Synthesis of two distinct interferons by human fibroblasts

VIROLOGY

89,

330-334

(1978)

Synthesis of Two Distinct lnterferons EDWARD Department

A. HAVELL,’

of Microbiology,

New

TERESA York

G. HAYES,

University Accepted

by Human Fibroblasts

School May

of Medicine,

AND

JAN New

York,

VILCEK’ New

York,

10016

6, 1978

Human interferon species Le and F can be distinguished on the basis of their distinct antigenic, biological, and physicochemical characteristics. Until now, Le interferon was thought to be produced only in cells of lymphoid origin. This study demonstrates that cultures of human fibroblast cell strains GM-258 and FS-4 produce both F and Le interferons. Le interferon constituted up to about 20% of the total interferon activity produced in fibroblast cultures after stimulation with Newcastle disease virus or vesicular stomatitis virus. In contrast, only F interferon was detected in preparations obtained after induction with polyinosinate-polycytidylate. These results indicate that the Le interferon gene is inducible in cells of nonlymphoid origin and that the expression of this gene depends on the nature of the inducing agent.

“Leukocyte” (Le) interferon forms the bulk of interferon activity present in preparations obtained from primary cultures of human buffy coat cells or from lymphoblastoid cell lines, while “fibroblast” (F) interferon is synthesized by a variety of nonlymphoid cells. Le and F interferons can be differentiated from each other with the aid of specific anti-Le and anti-F sera (l-5) and on the basis of different physicochemical and biological properties ( 6-9). A particularly useful distinguishing feature is the degree of antiviral activity in cells of heterologous species. Whereas Le interferon is highly active in bovine or porcine cells, interferon preparations derived from nonlymphoid cells generally show very little activity when assayed in these cells (10, 11). Recently, we concluded that separate genes exist for Le and F interferons ( 12). This conclusion is based on the demonstration that messenger RNA (mRNA) extracted from induced lymphoblastoid cells and mRNA from fibroblast cells directed the synthesis of the two distinct interferons on injection into Xenopus laevis oocytes. Although it was thought originally that Le interferon is elaborated only in cells ’ Present address: Trudeau Institute, New York, N. Y. 12983. ’ To whom reprint requests should

Saranac

Lake,

be addressed.

of lymphoid origin and F interferon only in nonlymphoid cells (3), we recently demonstrated the synthesis of F interferon in a line of lymphoblastoid cells (12, 13). The results of the present study show that both F and Le interferons can be synthesized in two human fibroblast cell strains of nonlymphoid origin. The GM-258 cell strain with trisomy 21 was obtained from the Human Genetic Mutant Cell Repository, Camden, N. J. Chromosome 21 is known to contain genetic information responsible for sensitivity to the antiviral action of interferon in human cells (14) and cells with trisomy 21 show increased sensitivity to the action of human interferons ( 25). However, chromosome 21 is not known to influence the synthesis of human interferons ( 16). Diploid cell strain FS-4 was originally derived in this laboratory from a neonate foreskin; this cell strain is widely employed for interferon production (I 7). Another foreskin cell strain, designated FS-7, was employed for interferon assays. GM-258, FS-4, and FS-7 cells were grown in Eagle’s minimal essential medium (MEM) supplemented with 5% fetal bovine serum (FBS). Vesicular stomatitis virus (VSV, Indiana type) was grown in primary cultures of chick embryo cells. Newcastle disease virus (NDV, Hickman strain) was grown in the allantoic cavity of U-day-old 330

