Chronic production of interferon in carrier mice congenitally infected with lymphocytic choriomeningitis virus

Chronic production of interferon in carrier mice congenitally infected with lymphocytic choriomeningitis virus

VIROLOGY 117,253-256 Chronic Production (1982) of Interferon MARIE-FRANCOISE Institut Pasteur, Received in Carrier Mice Choriomeningitis Con...

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VIROLOGY

117,253-256

Chronic

Production

(1982)

of Interferon

MARIE-FRANCOISE

Institut

Pasteur, Received

in Carrier Mice Choriomeningitis

Congenitally Virus

Infected

with

Lymphocytic

SARON,~ YVES RIVIERE, ARA G. HOVANESSIAN, AND JEAN-CLAUDE GUILLON 25, me July

du Lh- Roux,

75724 Paris

14, 1981; accepted

November

C&ex

15, France

1, 1981

Congenital infection of mice with lymphocytic choriomeningitis virus leads to a lifelong virus carrier state. Here we provide evidence for the presence and action of interferon in such mice. The level of circulating interferon in adult carrier mice is 8-16 NIH units/ ml of plasma. This interferon is acid stable and is capable of inducing 2-5A synthetase in mouse L 929 cells but not in human HeLa cells. Control, noninfected mice show less than 1 NIH unit/ml of plasma. In accord with these results, adult carrier mice have higher levels of the interferon-mediated pppA(2’p5’A), synthetase (2-5A synthetase) in their liver and spleen than normal mice. Congenitally infected newborn mice also have higher levels of 2-5A synthetase in their liver in contrast to newborn control mice. These results in congenitally infected newborn and adult mice suggest that interferon may play a role, at least in part, in the pathogenesis of infection.

In utero infection of mice (congenital infection) with lymphocytic choriomeningitis virus (LCMV) leads to a lifelong virus carrier state. This phenomenon is referred to as tolerated persistent infection (1,~). When LCMV infection is acquired at birth (neonatal infection), newborn mice develop an acute syndrome characterized by inhibition of growth, liver cell degeneration and death in the first few weeks of life (Z-4). Mice which survive such neonatal infection are LCMV carrier mice. Carrier mice from both congenital and neonatal infections have high titers of virus in their blood and tissues and develop an immune complex-glomerulonephritis (5~). Several investigators have suggested the presence of interferon in carrier mice (7) but no evidence has been provided for this. We have previously shown that injection of poly(1) * poly(C) to congenitally carrier C3H mice and to acutely infected C3H mice provokes an enhanced production of interferon (8). This priming effect suggested that congenitally carrier mice may produce interferon chronically. Here 1 To whom

reprint

requests

should

we have confirmed this by providing evidence for the presence of low levels of circulating interferon in LCMV carrier mice. Pathogen-free C3H/Ico mice were obtained from IFFA CREDO (France); congenitally carrier C3H/Ico mice were obtained from a C3H/Ico female who survived to neonatal infection with LCMV (strain CIPV 76001). After anesthesia, adult mice were bled from the axillary vessels and the blood was collected in polystyrene tubes containing heparin (100 u/ ml) and aprotinin (100 u/ml; Zymofren, Specia, France). Plasma was collected after centrifugation (2OOOg, 15 min) and stored at -80”. Titration for interferon activity was after treatment at pH 2 for 24 hr to inactivate virus. Interferon activity was measured by the cytopathic effect of vesicular stomatitis virus (VSV) on mouse L929 cells. One NIH unit of interferon was equivalent to 30 effective units (9). The plasma of both male (15 mice) and female (15 mice) LCMV carrier mice showed 8 to 16 NIH units (240 to 480 laboratory effective units) of interferon per ml of plasma. On the other hand the levels of interferon in the plasma of both male and female control mice (16 mice) were

be addressed. 253

0042~6822/82/030253-04$02.00/O Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

