IFN is strongly associated with their response to bacterial endotoxin.

In

fact, PM freshly isolated from Lps d mice are not capable of transferring an antiviral

state to susceptible target cells (Gessani et al.,

antiviral

state when cultivated

1987).

PM loose their

in v i t r o for a few days. This decay is accele-

rated in presence of antibodies to I FN-~II3. In this

paper

we summarize

the

IFN-13 gene expression and the ( 2 ' - 5 ' ) in PM during the in v i t r o aging.

results

obtained

oligc~-adenylate

on the modulation

of

(2-5A) synthetase levels

124 MATERIALS AND METHODS Mice.

C3HIHeN

Charles River,

(Lps n)

Italia.

mice

C3HIHeJ

(5

to

(Lps d)

8 weeks

old)

were

mice were obtained

purchased

from

from Jackson

Labo-

ratory (Bar Harbor, ME, USA). Preparation of PM culture. ritoneal

washings

were

For preparation of macrophage monolayers,

prepared

as previously

1984) and seeded in 24-well plates, cells

in

(FCS).

I

ml of

RPMI

described

each well containing

1640 medium supplemented

(Belardelli

approximately

with

10 % fetal

Cells were allowed to attach to the plastic culture

et

peal.,

I x 106

calf

serum

dish at 37~

for 3

h r and the non-adherent population was discarded. Virus titration.

The o r i g i n ,

stomatitis v i r u s (VSV)

methods of preparation and assay for vesicular

in mouse L929 cells have been previously described (Be-

lardelli et a l . , 1984). Determination

of 2-5A synthetase.

For determination

of 2-5A

synthetase,

the method described by Revel et al. (1982) was used. RNA polymerase chain

reaction

(PCR).

described elsewhere (Jacobsen et a l . , the reverse transcriptase

(RT) reactions,

Iug

with 300 ng of each antisense primer for dehydrogenase

(GAPDH)

(Jacobsen et a l . , into two parts,

1989).

one-third

in

The RNA PCR was performed

1989) with the following modification.

RT buffer

as For

of total cellular RNA was mixed

IFN-~ or glyceraldehyde-3-phosphate

and

processed

as described

For the PCR, each RT reaction for GAPDH and the t w o - t h i r d s

mixture

elsewhere

was divided

for IFN-I~. The reac-

tion conditions and the analysis of PCR products were performed as previously described (Jacobsen et a l . , 1989). Statistical analysis.

Results were analyzed by using S t u d e n t ' s

t test and

co-variance analysis.

RESULTS Effect of the in v i t r o cultivation

on VSV replication

phages from Lps n and Lps d mice: role of interferon.

in peritoneal

macro-

As shown in Figure I I A ,

PM from Lps n mice were nonpermissive for VSV replication when f i r s t placed in culture, donor

but became permissive during in v i t r o culture. Despite the fact that d peritoneal cells from Lps mice could not t r a n s f e r the antiviral state,

peritoneal macrophages from Lps d mice were nonpermissive for VSV replication when f i r s t placed in culture

(Figure l I B ) .

state occurred

in cultures

more rapidly

cultures of Lps n mice (Figure c u l t u r e , the antiviral to Lps n PM.

IIA

However, the decay of the antiviral of PM from these Lps d mice than

and l I B ) .

In fact,

state of Lps d PM had significantly

in

already after 24 hrs in decayed, as compared

125

B Ld v5 4 "H

3

1

-

anti IFN (]1i3

+

+

day 0

-

day I

+

-

day 2

+

-

day 3

+

day L,

1 -

+

day 0

-

+

day I

-

+

-

day 2

+

-

clay 3

+

clay 4

Time of in v/tro culture

d F i g u r e I . Decay of the a n t i v i r a l state in c u l t u r e s of PM from Lps n a n d Lps mice in the absence or in presence of antibody to mouse I FN-~II3. PM were h a r vested and seeded as described in methods section. PM were then incubated at 37~ with I ml of n u t r i e n t medium with (+) or w i t h o u t ( - ) a n t i b o d y to IFN-~I~ (R 5/4 a n t i b o d y , 1/20 d i l u t i o n ) . A t d i f f e r e n t times of i n c u b a t i o n , PM were infected with V S V , and v i r u s yields were determined. T h e r e were t h r e e macrophage c u l t u r e s for each e x p e r i m e n t . The results are presented as mean of VSV y i e l d l 0 . 2 ml (log10) + S.E. -O-O-O-O-O-O-O-O-O-