0042.6822/78/0891-0330$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

SHORT

331

COMMUNICATIONS

chick embryos. Titers of VSV and NDV were determined by the plaque assay in chick embryo cell cultures. Interferon preparations were made in cultures of GM-258 cells using three different conditions of induction. We also employed interferon made in cultures of FS-4 cells after stimulation with polyinosinatepolycytidylate [poly(I) t poly(C)] and “superinduction” with metabolic inhibitors (I 7), and a preparation of leukocyte interferon from primary cultures of buffy coat cells stimulated with Sendai virus (18). All interferon preparations were assayed for antiviral activity in human FS-7 and bovine MDBK cultures (Table 1). The ratio of FS7/MDBK activity was lowest with buffy coat interferon and highest with the preparations obtained from the two fibroblast cell strains after induction with poly(1) poly( C). These findings agree well with earlier results (10, II). GM-258 interferon preparations obtained after induction with VSV or NDV showed a FS-7/MDBK activity ratio higher than buffy coat interferon but considerably lower than the poly(1) poly(C)-induced firboblast interferon preparations. The same interferon preparations were examined in a neutralization assay with specific anti-F and anti-Le interferon sera (3, 11, 13) in cultures of human GM-258 cells and bovine MDBK cells (Table 2). Buffy coat interferon activity was consistently neutralized by anti-Le serum but not by anti-F serum. FS-4 or GM-258 interferons produced with poly(1). poly(C) were neutralized only by anti-F serum in both human and bovine cells. However, the activities of the virus-induced interferon preparations produced in GM-258 cells showed less consistent patterns of neutralization. In MDBK cells, both preparations were neutralized only by anti-Le serum, suggesting that activity in bovine cells was due to the presence of Le interferon. (The quantitative difference in the neutralizing titer of anti-Le serum against VSV- and NDV-induced interferons is probably insignificant as it was not seen in another test.) The activity of these preparations in GM-258 cells was not neutralized by either one of the two anti-interferon sera alone but a mixture of the two antisera did neutralize

TABLE ANTIVIRAL INTERFERON

ACTIVITIES PREPARATIONS

MDBK

BOVINE

Cell source of interferon GM-258

Inducing

1 OF VARIOUS IN HUMAN

HUMAN

FS-7

AND

CELLS

-___

agent”

Vesicular stomatitis virus Newcastle disease virus POIYU’POIY(C)

FS-4

Poly(1)

poly(C)

Buffy coat

Sendai

virus

“Confluent cultures of human GM-258 or FS-4 cells, grown in 490~cm’ plastic roller bottles (Corning Glass, Corning, N. Y.) were used for interferon induction. The growth medium was removed before induction and replaced with MEM containing VSV (multiplicity of infection, 10) or NDV (multiplicity of infection, 4). Cultures were incubated with the virus inoculum for 2 hr and then thoroughly washed. In the groups induced with poly(1) ‘poly(C), the cultures were first incubated with MEM containing poly(1) poly(C) (5 pg/ml) and cycloheximide (20 pg/ml) front 0 to 6 hr, actinomycin D (2 @g/ml) was added to the cultures at 5 hr, the inducing medium was removed 1 hr later, and the cultures were then thoroughly washed (7). After removal of the inducing agents, all groups of GM-258 and FS-4 cultures were incubated with 30 ml of production medium consisting of MEM with 0.5% (v/v) FBS. Production medium was collected 25 hr after the initiation of induction. Media collected from the virus-induced cultures were adjusted to pH 2.0 and kept at 4” for 5 days in order to inactivate the inducing virus; the fluids were then readjusted to pH 7.4 and centrifuged at 100,060 g for 1 hr to remove inactivated virions. The supernatants were used in interferon assays. Interferon derived by Sendai virus induction of buffy coat cultures from fresh blood (18) was obtained from Dr. Kari Cantell (Central Public Health Laboratory, Helsinki, Finland). ’ Interferon assays were performed simultaneously in cultures of the human FS-7 fibroblast strain and the bovine MDBK cell line, using cultures grown in 96. well plastic trays as previously described (27). The assay is based on the inhibition of cytopathic effect of VSV. Interferon titer is the reciprocal of the highest dilution of the tested materials which was found to protect at least 50% of the cell sheet from cytopathic effect in the respective cell type.

the activities; this result would be expected if these preparations contained a mixture of F and Le, with at least 10% of total

332

SHORT

COMMUNICATIONS TABLE

NEUTRALIZATION ANTI-Le

2

OFTHE ANTIVIRAL ACTIVITIES OFVARIOUS AND ANTI-F INTERFERON SERA IN HUMAN

Cell source of interferon

Inducing

agent”

HUMAN INTERFERON GM-258 AND BOVINE ~.

