functional and ultrastructural studies of the effects of human interferon on cell membranes of in...

Acta path. microbiol. scand. Sect. B, 84: 347-358, 1976

FUNCTIONAL AND ULTRASTRUCTURAL STUDIES OF THE EFFECTS OF HUMAN

INTERFERON ON CELL MEMBRANES OF IN P’ITRO CULTURED CELLS

MIKLOS DEGRO and TORSTEIN HOVIG

Capt. and Mrs. Wilhelmsen’s Institute of Bacteriology and Institute of Pathology, Electron Microscopic Laboratory, University of Oslo, Rikshospitalet, Oslo, Norway

DegrC, M. & Hovig, T. Functional and ultrastructural studies of the effects of human interferon on cell membranes of in vitro cultured cells. Acta path. microbiol. scand. Sect. B, 84: 347-358, 1976.

The effect of human leukocyte interferon on cultured U-amnion cells was examined, and several biological parameters were registered. Multiplication of Vesicular stomatitis virus and the virus-produced cytopathogenic effect was prevented. The growth rate of uninfected cells was reduced, as well as the spontaneous release of 3H-uridine. These effects were observed following treatment with 10 units of interferon per ml. No morphological alterations could be detected by scanning electron microscopy after 24 or 72 hours treatment with up to 2000 units interferon per ml.

Key words: Human interferon; cell membranes; function ; ultrastructure.

M. Degd, Kapt. W. Wilhelmsen og Frues Bakteriologiske Institutt, Rikshospitalet, Oslo 1, Norway.

Received 26.v.76 Accepted 22.vi.76 Besides its classical antiviral activity, inter-

feron seems to have a great variety of biological effects in the whole organism and on cultured cells: inhibition of growth of normal as well as of tumour cells (2, 8, 11, 17, 18), enhanced phagocytosis by macrophages (13), enhanced production of interferon by priming (20), enhanced cellular sensitivity to the toxic effects of vaccinia virus (12), and double stranded polynucleotides ( 2 1 ) , enhanced cytotoxicity of lymphocytes (16) and others. I t is generally accepted, although not finally proved, that the mechanism of antiviral activity is a preferential inhibition of transcription or translation of exogenous viral

genetic information. Very little is known of the mechanisms of the non-antiviral effects. The question has been raised whether or not these effects are interdependent at some level of the induction. I t has been proposed (15) that the cell membrane might be a candidate for a common target which, influenced by the interferon, acts as a trigger which initiates a series of biological responses.

Various consequences of the interferon interaction with cells, observed in different laboratories, have been interpreted to be attributable to alteration(s) of the cell membrane (1, 3, 14, 15, 22) . In the present com- munication we report some functional and scanning electronmicroscopical observations

347

of interferon effect on uninfected and virus infected human amnion cells, with special reference to the cell membrane.

M A T E R I A L S A N D M E T H O D S

Cells: The U-line of human amnion cells was received from D r K. Cantell, Helsinki. I t was grown in Eagle's Minimal Essential Medium (MEM) with addition of 5 per cent calf serum, NaHCO, and antibiotics. The cells were maintained in the same medium with 2 per cent calf serum. The same serum concentration was used in all experiments in this study.

Human embryonic lung cells (HEL) were prepared in this laboratory. They were grown in 50 per cent each of MEM and medium 199 with addition of 10 per cent calf serum, NaHCO, and antibiotics. The serum concentration was reduced to 5 and later to 2 per cent during maintenance. HEL cells were used between their 5th and 15th passage.

Virus: Vesicular stomatitis virus (VSV), Indiana strain, was grown in 2 days old cultures of L-F, mouse fibroblast cells. When the cytopathogenic effect was complete, usually after 2 days, the culture fluid was collected after freezing and thawing. Virus was stored at -7O"C, and a fresh ampoule was used in each experiment. Infectivity titres were assayed by the end point micromethod on L-F, and on U cells.

Interferon: Human interferon was produced in leukocytes, according to Cantell et al. ( 5 ) . The supernatants of the Sendai-virus-induced leukocytes were characterized as interferon by the standard criteria and were used without further purification. In some experiments, partially purified leukocyte interferon preparations, given to us by Dr K . Can- tell, were used. As the results obtained with this preparation were identical to those of the crude samples they are not presented separately.

T h e antiviral activity of interferon preparations was tested by the infectivity inhibition micromethod employing HEL cells and VSV, as described in detail elsewhere ( 6 ) . The titres were adjusted to the international standard preparation 69/19.