The decay of the a n t i v i r a l kedly This

enhanced

by

addition

state in c u l t u r e s of PM from Lps

of polyclonal

antibody-induced

antibody

increase was g r e a t e r d phage c u l t u r e s from Lps mice ( F i g u r e l I B ) .

to

n

IFN-~II3

in these c u l t u r e s

mice was mar(Figure than

IIA).

in macro-

Effect of the in v i t r o " a g i n g " on the level of 2-5A s y n t h e t a s e in peritoneal macrophages. expression

The 2-5A

in v i v o .

synthetase

As shown

mice e x p r e s s considerably

is considered

in F i g u r e 2,

high

intracellular

an important

PM f r e s h l y

marker

harvested

of

I FN

from normal

levels of 2-5A s y n t h e t a s e a c t i v i t y .

Moreover, an inverse correlation between the p e r m i s s i v i t y of these cells for VSV and the levels of 2-5A vitro

for

I

intracellular

day,

synthetase was found.

that were f u l l y

levels of this

resistant

enzyme.

In p a r t i c u l a r ,

to viral

On the o t h e r

PM c u l t i v a t e d

replication, hand,

contained

no enzymatic

in

high

activity

was detected in PM c u l t i v a t e d for 4-5 days when the cells became permissive to v i r a l replication. Expression of the IFN-B mRNA d u r i n g Lps d

PM.

Freshly

Table

harvested

I summarizes Lps n and

the

results

the in v i t r o c u l t i v a t i o n of

PCR analysis

of

of Lps n and IFN-~

mRNA.

Lps d PM expressed comparable amounts of I FN-13

mRNA. In Lps n PM, the levels of this mRNA remained stable for 6 h r s from the

126 coJ

oi_

3

I,O

so,.."

cn :L

~..I0o~ N

E

I._

2

E

TM

O

o

o

(Dn

_9.o

n

~'5-

/

A

-

-1 > U3

/

>

r r-

I

~0

I

0

I

I

1 2 3 4 Days of cultivation in vitro

5

F i g u r e 2. Relationship between the level of 2-5A synthetase in PM and the p e r missivity of these cells for VSV d u r i n g the in v i t r o 'Waging". PM were h a r v e s t e d and seeded as described in methods section. A t each time points, PM were infected with VSV. V i r u s yields and the levels of 2-5A synthetase were d e t e r mined as described in methods. -0-0-0-0-0-0-0-0-0Table

I

EFFECT OF THE IN V I T R O C U L T I V A T I O N ON THE EXPRESSION OF IFN-B mRNA IN PM Time ( h r )

Lps n PM

Lps d PM

0

+

+

6

+

-

12

-

-

Notes:

0 time means f r e s h l y

scribed in methods.

explanted

PM. I

To improve s e n s i t i v i t y

ug of RNA was amplified as deof the method,

d u c t s were reamplified by using nested primers.

the IFN-13 p r o -

127 beginning

of the in v i t r o cultivation.

At this time,

IFN-13 mRNA was not de-

tected in Lps d PM. A f t e r 12 h r s , the expression of IFN-13 mRNA was not detected in both Lps n and Lps d PM. DISCUSSION Freshly explanted PM from normal mice were resistant to VSV in vivo and when f i r s t placed in culture (Belardelli et a l . , evidence indicating

that IFN was responsible for this "intrinsic;'

may be summarized as follows. body

to

1984).

IFN-~IIB

Second,

1984; Proietti et a l . ,

were f u l l y

an inverse

1986). The

antiviral

state

F i r s t , PM harvested from mice injected with antipermissive

correlation

to viral

replication

was found

between

(Belardelli the

et al.o

levels of 2-5A

synthetase (generally considered a marker of IFN expression in v i v o ) in PM and t h e i r permissivity for VSV (Gresser et a l . ,

1985). Finally, the " i n t r i n s i c "

viral state of freshly explanted PM was gradually tivation

and this

(Gessani et a l . ,

decay was accelerated 1987).

lost d u r i n g the in v i t r o cul-

in presence of antibodies

to I FN-~/B

In this paper, we report that the spontaneous decay of

antiviral state of PM d u r i n g the in v i t r o cultivation the t u r n o v e r

anti-

of IFN-IB mRNA.