Neutralizing ~.._ GM-258

titer

PREPARATIONS MDBK CELLS

of anti-interferon

WITH sera in h

MDBK

Anti-F

Anti-Le

Anti-F

Anti-Le

GM-258

Vesicular stomatitis virus Newcastle disease virus PolY(I)‘PolY(c)

<50 <50 1600

<50 t50 t50

t50 <50 >400

13000 2400 t50

FS-4

PolYm’PolY(c)

3ooo

<50

>3oO

<50

Sendai

<50

1600

<50

3700

Buffy

coat

virus

-

(1See Table 1. bInterferon preparations were diluted to contain 20 units/ml as assayed in GM-258 and MDBK cells, respectively, and aliquots were mixed with equal volumes of a series of 2-fold dilutions of anti-F or anti-Le sera. MEM with 5% FBS was used as the diluent. The interferon-antiserum mixtures and the appropriate controls were incubated for 1 hr at 37” and then added to cultures of GM-258 or MDBK cells grown in 96-well plastic trays. After 18 hr incubation, the cultures were inoculated with VSV. The neutralizing titer is defined as the reciprocal of the highest dilution of antiserum that inhibited the antiviral action of 10 interferon units/ml (final concentration). The titer of the interferon preparations employed was checked at the time of the assay in each of the two cell types and an appropriate correction of the neutralization titer was made if the actual potency was either higher or lower than 10 units/ml. ’ Although anti-F and anti-Le sera alone failed to neutralize interferon activity at the indicated dilutions in these assays, a mixture of the same concentrations of the two antisera caused complete neutralization of the VSV- and NDV-induced interferon preparations in GM-258 cells.

activity expressed in the GM-258 cells due to Le interferon. (This conclusion is based on the fact that in the neutralization assay each interferon preparation is diluted to yield a final concentration of 10 units/ml but, by definition, as little as 1 unit/ml can produce inhibition of viral cytopathic effect in a culture. It follows that neutralization with any one of the anti-interferon sera alone will be demonstrable if at least 90% of the total interferon activity is of homologous antigenic specificity; neutralization will not be demonstrable if over 10%of total interferon activity present is of heterologous specificity. In practice, due to the variability of the biological assay, the actual interferon concentration used may be somewhat higher or lower than 10 units/ml and the level at which the presence of a heterologous interferon component becomes apparent may thus vary slightly from assay to assay.) The results shown in Tables 1 and 2 thus suggest that very little, if any, Le interferon was made in GM-258 cells stimulated by poly(1). poly(C) but that substantial amounts of Le interferon were produced

after inoculation with NDV or VSV. These tentative conclusions were further substantiated by direct isolation of the Le interferon moiety from a preparation of GM-258 interferon induced with NDV. Separation of the F and Le interferon moieties was accomplished by affinity chromatography, using specific anti-Le globulin coupled to Sepharose 4B. After passing the interferon preparation through the affinity column, interferon which did not bind to the immobilized antibody (“Unretained” fraction) no longer showed antiviral activity in bovine cells and it was specifically neutralized by anti-F serum alone (Table 3). Another portion of interferon activity was specifically bound to the immobilized anti-Le globulin from which it could be eluted by a pH 2.5 solution (“Retained” fraction). The latter fraction had the characteristics of Le interferon, i.e. it was active in bovine cells and was neutralized by anti-Le serum only. In view of the relative inaccuracy of interferon assays, the activity recovered in the two column fractions does not add up exactly to 100%of the original activity. It can be estimated, however, that about 20% of

SHORT TABLE

3

SEPARATION OF THE Le AND F SPECIES FROM “FIBROBLAST” INTERFERON BY AFFINITY CHROMATOCRAPHY Fraction”

Total

interferon activity”

FS-7

Neutralizing terferon

MDBK

FS-7 An$

Original Unretained Retained

-

AntiLe

-.