T h e interferon effect on cell growth was tested as described elsewhere ( 7 ) . Briefly, aliquots of lo5 U cells were seeded into at least 5 tubes per sam- ple in 1 ml medium, containing dilutions of interferon. Control tubes contained the same medium, but no interferon. The cells were incubated sta- tionary in 5 per cent CO, atmosphere at 37" C. After 3 days, incubation, the monolayers were trypsine-versene treated and the cells were counted in haemocytometer.

Assay of cell membrane integrity: The release of

348

soluble radioactive substances into the medium has earlier been used as indicator of damage to the cell membrane (23). Essentially the same method was used in the present study. Tubes were seeded with 105 U cells each. Three days old monolayers were incubated for 2 hours at 37" C in medium containing 1 pCi of 3H-uridine per ml. The cells were then washed thrice with fresh medium to remove extracellular radioactivity. Interferon dilutions in MEM were added to 6-8 cultures each. Control tubes contained MEM alone. After incubation at 37" C for various times, 0.1 ml of the supernatant was removed and transferred to a scintillation vial containing 10 ml of Aerosol. Samples were counted in a Packard liquid scintillator for 10 minutes. The spontaneous release from the control cultures was considered as 100 per cent and release from the interferon treated cultures were related to the controls.

The cells and the remaining medium in some experiments were further treated with 0.1 ml of the non-ionic detergent Triton-X 100 to a final concentration of 0.25 per cent (vJv). The tubes were agitated in a Vortex mixer and incubated at 37" C for 15 minutes. This treatment causes complete lysis of the cytoplasmic membrane, releasing all radioactivity into the medium. No intact cells could be seen by lightmicroscopic examination. One tenth of 1 ml of the lysate was transferred to a scintillation vial and counted, as described above.

Preparation of cells for scanning electronmicro- scopic examination: 105 U-cells were seeded into flat bottomed plastic trays, containing flying coverslip, in 1 ml MEM, containing various interferon concentrations. The cultures were incubated in 5 per cent CO, atmosphere at 37" C for 24 or 72 hours. No toxic effect on any of the interferon treated cultures could be observed by light microscopy.

The specimens were fixed in 2.5 per cent glutar- aldehyde in 0.1 M phosphate buffer ( p H 7.4) for 2 hours and postfixed in osmium tetroxide in Tyrode's solution ( p H 7.4) for 1 hour. The specimens were then dehydrated in graded ethanol solu- tions and dried by the critical point method in fluid CO, (Sorval critical point drying system). The mounted specimens were then coated with a thin layer of carbon and gold-palladium in an Edwards vacuum coating unit and examined in a Jeol JSM scanning electron microscope.

R E S U L T S

Antiviral Activity of Interferon in U Cells I t is known that U cells are highly sensitive

for antiviral activity of interferon. These cells are therefore routinely employed in assays of antiviral activities. The titre of interferon in

\\ I 5

P

2 5 3 3 5 4 ,n,erte.on t i t r o . ( loglo "", Ir )

\. \*

_\__\ 2 5 ,n,erte.on t i t r o . ( loglo "", Ir ) 3 5

Fig. I . Correlation between the antiviral titre of interferon and the titre of challenge virus as meas- ured by the infectivity inhibition microassay.

.~ 2 - 3 days

.~ 1

Fig. 2. Growth of U-amnion cells in culture with and without 100 units interferon per ml medium. 105 cells were seeded in each tube. Interferon was added simultaneously. Five interferon treated cultures 0-0 and five controls 0-0 were trypsinized and counted each day. Vertical lines indicate 1 S.D.

logarithmic units, tested in a micro-infectivity inhibition method is correlated along a straight line to the dose of infecting VSV (Fig. 1).

Treatment of U cells with 100 units of interferon 6 hours prior to inoculation with 10 TCID,, VSV reduced the titre of infec- tious virus after 18 hours' incubation by

more than 2 log,,. Extracellular and intracellular virus titres were reduced to the same extent.

Growth Inhibitory Activity of Interferon on U Cells

The growth rate of U cells was tested in the continuous presence of 100 units per ml of interferon. Six tubes were trypsinized each day, together with 6 control tubes, and the number of cells was counted. Interferon treated cells grow more slowly than the control cells (Fig. 2 ) . The differences were sig- nificant on the third day. This growth inhibitory effect was dose dependent, thus 10 units interferon per ml reduced the cell num- bers by ca 15 per cent, 100 units by practically 50 per cent, counted after incubation for 3 days.