Moreover,

is s t r o n g l y associated with

the decay of antiviral

state and the d degradation of IFN-B mRNA occur more rapidly in PM harvested from Lps mice than in normal Lps n PM, s t r o n g l y indicating

that an impaired capacity of Lps d

to stabilize the IFN-13 mRNA may account for the rapid decay of antiviral state in these PM. Several natural

stimuli may be responsible for the c o n s t i t u t i v e

of IFN under physiological conditions (reviewed by Bocci, 1981).

expression

In p a r t i c u l a r ,

it has been suggested by Vogel and Fertsch (1987) and by our group et a l . ,

1987) that bacterial

stitutive"

endotoxins may be involved

in stimulating

(Gessani a "con-

I FN expression in v i v o by PM.

In addition to its antiviral action, and functions.

IFN can d i f f e r e n t l y modulate cell division

It seems possible, therefore, that endogenous IFN may also e x h i -

bit more general effects on cell physiology. in this article emphasize that the in v i t r o PM may represent

a useful

model

for

In conclusion, "aging"

investigating

the results reported

of freshly

explanted mouse

the molecular

mechanisms

involved in the control of I FN expression under physiological conditions. ACKNOWLEDGEMENTS This work was supported by the CNR Special Project "Ingegneria Genetica" (contract No. 91.00054.99). P. Di Marzio was aided by a g r a n t from the "Fondazione Anna Villa Rustoni".

128 REFERENCES Belardelli, F., Vignaux, F., Proietti, E. and Gresser, I. (1984): Injection of mice with antibody to interferon renders peritoneal macrophages permissive for vesicular stomatitis virus and encephalomyocarditis v i r u s . Proc. Natl. Acad. Sci. USA, 81, 602-606. Bocci, V. (1981): Production and role of interferon in physiological conditions. Biol. Rev., 56, 59-85. Gessani, S., Belardelli, F., Borghi, P., Boraschi, D. and Gresser, I. (1987): Correlation between the lipopolysaccharide response of mice and the capacity of mouse peritoneal cells to transfer an antiviral state. J. Immunol., 139, 1991-1998. Gresser, I . , Vignaux, F., Belardelli, F., Tovey, M.G. and Maunoury, M.T. (1985): Injection of mice with antibody to mouse interferon ~16 decreases the level of 2"-5"-oligo-adenylate synthetase in peritoneal macrophages. J. V i r o l . , 53, 221-227. Jacobsen, H., Mestan, J . , Mittnacht, S. and Dieffenbach, C.W. (1989): 13 interferon subtype I induction by tumor necrosis factor. Mol. Cell. Biol., 9, 3037-3042. Morahan, P.S., Connor, J.R. and Learly, K.R. (1985): Viruses and the versatile macrophage. Br. Med. Bull., 41, 15-21. Proietti, E., Gessani, S., Belardelli, F. and Gresser, I. (1986): Mouse peritoneal cells confer an antiviral state on mouse cell monolayers: role of interferon. J. V i r o l . , 57, 456-463. Revel, M., Shattner, A . , Wallach, D., Merlin, G., Levavi, H., Hahn, T. and Levin, S. (1982): Monitoring of interferon therapy, diagnosis of viral diseases, and detection of interferon deficiencies by assay of an interferoninduced enzyme in human peripheral white blood cells. In: The Clinical Potential of Interferons, pp. 353-367. Editors: R. Kono and J. Vilcek. Tokyo University Press, Tokyo. Vogel, S.N. an~ Fertsch, D. (1987): Macrophages from endotoxin-hyporesponsive ( L p s ) C3HIHeJ mice are permissive for vesicular stomatitis virus because of reduced levels of endogenous interferon: possible mechanism for natural resistance to virus infection. J. V i r o l . , 61, 812-818.