8192 8192 1920

1536 t8 640

CRUDE

Arm-Le

ON IMMOBILIZED GLOBULIN

titer of anti-insera in’ __. MDBK r AntiAntiF

333

COMMUNICATIONS

Le

tlO0 1800

t50 <50


3300


2500


6600

” A preparation of NDV-induced GM-258 interferon (see Table 1 for the details of preparation) was concentrated lo-fold by dialysis against the hygroscopic reagent Ficoll-400 (Pharmacia, Uppsala, Sweden) and then dialyzed against 0.15 M phosphate-buffered saline (PBS, pH 7.4). This interferon preparation (“Original”) was then fractionated by affinity chromatography as follows. Gamma globulin, isolated from the anti-Le serum by ammonium sulfate precipitation, was covalently coupled to CNBr-activated Sepharose 4B (Pharmacia) by a modification of the method of Ankel et al. (19). Approximately 1 ml of the Sepharose beads with the immobilized anti-Le globulin was fust gently shaken for 20 hr at 4” with 1 ml of the concentrated interferon preparation. The Sepharose beads were then allowed to settle and the supernatant collected (“Unretained” fraction). The beads were resuspended in PBS and poured into a small column (1 ml bed volume). The column was successively washed with an excess of PBS and with PBS containing 0.5 M NaCl to remove nonspecifically bound interferon. Specific elution of bound interferon activity was done with a solution of 0.1 M acetic acid and 0.5 M NaCl, pH 2.5 (“Retained” fraction). ’ Interferon units/ml X total volume (milliliters) of sample. ’ See Table 2.

the FS-7 activity in the original preparation is accounted for by Le, with F interferon responsible for the rest. The finding that virus induction of GM258 friboblasts resulted in the synthesis of both F and Le interferons prompted us to examine the nature of interferons present in similar preparations derived from the FS-4 cell strain. An interferon neutralization assay was devised which measures the yield of infectious VSV progeny produced after a single cycle in GM-258 cells previously incubated with the tested interferons alone, with tested interferons in the presence of an excess of each neutralizing anti-

serum alone, or with an excess of both antisera combined. This method of assay enables the detection of an interferon species constituting less than 10% of the total interferon activity in a preparation (Table 4). As expected, the antiviral activky of virus-induced interferon preparations from GM-258 cells was not completely neutralized by anti-F or anti-Le serum alone, but neutralization was achieved with a mixture of the two antisera. A similar result was obtained with VSV-induced FS-4 interferon, indicating the presence of Le interferon. In the virus-induced preparations from GM-258 or FS-4 cells, anti-F serum alone caused partial neutralization, indicating that F interferon is the major component in these preparations. The presence of Le interferon was not clearly demonstrable in the poly(I) poly(C)-induced FS-4 prepTABLE

4

NEUTRALIZATION OF THE ANTIVIRAL ACTION OF INTERFERON PREPARATIONS FROM GM-258 OR FS-4 CELLS BY ANTI-F BY SINGI.F. CYCLE

OR ANTI-LP VSV YIELD 258

Cell source interferon

None GM-258 FS-4 Buffy

coat

01

Inducing --i-----

None NDV vsv vsv I’oly(I) Sendai

SERA, AS MEASURED INHIBITION IX GM-

CELLS

agent”

Log PFU/mi the presence

WV yield of antibody”

in

poly(C) virus

“See Table 1. VSV-induced FS-4 interferon was prepared in confluent dish cultures using a multiplicity of 10. Otherwise the procedures were the same as for the preparation of interferon in GM-258 cells. * Confluent cultures of GM-258 cells grown in 24. well plastic trays (17 mm diameter of each well) were incubated in duplicate with 50 units/ml of each of the listed interferon preparations. mixed with concentrations of anti-interferon sera sufficient to neutralize at least 200 units/ml of homologous interferon. After 1X hr, the interferon-antiserum mixtures were removed, the cultures were washed, and inoculated with VSV at a multiplicity of 3. The virus was allowed to adsorb for 1 hr, thereafter the cultures were washed 4 times with saline and replenished with MEM containing 2% FBS. Fluids were collected 8 hr after inoculation and the yield of VSV was determined by plaque assay.