Interferon Inhibition of Spontaneous Release of Radioactivity from Uninfected U Cells

After uptake of 3H-uridine into the cultured U cells, some of the radioactivity is

CPM

, I

I5 30 45 60 75 90 man

Fig. 3. Spontaneous release of 3H-uridine from cultured U-amnion cells into the medium. Cells were cultured for 2 hours in medium containing 1 pCi 3H-uridine per ml. After incubation the cells were washed thrice and supplied with fresh medium. Samples were removed at the indicated times and the released radioactivity was estimated. Other tubes were treated identically except that the medium was changed every 15th minutes following removal of the radioactive substance. Col- umns show release during the last 15 minutes, line shows total released radioactivity.

349

TABLE 1. Distribution of Radioactivity between Supernatant and Cells in Interferon Trea ted and Control U-amnion Cell Cultures

Cells

Total radioactivity (lysed cells + supernatant )

Released radioactivity (supernatant)

CPM CPM Per cent of total

Control cells 834 Cells treated with 100 unit I F 93 1

191 106

23 11

Cells were cultured in medium containing 1 pCi 3H-uridine per ml. After 2 hours' incubation the cells were washed 3 times and supplied with 1 ml fresh medium containing 100 units of interferon. Control cells did not receive interferon. After 30 minutes incubation supernatants and lysed cells were prepared as described under Methods.

Fig. 4. Influence of 200 units interferon 0-0 on spontaneous release of radioactivity from cultured U-amnion cells. Interferon was added immediately after removal of radioactive medium. Experimental details as described in Fig. 3.

released spontaneously into the medium (Fig. 3 ) . The rate of release is maximal immediately after removal of the 3H-uridine-containing medium. If the cell mebrane is dam- aged the leakage increases (23 ) . On the assumption that interferon affects the cell membrane we have tested whether interferon treatment influences the rate of spontaneous release of a soluble radioactive substance. Interferon, 200 units per ml, diluted in MEM, was added to the cells immediately after removal of "-uridine containing medium. Control tubes contained the same medium but no interferon. One tenth of the supernatant was removed after incubation for 15 and after 30 minutes and the radioactivity was assayed. The supernatant of inter-

350

I .I

5 2 5 a

I 1 5 10 5 0 200

1"I.rt.ron ""ill

Fig. 5 . Influence of various doses of interferon on spontaneous release of radioactivity from cultured U-amnion cells. Various concentrations of interferon were added to the cultures immediately after removal of radioactive medium. Samples were removed at 15 minutes (closed circles) and at 30 minutes (closed squares). The counts in the interferon treated cultures were related to controls without interferon (100 per cent).

Fig. 6 . Scanning electron micrograph of untreated U-line human amnion cells. Whereas most of the cells are polygonal of shape and somewhat flattened on the substrate, some spherical cells are also present. Note the numerous slender cytoplasmic ex- tensions (microvilli) . Some of the round cells show blebs.

Fig. 8. Control cells. Detail of a spherical cell with numerous blebs and some microvilli.

c Fig. 7. Control cells. Detail of a spherical cell with numerous microvilli.

23 Acta path. microbiol. scand. Sect. 8, 84, 6 353

fwon treated cultures contained less radioactivity indicating a reduced release (Fig. 4). The differences were reproducible and signif- icant. A control experiment indicated that the remaining radioactivity, approximately 80 per cent in the case of control cells and 90 per cent in the interferon treated cells, was associated with the cells (Table 1). The inhibitory effect of interferon on the spontaneous release of radioactive substance was dose-dependent, up to 10 units interferon per ml. Further increase of concentrations did not enhance the effect (Fig. 5). The effect seems to be maximal during the initial period. Longer incubation period seems to reduce the diferences between total release from interferon treated cells and control cells.

Scanning Electron Microscopic Observations Control cells. Control cells, as well as the

interferon treated cells were cultured under conditions as identical as possible. The cells were not removed from the coverslip (on which they had been growing) during the preparation procedure for scanning electron microscopy. As shown on Fig. 6, most cells were partly spread on the surface of the glass, but with a rounded shape in the region containing the nucleus. Long, flattened pseudo- podia were often seen. On the surface, numerous slender cytoplasmic projections with a diameter of about 0.1 micron were found. Some of them appeared to be bridging neigh- bouring cells (Figs. 6-8).

Rounded-up cell forms were also seen (Fig. 6) . These were covered either with numerous microvilli (Fig. 7) or with a mixture of microvilli and cytoplasmic blebs (Fig. 8).

After 72 hours’ incubation the number of cytoplasmic projections appeared to be slight- ly reduced as compared with the specimens incubated for 24 hours.