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routinely less than 1 NIH unit/ml (30 effective units/ml). It should be noted that here we measured the interferon activity after treatment at acid pH. Interferon in the plasma of carrier mice, therefore is of the type I, i.e., LY(fast migrating) or /3 (slow migrating) according to the new nomenclature (10). Low levels of interferon can reproducibly be detectable by the induction of 2-5A synthetase in cells in tissue culture (11). For this reason, therefore, we assayed the interferon activity in the plasma of LCMV carrier mice for its capacity to induce 25A synthetase in vitro, in mouse L929 and human HeLa cells (12, 13). Table 1 shows that treatment of L929 cells with the plasma of LCMV carrier mice results in an enhanced level of 2-5A synthetase whereas no such effect is observed after treatment of HeLa cells. Furthermore, the level of 2-5A synthetase in L929 cells treated with plasma of normal mice is similar to the background levels of the enzyme found in control L929 cells. These results confirm that the antiviral activity found in the plasma of LCMV carrier mice is indeed mouse interferon since in vitro it has the capacity to induce 2-5A synthetase in mouse cells but not in human cells. We and others have previously shown that the level of 2-5A synthetase in the liver and spleen of mice is enhanced by several fold on injection of mice with interferon or inducers of interferon, such as virus and double-stranded RNAs (9,14,15>. Thus, assay of 2-5A synthetase in different tissues of mice has served as a convenient marker for the presence and action of interferon since this enzyme could be detectable at a time when the level of circulating interferon is diminished or becomes undetectable. For this reason, therefore, we investigated the level of 25A synthetase in the tissues of control and LCMV carrier mice. Low but detectable levels of 2-5A synthetase were reproducibly obtained in extracts of liver and spleen of control mice. This level of 2-5A synthetase was enhanced by several fold in the liver (twoto fourfold) and spleen (six- to tenfold) of LCMV carrier mice (Table 2), a phenom-

TABLE

1

THE LEVEL OF 2-5A SYNTHETASE IN L929 AND HELA CELLS TREATED WITH PLASMA FROM NORMAL OR LCMV CARRIER MICE’ 2-5A synthetase (nmol/mg/hr) Treatment None None Mouse Mouse Carrier

interferon interferon plasma

(100 U/ml) (1000 U/ml) (10)

(20) Normal

plasma

(50) (10)

(20)

L929

HeLa

0.47 0.61 21.68 46.33 3.45 2.56 1.47 0.49 0.57

2.68 2.34 2.05 2.49 2.24 1.82 1.95 2.66 2.37

a Culture of L929 and HeLa cells, preparation of cell extracts, and treatment with interferon were as described previously (12, 13). L929 cells were treated with mouse interferon (24) at 100 or 1000 units/ml. Plasma from normal and LCMV carrier adult mice was first treated at pH 2 (24 hr, 4’) before addition to cell cultures at lo-, 20-, and 50-fold dilution. Assay of 2-5A synthetase was in a total reaction mixture (600 ~1) containing 200 pl of tissue extract (1 mg of protein); 10 mM Hepes pH 7.6; 50 mM KCl; 25 mM Mg(OAc)2; 7 mM 2-mercaptoethanok 5 mM ATP; 10 mM creatine phosphate; 0.16 mg/ml of creatine kinase; 0.1 mg/ml of poly(1) .poly(C); and 2 ~1 of [3H]ATP (1 mCi/ml) (Amersham, England). Incubation was for 90 min at 30” and was terminated by heating at 90” for 3 min. ‘H-labeled 2-5A was purified by DEAE-cellulose chromatography as described (24, 25) and the radioactivity of the entire sample was measured after dilution into 10 ml of liquid scintillant prepared according to Bray. The concentration of 2-5A in AMP equivalents was estimated from the percentage incorporation of the radioactivity from input [3H]ATP into 2-5A (3H-cpm). The 2-5A synthetase levels were calculated on this basis and are given as units corresponding to 1 nmol of 2-5A synthesized per hour per milligram of tissue extract (9, 15). The level of 2-5A synthetase in HeLa cells treated with 100 units/ml of human leukocyte interferon was 97.8 nmol/mg/hr.

enon which was in accord with the presence of interferon in the plasma of carrier mice (Table 1). Since in these experiments the level of 2-5A synthetase was measured by incubation of the crude tissue extracts then we assayed enzyme activity after its partial purification on poly(1) * poly(C)-Se-

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TABLE

2

THE LEVEL OF 2-5A SYNTHETASE IN THE LIVER SPLEEN OF CONTROL AND CARRIER MICE“

AND

2-5A synthetase (nmol/mg/hr) Mice Control

Sex Male

Female

Carrier

Male

Female

Liver

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Spleen

8.63 6.96 8.84 6.20 6.77 8.27

11.47 15.60 12.78 11.09 17.67 12.78

21.24 24.44 15.23 24.06 24.63 15.42 19.36 23.50 18.24 17.67 26.51 17.30 25.98

127.84 125.77 145.89 89.11 123.59 73.70 105.66 115.81 119.57 122.76 126.34 141.56 116.00

a Tissue extracts were prepared as described before (9, 15). The level of 2-5A synthetase in the liver and spleen of control and carrier mice (CIH/Ico; 8-9 weeks old) was determined as described in Table 1.