334

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aration, since anti-F serum alone caused virtually complete neutralization of the antiviral activity. In contrast, the experiment confirmed that buffy coat interferon is a mixture of Le and F, with F being the minor component. Earlier experiments showed that F interferon in preparations of Sendai virus-induced leukocyte interferon may account for up to about 1% of total activity (3-5). Because Le and F in’terferons do not cross-react antigenically, the primary structures of substantial parts of the two molecules must be different. Earlier work showed that separate mRNAs and, by inference, separate structural genes exist for Le and F interferons (12). Both genes are expressed in the Namalva line of lymphoblastoid cells after stimulation with NDV, with about 85% of the total interferon activity produced due to Le interferon and the rest to F (13). We now show that both genes can be simultaneously expressed in human fibroblasts, with Le interferon representing up to about 20% of total human interferon activity produced after induction with NDV. The expression of the Le and F interferon genes in different cells is apparently controlled by at least two factors, the cell type and the mode of induction. Cells of lymphoid origin make Le interferon preferentially, and nonlymphoid cells are stimulated more easily to make F interferon. In addition, the proportion of the two interferons made varies with the mode of induction: in both the GM-258 and FS-4 cell strains substantial quantities of Le interferon were produced after virus induction while Le interferon was not demonstrated after stimulation with poly(1). poly(C). ACKNOWLEDGMENTS We thank C. B. Anfinsen and D. Gurari-Rotman for antiserum against leukocyte interferon and K. Cantell for the preparation of leukocyte interferon. The work was supported by research Grants AI-07057, AI12948, and Contract NO1 AI-02169 from the National Institutes of Health. Teresa G. Hayes is supported by

Medical National

Scientist Institutes

Training Grant of Health.

GM-07308

from

the

REFERENCES 1. BERG, K., OGBURN, C. A., PAUCKER, K., MOGENSEN, K. E., and CANTELL, K., J. Immunol. 114, 640-644 (1975). 2. VILCEK, J., HAVELL, E. A., MOZES, L. W., and BERMAN, B., In “Proceedings of the 1st Intersectional Congress of the International Association of Microbiologists Society” Vol. 4, pp. 65-75. Science Council of Japan, Tokyo, 1975. 3. HAVELL, E. A., BERMAN, B., OGBURN, C. A., BERG, K., PAUKER, K., and VILCEK, J., Proc. Nat. Acad. Sci. USA 72, 2185-2187 (1975). 4. HAVELL, E. A., BERMAN, B., AND VILCEK, J., In “Proceedings of the Symposium on Clinical Use of Interferon,” pp. 49-61. Yugoslav Academy of Sciences and Arts, Zagreb, 1975. 5. PAUCKER, K., DALTON, B. J., OGBURN, C. A., and TERMS, E., Proc. Nat. Acad. Sci. USA 72, 4587-4591 (1975). 6. STEWART, W. E., II, DE SOMER, P., EDY. V. G., PAUCKER, K., BERG, K., and OGBURN, C. A., J. Gem Viral. 26, 327-331 (1975). 7. STEWART, W. E., II, and DF,SMYTF,R, J., Virology 67, 68-73 (1975). 8. KNIGHT, E., Proc. Nat. Acad. Sci. USA 73, 520-523 (1976). 9. VILEEK, J., HAVELL, E. A., and YAMAZAKI, S., Ann. N. Y. Acad. Sci. 284, 703-710 (1977). 10. GRESSER, I., BANDU, M.-T., BOUTY-BOY& D., and TOVEY, M. Nature 251, 543-545 (1974). Il. HAVELL, E. A., YIP, Y. K., and VILCEK, J., Arch. Virol. 55, 121-129 (1977). 12. CAVALIERI, R. L., HAVELL, E. A., VILCEK, J., and PESTKA, S., Proc. Nat. Acad. Sci. USA 74, 3287-3291 (1977). 23. HAVELL, E. A., YIP, Y. K., and VILCEK, J., J. Gen. Virol. 38, 51-59 (1977). 14. TAN, Y. H., TISCHFIELD, J., and RUDDLE, F. H., J. Exp. Med. 137, 317-330 (1973). 15. TAN, Y. H., SCHNEIDER, E. L., TISCHFIELD, J., EPSTEIN, C. J., and RUDDLE, F. H., Science 186, 61-63 (1974). 16. CRF.AGAN, R. P., TAN, Y. H., CHEN, S., and R~JDDLE, F. H., Fed. Proc. 34, 2222-2228 (1975). 17. VILEEK, J., and HAVELL, E. A., Proc. Nat. Acad. Sci. USA 70, 3909-3913 (1973). 18. STRANDER, H., and CANTELL, K., Ann. Med. Exp. Biol. Fenn. 45, 20-29 (1967). 19. ANKEL, H., CHANY, C., GALLIOT, B., CHEVALIER, M. J., and ROBERT, M., Proc. Nat. Acad. Sci. USA 70, 2360-2363 (1973).