Fig. 9. U cells treated with interferon, 200 units per ml for 24 hours. Both flattened and spherical cell types are present. The rounded cells contain microvilli as well as blebs.

23*

Interferon treated cells. In cell cultures treated with the different concentrations of interferon, no clear-cut differences could be demonstrated between the cells treated with different concentrations or without interferon. The growth pattern, the cell shape and surface with the numerous microvilli or blebs corresponded to those seen in the control specimens (Figs. 9 and 10).

D I S C U S S I O N

Our results confirm the earlier observations that interferon has profound effects on the homologous cells, effects which can be registered by a number of different biological parameters. Comparable amounts of interferon reduced the replication of VSV and the cytopathogenic effect caused by it, reduced the growth rate of uninfected cells, and reduced the spontaneous release of 3H-uridine from the cells.

Under the experimental conditions employed in this study interferon did not influence the process of virus release from the infected cells, but release of radioactive substances from uninfected cells was reduced. Reports from several laboratories indicate that, in some cases, especially those of onco- genic RNA viruse, a late step in the multiplication, maturation or release is affected by interferon (1, 9 ) . One of the alternative hypotheses forwarded is that interferon or the antiviral protein might interact with the cell membrane to inhibit virus release (1). Our results may support the notion of membrane stabilization by interferon. It may also be recalled, that there is no convincing evi- dence that interferon actually passes beyond the cell membrane (10).

To our knowledge, morphological alterations of the cell surface as a result of interferon treatment have not been reported, but none of the earlier observations included scanning electron microscopy. Our scanning electron microscopic studies revealed surface characteristics of both the controls and the interferon treated cells with numerous microvilli and rounded cells with blebs. Such blebs

355

are common on cells in mitosis (24) and it may be suggested that the rounded cells are in an early phase of mitosis. There was, how- ever, no clear difference in surface appearance of the interferon treated U cells and controls, even when the concentration of interferon was as high as 2000 units per ml, many times more than necessary to produce numerous functional alterations. Functional alterations can, of course, be present without interferring with the surface appearance and structures like microvilli and blebs formation. We believed that some data indirectly suggested that such changes might occur. Dibu- tyryl cyclic A M P treatment caused regression of the numerous long cell surface microvilli present on the L 929 cells (26). Further- more, several reports show that there is a relationship betwen the interferon system and cyclic AMP. Interferon treatment increases the cellular cyclic A M P levels both in uitro and in uiuo (4, 25). The interrelationship between cyclic A M P and interferon seems to vary in different cells, even in the same type of cells of different ages (19). I t is possible that, under different experimental conditions or in another cell type, the interferon treatment could produce visible changes, although mouse interferon treatment of L-F, fibroblast cells under the same conditions as in this study did not result in morphological changes that could be observed by light microscopy and scanning electron microscopy (Degre' & Houig, unpublished observations).

6.

7.

a.

9.

10.

11.

The excellent technical assistance of Mrs Solveig Beck and Mrs Sigrid Lystad is gratefully ac- knowledged.

R E F E R E N C E S

1. Billiau, A., Edy, V . G., Sobis, H . & DeSomer, P.: Influence of interferon on virus-particle synthesis in oncornavirus-carrier lines. 11. Evi- dence for a direct effect on particle release. Int. J. Cancer 14: 335-340, 1974.

12.

13.

14.

Fig. 10. Detail of interseron treated cell with both microvilli and blebs.

15.

Borecky, L., Fuchsberger, N. , Hajnickd, V., Stancek, D . & Zemla, I.: Distribution of antiviral and cell-inhibitory activity of interferon preparations. Acta Virol. 16: 356-358, 1972. Bourgeade, M.-F.: Interferon cell species specificity: Role of cell membrane receptors. Proc. S o c . exp. biol. Med. (N.Y.) 146: 820- 824, 1974. Braun, W. & Levy, H . B.: Interferon preparations as modifiers of immune responses. P m . SOC. exp. biol. Med. (N.Y.) 141: 769- 773, 1972. Cantell, K. , Hirvanen, S., Mogensen, K . E. & Pyhiilii, L.: Human leukocyte interferon: production, purification, stability and animal experiments. In V i m , Monograph no. 3: 35-38, 1973. Dahl, H . & Degrk, M . : A microassay for mouse and human interferon. Acta path. microbiol. scand. Sect. B, 80: 863-870, 1972. Dahl, H . & Degrk, M . : Human interferon and cell growth inhibition. I. Inhibitory effect of human interferon on the growth rate of cultured cells. Acta path. microbiol. scand. Sect. B, 84: 285-292, 1976. Fontaine-Brouty-Boyk, D . , Gresser, I . , Maci- eira-Coelho, A., Rosenfield, C . & Thomas, M . T.: Inhibition in vitro de la multiplication des cellules IeucCmiques murines L12 10 par des prkparations d'interferon. C. R. Acad. Sci. (Paris) 269: 406-408, 1969. Friedman, R. M . , Chang, E, H . , Ramseur, J . M . & Myers, M . W.: Interferon-directed inhibition of chronic murine leukemia virus production in cell cultures: Lack of effect on intracellular viral markers. J. Virol. 16: 569-