pharose (9). Once again, however, we obtained six- to tenfold higher levels of Z-5A synthetase in the spleen of carrier mice compared to that found in control mice, thus, suggesting that lower levels of 2-5A synthetase in the tissues of control mice (Table 2) were not due to the presence of interfering agents with the 2-5A synthetase assay. The results described above indicated the presence (Table 1) and action (Table 2) of interferon in the plasma of adult carrier mice. It was of interest to investigate the presence of interferon in newborn mice congenitally infected with LCMV. Because of inconvenience of collecting sufficient quantities of blood from individual newborn mice, we assayed interferon by its activity, i.e., the level of 2-5A synthetase in the liver. The level of 2-5A synthetase in the liver was measured on every day after birth until the mice were 20 days old

(Fig. 1). Once again as in Table 2, control newborn mice had lower levels of 2-5A synthetase in contrast to that observed in newborn mice congenitally infected with LCMV. In addition to this, there seemed to be a constant increase and decrease in the level of 2-5A synthetase in LCMV carrier newborn mice at different ages. This variation in the level of 2-5A synthetase in the carrier newborn mice was also observed in other experiments carried out under similar conditions. Whatever the level of 2-5A synthetase in the carrier mice, however, it was always several fold higher than that of control newborn mice (Fig. 1). The significance of this variation in the level of 2-5A synthetase in the liver of LCMV carrier newborn mice is not clear yet. It is possible that it is due to different levels of circulating interferon at different ages. Furthermore, such variations may also be due to individual differences. The results described above have shown for the first time that type I (cy or @) interferon is detectable in the plasma of LCMV adult carrier mice at levels which were reproducibly obtained either by titration for antiviral activity or by induction of 2-5A synthetase in vitro assays. This interferon activity is acid stable and is species specific as shown by its lack of activity on human HeLa cells (Table 1). In addition to the presence of interferon in

0

5

10

15

20

FIG. 1. Level of 2-5A synthetase in the liver of control and LCMV carrier newborn mice. Control (0 - - - 0) and carrier (0 0) newborn mice were sacrificed at different ages as indicated (abscissa) and the level of 2-5A synthetase (ordinate) was measured in liver extracts as described in Table 1. Five different litters were used in this experiment: Age 0 (at birth) to 8, 9 to 10, 11 to 13, 14, and 15 to 20 days old.

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carrier mice, such interferon is active in vivo since the tissues respond to it by the enhanced levels of 2-5A synthetase (Table 2). In the experiments concerning newborn mice, we used the assay of 2-5A synthetase in the liver as an indirect measurement for circulating interferon. Newborn mice congenitally infected with LCMV had several fold higher levels of 2-5A synthetase in their liver compared to control newborn mice (Fig. l), thus, suggesting the presence of circulating interferon in congenitally infected newborn mice. This chronic interferonemia in congenitally carrier mice may be responsible for the heterologous interference (7) and the priming effect for the induction of interferon (8) reported previously in such mice. It has been shown that interferon induced by LCMV following neonatal infection is responsible in large part for the early acute syndrome observed in suckling mice (16, 17) and favors the development of subsequent glomerulonephritis (18, 19). Thus it is possible that chronic interferonemia in congenitally carrier mice (Tables 1 and 2) may influence the development of the glomerulonephritis which is observed in such mice (2, 6, 20). It should be noted that this is not the only example of a disease in which interferon may be responsible for untoward effects. For example, mice with intermediate susceptibility to mouse hepatitis virus (MHVS) become virus carriers and develop a progressive neurologic disease (21) and these MHV3 carrier mice show a chronic production of interferon (22). Likewise, Hooks et al. (23) have reported the presence of a-interferon in the sera of patients with systemic lupus erythematosus. The results described here provide evidence for the presence and action of interferon in LCMV carrier mice. The precise role of circulating interferon in the course of virus replication and in the pathogenesis of kidney lesions in such carrier mice, however, remains to be determined. REFERENCES 1. TRAUB, E., J. Exp. Med. 63,847~861 (1936). 2. HOTCHIN, J., Cold Spring Harbor Symp. Bid 27, 479-499 (1962).

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