Friedman, R. M. & Sonnabend, J . A.: Mech- anism of action of interferon. Arch. intern. Med. 120: 51-63, 1970. Gresser, I . , Brouty-Boyk, D. , Thomas, M . T . & Macieira-Coelho, A. : Interferon and cell division. I. Inhibition of the multiplication of mouse leukemia L 1210 cells in vitro by interferon preparations. Proc. natl. Acad. Sci. 66: 1052-1058, 1970. Horak, I . , Iungwirth, C. & Bodo, G . : Poxvirus specific cytopathic effect in interferon-treated cells. Virology 45: 456-462, 197 1. Huang, K . Y., Donahoe, R . M . , Gordon, F. B. & Dressler, H . R. : Enhancement of phagocytosis by interferon containing preparations. Infect. Immunity 4: 581-588, 1971. Huet, C. , Gresser, I . , Bandu, M . T . & Lin- dahl, P.: Increased binding of concanavalin A to interferon-treated murine leukemia L 1210 cells. Proc. S o c . exp. biol. Med. (N.Y.) 147: 52-57, 1974. Katz , L . J., Lee, S . H . S . & Roree, K . R. : Interferon-mediated increased lysis of L-cells

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by vesicular stomatitis virus. Canad. J. Micro- biol. 20: 1077-1083, 1974.

16. Lindahl, P., Leary, P. & Gresser, I . : Enhance- ment by interferon of the specific cytotoxicity of sensitized lymphocytes. Proc. natl. Acad. Sci. 69: 721-725, 1972.

17. McNeill, T . A . & Gresser, I . : Inhibition of haemopoetic colony growth by interferon preparations from different sources. Nature, New Biol. 244: 173-174, 1973.

18. Paucker, K . , Cantell, K . & Henle, W. : Quan- titative studies on viral interference in sus- pended L cells. 111. Effect of interferring vi- ruses and interferon on the growth rate of cells. Virology 17: 324-334, 1962.

19. Reizin, F. N., Roikhel, V . M . & Chumakov, M . P.: The influence of substances changing the intracellular concentration of cyclic Ade- nosine 3’5’-monophosphate on interferon synthesis in chick embryo cell culture. Arch. Virol. 49: 307-315, 1975.

20. Stewart, W . E. ZI, Gosser, L. B. & Lockart, R . Z . jr.: Priming: a non-viral function of interferon. J. Virol. 7: 792-801, 1971.

21. Stewart, W . E. ZZ, DeClercq, E., Billiau, A., Desmyter, J . & DeSomer, P.: Increased sus- ceptibility of cells treated with interferon to

the toxicity of polyriboinosinic-polyribocytidylic acid. Proc. natl. Acad. Sci. 69: 1851-1854, 1972.

22. Stewart, W . E. ZZ, DeClercq, E. & DeSomer, P.: Specificity of interferon induced enhancement of toxicity for double-stranded ribo- nucleic acids. J. gen. Virol. 18: 237-246, 1973.

23. Thelestam, M. , Mollby, R . & Wadstrsm, T . : Effects of staphylococcal alpha-, beta-, delta-, and gamma-hemolysins on human diploid fibroblasts and HeLa cells: evaluation of a new quantitative assay for measuring cell damage. Infect. Immunity 8: 938-946, 1973.

24. Vesely, P . & Boyde, A.: The significance of SEM evaluation of the cell surface for tumor cell biology. Proc. Workshop on electron Mi- croscopy in Pathology. IIT Res. Inst. Chicago, 1973, pp. 689-696.

25. Weber, J . M . & Stewart, R . B.: Cyclic AMP potentiation of interferon antiviral activity and effect of interferon on cellular cyclic AMP levels. J. gen. Virol. 28: 363-372, 1975.

26. Wi!lingham, M . C . & Pastan, I . : Cyclic AMP modulates formation and agglutinability in transformed and normal mouse fibroblasts. Proc. natl. Acad. Sci. 72: 1263-1267, 1975.

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